gene function and cellular pathways in higher vertebrates, including humans, have increasingly shown to be highly conserved thr isj 6: 21, 2009 issn 1824-307x review erratum to: immunorecognition and immunoreceptors in the cnidaria [6: 7-14, 2009] sr dunn center for marine studies, university of queensland, australia in the above article affiliation was reproduced incorrectly, and it should have appeared as below: sr dunn center for marine studies and arc centre for excellence for coral reef studies, the university of queensland, st lucia, brisbane, qld 4072, australia 21 isj094.pdf isj 2: 28-31, 2005 issn 1824-307x short communication effect of pyridoxine on the reproduction of the mulberry silkworm, bombyx mori l. (lepidoptera: bombycidae) si faruki department of zoology, rajshahi university, rajshahi, bangladesh accepted march 30, 2005 abstract the present investigation reports the effects of vitamin b6, also known as pyridoxine supplemented feed on the reproductive potential of bombyx mori l. all the concentrations of the vitamin significantly reduced the fecundity. egg-viability was also reduced at all the concentrations, but the differences were not significant in comparison to control. key words: vitamin; reproduction; bombyx mori l introduction in silk industry, fecundity and fertility of the bombyx mori l female are two major factors because these are directly correlated with silk-production determining the number of offspring which offers the production of considerable amount of raw silk. nutrition is an important growth regulating factor in silkworm. it has been reported that the vitamins of b-complex group and certain essential sugars, proteins, amino acids, minerals etc. are responsible for the proper growth and development of the silkworm, b. mori (horie and ito, 1963; horie et al. 1966; sengupta et al., 1972; khan and saha, 1997a; faruki, 1998). a number of researchers works on the effects of vitamin enriched food on the reproduction of b. mori females (khalequzzaman and mannan, 1982; faruki et al., 1992; khan and saha, 1996,; saha and khan, 1996, 1999). pyridoxine also known as vitamin b6, is part of the b group vitamins and is water soluble. it stimulates growth and is important in protein metabolism, and its deficiency in mammals results in a decrease in phosphorylases (anonymous, 1998). the present work, therefore, was attempted to determine the effects of pyridoxine on the fecundity and egg-viability of the two strains of b. mori. corresponding author: saiful islam faruki department of zoology, rajshahi university, rajshahi, 6205, bangladesh e-mail: faruki64@yahoo.com materials and methods to determine the effect of pyridoxine, the eggs of the two multivoltine strains of b. mori viz. nistari-m and urboshi-1 were collected from the germplasm bank of the bangladesh sericultura research and training institute, rajshahi. they were then disinfected with a 2% formalin solution and kept in the rearing room for hatching. the rearing room and other appliances used in the present experiment were disinfected with a 5% formalin solution. newly-hatched larvae were brushed to wooden rearing trays (40 x 29 x 7.5 cm) and were reared on finely-chopped fresh, tender mulberry (morus alba l.) leaves up to the second moult. for this study the source of pyridoxine was pyrol® (pyridoxine hydrochloride hcl, 25) supplied by jayson pharmaceuticals ltd., bangladesh the concentrations of pyridoxine used in the present experiment, viz. 10, 100, 500 and 1000µg/ml were prepared by adding the requisite amounts of the vitamin in distilled water. the leaves were treated by dipping in these solutions and dried by fanning. treated leaves were then supplied to the silkworm larvae from the first day of third instar till spinning. the larvae of a control batch was simultaneously reared on fresh mulberry leaves dipping in distilled water only and dried by fanning. from the fourth instar onwards entire mulberry leaves of both treated and untreated groups were supplied to the larvae. feeding was supplied four times a day at six hour intervals. three replications, each with 50 larvae, were made for each pyridoxine concentration. the rearing trays were kept in fine-netted cabinets. 29 table 1. effect of pyridoxine on the fecundity of b. mori females (n = 15) concentrations (µg/ml) eggs laid mean ± sd prc f-ratio silkworm strains 0 (control) 365.53 ± 35.62 � 10 292.33 ± 13.96 20.03 nistari m 100 300.47 ± 19.90 17.80 4.78* 500 316.80 ± 18.79 13.33 1000 335.00 ± 29.89 8.35 0 (control) 474.87 ± 49.43 � 10 361.00 ± 35.83 23.98 urboshi 1 100 401.47 ± 17.37 15.46 3.73* 500 417.40 ± 37.78 12.10 1000 429.87 ± 35.56 9.48 note: * significant at p = 0.05; prc = percent reproduction control; f = variance ratio table 2. effect of pyridoxine on the viability (%) of b. mori eggs silkworm strains concentrations (µg/ml) mean ± sd prc f-ratio 0 (control) 81.31 ± 5.12 � 10 72.73 ± 3.90 10.55 nistari m 100 75.65 ± 4.14 6.96 1.08 ns 500 77.50 ± 4.33 4.69 1000 79.39 ± 2.94 2.36 0 (control) 97.44 ± 1.20 � 10 90.98 ± 3.45 6.63 urboshi 1 100 93.97 ± 3.25 3.56 2.09 ns 500 93.37 ± 2.98 4.17 1000 93.16 ± 1.92 4.39 note : ns = not significant; prc = percent reproduction control; f = variance ratio 30 mature larvae were transferred to bamboo-made mountages for spinning cocoons. after spinning and pupation the cocoons were harvested and stored according to their sexes. the sex was determined by cutting cocoons with a sharp blade and observing the external genitalia. freshly-emerged moths of opposite sexes were allowed to mate and the females were retained for oviposition. for each concentration, 15 females were observed for their fecundity. the number of eggs laid by individual female was carefully counted and the eggs were incubated to observe their viability. the number of larvae hatched out from the known number of eggs was carefully counted and viability (%) was calculated. data on fecundity and egg-viability were subjected to analyses of variance. here, the variance ratio f was calculated from the ratio between treatment mean square and residual mean square and the value was compared with the tabulated value for significance. the percent reproduction control (prc) was calculated by the formula of rizvi et al. (1980) as: prc = v1 – v2 / v1 x 100, where, v1 = eggs laid/ hatched from untreated control, v2 = eggs laid/ hatched from treated groups. all the experiments were conducted at a mean room temperature of 26 ± 1oc. results and discussion the average egg-productivity in the female b. mori moths of both the strains resulting from various concentrations of the vitamin was significantly (p < 0.05) reduced at the concentration used (table 1). the eggviability was reduced at all the concentrations in comparison to control but statistically the result was not significant (table 2). in present experiment, it was found that lowest number of eggs was produced at the lowest concentration (10µg/ml) and at higher concentrations it was increased but lower than control in case of both the strains. the same trend was also true for egg-viability. the present results are in agreement with the findings of faruki et al. (1992) who observed that para-amino benzoic acid (paba) significantly reduced the fecundity and egg-viability of b. mori females. pai et al. (1988) recorded a reduced reproductive potentials in the females of b. mori resulting from paba treatment. similarly, banerjee and khan (1992) observed that vitamin b6 enhance the oviposition of the bacillus thuringiensis var. kurstaki-infected b. mori, but the rate was lower than the controls. khan and saha (1997b), saha and khan (1997a) and khan et al. (2002) reported that the reproductive ability of female b. mori drastically reduced when reared on feed enriched with vertebrate sex-hormones. saha and khan (1997b) and khan and saha (2003) reported that higher concentrations of vitamins reduced the fecundity and fertility of b. mori. the fecundity and fertility of the silkworm females were reduced when they were reared on the feed supplemented with different artificial nutrients (khalequzzaman and mannan, 1982). the present work is a preliminary study on the use of vitamin b6 for the determination of the reproductive ability of the most commercially important insect. therefore, future more comprehensive works are very much solicited in this line with highest concentrations on this species is required. acknowledgements the author would like to extend the sincere thanks to the chairman of the department of zoology (rajshahi university) for providing necessary laboratory facilities and to the director of the bangladesh sericulture research and training institute (rajshahi) for kindly supplying the experimental eggs of the silkworm. references anonymous. instruction manual of pyrol®. jayson pharmaceuticals ltd., bangladesh. 1998. banerjee sk, khan ar. effect of vitamin b6 on the fecundity of bacillus thuringiensis var. kurstaki-treated bombyx mori l. bangladesh j. zool. 20: 361-362, 1992. faruki si, khan ar, mannan a. fecundity and fertility of the silkworm, bombyx mori l. fed on mulberry leaves supplemented with para-amino benzoic acid. bangladesh j. zool. 20: 351-353, 1992. faruki si. nutritive effects of thianomin® enriched mulberry leaves on the silkworm, bombyx mori l. univ. j. zool. rajshahi univ. 17: 39-44, 1998. horie y, ito t. vitamin requirements of the silkworm. nature 197: 98-99, 1963. horie y, watanabe h, ito t. nutrition of the silkworm, bombyx mori – xiv. further studies on the requirements for b vitamins. bull. seric. exp. sta., tokyo 20: 393-409, 1966. khalequzzaman m, manan ma. effect of artificial diet on certain bilological aspects of the silkworm, bombyx mori l. (bombycidae: lepidoptera). univ. j. zool. rajshahi univ. 1: 71-76, 1982. khan an, khan ar, rahman s. effect of lynestrenol® on the growth and development of the mulberry silkworm, bombyx mori l. (lepidoptera: bombycidae). sericologia 42: 573-578, 2002. khan ar, saha bn. nutritive effects of fe-plus® (ferous fumarate + folic acid) on the silkworm, bombyx mori l. bangladesh j. zool. 24: 195-203, 1996. khan ar, saha bn. nutrition of the mulberry silkworm, bombyx mori on feed supplemented with calcium lactate. j. ecobiol. 9: 53-58, 1997a. khan ar, saha bn. effect of vertebrate sex-hormones, mestranol and norethindrone, on growth and development of bombyx mori l. (lepidoptera). bangladesh j. zool. 25: 103109, 1997b. khan ar, saha bn. nutritive effects of thiamine on the silkworm, bombyx mori l. bangladesh j. zool. 31: 169-176, 2003. pai ik, hedge sn, krishnamurthy nb. paba induced genotoxic effects in silkworm, bombyx mori. proc. intl. symp. nature of genetic variation in man. xii annual conf. emsi, hyderabad, india, pp 54, 1988. rizvi sjh, pandey sk, mukerji d, mathur sn. 1,3,7trimethylxanthane, a new chemosterilant for stored grain pest, callosobruchus chinensis l. z. angew. ent. 90: 378381, 1980. saha bn, khan ar. effect of dietary supplementation of vitamins and minerals on the growth and development of bombyx mori l. bangladesh j. zool. 24: 125-131, 1996. saha bn, khan ar. effect of vertebrate sex-hormones on bombyx mori l. sericologia 37: 19-25, 1997a. saha bn, khan ar. the nutritive effects of sinafort®-b on bombyx mori l. entomon 22: 29-34, 1997b. 31 saha bn, khan ar. the growth and development of the silkworm, bombyx mori l. on feed supplemented with nicotinic acid. bangladesh j. life sci. 11: 103-109, 1999. sengupta k, singh bd, mustafi jc. nutrition of the silkworm, bombyx mori l. i. studies on enrichment of mulberry leaf with various sugars, proteins, amino acids and vitamins for vigorous growth of worms and increased cocoon crop production. indian j. seric. 11: 11-19, 1972. isj 5: 50-xx, 2008 isj 5: 50-53, 2008 issn 1824-307x short communication cytotoxic activity by the mussel mytilus galloprovincialis and the venus clam chamelea gallina in the adriatic sea in 2007 d malagoli, l casarini, f fiori1, e ottaviani department of animal biology, university of modena and reggio emilia, modena, italy 1coop m.a.r.e. soc. coop. a.r.l., cattolica (rn), italy accepted may 13, 2008 abstract given the ecological and economic importance of bivalve molluscs, the evaluation of their welfare is one of the primary aims for both biologists and people working in shell fishing. after a three year-long period monitoring the cytotoxic activity exerted by the hemolymph from the mussel mytilus galloprovincialis, we have concluded that cytotoxicity represents a useful parameter to evaluate the status of the immune activity and therefore the health of mussels in a specific period of the year. during 2007, we compared the mussel cytoxicity with that of the venus clam chamelea gallina from contiguous areas of the northern adriatic sea. our observations indicate that the cytotoxicity of the hemolymph of the two species follows a similar course during the year, suggesting that cytotoxic activity is primarily determined by the life/reproductive cycles. key words: mytilus galloprovincialis; chamelea gallina; mussel; venus clam; cytotoxicity; immunity; mollusc health introduction in view of their feeding behavior and their localization along the coasts or in internal waters, the identification of biomarkers able to represent reliably the welfare of bivalve molluscs is one of the primary goals for both biologists and people interested in the economic/environmental importance of these species (baršienė et al., 2006; pellacani et al., 2006). bivalves are usually considered as bioindicators, and their immune system is frequently chosen as the main target of these studies (ballarin et al., 2003; canesi et al., 2007a, b; malagoli et al., 2007b, 2008; novas et al., 2007a, b). as a result, the understanding of the parameters on which mollusc health relies has improved and led to a better evaluation of the real impact of exceptional situations connected with environmental catastrophes (laffos et al., 2006). since 2005, we have been studying the seasonal variations in the cytotoxic activity of the hemolymph from mytilus galloprovincialis grown in mussel farms in the northern part of adriatic sea, in order to understand ___________________________________________________________________________ corresponding author: davide malagoli department of animal biology via campi 213/d 41100 modena, italy e-mail: davide.malagoli@unimore.it whether this activity could be a useful parameter to evaluate the health of a specific mussel population (malagoli et al., 2006, 2007a). from these studies, we have concluded that cytotoxicity in m. galloprovincialis hemolymph is subject to cyclic modifications during the year (malagoli et al., 2006, 2007). to verify whether our considerations could also be extended to other bivalves, we here present data concerning the fluctuations of cytotoxic activity in both m. galloprovincialis and the venus clam, chamelea gallina, during 2007. moreover, to assess the impact of shell fishing on the health of molluscs, we have compared results collected from clams in areas in which the fishing was closed with those obtained from animals where fishing was regularly undertaken. materials and methods animals specimens of the bivalve mollusc, mytilus galloprovincialis, (minimum length 60 mm, except for september) were obtained monthly from local fishermen in the cesenatico area (fc, italy). immediately after collection, 40 animals were utilized to obtain the hemolymph as described below. a similar procedure was applied for 50 chamelea gallina (minimum length 25 mm), but the clams were sampled only four times in the year 2007: january (winter), may (spring), september (summer) and november (autumn). the c. gallina specimens came from the riccione area (rn, italy), about 23 miles from mussel farms in cesenatico. for each single period, 2 lots of 120 clams/each were subjected to hemolymph withdrawal. of the 2 lots, one came from a restricted area in which any kind of fishing was forbidden from august 2006 and for the whole period of the project (i.e. 15 months), while the second lot was collected about 1 mile away where clam fishing was regularly undertaken. hemolymph preparation and cytotoxicity assay the detailed procedure for the hemolysis assay in mussels has already been described elsewhere (malagoli and ottaviani, 2005), but there were some differences in procedure for m. galloprovincialis and c. gallina. briefly, the mussel hemolymph was collected by exerting gentle aspiration with a sterile syringe inserted between the mussel valves and filtered into sterile tubes (0.2 μm filter porosity). for c. gallina, the valves were opened with a razor blade, and the hemolymph was aspired with a syringe and subjected to centrifugation (13,000xg for 5 min). the supernatant was then carefully recovered in order to leave the pellet containing circulating cells and debris undisturbed. filtration was not used with c. gallina, since the collected volumes per animal where lower than 500 μl and therefore not compatible with the size of the syringe filters. the filtered/recovered hemolymph from each animal was kept separately and never pooled. in order to evaluate the hemolytic activity of the hemolymph from the two species, the cytolysis of human a positive erythrocytes following incubation for 1 h at 25 °c (hubert et al., 1997; malagoli and ottaviani, 2005) and subsequent centrifugation at 3000xg for 5 min at 4 °c was quantified using a spectrophotometer (abs 541 nm). samples with an optical density (od) exceeding the fixed threshold od level of 0,5 where considered cytotoxic (malagoli and ottaviani, 2005). for each lot of molluscs, the percentage of cytotoxic animals was then calculated. the experiments were repeated twice and in duplicate for each animal. results and discussion the monthly evaluation of the hemolytic activity of m. galloprovincialis hemolymph confirmed that significant fluctuations in the percentage of cytotoxic animals occur during the year (fig. 1a). the comparison of these data with those already collected in 2005 and 2006 confirmed that higher values are seen during the summer (malagoli et al., 2007a). the major discrepancies observed between data collected in 2007 and results in 2005 and 2006 (malagoli et al., 2007a) concerns the findings for january (nearly 100 % of animals positive in 2007) and september (nearly 0 % of animals positive in 2007) (fig. 1a). while for the data collected in january we have no indication of exceptional situations, the countertendency observed in september is most probably related to the introduction of new mussels (length < 20 mm). in that fig. 1 course state of mytilus galloprovincialis and chamelea gallina cytotoxicity of contiguous areas throughout 2007. a) course state of mussel grown in mussel farms of cesenatico; b) course state of venus clams coming form fishing or no fishing areas of cattolica. each histogram represent for every month the result of one out of a set of four independent experiments. period, hemolymph had to be taken from animals of significantly different size and age compared to the specimens usually sampled during the rest of the year. this result suggests the hypothesis that molecules responsible for hemolytic activity in m. galloprovincialis are less abundant in young animals. to our knowledge, there are no data in the literature concerning the cytotoxic activity of the hemolymph of c. gallina. consequently, the data we present here cannot be compared with other information related to this model. however, as with the mussel, we have also observed in the clam a fluctuation in the number of cytotoxic animals during the year (fig. 1b). no differences were seen between clams from restricted and open fishing areas (fig. 1b), and there were no differences between the characteristics of the water in the two areas during the year. therefore, normal fishing activity does not appear to have an impact on the health of the population. as far as the comparison between the m. galloprovincialis and c. gallina is concerned, it is worth noting that despite an always higher percentage of cytotoxic animals in the c. gallina population, the trend in the clams over the year is very similar to that observed for m. galloprovincialis (fig. 2). this last observation points to a possible similarity between the time course of cytotoxic activity in bivalves from contiguous areas. fluctuations in biological activity during the year are not new for mussels (cao et al., 2007; novas et 51 fig. 2 comparison between the course of hemolymph cytotoxic activity of m. galloprovincialis (mg) and c. gallina (cg) during the year. al., 2007b). in particular, variations in immune reactivity or the production of immune-related molecules have been recorded (barcia and ramosmartinez, 2008). various stress factors have been linked to alterations in molluscan immune responsiveness, particularly of the cellular component, i.e. the hemocytes (pampanin et al., 2002; ballarin et al., 2003; canesi et al., 2006; cao et al., 2007; novas et al., 2007b; malagoli et al., 2007b, 2008). however, cytotoxic activity is part of the humoral component of the immune system and is of protein origin (hubert et al., 1997). this means that cytotoxicity is not susceptible to rapid modification (i.e. within hours) as a consequence of a sudden alteration in an environmental parameter, because the rate of protein turnover and biosynthesis are the main causes of fluctuations in cytotoxic activity in the hemolymph. it remains to be established whether the component determining the time course of cytotoxicity is primarily environmental or rather connected to the mollusc life-cycle. from the present and previous data (malagoli et al., 2006, 2007a), it seems probable that environmental conditions may modify a state that is based on the life/reproductive cycle. thus, the cytotxic activity of the hemolymph has to be recommended as a valid parameter to determine the status of immune surveillance and welfare, especially for regular checks of mollusc welfare. indeed, for this purpose, the evaluation of cytotoxcity seems more reliable than rapidly changing parameters such as phagocytosis and lysosomal membrane stability, the levels of which may be influenced by manipulations and fishing procedures (pampanin et al., 2002; ballarin et al., 2003; malagoli et al., 2007b, 2008). acknowledgment this work has been in part supported by the centro di ricerche marine (cesenatico, fc, italy) and in part by the regione emilia-romagnaservizio economia ittica (italy) and the consorzio gestione pesca molluschi bivalvi (rimini division, italy) (project: “actions in chamelea gallina to define management strategies to improve production”). the authors also wish to thank the centro trasfusionale of the policlinico (modena, italy) for making available the blood and mr m marangoni who kindly provided the mussels. references ballarin l, pampanin dm, marin mg. mechanical disturbance affects haemocyte functionality in the venus clam chamelea gallina. comp. biochem. physiol. 136a: 631-640, 2003. barcia r, ramos-martinez ji. effects of interleukin2 on nitric oxide production in molluscan innate immunity. inv. surv. j. 5: 43-49, 2008. barsiene j, lehtonen kk, koehler a, broeg k, vuorinen pj, lang t, et al. biomarker responses in flounder (platichthys flesus) and mussel (mytilus edulis) in the klaipeda-būtinge area (baltic sea). mar. pollut. bull. 53: 422-436, 2006. canesi l, betti m, ciacci c, lo russo lc, pruzzo c, gallo g. cell signalling in the immune response of mussel hemocytes. inv. surv. j. 3: 40-49, 2006. canesi l, ciacci c, lorusso lc, betti m, gallo g, pojana g, et al. effects of triclosan on mytilus galloprovincialis hemocyte function and digestive gland enzyme activities: possible modes of action on non target organisms. comp. biochem. physiol. 145c: 464-472, 2007a. canesi l, lorusso lc, ciacci c, betti m, regoli f, poiana g et al. effects of blood lipid lowering pharmaceuticals (bezafibrate and gemfibrozil) on immune and digestive gland functions of the bivalve mollusc, mytilus galloprovincialis. chemosphere 69: 994-1002, 2007b. cao a, novás a, ramos-martínez ji, barcia r. seasonal variations in haemocyte response in the mussel mytilus galloprovincialis lmk. aquaculture 263: 310-319, 2007. hubert f, cooper el, roch p. structure and differential target sensitivity of the stimulable cytotoxic complex from hemolymph of the mediterranean mussel mytilus galloproíincialis. biochim. biophys. acta 1361: 29-41, 1997. laffon b, rabade t, pasaro e, mendez j. monitoring of the impact of prestige oil spill on mytilus galloprovincialis from galicia coast. env. int. 32: 342–348, 2006. malagoli d, ottaviani e. cytotoxicity as a marker of mussel health status j. mar. biol. ass. u.k. 85: 359-362, 2005. malagoli d, casarini l, ottaviani e. effects of the marine toxins okadaic acid and palytoxin on mussel phagocytosis. fish shellfish immunol. 24: 180-186, 2008. malagoli d, casarini l, ottaviani e. monitoring of the immune efficiency of mytilus galloprovincialis in adriatic sea mussel farms in 2005. inv. surv. j. 3: 1-3, 2006. malagoli d, casarini l, ottaviani e. monitoring of the immune efficiency of mytilus galloprovincialis in adriatic sea mussel farms in 2006: regular changes of cytotoxicity during the year. inv. surv. j. 4: 10-12, 2007a. malagoli d, casarini l, sacchi s, ottaviani e. stress and immune response in the mussel mytilus galloprovincialis. fish shellfish immunol. 23: 171-177, 2007b. 52 novas a, barcia r, ramos-martínez ji. after the prestige oil spill modifications in no production and other parameters related to the immune response were detected in hemocytes of mytilus galloprovincialis. aquat. toxicol. 85: 285-290, 2007a. pampanin dm, ballarin l, carotenuto l, marin mg. air exposure and functionality of chamelea gallina haemocytes: effects on haematocrit, adhesion, phagocytosis and enzyme contents. comp. biochem. physiol. 131a: 605-614, 2002. novas a, barcia r, ramos-martínez ji. nitric oxide production by haemocytes from mytilus galloprovincialis shows seasonal variations. fish shellfish immunol. 23: 886-891, 2007b. pellacani c, buschini a, furlini m, poli p, rossi c. a battery of in vivo and in vitro tests useful for genotoxic pollutant detection in surface waters. aquat. toxicol. 77: 1-10, 2006. 53 cytotoxic activity by the mussel mytilus galloprovincialis and the venus clam chamelea gallina in the adriatic sea in 2007 results and discussion the invertebrate blueprint of the connection between aging and immune neuroendocrine responses isj 6: 102-105, 2009 issn 1824-307x visions and perspectives the invertebrate blueprint of the connection between aging and immune neuroendocrine responses e ottaviani1, d malagoli1, c franceschi2 1department of animal biology, university of modena and reggio emilia, 41100 modena, italy 2department of experimental pathology, university of bologna, 40126 bologna, italy accepted july 13, 2009 abstract the present paper summarizes the main findings related to aging and immune neuroendocrine responses in invertebrates. in particular, the functional aspects and the genes involved are examined. a possible mechanism of correlation between aging and functioning of immune-neuroendocrinerelated genes is also discussed. key words: aging; longevity; immune neuroendocrine responses; gerontogenes introduction aging, or senescence, is a progressive somatic degeneration that provokes homeostatic modifications with damage to cellular functions and drastic inefficiency in survival. notwithstanding the presence of various stressors, such as bacteria, virus, radiations, heat, oxygen, and free radicals, the aging process is controlled by a variety of defense functions or anti-stress responses (franceschi et al., 2000). indeed, aging is a remodeling phenomenon that involves mainly the immune and neuroendocrine systems, while the aging phenotype can be interpreted as a global, adaptive response of the body to the age-related accumulation of nonrepaired damages, so allowing an optimal redistribution/utilization of the resources in order to survive (garinis et al., 2008; schumaker et al., 2009). in terms of mammalian immunosenescence, changes in adaptive and innate immunity have been observed. on the one hand, there is the involution of the thymus (nasi et al., 2006), a profound shrinkage in the t-cell repertoire (wack et al., 1998), and a massive increase of megaclones of memory cells directed against typical infections of old age (larbi et al., 2008). on the other, we see the activation of the macrophage that produces a large amount of cytokines and is responsible for the chronic inflammatory process in the aged organisms. this profound correlation between aging and inflammatory status has been described with the neologism “inflamm-aging” (franceschi et al., 2000). ___________________________________________________________________________ corresponding author: enzo ottaviani department of animal biology university of modena and reggio emilia via campi 213/d, 41100 modena, italy e-mail: enzo.ottaviani@unimore.it overall, during aging the immune system appears to concentrate on the recognition of external and internal environmental antigens met during life, while neglecting new encounters. furthermore, immunosenescence seems to be the result of a chronic hyper-stimulation of both adaptive and innate immunity (fagiolo et al., 1993). the appearance of adaptive clonotypical immunity from lower vertebrates onwards has given rise to great variability in the molecular structures devoted to the recognition of epitopes. in this context, the macrophage, a cell that plays a central role in the innate immunity, has long been neglected by the immunologists and has only recently assumed its own, correct, immune role. several data indicate that the macrophage has a profound evolutionary root, and in both vertebrates and invertebrates, the phagocytic capacity of these cells has always played a primary role in the stress response and inflammation. these activities share cells and mediators from vertebrates to invertebrates, suggesting a common origin (ottaviani et al., 2007). this idea is not completely new, but rather a reappraisal and extension of the phagocytosis theory of immunity proposed by elie metchnikoff, who was the first to propose the existence of an evolutionary mechanism devoted to the protection of organisms (metchnikoff, 1901). the macrophage is able not only to perform phagocytosis, but also to respond to different stimuli, such as bacterial products, neuropeptides, neurohormones, cytokines, and to release proinflammatory cytokines, e.g., nitric oxide, biogenic amines and neuropeptides, among others (ottaviani et al., 1997). in this light, various features and mechanisms of aging are a reshaping of old and conserved 102 functions of innate immunity (macrophage-centered) with the new functions of anticipatory immunity (lymphocyte-centered) and the neuroendocrine system (ottaviani, 1992; panerai and ottaviani, 1995). invertebrates functional age-related data aging has been extensively studied in two invertebrate models, drosophila melanogaster and caenorhabditis elegans. as far as we know, the freshwater snail lymnaea stagnalis is one of the first invertebrate models in which the relationship between aging and immune response was examined. in particular, juvenile samples of l. stagnalis presented fewer and less well developed circulating amoebocytes with more limited phagocytic capacity compared to adult specimens, suggesting that the immune system of juvenile snails is less competent (dikkeboom et al., 1984, 1985). this scenario could also explain the greater susceptibility to trichobilharzia ocellata infection of juvenile snails compared to adult specimens (meuleman et al., 1982; dikkeboom et al., 1985). the response to bacteria and caloric restriction are the main protocols in studying the effects of aging in flies and worms, respectively. it should be underlined that in these investigations, the researchers focused on the longevity aspect. although these studies are intriguing, some contradictions have been reported. it has been demonstrated that aged flies transcribe more antimicrobial peptide diptericin than young flies when exposed to septic bacterial infection, suggesting that this behaviour is due to immune senescence (zerofsky et al., 2005). aging also reduces the capacity to survive bacterial infection (ramsden et al., 2008). different findings were reported by ren et al. (2007), who showed that aged drosophila can tolerate a significant bacterial load and mount a large immune response without compromising survival. other experiments have demonstrated that caloric restriction counteracts aging and extends the lifespan of various organisms, including d. melanogaster and c. elegans (pletcher et al., 2002; lee et al., 2006). genetic age-related data in both d. melanogaster and c. elegans several genes have been seen to change their expression with age. in particular, chico, a drosophila homolog of vertebrate irs1-4, that plays an essential role in the control of cell size and growth is involved in the extension of lifespan in the flies (böhni et al., 1999; clancy et al., 2001). indeed, the use of mutants, such as chico1 homozygotes has resulted in increased median and maximum lifespan in females of up to 48 % and 41 %, respectively. chico1 heterozigotes showed an increase in median lifespan of up to 36 % and 13 % in females and males, respectively (clancy et al., 2001). in restricted diet conditions, elevated superoxide dismutase (sod) activity has been associated with an extension of lifespan, while increased sod activity (cuzn sod, but not mn sod activity) has been detected in chico1 homozygotes (kabil et al., 2007). when grown on a diet of escherichia coli, mutant alleles of age-1 and daf-2 increase the lifespan of c. elegans by 65 % and 100 %, respectively (friedman and johnson, 1988; kenyon et al., 1993). a further increase in lifespan is observed (100 % and 200 %, respectively) when the worms are maintained in axenic culture, and this increase is correlated to metabolic rate, suggesting that genes impact on longevity and senescence by regulating metabolic activities (vanfleteren and de vreese, 1995). the c. elegans germ-line directly influences its lifespan by modulating the activity of an insulin/igf-1 pathway that is involved in the regulation of worm aging. mutants able to reduce the efficiency of daf-2 (an igf-1 receptor homologue) extend the worm’s lifespan, and this process requires daf-16 activity (hsin and kenyon, 1999). the gerontogenes are also involved in immuneneuroendocrine responses. the daf-2, age-1 and age-2 genes in c. elegans have been seen to increase resistance to bacterial pathogens (laws et al., 2004; evans et al., 2008). moreover, such mutations, which normally enhance life expectancy, were also able to increase resistance to death caused by bacterial pathogens such as pseudomonas aeruginosa and salmonella enterica. these experimental results suggest that longevity is associated with stress resistance and that immunological challenges are an integral part of the spectrum of environmental stressors. with regards the neuroendocrine response, it has been demonstrated that in drosophila serotonin controls stress behaviour by regulating the daf-2 insulin/igf-1 receptor signalling to the daf16/foxo transcription factor. two classes of serotonergic neurons that present distinct serotonergic receptors are able to influence specific aspects of daf-16/foxo functions (liang et al., 2006). a recent finding suggests that in c. elegans, a distinct daf-16-dependent signalling pathway is involved in the activation of genes that provide resistance to bacterial pathogens (miyata et al., 2008). these findings offer a molecular and genetic basis for our hypothesis that the immune response and stress (neuroendocrine response) are both highly interconnected and conserved over the course of evolution (ottaviani and franceschi, 1997; ottaviani et al., 2007). concluding remarks the data reported above points to a relationship between aging and the efficacy of immune and stress responses. however, it is not easy to understand how these events are correlated. deveale et al. (2004) raise two questions: 1) why does the immune response change with age? 2) is this change aging-related or does it contribute to aging? with regards invertebrates, we can surmize an explanation. we have postulated that the immune and neuroendocrine systems have a common origin in which the macrophage is an immuneneuroendocrine effector system integrating innate 103 fig. 1 a schematic representation of the macrophage (ø) as the witness of the common origin of the immune and neuroendocrine systems. a) the normal homeostatic condition; b) during aging immunity, stress and inflammation (ottaviani and franceschi, 1997; ottaviani et al., 2007). this defense network may be interpreted as an equilateral triangle whose angles represent immunity, stress response and inflammation, while the macrophage sits in the middle (fig. 1a). in this unitarian perspective, these phenomena can be seen as an integrated network, with a common objective, i.e., the maintenance of body homeostasis. however, in different situations, one phenomenon may prevail over the others. as previously reported, the proinflammatory status of aging (inflamm-aging) is the result of the chronic activation of the macrophage. in this condition, the prevalence of inflammation over the other two parameters (immunity and stress) induces a change in the geometric shape to a scalene triangle (fig. 1b) representing the state of senescence. as far as invertebrates are concerned, old d. melanogaster present an inflammatory status (pletcher et al., 2007), suggesting that the causes and consequences may be the same in metazoans. from an ecoimmunology point of view, resources in the immune and neuroendocrine systems must be redistributed during aging in order to minimize the cost in terms of energy expenditure (ottaviani et al., 2008). our hypothesis of a reduced immune response as a consequence of a persisting inflammatory status could represent an answer to the questions raised by deveale et al. 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pirimiphos-methyl treatments alone or in combination ahm kamaruzzaman1, ams reza2, kamsh mondal3, s parween2 1institute of biological sciences, rajshahi university, rajshahi 6205, bangladesh 2 department of zoology, rajshahi university, rajshahi 6205, bangladesh 3member, bangladesh public service commission, agargaon, dhaka, bangladesh accepted november 03, 2006 abstract newly hatched (24 h old) larvae of tribolium castaneum and t. confusum were allowed to feed on different doses of cyromazine or pirimiphos-methyl, or on a combined dose of both compounds up to pupation. all the treatments produced deformities at all the life stages. cyromazine produced a number of abnormalities in the larval stage (p < 0.001) of the two species. both the compounds produced similar type of deformities in the adults, but the effect was slightly more in the female t. confusum. the combined action (10 ppm cyromazine + 0.1 ppm pirimiphos-methyl) of the compounds also produced deformities at each stage; and the effects were more pronounced than the effect caused by a single dose of either 10 ppm cyromazine or 0.1 ppm pirimiphos-methyl, and this will produce less stress on the environment and human health. key words: abnormalities; tribolium; cyromazine; pirimiphos-methyl introduction insects surviving insecticidal treatments very often become variously deformed, and the formation of chimeric individuals and elytral deformities in adults are very common. the organophosphorous insecticide pirimiphos-methyl has been reported to produce morphogenetic abnormalities in treated insects including tribolium species (khan, 1981; mondal, 1984; rahman, 1992). the insect growth regulators (igrs) are also able to produce various morphological abnormalities in treated insects (stall, 1975). among the igrs, chitin synthesis inhibitors (csis) interfere with the formation of new cuticle (hajjar, 1985), and disturb the process of ecdysis. a number of these compounds affects moulting in insects (hajjar, 1985), among which the triazine compounds are effectively used to control dipteran insects in poultry (bloomcamp et al., 1987), animal house (el-oshar et al., 1985) and public health (awad and mulla, 1984; nelson et al., 1986). cyromazine, derived from azido-triazine herbicides (shen and flapp, 1990), is effective against dipteran larvae (fox, 1990); but was ___________________________________________________________________________ corresponding author: dr. a.m. saleh reza department of zoology, rajshahi university rajshahi 6205, bangladesh e-mail: salehbgd@yahoo.com found to be active against the larvae of colorado potato beetle, leptinotarsa decemlineata (say) (bishop et al., 1990; sirota et al., 1993; sirota and grafius, 1994). as a csi compound, cyromazine affects growth and development in different insect species, including t. castaneum (herbst) (mondal and port, 1995). the present study is aimed to assess the abnormalities produced in flour beetles tribolium castaneum (herbst) and t. confusum duval due to the activities of cyromazine and pirimiphos-methyl alone or in combination, using low doses of both the compounds for protection of the stored products destined for human consumption. materials and methods insects used laboratory strains of tribolium castaneum and tribolium confusum were used in the present experiment. the insects were collected from the ipm laboratory, institute of biological sciences, rajshahi university. the stock cultures of these species were maintained in the laboratory for the last 15 years. compounds used the tested compounds were a chitin synthesis 97 mailto:salehbgd@yahoo.com table 1 larval deformities produced by cyromazine or pirimiphos-methyl alone and by their combination in t. castaneum (n = 250) treatment (ppm) pupal recovery (%) no. of abnormal larvae no. of larvalpupal intermediates total no. of abnormal larvae (%) control (0) 241 (96.40) cyromazine 10 20 30 228 (91.20) 215 (86.00) 197 (78.80) 09±0.23a 14±0.04b 17±0.31b 04±0.64a 07±0.5a 09±0.33ab 13±0.42 (5.20)a 21 ± 0.67 (8.40)b 26±0.34 (10.40)bc pirimiphos-methyl 0.1 0.2 0.4 230 (92.00) 196 (78.40) 180 (72.00) 07±0.41a 15±0.36b 19±0.1b 00 04±0.06a 05±0.72a 07± 0.48 (2.80)a 19 ± 0.39 (7.60)b 24±0.05 (9.60)b cyromazine + pirimiphos-methyl 10 + 0.1 190 (76.00) 16±0.7 08±0.48 24±0.55 (9.60)b note: data with the same letters do not differ significantly from each other (p > 0.05 dmrt) inhibitor, cyromazine and an organophosphorous insecticide pirimiphos-methyl. cyromazine was kindly supplied by the ciba geigy as larvedex ca 98.4% wp formulation. pirimiphos-methyl was purchased from the local agrochemical shop. doses used doses were prepared by mixing required amount of each of the compound and mixed with standard food medium (19:1, whole wheat flour: brewers’ yeast) to obtain the doses in ppm unit. the doses prepared for cyromazine were 10, 20 and 30 ppm and those for pirimiphos-methyl were 0.1, 0.2 and 0.4 ppm. the single combined dose used was prepared as 10 ppm cyromazine + 0.1 ppm pirimiphos-methyl (lowest doses of both the compounds). the doses of cyromazine and pirimiphos-methyl chosen were based on the mortality tests of the compounds on the larvae of t. castaneum and t. confusum (kamaruzzaman et al., 1999; kamaruzzaman, 2000). experimentation newly hatched larvae (12 h old) of both species were collected separately from sub-cultures of the beetles. the larvae were released in food medium treated with different doses of either cyromazine or pirimiphos-methyl or cyromazine + pirimiphosmethyl. the larvae were reared up to pupation. after every three days the food material was replaced by a fresh one treated with same dose and compound. the pupal recovery (%) was recorded. the pupae were sexed according to halstead (1963) and kept separately for emergence of adults. adult recovery (%) was recorded. the morphologically abnormal individuals were separated. the deformed characters were studied, and the number of abnormal individuals was counted for each life stage. a set of control larvae was reared similarly on untreated food. fifty larvae of each species were used for each treatment, each dose and control. the experiments were carried at 30±1 0c in an incubator, without controlling light and humidity, and replicated five times. statistical analyses the abnormalities produced by different treatments alone or in combination and in different species of tribolium were tested for significance using analysis of variance (anova). the effect of the different doses of each treatment with respect to control was tested with duncan’s multiple range test (dmrt). results abnormal characters produced cyromazine treatment produced various types of abnormalities in the larvae and adults of both species. abnormalities produced by cyromazine were recorded as follows: a) larval abnormalities i) reduced body size, ii) swelling in the integument/cuticular lesions, iii) stiffness of the cuticle, iv) incomplete metamorphosis: larviform pupae, pupal head with larval body, and pupa with larval skin. 98 table 2 larval deformities produced by cyromazine pirimiphos-methyl alone and by their combination in t. confusum (n = 250) treatment (ppm) pupal recovery (%) no. of abnormal larvae no. of larvalpupal intermediates total no. of abnormal larvae (%) control (0) 240 (96.00) cyromazine 10 20 30 230 (92.00) 221 (84.40) 209 (83.60) 10±0.88a 15±0.53ab 21±0.55b 05±0.72a 06±0.6a 08±0.46a 15±0.49 (6.00)a 21±0.55 (8.40)b 29±0.32 (11.60)c pirimiphos-methyl 0.1 0.2 0.4 227 (90.80) 198 (79.20) 195 (78.00) 08±0.52a 12±0.62ab 14±0.55b 00 05±0.46a 06±0.31a 08±0.51 (3.20)a 17±0.33 (6.80)b 20±0.05 (8.00)bc cyromazine + pirimiphos-methyl 10 + 0.1 200 (80.00) 15±0.43b 07±0.06a 22±0.71 (8.80)bc note: data with the same letters do not differ significantly from each other (p > 0.05 dmrt) b) adult abnormalities i) bent abdomen, ii) incomplete elytra. pirimiphos-methyl alone or in combination with cyromazine produced a similar type of deformities in both species, as follows: i) reduced larval body size, ii) larval-pupal intermediates, iii) adultoids and incomplete elytra in adults. the abnormal individuals with incomplete metamorphosis at larval-pupal transformation were categorized as larval abnormality and the adultoids as pupal abnormality. percentages of abnormalities produced cyromazine produced a higher percentage of abnormal larvae in t. confusum than t. castaneum (tables 1, 2). effect of cyromazine on the normal growth of larvae was found to be dose related (p < 0.001, f = 87.219), and was similar in both species (p > 0.05, f = 0.981). the number of abnormal larvae was higher than the number of larval-pupal intermediates, in both species of tribolium. pirimiphos-methyl produced comparatively less number of abnormal larvae in both species (p > 0.05, f = 1.31), though the effect varied with the doses (p < 0.001, f = 84.283) (tables 1, 2). the combined effect of the igr and the insecticide produced a greater effect on the morphogenesis of the larvae of both species (p > 0.05, f = 0.4). the effects of the combined treatment was more than the effects recorded with the lower doses of either cyromazine or pirimiphosmethyl (p < 0.001, f = 211.6) (tables 1, 2). cyromazine produced similar abnormal adults at same extent in both sexes of t. castaneum (table 3), whereas the effect was slightly greater in the females of t. confusum (table 4). the percentages of abnormal adults produced were not significant between the species (p > 0.05, f = 0.067); but significant differences of the effect was observed between the doses in t. confusum (p < 0.05, f = 9.25). pirimiphos-methyl treatments also produced similar percentage of adult abnormalities in both the species (p > 0.05, f = 0.72), which did not vary between the sexes of t. castaneum (table 3), but the females were more affected than the males of t. confusum (table 4). the adult morphogenesis of the beetles slightly varied among the doses (p < 0.01, f = 10.47). the combined treatment of cyromazine (10 ppm) and pirimiphos-methyl (0.1 ppm) produced a greater effect on the adult morphogenesis than the single treatment with either cyromazine or pirimiphos 99 table 3 adult abnormalities produced by cyromazine or pirimiphos-methyl alone and by their combination in t. castaneum (n = 250) no. of abnormal individuals (%) treatment (ppm) total adult recovery (%) male (no.) female (no.) male female control (0) 237 (94.80) 115 122 cyromazine 10 20 30 218 (87.20) 204 (81.60) 188 (75.20) 117 109 100 101 95 88 09±0.06 (7.69)a 09±0.2 (8.26)a 09±0.11 (9.00)a 06±0.21 (5.94)a 07±0.13 (7.37)a 12±0.31 (13.64)a pirimiphos-methyl 0.1 0.2 0.4 209 (83.60) 189 (75.60) 168 (67.20) 115 95 96 94 94 72 08±0.21 (6.96)a 09±0.05 (9.47)a 09±0.21 (9.37)a 07±0.07 (7.45)a 09±0.23 (9.57)a 13±0.15 (18.05)b cyromazine + pirimiphos-methyl 10 + 0.1 185 (74.00) 107 78 19±0.25 (17.76)b 15±0.22 (19.23)b note: data with the same letters do not differ significantly from each other (p > 0.05 dmrt) methyl at the same dosage (tables 3, 4). the combined treatment produced similar effects in both species of tribolium (p > 0.05, f = 0.034). discussion the mode of action of cyromazine is different from that of pirimiphos-methyl. being a chitin synthesis inhibitor, cyromazine affects the mechanical properties of the insect cuticle and produces abnormalities in the skin, and resists moulting (fox, 1990). inhibition of moulting results in increase of the internal body pressure in the larvae (fox, 1990), producing swellings on the cuticle (cuticular lesions) (kotze and reynolds, 1993; sirota et al., 1993; sirota and grafius, 1994). so, larval deformities are common in csi treated insects. the abnormal characteristics as noted in the present experiment, have been also reported in cyromazine treated colorado potato beele (sirota and grafius, 1994) and triflumuron (a csi compound) treated t. castaneum (parween, 1998). due to csi activity insect cuticle often becomes stiff (fox, 1990). consequently feeding is often hampered, as reported by neuman and guyer (1988), soltani (1984) and parween (1996) in different insects. even is some could survive, the starved larvae were reduced in body size. pirimiphos-methyl had been reported to avoid feeding on the treated medium tribolium species, and as a result adults loose weight and length (mondal, 1984b; rahman, 1992). pirimiphos-methyl affected the larval-pupal transformation and produced intermediate forms along with adult abnormalities. both cyromazine and pirimiphos-methyl affected growth of the emerged adults, which were superficially normal but with deformed elytrae. the combined action of cyromazine and pirimiphos-methyl to some extent was greater on the morphogenesis of tribolium species, than the action of single treatment of either compound. all the abnormal larvae and larval-pupal intermediates failed to survive long, and the abnormal adults were found to be uncapable to mate or oviposite. cyromazine is easily miscible with most of the standard insecticides and fungicides (fox, 1990), and in the present study was found to reduce the doses of insecticide used, producing the same effect. so, it can be used combined along with traditional insecticides at minimal doses for the protection of the stored products against beetle infestation, which may produce less stress on the environment and human health. both t. castaneum and t. confusum have become resistant against most of the insecticides used. for insect management in the grain and cereal stores, high doses of these insecticides are needed, which affect the human health, the environment and its biota. to overcome this problem, cyromazine could be used with pirimiphos-methyl against tribolium species, since this association would be able to produce abnormal individuals. the abnormal larvae or adults either would die or fail to 100 table 4. adult abnormalities produced by cyromazine or pirimiphos-methyl alone and by their combination in t. confusum (n = 250) no. of abnormal individuals (%) treatment (ppm) total adult recovery (%) male (no.) female (no.) male female control (0) 237 (94.80) 116 121 cyromazine 10 20 30 219 (87.60) 207 (82.80) 197 (78.80) 114 110 105 105 97 92 08±0.21 (7.02)a 08±0.03 (7.27)a 11±0.21 (10.48)b 06±0.04 (5.71)a 10±0.22 (10.31)b 14±0.5 (15.22)bc pirimiphos-methyl 0.1 0.2 0.4 221 (88.40) 194 (77.60) 188 (75.20) 116 101 102 105 93 86 06±0.06 (5.17)a 10±0.22 (9.90)b 14±0.22 (13.72)bc 10±0.15 (9.52)b 11±0.21 (11.83)b 10±0.03 (11.63)b cyromazine + pirimiphos-methyl 10 + 0.1 195 (72.78) 103 92 21±0.15 (20.39)c 11±0.3 (11.96)b note: data with the same letters do not differ significantly from each other (p > 0.05 dmrt) develop further or to reproduce, and ultimately the population would be controlled. moreover, as very low doses of these compounds could be used, the residues may not create hazard to the stored commodities as well as to the environment. references awad ti, mulla ms. morphogenic and 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d, mohamed aka. a laboratory study of cyromazine on aedes aegypti and culex quingfasciatus and its sensitivity on selected predators of mosquito larvae. j. am. mosq. control assn. 2: 296-299, 1986. neumann r, guyer w. a new chitin synthesis inhibitor cga 112 113: its biochemical mode of action as compared to diflubenzuron. proc. 10th 101 int. congr. plant prot. brighton, pp. 445-451, 1988. parween s. distribution and food consumption of larval and adult tribolium castaneum herbst on baycidal treated medium. j. biol. sci. 4: 113119, 1996. parween s. symptoms of triflumuron in toxicication in larvae of tribolium castaneum herbst (coleoptera: tenebrionidae). tribolium inf. bull. 38: 268-270, 1998. rahman asms. combined action of pirimiphosmethyl, synthetic methyl quinine and botanicals on tribolium confusum duval. ph. d. thesis, university of rajshahi, 1992. sher j, plapp fw. cyromazine resistance in the house fly (diptera: muscidae). genetics and cross resistance to diflubenzuron. j. econ. entomol. 83: 1689-1697, 1990. sirota jm, grafius e. effects of cyromazine on larval survival, pupation and adult emergence of colorado potato beetle (coleoptera: chrysomolidae). j. econ. entomol. 87: 577582, 1994. sirota jm, grafius e, ferrari b, kolarik p, seriber b,simstead s, boylan-pett w. control of colorado potato beetle with trigard. acarucide tests. 18: 155-156, 1993. soltani n. effects of ingested diflubenzuron on the longevity and the peritrophic membrane of adult mealworms (tenebrio molitor l.). pestic. sci. 15: 221-225, 1987. stall gb. insect growth regulators with juvenile hormone activity. ann. rev. entomol. 20: 417460, 1975. 102 introduction materials and methods insects used compounds used doses used experimentation results abnormal characters produced references prof isj 6: s3-s8, 2009 issn 1824-307x review professor giuseppe reverberi and the ascidian school in palermo f de bernardi department of biology, university of milan, milan, italy accepted march 13, 2009 giuseppe reverberi was born near perugia on 1901. after being ordained as a priest, he graduated in theology in 1924 from the pontificio ateneo lateranense. in the same year, he went to the university of rome, where he took another degree in natural science in the lab directed by federico raffaele, working on amphibian and chick embryos, under the guidance of professor pasquale pasquini. after the degree, he became an assistant at the zoological institute where started his studies in the field of experimental embryology, carrying out researches on the chick embryo-eye, on the centrifugation of the amphibian egg and on the potentialities of the amphibian tail bud: these researches were then continued in palermo by some of his collaborators. in 1930 he qualified to teach general embryology and became professor of experimental psychology and biology at the ateneo lateranense. in 1939 he was appointed at the university of perugia as director of the zoological institute. in the meantime, he worked at the zoological station of naples, a great attraction pole of international level for marine biology and a breeding ground of scientists. here he became director of the biological center of the national council for research and started to study the first stages of development of the ascidian embryos. in 1948, as a winner of a public competition, he was appointed as professor of zoology at the university of palermo, where he was director of the zoological institute and professor of biology at the medical school. in 1957 he founded the magazine acta embryologiae et morphologiae experimentalis, a latin title for an international journal published in english, the first in italy with an international editorial board. it represented a person whose broad knowledge was not limited to biology, but also covered philosophy, literature and theology. his researches, together with those carried out by the many research groups led by his collaborators were addressed essentially to developmental biology of several animals, overall marine invertebrates: annelids, ___________________________________________________________________________ corresponding author: fiorenza de bernardi department of biology university of milan via celoria 26, 20133 milan, italy e-mail: fiorenza.debernardi@unimi.it mollusca (dentalium) ctenophora, amphioxus and ascidians. in 1971 he published a fundamental textbook experimental embryology of marine and fresh-water invertebrates (north holland) which received a lot of quotations. for the above mentioned reasons, i wish to speak about the monumental scientific activity of professor reverberi, which lasted more than 50 years, and of his outstanding human personality. unfortunately, i did not have the honour to be one of his pupils, but i can remember very well two meetings. the first one was in 1968, at the zoological station of naples, during an experimental embryology course for young researchers: his tall figure, full of charisma, his availability to meet young people coming from different european countries for a dinner party. he was there with the teachers of the school, all the most outstanding embryologists of the 20th century: john runnstrom, sven horstadius, jean brachet, tryggve gustafson, gerhard czihak. i was very impressed by the friendly terms between professor reverberi and all those distinguished embryologists. it was a very important signal of his international reputation in a period (40 years ago) when not all the italian universities used to be so well-known abroad. i remember the meeting of the italian embryological group in 1977, an atypical club born in 1952 from the idea of some italian scientists on the model of the institut international d’embryologie (iie). the founders were pasquale pasquini, silvio ranzi, alberto monroy, alberto stefanelli, mario benazzi and, obviously, reverberi himself, who was a member of the iie. during this meeting, professor reverberi gave a lecture on the latest results and the perspectives of the ultrastructure and the cellular biochemistry of developing embryos, in which he compared and discussed the most recent results obtained in the palermo lab and in many other labs all over the world . i was very impressed by the fervour of his speech about the communication between nucleus and cytoplasm, one of his favourite subjects. ten years later, in 1987, i entered the institute of zoology in the historical place of via archirafi 18: this building was still characterized by his strong personality, even if his illness kept him away for many years. in fact, most researchers were working on ascidians and this was the demonstration that a real school had been created there; a place where the s3 fig. 1 prof. giuseppe reverberi at work in his room at the institute of zoology of palermo in 1965. same animal model was studied under manifold point of view. this fact impressed me very much as i was coming from the zoological institute of milan, where the researches used to be more heterogeneous. it was then inevitable to be overwhelmed by the contagious enthusiasm of his first collaborator and outstanding successor, giuseppina ortolani, for the manipulation of the embryos and eggs of the ascidians: one day i told her the technical troubles encountered when highlighting some rna markers in opaque xenopus embryos, and she said to me:“i worked a lot with xenopus eggs: my advice is to work with phallusia, which has big, transparent and wonderful eggs. try it and you’ll see!” so, i have been working for 20 years with ascidian embryos and i am feeling, even indirectly, an alumna of the famous ascidian school of palermo! reverberi’s scientific activity in palermo mainly involved the experimental embryology field, but the development of ascidian eggs always gained first priority among his interests. in the following sections i will try to define the main research lines concerning the ascidian development. development of egg fragments and isolated blastomeres when reverberi started to work on the development of ascidians, the current information on such matter was that developed by conklin, who wrote in the conclusion of his famous paper published in 1905: “the development of ascidians is a mosaic work because there are definitely localized organ-forming substances in the egg; in fact the mosaic is one of organ-forming substances rather than of cleavage cells. the study of ctenophores, nemertines, annelids, mollusks, ascidians and amphibians (the frog) shows that the same is probably true of all these forms and it suggests that the mosaic principle may apply to all animals.” it is interesting to follow the evolution during the time in which reverberi formed his opinion about the mosaicism based on experimental results. in one of his first papers, in 1931, reverberi describes the results obtained from the fertilization of egg fragments obtained by gentle crushing or by a small incision of the chorion. the fragments until 1/22 collected by a needle tip developed “as a whole” and gave normal larvae. his conclusion was “the ciona egg has to be classified among the regulative eggs” (reverberi, 1931). 1933: blastomeres separated at stages 2,4,8: the animal 4/8 developed blastulae; the vegetal 4/8 developed also gastrulae. “the cleavage is strictly partial (=incomplete), even if some aspects could make suspect of a regulation” (reverberi, 1933). 1936: “the partial cleavage appears only from the two blastomere stage onwards…; before this stage in fertilized egg the cleavage is of total type” (reverberi, 1936). in a frequently quoted paper, even if published only in italian (reverberi and minganti, 1946), we can find some very beautiful drawings, much more explanatory than modern photos. such drawings showed the results of the selective removal of two s4 blastomeres each time at 8-cell stage and the histological sections of the larvae obtained from the combination of the animal quartet with the two vegetal anterior blastomeres. this was the first paper unambiguously proving the existence of a “brain evocator” in the two vegetal anterior blastomeres and of a “ brain inhibitor” in vegetal posterior blastomeres. these figures became well known as reported in the book of nori satoh “developmental biology of ascidians” (1994), a textbook widely diffused in the world of ascidian researchers. in another paper of the following year (reverberi and minganti, 1947), the systematic combinations of blastomeres were described and it was shown that vegetal anterior blastomeres do not “induce” any presumptive epidermis to become brain, but they only “evocate” the neural presumptive ectoderm to become brain. these works were continued and reconsidered with other collaborators in the following years. they used the knowledge obtained in the meantime by ortolani about the cell-lineage of the single blastomeres until 64-cell stage and systematically removed the cells committed to give notochord or endoderm from stage 8 to 64. the single cells obtained were then transplanted under an isolated animal quartet. this great bulk of experiments, singularly evaluated, definitely showed that “the formation of the neural system in the ascidians is strictly directed by the same laws which spemann discovered in the amphibians. only slight differences are to be noticed between the inductive system in the amphibian and in the ascidians: in the amphibians, the inductor is chorda-mesoderm, in the ascidians it is (part of) the chorda-entoderm”. the observation that in the ascidians the inducing power of the chorda-endoderm was more restricted than in the amphibian led to support the idea of the “evocative” power instead of an “inducing” power, enclosed in a bright discussion among the embryologists at that time. these results were reported in another frequently quoted paper “the causal formation of the brain in the ascidian larva” (reverberi et al., 1960). it is to be noted that this discussion is coming close to a solution only in recent years, after the genome sequencing and the knowledge of the sequences involved in determination of embryonic territories. development of sense organs at the same time of the study on the induction of the nervous system and in the same papers was also studied the development of the principal sense organs: the otholith, the ocellus and the palps. a very elegant result was the cytochemical detection of the dopa oxidase, the enzyme activating the melanin precursor, only after the induction process at neurula stage. twenty years later, the problem of the sense organ development was resumed and in two papers was described the ultrastructure of the so-called “third sense organ”, probably dedicated to the hydrostatic pressure detection (reverberi, 1975, 1979). this organ is formed by a few cells bearing bulbous projections projected inside the cavity of the sensory vesicle, it was observed in many species and its function was related to the different ability of the larvae to move vertically along the water column. this topic was also innovative at that time: in fact the third organ was recently studied in several labs, and other hypotheses were developed for its function (e.g., neurosecreting cells or secondary sensorial neurons, imai and mainhertzhagen, 2007), but no hypotheses were considered more convincing than the hypothesis originally proposed by reverberi. causal analysis of embryonic development in a paper of 1939, published in commentationes pontificiae academiae scientiarum, with a latin abstract, reverberi resumed the experiments of fertilization of egg fragments obtained by centrifugation of the egg. the centrifuged eggs were divided in two parts, one completely hyaline and the other full of granules. only the latter, after fertilization, can develop even into larval stage, but the hyaline fragment cannot start any cleavage. from these observation, reverberi inferred that the construction of an organism starting from the zygote is the result of an ordered series of biochemical events. during the development of the ascidian egg the different constituent of the egg become more strictly segregated (plasm segregation) (reverberi and pitotti, 1939). the further development of these researches, carried out with collaborators, led to the cytochemical study of the segregation of rna and dna. in a review of 1960, published in advances in morphogenesis, reverberi states to trust the evidence that differentiating tissues have a higher concentration of rna (e.g., neural tissue at neurula stage), but he is sceptical about a different concentration of dna. in fact, at that time, the dispute about the constancy of the dna content was still going on. reverberi suggested that “the strong coloration of the nuclei of the nervous cells is not necessarily due to a higher content of dna, but, more likely, to the fact that the nuclei are more condensed”. interest was also shown in the mitochondrial respiratory enzymes. through a very accurate nadireaction and through various cytochemical reactions, it was observed that many enzymes accumulate following mitochondrial segregation in the yellow crescent, in vegetal posterior blastomeres, and in the musculature of the larva (reverberi, 1957a). the role of the enzymes in ascidian morphogenesis was established by blocking their activity with specific inhibitor. the main results were to ascertain a causal relation between the enzymatic activity and the differentiation of the muscular system, rich in mitochondrial enzymes. in fact the larvae obtained from the treatment with inhibitors showed a normal trunk, and a tail with various anomalies, all referred to a defective differentiation of the muscular fibers (reverberi, 1957b). also related to the previous ones, were the results of the cytochemical reaction for cholinesterase which can be noticed at neurulation not in the neural plate, as previously reported by other researchers, but in the muscular territories. s5 after the publication of these pioneer results (durante, 1956), the acetylcholinesterase as a marker of muscular differentiation was used by many authors in many labs (e.g., in usa by r whittaker, who visited several times the zoological institute in palermo, and in japan by n satoh). heterospecific hybridizations a lot of work was dedicated by reverberi to the heterospecific hybridizations. the results highlighted that, in order to obtain heterospecific fertilization, the eggs have to be naked, without chorion and follicular cells. the latter not only favour the floating of the eggs, but have also a function in speciesspecific recognizing.. the viable hybrid andromerogons (an enucleated egg of ascidia malaca fertilized with a sperm of phallusia mammillata) have only one set of paternal chromosomes. the morphological characters of the larva are, however, matroclinous, as the cytoplasm was “conditioned” by its nucleus much earlier, perhaps in the ovary. it must be noted that similar results have been observed in other labs on echinoderms. some studies carried out from 1955 to 1960 tried to explain this phenomenon by developing the embryos in sea water additioned with thymidin, adenine etc, in order to identify possible errors in the syntesis of dna and rna. but these experiments were too advanced at that time and the transcriptomic and proteomic were still far. very advanced were also the experiments on the fusion of the eggs of the same species or of two different species and subsequent fertilization with an heterospecific spermatozoon: giant triploid larvae were obtained and some time these larvae metamorphosed in juveniles (farinella et al., 1969). in order to better understand the great interest of reverberi for these studies it is important to remember that three chapters of his textbook “introduzione all’embriologia sperimentale” (1967), are dedicated to the heterospecific hybridizations. all the problems connected with the suppression of one of the two sets of chromosomes or their maintenance and integration, the “conditioning” of the diploid nuclei from differentiated cells and transplanted in the eggs, were debated in the sixties and had an intellectual appeal that can be grasped in the textbook. it is clearly resumed in the definition “the egg is an equipotential armonic system”. in the first chapter of the above mentioned book, entitled “birth and development of the embryology” a complex and fascinating historical account is outlined with clarity of style and the numerous latin quotations demonstrate that reverberi was a very cultured and intellectual person. effects of extraneous substances through a fusion of the two main research fields, the classical experimental biology and the chemical embryology, reverberi also tried a new way of manipulating the embryos: instead of using the glass needles, he used some chemical substances. the first of these new research lines on ascidian embryos was carried out with a classical, historical substance: lithium chloride (farinella ferruzza, 1955). treatment of unfertilized eggs produced normal larvae, but a treatment at stage 2 blastomeres gave rise to larvae lacking the trunk and all ectodermal derivatives, like nervous system and palps. the treatment at stage 64 gave apparently normal larvae, but deprived of nervous system and palps. the well known “vegetalizing” action of lithium chloride produced the absence of the neural induction (reverberi and farinella ferruzza, 1961). this research line was further developed and extended by some collaborators who also studied the effects of the environmental pollutants. another research line was the treatment of the embryos with chromomycin, actinomycin d or with aminoacids and their analogs, in order to modify the quality of the aminoacid pool leading the embryos to produce abnormal proteins. in all these cases a normal development was obtained when the treatment was carried out before the first cleavage. treatments in further stages until gastrula produced larvae with various degree of abnormalities (bramachary and reverberi, 1964). the results obtained by reverberi, together with the other results obtained in those years on amphibians, sea urchins and chickens were fundamental to establish that the transcription of embryonic rna starts at the blastula-gastrula stage and that the ascidians, even if traditionally considered examples of the mosaic development, “have many regulative aspects” as reverberi already wrote in 1932. the ascidian embryo ultrastructure in the fifties, electron microscopy was still in a pioneer phase when the first papers on the ultrastructure of the oocytes, of the unfertilized egg and of follicular and test cells were published. vincenzo mancuso and his collaborators worked actively in this research line. bearing in mind the lines of the previous researches, we shouldn’t be surprised that the electron microscopy was utilised to study the fine structure of the egg and of its constituents separated by centrifugation, in order to study directly what was inferred by cytological and cytochemical methods (reverberi and mancuso, 1960; mancuso, 1963). once established the unequal distribution of principal constituents of the cytoplasm, their segregation was observed during the cleavage in lineage of blastomeres defined by their morphogenetic commitment. in particular, the cell-lineage of the muscular cells, already outlined in the experiments of ortolani, was confirmed by the electron microscopy, from the richness in mitochondria of the committed blastomeres . i would like to mention a series papers on some “peroxidase cells”, so identified by ries through cytochemical reactions. these cells are present in two rows, between the ventral surface of the pharynx and the epidermis, in the mature larvae only in the species carrying a heavy tunic, such as a. malaca, p. mammillata, ascidiella aspersa. they have been considered of mesodermal origin, but materazzi and ortolani (1969) demonstrated that s6 they develop from the anterior vegetal blastomeres, an endodermal territory. these cells cross the ectoderm and join the tunic, where are probably active in the synthesis of mucopolissaccharids of the tunic. reverberi reported the electron microscopy observation of these cells, just sorted from the endoderm and rich in golgi apparatus and vesicles (reverberi, 1971). one of my first papers on the ascidians, carried out with the enthusiastic advice of ortolani, was about the cytoskeletal modifications of these “button cells” studied by immunofluorescence and confocal microscope while they penetrate the ectoderm (sotgia et al., 1993). immunobiology ascidian immunobiology has recently become very important in the department of animal biology in palermo but, surprisingly there are no studies on this matter in the papers of reverberi. precisely, this research line began with an idea of nicolò parrinello who decided to carry out the study on serum proteins through immunological techniques in order to solve some taxonomy problems. professor reverberi understood immediately the potentiality of this line and encouraged him to continue. in fact, one of the duties of a real master is also to support self-esteem and to encourage autonomy in young researchers! naturally, speaking of reverberi involves mentioning his collaborators and his school as well. in fact, all his students soundly developed their analytical skills thanks to their master, following and sometimes being ahead of the new research orientations suggested by the technical progress in biological research. in 2003, during the first international urochordate meeting, nori satoh, in the opening lecture “let’s move on ascidian biology with new ideas”, made an historical review on the ascidian researches and recalled the major scientists: chabry (1887), conklin (1905), reverberi (1931) and his collaborators ortolani and minganti. the meaning of this historical parade was clearly explained during the lecture: the school of palermo was leader in the experimental embryology for more than 50 years, mainly studying the ascidian development: its heraldic animal. references brahmachary rl., reverberi g. on the action of chromomycin on the eggs and embryos of ciona intestinalis. experientia 20: 621623,1964. conklin eg. mosaic development of ascidian eggs. j. exper. zool. 2: 146-223, 1905 durante m. cholinesterase in development of ciona intestinalis, experientia 12: 307-310, 1956 farinella ferruzza n. lo sviluppo embrionale delle ascidie dopo trattamento con licl pubbl. staz. zool. napoli 26: 42-54, 1955 farinella ferruzza n, reverberi g, single giant larvae of ascidia malaca double eggs. experientia 25: 651-652, 1969. imai jh, meinertzhagen ia. neurons of the ascidian larval nervous system in ciona intestinalis: i. central nervous system. j comp neurol 501: 335-352, 2007 mancuso v. distribution of the components of normal unfertilized eggs of ciona intestinalis examined at the electron microscope . acta embryol. morphol. exper. 6: 260-274, 1963. mancuso v. an electron microscopy study of the test cells and follicle cells of ciona intestinalis during oogenesis. acta embryol. morphol. exper. 8: 239-251, 1965. materazzi g, ortolani g. a study of the origin of the cells containing sulphated acid mucopolysaccharides in the cephalic portion of the larvae of phallusia mammillata and ascidia malaca. dev. biol. 20: 378-385, 1969. reverberi g. studi sperimentali sull’uovo di ascidia.i il comportamento e lo svilppo dei frammenti d’uovo di ciona nei diversi momenti compresi tra la deposizione e la segmentazione. pubbl. staz. zool. napoli 11: 170-193, 1931. reverberi g. studi sperimentali sull’uovo di ascidia. ii la segmentazione dei blastomeri isolate dell’uovo di ciona e di phallusia. pubbl. staz. zool. napoli 12: 385-406, 1933. reverberi g. la segmentazione dei frammenti dell’uovo non fecondato di ascidia. pubbl. staz. zool. napoli 15: 198-216, 1936. reverberi g. the embryology of ascidians. in adv. morphogen. 1: 55-101, 1960. reverberi g. introduzione all’embriologia sperimentale feltrinelli, milano, 1967. reverberi g. la citocromo-ossidasi lungo lo sviluppo delle ascidie.risultati che conseguono alla sua inibizione con azide sodico. pubbl. staz. zool. napoli 29:187-212, 1957a. reverberi g. some effects of enzymes inhibitors on the ascidian development . acta embryol. morphol. exper. 1: 12, 1957b. reverberi g. experimental embryology of marine and fresh-water invertebrates, north holland publ.co., 1971. reverberi g. osservazioni sulle cellule “a perossidasi” di alcune larve di ascidia col microscopio elettronico rend. acc. naz. lincei 50:795-798, 1971. reverberi g. on the third sensory receptor of ascidian tadpole. rend. acc. naz. lincei 58: 948-954, 1975. reverberi g. on the third sensorial organ of ascidian tadpoles., acta embryol. morphol. exper. 91-99, 1979. reverberi g, farinella ferruzza n. lithium ions and brain formation in the ascidian larvae. acta embryol. morphol. exper. 4: 239-249, 1961. reverberi g, mancuso v. the constituents of the egg of ciona intestinalis as seen at the electron microscope. acta embryol. morphol. exper. 3: 221-230, 1960. reverberi g, minganti a. fenomeni di evocazione nello sviluppo dell’uovo di ascidie. pubbl. staz. zool. napoli 20: 201-252, 1946. reverberi g, minganti a. la distribuzione delle potenze nel germe di ascidie allo stadio di otto blastomeri, analizzata mediante la combinazioni e i trapianti di blastomeri. pubbl. staz. zool. napoli 21:1-35, 1947. s7 reverberi g, ortolani g, farinella ferruzza n. the causal formation of the brain in the ascidian larva. acta embryol. morphol. exper. 3:296336, 1960. satoh n. developmental biology of ascidians. cambridge university press, cambridge, uk, 1994. sotgia c, fascio u, ortolani g, de bernardi f. behavior of endodermal “button cells” during metamorphosis of ascidian larvae. int. j. dev. biol. 37: 547-553, 1993. reverberi g, pitotti m. differenziazioni fisiologiche nell’uovo delle ascidie commentationes pont. acad. scientiarum 3: 471-488, 1939. s8 isj 3: 111-xx, 2006 isj 4: 13-17, 2007 issn 1824-307x short communication the effect of bmnpv infection on protein metabolism in silkworm (bombyx mori) larva k etebari 1, l matindoost 1, sz mirhoseini 1, mw turnbull 2 1dept. of sericulture, faculty of natural resources, university of guilan, somehe sara 1144, iran 2department of entomology, soils, and plant sciences, clemson university, 114 long hall, clemson, sc 296340315, usa accepted february 02, 2007 abstract grasseri is one of the most important diseases of silkworm with significant yield loss, which is caused by nuclear polyhedrosis viruses (npv). in the present research the effect of this disease on changes of biochemical compounds which are related to protein metabolism in 5th instar larvae were studied. the larvae that showed the grasseri symptoms after contamination with 5.5×10-4 polyhedral/ml were assumed as infected treatment. the hemolymph of infected and uninfected larvae in 3 and 5 days after 4th molting were collected and its total protein, urea, alanine aminotransferase (alt) and aspartate aminotransferase (ast) were measured. the results showed that the amount of all the compounds except urea were considerably different in both groups. total protein had decreased in infected larvae but activity level of two aminotransferases significantly increased. therefore, grasseri has a considerable effect on protein metabolism. key words: silkworm; nuclear polyhedrosis virus; protein metabolism; grasseri __________________________________________________________________________________________ introduction silkworm is susceptible to many diseases caused by viruses and this is one of the main problems in sericulture every year. generally, 70 % of damages caused by diseases are due to viruses (anonymous, 1976). among viruses, nuclear polyhedrosis viruses (npv) in recent years have caused the highest damage to silkworm (bombyx mori) in tropical regions (sivaprasad et al., 2003; biabani et al., 2005 a, b). bmnpv affect midgut epithelial cells, trachea system, hemolymph cells, fat body, etc. the nuclear of middle and inner cells of silk gland are also sometimes invaded by this virus (khurad et al., 2004). baculovirus infection starts when inclusion body is taken in by the sensitive insects. midgut fluid of lepidopteran larvae is totally alkali which digests the viral occlusion bodies. consequently, virions are released into alimentary system and cross the peritrophic membrane. they combine with midgut ___________________________________________________________________________ corresponding author: kayvan etebari dept. of sericulture, college of natural resources, university of guilan, somehe sara 1144, iran e-mail: etebari@guilan.ac.ir epithelial cells and enter nuclei starting the first cycle of viral production and replication. these processes cause many biochemical changes in larvae which respond to these biological phenomena with changing many of its metabolisms to defend against pathogen invasion. understanding and identifying these biochemical changes will be very important in discussing many biological stresses. additionally, silkworm and bmnpv make a good model for studying the interaction between insect and virus. so, determination of the biochemical responses in silkworm against bmnpv could facilitate the control of agricultural pests (gao et al., 2006). biochemical compounds of silkworm have been investigated under many stress conditions as an appropriate marker. serratia marcescens as one of the factors causing flasheri disease has been studied for the biochemical changes in silkworm hemolymph after infection and considerable decrease has been reported in total protein, carbohydrate and lipid (sam devdas et al., 1994). rami reddy et al. (1992) analyzed the effects of usi fly (exorista sorbillans) parasitism on activity levels of aminotransferases and showed that stress increases the activity of the enzymes in silkworm. the researches have shown that infection with npv does not have considerable effect on carbohydrates 13 mailto:etebari@guilan.ac.ir up to third day but with the time flow, the amount of carbohydrates is enhanced in infected larvae. however total lipid showed significant increase in the hemolymph from the first day of infection (sarma et al., 1994). the effects of npv on midgut enzymes of spodoptera litura were investigated by nathan et al. (2005) and it was demonstrated that alkaline phosphatase is decreased after infection by virus. conversely, matindoost (2006) showed that bmnpv had caused a considerable decrease in activity of this enzyme in silkworm after infection of a cell line established from silkworm embryo (bm-ek1). together with a drop in alkaline phosphatase activity, also many culture medium components such as cholesterol, urea and glucose showed considerable decrease. these findings confirm that viral infection has a significant effect on many cellular metabolisms. due to the importance of protein metabolism and its crucial involvement in many compensative and reductive processes involving biological stresses, in this study the effects of bmnpv infection on changes of some biochemical compounds which are related to protein metabolism was investigated. materials and methods bmnpv inoculum preparation larvae infected with grasseri were collected from a field in north of iran and their hemolymph was extracted into microtubes. the bmnpv in the larval hemolymph was confirmed by light microscopy of polyhedra. bmnpv polyhedra were purified as described by sugimori et al. (1990). samples were centrifuged at 10,000 rpm for 10 min to pelletize the bmnpv polyhedra. the polyhedra were then suspended in distilled water and quantified by hemocytometry as initial inoculums (biabani et al., 2005). silkworm rearing and hemolymph preparation the eggs of a japanese line, 103, were obtained from iran sericulture research centre (rasht, iran) and reared in the laboratory with standard rearing techniques under 25 ºc with rh of 75±5 % and photoperiod of 16l:8d (krishnasawam, 1978). after 3rd molting, the larvae were divided into two groups including bmnpv infected larvae and uninfected ones. in each treatment 500 larvae were reared in two part of rearing room and both groups were fed by the leaves of shinichenoise mulberry variety. a group of larvae were orally treated with 5.5×10-4 polyhedral/ml of bmnpv at the first day of 4th instar. since disease symptoms were not observed in 4th instar in order to be certain that larvae have been infected, for each treatment 20 random samples were selected from 5th instar larvae in their 3rd and 5th days. larval proleg was cut and hemolymph was collected in microtubes. one milligram phenylthiourea was added immediately to the tubes to prevent melanization. the samples were centrifuged at 14,000 rpm for 10 min. the supernatant was removed and kept in –20 ºc for analysis. biochemical analysis the method of lowry et al. (1951) was used for the total protein estimation. hemolymph was diluted with distilled water and was added to alkaline copper reagent in microtubes. after 10 min 0.5 ml of folin ciocalteu’s reagent was added to the mixture and microtubes were shaken thoroughly. the tubes were kept 20 min in room temperature for color development. the readings were taken on the spectrophotometer at 650 nm. for the reference, standard bsa was used. the concentration of urea was determined by measuring ammonia produced from urea, using a commercial urea assay kit (chemenzyme co., iran). alanine aminotransferase (alt) (ec 2.6.1.2) and aspartate aminotransferase (ast) (ec 2.6.1.1) were measured utilizing thomas (1998) procedure. the t-test in sas software was performed for determination of significant differences between the data in infected and healthy groups (sas, 1997). results and discussion the results of this study have been demonstrated in figs 1-4. as it is shown in fig. 1, total protein considerable decreased in infected larvae compared to control. this reduction was observed in both sampling times in a way that total protein in the third day of 5th instar was 36.5 mg/ml in healthy larvae while in the same day the amount of protein in infected larvae was 7.5 mg/ml. this trend continued through fifth day of 5th instar and the amount of total protein in infected larvae decreased to 50 % of the control (fig. 1). it was previously reported that the total protein of hemolymph decreases in npv infected silkworm larvae from the first to the seventh day of infection. this difference in the last larval day was more than 35 units decrease (sarma et al., 1994). reduction of protein caused by npv infection, which is called hypoproteinemia, not only occurs in silkworm but also it has been reported from many other lepidopteran species such as mamestra brassicae, trichoplusia ni, lymantria dispar, etc (young and lovell, 1971; pawar and ramakrishnan, 1977; mazzone, 1985). fig. 1 the changes of total protein in infected and healthy groups of larvae (mg/ml) 14 reduction of hemolymph protein could be due to the decrease of protein synthesis. it is assumed that virus activity is followed by the disruption of fat body cell and the lack of protein release to the hemolymph, hence, protein levels drop in hemolymph. in insects, the most important place for protein synthesis is fat body that is also the most sensitive tissue to npv in silkworm. on the other hand, protein decrease could be the consequence of absorption process in midgut, because after entrance of virus to larval body, epithelial cells of midgut are invaded and therefore absorption process is interrupted. although there are some researches that show there are not considerable changes in the quantity of some compounds in the insects infected by virus (miao, 2002), often biochemical studies of silkworm larvae show that most of the stresses drop the amount of total protein in their hemolymph. it is assumed that silkworm compensates the deficiency of energy caused by the stress with intensive breakdown of proteins to amino acids and entering them to tca cycle as a keto-acid (nath et al., 1997; etebari and matindoost, 2004a, b; etebari et al., 2006). it has been reported that high doses of baculovirus could increase the total protein in the cell culture of silkworm embryonic tissue. matindoost (2006) suggested that the reason for this could be due to the bursting of cell membrane after cell death and release of cellular proteins, however, other factors have also been outlined. baculoviruses cause intensive interruption in hormonal regulation of larvae by having egt gene. this gene codes ecdysteroid udp-glycosyl transferase and its expression inhibits in time ecdysis of larvae (liu and hou, 1985). this is while utilizing ecdyson and its derivatives affects protein metabolism in larvae infected by npv. liu and hou (1985) demonstrated that compounds containing this hormone when treated on npv infected larvae prevent intense changes in the amount and type of proteins within hemolymph. fig. 2 the changes of urea in infected and healthy groups of larvae (mg/ml) fig. 3 the changes of ast activity in infected and healthy groups of larvae (iu/l) hemolymph urea did not have significant changes in two experiment times although it showed little difference (fig. 2). urea fluctuated between 4.37 to 6 mg/ml in uninfected and infected larvae. urea as an excretory compound plays an important role in silkworm physiology and changes of its concentration in hemolymph of silkworm larvae are dependent to many factors such as larval stage, diet, etc (sumida et al., 1993). urea changes are directly related to nitrogen metabolism and amino acids (hirayama et al., 1996). in this study not only no difference was observed between infected and uninfected larvae in the amount of urea but also no difference was evident in two sampling times. two amino transferases, ast and alt, showed considerable increase in infected larvae (figs 3, 4). the mean activity of ast was more than five times higher than that in uninfected larvae. in detail, at third and fifth day of 5th instar the activity reached to 2,458.8 and 2,632.2 iu/l, respectively, while the activity in control at the same days was 294.7 and 474.5 iu/l. the changes of alt follow the same pattern and its activity in infected larvae was more than triplicate. it has been shown that silkworm larvae under different stress factors like parasitism by parasitoid flies, and/or after treatment with phosphorus pesticides and juvenile hormone analogues present fluctuations in the activities of amino transferase enzymes (rami reddy et al., 1992; nath et al., 1997; singh et al., 1997). etebari et al. (2005) used the activity levels of these two enzymes as an appropriate biochemical marker to study the biodiversity of silkworm strains. the transaminases are the important components of amino acid catabolism; which is mainly involved in transferring an amino group from one amino acid to another keto acid. the ast and alt serve as a strategic link between the carbohydrate and protein metabolism and are known to be altered during various physiological and pathological conditions (etebari et al., 2005). 15 fig. 4 the changes of alt activity in infected and healthy groups of larvae (iu/l) generally alt activity is mentioned as an index for breakdown of amino acids and ast as a sign for entrance of amino acid to glucogenesis process. glucogenesis is a main path for sugar synthesis from non carbohydrate substrates (lehninger, 1982). the carbon sources for glucogenesis in these series of reactions are amino acids and the activation of this metabolic pathway is usually associated by intensive decrease of free amino acids in fat body and hemolymph, because often with alt activity, alanine transforms to pyruvate and enters the above pathway for energy supply. although it has been reported that many factors such as larval strain, larval age, light period or rearing season and leaf type affect the changes of alt activity levels (khanikor et al., 1998; devi and sarma, 2000) viral infection is far more effective than other factors. references anonymous. fao agricultural services bulletin, manual of sericulture, fao of the united nations, rome, 1976. biabani mr, seydavi ar, gholami mr, etebari k, matindoost l. evaluation of resistance to nuclear polyhedrosis virus in commercial hybrids of silkworm (bombyx mori) formosan entomol. 25: 103-112, 2005. biabani mr, mirhoseini sz, etebari k, matindoost l. the effects of nuclear polyhedrosis virus infection in 9 commercial hybrids of silkworm (bombyx mori l.) xxth congress of the international sericultural commission, 15-18 dec. 2005 bangalore, india, 2005. devi d, sarma dk. aminotransferase activity of muga silkworm (antheraea assama ww.) proceedings of the third international conference on wild silk moths, india, 246-248, 2000. etebari k, matindoost l. a study on the effects of larval age on biochemical macromolecules abundance of haemolymph in silkworm bombyx mori l. 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biochem. physiol. 105a: 563-570, 1993. thomas l. clinical laboratory diagnostics. 1st ed. th books verlasgesellschaft, frankfurt, 1998. young sy, lovell js. hemolymph proteins of trichoplusia ni during the course of a nuclear polyhedrosis virus infection. j. invert. pathol. 17: 410-418, 1971. 17 bmnpv inoculum preparation << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice microsoft word isj192 isj 6: 138-143, 2009 issn 1824-307x minireview origins and functions of annelide immune cells: the concise survey v vetvicka1, p sima2 1university of louisville, department of pathology, 511 s. floyd, louisville, ky 40202, usa 2institute of microbiology, department of immunology and gnotobiology, czech academy of sciences, videnska 1083, 142 20 prague 4, czech republic accepted october 13, 2009 abstract immune cells and structures of annelida represent a complex subject that was studied for over 120 years. an overwhelming mountain of data have been accumulated during these studies, most of them being the subject of many excellent reviews and books. in the this paper we focused on a brief survay of old studies and some reflection on the findings during recent years. key words: annelida; cells; immunocytes; defense introduction annelids are considered to be the ancestral key assemblage in which important evolutionary novelties have originated. these animals represent virtually a new, highly progressive monophyletic group in which the emergence of new structures brought about better dynamics of locomotion and more effective functional and immune capabilities (clark, 1964). this allowed these animals an extensive adaptive radiation into all biocenoses of the earth with exception of the air. subsequently, evolutionary pressures within these different environments resulted in high diversification leading to approximately 9,000 estimated species which are ordinarily divided in two superclasses-the aclitellata with major group of polychaeta, and the clitellata consisting of oligochaeta and hirudinea (see for details sima, 1994a). compared to their predecesors, the annelids exhibit all three major advanced structural features, such as the metameric body arrangement, the secondary body cavity (the coelom) filled by coelomic fluid constituting the hydrostatic skeleton, and the blood-vascular system (in some taxa already closed). this means that all necessary requirements for more advanced eucoelomate basic body plan were finally present from space for development of more complex and hierarchized organs, to increased regional differentiation, and finally, to the ability to change duplicated parts in ways that might be advantageous for survival. all ___________________________________________________________________________ corresponding author: vaclav vetvicka department of pathology university of louisville 511 s. floyd, louisville, ky 40202, usa e-mail: vetvickavaclav@netscape.net these hallmarks enabled a more active way of life and a higher adaptive plasticity which are not found in any earlier bilateralian acoelomate animals. both the coelom and the closed vascular system are important for emergence of a new type and more effective immune strategy. this also contributed toward their successful survival from an evolutionary point of view. a celomic cavity is composed of paired chambers lying within the third germinal layer the mesoderm which represents a general source of all types of immunocompetent cells found in all eucelomate taxa including deuterostomia. morphological separation of celom from the vascular system allowed cytogenesis of mutually independent cell lineages and their functional specialization and higher degree of functional cooperation. consequently, a high number of free and sessile cell types can be found both in the celomic and vascular fluids. cell types differ in morphology among various annelide species. to this date, no clean-cut classification has been formed and widely accepted. thus, morphofunctional criteria still remain basic for distinguishing various annelide immunocyte lineages. roughly speaking, these cells are usually characterized as amoebocytes, eleocytes, erythrocytes and hemocytes (polychaeta), celomocytes, amoebocytes, vascular lymphocytes, eleocytes and macrophages (oligochaeta) and amoebocytes and chloragocytes in hirudinea. current literature on annelide colemocytes is vast and far beyond the scope of the review. therefore, we will focus on a survey of the main immunocyte categories in three annelide subclasses: polychaetes, oligocheates, and hirundineans. 138 table 1 main categories of polychaete amoebocytes (from dhainaut and porchet-henneré, 1988) granulocyte morphology responding to presence in families type i fusiform, spindleshape, granules/microfilaments in cytoplasm adherent hyaline fusiform leukocyte (1) arenicolodae, ophelidae, glyceridae, nephtyidae, nereidae type ii* spindleshape, granular, cytoplasm with vacuoles and without microfilaments adherent hyaline leukocyte (1) arenicolodae, nereidae, terebellidae, capitellidae type iii small, few granules, large nucleus small lymphocyte (1) precursor cell? in many families type iv typical macrophage common macrophage found in immunized animals type v large cytoplasmic spherules, stacks of rough endoplasmic reticulum eosinophil granulocyte (1) many families *similar cells also in sipunculids, arthropods and molluscs (1): romieu (1923) polychaeta the primary and most numerous defense cells are the amoebocytes that represent a rather heterogeneous population of cells. amoebocyes are present in almost all the polychete species studied. the most common nomenclature used for amoebocyte is granulocyte, convenient term proposed by baskin (1974). according dhainaut and porchet-henneré (1988), polychete granulocytes are divided into five types (table 1). it is important to note that not all the these call types are present in every polychaeta. despite decades of research, the relationship among the individual types is still not established. still unknown is whether they represent different stages of a single cell lineage or discrete series, or if ameobocytes have a common origin with other cell types, e.g., the eleocytes. similarly, we are not certain of their origin. in some species, such as aphrodite, the distinct “lymph glands” have been described (fordham, 1925), whereas in sabelliade, the longshaped extension of nephridia has been suggested as a possible source. discret tissues derived from celomic peritoneum in glycera might play similar role. in other polychaete species, the mesenchymal tissue agglomerations around the vessels and nephridia might also serve for hemopoiesis (dehorne, 1922). the major defense functions of these cells consist of phagocytosis, waste removal and pathogen clearing. they are involved in antibacterial defense and encapsulation, which was a detailed study in nereis (porchet-henneré et al., 1987). the high degree of the cooperation by individual subtypes of granulocytes during encapsulation reaction was documented via means of monoclonal antibodies (porchet-henneré, 1990). a second population of free cells within a polychaete celom is represented by the eleocytes (rosa, 1896), the relatively large cells (appr. about 40 μm) contained in their cytoplasm nutrition reserves (mainly lipids and glycogen). they have often been described as agranular lymphocytes, trephocytes, or adipo-spherular cells. eleocytes were found in a row of families belonging to both polychaete subclasses (the errantia and the sedentaria) but not in arenicolidae, glyceridae, nephyidae, and syllidae. celomic lining epithelia covering blood vessels and somatic peritoneal epithelium were documented as a source of eleocytes in nereidae (eckelbarger, 1976) and terebellidae (dhainaut, 1970). conversely, other authors supposed that they develop from phagocytic amoebocytes (romieu, 1923; dales and dixon, 1981). their role is mainly trophic, even if their active but non-defensive pinocytosis used for removing of damaged tissues has been documented (dhainaut and porchet-henneré, 1988). third cell type found in polychetes constitutes hemoglobin-containing-cells termed erythrocytes (rarely hemocytes, red celomocytes or blood cells). they are found in capitellidae, cirratulidae, glyceridae, and opheliidae, exclusively in their celomic cavity, (goodrich, 1898). according to dhainaut and porchet-henneré (1988) erythrocytes form two lineages differing in morphology and functional specialization. in general, eleocytes and erythrocytes are primarily involved in regeneration and respiration. as with other immunocytes in this phylum, the origin of these cells is unclear and probably differs among species. while the role of eleocytes in defense is most probably limited, hemoglobin-containing erythrocytes are actively phagocytosing (goodrich, 1898). a more questionable population of cells represent hemocytes (blood cells) closed in the vascular systems of various representatives of polychete families such as terebellidae, sabellidae, arenicolidae, and nereidae. the hemocytes resemble celomocytes in morphology but they are relatively small, around 3-10 μm. with the exception 139 of magelona papilliocornis (boilly, 1974), they never contain hemoglobin. their origin is unresolved and, for decades, they were considered to be an immigrant cell population. only later, electron microscopic studies (dhainaut and porchethenneré, 1988) provided clearer knowledge and allowed classification into three categories (types i to iii, from which the type iii comprises sessile or attached cells and may be morphologically analogized to blood folicle cells of oligochaetes). their role in defense reactions remains unknown. oligochaeta from a practical perspective, the oligochaeta, and the earthworms (of the family lumbricidae) in particular, have by far been the most studied annelids and have become the object of interest in various scientific fields such as evolutionary biology and invertebrate immunology as well as a variety of environmental diciplines (agriculture, pedology, environmental pollution, epidemiology, etc.). extensive studies accomplished during last 50 years have made them one of the most popular models of invertebrate immunity (bilej, 1994; cooper and roch 1994, 2003). due to the intense study of the earthworm species, our knowledge of cell population composition in this category is superior. current classification of free cells is based on old research (kukenthal, 1885; rosa, 1896). for detailed information on origin, classification and formation, the reader is asked to refer to the detailed reviews written by sima (1994b) and cooper and roch (1994). again, the primary source of most information were the lumbricus terrestris and eisenia foetida models and, on rare occasions, from limited representatives of other lumbricid species. therefore, they might not be universally valid for all oligochaete taxa. the rather complicated classification can be simplified into two basic categories: the celomocytes (amoebocytes and eleocytes) and the hemocytes (blood cells). amoebocytes can be, according their morphology, further subdivided into granulocytes (acidophils type i and type ii) and non-granular hyalocytes (basophils, amoebocytes forming a 70 % majority of free cells and a monolite population of neutrophils). granular amoebocytes are present in all representatives of lumbricids. some nomenclatures subdivide them further based on size or type of granules. it was generally supposed that, at least in lumbricids, free celomocytes originated from mesodermal peritoneal cells comprising the visceral or parietal epithelial lining of the coelom (splanchnopleure and somatopleure) (fischer, 1993; sima and slipka, 1995; sima et al., 1995). other possible sources are epithelial lining of the blood vessels and septa forming specialized poietic structures (liebmann, 1942; valembois, 1971). the third eventuality, the origin from specialized hemopoietic organs named blood glands or blood folicles described in several representatives of the lumbricullus sp. and genera sparganophilus, maoridrilus, pheretima and pontodrilus, must be taken into account as well (see for review sima, 1994b). it was further observed that losts of celomocytes after their depletion induced by irritation (via dorsal pores in the body wall) are followed by extensive cell proliferation in coelomic lining in the typhlosole and metanepridial regions (homa et al., 2008). the mesodermal origin of all subpopulations of lumbricid free cells was recently confirmed using different mammalian antigenspecific monoclonal antibodies reacting with distinct coelomocyte surface markers (engelmann et al., 2002, 2005). both types of amoeboid celomocytes form an extremely important part of the immune system of annelids. as with most invertebrates, they are involved in non-self recognition, transplantation reaction, cytotoxicity, encapsulation, endocytosis and enzymatic digestion of engulfed material. in addition, they actively participate in regenerative processes and wound healing. phagocytosis alone has been studied in earthworms since metchnikoff (1891). due to the availability, this model has been extensively studied and, in the last several decades, a vast amount of data has been obtained. those seeking detailed information should read these excellent reviews (cameron, 1932; davis, 1978; bilej, 1994; cooper and roch, 1994, 2003; salzet et al., 2006). two main different subpopulations among earthworm immunocompetent free celomocytes were identified by flow cytometric analyses (cossarizza et al., 1996; engelmann et al., 2002, 2005). a subpopulation of large celomocytes (25-50 μm in diameter) is active in phagocytosis and encapsulation of bacteria but no surface markers were identified using monoclonal antibodies. small celomocytes (10-25 μm) reacting with anti cd11a, cd45ra, cd45ro, cdw49b, cd54, cd90, and beta 2-microglobulin are cytotoxic. non-granular coelomocyte subpopulation recognize and neutralize foreign antigens. these cells exprime surface markers thy-1 (cd90) and beta 2microglobulin, both molecules belonging to the immunoglobulin superfamily (shalev et al., 1981; roch et al., 1983). eleocytes are again a heterogenous group of cells, usually divided into ergastoplasmic chloragocytes (which are probably the stem cells of this group), chloragocytes and free eleocytes. their morphology can vary with oval, round or oblong cell´s shape 10-60 μm in diameter. a majority of eleocytes possess spherical granules (1-3 μm) and chloragosomes within their cytoplasm. chloragocytes play a role in nutrition, excretion and osmotic balance. the defensive roles of these cells are less studied but they are certainly less pronounced. these cells proved inactive in phagocyting bacteria or other particles (dales and kalaç, 1992). in addition, they differ among individual species, e.g., they phagocytose in eisenia but not in lumbricus. with respect to hemocytes, the information is scarce and the current information has not substantially improved since the last century. other oligochaete families if someone gained the impression that the situation concerning the cells and cellular structures in earthworms is confusing, an even more complicated 140 table 2 main categories of coelomic cells of the theromyzon tessulatum (hirudinea) (from lefebvre et al., 2008) coelomocyte morphology responding to function large coelomic cell (chloragocyte) 100-150 μm free or attached to mesothelia granules in cytoplasm oligochaete chloragocyte non-phagocytic presence of recognition surface molecules encapsulation? vitellin production? granular amoebocyte 30-70 μm granules in cytoplasm numerous pseudopodia oligochaete granular amoebocyte chemotactic phagocyting g+ and gbacteria small coelomic cell 7-12 μm without cytoplasmic granules hyaline cytoplasm rich endoplasmic reticulum oligochaete hyaline cell non-phagocyting situation can be found in other oligochaeta families. even when these members commonly possess the same basic cell types, additional, more-or-less specialized or distinguished cell types have been described. pontodrilus bermudensis serve as an example (wamper and jamieson, 1986). on the other hand, genera such as achaeta, analycus, or grania, have only one type of celomocytes. buchholzia possesses two types (brinkhurst and jamieson, 1971). hirudinea general body anatomy responds to annelide basic body plan. however, these animals endured substantial modification when separate coleomic and vascular spaces were fused together into common hemocoel. these modifications of the vascular system and coelomic cavity led to the flow of hemocoelomic fluid through a highly complicated network of coelomic sinuses and channels. hirudidae can be characterized by the presence of the botryoidal tissue located within the parenchyma adjacent to the body wall. besides being involved in angiogenesis, this celothelium-derived tissue display myelo/erytroid function. it also can change its shape from a solid cord of cells to a prevascular structure when groups of closely associated cells became evident in the centre of the immature lumen. as the vessel growth, the precursors of circulating cells loose the cell-cell attachment and move freely within the lumen. unfortunately, cytology of hirudineans has attracted little attention, so our knowledge of the origin and function of their free cells are still incomplete. cell populations within the hemocel are commonly divided to amoebocytes and chloragogen cells, similar to the previous annelide classes. amoebocytes denominated also leukocytes or lymphocytes are usually of homogeneous size and morphology, exerting phagocytosis as the main defense activity (sawyer and fitzgerald, 1981). these cells are either free or attached to the hemocel wall lining. hypotheses about their origin are rather speculative. the size of chloragogen cells differs among individual species. their origin is clear, they arise from the hemocelomic epithelia (oka, 1894) and from the cilio-phagocytic organ of the nephridia (abeloos, 1925). recently, using human monoclonal antibodies, three cell populations, the macrophage-like, nk-like, and granular cells, were identified in glossiphonia complanata (de eguileor et al., 2000). similarly, immunohistochemical and ultramicroscopic studies demonstrated more precisely the presence of three basic cell populations in theromyzon tessulatum (lefebvre et al., 2008) (table 2). we must be also aware that free cells may be derived from different tissue sources in different species. on the contrary to family hirudidae, the celothelial botryoidal tissue in some species of the glossiphoniidae plays besides the angiogenetic functions the important role in production of free hemocelomic cells. prevascular structures are tightly associated with solid cell cords, from which circulating cells release loosing mutual contacts and pass freely into the vascular lumen (de eguileor et al., 2001). an increasing body of evidence indicates that in the leech hirudo medicinalis the angiogenic process is regulated and coordinated by the botryoidal tissue. hirudo medicinalis subjected to an angiogenic stimulus (e.g., wounding) responds with an extensive angiogensis that is accompanied by the production of free cells. these processes, moreover, could be influenced by means of mammalian activators of vascular cell growth, antiangiogenic peptides (angiostatin and endostatin) and even proliferation-inducing mitomycin. the surprising degree of similarity of invertebrate angiogenesis with neovascularization in vertebrates, both at the biochemical and cellular levels, suggests that both involve similar growth factors and their receptors, and common cell-cell or cell-extracellular matrix interactions (grimaldi et al., 2006, grimaldi et al., 2008). the reviewed data confirm phylogenetic kinship between hirudinean and vertebrate processes in wound healing. it suggests that cytogenesis of free cells within the vascular system in such remote phyla as protstomian annelids and deuterostomian chordates may share evolutionary common basic mechanisms. 141 summary defense reactions of annelids may be clearly defined as the two compound systems developed from and housed in the coelom in which cooperate the cellular (celomocytes) and humoral (celomic and vascular fluid) part of immunity. individual types of celomocytes play a role similar to vertebrate lymphocytes, leukocytes and macrophages. together, both parts are sufficiently effective in disposing of foreign material and pathogenic invaders. references abeloos m. recherches histochimique et physiologiques sur le parenchyme et les nephridies des huridinees rhynchobdelles. bull. biol. fr. belg. 59 : 436-456, 1925. baskin dg. the coelommocytes of nereid polychaets. contemp. topics immunol. 4: 5564, 1974. bilej m. cellular defense mechanisms. in: vetvicka v, sima p, cooper el, bilej m, roch p (eds), immunology of annelids, crc press, boca raton, pp 167-200, 1994. bilej m. humoral defense 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(france) 96: 59-65, 1971. wampler je, jamieson bgm. cell bound luminescence from pontodrilus bermudensis and its similarities to other earthworm bioluminiscence. comp. biochem. physiol. 84a: 81-87, 1986. 143 isj 7: yyy-xxx, 2010 isj 7: 149-156, 2010 issn 1824-307x review relationships between innate immunity in bivalve molluscs and environmental pollution mi girón-pérez laboratorio de inmunotoxicología, universidad autónoma de nayarit, boulevard tepic-xalisco s/n, cd de la cultura amado nervo s/n, 63190 tepic, nayarit, mexico accepted may 25, 2010 abstract the immune system of invertebrates, such as molluscs consists of innate mechanisms very effective against antigens commonly present in the environment. however, these defense strategies could be altered by pollutants. this review is focused mainly on the effect of metals, pcb, pesticides, pahs, and others environmental pollutant on immune response of molluscs. key words: molluscs; immune system; environmental pollution; metals; pesticides; pahs; pcb introduction immune system is strongly influenced by environmental conditions. successful host resistance is a major determinant in whether a pathogen will result in a disease outbreak. altered environmental conditions can affect immunity directly, by changing the concentration and efficiency of components including cytokines, cytokine receptors and cells of the immune response, or indirectly by inducing general stress response. subsequently, the relationship immunityenvironment is complex, but is an essential comprehended mechanistic aspect of it, and thus allow predictions on the potential effect of environmental factor on immune response (mydlarz et al., 2006). the bivalve molluscs have characteristics such as high distribution worldwide, sedentary and filter-feeding habits; hence these organisms accumulate large number of bacteria and chemical pollutants, which are both a source of nourishment and an immune challenge (bernal-hernandez et al., 2010). the immune response of molluscs has an important defense function against bacteria, fungi, and parasites. the immune system is constituted for a first line defense including physicochemical barriers as the cuticle, shell and mucus layer. moreover, in bivalves, cellular and humoral components ___________________________________________________________________________ corresponding author: manuel iván girón pérez laboratorio de inmunotoxicología, c.a. contaminación y toxicología ambiental, universidad autónoma de nayarit, bd tepic-xalisco s/n, 63190 tepic, nayarit, méxico e-mail: ivan_giron@hotmail.com are present and operate in a coordinated way (galloway and depledge, 2001). cellular response is carried out by circulating hemocytes that can kill microbes through phagocytosis and citotoxic reactions that include the release of lysosomal enzymes and anti-microbial peptides, and the respiratory burst which involves the production of oxygen metabolites, meaning superoxide anion, hydrogen peroxide, and intermediated compounds with high bactericidal activity (pruzzo et al., 2005). hemocytes are also involved in other physiological functions, such as wound and shell repair, digestion and transport of nutrients. hemocytes classification is controversial, but has hypothesize the existence of two circulating hemocytes cells: granulocytes (containing many citoplasmatic granules) and hyalinocytes (containing few or no granules). granulocytes are generally the most abundant cell type with higher phagocytic activity, while hyalinocytes are usually smaller than granulocytes, and have a high nucleus/cytoplasm ratio (hine et al., 1999; matozzo et al., 2007). the humoral components present in hemolymph are lectins, lysosomal enzymes and antimicrobial peptides. the presences of lectins have been shown in marine bivalves such as mussels, oyster, and clams. the role of lectins is induced agglutination of bacteria and act as a molecular bridge between the surface of bacteria and hemocytes (pruzzo et al., 2005). in spite of the efficiency of the immune system of molluscs in normal conditions, it may be altered by external factors (fig. 1). thus, this review is focused mainly on the effect of metals, pcb, pesticides, pahs, and others environmental pollutants on immune response of molluscs. 149 fig. 1 effect of the main contaminants in aquatic ecosystems on the immune system of molluscs xenobiotics and immune system of molluscs the presence of chemical contaminants in water is a major subject of concern, since many of these molecules are potent immunosupressors, even at a low concentration (table 1). a possible consequence for immunodeficient oyster could be an increased susceptibility to parasites and other pathogenic microorganisms (auffret et al., 2002). metal effects studies performed to understand the relationship between metals and immunotoxicity have been showed that in vitro cd exposure of hemocytes at sub-lethal concentrations up to 15 μm cdcl2 induce significantly increase in metallothionein (mt) and inhibition of ros generation (butler et al., 2000). studies realized with oyster (ostrea edulis) showed that exposure to cdcl2 (1, 10, 50, 100 µm) and co-exposure to cdcl2 and cucl2 (0.75 µm), induced non significant changes in the serum total protein level. moreover, the level of serum acid phosphatase, and hydrolytic enzymes remained unaltered. but a dose-dependent increase in total hemocytes was found in oyster exposed to cdcl2. on the other hand, the exposure to 1, 10 or 50 µm cdcl2 resulted in a dose-dependent decreased in the cell membrane potential, probably related to membrane alterations. phagocytic activity of o. edulis exposed to 1 or 10 µm cdcl2, or to 1 µm cdcl2/0.75 µm cucl2 showed a severe decrease compared with control group (auffret et al., 2002). the effect of copper exposure (0.02 and 0.05 ppm), alone or simultaneously with vibrio tubiashii at different temperature, was evaluated on mussels (mytilus edulis). results showed that 0.05 ppm copper induced a significant reduction on cellular content in hemolymph. when co-exposed mussels to bacterial challenge, the reduction in cell number was higher, compared with the effect of metal alone. in addition, intracellular superoxide decreased significantly by exposure to 0.02 and 0.05 ppm of copper to 10 °c. however, there was an increase of this parameter when analyzed at 15 °c. while, phagocytosis was increment by exposure at 0.02 ppm compared with control group (parry and pipe, 2004). another study evaluated the effect of cadmium, copper on ros production, and hemocyte viability from mytilus galloprovincialis. results showed a significantly decrease on viability of hemocytes (according xtt test) exposed to cd (1,120x10-5 μg/ml). exposure to cu (12.72 μg/ml) also induced 150 table 1 immunotoxic effect of pollutants on molluscs xenobiotic effect species reference metals hemocyte counts ↑ cd cell membrane potential ↓ ostrea edullis auffret et al., 2002 cd alone or cd/cu phagocytic activity ↓ ostrea edullis auffret et al., 2002 cd (in vitro) cd, and cu cu cu methalothionein↑ ros ↑ hemocyte viability ↓ phagocytosis ↓ hemocyte counts ↓ superoxide anion ↓ phagocytosis ↑ crassostrea virginica mytilus galloprovincialis elliptio complanata mytilus edulis butler et al., 2000 gómez-mandikute et al., 2003 gangé et al., 2008 parry et al., 2004 gas cellular viability ↓ o3 cox-activity ↑ elliptio complanata gangé et al., 2008 no2 ↑ estrogenic substances nonylphenol, monoethoxilate carboxylate, and 17α-ethynyl estradiol 17β-estradiol pharmaceutical drugs lysosomal enzyme release ↑ phagocytosis ↑↓ phagocytosis ↓ mytilus galloprovincialis corbicula fluminea canesi et al., 2007 champeau et al., 2006 benzafibrinate, gembibrozil, and trimetophin phagocytosis ↑ elliptio complanata gagné et al., 2006 novobiocin, and morphin phagocytosis ↓ elliptio complanata gagné et al., 2006 zulfamethazole, novobiocin, gemfibrocil, benzafibrate, and carbamazepine esterase activity ↓ elliptio complanata gagné et al., 2006 oxytetracycline, novobiocine, naproxen cell adherence ↓ elliptio complanata gagné et al., 2006 gemfibrozil, bezafibrate cell adherence ↑ elliptio complanata gagné et al., 2006 novobiocine,and sulfapyridine lipoperoxidation ↑ elliptio complanata gagné et al., 2006 coprostanol, and naproxen lipoperoxidation ↓ elliptio complanata gagné et al., 2006 151 pahs benzo[a]pyrene, and phenanthrene granulocyte cell (%) ↑ cell mortality, esterase and lysosome-positive cells ↓ crassostrea gigas gagnaire et al., 2006 benzo[a]pyrene benzo[a]pyrene phenanthrene phenanthrene hemocytes viability ↓ lysozyme activity ↓ phagocytic activity ↓ adhesion capability ↓ hemocyte mortality ↑ phagocytic cells ↓ superoxide generation ↓ hemocyte number ↑ cell membrane stability↓ phagocytosis↓ mytilus galloprovincialis chamelea gallina cerastoderma edule pecten maximus gómez-mandikute et al., 2003 matozzo et al., 2009 wootton et al., 2003 hannam et al., 2010 pcb pcb 77 lysosome-positive cells ↓ crassostrea gigas gagnaire et al., 2006 pesticides 2.4d cell mortality ↑ crassostrea gigas gagnaire et al., 2006 paroxon esterase and lysosome positivecell (%) ↓ crassostrea gigas gagnaire et al., 2006 ros positive-cell (%) ↑ chlorothalonil cell mortality and granulocyte (%)↑ crassostrea gigas gagnaire et al., 2006 pesticide mixture (atrazine, gliphosate, alachlor, metalachlor, fosetylaluminum, terbuthilazine, diuron, carbaryl) phagocytosis ↓ cell mortality, and ros production ↑ genes relationship with immune response (e.g., lyzozyme, defensines) ↓ crassostrea gigas gagnaire et al., 2007 susceptibility to bacteria challenge ↑ paraquat other hemocytes viability mytilus galloprovincialis gómez-mandikute et al., 2003 fuel oil no. 6 cellular viability↓ pinctada imbricate nusseti, et al. 2004 gst and cat↑ 4-nonylphenol gpx ↓ lysozyme concentration ↓ apoptitic index ↑ tapes philippinarum matozzo, et al. 2005 symbol: reduction (↓), increased (↑), biphasic effect (↑↓) 152 decrease in this parameter. the superoxide anion production (using nbt reduction test) in hemocytes was evaluated. the results indicated that cd exposure induced no changes, but cu exposure decrease the nbt reduction (gómez-mandikute et al., 2003). the studies have been showed that metals could modulate different immunologic parameters on molluscs. however, the immunomodulation is influenced by metal concentration, and other factors such as presence of potential pathogens and environmental variables, like temperature for instance. estrogenic substances effects others pollutant substances frequently present in aquatic ecosystem are estrogenic chemical. in this context, canesi, et al. (2007), evaluated the in vitro effect of endocrine disruptor compounds on mytilus hemocytes. the results showed that hemocytes incubated during 30 minutes, with estrogenic compounds, such as nonylphenol monoethoxylate carboxylate (np1ec) and 17αethynyl estradiol, increased the lysosomal enzyme release in 65 % and 45 %, respectively compared with control hemocytes. on the other hand, a biphasic effect was observed on phagocytosis, thus to lower concentrations (0.1 5 µm) a significant stimulations was detected, while to 25 100 µm a inhibitory effect was observed. the effect of 17β-estradiol (20, 200 and 2000 ng/l) was evaluated on asian clam corbicula fluminea, exposure during 15 or 30 days. results showed that this estrogenic substance did not affect the cell viability. however, the exposure to 200 and 2000 ng/l significantly inhibit the phagocytosis, in both evaluated times (champeau et al., 2006). the effects of endocrine disrupters, such as natural or synthetic steroids, on immune system of molluscs are not well known yet, in part by limited knowledge on invertebrate endocrine system and immunendocrine network. however, results suggest that immune system represents an important target of estrogenic compounds. thus, the study of these compounds and their effect on invertebrate physiology is necessary. pharmaceutical products effects municipal effluents represent a major source of pollution. these effluents could contain pharmaceutical products, xenobiotics that could modulate immune response of aquatic organisms. studies on mussels (e. complanata) hemocytes exposure in vitro to pharmaceutical drugs (benzafibrate, carbamazepine, fluoxetine, gemfibrozil, morphine, naproxen, novobiocin, oxytetracycline, sulfamethazole, sulfapyridine and trimethoprim) and urban waste (coprostanol, caffeine, cotinine) at 0, 2.5, 25, 50 and 100 μm, showed that some products as benzafibrate, gemfibrozil and trimethoprim, increased phagocytosis, while novobiocin and morphine reduced its activity. intracellular esterase activity was reduced with sulfamethazole, novobiocin, gemfibrozil, benzafibrate and carbamazepine. cellular adhesion was decreased by oxytetracycline, novobiocin and naproxen, and increased by gemfibrozil, bezafibrate and sulfapyridine. exposure to these products also modulated lipoperoxidation (lpo) in hemocytes. coprostanol and naproxen were more potent to reduce lpo while novobiocin and sulfapyridine were the most potent to induce lpo. on the other hand, on a parallel experiment, mussels were placed in aeration lagoons for the treatment of domestic wastewaters during 60 days. in mussels, a decrease of intracellular esterase and phagocytic activity was observed (gagne et al., 2006). pahs effects polycyclic aromatic hydrocarbons (pahs) are a ubiquitous class of organic contaminants generated as results of anthropogenic sources or natural constituents of crude oil. the effect of phenanthrene (50, 100, 200 or 400 µg/l) on immunological parameters of mytilus edulis, cerastoderma edule, and ensis siliqua, were evaluated after exposure during 7 and 14 days. phenanthrene exposure at 400 µg/l resulted in 100 % mortality of c. edule after 14 days exposure, while total mortality of e. siliqua was observed 7 days after exposure. nevertheless, no mortality on m. edulis was reported. in general terms, results of immunologic parameters showed that acid phosphatase concentration was increase in m. edulis after 7 days exposure to phenanthrene 50, 100 and 200 µg/l, but a diminish in this parameter was observed 14 days after exposure at same concentrations. phagocytic cells percentage and superoxide generation were significantly reduce in c. edule after 14 days exposure to 100 and 200 µg/l. comparative analysis between three different species suggests that e. siliqua is less sensible to alterations by exposure to phenanthrene (wootton et al., 2003). studies realized with scallop pecten maximus, showed that exposure during 7 days at 100 and 200 µg/l phenanthrene, increase hemocyte number. nevertheless, cell membrane stability, and phagocytosis were reduced when organism were exposed to 200 µg/l. in addition, oxidative stress parameters were evaluated, indicating that 200 µg/l phenanthrene provoke diminish of gsh activity, but significantly increased lipoperoxidation index (hannam et al., 2010). another pah is benzo(a)pyrene, effect of 0.5 mg/l of this substance was evaluated on immune response of clam chamelea gallina. exposure to this xenobiotic during 7 and 12 days significantly decreased lysozyme activity, phagocytic activity and adhesion capability (matozzo et al., 2009). another study showed that benzo(a)pyrene did not have significant effect on viability of hemocytes of mytilus galloprovincialis. but this substance induced a significant increased in superoxide anion production (gómez-mandikute et al., 2003). studies made with pacific oyster crassostea gigas exposed in vitro to benzo(a)pyrene and phrenanthrene showed that these substances significantly increased granulocyte percentage, but decreased cell mortality and esterase and lysosome-positive cells at doses of 200 and 300 μmol/l, respectively (gagnaire et al., 2006). atlantic pearl oyster (pinctada imbricata) exposure to fuel oil no 6 during 7 days, showed no 153 significantly changes in immunological parameters, such as hemocyte number, phagocytosis, and lysozyme concentration. however, cellular viability was reduced when oyster were exposed to this xenobiotic. antioxidant enzymes such as, glutathione s-transferase (gst), and catalase (cat) were significantly higher in the digestive gland. while in mantle, an increase of glutathione peroxidasa (gpx), and decrease gst activity was detected. this report suggests that these enzymes should be considered as potential tools for biomonitoring marine environmental contamination (nusetti et al., 2004). other studies have focused their efforts to study the immune system in organisms living at low temperature (-1 to 5 °c). camus et al. (2002), evaluated the effect of benzo(a)pyrene on oxyradical scavenging capacity (tosc), and cell membrane stability of hemocytes from arctic scallops (chlamys islandicus). results indicated a reduction of tosc, and cellular membrane stability when benzo(a)pyrene was administrated at 74 and 90.6 mg/kg. these alterations should be negative for the cellular immunity of bivalves by reducing the phagocytosis ability of hemocytes. pahs exposure could increase susceptibility to infections. some studies suggest that reduction in immunocompetence is related to stimulation of ros production induced by pahs. pesticides effects pesticides are often used in successful agriculture. however, the pesticide use leads to severe environmental pollution. this way, aquatic organisms are frequently affected by these xenobiotics. studies realized with pacific oyster crassostea exposure to pesticide, showed that 2,4dichlorophenoxyacetic acid (2,4d), increased cell mortality at 450 μmol/l after a 4 h incubation period. while, paroxon exposure induced decrease in percentage of esterase-positive cells after 4 and 24 h of incubation at 400 μmol/l. but paroxon at 40 and 400 μmol/l, after 24 h incubation period, decreased lysosome-positive cells percentage; in contrast ros-positive cells were significantly increased at 400 μmol/l after 4 h incubation period. the fungicide chlorothalonil at 2 μmol/l, significantly increased cell mortality and granulocyte percentage at 200 μmol/l after 4 h incubation period. in addition, a pesticide mixture (alachlor, metolachlor, terbutilazina, glyphosate, diuron, atrazine, carbaryl, and fosteyl aluminium) was realized, but interestingly enough none of this eight compounds generated significantly effect when tested individually on c. gigas, but the mixture indeed decreased phagocytic activity (gagnaire et al., 2006). other studies carried out with pesticides, showed that paraquat on mytilus galloprovincialis hemocytes, showed a significantly decrease on viability of hemocytes (according xtt test) exposed to paraquat (10 μg/ml). on the other hand, paraquat exposure, induced a significantly increase in superoxide anion (gómez-mandikute et al., 2003) in order to know the effect of pesticide on bacteria challenge, pacific oyster c. gigas were exposed to a mixture of pesticides (atrazine, glyphosate, alachlor, metolachlor, fosetylalumimium, terbuthylazine, diuron and carbaryl) at environmental relevant concentration over a 7-days period. as a first step, hemocyte parameters (cell mortality, enzymes activities, and phagocytosis) were evaluated. the results showed that phagocytosis was significantly reduced, while cell mortality, esterase and ros production, were not altered. however, real-time pcr analyses showed that 19 genes (involved with cell signaling, cytoskeleton function, phagocytosis and other defense mechanisms) were down-regulated in treated animals. moreover an increased susceptibility to a bacteria challenge was observed. as a second step, the interaction between pesticide exposure and bacteria challenge (vibrio splendidus, 4x107 ufc/oyter) was evaluated. in this coexposure condition, was observed that 10 of 19 genes (focolin, galectin, lbp, c-src, ankyrin, procl, sod, tmp, lysozyme, defensin) was up-regulated. the authors suggested that up-regulated genes could induce damage in host-tissue (gagnaire et al., 2007). ozone effects on the other hand, the municipal effluents then sometimes undergo disinfection. a common process involved is ozonation. however, ozone treatment might generate toxic products. studies carried out by gagné et al. (2008), showed that freshwater mussels (elliptio complanata) exposed to ozone (range 1 20 %), in laboratory condition for 7 weeks, significantly diminished phagocytosis and cellular viability. however, cell adherence suffered no changes when compared to control group. in contrast, cox-activity and nitrite levels were significantly increased. according to results, o3, at concentration evaluated, reduce microbial loading and completely remove citotoxicity, but increased inflammatory properties of the effluents. the observed effect could be related to the formation of carboxylic acid, aldehydes, and ketones which modifies the redox status of treated wastewaters. others substances the effect of 4-nonylphenol (np), final product of nonylphenol eyhoxylates, substances used as stabilizer, was evaluated on clam tapes philippinarum. results showed that exposure at sublethal concentrations (0.05 0.2 mg/l) during 7 days, significantly reduced lysozyme concentration and sod activity. in contrast, apoptotic index was increasing at same concentrations (matozzo et al., 2005). in another research, hemocytes from the pacific oyster crassostea gigas were exposed in vitro to polyclorinated byphenyls (pcb), such as pcb 77. the results showed that this substance significantly decreased lysosome-positive cells at 6 and 60 μmol/l after 4 h incubation (gagnaire et al., 2006). field studies laboratory studies showed some advantages, the principal being the experiments performed in controlled conditions. however, field researchers in ecotoxicology, permit to analyze parameters 154 according to conditions present at one particular moment, and with all the factors that have influence over an ecosystem. furthermore, these type of studies are more suited to distinguish a correlationship between factors present on specific sites. recently, our research group evaluated the presence and concentration of pahs (pyrene, naphtalene, and benzo(a)pyrene), metals (cu, pb, zn, mn, as, fe), and organophosphorus pesticide (acetylcholine inhibition) on mexican pacific estuarine zone, and the relationship of pollutants with immunological and oxidative stress parameters in oyster crassostrea corteziensis. results indicated that the main xenobiotic detected were cu and naphthalene. furthermore, the acethylcholine inhibition tests, suggest the presence of organophosphorus pesticide in the estuary. microbicidal activity was not altered, but a significantly decrease in hemocyte number was detected. on oxidative stress parameters, an increase of superoxide anion, hydrogen peroxide, catalase activity and lipoperoxidation were observed in gills from oyster (unpublished data). in other studies, a positive correlation between xenobiotic concentration presents in ecosystem and increase in defense mechanisms of molluscs has been reported (fisher et al., 2000; oliver et al., 2003).thus, experiment designing have been made to show if the deployment of eastern oyster (crassostrea virginica) from uncontaminated through contaminated sites would increase immune response parameters and vice versa. the results showed that hemocytes count and bactericidal activity were significantly elevated after 12-week deployment at contaminated sites (metals, pahs and pcb) from florida. however, when similar experiment were realized inverted, the results were ambiguous, thus lysozyme concentration was reduced, but hemocyte activities (principally bacteria killing index and hemocyte count) were not challenged. authors suggest that these results could be indicative of an acclimatization response with adaptative consequences of oyster to chronically polluted sites (fisher et al., 2003). conclusion the data presented here suggests that all groups of pollutants may be hazardous to molluscs defense system. in general terms, there are many examples of links between xenobiotic and susceptibility to diseases in wildlife species, principally vertebrates with economical importance. also laboratory tests allowed identifying potential hazard, mainly anthropogenic chemicals with immunosupressor properties. however, invertebrate organisms have ecological relevance besides only economical importance, as they represent around 95 % of all animal species. in this matter is very important to understand the immunologic mechanisms invertebrate as molluscs and relationship with environmental condition. this will allow acknowledging the susceptibility of each species to antigen challenges, mainly infectious agents, and if such conditions affects the intrinsic resistance of each organism. acknowledgments this review was conducted under fomix (conacyt-nayarit government) project (number 131614), and promep-sep founding. thanks to msc c rodríguez-cervantes for english correction style, and md student i parrao for figure design. references auffret m, mujdzic n, corporeau ch, moraga d. xenobiotic-induced immunomodulation in the european flat oyster, ostrea edulis. mar. environ. res. 54: 585-589, 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narbonne jf. effects of tributyltin and 17β-estradiol on immune and lysosomal systems of the asian clam corbicula fluminea (m.). environ. toxicol. pharmacol. 21: 323-330, 2006. fisher w, oliver l, winstead j, volety a. stimulation of defense factors for oysters deployed to contaminated sites in pensacola bay, florida. aquat. toxicol. 64: 375-391, 2003. gagnaire b, gay m, huvet a, ves daniel j, saulnier d, renault t. combination of a pesticide exposure and a bacterial challenge: in vivo effects on immune response of pacific oyster, crassostrea gigas (thunberg). aquat. toxicol. 84: 92-102, 2007. gagnaire b, thomas-guyon h, burgeot th, renault t. pollutant effects on pacific oyster, crassostrea gigas (thunberg), hemocytes: screening of 23 molecules using flow cytometry. cell. biol. toxicol. 22: 1-14, 2006. gagné f, andré c, cejka p, hausler r, fournier m, blaise c. immunotoxic effects on freshwater mussels of a primary-treated wastewater before and after ozonation: a pilot plant study. ecotoxicol. environ. safety 69: 366-373, 2008. gagné f, blaise c, fournier m, hansen pd. effects of selected pharmaceutical products on phagocytic activity in elliptio complanata mussels. comp. biochem. physiol. 143c: 179186, 2006. 155 galloway t, depledge m. immunotoxicity in invertebrates: measurement and ecotoxicological relevance. ecotoxicol. 10: 5-23, 2001. goméz-mandikute a, cajaraville mp. comparative effects of cadmium, copper, paraquat and benzo[a]pyrene on the actin cytoskeleton and production of reactive oxygen species (ros) in mussel haemocytes. toxicol. in vitro. 17: 539546, 2003. hannam ml, bamber s, galloway t, moody aj, jones m. effects of the model pah phenanthrene on immune function and oxidative stress in haemolymph of the temperature scallop pecten maximus. chemosphere 78: 779-784. 2010. hine pm. the inter-relationship of bivalves haemocytes. fish shellfish immunol. 9: 367385, 1999. matozzo v, marin mg. 4-nonylphenol induces immunomodulation and apoptotic events in the clam tapes philippinarum. mar. ecol. prog. ser. 285: 97-106, 2005. matozzo v, rova g, marin m. haemocytes of the cockle cerastoderma glaucum: morphological characterisation and involvement in immune responses. fish shellfish immunol. 23: 732-746, 2007. matozzo v, monari m, foschi j, cattani o, serrazanetti gp, marin mg. first evidence of altered immune response and resistence to air exposure in the clam chamelea gallina exposed to benzo(a)pyrene. arch. environ. toxicol. 56: 479-488, 2009. mydlarz ld, jones le, harvell d. innate immunity, environmental drivers, and disease ecology of marine and freshwater invertebrates. annu. rev. ecol. evol. syst. 37: 251-88, 2006. nusetti o, marcano l, zapata e, esclapés m, nusetti s, lodeiros c. respuestas inmunológicas y de enzimas antioxidantes en la ostra perla pinctada imbricate (mollusca: pteridae) expuesta a niveles subletales de fuel oil no 6. interciencia 29: 324-328, 2004. oliver l, fisher w, volety a, malaeb z. greater hemocyte bactericidal activity in oysters (crassostrea virginica) from a relatively contaminated site in pensacola bay, florida. aquat. toxicol. 64: 363-373, 2003. parry h, pipe r. interactive effects of temperature and copper on immunocompetence and disease susceptibility in mussels (mytilus edulis). aquat. toxicol. 69: 311-325, 2004. pruzzo c, gallo g, canesi l. persistence of vibrios in marine bivalves: the role of interactions with haemolymph components. environ. microbiol. 7: 761-722, 2005. wootton ec, dyrynda ea, pipe rk, ratcliffe na. comparisons of pah-induced immunomodulation in three bivalve molluscs. aquat. toxicol. 65: 13-25. 2003. 156 identification and preliminary characterisation of a haemagglutinin in the coelomic fluid of sipunculus nudus isj 7: 221-227, 2010 issn 1824-307x short communication identification and preliminary characterization of a ca2+-dependent hemagglutinin in the celomic fluid of sipunculus nudus l ballarin, m del favero department of biology, university of padua, padua, italy accepted october 5, 2010 abstract a soluble agglutinin was purified by affinity chromatography of the celomic fluid of the marine worm sipunculus nudus. this agglutinin requires metal cations for its activity and is specific for derivatives of d-galactose. it resulted lightly thermostable, with a ph optimum around 7.5. on sdspage, it was resolved in two bands, of 33 and 31 kda in reducing conditions and 29 and 26 kda in non-reducing conditions. this behavior is probably due to the presence of disulfide bridges between cysteine residues, which are required for the correct functioning of the hemagglutinin, as βmercaptoethanol completely abolish the agglutinating activity of cell-free celomic fluid. the purified lectin can influence in vitro phagocytosis of yeast by celomic leukocytes: in the presence of the molecule, ingestion of foreign particles results significantly decreased and yeast cells agglutinate and forms rosettes around the celomocytes. this suggests a role of the molecule in immunosurveillance. key words: sipunculus; invertebrates; celomic fluid; hemagglutinin; ca2+-dependent lectin introduction lectins are carbohydrate-binding proteins widely distributed in living organisms, animals included (barondes, 1981; yeaton, 1981a). animal lectins fulfill a variety of functions (yoshizaki, 1990; drickamer and taylor, 1993; gabius, 1997; dodd and drickamer, 2001) and many of them act as recognition molecules within the immune system, implicated in direct first-line defense against pathogens, cell trafficking and immune regulation (yeaton, 1981b; yoshizaki, 1990; drickamer and taylor, 1993; gabius, 1997). as regards the last point, lectins appear to participate in the tagging and exclusion of foreign organisms by invertebrate immunocytes, which are covered with different carbohydrate receptors (yeaton, 1981b; yoshizaki, 1990; gabius, 1997; kilpatrick, 2002). in the last two decades, the study of animal lectins has greatly increased and today we know that lectin activity in animals is found in association with a wide variety of primary structures which enable us to distinguish at least 12 families of sugar-binding proteins and a series of "orphan" lectins belonging to either some unknown lectin family or well-established protein families with the majority of the members unable to ___________________________________________________________________________ corresponding author: loriano ballarin department of biology, university of padua, via ugo bassi 58/b, 35100 padua, italy e-mail: loriano.ballarin@unipd.it bind sugars. in addition, many animal lectins also bind structures other than carbohydrates via proteinprotein, protein-lipid or protein-nucleic acid interactions (gabius, 1997; dodd and drickamer, 2001; kilpatrick, 2002; loris, 2002). sipunculans are marine worms devoid of circulatory system and with a large celomic cavity filled with celomic fluid containing various types of celomocytes, the majority of which are represented by hemerythrocytes, nucleated cells containing the red pigment hemerythrin (valembois and boiledieu, 1980; dybas, 1981). in addition, the celomic fluid contains wandering ciliated urn cell complexes, which secrete mucus able to trap foreign particles or cells (bang and bang, 1976, 1980; dybas, 1976; nicosia and sowinski, 1995), and leukocytes involved in defense responses against non-self materials. leukocytes are classically classified as granulocytes and hyalinocytes on the basis of the presence or absence of acidophilic or basophilic cytoplasmic granules (dybas, 1981). both granulocytes and hyalinocytes can engulf non-self material (brown and winterbottom, 1969; matozzo et al., 2001) and phagocytosis represents the main cell-mediated defense mechanisms against microbes in these organisms. both the cell types share a similar content of hydrolytic lysosomal enzymes and can produce superoxide anions as a consequence of the activation of a phagocytosisassociated respiratory burst. in addition, they contain lysozyme which can be released as a 221 mailto:loriano.ballarin@unipd.it consequence of a bacterial challenge (matozzo et al., 2001). as regards other humoral immune responses, the presence of various factors, such as lysins, agglutinins, opsonins, and antibacterial molecules has been reported in the celomic fluid of various sipunculan species (bang, 1966; weinheimer et al., 1970; evans et al., 1973; matozzo et al., 2001). however, up to now, no clear characterization of soluble molecules involved in immune responses has been carried out. with the aim of contributing to fill this gap, a search for soluble molecules with hemagglutinating activities in the celomic fluid of sipunculus nudus was undertaken. this reports presents some preliminary results on the identification and partial characterization of a ca2+dependent lectin with hemagglutinating activity from the celomic fluid of s. nudus with specificity for dgalactosides and able to influence phagocytosis. materials and methods animals specimens of sipunculus nudus were collected by hydraulic dredging in the sandy bottom (5-6 m depth) of the west coast of the northern adriatic sea, off the lagoon of venice (italy). they were transferred in 15 l aquaria containing abundant sand on the bottom and filtered (5 µm filter) seawater (fsw) at a temperature of 19 °c. celomic fluid collection celomic fluid (cf) was collected from the celomic cavity with a 1 ml plastic syringe. cfs from 10 animals were pooled and centrifuged at 780xg for 10 min and the supernatant was referred as cellfree celomic fluid (cfcf). hemagglutination (ha) assay rabbit erythrocytes were washed three times by centrifugation at 500xg for 10 min in tris-buffered saline (tbs: tris-hcl 50 mm, nacl 150 mm, ph 7.4) and incubated for 30 min at 37 °c in 0.1 mg/ml trypsin in tbs (ballarin et al., 1999). they were then washed again and resuspended in tbs containing 0.2 % gelatin to get a 1 % (v/v) solution. fifty µl of cfcf were serially diluted two-fold with tbs in the wells of u-bottomed microtiter plates and an equal volume of erythrocyte suspension was added to each well; fsw was used in controls. tbs containing 5 mm egta or 5 mm cacl2 (tbs-ca) was also used to assess the ca2+-dependency of the reaction. in another experimental series, cfcf was incubated for 30 min with 1 % rabbit erythrocyte to control the specificity of the interaction: the suspension was then centrifuged at 780xg for 10 min and the erythrocyte-absorbed supernatant was collected and used in the ha assay. plates were gently shaken, incubated for 1 h at 37 °c and then kept at 4 °c. the ha titer (ht), i.e., the reciprocal of the highest dilution giving positive ha was then evaluated. ha inhibition assay the following sugars were assayed for their effects on the agglutination of trypsinised erythrocytes: d-galactose, d-glucose, dgalactosamine, d-glucosamine, n-acetyl-dgalactosamine, n-acetyl-d-glucosamine, methyl-αd-galactopyranoside, methyl-β-dgalactopyranoside, methyl-α-d-glucopyranoside, 2deoxy-d-galactose, d-mannose, d-fucose, lrhamnose, d-melibiose, d-sucrose, d-lactulose, dlactose. they were purchased from sigma (st louis, mo, usa) and added to tbs-ca to yield 400 mm storage solutions. a total of 25 ml of cfcf were then added to an equal volume of two-fold serial dilutions of carbohydrates in the wells of ubottomed microtiter plates which were incubated for 30 min at 37 °c. erythrocytes were then added and, after a further incubation of 60 min at 37°c, the lowest carbohydrate concentrations able to inhibit agglutination were evaluated (modified from parrinello and canicattì, 1982). effects of periodate and β-mercaptoethanol on hemagglutinating activity to evaluate the importance of hemagglutininconjugated carbohydrates on ha, cfcf was incubated for 2 h at 4 °c with an equal volume of 0.08 m sodium meta-periodate in 0.2 m citrate buffer, ph 5.4 acid in order to oxidize sugars (millar and ratcliffe, 1987). the mixture was then dialyzed against tbs-ca for 3 h to remove periodate and used in ha assay. the importance of disulphide bridges in hemagglutinating activity was assessed by incubating cfcf with 20 mm of β-mercaptoethanol. the mixture was then dialyzed as described above and used in ha assay. lectin purification and characterization affinity chromatography of cfcf on acidtreated sepharose cl-6b (pharmacia, uppsala, sweden) was carried out as described by parrinello and canicattì (1982, 1983). the column (7 x 1.6 cm) was previously equilibrated with phosphate-buffered saline (pbs: 0.8 % nacl, 0.02 % kcl, 0.02 % kh2po4, 0.115 % na2hpo4, ph 7.2) containing 5 mm cacl2, loaded with 40 ml of cfcf, and washed with a solution of nacl 1 m and cacl2 5 mm. the flow rate was kept constant at 20 ml/h and 2-ml fractions were collected, the absorbance of which was measured, at 280 nm, with a kontron uvikon 930 uv-vis spectrophotometer. when absorbance resulted stable, at values close to zero, the column was eluted with 0.2 m d-galactose in 0.1 m nacl. a single absorbance peak was usually obtained after elution with d-galactose, and fractions corresponding to the peak were collected, dialyzed overnight at 4 °c against distilled water, lyophilized with a savant vacuum centrifuge, and stored at -20 °c until use. protein concentration was evaluated according to bradford (1976) using bovine serum albumin as standard. sds-page (12 % separating gel) of purified lectin was performed according to laemmli (1970). samples of lyophilized lectin were diluted to 1.0 mg/ml in sample buffer (0.5 m tris-hcl, ph 6.8, 10 % glycerol, 10 % sds, 0.5 % bromophenol blue with or without 5 % β-mercaptoethanol, for reducing and non-reducing conditions, respectively. proteins treated with β-mercaptoethanol were also boiled for 5 min. gels were calibrated with low molecular 222 weight marker proteins (biorad laboratories, hercules, ca, usa), run at a constant current of 18 ma/gel for approximately 3.5 h and stained with coomassie blue. effects of temperature and ph on hemagglutinating activity to study the effects of temperature on hemagglutinating activity, the sipunculus agglutinin (sa), at a concentration of 2.0 mg/ml in distilled water, was incubated for 30 min at 4, 25, 37, 60 and 80 °c, and then used in the ha assay as previously described. the stability of the lectin, at the above concentration, was tested by assaying its agglutinating activity, in tbs-ca, after incubation at room temperature for 0, 30, 60, 90, 120 and 180 min. fig. 1 presence and absence of hemagglutinating activity of s. nudus cfcf incubated in fsw (a) or fsw containing 5 mm egta (b). ht, hemagglutination titer fixed for 30 min at 4°c in a solution of 1 % glutaraldehyde and 1 % sucrose in fsw, and stained with 10 % giemsa for 5 min. at least 300 hemocytes per coverslip were observed under a leitz dialux 22 light microscope, in ten optical fields, at a magnification of 1250x, to determine the percentage of hemocytes with ingested yeast cells. each experiment was repeated three times with three different celomocyte pools. data are expressed as mean ± sd and were compared using the χ2 test. the effect of ph was evaluated using the following buffers in the ha assay, in the presence of 5 mm cacl2: 0.2 m tris-maleate (ph 6.0, 6.6, 7.0, 7.6, 8.0), 0.2 m glycine-naoh (ph 8.6, 9.0, 9.6, 10.0). data are expressed as mean ± sd. phagocytosis assay sixty µl of cf, collected as described above, were placed in the centre of culture chambers, prepared as described by ballarin et al. (1994) and cells were left to adhere to coverslips for 30 min at room temperature (rt). after adhesion, slides were repeatedly washed by dipping in a large volume of fsw in order to remove hemerythrocytes, which do not adhere to glass, and the remaining celomocytes were incubated with 60 ml of a suspension of yeast cell (yeast: hemocyte ratio = 10 : 1) in fsw in the presence or in the absence of 0.5 mg/ml of sa in fsw. in another series of experiments, yeast was previously incubated with sa (0.5 mg/ml in fsw) for 30 min before the assay. slides were then washed in fws to remove uningested yeast and cells were results cfcf shows hemagglutinating activity cfcf can agglutinate trypsinized rabbit erythrocytes (ht: 64; fig. 1a). ha was almost absent in erythrocyte-absorbed cfcf. it was inhibited in presence of various monosaccharides and disaccharides at various concentrations. galactose resulted the most powerful inhibiting sugar, showing the lowest minimum effective concentration, followed by other galactosides and galactose-containing disaccharides (table 1). table 1 effects of different sugars in hemagglutinating activity of s. nudus cfcf sugar minimum effective concentration (mm) d-galactose 50 d-glucose 200 d-galactosamine > 400 d-glucosamine > 400 n-acetyl-d-galactosamine — n-acetyl-d-glucosamine — methyl-α-d-galactopyranoside 100 methyl-α-d-glucopyranoside 200 methyl-β-d-galactopyranoside > 400 2-deoxy-d-galactose 400 d-fucose — l-rhamnose — d-mannose — d-sucrose — d-raffinose — d-lactulose 200 d-lactose 200 d-melibiose 400 cfcf, cell-free celomic fluid 223 fig. 2 affinity chromatography of s. nudus cfcf on acid-treated sepharose cl-6b. arrows indicate addition of galactose. the hemagglutinating activity required bivalent cations, as indicated by the absence of ha in the presence of egta (fig. 1b). treatment with periodate and β-mercaptoethanol completely abolished the ability of cfcf to agglutinate rabbit erythrocytes. hemagglutinin purification and physico-chemical characterization of the lectin sugar specificity was exploited to purify soluble sa by affinity chromatography on acid-treated sepharose cl-6b. we obtained a single peak showing hemagglutinating activity towards trypsinized rabbit erythrocytes (fig. 2). when sa was incubated at rt, its ht remained stable at the value of 64 after 60 min, but dropped to 16 after 120 min and conserved this residual activity in the following 4 h. the agglutinin resulted lightly thermostable as the ht remained stable at the value of 64 after 30 min exposure at temperatures ranging from 4 to 25 °c; decreased to 8 after 30 min at 37 °c and retained residual detectable activity (ht: 2) after 30 min at 80 °c (fig. 3a). the lectin was stable within ph ranging from 7.0 to 9.5, with maximum activity around 7.5 (fig. 3b). after sds-page, of the affinity-purified material, two bands were obtained with apparent molecular weight of 33 and 30 kda and 29 and 26 kda under reducing and non-reducing conditions, respectively (fig. 4). fig. 3 effects of ph (a) and temperature (b) on hemagglutinating activity of s. nudus cfcf. ht, hemagglutination titer 224 purified lectin can influence phagocytosis most of the s. nudus leukocytes can phagocytose foreign cells or particles (fig. 5a, table 2). incubation of celomocytes and yeast with the purified lectin resulted in a significant decrease of in vitro yeast phagocytosis (table 2). yeast cells appeared agglutinated and frequently form rosettes or clumps adherent to celomocyte surface without being ingested (figs 5b, c). when yeast was preincubated with the purified lectin, washed and incubated with celomocytes in fsw, no significant variation in the fraction of phagocytozing cells with respect to controls was observed (table 2). discussion in the present study, we have identified a lectin with agglutinating properties in the celomic fluid of s. nudus. d-galactose shows the highest inhibiting power and the molecule resulted specific for derivatives of d-galactose, which share the c4 hydroxyl in β position, whereas limited or no effects on ha were observed in the presence of glucosides, mannose, and rhamnose. the c6 hydroxyl is important for sugar-lectin interaction as its absence in fucose resulted in the lack of inhibition. the inhibition of agglutination is influenced by the addition of a methyl group to c1 hydroxyl: it leads to a light decreases if in position α (in methyl-α-dgalactopyranoside ),  but to a higher decrease when in position β (in methyl-β-d-galactopyranoside). the absence of c2 hydroxyl (in 2-deoxy-d-galactose) reduces the inhibitory power which is further decreased by its substitution as in d-galactosamine and n.acetyl-d-galactosamine: in this case the degree of inhibition depends on the steric hindrance, the latter compound being less effective than the former. among disaccharides, those containing galactose, i.e., lactulose and lactose, show inhibition of ha although to a less extent than the monosaccharide. fig. 4 sds-page of s. nudus purified lectin (b, c) and cfcf (d). lane a) molecular weight markers; lanes b and c) non reducing and reducing conditions, respectively. this excludes its belonging to galectin family, that includes the majority of ca2+-independent lectins, none of which are glycoproteins (kasai and hirabayashi, 1996); ii) the absence of any inhibitory effect on ha of rhamnose and melibiose. this indicates that sa is not a member of rhamnosebinding lectins, another family of sugar-binding proteins which do not require divalent cations for their activity (jimbo et al., 2007; terada et al., 2007; gasparini et al., 2008). two bands were present in the electrophoretic pattern of the affinity-purified material. similar patterns were reported for other purified lectins (parrinello and arizza, 1989; arizza et al., 1991; gasparini et al., 2008) and they can be interpreted as the consequence of the presence, in the pooled samples, of lightly different isoforms of the same molecule. the presence of different isoforms in the pooled hemolysate has been recently demonstrated the observed dependency on divalent cations suggests that the identified lectin belongs to the clectin family. this is further supported by: i) the nature of the protein which carries glycoconjugates required for the interaction with ligands, as indicated by the absence of ha after treatment with periodate. table 2 effect of the preincubation and incubation of yeast cells in the affinity-purified lectin on yeast phagocytosis by sipunculus leukocytes preincubation medium incubation medium percentage of phagocytosing cells fsw fsw (control) 54.8 ± 8.9 fsw sa 39.5 ± 5.3 ** sa fsw 47.1 ± 5.6 fsw, seawater; sa, sipunculus agglutinin ** = p < 0.01 225 fig. 5 a) phagocytes filled with ingested yeast cells (arrowheads); b) yeast cells clumped around a leukocyte (arrow) forming a rosette; c) agglutinated yeast cells in the proximity of a leukocyte (arrow) with ingested yeast cells (arrowheads). bar = 10 µm. in the compound ascidian botryllus schlosseri (gasparini et al., 2008). the apparent molecular weight of the two protein bands of 29 and 26 kda under non-reducing and 33 and 31 kda under reducing conditions suggests the presence of intramolecular disulphide bridges which keep the proteins in a highly folded form in non-reducing conditions so that they can move more rapidly in page. this is supported by the observation that βmercaptoethanol completely abolish the agglutinating capability of cfcf, which stress the importance of the disulphide bonds for the correct functioning of the hemagglutinin. in addition, similar electrophoretic behavior has been reported for various ascidian lectins (parrinello and arizza, 1988; cammarata et al., 2007; gasparini et al., 2008). despite the huge number of published papers on invertebrate lectins, their biological role is still a matter of debate. in most cases, they are thought to be involved in immune recognition, although only in few cases it has been clearly demonstrated. our lectin is involved in cell-cell interactions between: i) yeast cells enabling their agglutination and hindering their phagocytosis; ii) celomocytes and yeast cells leading to the formation of rosettes. the formation of large aggregates of foreign cells, although preventing their phagocytosis, may stimulate their encapsulation and subsequent melanization by wandering celomocytes, leading to the final formation of brown bodies, present in the hemolymphatic or celomic cavities of many invertebrates (hetzel, 1965; valembois et al., 1992, 1994; jans et al., 1996; pagliara et al., 2003). we have no direct evidences, at the moment, that this can occur in the case of yeast cells, but numerous brown bodies were frequently found in the celom of the animal used in the present experiments. future efforts are, therefore, required to better understand the role of the lectin, identify the source of the protein and complete its sequence for 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dev. comp. immunol. 5: 391-402, 1981a. yeaton rw. invertebrate lectins: ii. diversity of specificity, biological synthesis and function in recognition. dev. comp. immunol. 5: 535-545, 1981b. millar da, ratcliffe na. activity and preliminary characterization of a hemagglutinin from the hemichordate saccoglossus ruber. dev. comp. immunol. 11: 309-320, 1987. yoshizaki n. functions and properties of animal lectins. zool. sci. 7: 581-591, 1990. 227 isj 3: 97-102, 2006 isj 3: 118-124, 2006 issn 1824-307x minireview cytoskeletal proteins and morphogenesis in planarians a fagotti, f simoncelli, i di rosa, r pascolini department of cellular and environmental biology, university of perugia, perugia, italy accepted november 29, 2006 abstract regeneration processes employ a series of differentiative events related to various embryonic morphogenetic phenomena in which the cytoskeleton plays a fundamental role. planarians are an excellent model to study development mechanism because they show an exceptional physiological morphogenetic plasticity that is also at the basis of their extraordinary regenerative ability. in this paper we discuss results concerning two cytoskeleton components, actin and tubulin, during morphogenetic processes of the planarian schmidtea polychroa. comparative studies on cytoskeleton function during morphogenetic processes in evolutionary-distant animal models can offer important new insights in the fields of stem cell biology and regenerative medicine. key words: planarian morphogenesis; actin; tubulin __________________________________________________________________________________________ introduction the cytoskeleton of eukaryotic cells plays a basic role in many dynamic processes that are controlled by a network of cytoskeletal fibers constituted by three types of filaments: microfilaments made up of various actin isoforms, microtubules consisting of αand β-tubulin, and intermediate filaments. these cytoskeletal components are associated with hundreds of proteins that regulate a rich variety of structural or dynamic machineries, crucial for many essential cellular functions including cell migration, cell division, maintenance of cell shape, cell signalling, chromosome and organelle movements. the cytoskeleton also plays a pivotal role in cellsubstrate and cell-cell adhesions, supplying the structural support necessary for these contacts as well as providing the focal point for signal transductions. important advances have been made in the last years not only in characterizing the molecular and functional mechanisms of cytoskeleton but also in defining connections between cytoskeleton disorders and pathological conditions. cytoskeletal protein abnormalities are frequently the origin of many pathological conditions ___________________________________________________________________________ corresponding author: rita pascolini department of cellular and environmental biology university of perugia via a. pascoli 1, 06123 perugia, italy e-mail: ritapasc@unipg.it including several cardiovascular, muscle, neurodegenerative and skin diseases and cancer (ramaekers and bosman, 2004). many studies in various animal developmental models have also demonstrated the importance of cytoskeletal proteins in controlling cellular behavior during the morphogenetic and developmental processes. tissue building which occurs during both embryo development and wound-healing events, involves a series of cellular dynamics and movements that are driven by similarly orchestrated cytoskeletal machinery (martin and parkhurst, 2004; redd et al., 2004). embryonic epithelial wound repair shows striking similarities in cytoskeletal remodelling with morphogenetic events of dorsal closure in drosophila and ventral enclosure in caenorhabditis embryos (martin and parkhurst, 2004; wood et al., 2002). therefore, the study of cytoskeleton changes during wound repair can offer insights about evolutionary conserved cytoskeleton functions during embryo morphogenetic processes and viceversa. in this review we report and discuss results concerning the involvement of cytoskeletal proteins during the morphogenetic events underlying regeneration in freshwater planarians. planarians are a fascinating model for studying the development processes because they show a great physiological morphogenetic plasticity that is also at the basis of their exceptional regenerative ability. there is a high degree of molecular and functional conservation throughout the phylogeny in the basic 118 mechanisms of the developmental phenomena: the elucidation of molecular mechanisms underlying morphogenetic plasticity in animal models such as planarians will contribute to clarify key issues in the fields of wound healing and regenerative medicine. developmental plasticity in planarians the biological peculiarity of the planarian model is characterized by a continuous status of morphogenesis; its tissues are physiologically subjected to a continuous cell renewal. a striking example is its ability to either grow or de-grow depending on nutritional availability: the significant modifications in body size to which planarians are subjected always maintain the correct proportions and are reversible (baguñà and romero, 1981). the great developmental plasticity is also directly related to extraordinary regenerative power; it is known that planarians are able to rebuild a complete organism from a very small body region. the source of this morphological plasticity and regenerative properties is a stable population of totipotent stemcells, called “neoblasts”, that, in the adult, are the only proliferating cells (baguñà, 1976a, b). these stem cells are able to generate all different cell types present in the adult and are responsible for physiological homeostasis and tissue renewal. regeneration in planarians regeneration is widely distributed among metazoans: a large number of species have the capacity to replace injured or lost body parts and, in few cases, to rebuild the entire organism from a tiny portion of the body. models of regeneration are provided by cnidarian, platyhelminthes and echinodermata, that can regenerate a complete organism by bi-directional regeneration, and by urodele amphibians that can rebuild a complete appendage. morgan (1901) described two major types of regeneration: 1) morphallaxis, that involves remodelling and re-patterning of pre-existing tissues into newly organized structures as occurs in hydra, the classical example of this form of regeneration and 2) epimorphosis, that requires active cell proliferation at the wound site and the formation of a new growth zone from which the missing structure will be regenerated and correctly patterned. this mode of regeneration can involve the dedifferentiation of cells that then proliferate and redifferentiate to form new structures, such as occurs in amphibians, and/or the activation of preexisting stem cell populations to form a new stump, the blastema, that then differentiates into missing body parts. planarians are capable of blastemabased epimorphic regeneration. they are triploblastic acoelomates belonging to the platyhelminthes clade and are included as members of the lophotrochozoa (adoutte et al., 2000). although the epimorphosis is the prevalent regenerative process in planarians, morphallactic events also contribute to the restoration throughout the re-establishment of the exact body proportion and symmetry. the two phenomena can contribute differently to regeneration process depending on the starting-conditions. as mentioned before, epimorphic fig. 1 electron micrograph of a cluster of neoblasts. bar = 2 μm. regeneration in planarians is based mainly on a population of undifferentiated cells, the neoblasts. they are small (5-8 µm in diameter) with large nuclei and very little cytoplasm and are distributed throughout the parenchyma or mesenchyme along the whole body (fig. 1) (reddien and sànchez alvarado, 2004). when a planarian is injured a strong muscular contraction occurs immediately to minimize the wound area that is then rapidly covered by a thin layer of epithelium (pascolini et al., 1984; 1988a, b; baguñà et al., 1994; sànchez alvarado and newmark, 1998). sequentially, the neoblasts proliferate close to the site of injury and migrate distally accumulating under the wound epithelium. they form a regenerative blastema, the unpigmentated structure in which missing tissues regenerate in a week (dubois, 1949; baguñà, 1976b; saló and baguñà, 1984). once inside the blastema, the neoblasts no longer divide (saló and baguñà, 1984). there are two principal hypotheses concerning the source of blastema-forming cells: one is based on a transdetermination event that is supported by evidence that somatic cells can originate from undifferentiated cells committed to becoming germ cells (gremigni and miceli, 1980); the second is based on the totipotency of preexisting stem cells that proliferate in response to injury. the evidence that blastema is constituted by pre-existing stem cells came from experiments of xray irradiation after which planarians completely lost the ability to regenerate and died within several weeks. the irradiated animals only regained their regenerative ability when injected with an enriched fraction of neoblasts (brøndsted, 1969; baguñà et al., 1989). moreover, brdu labelling experiments of neoblasts confirmed that planarian stem cells contribute to the regeneration process through an active migration towards the wound region (newmark and sànchez alvarado, 2000). new molecular and genetic approaches have allowed to identify molecular markers of neoblasts and developmental genes important for both stemcell biology and regeneration. recently, proteins belonging to the argonaute/piwi family, smedwi-1 and smedwi-2, with a regulative role in the production of neoblast progeny have been isolated in schmidtea mediterranea (reddien et al., 2005). djpum, a member of evolutionary conserved puf 119 fig. 2 a) schematic representation of the head amputation in planarian. the blastema area at various times of regeneration is used for immunoblotting. b, c) immunoblotting of planarian blastema area at 0, 1, 2, 5, 7 days of regeneration decorated with anti-αsm-1 (b) and anti-total actin (c) mabs. family of rna-binding proteins, homologue of drosophila puf gene pumilio, also plays an essential function in dugesia japonica neoblast maintenance by supporting their mitotic proliferation (salvetti et al., 2005). the analysis of several neoblast markers, such as pcna protein and djpiwi-1 in dugesia japonica, have suggested that a neoblast population could be heterogeneous (ito et al., 2001; rossi et al., 2006). the molecular processes underlying regeneration in planarians are largely unknown. it is likely that a combination of chemical and mechanical cues stimulate blastema growth such as the triggers originating from wound epithelium, dorsal/ventral (d/v) pattern information and epidermis-mesenchyme interactions (chandebois, 1979; baguñà et al., 1988; kato et al., 1999, 2001). cytoskeletal proteins in planarians there is a high degree of molecular and functional conservation of the cytoskeletal proteins during animal evolution (doolittle, 1995). knowledge about the role of cytoskeleton proteins has increased due to immunochemical and molecular and genetic studies on animal models at different evolutionary levels such as yeast, dictyostelium, caenorhabditis, drosophila and mice. this paper discusses data concerning actin and tubulin, the most conserved cytoskeletal components, during morphogenetic processes of the planarian schmidtea polychroa. actin actin is the major component of the microfilament system of eukaryotic organisms and plays a central role in cell shape and motility processes. in most organisms it is encoded by a multigene family encoding different isoforms regulated in a tissue-, cell type-, and contextdependent fashion (vandekerckhove and weber, 1984; rubenstein, 1990; herman, 1993). the high evolutionary conservation reflects a stringent functional and structural constraint to interact with itself, as well as with a variety of other proteins (hennessey et al., 1993). among the higher vertebrates, six isoforms are present and grouped into two classes, muscle (α-skeletal, α-cardiac, αsmooth muscle, and γ-smooth muscle) and cytoplasmic (β and γ) actins (vandekerckhove and weber, 1978). the distinction between muscle and non-muscle actins is a characteristic of higher animals and insects; in other invertebrate the actin isoforms are mainly similar to cytoplasmic type. it has been hypothesized that muscle actins have appeared from non-muscle actins by gene duplication and divergence twice during animal evolution (mounier et al., 1992).the major differences among actin isoforms are essentially their nh2-terminal sequences. it has been hypothesized that this specific domain could be responsible for specialized functions due to the binding to specific actin-binding proteins. many expressing studies have demonstrated that actin isoforms show typical modulations during cell differentiation events characteristic of both celland tissue development and wound repair (sawtell and lessard, 1989; ruzicka and schwartz, 1988; darby et al., 1990; gunning et al., 1997). it has been demonstrated that in mammals α-smooth muscle isoactin is a marker of differentiation of both vascular smooth muscle cell during embryogenesis and regeneration, and myofibroblasts in wound healing (darby et al., 1990; serini and gabbiani, 1999). the development of striated muscle cells also shows in myotome the transient expression of the alpha-vascular smooth actin (woodcock-michell et al., 1988; sawtell and lessard, 1989). α-skeletal and α-cardiac actin isoforms are involved in cardiomyogenesis (ruzicka and schwartz, 1988). by using immunochemicaland pcr-based approaches various actin isoforms have been characterized in the planarian s. polychroa (pascolini et al., 1988a, b, 1992a, b; fagotti et al., 1998). a specific cytoplasmic isoform has been shown to be involved in cell differentiation and 120 fig. 3 immunoelectron micrographs of migrating neoblast stained with anti-αsm-1 mab. the labelling is mainly localized at the level of pseudopodia and filopodia (arrow). n, nucleus. bar = 0.5 μm (modified from pascolini et al., 1992b). morphogenesis (pascolini et al., 1992b; di rosa et al., 1994a). this isoactin was revealed by using the anti-αsm-1 mab that selectively recognized the nh2-terminus sequence ac-eeed, the specific domain of the endothermic vertebrate α-smooth muscle actin (skalli et al., 1986; chaponnier et al., 1995). in normal planarians, this anti-αsm-1 reactive actin is localized in cytoplasmic domains of migrating undifferentiated neoblasts as well as in epithelial and nerve cells (pascolini et al., 1992b; di rosa et al., 1994b). interestingly, when a planarian is induced to regenerate, the expression of antiαsm-1 reactive actin is up-regulated in the restricted region of blastema. there is a significant increase of this protein after two days of regeneration, it increases gradually to reach a maximum expression at about 5-6 days and then gradually decreases (fig. 2) (pascolini et al., 1992b). fine analysis of blastema has shown that the anti-αsm-1 reactive actin localization is associated with cytoskeletal re-arrangement that occurs during the activated neoblast migration and differentiation (pascolini et al., 1992b; di rosa et al., 1994a). in migrating neoblasts, whose activation is amplified during blastema growth, the labelling was mainly localized at the level of the filamentous structures of pseudopodia and filopodia (fig. 3). differentiating myoblasts, that are the neoblasts committed to originate muscle fibers, also transiently express this isoactin which disappears when the cellular phenotype is differentiated and the myofibers are organized (fig. 4). it is interesting to note that this specific expression pattern resembles those described for α-smooth muscle actin during the myogenesis process in higher vertebrates (woodcock-michell et al., 1988). the involvement of anti-αsm-1 reactive actin has also been well documented in the re-epithelialization process. the planarian epidermis consists of a single layer of columnar cells linked to one another in the apical portion by septate junctions and anchored to the basement membrane by hemidesmosomes (fig. 5) (hori, 1989; di rosa et al., 1994a). the basal region of epidermal cells form the characteristic processes, called ‘feet’, that are rich in intermediate filaments and microfilaments that are selectively labelled by an anti-αsm-1 mab (pascolini et al., 1992b; di rosa et al., 1994a). fig. 4 immunoelectron micrographs of differentiating myoblast stained with anti-αsm-1 mab. the labelling is not detected in the organized myofibers (arrow). bar = 0.5 μm (modified from pascolini et al., 1992b). because of the inability of epidermal cells to divide, renewal depends on neoblasts that migrate across basement membrane from the parenchyma (di rosa et al., 1994a; newmark and sanchez alvarado, 2000). this phenomenon is amplified during wound repair. in response to an injury, the wounded surface is first quickly covered by the cellular spreading of the old differentiated epidermal cells that lose their organized structure (hori, 1991; pascolini et al., 1984, 1988a, b; baguñà et al., 1994; sánchez alvarado and newmark, 1998). subsequently, the wound epidermis is renewed in about 1 week by the active migration and differentiation of stem cells from the blastema (newmark and sanchez alvarado, 2000). during their migration through basal lamina, the epidermal cell precursors over-express anti-αsm-1 reactive actin. they undergo a spatial and temporal cytoskeletal remodelling that involves the formation of stress-fibers in both lateral and basal cellular domains, that are specifically labelled by anti-αsm-1 (fig. 6) (pascolini et al., 1992b; di rosa et al., 1994a). fig. 5 electron micrograph of intact planarian monolayered epidermis (ep) showing basal ‘feet’ (arrow) anchored to the basement membrane (b m) and a differentiating epidermal cell ( ). m, myofiber. bar = 2 μm. 121 fig. 6 immunoelectron micrograph of regenerating planarian epidermis (ep) decorated with anti-αsm-1 mab: a differentiating epidermal cell ( ) appears markedly labelled. b m, basement membrane. bar = 1 μm (modified from pascolini et al., 1992b). these results demonstrate the modulation of anti-αsm-1 reactive actin in various planarian morphogenetic phenomena, such as blastema formation, myogenesis and re-epithelialization and strongly support the hypothesis of its specific role in cell differentiation. in particular, this specific actin is involved in neoblast activation and motility through a characteristic expression. tubulin microtubules are a ubiquitous cytoskeletal component present in all eukaryotic cells. they are formed by the self-assembly of αand β-tubulin heterodimers and are involved in various cellular functions such as cell division, intracellular transport, maintenance of cell shape and flagellar and ciliary motility. the variety of functions can probably be attributed to the expression of different genes that are selectively expressed in specific cell types or at specific stages of development. a tubulin cdna (sptub-1) has been isolated in planaria s. polychroa. sptub-1 encodes for a tubulin protein which shares the highest degree of similarity with the known α-tubulin sequences (simoncelli et al., 2003). the study of transcript expression shows that sptub-1 is selectively restricted to testis tissues that consist of many elliptical follicles organized in clusters that are located on the dorsal side of the animal (fig. 7a). our results show that sptub-1 mrna was expressed in the testis at the level of spermatogenetic cells as spermatogonia, spermatocytes and spermatids (fig. 7b). no signal was detected in the spermatozoa found inside the testis. the expression pattern suggests that sptub1 is a tubulin isotype specific of undifferentiated and differentiating germinal cells with a possible role in spermatogenesis. conclusions and perspectives morphogenesis includes processes in which cytoskeleton and cell migration are strongly involved. many studies on the molecular mechanisms of physiological and pathological processes, such as embryonic morphogenesis, wound healing, immune surveillance and cancer metastasis, indicate that a coordinated organization of cytoskeletal proteins is central to cell behavior, cell migration, intracellular signalling and cell-cycle control processes. our studies on planarian morphogenesis have demonstrated that cytoskeletal proteins are strongly implicated in the differentiation processes, that also occur in higher animals. in particular the findings reported in planarian show that: 1) a specific actin isoform is a marker of neoblast activation and differentiation. its modulation during morphogenetic phenomena suggests that this isoactin is necessary for regeneration. future studies should investigate the molecular dynamic of the anti-αsm-1 reactive actin. studies are in progress to confirm its specific involvement in cell locomotion processes. rna interference studies should also analyze the effects of loss-of-function of this isoactin gene during both physiological and regenerative conditions; fig. 7 a) in situ hybridization of sptub-1 mrna in planarian male gonad: the transcript is exclusively expressed in testis (te). bar = 10 μm (modified from simoncelli et al., 2003). b) in situ hybridization of sptub-1 mrna in planarian male gonad: higher magnification showing that the signal is restricted to spermatogonia, spermatocytes and spermatidis but not in the spermatozoa (sp). bar = 10 μm (modified from simoncelli et al., 2003). 122 2) a tubulin isoform is selectively expressed during spermatogenesis. further study should focus on the mechanisms underlying germ cell fate determination. the role of this tubulin should also be studied in relation to the expression of other genes implicated in planarian gametogenesis, such as vasa-like genes. greater knowledge about the cytoskeletal components and their function during morphogenetic phenomena in planarian sheds light the fundamental process of cell differentiation during animal evolution. comparative studies of molecular and functional cytoskeletal regulation among different animal models can provide important new insights on cytoskeleton function and reveal conserved mechanisms. acknowledgements we thank dr. nancy hutchinson for critical reading of the manuscript. references adoutte a. the new animal phylogeny: reliability and implications. proc.natl. acad.sci. usa 97: 4453-4456, 2000. baguñà j. mitosis in the intact and regenerating planarian dugesia mediterranea n. sp. i. mitotic studies during growth, 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morphogenesis in drosophyla embryos. nat. cell biol. 4: 907-912, 2002. woodcock-michell j, mitchell jj, low rb, kieny m, sengel p, rubbia l, et al. α-smooth muscle actin is transiently expressed in embryonic rat cardiac and skeletal muscles. differentiation 39: 161-166, 1998. 124 acknowledgements isj100.pdf 60 isj 2: 60-68, 2005 issn 1824-307x research report early suppression of immune response in heliothis virescens larvae by the endophagous parasitoid toxoneuron nigriceps r ferrarese1*, m brivio1, t congiu2, p falabella4, a grimaldi1, m mastore1, g perletti3, f pennacchio4, l sciacca1, g tettamanti1, r valvassori1, m de eguileor1 1department of structural and functional biology, university of insubria, varese, italy 2department of human morphology, university of insubria, italy 3department of structural and functional biology, university of insubria, busto arsizio, italy 4dipartimento di biologia, difesa e biotecnologie agro-forestali, università della basilicata, potenza, italy accepted april 28, 2005 abstract toxoneuron nigriceps is an endophagous parasitoid of larval stages of the noctuid moth heliothis virescens. as all parasitoids, this wasp avoid host immune reaction by a combination of several passive and active mechanisms. secretions injected by ovipositing females, which contain venom, calyx fluid and polydnaviruses, are the most probably factors actively disrupting heliothis virescens immune system. this paper describes the main alterations of the host immune response observed shortly after oviposition by t. nigriceps. a transient block of prophenoloxidase activity is registered along with changes in hemocyte number, adhesion and structure, which suggest the occurrence of apoptosis. in contrast, the host plasmatocytes appear structurally unaltered, but unable to produce a capsule in vitro. key words: insects; parasitoid; immune defenses introduction foreign objects entering insect hemocoel are recognized as non-self and elicit a variety of defense reactions, which are somewhat arbitrary divided into humoral and cellular responses (hoffman, 1995; gillespie et al., 1997; lavine and strand, 2002). humoral responses include the production of antibacterial/antifungal peptides (boman et al., 1991; hoffmann et al., 1993; hultmark, 1993; cociancich et al., 1994; lowenberger, 2001) and of reactive intermediates of oxygen or nitrogen (bogdan et al., 2000; vass and corresponding author: roberto ferrarese department of structural and functional biology, university of insubria, j.h. dunant 3, 21100 varese, italy e-mail: roberto.ferrarese@uninsubria.it nappi, 2001), as well as in the activation of enzymatic cascades regulating coagulation or melanization of hemolymph (gillespie et al., 1997). host cellular immune responses, like phagocytosis, nodulation and encapsulation (schmidt et al., 2001; lavine and strand, 2002) are mediated by different types of hemocytes, but the regulatory molecular mechanisms involved are much less understood than those controlling humoral responses (lavine and strand, 2002). endophagous parasitoids, entering the host body, have to protect themselves from these defense barriers (schmidt et al., 2001). non-permissive hosts typically eliminate endoparasitoids by encapsulation, which usually involves the binding of overlapping layers of hemocytes to the surface of parasitoid. larval endoparasitoids evade host immune defenses either passively, or by active suppression of the host immune system, or by a combination thereof (schmidt et al., 2001; lavine and strand, 2002). female secretions injected at the oviposition are the most common factors 61 actively disrupting the host immune response. these secretions include venom and calyx fluid, which may contain different types of viruses and virus-like particles, often involved in the suppression of the immune response (schmidt et al., 2001). among these viruses, the polydnaviruses are by far the most studied and their molecular characterization in different systems has allowed to shed new light on their role in the host regulation process (kroemer and webb, 2004; webb and strand, 2005). toxoneuron nigriceps (hymenoptera, braconidae) is an endophagous parasitoid of the larval stages of the tobacco budworm, heliothis virescens (lepidoptera, noctuidae). the bracovirus associated with this wasp (tnbv) is currently being studied and several genes expressed in parasitized host larvae have been isolated and their possible role partly elucidated (varricchio et al., 1999; pennacchio et al., 2001; falabella et al., 2003; malva et al., 2004; provost et al., 2004; lapointe et al., 2005). most of the isolated genes are actively expressed in the hemocytes of parasitized hosts and, then, probably involved in disruption and/or suppression. however, to better analyze the role played by these tnbv genes, and by other female secretions injected at the oviposition along with the egg, we need a better understanding of the alterations of the immune responses occurring in naturally parasitized hosts. this information is largely lacking, while for the passive evasion of host immune response it appears that the egg fibrous layer may play an important role (davies and vinson, 1986). the present paper aims at filling this gap by providing data on the most relevant precocious alterations associated with the suppression of host immune response observed in tobacco budworm larvae parasitized by t. nigriceps. materials and methods insect rearing toxoneuron nigriceps was reared in the laboratory according to the methodology described by vinson et al. (1973). heliothis virescens larvae were maintained on a modified artificial diet developed by vanderzant et al. (1962) (corn earworm diet, bioserve, frenchtown, nj, usa). rearing temperature was 29±1° c for both the host and parasitoid, whereas t. nigriceps adults were kept at 25±1 °c. in both cases a 16 h light photoperiodic regime was adopted and the relative humidity was 70±5 %. insect hosts h. virescens last instar larvae were staged according to webb and dahlman (1985) and synchronized as reported by pennacchio et al. (1992). enzymatic test for phenoloxidase (po) activity hemolymph was obtained by puncturing with a needle a proleg of last instar larvae cold anesthetized. all bleedings were done on ice-cold petri dishes and the hemolymph was transferred with a micropipette into icecold eppendorf tubes. hemolymph samples were then processed by low-speed centrifugation (1200 rpm for 3 min at 4 °c), to eliminate hemocytes and tissue debris. the supernatant (plasma) was immediately used or stored at –80 °c. time course analysis of po relative activity of plasma samples from parasitized and nonparasitized h. virescens larvae was carried out spectrophotometrically, by recording the formation of dopachrome from the l-dopa (dihydroxyphenylalanine) (sigma chemicals, st. louis, mo, usa) substrate. changes in absorbance were recorded at 490 nm (µa 490 nm/10 min) at 20 °c by a double-beam jasco v-560 spectrophotometer (jasco int. co., tokyo, japan). all assays were performed by adding 5 µl of plasma to 1 ml of l-dopa buffer (4 mm ldopa dissolved in 10 mm tris-hcl, ph 7.2). the ldopa buffer was used as a blank. to assess the effect of protease (ec 3.4.21.4) treatment on phenoloxidase activation, trypsin (15 iu) (sigma) was added to the substrate. in vitro encapsulation h. virescens hemocytes were obtained from last instar larvae 2 h after parasitization by t. nigriceps and from synchronous nonparasitized controls. insects were surface sterilized by rapid immersion in 70 % ethanol, washed in sterile distilled water, dried on sterile filter paper and bled by proleg amputation. samples of 40-60 µl hemolymph per larva were collected onto ice-cold petri-dishes, lined with 100 % ethanol washed parafilm. whole hemolymph samples were transferred in eppendorf tubes containing an equal volume of anticoagulant buffer mead (98 mm naoh, 145 mm nacl, 17 mm edta, 41 mm citric acid, ph 4.5). hemocytes were pelletted by centrifugation (1200 rpm for 10 min at 4 °c, in a sorvall rmc-14 refrigerated microcentrifuge) and twice washed in grace’s insect medium (sigma) containing 10 % fetal bovine serum (fbs), 1 % glutamine and 1 % antibiotic-antimycotic solution (sigma). the hemocytes were resuspended in 1 ml of the same medium and seeded at a final density of 2 x 105 per well and finally cultured in micro-wells (24-well culture plates, flat bottom, corning incorporated, costar, ny, usa). in vitro encapsulation assays were carried out immediately by adding chromatographic beads (dowex 1x2 mesh 100400) to cultured hemocytes, as described by lavine and strand (2001). hemocyte numbers and adhesion hemocytes were obtained as described in the above section from measured volumes of hemolymph extracted from nonparasitized and parasitized h. virescens larvae. cells were counted by using a burker chamber and subsequently cultured in micro-wells (24well culture plates), at 25 °c. after 16 h from cell seeding, the culture medium was removed and the nonadhering cells were counted. transmission and scanning electron microscopy hemocytes, obtained as described above, were fixed for 30 min in 0.1 m cacodylate buffer, ph 7.2, containing 2 % glutaraldehyde. cells were then washed in the same buffer and postfixed for 20 min with 1 % osmic acid in 0.1 m cacodylate buffer, ph 7.2. after a 62 standard step of serial ethanol dehydratation, cells were pelletted and embedded in an epon-araldite 812 mixture. sections were obtained with a reichert ultracut s ultratome (leica, wien, austria). thin sections were stained by uranyl acetate and lead citrate and observed with a jeol 1010 ex electron microscope (jeol, tokyo, japan). hemocytes for scanning electron microscopy (sem) were fixed and dehydrated as described above, treated with hexamethildisilazane and mounted on polylysinated slides. samples were then air dried and covered with a 9 nm gold film by flash evaporation of carbon in an emitech k 250 sputter coater (emitech, baltimore, md, usa). specimens were then examined with a sem-feg philips xl-30 microscope (philips, eindhoven, netherlands). immunocytochemistry hemocytes were collected as described above and plated on glass coverslips cultured in micro-wells (24well culture plates). cells were washed with pbs and then fixed for 10 min in pbs buffer, ph 7.6, containing 2 % sucrose and 3 % paraformaldehyde. cells were treated for 10 min at 4 °c with a permeabilizing solution (hepes 20 mm, ph 7.4, 300 mm sucrose, 50 mm nacl, 3 mm mgcl2, 0.5 % triton x-100), then washed with pbs buffer containing 2 % bovine serum albumine (bsa) and finally incubated 1.5 h at 37 °c with tetramethylrhodamine (tritc)-labeled phalloidin (sigma) (diluted 50 µg/ml) in pbs buffer containing 2 % bsa. coverslips were mounted in vectashield mounting medium for fluorescence (vector laboratories, burlingame, ca, usa); slides were examined with a confocal laser microscope (laser 568 nm; mrc 1024, bio-rad laboratories, hemel, hempstead, uk) and images were recorded with a delta vision microscope (deltavision real time system (applied precision, elcomind, italy). results prophenoloxidase(propo)-po cascade t. nigriceps oviposition determined a rapid inhibition of the propo cascade. the po activity was nearly abolished by 15 min after parasitization and then 4 h later gradually resumed, reaching levels recorded in nonparasitized controls (fig. 1). in order to assess if the inhibited po was still potentially functioning, a trypsin treatment of the host plasma samples was carried out. the restored po activity in plasma samples of parasitized larvae, previously found to be inactive, indicated that the host po was not damaged, since the pro-enzyme was converted into its active form by trypsin mediated cleavage (fig. 2). hemocyte number morphology and behaviour the total number of hemocytes in parasitized larvae started to decrease shortly after t. nigriceps oviposition. the lowest number of hemocytes was registered after 4 h and reached the 60 % of the value measured in synchronous nonparasitized controls (fig. 3). after 40 h from parasitization the hemocyte number returned to the control condition (fig. 3). sem observations of hemocytes extracted from nonparasitized host larvae allowed to easily discriminate between granulocytes and plasmatocytes. granulocytes were roundish and showed prominent nuclei while plasmatocytes were larger, with a highly ruffled surface and pseudopodia of varying size (fig. 4). both cell types displayed attachment and spreading behaviours (only 5 % of the total amount of cells did not adhere under in vitro condition). adhering cells showed bundles of actin filaments located close to the cell membrane and in the cytoskeleton of the pseudopodia (figs 5, 6). hemocyte morphology in h. virescens larvae rapidly changed after parasitisation. sem observation showed that granulocytes were evidently damaged, while plasmatocytes were apparently unaltered (figs 7-9). the major morphological alterations in granulocytes consisted of cytoplasmic vacuolization, which became more pronounced over time, membrane blebbing, chromatin condensation and nuclear envelope breakdown (figs 7-14). under in vitro condition after two hours from oviposition, about 33 % of the total cell number was unable to adhere to the substrate (fig. 15). non-adhesive hemocytes showed cytoskeleton disruption, with broken filaments visible close to the cell membrane (fig. 16), where actin oligomers were detected (figs 17, 18). encapsulation assay encapsulation and subsequent melanization of dowex beads rapidly occurred in presence of hemocytes extracted from non parasitized h. virescens larvae (figs 19-22). different types of haemocytes were involved in host defense and both plasmatocytes and granulocytes were recruited to form a developing capsule onto the surface of the dowex beads. when the beads were added to the culture of hemocytes extracted from parasitized h. virescens larvae, the capsule did not develop and just a few granulocytes were visible close to the beads, while plasmatocytes fig. 1 after parasitization instead of the expected durable activation of propo system, an inhibition of melanization and a reduction in the enzymatic activity was observed from 15 min to 4 h. data represent mean ± sd, *p< 0,01. 63 fig. 2 tryptic enzyme treatment restores propo activity (trypsin is usually used to check propo integrity). data represent mean ± sd, *p< 0,01. fig. 3 the number of hemocytes decreases in hemolymph of parasitized h. virescens. data represent mean ± sd, *p< 0,01. fig. 4 nonparasitized h. virescens: sem observations of collected hemocytes show different cell types: granulocytes (arrowheads) and plasmatocytes (arrows). figs 5, 6 nonparasitized h. virescens: in hemocytes, actin filaments, detected with phalloidin (arrowheads), are grouped in bundles under the membrane and in the pseudopodia. 64 figs 7-14 parasitized h. virescens: sem observations of collected hemocytes (figs 7-9) show that the granulocyte subpopulation is particularly damaged while plasmatocytes appear morphologically unaltered (arrows). several ultrastructural features typical of apoptotic cell death are visible in the granulocytes: surface blebbing, chromatin aggregation, broken nuclear envelope, altered cytoplasm with swollen vacuoles. granulocytes examined by tem show numerous and large vacuoles in the cytoplasm (figs 10, 14). 65 discussion the “host regulation” by insect parasitoids, as defined by vinson and iwantsch (1980), is the final result of the evolution of host-parasitoid relationship towards host control for the benefit of the parasitoid progeny. parasitoid has to colonize and use the host, that provides food and shelter for the progeny of parasitoid (vinson et al., 2001). the tactics for circumventing the host defense and for redirecting its physiology, growth and reproduction, to support the development of the parasitoid juvenile stages, are key-factors in successful parasitism. the study of the molecular details of the physiological mechanisms underlying host-parasitoid interactions in insects has generated a fairly large amount of information (vinson et al., 2001; schmidt et al., 2001; beckage and gelman, 2004), in particular relatively to the major categories of host regulation factors produced by parasitic hymenoptera, such as polydnaviruses (kroemer and webb, 2004; webb and strand, 2005) and venom (weaver et al., 2001; asgari et al., 2003; zhang et al., 2004a, 2004b) suppressing the host immune responses. the suppression seems to be largely associated with inhibition of serine protease cascades (brehelin et al., 1975; beckage et al., 1990; beck et al., 2000) and hemocyte disruption and/or death (strand and pech, 1995; schimdt et al., 2001). this article is focused on the in vivo and in vitro study of survival strategies adopted by t. nigriceps during the early time of parasitization (i.e. the most vulnerable period of development). t. nigriceps avoids the host immune reaction by a combination of both passive and active mechanisms. the outer fibrous layer of the egg seems to be the first protecting barrier (davies and vinson, 1986), which is effectively complemented by a very precocious and transient suppression of the po activity. this transient block could be interpreted as a mechanism to disrupt the early steps activated by the recognition of an invading organism. the observed reactivation of the enzyme in parasitized plasma samples by trypsin treatment indicates that the po retains its functionality. we can differently interpret this result: a parasitoid-derived molecule could bind the po, protecting the cleavage site, thus inhibiting its activation or a parasitoid factor involved in host regulation likely hit the protease cascade rather than the enzyme itself. this may inhibit both the melanization response and the possible production of signal molecules that would in turn activate the cellular immune reaction. in fact, it is reasonable to speculate that the propo-activating system (propo-as) plays a key-role in the regulation of these important mechanisms, as suggested for crustacea and lepidoptera by soderhall and cerenius (1998). the source of the host regulatory factors involved in the po inactivation is still unknown, but based on preliminary experimental data and on the fact that this alteration is triggered within minutes after parasitization, we can predict that venom and/or ovarian secretes in the calyx fluid may play an important role. as evidenced by several authors (lavine and beckage, 1995; doucet and cusson, 1996; hu et al., 2003), many parasitoids are able to suppress host immune defenses by altering the number and/or the behaviour of the circulating hemocytes. there are a number of studies available on this type of alteration, which, for example, can be induced by parasitoidassociated pdvs, as in choristoneura fumiferanatrasonema rostrale (doucet and cusson, 1996) and pseudoplusia includens-microplitis demolitor (strand and pech, 1995) associations, or by the combined action of pdvs and ovarian proteins, as in heliothis virescens-campoletis sonorensis (luckhart and webb, 1996), or by the venom alone, such as in lacanobia oleracea-pimpla hypochondriaca (richards and parkinson, 2000). in all these model systems, the numerical variation of hemocytes is always paired with an altered morphology and/or functionality of the cells. in h. virescens larvae parasitized by t. nigriceps, the total number of circulating hemocytes transiently decreases, with a minimum peak registered within few hours, followed by a slow recovery towards values normally registered in synchronous nonparasitized controls, which were attained by 40 h after parasitoid oviposition. during this interval, the hemocytes show different structural damages, evident actin cytoskeleton disruption and lost of adhesion properties, with general morphological changes, which suggest the occurrence of apoptosis. these hemocyte alterations seem to be selectively induced in granulocytes, while plasmatocytes appear to be morphologically unaltered. however, the apparently unaltered plasmatocytes do not start any encapsulation process of dowex beads in vitro. it remains to be studied if this is a consequence of granulocytes degeneration, which may prevents the plasmatocyte recruitment they regulate during encapsulation (lavine and strand, 2002), or if a more subtle functional alteration of plasmatocytes occurs. all these changes in hemocyte structure and function could be induced by venom and calyx fluid right after oviposition, and, after few hours, reinforced by the expression of tnbv genes. fig. 15 parasitized h. virescens: hemocytes loose their adhesion capability and the minimum was observed at two hour mark. data represent mean ± sd, *p< 0,01. 66 figs 16-18 parasitized h. virescens: in hemocyte actin filaments are disassembled and are visible, under transmission electron microscope, at the periphery of the cell (arrows). phalloidin evidences the oligomers of actin (red spots). figs 19, 20 hemocytes of nonparasitized h. virescens: both plasmatocytes and granulocytes are recruited onto the surface of beads (arrowheads) forming a capsule. figs. 21, 22 hemocytes of parasitized h. virescens: few granulocytes are visible near the beads and plasmatocytes, parallel disposed, are unable to adhere to dowex beads (arrowheads). 67 fig. 23 summary of different events transiently disabling host immune defenses. the thin lines indicate the interval in which a particular alteration occurs; the broad lines are darker where the inhibition is at the highest level. a few genes expressed in host hemocytes have been isolated and, then, are considered to be putatively involved in the host immune disguise (varricchio et al., 1999; pennacchio et al., 2001; falabella et al., 2003; malva et al., 2004; provost et al., 2004). at the present, only for the viral gene tnbv1, it has been demonstrated that an apoptosis-like degeneration is induced in insect cells (lapointe et al., 2005). detailed functional analyses are required to establish the role of other tnbv genes in the induction of the multifaceted immune syndrome recorded in parasitized h. virescens. however, we can predict that these genes, along with female secretion injected at the oviposition, coordinately induce a set of partially overlapping mechanisms, disabling cellular and humoral responses (fig. 23). acknowledgements we thank luisa guidali for her invaluable technical assistance. this work has been supported by a miur grant (cofin 2002-2004). references asgari s, zhang g, zareie r, schmidt o. a serin proteinase homolog venom protein from an endoparasitoid wasp inhibits melanization of the host hemolymph. insect biochem. mol. biol. 33: 1017-1024, 2003. beck m, theopold u, schmidt o. evidence of serine protease inhibitor activity in the ovarian calyx fluid of the endoparasitoid venturia canescens. j. insect physiol. 46: 1275-1283, 2000. 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endoparasitoid venom. insect biochem. mol. biol. 34: 477483, 2004b. isj 4: 38-xx, 2007 isj 4: 45-50, 2007 issn 1824-307x minireview the amphioxus immune system m pestarino, d oliveri, m parodi, s candiani dipartimento di biologia, università di genova, genova, italy accepted march 19, 2007 abstract the cephalochordate amphioxus is the closest living invertebrate relative of the vertebrates and therefore it can provide useful insights on the evolution of the adaptive immune system. in fact some components of the immune system are present in amphioxus but many other features are lacking. a proto-mhc region has been identified by chromosome walking and the presence of t cell receptor has been demonstrated despite the absence of true lymphocytes. moreover a further important step on the study of the amphioxus immune system is represented by the recent availability of the genome of branchiostoma floridae that will allow to better understand how many genes code for immunological molecules involved in adaptive immune system pathways of amphioxus. key words: evolution; protochordates; amphioxus; innate immunity; adaptive immunity __________________________________________________________________________________________ introduction protochordates, consisting of two subphyla (tunicates or urochordates and cephalochordates) sharing a common ancestry with the vertebrates, are useful model animals for the comprehension of the vertebrate phylogeny (swalla et al., 2000, schubert et al., 2006). in the last decades, several molecular data support the hypothesis that the cephalochordates are the sister group of vertebrates (holland et al., 2004), and therefore the cephalochordate amphioxus, also commonly known as lancelet, is particularly useful in order to study the evolution of the immune system and to identify the related genes. in fact, the recent availability of the genome assembly release v.1.0 (started march 2006 and freely available to the scientific community from january 2007) by the joint genome institute (us department of energy) facilitates the comparative studies of genomes of vertebrate and invertebrate species. on the other hand, the emergence of adaptive immunity has been placed at the jawed vertebrate stage but numerous recent comparative immunological findings suggest the presence in amphioxus not only of innate immunity but also of an ancestral adaptive immune system ___________________________________________________________________________ corresponding author: mario pestarino dipartimento di biologia università di genova viale benedetto xv, 5 16132 genova, italy e-mail: pesta@unige.it (sato et al., 2003; danchin et al., 2004; dong et al., 2005). even if the structure of the amphioxus vascular system has been extensively studied, free blood cells have not been clearly identified. only rhodes and coworkers (1982) described by electron microscope the presence in the perivisceral coelom of free cells able to phagocyte and similar to specialized leukocytes as described in ascidians. moreover, the blood vessels lack in a continuous endothelial layer, the blood is colourless and the vascular system is closed and consists of dorsal and ventral vessels from which vascular channels reach pharynx, intestine and gonads. multiple contractile and peristaltic pumps are present in the subintestinal vessel but few anatomical and histological data support their homology to the vertebrate heart even if amphioxus is the closest model for the vertebrate vascular plan (simõescosta et al., 2005). recently, holland and coworkers (2003) cloned in the amphioxus branchiostoma floridae the gene amphink2-tin, similar in sequence to vertebrate nk2 genes and the tinman gene of drosophila, both involved in cardiogenesis (simões-costa et al., 2005). amphink2-tin is firstly expressed in muscle cell precursors at level of the first five or six somites and afterwards in a ventral row of visceral peritoneal cells, containing non-striated myofibrils, and from which the wall of the contractile subintestinal vessel derives. therefore such data support the presence in amphioxus of a rudimentary cardiac system. 45 mailto:pesta@unige.it fig. 1 schematic representation of the evolution of the nine mhc paralogous region (labelled by an arrow and a roman number from i to ix) (modified from abi-rached et al. 2002 and vienne et al., 2003). for two gene families, duplications specific to the amphioxus lineage are evidenced by an asterisk (1: neuraminidase-like 2 and 3: neuraminidase-like 1; 6: udpgt-like 1 and 7: udpgt-like 2. all the other genes of amphioxus has at least an orthologue as shown for the amphioxus frequenin gene (20). two new predicted genes of amphioxus are orthologous to the human nadph (11) and mgc14327 (12). the yellow boxes show the anchor genes (5: rxra; 9: bat1/ddx39; 14: brd2,3,4,t; 15: c3,c4,c5; 16: cacna1a,b,e; 19: notch1,2,3,4; 24: psmb7; 26: psmb8; 27: prx1,2,3,4). the major histocompatibility complex (mhc) in humans mhc consists of a large genetic region containing more than 100 genes involved in graft rejection and clustered in a major chromosomal region named major histocompatibility complex. graft rejection is due to the recognition of mhc class i peptides that are recognized by t cells from the host. moreover, because also non-mhc genes are involved in the immune system, it has been possible to define a central region between the mhc class ii genes and the mhc class i genes, named mhc class iii region, which includes genes coding for the complement system and some members of the tumor necrosis factor (tnf) family (flajnik and du pasquier, 2004). moreover, further genes involved in mhc presentation have been found within the socalled mhc class i and class ii subregions (flajnik and du pasquier, 2004; danchin et al., 2004). as known, the adaptive immune response starts with the presentation of mhc-bound peptides to the tcell receptors. mhc class i and class ii proteins, like other transmembrane proteins, must be properly folded to be competent for exit from the endoplasmic reticulum. the assembly of the various constituents requires a mechanism that involves er-resident chaperones and the exit of mhc class i and class ii is mediated by housekeeping chaperones and dedicated proteins (cresswell, 1994; antoniou et al., 2003; paulsson and wang, 2003). an example of dedicated proteins in mhc class ii presentation are cd74 and the cathepsins. cd74 is a dedicated chaperone found only in the bony vertebrates (dijkstra et al., 2003) and no homologous of this gene has been found in nonvertebrate groups. the housekeeping chaperones have been co-opted by the neo-mhc molecules after the jawed/jawless vertebrate split, and some of the dedicated proteins have been co-opted directly after duplication (danchin et al., 2004). in fact, the emergence of adaptive immunity has been located for a long time at the appearance of gnathostome vertebrates, mainly because the major components 46 of the mammalian adaptive immune system such as mhc, t cell receptors (tcr), and immunoglobulin (ig) molecules, appear for the first time in the cartilaginous fish (cannon et al., 2004). recently, an ancestral adaptive immune system characterized by the presence of lymphocyte-like cells, has been described in lamprey (mayer et al., 2002; uinookool et al., 2002) and variable lymphocyte receptors (vlr) were also identified in hagfish (pancer et al., 2005). the adaptive immune system (ais) and peptide mhc presentation (fig. 1) probably appeared before the emergence of the last common ancestor of the gnatosthomes and after the jawless/jawed vertebrate split, but a possible proto-mhc region has been described in amphioxus (abi-rached et al., 2002), whereas according to danchin and coworkers (2004) the common ancestors of chordates did not possess an ais. it is known that some of the genes involved in the mhc system were co-opted after duplication (abi-rached et al., 2002). because amphioxus has a key position in chordate evolution, studies were performed in b. floridae in order to test the two rounds of en bloc duplication hypothesis for the mhc and its paralogous regions located on human chromosomes 1, 9, and 19 (abi-rached et al., 2002). nine highly conserved anchor genes located in the mhc were used as probes, and their orthologs were cloned from a b. floridae genomic library. ten cosmid clones containing the anchor genes were identified and sequenced, and 22 genes were detected in their surrounding genomic regions. the distribution of human and amphioxus orthologs in their respective genomes and the relationship between these distributions support the en bloc duplication events hypothesis and the phylogenetic analysis showed that all duplication events in these regions occurred after the divergence of cephalochordata and craniata and before the gnathostomata radiation. recently four new cosmids have been sequenced in the amphioxus mhc-like region, and their phylogenomic analysis supports the previously obtained results (vienne et al., 2003). furthermore, the physical linkage of these cosmids has been tested by two-colour fluorescent in situ hybridization (fish) to amphioxus metaphase chromosomes (castro and holland, 2002). in particular, six cosmids (corresponding to 27 genes) mapped to a single amphioxus chromosome, only the cosmid containing the orthologue amphic3 of the complement genes maps to a different chromosome of the 19 chromosome pairs in amphioxus. the obtained experimental data allow to reconstruct the protomhc in the ancestral chordate. such proto-mhc corresponds to the extant mammal class ii and class iii genomic regions and this correspondence shows that mhc class i genes were translocated recently in the mammal evolution and have colonized new genomic portions (flajnik and kasahara, 2001). it has been also hypothesized that the mhc genomic region is part of an ancient syntenic group present in the ancestor of protoand deuterostomes (trachtulec and forejt, 1999). even though the description of the conserved synteny was not supported by phylogenetic and statistical analysis (hughes and pontarotti, 2000), it has been possible to demonstrate that an mhc-like region was certainly present in the common ancestor of protoand deuterostomes (danchin et al., 2003). the immunoglobulin superfamily a multigene family containing ig-like variable regions, v region-containing chitin-binding protein (vcbp) and an immunoglobulin superfamily (igsf) gene homologous to cd47, have been identified in the intestine of amphioxus (cannon et al., 2002, sato et al., 2003). these studies suggest that some ancestral molecules involved in the adaptive immunity existed in protochordates (fig. 2). a fragment of est with significant similarity to a vertebrate tcr sequence was found in a cdna library of adult branchiostoma lanceolatum (sato et al., 2003). in particular an orf encoding a 351 aa long peptide has been found and the putative protein is named brla-vdb for “branchiostoma lanceolatum v-domain bearing”. brla-vdb consists of a n terminal domain starting with a putative leader peptide and followed by a sequence resembling the v domain of the cortical thymocytes of xenopus (ctx) protein. furthermore, the c terminal domain contains five hydrophobic segments separated by short hydrophilic stretches and therefore it may be considered a protein that crosses the plasma membrane five times. these findings support the hypothesis that v domain resembling those found in t cell receptors evolved in invertebrates before the emergence of the adaptive immune system and it may have been involved in non immunological functions (sato et al., 2003). some igsf members bearing the v or v-c structures have been found in amphioxus (cannon et al., 2002, sato et al., 2003). two kinds of igsf members have been described in an intestine cdna library of branchiostoma belcheri. the first one is a vcbp having approximately 70 % identities in amino acid sequence with the vcbp4 of b. floridae that encodes 338 amino acid residues. vcbp comprises a signal peptide, one v-type domain, one c-type domain, a transmembrane region, and a cytoplasmic region, and therefore named as v and c domain-bearing protein (vcp). as known in mammals, expression of recombination activating genes (rag) that are involved in the v (d) j recombination is regulated by the rag1 gene activator (rga) in mammals. recently, the sequence of a cdna clone from an amphioxus cdna library was found to be homologous to that of rag from mouse stromal cells (cannon et al., 2002). the full-length cdna sequence comprises 1119 bp and encodes a putative protein of 210 amino acid residues. characterization of the amino acid sequence revealed that two mtn3 domains and seven span transmembrane domains are present in this protein, indicating a potential role as a plasma membrane protein. a high expression level of rga, detected in gonads, gastrula embryos and adult stages of amphioxus suggests that the signal pathway required for the expression of rag could occur in cephalochordates (dong et al., 2005). therefore a 47 fig. 2 dendrogram showing bilaterian metazoan phylogeny. the possible origin time of the adaptive immune system is shown as well as the two en bloc genomic duplications: the first before the emergence of vertebrates and the second before the appearance of gnathostomes. during the evolutionary history of chordates, amphioxus acquires some of the immune molecules and mechanisms involved in pre-adaptive immunity. tcr, t cell receptor; igsf, immunoglobulin super family; vcbp, v region-containing chitin-binding protein receptors; gilt, ifn-γinduced lysosomal thiol; mhc synteny, major histocompatibility complex synteny (as described in the text). primitive adaptive immunity may have existed in amphioxus although the complete machinery of v(d)j rearrangement may be not formed. recently, a nucleotide sequence similar to ifnγ-induced lysosomal thiol (gilt) reductase have been found in amphioxus (yu et al., 2005). gilt is expressed constitutively in antigen presenting cells (apcs) and facilitates processing and presentation of ag peptide (zheng and chen, 2006). the amphigilt contains the conserved active site cxxc, and nine cysteins downstream of the active site just like the counterparts in human and mouse. the structure of amphi-gilt is vertebrate-like suggesting a closer function to its counterparts in vertebrates. furthermore, gamma-interferon (ifnγ)-inducible lysosomal thiol reductase (gilt) is involved not only in the internalization and delivering to lysosomes of exogenous antigens (watts, 1997), but also in negative regulation of t cell activation (barjaktarevic et al., 2006) and neutralization of extracellular pathogen (lackman and cresswell, 2006). gilt has been identified in several species of vertebrates and invertebrates (maric et al., 2001; woods et al., 2005; zheng and chen, 2006). recently, amphigilt has been demonstrated to have a conserved active domain probably involved in the innate immune responses in amphioxus (liu et al., 2007). innate immunity and humoral immune responses macrophage migration inhibitory factor (mif), a cytokine involved in host defenses and autoimmune diseases, has been found in b. belcheri in which unusually the mif gene is present in multi-copy per haploid genome (du et al., 2004). in particular two mif homologues, called bbt-mif-i and bbt-mif-ii, have been sequenced in amphioxus. mif exerts a crucial role in innate immunity and therefore it can be considered a key molecular marker of evolution of immune system, even if in amphioxus it has more broad function than in vertebrates (du et al., 2006). humoral fluids of b. belcheri contain lysozyme, microbial agglutinin and hemagglutinins before and after challenge with escherichia coli (pang et al., 2006), but also the coelomic fluid contains phenoloxidase, lectin and complement component c3. therefore it is possible to argue that amphioxus has a simple humoral immune defense system. as 48 known in vertebrates mannose-binding lectinassociated serine protease (masp) are involved in complement activation through the lectin pathway, in amphioxus a masp gene has been cloned (endo et al., 2003) and its structure is similar to the human masp1/3 (dahl et al., 2001). furthermore, the evolutionary history of the masp gene family seems to be parallel to that of their substrates such as c3 and c4. in fact, both c3 and masp homologue genes have been found in amphioxus (fujita, 2002), as well as a gene encoding a protein containing the membrane attack complex (mac)/perforin module, homologue to vertebrate c6 called amphic6 has been identified (suzuki et al., 2002). such evidences suggest that a primordial complement system is probably emerged after the cephalochordates. in conclusion several findings on the amphioxus immune system incline us to suggest that innate and adaptive immunity appeared early 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice exogenous il-8 induces phagocyte activation in the compound ascidian botryllus schlosseri isj 3: 18-24, 2006 issn 1824-307x research report exogenous il-8 induces phagocyte activation in the compound ascidian botryllus schlosseri a menin, l ballarin department of biology, university of padova, padova, italy accepted february 28, 2006 ______________________________________________________________________________ abstract we studied the responses of botryllus schlosseri phagocyte to human recombinant il-8 (hril-8) at three different concentrations (10, 25 and 50 ng/ml). both spreading ability and phagocytosis were significantly enhanced by the exogenous chemokine at 25 and 50 ng/ml in the culture medium and the effects are coupled to modifications of the actin cytoskeleton. the addition of the signal transduction inhibitors suramin, calphostin c and h-89 to the incubation medium, inhibits the above-reported effects and suggests that exogenous il-8 acts via protein kinase (pk) c and pka pathways through its binding to a g protein-coupled receptor. key words: botryllus schlosseri; immunocytes; il-8 _____________________________________________________________________________________________________________________ introduction interleukin-8 (il-8, cxcl8) is an inducible chemokine released during infections and inflammation in response to bacteria toxin or inflammatory cytokines like interleukin-1 (il-1) and tumour necrosis factor alpha (tnf-α) (rollins, 1997; baggiolini, 2001; casilli et al., 2005; esche et al., 2005). it causes rapid changes in the morphology of leucocytes, such as neutrophils, t-lymphocytes and natural killer cells (nk), as the consequence of a reorganisation of the actin cytoskeleton. in addition, il-8 induces the up-regulation and activation of integrins on leucocytes required for their adhesion to both endothelial cells, prior to extravasation, and extracellular matrix, during their way to inflammation sites (luster, 1998; imhof and aurrand-lions, 2004; casilli et al., 2005). as a chemokine, il-8 exerts its action by interacting with seven transmembrane, g protein-coupled receptors which, upon ligand binding, activate a heterotrimeric g-protein which dissociates into the gtp-bound α and the βγ subunits. the latter, ______________________________________________________________________________ corresponding author: loriano ballarin department of biology, university of padova via u. bassi 58/b, 35100 padova, italy email: loriano.ballarin@unipd.it in turn activates phospholipase c (plc) leading to the production of inositol triphosphate (ip3) and diacylglycerol (dag). in the case of il-8, the βγ subunit of the gprotein also recruits and activates phosphatidylinositol-3-kinase (pi3k) which has a central role in inflammation as it allows the recruitment and activation of phagocytes and nk cells (smith et al., 1992; hirsh et al., 2000; naccache et al., 2000; fuhler et al., 2005 maghazachi, 2005). as far as invertebrates are concerned, although the existence of molecules sharing homology with vertebrate cytokines is still a matter of debate (beschin et al., 2001, 2004), the presence of endogenous immunoregulatory cytokines has been indirectly suggested in various phyla (beck and habicht, 1991; hughes et al., 1990, 1991; ouwemissi-oukem-boyer et al., 1994; granath et al., 1994; cooper et al., 1995; ottaviani et al., 1995, 1996; franchini et al., 1996; beck, 1998). according to beschin et al. (2001, 2004), these molecules share with vertebrate cytokines some lectin domains which can explain the observed effects of mammalian cytokines on invertebrate immunocytes (kletsas et al., 1998; ottaviani et al., 2000, 2004). in tunicates, the presence of endogenous chemotactic molecules, is known from many studies. they are represented by either homologues of the 18 mammalian complement c3, as in styela plicata (raftos et al., 2002), halocynthia roretzi (nonaka et al., 1999), pyura stolonifera (raftos et al., 2003) and ciona intestinalis (pinto et al., 2003), or different molecules cross-reacting with anti-il-1 antibodies as in styela clava (raftos et al., 1998). in the colonial ascidian botryllus schlosseri, we have recently demonstrated that activated immunocytes release cytokines (i.e. immunomodulatory molecules, in the broad sense of the term) able to enhance yeast phagocytosis (menin et al., 2006). the above molecules are recognised by antibodies raised against mammalian il-1α and tnfα and are mainly produced by cytotoxic morula cells upon the recognition of non-self molecules (ballarin et al., 2001). these cells are known to be involved in the non-fusion reaction which occurs when genetically incompatible colonies contact each other and shares many features, such as chemotaxis, extravasation, degranulation and cytotoxicity, with vertebrate inflammation where chemokines play a key role (ballarin et al., 1995, 1998; rinkevich et al., 1998; cima et al., 2004). recently, we undertook a new research aimed to identify and characterise chemokines in our model. as a preliminary approach, we studied the effects of human recombinant il-8 (hril-8) on the activity of b. schlosseri immunocytes. results indicate that ascidian immunocytes can sense the presence of exogenous il-8 and consequently modify their activity. materials and methods animals colonies of botryllus schlosseri from the lagoon of venice were used. they were kept in aerated aquaria, attached to glass slides and fed with liquifry marine (liquifry co., dorking, england) and algae (dunaliella sp.). blood plasma preparation blood was collected with a glass micropipette after puncturing, with a fine tungsten needle, the tunic marginal vessels of colonies previously blotted dry. blood was centrifuged at 780 x g and the supernatant was referred as blood plasma (bp). the terms “autologous” and “heterologous” refer to bp from the same colony used for hemocyte collection or from a different, non-fusible colony, respectively. hemocyte collection and culture blood was collected, as described above, from colonies previously rinsed in 0.38 % na-citrate in filtered sea water (fsw), ph 7.5, as anti-clotting agent. it was then centrifuged at 780 x g for 10 min and pellets were finally resuspended in fsw to give a concentration of 5 x 106 cells/ml. sixty µl of hemocyte suspension were placed in the centre of culture chambers prepared as described elsewhere (ballarin et al., 1994) and left to adhere to coverslips for 30 min at room temperature. light microscopy of hemocytes after the adhesion of hemocytes to the coverslips, cells were exposed for 60 min to fsw containing hril8 (peprotech ec; 10, 25 and 50 ng/ml) in the presence or in the absence of 0.7 mm suramin, an antagonist of g protein (huang et al., 1990; ottaviani et al., 2000); fsw alone was used in controls. cells were then fixed for 30 min at 4 °c in 1 % glutaraldehyde in fsw containing 1 % sucrose, rinsed in phosphate-buffered saline (pbs: 1.37 m nacl, 0.03 m kcl, 0.015 m kh2po4, 0.065 m na2hpo4, ph 7.2) and stained with 10 % giemsa solution for 5 min. the morphology of hemocytes was observed under a leitz dialux 22 light microscope at a magnification of 1250 and the cell-spreading index, i.e. percentage of hemocytes with amoeboid shape, was finally calculated after counting at least 300 cells per coverslip. in another series of experiments, calphostin c, a specific inhibitor of protein kinase c (pkc; tamaoki, 1991) and h-89, a specific inhibitor of protein kinase a (pka; chijiwa et al., 1990) were added to the incubation media, in the presence or in the absence of hril-8, at the sublethal concentration of 0.1 and 1 µm, respectively. these concentrations were already used in experiments with invertebrate immunocytes (ottaviani et al., 2000). in addition, computer-assisted image analysis (casting image nt) was performed on fixed hemocyte monolayers, previously treated as described above, to evaluate the phagocyte shape factor, defined as in ottaviani et al. (1997). lower shapes factors indicate larger perimeters with respect to the areas and, therefore, an increased amoeboid shape. immunocytochemical assays for cytoskeleton hemocyte monolayers on coverslips, treated as described above, were fixed for 30 min at 4 °c in 4 % paraformaldehyde (serva electrophoresis gmbh, heidelberg, germany) and 1 % sucrose in isotonic salt solution (iso: 20 mm tris, 0.5 m nacl, ph 7.5; edds, 1985), washed in pbs plus 1 % sucrose and permeabilised with 0.1% triton x-100 (merck kgaa, darmstadt, germany) in pbs for 5 min. for the specific detection of f-actin, the monolayers were then incubated for 30 min at 25 °c in fitc-labelled phalloidin (sigma-aldrich co, st louis, mo, usa), 1 μg/ml in pbs. lastly, the coverslips were rinsed in 0.1 m carbonate buffer, ph 9.5, to amplify the fluorescent signal, mounted with vectashield (vector laboratories inc., burlingame, ca, usa) on glass slides, and observed at a magnification of 1250 under a leitz dialux 22 light and fluorescence microscope equipped with i2/3 filter block for fitc excitation. phagocytosis assay after adhesion, hemocytes were incubated with 60 µl of a suspension of yeast cell (yeast:hemocyte ratio = 10:1) in fsw containing hril-8 at the same concentrations indicated above, in the presence or 19 fig. 1 fixed b. schlosseri phagocytes. a, c-e) hyaline amoebocytes; b) univacuolated macrophage-like cell. a-c) giemsa’s stain; d-e) treatment with fluorescent falloidin to reveal f-actin. scale bar = 3 µm. absence of 0.7 mm suramin; fsw alone was used in controls. cultures were kept upside down for 60 min at room temperature. hemocyte monolayers were then washed by dipping the coverslips repeatedly in a large volume (100 ml) of fsw, fixed in a solution of 1 % glutaraldehyde and 1 % sucrose in fsw for 30 min at 4 °c and stained with 10 % giemsa for 5 min. fig. 2 a) cell-spreading index of botryllus hemocytes left to adhere for 60 min in fsw containing hril-8 in the presence or in the absence of 0.7 mm suramin; fsw alone was used in controls; b) phagocytic index of botryllus hemocytes exposed to yeast-containing fsw and hril-8 in the presence or in the absence of 0.7 mm suramin; fsw alone was used in controls. significant differences with respect to controls are marked by asterisks. * = p < 0.05. the coverslips were then mounted on glass slides with the aqueous medium “acquovitrex” (carlo erba reagenti spa, milan, italy) and at least 300 hemocytes were observed under the light microscope, in ten optical fields at a magnification of 1250, to determine the phagocytic index, i.e. the percentage of hemocytes with ingested yeast cells. in another series of experiments, calphostin c and h-89 were added to the incubation media, in the presence or in the absence of hril-8, at the sublethal concentration of 0.1 and 1 µm, respectively, as reported above. statistical analysis experiments were replicated at least three times; data are expressed as mean ± sd. at least 300 cells, in 10 optical fields at a magnification of 1250, were counted for each experiment aiming to determine the frequencies of amoeboid or yeast-containing cells; frequencies were subjected to angular transformation. the shape factor was calculated on 20 hyaline amoebocytes for each treatment. means were compared with the duncan’s test. results phagocyte spreading b. schlosseri phagocytes are represented by hyaline amoebocytes and macrophage-like cells. the former are motile cells able to spread on the substrate (fig. 1a) and actively engulf foreign particles. as a consequence of the ingestion, they change their morphology to globular, vacuolated macrophage-like cells (fig. 1b). in our controls, we had about 15 % of hemocytes with spreading morphology (fig. 2). similar results were obtained with the protein kinase inhibitors (fig. 3a). in the presence of hril-8 at the concentrations of 25 and 50 ng/ml, a significant (p < 0.05) increase in the cell spreading index was observed (fig. 1c) which was abolished by the presence of the g protein inhibitor suramin (fig. 2a). no significant effects were observed in the presence of 10 ng/ml hril-8 or suramin alone (data not shown). the shape factor of hyaline amoebocytes resulted significantly (p < 0.05) decreased by hril-8 at 25 and 50 ng/ml (table 1). no significant variation in the shape factor was observed in the presence of the protein kinase inhibitors. 20 control spreading phagocytes had a typical cytoskeletal organisation, with actin filaments organized in bundles of stress fibres without a preferential orientation. the same was true for pseudopodia which radiated from most of the cell contour and resulted strongly fluorescent (fig. 1d). in the presence of hril-8, phagocytes appeared remarkably spread with stress fibres oriented according to a well defined major axis and their cytoplasmic projections, rich in fluorescent actin, emerging from the apical endings (fig. 1e). phagocytosis about 15 % of hemocytes were able to ingest yeast cells. this fraction increased significantly (p < 0.05) in presence of hril-8 at the concentrations of 25 and 50 ng/ml, while the chemokine effect was suppressed by suramin (fig. 2b). in the absence of the chemokine, both calphostin c and h-89 significantly (p < 0.05) inhibit phagocytosis. the protein kinase inhibitors were able to significantly (p < 0.05) reduce the hril-8-stimulated phagocytosis when the chemokine was used at the concentration of 10 ng/ml. the inhibitory effect of h-89 only was still detectable at 25 ng/ml, while at the fig. 3 effects of 0.1 μm calphostin c (red) and 1 μm h-89 (yellow) on cell spreading index (a) and phagocytosis (b) in the presence or in the absence of hril-8. asterisks mark significant differences with respect to controls (incubation in yeast-containing fsw). * = p < 0.05. table 1 shape factors of hyaline amoebocytes in various experimental conditions ______________________________________________________________________________ treatment shape factor ______________________________________________________________________________ fsw (control) 0.21 ± 0.10 fsw + hril-8 10 ng/ml 0.19 ± 0.06 fsw + hril-8 25 ng/ml 0.14 ± 0.07 * fsw + hril-8 50 ng/ml 0.12 ± 0.04 *** ______________________________________________________________________________ asterisks mark significant differences with respect to controls. *: p < 0.05; ***: p < 0.001 concentration of 50 ng/ml the phagocytic index was not influenced by none of the inhibitors used (fig. 3b). discussion circulating immunocytes represent an important feature of coelomate metazoans as they represent the main component of the immune system. once sensed a non-self molecular pattern, immunocytes must be able to leak from the circulation into the tissues and migrate towards the source of the stimulus in order to kill and/or engulf it in the course of an inflammatory reaction. cytokines are immunoregulatory soluble molecules which modulate the activity of immunocytes (rubinstein et al., 1998; borish and steinke, 2003), whereas chemokines represent chemotactic cytokines, released by activated immunocytes and damaged tissues, which mediate immunocytes recruitment to the site of inflammation. several chemokines were identified in vertebrates, grouped in four families on the basis of their primary sequence (esche et al., 2005). the situation is less clear in invertebrates, even if the presence of chemotactic factors was reported (alvarez et al., 1995; nonaka et al., 1999; raftos et al., 2002; pinto et al., 2003; raftos et al., 2003; ottaviani et al., 2004). il-8, one of the most extensively studied vertebrate chemokine, is a key mediator in polymorphonuclear leucocyte, t lymphocyte and nk cell recruitment and activation (baggiolini, 2001; casilli et al., 2005; esche et al., 2005). it induces changes in the organisation of the actin cytoskeleton and expression of adhesion molecules on leucocytes required for their migration to the inflammation sites (luster, 1998; imhof and aurrand-lions, 2004; casilli et al., 2005). invertebrate immunocytes can change their functionality in the presence of exogenous mammalian cytokines (hughes et al., 1990; beck and habicht, 1991; granath et al., 1994, 2001; ottaviani et al., 1995; barcia et al., 1999). this study demonstrates that b. schlosseri immunocytes can respond to exogenous hril-8 with an increase in both phagocyte spreading, as indicated by the higher haemocyte cell spreading index and the reduced amoebocyte shape factor, and phagocytic index. 21 in vertebrates il-8-induced leucocyte modifications are mediated by both changes in the organisation of the actin cytoskeleton and upregulation of integrin expression (luster, 1998; imhof and aurrand-lions, 2004; casilli et al., 2005), the latter required for adhesion to substrata during migration and for the establishment of close contacts with foreign particles in the course of phagocytosis (matricon-gondran and letocart, 1999). we have already demonstrated the importance of cytoskeletal modifications and integrin expression in phagocyte spreading and phagocytosis in botryllus (ballarin et al., 2002). our results confirm previous data and indicate that exogenous il-8 can induce a cytoskeletal reorganisation which is related to the reported increase in phagocyte spreading and phagocytosis. il-8-induced modifications in immunocytes activity were also reported by ottaviani et al. (2000) in the bivalve mollusc mytilus galloprovincialis, suggesting that responsiveness to exogenous il-8 can be common among invertebrate immunocytes. in vertebrates, it is known that il-8 exerts its activity through its interaction with g protein-coupled receptors with seven α-helix transmembrane domains (esche et al., 2005). although no sequences sharing homology with vertebrate chemokine receptor genes have been identified in invertebrates so far (de vries et al., 2006), the presence of g protein-coupled receptors is firmly established and orthologues of the vertebrate receptors have been identified in both radiata and bilateria (brody and cravchik, 2000; bouchard et al., 2003; keating et al., 2003; kawada et al., 2004); in the compound ascidian ciona intestinalis a g protein-coupled receptor involved in intercellular signalling was recently described (elphick et al., 2003). the inhibition of the il-8-induced increase in cell spreading and phagocytosis by suramin, which is known to disrupt receptor-g protein coupling (chung and kermode, 2005), in botryllus (this report) and m. galloprovincialis (ottaviani et al., 2000), suggests that the chemokine interacts with some g protein-coupled receptor in invertebrate immunocytes, unrelated to vertebrate cytokine receptors, which can be the natural target of endogenous chemotactic molecules. vertebrate g protein-coupled receptors mainly act through two well known signal transduction pathways: the cyclic amp and the phosphoinositide pathways (gomberts et al., 2002). in the first case, the adenylate cyclase is activated with the consequent production of cyclic amp which, in turn, activates pka. in the phosphoinositide pathway, a plc is activated upon ligand-receptor interaction which results in the production of ip3 and dag, the former mobilising ca2+ from intracellular stores, the latter activating pkc. as suggested by experiments with calphostin c and h-89, both the transduction pathways are involved in il-8-induced botryllus cell spreading and phagocytosis. the observed decrease of the phagocytic index in the presence of the protein kinase inhibitors in the absence of the exogenous chemokine or in presence of il-8 at the ineffective concentration of 10 ng/ml indicates that both pka and pkc are routinely required for phagocytosis. in addition, in the presence of 25 ng/ml of il-8, h-89, but not calphostin c, reduces the phagocytic index to levels lower than the controls suggesting that the pka pathway is more important than that mediated by pkc in botryllus phagocytosis. similar effect were reported for il-8induced changes in cell shape in bivalve molluscs (ottaviani et al., 2000). further studies are now required to better characterise invertebrate g protein-coupled receptors, their natural ligands and the signal transduction pathways involved in immunocyte activation. acknowledgements authors wish to thank mr. m. del favero for technical help. this work was supported by the italian miur (prin, 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hypervariable immune-response genes of invertebrates l bowden, nm dheilly, da raftos, sv nair department of biological sciences, macquarie university, north ryde, new south wales, 2109, australia accepted december 14, 2007 abstract our understanding of invertebrate immune systems is undergoing a paradigm shift. until recently, the host defence responses of invertebrates were thought to rely on limited molecular diversity that could not tailor reactions toward specific microbes. this view is now being challenged. highly discriminatory defence responses, and hypervariable gene systems with the potential to drive them, have been identified in a number of invertebrate groups. these systems seem to be quite distinct, suggesting that pathogen-specific responses might have evolved on numerous occasions. here, we review evidence that inducible, disease-specific immunity might be commonplace in the animal kingdom. key words: adaptive immunity; host defence; immunization; invertebrate immunology; hypervariability introduction the continuing growth of invertebrate aquaculture worldwide, and its seemingly inherent problems with catastrophic disease outbreaks, is driving a renewed demand for research into invertebrate immune systems. much of this research revolves around two unresolved questions can invertebrate immune systems be fine-tuned to fight specific infections, and, can that fine-tuning provide protective immunity to re-infection? for the first time, powerful new molecular technologies are parallelling classical immunization experiments to provide evidence for disease-specific immunity among invertebrates. however, these two experimental approaches have rarely been unified to elucidate the mechanisms underpinning pathogen-specific defense. providing this synthesis will continue to drive the paradigm shift in invertebrate immunology. it could lay the foundation for the use of immunization as a powerful new method for disease control in invertebrate aquaculture, and provide fundamental insights into the adaptive selection pressures that drive the evolution of immune systems. in this article , we explore evidence for pathogen-specific immunity ___________________________________________________________________________ corresponding author: david a raftos department of biological sciences, macquarie university, north ryde, new south wales, 2109, australia e-mail: draftos@rna.bio.mq.edu.au among invertebrates, and the role that hypervariable gene families play in those responses. traditional views of invertebrate immune systems until recently, highly variable immune-response molecules were thought to be confined to the jawed vertebrates (gnathostomes) (raftos and raison, 1992; raftos, 1993; litman et al., 2005). adaptive immune responses among gnathostomes revolve around hypervariable antibodies and t-cell receptors (tcrs), which act as pathogen-specific recognition proteins. the enormous diversity of antigen-specific antibodies and tcrs is generated using enzymes encoded by somatic recombination activating (rag) genes. production of each unique antibody or tcr isotype is confined to individual lymphocyte clones, which are responsible for providing lasting immunity against specific pathogens via the process of clonal selection. since burnet (1959) put forward his theory of clonal selection, it has been assumed that invertebrates lacked highly specific immune responses based on pathogen-specific immunorecognition (raftos, 1993). this presumption was based, almost exclusively, on the failure to find hypervariable antibodies or tcrs amongst invertebrates (raftos and raison, 1992). remarkably, there is only a limited history of appropriately controlled immunization experiments 127 mailto:draftos@rna.bio.mq.edu.au being used to test whether antibody/tcrindependent pathogen-specific immune responses occur among invertebrates. the purported lack of pathogen-specific immunity among invertebrates has often been explained by the differential use of rand k selection strategies by invertebrates and vertebrates (klein, 1989, 1989; rinkevich, 1999). the terms, r and k, come from ecological algebra defined by verhulst's equation describing population dynamics (verhulst, 1838). dn/dt = rn (1-n/k), where "r" is the growth rate of a population, "k" is the carrying capacity of the environment and "n" is population size. the r/k-selection hypothesis has been used to distinguish invertebrates most often as r-selected, indicating that they are smaller, have higher fecundity, shorter lifespans and less stable populations than most vertebrates (pianka, 1970). this is seen as a high risk reproductive strategy that is more susceptible to chance events. it explains why organisms that are r-selected display higher fecundity. vertebrates, on the other hand, were seen to have lower reproductive outputs, longer lifespans and generally smaller, more stable populations that could be described as k-selected. the r/k-selection argument with respect to the evolution of pathogen-specific immune systems is simple invertebrates don't live as long as vertebrates, and they don't invest as much in each of their offspring. therefore, they do not require sophisticated immune responses that rely on pathogen-specific immunorecognition to maintain population stability. this is a simplistic argument that has always been flawed (stearns, 1977; getz, 1993). in terms of life history strategies, it is difficult to imagine that some form of adaptive immunity would not have evolved among invertebrates. based on the r/k selection paradigm, it has been taken for granted that vertebrates, with their presumed long life cycles and relatively lower reproductive outputs, had a greater evolutionary "need" for adaptive immunity than invertebrates (klein, 1989; medzhitov and janeway, 1997). however, r/k-selection does not neatly distinguish vertebrates from invertebrates (stearns, 1977; getz, 1993). both rand k-selection strategies can be observed in both taxa, including important stem groups. some invertebrates, such as bivalves, gastropods, echinoids and decapods, can live for many decades (up to 100 years in some cases), and clearly express features of k-selected strategies, including large body size and relatively low effective fecundity (powell and cummings, 1985). conversely, some gnathostomes, including fish, in which adaptive immunity appears to have first evolved, have short lifespans and high fecundity. for instance, coral fish (eviota sigillata) reach sexual maturity within weeks of birth and have a maximum lifespan of 59 days (depczynski and bellwood, 2005). so, using the r/k model to determine whether a broad taxonomic group "requires" highly specific responses to particular pathogens has never been well supported by the available evidence. factors other than r/k life history strategies are also likely to provide significant selection pressures for the evolution of pathogen-specific immune systems. many invertebrates occupy niches that place them in close contact with a broad range of pathogens. others establish high density populations that are prone to epizootic disease outbreaks (stow et al., 2007). many flies and other arthropods feed and/or breed in faeces or decomposing plant material, whilst some other insects, such as ants, bees and termites, live in highly social colonies that facilitate the transmission of virulent pathogens and parasites. eusociality, or the production of sterile workers to care for reproductive members of the population, is perhaps the most extreme example of a life history strategy that might necessitate pathogenspecific immunity. eusociality often involves asexual reproduction that limits genetic variability within populations, combined with high population densities in confined environments (hives or nests). according to van valen's (1973) red queen theory, which suggests that genetic diversity within populations is required to counter rapidly evolving pathogens, the genetic and social strictures of eusociality should make eusocial populations more susceptible to epizootic disease. as a result, there should be a strong link between eusociality and enhanced defensive capacity. this prediction fits well with recent data indicating that, among social insects, immunological capacity increases proportionately with the degree of sociality. increased eusociality and genetic relatedness among bees is strongly correlated with an increase in the production of antimicrobial compounds (stow et al., 2007). some eusocial species also show strong evidence for pathogen specific immunity (see below). new evidence for pathogen-specific immunity among invertebrates even though the r/k selection argument has been used since burnet's time to predict that invertebrates lack pathogen-specific recognition of the type provided by vertebrate antibodies and tcrs (klein, 1989), new data question this assumption. recent work indicates that arthropods and molluscs can generate fine-tuned responses against specific pathogens. these studies have used classical immunization experiments to test for pathogen specificity; usually by measuring host survival after inoculation and by comparing responses between primary and secondary inoculations using different pathogens (fig. 1). in these experiments, more rapid and powerful secondary immune responses against the original, inoculating pathogen are presumed to indicate that the host has developed an induced response targeting that pathogen (little et al., 2005). pathogen specificity is further implied if enhanced responses do not extend to microbes other than the one used in the initial, priming inoculation. this approach is not new. in the past it has been used to show that drosophila melanogaster can distinguish between broad groups of pathogens, such as fungi and bacteria (lemaitre et al., 1997). these broad responses are often mediated by the 128 fig. 1 a generic protocol for immunization experiments. host organisms are inoculated with one of two different pathogens (circles and squares). some time later, they are re-challenged either with the same pathogen used for the initial inoculation, or with the other pathogen. the intensity of host responses to the secondary challenge is then measured, often in terms of host mortality over time or the clearance rate of the pathogen (modified from little et al., 2005). toll and imd/relish signalling pathways and probably differentiate between highly conserved molecular structures on pathogens, such as bacterial lipopolysaccharides (lps) and fungal βglucans (leclerc and reichhart, 2004; beutler et al., 2006). janeway (1989, 1992) has designated these types of highly conserved target molecules as "pathogen-associated molecular patterns (pamps)". pamps define broad groups of pathogens for detection by generic "pattern recognition" receptors (prrs), such as toll-like receptors and lectins. pattern recognition, which lacks fine-scale differentiation between closely related microbes, is common throughout the animal kingdom and was thought to be the only molecular recognition paradigm available to invertebrates (medzhitov and janeway, 1997, 2002; janeway and medzhitov, 2002). however, more recent immunization experiments have shown that d. melanogaster is capable of much finer scale discrimination between microbes. pham et al. (2007) found that immune responses by d. melonogaster against streptococcus pneumoniae could be enhanced by inoculation with heat-killed s. pneumoniae. primed responses against s. pneumoniae did not provide protection against other pathogens, even closely related bacterial species. and prior exposure to a variety of other bacteria did not elicit protection against s. pneumoniae. specific protection could also be stimulated by injecting beauveria bassiana, a natural pathogen of d. melanogaster. however, inoculation with salmonella typhimurium, listeria monocytogenes and mycobacterium marinum, did not induce pathogen-specific protective responses. the enhanced responses to s. pneumoniae and b. bassiana in drosophila clearly have features in common with gnathostome adaptive immunity, yet they appear to be mediated by the same toll-like pathways and phagocytic responses that are associated with simple pattern recognition (pham et al., 2007). even more compelling evidence for pathogenspecific immunity among insects comes from studies of bumblebees (bombus terrestris). sadd and schmid-hempel (2006) immunized bees with a defined isolate of the gram-negative bacterium, pseudomonas fluorescens, and two closely-related gram-positive bacteria (paenibacillus alvei and paenibacillus larvae). bees were re-challenged either 8 or 22 days after the initial, priming injection, and their relative survival was recorded. the data showed that b. terrestris were capable of mounting highly specific immune responses that could differentiate even between the congeneric bacteria (sadd and schmid-hempel, 2006). such targeted responses only became evident when bees were left for 22 days before being re-challenged. pathogen-specific responses were masked by less specific reactions when bees were re-challenged within 8 days of the initial inoculation. these examples of pathogen-specific immunity in drosophila and b. terrestris are among a number of highly targeted immune responses so far identified among invertebrates. in 2006, kurtz cited five studies of either insects, crustaceans or molluscs, in which there is strong evidence for the induction of pathogen specific immunity by inoculation with trematodes, bacteria, tapeworms or trypanosomes (webster and woodhouse, 1998; schmid-hempel et al., 1999; carius et al., 2001; little et al., 2003; kurtz and franz, 2003). among these studies, kurtz and franz (2003) showed that infections of the copepod crustacean, 129 macrocyclops albidus, with the tapeworm, schistocephalus solidus, were far less severe if the hosts had been primed with siblings of the worms used for the subsequent infections. this acquired protection was not evident if the tapeworms used in the initial and subsequent challenges were genetically distinct. similar immunization experiments in the prawn, penaeus monodon, identified discriminatory responses to virulent white spot syndrome virus (wssv) (witteveldt et al., 2004). injecting the wssv envelope protein, vp28, provided substantial protection against white spot infection, whereas a closely related antigen, vp19, did not. in a another series of immunization experiments, it was shown that induced, specific protection against pathogens in the water flea, daphnia magna, can be transmitted from mother to offspring (little et al., 2005). the interpretation of these data is extremely controversial (hauton and smith, 2007). it does suggest that some protostomes can mount defensive responses that are fine-tuned toward some invading pathogens (schmid-hempel et al., 1999; kurtz and franz, 2003; schmid-hempel and ebert, 2003; little and kraaijeveld, 2004; kurtz, 2005; little et al., 2005; kurtz and armitage, 2006). however, there is still insufficient evidence to tell whether these systems are widespread throughout the metazoa. it is also unclear if they can generate targeted protection against many different pathogens, or whether they have been selected only to provide protection against pathogens that are the most relevant to the host species. hypervariable immune-response gene families of invertebrates at roughly the same time that traditional immunization experiments were revealing the existence of pathogen-specific responses in some invertebrates, new molecular technologies were being used to identify hypervariable immune response molecules that might underpin pathogenspecificity (flajnik, 2004; flajnik and du pasquier, 2004; loker et al., 2004; litman et al., 2005; du pasquier, 2006). it is now evident that a range of highly variable immune-response gene families are expressed among invertebrates and jawless vertebrates. some of these families have already been found to play key roles in immunological functions, such as self/non-self recognition and differentiation between broadly different pathogen types (i.e. fungi vs. bacteria). from the existing data it is clear that the gene families encoding highly variable immune-response proteins have arisen independently on numerous occasions in higher plants and in animals (flajnik, 2004; flajnik and du pasquier, 2004). none of the families that have been identified to date are closely related to each other, even though many incorporate immunoglobulin-superfamily (igsf) domains (flajnik, 2004; flajnik and du pasquier, 2004; litman et al., 2005). this suggests that there has been strong selection pressure within individual taxa to develop mechanisms that promote survival in the presence of rapidly evolving pathogens. a number of the highly variable immuneresponse families identified among invertebrates and "lower" vertebrates are described in more detail below. variable lymphocyte receptors in agnathans the two extant groups of agnathans (jawless fish; hagfish and lampreys) do not express highly diverse antibodies or tcr (raftos and raison, 1992). however, expressed sequence tag (est) analyses of lamprey lymphocytes revealed immuneresponse genes that encode highly variable lymphocyte receptors, or vlrs. these molecules contain numerous leucine-rich-repeat (lrr) modules, and may act as cell surface receptors implicated in immunological defence (flajnik, 2004; pancer et al., 2004; litman et al., 2005). vlrs have non-variable glycosyl-phosphatidyl-inositol (gpi) lipid anchors for attachment to the plasma membranes lymphocytes. these gpi anchors may become detached to free humoral forms of vlr molecules (litman et al., 2005). the high level of diversity displayed among vlrs arises from variation in their nucleotide sequences and in the number of lrr modules that they incorporate (pancer et al., 2004). there is some evidence that different lamprey lymphocytes express different and unique vlrs, implying a clonal expression system akin to that of gnathostome antibodies (flajnik, 2004). v region-containing chitin binding proteins in cephalochordates cannon et al. (2002) identified five families of highly variable immune-response molecules in the protochordate, amphioxus (branchiostoma floridae), that incorporate two n-terminal igsf v-region domains and one chitin-binding domain at the carboxy-terminus. these molecules have been designated v region-containing chitin binding proteins (vcbps). their v regions show high levels of diversification and might function as specific immune-recognition domains akin to the adaptive immunological receptors of gnathostomes. the five distinct families of vcbp transcripts identified by cannon et al. (2002) are highly diversified, showing only 27 % 41 % sequence identity among families. the chitin-binding region incorporates a diagnostic sequence of spaced cysteine residues that is characteristic of chitin-binding proteins in other species, and recombinant vcbps can bind chitin. in situ hybridisation revealed that vcbps are expressed exclusively in the intestine. given the filter feeding life history of amphioxus, this distribution fits well with a potential role in the control of pathogens in the gut. cannon et al. (2002) concluded that vcbps may have a dual function, combining pattern recognition by the chitin-binding domain with diversified specific immunorecognition afforded by the highly variable igsf v-regions. 185/333 proteins in sea urchins the 185/333 gene family from the purple sea urchin, strongylocentrotus purpuratus, has high levels 130 fig. 2 185/333 proteins are expressed primarily on the surface of one sea urchin (s. purpuratus) coelomocyte type, small phagocytes. in this image, 185/333 staining is shown in green and nuclei are counterstained red. note the expression of 185/333 on the extensive filopodia of small phagocytes. f, filopodia; n, nuclei. of both cdna and genomic dna sequence diversity (nair et al., 2005; terwilliger et al., 2006, 2007; buckley and smith, 2007). about 50-60 distinct genomic 185/333 loci have been estimated and almost 700 unique mrna transcripts have been sequenced to date (terwilliger et al., 2007; k buckley, george washington university, personal communication). there is also evidence for 185/333 gene sequences and gene expression in other sea urchin species (m roth, macquarie university, personal communication). unlike the highly variable gene families of other invertebrates, sequence comparisons have failed to reveal any similarities between 185/333 genes and those from other organisms (nair et al., 2005). however, based on their expression patterns, 185/333 proteins appear to be involved in immune responses. titres of both 185/333 mrna transcripts and proteins increase after immunological challenge (brockton et al., 2007; nm dheilly, unpublished data). the frequency of 185/333 mrnas increases 70-fold during bacterial infections, and 185/333 transcripts can comprise more than 60 % of the mrna specifically induced by immune responses (nair et al., 2005). there is also some evidence that different pamps elicit the expression of different 185/333 variants (nm dheilly, unpublished data; terwilliger et al., 2007). proteomics and transcriptome analysis have shown that 185/333 molecules have high levels of molecular diversity within and between sea urchins. individual sea urchins can express in excess of 200 distinct 185/333 proteins, and each animal has a distinctive suite of the proteins that differs from all other individuals (nm dheilly, unpublished data; terwilliger et al., 2007). 185/333 proteins are localized to the surface of a distinct class of coelomocytes, the frequency of which is substantially enhanced in coelomic fluid after immunological challenge (fig. 2) (brockton et al., 2007). the observed molecular weights of 185/333 proteins are much higher than those predicted from mrnas, suggesting that 185/333 proteins form strong associations with other molecules, or with each other (brockton et al., 2007; nm dheilly, unpublished data ). the variability of 185/333 genes can be explained by combinatorial diversity, single nucleotide polymorphisms (snps) and small insertions and deletions (indels). 185/333 mrna transcripts are comprised of 25 different blocks of sequence, or “elements”, that are either present or absent in many different combinations (nair et al., 2005; terwilliger et al., 2006; buckley and smith, 2007). snps and indels also occur frequently in all 185/333 transcripts, magnifying their diversity. nucleotide substitutions between 185/333 variants are often non-conservative, resulting in amino acid changes. this suggests that 185/333 diversity has been driven by positive selection of the type associated with pathogen-driven adaptation (nair et al., 2005; terwilliger et al., 2006). the processes that generate 185/333 diversity remain unclear, but they could include genomic or somatic recombination, alternative splicing, rna editing and post translational modifications (buckley and smith, 2007). the biological activities of 185/333 proteins have not yet been defined and their lack of homology with other molecules makes it difficult to predict structure/function relationships. however, evidence from sequence analyses is shedding more light on the structure of these proteins. 185/333 mrnas encode polypeptides with a hydrophobic leader, separate glycineand histidinerich regions, numerous n-linked and o-linked glycosylation sites, acidic sequence patches, and an arginine-glycineaspartic acid (rgd) motif (terwilliger et al., 2006). predicted polypeptides do not contain cysteines, transmembrane regions, gpi linkage sites or identifiable domains. the only regions with at least some similarity to other molecules are the rgd motif and one of the histidine-rich domains, which is comparable to histatins, a group of mammalian salivary proteins with powerful antifungal activities (xu et al., 1990). more detailed analysis of 185/333 sequences reveals many short amino acid sequence repeats that are found at numerous positions in the predicted proteins (nair et al., 2005; buckley and smith, 2007) (fig. 3). distinctive types of repeats are found in different regions of predicted polypeptides, most notably in the n-terminal glycinerich region and a c-terminal region that incorporates numerous potential n-glycosylation sites. in many cases, different types of short repeats are clustered together, and these clusters are duplicated throughout the polypeptide. distinct combinatorial patterns of repeats define eight distinct 185/333 sub-families, which phylogenetic analysis suggests arose by progressive recombination events (nm dheilly, unpublished data; k buckley, george washington university, personal communication). freps in molluscs highly variable fibrinogen-related proteins (freps) in the snail, biomphalaria glabrata, and in at least five other genera of gastropods, consist of 131 fig. 3 a) the pattern of amino acid repeats evident in a model 185/333 protein from the sea urchin, s. purpuratus. each colored shape represents a unique amino acid repeat. boxed areas show where different clusters of repeats are duplicated throughout the predicted polypeptide. distinct repeat clusters are found in the glycine-rich and n-glycosylated regions of the polypeptide. b) amino acid repeat patterns found in 8 distinct 185/333 sub-families. each family is distinguished by a unique combinatorial pattern of sequence repeats. either two or three domains. one is a c-terminal fibrinogen domain, while the other one or two domains are members of the igsf (fig. 4a) (zhang et al., 2004). freps are reported to have lectin-like (carbohydrate-binding) activity and their expression increases in response to challenge with the trematode parasites, echinostoma paraensei and schistosoma mansoni (adema et al., 1997). one strain of b. glabrata that is resistant to s. mansoni increases frep expression by up to 57-fold after exposure to the parasite, whilst frep expression in s. mansoni-susceptible snails is unaltered by immunological challenge (hertel et al., 2005). freps can precipitate antigens that are secreted by s. mansoni and can bind to the surface of e. paraensei. all of these observations suggest that freps are involved in host/parasite interactions and may act as highly diversified recognition and/or effector proteins (loker et al., 2004; hertel et al., 2005). substantial diversity is evident in the igsf domain(s) of freps. expressed sequence tag (est) analysis of transcripts enriched for the freps of s. mansoni-resistant snails by suppression subtractive hybridisation (ssh) identified 88 unique ssh-ests among the 112 clones sequenced (nowak et al., 2004). sequence diversity incorporates a number of distinct frep subfamilies, and additional variability is evident in the form of snps within each of these sub-families. diversity seems to be generated through a combination of alternative exon splicing, gene conversion and somatic hypermutation, meaning that different individuals express different suites of freps (fig. 4b) (zhang and loker, 2003; zhang et al., 2004). frep genes appear have diversified faster at the genomic level than other genes, and all of the variants identified to date seem to be derived from just nine ancestral or "mother" sequences (zhang et al., 2004). dscams in insects the down’s syndrome cell adhesion (dscam) molecules of d. melanogaster and other insects are members of the igsf. dscam genes comprise clusters of ig-like exons with high levels of sequence diversity that are flanked by relatively conserved regions (fig. 5) (schmucker et al., 2000). alternative splicing recombines exons by mutually exclusive excision, potentially generating more than 38,000 distinct extracellular domains. individual hemocytes can generate between 14-50 different isoforms of dscam and there are a total of about 19,000 hemocyte-specific isoforms (watson et al., 2005). hemocytes from mutant flies with impaired dscam expression, and hemocytes in which dscam expression had been specifically knocked down using interference rna (rnai), have significantly lowered phagocytic activities than hemocytes from normal flies. antibodies that specifically bind to the extracellular ig domains of dscams can also inhibit phagocytosis and dscam isoforms can bind onto pathogen surfaces (watson et al., 2005). this suggests 132 fig. 4 a) schematic polypeptide structure of the frep3 sub-family showing different domains (n-terminus on left). sp, signal peptide; igsf1 and 2, immunoglobulin superfamily domains; scr, small connecting region; icr, interceding region; fbg, fibrinogen domain. b) comparison of freps expressed by two different individuals of the same snail species (b. glabrata). the region of igsf1 shown by the black bar in a. was amplified from transcripts isolated from the two snails and sequenced. the numbers shown in the figure are the number of clones that were unique to each snail, and the number shared by the two snails (modified from zhang et al., 2004). that dscams are involved in the phagocytic uptake of pathogens, possibly acting as pathogen-specific recognition proteins (watson et al., 2005). interestingly, dscams also act as axon guidance receptors during neuronal development, with different dscam isoforms being expressed in the brain compared to hemocytes (watson et al., 2005). what role do highly variable gene families play in pathogen-specific immunity? the data on 185/333 genes, vcbps, freps and dscams imply that all of these gene families contribute to novel forms of inducible, pathogenspecific immune systems (flajnik, 2004; flajnik and du pasquier, 2004; litman et al., 2005). however, there is little direct evidence to support this conclusion. the molecular approaches that have been used to identify these genes have often lacked accompanying information about their physiological functions. in hindsight, resolving the biological activities of hypervariable proteins, particularly their contribution to pathogen-specific immune responses, is proving to be problematic and time consuming. in most cases, we still do not understand the biological relevance of these gene systems, or whether they help to combat infection. traditional immunization experiments have suffered from the opposite problem compared to molecular biological approaches. whilst immunization studies have been very useful in demonstrating the existence of acquired, pathogenspecific reactions, they have not provided information about the molecular mechanisms underlying induced responses (little and kraaijeveld, 2004; little et al., 2005). a new goal of invertebrate immunology will be to address this gap between molecular immunology and immunization experiments (hauton and smith, 2007). new work needs to combine classical immunization protocols with comprehensive molecular analyses to simultaneously identify pathogen-specific immune responses and the genes that control them. to date there are only a few convincing studies that have implemented this approach. dong et al. (2006) showed that particular dscam splice-variants in the mosquito, anopheles gambiae, are associated with resistance to malarial (plasmodium) and bacterial infections. rnai silencing of dscam expression significantly reduced survival rates from bacterial infections and increased plasmodium ooycst development in the midgut of mosquitoes. most significantly, the a. gambiae dscam (agdscam) variants produced in response to inoculation with particular bacteria, (escherichia coli and pseudomonas veronii) could bind onto these microbes with higher affinity than the agdscams produced in response to challenge with grampositive bacteria (staphylococcus aureus) or nonchallenged cell lines. silencing specific forms of agdscams associated with responses to different bacteria made mosquitoes more sensitive to infections by the target bacterial species, but not 133 fig. 5 gene organization of dscams from the mosquito anopheles gambiae showing clusters of highly variable igsf domains and constant domains. the numbers below the figure show the number of ig exons per cluster (modified from dong et al., 2006). others. these data imply a strong association between dscams and pathogen-specific immunity in insects (dong et al., 2006). similarly, robalino et al. (2005) have demonstrated a clear link between pathogenspecific systems in shrimp and protective immunity to wssv. they found that protection against wssv in litopenaeus vannamei could be elicited by injecting double-stranded rna (dsrna) (robalino et al., 2005). even though randomly generated dsrnas had some effect, much higher pathogenspecific protection against white spot disease was induced when dsrnas based on wssv sequences were used, indicating that shrimp can use pathogenspecific rnai systems to generate highly targeted protection against viral diseases. conclusions studies that combine immunization experiments with manipulative molecular analyses represent a new emphasis for invertebrate immunology. the data presented in this article suggest two things that pathogen-specific, inducible immune systems might be common among invertebrates, and that these systems have evolved independently on numerous occasions. this implies that many more pathogen-specific immune systems, and their associated hypervariable recognition molecules, await discovery. acknowledgments our work on immune-response proteins in sea urchins is funded by an australian research council discovery grant (dp0556486 with lc smith). l. courtney smith, k buckley, d terwilliger and v brockton from the george washington university in washington, dc, have been responsible for most of the work on sea urchin 185/333 molecules. our collaboration with them has helped to develop many of the ideas expressed in this article. we also thank courtney smith for helping to edit this article. the photomicrograph shown in fig. 4 was taken by da raftos in courtney smith's laboratory. our studies of disease resistance in molluscs are supported by an australian research council linkage grant in conjunction with the nsw department of primary industries (lp0453461). references adema cm, hertel la, miller rd, loker es. a family of fibrinogen-related proteins that precipitates parasite-derived molecules is produced by an invertebrate after infection. proc. natl. acad. sci. usa 94: 8691-8696, 1997. beutler b, jiang z, georgel p, crozat k, croker b, rutschmann s, et al. genetic analysis of host resistance: toll-like receptor signalling and immunity at large. annu. rev. immunol. 24: 353-389, 2006. brockton v, henson jh, raftos da, majeske aj, kim yo, smith lc. localization and expression of 185/333 proteins encoded by a diverse gene family in coelomocytes from the purple sea urchin, strongylocentrotus purpuratus (stimpson). j. cell sci. 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crayfish procambarus clarkii is used for the innate immune defense of crustaceans due to its convenience for laboratory culture and study. to know more about the transcriptome of the crayfish, we constructed and sequenced a cdna library from a mixture of hemocytes and hepatopancreas from white spot syndrome virus (wssv)-infected crayfish. by random sequencing, we obtained 9115 high-quality expressed sequence tags with a mean length of 370 bp, representing 3033 unigenes. most of the unigenes are first reports for the red swamp crayfish. besides the metabolic genes, many genes that may be involved in the innate immune system of the crayfish are also obtained from the library, such as antimicrobial peptides, pattern recognition receptors, proteases and protease inhibitors, signal transduction proteins, apoptosis-, antioxidant-, and rna interference-related proteins. we chose ten immune-related genes to analyze their expression pattern by quantitative real time polymerase chain reaction (qrt-pcr) from the hemocytes of normal and wssv-challenged crayfish. seven of them, including anti-lipopolysaccharide factor, astacidin, crustin 1, h3 histone family 3a, serine/threonine protein kinase, tgf beta-inducible nuclear protein, and tar rna binding protein, were upregulated after wssv injection, but the mrna expression levels of crustin 2, a lectin, and a digestive cysteine protease decreased after wssv infection. our results showed that the transcriptome analysis provides a useful resource for identification of immune related genes and understanding the immune responses of the crayfish. key words: hemocytes; hepatopancreas; expression sequence tags; white spot syndrome virus; red swamp crayfish; procambarus clarkii introduction the crustacean farming industry has been suffering serious problems and enormous economic losses from an outbreak of white spot syndrome virus (wssv) since 1993 (yan et al., 2007). wssv is a serious pathogen and can infect numerous crustaceans including shrimp, crab, and lobster (shi et al., 2005; zeng et al., 2009). the cultivation of red swamp crayfish (procambarus clarkii) has become an important economic activity in china as crayfish is a delicious food. p. clarkii can be infected by white spot syndrome virus (wssv) and is easy to be used as a laboratory model for immunity analysis. the investigation of the immune defense mechanisms of red swamp crayfish could be helpful in the control of the wssv disease of cultivated crustaceans by ___________________________________________________________________________ corresponding author: jin-xing wang school of life sciences shandong university jinan, shandong 250100, china e-mail: jxwang@sdu.edu.cn enhancing the defense activity. crustaceans lack an adaptive immune system and rely totally on the innate defense system to resist pathogen invasion. the innate immune reaction comprises cellular reactions such as phagocytosis, nodule formation and encapsulation performed by hemocytes, and humoral reactions mediated by antimicrobial peptides. crayfish hemocytes play important roles in the initiation of several immune responses and production of antimicrobial peptides (jiravanichpaisal et al., 2007). the hemocytes and hepatopancreas are crucial for the immune system in crustaceans as it is the main production site for immune recognition molecules, initiates the humoral reaction, and takes part in the cellular reaction by some specialized cells and phagocytes (gross et al., 2001). many immune-related genes have been found in hemocytes of wssv-challenged crayfish (jiravanichpaisal et al., 2007; zeng et al., 2009). expressed sequence tag analysis is widely used to identify novel and differentially expressed genes 120 (leu et al., 2007; jiang et al., 2008). liu (liu et al., 2006) firstly used suppression subtractive technique on crayfish to study transcripts after wssv infection. by suppression subtractive hybridization analysis, zeng and lu (zeng et al., 2009) found thirty-three differently expressed genes in wssv-infected hemocytes of crayfish. the transcriptome information for the hepatopancreas is still limited, so we constructed and sequenced a cdna library from wssv-challenged hemocytes and hepatopancreas in red swamp crayfish. the aim of this study is to identify and annotate more immune-related genes in the two immune organs and also provide some information to uncover the mechanisms of wssv pathogenesis in crustaceans. in this study, we obtained 3033 unigenes; most of them are novel for red swamp crayfish. many genes in the library were found to be related to the innate immune system in other species, such as pattern recognition receptors, antimicrobial peptides, proteases and protease inhibitors, signal transduction proteins, apoptosis-related proteins, antioxidant proteins, rna silencing-related proteins, molecular chaperones, cuticle proteins, and calcium-binding proteins. materials and methods wssv challenge of crayfish and collection of hepatopancreas and hemocytes red swamp crayfish (procambarus clarkii) (about 10-20 g each) were obtained from a seafood market in jinan, shandong province, china. they were cultured in 500 l tanks in fresh water at room temperature (~20 c) in the laboratory. wssv was extracted from the gills of infected crayfish, and diluted in pbs (phosphate buffered saline, containing 140 mm nacl, 2.7 mm kcl, 10 mm na2hpo4, and 1.8 mm kh2po4). using a 100 l syringe, 3.2×10 7 virus particles were injected into the abdominal segment of each crayfish. the preparation and quantification of viral inocula followed a previously described method (wang et al., 2009b). ninety-six hours after injection, the hemolymph was collected from the ventral sinus of ten crayfish and mixed with 1⁄10 volume of anticoagulant buffer (10 % sodium citrate, ph 7) containing 200 mm phenylthiourea as a melanization inhibitor. the hemolymph was then centrifuged at 800 g for 5 min (4 c) to collect the hemocytes. the hepatopancreas was also collected from the crayfish for rna extraction 96 h after wssv injection. rna extraction and cdna library construction the total rna was extracted from hemocytes and hepatopancreas of 96 h infected crayfish using unizol reagent following the manufacturer’s instructions (biostar, shanghai, china). messenger rna (mrna) was extracted with the polyatract mrna isolation system (promega, usa). the mrnas from hemocytes and hepatopancreas were mixed together and used to construct a cdna library. the creator smart cdna library construction kit (clontech, usa) was used for the cdna library construction following the manufacturer’s instructions. the double strand cdna was digested and ligated with the pdnr-lib vector, and then transformed into competent dh5 cells. individual colonies were randomly selected, and plasmids were extracted for sequencing from the 5'-ends. analysis and classification of the expression sequence tags (ests) high quality ests (more than 100 bp after removal of the vector sequence) were assembled by cap3 (http://seq.cs.iastate.edu./) in order to obtain contig sequences. unigenes (including contigs and singlets) were analyzed by blastx against the national center for biotechnology information (ncbi) protein database, blastn against the ncbi nucleotide database, and blastx against the swissprot protein database. the e-value of significant matches by the blastx and blastn is less than 0.001, and the alignment score is more than 30. the concrete functional information and metabolic profiles of unigenes were obtained using the kyoto encyclopedia for genes and genomes (kegg) (kanehisa et al., 2004). unigenes were assembled into different functional classes with clusters of orthologous groups of proteins (cogs) (http://www.ncbi.nlm.nih.gov/cog/). quantitative real time pcr (qrt-pcr) total rnas were extracted from hemocytes of ten normal and 96 h wssv-challenged crayfish using unizol reagent separately. then rnas were reverse transcribed to the first strand cdnas. the cdnas were diluted 100 fold and used as the template for qrt-pcr. ten genes including anti-lipopolysaccharide factor, astacidin, crustin 1, crustin 2, h3 histone family 3a, serine/threonine protein kinase (srk), tgf beta-inducible nuclear protein, tar rna binding protein (trbp), lectin and digestive cysteine protease from this cdna library were chosen for qrt-pcr analysis. these genes were chosen because they belonged to different groups and were firstly found in crayfish. we wanted to know whether they participate the antivirus innate immune reaction of crayfish. 18s rrna was used as the control. primer sequences used in this study are listed in table 1. qrt-pcr was performed following the manufacturer’s instruction with sybr premix ex taq (takara, japan) using a real-time thermal cycler (bio-rad, usa). the qrt-pcr was performed in 10 μl reactions containing 5 μl of 2×premix extaq, 1 μl of the 1:100 diluted cdna, 2 μl each of the forward and reverse primer (1 m). the amplification conditions were initial denaturation at 95 c for 5 min, 40 cycles of 95 c for 30 s, 60 c for 50 s, followed by a melting curve from 60 to 95 c. pcr products were detected by agarose gel analysis after each pcr reaction to confirm the specific gene amplification. data were analyzed using a comparative method (2 -∆∆ct ). qrt-pcr analysis was repeated three times for each sample. results and discussion general of the cdna library to get more information on the virus immunity-related proteins from crayfish, total rna was extracted from important immune tissues including hemocytes and hepatopancreas at 96 h http://www.ncbi.nlm.nih.gov/cog/ 121 table 1 primers used in the study after wssv challenge, and then a cdna library was constructed and randomly sequenced. as shown in table 2, a total of 10145 clones were sequenced, and among these, 9115 ests were equal to or longer than a cutoff length (100 bp) after removal of vector sequences. they are high quality ests with an average length of 370 bp. in total, 3033 unigenes were acquired including 859 contigs which have more than one est and 2174 singlets which have only one est; the singlet rate is 71.68 . the unigene hemocyanin has 213 ests, which is the largest unigene size (table 3). the novelty of the library is 33.27 . among the unigenes, only a small fraction is previously reported genes and most of them are novel genes in red swamp crayfish. we discovered many new genes from this library, and it also provides some information about the transcriptome of crayfish hemocytes and hepatopancreas. all the unigenes were aligned to the ncbi nr database using blastx, and to the ncbi nt database using blastn as a complementary analysis. moreover, the unigenes were repeatedly aligned against the blastx swissprot database. from the annotation of the alignments, we found that most of the ests shared high similarities with nucleotide sequences mainly from table 2 summary statistical analysis of the cdna library from hemocytes and hepatopancreas total ests: low and high quality ests; low quality ests: after remove empty ests and vector ests, the length of est less than a cutoff length (100bp); high quality ests: after remove empty ests and vector ests, the length of est more than a cutoff length (100bp); unigenes: contigs and singlets; contigs: the number of ests is equal to or bigger than 2; singlets: the number of ests is 1; novelty: unigenes/assembled ests; redundancy: 1-novelty gene primer sequence(5 -3 ) astacidin astacidin-f atgcgtcttctccatctcc astacidin-r ttacttgcctggacggta anti lipopolysaccharide factor alf-f ccgcctccttcaccccaca alf-r tccacctcaccgttccgcc crustin1 crustin1-f tattcctcgctgcacaaaca crustin1-r cacatagcacctccctctca crustin2 crustin2-f gggaagaaaagcacaatggt crustin2-r ggtatggaggtcgagacagg digestivecysteine protease dcysp-f aagtatgttgacgcagaggagg dcysp-r aaattggttcattgccaggttg h3 histone family 3a h3-f gtcaccatcatgcccaaggata h3-r gccagcactgcgaagtcaattc lectin lectin-f gttattgacgactccacctt lectin-r gtcttcccattgacccactt serine/threonine protein kinase spk-f tgctatgtgaagctcggctct spk-r: gcgatctgatgctcctcctct tgf beta-inducible nuclear protein tgfinp-f gcctgggtgctggtatcttgg tgfinp-r gttcgttctgtggcattgtgt tar rna binding protein trbp-f aaaatgtatcgtcaaccaccac trbp-r caccctctatctgcaacaagtc 18srna 18srna-f accgattgaatgatttagtgag 18srna-r tacggaaaccttgttacgac description number total ests 10145 low quality ests 1030 high quality ests 9115 unigenes 3033 contigs 859 singlets 2174 novelty (%) 33.27 redundancy (%) 66.73 122 fig. 1 the number of annotated unigenes with different e-values. crayfish (pacifastacus leniusculus), narrow-fingered crayfish (pontastacus leptodactylus), broad-fingered crayfish (astacus fluviatilis), and california spiny lobster (panulirus interruptus). about 68 of the unigenes have best hits and annotations against available databases, and the other 961 unigenes that have no hits may be undiscovered or unknown function genes in the database. a number of genes had identity scores of a given e value (1.00e 10). from the e-value distribution profile of all the annotated unigenes shown in fig. 1, we could see that about 90 of the annotated unigenes had an e-value 1.00e 10, so the annotation results of the blast searches in three databases are believable. the most highly expressed gene is hemocyanin 2, and its contig contains 213 ests. in arthropods, hemocyanin is a multifunctional protein which can transport oxygen in the hemolymph, shows phenoloxidase activity in its proteolytically cleaved n-terminal, and the c-terminal part sequence serves as antimicrobial peptides (lee et al., 2004). the hemocyanin of horseshoe crab could be directly activated by microbial protease and was enhanced by pathogen associated molecular patterns. the activated hemocyanin can generated highly reactive oxygen intermediates that kills microbes (jiang et al., 2007). therefore, hemocyanin plays important roles in the immune system of arthropods. here we obtained 213 ests of hemocyanin in our cdna library, and nine kinds of hemocyanin can be divided by alignment and phylogenetic analysis. this suggested that the hemocyanin might have function in antiviral immune response. megalin is also a highly expressed gene which contains 152 ests. megalin is a low-density lipoprotein receptor-related protein that is a cell surface endocytic receptor and could bind extracellular ligands for degradation (li et al., 2001). several proteases such as cathepsin l, trypsin, and zinc protease are all highly abundant expressed genes and have more than 100 ests. table 3 analysis of unigenes including contigs and singlets max unigene size：213 *unigene size: the number of ests in a unigene unigene size number of unigenes percent of unigenes(%) 1 2174 71.68 2 358 11.8 3 157 5.18 4–5 116 3.82 6–10 103 3.4 11–20 60 1.98 21–50 40 1.32 51–100 17 0.56 >100 8 0.26 123 genes potentially involved in the defense reactions hemocytes and hepatopancreas are important immune-related cells and organs in arthropods, and 33 differentially expressed genes have been discovered in hemocytes of wssv-challenged crayfish by suppression subtractive hybridization (ssh) and cdna microarrays (zeng et al., 2009). many upregulated genes in the ssh library such as serine protease inhibitor, tubulin, zinc finger protein, synaptosome-associated protein, fatty acid binding protein, superoxide dismutase precursor, arginine kinase, and heat shock protein were also found in our cdna library from hemocytes and hepatopancreas of wssv-challenged red swamp crayfish. among the newly discovered genes of our cdna library, there are many genes potentially involved in the defense reaction of crayfish (table 4). they could be assembled into seven groups such as antimicrobial peptides, pattern recognition receptors, proteases and protease inhibitors, signal transduction proteins, apoptosis-related proteins, antioxidant proteins, and others which could not be classified into groups. antimicrobial peptides in this cdna library, five kinds of antimicrobial peptides have been found, including anti-lipopolysaccharide factor (alf), astacidin, lysozymes, single wap domain-containing protein, and crustins. alfs are potential antimicrobial peptides which could bind to the lipopolysaccharide (lps) from the cell wall of gram-negative bacteria and inhibit the lps-mediated coagulation cascade in arthropod (rosa et al., 2008). alf could interfere with wssv replication in crayfish pacifastacus leniusculus and protect shrimp from wssv infection (liu et al., 2006). alf was upregulated in hemocytes 96 h after wssv challenge in qrt-pcr analysis (fig. 2a), and it may be related to the anti-wssv reaction in crayfish p. clarkii. astacidin 2 is a small proline/arginine-rich antibacterial peptide that shows strong antimicrobial activity against gram-positive and gram-negative bacteria, and has been purified from the hemolymph of freshwater crayfish p. leniusculus (jiravanichpaisal et al., 2007). it was upregulated in hemocytes 96 h after wssv challenge (fig. 2b). lysozymes are well known antimicrobial proteins that show lytic activity to a range of gram-positive and gram-negative bacteria (de-la-re-vega et al., 2006). both the single wap domain-containing protein (swd) and two crustins belong to the wap-containing proteins were also found in the crayfish. swd was upregulated in the wssv-challenged shrimp (amparyup et al., 2008; du et al., 2010). crustins are a large antimicrobial peptide family. they can be divided into three groups according to structural characters (smith et al., 2008). from an antimicrobial experiment in vitro, we could see that crustins mainly showed strong bactericidal activity against gram-positive bacteria and gram-negative bacteria so far (amparyup et al., 2008; smith et al., 2008). also in vivo experiment showed that after crustin was depleted, the mortality increased significantly after shrimp were infected by v. penaeicida (shockey et al., 2009). crustin 2 is a kind of carcinin like protein. crustin 1 and crustin 2 belong to different groups. crustin 1 was upregulated and crustin 2 was downregulated in hemocytes 96 h after challenge of wssv (figs 2c, d). pattern recognition receptors (prrs) in invertebrates, pattern recognition receptors can recognize and bind molecules present on the surface of pathogens such as -1,3-glucans, lipopolysaccharides, lipoteichoic acid, and peptidoglycans, which are called pathogen-associated molecular patterns (janeway, 1989). we discovered several kinds of pattern recognition receptors including c-type lectins, -1,3-glucan binding protein, lipopolysaccharide and -1,3-glucan binding protein, and lysm and putative peptidoglycan binding domain-containing protein. c-type lectins have been reported to participate in the innate defense reaction of nonself recognition by recognizing and binding to the molecules on the surfaces of cell walls and then inducing pathogen phagocytosis and agglutination (sun et al., 2007; ma et al., 2008). reports on chinese shrimp showed that c-type lectin served not only as a pattern recognition receptor but also an effector (sun et al., 2008). c-type lectin from the shrimp litopenaeus vannamei also has activity against wssv (zhao et al., 2009). a c-type lectin was downregulated 96 h after wssv injection, which was the same pattern as a c-type lectin, pmav, from penaeus monodon (leu et al., 2007) (fig. 2e). in crustaceans, -1,3-glucan binding protein (bgbp) and lipopolysaccharide and -1,3-glucan binding protein (lgbp) serve as pattern recognition receptors to recognize cell wall components of bacteria and fungi, such as -1,3-glucans and lipopolysaccharide. bgbp binding to -1,3-glucan induces hemocyte degranulation and subsequently activates the prophenoloxidase (propo) system (lin et al., 2008). lgbp from the crayfish p. leniusculus have been reported to participate in the activation of the propo system (lee et al., 2000). proteases and protease inhibitors the hepatopancreas was reportedly the site of synthesis of digestive enzymes in crustaceans. several protease and protease inhibitors were found in the crayfish. the proteases include astacin, zinc protease, zinc metalloproteinase, cathepsin, cysteine protease, carboxypeptidase, and trypsin. the protease inhibitors include three kinds of serine protease inhibitors such as hemocyte-specific kazal-type protease inhibitor, hepatopancreas kazal-type protease inhibitor, and the putative serine protease inhibitor serpin. astacin is a small zinc protease that can digest fibrillar collagen and other proteins (reyda et al., 1999). it was significantly upregulated by bacterial or lps challenge in oyster (roberts et al., 2009). zinc metalloproteinase is a protease family involved in growth factor activation, polypeptide degradation and extracellular protein processing (bond et al., 1995). trypsin is one of the most important proteases and contributes about 6 % of soluble protein in the 124 table 4 genes potentially included in innate immune response systems gene name species most similar to e-value no. of ests antibacterial peptide pl-crustin 1 pacifastacus leniusculus 2e-37 3 pl-crustin 2 pacifastacus leniusculus 3e-13 2 anti-lipopolysaccharide factor pacifastacus leniusculus 1e-19 3 astacidin 2 pacifastacus leniusculus 4e-20 1 lysozyme precursor crassostrea gigas 2e-06 2 single wap domain-containing protein marsupenaeus japonicus 1e-09 1 pattern recognition receptor c-type lectins fenneropenaeus chinensis 2e-06 72 -1,3-glucan binding protein penaeus monodon 1e-11 5 lipopolysaccharide and -1,3-glucan binding protein pacifastacus leniusculus 7e-56 1 proteases/protease inhibitors astacin astacus fluviatilis 5e-96 1 zinc proteases astacus astacus 4e-25 123 trypsin pacifastacus leniusculus 1e-12 124 cysteine protease homarus americanus 4e-11 60 cathepsin c marsupenaeus japonicus 7e-44 1 cathepsin d apriona germari 8e-34 1 cathepsin l nephrops norvegicus 2e-16 141 carboxypeptidase a1 scophthalmus maximus 4e-06 1 carboxypeptidase b astacus fluviatilis 7e-20 7 carboxypeptidase a5 precursor mus musculus 1e-06 8 serine carboxypeptidase precursor mus musculus 3e-10 3 semigranular hemocyte specific kazal-type protease inhibitor pacifastacus leniusculus 3e-13 1 hepatopancreas kazal-type protease inhibitor penaeus monodon 1e-07 17 zinc metalloproteinase caenorhabditis elegans 2e-07 1 putative serine protease inhibitor serpin pacifastacus leniusculus 4e-21 1 signal transduction proteins ras-related protein rab-1a lymnaea stagnalis 2e-40 3 ras-related protein rab-18 gallus gallus 2e-63 1 ras-related protein rab-5b pongo pygmaeus 2e-49 1 ras-related protein rab-7a rattus norvegicus 3e-34 1 ras-related gtp-binding protein c mus musculus 1e-10 1 ras-like gtp-binding protein rho1 isoform 1 apis mellifera 2e-25 2 125 rho family small gtp binding protein cdc42 nasonia vitripennis 6e-69 1 vacuolar atpase g subunit-like protein maconellicoccus hirsutu 2e-19 1 nucleolar gtp-binding protein 1 homo sapiens 2e-56 1 gtp-binding nuclear protein ran (gtpase ran) xenopus tropicalis 2e-45 3 ran-binding protein 16 mus musculus 7e-51 1 protein kinase n2 tribolium castaneum 2e-38 1 receptor for activated protein kinase c-like protein lepeophtheirus salmonis 9e-73 1 toll-like receptor anopheles gambiae 1e-06 2 relish litopenaeus vannamei 2e-25 1 insulin like growth factor binding protein mus musculus 2e-10 1 casein kinase i isoform alpha xenopus laevis 2e-06 1 mitogen-activated protein-binding protein-interacting protein nasonia vitripennis 5e-29 1 serine/threonine-protein phosphatase pp2a drosophila melanogaster 1e-25 1 arginine kinase homarus gammarus 3e-46 6 serine/threonine-protein kinase n2 homo sapiens 2e-38 1 apoptosis senescence-associated protein pisum sativum 1e-19 2 zinc finger protein xenopus laevis 2e-11 3 antioxidation thioredoxin-like protein maconellicoccus hirsutus 4e-33 1 thioredoxin domain containing 11 isoform 1 apis mellifera 1e-31 1 thioredoxin reductase 1 rattus norvegicus 9e-08 1 superoxide dismutase pontastacus leptodactylus 6e-18 1 glutathione peroxidase 5 mus musculus 1e-09 23 glutathione peroxidase 1 canis familiaris 4e-21 14 ferritin pacifastacus leniusculus 1e-09 11 glutathione s-transferase mu 2 mus musculus 1e-32 6 glutathione-s-transferase-like protein galleria mellonella 4e-40 1 glutathione s-transferase 1-1 (gst class-theta) tribolium castaneum 1e-36 1 glutamine synthetase panulirus argus 1e-42 1 microsomal glutathione s-transferase 3 mus musculus 1e-24 1 putative thiosulfate sulfurtransferase fmp31 saccharomyces cerevisiae 2e-13 1 omega class glutathione s-transferase crassostrea gigas 1e-27 1 farnesoic acid o-methyltransferase marsupenaeus japonicus 4e-08 6 o-methyltransferase fenneropenaeus chinensis 5e-25 1 acyl-coenzyme a oxidase 3 strongylocentrotus purpuratus 9e-07 1e-12 2 126 digestive gland. trypsins as important digestive proteases mainly take part in food digestion, hydrolysis, and activation of zymogens (muhlia-almazan et al., 2008). three kinds of cathepsins have been discovered in the crayfish, including cathepsin c, cathepsin d, and cathepsin l. cathepsin c is a multifunctional protease and is essential for the activation of other enzymes (turk et al., 2001). cathepsin d is an aspartic protease involved in several physiological functions such as protein degradation, apoptosis and autophagy (zaidi et al., 2008). cathepsin l could digest food in the gastrointestinal juice of crustaceans (hu et al., 2007). a digestive cysteine protease was downregulated 96 h after wssv injection (fig. 2f). carboxypeptidases are hydrolases that cleave in the c-terminus of proteins. carboxypeptidases a and b are digestive carboxypeptidases and serve in the degradation of proteins in the digestive tract. serine protease inhibitors of arthropods include the kazal, kunitz, -macroglobulin, and serpin families. they play important roles in prophenoloxidase and cytokine activation, blood coagulation, and pathogen digestion. three kinds of serine protease inhibitors including hemocyte-specific kazal-type protease inhibitor, hepatopancreas kazal-type protease inhibitor, and the putative serine protease inhibitor serpin have been isolated from this library. kazal-type protease inhibitors have been found in the hemocytes of shrimp, and were suggested to be involved in the host defense against wssv challenge (jarasrassamee et al., 2005). serpin from hemocytes cdna library of chinese shrimp has been shown to fluctuate after wssv and bacteria challenges (liu et al., 2009). proteins in signal transduction pathway ras-related protein rab, ran-binding protein, and ras-like gtp-binding protein rho were found in the cdna library; they all belong to the ras-related protein superfamily. the ras family of small gtpases plays roles in a series of cellular processes such as phagocytosis, vesicle transportation, and development. rab gtpase from japanese shrimp participates in the defense response to virus and might function as an intracellular virus recognition protein in virus-infected shrimp (wu et al., 2008). moreover ran from japanese shrimp is involved in antiviral defense immunity and could regulate hemocytic phagocytosis by interacting with myosin (liu et al., 2009). the studies of rho gtpase have mainly focused on phagocytosis and actin dynamics control (pan et al., 2005). toll is a receptor of the toll signal pathway responsible for antifungal and anti-gram-positive bacterial response in drosophila. toll like receptor from invertebrates could not bind pathogens directly, but it was activated by binding to spaetzle (hoffmann, 2003). it was also found in the crayfish cdna library. relish is a downstream nfb transcription factor in the imd signal pathway. the translocation of cleaved relish to the nucleus could activate the signal pathway (hoffmann, 2003). this means that the toll and imd pathways might existed in the crayfish, and the pathways might have functions in antiviral responses. insulin-like growth factor binding protein (igfbp) was also found in the crayfish. it is a member of the others lysm and putative peptidoglycan binding domain containing protein xenopus tropicalis 4e-09 1 megalin danio rerio 1e-16 152 hemocyanin 2 pacifastacus leniusculus 2e-15 213 atp-dependent rna helicase mus musculus 1e-23 6 paz and piwi domain protein/ piwi-like protein paramecium tetraurelia 5e-19 1 cuticle protein 6 blaberus craniifer 2e-11 1 crustacean calcium-binding protein 23 orconectes limosus 2e-46 1 heat shock 70 kda protein blastocladiella emersonii 4e-37 3 heat shock protein 27 drosophila melanogaster 2e-06 1 tgf beta-inducible nuclear protein brugia malayi 8e-07 1 h3 histone family 3a salmo salar 3e-29 1 tar rna binding protein fenneropenaeus chinensis 1e-09 1 chitinase fenneropenaeus chinensis 9e-14 43 127 insulin-like growth factor pathway. the insulin-like growth factor pathway takes part in growth and development, metabolic homeostasis, fecundity and stress resistance, and also lifespan in multicellular organisms (broughton et al., 2009). serine/threonine-protein kinases and phosphatases play roles in signal transduction of anoxia in the crayfish orconectes virilis (cowan et al., 2001). serine/threonine-protein kinase n2 and serine/threonine-protein phosphatase pp2a were isolated from our cdna library. casein kinase1 is a member of the serine/threonine protein kinase family, and it has seven isoforms in mammals and vertebrates. casein kinase1 could enhance receptor interacting protein-mediated nfb activation (wang et al., 2008). the mrna expression level of serine/threonine-protein kinase n2 from this cdna library increased 96 h after wssv injection (fig. 2g), and it may be involved in the antiviral innate immune reaction of crayfish. the transforming growth factor (tgf) signal pathway plays important roles in diverse biological processes. in caenorhabditis elegans, the tgf pathway participates in immune responses (schulenburg et al., 2004). tgf beta-inducible nuclear protein could be induced by stimulation of tgf, so it may be involved in the defense reaction. tgf beta-inducible nuclear protein was upregulated in hemocytes of crayfish after wssv challenge, so it may participate in the anti-virus immune response (fig. 2h). apoptosis-related proteins a senescence-associated protein was also found from this cdna library. the senescence-associated protein takes part in the early embryonic development of silkworm bombyx mori (hong et al., 2006). several zinc finger proteins were also found in the library, just as in the ssh library of hemocytes from wssv-challenged crayfish. one zinc finger protein could bind to viral mrna and prevent its accumulation in the cytoplasm (garcia et al., 2007). thus the zinc finger proteins of our cdna library may be involved in the anti-virus immune defense reaction of crayfish. antioxidant proteins reactive oxygen species, the products of normal aerobic metabolism, can cause oxidative damage to organisms. antioxidant proteins are needed to eliminate the reactive oxygen species and regulate the redox homeostasis. to protect against damage and regulate redox homeostasis, molecules in the glutaredoxin and thioredoxin systems are employed. glutaredoxins and thioredoxins are conserved proteins involved in many cellular processes including repair of oxidatively damaged proteins and protein refolding and regulation (grant, 2001). the thioredoxin system contains thioredoxin and thioredoxin reductase, and thioredoxin is catalyzed directly by thioredoxin reductase and electrons from nicotinamide adenine dinucleotide (nadh) (aispuro-hernandez et al., 2008). by the random sequencing of the cdna library, thioredoxin-like proteins and thioredoxin reductase were isolated from the wssv-challenged crayfish. thioredoxin was characterized in pacific white shrimp (l. vannamei) and the antioxidant activity of the recombinant protein was tested by reducing insulin disulfides using the trolox equivalent antioxidant capacity assay. the results showed that thioredoxin is an important antioxidant (aispuro-hernandez et al., 2008). glutathione peroxidase is a component of the glutaredoxin system. glutathione s-transferases belong to a multigene family that plays important roles in detoxification of xenobiotic compounds (rosa de lima et al., 2002). a selenium-dependent glutathione peroxidase and two glutathione s-transferases have been cloned from chinese shrimp and were involved in detoxification defense reactions (ren et al., 2009). a glutamine synthetase was also found in this library. superoxide dismutase is a kind of antioxidant enzyme. it was also found in the ssh library of wssv-challenged crayfish and upregulated after virus injection (zeng et al., 2009). ferritin is an iron storage protein that participates in iron metabolism and detoxification. ferritins from shrimp have been reported to be involved in the anti-virus defense reaction (zhang et al., 2006). others lysm and putative peptidoglycan binding domain-containing protein (pbp) has not been found in crustaceans so far, so this pbp may be the first one. it may also be involved in the innate immune reaction in crayfish. we found paz and piwi domain protein and atp-dependent rna helicase, which are involved in rna-mediated silencing and the defense response against viruses (cerutti et al., 2006). in humans, the trans-activation response (tar) rna-binding protein plays a role in passing small interfering rna to the rna-induced silencing complex and functions in rna interference(wang et al., 2009a). a tar rna binding protein was found in the cdna library; it was upregulated after wssv challenge in the hemocytes of crayfish (fig. 2i), so it may take part in the antiviral immune reaction. chitinases are members of the glycoside hydrolase family. it has been reported that in the mollusc crassostrea gigas, two chitinases are stimulated in response to lps challenge, which suggested that they are involved in the immune defense reaction of oyster (badariotti et al., 2007). chitinase was upregulated in hepatopancreas of wssv-resistant shrimp (pan et al., 2005). cuticle protein and crustacean calcium-binding protein were also found by an est approach in the wssv-infected shrimp p. mondon. calcium-binding proteins act as cytosolic ca 2 buffers and were significantly downregulated in wssv-injected shrimp (leu et al., 2007). a calcium-binding peptide from the exoskeleton of crayfish functions as a regulator of exoskeleton calcification (inoue et al., 2004). cuticle proteins were upregulated as a result of wssv infection, as the wssv mainly infects the cuticular epidermis in shrimp (leu et al., 2007). heat shock proteins are members of the chaperone family and take part in protein folding. in shrimp, heat shock protein 70 expression was influenced by wssv infection, and the association 128 fig. 2 qrt-pcr analysis of ten genes in total rna extracted from red swamp crayfish hemocytes 96 h after wssv challenge. the qrt-pcr data of the expression level of these genes in response to bacterial and viral challenge were calculated by 2 -∆∆ct . bars stand for the mean±sd. of three independent pcr amplifications and quantifications. the data obtained were subjected to the statistical analysis followed by an unpaired sample t-test. asterisks indicate significant differences (*p<0.05) when comparing to that in the healthy hemocytes (0 h). astacidin (genbank accession no. gq301199); crustin 1 (genbank accession no. gq301201); crustin 2 (genbank accession no. gq301202); anti-lipopolysaccharide factor (alf) hm005306; digestive cysteine proteinase hm005305; h3 histone family protein hm005304;serine/threonine protein kinase(ser) hm005303; tgf beta-inducible nuclear protein hm005302; tar rna binding protein (trbp)hm005301; lectin hm005300. 129 between the major envelope protein vp28 of wssv and heat shock protein 70 is direct and atp-dependent (xu et al., 2009). heat shock protein 70 was upregulated after wssv infection and involved in the anti-virus response in crayfish (zeng et al., 2009). another heat shock protein of this cdna library showed similarity to heat shock protein 27 from drosophila melanogaster, and it had anti-apoptotic activity by inhibition of cytochrome c and tnf-mediated cell death (arya et al., 2007). histones are basic protein components of chromatin. as early as 1942, some reports showed that histone possessed antibacterial properties (miller et al., 1942). recent work demonstrate that the histones and histone-derived fragments have antimicrobial activities in diverse range of organisms from shrimp to human (kawasaki and iwamuro, 2008). the h3 family contains two replacement histone genes, h3 histone family 3a and 3b. here we found a h3 histone family 3a. it was upregulated after wssv infection (fig. 2j). it was reported that histone h2b could mediate anti-virus immune defense 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investigating the effects of human proinflammatory cytokines on phagocytosis in the earthworm eisenia hortensis sl fuller-espie1, l goodfield1, k hill1, k grant2, n derogatis3 1 science department, cabrini college, radnor, pennsylvania, usa 2 college of graduate studies, thomas jefferson university, philadelphia, pennsylvania, usa 3 the children’s hospital of philadelphia, department of pathology and laboratory medicine, philadelphia, pennsylvania, usa accepted september 12, 2008 abstract this study was aimed at determining the influence of human proinflammatory cytokines on innate immune responses in the earthworm eisenia hortensis. preincubation of earthworm coelomocytes in vitro with either interleukin-1 beta (il-1 beta), granulocyte-macrophage colony stimulating factor (gmcsf), interleukin-2 (il-2), or tumor necrosis factor-alpha (tnf-alpha) followed by subsequent bacterial challenge was carried out to investigate whether human proinflammatory cytokines would induce a state of enhanced responsiveness in phagocytic cells derived from the coelomic cavity of e. hortensis. the effect on phagocytosis by large coelomocytes (hyaline amebocytes) was evaluated using flow cytometry where the uptake of escherichia coli expressing green fluorescence protein in the presence or absence of pretreatment with proinflammatory cytokines was measured. our results show that proinflammatory cytokines enhanced phagocytosis to a statistically significant (p ≤ 0.05) degree in 1018 % of earthworms tested for il-1 beta, 20 % for gm-csf, 20-27 % for il-2, and 27-30 % for tnfalpha, depending on the cytokine concentration used. our results favor the suggestion that receptorcoding genes have been conserved through evolution between vertebrates and invertebrates. key words: eisenia hortensis; proinflammatory cytokines; phagocytosis; innate immunity; flow cytometry; hyaline amebocytes introduction invertebrates possessing a coelomic cavity utilize innate immune responses consisting of highly effective cellular and humoral components (roch, 1996; cooper et al., 2002). for example, the coelomic fluid in earthworms consists of lytic and antimicrobial components such as eiseniapore (lange et al., 1997; lange et al., 1999), the cytokine-like cytolytic protein known as coelomic cytolytic factor 1 (ccf-1), and the highly homologous proteins fetidin and lysenin (bruhn et al., 2006; procházková et al., 2006). ccf-1 is functionally analogous with the mammalian cytokine tumor necrosis factor (bilej et al., 1995). when earthworms ___________________________________________________________________________ corresponding author: sheryl l fuller-espie science department cabrini college, 610 king of prussia road radnor, pennsylvania 19087-3698, usa email: sfuller-espie@cabrini.edu were injected intracoelomically with the endotoxin lipopolysaccharide (lps) both humoral and cellular levels of ccf-1 expression increased significantly (bilej et al., 1998), an effect also observed with tnf when mammals were injected with lps (carswell et al., 1975). despite functional analogy between ccf1 and tnf, it is interesting to note the absence of gene homology between the two (beschin et al., 2004). it has been suggested that ccf-1 is representative of a primitive inflammatory cytokinelike factor which participates in humoral and cellular inflammatory responses including the activation of prophenoloxidase activity (bilej et al., 1998; beschin et al., 1999). cytokine-like factors have also been reported in other invertebrates. prendergast et al. (1983), working with the starfish, asterias forbesi, isolated sea star factor (ssf) and demonstrated its ability to function across species by stimulating chemotaxis and macrophage activation in mammals. 124 mailto:sfuller-espie@cabrini.edu 0 10 20 30 40 50 60 fsc-h 0 10 20 30 40 50 60 s s c -h r1 r2 r3 a. 0 10 20 30 40 50 60 fsc-h 0 10 20 30 40 50 60 s s c -h r1 r2 r3 b. fig. 1 typical scatter profile of earthworm coelomocytes using flow cytometry. a two parameter dot plot measuring forward scatter (fsc) versus side scatter (ssc) of a typical earthworm coelomocyte population after extrusion and incubation overnight in sdmem medium is shown. this data shows the results obtained from ew14. three distinct populations are indicated; region 1 (r1) represents the eleocytes, region 2 (r2) represents the large coelomocytes (lc), and region 3 (r3) represents the small coelomocytes (sc). dot plots are shown without (a) and with (b) contour marks to emphasize densities of subpopulations. the fact that earthworms possess cytokine-like factors such as ccf-1 has prompted much attention on the biochemical activities of these cytokine-like factors, their evolution and conservation, and crossspecies activity using mammalian-derived cytokines. the studies of renzelli-cain et al. (1995) demostrated that mammalian interleukin-1 alpha (il-1 alpha) enhanced phagocytic activity of opioid-treated amebocytes of the earthworm lumbricus terrestris, and also influenced their aggregation and conformation. interleukin-8 (il-8) has been shown to mediate alterations in cell shape and increase chemotaxis and phagocytic activity in the mussel mytilus galloprovincialis (ottaviani et al., 2000). in the freshwater snails planorbarius corneus and viviparus ater, preincubation with il-1 and tnf-alpha caused a decrease in the release of biogenic amines when subsequently exposed to corticotrophin-releasing factor, a proto-type stress response inducer (ottaviani et al., 1995). in addition, the natural killer (nk)-like activity of p. corneus hemocytes was preserved in vitro when incubated with interleukin-2 (il-2) (franceschi et al., 1991). franchini et al. (2000; 2006) have also demonstrated that the giant leopard slug limax maximus had accelerated tissue repair when platelet-derived growth factor-ab (pdgf-ab) and transforming growth factor-beta (tgf-beta) were applied to wounds. investigation of the cellular immune responses of earthworms has resulted in the categorization of coelomocytes (leukocytes) which reside in the coelomic cavity into three major subpopulations, hyaline amebocytes (large coelomocytes), granular amebocytes (small coelomocytes), and chloragocytes (eleocytes). the coelomocytes are easily harvested from experimentally-induced earthworms following extrusion of coelomic fluid from the dorsal pores of the body wall, and can be manipulated in vitro to study their immune functions. the large coelomocytes (lc) are the major phagocytic cells, the small coelomocytes (sc) constitute the population exhibiting nk-like activity, and the eleocytes contain chloragosomes and do not participate in either phagocytic or nk-like activities (cooper, 1996; cossarizza et al., 1996; adamowicz et al., 2001; engelmann et al., 2002; engelmann et al., 2005). owing to the physical differences between coelomocyes including their granularity and size, flow cytometry permits lc, sc and eleocytes to be distinguished based on forward light scatter (fsc) and side light scatter (ssc) properties (cossarizza et al., 1996; engelmann et al., 2004; cossarizza et al., 2005; patel et al., 2007). in addition, flow cytometry can be used to selectively analyze immune functions of the subpopulations by drawing regions around and gating only those cells desired for investigative purposes. although light microscopy has been used by researchers to study phagocytosis in earthworms (adamowicz et al., 2001; kalaç et al., 2002), this approach lends itself to subjectivity, and places restrictions on the number of cells that can be included in quantitative assays based on time constraints. flow cytometry, in contrast, is an objective quantitative methodology which is capable of analyzing several thousands of cells per second, and limiting analysis to predetermined subpopulations. the selected examples cited above illustrate that mammalian cytokines can trigger immune responses in invertebrate models. we sought to investigate the effects of four proinflammatory cytokines, namely il-1 beta, gm-csf, il-2 and tnf-alpha, on the phagocytic activity of 125 coelomocytes isolated from the earthworm eisenia hortensis. our study used flow cytometry to follow the uptake of escherichia coli expressing green fluorescent protein after gating on coelomocytes that satisfied the fsc and ssc parameters characteristic of lc. our results show that preincubation of lc with the proinflammatory cytokines il-1 beta, gm-csf, il-2 and tnf-alpha induced a stimulatory effect on in vitro phagocytosis in 10-30 % of the earthworms tested in this study. materials and methods reagents general laboratory reagents and plasticware were purchased from fisher scientific unless otherwise noted. cell culture all cell culture reagents and phosphate-buffered saline (pbs) were purchased from invitrogen unless otherwise noted. dulbecco’s modified eagle medium (dmem) was supplemented with 10 % fetal calf serum, 100 μg/ml ampicillin (shelton scientific), 10 μg/ml kanamycin (shelton scientific), 10 μg/ml tetracycline, 5 μg/ml chloramphenicol (fluka biochemika), 1x penicillin/streptomycin/amphotericin b, 1x nonessential amino acids and 1x l-glutamine (gibco) to comprise super dmem (sdmem). earthworm husbandry eisenia hortensis (european nightcrawlers) were purchased from vermitechnology unlimited, orange lake, florida, usa, who import e. hortensis from star food, holland, scherpenzeelseweg 95, 3772me barneveld, the netherlands. species identity was determined by the united states department of agriculture, usda permit #52262 (vermitechnology, personal communication). shortterm colonies were maintained at room temperature (rt) in the dark on autoclaved shredded paper moistened with water and single grain rice cereal or rice with bananas cereal (gerber) until use. shredded paper was changed twice weekly. extrusion of coelomocytes prior to experimentation, earthworms were chosen based on their color and activity; earthworms with healthy deep coloration, lacking yellow appearance, and with high activity were placed overnight on moist paper towels saturated with 2.5 μg/ml fungizone (fisher scientific) to reduce the level of fecal material and other surface contaminants. to collect coelomocytes from an earthworm, earthworms were extruded according to engelmann et al. (2004) with minor modifications. briefly, earthworms were placed in a 100 mm petri dish containing 3 ml ice cold extrusion buffer (71.2 mm nacl, 5 % v/v ethanol, 50.4 mm guaiacol-glycerylether, 5 mm egta, ph 7.3). the coelomocytes were then transferred to 1 ml accumax (innovative cell technology) for a 5 min incubation period at rt. then the cells were washed with 5 ml lubricus balanced salt solution (lbss, 71.5 mm nacl, 0.3 mm nah2po4, 4.2 mm nahco3,4.8 mm kcl, 0.4 mm kh2po4, 1.1 mm mgso4 x 7 h2o, ph 7.3), prior to centrifugation at 150xg for 5 min at 4 oc. coelomocytes were resuspended in 1 ml sdmem and enumerated using a hemocytometer. only large coelomocytes (lc) and small coelomocytes (sc) were included in the cell count; eleocytes were not counted but did factor into a quality score. samples with large numbers of eleocytes compared to lc and sc were not used in phagocytosis assays. cytokines all cytokines were reconstituted and stored according to vendors’ recommendations. recombinant human il-1 beta was purchased from r & d systems (201-lb). recombinant human gm-csf, recombinant human il-2, and recombinant human tnf-alpha were purchased from prospec-tany technogene (cyt-221a, cyt-209 and cyt-223, respectively). in all cases purity was greater than 97 % and endotoxin levels were less than 1.0 eu/μg. il-1 beta was used at 20 ng/ml and 40 ng/ml; gmcsf was used at 2 ng/ml and 4 ng/ml; il-2 was used at 12.5 ng/ml and 25 ng/ml; and tnf-alpha was used at 2.5 ng/ml and 5 ng/ml. bacteria escherichia coli hb101 transformed with pglo (biorad) and expressing green fluorescent protein (gfp) were grown on tryptic soy agar containing 100 μg/ml ampicillin and 0.2 % arabinose at 32 °c for 24 h. cells were chemically fixed using 4 % paraformaldehyde for 1 h at rt, and then washed three times with pbs. centrifugation was carried out at 3273xg for 5 min at 4 oc, cells were resuspended in pbs, enumerated using a hemocytometer, and stored in the dark at 4 oc until used. these cells hereafter are referred to as e. coli-gfp. phagocytosis assay phagocytosis assays were carried out in sdmem. coelomocytes [20,000 per well for earthworm (ew) 1 and ew2; 50,000 per well for all others] were pretreated with individual cytokines at concentrations indicated above for 20 h, 5 % co2, at 20 oc in 96-well, v-bottom plates in 100 μl. depending on coelomocyte yield and scope of each experiment, the number of replicates differed between assays affecting the degrees of freedom in the statistical analyses. experiments were set up in duplicates for ew5 and ew15, in triplicates for ew1, ew4, ew6, ew8-14, and ew16, and quadruplicates for ew2, ew3, and ew5. the co2 incubator was placed in a 4 oc walkin cold room in order to obtain these conditions. following cytokine pretreatment, e. coli-gfp was added to each well at a multiplicity of infection of 1000 bacteria: 1 coelomocyte in 200 μl final volume per well. to control for non-specific binding of e. coli to the external surface of coelomocytes, 5 μm cytochalasin b was added to control wells 30 min before the addition of e.coli-gfp. incubation times for e. coli-gfp uptake ranged from 1-4 h at 30 oc. following e. coli-gfp uptake, trypan blue was used at a final concentration of 0.02 % for 30 min at room temperature in the dark, for quenching purposes to reduce background fluorescence (mosiman et al.,1997). the cells were centrifuged at 150xg for 5 min at 4 oc, washed once with pbs, and then resuspended in facs flow buffer (bd bioscience) for flow cytometry analysis. 126 cytokine (concentration) number of earthworms treated with cytokine number of responding earthworms (p ≤ 0.05) (ew identity number) statistically significant response rate to cytokine (p ≤ 0.05) il-1 beta (40ng/ml) 28 5 (ew 4,5,8,12,15) 18 % il-1 beta (20ng/ml) 20 2 (ew 3,5) 10 % gm-csf (4ng/ml) 10 2 (ew 4,5) 20 % gm-csf (2ng/ml) 50 10 (ew 1,2,5,6,8,9,10,11,14,16) 20 % il-2 (25ng/ml) 22 6 (ew 4,5,9,11,13,16) 27 % il-2 (12.5ng/ml) 10 2 (ew 4,5) 20 % tnf-alpha (5ng/ml) 22 6 (ew 4,5,9,11,13,14) 27 % tnf-alpha (2.5ng/ml) 10 3 (ew 5,6,7) 30 % table 1 summary of total number of earthworms used in this study and percent responders. the four cytokines and the concentrations used are indicated. the total number of earthworms pretreated with the cytokines, the number responding with p ≤ 0.05 to each cytokine, the identity of the earthworm responders, and the percentage of statistically significant responses are shown. flow cytometry fluorescence was measured using a facscalibur flow cytometer and cell quest software (bd biosciences) with the following specifications for all experiments described in this study: forward light scatter (fsc) e00-linear, side light scatter (ssc) 332v-linear, green fluorescent protein fluorescence (fl-1) 312v-log. listmode data was acquired with cell quest software, and analysis of data was carried out using winlist 5.0 (verity software house, inc.). only coelomocytes with the appropriate granularity and size corresponding to the large coelomocyte population were gated for analysis in two-dimensional dot plots of fsc vs fl-1. percent specific phagocytosis was determined by subtracting the autofluorescent background of the controls (coelomocytes incubated with cytochalasin b and e. coli-gfp) from the sample fluorescence measured by the fl-1detector. all histograms and dot plots were created using winlist 5.0. see figs 1-2 for representation of data acquisition and analysis. statistical analysis all graphs and data analysis were created and processed using microsoft excel 2007. only statistically relevant results with p ≤ 0.05 (as defined by student’s t-test) comparing untreated versus cytokine-treated samples are shown in figs 3-6. results enhanced phagocytosis was detected when coelomocytes were preincubated with the proinflammatory cytokines il-1 beta, gm-csf, il-2 and tnf-alpha fig. 1 illustrates a typical profile of earthworm coelomocytes obtained when measuring forward light 127 0 10 20 30 40 50 60 fsc-h 10 1 10 2 10 3 10 4 f l1 -h 4 5 6 7 r4 a. lr 96.57;ur3.43 0 10 20 30 40 50 60 fsc-h 10 1 10 2 10 3 10 4 f l1 -h 4 5 6 7 r4 b. lr 92.77;ur 7.23 0 10 20 30 40 50 60 fsc-h 10 1 10 2 10 3 10 4 f l1 -h 4 5 6 7 r4 c. lr 55.29;ur 44.71 0 10 20 30 40 50 60 fsc-h 10 1 10 2 10 3 10 4 f l1 -h 4 5 6 7 r4 d. lr 37.72;ur 62.28 fig. 2 characteristic profile of large coelomocytes (lc) before and after phagocytosis and proinflammatory cytokine treatment. two dimensional dot plots measuring forward scatter (fsc) on the x-axis versus green fluorescence (fl-1) on the y-axis were generated after gating on the r2 subpopulation (lc) as indicated in fig. 1. this data shows the results obtained for ew11. lr = % gated lc in lower right quadrant; ur = % gated lc in upper right quadrant. a. lc illustrating the level of autofluorescent background. b. lc in the presence of e. coligfp plus cytochalasin b, an antibiotic which interferes with microfilament activity and inhibits phagocytosis. c. lc incubated with e. coli-gfp. d. lc preincubated with il-2 (25 ng/ml) prior to the addition of e. coli-gfp (p = 0.016). e. coli-gfp control was not detected using the fsc, ssc and fl-1 settings employed (data not shown). scatter (fsc) versus side light scatter (ssc) properties. coelomocytes from ew14 are depicted. three characteristic populations were routinely observed and three regions, r1, r2, and r3, were drawn around the subpopulations corresponding to the eleocytes, large coelomocytes (lc) and small coelomocytes (sc), respectively. a dot plot represents the total population acquired (fig. 1a) while a contour plot shows the relative densities of each of the three subpopulations (fig. 1b). percent specific phagocytosis was determined for each earthworm treated with il-1 beta, gm-csf, il-2 and tnf-alpha. fig. 2 represents a typical profile of responding earthworm coelomocytes measuring forward scatter (fsc) versus gfp fluorescence (fl-1) before and after treatment with il-2 (25 ng/ml) (p = 0.016). coelomocytes from ew11 are depicted. fsc versus fl-1 dot plots were gated on the r2 subpopulation (lc) as illustrated in fig. 1. each dot plot was divided into four quadrants, upper left (ul) (4), upper right (ur) (5), lower left (ll) (6), and lower right (lr) (7). since analyses included r2-gated cells, events fall only in the ur and the lr quadrants. the lr quadrant corresponded to coelomocytes which did not phagocytose e. coli-gfp (i.e. nonfluorescent), and the ur corresponded to those coelomocytes which did, exhibited by the higher relative fluorescence intensity (fl-1) compared to controls. fig. 2a shows the large coelomocytes of ew11 incubated in the absence of e. coli-gfp illustrating the level of background autofluorescence observed. nonfluorescent cells comprised 96.57 % of the total gated lc population (lr), while autofluorescent cells comprised 3.43 % (ur). the lc had relatively low levels of autofluorescence in all samples analyzed, while the eleocytes had significantly higher levels, but the eleocytes were excluded from the analysis. fig. 2b shows the lc of ew11 incubated with e. coli-gfp together with cytochalasin b, an antibiotic which interferes with microfilament activity and thereby inhibits phagocytosis (axline et al., 1974). this control was important to exclude the possibility of nonspecific 128 earthworm il-1 β 40ng/ml il-1β 20ng/ml gm-csf 4ng/ml gm-csf 2ng/ml il-2 25ng/ml il-2 12.5ng/ml tnf-α 5ng/ml tnf-α 2.5ng/ml ew1 t t/r ew2 t t/r ew3 t/r t ew4 t/r t t/r t t/r t/r t/r t ew5 t/r t/r t/r t/r t/r t/r t/r t/r ew6 t t t t/r t t t t/r ew7 t t t t t t t t/r ew8 t/r t/r t t ew9 t t/r t/r t/r ew10 t/r ew11 t t/r t/r t/r ew12 t/r t t t ew13 t t t/r t/r ew14 t t/r t t/r ew15 t/r t t t ew16 t t/r t/r t table 2 summary of earthworms used in this study exhibiting a statistically significant response to at least one of the cytokines used in pretreatment. only worms exhibiting statistically significant responses to at least one of the cytokine treatments are shown. for each earthworm shown, the cytokines used are indicated. t = earthworm treated with cytokine at indicated concentration; t/r = earthworm treated with cytokine at indicated concentration and exhibiting a statistically significant response (p ≤ 0.05). binding of e. coli-gfp to the cell surface of the large coelomocytes and provided background values which were subtracted from the experimental values to obtain percent specific phagocytosis. in this example nonfluorescent cells comprised 92.77 % of the population (lr), while fluorescent cells made up 7.23 % (ur). percent specific phagocytosis was calculated by first subtracting the fluorescent background of the averaged cytochalasin b control samples (i.e., 7.23 %) from the fluorescence observed 129 fig. 3 effects of il-1 beta on phagocytosis in eisenia hortensis large coelomocytes. percent specific phagocytosis is shown for large coelomocytes incubated with e. coli-gfp in the absence (white) or presence of cytokine at (a) 20 ng/ml (black) or (b) 40 ng/ml (gray). percent specific phagocytosis represents cells exhibiting fluorescence above background autofluorescence levels. error bars represent ± sd. only earthworms with p ≤ 0.05 are shown. with e. coli-gfp only (fig. 2c, ur quadrant), or treated with e. coli-gfp plus cytokine (fig. 2d, ur quadrant). the % specific phagocytosis in this example for e. coli-gfp is 44.71 % 7.23 % = 37.48 %, and the % specific phagocytosis for e. coli-gfp plus il-2 is 62.28 % 7.23 % = 55.05 %. then statistical analyses were performed to determine if the differences between the averaged untreated and cytokine-treated samples were statistically significant. using these criteria analyses were carried out for assays investigating the effects of il1 beta, gm-csf, il-2 and tnf-alpha. this study analyzed the response of the coelomocytes extruded from 50 earthworms to il-1 beta, gm-csf, il-2 and tnf-alpha in 9 separate assays. cytokines were used individually in all cases, and not in combination. coelomocytes from 28 of these earthworms were treated with all four of these cytokines at one or both of the concentrations chosen for the assays. coelomocytes from the remaining earthworms were treated with gm-csf only, or gm-csf and il-1 beta. table 1 illustrates the number of earthworms tested for each cytokine and the corresponding concentrations. sixteen of the 50 earthworms exhibited a statistically significant (p ≤ 0.05) response to at least one of the four cytokines used and only those earthworms were included in our final analyses. table 2 lists the 16 responding earthworms, indicates which cytokines were used and the concentrations employed, and whether or not enhancement of phagocytosis was statistically significant. a figs 3-6 show the results for only those earthworms which exhibited statistically significant responses (p ≤ 0.05) to the cytokines employed. results for earthworms with p > 0.05 are not shown. when il-1 beta was used at 20 ng/ml, 2 out of 20 (10 %) responded significantly ( fig. 3a). at the higher concentration of 40 ng/ml, 5 out of 28 (18 %) responded significantly (fig. 3b). gm-csf used at 2 ng/ml induced a 20 % response rate with 10 out of 50 earthworms responding significantly (fig. 4a). at 4 ng/ml, 2 out of 10 (20 %) responded significantly. (fig. 4b). il-2 gave similar results with 2 out of 10 (20 %) responding at 12.5 ng/ml (fig. 5a), and 6 out of 22 (27 %) responding at 25 ng/ml (fig. 5b). higher response rates were observed with tnfalpha; 3 out of 10 (30 %) responded to 2.5 ng/ml (fig. 6a), and 6 out of 22 (27 %) responded to 5 ng/ml (fig. 6b). in summary, these results show an overall increase in phagocytosis in 10 30 % of earthworms subjected to proinflammatory cytokine treatment compared to untreated controls. b a b fig. 4 effects of gm-csf on phagocytosis in eisenia hortensis large coelomocytes. percent specific phagocytosis is shown for large coelomocytes incubated with e. coli-gfp in the absence (white) or presence of cytokine at (a) 2 ng/ml (black) or (b) 4 ng/ml (gray). percent specific phagocytosis represents cells exhibiting fluorescence above background autofluorescence levels. error bars represent ± sd. only earthworms with p ≤ 0.05 are shown. 130 fig. 5 effects of il-2 on phagocytosis in eisenia hortensis large coelomocytes. percent specific phagocytosis is shown for large coelomocytes incubated with e. coli-gfp in the absence (white) or presence of cytokine at (a) 12.5 ng/ml (black) or (b) 25 ng/ml (gray). percent specific phagocytosis represents cells exhibiting fluorescence above background autofluorescence levels. error bars represent ± sd. only earthworms with p ≤ 0.05 are shown. discussion innate immune cells utilize pattern recognition receptors (prrs) to recognize chemical entities that are common to different classes of pathogens. prrs bind to pathogen-associated molecular patterns (pamps) and initiate cell-signaling pathways of innate immunity. one type of prr is the evolutionarily conserved toll/toll-like receptor, which has the ability to recognize a broad spectrum of ligands. the toll receptor was originally discovered in drosophila during an investigation of dorsoventral patterning in embryonic development (reviewed in janssens et al., 2003). homologues of insect toll receptors have been identified in mammals and other animals and are known as tolllike receptors (tlrs). two examples of tlr signaling pathways are: 1) tlr2, which binds to peptidoglycan of both gram-positive and gramnegative bacteria, lipoteichoic acid of gram-positive bacteria, and zymosan of yeast; and 2) tlr4, which binds to lps of gram-negative bacteria, and certain viruses (reviewed in leulier et al., 2008). it has been shown that toll receptor and tlr signaling induces phagocytosis and the synthesis of anti-microbial compounds in vertebrates, insects, and the invertebrate caenorhabditis elegans, but it is not yet known if they are needed in order for these mechanisms to occur in earthworms (reviewed in cooper et al., 2006). a our results show that the proinflammatory cytokines, il-1 beta, gm-csf, il-2, and tnf-alpha significantly increased the levels of phagocytosis of lc in 10-18 % of earthworms tested for il-1 beta, 20 % for gm-csf, 20 27 % for il-2, and 27 30 % for tnf-alpha, depending on the cytokine concentration used in the assay. it is interesting to note that of the 28 earthworms that were treated with all four cytokines, 12 (43 %) responded significantly to at least one of the cytokines and in some cases to all four cytokines. specifically, 3 responded to only one cytokine (ew7 to tnf-alpha; ew12 and ew15 to il-1 beta), 5 responded to two cytokines (ew6 and ew14 to gm-csf and tnfalpha; ew8 to il-1 beta and gm-csf; ew13 to il-2 and tnf-alpha; and ew16 to gm-csf and il-2), 2 responded the three cytokines (ew9 and ew11 to gm-csf, il-2 and tnf-alpha), and 2 responded to all four cytokines (ew4 and ew5) (table 2). the overall responses to the cytokines used in our study reveal that the most efficient response was to tnfalpha. b possible reasons for the variation in sensitivity to il-1 beta, gm-csf, il-2 and tnf-alpha could be related to different batches of earthworms. our studies were carried out over a 7 month period and a b fig. 6 effects of tnf-alpha on phagocytosis in eisenia hortensis large coelomocytes. percent specific phagocytosis is shown for large coelomocytes incubated with e. coli-gfp in the absence (white) or presence of cytokine at (a) 2.5 ng/ml (black) or (b) 5 ng/ml (gray). percent specific phagocytosis represents cells exhibiting fluorescence above background autofluorescence levels. error bars represent ± sd. only earthworms with p ≤ 0.05 are shown. 131 used different batches of earthworms. seasonal variations may have also had an effect. in addition, the earthworm colony is an outbred population and genetic polymorphisms could contribute to cytokine sensitivities. it is also possible that the cytokine concentrations that were used in our studies were not in the appropriate range needed to stimulate immune responses in some of the earthworms. in addition, the fact that the cell populations were heterogeneous and included eleocytes, hyaline amebocytes and granular amebocytes, could explain variations due to different proportions of the three cellular subsets in each extruded sample. for example, a higher or lower number of eleocytes, known to contain riboflavin (plytycz et al., 2006) could influence the sensitivity of the amebocytes to the different cytokines used. methodologies are needed to improve subset purification, while preserving cell yield, to enable these types of studies to be conducted with hyaline amebocytes in isolation. perhaps the enhancement of phagocytosis observed in our experiments is due to the binding of human cytokines to primitive earthworm receptors. this may then stimulate the upregulation of prrs resulting in higher levels of bacteria being phagocytozed due to elevated levels of ligand receptors. kurt-jones et al. (2002) showed that gmcsf induced upregulated expression of tlr2 and cd14 on the surface of human neutrophils and also increased il-8 secretion and enhanced superoxide responses when gm-csf-primed neutrophils were stimulated with tlr2 ligands. further studies need to be conducted to determine whether a similar stimulatory pathway is induced by gm-csf in our model. selective pressures operating on the different evolutionary lines of descent include the pamps that make up the common pathogenic burden shared by both lineages. mandrioli et al. (2007), using a bioinformatic approach, suggested that invertebrate cytokine-like ancestral receptors may be promiscuous, and able to bind to different cytokines. their results favor the model which supports the conservation of receptor-coding, rather than ligand-coding genes between vertebrates and invertebrates, and that present-day receptors are more similar to their ancestor counterparts than the ligands for those receptors. gene duplications in vertebrates would permit receptor differentiation tailored for different ligands, with eventual specialization of a receptor towards a single ligand. the lack of gene duplication in invertebrates, however, would restrict receptor evolution, and favor the interaction of many ligands with a limited number of generalized receptors. this rationale would not only explain why molluscan immunocytes can bind to mammalian cytokines il-1alpha, il-1 beta, il-2, tnf-alpha and tnf-beta (reviewed in mandrioli et al., 2007), but would also explain the findings reported in this study for earthworm coelomocytes. it is interesting to note that although not yet reported in e. hortensis, the existence of a gene encoding a putative helical cytokine in drosophila melanogaster has been reported (malagoli et al., 2007) and shown to be upregulated following immune stimulation. these findings support the proposal that helical cytokines play a role in immune responses of invertebrates. perhaps similar genes will be identified in earthworms and other invertebrates. tnf-alpha, il-1 and tlr ligands have in common the ability to bind to cell surface receptors which share a conserved intracellular signaling motif. once engaged, the signal transduced culminates in the activation and nuclear translocation of nuclear factor-kappa b (nf-kb) and the subsequent upregulation of genes encoding tlrs, tnf-alpha, and il-1 (o’neill et al., 2000; muzio et al., 2000; haynes et al., 2001; hongxiu et al., 2006). in contrast to the nf-kb signaling pathway initiated by tnf-alpha and il-1 beta, gm-csf and il-2 stimulate the jak-stat pathway, which relies on janus kinases (jak) and signal transducers and activators of transcription (stat). interestingly, the jak-stat pathway components are molecularly and functionally conserved from the invertebrate drosophila to humans to a high degree (arbouzova et al., 2006). the signaling pathways operating in earthworms require elucidation to establish whether these conserved signal transduction mediators and adaptors are also involved in the signal transduction of innate immune responses in annelids. although not presented in this paper, we tested the effects of gm-csf in combination with il-1 beta on phagocytosis and preliminary evidence suggests a synergistic effect as seen by enhanced phagocytosis. these results need to include a larger number of respondents to verify synergistic effects of proinflammatory cytokines on phagocytosis. it would also be worthwhile to employ a wider variety of cytokines and different combinations. studies in our lab were also carried out to investigate whether il-1 beta, gm-csf, il-2 or tnf-alpha would have an effect on cell proliferation in vitro. the coelomocytes were incubated in vitro with cytokine for 48 h in order to provide sufficient time for any effects to be observed. cell proliferation was determined by measuring dna content using propidium iodide and flow cytometry. in contrast to the results obtained with phagocytosis, there was little to no effect on cell proliferation when coelomocytes were treated with the proinflammatory cytokines; only 4.1 % of cases exhibited increased proliferation in response to tnf-alpha, and no increased proliferation was observed in response to il-1 beta, il-2, or gm-csf (data not shown). therefore, our prelilminary results suggest that these proinflammatory cytokines do not enhance cell proliferation significantly in e. hortensis coelomocytes under the conditions used in this study. problems with fungal and bacterial contamination, despite the inclusion of a large number of antimicrobials in the culture medium, restricted our incubation period to a maximum of 48 h, however, longer incubation periods or increased temperature may be necessary to observe proliferative effects in vitro. enriching the different coelomocyte subpopulations may also lead to conditions warranting longer incubation periods needed to reveal proliferative responses to these cytokines. our lab also carried out preliminary experiments to study the in vitro effects of il-1 beta 132 and gm-csf on nk-like responses of small coelomocytes in e. hortensis using the nonradioactive flow cytometric procedure described by patel et al. (2007). interestingly the results obtained in these early experiments proved to be inhibitory, not stimulatory, to nk-activity with 3 out of 12 earthworms exhibiting statistically significant inhibition when pretreated with il-1beta, and 1 out of 12 exhibiting statistically significant inhibition when pretreated with gm-csf (data not shown). perhaps this difference can be attributed to the observation that nk-like activity in earthworms is mediated by sc, not lc which carry out phagocytosis (engelmann et al., 2004; salzet et al., 2006). perhaps these two different coelomocyte populations respond differently to proinflammatory cytokines through the use of distinct signal transduction pathways. it would be interesting to determine whether the signaling pathways associated with phagocytosis and proliferation in earthworms share signal transduction components which can be induced with similar cytokine or cytokine-like molecules. it would be worthwhile and of great importance to carry out a microarray analysis, or generate a cdna library to examine at the molecular level the changes occurring in earthworm coelomocytes in response to infection and exposure to human proinflammatory cytokines. for example, when highdensity oligonucleotide microarrays were generated in drosophila following microbial infection, 230 genes were induced and 170 were repressed, most of which had never before been associated with immune response, and many of the genes uncovered had unknown function (degregorio et al., 2001), providing new leads for innate immune activities in invertebrates. there is clearly a need to understand the signaling pathways operating in earthworms and microarrays would provide an invaluable tool to decipher the humoral, cellular and molecular interactions regulating innate immunity defenses in annelids. acknowledgements this work was supported by grants from the cabrini college faculty development grants committee (sf-e), and the pennsylvania academy of science (lg). references adamowicz a, wojtaszek j. morphology and phagocytotic activity of coelomocytes in dendrobaena veneta (lumbricidae). zoologica poloniae 46: 91-104, 2001. arbouzova ni, zeidler mp. jak/stat signaling in drosophila: insights into conserved regulatory and cellular functions. development 133: 26052616, 2006. axline sg, reaven ep. inhibition of phagocytosis and plasma membrane mobility of the cultivated macrophage by cytochalasin b. role of subplasmalemmal microfilaments. j. cell biol. 62: 647-659, 1974. bilej m, brys l, beschin a, lucas r, vercauteren e, hanušová r, et al. identification of a cytolytic protein in the coelomic fluid of eisenia foetida earthworms. immunol. lett. 45: 123-128, 1995. bilej m, rossmann p, šinkora m, hanušová r, beschin a, raes g, et al. cellular expression of the cytolytic factor in earthworms eisenia foetida. immunol. lett. 60: 23-29, 1998. beschin a, bilej m, brys l, torreele e, lucas r, magez s, et al. convergent evolution of cytokines. nature 400: 627-628, 1999. beschin a, bilej j, magez s, lucas r, debaetslier p. functional convergence of invertebrate and vertebrate cytokine-like molecules based on a similar lectin-like activity. prog. mol. subcell. biol. 34: 145-163, 2004. bruhn h, winkelmann j, andersen c, andrä j, leippe m. dissection of the mechanisms of cytolytic and antibacterial activity of lysenin, a defence protein of the annelid eisenia fetida. dev. comp. immunol. 30: 597-606, 2006. carswell ea, old lj, kassel rl, green s, fiore n, williamson b. an endotoxin-induced serum factor that causes necrosis of tumors. proc. natl. acad. sci. usa 72: 3666-3670, 1975. cooper el. earthworm immunity. in: rinkevich b, műller weg (eds), invertebrate immunology, springer verlag, heidelberg, pp 10-45, 1996. cooper el, kauschke e, cossarizza a. digging for innate immunity since darwin and metchnikoff. bioessays 24: 319-333, 2002. cooper el, dvell k, engelmann p, nemeth p. still waiting for the toll? 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tokai university, hiratsuka, kanagawa 259-1292, japan accepted september 2, 2010 abstract recently we biochemically determined the molecular recognition and regulatory mechanisms of how beetle’s larvae recognize gram-positive bacteria and fungi via toll signaling cascade. the biochemical analysis of newly identified molecules provides us how beetles recognize invading pathogenic microbes and how they defend their bodies using elegant innate immunity. here, we will focus on reviewing the biochemical analyses and biological functions of newly identified molecules involved in insect toll signaling cascade. key words: toll cascade; innate immunity; protease inhibitor; insects introduction innate immunity is an evolutionarily conserved first-line host defense that senses pathogenic microorganisms through “pattern-recognition” molecules that recognize the conserved molecular patterns on the surface of microbes (medzhitov and janeway, 1997). invertebrate’s innate immune response is a crucial host defense system to defend against microbial infection (hoffmann et al., 1999). the ability of a host to distinguish between self and non-self remains a central hallmark of innate immunity. the pathogenic microbes possess distinct pathogen-associated molecular patterns (pamps), such as peptidoglycans (pgs) of gram-positive bacteria and β-1,3-glucans of fungi. the recognition of pamps is achieved by a group of germ line-encoded receptors and soluble proteins (hoffmann et al., 1999). ___________________________________________________________________________ corresponding author: lee bok luel the national research laboratory of defense proteins, college of pharmacy, pusan national university, jangjeon dong, gumjeong gu, busan, 609-735, korea e-mail: brlee@pusan.ac.kr list of abbreviation: amp, antimicrobial peptides; sp, serine proteinase; pg, peptidoglycan; pgrp, peptidoglycan recognition protein; grp, β-1,3-glucan recognition protein; gnbp, gram-negative binding protein; msp, modular sp; modsp, modular sp; spe, spätzleprocessing enzyme; sae, spe activating enzyme; hp, hemolymph protease. in drosophila, the synthesis of antimicrobial peptides (amps) in response to microbial infections is under the control of the toll and immune deficiency (imd) signaling pathway (lemaitre and hoffmann, 2007). the toll signaling pathway responds mainly to the lysine (lys)-type pg of gram-positive bacteria and fungal β-1,3-glucan, whereas the imd pathway responds to mesodiaminopimelic acid (dap)-type pg of gramnegative bacteria and certain gram-positive bacilli. the biological significance of these two drosophila signaling pathways is demonstrated by the fact that mutations of the genes involved in these pathways dramatically decrease resistance to microbial infections, e.g., toll mutants are susceptible to fungal infections and relish, the nf-κb protein involved in imd pathway, mutants lose resistance to gram-negative bacterial infections ( lemaitre et al., 1996; hedengren et al., 1999). toll signaling cascade is amplified in hemolymph (insect blood) by a proteolytic serine protease (sp) cascade. the amplification of toll recognition signals results in an efficient host defense strategy in insects, which are devoid of an acquired immune system. drosophila genetic studies showed that lystype pg of gram-positive bacteria is recognized by two different protein complex consisted of the pg recognition protein-sa (pgrp-sa) and the gramnegative binding protein 1 (gnbp1) (michel et al., 2001; gobert et al., 2003; pili-floury et al., 2004). in contrast, fungal β-1.3-glucan is known to be recognized by gnbp3 (gottar et al., 2006). these lys-type pg and β-1.3-glucan recognition signals 181 that are mediated via pgrp-sa and gnbp3, respectively, are suggested to induce the activation of a sp proteolytic cascade, ultimately leading to the conversion of pro-spätzle into processed spätzle (lemaitre et al., 1996; weber et al., 2003; gay and gangloff, 2007). drosophila spätzle-processing enzyme (spe), a typical clip-domain-containing sp, has been identified as the terminal sp that induces the cleavage of pro-spätzle (jang et al., 2006; kambris et al., 2006). the processed spätzle functions as a native ligand for the toll receptor, and induces the expression of amp genes from the fat body (levashina et al., 1999; weber et al., 2003; gay and gangloff, 2007). therefore, drosophila toll signaling pathway is an elegant example showing of that pattern recognition proteins-mediated pathogen recognition signals are amplified by a proteolytic sp cascade. in addition, recent interesting study performed by shia et al. (2009) showed that knockdown of toll ligand spätzle in the drosophila hemocytes (insect blood cells) induced the inhibition of the expression of amp genes from fat body, suggesting that toll receptor-dependent amp responses in the fat body require spätzle secretion from hemocytes. also, this result indicates that the integration between humoral responses characterized by amp production by fat body and cellular response, such as phagocytosis, mediated by hemocytes depend on the cytokine-like spätzle production, a similar process with mammalian cytokine-mediated immune responses. the drosophila genetic studies are very powerful for characterizing and ordering the components in the drosophila toll signaling pathway (ligoxygakis et al., 2002a). however, there is a limit for this system in terms of determining the biochemical mechanisms involved in regulating this proteolytic cascade. drosophila has several alternative routes to the toll pathway, used in various developmental stages and infection protocols, and it seems difficult to determine the clear activation mechanism by the genetic approach only. for instance, drosophila persephone is another protease linked to the toll pathway and antifungal immunity, yet the biological functions of this molecule have been partially characterized by drosophila genetic studies. the proper identification of upstream or downstream factor(s) of drosophila persephone still awaits further investigations (levashina et al., 1999; gottar et al., 2006). to provide compelling biochemical data on how the lys-type pg recognition signal can be sequentially transferred to spätzle using purified pattern recognition proteins and sps, it is necessary to use a larger insect, which enables us to collect large amounts of hemolymph and to perform biochemical studies. recently, our group determined the activation and regulation mechanisms of tenebrio toll signaling cascade by approaching biochemical methods using a large beetle, tenebrio molitor (kim et al., 2008; jiang et al., 2009; roh et al., 2009). this large insect enabled us to collect large amounts of hemolymph for biochemical studies, allowing us to purify several different tenebrio sps. we demonstrated that the recognition of lys-type pg-mediated by the pgrp-sa/gnbp1 complex induces the processing of pro-spätzle to the cleaved spätzle through the sequential activation of three different tenebrio sps: modular serine protease (msp), spätzle-activating enzyme (sae) and spätzle-processing enzyme (spe) (kim et al., 2008). tenebrio spe has been shown as a terminal sp that cleaves pro-spätzle. additionally, we provided another biochemical evidences of the mechanism by which the gnbp3-mediated fungal β-1,3-glucan recognition signal is also converted pro-spätzle to spätzle after activation of the same three different sps that are involved in lys-type pg recognition signal (roh et al., 2009). therefore, it will be valuable to compare the biological diversity of toll signaling cascades in three different insects, fruit fly drosophila melanogaster, tobacco hornworm manduca sexta and mealworm t. molitor. this will shed further insight into biological diversity of the molecular mechanisms of pathogen recognition and amplification signals in insects. since excellent review papers focusing toll signaling and melanin synthesis cascades are available now (ferrandon et al., 2007; lemaitre and hoffmann, 2007; cerenius et al., 2008, 2010), we mainly focus on biochemical studies of toll signaling cascade using mealworm (t. molitor) and tobacco hornworm (m. sexta) larvae. pathogen recognition molecules of toll cascade pgrp family proteins are critical receptors in drosophila immune responses that are required for the recognition of pg and for subsequent activation of amp gene expression (lemaitre and hoffmann, 2007). pgrps were first characterized in the moths bombyx mori and trichoplusia ni (yoshida et al., 1996; kang et al., 1998) and proposed to be receptors that can trigger insect’s innate immune responses. pgrps share homology with nacetylmuramoyl-l-alanine amidases, which cleave pg at the lactylamide bond between the glycan backbone and the stem-peptides (kang et al., 1998). some non-catalytic pgrps, such as pgrp-lc, -le, -sa and -sd, lack a critical cysteine residue in the catalytic pocket and are not able to cleave pg (mellroth et al., 2003), but these pgrps can bind pgs and are necessary for the expression of amp genes, indicating that these pgrps directly recognize bacteria and activate innate immune responses. in contrast, catalytic pgrps, such as pgrp-sc1a, -lb and -sc2, include this cysteine residue in the active site and are potent enzymes that cleave pg. after digestion with pgrp-sc1b, staphylococcal pg exhibits less activation of the amp genes in a drosophila blood cell line, so it was hypothesized that catalytic pgrps may act as scavengers to limit an inflammatory response to free pg (mellroth et al., 2003). gnbp family, a 50-kda protein found in hemolymph of b. mori and named as a gramnegative binding protein (lee et al., 1996). however, the biochemical studies demonstrated that gnbp belong to the family of β-1,3-glucan recognition proteins (βgrp) that had first been purified on their ability to trigger the pro-phenoloxidase (melanin synthesizing enzyme in invertebrates) in response to fungal infections (yoshida et al., 1986). drosophila 182 genetic studies demonstrated that lys-type pg is recognized by a complex comprised of the pgrpsa and gnbp1 (gobert et al., 2003; pili-floury et al., 2004), while β-1,3-glucan from yeast is recognized by gnbp3 (gottar et al., 2006). both the pgrpsa/gnbp1 complex and gnbp3 are believed to mediate the activation of a sp cascade that ultimately leads to the cleavage of pro-spätzle into processed spätzle (lemaitre et al., 1996). native gnbp3 proteins were purified from three insects, silk worm (b. mori, ochiai and ashida, 2000), tobacco hormworm (m. sexta, ma and kanost, 2000) and mealworm (t. molitor, zhang et al., 2003). the purified proteins bound to 1,3-β-d-glucan but not to bacterial pg. subsequent molecular cdna clonings revealed that gnbp3 molecules contains a region with a sequence similar to bacterial glucanases. interestingly, two catalytically important residues in the glucanases had been replaced with nonhomologous amino acids in all three gnbp3, suggesting that gnbp3 molecules have evolved from an ancestral glucanase gene but retained only the ability to recognize β-1,3-glucan. native gnbp1 was only purified from mealworm and it was involved in pg-recognition reaction with pgrp-sa (kim et al., 2008). next, native pgrp-sa proteins were purified from three insects, silkworm, cabbage looper (t. ni) and mealworm (yoshida et al., 1996; kang et al., 1998; park et al., 2006). the purified pgrp-sa recognized bacterial pgs. in vitro reconstitution experiments demonstrated that tenebrio and bombyx pgrp-sas are recognition molecules of pg-dependent pro-phenoloxidase cascades (yoshida et al., 1996; park et al., 2006). until this time, pgrp proteins from tobacco hornworm were not purified yet. sp zymogens involved in the lys-type pgmediated toll signaling cascade drosophila genetic studies provided the evidence of that pgrp-sa and gnbp1 makes a complex upon recognition of lys-type pg and then recruits down-stream sps for the activation of toll signaling cascade (gobert et al., 2003). however, the identification of immediately downstream sps of the pgrp-sa/gnbp1 complex is not easily performed in drosophila system due to protein redundancy and a limitation of drosophila hemolymph collection. since tenebrio larvae also showed high antimicrobial activities to the challenge of gram-positive bacteria or fungi, it was supposed that tenebrio larvae may have all essential components necessary for the activation and regulation of tenebrio toll signaling cascade. therefore, we assumed that the immediate downstream sp of the pgrp-sa/gnbp1 complex can be purified using tenebrio hemolymph by biochemical approaches (park et al., 2007). also, we hypothesized that gnbp1 and an sp would be recruited to the lys-type pg/pgrp-sa complex when the soluble lys-type pg/tenebrio pgrp-sa complex was incubated with pgrp-sa-depleted tenebrio hemolymph. indeed, tenebrio pgrpsa/lys-type pg complex recruited a 50-kda protein and a 35-kda protein. the amino acid sequencing of the n-terminal residues identified the 50-kda protein as tenebrio gnbp1 and the 35-kda protein as tenebrio msp. interestingly, this msp protease does not contain a clip domain, which is commonly found in proteases upstream of the toll and pro-phenoloxidase cascades (piao et al., 2005). drosophila gnbp1 was known to physically interact with pgrp-sa and activates drosophila toll pathway. however, an interaction between gnbp1 and pgrp-sa has not been observed in vitro (gobert et al., 2003). our observation suggests that the binding of pgrp-sa to pg enhanced the interaction between pgrp-sa and gnbp1, and subsequently the active form of msp was recruited to the lys-type pg/pgrpsa/gnbp1 complex. subsequent cdna cloning demonstrated that t. molitor msp (tm-msp) contains four low-density lipoprotein receptor a repeat (ldl) domains, one complement control protein (ccp) domain and a chymotrypsin-like sp domain (kim et al., 2008). the substrate specificity pocket residues of the sp domain, ser-569 (c189; “c” for the chymotrypsinogen numbering), ser-596 (c216), and gly-610 (c226) indicate that tm-msp is a chymotrypsin-like sp. msp-like molecules was identified in other two insects, m. sexta hp (hemolymph protease)-14 (ms-hp-14) (ji et al., 2004) and the d. melanogaster msp protein (dmmsp, cg31217, buchon et al., 2009). the domain organizations of these three known msp orthologues are shown in fig. 1a and the biological functions of these three msp molecules are reported (ji et al., 2004; park et al., 2007; buchon et al., 2009). also, recently, bucheon et al reported that drosophila msp integrates signals originating from the circulating recognition molecules gnbp3 and pgrp-sa and connects them to the grass-spe-spätzle extracellular pathway upstream of the toll receptor (buchon et al., 2009). although we found that msp is recruited into the lys-type pg recognition complex, the lack of information regarding the identity of the immediate downstream factor(s) of msp limits our knowledge of the molecular details of pg recognition and the involvement of sps in the toll signaling pathway. therefore, the identification and characterization of the biological functions of sps involved in the tenebrio toll signaling pathway are essential for the elucidation of the molecular mechanism of innate host defense system. spe, a terminal sp that converts spätzle pro-protein into a processed form capable of binding the toll receptor, was identified in drosophila (jang et al., 2006). we purified the sps immediately downstream protease of tenebrio msp in order to obtain biochemical information regarding the lys-type pg recognition signaldependent activation of the toll cascade. two sps, tenebrio 41-kda and 44-kda protein, were purified to homogeneity by several column chromatographies and their cdnas were cloned (kim et al., 2008). in order to identify the spe molecule in tenebrio, we initially examined the active form of the 44-kda protease because the 44kda zymogen protein is similar to drosophila spe and easter. since the cdnas of tenebrio spätzle and the toll proteins have not been determined, we 183 fig. 1 the domain organizations of msp homologues (a) and tm-msp, tm-41 kda sp (tm-sae) and tm-44 kda sp (tm-spe) (b). a, comparison of domain organization between t. molitor msp (tm-msp), m. sexta hemolymph proteinase 14 (ms-hp14), and dm-msp homologue (cg31217). pink circles, rectangular symbols and boxes indicate the domains of ldla, ccp, and sp domains of msp homologues, respectively. b, green circles symbol indicates the clip domain. arrows represent the cleavage sites of sp zymogens during activation. the red and blue residues in the boxes indicate the specificity-related residue and catalytic triad ser residue, respectively. expressed the recombinant tribolium spätzle proprotein and the toll ectodomain in a baculovirus system. to address whether the purified active 44kda protease cleaves tribolium spätzle pro-protein in vitro, we incubated the pro-spätzle protein with trypsin, the active forms of tenebrio msp and the 41-kda and 44-kda proteases. only the active 44kda protease cleaved the spätzle pro-protein. under the same conditions, trypsin cleaved the prospätzle protein nonspecifically, and the active forms of tenebrio msp and the 41-kda protease did not cleave the pro-spätzle protein. after performing further biochemical experiments, we confirmed 44 kda protease as tenebrio spe. also, recombinant hp8 protease functioning as manduca spe was purified and its biological function was also determined (an et al., 2009). we next tried to identify the upstream activator of tenebrio spe. because tenebrio spe has a trypsin cleavage site, we hypothesized that the upstream sp is a trypsin-like sp. as mentioned above, the msp and the 41-kda zymogens were identified as chymotrypsin-like and trypsin-like sps, respectively, suggesting that the active form of the 41-kda protease may cleave the spe zymogen. to test this hypothesis, we incubated the active form of 41-kda protein with the purified spe zymogen. as expected, the spe zymogen was hydrolyzed into a 35-kda sp domain and a 15-kda clip domain. the 35-kda band was identified as the sp domain of spe. therefore, we designated the 41-kda protease as tenebrio spe-activating enzyme (sae). 184 because the sequence of the cleavage site in the sae zymogen is leu-124-ile-125, the upstream sp of sae is probably similar to chymotrypsin. this result suggests that the sae zymogen is cleaved by the msp. therefore, active tm-msp was incubated with the recombinant sae zymogen. the sae zymogen was hydrolyzed into a 35-kda sp domain and an 11-kda clip domain. the n-terminal amino acid sequence of the 35-kda band was ile-val-glygly-thr-asn. this sequence is identical to the amino acid sequence of the sae zymogen from ile-125 to asn-130, demonstrating that the msp induced a limited proteolytic cleavage between the clip domain and the catalytic sp domain of the sae zymogen. thus, the sae protease is an immediate downstream target of msp. the domain organization and the cleavage sites by upstream sps were summarized in figure 1b. recently, sae orthologue from manduca system is identified and its biochemical properties are demonstrated (an et al., 2009). drosophila sps required for the activation of toll signaling cascade were screened using rnai technology by kambris et al. (2006). they suggested that two d. melanogasrter catalytic sps (dm-grass and dmspirit) and two noncatalytic sp homologs (sphs), such as dm-spheroide and dm-sphinx1/2, have been involved in the pg-dependent drosophila toll pathway. by homology research with known sps, it turned out that dm-grass and dm-spirit are clip domain-containing trypsin-like sps. dm-spheroide and dm-sphinx1/2 each have a non-catalytic sp domain but no clip domains at the n-terminus. they also have gly and ile residues, respectively, instead of a ser residue in the catalytic site of sps. the reason why we do not identify similar sps and sphs in our studies is unclear, and further studies are necessary to answer this question. however, one plausible explanation for this can be that tenebrio sphs may exist with serine protease inhibitors (serpins) in the hemolymph and are not directly involved in the toll pathway activation. by performing rnai experiments against drosophila sps or sphs, there is a possibility that serpins can be released to the hemolymph by lack of sph and that catalytic sps, such as dm-grass and dm-spirit might be trapped by the released serpins leading to inhibition of the toll pathway activation. lys-type pg recognition signal is amplified via a three step proteolytic cascade finally, to confirm whether the spätzle proprotein is cleaved, lys-type-pg, pgrp-sa, gnbp1, zymogens of msp, sae, spe and pro-spätzle were incubated together in the presence of ca2+, and the cleavage of pro-spätzle was detected by western blot analysis. as predicted, a 14-kda band corresponding to cleaved spätzle was observed (kim et al., 2008). however, if any one of the components was omitted from the incubation mixture, no cleavage of the pro-spätzle occurred (kim et al., 2008). in conclusion, these experiments demonstrated that the pgrp-sa/gnbp1-mediated lys-type pg recognition signal is transferred by three different sps; the initiating enzyme is the 82kda chymotrypsin-like msp, and the other two enzymes are the 41-kda sae and 44-kda spe clip domain-containing trypsin-like sps. sp zymogens involved in the β-1,3-glucanmediated toll signaling cascade to identify the immediate downstream molecule(s) that is recruited by the β-1,3glucan/gnbp3 complex, insoluble β-1,3-glucan was incubated with the native tenebrio gnbp3. when gnbp3/insoluble β-1,3-glucan complex was incubated with the tenebrio hemolymph fraction, a 35-kda band was specifically enriched in the gnbp3/insoluble β-1,3-glucan complex. the nterminal amino acid sequence of the 35-kda protein perfectly matched that of the catalytic sp domain of activated tenebrio msp, suggesting that activated msp was recruited to the β-1,3-glucan/gnbp3 complex and that msp is an apical sp that functions as the immediate downstream molecule of the β1,3-glucan/gnbp3 complex (roh et al., 2009) as like the apical downstream sp of pgrp-sa/gnbp1 in response to lys-type pg (kim et al., 2008). because tenebrio pro-msp is activated in the presence of either the β-1,3-glucan/gnbp3 complex or the lys-type pgn/pgrp-sa/gnbp1 complex, we assumed that the downstream sps of the β-1,3glucan/gnbp3 complex would be identical to tenebrio sae and spe that are activated in response to lys-type pg. β-1,3-glucan recognition signal is also amplified via three-step proteolytic cascade we performed in vitro reconstitution experiments by using five purified proteins: gnbp3, pro-msp, pro-sae, pro-spe, and prospätzle (roh et al., 2009). western blot analysis revealed that processed spätzle was generated upon incubation of the five proteins, β-1,3-glucan, and ca2+ (roh et al., 2009). depletion of any of the components resulted in the loss of cleavage of the pro-spätzle. under the same conditions, the processing of prospätzle to the cleaved spätzle was also observed with pgrp-sa, gnbp1, msp, sae, spe, and spätzle in the presence of lys-type pg and ca2+ (roh et al., 2009). these results clearly demonstrate that gnbp3, in the presence of β-1,3glucan, induces the activation of a three-step proteolytic cascade involving msp, sae, and spe sequentially. this activation leads to the processing of pro-spätzle into the mature form that functions as a ligand for the toll receptor. in addition, these data show that the β-1,3glucan and lys-type pg recognition signals are sharing a common three step proteolytic cascade to transduce their recognition signals to the toll receptor (fig. 2). the effector molecules after toll signaling cascade activation although we have provided biochemical evidence elucidating the mechanism by which the lys-type pg and β-1,3-glucan recognition signals are transferred to the toll receptor, we have not demonstrated whether this sp cascade is present in vivo. we hypothesized that if this cascade is present 185 fig. 2 model summarizing the molecular events in the regulation of the tenebrio toll signaling cascade. the molecular activation mechanism of the tenebrio toll cascade leading to production of amps. processed spätzle induces the production of endogenous spn40 and spn55 as a negative feedback regulator of the toll cascade. in vivo, the same amp(s) will be produced in the insect hemolymph when the pathway molecules are injected into the tenebrio larvae since tenebrio toll signaling pathway is using a common proteolytic cascade. to address this hypothesis, we injected β1,3-glucan, lys-type pg, activated sae and processed spätzle into the tenebrio larvae. the hemolymphs collected after injection of above four molecules had high antimicrobial activities against s. aureus, e. coli and s. cerevisiae. we purified two amps, tenecin 1 and tenecin 2, by column chromatography from these four hemolymphs. tenecin 1 had a bactericidal activity against grampositive bacteria and was previously identified by our group (moon et al., 1994). the amino acid sequence of tenecin 1 and its disulfide bond arrangement are similar to the defensin protein from drosophila (bulet et al., 1999). tenecin 2 is highly homologous (65 % identity) to coleoptericin (bulet et al., 1991), which was purified from the coleopteran insect, zophobas atratus. tenecin 2 showed bactericidal activity against e. coli. taken together, tenecin 1 and tenecin 2 are induced by treatment with β-1,3-glucan, lys-type pg, sae and spätzle suggesting that β-1,3-glucan and lys-type pg activate toll receptors by the same three-step proteolytic cascade, which results in the production of tenecin 1 and 2 (fig. 2). 186 fig. 3 a comparison with drosophila, manduca and tenebrio extracellular proteolytic toll signaling pathways involving sp cascades. arrows indicate the activation of downstream components or steps in cascade. dashed arrows indicate steps that have not been experimentally verified or in which components of the pathway have not yet been determined. pg, peptidoglycan; pgrp, peptidoglycan recognition protein; grp, β-1,3-glucan recognition protein; gnbp, gram-negative binding protein; propo, prophenoloxidase; po, phenoloxidase; msp, modular sp; modsp, modular sp; spe, spätzle-processing enzyme; sae, spe activating enzyme; psh, persephone; sph, sp homologue; hp, hemolymph protease; pap, pro-po activating protease. diversity of toll proteolytic signaling cascades in drosophila, manduca and tenebrio system the outline of activation mechanism of toll signaling cascades in three different insects are summarized in figure 3. intensive biochemical studies performed in t. molitor and m. sexta, together with genetic analysis in d. melanogaster, reveal striking similarities in the mechanisms underlying sp activation by pattern recognition proteins. all involve the sequential activation of typical sps, such as modular sp and clip-domain containing sps. interestingly, tenebrio and drosophila used msp as an apical sp in the toll cascade, but, manduca hp14 corresponding to msp did not activate hp6 zymogen corresponding to tenebrio sae, suggesting that another direct downstream sp of pgrp-sa/gnbp1 complex might be existing in hemolymph in manduca system. in drosophila, epistasis analysis has demonstrated that an drosophila msp named modular sp (modsp), acts down stream of pgrp-sa and upstream of the sp grass (buchon et al., 2009). moreover, drosophila modsp does not participate in the persephone-dependent branch of the toll pathway, but is instead part of a linear pathway of sps connecting microbe recognition by pattern recognition proteins to the activation of spätzle by spe. tenebrio msp, manduca hp14 and drosophila modsp proteases all share a common structure comprising four or five ldl domains followed by a complement control domain and a c-terminal sp domain. the upstream pathogen recognition features of insect’s toll cascade are also reminiscent of the complement activation by the lectin pathway in mammals in which the recognition of carbohydrate by the mannose binding lectin (mbl) leads to the autoactivation of mbl-associated sps (masps, matsushita and fujita, 1992). masps also showed similar domain organization with those of insect msps. manduca toll cascade summarized that prohp6 becomes activated in response to microbial infection and participates in two immune pathways (fig. 3); activation of pap1, which leads to prophenoloxidase activation and melanin synthesis, and activation of hp8, which stimulates a toll-like pathway (an et al., 2009). hp8 activates spätzle to induce amp synthesis via toll receptor. in melanin synthesis cascade, an initiation proteinase precursor, prohp14, is autoactivated in response to grampositive bacterial or fungal infection. hp14 activates prohp21; hp21 activates propap2 or propap3; pap2 or pap3 then cleaves pro-phenoloxidase to form active phenoloxidase in the presence of sph1 and sph2. activation of pro-phenoloxidase can also be catalyzed by pap1 when the high mr sph complex is present simultaneously. pap1 also activates prosph2 directly and can indirectly lead to prohp6 activation (wang and jiang, 2007, 2008). tenebrio toll cascade support a model in which lys-type pg and β-1,3-glucan activate a common set of three sp zymogens sequentially (fig. 3). this three-step proteolytic cascade-dependent 187 processing of the extracellular pro-spätzle produces active spätzle, which then binds to the toll receptor, resulting in the induction of amp expression in the tenebrio larval hemocytes. each of the three sps has unique biochemical properties to regulate activation of the tenebrio toll pathway. the initial enzyme pro-msp is an 82-kda sp zymogen with an n-terminal chymotrypsin-like cleavage site and a cterminal chymotrypsin-like catalytic sp domain. prosae is a 41-kda sp with an n-terminal chymotrypsin cleavage site and a c-terminal trypsinlike catalytic sp domain, and pro-spe is a 44-kda protein with an n-terminal trypsin-like cleavage site and a c-terminal trypsin-like catalytic sp domain. these three different sp combinations enhance the specificity of the proteolytic sp cascade and prevent nonspecific cleavage of these sp zymogens. three serpins functioning as regulatory molecules of toll signaling cascade serpins act as suicide substrates by binding covalently to their target proteases and belong to a superfamily of sp inhibitors (gettins, 2002). serpins regulate various physiological processes and molecular defense systems, such as blood coagulation, fibrinolysis, inflammation and complement activation in mammals (gooptu and lomas, 2009). to date, four drosophila serpins are known to be involved in innate immunity (spn43ac, spn27a, spn77ba and spn28d) and have been extensively studied using a genetic approach. spn43ac mutant flies accumulate cleaved spätzle, resulting in the constitutive activation of the toll signaling pathway and expression of amps (levashina et al., 1999). spn27a and spn28d regulate the toll pathway during early development (hashimoto et al., 2003; ligoxygakis et al., 2003; scherfer et al., 2008) and are involved in the melanin biosynthesis.(de gregorio et al., 2002; ligoxygakis et al., 2002b) another serpin, spn77ba, was identified as a negative regulator of melanization in the drosophila respiratory system (the trachea) (tang et al., 2008). in manduca system, kanost’s group reported elegant results regarding serpin splicing: 12 different copies of manduca serpin 1 undergo mutually exclusive alternative splicing to produce 12 putative protein isoforms, which differ in their carboxyl-terminal 3946 residues including the p1 residue. these serpins inhibited manduca sps with different specificities (jiang and kanost, 1997; ragan et al., 2010). these serpins were characterized and suggested as negative regulators of the pro-phenoloxidase and toll signaling cascades (kanost et al., 2004). however, molecular regulatory mechanisms of how serpins regulate invertebrate’s innate immune responses are not well understood due to the uncertainty of the identity of the target sps by the serpins. we purified three novel serpins (spn40, spn55 and spn48) from the hemolymph of t. molitor (jiang et al., 2009) (fig. 2). these tenebrio serpins made specific serpin-sp complexes with three toll signaling cascade-activating sps, such as msp, sae, and spe and cooperatively blocked tenebrio toll signaling cascade and β-1,3-glucanmediated melanin biosynthesis. also, the protein expression levels of spn40 and spn55 were dramatically increased in vivo by the injection of a toll ligand, processed spätzle, into tenebrio larvae. this increase in spn40 and spn55 protein levels indicates that these two serpins function as inducible negative feedback inhibitors. surprisingly, tenebrio spn55 and spn48 were cleaved after tyr and glu residues of reactive center loops, respectively, despite being targeted by trypsin-like sae and spe proteases. these unexpected cleavage patterns are also highly similar to those of unusual mammalian serpins involved in blood coagulation and blood pressure regulation, indicating that they may contribute to highly specific and timely inactivation of detrimental sps during innate immune responses. taken together, our results showed the specific regulatory mechanisms of innate immune responses by three tenebrio novel serpins. a balance between activation and inhibition of the sp-mediated innate immune response must be maintained to avoid damage to the host (ferrandon et al., 2007). as described above, tenebrio three serpins targeting three toll cascade-activating sps act as negative regulators of toll signaling, indicating that each sp in a cascade may be regulated by a specific serpin. this is first biochemical evidences that sp-serpin pairs directly regulate the pattern recognition protein-dependent toll signaling cascade. to date, several cascade reactions including drosophila toll signaling cascade have been hypothesized to be regulated by a single “bottleneck” protease. however, our data show that the control of tenebrio toll proteolytic signaling cascades is more precisely regulated. furthermore, we have demonstrated that spn40 and spn55 function as inducible negative feedback regulators in vivo. these results, in combination with our other reports (kan et al., 2008; kim et al., 2008; roh et al., 2009) support a model in which the lys-type pg and β-1,3-glucan-dependent toll signaling and prophenoloxidase cascades are negatively regulated by three endogenous serpins (fig. 2). finally, our studies highlight the elaborate regulatory mechanism of invertebrate innate immune defense systems. conclusion the developments of genetics and molecular biology enable us to screen the drosophila mutants that had deficient immune responses against bacterial and fungal infection, subsequently to discover two conserved innate immune signaling cascades-toll and imd-both of which lead to the activation of nuclear factor kb (nf-kb) transcription factors (lemaitre and hoffmann, 2007). in this review, the activation and regulation mechanisms of toll cascade were discussed. however, molecular mechanism by microbe-derived protease-mediated toll cascade (so called danger signaling pathway) is not determined yet. the exact molecular activation and regulation mechanism of microbial proteasemediated toll pathway should be determined, such as how pro-persephone is cleaved by microbial proteinase and identity of tenebrio or manduca counter part protease of drosophila persephone proteinase, and whether really active form of 188 persephone specifically cleave pro-spe for the activation of spätzle in tenebrio or manduca system. also, dap-type pg-mediated imd signal pathway is unclear. even though the identities of pattern recognition receptors of drosophila imd pathway and recognition mechanism between tracheal cytotoxin (tct) and pgrp-le or pgrp-lc were determined (chang et al., 2006; lim et al., 2006), tct will not be generated naturally by all gramnegative bacteria and bacilli species. therefore, we should find a natural ligand of imd pathway and also should elucidate the dap-type pg recognition mechanism and extracellular signaling pathway of imd pathway in future. another unexploited area is how mycoplasma bacteria, which are deficient of pg in cell membrane, are recognized in insects. understanding the signaling pathway triggered by mycoplasma and how their recognition activates invertebrate innate immune responses will be big homework for invertebrate immunologist and biochemists. also, further challenging theme in insect immunity is the determination of recognition and activation mechanisms against intracellular pathogenic microbes, such as listeria monocytogenes. recently, kurata’s group nicely discussed the relationship between autophagy and insect innate immunity (yano et al., 2008; kurata, 2010). drosophila pgrp-lc and -le are known to sense the dap-type pg of extracellular and intracellular-infective bacteria and then to induce several innate immune responses, such as amp production, via activation of imd pathway. yano et al observed pgrp-le-dependent 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during prophenoloxidase activation. j. biol. chem. 278: 42072-42079, 2003. weber an, tauszig-delamasure s, hoffmann ja, lelievre e, gascan h, ray kp, et al. binding of the drosophila cytokine spatzle to toll is direct 191 apoptosis in molluscan immune defense isj 6: 49-58, 2009 issn 1824-307x review apoptosis in molluscan immune defense im sokolova department of biology, university of north carolina at charlotte, charlotte nc, usa accepted april 20, 2009 abstract apoptosis, or type i programmed cell death, is a fundamental biological process involved in cellular homeostasis in metazoans. apoptosis plays a key role in immune system homeostasis and function, defense against parasite and pathogens and self/non-self recognition. in this review, i present our current knowledge of the mechanisms and signaling pathways underlying apoptosis in mollusks and its roles in host-pathogen interactions and immune defense. both signaling and execution pathways of apoptosis appear to be highly conserved in mollusks, although there is evidence that some apoptotic mechanisms (e.g., caspase-independent cell death) may differ from model invertebrates such as caenorhabditis and drosophila and more closely resemble those seen in vertebrates. apoptosis is important for the functioning of the molluscan immune system as indicated by the high baseline apoptosis rates observed in circulating and resident hemocytes. apoptosis also plays a role in host protection against parasites by limiting the spread of the pathogen while preventing inflammatory damage of surrounding tissues. in molluscs, interaction between immune cells and parasites or pathogens usually triggers apoptosis; however, some pathogens (especially obligatory intracellular parasites that depend on the host cell for their survival and proliferation) can inhibit this response and prevent host cell death. currently, the mechanisms underlying pathogen-induced modulation of apoptosis in molluscs are not well understood. summarizing our current knowledge of the immune functions of apoptosis in molluscs, and comparing them with the more widely studied vertebrate systems, this review will delineate the gaps in our understanding of this critical cellular process in molluscs and assist in the ongoing search for evolutionary novel mechanisms of apoptosis in immune defense and self/non-self-recognition. key words: immune response; apoptosis; parasites; pathogens; mollusca introduction molluscs, the second most diverse group of animals (next only to arthropods) with about 93,000 extant species, are abundant in most marine, brackish, freshwater and terrestrial habitats. molluscs play vital roles in these ecosystems and are often used as bioindicators of the ecosystem health. in some ecosystems (most notably in marine and estuarine habitats), molluscs can function as ecosystem engineers creating and modifying habitats for other species and affecting the habitat quality by eliminating suspended particles or participating in the top-down control of algal blooms. molluscs have also been used by humans since the dawn of the human history as food resources or to ___________________________________________________________________________ corresponding author: inna m sokolova department of biology university of north carolina at charlotte 9201 university city blvd., charlotte nc, 28223 usa e-mail: isokolov@uncc.edu provide other valuable commodities such as pearls and mother-of-pearl, tyrian purple from muricid gastropods used to dye royal robes, non-opioid pain-killers derived from cone snails’ toxins and anticancer drugs from some gastropod peptides (mcfadden et al., 2003; faircloth and cuevas, 2006; han et al., 2008). moreover, molluscs serve as important vectors in the transmission of parasites and pathogens to humans and/or domestic animals. given the important ecological, economical and medicinal roles of molluscs, understanding the immune defenses of these animals is critical for improving disease resistance and increasing the survival of ecologically and economically important molluscs, and for improving their aquaculture production and/or sanitation to reduce parasite/pathogen transmission. studies of the molluscan immunity are also important from the viewpoint of evolutionary and comparative immunology. the study of the immune defense of molluscs may reveal immunobiological novelties 49 mailto:isokolov@uncc.edu thereby providing insights into the evolution of immunity, and may offer new solutions for the treatment of infectious and parasitic diseases or immunological disorders in humans and domestic animals. although molluscan immunity has been actively studied in the past few decades, our knowledge about the cellular and molecular mechanisms of molluscan immune defenses and their interactions with pathogens and parasites is still far from complete. significant progress has been made in our understanding of the phagocytic and bactericidal responses of molluscan immune cells and of the humoral factors involved in non-self recognition and pathogen killing; due to space limitations, i will only briefly mention these immune mechanisms in this review (see “overview of the molluscan immunity” below) and further refer the reader to a number of excellent reviews focusing on these topics (see anderson, 1996; kennedy et al., 1996; glinski and jarosz, 1997; canesi et al., 2002; paul, 2003; tiscar and mosca, 2004; vasta and ahmed, 2008 and references therein). the main goal of the present review is to draw attention of invertebrate immunologists to an important but currently understudied aspect of molluscan immunity – namely, the role of apoptosis, or type i programmed cell death (pcd i), in molluscan immune defense and host-pathogen interactions. given that the study of molluscan apoptosis is still in its infancy, i will draw on relevant examples from vertebrate literature to illustrate molecular mechanisms and possible immunological roles of apoptosis in protection against parasites, pathogens, and tumors in mollusks. it is my hope that by providing a comprehensive overview of our current knowledge of molluscan apoptosis, this review will provide a roadmap for future studies to understand this critical cellular process in mollusks and to potentially reveal novel mechanisms and roles of apoptosis in the immune defense and self/non-self-recognition in animals. overview of molluscan immunity the immune system of molluscs, as all other invertebrates, consists only of innate immunity and is lacking the adaptive immunity components. while the innate immune system is often regarded as an evolutionary more ancient and hence more primitive form of immunity than the adaptive responses seen in vertebrates, it is actually a surprisingly complex and efficient form of protection against many parasites and pathogens encountered by molluscs. indeed, successful adaptive radiation of molluscs and their ability to colonize a broad range of aquatic and terrestrial habitats clearly speaks to their ability to efficiently combat infections and parasitic invasions. external barriers (such as shells, mucus and epithelia) of mollusks constitute the first line of defense against pathogens and parasites; when these barriers are breached, the second, internal line of defense involving cellular and soluble (humoral) hemolymph components come into play. humoral components of the molluscan immune defense include lysosomal enzymes (such as βglucuronidase, acid and alkaline phosphatase, lipase, aminopeptidase and lysozyme), lectins including agglutinins, fibrinogen-related proteins (freps), and c-type lectins, and antimicrobial peptides that aid in recognition of pathogens and parasites by marking them for destruction via opsonizing or directly killing (review in anderson, 1996; kennedy et al., 1996; canesi et al., 2002; paul, 2003; tiscar and mosca, 2004; vasta and ahmed, 2008). agglutinins, freps and other lectins along with pattern recognition receptors (prrs, which in mollusks include toll-like receptors, imd pathway receptors and intracellular nucleotideoligomerization domain (nod) proteins) are responsible for the surprising degree of specificity shown by the innate immune system for pathogens and parasites (paul, 2003; yeretssian et al., 2008). fig. 1 transmission electron micrograph (tem) of resident hemocytes in gills (a) and hepatopancreas (b) of oysters c. virginica. hemocytes, h. 50 while humoral immunity is very important for host defense, it is indisputable that the cellular components of the hemolymph, hemocytes, play a central role in the innate immune responses of mollusks (fig. 1). hemocytes are the main effector components of the molluscan immune system and are responsible for phagocytosis of parasites, pathogens, and foreign particles (which represents a complex process including recognition, adhesion, ingestion, destruction or encapsulation and final elimination of foreign cells or materials). hemocytes are also directly engaged in pathogen and parasite killing via the oxidative burst reaction a rapid generation of toxic reactive oxygen species (ros) by a membrane bound enzyme nadh-oxidase. ros (especially hydrogen peroxide) can be further converted by hemocyte myeloperoxidase to hypochloric acid (hocl) that has strong bactericidal and viricidal properties (tiscar and mosca, 2004). molluscan hemocytes are comprised of two main types: hyalinocytes small cells with cytoplasm containing few or no granules, and granulocytes large phagocytic cells containing abundant numbers of cytoplasmic granules (kennedy et al., 1996; tiscar and mosca, 2004). these two major cell types are further classified into subtypes based on their size, morphology and (to the degree that it is known) function (e.g., acidophilic, basophilic and neutrophilic granulocytes, natural killer-like cells, etc.) (kennedy et al., 1996; takahashi and mori, 2000). overall, molluscan granulocytes strongly resemble vertebrate monocytes and macrophages both in structure and function and share with the latter such key properties as avid phagocytosis, pathogen-induced oxidative burst, production and release of nitric oxide and lysosomal enzymes (canesi et al., 2002). recent studies have revealed subpopulations of molluscan hemocytes that possess lymphocyte-like morphology and physiological properties similar to mammalian natural killer (nk) cells (monti et al., 1992). molecular mechanisms of apoptosis in molluscs the term “apoptosis” was coined by kerr, wyllie and curie in their seminal 1972 paper to describe a distinctive form of programmed cell death (pcd) defined by characteristic morphological features such as chromatin condensation, membrane blebbing and cell shrinkage (kerr et al., 1972 cited after feig and peter, 2007). since then, the study of apoptosis using both vertebrate and invertebrate model systems has led to the recognition that this is a multifunctional process, highly conserved evolutionarily, and plays a critical role in cellular and tissue homeostasis, embryonic development, and immune defense in multicellular organisms. unlike necrosis that may be considered as “accidental” cell death in response to major mechanical or chemical injuries, apoptosis is a highly orchestrated cellular process in which intracellular components are sequestered and disposed of in an orderly manner without the induction of inflammation. mechanisms of apoptosis have been based predominantly on the studies of vertebrates and invertebrate models such as caenorhabditis and drosophila. while these findings have been extensively reviewed previously (see reviews in hengartner, 1996, 2000; vermeulen et al., 2005; circu and aw, 2008; ow et al., 2008; suen et al., 2008 and references therein) and their detailed analysis is beyond the scope of the present review, a brief account of the major apoptotic pathways is in order to set the stage for a discussion of the mechanisms underlying molluscan apoptosis. briefly, the apoptotic cell death can be triggered through two major pathways – intrinsic (or mitochondrial), in response to internal cellular damage and extrinsic, in response to death clues received from the environment (fig. 2). although both pathways can function independently, there is substantial cross-talk that allows for amplification of the death signal (feig and peter, 2007). a key characteristic of the majority of apoptotic pathways is the involvement of a family of proteases called caspases that cleave target proteins at specific sites typically containing aspartic acid residues followed by a caspase-specific three amino acid sequence (creagh et al., 2003). caspases involved in apoptosis include initiator caspases (e.g., caspases 2, 8, 9 and 10) that cleave and activate the effector caspases (e.g., caspase 3, 6 and 7), which in turn act upon intracellular targets ranging from cytoskeleton proteins to specific cell structures such as mitochondria, nuclear lamina or chromatin (hengartner 2000; creagh et al., 2003). both extrinsic and intrinsic apoptotic pathways converge on the activation of caspases although it should be noted that caspase-independent apoptosis can also occur. in vertebrates, a hallmark of the intrinsic apoptotic pathway is a release of cytochrome c from mitochondria that is usually accompanied by the formation of a large-molecular size pore in the mitochondrial membrane (the mitochondrial permeability transition (mpt) pore) and collapse of the mitochondrial membrane potential (mignotte and vayssiere, 1998). factors that bring about activation of apoptosis via the mitochondrial pathway include dna damage, oxidative damage of mitochondrial or cytosolic proteins and lipids, viral stimulation and/or removal of pro-mitogenic signals (growth factor withdrawal) (circu and aw, 2008). once in the cytoplasm, cytochrome c interacts with apoptotic peptidase activating factor 1 (apaf-1), procaspase-9 and datp to form a complex called the apoptosome that activates the initiator caspase 9, which in turn activates the effector molecule, caspase 3. caspase 3 activation causes degradation of proteins, dna and other cellular components culminating in cell death (zimmermann et al., 2001). mitochondrial permeability transition and cytochrome c release are tightly regulated by the bcl-2 family of proteins that includes both proor anti-apoptotic members (circu and aw, 2008). the extrinsic pathway of apoptosis is initiated via binding of specific protein ligands to so called death receptors on the cell surface. in vertebrates, six major types of death receptors have been described including fas, trail (tnf-related apoptosis inducing ligand) receptor -1 and -2, tumor necrosis factor (tnf) receptor-1, tnf receptorrelated apoptosis-mediating protein (tramp), and death receptor-6 (dr6). activation of death receptors 51 fig. 2 schematic representation of major apoptotic signaling and execution pathways. extrinsic (death receptoractivated) pathways are shown on the right, and intrinsic (mitochondrial) including caspase-dependent and caspase-independent pathways are shown on the left of the diagram. note considerable cross-talk between the pathways. arrows indicate activation, and lines with black dots inhibition. red circles with the letter “m” indicate parts of the pathways that have been demonstrated in molluscs. abbreviations: permeability transition pore, ptp; cytochrome c, cyt c; procaspase, pro-casp; fas-associated death domain, fadd; apoptotic peptidase activating factor 1, apaf-1; apoptosis inducing factor, aif; inhibitor of apoptosis family of proteins, iap. causes rapid formation of a death-inducing signaling complex (disc) that initiates a cascade of caspase activation (notably initiated by the activation of caspase 8) leading to induction of apoptosis (zimmermann et al., 2001; schultz and harringto, 2003). homologues of death receptors and fasl have been also found in the genome of an ascidian ciona intestinalis (dehal et al., 2002; terajima et al., 2003). although previous studies have isolated several proteins containing death domains from other invertebrates, there are no unequivocal death receptor orthologs reported in drosophila, c. elegans or porifera genomes despite concerted efforts to find them (muzio, 1998; bridgham et al., 2003). this suggests that death receptor-mediated apoptosis may have evolved within the chordate lineage and may not be functional in non-chordate invertebrates. despite the central role of caspases in the intrinsic and extrinsic apoptotic pathways, caspase activation is not an absolute requirement for the induction of programmed cell death. the most prominent caspase-independent death effector molecules are apoptosis-inducing factor (aif), a phylogenetically conserved mitochondrial flavoprotein, and endonuclease g, a mitochondrionspecific nuclease (van loo et al., 2001; li et al., 2001; cande et al., 2002; tait and green, 2008). endonuclease g and aif are released from mitochondria upon a death signal and translocate directly into the nucleus where they bind to dna and induce caspase-independent chromatin condensation and dna fragmentation leading to cell death (van loo et al., 2001; li et al., 2001; cande et al., 2002). some non-caspase proteases such as cathepsins, calpains and serine proteases like granzyme a/b and omi/htr a can also trigger caspase-independent apoptosis (vermeulen et al., 2005). overall, the considerable redundancy (i.e. potential triggering of the multiple apoptotic pathways by a single stimulus), cross-talk, and mutual amplification between caspase-dependent 52 and -independent pathways of apoptosis stresses the critical importance of this cellular process in metazoans; it appears that once the death signal has been received, apoptosis proceeds at all costs to eliminate the damaged, malignant, infected or otherwise dangerous cells. programmed cell death with all the characteristic hallmarks of apoptosis including cell shrinkage and blebbing, chromatin condensation, dna fragmentation and translocation of a phospholipid phosphatydilserine into the outer leaflet of the cell membrane, have been described in a variety of mollusks (sunila and labanca, 2003; sokolova et al., 2004; buckland-nicks and tompkins, 2005). moreover, many of the molecular components of apoptotic cellular machinery have also been found in mollusks and appear to be highly structurally and functionally conserved. molluscan cells (including hemocytes) possess caspase 3-like activity (pirger et al., 2008), and similar to vertebrates, molluscan caspase 3 can be activated by cytochrome c and datp (sokolova et al., 2004). molluscan caspase-3 activity can also be specifically stimulated by staurosporin although, unlike in mammals, the mechanism of this activation in mollusks does not involve the activating cleavage of pro-caspase 3 (bravarenko et al., 2006). recent studies have demonstrated that the signaling pathways involved in the regulation of molluscan apoptosis share significant molecular and functional similarity with those seen in vertebrates. two central players in cell death and survival, tnfα and nf-κb transcription factors, appear to play a key role in cell signaling in mollusks. molluscan tnf-α shares considerable molecular and functional similarity with vertebrate homologs (terahara and takahashi, 2008). for example, its expression increases in response to bacterial infections and results in the induction of apoptosis in molluscan hemocytes (terahara and takahashi, 2008). in contrast to tnf-α, nf-κb transcription factor typically exhibits pro-survival, anti-apoptotic effects in vertebrates (aggarwal, 2004; dey et al., 2008). in mollusks, all the key components of nf-κb signaling cascade have been described and bear considerable similarity to the mammalian nf-κb signaling pathway (ouwe-missi-oukem-boyer et al., 1994; montagnani et al., 2004; zhu and wu, 2008). however, the exact role of nf-κb in molluscan apoptosis has not been studied and requires further investigation. many downstream elements of apoptotic signaling cascades show significant similarity between molluscan and vertebrate cell death. thus, similar to mammals, mitogen-activated protein kinases and rho, a member of the ras gtpase family, are involved in regulation of molluscan apoptosis and exhibit anti-apoptotic effects in molluscan hemocytes (lacoste et al., 2002). protein kinase a (but not protein kinase c) also appears to be involved in molluscan hemocyte apoptosis; in the case of noradrenaline-induced apoptosis, inhibition of pka but not pkc activity attenuated apoptosis levels (lacoste et al., 2002). another potential key effector of molluscan apoptosis is cyclic amp (camp); however, its role may be proor antiapoptotic, depending on the circumstances. for example, in oyster (crassostrea gigas) hemocytes, camp appears to promote apoptosis since the adenylate cyclase inhibitor 2’, 5’-dideoxyadenosine (dda) reduces levels of noradrenaline-induced pcd i (lacoste et al., 2002). in contrast, elevated levels of camp induced by pituitary adenylate cyclase activating polypeptide (pacap) have antiapoptotic effects in terrestrial snails (helix pomatia), significantly attenuating dopamineand colchicineinduced apoptosis in the salivary gland (pirger et al., 2008). further research is needed to determine whether these disparate effects of camp are speciesor cell type-specific, or whether they are differentially expressed depending on the physiological condition of an organism and/or the nature of the stimulus. a tumor suppressor p53 homolog also plays an important role in apoptosis signaling in mollusks. in normal mammalian cells, p53 suppresses the formation of tumors by arresting the cell cycle or by apoptosis in response to genotoxic stress-induced dna damage (böttger et al., 2008). apoptosis can be induced by p53 either via translocation into the nucleus where it upregulates transcription of proapoptotic genes or by translocation to the mitochondria where it binds to and inactivates bcl-2 and other antiapoptotic proteins (böttger et al., 2008). in clams (mya arenaria), p53 protein shares significant sequence and structural similarity to human p53 and localizes to the nucleus in response to apoptotic signals (holbrook et al., 2009). overexpression of mortalins (hsp70 family proteins) that bind p53 and prevent its translocation to the nucleus has been shown to inhibit the expression of pro-apoptotic p53-dependent genes and decrease levels of apoptosis in molluscan cells in a similar manner to that seen in vertebrate species (böttger et al., 2008). there is also evidence that the mitochondrial pathway of p53dependent apoptosis activation is functional in mollusks (böttger et al., 2008) but further studies are needed to confirm this suggestion. nitric oxide (no) is another signaling molecule that is involved in the regulation of apoptosis. the effects of no on apoptosis vary greatly depending upon the dose of no used, the cell type, and the physiological status of the cell, and can be either proor anti-apoptotic (brune et al., 1999). molluscan cells including hemocytes contain nitric oxide synthase and produce no (terahara and takahashi, 2008). however, the role of no in apoptotic regulation has not been extensively studied in mollusks. the only documented study on the effects of no on molluscan apoptosis shows that inhibition of nitric oxide synthase (nos) activity during ilyanassa obsoleta larvae metamorphosis induces apical ganglion cell apoptosis (gifondorwa and leise, 2006). in molluscan hemocytes, no production is elevated following exposure to parasites or pathogens, or following exposure to the cytokine, il-2 (barsia and ramos-martinez, 2008; terahara and takahashi, 2008), but it is not known whether this induction has an effect on the level of apoptosis in these cells or if yes, whether it involves a down-stream caspase activation. overall, the exact mechanisms and roles of no in mollusc cell apoptosis remain to be fully elucidated. 53 in contrast to some of the highly conserved elements of the caspase-dependent apoptotic machinery, little is known about the caspaseindependent apoptotic pathway in mollusks although its existence has been proposed. this is based on the observation that cadmium (cd)-induced apoptosis in molluscan hemocytes occurs in the absence of mitochondrial permeability transition or caspase 3 activation (sokolova et al., 2004). a recent study from our laboratory has also shown that a pancaspase inhibitor, z-val-ala-asp-fluoromethylketone (z-vad-fmk), fails to prevent apoptosis of crassostrea virginica hemocytes following infection with the intracellular parasite perkinsus marinus, suggesting the involvement of caspase-independent pathways (grewal and sokolova, unpublished data). in mammalian cells, caspase-independent apoptosis induced by trace metals (mn2+ and cd2+) or a toxic plant metabolite (allicin) is associated with protein kinase a-dependent increases in the expression and nuclear translocation of aif in the absence of caspase 3 activation or poly(adp-ribose) polymerase (parp) cleavage (ouabrahim et al., 2001; shih et al., 2003; park et al., 2005). the molecular mechanisms underlying caspaseindependent apoptosis in molluscs are not known and their determination would be an exciting avenue for future investigations. notably, a partial gene sequence of an aif homolog isolated from oysters c. virginica has been recently published in marine genomics database (www.marinegenomics.org, accession # mgid89694) that has significant similarity at the protein level to vertebrate aifs (55 % amino acid identity, e=10-74-10-72 by blastx, altschul et al., 1997). studies of caspaseindependent pcd i in molluscs hold an especially high promise of novelty because current information from model invertebrates (drosophila and caenorhabditis) suggest that caspase-independent apoptosis does not occur in these organisms (tait and green, 2008) raising a possibility that this pathway was either lost to ecdysozoa, or evolved independently in lophotrochozoa (including molluscs) and vertebrates ancestors. exposure to environmental stressors, such as high temperatures, changes in salinity, and the presence of pollutants has been shown to induce apoptosis in molluscan cells, presumably via the intrinsic pathway. for example, high salinities result in elevated levels of oyster (c. virginica) hemocyte apoptosis (goedken et al., 2005). similarly, exposure to elevated temperatures (28 ºc) stimulate apoptosis in oyster hemocytes whereas moderate temperature changes in the near-optimum range (between 10 and 25 ºc) has no effect (goedken et al., 2005; cherkasov et al., 2007). pollutants such as heavy metals, tributyltin (tbt), and pah, can also induce apoptosis in molluscan immune and non-immune cells, with the degree of induction depending on the dose and time of exposure to the pollutants, and on the exposure mode (in vitro or in vivo) (barsiene et al., 2008). given that most of the apoptosis-inducing environmental stressors described above are also known to induce elevated ros production and oxidative stress in mollusks (pruski and dixon, 2002; abele et al., 2002; lannig et al., 2006), it is reasonable to suggest that this stress-induced apoptotic cell death could be triggered by oxidative damage. however, the precise molecular mechanisms responsible for stress-induced apoptosis in mollusks are presently not known and require further study. apoptosis in molluscan cells can also be triggered by activation of surface receptors including hormone receptors and integrins. in oysters (c. gigas), activation of integrins (either by specific ligands or by integrin-binding rgd (arg-gly-asp)containing peptides) promotes apoptosis in a similar manner to that seen in mammalian neutrophils (terahara and takahashi, 2008). notably, unlike mammalian cells where rgd-induced apoptosis is due to anoikis (i.e. prevention of attachment of anchorage-dependent cells to extracellular matrix), the ability of rgd-containing peptide to induce apoptosis in non-adherent, adherent, and spreading molluscan hemocytes alike, indicates that the signaling pathways engaged by integrin activation are independent of hemocyte attachment (terahara and takahashi, 2008). finally, noradrenaline, a catecholamine produced by the neuroendocrine system and by the immune cells of mollusks, also can induce apoptosis of molluscan hemocytes via βadrenergic signaling pathways (lacoste et al., 2002). overall, current data suggests that the mechanisms underlying apoptosis in mollusks (to the degree that they are known) closely resemble those seen in vertebrates. this includes similarity of the major biochemical and molecular steps of apoptotic pathways, as well as redundancy and flexibility of the apoptotic program that can switch between caspase-dependent and -independent pathways in response to specific environmental or developmental stimuli. moreover, there is also evidence that some of the apoptotic pathways in mollusks may be sufficiently divergent from other model invertebrates (such as drosophila and caenorhabditis) so as to warrant further study in the hopes that the determination of the specific regulatory and execution pathways of molluscan apoptosis will discover novel mechanisms of pcd and shed light on the evolution of this crucial cellular process in metazoans. apoptosis as an immunomodulatory mechanism apoptosis has increasingly become a focus of study for biomedical immunologists with the discovery of the immunomodulatory and defense roles of apoptotic cell death and the recognition that high levels of apoptosis are essential for the normal functioning of immune system (hildeman et al., 2007; feig anf peter, 2007; birge and ucker, 2008). in molluscs and other non-model invertebrates, studies of the roles of apoptosis in immunity have been hampered by the scarcity of genetic information and available molecular tools. however, the high degree of evolutionary conservation seen in the apoptotic pathways between vertebrates and invertebrates provide both research tools and motivation for the students of molluscan immunity to venture into this exciting new field and to study the role of apoptosis in immune defense and hostpathogen relationships in mollusks. 54 http://www.marinegenomics.org/ the importance of cell death in the immune defenses of molluscs has long been proposed, even before the discovery of programmed cell death. thus, in an early paper by michelson (1963), cell death (which at that time was considered to be exclusively due to necrosis) is cited as an important immune defense mechanism of gastropods against a variety of pathological agents including trematodes, protozoa, and bacteria (cited by glinski and jarosz, 1997). the importance of apoptosis in the functioning of the molluscan immune system is reflected by the detection of high baseline apoptosis rates that range from 5 to 25 % in circulating hemocytes and can reach to up to 50 % in infiltrating tissue hemocytes (sunila and labanca, 2003; sokolova et al., 2004; goedken et al., 2005; cherkasov et al., 2007). typically, granulocytes show higher levels of apoptosis than hyalinocytes possibly due to the higher phagocytic and oxidative respiratory burst activity in granulocytes (sunila and labanca, 2003; goedken et al., 2005). apoptosis of immune cells can play an important role in protection against parasites and pathogens by the innate immune system. apoptosis is immunologically silent and does not induce inflammation (birge and ucker, 2008; yeretssian et al., 2008). thus, apoptosis of the infected cells is thought to dampen pathogen spread yet protect the integrity of surrounding tissues by limiting potentially damaging inflammation. recent studies in vertebrate models has shown that some pathogens, especially those obligate intracellular parasites that depend on survival of the host cells for their persistence, have evolved strategies to inhibit apoptotic cell death (review in böttger et al., 2008). inhibition of apoptosis in mammalian cells has been observed following infection with intracellular pathogens including leishmania donovani, trypanosoma spp. and theileria spp. (luder et al., 2001; bruchhaus et al., 2007). this parasite-induced inhibition of apoptosis is associated with decreases in caspase-3 activity, inhibition of pro-apoptotic protein kinase c–mediated c-fos gene expression, tnf-α induction and/or changes in nf-κb expression in host cells (nash et al., 1998; heussler et al., 2001; goebel et al., 2001; aga et al., 2002). bacteria, such as chlamidia, pneumococci, or rikketsia, and viruses can also prevent apoptosis of the infected host cells via interference with nf-κb signaling, caspase activation, or balance of proand anti-apoptotic members of bcl-2 family of proteins (böttger et al., 2008). furthermore, pharmaceutical or genetic inhibition of apoptosis renders hosts more susceptible to intracellular pathogens indicating that apoptosis triggered by these pathogens is protective for the host and could play a beneficial role in eliminating the infection (böttger et al., 2008). in molluscs, induction of apoptosis upon contact with pathogens or parasites, and parasite-induced inhibition of apoptosis have both been described. for example, hemocytes of the pacific oyster (crassostrea gigas) show an induction of apoptosis during phagocytosis of live or heat-killed marine bacteria planococcus citraeus (terahara and takahashi, 2008). elevated levels of apoptosis have been associated with the oxidative burst of hemocytes and are abolished by treatment with antioxidants suggesting that apoptosis may be induced by oxidative damage to hemocytes during bacterial killing (terahara and takahashi, 2008). symbiotic bacteria of some cephalopods (vibrio spp.) can also induce apoptosis in host cells (mcfall-ngai, 1999). importantly, our earlier studies have also shown an early increase in apoptosis of oyster hemocytes upon infection by the intracellular protozoan parasite, perkinsus marinus, which then returns back basal levels, perhaps due to parasiteinduced inhibition of the apoptotic response (fig. 3; sokolova, grewal and hughes, unpublished data). apoptosis levels in resident tissue hemocytes of oysters has also been shown to be dramatically reduced from approximately 50 % to 10-11 % in oysters naturally infected by p. marinus or another obligate intracellular protozoan parasite, haplosporidium nelsoni (sunila and labanca, 2003). notably, infection with p. marinus results in a significantly faster induction of apoptosis in hemocytes derived from p. marinus-resistant pacific oysters (c. gigas) than those from p. marinussusceptible eastern oysters (c. virginica) suggesting that faster induction of apoptosis may be an effective defense mechanisms against this intracellular parasite (goedken at al., 2005). overall, it is apparent that while apoptosis induction and the possible subversion of this response by successful parasites and pathogens may play a critical role in both disease resistance and parasite/pathogen development and transfer, our knowledge about the occurrence of, and mechanisms responsible for, apoptosis in infected mollusks is currently limited. clearly, further studies are urgently needed to determine how specific and wide-spread the apoptotic response to infections is fig. 3 parasite-induced apoptosis in molluscan hemocytes. hemocytes of oysters crassostrea virginica were infected with an intracellular pathogen perkinsus marinus, and tem obtained 30 min postinfection. (hemocyte, h; p. marinus cells, pm). arrows show apoptotic bodies of dying hemocytes. 55 in molluscs, and whether it differs in response to bacterial and viral pathogens, and to uniand multicellular eukaryotic parasites (especially schistosoma and other trematodes). summary and future directions recent advances in the fields of molluscan immunology and fundamental cell biology have brought about significant breakthroughs in our understanding of the mechanisms underlying apoptotic cell death in molluscan cells and, in particular, their immune cells. these studies reveal a high degree of evolutionary conservation of key signaling and execution pathways of apoptosis and indicate that programmed cell death likely plays a key role in homeostasis and functioning of the molluscan immune system. however, our knowledge about the molecular mechanisms of molluscan apoptosis and its immunomodulatory and immune defense roles is currently far from complete. further studies are urgently needed to delineate the mechanisms underlying apoptosis in molluscs. despite the high degree of evolutionary conservatism (attesting to the key role of this process in this organisms’ survival), some molluscan apoptotic pathways and induction mechanisms appear to be sufficiently different from those defined in invertebrate models such as drosophila and caenorhabditis, or in vertebrates, to expect that studies of molluscan apoptosis will yield important discoveries of the novel and evolutionarily distinct ways in which “molluscs do it”. our current studies of the abilities of parasites and pathogens to dysregulate molluscan apoptosis have also barely scratched the surface, and many exciting discoveries await researchers in the field. molluscan genome sequencing projects (such as those proposed for biomphalaria glabrata and c. gigas) and molluscan est libraries may provide essential new molecular tools for probing these mechanisms and their role(s) in the molluscan immune system. more studies are needed to determine the molecular mechanisms underlying pathogen-induced modulation of apoptosis in molluscan cells and these will provide significant insights into immune avoidance and the evolution of the host-parasite arms race that is apparent between molluscs and their parasites/pathogens. such studies will also help to identify the crucial physiological and biochemical pathways that may prove therapeutic targets to prevent proliferation and spread of the mollusc diseases. another interesting immunological aspect of apoptosis that has not been addressed in molluscs (or any other invertebrate model) is the suppression of the immune responses by apoptotic cells, and the role of parasite-induced apoptosis dysregulation in intra-host competition between parasites. in mammals, interaction of macrophages with apoptotic cells inhibits inflammatory cytokine and chemokine secretion thereby limiting immune responsiveness (birge and ucker, 2008). this immunosuppressive effect of apoptotic cell bodies appears to be conserved across metazoan evolution (birge and ucker, 2008) and so it would be interesting to determine whether parasite-induced suppression of apoptosis in molluscan hemocytes (such as seen in perkinsus spp. or haplosporidium spp. infections) not only assists in proliferation and spread of the parasites responsible for this inhibition, but may also prevent secondary infections by subsequent invaders thus reducing intraand interspecific competition between parasites within a host individual. unlike homeostatic mechanisms in mammals that closely regulate the organism’s body temperature, osmolarity, and ph, these parameters are allowed much wider variations in molluscs following changes in their environment. the ramifications of extrinsic and intrinsic changes in apoptosis on the immune system of mollusks are poorly understood and require further investigation. while excessive apoptosis due to an environmental stress may suppress the immune system, moderate stimulation may modulate host-parasite relationships and alter disease outcome. in either case, understanding these interactions will be crucial to predict the proliferation and dissemination parasites and pathogens in natural populations of molluscs and potentially, their role as vectors in the diseases of domestic animals and humans. knowledge of the molluscan apoptotic mechanisms could also provide critical information for the development of immortalized mollusk cell lines. currently, no such lines are available for marine mollusks, and the existing cell lines of biomphalaria are notorious for their difficulty to maintain in culture. development of new molluscan cell lines by suppressing apoptotic cell death in culture would provide critical new tools for advancement of experimental cell biology and physiology of mollusks. important as it is, apoptosis is not the only type of the programmed cell death in multicellular organisms. other highly conserved forms of pcd such as autophagy and pyroptosis (a proinflammatory, caspase-1-dependent pcd) have been described and may play a role in immune defense. currently, the role of the alternative forms of pcd in the immune defense has been underexplored, and their mechanisms, or even occurrence, in mollusks awaits further investigation. acknowledgements this work was supported by funds provided the national science foundation career award (ibn0347238), north carolina sea grant (award # 20062175-01), faculty research grant from unc charlotte and a unc charlotte advance program bonnie cone fellowship (through an nsf advance institutional transformation program grant, nsf-0548401). the author is grateful to drs marriott, gorbushin and sukhotin for their helpful comments on an earlier draft of this manuscript, and to mr cherkasov for his invaluable assistance with tem microscopy. references abele d, heise k, portner ho, puntarulo s. temperature-dependence of mitochondrial function and production of reactive oxygen species in 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identification of outer membrane protein ompr from rickettsia-like organism and induction of immune response in crassostrea ariakensis. mol. immunol. 45: 3198-3204, 2008. 58 http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22li%20ly%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22luo%20x%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22wang%20x%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22mcfadden%20dw%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus 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http://www.ncbi.nlm.nih.gov/pubmed/18568041?ordinalpos=101&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/18568041?ordinalpos=101&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/18559474?ordinalpos=102&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/18559474?ordinalpos=102&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22zhu%20b%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22wu%20x%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus javascript:al_get(this,%20'jour',%20'mol%20immunol.'); minireview isj 6: s35-s45, 2009 issn 1824-307x review genetic perspectives on the ascidian central nervous system a locascio*, f ristoratore*, a spagnuolo, l zanetti, m branno cellular and developmental biology laboratory, stazione zoologica “anton dohrn”, villa comunale, napoli, italy *equal contribution accepted march 13, 2009 abstract in 2002, date of publication of the ciona intestinalis genome, ascidians entered the post-genomic era. this tool had a fundamental role and has become the starting point for a series of new functional and genomic studies. recently, great efforts have been done to characterize the genetic cascades of genes having a key role in early embryonic development and to draw the regulatory networks in which they are involved. in this review, we focused our attention on the last advances obtained in the attempt to clarify the complex molecular events governing ascidian central nervous system development with a special interest for anterior neural and sensory structures. we discussed the more recent theories on its early induction and late regionalization. in particular, we used some conserved genes fully or partially characterized as examples to compare ascidian and vertebrate central nervous system (cns). by integrating the various results obtained with microarray, morpholino loss of function and promoter analyses, we showed that many progresses have been done to unravel the gene networks controlling early cns induction and formation. unfortunately, fewer advances have been done in the identification of the regulatory cascades controlling late cns regionalization and sensory organs differentiation. some results are discussed to point out the importance of fully characterizing also these specific regulatory cascades. key words: ascidian; nervous system; genetic pathway; genome; regulatory network   introduction situated at the base of the chordate lineage, ascidians possess many features shared with vertebrates; the larval form, in particular, has been recognized as evolutionarily significant because it reveals features of the early evolution of the vertebrate body plan. due to their relative simplicity and their crucial phylogenetic position, ascidians have the unique potential to illuminate molecular mechanisms underlying the ancestral body plan from which modern chordates diversified. in particular, the larval nervous system of ciona intestinalis, consisting of a brain vesicle, a visceral ganglion and a tail nerve chord, is simple but well differentiated. the ciona central nervous system (cns) can thus be viewed as a miniature prototype of the chordate brain and represents an exciting experimental model to understand ancestral features of the chordate nervous system, its development and physiology. ___________________________________________________________________________ corresponding author: annamaria locascio cellular and developmental biology laboratory stazione zoologica “anton dohrn” villa comunale, 80121, napoli, italy e-mail: anny@szn.it thanks to their transparent embryos, ascidians have been a model system for embryological studies for over a century. a number of new methodologies, developed in recent years and ranging from molecular, genomic, and physiological approaches, are greatly contributing to the knowledge of the complex regulatory networks underlying ascidian cns development. the sequencing of c. intestinalis (dehal et al., 2002) and ciona savignyi (http://www.broad.mit.edu/annotation/ciona/) genomes and the accumulation of molecular resources, that rival those available for fruit flies and mice, introduced ascidians into the post-genomic era. ciona embryos are readily amenable to experimentation in the laboratory and in vitro fertilization can produce thousands of synchronously dividing embryos that develop rapidly. ascidians are hermaphrodites, and the ability of ciona to self-fertilize provides a rapid means for identifying recessive zygotic mutations (moody et al., 1999; sordino et al., 2001). a screening for mutations naturally occurring in a large percentage of wild ciona populations (sordino et al., 2008), identified more than 40 % of the   s35 mailto:anny@szn.it mutations lead to morphological alterations at level of brain and sensory vesicle formation. this natural genetic polymorphism constitutes a valuable source of phenotypes for studying embryonic development. developmental mutants have also been obtained in both ciona species through chemical mutagenesis (moody et al., 1999; sordino et al., 2000; sordino et al., 2001) and, more recently, through insertional mutagenesis using the minos transposable element (sasakura et al., 2003). additionally, recently generated stable transgenics expressing gfp represent useful tools for investigations on mechanisms of tissue formation during embryogenesis and morphogenesis. both mutagenesis and stable transgenesis in ciona require the rearing of animals in the laboratory for several generations. this requirement has been assisted by the development of new culture techniques and maintenance protocols (cirino et al., 2002; hendrickson et al., 2004), and recently improved by introducing the use of closed, recalculating sea water systems to permit ciona culture in inland laboratories (joly et al., 2007). using electroporation, transient transgenesis is successfully performed in ascidians allowing characterization of cis-regulatory dnas (alfano et al., 2007; corbo et al., 1997; erives et al., 1998; ristoratore et al., 1999). the wide number of tissuespecific enhancers currently available allows both the production of mutant phenotypes, via ectopic expression of regulatory genes (spagnuolo and di lauro, 2002; takahashi et al., 1999), and disruption of gene activity through the overexpression of dominant negative proteins (corbo et al., 1998). injection of morpholino antisense oligonucleotides (satou et al., 2001) provides an alternative method for the analysis of loss of gene function. the use of comprehensive microarrays has revealed extensive temporal patterns of gene activity (azumi et al., 2007a) and assisted in the determination of spatial patterns of gene expression within individual blastomeres in sequentially staged embryos (yamada et al., 2005). recently, a simple method allowing the maintenance of dissociated cells from ciona in primary cultures has been developed (zanetti et al., 2007). cells that conserve their functionality have been successfully cultured, opening the possibility that this method could be used in a wide range of experiments in this animal model. in particular, this method allowed the identification of two types of neurons resembling motorneurons and large eminens cells (imai and meinertzhagen 2007a, b). taking into account that the ascidian nervous system is largely inaccessible to neurophysiological studies because of the tough outer tunic of the larvae, primary cell culture represents a useful system to overcome these limitations. collectively, all these technical advancements in ciona provide powerful tools for the study of the gene networks controlling chordate development. here we summarise the more recent advances on the genetic mechanisms underlying the development of nervous system structures, highlighting the similarities as well as the differences observed across chordates. we will focus primarily on c. intestinalis and, where appropriate, we discuss also findings from other ascidian species. ascidian cns in chordate evolution six years have passed from the publication in science of the c. intestinalis genome sequence (dehal et al., 2002) and since then much progress has been made in the knowledge of both genomic and developmental processes. the genomes of another ciona species, c. savignyi (http://www.broad.mit.edu/annotation/ciona/) and of the appendicularian oikopleura dioica (http://www.genoscope.cns.fr/spip/oikopleuradioica-whole-genome.html) have been sequenced and assembled. advances in genome annotation (ensembl), fish analysis (shoguchi et al., 2008) and identification of aflp markers (kano et al., 2006) greatly contributed to the construction of ciona chromosomal maps. shoguchi et al. (2008) mapped about 82 % of the genome sequence information on all arms of the ciona chromosomes. what emerged from these studies is that regulatory genes and their targets, belonging to the same developmental gene networks, are distributed over all fourteen chromosomes. thus, genes whose genomic regulation and functions are interconnected do not need physical clustering to be coordinately activated. all together, this new piece of information makes ascidians a very powerful model system for functional genomic studies. among the main novelties there is the new ascidian phylogenetic position as sister group to the vertebrates, a position long thought to be held by the cephalochordates. taking advantage of the genome sequence of the tunicate oikopleura dioica, a set of 146 genes from 14 deuterostome species have been aligned and phylogenetically analyzed. the results suggest that tunicates do not represent the earliest chordate lineage, as previously inferred, but in fact they are the closest living relatives of vertebrates (delsuc et al., 2006). recently the ciona genome release has been complemented by large scale cdna and est projects accompanied by expression analyses (http://ghost.zool.kyoto-u.ac.jp; imai et al., 2004; satou and satoh, 2005). cdna and oligo-dna microarrays allowed the construction of new urochordate expression map databases (azumi et al., 2007b; yamada et al., 2005) and detailed expression profiles are now available for most of the genes encoding cell signaling and regulatory proteins. a three dimensional computational software organizes and integrates gene expression data with the complex single cell developmental programs (http://crfb.univ-mrs.fr/aniseed/). a three dimensional ascidian body atlas database shows interactive developmental tables of 26 newly defined embryonic stages, accompanied by cell lineage descriptions and time laps photos (http://chordate.bpni.bio.keio.ac.jp/faba/1.2/top.html). all these data, together with the key position of ascidians at the base of vertebrate origin, give this system the power to clarify the complex gene networks governing chordate evolution and   s36 http://www.genoscope.cns.fr/spip/oikopleura-dioica-whole-genome.html http://www.genoscope.cns.fr/spip/oikopleura-dioica-whole-genome.html vertebrate development. the regionalized expression patterns in ascidians of specific markers of vertebrate forebrain, midbrain, mid-hindbrain boundary (mhb) and hindbrain, such as otx, pax2/5/8, en, and the hox genes, suggest a tripartite organization of the ascidian cns. in particular, the sensory vesicle, marked by the otx gene, is considered homologous to the vertebrate fore-midbrain; the neck region marked by pax2/5/8 and en is considered to be homologous to the vertebrate mhb; finally the visceral ganglion marked by hox genes corresponds to the vertebrate hindbrain (fig. 1) (imai et al., 2002; wada et al., 1998). the evolution of the cns and in particular of its anterior structures is a crucial point in the chordate lineage. vertebrates have a complex, segmented and highly organized cns that evolved from the same chordate ancestor of ascidians. many complex and still poorly defined regulatory networks governing nervous system development and differentiation can be clarified by taking advantage of recent progress in functional genomic studies and the compact and non redundant genome of ascidians. the ascidian larva is composed of only ~2600 cells of which about 330 cells constitute the cns. following the basic chordate body plan, neurulation gives rise to a sensory vesicle of ~215 cells, including two pigmented sensory organs and, in sequence along the antero-posterior axis, to the neck, the visceral ganglion and dorsal hollow nerve cord (nicol and meinertzhagen, 1991). more recently, the presence in ciona of a midbrain homologous region has been reconsidered based on a comparative analysis of the expression pattern of dmbx, a marker of the midbrain in vertebrates (takahashi and holland, 2004). takahashi and holland (2004) demonstrated that the ascidian dmbx gene is expressed only in the visceral ganglion, in a region that is posterior to the pax2/5/8 expression territory (mhb homologue) and coincident with rostral limit of hox gene territories (hindbrain homologue). the presence of the ciona dmbx gene only in the visceral ganglion could correspond to the expression of vertebrate dmbx in the hindbrain (fig.1), thus reinforcing visceral ganglion homology with the vertebrate hindbrain and suggests that a midbrain homologue is missing in ciona. data from amphioxus, where no dmbx expression is observed in the neural tube (takahashi and holland, 2004), further support the notion that the midbrain is a novelty that evolved specifically in the vertebrate lineage. despite the evident morphological differences between ascidian and vertebrate cns structures, there are clear homologies in nervous system patterning as deduced from the expression patterns of many developmental genes. the otx gene defines the forebrain of vertebrates, and marks also the ascidian larva sensory vesicle (fig. 1). this region has been compared to the vertebrate rostral cns and is considered analogous to the forebrain (wada et al., 1998). a series of vertebrate genes, foxh1, nkx2.1 and otp, are markers of the developing hypothalamus and specifically are involved in cell fate restriction, hypothalamus primordium delineation and neuroendocrine hypothalamic nuclei differentiation. the ascidian homologues (ci-foxha, ci-nkx2.1 and ci-otp) were found to be expressed in regionalized patterns within the ciona ventro-lateral sensory vesicle. these data suggest a possible correspondence between these vertebrate and ascidians structures (moret et al., 2005). supporting this suggestion of homology, the coronet cells on the left side of the sensory vesicle in ascidians have been structurally compared to the cells of saccus vasculosus in the vertebrate hypothalamus (katz, 1983). similarities can also be observed at the level of cns patterning, exemplified by the regionalized expression domains of hox genes. nine hox genes have been identified in the ciona genome (dehal et al., 2002; spagnuolo et al., 2003), which have undergone a significant reorganization, including rearrangement of the gene positions, dispersion, and breakage on separate chromosomes. despite the extensive shuffling of ciona hox gene cluster, some of the gene members maintain a coordinated expression in the larval cns. ci-hox1, 3, 5 and 10, indeed, show a restricted and colinear expression profile along the antero-posterior axis at the level of the visceral ganglion and nerve cord, which are considered homologous to vertebrate hindbrain and spinal cord respectively (gionti et al., 1998; ikuta et al., 2004; locascio et al., 1999). recently, a detailed analysis in ciona at various embryonic stages of the dynamic expression profiles of ciotx, pax2/5/8, cifgf8/17/18, en and hox genes in part supports the tripartite model but in part indicates that also a dipartite model, lacking the mhb region, may be consistent (ikuta and saiga, 2007). the expression profiles of pax2/5/8 and en in the neck region and the presence of an intervening gap between otx and hox gene expression territories in larval stages support the tripartite organization (fig.1). expression of fgf8/17/18 seems to be in contrast with this model. in vertebrates, fgf 8 is expressed in the mhb and play a pivotal role in the organizer activity (the capacity to change the fate of surrounding tissues when transplanted in other cns territory) of this region (liu and joyner, 2001). in ciona, the absence of fgf8/17/18 expression in the neck region and its presence only more posteriorly in the visceral ganglion (fig.1) could indicate that ascidians possess an ancestral mhb lacking organizer activity. on the other hand, the lack of a gap between otx and hox domains at earlier stages of development supports a dipartite organization. dufour (2006) proposed a third model based on ciphox2 expression at larva and adult stages. in vertebrates phox2 genes (phox2a and phox2b) mark the hindbrain and define cranial motorneurons of the branchiovisceral class. in ascidians, ciphox2 is restricted to the neck region at larva stage and will   s37 fig. 1 comparison of gene expression domains in the central nervous system of ascidians and vertebrates. homologous genes are indicated by the same colours. the most anterior regions are marked by otx (blue) genes and correspond to the sensory vesicle in ascidians and the forebrain and midbrain in vertebrates. dmbx gene (dark blue) is expressed in the midbrain only in vertebrates, while it is present in the hindbrain (visceral ganglion) in both chordate organisms. expression of pax2/5/8 (light blue) and en (green) in the intervening region between otx and hox genes (hox1, red; hox3, orange; hox 5, yellow; hox10, dark green) is common to both vertebrates and ascidians. fgf8/17/18 (lilac) is a marker of the mhb region, at the junction between midbrain and hindbrain, in vertebrates while it is expressed more posterior in the visceral ganglion in ascidians. d, dorsal; v, ventral. give rise to the adult motorneurons located in the cerebral ganglion. these results, together with hox genes expression profiles, suggested a possible homology of the posterior half of the neck with vertebrate hindbrain and of the visceral ganglion with spinal cord (dufour et al., 2006). unfortunately these controversial data, together with the absence in ascidian genome of another marker of vertebrate mhb, the gbx gene (castro et al., 2006), make it difficult to clearly establish which model most appropriately reflects the ascidian condition and thus, the appearance during chordate evolution of the mhb organizer. gene expression analyses in additional deuterostome organisms (eg. hemichordate) could help to elucidate the ancestral situation of the basic patterning of cns and the subsequent evolutionary steps that led to the differentiation of the highly complex vertebrate nervous system. cns regulatory networks ascidian neural specification is organized via conserved chordate gene networks. these networks have been expanded during vertebrate evolution to create more complex neural structures. in ascidian embryos, blastomere divisions are initially synchronous, and become partially asynchronous later in development. invariant cleavages and cell lineages give rise in ciona to about 330 neural cells, of which about 2/3 form the sensory vesicle, 47 the visceral ganglion and 95 the caudal nerve cord. at the 110 cell stage, most of the blastomere fates have been already determined; descendents of the   s38 a-line form the anterior neural structures, while the neural tube derives from the b8.19 and a7 cell lines (cole and meinertzhagen, 2004). it was already known that fgf9/16/20 has a fundamental role in inducing anterior neural differentiation in the a-line, while nodal and erk signaling are involved in caudal neural tube formation (imai et al., 2006; lemaire et al., 2002). imai et al. (2006) contributed much to our knowledge of gene networks involved in ciona neurogenesis. large scale morpholino assays and subsequent characterization by rt-qpcr of altered gene expression in the mutant phenotypes permitted the establishment of a series of regulatory relationships at the level of a single cell. in particular, they focused their attention on regulatory genes specifically localized in the blastomeres determined to acquire a particular cell fate. the analysis and comparison of significant changes in the expression profiles of genes permitted the description of the early networks that lead to the induction of different tissues and to the establishment of restricted cell fates (imai et al., 2006). unfortunately, morpholino studies do not permit the observer to unequivocally distinguish between direct and indirect target genes. the combination of morpholino “loss of function studies” of genes of interest together with promoter analyses of their candidate downstream genes has a great potential for the elucidation of regulatory networks and reconstruction of the precise genetic cascades leading to a specific tissue differentiation. ascidians represent indeed a very suitable model system to perform regulatory element assays given their compact genomes and the frequent location of minimal promoters approximately <1 kb upstream of the transcription start sites of the genes of interest. moreover, the opportunity to compare specific genomic fragments between closely related species represents a further advantage that these organisms offer. additionally, electroporation is a relatively simple and efficient method that gives hundreds of transgenic embryos (corbo et al., 1997). an interesting example of the results that can be achieved combining loss of function and gene promoter studies is offered by the reconstruction of the neural induction and otx genetic cascade during early embryonic development. ascidian ectodermal induction starts at the 8cell stage when ci-gataa expression is repressed in vegetal territories by β-catenin accumulation, and thus restricted to the animal hemisphere. at 816 cell stage, ci-gataa activates pan-animal pole genes, such as ci-fog, while fgf9/16/20 is active as a neural inducer in the a-line. at the 32 cell stage brain specification begins with the activation of the otx gene through fgf9/16/20 signaling. studies of the ci-otx promoter revealed the presence of seven ci-gataa sites for animal activation and two ets binding sites for its restriction to neural cells induced by fgf (bertrand et al., 2003; rothbacher et al., 2007). in particular, fgf9/16/20 activates otx and dmrt1 and both of them, on their own, activates transcription factor genes such as six1/2 and six3/6 (imai et al., 2006). nodal is a signaling molecule of the tgfβ factor superfamily, and acts as an organizing signal in the development of the nerve cord. at the 32 cell stage, fgf9/16/20 activates also nodal whose expression is restricted to the b6.5 cells through the activity of both soxc and foxa. nodal activates msxb, pax3/7, snail, delta-like and chordin in the b6.5 cell line and induces roof nerve cord differentiation. in the a7.8 lineage, nodal induces snail, delta-like and neurogenin to form the lateral ependymal cells of the nerve cord (imai et al., 2006). these studies have extended our understanding of the first phases of neural induction and formation; on the other hand, our knowledge on the gene networks acting later in development, during nervous system regionalization and differentiation, is still very poor. in most cases the roles of genes temporally expressed at various stages of development have been studied in detail only at early embryonic stages by morpholino loss of function experiments; their roles at later stages are still largely unknown. only few genes specifically expressed later in development, and potentially involved in neural structure differentiation, have been studied in detail. in most cases their function, and the genetic cascades in which they are involved, are only partially defined and remain unconnected to other known regulatory networks. to analyze the function of genes involved in late events of cns differentiation, a morpholino approach may give results difficult to interpret; studies of their transcriptional regulation could be more informative on the regulatory networks in which these genes are involved. here we offer three examples of genes expressed in the cns at late stages of ciona development; dissection of their promoter regions has already given some clues to the elements controlling their expression. further studies are needed to identify their upstream regulators in order to define some steps of the genetic circuits at the bases of cns regionalization. hox genes represent a good example of conserved regulatory genes involved in late cns regionalization. several ascidian hox genes show conserved expression patterns, indicating a role in patterning the antero-posterior axis, however most studies treat their expression profiles, genomic, and chromosomal organization adding little functional data. the study of cihox3 promoter, the only one deeply analysed, led to the characterization of an 80-bp element found to be sufficient to recapitulate the endogenous expression pattern of cihox3 (locascio et al., 1999). unfortunately this sequence did not show any likely recognition sequence for known transcription factors, this requires a deeper bioinformatics analysis to identify putative binding sites. the 80-bp cihox3 promoter fragment however served to address whether hox3 regulatory elements have been conserved during chordate evolution. the 80-bp cihox3 promoter fragment, when tested in mouse embryos, was unable to reproduce any neural-specific expression in transgenic mice, thus suggesting that the elements regulating hox3 expression are not conserved   s39 between ciona and mouse. nevertheless, it is interesting to note that, when a slightly larger enhancer fragment was tested, a reproducible segmental pattern of expression in the mouse hindbrain was obtained, suggesting that some elements acting in ciona are recognised by mouse transcriptional machinery (locascio et al., 1999). these early interesting results give rise to the possibility of identifying hox specific enhancer elements responsible for antero-posterior regionalization that have been conserved during chordate evolution. another example of an important regulatory gene, whose function has been remarkably well conserved during evolution, is the pax6 gene. pax 6 encodes a transcription factor that has been implicated in the development of eyes and portions of anterior nervous systems, throughout the animal kingdom. recently the regulatory region of the c. intestinalis pax6 has been analyzed and compared with that of mouse and drosophila pax6 (irvine et al., 2008). the results showed a similar level of complexity in cis-regulatory elements on a gross scale. the three species have regulatory elements located in a large intron near the 5' end of the gene, able to drive transcription in photoreceptors, brain and nerve cord. both drosophila and ciona have major enhancers for brain and nerve cord upstream of the transcription start site. despite these similarities in the genomic organization, no sequence similarities have been found using blast alignment, suggesting that after divergence of a common ancestry sequence similarities have been obscured by binding site turnover and rearrangements (irvine et al., 2008). a final example derives from studies of the msx family of transcription factors. members of the msx family are among the regulatory genes expressed from early to late embryonic stages and showing several different functions during development. msx genes show highly conserved functions during evolution in neural patterning and dorso-ventral subdivision of the embryonic neuroectoderm (d'alessio and frasch, 1996; bendall and abateshen, 2000). only one msx gene is present in ciona genome and it shows an interesting expression pattern in the mesenchyme and nervous system precursors starting from the early gastrula stage (aniello et al., 1999). in larval stages, the expression is maintained in the sensory vesicle and in the visceral ganglion. loss of function experiments, via morpholino oligonucleotide microinjection, gave some information on the role played by msx in muscle differentiation, but they failed to give crucial information on msx function in nervous system development, probably because this later function was masked by early expression (imai et al., 2006). in this case, the characterization of the regulatory region could give important insights on the regulation of this gene, in different tissues and at different developmental stages, and could help to place msx gene in the network leading to anterior cns regionalization. it has been demonstrated that the specific expression of msx in the neural precursors is regulated by a 30-bp region that is able to recapitulate the endogenous expression in the nervous system both at neurula and tailbud stages. comparison of the ci-msx promoter with that of murine msx-1 did not show any sequence homology, suggesting that these genes are regulated by different networks. unfortunately, although some putative binding sites have been identified in the 30-bp region, the factors responsible for msx activation have not yet been identified (russo et al., 2004). further studies are necessary to reconstruct its specific role in the nervous system regionalization and the genetic cascade in which it is involved. it is possible to argue from the fore mentioned examples that we have only begun to exploit the enormous potential of regulatory region analyses in ciona. recently, a database has been created containing information on regulation of tunicate genes collected from literature. it includes information regarding the minimal promoter length, the transcription factors involved and their binding sites, as well as the localization of the gene expression (sierro et al., 2006). further improvements, such as the inclusion of information on the regulation of halocynthia roretzi, c. savignyi and o. dioica genes, and continuous updating will greatly contribute to reconstruct genetic networks. cns sensory organ differentiation the brain vesicle of ascidian larvae contains two distinct pigmented sensory organs clearly visible through its transparent body. the more anterior pigmented sensory organ, the otolith, is involved in the perception of gravity, whereas the more posterior one, the ocellus, is involved in the perception of light stimuli. the two sensory organs are responsible for the swimming behaviour of the larva (tsuda et al., 2003). the otolith is composed of a large spherical cell attached to the ventral wall of the sensory vesicle by a narrow stalk. it contains a single pigment granule that occupies most of the cell body. the ocellus is composed of a cup-shaped pigmented cell, a number of photoreceptors and three lens cells (dilly, 1969; nicol and meinertzhagen, 1991). the ascidian ocellus is considered a simple eye. the presence of photoreceptors, of 3 lens cells and of a pigmented cell in the ocellus, suggests some similarity with vertebrate eye. the ascidian “simple eye”, with its close association of the pigment cell and photoreceptors, may thus offer a “unique possibility” to study the developmental programs bringing to both melanization and photoreceptors differentiation. the “unique possibility” is related not only to the simplicity of the system but also to the very well characterized lineages of photoreceptor and pigment cells. the molecular mechanisms regulating ascidian pigment cell development are not yet clear. the two pigment cells arise from the paired a8.25 blastomeres that are positioned bilaterally in the gastrula ascidian embryo and will give rise to the a9.49 pair at neural plate stage (fig. 2a, b). it is known that determination of a8.25 cells as pigment cell precursors requires direct inductive influence from the nerve cord precursor cells at the gastrula stage. at this stage the a8.25 blastomeres constitute   s40 fig. 2 cell lineage of pigment cell precursors (light blue) in ascidian embryos at 110 cell, neural plate and tailbud stages (a-c). expression pattern of chordin gene in halocynthia roretzi (d) and ciona intestinalis (h) embryos at tailbud stage. white arrow in d indicates chordin expressing cell in the sensory vesicle. image d is from darras et al., 2001. an equivalence group in the sense that both have the potential to form either an ocellus or otolith (darras and nishida, 2001). the choice to adopt either otolith or ocellus cell fate is made after neural tube closure at early tailbud stage and requires cellcell interactions. as the neural tube closes, the four cells derived from the division of the two a8.25 cells, the a10.97 and a10.98 pairs, converge and intercalate along the anterior-posterior axis in order to align along the midline (fig. 2c). the a10.98 pair gives rise to part of the brain vesicle, the anterior a10.97 forms the otolith while the posterior one differentiates into the ocellus pigment cell. in the ascidian h. roretzi, the choice to become ocellus or otolith seems to be influenced by the antagonistic effect of bmp, expressed at tailbud stage in the four tyrosinase positive cells (a10.98 and a10.97 pairs), and chordin, secreted from the adjacent posterior cell (fig. 2d) (darras and nishida, 2001). it seems that high doses of bmp inhibit pigmentation (as in the a10.98 pair), intermediate doses allow the development of an otolith (in the anterior a10.97) and low levels lead to ocellus development (in posterior a10.97) (darras and nishida, 2001). nevertheless this mechanism seems not to be universal, as suggested by the wide expression pattern of chordin in the anterior cns of c. intestinalis tailbud embryos (fig. 2e; personal unpublished data). this evident difference render it unlikely that in ciona bmp/chordin could be the sole responsible for the induction of the posterior a10.97 blastomere to form the ocellus. further studies are necessary to elucidate the molecular mechanisms underling early and late regulation of pigment cell formation. these studies will take advantage of the analysis of molecular markers specific for the pigment cell precursors at different stages of development. some genes involved in the process of melanization have been identified. tyrosinase expression, as well as its enzyme activity, has been detected in the two types of pigment cells in several ascidian species, suggesting that this enzyme is conserved as key enzyme for melanin synthesis and that melanin has similar chemical characteristics to the melanin of vertebrates. moreover, the expression pattern of a trp (tyrosinase related protein) has been analysed as well as a mitf gene (microphthalmia transcription factor); both show an expression pattern correlated, in some developmental stages, with restriction of pigment cell fate indicating their potential involvement in this process as in higher chordates. in the ascidian h. roretzi, as in higher chordates (camacho-hubner and beermann, 2000; camacho-hubner et al., 2002; camp et al., 2003; goding, 2000), tyrosinase gene family (tygf) has been used as a model to approach studies on “pigmentation programs” (kusakabe et al., 2001). firstly it has been identified a hrtyr promoter fragment able to direct lineage-specific and developmentally correct expression of hrtyr during embryogenesis. a search for conserved recognition sequences in this promoter has revealed the presence of several putative pax3-binding consensus sites; the data that over-expression of hrpax-3/7 is able to induce ectopic expression of hrtyr (halocynthia roretzi tyrosinase) have reinforced the hypothesis of a direct relation between hrpax-3/7 and hrtyr (toyoda et al., 2000). a parallel approach on the hrtrp (halocinthia roretzi thyrosinase related protein) promoter has permitted the identification of two otx binding consensus sites involved in hrtrp expression; here too, hroth overexpression can transactivate this promoter in an otx site-dependent manner,   s41 confirming a direct function exerted by hroth on hrtrp expression (dehal et al., 2002). the mechanisms of tyrosinase/trp activation seem therefore to be conserved on most aspects during chordate evolution (for a review see murisier and beermann, 2006). in higher chordates, members of the microphthalmia (mitf) transcription factor family play a central role in specification of pigment cell lineage (aksan and goding, 1998; hotta et al., 2000). the absence of mitf binding sites in hrtyr and hrtrp minimal promoter elements, compared with higher chordates, is a little bit surprising, given that molecular data from h. roretzi strongly supports the antiquity of the association of the mitf family members with pigment cells (yajima et al., 2003). one possibility is that mitf, at least in h. roretzi, is not involved in the regulation of hrtyr and hrtrp expression; or that hrmitf boxes, located in the far upstream region, are not necessary but can contribute to a full efficient expression of tyrosinase(s) during halocynthia embryogenesis; or that hrmitf/hrtyr-hrtrp interaction is not direct but mediated by other factors. the scenario emerging from the data collected so far in ascidians point to an evolutionary conservation of most factors involved in pigment cells differentiation. what about photoreceptors? the question is very intriguing given that photoreceptor and pigment cell precursors, in the ascidian c. intestinalis, share at the late gastrula stage common factors, as mitf and bmp5/7, that slightly later, at the neurula stage, become localized specifically in pigment cell precursors (data not shown). studies on photoreceptor differentiation in ascidians have produced very few data so far. opsin (ci-opsin1) (kusakabe et al., 2001) and arrestin (ci-arr) (nakagawa et al., 2002) genes have been isolated and characterized as photoreceptorspecific markers from the ascidian c. intestinalis; a 3kb ci-arr promoter region has been demonstrated to recapitulate the expression of the endogenous gene (ikuta et al., 2004). these genes are directly involved in the visual cycle; studies on their transcriptional regulation could therefore help to clarify the mechanisms involved in terminal differentiation of photoreceptors. only recently a key factor required for the differentiation of vertebrate eye, the rx gene, has been cloned from ciona (ci-rx). “loss of function” experiments have indicated that it is required for ocellus development. in particular, larvae lacking rx function do not develop ocellus pigment cell, lack photoreceptors and are unable to response to light stimuli (d'aniello et al., 2006). furthermore, a ci-rx regulatory region, that recapitulate the expression of the endogenous gene, has been identified (d'aniello et al., 2006). this ci-rx “eye enhancer” could provide interesting keys to unravel the genetic circuits controlling a step just prior to the terminal differentiation of photoreceptors. studies on the transcriptional regulation of genes as bmp and mitf in ascidians could finally be instrumental to shed light on the mechanisms/factors involved in the initial choice pigment cell-photoreceptor. recent work has suggested that pigmented cells of the ocellus and otolith are necessary for sensing light and gravity, respectively (sakurai et al., 2004; tsuda et al., 2003). the behaviour of ascidian larva has long been studied illustrating that ascidian larvae present phototactic and geotactic responses. the larvae become sensitive to light about 4 hours after hatching, responding to decreased light intensity by swimming more actively. a direct proof for the role of pigmentation in ascidian larval physiology have been obtained only recently, taking advantage of two mutant lines of c. savignyi that are unable to make melanin and thus lack pigment in the larval sensory structures. behavioural studies on non pigmented offspring demonstrated that unpigmented larvae are unable to detect source of light and consequently are unable to seek out the shaded location preferred by their wild type siblings (jiang et al., 2005). moreover, the c. savignyi mutant larvae lacking pigmentation do not behave properly in response to gravity. other information could come from the physiological studies of other naturally occurring mutants of c. intestinalis isolated in the gulf of naples, presenting several defects in pigmentation ranging from total absence of pigmented cells to presence of only one pigmented cell (otolith or ocellus) (sordino et al., 2008). conclusions the new molecular tools and the great progresses in deciphering ascidian genome and chromosomal mapping, allowed moving to a further and more advanced step in the comprehension of the complex chordate body organization. from the study of single genes or single genetic cascades, it is now possible to integrate and interconnect the regulatory networks that underlie the organization, function and development of the ascidian nervous system. these progresses refer overall to early steps of neural induction but represent the milestone for future reconstruction of late cns regionalization and differentiation. despite the obvious morphological differences and the divergence of regulatory sequences that, in some cases, have been observed, it seems that most of the main cns developmental programs are conserved between ascidians and vertebrates. this result strongly spurs to use ascidians, with their compact genome and relatively simple cns, as model system to characterize some of the conserved and fundamental steps of chordate neurogenesis. to this aim, it will be very important in the next years to integrate the functional studies (eg. morpholino loss of function) of genes 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silverman, n paquette, k aggarwal university of massachusetts medical school, worcester, usa accepted december 07, 2009 abstract the drosophila immune response is characterized by the rapid and robust production of a battery of antimicrobial peptides immediately following infection. the genes encoding these antimicrobial peptides are controlled by two nf-κb signaling pathways that respond to microbial infection. the imd pathway is triggered by dap-type peptidoglycan, from the cell wall of most gram-negative and certain gram-positive bacteria, and activates the nf-κb precursor protein relish. the toll pathway, on the other hand, is stimulated by lysine-type peptidoglycan from many gram-positive bacteria, β 1,3 glucans from many fungi, as well as by microbial proteases. toll signaling leads to the activation and nuclear translocation of dif or dorsal, two other nf-κb homologs. this review presents our current understanding of the molecular mechanisms involved in microbial recognition and signal transduction in these two innate immune pathways. key words: toll; imd; pgrp; peptidoglycan; antimicrobial peptides overview of drosophila immunity insects, such as drosophila, thrive in microberich environments. not surprisingly, they have evolved complex mechanisms to combat microbial infection. these defenses include structural barriers to infection, such as the cuticle and peritrophic membrane. insects also rely on inducible responses such as phagocytosis, the production of antimicrobial compounds, and homeostatic mechanisms that help repair the damage caused by infection (lemaitre and hoffmann, 2007). together, these defense mechanisms allow insects to be broadly resistant to a large range of pathogens without an acquired immune response. the inducible humoral insect immune response has been most widely studied in the favorite model system drosophila melanogaster, where microbial challenge leads to the rapid and robust production of a battery of antimicrobial peptides (amps). several families of amps have been described in drosophila, with antifungal and anti-bacterial (both anti-gram-negative or anti-gram-positive) activities. some of these amps appear to be unique to insects, e.g., diptericin, while others have homologs in mammals, e.g., defensins, cecropins and drosomycin (lee et al., 1989; simon et al., 2008). ___________________________________________________________________________ corresponding author: neal silverman divison of infectious diseases, department of medicine university of massachusetts medical school 364 plantation st, worcester, ma 01605, ma, usa e-mail: neal.silverman@umassmed.edu as best we know, production of amps is regulated at the transcriptional level. to date, nearly all amp genes have been found to be controlled by nf-κb family transcription factors. drosophila have two distinct pathways which activate nf-κb factors and drive transcription of amp genes following infection. the toll pathway responds to several different types of microbes, including fungi and many gram-positive bacteria, and leads to the activation of the nf-κb family members dif and dorsal. on the other hand, the imd pathway is activated by gram-negative and certain types of gram-positive bacteria and leads to the activation of the nf-κb precursor relish. the details of the how different microbes are detected and discriminated by these two pathways is the main focus of this review. first, the basic outline of these two signaling pathways will be summarized. toll and imd signaling as mentioned above, the toll pathway is able to activate two nf-κb homologs, dif and dorsal. both of these proteins are similar to mammalian p65, and are held in the cytoplasm of unstimulated cells by the drosophila iκb homolog cactus. like mammalian iκbs, cactus is phosphorylated and degraded upon stimulation (fernandez et al., 2001). one outstanding question within this pathway is the identity of the cactus kinase. to date, only the kinase pelle, homologous to the mammalian irak family of kinases, has been shown to function in the 163 mailto:neal.silverman@umassmed.edu figure 1 toll signaling pathway. the toll signaling pathway and its multiple modes of activation in the drosophila immune response. three distinct mechanisms for microbial recognition, leading to the cleavage of spätzle and activation of toll, are illustrated. the mechanisms include peptidoglycan detection, by pgrp-sa, pgrp-sd and gnbp1, β-glucan detection through gnbp3, and protease activity sensing via the serine protease persephone. all these detection modalities lead to the activation of the spätzle processing enzyme (spe) which converts this cytokine into its active form, for binding and activating toll. the intracellular signal transduction downstream of toll is very similar to the myd88-dependent pathway, which functions downstream of most mammalian tlrs. the key features of this pathway include a trimeric receptor associated complex, containing myd88, tube and pelle, which ultimately lead to the phosphorylation and degradation of the iκb homology cactus and the nuclear translocation of nf-κb homologs dif and dorsal. 164 toll pathway. in mammals, irak kinases are indirectly involved in the phosphorylation of iκbs, being required for the initiation of a kinase cascade that culminates in the activation of the iκb kinase (ikk) complex, which directly phosphorylates iκbα (skaug et al., 2009). in flies, a similar kinase cascade may be involved, although the components are not yet identified. alternatively, it remains possible that pelle directly phosphorylates cactus on the residues necessary for its degradation. this issue remains unresolved. it is clear that pelle is a component of a trimeric complex that associates with the active form of the transmembrane receptor toll. like all the mammalian toll-like receptors and the mammalian il-1 receptor, the cytoplasmic domain of drosophila toll contains a tir domain. this tir domain interacts with the drosophila myd88 homolog via a homotypic tir:tir interaction (tauszig-delamasure et al., 2002; charatsi et al., 2003; kambris et al., 2003). myd88 also contains a death domain (dd), which interacts with the protein tube. through its other face, the tube dd also interacts with pelle (towb et al., 1998; sun et al., 2002; sun et al., 2004). this trimeric myd88/tube/pelle complex is thought to interact transiently with the cytosolic tir domain of toll. this association likely leads to the auto-phosphorylation and activation of pelle, which in turn leads, directly or indirectly, to the phosphorylation of cactus. once cactus is degraded, the nf-κb factors dif and dorsal translocate to the nucleus where they control the transcription of target genes. in the adult fly, dif is critical for the activation of amp gene transcription (meng et al., 1999; rutschmann et al., 2000a; de gregorio et al., 2001, 2002; irving et al., 2001). in larvae, redundancy is observed between dif and dorsal, and only a double mutant fails to induce amp genes. in addition to controlling amp gene expression, dif and dorsal together also seem to regulate the survival of hemocytes in larvae (qiu et al., 1998; matova and anderson, 2006). dorsal (and much of the rest of the toll pathway) also plays a critical role in early embryonic development, in patterning the dorso-ventral axis. during development, phosphorylaztion of dorsal is linked to enhanced nuclear localization and transcriptional activation (drier et al., 1999, 2000). dif is not required in development but can partially substitute for dorsal in this process when expressed in the embryo (stein et al., 1998). the role of phosphorylation of dif, in the context of the immune response, has not been examined (for an overview of the toll signaling pathway, see fig. 1). the imd pathway culminates in the activation of a third nf-κb homolog, known as relish. like the mammalian nf-κb precursor proteins p100 or p105, relish contains an n-terminal rel homology domain and c-terminal iκb-like ankyrin repeats. in unstimulated cells, the c-terminal iκb-like domain is believed to hold full length relish in the cytoplasm (stöven et al., 2000). after immune stimulation relish is endoproteolytically cleaved, and the nterminal transcription factor module translocates into the nucleus while the c-terminal iκb-like domain remains in the cytoplasm. in addition to this proteolytic cleavage, full activation of relish also requires phosphorylation on two residues, serines 528 and 529, (erturk-hasdemir et al., 2009). this phosphorylation is not required for relish cleavage, but instead seems to be crucial for the transcriptional activation of some relish target genes. another protein, known as akirin, also functions in the nucleus for the induction of amp gene expression, but how it interacts with relish, if at all, is unclear (goto et al., 2008). relish cleavage and phosphorylation are controlled by two interconnected branches of the imd signaling pathway. serines 528 and 529 are directly phosphorylated by the drosophila ikk complex. this kinase complex includes drosophila ikkβ and ikkγ homologs, also known as ird5 and kenny, respectively (rutschmann et al., 2000b; silverman et al., 2000; lu et al., 2001). ikk activation, in turn, requires the map3k tak1 and its binding partner tab2. interestingly, both tab2 and ikkγ include conserved k63-polyubiquitin binding domains. these non-degratory ubiquitin chains are thought to function in the imd pathway, however the exact molecular mechanisms involved are not yet clear (zhou et al., 2005). the putative e3 ubiquitin ligase diap2 is also required in the imd pathway and may promote k63-chain formation (kleino et al., 2005; leulier et al., 2006; huh et al., 2007). further upstream, kinase activation also requires the imd protein, the drosophila fadd homolog, and the caspase-8 like dredd (leulier et al., 2000; georgel et al., 2001; leulier et al., 2002; naitza et al., 2002). these three proteins may form a trimeric complex as fadd can interact with both imd and dredd. how this complex signals the activation of tak1 is still under investigation. in addition to a poorly defined role in the activation of tak1, dredd is also required for the cleavage of relish. interestingly, the ikk complex is also required for relish cleavage, but its kinase activity is not involved in this function (stöven et al., 2003; erturkhasdemir et al., 2009). instead, the ikk complex may function as a scaffold facilitating the cleavage of relish by dredd (for an overview of the imd signaling pathway, see fig. 2). in addition to playing a key role in activation of the ikk complex and relish, tak1 also activates drosophila jnk signaling through hemipterous (mkk7) and basket (jnk) (sluss et al., 1996; holland et al., 1997; chen et al., 2002). thus, tak1 plays a crucial role at the nexus of jnk and nf-κb signaling in this innate immune signaling pathway. nf-κb (relish) plays a critical role in the induction of amp genes; without relish, no amp gene expression is detected. however, the role of the jnk pathway in amp regulation remains controversial. several reports have argued that jnk signaling actually down-modulates amp gene expression (kim et al., 2005, 2007), while another report has argued that jnk signaling is required for amp induction (delaney et al., 2006). more studies are required to resolve these conflicting conclusions. in addition to the possible inhibitory activity of jnk on nf-κb-responsive amp expression, the relish branch of the pathway also seems to generate an inhibitor of jnk signaling (park et al., 2004). the mechanism by which relish-induced gene products interfere with jnk 165 figure 2 imd signaling pathway. this pathway is preferentially triggered by dap-type peptidoglycan, common to gram-negative bacteria and certain gram-positives, especially bacillus spp. dap-type peptidoglycan can be recognized by different receptors, depending on its location and size. large insoluble peptidoglycan is recognized on the cell surface by pgrp-lcx, while smaller peptidoglycan fragments, like tct, can be recognized at the cell surface by a ligand-induced dimer of pgrp-lcx and pgrp-lca. in addition, dap-type peptidoglycan that reaches the cytosol can trigger another receptor, pgrp-le. both pgrp-lc and pgrp-le trigger a similar intracellular signal transduction pathway, as outlined here, that culminates in the activation of the nf-κb precursor relish. in addition, recognition of intracellular dap-type peptidoglycan by pgrp-le also triggers an autophagic response, which is important in the protection against intracellular bacteria. signaling has been suggested to involve the ubiquitin e3 ligase posh. posh is thought to target tak1 for degradation (tsuda et al., 2005), however it is not clear how this would preferentially inhibit jnk but not relish dependent responses. in addition to activating jnk and nf-κb/relish signaling, the imd pathway also induces the activation of the drosophila p38 pathway, which requires imd protein but not tak1 or any of the downstream components (zhuang et al., 2006). little is known about p38 signaling in the drosophila immune response, however it does appear to play a critical role in regulating the ros generating enzyme doux in the gut, thereby controlling the intestinal microflora (ha et al., 2009). microbial recognition both the toll and imd pathways are stimulated by bacterial peptidoglycans (leulier et al., 2003; kaneko et al., 2004). in addition, the toll pathway can be triggered by β-glucans, from fungal cell wall, or by proteases directly released from pathogens (gottar et al., 2006; el chamy et al., 2008). peptidoglycan (pgn) is the major structural component of the bacterial cell wall. pgn structures display a great deal of diversity, varying widely among different classes of bacteria. however, all pgns include a carbohydrate backbone, usually consisting of alternating nacetyl-glucosamine and n-acetyl-muramic acid residues and short stem-peptides containing both l and d amino acids. these stem-peptides are often cross-linked to each other to stiffen the cell wall; the precise structure of these cross-linking structures is highly variable. the stem-peptides also display a great deal of variation in their amino acid constituents. the carbohydrate backbone is more constant but also can be modified by various chemical substitutions, such as acetylation (schleifer and kandler, 1972; mengin-lecreulx and lemaitre, 2005) (see fig. 3 for a diagram of pgn structures). both the toll and imd pathways rely on peptidoglycan recognition proteins (pgrps) for sensing pgns (table 1). this family of proteins is structurally similar to type 2 amidases (nacetylmuramyl-l-alanine amidases), a class of enzymes that hydrolyze the bond between the lactyl group in acetylmuramic acid and l-alanine in the stem-peptide of pgn. in fact, some pgrps are type 2 amidases, while others lack the catalytic cysteine 166 figure 3 peptidoglycan structure. (a) as shown, peptidoglycan has a common core structure with a great deal of inherent variation. most notably, the constituent of the third position of the stem-peptide can vary, and are most commonly l-lysine or meso-dap. in addition, the amount and exact chemical nature of the crosslinking bridges can vary, with some examples noted in the box below. tct is a monomeric unit of the dap-type peptidoglycan chain, and is indicated in the dashed box. (b) structures of lysine and dap. and instead function by binding pgn (mellroth et al., 2003). drosophila encode for 13 pgrp genes, making about 17 distinct proteins through alternative splicing (werner et al., 2000). six of the drosophila pgrps (pgrp-sb1, -sb2, sc1a/b, sc2, -lb) are known or predicted type 2 amidases, that are involved in degrading pgn and dampening immune activation (mellroth et al., 2003; bischoff et al., 2006; zaidman-remy et al., 2006). the other seven lack type 2 amidase activity but function through binding pgn. in particular, 4 pgrps (pgrp-sa, -sd, -lc, and le) function as receptors in the imd or toll pathways, as detailed below. pgrp-lf seems to function as a decoy receptor, binding pgn but not activating immune signaling (persson et al., 2007; maillet et al., 2008), while the functions of pgrp-la and -ld remain elusive (royet and dziarski, 2007). initial studies suggested that the imd pathway was activated preferentially by gram-negative bacteria, which lead many to assume that lps, the most potent activator of the mammalian innate immune response, would be the main agonist of this pathway (samakovlis et al., 1990; werner et al., 2003). however, a careful analysis of published results suggested otherwise. in addition to gramnegatives, certain gram-positive bacteria, e.g., bacillus spp, were also imd pathway activators (lemaitre et al., 1997). subsequently, lemaitre’s group showed that diaminopimelic acid (dap)-type pgn, from escherichia coli or bacillus thurengensis, activated the imd pathway, while the silverman group demonstrated that purified lps samples were unable to trigger the imd pathway, and imd agonistic activity could be traced to dap-type pgn (leulier et al., 2003; kaneko et al., 2004). lemaitre’s group also showed that the toll pathway was activated by pgn, but in this case lysine-type pgn from gram-positives like m. luteus and e. fecalis was more potent. following these discoveries, a great deal has been learned about the molecular mechanisms involved in detecting various types of pgn. pgrpsa and pgrp-sd are required for the recognition of pgn and the activation of toll signaling (see below for more detail on the toll pathway) (michel et al., 2001; bischoff et al., 2004), while in the imd pathway either of two receptors, pgrp-lc or pgrp-le, are capable of recognizing dap-type pgn (gottar et al., 2002; ramet et al., 2002; kaneko et al., 2004, 2006; takehana et al., 2004; choe et al., 2005). pgn binding to either pgrp-lc or -le triggers the imd signaling pathway, described 167 table 1 functions and specificity of the pgrps retrieved in d. melanogaster * predicted specificity, based on the presence of arginine residue in key position for dap recognition. above, via a short conserved domain found in the nterminus of both receptors (kaneko et al., 2006). this conserved signaling domain has some similarity to the rhim domain found in the mammalian proteins rip1, rip3 and trif (meylan et al., 2004). however the molecular mechanisms involved in signaling by the pgrp-lc/le rhim-like domain still remain to be determined. regardless of the mechanisms involved, activation of these receptors leads to activation of both relish cleavage and relish phosphorylation. one key discovery, which enabled detailed molecular and biophysical analyses of pgrp-lc and pgrp-le, was that a monomeric fragment of pgn from gram-negative bacteria, known as trachael cytotoxin (tct), potently activates the imd pathway (kaneko et al., 2004; stenbak et al., 2004). tct is a disaccharide-tetrapeptide, featuring dap at the third position of its stem-peptide, isolated from culture supernatants of b. pertussis (fig. 3a). synthetic lactyl-tetrapeptides (substructures of tct) are able to serve as weak agonists of the imd pathway only if they contain dap at this third position, providing further demonstration that the dap residue is key to triggering the imd pathway (kaneko et al., 2004). pgrp-lc and pgrp-le preferentially bind dap containing pgn (takehana et al., 2002; swaminathan et al., 2006). the crystal structures of both pgrp-lc and pgrp-le bound to tct have been solved and show that a key arginine, arg254 in pgrp-le, provides the critical dap-specific interaction (chang et al., 2006; lim et al., 2006). compared to lysine, dap contains an additional carboxylate group (figure 3b), and the guanidinium group of arg254 forms a bidentate salt bridge with this moiety . in fact, this key arginine residue is common to all known dap-type recognizing pgrps and is found in the base of a deep cleft in which the pgn stem-peptide binds. in addition, dap binding pgrps also contain nearby glycine and tryptophan residues that are involved in other dap-specific interactions (swaminathan et al., 2006). pgrp-lc and pgrp-le are found in different subcellular compartments and detect microbes in these distinct environments. pgrp-lc is a single pass, type 2 transmembrane receptor that is found primarily on the cell surface (kaneko et al., 2004; mellroth et al., 2005). through alternative splicing, pgrp-lc encodes for 3 different receptors (pgrplca, -lcx, and -lcy) each with an identical cytosolic domain but distinct extracellular ligand binding pgrp domains. on the other hand, pgrple encodes for only one protein isoform, which lacks a transmembrane domain and functions as a cytosolic receptor (werner et al., 2000). while pgrp-lc is critical for recognizing extracellular bacteria (and extracellular pgn), pgrp-le surveils the cytosol for intracellular bacteria and/or small fragments of pgn that enter cells (kaneko et al., 2006; yano et al., 2008). rnai-based studies showed that the different splice isoforms of pgrplc are involved in recognizing different types of pgn. in particular, the recognition of polymeric name function pgn specificity pgrp-sb1 amidase daptype pgn pgrp-sb2 amidase daptype pgn* pgrp-sc1a/b amidase lys/daptype pgn pgrp-sc2 amidase daptype pgn* pgrp-lb amidase daptype pgn pgrpsa seml receptor for toll signaling lystype pgn pgrp-sd receptor for toll signaling daptype pgn pgrp-lc ird7 receptor for imd signaling daptype pgn pgrp-le receptor for imd signaling daptype pgn pgrp-lf decoy receptor daptype pgn pgrp-la unknown daptype pgn* pgrp-ld unknown daptype pgn* 168 pgn, as isolated from e. coli, requires only pgrplcx. moreover, pgrp-lcx mutants do not induce amp genes in response to e. coli infection. however, the response to extracellular monomeric pgn (tct) requires both pgrp-lcx and pgr-lca, while intracellular tct triggers pgrp-le. the finding that polymeric and monomeric dap-type pgns trigger distinct pgrp-lc receptors was explained, in part, by the crystal structure of pgrp-lca/x bound to tct. as mentioned above, most pgrps contain a deep cleft in which peptidoglycan fragments (sometimes referred to as muropeptides) bind. pgrp-lca is the exception; it contains two unique dipeptide sequences that disrupt the pgn binding cleft and occlude pgn binding. conversely, pgrp-lcx has a typical daptype pgn binding cleft and can avidly bind tct or polymeric pgn. upon binding tct, pgrp-lcx and pgrp-lca heterodimerize (chang et al., 2005; mellroth et al., 2005). the crystal structure of this ligand bound dimeric complex shows that the carbohydrate portion of tct makes key contributions to the dimerization interface, providing a clear explanation for the tct-induced dimerization (chang et al., 2006). it is reasonable to hypothesize that the ligand-induced heterodimerization of pgrplca and pgrp-lcx is critical for activation of downstream signaling, although this has not yet been demonstrated. this model of dimerizationinduced signaling does not explain how pgrp-lcx alone is sufficient for imd signaling triggered by polymeric pgn. the crystal structures suggest that pgrp-lcx is unlikely to form homo-multimers upon binding polymeric pgn, because of a steric clash at the putative dimerization interface. therefore, a distinct model must be proposed for signaling by polymeric pgn and pgrp-lcx. in this case, the ligand is polyvalent and likely binds to multiple individual pgrp-lcx receptors, perhaps creating a ‘cluster’ of receptors, thereby generating a density of pgrp-lcx cytosolic domains. this clustering, per se, may be sufficient to activate signal transduction, or perhaps the intracellular domains actually form higher order protein-protein interactions while the extracellular domains remain clustered on one large fragment of pgn, but not in direct contact with each other. future studies are required to examine these possibilities. like the pgrp-lca/x heterodimers, pgrp-le also multimerizes upon binding tct. however, the pgrp-le-tct complex forms very high order multimers in a ‘head to tail’ fashion. like the pgrplc structures, the pgrp-le structure was solved with the isolated pgrp domain, and we cannot be certain what quaternary structure the holo-receptor forms upon tct binding. however, these biophysical studies clearly demonstrate that tct causes pgrp-le to multimerize into large complexes (lim et al., 2006). as mentioned above, pgrp-le detects dap-type pgn that enters the cytosol. pgn may enter the cytosol from infection with intracellular bacteria, like listeria monocytogenes, or small pgn fragments, such as tct, appear to directly enter into cells. upon binding these pgns, pgrp-le likely forms higher order multimers and triggers imd signaling. in addition, pgrp-le activation can also induce an autophagic response that helps protect against intracellular microbes (yano et al., 2008). the role of ligand-induced receptor multimerization in the activation of imd signaling pathway, via pgrp-lcs and pgrp-le, requires further study. as mentioned above, these receptors signal through a rhim-like domain in their n-terminal domains. the mechanism by which the rhim-like domain functions in the context of dimerized, multimerized or clustered pgrp receptors is unknown. toll activation by pgn and beyond unlike the imd pathway, which is activated in a fairly specific manner by dap-type pgn, toll activation occurs indirectly by a wider array of immune stimuli. toll functions more like a cytokine receptor, binding a processed form of the cytokine spätzle, a member of the cysteine knot family of growth factors and cytokines (weber et al., 2003; hu et al., 2004; hoffmann et al., 2008). spätzle is made as a pro-protein that is found circulating in the hemolymph. upon immune activation (or developmental cues), serine protease cascades are triggered that culminate in the cleavage of spätzle. once processed, mature spätzle binds to and dimerizes the transmembrane receptor toll, initiating the intracellular signaling pathway described above. four different serine protease cascades appear to converge on the cleavage of spätzle. in early development, the protease easter is responsible for cleaving spätzle. during the immune response, bacterial pgn, fungal β-glucans, and microbial proteases are sensed by three distinct mechanisms, but converge upon activation of one serine protease, known as the spätzle processing enzyme (spe), which in turn cleaves and activates spätzle (fig. 1). the serine protease persephone appears to function as a sensor for proteases secreted by both fungal and bacterial pathogens (gottar et al., 2006; el chamy et al., 2008). persephone is likely activated by cleavage after a histidine residue, unlike the other proteases involved in the toll signaling pathways, and maybe a good target for subtilisin-like proteases produced by microbial pathogens (el chamy et al., 2008). the activation of the persephone-toll pathway by microbial proteases occurs independently of recognition of microbial cell wall material, which can also stimulate the toll pathway through more classical receptormediated recognition. for example, β-glucans from the cell wall of yeast are recognized by gram-negative binding protein 3 (gnbp3) (gottar et al., 2006), while two secreted pgrp receptors and gnbp1 are involved together in pgn recognition (gobert et al., 2003; bischoff et al., 2004; wang et al., 2006, 2008). [despite their name, none of the gnbps have been linked to the response to gram-negative bacteria, but gnbp1 and gnpb3 are involved in the recognition of fungal or gram-positive bacterial cell walls]. the n-terminus of gnbp3 binds to long β1,3 glucans, common to the cell walls of many types of fungi, especially yeast (mishima et al., 2009). gnbp1, on the other hand, is involved in pgn recognition, although its role is controversial. 169 lysine-type pgns, common to many grampositive bacteria, are potent agonist of the toll pathway. as in the imd pathway, pgrp receptors are critical for the recognition of lysine-type pgn. in particular, two secreted pgrps, pgrp-sa and -sd, are involved in the toll pathway. genetic studies have shown that some gram-positive bacteria and the pgn from these same species, such as m. luteus, are sensed through pgrp-sa (michel et al., 2001). in fact, the structure of pgrp-sa bound to a lysine-containing muropeptide has been solved. pgrp-sa can also bind dap-type pgn, albeit to a lesser degree. however, pgrp-sa appears to be able to specifically cleave dap-type muropeptides, removing the final amino acid in the stem-peptide. it has been postulated that this carboxypeptidase activity prevents dap-type pgn from stimulating the toll pathway via pgrp-sa (chang et al., 2004). interestingly, not all lysine-type pgn requires pgrp-sa to trigger the toll pathway. in particular, staphylococcus aureus, enterococcus faecalis, streptococcus pyogenes, and staphylococcus saprophyticus infections still produce strong amp gene responses in pgrp-sa mutant (seml) flies. response to these bacteria or their pgns instead requires either pgrp-sa or pgrp-sd (bischoff et al., 2004). the mechanism of pgrp-sd-mediated recognition of some, but not all, lysine-type pgn producing bacteria remains unclear. one possibility is a structural difference common to those pgns sensed by pgrp-sd, which prevents detection by pgrp-sa, or vice versa. however, biochemical studies of pgrp-sd do not support the notion that it is involved in recognizing lysine-type pgn. crystallographic studies show that pgrp-sd has a deep pgn binding cleft, typical of all pgrps, and this binding cleft includes an arginine (arg90) in the key position typical of dap-pgn specific recognition. in fact, binding studies confirm that pgrp-sd binds dap-type pgn, from b. subtilis, but not lys-type from s. aureus (leone et al., 2008). in addition, the moderate induction of drosomycin observed following either b. subtilis or e. coli infection, which is toll dependent (leulier et al., 2003), required both pgrp-sa and pgrp-sd. so, somehow pgrp-sa and pgrp-sd function together in the recognition of dap-type pgn, for moderate toll induction, but function in a more redundant manner for the recognition of certain lystype pgn, for robust toll induction. however, the recognition of m. luteus pgn appears to more simply require only pgrp-sa. the molecular mechanisms involved in the recognition of various pgns by pgrp-sa and/or pgrp-sd remain to be determined. as mentioned above, gnbp1 also functions in pgn recognition and toll signaling. in fact, pgrpsa, -sd, and gnbp1 form a trimeric complex together in the presence of pgn fragments (wang et al., 2008). some groups have reported that gnbp1 provides a critical pgn processing activity to this complex, required to generate small pgn fragments which are bound by pgrp-sa and/or pgrp-sd for toll activation (filipe et al., 2005; wang et al., 2006). however, another group has reported that they do not observe a similar pgn digesting activity associated with gnbp1, in drosophila or tenebrio molitor (buchon et al., 2009). instead, this group proposes that gnbp1 serves to link the pgrps with the downstream serine protease cascade, described below. thus, it appears that gnbp1 functions in a complex with the pgn sensing receptors pgrp-sa and pgrp-sd, but the biochemical mechanism by which it contributes to immune recognition or toll signaling are not yet clear. both the gnbp3-mediated recognition of βglucans and the pgrp-sa/sd/gnbp1-mediated recognition of bacterial pgns trigger toll signaling through the same serine protease cascade. this cascade involves the modular serine protease (modsp), which is probably directly activated by these microbial sensing receptor complexes, and at least two downstream clip-domain serine proteases grass and spe. as mentioned above, spe cleaves and thereby activates spätzle, the ligand for toll. another protease, known as spirit may function between grass and spe, and other non-catalytic serine-protease homologs, sphinx 1/2 and spheroide, were also implicated this pathway by rnai based studies (kambris et al., 2006). however, the assignment of these factors to this pathway requires further genetic and biochemical characterization. the pathways presented in fig. 1 suggest a protease cascade that is consistent with the genetic analysis of mutants in drosophila and the biochemical analysis of the cascade from the hemolymph of tenebrio. however, biochemistry of the drosophila serine protease cascade still requires further study, as several issues remain unresolved, including the role of spirit. in addition, the predicted specificity of the drosophila modsp does not match the predicted cleavage site of the downstream serine protease grass, and modsp does not cleave grass in vitro (buchon et al., 2009). thus, it remains possible that other factors may be involved. currently, it is not clear how (or even if) pgn binding to pgrp-sa/sd/gnbp1 leads to the activation of modsp, and, as mentioned above the exact biochemical role of gnbp1 remains controversial. concluding remarks the goal of this review is to summarize recent work on the toll and imd pathways, two important innate immune signaling pathways in drosophila, with an emphasis on the molecular mechanisms involved in microbial recognition in this model system. this review is not meant to be a comprehensive analysis of all aspect of the insect immune response. notably, many very exciting studies have been published recently on phagocytosis, anti-viral immunity, melanization and clotting all topics not covered here. instead, significant detail is presented on the biochemistry and genetics of bacterial recognition by the pgrp family of innate immune receptors. over the past 5 years, much progress been made in this area, and the basic underpinnings of how bacteria are recognized by pgrgp receptors, which bind the bacterial cell-wall derived compound peptidoglycan, has been resolved. in addition, the preferential binding of dap-type pgn by some of these 170 receptors, notably pgrp-lc and pgrp-le, provides a firm explanation on the specific activation of the imd pathway by gram-negative and certain gram-positive bacteria. however, the specificity, or lack thereof, in activating the toll pathway by different types of peptidoglycans still requires further investigation. moreover, 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martins-souza1, za andrade2, pmz coelho3,4 1departamento de parasitologia, instituto de ciências biológicas (icb), universidade federal de minas gerais (ufmg), belo horizonte,minas gerais, brazil 2centro de pesquisas gonçalo muniz (cpgm-fiocruz), salvador, bahia, brazil 3centro de pesquisas rené rachou (cprr-fiocruz), belo horizonte, minas gerais, brazil 4santa casa de misericórida de belo horizonte, belo horizonte, minas gerais, brazil accepted november 7, 2007 abstract digenetic trematodes use molluscs, almost always a gastropoda, in their evolutive cycle, as intermediary hosts. the genus schistosoma, with three main species that infect humans s. mansoni, s. japonicum, and s. haematobium – shows a prevalence of 200 million patients in various countries worldwide, and 600 million people are still at risk of infection. s. mansoni is the most prevalent species, and biomphalaria snails are its intermediary hosts. although the campaigns of schistosomiasis control based on chemotherapy have reduced the morbidity and prevalence of this disease, transmission continues in almost all the areas submitted to intervention. one of the factors that has influence on the susceptibility of biomphalaria to s. mansoni infection is ability of the host internal defense system (ids) to recognize and destroy the parasite. in biomphalaria, the ids is composed of cellular elements named hemocytes that act jointly with soluble components present in hemolymph, which could affect directly the larvae, or act in the recognition of the parasite, and activation of hemocytes. the susceptibility level of the mollusc has been attributed to the hemocyte capacity of involving and destroying the parasite, and this will be the centre of interest of this review. the study of s. mansoni and biomphalaria interaction in resistant snail strains is important not only due to the academic-scientific value of this fascinating research area, but also to the potentially possible alternatives for the control of this endemia. key words: schistosoma mansoni sporocysts; biomphalaria glabrata; biomphalaria tenagophila; circulating hemocytes; soluble factors of hemolymph introduction although the great majority of the living beings is represented by invertebrates, up to now the publications in mass dealing with the defense mechanisms against pathogens is practically restricted to interactions between pathogens of vertebrate animals. the invertebrate animals must necessarily reckon upon their defense system to recognize and destroy infectious agents, although this system are not able to generate the diversity of ___________________________________________________________________________ corresponding author: deborah negrão-correa departamento de parasitologia, instituto de ciências biológicas universidade federal de minas gerais avenida antonio carlos 6627, belo horizonte, mg, brazil campus pampulha – zip code: 31270-901 e-mail: denegrao@icb.ufmg.br recognition observed during the adaptative immune response of vertebrates (van der knaap and loker, 1990; loker et al., 2004). recent studies have demonstrated many similarities between the innate defense response of vertebrates and the internal defense system of invertebrates (hoffman et al., 1999; hoffman, 2003), being identified in various invertebrates organisms production of complement-like proteins, antimicrobial peptides, pattern-recognition receptors (prrs) such as toll-like receptor and c-type lectins, phagocytic cells, production of highly toxic metabolites of oxygen and nitrogen (loker et al., 2004). although many similarities were identified in the defense system of several groups of invertebrates, it is important to note that genomic studies have indicated varied defense mechanisms in invertebrate groups phylogenetically associated, 101 mailto:denegrao@icb.ufmg.br but with very different alimentary habits and habitat (loker et al., 2004). several groups of invertebrates, such as insects and molluscs, are important intermediary hosts of parasites species that are transmitted to humans and domestic animals. as an example we can mention the necessity of diptera insects genus lutzomya in the development of different species of leishmania, the anophelins in the transmission of malaria or the anophelins and culicides for the development of lymphatic filariosis. among the molluscs, gastropoda are obligatorily intermediary hosts in the development of the majority of digenetic trematode species, such as s. mansoni and fasciola hepatica. thus, it is of the utmost importance to get a better understanding of the effector mechanisms used by the internal defense system (ids) of these invertebrates for the development of new strategies related to the control of these parasite infections. these studies have also contributed significantly to a better knowledge about the innate response of vertebrates. this review will focus primarily on the response of biomphalaria during infection with s. mansoni. schistosoma mansoni infection in biomphalaria schistosomiasis is an important health problem that affects over 200 million people worldwide. among the schistosomes species that infect human beings, s. mansoni is transmitted by biomphalaria snails and causes intestinal and hepatic schistosomiasis in africa, arabian peninsula, and south america (gryseels et al., 2006). recent estimative indicates that 6-7 million people are infected by s. mansoni only in brazil (katz and peixoto, 2000). s. mansoni infects biomphalaria by means of active penetration of the parasite´s ciliated larvae, named miracidia, at any site of the snail’s exposed parts, frequently the base of the antennae and cephalopodal mass. in the process of penetration, the parasite undergoes morphological and physiological changes, being transformed into primary sporocyst that remains in the fibro-muscular tissue of the host´s cephalopodal region near the penetration site. the primary sporocysts generate the secondary ones, which migrate from the cephalopodal musculature to the digestive glands or hepatopancreas of the mollusc, where they undergo profound anatomic changes and their germinative cells can generate the cercariae (maldonado and acosta-matienzo, 1947; pan, 1965; pereira et al., 1984). in brazil, out of ten species of molluscs genus biomphalaria described, only three were found naturally infected by s. mansoni: b. glabrata, b. tenagophila and b. straminea (paraense, 2001). the susceptibility level of these different species of biomphalaria to infection with the same lineage of s. mansoni is much diversified, and b. glabrata may present up to 75.3 % of susceptibility in experimental infections, b. tenagophila 32.6 %, and b. straminea 11.3 %, as demonstrated by souza et al. (1997). besides the difference in susceptibility observed among biomphalaria species compatible with the parasite, some lineages or geographic isolates of a same species of biomphalaria also present a great variation of susceptibility to the parasite. as far as b. tenagophila is concerned, the geographic lineage isolated at the biological reservoir in taim (rio grande do sul, brazil) was found to be completely resistant to the development of all s. mansoni isolates already tested. the character of resistance of this b. tenagophila lineage has been explored at our laboratory, aiming at studying the possible mechanisms of the parasite’s destruction, representing a potential model for the control of transmission in endemic areas, where b. tenagophila is the unique transmitter agent of the disease (coelho et al., 2004). genetic control of resistance of biomphalaria to s. mansoni infection the compatibility between s. mansoni and its intermediary host is influenced by behavioral and physiological factors of the mollusc. once found a suitable host, the susceptibility level of biomphalaria to s. mansoni can be determined by the genetic differences of the molluscs, as well as by the genetic constitution of schistosoma (basch, 1976). newton (1952, 1953) demonstrated for the first time that the susceptibility of b. glabrata snail to s. mansoni depends largely upon genetic factors. later, these results were corroborated by richards (1970), who demonstrated that the resistance character, acquired at the maturity phase, is determined by a single dominant gene, with mendelian inheritance. nevertheless, in b. glabrata, age is a determinant factor of the snail susceptibility; juvenile snails being more susceptible to infection even in lineages where the adult snail is resistant to s. mansoni infection. thus, the susceptibility of juvenile b. glabrata to s. mansoni infection is also regulated by genetic factors, being estimated that four or more genes affect this character (richards, 1977). b. tenagophila taim is a lineage that presents absolute resistance to infection by all s. mansoni strains tested at any phase of the mollusc development (santos et al., 1979; martins-souza et al., 2003; coelho et al., 2004). crossbreedings between b. tenagophila taim and b. tenagophila bh (susceptible lineage) showed that the character of resistance to s. mansoni infection is dominant (santos et al., 1979). the dominance of the resistance character of b. tenagophila taim was confirmed in crossbreedings with the susceptible joinville lineage (rosa et al., 2005), showing that 100 % of the f1 offspring were resistant and only 8 % of the f2 offspring were susceptible to infection by the parasite (table 1). this study suggests that at least two dominant genes would be responsible for the resistance to s. mansoni observed in b. tenagophila taim, one of them being the most important since it was expressed in all f1 offspring (rosa et al., 2005). the dominance of the resistance character in b. tenagophila taim added to the reproductive success of this lineage in the presence of susceptible snails of the same species (rosa et al., 2006), led us to suggest the use of this lineage as an alternative for biological control in 102 table 1 susceptibility rates of b. tenagophila joinville, b. tenagophila taim, f1 and f2 when submitted to infection with 25 miracidia of le strain of s. mansoni experiment group number of snails exposed to s. mansoni number of surviving snails number of infected snails eliminanting cercariae (%) 1 taim 64 60 0 (0) joinvillle 35 12 7 (58.3) f1 170 150 1 (0.6) f2 110 87 7 (8) 2 taim 30 25 0 (0) joinville 30 15 9 (60) f1 50 44 0 (0) f2 50 38 2 (5.3) some areas of schistosome transmission (coelho et al., 2004). one of the factors that influence the susceptibility and may be genetically determined is the activity of the snails ids. experimental infections in b. tenagophila taim have shown that s. mansoni miracidia are able to penetrate this snail lineage, however the parasites induce an intense cellular infiltration and are rapidly destroyed, suggesting an important participation of the ids on determination of resistance to s. mansoni in b. tenagophila taim. internal defense system (ids) of the mollusc the ids of snails is composed of cellular elements constituted by hemocytes, and by soluble factors present in hemolymph. the hemocytes may be circulating in hemolymph or fixed in tissues. in planorbids the hemolymph circulates in a semi-open system impelled by the heart. the hemoymph leaves the heart through the aorta reaching the tissues, draining in the venous sinus and returning to the heart via the pulmonary and renal veins, after being re-oxygenated in the pulmonar wall (baker, 1945). the heart, enclosed by the pericardium membrane, is divided into two chambers, the auricula, which receives hemolymph from the pulmonary cavity, and the ventricule that impels the hemolymph through the aorta. the aorta is divided into two arteries: the visceral artery, which irrigates the posterior part of the snail´s body, including the digestive and genital systems, and the cephalic artery, that reachs all the cephalopodal region. the arteries are exhausted in the pseudovascular spaces of the tissues, accumulating hemolymph in three venous sinuses: cephalopodal, visceral and sub-renal, returning to the heart after circulating through the kidney and lung (baker, 1945; paraense, 2001). in b. glabrata and bulinus sp a well defined region, located between the pericardium and the posterior epithelium of the mantle cavity (fig. 1a), also called amebocyte producing organ (apo), was identified as the main site for the production of hemocytes (lie, 1976). recent observations (sullivan et al., 2004; sullivan and castro, 2005) showed an increase of mitoses in the cells of this region, ranging from 24-72 h after inoculation of antigens of s. mansoni miracidia or cercariae, this being more evident in resistant lineage of b. glabrata. nevertheless, some authors (matricongondran, 1990; souza and andrade, 2006) demonstrated that b. glabrata hemocytes may present multi-centric origin, and sites with proliferation of hemocytes were detected also at the saccular portion of the renal tubules and in the ventricular cavity of the heart (fig. 1b). the circulating hemocytes of different species of molluscs present morphological and functional heterogeneity. according to ottaviani (1992; 2006), the population of circulating hemocytes of the majority of gastropod molluscs is constituted by two cellular types: the starry hemocytes that emit pseudopodes, and the roundish hemocytes. in planorbarius corneus, the starry hemocytes are cells that present phagocytic activity, adhere to glass and express proteins that are recognized by pro-inflammatory anti-cytokine antibodies of vertebrates. on the other hand, the roundish hemocytes are not endowed with phagocytic activity, they are not able to adhere to glass, and besides they proliferate in the presence of phytoagglutinin (ottaviani, 1992; ottaviani et al., 1993). similarly to p. corneus, the majority of the authors (harris, 1975; lie et al., 1987; barraco et al., 1993; borges and andrade, 2003) also distinguish two sub-populations of circulating hemocytes in hemolymph of b. glabrata. these subpopulations are called granulocytes, i.e., the hemocytes that emit pseudopodes and produce phagocytosis, and hyalinocytes that are the small and roundish hemocytes (fig. 2a). granulocytes can be easily identified by the uptake of neutral red stain into the cell vesicles (fig. 2b), showing that s. mansoni infection induce cellular proliferation (martins-souza et al., 2003). however, ultrastructural studies (matricon-gondran and letorcart, 1999 a,b), analyses of distribution and abundance of lysosomal enzymes (granath and yoshino, 1983), as well as of expression of lectin-ligants on the cellular surface (joky et al., 1983; martinssouza et al., 2006) suggest that the circulating hemocytes of biomphalaria constitute a cellular population significantly more heterogenous than that previously described (fig. 2b). the phenotypical and functional definition of biomphalaria hemocytes is of fundamental importance to understand the participation of these cells, or of any cellular subpopulation, in the destruction mechanism of s. mansoni larvae or other parasites. even though hemocytes are the main component of the mollusc ids, t here are some experimental 103 fig. 1 photomicrographs of biomphalaria glabrata heart region. (a) snail heart tissue in close contact with the pericardial membrane. the arrows indicate the amebocyte-forming organ (apo), a narrow and long band of epithelial-like cell along the pericardial membrane (100x). (b) heart tissue with a dense collection of hemocytes (arrow) (200x). hematoxylin-eosin stain evidences indicating that soluble elements of the hemolymph would participate in the protective mechanism against pathogens. soluble components of the hemolymph of molluscs can directly interact with pathogenic agents, by means of production of toxic substances or lytic peptides, or indirectly through mediator molecules for recognition of the pathogen or hemocyte activators. peptides with anti-microbial function, called mytilines, are produced and stored in hemocyte granules, and they are secreted in hemolymph of mytilus galloprovincialis (mitta et al., 2000) notwithstanding the participation of these peptides in the destruction of bacterial infections, there are no evidences of the participation of these peptides in the interaction of mollucs with metazoan parasites. fryer and bayne (1996) show that particles of polystireno treated with soluble factors of hemolymph of b. glabrata are significantly more phagocyted by hemocytes than the untreated particles, demonstrating that soluble factors of hemolymph may participate of the recognition mechanism and opsonization of particles by hemocytes. johnston and yoshino (1996) demonstrate that lectins similar to those of conavalia ensiformis (cona), erythrina corallodendrom (eca), glycine max (sba), tetragonolobus purpureas (tpa), and triticum vulgaris (wga) are present in hemolymph of b. glabrata. in molluscs, lectins are synthesized by hemocytes and released in hemolymph, where they immobilize the material particularized by agglutination, or are expressed at the surface of circulating hemocytes, where apparently they act as cytophylic receptors (richards and renwrantz, 1991; fryer and bayne, 1989). besides the lectins, other proteins with homologous function to cellular mediators, and already characterized in vertebrates, have been identified in hemolymph of molluscs and may be involved in the activation of hemocytes during infection by digenetic trematodes (ottaviani et al., 1993, 1995). ottaviani and co-workers (1993) reported the presence of a variety of proteins similar to pro-inflammatory cytokines of vertebrates, including interleukin-1 alpha (il-1α), interleukin-1 beta (il-1β), interleukin-2 (il-2), interleukin-6 (il-6), and alpha tumoral necrosis factor (tnf-α) in hemocytes of two species of molluscs, planorbarius corneus and viviparus ater, being present only in hemocytes with phagocytic activity. in further studies, ottaviani et al. (1995), relate the production of homologous proteins to cytokine with an increase of phagocytic activity and induction of nitric oxide synthase (nos) of mollusc hemocytes. these results suggest that cytokines-like may participate in the activation of hemocytes. functional mechanisms in parasite-mollusc interaction in the last years, many aspects of the interaction between the digenetic trematode larvae and the internal defense system of molluscs have been elucidated. nevertheless, the possible mechanisms responsible for destruction of the majority of larvae in resistant snails remain to be totally understood. the results reported up to now suggest that the hemocyte could be the effector element in the destruction mechanism of trematodes, being directly involved in the death of some encapsulated parasites (van der knaap and loker, 1990; bayne et al., 2001) or in the production of soluble factors which could be cytotoxic (connors et al., 1995). the majority of the authors (connors et al., 1995; bayne et al., 2001; martins-souza, 2003) agree that the snails´ defense generally occurs by means of destruction, total or partial, of the primary sporocyst at the first few hours following the penetration of the miracidium. the existence of a cellular defense mechanism deployed by molluscs against trematode infection was initially suggested by the finding of histological reactions around parasite sporocysts (newton, 1952). further studies have shown that hemocytes infiltration around parasite larvae in s. mansoni 104 fig. 2 morphological aspects of circulating hemocytes from biomphalaria tenagophila. (a) phase contrast photomicrography showing a granulocyte (g) and a hyalinocytes circulating in b. tenagophila hemolymph (400x). (b) bright field photomicrography of b. tenagophila circulating hemocytes after addition of neutral red staining, showing the heterogeneous granulocyte population stained in red (g) and the non-stained cells designated as hyalinocytes (h) (200x) infected biomphalaria was stronger in snail species that are more resistant to parasite infection, such as b. tenagophila and b. straminea (souza et al., 1997). in highly susceptible b. glabrata, confirmed by the great quantity of cercariae eliminated during a long period of time, sporocysts and cercariae at different developmental stages are found in abundance in the inner of the host, resulting in compression of the host´s structures, mainly in the interstice of the digestive glands, ovotestis and renal tubules. however, the presence of a great number of parasites did not induce cellular reaction in the parasite susceptible snail strains (godoy et al., 1997). on the opposite extreme appear the resistant snails, which shed a few cercariae and show an extensive infiltration in tissues with numerous hemocytes, frequently placed around the parasite structures in disintegration. the focal reactions frequently assumed a granuloma-like appearance (godoy et al., 1997). in our experimental model, s. mansoni infection in b. tenagophila of taim strain resulted in intense, diffuse or granuloma-like cellular infiltration in the infection site, mainly in the connective tissue of the snail cephalopodal region and antennae (fig. 3). the cellular infiltration was detected few hours after the parasite infection and no viable sporocysts were recovered from these infected snails. direct evidence of the hemocyte participation in s. mansoni infection control was obtained with experiments that transferred the apo from resistant to susceptible snail strains. in b. glabrata, the transplantation of apo from miracidiaexposed resistant strain to susceptible nih snails resulted in significantly more killed sporocysts than as observed during s. mansoni infection in nih snails (sullivan and spencer, 1994). recently, barbosa et al. (2006) showed in b. tenagophila that transplantation of the hematopoietic organ from taim lineage (totally resistant) to a susceptible s. mansoni strain resulted in an absolute resistance in the receptors whose transplant was successful. moreover, inoculation of silica particles in b. tenagophila cabo frio resulted in transitory reduction of a macrophage-like cell population of circulating hemocytes. the cellular depletion induced by silica-treatment in b. tenagophila cabo frio was accomplished by enhanced susceptibility to s. mansoni infection, shortening the intramolluscan phase of the parasite and increasing the number of sporocysts and cercariae produced (martins-souza et al., 2003). the process of destruction of s. mansoni larvae by hemocytes initiates with the recognition and encapsulation of the newly-penetrated sporocyst. bayne and co-workers (1980b) demonstrated that cell-free hemolymph obtained from susceptible and resistant b. glabrata strains are unable to change visibly the morphology of s. mansoni in vitro, the same occurring with hemolymph containing hemocytes of susceptible lineages. however, hemocytes of susceptible strains associated with soluble factors of hemolymph of resistant b. glabrata lineages acquire the ability to destroy s. mansoni sporocysts. the importance of the soluble fraction of biomphalaria hemolymph in the destruction mechanism of s. mansoni sporocysts was also confirmed in vivo in studies dealing with the transference of this fraction obtained from resistant b. glabrata snails to other ones susceptible to the parasite (granath and yoshino, 1984). recent results obtained with b. tenagophila taim also showed that addition of the cell-free hemolymph of this resistant snail strain significantly increased the ability of hemocytes from susceptible strain of b. tenagophila (cabo frio or joinville) to destroy s. mansoni sporocysts, in vitro. moreover, the increased mortality of sporocysts was associated with higher number of hemocytes on the parasite tegument (fig. 4), suggesting that cell-free hemolymph from resistant snail strain increased parasite recognition by hemocytes. however, in contrast with b. glabrata (bayne et al., 1980), cell-free 105 fig. 3 intense hemocyte infiltration in schistosoma mansoni infected biomphalaria tenagophila taim, a parasite-resistant snail strain. photomicrograph of a transversal section of antennae tissue from b. tenagophila taim 15 days after s. mansoni infection (20 miracidium/snail), showing a focal cellular reaction (arrow) with a granuloma-like appearance (200x). diffuse and focal cellular infiltration is observed in cephalopodal tissue of parasite infected resistant snails and has been associated with the parasite penetration and destruction. hematoxylineosin stain hemolymph from b. tenagophila taim was able to destroy a small, but statistically significant, percentage of s. mansoni sporocysts even in absence of hemocytes (pereira, 2005). the importance of b. tenagophila taim cell-free hemolymph in s. mansoni control was also confirmed in vivo, since susceptible snails treated with b. tenagophila taim cell-free hemolymph had lower percentage of infectivity (pereira, 2005; coelho and bezerra, 2006), and the snails that got infection produced lower number of sporocysts and cercariae (pereira, 2005). the main components of s. mansoni sporocysts tegument is glicoproteins and glicolipides (zelck and becker, 1990; uchikawa and loker, 1991). johnston and yoshino (1996) showed that lectins from b. glabrata cell-free hemolymph bind to glicoproteins extracted from the parasite tegument. more recently (adema et al., 1997a,b) a group of proteins, homologous to fibrogen and that has been associated with recognition, was identified in b. glabrata hemolymph, being its expression enhanced after infection of the mollusc with echinostoma paraensei, another digenetic trematode. these results suggested that lectins would serve as cell surface receptors for carbohydrate structures from trematode parasites. moreover, soluble lectins would also participate in the recognition mechanism by binding to carbohydrate structures from both hemocytes and parasite tegument (van der knapp et al., 1990). besides participating in the recognition of s. mansoni, lectins can also activate hemocytes. hemocytes of susceptible and resistant b. glabrata strains were stimulated with bovine albumin associated with one of the six carbohydrates: mannose, galactose, fucose, n-acetyl-glucosamine, n-acetyl-galactosamine and lactose, that are present in the tegument of s. mansoni sporocysts. hemocytes stimulated with bsa-galactose, bsamannose, and bsa-fructose were able to produce reactive oxygen-species (ros) (hahn et al., 2000). our results also confirmed the participation of soluble lectins in s. mansoni-sporocysts recognition mechanisms by b. tenagophila species. circulating hemocytes recovered from b. tenagophila both taim and cabo frio strains were intensively labelled by fitc-conjugated lectins, such as pna, sba, and wga. moreover, s. mansoni infection in resistant snail strain (taim) resulted in initial reduction in number of labelled-hemocytes in circulation (martins-souza et al., 2006). the reduction of circulating hemocytes during the first few hours after s. mansoni infection has been associated with the cell recruitment to the infection site (bezerra et al., 1997; martins-souza, 2003). other proteins similar to pro-inflammatory cytokines of vertebrates have been identified in hemolymph of molluscs and can participate in activation of hemocytes during the destruction process of parasites. specifically in b. glabrata was identified a protein with immunological and functional similarity to interleukin-1-like (il-1 like). in this snail, il1-like protein is induced by s. mansoni infection, and the level was significantly higher in resistant snail strains (granath et al., 1994). the inoculation of human recombinant il-1α in susceptible strain of b. glabrata resulted in increased production of ros by circulating hemocytes and reduced number of s. mansoni cercariae upon parasite infection (connors et al., 1995). these authors confirmed, in vitro, that cellfree hemolymph recovered from rhil-1α-treated snail, but not only rhil-1α, was capable of destroy s. mansoni sporocysts, suggesting that il-1 would activate b. glabrata-hemocytes to produce and secrete soluble cytotoxic mediators (connors et al., 1998). the effector mechanisms by which activated hemocytes are able to kill trematode larvae are not fully understood yet. dikheboom et al. (1988a,b) showed for the first time that gastropod hemocytes do produce reactive oxygen species (ros) in response to trematode infection. the initial reduction of o2 to superoxide anion (o2) is catalyzed by nadph-oxidase and o2 can be converted to other ros, including hydrogen peroxide (h202) and hypochlorous acid (hoci) (hampton et al., 1998; bayne et al., 2001). nadph oxidase-like activity has been identified in gastropod hemocytes (adema et al., 1993) and ros production 106 fig. 4 interaction of schistosoma mansoni sporocyst and hemocytes from biomphalaria tenagophila taim (b) or biomphalaria glabrata bh (c). (a) s. mansoni sporocyst in chernin’s balanced salt solution, showing an intact parasite larva (200x). (b) s. mansoni sporocyst completely encapsulated by hemocytes from b. tenagophila taim, a snail strain totally resistant to parasite infection (100x). (c) s. mansoni sporocyst incubated with hemocytes from b. glabrata bh, snail highly susceptible to parasite infection, showing an intact parasite with very few cell attached, x100 by b. glabrata hemocytes has been associated with s. mansoni resistance (adema et al., 1994). additionally, molluscan hemocytes also generate nitric oxide (no) from molecular oxygen and arginine (conte and ottaviani, 1995; arumugan et al., 2000). experimental evidences of ros and/or nos participation in killing of s. mansoni sporocysts by b. glabrata hemocytes were obtained using specific oxidant scavengers or enzyme inhibitors during the in vitro association. the results showed that inhibition of h2o2 and no production do favor sporocysts survival, indicating that this reactive species would be toxic to trematode larvae (hahn et al., 2001 a,b). however, attempts to associate h2o2 or no production with snail strain susceptibility to s. mansoni infection have not been conclusive (hahn et al., 2000; bayne et al., 2001). similarly, we were able to estimate no production by hemocytes isolated from b. glabrata or b. tenagophila at different time after s. mansoni infection and restimulated in vitro with s. mansoni egg antigen (sea). at each experimental point, as well as for each snail strain, analyses were performed in triplicate with total hemolymph collected and gathered together from 3 snails. after centrifugation, the hemocyte pellet was ressuspended in chernin´s balanced salt solution (5 x 105/ml of cbss) containing 100 μg/ml of sea, plated in 96-well tissue culture plates and incubated at 26 °c and 5 % co2. after 18 h of incubation, no presence was assayed directly in the cell supernatant by the greiss reaction (green et al., 1982) that quantifies the nitrite contents of the supernatants, as detailed by perreira et al. (2006). the no level increased in hemocytes recovered after the first few days of s. mansoni infection, however there were no detectable differences in no level between the snails, although each species or strain shows remarkable difference in parasite susceptibility (fig. 5). however, it is important to confirm if supernatant level do reflect the local production in hemocytesporocyts interaction. besides producing reactive species of oxygen and nitrogen, microscopy analyses indicate that hemocytes from parasiteresistant snails would phagocvte portions of sporocyst tegument leading to mechanical lesion that may be also lethal to the parasite (van der knaap and loker, 1990). finally, in order to evaluate the efficiency of biomphalaria spp defense system in the destruction of s. mansoni larvae, one must consider that there are many evidences indicating that the parasite is able to develop strategies to allow its evasion. thus, it has been described that the primary sporocyst of s. mansoni acquires quickly the antigens present in the host´s hemolymph (bayne et al., 1986), as well as express in the tegument antigens similar to those expressed by the host´s cells (yoshino and bayne, 1983) hindering the recognition process of the parasite by hemocytes. it has been also reported that components of the excreted/secreted material by the miracidium during the transformation process may reduce the motility of hemocytes, as well as their phagocytic capacity (connors and yoshino, 1990; lodes and yoshino, 1990), thus justifying the cellular reaction almost inexistent observed around s. mansoni sporocysts present in the tissue of b. glabrata susceptible strains. in addition to avoid the hemocytes´ approach, it has been also reported that sporocysts incubated in vitro with hemocytes of susceptible strains may be encapsulated, but not destroyed (boehmler et al., 1996; hahn et al., 2001a), suggesting the existence of anti-oxidant mechanisms. perspectives the most recent studies related to the internal defense system of invertebrates have shown new aspects of the invertebrate-pathogen relationship. these aspects afforded us a better understanding of the recognition and cellular activation mechanisms, which are also present in the innate defense response of vertebrates. biomphalaria-schistosoma mansoni interaction, besides being an important model in human health, constitutes an experimental approach that may add important information to the knowledge of the defense mechanism utilized by invertebrates, as well as of phylogenetic evolution of these mechanisms. in this context, our group has carried out researches dealing with the phenotypic and functional 107 fig. 5 nitric oxide (no) level in supernatant of circulanting hemocytes recovered from biomphalaria glabrata, b. tenagophila cabo frio e b. tenagophila taim during the s. mansoni infection. no levels was indirectly estimated by quantification of nitrite, using the greiss reaction, in hemocyte supernatant recovered after 18 h of in vitro restimulation with soluble egg antigen (sea 100 μg/ml). *** for p < 0.001 when comparing with nitrite level obtained after stimulation of non-infected hemocytes from the same snail strain. at each time point, there was no statistically significant difference in the nitrite level between the snail strains characterization of the hemocytes and hemolymph from different strains of b. glabrata and b. tenagophila. our goal is to identify possible mechanism of trematode recognition and hemocyte activation in b. tenagophila taim that would be responsible to the fast parasite destruction and consequent resistance against s. mansoni infection observed in this snail strain. in parallel, we are investigating the heritage of the resistance character from b. tenagophila taim to the offspring resulted of crossbreeding with the susceptible snail strains. a more comprehensive identification of these mechanisms would expand the theorical base that gives support to mass introduction of b. tenagophila resistant strain from taim in areas where transmission is maintained by this species (coelho et al., 2004). acknowledgement this work was supported by grants from fundação de amparo à pesquisa do estado de minas gerais (fapemig), and pronex (cnpq/fapemig). acknowledgement is also due to jeferson do carmo bernardes, josé carlos reis dos santos and selma fernandes for the technical support in the experiments. the authors are also grateful to mrs vera de paula ribeiro for reviewing the text and dr ary corrêa jr for the support in image production and documentation. references adema cm, arguello df, stricker sa, loker es. a time-lapse study of interactions between echinostoma paraensei intramolluscan larval stages and adherent hemocytes from biomphalaria glabrata and helix aspersa. j. parasitol. 80: 719-727, 1997a. adema cm, hertel la, miller rd, loker es. a family of fribrinogen related proteins that precipitates parasite derived molecules is produced by an 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517-522, 1997. souza s and andrade za. on the origin of the biomphalaria glabrata hemocytes. mem. inst. oswaldo cruz 101 (suppl. 1): 213-218, 2006 sullivan jt, castro l.mitotic arrest and toxicity in biomphalaria glabrata (mollusca: pulmonata) exposed to colchicine. j invertebr. pathol. 90: 32-38, 2005. sullivan jt, pikios ss, alonzo aq. mitotic responses to extracts of miracidia and cercariae of schistosoma mansoni in the amebocyte-producing organ of the snail intermediate host biomphalaria glabrata. j. parasitol. 90: 92-96, 2004. sullivan jt, spence jv. transfer of resistance to schistosoma mansoni in biomphalaria glabrata by allografts of amoebocyte-producing organ. j. parasitol. 80: 449-453, 1994. uchikawa r, loker es. lectin-binding properties of the surfaces of in vitro-transformed schistosoma mansoni and echinostoma paraensei sporocysts. j. parasitol. 77: 742-748, 199. van der knaap wp, loker es. immune mechanisms in trematode-snail interactions. parasitol. today 6: 175-182, 1990. yoshino tp, bayne cj. mimicry of snail host antigens by miracidia and primary sporocysts of schistosoma mansoni. parasite immunol. 5: 317-328, 1983. zelck u, becker w. lectin binding to cells of schistosoma mansoni sporocysts and surrounding biomphalaria glabrata tissue. j. invertebr. pathol. 55: 93-99, 1990. 111 corresponding author: group << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice isj108.pdf 142 isj 2: 142-151, 2005 issn 1824-307x review insect immunorecognition e ottaviani department of animal biology, university of modena and reggio emilia, modena, italy accepted october 14, 2005 abstract the mechanisms of the innate immunity in the insects have been reviewed. in particular, the cellular component (phagocytosis, encapsulation, melanization, nodule formation, wound healing, hemolymph clotting and transplantation) and the humoral component (lectins, cytokine-like molecules and anti-microbial peptides) of the hemolymph have been investigated. key words: insects; immune and neuroendocrine responses introduction the first line of defence in insects is the cuticle. this external barrier of carbohydrate-protein material protects the animal from both physical damage and pathogen attack. the second and more important line of defence is the internal defence system, with a welldeveloped capacity to discriminate between self and non-self. as other coelomatic animals, insects implement this recognition through the cellular and humoral components of the hemolymph. cellular component with regard to the cellular component, there is a general problem in defining the number of immunocytes present in the invertebrate hemolymph and this is a subject of great debate. one reason for this situation is that in most cases there is no hemopoietic organ. when, however, the organ is present, as for example in insects, it supplies immunocytes during the animal’s development, and these continue to differentiate in the circulation (nappi and carton, 1986). however, the immunocyte proliferation occurs mainly in circulating hemolymph, thus containing different stages of maturation of the same cell. corresponding author: enzo ottaviani department of animal biology, university of modena and reggio emilia, via campi 213/d, 41100 modena, italy e-mail: ottaviani.enzo@unimore.it to avoid describing these different stages of maturation as specific cell types, morphological studies should be performed in parallel with functional analyses. this problem is also seen in insect immunocytes. these derive from the mitotic division of circulating blood cells and hemopoietic organs (jones, 1977). the hemopoietic organs have been described in different species and present peculiar locations and morphological characteristics. in particular, five pairs of lymph glands located along the anterior portion of the dorsal blood vessel are the sources for immunocytes in drosophila melanogaster (nappi and carton, 1986). hoffmann (1973) reported two possible organizations for hemopoietic organs. one is structured as dissociated accumulations of immunocytes that could be referred to as the “haemocyte reservoirs” described by jones (1977), while the other is considered a truly differentiated hemopoietic organ. in their elegant treatise, rowley and ratcliffe (1981) reported the presence in the insect hemolymph of the following cell types: prohaemocytes, plasmatocytes, granular cells, cystocytes, spherule cells and oenocytoids. subsequently brehélin and zachary (1986) proposed a new classification of blood cells, based mainly on the ultra-structural observations, and nine cell types are described: prohaemocytes, plasmatocytes, oenocytoids, spherule cells, thrombocytoids and four types of granular haemocytes. among the different types of cells described from different species, some have an immune function, while in others either the function is unknown or it is not directly involved in defence. prohaemocytes are present in all the species examined. they are small and rounded, with a large, round nucleus and seem to function as stem cells 143 (brehélin and zachary, 1986; franchini et al., 1996) (fig. 1). plasmatocytes share the typical functions of a macrophage, i.e. glass adhesion with the emission of pseudopodia allowing amoeboid movement, phagocytic capacity, encapsulation, nodule formation and wound repair (rowley and ratcliffe, 1981; franchini et al., 1996). despite the morphology, granular cells also present the immune functions described in plasmatocytes, even if to a lesser extent. spherule cells do not seem to be involved in immune functions, but the available data suggest a role in the synthesis of blood mucopolysaccharides (gupta and sutherland, 1967; akai and sato, 1973) and in silk production in bombix mori (nittono, 1960). the function of the oenocytoids is also as yet not established, even if the cytoplasmic presence of phenoloxidase (propo) activity may support an involvement in melanization processes. the crystal cells containing a crystal of tyrosine belong to this cell type and are found in some diptera (rizki and rizki, 1959; drif and brehélin, 1983). despite attempts to establish a correct, comparative classification of insect blood cell types, problems still remain. apart from the prohaemocytes, probably only two immunocytes, plasmatocyes and granular cells, appear to play a role in immune functions. however, some exceptions exist. d. melanogaster, for instance, only shows the plasmatocyte together with the specialized crystal cells (nappi and corton, 1986). this result reflects findings in other invertebrates such as molluscs and annelids (yoshino, 1976; cheng and guida, 1980; ottaviani, 1992; cooper et al., 1995). phagocytosis this phenomenon is of pivotal importance for nutrition and defence, and it is present throughout the animal kingdom. as mentioned before, both plasmatocytes (fig. 2) and granular cells are involved in phagocytosis. the process is characterized by various steps: recognition (chemotaxis and attachment), ingestion and killing. chemotaxis, i.e. non-random locomotion, allows cells to move towards a chemoattractant which has been recognized. the data on insect chemotaxis are controversial: some authors have demonstrated a chemotactic action of aspergillus flavus conidia towards immunocytes from galleria mellonella (vey et al., 1968; vey, 1969), while others failed to see such an action (salt, 1970; jones, 1956). furthermore, plasmatocyte chemotactic activity has been seen to be involved in encapsulation, nodule formation and wound repair (nappi, 1973; ratcliffe and gagen, 1977; rowley and ratcliffe, 1978). the study of the mechanism by which invertebrate immunocytes recognize non-self material is also still in its infancy. it should be remembered that the immune system involves two types of response: innate (or natural) and adaptive (or acquired). innate immunity is conserved from invertebrates to vertebrates, while adaptive immunity, based on antigen-specific t and b cells, only appears in vertebrates. in the latter, the old, innate immune system is able to discriminate between self and nonself by means of pattern-recognition receptors (prrs), able to recognize conserved pathogenassociated molecular patterns (pamps) produced by microorganisms (fearon, 1997; medzhitov and janeway, 2000). a similar scenario could also be present in invertebrates. mammalian immune cells express several toll-like receptors that are considered prrs (akira, 2001). in drosophila, the innate immune recognition of micro-organisms is mediated by signalling through toll receptors (hoffmann et al., 1999; takeda and akira, 2001). peptidoglycan recognition proteins (pgrfs) are able to recognize bacteria and their cell wall component, the peptidoglycan, and are well conserved from insects to mammals (dziarski, 2004). the mosquito anopheles gambiae secretes a thioester-containing protein (tep), αtep1, which is related to vertebrate complement factors and α2-macroglobulins (levashina et al., 2001). the reported studies are some examples of the extensive literature seeking to complete the mosaic of mechanisms involved in invertebrate innate immunity. immunocytes also recognize abiotic material suggesting that these cells exert their recognition not only on micro-organism pamps, but it is unknown how this happens. lavine and strand (2002) surmise the presence in invertebrate immunocytes of receptors with promiscuous capacities that can interact with a wide range of molecules normally not encountered in the hemocoel. in summary, as suggested by medzhitov and janeway (1997), innate immunity is based on the recognition of invariant modules in different bacterial and viral species. consequently, the limited number of recognition systems is mirrored by a limited number of molecular structures, which are recognized. paradoxically, from an invertebrate point of view, few are the microorganisms making the small number of recognition units (an extremely restricted repertoire) perfectly adequate for the small number of foreign modules to be recognized. the small number of recognition units induces a complex and efficient reaction by the ancestral defence based on the immune-neuroendocrine effector system present in invertebrates. fig. 1 cytospin preparation of immunocytes from calliphora vomitoria: a) plasmatocyte, b) granular cells (bar = 10 µm). 144 fig. 2 in vitro bacterial phagocytosis from calliphora vomitoria plasmatocyte (asterisk) (bar = 10 µm) (modified from franchini et al., 1996). innate immunity, stress and inflammation are interconnected in this system that has been conserved throughout evolution (ottaviani and franceschi, 1997; ottaviani et al., 1998). the last steps in phagocytosis are ingestion and killing. as in vertebrates, the ingested phase shows the formation of phagosomes and subsequent lysosomal fusion, as reported in the plasmatocytes of g. mellonella (rowley and ratcliffe, 1979). studies in the fruit fly ceratitis capitata suggest that signal transduction pathways that modulate phagocytosis are conserved in insects and mammals (foukas et al., 1998; metheniti et al., 2001). furthermore, teps seem to have an opsonic effect on the drosophila phagocytosis in a similar manner to mammalian complement factor c3 (lagueux et al., 2000). similar to mammals, the ingested phase needs energy that derives from the glycolitic pathway, as demonstrated by anderson et al. (1973) in the blaberus craniifer immunocytes. the authors also report an antimicrobial system. indeed, reactive oxygen intermediates (roi), reactive nitrogen intermediates (rni) and nitric oxide (no) are found in immunocytes of insects (whitten and ratcliffe, 1999; luckhart et al., 1998; nappi et al., 2000). in d. melanogaster and d. teissieri, no activates the gene encoding the anti-microbial peptide diptericin (nappi et al., 2000). encapsulation cellular encapsulation is a mechanism present in all the insect groups examined (rowley and ratcliffe, 1981). this means of defence involves immobilizing insect parasites, fungi and large protozoans that escape the phagocytic activity of the single immunocyte. the reaction is strong against living organisms and is followed by melanization, while it is weak against abiotic material and not always followed by melanization. the encapsulated material is surrounded by multi-cellular sheaths, hence the term capsule. götz (1986) defines ten steps in this cellular defence, with granular cells and plasmatocytes as the main actors. briefly, the first event in the encapsulation process is the contact of the immunocytes with foreign material and the degranulation of the granular cells. the material released from granules is sticky and adheres to foreign surfaces. the degranulation and disintegration of granular cells activate the plasmatocytes that participate in the formation of the capsule. subsequently, the process of melanization, originated in the granular cells, takes place. the approximate time of the encapsulation is about 1-3 days (fig. 3). role of melanization melanization is involved not only in encapsulation, but also in cuticular defenses, e.g. against bacteria, wounding and sclerotization, as well as in other defense responses such as the killing of bacteria or parasites and cytotoxicity (söderhräll and smith, 1986; ashida and brey, 1995; nappi et al., 1992; nayar et al., 1992; nappi and ottaviani, 2000). the melanin is synthesized by the activation of the propo-activating system (propo system) (söderhräll, 1982) comprising a complex cascade of serine proteases that allows the conversion of propo to phenoloxide (po), which, in turn, acts on substrates such as tyrosine and its derivatives (dopa and dopamine) to form melanin. this process was extensively studied for the first time in crustacean astacus astacus (söderhräll, 1982) and in the insect bombyx mori (ashida, 1971, ashida and ohnishi, 1967, ashida et al., 1983). the activation of propo has now been described in several invertebrate taxa (arizza et al., 1995; beschin et al., 1998; frizzo et al., 1999; tujula et al., 2001). the propo system has also been seen as a recognition system activated by different foreign materials, such as β-1,3-glucans from fungal and algal cell walls, and lipopolysaccharides and peptidoglycans from microbial cell walls (söderhräll and cerenius, 1998). the cdna encoding pgrps has been cloned from the silkworm b. mori fat body cdna library (ochiai and ashida, 1999). the specific binding of pgrp to the peptidoglycan induces the propo cascade. furthermore, these studies show that the means of extracellular recognition of microorganisms is conserved from insects to mammals. with regard the role of the propo system in the cuticular site, it has been demonstrated that propo originates from the circulating immunocytes in the silkworm b. mori and from immunocytes and epidermal cells in the caterpillar calpodes ethlius. subsequently, it is actively transported across the epidermis to the cuticular matrix (ashida and brey, 1995; sass et al., 1994). the propo cuticular silkworm is activated through a limited proteolysis by the serine proteinase, termed propo activating enzyme, which itself exists as a zimogen (ashida and brey, 1995). cuticular po is normally considered to be injury po, however, other two types of po are present in the cuticle of insects: granular po involved in the body colour and laccase-type po involved in sclerotization of a newly ecdysed cuticle (barrett, 1991). recently, 145 fig. 3 encapsulation of the egg of the wasp parasitoid leptopilina boulardi in drosophila melanogaster host (courtesy prof. nappi aj). asano and ashida (2001) studying cuticular po and its zymogen (propo) in b. mori purified two propo isoforms. in the same animal, the authors also found two propo isoforms in the hemolymph. these latter isoforms differ from the cuticular isoforms for the presence of five aminoacid residues. all the isoforms are activated by specific enzymes, and the hemolymph isofoms are transported to the cuticle. nodule formation this type of cellular defence takes place when phagocytosis is inadequate in the face of a large number of small non-self particles such as bacteria. rowley and ratcliffe (1981) divide nodule formation into two phases. in the first, the bacteria are entrapped in the material released by exocytosis from the granular cells and melanization then occurs externally. in the second, plasmotocytes, adhere and flatten the necrotic core forming the typical multicellular sheath. wound healing, hemolymph clotting and transplantation invertebrates respond to an external injury in order to avoid the loss of biological liquids. different mechanisms, such as fat or intestine extrusion, muscular contraction, hemolymph clot formation, cellular aggregation and melanin deposition, have been described (theopold et al., 2004, 2002; bidla et al., 2005). the hemolymph clot is well-known in the american cockroach, leucophaea maderae (bohn and barwig, 1984). the process involves clotting proteins present in the hemolymph plasma (plasma coagulogen) that are released from immunocytes (hemocyte coagulogen). the immunocytes migrate and aggregate around the altered region, and after their rupture the clotting starts. the process involves an enzymatic cascade which is activated by ca2+. the clotting reaction is very fast and in less than 3 minutes the clot is completely insoluble. a different clotting mechanism is described for locusta migratoria (brehélin, 1979). in this case, coagulation involves only the plasma (by means of the coagulogen), and this seems to be a functional equivalent in clotting of mammalian fibrinogen. lackie (1986) raised the question of how the damaged self is recognized, but following a study of the possible mechanisms involved, i.e. a non-specific response to the altered physiochemical surface proprieties of the connective tissue layer, a specific response via receptor-ligand interactions and the release of wounding factors by immunocytes, found no conclusive answer. tissue graft transplantation is a technique to test immunological specificity and memory in insects and, in general, in invertebrates, even if the mechanisms by which the host recognizes self and non-self implanted material have not yet been clarified. autoand allografts of integument in the insects b. craniifer and extatosoma tiaratum were accepted, while xenografts were rejected after melanization. all these phenomena showed an accumulation of immunocytes around the grafts which was more marked in the xenografts (thomas and ratcliffe, 1982). a different result was obtained with allogeneic cuticle and xenogeneic cuticle from blatta orientalis. both grafts were recognized as foreign by periplaneta americana, while implanted xenogeneic tissue from b. craniifer was not rejected by p. americana (lackie, 1983). the transplantation experiments by karp and meade (1993) using the same species, i.e. p. americana and b. orientalis, found that p. americana recognizes b. orientalis as foreign and mounts a rejection response against integumentary grafts. humoral component although lacking immunoglobulins, insects, as all other invertebrates, possess a variety of factors of varying degrees of specificity, including lectins, cytokine-like molecules and anti-microbial peptides. 146 lectins lectins are sugar-binding proteins or glycoproteins that agglutinate cells and/or precipitate glycoconjugates (goldstein et al., 1980). in insects and other invertebrates, the lectins are also called agglutinins and hemagglutinins. many insect species contain natural agglutinins (yeaton, 1981; ratcliffe and rowley, 1983; chen et al., 1993) that can be induced by antigenic stimulation (komano et al., 1980; ratcliffe and rowley, 1983). these are mainly produced by immunocytes (whitcomb et al., 1974) and fat body (komano et al., 1983). biomolecular studies have allowed the cdna from lectins of arachis hypogaea (shanker and das, 2001) and from pinellia ternata (yao et al., 2003) to be cloned. even if conflicting data have been reported, these substances appear able to agglutinate foreign material (komano et al., 1980; ratcliffe and rowley, 1983), they present opsonic properties (pendland et al., 1988; kawasaki et al., 1993), they increase phagocytic activity (wilson et al., 1999) and they have a toxic effect (powell et al., 1998). with regards this latter effect, galanthus nivalis agglutinin (gna) added to the diet of the insect pest, nilaparvata lugens, has been seen to cross the midgut epithelial barrier and pass into the circulatory system of the insect, so exerting its toxic effect. lectins from p. ternata and pinellia pedatisecta present insecticidal activity towards cotton aphids (aphis gossypii) and peach potato aphids (myzus persicae) when incorporated into artificial diets (huang et al., 1997; pan et al., 1998). cytokine-like molecules cytokines belong to the broad family of soluble factors that by communicating among different cell types induce the complex interactions that allow immune responses. these molecules have been called by different names according to their origin or function. nowadays, the general term cytokines is used for these signal molecules. interleukins (il), tumour necrosis factor (tnf), interferons (ifn), transforming growth factor (tgf)-β, platelet-derived growth factor (pdgf), etc. are the most common examples in mammals. cytokine-like molecules have been reported in various tissues of a number of invertebrates, including insects (table 1). the first findings on cytokine-like molecules in invertebrates reported a so-called "growth promoting substance" in insects and, in particular, in b. mori (aizawa and sato, 1963; vaugghn and louloudes, 1978) and samia cynthia (williams and kambysellis, 1969). cherbas (1973) indicated haemokinin as a fraction partially purified from both plasma and epidermal tissue of saturniid pupae and able to stimulate immunocyte activation. subsequently, the presence of epidermal growth factor (egf), fibroblast growth factor (fgf) and others has been demonstrated in different cells and tissues (table 1). particular attention has been paid to insulin-like growth factors, which have been described in different taxa, including insects (joosse et al., 1983; ebberink et al., 1989). moreover, genomic dna clones encoding brain secretory peptides that have been isolated from insects share substantial homologies with vertebrate preproinsulins. (adachi et al., 1989; iwami et al., 1989; lagueux et al., 1990). il-1α-, tnf-α-, pdgf-aband tgf-β1-like molecules have been detected in the plasmatocytes and granular cells of calliphora vomitoria and g. mellonella (franchini et al., 1996; wittwer et al., 1998). similar results have also been reported in the cell lines derived from the hemolymph of estigmene acraea and from the fat body cell of lymatria dispar (wittwer et al., 1998; ottaviani et al., 2000). the presence in insects of pro-inflammatory cytokines, i.e. il-1 and tnf-α, suggest an important role of these molecules in animal host defence responses. even if few functional data are present in the literature regarding insects, studies from our laboratory dealing with cytokines in invertebrates (mainly molluscs) have demonstrated that mammalian cytokines affect immune and neuroendocrine functions, wound repair and programmed cell death, while, at the same time, presenting the pleiotropicity, functional redundancy and receptor promiscuity seen in vertebrates (ottaviani et al., 2004). these findings suggest that the same frame may also exist in insects. furthermore, the insect cytoplasmic domain of the toll family proteins is homologous with the cytoplasmatic domain of the il-1 receptor family (kopp and medzhitov, 1999). given the lack of molecular biology data, it has been hypothesized in the literature that a correlation between invertebrate and vertebrate cytokine genes does not exist (beschin et al., 2001). however, to claim that mammalian cytokines intervene in the main invertebrate functions merely as a result of functional convergence does seem reductive. different research groups have found that the main molecules involved in the basic and fundamental functions of cell survival, for example adenocorticotropic hormone (acth), corticotrophin-releasing hormone (crh) and nitric oxide synthase (nos), show a gene homology with their vertebrate counterparts (duvaux-miret and capron, 1991; yuda et al., 1996; salzet et al., 1997). anti-microbial peptides anti-microbial peptides that are expressed constitutively or are readily inducible have been extensively studied in insects (boman, 1995; bulet et al., 1999). the main production site is the fat body, a functional equivalent of the mammalian liver (fehlbaum et al., 1994), but immunocytes (boman, 1991), the cuticular epithelial cells (brey et al., 1993) and the reproductive tract (rosetto et al., 1996) are also involved. bulet et al. (1999) classified the antimicrobial peptides into three classes on the basis of the sequence and structural characteristics: 1. linear peptides forming α-helices and devoid of cysteine residues; 2. cyclic peptides containing cysteine residues; 3. peptides with an over-representation in proline and/or glycine residues. the different anti-microbial peptides are named attacins, cecropins, defensins, drosomicins, etc. according to the bulet et al. (1999) classification, cecropins belong to the first class of anti-microbial 147 table 1 insecta cytokine-like molecules references ______________________________________________________________________ bombyx mori growth promoting factor aizawa and sato (1963); vaughn and louloudes(1978) samia cynthia growth promoting factor williams and hemokinin kambysellis (1969) cherbas (1973) antheraea polyphemus hemokinin cherbas (1973) hyalophora cecropea hemokinin cherbas (1973) drosophila melanogaster egf wharton et al. (1985); tgf-α, β kelley et al. (1987); neurotrophic factor padgett et al. (1987); imaginal disc gfs bryant (1988); kopczynski et al. (1988); hayashi et al. (1992); neuman-silberberg and schupback (1993); kutty et al. (1998); kawamura et al. (1999) manduca sexta hemolymph trophic factor wielgus et al. (1990) calliphora vomitoria tnf-α, pdgf-ab, tgf-β1 franchini et al. (1996) galleria mellonella il-1α, tnf-α wittwer et al. (1999) estigmene acraea il-1α, tnf-α wittwer et al. (1999) lymantria dispar pdgf-ab, tgf-β1 ottaviani et al. (2000) ______________________________________________________________________________ peptides, while defensins are ascribable to the second class. cecropins were the first anti-microbial peptides to be isolated and characterized (hultmark et al., 1980). they are small 4 kda peptides found in diptera and lepidotera and present an anti-bacterial activity against both gram positive and gram negative bacteria. cecropins share common features, i.e. molecular size (31-39 aminoacids), a strong basicity in the n-terminal region and a hydrophobic portion in cterminal part. they are induced by bacterial infection and are synthesized as preproproteins of 62-64 residues (boman, 1991). the precursors are first cleaved by a signal peptidase, and the pro-portion is then removed by a dipeptidyl aminopeptidase (boman et al., 1989). in contrast to cecropins, defensins mainly attack gram positive bacteria (boman, 1991). they are 4 kda cationic peptides with a characteristic six cysteine/three disulfide bridge pattern and three domains, a flexible amino-terminal loop, a central αhelix and a carboxy-terminal anti-parallel β-sheet (hanzawa et al., 1990; bonmatin et al., 1992; bulet et al., 1999). several reports have described the presence of defensins in different insect species (bulet et al., 1999), but not in lepidoptera. in our laboratory we recently identified for the first time the presence of a defensin active against gram positive bacteria in lepidoptera (izd-mb-0503 cell line derived from immunocytes of mamestra brassicae) (mandrioli et al., 2003). the biomolecular study revealed the presence of the defensin gene of 294 pb (fig. 4). alignment of m. brassicae defensin with homologous sequences from the insect defensin a genes revealed identity ranging from 43 % to 59 % (fig. 5). the analysis of the putative protein indicated the presence of 98 aminoacids, including 8 cysteine residues. in particular, cysteine residues 3-8 were highly conserved, suggesting their involvement in the formation of three disulfide bridges. northern blotting experiments showed a constitutive expression of the defensin gene in m. brassicae cells. its expression was increased by gram positive, but not by gram negative bacteria (fig. 6). fig. 4 complete sequence of m. brassicae defensin gene. upper lines show the nucleotide sequence, while the putative amino acidic sequence is indicated in the lower lines (modified from mandrioli et al., 2003). 148 fig. 5 defensin peptide alignment of mamestra brassicae (af465486) (a) with aedes aegypti (af156093) (b), apis mellifera (d17670) (c), phormia terranovae (af182164) (d), drosophila melanogaster (ac007414) (e) and anopheles gambiae (af063402) (f). highly conserved aminoacids are boxed (modified from mandrioli et al., 2003). fig. 6 northern blotting with the defensin probe on rna samples extracted at different times from m. brassicae cells after induction with heat-killed (a) and live bacteria (b). northern blotting indicates that defensin expression is increased by gram positive bacteria, in particular by live specimens, but not by candida albicans and saccharomyces cerevisiae (c) (modified from mandrioli et al., 2003). defensin induction was greater when using live bacteria rather than heat-killed specimens (fig. 6). no defensin gene induction was observed in the presence of candida albicans and saccharomyces cerevisiae (fig. 6). microbial killing by antimicrobial peptides involves both the charge and the structure of the latter. in cecropins and defensins, the mechanism is thought to relate to the peptides’ ability to assemble in the target membrane and form a pore (boman, 1991). the means by which cecropins associate with the plasma membrane is called “carpet-like” and involves: i) peptide arrangement parallel to membrane surface by binding to the phospholipid head groups; ii) rotation of peptides in order to re-orientate their hydrophobic residues toward the membrane hydrophobic core; iii) disruption of the membrane 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1976. yuda m, hirai m, miura k, matsumura h, ando k, chinzei y. cdna cloning, expression and characterization of nitricoxide synthase from the salivary glands of the bloodsucking insect rhodnius prolixus. eur. j. biochem. 242: 807-812, 1996. title: isj 7: 32-44, 2010 issn 1824-307x review the complexity of drosophila innate immunity a reumer1, t van loy2, l schoofs1 1 department of biology, functional genomics and proteomics unit, k.u.leuven, naamsestraat 59, 3000 leuven, belgium 2 department of biology, molecular developmental physiology and signal transduction unit, k.u.leuven, leuven, belgium; current address: institut de recherche interdisciplinaire et biologie humaine et moléculaire (iribhm), faculty of medicine, université libre de bruxelles (belgium) accepted january 11, 2010 abstract metazoans rely on efficient mechanisms to oppose infections caused by pathogens. the immediate and first-line defense mechanism(s) in metazoans, referred to as the innate immune system, is initiated upon recognition of microbial intruders by germline encoded receptors and is executed by a set of rapid effector mechanisms. adaptive immunity is restricted to vertebrate species and it is controlled and assisted by the innate immune system. interestingly, most of the basic signaling cascades that regulate the primeval innate defense mechanism(s) have been well conserved during evolution, for instance between humans and the fruit fly, drosophila melanogaster. being devoid of adaptive signaling and effector systems, drosophila has become an established model system for studying pristine innate immune cascades and reactions. in general, an immune response is evoked when microorganisms pass the fruit fly’s physical barriers (e.g., cuticle, epithelial lining of gut and trachea), and it is mainly executed in the hemolymph, the equivalent of the mammalian blood. innate immunity in the fruit fly consists of a phenoloxidase (po) response, a cellular response (hemocytes), an antiviral response, and the nf-κb dependent production of antimicrobial peptides referred to as the humoral response. the jak/stat and jun kinase signaling cascades are also implicated in the defence against pathogens. key words: drosophila; innate immunity; signaling cascades introduction immune responses are typically distinguished in two main systems, the adaptive and the innate immune response. adaptive immunity is specific, has memory and is generally considered to be restricted to vertebrates. it relies on the generation of immune receptors, like immunoglobulins and tcell receptors, through somatic gene rearrangements in specified blood cells and on the clonal expansion of activated lymphocytes (b and t cells). innate immunity, on the other hand, refers to the evolutionary ancient and presumably conserved first-line host defence against the early phases of microbial infection, and it is believed to be naive in recognizing broadly conserved microbial moieties. the model organism drosophila melanogaster only seems to rely on this innate immune system for its ___________________________________________________________________________ corresponding author: ank reumer department of biology functional genomics and proteomics unit k.u.leuven, naamsestraat 59, 3000 leuven, belgium e-mail: ank.reumer@bio.kuleuven.be defence against pathogens. drosophila therefore has physical barriers (exoskeleton, chitinous epithelial lining of gut and trachea) to prevent microbial entrance into its body cavity. additionally, specialized blood cells, called hemocytes, floating in the hemolymph participate in phagocytosis and encapsulation of foreign invaders and some of their components are needed for their subsequent melanization. the larval fat body, a functional analog of the mammalian liver and the main larval energy reservoir, is the main site of the fruit fly larva’s humoral (or systemic) immune response, while in adults other tissues can take on major roles for immune surveillance (hoffmann, 2003). humoral response the humoral response is the most intensively studied part of drosophila innate immunity. it consists of recognition systems in the hemolymph which signal the presence of pathogens to the fat body, one of the main immune tissues. this leads to the activation of the toll and/or the immune deficiency 32 mailto:ank.reumer@bio.kuleuven.be fig. 1 the drosophila imd pathway. grambacterial cell wall components (dap type pgn) in the hemolymph are recognized by transmembrane or extracellular pgrp-le and pgrp-lc. imd pathway activation then is initiated by multimerization of transmembrane pgrp-lc and intracellular pgrp-le both containing a rhim-like [receptor interacting protein (rip) homotypic interaction motif] motif. pgrp-sc and pgrp-lb mediate the intensity of this activation. the adaptor protein that links this rhim-like motifs containing complex to imd is still unknown. downstream of imd, several processes can be distinguished of which some probably depend on k63 linked ubiquitin chains for the assembly of active protein complexes. 1) genetic evidence suggest the combined action of ubc-13 (ubiquitin conjugating enzyme), diap-2 (drosophila inhibitor of apoptosis protein 2) and uev1a (ubiquitin conjugating enzyme e2 variant 1) to be needed for the formation of these ubiquitin chains. 2) the complex formed between tak1 and tab2 mediates both jnk pathway and ikk signaling activation. the ikk complex composed of the regulatory subunit kenny and the catalytic subunit ird5 then phosphorylates relish. 3) the unit consisting of imd, fadd and dredd, which interact with each other through their death domains (dd), respectively death effector domains (ded), is supposed to be involved in the activation of tak1. dredd further is presumed to cleave phosphorylated relish resulting in the freeing of the rel transcription factor. further processing steps of the phosphorylated inhibitory part of relish are unknown. dnr-1 (defence repressor 1) probably is part of a negative regulatory loop that keeps the imd-fadd-dredd complex inactive until imd pathway activation upon infection while sickie positively regulates dredd mediated cleavage of relish (foley and o'farrell, 2004). the final result of all these processes is the translocation of the nf-κb transcription factor rel to the nucleus to start transcription of target genes. caspar and the ubiquitin-proteasome pathway further negatively regulate imd pathway signaling. 33 (imd) pathway resulting in the synthesis of amps as well as other effector molecules, and their subsequent release into the hemolymph to fight infection (reviewed in ferrandon et al., 2007). recognition of pathogens is mainly achieved by a diverse array of pattern recognition receptors (prrs) belonging to the peptidoglycan recognition protein (pgrp) and gram negative binding protein (gnbp) families which can discriminate between distinct classes of microorganisms (lemaitre et al., 1997). detection of the diaminopimelic (dap) type of peptidoglycan (pgn) constituting the inner membrane of gbacteria is mediated by pgrp-lc isoforms and pgrp-le resulting in the activation of the imd pathway (fig. 1) (wu et al., 2001; choe et al., 2002; gottar et al., 2002; ramet et al., 2002; takehana et al., 2004; kaneko et al., 2006). lysine (lys) type pgn of g+ bacteria on the other hand is recognized by pgrp-sa, pgrp-sd and gnbp1 leading to toll pathway activation (fig. 2) (michel et al., 2001; gobert et al., 2003; bischoff et al., 2004). sensing of fungal presence relies on the detection of glucan, a fungal cell wall constituent, by gnbp3 (gottar et al., 2006) and on the activation of the serine protease persephone (psh) in the hemolymph by virulence factors like fungal proteases (ligoxygakis et al., 2002). both fungal infection detection cascades converge on toll pathway activation (fig. 2). the ability to form several prr complexes furthermore expands the repertoire of microbial species of which the presence can be sensed (bischoff et al., 2004). recent findings also suggest that these recognition systems presumably detect proliferation of bacteria rather than their presence (ferrandon et al., 2007). furthermore, some microbial species can elicit both toll and imd signaling through a yet unknown process (de gregorio et al., 2002b). upon detection of gram negative bacterial infection, activation of the imd pathway (fig. 1) is probably achieved through cooperation of transmembrane pgrp-lc and intracellular pgrple (takehana et al., 2004; chang et al., 2005; mellroth et al., 2005; kaneko et al., 2006). pgrpsc and pgrp-lb, which reside in the hemolymph, further modulate the intensity of the imd pathway stimulation (mellroth et al., 2003; mellroth and steiner, 2006; zaidman-remy et al., 2006). this then results in the induction of several cascades downstream of the imd protein leading to activation of the relish derived nf-κb transcription factor rel (hedengren et al., 1999), as well as to jun nterminal kinase (jnk) pathway activation (boutros et al., 2002; park et al., 2004). both phosphorylation and cleavage of relish are needed to liberate the transcription factor fragment rel from its inhibitory part. phosphorylation is performed by the iκb kinase (ikk) signaling complex composed of kenny and ird5 (immune response deficient 5) whereas cleavage involves the coordinated action of at least three proteins, namely imd, fadd (fas-associated death domain) and dredd (death-related ced3/nedd2-like protein) (rutschmann et al., 2000; silverman et al., 2000; stoven et al., 2000; lu et al., 2001; leulier et al., 2002;). ikk and jnk activation furthermore is mediated by tak1 and tab2 (tak1binding protein 2) (vidal et al., 2001; boutros et al., 2002; silverman et al., 2003; gesellchen et al., 2005; geuking et al., 2005; kleino et al., 2005). rel-induced gene activation then results in the synthesis and release of amps and other effector proteins. among the drosophila amps, diptericin (dipt), attacin (att), drosocin (dros) and cecropin (cec) were identified as being imd pathway induced (hoffmann, 2003). negative regulation of the imd pathway furthermore involves caspar and the ubiquitin-proteasome pathway for controling relish and dredd activation levels (khush et al., 2002; kim et al., 2006). the physiological relevance of jnk signaling in the innate immune response remains elusive although it has been suggested to be involved in the control of the expression of some amps, and to regulate wound healing and melanization (boutros et al., 2002; igaki et al., 2002; silverman et al., 2003; kim et al., 2005) jnk signaling furthermore is shut off upon relish activation. the jnk-dependent response thus is transient and it precedes the sustained induction of relish-dependent innate immune loci (park et al., 2004). induction of the toll pathway (fig. 2) upon g+ and fungal infection is preceded by the cleavage of spätzle (spz) by the spätzle processing enzyme (spe) (jang et al., 2006). spz then binds to the membrane bound toll receptor thereby initiating the assembly of the toll induced signaling complex (tisc) which is composed of myeloid differentiation primary response gene 88 (myd88), tube and pelle (tauszig-delamasure et al., 2002; weber et al., 2003). tisc subsequently targets the cactus-dif (dorsal related immunity factor) complex in an unknown way to induce the degradation of the nfκb inhibitor cactus resulting in the release of the nf-κb transcription factor dif. gene activation is then accomplished upon translocation of dif to the nucleus (belvin et al., 1995; fernandez et al., 2001). drosomycin (drom), defensin (def) and metchnikowin (metch) are the amps of which the synthesis is induced by toll pathway activation (hoffmann, 2003). both the induction of the toll and the imd pathway thus leads to the activation of nf-κb transcription factors, dif and rel, which recognize distinct κb binding sites. many more putative toll and imd pathway components were discovered using several distinct assays, e.g., rnai on cultured s2 cells (avila et al., 2002) and loss-off-function screens (wu and anderson, 1998; wu et al., 2001), but their sites of action in toll and/or imd signaling still need to be explored. constitutive amp activation furthermore has been demonstrated in several epithelia in close contact with the environment. their expression however seems to be independent of both toll and imd signaling. upon infection, on the other hand, amp expression in these epithelia solely relies on imd pathway activation (ferrandon et al., 1998; tzou et al., 2000). cellular response the blood cells, or hemocytes, of drosophila participate in the immune response through the production of amps, the phagocytosis of bacteria, the 34 fig. 2 the drosophila toll pathway. structural components of the cell wall of g+ bacteria and fungi are detected by soluble pattern recognition receptors in the hemolymph. pgrp-sa, pgrp-sd and gnbp1 recognize lys type pgn probably upon formation of prr complexes, and gnbp3 binds to fungal derived β-glucans. virulence factors like fungal proteases [e.g., pr1 (gottar et al., 2006)] also can initiate an immune response through activation of the clip serine protease psh. the protease inhibitor necrotic (not shown) furthermore seems to be involved in sensing fungal infection in the same proteolitic pathway as psh (levashina et al., 1999). recently a second proteolitic cascade acting downstream of circulating prrs and including the activity of grass (gram positive specific serine protease) was uncovered (not shown) (el chamy et al., 2008). both the psh and the grass dependent pathways are required for full activation of toll upon fungal and g+ infection. all these diverse pathogen recognition systems converge in an largely unknown way to the cleavage of spätzle by spe resulting in the release of its 106 amino acid c-terminal fragment (c106). recently the modular serine protease (modsp) was shown to integrate signals originating from gnbp3 and pgrp-sa and to connect them to spe activity on spz (buchon et al., 2009b) spätzle c106 then binds to toll thereby inducing conformational changes in the receptor resulting in its activation. activated toll induces the assembly of the tisc composed of three members bearing dds. tube links pelle to myd88 through dd interactions, and myd88 probably interacts with toll through its tir (toll/il-1r) domain. tisc formation results in the activation of the kinase domain (kd) of pelle, and in an unknown way in the phosphorylation of the nf-κb inhibitor cactus thereby initiating its rapid polyubiquitylation and subsequent degradation. this allows the liberated nf-κb transcription factor dif to translocate to the nucleus and to induce gene activation upon binding to nf-κb elements. a cell culture study furthermore suggest the existence of a second branch downstream of toll activation consisting of atypical protein kinase c (apkc) and ref(2)p signaling components to aid in inducing dif activation (avila et al., 2002). 35 encapsulation of larger foreign particles such as parasitic eggs as well as the signalling of pathogen presence to the fat body (evans et al., 2003). at the larval stage, three types of circulating hemocytes can be distinguished originating from the embryonic head mesoderm (holz et al., 2003), the larval lymph glands (sorrentino et al., 2002) as well as from sessile hemocytes attached to larval epithelial tissues (markus et al., 2009). among these, the predominant type (90-95%) consists of small round plasmatocytes which essentially are phagocytic cells or macrophages. these engulf and degrade apoptotic and dead cells, and cellular detritus as well as microbial pathogens occurring in their hemolymph in a process known as phagocytosis. phagocytosis is initiated by the recognition of these targets mainly by four classes of molecules: the complement-like opsonin family of teps (thioestercontaining proteins), the class b scavenger receptors cd36, peste and croquemort, the egflike repeat containing receptors eater, nimrod c1 and draper, and the dscam (down syndrome cell adhesion molecule) isoforms (reviewed in stuart and ezekowitz, 2008). most of these components as well as a plethora of other intercellular phagocyte gene products were discovered with studies using a variant of drosophila embryonic s2 (schneider’s line 2) cells combined with rnai (pearson et al., 2003;ramet et al., 2001). additional studies are required to explore their function(s) in vivo. plasmatocytes also secrete amps as well as other immune signaling components. upd3 (unpaired 3), one of these molecules, has been shown to signal an infection induced by septic injury to the fat body thereby activating the jak/stat pathway in a cytokine-like manner (agaisse et al., 2003). a smaller proportion of circulating hemocytes is formed by the crystal cells which are recognized by pronounced crystal-like inclusions in their cytoplasm. these contain the enzymes necessary for humoral melanization which accompanies many immune reactions (see below). the third circulating hemocyte type, the lamellocyte, is normally not present in healthy larvae. lamellocytes are large flat cells of which the amount substantially increases after specific stimuli, e.g., presence of parasitic wasp eggs, through a small burst of mitosis and subsequent differentiation of sessile cells in the lymph glands (sorrentino et al., 2002) and through differentiation of a subepidermal lamellocyte precursor population (markus et al., 2009). the balance between the multipotent prohemocytes and the differentiating blood cells upon infection is controlled by a small cluster of signalling cells in the lymph glands, termed the posterior signalling centre (psc) (krzemien et al., 2007). lamellocytes mediate the encapsulation of invaders, e.g., parasitoids, that are often too large to be phagocytized. integrins seem to be involved in the strengthening of the capsule (irving et al., 2005). encapsulation furthermore is often accompanied by a localized melanization reaction and an augmented nitric oxide (cytotoxic) production, resulting in the killing of the parasite within the black capsule (carton and nappi, 1997, nappi et al., 2000). the hemocytes detected in drosophila adults originate from the embryonic and larval lineages that persist during metamorphosis to populate this developmental stage. no adult hematopoietic organ has been described so far (holz et al., 2003). recently, it was found that priming drosophila with sublethal doses of streptococcus pneumoniae or the natural fly pathogen beauveria bassiana has protective effects during subsequent challenges, and this persisted for the life of the fly. phagocytes as well as toll pathway activation, however independent of amp synthesis, were required for this presumably species specific but not generally observed effect. these findings raise questions about the absence of memory in drosophila’s innate immune responses (pham et al., 2007). for a more in depth description of the cellular immune response, see for review evans et al. (2003) and stuart and ezekowitz (2008). the phenoloxidase response the melanization reaction seems to be the most immediate immune response against invading pathogens in drosophila (cerenius and söderhäll, 2004). it frequently assists the encapsulation reaction in the killing of microbial pathogens (nappi and christensen, 2005). melanization is visible by the blackening of a wound site or of the surface of invaders, which results from the synthesis and deposition of melanin (tang et al., 2006). the melanization reaction starts off with the activation of prophenoloxidases (propo) to active phenoloxidase (po) enzyme in the hemolymph of arthropods, hence it is also referred to as po response. this system had been extensively studied in large insects such as manduca sexta (cerenius and söderhäll, 2004; söderhäll and cerenius, 1998). this has led to the present model (fig. 3) in which the recognition of microorganisms triggers a propoactivating enzyme (ppae) proteolytic cascade dedicated to the activation of po. the activated po then catalyses the oxidation of tyrosine-derived phenols to quinones. quinones then polymerize non-enzymatically to form insoluble melanin. quinones as well as melanin and its biosynthetic byproducts, hydrogen peroxide and nitric oxide amongst others, are directly toxic to microorganisms (nappi et al., 2000, nappi and ottaviani, 2000). melanin deposition is observed at all infection sites, where it possibly contributes to wound healing and to the control of microorganism growth as well as to their killing (leclerc et al., 2006, nappi et al., 1995, carton et al., 2009, nappi and christensen, 2005). of note, nearly all arthropod propos are devoid of a secretion signal sequence. there presence in the hemolymph is therefore assumed to result from hemocyte rupture, as reported for some drosophila propos (bidla et al., 2007). in drosophila, induction of the melanization response upon ginfection involves pgrp-le, which is also one of the key prrs of the imd pathway (fig. 1). the crystal cells furthermore are shown to express propo a1 (or 54) as well as propo45. the third drosophila propo is expressed exclusively in lamellocytes. the cells that mediate the encapsulation response thus also can provide 36 fig. 3 overview of the arthropod melanization cascade. the system is activated upon recognition of bacterial and fungal cell wall components by prrs as well as by some endogenous factors produced upon tissue damage, e.g., during wounding. a cascades of serine proteases presumably will result in the cleavage of proprophenoloxidase activating enzyme (pro-ppae) thereby activating it. cleavage of propo by active ppae results in the activation of po. next, po catalyzes the oxidation of phenols to quinones, which subsequently can polymerize to melanin. control of po activity is presumed to result from its synthesis as inactive precursor as well as from the presence of proteinase inhibitors which probably avoid excessive or premature activation (adapted from cerenius & söderhäll, 2004). one of the key enzymes for melanization (irving et al., 2005). the activation of propo in drosophila is partially controlled by the serine protease inhibitor serpin 27a (spn27a) (nappi and christensen, 2005). the target of spn27a is thought to be ppae since recombinant spn27a is able to inhibit beetle ppae (de gregorio et al., 2002). this however has not been demonstrated to occur in vitro or in vivo with drosophila ppae. two immune inducible serine proteases, mp1 (cg1102) and mp2 (cg3066) (melanization protease 1 and 2), furthermore were identified which act in the po cascade regulated by spn27a. mp1 seems to be required for activation of the melanization process upon bacterial and fungal infection whereas mp2 is only involved in fighting fungal infection and thereby acting upstream of mp1. mp2 is furthermore able to induce drosomycin expression independent of toll pathway activation (tang et al., 2006). mp2 (or ppae1) was furthermore reported to be a drosophila ppae as constitutive ppae mutants showed constitutive po activation (leclerc et al., 2006). further elucidation of the drosophila po response thus is required to explore whether it is consistent with the proposed model (fig. 3). recently, colinet et al. (2009) identified the first serpin used as a virulence factor from the parasitoid wasp leptopilina boulardi and they showed that it targets the drosophila phenoloxidase cascades. the jak/stat cascade in innate immunity the evolutionary conserved janus kinase (jak)/signal transducer and activator of transcription (stat) cascade plays a key role in a wide variety of biological processes (arbouzova and zeidler, 2006). in drosophila, only one stat protein, stat92, seems to exist and hopscotch (hop) is the homolog of vertebrate tyrosine kinase jak (binari and perrimon, 1994; hou et al., 1996; yan et al., 1996). loss-of-function mutants of both stat92e and hop show a severe decrease in immune response activation upon infection, implicating the drosophila jak/stat pathway in the innate immune defense (sorrentino et al., 2004). stat92e furthermore is activated in the fat body upon immunization (kwon et al., 2000). jak/stat pathway activation (fig. 4) requires binding of an extracellular ligand, unpaired (upd), to a transmembrane receptor, domeless/master of marelle (dome/mom). upd, upd2 or upd3 constitute the drosophila upd family of which only the latter is implicated in the fruit fly’s immune response (agaisse et al., 2003). ligand binding then results in the activation of receptor-associated jaks which recruit stats after their phosphorylation. next, the stats are phosphorylated and they will form the dimers that are responsible for gene activation upon translocation to the nucleus (see for review 37 fig. 4 the jak/stat pathway. upd binding onto the dome receptor dimer activates jak kinases. jaks phosphorylate (p) one another and subsequently attract stats. after jak dependent phosphorylation and subsequent dimerization, stat dimers are translocated to the nucleus through nuclear pore complexes to act as activators of gene transcription. arbouzova and zeidler, 2006). upon infection, turandot a (tota), totm and totc, which normally accumulate in the hemolymph in response to various stress conditions including immune challenge, are expressed in the fat body thereby requiring jak activity (agaisse et al., 2003). gene activation of vir1 (virus induced rna 1) furthermore is also attributed to jak/stat signaling since stat92e binds to its promoter (dostert et al., 2005). in drosophila, several negative regulators of jak/stat signalling were identified (fig. 4). among these, soc36e (suppressors of cytokine signaling 36e) and tyrosine phosphatase ptp61f (phosphotyr phosphatase 61f) both are a transcriptional target as well as a negative regulator, thereby forming a negative feedback loop to down-regulate pathway activity (baeg et al., 2005; muller et al., 2005). furthermore, the drosophila homologs of ranbp3 and ranbp10 control the nucleocytoplasmic transport of stat92e (baeg et al., 2005). ken and barbie (ken), a transcriptional repressor, selectively regulates stat92e activity (arbouzova et al., 2006). jak/stat pathway activity furthermore is also detected in hemocytes where it modulates hemocyte proliferation and differentiation, e.g., into lamellocytes in cooperation with the ras and toll pathway (evans et al., 2003). biological functions of jak/stat, besides its implication in the innate immune response, are summarized in agaisse and perrimon (2004) and in arbouzova and zeidler (2006). the antiviral response: implication of rna interference in the innate immune system rnai probably originated as an innate immune mechanism for fighting viral infections (fig. 5). viral dsrna thereby is used to trigger host-mediated degradation of viral rna. the identification of viral proteins, e.g., b2 of flock house virus and 1a from dcv (drosophila c virus), that are able to inhibit the rnai pathway (li et al., 2004) and the presence of viral small interfering rnas in infected cells (aliyari et al., 2008) support this rnai origin hypothesis. in drosophila, three rnai pathways are described of which at least two, an argonaute 2 (ago2) dependent and the piwi pathway, seem to be implicated in the anti-viral defence although both are elicited upon different viral infestations (van rij and berezikov, 2009). of note, dicer 2, argonaute 2 and r2d2 are encoded by the 3 % fastest evolving genes in drosophila. this probably is driven by the likewise fast evolving viral inhibitor proteins (obbard et al., 2006). in addition, studies using drosophila x virus (dxv, a dsrna virus) implied both toll and imd pathway signaling in the detection of viral infection, although only a toll induced nf-κb activation, dissimilar to the one described for amp production (fig. 2) seems to confer protective effects (zambon et al., 2005). dcv infection, adversely, results in jak/stat-induced immune signaling but not either tollnor imd-mediated immune responses (dostert et al., 2005). viruses thus evoke different defense 38 fig. 5 defense response against viral infection. left: viral infection (mainly through endocytosis) as well as viral replication in infected cells depend on endogenous cellular factors of the infected host (depicted in green). many viruses depend on dsrna production for replication. the hosts rnai machinery, composed of dicer-2 (dcr-2) and its cofactor r2d2, mediate cleavage of the viral dsrna into sirnas. these are subsequently incorporated into an ago2 containing risc complex which is devoted to the targeted destruction of analogical sequence specific viral rna (depicted in blue). viruses object this rnai mediated defence response by encoding rnai inhibitor components (depicted in red). some viral infections furthermore lead to the activation of a toll induced, yet unknown signaling cascade that results in nf-κb induced (dif) antiviral gene activation (depicted in blue). recently, the amino terminal dexd/h box helicase domain of dicer 2 was implicated in the inducible antiviral response thereby suggesting a connection between this and rnai (deddouche et al., 2008). right: uninfected cells may be induced to produce and release antiviral factors by an unknown mechanism probably involving recognition of viral products, cell debris or other host antiviral factors. both jak/stat and toll pathway activation have been implicated in the production of the proteins of which some are assumed to have antiviral proporties, e.g., vago (deddouche et al., 2008) and vir-1 (dostert et al., 2005). responses probably depending on differences in their pathogenesis and replication cycles. the exploration of the antiviral response in drosophila commenced about five years ago resulting in a yet limited understanding which is even more complicated by the different defense responses evoked by various viruses (see for reviews kemp and imler, 2009; van rij and berezikov, 2009). immune defence systems in epithelial tissues the innate immune responses described above were all explored in the two main immune tissues of drosophila, the fat body and the hemocytes. they focus on the recognition and signaling of a pathogenic invader in the body cavity, and the subsequent production and release of immune effectors into its main battlefield, the hemolymph. this is referred to as the systemic immune response. as is the case in mammals, also the epithelial linings of the digestive, respiratory and reproductive system that constitute the physical barrier for pathogen entrance into the body cavity do seem to rely on an effective immune defense system to try to prevent this invasion. epithelial expression of antimicrobial peptides, for example was explored using reporter flies in which the promoter sequence of each of the seven amps (att, cec, def, droc, dipt, drom and metch) was fused to the green fluorescent protein coding sequence. this way, the expression pattern of the amps was explored both in larvae and in adults (ferrandon et al., 1998; tzou et al., 2000). this led to the finding that amps can be induced in surface epithelia in a tissue-specific manner and that imd plays a critical role in the activation of this local response to infection (tzou et al., 2000). malphigian tubule epithelia, furthermore, shown expression of pgrps, e.g., pgrp-le when induced by tct (tracheal cytotoxin), disaccharide-tetrapeptide fragment of pgn (kaneko et al., 2006). furthermore, the drosophila gut lumen is considered to be hostile to transient microbial 39 colonization due to physical (acidity) and physiological (peristalsis of the gut) properties and the presence of lysozymes. and the gut epithelium further was shown to express amps and to catalyze the generation of ros that together most often provide an effective barrier against ingested microbes. next, global gene expression analysis of drosophila intestinal tissue, i.e., the gut minus the malpighian tubules and gastric caeca, to oral infection with the gbacterium erwinia carotovora recently revealed that immune responses in the gut are regulated by the imd and jak-stat pathways, but not the toll pathway (buchon et al., 2009a). in addition, the malpighian tubules also possess cellautonomous, immune-sensing capabilities. all components of both the imd and the toll pathway are expressed herein, and they are expected to mainly lead to the production of diptericin, cecropin and metchnikowin. the tubules furthermore produce nitric oxide (no) which was also shown to be extremely important for amp production as significant improvement in survival rates of the whole animal upon immune challenge was observed after forced no production (davies and dow, 2009). similarities between mammalian and insect immune responses insects and vertebrates display considerable overlap in the signalling pathways that regulate innate immunity and in some of the effector mechanisms used against microbes. the mode of detection of microbial patterns through activation of pattern recognition receptors and nf-κb signalling cascades have been conserved throughout evolution. the drosophila toll pathway, for example, has some parallels to the mammalian signalling systems downstream of the interleukin 1 receptor (il-1r) and the toll-like receptors (tlrs). the main difference seems to be the fact that the drosophila toll receptor does not sense microbial inducers directly, as most mammalian tlrs do, but instead relies on an upstream recognition system. furthermore, the mammalian system seems to be able to use the same nf-κb inducing cascade after recognition of both g+ and gbacterial infection, while drosophila uses two distinct systems, i.e., toll and imd signaling. the drosophila imd cascade furthermore is similar to the mammalian tumour necrosis factor receptor (tnfr) pathway (hoffmann, 2003; wang and ligoxygakis, 2006). a number of similarities between drosophila hemocytes and mammalian blood cells such as amp synthesis and cytokine production were also reported, as well as some specifics of invertebrate cellular immunity. the melanisation response, for example, has no clear counterpart in mammals but instead uses molecular building blocks (proteolytic cascades, integrins) that are conserved among phyla (irving et al., 2005). drosophila blood cells consist of only a few terminally differentiated types whose functions resemble those of the cells of the vertebrate myeloid lineage which gives rise to macrophages among others (evans et al., 2003). a proteomic analysis of the phagosome content of drosophila plasmatocytes, for example, has revealed that 70 % of its protein content (600 identified proteins) has a mammalian orthologue thereby validating fruit fly phagosomes as a model to study phagocytosis (stuart and ezekowitz, 2008). the encapsulation reaction furthermore has a similar function as the formation of granuloma in vertebrates (markus et al., 2009). in addition, the activation of tota in the drosophila fat body as a response to the release of upd3 by hemocytes, for example, is very similar to some aspects of the mammalian acute-phase response mediated by cytokines (agaisse and perrimon, 2004). next, the human genome encodes all major jak/stat pathway components. molecular and functional data clearly indicate that a high level of conservation exists between the structural components of both the insect/drosophila and the mammalian pathway (arbouzova and zeidler, 2006). for fighting off viral infections, the mammalian system seems to depend largely on recognition of dsrna, as does drosophila, but in mammals this mainly leads to the production of interferons while in drosophila rna interference is the main immune effector (cherry and silverman, 2006). the study of the drosophila antiviral immunity only just recently has gained serious interest. further similarities and differences thus are expected to be uncovered in the near future. overall, discoveries made through research in the fruit fly, drosophila melanogaster may be applicable to the study of innate immunity in humans. the exploration of innate immunity in insects has also garnered increasing attention because of the role of many insects in transmission of human disease agents. understanding how the insect immune system interacts with pathogens may contribute to the development of new strategies to block transmission of disease agents (shi and paskewitz, 2006). recently, questions were raised regarding the absence of memory and the absence of the diversity of immunoglobulin like recognition receptors in drosophila innate immunity as seen in mammals. induction of resistance to lethal doses of a pathogen by priming with sublethal doses was observed for s. pneumoniae and b. bassiana and required both the toll pathway and phagocytosis (pham et al., 2007). the discovery that the neuronal immunoglobulin superfamily member dscam, which is encoded by a gene that can potentially generate 18,000 splice isoforms, is expressed by hemocytes and by cells in the fat body has also raised considerable interest as to the possibility of the generation of a large receptor repertoire in d. melanogaster (watson et 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nature 392: 93-97, 1998. 44 conclusion 18 isj 14: 18-31, 2017 issn 1824-307x research report an integrated approach to study the biomarker responses in marine gastropod nerita chamaeleon environmentally exposed to polycyclic aromatic hydrocarbons j bhagat1, a sarkar2#, v deepti2, v singh2, l raiker2, bs ingole1 1biological oceanographic division, csir-national institute of oceanography, dona paula, goa 403004, india 2chemical oceanographic division, csir-national institute of oceanography, dona paula, goa 403004, india # present address: global enviro-care, kevnem, caranzalem, goa 403002, india accepted december 28, 2016 abstract ecological risk assessment using multiple biomarkers produce a large amount of data that is hard to interpret and the result are often contradictory. in this context, integrated biomarker response (ibr) index was used to integrate the biomarkers effects to assess the impact of environmental contaminants in marine gastropod nerita chamaeleon from goa, india. genotoxic (dna damage as measured by comet assay and alkaline unwinding assay) and biochemical [superoxide dismutase, catalase, glutathione s-transferase, lipid peroxidation and acetylcholinesterase] biomarkers were measured in snails collected from different sites (arambol, anjuna, sinquerim, dona paula, velsao, betul and palolem). total polycyclic aromatic hydrocarbons in snail tissue were in the range from 5.29 12.14 µg/g wet weight. standardized values of biomarker response were visualized using star plots, which show unique patterns for different biomarkers. the mean ibr value was found to be highest at dona paula (8.07 ± 0.91) followed by sinquerim (6.95 ± 0.91), velsao (4.48 ± 0.68), anjuna (3.28 ± 1.05), palolem (2.53 ± 0.73), arambol (1.81 ± 0.21) and betul (0.88 ± 0.77). additionally, the ibr values were found to be positively correlated with pah concentration in snail tissues. these results suggest that integration of biomarkers effects using ibr along with chemical analysis can be a useful tool for the assessment of environmental pollution and to identify spatial patterns of contamination in the aquatic ecosystem. key words: integrated biomarker response; oxidative stress; genotoxic damage; polycyclic aromatic hydrocarbon; comet assay introduction polycyclic aromatic hydrocarbons (pah) are persistent and ubiquitous environmental contaminants found in air, water, and soil. they are studied extensively due to their carcinogenic properties for human as well as animals. the international agency for research on cancer (iarc) has classified pahs as possible and probable carcinogen to human (iarc, 2010). the lipophilic and hydrophobic nature allows pahs to accumulate in the marine organism (mashroofeh et al., 2015). the accumulation of pahs in a marine organism can negatively affect their health (frouin et al., 2007; grintzalis et al., 2012). pahs and their metabolites interact with dna and form dna adducts. pah activation process also generates ___________________________________________________________________________ corresponding author: jacky bhagat biological oceanographic division csir-national institute of oceanography dona paula, goa-403004, india e-mail: bhagatjack@gmail.com reactive oxygen species (ros) which can induce genotoxic damage by modifying integrity of dna (mattson et al., 2009). biomarkers are an important tool to detect exposure and adverse effects of human-made or natural contaminants on aquatic organisms. some biomarkers are specific to chemicals or group of chemicals while other are non-specific and induces upon exposure to broad range of pollutants. due to the complexity of contaminants, use of multibiomarker has become an increasingly popular tool to study the environmental parameters as well as organism health. comet (or single cell gel electrophoresis) assay is the most commonly used as a biomarker of dna damage in various research areas because of it sensitive and reliable nature. dna damage as measured by comet assay has been reported in mussels, clams and several other aquatic organisms (martins et al., 2013; dailianis et al., 2014; sarkar et al., 2014). another technique is known as dna-alkaline unwinding assay (daua) is also widely used to detect dna damage in aquatic 19 fig. 1 sampling site located along goa, west coast of india. organisms (sarkar et al., 2008, 2013; oliveira et al., 2010). several antioxidant enzymes are induced to combat the excessive ros produced as a result of pah metabolism. overproduction of ros can cause oxidative damage in a cell leading to damage to proteins, molecules, and dna. studies on antioxidant enzymatic defenses as biomarkers of oxidative stress are well documented in marine organisms (niyogi et al., 2001a, b; pan et al., 2006). among the oxidative stress biomarkers superoxide dismutase (sod), catalase (cat), glutathione stransferase (gst), and lipid peroxidation (lpo) have been widely used as an environmental biomarker in gastropods (abdel-halim et al., 2013; zheng et al., 2013; wang et al., 2014). gst helps in detoxification of the reactive products produced as a result of lipid peroxidation (olsvik et al., 2010). correlation between gst activity and pah in the tissues has been reported in several studies on mussels mytilus edulis (gowland et al., 2002). pan et al. (2006) have studied lipid peroxidation in scallop chlamys ferreri exposed to pah, benzo(a)pyrene (bap). catalase activity was measured in clam (frouin et al., 2007) and fishes (nahrganga et al., 2009) exposed to pah. significant induction of lipid peroxidation was detected in mussels collected near oil spillage site (porte et al., 1991). the author also showed a strong correlation between lipid peroxidation and total body pah in mussels. acetylcholinesterase (ache) activity is another very useful biomarker of neurotoxic contaminants in marine organisms (gaitonde et al., 2006; sarkar et al., 2006). ache has been considered as specific biomarkers for organophosphate and carbamate pesticides. recent studies also report inhibition of ache in gastropods exposed to heavy metals and biocides (gaitonde et al., 2006; ma et al., 2014). the combination of biomarkers yields a complicated and vast data which is hard to interpret. integrated biomarker response (ibr) integrates results from individual biomarkers into a single index, called ibr index which provides a comparison between stations and also between biomarkers. it is widely used in aquatic organisms exposed to contaminants(barda et al., 2014; turja et al., 2014). ibr can also be summarized into a star plot where radius values are ibrs estimated at each station. star plots provide corresponding information regarding mechanisms of biological effects of contaminants (marigómez et al., 2013). beliaeff and burgeot (2002) were the first to construct star plot using ibr values in flounder platichthys flesus using erod, gst, cat, ache enzymatic activities and dna damage biomarkers along with pah and pcb concentrations in tissues. pah contamination in caged mussel mytilus trossulus and mytilus galloprovincialis was also studied using ibr index (tsangaris et al., 2011; dabrowska et al., 2013). molluscs have been widely used in environmental monitoring due to their economical and ecological importance. gastropods have received great attention in recent years thanks to the discovery of imposex (smith, 1981). environment contaminants such as organotin compounds are known to cause imposex in snails that lead to a decline in pollution because of sterility and reproduction abnormality (garaventa et al., 20 2008). nerita is among the oldest molluscan names, dating to linnaeus and is a potential biological monitor for environment monitoring (kumar and devi, 1997; kumar, 1990). in this study, a battery of biomarkers for genotoxic damage (comet assay and alkaline unwinding assay), oxidative stress (sod, cat, gst, lpo) and neurotoxicity (ache) were considered. ibr integrates the biological response of multiple biomarkers and provides a single value; hence, in this study, ibr was applied to study the spatial variation in biomarkers in n. chamaeleon. pah content in the tissues of snail was also determined to relate to the variability of the biomarker response. materials and methods chemicals 1-chloro-2, 4-dinitrobenzene (cdnb), acetylcholine bromide, bromothymol blue, calf thymus dna, epinephrine, ethidium bromide, ethylene glycol-bis (2-aminoethylether)-n,n,n',n'tetraacetic acid (egta), guaiacol glycerol ether, hydrogen peroxide (h2o2), l-glutathione reduced, trypan blue and sephadex g-50 were purchased from sigma-aldrichpvt. ltd, india. frosted slides and cover slips were supplied by himedia, goa, india, dimethyl sulfoxide was obtained from qualigens, goa. tris buffer and triton x-100 were obtained from merck, goa. sampling site and gastropods nerita chamaeleon (around 30 snails from each site) were collected during post-monsoon (2011) period from the intertidal rocks along the seven sites (anjuna, arambol, sinquerim, dona paula, velsao, betul and palolem) in goa, west coast of india (fig. 1). arambol is situated at the northern tip of goa near tiracol estuary and is chosen as reference site because of its comparatively uncontaminated environment (sarkar et al., 2014). anjuna and sinquerim are most popularly known for their touristic activities around the world and hold a large number of restaurants, resorts, and shacks in the close proximity of the beach. discharge of contaminated water from the shacks and restaurant might contribute largely towards the pollution at these sites. it should be noted that mv river princess has been grounded on sinquerimcandolim beach since 2000. the grounding of the ship for such a long time release of huge amount of petroleum hydrocarbon from the ship might also contribute to coastal pollution (ingole et al., 2006). dona paula is situated between mandovi and zuari estuaries, just opposite to mormugao harbor. excessive shipping activities such as tourist boats, cargo ships, fishing trawlers, casinos, and barges carrying iron ores from the mines and water scooters are major contributors to the rapid increasing water pollution at this site. velsao beach lies in the vicinity of one of the leading agrochemical industries (zuari agrochemicals) of india in verna industrial state region. during the sampling at velsao, the prevalence of pungent smell from the region of discharge outlet clearly indicated the prevailing state of coastal pollution. betul and palolem are situated in the southern part of goa. the collected snails were identified using the certified reference sample from zoological survey of india; kolkata, india (subba rao et al., 1992). snails of similar size (around 18 30 mm) irrespective of their sex were used in this study. snails were transported in a plastic container to the lab within 3 h of collection. the collected snails were acclimatized in 4 liters plastic aquaria for 48 h in aerated seawater at room temperature. the shells of the gastropods were gently broken and whole body tissue was carefully excised out. soft tissues from three to four snails were pooled together (1 gram) and used for biochemical, alkaline unwinding assay and comet assay. all the measurements were carried out in triplicate. measurement of physicochemical parameters physicochemical parameters such as ph of the water from the sampling site were measured using ph analyzer elico model li-612, whereas turbidity and conductivity were measured using turbidimeter (systronics type 132) and conductivity meter (systronics digital direct model: 304). nutrients (nitrate, nitrite, and phosphate) were measured spectrophotometrically using shimadzu uv 1800 spectrophotometer whereas dissolved oxygen (do) and biochemical oxygen demand (bod) were determined following the standard methods of grasshoff et al. (1983). measurement of dna damage dna damage in snails has been evaluated by comet assay and alkaline unwinding assay. comet assay was performed in n. chameleon using the methods as described in our previous studies (sarkar et al., 2015). the dna integrity was measured in n. chameleon using partial alkaline unwinding assay following the methods of sarkar et al. (2013). biochemical assays for extraction of sod, one gram of tissue was homogenized (using ultra-turrax t 25 basic 1ka werke homogenizer) in 1 ml of 0.1 m phosphate buffer (ph 7.4). following that 0.2 ml of ice cold chloroform, 0.15 ml of ice-cold ethanol, and 1 ml of distilled water was added and the whole solution was shaken thoroughly. the mixture was then centrifuged at 3000 rpm for 10 min at 4 °c (using eltek refrigerated centrifuge rc 4100d). sod activity was determined by the rate of auto-oxidation of epinephrine to adrenochrome (misra and fridovich, 1972). the reaction volume (1 ml) contained 10 mm epinephrine, 50 mm sodium carbonate buffer (ph 10.2) and 10 mm edta. sod activity is reported in per milligram of protein (u mg 1); where 1 u of sod is defined as the amount of sample causing 50 % of inhibition of epinephrine auto-oxidation. for catalase, one gram of tissue was homogenized with 4 ml of phosphate buffer (0.1m, ph7.4) using high-speed ultra turrax homogenizer for 1 min and centrifuged at 18,000 rpm, 1 h, 4 ºc. catalase activity was measured following the methods of sinha et al. (1972). 1 ml of sodium phosphate buffer (0.01 m, ph 7.0), 0.5 ml of 0.2 m hydrogen peroxide (h2o2), and 0.4 ml distilled water 21 were mixed to make the reaction mixture. the reaction was stopped by pouring 2 ml of dichromate-acetic acid reagent (containing potassium dichromate 1 part and glacial acetic acid 3 parts). it was then heated for 10 min and allowed to cool. after the mixture cools down, the absorbance was read at 583 nm against blank on a spectrophotometer. the activity of cat was expressed as mm of h2o2 consumed/min/mg protein. glutathione s-transferase (gst) activity was measured according to the method of habig et al. (1974). it is based on conjugation of 1-chloro-2,4dinitrobenzene (cdnb) solution (30 mm) and reduced glutathione (gsh) solution (30mm) in reaction buffer (0.1 m k2hpo4, edta-na2, ph 6.5). the change in absorbance was measured at 340 nm for every 30 seconds for 5 min using a uv-vis spectrophotometer. the activity of gst was determined using extinction coefficient of 9.6 mm-1 cm-1 for cdnb. gst activity was expressed as nm/min/mg of protein. lipid peroxidation was measured by the method adapted from ohkawa et al. (1979). briefly, 1 g of soft tissue was homogenized with 9 ml of 0.25 m sucrose using ultra turrax homogenizer for 1 min.0.2 ml of 8 % sds, 1.5 ml of 20 % acetic acid and 1.5 ml of 0.8 % tba was added to 0.2 ml of the tissue homogenate. it was then made up to 4 ml using distilled water and heated at 95 ˚c for 60 min. it was then cooled and centrifuged at 3,000 rpm for 10 min. following that, the absorbance was read at 532 nm. lpo value was measured as malondialdehyde (mda) equivalent and expressed as nm of mda min-1 mg-1. extraction of ache from tissue was performed in phosphate buffer (100 mm; ph 7.4) mixed with sucrose solution (250 mm) (1 ml of both per gram of tissue) spiked with 100μl tritonx-100 in order to break the tissue. the homogenate was centrifuged at 14,000 rpm at 4° c for 45 min. ache activity in snails was measured using the ∆-ph-metric method as described by sarkar et al. (1992) and gaitonde et al. (2006). briefly, 0.1 ml of sample enzyme was incubated with 0.2 ml of substrate (acetylcholine bromide) in phosphate buffer (0.01m, ph-8.0 ±0.10) with bromothymol blue as an indicator. the changes in ph (∆-ph) was measured at an interval of 10 min over a period of 1 h of incubation corresponding to the amount of acetic acid liberated due to interaction with the acetylcholinesterase enzyme. the unit of activity of ache was expressed as micromoles of acetic acid liberated per minute per mg of protein. proteins were determined according to the method of lowry et al. (1951), using bovine serum albumin as standard. estimation of polycyclic aromatic hydrocarbons (pah) pah were extracted by homogenization of tissues of snails with bi-distilled hexane using ultra turrax homogenizer. the moisture content in the solvent extracts was removed by anhydrous sodium sulphate and the solvent extracts were concentrated to 1 ml using kuderna danish evaporator followed by purification of aliquots through alumina (10 % deactivated) column using bi-distilled hexane (grasshoff et al., 1983). the final 10 ml of aliquots thus obtained were measured by a spectrofluorometer (shimadzu rf-5301 pc) with excitation at 310 nm and emission at 360 nm (burns, 1993; sarkar et al., 2008, 2014). kuwait oil was used as the standard. total pah in tissues was represented as µg/g wet weight. statistical analysis the data were expressed as mean ± standard deviation. results from biochemical assays and genotoxic damage were analyzed by analysis of variance (anova) followed by tukey hsd posttest. kolmogorov-smirnov test for normality of distribution was used prior to anova. spearman correlation matrix was also calculated to study the relationships between the different biomarkers measured. biomarker values were compared with the reference site, arambol. three levels were significance is reported: (a) p < 0.05, (b) p < 0.01, and (c) p < 0.001. all statistical comparisons were performed using originpro 8.5.0. principal component analysis (pca) was conducted to determine physicochemical water parameters association with biochemical using xlstat. integrative biomarker response (ibr) integrated biomarker response (ibr) was calculated as described by beliaeff and burgeot (2002) with modification by guerlet et al. (2010) and devin et al. (2014). briefly, the biomarker response data for each site was standardized using the formulae yi = (xi-m)/s where yi is the standardized biomarker response, xi is response value of each biomarker, m, and s are mean value and standard deviation for all sites respectively. the mean of standardized biomarker response (zi) was then calculated using the formulae as zi = yi or zi = –yi for biomarker responding to contamination by induction or inhibition, respectively. the minimum value for each biomarker at all station was also calculated from the standardized biomarker response. the scores for the biomarker was computed as si = zi+|mini|. individual areas ai connecting the ith and the (i + 1)th radius coordinates of the star plot were obtained according to the formulae ai = si * si+1 *sin (2π/k)/2, where si and si+1 represent the individual biomarker scores (calculated from standardized data) and their successive star plot radius coordinates and k represent the number of radii corresponding to the biomarkers used in the survey. biomarkers were ranged clockwise from sub-cellular level as follows: percentage tail dna (tdna), dna integrity value (dna-f), sod, cat, gst, lpo and ache (serafim et al., 2012).and the ibr value is calculated as follow: ibr= , where ai is the triangular area represented by two consecutive biomarker scores on the star plot and n is the number of biomarkers used in the ibr calculation. results water quality parameters physico-chemical water parameters varied significantly across all the sampling sites along the coast of goa (table 1). ph of the seawater was in the 22 table 1 physico-chemical properties of water sampled at different sites along the coast of goa, india sites ph temp. (˚c) turbidity (ntu) conductivity ms/cm nitrite µm/l nitrate µm/l phosphate µm/l do (mg/l) b.o.d (mg/l) arambol 7.97±0.01 31.75±0.35 5.60±0.14 40.50±0.71 0.47±0.12 1.66±0.06 0.54±0.14 4.29±0.00 1.19±0.08 anjuna 8.37±0.02*** 33.00±0.00*** 2.35±0.49*** 41.50±0.71 0.34±0.05 1.69±0.06 0.38±0.08 5.93±0.08*** 1.58±0.08** sinquerim 8.09±0.04* 30.00±0.35*** 5.15±0.49 36.50±0.71* 0.91±0.04** 9.14±0.20*** 0.73±0.15 5.87±0.32*** 1.69±0.08*** dona paula 7.75±0.01*** 30.00±0.35*** 10.05±0.35*** 40.00±1.41 1.11±0.01*** 11.08±0.02*** 0.80±0.02 4.40±0.16 0.56±0.02*** velsao 8.06±0.01 33.00±0.25*** 2.25±0.35*** 64.50±0.71*** 9.64±0.01*** 32.76±0.80*** 3.48±0.05*** 4.12±0.08 2.20±0.04*** betul 8.21±0.02** 32.00±0.25 1.24±0.02*** 39.50±0.71 0.23±0.03* 1.44±0.10 0.28±0.02 5.14±0.08** 4.01±0.02*** palolem 7.90±0.04 31.00±0.00** 4.15±0.07* 42.50±0.71 0.43±0.04 0.07±0.02 0.42±0.00 4.52±0.00 3.95±0.05*** values are represented as means± standard deviation. maximum values were shown in bold. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001 significantly different from the reference site arambol (anova, tukey hsd post-test). range from 7.75 to 8.37. maximum values for seawater temperature was observed at anjuna (33 ˚c), and minimum at palolem (31 ˚c). the highest turbidity was found at dona paula (10.05 ± 0.35 ntu) and the least at betul (1.24 ± 0.02 ntu). seawater from velsao showed significant variation in nutrients as compared to all other stations (p < 0.001). velsao also showed the maximum values for conductivity (64.50 ± 0.71 ms/cm). the values for nitrite, nitrate, and total phosphate at velsao were found to be 9.64 ± 0.01 µm/l, 32.76 ± 0.80 µm/l and 3.48 ± 0.05 µm/l respectively. dissolved oxygen (do) also varied significantly between the sites, with maximum values observed at anjuna (5.93 ± 0.08 mg/l). the highest biological oxygen demand (bod) was measured at betul (4.01 ± 0.02 mg/l) and the least at dona paula (0.56 ± 0.02 mg/l). biomarker responses dna damage the impairment of dna in whole body tissue of marine gastropods is clearly indicated by the decrease in the integrity of dna in n. chamaeleon exposed to various types of genotoxic contaminants prevalent at different sites along the coast of goa (fig. 2). the highest value of tdna was observed at sinquerim (55.86 ± 4.09). a significant difference in tdna was observed between sinquerim and arambol (p < 0.01) and sinquerim and anjuna (p < 0.01). the dna integrity at the reference site arambol was found to be relatively quite high (0.71 ± 0.03) as compared to the other sampling sites. except for betul and palolem, all the sampling sites showed a significant decrease in dna integrity as compared to arambol. the mean value of dna integrity in snails was found to be 0.48. fig. 2 (a) mean percentage dna in the tail (tdna) and (b) dna integrity (f value) in marine gastropod nerita chamaeleon collected from different sites along the coast of goa, india. values are means ± standard deviation (*) p < 0.05, (**) p < 0.01, (***) p < 0.001 significantly different from the reference site, arambol (anova, tukey hsd post-test). 23 fig. 3 (a) superoxide dismutase (sod), (b) catalase (cat), (c) glutathione s-transferase (gst), (d) acetylcholinesterase (ache) activities, and (e) lipid peroxidation (lpo) level in marine gastropod nerita chamaeleon collected from different sites along the coast of goa, india. values are means± standard deviation (*) p < 0.05, (**) p < 0.01, (***) p < 0.001 significantly different from the reference site, arambol (anova, tukey hsd post-test). biochemical assays all biomarkers varied across sites. sod activity in whole body tissue of snails was in the range from 1.57 to 17.27 u/mg of protein (fig. 3). significant differences in sod activity was observed for sinquerim, dona paula, velsao and palolem as compared to the reference site arambol. cat activity also showed significant variations among the sampling sites. the highest cat activity was observed at dona paula (0.39 ± 0.05 mm/min/mg) and the least at palolem (0.04 ± 0.01 mm/min/mg). all the other site except dona paula, showed significant decrease in cat activity as compared to arambol. cat activity between sinquerim and palolem were not significant, however, a significant change was observed between palolem and dona paula (p < 0.001). gst activity showed maximum value recorded at dona paula (191.54 ± 18.7 nm/min/mg) and minimum at sinquerim (28.11 ± 0.06 nm/min/mg). gst activity at dona paula was significantly (p < 0.001) higher than arambol (74.21 ± 1.47 nm/min/mg).the lowest lpo value (0.09 ± 24 0.01 mm mda/min/mg) was observed in snails collected at palolem whereas dona paula exhibited the highest value (1.18 ± 0.16 mm mda/min/mg). ache activity were significantly lower (p < 0.01) in snails from the sinquerim (10.44 ± 0.07 u/min/mg) in comparison to arambol (39.68 ± 0.18 u/min/mg). pca analysis showed that nutrients (phosphate, nitrate and nitrate) group together with conductivity. a strong positive correlation between lpo and turbidity (r = 0.84), and ache and turbidity (r = 0.801) was observed. dissolve oxygen showed negative correlation with dna integrity value, whereas ph showed negative correlation with all the biomarkers. measurement of pah pah concentrations in tissue of snail showed wide variation among the sites. sinquerim showed the highest value for total pah (12.14 ± 0.27 µg/g wet weight) in snail tissues whereas it was found to be least at arambol (5.29 ± 0.67 µg/g w.w.). the entire sampling site except anjuna showed significant variations in the pah content in snails (fig. 4). table 2 shows the pah content in molluscs from a different part of the world. relationship between biomarker response and pah content a moderate positive correlation was observed between tdna and pah (r = 0.626, p = 0.017) contents in snails (table 3). sod activity also showed positive correlation with pah (r = 0.604, p = 0.022) while ache activity was found to be negatively correlated with pah (r = 0.542, p = 0.045). strong positive correlation between gst activity and ache activity (r = 0.534, p = 0.049), gst and cat activity (r = 0.534, p = 0.047) were also observed. similar trend was observed between cat and sod activity (r = 0.613, p = 0.20,) and lpo value and sod activity (r = 0.942, p = 0.022). in contrast, dna-f was shown to be negatively correlated with sod activity (r = -0.617, p = 0.019) and lpo value (r = 0.679, p = 0.008). integrative biomarker response (ibr) ibr values for dona paula showed the maximum value for tdna (9.31 ± 0.91) as measured by comet assay and the minimum value was observed at palolem (0.25 ± 0.063) (fig. 5). the ibr values were significant for dna-f when sites arambol and dona paula were compared (p < 0.001). a very similar pattern was observed in star plots for all the oxidative stress biomarkers. the maximum ibr values for sod were measured at dona paula (7.17 ± 0.63) and the minimum values occurred at betul (0.91 ± 0.87). the ibr values for lpo at sinquerim and dona paula were significantly different from those of arambol (p < 0.001). however, no significant differences existed in any biomarker when ibr values for anjuna, betul and palolem were compared with arambol. sinquerim and dona paula showed significant differences in all the biomarkers studied with compared to arambol. the mean ibr values calculated from seven biomarkers was found to be highest at dona paula (8.07 ± 0.91) followed by sinquerim (6.95 ± 0.91), velsao (4.48 ± 0.68), anjuna (3.28 ± 1.05), palolem (2.53 ± 0.73), arambol (1.81 ± 0.21) and betul (0.88 ± 0.77). ibr values for genotoxic (tdna and dna-f), oxidative stress (sum of sod, cat, gst and sod) biomarker and total pah concentration in tissues were represented as star plot in figure 6. comparison of ibr for genotoxic, oxidative biomarkers and pah concentration shows that along with pah there might be other genotoxicants which are responsible for the impairment of dna in snails from different sites. fig. 4 polycyclic aromatic hydrocarbon (pah) content in marine gastropod nerita chamaeleon collected from different sites along the coast of goa, india. values are means± standard deviation (*) p < 0.05, (**) p < 0.01, (***) p < 0.001 significantly different from the reference site, arambol (anova, tukey hsd post-test). 25 table 2 polycyclic aromatic hydrocarbon (pah) concentrations in molluscs reported from different parts of the world organisms group location tissues no. of pah wet weight (µg/g) references nerita chamaeleon gastropod goa coast, india whole body tissues total pah 5.29-12.14 this study cronia contracta gastropod goa coast, india muscle tissue total pah 22.32–53.78 sarkar et al., 2008 turbo cornutus gastropod japan coast, japan soft tissue 8 0.044 koyama et al., 2004 sunetta scripta clam cochin harbor, india soft tissue total pah 13.35-21.49 menon & menon, 1999 donax trunculus clam comunidad valenciana coast soft tissue 8 0.43-10.09 bouzas et al., 2011 donax trunculus clam abu qir bay, egypt soft tissue 17 1137.07 el-deeb et al., 2007 saccostrea cucullata oyster hooghly estuary, india soft tissue total pah 0.8-12.5 niyogi et al., 2001a mytilus galloprovincialis mussel marmara sea, izmit bay soft tissue 16 5.67-14.81 telli-karakoç et al., 2002 mitylus galloprovincialis mussel adriatic sea, italy soft tissue 13 0.034 perugini et al., 2007 mytilus galloprovincialis mussel gulf of rijeka, croatia. soft tissue 10 0.049-0.134 bihari et al., 2007 mytilus galloprovincialis mussel gulf of trieste soft tissue 644-685 notar et al., 2001 mytilus galloprovincialis mussel comunidad valenciana coast soft tissue 8 0.21-8.95 bouzas et al., 2011 mytilus edulis mussel baltic sea, poland soft tissue 14 8.64–29.7 potrykus et al., 2003 palaeomonetes sp. shrimp norco, usa soft tissue total pah 7.18-10.86 oberdorster et al., 1999 penaeus japonicus shrimp gulf of suez muscle tissue 16 2.01 ali et al., 2014 sepia species cuttlefish gulf of suez muscle tissue 16 4.09 ali et al., 2014 portunus pelagicus crab gulf of suez muscle tissue 16 8.10 ali et al., 2014 nephrops norvegicus lobster adriatic sea, italy soft tissue 13 0.015 perugini et al., 2007 discussion in this study, seven biomarkers were measured in marine snail n. chamaeleon collected from goa coast and the obtained results show differences in individual biomarker responses. significant variations in genotoxic as well as biochemical biomarkers were observed and this can be associated with pollutant exposure. biomarker responses in snails showed clear spatial variations. the range of pah in snails in this study lies within the same range as reported in clams (menon and menon, 1999) and oysters (niyogi et al., 2001a) in indian coastal waters. however, higher values of pah were reported marine gastropods croniacontracta by sarkar et al. (2008) (table 2). total pah concentrations in snails from goa region range from the lowest value at arambol (5.29 ± 0.67 µg/g wet weight) to highest value at sinquerim (12.14 ± 0.27 µg/g wet weight). sinquerim also showed the highest values for tdna (55.86 ± 4.09) and lower dna-f (0.46 ± 0.04). accumulation of pah has been linked to dna damage through production of ros (jarvis et al., 2013). the ros produced as a result of pah exposure can cause single or double strand breakage in the dna (kaloyianni et al., 2009). sarkar et al. (2008) studied the seasonal variation of pah in cronia contracta collected from six sites along the goa coast and reported a positive correlation between the pah and dna damage. such a huge impairment in dna integrity in the gastropod at sinquerim can be attributed to genotoxic pollutants like polycyclic aromatic hydrocarbons being discharged extensively from various types shipping activities such as cargo ships, research vessel, tourist vessel, motor boats, fishing trawler, water scooters, barges sailing through this site as well as accidental oil 26 table 3 spearman’s correlation matrix on all the biomarkers and polycyclic aromatic hydrocarbon (pah) content in marine gastropod, nerita chamaeleon t-dna dna-f sod cat gst lpo ache dna-f -0.029 sod 0.332 -0.617* cat -0.143 -0.007 0.613* gst -0.398 -0.244 0.257 0.538* lpo 0.279 -0.679* 0.942* 0.495 0.182 ache -0.442 -0.152 0.099 0.327 0.534* 0.200 pah 0.626* -0.345 0.604* 0.095 -0.002 0.516 -0.542* (*) p < 0.05 significantly different(anova, tukey hsd post-test) spills, etc. (desai, et al, 2010). ingole et al. (2006) have reported a high level of total petroleum hydrocarbon at sinquerim-candolim beach due to the grounding of mv river princess. in velsao, the pah concentrations increase by two-fold as compared to arambol, such an increase in pah in snails at velsao revealed the severity of pollution in velsao. during sampling at velsao pungent smell from the discharge outlet from the industry has been observed, that indicates the severity of pollution at this site. there were no significant differences between pah concentration at velsao, sinquerim and dona paula. oxidative stress biomarker in n. chamaeleon showed spatial variability, with significant induction in sod activity at dona paula and sinquerim.sod activity at both of these sites is strongly correlated with the high values of pah measured in snails. numerous studies have also documented significant relationships between antioxidant activity and heavy metal/pah body burdens (giguere et al., 2003; manduzio et al., 2003). dona paula has been previously reported for heavy metal and pah contamination (sarkar et al., 2008). high values of tbt and other organotin compounds were also reported in dona paula (meena et al., 2009). catalase is well known to play an important role in scavenging h202 (di giulio and meyer, 2008), which is produced as a result of scavenging of superoxide radicals by sod. positive relationships between cat activity and pah levels were observed in oyster (niyogi et al., 2001a), barnacle (niyogi et al., 2001b), and in the gills of the mussel (cheung et al., 2001). in this study, a significant positive correlation was observed between sod and cat activity. the increase in sod and cat activity may be due to increasing in cellular ros produced due to exposure to pah (au et al., 1999). niyogi et al., (2001a) has also reported a positive correlation of cat and sod activity with pah tissue content in fig. 5 integrated biomarker response (ibr) represented by star plots for each sampling sites. 27 fig. 6 comparison of ibr index for genotoxic (dna damage as measured by comet assay and alkaline unwinding assay) and oxidative stress [superoxide dismutase (sod), catalase (cat), glutathione s-transferase (gst) and lipid peroxidation (lpo)] biomarkers with average polycyclic aromatic hydrocarbon (pah) value in snails from different sites of goa, india. s. cucullata. such a relationship has also been reported for different bivalve species exposed to hydrocarbons (richardson et al., 2008) and suggests that hydrocarbons induce oxidative stress by producing ros such as o2 -. the activity of cat is dependent on the level of h2o2 in the cell. another enzyme, gpx (glutathione peroxidase) eliminates h2o2 while carrying out gsh to gssg conversion. increase in gpx activity might lead to reduction of h2o2 that in turn can affect the activity of catalase. in our study high level of cat activity was observed at the reference site. in spite of high amount of pah in the other sites, a decreasing trend in cat activity was observed. wu et al. (2011) has also found that cat activity in eisenia fetida was unaltered suggesting that pah exposure does not induce increased cat activity. jifa et al. (2006) also reported unaltered changes in lateolabrax japonicus cat activity after (b[a]p) exposure. gst is a phase ii metabolizing enzyme and catalyzes the conjugation of reduced glutathione (gsh) with pah derivatives. the activity of gst depends on the availability of gsh. contrarily to other antioxidant enzymes, gst showed a negative correlation with pah in this study. decrease in gst activity after long exposure to pah can be due to the reduction of gsh levels. gsh can also be converted to its oxidized state (gssg) by glutathione peroxidase (gpx). decrease in gsh has been reported in molluscs exposed to pahs (grintzalis et al., 2012). studies with gst in aquatic organisms exposed to environmental or anthropogenic contaminants has shown increase (zheng et al., 2013; cabecinhas et al., 2014), unaltered (bianco et al., 2013; rivadeneira et al., 2013) or decrease (ma et al., 2014; ali et al., 2015) enzyme activities. free radicals produced as a result of metabolic activities can react with polyunsaturated fatty acids in the cell membrane, resulting in an increase of lipid peroxidation (livingstone et al., 2001). lpo is well known oxidative stress biomarkers in bivalves exposed to pah (kaloyianni et al., 2009). lpo results in production of mda which can react with dna and form dna adduct. increased formation of dna adduct in gill cells of mytilus galloprovincialis after exposure to pah, benzo(a)pyrene have been reported by venier and canova (1996). enhancement of lipid peroxidation and inhibition of ache were detected in gills of mussels m.galloprovincialis exposed to phenanthrene (grintzalis et al., 2012). elevated levels of cat, lpo and sod were observed in mussels (mytilus galloprovincialis) collected from sites affected by the oil spill (sureda et al., 2011). in this study significant decrease in ache activity was observed in sinquerim. this may be due to the prevalence of hydrocarbon pollution as observed by high amount of pah reported in the gastropods. presence of high amount of pah in snails from sinquerim may be due to extensive shipping activities as well as accidental oil spills (desai et al., 2010). enhancement of lipid peroxidation by-products and inhibition of ache in mussels exposed to phenanthrene and/or anthracene were reported by grintzalis et al. (2012). pca indicated that biochemical biomarkers tend to be increased under condition of higher turbidity and lower ph. pca analysis provided an important association between the physicochemical parameters and biomarker response. the fact that the biomarkers assessed were moderately associated with the environmental variables suggest that other contaminants, besides those measured here, were also contributing to the lower health status of the snails. ibr with star plots showed higher biomarker response in snails from dona paula, sinquerim, and velsao, and moderate at anjuna and palolem. thus higher values of pah and oxidative stress biomarkers at velsao, sinquerim and dona paula shows critical unbalance ros formation and the severity of pollution at these sites. several authors have reported higher values of ibr in contaminated sites as compared to the reference site (tankoua et al., 2013; turja et al., 2014). in this study elevated the level of pah as well as oxidative stress biomarkers response was observed in snails from sinquerim. vega-lópez et al. (2013) has investigated t h e r el a t i o n b et w e e n o xi d a t i v e s t r e s s a n d 28 fig. 7 principal component analysis (pca) on the data set with physicochemical water parameters [ph, temperature (temp.), turbidity (turb.), conductivity (cond.), nitrite (no2 -), nitrate (no3 -), phosphate (po4 3-), dissolve oxygen (do), biological oxygen demand (bod)] and biomarkers response [dna integrity (dna-f), percentage tail dna (tdna), superoxide dismutase (sod), catalase (cat), glutathione s-transferase (gst), acetylcholinesterase (ache) activities, and lipid peroxidation (lpo)] in nerita chamaeleon. antioxidant defenses in phytoplankton with heavy metal and pah. the author stated that oxidative damage was related with pah (benzo[b]fluoranthene) using ibr. pah are suspected of having an oxidative damage leading to damage in the genetic material and some of the pah such as benzo(a)pyrene, indeno[1,2,3c,d]pyrene, benzo[g,h, i]perylene etc. are well known for such actions (perez-cadahia et al., 2004; woo et al., 2006). there is strong evidence that some of them are carcinogenic (diguilio et al., 1995) with the capacity to cause various types of oxidative stress and dna damage. these results suggest that integration of genotoxic and biochemical biomarker can serve as a useful tool in environmental monitoring programs. conclusion the present study showed that tdna, dna-f, sod, cat, gst, lpo and ache levels in snails varied along different sites. integrated biomarker response index based on a battery of biomarkers proved a useful tool for visualization of biological responses in snails, facilitating comparisons between different sites. increased value of ibr index at sinquerim and dona paula can be attributed to exposure of snails to contaminants prevalent at these sites. our study demonstrated, the sensitivity of marine snail n. chamaeleon as a good candidate species for pah contamination. this study demonstrates the usefulness of multibiomarker approach 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pollut. bull. 52: 1768-1775, 2006. wu s, wu e, qiu l, zhong w, chen j. effects of phenanthrene on the mortality, growth, and anti-oxidant system of earthworms (eisenia fetida) under laboratory conditions. chemosphere 83: 429-434, 2011. zheng s, wang y, zhou q, chen c. responses of oxidative stress biomarkers and dna damage on a freshwater snail (bellamya aeruginosa) stressed by ethylbenzene. arch. environ. contam. toxicol. 65: 251-259, 2013. societ italiana di immunobiologia comparata isj 5: 30-40, 2008 issn 1824-307x report of meeting ixth scientific meeting of the italian association of developmental and comparative immunobiology (iadci), 27 29 february 2008, biological departments, university of insubria, varese, italy organizers: m de eguileor, a grimaldi, g tettamanti, r valvassori department of structural and functional biology, university of insubria, varese, italy session 1. chairman: m cammarata, university of palermo, italy the immune system of compound ascidians l ballarin department of biology, university of padua, padua, italy differently from vertebrates, having an adaptive immunity, ascidians are chordates with a germ-line based innate immunity in which phagocytosis and cytotoxicity are the main effector systems. in recent years, the comprehension of the immune system of compound ascidian has greatly improved. most of the research was carried out in species of the genus botryllus and botrylloides. in these organisms, immunocytes constitute a considerable fraction of circulating haemocytes and are represented by phagocytes and phenoloxidase (po)-containing, cytotoxic cells. phagocytes include hyaline amoebocytes (ha) and macrophage-like cells (mlc). the former are wandering cells which quickly recognise and engulf foreign particles or cells: after the ingestion, they withdraw their cytoplasmic projections and acquire a globular shape, turning to mlc. phagocytosis requires the recognition of molecular patterns on the surface of target particles by receptors on phagocytes and is greatly influenced by the nature of the particle. the recognition triggers signal transduction pathways which lead to the activation of the map-kinase cascade and of nf-kb. soluble lectins can increase the phagocytosis of foreign particles acting as opsonins. phagocytes play an important role during the colonial generation change (take-over), when the old adult zooids stop their filtering activity and are progressively resorbed. during this period, tissues of adult zooids undergo diffuse apoptosis and are rapidly infiltrated by blood phagocytes which rapidly and massively engulf senescent cells. cytotoxic morula cells are characterised by the presence of vacuoles containing inactive pro-po and its polyphenol substrata. they are the effectors of the rejection reaction between contacting, genetically incompatible colonies. these cells crowd inside the ampullae contacting the alien colony, cross the epithelium of the ampullar tips and enter the tunic where they degranulate and release the content of their vacuoles. the activated po acts on substrata and induce necrotic death through the induction of an oxidative stress. there is an interesting cross-talk between cytotoxic an phagocytic cells: i) phagocytosis can be modulated by soluble molecules (cytokines) released upon the recognition of foreign particles (yeast cells and bacterial spores) by the cytotoxic cells; ii) some ha are exposed to seawater, on the internal side of the siphons, where they act as guard cells. once recognised foreign materials, they activate morula cells in the tentacular lacunae which, in turn alert the whole immune system towards the potentially dangerous particles that can be phagocytosed or degraded. further insights on siphonal guard cells of ascidians f cima department of biology, university of padua, padua, italy in the oral siphon of the colonial ascidian botryllus schlosseri, hyaline amoebocytes directly exposed to the sea-water flow entering into the pharynx have been recently observed and described. these cells, named “siphonal guard cells” (sgc), are free of moving on the surface of the tunic that internally covers the siphons. our previous observations by means of histochemical, histoenzymatic and immunohistochemical techniques showed that they share many morphofunctional characteristics with the phagocytic blood cell line, from which probably they originate, and are able to recognise and phagocytise various foreign particles. after exposure of colonies to bacterial spores, the observations at both light and electronic microscope revealed that these cells are involved in a complex and unusual series of local and 30 systemic immune events. already after 5 min, the sgc showed bacteria inside their heterophagic vacuoles. after 10-15 min, as a transitory plug of floccular and colloidal material formed in the lumen of the siphon by exocytosis of some sgc, other ones with engulfed bacteria crossed the epidermis of the siphon reaching the siphonal sinus; cells of the cytotoxic blood cell line (morula cells) were drawn and crowded into the siphonal sinus, where most of them were positive to antitnf-α and anti-cd57 antibodies and degranulated stimulating, after this time and until 12 h, large scavenger phagocytes. the latter showed bacteria engulfed in their large phagosomes, increased in number in the blood circulation and were continuously eliminated through the peribranchial chamber with a mechanism which was never previously described. as regards the ability to transfer an alert signal, the role of sgc appears important as regards the immunosurveillance of the opening of the alimentary canal, similarly to what occurs in the vertebrate oropharyngeal lymphatic tissues. apoptosis signalling pathways in the compoud ascidian botryllus schlosseri during the colonial blastogenetic cycle a menin, l comini, l ballarin department of biology, university of padua, padua, italy in the colony of the ascidian botryllus schlosseri, three blastogenetic generations are usually present: adult zooids, primary buds on zooids and secondary buds on primary buds. colonies undergo recurrent generation changes in which adult zooids are gradually resorbed and replaced by new blastogenetic generations. it is possibile, therefore, to define a colonial blastogenetic cycle that begins with the appearance of a new generation, and ends with the generation change, during the take-over phase, in which programmed cell death by apoptosis is largely diffuse. using the haemocytes as reference tissue we investigate the extent of cell death during the colonial blastogenetic cycle . our results confirm the expression, on cells surfaces, of fas receptors and theirs ligands fasl. moreover, we showed the presence of members of the caspase family: the initiator caspases 8 and 9 and the executioner caspases 3 and 7. the activated executioner caspases can subsequently cleave distinct cellular proteins such as parp: using immunoblot assay we observed the cleaveage of proteins recognised by anti-parp. in vertebrates, intrinsically mediated initiation begins with mitochondrial membrane disruption resulting in cytochrome c (cyt c) release. we observed an encrease of h2o2 in cytoplasmatic contents and a different expression of cyt c during the take-over. these results confirm botryllus an interesting model organism for the study of apoptosis. a novel rhamnose-binding lectin from the colonial ascidian botryllus schlosseri n franchi, f gasparini, b spolaore1, l ballarin department of biology, university of padua, padua, italy 1cribi, university of padua, padua, italy lectins are carbohydrate-binding proteins which agglutinate cells and/or precipitate glycoconjugates. the family of rhamnose binding lectins (rbls) includes various proteins, previously classified as galectins, with common sugar specificity and one to three homolougous carbohydrate-recognition domains (crds), about 100-aminoacids long and characterised by eight highly conserved cysteine residues. from a fulllength cdna library from the compound ascidian botryllus schlosseri we identified five complete transcripts homologous to known rbls. comparisons of the predicted amino acid sequences (118 aa) suggest that they represent different isoforms of a novel rbl, called bsrbl-1-5 with only one crd. reverse-phase hplc and mass spectrometry of the affinity-purified material confirmed the presence of four of these isolectins in botryllus homogenate. analysis of both molecular masses and tryptic digests of bsrbls indicated that the n-terminal sequence of the purified proteins starts from residue 22 of the putative amino acid sequence, so that residues 1-21 represent a signal peptide. analysis by mass spectrometry of v8protease digests confirmed the presence and alignments of the eight cysteines involved in the disulphide bridges characterising rbls. functional studies confirmed the enhancing effect on phagocytosis of the affinity-purified material. the phylogenetic relationship of bs-rbls with orthologous molecules from protostomes and deuterostomes was also studied. enhanced expression of a cintnf gene in the lps challenged inflammatory responses of the ascidian ciona intestinalis n parrinello, a vizzini, v arizza, m cammarata, d parrinello, f giaramita, g salerno, m pergolizzi, ma sanfratello, m vazzana marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy in invertebrates immune system, cell proliferation, phagocytosis and chemotaxis are regulated by cytophilic humoral molecules with functional similarities to vertebrate cytokines. these molecules modulate defense responses to exogenous and endogenous insults, tissue repair and recovery of homeostasis by ligand-specific receptor interactions required to initiate and regulate immune responses. tumor necrosis factor (tnf) is a pro-inflammatory cytokine produced as part of the innate response. in invertebrates tnf-like molecules have been identified by using various methods. since in ciona intestinalis genome (ensembl) tnf gene has been identified (cintnf), 31 real-time pcr analysis was carried out. results showed a prompt (2-4 hrs) enhanced cintnf gene expression in the inflamed body wall after intratunic lps injection. in situ hybridization assays supported the involvement of pharynx hemocytes in the inflammatory response, and transcript was mainly found in morula cells and in unidentified cells associated with epidermis. similar results were found by examining hemocytes from the hemolymph. immunoblotting assay with anti-cintnf specific antibodies revealed that a 17 kda cintnf is released in the hemolymph. sphingomyelin as well as carbohydrates are involved in the mechanism of cytotoxic molecules contained and released in vitro by ciona intestinalis granulocytes v arizza, d parrinello, f giaramita, a vizzini, m cammarata, m vazzana, n parrinello marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy the immune-system of invertebrates recognizes and then reacts to foreign particles potentially pathogen by means of cytotoxicity, phagocytosis, encapsulation, and humoral effector mechanisms (agglutination, lysis). recent studies have shown the presence in invertebrates, including tunicates, of hemocyte/coelomocyte cytolytic molecules (lysins) that can be released into the hemolymph or coelomic fluid, and their lytic activity depends on their integration inside target cell membranes. in some cases membrane lesions appear as circular pores as shown by electron microscopy observations. there are few data on ascidian lysins and their lytic mechanisms. in ciona intestinalis a cytotoxic activity towards mammalian erythrocytes has been reported. the lytic activity, examined using a tris-buffered saline containing calcium ions and isosmotic to the hemolymph, was inhibited by sphingomyelin (25 µg/ml). to identify the lysin-releasing cells, hemolymph was separated through a discontinuous percoll gradient. the hemocyte populations were separated in 6 bands, each of them appeared to be enriched with a particular hemocyte population. the hemocytes from band 5 (b5) mainly composed with unilocular refractile granulocytes were responsible for the lysis of rabbit erythrocytes (re). to characterize the activity, the supernatant of the b5 hemocyte lysate supernatant (b5hls) or b5 hemocyte culture supernatant (b5hcs) was assayed with different mammal targets. both b5hls and b5hcs contain at various extent lytic activity against re and k562 tumor cell line. a lower level of cytotoxicity was found against sheep erythrocytes. such an activity was calcium-dependent, thermo-stable (56 °c), inhibited by sphingomyelin (25 µg/ml), phospholypase a2 inhibitors e.g. dibucain and quinacrine, as well as by d-galactose and cell-free hemolymph. present results suggest that a complex lytic mechanism dependent on sugar-crd interaction may be involved in innate immune response of ciona intestinalis. session 2. chairman: l abelli, university of ferrara, italy leech neuroimmunity: a crossing point between injury and nerve repair j vizioli, pe sautière, c lefebvre, f croq, m tahtouh, m salzet, j pestel laboratoire de neuroimmunologie des annélides fre 2933 cnrs. university of lille 1-59655 villeneuve d’ascq, france lophotrochozoans (annelids and molluscs) are increasingly contributing, along with vertebrates and ecdysozoans, to a deeper understanding of important biological processes like innate immunity or neurogenesis. among the annelids, the medicinal leech hirudo medicinalis, is one of the most extensively used model organism since the xix century for neurobiology studies. indeed, the development, the anatomy and the physiology of many nerve cells are now well characterized. in addition, the medicinal leech is a recognized model in central nervous system (cns) regeneration studies because of its capacity to get in a few weeks a complete morphological and functional repair of the injured nerve cord. recent results showed that leech cns is able to activate an innate immune response upon bacterial challenge and that several induced molecules are also known to be involved in regeneration events. early alert signals, glial cells recruitment, neuronal growth and axonal guidance are the major events occurring during cns regeneration in hirudo, but the basic molecular mechanisms of these processes are little known. we presently develop a research project aimed to the comprehension of such mechanisms and the characterization of some protagonist of this complex phenomenon. the main points of interest and the guidelines of our research are here briefly exposed to discuss the role of neuroimmunity during leech nerve repair. leech hematopoietic cells involved in muscle regeneration a grimaldi, g tettamanti, s banfi, ml guidali, g greco, c bianchi, r valvassori, m de eguileor department of structural and functional biology, university of insubria, varese, italy recently, myogenic and endothelial (myoendothelial) cell progenitors were identified in the interstitial spaces of murine skeletal muscle. these are primitive cells, distinct from satellite cells (potent myogenic stem cell population residing in the muscle of vertebrates and responsible for postnatal muscle regeneration and growth), they are located outside from the basal lamina and expressing the hematopoietic precursor marker cd34. these cd34+ precursors cells play a fundamental role during muscle regeneration, differentiating into myogenic cells and contributing to new fibres formation. hematopoietic and endothelial precursors cells of leeches show an impressive conservation in 32 morphofunctional and molecular mechanisms compared to vertebrates. for this reason leeches can be considered a simple model to better understand several mechanism regulating musclederived hematopoietic stem cell biology. we have previously demonstrated that in leeches endothelial and circulating precursor cells, express cd34 and the vegf receptor flk1 that in vertebrate are specific for both the endothelial and the myo-endothelial cells involved in muscle regeneration. initially, leeches show in the area of lesion a plug that is in a short time replaced by a pseudoblastema only made of fibroblasts and macrophages without any type of muscle fibres. after 2 months from injury, new muscle are present in the cicatricial area and the damaged body sac is regenerated. we focus on the origin of the new muscle fibres since, unlike from vertebrates, no satellite cells are present in the adult muscle body. our findings suggest that cd34+ circulating precursors cells in leeches, as muscle-derived hematopoietic vertebrate stem cell, can be a source of myogenic precursors, directly recruited from vegf in the regenerative area. self/non-self recognition in the ciliate euplotes raikovi: characterization of er-mapk1, a downstream component of the autocrine signal transduction pathway a vallesi, b di pretoro, p luporini department of biology, university of camerino, camerino, italy in the ciliate euplotes raikovi, cell typespecific, water-borne signal proteins (pheromones) control self/non-self recognition phenomena by binding their target cell-surface receptors and activating downstream signal transduction pathways. immunorecognition analyses of e. raikovi cell extracts revealed that at least three distinct protein kinases are activated (phosphorylated) in functional association with the autocrine pheromone-receptor loop that promotes the vegetative (mitogenic) cell growth. one of these kinases, designated as er-mapk1 (from mitogen-activated protein kinases), was structurally characterized by molecular cloning of the relevant gene. the er-mapk1 n-terminal half of 300 amino acids bears unmistakable structural homology with the “intestinal cell kinase” and the “male-germ cell associated kinase”, that are involved in the regulation of proliferation and differentiation of specialized animal cells. it contains all the basic structural features that are required for a mapk catalytic activity, in particular the dual phosphorylation site represented by the thr-asp-tyr motif in the activation loop. in contrast, the er-mapk1 c-terminal half of 331 amino acids appears to be structurally unique. it is particularly rich in glycine residues and potential sites of regulatory activities, and shows sequence motifs that clearly predicts a nuclear localization of er-mapk1. old and new immunomarkers to assess health status of clams (tapes philippinarum) from the lagoon of venice v matozzo, mg marin department of biology, university of padua, padua, italy the aim of the present study was to examine the health status of clams (tapes philippinarum) from the lagoon of venice by means of immunomarkers. bivalves were collected in june 2007 in 8 sites of the lagoon characterised by differing contamination levels. immunomarkers included total haemocyte count (thc), lysozymelike activity in cell-free haemolymph (as measure of cell membrane stability), and vitellogenin (vg)-like protein levels in both digestive gland and cell-free haemolymph. the results showed that clams sampled at marghera and fusina (highly polluted sites) had significantly reduced thc values with respect to those of animals from the other sites. conversely, significantly increased thc was observed in clams from valle di brenta and cà roman, influenced by wastewater from agricultural areas and intense fishing and passage of ships, respectively. significantly increased lysozyme-like activity was also recorded in cell-free haemolymph of clams collected at the most polluted sites (campalto, marghera, cà roman), suggesting that destabilisation of cell membranes occurred in haemocytes. interestingly, altered vg-like protein levels were observed in digestive gland and haemolymph of both male and female clams from contaminated sites. vg induction is generally recognised as a biomarker of exposure to estrogenic compounds. however, in the light of recent findings concerning involvement of vg in immune responses (zhang et al. fish shellfish immunol. 19: 93-95, 2005), application of vg as potential immunomarker in future laboratory and field studies is suggested. on the basis of the immune responses analysed in the present study, we can conclude that the health status of t. philippinarum at contaminated sites is poor. however, influence of both exogenous (i.e., water temperature and salinity, food availability) and endogenous factors (i.e., reproductive cycle) on cell functional responses investigated have to be taken into proper account. a new hemagglutinin from the coelomic fluid of the sea urchin paracentrotus lividus (lamarck, 1816) (echinodermata) f drago department of animal biology, “ marcello la greca”, university of catania, catania, italy agglutinins or lectins are glycoproteins that play a fundamental role in the innate immune responses in invertebrates. they are usually detected in cellfree coelomic fluid or hemolymph by the ability to agglutinate particles such as vertebrate erythrocytes (hemagglutinin), bacteria, protozoa or fungal cells. hemolymph lectins have been isolated from several 33 species of invertebrate taxa, while few data are available in echinodermata. in the present study a hemagglutinin was purified from the coelomic fluid of the sea urchin paracentrotus lividus, by ionexchange chromatography on deae-sephadex. a fraction, corresponding to a band at 11 kda in sds page, was found to have hemagglutinating activity. the amino acid composition of the lectin is in progress by the use of a 2d electrophoresis followed by maldi tof analysis. session 3. chairman: r valvassori, university of insubria, varese, italy immunosuppression and host regulation by parasitic hymenoptera f pennacchio department of entomology “filippo silvestri”, university of naples “federico ii”, portici, naples, italy the success of parasitism in hymenoptera is largely influenced by molecules and genes that the ovipositing female injects along with the egg into the host’s body or that offspring produces during the course of development. here we analyze how the basal “toolkit” of gene products in ancestral ectoparasitic idiobionts has changed with the evolution of more intimate developmental strategies, which require very efficient mechanisms of evasion and/or suppression of host immune response. the functional and molecular bases of the major alterations of host immune system, endocrine balance, development and reproduction induced by regulation factors, both of maternal and embryonic/larval origin, are presented and the impact of these changes on host suitability and parasitoid fitness analyzed. hril-8 stimulates unpaired (upd)-3 expression and other immune-related activities in drosophila melanogaster sl2 cell line d malagoli, s sacchi, e ottaviani department of animal biology, university of modena and reggio emilia, modena, italy invertebrate innate immunity relies on both cellular and humoral components. among humoral factors, there is less information on soluble molecules able to act as signals during the immune response (i.e. cytokines). to date, only a few cytokines have been observed in different arthropod taxa such as insects (spätzle and unpaired [upd]-3) and crustaceans (astakine). as it has been proposed that a possible involvement of chemotactic factors may increase the expression of upd-3, we have studied the effects of human recombinant ilnterleukin-8 (hril-8) on the immune functions of drosophila melanogaster sl2 macrophage-like cells. we have found that hril-8 enhances the expression of upd-3 and of the putative cytokine, drosophila helical factor (dhf). furthermore, hril-8 promotes the transcription of the antimicrobial peptide genes defensin and cecropin a1. beside these evidences on humoral factors, hril-8 also increases the phagocytic activity of sl2 cells. our data suggest the existence in d. melanogaster of one or more soluble factors that possibly share some structural similarity with il-8, eliciting an immune response involving simultaneously cellular and humoral components. il-1 system in reproduction of invertebrates a pellegrini, s jantra, f ietta, n bechi, e bigliardi1, l paulesu department of physiology, university of siena, siena, italy 1department of evolutionary biology, university of siena, siena, italy interleukin-1 (il-1) is a key regulator of the inflammatory response and an important mediator of materno-fetal immunotolerance in mice. we recently showed that il1-β and its functional membrane receptor type i (il-1r ti) are also expressed in the female reproductive tract of vertebrate species with different reproductive strategies, i.e. viviparity and oviparity. in both cases, there are mechanisms of materno-fetal immunotollerance since the sperm enter and cross the female genital tract and then, the fertilized egg/zygote/embryo is transported or retained in the oviduct. to date, research available is limited to vertebrates while no data are reported in invertebrates. since the cytoplasmic domain of il1r ti shares high homology with the cytoplasmic region of toll protein that recognize many pathogen associated molecular patterns in drosophila, we investigated the expression of toll-il1r ti (tir) domain in reproductive tissues of this invertebrate. by using a specific anti-human antibody against the tir domain, western blot analysis revealed a band of 117 kda in membrane lysates corresponding to drosophila toll protein. immunohistochemistry showed protein expression in the epithelial cells of oviductal tissues and in the spermathecae. these findings suggest a potential ubiquitous role of il-1 system in reproductive tissues of vertebrates and invertebrates. differential responses of mussel hemocytes to bacterial challenge c ciacci 1, m betti1, p roch2, l canesi3 1institute of physiological science, university of urbino “carlo bo”, loc. crocicchia, urbino, italy 2umr ecosystèmes lagunaires, universitè de montpellier 2-34099 montpellier, cedex 5, france 3department of biology, university of genoa, genoa, italy marine bivalves are widespread molluscs in coastal waters at different latitudes. due to their filter-feeding habits, they accumulate large number of both autochthonous and allochtonous bacteria from the harvesting waters. in this work the functional responses of mytilus hemocytes to in vivo challenge with different 34 bacteria were evaluated. two gram-negative bacteria, vibrio splendidus and vibrio anguillarum, and one gram-positive bacterium, micrococcus lysodeikticus, were used. mussels have been injected with heat-killed bacteria and hemocytes sampled at different time post-injection (from 3 to 24 h). the results demonstrated that different bacteria elicited significant functional responses in mussel hemocytes. all bacteria lead to a significant lysosome membrane destabilisation (v. splendidus > v. anguillarum > m. lysodeikticus) at short time post-injection followed by recovery at longer times. similar effects have been observed in mussels collected from two different populations (adriatic sea and ligurian sea). bacterial challenge also induced a significant increase (about 100 %) in serum lysozyme activity. moreover, challenge with v. anguillarum significantly stimulated the bactericidal activity of hemocytes towards e. coli. the results indicated differential functional responses of mussel hemocytes to different bacteria. the application of this knowledge to the understanding of the actual adaptive responses of bivalve when exposed to micro-organisms in their natural environment can represent significant ecological, economical and public health-related interest. this work was partially supported by the eu program imaquanim (food-ct-2005-007103). morphological changes of mytilus galloprovincialis hemocytes after bacterial interaction p pagliara, p roch1 department of biological and environmental sciences and technologies, university of salento, lecce, italy 1umr ecosystèmes lagunaires, université de montpellier 2-34099 montpellier, cedex 5, france apoptosis represents a physiological process used by multicellular organisms to regulate the cell number (tissue homeostasis), to remove damaged or unwanted cells or to eliminate different pathogens. protozoan parasites can induce or inhibit apoptosis in host cells, whereas bacteria increased apoptosis in human neutrophils and macrophages, interfering with such essential components of the innate immunity. so, upregulation of apoptosis due to infection may result in immune suppression. mainly studied in vertebrates, only few data are available on the relationships between apoptosis and immunity in invertebrates. in the present study, we investigated the response of the marine bivalve mytilus galloprovincialis hemocytes to different bacteria species, as well as the morphological modifications of their nucleus using microscopic observations.. the numbers of hemocytes that detached from the substrate and died by necrosis increased after bacteria contact. the detached hemocytes became round losing their typical cytoplasm extensions. they were also evidences for morphological changes of nucleus with chromatin condensation and bordering at the nucleus periphery, and sometimes its fragmentation. control of apoptotic induction in mussel has been done on adhering hemocytes treated with h2o2 and puromicin. in conclusion, the changes in nucleus morphology and the rounding of cells suggested apoptosis activation, being more evident with vibrio splendidus than with vibrio anguillarum. session 4. chairman u oreste, cnr, naples, italy immune defences and interactions with the environment g scapigliati, e randelli, d casani, f buonocore department of enviromental sciences, university of tuscia, viterbo, italy animal species live in a condition of homeostasis with the microbiome present in their body and in the environment. this microbiome is constituted by a great number of microorganism species, many of which are potential pathogens. the homeostatic condition describes a continuous and dynamic equilibrium present between aggression strategies developed by microorganisms, and defence strategies invented by animal eukaryiotes. during millenia, the continuous development of aggression and defence strategies became an evolutionary machine that produced a great number of orthologous and paralogous genes. also, the evolution of animal behaviours has been possible by the evolutive capabilities offered by the immune system, that must guarantee full immune defence in every environmental condition. the research performed in our lab considered the morphological and functional organisation of immune defences initially in insects, subsequently in teleost fish and amphibians. teleost fish represent an experimental model widely employed, since are oldest living vertebrates with a functional platform of immune system conserved in all its components until mammals, they are present in every aquatic environment of the planet, and are of interest for animal and environmental biotechnolgy. in addition, teleosts have a free-swimming larval stage, and it’s possible to divide vital stages when only innate defences are present, and when both innate and acquired defences are functional. employing as main investiagted species the sea bass dicentrarchus labrax, we defined in a teleost species the panel of distribution and the ontogenesis of lymphocytes, the involvement of lymphocytes during antigenic stimulation “in vivo” and “in vitro”, the cloning of genes coding for immunoregulatory molecules, and the expression of these genes during immune responses. in the imaquanim consortium, we are developing systems of transcriptomic analysis by using pcrarrays, to evaluate immune responses through the simultaneous expression of groups of genes coding i) for the whole sea bass immunome (ca 50 genes), ii) for the “innate” immunome (ca 15 genes), and iii) for the “acquired” immunome (ca 35 genes). of great interest for immune studies is gut-associated 35 immune system, whose functional organisation could be also related to the intestinal microbiome and food habits. many t cells are present in the intestinal epithelium of predatory teleosts, and current research is in progress to extend these observations to herbivorous and plancton-eating fish species. research funded by european union (6fp, n° 000173). thymocyte decisions in sea bass, dicentrarchus labrax (l.): cd4 or cd8? gene expression profiling and in situ hybridisation studies l guerra, f buonocore, e randelli, am fausto, l abelli1, s picchietti department of environmental sciences, university of tuscia, viterbo, italy 1department of biology and evolution, university of ferrara, ferrara, italy in teleosts no precise information is still available on differentiation and selection of t cells, crucial steps to establish a functional immune system, although the role of thymic microenvironment in t-cell development has long been recognized in mammals. this work first defines the expression levels of cd4, cd8-α and tcr-β, three important genes in t cell function, in the thymus of the marine teleost dicentrarchus labrax (l.). specific primers were used to analyse by real time pcr gene expression in the thymus of one-year-old specimens. the results reported high tcr-β and cd8-α transcripts, while cd4 transcripts were lower (p<0.05 vs. tcrβ). the in situ hybridization with rna probes identified cd4 and cd8-α expressing cells in each thymic lobe, cd8α+ and cd4+ thymocytes almost filled the cortex drawing a cortex-medulla demarcation and extended in large cords in the medulla. in sea bass thymus, the expression pattern of cd4 and cd8-α largely overlapped that of tcrβ, except in the subcapsular zone where tcrβ+ double-negative thymocytes were detected. these results provide new information about the thymic compartmentalization in teleosts and leads to new hypotheses about thymocyte differentiation pathways. gene expression and functional studies on gut immune system of the sea bass, dicentrarchus labrax (l.) s picchietti, l guerra, e randelli, f bertoni1, am fausto, f buonocore, l abelli1 department of environmental sciences, university of tuscia, viterbo, italy 1department of biology and evolution, university of ferrara, ferrara, italy morphological and molecular data have evidenced in a few teleost species a gut-associated lymphoid tissue (galt), consisting mainly of t cells, whose origin, selection and functions are still unclear. this work reports about tcr-β, cd8-α and cd4 transcription in the intestine of the marine teleost dicentrarchus labrax (l.) and localization of t cells expressing such genes. anterior, middle and posterior intestine segments of one-year-old specimens were sampled for rq-pcr and in situ hybridization (ish) studies. tcr-β and cd4 transcription did not differ along the intestine, while cd8-α transcripts rose in the posterior segment (p<0.05 vs. middle). in the whole intestine tcr-β and cd8-α transcripts were higher than cd4 ones (p<0.001). in anterior and middle segments tcr-β expression was higher than cd8-α (p<0.001). the ish with rna probes identified numerous tcr-β+ and cd8-α+ cells intraepithelially and in the lamina propria of mucosa, the latter forming aggregates, and rare cd4+ cells. percoll-purified leucocytes from the whole intestine showed innate cytotoxic activity against a xenogenic tumour cell line. there results confirm that the sea bass galt consists mainly of t cells with significant cytotoxic activity. early administration of probiotic strains stimulates the gut immune system of the marine teleosts dicentrarchus labrax and sparus aurata l abelli, s picchietti1, e randelli1, o carnevali2 department of biology and evolution, university of ferrara, italy 1department of environmental sciences, tuscia university, viterbo, italy 2department of marine sciences, marche polytechnic university, ancona, italy early feeding (started during gut metamorphosis) with probiotic-supplemented diets, besides modifying the intestinal microflora, provoked profound effects on physiology of fish larvae. using rotifers and artemia as living vectors, the autochthonous bacterium lactobacillus delbrueckii or a multispecies probiotic formulation (autochthonous lactobacillus fructivorans + lactobacillus plantarum from human faeces) were orally administered to sea bass and gilthead seabream larvae, respectively. the treatments stimulated the larval rearing (significantly increased body weight, decreased cortisol levels and improved stress response) and the immune system. in sea bass, the probiotic raised intestinal tcells (+105 %), in keeping with increased total body tcr-β transcript (+41 %), and acidophilic granulocytes (+118 %) concomitant to lower transcription of pro-inflammatory genes (il-1β, tgfβ, il-10, cox-2). in seabream, the multispecies formulation raised intestinal ig+ cells (+51 %) and acidophilic granulocytes (+284 %), mainly belonging to the mab g7+ phagocytic population (+536 % vs. control). 36 these results point to stimulatory effects of probiotics on the gut immune system that correlates with improvement of fry survival. a cd4 homologue in sea bass (dicentrarchus labrax): molecular characterisation and structural considerations e randelli, f buonocore, d casani, s costantini1, am facchiano1, jj zou2, cj secombes2, g scapigliati department of environmental sciences, tuscia university, viterbo, italy 1laboratorio di bioinformatica e biologia computazionale; institute of food sciences, cnr, avellino, italy 2scottish fish immunology research centre, aberdeen university, aberdeen, uk the cd4 is a transmembrane glycoprotein fundamental for cell-mediated immunity. its action as a t cell co-receptor increases the avidity of association between a t cell and an antigenpresenting cell by interacting with portions of the complex between mhc class ii and tr molecules. the sea bass cd4 cdna consists of 2071 bp that translates in one reading frame to give the entire molecule containing 480 amino acids. the analysis of the sequence shows the presence of four putative ig-like domains and that some fundamental structural features are conserved from sea bass to mammals. by real time pcr analyses very high levels of cd4 mrna basal transcripts have been evidenced in thymus, followed by gut and gills. in vitro stimulation of head kidney leukocites with lps and pha-l gave an increase of cd4 mrna levels after 4 h and a decrease after 24 h. homology modelling has been used to create a 3d model of sea bass cd4 and to investigate its interaction with sea bass mhc-ii. our results will add new insights in sea bass t cell immune response and will help to identify t cell subsets in teleost fishes to better understand the evolution of cell-mediated immunity. this work was supported by the european commission within the project imaquanim (ec contract number food-ct-2005-007103). effects of exogenous cortisol on the expression of glucocorticoid receptor (dlgr1) and hsp70 in head-kidney and peritoneal cavity cells from dicentrarchus labrax a vizzini, m vazzana, g salerno, m celi, ma sanfratello, n parrinello marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy fish are constantly exposed to stressful conditions, and have evolved responses including the prompt release of corticosteroids hormones. the physiological effects of corticosteroids are regulated by cellular glucocorticoid receptors (gr), that mediate gene expression influencing a variety of physiological function related to metabolism, immunity, beahaviour, osmoregulation and cardiovascular transport. the assembly, functionality and transport of grs are dependent upon the activity of heat shock proteins (hsp) including hsp 90, hsp 70 and hsp 56. in previous papers we showed that high plasma cortisol level due to fish confinement affects sea bass innate immunity, then we cloned and sequenced a gr from sea bass peritoneal cavity leucocytes (dlgr1). in this study we examined the expression of dlgr1 as related with hsp 70 tissue content in the presence of high exogenous plasma cortisol level. cortisol (20mg/kg body mass) was injected into fish peritoneal cavity, and, at various times after the injection, dlgr1 gene expression was evaluated by real time pcr. hsp 70 was identified by specific antibodies an densitometry analysis of western blot pattern was carried out. results showed that, despite a drop in dlgr1 protein content, there was a significantly enhanced dlgr1 mrna expression in head kidney cells due to stress for manipulation procedures, whereas in leucocytes from peritoneal cavity the enhanced expression of dlgr1 could be related to a cortisol effect. an increased level of hsp 70 protein was also shown. research are in progress to evaluate hsp 70 gene expression. session 5. chairman: n parrinello, university of palermo, italy longevity genes across species: conservation versus evolvability s salvioli1,2,3, ptieri1,2, g castellani2,4, m capri1, c barbi1, a santoro1,2, s altilia1,2, l invidia1,2, m pierini1,2, e bellavista1,2, d monti5, c franceschi1,2,3,6 1 department of experimental pathology, university of bologna, 40126 bologna, italy 2interdepartmental center “l. galvani” (c.i.g.), university of bologna, 40126 bologna, italy 3er-gentech laboratory, 44100 ferrara, italy 4dimorfipa, university of bologna, 40064 ozzano dell'emilia, italy 5department of experimental pathology and oncology, university of florence, 50134 florence, italy 6department of gerontological science, italian national research centre on aging (inrca), 60131 ancona, italy the search for longevity genes has greatly developed in recent years basing on the idea that a consistent part of longevity is determined by genetics. the ultimate goal of this research is to identify possible genetic determinants of human aging and longevity, but studies on humans are limited by a series of critical restrictions. for this reason, most of the studies in this field have been, and still are, performed on animal models, basing on the assumption that fundamental biological mechanisms are highly conserved throughout evolution and that, accordingly, extrapolation from model systems to humans is quite reasonable. 37 indeed, many comparative data obtained on single genes or gene families fit with this assumption. however, it is also clear that, despite such a basic conservative scenario, major changes also occurred in evolution, particularly regarding biological regulatory processes and integration between and among pathways. this consideration raises the fundamental question of the transferability of the results obtained from model systems to humans. indeed, many data obtained on animal models were not confirmed on humans or, on the contrary, some contributions to the genetics of longevity were discovered in humans and not always have been replicated in animals. recent conceptualizations stressed the importance of robustness as a fundamental property of living organisms, a characteristic acquired during evolution that allow them to be error-tolerant and to easily respond to external perturbations. a “bow-tie” organizational architecture is likely to be a common feature of highly organized, robust systems. a bow-tie model is present when many inputs converge on, and are integrated by, few elements, and many different outputs come out as the product of the integration. the elements composing the core (“knot”) of the bow-tie in a biological system are “hub proteins” and the genes that encode them are likely to be in many cases robustness genes. much of the core of a bowtie is often conserved throughout evolution, but this conservation does not prevent, but rather facilitates the variability of the possible outputs. thus, a bowtie structure allows both robustness and evolvability. we propose that longevity genes should participate with a core position to a bow-tie metabolic structure and that a new way to identify putative genetic determinants of longevity could be thus the conservation of their products in bow-tie structures all along evolution. thymus development is influeced by hypophyseal and thymic allograft after hypophysectomy: expression of pcna and cd3 markers m aita, e caccia1, n romano1 department of human physiology and pharmacology, university “la sapienza”, rome, italy 1department of environmental sciences, university of tuscia, viterbo, italy experiments of hypophysectomy, followed by hypophyseal or thymic allograft were performed in chicken embryos in order to study the influence of thymus or hypophysis on the thymus development. experimental and control thymuses, collected at the 18th day of incubation, were tested for antipcna (proliferating cell nuclear antigen) and anticd3 immune reaction. the immunoreactive cells were evaluated by computer-assisted statistical analysis. in the first series of experiments, hypophysectomy was performed on chick embryos at 36-40 hr of incubation. the thymuses collected evidenced in the cortex, a significant reduction of pcna immunereactive thymocytes (p<0,05). in the second series of experiments hypophysectomized embryos received at the 12th day of incubation or a hypophyseal allograft or a thymic allograft onto the chorio-allantoic membrane from 18 day-old donor embryos. the hypophyseal allograft allowed an increase of the cd3 expression of the cortical and medullary thymocytes but no improvement of pcna expression in cortical thymocytes. the thymic allograft let a very good recovery (p<0,001) both in pcna and cd3 expressions in the cortical thymocytes and a good recovery, of cd3 similar to that found in hypophyseal allograft, in the medullary thymocytes. these findings confirm that the lack of hypophysis decreases the possibility of cortical thymocytes to proliferate, but not to differentiate further on and that a thymic allograft may influence the recovery of the thymus of a hypophysectomized embryo, probably also by an emigration of thymocytes from the thymic graft. comparative analysis of fucose binding lectins isolated and characterized from different teleost species, and distribution of a f-lectin during dicentrarchus labrax ontogenesis m cammarata, mg parisi, g benenati, v mangano, a vizzini, g salerno, d parrinello, m vazzana, n parrinello marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy fucose-binding lectins (fbl) are contained in tissues and fluids from invertebrates and vertebrates. the lectin repertoire in teleost fish is highly diversified and, recently, the fbl structure with a novel lectin fold of f-type carbohydrate recognition sequence (the eel “f-type”) has been described (bianchet et al. nat. struct. biol. 8: 628634, 2002). such a lectin shared an unique fucosebinding sequence motif contained in carbohydratebinding domain of lectins and unrelated proteins. recently, we have shown that a serum fbl from sea bass dicentrarchus labrax (dlfbp) and sea bream sparus aurata (safbp) are involved in innate immunity. both lectins were composed of two tandem domains that exhibit the eel “f-type” motif. these lectins were purified, characterized, cloned and sequenced, and biological activity and tissue distribution were examined. a complete coding of teleost f-type lectin crds showed that dlfbp and safbp are members of the f-lectin family. the phylogenetic sequence analysis from bacteria to vertebrates, showed that the f-lectin motif is broadly distributed, and significantly diversified in terms of molecular organization and number of domains. preliminary results on comparative analysis of purified and characterized lectins from different teleost species are also reported. these lectins, that showed calcium independent hemoagglutinating activity against rabbit erythrocytes, have been purified using a fucose-agarose affinity chromatography. serum fucose binding lectins are present in all the studied species with a molecular size range from 32 to 41 38 kda, and share shrinkage property in sds-page. the relationship with the f-lectin family has been supported with western blot analysis using dlfbp antibody. finally, we examined distribution and expression of dlfbp during fish ontogeny and showed lectins are also present in eggs. cloning and expression analyses of an interferon (ifn) in sea bass (dicentrarchus labrax l.) after poly i:c induction d casani, f buonocore, e randelli, v scala, jj zou1, cj secombes1, g scapigliati department of environmental sciences, tuscia university, viterbo, italy 1scottish fish immunology research centre, aberdeen university, aberdeen, uk interferons (ifns) are a multigene family of inducible cytokines and posses antiviral activity through a jak/stat signalling pathway. ifn action induces the synthesis of ifn-regulated proteins, as mx, that collectively constitute the antiviral response responsible for the inhibition of virus multiplication. ifn cdna cloning was possible after in vitro stimulation of head kidney sea bass (dicentrarchus labrax l.) leukocytes with polyinosinic: polycytidilic acid (poly i:c). poly i:c is a synthetic ribonucleotide that acts as a viral rna to induce the ifn system. we used the “homology cloning” strategy: with degenerate primers we obtained an initial fragment of 189 bp. from this fragment we designed specific primers for 3’ and 5’ race pcr to complete the cdna sequence. an alignment was performed using available ifn amino acid sequences to study the conservation of characteristic features and a phylogenetic tree was generated using the same sequences. finally, specific primers were used to analyse by real time-pcr the ifn and mx expression in head kidney leukocytes stimulated with poly i:c for 6 and 24 h. the ifn expression after 6 h is higher with respect to 24 h expression, whereas for mx it is the contrary. this is an agreement with the teory that ifn is produced after viral infection and, successively, it induce mx synthesis. the same cloning procedure will be used on genomic dna, as it is important to determine the number of introns in the ifn sequence to investigate its evolution from fish to mammals. this work was supported by the european commission within the project imaquanim (ec contract number food-ct-2005-007103). phylogenetic analysis of teleost light chain immunoglobulin u oreste, s varriale, m r coscia cnr, institute of protein biochemistry, napoli , italy in the last decade a large number of cdna igl sequences have been obtained from teleosts. because of the extensive igl diversity within the genome of each species and the very wide phylogenetic distance among teleost orders, the classification of igl isotypes from teleost species remains questionable. at present, because the number of igl isotypes identified so far varies in different teleost species, it remains unclear whether all possess the same number of igl isotypes. the discrepancy in the number of igl isotypes identified in each species may be explained by incomplete screening of differently expressed isotype specific mrnas and by a different degree of diversity between sequences identified in each species. to date, igl genes have been sequenced from 27 different species, two of them being antarctic species investigated by us: trematomus bernacchii and gymnodraco acuticeps. in the present work we compared all the available teleost immunoglobulin light chain sequences in order to evaluate the evolutionary relationships among them. we searched the data banks for all teleost immunoglobulin light chain, either genomic or cdna, sequences. for each species we aligned the sequences by clustalx, distinguished different isotypes and constructed a consensus sequence for each isotype. then we calculated the distances from the consensus of individual sequences and used the closest one in a data set. the sequences of the data set were aligned and phylogenetic trees were constructed by neighbor-joining and maximum-likelihood methods. our results indicated that three different isotypes can be identified in teleosts. in the acanthopterygean species one isotype can be distinguished into two subisotypes one of them carrying a dna microsatellite insertion. specific sequential features of each group of isotypes have also been observed. work supported by the pnra project 2005.1.2 screening for antimicrobial peptides in zebrafish (danio rerio) el caccia, g vasta1, ag ficca, p strickler1, n romano department of environmental sciences, university of tuscia, viterbo, italy 1comb, university of maryland, baltimore, maryland, usa antimicrobial peptides (amps) are small-sized (from 12 to 45 amino acids in length), cationic and amphipathic molecules able to neutralize pathogenic microrganisms. the amp are very ancient in evolution life and wide expressed in methazoan species. they were described as being inducible and exert the killing or slow growth of bacteria, thus representing an old immune system that share adaptive and natural characteristics. probably, the amps are early expressed during ontogeny of vertebrate as “evolution memory” before more complicate immune system has to differentiate. with the emergence of antibiotic resistant bacteria the amps represent good candidate for therapeutic strategies to counter bacterial infection in animals. the aim of this study is to identify the genes encoding for amps in 39 zebrafish. by analysing zebrafish est and genome databases we identified those sequences homologous to known antimicrobial peptides genes from higher vertebrates: 1) mucosal (parasin i, chatepsin d3 and d5); 2) liver/organs expressed (defensin beta-db1, leap-2); 3) circulating phagocytes (bactericidal permeability-increasing protein, bpi); 4) the neuroendocrine/immune system–related peptides, chromogranin a and b (cga, b). we designed specific primers sets that allowed the amplification of the full or partial length of these genes. the lengths of the seven cdnas (obtained by retro-trascription of total rna extracted from adult samples) range from 242 bp (dbi) to 504 bp (cgb). they have been cloned and sequenced and the deduced amino acids sequences have been aligned to the well known antimicrobial peptides to confirm successful amplification of the genes of interest. here we report the comparison analysis the deduced amps amino acid sequences from danio rerio with those of other fishes. further analysis will be performed in order to investigate the expression of these molecules during ontogeny. more than three decades since the use of mussels as pollution biosensors, their genes are still almost unknown. in the frame of the european integrated project food-ct-2005-007103, est sequencing and gene expression profiling are providing new interesting evidences. the former refers to the extension of the dna microarray of m. galloprovincialis defined at cribi. (university of padova) and currently used for investigating mussel gene transcription. among a number of selected stress factors, mussels of different origin have been challenged with bacterial antigens and the total rna subsequently purified from haemolymph samples was used to build primary cdna libraries. the abundant expression and molecular variability of transcripts identified as antimicrobial peptides emphasize the importance of such molecules in the response to bacterial pathogens. in silico processing and robust sequence annotation is in progress on the whole bulk of the new mussel ests. parallel evaluation of gene expression is performed by dna microarray analysis on indigenous mussels from the venice lagoon, a coastal transition system (4 sites, summer samplings 2005-2007). a relatively small number of immune-specific probes is present in the available cdna microarray and, with exception of one defensin, they appear down-regulated in the lagoon compared to offshore mussels. results will be discussed according to the published literature. experimental evidences from est sequencing and gene expression profiling in mytilus galloprovincialis l varotto1, a pallavicini2, f bernante3, s domeneghetti1, s cagnin3, g lanfranchi1,3, p venier1 1department of biology, university of padua, padua, italy this work was also supported in part by corila (consortium for the management and coordination of the activities related to the venice lagoon system). 2department of biology, university of trieste, trieste, italy 3cribi, university of padua, padua, italy 40 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice isj105.pdf 64 isj 3: 64-76, 2006 issn 1824-307x review regeneration in echinoderms: repair, regrowth, cloning md candia carnevali department of biology, university of milan, milan, italy accepted june 16, 2006 abstract regenerative potential is expressed to a maximum extent in echinoderms. it is a common phenomenon in all the classes, extensively employed to reconstruct external appendages and internal organs often subjected to amputation, self-induced or traumatic, rapidly followed by complete successful re-growth of the lost parts. regeneration has been studied in adult individuals as well as in larvae. in armed echinoderms, regeneration of arms is obviously frequent: in many cases, the detached body fragments can undergo phenomena of partial or total regeneration independently of the donor animal, and, in a few cases (asteroids), the individual autotomised arms can even regenerate to produce new complete adults, offering superb examples of cloning strategies. in the species examined so far most results throw light on aspects related to wound healing, growth, morphogenesis and differentiation, even though in most cases many crucial questions remain unanswered. the present paper provides an overview of the current understanding of the phenomenon and covers the main biological aspects of regeneration giving an idea of the “state of the art” across the phylum in terms of experimental approaches and representative models. key words: regenerative development; repair; regrowth; cloning introduction to regeneration development is a broad-spectrum process intrinsic of life which does not always start from the egg, fertilized or not, but can involve significantly all the stages of the life cycle, including not only the embryonic and post-embryonic periods, but also the adult phase. regeneration is in fact a distinct type of developmental process typically occurring in adults or larvae: it can involve limited processes of cell turnover and tissue repair, replacement of lost parts or organs, cast off following self-induced or traumatic mutilations, and even complete regrowth of whole individuals from body fragments (thus contributing to typical asexual reproductive processes). corresponding author: md candia carnevali dipartimento di biologia, universita’ degli studi di milano via celoria 26, 20133 milano, italy e-mail: daniela.candia@unimi.it in terms of general significance, regeneration is an important regulatory phenomenon with wide implications on reproductive biology, as previously suggested by spallanzani in his historical studies on head regeneration in snails (1768). it does not represent the first expression of new combination of genes resulting from sexual processes, but starts from cells, which have an already tested genetic program. it is fundamentally a conservative asexual process. in fact, thanks to its evident close relation with fission phenomena and cloning processes, regeneration can be regarded as the necessary and specific developmental complement to asexual reproduction, in analogy, and in parallel, to what happens for embryogenesis which is the established developmental strategy complementary to sexual gametic reproduction. therefore, although regeneration unavoidably involves analogous problems in terms of basic mechanisms and often superficially resembles embryogenesis, fundamental differences in its intrinsic asexual start and meaning makes regeneration a significantly diverse biological process. in embryogenesis, the whole organismic structure is totally created de novo; in contrast, in regeneration an anatomically defined part of the 65 organism, small or large, is reformed after its loss or severe injury and the new cells develop in an established context of mature tissues and differentiated cells in individuals (adult or larval) well characterized in terms of morphology and functions. therefore, in all animals, the regenerative processes related to different organs and structures should be regarded as fundamentally distinct developmental processes, which can not be considered an accelerated version of ontogenetic processes (candia carnevali and bonasoro, 2001a; reik and dean, 2002). although a response to injury is evoked in all animals, there is a remarkable variability in terms of degree of morphological and functional recovery, not only between unrelated groups, but also between closely related species, and even between organs and parts of the same individual. in contrast to old traditional views regarding the regenerative potential as a unique prerogative of the simplest and most primitive animals, regeneration is actually a common and widespread phenomenon through phylogeny, and its quite heterogeneous distribution from the lowest to the highest phyla appears to be independent of their organization and complexity level (ferretti and géraudie, 1997; thouveny and tassava, 1997; candia carnevali and bonasoro, 2001a). in fact, the regenerative capabilities appear to depend upon the individual potential for histogenetic and morphogenetic plasticity expressed in terms of recruitment of stem cells and/or dedifferentiated cells, cell proliferation and migration, supply of specific regulatory/trophic factors, and finally expression, or reexpression, of a specific developmental program (weissman, 2000; wadman, 2005). despite the differences between the species, repair and regrowth are common to all organisms, and differentiated tissues (liver, muscles) can be replaced de novo even in adult mammals, at least to a certain degree. obviously the regenerative potential can vary significantly with the stage of the life cycle (embryonic, larval, adult) and the age of the individual, the regenerative capabilities being higher in larval tissues and organs in comparison with those of adults, and in many cases present only in the early embryonic stages (mammals). it is not a case that traditional studies of regeneration in vertebrates have often employed larval stages of amphibians. if a close correlation between the regenerative potential of the individual and its possibility of survival can be inferred easily, self-repair abilities appear not only an obvious advantage for the individual, but gives a fundamental contribution to the adaptive capacities of the species and its fitness, since they increase the individual’s chances of reproducing, sexually or asexually, even when its body integrity is dramatically compromised. the universal character of regeneration is expressed in the famous aphorism by goss (1969): “if there were no regeneration there could be no life, if everything regenerated there would be no death. all organisms exist between these two extremes “. on the other hand, there are still unsolved fundamental questions concerning why regenerative potential is expressed to such different extents in different organisms and whether this phenomenon has been selected for or against in the course of evolution. according to a modern authoritative hypothesis (goss, 1988, 1992; thouveny and tassava, 1997) which takes into account the evolution of regeneration abilities in the organisms, regeneration should be regarded as a primary attribute of life: all organisms in principle possess a latent potential for regeneration, that could have been suppressed in response to specific selection pressures, i.e. inhibited/eliminated wherever its expression does not help survival and reproduction and does not counterbalance the disadvantages of its high metabolic cost. in other words, as far as the animal kingdom is concerned, the suppression of regeneration should not be regarded as a negative adaptation but rather as a pleiotropic epiphenomenon related to specific constraints and linked to more useful but incompatible adaptations (goss, 1988, 1992). in spite of the wide choice of potential models for studying regeneration, this phenomenon has been appropriately explored only in a few animal models traditionally and successfully employed by developmental biologists (hydrozoans and planarians among invertebrates and amphibian urodeles among vertebrates). surprisingly, with regard to many other animal groups well known for their spectacular regenerative capabilities, the actual knowledge is very poor not only in terms of cellular and molecular aspects of regeneration, but also in terms of basic mechanisms (candia carnevali and bonasoro, 2001a). with regard to these latter, according to a traditional view, regeneration involves two alternative basic mechanisms, epimorphosis and morphallaxis (following the terminology introduced by morgan, 1901). in epimorphosis, new tissues arise from undifferentiated cells (stem cells or de-differentiated cells), which are recruited to develop a typical blastema. this pool of new cells represents a discrete centre of proliferation activity from which all the regenerated structures are derived. in morphallaxis extensive phenomena of rearrangement/recycling from differentiated tissues take place and no blastema is formed: only limited and localized proliferation activity involves cells derived from existing tissues by reversal of differentiation and/or migration. in spite of this schematic and apparently well-established difference between epimorphosis and morphallaxis, there is recent experimental evidence (candia carnevali and bonasoro, 2001b) that the mechanisms at the tissue/cellular level can be much more flexible and largely overlapped to each other, and that the dichotomic view of these two alternative processes is too reductive. the regenerative potential in echinoderms regeneration is a physiological phenomenon in echinoderms, common in all classes (hyman, 1955). thanks to their spectacular regenerative capabilities, echinoderms were favourite models for the pioneer regenerationists of the 19th and early 20th centuries. after an unexplainable long scientific oblivion, they were recently re-proposed to the attention by a series of papers (for review see candia carnevali, 2005; candia carnevali and bonasoro, 2001b; thorndyke and candia carneval, 2001) exploring the basic 66 mechanisms of the regenerative phenomenon, its cellular and molecular aspects and also its potential for applied research. regeneration is extensively employed to reconstruct external parts (arms or appendages such as spines or pedicellariae) and internal organs (gonads, gut, whole visceral mass) often subjected to predation or amputation, selfinduced or traumatic, rapidly followed by complete regrowth of the lost parts; it is also part of the life cycle as an indispensable complement of the programme of asexual reproduction and is extensively employed in cloning processes. repair, regrowth and cloning processes can involve both the adult and the larval stages (eaves and palmer, 2003). regenerative potential in echinoderms is developed to a very broad extent: contradicting goss’s paradigm about the “non-regenerability” of the truly vital structures (1965, 1969), it enables vital organs such as the whole visceral mass to be regenerated completely after evisceration. it is largely a predicted phenomenon and in most cases follows autotomic self-induced mutilations, which can be considered the most important proximate cause of structural loss and depends on the presence and unique properties of “mutable collagenous tissues” (mcts, see for a review wilkie, 2001, 2005) at the level of the autotomy plane. it is relevant that in physiological conditions regeneration is always prompted by autotomy and proceeds from the retained side of a fractured autotomy plane. adults. the capabilities to regenerate body parts represent an obvious advantage for echinoderms. firstly, for the replacement of tissues following predation, an ability that echinoderms show to a greater extent than other invertebrates. reconstitutive regeneration is particularly frequent and extensive in crinoids and ophiuroids which both have long, fragile arms often subjected to predation or mutilations, selfinduced or traumatic, rapidly followed by complete regrowth of the lost parts. these regenerative phenomena are so frequent that specimens collected in natural environments always have two or more regenerating arms at different regrowth stage. in many cases, the detached body fragments can survive for a long time and undergo phenomena of partial or total regeneration independently of the donor animal (candia carnevali et al., 1998). these phenomena, quite common also in asteroids, provide clear evidence for the wide exploitation and implications of regenerative potential in echinoderms. in particular in asteroids, besides the extensive application in common repair mechanisms, arm regeneration offers in fact the most complete examples of cloning strategies carried out by adult specimens. as well known, in some starfish (linkia sp., coscinasterias sp.), new complete adults can be regenerated from individual autotomised arms. this extreme case clearly shows that, in echinoderms, regeneration is employed as part of a programme of asexual reproduction leading to the development of new individuals through specific fission mechanisms (emson and wilkie, 1980; mladenov and burke, 1994). besides asteroids, also many ophiuroids and holothuroids undergo asexual propagation involving the splitting of adults into two or three pieces with subsequent regenerative development of new complete individuals from each isolated portion. fission phenomena appear to be correlated with seasonal and metabolic factors as well as to ecological factors, and also depend on the age and size of the individuals: they are particularly frequent in small specimens, whereas the larger ones appear to prefer sexual processes. in terms of epimorphosis versus morphallaxis, echinoderms appear to employ in regeneration one or the other of these two processes according to apparently established criteria which depend on the specific start-conditions and requirements; however, just in echinoderms there is the clearest evidence that epimorphic regeneration often involves a significant contribution of morphallactic processes and that the borderline between these two processes is not so defined (candia carnevali and bonasoro, 2001a, b). if we consider the distribution of typical epimorphic or morphallactic processes in the different echinoderm groups (bonasoro et al., 1998, candia carnevali and bonasoro, 2001a; thorndyke et al., 1999), the emerging pattern can be summarized as follow. typical epimorphic processes with blastema formation appear to be employed in those situations where regeneration is a widely predicted, rapid and effective phenomenon, which takes place following autotomy (for instance in crinoids and ophiuroids, candia carnevali and bonasoro, 2001a, b; thorndyke et al., 2001b). in contrast, morphallactic regeneration seems to be a more complicate and slower process, which tends to follow traumatic mutilations (for instance in arm tip regeneration of asteroids, mladenov et al., 1989; moss et al., 1998): in this case amputation is not a predictable event and the regenerative mechanisms imply phenomena of substantial rearrangement of the old structures. in spite of their apparently different meaning and alternative employment in echinoderm regeneration processes, recent results (see crinoid regeneration in different experimental conditions, candia carnevali and bonasoro, 2001b) show that the same individual can employ both epimorphic and morphallactic mechanisms, modulating their different contributions according to its specific needs. this suggests that previous interpretations of the mechanisms at the tissue/cellular level need to be revised substantially. although occurring in all echinoderm classes, regeneration was investigated by a modern approach only in a few echinoderm models. rather detailed investigations are related to crinoids, ophiuroids, asteroids, and recently also holothuroids, which were explored both in terms of mechanisms at the tissue/cellular level and ecological significance (thorndyke et al., 1999; candia carnevali and bonasoro, 2001b; thorndyke and candia carnevali, 2001; dolmatov and ginanova, 2001; garcia-arraras and greenberg, 2001). regeneration also occurs in echinoids, but is less spectacular in terms of extent and degree of capabilities and only a few examples have been investigated so far (bonasoro et al., 2004; dubois and ameye, 2001). larvae. it is well known that traditional studies of regeneration in vertebrates have often employed larval stages of amphibians. this interest in larval stages is justified by the much higher regenerative potential of larval tissues and organs in comparison with those of 67 adults. as a general rule, any regeneration, which begins during larval life, appears to be inhibited or retarded after metamorphosis (wallace, 1981). in spite of the many obvious reasons for exploring the problem of the regenerative potential of echinoderm larvae, and apart from some fascinating descriptions provided by historical reports (mortensen, 1921; hörstadius, 1925a, b , c), this aspect of echinoderm regeneration has been neglected until recent times, when some exciting results have renewed interest in this phenomenon and in particular have focused attention on its possible implications for evolution, phylogeny and clonal populations. current research has been specifically addressed to regeneration of larvae: the comparative analysis of the different potential exploited by the diverse groups shows that the phenomenon of regeneration has an unexpected plasticity in the larvae and indicates its direct and close relationship to asexual reproduction and cloning (vickery et al., 2001). as is the case for all other regenerating systems, the basic goal in echinoderm regeneration research is to answer a few crucial questions regarding how regeneration processes are initiated, which sets of genes are activated (or reactivated), what is the origin of the cells involved in reconstruction or repair of the damaged or lost structure, and which factors (morphogens and/or mitogens) regulate growth, morphogenesis and differentiation at the right time and at the correct place to ensure a complete reestablishment of anatomical pattern and functional integrity (carlson, 1998; ferretti and géraudie, 1998; thouveny and tassava, 1998). in the species examined so far most results throw light on aspects related to wound healing, growth, morphogenesis and differentiation, but in most cases many fundamental questions remain unanswered, especially those related to specific cellular and molecular aspects and much work is still requested. in spite of the widespread and successful employment of echinoderms for molecular studies based on embryonic or larval development, there is at present a large gap in our knowledge of regeneration and only a few recent data are available. the following synthetic overview, although far to be exhaustive, offers an account of the phenomenological aspects of regeneration through the echinoderm phylum, presenting a summary of results obtained by diverse perspectives and different experimental approaches in representative models, and highlighting the biological relevance and the evolutionary implications of the phenomenon. crinoids adult: regeneration of body parts (crown and stalk) following self-induced or traumatic amputation in sealilies; regeneration of appendices (arms, pinnulae, cirri) or body parts following self-induced or traumatic amputation in feather stars; regeneration of individual internal organs (gonads, gut) or whole visceral mass following self-induced or traumatic mutilation/evisceration in feather stars. the main cellular and molecular aspects of the regenerative processes are summarized in table 1. crinoids are well known for their spectacular regenerative potential. feather stars extensively employ regeneration to reconstruct both external parts, namely arms, pinnules and cirri, and internal organs, such as digestive apparatus, gonads, and even complete visceral mass, which can be frequently lost following traumatic injury, predation or spontaneous autotomy (perrier, 1873; minckert, 1905; reichensperger, 1912). specimens collected in nature always show regenerating arms at different stages of growth. these regenerative phenomena can be easily reproduced in the lab mimicking the autotomy conditions and amputating the arms at the level of the autotomy plane (sutures). the overall regenerative potential of comatulids was accurately tested by przibram (1901) in experiments of severe mutilations. according to these studies, the animal appears to be able to survive and subsequently regenerate the lost parts in a number of traumatic conditions: for instance, when the body is halved and even when reduced to only one fifth. crinoid sea lilies (metacrinus rotundus) are also spectacular in their regeneration powers, and not only a new stalk can be regenerated following partial or complete removal (nakano et al., 2004), but also a completely new and functional crown can be regenerated following total ablation (amemiya and oji 1992). regeneration is also documented in fossil crinoids, particularly in extinct crinoids of the paleozoic, mesozoic and cenozoic, which provide evidence of additional branchings in regenerated arms, underlining the biological, ecological and evolutionary implications of the regenerative phenomenon in echinoderm phylogeny (oji, 2001). arm regeneration in feather stars represents the most thoroughly explored model in echinoderm regeneration studies (candia carnevali and bonasoro, 2001b). in these recent years a comprehensive study of the overall process of arm regeneration was carried out in antedon mediterranea, a flexible experimental model previously successfully employed in old classical studies (minckert 1905; perrier 1873; reichensperger 1912), which was re-explored in all its aspects from the macroscopic to the molecular level. this phenomenon can be described on the whole as a typical blastemal regeneration in which new structures develop from migratory pluripotent, actively proliferating cells in the presence of presumptive regulatory factors, which are responsible for both repair and regenerative phenomena. the overall process can be subdivided into three main phases: a repair phase, an early regenerative phase and an advanced regenerative phase, whose crucial aspects are related to common fundamental mechanisms such as 1) intervention of stem cells and/or dedifferentiated cells; 2) cell migration and proliferation; 3) contribution of putative growth factors, particularly in terms of specific neurally derived factors; 4) mechanisms of pattern formation. the data obtained so far are derived from an integrated approach, which utilizes different methods on experimentally induced arm regenerations (standard or abnormal) obtained in significantly different experimental conditions, including extreme mutilations (explants). the regenerative response was also employed in applied research as a new valuable model for ecotoxicological studies addressed to the e f f e c t s of the e x p o s u r e t o s p e c i f i c c l a s s e s o f 68 table 1. crinoids. summary of main cellular and molecular aspects of regeneration mechanisms responsible cells recruitment growth factors genes involved -epimorphic process with main contribution of undifferentiated cells -blastema -nerve-dependent regeneration -stem cells (amoebocytes, coelomocytes) -differentiated cells (phagocytes, (granulocytes) -dedifferentiated cells (myocytes) -differentiation -dedifferentiation -(re)differentiation -transdifferentiation -extensive migration -extensive proliferation -neurotrasmitters (dopamine, serotonin) -neuropeptides (s1, s2, substance-p) neural growth factors (tgfß, bmp, ngf, fgf-2) anbmp2/4 (tgf-ß superfamily environmental contaminants (candia carnevali, 2005). in particular, the normal mechanisms and pattern of the regenerative processes in standard conditions have been established in a series of experiments on regeneration at different stages following pseudo-autotomic amputations (candia carnevali et al. 1993; 1995; 1997). a parallel analysis has been carried out on the regenerative processes of both the normal regenerating arms and the respective amputated arm segments (explants) (candia carnevali et al., 1998; bonasoro et al., 1999; candia carnevali and bonasoro, 2001b): the explants can be maintained in good living conditions for weeks and represent excellent models for testing the arm regenerative potential in terms of autonomy of resources and control and for comparing regenerative mechanisms in a same individual. different types of isolated explants have been successfully employed: during the culture period they are able to undergo extensive repair and regenerative processes in parallel with their donor arms. comparison between the regenerative processes of arm explants and normal regenerating arms of corresponding stages highlights that beside general similarities in the basic regenerative processes there are some meaningful differences in terms of mechanisms employed and cellular/tissue elements involved. in terms of mechanisms, there is clear evidence that the epimorphic blastemal regeneration can involve a significant contribution of morphallactic processes (candia carnevali and bonasoro, 2001a, b). the regenerative potential, mechanisms and pattern have been also explored and compared with regard to aberrant regenerations resulting from arms deliberately subjected to traumatic mutilations which do not reproduce autotomy (candia carnevali and bonasoro, 2001b). the bulk of the results obtained so far in crinoids not only contribute to throw light on the most relevant aspects related to wound healing, morphogenesis, differentiation and growth in echinoderm regeneration, but also strongly suggest to employ this fascinating and promising experimental model for a successful applied approach. in terms of the specific cellular contribution, most of the cell types involved in regeneration are morphologically undifferentiated migratory elements, which are produced at the level of a) the brachial nerve (amoebocytes) and b) the coelomic epithelium (coelomocytes) respectively. available evidence suggests that the migratory amoebocytes produce the blastemal cells and all the blastema-derived differentiated cells, whereas the coelomocytes give rise to all the differentiated elements related to coelomic tissues. other types of migratory cell involved include phagocytes and granulocytes (“wanderzellen”, reichensperger, 1912). these latter are considered to be a source of putative growth factors. the growth processes are supported by extensive cell cycle activity as evidenced by brdu incorporation studies showing sites of extensive cell proliferation in the blastema and in the coelomic epithelium (candia carnevali et al., 1995, 1997, 1998). the nervous system with its differentiated components (ectoneural, entoneural, hyponeural) plays a crucial role in regeneration: this is related to its striking capacity to regenerate itself, to its pilot-action as a promoter/inductor of the overall regenerative processes, particularly for the development of the musculo-skeletal components, and finally to its contribution in terms of release of regulatory factors (candia carnevali et al., 1989, 1996; thorndyke and candia carnevali, 2001; patruno et al., 2002, 2003). a number of neurohumoral factors with paracrine or autocrine action are involved in regenerative development: neurotransmitters, particularly monoamines such as dopamine and serotonin; neuropeptides, such as substance-p, salmfamide 1 (s1), salmfamide 2 (s2), nerve-derived growth factors, particularly tgf-ß and related peptides (bmp), ngf, fgf-2. recent specific results are related to differential localization and levels of tgf-β1 and tgf-β-type ii receptor expression during regeneration in a. mediterranea (patruno et al., 2002), and to the cloning of native growth factors in crinoids and their implications for regenerative processes (patruno et al., 2003). in particular, the gene identified so far in antedon bifida, is a new member of the tgf ß superfamily, anbmp2/4, which shows a sequence similarity with other echinoderm and human bmps. according to the expression pattern of this gene a plausible role can be suggested in specification of migratory stem cells, blastemal growth and tissue differentiation, particularly skeletogenesis (patruno et al., 2003). it is relevant to remind that, in general, the active gradient established by bmp ligands is considered as one of the main factors responsible for 69 table 2. asteroids. summary of main cellular and molecular aspects of regeneration mechanisms responsible cells recruitment growth factors genes involved -morphallactic processes: substantial contribution of old tissues -no blastema nerve-dependent regeneration -dedifferentiated cells -stem cells (coelomocytes?) -dedifferentiation -(re)differentiation -transdifferentiation -extensive migration -limited proliferation -neurotransmitters (dopamine, serotonin) -neuropeptides (s1, s2) -arhox1 (homeoboxcontaining gene) generating the positional information during development, particularly in regeneration. visceral regeneration. if arm regeneration in crinoids can be considered the representative model of complete regeneration of a complex body part, the regenerative potential of the internal organs is well illustrated by the visceral regeneration process. in all crinoids, the loss of the visceral mass does not seem to be a very traumatic event and, in spite of the apparent complexity of the organs and tissues involved, can be easily repaired by prompt regeneration (dendy, 1886; clark, 1921; hyman, 1955). current research (dolmatov et al., 2003; mozzi et al., 2004) is actually focusing on the overall process of visceral regeneration (involving gut and associated tissues and organs) in a. mediterranea, a phenomenon which was explored in the past by the historical study by dendy (1886). the preliminary results collected so far show that visceral regeneration is a very rapid and effective process during which a small gut, functionally and anatomically complete, is reformed de novo in a loose context of new tissues (mainly coelomic cavities and hemal lacunae). in terms of responsible cellular elements, the migratory cells usually employed in regeneration (see arm regeneration) are involved (amoebocytes, coelomocytes, granulocytes, phagocytes). in addition, recent experiments on transplantation of the tegmen and related viscera also show an unexpected plasticity and adaptability of tissues and organs to extremely traumatic conditions. in terms of repair and reconstitutive processes at the tissue level, the mechanisms involved appear to be only partly comparable to those described in visceral regeneration: all the migratory cells seen above contribute to regeneration and are involved in mutual cell exchange between donor and acceptor tissues (mozzi et al., 2004). these results show a striking potential of cell plasticity and tissue histocompatibility in crinoids and confirm their remarkable repair/regenerative capabilities. on the whole, regeneration appears to be a quick and effective process when the mutilation involves a vital organ or the injury is particularly traumatic: in contrast, it seems to be a slower and less effective phenomenon when the part involved is not so indispensable for survival (reichensperger, 1912). interestingly, there is no apparent correlation between availability/assimilation of food and regenerative capabilities which seem to be comparably rapid and effective in both well-fed and starved animals. larvae: partial regeneration of body parts following traumatic amputations. surprisingly, larval regeneration was only rarely recorded in crinoids. the available data refer to phenomena of partial regeneration of body parts following traumatic amputations in both swimming and sessile larval stages (runnström, 1915, 1925), which have been re-explored also in recent preliminary studies (barbaglio et al., unpublished). nothing is known at the moment about possible phenomena of larval cloning, which is actually a field of topical interest and of expanding potential for future research. asteroids adults: regeneration of body parts ( arms) following selfinduced or traumatic amputation; regeneration of internal organs (pyloric caeca, cardiac stomach) following self-induced or traumatic mutilation; fission processes. the main cellular and molecular aspects of the regenerative processes are summarized in table 2. in asteroid arm regeneration (for instance in leptasterias hexactis and asterias rubens), no discrete blastema as a centre of cell proliferation is evident. this appears to apply to both post-traumatic and post-autotomic regenerations (mladenov et al., 1989; moss et al., 1998). typical morphallactic processes seem to be employed and most cell cycle activity is concentrated in the epidermal layer and in the epithelium of the coelomic canals, the two different populations of cells, epidermal and coelomicmesothelial, providing a significant contribution in terms of development/regrowth of the tissues. migratory cells (coelomocytes or others) recruited at distance contribute to a minor extent to tissue 70 regeneration. as noted above, the morphallactic process in asteroids is rather slow when compared to the epimorphic processes of ophiuroids and crinoids; in particular, the initial repair phase may last for a week or more depending on temperature and species, the cell cycle activity being rather low at this stage (moss et al., 1998) and more effective at more advanced stages of arm regrowth. it is also at this period that the regenerating tip begins to display the organization and features of an adult arm complete with distal optic cushion and terminal tentacle. the results obtained so far suggest: a) reversal of differentiation, proliferation and redifferentiation and/or b) direct transdifferentiation of committed cells which can switch from their ‘default' pathway to another and generate all the other cell types (moss et al., 1998). asteroid arm regeneration is a nerve-dependent process. in model species (asterina gibbosa), arm regeneration can not occur if the radial nerve has been removed and the neurotrophic action of the nervous system is needed throughout the whole course of regeneration (huet, 1975; huet and franquinet, 1981; thorndyke and candia carnevali, 2001). in some species, as stated above, following autoamputation isolated arms (comets) can regenerate to produce completely new adults. in coscinasterias muricata the phenomenon is reproducible in the lab: two isolated arms, including a minimum portion of the original central disk, are able to regenerate two complete new starfishes following a process which is perfectly simultaneous in both uniparental comets and similar in terms of regeneration rate and histological aspects (ducati et al., 2004). this phenomenon is regulated by both exogenous (seasonal changes, environmental stimuli) and endogenous factors (body size, humoral and nervous factors). recent results in asterias rubens (thorndyke et al., 2001a) give a further insight in the promising field of the molecular approach to echinoderm regeneration, with particular reference to the characterization and implication of homeoboxcontaining genes (arhox1) and their spatial and temporal expression patterns during starfish arm regeneration. larvae ( all stages): regeneration of body parts following traumatic amputation; processes of larval cloning . in asteroid larvae, post-traumatic regeneration was explored in several past and recent studies (hörstadius, 1925a, b, c, 1973; vickery and mcclintock, 2000; vickery et al., 2001). in some species (luidia foliolata), more substantial regeneration can take place whereby two new, completely independent larvae can be produced following surgical bisection of the original (vickery and mcclintock, 1998). in planktonic asteroid larvae, there is also convincing evidence for regeneration in terms of true asexual reproduction and potential for clonal growth (bosch, 1988; vickery et al., 2001; eaves and palmer, 2003). planktotrophic bipinnaria larvae of some starfish species have been shown to undergo clonal reproduction whereby posterolateral arms transform into new larvae and are released by fission, leaving the arm stump to regenerate in its entirety (bosch et al. 1989). in optimized laboratory conditions, up to 24% bipinnaria larvae of pisaster ochraceus undergo asexual reproduction (vickery and mcclintock, 2000); in samples of field collected larvae of other species up to 90% cloning was recorded (eaves and palmer, 2003). ophiuroids adults: regeneration of body parts (arms) following selfinduced or traumatic amputation; regeneration of internal organs (pyloric caeca, cardiac stomach) following self-induced or traumatic mutilation; fission processes. the main cellular and molecular aspects of the regenerative processes are summarized in table 3. ophiuroids also show a great capacity for regeneration, since they frequently cast off their arms at all levels, with breakage occurring at all the autotomy planes after damage, predation, or handling. regeneration is usually rather rapid and can involve several arms. the cellular aspects of the regeneration process in ophiuroids are poorly understood. most recent work has focused on the ecological benefits and impacts of the extensive regeneration seen in many species (stancyk et al., 1994; thorndyke et al., 2001b). these animals are certainly ideal subjects for studying the implications of regrowth on other physiological processes. they are also valuable models for experiments concerning the cellular basis of regeneration: present evidence suggests that a combination of morphallactic and epimorphorphic processes is responsible for regeneration in ophiuroids. thus morphallactic wound-healing processes involving initial expansion and migration of epidermal cells are followed by the rapid formation of an epimorphic blastemal structure due to a local accumulation of coelomocytes (dobson, 1988, thorndyke et al., 2001b; bannister et al., 2005). several specific studies on cell cycle activity (dobson 1988; thorndyke et al., 2001b) clearly indicate the importance of cell division in this regenerative process. moreover, the primary location of the blastema at the end of the severed nerve cord provides clear support for the idea of an important role for the nervous system in regrowth, as previously suggested by early studies (zeleny, 1903; morgulis, 1909;). recent molecular studies in amphiura filiformis (bannister et al., 2005) has led to identify and clone afuni, a novel tgf-ß gene, expressed in regenerating tissues and showing a sequence similarity with sea urchin univin (85 % identity). on the basis of the differential spatiotemporal expression of this gene in regenerating arm of advanced stages, a presumptive role in arm growth and segmentation, as well as in neurogenesis and skeletogenesis was suggested. larvae (pre-metamorfic stages): regeneration of body parts following traumatic amputation; 71 table 3. ophiuroids. summary of main cellular and molecular aspects of regeneration mechanisms responsible cells recruitment growth factors genes involved -epimorphic regeneration with main contribution of undifferentiated cells -blastema formation nerve-dependent regeneration -stem cells (coelomocytes) -dedifferentiated cells (? ) -dedifferentiation -(re)differentiation -transdifferentiation -extensive migration -extensive proliferation -neural factors (neuropeptides s1, s2) afuni (novel tgf-ß gene cloned) table 4. holothuroids. summary of main cellular and molecular aspects of regeneration mechanisms responsible cells recruitment growth factors genes involved -epimorphic-like regeneration with presumptive contribution of undifferentiated cells pseudo-blastema formation nerve-dependent regeneration -dedifferentiated cells (myocytes) stem cells (coelomocytes) -dedifferentiation -(re)differentiation -transdifferentiation -extensive migration -limited local proliferation -neural factors (?) -ependimin-related protein (?) epenhg (ependymi n-related gene) processes of larval cloning (secondary larvae) from larval posterolateral arms detached from primary ophiopluteus. in ophiuroids, larval regeneration has been described in pre-metamorphic stages. the most frequent phenomenon is the regeneration of secondary larvae from the posterolateral arms which are lost or released on settlement of the metamorphosed juvenile (mortensen, 1921; balser, 1996, 1998; eaves and palmer, 2003), which can even involve the continuous and progressive development of serial clones of regenerating larvae (tertiary larvae, etc.) in a sort of strobilation process. the overall phenomenon appears to involve also pseudogastrulation morphogenetic events, whereby cell proliferation, de-differentiation, migration and differentiation take place to produce larval clones (balser, 1998). holothuroids adults: regeneration of body parts and appendages (tentacles) following self-induced or traumatic amputation; regeneration of whole visceral mass or individual internal organs (gut, gonads, haemal system, respiratory trees, cuvierian tubules) following self induced or traumatic amputation; asexual fission processes. the main cellular and molecular aspects of the regenerative processes are summarized in table 4. in holothurians the regenerative potential can differ a lot between different groups (dendrochirota, aspidochirota, apoda) and also vary with the age of the individuals. since the first half of the 20th century, much attention has been focused on visceral regeneration (gut and related structures, bertolini, 1930; dolmatov, 1992; garcia-arraras et al., 1998; 1999; garcia-arraras and greenberg, 2001) and on muscle regeneration (dolmatov et al., 1996). visceral regeneration. recent work (garcia-arraras et al., 1998, 2001; garcia-arraras and greenberg, 2001; dolmatov, 1992, 1996) has indicated that in holothuria glaberrima, following evisceration the new digestive tube develops from the mesentherial lamina, which anchored the original gut to the body wall. this phenomenon is apparently a typical epimorphic process, where a blastema-like structure is formed as a thickening of the mesentherial edge of the lamina. in the following stages the regrowth of the intestinal tract can imply two alternative mechanisms of cell recruitment (garcia-arraras and greenberg, 2001): a) from the remnants of the oesophagus and cloaca, through morphallactic mechanisms of tissue 72 rearrangement and migration/proliferation of endodermally derived cells (garcia-arraras et al., 1998; mashanov and dolmatov, 2001); b) from coelomic epithelium, through direct and exclusive proliferation/migration of new mesodermally derived progenitor cells, this phenomenon typically occurring when endodermally derived tissues are completely lost by evisceration (mosher, 1956; mashanov et al., 2004). on the basis of indirect evidence, the overall process is considered to be a nerve-dependent regeneration. the employment of immunocytochemical methods underpin specific activities in the mesentherial septum: at early stages, extensive ecm (extracellular matrix) degradation (collagen, fibronectin, laminin) and metalloproteinase (mmps) activity or effects of their inhibitors; at advanced stages, extensive rearrangement of the ecm (collagen, fibronectin, laminin) and myocyte dedifferentiation (quinones et al., 2002; garciaarraras et al., 2001). although more specific mechanisms at cellular level have still to be explored, particularly by developing appropriate methods for cell culture (odintsova et al., 2005), a plausible interpretation about elements involved and their possible roles has been proposed, with particular reference to the fundamental role of the mesothelium and the organogenetic potential of the coelomic epithelium, which also implies a presumptive mesodermal derivation of the luminal epithelium of the new gut. recent work was carried out on the molecular aspects of regeneration and has allowed identification of at least one gene, epenhg (ependymin-related gene), which is expressed in many tissues and overexpressed during regeneration. this gene shows a sequence similarity with holothurian, sea-urchin and vertebrate (frog and mammalian) ependiminrelated sequences. in terms of specific roles, its presumptive involvement as promotor of proliferation and differentiation, neurotrophic factor and inductor of axonal regrowth was proposed (suarez-castillo et al., 2004) muscle regeneration. a specific aspect of regeneration which is extremely interesting per se in terms of morphogenetic and histogenetic processes and particularly relevant for the functional recovery of the injured individual, is the regenerative development of the muscles. this problem has been explored in holothurians, the echinoderm group in which muscles are most developed, as far as myogenesis of somatic and visceral muscles is concerned (dolmatov et al., 1996; dolmatov and ginanova, 2001). here the regeneration process appears to be due to the coelomic epithelial cells which apparently dedifferentiate, migrate and invade the muscle bands where they differentiate into muscle bundle rudiments, finally giving rise to new muscle cells. the myogenesis appears to start from two different cell precursors: with regard to somatic muscles, myogenesis starts from undifferentiated coelomocytes, in their turn derived from epithelial cells of the coelothelium (perytoneocytes) via migration, proliferation, differentiation; with regard to visceral muscles, from myocytes of the coelothelium, via partial dedifferentiation/transdifferentiation and migration (dolmatov and ginanova, 2001; murray and garciaarraras, 2004). on the basis of the resulting pattern in terms of occurrence and distribution of dedifferentiation, migration, proliferation and redifferentiation at tissue and cellular level, a basic mechanism for holothurian muscle regeneration is proposed which excludes a significant role for cell division but is based fundamentally on the recycling and transdifferentiation of cells from the coelomic epithelium. larvae (different stages): regeneration of body parts following traumatic amputations; processes of larval cloning. larval regeneration after surgical transection was previously described in holothurians in earlier and recent papers (hörstadius, 1925a, b, c, 1973; dolmatov, 1991, 1996). in eupentacta fraudatrix, the pentactula juvenile stage, after bisection, can regenerate the posterior part including the intestinal tract (mashanov and dolmatov, 2001). besides these processes of post-traumatic regeneration, frequent phenomena of larval cloning have been also observed (eaves and palmer, 2003), particularly development of secondary auricularia larvae (parastichopus californicus). in addition, asexual budding is also frequently observed in early pentactulae. echinoids adult: regeneration of external appendages (spines, pedicellariae) post-fracture, removal or self-induced release; regeneration of test after surgical treatment or traumatic mutilations. the main cellular and molecular aspects of the regenerative processes are summarized in table 5. echinoids show an apparent reduced requirement for regeneration as they do not reproduce by fission and do not show large exposed appendages vulnerable to predation. nevertheless, if arm regeneration is a typical feature of long-armed echinoderms, traumatic or self-induced loss and subsequent regeneration of external appendages, such as spines and pedicellariae, are frequently occurring in sea urchins and recent studies have concentrated on regeneration of spines and pedicellariae in the common sea urchin paracentrotus lividus (carpenter, 1847; dubois and ameye, 2001) after fracture or total removal. interestingly, a pattern emerges from the detailed comparative analysis of diverse situations, which suggests that the regenerative processes in these external appendages can vary significantly in terms of basic mechanisms and processes and can be modified in response to different types of injury. these results are strongly consistent with the idea of a fundamentally different significance of post-traumatic and post-autotomic regeneration and confirm the wide plasticity/adaptability of the repair and regenerative processes in echinoderms in relation to the specific conditions of local damage. 73 table 5. echinoids. summary of main cellular and molecular aspects of regeneration mechanisms responsible cells recruitment growth factors genes involved -morphallactic processes with substantial contribution of migratory cells -annular blastema, centripetal growth -dedifferentiated cells -stem cells (coelomocytes?) -dedifferentiation -(re)differentiation -transdifferentiation -extensive migration -limited local proliferation only a few reports have been focused on test regeneration after injury (bonasoro et al., 2004; ameye and dubois, 1995; shimizu et al., 1994) and the process has still to be explored in its detail. in terms of timing and modalities, sea-urchin test regeneration appears to vary a lot, depending on many intrinsic and extrinsic factors. nevertheless, the main events tend to follow a constant sequence: in paracentrotus, after surgical removal of a small test portion (one or two skeletal plates), the experimental animals are able to repair their wounds by employing regenerative mechanisms which involve three main stages (repair, early and advanced regeneration) and lead to the complete closure of the wound. the overall regeneration process implies the formation of an annular blastema and follows a concentric centripetal regrowth (bonasoro et al., 2004): the new tissues are progressively reformed due to the substantial contribution of migratory cells presumably derived through a morphallactic rearrangement of old tissues, with only a minor local employment of cell proliferation. larvae: regeneration of body parts after surgical division or amputation; larval cloning. the regenerative potential of sea-urchin larvae after surgical or traumatic amputation was described in historical and recent papers (runnström, 1915; 1925; hörstadius, 1925a, b, c, 1973; vickery et al., 1999, 2001). in lytechinus variegates, after bisection, different larval stages, particularly echinoplutei, can completely regenerate the anterior or posterior body portion (vickery et al., 2001). in addition, in different larval stages frequent phenomena of larval cloning by budding have been also described (for instance in strongylocentrotus purpuratus, eaves and palmer, 2003), leading also in echinoids to the regular formation of secondary larvae. concluding remarks the extensive and strategic employment of regenerative phenomena throughout the phylum indicates that in echinoderms regeneration actually represents an essential component of the life-cycle and has a wide range of relevant biological implications: not only it increases the potential of survival of the individuals, but it can also be considered the indispensable requisite for performing fissiparous reproduction, thus allowing the rapid colonization of new environments through the production of multiple copies of genotypes (clones) well adapted to local conditions. on the basis of its evident contribution to the adaptive capacities of the species and of its biological and ecological implications for echinoderm phylogeny the regeneration phenomenon must be considered one of the most important responsible factor for the evolutionary success of the groups and for the striking success of the whole echinoderm phylum throughout the marine ecosystem. unfortunately what we actually know about the biology of echinoderm regeneration is far to be exhaustive. much work is requested and many questions are still to be answered. results obtained so far strongly encourage a wider employment of molecular approaches in regeneration research (odelberg, 2004) and promote the hope that, with the powerful tools provided by molecular biology, the key of the striking regenerative performances of echinoderms can be soon appropriately studied and completely explained. we should not forget that what we learn from echinoderms can be relevant in applied research: regenerative medicine can in fact receive a significant improvement if echinoderm models are extensively studied in parallel with traditional mammal models, in the reasonable hope that what echinoderms can do so easily may eventually become easy also for other animals, humans included (lagasse et al., 2001). acknowledgements this work was carried out thanks to the support of a number of specific projects financed through the years by cnr, murst (cofin) and university of milano (first) programs. the author is particularly grateful to the following colleagues for their indispensable help and valuable collaboration during the research work: a barbaglio, a biressi, f bonasoro, c di benedetto, d mozzi, l parma, m patruno and m sugni. 74 references ameye l, dubois p. resorption and calcification during regeneration of echinoid test. in: emson rh, smith ab, campbell ac (eds), echinoderm research 1995, rotterdam, balkema, pp 231-235, 1995. amemiya s, oji t. regeneration in sea lilies. nature 357: 546-547, 1992. balser ej.mortensen vs mcbride: evidence for asexual reproduction in ophiuroid larvae. am.zool. 36: 70a, 1996 balser ej. cloning by ophiuroid echinoderm larvae. biol. 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and physiological events. here, we characterized a ste20-like kinase gene (djslk) in planarian dugesia japonica. whole-mount in situ hybridizations revealed that djslk was expressed in the central nervous system (cns) including cephalic ganglia and ventral nerve cords (vncs) in intact and regenerating animals. after rna interference (rnai) of djslk, adult planarians became immobilized and wrinkled, then swelled and lysed eventually. djslk rnai treated regenerating planarians could form the entire animals, and then displayed the similar phenotype transformation. these results suggest that loss of function of djslk leads to morphogenetic malformation of planarian d. japonica probably via regulating cell volume instead of disrupting the balance between cell proliferation and apoptosis. key words: cns; djslk; expression; morphogenetic malformation; cell volume introduction endowed with abundant pluripotent stem cells named neoblasts which proliferate and differentiate to all cell types in response to amputation and/or injury ， planarians can regenerate the complete worms with all organs from almost any tiny fragments (newmark and sánchez alvarado, 2002; reddien and sánchez alvarado, 2004). neoblast is the only source to provide new cells during turnover and regeneration (wagner de et al., 2011). elimination of neoblasts by irradiation, planarians demonstrate the typical phenotype defects such as head regression, dorsal curling and lesions during homeostasis and fail to shape the whole animals for amputated pieces (newmark and sánchez alvarado, 2002; reddien and sánchez alvarado, 2004; saló et al., 2009; scimone et al., 2010). planarians can acquire similar phenotypes by silencing some genes related to neoblast maintenance, proliferation, differentiation, and apoptosis (reddien et al., 2005; guo et al., 2006; pearson and sánchez alvarado, 2010; li et al., 2011). ___________________________________________________________________________ corresponding author: bosheng zhao school of life sciences shandong university of technology 266 xincun w estern road, zibo 255049, p.r. china e-mail: zhaobosheng@sdut.edu.cn sterile 20 (ste20) is originally found as mitogen-activated protein kinase kinase kinase kinase (map4k) involved in the mating response of haploid yeast saccharomyces cerevisiae (wu et al., 1995). its homologs in other organisms form the ste20-like kinase (slk) superfamily and are mainly regarded as upstream regulators of the mapk pathways (dan et al., 2001; ling et al., 2008). ste20-like kinases play crucial roles in various cellular processes including cell growth, cell migration, apoptosis, cell-cycle control, cell shape change and stress responses (dan et al., 2001; strange et al., 2005; ling et al., 2008). in this paper, we identified a slk gene in planarian dugesia japonica and studied its tempospacial expression pattern and loss-of-function phenotype in intact and regenerating animals. materials and methods animals the planarians dugesia japonica collected in boshan, shandong, china, are maintained in autoclaved tap water at 20 ℃ and 6 10 mm long animals were starved for at least one week before use in experiments. cloning and sequence analysis of cdna the djslk cdna was derived from random mailto:zhaobosheng@sdut.edu.cn 2 sequencing of a planarian cdna library. comparison against the genbank protein database was performed using the blast network server at the national center for biotechnology information (ncbi). multiple protein sequences were aligned using the megalign program by the clustal method in dnastar software package (burland, 2000). whole-mount in situ hybridization whole-mount in situ hybridizations were performed as described previously (pearson et al., 2009). the digoxigenin (dig)-labelled antisense rna probes were synthesized in vitro. hybridizations were carried out at 56 ℃ for 17 h, the bcip/nbt mixture solution (roche) was used for color development. for regeneration experiments, animals were amputated preand post-pharyngeally and left to regenerate, the head-, trunk-, and tail-pieces were collected at the times indicated. quantitative real-time pcr quantitative real-time pcr was used to monitor the quantitative expression of the djslk as described previously (yu et al., 2015) in intact planarians, regenerating trunk fragments， and regenerating trunk fragments of rnai-treated planarians at different times after amputation. the cdna was synthesized using a first-strand system kit from invitrogen after total rnas were extracted using rnaiso reagent (takara). qpcr reactions were performed using fast start universal sybr green master (rox) (roche, switzerland) according to the manufacturer’s protocol. three samples for each condition were run in parallel by a 7,500 real time pcr system (applied biosystems). data were normalized to the expression of the internal control djef2. the following sets of specific primers were used: djslk mrna, 5′-cgaaggacaaaggcacat-3′, 5′-gagcgaacaccaggaact-3′. djef2 mrna, 5 ′ t t a a t g a t g g g a a g a t a tg t t g 3 ′; 5 ′ g t a c c a t a g g a t c t g a t t t t g c 3 ′. the data were analyzed using spss 16.0 software. the significance of differences was analyzed by one-way analysis of variance (anova) followed by a tukey's post-hoc analysis to identify differences between the experimental and intact planarians. data presented are means ± sd. values of p < 0.05 were considered to be significant. rna interference djslk was cloned into the l1440 plasmid with two t7 primers, and then dsrna was synthesized according to the manufactrue’s instructions (megascript® rnai kit). animals were injected at day 0, day 2, 4, and 6. for regeneration studies, animals were cut at day 10. control animals were injected with deionized sterile water. immunostaining animals were killed in 2 % hcl for 5 min at rt and fixed in 4 % paraformaldehyde solution for 3 h at 4 °c, then dehydrated in 100 % methanol solution for 1 h at -20 ℃. the following procedures were processed as described elsewhere (robb and sánchez alvarado, 2002; inoue et al., 2007; cebria, 2007, 2008). the primary antibody anti-synorf1, a mouse monoclonal antibody specific for synapsin (developmental studies hybridoma bank) was used at a dilution of 1:25. the secondary antibody dylight 594 affinipure goat anti-mouse igg(h+l) (earthox) was used at a dilution of 1:200. results sequence analysis of djslk the cdna clone obtained from planarian d. japonica cdna library is about 1,100 bp with the longest open reading frame of 879 bp. it encodes for a deduced protein of 292 amino acids with predicted molecular mass of approximately 32.4 kda (fig. 1) fig. 1 the nucleotide and deduced amino acid sequence of djslk. 3 fig. 2 alignment of subdomain xi of ste20-like kinases, including djslk using the megalign program (dnastar) by the clustal w method. shaded (with solid black) residues are the amino acids that match the consensus. genbank accession numbers: aplysia californica (xp_005111485.1), ailuropoda melanoleuca (xp_002914907.1), crassostrea gigas (ekc21462.1), canis lupus (xp_003433112.1), clonorchis sinensis (gaa52112.1), drosophila melanogaster (nm_142339.2), danio rerio (xp_005165982.1), felis catus (xp_003980538.1), latimeria chalumnae (xp_005992350.1), loa loa (ejd76726.1), schistosoma japonicum (cax753595.1), salmo salar (aci33699.1). initial blastp search at ncbi revealed that this gene belongs to ste20-like kinase. however, its 5′ end is missed and only subdomain xi is entire (hanks and hunter, 1995). the deduced amino acids aligned with other subdomain xi of ste20-like kinases showed that djslk shares 39.2 % 72.2 % similarity with its homlogs in other organisms (fig. 2). ste20 kinases consist of the p21-activated kinase (pak) and germinal center kinase (gck) families according to the relative location of kinase domain, these two families can be further subdivided into pak i and pak ii and gck i to viii subfamilies, respectively (dan et al., 2001; strange et al., 2005; ling et al., 2008). due to loss of most subdomains, the closest homlogs can not be ascertained and the gene is termed ste20 like kinase (djslk) in this study. djslk expression pattern in adult and regenerating planarians in order to analyse the expression pattern of the planarian djslk gene we performed whole mount in situ hybridization on intact and regenerating animals. in intact planarians, djslk was expressed in central nervous system which possesses an inverted u-shaped pair of cephalic ganglia and two longitudinal ventral nerve cords that project posteriorly along the worm (cebrià et al., 2002; cebrià, 2007; agata and umesono, 2008) (fig. 3b). and djslk localized in both the central spongy region and the lateral branches in the cephalic ganglia (fig. 3b). in regenerating animals, djslk transcripts could always be detected in cns in the head-, trunk-, and tail-pieces. during the initial regeneration stages after amputation, djslk expression was not detected fig. 3 expression of djslk in intact planarian (a) an intact planarian processed and hybridized using the djslk sense probe. no signal was seen in the control. (b) ventral view of intact planarian, expression of djslk is mainly present in the cns. anterior is to the left. scale bar: 500 μm. 4 fig. 4 expression of djslk during regeneration. expression of djslk in regeneration of day 1 (a c), day 3 (d f), day 5 (g i), day 7 (j l), and day 9 (m o) after amputation. djslk is detected in the pre-exiting and newly regenerated cns. anterior is to the left. scale bar: 300 μm. within the head and tail blastema, but it was detected in the preexisting cns (figs 4a c). at 3 and 5 days of regeneration, new neural cells in front of the commissure differentiated, and cns recovered most of its function (cebria, 2007). djslk expression was detected in the preexisting and newly regenerated cns (figs 4d i). with the development of regeneration, the original expression was gradually reestablished (figs 4j o). the relative quantitative real-time pcr analysis was performed to investigate the change of expression of djslk mrna during planarian regeneration. we examined rna samples from normal intact planarians and trunk fragments regenerated for 1 day, 5 fig. 5 qrt-pcr analysis of djslk expression in intact and regenerating truck fragments at 1, 3, 5, 7, 9 days after amputation. data was expressed as the ratio of djslk to djef2α mrna. error bars represent the  sd for three independent pcr amplifications and quantifications. *p < 0.05 or **p < 0.01 compared to control intact planarians. fig. 6 qrt-pcr analysis of djslk rnai efficiency in adult intact planarians. error bars represent the  sd for three independent pcr amplifications and quantifications. **p < 0.01 is the comparison between control intact animals and rnai intact animals. template 6 fig. 7 abnormal appearance change in intact (b-e) and regenerating planarians (b′-e′) after djslk rnai. (a) control animal, microinjection of water in adult animal after 2 months. (b-e) after rnai, planarians became immobilized and wrinkled (b), swelled (c), lysed (d), and died (e). (a′) contral animal, day 30 of regeneration after amputation. (b′-e′) the appearance transformation in regenerating trunk fragments after rnai-treated planarians. anterior is to the top. scale bar: a e = 800 μm, a′e′ = 200 μm. 3 days, 5 days, 7 days, and 9 days, respectively. the resluts indicated that djslk mrna was gradually increased during regeneration compared to normal intact planarians and achieved to the maximal leval at 7 days (p < 0.01) after amputation. then it declined to almost normal level at regeneration day 9 (fig. 5). djslk rnai induces morphogenetic defects and death of planarians to study the role of djslk gene in the homeostasis and regeneration of the planarian, we knocked down the endogenous expression of djslk using rnai. real-time pcr analysis of djslk mrna showed that the rnai-treatment efficiently down-regulated the expression of djslk in intact planarians ( p< 0.01) (fig. 6). after 5 days of injecting djslk dsrna, the intact animals became immobilized and wrinkled (fig. 7b, n = 6/13). then the planarians swelled and started to lyse at day 9 and day 15, respectively (figs 7c and d, n = 6/13). and the animals completely lysed at about 25 days after the treatment (fig. 7e, n = 6/13). in contrast, control animals lived without any abnormal change even for 2 months (fig. 7a, n = 10/10). djslk rnai treated trunk fragments could regenerate the whole bodies, and then showed the same appearance change starting at 14 days after amputation (figs 7b’ e’). immunostaining with an anti-synorf1 antibody against synapsin revealed that djslk rnai didn′t interfere with cns intactness and regeneration (fig. 8). discussion ste20 kinases function in morphological events in different organisms (strange et al., 2005). in yeast mating pathway, cell shrinkage activates ste20 kinase and suppresses mating defects (strange et al., 2005). one ste20 kinase named proline-alanine-rich ste20-related kinase (pask) is strongly expressed in neurons and transporting epithelia in rats (ushiro et al., 1998). pask can interact with and phosphorylate na-k-2cl (nkcc) and k-cl (kcc) cotransporters to regulate cell volume (piechotta et al., 2002; dowd and forbush, 2003; strange et al., 2005). during cell swelling, loss of kinase activity of pask results in dephosphorylation of both cotransporters which lead to inhibit nkcc and activate kcc (strange et al., 2005). in this study, djslk was expressed in cns and rnai enventully induced swelling and lysing of planarians. and the defects probably resulted from cell swelling and osmotic lysis after loss of kinase 7 fig. 8 immunostaining with anti-synorf1 in djslk-rnai-treated planarians during homeostasis (a b and a' b') and regeneration (c d and c' d'), the defects of cns including brain and vnc were not detected. (a) intact planarian was injected with water as control. (b) djslk-rnai-treated intact planarian at 9 days. (c) immunostaining of normally regeneration of the cutting head sample was detected at 15 days after amputation. (d) djslk-rnai-treated intact planarian was cut head to regenerate at 15 days. a ′d′ the magnification of head in a d, respectively. anterior is to the up. scale bars: a d = 500 μm; a′ d′ = 100 μm. activity of djslk. meanwhile, the irradiation-treated typical phenotype change did not occur in intact planarians, and amputated fragments could regenerate the whole bodies after silencing djslk. all these results suggest that, just like pask, djslk regulates cell volume instead of involving in neoblast maintenance, proliferation, differentiation, and apoptosis like other neoblast-related genes. acknowledgments we thank dr. yuqi huo from translational medicine center, the sixth people′s hospital of zhengzhou, for reading and discussing the manuscript. this work was supported by the national natural science foundation of china (grant numbers 31572263, 31172074) and shandong province natural science foundation of china (grant number zr2013cm011). references agata k, umesono y. brain 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yadav s, hertz nt, oses-prieto ja, claxton s, burlingame al, et al. mst3 kinase phosphorylates tao1/2 to enable myosin va function in promoting spine synapse development. neuron 84: 968-982, 2014. wagner de, wang ie, reddien pw. clonogenic neoblasts are pluripotent adult stem cells that underlie planarian regeneration. science 332: 811-816, 2011. wu c, whiteway m,thomas dy, lerberer e. molecular characterization of ste20p, a potential mitogen-activated protein or extracellular signal-regulated kinase kinase (mek) kinase kinase from saccharomyces cerevisiae. j. biol. chem. 270: 15984-15992, 1995. yu s, chen x, yuan z, zhou l, pang q, mao b, et al. planarian myosin essential light chain is involved in the formation of brain lateral branches during regeneration. mol. genet. genomics 290: 1277-1285, 2015. isj096.pdf 32 isj 2: 32-40, 2005 issn 1824-307x review flow cytometry as a tool for analysing invertebrate cells a cossarizza1, m pinti1, l troiano1, el cooper2 1 department of biomedical sciences, university of modena and reggio emilia, modena, italy 2 department of neurobiology, ucla school of medicine, los angeles, ca, usa accepted april 8, 2005 abstract flow cytometry (fcm) is a powerful tool that allows analysis of thousand of cells in a few seconds, at the single cell level. in the last 15 years, researchers have used fcm to investigate the cellular machinery of invertebrates. analyses have focused on functions linked to innate immunity, such as phagocytosis and natural killer cell activity, as well as on the sensitivity of invertebrate cells to a particular stress or to a toxic agent. further, fcm has been employed to recognize antigens, or at least immunodominant epitopes, shared in common with mammalian cells, including human leukocytes. in this review, main studies that have utilized fcm to investigate either phenotype and functions of invertebrate cells are reported and discussed. keywords: invertebrate; flow cytometry; immunology introduction: diversity of approaches and principles of flow cytometry (fcm) recently, a consistent number of scientists working in different fields have utilized flow cytometry (fcm) for numerous analyses. fcm has indeed impacted both basic cell biology and clinical medicine in a significant manner, and instruments are present almost everywhere. according to the essential principle of fcm, single cells (or particles, or organelles) suspended within a stream of liquid pass through a light source (usually a laser beam tuned at 488 nm) focused on a minute region. during this passage, cells are interrogated individually thus providing relevant numbers of information. basically, signals generated by cells passing through the laser beam are spectral bands of light in the visible spectrum, which represent the detection of various chemical or biological components, mostly fluorescence. since flow cytometers can analyze single particles or single cells, it is possible to separate them into populations based upon differences in any of the variables that can be measured on each particle/cell. corresponding author: andrea cossarizza dept. biomedical sciences, university of modena and reggio emilia,via campi 287, 41100 modena, italy. e-mail: cossarizza.andrea@unimore.it currently, the number of these variables is consistently high, and several instruments allow identification of more than a dozen different parameters in the same particle/cell. it is then possible to separate these populations electronically, and to identify them using multivariate analysis techniques. fcm uses several fluorescent molecules that are attached by one means or another to the particle or cell of interest. the fluorescent probe might be membrane bound, cytoplasmic, or attached to nuclear material. to recognize specific receptors present on the plasma membrane, as well as to identify intracellular antigens or to calculate the amount of a given molecule within a cell, it is a common practice to use monoclonal or polyclonal antibodies directly conjugated to fluorescent dyes. particles of almost any nature can be evaluated by flow cytometry. they can be very small, even below the resolution limits of visible light, because they can be detected by their fluorescent signatures. similarly, depending on the structure of the flow cell and fluidics, particles as large as several thousand microns can be evaluated. fcm allows the evaluation of thousands of events in a very short time. for example, the most common instruments can detect hundreds of cells per second, measuring up to 5 parameters; sophisticated systems exist that can run particles at rates approaching 100,000 events per second while collecting 10 to 20 parameters from each particle. finally, it is possible to separate single particles/cells physically from mixed 33 populations and, by using the sorting option eventually present in the instrument, to place them into a defined location for cloning, or for further molecular analysis. fcm analysis of cells from planorbarius corneus using antisera to vertebrate bioactive peptides the first studies that employed fcm in invertebrates were devoted to the analysis of epitopes/molecules present in cells from the mollusc p. corneus that were able to cross-react with human molecules. the starting point was represented by immunohistochemical data, when the attention was devoted to the detection of immunoreactive molecules in round and spreading hemocytes of p. corneus (ottaviani and cossarizza, 1990). antisera to vertebrate (mainly human) bioactive peptides were used, and it was found that spreading hemocytes had alpha 1-antichymotrypsin-bombesin-, calcitonin-, cck-8 (inc)-, cck-39-, gastrin-, glucagon-, metenkephalin-, neurotensin-, oxytocin-, somatostatin-, substance p-, vip-, and vasopressin-immunoreactive molecules, while round hemocytes were only positive to anti-bombesin, anti-calcitonin, anti-cck-8, anticck-39, anti-neurotensin, anti-oxytocin, antisubstance p and anti-vasopressin antibodies. fcm, whose sensibility is clearly much higher than that of a microscope, was then used to confirm the presence of acth and â-endorphin immunoreactive molecules in the same invertebrate (ottaviani et al., 1990). in the hemolymph of these animals, two main types of cells exist, i.e. round (rh) and spreading hemocytes (sh) (ottaviani, 1991). sh but not rh were positive to antisera recognizing vertebrate acth and âendorphin. interestingly, only by means of analyzing physical characteristics (dimension, granularity) of cells from invertebrate hemolymph, fcm allowed identification of the same cell types that had been described morphologically long before (fig. 1). interestingly, similar data on the presence of acthimmunoreactive molecules were obtained in another mollusc, such as lymnea stagnalis (ottaviani et al., 1991), and in leukocytes from the frog rana esculenta (ottaviani et al., 1992). the functional capacity of p. corneus spreading hemocytes, along with the presence of acth-like molecules (i.e., reactive with antibodies directed against human acth) in other species, including carassius carassius var. auratus and coris julius (pisces), hymenochirys gillii (amphibia), podarcis muralis (reptilia), and gallus domesticus (aves) suggested that cells with phagocytic activity, likely the oldest natural immune response, may represent a suitable model to unravel the tangled web of the common ancestor of the immune and the neuroendocrine systems. evidence of cytotoxicity in molluscs the investigation on natural immunity in p. corneus continued by evaluating the capacity of its cells to kill targets in a non-mhc restricted manner (franceschi et al., 1991). natural killer (nk) cells represent an important form of cell recognition that results in cytotoxicity, and it was predicted that nk-like activity had to be preserved throughout phylogenetic development (cooper et al., 2002). nonadherent round hemocytes were able to lyse the k562 human target cell line in a short-term nk cytotoxicity test. this nk-like activity, severely reduced after 18 hour incubation at 24° c, was maintained by the presence of human recombinant interleukin-2 (il-2). a further analysis performed by mouse anti-human monoclonal antibodies and cytofluorimetric analysis revealed that both sh and rh reacted with several antibodies, including those directed against epitopes typical of mammalian nk cells, and against different cell adhesion molecules (fig. 2). these data suggested the hypothesis that a primitive nk-like activity appeared early in evolution, and is not shared by phagocytic cells. possible role of interleukins a support for this hypothesis came from the cytofluorimetric observation that il-2 could compete with corticotropin releasing factor (crf) by binding to the receptor that is able to bind human il-2, present on the plasma membrane of molluscan hemocytes (fig. 3). at the functional level, pre-incubation of hemocytes with il-2 or anti-il-2 monoclonal antibody (mab) significantly reduced or completely eliminated the crf-induced release of biogenic amines. these findings are compatible with the presence of a unique (ancestral?) receptor on molluscan hemocytes, capable of binding both crf and il-2, two key molecules of the neuroendocrine and immune system (ottaviani et al., 1994). subsequently, using fcm it was observed that spread cells present in the hemolymph of the mussel mytilus galloprovincialis expressed three il-2 receptor (il-2r) subunits: á, â and ã. mussel il-2rá and il2râ subunits displayed a molecular weight similar to those in vertebrates tissues, whereas mussel il-2rã was smaller than that in vertebrates. both lipopolysaccharide (lps) and il-2 induced il-2rá expression, and such induction depended on the concentration of both agonists (barcia et al., 1999). stimulation with lps, il-2 or platelet-derived growth factor also resulted in an increased release of dopamine, adrenaline and noradrenaline in the culture medium (cao et al., 2004). identification of cell shape and surface antigens in other species, and relevance to tumor cells and environmental chemicals fcm proved to be a powerful technique for the analysis of the heterogeneity of cell populations in a variety of invertebrates. studies were performed on hemocytes derived from individual l. stagnalis, and revealed that cell sizes were comparable in all 40 specimens studied, and was not affected by age or by infection with trichobilharzia ocellata (amen et al., 1992). a monoclonal antibody of the clam was generated to normal hemocytes of mya arenaria (white et al., 1993). the antibody, designated 2a4, was evaluated by elisa, immunocytochemistry, western blotting, and finally fcm. m. arenaria is a very interesting model, because it develops a leukemia 34 fig. 1 presence of immunoreactive acth molecules on the cell surface of spreading (sh) and round (rh) hemocytes from planorbarius corneus and from lymnea stagnalis. a= control, b= stained cells. modified from ottaviani et al., 1991. fig. 2 expression of epitopes recognized by mouse anti-human monoclonal antibodies in round (rh) or spreading (sh) hemocytes from planorbarius corneus. a= control, b= stained cells. modified from franceschi et al., 1991. 35 fig. 3 expression of il-2 like molecules on the plasma membrane of planorbarius corneus revealed by anti-il-2 mabs, and inhibition of the staining with exogenous il-2 or crf. modified from ottaviani et al., 1994. detected first in the hemolymph and, as the disease progresses, in solid tissue. the 2a4 antigen was detected on 87 % normal adherent cells, but was lost as nonadherent leukemia cells proliferated. the mature leukemia cell neither expresses 2a4 nor can 2a4 be detected in the leukemia cell lystate. since 2a4 reacts with a 130-kda protein, it was suggested that p130 may be involved in the regulation of cell adhesion. subsequently, the same investigators evaluated the reactivity of both normal and tumor cells from m. arenaria to a polyclonal antibody to polychlorinated biphenyls (pcbs) (harper et al., 1994). fcm was indeed used to ascertain both leukemia prevalence and pcb reactivity. analytical chemistry was used to quantitate the amount of aroclor 1254 (a widely studied pcb), per tumor cell population and compared directly to fcm results. the prevalence of leukemia consistently exceeded 60 % when clams were retrieved from new bedford harbor, a site heavily contaminated with pcbs. both normal circulating cells and tumor cells were extremely reactive with the pcb antibody. when clams from two other sites were compared with clams from new bedford harbour, both disease prevalence and cell reactivity to the pcb antibody were significantly reduced. this study was the first that demonstrated the presence of pcbs in cell populations of marine invertebrates, and showed that the presence of pcbs in vivo was directly correlated with environmentally linked leukemia. exposure to ubiquitous environmental chemicals, such as polycyclic aromatic hydrocarbons, may affect the earthworm cellular immune-defense system. after exposure to soil contaminated with 7,12dimethylbenzanthracene (dmba), cellular functions of coelomocytes from the earthworm eisenia fetida were examined by flow cytometry, that allowed to classify them as small and large cells (komiyama et al., 2004). sexually mature animals were kept in dark at 18° c with various doses of dmba contaminated artificial soil for 7 days. coelomocytes were harvested from earthworms and processed for in vitro phagocytosis and h2o2 activity. phagocytosis was assessed by ingestion of fluorescence beads and h2o2 activity examined using 2’,7’-dichloro-fluorescin diacetate. cell functions were down regulated in a dose dependent manner after exposure to sublethal doses of dmba. this study demonstrates the utility of flow cytometry to evaluate the biological activity of earthworm cells, and the effect of soil contamination for ecotoxicological studies. age-related changes in the immunocyte population in m. galloprovincialis have been reported (ottaviani et al., 1998). in this mollusc, a young and an old stage belonging to the same cell type have been described. interestingly, either in young or adult animals these cells were positive to the staining with mouse anti-human mabs anti-cd5, -cd11b and cd16. other molecules, such as cd10, a surface antigen known to be identical to neutral endopeptidase-24.11 (nep), have been recently found in immunocytes of m. galloprovincialis (caselgrandi et al., 2000). computer-assisted microscopic image analysis revealed nep functional activity, i.e. the capacity to deactivate the pdgf-aband tgf-â1induced changes in immunocyte shape. cytotoxicity in earthworms: the model of eisenia foetida a cytometric approach has been used to investigate the main features of natural cytotoxicity in coelomocytes from e. foetida, that were used as effector cells against the human tumor target cell line k562 (cooper et al., 1995). to first assess the viability of effectors, dna synthesis was tested and was higher in autogeneic (cells from one animal) than in 36 fig. 4 allogeneic inhibition of cell proliferation. left panel: cell viability in freshly collected coelomocytes from eisenia fetida. right panels: a, proliferation of coelomocytes from single animals or pooled from different animals after stimulation with phytohemagglutinin (pha), concanavalin a (cona) or inhibition with mitomycin-c (mmc). note that pooled cells showed an allogeneic inhibition. b and c: cytofluorimetric analysis shows the comparison between unstimulated cells from a single animal (b) or cells collected and pooled from different animals (c). note the difference in the percentages of cells in the s or g2/m phases of the cell cycle. modified from cossarizza et al., 1996. fig. 5 expression of epitopes recognized by mouse anti-human monoclonal antibodies in small and large cells from eisenia fetida. k= control, unstained cells. modified from cooper et al., 1995. 37 allogeneic (cells from two animals) coelomocytes. cell cycle analysis revealed that autogeneic cultures showed significantly greater numbers of cells in s, g2, or m phases than allogeneic ones (fig. 4). when autogeneic or allogeneic cells were kept together in culture, no significant cell killing occurred in either, while autogeneic but not allogeneic cultures could kill k562 target cells. cytotoxicity was dependent upon membrane binding between small, electron-dense coelomocytes and targets; it was enhanced by adding the lectin phytohemagglutinin. the heat labile supernatant from autogeneic but not allogeneic cultures killed k562 targets. recognition of, binding to, and killing of foreign cells in a natural killer cell-like reaction may reflect natural immunity in earthworms. subsequently, it was found that earthworm coelomocytes effect cytotoxicity at significantly high levels also against the nk-resistant target cells (such as the u937, bsm, and cem cell lines) (cossarizza et al., 1996). then, using fcm and mouse anti-human monoclonal antibodies, the two main types of cells present in e. foetida coelom were better characterized: small (8-11 µm), electron-dense cells (sc) were: cd11a+, cd45ra+, cd45r0+, cdw49b+, cd54+, hla class ii (dr)+, â2-µ+ and thy-1+ (cd90+) (fig. 5); large (12-15 µm), electron-lucent cells (lc) were negative for these markers. both cell types were negative for other cd and mhc class i and class ii markers. subsequently, it was found that coelomocytes were positive to the staining with rat anti-mouse mabs for cd90, cd5, cd8, cd45ra, cd45r0 and anti-perforin, negative for ia, cd4 and cd11c (komiyama et al., 2002). in general, only sc and to a lesser extent, lc reacted with these antibodies. sc were active during recognition, rapidly binding to targets; lc were phagocytic (cossarizza et al., 1996). sc were able to exert rapid, significant, and equal levels of killing at 4, 20, and 37°c, suggesting that, as for phagocytosis, also primitive nk-like activity appeared early in evolution. fcm can be used to analyze cell organelles and different cellular functions the presence and functionality of mitochondria in terms of mass and membrane potential (mmp) have been investigated in both sc and lc from e. foetida at the single cell level by different fcm techniques (cossarizza et al., 1995). in comparison with sc, lc have a higher number of mitochondria, and, accordingly, showed a greater fluorescence intensity when mitochondrial mass was measured by nonyl acridine orange. to measure mmp, both the lipophilic cationic probe jc-1 and rhodamine (rh) 123 were used. using jc-1, mmp was analyzed separately on sc and lc, and significant percentages of coelomocytes (> 95 % of sc and about 90 % of lc) displayed a high mmp. adding 0.1 µm valinomycin (val) caused most sc to depolarize, while this occurred in only a few lc. rh123 gave different results: no effects of val were observed either in sc or in lc. it was concluded that in coelomocytes there may be several energy-independent rh123-binding sites, and that it is possible to analyze mitochondrial parameters by fcm in intact invertebrate cells. earthworms possess also non-specific responses found in other complex metazoans. coelomocytes from the e. foetida were further characterized by electron microscopy and fcm analyses, and the structural changes that occur when effector coelomocytes and target cells interact during cytotoxic activity were deeply investigated (quaglino et al., 1996). it was found that using in vitro cultures: 1) the two aforementioned earthworm cell types retained their morphological features; 2) their dna content was significantly less than that of human lymphocytes and the erythromyeloid human tumor cell line k562; 3) significant percentages of coelomocytes were found to be in s or g2/m phases of the cell cycle. it is noteworthy that these two parameters were investigated by direct staining of dna with fluorescent probes, as well as by the classical staining with bromodeoxyuridine (dolbeare et al., 1983). when cultivated alone, coelomocytes formed no aggregates. however, when mixed with k562 target cells, as described above, coelomocytes spontaneously killed tumor cells, and cytotoxic reactivity was accompanied by the formation of multiple aggregates similar to granulomas. these results are the first to describe this type of earthworm non-specific "inflammatory" response in vitro against xenogeneic tumor cells. fcm analysis of dna content and cell cycle fcm has been used to quantify dna content in invertebrate cells (ulrich, 1990). cell material from the diptera species chironomus thummi, drosophila melanogaster, calliphora vicina and musca domestica was analyzed using an impulse cytophotometer with a special quartz objective, that was especially manufactured for cytofluorometric investigations with the dna-specific fluorochrome dapi in combination with the protein fluorochrome sulforhodamine 101. the occurrence of heterogenous cell populations with aneuploid and polyploid dna content within the cell material of different developmental stages of diptera species have been determined, whereby in larvae polyploid cell populations and in imagos aneuploid cell populations predominate. more recently, studies were carried out to determine the alteration in dna cell cycle characteristics of hemocytes from m. galloprovincialis collected at 17 different locations along the adriatic coast, croatia (bihari et al., 2003). fcm was used to connect possible genomic manifestation to urban and/or industrial waste, and the incidence of altered dna profiles was investigated. different alterations in cell cycle, mirroring either acute or cumulative genotoxic effects of the surrounding environment on mussel hemocyte dna, were found. among these, the most relevant were intraindividual genome size variability, aneuploidy and accidental apoptotic processes; normal cell cycle dna profiles were obtained in 60.9 % individuals from all 17 sites. molecular assays were used along with cytofluorimetric analysis of cell cycle, and confirmed the presence of dna damages, such as alkali-labile sites and single-strand breaks, interstrand cross-links and dna-protein cross-links (bihari et al., 2002). 38 a cell line from the insect spodoptera frugiperda (sf9) was used to investigate the capacity of azadirachtin to alter the mitotic index (salehzadeh et al., 2003). fcm demonstrated that cells accumulated in the g2/m phase of the cell cycle, and that the effect was concentration-dependent. azadirachtin had the same effect on c6/36 mosquito cells, but failed to affect l929 murine fibroblasts even at high concentrations. experiments with colchicine and taxol showed similarities of action between azadirachtin and colchicine, and azadirachtin was apparently able to displace colchicine from specific binding-sites present in living insect cells. in vitro analysis of the effects of 20hydroxyecdysone (20e) on the cell cycle in ial-pid2 cell line established from imaginal wing discs of plodia interpunctella were reported (mottier et al., 2004). it was found that 20e induced an arrest of cells in g2 phase, accompanied by a sharp decrease in the levels of cyclin a and b expression. studies on dna damages and apoptosis several groups have investigated by fcm either dna damages or the induction of apoptosis in invertebrate cells. the terminal dutp nick-end labeling technique (tunel) was used to detect m. galloprovincialis cells displaying dna fragmentation within gill structures after treatment with tri-n-butyltin (tbt) (micic et al., 2001). dna degradation as well as decreased number of cells in g0/g1 were detectable after 1.5 hour of tbt incubation. presence of apoptotic cells in mussels' gills was indicated by the selective loss of g2/m cells concomitant with the appearance of cells with decreased dna content in the s and g0/g1 regions of cell cycle. the effect of the tbt on cell cycle in a mussel gill was depending upon dose and exposure time. metabolites of salsolinol (sal), an intraneuronal, dopamine-derived tetrahydroisoquinoline (tiq), induce apoptosis in human dopaminergic neuroblastoma cells, and likely contribute to the killing of nigrostriatal dopaminergic neurons occurring in parkinson's disease. since insects employ dopamine and related catecholamines in a variety of processes including cuticular sclerotization and cellular immune reactions, the capacity of their cells to metabolize exogenous sal was investigated (ottaviani et al., 2002). iplb-ldfb cells from lymantria dispar exhibited no significant increase in apoptosis when incubated for 48 hours with low concentrations of sal (up to 1 mm). apoptosis was observed only with the highest concentration of sal tested (5 mm), but only 12.4 % of the cells manifested this form of cell death. the resistance of iplb-ldfb cells to sal-induced apoptosis was attributed to the ability of these insect cells to metabolize and/or detoxify the dopaminederived catecholic tiqs. the toxicity of the mycotoxins nivalenol (niv), deoxynivalenol (don), and fumonisin b1 (fb1) was studied in the lepidopteran s. frugiperda sf-9 cells by fcm (fornelli et al., 2004). niv was significantly more toxic than don, and both were significantly more toxic than fb1. cell cycle distribution showed an arrest of cells in the g0/g1 phase in the presence of all of the three compounds. morphological evidence of apoptosis was related to the toxicity of the substances in that the more toxic niv induced late apoptosis, whereas don and fb1 produced less-severe morphological changes characteristic of early apoptosis. it was concluded that niv is more toxic than don, which in turn is more toxic than fb1, and that these mycotoxins can modify the normal progression of the cell cycle and induce an apoptotic process. assessing the response of invertebrate cells to fungi and bacteria innate immunity is strongly regulated by microbial products, and fcm can be added to the list of available methods to investigate this activity. furthermore, fcm can provide a simple, reproducible, and sensitive method for evaluating invertebrate hemocyte responses to immunological stimuli. the response of hemocytes from the white river crayfish procambarus zonangulus to stimulation by fungal cell walls (zymosan a) were recently measured by fcm (cardenas et al., 2000). changes in physical characteristics were assessed using forwardand side-scatter light parameters, and viability was measured by two-color fluorescent staining with calcein-am and ethidium homodimer 1. the main effects of zymosan a on crayfish hemocytes were reduction in cell size and viability. adding trypsin inhibitor in reaction mixtures further delayed the reduction in hemocyte size and cell death, thereby indicating that a proteolytic cascade played a key role in generating signal molecules which mediate these cellular responses. also lps from gram-negative bacteria strongly stimulate hemocytes from p. zonangulus in vitro (cardenas et al., 2004). fcm revealed that treating hemocytes with lps caused a conspicuous and reproducible decrease in cell size as compared to control hemocytes. this physical modification was accompanied by a reduction in hemocyte viability, that was assessed as described above. the onset of cell size reduction was gradual and occurred prior to cell death. interestingly, hemocytes treated with lps from salmonella minnesota without the lipid a moiety (detoxified lps) decreased in size without a reduction of viability, while the addition of trypsin inhibitor to the lps treatments caused noticeable delays in cell size and viability changes. it was concluded that crayfish hemocytes react differently to the polysaccharide and lipid a moieties of lps, where lipid a is cytotoxic and the polysaccharide portion is stimulatory. these effects concur with the general pattern of mammalian cell activation by lps, thereby indicating common innate immune recognition mechanisms to bacterial antigens between cells from mammals and invertebrates. fcm reveals cellular responses to stress such as environmental pollution the use of invertebrates as bioindicators in environmental monitoring is of relevant interest for its practical implications. tunicates are filter feeding marine invertebrates that are susceptible to 39 environmental contamination by toxic metals and polyaromatic hydrocarbons (radford et al., 2000). their immune reactions are profoundly affected by exposure to tributyltin (tbt) and copper, both of which are components of marine antifouling paints. immunofluorescence labeling with an anti-hemocyte monoclonal antibody whose binding to plasma membrane was revealed by fcm demonstrated that the antigenic structure of the circulating hemocyte population was substantially affected by tbt and copper. antigen-positive hemocytes were also found to accumulate in the pharyngeal papillae of tbtexposed tunicates, confirming that exogenous metals can have profound effects on tunicate hemocytes. the effects of mercuric chloride and methylmercury on the phagocytic capacities of m. arenaria hemocytes were evaluated (fournier et al., 2001). clams were exposed to single metal in water for up to 28 days at different concentrations (10-9 to 10-5 m), and phagocytic activity of hemocytes was determined by uptake of fluorescent microspheres and fcm. the phagocytic capacity is clearly proportional to ingestion of these particles, and thus to the total fluorescence that can be detected from each cell. clams exposed to high concentrations died by day 7 of exposure, while the viability of hemocytes was decreased only in clams exposed for 28 days to a 10 times lower concentration. a significant decrease in phagocytic activity of hemocytes was also present, and a clear correlation was established between body burden of mercury and effects on phagocytic activity of hemocytes. recently, the presence of immunoreactive inducible nitric oxide synthase molecules (ir-inos) has been demonstrated in the l. dispar iplb-ldfb cell line. in these cells, ir-inos were induced after 18 hour-incubation with sodium nitroprusside (snp). the increase in no provoked by snp in turn induced apoptosis, that was further increased by n-acetyl-lcysteine. apoptosis was quantified by fcm through the identification of nuclei showing the typical hypodiploic peak present in cells with less dna content, after staining with the fluorescent probe propidium iodide (ottaviani et al., 2001). the analysis of apoptosis by fcm is a very simple assay, that can be performed in almost all cells from all species, since the basic principle is that of staining dna with a stoichiometric fluorescent molecule. changes in fluorescence, along with modifications of the physical parameters of the cell, typically occur in those that lose dna, as during apoptosis. conclusions and perspectives on analysis of invertebrate cells by fcm a variety of fcm techniques currently exist that allow analysis of almost all cellular functions and parameters, with all of the possible advantages of this approach. from this point of view, until now the world of invertebrates has received a poor attention, and is almost completely virgin territory for more research. since invertebrate cells are obviously eukaryotic, researchers have indeed the possibility to use all of the methodologies that have been developed for studies on mammalian and human cells. similarly, almost all reagents and fluorescent dyes that have been developed to investigate different cellular activities can be successfully used in invertebrates. even if a variety of functional parameters have still to be investigated, the few data available already provide relevant information not only on the phenotype or the physical characteristics of a given invertebrate cell, but also on its capacity to perform a given function or to respond to a given stress. considering the relevant importance that comparative studies are assuming, either for the importance of these biological models and their low cost, and the relevant decrease in the cost of flow cytometers (that fortunately are becoming always more affordable), it is easy to predict that in the next few years we will assist in an unprecedented development of cellular studies in the field invertebrate biology and immunology. references amen ri, aten ja, baggen jm, meuleman ea, de lange-de klerk es, sminia t. trichobilharzia ocellata in lymnaea stagnalis: a flow cytometric approach to study its effects on hemocytes. j. invertebr. pathol. 59: 95-98, 1992. barcia r, cao a, arbeteta j, ramos-martinez ji. the il-2 receptor in hemocytes of the sea mussel mytilus galloprovincialis lmk. iubmb life. 48: 419-423, 1999. bihari n, hamer b, jaksic z, fafandel m, micic m, batel r. application of alkaline elution, fast micromethod and flow cytometry in detection of marine 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(gastropoda, pulmonata). boll zool. 59: 129-139, 1991. ottaviani e, barbieri d, malagoli d, franchini a. nitric oxide induces apoptosis in the fat body cell line iplb-ldfb from the insect lymantria dispar. comp. biochem. physiol. 128b: 247-254, 2001. ottaviani e, cossarizza a. immunocytochemical evidence of vertebrate bioactive peptide-like molecules in the immuno cell types of the freshwater snail planorbarius corneus (l.) (gastropoda, pulmonata). febs lett. 267: 250-252, 1990. ottaviani e, cossarizza a, ortolani c, monti d, franceschi c. acth-like molecules in gastropod molluscs: a possible role in ancestral immune response and stress. proc. r. soc. lond. b biol. sci. 245: 215-218, 1991. ottaviani e, franchini a, barbieri d, kletsas d. comparative and morphofunctional studies on mytilus galloprovincialis hemocytes: presence of two agingrelated hemocytes stages. it. j. zool. 65: 349-354, 1998. ottaviani e, franchini a, caselgrandi e, cossarizza a, franceschi c. relationship between corticotropinreleasing factor and interleukin-2: evolutionary evidence. febs lett. 351: 19-21, 1994. ottaviani e, franchini a, cossarizza a, frenceschi c. acthlike molecules in lymphocytes. a study in different vertebrate classes. neuropeptides 23: 215-219, 1992. ottaviani e, nappi aj, vass e. resistance of the insect cell line iplb-ldfb to salsolinol-induced apoptosis. arch. insect biochem. physiol. 49: 1-9, 2002. ottaviani e, petraglia f, montagnani g, cossarizza a, monti d, franceschi c. presence of acth and beta-endorphin immunoreactive molecules in the freshwater snail planorbarius corneus (l.) (gastropoda, pulmonata) and their possible role in phagocytosis. regul. pept. 27: 1-9, 1990. quaglino d, cooper el, salvioli s, capri m, suzuki mm, ronchetti ip, franceschi c, cossarizza a. earthworm coelomocytes in vitro: cellular features and "granuloma" formation during cytotoxic activity against the mammalian tumor cell target k562. eur. j. cell. biol. 70: 278-278, 1996. radford jl, hutchinson ae, burandt m, raftos da. effects of metal-based environmental pollutants on tunicate hemocytes. j. invertebr. pathol. 76: 242-248, 2000. salehzadeh a, akhkha a, cushley w, adams rl, kusel jr, strang rh. the antimitotic effect of the neem terpenoid azadirachtin on cultured insect cells. insect biochem. mol. biol. 33: 681-689, 2003. ulrich w. aneuploid and polyploid cellular dna heterogeneity in insect cell material of diptera species analyzed by flow cytometry. z. naturforsch. 45: 1027-1030, 1990. white mk, miosky d, flessas da, reinisch cl. the expression of an adhesion-related protein by clam hemocytes. j. invertebr. pathol. 61: 253-259, 1993. isj 6: sxx-syy, 2009 isj 6: 22-31, 2009 issn 1824-307x report of meeting xth scientific meeting of the italian association of developmental and comparative immunobiology (iadci), 18 20 february 2009, department of human, environmental and natural sciences, university of urbino “carlo bo”, urbino, italy organizers: m betti, m balsamo, s papa department of human, environmental and natural sciences, university of urbino “carlo bo”, urbino, italy session 1 1999-2009: a 10-year apprenticeship in comparative immunology. the immunocytes as leading actors in the responses of the bivalve mytilus to environmental challenge l canesi1, m betti2, c ciacci2, c pruzzo1, g gallo1 1department of biology, university of genoa, genoa, italy 2department of human, environmental and natural sciences, università di urbino “carlo bo”, urbino, italy in bivalve molluscs, circulating hemocytes are responsible for the innate immune defence, and are also involved in the transport of nutrients from the digestive gland to the gonad during gametogenesis. although it has long been known that the activity of these cells can be modulated by multiple signals, only in recent years the mechanisms involved in the responses of hemocytes to different stimuli, from both the internal and the external environment, have been investigated. in the marine bivalve, the edible mussel mytilus, studies carried out both in vitro and in vivo on the effects of challenge with different bacterial species and strains, heterologous cytokines, as well as with a number of environmental contaminants, lead to the identification of the main signaling pathways involved in immune activation, as well as of the main mechanisms of cellular damage and consequent immunodepression. moreover, endogenous estrogens were identified as physiological modulators of the hemocyte function. the results obtained added force to the hypothesis that the mechanisms of innate immunity are extremely conserved between invertebrate and mammalian systems. overall, the data collected so far allowed the functional characterization of mussel hemocytes as a model for investigating both the basic processes of the immune response, and the mechanisms toxicity of emerging environmental contaminants. moreover, these studies demonstrated that mytilus hemocytes represent a sensitive target for environmental stimuli, and that changes in their function are related to significant changes in the physiological status of bivalves, an ecologically and economically relevant group of invertebrates. effects of bacterial challenge on mytilus digestive gland biomarkers c ciacci1, b citterio2, r fabbri1, c barmo3, m betti1, p roch4, l canesi3 1department of human, environmental and natural sciences, università di urbino “carlo bo”, urbino, italy 2dipartimento di scienze biomolecolari, università degli studi di urbino “carlo bo”,urbino, italy 3department of biology, university of genoa, genoa, italy 4jru ecosystèmes lagunaires, cnrs-universitè de montpellier 2-france in bivalve molluscs, responses to bacterial challenge have been largely investigated in the immune cells, the hemocytes, whereas little is known about the possible effects at the tissue level. in this work, the effects of in vivo challenge of mussels (mytilus spp.) with different bacteria (micrococcus lysodeikticus, vibrio anguillarum, vibrio splendidus) on different biomarkers of stress were evaluated in the digestive gland at different times post injection (p.i.). all bacteria induced a significant decrease in lysosomal membrane stability lms (about -50 %) with respect to controls (pbs-nacl-injected mussels) from 3 to 24 h p.i. when expression of antioxidant genes was evaluated by quantitative rtpcr with both m. lysodeikticus and v. anguillarum a general downregulation of both metallothionein isoforms, mt10 and mt20, was observed, with the exception of a large increase in mt20 mrna induced by v. anguillarum and a smaller increase in mt10 by m. lysodeikticus at 6 h p.i. both bacteria downregulated glutathione s-transferase gst-π at all time p.i. v. anguillarum decreased the expression of catalase, wherease no effects were observed with m. lysodeikticus. at 48 h p.i. the activity of 22 catalase was increased (2-fold with both m. lysodeikticus and v. anguillarum; +60 % with v. splendidus) and total gst activity was decreased (30, -40 %) with all bacteria. both m. lysodeikticus and v. splendidus also decreased gsh reductase gsr activity (about -24 %) and total glutathione content (-47 %) whereas no effects were observed with v. anguillarum. flow cytometry as a tool for analyzing invertebrate immunocytes: our experience with mytilus hemocytes b canonico, m betti, c ciacci, m arcangeletti, s papa, l canesi1 department of human, environmental and natural sciences, university of urbino “carlo bo”, urbino, italy 1department of biology, university of genoa, genoa, italy flow cytometry (fc) represents a powerful tool generally utilized in biomedical research. fc has been also increasingly applied in invertebrate immunology; in particular, in bivalve molluscs, fc has been largely utilized to characterize hemocyte populations and to evaluate total cell count and oxyradical production. in addition, fc can be employed to recognize antigens, or at least immunodominant epitopes, shared in common with mammalian cells, including human leukocytes. in addition to traditional methods such as microscopy and protein chemistry, fc can provide a simple, reproducible, and sensitive method for evaluating invertebrate hemocyte responses to different immunological stimuli. data are here summarized obtained in different in vitro and in vivo studies carried out on the hemocytes of the marine bivalve mytilus galloprovincialis. fc was successfully utilised to evidentiate, utilizing annexin v binding and mitochondrial markers, pre-apoptotic processes in mussel hemocytes in response to cytokines (tnfα) and emerging contaminants (nanoparticles), as well as proliferation/differentiation processes in response to growth factors (stem cell factor-scf). furthermore, we have recently investigated the presence of different antigens (cd34, cd117 and cd11b) in mussel immunocytes and changes in their expression following in vivo challenge with both gram(+) bacteria and autoctonous vibrio species. these data support the importance of developing the utilization of fc in comparative and environmental studies on invertebrate immunocytes. new knowledge of antimicrobial peptides in mediterranean mussel u rosani1, l varotto1, s domeneghetti1, a pallavicini2, g lanfranchi1,3, p venier1 1department of biology, university of padoa, padoa, italy 2department of biology, university of trieste, trieste, italy 3c.r.i.b.i., university of padoa, padoa, italy marine invertebrates are constantly surrounded by potentially invading microorganisms. their defence mechanisms involved circulating haemocytes that infiltrate injured tissues, encapsulate or phagocyte microbial cells and release cytotoxic factors such as lectins, complement factors and antimicrobial peptides (amps). mussels especially seem more resistant to infections and diseases than other edible bivalves. in the frame of the european integrated project food-ct-2005-007103, est production and gene expression profiling in m. galloprovincialis (lmk., 1919) are providing new interesting evidences. massive est sequencing of primary cdna and normalized libraries of immuno-stimulated mussels revealed a high sequence variability of amps, especially in haemolymph. these results confirm the importance of such molecules in innate immune response of marine invertebrates. selected immune-specific and immune-related trancripts were arrayed in the first mussel immunooligochip. preliminaries microarray and real time experiments showed various gene expression changes in mussels injected with bacterial antigens. moreover, the new generation sequencing technologies provide us plentiful and precise knowledge of the variability of these natural antibiotics, that can explicate better the ancient host-pathogen interactions and the adaptation strategies. proliferation of mussel haemocytes: effects of stem cell factor (scf) m betti, p gobbi, b canonico, c ciacci, s papa, l canesi1 department of human, environmental and natural sciences, university of urbino “carlo bo”, urbino, italy 1department of biology, university of genoa, genoa, italy stem cell factor (scf) is a member of mammalian haematopoietic cytokines, a group of glycoproteins that regulate the growth and differentiation of haematopoietic cells and functionally activate mature neutrophils or macrophages. in bivalve molluscs, circulating haemocytes resemble the monocyte/macrophage lineage. however, in these organisms, no information is available on the physiological control of haematopoiesis. in this work, the in vitro effects of heterologous scf on the haemocytes of mytilus sp. were investigated. flow cytometry (fc) analysis of control haemocytes identified 3 main cell subpopulations: granulocytes, agranulocytes, blast-like cells. a significant proportion of total haemocytes showed immunoreactivity towards anti-cd34 and anticd90 antibodies, utilised as markers of haematopoietic stem cells. scf induced significant changes in haemocytes functional parameters, indicating increase in phagocytic activity and reduction in lysosomal membrane fusion processes. moreover, fc analysis showed that scf significantly affected 23 both haemocyte number and cell cycle, mitochondrial activity, and immunoreactivity towards different anti-cd-antibodies. in particular, increases in the number of granular (phagocytic) haemocytes were observed; the effects of scf involved the activation of c-kit tyrosine kinase-like receptors. ultrastructural morphological studies in colcemide-treated haemocytes confirmed that the large majority of cells are able to enter the mitotic phase of the cell cycle. the results support the hypothesis that common pathways involved in modulating activity, proliferation and differentiation of immune cells are conserved from molluscs to mammals. effects of the protein pheromone er-1 isolated from the ciliate euplotes raikovi, on the phagocytic activity of the bivalve mytilus galloprovincialis l casarini, d malagoli, a vallesi1, e ottaviani department of animal biology, university of modena and reggio emilia, modena, italy 1dipartimento di biologia molecolare cellulare e animale, university of camerino, camerino, italy phagocytosis plays a central role in the cellmediated immune response and the study of cytokine-like molecules capable of modulating the phagocytic activity of immunocytes may contribute insightful information on the evolution of the immune response. previous experiments on the immunocytes of the mussel mytilus galloprovincialis suggested that regulation of the phagocytic activity involves campand pka-pathways. we have now analyzed the response of these immunocytes to the effects of a protein pheromone, denoted er-1, produced by the protozoan ciliate euplotes raikovi and characterized by an exclusively helical structure like il-2 and its cytokine family members. our results indicate that er-1 increases the immunocyte phagocytic activity, and this increase follows camp and pka-dependent signal transduction pathways. this indication is consistent with the activity of ciliate pheromones in mechanisms of self-nonself recognition and cell-cell adhesion. mytilus hemocytes as a model for nanoparticle toxicology in marine invertebrates c ciacci1, b canonico1, m betti1, g pojana2, a marcomini2, l canesi3 1department of human, environmental and natural sciences, university of urbino “carlo bo”, urbino, italy 2department of environmental sciences, university ‘ca foscari’ of venice, venice, italy 3department of biology, university of genoa, genoa, italy the potential for human and ecological toxicity associated with nanomaterials is a growing area of investigation. in mammalian cells, nanoparticles (nps) have been shown to induce inflammation and oxidative stress, and changes in cell signalling and gene expression. as the nanotechnology industries increase production, nanoscale products and by products will enter the aquatic environment, posing a possible threat to aquatic organisms. in particular, filter-feeding invertebrates may represent a unique target group for nanoparticle toxicology, since they have highly developed processes for the cellular internalisation of nanoand microscale particles, endocytosis and phagocytosis, that are integral to key physiological functions such as intracellular digestion and cellular immunity. in this work we show that in the hemocytes of the marine bivalve mytilus different types of engineered nanoparticles (carbon black, c60 fullerene, tio2, sio2) induce activation of different immune parameters (lysozyme release, activation of oxidative burst and no production) to a different extent depending on the np type and concentration, without significant cytotoxicity. only at higher concentrations mitochondrial damage was observed, this indicating pre-apoptotic processes. the inflammatory effects of nps were mediated by rapid activation of the stress activated p38 mapk. the results indicate that bivalve immunocytes represent a suitable model for investigating the potential effects of nps in aquatic organisms. session2 are really males the sterner sex? the immune responses of the clam tapes philippinarum as a case study v matozzo, m marin department of biology, university of padoa, padoa, italy for the first time, gender-related differences in immune responses of the clam tapes philippinarum were investigated. haemocytes from male, female and sexually undifferentiated clams were collected, and total haemocyte count (thc), haemocyte volume, capability of haemocytes to assume the vital dye neutral red (indicative of endocytotic activity), acid phosphatase and lysozyme-like activities in both haemocyte lysate (hl) and cell-free haemolymph (cfh), were evaluated. no statistically significant differences in thc values were observed. however, differing haemocyte size frequency distribution was found: the fraction of larger haemocytes (6-8 µm diameter, 200 fl volume) markedly increased in females, whereas the fraction of smaller haemocytes (< 5 µm diameter, < 200 fl volume) increased in both male and undifferentiated clams. significantly increased neutral red uptake was recorded in haemocytes from females. this was most likely related to the higher fraction of larger haemocytes in females, these cells being more actively involved in phagocytosis. no significant variations in lysozymelike activity was observed in hl, whereas in cfh enzyme activity resulted significantly higher in females with respect to male and undifferentiated animals. hl acid phosphatase activity was significantly higher in males with respect to females and undifferentiated clams, whereas no significant variations in enzyme activity was observed in cfh. overall, results obtained demonstrated that gender 24 related differences in immune responses occurred in t. philippinarum, and indicated that females had more active haemocytes than both male and undifferentiated clams, at least on the basis of the cellular parameters investigated. rhamnose-binding lectins in the compound ascidian botryllus schlosseri as multifaceted immune molecules n franchi, f schiavon, l ballarin department of biology, university of padoa, padoa, italy rhamnose-binding lectins are characterised by the presence of common aminoacid motifs (ygr, dpc, kyl) and the presence of eight conserved cysteine residues involved in four disulphide bridges responsible of the compact structure of the proteins. they have been described in many fish and few invertebrates. recently, in the compound ascidian botryllus schlosseri, we identified and characterised a rhamnose-binding lectin (bs-rbl) with opsonic activity, able to enhance phagocytosis of foreign target cells. in the present work, we continued our investigation on bs-rbl and studied the expression of the molecule through immunohistochemical and immunocytochemical analysis, using specific polyclonal antibodies, and in situ hybridisation on both colony sections and haemocyte monolayers. results obtained indicate the phagocytes as the site of synthesis of this kind of molecules as both the protein and the corresponding mrna were located in this cell type. with immunoblot analysis of colony lysates, we studied the expression of bs-rbl during the colonial blastogenetic cycle. the electrophoretic bands recognised by the anti-bsrbl antibody were of higher intensity during the cyclical colony generation change (take-over), characterised by massive apoptosis of zooid tissues, in addition, immunocytochemical analysis showed that that the frequency of haemocyte recognised by the anti-bsrbl antibody significantly increased during the take-over, and that, in addition to phagocytes with a labelled cytoplasm, the plasma membrane of other cell types (e.g., morula cells) resulted immunopositive, indicating that this molecule can recognise sugars on the surface of senescent cells, unexposed in healthy cells, and suggesting its involvement in efferocytosis. the incubation of haemocyte monolayers with purified bs-rbl results in the release in the culture medium of molecules recognised by antibodies raised against mammalian il-1α and tnfα, we have already demonstrated to have an immunomodulatory role. as these molecules are synthesised and released by cytotoxic morula cells (mc), the above observation indicate an immunomodulatory role of bs-rbl on mc activity. in addition, it has been recently demonstrated that fish rbl can recognise lps and lipoteichoic acid, basic components of gram(–) and gram(+) bacterial cell wall, respectively, suggesting an antibacterial role of these proteins. we are carrying out new experiments to verify this aspect in bsrbl. evolutionary analyses of sequence and intronexon structure of two lectin genes in ascidians c gissi, f griggio, f iannelli department of biomolecular sciences and biotechnology, university of milan, milan, italy lectins are a group of sugar-binding proteins that recognize specific carbohydrate structures and agglutinate cells by binding to cell-surface glycoconjugates. these proteins are involved in a variety of distinct role in different cells and species, and in invertebrates they are considered as recognition molecules that trigger defensive activities. lectins show a wide variety of protein architecture, thus they are classified into several families depending on the sugar-binding specificities and the sequence similarities of the carbohydraterecognition domain (crd). we have analysed the evolutionary history of the crd of two lectin genes, the galectin and the rhamnose-binding lectin (rbl), within chordates. in particular, these genes have been identified in the publicly available genomic sequences of ciona, oikopleura (tunicata) and branchistoma (cephalochordata), all protochordate species, and have been annotated using several bioinformatics methods and also with the help of est data. the evolutionary analyses have been carried out in a wide taxonomic sample including the homologous functionally characterized sequences of other deuterostome species, and the human annotated homologs. in addition to the traditional phylogenetic reconstructions based on amino acid sequences, we have also investigated the conservation of the intron-exon structure and evaluated the congruency between the results provided by these two different data types. our results show that the in most cases the single crds are delimited by introns, with linkers and additional protein domains encoded by distinct exons, and that tunicate genes evolve always very fast both at level of sequence and gene structure. individuation of a new metallothionein from the urochordate ciona intestinalis n franchi, l ballarin, e piccinni department of biology, university of padoa, padoa, italy metallothioneins (mts) are metal-chelating proteins occurring in animals, plants and prokaryotes, involved in detosification and immunity. they do not constitute a monophyletic protein family, but rather a superfamily of heterogeneous, low molecular weight, cysteine-rich peptides. current knowledge on mts comes mainly from vertebrate molecules which are composed of approximately 60 amino acids, including 20 cysteine residues, and folded into two independent domains when coordinating divalent metal ions. up to now, there are no descriptions of mts in invertebrate chordates althought it seems that the vertebrate structure is maintained also in other deuterostomes such as the echinoderms. 25 in this study, we present some data on mts of the solitary urochordate ciona intestinalis. we have cloned the transcript and characterized the gene of a new mt, codifying for 39 amino acids, including 12 cys residues (30 % of total amino acids, in accordance with other mts); moreover, the typical organization of cysteine residues in c-x-c motifs is conserved. the gene is composed of two introns (one inside the coding region and the other inside the 3’ utr region) and three exons. the 5’ untrascribed region contains several cis elements similar to those found in vertebrate mt genes such as: metal responding elements (mre), antioxidant responding elements and stat3, which are involved in constitutive and metal-related, ros and cytochines induction, respectively. the amino acid sequence of c. intestinalis mt shows only limited similarity with other mts, e.g., mytilus edulis mt (28.8 % identity), strongylocentrotus purpuratus mt (23.4 % identity) and sparus aurata mt (36.7 % identity). phylogenetic analyses with various methods are in progress. a preliminary study by atomic force microscopy of cytogenetic stability in a mdvintegrated chicken lymphoblastoid cell line s di bucchianico, mf giardi, d botti department of basic and applied biology, university of l’aquila, l’aquila, italy in previous works we have demonstrated that the chicken lymphoblastoid t cell line mdcc-msb1 is able to produce ovotransferrin and nitric oxide to a higher extent than cef (chicken embryo fibroblasts). this ability can be tentatively related to the integration of marek’s disease virus (mdv) genome. in addition, it is well known from literature that lymphocytes infected by mdv produce vil-8, with a chemotactic function towards other lymphocytes, to amplify viral spreading. on the other hand, it is also known that the virus undergoes a fragmentation prior to a random integration, preferentially in subtelomeric regions. the viral integration may induce a novel organization of chromatin architecture, with a modified gene expression. in our opinion it is worthwhile trying to relate cytogenetic stability to functional modifications. recently, atomic force microscopy (afm) technique was applied to study the structure of chromosomes at nanoscale level. structural analysis at high resolution of high molecular complexes provide detailed information such as 3dimensional topological data, mechanical behaviours, dynamic processes and molecular interactions. when scanning soft biological surfaces, the afm can achieve a resolution of about 1 nm. the vertical resolution is mostly determined by the afm scanner sensitivity, and typically is 0.01 nm. these features allow to investigate the different structure of chromatin in the regions of the viral insertion. on the basis of these data, the correlation between the localization of viral insertion and the different gene expression due to marek’s disease virus localization is investigated. session 3 self/nonself recognition in the ciliated protozoa: characterization of the pheromone gene family of euplotes nobilii a vallesi1, c alimenti1, a la terza1, g di giuseppe2, f dini2, p luporini1 1dipartimento di biologia molecolare cellulare animale, university of camerino, camerino, italy 2dipartimento di biologia, university of pisa, pisa, italy a family of seven allelic genes encoding self/nonself signal proteins (pheromones) in the polar (cold-adapted) marine hypotrichous ciliate, euplotes nobilii, were cloned from the somatic subchromosomic genome of the cell macronucleus. the determination of their full-length nucleotide sequences shows that their open reading frames specify proteins of 83 to 94 amino acid residues which represent the cytoplasmic pheromone precursors (pre-pro-pheromones). two proteolytic steps would thus remove the pre and pro segments formed by tightly conserved sequences, before the secretion of the structurally more variable mature proteins. at odds with respect to the general structural organization of the macronuclear genes of the hypotrichous ciliates, the 5’ region of all the cloned pheromone genes is significantly longer than the respective coding region (approximately, 350 versus 250 nucleotides). considered jointly with the tight sequence conservation, this feature implies that the 5’ region is site of specific activities in the mechanism of pheromone gene expression. antimicrobial peptide transcription is modulated after repeated exposure to heat-inactivated escherichia coli in drosophila melanogaster sl2 cell line d malagoli, s sacchi, e ottaviani department of animal biology, university of modena and reggio emilia, modena, italy recent findings in drosophila melanogaster indicate that the activation of the immune system immediately before an infection increases the resistance to pathogens, suggesting the possibility that a first exposure to an immunogen may strengthen the immune response to a second challenge. by rt-pcr experiments, we have observed in the d. melanogaster sl-2 hemocyte cell line that a second 6 h exposure to heat inactivated escherichia coli significantly increases the expression of the antimicrobial peptides (amps) drosomycin and diptericin, with respect to the first 24 h exposure. conversely, expression levels reached after the first 24 h exposure are not exceeded after a second 6 h immune challenge by the amps defensin and cecropin a1 and by the putative helical cytokine, dhf. surprisingly, the expression of the imd-related kinase dtak1, is increased by the first incubation with bacteria, while its expression is lower than in controls after the second exposure. the augmented expression 26 observed for drosomycin and diptericin is the consequence of the additive effect of the second exposure. in accordance with data present in literature, our observations suggest the existence in d. melanogaster of mechanisms devoted to control the intensity of the immune response. this regulation involves the possibility to modulate the expression of amps, in our case drosomycin and diptericin, and signal transduction regulators, e.g., dtak1. effects of bacterial injection and salinity stress on the humoral immune response of paracentrotus lividus (echinodermata) f drago department of animal biology “marcello la greca”, university of catania, catania, italy the altered expression of inos and lysozyme in the coelomocytes present in the wall of the gut of p. lividus, was observed after bacterial injection and osmotic stress by histochemical and immunocytochemical procedures. after 3 h from injection of bacteria a significant increase expression of inos was observed. this increase remained at the same level until 24 h post inoculum. conversely, the expression of lysozyme in the coelomocytes showed a relevant increase only after 24 h p.i. we also observed that the expression of inos and lysozyme after osmotic stress increased following the pattern registered after bacterial injection. in accordance with data present in literature, the present study identifies the coelomocytes of the sea urchin as the main effectors of defense response and indicate that the expression of inos increases faster than that of the lysozyme. does trichoplax (placozoa) produce resistance stages? l guidi, e cesarini, m balsamo department of human, environmental and natural sciences, university of urbino “carlo bo”, urbino, italy trichoplax adhaerens (placozoa) is currently regarded as the most primitive living metazoan. its mitochondrial and nuclear genomes have been wholly sequenced but its life cycle is still little known. placozoa reproduce asexually, but molecular evidences suggest the occurring of sexual events. under stress conditions, animals enter a degenerative phase (d-phase), and produce few, large oocytes. the existence of spermatozoa has not yet proved with certainty. the d-phase was induced in laboratory cultures of trichoplax, and after four weeks the production of oocytes was observed in vivo. after at least another week eggs were released through animal body disgregation. animals containing oocytes, and isolated eggs at 5, 6 and 10 weeks of age were studied under optical microscopy, sem and tem. the oocyte in the animal body at first shows only the oolemma, then produces an external envelope made up of 2-3 layers of different thickness and nature: at that stage it is released. early stages of cleavage were occasionally seen, in rare cases even in the animal body. very few isolated eggs in cleavage phase and many undivided ones were observed in the cultures: all showed an envelope with an identical morphology. that suggests that sexual reproduction is performed only in adverse environmental conditions, and that both quick-hatching and latehatching eggs might be produced. the formation process of the thick egg envelope and its fine structure appear very close to those reported for the asexual cysts of some ciliata. that supports a possible resistance function of the trichoplax eggs to stress conditions like those induced in the cultures, or those occurring in natural unstable habitats. trichoplax lives as an epibenthic form in the littoral marine environment, where the ecological conditions are quite variable and resistance stages are produced by a number of colonizing organisms (e.g., cysts of protists, gemmules of sponges). identification of a functional motif mobilized by retroelements in mrna from activated human lymphocytes e capelli1, s panelli2, g damiani3 1dipartimento di biologia animale, università di pavia, pavia, italy 2parco tecnologico padano, lodi 3igm-cnr, pavia, italy 3’ untranslated regions (3’ utrs) of eukaryotic mrnas commonly contain conserved repeated motifs involved in the control of mrna stability, i.e., through micro-rna (mirna) mediated regulation. following alignment of functionally related genes (i.e., of genes co-regulated in response to the same stimuli), we identified a number of conserved motives shared by genes involved in the same process (in particular: lymphocyte activation). primers were designed on these putative regulatory motives and used for rt-pcr fingerprintings on cdna from human t lymphocytes before and after pha pulse and il-2 activation. we noticed complex patterns of bands only when using cdna from activated cells. bands were cloned and sequenced. quantitative rt-pcr confirmed that the identified genes, containing the putative regulatory motives, were expressed only in activated cells. searches in mirna databases (mirbase) showed that one of our motives, cactn (3,4,3), corresponds to a sequence of a known mirna family, and thus confirmed its regulatory role. this motif, highly conserved across evolution, in ruminants is part of a short interspersed nuclear element (sine) called bov-a2. this sine is integrated in regulatory regions (promoter, 5’ and 3’ utr, introns) of many immune-related genes. these results are suggestive of connections among retroelements and the sharing of common regulative motives among functionally related genes. in particular, the role of retroelements could be to carry these important motives and help in their spreading among genes (seed) as already suggested by several authors. 27 our results, in turn, suggest a deep involvement of these processes in immunity and in the regulation of genes involved in the response of immune subsets like lymphocytes to external stimuli. session 4 an attempt to re-examine the immune role of ciona intestinalis hemocytes n parrinello, v arizza, m cammarata, a vizzini, d parrinello, m vazzana, tf giaramita, g salerno, m pergolizzi, ma sanfratello department of animal biology, university of palermo, palermo, italy in the last few years, interest in the mutual relationships between ascidians hemocytes and products of innate immunity gene repertoire has led to a more clear-cut knowledge of multifunctional role of hemocytes in ciona intestinalis immunity. this is a suitable approach that may include morphofunctional screening methods allowing us to disclose differentiation of the hemocytes, their activities in immune responses leading to a more precise and reasonable classification taking in account a multi-parameter approach. the genome sequence provided new insight in studying the innate immunity. bioinformatic approach and extensive in silico search have concerned immunorelevant molecules, gene expression patterns and some specific immune properties that contribute to clarify morpho-functional aspects. the involvement in immunity of hemoblasts, lymphocyte-like cells, hyaline amebocytes, and various types of granulocytes have been described. the ciona intestinalis prophenoloxidase activating system during lps inflammatory reaction m cammarata, v mangano, v arizza, c cianciolo, d parrinello, m vazzana, a vizzini, g salerno, n parrinello department of animal biology, university of palermo, palermo , italy phenoloxidases initiate melanin synthesis in almost all organisms, and are involved in different biological activities. in many invertebrates, defense reactions are linked to phenoloxidase activity and/or melanization, an innate response generally observed in wounded tissues. contact with a foreign molecule is able to trigger the prophenoloxidase (propo) system that requires serine protease cleavage for activating the zymogen to phenoloxidase (po). it is generally accepted that the propo system is fully expressed in arthropods, and, recently, progress in the regulation of crustacean and insect propo activation steps has been made. after cells are stimulated by components of the pathogen associated molecular pattern (pamp), propo activation takes place via zimogenic serine proteinase in turn activated by pamp. in the present paper, we report on the ciona intestinalis propo system and related molecules, with particular focus on the biochemical, cellular and molecular components of the propo system in the tunic tissue following lps intratunic injection. tunic homogenate supernatant (ths), assayed with the dopa-mbth reaction, displayed ca2+-independent po activity that was increased by lps and further enhanced by proteases. in vivo experiments were performed by injecting isosmotic medium or lps, and ths was assayed for its po activity. to determine the po response at the injured site, an assay with dopa-mbth was performed in vitro. quinones were mainly contained in the tunic matrix area enriched with inflammatory cells around the injection site. microscopy observations and immunohistochemistry with anti-cinpo2 antibodies showed granulocytes and unilocular refractile granulocytes containing po. immunoblotting with anti-cinpo2 and sds-page zymograms demonstrated po activity linked to different bands as an effect of lps injection. these pos distinguishable by their size are contained and presumably released by tunic inflammatory cells and hemocytes of the pharynx bars. proliferating and propo activity of the hemocytes of ciona intestinalis m pergolizzi¹, i soderhall², h liu², k soderhall², n parrinello¹ ¹department of animal biology, university of palermo, palermo, italy ²department of comparative physiology, evolutionary biology centre, uppsala university, norbivagen 18a, uppsala 75236, sweden hemocytes are essential in tunicate immunity, performing functions such as phagocytosis, encapsulation and lysis of foreign cells. because of the ciona intestinalis phylogenetic taxonomic position between invertebrate and vertebrate, in the last decades, a molecular biology approach has been used to identify the gene expressed in the hemocytes of this tunicate. here, we tried to throw light on hematopoiesis in c. intestinalis. therefore, we used circulating hemocytes, as source of hpt stem cells, and separated them by discontinuous percoll gradient. we characterized the hemocytes with a blood cell staining method, such as maygrunwald-giemsa staining. then we cloned 2 predicted hemocyte genes: pcna, as marker to identify hpt stem cells and their progenitors; propo2, as marker for differentiation and for studying a well known innate defense mechanism of the hemocytes of c. intestinalis. we valued the expression for both the genes by rtpcr, and for pcna gene, by ish. we made primary culture attempts of the circulating hemocytes, and valued their vitality and proliferating activity by mtt assay. the results of the rt-pcr, showed that, the hemocytes of the percoll second band expressed more than those of the other bands both pcna and propo2 genes, whereas, by ish, the transcript for pcna gene was present in 4 of all the hemocyte types: hyaline and granular amoebocytes, urgs and lymphocyte-like cells. the primary culture attempts showed that the hemocytes of the second band seem to proliferate, arrange and form clumps 28 already 30 min after plating, and that these formations become brown within 24 h. session 5 characterisation of a progranulin in the medicinal leech, hirudo medicinalis j vizioli1, a grimaldi2, f croq1, c lefebvre1, c van camp1, g tettamanti2, m salzet1, j pestel1, m de eguileor2, p-e sautière1 1laboratory of annelids neuroimmunology fre 2933 cnrs. university of de lille 1, villeneuve d’ascq, france 2department of biotechnology and molecular science, university of insubria, varese, italy progranulin (pgrn) is a pluripotent growth factor expressed in many animal groups and involved in development, tumorigenesis, inflammation and wound repair. its mechanisms of action remain largely unknown. in animals, the progranulin gene encodes a cystein rich glycoprotein containing several repeated motifs called granulins (grn). granulins are 6 kda peptides produced by proteolytic cleavage of the precursor pgrn. in vertebrates, pgrn is expressed in leucocytes and is involved in defence as well as in wound healing events. its processing is linked to regulation mechanisms of inflammation. to better understand the function(s) of (pro)granulin, we investigated the role of this molecule in an invertebrate model, the medicinal leech hirudo medicinalis. the hirudo progranulin (hmpgrn) gene codes a 150 kda protein containing 16.5 putative grn motifs and showing a high similarity with human and other animal pgrn. hmpgrn is constitutively expressed in leech body and 24 hours after injury the protein accumulates as granules in granulocytes, belonging to fibrovascular tissue (a peculiar hirudinean tissue). these cells, after surgical lesion, increase in number and migrate towards the wound site. in central nervous system (cns) of mammals this molecule is expressed by neurons and microglial cells and probably acts as a neurotrophic factor. in human, mutations of pgrn gene are linked to frontotemporal lobar degeneration and other neurodegenerative pathologies. in leech cns, hmpgrn gene is constitutively expressed in neurons and is upregulated during nerve cord repair. immunohistochemical analysis revealed the accumulation of the protein in neurons cell bodies after injury. taken together, these data suggest that hmpgrn might play a neurotrophic role and contribute to the repair process. since progranulin structure is highly conserved in animals, its study in leech can increase our knowledge on its functions in normal or injured tissues and lead to new therapeutic investigations. session 6 cellular and molecular responses of the teleost fish dicentrarchus labrax against in vivo challenge with nodavirus and photobacterium damselae g scapigliati, f buonocore, e randelli, d casani, s meloni, d pietretti, imaquanim1 consortium dipartimento di scienze ambientali, università della tuscia, viterbo, italy 1www.imaquanim.dfvf.dk the need of high quality farmed fish production in conditions to be safe for the consumer and for the environment requires much efforts to maintain fish health and to avoid spreading of infectious diseases. this, in turn, requires much knowledge of immune responses against most dangerous pathogens that, in the case of mediterranean sea bass (dicentrarchus labrax) are a nodavirus (retrovirus) (nnv) and the gram-negative photobacterium damselae subsp. piscicida (phda). in a concerted scientific action aimed to study cellular and molecular responses of sea bass against these pathogens, fish (20-30 grams) have been challenged intraperitoneally with a modest dose of nnv that caused 12 % mortality (at 30 days), then treated again after 48 days with same virus. alternatively, other fish groups have been treated with virulent phda administered in water, followed by a similar treatment after 30 days. fish from all experimental groups were sampled for molecular analysis by preparing rna from homogenised organs and tissues, copying in cdna and analysing by quantitative pcr for immunomodulatory expressed genes. this analysis was performed through pcr array using a protocol developed by our group. fish were also sampled to obtain leucocytes for cellular analysis to investigate proliferation, lymphocytes profile, and sera analysed for the presence of pathogenspecific antibody by elisa. proliferation of leucocytes in response to inactivated nnv was detected in vitro at day 43 in pbl and gills, and at day 73 in pbl, gills, and head kidney. no detectable in vitro proliferation was observed when adding phda. serum analysis by indirect elisa of pathogenspecific antibody showed that the treatment with nnv induced a specific response observed from day 47 onwards. elisa analysis of phda-treated fish showed the presence of specific antibody at days 10 and 44. however, the observed presence of detectable amounts of antibody against nnv and phda in control fish raises interesting questions. several genes cloned in sea bass have been used for expression array analysis using pcr methodologies, and results obtained with fish challenged with nnv showed modulation of antiviral proteins interferon (type i) and mx, of inflammation-related proteins il-1, cox-2, il-10, and tgf-beta, whereas t cell genes tcr, cd4, and cd8 were not significantly affected. when analysing transcripts from fish challenged with phda, proinflammatory peptides il-1 and cox-2 showed a potent stimulation at 6 h after challenge, whereas t cell genes were only slightly upregulated after boosting. interestingly, none of the immunomodulatory genes analysed was downregulated by nnv and phda challenges. 29 developmental expression of mhc class ii in sea bass dicentrarchus labrax (l) l guerra, l abelli1, e randelli, f buonocore, am fausto, s picchietti department of environmental sciences, tuscia university, viterbo; italy 1department of biology & evolution, section comparative anatomy, university of ferrara, ferrara, italy the major histocompatibility complex (mhc) class i and class ii molecules play a pivotal role in vertebrate immune response to antigenic peptides. mhc class ii genes have been identified in various teleost fish species, but no reports on their expression in sea bass development are still available. sea bass eggs, larvae from 2 to 92 days posthatching (dph) and juveniles, were analysed for the occurrence of mhcii-β transcripts. these were first detected in 4 dph larvae by rt-pcrs, while q-pcr revealed their significant increase until 92 dph (p<0.001). an early role of the mhcii-β molecules in the larval development is therefore suggested. in fact, cd4+ t cells appear later in sea bass (51 dph), when the thymus is well developed. at this stage the in situ hybridization of mhcii-β mrna labelled cells in the inner zones of the thymic paired glands. from 75 dph on, the signal was detected in the thymic cortex and mainly in the outer and inner medulla. in one year old thymus numerous stromal cells were mhcii-β+. this report enlightens possible roles of stromal components in the mechanisms of thymocyte selection in fish. mx protein and interferon in sea bass (dicentrarchus labrax l.): an evolutionary perspective f buonocore, d casani, e randelli, s costantini1, am facchiano1, j zou2, cj secombes2, g scapigliati department of environmental sciences, university of tuscia, viterbo, italy 1laboratory of bioinformatics and computational biology cnr, istituto di scienze dell'alimentazione, avellino, italy 2scottish fish immunology research centre, aberdeen university, aberdeen, uk the interferons (ifns) are a large family of soluble cytokines involved in the immune response against viral pathogens. three families of ifns have been identified in mammals (type i, type ii and type iii) and, recently, homologues of type i and type ii genes have been found in various teleost fish species. our work has been focused on the identification of ifn and mx homologues from sea bass (dicentrarchus labrax), a fish of high economic impact for south mediterranean countries. moreover, we analysed the ifn gene structure and the expression of both ifn and mx by real time pcr after stimulation with poly i:c. finally, we predicted by template-based modelling the 3d structure of ifn and hypothesized its possible residues of interaction with the putative sea bass ifn receptor on the basis of the correspondent human complex. the sea bass ifn cdna consists of 1047 bp that translates in one reading frame to give the entire molecule containing 185 amino acids. the analysis of the sequence revealed the presence of a putative 22 amino acid signal peptide, two cysteine residues and three potential n-glycosylation sites. four mx protein cdnas were obtained and they translated in a putative protein of 652 amino acids. the sea bass ifn gene contains four introns as with other type i ifn teleost genes, except medaka that contains three introns. real time pcr was performed after poly i:c stimulation of dlec cell line and head kidney leukocytes to investigate the expression of sea bass ifn and mx and an induction was observed for both genes. the predicted 3d structure of sea bass ifn is characterized by an “all-alpha” domain that shows an “up-down bundle” architecture made of six helices (abb’cde). the two cysteine residues present in the sequence (i.e. cys23 and cys126) are in a position and at a distance that suggest the possible formation of a disulfide bridge that may stabilize the structure. these data add new insights on the evolution of the ifn system in teleosts and vertebrates more generally. identification of vaccine candidates against photobacterium damselae subsp. piscicida f andreoni, r boiani, i bianconi, g serafini, f gorini, m magnani department of biomolecular science, university of urbino “carlo bo”, urbino, italy photobacterium damselae subsp. piscicida is the etiological agent of pasteurellosis, which is one of the most devastating bacterial diseases affecting the culture of gilt-head seabream and seabass in mediterranean area. to date, efficient vaccines against the pathogen are not available. the aim of this study is the identification of vaccine candidates against p. damselae subsp. piscicida, using the reverse vaccinology, a new approach which starts from the in silico analysis of the genome sequence. a genomic cosmid library of p. damselae subsp. piscicida strain ncimb 2058 was constructed. sequences, obtained by the shotgun method, were analyzed in silico by glimmer 2.13 and blastp for the identification of the proteins potentially encoded by the bacterium. protein localization was predicted by psortb, tmpred and signalp softwares, to select surface-exposed or secreted proteins. the analysis of 12 cosmid clones (421575 bp) identified 297 orfs, 138 of which have a cellular localization spanning from the inner membrane to outside the bacterium. among these proteins, 35 potential vaccine candidates were selected for expression as recombinant proteins. orfs were cloned into pet-21b vector, expressed as c-terminal his6-tag proteins in e. coli and purified by metal-affinity chromatography. the potentiality of each antigen to become a vaccine ought to be tested by in vitro assay and by challenge experiments in fish. the complete genome sequencing of p. damselae subsp. piscicida will provide us with the inclusive set of 30 proteins potentially encoded by the bacterium and new antigenic molecules to use for vaccination. analysis of the immunoglobulin heavy chain gene locus of the antarctic teleost chionodraco hamatus u oreste, s varriale, v avagliano, mr coscia istituto di biochimica delle proteine, cnr, napoli, italy in teleosts, as in mammals, immunoglobulin heavy chain (igh) primary transcripts are alternatively spliced into mature transcripts encoding either the membrane receptor or the secreted antibody. however, the cryptic donor splice site in the last heavy chain constant domain (ch4), used in mammalian igh gene, is absent in teleosts igh. thus, the exon for the transmembrane region splices to the 3’ end of the ch3 exon. as a result, the synthesized protein lacks the entire ch4 domain. we have previously reported that in chionodraco hamatus, as in the majority of antarctic teleosts, an atypical splicing mechanism generates the membrane form, excluding two entire domains and including two additional 39-nt exons. to investigate what makes this specific splicing type possible, a c. hamatus genomic dna fragment was sequenced. this analysis revealed that the two 39-nt exons are part of reverse complement sequences (rcs) of an upstream gene region (ch3 exon). three rcs regions are present in the gene locus (rcs1, rcs2 and rcs3). each rcs shares, on average, 89.7 % of nucleotide identity with the respective counterpart that is present in the ch3 exon. although sharing all the splicing signatures, the 39-nt sequence, present in rcs1, was missing in the mature transcript. to explain the reason why the rcs1 39–nt element is spliced out from the transcript, the folding of the partial primary transcript encompassing the sequenced genomic region was predicted by using the mfold computational tool. the results showed a very compact structure stabilized by a long duplex comprising the entire ch2 and ch4 exons bound to the rcs1 sequence. the presence of both ch3 exon and the rc1 39-nt element in the duplex may account for the splicing mechanism observed. c. hamatus belongs to the radiation occurred during the most relevant cooling geological period. this may suggest that the environmental changes directed the adaptive evolution of immunoglobulin gene locus. an immunoglobulin transmembrane dimerization motif conserved throughout vertebrate evolution s varriale1.2, a merlino2, mr coscia1, v avagliano1, l mazzarella2, u oreste1 1istituto di biochimica delle proteine, cnr, napoli, italy 2dipartimento di chimica, università di napoli ‘federico ii’, napoli, italy immunoglobulins (ig), the key molecules of the adaptive immune system, are present in all vertebrate classes, but sharing very low sequence identity and differing in many features such as heavy and light chain isotype number, polymeric assembly, heavy chain domains number, carbohydrate content. however ig molecules from different species do share a few important molecular characteristics. these include diversity of the variable domains achieved by somatic recombinatorial events, dimerization of the heavy chain, alternative splicing of premrna to synthesize the secretory or the membrane-bound ig form. the functionality of the membrane-bound ig depends on its ability to link the extracellular antigen recognition event to the cytoplasmic signal transducing machinery. in the present work we are aimed at identifying the structural motif that is universally conserved in vertebrate igs, and is responsible for this activity. structural models of the igtm homodimer were obtained using ig from species of different classes: heterodontus francisci, chionodraco hamatus, pleurodeles waltl, anas platyrhinchos, and homo sapiens. several molecular dynamic simulations were performed in a lipid bilayer using two copies of models of ig transmembrane (igtm) sequences from each species, obtained by homology modeling. all predicted structures of the igtm homodimers displayed similar packing interfaces, characterized by a high degree of surface complementarity. from the analysis of the models, we identified fxxxf as the motif presumptively responsible for the interaction and, in consequence, for the receptor stability and functionality. 31 isj 4: 51-xx, 2007 isj 4: 51-54, 2007 issn 1824-307x visions and perspectives evolution game: which came first, the receptor or the ligand? m mandrioli, d malagoli, e ottaviani department of animal biology, university of modena and reggio emilia, modena, italy accepted april 24, 2007 abstract on the basis of a bioinformatic approach, we suggest that in invertebrates many ligands interact with a single, ancestral and generalized receptor driving ligand evolution. in vertebrates, on the other hand, the occurrence of gene/genome duplications induced the shift to a ligand-directed evolution of receptors. key words: cytokines; cytokine receptors; ligand-receptor evolution immunocytochemical approaches revealed that a number of invertebrate species contains mammalian cytokine-like molecules (ottaviani et al., 2004; malagoli et al., 2007). moreover, mammalian cytokines influence some invertebrate immune functions, such as cell motility, chemotaxis, phagocytosis and cytotoxicity (ottaviani et al., 2004) suggesting the presence of cytokine receptors conserved during evolution from invertebrates to vertebrates. il-7 receptor complex (kondo et al., 1993; noguchi et al., 1993; russell et al., 1993). the possibility that receptors able to bind different cytokines may be present in invertebrates prompted us to look at the evolutionary interrelationships between cytokines and their receptors from invertebrates to vertebrates. we took a comparative bioinformatic approach based on the screening of the wholly sequenced drosophila melanogaster, anopheles gambiae and caenorhabditis elegans genomes for the presence of cytokineand related receptor-coding genes. standard tools such us blast and clustalw working at both dna and protein sequence level were used. the presence of cytokine receptors has been reported for platelet-derived growth factor (pdgf)ab and transforming growth factor (tgf)-β1 (kletsas et al., 1998) in molluscan immunocytes, for interleukin (il)-1-, il-2-, il-6and interferon (ifn)-γ in sea star cells (legac et al., 1996) and for ifn-γ in tobacco hornworm larvae (parker and ourth, 1999). interestingly, studies performed on il-2 and corticotrophin-releasing hormone (crh) revealed that the mammalian cytokines il-1α, il-1β, il-2, tumor necrosis factor (tnf)-α, tnf-β and crh may bind the same receptor in molluscan immunocytes, suggesting the existence of an ancestral common receptor on the invertebrate cell membrane (ottaviani et al., 1994, 1995). this result indicates that different cytokines could interact with a single receptor in invertebrates. the question about the lead role of either receptor or the ligand during evolution could have two different replies. the first consists in a liganddirected evolution of receptors, where the former acts as selective agent in the evolution of the latter. in this case, a single ligand may have interacted with different receptors, whose structure has been optimized during evolution in order to increase the specificity of the interaction between ligand and receptor (fig. 1a). if this hypothesis is true, we should find ligands that have been strongly conserved from invertebrates to vertebrates. the second reply envisages the evolution of ligands depending on receptor structure and the receptor acting as a selective agent in the evolution of ligands. in this view, different ligands can interact with a single receptor that serves as a selective agent driving ligand evolution. this would mean that receptors which have been strongly conserved from invertebrates to vertebrates could be identified (fig. 1b). the hypothesis that a single ancestral receptor might bind different ligands is strengthened by data reporting that the mammalian γ chain of the il-2 receptor is also functionally involved in the il-4 and ___________________________________________________________________________ corresponding author: enzo ottaviani department of animal biology via campi 213/d in order to prove these hypotheses, we studied the evolutionary relationship between crh, tgf-β 41100 modena, italy e-mail: ottaviani.enzo@unimore.it 51 mailto:ottaviani.enzo@unimore.it b. receptor-directed ligand a. ligand-directed receptor a single ligand binds different receptors. a single receptor recognizes and binds different ligands. strong functional constriction of ligand sequence strong ligand conservation during evolution strong functional constriction of receptor sequence strong receptor conservation during l r r r r l l l fig. 1 comparison of the putative mechanisms involved in the evolution of ligands (l) and receptors (r) in invertebrates. family ligands and epidermal growth factor (egf) and their specific receptors. the analysis of genes coding for crh and the two related receptors showed that crh receptors are found in invertebrates with a similarity to mammalian homologues ranging from 43 % to 51 %, whereas the only conserved crh-coding gene was identified in the lepidopteran mamestra brassicae (malagoli et al., 2002). a similar evolutionary pattern was obtained with the tgfβ receptors, which showed a 49 % to 69 % similarity to mammalian homologues. the present data are in agreement with those reported in raftery et al. (1999), who confirmed that more anopheles and drosophila genes code for putative tgf-β family ligands (raftery et al., 1999). overall, these findings indicate that there are far fewer receptor-genes than ligand-coding ones (raftery et al., 1999). egf analysis gives a similar picture: egf receptorcoding genes are conserved in invertebrates with a sequence similarity ranging from 45 to 67 %. our bioinformatic analysis suggests that receptor-coding rather than ligand-coding genes are conserved between vertebrates and invertebrates and that present receptors are more similar to their ancestor than ligands. moreover, data from invertebrates suggest that more ligands may interact with a single receptor (ottaviani et al., 1994), supporting the hypothesis that ligand evolution is receptor-dependent. our assumption founded on cytokineand cytokine receptor-coding sequences is strengthened by data on the evolution of estrogen receptors (schwabe and teichmann, 2004). in particular, schwabe and teichmann (2004) suggested that the steroid receptors are much more ancient than previously thought and that the evolution of nuclear receptors is not liganddirected. using a new structure prediction algorithm, developed to find helical cytokines in human databases (conklin, 2004), we recently have found in d. melanogaster a molecule with a structure similar to that of mammalian helical cytokines (malagoli et al., 2007). this molecule may be involved in the fly immunity, however no information on its receptor is available at present. we, therefore, suggest that receptor structure undergoes a more tights constraint than ligand origin and that receptors drive ligand evolution in invertebrates. this tendency could also be valid in vertebrates, even if, given the occurrence of gene/genome duplications in vertebrate lineage (ohno, 1970; panopoulou et al., 2003), a new element has to be added to the previous receptorligand scenario. the drastic increase in genome size and gene number occurred in vertebrates as a result of two rounds of whole-genome duplication (2r hypothesis) or one complete genome duplication plus many segmental duplications (ohno, 1970; panopoulou et al., 2003). this led to the creation of additional gene copies and the evolution of new protein functions (ohno, 1970; hughes, 1999). the gene/genome duplications at the boundary between invertebrates and vertebrates are responsible for the presence of more genes coding 52 genome duplication receptor specialization for a unique ligand duplication of receptor gene brings to the presence of different receptors allowing the specialization of a receptor towards a single ligand. co-evolution of ligand and receptors r1 l1 l2 l3 r1 r1r1 r1a r1b r1c genome duplication receptor specialization for a unique ligand duplication of receptor gene brings to the presence of different receptors allowing the specialization of a receptor towards a single ligand. co-evolution of ligand and receptors r1 l1 l2 l3 r1 r1r1 r1a r1b r1c fig. 2 transition of the evolution mechanism of ligands (l) and receptors from a receptor(r)-directed mechanism to a ligand-directed one in vertebrates. for receptors in the vertebrate lineage, so allowing the differentiation of receptors towards different ligands. in vertebrates, therefore, we observe a transition from common receptors for different ligands to a specific receptor for each ligand. this phenomenon modifies the invertebrate relationship between receptors and ligands, and permits ligands to drive the evolution of newly duplicated receptors (fig. 2). the transition from a protein with a generalized recognition to more specialized proteins is a common model in evolutionary biology, as observed in the evolution of enzymes (jensen, 1976; o'brien and herschlag, 1999). hughes al. phylogenies of developmentally important proteins do not support the hypothesis of two rounds of genome duplication early in vertebrate history. j. mol. evol. 48: 565–576, 1999. jensen ra. enzyme recruitment in evolution of new function. annu. rev. microbiol. 30: 409-425, 1976. kletsas d, sassi d, franchini a, ottaviani e. pdgf and tgf-β induce cell shape changes in invertebrate immunocytes via specific cell surface receptors. eur. j. cell biol. 75: 362366, 1998. kondo m, takeshita t, ishii n, nakamura m, watanabe s, arai k, et al. sharing of the interleukin-2 (il-2) receptor gamma chain between receptors for il-2 and il-4. science 262: 1874-1877, 1993. in conclusion on the basis of the present bioinformatic findings and reports in the literature, we suggest that in invertebrates many ligands interact with a single, ancestral and generalized receptor driving ligand evolution. in vertebrates, on the other hand, the occurrence of gene/genome duplications induced the transition to a liganddirected evolution of receptors. this transition represented an evolutive advantage because it couples a more refine signalling to a minor sensitivity to receptor gene mutation. legac eg, vaugier l, bousquet f, bajelan m, leclerc d. primitive cytokines and cytokine receptors in invertebrates: the sea star asterias rubens as a model of study. scand. j. immunol. 44: 375-380, 1996. malagoli d, mandrioli m, ottaviani e. cloning and characterisation of a procorticotrophin-releasing hormone in the izd-mb-0503 immunocyte line from the insect mamestra brassicae. peptides 23: 1829-1836, 2002. acknowledgements this work was supported by miur (italy) grants to eo. malagoli d, conklin d, sacchi s, mandrioli m, ottaviani e. a putative helical cytokine functioning in innate immune signalling in drosophila melanogaster. biochim. biophys. acta 1770: 974-978, 2007. references conklin d. recognition of the helical cytokine fold, j. comput. biol. 11: 1189-1200, 2004. 53 noguchi m, nakamura y, russell sm, ziegler sf, tsang m, cao x, et al. interleukin-2 receptor gamma chain: a functional component of the interleukin-7 receptor. science 262: 1877-1880, 1993. panopoulou g, hennig s, groth d, krause a, poustka aj, herwig r, et al. new evidence for genome-wide duplications at the origin of vertebrates using an amphioxus gene set and completed animal genomes. genome res. 13: 1056-1066, 2003. o'brien pj, herschlag d. catalytic promiscuity and the evolution of new enzymatic activities. chem. biol. 6: r91-r105, 1999. parker ms, ourth dd. specific binding of human interferon-gamma to particulates from hemolymph and protocerebrum of tobacco hornworm (manduca sexta) larvae. comp. biochem. physiol. 122b: 155-63, 1999. ohno s. evolution by gene duplication. springerverlag, new york, 1970. ottaviani e, caselgrandi e, franceschi c. cytokines and evolution: in vitro effects of il-1α, il-1β, tnf-α and tnf-β on an ancestral type of stress response. biochem. biophys. res. commun. 207: 288-292, 1995. raftery la, sutherland dj. tgf-beta family signal transduction in drosophila development: from mad to smads. dev. biol. 210: 251-268, 1999. russell sm, keegan ad, harada n, nakamura y, noguchi m, leland p, et al. interleukin-2 receptor gamma chain: a functional component of the interleukin-4 receptor. science 262: 1880-1883, 1993. ottaviani e, franchini a, caselgrandi e, cossarizza a, franceschi c. relationship between corticotropin-releasing factor and interleukin-2: evolutionary evidence. febs lett. 351: 19 -21, 1994. schwabe jw, teichmann sa. nuclear receptors: the evolution of diversity. sci. stke 217: pe4, 2004. ottaviani e, malagoli d, franchini a. invertebrate humoral factors: cytokines as mediators of cell survival. prog. mol. subcell. biol. 34: 1-25, 2004. 54 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice isj 5: xxx-yyy, 2008 isj 5: 162-179, 2008 issn 1824-307x review the antimicrobial peptides of the immune response of shrimp xf zhao, jx wang school of life sciences, shandong university, jinan, shandong, china accepted october 23, 2008 abstract the cultivation of penaeid shrimp is a worldwide economic activity which has the potential to contribute to increasing shrimp production. however, penaeid shrimps are susceptible to bacterial and viral diseases, and may thus cause significant losses to the aquaculture industry. in view of this, it is imperative to understand the immune response of shrimp against pathogens as this could help in devising efficient strategies to control, and eventually eradicate, shrimp diseases. at present, a considerable number of research studies on the identification and characterization of antimicrobial peptides/proteins (amps) in penaeid shrimps. such research activities will contribute to finding solutions to shrimp diseases. amps are widespread in animals and plants, involved in their innate immunity, and considered as the front liners of host defense against pathogens. in penaeid shrimps, eight kinds of amps have been found. these are the penaeidins, whey acidic protein (wap) domain containing proteins [crustins and single wap domain containing peptides (swd)], antilipopolysaccharide factors (alfs), lysozymes, a c-type lectin, histones, anionic hemocyanins, and peritrophins. in this study, the structures, distributions, expression profiles, phylogenetic evolution, and functions of some amps are discussed, focusing on the wap-domain containing peptides and alf in penaeid shrimp. key words: antimicrobial peptides; innate defense effectors; innate immunity; penaeid shrimp introduction the cultivation of penaeid shrimp is an important economic activity in the world. this industry, however, has been suffering serious problems brought by viral and bacterial diseases. one specific disease is the white spot syndrome virus (wssv) infection which has caused a drastic decline in production and multi-national economic losses. based on the report of the fisheries and aquaculture department of food and agriculture organization (2007), there is an exceeding 2.4 million tons per annum of shrimp global aquaculture production. however, up to 25 % of this production was estimated to have been lost due to diseases. given that pathogenic diseases are one significant cause of production and economic loss in the shrimp industry, this phenomenon calls for an urgent understanding of the immune defenses of shrimp. at present, research studies are being conducted to ___________________________________________________________________________ corresponding author: jin-xing wang school of life sciences shandong university jinan, shandong 250100, china e-mail: jxwang@sdu.edu.cn examine the innate immunity of shrimp, and such activities have been continuously contributing to the development of shrimp aquaculture. in fact, it has been found that comparable to insects, the innate defense of shrimp is triggered by the pattern recognition receptors, such as the gram-negative binding proteins (vargas-albores et al., 1997; yepizplascencia et al., 1998; jimenez-vega et al., 2002; roux et al., 2002; sritunyalucksana et al., 2002; romo-figueroa et al., 2004; cheng et al., 2005; du et al., 2007; lin et al., 2008) and the c-type lectins (luo et al., 2006; liu et al., 2007; sun et al., 2008). generally, the recognition of non-self activates a proteolytic cascade of serine proteases that amplify the signal and trigger downstream effector responses. this becomes possible through the signal transduction pathways which lead to the elimination of the invader. moreover, the serine proteinase and its inhibitors was found in shrimp (okumura, 2007), and the toll-like receptors and other signal pathway molecules also reported in several shrimp (arts et al., 2007; yang et al., 2007; yang et al., 2008). anti-microbial peptides (amps) are a diverse group of innate immune effector molecules in multi162 cellular organisms. they are considered as effector molecules for immune cells that prevent or withstand microbial infection. similar to those found in other animals, amps are also key factors in the innate immunity of shrimp (bachère et al., 2004). since amps play a significant role in the inherent immunity of shrimp, research studies have been actively focusing on the identification and characterization of amps in penaeid shrimp. in fact, eight kinds of amps have been found in penaeid shrimps. these are the penaeidins (destoumieux et al., 1997, 1999, 2000; cuthbertson et al., 2004; muñoz et al., 2004; kang et al., 2004, 2007), whey acidic protein (wap) domain containing proteins [crustins and single wap containing peptides (swd)] (gross et al., 2001; amparyup et al., 2008a; jia et al., in press), antilipopolysaccharide factors (alfs) (gross et al., 2001; liu et al., 2005; liu 2006; somboonwiwat et al., 2005, 2008; tharntada et al., 2008), histones (patat et al., 2004), hemocyanin (destoumieux-garzon et al., 2001; zhang et al., 2004), lysozymes (hikima et al., 2003 sotelo-mundo et al., 2003; bu et al., 2008; de la re vega et al., 2006; burge et al., 2007; xing et al., in press), a c-type lectin (sun et al., 2008), and peritrophins (loongyai et al., 2007). this study presents a discussion of the structures, distributions, phylogenetic evolution, expression profiles, and functions of some amps, particularly on the wap-domain containing peptides and alf from penaeid shrimp. whey acidic protein (wap)-domain containing peptides (wdps) a major milk protein in most mammals, wap, has eight cysteine residues arranged to form a tightly packed structure called a four-disulphide core (4-dsc) at the carboxyl terminus (hennighausen and sippel, 1982). these wap domain-containing proteins are found to prevail among metazoans (beg, 1995; devinoy et al., 1988; ali et al., 2002; carro et al., 2004; furutani et al., 2004). they are further found to have highly diverse biological functions, including proteinase inhibition (ranganathan et al., 1999; schalkwijk et al., 1999; ota et al., 2002), antimicrobial activity (relf et al., 1999; hagiwara et al., 2003), and association to ovulation (garczynski et al., 1997). in addition, wap-domain proteins have antiviral functions, specifically against the human immunodeficiency virus (alvarez et al., 2008). crustins, which are anti-microbial peptides containing a wap-domain, were first identified in the shore crab carcinus maenas, characterized as cysteine-rich 11.5 kda antimicrobial peptides which function against gram-positive bacteria (relf et al., 1999). there have been more than 50 crustins or crustin-like peptides reported to have been found from a variety of decapods, including crabs, lobsters, shrimp, and crayfish (refer to the review of smith et al., 2008). in this study, they were termed as wap-domain containing peptides (or wdps) and were classified into two sub-families, namely, crustins and swd (the justification for such is discussed below). these wdps are apparently a large family of antimicrobial peptides ubiquitous among penaeid shrimp. in fact, the cdnas of crustins and wdps have been reported to be present in a variety of penaeid shrimp, including litopenaeus vannamei, litopenaeus setiferus (gross et al., 2001; bartlett et al., 2002; vargasalbores et al., 2004), penaeus monodon (chen et al., 2004; supungul et al., 2004, 2008; amparyup et al., 2008a, b), marsupenaeus japonicus (rattanachai et al., 2004), fenneropenaeus chinensis (zhang et al., 2007; jia et al., in press), farfantepenaeus paulensis, farfantepenaeus subtilis, farfantepenaeus brasiliensis, and litopenaeus schmitti (rosa et al., 2007). accordingly, the crustins in shrimp are diverse in amino acid sequences. however, they are conserved with the c-terminus of 12 cysteine residues, thereby leading it to be termed as crustindomain, in which a single wap domain is contained. the swds have no crustin domain, and only contain a single wap-domain (8 cysteine residues). classification of shrimp wdps recently, the crustins (wpds) in crustaceans were comprehensively reviewed by smith et al. (2008). in the review they discussed three main types of crustins (crustin type i, ii and iii) in crustaceans. here, we focused on the crustins and swds in penaeid shrimp, including the new functions of the peptides. there have been several studies that have revealed the presence of crustins and swds in different penaeid shrimp species (refer to the above citations), in this study, most of the sequences wdps in penaeid shrimp and in other crustaceans were collected, including some expressed sequence tags (est) from the genbank database. a multiple alignment analysis for the amino acid sequences (fig. 1) and the phylogenetic analysis of the proteins were performed. the neighbor-joining tree revealed that the wdps in crustaceans could be divided into four different clusters (fig. 2), namely, crustins i and ii, carcinin and carcinin-like peptides, and the swd. it is noteworthy to mention that in this study, our classification is considerably different from that of smith et al. (2008). the crustin type i that they discussed in their study is similar to carcinin and carcinin-like peptide discussed in this study. similarly, the crustin type ii is equivalent to our crustins i and ii, and the crustin type iii is equivalent to our swds. the wdps in the penaeid shrimp are divided into three classes, namely, crustin i, crustin ii, and swds. the first class is characterized by the following: (1) a relatively conserved signal peptide, (2) an n-terminal glycine-rich domain, and (3) a cterminal cysteine-rich domain (12-cysteine crustin domain) with the following signature: c1(x3)c2(xx)c3c4(x16)c5(x6)c6(xn)c7(x5)c8(x5) c9(x5)c9c10(x3)c11(x5)c12. the second class of crustins have similar sequence domains as class i, but in terms of signal peptide, there are sequence differences between them. another difference between them lies in the sequence of their crustin domain, as crustin ii have the following signature: c1(x2)c2(x7)c3c4(x4)c5(x6)c6(xn)c7(x5)c8(x5)c9 c10(x3)c11(x5)c12, specifically in the residue numbers between c4 and c5 (fig.1a). finally, the 163 164 fig. 1 alignment of amino acid sequences of crustins (a), and swds (b), and the domain signature of penaeid shrimp and other crustacea (c). fch, fenneropenaeus chinensis; lse, litopenaeus setiferus; lva, litopenaeus vannamei; mja, marsupenaeus japonicus; pmo, penaeus monodon; the sequences of signal peptides are presented in yellow, the identical cysteines that characterize crustin or wap domain are in purple, and the wap domains are shown in blue. class iii wdps are single wap domain-containing peptides (swd) which are characterized by the following: (1) a highly conserved signal peptide, (2) a prolineand arginine-rich motif between the signal peptide and the wap domain, and (3) a wap-domain (8 cysteine residues) in the c-terminus with the signature: c1(x9)c2(x6)c3(x5)c4(x5)c5c6(x3)c(x3)c (fig.1b). in crustaceans, carcinin and carcinin-like peptides have a signal peptide and a crustin domain, without the n-terminal glycine-rich domain. moreover, swds are significantly different from crustins i and ii, and carcinins because they have no crustin-domain in their sequences. we therefore consider the idea that the four groups of wdps in crustaceans should be divided into two sub-families, namely, the crustins (which present crustin-domain in their sequences) and the swds (which only have wap-domain). as such, these two sub-families also have different functions in vitro (as discussed below). structure comparison of shrimp wdps with other amps to date, a wide variety of amps in metazoans have been identified. on the basis of sequence and structural features, these cationic amps can be grouped into three classes: (i) the linear peptides which form α-helices and do not contain cysteine residues; (ii) the cyclic peptides which contain cysteine residues; and (iii) the peptides with an over-representation in one or two residues, such as proline, glycine, arginine, and tryptophan (bulet et al., 2004). several antibacterial glycine-rich polypeptides have been isolated from various insect species. they are actually considered effective against gram-negative bacteria and are inactive against gram-positive bacteria, yeasts and mammalian cell lines (mackintosh et al., 1998). another example is that of short-chain proline-rich peptides, which are mostly active against gram-negative bacteria, while the gram-positive cells remain generally unaffected (bulet et al., 1999). furthermore, the cyclic peptides containing cysteine residues, like insect defensins, are active against a wide range of gram-positive bacteria and only for a few gramnegative bacteria, fungi and yeasts (bulet et al., 1999). crustins i and ii are composed of an n-terminal glycine-rich domain, and a c-terminal region which contains 12 cysteine residues (crustin domain) organized in two doublets. these crustins are similar with the two classes of insect amps, that is glycine-rich peptides and cyclic peptides containing cysteine residues (bulet et al., 1999). on the other hand, the swds are composed of a short prolinearginine-rich region and a c-terminal region containing 8 cysteine residues (the wap domain). they are also similar in terms of the two classes of insect amps, particularly the cyclic peptides containing cysteine residues and the prolineand arginine-rich peptides. similar to the penaeidins found in shrimp, the crustins and the swd are chimera molecules of glycineor proline-rich amps and cysteine-rich amps. chimera-like features often reflect the multifunctional properties of a molecule, such that each domain performs different functions. for example, crustins i and ii have glycine-rich and crustin domains, and therefore should have anti-gram positive and negative bacterial activities. supungul et al. (2008) reported that crustinpm1 (which belongs to crustin i) exhibited anti-microbial activity against only a gram-positive bacteria, whereas the rcrus-likepm (crustin ii) showed remarkable antimicrobial activity against both gram-positive and negative bacteria (amparyup et al., 2008b). likewise, the crufc (crustin ii) from f. chinensis exhibited high activity against gram-positive bacteria but low activity was exhibited against gram-negative bacteria and fungi (zhang et al., 2007). the swds have proand arg-rich and wap domains. they exhibit the following activities: relatively high against gram-positive and/or negative 165 lsecrus2 af430078 lvacrus2 af430072 lvacrus ay488497 lvacrus ay488494 lvacrus3 af430073 lvacrus ay488495 lvacrus ay488496 lvacrus ay488493 lvacrus ay488492 lvacrus1 af430071 fpacrus ef182747 fsu crusef450744 lsecrus ef182748 fbrcrus ef601055 mjacrus1 ab121740 mjacrus3 ab121742 mjacrus5 ab121744 mjacrus2 ab121741 mjacrus4 ab121743 lsecrus1 af430077 lsecrus3 af430079 fchcrus ay871268 pmocrus3 bi018073 pmocrus1 cf415873 pmocrus4 cf415873 pmocrus2 bi018072 pmocrusee661627 plecrus2 ef523613 cmacar aj237947 cmacari aj821886 cmacarii aj821887 cmacariii aj821888 cmacar aj427538 cmacariv aj821889 fchcrus dq097703 pmocrus ef654658 pmoswd eu623981 mjaswd au176270 lvaswd ay465833 pmoswd eu623980 fchswd ef216349 pmoswd ay464465 pmoswd eu62397973 89 56 77 96 99 68 52 100 9099 100 89 98 84 60 71 76 99 95 56 64 5444 12 10 39 0.2 c ru st in i c ar ci ni nlik e c ru st in ii sw d fig. 2 phylogenetic analysis of wap containing proteins/ peptides in penaeid shrimps by mega 4. five thousand bootstraps were performed for the neighbour-joining trees to verify the reliability of the results. cma, carcinus maenas; ham, homarus americanus; hga, homarus gammarus; par, panulirus argus; ple, pacifastacus leniusculus. others are the same with fig. 1. 166 bacteria, moderate against fungi, and strong antiproteinase activity, especially against the bacterial proteinases (amparyup et al., 2008a; jia et al., in press). therefore, they are bi-function peptides. from above results, we can see that the primary structures of amp are not corresponding to their functions. it need further study for their tertiary structures. expression profiles and functions of shrimp wdps the spatio-temporal expression of wdps in shrimp was not well-understood. most of them seem to be constitutively expressed in the hemocytes. apparently, the expression patterns was only reported during the development of the larvae of shrimp, p. monodon. high level expression of a crustin are recorded at all stages of development from the nauplii stage iv to juvenile period(jiravanichpaisal et al., 2007). furthermore, the expression patterns of wdps to bacterial challenge were reported in several shrimps, but the results showed no consistent patterns of change in expression subsequent to bacterial injection. vargas-albores et al. (2004) found two isoforms of crustin i in l. vannamei, which showed different expression patterns after bacterial inoculation. first, crustin-p seems to be constitutively expressed, and second, the crustin-i mrna concentration drops after 6 h. results also revealed that there was a decrease in the transcribed expression of crustin in p. monodon subjected to bacterial challenge (supungul et al., 2004). in hemocytes, the m. japonicus crustin-like peptide mrna was identified, and the expression level of this peptide mrna increased significantly 1, 3, and 7 days after peptidoglycan feeding (rattanachai et al., 2004). the crus pm1 (crustin i) in p. monodon was also expressed in hemocytes, but the expression profile was not analyzed (supungul et al., 2008). the mrna transcript of a crus-like pm2 (crustin ii) in p. monodon was found to be abundantly expressed in hemocytes and was significantly up-regulated after vibrio harveyi injection (amparyup et al., 2008b). jiménez-vega et al. (2004) reported that the expression of the swd gene in l. vannamei hemocytes increased after 3 to 6 h it was inoculated with v. alginolyticus, but slowly returned to nonstimulated levels within 12 to 24 h. the fc-swd from chinese shrimp is constitutively expressed and increased in hemocytes 24 h after bacterial challenge (staphylococcus aureus and vibrio anguillarum). the results of the reverse transcriptase-polymerase chain reaction (rtpcr) analysis revealed a weak expression in heart and gill and challenged stomachs in addition to hemocytes. moreover, results showed that the signal from challenged tissues was stronger than from those unchallenged. consequently, these results suggest that fc-swd is an inducible gene and is essential in responding to bacterial infection (jia et al., in press). the tissue distribution of swds in p. monodon was as well analyzed through rtpcr. results indicated the presence of all three swd transcripts in hemocytes. the transcript expression of swdpm1 was down-regulated upon injection with s. aureus while no change was recorded in temrs of swdpm2 and swdpm3 expressions. contrastingly, the results obtained from the wssv injection showed that in a biphasic response, there was an up-regulation of the swdpm1 and swdpm2 transcripts at 6 h followed by a significant down-regulation by 24 h after infection (amparyup et al., 2008a). the wdps are a large family of antimicrobial effectors in shrimp immunity. in fact, more than 30 wdps, including isoforms and ests, have been found in shrimp. despite this, many of them are poorly characterized for their functions. one of the p. monodon crustins (crustin i), recombinant expressed in e. coli, exhibited an antimicrobial activity against only gram-positive bacteria, specifically with strong inhibition against s. aureus and streptococcus iniae (supungul et al., 2008). zhang et al. (2007) similarly reported that the recombinant crusfc (crustin ii) had relatively high activities against gram-positive bacteria and low activities against gram-negative bacteria and fungi. moreover, another recombinant crustin ii (ef654658) from p. monodon has been recently reported to have a strong activity not only against gram-positive bacteria, but also against gramnegative bacteria, such as escherichia coli 363 and vibrio harveyi (amparyup et al., 2008b). in penaeid shrimp, there were less than 10 swds found, and two of them were studied for their functions in vitro. the functions of swd molecule from chinese shrimp were analyzed (jia et al., in press). the recombinant fc-swd has manifested antimicrobial activities against gram-positive and gram-negative bacteria and fungi, as well as a strong inhibitory activity against subtilisin a and protein k with an inhibition constant (ki) of 2.14 nm and 2.27 nm, respectively; but a much lesser activity against trypsin was recorded. amparyup et al. (2008a) also analyzed the biological functions of recombinant swdpm. based on the results they obtained, recombinant swdpm exhibits activity against several gram-positive, but not gramnegative bacteria and is a competitive inhibitor of subtilisin a with an inhibition constant (ki) of 1.98 nm. this phenomenon indicates the dual functions of swds, that is antimicrobial activity and antiproteinase activity against pathogenic proteinase. therefore, these swds might have an important role in the immunity of shrimp in vivo. so far, it is generally accepted that both activities could not co-exist in the same (unique) domain. why does swd show both anti-microbial and anti-proteinase activities? in fact, similar situations were found in some peptides with a single domain, which is the avian wap (awap iv) originally found in chicks (townes et al., 2006). this has a broad-spectrum of antibacterial activities against both gram-positive and gram-negative bacteria. in addition to that, the avian wap lysate significantly inhibited the activities of the microbial serine proteinases subtilisin and proteinase k. furthermore, li et al. (2007) reported the occurrence of a small serine proteinase inhibitor with antimicrobial capability in a diskless-fingered odorous frog, odorrana grahami. based on a disulfide-bridged hendecapeptide loop of this serine 167 crustin ii swd double wap domain protein wap domain crustin i fig. 3 the possible divergent evolution of wpds in crustaceans. proteinase inhibitor, a series of peptides have been synthesized. they found that seven synthetic peptides exhibited trypsin inhibitory activity, while the other five have both the trypsin inhibitory and antimicrobial activities. in terms of swds, the findings showed that they have high anti-proteinase activities to bacterial serine proteinases (subtilisin a and proteinase k) and antimicrobial activities. the recombinant wap domain (fc-wapd) shows relatively low activities against the bacterial serine proteinases (jia et al., in press), and manifests quite a low activity against bacteria. comparing their sequence, it was found that fc-swd contain higher positively charged amino acids than the fc-wapd. the net charge of fc-swd is +4, while that of fc-wapd is − 2. in addition, it is generally known that most antimicrobial cationic peptides have the same unique features, that is, they are both polycationic (having a net positive charge of more than +2) and fold into amphipathic structures (having both a hydrophobic and a hydrophilic domain). as such, these characteristics enable them to interact with the negatively charged surface molecule of bacteria and to interact with and penetrate into the negatively charged cytoplasmic membranes of most bacteria (hancock, 1997). this is therefore the reason for the high anti-microbial activity of fc-swd compared to that of fc-wapd’s low activity against bacteria (jia et al., in press). the possible divergent evolution of the wap domain in crustaceans the wap domain was initially identified in the primary milk protein of rats and mice (hennighausen and sippel, 1982). wap proteins have one or more wap domains containing about 50 amino acids with eight highly conserved cysteine residues that form a four-disulphide core (4-dsc). amino acids in the wap domain, except for the conserved cysteine residues, are significantly diverse, and proteins with a wap motif perform variety of functions. in fact, a large biological diversity exists between the proteins that contain one or two wap domains, with many being identified as proteinase inhibitors or amps. the most studied wap proteins, for example, are the elafin and antileukoproteinase, which are two serine-proteinase inhibitors with anti-microbial and inflammatory activity (bouchard et al., 2006). in crustaceans, several wap domain-containing proteins were found. in addition to the aforementioned four wap-containing proteins, double wap domain containing proteins were also reported in shrimp [the l. vannamei secretory leukocyte proteinase inhibitor, ef467169; the m. japonicus double wap domain-containing protein, eu095018; the f. chinensis double wap domaincontaining protein (our unpublished data)]. many identified genes that code for wap proteins in human are clustered on chromosome 20q12–13.1 (bouchard et al., 2006). the results of southern analysis show that a large family of sequences related to the crustins is present in l. vannamei genome (bartlett et al., 2002). this may indicate some similarities in gene locations. based on the domain structure and functions of the wapcontaining proteins in crustaceans, we therefore propose the possible divergent evolution of wap domain in crustaceans (fig. 3). in this proposal, the different groups may have different functions, including antimicrobial and anti-proteinase activities among others. alf factors alf, a basic peptide, was initially found as a potent anticoagulant from horseshoe crabs, limulus polyphemus and tachypleus tridentatus, which inhibited the endotoxin mediated activation of the coagulation cascade (tanaka et al., 1982). thereafter, several studies demonstrated that the alf from hemocytes of the horseshoe crab l. polyphemus have similar characteristics with that of the binding and neutralizing lipopolysaccharide (lps). additionally, alf was indicated to have a strong antibacterial activity, particularly on the growth of gram-negative bacteria (morita et al., 1985; aketagawa et al., 1986; muta et al., 1987). in shrimp, the cdna clones homologous to the horseshoe crab alfs were initially identified in hemocytes of p. monodon and l. setiferus by means of est analysis (gross et al., 2001; supungul et al., 2004). in recent years, there have been a growing number of studies on shrimp alf available to provide pertinent information. to note, several alfs have been isolated and characterized from hemocytes in penaeid shrimp, f. chinensis (liu 168 http://www.ncbi.nlm.nih.gov/pubmed/9033483?ordinalpos=14&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvdocsum fig. 4 alignment of alfs-based amino acid sequence. all sequences of alfs are from genbank. fch, fenneropenaeus chinensis; fpa, farfantepenaeus paulensis; ham, homarus americanus; lse, litopenaeus setiferus; lsc, lst; litopenaeus stylirostris:litopenaeus schmitti; lva, litopenaeus vannamei; mja, marsupenaeus japonicus; mol, macrobrachium olfersii; pmo, penaeus monodon; and ple, pacifastacus leniusculus, lpo, limulus polyphemus. 169 et al., 2005; zhou et al., 2008) m. japonicus (nagoshi et al., 2006), p. monodon (supungul et al., 2004; somboonwiwat et al., 2005; tharntada et al., 2008), l. vannamei (de la vega et al., 2008), l. setiferus (gross et al., 2001), f. paulensis (ef601051, ef601054), l. schmitti (dq991357), and l. stylirostris (dq010421). classification of shrimp alf twenty-nine crustacean alf sequences collected from genbank were aligned using the alignment editor of the mega 4 software (tamura et al., 2007). the results obtained by the multiple alignment (fig. 4) revealed that all the molecules of alfs contain two preserved cysteine residues which form a disulfide bridge. it was also found out that alf contained a relatively conserved sequence of a positively-charged amino acid residue cluster within the disulfide loop. this structure of the βhairpin loop in shrimp alf suggests a conservation of the lps binding activity. with the use of neighbor joining method of mega 4, a phylogenetic tree was constructed (tamura et al., 2007). to attain and assess the reliability of the tree, bootstrapping using 5,000 replications was as well performed. the phylogenetic tree analysis categorized the various alf proteins into three main groups. accordingly, most alfs from shrimp belong to cluster i, including the alfs from l. setiferus (white gulf shrimp); then cluster ii contains the l. stylirostris (blue shrimp) and eriocheir sinensis (chinese mitten crab). finally, cluster iii contains the alfs from l. vannamei (pacific white shrimp), p. monodon (black tiger shrimp) and other crustacean pacifastacus leniusculus (signal crayfish), l. polyphemus (atlantic horseshoe crab), and tachypleus tridentatus (horseshoe crab) (fig. 5). it can be observed that in l. vanamei and p. monodon, two kinds of alfs. expression profiles and functions in shrimp innate immunity the transcription of alfs in some species was specified in tissues. in chinese shrimp (f. chinensis), for example, the alf (cluster i) has high expression in hemocytes, gills, and intestine but exhibited low expression in ovary, hepatopancreas, and muscle (liu et al., 2005). similar observations had been reported in p. monodon, in which alf (cluster i) was constitutively expressed in hemocytes, hearts, gills, intestines, and lymphoid organs, while there was no observed transcription in the hepatopancreas (supungul et al., 2004). another example is that in the kuruma shrimp (m. japonicus), in which the alf (cluster i) was reported to be expressed at higher levels in hemocytes, lymphoid organs, hearts, intestines, and gills. the expression of alfs, on the other hand, was found to be at lower levels in stomachs, hepatopancreas, and muscles (nagoshi et al., 2006). in l. vannamei, alf1 (which belongs to cluster iii ) was found to have high mrna levels in the lymphoid organ and heart, intermediate levels in the gills, eyestalk, and hemocytes, and very low levels in the muscle and hepatopancreas (de la vega et al., 2008). the aforementioned reports are considerably in contrast with the patterns of many other amps in shrimp which are carried out primarily in hemocytes (munoz et al., 2002; bachère et al., 2004; kang et al., 2004). it is important to note that in shrimp, however, it is clear that alf transcription, although it is tissue-specific, occurs in multiple organs and could thereby provide systemic protection against pathogens. the transcription of the alf is induced upon bacterial challenge in several shrimp (supungul et al., 2004; liu et al., 2005; nagoshi et al., 2006). in a study, alf was induced upon wssv infection or exposed to uv-inactivated wssv (liu et al., 2006). on the other hand, in l. vanname, the infection with pathogenic bacterium, vibrio penaeicida or fungus, fusarium oxysporum, did not cause any significant change in the lvalf1 mrna levels compared to saline-injected controls. an considerable increase was recorded in the lvalf1 mrna expression in wssv-infected shrimp at 54-h time point (de la vega et al., 2008). similarly, in l. vannamei, alf was significantly up-regulated in the infected viral hepatopancreas (robalino et al., 2007). this upregulation of antimicrobial proteins as a response to viral infection has also been reported in drosophila (zambon et al., 2005). despite these multitude studies, the mechanism in anti-viral responses of alf still needs to be clarified. the results reported by different authors indicate the inconsistent expression patterns of the alfs after the shrimps have been injected with bacteria or viruses. as such, this further indicates the different functions in vivo of the alfs. to date, only a few alfs and their characteristics have been analyzed. in p. monodon, alfpm3, a predominant antimicrobial peptide, was identified in both the unchallenged and v. harveyichallenged shrimp (supungul et al., 2004). in their study, a strong activity against multiple grampositive and gram-negative bacteria and filamentous fungi (somboonwiwat et al., 2005) has been recorded. another study found out that a synthetic peptide, corresponding to the lps-binding domain of mj-alf from m. japonicus, exhibited a lps-neutralizing and hemolytic activities on lpssensitized human red blood cells (nagoshi et al., 2006). moreover, alf was found to have a potential in interfering with the replication of wssv in crayfish p. leniusculus (liu et al., 2006). the in vivo function of alf in protecting shrimp from bacterial, fungal, and viral infections was also studied in l vanamei through the rna interference (rnai) method (de la vega et al., 2008). the injection of double-stranded rna (dsrna) corresponding to the lvalf1 message resulted in a significant reduction of the abundant lvalf1 mrna levels. following knockdown of the lvalf1, the shrimps were challenged with low pathogenic doses of pathogenic v. penaeicida, or f. oxysporum. the results showed a significant increase in the mortality among the lvalf1 low-level shrimps, specifically those with v. penaeicida and f. oxysporum infections, compared to the control shrimps. the result showed that this gene functions in protecting shrimp from both bacterial and fungal infections. in the viral challenge using wssv, the alf dsrna injection caused no significant increase in mortality 170 fig. 5 phylogenetic analysis of alfs from shrimp using the sequence information from fig. 4. compared to the non-specific dsrna controls in the l. vannamei (de la vega et al., 2008). other experiments that focused on alf rnai in freshwater p. leniusculus indicated that alf can protect against wssv infection, as observed in the alf low-level through rnai, which specifically resulted in higher rates of viral propagation (liu et al., 2006). the differences in the two above-mentioned reports emphasize the diverse taxonomic groups of crustacea and their varying responses to infection and immunity mechanisms (de la vega et al., 2008). possible mechanisms of lps binding and antimicrobial activity alfs contain two conserved cysteine residues which form a disulfide bridge. they also contain a relatively conserved sequence of a positively charged amino acid residue cluster within the disulfide loop, which is regarded as alf’s functional domain. hoess et al. (1993) reported the high resolution structure of a recombinant limulus-alf (l-alf). in their report, they stressed that it contains an n-terminal α-helix (that opens into a α-helix in its final turn), a simple four-stranded anti-parallel βsheet, and two c-terminal α-helices. the three helices form a bundle that packs against the β-sheet and encloses a hydrophobic and highly-aromatic core. their study performed a structural analysis of l-alf through the x-ray crystallography, and the results demonstrated the β-hairpin loop with an alternating series of hydrophilic (mainly basic amino acids) in the central disulfide-bonded loop region thus obtaining an amphipathic protein molecule (hoess et al., 1993). this amphipathic loop structure is believed to be a lps-binding motif which can bind a single fatty acid with the phosphoglucosamine portion of lipid a (the membrane anchor of lps). this amphipathic loop of l-alf were compared to the alf sequences (cys1st to cys2nd ) of shrimp, and the results revealed similarity between the alternating residues pattern (fig. 4). as such, the structure of the β-hairpin loop in shrimp alf suggests that there is a conservation of lps binding activity. somboonwiwat et al. (2008) also studied the binding activity of ralfpm3 and found that it could strongly bind to both gram negative and gram positive bacterial cells. further analysis demonstrated that alfpm3 could bind to both the immobilized lps and lipoteichoic acid (lta), with a high dissociation constant (kd) of 1.26×10 −8 and 1.34×10−8 m, respectively. this suggested that they lva dq208701 lva dq208706 lva dq208702 lva dq208705 lva dq208703 lva dq208704 lsc dq991357 fpa ef601054 fpa ef601051 fpa ef601052 fpa ef601053 fch ay859500 fch-alf-2 pmo eu617325 pmo ef523562 pmo ef523559 pmo ef523563 ham eu625516 mol eu289220 ham eu625517 lva alf2 mja ab210110 esi dq793214 lse be846661 lst dq010421 ple ef523760 lpo 1307201a ttr p07087 lva alf1 pmo ef523561 100 100 100 99 96 55 76 58 78 39 76 91 44 25 80 93 93 96 58 51 29 60 71 39 80 89 0.1 171 i ii iii lva af387661 lva ax006138 lva af387662 lva af390145 lva af390139 lva y14926 lva af387663 lva dq206403 lva y14928 lva y14927 fpe eu333491 lva af387660 fpe eu333492 lva af390143 lva dq211700 lva af390142 lva af390140 lva af390144 lva af390141 lst dq010422 lva ay351656 lse ay039204 lse ay039206 lse ay039202 lse ay039203 fch ay260151 fch dq308408 pmo af475082 pmo ay326471 lsc ay956420 lse ay039207 lva dq211701 lva af390149 lva af390147 lva dq206402 fbr ef450745 fsu ef450742 fpa ay956416 fpa ay956417 lsc ay956418 lse ay039205 lsc ay956419 lst ay351655 lva dq211699 lva af390146 lva dq206401 lva ax006136 lva y14925 fch dq153253 fch dq308407 fch ay669323 fch dq154152 64 75 58 99 43 88 99 12 67 29 10 99 97 97 46 99 60 11 2 99 39 19 99 53 31 47 99 99 80 76 92 85 71 67 59 86 50 25 0.000.050.100.150.200.25 iv ii v iii fig. 6 phylogenetic analysis of penaeidins from shrimp using the sequence information from genbank. the upgma tree was obtained using mega with complete deletions of gaps. bootstraps (5000) were performed for the upgma trees to verify repeatability and reliability of results. fch, fenneropenaeus chinensis; fpa, farfantepenaeus paulensis; fpe, fenneropenaeus penicillatus; fsu, farfantepenaeus subtilis; fbr, farfantepenaeus brasiliensis; lva, litopenaeus vannamei; lst, litopenaeus stylirostris; lsc, litopenaeus schmitti; lse, litopenaeus setiferus, and pmo, penaeus monodon 172 are at least one of the target molecules for the alfpm3 on gram-negative and gram-positive bacteria, respectively. assuming that alf binds to bacterial cells before they exterminate the cells, it is still unknown as to how such extermination is performed. alf has both the ability to inhibit the endotoxin or lps mediated coagulation system and to exhibit strong anti-microbial activity against the gramnegative and gram-positive bacteria and fungi. thus, alf is also one of the pivotal effectors in shrimp immunity. nes (bu t al., 2008). -type lectin molecule in the nate immunity of chinese shrimp. ther antimicrobial peptides/proteins penaeidins penaeidins, initially isolated from the pacific white shrimp l. vannamei (destoumieux et al., 1997), are also a large family of amps that have been detected in several penaeid shrimp, including l. setiferus, m. japonicus, p. monodon, f. chinensis (bachère et al., 2004; kang et al., 2004, 2007; cuthbertson et al., 2008), l. stylirostris (aay33770), l. schmitti (aax58698; aax58697), fenneropenaeus penicillatus (aby56821), f. paulensis (aax58696), f. subtilis (abo93321), and f. brasiliensis (abo93324). in fact, among the amp family, the penaeidins are considered the most well-characterized in terms of the level of gene expression and biological activities. there are four classes of penaeidins (penaeidins 2, 3, 4, and 5) that have been characterized so far (bachère et al., 2004; kang et al., 2007) (fig. 6). this classification and characterization of penaeidin isoforms have been summarized in the database, penbase (gueguen et al., 2006) and were reviewed by several authors (bachère et al., 2004; cuthbertson et al., 2008). the evolution pattern of these peptides was likewise analyzed (padhi et al., 2007). table 1 presents the primary characteristics and functions of penaeidins. lysozymes lysozyme (muramidase, ec.3.2.1.17), an important antibacterial protein, catalyzes the hydrolysis of bacterial cell walls and acts as a nonspecific innate immunity molecule against the invasion of bacterial pathogens (jollés and jollés, 1984). initially found among eukaryotes and prokaryotes, the lysozymes are classified into six types. these are the chicken-type lysozyme (ctype), goose-type lysozyme (g-type), plant lysozyme, bacteria lysozyme, t4 phage lysozyme (phage-type), and invertebrate lysozyme (i-type) (hikima et al., 2003). similarly, these lysozymes had been reported in several shrimps, such as l. vannamei (sotelo-mundo et al., 2003; de-la-revega et al., 2006; burge et al., 2007; xing et al., in press), m. japonicus (hikima et al., 2003), p. monodon, (xing et al., in press), p. semisulcatus (xing et al., in press) and f. chinensis (bu et al., 2008). furthermore, most lysozymes identified in shrimps belong to a c-type lysozyme, like those from l. vannamei (af425673), m. japonicus (bac57467), p. monodon (b1784440), and f. chinensis (aav83994). in addition, there were i-type lysozymes identified in shrimp, such as lysozymes from l. vannamei (bf023863, bf024192) and l. setiferus (bf024309) (hikima et al., 2003). in some penaeid shrimp, the lysozymes are well-characterized, and they possess lytic activity against a range of gram-positive and gramnegative bacterial species, including pathogenic vibrio spp. (hikima et al., 2003; de-la-re-vega et al., 2006). hikima et al. (2003) used the rt-pcr analysis and reported that the lysozyme from m. japonicus was strongly expressed in samples from hemocytes, moderately expressed in the epidermis, and weakly expressed in the gills, midgut, and muscle. furthermore, the post-infection expression profile of a lysozyme est was analyzed using the macro-array, northern blot, and real-time pcr. the results revealed that the hemocyte lysozyme expression during the v. penaeicida challenge was significantly lower at 12 h after the infection, but had returned to control levels within 24-96 h after the challenge as observed in the surviving shrimps (de lorgeril et al., 2005). similarly, the lysozyme mrna from chinese shrimp (fclyz) was analyzed by semiquantitative rt-pcr. the lysozyme was actually expressed in various tissues of the unchallenged shrimp and the expression of fclyz was increased in the bacterial-challenged tissues of the hemocytes, heart, hepatopancreas, and gills as compared to the mock (saline)-challenged o e c c-type lectins have diverse functions. aside from their agglutinating activity and opsonic effects, some c-type lectins perform antimicrobial activities. a hepatopancreas specific c-type lectin, designated fc-hsl, has been found from the hepatopancreas of the chinese shrimp, f. chinensis (sun et al., 2008). this type of lectin was constitutively expressed in the hepatopancreas of normal shrimp, and its expression was up-regulated after the bacterial and viral challenge. in addition to fc-hsl’s calciumdependent agglutinating and binding activity to some gram-positive and gram-negative bacteria, it also has high anti-microbial activity against some gram-positive and gram-negative bacteria and fungi. therefore, fc-hsl may act as a pattern recognition receptor and an effector in o hemocyanin, as the main protein component of hemolymph, is prevalent in several invertebrate animals. it typically represents up to 95 % of the total amount of protein (sellos et al., 1997). meanwhile, the hexamer is the predominant form in the most primitive crustacean decapoda, such as penaeus setiferus or p. monodon (sellos et al., 1997). the hemocyanin, in relation to crustaceans, primarily functions as anthropods’ oxygen carrier. it is also the multi-functional proteins involved in physiological processes, such as osmoregulation, protein storage or enzymatic activities. in some reports, the fragments generated from the cterminus of hemocyanin of l. vanamei and l. stylirostris have exhibited a high anti-fungal activity (destoumieux-garzon et al., 2001). contrary to most 173 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=63109228 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=62002137 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=62002135 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=164451887 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=62002133 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=143598606 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=143598723 table 1 antimicrobial peptides/proteins in penaeid shrimp amps expression tissues functions references 1. penaidins penaeidin ii penaeidin iii penaeidin iv penaeidin v primarily in hemocytes and in highly vascular tissues broad spectrum of anti-grampositive and anti-fungal activities and weak activity against gram-negative strains destoumieux et al., 1997, 1999, 2000; bachère et al., 2004; cuthbertson et al., 2004, 2008; kang et al., 2004, 2007 2. wdps crustin i hemocytes anti-gram-positive activity supungul et al., 2008 crustin ii hemocytes anti gram-positive and antigram-negative activities zhang et al., 2007; amparyuo et al., 2008 swd hemocytes anti-gram-positive, antigram-negative, anti-fungal and anti-proteinase activities amparyup et al., 2008; jia et al., in press 3. alfs alf i high expression in hemocytes, gills and intestine and low expression in ovary, hepatopancreas and muscle anti-gram-positive, antigram-negative and antifungal activities; lpsneutralizing and hemolytic activities; interference with the replication of wssv gross et al., 2001; supungul et al., 2004; liu et al., 2005; nagoshi et al., 2006 alf ii ? ? (dq010421, be846661) alf iii high level in lymphoid organ and heart, intermediate levels in the gills, eyestalk and hemocytes and very low levels in the muscle and hepatopancreas anti-gram-positive, antigram-negative and antifungal activities de la vega et al., 2008; somboonwiwat et al., 2005; zhou et al., 2008 4. lysozymes c-type lysozyme hemocytes, lymphoid organ, hepatopancreas, gill, heart, midgut, muscle, epidermis, and eyestalk anti gram-positive and antigram-negative activities sotelo-mundo et al., 2003; hikima et al.,2003; de-lare-vega et al., 2006; burge et al., 2007; xing et al., in press; bu et al., 2008 i-type lysozyme ? ? (bf023863, bf024192) (bf024309) 5. c-type lectin hsl hepatopancreas anti-gram-positive, antigram-negative and antifungal activities sun et al., 2008 6. hemocyanin-derived peptides c-terminal fragment 7.9 kda, 8.3 kda hepatopancreas anti-fungal activity destoumieux-garzón et al., 2001 hemocyanin subunits hepatopancreas anti-viral activity zhang et al., 2004 7. histones h2a, h2b, and h4 hemocytes anti-gram-positive activity patat et al., 2004 8. peritrophin ovary anti gram-positive and antigram-negative activities loongyai et al., 2007 174 hemocytes: penaeidins crustins swd, alf lysozyme histones hepatopancreas: hemocyanin c-type lectin alf lysozyme lymphoid organ: alf lysozyme intestine: lysozyme penaeidins alf gill: lysozyme alf penaeidin epidermis: lysozyme penaeidin fig. 7 distribution of anti-microbial peptides/proteins in shrimp. amps with highly-cationic charges, the antifungal substances present a negative net charge at physiological ph with a pi ranging from 5.65 to 6.54. zhang et al. (2004) also reported that shrimp hemocyanin had antiviral property as it inhibited the virus replication. histone proteins are primarily involved in dna packaging and regulation of dna replication and transcription. a number of reports have shown that histone proteins or histone-derived peptides from various vertebrates possess antimicrobial activity (hirsch, 1958; robinette et al., 1998; fernandes et al., 2002). patat et al. (2004) as well reported that the hemocyte histone h2a, a mixture of histones h2b and h4 and an h1-derived fragment in l. vannamei, have anti-microbial activity against the tested bacterium. they believed that histone proteins or histone-derived peptides can be secreted to the cytoplasm from the nucleus and be localized in it along with other antimicrobial peptides. shrimp peritrophin, a major protein in jelly layer (jl) and cortical rods (crs) (du et al., 2006; loongyai et al., 2007), was inducing expression in hemocytes, heart, stomach, intestine and gill, and was constitutively expressed in ovary (du et al., 2006). the recombinant peritropnin exhibited a chitinase activity and efficiently inhibited the growth of vibrio harveyi and s. aureus, with minimum inhibitory concentrations of 2.4 and 15.7 μm, respectively (loongyai et al., 2007). conclusions to summarize, shrimps have efficiently developed and used their innate immune system in defense against pathogenic microorganisms. to date, eight kinds of amps have been identified in penaeid shrimp, namely, penaeidins, wdps (crustins and swds), alfs, lysozymes, anionic hemocyanin, histones, and a c-type lectin. these kinds of amps have different distribution and expression profiles and functions in shrimp (table 1 and fig. 7). furthermore, there are several subclasses or isoformes for each kind of amps in one species. an example is the three subgroups of wdps (crustin i, ii, and swd) in chinese shrimp. this only suggests that they had different functions in vivo in the shrimp. even though large groups of amps were found in penaeid shrimp, there is still a limited number of studies conducted about the amps’ antibacterial properties and their other functions. in view of this, there is a need to have more studies that will delve on the functions and the anti-bacterial properties of amps, especially with respect to the diverse bioactivities of the natural proteins in vivo. as such, some amps in shrimp can be better 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giessen, d-35392 giessen, germany accepted july 29, 2009 abstract hydra vulgaris is currently receiving increased attention as a genetically tractable invertebrate model system for studying important processes of life such as the innate immune defense. similar to complex animals, h. vulgaris polyps respond to injury by abrupt muscle contraction, by limited escape behavior, and by healing the damaged tissue. simultaneously, cellular processes such as phagocytosis and programmed cell death as well as the massive production of antimicrobial peptides are induced. recent studies identified several molecular pathways controlling these responses; however, the interdependence of innate immunity and, for example, regeneration and tissue remodeling is not well elucidated yet. h. vulgaris belongs to the cnidaria representing the phylogenic sister group of bilaterian animals; hence, a better understanding of evolutionarily conserved as well as hydra/cnidaria-specific immune responses will provide deep insight into both origin and evolution of the animal innate immune system. key words: cnidaria; danger signaling; hydra vulgaris, innate immunity; regeneration; model organism introduction a major evolutionary split occurred in the animal kingdom between so-called basal animals, which include cnidaria, and the deuterostomia/protostomia, which include most other animals (fig. 1) (finnerty et al., 2004). the freshwater cnidarian hydra vulgaris has a simple body plan with two cell layers, the ectoderm and the endoderm, separated by an extracellular matrix (mesoglea). hydra is prominent for its ability to regenerate a complete organism from small body fragments. therefore, h. vulgaris has widely been used as feasible model system to study pattern formation, cell differentiation, morphogenesis, and regeneration (trembley, 1744; campbell, 1967; gierer et al., 1972; bode et al., 1986; holstein et al., 1991; bosch, 1998; hassel, 1998; galliot and schmid, 2002; steele, 2002; bode, 2003; holstein et al., 2003; bosch, 2007). this review focuses on recent studies investigating the innate immune system in h. vulgaris. ___________________________________________________________________________ corresponding author: boran altincicek interdisciplinary research center institute of phytopathology and applied zoology justus-liebig-university of giessen d-35392 giessen, germany e-mail: boran.altincicek@agrar.uni-giessen.de hydra vulgaris as model system to study innate immunity in higher animals, immunity is a complex and highly developed system of specialized cells and organs that protects an organism against bacterial, parasitic, fungal, and viral infections. besides the conserved innate immunity, the adaptive or acquired immunity is present in vertebrates representing a novel evolutionary acquirement which enables the somatic generation of highly variable t-cell receptors and b-cell derived antibodies by recombination-activating gene dependent genomic rearrangements (germain, 2004; akira et al., 2006; medzhitov, 2007). these molecules provide pathogen-specific antigen recognition and create the so-called immunological memory. invertebrate immunity shows many parallels to the innate immunity of vertebrates, which complements the adaptive immunity. it includes cellular phagocytosis, antiviral rna-interference, and killing of pathogens by antimicrobial peptides, lysozymes, and reactive oxygen species (boman, 2003; kim and ausubel, 2005; cherry and silverman, 2006; lemaitre and hoffmann, 2007) (fig. 1). both vertebrate and invertebrate innate immunity recognize invading pathogens by hereditary pattern recognition receptors including 106 mailto:boran.altincicek@agrar.uni-giessen.de fig. 1 origin and evolution of immunity in metazoa. many mechanisms of innate immunity such as toll-like receptors (tlr), rna interference (rnai), antimicrobial peptides and toxic compounds are conserved across all animals. however, some aspects such as acquired immunity have only been evolved in vertebrates. several animal species that we examined for immune inducible genes to further understanding of the evolution of innate immunity are written in gray (altincicek and vilcinskas, 2007a, b, c, 2008b, c; altincicek et al., 2008). nfκb, nuclear factor kappa b, myd88, myeloid differentiation primary response gene 88; ig-like, immunoglobulin-like; srcr, scavenger receptor cysteine rich; gnbp, gram-negative bacteria-binding protein; ppo, prophenoloxidase. toll receptors, peptidoglycan recognition proteins, and lectins which bind to microbial components such as bacterial lipopolysaccharides, peptidoglycans, or fungal ß-1,3 glucans (royet, 2004; akira et al., 2006; medzhitov, 2007). moreover, immune related signaling is achieved in animals ranging from sponges to humans mainly by myeloid differentiation primary response gene 88 (myd88), nuclear factor kappa b (nfκb)-like factors, and stress-activated protein kinases such as p38 and jnk (böhm et al., 2002; wiens et al., 2005; akira et al., 2006; wiens et al., 2007) (fig. 1). this suggests a shared root for invertebrate and vertebrate immune pathways conserved over hundreds of millions of years. to elucidate origin and evolution of animal innate immunity, immune defense reactions have been studied in basal animals such as poriferans and cnidarians. recently, methods like dsrnamediated gene silencing (lohmann et al., 1999; chera et al., 2006; miljkovic-licina et al., 2007) and of transgenesis allowing stable genetic manipulation have been established in h. vulgaris (wittlieb et al. 2006; khalturin et al., 2007). hence, h. vulgaris represents a complementary invertebrate model system to the fruit fly drosophila melanogaster and the nematode caenorhabditis elegans for studying important processes of life including the innate immune defense. moreover, whole genome sequences have become available for two cnidarians, the hydrozoan hydra magnipapillata and the anthozoan nematostella vectensis (technau et al., 2005). this allowed recent comparative investigations of gene sets regarding innate immunity proteins in these two basal eumetazoans (hemmrich et al., 2007; miller et al., 2007). regarding the use of h. vulgaris as model system to study innate immunity, it should be mentioned that numerous hydra species live in mutualistic symbiosis with algae (pardy and heacox, 1976). moreover, symbiosis with algae seems to represent a common feature of the hydra group since symbiosis can be experimentally induced in non-symbiotic hydra species (rahat and reich, 1984, 1985, 1986). a tight association also exists with bacterial symbionts or commensals (wilkerson, 1980; rahat and dimentman, 1982; fraune and bosch, 2007; fraune et al., 2009) indicating that the immune system of hydra polyps is capable to distinguish between beneficial and pathogenic microbes. theory predicts that hydra may induce sophistically regulated and probably specific immune responses, which control interacting microbes. yet, specific receptors or signaling pathways have not been identified, but it is obvious that the evolutionarily conserved programmed cell death (cikala et al., 1999; böttger and alexandrova, 2007) plays a major role in controlling mutualistic symbionts or pathogens probably similar to observations from other cnidarian species (dunn et al., 2002; dunn et al., 2004; ainsworth et al., 2007; dunn, 2009; dunn and weis, 2009). 107 fig. 2 early phase responses of h. vulgaris polyp to bisection: (a) h. vulgaris is organized as body column with a mouth and ring of tentacles at the apical pole and a foot process with a basal disk at the distal pole. buds are formed for asexual reproduction. (b) bisection of a polyp results in abrupt muscle contraction and subsequently in limited escape behavior. (c) the contraction reduces the area of injury to a minimum which is indicated by a circle. (d) within several minutes post bisection mucus is produced and motile cells appear at the wounding site which phagocytize debris form damaged cells. nc, nematocyst. bars = 200 µm (a, b); 20 µm (c, d). immunorecognition and effector molecules h. vulgaris polyps show many parallels to complex animals regarding to their innate immune reactions. bisection of h. vulgaris polyps, for example, leads to an abrupt muscle contraction and escape reaction (figs 2a, b). these behavioural reactions are accompanied by the massive production of mucus at the wounding site and the appearance of phagocytic cells eliminating cell debris and dying cells (figs 2c, d). significantly, homogenates obtained from bisected animals exhibited a significant higher level of antimicrobial activity than homogenates obtained from untreated animals as determined by an inhibition zone assay with live bacteria (fig. 3). this is in line with recent observations that the hydra innate immune system senses tissue injury itself or along with bacterial contamination from the environment to induce the massive production of antimicrobial peptides along 108 with further potential bactericidal or bacteriostatic molecules (altincicek and vilcinskas, 2008a; jung et al., 2008; bosch et al., 2009). the finding that the drosophila transmembrane protein toll, which is essential for the development of embryonic dorsoventral polarity in the fruit fly, also mediates immune responses to infection had a pioneering role in the identification of toll-like receptors as evolutionarily conserved and essential regulators of immunity (beutler, 2004). mammalian toll-like receptors recognize a highly divergent collection of ligands such as lipoproteins, bacterial lipopolysaccharide and peptidoglycans, fungal zymosan, plant diterpene paclitaxel, viral proteins, and endogenous molecules such as heat-shock proteins and nucleic acids, all of which have different structures (akira et al., 2006; karin et al., 2006). in nematostella, toll-like receptors and a nfκb homolog have been identified but, surprisingly, not in hydra suggesting that a substantial immune gene loss or significant gene diversification of these factors have occurred during hydra evolution (miller et al., 2007). consistent with this hypothesis, a recent study indicated that in hydra instead of typical toll-like receptors two related toll-il-1 receptor (tir) domain transmembrane proteins interact with leucine-rich repeat and epidermal-growth factor domain transmembrane proteins to induce the expression of antimicrobial peptides (bosch et al., 2009); however, a corresponding transcriptional activator of this signaling pathway has yet not been identified. our recent studies with h. vulgaris revealed several immune-relevant inducible genes including, for example, a potential antimicrobial peptide, major vault protein and p47phox protein or neutrophil cytosolic factor 1 (altincicek and vilcinskas, 2008a). in mammals, major vault protein has been demonstrated to be essential for optimal epithelial cell phagocytosis of the pathogenic bacterium pseudomonas aeruginosa (kowalski et al., 2007). phagocyte napdh-oxidase (phox) is responsible for the generation of superoxide with secondary production of other microbicidal reactive oxygen species (ros) in human neutrophils and macrophages. phox consists of the catalytic subunit gp91phox, along with the regulatory subunits p22phox, p67phox, p40phox, the small gtpase rac, and p47phox or neutrophil cytosolic factor 1 (el-benna et al., 2009). since both molecules, major vault protein and p47phox protein, are reported to be important in mammalian immunity to eliminate invading pathogens investigation of their counterparts in hydra immune responses may further our understanding of human immune defense reactions. injury induces immune responses and regeneration processes the ability to respond to injury and to repair tissue is a fundamental property of all multicellular organisms. injury of the vertebrate skin, for example, initiates a complex sequence of events involving inflammation as well as the formation and remodeling of new tissue (schäfer and werner, fig. 3 bisection induces expression of antimicrobial factors in h. vulgaris polyps. homogenates obtained from 10 animals 24 h post bisection exhibited a significant higher level of antimicrobial activity than homogenates obtained from 10 untreated animals. antibacterial activity was determined with live micrococcus luteus as described (altincicek and vilcinskas, 2008c). results represent mean values of three independent repetitions ±sd. statistical significance was determined by student’s t-test (p<0.01). 2007). at the wounding site, multiple biological pathways immediately become activated and are synchronized to respond. the mammalian immune system is triggered by both microbial pattern molecules and endogenously derived danger signals such as extracellular heat shock proteins, uric acid, and hmgb1, which are released during tissue injury (matzinger, 2002; akira et al., 2006; karin et al., 2006). similarly, hydra is capable in sensing microbial molecular pattern such as lipopolysaccharide (altincicek and vilcinskas, 2008a; bosch et al., 2009) and flagellin (bosch et al., 2009) as well as danger molecules derived from dying or damaged cells such as extracellular nucleic acids and uric acid to induce expression of antimicrobial peptides (bosch et al., 2009). in vertebrates, wound healing requires dickkopf and wnt/β-catenin signaling and, additionally, the action of tgf-β/bmp and matrix metalloproteinases (mmps) (stoick-cooper et al., 2007). homologs of all of these factors have also been described to regulate hydra regeneration processes (galliot and schmid, 2002; holstein et al., 2003). recent studies, for example, demonstrated that the wnt and tgf-β/bmp signaling pathway components are transcriptionally up-regulated early during regeneration in hydra (holstein et al., 2003) and that the hydra dickkopf-like protein expression that antagonizes wnt/β-catenin signaling is stimulated by the injury signal itself (holstein et al., 2003). it has furthermore been shown that hydra astacinclass metalloproteinases are crucial for both foot and head regeneration (yan et al., 2000a, b) and that 109 fig. 4 phylogenic analysis of cnidaria mmps. phylogenic reconstruction was performed with the software package mrbayes 3.1.2 similar as described (knorr et al., 2009). the model with the overall highest posterior probability was wag. the average standard deviation of split frequencies at 106 generations was 0.01 and therefore indicated that the two chains that were run converged on similar results. the tree was drawn with figtree (tree.bio.ed.ac.uk/software/figtree). posterior probabilities are plotted at the nodes, which can be interpreted as the probability that the tree or clade is correct. the scale bar represents the substitutions per site. accession numbers of investigated mmps are as follows: t. castaneum (tribolium1, xp_968822; tribolium2, xp_969495; tribolium3, xp_972146); d. melanogaster (drosophila1, np_523852; drosophila2, np_995788), homo sapiens (human19, q99542; human28, q9h239; human11, p24347; human21, q8n119; human23, o75900; human17, q9ulz9; human25, q9npa2; human14, p50281; human15, p51511; human16, p51512; human24, q9y5r2; human20, o60882; human12, p39900; human13, p45452; human1, p03956; human8, p22894; human3, p08254; human10, p09238; human7, p09237; human26, q9nre1; human2, p08253; human9, p14780); h. vulgaris mmp (aad45804); h. magnipapillata (hyd1, xp_002158607; hyd2, xp_002163047; hyd3, xp_002163794; hyd4, xp_002160477; hyd5, xp_002170324; hyd6, xp_002164979; hyd7, xp_002168433; hyd8, xp_002168462; hyd9, xp_002155089; hyd10, xp_002161129; hyd11, xp_002166835; hyd12, xp_002168334; hyd13, xp_002155205; hyd14, xp_002154985; hyd15, xp_002165116; hyd16, xp_002163948; hyd17, xp_002164594; hyd18, xp_002165203; hyd19, xp_002163814); n. vectensis (nema1, xp_001640669; nema2, xp_001640565; nema3, xp_001633230; nema4, xp_001629775; nema5, xp_001636910; nema6, xp_001635715; nema7, xp_001640426; nema8, xp_001638726; nema9, xp_001640819; nema10, xp_001630740; nema11, xp_001626901; nema12, xp_001640427; nema13, xp_001622031; nema14, xp_001630704; nema15, xp_001642037; nema16, xp_001635184; nema17, xp_001628710). 110 http://tree.bio.ed.ac.uk/software/figtree a hydra matrix metalloproteinase (mmp) represents a central effector molecule in extracellular matrix degradation, epithelial morphogenesis, and probably in cell differentiation (leontovich et al., 2000; sarras et al., 2002; shimizu et al., 2002). in humans, more than 20 different mmps have been identified which degrade virtually all extracellular matrix proteins and release or degrade bioactive fragments and growth factors (pagemccaw et al., 2007). they influence fundamental biological and pathological processes like embryonic development, angiogenesis, tissue homeostasis and morphogenesis, wound repair, inflammation, and tumor progression (page-mccaw et al., 2007). the recently investigated h. vulgaris mmp (leontovich et al., 2000; sarras et al., 2002; shimizu et al., 2002) has a well-conserved overall domain structure to that of their invertebrate and vertebrate counterparts. h. vulgaris mmp includes a propeptide sequence containing a conserved inhibitory cysteine switch sequence and the catalytic domain with the conserved hexxhxxgxxh sequence critical for binding the catalytically active zinc ion (nagase and woessner, 1999). interestingly, examination of the cnidarian whole genome sequences revealed that 19 h. magnipapillata and 17 n. vectensis mmp homologs exist which most of them clade together with the identified h. vulgaris mmp (fig. 4). these observations suggest that mmps fulfill complex and overlapping functions in cnidaria probably similar to mammalian mmps (page-mccaw et al., 2007). interestingly, two nematostella mmps grouped together with human mmp19, 21, 23, 28 and insect mmp1s indicating possible evolutionarily conservation of these mmps in n. vectensis and loss in h. magnipapillata (fig. 4). mmps have further been recognized as important modulators of immunity in both mammals (parks et al., 2004; vanlaere and libert, 2009) and insects (altincicek and vilcinskas, 2008c; knorr et al., 2009); hence, we believe that their investigation in hydra will help to unravel the complex interdependence of the immunity with regeneration processes. altincicek b, vilcinskas a. identification of immunerelated genes from an apterygote insect, the firebrat thermobia domestica. insect biochem. mol. biol. 37: 726-31, 2007c. future priorities in the field of studying hydra innate immunity goals in the field of studying innate immunity in the h. vulgaris model system will be the elucidation of both genetic mechanisms driving host-parasite as well as host-symbiont co-evolution and principles of epithelial immune mechanisms in an animal that lacks a coelomic cavity. examination of the hydra immunity may further help to understand interactions of reef-building cnidaria with their pathogens as well as their symbionts (bosch, 2008) helping to keep these ecosystems alive. in this context, however, it should also be noted that the depth of the split between the cnidarian classes anthozoa (nematostella) and hydrozoa (hydra) has been determined to be as great as that between protostomia and deuterostomia (miller and ball, 2008). this observation indicates that immune mechanisms may be highly derived within cnidaria as, for example, found for the toll and nfκb signaling as mentioned above. in conclusion, the investigation of innate immune defense of basal animals with h. vulgaris as a genetically tractable model system is an exciting field, which will give valuable insight into the evolution of animals. combining mechanistic understanding of immunity along with processes of regeneration and of development will be, without doubt, a 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dexter s, sarras mp jr. identification and characterization of hydra metalloproteinase 2 (hmp2): a meprin-like astacin metalloproteinase that functions in foot morphogenesis. development 127: 129-141, 2000. yan l, leontovich a, fei k, sarras mp jr. hydra metalloproteinase 1: a secreted astacin metalloproteinase whose apical axis expression is differentially regulated during head regeneration. dev. biol. 219: 115-128, 2000. 113 ii incontro degli ascidiologi italiani isj 5: 83-96, 2008 issn 1824-307x report of meeting ii scientific meeting of the italian ascidiologists, 30 june – 1 july 2008, department of animal biology, university of palermo, palermo, italy organizers: n parrinello, v arizza, m cammarata, m vazzana, a vizzini marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy session 1. development and morphogenesis sensory organs in the oral siphon of pyrosoma p burighel, l manni f caicci department of biology, university of padua, padua, italy in previous papers we studied the sensory structures in the branchial siphons of ascidians evidencing the presence of sparse primary sensory cells and secondary sensory cells. the latter constitute an extended mechanosensory organ (the coronal organ) presents in the oral siphon of all the examined ascidians. for position, origin and ultrastructural features these sensory cells have several correspondences with the hair cells of vertebrates. thus, the possibility that they derive from embryonic territories sharing common aspects to the neural placodes of vertebrates was proposed. in order to verify whether the secondary sensory cells can be considered a common feature of all tunicates, we studied a representative of the pyrosomatida (thaliacea). pyrosomes are planktonic free-swimming tunicates forming tubular colonies. the individuals are embedded in the wall of the tube, with their oral siphon opened at the outer surface and their cloacal siphon opened into the common, central cavity. a water current passes each individual from the oral to the cloacal siphon. the oral siphon is rich in musculature and is covered by the epidermis also in its interior surface. a crown of tentacles run at the base of the siphon; in particular, a long ventral tentacle extends down toward the branchial cavity. with series of thick (1 µm) and thin sections we have evidenced that two kind of sensory cells are present in the oral siphon of pyrosomes. one is represented by the sensory cells of small, scattered cupular organs, which are formed of the central sensory cell accompanied by two supporting cells. the sensory cell is covered by a tunic-like cupula and its apical region has an extracellular canal containing a long cilium emerging among numerous long microvilli. the organisation of these cupular organs strictly recall the mechanoreceptor capsular organs of some ascidians. the second kind is represented by secondary mechanosensory, ciliated cells whose base contact neurites directed to (or arriving from) the brain. these cells run in a continuous row along the border of all the tentacles, forming a coronal organ corresponding to that of ascidians and resemble vertebrate hair cells for the presence of one-two cilia surrounded by a dozen of microvilli. the data suggest that the coronal organ is a common feature of tunicates. differentiation of sensory papillae in the embryo and larva of botryllus schlosseri f caicci, p burighel, l manni department of biology, university of padua, padua, italy in ascidians, the main class of the tunicates, the primary sensory cell constitutes the basic cellular element of most sensory structures, both in the freeswimming larva and in the sessile adult. the larva has sensory structures specialized for reception of gravitational, light, tactile and chemical stimuli. in particular, the sensory papillae are considered chemoreceptive structures, localized in rostral position of larval cephalenteron, which recognize and select the suitable substrate for adhesion at the onset of metamorphosis. by light and electron microscopy, we have studied the development and the structure of the sensory papillae in the embryo and larva of botryllus schlosseri. in this species, the papillae are three in a triradial arrangement and were defined in the past as “ganglionated type” for the presence of a ganglion to their base. the first rudiments of the papillae appear in early embryo in form of three distinct ectoderm evaginations, delimiting a small cavity in communication with the underlying mesenchymal space. in the successive stages, the cavity of each papilla enlarges to receive the papillary ganglion, which is constituted of primary sensory cells. these latter are bipolar neurons possessing receptor terminations in the tunic and a long axon extended to the central visceral ganglion. the papillary ganglion becomes recognizable under the epidermis owing to extension of the sensory neuron bodies into the papillary cavity. in the swimming larvae, the papillae are formed of three cell types: central sensory cells of papillary ganglion, peripheral sensory cells and cubical parietal cells. immediately before the adhesion to substrate, the papillae modify their shape: they elongate, the 83 ganglion becomes embedded in the papillary ectoderm, and the receptor terminations, crossing the tunic layer, are exposed to external side through fissures of the tunic. after adhesion, the papillae are retracted, while the central interpapillary area reaches the substrate; at the same time, the larva protrudes the blood ampullae for the definitive attach to the substrate. developmental expression and gene organization of protochordate synapsins: highlights on basic functions s candiani1, m parodi1, d oliveri2, f benfenati1, m pestarino1 1department of biology university of genoa, genoa, italy 2department of experimental medicine, university of genoa, genoa, italy synapsins belong to a family of neuron-specific phosphoproteins and are involved in several functions correlated with neurotransmitter release and synaptogenesis (reviewed in hilfiker et al. phil.trans. r. soc. lond. b 354: 269-279, 1999). the comprehension of the basal role of the synapsin family is hampered in vertebrates by the presence of multiple members. then, the study of homologous genes in protochordates, such as ascidians and amphioxus, lacking the whole genome duplications characteristic of vertebrates, could help to better understand the complex functions of synapsins. therefore, we have cloned and analyzed two synapsin genes, ci-syn in the ascidian ciona intestinalis and amphisyn in the amphioxus branchiostoma floridae. our results show the presence of a single synapsin gene in both organisms, and the occurrence of a second transcript by alternative splicing in b. floridae. our genomic analysis reveals an high homology degree with vertebrate synapsins, in particular with synapsin iii, the occurrence of three conserved domains a, c and e, and the presence of the classical nested organization of a timp (tissue inhibitor of metalloproteinase) gene in the intronic sequence. moreover, we have localized by in situ hybridization ci-syn and amphisyn transcripts exclusively at neuronal levels and in particular in a relevant portion of developing neurons, suggesting that the basic role of synapsins as regulators of neurotransmission and synaptogenesys has been conserved during evolution. sex hormone profiles in ciona intestinalis (ascidiacea urochordata) and their modulation by tributyltin (tbt) mv cangialosi1, e puccia1, a mazzola2, a arukwe3 1department of animal biology “g. reverberi”, university of palermo, palermo, italy 2department of ecology, university of palermo, palermo, italy 3department of biology, norwegian university of science and technology (ntnu), høgskoleringen 5, 7491 trondheim, norway regardless of species, steroid hormones are derived from cholesterol as a common precursor. in chordate, genome sequencing showed that cytochrome p450 (cyp) enzyme system play integral roles steroidogenic pathways and these processes are well conserved in many species. interestingly, in the protochordate ciona intestinalis, several rate-limiting steps involving cyp enzymes are lacking, although steroid hormones have been detected. the aim of this work is investigate the synthetic pathway of steroid hormones in c. intestinalis. given the lack of integral cyp enzyme genes, our hypothesis is that steroid hormone synthesis in c. intestinalis are probably mediated through an alternative pathway involving second messenger (camp) that activate protein kinases to regulate acute steroid production at level of substrate (cholesterol) availability. our preliminary data showed an increase in cholesterol and ca2+ concentrations in ovary of c. intestinalis after in vitro tbt treatment. based on these results, we started the investigation of steroid hormones levels in c. intestinalis after in vitro tbt treatment. analysis on testosterone, 17β-estradiol, aldosterone, progesterone and cortisol concentrations has shown appreciable variations in these hormones. thus, we are currently evaluating whether these responses could be activated or enhanced in the presence of camp or second messenger activator (forskolin). ion current and molecules involved in meiotic progression and fertilization of ciona intestinalis oocytes a cuomo1, f silvestre1, s bilotto1,3, gl russo2, e tosti1 1stazione zoologica “anton dohrn”, napoli, italy 2istituto di scienze dell’alimentazione, cnr, avellino, italy 3dipartimento di biologia, università di napoli “federico ii”, napoli, italy we performed an electrophysiological characterization of immature oocytes of the ciona intestinalis at different stages of growth and meiotic competence. oocytes showing a clear germinal vesicle (gv) have been classified on the basis of diameter and cytoplasm pigmentation as follows: stage a: <70μm diameter with a clear cytoplasm; stage b: 70-120μm diameter with a yellow cytoplasm; stage c: >120μm diameter with a brown cytoplasm. using the whole-cell voltage clamp technique on the oocytes collected from the ovary and deprived of accessory membranes, we showed for the first time a high l-type calcium currents activity at the stage a, these currents significantly decreased through meiosis up to the metaphase i (mi). oocytes at stage b showed the first appearance of sodium current activity that are still active at the mi phase. in vitro maturation experiments in calcium free sea water showed a significant reduction of maturation of oocytes at the stage c. intracellular calcium release was higher in mi than in previous stages; at last by using cyclic amp activators we showed a significant decrease of the germinal vesicle breakdown. fertilization current generated in sodium free sea water was significantly lower than the control, furthermore, oocytes fertilized in absence of sodium showed high development of the anomalous “rosette” embryos. 84 taken together these results imply: i) an involvement of external calcium in modulating oocytes growth, meiotic competence acquisition, prophase/metaphase transition and calcium stores refilling necessary for oocyte contraction at fertilization ii) a role of cyclicamp in maintaining the meiotic arrest as it happens in mammals; iii) a role of na+ currents during electrical events at fertilization and subsequent embryo development. a morpho-functional analysis of the adhesive papillae of ascidian larvae f de bernardi, g zega, s groppelli, c sotgia, r pennati dipartimento di biologia, università di milano, milano, italy most ascidian larvae adhere to the substrate by mucous secreted by the adhesive papillae, which in nearly all species are made of elongated secretory cells and primary sensory neurons. the exceptions are the papillae of the species of the genus botrylloides, which are considered to have only a sensorial function (grave, 1934) and that of genus clavelina, which are reported to be only secretory (turon, 1991). we analysed the adhesive papillae of botrylloides leachi, clavelina lepadiformis and diplosoma listerianum by histological analysis and by immunolocalization of serotonin and β-tubulin. we demonstrated that in all analysed species the adhesive papillae have both sensorial and secretory function. in particular, larvae of b. laechi have a lot of secretory cells gathered in the centre of a small area between the three adhesive papillae, forming a glandular organ. this organ could allow the adhesion of the larva to the substrate also by a sucker mechanism. the adhesive papillae of c. lepadiformis contain, in addition to the secretory cells, many primary neurons, the axons of which gathered to form papillary nerves. all analysed species contain two different types of neurons with different localisation and possibly a different function. the first type neurons, likely mechanoceptors or chemoceptors, emerge from the tunic at the apex of the papillae and play a role in the substrate choice. the second type neurons are localised in more lateral position in the papillae and do not contact the substrate until the papillae are retracting owing to a permanent adhesion. these neurons contain serotonin and they could play a role in triggering metamorphosis. on the basis of the role of neurotransmitters in ascidian metamorphosis, we hypothesize that serotonin released by the peripheral neurons after the permanent adhesion may acts as an internal signal triggering the metamorphosis process. the adult musculature in botryllus schlosseri: differentiation and gene expression v degasperi, f gasparini, l manni p, burighel department of biology, university of padua, padua, italy typically, ascidians have three types of muscles: skeletal in the larva, cardiac and smooth in the postmetamorphic sessile organism. the larval and cardiac muscles are striated, with vertebrate-like arrangement of myofilaments. instead, the smooth body-wall musculature has intermediate characters between smooth and striated muscle of vertebrates. we studied the musculature in the blastozooids of botryllus schlosseri analysing its organisation, differentiation and gene expression. we isolated and characterised two transctipts resulted homologous to muscle genes of other adult ascidians: a muscle-type actin (bsma2) and troponin t (bstnt-a); moreover, we obtained also the genomic sequence coding for bsma2. phylogenetic analyses showed a close relationship between urochordates and vertebrates muscle genes. the bsma2 genomic sequence was compared in the exon-intron organization with other muscle and cytoplasmic–type actin genes of both invertebrates and vertebrates. our analysis showed that intron positions are conserved in ascidian and in the other deuterostomes. we detected the expression of the two genes by in situ hybridization (ish), in order to follow the muscle development throughout the blastogenetic cycle of b. schlosseri. the ish, in parallel with phalloidin staining experiments, showed that the first diffuse signal of muscle differentiation appears in the intersiphonal epidermis of young buds. then, the muscle fibers differentiate into the body-wall, while an intense expression of bsma2 marks the heart myocardium just when it begins contractions. the ultrastructure of smooth muscle cells was also investigated during differentiation from mesenchymal cells in the bud, in the adult, and in the fibers contraction during regression of zooids. preliminary data on antiproliferative effects in haemocyte extracts of the ascidian styela plicata (stolidobranchiata, styelidae) ma di bella1, mc carbone1, r alessandro1, e fattorusso2, m menna2, g de leo1 1dipartimento di biopatologia e metodologie biomediche, università degli studi di palermo, palermo, italy 2dipartimento di chimica delle sostanze naturali, università degli studi di napoli “federico ii”, napoli, italy several marine invertebrate are among the most important sources of biomedically relevant natural products. a recent expansion of the studies of marine natural products involves the search for compounds with antitumor properties. in particular, among the compounds that have been isolated from sponges, bryozoans, and tunicates, several have been widely described (discodermolide, bryostatin-1, et-743), and have progressed to pre-clinical or clinical-trial phases. tunicates, organisms having a wide geographic distribution, have increasingly become the target of natural products research and have been reported to harbour metabolites with a wide range of biological activities such as metal accumulation, sclerotization of their extracellular matrices, and antimicrobial activity. some of these activities in marine invertebrate are also carried out by the circulating hemolymph that presents different types of haemocytes embedded in the liquid plasma and contains biologically active substances that contribute to the innate immune mechanisms. recent researches have shown that the extracts of the total haemocytes population from different ascidians 85 contain several compounds with an antimicrobial activity such as antibacterial, antifungal and antiviral ability. the aims of the present study was to extend the knowledge on the biological activities of these compounds testing the antiproliferative potential of the haemocyte extracts from the solitary ascidian st. plicata on leukaemia cell lines. comparative ultrastructural investigation on the adhesive papillae of the swimming larvae of three ascidiae species g dolcemascolo1, r pennati2, f de bernardi2, f damiani2, m gianguzza1 1dipartimento di biopatologia e metodologie biomediche, sez. di biologia e genetica, università degli studi di palermo, palermo, italy 2dipartimento di biologia, università degli studi di milano, milano, italy ascidian swimming larvae bear three peculiar organs of ectodermic origin, named “palps” or adhesive papillae, located in the anterior region of cephalenteron. term “adhesive” is correlated to one of the function of these structure based on secretion of an adhesive substance which enables swimming larvae to adhere to a substratum. recently a sensory function has also been described in some phlebobranchia papillae with a simple morpho-functional organization. there are few ultrastructural investigations in literature, sometimes disputed, able to make clear papillae cells functions. to clarify this problem, a comparative investigation has been carried out, just in this work, about ultrastructural and morpho-functional organizations of adhesive papillae of three ascidian swimming larvae, ascidia malaca, phallusia mammillata and ciona intestinalis. the investigations has been carried out by transmission electronic microscopy and confocal microscopy. papillae of above-mentionated ascidian swimming larvae, are three in number and they are located at the vertices of a triangular field. according to a recent classification scheme by burighel and cloney, they are coniform and non-eversible simple type papillae. in the apex of each papilla there is an hyaline cap with an electron-dense substance made of proteoglycan component. it’s quite certain that this substance is used by ascidian larvae to adhere to the substratum. ultrastructural investigations carried out on the adhesive papillae of a. malaca and p. mammillata have not emphasize significant ultrastructural differences in the papillae cells body. a. malaca and p. mammillata papillae are made of three types of cells that characterize their functions: a) collocytes, b) axial columnar cells, c) sensory cells. collocytes, whose ultrastructure is typical of cells with secretory activity, are certainly deputed to form adhesive secretion. they lie in a median or semilateral side of the papillar body. axial columnar cells that show an elongated shape, lie in the mid-central papillae region and they are characterized by the presence, in their apical part of long microvilli that run along the whole of hyaline cap length. above a structural supporting role, it is supposed a possible sensory function concerning these cells. sensory cells presents an ovoidal and elongated shape holding on tight at their base like a funnel lying a lateral and marginal position in papillar body. these type of cells have been described in a. malaca which can be classified as primary sensory neurons. these are characterized by the presence of a single cilium in the apical end and by an axonic process at their base extending over papilla. ultrastructural investigations on the adhesive papillae of c. intestinalis confirmed, even if showing some differences, ultrastructural likeness the two above-mentioned species of adhesive papillae. collocytes with secretory activity, sensory cells and columnar axial cells come into sight also in c. intestinalis papillae. the columnar axial cells ultrastructure is different from the a. malaca and p. mammillata papillae. in the apical part of these cells there are not microvilli but digitiform protusions with numerous microtubules inside running paralleling along longitudinal cell axis. the ultrastructure of these cells suggest their lengthening ability to allow digitiform protrusions to put into contact with the cuticular layer of hyaline cap apex, during step before the adhesion. ultrastructural characteristics suggest that they can also perform a sensorial duty but their mechanism is still unclear. the observation was carried out by confocal microscopy on the ascidian larvae just before or at the beginning of adhesion to the substratum (6-7 hours from hatching). the use of anti-β-tubulin antibody, have shown an extensive nervous network in all three ascidiae species starting from the papillar base and converging into only one nerve extending up to the cerebral vescicle. these data confirm previous observations carried out with confocal microscopy on a. malaca and p. mammillata. larvae. in c. intestinalis adhesive papillae marked with anti-β-tubulin antibody, the fluorescence is more bright than p. mammillata and a. malaca. probably this result is correlated with apical protrusion rich in microtubule. ultrastructure observations of some neurons being in the anterior region of cephalenteron, extending from the base of papilla to the cerebral vescicle, have been emphasized in this work. these neurons are in the same position of the ones described like rten (rostral trunk epidermal neuron) by imai and meinertzhagen and they are formed by cellular ovoidal body extending into a long axon. ultrastructural of these neurons have pointed out likeness with vertebrate neurons. concluding, ultrastructural investigations have emphasize only some difference between papillar cells of a. malaca, p. mammillata and c. intestinalis larvae, confirming adhesive and sensory functions. evolution of anterior hox regulatory elements among chordates a locascio1, m manzanares2, ml chiusano3, a amoroso1, e d’aniello1, r krumlauf4, m branno1 1laboratory of biochemistry and molecular biology, stazione zoologica “anton dohrn”, villa comunale, naples, italy 2department of cardiovascular developmental biology, centro nacional de investigaciones cardiovascularescnic, madrid, spain 3department of structural and functional biology, università degli studi di napoli “federico ii”, monte s. angelo, naples, italy 4stowers institute for medical research, kansas city, mo 64110, usa hox genes determine ap identities from insects to 86 vertebrates in all the animal species where they have been studied. their organization in cluster and spatiotemporal colinearity of expression have usually been considered their more significant characteristics. the sequencing of the genomes of even more species has helped to understand various aspects of hox gene organization. among chordates, particularly interesting are the anterior groups of hox genes since their expression is coupled to the control of regional identity in the anterior nervous system, where the highest structural diversity is observed. hox genes exert a fundamental role in vertebrate hindbrain formation and segmentation. both ascidians and amphioxus lack a segmented hindbrain but at the same time show restricted expression patterns of anterior hox genes like their vertebrate counterparts. changes in gene regulatory regions are considered a driving force for the evolution of more complex body plan structures. to investigate how hox gene regulation changed and evolved in the chordate lineage, we have analysed and compared various hox regulatory regions of three chordate species, amphioxus, ascidian ciona intestinalis and mouse. in particular, we focused our attention on elements controlling anterior hox genes expression and active in the anterior nervous system. we have studied the ability of amphioxus and mouse regulatory elements to function in ciona embryos and to reproduce the segmental expression pattern typical of hox genes. conversely, we have also analysed the ability of ciona nervous specific enhancers to be recognized by the mouse regulatory mechanisms. anatomy of ciona intestinalis: larval, metamorphic and juvenile phases l manni, f gasparini, p burighel department of biology, university of padua, padua, italy the solitary ascidian ciona intestinalis is a model organism frequently exploited for comparative investigations of chordate development and for unravelling the molecular mechanisms underlying morphogenesis and cell fate specification. this species has an indirect development comprising the embryonic phase, free swimming larval stage, metamorphosis to form a sessile juvenile and, ultimately, the adult with the mature reproductive organs. some of the first developmental stages have been described in detail using different classical and modern techniques. thank to these lines of study, cell lineages in ascidian embryo were thoroughly described from fertilized egg until the larval stage. recently, also successive developmental stages, such as the metamorphosis and the juvenile differentiation, has begun to be investigated, although less extensively. all these studies concur to improve our knowledge on ciona development, but several questions remain to be solved, because each technique has intrinsic advantages and limits. in this light, the authors discuss recent data from other laboratories examining also results obtaining recently by confocal microscopy. despite this technique permits the three dimensional reconstruction of the main developmental events in young embryos, it remain unsatisfactory in resolving precise aspects of interaction and differentiation between cells and/or tissues in successive stages. this is particularly relevant for late larvae and during metamorphosis, when the complex organogenesis of new rudiments and body reorganization occur accompanied by tissue shrinkage and disaggregation of embryonic structures,. the authors exemplify, analysing by means accurate reconstruction of specimens with histology and ultrastructure, critical stages of ciona development. the data show that also today the classical histology and the ultrastructure are complementary and essential techniques for the understanding of the anatomical organization of tissues, organs and the organism as a whole, and can represent fundamental reference for interpretations of sections in which tissues are partially labelled, as in in situ-hybridization or in immunocytochemistry. distribution of neural phenotypes in the larva of ciona intestinalis: comparison with cephalochordata and vertebrata r pennati1, g zega1, s candiani2, s groppelli1, m pestarino2, f de bernardi1 1department of biology, university of milan, milan, italy 2department of biology, university of genoa, genoa, italy the complex organisation of vertebrate nervous system is not only the product of an ontogenetic process, but it is the result of a long evolutionary history. a disclosure of this history can help to understand i) which developmental processes are more ancient and constitute the basis of vertebrate nervous system formation; ii) which processes evolved and/or superimposed in a second time. ascidians belong to tunicata subphylum are a very good model for these studies, because they are chordata and, according to recent studies, may be considered the group phylogenetically closest to vertebrates. ascidians and vertebrates share a tripartite organisation of the central nervous system, including an anterior region, domain of otx genes, a central region which express pax 2/5/8 and a posterior region regulated by the expression of hox genes. within this structural regionalization, during differentiation single neurons acquire an important phenotypic character given by the neurotransmitter released to excite or inhibit the target cells. we identified the serotonergic, gabaergic and dopaminergic neurons in developing central nervous system of ciona intestinalis by studying the expression pattern of genes codifying the neurotransmitter synthesis enzymes. we showed that some functional regions are conserved among the cns of ascidian larva, of the amphioxus and of vertebrates. this allowed us to hypothesize that these regions were already present in the ancestral chordate. citcf and pigment organs formation during ciona embryogenesis e riano, p d’ambrosio, a spagnuolo laboratory of biochemistry and molecular biology, stazione zoologica “anton dohrn”, villa comunale, naples, italy 87 the wnt signaling cascade plays an important role during embryonic patterning and cell fate determination and is highly conserved throughout evolution. factors of the tcf/lef hmg domain family (tcfs) are the downstream effectors of this signal transduction pathway. to study the function of tcf during ascidian embryogenesis, we attempted to isolate the ciona counterpart(s). our data indicate that ciona genome contains a single tcf gene, citcf (as confirmed by the analysis of the annotated genome) compared to the mammalian genome that harbours four tcf family members. in situ hybridization experiments indicate that citcf is broadly expressed at the early stages of development; from early neurula stage citcf signal becomes localized to the two pigment cell precursors. block of citcf function, by morpholino-antisense injection, strongly suggests its involvement in pigment cells formation; on the other end, transcriptional regulation analysis, by electroporation method, demonstrates that a 2.0 kb citcf promoter region, upstream from the tata box, is able to drive the tissue specific spatial expression of reporter gene. by a comparative analysis with ciona savigny we have identified a shared element of about 0.3 kb, upstream from the ci/cstcf genes, able to reproduce the pattern obtained with the 2.0 kb region. deletion constructs are currently under investigation, in order to further restrict the 0.3 kb minimal promoter fragment and to identify the core elements, controlling ci-tcf expression, and then the transcription factor(s) able to bind to this sequence. the homologous ciona factors will then be searched, by blast analysis against the annotated genome, fished in the cdna collection, present in our laboratory, and tried both in vitro and in vivo for their ability to interact with ci-tcf promoter and transactivate the reporter gene angiogenic-like mechanism in the colonial circulatory system of botryllus schlosseri g zaniolo, f gasparini department of biology, university of padua, padua, italy the colonial circulatory system (ccs) of the ascidian botryllus schlosseri runs in the common tunic and forms an anastomized network of vessels, defined by simple epithelium, connected to the open circulatory system of the zooids. the ccs originates from epidermal evagination, grows and increases its network accompanying colony propagation by means of mechanisms of tubular sprouting. we evidenced that the regeneration of experimentally ablated areas of ccs occurs by the same sprouting mechanism. in the two cases (normal growth and regeneration, the same histogenetic mechanism and homologous factors and receptors are shared. strong similarities in organization (e.g., simple cubic epithelium) and cell structure (e.g., extension of filopodia) in the apexes of sprouting vessels during normal growth and regeneration were observed. immunohistological responses to anti-pcna a marker of cell proliferationand antibodies against vertebrate angiogenic growth factors (vegf, egf, fgf-2) and receptors (vegfr-1, vegfr-2, egfr), reveal that cell proliferation and the same angiogenic signals take part in corresponding sprouting regions of ccs during normal development and regeneration. sprouting is the most common and best-known mechanism of vertebrate angiogenesis, but it is also found in other developing structures, such as nerves, and in drosophila tracheas. all our observations show that correspondences exist between the ccs sprouting modality of b. schlosseri and angiogenic sprouting in vertebrates, during both normal development and regeneration, and support the idea that this morphogenetic mechanism was co-opted during the evolution of various developmental processes in different taxa. effects of paraquat on the development and metamorphosis of phallusia mammillata and ciona intestinalis (ascidiacea, tunicata) g zega, s groppelli, f de bernardi, r pennati department of biology, university of milan, milan, italy paraquat (1,1’dimethyl-4,4’-bipyridylium) is an herbicide largely employed in agriculture, the use of which has been authorized in 120 countries. several toxicological studies demonstrated that this substance has neurotoxic effects on numerous animal models and causes symptoms similar to those observed in patients with parkinson’s disease. we exposed phallusia mammillata embryos at different concentrations of paraquat until they reached the swimming larva stage, then we immunolabeled their nervous system with anti β tubulin antibody, in order to analyzed the malformations eventually induced by the treatments. treated larvae showed dosedependent alterations of the ocellus and of the fibres that innervate the otolith. the gravity of the malformations decreased when the embryos were treated with both paraquat and ascorbic acid, suggesting that oxidative stress is involved in the onset of the observed malformations. moreover, we observed that treated larvae survived for more than four days, but they could not metamorphose. exposure to paraquat had similar effects on ciona intestinalis embryos. we characterized paraquat induced phenotype in c. intestinalis larvae by in situ hybridization with marker genes of different neural populations in order to evaluate the specificity of action of this neurotoxic agent. treated larvae showed a drastic reduction of gabaergic neurons, while the number of dopaminergic neurons was not affected by the treatments. these results are in contrast with the supposed effects of paraquat on humans. in fact this herbicide is suspected to be involved in the etiology of parkinson’s disease, a pathology characterized by progressive loss of dopaminergic neurons of the substantia nigra. role of nitric oxide during ciona intestinalis development a palumbo, d d’esposito, e ercolesi, g fiore, a locascio, m branno laboratory of biochemistry and molecular biology, stazione zoologica “anton dohrn”, villa comunale, naples, italy 88 nitric oxide (no) plays an important role in fertilization and development of some marine organisms, including sea urchins, marine snail and ascidians. we have recently reported that in ciona intestinalis no is involved in larval development and metamorphosis. the spatial patterns of nitric oxide synthase expression, as well as no detection, during larval development are very dynamic, moving rapidly along the body in very few hours, from the anterior part of the trunk to central nervous system, tail and juvenile digestive organs, thus suggesting the involvement of no in many processes. in particular, no regulates tail regression acting on caspase-dependent apoptosis. to further investigate the role of no during c. intestinalis development we focused our attention on no signalling during metamorphosis. experiments have been carried out to examine how the modulation of this signalling affects tail resorption and subsequent juvenile development. parallel experiments have been also performed to investigate the possible involvement of no in the embryonic development. to this aim we have examined the spatial expression patterns of nitric oxide synthase and no detection in embryos at different stages as well as the effect of endogenous no levels modulation on development mathematical models for excitability of egg cells pg reas department of mathematics and applications, university of palermo, palermo, italy the excitable systems play a very important role in biology and medicine. phenomena such as transmission of impulses between neurons, the cardiac arrhythmia he aggregation of amoebas, the appearance of organized structures in the cortex of egg cells, all derive from the activity of excitable media. in the first part of this work a general definitions of excitable system is given; we then analyze some cases of excitability, distinguishing between electrical ,chemical and mechanical excitability and comparing experimental observations with simulations carried out by appropriate mathematical models. such models are almost always formulated by partial differential equations of “reaction-diffusion” type and they have the characteristic to describe propagations of electrical waves or chemical and mechanical waves (propagation of ca waves and mechanical waves in the endoplasmic reticulum). the aim is to put in evidence that the biological systems can show not only excitability of electrical type, but also excitability of chemical and mechanical nature, which can be observed in the first steps of development of egg cells. session 2. phylogenesis and microevolution the fast evolutionary dynamics of ascidian mitochondrial genome: an exception to the general deuterostome evolutionary trend c gissi1, f iannelli1, f griggio2, g pesole1 1dipartimento di scienze biomolecolari e biotecnologie, università di milano, milano, italy 2dipartimento di biochimica e biologia molecolare “e. quagliariello”, università di bari, bari, italy the availability of more than one thousand sequences of mitochondrial genomes (mtdna) of metazoa provides an almost unique opportunity to decode the mechanisms of evolution of an entire genome in a phylogenetic framework. we have compared several structural features of the ascidian mtdna (gene content, genome size and architecture, and gene strand asymmetry) to that of other metazoans, focusing also on comparisons at congeneric level, as analyses at short evolutionary distances reduce the risk of saturation effects in the observed genomic changes. the current data show that ascidians exhibit a degree of mtdna variability very high and comparable to that of non-deuterostome groups. indeed, a trend toward stabilization of the mt genomic features has occurred in most deuterostomes and has been exacerbated in vertebrates, where gene content, genome architecture and gene strand asymmetry are almost invariant. on the contrary, ascidians show a gene strand asymmetry and a variability in mtdna architecture similar to nonbilaterians and lophotrochozoans, suggesting that these features are primitive, rather than a derived traits of the mtdna. in addition, our data highlight that the high degree of rearrangements in mtdna architecture found in ascidians, as well as in enoplean nematodes, is due to translocations of both rna and proteincoding genes, while changes in genome architecture are mostly due to the variation of number/position of trnas in taxa with moderate/low rearrangements. the variability observed in congeneric species significantly recapitulates the overall mtdna evolutionary dynamics observed at higher taxonomic ranks, especially in taxa with high genome plasticity and/or fast nucleotide substitution rate. moreover, congeneric comparisons appear quite promising to investigate in detail mtdna evolutionary mechanisms. one ring to divide them all: mitochondrial genomics unveils two cryptic species in ciona intestinalis f iannelli1, g pesole2, p sordino3, c gissi1 1dipartimento di scienze biomolecolari e biotecnologie, università di milano, milano, italy 2dipartimento di biochimica e biologia molecolare “e. quagliariello”, università di bari, bari, italy 3laboratory of biochemistry and molecular biology, stazione zoologica “a. dohrn”, naples, italy the circular mitochondrial genome (mtdna) of metazoans represents a rich source of genetic markers for phylogenetic analyses at many taxonomic levels. both sequences and genome-level features, such as gene arrangement, have been used to resolve deeplevel phylogenetic relationships, whereas single mitochondrial genes or regions are commonly analysed in population genetic studies. we used a mitogenomic approach, based on the comparison of several genome-level mitochondrial features, to unambiguously demonstrate the existence of two cryptic species in the ascidian ciona intestinalis, a model chordate whose taxonomic status of a single species has been recently questioned. a comprehensive comparative analysis between the mtdna of the two putative cryptic species revealed significant differences in gene order, size and number of non-coding regions, 89 compositional features, and evolutionary rate of protein-coding genes. these mitochondrial features are clearly incompatible with intra-species variability, and strongly suggest the existence of the two cryptic species. furthermore, our approach allowed to set two pcr-based diagnostic tests for the discrimination of the cryptic species without recourse to morphological analyses, demonstrating that mtdna represents an accessible and powerful tool to be used both in routine analyses and in high-throughput screenings. phylogenetic conservation of csf-related genes in the ascidian ciona intestinalis gl russo1,2, s bilotto1,2,3, g ciarcia3, e tosti1 1stazione zoologica ‘anton dohrn’, napoli, italy 2institute of food sciences, national research council, avellino, italy 3department of biology, section of zoology, university of naples, italy in all vertebrates, mature oocytes arrest at the metaphase of the ii meiotic division, while some invertebrates arrest at metaphase i, others at pronucleus stage. fertilization induces completion of meiosis and entry into the first mitotic division. how the different mechanisms underlying meiotic regulation evolved is very far from being clarified. in the past decades, several experimental models have been considered from both vertebrates and invertebrates in order to shed light on the peculiar aspects of meiotic division, such as the regulation of the cytostatic factor (csf) and the maturation promoting factor (mpf) in metaphase i or ii. some of these questions remain elusive and can be approached by the introduction of new experimental models. in the recent past, we proposed the oocytes of ascidian ciona intestinalis as a new model to study the meiotic division both at the physiological and molecular level. here, taking advantage of the recent publication of a draft copy of c. intestinalis genome, we present a phylogenetic analysis of key genes involved in the control of meiotic completion after fertilization, such as cdc2/cyclin b and mapk/mos components of mpf and csf, respectively. the presence of c-mos homolog in c. intestinalis genome suggests a regulation of metaphase arrest in ascidians different than in other invertebrates, where this gene is not conserved. we further investigated the regulation of csf by demonstrating that both csf and mpf inactivation, at the exit of metaphase i, are independent from protein synthesis. in fact, oocytes loaded with emetin completed meiosis i after fertilization, similarly to control oocytes, as demonstrated by mpf decrease and extrusion of the first polar body. this result indicates the absence of short-lived factors that regulate metaphase stability, as in other invertebrate species. in addition, antibody raised against the xenopus homolog of mos are able to abolish c. intestinalis csf activity. finally, mapk enzymatic activity sharply decreases after fertilization, as confirmed by a mapk specific antibody that was able to recognize the active, phosphorylated form of the kinase. the results obtained indicate that meiotic regulation in c. intestinalis resembles that of vertebrates, such as xenopus, more than those of other invertebrates. advancing forward genetics in ciona intestinalis sp. a. p sordino, n andreakis, l caputi laboratory of biochemistry and molecular biology, stazione zoologica “a. dohrn”, villa comunale, naples, italy our aim is to elucidate the cellular, molecular and evolutionary basis of pattern and differentiation in animal body plans. we combine classical and modern techniques for understanding gene functions during embryonic development by centering our activity on ciona intestinalis (ascidiacea). herein, we report about the implementation of forward genetics approaches. the primary objective of forward genetics is a low-cost resource of mutants with interesting phenotypes to be rapidly mapped and identified at the molecular level, a concept which applies to a limited number of model species in which mutagenesis techniques allow to compare phenotype and genotype in great detail. we are currently analyzing several aspects that are essential for the establishment of a phenotype-driven methodology. among them are the existence of two cryptic species, as shown by independent species concepts, the identification of a source of mutants, the construction of high-resolution genetic linkage map, the generation and culturing of inbred or semi-inbred strains, a protocol of sperm cryopreservation for the optimization of line management, and the characterization of genetic and morphological variation in natural populations. also, we are studying the phylogeographic structure of c. intestinalis at global and local scale by using different molecular markers, with the aim to examine how historical, geographical and environmental factors influence the distribution of morphological and genetic polymorphism in a model chordate. shuttling and rrna processing of pre-ribosomal subunits in the ascidian ciona intestinalis r barbieri dipartimento di biologia cellulare e dello sviluppo, università di palermo, palermo, italy ribosome biogenesis in eukaryotes is one of the most challenging topics in molecular and cellular biology of the last ten years. although different steps of pre-ribosome maturation has been elucidated in the last years, some intriguing facets remains still unresolved. among these questions, the different fates of the two sub-units during ribosome biogenesis, and the reason why different maturation steps of these particles occur in different cellular compartments. it is generally acknowledged for all the eukaryotes that the two different ribosomal pre-subunits (pre-40s and pre-60s) undergo to different fates during their late maturation steps. the smaller one (containing a 21s rrna) is exported to the cytoplasm where its maturation is completed, whereas the pre-60s particle (containing a 28s rrna) completes its maturation in the nucleus, and successively is exported in the cytoplasm as a mature particle. to the contrary of the ribosome maturation pattern up to now proposed, we found in the ascidian ciona intestinalis, as well as in sea urchin and human cells, that pre-ribosomal subunits maturation is a concerted, parallel processing 90 and shuttling mechanism involving both the two preribosomal particles; this mechanism includes a cytoplasmic rrna processing event never described before. primer extension experiment on total rna extracted separately from nuclei and cytoplasm of ciona intestinalis 4/8 blastomeres embryos, reveals the presence of the same “large” rrna precursors both in the nucleus and in the cytoplasm of these cells. the comparison with what occurs in the sea urchin p. lividus (left panel) and in human white blood cells (not shown), allowed us to demonstrate both that the shuttling/processing model is substantially different with respect to the one proposed, and that the shuttling/processing events we demonstrated work probably in all the eukaryotic species. frank homologies between invertebrate and vertebrate chordates revealed through the neurophysiology of swimming in ciona tadpoles brown er1, piscopo s1, nishino a2, okamura y3 1 laboratorio di fisiologia animale ed evoluzione, stazione zoologica anton dohrn, villa comunale, 80121 napoli, italy 2 department of biological sciences, graduate school of science, osaka university, machikaneyama 1-1, toyonaka, osaka 560-0043, japan 3 department of integrative physiology, graduate school of medicine, osaka university, yamada-oka 2-2, suita, osaka, 565-0871, japan with the recent sequencing of the genome of ciona intestinalis there has been an increase in interest in ascidians as models to study the evolution of chordates. one way to do this is to look at the similarities and differences between genes and families of genes at the genomic and functional level. however deeper homologies may be revealed by looking at (in addition) the function of proteins in a given physiological system which may shed light on their evolutionary pathways. here we report progress in our understanding of chordate locomotion and physiology through study of the neurophysiology of swimming in c. intestinalis. although ciona has a sessile adult form, the tadpole-like larva swims with rapidly alternating tail beats that superficially resemble vertebrate swimming. in vertebrates, such alternating activity during swimming is generated in spinal segments and is controlled by a combination of glutamatergic excitation of motorneurones and inhibitory glycinergic interneurons which provide contralateral inhibition. modulation of the period of swimming is achieved through gabaergic inhibition. these networks of excitatory and inhibitory neurons are known as central pattern generators (cpgs). we show that: 1) a cpg exists in the visceral ganglion and nerve chord and is excited by glutamate, 2) precise alternation of tailbeats is controlled by a glycinergic system consisting of interneurons and glycine receptors (ciglyr), 3) a gabaergic system modulates swimming but does not control alternating movements. we conclude that the ‘spinal-like’ cpg and the ‘use’ of glycinergic and gabaergic inhibition for the control and modulation of swimming were ancestral chordate innovations. session 3. immunity stem cells and chimerism in colonial ascidian a voskoboynik institute of stem cell biology and regenerative medicine, department of pathology, stanford university school of medicine, stanford, ca 94305, usa, and department of developmental biology, stanford university hopkins marine station, pacific grove, ca 93950 usa natural chimerism is the coexistence of cells of two genetically distinct organisms in one individual. it is a common phenomenon, which can be detected in a wide variety of multicellular organisms, including vertebrates. in mammals, natural chimerism is usually established during pregnancy between the mother and the fetus or between fetuses in multiple embryos pregnancy. colonial marine ascidians, like botryllus schlosseri, may serve as evolutionary model system to vertebrates chimeras. in these organisms, pairs of allogeneic colonies can establish a natural chimerism upon physical contact. the ability to create a chimeric entity between these colonies is determined by a single, highly polymorphic, fusion / histocompatibility locus (fu/hc). colonies that share at least one allele in their fu/hc locus (mainly kin under in situ conditions) would fuse upon contact. a pair that does not share any fu/hc allele would not. following fusion, cells transmigrate between colonies and, in some cases, replace the germline and/or the somatic tissues of the host (termed as germline and somatic cell parasitism respectfully). the replacement of host tissues by a donor genotype is pre-determined genetically and follows hierarchies of “winner strains” replacing “loser strains” tissues. in both mammals and ascidians, natural creation of a chimera entity is restricted to kin; longterm chimerism can be established by stem cells; and tolerance or intolerance state to donor tissues can be mediated by chimerism. while several studies and observations across different species, tissues and systems link chimerism to tolerance, its actual role in tolerance induction or maintenance is yet unknown. here i’ll review the chimerism phenomenon in mammals and ascidians, discuss the possible role of stem cells as mediators of chimerism and the possible role of cells chimerism as mediators of tolerance. future studies, which monitor the dynamics of chimeric donor cells within the host, identify the factors that support or inhibit survival and proliferation of donor cells and its affects on tolerance or intolerance should shed new light on this widespread natural phenomenon. morula cells, phenoloxidase and dopacontaining proteins in the compound ascidian botryllus schlosseri l ballarin1, s scippa2, f cima1 1department of biology, university of padua, padua, italy 91 2department of biological sciences, section of genetics and molecular biology, university of naples, ”federico ii”, naples, italy morula cells (mcs) represent the most abundant circulating haemocyte type in the compound ascidian botryllus schlosseri. they are involved in defence reactions as they: i) can recognise alien substances and cells and induce cytotoxicity; ii) are the effectors of the cytotoxic rejection reaction which occurs between contacting, genetically incompatible colonies. a main role in mc-related cytotoxicity is exerted by the enzyme phenoloxidase (po) which converts polyphenol substrata to quinones; the latter, in turn, polymerise to form melanins. in the present research, we carried out new spectrophotometrical and cytochemical analysis to investigate further the behaviour of po and the nature of its substrates. results confirm that po is located inside mc vacuoles. in addition, immunocytochemical analysis indicate that mcs contain quinones which probably represent ready-to-use cytotoxic molecules, likely deriving from the oxidation, by po, of dopacontaining proteins. in addition, small dopacontaining peptides, called tunichromes, are likely present inside mcs. vcbp genes in ciona intestinalis. i. structural analysis s giacomelli1, r de santis1, w litmang2, d melillo1, mr pinto1, i zucchetti1 1laboratory of cell biology, stazione zoologica “anton dohrn”, napoli, italy 2department of pediatrics, university of south florida, saint petersburg, florida, usa the pivotal genes encoding for adaptive immunity molecules, such as the major histocompatibility complex (mhc) class i and ii, t-cell receptors, or dimeric immunoglobulins, have not been identified in jawless vertebrates, protochordates and in nonchordate invertebrates. however, in an attempt to identify adaptive immune-like genes, a secretionsignal-peptide-selection-based approach in the amphioxus branchiostoma floridae, allowed the identification of five gene families encoding secreted proteins characterized by a pair of n-terminal immunoglobulin variable domains (v) and a single cterminal chitin-binding domain. these families are distinguished by sequence differences in their v regions. the high degree of germline polymorphism, the chimeric immunoglobulin-lectin structure of vcbps and their tissue-specific expression, are all consistent with involvement of these molecules in immune recognition. vcbps may reflect structural characteristics of an important transition between nonrearranging innate pattern-recognition molecules and the conventional adaptive immune receptors. in an attempt to add further evidences for the presence of alternative solutions for both innate and adaptive immunity along the phylogenetic tree, a search for vcbp genes has been carried out in ciona intestinalis genome and est libraries. this resulted in the identification of three vcbp-like genes, ci-vcbp1, ci-vcbp 2 and ci-vcbp 3, encoding three proteins of 349 (ci-vcbp1) and 338 (ci-vcbp2 and ci-vcbp3) aminoacids. the analysis of the deduced aminoacid sequences indicates that ci-vcbp2 and 3 exhibit 72 % identity, which is about 29 % when ci-vcbp1 is compared with ci-vcbp2 or ci-vcbp3. the domain structure analysis of the three gene products revealed the presence of a signal peptide sequence, two immunoglobulin domains and a chitin-binding domain. a very preliminary analysis carried out by pcr with degenerate oligonucleotides, designed on the alignment of the three nucleotide sequences, allows us to exclude the presence of other paralogous genes in c. intestinalis genome. furthermore, pcr analysis performed with cdna from different individuals evidenced the presence of limited allelic polymorphism. molecular events triggered by the interaction between the ciona intestinalis anaphylatoxin and its specific receptor d melillo, r de santis, s giacomelli, mr pinto laboratory of cell biology, stazione zoologica “anton dohrn”, naples, italy many molecules belonging to the arms of the innate immune system have been found scattered in invertebrates at different levels of the phylogenetic tree. in this context, a major breakthrough has been the identification in the echinoderms, in the urochordates and in the cephalochordates of gene homologs of c3, a multifunctional protein that plays a central role in the complement system. in mammals, c3 activation produces two bioactive proteolytic fragments: c3b responsible of the opsonic pathway, and the anaphylatoxin c3a, a potent mediator of inflammatory reactions. we have demonstrated that in ciona intestinalis, like in mammals, the bioactive fragment c3a, released from c3 during complement activation, promotes hemocyte chemotaxis, thus suggesting an important role for this molecule in inflammatory processes. cic3a exerts its functional activity through the specific binding to a cell surface g protein-coupled seven-transmembrane receptor (cic3ar), present on granular and hyaline amoebocytes, which has been recently identified and sequenced for the first time in an invertebrate species by our group. in an attempt to better characterize the cellular and molecular events triggered by the ligand-receptor (cic3a-cic3ar) interaction, we have focused our attention on the receptor internalization process, which is an important control mechanism described for g protein coupled receptors. we have analyzed by confocal microscopy the dynamic process of internalization of the cic3ar induced by the binding of cic3a in the form of the c-terminal eighteen aminoacid peptide (cic3a59-76) of the cic3a anaphylatoxin. the kinetic of the overall process has been monitored by fixing hemocyte samples at intervals between 0 and 30 minutes, and by immunostaining, performed with an anti-cic3ar antibody and an anti-rabbit igg fitcconjugated secondary antibody. preliminary results indicate that cic3ar, following the cic3a59-76 stimulus, is internalized in few minutes, and, in 30 minutes, recycled to the cell surface, made again available for cic3a binding. immunomodulatory soluble factors in the compound ascidian botryllus schlosseri 92 a menin, l ballarin department of biology, university of padua, padua, italy cytokines are proteins with immunomodulatory activity acting in a paracrine or autocrine way. most of them are produced and secreted by activated immunocytes after the recognition of non-self and are involved in several immunological processes such as inflammation, apoptosis, clearance of effete cells and corpes, cytotoxicity and phagocytosis. today the characterization of invertebrate cytokines represents one of the major topics of interest in immunobiology and, although the presence of molecules sharing homologies with vertebrate cytokines is still a matter of great debate, the presence of endogenous immunomodulatory molecules was indirectly demonstrated in various invertebrate taxa such as molluscs, anellids, echinoderms and tunicates. in tunicates, the presence of molecules able to influence the behaviour of immunocytes has been reported in both solitary and colonial ascidians. in the compound ascidian botryllus schlosseri, the presence of soluble opsonins released by phagocytes as well as the presence of molecules able to enhance yeast phagocytosis, modulate cytotoxicity and cross-reacting with antibodies raised against mammalian proinflammatory cytokines such as tnf-α and il-1α has already been described. in the present report, we investigated the presence and the behaviour, under various conditions, of molecules recognised by antibodies raised against mammalian cytokines. results confirm the presence of soluble peptides, upon the recognition of foreign molecules, able to increase the phagocytic index of b. schlosseri and recognised by antibodies antitnf-α and il-1α. the rise of phagocytosis is significantly decreased in presence of specific sugars such as galactose, rhamnose and sorbitol. the same behaviour was observed in haemoagglutination assays where the presence of glucose and rhamnose lowered the haemoagglutination titre. this suggests that lectins, able to modulate the immune responses, are secreted by stimulated immunocytes. vcbp genes in ciona intestinalis. ii. gene expression analysis i zucchetti1, r de santis1, s giacomelli1, gw litman2, d melillo1, mr pinto1 1laboratory of cell biology, stazione zoologica “anton dohrn”, naples, italy 2department of pediatrics, university of south florida, saint petersburg, florida, usa it has been found that vcbp genes of branchiostoma floridae are selectively expressed in scattered cells of the intestine, a digestive tract continuously exposed to a diverse range of potential pathogens. the overall size of these gene families together with their extensive polymorphism may be associated with specific v-directed recognition of foreign antigens. furthermore, it has been reported that some chitin-binding proteins found in invertebrates have an antimicrobial and antifungal role. hence, vcbps could be considered bifuctional molecules with features of the adaptive immune receptors combined with innate immune functions. in this context, due to its phylogenetic position, the study of ciona intestinalis vcbps may provide interesting indications. this, together with the possibility of the easy handling of both embryonic stages and adult tissues in c. intestinalis could allow a more extensive analysis of the gene expression and function. whole mount in situ hybridization experiments of ci-vcbp1, ci-vcbp2 and ci-vcbp3 carried out on young adults have demonstrated that the genes are exclusively expressed in the stomach. in situ hybridization on stomach sections confirms and extends these results providing also a more exhaustive analysis at cellular level. ci-vcbps expression is enhanced by challenging the animals, or blood cells, with bacterial lipopolysaccharides. preliminary whole mount in situ hybridization experiments carried out on larva stage indicate that ci-vcbps expression is localized in defined regions of the endoderm. infammatory responses of the ascidian ciona intestinalis n parrinello marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy innate immune system responses include acute inflammatory and granulation tissue production phases. cell migration, phagocytosis, encapsulation, tissue injury, matrix production, endothelial and epidermis activity, coordinate hemocyte stimulation. and wound repair have been described in ciona intestinalis following body wall challenge with lps. several reports suggest that in invertebrates immune system, cell proliferation, phagocytosis and chemotaxis are regulated by cytophilic humoral molecules with functional similarities to vertebrate pro-inflammatory cytokines (il1, il6, tnf) and galectins. here we show that like in mammals this inflammatory agent promptly challenges several cellular and molecular responses including enhanced serum lectins with il-1 epitopes, tnf, and type ix-like collagen. galectin-like molecules (ca2+-independent, specific for d-galactose and β-galactosides) with opsonic property can be enhanced as a response to a body wall wound and their release further stimulated by lps. the western blot pattern and immunohistochemical methods display oligomers with hril1α epitopes expressed by hemocytes, endothelial tissue and present in hemopoietic sites. these lectins can be promptly released by hemocytes challenged in vitro with lps; both cell lysate supernatants and hemocyte culture medium displayed hemagglutinating and opsonic activities inhibited by β-galactosides, and anti-hril1 antibodies. although a percoll density gradient separation method showed that several hemocyte types contain and release β-galactosidespecific molecules, assay of enriched hemocyte populations suggest that amoebocytes are the primary source of these molecules. for the first time we show citnfα to be constitutively expressed in the ascidian inflamed body wall and hemolymph, and upregulated by in vivo lps inoculation. cdna and deduced aminoacid sequence as well as lps challenged gene expression demonstrate unequivocally the involvement of soluble and cell bound citnfα forms in the ascidian 93 inflammatory response. sequence similarities with vertebrate tnfs include this cytokine into the tnf family. a prompt (2-4 h) enhanced citnf gene expression in the inflamed body is shown: in situ hybridization assays, cytometry and immunohistochemical methods support the involvement of pharynx and circulating hemocytes in the inflammatory response. immunoblotting assay with anti-citnf specific or anti-hrtnf antibodies revealed that hemocytes contains a 43 kda citnf whereas a 15 kda tnf is released into the serum hemolymph suggesting that, like in mammals and fish, citnfα cell bound homotrimer may be processed to a monomeric soluble form. densitometry analysis confirmed that citnfα protein expression is modulated by the lps challenge. enhanced transcript of a collagen with facit structural features (ci-typeix-col), in hemocytes, epidermis and migrating cells is also shown, while flow cytometry with specific antibodies, raised against an opportunely chosen ci-typeix-col synthetic peptide, displays fibroblast-property of hemocytes challenged in vitro with lps (at 4 hrs). in situ hybridation assay and immunocytochemistry identified collagen expressing hemocytes and disclosed epidermis gene expression in the time-course of the inflammatory reaction presumably involved in the inflammatory granulation phase. finally, the expression of a phenoloxidase component appears to be stimulated by inflammatory stimuli. in conclusion, the inflammatory response of c. intestinalis is characterized by components homologous (tnf, c3-like, collagens) and analogous (galectin-like) to the vertebrate ones. tunicate immunocytes can be cytotoxic toward foreign cells v arizza, ft giaramita, d parrinello , m vazzana, a vizzini, g salerno, m cammarata, n parrinello marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy tunicate immunocytes can be cytotoxic toward foreign cells, and cytolytic molecules (“lysins”) have been revealed in vitro by using erythrocyte targets. in ciona intestinalis the hemocyte cytotoxic activity has been examined towards mammalian erythrocytes in a medium isosmotic to the hemolymph containing 10 mm ca2+ (tbs). unilocular refractile hemocytes (urgs) release cytotoxic factors inhibited by sphingomyelin in a plaque-forming assay. to separate the lysinreleasing cells from the hemolymph and characterize lysins, a discontinuous percoll gradient was performed and hemocyte populations were separated in 6 bands. urgs cytotoxic for re were enriched (~40 %) in the band 5 (b5) and then lysed to obtain the supernatant. the b5-lysate supernatant (b5-hls) showed lytic activity (~84 %) specific for re and k562 cells whereas a lower level of cytotoxicity was found against sheep erythrocytes (se). such an activity was ca2+dependent and thermostable at 56 °c, b5-hls also showed a ca2+-independent hemagglutinating activity against trypsinized re (ht 4.3) but not toward trypsinized se. inhibition experiments displayed that lysins and lectins could be inhibited by carbohydrates (galactose, thio-digalactoside, lactose, lactulose) whereas sphingomyelin (2.5 µg/ml) only inhibited the lytic activity, suggesting that lectins may be involved in membrane sphingomielyn-lysin interactions. to check for the enzymatic nature of the lysins, phospholypase a2 inhibitors such as dibucain and quinacrine were assayed. the experiments showed that both the molecules were inhibitors of b5-hls cytotoxic activity suggesting the involvement of a ca-dependent phospholypase a2 activity. a cytotoxic mechanism based on phospholypase a2 activation due to lectinsugar interactions is discussed as a model. lysins against re and k562 cells with the same properties including sugar and phospholypase a2 inhibition, can be promptly (within 3 h) released in vitro by urgs in a culture medium suggesting that activated cells could participate in the defence response exerting a cytotoxic role. the prophenoloxidase system is activated during the tunic inflammatory reaction of ciona intestinalis m cammarata, v arizza, c cianciolo, d parrinello, m vazzana, a vizzini, g salerno, n parrinello marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy in invertebrates, the "prophenoloxidase activating system" involved in immune responses, is challenged by ß-1,3 glucans or lipopolysaccharides through a limited proteolysis due to serine proteases. phenoloxidase (po) is usually synthesized as proenzyme (prophenoloxidase, propo) and, upon activation, it plays a key role in humoral immune response and melanization processes. po activity in the tunic tissue of ciona intestinalis following lps intratunic injection was examined. tunic homogenate supernatant (ths), assayed with dopa-mbth reaction, displayed ca2+-independent po activity that was raised by lps and further enhanced by proteases. specific inhibitors (tropolone, phenylthiourea, diethylthiocarbamate) supported the specificity of the reaction. assay with soybean trypsin inhibitor suggests that, in the tunic, proteases diverse from serine proteases could also be involved in the activation pathway. in vivo experiments were carried out by injecting isosmotic medium or lps, and ths assayed for its po activity. anova analysis of the time course profiles showed that lps was more effective in activating propo. to disclose the po response at the injured site, an assay with dopa-mbth was performed in vitro. quinones were mainly contained in the tunic matrix enriched with inflammatory cells around the injection site. microscopy observations and immunohistochemistry with anti-cinpo-2 antibodies showed granulocytes and unilocular refractile granulocytes containing po, whereas rare morula cells were stained. in ths zymograms (sds-page), po activity linked to 90 and 120 kda bands was observed as an effect of lps injection, whereas the density of the 170 kda po was weak. in addition a third presumptive po enzyme (cinpo-3) containing the cinpo-2 peptide was identified in the recent ciona genome version. the possible involvement of a presumptive cinpo-3 similar to cinpo-2, as predicted by in silico analysis with blast (pam 30 scoring matrix) of ciona genome sequences (jgi v2), is 94 discussed. presumably, lps stimulated the production and dimerization (120 kda) of cinpo-3 (66 kda). in conclusion, the activated propo system includes several pos distinguishable in their size contained and presumably released by tunic inflammatory cells and hemocytes of the pharynx bars. isolation, characterization and expression analysis of a collectin in tunicate ciona intestinalis a bonura1, a vizzini2, g salerno2, d parrinello2, n parrinello2, v longo1, p colombo1 1institute of biomedicine and molecular immunology "a. monroy". the national research council (cnr) palermo, italy 2department of animal biology, university of palermo, palermo, italy collectins are a family of calcium-dependent (ctype) lectins characterized by four functional domains: a short amino terminal ‘tail’ domain, a collagen-like domain that is typified by its repeating pattern of glycine-x-y (gly-x-y) amino acid triplets, a distinct neck region and the carboxy-terminal c-type carbohydrate recognition domain (crd). six distinct classes have been identified in vertebrates: mannose binding lectins (mbl), lung surfactant proteins a and d (sp-a and sp-d), conglutinins, serum collectin-43 (cl43) and serum collectin-46 (cl-46). mbl can recognize carbohydrates on the surface of pathogens and then activate the central complement component, via a mannose binding lectin-associated serine protease (masp), during acute phase responses to infection. in order to study molecules with differential activation of gene expression during the immune response, an injection of lipopolysaccharide (lps) into the tunic tissue at the median body region of ciona intestinalis was performed. one hour following injection animal was sacrified and rna was extracted and used for subtractive hybridization. rna was reverse transcribed and cloned using e. coli. sequencing analysis identified a cdna of 863 nucleotides encoding a protein containing 221 amino acids for a molecular weight of 24426 daltons. the sequence of this protein showed structural domains typical of collectin and a high degree of similarity with the mbl of gallus gallus and human mbl 2. in addition, studies of gene expression conducted through analysis of real time and in situ hybridization, have highlighted the increase of expression of this mrna compared to control animals that an increase of cells that express the mrna collectin near the site of lps inoculation. morphological characterization and acetylcholinesterase activity in ciona intestinalis hemocytes v mansueto, v arizza, n parrinello marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy the ascidian hemocytes have been reported to be involved in various functions including coagulation, nutrition, defense, infiammatory-like reaction, allogeneic reaction, tunic, gonad and germ cells formation. in addition, some of them are known to be involved in vanadium accumulation. hemocytes have been classified as stem cells, pigment cells, hyaline amoebocytes, granular amoebocytes, unilocular refractile granulocytes (urg), compartment cells, signet-ring cells and morula cells. cell lineage analysis showed that hemocytes may originate from a pair of a7.6 blastomeres of a 64-cell embryo, which give rise to trunk lateral cells (tlcs). our previous researches showed an acetylcholinesterase (ache) activity in the tlcs of ciona intestinalis swimming larva. in mammals, this enzyme has a non-classical control role in stem cell differentiation, apoptosis, defense responses and haematopoiesis. several c. intestinalis circulating hemocytes were characterized by means of different staining methods, and ache activity was identified in morula and signet ring cells. in c. intestinalis , this enzyme could function as an inhibitor of stem cells proliferation also related to cell differentiation. we show once again that several hemocyte forms circulate in c. intestinalis hemolymph some of them presumably due to differentiation steps from stem cells. session 4. enviromental stress tributyltin-induced effects on mapk signaling in ascidian embryos f damiani, m gianguzza, g dolcemascolo dipartimento di biopatologia e metodologie biomediche, sez. di biologia e genetica, università degli studi di palermo, palermo, italy among the class of organotin compounds, the most well known is tributyltin (tbt). organotin have many applications, which include use in pvc, as catalyst in chemical reactions, agricultural pesticides and antifungal treatments for textile polymers. in particular tbt is used in marine antifoulant paints to prevent the growth of organisms such as barnacles on the hull of ships. extensive use in antifouling paints led to the widespread distribution of tbt and its breakdown products in the global marine, sediment and biota. high levels of tbt in the waters were found to have impaired reproduction, by inhibiting embryogenesis and larval development in a variety of marine organisms. symptoms of the exposure to high levels of tbt in some invertebrates includes the development of male sexual characteristics as a penis and vas deferens by females (imposex). ascidians are a good model for the study of embryonal development. they are also sensitive bioindicators of habitat degradation. the effects of tributyltin (iv) chloride (tbt chloride) solutions on ascidian embryos of ciona intestinalis at different stages of development have been described. previously, we carried out observations with both the light and the electron microscope on ciona intestinalis embryos and larvae incubated in tbt solutions. this studies showed morphological and ultrastructural modifications of the embryos and larvae after incubation in tbt chloride at different concentrations. to understand molecular effects of tbt-induced on ascidians embryogenesis we have set out to study the effects of tbt at different concentrations, testing the activity of some protein with a basic role in embryonic development. in ascidian 95 embryos, a fibroblast growth factor (fgf)-like signal has been proposed to be involved in induction of notochord and mesoderm formation. a main pathway is a protein kinase transduction pathway, which includes ras, raf, mitogen-activated protein (map) kinase and extracellular signal-regulated kinase/map kinase (erk). the aim of this work in progress is to understand whether the tbt exposure on ascidian embryos at different stage of development cause alterations in tyrosine phosphorylation pattern and in mapk activity. tyrosine phosphorylation promotes cell growth, differentiation and apoptosis, due to activity of receptor tyrosine kinases and furthermore different stressors are known to stimulate tyrosine kinase activity. at first we focused our attention on tyrosine phosphorylation pattern after ascidian embryos to different stage of development tbt treatment. phosphorylated proteins pattern is evaluated by sdspage electrophoresis and western blotting on protein extract of ascidian embryos incubated with tbt, using anti-phosphotyrosine-antibody directed against mammalian phosphotyrosine. preliminary results showed a different pattern on protein phosphorylation in response to the incubation with tbt in µm range. since mapks play a key role in animal responses to a wide variety of environmental stresses, we have thought to test the role of mapk pathway proteins such as mapk p38 (thr 180 and tyr 182), p44/42 (thr 202/tyr 204) and c-jun n-terminal kinases (jnk) after tbt treatment. effects of cadmium on the functionality of haemocytes from the compound ascidian botryllus schlosseri n franchi, m di silvestro, l ballarin department of biology, university of padua, padua, italy ascidians share a variety of circulating haemocytes differing in their morphology and involvement in various biological functions. most of them are immunocytes, able to mount defence reactions against foreign, potentially dangerous, cells or molecules. metallothioneins are ubiquitary, cysteines-rich proteins which exert fundamental roles in the detoxification of trace metals such as ag, hg, cd, cu and zn, some of which are required for normal cell metabolism. up to now, no molecular data are available in both solitary and compound ascidians, included the species botryllus schlosseri, an important model organism for a wide variety of studies. in the attempt to study the involvement and the role of metallothioneins in botryllus immunobiology, we investigated the effects of acute exposure of haemocytes to cd on cell functionality. preliminary results indicate a dose-dependent negative effect on the ability to adhere of haemocytes and a decrease of the capability of phagocytes to spread on the substrate and to trigger a respiratory burst when matched with yeast cells. a cdna library from cd-exposed haemocytes is now under construction from which we expect to find metallothionein sequences toxic effect of methylmercury on ascidian (styela plicata) immunocyte responses mg parisi, m cammarata, g benenati, v arizza, t cillari, d piazzese, a gianguzza, m vazzana, a vizzini, n parrinello marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy pollution by heavy metals is one of the major risk in aquatic ecosystem, where high concentration cause adverse biological effects, including changes in immune function of invertebrate and vertebrate species. in marine environment, although mercury concentration in water column and sediments may be low, filter feeding invertebrates highly accumulate this metal in their tissues. in addition, biological processes mediate the mercury methylation transforming the metal in methylmercury which is the most toxic form due to the methyl group that facilitates cell penetration and interaction with proteins interfering with their synthesis and leading to lipid peroxidation. this study shows that high methylmercury concentrations are cytotoxic for styela plicata haemocytes, whereas subletal concentrations promptly affect immunocyte responses. moreover, haemocytes exposed to the xenobiotic present a significantly enhanced phenoloxidase activity as revealed in the haemocyte lysate supernatant compared to the control. although the cytotoxic activity of s. plicata haemocytes toward rabbit erythrocytes is a po-dependent cell-target reaction due to quinone products, it was significantly decreased by suitable methylmercury concentrations in the medium. the same xenobiotic concentrations decreased the haemocyte phagocytic activity toward yeast. in both the responses cell-target contacts could be affected by methylmercury, whereas the releasing capacity appeared to be unchanged as indicated by haemocyte po-release in the medium. finally, changes in haemocyte shape and spreading capacity were shown. effects on cytoskeleton could be responsible of changes in the haemocyte morphology and spreading capacity as revealed by the microplate assay we performed. on the basis of the present results, s. plicata could be an additional sentinel species for heavy metal environmental pollution by using immunotoxicology tests and a microplate method that reveals cell morphological changes and spreading capacity. similar results on the cells were obtained by assaying polluted sea water from different sicilian coastal sites. 96 isj102.pdf 91 isj 2: 91-104, 2005 issn 1824-307x review the point about oxidative stress in molluscs h manduzio, b rocher, f durand, c galap, f leboulenger laboratory of ecotoxicology (lema), upres ea 3222, ifrmp 23, ufr of sciences and techniques, university of le havre, france accepted july 8, 2005 abstract in the normal metabolism of the aerobic cell, oxygen is used for various biochemical reactions. because of its two lone electrons of parallel spins, the molecular oxygen is stable. however, oxygen generates reactive oxygenated species or ros by successive transfer of electrons. the ros have a strong reactivity and can potentially interact with all other cellular components (lipids, proteins, dna). they are at the origin of oxidations in chain by creating radicals. the cell has antioxidant systems which limit the effects of the ros. these systems are composed of enzymes such as glutathione reductase, glutathione peroxidase, etc., and molecules of nonenzymatic nature like the reduced glutathione or vitamins. the production and the destruction of the radicals of oxygen coexist in a weak balance. if this balance is broken in favour of the ros, an oxidative stress is generated. xenobiotics could influence this balance by catalysing production of ros. key words: oxidative stress; molluscs; ros; antioxidant; xenobiotics introduction face to chemical stress, each type of organisms, and for any species has a capacity of adaptation, based on regulating processes. these processes make it possible to maintain physiological homeostasis and the integrity of the individual, structural or functional deteriorations remainder entirely reversible or reparable. corresponding author: hélène manduzio, laboratory of ecotoxicology (lema), upres-ea 3222, ifrmp 23, ufr of sciences and techniques, university of le havre, 25 rue philippe lebon – bp 540, 76058 le havre cedex, france e-mail: helene.manduzio@univ-lehavre.fr list of abbreviations: cat: catalase; dt-d: dt-diaphorase; gpx: glutathione peroxydase; grd: glutathione reductase; gsh: reduced glutathione ; gst: glutathione s-transferase; mda: malondialdehyde; pah: polyaromatic hydrocarbons; h202: hydrogen peroxide; mda: lipoperoxydation; pcb: polychlorinated biphenyls; pco: peroxidation of proteins; segpx: glutathione peroxydase seleno-dependent; sod: superoxide dismutase passed the threshold of toxicity, the more marked irreversible attacks lead to a pathological state which results in a significant deterioration of the individual performances and later lead to death of the organism. now, many xenobiotics are recognized like exerting their harmfulness by catalysing production of oxygenated radicals (winston and di giulio, 1991). so the impact study of the toxic effects of the contaminants rejected into the environment requires a preliminary knowledge of the normal physiological mechanisms of adaptation (ecophysiology) and the comprehension of deteriorations of these processes induced by the contaminants (ecotoxicology). oxygen holds a capital place in the diversification of the species and their occupation of a majority of ecosystems. being at the base many biochemical processes of the metabolism of the aerobic organisms, oxygen is an essential molecule. however, its oxidizing capacities make of it a potentially aggressive element for the majority of the bio-molecules. in order to limit its harmful effect, the antioxidant mechanisms were set up which make it possible the organism to maintain the rate of radicals on a low basal level. in the event of oxidative stress, the antioxidant systems can be exceeded, then causing the oxidation of different molecules and leading to cellular dysfunctions. 92 in a context of a multiple contamination in particular in water ecosystem, the study of oxidative stress is very used in biomonitoring. the molluscs are especially used in this type of study on account of their characteristics. we will try to resume the data about oxidative stress in these organisms. nature and origin of the reactive oxygenated species (ros) the free radicals are atoms or molecules unstable presenting one or more lone electrons. to reach a better level of stability, they will yield or tear off electrons from molecules met. ros create new radical species thus, causing oxidations in chain. all the bio-molecules of the cell (nucleic acids, lipids, proteins, polysaccharides) are potential substrates of ros. a significant criterion in the characterization of the radicals is their diffusion capacity, which reflects the level of stability of the ros. a little reactive form tends to act far from its site of production and thus has a significant diffusion. on the contrary, a very reactive species acts very quickly and its diffusion is so limited. molecular oxygen o2 can be regarded as a radical species since it has two lone electrons; however, this molecule has a significant stability, the simultaneous addition of two electrons being difficult. in order to carry out this reaction, enzymes will create intermediates, which constitute the ros in particular during respiration and photosynthesis. the superoxide anion radical o2 -· is produced during endergonic reduction of molecular oxygen by capture of an electron. this reaction can be spontaneous in aerobic medium. o2 -· is then generated primarily in membranes because of the high solubility of oxygen in hydrophobic medium (gutteridge and halliwell, 1993). however, it is produced during various reactions. metals of transition such iron and enzymes are implied in its formation. the flavoenzymes and the xanthine oxidase activated by ischemia produce it (fukai et al., 2002). in addition, the phagocytic cells generate some for the degradation of the immune complexes by the means of four enzymes: nadph oxidase, superoxide dismutase, nitric oxide synthase and myeloperoxydase (babior, 1978a, 1978b). the superoxide anion radical is characterized by a low reactivity. moreover, it does not have the capacity to pass the membranes; it remains limited to the compartment where it was produced. but it is at the origin of the oxidation of lipids. in fact, the deterioration of the membrane structures is carried out by nucleophilic attack between fatty acids and glycerol of phospholipids. o2 -· can act at the same time like an oxidant and a reducer. in the presence of some metals (manganese or vanadium), it catalyses reactions of oxidation in chain for example oxidation of many molecules of nad(p)h (liochev and fridovich, 1989). on the contrary, within the framework of metals of transition (iron or copper) present at the active sites of enzymes, it presents a reducing behaviour (liochev and fridovich, 1989). hydroxyl radical oh· can be produced during the thermal reactions or under the effect of ionizing radiations. it can also be generated during a reaction implying iron and which is translated by the fenton’s reaction. the hydroxyl radical can be also produced by homolytic fission of the h2o2. this reaction of haberweiss is catalysed by metals of transition. the first stage is the reduction of the superoxide anion radical and the second corresponds to the reaction of fenton. the haber-weiss reaction can be inhibited by chelating of metals, in particular the desferrioxamine in the case of iron. the hydroxyl radical is very reactive and thus its diffusion is limited. it will interfere with the first molecules met, generally on the level of the site of production. it acts, either by addition or by wrenching of hydrogen from the target molecule, or by transfer of electrons. it is at the origin of the lipid peroxidation. the h2o2 is produced by the dismutation of the superoxide anion radical. this anion leads spontaneously to h2o2 under the conditions of physiological ph. this reaction can be accelerated by action of superoxide dismutases (fridovich, 1975). the h2o2 is moderately reactive but its diffusion is high, having the capacity to cross the membranes. its intracellular concentration is very weak between 0.001 and 0.1 µm (sies, 1991). however, in mitochondria and peroxysomes, these concentrations can reach higher levels (boveries et al., 1972). the h2o2 holds a significant place among the ros because it plays the role of intermediate in the production of other reactive radicals. by the means of metals of transition (copper, iron), it gives rise to the hydroxyl radical. moreover, the h2o2 is an intracellular signalling factor (sundaresan et al., 1995). the nitric oxide no· is not always regarded as a ros. indeed, it plays at the same time a role in the destruction and the production of radicals. moreover, it is not very reactive with the cellular components and reacts with radicals generating of less reactive species. it is thus able to inhibit lipid peroxidation (rubbo et al., 2000). conversely, the nitric oxide combined with the superoxide anion radical involves the formation of peroxynitrite, highly toxic radical (beckman and koppenol, 1996). the no is produced from endogenous or exogenous no donors or thanks to a reaction of oxidation of the arginine in the presence of oxygen and nadph. this reaction is produced in particular on the level of the phagocytic cells (marletta, 1994). the nitric oxide and the radicals that result from this are gathered under the term of reactive nitrogenized species rns. the hydroperoxyl (roo· ) and alcoxyl (ro· ) radicals rise from the peroxidation of the lipids. these radicals allow the propagation gradually of the lipid peroxidation. after degradation, they lead to aldehyde formation organized in three major groups: 2acetaldehydes (e.g. 2-hexenal), 4-hydroxy-2acetaldehydes (e.g. 4-hydroxynonenal) and ketoaldehydes (e.g. malondialdehyde) (uchida, 2003). origin and role of ros the major role of the endogenous production of ros is an activity of regulation. indeed, the radicals can interact directly with the molecules containing sulfhydryl groups and thus change their conformation. this type of regulation can in particular affect molecules implied in the mechanisms of signal transduction like protein kinase c (dalton et al., 1999). 93 table 1. compounds generating ros and biomarkers of oxidative stress measured in bivalves contaminants organisms measures authors menadione mytilus edulis sod, gpx, cat (livingstone et al., 1990) dt-d, mda mytilus edulis mda, gsh (ribera et al., 1991) b(a)p mytilus edulis sod; gpx, (livingstone et al., 1990) cat, dt-d, mda paraquat geukensia demissa sod, cat, (wenning et al., 1988) gsh, mda thiram unio tumidus gpx, grd, sod (doyotte et al., 1997) cat, gsh, mda nitrofurantoine mytilus edulis ros (martinez et al., 1995) h2o2 mytilus cat, sod, (cavaletto et al., 2000) galloprovincialis gpx, gsh, mda aroclor 1254 chamaelea gallina cat, grd, (rodriguez-ariza et al., 2003) gpx, gsh table 2. metals generating ros and biomarkers of oxidative stress measured in bivalves contaminants organisms measures authors copper mytilus edulis mda, gsh (viarengo et al., 1989) mytilus edulis sod (manduzio et al., 2003) mytilus sod, grd, gpx (regoli and principato, 1995) galloprovincialis cat, gsh mytilus mda, gsh (viarengo et al., 1990) galloprovincialis lysosomal stability mytilus gsh (canesi et al., 1999) galloprovincialis unio tumidus gpx, grd, sod (doyotte et al., 1997) cat, gsh, mda ruditapes sod, cat, segpx (geret et al., 2002) decussatus gpx, mda cadmium mytilus mda, gsh (viarengo et al., 1990) galloprovincialis lysosomal stability zinc mytilus mda, gsh (viarengo et al., 1990) galloprovincialis lysosomal stability mercury anadara granosa grd, gsh (patel et al., 1990) mytilus gsh (canesi et al., 1999) galloprovincialis selenium anadara granosa grd, gsh (patel et al., 1990) complex mytilus gpx, cat, sod, (regoli and principato, 1995) contamination galloprovincialis grd, gsh 94 table 3. in situ contamination studies and biomarkers of oxidative stress measured in bivalves contaminants organisms measures authors hap perna viridis sod, cat, gpx (cheung et al., 2001) dt-d, gsh, mda mytilus edulis sod, cat (eertman et al., 1995) saccostrea cucullata cat, sod (nyogi et al., 2001b) gpx, dt-d hap + pcb mytilus sod, segpx, cat (solé et al., 1995) galloprovincialis unio tumidus grd, segpx, gpx, (cossu et al., 1997), gsh, cat, sod, mda (cossu et al., 2000) mytilus cat, sod (porte et al., 1991) galloprovincialis gpx, dt-d complex crassostrea virginica gsh, mda (ringwood et al., 1999) contamination lysosomal stability mytilus gsh, grd, gpx (regoli and principato, 1995) galloprovincialis cat, sod dreissena mda, dna (de lafontaine et al., 2000) polymorpha mytilus cat, dt-d, sod (livingstone et al., 1995) galloprovincialis mytilus edulis sod, gpx, gst (manduzio et al., 2004) metals mytilus grd, segpx, gpx (regoli and winston, 1998) galloprovincialis cat, sod, gsh mytilus lysosomal stability (domouhtsidou galloprovincialis mda and dimitriadis, 2001) they allow the regulation of many other molecules (babior et al., 1997). studies showed that the h2o2 can replace insulin in its role of growth promoter (allen and tresini, 2000). thereafter, other experiments showed the stimulation of the production of h2o2 by insulin and nerve growth factor (mukherjee et al., 1978; mukherjee and mukherjee, 1982). the nitric oxide plays itself a role in the vasodilatation and the neurotransmission by activation of enzymes (allen and tresini, 2000). the radicals also play of the roles of control of various factors of transcription (sen and packer, 1996). a significant source of free oxygenated radicals comes from the redox cycles and of the oxidation catalysed by cytochrome p450 monooxigenases. these enzymes allow the addition of a functional group to the exogenous compounds. the redox cycles pass by reactions of oxidation, reduction and hydrolysis, each mediated by transfers of electrons. at the time of each reaction, ros are formed. many exogenous compounds can stimulate the production of ros (tables 1-3). several modes of action were described. many xenobiotics catalyse the microsomal transfer coming from the nad(p)h towards oxygen of electrons and then involving ros formation. it is the case of the nitroaromatic compounds (e.g. nitrofurantoin), quinones (e.g. menadione), and derived from the bypiridium (e.g. paraquat). various studies showed a production nad(p)h-dependent of ros stimulated by contaminants (lemaire et al., 1994; peters et al., 1996; lemaire and livingstone, 1997; livingstone et al., 2000). a number of compounds will involve the formation of active species of oxygen after metabolisation during which they become themselves of the radicals as quinones. in this case, xenobiotic is first reduced by a nadph-dependent reductase during the reaction on phase i producing a radical. this last can then transfer an electron to oxygen. a superoxide anion radical is generated. in each cycle, two potentially harmful compounds can thus be produced (winston and di giulio, 1991; goeptar et al., 1995). a deficiency in metal can also lead to an oxidative stress. indeed, several metals are integrated into proteins; it is the case of the copper in cu/zn-sod (l’abbe and fischer, 1984; taylor et al., 1988). in the rat, a feeding without coppers leads to a rise of the quantity of oxidized proteins and to a fall of the activity of cu/zn-sod in erythrocytes (sukalski et al., 1997). however, in excess, copper can increase the rate of malondialdehyde, marker of the lipid peroxidation, and induce a reduction in the rate of glutathione. indeed, copper is suitable for catalyse the production of hydroxyl radicals via the reaction of haber-weiss (kadiiska et al., 1993; bremmer, 1998). 95 the ionizing and ultraviolet rays are also of significant sources of radicals of oxygen by break of the molecules. origin of ros in molluscs in ecotoxicology, many compounds such as pah, pcb and metals were implicated in the induction of the production of radicals at the laboratory like in situ (tables 1-3). however, it has a lack of data about the exact mechanism of action of these compounds. the measurement of the oxidative stress is generally carried out by the follow-up of the modifications of the activity levels of enzymes (cat, sod, etc.) and of the rates of the molecules (gsh, etc.) implied in the antioxidant defence in two main tissues, digestive gland and gills. these studies involved a hierarchical organization of the cellular answers and more specifically of the antioxidant enzymes. thus, cat is regarded as an enzyme presenting a clear and early response to contamination (wenning et al., 1988). the induction of the gpx is generally noted in a concomitant way to that of the cat and sometimes to that of sod (rodriguez-ariza et al., 1993). in molluscs, as in the mussel mytilus galloprovincialis, sod seems to be a stable enzyme, seldom presenting variations of activity (livingstone et al., 1995). the various studies can however show contradictory results. thus, the sod is sometimes described like presenting a modification of activity in a concomitant way at the cat as in geukensia demissa after 12 h of exposure to the paraquat (wenning et al., 1988). géret et al. (2002) describe a reduction in the levels of seleno-dependent and total gpx activities in ruditapes decussatus exposed to copper (0.5; 2.5 and 25 µg.l-1) after one day of exposure. at the reverse, the carp cyprinus carpio morpha presents an increase in the gpx activity after 1 day of exposure to copper (5, 10, 25 and 50 µg.l-1) (radi and matkovics, 1988). other molecules implied in antioxidant defences are measured, most current being gsh. the reduction in the rate of reduced gsh was observed at bivalves m. galloprovincialis and unio tumidus in correlation with the presence of pah and pcb in the medium (regoli and principato, 1995; doyotte et al., 1997; cossu et al., 1997, 2000). the modification of the rate of reduced gsh as well as balance between the rates of reduced and oxidized glutathione (gsh/gssg) can be correlated with the variation of grd activity. patel et al. (1990) observed a reduction in the grd activity and an increase in the rate of oxidized glutathione during the exposure of bivalves (anadara granosa) to mercury. in this context, the grd activity also constitutes an interesting enzyme in the study of the oxidative stress. the reduction in the rate of reduced gsh was connected to the induction of the lipid peroxidation in particular in the mussel exposed to metal contaminants (viarengo et al., 1988). nevertheless, the lipid peroxidation is generally correlated with the reduction in the whole of the antioxidative enzymes (doyotte et al., 1997; cossu et al., 1997, 2000; géret et al., 2002). the environmental parameters are also suitable for induce a variation of pro-oxidant/antioxidant balance. many studies showed seasonal variations of the antioxidant activities at marine species (viarengo et al., 1991; orbea et al., 2002). these variations are due to the fluctuations of temperature, salinity, the oxygen rate and the quantity of food available (viarengo et al., 1991). changes of seasonal nature were also studied in order to understand their implication in the answers of the antioxidant systems to the contaminants. this kind of studies allows establishing the link between ecophysiologic parameters and ecotoxicological reactions (niyogi et al., 2001a, 2001b). effects of ros and diseases ros can act on the whole of the cellular components. the variations of level of these radicals thus have significant effects on cellular functions. the ros influence in particular the thiol groups of proteins, leading to the formation of intraor inter-molecular disulphide bridges. the most studied action of ros is the lipid peroxidation. this reaction is mainly carried out by the hydroxyl radical (stegeman et al., 1992a; steinberg, 1997). this process corresponds to reactions in chain. after rearrangement and addition of oxygen, peroxyl (roo· ) and alcoxyl (ro· ) radicals are generated. oxidation is propagated thereafter with other unsaturated lipids and can even reach proteins. the oxidation of phospholipids membranes involves disturbances of these structures. as a first consequence, we can observe a reduction in fluidity of the membranes and the inactivation of the receptors and enzymes located at their level (snell and mullock, 1987). in a second time, this oxidation and particularly oxidized products increase the permeability of the membranes, in particular with the calcium ions leading to cellular death (gutteridge and halliwell, 1990). on the level of the mitochondria like lysosomes, the lipid peroxidation results in the lysis of these organelles and the release of enzymes. these enzymes then catalyse the decomposition of proteins, nucleic acids and cellular polysaccharides (horton and fairhurst, 1987; snell and mullock, 1987; pre, 1991). as example, the oxidation of the polyinsaturated lipids can induce the appearance of cardiovascular diseases (wattanapitayakul and bauer, 2001). in a general way, during this reaction, various compounds are produced such malondialdehyde (mda) and 4-hydroxynonenal (hne), both able to bind to proteins and to form adducts. indeed, these compounds react in a spontaneous way with cysteines of proteins and with glutathione. the 4hydroxynonenal can inhibit the synthesis of the nucleic acids and proteins, and block the cellular proliferation (benedetti et al., 1982, 1986; esterbauer and cheeseman, 1990; esterbauer, 1993). another action of the ros relates to proteins. the oxidation of proteins derives from direct action of ros or indirect interaction with the alcoxyl (ro· ) or peroxyl (roo· ) radicals generated at the time of the lipid peroxidation. the amino acids most sensitive are those including sulfhydryl groups such methionine and tryptophan. this oxidation can involve of: (i) change of protein conformation by modification of some amino acids; (ii) generate bridges between proteins and proteins and lipids; (iii) cuts (levine et al., 1994). the structural modifications induce functional changes in 96 particular cellular metabolism (shacter et al., 1994). indeed, the oxidation of proteins can result in a disturbance of ionic transport, enzymatic activities and calcium homeostasis. the damaged proteins are then more sensitive to the proteases action, and thus destroyed more quickly, this being able to induce tissue degradation (rice-evans et al., 1991). the nucleic acids are also targets for the free oxygenated radicals. the damage is not specific: simple or double cuts, formation of abasic sites, covalent bonds between dna or dna and proteins, and modifications of the bases, the most reached being the deoxyguanosine oxidized in 8-hydroxy-2'deoxyguanosine (8-ohdg) (meneghini, 1988; dizdaroglu, 1991; spencer et al., 1996). the dna damages are mainly caused by the hydroxyl radical (oh· ). superoxide anion radical can cause also cuts of dna and lesions of the bases. if guanine is the majority target, each base can undergo these attacks (halliwell and dizdaroglu, 1992). finally, the glucid oxidations in presence of metals involve protein cuts. this reaction is initiated by the hydroxyl radical, which tears off a hydrogen atom to the one of carbons close to the glycoprotein. other radicals are produced such as the peroxyl radical. the cytotoxicity of the radicals of oxygen takes part in the development of much pathology. the oxidative stress is thus implied in the disease of alzheimer (bowling and beal, 1995; ihara et al., 1997). the damage caused by the radicals of oxygen among parkinsonian patients was shown and would be related to a deficiency of the system of defence in brain, in particular in sods activity (radunovic et al., 1997). conversely, an overproduction of radicals of oxygen is implicated in the development of some diseases. at the time of the respiratory syndrome of distress, an infiltration of fluid is observed in the air cells resulting from damage of the endothelium of the capillaries. at the people reached of this syndrome, the lungs contain a significant number of neutrophils (weiland et al., 1986). the production of ros is also implied in rheumatoid arthritis. the therapies against this pathology include antioxidant components (reglinski et al., 1997). balance between pro-oxidants and antioxidants systems would be also implied in the phenomenon of cellular ageing (sagar et al., 1992). the oxidative stress would increase during cellular differentiation and ageing (sohal et al., 1990). however, the implication of the radicals in cellular ageing is not cleared up. indeed, the studies do not show all the same variations according to the age (e.g. mizuno and ohta, 1986; sohal et al., 1990; hussain et al., 1995; sahoo and chainy, 1997; kim et al., 2002). effects in molluscs at the marine molluscs, the physiological and morphological modifications in response to the chemical stress are not much studied. indeed, the appearance of pathologies is generally synonymous with irreversible damage. the major observations related to hepatic pathologies such as the increase in the occurrence of parasitic infections, ignition and necrosis in the fish (vethaak, 1992). however, in molluscs, the majority of the studies evaluate the impact of pollution by the appearance of neoplasia (malins et al., 1988; kinae et al., 1990). in molluscs, the observation of pathologies does not correspond to a major axis of ecotoxicological studies. nevertheless, in situ works bring back the observation of a blood neoplasm, haematopoietic neoplasm, disseminated neoplasia, hemic neoplasia, leukaemia or proliferate cellular disorder (krishnakumar et al., 1999). this syndrome was observed overall on 15 species of bivalves including 4 of oysters, 6 of clams and 5 species of moulds (re-examined of elston et al., 1992). this disease is characterized by the proliferation of circulating haemocytes. they present a significant core of lobed form which compared fills the major part of the cell to the cytoplasm. moreover, one or more micronuclei are observed as well as a high frequency of mitoses (farley, 1969; mix, 1983). the origins of this neoplasia remain discussed. indeed, some authors advance a potential implication of carcinogenic compounds (lowe and moore, 1978; farley et al., 1991). other studies connect on the contrary, the appearance of this pathology to a retrovirus or to a genetic disposal of the individuals (couch and harshbarger, 1985; elston et al., 1988). nevertheless, lowe and moore (1978) showed the appearance of neoplasms in the mussel mytilus edulis subjected to a pollution of domestic and industrial nature including hydrocarbons. other authors blame these same xenobiotics in the appearance of these tumours in different bivalves, in particular of oysters, subjected to the pollution generated by the shipwreck of amoco cadiz in france (balouet et al., 1986; barry and yevich, 1975; yevich and parszcz, 1977). conversely, mix and schaffer (1983) do not note any incidence of pah on the frequency of appearance of neoplasia in m. edulis. in addition, farley et al. (1991) showed a linear correlation between the appearance of neoplasms and the tissue concentration in chlordane. the exposures in laboratory lead in the same way to contradictory results, with either inductions of tumours or no incidence following treatments with the pah or other xenobiotics (khudoley and syrenko, 1978; rasmussen et al., 1983a, b, 1985; winstead and couch, 1988; krishnakumar et al., 1999). however, the implication of the oxidative stress is however not proven in a sure way (elston et al., 1988; moore et al., 1991; krishnakumar et al., 1994, 1999). moreover, in m. edulis and mya arenaria, an increase in the frequency of appearance of the neoplasms is observed during the coldest months of the year. these observations can be related to the reduction in the activities of the antioxidant enzymes at low temperatures (elston et al., 1992). defences of the organisms the production and the action of the ros must be controlled in order to limit the cellular damage. this limitation is carried out initially by sequestration, even the destruction, of the systems pro-oxidants such as the complexation of free metals by metallothioneines. the antioxidant systems also include enzymes whose activity involves the destruction of the reactive oxygenated species. these enzymes can act by the 97 means of metals of transition. a last means of fight against the oxidative stress is the stop of oxidations in chain of the cellular components. the antioxidant capacities are variable from one species to another. moreover, it is allowed that these activities vary according to the seasons. lastly, another adaptation of the organisms to the increase in the production of ros is the induction of the synthesis of antioxidant molecules. two categories of antioxidant systems are generally defined: antioxidant enzymes and molecules without enzymatic activity. three major enzymes act jointly for the destruction of the ros in the cell: sods, cat and gpxs. the grd can be added to these enzymes even if it does not present a direct role in the catabolism of the oxygenated radicals. sods (sod; ec. 1.15.1.1) will allow thereafter the destruction of the superoxide anion radical by dismutation out of h2o2 dealt by cat. the two enzymes, sod and cat, have the same principal localization in the cell, the peroxysomes. isoenzymes of the sod are found in the various compartments of the cell, but their active site has a tertiary structure overall good preserved, made of a hydrophobic well where the superoxide anion radical fits. the reaction of dismutation is catalysed by a metal from which nature makes it possible to distinguish three types of isoenzymes. during the reaction, the metal ion captures an electron of the superoxide anion radical. sods seem to be very significant enzymes because of their ubiquity and of their localization at the same time intraand extra-cellular (stegeman et al., 1992b). cu/zn-sod (35 kda) was identified for the first time in 1968, in bovines erythrocytes by mccord and fridovitch (mccord and fridovich, 1969). in addition to its localization in the cytoplasm, its presence was also shown on the external face of the endothelial cells and in the blood plasma. later on, it was also detected in the peroxysomes, the lysosomes and the core of the eukaryotes cells (beyer et al., 1991). this isoenzyme is made up of two identical subunits from approximately 15 000 da each one, to which two metal atoms are added: copper and zinc. the function of destruction of the superoxide anion is provided by copper whereas zinc would have only one structural role. cu/zn-sod was described at the vertebrate ones, the aquatic and terrestrial invertebrates, like at the plants on the chloroplastic and cytosolic level. more recently, an extra-cellular form noted ec-sod, was characterized at the vertebrate ones, then at the invertebrates, and more precisely the nematode caenorhabditis elegans (hjalmarsson et al., 1987; wilson et al., 1994; folz et al., 1997). indeed, this extracellular copper/zinc-sod was detected in the fluids circulating like plasma, the lymph and the synovial liquid (marklund, 1982; fridovich, 1995). however, it would be mainly related to the proteoglycanes of the cellular membrane and only less than 1 % would be present in circulating form (karlsson and marklund, 1987; karlsson et al., 1988; adachi et al., 1995). the mitochondrial matrix contains mn-sod in eukaryotes and bacteria and fe-sod in plants and bacteria. these two isoenzymes are also present in lysosomes, peroxysomes and nuclear compartment. fe-sod is localised in the chloroplasts of plants and constitutes for those the most significant form. these two shapes of sods present analogies of structure; however, it is allowed that the mn-sod is inducible by the superoxide anion radical, whereas it would not be the case of fe-sod. various substances are able to inhibit the sod with for some, specificity with respect to an isoenzyme. thus the cu/zn form is inhibited by cyanide (weisiger and fridovich, 1973) and mn-sod by a treatment to sodium dodecyl sulfate. the h2o2 inactivates fe-sod (hodgson and fridovich, 1975). however, yim et al. (1990) bring back an inhibition at the same time of cu/zn-sod and fe-sod by h2o2. all these enzymes are also inhibited by elimination of their metal of transition, by chelators. however, this inhibition is reversible. lastly, the activity of mn-sod is inhibited with ph 9 whereas that of cu/zn-sod is not influenced by the ph. deficiency in sods or their inhibition increases the sensitivity of the organisms to oxidants. in this general context, it was shown in particular that the mitochondrial mn-sod is essential for the life. indeed, mn-sod deficiency was implicated in the appearance of serious pathologies, the production of superoxide anion becoming very significant. thus, the knockout mice for mn-sod die after the birth or suffer from neuro-degenerative diseases (melov et al., 1998). the form of mn-sod, contrary to that of cu/zn-sod, would be controlled by the superoxide anion and in a general way by the radicals of oxygen (liu et al., 2000). in the bacteria, this induction brings into play a locus soxr which controls the transcription of nine genes implied in the synthesis of enzymes for the production of nadph, the repair of the dna, the protein synthesis and the membrane permeability (harris, 1992). the form of mn-sod is thus inducible by cytokines in various cellular types and by ionizing radiations (masuda et al., 1988). studies showed that the tumoral necrosis factor tnf-á could induce the expression of the manganese-sod (wong et al., 1989, 1995). in the same way, otieno et al. (2000) showed the transcriptional regulation of mn-sod by the chemoprotective 3h-1,2-dithiol-3-thiol. conversely, the rates of mrna of cu/zn-sod do not vary following this treatment. in fact, cu/zn-sod cytosolic appears less significant in the limitation of the oxidative stress. indeed, the transgenic animals not expressing this enzyme present a normal phenotype (ohlemiller et al., 1999). cat (cat; ec. 1.11.1.6) is present primarily at peroxisomial level. this inducible enzyme allows the destruction of h2o2 out of water and oxygen. it is a hemoprotein including an iron atom per unit, the number of units varying according to species. it catalyses a two stages reaction corresponding to a catalasic activity. however, the cat can also present a peroxidasic activity (leguille-cossu, 1996). the cat has other functions during the normal function of the cell. thus, this enzyme catalyses the detoxication of substrates such alcohols and phenols in connection with the reaction of reduction of hydrogen peroxide (akyilmaz and dinckaya, 2003). however, generally, this enzyme is regarded as being able to catalyse only the destruction of hydrogen peroxide (stegeman et al., 1992b). it was shown in the rat, the possibility of a transcriptional induction of cat under treatment by a chemoprotective, 3h-1,2-dithiole-3-thione (otieno et al., 2000). its localization makes it possible this 98 enzyme to carry on an activity complementary to the gpx. in the bacteria, its activity is induced by h2o2 on the level of the locus oxyr (harris, 1992). one second way of destruction of h2o2 utilizes the gpxs (gpx; ec.1.11.1.9). the enzymatic activity is coupled with the oxidation of the gsh and generates alcohols. the gpxs are also able to reduce other peroxides. these enzymes are localised in the cytoplasm and the mitochondrial matrix of the cells. they include two categories: the segpx and a seindependent form, which corresponds in fact to a glutathione s-transferase with a peroxidasic activity. this last form is a dimer of 50 kda localised mainly in the microsomes and catalyses only the reduction of organic peroxides. the segpx is a tetrameric metalloenzyme (80 kda) of which each subunit comprises a selenium atom in the form of a selenocysteine residue and incorporated in the active site thanks to a selenocysteine-specific rna (spallholz and boylan, 1991). a selenium deficiency thus will involve an inhibition of this enzyme. there are other inhibitors of gpx whose metals by a thioloprive action (cadmium and lead) and in a general way the detergents (triton, etc.). the catalytic mechanism proposed for the reduction of peroxides by gpx passes by the oxidation of the active site in selenic acid (seoh). the seoh is transformed by adjunction of a molecule of reduced gsh. the addition of one second molecule of gsh regenerates the active site of the enzyme and the oxidized glutathione (gssg). moreover, ursini et al. (1982) described another form of se-dependent gpx in the liver on pig, the phospholipid hydroperoxide peroxidase (plgpx; ec. 1.11.1.12). this enzyme is a monomer (22 kda) and is implied in the protection of the liposomes and membranes against the oxidative damage. contrary to the preceding enzyme, its selenium requirement is less strict (spallholz and boylan, 1991). the grd (grd; ec. 1.8.1.7) is not always recognized as an antioxidant enzyme. it can nevertheless be included in this category because it makes it possible to reduce the oxidized glutathione (gssg) according to a nadph-dependent process, and it is thus at the base of the regeneration of reduced gsh necessary to the operation of the gpxs and of many other enzymes of the cell. balance between gssg and gsh is capital in the maintenance of cellular homeostasis (winston and di giulio, 1991). in the cell, all these enzymes will intervene in concert, each one according to a specific cellular under-localization, in order to control the quantity of free radicals. other enzymes are regarded as having an antioxidant action. it is the case of dt-d and the glutathione s-transferase. indeed, the glutathione stransferase presents a peroxidasic activity with respect to organic peroxides and belongs to the group of the se-independent gpx. dt-d, also indicated as nad(p)h oxidoreductase 1 (nqo1, ec. 1.6.99.2) catalyses the reduction of quinones by addition of two electrons, thus generating hydroquinones which are more easily excreted after conjugation with sulfates groups or glucuronide (cadenas, 1995). this enzyme thus makes it possible to produce a quinoline stable form without passage by radicals intermediates. in this direction, dt-d can be regarded as an antioxidant enzyme. however, hydroquinones are also able to generate radical species of oxygen, or to react directly with the dna in reactions of alkylation (cadenas, 1995). in this last case, dt-d constitutes an enzyme of bioactivation. more recently, a new family of antioxidant enzymes, the peroxiredoxines, was described in some procaryotic organisms and mammals (chae et al., 1994). these proteins have homologies of sequence with the thioredoxine peroxidase of yeast and they have a peroxidasic function. six groups were defined according to their sequences and their immunological properties (kang et al., 1998; chae et al., 1999; seo et al., 2000). in human, they are expressed in the brain, each of the six groups presenting of the particular localizations (kang et al., 1998; chae et al., 1999; seo et al., 2000). their differential expression was connected to neurodegenerative disorders such as the disease of alzheimer (krapfenbauer et al., 2003). antioxidants of nonenzymatic nature exist too. an antioxidant capacity is conferred on the gsh (l-γglutamyl-l-cysteinyl glycin) by the presence of the thiol group carried by the cysteinic residue. the gsh is a tripeptide of glutamate (l-glu), cysteine (l-cys) and glycin (gly), the glutamate and cysteine being connected by γ-peptide connection. it is synthesized by the consecutive action of two enzymes, γglutamylcysteine synthetase and the gsh synthetase. in the cells, the gsh is present mainly in its reduced form gsh and represents the most significant thiol in eukaryotes cells (0,2 to 10 mm). an increase in the proportion of oxidized form (gssg) translates an oxidative stress. the gsh exerts many functions in the cell. it intervenes in the processes of reduction such as the synthesis and the degradation of proteins, the formation of deoxyribonucleotides, the regulation of the enzymes and the protection of the cells against the free radicals of oxygen. the gsh also plays the role of co-enzyme for various reactions and it is combined with compounds either endogenous (oestrogens, prostaglandins and leucotrienes) or exogenous (drugs and xenobiotics), thus taking part in their metabolism. the gsh thus indirectly supports the detoxication of the radical compounds by its function of co-substrate of the antioxidant enzymes such as the gpxs. moreover, it is the co-substrate of a significant enzyme in the process of detoxication: the gst. the gsh is thus regarded as a central element of antioxidant defences. indeed, a depletion in gsh induces an increase in the sensitivity of the organisms to xenobiotics or overall, with generating processes of radicals (jones et al., 1995; conners, 1998). other compounds known as low-weight molecular have an antioxidant role. thus, the lipoic acid in reduced form can reduce the gsh and the peroxyl radicals. it also has a chelating capacity of metals, quenching of free radicals (kagan et al., 1992) and of regeneration of others antioxidants like the ascorbic acid and the vitamin e (packer et al., 1995). other protective elements are brought by the food: vitamin e, vitamin c and pigments such carotenoids. these antioxidant systems make it possible to stop the chain reactions, in particular those of the lipid peroxidation. indeed, these substances are localised on the level of the membranes and destroy the free radicals by collecting the lone electron. other natural compounds 99 also have an antioxidant character: urea, thiourea, mannitol and dimethyl sulfoxide. in substitution for iron, zinc exerts also an antioxidant action. metals constitute however a particular case because they can at the same time generate radicals and destroy them. antioxidants in molluscs the antioxidant systems known in the mammals are found in the marine organisms. all in all, the antioxidant activities are lower at the aquatic species compared to those of the mammals. in particular, the mn-sod is little expressed in tissues of m. edulis, cu/zn-sod being the main form (livingstone et al., 1992; manduzio et al., 2003). furthermore, the expression of the cu/zn-sod is modulated by xenobiotics. manduzio et al. (2003) described the induction of expression of a cu/zn-sod isoform in mussel m. edulis exposed to contaminants in field and laboratory. however, the bivalve molluscs have levels of activity in digestive gland of the same order as those measured in the liver of fish. at these organisms, the antioxidant activities however are influenced by various factors: (i) an anaerobic respiration gives rise to a reduction in the enzymatic activities and lipid peroxidation, levels returning to normal when oxygenation is restored (viarengo et al., 1989); (ii) the laying involves an increase in the antioxidant activities in march-april, followed by a progressive reduction at spring whereas the availability in food and the temperature increase (solé et al., 1995); (iii) the age sensitizes with the oxidizing effects by reduction of the antioxidant capacities what results in an increase in the rates of lipid peroxidation (viarengo et al., 1991). in the same way, the seasonal fall in antioxidant enzymes is concomitant to an increase in the rate of lipid peroxidation. however, this decrease could be compensating by an augmentation of gst activity in gills of mussels (power and sheehan, 1996; sheehan and power, 1999; manduzio et al., 2004). this observation is all the more pronounced since the water is polluted as described in the harbour of le havre, which is characterized by a general contamination by various compounds such as pahs, pcbs and heavy metals (manduzio et al., 2004). conclusion in spite of many studies about oxidative stress in molluscs, there still exists many questions. this could be explaining sometimes contradictory data. it would be interesting to study thoroughly physiological natural factors which could induce modification between prooxidant and antioxidant systems. in particular, among these factors, the phenomena of hypoxia/anoxia could have an important impact. moreover, the disappearance of environment being able to be considered as free from pollution and so 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usa 87: 5006-5010, 1990. isj 4: x-y, 2007 isj 4: 18-23, 2007 issn 1824-307x research report identification of a putative rnase iii (dicer homolog) gene in silkworm bombyx mori km ponnuvel, b mohana sundari, r saravana kumar, rk sinha, ck kamble biotechnology laboratory, central sericultural germplasm resources centre pb no 44, thally road, hosur 635 109, india accepted february 26, 2007 abstract like other invertebrates, silkworms also encounter a problem from microbial infection including from rna viruses. in insects, rna interference acts as a natural anitiviral response to rna virus infection. especially in drosophila, it is proved that dicer mediated rna interference directs innate immunity against rna viruses. this information prompted us to identify similar rnase iii (dicer) gene in mulberry silkworm bombyx mori. the drosophila dicer gene was blast-searched with b. mori genome and single contig (genbank accession n° aadk01001038) showed maximum homology with dicer gene, through which the rnase iii gene sequence was identified in the genome of silkworm b. mori. the rnase iii domain was present in the three regions with the length of 278 bp, 277 bp and 185 bp in the contig, possibly these three regions form exons. the primers were designed for three b. mori rnase iii regions and amplified through pcr. the region i was amplified only in pure mysore silkworm strain whereas all three regions were amplified in daizo strain. the pcr product sequences were translated and showed rnase iii domain with in the amplified product. the predicted b. mori rnase iii domain had phylogenetic relationship with other insect dicer genes. we presume that this rnase iii (dicer) would protect b. mori larvae from invading rna viruses, which exists in the other insects. key words: bombyx mori; rnase iii; dicer; domain __________________________________________________________________________________________ introduction mulberry silkworm bombyx mori, is highly susceptible to various diseases which adversely affects the silk production. among silkworm diseases, viral diseases are very severe in tropical countries due to unfavorable climatic conditions coupled with improper hygiene in rearing environment (subbarao et al., 1991). b. mori nuclear polyhedrovirus (bmnpv) is a major viral pathogen responsible for grasserie disease followed by other viral pathogens like cytoplasmic polyhedrovirus (cpv) and infectious flacherie virus (ifv) which are known to equally contribute to silkworm crop loss (samson et al., 1990). there are many reports on b. mori immune response against invading microbial pathogens especially against bacterial pathogens (ponnuvel and yamakawa, 2002). ___________________________________________________________________________ corresponding author: km ponnuvel biotechnology laboratory central sericulture germplasm resources centre pb no 44, thally road hosur 635 109, tamil nadu, india email: kmpvel@yahoo.com however, the information on viral disease resistance remains scanty. the red florescence protein, lipases and serine protease are involved in antiviral response especially against bmnpv, a dna virus (ponnuvel et al., 2003). at present, the molecular immune response existing in b. mori against rna viruses has not been reported. among silkworm viral pathogens, cytoplasmic polyhedrovirus (cpv) and infectious flacherie virus (ifv) are rna viruses. in cpv, the genome is a segmented double strand rna where as ifv is a positive single strand rna virus and both viruses do not have dna stage in their replication cycle. the ifv belongs to the picorna virus and rna dependent rna polymerase (rdrp) of the virus helps viral replication that converts genomic single strand rna into a double stranded rna (isawa et al., 1997). these two viruses possess the double stranded rna in their genome or in the replication stage. rnase iii is a double stranded rna-specific endonuclease. in prokaryotes, rnase iii is important in post-transcriptional control of mrna 18 stability and translational efficiency. it is involved in the processing of ribosomal rna precursors. prokaryotic rnase iii also plays a role in the maturation of trna precursors and in the processing of phage and plasmid transcripts (kharrat et al., 1995; nicholson 1999). while, the eukaryotic rnase iii's participate (through direct cleavage) in rrna processing, in processing of small nucleolar rnas (snornas) and snrnas (components of the spliceosome). in eukaryotes, rnase iii enzyme such as dicer is involved in rnai (rna interference) and mirna (micro-rna) gene silencing (hannon, 2002). eukaryotes have evolved many different systems to resist virus infection. identification of specific virus encoded molecules or recognition of nucleic acid structures that are present only in infected cells could induce antiviral responses (lichner et al., 2003). as long double-stranded (ds) rnas do not occur in the cytoplasm of eukaryotic cells, the accumulation of ds replicative intermediates of rna viruses, like cytoplasmic polyhedrovirus (cpv) and infectious flacherie virus (ifv) could activates antiviral responses as rna interference (rnai) or translation inhibition and apoptosis. rnai is an ancient defence mechanism that degrades dsrnas and cognate mrnas in a sequence-specific manner (bernstein et al., 2001, zamore et al., 2000). viral dsrnas are first processed by an rnase iii-like nuclease (dicer) into 21–26 nt dsrnas (sirnas) that guide another nuclease complex (risc) to cleave homologous single-stranded (ss) viral rnas (ding, 2000). sirnas also serve as guides for an rna-dependent rna polymerase to transform the target ssrna into dsrna (fire et al., 1998; vance and vauchert, 2001). rnai was shown to act as an efficient antiviral system in plant and insect cells (adelman et al., 2001; li et al., 2002) and might also play an antiviral role in mammalian cells. cell-autonomous rnai generates an unidentified mobile signal, thereby directing sequence-specific rna degradation in distant tissues (baulcombe, 1999). to inhibit the antiviral effect of rnai, plant (anandalakshmi et al, 1998) and insect (li et al., 2002) viruses express different rnai suppressor proteins. the above findings clearly indicate existence of host virus interaction and sequence specific antiviral defence mechanism against rna viruses in eukaryotic organisms. numerous experiments have already proved rnai activity in silkworm b. mori, whereas the characterization and sequence analysis of dicer gene is yet to be studied. the present paper describes identification and genomic organization of a putative rnase iii (dicer) gene in silkworm b. mori. materials and methods silkworm strains selected two multivoltine silkworms namely, pure mysore and daizo were selected in the present work. these two strains are polyvoltine breeds and showed tolerant response to different silkworm diseases. identification of dicer gene homolog in drosophila the dicer gene was already identified and their domains were well predicted (lee et al., 2004). the dicer gene sequence was blast (tblastn) searched with b. mori genomic dna database for identification homologous sequence of dicer gene. the genomic dna sequence showing homologous sequence to drosophila rnase iii gene was identified and subsequently translated to determine putative amino acid sequence. the amino acid sequence was further analysed through conserved domain search for the presence of the rnase iii domain in dicer protein. selection of primers the up and down gene specific primers were designed for all three rnase iii domains existing in the b. mori genomic contig using the software programme of primer3 (http://frodo.wi.mit.edu/cgibin/ primer3). based on the software programme the primer binding site and the pcr product size were determined. the three different regions located in the genomic contig were amplified by pcr using genomic dna as template. the forward primer in rnaseregion i was (5’-tgtttgaaggttgggacagc–3’) and reverse primer was (5’-cgaaaataggac acgcgaaa–3’). similarly in rnase iii, the region ii forward primer used was (5’-ggagaaagcacggt ttgataa-3’) and reverse primer was (5’-cgcg ttcaaacaaacaaact-3’), while in region iii forward primer was (5’-cgaactgttgatgctaag acca) and reverse primer was (5’-cctcagcgc gtaacagtaca-3’). the pcr products were then cloned into the ta cloning vector with m13 primer sequence in both 5’ and 3’ ends. pcr and analysis of amplified product the genomic dna was isolated from silk moths using standard protocols and used as template dna in pcr reactions (nagaraja and nagaraju 1995). the reaction was done in an mj research thermal cycler, ptc200, using 20 μl reaction mixture containing 50-100 ng of genomic dna as template, 2.0 μl of 10xpcr buffer, 0.2 mm dntps, 1.5 mm mgcl2, 66 ng of forward and reverse primer each and 0.3 u of taq dna polymerase (mbi fermentas). the pcr schedule was 94 °c for 3min followed by 30 cycles of 94 °c for 30 s, 50 °c for 30 s, 72 °c for 2 min and a final extension of 7 min at 72 °c. the pcr products were resolved on 1.5 % agarose gel in tris-acetic acid/edta buffer (1xtae) and electrophoresis was carried out with a constant voltage of 80 v in parallel with molecular weight markers. gel was stained with ethidium bromide (0.5 μg/ml) and photographed with gel documentation. dna sequencing the pcr amplified products were purified through gel-spin column (bangalore genei). in sequencing reaction, the gene specific positive sense primer was used. 19 table 1 rnase iii (dicer) gene biological significance results and discussion in invertebrates, receptors of the innate immune system recognize pathogen-associated molecular patterns in order to activate early defense mechanisms (hoffmann, 2003). in insects, innate immune recognition plays an important role in clearing the majority of invading pathogens through activation of the toll and immune deficiency (imd) pathways. although the b. mori immune system has been well studied in the context of antibacterial response, there is no information at the cellular or molecular level regarding the immune response directed against rna viruses. keene et al. (2004) hypothesized that rnai may also act as an antagonist to alphavirus replication in anopheles gambiae because rna viruses form dsrna during replication and they proved that rnai is a mechanism to protect mosquitoes from viral infection. silencing agago2 expression would make a. gambiae mosquitoes more permissive to virus infection. the findings gave direct evidence that rnai is an antagonist of o’nyong-nyong virus replication in a. gambiae. in india, flacherie disease of the silkworm b. mori is a major factor causing serious loss in cocoon production to sericulture farmers. based on pathological symptoms, the causative agent of this disease is an infectious flacherie virus (ifv), which is an insect picorna virus (isawa et al., 1997). the target of ifv is the goblet cells of the midgut epithelium, and the virus multiplies in the cytoplasm. the complexity of the biological properties of dsrna in vivo became more apparent with the discovery of dsrna mediated post-transcriptional gene silencing (ptgs or rna interference [rnai]). the phenomenon has been described in both plants and animals, including nematodes, insects, and mammals (hannon, 2002). the sequence-specific effects of dsrna that result in endogenous rna degradation are widely conserved and probably present in most invertebrates including b. mori. wang et al (2006) selected drosophila model for studying the innate immunity against rna viruses in animals. they demonstrated that a rna interference pathway protects adult flies from infection by two evolutionarily diverse viruses. their findings describes a molecular framework for the viral immunity, in which viral double stranded rna produced during infection acts as the pathogen trigger, whereas drosophila dicer-2 and argonaute2 act as host sensor and effector respectively (lee et al., 2004). our paper reports that b. mori genome possess rnase iii (dicer) gene sequence and has maximum homology to drosophila dicer. further, the rnase iii domain of b. mori is conserved from plant to animal and it indicates that antiviral immunity in insects shares some of the molecular features of vertebrate antiviral responses. initially the drosophila sequence was compared with genomic dna database of silkworm b. mori. it was found that a single genomic contig (genbank accession n° aadk01001038) possessing the dicer gene homologous sequence (table 1). further, the genomic contig was analyzed for the presence of rnase iii domain using conserved domain search. using the program, distinct functional and structural unit of the translated protein was identified (http://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb/cgi). the protein query sequence submitted to ncbi's protein blast search service was scanned for conserved domain signatures and results showed fig. 1 putative exons and introns of rnase iii gene present in silkworm genome (genomic contig accession no: aadk01001038). the arrow mark indicate forward and reverse primer binding site target genes nature of protein biological activity expressed in contig genbank accession n° dicer rnase iii development, genome organization, viral and transposon defense ubiquitous aadk01001038 20 table 2 details of genomic contig and amplicon size of dicer gene in silkworm bombyx mori target genes primer sequence (5’-3’) primer binding location in genomic contig. amplicon size rnase iii domain region corresponding dna contig accession number forward primer tgtttgaaggttgggacagc 451 bp rnaseregion i reverse primer cgaaaataggacacgcgaaa 859 bp 398 bp 523 bp to 801 bp ef117689 forward primer ggagaaagcacggtttgataa 19214 bp rnaseregion ii reverse primer cgcgttcaaacaaacaaact 19613 bp 400 bp 19335 bp to 19562 bp ef117690 forward primer cgaactgttgatgctaagacca 20797 bp rnaseregion iii reverse primer cctcagcgcgtaacagtaca 21176 bp 358 bp 20936 bp to 21121 bp ef117691 the rnase iii domain was present in the translated protein sequence. it was found that rnase iii domain was present in the contig in the three different regions, which were designated as region i, region ii and region iii. the first region was present from 523 bp to 801 bp whereas other two regions were at 19335 bp to 19562 bp and 20936 bp to 21121 bp (fig. 1, table 2). we believe that all three domains are partial and are scattered in the different exons and after splicing, it may form a dicer enzyme with single domain of rnase iii. in order to confirm the splicing the all three domains were joined together and analyzed for the conserved domain search and it was found that it formed a single protein with rnase iii domain (fig. 2). further, the primers were designed for the putative rnase iii regions based on the sequence available in the all three regions present in the single contig (genbank accession n° aadk01001038). in order to amplify rnase iii domain present in the genomic contig, the pcr was performed with three sets of primers, which were binding site at region i (451f – 859r), region ii (19214f – 19613r) and region iii (20797f – 21176r) (fig. 1). the primers designed in region i yield the pcr product of 398 bp, region ii amplified pcr product size is 400 bp, while region iii pcr product showed molecular weight of 358 bp (table 2). the first region was amplified in pure mysore strain, while the remaining two regions were not amplified due to lack of primer binding sequence in pure mysore. this is due to the fact that genomic dna database was derived from the silkworm strain of daizo and pure mysore strain sequence may not have homologous sequence with daizo. hence, the genomic dna of daizo was used as template and as a result all three regions were amplified in silkworm daizo strain (fig. 3). this finding indicates that the identified rnase iii is located in specified manner in genomic dna, presumably in a single chromosome. uhlirova et al. (2003) used a recombinant sindbis virus as a tool to silence brc expression in the silkmoth bombyx mori. the virus expressing a br-c antisense rna fragment reduced endogenous br-c mrna levels in infected tissues (adult wing and leg primordia) via rna interference (rnai). their findings support that the b. mori also have the rnase iii enzyme which degrade double stranded target rna in a sequence specific manner. the amplified pcr fragment was further sequenced to confirm the presence of rnase iii domains. the sequence was translated and also analyzed for the presence of rnase iii domain in the translated region. it clearly indicates that rnase iii domains exist in the genome of silkworm strains of pure mysore and daizo. the putative amino acid sequence was further analysed for phylogenetic relationship with other eukaryotic organisms. the rna silencing is rna guided gene regulatory mechanism that include post transcriptional gene silencing (pgts) and it is conserved from fission in yeast, plants to animals (ding et al., 2004). phylogenetic analysis was performed using clustalw and mega3 programmes and results showed that the bombyx rnase iii is strongly aligned with rnase iii sequences of insects, especially in tribolium castaneum, a red flour beetle (bucher et al., 2002). robaino et al. (2004) demonstrated that in invertebrate immune system, fig. 2 pcr products amplified in all three regions were joined together, analyzed for the conserved domain in translated amino acid sequence and formed a single protein with rnase iii domain of bombyx mori 21 fig. 3 pcr amplification of rnase iii gene in silkworm bombyx mori like its vertebrate counterparts, could recognize dsrna as a virus-associated molecular pattern, resulting in the activation of an innate antiviral response. interestingly, all the vertebrate rnase iii formed a major group and plant rnase iii gene is more close to bacterial rnase iii. down regulation of dicer makes insects vulnerable to get viral infection (wang et al., 2006). all these findings clearly indicate that dicer enzymes are involved in disease resistant mechanism of insects, especially against rna viruses. silkworms are also known to get infection from rna viruses like cpv and ifv and it is reasonable to expect rnai mediated disease resistant mechanism may also prevail in b. mori. in future studies, the disease resistant mechanism of b. mori against the rna viruses will be deciphered by designing dsrna against b. mori dicer and other proteins involved in risc complex. nucleotide sequence accession number the nucleotide sequences of rnase iii regions have been deposited with ncbi, ddbj and embl libraries under accession n° ef117689, ef117690 and ef117691. acknowledgment this research work was supported by grants from department of biotechnology, ministry of science and technology, government of india (bt/pr4503/pbd/19/099/2003 dated 26th march 2004). references adelman zn, blair cd, carlson jo, beaty bj, olson ke. sindbis virus induced silencing of dengue viruses in mosquitoes. insect mol. biol. 10: 265273, 2001. anandalakshmi r, pruss gj, ge x, marathe r, mallory ac, smith th, et al. a viral suppressor of gene silencing in plants. proc. natl. acad. sci. usa 95: 13079-13084, 1998. baulcombe d. viruses and gene silencing in 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rnai in a marine invertebrate. j. gen. virol. 78: 10442-10448, 2004. uhlirova m, foy bd, beaty bj, olson ke, riddiford lm, jindra m. use of sindbis virus-mediated rna interference to demonstrate a conserved role of broad-complex in insect metamorphosis. proc. natl. acad. sci, usa 100: 15607-15612, 2003. vance v, vaucheret h. rna silencing in plants defense and counter defense. science 292: 2277-2280, 2001. wang xh, aliyari r, li wx, li hw, kim k, carthew r, et al. rna interference directs innate immunity against viruses in adult drosophila. science 312: 452-454, 2006. zamore pd, tuschl t, sharp pa, bartel dp. rnai: double-stranded rna directs the atpdependent cleavage of mrna at 21 to 23 nucleotide intervals. cell 101: 25-33, 2000. 23 abstract materials and methods isj 3: 89-96, 2006 isj 3: 103-110, 2006 issn 1824-307x minireview the multiple functions of the pgrp family in drosophila immunity a goto, s kurata graduate school of pharmaceutical sciences, tohoku university, japan accepted november 13, 2006 abstract the innate immune system discriminates between infectious non-self and self using germ-lineencoded pattern recognition receptors (prrs) that are highly conserved from insects to mammals. peptidoglycan recognition protein (pgrp) is one of the hallmark pattern recognition receptors responsible for detecting unique bacteria-derived peptidoglycans. the pgrp family comprises several members (13 in drosophila, 7 in anopheles, and 4 in mammals) and are differentially expressed on immune-responsive organs. some pgrps have amidase or bactericidal activities and function as immune modulators, whereas others have lost their enzymatic activity, but still have crucial roles in the activation of innate immune signaling. evidence from recent drosophila studies suggests that pgrps have a role in a variety of immune reactions, such as in the activation of the prophenoloxidase cascade, the production of antimicrobial peptides through the activation of the toll and imd pathways, intracellular bacteria recognition, and phagocytosis. key words: innate immunity; pgrp (peptidoglycan recognition protein); prr (pattern recognition receptor); pamp (pathogen-associated molecular pattern); amidase; antimicrobial peptide; phagocytosis __________________________________________________________________________________________ introduction insects are diverse group of organisms and comprise more than 80 % of the animal kingdom. to prevail as such a vast majority of species among other organisms, insects have developed excellent defense mechanisms against bacterial infection. in contrast to adaptive immunity, which requires highly specific receptors created by somatic gene rearrangement, the innate immune system is a defense mechanism that exploits germ-line encoded gene products. the principal mechanisms of innate immunity are difficult to study in vertebrates largely due to the effects of the adaptive immune response. thus, insects have become a favorite model system because, as invertebrates, they depend solely on the innate immune system to fight off infection. the powerful genetic and molecular techniques with the complete genome sequencing make drosophila an attractive model for deciphering the precise mechanisms of the innate immune response (hoffmann, 2003; hultmark, 2003). ___________________________________________________________________________ corresponding author: shoichiro kurata graduate school of pharmaceutical sciences, tohoku university, sendai 980-8578, japan e-mail: kurata@mail.pharm.tohoku.ac.jp although the innate immune system is composed of multiple complex processes including cellular and humoral responses, much of the attention in drosophila has focused on humoral reactions. the primary response is the most immediate response (within minutes) against microorganism invasion and depends on constitutively present endogenous molecules in the hemolymph, such as coagulation factors, cell surface recognition receptors or prophenoloxidase (propo) for wound healing, phagocytosis and melanization which confine microorganisms in small spaces, respectively (theopold et al., 2003; goto et al., 2003; ashida, 2004; kurata et al., 2006). shortly after the primary response, the secondary response begins and ultimately leads to the production of antimicrobial peptides (amps) from the fat body, the functional equivalent of the mammalian liver. to date, there are seven known distinct antimicrobial peptides in drosophila and they are basically small cationic molecules with specific membraneattackable bactericidal activity against various types of microorganisms. the transcriptional regulation of amp expression is under the control of two distinct pathways, the toll and immune deficiency (imd) pathways (kurata, 2004; kurata et al., 2006). the toll pathway was originally identified as a 103 mailto:kurata@mail.pharm.tohoku.ac.jp signaling cascade involved in dorsoventral patterning in embryos (morisato and anderson, 1995; roth, 2003). subsequent genetic analysis serendipitously led to the discovery that it also controls the expression of the antifungal peptide drosomycin, which is predominantly triggered by gram-positive or fungal infection (lemaitre et al., 1996). activation of the toll pathway is initiated through the binding of the proteolytically cleaved cytokine spaetzle to the toll receptor, which then transduces its signal to the cells. in the cell, a toll receptor-adaptor complex composed of dmyd88, tube, pelle, and other unidentified factors induces the phosphorylation and degradation of the ankyrinrepeat inhibitor protein cactus. the nuclear factor nf-κb protein dif, which is normally retained in the cytoplasm by binding to cactus, is then translocated into the nucleus and triggers the expression of the antifungal peptide drosomycin together with numerous effector molecules (hoffman and reichhart, 2002; hoffmann, 2003). this discovery unambiguously led to the identification of the tolllike receptor (tlr) family in mammals (takeda et al., 2003; royet et al., 2005). in contrast, the imd pathway is predominantly activated by gram-negative bacterial infection. while analyzing the expression of antibacterial genes in a phenoloxidase cascade mutant, imd was serendipitously discovered as the first recessive mutation that impairs the inducibility of all antibacterial peptides in the drosophila immune response (lemaitre et al., 1995). six years later, the imd gene product was identified. it is a 25-kda protein containing a death domain with significant sequence similarity to that of the mammalian tumor necrosis factor-receptor interacting protein rip (georgel et al., 2001). activation of the imd pathway triggers the kinase cascade of dtak (a map 3 kinase homologue) and the ikk complex, and ultimately leads to phosphorylation of the rel protein relish (leulier et al., 2002; naitza et al., 2002; vidal et al., 2001). phosphorylated relish is then cleaved by dredd (a mammalian homolog of caspase-8), and its dna binding domain leaving the i-κb domain in the cytoplasm is translocated into the nucleus and triggers amp expression (leulier et al., 2000; stoven et al., 2003). thus, over the past decade, our understanding of drosophila innate immunity has dramatically centered on the field of intracellular signaling mechanisms. there remain crucial questions, however, about how the innate immune system recognizes microorganisms, and discriminates between self and non-self infections, and how the signals from outside the cells subsequently reach the cognate innate receptors to activate the toll or imd pathway. the discovery of pattern recognition receptors (prr) such as peptidoglycan recognition proteins (pgrps) that recognize unique bacterial cell component peptidoglycans (pgns) led to our deeper understanding of the innate immune recognition concept (medzhitov and janeway, 2002; royet, 2004). here, we review recent advances in the understanding of the multifunctional pgrp family, a representative pattern recognition molecule, in drosophila. discovery of the pgrp family the name ”peptidoglycan recognition protein” was first introduced by ashida’s group (yoshida et al., 1996). they purified a 19-kda protein from the hemolymph and cuticles of silkworm (bombyx mori) and demonstrated that it binds to gram-positive bacteria and pgn, and activates the prophenoloxidase cascade (yoshida et al., 1996). later, its corresponding cdna was cloned (ochiai et al., 1999). pgrp gene homologs from a moth (trichoplusia ni) and subsequently from mouse and human were in turn cloned, indicating that the pgrp family is highly conserved from insects to mammals (kang et al., 1998). completion of various genome projects (e.g., drosophila melanogaster, anopheles gambiae, human, mouse, etc.) also boosted the discovery of diverse pgrp family. the drosophila genome encodes at least 13 pgrp family members, which are subdivided into seven short transcripts (pgrp-s; ~200 amino acids long) and six long transcripts (pgrp-l; from 200 to 600 amino acids long). the short transcripts are predicted to be secreted molecules due to the presence of signal peptides and are induced in response to bacterial infection, whereas the long transcripts are constitutively present on the cell surface or integrated into the plasma membrane, except for pgrp-lb (werner et al., 2000). extracellular recognition by the pgrp family pgrp roles in the immune response a significant breakthrough with regard to the role of pgrp in the immune response came through a large-scale ethylmethane sulfonate mutagenesis screen from drosophila. michel et al isolated a mutation called semmelweis (seml), named after the hungarian physician ignaz phillipp semmelweis who is a pioneer of antiseptic treatments (celine, 1999; michel et al., 2001). the pgrp-saseml mutants lead to defects in amp expression and render flies more susceptible to several gram-positive bacterial infections. the mutated region was found to be on a highly conserved cysteine residue that is changed to a tyrosine in pgrp-sa. hemolymph transfer experiments revealed that pgrp-sa is secreted into the hemolymph. surprisingly, activation of the toll pathway by fungi and of the imd pathway by gram-negative bacteria is normal compared to wildtype, raising the possibility that there are other prrs specific for fungi and gram-negative bacteria (michel et al., 2001). consistent with this hypothesis, three independent groups identified pgrp-lc mutations (ird7 or totem) (choe et al., 2002; gottar et al., 2002; ramet et al., 2002). pgrp-lc mutant flies are highly susceptible to some gram-negative bacterial infections and do not express imd-mediated amp genes (such as diptericin and attacin). epistatic analysis indicates that pgrp-lc encoding a single transmembrane protein acts upstream of imd, suggesting that pgrp-lc functions in the activation of the imd pathway in response to gram-negative bacterial infection. pgrp-sa binds to lysine (lys)-type pgn 104 from gram-positive bacteria, whereas pgrp-lc binds to meso-diaminopimelic (dap)-type pgn from gram-negative bacteria, reinforcing the structural basis for the recognition mechanisms (leulier et al., 2003; kaneko et al., 2004). cooperative roles of pgrps for detecting pathogens curiously, the pgrp-lc mutant phenotypes are not as severe as that of ikk� or relish null mutants in terms of both amp expression and survival rates upon some gram-negative bacterial infections (gottar et al., 2002). accordingly, some gram-positive bacteria such as staphylococcus saprophyticus, staphylococcus aureus, or enterococcus faecalis induce normal activation of the toll pathway in a pgrp-sa mutant background, but this activation is completely blocked in dif mutants (bischoff et al., 2004). those findings led to the observation that the one to one prr/ pathogen associated molecular pattern (pamp) concept is inappropriate for innate immune sensing mechanisms. this interpretation was soon validated by the discovery of multi-functional pgrp-le, which contains a unique acidic domain, functions in the activation of the imd pathway and also triggers the propo cascade when it is ectopically expressed (takehana et al., 2002). more importantly, pgrplc/pgrp-le double mutants are more susceptible against gram-negative bacteria (e. coli) infection than either single mutant alone, suggesting that these two proteins cooperate to recognize gramnegative bacteria and lead to the activation of the imd pathway (takehana et al., 2004). similarly, in the case of gram-positive bacteria sensing, other cooperative receptor complexes are required upstream of toll. gram-negative bacteria binding protein (gnbp), which binds gram-negative bacteria, was first identified from silkworm (lee et al., 1996). gobert et al (2003) subsequently reported that gnbp1 mutants are not resistant against some gram-positive bacterial infections, showing a similar immune defective phenotype as the pgrp-sa mutant. the finding that gnbp1 physically interacts with pgrp-sa in the circulation to activate the toll pathway supports this observation (gobert et al., 2003; pili-floury et al., 2004; wang et al., 2006). moreover, the generation of another pgrp family member mutant, the pgrpsd loss-of-function mutant, has been reported (bischoff et al., 2004). pgrp-sd mutants are highly susceptible to some types of gram-positive bacterial infection, and this mutation exacerbates the pgrpsa and gnbp1 mutant phenotypes, suggesting that pgrp-sd is another gram-positive bacteria recognition molecule involved in the activation of the toll pathway. taken together, there are four known pgrp family members, pgrp-sa, -sd, -lc, and –le, that are crucial for microbe recognition and for activating the innate immune signaling pathways (toll, imd , and propo cascades) (fig. 1). fig. 1 extracellular recognition by pgrp family. in hemolymph, meso-diaminopimelic (dap)-type peptidoglycans (pgns) from gram-negative and some gram-positive bacteria (bacillus species) are recognized by pgrp-lc or pgrp-le and activate the imd pathway to trigger diptericin or attacin expression. in the case of lysine (lys)-type gram-positive bacterial infection, either pgrp-sc1a, -sd, -sa, gnbp1 alone or in combination is responsible for the activation of the toll pathway through binding of the endogenous ligand spaetzle (spz). 105 intracellular recognition by pgrp-le large-scale gain-of-function genetic screening using amp reporters led to the identification of pgrp-le. pgrp-le overexpression leads to constitutive activation of the imd pathway as well as the propo cascade, which ultimately leads to the formation of large melanotic tumors in the hemolymph (takehana et al., 2002). pgrp-le lossof-function mutants were isolated in subsequent studies 2 years after the identification of pgrp-le (takehana et al., 2004). the phenotypes of a single pgrp-le gene mutation are fairly weak in terms of their survival rates against e. coli infection and amp expression. however, when combined with the pgrp-lc mutation, the pgrp-le/pgrp-lc double mutants exhibit high susceptibility to e. coli and to other dap-type pgn-containing bacteria (takehana et al., 2004). this finding clearly suggests that pgrp-le synergistically or redundantly acts with pgrp-lc to activate the imd pathway. to gain a better understanding of the cooperative recognition mechanisms between pgrp-lc and pgrp-le, two independent groups recently conducted very exciting experiments using monomeric dap-type pgn tracheal cytotoxin (tct) (kaneko et al., 2006; lim et al., 2006). tct is a disaccharide-tetrapeptide fragment of pgn that contains a 1,6-anhydro-arranged muramic acid and a dap residue at the third position of the stem peptides (cookson et al., 1989), and is continuously released from gram-negative bacteria as a turnover product of the pgn in the cell wall (park, 2001). the presence of such a small diffusible monomeric pgn is an ideal signature for the innate immune recognition system by prrs, as the pgn layer of gram-negative bacteria is basically hidden under the outer lipopolysaccharide (lps) layers. in vitro cell culture studies demonstrated that tct potently activates the imd pathway via two different pgrplc isoforms: pgrp-lca and pgrp-lcx (kaneko et al., 2004). deletion mutant studies demonstrated that the rhim-like motif of pgrp-lc is responsible for the signaling, but is dispensable for the interaction with imd (kaneko et al., 2006; choe et al., 2005). whereas a single pgrp-lc or pgrp-le mutant responds normally to tct, e. coli pgn, or live e. coli, the response of pgrp-lc/pgrp-le double mutants is completely abolished. clonal expression studies demonstrated that pgrp-le acts in a cell-autonomous manner on some immune responsive tissues, such as malpighian tubes. direct delivery of tct into the cytosol by calcium phosphate transfection triggers an enhanced amp expression that is dependent on pgrp-le, suggesting that pgrp-le is a second receptor for tct (kaneko et al., 2006). this idea is supported by analysis of the crystal structure of pgrp-lepg (pgrp domain of pgrp-le)/tct complex, which indicates that tct strongly binds to pgrp-lepg with an apparent kd of 27 nm, and induces pgrp-le multimerization through head-to-tail dimer formation (lim et al., 2006). collectively, these results indicate that pgrple potentially has dual functions depending on its localization; on the outside of the cells, it cooperates with pgrp-lc through its pgrp domain and triggers the propo cascade, and inside the cells, it acts as an intracellular recognition receptor for activation of the imd pathway (fig. 2). fig. 2 recognition mechanisms of pgrp-le. monomeric dap-type pgns (tct) binds to the pgrp domain of pgrp-le. in the hemolymph, this complex triggers the propo cascade. tct can also bind to pgrp-lcx to form three potential types of receptor complexes (lepg/tct/lc, lcx/tct/lca or lcx/pgn/lcx) on the cell surface. the rhim-like motif of pgrp-lcx and lca is responsible for the activation of the imd pathway. the pgrp-le/tct complex also functions in intracellular recognition. immune modulator activities of the pgrp family a representative feature of the pgrp family is that all pgrps in insects and mammals have in common an approximately 160 amino acid-long pgrp domain with similarity to the bacteriophaget7 lysozyme, a zinc-dependent n-acetyl-muramyl-lalanine amidase (steiner, 2004; royet et al., 2005). among the 13 pgrp family members in drosophila, the first group is categorized as non-catalytic pgrps, which lack the zinc binding residues required for amidase activity, but retain the binding capacities to pgn (pgrp-sa, -sd, -la, -lc, ld, le, -lf). the second group is the catalytic pgrps, which have zinc-dependent amidase activity that either reduces or eliminates the biological activities of the pgns (pgrp-sc1a, -sc1b, -lb, -sb1, sc2). the first striking discovery of the potential role of catalytic pgrps in the drosophila immune response was reported for pgrp-sc1b (mellroth et al., 2003). in vitro studies performed by mellroth et al. (2003) demonstrated that recombinant sc1b hydrolyzes the lactylamide bond between the glycan strand and the stem peptides of pgn, and the degraded pgn has less immune stimulatory activity compared with undigested pgn, indicating that 106 pgrp-sc1b has scavenger activity. unexpectedly, chang et al. (2004), in the course of crystal structure analysis, demonstrated that recombinant pgrp-sa has l,d-carboxypeptidase activity that cleaves the dap-type muropeptide, but not the lystype compound. moreover, three independent groups reported the roles of catalytic pgrps: pgrp-sc1/2 (bischoff et al., 2006), pgrp-lb (zaidman-rémy et al., 2006), and pgrp-sb1 (mellroth and steiner, 2006). first, bischoff et al. (2006) used rna interference techniques to generate pgrp-sc1/2 loss of function mutants and reported that the mutants overactivate the imd pathway after bacterial infection, and feeding-induced infection by erwinia carotovora carotovora is enhanced compared to controls. thus, pgrp-sc1/sc2 might modulate the activation of the imd pathway in the gut, which is constantly threatened by bacterial infection (bischoff et al., 2006). the effects of phenotypes of recently isolated pgrp-sc1a mutants (picky) on the toll pathway are confusing with respect to the complex immune modulator mechanisms (garver et al., 2006). second, biochemical analysis indicates that pgrp-lb has specific amidase activity for daptype pgn, including tct. similar to the rnainterference generated pgrp-sc1/2 mutants, the analysis of the time course of amp expression induced by gram-negative bacteria or tct infection revealed a strong immune response by the rnainterference generated pgrp-lb mutants compared to wild-type controls (zaidman-rémy et al., 2006). therefore, they concluded that pgrp-lb negatively regulates the imd pathway and functions as a scavenger receptor for gram-negative bacterial pgn. finally, recent biochemical analysis demonstrated that pgrp-sb1 is an nacetylmuramoyl l-alanine amidase that preferentially hydrolyzes dap-type pgn. in contrast to pgrp-lb, pgrp-sb1 lacks enzymatic activity against tct. in addition, this report first demonstrated that pgrp-sb1 possesses bactericidal activity against b. megaterium (mellroth and steiner, 2006). taken together, these findings suggest that non-catalytic pgrps preferentially function in the detection of pgn, and catalytic pgrps serve as scavenger receptors to detoxify harmful pgn and modulate the signaling pathways, although catalytic pgrps that specifically cleave lys-type pgn have yet to be identified. roles of pgrps in phagocytosis phagocytosis is a phenomenon whereby invading microbes or altered self-like apoptotic or infected cells are engulfed and, eventually, digested by host cells called phagocytes (aderem and underhill, 1999). in drosophila, most humoral immune responses leading to toll and imd pathway activation are mainly mediated by the fat body, whereas cellular responses such as phagocytosis, encapsulation, or melanization are mediated by blood cells called hemocytes (hoffmann and reichhart, 2002). there are three known hemocyte types in drosophila. plasmatocytes account for approximately 90 % of hemocytes and are the main executors of phagocytosis. crystal cells, approximately 5% of the hemocyte population, contain a set of substrates and enzymes responsible for propo-mediated humoral melanization. lamellocytes, the third hemocyte type, show a large flattened morphology. under non-infected conditions, lamellocytes are absent, but once large invaders, such as wasps, are infected, lamellocytes are differentiated from the lymph glands, the main hemocyte-producing organs in larvae (meister and lagueux, 2003). in addition to several phagocytic receptors, such as croquemort (“catcher of death” ) (franc et al., 1996, 1999; stuart et al., 2005), scavenger receptor (sr) -ci (ramet et al., 2001; ulvila et al., 2006), peste (philips et al., 2005), eater (kocks et al., 2005), and dscam (watson et al., 2005), pgrp family members are also involved in phagocytosis. pgrp-lc was the first pgrp family member discovered to be involved in phagocytosis. a double-stranded (ds)rna interference-based screen of the drosophila macrophage cell line s2 identified 34 genes, including pgrp-lc, that are essential for phagocytosis. fluorescence activated cell sorting analysis indicated that s2 cells with pgrp-lc knock-down had substantially decreased phagocytic activity towards gram-negative bacteria e. coli, but not gram-positive bacteria s. aureus. they also demonstrated that long transcripts of the same types of pgrp family, pgrp-la or pgrp– ld, are not involved in phagocytosis (ramet et al., 2002). conversely, another group recently reported that pgrp-sc1a mutation (picky) impairs phagocytic ability s. aureus, but not e. coli or s. cerevisiae (yeast), based on the results of a forward genetic ethylmethane sulfonate screen. moreover, activation of the toll pathway is significantly impaired in pgrp-sc1a mutants. transgenic rescue experiments indicated that the amidase activity of pgrp-sc1a does contribute to toll pathway activation, but is essential for the uptake of s. aureus (garver et al., 2006). conclusion over the past 5 years, the concept of the prr/pamp theory originally introduced by charles janeway has been verified experimentally using versatile molecular and genetic strategies (janeway and medzhitov, 2002). in mammals, functional characterization of toll-like receptor family has considerably deepened our understanding of the recognition mechanisms of innate immunity (takeda and akira, 2005). in contrast, it seems that insect tolls are not involved in immune functions as pattern recognition receptors, but are more likely to have other functions. drosophila toll is a kind of cytokine receptor that is activated by the endogenous proteolytically cleaved ligand spaetzle (imler and hoffmann, 2002). now, the pgrp family members are coming to the forefront as molecular sensors against microbial infection in insects. recent functional characterization of the pgrp family members in drosophila has enabled us broaden the partial picture of pattern recognition mechanisms: 1) specific types of pgn (lys-type or dap-type) can be detected by different pgrps; 2) 107 catalytic amidase pgrps act as immune modulators; 3) microbe detection by pgrps triggers the activation of innate immune signaling pathways; and 4) pgrps possess receptor functions for phagocytosis. still, a fundamental question remains as to how the signals are transmitted into the cells to kill microbes after pgns are recognized by pgrps. further studies will address this issue to complete the picture of the prr/ 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ballarin department of biology, university of padova, padova, italy accepted december 20, 2006 abstract we studied colony specificity in the colonial ascidian botrylloides leachi which, as in other botryllid ascidians, leads to either fusion or non-fusion between contacting colonies. fusion requires the prior disappearance of contacting tunic cuticles and contact between facing ampullar epithelia. the epithelial cells of the ampullar tip show “pad regions” rich in ribosomes, which contribute to the synthesis of new tunic and cuticle. blood cells, mainly phagocytes and pigment cells, increase their concentrations inside the ampullar lumen and phagocytes can cross the ampullar epithelium and enter the tunic, where they can contribute to the digestion of tunic cuticles and cells of the ampullar epithelium in order to establish a common circulation. non-fusion reaction, as studied in the colony allorecognition assay, resembles the subcuticular rejection described in japanese botrylloides, characterised by limited tunic fusion, hemocyte leakage, and necrotic spots. conversely, in the cut surface assay, a more intense cytotoxic reaction is observed along the contact border. in this case, morula cells crowd massively inside the facing ampullae, enter the tunic, and release their vacuolar contents which are probably required for the formation of necrotic spots. key words: botrylloides; ascidians; colony specificity; allorecognition _________________________________________________________________________________________________________________ introduction the term allorecognition defines the capability of intraspecific non-self recognition; in clonal organisms, it is known as colony specificity and has been described in many species of compound ascidians, in which it leads to either fusion of facing, genetically compatible colonies into a larger chimerical colony, or non-fusion, when contacting colonies are genetically incompatible (taneda et al., 1985). botryllid ascidians are a group of compound tunicates, characterised by palleal budding, with colonies formed of many zooids embedded in a common tunic containing a vascular network, which connects and synchronises all the zooids. a marginal vessel runs along the border of the colony, from which many sausage-like blind endings, known as ampullae, sprout. ampullae sited at the growing edge allow the adhesion of the colony to the substratum ___________________________________________________________________________ corresponding author: loriano ballarin department of biology, university of padova via ugo bassi 58/b, 35100 padova, italy e-mail: loriano.ballarin@unipd.it and have a columnar epithelium on their tips, provided with pad cells (katow and watanabe, 1980; rinkevich et al., 1998). the outcome and intensity of the non-fusion reaction in botryllid ascidians depends on the tissues involved in the recognition. according to rinkevich (1992) and saito et al. (1994), allorecognition can occur: i) after the disappearance of most of the contacting cuticular layers, and consequent fusion of the tunics. in this case, the ampullae penetrate the opposite tunic and face the basal side of alien ampullae (tip-to-side interaction); massive crowding of hemocytes inside the ampullar tips then occurs, followed by leakage of cells into the tunic, contributing to the formation of a clear necrotic area. the ampullar tips then shrink, are amputated, and new tunic cuticles are formed by the two colonies to isolate the necrotic region along the contact border. this is typical of botryllus primigenus (taneda and watanabe, 1982); ii) after partial fusion of the tunic, restricted to regions in front of the facing (tip-to-tip) ampullae, which then can (woods hole botryllus schlosseri) or 125 fig. 1 a) living colony of botrylloides leachi from the dorsal side. zooids and buds are embedded in the common tunic, crossed by vessels with terminal ampullae. b) interacting ampullae of fusible colonies (a, b). arrows indicate the contact region. c-d) contacting ampullar tips of fusible colonies (a, b). the ampullar tips can contact either the tip or the apical side of the facing counterparts. bar = 150 µm. e) megaloampullae (m) in one of the contacting colony. bars = a, 1 mm; b-e, 150 µm. 126 fig. 2 interacting ampullae of contacting fusible colonies (a, b). a, c) contacting tips, semithin sections. bar = 50 and 15 µm, respectively. b, d) contact between tip and ampullar apical side. bars = 50 and 15 µm, respectively. e) contact between fusible colonies, ultrathin section. the two cuticles show numerous papillae (inset) and are separated by a narrow space; cells of the ampullar epithelium are cylindrical, with large nuclei (n) and welldeveloped rough endoplasmic reticulum (rer). bar = 2 µm (inset: 0.6 µm). f, highly folded plasma membrane in baso-lateral region of epithelial cells of the ampullar tip. ph, phagocytes; pc, pigment cells. bar = 2 µm. 127 cannot (monterey and mediterranean b. schlosseri) enter the facing tunic. cells crowd inside the ampullar tips, leak into the tunic, and contribute to the formation of a series of necrotic spots along the contact border. the ampullae then shrink and withdraw; in the woods hole population, their tips are amputated near the colonial boundary (boyd et al., 1990; sabbadin et al., 1992); iii) at the outer part of the tunic, just underneath the cuticle (subcuticular), which dissolves in very narrow regions in front of the facing ampullae. a few hemocytes leak from the ampullar lumen into the tunic, and contribute to the formation of small necrotic regions, which are not recognisable under the binocular microscope, along the contact border. this is reported in botrylloides simodensis, botrylloides fuscus and botrylloides violaceus (hirose et al., 1988, 1997); iv) after the fusion of the facing ampullae and an initial blood exchange. this characterises the non-fusion reaction of botryllus scalaris (saito and watanabe, 1982; shirae et al., 1999). non-fusion reaction has been widely investigated in b. schlosseri, in which morula cells (mc), representing the majority of the circulating hemocytes, massively crowd inside the tips of facing ampullae before crossing the ampullar epithelium and entering the tunic, where they degenerate and contribute to the formation of the necrotic masses observed along the contact border (ballarin et al., 1995, 1998). these cells degranulate and release the enzyme phenoloxidase, which is responsible for the cytotoxicity observed, due to the induction of oxidative stress (ballarin et al., 2002). botrylloides leachi is a compound ascidian, common in the mediterranean, and it can easily grow and reproduce in laboratory conditions. unlike from the sympatric species b. schlosseri, in which zooids are grouped in star-shaped systems and colonies are characterised by high chromatic polymorphism, b. leachi is easily distinguishable, thanks to the organisation of zooids in laddershaped systems and their orange to brown colonies (fig. 1a) . in spite of its abundance, colony specificity has been poorly investigated in this species (rinkevich et al., 1994; rinkevich, 1995) to fill this gap, we carried out a detailed investigation, at morphological and cellular level, of fusion and non-fusion in b. leachi, using both colony allorecognition assay (caa) and cut surface assay (csa) as defined by rinkevich (1992). results are discussed in the context of colony specificity in botryllid ascidians, and cellular events compared with those known in b. schlosseri. materials and methods animals b. leachi colonies, collected in the lagoon of venice, were allowed to adhere to glass slides (5 x 5 cm) and reared in aerated aquaria, filled with filtered seawater (fsw), at a temperature of 19 °c. they were fed with liquifry marine (liquifry co., dorking, england) and water was changed every other day. colony specificity assays in colony allorecognition assay (caa), colonies were juxtaposed on a supporting glass slide at a distance of 1-2 mm, their growing edges facing each other, in a moist chamber for 30 min, before being returned to the aquarium. within 24-48 h, the colonies extended towards each other and their facing ampullae contacted. cut surface assay (csa) was carried out to better investigate the non-fusion reaction: in this case, colonies were cut with razor blades and pieces of the same size from different colonies were brought into contact at their cut surface on a supporting glass slide and allowed to adhere for 30 min in a moist chamber. they were then returned to the aquarium. in each case, colonies were observed under a binocular microscope for up to 48 h, until either fusion or nonfusion occurred. light microscopy contacting colonies, at various stages of both fusion and non-fusion, were fixed for 2 h in 4 % paraformaldehyde plus 1 % glutaraldehyde in saline buffer (0.2 m na-cacodylate buffer, ph 7.4, plus 1.7 % nacl and 1 % sucrose), rinsed in saline buffer, dehydrated and embedded in paraplast x-tra (oxford labware). sections (7 µm thick) were obtained with a leitz 1212 microtome and stained with either haematoxylin/eosin or 2 % eosin g (shirae et al., 2002). electron microscopy fusing and non-fusing colonies were fixed for 2 h in 1.5 % glutaraldehyde in saline buffer, post-fixed for 1 h in oso4 in saline buffer, dehydrated and embedded in epon. thick sections (1 µm) were obtained with a lkb 2128 ultratome, stained with a hot solution of 1 % toluidine blue and 1 % borax in distilled water, and observed under the light microscope. thin (60 nm) sections were collected on copper grids, stained with uranyl acetate and lead citrate, and observed under a hitachi h 600 transmission electron microscope. results fusion interacting colonies extend the ampullae of their growing edges towards those of the alien colony to a close contact, in which ampullar tips are separated only by a thin layer of tunic (figs 1b-d). the ampullar tips can contact either the tips (figs 1c, d, 2a, c) or the apical sides (figs 1c, d, 2b, d)) of the alien counterpart. in both cases, facing tunic cuticles of early contacting colonies appear separated by a narrow space (figs 1c, d, 2c-e); they present numerous protruding papillae, 90 nm in height (fig. 2e (inset)). the tunic matrix appears rich in fibres, and tunic cells can be observed in the contacting region (figs 2c, e). the cells of the epithelium of the ampullar tips have a central nucleus, many mitochondria and a well-developed rough endoplasmic reticulum (rer), organised in a series of overlapping cisternae (fig. 2e); the basolateral plasma membranes are tightly folded (fig. 2e). the epithelium appears cylindrical in shape, 128 fig. 3 interacting ampullae of contacting fusible colonies (a, b), ultrathin sections. a) tight contact between cuticles. the epithelium appears fenestrated (asterisks) and cells show tight junctions in their apical region. b) magnification of a tight junction of the ampullar epithelium. c) pad regions (arrows) in epithelial cells of the ampullar tips. rer, rough endoplasmic reticulum. bars = 2 µm in a, c, d; 0.2 µm in b. 129 fig. 4 interacting fusible colonies. a-c) electron micrographs of the contact region between fusible colonies showing phagocytes (ph) in the lumen of facing ampulla (a), a phagocyte crossing the ampullar epithelium (b) and the complete fusion of contacting tunics of contacting colonies a and b in the framed region (c). bars = 3 µm for a and b; 1 µm for c. d-f) light micrographs of the interacting region between fusible colonies showing close contact between the tips of two facing ampullae (d) and formation of a new vessel allowing blood exchange between the two colonies a and b (e, f; arrows). bar = 250 µm. 130 fig. 5 a) light micrograph of the contact region between non-fusible colonies in caa. arrows indicate the region of close adhesion between the cuticles. bar = 0.5 mm. b) electron micrograph of the tip epithelium of interacting ampullae, cylindrical, rich in rer, and fenestrated (asterisks). scale bar: 1.5 µm. c-d) semithin sections of interacting ampullae showing crossing hemocytes (c; arrow) and fenestrated epithelium (d). bar = 20 µm. e) degenerated cells in the common tunic in front of interacting ampullae. electron-dense granules leaked from morula cells are indicated by arrowheads. bar = 2 µm. 131 lying on a thick basal lamina and provided with wide tight junctions in the apex region of the cells (figs 2e, 3a, b). once contact is established, the ampullae can grow to form megaloampullae (fig. 1e), as defined by rinkevich et al. (1994). no “wavy” epithelium, as described by rinkevich et al. (1994) in botrylloides from the mediterranean coast of israel, was observed in interacting ampullae. within 24-48 h from the first touch, a tight contact between the cuticles is observed (figs 3a, c). the epithelium of the ampullar tips is always continuous, although it now appears fenestrated (figs 3a, c); cells form irregular expansions at their apex (pad regions, according to katow and watanabe (1980)), containing ribosomes, finely granular material, and some small vesicles (fig. 3d). an increase in the concentration of various hemocytes is observed inside the facing ampullar tips (figs 2a-d), mainly represented by phagocytes and pigment cells (figs 2c, d, 4a). blood cells, particularly phagocytes, can be seen crossing the epithelial cells towards the tunic (fig. 4b). within 24-48 h of contact, the cuticles disappear, and tunic fusion is attained along the whole contact border (fig. 4c). tunic fusion is followed by fusion of the ampullar tips, so that blood can now flow from one colony to the other (figs 4d-f). non-fusion, caa at the beginning of the reaction, the tunic cuticles are well developed and separate the two contacting colonies (fig. 5a). the morphology of the ampullar tip epithelium is comparable to that observed in fusing colonies. megaloampullae may form (fig. 6a). in later stages, the contact between tunic become tighter, and the cuticles disappear in a narrow region in front of the facing ampullae where fusion of the tunic occurs; crowding of hemocytes is observed inside the facing ampullae. at about 24 h from contact, hemocytes begin to cross the ampullar tip epithelium (fig. 5c), which appears fenestrated (figs 5b, d) and, on entering the tunic, they degenerate and contribute to the formation of small, dark cytotoxic spots (fig. 6b). some of these cells are easily recognisable (e.g., phagocytes and mc; figs 5c, d); others appear degenerated, and granules with electron-dense contents are visible in the tunic (fig. 5e). leakage of cells is followed by the withdrawal of ampullae. non-fusion, csa in early stages of csa (fig. 6c), tunics are still separated, and several ampullae are observable in the region pushing towards the contact border. their lumen are crowded with hemocytes, particularly mc (figs 6d; 7a), which are easily recognisable due to their morphology and eosinophily, and are characterised by the presence of many small vacuoles, 2 µm in size, which give them a typical mulberry shape. the epithelium of the ampullar tips is flattened (figs 6d; 7a-c), with large nuclei and a well-developed rer (fig. 7b). within 24 h, tunic fusion occurs in limited regions and, over the next 24 h, many blood cells, mainly mc, leak from the ampullae (figs 7b, c) and crowd in the tunic of the contact region (fig. 6e), together with some granular cells (fig. 6f), as defined by cima et al. (2001). in this phase, mc alter their morphology and show empty vacuoles of larger size (fig. 6g). filamentous eosinophilic material is visible around the mc (fig. 6h). in advanced stages of the nonfusion reaction, mc aggregate along the contact border and contribute to the formation of a series of clearly visible, pigmented necrotic spots (fig. 6i). discussion as compound organisms, botryllid ascidians share the ability for intraspecific recognition, observable when colonies come into contact, leading to either fusion or non-fusion of genetically compatible or incompatible colonies, respectively (saito et al., 1994). the genetic bases of allorecognition have been deeply studied in b. schlosseri and b. primigenus, in which colonies can fuse when they share at least one allele at a highly polymorphic fu/hc locus; the absence of common alleles results in non-fusion (oka and watanabe, 1957, 1960; sabbadin, 1962; oka, 1970;). other botryllid species seem to follow the same kind of genetic control (saito et al., 1994). we studied fusion and non-fusion reactions between contacting colonies of the compound ascidian b. leachi. both tip-to-tip and tip-to-side interactions occur between the ampullae of the growing edges, although they never enter the opposite tunic in advanced stages of the reactions. a typical feature of this species is the frequent enlargement of contacting ampullae to form megaloampullae, already described in the case of non-fusion (rinkevich et al., 1994), and now described also for fusion. in the case of fusion, vascular anastomosis is preceded by the disappearance of the contacting cuticles and fusion of the thin layer of tunic covering the apex of growing-edge ampullae, which begins in front of the facing ampullae and extends rapidly to the whole contact border. blood cells, especially phagocytes, crowd inside the ampullar lumen and, after crossing the epithelium, enter the tunic. they can contribute to the digestion of tunic cuticles and ampullar tips, in order to allow the establishment of a common circulation. the epithelium of the ampullar tips changes its morphology, as reported by katow and watanabe (1980) in b. primigenus: numerous fenestrae appear between cells which remain closely adherent through well-developed tight junctions, and their apexes form pad regions which may contribute to the synthesis of new tunic and cuticle (katow and watanabe, 1980). the non-fusion reaction has been widely investigated in b. schlosseri: in this species incompatible colonies lead to limited fusion of the tunic in front of the ampullae facing tip-to-tip. soluble factors diffusing from the alien tunic and activated hemocytes attract blood cells, mainly mc, which crowd inside the apex of the facing ampullae, before crossing the epithelium of the ampullar tips and entering the tunic. during this process, mc degranulate, release their vacuolar contents, and contribute to the formation of a series of necrotic spots scattered along the contact border (sabbadin et al., 1992; ballarin et al., 1995, cima et al., 2006). according to our results, the non-fusion reaction in 132 fig. 6 a-b) caa. megaloampullae (m) in contact region between non-fusible colonies (a) and dark, cytotoxic spots in the contact area (b). bars = 250 and 150 µm, respectively. c-i) csa between two colonies (a, b) showing several ampullae in the contact region (c). the lumen of the ampullae in the contact area is filled with hemocytes, mainly morula cells (d), which leak in the tunic and crowd in the contact area (e), together with some granular cell (f, arrowhead), where morula cells degranulate, changing their morphology and showing large, empty vacuoles (g). eosinophilic material (arrowheads) is visible around morula cells (h). in advanced stages of the non-fusion reaction, diffuse necrotic regions are visible along the contact area (i). bars = 1 mm for c and i, 25 µm for d, f, g; 50 µm for e. 133 fig. 7 interacting ampullae in csa. a) semithin section of ampullae with flattened epithelium and blood cells, many morula cells (arrowheads) are visible inside their lumen. bar = 50 µm. b-c) electron micrographs of a morula cell (mc) interacting with the epithelium of the ampullar tip (b) and crossing it (c). bar = 1 µm b. leachi, as observed in caa, is characterised by limited tunic fusion and blood cell leakage from facing ampullae which do not penetrate the opposite tunic. giant ampullae sometimes, appear, but the outcome of the reaction seems to be similar in both the presence and absence of megaloampullae: hemocytes crowd inside the facing ampullae, and later move into the tunic through the fenestrated epithelium of the ampullar tips. the ampullae then, withdraw, and the colonies change their preferential direction of growth. in any case, the extent of the leakage of hemocytes and the size of the necrotic spots are very small when compared with the case in b. schlosseri (sabbadin, 1982; scofield and nagashima, 1983; sabbadin et al., 1992), and the reaction resembles the subcuticular rejection described in japanese species of the genus botrylloides (hirose et al., 1988, 1997), in which a few blood cells leak through the ampullar epithelium in front of the ampullar tips, and cytotoxic foci are poorly visible. in order to study better the role of blood cells in the non-fusion reaction, we used csa, which gives a more intense cytotoxic reaction in those species characterised by subcuticular rejection in caa (hirose et al., 1990, 1997, 1998). with this assay, we were able to demonstrate that mc are involved in the non-fusion reaction in b. leachi. similarly to what occurs in b. schlosseri (ballarin et al., 1995, 1998; rinkevich et al., 1998; cima et al., 2006), mc selectively accumulate inside 134 facing ampullae. this suggests chemotactic activity by soluble factors from the alien colony, which may be reinforced by the release of chemotactic chemokines by activated hemocytes. similar behaviour by activated mc has recently been demonstrated in b. schlosseri (cima et al., 2006). once inside the ampullae, mc leak into the tunic, together with some granular cells, and change their morphology: the latter event is probably related to the release of vacuolar contents which, as in b. schlosseri (ballarin et al., 1995, 1998), may be responsible for the induction of cytotoxicity. the presence of filamentous material, sharing staining affinity with the contents of morula cell vacuoles in the tunic surrounding morula cell aggregates, fits the above hypothesis. the intense dark pigmentation along the contact region suggests that phenoloxidase is involved in the induction of cytotoxicity in csa, as reported for b. schlosseri and other ascidian species (ballarin et al., 1995, 1998; shirae and saito, 2000; shirae et al., 2002). future investigations will be directed towards both better comprehension of the role of phenoloxidase in the non-fusion reaction of b. leachi and the search for the immunomodulatory 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biotechnology, maria curie-skłodowska university, akademicka 19 st., 20-033 lublin, poland 2institute of agrophysics, polish academy of sciences, doświadczalna 4 st., 20-290 lublin, poland 3department of biotechnology, human nutrition and science of food commodities, university of life sciences in lublin, skromna 8 st., 20-704 lublin, poland accepted december 16, 2016 abstract the black soldier fly hermetia illucens is an ecological decomposer used for biodegradation of organic waste. its larvae can develop on a wide range of decaying plant and animal matter, including manure and food scraps, i.e., habitats that are extremely rich in various microorganisms. living in such conditions requires very well-functioning immune mechanisms. however, the immune response processes have not been examined so far in h. illucens larvae. in order to shed light on the immune system in the black soldier fly, in the present study we have examined h. illucens hemocytes and analyzed the effects of immune challenge of h. illucens larvae on the activity of the key components of insect humoral immune response, i.e., phenoloxidase, lysozyme, and antimicrobial peptides. key words: hermetia illucens; innate immunity; hemocytes; antimicrobial peptides; lysozyme; phenoloxidase introduction the black soldier fly hermetia illucens is an ecological decomposer used for biodegradation of organic waste (čičkova et al., 2015). its larvae develop through six larval instars on a wide range of decaying plant and animal matter, including manure, food scrapes, municipal garbage, and rotting plant material (sheppard et al., 1994, 2002; diener et al., 2011). living in an environment that is extremely rich in various microorganisms, including many pathogenic ones, requires a very well-functioning immune system. insect immunity relies on cellular and humoral innate mechanisms, which have been well characterized in e.g., drosophila melanogaster, a dipteran model organism (lemaitre and hoffmann, 2007; buchon et al., 2014; kleino and silverman, 2014; lindsay and wasserman, 2014; cytryńska et al., 2016). the hemolymph cells, hemocytes, are involved in processes of the cellular immune response, i.e., phagocytosis, nodulation, and encapsulation (lavine and strand, 2002; dubovskiy et al., 2016). in addition to prohemocytes, three types of hemocytes, i.e., plasmatocytes, crystal cells, ___________________________________________________________________________ corresponding author: agnieszka zdybicka-barabas department of immunobiology faculty of biology and biotechnology maria curie-skłodowska university akademicka 19 st. 20-033 lublin, poland e-mail: barabas@poczta.umcs.lublin.pl and lamellocytes were characterized in d. melanogaster (ribeiro and brehélin, 2006), whereas those in anopheles gambiae and aedes aegypti were classified as granulocytes and oenocytoids (strand, 2008; hillyer and strand, 2014). an important role in humoral immune response against pathogens is played by phenoloxidase (po) as well as antimicrobial peptides and proteins. po activity is a result of fast activation of the po system by pathogen-associated molecular patterns (pamps), including components of microbial cell walls, i.e., bacterial lipopolysaccharide (lps), peptidoglycan (pgn), and fungal β-1,3-glucan (cerenius et al., 2008; bidla et al., 2009; lu et al., 2014). po activity leads to formation of quinones and other cytotoxic intermediates of melanin, and finally to melanin deposition at the site of injury and around invading pathogens. melanin as well as cytotoxic intermediate products exhibit strong antimicrobial activity and prevent spreading of the pathogens in an insect organism (suguraman, 2002; lee and miura, 2014). in addition to activation of the po system, recognition of pathogens results in induction of synthesis of defense peptides able to kill the invaders. the inducible antimicrobial peptides are mainly synthesized in the insect fat body and released into hemolymph, where they are essential components of systemic immune response. seven families of defense peptides with different biochemical and antimicrobial properties have been 10 described in d. melanogaster, i.e., attacins, cecropins, insect defensin, diptericins, drosocin, drosomycins, and metchnikowin (uvel and engström, 2007). in other insect species, a number of various defense peptides have been characterized to date, including anionic antimicrobial peptides (cytryńska et al., 2007a; scocchi et al., 2011; mylonakis et al., 2016). beside the defense peptides, hemolymph lysozymes play an important role in antimicrobial immune response in insects largely due to enzymatic muramidase activity (hultmark, 1996). lysozymes are usually constitutive components of insect hemolymph; however, they can act synergistically with defense peptides, and pathogen recognition may also result in a dramatic increase in their level and activity in hemolymph, which contributes considerably to effective antimicrobial defense (yu et al., 2002; chapelle et al., 2009; zhang et al., 2009; zdybicka-barabas et al., 2012, 2013; sowa-jasiłek et al., 2014; beckert et al., 2015). the habitat of h. illucens larvae, which is extremely rich in various microorganisms, implies that the immune system of this insect species functions very efficiently and effectively. recently, park et al. (2015) have reported on purification and characterization of a h. illucens defensin-like peptide with activity against gram-positive bacteria. however, although h. illucens larvae are used in composting piles, the immune response processes in these insects have not been examined so far and there are no data currently available in this area. in order to shed light on the immune system in the black soldier fly, in the present study we have examined h. illucens hemocytes and analyzed the effects of immune challenge of h. illucens larvae on activity of the key components of insect humoral immune response, i.e., phenoloxidase, lysozyme, and antimicrobial peptides. materials and methods insect culture conditions the larvae of hermetia illucens (diptera: striatomyidae) were reared in laboratory conditions at 29 oc and substrate humidity of 50 80 % in the institute of agrophysics of the polish academy of sciences in lublin, poland. the larvae were fed with feed consisting of protein 25 %, fat and oil 5 %, crude fiber 5.8 %, ash 5.7 %, lysine 1.25 %, calcium 1 %, phosphorus 0.97 %, methionine 0.4 %, and sodium 0.05 %. last instar larvae were used in the experiments. microorganisms gram-negative bacterium escherichia coli d31 and gram-positive bacterium micrococcus luteus atcc 10240 were grown in lb (lysogeny broth) at 37 c and 28 c, respectively, until the logarithmic growth phase. filamentous fungus aspergillus niger was grown on solid pda medium (5 % potato extract, 0.5 % dextrose, 1.7 % agar) at 28 oc until conidial spores were obtained and then stored at 4 oc. a conidial suspension for the antifungal activity assay (see below) was prepared as described in our previous paper (mak et al., 2010). insect immune challenge, hemolymph collection, and preparation of hemolymph methanolic extracts an immune challenge with live gram-negative bacterium e. coli or gram-positive bacterium m. luteus was performed with a piercing method, essentially as described previously for galleria mellonella larvae (mak et al., 2010). before immunization, the larvae were washed with sterile water and the puncture sites were disinfected with 70 % ethanol. the larvae were punctured with a sterile needle (control) or with a needle dipped in a pellet of live bacteria (40 larvae per group). next, the larvae were stored in petri dishes provided with food or in sterile conditions, and the hemolymph was collected 6 h, 24 h and 48 h after the treatment as well as from the unchallenged (naive) larvae. the hemolymph (5 µl per larva) was combined to obtain pooled samples. hemocyte-free hemolymph was obtained by centrifugation at 200xg for 5 min and subsequently at 20,000g for 15 min at 4 c (mak et al., 2010). an acidic/methanolic extraction method was used for partial purification of antimicrobial peptides. the hemolymph was diluted ten times with the extraction solution (methanol/acetic acid, glacial/water; 90:1:9), mixed thoroughly, and centrifuged (20,000×g, 30 min 4 oc) in order to pellet precipitated proteins. the supernatant containing mainly proteins of mr below 30kda and peptides was collected and vacuum dried, and the pellet was stored at -20 oc until needed (cytryńska et al., 2007a; mak et al., 2010). antimicrobial activity assays well diffusion assay the hemolymph antibacterial activity against e. coli d31 and m. luteus was detected by a growth inhibition zone assay on lb agar plates, essentially as reported (mak et al., 2010). to improve the sensitivity of the method against gram-negative bacteria, egg white lysozyme (ewl) at the final concentration of 2.5 mg/ml was added (cytryńska et al., 2001). the hemolymph antifungal activity was detected using pda agar plates containing a. niger conidia, as described previously (mak et al., 2010). lysozyme activity in the hemolymph was estimated using agarose plates containing freeze-dried m. luteus as reported (jarosz, 1995). the activity of lysozyme was calculated from a standard curve made with egg white lysozyme (ewl, ec 3.2.1.17; sigma-aldrich). each well on the petri dish was filled with 4l of five times diluted hemolymph and the plates were incubated at 37 oc (e. coli) or 28 oc (m. luteus, a. niger). the diameters of bacterial and fungal growth inhibition zones were measured after 24-h and 48-h incubation, respectively. the level of anti-e. coli activity was calculated using the algorithm described previously (hultmark et al., 1982) with cecropin b of hyalophora cecropia (sigma-aldrich) as a standard. bioautography (sds gel overlay method) detection of antibacterial activity in the hemolymph after sds/page and subsequent renaturation of polypeptides was performed as described previously (cytryńska et al., 2001). 11 briefly, after separation of proteins (300 µg of total protein per sample), the gels were washed in 2.5 % triton x-100 (bio rad) for removal of sds. next, the polypeptides were renatured in 50mm tris-hcl ph 7.5 and subsequently in lb. finally, the gels were overlaid with nutrient agar containing e. coli d31 cells and 2.5 mg/ml ewl, and the zones of bacterial growth inhibition were observed after incubation for 6 12 h at 37 oc. phenoloxidase activity assay the phenoloxidase activity in the hemolymph was determined on the basis of melanin formation according to the method described previously (park et al., 2005; zdybicka-barabas et al., 2014). briefly, 2 μl of non-diluted hemolymph was added to 18 μl of tbs (50 mm tris-hcl ph 7.4, 150 mm nacl) containing 5mm cacl2 in the wells of a 96-well plate and the mixture was incubated for 20 min at room temperature. next, 180 μl of 2 mm dopamine in 50 mm sodium phosphate ph 6.5 was added and absorbance of the mixture was measured at 490 nm over 90 min at a 5-min interval using a microtiter plate reader (biorad). the experiment was performed in triplicate on three independent occasions. other methods polyacrylamide gel electrophoresis of proteins was performed by 13.8 % glycine sds/page according to laemmli (1970). separation of proteins below 30 kda and peptides was carried out by tricine sds/page (16.5 % t, 3 % c) (schägger and von jagow, 1987). the proteins and peptides were stained using coomassie blilliant blue r-250 (0.25 %) or g-250 (0.025 %), respectively, after glycine sds/page and tricine sds/page. the protein concentration was estimated by the bradford method using bovine serum albumin (bsa) as a standard (bradford, 1976). for microscopic observations of hemocytes, the samples of freshly collected hemolymph (5µl) were placed onto microscopic slides, covered with coverslips, and the hemocytes were observed immediately using a contrast-phase microscope olympus bh-2 (lens magnification 40×). results and discussion h. illucens hemocytes the microscopic examination of the hemolymph revealed the presence of at least three types of morphologically different hemocytes, i.e. crystal celllike, plasmatocyte-like, and granule-containing hemocytes (fig. 1a). the oval shaped crystal celllike hemocytes (approx. dimensions 18 µm × 15 µm) were non-adherent cells and contained evident crystal-like inclusions (approx. 12 µm in length and 2.5 µm in width), similar to those reported in d. melanogaster crystal cells (ribeiro and brehélin, 2006). based on these characteristics, these cells may be involved in the melanization process. the two other types were adherent cells that formed filopodia-like projections, a feature that implicates their engagement in cellular immune response processes. the plasmatocyte-like cells were morphologically similar to some lepidopteran plasmatocytes (cytryńska et al., 2007b; hori et al., 2013; wu et al., 2016) and exhibited a tendency to aggregate (fig. 1b). the granule-containing hemocytes resembled morphologically culex pipiens quinquefasciatus granulocytes. interestingly, in c. pipiens quinquefasciatus, a dipteran species, three hemocyte types, i.e., oenocytoids, plasmatocytes, and granulocytes, were identified beside prohemocytes (wang et al., 2011). although the h. illucens granule-like cells resembled c. pipiens quinquefasciatus granulocytes, the plasmatocyte-like cells were morphologically distinct from those in c. pipiens quinquefasciatus. in addition, hemocytes with undefined features were observed (fig. 1b). notably, nodule-like structures with a diameter approx. 25 35 µm were observed in the hemolymph of the bacteria-challenged h. illucens larvae (fig. 1c), suggesting that nodulation may be one of the cellular immune response processes involved in fast elimination of invaders in h. illucens larvae. phenoloxidase activity in h. illucens hemolymph the immune challenge of h. illucens larvae with the gram-negative and gram-positive bacteria led to a considerable increase in hemolymph po activity. in comparison with the level of po activity in the hemolymph of naive larvae (control), the enzyme activity increased 1.27-fold and 1.6-fold after the challenge with e. coli and m. luteus, respectively (fig. 2a). interestingly, the po activity after sterile puncturing was by approx. 22 % lower than in the control hemolymph. if one takes this into consideration, the po activity level after the challenge with e. coli and m. luteus was 1.6-fold and 2-fold higher, respectively (fig. 2a). in addition to the changes in the hemolymph po activity, effects of local po activation that led to melanin deposition at the site of injury were observed on the surface of larval body (fig. 2b). the results clearly indicate an important role of po activity in h. illucens immune response against invading gramnegative and gram-positive bacteria. antimicrobial activity in h. illucens hemolymph in addition to the evident changes in the po activity level, the immune challenge of the h. illucens larvae induced antimicrobial activity in the hemolymph. the lysozyme and anti-gram-positive bacterium m. luteus activity, both detected in the hemolymph of the naive larvae, increased considerably after the challenge and reached the highest level in the e. coli-challenged larvae (tables 1, 2). in contrast, antibacterial activity measured against gram-negative bacterium e. coli d31 was detected only in the hemolymph of the challenged larvae. it was induced by the bacterial challenge and by the sterile puncturing of the larvae. the level of anti-e. coli activity in the hemolymph of the sterile punctured as well as m. luteusand e. colichallenged larvae corresponded to the activity of 0.34 µm, 0.55 µm, and 1.2 µm of a cecropin b solution, respectively (table 2). no antifungal activity against a. niger was detected in h. illucens hemolymph in our experimental conditions. on the other hand, the lysozyme activity in the hemolymph of naive larvae suggests constitutive synthesis of 12 fig. 1 hermetia illucens hemocytes. the hemocytes (a, b) and cell aggregates or nodules (c) were observed in a contrast-phase microscope olympus bh-2. the white arrows and arrowheads indicate, respectively, crystal-like intrusions and filopodia-like projections. bar = 10 µm. 13 fig. 2 phenoloxidase activity in h. illucens larvae. (a) po activity in the hemolymph. the larvae were immunized with sterile injury or bacteria-challenged, and the phenoloxidase activity was determined in the hemolymph collected 24 h after the treatment. the results are presented as ±sd from three independent experiments. the inset demonstrates melanin formed in the wells after 90 min incubation with dopa as a substrate. c naive larvae; in sterile-injured larvae; ml and ec m. luteusand e. coli-challenged larvae, respectively. (b) melanization localized at a site of injury is indicated by the red arrowheads. this antibacterial factor in h. illucens larvae and its role in elimination of gram-positive bacteria (e.g., m. luteus). it should be noted that a considerable increase in the lysozyme activity and induction of anti-e. coli activity was detected in the hemolymph of the larvae stored in the non-sterile conditions (i.e., provided with food) after the sterile puncturing (table 2). such conditions reflect a possibility of invasion of pathogens present in food through mechanical injury in the natural habitat of h. illucens larvae. the results also indicate great adaptation of the h. illucens immune system to fight against pathogens. interestingly, when the immune-challenged larvae were incubated in the sterile conditions, the challenge with e. coli induced the lysozyme, anti-m. luteus, and anti-e. coli activity, whereas the challenge with sterile puncturing and m. luteus caused only an increase in the lysozyme and antim. luteus activity. in the hemolymph of the sterile punctured and m. luteus-challenged larvae, no antie. coli activity was detected (table 1). this suggests discrimination between gram-positive and gram-negative bacteria by the h. illucens immune system, similarly to d. melanogaster, in which such a phenomenon is well documented (goto 14 table 1 antimicrobial activity in the hemolymph of h. illucens larvae kept in sterile conditions antimicrobial activity time after challenge (h) experimental group control larvae sterileinjured larvae m. luteuschallenged larvae e. colichallenged larvae lysozyme activity [µg/ml] 6 24 48 2.24±0.2 2.63±0.27 3.98±0.95 2.82±0.18 3.98±0.7 6.03±1.1 3.08±0.18 12.59±0.8 4.47±0.34 4.08±0.27 20.42±0.3 8.51±0.4 anti-m. luteus activity (mm) 6 24 48 12.6±0.3 10.4±0.25 12.9±0.2 16.4±0.1 17.0±0.2 16.4±0.3 16.0±0.15 18.6±0.25 17.2±0.1 16.3±0.3 20.4±0.15 18.0±0.5 anti-e. coli activity [µm] 6 24 48 nd nd nd nd nd nd nd nd nd 1.3±0.26 1.66±0.42 nd h. illucens larvae were sterile-injured or immunized with e. coli or m. luteus and kept in sterile conditions for 48 h. the hemolymph was collected 6 h, 24 h, and 48 h after the challenge. next, the lysozyme, anti-m. luteus, and anti-e. coli activity was determined by a well diffusion assay. the results are presented as ±sd from three independent experiments. nd = not detected and kurata, 2006; silverman et al., 2009; reumer et al., 2010). the hemolymph antibacterial activity estimated against e. coli with the diffusion well assay was determined by the presence of inducible antimicrobial peptides with molecular mass corresponding to cecropin b, as revealed by bioautography (fig. 3a) and tricine sds/page of the hemolymph methanolic extracts (fig. 3b). electrophoretic analysis of hemolymph proteins indicated that, despite induction of defense peptides, the bacterial challenge of h. illucens larvae led to appearance of an additional protein with molecular mass approx. 43 kda. interestingly, the e. coli challenge resulted in appearance of two other proteins with molecular masses approx. 58 kda and 27 kda (fig. 3c). determination of the identity of these proteins and their role in h. illucens immune response requires further study. in conclusion, it is important to note that the immune challenge of h. illucens larvae with grampositive m. luteus induced only anti-gram-positive bacterial activity, possibly resulting from increased lysozyme activity. in contrast, in the hemolymph of larvae immunized with gram-negative e. coli, besides anti-gram-positive bacterial activity also anti-gram-negative bacterial activity was detected. this is consistent with the data reported recently (park et al., 2015). these authors demonstrated that a defensin-like peptide purified from the hemolymph of h. illucens larvae challenged with gram-positive staphylococcus aureus was active against gram-positive bacteria and not against gram-negative ones (park et al., 2015). our results suggest that, depending on the bacteria used for the immune challenge (containing diaminopimelic-type pgn or lysine-type pgn in the cell wall), synthesis of different sets of antimicrobial peptides is induced in h. illucens, as in the case of d. melanogaster (silverman et al., 2009; lindsey and wasserman, 2014; kleino and silverman, 2014) and g. mellonella (mak et al., 2010) reported previously. table 2 antimicrobial activity in the hemolymph of h. illucens larvae kept in non-sterile conditions experimental group antimicrobial activity lysozyme activity [µg/ml] anti-e. coli activity [µm] control larvae 7.2±3.6 nd sterile-injured larvae 11.47±0.73 0.34±0.06 m. luteus-challenged larvae 26.12±5.84 0.55±0.09 e. coli-challenged larvae 35.48±10.83 1.2±0.48 h. illucens larvae were sterile-injured or immunized with e. coli or m. luteus and kept in non-sterile conditions for 24 h. next, the hemolymph was collected, and the lysozyme and anti-e. coli activity was determined by a well diffusion assay. the results are presented as ±sd from three independent experiments. nd = not detected. 15 fig. 3 antimicrobial activity (a) and changes in peptide (b) and protein (c) patterns in the hemolymph of immunized h. illucens larvae. the hemolymph was collected from naive larvae (control c) and from sterileinjured (in), m. luteus (ml)-, or e. coli (ec)-challenged larvae 24 h after the treatment. (a) antimicrobial activity detected by bioautography after separation of the hemolymph polypeptides (300 µg of total protein) in 13.8 % polyacrylamide gel and subsequent renaturation. cecropin b (1µg) was used as a control peptide. a gel fragment presenting clear zones (darker areas) of e. coli growth inhibition is shown. (b) electrophoretic analysis of h. illucens hemolymph peptides. the peptides extracted from 5 µl of the hemolymph with the acidic/methanolic extraction method were diluted in 20 µl of sample buffer and separated by tricine sds/page. additional peptide bands appearing in the larval hemolymph after the bacterial challenge are encircled by a red frame. 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mendyk e, cytryńska m. synergistic action of galleria mellonella apolipophorin iii and lysozyme against gram-negative bacteria. biochim. biophys. acta 1828: 1449-1456, 2013. zhang y, huang j, zhou b, zhang c, liu w, miao x, et al. up-regulation of lysozyme gene expression during metamorphosis and immune challenge of the cotton bollworm, helicoverpa armigera. arch. insect biochem. physiol. 70: 18-29, 2009. isj 5: 41-42, 2008 isj 5: 41-42, 2008 issn 1824-307x visions and perspectives developed to cull: how a master control gene of development turned into a regulator of innate immune homeostasis v zappavigna department of animal biology, university of modena and reggio emilia, modena, italy accepted march 13, 2008 abstract a striking novel role for the caudal "master control" gene of development in the regulation of innate immune functions in insects has emerged. a recent study now adds further insight into the function of this homeobox gene in the maintenance of the immune homeostasis that is required to preserve the normal commensal community within the drosophila gut. these results point to a possible more widespread co-option of developmental regulatory genes during evolution to add tissue and/or organ-specific regulatory plasticity to innate immune systems. key words: innate immunity; homeostasis; insects; drosophila melanogaster; homeobox genes; caudal in metazoa innate immunity represents the first barrier of defense against infections caused by various types of microorganisms. insects, for instance, rely primarily on innate immunity to fight infectious microbes. an important branch of the immune defence system in these organisms is based on the production and secretion of antimicrobial peptides (amps). the inducible production of amps is one of the best-studied mechanisms of immune defence in insects. in drosophila melanogaster the synthesis of amps can be triggered by the activation of three main signalling pathways: the toll, immune deficiency (imd), and jnk pathways. both the toll and imd pathways control the transcription of amp genes via the activation of two drosophila homologs of the nfkb transcription factor, dorsal and relish. amps, however, are synthesized not only in response to infection, but are also produced constitutively in healthy individuals in a tissue-, sex-, and genespecific manner (reviewed in uvell and engstrom, 2007). understanding the molecular mechanisms underlying the constitutive and inducible expressions of amps in various tissues represents thus an intriguing novel field of study. one of the innovative concepts that have already begun to emerge in this field is that the regulation of amp expression may rely on the same gene regulatory ___________________________________________________________________________ corresponding author: vincenzo zappavigna department of animal biology university of modena and reggio emilia via campi 213/d, 41100 modena, italy e-mail: vincenzo.zappavigna@unimore.it networks that control cell fate during developmental processes. a beautiful example of this was published in a recent issue of science by ryu et al.(2008). in their work ryu and co-wokers analyzed at the molecular level the immune interactions between commensal microbiota and the drosophila gut. they found that, despite the chronical imdmediated high-level activation of relish by gut commensal microorganisms, only a subset of its target genes was expressed, remarkably excluding amps. the reason for this selective exclusion of amp gene expression was found in the specific repressive action of the caudal (cad) homeodomain transcription factor, as ryu et al. (2008) elegantly demonstrate. caudal had been previously shown to act as a master control gene in several crucial developmental processes in drosophila, including the definition of the anteroposterior axis and gut development (mlodzik and gehring, 1987; moreno and morata, 1999; lengval and iwaki, 2002). in addition, cad has been recently found to be crucial for constitutive amp expression in salivary glands and ejaculatory duct epithelia, implicating for the first time this developmental regulatory gene in a constitutive innate immune strategy (ryu et al., 2004). ryu et al. (2008) now show that the knockdown of cad expression in gut cells via transgenic rnai restores the production of amps in the gut, indicating that cad acts as a gut-specific transcriptional repressor of commensal-induced nfkb-dependent amp expression. strikingly, cad knockdown in intestinal cells was also accompanied by a significant rise in gut epithelial cell apoptosis. as it turned out, gut cell death was induced as a secondary effect to amp hyperactivation due to 41 drastic changes in the gut commensal community structure. in particular two bacterial strains were shown to vary considerably in their abundance upon cad knock-down. a911 bacteria, which represents the dominant strain in the gut microbial community, were significantly reduced in number in cad knockdown flies, whereas the g707 strain, which is a normally a minor member of the commensal community in the drosophila gut, emerged as a dominant commensal. g707 bacteria revealed to be responsible for the observed induction of apoptosis in gut cells and the consequent rise in mortality of host flies. interestingly, g707 bacteria fed to animals with a normal gut commensal community did not induce apoptosis, and germ-free animals first colonised with a911 bacteria prevented the growth of g707 bacteria, indicating that the normal gut microbial community is sufficient to suppress the growth of pathogenic bacteria. a911 bacteria were furthermore found to be sensitive to amps, whereas g707 bacteria were much less so, thus explaining their rise in number in cad knock-down ampexpressing drosophila guts. overall, the results by ryu et al. (2008) show that the maintenance of the immune homeostasis required for the preservation of the normal commensal community of the drosophila gut ultimately rests on the gut-specific repressive action of the cad homeodomain transcription factor on amp genes. drosophila intestinal epithelia have thus evolved a remarkable immune strategy, which entails the recruitment of a developmental regulatory gene whose repressive action allows the selective survival of non-pathogenic commensal bacteria capable of maintaining homeostasis via colonization resistance. on a broader perspective, it is tempting to speculate that the co-option of developmental regulatory genes expressed in a tissueand/or organ-specific manner may offer the unique evolutionary advantage of adding regulatory plasticity to the innate immune system. indeed, the transcriptional control of amps by tissue-specific transcription factors would meet the needs for an infection-independent costitutive expression (or repression, as in the case of the drosophila gut) that is tailored to obtain a spatially-differentiated immune defense. it is therefore not unlikely that cad will represent the first example of a whole series of developmental "master control" genes that will reveal in the near future to play fundamental roles in the tissueand/or organ-specific regulation of the innate immune system function. references lengyel ja, iwaki dd. it takes guts: the drosophila hindgut as a model system for organogenesis. dev. biol. 243: 1-19, 2002. mlodzik m, gehring wj. expression of the caudal gene in the germ line of drosophila: formation of an rna and protein gradient during early embryogenesis. cell 48: 465-478, 1987. moreno e, morata g. caudal is the hox gene that specifies the most posterior drosophile segment. nature 400: 873-877, 1999. ryu jh, kim sh, lee hy, bai jy, nam yd, bae jw, et al. innate immune homeostasis by the homeobox gene caudal and commensal-gut mutualism in drosophila. science 319: 777782, 2008. ryu jh, nam kb, oh ct, nam hj, kim sh, yoon jh, et al. the homeobox gene caudal regulates constitutive local expression of antimicrobial peptide genes in drosophila epithelia. mol. cell. biol. 24: 172-185, 2004. uvell h, engstrom y. a multilayered defense against infection: combinatorial control of insect immune genes. trends genet. 23: 342-349, 2007. 42 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice isj101.pdf 69 isj 2: 69-74, 2005 issn 1824-307x minireview are matrix metalloproteinases the missing link? f mannello1, g tonti1, s papa2 1istituto di istologia e analisi di laboratorio, università studi “carlo bo”, urbino, italy 2istituto di scienze morfologiche, università studi “carlo bo”, urbino, italy accepted may 25, 2005 abstract it is more and more evident that the matrix metalloproteinase (mmp) system is not a characteristic feature of vertebrate animals only, as it can also be found in many invertebrate organisms. this endopeptidase family has been widely studied since its first member was described 40 years ago during metamorphosis in tadpole tails. many researches have been carried out in mammals in order to elucidate and analyze the several and important roles these endopeptidases play, both in physiological pathways and in pathological processes. the evolving researches of these multifaceted enzymes enter the very interesting and fascinating world of the invertebrates, where these enzymes seem to be in the front line during important biological events. mmp-like enzymes and their inhibitors have been found in insects, crustaceans, mussels, sea urchins and also in organisms as simple as hydra. in these species mmps partake in several fundamental processes, such as extracellular matrix (ecm) remodelling, embryonic development, cell growth and differentiation and also in defense mechanisms thus highlightening their intriguing and unexpected functional importance in invertebrate life too. key words: development; embryogenesis; extracellular matrix; invertebrates; metalloproteinases; tissue inhibitors of metalloproteinases introduction the discovery of the first member of the matrix metalloproteinase (mmp) family set the way to a new line of research dealing with this novel class of enzymes. an extraordinary number of new discoveries are contributing to put together the many pieces of this intruiging mmp puzzle world, adding more and more informations on how these enzymes work. the first enzyme with the capacity of degrading interstitial collagenase was found in the tail of a tadpole (gross and lapiere, 1962) and from then onwards the search continued and brought to the identification of more than 25 vertebrate mmps (brinckerhoff and matrisian, 2002; mott and werb, 2004; mannello et al., 2005a). corresponding author: ferdinando mannello istituto di istologia ed analisi di laboratorio, via e. zeppi, snc, università studi “carlo bo”, 61029 urbino (pu), italy e-mail: f.mannello@uniurb.it at the beginning there were the “vertebrate mmps”... mmps are endopeptidases characterized by their zinc-dependece and by a highly conserved sequence that contains three histidines necessary for binding the zinc ion at the catalytic site, and a conserved methionine turn that lies beneath the active site zinc. (stöcker et al., 1995). mmps are classified according to their substrate specificity and are subdivided into collagenases, gelatinases, elastases, stromelysins and membrane-type mmps; moreover, they are also classified depending on their domain structure. the nterminal portion of all mmps (pre-domain) is a signal sequence due to be removed whose function is to guide the mmp synthesis to the endoplasmic reticulum and their following secretion in the extracellular environment. mmps are secreted in a zymogenic form and the latency of these enzymes is maintained by a mechanism known as the “cysteine switch”; the unpaired cysteine in the pro-domain of latent mmps forms a bridge with the catalytic zinc, thus maintaining the enzyme inactive as zymogen until this interaction is abolished by mechanical disruption or cleavage by 70 other proteinases yielding a fully active enzyme (vu and werb, 2000). the third fundamental domain of mmps is the catalytic domain where the zinc ion is coordinated with three histidine residues and its fourth ligand is represented by a water molecule (overall, 2004). most mmps are characterized by a hemopexin/vitronectin-like sequence that is linked to the catalytic domain by a hinge region that can vary in length (baragi et al., 1994). two mmps, known as gelatinases, are peculiar as they show the insertion of three fibronectin-like repeats within their catalytic domain which are necessery for binding and degrading specific substrates (shipley, 1996). mmps are not only secreted enzymes, as there are membrane-type mmps which have a single-pass transmembrane domain and a short cytoplasmic cterminal tail, or a short c-terminal hydrophobic region (itoh et al., 1999). mmp activity is specifically inhibited by endogenous tissue inhibitors of metalloproteinases (timp) which reversibly bind previously activated mmps in a 1:1 stoichiometric ratio and differ in their expression patterns and mmp affinity (gomez et al., 1997; mannello and gazzanelli, 2001). also nonspecific inhibitors (e.g., á2-macroglobulin) can control mmp activity (mannello et al., 2005b). the level of expression of mmps by unstimulated cells and in intact tissues is generally low. the mmp expression is inducible by cytokines, growth factors, physical stress, oncogenic transformation, by cell-cell and cellmatrix interactions (stamenkovic, 2003), even if their expression is regulated primarily at the level of transcription and their proteolytic activity requires zymogen activation (mannello et al., 2005b). mmps can degrade almost every component of the extracellular scaffold but their role is not limited only to the breakdown of structural components, as they have been found to be strongly involved in many physiological processes, such as cell migration, tissue morphogenesis, wound healing and in the modulation of the bioavailability of active molecules (vu and werb, 2000), and in pathological situations such as inflammation, cancer development and metastasis (stamenkovic, 2003; mannello et al., 2005b). ...but something else emerges from the depth of the sea world... one of the first invertebrates that has been analyzed in this context is the sea urchin in its different embryogenetic stages and it has been demonstrated that normal developmental processes (i.e. spiculogenesis and gastrulation) necessitate collagen deposition and ecm modifications (spiegel et al., 1989), thus evidencing the necessity of enzymes with proteolytic activity (wessel et al., 1984). during the blastula early stages the sea urchin regulates the transcription and secretion of the hatching enzyme (envelysin) which degrades the protective envelope. this collagenase-like enzyme is structurally very similar to the vertebrate counterpart as it is characterized by domains displaying the same functional roles found in mammalian collagenases; in addition, it posesses its own distinctive sequences. (lepage and gache, 1990; roe and lennarz, 1990; nomura et al., 1997). during the following embryonic developmental stages, the sea urchin expresses a 41 kda protease which shows substrate specificity towards gelatin and extraembryonic collagen components (mayne and robinson, 1996; mayne and robinson, 1998). this gelatinase has been found in both the hyaline and basal lamina, which contain components similar to those found in vertebrate ecms (wessel et al., 1984). this 41 kda enzyme is secreted on the apical surface of the embryo and prior to its secretion it can be detected in the cortical and in the yolk granules (mayne and robinson, 1998). the structural organization of this collagenase is closely related to that of vertebrate mmps as it has a signal and a pro-peptide, a zn2+-binding catalytic domain and a hemopexin-like c-terminal domain (nomura et al., 1991). in the gastrula and pluteus stages the sea urchin expresses an 87 kda protease which can specifically cleave gelatin and can control shapechanges, cell-cell and cell-ecm interactions during the gastrula and pluteus stages through the regulation of ecm composition (robinson, 1997). this enzyme is ca2+ and zn2+ dependent, but its activation mechanism may be different from that of the known “cysteine switch”, as demonstrated by inhibitory and activating studies. the two proteinases (41 and 87 kda) described up to now are both zn2+-dependent and also need low affinity ca2+ binding for activity; moreover, mg2+ seem to have an inhibitory effect on the enzyme (robinson, 2000). this contrasting effect of ca2+ and mg2+ on the gelatinase activity could cause the fine regulation and modulation of the enzymatic activity on the cell surface, as small variations in the ion concentrations, obtained through the binding capacity of extraembryonic matrix, can regulate the enzyme activity (robinson and mayne, 1998). the hyaline layer of the sea urchin was analyzed for mmp activity during the transition from early to late stage embryos and enzymes (initially secreted as proenzymes and subsequently proteolytically activated) with molecular masses of 94/117, 90 and 45 kda with gelatin specificity and ca2+-zn2+ dependence were found and suggested to be matrix metalloproteinases (flood et al., 2000). the exact relationship between the 94/117 kda and 90 kda species and those found in the sea urchin embryo is still unclear. also the process of skeleton formation and the process of spiculogenesis seems to be correlated to the action of metalloproteinases, as inhibitors of these enzymes block these morphological events (ingersoll et al., 2003). in echinoderms, mmps are not only involved during embryonic development, but play important roles also during tissue/organ regeneration due to the modifications that need to occur in the ecm. evidence of this is the expression and activity of mmps during early stages of intestinal regeneration in the sea cucumber holothuria glaberrima (quinones et al., 2002). a gelatinase has also been found in the digestive tissues of the crab scylla serrata. all the identified crustacean collagenases belong to the serine protease family, while this novel enzyme results as a metalloproteinase with gelatin specificity. this enzyme has high proteolytic activity at low temperatures and acts on a wide range of substrates (this could be due to the fact that lower animal collagenases are necessary for the digestion of collagen containing tissues of the prey that these animals feed on) but its 71 preferential substrate is represented by denatured collagen (sivakumar et al., 1999). the barnacle balanus amphitrite is a thoracican cirriped crustacean that is due to undergo many complex and substantial morphological changes before it metamorphoses to the final stage of larval development: the lecithotrophic cypris larva. these developmental processes depend on the activity of specific extracellular matrix-degrading enzymes as b. amphitrite naupliar stages contain several proteinases specific towards different gelatin substrates evidencing their involvement in all phases of larval growth and development. substrate and activity analyses collocate these proteolytic enzymes in the mmp family as they are specific towards gelatin substrates and dependend on zn2+ and ca2+ ions (mannello et al., 2003). these enzymes could also be dependent on mg2+ suggesting that the enzyme activity could be regulated by these cations, as can be seen also in the sea urchin embryo (robinson, 2000) and in the mussel mytilus galloprovincialis (mannello et al., 2001). even though the activation mechanism of the barnacle mmps is quite similar to the one observed for mammals it is probably not regulated by the same cysteine switch trigger but has its own unique setup (robinson, 1997; mannello et al., 2001). the serum and hemocytes of the eastern oyster crassostrea virginica have been analyzed for ecmdegrading activity underlining the role of ecm ptoteolytic enzymes in both normal and diseased molluscs (ziegler et al., 2002). a mmp-like enzyme has been fonud in the hemocytes, but not in the serum of c. virginica. this enzyme has been proven to be an mmp due to its gelatin and collagen degrading activity and to its inhibition profile. this mmp is not involved in the degradation of interiorized phagocytosed materials, but it is probably active in the external environment. these findings support the hypothesis that hemocyte derived mmp-like activity may control the remodelling and development of ecm and may be also involved in the response towards pathogen invasion (ziegler et al., 2002). the new exciting role mmp may have during deseased or damaged states has been further investigated in the pacific oyster crassostrea gigas and a mmp inhibitor named cg-timp with functional characteristics extremely similar to vertebrate was identified (montagnani et al., 2001). this inhibitor binds and blocks mmps but posesses other functions probably regulated by an additional pair of cysteine residues in the carboxy-terminal domain (this could be a characteristic of invertebrate timps as it is present also in drosophila timp). cg-timp could be strongly involved in pathogen protection or in wound healing processes as it was only expressed in hemocytes and showed increased activity after shell damage or bacterial infection, suggesting new insights for the anti-microbial defense of marine invertebrates (bachere et al., 2004). gelatinolytic activity similar to that found in c. virginica was discovered in the hemocyte and serum homogenates of the mussel m. galloprovincialis (mannello et al., 2001). this proteolytic enzyme was similar to known mmps due to its gelatinase and collagenase activity and to its ionic requirements, but exhibited different activating and inhibiting processes suggesting that in molluscs the activation mechanism differs from the vertebrate “cysteine switch”. in healthy mussels this gelatinase may regulate normal physiological functions such as cell migration and tissue infiltration, but mmp activity goes beyond these roles being of great importance during cell-mediated and humoral immune responses (chen and bayne, 1995) and moreover in wound repair, inflammation, internal defense and also in pathological conditions as hematopoietic neoplasia (riginos and cunningham, 2005). it is clearly emerging that these mmp-like enzymes touch several aspects of oyster and mussel biology as they take part to tissue homeostasis and are also strongly involved in defence mechanisms, or because they get produced directly by the pathogenic agent (norqvist et al., 1990; lepore et al., 1996) or because they are stimulated to be secreted by the pathogen itself (okamoto et al., 1997). mmp-like enzymes have been found in a member of the cnidaria family as hydra; the entire body wall of this organism is structurally reduced to an epithelium bilayer (ectoderm and endoderm) with an intervening extracellular matrix that contains basement membrane components such as laminin and interstitial matrix components such as a unique type i fibrillar collagen. the simple structure of this metazoan and the relation between developmental processes and cell-ecm interaction led to the search for mmp-like enzymes (zhang and sarras, 1994). a single hydra matrix metalloproteinase, hmmp, with a strong sequence similarity to human mmps was identified, (even though it contains some unique amino acid stretches) (leontovich et al., 2000). hmmp is not only structurally similar to vertebrate mmps, but it also shares functional characteristics as inhibition by specific mmp-inhibitors and substrate specificity towards hydra ecm molecules and gelatin. activation studies on hmmp demonstrate that it can be activated intracellularly by a furin-like enzyme and that there can be an intermediate step before reaching the fully active enzymatic form. hmmp has been studied during foot and head regeneration processes (leontovich et al., 2000). these results clearly evidence a direct implication of this enzyme during morphogenetic events as hmmp is involved in cell transdifferentiation (werb and chin, 1998) and regenerative processes being implicated in biogenesis (shimizu et al., 2002). hmmp is secreted by the cells belonging to the endoderm evidencing that although hydra ecm has a symmetrical structure, its components are synthesized in a non-symmetrical manner (shimizu et al., 2002). besides having a important role during regenerative events and also in the maintainance of the differentiated state of certain cells (leontovich et al., 2000), hmmp may participate in the regulation of the bioavailability of signalling molecules which are sequestered by the ecm, and can be released by a hmmp dependent proteolytic cleavage (muller, 1996). hmmp demonstrates to be a fundamental element in several processes of the hydra underlining the importance of ecm-related mechanisms. molecules similar to mmp inhibitors have been found in low metazoans such as sponges; callyspongia truncata produces callysponginol sulphate a (fujita et al., 2003a) and agelas nakamurai expresses a novel mmp inhibitor, ageladine a which also possesses antiangiogenic activity (fujita et al., 2003b). 72 ... and spreads into the sky... drosophila melanogaster expresses two matrix metalloproteinases: dm1-mmp and dm-2-mmp. dm1mmp, as vertebrate mmps, contains a signal sequence necessary for secretion, a pro-peptide with a cys residue that maintains the enzyme in an inactive zymogenic form, a catalytic domain with the zinc-binding site and finally a hinge region and a nterminal hemppexin domain. one more aspect of this newly discovered proteinase is that its activation could also be regulated by furin-like proteases, due to the presence of a furin-like cleavage site located at the end of the pro-peptide (roebroek et al., 1993; llano et al., 2000). studies on substrate specificity of recombinant dm1-mmp evidenced proteolytic activity towards extracellular matrix and basement membrane proteins, such as fibronectin and type iv collagen which are present in drosophila (fessler and fessler, 1989). on the bases of several reports it can be to hypothesized that this mmp could be directly involved in the guidance and extention of axons during the nervous system development, as it was found to be secreted by the glial cells of larval tissues (menne et al., 1997; llano et al., 2000); this could depent or on the brakdown of extracellular barriers or on the release of hidden, not yet available signal molecules. dm2-mmp, is similar to dm1-mmp by posessing the mmp distinctive structural domains, but it differs from dm1-mmp because of the presence of a 200 amino acid-long insertion in the hinge region (llano et al., 2002) and also because this enzyme is expressed in all of the developmental stages of the fly, while dm1mmp is present in the developing embryo at stages 12 and 13 (llano et al., 2000). one more difference is that dm1-mmp is a secreted enzyme, while dm2mmp is a membrane bound proteinase. due to the differential pattern of expression of the two mmps it could be possible that they have different functional roles. it has been hypotesized that dm2-mmp may be involved in photoreceptor growth cone guidance and cell rearrangement in the retina and nervous system (llano et al., 2002). dm1-mmp seems to be determinant for larval tracheal growth and pupal head reversion, while dm2-mmp participates to larval tissue histolysis and epithelial fusion during metamorphosis and both enzymes seem to be required for tissue remodeling (page-mccaw et al., 2003). for a regulated mmp activity the presence of mmp inhibitors is fundamental, and infact one inhibitor of matrix metalloproteinases has been found in the fly (pohar et al., 1999) and it is structurely closely related to mammalian timps (wei et al., 2003). it is clear that drosophila in its relative semplicity unravels a highly regulated functional proteolytic system involved in several biological and physiological processes which could account for a common origin with the fully evolved vertebrate mmp system (wei et al., 2003). the larvae of the greater wax moth, galleria mellonella, is peculiar in that it containes the first insect inhibitor of metalloproteinases (impi) with no similarity at all with other known vertebrate or invertebrate counterparts. it is possible that this impi is implicated during the response of g. mellonella to invading pathogens as it is released during the humoral immune response (probably stimulated by particular peptidic fragments) and is able to protect the insect from exogenous metalloproteinases of pathogen origin such as bacterial thermolysin (wedde et al., 1998; vilcinskas and wedde, 2002). ... and into every nook and craggy ... three gene products (mmp-c31, h19 and y19) encoding matrix metalloproteinases have been found in the nematode caenorhabditis elegans. these enzymes have a domain organization very similar to human mmps, in particular the catalytic sites of these newly discovered mmps show high sequence homology with human interstitial collagenase. interestingly, two of these nematode mmps were inhibited by specific human mmp inhibitors evidencing a similar mmp system between mammalian and c. elegans mmps (wada et al., 1998). a glycoprotein similar to c. elegans mmps was analyzed in the larvae of gnathostoma spinigerum and this protein showed to possess an n-terminal signal peptide necessary for its secretion and the catalytic domain which are two elements distinctive of other known mmps (uparanukraw et al., 2001). considerations it is evident that in all organisms the role of the ecm goes beyond that of a mere scaffold having only structural functions, but controls many more complex and important processes such as cell shape, growth, migration and differentiation as a consequence of its capacity of sequestering signal molecules. the involvement of the ecm components in such biological pathways led to a great amount of rersearch in order to better understand the mechanisms that regulate the organization of extracellular environment. key elements in the regulation of ecm components are the proteinases belonging to the mmp family; these enzymes seem to hide many surprises as their functional roles touch many aspects of physiological and pathological processes and have been widely studied not only in vertebrate models but also in invertebrates (massova et al., 1998). mmp-like enzymes with their relative inhibitors have been discovered in animals belonging to distantly related taxa, evidencing the existence and importance of such a proteolytic system also in relatively simple organisms. even though mmps and relative inhibitors are present in such a varied range of organisms, they share many features characteristic of the vertebrate mmp family, suggesting a possible common ancient origin. these vertebrate and invertebrate enzymes may share structural similarities, but it is amazing to note that invertebrate mmps take part in many processes such as embryogenesis, differentiation, cell migration, wound healing and immune defense, evidencing that also in these organisms mmps are far more than simple structure destroyers. it is evident that in all these organisms mmps and their inhibitors are closely linked to ecm remodelling displaying multiple roles that touch many aspects of animal physiology and demonstrating that in such evolutionary distant organisms several biological pathways follow similar laws. 73 finally, this overview add force to the emerging concept that mmps and timps are universal and ubiquitous in animals, and that invertebrates may provide further novel information for the understanding of the multifaceted physiological roles of this ancient proteolytic system (mannello et al., 2005a); numerous evidences suggest that both mmp and timp expression and their remodelling functions appear well conserved in invertebrates, laying the hypothetical 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crassostrea virginica. comp. biochem. physiol. 131b: 361-370, 2002. minireview isj 7: 157-164, 2010 issn 1824-307x minireview defensins and cystein rich peptides: two types of antimicrobial peptides in marine molluscs g arenas díaz laboratorio de genética e inmunología molecular, instituto de biología, campus curauma, pontificia universidad católica de valparaíso, chile accepted june 3, 2010 abstract this review focuses on defensins and cystein rich peptides, which are the most abundant natural antimicrobial peptides (amps) described in molluscs. these are compact peptides, 3-5 kda in molecular mass, cationic and amphipatic; the presence of at least six cysteine residues forming three or four disulfide bridges is their prime structural characteristic. a 3-d structural characterization of these molecules has been included in recent investigations, using currently-available techniques. amps have been purified from hemocytes, epithelial tissue and plasma as well as cloned and chemically synthesized. their antibacterial activity against gram-positive and gram-negative bacteria and fungi has been shown; only a synthetic mytilin fragment has displayed activity against viruses. key words: defensins; cystein rich peptides; antimicrobial peptides; innate immunity; marine molluscs introduction marine molluscs are exposed to microbial pathogens in their environment, which can number up to 106 bacteria/ml and 109 virus/ml of seawater (ammerman et al., 1984). in order to defend themselves against such condition, molluscs have developed very effective mechanisms that are part of their innate immunity (tincu and taylor, 2004). antimicrobial peptides (amps) are the major component of the innate immune system in marine invertebrates (destoumieux et al., 1997, mercado et al., 2005; arenas et al., 2009; de zoysa et al., 2009). the first research on amps in bivalve molluscs through reverse genomics was done at the end of the 90s (hubert et al., 1996). amps are distinguished by their net positive charge and amino acidic residue amphipathic distribution; these key features explain their mode of action with the membrane of target microorganisms (marshall and arenas, 2003). in order for the synthetic peptides to maintain the antimicrobial activity, they must be able to form an amphipathic structure, i.e., they must be organized in hydrophobic and hydrophilic amino acid zones (zasloff, 2002; arenas et al., 2009). a general mechanism of action has been proposed describing the sequence of associated ___________________________________________________________________________ corresponding author: gloria arenas díaz laboratorio de genética e inmunología molecular, instituto de biología, campus curauma, pontificia universidad católica de valparaíso, chile e-mail: garenas@ucv.cl events occurring once the peptides are initially attracted to the target membrane of microorganisms by electrostatic attraction. then, hydrophobic interactions with the membrane ensue, followed by accumulation of the peptide until a threshold concentration is achieved. the peptide then adopts a new dynamic conformation that causes a deformation of the membrane, followed by a transient peptide conformation which enables it to insert into the membrane. in the next step the peptides multimerize forming complexes such as barrel-staves or toroid pores. in the final stages the peptide is translocated to the cytoplasmic face of the membrane to exert its action on membranous cytosolic components. different types of amps follow some or all of the steps described above. (cudic and otvos, 2002; zasloff, 2002; yeaman and yount, 2003). the purification procedure is summarized as follows: the homogenized samples are suspended in cold acetic acid 11 % (1:10) in order to solubilize cationic molecules and sonicated for 3x30 sec at 11 rms in ice. the crude extract is centrifuged at 11,000xg, 35 min at 4 °c and the pellet is discarded. the supernatant is called acid extract (ae) and is further shaked at 37 °c for 1 h to favor sugar hydrolysis. the acid extract is loaded on a sulfoethyl (se) sephadex c-50 cation-exchange chromatography column (biorad), eluted with 1 m nacl 1 % acetic acid ( ph 3.0), in order to enrich cationic peptides. the eluate is applied onto a seppak c18 vac cartridge (waters associates) 157 equilibrated in acidified water (0.05 % trifluoroacetic acid in upw (ultra pure water). after a wash with acidified water, the peptides are eluted with 5 %, 20 %, 40 %, 60 % and 80 % acetonitrile (acn), to obtain several hydrophobic fractions. the samples obtained are lyophilized and reconstituted in milliq water, total protein content determined by the bicinchoninic acid (bca) microplate assay (pierce) and tested for antibacterial activity. only those with antimicrobial fraction activity are subjected to reversed phase hplc. all purification steps are performed on a rp-hplc model lachrom d-7000 with a lachrom model l-7455 photodiode array detector. column effluents are monitored by uv absorption at 225 nm. eluates are selected for further purification and loaded on a sephasil c-18 (250x4.1 mm) column (lichrocart). elution is performed with a linear gradient of 5-60 % acn in acidified water over 90 min at a flow rate of 0.6 ml min-1. the resulting fractions are collected, lyophilized, reconstituted in ultra-pure water (upw ) and frozen at -20 °c until antimicrobial activity testing (bulet et al., 1991; charlet et al., 1996; mercado et al., 2005). among the different natural amps, those containing pairs of cysteine residues forming intramolecular disulfide bridges are particularly common (dimarcq et al., 1998; bulet et al., 2004; reddy et al., 2004; yount et al., 2006). this highly complex 3 5 kda group has been extensively studied in mussels, mytilus edulis and mytilus galloprovincialis, where they were classified into four groups: defensins, mytilins, myticins and mytimycin (charlet et al., 1996; mitta et al., 1999a; pallavicini et al., 2008; costa et al., 2009; parisi et al., 2009). defensins have been also recently described in oysters crassostrea virginica and crassostrea gigas (seo et al., 2005; gueguen et al., 2006; gonzález et al., 2007) and abalone haliotis discus discus (de zoyza et al., 2010); mytilins and myticins, on the other hand, have been also described in clams ruditapes decussates (gestal et al., 2007). defensins and cystein rich peptides from marine molluscs express a stronger activity against gram-positive and gram-negative bacteria and fungi (charlet et al., 1996; mitta et al., 1999a, b; seo et al., 2005; gueguen et al., 2006; gestal et al., 2007) and one synthetic mytilin fragment displayed activity against the white spot syndrome virus (dupuy et al., 2004; roch et al., 2008). defensins and cystein rich peptides from mussels for the summary of the amps described in mussels and the relative alignments see table 1 and fig. 1. defensins a (4314.3 da) and b (4392.4 da) were purified from the hemolymph of m. edulis using chromatographic methods. both exhibited six cysteines, forming three intramolecular disulfide bridges positioned in a highly conserved array, thus allowing a complex three-dimensional structure the cysteine consensus motif is identical to that found in the large family of arthropod defensins, phormia defensin, described in detail for the phormia terranovae (charlet et al., 1996). the latter corresponds to a central amphipathic α-helix with an extended nh2-terminal loop and a cooh-terminal antiparallel β-sheet. the helix is stabilized through two disulfide bridges to the β-sheet and the nh2terminal loop is linked to one of the strands of this sheet via the third disulfide bridge (cornet et al., 1995). using the liquid growth inhibition method (bulet et al., 1993) it was determined the mytilus defensins a and b were consistently more active against the gram-positive strain m. luteus (mic: 0.6 1.2 μm) than to the gram-negative strain e. coli (mic: 2.5 10 μm). the minimal inhibitory concentration (mic) values are expressed as an interval (a b), where (a) represents the highest peptide concentration tested at which bacteria are still growing and (b) the lowest concentration that causes 100 % growth inhibition (charlet et al., 1996). the defensin isoforms mgd-1 and mgd-2 (4 kda), containing eight cysteines, were purified from the plasma and hemocytes of mussels, m. galloprovincialis, using conventional chromatographic methods. two extra cysteines and one modified amino acid suggested that these molecules are new members of the arthropod defensins family (mitta et al., 1999b). the 3-d structure of mgd1 was established using nmr analysis, which mainly consists on the classical csαβ structural motif (cys4 cys25, cys10 cys33 and cys14 cys35 disulfide bonds). the two extra cysteines (cys21 cys38) form an original fourth disulfide bond. synthetic mgd1, correctly folded to form the four disulfide bonds, retains the antibacterial activity of the native molecule and presents a similar effect than the insect defensin a, thus proving the fourth disulfide bond of mgd1 was not significant for the biological activity (yang et al., 2000). a series of synthetic peptides, conforming the main known secondary structures of mgd1, allowed the location of the nonapeptide cggwhrlrc corresponding to the residues between cys25 and cys33. the bacteriostatic activity of such sequence was strictly dependent on the bridging of cys25 and cys33. the antibacterial activity of this synthetic nonapeptide clearly evidenced an effect on grampositive when tested against the gram-positive bacteria micrococcus lysodeikticus, staphylococcus aureus, staphylococcus epidermidis, bacillus megaterium (mic 0.6 0.8 μm) and the gramnegative bacteria vibrio alginolyticus, vibrio metschnikowii, escherichia coli 363, salmonella newport (mic>75 μm) and fusarium oxysporum (mic 5 μm) (romestand et al., 2003). modelling studies evidenced that positively charged and hydrophobic residues of mgd1 were organized in two discrete domains; this feature would support the hypothesis that positive charges allow the initial attraction of the peptide with the membrane, as well as the hydrophobic domain insertion into the bacterial lipid bilayer (romestand et al., 2003). the tissue location analysis through optical and ultrastructural levels showed mgds were mainly located in the granular structures of the hemocytes and enterocytes, where they were synthesized as an 81 amino acid precursor and processed as active 158 table 1 shows a summary of the amps described in marine bivalves peptide origin acces number mw da sequence reference defensin a m. edulis sp | p81610 4314.3 gfgcpndypchrhcksipgrxggycggxhrlrctcyr charlet et al., 1996 defensin b m. edulis sp | p81611 4392.4 gfgcpndypchrhcksipgryggycggxhrlrctc charlet et al., 1996 mgd-1 m. galloprovincialis sp | p80571 4000 gfgcpnnyqchrhcksipgrcggycggwhrlrctc mitta et al., 1999b mgd-2 m. galloprovincialis gb | aad52660 4000 gfgcpnnyachqhcksirgycggycaswfrlrctc mitta et al., 1999b aod c. virginica sp | p85008 4265.0 gfgcpwnryqchshcrsigrlggycagslrltctcyrs seo et al., 2005 cg-def c. gigas gb | aj565499 (est) emb | caj19280 n.a. gfgcpgnqlkcn nhcksiscragycdaatlwlrctc gueguen et al., 2006 cg-defh1 c. gigas gb |dq400101 n.a. gfgcprdqykcnshcqsigcragycdavtlwlrctc gonzalez et al., 2007 cg-defh2 c. gigas gb |dq400102 n.a. gfgcpgdqyecnrhcrsigcragycdavtlwlrctc gonzalez et al., 2007 abalone defensin h. discus discus gb |fj864724 4900 krvtcdllslqimgnsfgdsacaahcig lhhsgghcsggvcvcr de zoyza et al., 2010 mytilin a m. edulis sp | p81612 3773.7 gcasrckakcagrrckgwasasfrgrcyckcfrc charlet et al., 1996 mytilin b m. edulis sp | p81613 3974.3 scasrckghcrarrcgyyvsvlyrgrcyckclrc charlet et. al., 1996 mytilin b m. galloprovincialis gb |aad52661 n.a. spsdmmpqmnenentefgqdmptgeteqgetgi mitta et al., 2000 mytilin c m. galloprovincialis gb |aad45013* n.a. scasrcksrcrarrcryyvsvryggfcycrc mitta et al., 2000 mytilin d m. galloprovincialis gb |acf21701 n.a. gcasrckakcagrrckgwasasfrrrcyckcfrc mitta et al., 2000 mytilin g1 m. galloprovincialis n.a. n.a. tcgslckahctfrkcgyfmsvlyhgrcycrcllc mitta et al., 2000 myticin a m. galloprovincialis gb |aad47638 4438 hshactsywcgkfcgtakmcacvhcsrvnnpfrvnqvaksindldytpim mitta et al., 2000 myticin b m. galloprovincialis gb |aad47639 4562 hphvctsyycskfcgtaklcfclhcsrvkfpfgatqdaksmneleytpim mitta et al., 2000 myticin 1 r. decussatus n.a. n.a. qsvactsyycskfcgsakicyclhcrraesplalsgsarnvndknnemdnspvm gestal et al., 2007 myticin 2 r. decussatus n.a. n.a. vpcastycarfcgsakicyclhcrraesplalsgsarnvndknnemdnspvm gestal et al., 2007 myticin 3 r. decussatus n.a. n.a. vpcastlcsrfcgsakicyclhcrraesplalsgsarnvndqnkemdnspvm gestal et al., 2007 mytimycin m. edulis n.a. 6233.5 dccrkpfrkhcwdctagtpyygystrnifgctc charlet et al., 1996 aminoacidic sequences of the amps presented in the text with their accession number (from swissprot and genbank data bases), molecular weight and references. *the amino acids different in genbank database are highlighted in grey. n.a.: not available. peptides. the bacterial challenge caused the release of mgd-1 and mgd-2 from the stimulated hemocytes (mitta et al., 1999b, 2000). the mgd-1 and mgd-2 antibacterial test at 10ul/100ul against m. luteus, using microtitration plates that were measured 24 h post incubation at 30 °c (od 600 nm), showed inhibitory effects. antifungal activity was monitored against spores from f. oxysporum; growth inhibition was observed microscopically after 24 h incubation at 30 °c with 10ul/80ul, and quantified by measurement of optical density at 600 nm after 48 h (felhbaum et al., 1994, mitta et al., 1999b). mytilin comprise isoforms a, b, c, d and g1, containing eight cysteines, represent the second group of amps described in mussels. isoforms a and b were isolated from m. edulis plasma (charlet et al, 1996); isoforms b, c, d and g1, on the other hand, were isolated from m. galloprovincialis hemocytes (mitta et al., 2000). mytilins a (3773.7 da) and b (3974.3 da) were isolated by conventional chromatographic methods. the concentration of mytilins in the blood of mussels may be estimated at approximately 2 μm. this is a range of the mic determined for most of the tested bacteria. mytilins appeared primarily effective against gram-positive bacteria (0.6-1.2 um) and less active against gram-negative bacteria (2.5 10 um). minimal inhibitory concentration (mic) values are expressed as an interval (a b), where (a) represents the highest peptide concentration tested at which bacteria are still growing and (b) the lowest concentration that causes 100 % growth inhibition (charlet et al., 1996). 159 fig. 1 clustalw alignment of the peptides sequences from defensin, mytilin and myticin from m. edulis, m. galloprovincialis, c,virginica, c. gigas, h. discus discus and r. decussatus. the color code is basic blue, acidic red, polar without charge green and hydrophobic white, the cys are highlighted in yellow. the figures were created with jalview (clam et al., 2004) mytilins b, c, d, and g1 were isolated from m. galloprovincialis (mitta et al., 1999a). they were synthesized as precursors and processed as active peptides within the hemocytes. they were found, by confocal microscopy analysis in two subclasses of circulating granulocytes, one containing small granules and one with large clear granules (mitta et al., 2000). the 3-d solution structure of synthetic peptides derived from the structure of mytilin b (34-residue) was established by 1h nmr. this structure consists of the common cysteine-stabilized α β motif (cs αβ) closely related to the one observed in the mussel defensin mgd-1. the 8 cysteines formed four disulfide bonds (2 27, 6 29, 10 31, and 15 34) only involving the β-strand ii. the percentage of the hydrophilic and hydrophobic areas from mytilin and mgd-1 are closely related (63 % 37 % and 64 % 36 %, respectively). the c10c (accvcyrgrcycnh2) mytilin fragment showed antiviral activities again with white spot syndrome virus (wssv). this peptide was precipitated in salty water, although it was able to recover its original structure once the salt content was lowered, and appeared to be soluble in a high ionic strength environment. this fact anticipates its potential application as an antiviral agent in both aquatic and terrestrial animals and in humans. the small size and ease of synthesis of c10c enables its biotechnology development (roch et al., 2008). diversity of mrnas from mytilin b in m. galloprovincialis has been studied from circulating hemocytes, thus defining 10 individual dgge (denaturing gradient gel electrophoresis) patterns in untreated mussels (parisi et al., 2009). further analysis is required on amps polymorphism to determine the role of the environment on the polymorphism of these molecules in molluscs. mytimycin isolated from the plasma of m. edulis (6233.5 da) involves twelve cysteines engaging in the formation of six intra-molecular disulfide bridges. mytimycin demonstrated to be strictly anti-fungal when tested against the strains neurospora crassa and f. culmorum (charlet et al., 1996). myticin isolated from m. galloprovincialis is a cysteine-rich peptide produced in two isoforms, a and b. myticins a and b were isolated from the hemocytes (a 4.438 da and b of 4.562 da); myticin a was also isolated from the plasma of the mussel. the mature molecule consists of 40 residues, with four intramolecular disulfide bridges and a cysteine array in the primary structure, which is different from that of previously characterized cysteine-rich antimicrobial peptides (mitta et al., 1999a). the sequence analysis of the cloned cdnas revealed that myticin precursors comprise 96 amino acids. 160 myticins a and b displayed antibacterial activity against gram-positive bacteria, and myticin b is active against the fungus f. oxysporum and the gram-negative bacteria e. coli d31 (mitta et al., 2000). myticin c, a novel antimicrobial peptide from m. galloprovincialis (pallavicini et al., 2008), appears to be extremely polymorphic. seventy four variants with nucleotide mutations were identified using dgge (costa et al., 2009). defensin from oyster for the summary of the amps described in oysters and the relative alignments see table 1 and fig. 1. the first mollusc defensin isolated from an oyster species was named american oyster defensin (aod) (seo et al., 2005). it was purified from a gill extract of c. virginica using classical chromatography for cationic peptides. electrospray ionization mass spectroscopy (esi-ms) of aod evidenced a mass of 4265.0 da. the aod (38 amino acids) has common structural features with arthropod defensins: i.e., 1) six cysteine residues; 2) at least 4 basic amino acid residues; 3) a hydrophobic loop in the amino terminal; 4) a tetraamino acid motif gly-gly-tyr-cys; and 5) a carboxy terminal penta amino acid motif cys-thr-cys-tyrarg. aod has high sequence homology (62 73 %) with the defensins from m. edulis and m. galloprovincialis, respectively (charlet et al., 1996; hubert et al., 1996; mitta et al., 1999b). sequence homology search of the purified peptide was performed using blastp 2.2.10 and tblastn 2.2.10 on genome net (http://www.ncbi.nlm.gov/blast). the theoretical isoelectric point (pi) and molecular mass were estimated by expasy (http://www.expasy.ch tools/peptide-mass.html). sequence alignment was performed using the clustalx program (thompson et al., 1997). the antibacterial activity of the purified peptide was tested using a double-layer radial diffusion assay (lehrer et al., 1991). using the minimal effective concentration (mec) as a parameter, significant activity against the gram-positive bacteria lactococcus lactis subsp.lactis and s. aureus at 2.4 and 3.0 ug/ml, respectively, was detected,. on the other hand, a lower effect on the gram-negative bacteria e. coli d31 and vibrio parahemolyticus, mec at 7.6 and 15.0 ug/ml, respectively, was observed. mec was calculated as described (zhao et al., 2001). another defensin from an oyster was identified in the mantle of c. gigas (cg-def) (gueguen et al., 2006). the cg-def gene is continuously expressed in the mantle. the structure of the recombinant peptide in e. coli is cs-αβ like arthropod defensins, but it includes an additional disulfide bond as the mussel defensin mgd-1. nonetheless, the difference with mgd-1 is the size of their loops and the presence of two aspartic residues. the oyster cg-def cdna contained 323 bp. the 195 bp coding region encoded a 65 amino acid propeptide (genbanktm caj19280) (gueguen et al., 2003). cg-def is not synthesized as a precursor. the antimicrobial activity of the recombinant cg-def was determined against gram-positive and gram-negative bacteria and filamentous fungi, resulting mainly effective against the gram-positive strains micrococcus lysodeikticus, s. aureus brevibacterium stationi, and microbacterium maritypicum at 0.005 1.25 um (mic values). the activity of cg-def was retained in vitro at a salt concentration similar to that of seawater (gueguen et al., 2009). it has been additionally established that the two isoforms of the defensin from the hemocytes of the oyster c. gigas, cg-defh1 and cg-defh2 (43 amino acids), have four disulfide bridges. this feature is also present in the defensins mgd-1 and mgd-2 from m. galloprovincialis hemocytes and cg-def from the c. gigas mantle (gonzalez et al., 2007). a quantitative rt-pcr (qrt-pcr) analysis indicated that cg-defh2 was continuously expressed in the hemocytes of c. gigas. in addition, the level of cg-defh2 transcripts decreased drastically in the circulating hemocyte population after a bacterial challenge, thus suggesting a possible migration of the hemocytes towards the gill and mantle tissue. this fact may be correlated with an increase of cgdefh2 transcripts in such tissues (gonzález et al., 2007). rich cystein antimicrobial peptides from clams for the summary of the amps described in clams and the relative alignments see table 1 and fig. 1. myticin isoforms 1, 2 and 3 were identified and characterized for the first time in clams, ruditapes decussatus (gestal et al., 2007), using the suppression subtractive hybridization technique (ssh). suppression subtractive hybridization libraries may facilitate the identification of genes involved on bivalve immune response (tanguy et al., 2004). clam myticins (40 aa) and mytilin (34 aa) are similar to the myticins previously reported from m. galloprovincialis, as both conserved the cystein array with four intramolecular disulfide bridges, which are characteristic of the myticin family (gestal et al., 2007). clams challenged with bacteria showed that clam myticin and clam mytilin increased the expression levels 48 h post-infection using qpcrs performed on hemocytes. during the challenge, specimens were injected 100 μl (containing 107 cells/ml) of a dead bacteria mixture into the adductor muscle, which included micrococcus lysodeikticus, vibrio splendidus and vibrio anguillarum (gestal et al., 2007). aminoacidic diversity of the clam myticin was found with the previously described mussel myticins a and b and with myticin c (pallavicini et al., 2008). the variation in aminoacidic residues of the different amps may occur as a response to the recognition of different pathogens in their environment (gestal et al., 2007). 161 defensins from gastropods for the summary of the amps described in abalone and the relative alignments see table 1 and fig. 1. an abalone defensin, with 66 aa, lower molecular mass of 4.9 kda, positive charge +5, hydrophobic residue ratio 46 %, α-helical structure and with an arrangement of six cysteine residues forming three disulfide linkages in c1-c4, c2-c5 and c3-c6, was characterized from the h. discus discus. the complete coding sequence of the defensin was obtained from the abalone cdna library est database (de zoyza et al., 2010). hemocytes were collected for rna extraction. a domain sequence was identified, 24 66 aa, exhibiting the same basic characteristics of the arthropod defensin family members. it is expressed constitutively in hemocytes, gills, muscle and digestive tract; however, the transcripts in tissues were significantly induced 48 h post-infection in abalones injected into the adductor muscle with 100 μl of a bacterial mixture containing v. alginolyticus, v. parahemolyticus and lysteria monocytogenes (5x107 cells/ml). conclusions different research groups have focused on amps since they are molecules of the innate immune system of a wide range of organisms, displaying an efficient activity against pathogenic microorganisms without cytotoxic effects on eukaryotic cells. they were discovered in marine molluscs almost 20 years ago and they have been primarily identified in mussels mainly as defensins and cys-rich peptides. since then, they have been purified from tissues and the genes associated to their expression have been identified; furthermore, the phylogenetic relationships with existing molecules from other invertebrates have also been established. all these aspects have contributed to the knowledge of the immune responses of this phylum. the study of the structures and antimicrobial effects of the different amps, on the other hand, has allowed achieving significant progress in the elucidation of the different action mechanisms, thus suggesting specific models that have been integrated as part of a sequence of events of a more general mechanism. various trends have also emerged where amps are regarded as alternative molecules to classical antibiotics due to their structural and physiological characteristics, stressing the fact that in vitro assays and clinical tests until phase iii have confirmed that resistance evolution against antimicrobial peptides is less probable than that observed for conventional antibiotics, although the broad therapeutic use of amps is still unconsolidated and a long path must be covered to overcome the technical problems limiting such aspirations. although the diversity of marine molluscs is quite large, amps have been explored only in a few species; therefore, there is still a great potential to unveil new molecules in this phylum. the development of molecular biology, bioinformatics and genetic engineering techniques have allowed to produce amps in such quantities as to be used as drugs to curtail human, animal and plant diseases (zasloff, 2002; gordon et al., 2005; keymanesh et al., 2009). genetic manipulation using antimicrobial peptide 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chemicals whose environmental hazard is actually poorly determined. the peculiar behavior of nanomaterials makes them much more similar to new chemicals than to the corresponding bulk materials; this feature imposes reliable and standardized evaluation protocols for toxicity and ecotoxicity assessments. general rules for assessing nanotoxicity and the state of the art are periodically published in reports by control agencies. this review highlights the role of invertebrates as valuable and validated test organisms for assessing ecotoxicity of new and/or untested chemicals. the general scarcity of experimental data, their unequal distribution among the different nanomaterials and environmental conditions, the difficulties in manipulating nanomaterials and obtaining stable and homogeneous suspensions, the confusion arising from a not well defined metrics are discussed. key words: engineered nanomaterials; nanotoxicology; invertebrates introduction assessing the ecotoxicity of previously untested substances, as in the case of nanomaterials, is a challenging task. therefore, inexpensive, rapid and reproducible methods are preferred, and a coordinated standardization could help in avoiding the waste of resources. nanomaterial is a material having at least one dimension 100 nm or less. nanomaterials can be nanoscale in one dimension (e.g., films), two dimensions (e.g., fibers and tubes), or three dimensions (e.g., particles). nanoparticles constitute a sub-fraction of what is defined as “colloids” (christian et al., 2008). chemicals fitting these requirements share protean physical chemistry, which confers very unusual properties to them. the colloid nature, the ability to form aggregates and an appealing potential for practical applications remain nevertheless common features, explaining the growing interest in engineered nanomaterials. scientists studying this field were awarded twice with the nobel prize for chemistry. this exclusive acknowledgment was firstly given in 1996 ___________________________________________________________________________ corresponding author: anna giulia cattaneo department of biotechnology and molecular sciences (dbms) university of insubria via j-h dunant 3, 21100 varese, italy e-mail: annagiulia.cattaneo@uninsubria.it for the fullerene synthesis discover (curl, 1996; kroto, 1996; smalley, 1996), and then in 2000 for research on conductive and semi-conductive nanopolymers (heeger, 2000; macdiarmid, 2000; shirakawa, 2000). since then, the production of newer engineered nanomaterials and their applications exponentially increased, spanning from cosmetics, drug delivery systems and food additives, to products for waste remediation and fuel and energy production, the so called environmentally friendly nanotechnologies (tungittiplakorn, 2005; hollins, 2007). space and military applications of nanomaterials range at the present from protective clothing, sensors and signals, to propellants and explosives, and more (for reviews see ruffin, 2004; glenn, 2005). at the same time, basic research in the fields of nanoscience and nanotechnology improved rapidly: manufactured and natural nanomaterials branched out as distinctive fields, theories and models developed about their ability to actively interact with environmental and biological systems and the issue of their potential hazard for health and environment has been posed (oberdörster, 2004; oberdörster et al., 2005; moore, 2006; chun ke and qiao, 2007; oberdörster et al., 2007; christian et al., 2008; handy et al., 2008a, 2008b; di gioacchino et al., in press). nevertheless, nanomaterials remain very poorly tested potential pollutants, in contrast with their 78 mailto:annagiulia.cattaneo@uninsubria.it large diffusion: main difficulties in assessing toxicity are a consequence of their colloidal nature and dynamics, as systems in which smaller or larger aggregates can form in poorly predictable ways, making it difficult to measure shape, size and concentration in the final sample (service, 2004; nowack and bucheli, 2007; blaser et al., 2008; diegoli et al., 2008; hassellöv et al., 2008; tiede et al., 2008, 2009). the ability of nanomaterials to interact with natural soils and porous or colloid substrates allows a long, often unexpected, passive transport to the groundwater (nowack and bucheli, 2007; loux and savage, 2008; farré et al., 2009). important tools lacking in assessing nanotoxicity are sample-related certified standards, reliable measure units and analytical chemical procedures to measure nanomaterials in the environment. despite a great concern about waste and pollution, the presence of nanoparticles in effluents, sediments or surface waters near urban areas supporting a nanotechnology industry is not yet documented. the use of approved or certified standards, a basic requirement for good practice in toxicology laboratories, seems far from fulfillment in the case of nanomaterials. in all naturally occurring systems (water, soil, air and their combinations), the organization of the dispersed nanophase depends equally from the physical-chemistry of the manufactured nanomaterials and from that of the environment, as well as from the modalities of suspension. obtaining a gold standard for every case is far-fetched, redundant and expensive, while the use of a stable internal standard could attain a satisfactory level of laboratory practice. furthermore, interlaboratory comparisons will improve the characterization and overcome the complexity of nanometrology (hassellöv et al., 2008). theoretical prediction of equilibrium represents a pivotal characterization of nanocolloids. indeed, nanoparticles partially elude the rules of derjaguin, landau, verwey and overbeek (dvlo) theory requiring remodelling, nernst equilibrium does not apply, and the charge density at the surface cannot be calculated when the surface area is unknown (loux and savage, 2008). the specific surface area exponentially increases as a function of small size, and amplifies the energy of collision between particles due to the brownian motion, a major event affecting the colloid stability (casey and rustad, 2007; christian et al., 2008; tenne and seifert, 2009). experimental evidences, positively relating this parameter to toxicity, endorse speculations about its importance in toxicology (oberdörster et al., 2005; stoeger et al., 2006). another feature characterizing the colloid stability is the zeta potential, i.e. the diffuse surface charge, linked to the chemical nature of the dispersed nanomaterials and to the properties of the continuous phase (ph, dilution, temperature and interatomic distance between others). its measure gives good information on nanomaterials mobility, aggregation rates and interactions with surfaces: when its value approaches 0 mv massive aggregation occurs (dunphy guzman et al., 2006a; loux and savage, 2008). moreover, for magnetically charged particles, the dipole moment is a key feature in their characterization and appears to be related to the toxicity potential (kumar et al., 2006; malvindi et al., 2008). the critical coagulation concentration (ccc) of electrolytes (mol/l) is particularly interesting in evaluating the stability of colloid suspensions in hard and salt water: the stability of suspension is strongly dependent from counterion valence and electrostatic potential at the interface, at least in nearly spherical nanometals (loux and savage, 2008). the recently released notes of the organization for economic co-operation and development (oecd, 2008; table 1) include these properties in the endpoints for phase one characterization of manufactured nanomaterials. in addition to them, water solubility and stability of dispersions, crystalline phase, dustiness, crystallite size, tem picture(s), particle size distribution, surface chemistry, photocatalytic activity, pour density, porosity, octanol-water partition coefficient, redox potential, and radical formation potential are listed. the second tool, i.e., a reliable measuring unit expressing toxicity, gave matter of discussion. particle size, firstly suggested as a highly appropriate unit of measure related to toxicity, declined in popularity in recent years: the size of aggregate, not particles, seems to be more informative (pauluhn, 2009). an alternative method, expressing nanomaterials toxicity based on mass or on surface area, seemed to be satisfactory (oberdörster et al., 2005; stoeger et al., 2006). however, a revision of published data in an attempt to explain non-linear dose-response toxicity of some nanomaterials revealed that surface-to-mass area seemed to be preferred to surface-to-size, and the number of particles performed as well (wittmaack, 2006). a lively discussion followed with every scientist supporting his or her own reasons (oberdörster et al., 2007; stoeger et al., 2007; wittmaack, 2007). the problem, as claimed in many works, is probably more complex, and additional information is needed to obtain optimally informative metrics (kandlikar et al., 2007; teeguarden et al., 2007; gornati et al., in press). nevertheless, the number-based metrics proposed by wittmaack (2006) adds predictive value to the existing experimental data on nanomaterial ecotoxicity, which remain mainly, if not exclusively, expressed as concentrations (ppm, molarity or w/v). this method is simple, generally accepted and comparable with the conventional units for corresponding bulk chemicals and should be used at least until adequate standard metrics and samples are available. another regrettable lacking matter is the systematic measure of elements released by dissolution. dissolution is dependent from the chemical nature and size of the nanoparticle, as well as from environmental variables, such as ph and temperature (meulenkamp, 1998; vogelsberger, 2003; hardman, 2006). consequently, the measured effect and the eventually observed toxicity could be not a feature of the nanomaterial itself, but of its degrading products. dissolution has been very rarely documented, while these kinds of events are well known and frequently postulated 79 table 1 guidelines for assessing toxicological risk of nanomaterials. national and supranational agencies agency country document id. year cst uk nanosciences and nanotechnologies: a review of government’s progress on its policy commitments (http://www2.cst.gov.uk/cst/business/files/nano_review.pdf) 2007 defra uk nanotechnologies research reports (http://www.defra.gov.uk/environment/nanotech/research/reports/index.htm) 2009 rcep uk novel materials in the environment: the case of nanotechnology (http://www.rcep.org.uk/reports/27-novel%20materials/documents/novelmaterials-report.pdf ) 2008 european commission eu european activities in the field of ethical, legal and social aspects (elsa) and governance of nanotechnology. (http://cordis.europa.eu/nanotechnology/) 2008 oecd series on the safety of manufactured nanomaterials – nr. 6 (http://www.olis.oecd.org/olis/2008doc.nsf/linkto/nt000034c6/$file/jt 03248749.pdf) 2008 oecd series on the safety of manufactured nanomaterials – nr. 8 (http://www.olis.oecd.org/olis/2009doc.nsf/linkto/nt000029e6/$file/jt 03263204.pdf) 2009a oecd nanotechnology research resources by country (http://www.oecd.org/countrylist/0,3349,en_21571361_41212117_42325 621_1_1_1_1,00.html) 2009b oecd guidelines for the testing of chemicals bioaccumulation in terrestrial oligochaetes (http://www.oecd.org/dataoecd/11/8/42551309.pdf) 2009c iso, iec, nist and oecd international workshop on documentary standards for measurement and characterization for nanotechnologies (http://www.standardsinfo.net/info/livelink/fetch/2000/148478/7746082/in dex.html) 2008 us epa usa nanoscale materials: stewardship program. interim report (http://www.oecd.org/dataoecd/33/55/42061387.pdf) 2009 scenihr eu risk assessment of products of nanotechnologies (http://nanotech.lawbc.com/tags/scenihr/) 2009 cst: the council for science and technologies; defra: the department for environment, food and rural affairs; us epa: environmental protection agency, usa; scenhir: scientific committee on emerging and newly identified health risks; ue: united europe; oecd: organisation for economic co-operation and development; iso: international organization for standardization; iec: international electrotechnical commission; nist: national institute of standards and technology; rcep: royal commission on environmental pollution (lovern and klaper, 2006; biju et al., 2008; handy et al., 2008b; heinlaan et al., 2008). the common efforts of the worldwide control agencies and governmental organisms finally achieved their goal in coordinating the resources, and periodically updated guidelines for risk assessment of nanomaterials have been published (table 1). the policy applied by control agencies to expert recruitment, research funding and targeting the point of interest have been critically reviewed over time (oberdörster et al., 2005; dunphy guzman et al., 2006b; rickerby and morrison, 2007; paradise et al., 2008; wilhelmi, 2008). the us epa in its recently released nanoscale materials stewardship program (nmsp), elaborated an assessment of knowledge about nanomaterials: data scheduled in databases (nanowerk nanomaterials database and wilson center project on emerging nanotechnologies (pen) inventory of nanomaterials in consumer products), or directly submitted to the nmsp were collected and compared. sharing a part of commercially available products from those for research use only or under development, the engineering process standardization and description was the best characterized (epa, 2009; table 1). an overall picture of the statistical elaboration of the data set evidenced that morphology, physical chemistry and production processes have been characterized by 50-90 % in all nanomaterials. this means that validation of producing processes, availability of approved standard for pristine materials and approved nomenclature are near to be reached purposes for the largest part of known engineered nanomaterials. the following best characterized point was the knowledge about professional risk factors in handling and manipulating nanomaterials. the risk was assessed for 50 % of known materials. rapid improvement in this field should be expected from the protocol planned for the period 2003-2013 and involving risk insurance companies (lauterwasser, 2003; oecd, 2009a; table 1). 80 experimental data about acute and chronic ecotoxicity and environmental fate score lower by far: tested materials do not exceed 20 % in this respect. any effort to bridge the gap with an adequate set of measures in this field should be encouraged in the near future. while natural systems represent the real target of these investigations, artificial laboratory conditions can be preferred for practical reasons. first, the steps of knowledge can speed up by using a combination of test media mimicking the natural environment, and test species adapted to the laboratory, representative of the field. acute lethality should be conveniently studied first, long-term toxicity tests following as needed, focused on growth and reproduction as well as morphology and behavioural changes. simplified or natural food webs should be tested to identify the levels and the risks for bioaccumulation and biomagnification (crane et al., 2008), and modelling of exposure has been proposed (mueller and nowack, 2008). endpoints for phase one assessment of environmental toxicity of nanomaterials comprise a list of short and long term effects on species inhabiting pelagic, sediment/benthic, soil and other terrestrial habitats (gourmelon and ahtiainen, 2007). invertebrates as test organisms for environmental pollution studies the invertebrates represent valuable organisms for environmental pollution studies. in addition to be among the most widely distributed living organisms on the earth and offer the opportunity to explore nearly every ecological niche, they have a relatively short life span, reproduce quickly at higher rates and are sensitive to pollutants. invertebrate-based tests are cost-effective, reasonably quick and easy to perform with reproducible standard protocols for multicentered trials. these organisms are indeed particularly convenient as test species to pioneering ecotoxicity studies on newer chemicals, and nanoparticles among others (gourmelon and ahtiainen, 2007). other physiological features of some invertebrates are amictic reproduction, warranting a large number of identical clones, and production of resting eggs, exploited to produce commercial test kits. wild specimens generally adapt quickly to laboratory conditions, making standardization easily obtainable. additional advantages are the highly reproducible staging for larval progression, the small size of adult individuals with transparent bodies, and a stereotyped behavior with easily recognizable disruption. these features facilitate the collection of valuable statistical data at the three levels important for species threatening: survival (quantified by lc50 or median lethal concentration), growth rate, and fertility (expressed as latency and length of reproductive life, number of offspring per life cycle, and offspring viability rates). chronic studies provide information on sub lethal effects, such as active swimming, feeding and avoiding predation capabilities: noec (non observed effect concentration) and loec (lowest observed effect concentration) are the mostly used in this type of approach. besides, an array of changes at cellular and sub cellular level can be followed in invertebrates to inform on the mode of actions of chemicals. despite their wide use in other fields, environmental and ecotoxicological genomics, proteomics and metabolomics (snape et al., 2004) in invertebrates are at the very beginning. the increasing interests in these areas raise the hope for a rapid enhancement in the near future, with useful applications to nanoparticle studies (snape et al., 2004; hines et al., 2007; iguchi et al., 2007; poynton et al., 2007; soetaert et al., 2007; ralstonhooper el al., 2008; steinberg et al., 2008). differently from prokaryotes (fang et al., 2007; heinlaan et al., 2008; holbrook et al., 2008), invertebrates are able to intake nanomaterials dispersed in the environment by different ways: direct ingestion or from contaminated preys, water filtration, inhalation, and surface contact. some degrees of biomodification occur, at least in daphnids (oberdörster et al., 2006a; roberts et al., 2007; baun et al., 2008; filella et al., 2008), and compartmentalization of nanosized contaminants in selected tissues and intracellular organelles has been documented (moore et al., 1997; leeuw et al., 2007; tortiglione et al., 2007; ingle et al., 2008; koehler et al., 2008; oughton et al., 2008; tedesco et al., 2008). invertebrates largely enter the food chain mainly at intermediate levels, and represent a powerful vehicle for recycling pollutants deposited in the sediments. as predator of bacteria, plants, algae and other invertebrates, or feeding on substrates, they become the preferential prey of larger organisms: humans, fish and birds, which, in turn, represent a great deal of the human diet. the risk for humans is possibly related to the doses, therefore, bioconcentration, bioaccumulation and biomagnification raise a matter of concern. their rates, clearance time and fate of the contaminants through the food chain should be measured whenever possible (allen et al., 2005; rocha et al., 2005; emerich and thanos, 2006). studies on ecotoxicological risk assessment for nanomaterials should follow rules and protocols accepted for previously untested chemicals; whenever possible comparison with comparable bulk materials should be considered. the weak stability of nanocolloids represents a problem to be solved: a comprehensive and recent paper in this particular issue reports the generally accepted approaching scheme (crane et al., 2008). briefly, a tired protocol evaluates first acute lethality tests of contaminated waters and sediments on simple organisms: bacteria, monocellular algae and invertebrates. long term tests should follow, if convenient, and data should be predictive of lethality, growth, reproduction and offspring viability. organisms of greater complexity (from bacteria to algae, invertebrates and eventually fish and birds) will be included as needed by the main aim of the study. the preference should be given to standardized test and to test species ecologically representative and well adapted to standard laboratory conditions: the control agencies for environmental contaminant surveillance schedule tests on daphnids, in addition to bacteria, algae and 81 fig. 1 percentage of nanotoxicological studies performed in different test media and species. fw: freshwater; sw: salt (marine or artificial sea) water. other*: liquid media (c. elegans), foods (d. melanogaster) fish, as key step for assessing aquatic toxicity. other invertebrates are recommended as standard test organisms to test benthic waters, sediments, soil, ingestion or skin contact, or for artificial food web analysis (crane et al., 2008). once the material under investigation has shown aquatic toxicity and a trend to accumulate in soils and sediments, tests on these substrates should follow. finally, a set of experiments on organisms playing a higher role in the alimentary chain should be appropriate, in the case of suspected or documented bioaccumulation or biomagnification interesting the food chain. invertebrates used in toxicity studies on nanomaterials: main characters and habitats this section will present the features which characterizes the species of invertebrates which underwent nanotoxicity assessment up until today and the rational for their selection. as shown in fig. 1, species entering nanotoxicity studies are few and unevenly distributed in different environmental samples. test on the water column are prevalent, mainly conducted on daphnids and other cladocerans, while studies on sediments are neglected, despite the emphasized trend of aqueous nanocolloids to be unstable and precipitate. again, in some cases practical reasons (kit availability, lab expertise, others) seems to prevail on recommended protocols in determining the choice of test species. the main taxonomic classification of invertebrates considered in the studies on nanotoxicity is presented in table 2, together with codes of standard tests validated by at least one of the control agencies listed in the legend of table 1. testing the water column: pelagic and littoral organisms tests on water column or water-suspended sediments should be performed in standardized media. commercial mixtures, like those used for aquaria or provided in kits, should be preferred to comparable solutions obtained at the lab bench by mixing high-grade chemicals in demineralized water. these solutions, in fact, are expensive, poorly reproducible and at higher risk of leaving unsafe concentrations of heavy metals in the test medium (ecetoc, 2003). control agencies recognize daphnids (daphnia magna, daphnia pulex and ceriodaphnia dubia) as “first choice” test organisms for validated ecotoxicological tests (joncxyk and gilron, 2005). the daphnia genomics consortium (dcg, http://daphnia.cgb.indiana.edu) promotes sequencing projects of the entire genome of daphnia sp. as model organisms for ecology. these small crustaceans, widely distributed in all aquatic habitats with the only exception for extreme environments, show all the features mentioned before as highly convenient for testing. if maintained in optimal conditions (ph = 7.2-8.5; t = 20 °c; hard water=160-180 mg/l or 80-90 mg/l caco3, related to species), daphnids adapt quickly in the laboratory. stereotype behaviors, which involve filtration feeding, jerky motion in swimming and anti-photo tactic movements, are in part genetically determined and appear to be sensitive to chemical pollution. 82 table 2 list of invertebrate species used in testing nanoparticles toxicity the tests approved by control organisms and test codes, whenever available, are reported (see even burton et al., 2003; crane et al., 2008). asterisks: not yet validated by control agencies. species class order codes of validated tests approved by astm/epa/oecd/eu/i so freshwater crustaceans: daphnia magna and d.pulex, caeriodaphnia dubia acute: epa850.1010; epa821r02.013, oecd202, astme-1209501. chydorus sphaericus* branchiopoda diplostraca sublethal: epa850.1300; oecd211, astme1193-97, astme-12095-01 all (only daphnids) crustaceans: thamnocephalus platyurus* branchiopoda anostraca none rotifera: brachionus calyciflorus monogononta ploimida acute: astme-1440-91 sublethal: astme-2317-04 astm/epa cnidaria: hydra attenuata hydrozoa hydroida astm: stp921-eb astm/epa molluscs: elliptio complanata* bivalvia unionoidea none salt (estuarine, sea water) crustaceans: harpacticoida copepods maxillopoda harpacticoida astme-2317-04, oecd 254 astm/epa/oecd molluscs: mytilus edulis bivalvia mytiloidea astme-2122-02, epa850.1050 astm/epa/oecd/eu freshwater sediments crustaceans: hyalella azteca malacostraca amphipoda astme-1706-00, oecd 251, epa850.1735, epa600/r99.064 astm/epa/oecd worms: lumbriculus variegatus oligochaeta lumbriculida astme1688-00, epa 823-f-00-002; oecd2007: new proposal astm/epa/oecd sea water sediments crustaceans: leptocheirus plumulosus malacostraca amphipoda astme1367-99, epa850.1735; epa 600/r01/020, oecd 252 astm/epa/oecd soil earthworms: eisenia sp. oligochaeta haplotaxida astme1676-04 (toxicity and bioaccumulation); epa850.6200; oecd207/211 (acute/chronic) all potworms: enchytraeus crypticus oligochaeta enchytraeidae astme1676-04 (toxicity and bioaccumulation); iso 16387:2004; oecd 207/fkz: 204 67 458 astm/iso/oecd crustaceans: porcellio scaber* isopoda oniscidea none other nematodes: caenorhabditis elegans* chromadorea rhabditida model organism arthropods: drosophila melanogaster* insecta diptera model organism 83 short life cycles and amictic reproduction are the rule: deposition of non-resting eggs in the dorsal brood chamber (from which juvenile female are released) gives rise to large, rapidly evolving clones. a great level of body transparency permits noninvasive exploration of brood chamber and of gut content. sexual cycle follows non favorable changes in natural habitats (freezing, drought and others): 12 resting eggs are deposited in the ephyppium, a detachable modification of the carapace, and male individuals are produced, whose sperm is haploid, tailless but motile. sexual cycles are more frequent in c. dubia (ebert, 2005). commercial kits, based on standardized acute and chronic toxicity tests on daphnids, contain “cysts” (ephyppia) and use hard freshwater as the test medium (persoone et al., 1994; centeno et al., 1995; ruck, 1998; lazorchak et al., 2008). testing the water column: pelagic (or littoral)/ benthic organisms chydorus sphaericus is a daphnid-like cladoceran whose small spherical body is transparent. the dorsal brood chamber is present only in adult females, and reproduction is mainly amictic. feeding and swimming patterns are similar to those described for daphnids. its ability to change habitat from pelagic to bentic confers to him the property of an useful alternative to daphnid testing, used since 1991 (havens, 1991). while not yet approved by control agencies a test for acute toxicity on c. sphaericus has been recently proposed (pieters et al., 2008; velzeboer et al., 2008). copepods are other organisms in this group, perhaps the dominant ones, at least for the very large number of species and the ability to colonize a wide range of aquatic ecosystems. studies on nanomaterials have been performed only in marine species, by far the most numerous species. different from daphnids, their body is segmented and poorly transparent. pelagic larvae (nauplia) develop slowly (within months) into sexually (male and female) differentiated adults. the life span is comparably long and variable between species. no resting eggs are produced, but in unfavorable environmental conditions adults enter diapause. benthic habits are restricted to the adult life. swimming and crawling-swimming movements on substrates are powerful (90 m/h). copepods feed on suspended particles (like algae, bacteria and organic matter), and are validated test organisms for ecotoxicology (anderson et al., 2001). thamnocephalus platyurus is a big freshwater crustacean, with a segmented and poorly transparent body. while two distinct sexes have been described (byron and ponder, 1949), resting eggs can be produced. a commercial kit supplies this material to perform acute studies. however, the test time (24 h for hatching plus 1 h for in vitro exposure to contaminants) is too short to allow a full maturation of the larvae (williams, 2007). the test is widely used, but not included in standard validated methods (centeno et al.,1995). other benthic organisms are useful for longterm toxicity studies, such as the cnidarian hydra attenuata, a freshwater, relatively small organism. lacking a mesoderm hydras belong to the group of diploblastic animals; their tubular body develops a head, terminated by an oral apparatus with a crown of tentacles, and a basal disc (adherent to the substrate) at opposite extremities. reproduction proceeds by clones; they are generated by budding from the distal third of their body, near the basal disc. a gradient of activators regulates head formation and regeneration by morphallaxis, and inhibits budding in younger individuals. differentiation into true hermaphrodite and sexual reproduction are induced by unfavorable conditions. toxicity tests have been described: clubbing movements of tentacles are early signs of exposure to toxic agents, before reproductive changes or death (davies and freeman, 1995; blaise and kusui, 1997; holdway, 2005). hydras are interesting models for aging: no tissue aging processes or age-related enhanced mortality have been observed in hydras over a period of 4 years. however, some degeneration follows the induction of sexual reproduction, at least in some species, but not in h. attenuata (martinez, 1998; austad, 2009). chronic toxicity was assessed on the bivalves, mytilus edulis (blue mussel) and elliptio complanata, two species feeding while filtering large amounts of sea and freshwater, respectively. the model was restricted to quite large, long living and mainly sessile organisms. the sea species is long 3-7 cm, and inhabits the intertidal, infra and circa-littoral zones. e. complanata, whose habitat is restricted to freshwater, can reach 25 cm in length and 10 years of age. both species reproduce sexually. their importance as test organisms for ecotoxicology is linked to the role played in the food chain. the rotifer brachionus calyciflorus was studied under exposure to nanomaterials as a component of a simplified food web: algae and bacteria were predated by a protozoan, which in turn was the rotifer prey; both fed fish (holbrook et al., 2008). testing artificial food web containing rotifers is a validated procedure (snell, 2005). the acute test has been validated, and a commercial kit for acute (24 h) and short-chronic (48 h) toxicity tests is available: the kit supplies resting eggs and the test is performed on newly hatched larvae (persoone et al., 1993). brachionidae are eutelic, small loricate organisms, with one-pieced, thin and transparent lorica, a small corona (the oral apparatus), a single gonad, and a long “foot” used for anchoring. both free swimming and sessile forms are present. amictic and sexual reproductive cycles can alternate in relation to environmental conditions. in favorable conditions, non-resting eggs externally attached to the root of the maternal foot hatch within 12 h. immediately before periods of freezing or drying, small defective males lacking a digestive system appears. insemination of females leads to deposition of resting eggs, from which a new generation of diploid females hatches when environmental conditions improve. the life span is short (ca. 2-3 weeks), but the total number of offspring is large. 84 testing sediments: fresh-water and marine benthic organisms these animals inhabit the deepest zone of water bodies and are useful to study sediments, on which they are feeding. three species have been tested in nanoecotoxicology, all representing validated test organisms. hyalella azteca (family: hyalellidae) and leptocheirus plumulosus (family: aoridae) are small shrimps inhabiting freshwater and estuarine sediments respectively, and feed on sediments or suspended particles. sexual reproduction is the rule, with two dimorphic sexes (larger males). tests for acute and chronic toxicity are performed in younger individuals and the experimental time should not exceed 4 weeks for h. azteca and 10 days for l. plumulosus (borgmann et al., 2005). the worm lumbriculus variegatus (annelida, oligochaeta, lumbriculidae) inhabits freshwater sediments in ponds, lakes and marshes, where it feeds on decaying vegetation and microrganisms. it can be occasionally found in silty sediments in deeper waters. sexual reproduction begins between true hermaphrodites, with formation of transparent cocoons containing 4-11 fertilized eggs. small worms hatch in 2 weeks without a previous larval stage. in laboratory conditions, the worms are smaller (4-6 cm), never reach sexual maturity and reproduce by spontaneous asexual fragmentation followed by rapid regeneration of a complete worm from surviving fragments. regeneration is also described in natural conditions, fragmentation is a rapid and common response to injuries, like body compression. its use as a test organism in ecotoxicology is validated (epa 823-f-00-002). testing soils: terrestrial organisms the earthworms eisenia sp. (eisenia foetida and eisenia veneta, annelida, oligochaeta, lumbricidae) and the potworm enchytraeus crypticus (oligochaeta, enchytraeidae) strictly depend on soil, as their habitat and food source, are easy available, growth rapidly and enter the food chain through fish and higher vertebrates. earthworms and potworms have a segmented body with cephalo-caudal symmetry. the cephalic portion ends in a clitellum, characterizing sexually mature individuals. toxicity tests, however, require younger individuals. both groups are true hermaphrodite, each individual developing a complete male and female set of reproductive organs, with two pairs of testes and one pair of ovaries. while able to completely regenerate from fragments if injured, sexual reproduction is the rule (dominguez et al., 2003). eisenia sp. and enchytraeidae are validated test organisms for soil ecotoxicological studies (epa850.6200; oecd207/211; iso 16387:2004; egeler et al., 2009). the isopod porcellio scaber is another terrestrial organism. it is heavily pigmented and has a ventral brood pouch which characterizes sexually mature females. it hosts fertilized eggs (by sexual reproduction with internal copulation) and hatched larvae as only young complete individuals are released. this crustacean is common in the wooded soil; its diet is mostly on vegetal debris. life span (up to 3 years) and reproduction rates are sensitive to a number of environmental changes and pollutants. the oecd recently released updated guidelines for testing the bioaccumulation of chemicals in soils, using earthand pot-worms as test organisms (oecd 2009c). p. scaber, instead, is a test species not yet validated; however, it is the only terrestrial crustacean useful for soil pollution testing. model organisms: caenorhabditis elegans and drosophila melanogaster studies on the toxicity of nanomaterials have been performed on the larvae of two species considered to be model organisms for developmental and cell biology and genetics: the nematode c. elegans and the fruit fly d. melanogaster. d. melanogaster is perhaps the prototype of model animal organisms and its genome has been completely sequenced (adams et al., 2000). it is a small fly, with sexual dimorphism and sexual reproduction. eggs mature outside the female body; larvae development is by stages. the soil nematode c. elegans is easily cultured in laboratory conditions on agar plates. viable worms can even be stored in frozen stocks indefinitely. the worm body is tiny (about 1 cm long) and transparent, eutelic (100 cells ca) and moving with swimming motion. two sexes are described: a self-fertilizing true hermaphrodite, with two intra-abdominal gonads, one producing sperm, the other eggs. it is able to give rise to about 300 larvae in its life course. males are occasionally and rarely produced (1/103). they are slightly smaller than hermaphrodites, from which they differ for the presence of the only male gonad in the abdomen and the shape of the tail, carrying the organ for the internal copulation, not always followed by fertilization. sex is genetically determined (hermaphrodites are xx, males x0), however environmental or dietary changes can convert hermaphrodites born from mating into males, but it never occurs with amictically produced hermaphrodites (hart, 2006; the c. elegans sequencing consortium, 1998). toxicity of engineered nanomaterials to invertebrates while natural and engineered nanomaterials can overlap and share common properties, this review has been limited to manufactured nanomaterials and to the related ecotoxicological risks. nanotoxicology studies, in which the invertebrate plays a significant while intermediate role, remain poorly and unevenly distributed, in spite of great attention reserved to nanotoxicity in humans, cultured cell lines and vertebrate organisms (chun ke and qiao, 2007; duffin et al., 2007; farrè et al., 2009; shvedova et al., 2009; di gioacchino et al., in press; gornati et al., in press). environmental nanotoxicology remains almost confined to the aquatic environments, mainly freshwater, while studies on contaminated sediments and non aquatic environments are scarce, accounting altogether for 30 % of the total, 85 fig. 2 percent of ecotoxicological studies on different nanomaterials. fullerene: c60 and carbon nanotubes (cnt); metal oxides: tio2 and metal oxides plus tio2 plus (*), qds: quantum dots; others: organic (sucrose polyester oil, polymethyl-methacrylate, nipam/bam) and inorganic (upconverting phosphors, glass wool, nanometals) nanomaterials not included in the chemicals classes mentioned before. including observations on model organisms maintained in artificial aqueous medium (c. elegans and d. melanogaster). the most studied test species by far are daphnids (d. magna, d. pulex and c. dubia) (fig. 1). the most studied materials are fullerenes and metal oxides that altogether represent 70 % of the available literature in this field (fig. 2). fullerenes carbon nanomaterials captured great attention for both historical reasons and convenience. fullerenes are organized as nearly spherical nanoparticles, the so called buckminsterfullerenes (nc60, nc70, and fullerols, their hydroxylated derivatives) or as single, double or multi-walled carbon nanotubes (swcnt, dwnct and mwcnt, respectively). these materials can be toxic for higher organisms: they are able to cross the membrane of eukaryote cells, accumulating in lysosomes and mitochondria, as well the bloodbrain barrier, reaching the olfactory bulb in fish and mammals via the olfactory nerve (oberdörster, 2004; oberdörster et al. 2005; tin-tin-win et al. 2008). moreover, in human cell cultures, swcnt determine changes in the expression of several genes, mainly involved in apoptosis (cui, et al., 2005; sarkar et al., 2007). fullerenes have a very wide application range, from cosmetics to computer engines, and tons are produced yearly. their waste in the environment is significant and safety is poorly granted by their hydrophobicity, which partially prevents miscibility with superficial water (heymann et al., 1996; deguchi et al., 2001; fortner et al., 2005). while protective effects of fullerenes against other more powerful contaminants were sporadically described (baun et al., 2008), the potential risk of heavy pollution with fullerene poses major environmental concern and need for studies on adequate models, invertebrates among others. the toxicity of fullerene in invertebrates (table 3) is variable and related not only to nanoparticle properties, but even to the methods adopted for suspension in water and to the test species. the link between lethality rates and methods for obtaining suspension was determined for nc60 in d. magna, perhaps the most sensitive species. suspensions prepared by long stirring alone seemed to be safe: the cmax ≅ 35 ppm did not reach the lc50 even when the test time was prolonged to 96 h. stirring plus sonication reached an intermediate toxicity, while the solution retaining some traces of thf, added as solvent, was the most toxic. this toxicity apparently is not linked to the residual solvent as, in the absence of fullerene, traces of thf were not toxic (lovern and kapler, 2006; oberdörster et al., 2006a; zhu et al., 2006; lovern et al., 2007; spohn et al., 2009; zhu et al., 2009). lc50 was higher for d. pulex while the test time in this case was shorter (klaper et al., 2009). other species like t. platyurus, h. azteca and harpacticoida copepods, seemed to be more resistant and did not show acute toxicity (oberdörster et al., 2006a; blaise et al., 2008). fullerenes added to soils seemed to be ineffective on survival rates of earthand pot-worms (baird 2007; scott-fordsmand et al., 2008). d. magna appeared to be the most sensitive species even to sublethal effects. exposure to nc60 86 table 3 summary of lethality data for fullerenes lc50: median lethal concentration; cnt: carbon nanotubes: sw: single walled, dw: double walled, mw: multi walled. (10 ppm, 48 h) increased the heart rate and amplified (and partially disrupted) stereotypical movements in swimming and feeding. fullerols had only fable effects, reversible within 30 min (lovern and klaper, 2006; lovern et al., 2007). long-term (21 days) exposure to stirred nc60 solutions reduced reproductive rates. moreover, the final population was significantly reduced, despite some degree of adaptation (noec = 1.0 ppm, loec = 2.5 ppm; oberdörster et al., 2006a). oxidative stress was observed in d. pulex (klaper et al., 2009). chronic exposure of h. attenuata to nc60 was associated with clubbing and retraction of tentacles (ec50 < 10 ppm, 96 h) (blaise et al., 2008), while d. melanogaster exposed during the larval stages showed only slight effect at smart (somatic mutation recombination test) on wing cells (loec = 2.24 ppm, noec = 0.45 ppm). fullerols were not effective (noec = 2.46 ppm) (zakharenko et al., 1997). nc60 (0.1-1 % in soil) did not affect reproduction in the potworm enchytraeus crypticus (baird, 2007), but was able to inhibit cocoon formation in e. veneta (scott-fordsmand et al., 2008). bioavailability and bioaccumulation of nc60 were measured in d. magna. the maximum intake of carbon was greater than 2 ppm/mg of tissue after a 48 h exposure (oberdörster et al., 2006a), mainly localized in the gut. a near to complete excretion of carbon clumps organized at micrometer level was recorded after 48 h clearance, and no bioaccumulation of nanoparticles seemed to occur (baun et al., 2008). the complete clearance was accompanied by a complete recovery from toxic effects (lovern et al., 2007). the toxicity of cnt seemed to be lower, or even absent, mw proved to be generally more aggressive than dw and swcnt, which moderately affected the mortality rate of d. magna fed on algae. however, swcnt coated with phospholipoproteins (pl-swcnt) protected d. magna from mortality due to starvation. cladocerans seemed to be able to metabolize the lp-swcnt: when starved, they ate the lipid coating, causing aggregation and precipitation of nanotubes. nanocarbon, mainly internalized in the gut within 45 min, was completely excreted in clumps of amorphous carbon after 20 h, or sooner if fullerene species lc50 time notes d. pulex 0.5-5.0 (lc30/75) 24 h nc60 in thf 0.8, 0.46 ppm nc60 stirred/sonicated 7.9, 10.51 ppm d. magna >35 ppm 48-96 h d. pulex lc40 = 100 ppm 24 h harpacticoida copepods never reached 48-96 h h. azteca never reached 96 h nc60 stirred t. platyurus never reached 24 h no mortality effects e. veneta never reached no mortality effects nc60 added to soil e. crypticus never reached no mortality effects nc60/70 d.pulex never reached 24 h no mortality effects fullerols d.pulex lc40 = 100 ppm 24 h d.magna 2.42 ppm 48 h t. platyurus 24 h h.attenuata 96 h a. tenuiremis 35 days l.variegatus 28 days swcnt d.melanogaster never reached no mortality effects dwcnt e. veneta never reached 28 days no mortality effects d .magna 22.75 ppm 48 h c. dubia 7 % survival at 40 ppm 48 h h. azteca >264 ppm l. plumulosus 68 ppm 10 days mwcnt l.variegatus never reached 28 days no mortality effects 87 table 4 summary of sublethal toxicity for fullerenes fullerene species noec dose time effect 0.18 ppm loec = 0.26 ppm 60 min swimming and feeding movements d. magna 1.0 ppm loec = 2.5 ppm 21 days reduced reproduction d. pulex 20 ppm loec = 100 ppm 24h oxidative stress h. attenuata ec50 < 10 ppm 96 h morphological changes e. veneta 1000 mg/kg d.w.f. weight gain reduction (20 %) cocoon formation, not hatchability (by 78 %) enchytraeus crypticus 2000 ppm 14 days reproduction not affected nc60 stirred 0.45 ppm loec = 2.24 ppm 2.46 ppm d.melanogaster 24 ppm smart fullerols d.pulex 7.5 ppm loec = 20 ppm 24 h oxidative stress nc60/nc60 d.pulex 5.0 ppm loec = 7.5 ppm 24 h oxidative stress h.attenuata ec50: 10-100 ppm 96 h swcnt a.tenuiremis 10 ppm 35 days ec50: 94 mg/kg d.w.f. 28 days weight gain reduction (20 %) dwcnt e. veneta ec10 = 37 and ec50 = 176 mg/kg d.w.f. 28 days cocoon formation, not hatchability (by 60 %) ec50: median effective concentration; noec: non observed effect concentration; loec: lowest observed effect concentration; cnt: carbon nanotubes: sw: single walled, dw: double walled, mw: multi walled; smart: somatic mutation recombination test; d.w.f.: dry weight food. algae was provided for feeding (roberts et al., 2007). exposure to maximal concentrations of swcnt did not affect mortality rates in t. platyurus (24 h), h. attenuata (96 h) and in copepod amphiascus tenuiremis (28-35 days) (templeton et al., 2006; leeuw et al., 2007; blaise et al., 2008). clubbing of tentacles were present in h. attenuata exposed to sublethal doses (ec50: 10-100 ppm, 96 h; blaise et al., 2008). a. tenuiremis tolerated without side effects the presence of purified swcnt (≤10 ppm) along its entire life cycle (up to 35 days). mortality and fertility rates, sex ratio and viability of the offspring were not affected, and a delay of 1 day in offspring development was the only side effect registered. however, the presence of fluorescent byproducts of fullerenes in the solution, possibly related to procedures adopted to obtain suspension, led to an 80 % mortality rate (templeton et al., 2006). d. melanogaster larvae well tolerated the ingestion of swcnt mixed with yeast feeding paste, and the mortality and fertility rates were not affected, notwithstanding the occurrence of nanocarbon accumulation in tissues and fluids (leeuw et al., 2007). dwcnt (10-30 nm diameter, 5-15 µm length) were tested on e. veneta. nanotubes were mixed with food (50-495 mg/kg dry weight food, d.w.f.) and administered to worms according to standard protocols (10 g mixed food every 7th day for 28 days, astme1676-99, epa850.6200; oecd211). no lethality was observed, but the exposure to the highest dose reduced weight gain (ec10 = 94 mg/kg d.w.f.) and cocoon formation (ec10 = 37 and ec50 = 176 mg/kg), but not hatchability. mwcnt retained the highest toxicity, among different types of cnt: lethality rate was enhanced in c. dubia, h. azteca and l. plumulosus (kennedy et al., 2008, zhu et al., 2006). the only exception was d. magna (lc50 = 22.75 vs 2.42 ppm, mw vs swcnt, 48 h). these values were obtained in a recent work, in which disagreement with previous results were commonly observed with all the materials tested (tables 3-5): the reason, as discussed by the authors, could be found in the different modalities for suspension preparation, and especially in continuous stirring and shaking of test medium during the exposure period (zhu et al., 2009). hydroxylation, carboxylation and coating with natural organic matter (nom) reduced mortality rate, at least in c. dubia. survivors showed anomalies of the carapace, to which nanoparticles can adhere (baun et al., 2008). carbon accumulated mainly in the gut; it was completely excreted after 24 h washing-out and refeeding. (kennedy et al., 2008). 88 table 5 summary of lethality data collected for metal oxides organized at nanoscale nano-compound test species and medium lethality reference tio2: <20 nm 25-30 nm d.magna d magna d.pulex c.dubia c.elegans p.scaber 48 h lc50 = 143 ppm 48 h lc50 = 5.5 ppm 48 h lc50> 10 ppm 48 h lc50> 10 ppm 24 h lc50 = 80 ppm 14 d noec >1000 ppm zhu et al., 2009 lovern and klaper, 2006 griffitt et al., 2008 wang et al., 2009 drobne et al., 2009 al2o3 (60 nm) c.elegans 24 h lc50 = 82 ppm wang et al., 2009 ag oxide (20-30 nm) d.magna d.pulex c.dubia 48 h lc50 = 0.04 ppm 48 h lc50 = 0.067 ppm griffitt et al., 2008 al oxide: 20-30 nm; or 51 nm d.magna d.pulex c.dubia 48 h lc50> 162 ppm 48 h lc50> 10 ppm 48 h lc50 = 3.99 ppm zhu et al., 2009 velzeboer et al., 2008; griffitt et al., 2008 co oxide (10-20 nm) d.pulex c.dubia 48 h lc50> 10 ppm 48 h lc50 = 1.67 ppm griffitt et al., 2008 cuo: 30 nm, or 15-45 nm d.magna t. platyurus d.pulex c.dubia 48 h lc50 = 3.2 ppm 48 h lc50 = 2.1 ppm 48 h lc50 = 0.06 ppm 48 h lc50 = 0.419 ppm heinlaan et al., 2008 griffitt et al., 2008 ni oxide (5-20 nm) d.pulex c.dubia 48 h lc50 = .89 ppm 48 h lc50 = 0.674 ppm griffitt et al., 2008 sio2 (205>4,700 nm) d.magna 48 h, lc70 = 10 ppm adams et al., 2006 zno: 20 nm 50-70 nm 480>4,000 nm d.magna d.magna t. platyurus d. magna c.elegans 48 h lc50 = 1.5 ppm 48 h lc50 = 3.2 ppm 48 h lc50 = 0.18ppm 48 h lc73 = 0.2 ppm 24 h lc50 = 2.3 ppm zhu et al., 2009 heinlaan et al., 2008 adams et al., 2006 wang et al., 2009 cuznfe2o3/indium tin oxide/ho2o3 t. platyurus h. attenuata 48h, lc50 = 0.1-1.0 ppm 96 h, ec50 = 10-100 ppm blaise et al., 2008 niznfe2o3/o3sm2 / er2o3 t. platyurus 48h, lc50 = 1-10 ppm blaise et al., 2008 srfe12o19/tio2/fe5o12y3 t. platyurus 48h, lc50 = 10-100 ppm blaise et al., 2008 kinetic of intake and depuration rates after ingestion of mwcnt (diameter: 30-70 nm) and swcnt (1-2 nm diameter) mixed with sediments were studied in l. variegatus. data were expressed as basfs (biota–sediment accumulation factors, calculated as the ratio of the concentration of a substance in an organism normalized by the organism lipid fraction to its concentration in the sediment normalized by its organic carbon fraction, mg/g dry sediment (petersen et al., 2008b). at the time (7 days) of first observation, the accumulation had reached maximal levels and remained stable for the entire 28 days period of observation. values were slightly larger for mwcnt (0.40 vs 0.28 mg/g dry sediment). mortality did not increase; sub-lethal toxicity was not tested. clean sediments added to water accelerated the clearance, nearly complete in about 2 days, and still incomplete after 60 h in clean water without sediments (petersen et al., 2008b). a similar work performed in e. foetida gave similar results (petersen et al., 2008a). metal oxides these compounds behave differently from fullerenes. particle and aggregate size are more uniformly distributed, and their hydrophobicity is generally lower, allowing easier standardization in preparing aqueous suspensions. table 5 shows lethality data for metal oxides. it is evident that tio2 is by far the most studied among this group of compounds (fig. 2). two freshwater invertebrates, t. platyurus and h. attenuata, permitted to group metal oxides into three toxicity degrees: cuznfe2o3, indium-tin oxide and ho2o3 scored highest, niznfe2o3, o3sm2, and er2o3 retained an intermediate toxicity level, while tio2, srfe12o19, and fe5o12y3 scored the lowest. moreover, compounds listed in the two last groups did not show toxic sub lethal effects on h. attenuata (blaise et al., 2008). when metal oxides (size range: 5-50 nm) were compared in different organisms, the toxicity degree was ag> cu> ni> co = al = ti in d. pulex, and ag> cu> ni> co> al> ti in c. dubia. nanoparticles toxicity in comparison with that of the corresponding bulk salts gave contrasting results in different species: in aquatic organisms (d. pulex, c. dubia) toxicity was greater for nanoparticles, in c. elegans for bulk salts (griffitt et al., 2008; wang et al., 2009). the toxicity degree of larger particles (>200 nm) was zno> sio2> tio2 in d. magna 89 (adams et al., 2006), partially confirmed in a recent study which found the toxicity degree to be zno> tio2> al2o3 (zhu et al., 2009). highly sized (>500 nm) tio2, alo2 and ceo2 did not retain toxicity against c. sphaericus: the solutions containing the higher concentrations (10 and 100 ppm) however were cloudy and unstable (velzeboer et al., 2008). reproduction was negatively affected in c. elegans (wang et al., 2009). here again, not only the chemical species, but also the modalities of suspension preparations and the size of nanoparticles were influent on toxicity. preparing tio2 suspension by filtration lead to ultra fine particulate (25-30 nm): this suspension is much more toxic than those obtained by sonication (particle size: >100 nm). particles larger than 100 nm are poorly or nontoxic for d. magna, t. platyurus and c. sphaericus (adams et al., 2006; heinlaan et al.,2008; lovern and klaper, 2006; warheit et al., 2007; blaise et al., 2008; velzeboer et al., 2008). sublethal toxicity tests were performed in two crustaceans, d. magna, inhabiting freshwater, and p. scaber, adapted to wooden soil. filtered tio2 particles (30 nm) did not alter stereotypical movements of d. magna after 60 min exposure to the loec = 2.0 ppm (lovern and klaper, 2006; lovern et al., 2007). an immobilization test of d. magna after 48 h exposure to 100 % anatase tio2 particles (30 and 100 nm) gave unclear results: smaller and photo-catalysed particles seemed to be more toxic, but statistical difference was never reached (hund-rinke et al., 2006). nanotio2 (10, 100 and 1,000 ppm) was not toxic when mixed with food for 14 days to p. scaber. enhanced feeding rate, assimilation efficiency, levels of catalase and glutathione-s-transferase where observed: these effects appeared earlier at higher dose (3,000 ppm). again, sonicated and bigger particles were ineffective (jemec et al., 2008; drobne et al., 2009). quantum dots, qds in addition to a number of possible interesting uses in optical and computer sciences, quantum dots represent a flexible and interesting dye for living cells and organisms. their toxicity, bioaccumulation and clearance efficiency in invertebrate organisms have been tested in only a few species: c. dubia, h. attenuata and elliptio complanata, in addition to a simplified food web including rotifers. the potential for lethality of qds seems to be determined by their metal core, its position inside the shell and dissolution rate. qds with a cdse crystalline core of 4 nm inside a shell of zns coated with organic polymer (total size: 15-20 nm) and registered qds (545 itk carboxyl quantum dots) were diffusely internalized in c. dubia, whose body, and especially the gut, showed intense fluorescence. the toxicity was nevertheless absent (noec = 600 or 110 ppt, respectively; bouldin et al., 2008; ingle et al., 2008). e. complanata was sensitive to toxic effects of qds with cdte (instead of cdse) crystal core. mussels, collected in the field and exposed for 24 h to qds dispersed in tanks of freshwater, showed lipid peroxidation of gills and gut, reduced viability and immune activity of hemocytes (ec50 = 2-4 ppm; gagné et al., 2008). qds formed by cdse core asymmetrically sited inside a rod-shaped shell of cds induced nonsynchronous tentacle retraction, a behaviour anticipating the beginning of sexual reproduction in h. attenuata. the test substance was not internalized, and the dose-independent effect developed only in the presence of functioning nerves in the test organisms (intact animals or segments of their bodies). the dipole moment retained by asymmetrical qds was probably a key requisite for inducing the anomaly: particles in which the core was symmetrically disposed inside the shell were lacking both qds properties and toxicity (malvindi et al., 2008). identical particles, solubilized by coating with polymers, instead were able to enter the test species: after localization in the head (within 1 h), diffusion to the entire body followed within 24 h. these nanoparticles were able to enter dissected cells of hydra sp. (tortiglione et al., 2007). finally, a simplified food web did not reveal bioaccumulation or biomagnification: e. coli was unable to internalize qds, however those biotynilated or carboxylated adhered to the bacterial surface, and caused cells aggregation. consequently, the ciliate predator (tetrahymena pyriformis) avoided eating aggregates; but internalized qds with water ingestion. neither degradation occurred nor release of toxic metals from the core. the bioaccumulation was low and the excretion rate efficient (t1/2 = 1.5-3.6 days). the biomagnification in the rotifer predating ciliate was very low (0.29-0.62), the t1/2 = 14-21 h. no toxicity was found in protozoans or in predating rotifers (holbrook et al., 2008). other nanosized materials a pioneering study on material possibly organized at nanoscale was carried out in 1997, using as test substance, a sonicated suspension of sucrose polyester oil, proposed as food additive for humans. droplets were not measured. after having been exposed in vivo and in vitro to this suspension, m. edulis showed signs of oxidative stress, persistent lipofuscin formation and reduced lysosomes membrane stability (moore et al., 1997). nanopt coated with polyvinylpyrrolidone (size: 2.4 nm) acts as an antioxidant agent in larvae of c. elegans at l4 development stage. the mean life span was prolonged in wild-type worms and in short-living mutants mev-1, and the oxidative stress induced by paraquat was partially counteracted at the loec = 5 mm. (kim et al., 2008). m. edulis reacted to 24 h exposure to nanoau (gnp, gold 0 (stable), 13 nm, and 0.75 ppm) with enhanced stress parameters in digestive glands, mantle and haematocytes. paradoxically, gnp partially protected from the oxidative stress due to menadione (tedesco et al., 2008). nanoag (lc50 = 125 ppm, 24 h) accumulated in the gut of d.magna and on antennae in individuals near to death (oberdörster et al., 2006b). nano60co was tested on young adults e. foetida maintained on standard soil. radioactive particles obtained by neutron activation emitted 90 beta and gamma radiation (0.157 kbq/μg) used as a tracer, and were administered mixed with food for 7 days (667 g horse manure containing 87 μg nanoco, or 13.7 kbq, per g). no toxicity was observed, while radioactivity was found in many tissues: spermatogenic cells, clitellum, cocoons and blood among others. a low intestinal clearance followed the depuration period (20 % within 8 weeks): the authors considered this work a pilot study on the kinetics of nanoparticles in biological systems (oughton et al., 2008). upconverted phosphors (ucp) are a promising new class of dyes with application, as an example, in biomedical researches. these particles consist of trivalent ions of lanthanides embedded in a lattice of crystalline chromophores. these last act as an antenna and permit the transfer of one-two photons to the ions, otherwise unable to absorb light. decaying from the excited state, the trivalent ions emit a luminescence characterized by its brightness and long-decay time. ucp sized 150 nm were proved non-toxic within 24 h in c. elegans immobilized with na azide (noec = 0.5 ppm). nanospheres were internalized in the gut, and completely excreted after 2 h only in re-fed worms (lim et al., 2006). glass wool, used to absorb material from floating oil spill barriers, is a form of silica oxide organized in nanowires. those used in the study (diameter: 5-25 nm, length: several microns) accumulated in lysosomes and endosomes of gills, and in mitochondria of the hepato-pancreas of m. edulis as shorter fibres, 100-200 nm and 60 nm, respectively. membrane stability of lysosomes was reduced by 70 % within 24 h, and chronic exposure for 16 days enhanced lipofuscin in the hepatopancreas (koelher et al., 2008). the commercially available polymethylmethacrylate (pmma, diameter: 60 nm-1.08 µm) retained toxicity independent from particle size when tested by the chidotox test, on chidorus sphaericus: (lc50> 100 ppm, 48 h) (velzeboer et al., 2008). two organic nano-copolymer particles used in medicine (poly n-isopropylacrylamide, pnipam and n-isopropylacrylamide-co-n-tert-butylacrylamide, nipam/bam, with three different ratios of the comonomers) were tested in freshwater organisms, d. magna and t. platyurus in addition to primary consumers. the stability of suspension in water was greatly dependent from temperature, in the range 10-25 °c. nipam/bam, 50:50 showed the higher toxicity, and d.magna was even in this case more sensitive than t.platyurus, (noec = <50 vs <200 ppm; loec = 50 vs 200 ppm; lc50 = 61 vs 353 ppm, respectively; naha et al., 2009). conclusion the importance of invertebrates to test the potential toxicity of nanomaterials can be educed from the data discussed in this paper. invertebrate tests should not be considered as opposed to tests on other systems; instead, invertebrates add valuable information, at an intermediate level between prokaryotes and vertebrates, on pollution affecting the environment. moreover, the wide degree of standardization, time and cost effectiveness, and suitability to study acute lethality as well as long-term toxicity represent additional advantages. in conclusion, nanoparticles showed their elusive nature in ecotoxicological studies, leading to partially inhomogeneous results. the chemical nature of the compound, the sensitivity of the test species, the dose and the time scheduling obviously played a significant role. to them, however, the effects exerted by the nanoparticle properties should be added, so that different manipulations of pristine material and suspension generated confusion. the aggregates shape and dimension greatly influenced the results, in strict dependence from test media composition and physical-chemistry variable, ph and temperature among others. (lovern and klaper, 2006; oberdörster et al., 2006, zhu et al., 2006; baun et al., 2008). functionalized surfaces, by coating, or by hydroxylation or carboxylation, generally reduced the toxicity of fullerenes (lovern et al., 2007; kennedy et al., 2008; klaper et al., 2009). even more impressive were the results obtained by introducing apparently innocent variations, like stirring and shaking during the entire exposure time. these simple modifications were associated with unexpected changes (zhu et al., 2009). while metal oxides seemed to retain higher ecotoxicity for invertebrates, followed by buckminsterfullerenes and mwcnt, generalization is difficult and with some degree of arbitrariness. nanomaterial interactions with living systems are quite complicated: they can act as carriers for drugs, toxic chemicals, and dissolved substances, and enhance the bioavailability of other molecules (zhang et al., 2007; sun et al., 2008). daphnid sensitivity in acute testing was high with all tested substances. a dose-dependent response to concentration or exposure time was frequent, but not always present. the concentration was indeed self-limiting, being that these compounds were poorly soluble. chronic toxicity was a rare event, but persistent, and able to affect the population size. in the case of qds, generalization is difficult. the apparent low or even absent toxicity of tested formulations on invertebrates is contrasting with data from other systems (tang et al., 2008; kingheiden et al., 2009). evidences in invertebrates, however, remain quite incomplete. only very recent papers started a systematic study on bioavailability, bioaccumulation, compartmentalization and degradation of qds in exposed invertebrates (jackson et al., 2009; peyrot et al., 2009). biodegradation and bioaccumulation were poorly studied. both fullerene and nano-metal oxide mainly accumulated in the gut, at least in daphnids: a nearly complete faecal excretion of digested bulk material within 20-24 h prevented bioaccumulation, as demonstrated at least in d. magna and c. dubia. great amounts of nanoparticles (fullerene and metal oxide ot nanoag) were instead found accumulated in all tissues of dead daphnids (oberdörster et al., 2006b; roberts et al., 2007; kennedy et al., 2008). they were able to adhere to the carapace of daphnids and copepods, while the link to toxic physiological or behavioral effects was 91 only postulated, not documented (baun et al., 2008). validation protocols for assessing bioaccumulations of soil pollutants in terrestrial organisms have been planned by the oecd (egeler et al., 2009). a need for better standardization and supervision of studies seems urgent, to avoid dispersal of efforts and accumulation of anecdoctical and poorly descriptive results. planning research under a rational of feasibility and finding easy methods is a positive trend and should be encouraged, with the limit that the comparison with different and previous studies will not be compromised, and the conventional protocols fulfilled. another regrettable point is the complete lack of molecular data: 48 % of about 30 genes found overor under-expressed in human cells exposed to carbon nanotubes (cui et al., 2005; sarkar et al., 2007) are conserved in bilateria and the correspondent genes are known at least in the model organisms d. melanogaster and c. elegans. data on gene expression changes caused by exposure to nanomaterials in invertebrates should clarify the modalities of action and the underlying pathogenesis mechanisms; a wide place for new researches seems to be available in this field. acknowledgments m chiriva-internati's stay at the dbsm was partially supported by a cariplo grant. references adams lk, lyon dy, mcintosh a, alvarez pj. comparative toxicity of nano-scale tio2, sio2 and zno water suspensions. water sci. technol. 54: 327-334, 2006. adams md, celniker se, holt ra, evans ca, gocayne jd, amanatides pg, et al. the genome sequence of drosophila melanogaster. science 287: 2185–2195, 2000. allen sn, edge m, sandoval g, verran j, stratton j, maltby j. photocatalytic coatings for environmental applications. photochem. photobiol.81: 279-290, 2005. anderson hr, wollenberger l, halling-sorensen b, kusk ko. development of copepod nauplii to copepodites: a parameter for chronic toxicity including endocrine disruption. environ. toxicol. chem. 20: 2821-2829, 2001. austad sn. is there a role for new invertebrate models for aging research? 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type of tissue repair mechanism. in some species, the capacity to repair tissues is limited to the healing of wounds, but others posses a striking repair capability to replace the entire organs. it has been reported that some mechanisms, namely extracellular matrix remodeling, appear to occur in most repair processes. however, it remains unclear to what extent the process of wound healing is similar to organ regeneration. key words: annelid; wound healing; mechanism introduction the phylum annelid (13,000 living species) is a line of invertebrate life dating back 540 million years. their elongated, segmentated body plan and other body features have allowed annelids to specialize at burrowing through substrates, and to radiate into other ecological niches (class polychaeta segmentated worms in marine environments; class oligochaeta in fresh water and on land; class hirudina leeches parasites). animal species possess different capability to replace lost body parts through regeneration (herlant-meewis, 1964; thouveny and tassava, 1998; brusca and brusca, 2003). some species such as planaria, hydra or starfish can readily regenerate lost organs or body parts. in contrast, other species, such as most vertebrates have limited regenerative capacities. but, all species have some capacity to heal wounds produced by external factors during their existence. many researchers attempted to find the relationship between the process of wound healing and regeneration. there are some indications that these processes might differ at the cellular and molecular levels. in see urchins has been suggested that healing of broken spines occurs by a morphallactic mechanism involving recruitment of differentiated cells, while regeneration of removed spines and pedicellaria occurs by an epimorphic process involving undifferentiated precursors (dubois and ameye, 2001). also in zebra fish mutant (dobdevoid of blastema) was found that it failed to regenerate the ___________________________________________________________________________ corresponding author: mira grdisa division of molecular medicine, rudjer boskovic institute, bijenicka 54, 10 000 zagreb, croatia e-mail: grdisa@irb.hr fin but the response in wound healing was normal (whitehead et al., 2005). recently, it has been shown that both wound healing and regeneration in axolotl are dependent on epithelial/mesenchymal interactions, the formation of the wound epidermis, the restructuring of the extracellular matrix and other cellular/molecular events (roy and levesque, 2006). to see if the cellular mechanisms during wound healing and organ regeneration are similar, the sea cucumber holothuria glaberrima was investigated (miguel-ruiz and garcia-arraras, 2007). animals were lesioned and damaged tissue included muscle, nerve, water canal and dermis. the authors founded that cellular events associated with wound healing correspond to those occurred during organ regeneration. these include: an increase in the number of spherule-containing cells, remodeling of extracellular matrix, formation of spindle-structures that signal dedifferentiation of muscle cells and intense cellular division. thus, it is possible that regenerative limitations in some organisms are due to the absence of particular mechanisms associated with repair or the inability of activating the repair process in some tissues or stages. annelids: wound healing and regeneration annelids have a reputation for impressive regenerative abilities, but this ability varies widely. manny annelid worms are limited in their ability to regenerate anterior body parts, whereas posterior segment regeneration is much more common (bely, 2006). in addition to their ability to regenerate body segments, annelids generally have a marked capacity for wound healing (zoran and martinez, 2009). the remarkable ability of some annelids to reconstruct their entire body require coordinated 192 mailto:grdisa@irb.hr activation of multiple developmental, regenerative and wound healing processes in response to injury. on the other hand, leeches are incapable to regenerate lost segments, whereas they generate an effective response to injury by assembling an extracellular scaffold of proteins that facilitates the restoration of traumatized structures (tettamanti et al., 2004). wound healing in the earthworms could be used as a biomarker for assessing chemical toxicity. wound healing in earthworms is continuing process from open to healed wound with different stages and activities (cooper and roch, 1986). it involves the inflammatory response and various cell types, including immunoactive macrophage-like celomocytes. also the influence on cell membranes, cell division, energy production or use, synthesis of dna or rna and enzymatic pathways should be sufficient to interfere with the complex processes required to heal damaged tissue. wound healing in earthworms lumbricus terrestris (cikutovic et al., 1999) was monitored after cutting of three-sided patch of integument of l. terrestris during 5 days, after exposure to variety chloride compounds. the authors found that both, concentration and duration of exposure significantly reduced wound healing. similar finding was observed earlier (cooper and roch, 1992; ville et al., 1995). suppression of healing could be the consequence of interfering chloride compounds with the membranes of macrophages and other cells that function in healing process. it was found (ville et al., 1995; goven et al., 1993, giggleman et al., 1998) that some organics (polychlorinated biphenyl, pentachlorophenol, chlordane) suppress phagocytosis in earthworm celomocytes, probably by affecting their cell membranes. another chlorinated pesticide, lindane, was reported to reduce rna synthesis in mammals (lewis and adams, 1985; thomas and faith, 1985), which could also interfere with tissue repair during wound healing. cell division, which is also important in the wound healing process, might be affected with pollution in soil (cikutovic et al., 1993). heavy metals, such cd2+ or cu2+ may affect enzyme activity necessary for wound healing. it was shown (chen et al., 2001) that cu2+ interferes with an enzymatic pathway in celomocytes leading to production of superoxide (o2-), which is responsible for killing phagocytosed microorganisms in many animal species. it has been shown that exposure to cd2+ in mammals increased infections (lawrence, 1985), inhibited rna and dna synthesis (daun et al., 1993) and atp utilization (graham et al., 1975), whereas in l. terrestris celomocytes suppressed phagocytosis (roy and levesque, 2006). suppression of the healing process portends pathological effects in wildlife exposed to environmental toxicant if they are wounded during natural activities. healing of lacerations, punctures and abrasions of the integument or digestive tract may be compromised, resulting in microbial infection or parasite infestation. injured animals may be even more susceptible to pathogens if the chemicals that suppress the healing process also affect immunoactive cells responsible for phagocytizing and killing microorganisms. during wound healing the extracellular matrix (ecm) is produced, as well as its structural component, collagen. collagen also plays a major role in the modulation of several cell functions, including adhesion, migration, growth and differentiation (hay, 1991; birk and zycband, 1994; lim et al., 1994). in addition, fibrillar collagens are also involved in numerous processes, including stabilization of tissue shape and form during both vertebrate development and tissue regeneration (birk and trelstad, 1984; ingberg, 1994; fraizer et al., 1996; kletsas et al., 2000; badylak, 2002). collagen fibrils are present in both vertebrates and in lower invertebrates (bradbury, 1958; matsudanakagava and nicholls, 1991; sicot et al., 1997). previously was demonstrated that hirudo medicinalis (annelida, hirudinea) could be a very good animal model for investigation of tissue repair and wound healing (de eguileor et al. 2001, 2004; grimaldi et al. 2010). the body of leech has a simple organization. tissue repair in leeches shows a high degree of similarity to wound healing in vertebrates in biochemical and structural-functional points of view. the wound healing process in leeches can be divided into three stages: inflammation, granulation tissue and scar tissue remodeling. granulation stage in leeches is characterized by re-epithelization, angiogenesis and fibroplasias. during these steps occurs the formation of new epithelium, followed by the blood vessels and then by connective tissues (de eguileor et al. 2004; tettamanti et al. 2004) reorganization of collagen was studying in leech wound healing (tettamanti et al., 2004). after surgical lesion of the leeches it was shown that newly synthesized collagen acting as an extracellular scaffold. it directs and organizes the outgrowth of new vessels and the migration of immune cells towards the tissue lesions. in these animals, the collagen fibrils generated during tissue regeneration, showed similarities to both the structural pattern of collagen bundles and assembly processes observed in several vertebrate systems (fish scales, amphibian skin, human cornea). thus, leeches respond to surgical lesions with the same sequence of wound healing and tissue regeneration events as that described for vertebrate (tettamanti et al., 2005). it was found that the general architecture of leech collagen fibril organization and bundle orientation is identical with the structural pattern of collagen bundles observed in vertebrate cornea (birk and trelstad, 1984). thus, it could be hypothesized that collagen structures, characterized by a striking structural complexity and multifunctional purposes, are anatomical system highly conserved throughout evolution. to support the observation that “nature has followed economic and conservative strategies based on the conservation of a lot of molecules and related functions” (ottaviani et al., 2004), probably, evolution preserved the primitive models because of their excellent functional utility and effectiveness. the phases which could be involved in annelid wound healing is depicted on figure 1. if the cellular mechanisms during wound healing are similar to those occurred during organ regeneration, these report could be expanded with additional data. regeneration of annelids was studied 193 fig. 1 phase during wound healing in annelids in few groups, even at molecular level, with identification of the genes involved in the regeneration process (bely and sikes, 2010). in review bely (2006) has described that the most annelids have the ability to regenerate posteriorly. the ability to regenerate anteriorly is common but less widespread. the molecular mechanisms for regeneration process have not been yet completed. some annelids exhibit regenerative abilities very similar to planarians (bode et al., 1973; newmark et al., 2000), which can completely regenerate a new organism from small body fragment. however, the regeneration mechanisms are thought to be quite different between planarians and annelids. the planarians regenerate via totipotent stem cells (neoblasts) that are widely distributed throughout their bodies (redien and alvaro, 2004). the annelids regeneration is thought to occur primarily by cellular dedifferentiation and redifferentiation, without the contribution of totipotent cells (thouveny and tassava, 1998), but lately more date point on the involvement of stem cells (totipotent cells) in regeneration process (grimaldi et al. 2008, 2010). to better understanding the regeneration mechanisms in annelids, the genes that are expressed specifically during the course of regeneration, were identified on model animal enchytraeus japonensis (myohara et al., 2006). besides the known genes which play the roles in development (e.g genes for ecm, glutamine synthetase, nice-5, glucosidase), the new genes ejrup1-5 upregulated during regeneration, were isolated. after structural analyses of the products of these genes, a variety of putative functions that can be associated with their protein products were noticed. these functions include transportation and binding, transcriptional regulation, protein interaction and cell adhesion and perhaps some of them play an important role in regeneration. using in situ hybridization (niv et al., 2008), a strong expression of glutamine synthetase gene occurs in the blastemal regions of regenerating e. japonensis. strong expression was detectable at the cell layer covering the wound and was found to persist in the epidermal cells during the formation and elongation of the blastema. thus, according the results the authors suggested that e. japonensis glutamine synthetase may play roles in regeneration, nerve function, cell proliferation, nitrogenous waste excretion, macromolecule synthesis and gametogenesis. regenerative phenomenon has been explored also on the echinoderms and many details are shown in the reviews (candia-carnevali, 2006; kondo and akasaka, 2010). most explored model in echinoderm regeneration studies is the process of arm regeneration after lost following traumatic or self-induced amputation (candia-carnevali and bonasoro, 1994, 2001; bonasoro et al. 1995, 1998; candia-carnevali et al. 1995, 1998; patruno et al. 2001) using the feather star (crinoid antedon mediterranea). in this process new structures develop from migratory actively proliferating cells. different type of cells is involved in regeneration, including those that are considered to be stem cells. during regeneration, coelomocytes from coelomic canal and amoebocytes from brachial nerve, migrate to the distal wound area and are involved in regenerative process. from migratory amebocytes is formed a blastema. on the other hand, migratory coelomocytes contribute to regenerate the celomic system. cells proliferate at the blastema, coelomic canals and brachial nerve. since the migrating cells differentiate into new structure of the arm, they are presumably undifferentiated multipotent stem cells. but the knowledge about stem cells in crinoids 194 would be further support with molecular analyses. recently, similar results have been reported on the study of wound healing and arm regeneration in ophiderma longicaudum and amphiura filiformis (biressi et al. 2010). the earthworm provides a unique and valuable model to investigate the mechanism of regeneration because this process is rapid and it regeneration of a complete head and tail requires the reformation of various tissues and organs. to study the head and tail regeneration on annelid perionyx excavatus, the expression pattern of three labial genes (pex-la-01, pex-lab02, pex-lab03) was monitored (cho et al., 2009). the results indicated that these genes were expressed only in the head-regenerating tissues. also, the authors found that the expression of pexlab01 and pex-lab02 was up-regulated, and this indicated their involvement in wound healing and the blastema formation process during early head regeneration. on the other hand, the leeches do not possess ability to regenerate segments posteriorly or anteriorly (hyman, 1940). only some leeches can wound heal (le gore et al., 1971; huguet and molinas, 1996) and undergo limited nervous system repair (von bernhardi and muller, 1995), without any tissue or segment regeneration. data about wound healing in annelids are very obscure. the annelids have a big potential for regeneration of different body part, because they produce unique and potent molecules. probably, it is a reason that they are also investigated as a wound healing agent (matausic-pisl et al., 2010). the elucidation of the annelid regeneration mechanisms is thus expected to provide valuable information that may allow us in the future to explore strategies to enhance the regenerative capabilities in vertebrates. the 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185204, 2009 197 isj 4: xx-yy, 2007 isj 4: 86-91, 2007 issn 1824-307x minireview a review on nutritive effect of mulberry leaves enrichment with vitamins on economic traits and biological parameters of silkworm bombyx mori l. r rajabi kanafi1, r ebadi1, sz mirhosseini2, ar seidavi3*, m zolfaghari4, k etebari5 1plant protection department, agriculture college, isfahan university of technology, isfahan, iran 2animal science department, agriculture college, guilan university, rasht, iran 3islamic azad university, science and research branch, teheran, iran 4plant protection student, agriculture college, bu-ali sina university, hamedan, iran 5sericulture department, natural resources college, university of guilan, iran accepted august 02, 2007 abstract sericulture depends on rearing of silkworm on mulberry leaves; for this reason, silk production has direct relationship with larval growth on mulberry. the quality and quantity of mulberry leaves change due to climatic conditions and field practices. one of the alternative ways of improvement of larval feeding is enrichment of mulberry leaves with supplementary nutrients such as vitamins. many studies are accomplished on the effects of mulberry leaves enrichment with vitamin on economic traits and biological parameters, and this review is the first trial for organization of all available data related to vitamins for elucidation of this topic of science. key words: silkworm; mulberry leaves; enrichment; vitamin; economic traits introduction nutrition plays an important role in improving the growth and development of the silkworm, bombyx mori l. like other organisms. legay (1958) stated that silk production is dependent on the larval nutrition and nutritive value of mulberry leaves plays a very effective role in producing good quality cocoons. seki and oshikane (1959) observed better growth and development of silkworm larvae as well as good quality cocoons when fed on nutritionally enriched leaves. silkworm obtains its entire nutritional requirement from mulberry leaves because this insect is monophagous and can complete the life cycle on mulberry leaves exclusively. studies of ito (1978) determined that generally vitamins present in the mulberry leaves satisfy minimum needs of silkworm but the amount of vitamins present in mulberry leaves varies on the basis of environmental conditions, usage of fertilizers in field and mulberry varieties and other field practices. sengupta et al. (1972) showed that b. mori requires specific essential sugars, amino acids, proteins and vitamins for its normal growth, ___________________________________________________________________________ corresponding author: alireza seidavi islamic azad university, science and research branch, teheran, iran e-mail: alirezaseidavi@yahoo.com survival and also for the silkgland growth. akhtar and asghar (1972) found that vitamins and mineral salts played an important role in the nutrition of silkworm. the effect of vitamin supplementation on the growth of b. mori have been investigated by many researchers (majumdar and medda, 1975; bhattacharyya and medda, 1981; das and medda, 1988; faruki, 1990, 1998, 2005; babu et al., 1992; faruki et al., 1992; khan and saha, 1996; nirwani and kaliwal, 1996, 1998; mosallanejad, 2002; etebari et al., 2004; rajabi et al., 2006a,b). keeping the importance of vitamins on silkworm nutrition in mind, following review was accomplished in order to determinate enrichment efficacy of mulberry leaves by vitamins. a list of vitamin quoted in international research papers are showed in table 1. water soluble vitamins water-soluble vitamins consist of members of the vitamin b complex and the vitamin c. ascorbic acid (vitamin c) ascorbic acid has many important functions in the animal body. it is a powerful antioxidant, protecting against oxidative damage to dna, membrane lipids and proteins. antioxidant activity of 86 mailto:alirezaseidavi@yahoo.com table 1 list of used vitamins and its derivation associated with related location. references location derivative vitamin etebari et al. (2004) iran vitamin c sarkar et al. (1995) bangladesh el-karaksy and idriss (1990) egypt chauhan and singh (1992) babu et al. (1992) gomma et al. (1977) india das and medda (1998) majumdar and medda (1975) bhattacharya and medda (1981) india cyanocobalmin (b12) rajabi et al. (2006b) iran pyridoxine (b6) faruki (2005) bangladesh rajabi et al. (2006a) iran riboflavin (b2) nirwani and kaliwal (1998) khan and saha (2003) india thiamine (b1) faruki (1998) bangladesh thianomin nirwani and kaliwal (1996) india folic acid (b9) faruki and khan (1992) khan and faruki (1990) bangladesh paba khan and saha (1996) bangladesh fe-plus etebari and matindoost (2004) saha and khan (1996) iran niacin (b3) etebari and matindoost (2005) iran multi-vitamin compounds muniandy et al. (1995) india saha and khan (1996) bangladesh evangelista et al. (1997) brazil sarkar et al. (1995) bangladesh b-complex mosallanejad (2002) iran vitamin e ascorbic acid decreases reactive oxygen species and oxidative pressure, and, as a result, the absorption of nutritious substances in the midgut would increase (felton and summers, 1993). ascorbic acid shows a particular behaviour as it is very susceptible to degradation, especially when in solution, and/or exposed to light, oxygen, and free radicals. ito (1961) recorded relationship of ascorbic acid supplementation and growth of silkworm. the absence of ascorbic acid in the diet of first and second instar larvae postponed growth and development of silkworm. there is enough vitamin c in mulberry leaves and ascorbic acid content of growing larvae is dependent on amount of this vitamin in diet. supplementation of mulberry leaves more than any other vitamin ascorbic acid has been used (etebari et al., 2004). several research demonstrated phagostimulatory effect of ascorbic acid for insects (ito, 1978; dobzhenok, 1974). in silkworm a gustatory stimulating activity have been observed to some extent (ito, 1961). gomma et al. (1977) observed that ascorbic acid significantly increased the weight of silkworm larvae. babu et al. (1992) observed that the first and second instar larvae reared on 1.5 % ascorbic acid enriched mulberry leaves resulted in higher silk filament length, weight and denier values. sengupta et al. (1972) reported that silk production increased with 1% ascorbic acid in the diet of silkworm. etebari et al. (2004) demonstrated that feeding on mulberry leaves enriched with ascorbic acid at 3% concentration decreased larval weight due to hypervitaminosis. chauhan and singh (1992) showed that 1% concentration of ascorbic acid could increase the number of eggs in the silkworm. although its lower concentration the leaves in the first and second generation also did not have positive effects on the fecundity in the silkworm. vitamin b complex the vitamin b complex is traditionally made up of 10 members (listed below) that differ in their biological actions, although many participate in energy production from carbohydrates and fats. the optimal levels of essential vitamins such as biotin, 87 table 2 comparison of quantitative requirements for bvitamins of the silkworm with the amounts in mulberry leaves (from ito, 1978) amount in mulberry leaves mg/g of dry matter minimum amount required mg/g of dry diet vitamin 0.2-0.8 1 biotin(b8) 930-1550 750 choline 4000 1000 inositol 69-99 20 nicotinic acid(b3) 16-35 20 pantothenic acid(b5) 43-50 5 pyridoxine(b6) 13-21 5 riboflavin(b2) 6.7 0.5 thiamine(b1) choline, pyridoxine, panthotinate, inositol, riboflavin, thiamine, nicotinic acid have been determined by horie and ito (1963, 1965) (table 2). riboflavin (b2) riboflavin is important in promoting the release of energy from carbohydrates, fats and proteins “i.e. in the metabolic pathway for atp production”. the enrichment of mulberry leaves with riboflavin at 77 ppm enhanced certain economic characters of silkworm, and improved silk production in north climatic conditions of iran (rajabi et al., 2006a). male cocoon weight (1.195 g) was greater at 77 ppm while female cocoon weight (1.622 g) was greater at 127ppm. maximum male pupal weight was recorded at 37 ppm (0.895 g) compared to 127 ppm for the female (1.169 g). male and female shell weight (0.311 and 0.318 g) had significant increase at 77 ppm compared to control (0.276 and 0.277 g). male and female cocoon shell percentage reached their maximum at 77 ppm treatment, which were 26.06 % and 21.46 % respectively. average of 50 egg weight, number of eggs for every female and hatchability percentage did not show significant difference among the treatments and the control. similar improvements were not obtained in natanz, in the center of iran, place which has dry climatic conditions. folic acid (b9) folic acid plays a major role in cellular metabolism including the synthesis of some of the components of dna and pigment precursor (national research council (u.s), 1987, chapman, 1998). yosuhiro and sholchi (1971) noted that the silkworm growth decreased when folic acid was eliminated from artificial diet. nirwani and kaliwal (1996) determined that folic acid has phagostimulatory effects with significant increase in female and male cocoon weight and shell weight. para-amino benzoic acid (paba) is a growth regulator and represents one of the forms of folic acid. paba is one of the substances belonging to the vitamin b-complex group and supports vital function in insects and especially in silkworm (pai et al., 1988). paba supplementation has no significant effects on adult weight whereas it caused deleterious effects on their length (p<0.001) and wing-span (p<0.001) (faruki and khan, 1992). feplus® (ferrus fumarate+folic acid) supplementation significantly increased larval, pupal and adult weight in comparison with controls with lowest and highest growths obtained at the concentrations of 0.32 and 0.64 %, respectively (khan and saha, 1996). larval and pupal periods decreased at lower doses (0.08 and 0.16 %) while they increased after exposure to higher doses (0.32 and 0.64 %). fertility increased significantly in all treatments when compared to control except for 0.64 % concentration (khan and saha, 1996). pyridoxine (b6) pyridoxine is necessary for the proper functioning of over 60 enzymes that participate in amino acid metabolism. it is also involved in carbohydrate and fat metabolism. without pyridoxine or its derivatives no larva reached the third instar under aseptic condition. pyridoxine is important in protein metabolism and its deficiency in mammals results in decrease in phosphorylases (national research council (u.s), 1987). faruki (2005) reported that mulberry leaf fortification by pyrol® (pyridoxine hydrochloride hcl.25) in various concentrations from the third instar significantly reduced the fecundity. in this experiment it was found the lowest number of eggs was produced at the lowest concentration (10 µg/ml) and at higher concentrations it remains lower than in the control. banerjee and khan (1992) observed that vitamin b6 enhances the oviposition of the silkworm infected by bacillus thuringiensis var. kurstaki but the rate was lower than the control. faruki (2005) reported that higher concentration of vitamins reduced the fecundity and fertility of silkworm. further, the percent reproduction control (prc) in lower concentrations was more than higher concentrations (faruki, 2005). the reproductive success in lower concentrations was prominent with compared with the higher concentrations (faruki, 2005; etebari and matindoost, 2005). rajabi et al (2006b) showed that in north climatic conditions of iran, larval weight reached a maximum 2.601 g at the end of 5th instar. effective rate of rearing (err) was higher (75.33 %) at 100 ppm concentration compared with other concentrations (10, 500 and 1000 ppm). larval and silk indices reached their maximum at 100 ppm concentration while pupal and adult indices reached their maximum at 1000 ppm concentration in male and female. larval duration was longer (622.5 h) in 88 treatments against control (604.5 h). treatment differences were recorded also in respect of other economic characteristics (rajabi et al., 2006b). nicotinic acid (b3, niacin) niacin is important for the release of energy from carbohydrates and fats, the metabolism of proteins and production of several hormones (national research council (u.s), 1987). horie and ito (1965) showed the required level of niacin for silkworm is highly regulated to the most appropriate level of 33 μg/l of dry weight and the increase of niacin reduced the larval weight. horie (1995) showed a reduction of requirement pattern with increasing larval weight. niacin caused significant deleterious effects on larval growth. cocoon parameters such as cocoon weight, pupal weight and cocoon shell weight also showed significant decrease in all treatments (etebari and matindoost, 2004). furthermore, mulberry leaf enrichment with nicotinamide (10, 20 and 30 g/l) from first instar caused intensive mortality in the larval growth and only 1.2% larvae could reach the 5th instar. high doses (20 and 30 g/l) of nicotinamide killed all larvae before entering the 5th instar (etebari and matindoost, 2004). horie and ito (1965) reported that the analogues of niacin, 4-acetyl pyridine interrupts the larval growth when added to mulberry leaves and acted as an antimetabolite. niacin with 0.5 g/l acted as an antifeedant for silkworm larvae and decreased their metabolism (etebari and matindoost, 2004). thiamine (b1) thiamine is important for energy metabolism (national research council (u.s), 1987). nirwani and kaliwal (1998) reported that the weight of larvae and silk glands in all the thiamine fed groups had not shown any significant changes. on the other hand, larval duration, cocoon weight, shell weight and fecundity increased significantly. in opposition with what observed for ascorbic acid and folic acid, it has been found that thiamine has no phagostimulatory effect on silkworm (horie and ito, 1963; nirwani and kaliwal, 1996). faruki (1998) reported that the thiamine derivative thianomin enhanced the growth of silkworm larvae, pupae and adults at all concentrations used (50, 100, 500 and 1000 ppm). mulberry leaf enrichment with thianomin increased the growth indices such as larval, pupal and adult weight. silk index increased too in all treatments. thianomin increased cocoon characters such as cocoon weight, shell weight, cocoon shell length and breadth significantly (p< 0.05). cyanocobalamin (b12, cobalamin) cyanocobalamin plays important role in silkworm because it is a co-factor of propionate metabolism which is important substrate for biosynthesis of juvenile hormone in insects (halarnkar and blomquist, 1989). vitamin b12 does not occur in mulberry leaves but considerable amount of this vitamin was observed in larvae and pupae. it is believed that actinomycetes in the gut lumen produce vitamin b12. das and medda (1998) reported that supplementation of mulberry leaves with b12 vitamin could increase the synthesis of nucleic acid and proteins in the silkglands of the silkworm. pantothenic acid (b5) pantothenic acid is the precursor of coenzyme a that is vital for the metabolism of carbohydrates, the synthesis and degradation of fats, the synthesis of sterols and the resultant steroid hormones, and the synthesis of many other important compounds (national research council (u.s), 1987). biotin (b8, vitamin h) biotin has an important role in carbohydrate and fat metabolism. it has been showed that biotin is one of the essential vitamins for the silkworm b. mori (horie et al., 1966). it has important role in the synthesis of fatty acids in the silkworm and it is confirmed that minimal optimal level of biotin for growth and survival of the silkworm was much lower than those of other vitamins including nicotinic acid, pantothenic acid and pyridoxine (horie and ito, 1965). it is identical with the minimal threshold for alteration of fatty acid composition (horie and nakasone, 1968). however, to the best of our knowledge, research reports on enrichment mulberry leaves with biotin are not available. choline and inositol choline is traditionally not a vitamin; however it was identified as part of vitamin b complex. inositol is an important part of signaling mechanism that transmits information from outside to the inside of cells. it is generally considered dispensable in insect diets. choline and inositol are required by silkworm in higher level because they are lipogen substrate, also involved in the production of cell membranes. however, research reports on enrichment mulberry leaves with choline and inositol do not seem to be available. multi-vitamin compounds saha and khan (1996) described the extensive effects of multi-vitamin compounds as diet factors on growth interruption and the decrease of cocoon economical characteristics. it is showed that multivitamin and mineral compounds could increase the food intake, growth and conversion efficiency of silkworm (muniandy et al., 1995). evangelista et al. (1997) reported that the larval and cocoon weight increase under multi-vitamin compound treatment, but did not have any positive effects on cocoon shell weight. feeding with multi-vitamins in the larval stage adversely affected the hatchability of eggs. multi-vitamins even though could increase some biological characteristics in silkworm, did not influence the economical and yield contributing parameters. etebari and matindoost (2005) reported that feeding of silkworm on mulberry leaves enriched by with multi-vitamins from 4th instar increased female cocoon shell weight in 2.5% 89 concentration, while female pupal weight increased in 1% concentration. male and female shell ratio did not increase compared to controls. fat-soluble vitamins fat-soluble vitamins consist of the a, d, e and k vitamins. among these, enrichment of mulberry leaves with vitamin a, d or k for silkworm do not seem to have been studied. vitamin e α-tocopherol is slightly effective in increasing the number of eggs laid by moths and β-carotene has also some growth-promoting effect (ito, 1978). enrichment of mulberry leaves with e vitamin did not have significant effect on food consumption in silkworm larvae (mosallanejad, 2002). concluding remarks this review summarizes data showing that enrichment of mulberry leaves with various vitamins have different effects on economic traits and biological parameters of the silkworm. however, reported effects depend also on weather condition, larval stage treated, type of vitamin, varieties of mulberry and silkworm race studied. this possibly indicates the need for elaborating comprehensive studies on the subject. it is advisable that we keep in mind the negative effects of enrichment beside its positive effects on economic traits and biological parameters. moreover, each intervention on the natural content of silkworm food should take into account also costs, environmental safety and large scale feasibility. acknowledgements our studies cited in this paper were supported by the biotechnology institute and iran silkworm research center. references akhtar m, asghar a. nutritional requirement of silkworm bombyx mori. pakistan j. zool. 4: 101-107, 1972. babu m, swamy mt, rao pk, rao ms. effect of ascorbic acid-enriched mulberry leaves on rearing of bombyx mori l. indian j. seric. 31: 11-114, 1992. banerjee sk, khan ar. effect of vitamin b6 on the fecundity of bacillus thuringiensis var. kurstaki treated bombyx mori l. bangladesh j. zool. 20: 361-362, 1992. bhattacharyya a, medda ak. effect of cyanocobalmine and cobalt chloride on glycogen of silkgland of bombyx mori l.nistari race. sci. cult. 77: 268-270, 1981. chapman, r. f. the insect structure and function, cambridge university press, cambridge. 1998. chauhan t, singh k. studies on the effect of ascorbic acid on the fecundity in the mulberry silkworm. sericologia 32: 567-574, 1992. das s, medda a. effect of cyanocobalamine on protein and nucleic acid contents of ovary of silkworm, bombyx mori l., during larval, pupal and adult stages of development. insect sci. appl. 9: 641-646, 1988. dobzhenok nv. the effect of ascorbic acid on the physiological condition of the codling moth and its resistance to fungus and bacterial infection. zakhist roslin. 19: 3-7, 1974. el-karaksy, i. r. and m. idriss. ascorbic acid enhances the silk yield of the mulberry silkworm, bombyx mori l. j. appl. entomol. 109: 81-86, 1990. etebari k, ebadi r, matindoost l. effect of feeding mulberry’s enriched leaves with ascorbic acid on some biological, biochemical and economical characteristics of silkworm bombyx mori l. int. j. indust. entomol. 8: 81– 87, 2004. etebari k, matindoost l. application of multivitamins as nutrients on biological and economical characteristics of silkworm bombyx mori l. j. asia-pacefic entomol. 8: 16, 2005. etebari k, matindoost l. effects of hypervitaminosis of vitamin b3 on silkworm biology. j. biosci. 29: 417–422, 2004. evangelista a., carvalho ad, takahashi r, de carvalho ad. performance of silkworm (bombyx mori l.) fed with vitamin and mineral supplement. rev. agricul. pirac. 72: 199-204, 1997. faruki si, khan ar, mannan a. fecundity and fertility of the silkworm, bombyx mori l. fed on mulberry leaves supplemented with paraamino benzoic acid. bangladesh j. zool. 20: 351-353, 1992. faruki si. effect of pyridoxine on the reproduction of the mulberry silkworm, bombyx mori l. 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enrichment of mulberry leaf with various sugars, proteins, amino acids and vitamins for vigorous growth of the worm and increased cocoon crop protection. indian j. seric. 11: 1127, 1972. yasuhiro h, sholchi n. effects of levels of biosynthesis of fatty acids and carbohydrates in a diet on the biosynthesis of fatty acids in larvae of silkworm. j. insect physiol. 19: 14411450, 1971. 91 http://worldcat.org/search?q=au%3anational+research+council+%28u.s.%29.+subcommittee+on+vitamin+tolerance.&qt=hot_author http://worldcat.org/search?q=au%3anational+research+council+%28u.s.%29.+subcommittee+on+vitamin+tolerance.&qt=hot_author http://worldcat.org/search?q=au%3anetlibrary%2c+inc.&qt=hot_author << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice review isj 7: 228-238, 2010 issn 1824-307x review insect immunity and its signalling: an overview s tsakas, vj marmaras department of biology, university of patras, 26500 patras, greece accepted october 21, 2010 abstract the innate immunity is the immediate and sole response of invertebrates for the protection against foreign substances and pathogens. in insects, it relies on both humoral and cellular responses that are mediated via certain recognizing receptors and activation of several signalling pathways. fat body and hemocytes are the origins for the production and secretion of antimicrobial agents and activators/regulators of cellular response, while cell mediated immunity in insects is performed by hemocytes. in the last years, research has focused on the mechanisms of microbial recognition and activation of intracellular signalling molecules in response to invaders. in this review, we summarize the mechanisms of the innate immunity in insects and refer to potential interactions between humoral and cellular responses, combined with the involving signalling pathways and their cross talk. key words: insects; innate immunity; signalling pathways introduction living creatures are surrounded by a basically hostile environment. in order to survive, they have developed several defense mechanisms, including the immune system. these mechanisms protect organisms against foreign substances and pathogen invasion. in case of such an invasion, the first line of defense is available immediately and involves mechanisms, either humoral or cellular, that are non specific. the discrimination between humoral and cellular responses is, up to a point, arbitrary, since they all share same signalling pathway that are activated by different stimuli (lavine and strand, 2002; marmaras and lampropoulou, 2009).these mechanisms are embraced under the term innate immunity, which is the sole immune response in invertebrates. vertebrates have developed a second line of defense, the acquired immunity, which is highly specific and contains mechanisms targeting to a particular threat, each time. insects, the most widespread metazoans on earth, have a well-developed innate immune system that allows general and rapid responses to infectious agents while they lack an acquired immune system. the protection against pathogens begins primarily with certain barriers such as cuticle, gut and trachea, tissues that are difficult to be penetrated, while immune response is originated by ___________________________________________________________________________ corresponding author: vassilis j marmaras department of biology, university of patras, 26500 patras, greece e-mail address: marmaras@upatras.gr the fat body and the hemocytes. fat body is the largest organ of the hemocel, the insect body cavity, and is a major site for the production and secretion of antimicrobial peptides (hoffmann, 2003). hemocytes circulate in insect hemolymph. they derive from stem cells that differentiate into specific lineages. however, certain hemocyte types are not common in all insects and differ among species (charalambidis et al., 1995; meister and lagueux, 2003). the humoral immune response is based on the products of characterized immune genes induced by microbial infection and encode antimicrobial peptides, which are synthesized predominantly in fat body and released into hemolymph (hoffmann, 1995; gillespie et al., 1997; nakatogawa et al., 2009; shia et al., 2009). hemocytes and epithelial layers of the integument and the gut are also sites for the synthesis of such molecules. these genes are either not expressed or are constitutively expressed at a low rate prior to infection (hoffmann, 1995; engström, 1998). in addition, humoral immune responses include activation of enzymic cascades that regulate coagulation and melanization of hemolymph, and production of reactive oxygen and nitrogen species (ros-rns) (gilespie et al., 1997; bogdan et al., 2000; nappi and vass, 2001; hoffmann, 2003; mavrouli et al., 2005). cellular responses are performed by hemocytes and include phagocytosis, nodulation and encapsulation (schmidt et al., 2001; nappi et al., 2004; lamprou et al., 2005; mavrouli et al., 2005; sideri et al., 2007). 228 mailto:marmaras@upatras.gr there are a lot of important review papers in the literature which present, in details, specific groups of signalling pathways or mechanisms of innate immunity in insects. this paper is an overview of these mechanisms, describing, in general, humoral and cellular responses along with major signalling transduction pathways, emphasizing on their cross talk. origins of innate immunity in insects fat body the larval fat body is the major site of the intermediate metabolism of insects and plays functions analogous to those of the vertebrate liver. it consists of thin layers or strings, generally one or two cells thick, or small nodules suspended in the hemocel and distributed throughout insect body (roma et al., 2010). the majority of proteins of the hemolymph are synthesized in this tissue, which also serves as lipid, carbohydrate and protein storage. the fat body is a target tissue for all principal insect hormones such as neural hormones, juvenile hormone and ecdysone (keeley, 1985) and is also a site of response to microbial infection. characterized immune genes, in the fat body, are induced by microbial infection and encode antimicrobial peptides which are then released into the hemolymph (hoffmann, 1995; engström, 1998). in drosophila, seven antibacterial peptides have been characterised namely, cecropin, attacin, defensin, drosocin, diptericin, metchnikowin and also an antifungal peptide called drosomycin (lemaitre and hoffmann, 2007). in addition, lepidopreran fat body synthesizes and releases several other proteins, such as pattern recognition protein hemolin and two immulectins serine proteinases: prophenoloxidase activating proteinase and a serine proteinase inhibitor from the serpin family (zhu et al., 2003). hemocytes in insects, there are no blood vessels. blood and interstitial fluid are indistinguishable and are collectively referred as hemolymph which bathes all internal tissues, organs and hemocytes, and facilitates the transport of nutrients, waste products and metabolites. the most common types of circulating hemocytes, in the hemolymph of lepidoptera (manduca sexta, bombyx mori) and diptera (drosophila melanogaster, ceratitis capitata) are granulocytes and plasmatocytes (lavine and strand, 2002; kanost et al., 2004). however, these haemocyte types are not common in all insect species (lavine and strand 2002; meister and lagueux, 2003; lamprou et al., 2007). in addition, the terminology used to designate each haemocyte type is often different from one insect species to another (ribeiro and brehelin, 2006), although, there have functional similarities among different insect species. in drosophila, plasmatocytes are professional phagocytes and are the equivalent of mammalian cells from the monocyte/macrophage lineage. phagocytosis permits rapid removal of dead cells, during embryogenesis and metamorphosis and pathogens during infections. plasmatocytes also synthesize and secrete antimicrobial peptides and signal to the larval fat body, the functional equivalent of the mammalian liver, in response to an infection (agaisse et al., 2003). based on morphological criteria, hemocyte types similar to drosophila have been recognised and classified in c. capitata larvae, although they may differ significantly in function. medfly plasmatocytes, besides phagocytic activity, are involved in nodule formation and melanization, as they contain the precursors of the prophenoloxidase cascade (mavrouli et al., 2005; sideri et al., 2007; marmaras and lampropoulou, 2009). pattern recognition proteins/receptors the first step for the initiation of immune response, either humoral or cellular, is the recognition of the pathogen. this is achieved by the pattern recognition proteins/receptors (prps), that recognize and bind conserved domains (patterns) located on the pathogen surface, which are called pathogen-associated molecular patterns, (pamps) (medzhitov and janeway, 1997) the most characterized prps are the type c lectins, the peptidoglycan recognizing proteins, the β-1,3-glucan proteins, the hemolin and the integrins (bettencourt et al., 1997; michel et al., 2001; bettencourt et al., 2004). these proteins are present on the plasma membrane of fat body cells and hemocytes or they are soluble in the hemolymph. they bind on lipids and carbohydrates which are synthesized by microorganisms and are exposed on their surface, such as lipopolysaccharites (lps) of gram negative bacteria, lipoteichoic acids and peptidoglycans of gram positive bacteria and β-1,3-glucans of fungi (nappi et al., 2000). insights on the characterization of prps have been obtained mainly from studies in drosophila. certain hemocyte protein recognition receptors appear to be unique to drosophila whereas others have direct homologues to other insect species or even mammals (marmaras and lampropoulou, 2009). the binding of invaders’ pamps on prps induces the synthesis of antimicrobial proteins or initiates the proteolytic activation of phenoloxidase cascade or activates cellular immune response, leading to phagocytosis, nodule formation and encapsulation of the invaders (yu xq et al., 2002; marmaras and lampropoulou, 2009). immunolectins lectins are sugar recognition molecules and play an important role in immune-related reactions enabling an organism to distinguish self from nonself or modified-self determinants. they are characterized by a wide range of binding activities. nineteen genes were originally identified in drosophila as members of the c-type lectin family, although the distinct function, for each one of them, had not been clarified (theopold et al., 1999). ctype lectins had been classified into seven groups based on their overall domain structure. analyses of the superfamily representation in several completely sequenced genomes have added 10 new groups (zelensky and gready, 2005). in lepidopterans, immunolectins are involved in prophenoloxidase 229 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t79-4899v7y-3&_user=83471&_coverdate=05%2f31%2f2003&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_acct=c000059670&_version=1&_urlversion=0&_userid=83471&md5=78b2191eae38a0987be919748d9327b8#bib22#bib22 activation, phagocytosis and nodule formation (yu et al., 2003; yu and kanost, 2004). peptidoglycan recognizing proteins peptidoglycan (pgn) consists of a sugar backbone and a stem-peptide of three to five amino acids, found mostly on the surface of gram positive bacteria (kaneko and silverman, 2005; little and cobbe, 2005). the peptidoglycan recognizing proteins (pgrps) are small extracellular proteins (20 kda) which are synthesized and secreted by the fat body, integument, gut, and in a minor degree by the hemocytes (kaneko and silverman, 2005). they are defined by a domain with homology to an enzyme called amidase. several pgrps, from flies and mammals, present amidase activity, and several others are predicted to have amidase activity. in addition, some pgrps lack a critical cysteine required for the active site, and are therefore thought to function only as recognition proteins without enzymic activity (mellroth et al., 2003). recognition of pgns on gram-positive or gram-negative bacteria, by circulating pgrps, activates the toll or imd intracellular signalling pathways, respectively, leading to the nuclear translocation of two nf-κb/rel proteins and drives anti-bacterial peptide gene expression. the details of these intracellular signalling pathway have been reviewed previously (silverman and maniatis, 2001; aggrawal and silverman, 2007). β-1,3-glucan recognising proteins in drosophila there is a gram-negative bacteria-binding protein (dgnbp) family, (kim et al., 2000). dgnbp-1 exists in both soluble and glycosylphosphatidylinositol-anchored membrane and functions as a pattern recognition receptor for lps from gram-negative bacteria and β-1,3-glucan from fungi and mediates innate immune signalling for the induction of antimicrobial peptide gene induction in cultured drosophila immune cells. (kim et al., 2000). two biosensor for fungal and bacterial infection, called β-1,3-glucan recognition proteins (βgrps), are present in the hemolymph of m. sexta (wang and jiang, 2010). both βgrps specifically recognize soluble or insoluble β-1,3-glucan and lps, bind onto a hemolymph proteinase-14 precursor (prohp14) through specific protein– protein interactions and initiate propo activation system.(wang and jiang, 2010). similar activation of propo system occurs in the beetle tenebrio molitor (zhao et al., 2007). hemolin the hemolin is a member of the immunoglobin superfamily and is synthesized by the fat body. hemolin has been found in the hemolymph of two lepidopteran species hyalophora cecropia and m. sexta and its concentration increases 20-fold, upon bacterial infection, although it has no direct antimicrobial activity (bettencourt et al., 1997; eleftherianos et al., 2007). in m. sexta, hemolin recognizes and binds lps on gram negative bacteria and lipoteichoic acid on gram positives bacteria, leading to their aggregation. (daffre and faye, 1997; yu and kanost, 2002). it must be noted that lps and lipoteichoic acid bind on the same site on the hemolin molecule. hemolin may also bind on glycolipids of the bacterial wall, showing that it acts as a wide range prp against infection. in h. cecropia, hemolin binds on bacterial lps and then binds on hemocytes, in a calcium dependent manner, thus activating protein kinase c, which initiates phagocytosis (bettencourt et al., 1997; daffre and faye, 1997). integrins integrins are surface proteins, widely expressed in metazoans (sponges to humans), and participate in adhesion, migration and tissue organization (hughes, 2001). integrins recognize and bind rgd motifs (amino-acid triplet arg-gly-asp) in specific cell-surface, or extracellular matrix (ecm) or soluble proteins (collagen, laminins, fibronectins) (hynes, 2002; humphries et al., 2004). integrins are primary molecules for the recognition of foreign agents and the initiation of immune response. in the medfly c. capitata, they are involved in bacteria (grampositive and gram-negative) phagocytosis by plasmatocytes, but not in lps or abiotic targets uptake (lamprou et al., 2007; mamali et al., 2009). in m. sexta, integrins play a key role in stimulating hemocytes adhesion leading to encapsulation (zhuang et al., 2008). humoral responses the recognition of invading pathogen either as bacteria or fungi or even viruses is followed by the immediate de novo synthesis of antimicrobial peptides (amps) and their secretion into the hemolymph (zasloff, 2002; bulet et al., 2004). these peptides are mainly synthesized by the fat body and in a lesser degree by the hemocytes, integument, gut, salivary glands and reproductive structures (nappi and ottaviani, 2000). antimicrobial peptides over 150 antimicrobial peptides (amps) have been isolated and characterized in insects. these molecules are small, 12-50 amino acids, cationic peptides, which bind anionic bacterial or fungal membranes leading to disruption and cell death (zasloff, 2002; yount and yeaman, 2006). although they have different structure and target organisms (bacteria or fungi), the amps are classified in four groups; a) cecropins, b) cysteine-rich peptides, c) proline-rich peptides, and d) glycine-rich peptides. cecropins were firstly isolated in h. cecropia after injection with bacteria (hultmark et al., 1980; steiner et al., 1981). these peptides are produced in response to septic injury by either gram positive or gram negative bacteria bacteria and affect on cellular proliferation by inhibiting the synthesis of proteins of the cell membrane. defensins and drosomycin are cysteine-rich peptides. defensins, destroy mostly gram-positive peptides by forming channels in the plasma membrane which leads to cell lysis, while drosomycin has an antifungal activity. diptericin is an antibacterial peptide that has been found only in diptera species and is induced upon gram negative bacteria infection in a way similar to attacines (nappi and ottaviani, 2000). lysozymes are enzymes that 230 http://www3.interscience.wiley.com/cgi-bin/fulltext/118714469/main.html,ftx_abs#b3#b3 degrade peptidoglycans of the bacterial cell wall. they are also found in other animals, plants, fungi and bacteriophages (bulet et al., 1999). enzymic cascades coagulation of hemolymph insects have developed mechanisms for the coagulation of hemolymph, in case of wounding, to prevent loss of body fluids (theopold et al., 2002).in the cockroach leucophaea maderae, hemocytes secrete a calcium dependent transglutaminase that catalyzes the polymerization of lipophorins and vitellogenin-like proteins. these last proteins have a domain homologous to the von willebrand clotting factor in mammals (βohn et al., 1994). the most characterized mechanism is the one in lymulus polyphemus, which appears to be similar in drosophila (vimlos and kurucz, 1998). according to this, lps and β-1,3-glucan trigger a serine protease chain reaction, finally leading to the coagulation of the hemolymph. in addition, serine protease activates melanization cascade (nappi et , al., 1995; mavrouli et al., 2005; sideri et al., 2007). it must be noted the dual role of serine protease in the insect immunity since intermediate metabolites of these two cascades, preclotting enzymes, melanin derivatives and reactive oxygen species, are toxic invading pathogens. melanization of hemolymph melanization, the pathway leading to melanin formation, has a central role in defense against a wide range of pathogens and participates in wound healing as well as in nodule and capsule formation in some lepidopteran and dipteran insects, (lavine and strand, 2001; lavine and strand, 2003; mavrouli et al., 2005). melanization depends on tyrosine metabolism. briefly, tyrosine is converted to dopa, an important branch point substrate, by activated phenoloxidase (po). dopa may be either decarboxylated by dopa decarboxylase (ddc) to dopamine or oxidised by po to dopaquinone. dopamine is also an important branch point substrate, because dopamine-derived metabolites either via po or through other enzymes are used in several metabolic pathways, participating in neurotransmission, cuticular sclerotization, crosslinking of cuticular components via quinone intermediates, phagocytosis, wound healing and melanization in immune reactive insects (fearon, 1997; aderem and underhill, 1999; ling and yu, 2005; marmaras and lampropoulou, 2009). cellular responses hemocytes are responsible for a number of defense responses in insects, among which phagocytosis, nodulation, encapsulation and melanization have been documented. these processes appear to be discrete immune responses in terms of gene expression and outcome. however, these certain immune responses share a number of common elements that function in concert to clear pathogens from the hemolymph. below we have outlined the current data on these defense responses and their relationships. phagocytosis phagocytosis initiates with the recognition of the invading pathogens, engulfment and is completed with their intracellular destruction, by individual hemocytes. in insects, phagocytosis is achieved mainly by the circulating plasmatocytes or granulocytes, in the hemolymph (gillespie et al., 1997; meister and lagueux, 2003; lamprou et al., 2005, 2007). the uptake of a microbe by a phagocytic cell is an extremely complex and diverse process which requires multiple successive interactions between the phagocyte and the pathogen as well as sequential signal transduction events. phagocytosis is induced when phagocyte surface receptors, are activated by target cells. it must be noted that the hemocyte response to various bacteria differs. for example, in a. aegypti hemocytes respond to e. coli with phagocytosis, whereas to micrococcus luteus with melanization (hernandez-martinez et al., 2002; hillyer and schmidt 2003a, b). furthermore, differences exist in the efficiency and speed of phagocytosis among different bacteria. it has been shown that e. coli is more readily phagocytosed than s. aureus, in a. gambiae as well as in isolated medfly hemocytes (levashina et al., 2001; moita et al., 2006; lamprou et al., 2007). these results strongly suggest that several distinct molecular mechanisms regulate phagocytosis in insects. nodulation nodulation refers to multicellular hemocytic aggregates that entrap a large number of bacteria. melanized or non-melanized nodules are formed in response to a number of invaders. nodule formation appears to be related with eicosanoids in many insect species (miller et al., 1999) or prophenoloxidase (po) and dopa decarboxylase (ddc) in medfly hemocytes (sideri et al., 2007). encapsulation encapsulation refers to the binding of hemocytes to larger targets, such as parasites, protozoa, and nematodes. encapsulation can be observed when parasitoid wasps lay their eggs in the hemocel of drosophila larvae. hemocytes after binding to their target they form a multilayer capsule around the invader, which is ultimately accompanied by melanization. within the capsule the invader is killed, by the local production of cytotoxic free radicals ros and rns, or by asphyxiation (nappi et al., 1995; nappi and ottaviani, 2000). antiviral response viruses are intracellular pathogens that infect all forms of life. the first potent antiviral defense mechanism was identified in plants, through rna silencing (ding and voinnet, 2007). recently, rnai was found to play an important role in the control of viral infection in drosophila. this mechanism of gene silencing depends upon small rnas that are 21-30 nucleotides. central to the rnai mechanism are the slicing enzymes of the argonaute (ago) family, which mediate highly specific cleavage of target rna molecules. the specificity of ago enzymes is achieved by their association with small rnas, 231 which guide them to complementary sequences. three rnai pathways, involving different members of the ago family, have been defined in drosophila: first, the small interfering (si)rna pathway involves ago-2, and is activated by double-stranded (ds)rna. sirnas are produced by the rnaseiii enzyme dicer-2, which forms a complex with the dsrna-binding protein (dsrbp) protein r2d2; second, the micro (mi)rna pathway involves ago1, dicer-1, and its dsrbd cofactor r3d1, and regulates expression of drosophila genes, in particular during development; third, the piwiassociated rna (pirna) pathway involves the three other ago proteins encoded by the drosophila genome, namely piwi, aubergine, and ago3. pirnas are involved in the control of mobile genetic elements, including the retrovirus gypsy, in the germ-line (brennecke et al., 2007; ding and voinnet, 2007; kemp and imler, 2007). signalling pathways in innate immunity from the overview of the mechanisms concerning the innate immunity, three major responses can be summarized. the production of antimicrobial peptides due to specific receptors, either soluble or membrane, the internalizationphagocytosis, which follows the attachment of bacteria on the cell membrane and the role of rna interference in the antiviral immunity. the hallmark of the drosophila humoral immune response is the production of antimicrobial peptides in the fat body and their release into the circulation (aggarwal, and silverman, 2008; feldhaar and gross, 2008). two recognition and signalling cascades regulate expression of these antimicrobial peptide genes. the toll pathway is activated by fungal and many gram-positive bacterial infections, whereas the immune deficiency (imd) pathway responds to gram-negative bacteria. both of these are initiated by peptidoglycan recognition proteins (pgrps) and complete their action via the conserved nf-κb signalling cascades for the control of immune-induced gene expression (aggarwal and silverman, 2008). phagocytosis is triggered by certain transmembrane proteins on the hemocyte surface. the most common classes of such receptors in insect plasmatocytes are the scavenger receptors, the egf-like-repeat-containing receptors, the integrins and the pgrps (feldhaar and gross, 2008; marmaras and lamproulou, 2009). the key intracellular molecules that promote signals from pathogens that attach on cell-surface receptors, are the scaffold and adaptor proteins. scaffold proteins are proteins that bind other proteins that usually function in sequence. adaptor proteins are proteins that augment cellular responses by recruiting other proteins to a complex. these molecules function as organizing platforms that bring together both the enzymes and the substrate proteins, in the same complex (marmaras and lampropoulou, 2009). we show that antimicrobial peptide synthesis and bacterial internalization share a lot of signalling molecules and pathways. antiviral response, although it consists of a totally different procedure targeting to the degradation of viral nucleic acids by rna interference it includes three classical immune signalling pathways (toll, imd, and jak-stat) responsive to infection by different viruses (kemp and imler, 2009; sabin et al., 2010). the toll pathway insects respond to gram-positive bacterial and fungal infections via the toll pathway. its basic component is the transmembrane receptor toll and the intracellular adaptors tube and myd88 (lemaitre and hoffmann, 2007; aggarwal and silverman, 2008), toll is not a pattern recognition receptor since it does not bind pathogens or pathogen-derived compounds, directly and is activated by the extracellular cytokine spätzle. to activate toll pathway, microbial recognition must be preceded. the detection of gram-positive bacterial peptidoglycans and fungal betaglucans by specific pgrps and gnbps, respectively, activate serine protease cascades from the fat body that culminate in spätzle cleavage, thus, liberating the c-terminal 106 amino acids of spätzle, the mature toll ligand. the cleaved spätzle binds the toll receptor which recruits the tube/myd88 complex, followed by the kinase pelle activation. pelle kinase triggers an intracellular signalling cascade involving several factors resulting in the activation of the transactivator proteins dorsal and dif belonging to the nf-kb family. after their translocation in the nucleus they induce transcription of the respective genes encoding for instance defensins, drosomycin, cecropins. in addition to dorsal and dif a drosophila ikb homolog called cactus is activated which is an inhibitory factor that negatively modulates the tollmediated immune response (feldhaar and gross, 2008). the imd pathway the gram-negative bacteria activate antimicrobial peptide synthesis via the imd pathway (nappi et al., 2004; lemaitre and hoffmann, 2007; aggarwal and silverman, 2008). this pathway was initially defined by the identification of a mutation named immune deficiency (imd) that impaired the expression of several antibacterial peptide genes (lemaitre and hoffmann, 2007). the bind of bacterial monomeric or polymeric dap-type pgn on the single-pass transmembrane cell surface receptor pgrp-lc, results the recruiting of the intracellular adaptor imd (aggarwal and silverman 2008). signal transduction leads to relish cleavage and the rel domain translocates to the nucleus, whereas the inhibitory domain remains stable in the cytoplasm. diptericin gene is an imd target in response to injection of e. coli (gram-negative bacteria). the jak/stat pathway the jak/stat pathway, has three main cellular components: the receptor domeless, the janus kinase (jak), and the stat transcription factor (lemaitre and hoffmann, 2007). bacterial 232 fig. 1 humoral immune response in insect fat body. secreted cytokines as well as pathogens, either bacteria or fungi, bind on several immune-related receptors in a non specific way, among insect species. this leads to the expression of antimicrobial protein genes and secretion of their respective peptides, via certain cytoplasmic pathways either specific (jak/stat for domeless or imd for peptidoglycan recognizing proteins-pgrp) or non specific (toll receptor) for each receptor. infections induce hemocyte to produce cytokine unpaired-3 (upd3), which is the ligand of domeless. the result of this pathway, after immune challenge, is the stat protein accumulation in the nucleus and the activation of gene expression. the transcriptional regulation is complex, with additional inputs from both the imd and mapk (mitogenactivated protein kinase) pathways (aggarwal and silverman, 2008). rna interference pathway rna interference (rnai) has been found to play an important role in the control of viral infection in drosophila (kemp and imler, 2009). central to the rnai mechanism are the slicing enzymes of the argonaute (ago) family, which mediate highly specific cleavage of target rna molecules. these enzymes associate with small rnas, which guide them to complementary sequences. three rnai pathways, involving different members of the ago family, have been defined in drosophila: • the small interfering (si)rna pathway involves ago-2, and is activated by double-stranded (ds)rna. sirnas are produced by the rnaseiii enzyme dicer-2, which forms a complex with the dsrna-binding protein (dsrbp) protein r2d2 (ding and voinnet, 2007) • the micro (mi)rna pathway involves ago-1, dicer-1, and its dsrbd cofactor, and regulates expression of drosophila genes • the piwi-associated rna (pirna) pathway involves the three other ago proteins encoded by the drosophila genes, namely piwi, aubergine, and ago3. pirnas are involved in the control of mobile genetic elements, including the retrovirus gypsy, in the germ-line (brennecke et al., 2007) demonstration of the critical role of rnai as a potent antiviral mechanism in drosophila is based on three lines of evidence: genetic data indicating that rnai pathway mutants are hypersensitive to rna virus infections, identification of viral suppressors of rnai (vsrs), which counteract the immune defense of the fly, and the presence of sirnas of viral origin in infected cells/flies (kemp and imler, 2009). integrin pathway integrins are heterodimeric transmembrane receptors consisting by an α and a β subunit. integrins recognize and bind rgd motifs (aminoacid triplet arg-gly-asp) in specific cell-surface, or extracellular matrix (ecm) or soluble proteins such as collagen, laminin and fibronectin (hynes, 2002; humphries et al., 2004). this ability leads to intracellular signal transduction (outside-in signalling) via activation fak/src pathways and mapk (lamprou et al., 2007; marmaras and lampropoulou, 2009). in the medfly c. capitata, integrins are involved in phagocytosis of bacteria, but not lps, by hemocytes (lamprou et al., 2005; lamprou et al., 2007; mamali et al., 2009), due to the activation of p38 via ras/rho/actin remodelling pathway, while in m. sexta, they lead to stimulate encapsulation by the stimulation of hemocyte adhesion (zhuang et al., 2008). pathway cross talk signal transduction is based on several pathways which form a complicated network, cross talking to each other in order to lead to the appropriate response, due to extracellular stimuli. such interactions may appear in every level of these pathways either in recognition (fig. 1) or signal transduction or even the final response (fig. 2) (garcia-lara et al., 2005). 233 fig. 2 humoral and cellular immune response in insect hemocytes. the bind of bacteria on a β-integrin transmembrane subunit, triggers cytoplasmic signalling via focal adhesion kinase (fak) and mitogen activated protein kinases (mapks) activation, major key point pathways. this leads to cellular responses such as phagocytosis, nodulation and encapsulation. the activation of fak and mapks may also lead to humoral response such as melanisation and wound healing, through the activation of cell surface prophenoloxidase (propo). different peptidoglycan recognition proteins (pgrps) show strong preference, but not exclusivity, towards specific pathogen–associated molecular patterns (pamps) and on the other hand these pathogens may be concerted with other protein recognition patterns (prrs). in drosophila hemolymph, certain soluble pgrp, which recognizes not only a pgn, common to s. aureus and other gram-positive bacteria but even a gnbp1, activate the toll signal transduction pathway (michel et al., 2001; gobert et al., 2003). the result is the synthesis of antimicrobial peptides (amps), such as drosomycin. on the other hand, a membrane pgrp (choe et al., 2002; gottar et al., 2002; ramet et al., 2002) and a soluble pgrp recognize peptidoglycans and activate the imd pathway (lemaitre et al., 1997; takehana et al., 2002). other pgrps may also weakly recognize gram-positive-type pgn or can bind with low affinity gram-negative-type pgn as well as lipopolysaccharide (lps) and lipotteichoic acid (lta) (dziarski, 2004). it becomes obvious that an initial signal does not guarantee a specific outcome, since there is no exclusivity for the activated receptor. in addition, the transduction of the signal, from the cell membrane into the cytoplasm, does not necessarily follow a single pathway but it may change course to another, by unspecific intracellular pathways such as these of src family or mapks namely, erk, p38 and jnk (garcia-lara et al., 2005). these enzymes appear to have overlapping and complementary functions in many pathways. perhaps the function of these enzymes is to modulate the overall intracellular signalling network in the fat body and hemocytes, rather than operating as exclusive signalling switches for defined pathways. thus, the final product differs among species and tissues. drosophila responds to gram-positive bacteria by the induction of drosomycin synthesis, through the toll pathway, while in gram-negatives by the induction of diptericin, attacin and cecropin through the imd pathway (leclerc and reichhart, 2004). however, it has also been reported that s. aureus, may induce the production of cecropins, while e. coli may induce the expression of drosomycin, giving additional evidence for a cross-talk between the two pathways (hedengren-olcott et al., 2004). the jak/stat pathway is activated in response to gram-negative bacteria and appears to branch out from the imd pathway in fruit flies and mosquitoes (agaisse and perrimon, 2004). the production of amps is not the only final response to the same initial stimulus. other humoral as well as cellular responses may be triggered, indicating an extracellular response network of innate immunity, too. immune response cross talk fat body and hemocytes are the major components of the innate immune response in insects. they possess a diverse repertoire of receptors that allow cells to respond to external stimuli such as cytokines and pathogen-associated molecules. signals resulting from these stimuli 234 activate the synthesis of antiviral peptides and the synthesis and secretion of antimicrobial peptides by the fat body. the functional responses of hemocytes are adhesion, cytokine release, melanization, phagocytosis, nodule formation and encapsulation. hemocyte challenging, by a pathogen, triggers all these humoral and cellular responses, which do not function separately, but they seem to cooperate with each other, in order to block pathogen invasion (fig. 2). in medfly and mosquito, phagocytosis begins with the binding of e. coli on an integrin β-subunit of the hemocyte surface (humphries et al., 2004; mavrouli et al., 2005; moita et al., 2006; mamali et al., 2009). integrins transmit signal to focal adhesion kinase/sarcoma (fak/src) and mitogen activated protein kinase pathways (mavrouli et al., 2005). this signal transduction leads to the secretion of serine proteases which convert the surface inactive prophenoloxidase to the active phenoloxidase and initiate melanization. in parallel, phagocytosis and nodule formation are triggered. although these responses seem to be distinct, they appear to cooperate since blockade of one of them inhibits the other (sideri et al., 2007). abiotic latex beads and lipopolysaccharide (lps) phagocytosis do not depend on propo activation (mavrouli et al., 2005; lamprou et al., 2007). the propo activation system is composed of proteins recognizing several pattern-recognition proteins, serine proteases, propo, as well as proteinase inhibitors that function as regulatory factors (cerenius and söderhall, 2004). propo is synthesized in hemocytes and appears to be distributed ubiquitously in the cytoplasm as well as on the surface of hemocytes (ling and yu, 2005; mavrouli et al., 2005). the propo activation system is triggered by several microbial components, such as lps and peptidoglycans. activated po catalyses the hydroxylation of tyrosine to 3,4-dihydroxyphenyl-alanine (dopa). dopa can be oxidized by po to dopaquinone, which, via po and the dopachrome conversion enzyme, ultimately result in melanin. dopa may also be decarboxylated by dopa decarboxylase (ddc) to form dopamine (marmaras and lampropoulou, 2009). ddc is involved in wound healing, parasite defense, cuticle hardening and melanisation (hodgetts and o’keefe, 2006). a pobased oxidation of dopamine leads to dopaminequinone and finally the cross-linking and melanization of proteins. the expression of ddc mrna in the hemocytes of pseudaletia separata was enhanced by injection of an insect cytokine, growth-blocking peptide (noguchi et al., 2003). melanization is the process that leads to melanin formation in both hemocyte-free hemolymph as well as on hemocyte surface after wounding or upon invasion with pathogens. ddc is a key enzyme between melanization and phagocytosis, two unrelated procedures that are linked and facilitate each other. the activity of ddc is elevated during melanotic responses in drosophila and in the mosquito armigeres subalbatus (nappi et al., 1992; huang et al., 2005). melanization is also a critical process in defense against bacteria, and several reports link components of the melanization process with phagocytosis (johansson and söderhall, 1996; hillyer et al., 2004; mavrouli et al., 2005) it has been proposed that pathogens might be killed by toxic reactive oxygen metabolites produced in the process of melanisation (nappi et al., 1995). however, ddc based melanization, appears to be distinct from the pathway leading to phagocytosis (sideri et al., 2007). these two unrelated procedures share a number of substrates (tyrosine, dopa, dopamine) and enzymes (po, ddc). nodulation, as stated in the introduction, refers to multicellular hemocyte aggregates that entrap a large number of bacteria, and po and ddc are key enzymes in this process (mavrouli et al., 2005). nodules may be attached to tissue or surrounded by hemocytes. nodule formation has not been fully characterized, although it is known that it is lectinmediated. melanization and nodulation are two distinct pathways which share a number of substrates and enzymes. 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expressed in manduca sexta fat body. insect biochem. mol. biol. 33: 541-559, 2003. yu xq, zhu yf, ma c, fabrick ja, kanost mr. pattern recognition proteins in manduca sexta plasma. insect biochem. mol. biol. 32: 12871293, 2002. zhuang s, kelo l, nardi jb, kanost mr. multiple alpha subunits of integrin are involved in cellmediated responses of the manduca immune system. dev. comp. immunol. 32: 365-379, 2008. zasloff m. antimicrobial peptides of multicellular organisms. nature 415: 389-395, 2002. 238 http://www.ncbi.nlm.nih.gov/pubmed/12590970 http://www.ncbi.nlm.nih.gov/pubmed?term=%22zelensky%20an%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22gready%20je%22%5bauthor%5d javascript:al_get(this,%20'jour',%20'febs%20j.'); http://www.sciencedirect.com/science/journal/09651748 http://www.sciencedirect.com/science/journal/09651748 http://www.sciencedirect.com/science?_ob=publicationurl&_tockey=%23toc%235053%232003%23999669994%23418957%23fla%23&_cdi=5053&_pubtype=j&view=c&_auth=y&_acct=c000059670&_version=1&_urlversion=0&_userid=83471&md5=65ed6b099f154363a090bfeb4ca63bde http://www.ncbi.nlm.nih.gov/pubmed?term=%22zhuang%20s%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22kelo%20l%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22nardi%20jb%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22kanost%20mr%22%5bauthor%5d javascript:al_get(this,%20'jour',%20'dev%20comp%20immunol.'); humoral responses the recognition of invading pathogen either as bacteria or fungi or even viruses is followed by the immediate de novo synthesis of antimicrobial peptides (amps) and their secretion into the hemolymph (zasloff, 2002; bulet et al., 2004). these peptides are mainly synthesized by the fat body and in a lesser degree by the hemocytes, integument, gut, salivary glands and reproductive structures (nappi and ottaviani, 2000). johansson mw, söderhall k. the prophenoloxidase activating system and associated proteins in invertebrates. prog. mol. subcell. biol. 15: 46-66, 1996. shia ak, glittenberg m, thompson g, weber an, reichhart jm, ligoxygakis p. toll-dependent antimicrobial responses in drosophila larval fat body require spätzle secreted by haemocytes. j. cell sci. 122: 4505-4515, 2009. zelensky an, gready je. the c-type lectin-like domain superfamily. febs j. 272: 6179-6217, 2005. 52 isj 15: 52-60, 2018 issn 1824-307x research report molecular cloning and characterization of rheb from white shrimp (litopenaeus vannamei) x liu1,2,3, m wang1,2, j shao1,2,3, b wang1,2, k jiang1,2, m liu1,2*, l wang1,2* 1key laboratory of experimental marine biology, institute of oceanology, chinese academy of sciences, qingdao 266071, china 2laboratory of marine biology and biotechnology, qingdao national laboratory for marine science and technology, qingdao 266237 3university of chinese academy of sciences, beijing 100049, china accepted february 11, 2018 abstract the ras family gtpase rheb, a gtp-binding protein, binds specifically to the mtor catalytic domain and induces activation of the mtor catalytic function. furthermore, rheb is related to a stable modification of the configuration of mtorc1 that increases access of substrates to their binding site on the raptor polypeptide. in the present research, a cdna of 898 bp for the litopenaeus vannamei rheb was cloned via rapid amplification of cdna ends (race) technique. the complete cdna sequence of rheb contained an open reading frame (orf) of 549 bp, which encoded a protein of 182 amino acids. the amino acid sequence of rheb shared more than 60% similarity with other identified rheb proteins. a ras domain (from p3 to a169) was found in the amino acid sequence of rheb that can react selectively and non-covalently with gtp. the mrna transcripts of rheb were consistently expressed in all the tested tissues, including muscle, gill, hepatopancreas, eyestalk, intestine and stomach. the mrna expression profiles of rheb in muscle after the stimulation with rapamycin were promoted, which further proved that rheb protein could be a feedback regulator to mtor signaling pathway. furthermore, the results of the present study indicated that rheb played an important role in the regulation of mtor signaling pathway during the stimulation of dietary restriction, amino acid supplementation and rapamycin stimulation in shrimp. key words: litopenaeus vannamei; molecular cloning; mtor; rheb introduction mechanistic target of rapamycin (mtor) is a fundamental regulator of cell growth and proliferation in all eukaryotes (wullschleger et al., 2006). mtor can sense stress, oxygen, amino acids, energy levels and growth factors to perform cell function (laplante et al., 2012). the functions of mtor were performed by two independent complexes. the mtor complex 1 (mtorc1) is comprised of mtor ___________________________________________________________________________ corresponding authors: mei liu key laboratory of experimental marine biology institute of oceanology chinese academy of sciences qingdao 266071, china e-mail: liumei@qdio.ac.cn lei wang key laboratory of experimental marine biology institute of oceanology chinese academy of sciences qingdao 266071, china e-mail: wanglei@qdio.ac.cn in association with raptor, lst8, deptor, ttl1/tel2 and ppras40, which could be inhibited by rapamycin (rapa) (kunz et al., 1993; hara et al., 2002; kim et al., 2002; loewith et al., 2002). the mtor complex 2 (mtorc2) shares include mtor, mlst8, deptor, and tti1/tel2 with mtorc1. in addition, rictor, msin1, and ppr5/protor are specific for mtorc2 (loewith et al., 2002; jacinto et al., 2004; sarbassov et al., 2004). its output is insensitive to rapa. in mammalian cells, mtorc1 output is sensitive to amino acid (especially leucine and arginine) sufficiency (long et al., 2005a). amino acid withdrawal can lead to the inhibition of mtorc1 signaling (demetriades et al., 2014). the overexpression of rheb can rescue mtor from inactivation in vivo caused by amino-acid withdrawal (long et al., 2005a). what’s more, genetic evidence from drosophila indicated that the rheb is an indispensable activator of mtorc1 in the insulin/igf-i receptor pathway. the studies indicate that rheb is a key regulator of the output of mtorc1. mailto:liumei@qdio.ac.cn mailto:wanglei@qdio.ac.cn 53 rapa is a specific inhibitor of mtorc1 and the effects of rapa on mtor signaling are complicated. many mtorc1 functions are highly sensitive to rapa, for example, mtor-mediated protein synthesis, lipogenesis, energy metabolism, and lysosome biogenesis (thomas et al., 1997; schmelzle et al., 2000; brugarolas et al., 2003; laplante et al., 2009; settembre et al., 2012). rapa forms a complex with the intracellular 12-kda fk506-binding protein (fkbp12) (brown et al., 1994; sabatini et al., 1994). this complex directly combines with and inhibits mtorc1. rheb can relieve the inhibition of the mtorc1 (ma et al., 2008; sun et al., 2008). the influence and mechanism of rheb to control the mtorc1 are as yet unknown. rheb can bind specifically to the mtor catalytic domain in vivo and in vitro (avruch et al., 2014). the mtor–rheb interaction can promote the activation of mtor kinase (long et al., 2005a). amino acids appear to control the efficacy of rheb-gtp towards mtorc1 (avruch et al., 2009). besides, rheb promotes a reconfiguration of the mtorc1 complex that enhances the accession and binding of substrates to raptor. a previous research showed that the mtorc1 substrates s6k1 and 4e-bp bound directly to raptor (hara et al., 2002). the process can promote the phosphorylation of translational regulators eukaryotic translation initiation factor 4e (eif4e)-binding protein 1 (4e-bp1) and s6 kinase 1 (s6k1), which, in turn, promote protein synthesis (ma et al., 2009). however, further work is needed to elucidate the mechanisms of rheb–mtor interaction that promotes mtor kinase activity. the researches about rheb functions concentrates upon mammals and some insects. su et al. found that rheb mrna expression was increased in wssv-infected shrimp (su et al., 2014). beyond that, very few studies have been done on its functions in crustaceans. what’s more, a relatively small number of studies explore the interaction between rheb and mtorc1. so, the mechanism of activation is not fully understood. therefore, studies to elucidate the rheb-mtor signaling pathway are of great importance. litopenaeus vannamei has proven to be a useful decapod crustacean model system for the study of evolution, in addition to its importance as a food source (sakthivel, 2014). the research about mtor signaling pathway in l. vannamei can deepen our understanding of the important signaling pathway that regulate growth and metabolism in eukaryotes and reveal the evolutionary traces of mtor signaling pathway. meanwhile, the studies about growth mechanism in shrimp can lead to more reliable way to the selection of the new breed and optimizing the feed formula. materials and methods experimental animals litopenaeus vannamei (5 ± 0.5 g) from the qingdao ruizi aquaculture base (shandong, china) were used in this study and were stocked in two tanks at a density of 100 shrimps per tank (1000 l) at 30‰ salinity. shrimp were maintained for 1 week and fed commercial pellets (42.3% protein, 7.2% fat, 11.6% water, and 15.5% ash, supplied by da le co., ltd, yantai, china). the water quality parameters were evaluated 2−3 times per week and maintained at ph 7.5−8.2, temperature 25−29 °c, dissolved oxygen 5.0−6.5 mgl-1 during the trial. rapamycin injection in the first tank, we injected shrimps on the sixth uromere with 100 μl rapa (500 mm), and shrimps in the other tank were injected with 100 μl dmso diluted by phosphate-buffered saline (pbs) (0.01 m, ph 7.4) on the ratio of 1:1000 (the solvent of rapa). shrimps were dissected at different time-points (0, 0.5, 1, 2, 4 and 6 h) after injection with rapa (experimental group) or dmso diluent (control group) to obtain muscle tissue. the control and experimental groups (n = 9 shrimps/group, conducted in triplicate) obtained at each time-point were used to obtain muscular tissue for real-time (rt)-pcr analysis. the muscle tissue was preserved in rna store solution (beijing comwin biotech co., ltd., beijing, china). amino acid injection the shrimps were divided into six groups. the shrimps in the first group injected with 100 μl pbs (0.01 m, ph 7.4) were the control group. the last group srhimps were injected with 100 µl rapa (500 mm). the remaining 4 groups were starved for 3 days. after the dietary restriction (dr), shrimps in the second group were injected with 100 μl pbs (0.01 m, ph 7.4). the shrimps in the third and fourth groups were injected with 100 μl 0.1 m leucine or 100 μl 0.1 m arginine, respectively. the shrimps in the fifth group were injected with 100 μl leucine (0.1 m) and 100 μl rapa (500 mm). the control and experimental groups (n = 9 shrimps/group, conducted in triplicate) were used to obtain muscular tissue for rt-pcr analysis 30 min after injection. the muscle tissue was preserved in rna store solution (beijing comwin biotech co., ltd., beijing, china). rna preparation and cdna synthesis total rna was extracted using a minibest universal rna extraction kit (takara, dalian, china) according to the manufacturer’s instructions. rna degradation and contamination was monitored on 1% agarose gels. the cdna synthesis was carried out by transscript ii one-step gdna removal and cdna synthesis supermix (ah311-02, transgen biotech, china). the reactions were performed at 42 °c for 30 min, terminated by heating at 85 °c for 5 min and then stored at -80 °c. cloning the cdna of rheb the partial sequence of rheb cdna was obtained from the transcriptome database of l. vannamei. standard procedures were used for cdna cloning. two pairs of gene-specific primers, 3r1/2 and 5r1/2, were designed based on this partial sequence to clone the 3’ end and 5’ end of rheb cdna by rapid-amplification of cdna ends (race) technique. the full-length cdna of the rheb from l. vannamei was amplified by pcr using the primers rheb3'-f and rheb5'-r. the primers were designed by ncbi primer blast and were given in table 1. all pcr amplification was performed in a mj 54 table 1 oligonucleotide primers used in the current experiments. name sequence (5’-3’) brief information 3r1 agcgtgggcaaatcctcctt gene specific primer for race 3r2 atgccccgaccatcgagaac gene specific primer for race 5r1 ccctcgggactgatgttgcc gene specific primer for race 5r2 gttgcccaccaagacaatggg gene specific primer for race qrhe-f aggaaagtggccgttatggg gene specific primer for real-time pcr qrheb-r taccagctccaggccatact gene specific primer for real-time pcr qβ-actin-f gcccatctacgagggata internal control for real-time pcr qβ-actin-r ggtggtcgtgaaggtgtaa internal control for real-time pcr rheb3'-f tgtctctcccttcccttcgg gene specific primers used to amplify full-length rheb rheb5'-r aaggtccatcctataacccagg gene specific primers used to amplify full-length rheb nup aagcagtggtatcaacgcagagt universal primers for race upml ctaatacgactcactatagggcaagcagtggtatcaacgcagagt universal primers for race upms ctaatacgactcactatagggc universal primers for race 3’cds aagcagtggtatcaacgcagagtac(t)30v n oligo (dt) for cdna synthesizing 5’cds aagcagtggtatcaacgcagagtgggggggggghn anchor primer for 5’ race m13f tgtaaaacgacggccagt vector primer for sequencing m13r caggaaacagctatgacc vector primer for sequencing mini personal thermal cycler (bio-rad, usa), and the pcr products were purified using dna gel extraction kit (dp210, tiangen, china) and cloned into the peasy-t1 cloning vector (ct101, transgen biotech, china). after being transformed into the trans5α chemically competent cell (cd201, transgen biotech, china), the positive recombinants were identified via anti-ampicillin selection. sequence characterization and multiple sequence alignment the protein sequence similarities were discovered by protein blast at the national center for biotechnology information (ncbi). the physicochemical property of protein rheb were analyzed by protparam tool (https://www.expasy.ch/tools/protparam.html). signalp 4.1 program was utilized to predict the presence and location of signal peptide (http://www.cbs.dtu.dk/services/signalp/). the protein domain features of rheb were predicted by simple modular architecture research tool (smart) 7.0 (http://smart.emblheidelberg.de/). multiple sequence alignment of rheb and other rhebs was performed with clustalw multiple alignment program 2.1 (http://www.ch.embnet.org/software/clustalw.html) and multiple alignment show program 2.0 (http://www.bioinformatics.org/sms2/color_align_con s.html). a neighbor-joining (nj) phylogenic tree of rheb was constructed with mega 6.0 software package. to derive confidence value for the phylogeny analysis, bootstrap trials were replicated 1000 times. real-time pcr analysis of rheb mrna expression the mrna transcripts of rheb in muscle, gill, hepatopancreas, eyestalk, intestine and stomach were quantified by rt-pcr, and its temporal expression profiles in muscle of l. vannamei stimulated with rapa were determined by rt-pcr. pcr amplification was performed using the following cycling conditions: denaturation for 30 s at 94 °c, followed by 40 cycles of 5 s at 94 °c, and 30 s at 60 °c. to confirm that only one pcr product was amplified and measured, dissociation curve analysis was performed at the end of each pcr. all rt-pcr was performed with the sybr premix ex taq kit (takara biotechnology co., dalian, china). the information of all primers used in this assay was shown in table 1. the primers were designed by ncbi primer blast. the expression of rheb was normalized to the expression of β-actin gene for each sample. the comparative ct method (2-δδct) was used to analyse the expression level of rheb (schmittgen et al., 2008). statistical analysis results are expressed as means ± sd. the statistical analysis were performed by one-way analysis of variance (one-way anova) using spss software to detect significant intergroup differences. the p values less than 0.05 were considered statistically significant. results the molecular features, sequence alignment and phylogeny relationship of rheb a nucleotide sequence from the l. vannamei transcriptome is homologous to rheb identified previously confirmed by sequencing and blast analysis. based on this fragment, a fragment was amplified with nested primers upm/3r1 and nup/3r2 to obtain the 3’ end of the sequence. a fragment was amplified with nested primers upm/5r1 and nup/5r2 to obtain the 5’ end of the sequence. a 898 bp nucleotide sequence representing the complete cdna sequence of rheb 55 fig. 1 nucleotide and deduced amino acid sequences of rheb. the nucleotides and deduced amino acids are numbered along the left margin. the ras domain was in shade. conserved amino acids involved in mtor binding activity are boxed. conserved amino acids involved in guanyl nucleotides binding activity are circled. the asterisk indicated the stop codon. the amino acid sequences of rheb has been submitted to genbank and the accession number is mg696863. of l. vannamei was assembled. the complete cdna sequence of rheb contains a 208 bp 5’ untranslated region, a 141 bp 3’ untranslated region with a poly (a) tail and the complete sequence of an open reading frame (orf) of 549 bp (fig. 1). the orf encoded a polypeptide of 182 amino acid residues with a calculated molecular mass of approximately 20.55 kda. the theoretical isoelectric point is 5.67. no signal peptide was predicted in the deduced amino acid sequence of rheb by signalp program. a ras domain (from p3 to a169) was found in the amino acid sequence of rheb. the deduced amino acid sequences of the six crustacean rheb proteins were highly conserved, showing high identity and similarity to each other (fig. 2). sequence identity was particularly high within the ‘g box’ motifs (g1–g5) in all the rheb proteins, particularly in the g2 motifs (fig. 2). the effect domain (switch i) is highly conservative region too. the deduced amino acid sequence of rheb exhibited high similarity with other reported rhebs, such as 91% with that from homarus americanus (adv76255), 86% with that from carcinus maenas (adv76253) and 66% with that from homo sapiens (np_005605) (fig. 3). the nucleotide sequence of rheb has been submitted to genbank and the accession number is mg696863. the tissue distribution of rheb mrna the rt-pcr analysis was employed to detect the tissue distribution of the rheb mrna in different tissues and the β-actin gene as internal control. the lowest expression level of rheb transcripts was present in intestine. in other detected tissues, the rheb mrna transcripts were significantly higher 56 fig. 2 multiple alignments of deduced amino acid sequences for rheb proteins. the black shadow region indicated positions where all sequences share the same amino acid residue. similar amino acids are shaded in grey. g boxes and effector domain (switch i region) has been marked in the figure. the effector switch i region is responsible for interactions with the mtor protein, fkbp38 and other proteins (aspuria and tamanoi, 2004; ma et al., 2008). species and gene accession numbers are as follows: litopenaeus vannamei (mg696863), homarus americanus (adv76255), carcinus maenas (adv76253), zootermopsis nevadensis (xp_021934312), danio rerio (np_957023) and homo sapiens (np_005605). fig. 3 neighbor-joining (nj) phylogenic tree of rheb constructed using mega 6.0 software package based on the amino acid sequences of rhebs from different organisms. to derive confidence value for the phylogeny analysis, bootstrap trials were replicated 1000 times. species and protein sequences id are as follows: litopenaeus vannamei (mg696863), drosophila melanogaster (np_730950), danio rerio (np_957023), homo sapiens (np_005605), mus musculus (np_444305), homarus americanus (adv76255), carcinus maenas (adv76253), cimex lectularius (xp_014260514), zootermopsis nevadensis (xp_021934312), xenopus laevis (np_001080494), taeniopygia guttata (np_001232539), the rheb protein of l. vannamei is indicated with a black triangle. the numbers at the forks indicated the bootstrap value. the scale bar represents the proportion of amino acid differences between sequences based on nucleotide substitutions per site. than those in intestine. what’s more, the expression level of rheb among muscle, gill, hepatopancreas, eyestalk, intestine and stomach showed no significant difference (fig. 4). the temporal expression profile of rheb mrna post rapa stimulation the temporal mrna expression profile of rheb in muscles after rapa stimulation was examined via rt-pcr. the mrna expression of rheb in muscles increased significantly during 0.5 h-6 h (p < 0.05) after the stimulation of rapa. the mrna transcripts increased to the peak level at 1 h post stimulation (19.53-fold, p < 0.05). in the control group, no significant change of rheb mrna expression was observed after dmso injection during the whole experiment (fig. 5). the temporal expression profile of rheb mrna post dietary restriction, amino acid and rapa stimulation rt-pcr analysis was employed to examine the mrna expression profile of rheb in muscles after dietary restriction and at 30 min after injection of amino acid and rapa. as shown in fig. 6, the mrna 57 fig. 4 tissue distribution of rheb mrna transcripts detected by rt-pcr technology. β-actin gene was used as an internal control. rheb mrna transcripts come from stomach, eyestalk, hepatopancreas, muscle, intestine and gill of five adult shrimp. vertical bars represented mean ± s.d. (n = 5), and bars with different characters indicated significantly different (p < 0.05). transcripts of rheb in muscles increased after the shrimps deprived of feed for 3 days (3.2-fold compared with the origin level, p < 0.05). the leucine and arginine injections can relieve the influence caused by hunger and make the expression of rheb decrease to the original expression level. the injection of rapa can hinder the ability of leucine injection to relieve the increase expression level of rheb due to dietary restriction. there was still a significant 3.0-fold increase in rheb mrna in the group injected with leucine and rapa compared to control group (p < 0.05) (fig. 6). discussion the small gtpase rheb protein that can bind directly to mtorc1 and play a positive role in the regulation of mtor signaling pathway is involved in the activation of protein synthesis and growth (sato et al., 2008). in the present study, the full-length cdna of rheb was cloned from l. vannamei. the deduced polypeptide of rheb consisted of 182 amino acids, and its calculated molecular weight was 20.55 kda, which was close to those from vertebrate and invertebrate (fig. 1). the amino acid sequence of rheb shared over 60% similarities with other identified rhebs (fig. 2). moreover, a typical ras domain (fig. 1) was found which can react selectively and non-covalently with gtp (akashi et al., 2007). the switch i domain (switch i) is a highly conservative region that is crucial for the direct bond between mtor and rheb (long et al., 2007). our work also verified the viewpoint that the decapod crustacean rheb contained the five highly conserved g boxes which are related to gtp binding and gtpase activity (maclea et al., 2012). as shown in fig. 3, the rheb is a very conservative protein. the amino acid sequence of rheb in l. vannamei shared more than 60% similarity with other identified rheb proteins. to investigate the function of rheb in controlling cell function of shrimp, the distribution of its mrna in different tissues was detected by rt-pcr technique. the rheb mrna transcripts were observed consistently expressed in all the detected tissues. the analogous expression of rheb in the tissues indicated that it played vital role in the regulation of cell function. the reason for lower expression of rheb in intestine maybe was that intestine is the main tissue absorbing amino acids that makes the concentration of amino acids in intestine is higher than other tissues. in order to prevent the irrational regulation of mtor signaling pathway caused by amino acids, the low expression of rheb could be essential for the cell function regulation in intestine. to further understand the regulative roles of rheb in mtor signaling pathway, we depressed the mtor signaling pathway by rapa. the temporal expression profile in muscle post rapa stimulation was detected by rt-pcr technique. we found the expression of rheb increased when the mtor signaling pathway was depressed by rapa. the highest expression was found when we injected rapa into shrimps for 1 h. which was 19.53-fold (p < 0.05) of that in control group. previous studies found that rheb overexpression activates torc1 signaling (sun et al., 2008). ma et al. (2008) identified that rheb binds specifically to fkbp38 58 fig. 5 temporal mrna expression profiles of rheb detected by rt-pcr in shrimp muscle at 0, 0.5, 1, 2, 4 and 6 h post rapa stimulation. the shrimps injected with dmso were employed as control groups. β-actin gene was used as an internal control to calibrate the cdna template for all the samples. each value was shown as mean ± s.d. (n = 5), and bars with different characters indicated significantly different (p < 0.05). (highly similar to fkbp12) in a gtp-dependent manner through its switch 1 region in hek293 cells. the combination between rheb and fkbp38 can displace fkbp38 (an mtor inhibitor) from the fkbp/rapa-binding domain and contributes to mtorc1 activation (avruch et al., 2009). our work suggested that the feedback regulations between rheb and mtor were existing in shrimp. the depression of mtor signaling pathway caused by rapa can promote the expression of rheb. rheb overexpression might be able to activate torc1 signaling in shrimp, too. however, as yet unknown and the considerable additional work is needed to elucidate the feed-back mechanisms by which the rheb–tor interaction promotes tor kinase activity. mtor output is sensitive to amino-acid (especially leucine and arginine) (long et al., 2005a). furthermore, kimball et al. found leucine caused the most obvious stimulation of the tor signaling pathway compared with other amino acids (jefferson et al., 2001). so, we injected shrimps which were deprived of food for three days with leucine, arginine or rapamycin alone or leucine and rapamycin combination to explore the regulation of rheb expression related to mtor pathway under these circumstances. previous works have found dietary restriction (dr) can reduces mtorc1 activity (mejia et al., 2015; garratt et al., 2016). in our work, the dr group expression level of rheb was obviously increased. this result further proved the existence of feedback regulation between rheb and mtor. previous research has proved the inhibition of mtorc1 signaling path caused by leucine withdrawal can be completely reversed by overexpression of rheb (avruch et al., 2009). in our work, the adding of leucine and arginine to shrimp deprived food for three days can relieve the accelerated expression of rheb. the result proved that rheb was regulated by amino acids, and the regulation play an important role in mtor signaling path in shrimp. in “leu + rapa” group, we injected leucine and rapa into shrimps. we found that rapa can totally hinder the regulation of leucine to rheb compared with leucine group in shrimp. long x et al. found that rheb-mtor interaction can activate mtorc1 activity in vitro and the interaction appears to be regulated by amino acid (long et al., 2005a; long et al., 2005b). some work has been proved that rheb can relieves the inhibition mtor signaling path caused by rapa (ma et al., 2008). our results are consistent with these findings. so, we suspect that the inhibitory effect of rapa-fkbp12 complex and the positive impact of rheb on mtor signaling pathway are mutually inhibited in shrimp. the dominant rapa hamper the activation of mtor caused by amino acid. so, the rheb expression of “leu + rapa” group don’t have obvious change compared with dr group. and it was shown that the dr and rapa injection exhibited a similar effect on the expression of rheb (fig. 6). 59 fig. 6 the mrna expression profiles of rheb detected by rt-pcr post dietary restriction (dr), amino acid and rapa stimulation. the shrimps injected with pbs was the control group. the expression profiling of second group was done 3 days after dr and 30 min after injection with pbs. the mrna expression profiles of other groups were done 30 min after injection with leucine (leu), arginine (arg), rapamycin (rapa) and “leucine + rapamycin” (leu + rapa). β-actin gene was used as an internal control to calibrate the cdna template for all the samples. each value was shown as mean ± s.d. (n = 5), and bars with different characters indicated significantly different (p < 0.05). acknowledgement this research was supported by the national natural science foundation of china (41406151), and the chinese academy of sciences sts major deployment project (kfzd-sw-106). we are grateful to all the laboratory members for their technical advice and helpful discussion. competing financial interests the authors declare no competing financial interests. reference akashi s, shirouzu m, yokoyama s, takio k. detection of molecular ions of non-covalent complexes of rasgdp and rasgppnp by maldi-tofms. j. mass. spectrom. soc. jpn. 44: 269-277, 2007. aspuria pj, tamanoi f. the rheb family of gtp-binding proteins. cell. signalling 16: 1105-1112, 2004. avruch j, long x, lin y, ortiz-vega s, rapley j, papageorgiou a, et al. activation of mtorc1 in two steps: rheb-gtp activation of catalytic function and increased binding of substrates to raptor. biochem. soc. trans. 37: 223-226, 2009. avruch j, 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signaling in growth and metabolism. cell. 124: 471-484, 2006. accepted february 11, 2018 hypothetical photosensory structure in ciliated protozoan, blepharisma isj 3: 77-83, 2006 issn 1824-307x research report cyst wall formation in the ciliated protozoan colpoda cucullus: cyst wall is not originated from pellicle membranes a kida, t matsuoka institute of biological science, faculty of science, kochi university, kochi 780-8520, japan accepted 18 july 2006 abstract ultrastructural changes during encystment (resting cyst formation) of colpoda cucullus were observed with special reference to cyst wall formation. within 1.5 h after encystment induction, fragmentation of some of the mitochondria occurred, followed by the appearance of a number of net-like globules in the cytoplasm, which were expelled to outside and then involved in cell-to-cell or cell-to-substratum adhesion. the cells were transformed into a spherical shape, and a number of ellipsoidal vacuoles in which ectocyst precursor (amorphous substance) was contained appeared near the cell surface (some of these opened to the outside). in this stage, the ectocyst (outermost layer) was completed. in 3 ~ 10 h toluidine blue-stained substance (tbs), which was probably the precursor for the first synthesized layer of endocyst (en-1), was released from a point near the cell surface and diffused over the cell surface (diffused into between the ectocyst and plasma membrane). thereby, the ectocyst was lined by the en-1. thereafter, several layers of endocyst were periodically formed for 1 ~ 2 weeks. finally a number of reserve grains were accumulated, and cilia were resorbed. key words: colpoda; encystment; cyst wall; ectocyst; endocyst _________________________________________________________________________________________________________ introduction when the protozoans face hazardous environments such as dessication, lack of food organisms and overpopulation, some of them are transformed into resting cysts which can survive hostile environments. the species of genus colpoda (foissner, 1993) are one of the protozoans that have been widely studied with regard to the resting cyst formation (encystment). since the beginning of the last century (for review see corliss and esser, 1974), and especially for the last 50 years, ultrastructural research with ______________________________________________________________________ corresponding author: tatsuomi matsuoka institute of biological science, faculty of science, kochi university, kochi 780-8520, japan email: tmatsuok@cc.kochi-u.ac.jp special reference to the cyst wall formation has been reported (kawakami and yagiu, 1963a, b; tibbs, 1968; janisch, 1980; ruthmann and kuck, 1985; martín-gonzález et al., 1992, 1994; frenkel, 1994; delmonte corrado, 1996; chessa et al., 2002). the knowledge about the structure of the cyst wall of colpodid ciliates and the process of wall formation reported in the previous studies could be summarized as follows: (1) the cyst wall of the resting cyst is composed of a single outermost layer (ectocyst) and several inner layers (endocyst) [classified into ecto-, mesoand endocysts in some reports]; (2) in some studies, it has been suggested that the cyst wall may originate from pellicular membrane (kawakami and yagiu, 1963a; ruthmann and kuck, 1985); (3) in the precystic cells, ellipsoidal vesicles that are believed to be mucocysts 77 (frenkel, 1994) open to the outside, and the their content, which is presumably cyst wall precursor, is excreted (martín-gonzález et al., 1992, 1994; frenkel, 1994). however, we have neither evidence for membrane-derived cyst wall formation nor any images of electron micrographs showing that the ectoand/or endocyst are just being formed from materials excreted from presumed precursor-containing vacuoles. the present study revealed that ectocyst and endocyst of c. cucullus did not originate from pellicle membranes but instead were synthesized with different precursors excreted from the different types of vacuoles. materials and methods colpoda cucullus was cultured in a 0.1 % (w/v) infusion of dried cereal leaves inoculated with bacteria (enterobacter aerogenes) at 23 oc in the dark. bacteria were cultured on agar plates containing 1.5 % agar, 0.5 % polypeptone, 1 % meat extract and 0.5 % nacl. the cultured vegetative cells were collected by centrifugation (1,000 xg, 1 min) and subsequently suspended in a standard saline solution containing 1 mm cacl2, 1 mm kcl and 5 mm tris-hcl (ph 7.2). the cells were rinsed 2-3 times by repeating the sedimentation and suspension in standard saline solution, and finally suspended in the solution to induce encystment. for vital staining of precystic cells with toluidine blue, 0.1 % toluidine blue dissolved in the standard saline solution was added to an equal volume of cell suspension, and kept for 5 ~ 10 min. for prefixation of the precystic cells (0 ~ 1 h after encystment induction), one volume of the suspension of cells was mixed with 6 volumes of a glutaraldehyde (ga) fixative containing 6 % glutaraldehyde, 1 % oso4, 100 mm cacodylate buffer (ph 7.2) and 4 mm sucrose. after 10-min incubation, the prefixed samples were rinsed 5 times in 100 mm cacodylate buffer (ph 7.2) and then postfixed for 2 h in a fixative containing 1 % oso4, 100 mm cacodylate buffer (ph 7.2) and 2 mm sucrose. the precystic cells and mature cysts (1 h ~ 2 weeks after encystment induction) were prefixed with ga fixative without oso4 for 6 h and postfixed with a fixative containing 1 % oso4, 100 mm cacodylate buffer (ph 7.2) and 2 mm sucrose for 1 week. the postfixed samples were rinsed several times in distilled water, dehydrated through a graded ethanol series (30, 40, 50, 60, 70, 80, 90 and 100 % ethanol) for 15 min each and finally suspended in acetone. the dehydrated samples were embedded in spurr’s resin. ultrathin sections were stained with 3 % uranyl acetate and then with lead citrate (10 min each). the sections were observed under a transmission electron microscope (jeol, 1010t). fig. 1 transmission electron micrographs of a vegetative cell of c. cucullus (fig. 1a) and the precystic cell in the early phase of stage 1 (0 ~ 1 h after encystment induction) (figs 1b-d). fig. 1a (inset): a magnified picture of the cortical region, showing the alveolus (al). fig. 1c: arrowheads, mitochondria that has just been torn and fragmented. fig. 1d: fr, mitochondria fragmented into small pieces; ap, autophagosome-like structure. results the morphological events during encystment are described by dividing them into stage 1 ~ 4. the parentheses show the typical terms after encystment induction in which the cytoplasmic events in each stage are observed in most cells. stage 1 (0 ~ 1.5 h after encystment induction). fig. 1a is a longitudinal section of a vegetative cell, showing a number of small-sized vacuoles or vesicles in the cytoplasm and alveoli in the cortical region. within 1 h after encystment induction, some of the mitochondria were fragmented (figs 1b, c, d) and a number of autophagosome-like structures appeared (fig. 1d). during the latter part of this stage, a number of net-like globules that had been described previously (kawakami and yagiu, 1963a) appeared in the cytoplasm (figs 2a, b) and were subsequently released into extracellular space. stage 2 (1.5 ~ 3 h). the precystic cells became round, and cell movement was gradually slower and finally stopped. a number of spherical or ellipsoidal vacuoles, which were possibly similar organelles (mucocysts) previously reported in precystic cells of tillina magna (frenkel, 1994), were localized in the vicinity of the cell surface and opened to the extracellular space to excrete amorphous precursor material (fig. 3b) for an outermost layer (ectocyst) (figs 3a, b). the excreted materials were gradually deposited (fig. 3c) and finally formed an electron-dense single layer (ectocyst) (fig. 3d). stage 3 (3 ~ 10 h). by the time ectocyst completion occurred (figs 4a, b), the cells had ceased swimming, but cells continued to rotate 78 fig. 2 transmission electron micrographs of the precystic cells in the latter phase of stage 1 (1 ~ 1.5 h), showing net-like globules (gl) in the cytoplasm. fig. 2a, a total view of the cell; fig. 2b, a magnified picture and highly magnified one (inset) of net-like globules. for a while (10~30 min) by ciliary movement inside the ectocyst envelope. during this stage, the number of electron-lucent vacuoles was reduced, while numerous endoplasmic reticula and mottled or electron-dense granules appeared (figs 4a, b), which may be responsible for the next event, the synthesis of endocyst. a drastic event was visualized in toluidine blue-stained cells just after the precystic cells ceased to rotate inside the ectocyst envelope (figs 4c, d). an extremely large vacuole (fig. 4c) opened near the cell surface, and substance deeply stained with toluidine blue (tbs) diffused over the entire cell surface within a few minutes (figs 4c, d). the tbs release occurred at 1.5 h in cases when the encystment was most quickly induced but proceeded much more slowly in the case of typical cells. the cells whose cortical region was deeply stained by the diffusion of tbs could hardly be crushed by a mechanical press. in the electron micrograph of this stage, fibrous materials constituting the first layer (en-1) of endocyst were observed between the ectocyst and plasma membrane (fig. 4e), which probably correspond to tbs. fig. 3 transmission electron micrographs of precystic cells in stage 2, showing ectocyst formation. figs 3a-c: ec, ectocyst that has just been formed with amorphous precursor materials (fig. 3b, pr) excreted from spherical or ellipsoidal vacuoles. ma, macronucleus; v, vacuoles containing amorphous materials; ci, cilia; al, alveolus. fig. 3b (inset), a magnified picture of just excreted amorphous materials; fig. 3c (inset), magnified image of ectocyst just being formed. fig. 3d, an image showing a just completed ectocyst envelope (ec). fig. 4 transmission electron micrographs (figs 4a, b, e) of precystic cells and photomicrographs of toluidine blue-stained living cells (figs 4c, d), showing the process of first layer (en-1) formation of endocyst. fig. 4f [stage 4]: a transmission electron micrograph of the cell at 24 h after encystment induction, showing a completed first layer of endocyst (en-1). figs 4b, e, f: er, endoplasmic reticulum; ci, cilia; g, mottled granules; ec, ectocyst; en-1, endocyst-1 (first synthesized endocyst layer); al, alveolus. figs 4c, d: v, vacuole containing presumed precursor for endocyst layer (en-1); arrowhead, a point where toluidine blue-stained substance (tbs) was being released. 79 fig. 5 transmission electron micrographs of an immature cyst in the early phase of stage 4 (2 days after encystment induction), showing a third layer (en-3) of endocyst are being formed. fig. 5a (inset), a magnified picture showing the ectocyst (ec) lined by endocyst-1 (en-1). en-2, second synthesized layer of endocyst; en-3, third synthesized layer of endocyst; v, vacuole containing presumable precursor for endocyst; gl, net-like globule attached to surface of ectocyst; g, electron-dense granule; arrow (fig. 5a), a boundary of two cysts adhered to each other with crushed net-like globules; arrowheads in fig. 5b, vacuoles just beginning to excrete the precursor for endocyst. stage 4 (10 h~ 2 weeks). in most cells, the formation of en-1 was completed within 1 day, and the ectocyst was lined by the en-1 (fig. 4f). during this stage, several layers of endocyst were formed. as shown in fig. 5, in the 2-day-old immature cyst, a third layer (en-3) of endocyst was observed to be just forming. in this case, an extremely large vacuole (figs 5 a, b) associated with many electron-lucent small-sized vacuoles or ducts appeared near the cortical region, some of which opened to the space between the plasma membrane and the already synthesized fig. 6 a photomicrograph (fig. 6c) and transmission electron micrographs (figs 6a, b, d, e) of immature cysts at 1 week after encystment induction and in a 2-week-old mature cyst (fig. 6f). fig. 6a: 1-week-aged cyst. a vacuole (v) containing endocyst precursor opened to the space between the plasma membrane and the already synthesized innermost endocyst layer. fig. 6b: a magnified picture of cyst wall. inset, a highly magnified picture of endocyst showing that endocyst layers are composed of fine fibrous materials. fig. 6c: a photomicrograph of 1-week-aged living cyst. fig. 6d: a section of 1-week-aged cyst showing that cytoplasmic reconstruction rather progressed than the cyst shown in fig. 6a. fig. 6e: a magnified picture of the section shown in fig. 6d. fig. 6f: a section of 2-week-aged mature cyst. figs 6a ~ f: rg, reserve grains; ec, ectocyst; en, endocyst; ci, cilia; gl, net-like globules; gr, small grains surrounding macronucleus; ma, macronucleus; mt, mitochondria; en-3, third synthesized endocyst layer; al, alveolus; arrowhead in fig. 6e, electron-lucent amorphous materials. layer (en-2) of endocyst, and endocyst precursor was excreted (fig. 5b, arrowheads). in addition, a number of electron-dense granules were seen inside and in the vicinity of the large vacuole (figs 5b, g). the complex of the large vacuole and many small-sized vacuoles or ducts leading to the cell surface (fig. 5 b) may correspond to the vacuole (figs 4c, d) observed by light microscope. fig. 6 show a photomicrograph of living cell (fig. 6c) and electron micrographs (figs 6a, b, d, e) of 7-day-old and 2-week-old cysts (fig. 6f). some of the cysts were still immature, and endocyst precursor was observed being released from a large vacuole (fig. 6a) 80 fig. 7 schematic diagrams showing the entire encystment process (fig. 7a) and two different ideas for cyst wall formation (fig. 7b). fig. 7b-1: a hypothesis for ectocyst formation in which it originates pellicle membranes. fig. 7b-2: a schematic diagram showing ectoand endocyst formation based on the present results. ec, ectocyst; en, endocyst; gl, net-like globules; tbs (en), toluidine blue-stained substance (endocyst precursor); rg, reserve grains; al, alveolus, m, plasma membrane; p (ec), precursor for ectocyst. forming the innermost layer of endocyst. during this stage, the compact and narrow layers and broad layers of endocyst were observed to be alternatively formed (figs 6a, b). both of the layers seem to be composed of identical fibrous materials (fig. 6b). the broad layers of endocyst might not be produced by the fixation and dehydration processes for electron microscopy, because a broad space (layer) was seen in a living cyst (fig. 6c). after the endocyst layers were completed, the reserve grains gradually accumulated in the central region (fig. 6d), and 81 the cytoplasm became dappled by the appearance of amorphous electron-lucent structures (figs 6d, e), which seem to develop into the reserve grains. in addition, the macronucleus was surrounded by a number of small grains (fig. 6d). finally, electron-lucent amorphous structures were replaced by a large amount of regularly stacked ellipsoidal reserve grains (fig. 6f), and, as a result, the central region of cytoplasmic space was occupied mainly by the reserve grains and mitochondria located in the peripheral region. in the final stage (figs 6d-f), cilia were resorbed. discussion the outline of the encystment process with special reference to cyst wall formation is schematically summarized in fig. 7. the first drastic events in the stage 1 are the fragmentation of mitochondria and appearance of autophagosome-like structures (figs 1b, c, d, 7a). the fragmented mitochondria are probably digested inside the autophagosomes. the net-like globules appeared and excreted in the latter phase of this stage (figs 2, 7a) are probably involved in a cell-to-cell adhesion (see fig. 5a, arrow) or adhesion of the cells to the substratum. if the ectocyst originated from pellicle membranes, as suggested in the previous reports (kawakami and yagiu, 1963a; ruthmann and kuck, 1985; watoh et al., 2005), alveoli might be fused with one another to produce new plasma membrane and outermost double membranes developing into the ectocyst which are deposited with a precursor substance (fig. 7b-1). although electron microscopic images implying the fusion of alveoli are observed occasionally (watoh et al., 2005), no images showing that the outer double membranes produced by fusion of alveoli further developed into ectocyst or endocyst have been observed in the previous study (watoh et al., 2005). on the other hand, in the present study, we succeeded in obtaining successive images showing that the ectocyst was just being formed with amorphous substance excreted from vacuoles (figs 3a-d, ref. fig. 7b-2). in this process, neither the fusion of alveoli nor the appearance of new alveoli was detected. as a result, we believe that the images of fused alveoli observed in the previous report (watoh et al., 2005) may have been artifacts produced by the fixation and/or dehydration processes. the ectocyst formation is followed by a diffusion of tbs which is probably endocyst precursor (figs 4c, d). in the previous report (watoh et al., 2005), the tbs is suggested to diffuse between the first-formed layer of endocyst (en-1) and the plasma membrane based on the photomicroscopic observation. this idea should be modified, because the present electron microscopy revealed that during this stage, fibrous endocyst materials probably corresponding to tbs were observed in the space between the ectocyst and the plasma membrane (fig. 4e, ref. fig. 7b-2). the formation of endocyst-1 (en-1) is followed by the formation of several layers of endocyst, the precursor of which is probably supplied by a periodic excretion into the space between the plasma membrane and the innermost endocyst layer (figs 5, 6a). in this case, the newly formed endocyst shows the replica-like configuration of the cell surface, i.e. the smooth endocyst layers beneath the smooth cell surface (figs 6a, b) and undulate layers for furrowed cell surface (figs 6d, e). the alternative formation of the narrow and broad layers of endocyst may be responsible for the periodic shrinkage of the cell body. that is, broad endocyst layers may be formed when the endocyst precursor is excreted into the broad space between the plasma membrane and innermost endocyst layer which is produced by the cell shrinkage. every layer of endocyst is composed of fine fibrous materials (fig. 6b), indicating that it is derived from a common precursor material, which is expected to be deeply stained with toluidine blue. in the present study, we defined a “mature cyst” of c. cucullus as a cyst in which every cytoplasmic event (formation of ecto-and endocysts, resorption of cilia, accumulation of reserve grains, aggregation of mitochondria in the peripheral region of cytoplasm) during the resting cyst formation has occurred and further prominent change is not observed (see fig. 6f). it has been reported that in other species of colpoda, the silver impregnation method indicates that some pairs of kinetosomes disappear during resting cyst formation (martín-gonzález et al., 1991). unfortunately, the present microscopy failed to conclude whether the kinetosomal structures completely disappear in the mature cysts, because the cytoplasm of mature cysts is compacted and highly electron-dense. acknowledgement we thank ms watoh t for her technical assistance in electron microscopy. references chessa mg, largana i, trielli f, rosati g, politi h, angelini c, delmonte corrado mu. changes in the ultrastructure and glycoproteins of the cyst wall of colpoda cucullus during resting encystment. eur. j. protist. 38: 373-381, 2002. corliss jo, esser sc. comments on the role of the cyst in the life cycle and survival of free-living protozoa. trans. amer. micros. soc. 93: 578-593, 1974. delmonte corrado mu, chessa mg, pelli p. ultrastructural survey of mucocysts throughout the life cycle of colpoda cucullus (ciliophora, colpodea). 82 acta protozool. 35: 125-129, 1996. foissner w. colpodea (ciliophora). gustav fischer verlag, stuttgart, 1993. frenkel ma. the cyst wall formation in tillina magna (ciliophora, colpodidae). arch. protistenkd. 144: 17-29, 1994. janisch r. a freeze-etch study of the ultrastructure of colpoda cucullus protective cysts. acta protozool. 19: 239-246, 1980. kawakami h, yagiu r. an electron microscopical study of the change of fine structures in the ciliate, colpoda cucullus, during its life cycle. ii. from the preencystment stage to the early stage of the formation of the first layer of resting cyst membrane. zool. mag. 72: 146-151, 1963a. kawakami h, yagiu r. the electron microscopical study of the change of fine structure in the ciliate, colpoda cucullus, during its life cycle. iii. from the stage of completion of the first layer of resting cyst membrane to the completion of the resting cyst. zool. mag. 72: 224-229, 1963b. martín-gonzález a, benítez l, gutiérrez jc. cortical and nuclear events during cell division and resting cyst formation in colpoda inflata. j. protozool. 38: 338-344, 1991. martín-gonzález a, benítez l, gutiérrez jc. ultrastructural analysis of resting cysts and encystment in colpoda inflata. 2. encystment process and a review of ciliate resting cyst classification. cytobios 72: 93-106, 1992. martín-gonzález a, palacios g, gutiérrez jc. cyst wall precursors of colpoda inflata: a comparative ultrastructural study and a review of ciliate cyst wall precursors. cytobios 77: 215-223, 1994. ruthmann a, kuck a. formation of the cyst wall of the ciliate colpoda steinii. j. protozool. 32: 677-682, 1985. tibbs j. fine structure of colpoda steinii during encystment and excystment. j. protozool. 15: 725-732, 1968. watoh t, sekida s, yamamoto k, kida a, matsuoka t. morphological study on the encystment of the ciliated protozoan colpoda cucullus. j. protozool. res. 15: 20-28, 2005. 83 introduction materials and methods results discussion acknowledgement review 31 isj 15: 31-38, 2018 issn 1824-307x research report differential impact of pesticides and biopesticides on edaphic invertebrate communities in a citrus agroecosystem mz majeed1,2, m naveed2, ma riaz2,3, c-s ma1, m afzal2 1state key laboratory for biology of plant diseases and insect pests, institute of plant protection, chinese academy of agricultural sciences, beijing 100193, pr china 2department of entomology, college of agriculture, university of sargodha, 40100, sargodha, pakistan 3department of entomology, university of georgia, athens, ga 30602, usa accepted january 08, 2018 abstract edaphic invertebrate fauna is usually exposed directly or indirectly to a wide range of pesticides in agroecosystems worldwide. very few studies have assessed the negative effects of these pesticides on the diversity and population dynamics of soil invertebrates. in this study, the effect of most commonly used pesticides viz; bifenthrin (a synthetic pyrethroid), spinosad (a bio-insecticide), aliette (a synthetic fungicide) and trichoderma harzianum formulation (30x106 cells ml-1; a bio-fungicide) was assessed on soil invertebrate fauna in a citrus agroecosystem. secondary objective was to compare the impact of synthetic versus biological pesticides and insecticides versus fungicides. there was a significant effect of all pesticides on the population abundance of springtails (f4,14 = 16.53; p<0.001), mites (f4,14 = 12.07; p<0.001) and ants (f4,14 = 16.28; p<0.001). by and large, soil fauna got recovered after two to three weeks post-treatment. insecticides were more suppressive for soil invertebrates than fungicides. overall, biological pesticides i.e. spinosad and t. harzianum formulation were less disruptive to soil invertebrate fauna than synthetic conventional pesticides. hence, keeping in view the key role of soil invertebrates in soil sustainability and crop productivity, the utilization of biopesticides should be encouraged. key words: bio-pesticides; fungicides; insecticides; population abundance; soil invertebrates introduction citrus is an important fruit crop around the globe. however, its production is hampered by numerous species of insect pests including psyllids, leafminers, fruit flies and scales, and diseases including canker, greening and downy mildews (anjum and javaid, 2005; tahir et al., 2015). in order to control these pests and to protect their crop and yield, farmers indiscriminately and recurrently use a wide range of synthetic pesticides including insecticides and fungicides (monzo et al., 2014). although these pesticides provide the control of insect pests and diseases because of their rapid knockdown effect and save considerable yield loss by reducing the pest infestation, but concomitantly these agro-chemicals have many non-target effects ___________________________________________________________________________ corresponding author: muhammad zeeshan majeed state key laboratory for biology of plant diseases and insect pests institute of plant protection chinese academy of agricultural sciences beijing 100193, pr china e-mail: zeeshan.majeed@uos.edu.pk including disruption of beneficial organisms, insecticide resistance, pest resurgence and human health hazards (edwards, 2013; monzo et al., 2014). most of the pesticides being used by citrus growers in indo-pak region are broad spectrum and highly persistent in the environment diminishing many beneficial fauna along with the target pests (ashraf et al., 2014). regarding pesticide side effects, soil invertebrates are one of the non-target organisms which may be exposed to all pesticides either directly to spray splashes and drift or indirectly by contacting pesticidal residues on foliage, soil, litter and thatch (bünemann et al., 2006; larson et al., 2014). agricultural soils harbor a considerable diversity of invertebrates often classified as micro and mesofauna including collembolans, mites and tiny insects etc., and macro-fauna including earthworms, termites, ants, beetles and spiders etc. (lavelle et al., 1997; kocourek et al., 2013). these soil invertebrates play a crucial role in different ecological processes such as organic matter decomposition and nutrients cycling, and are indispensable for soil biological functioning and mailto:zeeshan.majeed@uos.edu.pk 32 table 1 list of treatments used in this study *water used in control plots was same as used for preparation of pesticides spray mixtures. sustained crop productivity in agro-ecosystems (lavelle et al., 1997; lardo et al., 2012; majeed, 2012; bagyaraj et al., 2016). soil fauna, particularly mesoand macro-fauna, improve soil physicochemical conditions through their feeding (ingestion, digestion and ejection) and foraging (tunneling, boring, mining, movement) activities (lee and pankhurst, 1992; edwards and bohlen, 1996; lavelle at al., 1997; jouquet et al., 2006; coleman and wall, 2015; bagyaraj et al., 2016). keeping in view the ecological importance of soil invertebrates and lack of information regarding the side effects of most commonly and widely used pesticides in indigenous citrus agroecosystems on the soil invertebrate fauna, this study sought to compare the impact of different type of pesticides (insecticides and fungicides) on the density (abundance) and diversity (community assemblage) of non-target soil invertebrates. our a priori hypothesis was that conventional pesticides would have more severe effects on soil fauna than biopesticides. to achieve these objectives, different pesticides were applied according their labelrecommended dose rates on the under canopy area of citrus plants and preand post-application data regarding soil invertebrate fauna was collected for different time intervals up to two months. materials and methods the study area is situated in the district sargodha of the province of punjab (pakistan) and is characterized by semi-arid sub-tropical climatic conditions with mean annual temperature and precipitation of 23 °c and 450 mm, respectively. citrus is the principal fruit crop of sargodha region. study was conducted in private citrus orchards of kinnow mandarin (cv. citrus reticulata) (32°05'n and 72°40'e) situated in the vicinity of the college of agriculture, university of sargodha. orchard age was approximately 12 years and was not treated with any pesticide for last nine weeks. fifteen healthy and equal sized citrus trees were selected and tagged at random leaving rows of plants on all sides of the orchard as buffer zone to avoid edge effect. five treatments as given in table 1 including four most commonly used pesticides by indigenous citrus growers and one control (sprayed only with tap water) were applied on 30 april, 2017 on the under-canopy soil area of the citrus trees according to randomized complete block (rcb) design with five replications for each treatment. pesticides were applied according to their label-recommended dose rates using a back-mounted knapsack pump sprayer from a height of 1.5 m to mimic the pesticide spray drift. physico-chemical characteristics of the study soil were also determined for pre-treatment soil samples (table 2). pesticides included bifenthrin (a synthetic pyrethroid insecticide), spinosad (a microbial bioinsecticide), aliette (an aluminium based (fosetyl-al) synthetic fungicide) and formulation of trichoderma harzianum (a bio-fungicide). there were two motives behind the selection of these pesticides. first was to have a comparative assessment of the impact of different pesticides (insecticides and fungicides) and different pesticide groups (i.e. synthetic and biological) on soil non-target fauna. table 2 physico-chemical characteristics of the soil of citrus orchards studied soil characteristic condition/content soil texture sandy loam ph 7.8 (0.03) ece ( µs cm-1) 2410.0 (221.4) soil organic matter (g kg-1) 8.1 (0.29) soil organic-c (g kg-1 soil) 4.7 (0.16) total soil n (mg kg-1) 408.6 (15.01) nahco3 extractable-p (mg kg -1 soil) 7.9 (0.40) extractable-k (mg kg-1 soil) 163.4 (5.65) values are means of five independent soil composite samples along with standard errors within parenthesis. treatment no. treatment application rate 1 bifenthrin (synthetic pyrethroid) 625 ml ha-1 2 spinosad (microbial bio-insecticide) 150 ml ha-1 3 aliette (fosetyl-al; synthetic fungicide) 7 kg ha-1 4 trichoderma harzianum (bio-fungicide) 30x106 conidia ml-1 5 control (water)* 33 table 3 diversity indices of different edaphic faunal (invertebrate) groups in citrus orchard soils treated with different types of pesticides dbt: days before treatment; dat = days after treatment second criterion was that these pesticides were the most commonly used by citrus famers in sargodha region as assessed from a preliminary survey of local pesticide dealers and citrus growers. three 0 15 cm deep soil samples were collected from each treatment 2 days before, 3, 15, 30 and 60 days after application of treatments. each sample (weighing about 1,750 g) was the composite of four sub-samples taken randomly from four sides of the treated plant using 10x10 cm metallic soil corer. these samples were brought to the laboratory of the department of entomology and data regarding soil invertebrate fauna was taken. macro-invertebrates were collected and enumerated from each sample manually, while mesoand micro-invertebrates were extracted from samples by installing them on tullgren-berlese funnel for 24 h. extracted invertebrates were preserved in 50% ethanol solution in transparent 20 ml plastic vials for their further identification and enumeration under light microscope up to higher taxonomic (order, genus or family) level. statistica® version 7.1 (statsoft®, france) was used for statistical interpretation of data. normality of data was checked and data were transformed by log10 (x+2) before further analyses where normality was not met. data regarding soil fauna was subjected to 2-way factorial analysis of variance with treatment and time interval as factors and twosample student’s t-tests were used to compare pesticides and/or their groups. for effect of each pesticide on soil invertebrates, one-way anova was applied at 95% confidence level followed by tukey’s highest significant difference (hsd) tests to compare treatment means. for faunal (invertebrates) community assemblage determination, shannon-wiener’s index, faunal group richness and evenness indices were calculated as described by ahmed et al. (2017) along with the graphical presentation (pie charts) of data. moreover, multivariate analysis of significance was also run to assess the impact of pesticides on soil invertebrate fauna. results impact of pesticides on the diversity of soil faunal groups results have demonstrated that shannonwiener diversity index, which estimates the relative richness and abundance of different groups or species of organisms collected and sampled from different locations (shannon-wiener, 1963), fluctuated among different time intervals for all treatments (table 3). maximum diversity index (1.90) was found for control treatment at 30dat (days after treatment) while minimum was recorded for bio-fungicide at 15dat (table 3) for all pesticide treatments, diversity of soil invertebrate faunal groups reduced for the first two weeks posttreatment as compared to the diversity index of control treatment which was least perturbed during the entire period of experiment. after 15 days posttreatment, shannon-wiener index increased to its maximum at 30 dat for all treatments but then reduced to normal level at 60dat. pesticide treatments had a differential impact on the evenness index of soil faunal groups. evenness index measures the relative abundance of species or organismal groups of an area. in case of insecticide (bifenthrin), maximum evenness (0.79) was observed just after three days of spray but then reduced slightly as compared to control (before spray) value. in case of bio-insecticide (spinosad), minimum evenness value (0.58) was observed at 30dat and then returned to normal (control) value after one month. in case of fungicides, both synthetic (aliette) and bio-fungicide (t. harzianum formulation) showed similar response regarding their effect on fauna group evenness with minimum values (0.55 and 0.53, respectively) at 15dat (table 3). similarly, soil faunal (invertebrate) groups’ richness was reduced up to 15dat, then increased to a maximum value of 11.00 at 30dat, and again decreased up to normal level at 60dat (table 3). among treatments, synthetic fungicide showed minimum values of richness of soil faunal groups as compared to others, while insecticide (bifenthrin) showed maximum fluctuation in faunal group richness index (table 3). effect of pesticides on community assemblages of invertebrate faunal groups gross higher-level taxonomic composition of treated soil samples have been presented in the form of pie charts (fig. 1). according to this graphical presentation of invertebrate fauna encountered in soil samples, collembolans and mites were the most abundant faunal groups found in all samples, followed by ants, spiders and rove beetles. the least dominant groups were oligochaeta (earthworms) and carabid beetles. the diversity indices shannon w iener's diversity index richness index evenness index treatments 1dbt 3dat 15dat 30dat 60dat 1dbt 3dat 15dat 30dat 60dat 1dbt 3dat 15dat 30dat 60dat insecticide 1.56 1.54 1.22 1.44 1.40 10.00 7.00 7.00 10.00 7.00 0.68 0.79 0.63 0.62 0.72 bio-insecticide 1.39 1.53 1.13 1.23 1.51 9.00 9.00 7.00 10.00 9.00 0.63 0.70 0.58 0.53 0.69 fungicide 1.44 1.35 1.20 1.59 1.51 9.00 8.00 9.00 9.00 10.00 0.65 0.65 0.55 0.72 0.66 bio-fungicide 1.44 1.44 1.04 1.70 1.46 10.00 9.00 7.00 11.00 8.00 0.63 0.66 0.53 0.71 0.70 control 1.47 1.55 1.74 1.90 1.68 9.00 9.00 9.00 11.00 10.00 0.67 0.71 0.79 0.79 0.73 34 fig. 1 pie-charts showing community assemblages of edaphic faunal (invertebrate) groups in a citrus agroecosystem at different time intervals in response to application of different types of pesticides. (dbt = days before treatment; dat = days after treatment). most prominent change occurred in the community assemblages of soils treated with insecticide (bifenthrin) and bio-insecticide (spinosad) till 15 days post-exposure. the most effected faunal groups were ants and collembolans (fig. 1). for the first month of experiment, the faunal community assemblages remained dominant by collembolans (springtails) in all samples while oribatid mites (cryptostigmata) outnumbered all other invertebrate faunal groups in the last observation at 60 days post treatment. impact of pesticides on population abundance of different soil faunal groups collembola (springtails), cryptostigmata (oribatid mites), formicidae (ants) and araneae (spiders) were the most abundant and dominant faunal groups in the study soils of citrus orchard. therefore, the response of only these faunal groups towards different pesticides was further analyzed statistically apart from its graphical representation (fig. 2). in case of micro-invertebrates (collembola and cryptostigmata), insecticide (bifenthrin) reduced three times the average population of springtails and oribatid mites at 3dat than their control population (i.e. 15.0 springtails and 13.3 mites sample-1). the maximum population of springtails and mites reached at 30dat (i.e. 35.0 springtails and 16.3 mites sample-1). however, in case of all treatments including control, population of springtails suddenly decreased at 60dat except mites (fig. 2). similarly, bio-insecticide (spinosad) had a little but significant effect while fungicide and bio-fungicide had no significant effect on population abundance of springtails and oribatid mites. similarly, regarding macro-invertebrates (formicidae and araneae), a similar trend had been observed for ants. while in case of spiders, there was no clear-cut trend regarding the impact of pesticides on their population dynamics. maximum population of ants and spiders (i.e. 8.67 ants and 3.67 spiders sample-1) were recorded at 30dat and 60dat respectively, while the minimum ones (i.e. 0.67 ants and 0.33 mites sample-1) at 15dat and 60dat, respectively. in general, population of all faunal groups in control soils, which were treated with water only, gradually increased till 30dat, and then decreased at 60dat for all groups except oribatid mites which continued to increase till 60dat. however, in control (water) treatment, spider population increased gradually from start to the end of experiment at 60dat (fig. 2). 35 fig. 2 impact of different types of pesticides on the population abundance of major edaphic invertebrate groups in a citrus agroecosystem. data points represent population means ± standard error (n = 4). for each faunal group, treatments bearing same superscripted letters are not statistically different from each other (factorial (two factor) anova; α = 0.05). (dbt = days before treatment; dat = days after treatment; bf = bio-fungicide; f = fungicide; bi = bio-insecticide; i = insecticide; c = control). according to two-way factorial analysis of variance, insecticide (bifenthrin) had the most drastic and significant effect on population abundance of all edaphic faunal groups followed by bio-insecticide (spinosad), while the least disturbing pesticide for soil invertebrate fauna was formulation t. harzianum (bio-fungicide) (fig. 2). fungicide (aliette) exhibited an intermediate response (fig. 2). overall, there was a significant effect of pesticides on the population abundance of springtails (f4,14 = 16.53; p<0.001), oribatid mites (f4,14 = 12.07; p<0.001), formicid ants (f4,14 = 16.28; p<0.001) but non-significant for the population abundance of spiders (f4,14 = 1.51; p=0.212). similarly, observation times had a significant effect on the population abundance of springtails (f4,14 = 4.46; p<0.01), oribatid mites (f4,14 = 3.01; p=0.027), formicid ants (f4,14 = 6.60; p<0.001) and spiders (f4,14 = 9.27; p<0.001). however, the interaction of treatment and time exhibited significant effect on the population of all faunal groups except spiders (araneae). nevertheless, according to multivariate analysis, all treatments (pesticides) and time intervals exhibited a significant (p < 0.05) effect on overall population abundance of soil invertebrate fauna encountered in soil samples of different treatments, except for time effect of bio-fungicide (p = 0.2455; table 4). discussion in sustainable agriculture, one of the contemporary ecological issues regarding extensive use of pesticides is their deleterious effects on nontarget organisms including soil invertebrate fauna (edwards, 2013; pisa et al., 2015). beneficial edaphic invertebrate fauna is usually exposed directly or indirectly to a wide range of pesticides (frampton and van den brink, 2006; adamski et al., 2009; larson et al., 2014). this study was carried out to determine the impact of different types of pesticides on edaphic soil fauna in a citrus orchard. two types of pesticides i.e. insecticides and fungicides with different origins i.e. synthetic and biological were evaluated under field conditions. most commonly used insecticides selected in this study were bifenthrin and spinosad, while fungicides were aliette and t. harzianum formulation containing 30x106 cells ml-1. soil fauna was sampled, identified up to group or order level and enumerated at regular time intervals for two months post treatment. the overall effect of these pesticides on diversity of soil invertebrate fauna was evaluated by calculating three diversity indices i.e. shannonwiener diversity index, faunal group evenness index and faunal group richness index, while the abundance of major soil faunal groups was compared 36 table 4 multivariate analysis of significance for the effect of different types of pesticides on the abundance of edaphic faunal (invertebrate) groups in citrus orchard soils treatments/effect wilk’s lambda value f-value hypothesis df error df p-value insecticide 0.000182 2061.87 8 3.00 0.0000 *** observation time 0.000026 6.51 32 12.66 0.0005 *** bio-insecticide 0.001722 331.34 7 4.00 0.0000 *** observation time 0.002005 2.60 28 15.84 0.0246 * fungicide 0.000939 608.02 7 4.00 0.0001 *** observation time 0.001874 2.66 28 15.84 0.0222 * bio-fungicide 0.001143 499.34 7 4.00 0.0000 *** observation time 0.011285 1.39 28 15.84 0.2455 *** p < 0.001; * p < 0.01; * p < 0.05; multivariate analysis (effective decomposition of the hypothesis) at α = 0.05 with control (water) treatment and with pretreatment data of soil fauna as well. study results revealed that the insecticide bifenthrin, a synthetic pyrethroid, exerted considerable reduction of soil invertebrate fauna particularly of springtails, mites, ants and earthworms. similar findings have been reported by frampton and van den brink (2007) and larson et al. (2014) that pyrethroid insecticides like cypermethrin and bifenthrin had more pronounced negative effects on soil-dwelling fauna as compared to organophosphate (chlorpyriphos) and neonicotinoids (clothianidin). similar effects of pyrethroid insecticides (deltamethrin and cypermethrin) on soil-inhabiting spiders have been described by pekár and beneš (2008). nevertheless, jänsch et al. (2006) also reported the most pronounced deleterious effects of pyrethroid and organophosphate insecticides to earthworms, spiders, mites and springtails. hence, the continuous use of such toxic insecticides may reduce the diversity and abundance of soil nontarget invertebrates (bünemann et al., 2006; frampton and van den brink, 2006; adamski et al., 2009). although spinosad, which is a bio-insecticide derived from metabolites of soil-borne actinomycete saccharopolyspora spinosa, reduced the diversity and abundance of soil fauna particularly of springtails, mites and ants, however, it exhibited relatively less effect on soil fauna as compared to bifenthrin. insecticides of biological origin such as spinosad are replacing conventional insecticides due to their higher target specificity, quick environmental biodegradation and more biorational and eco-friendly nature (williams et al., 2003; biondi et al., 2012). nevertheless, the combined effect of insecticide and bio-insecticide on soil non-target fauna was also significant. both type of chemicals suppressed population abundance and community assemblage of all soil faunal groups up to 15 days post-application. these observations are in accordance with those of pekár and beneš (2008) who demonstrated up to 100 % mortality of soilinhabiting spiders by their exposure to few hours to 20 days old residues of insecticides (chlorpyrifos, cypermethrin and deltamethrin). on the other hand, fungicide aliette (fosetyl aluminum) exerted very little reduction of some soil fauna only for three days post-application while t. harzianum formulation did not affect soil fauna as much as insecticides. al-assiuty et al. (2014) described similar findings that bio-fungicides have less severe effect on community structure and population size of oribatid mites than synthetic fungicides. however, possible reasons for the observed little or no significant effects on nontargets in case of fungicides might be due to the poor diversity of invertebrates in study soils as well as the application of treatments on limited soil patches close to citrus plants as manifested by babendreier et al. (2015). nevertheless, the effects of both fungicides were non-significant. this observation is in context with the findings of moreby et al. (1997) and jänsch et al. (2006) who found that fungicides have less negative effect on soil dwelling invertebrates than insecticides. however, prolonged exposure to fungicides may reduce the population abundance of soil fauna either by exerting immediate changes in soil food web system (jänsch et al., 2006) or by affecting the behavior and longterm survival of soil invertebrates (evans et al., 2010). regarding community assemblages of different soil faunal groups, the most prominent changes were observed in case of micro-invertebrates (springtails and mites) and ants in response to insecticides. in case of fungicides, community assemblages of soil fauna remained almost identical at all sampling dates. this trend corroborates the findings of moreby et al. (1997) and al-assiuty et al. (2014). nevertheless, diversity of different edaphic faunal groups collected and identified during the study was influenced by the application of pesticides. in general, all diversity indices i.e. shannon-wiener’ index, species (faunal group) evenness and richness indices were lowered in the first two weeks of application and then either 37 recovered to normal or increased from 15dat to 30dat levels. the same pattern of recovery was dominant in both micro-invertebrates (springtails and mites) and macro-invertebrates (ants, spiders, rove beetles and earthworms). soil invertebrates usually have high recovery rates following the pesticidal application but repeated use of pesticides may exacerbate the situation of soil biological functioning (desneux et al., 2007; iloba and ekrakene, 2008; evans et al., 2010). however, there was a sudden decline in the diversity and abundance as well for all the treatments including control one. this trend might be due to the increased temperature in the month of june. only mite population exhibited an increasing trend till 60dat, which may be due the fact that mites survive better in hot and dry environment than other invertebrates (behan et al., 2003; al-assiuty et al., 2014). nevertheless, some insecticides such as neonicotinoids have shown a positive effect on the population abundance of mites (pisa et al., 2014). apart from four major faunal groups, i.e. springtails (collembola), mites (cryptostigmata), ants (formicidae) and spiders (araneae), rove (staphylinidae) and ground (carabaiedae) beetles were also encountered in all soil samples. nevertheless, rove beetles showed the minimum impact of pesticides while ground beetles population showed a decreasing trend in all pesticidal treatments. these results are in line with those of huusela-veistola et al. (1996) who demonstrated that ground beetles are very sensitive to agrochemicals than other macro-invertebrates, particularly staphylinidae beetles (honěk et al., 2012). conclusion the principal finding of this study is that synthetic pesticides (insecticides and fungicides) have more adverse effects on soil biological components such as soil invertebrate fauna than pesticides of biological origin. therefore, keeping in view the key role of soil fauna in soil sustainability and crop productivity and the ecological implications of pesticides in citrus agroecosystems, farmers should opt for more eco-friendly plant protection options such as biopesticides. references adamski z, błoszyk j, piosik k, tomczak k. effects of diflubenzuron and mancozeb on soil microarthropods: a long-term study. biol. lett. 46: 3-13, 2009. al-assiuty ani, khalil ma, ismail awa, van straalen nm, ageba mf. effects of fungicides and biofungicides on population density and community structure of soil oribatid mites. sci. total environ. 466: 412-420, 2014. anjum t, javaid a. major diseases of citrus in pakistan: a review. int. j. biol. biotechnol. 2: 793-796, 2005. ashraf s, khan ga, ali s, iftikhar m, mehmood n. managing insect pests & diseases of citrus: on farm analysis from pakistan. pak. j. phytopathol. 26: 301-307, 2014. babendreier d, jeanneret p, pilz c, toepfer s. non‐target effects of insecticides, entomopathogenic fungi and nematodes applied against western corn rootworm larvae in maize. j. appl. entomol. 139: 457-467, 2015. bagyaraj dj, nethravathi cj, nitin ks. soil biodiversity and arthropods: role in soil fertility. in: chakravarthy ak, sridhara s. 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[phd thesis.] universite montpellier ii, montpellier, france: pp 235, 2012. monzo c, qureshi ja, stansly pa. insecticide sprays, natural enemy assemblages and predation on asian citrus psyllid, diaphorina citri (hemiptera: psyllidae). bull. entomol. res. 104: 576-585, 2014. moreby sj, sotherton nw, jepson pc. the effects of pesticides on species of non-target heteroptera inhabiting cereal fields in southern england. pestic. sci. 51: 39-48, 1997. pekár s, beneš j. aged pesticide residues are detrimental to agrobiont spiders (araneae). j. appl. entomol. 132: 614-622, 2008. pisa lw, amaral-rogers v, belzunces lp, bonmatin jm, downs ca, goulson d, et al. effects of neonicotinoids and fipronil on nontarget invertebrates. environ. sci. pollut. res. 22: 68-102, 2015. stork ne, eggleton p. invertebrates as determinants and indicators of soil quality. am. j. alternative agr. 7: 38-47, 1992. tahir hm, nazarat i, naseem s, butt a, yaqoob r, mukhtar mk, et al. seasonal dynamics of spiders and insect pests in citrus orchards of district sargodha, pakistan. pak. j. zool. 47: 1673-1681, 2015. williams t, valle j, viñuela e. is the naturally derived insecticide spinosad® compatible with insect natural enemies? biocontrol sci. technol. 13: 459-475, 2003. chimerism a natural ability to tolerate kin, evolutionary traits connecting mammalian and protochordates isj 6: s9-s20, 2009 issn 1824-307x review chimerism a natural ability to tolerate kin, evolutionary traits connecting mammalian and protochordates a voskoboynik institute of stem cell biology and regenerative medicine, department of pathology, stanford university school of medicine, stanford, usa and department of developmental biology, stanford university hopkins marine station, pacific grove, usa accepted march 13, 2009 abstract in the middle of the 20th century, owen (1945, 1954) and billingham et al. (1953) immunological studies suggested that fetal exposure to foreign antigens during pregnancy induce immunologic tolerance in the fetus. recently, mold et al. found that a substantial number of maternal cells crosses the placenta to reside in fetal lymph nodes and induces the development of regulatory t cells (tregs) that suppress fetal anti-maternal immunity. these tregs cells persist till, at least, early adulthood. this result demonstrates how chimerism induces fetal tolerance to maternal antigens during mammalian pregnancy. natural chimerism is the coexistence of two or more genomic lineages within the same individual. it is a common phenomenon which can be detected in a wide variety of multi-cellular organisms. in mammals, natural chimerism can be established during pregnancy between the mother and the fetus or between fetuses in a multiple embryos pregnancy. restriction of natural chimerism mainly to kin is also observed in colonial marine protochordates. in protochordates, like botryllus schlosseri, natural chimerism can be established through fusion of vasculature, between a parent colony and its progeny or between siblings (adult distinct colonies).the ability to tolerate a partial allogeneic individual and to create a chimeric entity between these colonies is determined by a single, highly polymorphic, fusion/histocompatibility locus (fu/hc). colonies that share at least one allele in their fu/hc locus would fuse upon contact. a pair that does not share any fu/hc allele would not. in the chimera, cells transmigrate between partners and in some cases, replace the germline and/or the somatic tissues of the host. this genotype replacement is mediated by stem cells (termed somatic/germ cell parasitism). botryllus colonies propagate asexually through budding, therefore somatic stem cell parasitism in host colonies can induce the development of a partial allogeneic entity (buds) within the host colony. in this way, chimerism in protochordates serves as a state that enables the development of a “virtual embryo” within the host colony. in light of mold et al., study, which demonstrates a role to chimerism in tolerance induction during pregnancy, studying the immunological mediators for natural acceptance of partial allogeneic allograft in protochordates may reveal the evolutionary precursors to the tolerance state during mammalian pregnancy. key words: chimerism; immunologic tolerance; stem cells; tunicate; mammalian pregnancy; fu/hc; uterine nk chimerism in pregnancy an increasing number of studies have detected ___________________________________________________________________________ corresponding author: avelet voskoboynik institute of stem cell biology and regenerative medicine department of pathology stanford university school of medicine stanford, ca 94305, usa department of developmental biology stanford university hopkins marine station pacific grove, ca 93950 usa email: ayeletv@stanford.edu natural chimerism in a wide variety of multi-cellular organisms, including vertebrates. this suggests that chimerism is a common phenomenon in the wild (e.g. buss, 1982; van dijk et al., 1996; bianchi et al., 1996; marleau et al., 2003; pineda-krch and lehtila, 2004; rinkevich 2004a, b; kaplan and land, 2005; khosrotehraniet et al., 2005; loubiere et al., 2006; bianchi, 2007; mold et al., 2008). studies demonstrated that chimerism can be experimentally initiated by engraftment of a single stem cell (e.g. cao et al., 2004; laird et al., 2005). human chimerism which originated from twins was reported already in 1953. with the advent of blood typing, it was discovered that some people have blood cells s9 mailto:ayeletv@stanford.edu of more than one type (dunsford et al., 1953). until 1996, twins chimerism seems to be very rare in humans, only 40 cases of twins chimerism were reported (trippet, 1983). these cases were found during routine blood grouping, a procedure which detects a mixture of red blood cells only when the percentage of allogeneic cell in the blood is above 5 % of all blood cells (van dijk et al., 1996). van dijk et al. (1996) applied a more sensitive method and found that in humans, 8 % of non-identical twins and 21 % of triplets are chimeric (van dijk et al., 1996). twin chimerism with high levels of blood cells from the other twin yields tolerance to donor antigens. all chimeras twins which were identified in a routine blood grouping (frequency >5 % of chimeric cell population) had mutual tolerance to blood transfusion and skin grafts from their chimeric partner (trippet, 1983). chimeras with low frequencies of foreign cell population (like those identified by van dijk et al. (1996), with chimeric cell population frequency of ~0.1 %) were not tolerant to their twin foreign antigens and rejected blood grafts. fetal-maternal micro –chimerism is another natural chimerism that is developed during human pregnancy. cell trafficking, during normal human pregnancy, between the mother and the fetus, leads to the establishment of a chimeric state in both. fetal cells were identified in the maternal circulation (herzenberg, 1979; lo et al., 1989, 1996, 1998; petit et al., 1997; bianchi et al., 1997, 2002). maternal cells were found in the umbilical cord and in fetal blood samples (socie et al., 1994; hall et al., 1995; lo et al., 1996; petit et al., 1995; 1997; bauer et al., 2001). during human pregnancy, 20 %-50 % of the erythroblasts in maternal blood are originated from the fetal (sekizawa et al., 1996; von eggeline et al., 1997; oosterwijk et al., 1998; wachtel et al., 1998; troeger et al., 1999). these percentages indicate relatively high levels of chimerism between mother and fetal during pregnancy in the host mother. persistence of allogeneic fetal progenitor cells were detected in chimeric mothers (humans) as long as 27 years after birth (termed as microchimerism, mc; bianchi et al., 1996). fetal mesenchymal stem cells were detected in maternal bone marrow and in mother’s ribs even 35 years after delivery (1 fetal cell in ~105 maternal cells; o’donoghue et al., 2004a, b). long term persistence of maternal cells in progeny (maternal mc) was described too (maloney et al., 1999). loubiere et al. (2006) found that high percent of healthy adult women harbor maternal and fetal mc among their t and b lymphocytes, monocyte/macrophages and nk cells. 78 % (21/27) of healthy tested women carried fetal mc (tested women’s children average age 12.6 years), 39 % carried maternal chimerism (12/31 average age = 39; loubiere et al., 2006). maternal / fetal mc is not limited to blood cells, it has been identified in many organs including normal kidney, liver and heart of women with or without pregnancy history (chimerism in different organs was detected in 23 out of 75 tested women, ages 29-93 at death; koopmans et al., 2005). since stem cells are the only cells in the tissues with a self-renewing capability, the detection of maternal and fetal mc up to four decades following delivery indicates that healthy humans harbor long-surviving populations of maternal/fetal hematopoietic stem cells. the above studies suggest that during pregnancy fetal/maternal stem cells enter maternal/fetal blood engraft, differentiate in host tissues and remain presence throughout its entire life. tolerance in pregnancy acceptance of a fetus, which expresses paternally inherited alloantigens, by the mother during pregnancy is a unique example of the way immune system reshapes a destructive alloimmune response to a state of tolerance (guleria and sayegh, 2007). intolerance of the embryo, as an antigenetically foreign body growing within the female, can represent a significant obstacle to reproduction. in humans, it is estimated that 70 % of conceptions fail (hill, 1992). similar estimates of early fetal loss, ranging from 10 % to 60 % have been obtained for other mammalian species (baker and bellis, 1995). in human, approximately 45 % of the couples experiencing primary recurrent spontaneous abortion have been linked to immunological factors (clark, 1999). on the other hand, during successful mammalian pregnancy, the semiallogeneic fetus resides comfortably within the maternal uterus and is protected from response of maternal graft rejection. the maternal immune system is active during pregnancy, yet mothers tolerate and do not reject their genetically disparate fetuses (hunt et al., 2005; lightner et al., 2008). there is growing evidence that viviparous reproduction depends on a two-way interactions between fetal and maternal tissues that involve humoral, cellular, innate and adaptive immune responses (hill, 1995; guleria and sayegh 2007; mold et al., 2008). these include, expression of non-classical mhc molecules by trophoblast cells (king et al., 2000; ishitani et al., 2003; hunt et al., 2005; lightner et al., 2008), tryptophan catabolism by the enzyme ido (munn et al., 1998), t cell apoptosis (hunt et al., 1997), the complement system, regulatory t cells (tregs) and inhibitory costimulatory molecule programmed death ligand (pdl) 1 (guleria et al., 2005; lightner et al., 2008; mold et al., 2008). it was also suggested that dendritic cells have a potential role in reproductive immunology and chemokine decoy receptors (borroni et al., 2008; kammerer et al., 2008). the classic, highly polymorphic mhc loci has a minimal expression, immediately following implantation, but it is increased steadily in placental and extravillous cytotrophoblast tissues during pregnancy (kurpisz et al., 1995). restriction of polymorphic mhc gene expression to later stages of embryonic development is probably useful to limit maternal rejection of the fetus. the contribution of each mechanism and the potential interactions among the various pathways are just beginning to emerge (guleria and sayegh, 2007). liegeois (1983) hypothesized that chimerism has a role in tolerance induction and maintenance during placental pregnancy. this hypothesis, although never been tested directly, is consistent with historical and recent findings which connect s10 induction of donor specific tolerance in fetal with cell chimerism during pregnancy. in 1945, owen found that bovine fraternal twins shared for life the blood cells types of both calves. based on earlier observations, indicating that bovine twins share a single placenta and blood circulation during fetal phase (lillie, 1916), owen conjectured that blood cells and their precursors (which he called “embryonal cells ancestral to the erythrocytes”) move from one twin to the other in the uterus, allowing mixing of blood cells from both genotypes (owen, 1945). bovine fraternal twins frequently exhibit a complete lifelong mutual tolerance to each other’s leukocyte and a temporary (up to 15 months) tolerance to each other’s skin grafts (anderson et al., 1951; billingham et al., 1952; stone et al., 1965, 1971; emery and mccullagh 1980a, b). in 1954, owen et al. (1954), further detected another remarkable acquired tolerance phenomenon, connected to embryonic exposure. they reported that rhesus d negative pregnant mothers are less likely to produce antibodies against rhesus d positive child, in cases where the grandmother is rhesus d positive. this observation led to the discovery that a high percent of individuals tolerate non-inherited maternal hla antigens (nima), better than non-inherited paternal hla antigens nipa (claas et al., 1988, 1989). then, it was hypothesized that exposure of a child to nima during pregnancy may lead to nimaspecific tolerance later in life. chimerism has probably an important role in this acquired tolerance effect (owen et al., 1954; andrassy et al., 2003; van den boogaardt et al., 2006). billingham et al. (1953) were the first to induce specific tolerance to solid organs by exposing fetal and neonatal mice to the donor hematopoietic cell infusions. engrafted mice did not reject allogeneic material, accepting skin from mice of the leukocyte donor strain, but not from any other strain (6-8 weeks after birth; billingham et al., 1953; billingham and brent, 1956). tolerance to skin allograft was permanent or transient, depending on the mouse strain (billingham and brent, 1956). this acquired tolerance to donor tissues was associated with leukocyte chimerism, which was detected in the animals’ lymphoid organs (billingham et al., 1953; billingham and brent, 1956). since then, there have been numerous reports suggesting that transfer of foreign antigens from the mother to the fetus is common (review in adams and nelson, 2004). recently, mold et al. (2008) found that substantial numbers of maternal cells cross the placenta to reside in fetal lymph nodes, inducing the development of regulatory t cells (tregs) that suppress fetal anti-maternal immunity and persist at least until early adulthood (mold et al., 2008). the above study reveals a form of antigen-specific tolerance in humans induced in utero via chimerism and highlights the potential important role of cell chimerism for induction of fetal tolerance to maternal tissues. colonial protochordates may offer a unique evolutionary perspective on the maternal-fetal relationship during pregnancy. in these organisms, a genetically controlled allorecognition system, similar to the natural killer missing self model system in vertebrate, enables the creation of chimeric state (through vasculature fusion) with kin and prevents chimerism with non-related individuals (oka and watanabe, 1957; sabbadin, 1962; grosberg, 1988; buss, 1982, 1990; scofield et al., 1982). protochordates, are considered as the closest invertebrate relative of vertebrate (delsuc, 2006). studying the immunobiology of the tolerance to partial allogeneic allograft in these organisms may reveal the evolutionary precursors to the maternalfetal relationship during pregnancy. genetic basic for allograft acceptance or rejection in protochordate in colonial protochordate, like the botryllus schlosseri, homeostasis is defined by generation of all organ systems every week. colonies are initially formed by asexual reproduction of founder individuals, a product of sexual reproduction (review in manni and burighel, 2006; manni et al., 2007). the progeny clone members are united under a single gelatinous tunic by a network of anastomosed extracorporeal blood vessels. throughout adult life, the b. schlosseri generates its entire body every week. this cycle of development, includes the formation of all body organs including heart, respiration system, digestive system, and neural complex. ovary and testis are formed within each individual when sexual reproduction commences (burighel and cloney, 1997; manni and burighel, 2006; manni et al., 2007). in addition to their extraordinarily high developmental activity, allogeneic botryllus colonies can fuse and create a chimera. when co-specific botryllid colonies contact each other, several morphological and cellular allogeneic processes and reactions are developed, including: fusion, rejection, indifference (no reaction) and a temporary fusion followed by disconnection (see fig 1 for detailed fusion process; bancroft 1903; oka and watanabe 1957, 1960, 1967; sabbadin 1962, 1982; mukai 1967; tanaka and watanabe 1973; scofield et al., 1982; saito and watanabe 1982, 1984; scofield et al., 1983; hirose et al., 1988; 1990; boyd et al., 1990; sabbadin et al., 1992; rinkevich and weissman 1992a, b; rinkevich et al., 1994a, b, 1998; saito et al., 1994; chadwik-furman and weissman 1995; ballarin et al., 1995, 1998, 2002; cima et al., 2004, 2006; rinkevich, 2005). fusion or non fusion response between adjacent colonies is determined by a single, highly polymorphic, fusibility/histocompatibility (fu/hc) locus (oka and watanabe, 1957; sabbadin, 1962; scofield et al., 1982) which was isolated and characterized (de tomaso et al., 2005). colonies that share at least one allele in their fu/hc locus would fuse upon contact, and would become a chimera, while those that don’t share any allele would not (oka and watanabe, 1957). burnet (1971) suggested studying botryllus as a model for the evolution of self-recognition. he wrote, “although self recognition in ascidians is not analogous to the immunological processes of vertebrates, it presents a primitive type of ’self and not self‘ recognition from which adaptive immunity may have evolved” (burnet, s11 fig. 1 fusion process between kin. using time laps microscopy (imagexpress as described in voskoboynik et al., 2008), we followed and documented the fusion process between 6 different pairs of compatible b. schlosseri colonies. these observations revealed a more elaborate fusion process than the one described before. a) a mother colony and its embryo. b) larva seeks for a settlement site near its mother colony. c) initial contact is established between the tunics and ampullae of the mother colony and its offspring colony. d) following partial fusion of the tunics the offspring ampullae 1 and 2 penetrate into the tunic of the mother colony. e) the offsprings’ penetrating ampullae change the shape of their tips into a cone like shape and through dynamic cycles of extension and retraction further penetrate and explore the tunic of the mother colony. f) 64 hours later, these 2 navigating ampullae fuse with the marginal blood vessel of the mother colony. g) following partial fusion of the tunics, colony a ampulla number 3 penetrates into the tunic of its sibling colony b. h) penetrating ampulla, of colony a changes its tip shape into a cone like shape and through dynamic cycles of extension and retraction further penetrates and explores the tunic of its sibling colony. i) 16 hours later, ampulla 3 fuses with the marginal blood vessel of its sibling colony b. j) only a navigator ampulla fuses and creates a common blood vessel. once created, the number of common blood vessels in the chimera remains constant throughout the chimera life. k-l) 360 hours following ampullae penetration, colony b got resorbed by its sibling colony a. amp, ampulla; h, hours following ampullae contact; bv, blood vessel; mbv, marginal blood vessel; ampullae and common blood vessels are outlined by a dotted line. bar = 75 µm. s12 1971). an extensive study of the botryllid ascidians self-nonself recognition system in the last 25 years revealed that, while the colonial ascidian fu/hc and the mammalian major histocompatibility complex (mhc) share phenomenological features, including polymorphism and specificity, their molecular structure is different. similar to the mhc, the fu/hc is highly polymorphic (karakashian and milkman 1967; scofield et al., 1982; 1983; grosberg and quinn, 1986; grosberg 1987; rinkevich et al., 1995; paz et al., 2003; de tomaso et al., 2005; benshlomo et al., 2001, 2006, 2008). however, structural homologies were not found (weissman et al., 1990; rinkevich and weissman, 1992b; fagan and weissman, 1997; pancer et al., 1993, 1996a, b, c, 1997; muller et al., 1994; khalturin et al., 2003; de tomaso et al., 2005; nyholm et al., 2006). the botryllus fu/hc is not homologous to any molecules of the vertebrate mhc-based histocompatibility system. moreover, the whole botryllus fu/hc locus does not have a syntenic region in the ciona genome or in the genomes of vertebrates (de tomaso et al., 2005). genes involved in adaptive immunity, which include the polymorphic mhc class i and ii glycoproteins that present internal peptides to t cells, the clonally expressed t-cell receptors (tcrs), immunoglobulins (igs) and the recombination activating genes (rag1, rag2), have not been identified in protochordates (klein 1989, laird et al., 2000; dehal et al., 2002; kaufman, 2002; azumi et al., 2003; khalturin et al., 2003; de tomaso et al., 2005; litman et al., 2005, 2007). in both mice and humans, the mediators of the adaptive immune system are thought to be responsible for the rejection of a transplant. the adaptive immune t cell system rejects grafts even if one of two alleles is shared, as t cells make an immune reaction against non-self allele gene products. in contrast, allogeneic botryllus colonies tolerate each other and create a chimera even if only one of the two fu/hc alleles is shared. this is consistent with immune systems, like the nk system in vertebrates, wherein recognition of self prevents an immune reaction. recently, nk cells have been recognized as active participants in the acute and chronic rejection of solid tissue grafts (reviewed in kitchen et al., 2005). the importance of nk cell attributes has been first recognized in bone marrow transplantation, where nk cells are fully sufficient to reject hematopoietic cell transplants, even in the absence of t or b cell responses (lethally-irradiated or lack mhc class i expression recipients; hoglund et al., 1991; bix et al., 1991; manilay and sykes, 1998). recent studies suggest that nk contribute to organ rejection indirectly, by activating or helping effector cells, such as cytotoxic and helper t cells (reviewed in kitchen et al., 2005). studies also point to a potential involvement of nk in the induction of tolerance to solid graft in mix chimerism, a tolerance regimen that employs the generation of mixed hematopoietic chimerism through donor stem cell engraftment (zhao et al., 2003). another aspect that might connect mammalian nk and the botryllus fu/hc was recently raised by lighter et al. (2008). studying uterine nk, lighter et al., pointed to a unique phenotype that uterine nk and the botryllus fu/hc might share. uterine nk cells produce angiogenic growth factors and are potential regulators of decidual angiogenesis in early pregnancy (ashkar and croy, 2001; hanna et al., 2006; manaster and mandelboim, 2008). they suggested that, as botryllus fu/hc is probably involved in the generation of a common vascular system between two individuals, uterine nk may share the same evolutionary roots as the botryllus fu/hc. cell parasitism induces development of a foreign entity within host colony inspired by the genetic control for allograft acceptance and creation of chimerism within kin in botryllus, burnet (1971) hypothesized the emergence of intraspecific parasitism along the evolution. indeed, his hypothesis was later confirmed in botryllus chimeras, where the replacement of host germline by a donor genotype was demonstrated (sabbadin and zaniolo, 1979). in a remarkable set of experiments sabbadin and zaniolo (1979) demonstrated this kind of parasitism in b. schlosseri. years later pancer et al. (1995), stoner and weissman (1996) and stoner et al. (1999) confirmed these results and further showed that in a chimera, the blood, soma and germ cells, demonstrated the combine genotypes of both chimeric partners. moreover, in many cases the circulating pluripotent cells of one partner parasitized either the soma or the germ line of the other partner and replaced the whole mass of gonads or the soma (bud/zooid) of several individuals in the host colony (termed gonads or somatic cell parasitism; g/scp). in a few cases, a complete takeover of donor genotype occurred and the whole mass of gonads in the chimeric colony expressed solely the donor’s genotype (sabbadin and zaniolo 1979; pancer et al., 1995; stoner and weissman 1996; stoner et al., 1999). under invariant environmental conditions, both germline and somatic cell parasitism followed repeatable hierarchies of “winner strains” and “loser strains” (stoner et al., 1999). however, breeding experiments proved that only the hierarchical position of germ cell parasitism is sexually inherited (stoner et al., 1999). the hierarchy of somatic parasitism in botryllus chimeras is a plastic trait, as variations in the environmental conditions (such as seawater temperature) can be reversed; the winner – loser hierarchy at the somatic parasitism level (rinkevich and yankelevich, 2004). botryllus colonies propagate a-sexually through budding, therefore somatic stem cell parasitism in host colonies can induce the development of partial allogeneic entities (buds) within the host colony. as a result, chimerism in protochordate could serve as a state that enables the development of a “virtual embryo” within the host colony (voskoboynik et al., in press; fig. 2). beside cell parasitism chimerism may alter tolerance and intolerance state in the colonies. chimeras might fuse with colonies that they used to reject (on their non chimeric phase) or reject colonies that they used to fuse with (mukai 1967; sabbadin and astorri, 1988). moreover, in some cases, chimeric colonies will simultaneously fuse s13 fig. 2 virtual pregnancy: a development of a semi allogeneic entity in a host colony through asexual budding in a chimera of a mother colony and its offspring. botryllus colonies propagate asexually through budding, therefore, somatic stem cell parasitism in host colonies can induce the development of a partial allogeneic entities (buds) within the host colony. in this illustration, an offspring colony (red) is fused with its mother colony (blue). genetic analysis of the chimera’s buds and gonads can reveal several cell chimerism patterns. either both genotypes are detected or only one genotype is detected. bv, blood vessel; mbv, marginal blood vessel; red, offspring genotype; blue, mother genotype. and reject another colony (taneda, 1985; sabbadin and astorri, 1988). the presence, of a simultaneously fusion and rejection with a genotype that one of the chimera partner used to fuse with and the other partner used to reject, prove the persistence of both genotypes and suggest an uneven distribution of each genotype within the chimera. different fusibility patterns that the chimeric entity presents on different time points suggests genomes fluctuation and competitive interaction of the different genomes within the chimera (sabbadin and astorri, 1988). sabbadin and astorri observed changes in the tolerance state of the chimeric colonies and linked it to changes in the dynamic of the chimeric cells within the chimera. genetic analysis of the dynamic of chimeric cell revealed that chimeras exhibit either a sectorial pattern in which both genotypes are detected within some systems but not others, or a uniform pattern in which tissues throughout the entire chimera exhibit both genotypes (stoner and weissman, 1966). colonies which showed rejection and fusion on the same time probably expressed sectorial pattern and the others expressed uniform pattern. the dynamic of chimeric cells within the host is changing with time, as different patterns are observed during different time points (pancer et al., 1995; stoner and weissman, 1966; stoner et al., 1999). these studies show that the genetically controlled ability of botryllus colonies to tolerate or reject other colonies can be altered by chimerism. the temporal and spatial dynamic of the chimeric cells, patterns of host/donor cells competition, niche occupation and immunoregulatory mechanisms for routing, timing and frequencies of chimeric cells have probably important role in the induction of tolerance or intolerance. stem cells mediated chimerism the long-term ability of cells from one genotype to replace the germline and somatic cells of the host, led to hypothesize that cell parasitism in the chimeras is mediated by stem cells (sabbadin and zaniolo, 1979; rinkevich and weissman, 1987; pancer et al., 1995; stoner and weissman 1996; stoner et al., 1999). by transplanting a single cell, which expresses high enzymatic activity of aldehyde dehydrogenase and a set of serial engraftment assays, laird et al. (2005) revealed that multipotent stem cells are responsible for a stable long-term chimerism in adult b. schlosseri colonies. yet, the location of these cells remained unknown (laird et al., 2005). recently, by using cell engraftments, chimeric fusion techniques, and in vivo cell tracking by automated time lapse fluorescence microscopy, s14 we (voskoboynik et al., 2008) have demonstrated that the anterior ventral region of the endostyle (termed endostyle niche) harbors adult stem cells and exports them to developing and regenerating organs, wherein they participate in tissue formation. as few as 5-20 engrafted cells transplanted from the donor endostyle niche sufficed to generate a somatic chimerism in compatible hosts; however, no germline chimerism was demonstrated. the induction of somatic chimerism demonstrates a remarkable stemness capacity of the cells in the endostyle niche (voskoboynik et al., 2008). the endostyle produces thyroid hormones and serotonin and expresses several variety of factors that are involved in development and stem cell regulation like wnt, hox1, pax 2/5/8, pl10, cadherin, raldh and pcna (canestro et al., 2008; dunn, 1980; hiruta et al., 2005; nilsson et al., 1988; pennati et al., 2001; rosner et al., 2006; rosner et al., 2007; voskoboynik et al., 2008). the endostyle niche is the first somatic stem cell niche ever described in a protochordate and is one of the most accessible stem cell niches for in vivo studies. the discovery of a major stem cell niche in an organism with fu/hc controlled chimerism, pluripotent stem cell parasitism, and tolerance or intolerance induction via chimerism promotes the botryllus as an evolutionary model for studying molecular regulations of tolerance induction by cellular chimerism in the absence of an adaptive immune system. acknowledgements i thank a voskoboynik for critical review, il weissman and b rinkevich for discussion, p brown, r marinelli, r pesich l jerabek, kj ishizuka, and kj palmeri for 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rev. 113: 227-241, 1990. voskoboynik a, soen y, rinkevich y, ueno h, rosner a, reshef r, et al. identification of the endostyle as a stem cell niche in a colonial chordate. cell stem cell 3: 456-464, 2008. zhao y, ohdan h, manilay jo, sykes m. nk cell tolerance in mixed allogeneic chimeras. j. immunol. 170: 5398-5405, 2003. s20 antimicrobial peptides in echinoderms isj 7: 132-140, 2010 issn 1824-307x minireview antimicrobial peptides in echinoderms c li, t haug, k stensvåg norwegian college of fishery science, faculty of bioscience, fisheries and economics, university of tromsø, breivika, n-9037 tromsø, norway accepted may 5, 2010 abstract antimicrobial peptides (amps) are important immune effector molecules for invertebrates, including echinoderms, which lack a vertebrate-type adaptive immune system. here we summarize the knowledge of such peptides in echinoderms. strongylocins are a novel family of cysteine-rich amps, recently identified in the sea urchins, strongylocentrotus droebachiensis and s. purpuratus. although these molecules present diverse amino acid sequences, they share an identical cysteine arrangement pattern, dissimilar to other known amps. a family of heterodimeric amps, named centrocins, are also present in s. droebachiensis. lysozymes and fragments of larger proteins, such as beta-thymocins, actin, histone 2a and filamin a have also been shown to display antimicrobial activities in echinoderms. future studies on amps should be aimed in revealing how echinoderms use these amps in the immune response against microbial pathogens. key words: sea urchin; host defence peptides; celomocyte; innate immunity introduction antimicrobial peptides (amps) are evolutionarily conserved small molecular weight proteins of the innate immune response, with a broad spectrum of antimicrobial activities against bacteria, viruses, and fungi (reviewed by mookherjee and hancock, 2007). amps appear naturally throughout all three domains of life from unicellular to multicellular organisms (zasloff, 2002; riley and chavan, 2007; wang et al., 2009). by february 2010, more than 1,500 amps have been identified (http://aps.unmc.edu/ap/main.php). amps are characterized as having less than 100 amino acids and are usually cationic in nature. nearly all antimicrobial peptides form amphipathic structures which reflect the relative abundance and polarization of hydrophobic and hydrophilic domains within the peptides. the hydrophobicity enables the water-soluble antimicrobial peptides to interact with the hydrophobic lipid bilayer of the microbial membranes. amps are likely to be attracted by and attach to the negatively charged bacterial surfaces because of their cationic nature. the mechanism by which amps interact with the cell wall structures of gram ___________________________________________________________________________ corresponding author: klara stensvåg norwegian college of fishery science faculty of bioscience, fisheries and economics university of tromsø, breivika, n-9037 tromsø, norway e-mail: klara.stensvag@uit.no negative and gram-positive bacteria has rarely been addressed and is therefore not yet understood (brogden, 2005). however, once the peptides come into contact with the outer leaflet of the cell membrane and the peptide/lipid ratio increases, the peptides start forming multimers or self-associating on top of the membrane (yang et al., 2001). at sufficiently high concentrations of the peptides orientate perpendicularly and insert into the bilayer, thereby interfering with membrane integrity. however, new paradigms imply that pore-forming is not the only mechanism of antimicrobial activity. some peptides are also able to interact with intracellular targets without disrupting the membrane integrity. intracellular targets of antimicrobial peptides vary from nucleic acids to enzymatic proteins (brogden, 2005). amps are crucial immune effector molecules for invertebrates which lack a vertebrate-type adaptive immune system. they have been identified both in plasma and in various blood cells and their distribution in the host can be site-specific or systemic. they are either expressed constitutively or the expression is induced by exposure to pathogens. they do not only inactivate bacteria in vitro and in vivo , thereby protecting host organisms against a variety of infections, but they also modulate immunity ( hancock and diamond, 2000; zasloff, 2002; hancock et al., 2006). it is worth mentioning that a few amps play a role as anti-endotoxins (scott et al., 2002; bowdish et al., 2005) and chemokines in vertebrates (durr and peschel, 2002; 132 http://aps.unmc.edu/ap/main.php mailto:klara.stensvag@uit.no davidson et al., 2004) and might also induce production of cytokines and chemokines (bals and wilson, 2003). these immunomodulatory functions do not directly kill microbes, but recruitment and activation of immune cells and signalling molecules improves the host defence system. in this review we will present an overview of amps identified and characterized from echinoderms and present their characteristics. in particularly, we will describe their antimicrobial activities, their primary structure, and posttranslational modifications of these molecules. finally, we will address some important questions regarding future amp research on echinoderms. antimicrobial activities in echinoderms the phylum echinodermata is a large phylum of deuterostomes containing approximately 7,000 living species divided into the groups crinoidea (sea lilies), ophiuroidea (brittle stars), asteroidea (starfishes), echinoidea (sea urchins) and holothuroidea (sea cucumbers). the adult echinoderms have a few major organs such as mouth parts, intestine, nerve ring, and gonad. these organs are located in the cavity, named celomic cavity. the remaining space of the celomic cavity is filled with celomic fluid which contains various “blood cells”, the celomocytes. these cells are though to mediate the main immune functions in echinoderms, including antimicrobial activities. many studies have been conducted to examine the activity of celomocytes lysates and celomic fluid against bacteria, fungi and even tumour cells. celomic fluid from echinus esculentus possess bactericidal activity against marine pseudomonas sp (wardlaw and unkles, 1978). gerardi et al. (1990) found that the highest bacterial growth inhibition against several vibrio sp. is shown by phagocytes (called amoebocytes) and red spherule cells of paracentrotus lividus.. stabili et al. (1996) also discovered antibacterial activity against v. alginolyticus in celomocyte lysates and celomic fluid of p. lividus. recently, it was reported that celomocytes of p. lividus show a cytotoxic activity against rabbit erythrocytes and the k562 tumour cell line (arizza et al., 2007). antibacterial activity was detected in extracts of several tissues from the green sea urchin s. droebachiensis, the common sea star asterias rubens, and the sea cucumber cucumaria frondosa (haug et al., 2002). some of the activities detected (in extracts of celomocytes and body walls) were caused by compounds of protein nature. several drug discovery projects have screened echinoderms for antibiotic activities. an early study by rinehart et al (1981) showed that antimicrobial activity was present in 43 % of 83 unidentified species of echinoderms (collected from the west coast of baja california and the gulf of california) and 58 % of 36 unidentified caribbean species displayed antimicrobial activities. in the northern gulf of mexico, 80 % of 22 echinoderm species showed antimicrobial activity (bryan et al., 1994). body wall extracts of echinoderms displayed activity against marine bacteria, but did also inhibit settlement of marine larvae (bryan et al., 1996). several antimicrobial molecules (other than amps) have been isolated from echinoderms, including echinochrome a (kuwahara et al., 2009; service and wardlaw, 1984), steroidal glycosides (andersson et al., 1989; chludil et al., 2002; levina et al., 2009), and polyhydroxylated sterols (iorizzi et al., 1995). although these results indicate that echinoderms present remarkable activity against microbes, only a few amps have been reported. antimicrobial peptides in echinoderms lysozymes are widely distributed throughout the animal kingdom and catalyze the hydrolysis of peptidoglycans of bacterial cell walls of especially gram-positive bacteria, and acts as a nonspecific innate immunity molecules against the invasion of bacterial pathogens (jollès and jollès, 1984). they are characterized as basic proteins of low molecular weight (15-25 kda) with an isoelectric point of 10.5– 11.0, stable at acidic ph and even at high temperatures. there are several lysozymes or lysozyme-like proteins identified from celomic fluid, celomocytes and other tissues of echinoderms (jollès and jollès, 1975; canicatti and roch, 1989; canicatti, 1990; canicatti and d´ancona, 1990; gerardi et al., 1990; stabili et al., 1994; stabili and canicatti, 1994; stabili and pagliara, 1994, 2009; shimizu et al., 1999; bachali et al., 2004; cong et al., 2009) (table 1). a study by beauregard and co-workers indicated the presence of amps in the celomic fluid of the orange-footed sea cucumber, c. frondosa (beauregard et al., 2001). the celomic fluid extract was purified by molecular sieve chromatography and antibacterial activity was detected in separate fractions. a peptide of approximately 6 kda was identified, but no sequence was reported (table 1). in a celomocyte extract from the starfish a. rubens showing antimicrobial activity, several protein/peptides with molecular mass around 2 kda were isolated (maltseva et al., 2004; maltseva et al., 2007) (table 1). two peptides were identified as part of the histone molecule, h2a. two other peptides were identified as fragments of actin, while one peptide was a fragment of filamin a. in addition, four other amps were detected, but not characterized. it had been known for long time that histones have antibacterial properties (hirsch 1958). histone-derived fragments (h1, from h2a and h2b) have shown to possess antimicrobial activity (from et al., 1996; robinette et al., 1998; patrzykat et al., 2001; richards et al., 2001; fernandes et al., 2002). although maltseva et al. (2004, 2007) also identified fragments of actin and filamin a in the active extract, the antimicrobial activity of these fragments need to be further investigated. recently, schillaci et al. (2009) reported that a 5 kda peptide fraction from the celomocytes of p. lividus presents activity against both gram-positive and gram-negative bacteria and fungi (table 1). the peptide fraction also showed the ability to inhibit the formation of staphylococcus biofilms. the authors suggested that the antimicrobial and antistaphylococcal biofilm activity of this peptide 133 table 1 antimicrobial peptides and proteins characterized in echinoderms class and genus origin (tissue/cell type) compound mw (kda) reference asteroidea asterias forbesi cell-free celomic fluid complement-like leonard et al., 1990 a. rubens body wall peptides/proteins <20 haug et al., 2002 celomocytes fragments of actin, histone h2a, and filamin a 1.82.4 maltseva et al., 2007; maltseva et al., 2004 celomocytes peptides 2.0-4.7 maltseva et al., 2007; maltseva et al., 2004 lysozyme 15.5 jollès and jollès 1975; bachali et al., 2004 marthasterias glacialis eggs lysozyme-like stabili and pagliara, 1994, 2009 body wall mucus lysozyme-like canicatti and d´ancona, 1990 echinoidea holothuria scabra celomic fluid lectin >10 gowda et al., 2008 h. polii celomocytes lysozyme-like canicatti and roch, 1989 various tissues lysozyme-like canicatti, 1990 paracentrotus lividus celomocytes lysozyme-like gerardi et al., 1990 celomocytes protein ~ 60 stabili et al., 1996 celomocytes peptide ~ 5 schillaci et al., 2009 larvae lysozyme-like stabili et al., 1994 seminal plasma lysozyme-like stabili and canicatti, 1994 various tissues lysozyme-like canicatti, 1990 strongylocentrotus droebachiensis celomocytes stongylocins 5.65.8 li et al., 2008 celomocytes centrocins 4.4-4.5 li et al., 2010b s. intermedius celomic fluid lysozyme 14 shimizu et al., 1999 s. purpuratus celomocyte cdna spstrongylocinsa 5.6-6.1 li et al., 2010a holothuroidea cucumaria echinata whole body fragments of lectin cel-iii 2.04.2 hatakeyama et al., 2004; hisamatsu et al., 2008 c. frondosa celomic fluid peptides ~ 6 beauregard et al., 2001 various tissues peptides/proteins <20 haug et al., 2002 parastichopus califormicus celomic cavity lps-binding compound dybas and fankboner, 1986 stichopus japonicus intestine cdna lysozymea 14 cong et al., 2009 a recombinantly produced and tested for activity. fraction is associated with beta-thymosin like fragments. beta thymosins have been reported to induce the production of metalloproteinases, display chemotactic and anti-inflammatory activities (huff et al., 2001) and even function as amps released by human platelets (tang et al., 2002). several amps have been isolated and characterized from the green sea urchin, s. droebachiensis. one group is the cysteine rich peptides named strongylocins (li et al., 2008). homologous genes, named spstrongylocins, were also identified in the sister species s. purpuratus (li et al., 2010a). all these native peptides or recombinant equivalents show antibacterial activity against both gram positive and gram negative bacteria (table 2). in addition, there are several heterodimer structured peptides, named centrocins, identified from s. droebachiensis (li et al, 2010b). they show strong potent activity, not only against gram-positive and gram-negative bacteria, but the longest peptide chain of the dimers also show strong activity against fungi and yeasts. in silico analysis of strongylocins and comparison of the native peptide sequences and the ones deduced from cdna sequences, show that these peptides contain three regions: a signal peptide, a prosequence and a native sequence (fig. 1). analysis , using both the neutral network model and the hidden markov model (bendtsen et al., 2004; http://www.cbs.dtu.dk/services/signalp/), indicates that a signal peptide cleavage site is located between the amino acid number 22 and 23 for all these peptides. 134 http://www.cbs.dtu.dk/services/signalp/ table 2 susceptibility of bacterial strains to the antibacterial peptides, strongylocins and centrocins, isolated from s. droebachiensis celomocytes, and recombinant spstrongylocins (li et al., 2008; li et al. 2010a, b) minimal inhibitory concentration (μm) peptide listonella anguillarum escherichia coli corynebacterium glutamicum staphylococcus aureus strongylocin 1 a 2.5 5.0 2.5 2.5 strongylocin 2 a 1.3 5.0 2.5 2.5 recombinant spstrongylocin 1 b 15.0 7.5 7.5 15.0 recombinant spstrongylocin 2 b 15.0 7.5 3.8 15.0 centrocin 1 a 2.5 1.3 1.3 2.5 centrocin 2 a 2.5 2.5 1.3 5.0 a minimal inhibitory concentration (mic) was determined as the lowest concentration of peptide causing an optical density less than 50 % of the growth control without any peptide present. b minimal inhibitory concentration (mic) was determined as the lowest concentration of peptide causing 100 % of the growth inhibition of the test organism compared to the growth control. it is not surprising that strongylocin 1 and spstrongylocin 1 contain an identical signal peptide sequence since they share high amino acid sequence similarity. however, spstrongylocin 2 does not share an identical signal peptide with strongylocin 2, but with strongylocin 1. although no experimental data show which mechanism assists the migration of these precursors of strongylocins and spstrongylocins within the cells, the diversity of the signal peptides suggests that strongylocin 2 likely employs a divergent intracellular trafficking pathway than the other sea urchin amps. analysis of strongylocins and spstrongylocins show that their prosequences are located at the nterminal region, following the signal peptide. it is common that prosequences are located at the nterminal or the c-terminal region, or even in between parts of the mature sequences. the prosequence is considered to act as an intracellular steric chaperone during the folding process (inouye, 1991), and it has been shown that prosequencesupported protein folding is crucial for processing of the proper protein having antifungal activity (reichhart and achstetter, 1990). in addition, it has been shown that the prosequences in some proteolytic enzyme precursors inhibit the activity of the mature proteins (neurath, 1989). the prosequences of strongylocins and spstrongylocins are negatively charged sequences which may neutralize the positive charges of the mature sequences (li et al., 2008; li et al., 2010a, b). therefore, the precursor molecules are likely less toxic to the host cells than the mature peptides. the peptides will obtain their activity during their maturation once the prosequences are removed. strongylocins contain six cysteine residues, which form three disulfide bridges (li et al., 2008; li et al., 2010a). cysteine-rich amps are found in various phyla, such as the defensin families in both vertebrates and invertebrates, tachystatins in horse shoe crabs and circulin a in plants (daly et al., 1999; wang et al., 2009). table 3 shows that strongylocins and spstrongylocins display a novel cysteine location pattern compared to other known cysteine-rich peptides containing 6 cysteines. this indicates that these amps may not form the same disulfide bridge linkages as the other peptides containing six cysteines, since proteins that have the same cysteine residue pattern tend to have identical disulfide connections (bania et al., 1999; bontems et al., 1991; pallaghy et al., 1994). disulfide bridges are crucial for the antimicrobial activity of most cysteine-containing amps (daher et al., 1986; mandal and nagaraj, 2002), and they may also stabilize the conformation of the molecules (selsted and ouellette, 2005). therefore, the disulfide linkages may aid strongylocins and spstrongylocins to resist proteolysis within the celomocytes, and/or after their release into protease-containing environments. strongylocin 2 contains a tryptophan in the mature sequence which is likely brominated (li et al., 2008). several amps with bromotryptophan have also been isolated from other marine organisms, for example styelin d from the tunicate, styela clavata (taylor et al., 2000), cathelicidin from the hagfish, myxine glutinosa (shinnar et al., 2003), and hedistin from the annelid, nereis diversicolor (tasiemski et 135 fig. 1 alignment of strongylocins from s. droebachiensis and spstrongylocins from s. purpuratus. the predicted cleavage site between the signal peptide and the proregion is shown (▼). the first amino acid in active strongylocin 2 and spstrongylocin 2 are likely a modified tryptophan (♦). identical amino acids are shown in boxes, similar amino acids are shaded in grey, and cysteines are highlighted in black. al., 2007). brominated amino acids may protect the peptides from proteolysis, and/or increase the biological activity of peptides (bittner et al., 2007). although spstrongylocin 2 also contains a tryptophan residue in the same position as strongylocin 2, the recombinantly produced spstrongylocins, having a non-brominated trp residue, are still antimicrobial active (li et al., 2010a). therefore, we speculate that the brominated tryptophan affects the peptides by enhancing their stability, but does not affect their antimicrobial activity (li et al., 2010a). this suggestion is supported by results showing no difference in the antimicrobial activity between the heavy chain of the dimeric peptide centrocin 1, with or without the brominated modification (stensvåg, unpublished). future studies on amps in echinoderms amps are important immune defence molecules for echinoderms that lack a vertebratetype adaptive immune system (reviewed by smith et al., 2010). it is therefore interesting to know whether these amps are secreted into the celomic fluid or stored in the granula of certain cell types. it has been documented that neutrophilic granules packed with human cathelicidin and defensins are fused with phagocytic vacuoles, where they contain certain concentrations of amps to eliminate phagocytosed pathogens (reviewed by ganz, 2003; lehrer, 2004; brogden, 2005). for penaeidins in shrimp, it seems that haemocytes migrate towards the infection sites and then release the amps presumably by lysis of the cells (munoz et al., 2002). echinoderms have a large celomic cavity filled with circulating fluid which could dilute amps if they were secreted or released by the cells. therefore, further investigation on the distribution of amps in echinoderms might uncover whether the peptides are involved in 1) intracellular elimination of pathogens; 2) local release of amps at the infection sites; or 3) massive release of amps to regulate other immune activities. although several studies have documented mammalian amps as immune regulatory molecules (reviewed by hancock et al., 2006 and diamond et al., 2009), unfortunately, there are not many investigations on invertebrate amps. it has been reported that tachypleus tridentatus haemocyte granules can release tachyplesin after 136 table 3 comparison of cysteine location patterns in amps containing six cysteines peptide family cysteine location patterns 1 animals strongylocins c – c – c – cc – c s. droebachiensis spstrongylocins c – c – c – cc – c s. purpuratus βeta-defensins c – c – c – c – cc bos taurus alpha-defensins c – c – c – c – cc homo sapiens tachystatins c – c – cc – c – c t. tridentatus knottin-type amps c – c – cc – c – c phytolacca americana thionins type iii and iv amps cc – c – c – c – c sorghum bicolor insect defensins c – c – c – c – c – c rhodnius prolixus 1 adjacent double cysteine residues are highlighted in yellow. information regarding cysteine arrangements in the different peptides was obtained from the antimicrobial peptide database (wang et al., 2009). encountering microbial endotoxins and form a complex (iwanaga et al., 1998; hirakura et al., 2002). this complex will then block the activation of factor c, which is crucial for haemolymph coagulation (nakamura et al., 1988). pancer et al. (1999) identified an nf-κb homologue, transcription factor in s. purpuratus celomocytes according to analysis of the immune related gene repertoire of s. purpuratus (hibino et al., 2006; rast et al., 2006), toll-like receptor (tlr) genes, interleukin (il)-17 genes, il receptors, and tumor necrosis factor (tnf) family members are present in the sea urchin genome. it is possible that amps are involved in the endotoxin-induced signalling pathway from the tlr to nf-κb, and/or suppress inflammation. therefore, it is important to keep in mind that amps may play multiple immune roles in the echinoderm´s immunity as in other species (hancock and diamond, 2000; lehrer, 2004; durr et al., 2006; hancock et al., 2006; mookherjee and hancock, 2007; cuthbertson et al., 2008; smith et al., 2008). the discovery of amps in echinoderms has attracted attention from both pharmaceutical and academic sectors. however, there should be more focus on searching for new amps in crinoidea and ophiuroidea by newly developed high-throughput screening systems or other proteomic techniques since there is limited information about antimicrobial compounds from these echinoderm classes. in summary, amps are important host defence molecules in invertebrates. investigations of antimicrobial peptides/proteins in echinoderms have revealed two novel families of amps in strongylocentrotus, lysozymes in various species and fragments of larger proteins having antibacterial activity. these amps do not only attract interest as potent drugs or drug leads, but may also become useful in studying the echinoderm immune system. acknowledgements writing this review was supported by grants from the university of tromsø, the marine biotechnology in tromsø (mabit) research program (no. bs0034) and the norwegian research council (nos. 178214/s40 and 184688/s40). oleg sokolov is appreciated for translating the russian papers. references andersson l, bohlin l, iorizzi m, riccio r, minale l, moreno-lópez w. biological activity of some saponins and saponin-like compounds from starfish and brittle-stars. toxicon 27: 179-188, 1989. arizza v, giaramita ft, parrinello d, carnmarata m, parrinello n. cell cooperation in coelomocyte cytotoxic activity of paracentrotus lividus, coelomocytes. comp. biochem. physiol. 147a: 389-394, 2007. bachali s, bailly x, jolles j, jolles p, deutsch js. the lysozyme of the starfish asterias rubens. eur. j. biochem. 271: 237-242, 2004. bals r, wilson jm, cathelicidins--a family of multifunctional antimicrobial peptides. cell. mol. life sci. 60: 711-720, 2003. bania j, stachowiak d, polanowski a. primary structure and properties of the cathepsin g/chymotrypsin inhibitor from the larval hemolymph of apis mellifera. eur. j. biochem. 262: 680-687, 1999. beauregard ka, truong nt, zhang h, lin w, beck g. the detection and isolation of a novel antimicrobial peptide from the echinoderm, cucumaria frondosa. adv. exp. med. biol. 484: 55-62, 2001. bendtsen jd, nielsen h, von heijne g, brunak s. improved prediction of signal peptides: signalp 3.0. j. mol. biol. 340: 783-795, 2004. bittner s, scherzer r, harlev e. the five bromotryptophans. amino acids 33: 19-42, 2007. bontems f, roumestand c, gilquin b, menez a, toma f. refined structure of charybdotoxin: common motifs in scorpion toxins and insect defensins. science 254: 1521-523, 1991. bowdish dm, davidson dj, lau ye, lee k, scott mg, hancock re. impact of ll-37 on antiinfective immunity. j. leukoc. biol. 77: 451-459, 2005. brogden ka. antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? nat. rev. microbiol. 3: 238-250, 2005. 137 bryan pj, mcclintock jb, watts sa, marion kr, hopkins ts. antimicrobial activity of ethanolic extracts of echinoderms from the northern gulf of mexico. in: b david, a guille, j-p feral, m roux (eds), echinoderms through time, balkema, rotterdam pp 17-23, 1994. bryan pj, rittschof d, mcclintock jb. bioactivity of echinoderm ethanolic body-wall extracts: an assessment of marine bacterial attachment and macroinvertebrate larval settlement. j. exp. mar. biol. ecol. 196: 79-96, 1996. canicatti c, distribution d´une activite lysozymiale dans un echinoderme holothuroide, holothuria polii, et dans les oeufs et les larves d´un echinoderme echinoide, paracentrotus lividus. eur. arch. biol. 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14751485, 2001. zasloff m. antimicrobial peptides of multicellular organisms. nature 415: 389-395, 2002. 140 effects of ocean acidification and warming on the covering behavior of sea urchins glyptocidaris crenularis and strongylocentrotus intermedius 14 isj 15: 14-18, 2018 issn 1824-307x short communication effects of water temperature and ph value on covering behavior of the sea urchin glyptocidaris crenularis mf yang#, sb luo#, jn sun, dt shi, jy ding, yq chang, c zhao # these authors contributed equally to this work key laboratory of north mariculture and stock enhancement, ministry of agriculture, dalian ocean university, dalian, china, 116023 accepted november 28, 2017 abstract as marine calcifying organisms, sea urchins are sensitive in the changing ocean. more information is needed about the effects of ph value and water temperature on behaviors of sea urchins. here, we reported that ph value and water temperature significantly affected the covering behavior of sea urchins glyptocidaris crenularis. lower ph value (ph = 7.4) significantly reduced the time to first covering (p = 0.026), while significantly decreased number of covered sea urchins (p = 0.029) and number of shells used for covering (p = 0.007) in g. crenularis. water temperature did not significantly affect the time to first covering (p = 0.180) or number of covered sea urchins (p = 0.157), though significantly affected number of shells used for covering (p = 0.042). the present study provides preliminary information on behavioral ecology of sea urchins. notably, the effects of co2 induced acidification should be further investigated in future. key words: sea urchin; covering behavior; ph value; water temperature introduction over the past 250 years, atmospheric carbon dioxide (co2) levels increased by approximately 40 % (solomon et al., 2007), one third of which has been taken up by the oceans (sabine et al., 2004). due to absorbing increasing amounts of co2, ocean warming and acidification have been identified as the greatest anthropogenic threat to marine ecosystems (halpern et al., 2008; de madron et al., 2011). this highlights the risks to marine calcifying organisms in future (orr et al., 2005). as marine calcifying organisms, sea urchins are sensitive in the changing ocean (dupont et al., 2013). ocean warming and acidification have showed greatly impacts on reproduction (reuter et al., 2011), gamete health (morgan and galione, 2007), larval development (brennand et al., 2010; stumpp et al., 2011a, b) calcification (byrne et al., 2011) and gene expression (o'donnell et al., 2010) of sea urchins. effects of ph value and water temperature on ___________________________________________________________________________ corresponding author: chong zhao key laboratory of north mariculture and stock enhancement ministry of agriculture dalian ocean university email: chongzhao@dlou.edu.cn behaviors of sea urchins remain largely unknown, although the fragility has showed in various of behaviors of marine fish (munday et al., 2009; cripps et al., 2011; simpson et al., 2011) and bivalves (chan et al., 2011). covering behavior refers to echinoids using their tube feet and spines to manipulate environmental objects, such as shells, stones and algae fragments, to put onto their dorsal surface (verling et al., 2002). covering behavior has been well proposed to have multiple functions of protection from lights (verling et al., 2002), predation (agatsuma, 2001), floating sand (richner and milinski, 2000) and wave surge (dumont et al., 2007). recently, effects of elevated temperature and acidification have been investigated separately (challener and mcclintock, 2013; zhao et al., 2014; brothers and mcclintock, 2015; zhang et al., 2017). the effects of water temperature and ph value on covering behavior, however, were not simultaneously investigated. acidification can be experimentally imitated by co2 (for example, dupont et al., 2013) or hcl (for example, yamada and ikeda, 1999) in laboratory. a comparative study indicates that hcl-induced acidification showed lower acute toxicity to aquatic animals than that induced by co2 (zhang et al., 2011). mailto:chongzhao@dlou.edu.cn 15 table 1 characteristics of sea urchins and covering materials water temperature 15°c 25°c ph value 7.4 8.2 7.4 8.2 glyptocidaris crenularis test diameter (mm) 16.55 ± 1.98 16.44 ± 1.76 16.58 ± 2.04 16.50 ± 1.89 test height (mm) 7.87 ± 1.14 8.20 ± 0.98 8.09 ± 0.98 8.16 ± 1.23 body weight (g) 2.04 ± 0.73 2.10 ± 0.71 2.10 ± 0.74 2.06 ± 0.74 mytilus galloprovincialis shell length (mm) 11.82 ± 0.81 11.91 ± 0.85 11.74 ± 0.84 11.59 ± 0.76 shell height (mm) 19.84 ± 2.61 20.31 ± 1.45 19.96 ± 1.39 19.45 ± 2.02 shell weight (g) 0.12 ± 0.02 0.13 ± 0.02 0.12 ± 0.02 0.12 ± 0.02 (mean ± sd, n = 36 for glyptocidaris crenularis, n = 40 for mytilus galloprovincialis) thus, hcl-induced acidification can be used to provide preliminary information on behavioral response of sea urchins to ocean acidification. the aim of the present study is to investigate the effects of water temperature and ph value on the covering behavior of the sea urchin glyptocidaris crenularis. materials and methods sea urchins glyptocidaris crenularis were originally reared at dalian haibao seafood company in lvshun of dalian, china. individuals from one cohort were transported to key laboratory of north mariculture and stock enhancement, dalian ocean university, china. all sea urchins were acclimated for 7 days at 19 21 °c of water temperature, 30 31 ‰ of salinity and 8.2 of ph before the behavioral experiment. there was no significant difference of test diameter, test height and body weight among experimental groups in g. crenularis (p > 0.05, table 1). experimental design geochemical models indicates that ocean acidification will be over 1.4 ph units in the next 300 years (caldeira and wickett, 2003). consequently, two ph values (7.4 and 8.2) were comparatively studied in the coral oculina patagonica (fine and tchernov, 2007). we accordingly set ph = 7.4 and 8.2 in the present study. the low ph value (ph = 7.4) was simply produced by adding hydrochloric acid to the natural seawater (ph = 8.2). to maintain water temperatures, the behavioral experiments were carried out in buckets (diameter of the bucket bottom = 22 cm) bathed in temperature-controlled tanks, where two water temperatures (15 °c and 25 °c) were set. forty shells of mytilus galloprovincialis were randomly selected and put into each bucket according to our previous study (zhao et al., 2014). there were no significant differences in shell length, shell width and body weight of m. galloprovincialis among experimental groups (table 1, p > 0.05). we then placed 144 sea urchins into the 12 buckets (12 individuals per bucket, 4 treatments, 3 replicates). because the experimental duration was relatively short, ph values were well maintained. time to first covering refers to the time of covering by the first sea urchin that covered, indicating the behavioral reaction. the number of covered sea urchins and number of shells used for covering were measured every 10 min during 90 min, indicating the behavioral capability. fig. 1 time to first covering of glyptocidaris crenularis at different water temperatures and ph values (n = 3, mean ± sd). 16 fig. 2 number of covered glyptocidaris crenularis at different water temperatures and ph values (n = 3, mean ± sd). statistical analysis all original data were tested for normal distribution and homogeneity of variance. repeated measured anova was used to detect the differences of number of covered sea urchins and number of shells used for covering among experimental groups. time to first covering was analyzed using mann-whiney u test because the data showed heterogeneity of variance. all analysis was carried out using spss 13.0. a probability level of p < 0.05 was considered statistically significant. results time to first covering time to first covering was significantly lower in sea urchins exposed to lower ph (p = 0.026, fig. 1). however, water temperature did not significantly affect time to first covering, although g. crenularis showed obviously quicker reaction at 25 °c than those at 15 °c (p = 0.180, fig. 1). number of covered sea urchins and number of shells used for covering lower ph significantly decreased number of covered sea urchins (p = 0.029, fig. 2) and number of shells used for covering (p = 0.007, fig. 3). water temperature did not significantly affect number of covered sea urchins (p = 0.157, fig. 2), though significantly affected number of shells used for covering (p = 0.042, fig. 3). interaction between water temperature and ph value showed no significant effect on number of covered sea urchins (p = 0.752) or number of shells used for covering (p = 0.989). observational duration significantly affected both number of covered sea urchins (p < 0.001) and number of shells used for covering (p = 0.01). both number of covered sea urchins and number of shells used for covering increased in the first 20 or 30 min. and then obviously decreased in lower ph groups (figs 2, 3). discussion water temperature and ph value significantly affected calcification (byrne et al., 2011), growth (albright et al., 2012) and behavior (cripps et al., 2011) of marine organisms, ultimately impacting the entire ecosystem (strandberg et al., 2012). considering their roles in structuring marine benthic communities, it is critical to understand ecological response of sea urchins to water temperature and ph value. the present study investigated the behavioral response of sea urchins to water temperatures and ph values. it provides preliminary information on behavioral ecology of sea urchins. in the present study, we found that lower ph value (ph = 7.4) significantly affected covering behavior of g. crenularis. this result is in agreement with previous reports on the behavioral response of marine organisms to ph values. in reef fish pseudochromis fuscus, for example, ocean acidification significantly disturbed its prey detection (cripps et al., 2011). lower ph value significantly decreased the number of covered sea urchins and number of shells used for covering. this result is not in agreement with the finding of challener and mcclintock (2013) that ocean acidification did not significantly impact covering behavior of juvenile sea 17 fig. 3 number of shells used for covering of glyptocidaris crenularis at different water temperatures and ph values (n = 3, mean ± sd). urchins lytechinus variegatus. the disagreement is probably due to the difference of behavioral response to ocean acidification between the two species. interestingly, we found that lower ph value significantly increased the speed of covering reaction. a reasonable explanation is that the acute stress increased the covering reaction. consistently, brothers and mcclintock (2015) found that acute exposure to elevated temperature significantly increased covering behavior of l. variegatus, compared to chronical exposure. water temperature did not significantly affect the time to first covering and number of covered sea urchins. crook (2003) found that paracentrotus lividus covered more quickly during summer time and obviously less in winter time. these results confirm the difference of the covering behavior among different species (verling et al., 2004). interestingly, we found number of shells used for covering was significantly different between sea urchins at 15 °c and 25 °c, although the p value was very close to 0.05 (p = 0.042). this result is not consistent with our previous study, in which we found no significant difference of number of shells used for covering between g. crenularis in 15 °c and 25 °c (zhao et al., 2014). this disagreement might be due to limited sample size in both studies and strongly highlights the individual variation of covering behavior of sea urchins. considering the multiple functions of 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comp. biochem. physiol. 160a: 331-340, 2011b. verling e, crook a, barnes d. covering behaviour in paracentrotus lividus: is light important? mar. biol. 140: 391-396, 2002. yamada y, ikeda t. acute toxicity of lowered ph to some oceanic zooplankto n. plankton biol. ecol. 46: 62-67, 1999. zhang dj, li sj, wang gz, guo dh. comparative study on the acute toxicity of ocean acidification driven by co2 and hcl on several marine copepods. j. xiamen univ. 50: 631-636, 2011. zhang l, zhang l, shi d, wei j, chang y, zhao c. effects of long-term elevated temperature on covering, sheltering and righting behaviors of the sea urchin strongylocentrotus intermedius. peer j. 5:e3122, 2017. zhao c, luo s, zhou h, tian x, chang y. effect of temperature on covering behavior of the sea urchins glyptocidaris crenulars and strongylocentrotus intermedius. oceanologia et limnologia sinica 45: 522-528 (in chinese with an english abstract), 2014. http://apps.webofknowledge.com/oneclicksearch.do?product=ua&search_mode=oneclicksearch&colname=wos&sid=4bcmmam72j5ledn3edp&field=au&value=byrne,%20m&ut=1963402&pos=%7b2%7d http://apps.webofknowledge.com/oneclicksearch.do?product=ua&search_mode=oneclicksearch&colname=wos&sid=4bcmmam72j5ledn3edp&field=au&value=ho,%20m http://apps.webofknowledge.com/oneclicksearch.do?product=ua&search_mode=oneclicksearch&colname=wos&sid=4bcmmam72j5ledn3edp&field=au&value=ho,%20m http://apps.webofknowledge.com/oneclicksearch.do?product=ua&search_mode=oneclicksearch&colname=wos&sid=4bcmmam72j5ledn3edp&field=au&value=soars,%20na http://apps.webofknowledge.com/oneclicksearch.do?product=ua&search_mode=oneclicksearch&colname=wos&sid=4bcmmam72j5ledn3edp&field=au&value=selvakumaraswamy,%20p&ut=14338795&pos=%7b2%7d http://apps.webofknowledge.com/oneclicksearch.do?product=ua&search_mode=oneclicksearch&colname=wos&sid=4bcmmam72j5ledn3edp&field=au&value=shepard-brennand,%20h characterization of a natural hemagglutinin in the serum of a mud crab scylla serrata isj 7: 79-88, 2010 issn 1824-307x research report physico-chemical characterization of bacterial and hemagglutinins from the serum of the mud crab scylla serrata ss jayaraj1, r thiagarajan2, m arumugam2, s vincent1 1unit of environmental health and biotechnology, department of advanced zoology and biotechnology, loyola college, chennai 600 034, india 2unit of pathobiology, department of zoology, university of madras, guindy campus, chennai 600 025, india accepted february 15, 2010 abstract a naturally occurring hemagglutinin (ha), with activity against bacteria and yeast cells were detected in the serum of scylla serrata using mammalian erythrocytes (rbc), various bacteria and yeast as indicator cells. the serum gave highest ha titer with rabbit rbc, tripsinized yeast and vibrio fluvialis. an analysis of the physico-chemical properties of the ha showed it to be specifically dependent on the presence of ca2+ for its activity, stable between ph 7 to 9 and showed thermal stability between 10 to 30 °c. further studies demonstrated that the ha is precipitable by ammonium sulphate and tca. hainhibition assays performed with carbohydrates revealed that the serum ha was specific for non-reducing terminal glucose with 1-2 glucosidic linkages. thus this agglutinin appears to be unique among all the known crustacean agglutinins. key words: hemagglutinin; bacterial agglutinin; yeast agglutinin; serum; mud crab; scylla serrata introduction mud crab scylla serrata is an economically important decapod crustacean in aquaculture in india. understanding the defence system of s. serrata at humoral and cellular levels is indispensable for preventing disease occurrence. lectins are carbohydrate-binding proteins and in invertebrates, lectins are vital means for non-self recognition and clearance of invading microorganisms. in invertebrates, phagocytosis is considered to be the primary mechanism of innate defense against foreign invaders (ratcliffe and rowley, 1981; coombe et al., 1984; ratcliffe et al., 1985). in this process, an intimate interaction of humoral substances, particularly as recognition factors, has been implicated (richards and renwrantz, 1991; zelck and becker, 1992). a variety of humoral factors, naturally occurring and/or formed after antigenic stimulation, have been detected in the serum of invertebrates and they include agglutinins (cornick and stewart, 1973; renwrantz, 1986; nalini et al., 1994; murali et al., 1999; jayasree, 2001; jayaraj et al., 2008a), lysins (osada et al., 1993), antibacterial (xylander and neverman, 1990), and antifungal proteins (iijima et al., 1993), phenoloxidase system (söderhäll, 1982), lps binding protein (jomori and natori, 1992) and β ___________________________________________________________________________ corresponding author: ss jayaraj department of advanced zoology and biotechnology loyola college, chennai, india e-mail: jayarajss@gmail.com 1, 3 glucan binding protein (jayaraj et al., 2008b). due to the probable functional similarities between agglutinins and vertebrate antibodies and the indications that agglutinins serve a defensive function (ofek and sharon, 1988), invertebrate agglutinins have been extensively studied. agglutinins (= lectins) are dior multivalent carbohydrate-binding proteins with the ability to agglutinate cells with complementary carbohydrates on their surfaces (sharon and lis, 1972; barondes, 1988). they are known to specifically recognize the whole sugar (bretting and kabat, 1976), a specific site in a sugar (shimizu et al., 1977), a sequence of sugars (kobiler and mirelman, 1980), or their glycosidic linkages (koch et al., 1982). the agglutinating molecules are widely distributed in microorganisms (sasmal et al., 1992), plants and animals (gold and balding, 1975). the body fluid or hemolymph of almost all invertebrate species tested contains agglutinins (yeaton, 1981; ratcliffe et al., 1985; renwrantz, 1986; nalini et al., 1994; murali et al., 1999; jayasree, 2001; jayaraj et al., 2008a). the presence of agglutinins has also been detected in the mucus as well as in certain tissues of invertebrates (renwrantz, 1986; mullainadhan and renwrantz, 1989; suzuki and mori, 1991). however, its immunological role is best understood in the hemolymph, and recent studies have shown that purified, hemolymph-derived agglutinins served as opsonin in a few insects and molluscs (renwrantz and stahmer, 1983; pendland 79 mailto:jayarajss@gmail et al., 1988; fryer et al., 1989; richards and renwrantz, 1991; jayaraj et al., unpublished observations). although a number of studies have demonstrated the presence of humoral agglutinins in several crustacean species, it can be noted that the immunological roles of these agglutinins remain largely unknown and that the carbohydrate specificity of serum agglutinins from crustaceans have been elucidated only in a few species (vasta et al., 1983; smith and chisholm, 1992; nalini et al., 1994; kondo et al., 1998; jayasree, 2001). this study thus describes rbc and bacterial binding activities, physico-chemical properties and carbohydrate specificity of a naturally occurring agglutinin in the serum of the marine crab s. serrata. material and methods experimental animals and laboratory maintenance the marine crab scylla serrata weighing 150 to 200 g were obtained from muttukadu estuary, chennai. in the laboratory, these crabs were maintained in plastic tanks (90 x 45 x 60 cm) containing aerated natural seawater and the medium was changed every day. the crabs were fed with donax spp. during the period of acclimation (24 h) and only male crabs were used. preparation of serum hemolymph samples (1 to 2 ml) from individual crabs were collected from the cut end of the dactylus region of the walking leg. the samples were collected in clean polystyrene plastic tubes held on ice and allowed to clot at room temperature (rt: 28 ± 2 °c for 20 min). serum was separated by centrifugation (400xg, 10 min, rt) and the resulting clear supernatant (=serum) was used immediately. preparation of erythrocyte (rbc) suspension human and other mammalian blood samples were obtained by venous or cardiac puncture and collected in sterile alsever’s solution (garvey et al., 1979) containing 10 µg/ml of streptomycin. prior to use, the rbcs were washed thrice with 0.9% saline and once with tbs-i (50 mm tris-hcl, 115 mm nacl, 10 mm cacl2, 300 mosm) by centrifugation (400xg, 5 min, rt). unless specified, the rbc pellet was finally resuspended in tbs-i as 1.5% suspension (v/v). preparation of yeast cell suspension a hundred mg commercial grade baker’s yeast (saccharomyces cerevisiae) purchased from local market were suspended in 10 ml of 0.9 % saline, washed extensively with saline by centrifugation (400xg, 5 min, rt) and suspended in the same medium. the yeast cell suspension was heatinactivated by autoclaving the suspension for 15 min at 15 psi. after cooling the suspension to rt, the heat-inactivated yeast cells were washed extensively with 0.9 % saline and finally resuspended in tbs-i as 0.5 % (v/v) suspension. trypsinization of yeast cells five μl of washed yeast cells were suspended in 1 ml of tbs-i containing trypsin (0.5 %) to give a final concentration of 0.5 % yeast. this suspension was incubated for 1 h at 37 oc with occasional gentle shaking. after incubation, the trypsinized yeast cells were washed once with tbs-i by centrifugation (400xg, 5 min, rt) and finally resuspended in tbs-i as 0.5 % (v/v) suspension. hemagglutination (ha) assay ha assays were performed in v-bottom microtiter plates (greiner, nürtingen, germany) by serial two-fold dilution of a 25 µl serum sample with an equal volume of tbs-i. after dilution, 25 µl rbc suspension was added to each well and incubated for 45 min at rt. the ha titers were recorded as the reciprocal of the highest dilution of the sample causing complete agglutination of rbc (garvey et al., 1979). controls for all assays consisted of the substitution of the sample by tbs-i. all the ha assays were performed in duplicate. yeast agglutination assay the agglutinating activity of serum against yeast cells was performed in v-bottom microtiter plates by serial two-fold dilution of 25 µl serum with an equal volume of tbs-i. after dilution, 25 µl of 0.5 % native or trypsinized yeast cell suspension was added to each well and incubated for 45 min at 26 °c. control consisted of substitution of serum with tbs-i. the agglutination of yeast cells by serum was assessed under microscope (40 x) and the agglutination titers were recorded as the reciprocal of the highest dilution of the sample causing complete agglutination. the experiment was performed using duplicates. bacterial agglutinating activity frozen stock culture of bacteria were inoculated in tbs-i and incubated for 6 h. the broth cultures were then centrifuged (5,000xg, 10 min). the pellet was collected and washed 3 times by centrifugation with tbs-i. the final concentration was adjusted to 1x108 cells ml-1 in tbs-i before use. two-fold serial dilutions of serum samples (in duplicates) were made in tbs-i. then, 25 µl of each serum dilution was incubated with 25 µl bacterial suspension. the reaction mixture was incubated at 20 ± 2 °c for 1 h. the appearance of clumps of bacteria was then recorded by microscopic examination (40x). agglutination titer was defined as the reciprocal of the last dilution giving evidence of agglutination after incubation. the negative controls comprised mixed equal volumes of bacterial suspension and tbs-i. divalent cation dependency and edta sensitivity serum samples (each 300 µl in duplicates) were dialysed (mw exclusion limit <10,000) extensively at 20 °c against divalent cation-free tbs-ii (50 mm tris-hcl, 135 mm nacl, 300 mosm) to examine cation dependency or in tbs-iii containing 50 mm edta (50 mm tris-hcl, 72 mm nacl, 40 mm cacl2, 300 mosm) to test edta sensitivity of the agglutinating activity of serum. the samples dialysed against tbs-iii were subsequently re-equilibrated by dialysis in tbs-ii. all the resulting dialysates were centrifuged (400xg, 10 min, 20 °c). the supernatant was tested for hemagglutinating activity using rabbit rbc in the presence of tbs 80 that did or did not contain 10 mm cacl2, mgcl2 (or) mncl2. a serum sample (300 µl) concurrently dialysed against, tbs containing 10 mm cacl2 (tbs-i) was also tested for the hemagglutinating activity against rabbit rbc in tbs-i. ph and thermal stability the stability of serum ha activity (in duplicates) at different ph was examined by dialyzing (24 h, 4 °c) 300 μl serum samples against the following buffers at ph ranging from 3 to 12 (lillie, 1954; pearse, 1968): 0.2 m acetate buffer (ph 3 to 6), 0.2 m tris-hcl buffer (ph 7 to 9) and 0.1 m glycinenaoh buffer (ph 10 to 12). after dialysis, all the samples were finally re-equilibrated by dialysis against tbs-i and the ha titer was determined with rabbit rbc. in another experiment designed to study the thermal stability of ha, 300 μl serum samples were held for 30 min at temperatures ranging from 10 to 100 °c, centrifuged and tested for ha activity with rabbit rbc. precipitation by ammonium sulphate and trichloro acetic acid precipitation of ha activity from serum (in duplicates) was attempted using 25, 50 and 75 % ammonium sulphate [(nh4)2so4] solution as well as 10 % trichloroacetic acid (tca) as described previously (millar, 1987). the ha activity was finally measured using rabbit rbc. ha-inhibition assays several carbohydrates were tested for their ability to inhibit serum ha activity. they were dissolved in tbs-iii (50 mm tris-hcl, 115 mm nacl, 50 mm edta, 300 mosm) and if necessary, the ph was adjusted to 7.5 using concentrated naoh. serum samples were diluted with tbs-iv (50 mm trishcl, 5 mm nacl, 30 mm cacl2, 135 mosm) to a ha titer of 4 against rabbit rbc. the inhibitor to be tested (25 μl) was serially diluted two-fold with an equal volume of diluted sample in microtiter plates and incubated for 1 h at rt. rabbit rbc suspension (25 μl) was added to each well and kept for 3 h at rt. the minimal concentration of carbohydrate that completely inhibited ha activity was recorded. the experiments were performed in duplicates. protein determination total protein concentration was measured using bovine serum albumin (bsa) as a standard (lowry et al., 1951). result serum ha profile the serum of mud crab scylla serrata agglutinated a variety of mammalian rbc types. among the various rbc types tested, the highest titer of 128 was obtained with rabbit erythrocytes. the serum did not discriminate human a, b and o rbc types and agglutinated them to the same degree. sheep and goat rbc were agglutinated at relatively low titers (table 1). however serum did not agglutinate ox rbc and the serum showed highest agglutinating activity against tripsinized yeast cells when compare to native yeast cells (table 2). table 1 hemagglutinating (ha) activity of serum from the mud crab s. serrata against various mammalian erythrocyte (rbc) types rbc types tested ha titer* rabbit 128 mice 64 rat 32 human b 32 buffalo 16 human a 8 human o 8 horse 4 goat 2 sheep 2 ox 0 * based on 20 determinations for each rbc type bacterial agglutination the serum strongly agglutinated vibrio fluvialis (titer 8), weekly agglutinated vibrio parahemolyticus, vibrio mimicus, escherichia coli, pseudomonas spp. and aerobacter aerogenes. the results of bacterial agglutination was assessed using a phase-contrast microscope (table 3). divalent cation dependency an edta sensitivity the serum tested in tbs containing 10 mm cacl2 (tbs-i) gave a hemagglutianation titer of 128 against rabbit rbc. when the serum was dialysed against tbsi and then tested in the absence of divalent cation, the agglutination titer reduced to 16. but, this serum sample recovered it’s ha activity only upon addition of ca2+ to the reaction mixture. further, substitution of ca2+ with mg2+ showed a considerable improvement in ha titer, while mn2+ could not reverse the effect of edta treatment. the serum dialyzed against tbs-iii containing 50 mm edta and tested in the absence of divalent cation, considerably lost its agglutinating activity against rabbit rbc (table 4). further the addition of mg2+ or 81 mn2+ to this sample could not restore the original ha activity and addition of ca2+, rescued the activity to 64 (table 4). ph and thermal stability the serum hemagglutinating activity of the marine crab s serrata was tested in the ph range of 3-12. as shown in figure 1, the hemagglutinating activity against rabbit rbc was found to be relatively stable between ph 7 and 9 reduced at ph below or above this ph range and completely lost at ph 11 and 12. the activity of the serum against rabbit rbc was unaffected only up to 30 °c but it was reduced considerably at 40 °c and 50 °c and completely inactivated at 60 °c and above (fig. 2). precipitation of serum ha activity by ammonium sulphate and tca serum was incubated with ammonium sulphate (25, 50 and 75 %) for 3 h. the 50 % concentration of ammonium sulphate completely precipitated ha activity from the serum, whereas ammonium sulphate concentration at 25 and 75 %, moderately precipitated the serum ha activity. by contrast 10 % tca failed to precipitate serum ha activity (table 5). carbohydrate binding specificity among the 24 carbohydrates tested, as many as 15 carbohydrates were found to inhibit serum hemagglutinating activity at concentrations ranging from 50 to 100 mm. all the three acetylated hexosamines (glcnac, galnac and mannac), but not their hexoses and hexosamine counterparts, were inhibitory at 50 or 100 mm. but the few sialic acids examined in this study and 9 other carbohydrates were not inhibitory when tested up to concentrations from 20 to 200 mm (table 6). among the six different polysaccharides tested (table7), only mannan and laminarin inhibited the ha activity at 0.25 and 0.50 mg/ml, respectively. among all the inhibitory carbohydrates, mannan was found to be most potent. table 2 agglutinating activity of s. serrata serum against native and trypsinized yeast cells yeast cells tested ha titer* native 16 trypsinized 64 * based on 20 determinations for native and trypsinized yeast cells discussion the serum of the marine mud crab s. serrata was found to possess naturally occurring agglutinating activity which showed the highest reactivity with rabbit rbc among the rbc types tested. these results also suggest that the rbc types agglutinated by the serum of s. serrata probably share a common surface receptor but with a quantitative difference in its ha binding sites. the serum agglutinated a variety of bacteria including gram + and gramtypes and the species of vibrio tested are known to be the most frequent opportunistic pathogens of aquatic crustaceans (equidius, 1987; vargas-albores et al., 1993) and the plasma showed highest agglutinating activity against trypsinized yeast cells (jayaraj et al., 2008b). the ability of the serum of s. serrata to agglutinate bacteria, particularly the potential pathogens, implicates a possible involvement of the humoral agglutinins in host defense response. table 3 agglutinating activity of s. serrata serum against various bacterial species bacterial species tested bacterial agglutination* (o.d: 0.8) vibrio fluvialis 16 vibrio alginolyticus 8 vibrio vulnificus 8 vibrio anguillarum 4 vibrio parahemolyticus 4 vibrio mimicus 2 escherichia coli 2 pseudomonas sp 2 bacillus subtilis 4 aerobacter aerogenes 2 * the assay was repeated six times for each bacterial species with identical results using samples from different preparations 82 table 4 effect of divalent cations and edta on the hemagglutinating (ha) activity of serum of s. serrata serum sample tested cation (10 mm) in sample diluting and rbc suspension ha titer* before dialysis cacl2 128 after dialysis against divalent cation free tbs (tbs-ii). none cacl2 mgcl2 mncl2 16 128 64 16 after dialysis against tbs+10 mm cacl2 (tbs-i) cacl2 128 after dialysis against tbs+50 mm edta (tbs-iii) followed by dialysis against tbsii none cacl2 mgcl2 mncl2 8 64 4 4 * determination using rabbit rbc and the results based on six determinations the serum agglutinin was heat-labile and susceptible to ph extremes. its ph stability was comparable to that found in one earlier report (nalini et al., 1994) and the proteinaceous nature of agglutinin has been well demonstrated (mckay and jenkin, 1969; acton et al., 1969; miller et al., 1972). the serum lost most of it’s ha activity after dialysis against cation-free tbs and when tested in the absence of cations. however, the activity in this sample completely regained only upon addition of ca2+ and the ha titer of serum did not change after dialysis against tbs containing ca2+. these observations demonstrated that the serum agglutinin of s. serrata specifically requires ca2+ for it’s ha activity. furthermore, the activity was sensitive to edta treatment, since dialysis of serum against tbs containing edta resulted in a significant reduction in the ha activity. none of the cations tested could restore the ha activity, albeit ca2+ moderately rescued the activity in these samples, thereby indicating that the ha of s. serrata appears to be irreversibly sensitive to edta which is in contrast with other crustacean agglutinins (hall and rowlands, 1974a; ravindranath et al., 1985; kamiya et al., 1987). crustacean serum agglutinins were shown to be specific for fucose (amirante and basso, 1984), glucose (umetsu et al., 1991), galactose (kamiya et al., 1987; umetsu et al., 1991), galnac (amirante and basso, 1984; vargas-albores et al., 1993), or sialic acids such as neuac (hall and rowlands, 1974b; vasta et al., 1983; vasta and cohen, 1984; cassels et al., 1986; ratanapo et al., 1990), 4and 9-0-acetyl neuac (ravindranath et al., 1985), 9-0acetyl neuac (vazquez et al., 1993) and neugc (mercy and ravindranath, 1993). the hemagglutination-inhibition test performed in this study using different carbohydrates, encompassing several diverse, unrelated monosaccharides and their derivatives as well as diand oligo-saccharides inhibited the serum agglutinating activity. furthermore, all the three acetylated hexosamines tested consistently inhibited the ha activities of crab table 5 ammonium sulphate and tca precipitation of haemagglutination (ha) activity against rabbit rbc from the serum of s. serrata ammonium sulphate and tca (%) saturation ha titer* (rabbit rbc) untreated sample 128 25 % 16 50 % 128 75 % 32 tca 10 % 0 * determination using rabbit rbc and the results based on six determinations 83 table 6 inhibition of agglutinating activity (titer = 4) of serum from the mud crab s. serrata by various carbohydrates carbohydrates tested maximum concentration tested (mm) minimum inhibitory concentration (mm)* monosaccharides simple sugars d-mannose 200 100 l-sorbose 100 100 d-fucose 100 100 l-fucose 100 50 deoxy sugars l-rhamnose 200 100 n-acetyl sugars n-acetyl-d-glucosamine (glcnac) 200 100 n-acetyl-d-galactosamine (galnac) 200 100 n-acetyl-d-mannosamine (mannac) 200 50 disaccharides trehalose (glcα1 → 1 glc) 200 50 cellobiose (glcβ1 → 4 glc) 200 100 β-gentiobiose (glcβ1 → 6 glc) 200 50 sucrose 200 100 palatinose (glcα1 → 6 fruc) 200 50 melibiose (galα1 → 6 glc) 200 100 lactose (galβ1 → 4 glc) 200 100 the following carbohydrates also did not inhibit the agglutinating activity and unless otherwise stated, all carbohydrate was tested at concentrations upto 200 mm: d-glucose, d-galactose, β-allose, d-fructose, dglucosamine (glcn), d-galactosamine (galn), mannosamine (mann), maltose (glcα1 → 4 glc), turanose (glcα1 → 3 fruc). * the assay was repeated five times for each carbohydrate with identical results table 7 agglutination–inhibition of s. serrata serum (agglutination titer = 4) by polysaccharides against rabbit rbc polysaccharides tested structural linkages maximum concentration tested (mg. ml-1) minimum inhibitory concentration (mg. ml-1)* mannan (α 1-6 homopolymer of mannose) 1 0.25 laminarin (β 1-3 homopolymer of glucose) 1 0.50 dextran t70 (α 1-6,3,2 homopolymer of glucose) 2 ni dextran t500 (α 1-6,3,2 homopolymer of glucose) 2 ni inulin (α 2-6 homopolymer of fructose) 5 ni colominic acid (α 2-8 homopolymer of neu5ac) 5 ni * the assay was repeated three times for each polysaccharide with identical results using samples from different preparations ni: no inhibition 84 fig. 1 ph stability of hemagglutinating activity of serum of s. serrata against rabbit rbc serum and this has been earlier demonstrated (amirante and basso, 1984; vargas-albores et al., 1993; mercy and ravindranath,1993). the serum ha activity of s. serrata was not inhibited by the amino sugar tested. but it was inhibited by the simple hexoses namely mannose, l-sorbose, dfucose and l-fucose. this finding is supported by previous studies wherein simple hexoses were shown to inhibit agglutinating activity, including fucose (amirante and basso, 1984), glucose (umetsu et al., 1991). the c-l position of these hexoses is essential for interaction with the agglutinin. the amino derivatives (glcn, galn and mann) did not inhibit the ha activity. however, their n-acetyl derivatives (glcnac, galnac and mannac) were able to inhibit the serum ha activity. recently, alpuche et al. (2005) have shown the inhibition of purified shrimp lectin by nacetylated sugars and this lends support to our study. the disaccharides d-maltose and turanose failed to inhibit the ha activity but all other disaccharides were inhibitory. fig. 2 thermal stability of hemagglutinating activity of serum of s. serrata against rabbit rbc 85 all these observations clearly demonstrate that the presence of acetyl group at c-2 position of hexosamines does not favour the interaction with agglutinin whereas this position with a free hydroxyl group or its substitution with amino group is essential for the interaction. ha inhibition tests employing polysaccharides indicated that only laminarin and mannan inhibited the serum agglutinating activity. this indicates that the agglutinin molecules in crab serum tend to exhibit affinity for extended structures particularly for polysaccharides with β-linked hexoses. this is significant and shows the ability of the serum agglutinating activity to recognize a wide range of carbohydrates that will potentially help the animal to recognize a variety of pathogens based on their surface molecules (zhang et al., 2009). thus, all the results obtained from the inhibitory effects of various carbohydrates and glycoproteins taken together clearly indicate that the agglutinins present in the serum of s. serrata interact with a wide range of carbohydrates including acetylated hexosamines, acetylated or non-acetylated sialic acids and several other carbohydrates, though their preference for a specific carbohydrate structure could not be ascertained. however, these findings in turn strongly suggest the natural occurrence of multiple agglutinins in the serum of this crab. the agglutinins in several crustaceans have been characterized (cornick and stewart, 1968; mckay and jenkin, 1969, 1970; huang et al., 1981; vasta et al., 1983; ratanapo et al.,1990; adams, 1991; nalini et al., 1994; jayasree, 2001). this agglutinin appears to be unique among all the known crustacean agglutinins. thus, based on the hemagglutination and bacterial agglutinating activity of the serum agglutinin, it is possible that this component of the crab is probably involved in non-self recognition and eliciting immune response in the mud crab s. serrata, against invading pathogens. the identification of immune effectors like agglutinin and the understanding of their regulation in response to infection will open the way to the selection of pathogen resistant animals. this can be achieved through the characterization and purification of this novel agglutinin of s. serrata as a prerequisite to elucidate the immunological roles of crustacean agglutinins. references acton rt, bennet jc, evans ee, schrohenloher re. physical and chemical characterization of an oyster haemagglutinin. j. biol. chem. 244: 4128-4135, 1969. adams a. response of penaeid shrimp to exposure to vibrio species. fish shellfish immunol. 1: 5970, 1991. alpuche j, pereyra a, agundis c, rosas c, pascual c, slomianny m-c, vázquez l, zenteno e. purification and characterization of a lectin from the white shrimp litopenaeus setiferus (crustacea decapoda) hemolymph. biochim. biophy. acta 1724: 86-93, 2005. amirante ga, basso v. analytical study of lectins in squilla mantis l. 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http://www.sciencedirect.com/science?_ob=publicationurl&_tockey=%23toc%235121%232009%23999539991%231045056%23fla%23&_cdi=5121&_pubtype=j&view=c&_auth=y&_acct=c000050221&_version=1&_urlversion=0&_userid=10&md5=0e26e5290e78608baf0c21c302be43e2 monosaccharides disaccharides isj109.pdf 152 isj 2: 152-158, 2005 issn 1824-307x review insights into brown spider and loxoscelism mh appel1, r bertoni da silveira1,3, w gremski1,2, ss veiga1 1department of cell biology, federal university of paraná, jardim das américas, curitiba, paraná , brazil 2catholic university of paraná, health and biological sciences institute, curitiba, paraná brazil 3department of biochemistry, federal university of são paulo, são paulo, brazil accepted december 27, 2005 abstract loxosceles is a genus of cosmopolitan spiders comprising several species, and popularly known as brown spiders or brown recluses. brown spider bites can cause dermonecrotic lesions and systemic reactions known as loxoscelism. systemic effects are less common but may be severe or even fatal in some patients. systemic manifestations include intravascular hemolysis, disseminated intravascular coagulation and acute renal failure. a rapid diagnosis and an understanding of the venom’s molecular activity are crucial for satisfactory treatment. mechanisms by which venoms exert their deleterious effects are under investigation, and searches are underway for diagnostic envenomation assays. molecular biology is being used to produce quantities of several of the most important venom molecules and has contributed to the study and understanding of their mechanisms of action. key words: brown spider; loxoscelism; venom; recombinant toxins; dermonecrosis introduction more than 40,000 spider species exist, with probably 100,000 to be described, but only 3 taxa are recognized as dangerous, namely theridiidae, loxoscelidae and ctenidae. moreover, only the genera atrax, lactrodectus and loxosceles are associated with human deaths (escoubas et al., 2000; rash and hodgson, 2002). early european tales during the middle ages linked injuries or illness to spider bites (schienle et al., 2005). for example the tarantula bite was associated with a disease (tarantism) for which the cure was a frenetic dancing for 3-4 days. this energetic dance, called tarantella, is now a typical italian dance (isbister, 2004). today, as a consequence of mistaken diagnoses of spider bites, scientists are looking for methods to characterize and identify spider bites and their manifestations as well as to better understand the biological and molecular corresponding author: silvio s. veiga department of cell biology, federal university of paraná, jardim das américas,81531-990, curitiba, paraná, brazil e-mail: veigass@ufpr.br mechanisms of envenomation. the genus loxosceles (variously known as the brown spider, brown recluse, fiddleback, or gaucho spiders) is important in these studies because of its commonness in and around human dwellings. their bite is characterized by dermonecrosis and systemic effects known as loxoscelism (hogan et al., 2004). the first case of documented loxoscelism occurred in 1879 in tennessee. however, consistent data traced back about 50 years ago and were collected in chile, then other observations were made in brazil followed by the united states. these reports linked brown spider bite with necrotic skin lesions (macchiavello, 1947; atkin et al., 1958; sams et al., 2001). spiders’ habits have caused a close association with humans, and the number of bites is increasing and has become a public health problem in brazil, chile and the united states (da silva et al., 2004). most bites occur during sleep or dressing, and women are bitten more often than men. thighs, trunk, hands and arms are more often bitten (hogan et al., 2004). loxosceles spiders loxosceles spiders are known as violin (fiddleback) spiders due to a characteristic violin 153 shape on their cephalothorax (futrell, 1992). they are also known as brown spiders because their colour varies from a pale (l. laeta) to a dark brown (l. gaucho). loxosceles body length ranges from 8 to 15 mm with legs measuring from 8 to 30 mm (da silva et al., 2004). they are sedentary and nocturnal (andrade et al., 1999) with a lifetime of 3 – 7 years (andrade et al., 2000). brown spiders have three pairs of eyes (an important characteristic useful to identify the genus) (vetter and visscher, 1998). they build irregular, cottony webs (futrell, 1992) and normally prefer dead scavenged prey rather than live preys (sandidge, 2003). they can survive months without food or water and withstand temperatures ranging from 8 °c to 43 °c. they are not aggressive and prefer dark dry places (futrell, 1992; málaque et al., 2002; vetter and barger, 2002; da silva et al., 2004). the sexes produce venom with differences in volume, toxicity and compounds proportion (oliveira et. al., 1999). comparative analysis of sex and species in l. laeta and l. intermedia venom showed some biological activities (complement-dependent hemolysis and dermonecrosis) more prominent in venom from female spiders, especially from l. laeta (oliveira et al., 2005). epidemiology loxosceles spiders can be found distributed all over the world. in north america, the most important species are l. reclusa, l. deserta, l arizona, l. rufences (united states and mexico) and l. laeta (canada) (sams et al., 2001; vetter and bush, 2002a). europe, africa, middle east, some parts of asia, israel, and australia are hosts to some loxosceles species (futrell, 1992; borkkan et al., 1995; young and pincus, 2001; nicholson and graudins, 2003). in brazil, seven species have been described but three are the most implicated in human bites l. intermedia, l. gaucho and l. laeta (sezerino et al., 1998). from 1990 to 1993, the brazilian ministry of health received 17.781 reports of spiders’ bites, of which 36 % were due to loxosceles (sezerino et al., 1998). in the metropolitan area of curitiba, in the state of parana (southern brazil) about 3.000 brown spider bites are reported annually (málaque et al., 2002). in a retrospective study in florianopolis, in the state of santa catarina, brazil, 487 suspected cases of brown spider bites were found, 267 of which fulfilled the criteria for inclusion in the study (sezerino et al., 1998). in 359 cases of loxoscelism between january 1985 and december 1996 at butantan intitute, são paulo, brazil, 14 % of patients captured the spiders so that 28 were classified as l. gaucho, 5 as l. laeta and 18 as non-classified loxosceles (málaque et al., 2002). more bites occur in warmer months (schenone, 1996). in curitiba, from 1998 to 2001 the incidence of loxosceles bites was 1.4 cases per 1,000 habitants. of these, 23 % were in the thigh, 16.7 % in the trunk, 14 % in the arm and 13 % in the lower leg. only 1 % of cases were severe (health secretary, curitiba, parana, brazil, 2002). pathophysiology of loxoscelism dermonecrosis is the hallmark of loxoscelism (fig. 1). histopathology and clinical data are obtained from biopsies of human patients after brown spider bites. rabbit skin artificially injected with loxosceles venom is used for more controlled investigation since this animal model reproduces human skin lesions that follow envenomation (ospedal et al., 2002). systemic effects, such as renal failure, are less common and are usually reproduced in mouse (luciano et al., 2004). observation of human skin biopsies showed an inflammatory infiltrate, thrombosis, hemorrhage, dermatitis, erythema, induration of affected area and liquefactive necrosis of the epidermis and dermis consistent with pyoderma grangrenosum (futrell, 1992; yannias and winkelmann, 1992). symptoms in an experimental study in rabbits showed that after 4 h oedema, hemorrhage, degeneration of blood vessel walls, plasma exudation, thrombosis, neutrophil accumulation in and around blood vessels with an intensive diapedesis, a diffuse collection of inflammatory cells (polymorphonuclear leucocytes) in the dermis, and subcutaneous muscular oedema all occur. over the following hours and up to 5 days after envenomation, the changes progressed to a massive neutrophil infiltration into the dermis and even into subcutaneous muscle tissue, destruction of blood vessels, thrombosis, hemorrhage, myonecrosis, and coagulative necrosis on the 5th day (ospedal et al., 2002). neutrophil participation and the inflammatory response seem to be dependent on an endothelial cell agonist effect triggered by the venom that leads to an indirect and dysregulated neutrophil activation involved in dermonecrosis (patel, 1994). envenomation of rabbit skin with l. reclusa venom after 14 days results in a mixed inflammatory cell infiltrate, coagulative tissue necrosis, vasculitis and a dense band of neutrophils bordering the zone of necrosis (elston et al., 2000). l. intermedia venom damaged vessel endothelia, as shown by vessel instability, endothelium cell vacuolization in biopsies of rabbit skin (veiga et al., 2001a; zanetti et.al., 2002). in vitro experiments on rabbit aorta endothelium cell cultures showed cytotoxicity of l. intermedia venom associated with loss of cell adhesion to the culture substrate and the shedding of proteoglycans from the extracellular matrix and cell surface into the medium (veiga et al., 2001a). in human umbilical vein endothelial cell (huvec) cultures treated with l. reclusa venom, agonist activity ensued, inducing endothelial cell expression of e-selectin and the release of interleukin (il)-8 and granulocyte macrophage colony-stimulating factor, resulting in dysregulated inflammatory response (patel et al., 1994). huvec exposed to l. deserta venom produced il-8, growth-related oncogene-α and monocyte chemoattractant protein-1 via an nf-κbdependent pathway (desai et al., 1999; gomez et al., 1999). l. deserta venom induces the expression of vascular endothelial growth factor (vegf) in human keratinocytes, suggesting that keratinocyte-derived vegf may contribute to vasodilatation, oedema and erythema in brown spider envenomation (desai et al., 2000). primary cultures of keratinocytes exposed to 100 ng/ml of l. gaucho venom release tumour necrosis factor (tnf)-α into the medium after 6 h (málaque et al., 1999). mice injected with l. reclusa venom developed local hemorrhage after 6 h accompanied by blistering of the ear skin (sunderkötter et al., 2001). 154 fig. 1 cellular and molecular aspects of brown spider and loxoscelism. (a) loxosceles intermedia (brown spider) male. (b) l. intermedia (brown spider) female. (c) sds-page 3-20 % venom profile stained by coomassie blue dye. (d) dermonecrotic lesion on rabbit skin after 24 h post-l. intermedia venom (10 µg) exposure. arrowhead indicates the site of venom injection with characteristic black and white eschar named marble plate. black arrow points an erythema surrounding the lesion and white arrow shows the gravitational spreading of lesion (a hallmark of dermonecrotic loxoscelism). (e) microscopical view of dermonecrotic lesion showing inflammatory leukocytes accumulated in the connective tissue (arrowhead) and disorganization of collagen fiber and oedema (black arrow) (magnification 400x). the inset shows inflammatory cells of the infiltrate represented by neutrophils (white arrow) (magnification 1.000x). histopathology showed a vasculitis reaction 2 h after exposure. the microscopical analysis of some mouse organs injected with different doses of l. intermedia venom revealed remarkable kidney alterations. acute tubular necrosis accompanied by deposition of eosinophilic material inside the proximal and distal renal tubules was seen in several nephrons (tambourgi et.al., 1998). mouse kidneys, treated with l. intermedia venom showed hyalinisation and erythrocytes in the bowman’s space, glomerular collapse, tubular epithelial cell cytotoxicity and deposition of eosinophilic material within the tubular lumen (luciano et al., 2004). confocal microscopy observations of double staining immunofluorescence against type iv collagen or laminin and l. intermedia venom showed that toxins deposit and bind along the tubular and glomerular basement membrane of mice kidneys. ultrastructural observations showed glomerular epithelial and endothelial cell cytotoxicity, the collapse and destruction of glomerular basement membrane and tubular epithelial cell degeneration. the basement membrane is a target for brown spider venom, as shown administrating l. intermedia venom to murine tumor engelbreth-holm-swarm (ehs), which is rich in basement membrane molecules. l. intermedia venom degraded and fragmented the basement membrane (veiga et.al., 2000a). venom 29 44 12 116 205 kd a male female a b c d e 1cm 1cm 1cm 155 displays hydrolytic activity for entactin and heparan sulphate proteoglycan, two important constituents of basement membranes, while having no apparent activity on purified type iv collagen and laminin (veiga et al., 2000a, 2001a,b). in the bone marrow and peripheral blood cells, l. intermedia initially causes a decrease in the number of nucleated red cells, bone-marrow depression of megakariocytes with thrombocytopenia in peripheral blood and decrease of platelet count (da silva et al., 2003). neutropenia in peripheral blood and low neutrophil counts were observed as consequence of bone-marrow depletion, which may reflect an extensive neutrophil influx to the tissues. eosinophils are apparently unaffected. brown spider venom toxins l. intermedia and l. laeta have different protein patterns of glycosylation and the same is between sexes of the same species (oliveira et al., 2005). hemolytic and dermonecrotic activities have been described for l. similes venom. sphingomyelinase d molecules, with molecular mass ranging from 30 to 35 kda and having hemolytic, necrotic and platelet aggregation activity were found in l. reclusa, l. rufescens, l. gaucho, l. laeta and l. intermedia venoms (futrell 1992; barbaro et al., 1994; mota and barbaro, 1995; tambourgi et al., 1995; barbaro et al., 1996a,b, 1997). three sphingomyelinase d isoforms were purified from l. boneti venom (lb1, lb2 and lb3). only lb1 and lb2 had dermonecrotic activity (ramos-cerrillo et al., 2004). an alkaline phosphatase was described in l. reclusa venom (futrell, 1992). hyaluronidase (32.5 kda) was found in l. refescens and l. reclusa (futrell, 1992; young and pincus, 2001). l. deserta, l. gaucho, l. intermedia, l. laeta and l. reclusa venoms contained an enzyme of similar molecular size (44 kda), which digested hyaluronic acid (barbaro et al., 2005). a 5’ribonucleotide phosphohydrolase was found in l. reclusa venom (futrell 1992). loxnecrogin a (31.4 kda) and loxnecrogin b (31.6 kda) with necrotic activity on rabbit skin were found in l. gaucho venom (cunha et al., 2003). l. intermedia has a range of proteases described in its venom: loxolysin a (20-28 kda) with fibronectinolytic and fibrinogenolytic activity; loxolysin b (32-35 kda) with gelatinolytic activity (feitosa et al., 1998); a serin protease (85 kda) with gelatinolytic activity (veiga et.al., 2000b) and proteases able to hydrolyse entactin, heparan sulphate proteoglican and basement membrane (veiga et al., 2000b, 2001a,b). l. rufescens also has a broad molecular range of caseinolytic, gelatinolytic and fibrogenolytic metalloproteases (young and pincus, 2001). to test whether proteases in l. intermedia venom were due to natural constitution and not a digest fluid contamination, da silveira et al., (2002) compared the proteolytic activity of the venom obtained directly from venom glands with that obtained by electroshock. both protein profiles showed very similar electrophoretic and enzymatic characteristics. at present, a new generation of molecules developed through cloning techniques is under study. l. intermedia lid1 recombinant protein (31.4 kda) is a sphingomyelinase d family molecule without dermonecrotic activity but with antigenic activity (kalapothakis et al., 2002). l. laeta recombinant protein (33 kda) is a sphingomyelinase isoform able to degrade sphingomyelin (pedrosa et al., 2002). this recombinant protein induced complement susceptibility, release of glycophorins and had dermonecrotic activity. l. intermedia recombinant protein (lirecdt, 34 kda) has dermonecrotic activity and was able to directly induce nephrotoxicity in mice (chaim et al., 2005). l. laeta recombinant phospholipase d generated lysophosphatidic acid and was hemolytic (lee and lynch, 2005). clinical features, diagnosis and treatment of brown spider bites diagnosis of loxoscelism is rarely based on spider identification and therefore clinical features, epidemiological and historical findings must be well known (wright et al., 1997; vetter, 1999; málaque et al., 2002). lesion recovery improves once the patient is treated. however, brown recluse bites have been misdiagnosed in north america because they occurred in regions of non-endemicity (vetter, 1999; nishioka, 2001; vetter and barger, 2002; vetter and bush, 2002a,b; vetter et al., 2003). a typical necrotic skin lesion begins soon after the spider bites the victim, followed by gravitational spreading (da silva et al., 2004). the bite is painless, hence the patient is often unaware that he has been bitten (futrell, 1992), and the delay between the bite and when the victim pursues help makes the treatment less effective. from mild to severe pain begins 2-8 h after the bite. at the bite a small puncture wound may appear, associated with transient erythema with itching and swelling and mild to severe tenderness (futrell, 1992; da silva et al., 2004). blebs or blisters appear (12-24 h), may become hemorrhagic, and surrounded by a halo of ischemic tissue. in the following days, necrotized lesions become a dull blue-violet, the area of the gravitational spread turns blue, and the size of the blue area increases. within three to seven days an eschar may form, after which the lesion hardens. the eschar may drop off leaving an ulcer that may require a skin graft (schenone, 1996; sezerino et al., 1998; málaque et al., 2002; da silva et al., 2004). success of therapy depends upon a correct and rapid diagnosis, the volume of the venom injected, and the patient susceptibility to the venom (futrell, 1992; da silva et al., 2004). phentolamine, heparin, topical nitro-glycerine, cyproheptadine and hyperbaric oxygenation have been used for therapy, but the efficacy of these therapies is inconclusive and their use is not recommended (futrell, 1992; wendell, 2003; da silva et al., 2004). the established therapy is dapsone, acetylsalicylic acid (aspirin), antibiotics (erythromycin and cephalosporin), ice and elevation, avoidance of strenuous activity and heat and, when necessary, surgery. early surgical excision has not been shown to be effective and often delays healing (futrell, 1992; merigian and blaho, 1996; goddard, 1998, monteiro et al., 2002; da silva et al., 2004). serum anti-loxosceles venom is used only in severe cases and effectiveness is doubtful especially against local manifestation. systemic envenomation studies in 156 animals and humans have demonstrated that antivenom neutralizes the deleterious effects of the venom and reduces paediatric mortality (isbister et al., 2003). effectiveness of antivenom to prevent dermonecrotic lesions seems to be time dependent and usually patient looks for medical help 4 h after the bite when lesions is already established (ospedal et al., 2002; nicholson and graudins, 2003). some local and systemic noxious activities of the venom are attributed to proteolytic toxins that degrade fibrinogen, fibronectin, entactin and heparan sulphate proteoglycan and disrupt basement membrane structures, thereby causing local hemorrhage, gravitational spreading, disseminated intravascular coagulation and renal failure (feitosa, et al., 1998; veiga et al., 1999, 2000b, 2001a,b; luciano et al., 2004; chaim et al., 2005). biotechnological products from brown spider venom recently developed technologies are being used to produce biotechnological products from loxosceles venom. arachnase (hemostasis diagnosis international co., denver, co, usa), normal plasma containing l. reclusa venom, mimics a lupus anticoagulant and may provide a positive control for anticoagulant testing (mcglasson et al., 1993). an antiserum against venoms of l. gaucho, phoneutria nigriventer, tityus serrulatus, and tityus bahiensis that reacts with l. intermedia and l. laeta toxins is produced by the butantan institute, são paulo, brazil. the ccpi (production center of immunobiologic products, parana, brazil) has also produced antiserum using l. intermedia venom that is able to neutralize some activities of loxosceles venom. l. laeta antiserum is produced by the national institute of health (peru) (da silva et al., 2004). these antisera have all been used as bioproducts for serum therapy (roodt et al., 2002; barbaro et al., 1994; 1996a; health secretary, curitiba, parana, brazil). guilherme et al. (2001) produced monoclonal antibodies recognising l. gaucho venom toxins, which were able to neutralize the dermonecrotic effect and lethal activities of this species venom but not those of heterologous venoms. monoclonal and polyclonal antibodies are not only powerful tools for neutralizing the effects of venom; they are also useful for research. they can be used to purify toxins from venom by affinity chromatography. they can be used on location of specific toxins on cell and tissue treated with venom toxins. immunofluorescence techniques such as confocal microscopy and flow cytometry are modern techniques based on antibody specific binding. in contrast to the collection of snake venom, spiders provide very little venom, which limits the ability to study spider venom toxins. protein cloning techniques are helping to solve this problem. after cloning it is possible to have milligrams of the same protein thereby improving the quality of research work and allowing more controlled experimental studies. today several spider venom recombinant proteins are under investigation most of which are in the sphingomyelinase protein family (kalapothakis et al., 2002; pedrosa et al., 2002; chaim et al., 2005). future perspectives toxins from loxosceles spiders are a group of proteins with a great range of different activities. each toxin may be used to investigate molecular and cellular effects of venom. also each of these proteins is a putative molecular model for drug design and to develop knowledge on some effects not yet fully understood such as the inflammatory reaction of dermonecrosis and platelet aggregation. acknowledgements this work was supported by cnpq, capes, fundação araucária and secretaria de estado de ciência, tecnologia e ensino superior do parana, brazil. references andrade rmg, oliveira kc, giusti al, silva wd, tambourgi dv. ontogenetic development of loxosceles intermedia spider venom. toxicon 37: 627-632, 1999. andrade rmg, lourenço wr, tambourgi dv. 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k.u.leuven, leuven, belgium accepted january 19, 2010 abstract the nematode caenorhabditis elegans is one of the most successful model species for experimental research because of its sequenced genome, the versatile genetic toolkit and the straightforward breeding among others. in natural conditions however, this tiny worm is constantly surrounded by micro-organisms, simultaneously a source of indispensable nutrition and inevitable pathogens. lacking an adaptive immune system, the worm solely relies on its innate immune defence to cope with its challenging life style. hence c. elegans is an excellent model to gain more insight in innate immunity, which is remarkably preserved between invertebrate and vertebrate animals. the innate defence consists of receptors to detect potential pathogens, a complex network of signalling pathways and last but not least, effector molecules to abolish harmful microbes. in this review, we focus on the antimicrobial peptides, a vital subgroup of effector molecules. we summarise the current knowledge of the different families of c. elegans antimicrobial peptides, comprising nlps, caenacins, abfs, caenopores, and a recently discovered group with antifungal activity among which thaumatinlike proteins. key words: caenacins; abfs; caenopores; insulin signalling; immunity; host-pathogen interaction introduction as a free living soil nematode, caenorhabditis elegans forms an extremely interesting model to study the interaction with bacteria, its main food source and substrate. as the distribution of bacteria is mixed in natural conditions, worms should continuously search for regions in the soil where the benefit of energy-rich and benign bacteria exceeds the possible presence of harmful bacteria, be it slow or fast killers (shtonda and avery, 2006; abada et al., 2009). it is clear that discerning different types of bacteria and employing an effective battery of antibacterial molecules are crucial for worms. c. elegans has a tremendous variety of chemosensory receptors to detect both interesting and harmful bacteria, the latter provoking pathogen avoiding behaviour (zhang et al., 2005; pradel et al., 2007; schulenburg and ewbank, 2007). in contact with harmful bacteria, recognition molecules activate specific signalling pathways which ultimately induce the release of immune molecules. in this review we specifically focus on the variety of antimicrobial ___________________________________________________________________________ corresponding author: annelies bogaerts research group of functional genomics and proteomics k.u.leuven, zoological institute naamsestraat 59, 3000 leuven, belgium e-mail: annelies.bogaerts@bio.kuleuven.be peptides (amps), produced by c. elegans as part of its defence system. amps are defined as relatively short molecules with a low molecular weight (below 5 kda), often containing 10 up to 150 amino acids (bulet et al., 1999; jenssen et al., 2006). their expression can be either constitutive or inducible at the time of infection (kato et al., 2002; alegado and tan, 2008). amps posses a natural antimicrobial activity often thanks to their cationic and amphipathic structure which facilitates the disruption of anionic cell walls and phospholipids membranes of microbes, although other microbicidal mechanisms have also been proposed (bulet et al., 2004; brogden, 2005). worms have an innate immune system which constitutively expresses certain amps whereas complex mixtures of amps are induced upon encounter with different pathogens. note that by unfolding a specific mixture of ‘antibiotics’ the worm can prevent a straightforward development of resistant pathogenic strains. therefore, studying amps in an experimentally favourable immunological model such as c. elegans, forms a lead for the development of new strategies to deal with pathogenic infections in the future. we highlight the diverse amp families in c. elegans and summarise the evidence for their biological function, specificity, expression and the pathways involved as far as known. 45 mailto:annelies.bogaerts@bio.kuleuven.be neuropeptide like proteins the c. elegans genome encodes at least 42 neuropeptide like proteins (nlps) which can be divided into minimal 11 subgroups according to their unique bioactive motifs (nathoo et al., 2001). although most of the nlp genes are translated into conventional neuropeptides, others may possess distinct or additional functions. the first indication of an antimicrobial role for nlp genes came from a study in 2002 by mallo et al. in which they observed the induced expression of nlp-29 upon infection with the gram-negative bacterium serratia marescens. later on in 2004, ewbank’s group demonstrated an antifungal activity for nlp-31 against drechmeria coniospora in vitro (couillault et al., 2004). in c. elegans, most of the infection-inducible nlp genes were named as such because of their limited sequence similarity with yggxamide neuropeptide genes, sharing yggwg and yggyg motifs (nathoo et al., 2001). they form a monophyletic group, found in a 12 kb region on the left arm of chromosome v, referred to as “the nlp-29 cluster” (pujol et al., 2008b). this cluster comprises nlp-27 to 31 and the adjacent gene nlp-34. two other caenorhabditis species c. briggsae and c. remanei possess orthologous genes. in c. brigssae, these genes are orientated in different clusters: cbrnlp-27 and cbr-nlp-34.1, cbr-nlp-34.2 and cbr-nlp34.3. phylogenetic analysis indicates that gene duplications driven by natural selection form the basis for the evolutionary diversification of these clusters. at the time of divergence of the different caenorhabditis lineages from a common ancestor, two genes were at the nlp locus: nlp-27 and nlp-34. via gene duplication, nlp-27 gave rise to 5 genes in c. elegans whilst nlp-34 gave rise to 3 genes in c. brigssae (pujol et al., 2008b). expression of the genes belonging to the nlp-29 cluster is predominately limited to the epidermis and the intestine and is controlled by a diverse interplay of mechanisms. besides infection, physical injury of the epidermis as well seems to have an influence on expression of nlp-29 and nlp-31 (pujol et al., 2008a). in both conditions, the expression levels are controlled by distinct pathways that converge in a conserved signalling cascade: the p38 mitogen activated kinase (mapk) pathway. this pathway, involving mapk pmk-1, mapk kinase (mapkk) sek-1 and mapkk kinase (mapkkk) nsy-1, lies downstream of the tir (toll-interleukine 1 receptor) adaptor protein tir-1, an ortholog of the human protein sarm (selective androgen receptor modulator). involvement of tir-1 in the control of nlp expression upon fungal infection was established in 2004 by couillault et al. these researchers generated transgenic worms expressing gfp (green fluorescent protein) under control of the nlp-29 promotor and observed an increased fluorescent signal in the hypodermis upon infection with d. coniospora and s. marescens. rnai of tir-1 in the pnlp-29::gfp reporter lines diminished the constitutive and infection-induced expression of pnlp-29::gfp. moreover, tir-1 (rnai) worms are more susceptible to the deleterious effects of both fungi (d. coniospora) and bacteria (s. marescens). further studies have unravelled additional components of the immune signalling pathway. expression of nlp-29 is regulated by the upstream factor tpa-1, homologous to the mammalian protein kinase c (pkc) delta (ziegler et al., 2009). in the epidermal response to fungal infection, pkc delta is activated by a tribbles-like kinase nipi-3. however, upon wounding nipi-3 is not required for nlp-29 induction, supporting the existence of a pathogenspecific reaction, in addition to a non-specific protective response (pujol et al., 2008a). as mentioned above, also bacteria can trigger the nlp gene expression. in contrast to fungal infection, expression predominately occurs in the intestinal epithelium and is regulated by a c. elegans protein kinase d (dkf-2) which lies downstream of tpa-1, and acts in pmk-1 dependent and independent ways (ren et al., 2009). to make things even more complicated nlp-28 and nlp-29 expression is also induced as a consequence of osmotic stress and this by yet another transcriptional response which is pmk independent (pujol et al., 2008b). caenacins similarly to the neuropeptide like proteins, specific genes belonging to the caenacin family are induced upon infection with d. coniospora amongst other pathogens. cnc-1 up to cnc-5 and cnc-11 form a genomic cluster, also situated on the left arm of chromosome v, referred to as “the cnc-2 cluster”. mature peptides belonging to the nlp and cnc classes are rich in glycine and aromatic amino acids and most of them can be distinguished by the qwgyg motif present just c-terminal to the predicted signal sequence cleavage site. despite the fact that they are structurally and phylogenetically related to the nlps, these cnc genes are regulated in a very distinct way (zugasti et al., 2009). unlike the nlp genes, osmotic stress has only little effect on the expression of cnc-11 and no effect on the other members of the cnc-2 cluster. while induction of genes, belonging to the nlp-29 cluster, upon wounding or infection relies almost entirely on a p38 mapk signalling cascade, only physical injury and not infection seems to have a lowering effect on induction of cnc-2 cluster genes in pmk-1 mutants. this indicates that the expression of cnc genes in the epidermis upon fungal infection is dependent on a different immune pathway (zugasti et al., 2009). as the expression of cnc-2 was more strongly induced upon infection than upon wounding, researchers focused on this gene. they constructed transgenic strains expressing either gfp (pcnc2::gfp) or the ‘mcherry’ fluorescent protein (pcnc2::mcherry) under control of the cnc-2 promotor and reported that the cnc-2 gene was exclusively expressed in the epidermis. furthermore, induced expression of reporter genes was observed upon infection with d. coniospora but not upon bacterial infection with s. marescens and pseudomonas aeruginosa (zugasti et al., 2009). searching for the specific signalling pathway involved in cnc-2 gene expression they found that the transcription is controlled in a paracrine way by 46 the c. elegans transforming growth factor β ortholog dbl-1 as in dbl-1 mutant worms the induced expression of the cnc-2 reporter was much lower (zugasti et al., 2009). antibacterial factor (abf) peptides the c. elegans genome encodes six homologues (abf-1 to abf-6) of the ascaris suum antibacterial factor (asabf) peptides, which are microbicidal factors that were first discovered in the body fluid of the parasitic nematode a. suum (kato and komatsu, 1996). sequence identity and structural commonality reveals that these nematode abfs are genetically related: all known abf peptides share a cysteine-array consisting of eight conserved cysteine residues and a secretory signal sequence at the n-terminus. high homology appears in the region encompassing the eight cysteines with 25-95% similarity. in contrast, the region c-terminal to the last cysteine is divergent and varies in length (froy, 2005). most likely this part is cleaved off post-translationally, as was shown for asabf-α (kato and komatsu, 1996; zhang et al., 2000). apart from their sequence similarity, several other properties support a direct role for abfs in the innate immune response of c. elegans. recombinant abf-2 exhibits antimicrobial activity in vitro against a broad range of gram-positive, gramnegative and fungal pathogens. however, grampositive bacteria tend to be more sensitive and some gram-negative and fungal strains are resistant to abf-2 (kato et al., 2002). asabf-α possesses a similar antimicrobial specificity (zhang et al., 2000). the exact bactericidal mechanism of abf peptides remains to be elucidated, but based upon similarities in primary structure and antimicrobial effects, the killing of microbes could be caused by disruption of their cytoplasmic membrane (zhang et al., 2000; kato, 2007). abf peptides are constitutively expressed under normal growth conditions when c. elegans is cultivated on a lawn of the non-pathogenic strain e. coli op50, as was shown for abf-1, abf-2 and abf-3 (kato et al., 2002; alper et al., 2007; alegado and tan, 2008). the pharyngeal tissue represents the main production site for abf-1 and abf-2 (kato et al., 2002). together with abf-3, abf-1 is also produced in the intestine (alper et al., 2007). in this way, the constitutive expression of abfs may be part of a general defence mechanism in the worm that protects the digestive tract from microbial infection, an important threat since c. elegans mainly feeds on bacteria. expression of abf-2 and abf-3 was also observed in the excretory cells of the worm, presumably to protect the openings to the exterior (e.g., anus and excretory pores) that are continuously in contact with potential pathogens from the environment (kato et al., 2002; alper et al., 2007). recent evidence indicates that on top of the general defences against various microbes abfs also participate in a specific immune response induced upon infection (alper et al., 2007; alegado and tan, 2008; means et al., 2009). in a. suum a number of the asabf peptides were demonstrated to be induced after injection of heat-killed bacteria in the pseudocoelom (pillai et al., 2003; minabi et al., 2009). this induction was also proven for some of the asabf-type homologues in c. elegans. worms infected with the gram-negative pathogen salmonella typhimurium displayed an increase in abf-2 transcript levels by a hundred-fold. deficiencies in abf-2 after rnai treatment correlated with a significantly higher bacterial load in the intestine after exposure to s. typhimurium in comparison with control animals. these findings suggest that c. elegans induces the expression of abf-2 as part of an immune response to s. typhimurium infection that is essential for limiting bacterial growth in the worm’s digestive tract. however, transcript levels were initially indistinguishable within the first 24h of exposure which implies that the induced amp response may not be due to direct sensing of pathogenic microbes but due to later events such as the production of specific bacterial factors or damage to host tissues (alegado and tan, 2008). upregulation of abf-1 and abf-2 was also observed in wild type c. elegans after infection with the yeast cryptococcus neoformans (means et al., 2009) and exposure to the gram-positive bacteria staphylococcus aureus elicited a weak induction of abf-3 (alper et al., 2007). therefore, we can conclude that nematode abfs play a crucial role in the general and more specific induced immune response to pathogenic attack. the signalling pathways responsible for the upregulation of abf peptides upon infection of c. elegans have not yet been fully elucidated. tol-1, the single homologue of the toll-like receptor encoded in the worm’s genome, seems to be required for the correct expression of abf-2 since quantitative reverse transcription-pcr analysis indicates a decreased level of abf-2 transcripts in tol-1 mutants (tenor and aballay, 2008). moreover tol-1 mutants display a reduced lifespan on salmonella enterica due to a rapid invasion of the pharynx, which is the main expression site for the antibacterial abf-2 peptide (tenor and aballay, 2008). these results demonstrate that tlrmediated signalling probably contributes to the elicitation of a specific immune response in c. elegans on top of its established role in the pathogenic avoiding behaviour of the worm (pujol et al., 2001). in addition neurons expressing g proteincoupled receptors (gpcrs) also participate in the regulation of abf expression levels. a deficiency in the neural circuit involving npr-1, which encodes a gpcr related to the mammalian neuropeptide y receptors, reduces the expression level of abf-1 in response to infection by p. aeruginosa (styer et al., 2008). recently an evolutionary conserved pathway consisting of ced-1 and c03f11.3, orthologs of the mammalian scavenger receptors scarf1 and cd36, was shown to activate antimicrobial peptides including abf-1 and abf-2 upon yeast-infection (means et al., 2009). up till now, the phylogenetic relationship between nematode abfs and other metazoan amps remains unclear. asabf-type peptides contain a cysteine-stabilised α/β (csα/β) consensus motif consisting of an α-helix and two antiparallel β 47 strands stabilised by four internal disulfide bridges. recently, a novel member of asabf-type peptides was discovered in a. suum containing only six cysteines. so far this amp forms the single exception and probably arose after the divergence of ascaridida and rhabditida (minaba et al., 2009). structural similarities between nematode abfs and invertebrate defensins suggest a common ancestry in the evolution of these antimicrobial factors. first of all, the csα/β motif of asabf-type peptides is also found within invertebrate defensins that are further classified according to the number of cysteine residues contributing to the intramolecular disulfide bonds: the cysteine array of insect/arthropod defensins typically comprises six cysteine residues, mollusk defensins are characterized by eight cysteines (froy, 2005). secondly, the primary structure of asabf-type peptides includes an insect/arthropod consensus sequence (cys1-[…]-cys2-xaa-xaa-xaa-cys3-[…]gly-xaa-cys4-[…]-cys5-xaa-cys6) with six conserved cysteines and one glycine residue (kato and komatsu, 1996; kato et al., 2002). as a third argument zhang and kato (2003) showed that nematode abfs share even more characteristics with two mollusk defensins: myticin and an amp isolated from the mediterranean mussel mytilus galloprovincialis (mgd-1). asabf-type peptides and mollusk defensins both contain eight cysteine residues with an identical pairing and a similar precursor organization consisting of an n-terminal secretory signal sequence, followed by the mature polypeptide and a cleavable “pro-region “ at the cterminus. in classical csα/β type amps, such as insect defensins, this “pro-region” is located directly after at the n-terminus. in summary, nematode abfs and mollusk defensins share several structural properties and could therefore be generated from a common ancestor. however, the absence of highly reliable evidence such as a significant sequence similarity or a conserved genomic organization (exon-intron structure) cannot exclude that these two groups of amps developed through convergent evolution (froy, 2005). hopefully, the identification of new csα/β type amps in different phyla will clarify the evolutionary trajectory of nematode and invertebrate defensins (froy, 2005; rodriguez de le vega and possani, 2005). caenopores caenopores are the saposin-like proteins (spp) of c. elegans. saposins form a multifarious protein family characterized by an alpha helix bundle stabilized by 3 unique disulphide bonds and the ability to interact with phospholipid membranes (see for review bruhn, 2005). based on these hallmarks, patthy (1996) designated the putative protein product of gene t07c4.4 as the first c. elegans saposin-like protein. two years later, 5 additional spp genes were found in this nematode. all six predicted spps appeared similar to the amoebapores of entamoeba histolytica and granulysin from human cytotoxic t lymphocytes as they consist of a secretory signal peptide followed by a single saposin-like domain (banyai and patthy, 1998). note that amoebapore-like spps might have been the first antimicrobial peptides since this protein family emerged even before the advent of metazoans (leippe 1999). banyai and patthy (1998) recombinantly expressed t07c4.4 (spp-1) which allowed to demonstrate the characteristic helix bundle structure and an antibacterial effect on e. coli (jm-109). in addition, the three-dimensional structure of spp-5 was studied in great detail by mysliwy et al. (in press). they confirmed that spp-5 has five amphiphatic helices, connected by three disulfide bonds, arranged in a folded leaf typical for the saposin-like protein family. further examination of the worm’s genome lead to the discovery of 28 different spp genes coding for 33 saposin-like proteins which were named caenopores because of their structural and functional resemblance with amoebapores (roeder et al., 2010). indeed, like amoebopores, at least spp-5 was shown to display pore-forming activity capable of killing bacteria by permeabilizing their cytoplasmic membrane. a phylogenetic analysis of the 33 spps shows different clusters. in case of the cluster comprising spp-2 to spp-6, all corresponding genes are located in the same chromosomal region, consistent with a series of gene duplications. roeder et al. (2010) investigated the functional significance of spp-1, spp-3, spp-4, spp-5 and spp-6 by means of rnai-mediated gene silencing. one gene, spp-5, significantly affected the overall fitness of worms measured as the number of laid eggs and the deposition of fat tissue. silencing this same gene, contrary to the other genes tested, had a huge impact on the number of e. coli that could survive in the intestinal lumen (roeder et al., 2010). when grown on standard culture medium ngm, wild type worms consequently expressed spp-5 for all the bacteria/conditions tested. the spp-3 gene, on the other hand, was induced only when confronted with b. megaterium, m. luteus and starvation. further analyses of spp-5 learnt that it is as potent as spp-1 against the gram-positive b. megaterium and even more active against the gramnegative e. coli (roeder et al., 2010). the genome wide microarray analysis of wong et al. (2007) searched for pathogen specific signatures in the immune response of c. elegans. no expression change of any spp gene was observed against erwinia carotovora, whereas spp-18 was upregulated upon infection with photorhabdus luminescens as well as spp-5, spp-8, spp-14 and spp-21 in case of an enterococcus faecalis infection (wong et al. 2007). moreover, spp-1 was shown to be induced upon infection with s. typhimurium. it was found that salmonella strains lacking, among others, the virulence factor spi-2 had difficulties to persist in c. elegans intestine. interestingly, such persistence deficiencies could be rescued when spp-1 of the worm was reduced by rnai (alegado and tan, 2008). however, evans et al. (2008) showed that spp-1 is repressed upon infection with p. aeruginosa. next, they showed that downregulation of spp-1 expression, among others, is a key strategy to overcome the immune system of its host. this downregulation depends on the response regulator gaca and the quorum sensing 48 regulators lasr and rhlr; and interferes with the insulin-like signalling via the daf-2/daf-16 axis, which is important for the regulation of stress response, longevity and immune function (evans et al., 2008). this discovery correlates perfectly with the observation that spp-1 (and spp-12) expression is downregulated by loss of insulin signalling. decreasing the expression of either of these spp genes by rnai reduced the lifespan of c. elegans on e. coli (murphy et al., 2003). the exact mechanism of how the diverse spp genes are controlled is still not known. the only additional information currently available is that expression of spp-9 and spp-18, along with more than 80 defencerelated genes, appears to be regulated by the protein kinase d (ren et al., 2009). worth mentioning is that spp-5 is exclusively expressed in the gut (roeder et al., 2010). the same accounts for spp-1, but spp-7 is also expressed in the pharyngeal muscles and head neurons (alper et al., 2007). given that most caenopore genes (except spp-2, spp-12, spp-16 and spp-19) have the intestine specific transcription factor elt-2 binding domain in their putative promotor regions, it can be assumed that most caenopores will be expressed in the intestine. this finding prompted roeder et al. (2010) to state that “caenopores or spps are most likely the only candidates to tackle the diverse mixture of microbes c. elegans is confronted with in the natural environment”. in their opinion, other antimicrobial peptides described for c. elegans are either not numerous enough (abfs), or they are expressed at the wrong location (hypodermis: cncs and nlps). although roeder is perhaps correct when postulating that the spps are the most significant group of amps, this statement should be relativized as the importance of other types of amps, functioning alone or in cooperation with other defence molecules of the immune system of c. elegans, should not be underestimated. other potential amps besides the above mentioned members of the nlp, cnc, abf and spp families, which are induced upon infection with different types of pathogens (troemel et al., 2006; muir et al., 2008), pujol et al. (2008b) found other previously uncharacterised genes that seem to be specifically induced upon fungal challenge. hence they were named: fungus-induced proteins (fip), fip related proteins (fipr) and glycine-rich secreted protein 2 (grsp-2). a comparison to peptides with known antimicrobial activity (fjell et al., 2007) revealed that each of the fip, fipr and grps genes could potentially encode amps (pujol et al., 2008b). however, further biochemical and functional analysis will be required to confirm this statement. recently, homologs of thaumatins and other pathogenesis-related plant proteins have been discovered in the c. elegans genome (brandazza et al., 2004; shatters et al., 2006). thaumatins were originally isolated from the fruits of thaumatococcus danielli and extensively studied because of its sweetening properties. later studies have demonstrated antifungal activity for thaumatin-like proteins such as stimulating microbial membrane permeability (vigers et al., 1991), beta-1,3glucanase activity (grenier et al., 1999) and alphaamylase inhibiting properties (svensson et al., 2004). moreover, recent work from the tan group has implicated an antimicrobial role for a member of the thaumatin family: thn-2. knockdown of thn-2 by rnai enhances the susceptibility of c. elegans to a p. aeruginosa infection (evans et al., 2008). as for spp-1 (see above), the expression of thn-2 is downregulated upon infection with this pathogen (shapira et al., 2006; evans et al., 2008). these findings suggest that homologs of these plant proteins could well represent potential antimicrobial proteins in c. elegans. note that thaumatins are not indisputably antimicrobial peptides as they count around 200 amino acid residues. conclusion to feed on and fight off a smorgasbord of bacteria and pathogens, c. elegans developed a diverse armory of immune defence molecules. this feed versus fight paradox is most certainly an intriguing issue. whilst on the one hand worms are completely dependent on bacteria/fungi for their survival, some of these micro-organisms might on the other hand contribute to their death. numerous chemoreceptors allow c. elegans to distinguish between benign and harmful bacteria. however, a continuous cost-benefit analysis is necessary for making the correct choice between for example a slightly pathogenic bacterium with a high nutritional value or a non-pathogenic bacterium with low nutritional value. the amps constitute the most numerous and versatile group of immune effector molecules, allowing specific and effective responses against different pathogens. even the expression of related amp genes can be regulated by very distinct pathways, providing the host specific alternative defence possibilities against the potentially detrimental effects of different types of pathogen invasions. the important role of amps in the early immune response has already been proven by the isolation of numerous animal amps, mostly from higher organisms such as vertebrates and arthropods. nonetheless, the c. elegans amps still form a poorly studied group. as described earlier in this review many putative amps were yet identified thanks to their induced expression upon infection or their structure/sequence similarities with closely related amps (kato et al., 2002; mallo et al., 2002; couillault et al., 2004). the strategy of homologybased searches starting from known amp sequences (e.g. vertebrate or arthropod amps), however, is limited due to the short length and rapid molecular evolution of these peptides (kato et al., 2002). this constant evolution of the immune defences compensates for the renewing virulence mechanisms of a pathogen and is a necessity to ensure the survival of a species. in fact, to our knowledge, none of the natural occurring amps were yet directly purified from c. elegans. therefore, we assume that not all c. elegans amps are identified yet which makes the identification of additional amps a first great challenge to better 49 understand the worm’s innate immunity. in addition, peptidomics based approaches for the identification of c. elegans amps might help to address the question of which amps might cooperate and contribute to target specific pathogens. since multiple amps are expressed in response to infection by a single pathogen a certain level of cooperativity might exist between different amps and/or other defense molecules. cases of cooperative activity among amps and/or other immune effectors have already been reported in mammals, e.g. the synergy between different murine defensins or the enhanced antibacterial effects of human β-defensin and lyzosyme (chen et al., 2005; wu et al., 2009). in c. elegans the field of synergistic effects in innate immunity thus remains an important research objective. to date, many aspects of the regulation of amp expression and their mode/site of action remain elusive. it was shown that c. elegans can activate multiple immune pathways upon encountering a single pathogen (alper et al., 2007; schulenberg et al., 2007) and assumed that this network of interacting signalling cascades results in the expression of an appropriate set of antimicrobial genes. indeed, another type of pathogen can elicit the expression of a different set of amps indicating that c. elegans distinguishes between pathogens and suppresses infections in a specific manner (alper et al., 2007; wong et al., 2007). yet, the exact role of many putative upstream regulators of amp expression, as suggested by e.g. genomewide transcriptomic analyses, has not yet been characterised. given that c. elegans is a versatile model with an extended experimental toolbox, e.g. the possibility to perform (large-scale) rnai and fluorescence studies (couillault et al., 2004; 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expression by a noncanonical tgf-beta signaling pathway in caenorhabditis elegans epidermis. nat. immunol. 10: 249-256, 2009. 52 isj 5: 1-xx, 2008 isj 5: 1-11, 2008 issn 1824-307x review nongenomic and genomic actions of an insect steroid coordinately regulate programmed cell death of anterior silk glands of bombyx mori m manaboon, m iga, s sakurai division of life sciences, graduate school of natural science and technology, kanazawa university, japan accepted february 4, 2008 abstract the insect steroid hormone 20-hydroxyecdysone (20e) induces programmed cell death of larvaspecific tissues at pupal metamorphosis. in the silkworm bombyx mori, the anterior silk gland undergoes cell death in response to the metamorphic peak titer of ecdysteroids in vivo and also to 20e in vitro. although 20e elicits early gene activation, an additional 20e stimulus is required for completion of cell death. this additional stimulus involves caspase-3-like protease activation, indicating that 20e also acts through a nongenomic mechanism. studies using various inhibitors, agonists, and antagonists have shown that cell condensation is under the control of 20e genomic action, and that 20e nongenomic action begins with 20e binding to the putative membrane-bound ecdysone receptor, which is probably a g-protein-coupled receptor. this step is followed by a signaling pathway comprising phospholipase c/inositol 3,4,5-triphosphate/ca2+/protein kinase c/caspase-3-like protease, which induces dna and nuclear fragmentation. nuclear condensation is regulated by signaling of calmodulin/calmodulin-dependent protein kinase ii (camkii), but camkii activation is independent of intracellular ca2+ elevation. in addition, the genomic action of 20e is indispensable for driving its nongenomic action, indicating that crosstalk between genomic and nongenomic action plays a significant role in 20e-induced cell death. key words: ecdysone; programmed cell death; nongenomic; genomic; membrane ecdysone receptor; calcium; protein kinases introduction steroid hormones regulate development, reproduction, metabolism, and homeostasis in insects and mammals. in vertebrates, steroids including estrogens, androgens, progesterone and glucocorticoids control cell death (herold et al., 2006), and in insects, the steroid 20hydroxyecdysone (20e) induces apoptosis (terashima et al., 2000; fahrbach et al., 2005). regulation of those steroids has been studied primarily in conjunction with steroid receptor function. steroids regulate cell death by two major mechanisms. some, like estrogens, act as survival factors that trigger cell death when withdrawn, while ___________________________________________________________________________ corresponding author: sho sakurai, ph.d. division of life sciences graduate school of natural science and technology kanazawa university kakumamachi, kanazawa 920-1192, japan e-mail: ssakurai@kenroku.kanazawa-u.ac.jp others, such as glucocorticoids and 20e, actively trigger cell death. although little is known about the mode of action of steroids in cell death, recent studies have provided insight into the genetic mechanisms underlying 20e-induced programmed cell death in the salivary glands of drosophila melanogaster. it is well understood that steroid hormones regulate gene expression by binding to a nuclear receptor. thus, this genetic mechanism has been the major topic of study in relation to cell death. the genetic aspects, however, do not sufficiently account for the molecular mechanisms underlying steroid-induced cell death, which is accompanied at the later stages by caspase-3 activation (woo et al., 1998). parts of this pathway are also used in liganddependent cell death, such as that observed for fas-ligand-dependent cell death in lymphocytes (winoto and littman, 2002). similarly, 20e-induced cell death in drosophila salivary glands includes serial activation/inhibition of a protease cascade, including diap1/dronc (a homologue of caspase 1 mailto:ssakurai@kenroku.kanazawa-u.ac.jp 9) pathway leading executioner caspase activation such as drice and decay (dorstyn et al., 1999; yu et al., 2002; leulier et al., 2006; callus and vaux, 2007). both the genomic pathway that regulates early genes, leading to activation of death genes (yin and thummel, 2005), and the nongenomic pathway that activates caspase-3, are required for successful execution of programmed cell death (martin and baehrecke, 2004); however, little is known about how the two pathways are linked to each other. regulation of programmed cell death by 20e post-embryonic development of insects is associated with several molting cycles. in holometabolous insects, the larvae undergo larvalpupal ecdysis after growth. bombyx mori (silkworm) and d. melanogaster undergo four and two larvallarval molts, respectively. pupal ecdysis is accompanied by various developmental changes in tissues at the cellular and molecular levels (riddiford, 1994; henrich et al., 1999). larval-pupal transformation begins at the end of the penultimate instar (fourth larval instar in bombyx), when wing imaginal discs and leg primordia are committed to undergoing pupal metamorphosis at the following ecdysis (obara et al., 2002; koyama and sakurai, unpublished). in the feeding period of the last larval instar (fifth instar), wing discs and epidermal cells are pupally committed (riddiford, 1996; obara et al., 2002), while silk glands are committed to die after pupal ecdysis (kakei et al., 2005). all of these events are under the control of a single steroid hormone, 20e. silk glands, which are the largest tissues in bombyx last instar larvae, degenerate soon after pupation through 20e-induced programmed cell death. at the end of the feeding stage, the ecdysteroid concentration in the hemolymph increases slightly to a small peak known as the “commitment peak”. this rise causes feeding to cease and induces spinning of silk thread from the silk glands. silk glands consist of three parts: the anterior, middle, and posterior silk glands. the middle silk gland produces sericin, the “glue” protein, and the posterior silk gland produces fibroin, the silk thread protein itself. in the anterior silk gland (asg), the middle and posterior glands join to form a duct where the silk thread is spun by coating of the amorphous fibroin protein with sericin. the asg is entirely covered with a thick basement lamina and lined with a thin cuticle layer (cuticular intima) on the lumen-side surface (akai, 1983). it consists of hundreds of a single type of cell, which is rather flat and irregularly hexagonal and possesses highly branched, thread-like nuclei (fig. 1). the asg undergoes programmed cell death in response to a large metamorphic peak of ecdysteroid in the hemolymph. detachment at the cell boundary is the first morphological change (chinzei, 1975; terashima et al., 2000) and occurs on the third day (g2) of the prepupal period that begins with gut purge and lasts for 3-4 days. then, the cells condense and become round in shape (cell condensation), and the nuclear branches thicken (nuclear condensation). this step is followed by dna fragmentation, which is detectable as a ladder pattern on agarose gel electrophoresis, and by nuclear fragmentation (iga et al., 2007). finally, small granules containing the fragmented dna, which probably correspond to apoptotic bodies, are formed (fig. 2). the cell death process begins in response to the small ecdysteroid peak at the molecular level and is fully activated by the metamorphic peak, when the cells are slightly rounded in shape. further 20e stimulus is not needed after this point for cell death, ending with apoptotic body formation (terashima et al., 2000). although the drosophila salivary gland is not a homologue of the silk gland, its cells do undergo cell death in response to a metamorphic ecdysteroid peak in the prepupal period. bombyx larvae have salivary glands that survive until adult stage. these glands produce cocoonase, which opens a window in the cocoon at eclosion through which the adult escapes (kafatos et al., 1967). the salivary gland cells of drosophila are small and have round nuclei, which is disadvantageous for following changes in cellular and nuclear morphologies. hence, morphological descriptions of cell death in these glands have been focused on dna fragmentation as indicated by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphatebiotin nick end labeling (tunel) method (daish et al., 2004), since the dna is difficult to recover in sufficient quantity to observe the ladder pattern on a gel (see martin and baehrecke, 2004). genetic regulation of 20e-induced programmed cell death in drosophila salivary glands has been the subject of extensive study. however, only the pathway leading to dna fragmentation has been studied. in contrast, the flat shape and highly branched nuclei of asg cells are conducive to the following changes in cellular and nuclear morphology and allow discrimination of the pathways leading to changes in cell morphology, nuclear condensation, dna fragmentation, nuclear fragmentation, and apoptotic body formation (fig. 1). ecr isoform involved in programmed cell death the nuclear receptor for 20e is a heterodimer of the ecdysone receptor, ecr, and its partner protein, ultraspiracle (usp) (yao et al., 1992; henrich et al., 1999). binding of 20e to its receptor regulates the expression of early genes and then of effecter genes (yin and thummel, 2005). the genomic effects of 20e action, beginning with serial activation/inhibition of gene expression, have been studied extensively using drosophila salivary glands (lee and baehrecke, 2001; baehrecke, 2003; yin and thummel, 2005), which are larva-specific. at the time of pupal metamorphosis, these glands undergo cell death in response to 20e, which induces pupariation and then pupation. the hierarchical control of expression of early genes in the salivary glands begins with 20e activation of ecr-b1, which encodes an ecr isoform involved in cell death. in drosophila, knockout of individual ecr isoforms has shown that mutations in ecr-a arrest development at the end of the last (third) larval instar 2 fig. 1 (a) in vivo progression of programmed cell death of bombyx anterior silk glands (asgs) after gut purge. g0-g3, 0-3 days after gut purge; p0-p2, 0-2 days after pupation. for each pair of panels representing an individual day, images were obtained by light microscopy (left panel) and by fluorescence microscopy after staining with acridine orange (right panel). note that the g0 cells are hexagonal in shape with finely branched nuclei. from g3 to p1, cellular and nuclear condensation occurs. finally, in p2, numerous granules are formed. inset: enlargement of acridine orange-stained image showing the small granules. at p2, the asgs are lined with thick basal membranes, which serve to maintain the outer shape, but soon after p2, the asgs degenerate completely and disappear from the pupal body. (b) in vitro progression of programmed cell death. asgs were cultured with 20e (0.5 µg/ml) and stained with dapi. in the far right panels for individual scores, bottom row of panels, the dapi-stained images are enlarged to show the shape of the nuclei. see fig. 2 for detailed progression. and prevent pupation, with the tissues that normally form adult structures remaining in their larval forms (davis et al., 2005). ecr-b1 mrna predominates in tissues (including salivary glands) destined to undergo programmed cell death at pupal metamorphosis (talbot et al., 1993). in an ecr-b1 loss-of-function drosophila mutant, the ecdysoneinducible genes in the larval salivary glands failed to activate (bender et al., 1997). furthermore, an ecrb1 knockout mutation prevents programmed cell death 3 fig. 2 death sequence in vitro. solid lines indicate the time period during which individual cellular events occur (see terashima et al., 2000 and iga et al., 2007 for details). modified from iga et al. (2007). in pupal bodies but does not affect the larval-pupal transformation. thus, ecr-b1 is the ecr isoform involved in metamorphic changes in larva-specific tissues that undergo cell death. in insect species other than drosophila, the roles of the ecr isoforms appear to be reversed. swallowtail butterflies commonly possess wings with intricately “cut” edges, especially in the posterior region of the hind wings. immediately after elongation of the wing imaginal discs at pupation, the wing edge is smooth. the swallowtail shape is then formed by elimination of the outer regions of the pupal wings by programmed cell death (suyama et al., 2003). in the pupal wings of the swallowtail papilio xuthus, two ecr isoforms are expressed: ecr-b1 is expressed exclusively in the proximal part of the margins between the cells that survive and form adult wings and the cells that undergo cell death, while ecr-a is expressed in the distal region that is eliminated in response to 20e. thus, ecr-a is the ecr isoform involved in region-specific programmed cell death in the pupal wings of p. xuthus. similarly, in blattella germanica (cockroach), ecr-a mediates programmed cell death of the prothoracic glands (cruz et al., 2006). in bombyx asgs, ecr-a is induced at the beginning of the prepupal period, and its mrna level increases until day 2 of the prepupal period (kamimura et al., 1997; sekimoto et al., 2006). in other tissues, such as wing discs, epidermis and midguts that do not undergo cell death, ecr-b1 is the predominant ecr isoform (kamimura et al., 1997). accordingly, although there is no knockout or knockdown data in lepidopterans, ecr-a is likely to be the isoform responsible for induction of programmed cell death in lepidopteran insects. genetic hierarchies downstream of ecr genetic regulation downstream of ecr is well documented in drosophila salivary glands (jiang et al., 2000; yin and thummel, 2005). the genes thus far shown to be involved in programmed cell death are the early genes ecr, broad complex (br-c), e74, e75, and e93; the late gene βftz-f1 (the drosophila homologue of fushitarazu); the death activator genes reaper (rpr) and head involution (hid); and dronc and drice, which are involved in the pathway leading to caspase activation (jiang et al., 2000; yin and thummel, 2005, 2007). there are two ecdysteroid surges during the drosophila third instar period, as in bombyx (sakurai et al., 1998); the first surge induces pupariation, and the second, large surge induces pupation. the surge that begins shortly before pupariation induces br-c, e74a, and e75a, but not the death activators, rpr and hid. the genes e75a, e75b, e74a, and br-c are upregulated in response to the second ecdysone surge, leading to death activator gene induction (jiang et al., 2000; dubrovsky, 2005; yin and thummel, 2005). in e93, and br-c mutants, the salivary glands fail to undergo cell death in response to 20e. this dysfunction is probably due to the lack of active caspase-3/drice, as indicated by reduction of drice expression in e74 mutant salivary glands (lee et al., 2003) and by suppression of dna/nuclear fragmentation (martin and baehrecke, 2004). these data provide further support for an interaction between genomic and nongenomic actions of 20e. direct evidence for early gene regulation has also been found in b. germanica (cruz et al., 2006, 2007). knockdown of b. germanica hormone 4 receptor 3 (bghr3) by a single injection of doublestranded rna into early last (sixth) instar b. germanica nymphs induces an arrest of developmental to adult, showing that bghr3 is involved in hierarchical stimulation of the early genes. bgecr-a knockdown by rna interference prevents programmed cell death of the prothoracic glands, which normally occurs after adult eclosion, at the end of the same instar. it also markedly reduces the bghr3 level in the glands, indicating an involvement of bghr3 in 20e-induced programmed cell death. e75 knockdown induces premature degeneration of the prothoracic glands, indicating that e75 acts as a “suppressor” of programmed cell death (mane-padros et al., 2008). therefore, in b. germanica, 20e stimulates the ecra/bghr3 pathway to activate the death activator genes, while also suppressing e75 expression in the prothoracic glands. this mechanism is similar to that found in drosophila salivary glands, where e75 is required to repress the death inhibitor gene diap2 (jiang et al., 2000; palanker et al., 2006). in bombyx asgs, the temporal profiles of e75 and bombyx hormone receptor 3 (bhr3) gene expression after 20e challenge (sekimoto et al., 2006) are very similar to those in drosophila salivary glands (lam et al., 1999), an indication for the presence of a similar transcription hierarchical control to b. germanica prothoracic glands and drosophila salivary glands. gene expression profiling has provided insight into the early gene regulation of 20e-induced cell death of bombyx asgs. the developmental expression profiles of early and early-late genes indicate that early gene regulation in bombyx is similar to that in drosophila (jiang et al., 2000; sekimoto et al., 2006, 2007). the times at which upregulation begins and expression peaks during the feeding and prepupal periods of bombyx are very similar to those in the drosophila prepupal period, with two exceptions described below (sekimoto et al., 2006) in drosophila, there are two peaks in the hemolymph ecdysteroid titer, one for pupariation and the other for pupation (riddiford, 1996). in bombyx, there are also two peaks, one for entering the prepupal period and the other for pupation. however, the two peaks are not separated by a distinct time interval; rather, the titer gradually increases with substantial fluctuations from the rise of the small peak to the maximum peak titer (sakurai et al., 1998). nongenomic action of vertebrate steroid hormones the various tissues and cells of vertebrates respond rapidly to steroid hormones (wehling, 1997; lösel and wehling, 2003; lösel et al., 2003; sergeev, 2005; tasker et al., 2006). on the other hand, the genomic effects of steroid hormones are not fully realized for several hours or days. the rapid responses are mediated by ligand-dependent channel proteins in the plasma membrane (qiu et al., 2006), by nuclear receptors localized to the plasma membrane (simoncini et al., 2002, 2004), or by membrane-bound ligand receptors (revankar et al., 2005; boonyaratanakornkit and edward, 2007). although the nongenomic actions of glucocorticoids have been well elucidated (herr et al., 2007), the identity of the glucocorticoid receptor is, as yet, unknown. it is assumed to be a g-protein coupled receptor (gpcr). studies of the novel mechanisms by which glucocorticoids suppress the immune response of t-lymphocytes have suggested that glucocorticoids have rapid effects on transmembrane currents, the t-cell receptor complex, and mitogen-activated protein (map) kinase signaling pathways, in addition to elevating the level of intracellular ca2+ (löwenberg et al., 2007). the steroid compound 17α,20β-dihydroxy-4pregnen-3-one (17α,20β-dp) acts on the sea trout oocyte to stimulate ovarian maturation (zhu et al., 2003). it brings about a decrease in the level of camp via binding to the membrane-bound progestin receptor (mpr), the first steroid membrane receptor identified as a gpcr (zhu et al., 2003; thomas et al., 2007). by coupling to g-protein αi subunit, mpr inhibits adenylyl cyclase, thus decreasing the intracellular camp level (pace and thomas, 2005). unlike other steroids with specific nuclear receptors, 17α,20β-dp does not have a nuclear receptor. therefore, it must elicit its effects only through nongenomic action. the nongenomic action of estrogen is mediated by a membrane-bound receptor or by an alternative pathway initiating with the cytoplasmic estrogen receptor. binding of estrogen to the membranebound estrogen receptor (gpr30) increases intracellular camp levels by activating adenylyl cyclase (lösel et al., 2003; rønnekleiv and kelly, 2005; thomas et al., 2005; zivadinovic et al., 2005). estrogen also binds with estrogen receptor α in the cytoplasm and activates phosphatidylinositol 3kinase (pi3k), thus activating a protein kinase cascade (simoncini et al., 2002). in insects, immunohistochemical analysis in rhodnius prolixus (schlattner et al., 2006) and bombyx (hossain et al., 2006) has identified an ecdysone receptor in the cytoplasm beneath the plasma membrane of brain neurosecretory cells, suggesting that ecr is localized close to the plasma membrane of other tissue cells, and therefore would be involved in asg cell death. however, inhibitors of pi3k and the map kinase/extracellular signal-regulated kinase (erk) kinase do not interfere with the 20e-triggered death sequence (iga et al., 2007; iga and sakurai, unpublished). thus, 20e-induced programmed cell death might not involve the ecr/pi3k pathway, although this pathway may be present in insect cells. possible nongenomic action of 20e arthropods respond rapidly to 20e, which is believed to affect na+-k+ atpase, na+-h+ exchangers, k+ channels, ecdysone transport, electrolyte (na+, k+, h+) transport, and second messenger (camp, ca2+) levels, and acts as a neuromodulator (see tomaschko, 1999 for review). in the wing imaginal discs of hyalophora gloveri (giant silk moth), 20e increases camp levels (applebaum and gilbert, 1972), indicating a 5 fig. 3 possible interaction between genomic and nongenomic actions of 20e. see text for details. modified from iga et al. (2007). nongenomic activity of 20e. it stimulates nomediated cell proliferation in the pupal eye of manduca sexta (tobacco hornworm) (champlin and truman, 1998, 2000). the no-mediated action of 20e is thought to involve activation of the nuclear ecr, which upregulates the gene for no synthase (nos), thereby increasing the activity of the enzyme. in the wing imaginal discs of bombyx fifth instar larvae, 20e stimulates cell proliferation (koyama et al., 2003). the presence of α-amanitin, a transcription inhibitor, does not affect this action of 20e (koyama and sakurai, unpublished data), indicating that de novo gene expression is unnecessary. thus, 20e probably elicits its effect on cell proliferation through a nongenomic pathway, like the estrogen/erα/pi3k/akt/nos pathway (simoncini et al., 2002). the molecular mechanisms that stimulate the 20e nongenomic pathway are largely unknown, but in drosophila, stimulation of this pathway is known to involve the catecholamine receptor (dopamine/ecdysone receptor; dmdopecr) (srivastava et al., 2005). dmdopecr is a gpcr that binds 20e and dopamine in different binding pockets; binding of 20e to its pocket on dmdopecr elevates the intracellular camp level. although dmdopecr acts as a 20e membrane receptor, it does not appear to be the membrane receptor involved in 20e-induced programmed cell death, since neither camp nor protein kinase a mediates 20e signaling up to asg cell death (iga et al., 2007). in the sections that follow below, i will present some details of the programmed cell death of bombyx asgs and discuss the 20e signaling pathway. because our information concerning 20e signaling derives entirely from our own studies, the following discussion is based on our unpublished data, except where a reference is cited (see fig. 3 for the following sections). involvement of the ecdysone membrane receptor in completion of cell death exposure to 20e induces programmed cell death of bombyx asgs. apoptotic body formation occurs 120 to 144 h after 20e challenge (fig. 1), but timed additions of α-amanitin have shown that de novo expression of the genes needed for execution of cell death is complete within 8 h of challenge (terashima et al., 2000). similarly, studies using cycloheximide, a translation inhibitor, have shown that protein synthesis is completed within 18 h of 20e challenge (terashima et al., 2000). in the classical model for steroid hormone action through nuclear receptor binding, these data would suggest that, after 18 h of challenge, the 20e stimulus is no longer needed for the cell death to occur. nevertheless, cell death is not fully realized unless 20e is present continuously for 42 h (terashima et al., 2000), suggesting that 20e activates a nongenomic pathway in addition to exerting its genomic action (fig. 2). since activation of the genetic pathway required for programmed cell death of bombyx asgs is complete within 18 h of the 20e challenge (fig. 2), we examined the nongenomic action of 20e using asgs that had been incubated with 20e for 18 h. under these culture conditions, 20e increased the intracellular level of camp (elmogy et al., 2006), suggesting the presence of an ecdysone membrane receptor (mecr; elmogy et al., 2004, 2007). [subsequently, cell death was found not to involve camp (iga et al., 2007).] the plasma membrane fraction prepared from the pre-cultured asgs bound to ponasterone a, a plant ecdysteroid with a km of 18 nm (elmogy et al., 2004), a value being comparable to the km for dmdopecr (srivastava et al., 2005). this value is approximately 10 times higher than the km for the lepidopteran ecr/usp complex (minakuchi et al., 2002) the binding protein(s) in bombyx asgs are integral plasma membrane proteins (elmogy et al., 2004, 2007). in addition, the bisacrylkydrazine ecdysone agonists, which bind with a high affinity to ecr/usp, exhibit very low affinities to the putative mecr and also dmdopecr, showing that the mecr is not the classical ecr. these biochemical and topological evidence indicates the presence of mecr in bombyx asgs. calcium mobilization and inositol triphosphate agonists, antagonists, and inhibitors are powerful tools for studying signal transduction pathways. since serial activation of protein kinases is a major feature of these pathways, we used various kinase inhibitors to search for the kinase involved in 20e signaling. inhibition of protein kinase c (pkc) suppressed the progression of death sequence except for nuclear condensation, suggesting that ca2+ is the second messenger. in fact, a ca2+ ionophore added to the asg culture after 18 h of 20e challenge mimicked the dna and nuclear fragmentation-inducing effects of 20e. when the ionophore was added at the beginning of the culture, however, it elicited no change in the asgs, indicating that the genomic effects of 20e are 6 a prerequisite for driving the ca2+ pathway (iga et al., 2007). verapamil, which blocks voltage-activated ca2+ channels, had no effect on the death sequence, indicating that 20e does not activate ltype ca2+ channels (lieberherr and grosse, 1994). flunarizine, which blocks ligand-dependent (ttype) ca2+ channels (qui et al., 2006), inhibited dna and nuclear fragmentation, but allowed other cellular responses (cell and nuclear condensation) to occur normally. the inhibitory effect of flunarizine indicates that the mecr could be a ltype ca2+ channel-coupled receptor (manaboon and sakurai, unpublished). in this case, the ca2+ influx could come from the medium. however, 20e induced complete cell death in ca2+-free medium, and flunarizine exhibited the same inhibitory effects in ca2+-free medium as in normal medium. thus, the ca2+ reservoir must be inside the cell, most likely in the endoplasmic reticulum (er), which provides ca2+ for many signal transduction pathways. in fact, 2-aminoethyl diphenylborinate, an inhibitor of the inositol 3,4,5triphosphate receptor (ip3r) on the er membrane inhibits dna and nuclear fragmentation (manaboon and sakurai, unpublished), consistent with the er being the reservoir for intracellular ca2+ elevation. flunarizine may not act as a ca2+ channel blocker in bombyx asgs but inhibit calmodulin/camkii pathway, which may mediate the 20e-induced dna and nuclear fragmentations (see below), as demonstrated in bovine brains (kubo et al., 1984). upstream of ca2+ suramin, an inhibitor of g-proteins, suppresses the cellular responses to 20e (dna and nuclear fragmentation) underlying the nongenomic pathway. u73122, an inhibitor of phospholipase c (plc), also inhibits these responses, indicating that the gprotein αq subunit (gαq)/plc/ip3 pathway acts downstream of the mecr (manaboon and sakurai, unpublished). in the prepupal period, the intracellular camp level increases transiently at the third day of gut purge, when the asgs are fully stimulated to complete programmed cell death with no further 20e stimulus (elmogy et al., 2007). in asgs cultured with 20e, intracellular camp also increases, beginning 24 h after 20e challenge, indicating an involvement of g-protein αs subunit (gαs). however, dibutyl camp, a membrane-permeable camp analogue, does not mimic 20e action, and an inhibitor of protein kinase a (pka) has no effect on 20e signaling (iga et al., 2007), demonstrating camp/pka is not involved in induction of cell death. in chinese hamster ovary cells expressing histamine h1 receptor, histamine increases intracellular camp levels through activation of the receptor, which releases g-protein βγ subunits (gβγ) that activate adenylyl cyclase (maruko et al., 2005). if gβγ/adenylyl cyclase pathway occur in the asgs, the mecr may be coupled with both gαq, but the role of gβγ and diacylglyceride in cell death remains to be seen. downstream of ca2+ as described above, inhibition of pkc suppresses dna and nuclear fragmentation. pkc inhibitor suppresses these cellular responses when added 24 h, but not 48 h, after 20e challenge, showing that the pkc activation required for inducing these responses is complete by 48 h. however, when pkc activity was assessed using a fluorescence labeled pkc substrate, it was shown to be at substantial levels with small fluctuations (iga et al., 2007). since the substrate is phosphorylated by a broad spectrum of pkc isozymes, the isozyme involved in 20e signaling remains to be identified. in rat, phorbol esters increase intracellular antiapoptotic protein, phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (ped/pea)-15 by reducing its proteasomal degradation, thereby enhancing its anti-apoptotic action. pkc-ζ and calmodulin/calmodulin-dependent protein kinase ii (camkii) activities are necessary for phorbol ester-dependent phosphorylation of ped/pea-15 (perfetti et al., 2007), indicating that whether pkc induces or inhibits apoptosis depends on the particular subtype. therefore, subtype identification is important for understanding the 20e signaling pathway. caspase-3, an apoptosis-related cysteine protease, is a key enzyme commonly found in the cell death pathways that respond to extracellular signals (martin and baehrecke, 2004). in vertebrates, caspase-3 activation releases caspaseactivated dnase (cad) from its inhibitor (inhibitor of caspase-activated dnase; icad), and the liberated cad induces dna fragmentation (liu et al., 1997; enari et al., 1998; sakahira et al., 1998). since caspase-3 is highly conserved throughout the animal kingdom, caspase-3-like protease activity can be measured using a colorimetric substrate for human caspase-3 (ilangovan et al., 2003). an antihuman caspase-3 antibody exhibits strong crossreactivity to the caspase-3-like protease of drosophila (yu et al., 2002). using these tools, we showed that 20e-induced cell death includes activation of the caspase-3-like protease. a caspase-3 inhibitor inhibited dna and nuclear fragmentation, showing that the caspase-3like protease is involved in 20e signaling, as it is in cell death in other animals. timed addition of the inhibitor showed that addition at 72 h, but not at 96 h, of 20e challenge prevented dna and nuclear fragmentation. caspase-3 activity measured using a colorimetric substrate also began to increase after 72 h and peaked at 96 h. thus, the caspase-3-like protease may be activated between 72 and 96 h, in accordance with the timing of dna fragmentation beginning at 96 h (iga et al., 2007). the caspase-3-like protease involved in 20einduced cell death appears somewhat different from human caspase-3. western blot analysis of cultured bombyx asgs using anti-human caspase-3 antibody that recognizes the active fragment of caspase-3 revealed a single immunoreactive band. the intensity of this band increased dramatically between 72 and 96 h of 20e challenge, in accordance with the above-mentioned results. in human and drosophila, proteolytic cleavage of 7 caspase-3 yields active fragments of 17 kda (han et al., 1997) and 45 kda, respectively; in bombyx, the molecular weight of the fragment, estimated based on western blotting, was 66 kda (kaneko and sakurai, unpublished). these data indicate that the bombyx caspase-3-like protease functions similarly to human caspase-3, but further understanding of the caspase-3-like protease awaits its identification and characterization. calmodulin/calmodulin-dependent protein kinase pathway the 20e signaling pathway leading to nuclear (chromatin) condensation in bombyx asgs is distinct from the pathway leading to caspase-3-like protease-dependent dna and nuclear fragmentation (iga et al., 2007). similarly, in the drosophila adult egg chamber, chromatin condensation occurs independently of any caspase-3-like protease activation in ovarian nurse or follicle cells and is controlled independently of dna fragmentation (nezis et al., 2006). induction of the apoptotic chromatin condensation has been suggested to result from acinus activation by active caspase-3 (sahara et al., 1999). in bombyx asgs, the calmodulin antagonist w7, when added at 18 h of 20e challenge, inhibits induction of nuclear condensation as well as of dna and nuclear fragmentation. similarly, kn-93, an inhibitor of camkii, inhibited the condensation, indicating that calmodulin/camkii activation leads to nuclear condensation (manaboon and sakurai, unpublished). calmodulin/camkii is usually activated by ca2+, but in 20e-induced cell death, a ca2+ ionophore mimicks 20e action by inducing dna and nuclear fragmentation but not nuclear condensation. this result indicates that 20e signaling activates calmodulin/camkii independently of ca2+ to regulate nuclear condensation. a calmodulin agonist and camkii inhibitor suppresses dna and nuclear fragmentation to the same degree as that of the caspase-3 inhibitor, indicating that activation of the caspase-3-like protease in bombyx may be dually regulated through pkc and camkii, although this issue is far from settled. the calmodulin/camkii pathway is involved in cell death in mammals and insects. camkii activation is regulated by ca2+/calmodulindependent protein (cam) binding or by autophosphorylation, which persists independently of ca2+. although camkii autophosphorylation is initiated by binding of ca2+/cam, ca2+/camindependent activity is substantial and is postulated to be important for synaptic and cellular plasticity in rat and drosophila (griffith, 2004; elgersma et al., 2004). in the central nervous system of drosophila, the autophosphorylation ability of ca2+/camkii allows camkii to become ca2+-independent (mehren and griffith, 2004). the protein phosphatase 1 and 2a (pp1/pp2) inhibitor calyculin a induces apoptosis, and the camkii inhibitor kn-93 blocks this effect of calyculin a. calyculin a induces apoptosis by hyperphosphorylating camkii, suggesting that stringent limitation of camkii autophosphorylation restricts apoptosis (fährmann et al., 2007). although the mechanism of caspase-3-like protease activation in asgs is unknown, ca2+-independent, cam-dependent activation of camkii may be involved. isoform-specific suppression of camkiiδc with the dominant negative-camkiiδc mutant, as well as nonselective camkii inhibition by kn-93, inhibits β1adrenergic receptor-mediated stimulation of camkiiδc-mediated apoptosis in rat cardiomyocytes (zhu et al., 2007). in drosophila, dmdopecr is a homolog of the vertebrate γ-adrenergic receptors and is activated by 20e as well as dopamine. it has both a 20e-binding pocket and a dopamine-binding pocket. the receptor is coupled with gαs, and 20e activates the receptor as powerfully as dopamine (srivastava et al., 2005). these independent results indicate an involvement of mecr in activation of camkii pathway in bombyx asg cell death. camkii activation obviously mediates extracellular signal transduction in the cell death sequence, and ca2+/cam-independent activation is brought about through autophosphorylation of camkii, although the initial phase of autophosphorylation requires mobilization of intracellular ca2+. in the death of bombyx asgs, a ca2+ ionophore may not activate camkii, since it does not mimic 20e by inducing nuclear condensation, but inhibition of either calmodulin or camkii suppresses nuclear condensation (iga et al., 2007; manaboon and sakurai, unpublished), indicating that the mechanism involves ca2+independent, calmodulin-dependent camkii activation. concluding remarks although our understanding of the nongenomic action of steroid hormones is limited, elucidation of its molecular mechanisms is important for understanding how steroid hormones exert their effects at quite different levels of biological events, including embryonic and post-embryonic development, reproduction, metabolism, homeostasis, and biological defense. in eliciting its effects, 20e has interacting nongenomic and genomic actions. in choristoneura fumiferana (spruce budworm), dequalinium-14;1,1′decamethylenebis-4-aminoquinaldinium diiodide (deca), an inhibitor of receptor of activated c kinase 1 (rack1) binding to pkc, blocks 20einduced expression of the transcription factor chr3 by inhibiting translocation of ecr, probably by preventing its phosphorylation (quan et al., 2006). chr3 is an early-late gene and is a homologue of bhr3 in bombyx and bghr3 in b. germanica, the latter of which is involved in prothoracic gland cell death (cruz et al., 2006, 2007). activation of the rack1/pkc pathway is probably one of the nongenomic actions of 20e mediated by mecr, and an example of the interaction between the genomic and nongenomic actions of 20e. nongenomic action of steroids may be involved, directly or indirectly, in regulating a variety of biological events. elucidation of its underlying molecular mechanisms will contribute to the development not only of chemicals for insect 8 population control but also of therapies and drugs for syndromes that respond to steroid hormones. until now, the molecular mechanisms underlying steroid action have been mostly understood as separate genomic and nongenomic mechanisms. we now recognize that this paradigm is inadequate. the elucidation of the interaction between the two signaling pathways may herald a new era for the study of steroid hormones. 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plant protection, college of agriculture, university of guilan, rasht, iran accepted july 21, 2008 abstract the resistance mechanisms to oxydemeton-methyl were surveyed in two iranian strains of the two spotted spider mite, tetranychus urticae koch. bioassay was carried out on two strains, collected from tehran and rasht using dipping method. the results of bioassay indicated that resistance ratio was 20.47 for resistant strain. the activity of esterase and glutathione s-transferase in resistant and susceptible strains showed that one of resistance mechanisms to oxydemeton-methyl was esterasebased resistance and glutathione s-transferase. the esterase activity of the resistant strain was 2.5 and 2.14-fold higher than those of the susceptible strain for α-naphtyl acetate (α-na) and β-naphtyl acetate (β-na) respectively. the kinetic characteristics acetylcholinesterase (ache) showed that the ache of resistant strain had lower affinity to artificial substrates; acetylthiocholine and butyrylthiocholine than that of susceptible strain. i50 of oxydemeton-methyl for resistant and susceptible strains were 2.68×10-6 m and 7.79×10-7 m respectively. the results suggested that ache of resistant strain is insensitive to oxydemeton-methyl and ratio of ache insensitivity of resistant to susceptible strain were 3.49 and 7.8-fold to oxydemeton-methyl and paraoxon, respectively. key words: tetranychus urticae; oxydemeton-methyl; esterase; insensitive acetylcholinesterase; glutathione s transferase introduction the two-spotted spider mite, tetranychus urticae koch (acari: tetranychidae), is an important agricultural pest with a global distribution. its phytophagous nature, high reproductive potential and short life cycle facilitate rapid resistance development to many acaricides often after a few applications (cranham and helle 1985; keena and granett, 1990; devine et al., 2001; stumpf and nauen, 2001). so far resistance have been reported in several countries for compounds, such as organophosphates (ops) (sato et al, 1994; anazawa et al., 2003), dicofol (fergusson-kolmes et al., 1991), organotins (edge and james, 1986); hexythiazox (herron and rophail, 1993), clofentezine (herron et al., 1993); fenpyroximate (sato et al., 2004) and abamectin (beers et al., 1998). ___________________________________________________________________________ corresponding author: m ghadamyari department of plant protection college of agriculture university of guilan, rasht, iran e-mail: ghadamyari@guilan.ac.ir insensitive ache causing op resistance is widespread and has been detected in t. urticae strains from germany (matsumura and voss, 1964; smissaert et al., 1970), japan (anazawa et al., 2003) and new zealand (ballantyne and harrison, 1967) and in a few other tetranychid pest species, including t. cinnabarinus from israel (zahavi and tahori, 1970) and t. kanzawai from japan (kuwahara, 1982 and aiki et al., 2005). also the insensitivity of ache to demeton-s-methyl, ethyl paraoxon, chlorpyrifos oxon and carbofuran was identified in a german laboratory strain of t. urticae and a field collected strain from florida (stumpf et al., 2001). however, insensitive ache was not the only mechanism of op resistance in spider mites described, as some resistant strains of t. urticae showed an enhanced degradation of malathion, malaoxon, and ethyl parathion to nontoxic products (matsumura and voss, 1964; herne and brown, 1969). op-resistant strains of t. kanzawai rapidly degraded malathion in vitro and the resistance was obviously attributed to high nonspecific esterase activity (kuwahara, 1981, 1982). pilz et al. 97 mailto:ghadamyari@guilan.ac.ir (1978) showed that a german dimethoateselected laboratory strain of t. urticae possessed multiple mechanisms of op resistance. in addition to an ache insensitive to dimethoxon, the toxicity of dimethoate was enhanced by synergists, such as piperonyl butoxide indicating the involvement of cytochrome p-450-mediated oxidative detoxication. oxydemeton-methyl is currently used in iran to control some pests, such as aphids and t. urticae in several crops. the intensive use of oxydemetonmethyl to control of t. urticae and aphids in greenhouse facilitates resistance development in some populations of t. urticae in iran. there is no information about oxydemeton-methyl resistance in this pest in iran. resolution of the underlying biochemical mechanisms of resistance can play an important role in circumventing problems associated with pesticide resistance and assist in rational choices of chemicals for pesticide mixtures and rotations. the purpose of this study was to collect information about the presence of esterases, gluthathion s-transferase and insensitive acetylcholinesterases in the resistance of t. urticae by bioassays and biochemical assays. material and methods two spotted spider mite strains the resistant strain was collected from infected been plants grown in the research greenhouse in plant pests and disease research institute of iran, tehran. a strain from rasht was considered as a strain susceptible to oxydemeton-methyl which had no previous exposure to pesticides and was collected from convolvulus sp. in university of guilan. the mites were reared routinely on been plants (phaseolus vulgaris) grown under greenhouse conditions [25 ± 4 °c, 60 ± 20 rh (relative humidity)]. pesticide oxydemeton-methyl was used as the commercial formulation in the bioassay (ec 25 %) and was purchased from bayer crop science, germany. chemicals acetylthiocholine iodide (atc), sbutyrylthiocholine iodide (btc), 5,5 ́-dithiobis-(2nitrobenzoic acid, dtnb), triton x-100 were purchased from sigma. fast blue rr salt, α-naphtyl acetate (α-na) and β-naphthyl acetate (β-na) were obtained from fluka, and oxydemeton-methyl from accustandard. 1-chloro-2,4-dinitrobenzene (cdnb), 1,2-dichloro-4-nitrobenzene (dcnb) were purchased from merck, germany. bioassay the toxicities of oxydemeton-methyl to the susceptible and resistant strains of two-spotted spider mite were assayed using the dipping method. the formulated oxydemeton-methyl was diluted with distilled water to generate five serial dilutions. the leaf disc (diameter 3.5 cm) was immersed in the dilutions for 45 s. after drying, adult mites were placed on each treated leaf disk on wet cotton in a petri dish. up to 10 adults were placed on each leaf disk. mortality was assessed after the treated mites were maintained at 25 ± 2 °c, 70 ± 10 rh and 16:8 (light: dark) for 48 h. mites that could walk at least one body length after a gentle probe with a fine brush were scored alive. bioassay data were analyzed for ld50 values and their 95 % confidence intervals (95 % cl) using the polo-pc computer program (leora software 1987). resistance factors (rf) were calculated by dividing the ld50 value of the resistant strain by the ld50 value of the susceptible strain. determination of esterase activity adults were homogenized in ice-cold 0.2 m phosphate buffer (ph 7.0) containing 0.05 % triton x-100. after the homogenates were centrifuged at 10000 g for 12 min at 4 °c. the esterase activity was measured according to van asperen’s method (van asperen, 1962). the substrate was α-na and β-na. fifteen µl of supernatant was added to a microplate containing 35 µl 0.2 m, ph 7.0, phosphate buffer per well. the addition of 100 µl substrate per well (0.65 mm in buffer) initiated a reaction. after incubation for exactly 10 min at room temperature, 50 µl of fast blue rr salt was added and the microplate left in the dark for 30 min. absorbance at 450 nm (od450) was then measured in a microplate reader (awareness stat fax® 3200). determination of glutathione s-transferase (gst) adults were homogenized in ice-cold 0.2 m phosphate buffer (ph 7.0). after the homogenates were centrifuged at 10,000xg for 12 min at 4 °c. gst activity was measured using 1-chloro-2,4dinitrobenzene (cdnb), 1,2-dichloro-4-nitrobenzene (dcnb) and reduced gsh as substrates with slight modifications according to habig et al. (1974) in 96well microplates. the total reaction volume per well of a 96-well microplate was 300 µl, consisting of 100 µl, supernatant, cdnb (or dcnb) and gsh in buffer, giving final concentrations of 0.4 and 4 mm of cdnb (or dcnb) and gsh, respectively. the non-enzymatic reaction of cdnb (or dcnb) with gsh measured without supernatant served as control. the change in absorbance was measured continuously for 10 min at 340 nm in a thermomax kinetic microplate reader (awareness stat fax® 3200). ache kinetics mites were homogenized in ice-cold 0.2 m phosphate buffer (ph 7.0) containing 0.1 % triton x-100. after the homogenates were centrifuged at 10000 g for 15 min at 4 oc. ache activity was measured according to the methods of stumpf et al (2001) with some modifications. fifty microliters of the enzyme source was added to each well of microplate containing 140 µl of 0.2 m phosphate buffer (ph 7.0) and 20 µl dtnb solution. then 40 µl of atc was added to each well. the concentrations of the substrate were changed from 0.01 mm to 10 mm to evaluate the michaels’s constant (km). optical density was measured at 415 nm with a microplate reader (awareness stat fax® 3200). 98 table 1 log dose probit-mortality data for oxydemeton-methyl against susceptible and resistant strain of t. urticae strain insecticide n ld50 (95 % ci) a slope ± se χ2 b rrc resistant oxydemeton-methyl 245 4675.9 (44734892) 10.79±1.36 0.88 20.47 susceptible oxydemeton-methyl 250 228.6 (191-268) 2.5 ± 0.27 1.11 ald50 values and their ci are expressed in ppm formulated pesticide bvalues of χ2 smaller than 7.81 (p < 0.05) considered to be represented satisfactory agreement between observed and expected results cresistance ratio, ld50 of resistant strain/ld50 of susceptible strain inhibition assay the enzyme was preincubated with inhibitor at 37 °c for 15 min. after preincubation, the atc substrate was added to the mixture (containing 0.2 m phosphate buffer (ph 7.0) and dtnb). the remaining activity was determined at 30 min following preparation of the reaction mixture. optical density was measured at 415 nm with a microplate reader (awareness stat fax® 3200). i50 values for the ache of susceptible and resistant strains were estimated by probit analysis using the polo-pc computer program. results resistance levels in bioassay table 1 summarizes the toxicological data for susceptible and resistant strains exposed to oxydemeton-methyl. the resistance ratio of the resistant strain was 20.47. fig. 1 esterase activity in resistant and susceptible strains of t. urticae. the asterisk (*) indicates significant differences between the two strains at p<0.01 (t-test) activity of esterase the measured esterase activity of the resistant strain was significantly higher than that of the susceptible strain (t-test p < 0.001). the esterase activity of the resistant strain was 2.5 and 2.14-fold higher than those of the susceptible strain for α-na and β-na respectively (fig. 1). activity of gst the measured glutathione s-transferase activity of the resistant strain was significantly higher than that of the susceptible strain (t-test p < 0.001). the glutathione s-transferase activity of the resistant strain was 1.75 and 1.27-fold higher than those of the susceptible strain for cdnb and dcnb, respectively (fig. 2). kinetic analysis of ache the effect of substrate concentrations on ache activity were investigated using atc and btc. the different specificities of ache in resistant and susceptible strains toward two substrates are summarized in table 2. km values suggest that ache in resistant strain was kinetically different from that in susceptible strain, indicating qualitative differences among enzymes in two strains. the kinetic study indicated that ache from the resistant strain had 1.55 and 2.16fold lower affinities to substrates atc and btc than susceptible strain respectively. ache of susceptible strain showed significantly higher affinity toward btc than ache of resistant strain, suggesting that a modification of the enzyme catalytic site might be present in the ache from the resistant mite. inhibition of ache by oxydemeton-methyl and paraoxon a comparison of the i50 values of the susceptible and resistant strains showed 3.49 and 7.8-fold resistance to oxydemeton-methyl and ethyl paraoxon, respectively (table 3; fig. 3). discussion metabolic resistance mechanisms seem to be most important in arthropod species exhibiting resistance to organophosphate and carbamate pesticides (devonshire et al., 1982; kono and tomita, 1992; moores et al., 1994; ghadamyari et 99 table 2 km and vmax values of ache in resistant and susceptible strains of t. urticae vmax (δod/30 min/mite) (±sd) km (µm) (±sd) strain substrate 5 ± 0.4 95± 5.2* resistant atc 4.33 ± 0.31 61± 4.1 susceptible 3.2±0.27 337±32* resistant btc 2.9±0.23 156±23 susceptible the asterisk (*) indicates significant differences between the two strains at p<0.01 (t-test) al., 2008a, b). our results showed that probably glutathione s-transferase was related to oxydemeton-methyl resistance in t. urticae, and there is 1.75and 1.27-fold increase in glutathione s-transferase activity in the resistant strain, when cdnb and dcnb were used as substrate respectively. gsts are detoxification enzymes frequently associated with insecticides resistance, particularly op resistance (soderlund and bloomquist, 1990; yu, 1996). these enzymes may act as binding proteins increasing the activity of other pesticide detoxification enzymes such as esterases (grant and matsumura, 1994). also esterases have a role in resistance of t. urticae to oxydemeton-methyl (fig.1). these enzymes probably sequester or degrade insecticide esters before they reach their target sites in the nervous system. this mechanism seems to be important in the insecticide resistance of culex mosquitoes (mouches et al, 1986; kono and tomita, 1992; tomita et al., 1996) and aphis gossypii (suzuki et al., 1993). the relationship between the enzymes which catalyze hydrolysis of β-na and degradation of malathion was studied in resistance fig. 2 gst activity in resistant and susceptible strains of t. urticae. the asterisk (*) indicates significant differences between the two strains at p<0.01 (t-test) and susceptible strains of t. kanzawai kishida by kuwahara (1981). their results showed that resistance to malathion was associated with increased esterase activity at e3 and e4 bonds on which the main peak of malathion degradation was detected. further experimental data are required to evaluate the importance of these two degradation pathways and to clarify the existence of general esterase and glutathione transferase for oxydemeton-methyl resistance in t. urticae. although metabolic detoxification mechanisms are implicated, insensitive ache is considered one of the mechanisms of resistance to oxydemetonmethyl in t. urticae. the occurrence of pesticideinsensitive ache in spider mite was first demonstrated by smissaert (1964). the present study indicates that the resistant strain possesses an altered ache with decreased sensitivity to inhibition by oxydemeton-methyl and paraoxon and decreased affinity to atc and btc substrates. the km values for atc determined in our study were 95 and 61 µm for the insensitive and sensitive forms of ache, respectively (table 2). the maximum velocities of ache from resistant and susceptible strains were equal and only differed in terms of the affinity toward atc and btc (table 2). our results agree well with those reported by anazawa et al. (2003) with respect to the involvement of insensitive ache in conferring op resistance in t. urticae. because ache from the resistant strain had reduced affinity to atc and btc (i.e., increased km values) and reduced sensitivity to inhibition by oxydemetn-methyl and paraoxon (i.e., increased i50 values) compared with ache from susceptible strain, it is clear that the resistant strain possesses qualitatively altered ache. recent molecular investigations suggest that some amino acid substitutions in the ache of t. urticae may result in different responses of the altered aches to different substrates and inhibitors (anazawa et al., 2003). therefore the amino acid sequences of ache in iranian strains need to be analyzed. the mechanisms of oxydemeton-methyl resistance in t. urticae vary and belong to different classes of biochemical mechanisms and both detoxification and target alteration are involved in resistance of t. urticae to oxydemeton-methyl. zhu and gao (1999) showed that the modified ache alone was not sufficient to cause a high degree of resistance in insects. it seems that these two mechanisms (esterase, gst and ache insensitivity) have an additive interaction in t. urticae. on the basis of these data, it can not be 100 table 3 i50 values of oxydemeton-methyl and paraoxon on ache from susceptible and resistant strains of t. urticae i50 (m) (95 % ci) inhibitor resistant susceptible ir (95 % ci)a 2.68×10-6 (2.3×10-63.15×10-6) 7.79×10-7 (6.6×10-79×10-7) 3.49 (2.82-4.37)* oxydemeton-methyl paraoxon 6.5×10-6 (5.4×10-67.8×10-6) 8×10-7 (5.2×10-7-12.2×10-7) 7.8(5.2-11.8)* ainsensitivity ratio = i50 for resistant strain/susceptible strain and confidence interval (ci) the asterisk (*) indicates significant differences between the two strains at p<0.05 (t-test) decided which mechanism is the dominant factor in the oxydemeton-methyl resistance. resistant strain has high potential to develop cross-resistance to parathion since the ache from resistant strain showed 7.8-fold insensitivity to ethyl paraoxon and this strain had no previous exposure to parathion. it may be inferred that probably altered ache due to extensive use of oxydemeton methyl might have caused insensitivity to paraoxon. in conclusion, oxydemeton-methyl is the most commonly used insecticide and acaricide for controlling t. urticae and aphids in iran and therefore the present findings may be regarded as a future strategy for controlling t. urticae. fig. 3 inhibition of ache from t. urticae by oxydemeton-methyl and ethyl paraoxon acknowledgments this work was supported by university of guilan (rasht, iran). references anazawa y, tomita t, aiki y, kozaki t, kono y. sequence of a cdna encoding acetylcholinesterase from susceptible and resistant two-spotted spider mite, tetranychus urticae, insect biochem. mol. biol. 33: 509-514, 2003. aiki y, kozaki t, mizuno h, kono y. amino acid substitution in ace paralogous acetylcholinesterase accompanied by organophosphate resistance in spider mite tetranychus kanzawai. pestic. biochem. physiol. 82: 154-161, 2005. ballantyne gh, harrison ra. genetic and biochemical comparisons of organophosphate resistance between strains of spider mites (tetranychus species: acari), entomol. exp. appl. 10: 231-239, 1967. beers eh, riedl h, dunley je. resistance to abamectin and reversion to susceptibility to fenbutatin oxide in spider mite (acari: tetranychidae) populations in the pacific northwest. j. econ. entomol. 91: 352-360, 1998. cranham je, helle w. pesticide resistance in tetranychidae. in: helle w, sabelis mw (eds), spider mites: their biology, natural enemies, and control. vol. 1b, amsterdam, elsevier, pp 405-421, 1985. devine gj, barber m, denholm i. incidence and inheritance of resistance to meti-acaricides in european strains of the two-spotted spider mite (tetranychus urticae) (acari: tetranychidae). pest manag. sci. 57: 443-448, 2001. devonshire al, moores gd. a carboxylesterase with broad substrate specificity causes organophosphorus, carbamate, pyrethroid resistance in peach potato aphid (myzus persicae). pestic. biochem. physiol. 18: 235246, 1982. edge ve, james, dg. organotin resistance in tetranychus urticae (acari: tetranychidae) in australia. j. econ. entomol. 79: 1477-1483, 1986. fergusson-kolmes la, scott jg, dennehy tj. dicofol resistance in tetranychus urticae (acari: tetranychidae): cross-resistance and pharmacokinetics. j. econ. entomol. 84: 41-48, 1991. 101 ghadamyari m, mizuno h, oh s, talebi k, kono y. studies on pirimicarb resistance mechanisms in iranian populations of the peach-potato aphid, myzus persicae. appl. entomol. zool. 43: 149157, 2008a. ghadamyari m, talebi k, mizuno h, kono y. oxydemeton-methyl resistance, mechanisms and associated fitness cost in green peach potato aphids (homopotera: aphididae). j. econ. entomol. 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aphididae). pestic. sci. 55: 11-17, 1999. 102 lectins and cytokines in celomatic invertebrates: two tales with the same end isj 7: 1-10, 2010 issn 1824-307x minireview lectins and cytokines in celomatic invertebrates: two tales with the same end d malagoli1, s sacchi2, e ottaviani1 1department of animal biology, university of modena and reggio emilia, modena, italy 2department of biological sciences, george washington university, washington dc, usa accepted december 18, 2009 abstract the paper presents the principle data regarding the presence and the roles of lectins and cytokines in invertebrates. the former have been described in the main invertebrate taxa, such molluscs, annelids, arthropods, echinoderms and tunicates, while convincing evidence for cytokines was found only in the insects, drosophila melanogaster and pseudaletia separata, and the freshwater crayfish, pacifastacus leniusculus. lectins and cytokines share convergent and common functions, and one of the multiples roles of these messenger molecules is their participation in fighting against non-self. kew words: invertebrate immunity; lectins; cytokines; evolution introduction according to barondes (1988) e yoshizaki (1990), lectins are sugar-binding proteins or glycoproteins bearing one or more sugar-binding sites and capable of agglutinating cells and/or precipitating glycoconjugates. the specificity of lectin is usually defined in terms of the monosaccharide(s) or simple oligosaccharides that inhibit lectin-induced agglutination. although lectins were first discovered in plants, they are present in all kingdoms, including bacteria and animals. cytokines constitute a more heterogeneous group of soluble mediators, but despite significant differences in terms of structure and function, some common characteristics are evident. cytokines are mainly produced by the cells of the immune or neuroendocrine systems (nisticò, 1993; schöbitz et al., 1994). described principally in mammalian models, cytokines are glycoproteins of a relatively small molecular weight, and in most cases they are synthesized de novo by activated cells during the efferent phase of immune response. the main role of cytokines is that of mediator and modulator of immune responses and inflammation, but they are also involved as signal molecules in the neuroendocrine system (nisticò, 1993; blalock, 1994; schöbitz et al., 1994). cytokines are characterized by pleiotropicity and redundancy (i.e., the same cytokine can have different effects on ______________________________________________________________________________ corresponding author: enzo ottaviani department of animal biology university of modena and reggio emilia via campi 213/d, 41100 modena, italy e-mail: enzo.ottaviani@unimore.it diverse cellular targets, and the same function can be performed by different cytokines), they act on target cells by autocrine, paracrine and endocrine mechanisms, and they bind to specific plasma membrane receptors which show a certain degree of promiscuity (kishimoto et al., 1994; paul and seder, 1994). notably, several human cytokines also display a lectin-like activity, and this may be essential to explain some of their biological properties (cebo et al., 2002). immune recognition in celomatic invertebrates is principally carried out by cells and humoral components that include lectins and cytokines and are present in the hemolymph (ottaviani, 2005, 2006). from the literature, it emerges that comparative immunologists have devoted their attention mainly to invertebrate lectins (table 1) rather than to cytokines (table 2). this may be related to the fact that, usually, lectins are more abundant, stable and functionally recognizable than cytokines and it is possible to purify and characterize a lectin even in absence of a conspicuous molecular dataset. conversely, the isolation and characterization of a cytokine needs several molecular biology-based investigations. now that molecular databases are becoming available for several invertebrate models, cytokines are, not surprisingly, receiving the appropriate attention. lectins lectins may be classified by structural or functional criteria. lectins play an important role in cell-to-cell or cell-to-matrix interaction, glycoprotein 1 mailto:enzo.ottaviani@unimore.it table 1 examples of lectins described in invertebrates taxon species reference mollusca helix pomatia hammarström and kabat, 1969 helix pomatia hu et al., 2008 aplysia californica pauley et al., 1971 mercenaria mercenaria arimoto and tripp, 1977 biomphalaria glabrata stein and basch, 1979 biomphalaria glabrata boswell and bayne, 1984 mytilus edulis renwrantz et al., 1985 octopus vulgaris rögener et al., 1985 planorbarius corneus ottaviani and tarugi, 1986 planorbarius corneus ottaviani and tarugi, 1986 crassostrea virginica yamaura et al., 2008 annelida lumbricus terrestris stein et al., 1982 eisenia fetida eue et al., 1998 caenorhabditis elegans cooper and barondes, 1999 eisenia fetida bloc et al., 2002 arthropoda sarcophaga peregrina komano et al., 1980 sarchophaga peregrina takahashi et al., 1985 rhodnius prolixus pereira et al., 1981 limulus polyphemus rostam-abadi and pistole, 1982 limulus polyphemus muta et al., 1991 limulus polyphemus amstrong et al., 1996 aphonopelma chalcodes vasta and cohen, 1984 aphonopelma cochise vasta and cohen, 1984 aphonopelma chiricawa vasta and cohen, 1984 cancer antennarius ravindranaths et al., 1985 spodoptera exigua pendland and boucias, 1986 megabalanus rosa muramoto and kamiya, 1990 periplaneta americana jomori and natori, 1991 calliphora vomitoria mckenzie and preston, 1992 pacifastacus leniusculus kopáček et al., 1993 tachypleus tredenatus saito et al., 1997 tachypleus tredenatus kawabata and iwanaga, 1999 pinellia ternata yao et al., 2003 anopheles gambiae pace and baum, 2004 drosophila melanogaster pace and baum, 2004 echinodermata anthocidaris crassispina giga et al., 1985 anthocidaris crassispina giga et al., 1987 anthocidaris crassispina ozeki et al., 1991 holothuria polii canicattì and rizzi, 1991 asterina pectinifera kamiya et al., 1992 paracentrotus lividus canicattì et al., 1992 paracentrotus lividus drago et al., 2009 stichopus japonicus hatakeyama et al., 1993 stichopus japonicus himeshima et al., 1994 2 table 1 (continue) examples of lectins described in invertebrates taxon species reference echinodermata stichopus japonicus matsui et al., 1994 cucumaria echinata hatakeyama et al., 1994 cucumaria japonica bulgakov et al., 2000 strongylocentrotus purpuratus hibino et al., 2006 holothuria scabra gowda et al., 2008 tunicata botrylloides leachii coombe et al., 1982 didemnum candidum vasta et al., 1986 phallusia mamillata parrinello and arizza, 1989 styela clava kelly et al., 1992 clavelina picta elola and vasta, 1994 clavelina picta vasta et al., 1999 botryllus schlosseri ballarin et al., 1999 botryllus schlosseri gasparini et al., 2008 halocynthia roretzi sekine et al., 2001 pyura stolonifera pearce et al., 2001 ciona intestinalis azumi et al., 2003 ciona intestinalis parrinello et al., 2007 ciona intestinalis bonura et al., 2009 trafficking, protein folding, signal transduction, fertilization, development and self/non-self discrimination (vasta et al., 2004). with regards the structural composition, at least seven families have been identified in animals on the basis of the carbohydrate-recognition domain (crd): 1. p-type lectins; 2. s-type lectins; 3. c-type lectins; 4. pentraxins (sharon, 1993; drickamer and taylor, 1993); 5. i-type lectins (gabius, 1997; angata et al., 2002); 6. fucolectins (bianchet et al., 2002); 7. rhamnose-binding lectins (jimbo et al., 2007; terada et al., 2007). further studies have revealed others, e.g., galectins (formerly included among stype lectins) (barondes et al., 1994), calnexin, calreticulin (trombetta and helenius, 1998; parodi, 2000), collectins, ficolins (lu et al., 2002), immulectins (ascribable to c-type lectins) (yu et al., 2002) and mannose-binding lectins (ascribable to ctype lectins) (ip et al., 2009). vasta and colleagues (2004) report that only some of the above mentioned lectins are present in invertebrates. the c-type crds have been reported in several invertebrates, such as the flesh fly sarchophaga peregrina (takahashi et al., 1985), the sea urchin anthocidaris crassispina (giga et al., 1987), the acorn barnacle megabalanus rosa (muramoto and kamiya, 1990), the tunicate polyandrocarpa misakiensis (suzuki et al., 1990), the horseshoe crab limulus polyphemus (muta et al., 1991), the cockroach periplaneta americana (jomori and natori, 1991), the sea urchins paracentrotus lividus (canicattì et al., 1992) and strongylocentrotus purpuratus (smith et al., 1996) and the sea cucumber stichopus japonicus (himeshima et al., 1994). pentraxins have been reported in the tunicates clavelina picta (elola and vasta, 1994), the horseshoe crabs l. polyphemus (amstrong et al., 1996) and tachypleus tridenatus (saito et al., 1997). galectins were found in the dipterans drosophila melanogaster and anopheles gambiae (pace and baum, 2004), the nematode caenorhabditis elegans (cooper and barondes, 1999) and the ascidian, clavelina picta (vasta et al., 1999). fucolectins have been retrieved in bivalves (yamaura et al., 2008) and rhamnose-binding lectins have been observed in bivalves (naganuma et al., 2006), echinoderms (ozeki et al., 1991) and tunicates (gasparini et al., 2008). if the increased availability of molecular information has improved our knowledge of invertebrate cytokines, this is also the case for lectins. for instance, two galectins have been characterized in c. elegans and a screening of the genbank database ten years ago retrieved 26 putative galectins (cooper and barondes, 1999). in the fruit fly d. melanogaster a galectin homologue (dmgal, genbank accession number af338142) has been identified (pace et al., 2002), and in the solitary ascidian ciona intestinalis nine collectin-like genes have been retrieved (azumi et al., 2003). collectin gene expression has been found to change after lps injection in c. intestinalis (bonura et al., 2009). in the colonial ascidian botryllus schlosseri ballarin and collaborators have identified 5 transcripts from a cdna library, each with a complete coding sequence homologous to known rhamnose-binding lectins (gasparini et al., 2008). in the fully sequenced genome of s. purpuratus were identified 104 genes that encode for small c-type lectins composed of one or two domains that can 3 table 2 list of the cytokines described in invertebrates, including dhf taxon species cytokine name reference arthropoda drosophila melanogaster spätzle morisato and anderson, 1994 drosophila melanogaster upd-3 agaisse et al., 2003 drosophila melanogaster dhf malagoli et al., 2007 pseudaletia separata hcp nakatogawa et al., 2009 pacifastacus leniusculus astakine söderhäll et al., 2005 bind a wide range of oligosaccharide (hibino et al., 2006). one of these c-type lectins called spechinoidin was well characterized and it was shown a possible function in the immune defense of the sea urchin because its expression is exclusively in the phagocytes after lps-challenge (multerer and smith, 2004; terwilliger et al., 2004). molecular recognition is carried out by lectins through specific carbohydrate binding motifs. the carbohydrates exhibit several folds corresponding to different carbohydrate-binding motifs (vijayan and chandra, 1999). according to vasta and colleagues (1994), c-type lectins and pentraxins play an important role in innate immune functions, since they are probably the most ancient non-self recognition/defense mechanism. recently, a large number of c-type lectin domain-(ctld) containing proteins has been reported in c. elegans, many of which show a pathogen-specific response during infection (schulenburg et al., 2008). among the ctype lectins, mannose-binding lectins (mbl) deserve particular attention. indeed, mbl are involved in innate immune protection and work with epithelial barriers, cellular defenses such as phagocytosis (they can act as opsonins), and pattern-recognition receptors that trigger pro-inflammatory signalling cascades. in particular, ip and colleagues (2009) have found that mbl play a role as a co-receptor of toll-like receptors (tlrs), since they are linked by their spatial localization on the phagosome. furthermore, a novel involvement of mbl as a tlr co-receptor has been found, and a new paradigm for the role of these opsonins has been defined: mbl may function not only to increase microbial uptake but also to coordinate spatially, amplify, and synchronize innate immune defense mechanisms. chemical analysis and immunocytochemical reactions have demonstrated the presence of nacetylmuramic acid (nam) and the absence of sialic acid in the glycoconjugates in different tissue from mollusca gastropoda (bolognani et al., 1981; ottaviani and montagnani, 1989; bolognani fantin and ottaviani, 1990; ottaviani et al., 1990). accordingly, nam has also been found in the carbohydrate fraction of a lectin present in the freshwater snail, planorbarius corneus (ottaviani and tarugi, 1986). cytokines as far as cytokines in invertebrates are concerned, several authors have reported the presence of cytokine-like molecules in molluscs, insects, annelids, echinoderms and tunicates. together with morphological evidence, functional experiments have also suggested the presence of invertebrate cytokines that are homologues to those in mammals. indeed, several mammalian cytokines, e.g., il-2, il-8 and growth factors, stimulate cell motility, chemotaxis, phagocytosis, cytotoxicity, stress response, wound repair and the regulation of cell death in invertebrate immune cells (ottaviani et al., 2004). most of these findings were recorded in the 1980s and 1990s and principally concerned il-like molecules. however, the scientific community was sceptical about the existence of invertebrate homologues of vertebrate interleukins, given the absence of experimental evidence for the presence of a real gene similarity. since the immune molecules, celomic cytolitic factor (ccf), from the annelid eisenia foetida and human tumor necrosis factor (tnf)-α present a relevant functional similarity as a result of a shared lectin-like activity, beschin and colleagues (2001) surmized that the evidence of invertebrate immune molecules that were hypothetically homologous to vertebrate cytokines was essentially the consequence of a functional convergence on a lectin-like activity by both the invertebrate immune factors and the vertebrate cytokines. in other words, the elusive, invertebrate cytokine-like immune factors were suggested to be lectins or, alternatively, molecules endowed with lectin-like activity, whose effects were similar to those displayed by some vertebrate cytokines (beschin, 1999; beschin et al., 2001, cebo et al., 2002). this hypothesis was reinforced not only by the data on ccf, but also by the absence of molecular evidence (immunoblot or pcr-derived data (beschin et al., 2004) supporting the existence of invertebrate cytokines. a drawback of this analysis is, however, the extreme variability of the cytokine sequences, especially of interleukins, that makes the application of a typical sequence-based algorithm to find cytokine gene homologues almost impossible (huising et al., 2006). molecular biology and functional studies have demonstrated the presence of cytokines in invertebrates: spätzle (morisoto and anderson, 1994) and upd3 (harrison et al., 1998; agaisse et al., 2003) in d. melanogaster, hemocyte chemotactic peptide (hcp) from the moth pseudaletia separata (nakatogawa et al., 2009) and 4 fig. 1 distribution of helical motifs in the preprotein form of dhf (a) and a mammalian helical cytokine (b). the name of the helices (a to d) and their position between the first methionine (m) and the last aminoacid (stop) are given accordingly to conklin (2004) and conklin et al. (2005). the extension of the signal peptide (signal) is also reported. boxes of similar colors (e.g., azure bars and solid azure) indicate correspondent helices with unrelated amino acidic sequences. astakine 1 in the freshwater crayfish pacifastacus leniusculus (söderhäll et al., 2005). however, there is no indication of gene homology or structure similarity between these molecules and cytokines in vertebrate species. more precisely, the conformation of spätzle resembles that of vertebrate ngf and coagulogen in the horseshoe crab (mizuguchi et al., 1998). upd-3 and hcp has no homology or similarity with vertebrate cytokines and immune molecules while astakine 1 possesses a prokineticin (pk) domain found in vertebrates in molecules with many different functions, including angiogenesis and spermatogenesis (söderhäll et al., 2005). while the cited invertebrate cytokines show little or no conservations with their functional counterparts in vertebrates, signal transduction pathways appear to be well conserved. spätzle activates toll signalling that is considered to share significant similarity with the pathway activated by il-1 in mammals (lemaitre and hoffmann, 2007), while the hemocyte-derived upd-3 activates the jak/stat pathway in the fat body (agaisse and perrimon, 2004). in terms of function, the gene spätzle encodes for a secreted protein that requires proteolytic processing for activity (morisato and anderson, 1994). the protein spätzle acts immediately upstream of the receptor toll. spätzle mutant flies can recover the inducibility of drosomycin after injection of either recombinant full-lengh spätzle or hemolymph from wild-type flies. however, the recovery of drosomycin induction is always subsequent to an immune challenge with mycetes or gram positive bacteria. these results demonstrate that spätzle is a cytokine present in the hemolymph as an inactive precursor which is converted to its active form in response to infections (ferrandon et al., 2004). upd3 has been characterized as a member of the unpaired (upd) family that activates the jak/stat pathway during the embryogenesis of drosophila (harrison et al., 1998). upd3 is secreted by hemocytes and subsequently activates tota expression in the fat body (agaisse and perrimon, 2004). rna interference data suggest that upd-3 is not induced by activation of the imd-pathway, or at least not by the branch controlled by the kinase dtak1 (malagoli et al., 2008). hcp is a chemotactic factor that displays several characteristics of a mammalian chemokine. it is a small secreted peptide, present in epidermis, granulocytes and nervous system of the larvae of the lepidopteron p. separata. hcp displays a strong chemotactic activity and recruits circulating hemocytes to the wound where it is supposed to enhance clotting. at present no information is available on the receptor bound by hcp and on the signalling pathway activated by this insect chemokine (nakatogawa et al., 2009). finally, astakine 1 induces a strong hematopoietic response by interacting with a f1atp synthase receptor present exclusively on the hemopoietic tissue and not on the surface of circulating hemocytes (lin et al., 2009). a similar molecule, astakine 2, has been identified in another crustacean, the shrimp penaeus monodon, but there is still scant information on this finding (söderhall et al., 2005). the discovery of the above mentioned cytokines has contributed to the general acceptance of the existence of cytokines in invertebrates; however, the findings offer little help in understanding whether homologues for vertebrate interleukins can be retrieved in invertebrate models. a significant advance in this field was the discovery of the first gene predicted to encode for a helical cytokine in d. melanogaster labelled dhf (drosophila helical factor) (malagoli et al., 2007). dhf was recorded following the utilization of an algorithm (qt method) specifically developed to scan protein and cdna databases and recognize sequences encoding for helical cytokines (conklin, 2004). in vertebrates, helical cytokines represent one structural class of cytokines that include il-2, il-6, il-11, il-23, interferon α-1 and gm-csf (granulocyte-macrophage colony-stimulating factor). as mentioned above, helical cytokines are a divergent protein family, and their phylogenic relationship arises from the conserved protein structure, intron phases and broadly similar receptor families (conklin et al., 2005). accordingly, no sequences similar to that of dhf have been retrieved in databases of other insects such as a. gambiae and apis mellifera. 5 dhf (genpept accession no. aaf53861) is a peptide of 214 amino acids, and the qt method predicts that this sequence has a helical cytokine fold with 4 core amphipathic helices (fig. 1). functional experiments demonstrate that dhf expression is significantly increased after immune stimulation, suggesting the involvement of this putative helical cytokine in the innate immune response of invertebrates (malagoli et al., 2007, 2008). furthermore, using the anti-rdhf antibody, the macrophage-like drosophila embryonic hemocytes (sl2 cell line) have been found to promote the secretion of dhf following exposure to heat-inactivated bacteria and after the administration of the recombinant peptide rdhf (malagoli et al., submitted). although present data do not allow us to conclude that dhf is a homologue of vertebrate helical cytokines, the results do point to the first invertebrate candidate that could prove of some help in describing the evolution of one of the major classes of immune-related molecules. concluding remarks the major new insight into the invertebrate immunological system suggested by the above data is that, as in vertebrates, both lectins and cytokines are involved in the chemical communication among immunocompetent cells. the functions fit into the same framework, indeed these molecules share convergent and common functions, and one of the major roles of these messenger molecules is their participation in fighting against non-self. even though these conclusions may appear limited, this is a synopsis of what we know about lectins and cytokines in invertebrates. these two classes of molecules have been studied with a quite different attitude in the last 25 years. lectins have been essentially characterized from a functional and biochemical point of view. this has led to the identification of a plethora of factors, all indicated as lectins, among which it is quite difficult to find a starting point for an evolutionary analysis. conversely, considerable attention has been paid to cytokines for purposes of molecular characterization. the continuing search for elements of conservation between invertebrate and vertebrate mediators has moved towards sequence and domain analysis as a first step, maintaining the functional characterization as a necessary but not sufficient task. as such, we can say that currently only three cytokines, i.e., spätzle, upd-3 and astakine 1, are known in invertebrates. dhf is a likely further candidate, but it has still to be considered as a putative cytokine as a consequence of the sceptical attitude mentioned before. finally, we have not referred to other cytokine-like molecules that have been found in recent years in different invertebrate taxa. in absence of the required molecular, structural and functional characterization, the respective discoverers propose these as cytokine-like molecules. in conclusion, we would say that in the case of lectins, the adopted perspective has allowed the identification of an enormous number of family members, while the approach to cytokines has produced only three accepted members in the last 25 years. overall and for opposite reasons, present knowledge of both lectins and cytokines must be considered inadequate for evolutionary studies. acknowledgements we gratefully acknowledge prof l ballarin (university of padua, italy) for helpful discussion on 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zool. sci. 7: 581–591, 1990. yu xq, zhu yf, ma c, fabrick ja, kanost mr. pattern recognition proteins in manduca sexta plasma. insect biochem. mol. biol. 32: 12871293, 2002. 10 isj 5: 179-yyy, 2008 issn 1824-307x isj 5: 180-189, 2008 issn 1824-307x research report lipase and invertase activities in midgut and salivary glands of chilo suppressalis (walker) (lepidoptera, pyralidae), rice striped stem borer a zibaee, ar bandani, s ramzi plant protection department, faculty of agriculture, university of tehran, karaj 31584, iran accepted november 4, 2008 abstract the rice striped stem borer, chilo supprressalis, was introduced to iran in 1973 where it is now widely distributed and causes severe damages. lipases, which catalyses the hydrolysis of fatty acid ester bonds, are widely distributed among animals, plants and microorganisms. invertases (βfructofuranosidase) are glycosidehydrolases that catalyze the cleavage of sucrose (β-dglucopyranosyl-s-d-fructofuranoside) into the monosaccharides glucose and fructose. laboratoryreared 4th instar larvae were randomly selected, their midgut and salivary glands were removed by dissection under a light microscope and lipase and invertase activities were assayed. the activity of lipase/invertase in the midgut and salivary gland were 0.49/0.27 and 0.35/0.23 µmol/min/mg protein, respectively. the optimum ph and temperature for both the two enzymes were determined to be 1011 and 37-40 °c, which is consistent with ph and temperature values already observed in lepidoptera. the enzyme activity was reduced by addition of nacl, kcl, mgcl2, sds, urea and plant extracts from artemisia annua, but not by cacl2 which enhanced enzyme activity. pest control with usage of resistant varieties of plants is one of the most important practices that are dependant on inhibitors already present in nature. hence, characterization of insect digestive enzymes, especially examination of inhibition effects on enzyme activity, could be useful in developing new strategies for pest control. key words: α-amylase; rice striped stem borer; midgut; salivary glands introduction the rice striped stem borer (chilo supprressalis, walker) is a cosmopolitan and destructive pest in rice fields of the world (zibaee et al., 2008). this pest was introduced in iran in 1973 and since then has been widely distributed in the country rice fields. it causes severe damages in all rice fields and its present density is superior than the economic injury level (eil) (dezfoulian and moustofipoor, 1972). the chemical control by using organophosphorus compounds, has been a common control procedure, although other methods based on agricultural practices such as ploughing, usage of resistant varieties of plants, weed control as overwintering sites and biological control with trichogramma spp. have been incorporated. in recent ___________________________________________________________________________ corresponding author: ali reza bandani plant protection department college of agriculture and natural resources university of tehran, karaj, 31584 iran e-mail: abandani@ut.ac.ir years, resistant varieties and pheromones also have been added to control the diffusion of c. suppressalis like in other places of the world (muralidharan and pasaalu, 2006). a study on 78 different varieties of rice showed that binam with 15 % white head is the most resistant variety. germplasts studies showed that khazar variety is resistant to the first generation of rice striped stem borer. however, it is susceptible to the second generation. lipases (triacylglycerol–acyl-hydrolase ec 3.1.1.3), which catalyzes the hydrolysis of fatty acid ester bonds, are widely distributed among animals, plants and microorganisms (naumff, 2001). it has been showed that lipases can also hydrolyze a variety of esters in organic solvent systems and thus they can be widely used in many industrial areas, e.g., dairy, food, detergent and biofuel industries (ishaaya and swirski, 1970; henrissat and bairoch, 1993; grillo et al., 2007). the most characteristic property of lipases is that they act on substrate at the interface between the aqueous and the lipid phase (grillo et al., 2007). 180 to date, many research groups have carried out the isolation and purification of lipases from various sources, mainly microorganisms, fish, fungi, milk and plants (cherry and crandall, 1932; henrissat and bairoch, 1993; degerli and akpinar 2002; grillo et al., 2007). however, lipid biochemistry studies in insects is time-consuming and moved on very slowly due to high diversity of insects and changes in the lipid composition and lipophorin present in hemolymph during metamorphosis from larva to pupa (degerli and akpinar, 2002). recently, lipid mobilization and transport in insects is under investigation, especially lipases and lipophorin (a reusable lipoprotein particle in insect systems) because of their roles in energy production and transport of lipids at flying activity (ayre, 1967). although stored lipids in vertebrate adipose tissue are released as free fatty acids, in insects most fatty acids are released as 1,2-diacylglycerols and mobilization of lipid reserves from insect fat body is under the control of adipokinetic hormone (grillo et al., 2007). invertases (β-fructofuranosidase, ec3.2.1.26) also termed fructosidase, saccharase, or sucrase, are glycosidehydrolases (ec 3.2.1) that catalyze the cleavage of sucrose (β-d-glucopyranosyl-s-dfructofuranoside) into the monosaccharides, glucose and fructose (henrissat and bairoch, 1993; sturm and tang, 1999; naumoff, 2001). invertase, thus, appears to be a particularly important enzyme for plants and animals. given this general importance, a surprisingly limited number of studies have tried to quantify invertase activity in ants (ayre, 1963, 1967; ricks and vinson, 1972) or other animals (martinez del rio, 1990; zhang et al., 1993). this might be due to the particular methodological problems arising from the quantification of invertase in animals whose carbohydrate metabolism is highly active. digestion is a phase of insect physiology on which little research has been performed, despite the economic importance of the food of insects and the fact that the most important control measures involve the action of digestive juices on poisons taken into the digestive tract. a better understanding of enzyme catalysis is essential in order to develop methods of insect control (bandani et al., 2001; ghoshal et al., 2001; maqbool et al., 2001). the purpose of the present study is to identify and characterize the lipase and invertase activities from midgut and salivary glands (sg) of rice striped stem borer larvae to gain a better understanding of the digestive physiology. this understanding will hopefully lead to new management strategies for control of this pest. materials and methods insects to decrease the side effects of laboratory mass culture, 400 pupae were collected from fields and reared on the same variety seedling (taroum) as sampling sites. insects were reared based on the method mentioned by kammano and sato (1985) in 28 ± 1 °c, light cycle 16l:8d and rh > 80 %. when the larvae grow up to 4th instar larvae, 30 larvae were randomly selected for biochemical analysis. for 4th instar determination, dayer's formula was used which had been described by majidi et al. (2002). sample preparation and enzyme assays briefly, larvae were randomly selected and total midgut and sg were removed by dissection under a stereo microscope in ice-cold saline buffer (6 μm nacl). the midgut and sg were separated from the insect’s body, rinsed in ice-cold buffer, placed in a pre-cooled homogenizer and ground in 1 ml of universal buffer containing succinate, glycine and 2morpholinoethanesulfonic acid (ph 7.2) (hosseinkhani and nemat-gorgani, 2003). the homogenates from both preparations (midgut and sg) were separately transferred to 1.5 ml centrifuge tubes and centrifuged at 20,000 x g for 20 min at 4 °c. the supernatants were pooled and stored at -20 °c for subsequent analyses. lipase activity the enzyme assays were carried out as described by tsujita et al. (1989). thirty µl of gut and salivary glands tissue extracts and 100 µl of pnitrophenyl butyrate (50 mm), as substrate, were incorporated, mixed thoroughly and incubated at 37 °c. for negative control tubes, samples (midgut and salivary glands) were placed in a boiling water bath for 15 min to destroy the enzyme activity and then cooled. after 1 min, 100 µl distilled water were added to each tube (control and treatment) and absorbance was read at 405 nm. one unit of enzyme 0 0.1 0.2 0.3 0.4 0.5 0.6 midgut salivary glands a ct iv ity o f l ip as e (µ m ol /m in /m g pr ot ei n) 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 midgut salivary glandsa ct iv ity o f i nv er ta se ( µm ol /m in /m g pr ot ei n) fig. 1 activity level of lipase (up) and invertase (down) in 4th instar larvae midgut and salivary glands of rice striped stem borer. 181 http://www.citeulike.org/user/biblio24/author/tsujita y = 0.0091x + 0.0556 r2 = 0.9693 0 0.1 0.2 0.3 0.4 0.5 0.6 0 10 20 30 40 50 concentration of p-nitrophenol a b so rb an ce a t 40 60 5 n m y = 0.2011x + 0.008 r2 = 0.9922 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0 0.5 1 1.5 2 2.5 3 3. concentration of glucose a b so rb an ce a t 34 0 nm 5 fig. 2 standard calibration curve for the determination of p-nitrophenol and glucose released in the lipase (up) and invertase (down) assay. will release 1.0 nmol of p-nitrophenol per min at ph 7.2 at 37 °c using p-nitrophenyl butyrate as substrate. standard curve was used to calculate the specific activity of enzyme. invertase activity samples were transferred to 100 ml flasks and 1 ml toluene was added to arrest the enzyme activity. after 15 min, 6 ml of 0.2 m glycine buffer (ph 7.2) containing 18 mm sucrose was added to the samples and the flasks were closed with cotton plugs then held for 24 h at 30 °c. samples were passed through whatman filter paper and glucose in the filtrate was assayed at 340 nm (nelson, 1994). kinetic parameters measurements twenty µl of appropriately diluted enzyme preparation was used in each assay. final concentrations for substrate were 20, 30, 40, 50 and 60 mm for lipase and 0.09, 0.18, 0.36, 0.72 and 1.54 mm for invertase, respectively. the michaelis constant (km) and the maximum velocity (vmax) were estimated by sigmaplot software version 11 (systat software inc., chicago, il, usa) and the results of km and vmax were the means ± se of three replicates for every population. effect of ph and temperature on enzyme activity the effect of temperature and ph on lipase and invertase activity were examined using enzyme extractions from the larval midgut and sg. the effect of temperature on enzymes activity was determined by incubating the reaction mixture at 20, 25, 30, 35, 37, 40, 45, 50, 55, 60 and 70 °c for 24 h, followed by measurement of activity. optimal ph for their activities was determined using universal buffer with ph set at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13. effect of activators and inhibitors on enzyme activity to test the effect of different ions on the enzymes, midguts and sg were dissected in distilled water. enzyme assays were performed in the presence of different concentrations of chloride salts of na+ (5, 10, 20 and 40 mm), k+ (5, 10, 20 and 40 mm), ca2+ (5, 10, 20 and 40 mm), mg2+ (5, 10, 20 and 40 mm), and ethylenediaminetetraacetic acid (edta; 0.5, 1, 2 and 4 mm), sodium dodecylsulfate (sds; 1, 2 and 4 mm), urea (0.5, 1, 2, 4, 6 and 8 mm) and artemisia annua extract (10, 15 and 25 % concentrations). these compounds were added to the assay mixture, and activity was measured after 30 min incubation. a control was also measured (no compounds added). effect of a. annua extract on enzymatic parameters of invertase and lipase for this experiment, 20 µl of appropriately diluted enzyme preparation was used in each assay. final concentrations for substrate were 20, 30, 40, 50 and 60 mm for lipase and 0.09, 0.18, 0.36, 0.72 and 1.54 mm for invertase, respectively. finally, 20 % of plant extract added to each well. km and vmax were estimated by sigmaplot software version 11 (systat software inc.) and the results of 0 0.2 0.4 0.6 0.8 1 3 4 5 6 7 8 9 10 11 12 13 ph a ct iv ity o f l ip as e (µ m ol /m in /m g pr ot ei n ) midgut salivary glands 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 3 4 5 6 7 8 9 10 11 12 ph in ve rt as e ac tiv ity (µ m ol /m in /m g pr ot ei n ) midgut salivary glands fig. 3 effect of ph on activity of lipase (up) and invertase (down) extracted from midgut and salivary gland of rice striped stem borer. 182 0 0.1 0.2 0.3 0.4 0.5 0.6 20 30 35 37 40 45 50 55 60 70 tem perature (°c) a ct iv it y o f l ip as e (µ m o l/m in /m g p ro te in ) midgut salivary glands results lipase and invertase activities studies showed that lipase and invertase are present in the midgut and salivary glands of adult c. suppressalis (fig. 1). the activity of lipase was 0.486 µmol/min/mg protein and 0.27 µmol/min/mg protein in midgut and sg, respectively. the invertase activity in midgut and sg was 0.35 and 0.23 µmol/min/mg protein, respectively. there was a significant difference in the degree of enzyme activity between midgut and salivary glands (figs 1, 2). effect of ph and temperature on enzyme activity the in vitro evaluation of c. suppressalis lipase and invertase from midgut and sg indicated that enzyme activity increased steadily from ph 3 to 11 and from 3 to 10, respectively. after reaching the threshold ph level, enzyme activity decreased with the increasing of ph and there were significant differences among measured values for each ph (fig. 3). both enzymes were considerably active over a broad range of temperatures. as the results show, the optimum temperature for lipase and invertase activities were 37 and 40 °c for midgut and sg, respectively (fig. 4). -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 20 30 35 37 40 45 50 55 60 70 tem perature (° c) a ct iv it y o f in ve rt as e (µ m o l/m in /m g p ro te in ) midgut salivary glands effect of activators and inhibitors on enzyme activity several molecules and chemical compunds affects the activity of lipase and invertase in midgut and sg of rice striped stem borer, although they had a similar effect on both enzymes (tables 1 and 2). activity level of enzymes in midgut and sg elevated due to increasing of cacl2 and edta concentrations for lipase and just cacl2 for invertase (table 1 and 2). activity level of enzyme decreased in presence of nacl, kcl, edta, mgcl2, sds, urea and a. annua extract in both midgut and sg (tables 1 and 2). fig. 4 effect of temperature on activity of lipase (up) and invertase (down) extracted from midgut and salivary gland of rice striped stem borer. km and vmax were the means ± se of three replicates for every population. for determination of a. annua extract effect on enzymatic parameters of lipase and invertase, 20 % of plant extract was added to each well. kinetic parameters as can be seen in table 3, lipase vmax of midgut and sg were 0.5 and 0.35 µmol/min/mg protein, respectively. lipase km of midgut and sg were 15 and 19 mm, respectively. invertase vmax was 0.9 and 0.5 µmol/min/mg protein in midgut and sg, respectively, while invertase km was 0.31 and 0.39 mm in midgut and sg (table 3, fig. 5). polyacrylamide gel electrophoresis (page) in order to determine the molecular mass of native lipase, native polyacrylamide disc-gel electrophoresis was carried out using the method of parish and marchalonis (1970) using 2.7 % and 7.7 % polyacrylamide for the stacking and resolving gels, respectively. the gel was stained with 1.5 % (w/v) coomassie brilliant blue g-250 and distained in glacial acetic acid-methanol-water 7.5: 5.0: 87.5. effect of a. annua extract on enzymatic parameters of invertase and lipase enzymes parameters changed due to using of a. annua extract. as it is shown in table 4, lipase kinetic parameter, vmax-km were 0.35 µmol/min/mg protein-37.5 mm in midgut and 2.12 µmol/min/mg protein-21 mm in sg. as well as invertase is concerned, vmax-km were 0.49 µmol/min/mg protein0.30 mm in midgut and 0.81 µmol/min/mg protein0.92 mm in sg (table 4, fig. 6). protein determination protein concentration was measured according to the method of bradford (1976), using bovine serum albumin (bio-rad, münchen, germany) as a standard. statistical analysis native page data were compared by one-way analysis of variance (anova) followed by tukey's studentized test when significant differences were found at p=0.05. enzyme kinetic parameters were analyzed by using the sigmaplot software version 11 (systat software inc.). analysis of midgut and sg lipase and invertase enzyme from homogenates of c. suppressalis by vertical slab electrophoresis on 8 % polyacrylamide gels indicated one band in all samples except for midgut's lipase (fig. 7). 183 table 1 relative activity of c. suppressalis lipase toward different compounds compounds concentration (mmol/l) relative activity (midgut) relative activity (salivary gland) control 100 100 nacl 5 102.77* 189.47* 10 77.77* 157.9* 20 33.33* 89.47* 40 6.38* 52.63* cacl2 5 50* 57.89* 10 77.77* 121.05* 20 88.88* 173.68* 40 119.44* 210.52* kcl 5 97.3* 100* 10 76.54* 87.36* 20 55.89* 68.66* 40 32.77* 45.25* mgcl2 5 247.22* 421.05* 10 208.33* 363.15* 20 91.66* 236.84* 40 44.44* 131.57* edta 0.5 38.88* 48.84* 1 72.22* 73.68* 2 105.55* 157.89* 4 231.55* 207.89* sds 2 113.88* 194.73* 4 80.55* 121.05* 6 44.44* 52.63* 8 26.66* 45.26* urea 1000 116.66* 205.23* 2000 102.77* 142.1* 4000 69.44* 115.78* 5000 27.77* 89.47* 6000 0.036* 31.57* plant extract 10 % 78* 86* 15 % 59* 63* 25% 34* 39* *p < 0.05 vs control 184 table 2 relative activity of c. suppressalis invertase in presence different compounds compounds concentration (mmol/l) relative activity (midgut) relative activity (salivary gland) control 100 100 nacl 5 102.77* 189.47* 10 77.77* 157.9* 20 33.33* 89.47* 40 6.38* 52.63* cacl2 5 50* 57.89* 10 77.77* 121.05* 20 88.88* 173.68* 40 119.44* 210.52* kcl 5 97.3* 100* 10 76.54* 87.36* 20 55.89* 68.66* 40 32.77* 45.25* mgcl2 5 247.22* 421.05* 10 208.33* 363.15* 20 91.66* 236.84* 40 44.44* 131.57* edta 0.5 38.88* 48.84* 1 72.22* 73.68* 2 105.55* 157.89* 4 231.55* 207.89* sds 2 113.88* 194.73* 4 80.55* 121.05* 6 44.44* 52.63* 8 26.66* 45.26* urea 1000 116.66* 205.23* 2000 102.77* 142.1* 4000 69.44* 115.78* 5000 27.77* 89.47* 6000 0.036* 31.57* plant extract 10 % 78* 86* 15 % 59* 63* 25% 34* 39* *p < 0.05 vs control 185 lipase-midgut 1/s -0.05 0.00 0.05 0.10 0.15 1/ v 0 1 2 3 4 5 6 lipase-salivary glands 1/s -0.05 0.00 0.05 0.10 0.15 1/ v 0 2 4 6 8 10 invertase-salivary glands 1/s -4 -2 0 2 4 6 8 10 12 1/ v 1 2 3 4 5 6 7 8 invertase-midgut 1/s -4 -2 0 2 4 6 8 10 12 1/ v 0 1 2 3 4 5 6 fig. 5 lineweaver-burk plot (vmax and km) of lipase and invertase extracted from 4th instar larvae of rice striped stem borer. discussion the present study shows that the larvae of c. suppressalis present lipase and invertase activities both in the midgut and in the sg. reports concerning lipase characterization have been obtained from several species of insects. metcalf (1945) found amylase, protease, and lipase to be absent from the sg of the mosquito anopheles quadrimaculatus while he observed that invertase is present in both midgut and crop homogenates. fisk and shambaugh (1954) found no activity of lipase in the sg of a. quadrimaculatus. a total body lipase was partially purified from abdomen homogenate of gryllus campestris l. (orthoptera, gryllidae) (orscelik et al., 2007). current study showed that both lipase and invertase are present in c. suppressalis and that the optimal ph for both enzymes are in alkaline condition (around ph 10). optimal temperatures for both enzymes are 37-40 °c in midgut and sg, respectively. ishaaya and swirski (1970) demonstrated that the optimum ph and temperature for invertase activity in the hemipteron chrysomphalus aonidum are 5.5 and 30 °c, respectively. degleri and anuipor (2002) showed that the optimal ph of lipases activity in the teleost fish cyprinion macrostomus is 7.5. studies on lipase properties of yeasts revealed that optimum ph and temperature for lipases activity are 7.5-8.2 and 3040 °c, respectively (vakhlu and kour, 2006). the utilization of dietary lipids was studied in adult females of the blood-sucking bug rhodnius prolixus with the use of radiolabeled triacylglycerol (grillo et al., 2007). these researches indicates that lipase activity is affected by ph and shows an optimal activity at a ph of 7.0-7.5. the optimal ph generally reflects the ph of the environment in which the enzyme normally functions. one way in which ph affects reactions rates is by altering the charge state of the substrate or of the active site of the enzyme. extreme phs can also disrupt the hydrogen bonds 186 lipase-midgut 1/s .08 -0.06 -0.04 -0.02 0.00 0.02 0.04 0.06 0.08 0.10 0.12 1/ v 0 2 4 6 8 10 12 14 16 invertase-midgut 1/s .0 -0.5 0.0 0.5 1.0 1.5 2.0 1/ v 0.0 0.2 0.4 0.6 0.8 1.0 invertase-salivary glands 1/s -4 -2 0 2 4 6 8 10 12 1/ v 0 2 4 6 8 10 12 14 16 fig. 6 lineweaver-burk plot (vmax and km) of lipase and invertase extracted from 4th instar larvae of rice striped stem borer due to using a. annua extract. that hold the enzyme in its three-dimensional structure, denaturing the protein (zeng et al., 2000). biological reactions occur faster with increasing temperature up to the point of enzyme denaturation, above which temperature enzyme activity and the rate of the reaction decreases sharply (applebaum 1985; agblor et al., 1994; zeng et al., 2002). current study results showed that ca2+ ions have activatory effects on the lipase and invertase activities of rice striped stem borer. podolor and applebaum (1971) reported that ca2+ ions have activatory effects on the lipase and invertase activities of the coleopteron callosobruchus chinensis. these results showed that these enzymes are metalloproteins which require calcium for maximum activity. researches have shown a significant correlation between the activity level of digestive enzymes in the hemolymph and the midgut. saleem and shakoori (1987) showed that sublethal concentrations of pyrethroids decrease amylase activity in larval gut of the beetle tribolium castaneum. lee et al. (1994) showed that some insect growth regulators decreased the activity level of alpha-amylase and esterase in treated larvae of c. suppressalis. ascher and ishaaya (2004) showed that the activity level of this enzyme increased 30 % in the noctuid moth spodoptera littoralis treated with phentine acetate compared with control. shekari et al. (2008) suggest that its activity level decreases 24 h after treatment and sharply increases at 48 h. digestive enzyme inhibitors occur naturally in many food plants and are particularly abundant in cereals and legumes (franco et al., 2002). insects gain access to food sources when they evolve enzymes that are not affected by inhibitors present in the food source, and plants become resistant when they evolve inhibitors effective against these 187 table 3 kinetic parameters of lipase and invertase enzymes extracted from midgut and sg of 4th instar larvae of c. suppressalis midgut* sg* enzymes vmax (µmol/min/mg protein) km (mm) vmax (µmol/min/mg protein) km (mm) lipase 0.5 ± 0.06 15 ± 3.74 0.35 ± 0.09 19 ± 6.45 invertase 0.9 ± 0.036 0.31 ± 0.047 0.5 ± 0.078 0.39 ± 0.77 *means ± se, n = 3 table 4 kinetic parameters of lipase and invertase enzymes extracted from midgut and sg of 4th instar larvae of c. suppressalis after exposure to a.annua extract midgut* sg* enzymes vmax (µmol/min/mg protein) km (mm) vmax (µmol/min/mg protein) km (mm) lipase 0.35 ± 0.087 37.5 ± 15.24 2.12 ± 0.68 21 ± 5.8 invertase 0.49 ± 0.087 0.30 ± 0.063 0.81 ± 0.021 0.92 ± 0.77 *means ± se, n = 3 insect enzymes. when the action of digestive enzymes is inhibited, insect’s nutrition is impaired, growth and development are retarded and eventually death occurred due to starvation. genes encoding for digestive enzyme inhibitors have been used to make transgenic crops by gene transfer technology. in transgenic pea expressing the αamylase inhibitor, the expression of digestive enzyme inhibitors makes plants harmful to target insects and pests, interfering with their digestive and fig. 7 native-page gel electrophoresis of midgut and sg from c. suppressalis. absorption processes, whereas neither antinutritional nor toxic effects were observed in rats (pusztai et al., 1999). the primary reason for producing insect-resistant transgenic crops is to reduce the use of chemical pesticides, which by one side lowers production costs and in the meantime reduces the insecticide loads in the environment. making insect-resistant plants requires the characterization of α-amylase and other digestive enzymes of the target insect and the identification of suitable inhibitors from plants or other sources. in our opinion, the purification and characterization of more insect digestive enzymes will greatly facilitate the understanding of the mechanisms responsible for this selectivity and will help to design new and more specific strategies for insect control. acknowledgment this research was supported by a university of tehran grant. we are really appreciated mr belbasi and ramadzan-khani for their assistances. we have special thanks to three anonymous reviewers for their helpful comments that resulted in a significant improvement of the article. references agblor a, henerson hm, madrid fj. characterization of alpha-amylase and polygalacturonase from lygus spp. (heteroptera: miridae). food res. inter. 27: 321-326, 1994. applebaum s w. biochemistry of digestion. in: ga kerkut, ll gilbert (eds.). comprehensive insect physiology, biochemistry and pharmacology. vol.4: regulation, digestion, excretion. pergamon press., pp. 279-307, 1985. 188 ascher krs, ishaaya i, antifeeding and protease and amylase inhibiting activity of phentin acetate in spodoptera littoralis larvae, pestic. biochem. physiol. 75: 326-336, 2004. ayre gl. feeding behaviour and digestion in camponotus herculeanus (l.) 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these three have been found in all taxa investigated. spherulocytes and coagulocytes were only found in some taxa. adipohemocytes occur quite infrequent, oenocytoids have not been detected but may be restricted to specific stages of the moulting cycle. cystocytes described earlier seem to be a preparation artefact. granular hemocytes are the most frequent hemocytes followed by the plasmatocytes. in general, diplopoda have fewer hemocytes than chilopoda. the differences in the hematogram and the functions of hemocytes are discussed. key words: arthropoda; myriapoda; chilopoda; diplopoda; hemocytes; immune reactions; plasmatocytes; granular hemocytes; phenoloxidase introduction in arthropoda, hemocytes have numerous functions: they are responsible for hemolymph clotting after cuticle and epidermis rupture, wound healing, self recognition, general and specific immune response and opsonisation (e.g., gupta, 1986; millar and ratcliffe, 1989; xylander, 1992, 1994). they phagocytise smaller pathogens like bacteria or fungal spores and encapsulate larger ones like metazoan parasites or xenografts. hemocytes, furthermore, produce and store substances which may be discharged after infections such as antibacterial substances and elements of the phenoloxidase system, as well as antibacterial substances, lectins and hemolysins. all these functions which were discovered in other groups of arthropoda have also been found to occur in the myriapoda (xylander and nevermann, 1990, 1993; xylander, 1992, in press; xylander and bogusch, 1992, 1997; nevermann, 1996). in some arthropods hemocytes are, furthermore, responsible for production and storage of the respiratory pigments. the number of hemocyte types investigated differs according to the subtaxon of arthropoda. additionally, the techniques and procedures of preparation, e.g., obtaining of hemolymph, in vitropreparation, fixation and staining techniques have ___________________________________________________________________________ corresponding author: willi xylander senckenberg museum für naturkunde görlitz postfach 300 154, 02806 görlitz, germany e-mail: willi.xylander@senckenberg.de impact on the reactions of hemocytes; as a consequence, the characteristics on which the nomenclature of hemocytes was based became highly diverse. as a consequence, the nomenclature of hemocytes in arthropods, especially in insects, became more and more confusing over the 100 years since the investigations started using more than 50 termini, often for the same cell types. with the aim to clarify at least in part this confusing situation, jones (1962), and ratcliffe and price (1974), presented a new nomenclature applicable more or less to hemocytes of all arthropod taxa. also in myriapoda the situation was quite complicated. early light microscopic investigations by gregoire (1955), ravindranath (1973, 1977, 1981) and rajulu (1971) resulted in the description of 7 different hemocyte types (prehemocytes, granular hemocytes, plasmatocytes, cystocytes, spherulocytes, adipohemocytes and oenocytoids). transmission electron microscopy (tem) investigations have described hemocyte types of chilopods and diplopods directly taken from the haemolymph, after in vitro preparation and partly during immune defense reactions (nevermann et al., 1991, 1996; nevermann, 1996; nevermann and xylander, 1996, 2006; hilken et al., 2003). by using light-microscopy, xylander (1992), xylander and nevermann (1993), and xylander and bogusch (1997), gave further characteristics applicable to differentiate the haematocyte types of myriapods. the intention of this paper is to review the recent results on myriapod hemocytes and their nomenclature. 114 mailto:willi.xylander@senckenberg.de fig. 1 (a-e) hemocytes of chicobolus spp., pure haemolymph, unfixed, spread on glass slides for different periods, observed by phase contrast microscopy and (f-j) r. virgator hemocytes, spread on glass slides for 10 min, fixed and stained. (a) various hemocytes (p= plasmatocytes, gl = granular hemocytes of type i, gii = granular hemocytes of type ii, the arrowhead points to cell proliferations). (b) plasmatocyte and prehemocyte (pr) (10 min). (c) two plasmatocytes with few grana spreading (“star-type”). (d) plasmatocytes representing various spreading-types (above: “star-type” with pseudopodia, below: “fried-egg-type” without pseudopodia, 20 min). (e) hemocyte with large grana in close vicinity to two different granular hemocytes (gi and gii, the arrowhead indicates cell proliferations of gii ,10 min). (f) may-grünwald and giemsa staining observed by phase contrast. (g) may-grünwald and giemsa staining observed in bright field. the prehemocytes are burgundy-coloured , the plasma of plasmatocytes is greyish and that of granular hemocytes (gi and gii) violet. (h) sudan-black-staining. several granular hemocytes are stained dark (bright field). (i) giemsa-staining. the more translucent plasma clearly differentiate plasmatocytes from granular hemocytes. (j) may-grünwald and giemsa staining. the plasma of granular hemocytes of type ii (gii) is bluish, their nuclei are blue. granular hemocytes of type i (gi) are faint purple , plasmocytes (p) are deep purple. (dark field). bars: 50 µm (a, c-e); 25 µm (b, f-j). 115 material and methods animals animals of different species were investigated. description of collecting sites, rearing conditions and other species specific informations have already been given in detail (xylander and nevermann, 1990; xylander, 1992; nevermann, 1996; nevermann and xylander, 1996). in vitro and staining techniques as well as hemocyte counts were described by xylander and nevermann (2006). electron microscopy preparations hemocytes were obtained by opening the cuticle of living animals. they spread and thus became attached to different substrates (e.g., cellophane) kept in cold ringer-solutions prior to fixation. such samples were fixed in the cold for at least 2 h in a modified karnoffsky´s fixative or in 2.5 % glutaraldehyde and postfixed in 2 % (w/v) oso4 and dehydrated in acetone [see xylander and nevermann 2006 for further details of the procedures]. after oso4-fixation samples were subdivided and (a) embedded for transmission electron microscopy samples in araldite or (b) critically point dried and sputtered with gold for scanning electron microscopy. tem-investigations were mainly done on a zeiss em 9a, sem investigations on a jeol or cambridge stereoscan s4 se-microscope. results hemocyte types six hemocyte types were found by light and electron microscopy, cytochemical and in vitro techniques in the two diplopod and three chilopod taxa. three of these occur in all taxa investigated: prehemocytes, plasmatocytes and granular hemocytes. in a limited number of chilopoda investigated, spherulocytes, coagulocytes and discoid cells were described (xylander and nevermann, 2006). prehemocytes three to 10 % of the hemocytes in the diplopods and chilopods investigated are prehemocytes. they are the smallest cells of all hemocytes and spherical in shape (figs 1b, f, g; 2c, g). their nucleus is large compared to the volume of cell plasma and located centrally (fig. 2g). in vitro, these cells spread later than other cell types keeping their shape for several minutes. only a few small grana may be found in the plasma of these prehemocytes and their phenoloxidase activity is weak (xylander and bogusch 1997). plasmatocytes plasmatocytes are significantly larger than the prehemocytes (fig. 1b) with a mean diameter of 30 to 70 µm. this cell type is characterized by intense spreading (figs 1, 2). furthermore, they contain no or just a few grana (figs 3a, c, d). their plasma is greyish in phase contrast (figs 1a-d). after activation of hemocyte cultures for phenoloxidase staining and incubation with dopa (xylander and bogusch 1997) there is no or just little reaction in the plasmatocytes. the nucleus of spread plasmatocytes is located eccentrically (figs 1b, d) and it occurs larger than in any other hemocyte type. directly after obtaining the haemolymph plasmatocytes are spherical to spindle-shaped but flatten and attach to glass slides within 8 to 10 min (in an undiluted haemolymph sample) forming pseudopodia (figs 2a, b, d; 3b, e). in the diplopoda, two types of spreading were observed in this cell type resulting in different hemocyte morphology (xylander and nevermann 2006): a) the “fried-egg-type”. in this type of spreading cells flatten more or less homogeneously not forming many pseudopodia; plasmatocytes showing such spreading behaviour contain no or just a few grana (fig. 1d) b) the “star-type”. here many thin pseudopodia grow centrifugally during spreading; in plasmatocytes of this type normally several small grana occur which are bluish in phase contrast (fig. 1d). tem observations show that in the plasmatocytes of chilopoda electron translucent vacuoles with moderately electron dense filamentous material occur (nevermann et al., 1991; hilken et al., 2003). in plasmatocytes of the spirobolid diplopod rhapidostreptus virgator comparable vacuoles were found containing (figs 3a, c, d) which, however contain flocculent rather than filamentous material; this material is discharged after some time during vitro-incubation. the material seems to attract other cell types thereby becoming involved in more complex aggregation (the immune reaction); such attraction of other cellular components of the immune system (against non-self material) is called opsonisation effect. granular hemocytes granular hemocytes are the most numerous hemocytes in myriapoda (xylander and nevermann 2006). they contain numerous electron dense grana of variable size and shape and some vacuoles (figs 1f-j; 2c, e; 4; 5). granular hemocytes are medium sized after spreading (larger than prohemocytes but smaller than plasmatocytes) (figs 1f-j). all cells of this type are phenoloxidase active (xylander 1996, xylander and bogusch 1997, xylander and nevermann 1993, 2006). briefly, after obtaining the haemolymph, granular hemocytes are more or less spherical (figs 1i, j). then they start to flatten in vitro and attach to the substrates within 15 min (figs 1f, g). they can, however, be distinguished from plasmatocytes for more than 1 h in light microscopy by their significantly lower spreading capabilities. during spreading, granular hemocytes often develop a unidirectional process (xylander and nevermann, 2006). intermediate forms of granular hemocytes and plasmatocytes have been found in a few cases (fig. 3d) indicating that granular hemocytes (at least of type i, see below) may constitute a transformation product of plasmatocytes. in the two species of diplopods investigated (chicobolus spp. and r. virgator) two types of granular hemocytes could be differentiated. granular 116 fig. 2 hemocytes of r. virgator. (a) hemolymph preparation with various hemocytes (pure haemolymph, 20 min after puncture, phase contrast. p= plasmatocyte, gi and gii= granular hemocyte type i and type ii.). (b) various hemocytes in a hemolymph smears (pure hemolymph, 30 min, phase contrast). (c) overview of various hemocytes observed by tem. (d) hemocyte aggregation in vitro observed by scanning electron microscopy (sem). the hemocytes with short extensions on the cell surface are plasmatocytes. (e) granular hemocytes of type i and type ii observed by tem. the grana of type ii are larger, more spherical and form fewer pseudopodia than type i. (f) tem observation of a granular hemocyte forming pseudopodia with contact to the substrate (g= granulum). (g) tem photograph of a prehemocyte. few small grana (g), high numbers of free ribosomes, rer and the nucleus (nu) are visible. bars: 50 µm (a, b); 5 μm (c, e); 10 µm (d); 0.2 µm (f); 1 µm (g). 117 hemocytes of type i show significantly higher spreading capabilities (fig. 1a). they contain fewer and smaller grana (figs 2c, i; 4a) which occur blue or green in phase contrast microscopy. in transmission electron microscopy their grana show to be irregular in shape (figs 2c; 4 a-c). after spreading the grana of type i granular hemocytes are predominantly located at the periphery of the nucleus; so mostly the nucleus is clearly visible. phenoloxidase activity is moderate in granular hemocytes of type i (and significantly lower than in type ii) (xylander and bogusch, 1997; fig. 3 plasmatocytes of r. virgator fixed after in vitro incubation on gelatine with i-ringer. (a) tem observation of a prehemocyte (with grana, left) and plasmatocyte (right). (b) two plasmatocytes observed by sem. (c) plasmatocyte. peripheral in the cytoplasm only few grana are visible. in the centre of the cell vesicles filled with flocculent material have gathered. (er= rough endoplasmic reticulum, m= mitochondrion, nu= nucleus, *: glycogen like inclusions). (d) presumed transitory stage between plasmatocyte and granular hemocyte type i. the number of grana increase, but they are less frequent and electron dense as in granular hemocytes. (e) plasmatocyte observed by sem. bars: 1 µm (a, c, d); 5 μm (b, e). 118 fig. 4 granular type i hemocytes of r. virgator under different types of fixation. hemocytes in a and c were fixed after in vitro incubation, 60 min in wenning-ringer on cellophane. hemocytes in b and d were fixed after in vitro incubation 60 min in i-ringer on gelatine. (a) for comparison: granular hemocytes of type i and type ii (nu= nucleus). (b) granular hemocyte of type i. at the periphery of the cell there are rer-cisterns and in the centre there are vacuoles. one of the vacuoles is filled with material resembling glycogen-rosettes (*). (c) in gr i there are fewer vacuoles after incubation in wenning-ringer on cellophane. (d) in unstained tem-sections (no double staining with uranyl acetate and lead citrate) the grana occur to be subdivided into a more electron dense and a more translucent part. bars: 1 µm. 119 xylander and nevermann 2006). in unstained temsections the grana of this type occur to be “subdivided” into a more electron dense and a more translucent part; the more electron dense part is smaller and enclosed eccentrically by the more translucent one (fig. 4d) (see also xylander and nevermann, 2006). granular hemocytes of type ii occur even smaller after spreading than plasmatocytes and granular hemocytes of type i and they spread less intensively (figs 1a, f, g). their volume, however, is about the same of the other two types. in many cases granular hemocytes of type ii also form a unidirectional process. in phase contrast they appear yellowish mostly due to the higher number of grana (fig. 1a). these grana are larger and spherical (figs 2c, e; 4a; 5a, c-e). when located peripherally in the granular hemocytes these grana may bulge their surface (figs 5a, b, f). granular hemocytes of type ii react stronger than the other hemocytes after prophenoloxidase activation and substrate incubation and stain dark brown (xylander and bogusch, 1997, xylander and nevermann, 2006). spherulocytes a fourth hemocyte type has been described for the chilopods lithobius forficatus and scutigera coleoptrata by nevermann et al. (1991) and hilken et al. (2003): the spherulocyte. it is characterised by regularly shaped spherical grana, significantly lower spreading capability and – as a major characteristic for differentiation from other hemocyte types the complete lack of phenoloxidase inactivity. these cells tend to form a single cytoplasmic protrusion which never bears grana. although spherulocytes may form such protrusions, contain grana, spread just moderately and therefore resemble the granular hemocytes intermediate forms between spherulocytes and granular hemocytes or plasmatocytes have never been detected. up to now, spherulocytes were only found in the two species of chilopoda. in diplopoda, as well as in scolopendra cingulata, this cell type does not occur (xylander 1992; nevermann, 1996; xylander and nevermann, 2006). in spite of the similarities, xylander and nevermann (2006) considered the spherulocytes to be a distinct hemocyte type possibly restricted to a subtaxon of chilopoda. coagulocytes in haemolymph preparations from lithobius forficatus, nevermann et al. (1991) described zones with typical plasma coagulations and postulated the occurrence of a fifth extremely fragile hemocyte type, which immediately disintegrates in vitro, the coagulocyte. nevermann (1996) described disintegrating hemocytes when dropping haemolymph directly into a fixative. these hemocytes resembled plasmatocytes containing only few electron dense grana and vacuoles but were characterised by large vacuoles containing flocculent material (indicating the disintegration process). nevermann (1996), therefore, considered the coagulocytes found just to represent “stressed plasmatocytes”. more recently, xylander and nevermann (2006), however, considered the coagulocytes to be one of the “valid” hemocyte types of myriapoda. which, due to their immediate disintegration during the procedures of obtaining the haemolymph, is just very rarely found in hemocyte preparation. naked nuclei and isolated membranes (most possibly remnants of hemocytes after disintegration) were also found in hemocyte aggregations (“capsules”) surrounding xenografts in the diplopod rhapidospreptus virgator (xylander, unpublished). they could also be remnants of coagulocytes indicating that this hemocyte type is more widely distributed throughout the myriapoda than considered earlier and just hard to detect with the methods usually applied. discoid hemocyte nevermann (1996) described a sixth hemocyte type by tem studies characterised by peripheral circular bundles of microtubules for s. cingulata. these microtubules disintegrate when the hemocytes attach to the substrate and start to spread. then they become invisible. bundles of microtubules equivalently arranged were also described from granular hemocytes of scutigera coleoptrata (hilken et al., 2003). so peripheral bundles of microtubules arranged in circles may represent a general characteristic of native plasmatocytes and granular hemocytes in vivo. cystocytes ravindranath (1981) in an early review of hemocytes in myriapoda described cystocytes for diplopoda. subsequently nevermann et al. (1991) showed, however, that this description was due to a preparation artefact: such hemocytes were exclusively found in cell preparations after mechanical stress (e.g., after sucking hemocytes into the narrow slit between a microscopic slide and cover slip). therefore, cystocytes are considered not to be a “valid hemocyte type” for the myriapoda. adipohemocytes in very few cases cells containing large lipid grana were found circulating in the hemolymph (fig. 1e). such numerous lipid grana (stainable with sudan black) are typical for so called “adipohemocytes”. but such observations were not reproducible with other specimens of the same species. therefore, it seems probable that such cells originated from the subepidermal fat body and are set free when obtaining the haemolymph by puncture. therefore, they represent rather an artifact originating from preparation and than a genuine hemocyte type in myriapoda. oenocytoids during all investigations performed in our working group with various diplopod and chilopod species, oenocytoids were never observed. however, oenocytoids are considered to be involved in the moulting process and their occurrence circulating free in the haemolymph may be restricted to a short period prior to moulting (which is a rare event in mature specimens of the species of diplopoda and chilopoda investigated during our 120 fig. 5 granular type ii hemocytes of r. virgator. hemocytes in a, d and e after in vitro incubation in wenningsaline for 60 min on cellophane; b, c and f, after in vitro incubation in i-ringer on gelatine for 60 min. (a) granular hemocyte of type ii. tem image displaying that grana are mostly spherical and bulge the plasma membrane at different sites (arrowheads); nu = nucleus. (b) grana responsible for extension of the plasma membrane are visible also by sem. (c) a few grana are surrounded by rer (arrowheads). (d) cisternae of rer could frequently been found at the cell periphery (er). free ribosomes are also widely distributed in the cytoplasm. note that mitochondria (mi) with electron dense matrix and translucent cristae are visible. (e) golgi complexes (di) with electron dense grana were occasionally observed in vicinity to the nucleus (nu). (f) sem micrograph of gii. bars: 1 µm (a, c); 5 μm (b, f); 0.2 µm (d, e). 121 studies). in fact, during our investigations specimens in the moulting process (which are extraordinary sensitive to cuticle rupture and may die briefly after injury) were excluded. this may be the reason for the absence of oenocytoids in our samples. therefore, definitive conclusions regarding the existence of oenocytoids in chilopods and diplopods are not possible. differential hemocyte counts for two diplopod species, r. virgator and chicobolus spp., the percentage of different hemocyte types were determined (xylander and nevermann, 2006). the granular hemocytes of type i was most frequent comprising 38 and 39 % of all hemocytes, respectively. granular hemocytes of type ii represented 30 % of all hemocytes in r. virgator and 17 % in chicobolus. in r. virgator 27 % of the hemocytes were plasmatocytes and 39 % in chicobolus. in both species prehemocytes represented about 5 %. total hemocyte counts the total number of hemocytes per hemolymph volume varies extremely between chilopoda and diplopoda: in the species investigated centipedes had a tenfold higher numbers of hemocyte than millipedes. in lithobius forficatus xylander and nevermann (2006) found 45,000 hemocytes µl-1 hemolymph, in scolopendra cingulata 31,500 and in s. oraniensis about 50.000. in contrast, there were only 2,450 and 6,500 hemocytes µl-1 hemolymph, respectively, in the diplopods r. virgator and chicobolus spp. discussion hemocyte types – a comparison within and outside the myriapoda in the arthropods at least 9 different hemocytes types can be differentiated (table 1). within the three major taxa of myriapods (chilopoda, diplopoda and symphyla) investigated with regard to their hemocytes until now prehemocytes, plasmatocytes and granular hemocytes occur in all three. other types described so far seem to be restricted to specific subtaxa or have to be considered as preparation artefacts. with regard of their microscopic and submicroscopic morphology the hemocytes of myriapod correspond to that of other arthropods (see for review jones, 1962; ravindranath, 1974; bauchau, 1981; sherman, 1981; xylander, 1992). this similarity of characters even on the electron microscopic level allows to use the generalised nomenclature for arthropod hemocytes not only for pragmatical reasons but also due to comparative morphology and function. however, the hemocyte types of decapod crustaceans which are separated into those bearing many granules (“granular cells”) and those bearing few (“semi-granular cells”) (bauchau, 1981; xylander et al., 2003) “resist” the inclusion into this system up to now. so further investigations are necessary to clarify the homology of the different hemocyte types. xylander and nevermann (2006) considered at least 4 hemocyte types to belong to the ground pattern of arthropods: the prohemocytes, the plasmatocytes, the granular hemocytes and the cyanocytes, the latter responsible for production and storage of the respiratory pigments. after the evolution of terrestrial arthropods with trachea, respiratory pigments and the hemocytes producing them were reduced. subsequently, immune defense, wound closure and hematopoiesis represented the major tasks of the remaining hemocytes. total hemocyte counts the range of hemocytes number per volume is extremely variable reaching from 500 µl-1 in decapod crustaceans to 60,000 µl-1 in cockroaches (overview in xylander and nevermann, 2006). diplopoda are among those taxa with a low number table 1 a comparison of the presence of different hemocyte types (listed in the results section) within the main taxa of arthropoda. myriapoda species are singularly reported. modified from xylander and nevermann (2006). taxon ph pl gr sph coa disc adi cyst oen cyan rhapidostreptus virgator x x x ? ? chicobolus spp. x x x ? lithobius forficatus x x x x (x) ? scolopendra cingulata x x x x ? scutigera coleoptrata x x x x insecta x x x x x x x crustacea x x x x x xiphosura ? x x x scorpiones x x x x x x ? aranea x x x ? x x 122 table 2 total hemocyte counts (hemocytes µl-1) [h (hc/µl)] for various arthropods. modified from xylander and nevermann (2006). myriapoda h (hc/µl) references rhapidostreptus virgator 2,500 xylander and nevermann, 2006 chicobolus spp. 6,500 xylander and nevermann, 2006 scolopendra cingulata 31,000 nevermann, 1996 scolopendra oraniensis ~50,000 xylander and nevermann, 2006 lithobius forficatus 45,000 nevermann, 1996 crustacea astacus leptodactylus ~500 ullrich, 1993; xilander et al., 1997 procambarus clarkii ~580 ullrich, 1993; xilander et al., 1997 crangon crangon 800-1,200 smith and johnston, 1992 insecta blatella germanica 23,000 gupta, 1986 periplaneta americana 60,000 crossley, 1975 chironomus thummi 1,000-3,000 götz and vey, 1974 drosophila melanogaster (2d) 2,000 rizki and rizki, 1992 drosophila melanogaster (4d) 23,000 rizki and rizki, 1992 manduca sexta (l5) 4,500 horohov and dunn, 1982 galleria melonella (l5) 25,000 chain and anderson, 1982 arachnida limulus polyphemus 30,000 sherman, 1981 arenea spp. 11,000 sherman, 1981 of hemocytes, whereas chilopoda range among those with quite high numbers. when looking at hemocyte numbers more generally, however, it should be taken into account that in those few species in which the topic was investigated, hemocyte numbers often differed significantly during ontogenetical stages. the variability of the hemocytes counts cannot be explained on the basis of the available data. but, as a tendency, those species with hard and strongly calcified cuticles or passive protection strategies against predators (such as glandular defense secretions) have fewer hemocytes (e.g., decapod crustaceans and diplopods). in these species the risk of injuries and infections and, therefore, the demand for wound closure and immune defense is most probably lower. in predatory groups with a thin cuticle (reducing body weight and enabling effective scavenging with lower energy demand than in those groups with calcified cuticles) or taxa with higher risk of injury due to their life style (e.g., chilopods, cockroaches, dipteran larvae) hemocyte numbers are often high. in this context, fründ (1992) showed that 28 to 60 % of the specimens of lithobius forficatus in his study had melanised scares or lost legs. thus the high total hemocyte count probably constitutes an adaptation to their predatory life style (xylander and nevermann, 2006). acknowledgements the author would like to thank dr l nevermann, dr h-u jahn and o bogusch for their support during the investigations on the immune system of myriapoda conducted at the justus-liebig university (germany). acknowledgments are also extended to prof dr g hilken and prof dr g seifert who contributed their part to this publication. references bauchau ag. crustaceans. in: ratcliffe na, rowley af (eds), invertebrate blood cells. vol. 2. academic press, london, pp 385-420, 1981. chain bm, anderson rs. selective depletion of plasmatocytes in galleria mellonella following injection of bacteria. j. insect physiol. 28: 377384, 1982. crossley ca. the cytophysiology of insects. adv. insect physiol. 11: 117-222, 1975. fründ h-c. the occurrence and frequency of scars in centipedes. ber. nat.-med. verein innsbruck, suppl. 10: 269-275, 1992. 123 götz p, vey a. humoral encapsulation in diptera (insecta): defence reactions of chironomus larvae against fungi. parasitology 68: 193-205, 1974. gregoire c. blood coagulation in arthropods. vi. a study by phase contrast microscopy of blood reactions in vitro in onychophora and various groups of arthropods. arch. biol. (liege) 66: 489-508, 1955. gupta ap. hemocytes of scutigerella immaculata and the ancestry of insecta. ann. entomol. soc. am. 61: 1028-1029, 1968. gupta ap. arthropod immunocytes. identification, structure, functions, and anologies to the functions of vertebrate band t-lymphocytes. in: gupta ap (ed), hemocytic and humoral immunity in arthropods. wiley and son, new york, chichester, brisbane, toronto, singapore, pp 3-59, 1986. hilken g, brockmann c, nevermann l. hemocytes of the centipede scutigera coleoptrata (chilopoda, notostigmophora) with notes on their interactions with the tracheae. j. morphol. 257: 181-189, 2003. horohov dw, dunn pe. phagocytosis and nodule formation by hemocytes of manduca sexta larvae following injection of pseudomonas aeruginosa. j. invert. pathol. 41, 203-213, 1982. jones jc. current concepts concerning insect hemocytes. amer. zool. 2: 209-246, 1962. millar da, ratcliffe na. the evolution of blood cells: facts and enigmas. endeavour 13: 72-77, 1989. nevermann l. untersuchungen an haemozyten von scolopendra cingulata und lithobius forficatus unter dem aspekt zellulärer abwehrreaktionen. doctoral thesis, university of giessen. www.nevermanns.de/hemocytes/, 1996 nevermann l, kaiser he, xylander wer. microbial induced haemocytic immune reactions in chilopods. in vivo 10: 161-168, 1996. nevermann l, xylander wer. in vitro cellular immune reactions of hemocytes against bacteria and their differential degradation in myriapods. geoffroy, jj, mauries l, nguyen duy-jacquemin m (eds), acta myriapodologica. mem. mus. natn. hist. natur. 169: 421-430, 1996. nevermann l, xylander wer, seifert g. the hemocytes of the centipede lithobius forficatus (chilopoda, lithobiomorpha): light and electron microscopic studies using in-vitro techniques. zoomorphology 110: 317-327, 1991. rajulu gs. a study of haemocytes in a centipede scolopendra morsitans (chilopoda: myriapoda). cytologia 36: 515-521, 1971. ratcliffe na, price cd. a reappraisal of insect haemocyte classification by the examination of blood from fifteen insect orders. z. zellforsch. 147: 537-549, 1974. ravindranath mh. the hemocytes of a millipede, thyropygus poseidon. j. morphol. 141: 257268, 1973. ravindranath mh. the hemocytes of a scorpion palamnaeus swammerdami. j. morpohol. 144: 1-10, 1974. ravindranath mh. a comparative study of the morphology and behaviour of granular haemocytes of arthropods. cytologia 42:743751, 1977. ravindranath mh. onychophorans and myriapods. in: ratcliffe na, rowley af (eds), invertebrate blood cells, vol. 2, academic press, london, pp 327-354, 1981. rizki tm, rizki rm. lamellocyte differentiation in drosophila larvae parasitized by leptopilina. dev. comp. immunol. 16: 103-110, 1992. sherman rg. chelicerates. na, rowley af. invertebrate blood cells. invertebrate blood cells, vol. 2, academic press, london, pp 335384, 1981. smith vj, johnston pa. differential haemotoxic effect of pcb congeners in the common shrimp, crangon crangon. comp. biochem. physiol. 101c: 641-949, 1992. ullrich g. antibakterielle substanzen bei astacus leptodactylus (crustacea, decapoda). 60 pp. thesis, university of giessen, 1993. xylander wer. immune defense reactions of myriapoda a brief presentation of recent results. in: thaler k, meyer e, schedl w (eds), advances in myriapodology (proceedings of the 8th international congress of myriapodology). ber. nat.-med. verein innsbruck, suppl. 10: 101-110, 1992. xylander wer. immunabwehr bei gliederfüßern wie sich spinnentiere, krebse, insekten und tausendfüßer gegen krankheitserreger schützen. spiegel der forschung 11: 27-30, 1994. xylander wer. antibacterial substances and characteristics from the haemolymph of chilopoda and diplopoda (myriapoda, arthropoda). soil organisms [in press]. xylander wer, bogusch o. investigations on the phenoloxidase of rhapidostreptus virgator (arthropoda, diplopoda). zool. jb. physiol. 96: 309-321, 1992. xylander wer, bogusch o. granular hemocytes as the main location of prophenoloxidase in the millipede rhapidostreptus virgator (diplopoda, spirostreptida, spirostreptidae). ent. scand. suppl. 51: 183-189, 1997. xylander wer, nevermann l. antibacterial activity in the hemolymph of myriapoda (arthropoda). j. inv. pathol. 56: 206-214, 1990. xylander wer, nevermann l. phenoloxidase-active hemocytes in lithobius forficatus, scolopendra cingulata (chilopoda) and chicobolus spec. (diplopoda). verh. dtsch. zool. ges. 86.1: 197, 1993. xylander wer, nevermann l. haemocytes in diplopoda and chilopoda (arthropoda, myriapoda) -types, structures, and numbers. scand. j. entomol. 53: 195-210, 2006. xylander wer, ullrich g, kaiser he. antibacterial immune response in astacus leptodactylus (decapoda, crustacea). in vivo 11: 195-200, 1997. xylander wer, ullrich g, nevermann l, eichelberg d. discrimination of haemocytes of astacus leptodactylus (decapoda: crustacea) by differential spreading and cytochemical staining. abh. ber. naturmuseum görlitz 75: 83-90, 2003. 124 http://www.nevermanns.de/hemocytes/ spherulocytes coagulocytes discoid hemocyte cystocytes adipohemocytes oenocytoids h (hc/µl) references crustacea insecta arachnida isj103.pdf 105 isj 2: 105-113, 2005 issn 1824-307x minireview toll-like receptors in invertebrate innate immunity l zheng1, l zhang1, 2, h lin1, 3, mt mcintosh1, ar malacrida4 1 yale university school of medicine, epidemiology and public health, 60 college street, new haven, ct 06520, usa 2 department of parasitology, medical college, jinan university, shipai, guangzhou 510632, guangdong, p. r. china 3 fujian provincial centers for disease control and prevention, 76 jintai avenue, fuzhou, fujian, 350001, p.r.china 4 dipartimento di biologia animale, università di pavia, piazza botta 9, 27100 pavia, italy accepted august 4, 2005 abstract among invertebrates, innate immunity is the only defense mechanism against harmful non-self agents. in response to recognition of microbial pattern molecules, drosophila melanogaster activates either the toll or imd pathway, leading to the translocation of nf-kb (or rel) transcription factors from the cytoplasm to the nucleus and the subsequent production of antimicrobial peptides, which provide systemic innate immunity. toll-like receptors (tlrs) are characterized by an extracellular leucine rich repeat (lrr) domain and an intracellular toll/interleukin-1 receptor (tir) domain. tlrs are found from cnidarians to mammals. here we argue that tlr mediated innate immunity developed during an early stage of evolution when organisms acquired a body cavity. this is supported by the distributions of tlr and rel genes in the animal kingdom. further, tlr mediated immunity appears to have developed independently in invertebrates and vertebrates. recent studies have shown that microbial molecules, with the potential to signal through tlr, can be beneficial to host survival. studies on this signaling pathway could open doors to a better understanding of the origins of innate immunity in invertebrates and potential transmission blocking strategies aimed at ameliorating vector-borne diseases. key words: toll; innate immunity; antimicrobial peptides; invertebrates; coelom introduction in invertebrates, innate immunity is the sole defense mechanism against infectious non-self agents. there are three manifestations of innate immunity: phagocytosis, activation of humoral responses leading to coagulation, melanization or opsonization, and the systemic production of antimicrobial peptides, or amps (hoffmann et al., 1999). production of amps may represent a more recent evolutionary adaptation of innate immunity, allowing for an effective and efficient means of protection against systemic infection. corresponding author: liangbiao zheng yale university school of medicine, epidemiology and public health, 60 college street, new haven, ct 06520, usa e-mail: liangbiao.zheng@yale.edu phagocytosis, being the most primitive feature, developed probably as a means of acquiring nutrients and was probably co-opted later as a defense mechanism. while origins of the other two forms of innate responses are not clear, it is likely they too developed quite early when life forms co-existed in primordial oceans and had to contend with a milieu of diverse microbial agents (friends and foes). that said, for primitive aquatic organisms lacking a real body cavity it would not have been efficient to produce and secrete amps onto the surfaces as these would be subjected to dilution by the environment and thus never reach the critical concentration required for effective clearance of microbes. it is therefore likely that systemic immunity in the form of production of amps mediated by toll-like receptors (tlrs) first developed in organisms with a body cavity. 106 systemic immunity in the model system drosophila melanogaster systemic production of amps is best characterized in the fruit fly, drosophila melanogaster (reviewed in hoffmann, 2003). generally speaking, immunity against fungi and gram(+) bacteria is mediated by the tolldif/dorsal pathway while anti gram(-) bacterial responses are mediated by the imd-relish pathway, leading to the activation and expression of a different set of antimicrobial peptide genes. imd was first identified genetically as a key factor for survival against bacterial challenge (lemaitre et al., 1995). how gram(-) bacteria are recognized remains to be determined; though it is known that the recognition eventually leads to the activation of a member of the peptidoglycan recognition protein (pgrp) family, pgrp-lc. pgrp-lc interacts directly with imd (choe et al., 2005), which then sends the signal through two separate channels. one leads to the activation of a signalsome consisting of ikkβ and ikkγ via tak1. the other channel leads to the activation of dredd, a member of the caspase family. both channels converge on the nf-kb factor, relish. ikkβ and ikkγ complex phosphorylate the i-kb domain of relish, while dredd presumably cleaves the i-kb domain (stoven et al., 2000, 2003). this allows the amino terminal portion of relish, carrying both dna binding and transcriptional activation domains, to enter into the nucleus and activate expression of antimicrobial peptide genes such as diptericin and cecropin. in d. melanogaster, fungal pathogens lead to an activation of an extracellular protease, persephone, while anti-gram(+) bacterial responses require the products of pgrp-sa and gnbp1, members pgrp and gram negative binding protein (gnbp) families, respectively. both fungal and gram(+) pathogens eventually converged on the cytokine-like molecule spaetzle, a protein with cysteine knots. proteolytic cleavage of pre-protein spaetzle produces the ligand for toll. toll then sends the signal through a multimeric protein complex, consisting of myd88, tube and pelle, leading to the phosphorylation of cactus, an i-kb like molecule, and subsequent activation and nuclear translocation of dif or dorsal, both belonging to the nfkb family of transcription factors. in adult flies, dif activates transcription of antimicrobial peptide genes such as drosomycin and metchnikowin (reviewed in hoffmann, 2003). toll was originally identified as a gene required for dorsal ventral patterning (nusslein-volhard et al., 1980) but was then found to be required for antifungal immunity in adults (lemaitre et al., 1996). toll is one of nine tlr genes in d. melanogaster that encode proteins with extracellular leucine rich repeat (lrr) arrays and an intracellular toll-interleukin-1 receptor (tir) domain (tauszig et al., 2000). thus far, genetic studies have shown that toll (or toll1) is the primary receptor in innate immunity. however, experiments in cell lines also suggest that toll5 (luo et al., 2001) and toll9 (ooi et al., 2002) can activate expression of antimicrobial peptide genes. toll1 and toll5 (also known as tehao) are similar in the tir domain, though toll1 carries a carboxyl terminal extension which toll5 lacks (luo and zheng, 2000). to date, screening by whole genome rna interference has not elucidated the functions of the other eight tlr genes in d. melanogaster. toll and imd signaling pathways in insects the origin of the innate immune systems will be better understood as more and more genomes of invertebrates are determined. the completion of the genome sequencing of anopheles gambiae, the main vector for human malaria (holt et al., 2002), in addition to the resolved genome of d. melanogaster, allows for comparative immunology to be examined between these two dipterans. although most of the toll and imd signaling components are conserved in a. gambiae, significant differences have been found (christophides et al., 2002). similar conclusions can be drawn from the ongoing genome sequencing of another major vector of human diseases, aedes aegypti, which transmits dengue and yellow fever viruses (unpublished observations). for the imd pathway, it was found that rel2, in both a. gambiae and a. aegypti, is both structurally and functionally different from its orthologue, relish, in d. melanogaster. rel2 in mosquitoes contains an additional death domain which is absent in the fly orthologue (shin et al., 2002). furthermore, the rel2 gene in mosquitoes produces different spliced variants. in a. aegypti, rel2 produces three spliced forms, yielding a full length protein, with both rel-homology domain (rhd) and i-kb ankyrin repeats, and partial length polypeptides, containing either the i-kb domain or the rhd domain, respectively (shin et al., 2002). in a. gambiae, two spliced variants have been found thus far, corresponding to the full length and rhd domain containing polypeptides from a. aegypti, respectively. more importantly, it has been shown in a. gambiae that the imd pathway functions in immune responses against both gram(+) and gram (-) bacteria (meister et al., 2005). components of the toll pathway are also found in a. gambiae, though once again significant differences have been found between it and d. melanogaster. the most conspicuous being the absence of a dif orthologue in both a. gambiae and a. aegypti. it was found recently that the dorsal homologue, rel1 from a. aegypti also produces spliced variants, rel1a and rel1b, differing at the carboxyl terminal domains (shin et al., 2005). similar spliced variants have been found for drosophila dorsal. concomitant over-expression of the two variants rel1a and rel1b from a. aegypti leads to the up-regulation of defa and ceca in a cell line (shin et al., 2005). alternative splicing also appears to occur in the a. gambiae dorsal homologue known as gambif (barillasmury et al., 1996) or rel1. one expressed sequence tag (ensangestt00000367694) of rel1 from a. gambiae represents a partial cdna clone which uses different splice sites than the full-length transcript rel1 transcript characterized previously (barillas-mury et al., 1996). at present, whether or not spliced forms similar to the 107 aedes rel1a and rel1b exist in a. gambiae remains to be determined. another major difference is the presence of four genes (toll1a, 1b, 5a and 5b) that share similarities in the tir domain with d. melanogaster toll1 and toll5. toll1a and 5a are arranged in tandem on the x chromosome, while toll1b and 5b are situated in tandem on the third chromosome (christophides et al., 2002). while it has been shown that over-expression of toll related genes from mosquitoes in d. melanogaster cell lines leads to elevated expression from the promoter of drosomycin (luna et al., 2002, 2003), there remains no additional functional evidence for the involvement of mosquito tolls in innate immunity. structure and functions of toll-like receptors (tlrs) as indicated above, toll receptors are transmembrane proteins with an extracellular lrr domain interspersed with cysteine knots. the intracellular domain shares sequence similarities with the interleukin-1 receptors from mammals, and is called the toll/interleukin-1 receptor (tir) domain (reviewed in imler and zheng, 2004). the tir domain is composed of approximately 150 amino acid residues. it is present not only in tlrs, but also in other intracellular signaling components, such as myd88. these intracellular tir-containing adaptor molecules, however, do not contain a leucine rich repeat. in addition, tir domains and leucine rich repeats are also present in some plant resistance (r) genes, typified by rpp5, which also has a nucleotide binding domain (nbd) (parker et al., 1997). in contrast to tlrs, the plant tir-nbd-lrr resistant proteins are intracellular. furthermore, their tir domains are phylogenetically distinct from tlrs. thus, it seems likely that the tir domain-containing adaptor and plant tir-nbd-lrr genes evolved independently from tlrs. the molecular structures of the tir domains of human tlr1 and tlr2 have been determined and show a five-stranded parallel β-sheet surrounded by five helices on both sides (xu et al., 2000). there is a conserved surface which primarily consists of a loop, known as the bb loop, which protrudes from the rest of the structure. mutations in the bb loop result in a loss or significant reduction in activity of tlrs from both man and drosophila. within this region are a few highly conserved charged amino acid residues, including an arginine at loop position 3 and an aspartic acid at loop position 4. a proline residue at loop position 7 is also known to be essential for lps signaling by human tlr4. as eluded to previously, this proline residue is not conserved in all tlrs (luna et al., 2003) and functional studies suggest that it is not a key structural determinant (xu et al., 2000). instead, substitution of this residue may disrupt interactions with other signaling components needed for activation of the pathway. mutation at a histidine residue in the bb loop position 1 in d. melanogaster, represented by allele tlrb1 or tlr6 in the flybase database (www.flybase.org), also results in a loss of function of toll in dorsal ventral formation in early embryogenesis (schneider et al., 1991). this histidine residue is highly conserved in insect tlrs, but not in vertebrate tlrs. loss of function mutations outside the bb loop yet still within the tir domain have also been documented in d. melanogaster. how these residues fully contribute to the function of toll signaling has yet to be determined. as tir domains of myd88 genes from different insects vary widely in sequence, variation on this interacting surface may contribute to species specific signaling in insects (unpublished observations). distribution of tlrs in the animal kingdom thus far, on the tree of life, tlrs have been found to be present in animals ranging from cnidarians to mammals, though they appear to be absent in platyhelminths (table 1). tlr genes have been found in all vertebrates where they also play an important role in bridging innate and adaptive immunity, reviewed in (pasare and medzhitov, 2004). for example, human and mouse genomes contain 10 and 12 tlr genes, respectively. these tlrs are activated, individually or in combinations, by different bacterial pattern molecules, such as lipopolysaccharide, peptidoglycan, or naked dna (for a recent review see kaisho and akira, 2004). tlrs are also found in invertebrates that have body cavities (table 2). d. melanogaster possesses 9 tlrs while a. gambiae encodes 10. thus far, at least one tlr gene has been found in all insect genomes. within the phylum arthropoda, one tlr gene has been found in the horseshoe crab tachypleus (inamori et al., 2000, 2004) and the lobster homarus americanus (accession number: cn852754, unpublished observation). limited genomic information has shown that at least one tlr gene is present in euprymna, the hawaiian squid which belongs to the phylum mollusca (hoa nt et al., unpublished observations). likewise, nematoda genome sequencing has revealed that caenorhbiditis elegans and caenorhbiditis briggsea each encode one tlr. interestingly, functional studies showed that this unique tlr gene (cetol-1) appears to have no function in innate immunity in c. elegans (pujol et al., 2001). a tlr gene has yet to be found in the phylum platyhelminth, though significant genomic information is available for schistosoma japonicum, schistosoma mansoni and schmidtea mediterranea. surprisingly though, on-going genome sequencing of a cnidarian recently showed that hydra magnipapillata has at least 3 tlr genes. these expressed sequence tags (accession number cb889349, cn630662 and cn630303) encode proteins with a tir domain and a transmembrane segment, thus establishing them as likely tlr genes. phylogenetic analysis provides further evidence that these tir domains cluster with animal tlrs, rather than with the intracellular tir domain containing genes. while it is conceivable that tlrs were lost during the evolutionary period leading to platyhelminths, as apparent in schistosoma and schmidtea, further genome 108 table 1. distribution of toll related receptors (tlr) and nf-kb genes in the animal kingdom phyla representative toll rel cnidaria hydra 3 -? platyhelminth schistosoma -? -? nematoda caenorhbiditis 1 annelida tubifex ? 1 mollusca euprymna/crassostrea 1? (+) arthropoda drosophila, 9 3 anopheles 10 2 tachypleus 1 ? homarus 1 ? echinodermata strongylocentrotus 2 1 chordata mus 12 5 homo 10 5 table 2. toll like receptor genes in arthropods† subclass infraclass order suborder # of genes representative neoptera orthopteroida orthoptera caelifera 2* schistocerca caelifera >1* locusta paraneoptera hemiptera 1* homalodisca endopterygota hymenoptera aculeata 5* apis coleoptera polyphaga 1* tribolium lepidoptera glossata 7* bombyx glossata 1* manduca diptera brachycera 9 drosophila brachycera 1* ceratitis brachycera 1* glossina nematocera 4* aedes nematocera 1* culex nematocera 3* clogmia nematocera 10 anopheles * indicates genome has not been fully sequenced or annotated. †not included here are one tlr gene each found in a lobster and a horseshoe crab, respectively. sequencing may yet reveal tlrs within the phylum platyhelminth. phylogeny of tlr genes the numbers of extracellular lrr arrays in different tlrs are different, making sequence alignment in this region highly subjective. therefore, we based the phylogenetic relationships among tlrs on the intracellular tir domain. the tir domain was identified using the smart algorithm (schultz et al., 2000; letunic et al., 2004) and aligned by clustal x (thompson et al., 1997). neighbor-joining showed that all tlrs from mollusca and arthropoda form a clade, except toll9s from drosophila and anopheles, which cluster with mammalian tlrs. tlrs from nematoda form a separate clade (fig. 1). this is consistent with the proposal that innate immunity developed independently in invertebrates and vertebrates (hughes, 1998; friedman and hughes, 2002). interestingly, the tlrs from cnidarian cluster with mammalian tlrs (fig. 1). distribution of rel transcription factors in the animal kingdom from studies in drosophila and mammals, toll signaling pathways in innate immunity require key transcriptional factors belonging to the nf-kb (or rel) family. these proteins are characterized by a rhd domain. thus far, nf-kb factors have been found in an 109 fig. 1 phylogenetic relationships among the tlrs. tir domains were identified using the smart algorithm and sequences before the conserved motif (f/y)da and after the motif fw(e/d) were removed. alignment was performed by clustal x, followed by visual inspection. edited alignment was used for tree generation using 1000 bootstrap iterations. nodes with bootstrap values >700/1000 are highlighted with filled circles. scale represents 0.1 amino acid change/residue. abbreviations are as follows: hu (human), dm (drosophila), aa (aedes), ag (anopheles), am (apis), at (arabidopsis), bf (branchiostoma), bm (bombyx), bom (boophilus), ca (clogmia), cc (ceratitis), ce (caenorhbiditis), ci (ciona), cp (culex), es (euprymna), ha (homarus), mx (manduca), sa (schistocerca), ss (strongyloides), tc (tribolium), tt (techypleus). due to the high throughput nature of the sequences analyzed, some insect tlr genes may be allelic. for simplicity, arthropod tlrs are indicated by a number after the species abbreviation, while the single tlr genes from caenorhbiditis, strongyloides and euprymna each are designated as tol-1. the alignment file is available upon request. 110 fig. 2 coelomata (panel a) versus ecdysozoa (panel b). if genes shared among organisms with a body cavity are absent in nematodes (black filled circle), they can be easily explained by the coelomata hypothesis (panel a). the ecdysozoa hypothesis would need to invoke a loss of genes in the nematodes (dashed open circle). genes shared among coelomates but absent in one of the two nematode species (filled rectangle) can be explained equally well by either the coelomata or ecdysozoa hypotheses (where gene losses are represented by dashed rectangles with different fill patterns for the two nematode species). the distribution of tlr and rel genes (last two columns in panel a) is consistent with the coelomata hypothesis. an asterisk indicates the absence of a corresponding gene, though it is conceivable that the gene could be found when the complete genome is sequenced. for simplicity, the rel gene from another coelomate annelid, tubifex is not shown here. 111 annelid, mollusks, arthropods and vertebrates, all of which possess real body cavities. none have been found in nematodes, platyhelminths or cnidarian (the former being pseudocoelomates while the later two acoelomates; see table 1 and fig. 2). this was based on a tblastn analysis (altschul et al., 1997) performed on april 7, 2005, using a. gambiae rel1 and d. melanogaster relish as queries against the updated non-redundant genbank database. tlr mediated systemic immunity developed in coelomates: a hypothesis among invertebrates, antimicrobial peptide production is the main form of systemic immunity. we argue that such immunity developed in coelomates (kanzok et al., 2004). before animals became terrestrial, it would not have been efficient to produce antimicrobial peptide on the surface, where the activity would have been diluted by sea water. when a coelom was developed in an animal, it provided a nutrient-rich and sterile environment that would have been envied by microbes. to protect against internal infection, the first coelomate patched together a signaling pathway that includes the existing tlr genes and may have acquired a novel transcription factor of the nf-kb family. this is consistent with the presence of both tlr and nf-kb genes in the phyla where sufficient genomic sequences are available: echinodermata, chordata, arthropoda and mollusca. further, antimicrobial peptides such as defensin have also been found in mammals, arthropods and mollusks. although one tlr gene each has been found in c. elegans and c. briggsae, no nf-kb-like transcription factor has been found in nematodes. further, genetic evidence with mutations in the tlr gene of c. elegans suggests that it is essential for development and is not involved in innate immunity (pujol et al., 2001). coelomata and ecdysozoa traditional classification of metazoans (or coelomata hypothesis) is based on the presence of a coelom and divides bilaterians into acoels, pseudocoels and coels (hyman, 1951). our hypothesis implies that classical division of the animal kingdom based on the presence or absence of a coelom (body cavity) is correct and promoted us to examine the relationships among taxa at the deep root of the evolutionary tree. both mollusca and arthropoda are protostomia coels and are considered more closely related to each other than they are to a pseudocoel phylum, nematoda. likewise, newer morphology-based classifications recognize protostomia and deuterostomia, with protostomia divided into spiralia and cycloneuralia, and these place mollusca and arthropoda as more closely related to each other than to nematoda (nielsen, 2001). a radical view proposed recently separates mollusca from arthropoda and groups arthropoda with nematoda and others, forming a clade named ecdysozoa. a separate clade, lophotrochozoa includes organisms such as annelids, mollusks and platyhelminths. this later view is primarily based on phylogenetic analysis of nuclear 18s ribosomal rna sequences (aguinaldo et al., 1997). evidence since has been accumulated in favor of (e.g. dopazo and dopazo, 2005; roy and gilbert, 2005) and against (e.g. hughes and friedman, 2004; steinauer et al., 2005) the ecdysozoa hypothesis. instead of examining sequence variations over time, which are subjected to varying evolutionary rates for different genes and homoplasy, recent studies show that large numbers of genes are shared between mollusks and arthropods yet are absent in the nematodes c. elegans and c. briggsae (hoa nt et al, unpublished observations). even taking into consideration gene loss that is known to occur in c. elegans, coelomata theory provides the most parsimonious explanation for the sharing of genes between mollusks and arthropods. if the coelomata theory is indeed correct, it would explain with the distributions of tlr and nf-kb genes in the animal kingdom. tlr genes first appeared in cnidarian and were subsequently lost in platyhelminths. the nf-kb transcription factor evolved once in coelomates and was retained in organisms such as annelids, mollusks, arthropods and vertebrates. the combination of tlr and nf-kb transcription factor genes provided the first coelomate a systemic immune response to internal infection by harmful bacteria. future perspectives we argue here that tlr mediated innate immunity developed after the appearance of coelomates. whether the imd pathway evolved in the same way remains to be examined. as more and more genome sequences become available, comparative immunology will shed further light on the origin of the systemic innate immune responses. interactions between microbes and their animal hosts range from pathogenic, to mutualistic, to symbiotic. thus far, we have examined host immune responses to microbes as non-self. in invertebrates, symbiotic microbes are essential to the survival and success of their hosts (rio et al., 2004). in a mutualistic interaction, the mollusk, euprymna scolopes or hawaiian squid, depends on its microbial partner, vibrio fisheri, to build a light-emitting organ. recent studies suggested that microbial pattern molecules are essential to the generation of this organ (koropatnick et al., 2004), though how these pattern molecules trigger organ development remains a mystery. whether toll or imd, both or additional pathways are involved in the formation of this unique organ or even other bacterial associated structures in invertebrates are questions which also remain unsolved. it is worth noting however that recent studies in mammals have shown that commensal microbes can activate host tlr receptors which in turn are required to maintain intestinal homeostasis (rakoffnahoum et al., 2004). in summary, understanding how microbial organisms interact with their hosts will provide 112 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proc. natl. acad. sci. usa 97: 10520-10525, 2000. thompson jd, gibson tj, plewniak f, jeanmougin f, higgins dg. the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. nucleic acids res. 25: 4876-4882, 1997. xu y, et al. structural basis for signal transduction by the toll/interleukin-1 receptor domains. nature 408: 111-115, 2000. isj 3: 111-xx, 2006 isj 3: 137-145, 2006 issn 1824-307x research report brush border membrane vesicles from dipteran midgut: a tool for studies on nutrient absorption mg leonardi, s caccia, b giordana dipartimento di biologia, università degli studi di milano, milano, italy accepted december 20, 2006 abstract brush border membrane vesicles (bbmv) from insects midgut can be successfully used to study several membrane phenomena, including nutrient absorption, ions permeability and insecticides mode of action. midgut bbmv, purified from musca domestica whole larvae, were used for the functional characterization of leucine transport. the amino acid uptake was accelerated in the presence of sodium or potassium and increased significantly when the extravesicular ph was 5.0, in agreement with the luminal ph in vivo. radiolabelled leucine uptake was significantly reduced by an excess of cold leucine, histidine, serine and glycine, suggesting that the amino acid transporter is a broad scope carrier that does not recognize proline, glutamine and the dibasic amino acids lysine and arginine. midgut bbmv were also obtained from homogenization of m. domestica and bactrocera oleae adults. the final preparations showed a high enrichment in the specific activity of the bbm marker enzymes aminopeptidase n and γ-glutamyl transpeptidase, and were poorly contaminated by basolateral membranes, as indicated by the low specific activities of their marker enzyme na+/k+ atpase. electron microscopy of b. oleae bbm fraction showed the presence of closed vesicles. similar sds-page patterns, with numerous distinct bands, were detected for both b. oleae and m. domestica bbmv. key words: dipteran midgut; brush border membrane vesicles; amino acid absorption; bactrocera oleae; musca domestica _____________________________________________________________________________________________ introduction amino acids play a key role in several physiological processes and for this reason intestinal amino acid absorption, a crucial step in nitrogen metabolism, affects the biological development of the whole organism. the functional and regulative properties of the amino acid transport systems expressed in the insect midgut epithelium has been extensively studied in lepidopteran larvae (giordana et al., 1989, 1998, 2002; wolfersberger, 1996; leonardi et al., 1998a, 2001a, b; casartelli et al., 2001; ______________________________________________________________________________ corresponding author: m. giovanna leonardi dipartimento di biologia univerisity of milan via celoria 26, 20133 milano, italy e-mail: mgiovanna.leonardi@unimi.it parenti et al. 2002) while little is known on intestinal amino acid absorption in other insect orders (hong et al., 1995, 1997; neal, 1996; neal et al., 1996; parenti et al., 1986, 2001). brush border membrane fragments form sealed vesicles spontaneously maintaining their correct orientation (haase et al., 1978), that allow to define the internal and external environments according to the experimental needs. the brush border membrane vesicles (bbmv) isolated from lepidopteran midgut epithelium have been successfully used for the functional characterization of amino acid transport proteins (giordana et al., 1989, 1998; wolfersberger, 1996; leonardi et al., 1998a; casartelli et al., 2001), to analyze in vitro the activity of the insecticide fenoxycarb (leonardi et al. 1996, 1998b, 2001a) and, far more extensively, to study the mode of action of 137 whole dipteran adults homogenized in 1:10 w/v buffer a (300 mm mannitol, 2 mm tris-cl at ph 7.1) in polytron (2 periods lasting 1 min) and filtered through gauze homogenate centrifuged at 1000xg for 10 min s1 fig. 1 procedure for the preparation of bbmv from adults m. domestica and b. oleae (described in detail in materials and methods). p1 centrifuged at 20000xg for 15 min s2p2 resuspended in 30 ml of buffer a plus percoll 10.8%, with 3 strokes in a loose dounce and centrifuged at 40000xg for 35 min band collected, diluted to 30 ml with buffer a and centrifuged at 100000xg for 60 min p3b – fluffy pellet p3a – hard glassy percoll pellet s3 p4 s4 centrifuged at 48000xg for 20 min s5 p3b resuspended in 20 ml buffer a, mixed with cacl2 to10 mm final concentration and centrifuged at 1000xg for 15 min bbmv p5 138 cry1 toxins of bacillus thuringiensis (sacchi et al. 1986; gill et al., 1992; giordana et al., 1993; knowles, 1994; leonardi et al., 1997; bravo et al., 2002). the preparation of purified bbmv from the midgut of insects of interest is therefore fundamental to investigate several membrane phenomena. in the present paper we describe the preparation of purified midgut bbmv using whole musca domestica larvae as a starting material. the vesicles were then used to describe the functional properties of amino acid uptake in this dipteran larva. we also describe a method to obtain midgut bbmv from adults of m. domestica and bactrocera oleae, preparations that can be used to study the physiological processes active at the apical membrane of midgut cells or the effect on the same membrane of insecticides potentially active against the adults of these two dipteran pests. materials and methods materials l-[4,5-3h]leucine (71 ci/mmol) was purchased from amersham biosciences europe, italy. all the other reagents were supplied by sigma-aldrich s.r.l., italy. experimental animals m. domestica larvae, kindly provided by dr. marcello verdinelli (istituto di ricerca sul controllo biologico dell’ambiente (ircoba), cnr, sassari), were immediately frozen and preserved in liquid nitrogen for few weeks. pupae of m. domestica and b. oleae, also provided by ircoba, were maintained at 25 ± 1 °c, 65-70 % r.h. and 12l:12d photoperiod. immediately after eclosion, the adults were anaesthetized at 4 °c, then frozen in liquid nitrogen and there preserved for few weeks. bbmv preparation from larvae of m. domestica larvae of m. domestica, removed from liquid nitrogen, were placed in 1:10 w/v of an ice-cold buffer composed of 100 mm mannitol, 10 mm hepes-tris at ph 7.1, 1 mm pmsf and homogenized on ice with polytron (kinematica ch-6010 kriens-lu) for 3 periods lasting 30 s at velocity 5. the homogenate was filtered through 2 gauze layers and bbmv were prepared by ca++-precipitation and differential centrifugation as reported by giordana et al. (1982). the filtered homogenate (h) was added with cacl2 to a final concentration of 10 mm, the suspension was stirred on ice for 15 min and then centrifuged at 3000 x g for 15 min. the supernatant was decanted and kept on ice. the pellet was resuspended in half of the original volume of buffer (100 mm mannitol, 10 mm hepes-tris at ph 7.1) with the aid of a glass-teflon homogenizer (ika-labortechnick re 16) with five strokes at 1500 rpm. the suspension was mixed with cacl2 to a final concentration of 10 mm, blended on ice for 15 min and then centrifuged at 3000 x g for 15 min. the second pellet was discarded. the first and the second supernatants were pooled and centrifuged at 48000 x g for 20 min. the supernatant was discarded and the pellet resuspended in the original volume of buffer with five strokes at 1500 rpm in the glass-teflon homogenizer. the suspension was centrifuged at 48000 x g for 20 min and the final pellet, containing the brush border membranes, was resuspended in a suitable amount of buffer by 10 passes through a 22 gauge needle. protein concentration was assessed with the coomassie brilliant blue g-250 protein assay (pierce, rockford, il, usa) with bovine serum albumin as standard. bbmv preparation from adults of m. domestica and b. oleae the procedure is schematically presented in fig. 1. all the steps were performed at 4 °c. the frozen adults were placed in ice-cold buffer composed of 300 mm mannitol, 2 mm tris-cl at ph 7.1, in a 1:10 w/v proportion, and homogenized with a polytron (kinematica ch-6010 kriens-lu) for 2 periods lasting 1 min at velocity 5. the homogenate was filtered through 3 layers of gauze to remove large debris. the filtered homogenate (h) was centrifuged at 1000 x g for 10 min. the first pellet p1 was discarded and the supernatant s1 was centrifuged at 20000 x g for 15 min. the supernatant s2 was discarded and the pellet p2 resuspended in 30 ml of buffer containing 10.8 % of percoll with 3 strokes in a loose dounce homogenizer. the suspension was centrifuged at 40000 x g for 35 min with no brake. the plasma membranes were present as a band at the third upper portion of the continuous density gradient formed by percoll. the membrane fraction was carefully removed with a pasteur pipette, diluted to 30 ml with buffer and centrifuged at 100000 x g for 1 h. the supernatant s3 was carefully decanted and the fluffy membrane layer p3b, separated from the glassy percoll pellet p3a, was resuspended in 20 ml of buffer. cacl2 was added to the suspension to a final concentration of 10 mm, the mixture was blended on ice for 15 min and then centrifuged at 1000 x g for 10 min. the pellet p4 was discarded and the supernatant s4 centrifuged at 48000 x g for 20 min. the final pellet p5, containing the brush border membranes, was resuspended in a suitable volume of buffer by means of 10 passes through a 22 gauge needle. enzyme assays enzyme activities were measured in the filtered homogenate (h) and in bbmv. aminopeptidase n (ec 3.4.11.2) was determined by measuring the release of p-nitroaniline from l-leucine-p-nitroanilide in 40 mm trishcl at ph 7.5 and γ-glutamyl transferase (e.c. 2.3.2.2) by the release of 5-amino-2-nitrobenzoate from l-γ-glutamyl-glycilglycine in 100 mm trishcl at ph 8. incubations were carried out in a spectrophotometer (ultrospec 3000 pharmacia biotech, cambridge uk) with a thermostatic (25 °c) cuvette holder, in conditions in which activity was proportional to the protein concentration and time. 139 table 1 protein yield and activities of plasma membrane marker enzymes in homogenate and bbmv from m. domestica larvae protein yield (mg protein/g animals) bbmv 0.31 ± 0.09 (5) enzyme activity (mu/mg protein) aminopeptidase n γ-glutamyl transferase na+/k+ atpase homogenate 84.1 ± 3.0 (5) 2.0 ± 0.1 (3) 121 ± 21 (3) bbmv 642 ± 119 (5) 20.6 ± 0.6 (3) 189 ± 8 (3) enrichment factor* 7.5 ± 1.2 (5) 10.3 ± 0.2 (3) 1.7 ± 0.4 (3) values are means ± se in brackets are reported the number of independent preparations assayed *ratio of the enzyme specific activity in bbmv and in the homogenate one unit of enzyme activity corresponds to the hydrolysis of 1 μmol of substrate/min. na+/k+-atpase (e.c. 3.6.1.3.) activity was measured according to quigley and gotterer (1969). electrophoresis brush border membrane proteins were analyzed under denaturing conditions by sds-page (laemmli, 1970) in a mini protean ii bio-rad electrophoresis system. 4 % and 10 % acrylamide concentrations were used for stacking and running gels respectively. samples were prepared by mixing 1:1 (v/v) h or bbmv with the loading buffer 2x (4 % sds, 20 % glycerol, 10 % 2-mercaptoethanol, 0.004 % bromphenol blue, 0.125 m tris-hcl at ph 6.8) (sigmaaldrich s.r.l., italy). the mixture was incubated at 100 °c for 4 min before loading the samples. the bands were stained with coomassie blue r-250. transmission and scanning electron microscopy bbmv were fixed for 30 min in 0.1 m cacodylate buffer, ph 7.2, containing 2 % glutaraldehyde. samples were then washed in the same buffer and postfixed for 20 min with 1 % osmic acid in 0.1 m cacodylate buffer, ph 7.2. after a standard step of serial ethanol dehydratation, bbmv were pelletted and embedded in an epon-araldite 812 mixture. sections were obtained with a reichert ultracut s ultratome (leica, wien, austria). thin sections were stained by uranyl acetate and lead citrate and observed with a jeol 1010 ex electron microscope (jeol, tokyo, japan). measurements of leucine uptake in bbmv from m. domestica larvae transport experiments were performed in triplicate at room temperature by rapid filtration (hanozet et al., 1980). to measure leucine uptake, 1 volume of the bbmv suspension was mixed to 4 volumes of the radiolabelled incubation medium, whose final concentration is reported in the legend of the figures. uptake was terminated by diluting the incubated mixture with 50 volumes of ice-cold stop solution (150 mm nacl, 10 mm hepes-tris at ph 7.2). the suspension was then filtered through a prewetted mixed cellulose ester filter (0.45 µm pore size, micro filtration systems, dublin, ca), then counted for radioactivity in a scintillation spectrometer (tri-carb, packard, model 1600 ca). results and discussion in the 1980’s two methods were published to prepare bbmv from the midgut of lepidopteran larvae (hanozet et al., 1980; giordana et al., 1982; wolfersberger et al., 1987). these procedures required that the midgut tissues were isolated from the larvae and were based on the calciumor magnesiumprecipitation of the membrane fragments obtained from the tissue homogenization, followed by differential centrifugations. later, modifications of the original protocols were introduced to prepare midgut bbmv sufficiently purified starting directly from the entire larva of the lepidoptera plutella xilostella (macintosh et al., 1994) or the diptera aedes aegypti (abdul-rauf and ellar, 1999) and chironomus riparius (parenti et al., 2000). the use as a starting material of the entire animal instead of the isolated midguts reduces enormously the time necessary to obtain bbmv. we applied the ca++-precipitation method (giordana et al., 1982), conveniently modified, to the larval stage of the important pest m. domestica. a satisfying homogenization of all the larval tissues could be obtained only by using a polytron homogenizer, and an adequately purified final preparation was achieved by adding a second cycle of ca++-precipitation and 140 fig. 2 initial rates (panel a) and time course (panel b) of leucine uptake in bbmv from m. domestica larvae. bbmv, resuspended in 100 mm mannitol, 10 mm hepes-tris at ph 7.2, were diluted 1:5 to obtain the following final composition: 0.1 mm l-[4,5-3h] leucine 30 μci/ml, 100 mm nascn (na+, ▼�) or kscn (k+, ○), or 200 mm sucrose (none, ●), 20 mm hepes-tris at ph 7.2 or 20 mm mes-tris at ph 5.0 (na+, �). values are means ± se of a typical experiment performed in triplicate. initial rates (panel a) and time course (panel b) of leucine uptake in bbmv from m. domestica larvae. bbmv, resuspended in 100 mm mannitol, 10 mm hepes-tris at ph 7.2, were diluted 1:5 to obtain the following final composition: 0.1 mm l-[4,5-3h] leucine 30 μci/ml, 100 mm nascn (na+, ▼�) or kscn (k+, ○), or 200 mm sucrose (none, ●), 20 mm hepes-tris at ph 7.2 or 20 mm mes-tris at ph 5.0 (na+, �). values are means ± se of a typical experiment performed in triplicate. differential centrifugation. the purity was verified by measuring the specific activity in the homogenate and in the final pellet of aminopeptidase n and γ-glutamyl transferase, two enzymes exclusively localized in the apical membrane of midgut cells, and of na+/k+ atpase, the marker enzyme of the basal plasma membrane. the enrichment factor was calculated as the ratio between the enzyme specific activity in the final preparation and in the homogenate. the brush border marker enzymes were enriched sevenfold and tenfold, respectively, with a negligible enrichment of na+/k+ atpase (table 1), so the final bbmv preparation was essentially free of contaminating basal membranes. altogether, the enrichment factors of this study are consistent with previous results, since a tenfold enrichment of the bbm marker enzymes were obtained for bbmv prepared from whole aedes aegypti larvae (abdul-rauf and ellar, 1999), and enrichment factors varying between 3 and 5 were calculated for bbm purified from the isolated larval midguts of m. domestica as a starting material (lemos and terra, 1992; jordao et al., 1995). we performed transport experiments in m. domestica bbmv to identify the main features of amino acid absorption in this insect. the time course of 0.1 mm leucine uptake into bbmv was measured in the absence of cations, or in the presence of an inwardly directed sodiumor potassium-gradient. if an amino acid is transported by a transport protein that also binds and translocates a cation, i.e. by a cotransporter, a transient accumulation of the amino acid in the intravesicular space should occur, due to the flow of the cation along its electrochemical gradient. otherwise, the uptake is merely equilibrative and the equilibrium will be attained at different times, according to the amino acid transport rate. when the extravesicular ph was 7, leucine initial uptake rate at 15 s increased fourfold in the presence of k+ and eightfold with na+ (fig. 2, panel a). the time course of leucine uptake in the absence of cations was very slow and the equilibrium was not yet reached after 120 min (fig. 2, panel b). in the presence of a k+or a na+-gradient, leucine uptake was accelerated but no transient accumulation of the amino acid was observed 141 fig. 3 inhibition of radiolabelled leucine uptake by a large excess of the indicated amino acids in bbmv from m. domestica larvae. bbmv, resuspended in 100 mm mannitol, 10 mm hepes-tris at ph 7.2, were diluted 1:5 to obtain the following final composition: 0.4 μm l-[4,5-3h]leucine 30 μci/ml, 100 mm nascn, 10 mm inhibitors or mannitol (control), 20 mm hepes-tris at ph 7.2. values are means ± se of a typical experiment performed in triplicate. the statistical analysis of the data was performed by student’s t-test. ** p < 0.01; *** p < 0.001. (panel b). therefore, na+ and k+ are able to activate leucine transporter in m. domestica midgut, but no evidence is given that the protein is a cotransporter. in coleopteran larval midgut, the uptake of tyrosine and methionine, but not that of leucine, was also stimulated by na+ and k+ (hong et al., 1995) and, in agreement with our results, no accumulation of the two amino acids was observed with a na+or a k+ gradient (neal, 1996). the na+-dependent leucine uptake increased markedly when the extravesicular ph was 5.0 (fig. 2, panels a and b), suggesting that leucine transporter is adapted to the physiological environment of m. domestica larval midgut, where the lumen contents is weakly acidic in the anterior and posterior regions and highly acidic in the middle region (terra, 1988; dubreuil, 2004). interestingly, leucine uptake in midgut bbmv of lepidopteran larvae is mediated by a transport system that is, in that case, strongly activated by an extravesicular alkaline ph, as tipically present in the luminal fluids of the larval midgut: the ph-gradient can be exploited to perform leucine intravesicular accumulation (giordana et al., 1998). therefore, the ability to operate more efficiently in the presence of the physiological ph is a functional property in common between dipteran and lepidopteran leucine transport systems. as expected for a carrier-mediated process, a 87 % reduction of radiolabelled leucine uptake was observed in the presence of a large excess of cold leucine (fig. 3). the transporter recognized other neutral amino acids, since a 56 %, 34 % and 37 % inhibition was observed with an excess of histidine, serine and glycine, respectively (fig. 3). the percent inhibitions were calculated with respect to the control value after subtraction of the residual uptake in the presence of an excess of leucine. the uptake was not affected by proline, glutamine or by the dibasic amino acids lysine and arginine, suggesting the presence of other transporters for the intestinal absorption of these amino acids. while protocols for the preparation of bbmv from whole larvae are now available (abdul-rauf and ellar, 1999; parenti et al., 2000; this paper), no procedures are actually known to obtain bbmv directly from adult diptera, a severe limit to the investigation of the physiological processes occurring at the apical membrane of midgut cells and to the detection of new drugs potentially noxious to the brush border, that may be used as pesticides. we have, therefore, developed a new procedure based on the fractionation of the homogenate on a continuous density gradient of percoll, followed by calcium-precipitation (fig. 1). a complete homogenization of b. oleae and m. domestica adults was obtained with the polytron homogenizer. the brown-black color developed during homogenization remained in the pellet of the first lowspeed centrifugation, that is discarded, so that no dark coloration was observed in the following steps of the procedure. the plasma membranes stratified as a distinct diffused band located at the third upper portion of the continuous density gradient formed by percoll. the residual percoll present in the collected fraction was later removed as a glassy pellet after the high speed centrifugation. 142 table 2 protein yield and activities of the plasmamembrane marker enzymes in homogenate and bbmv from m. domestica and b. oleae adults protein yield (mg protein/g insects) b. oleae m. domestica bbmv 0.03 ± 0.01 (3) 0.13 (2) enzyme activity (mu/mg protein) b. oleae aminopeptidase n γ-glutamyl transferase na+/k+ atpase homogenate 19.0 ± 0.04 (3) 2.0 ± 0.1 (3) 83 ± 7 (3) bbmv 531 ± 18.2 (3) 58.3 ± 6.8 (3) 275 ± 24 (3) enrichment factor* 31.9 ± 9.6 (3) 28.8 ± 3.2 (3) 3.3 ± 0.2 (3) m. domestica aminopeptidase n γ-glutamyl transferase na+/k+ atpase homogenate 17.2 (2) not detectable 380 ± 40 (2) bbmv 65.0 (2) 52.2 (2) not detectable enrichment factor* 3.8 (2) values are means ± se in brackets are reported the number of independent preparations assayed *ratio of enzyme specific activity in bbmv and in the homogenate as shown in table 2, the final pellet obtained from b. oleae was enriched 30-fold in aminopeptidase n and γ-glutamyl transferase activities with a very low enrichment for na+/k+ atpase, a reliable indication that the protocol leads to a highly purified brush border membrane preparation. however, bbmv protein yield was extremely low (table 2), a result more likely related to the fact that the intestinal epithelium represents a very small portion of the starting material rather than to the complexity of the procedure. the microphotograph of the final pellet (p5) indicated the presence of closed vesicles and of some amorphous material (fig. 4). the analysis of the purity of the bbmv from m. domestica (table 2) confirms that this final preparation is also fairly purified. the enrichment factor of aminopeptidase n was lower than that obtained for b. oleae but the final pellet was not contaminated by basal membranes, since no na+/k+ atpase activity was detectable. the enrichment in brush border membranes was also confirmed by the emergence in the bbmv pellet of a γ-glutamyl transferase activity, too diluted in the homogenate to be detected. a characterization of b. oleae and m. domestica bbmv was performed by sds-page (fig. 5). numerous distinct bands were resolved by the gel. the patterns produced by the bbmv of the two species were very similar (lanes 3 and 5), while they fig. 4 electron micrograph of the final membrane pellet from b. oleae whole adults. 143 fig. 5 comparison of sds-page profile of bbmv prepared from adults of b. oleae (lane c) and m. domestica (lane e). lane a) molecular weights (kda); lane b) bbmv prepared from bombyx mori larval midgut; lane d) homogenate from adults of m. domestica. were evidently different from that obtained with midgut bbmv of a lepidopteran larva (lane 1) here presented for comparison. the apparent molecular masses of some major bands, present in the two dipteran bbmv and lacking in the homogenate (lane 4), correspond to a number of proteins involved in the architecture of microvilli cytoskeleton, such as actin (43 kda), myosin i (110 kda) and fimbrin (68 kda). in conclusion, our experimentation provides a midgut preparation from larvae of a pest insect, that can be used to characterize fundamental physiological processes such as amino acid absorption. the in depth knowledge of these processes may provide targets for new pesticides. the novel preparation of midgut bbmv from dipteran whole adults, obtained for the first time, may contribute to solve the problem of the identification of the mechanism of action of insecticidal molecules acting on midgut brush border membrane in insects of small dimension. acknowledgements we are grateful to prof. m de eguileor (dipartimento di biologia strutturale e funzionale, università dell'insubria, italy) for the tem image of bbmv pellet. references abdul-rauf m, ellar 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brush border membrane of pieris brassicae midgut cells. febs lett. 204: 213-218, 1986. terra wr. physiology and biochemistry of insect digestion: an evolutionary perspective. brazilian j. med. biol. res. 21: 675-734, 1988. wolfersberger mg, lüthy p, maurer a, parenti p, sacchi vf, giordana b, et al. preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (pieris brassicae). comp. biochem. physiol. 86a: 301-308, 1987. wolfersberger mg. localization of amino acid absorption systems in the larval midgut of the tobacco hornworm manduca sexta. j. insect physiol. 42: 975-982,1996. 145 dipartimento di biologia, università degli studi di milano, milano, italy accepted december 20, 2006 transmission and scanning electron microscopy isj114.pdf 25 isj 3: 25-39, 2006 issn 1824-307x review the sea urchin immune system lc smith1, jp rast2, v brockton1, dp terwilliger1, sv nair3, km buckley1, aj majeske1 1department of biological sciences, george washington university, washington dc, usa 2division of molecular and cell biology, sunnybrook and women's research institute, department of medical biophysics, university of toronto, toronto, canada 3department of biological sciences, macquarie university, sydney, australia accepted may 10, 2006 abstract metchnikoff’s use of sea star larvae to observe encapsulation and phagocytosis, which was followed much later by allograft rejection kinetics, revealed that echinoderms had an innate immune system that was lacking of adaptive attributes. larval sea urchins mount defenses in response to contact with microbes, which are mediated by phagocytic blastocoelar cells and pigment cells. in the adult, the coelomocytes mediate immune responses through phagocytosis and encapsulation of foreign particles in addition to degranulation of antimicrobial molecules. molecular analysis of immune functions in the sea urchin has demonstrated a complement system that appears to have multiple alternative pathways and several activators of the lectin pathway, but may be missing the terminal pathway. other genes and proteins involved in the sea urchin immunity include expanded sets of lectins, proteins with scavenger receptor cysteine-rich repeats, toll-like receptors and associated signalling proteins. a vast array of proteins belonging to the 185/333 family are expressed in coelomocytes in response to lipopolysaccharide and show a surprising level of diversity. the sea urchin innate immune system has a number of large gene families with unexpected complexities and elevated levels of diversification. key words: evolution; deuterostome; echinoderm; coelomocyte; innate immunity; diversification; complement; 185/333 genes introduction for many years, it was thought that invertebrates did not possess a recognizable immune system, a viewpoint which ignored metchnikoff’s seminal research on the mechanisms of inflammation (metchnikoff, 1893). he was the first to demonstrate cellular encapsulation of a foreign body inserted into an invertebrate, and employed the larval sea star, astropecten pentacanthus. metchnikoff extended this observation to corresponding author: l. courtney smith department of biological sciences, george washington university, 2023 g st nw, washington dc 20052, usa email: csmith@gwu.edu a phylogenetically diverse array of animals and was able to establish that phagocytic cells are a basic characteristic of metazoan biology. these observations formed the basis of the emerging field of cellular immunity for which he was awarded the nobel prize in 1908. however, it was not until seventy years later that hildemann and colleagues demonstrated the ability of several echinoderm species to differentiate self from nonself tissues through allograft rejection studies (hildemann and dix, 1972; karp and hildemann, 1976). these initial observations were extended in studies of sea urchins, lytechinus pictus and strongylocentrotus purpuratus, in which the non-specific nature of innate immune responses of the sea urchins was defined (coffaro and hinegardner, 1977; coffaro, 1979, 1980; smith and davidson 1992). these and other studies identified the cells of the open circulatory system in adult echinoderms, the coelomocytes, as the main effectors of defense 26 responses and as the primary mediators of allograft rejection (hildemann and dix, 1972; coffaro and hinegardner, 1977), response to injury or infection, and the clearance of foreign substances (reinisch and bang, 1971; bertheussen, 1981; yui and bayne, 1983; plytycz and seljelid, 1993). of the echinoderms, the genus strongylocentrotus is one of the most comprehensively studied with respect to immunological defense capabilities, particularly in the adult (reviewed by gross et al., 1999). yet, the embryonic and larval forms also must contend with microbes in marine waters (reviewed by smith, 2005), and studies to understand the immune capabilities at those life stages are underway. larval immunity the life cycle of most sea urchin species begins with an egg in which fertilization and development are initiated after release into the water column. although there is significant variation among species, the resulting ciliated embryo typically hatches from the fertilization envelope just prior to gastrulation and develops into a simple feeding larva that lives in the plankton and bears little resemblance to the adult form (fig. 1). pluteus larvae are bilateral with an internal calcite skeleton that supports the overall structure of the larva including the gut, the blastocoelar spaces, and the “arms” that extend out from the oral side of the organism. cilia cover the animal but are concentrated in bands at the intersection of the oral and aboral ectoderm (strathmann, 1975). these ciliated bands enable larvae to swim and remain within the plankton near the surface, as well as to sweep food particles into the mouth and down the esophagus. planktonic larvae eat a variety of organisms including bacteria, single celled and multicellular eukaryotes. both the embryos and the larvae live in environments teeming with potential microbial pathogens, including those that are introduced into the gut. consequently, these animals must have mechanisms for protecting themselves against microbial colonization and invasion of the two major potential sites of pathogen entry; the ectodermal surfaces and along the internal surfaces of the gut (reviewed by smith, 2005). in a similar but more recent study than those by metchnikoff, silva (2000) demonstrated that embryonic mesenchyme cells were capable of phagocytosing yeast that had been injected into the blastocoelar cavity. although the identity of these cells was left unclear, possible candidates for embryonic phagocytes include the blastocoelar cells that populate the blastocoelar spaces of the larva and pigment cells that are found within and near the ectoderm (fig. 1) (gibson and burke, 1985, 1987). both of these cell types originate from the secondary mesenchyme cells that are specified from a mesodermal ring in the gastrulating embryo. during gastrulation ~30 pigment cell precursors migrate to positions in the aboral ectoderm (gibson and burke, 1985, 1987), and after differentiation, they are filled with granules containing red pigment and express a set of enzymes required to produce echinochrome (castellani et al., 2003). echinochrome a has been shown to have effective antibacterial properties (service and wardlaw, 1984; see below), suggesting that pigment cells may have protective functions within the larval ectoderm. the blastocoelar cells develop from the secondary mesenchyme cells that migrate from the tip of the extending archenteron at late gastrulation (tamboline and burke, 1992). approximately 20 of these cells take up residence in the blastocoel, form long pseudopodia, and are particularly concentrated around the gut (fig. 1). recently, both of these cell types have been shown to be capable of bacterial recognition and phagocytosis (hibino and rast, unpublished). the combination of these two cell types thus may ‘patrol and protect’ the larval ectoderm and gut from microbial colonization and invasion. commonly, phagocytosis either requires, or is significantly augmented by, molecular interactions between the phagocyte membrane and foreign particles. this interaction may be mediated by either an opsonin deposited on the foreign particle for which the phagocyte has a receptor, or by the presence of a receptor on the phagocyte that directly recognizes a microbial molecular pattern. no information is available as to whether opsonins or specific receptors are required for microbial phagocytosis by blastocoelar cells or degranulation of echinochrome by pigment cells. however, silva (2000) suggested that contact with blastocoelar fluid might be required for phagocytosis of yeast by cells in the embryos and larvae of the sea urchin, lytechinus variagatus. it is therefore interesting that two homologues of sea urchin complement c3 (spc3 and spc3-2; see below for more details) are expressed in the embryos and larvae. furthermore, the gene encoding spc3 shows low expression in embryos, but is elevated in response to contact with bacteria just prior to gastrulation (shah et al., 2003). spc3 is known to function as an opsonin in adult sea urchins (clow et al., 2004), and may have a similar function in the embryos and larvae. the second sea urchin c3 homologue, spc3-2, is not well characterized but is expressed at higher levels in the gastrulating embryo and larvae, whereas the transcripts are much less prevalent in adult coelomocytes (rast, unpublished). because spc3 is highly expressed in coelomocytes and spc3-2 is more highly expressed in embryos and larvae, these two c3 homologues may represent the core of two alternative complement pathways that operate at two different developmental stages of the sea urchin (see below). metamorphosis and adult coelomocytes the larvae of indirect-developing sea urchins feed in the plankton for weeks to months in a developmental process, which serves, disperse the larvae far distances from their benthic parents. after a period of feeding, the adult rudiment forms within left side of the larva (okazaki, 1975) and upon full development, the larvae leave the plankton and metamorphose if they settle on an appropriate substrate. the adult rudiment is everted into 27 fig. 1 cells with immune function in the sea urchin pluteus larva. blastocoelar cells with pseudopodia (some marked with white asterisks) occupy the blastocoel and surround the gut. pigment cells (some marked with red asterisks) are usually in close apposition to the aboral ectoderm. a single pigment cell with vesicles is shown among cells of the aboral ectoderm (left inset). the same pigment cell is shown in outline in the right inset. subdivisions of the gut are indicated for orientation. f, foregut; m midgut; h, hindgut. the spicular skeleton is birefringent in the preparation. a juvenile, leaving a “turban” of larval tissues on the dorsal side of a tiny sea urchin that has five tube feet and five spines. this process transforms the animal from a bilateral larval form to a pentamerous juvenile. the anatomy of the fully metamorphosed sea urchin is essentially the same as the adult with aristotle’s lantern (mouth parts), gut, and gonad being the major organs in the coelomic cavity. filling the spaces between the organs within the coelomic cavity is coelomic fluid that contains coelomocytes. there are a variety of coelomocyte types in s. purpuratus that can be differentiated based on their structural attributes (johnson, 1969; bertheussen and seljelid, 1978; edds, 1993; gross et al., 2000). four categories are currently recognized; phagocytes, red spherule cells, colorless spherule cells and vibratile cells (table 1; fig. 2). the proportions of each type of coelomocyte in the coelomic fluid vary considerably (table 1) at both the interand intra-individual level (smith, unpublished). this variability may reflect the nutritional, immunological and homeostatic status of the individual sea urchin from which the coelomocyte samples were taken (smith and davidson, 1994; reviewed by gross et al., 1999). the phagocytes constitute the majority of coelomocytes and have been reported to be involved in graft rejection, chemotaxis, phagocytosis, encapsulation, immune gene expression, agglutination (gross et al., 2000; clow et al., 2004; reviewed in gross et al., 1999) and clotting reactions (hillier and vacquier, 2003). phagocytes also appear to be the major cell type to express immune genes including complement homologues, a c-type lectin and a homologue of nfkb (fig. 3). phagocytes can be sub-categorized into three types based on morphology. type 1 phagocytes (fig. 2a, b), also known as discoidal cells, can be readily differentiated from type 2 phagocytes (fig. 2c, d), or polygonal cells, by their distinct cytoskeletal morphologies (edds, 1993; henson et al., 1992; 1999). discoidal cells have radially arranged actin filaments emanating from the central nuclear region of the cell, whereas polygonal cells have their actin filaments arranged laterally and lie along the length of the cell membrane forming irregular polygonal shapes (edds, 1993). the discoidal cells are stationary in vitro, while the polygonal cells show greater motility (henson et al., 1992) and can translocate across glass like fibroblasts when in contact with coelomic fluid proteins (edds et al., 1983). differences in these two cell types also include subcellular localization of kinesin, microtubules, and myosin ii, in addition to positioning of the mitochondria (henson et al., 1999). the possible developmental relationships between these two subtypes of phagocytes have not been described. small 28 phagocytes (fig. 2e), a third type of phagocyte identified by gross et al. (2000), are smaller with less cytoplasm than either of the other two cell types. they have not been reported previously, perhaps because of their small size and low numbers. to date, the function of these cells is unknown. there are two types of spherule cells, those with red spherules, also called morula cells (fig. 2f), and those with colorless spherules (fig. 2g). they are similar in size and are both amoeboid (matranga et al., 2006), however the red spherule cells are significantly more dense than the colorless type, enabling separation by density centrifugation (bertheussen and seljelid, 1978; gross et al., 2000). the red spherules contain echinochrome a, a naphthoquinone which gives the cells their characteristic red color. echinochrome a is degranulated in the presence of bacteria (johnson, 1969) and has antimicrobial properties against both gram positive and gram negative bacteria (service and wardlaw, 1984; gerardi et al., 1990; haug et al., 2002). red spherule cells accumulate around injuries and sites of infection (johnson, 1970; coffaro and hinegardner, 1977), suggesting that these cells and echinochrome a play a role in the immune response in adult sea urchins. the functions of the colorless spherule cells have yet to be identified. vibratile cells (fig. 2h) are spherical and show no amoeboid movement but have a single flagellum, which may propel them through the coelomic fluid. they have been associated with clotting reactions (bertheussen and seljelid, 1978), which is an important response to injury. upon immediate removal from a sea urchin, the phagocytes appear as bladder amoebocytes (bertheussen and seljelid, 1978) or petaloid phagocytes (edds, 1979; 1983), which is a description of lamellipodia that extend from the cell body in all dimensions. this may be the normal morphology of phagocytes while in the coelomic cavity, but upon settling on glass, the petaloid table 1 coelomocyte cell types and functions in the purple sea urchin strongylocentrotus purpuratus. cell type % in coelomic fluid morphology function type 1 discoidal cell dense nucleus with clear disc shaped cytoplasm, devoid of organelles with actin striations radiating from the center (fig. 2a, g) immune function: encapsulation, opsonisation (clow et al., 2004), chemotaxis and phagocytosis (reviewed by gross et al., 2000), graft rejection (reviewed by edds 1993) type 2 polygonal cell clear nucleolus (granular nucleus) and obvious organelles in the surrounding cytoplasm. forms large thin irregular shapes. actin bundles run parallel to straight edges of cell (fig. 2g, h) immune function: chemotaxis, opsonisation (clow et al., 2004), phagocytosis (reviewed by gross et al., 2000), antibacterial activity due to lysozyme (gerardi et al., 1990), clotting and encapsulation, graft rejection (reviewed by edds 1993) type 3 small phagocyte 40-80 % (total phagocytes) (edds 1993, gross et al., 2000 and smith l.c., unpublished) smaller than both type 1 and type 2 with little cytoplasm (fig. 2c) unknown red spherule 7-40 % (gross et al., 2000 and smith l.c., unpublished) amoeboid cells with red spherical inclusions, (fig. 2d) antibacterial activity not due to lysozyme (gerardi et al., 1990) contains echinochrome a (service and wardlaw 1984) colorless spherule 3.7-25 % (gross et al., 2000 and smith l.c., unpublished) colorless amoeboid cells with spherical inclusions; may be several types in other echinoderms (fig. 2e) unknown vibratile (boolootian and geise 1958, johnson 1969, karp and coffaro 1980, smith 1981) 11.9-20 % (gross et al., 2000 and smith l.c., unpublished) round colorless cells with a single flagellum (fig. 2f) movement or agitation of coelomic fluid? associated with clotting (bertheussen and seljelid, 1978) 29 fig. 2 coelomocyte types in the sea urchin, s. purpuratus. phase contrast images of live cells are shown for type 1 discoidal phagocyte (a), type 2 polygonal phagocyte (c), small phagocyte (e), red spherule cell (f), colorless spherule cell (g) and vibratile cell (h). fluorescent images of a type 1 discoidal cell (b) and a type 2 polygonal cell (d) were obtained after settling the cells on poly-l-lysine (0.1 �g/ml) coated coverslips for 5 min in calcium magnesium-free seawater with 30 mm edta, and then in coelomocyte culture media (henson et al., 1999) for 30 min. the cells were fixed and stained with a monoclonal anti-actin antibody (icn) followed by goat anti-mouse ig labeled with fluorescein and counterstained with hoescht 33258 (molecular probes) nuclear stain. all images were collected using a zeiss axioplan fluorescence microscope. scale bar in a and c is 15 µm. scale bar in e, f and g is 12 µm. scale bar in h is 10 µm. lamellipodia transform into discoidal or polygonal morphology and may show ruffling (henson et al., 1992; edds, 1993). this cellular behaviour has been considered akin to encapsulation, because the glass slide is perceived as foreign (edds, 1993). upon exposure to hypotonic media, lamellipodia of both discoidal and polygonal cell types transform into filopodia starting with the formation of serrations at the lamellipodial edge followed by cytoplasmic retraction and filopodial extension (edds, 1980). the filopodia have been described as forming physical links among several phagocytes, or filopodial intertwining, to result in a cellular clot, which is retracted through filopodial shortening (edds, 1977; smith, 1981). a complement system in sea urchins coelomocytes are the central mediators of immune responses in sea urchins. besides graft rejection, encapsulation, and cellular clot formation (smith and davidson, 1994; reviewed by gross et al., 1999), coelomocyte activities include the efficient clearance of a broad array of foreign cells or particles injected into the coelomic cavity, including bacteria (bertheussen, 1981; yui and bayne, 1983; plytycz and seljelid, 1993), xenogeneic cells (reinisch and bang, 1971), latex beads and yeast (bertheussen, 1981), bacteriophage (coffaro, 1979), and red blood cells (kaplan and bertheussen, 1977; bertheussen, 1981; for reviews see smith and davidson, 1994; gross et al., 1999). fig. 3 phagocytes are the major expressors of immune response genes. expression of several immune genes were analyzed in fractions of coelomocytes by reverse transcriptase – pcr. the genes analyzed encode complement proteins spc3 (clow et al., 2000) and spbf (smith et al., 1998; terwilliger et al., 2004), a c-type lectin, sp056, called spechinoidin (genbank accession number; aar02404) and the transcription factor spnfkb (pancer et al., 1999). actin expression was used as the control. phagocytes show the strongest expression of all genes while the red spherule cells also express spnfkb. total rna was isolated from density separated coelomocytes (see gross et al., 2000), reverse transcribed with a random hexamer and employed in pcr with primers specific for the genes listed to the right of the image. lane 1, phagocytes; lane 2, mix of vitratile cells and colorless spherule cells; lane 3, red spherule cells. lanes 2 & 3 contain < 2.1% phagocytes, and may account for the minor bands in those lanes. 30 phagocytosis of sheep rbcs (srbcs) by coelomocytes was not efficient until the srbcs were opsonized with human igm and c5-depleted serum (which blocked the lytic pathway) (kaplan and bertheussen, 1977; bertheussen, 1981) and led to further investigations. using isolated human complement components for controlled srbc opsonization also augmented phagocytosis of srbcs and suggested that both c3b and the inactivated form, c3bi, were involved in phagocytosis (bertheussen, 1982). these results inferred that type 3 complement receptors were present on the phagocytes and lead to the speculation that sea urchins might have a complement system (bertheussen, 1983). molecular identification of sea urchin complement components an early molecular investigation of genes expressed in sea urchin coelomocytes showed that profilin transcripts were up-regulated in response to injections of lipopolysaccharide (lps) into the coelomic cavity (smith et al., 1992, 1995). the implication was that because profilin functions in cytoskeletal modifications (paavilainen et al., 2004), its elevated expression indicated immune activation in coelomocytes as reflected by increased amoeboid movement, phagocytosis and secretion, all of which requires changes in cell shape. consequently profilin was used as a marker for cell activation, and pools of coelomocytes were employed in an expressed sequence tag (est) study to generate a snapshot of gene expression in the coelomocytes responding to lps and to provide a basic understanding of the molecular immunological functions of these cells (smith et al., 1996). this study yielded 307 ests, of which 89 matched to known sequences derived from 55 distinct genes. of those ests, two matched to mammalian complement proteins as had been predicted by bertheussen (1982). spc3 identification est064 was a new member of the thioestercontaining complement protein family that includes c3, c4, and c5 (smith et al., 1996). alignments of the full length sequences showed that the deduced sea urchin protein matched most closely to other c3 proteins and was therefore called spc3 (al-sharif et al., 1998). the spc3 protein contained a conserved α/β cleavage site to produce a mature protein with two chains and a conserved thioester site in the α chain. sequence analysis of spc3 identified n-linked glycosylation sites, cleavage sites for factor i and binding sites for factor h and factor b, leading to speculation that spc3 functioned similarly to other c3 homologues (al-sharif et al., 1998; smith et al., 1999, 2001; smith, 2001). immunoquiescent sea urchins have either undetectable or very low levels of spc3 in the coelomic fluid (gross et al., 1999). upon immune challenge with lps, however, spc3 protein appears in the coelomic fluid within 15 minutes (clow et al., 2000). furthermore, spc3 is produced by a subset of phagocytes that appear to maintain the protein within small cytoplasmic vesicles (fig. 4). in addition to the spc3 secretion response, there is also an increase in number of phagocytes containing spc3 after challenge (gross et al., 2000). spc3 was the first complement component identified in an invertebrate, which galvanized others to search for and to find complement systems in a wide variety of animals. phylogenetic analysis of the thioester-containing protein family placed spc3 basal to the chordate complement proteins and therefore identified it as a homologue of the common ancestor of the c3, c4 and c5 family in deuterostomes (al-sharif et al., 1998). c3 homologues have also been identified in the tunicates, halocynthia roretzi, styela plicata and ciona intestinalis (nonaka and azumi, 1999; marino et al., 2002; raftos et al., 2002) and until recently, only deuterostomes were thought to contain complement-like proteins. however, c3 homologues have been found in the horseshoe crab, carcinoscorpius rotundicauda (zhu et al., 2005) and in the gorgonian coral, swiftia exerta (dishaw et al., 2005), indicating that c3 appeared prior to the protostomedeuterostome split. thioester-containing proteins have also been identified in both drosophila (lagueux et al., 2000) and the mosquito, anopheles gambiae (levashina et al., 2001), but phylogenetic analysis of these proteins indicates that they fall into a different clade than either the complement clade or the α2-macroglobulin clade proteins (blandin and levashina, 2004). fig 4. spc3 is localized in small vesicles in phagocytes. confocal fluorescent micrographs of spc3 localization in the type 1 and type 2 phagocytes of s. purpuratus. density separated phagocytes were centrifuged onto slides, fixed and stained with spc3-anti-peptide antiserum followed by goat anti rabbit ig conjugated to alexa (gárig-a, pierce). images were captured using a bio rad mrc 1024 confocal laser scanning system attached to an olympus imt2-rfc inverted microscope (for details, see gross et al., 2000). scale bar is 15 µm. 31 the identification of thioester containing proteins, which appear to be present throughout the animal kingdom, suggests that this mechanism for recognizing, binding to, and eliminating pathogens is very ancient. spc3 function sequence analysis had indicated that spc3 was a c3 homologue, but it was important to determine whether the function of spc3 was conserved. based on the structure of c3 and c4, the active thioester site is recessed in a pocket and partially protected from deactivation (reviewed in sim and sim, 1983). however, when activated c3 proteins are denatured and heated under alkaline conditions, thioester sites undergo autolysis causing α chain cleavage (sim and sim, 1981, 1983). autolysis does not occur when the thioester has either not been activated or has been deactivated, and therefore the chemical reaction can be used to assess the fraction of proteins containing active thioester sites. alternatively, thioester function can also be demonstrated by binding small nucleophiles such as methyl amine or hydroxylamine. when these approaches were used to analyze spc3, autolysis was found to occur in a fraction of the proteins that varied among individual sea urchins (smith, 2002). furthermore, spc3 bound both methylamine and yeast, which blocked autolysis, and suggested that spc3 could function as an opsonin. consequently, when yeast were opsonized using coelomic fluid from immune activated sea urchins, phagocytosis was augmented (clow et al., 2004). overall, these data indicate that the thioester site is functional on spc3 and that it is a major opsonin in sea urchin coelomic fluid (smith, 2001, 2002). spbf identification factor b (bf), which has been found throughout the deuterostomes (ishiguro et al., 1992; nakao et al., 1998; smith et al., 1998; nonaka and azumi, 1999; azumi et al., 2003), is the second component of the alternative pathway, and the second complement homologue identified in the sea urchin (smith et al., 1996, 1998). bf is a mosaic protein containing several short consensus repeats (scrs), a von willebrand factor (vwf) domain and a serine protease domain. most bf proteins have three scrs, however, exceptions include the carp bf homologue with four scrs (nakao et al., 1998), and the sea urchin bf protein (spbf) with five scrs (smith et al., 1998). the bf protein in the tunicate, c. intestinalis, is quite different and has three scrs and two cub (c1r, u-epidermal growth factor, bone morphogenic protein) domains (azumi et al., 2003). expression of the gene encoding spbf (sp152) was constitutive and not affected by challenge with lps (terwilliger et al., 2004). along with the full-length sp152 transcript containing five scrs, some sp152 transcripts were alternatively spliced into remove the first and/or the fourth scr (terwilliger et al., 2004). however, only those messages that were missing the fourth scr encoded an in-frame protein. messages in which the first scr was deleted had a frame shift which resulted in an early stop codon and a truncated protein. therefore, it appears that in most higher deuterostomes, bf proteins have three scrs, while in lower deuterostomes, including s. purpuratus and the tunicate c. intestinalis, bf proteins have four or five scrs (azumi et al., 2003; terwilliger et al., 2004). it is not clear from the phylogenetic analyses of bf proteins whether the evolutionary process has selected for the loss of scrs in higher deuterostomes from an ancestral protein structure containing five scrs, or if the lower deuterostomes have undergone domain duplication more recently to result in bf proteins with five scrs (terwilliger et al., 2004). complement systems in lower deuterostomes in higher vertebrates, the complement system is composed of about 35 serum and cell surface proteins (volanakis, 1998) and is categorized into three activating pathways, called classical, alternative and lectin, which unite to trigger the terminal pathway (dodds and law, 1998) (fig. 5). in addition, there are a number of regulatory proteins located in both the serum and on cell surfaces, and complement receptors located on many cell types including phagocytes. analysis of the c. intestinalis genome has shown preliminary identification of many complement components in this invertebrate including duplications of proteins in the alternative and lectin pathways, and several matches to c6 in the terminal pathway (azumi et al., 2003; reviewed in nonaka and yoshizaki, 2004). although cloning and sequencing have identified c3 and bf homologues in the sea urchin, searches of the first build of the sea urchin genome (7/18/05 assembly) have uncovered additional gene models that match to complement homologues. these include four members of the c3/4/5 family, three members of the bf/c2 family, mannose binding lectin (mbl), and several matches to c1q (fig. 5) (unpublished). in addition, a number of matches were identified to variant forms of mbl-associated serine proteases (masps) and to proteins with a perforinmembrane attack complex (macpf) domain. with three gene models that encode c3 homologues and three that encode bf homologues, the sea urchin complement system appears as an expansion of the alternative pathway, perhaps with activation through an expanded lectin pathway using mbl, c1q and masps, plus a possible terminal pathway. however, without clear identification of members of the terminal pathway, the major function of this system may be opsonization. a complement system consisting of the alternative and lectin pathways and functioning in opsonization can be of significant value in combating microbial invasion. the alternative pathway in higher vertebrates has been considered to be the core of the complement system, and the current characterization of the sea urchin system emphasizes this notion. the positive feedback loop significantly augments the rate at which foreign particles can be opsonized and is therefore more effective and efficient than individual opsonins such as simple lectins that rely on diffusion to bind to the target. the presence of a c3-convertase in the sea urchin, composed of a complex of spc3 and spbf, and the presence of a positive feedback loop has been suggested previously 32 fig. 5 the complement cascade. the mammalian complement cascade is shown with the known (green circles) and predicted (green striped circles) sea urchin complement proteins mapped onto it. complement proteins known in ciona intestinalis are circled with a bold line. the dotted line represents the positive feedback loop in the alternative pathway. the dashed line suggests the possible activation of masps by c1q homologues in both sea urchins and tunicates. and might be of significant benefit to the species (fig. 5) (smith et al., 1999, 2001; smith, 2001). the importance of the complement system within the immune response of the sea urchin is reflected by multiple alternative pathways that may be activated through mbl or c1q. these results infer the importance of opsonization followed by phagocytosis by coelomocytes for removing and destroying invading microbes. regulatory proteins and receptors in the complement system the amount of functional c3 in body fluids depends on the rate of thioester activation vs. deactivation either by the formation of covalent bonds with a target or by hydrolysis. the thioester site is an intra-chain bond between the side groups of cysteine and a nearby glutamic acid (dodds and law, 1998) and when activated, becomes available for ester or amide bond formation with nearby substrate molecules. normally, if covalent bonds are not formed, the reactive thioester undergoes hydrolysis due to the abundance of surrounding water, thereby limiting the spread of reactive thioester-containing molecules (dodds and law, 1998). the short time frame of thioester reactivity helps to ensure that covalent bond formation occurs near where complement activation was initiated, usually on the surface of a pathogen rather than in the plasma or on host cell surfaces. however, autoactivation of c3 and augmented c3 activation by c3-convertase functioning within the positive feedback loop can lead to large amounts of activated c3 and inappropriate deposition of c3 fragments on host cells. consequently, to protect self tissues from autologous complement attack, a number of regulatory proteins are required both in body fluids and on cell surfaces. many of the regulatory proteins and complement receptors in higher vertebrates are constructed of short consensus repeats (scrs) or complement control protein (ccp) modules. searches of the sea urchin genome reveal 247 proteins with scr domains (7/18/05 assembly). it is feasible that some of these gene models will be characterized in the future to encode complement receptors and regulatory proteins that function to protect self. two examples of expressed genes have been identified that encode proteins with putative complement regulatory function or may be members of a primitive terminal pathway (multerer and smith, 2004). they are spcrl (s. purpuratus complement related protein, long form) and spcrs (short form) and both have multiple scrs and a fimac (factor i-membrane attack complex) domain. both share domains with factor h and factor i, which have regulatory functions, and c6 and c7, which are members of the terminal pathway. other genes expressed in coelomocytes lectins in addition to the complement system, c-type lectins may also be of significant importance in immune defense p c3 c3b c5 c6 c7 c8 c9 c3a c5a c4 masps microbes terminal pathway lectin pathway alternative pathway d c1q c1r, c1s c4b2bc4b c2 c2a c4a c4b2b3b classical pathway antigen antibody c3 c3b c3bbb c3bbb3b c3a bf ba mbl ficolin ? p c3 c3b c5 c6 c7 c8 c9 c3a c5a c4 masps microbes terminal pathway lectin pathway alternative pathway d c1q c1r, c1s c4b2bc4b c2 c2a c4a c4b2b3b classical pathway antigen antibody c3 c3b c3bbb c3bbb3b c3a bf ba mblmbl ficolinficolin ? 33 in the sea urchin. c-type lectins are carbohydrate binding proteins that require ca2+ for proper conformation and function of the carbohydrate recognition domain (crd). an est that matched to a c-type lectin, shows significant sequence similarity to echinoidin (smith et al., 1998), which was previously characterized in the sea urchin, anthocidaris crassispina (giga et al., 1987). spechinoidin (echinoidin from s. purpuratus; genbank accession ay336600) has six cysteines in conserved positions and a seventh that suggests that it may form a homodimer (smith et al., unpublished). the carbohydrate binding motif composed of three amino acids within the crd, predicts that spechinoidin may bind galactose or its derivatives. the gene encoding spechinoidin, sp056, is expressed exclusively in the phagocyte class of coelomocytes and only after immune challenge (multerer and smith, 2004; terwilliger et al., 2004; smith et al., unpublished). analyses of small lectins in the coelomic fluid of s. purpuratus suggest that there is vast array that have differing carbohydrate binding specificities and are expressed in response to immune challenge (smith et al., unpublished). this is in agreement with searches of the sea urchin genome (7/18/05 assembly) in which a large number of gene models matched to single domain c-type lectins and have motifs for binding an array of carbohydrates. srcrs coelomocytes express a large, complex family of transcripts that contain domains called scavenger receptor cysteine-rich (srcr) repeats (pancer et al., 1999; pancer, 2000) and comprise a protein superfamily in metazoans. srcr domains are 110 amino acids in length and exhibit a conserved spacing of six to eight cysteine residues, which is important for intradomain disulfide bonds (freeman et al., 1990). srcr-containing proteins have been identified in a wide variety of animals (sarrias et al., 2004) and in invertebrates, have been shown to function as an aggregation receptor in a marine sponge (blumbach et al., 1998), or a sperm activation receptor in a sea urchin (dangott et al., 1989). in vertebrates, however, srcrcontaining proteins play a critical role in the regulation and development of the immune response. srcrs are expressed in both hematopoietic and non-hematopoietic vertebrate immune cells (sarrias et al., 2004), and down-regulate antigen receptor signalling on t and b cells (perez-villar et al., 1999), down-regulate host response to endotoxin (trahey and weissman, 1999), regulate apoptosis (miyazaki et al., 1999), inhibit endocytosis (takito et al., 1996), and bind pulmonary lectins (holmskov et al., 1999). relevant functions of srcr proteins within innate immunity include binding repetitive polyanionic structures such as modified lipoproteins, bacterial lipids and certain nucleotide aggregates (sarrias et al., 2004). in the sea urchin, a diverse set of srcr-containing transcripts was isolated from coelomocytes, revealing six types of srcr molecules (pancer et al., 1999; pancer, 2000). the proteins have between 2 and 20 srcr domains plus a variety of other domains, including a von willebrand factor (vwf) domain, epidermal growth factor (egf) domains, scrs, and an extracellular-matrix-like domain (ecm). the srcr transcripts are derived from a large gene family in the sea urchin and genome blots revealed complex hybridization patterns suggesting that srcr genes may exist in multiple forms and may be clustered. furthermore, the diversity of srcr genes is high both within individuals and within the population, which is supported by a preliminary analysis of the sea urchin genome (7/18/05 assembly), which indicates that there are 228 srcr gene models present. the srcr transcript repertoire of individual sea urchins is highly transient in unchallenged animals and is greatly altered after injury and bacterial or fungal challenge (pancer, 2000). unchallenged animals display changes in srcr expression of 20to 30-fold over a period of three months. animals challenged with pathogens also display variations in transcript expression of this magnitude, although there is no specific expression pattern observed among the animals. although the regulation of these genes is unknown, a high degree of conservation in the 5’ flanking region suggested possible coordinated regulation of srcr gene transcription. many of the srcr-containing genes in the sea urchin may have immunological relevance, although the specific functions of the proteins encoded by the individual genes are unknown. toll receptors and transcription factors animal toll receptors are characterized by extracellular leucine rich repeats (lrr), which function to recognize molecular pathogen signatures (pasare and medzhitov, 2005), and cytoplasmic tir (toll-il1 receptor) domains, which are important for signalling. drosophila has eight toll genes and humans have eleven, however, plant genomes have hundreds of r genes which have lrrs. similar to the plant system, the purple sea urchin has a very large array of predicted toll genes (pancer and cooper, 2006). although expression of most of these genes is currently unknown, a few are expressed in coelomocytes (see genbank accessions aak25761, aak25762). in correlation with the expansion of the toll genes in the sea urchin genome, there is a moderate expansion of genes encoding proteins that initiate the signalling pathways that are activated by toll (rast, unpublished). these include myd88 homologues, which are cytoplasmic proteins that interact with the tir domains. the end of the toll signalling pathway results in the release of rel transcription factors from their cytoplasmic inhibitors, followed by their translocation into the nucleus. spnfkb (s. purpuratus nuclear factor kappa b) was the first rel protein described in the sea urchin and is homologous to drosophila relish and vertebrate p105 and p65 proteins (pancer et al., 1999). other rel proteins have been identified in the sea urchin genome including a second nfkb homologue and one nfat homologue (unpublished). spnfkb and sprunt expression in non-activated coelomocytes is undetectable, but both transcription factors are highly expressed 6 to 12 hrs after bacterial challenge or injury (pancer et al., 1999). on the other hand, spgatac has 34 the opposite expression pattern in coelomocytes. its expression in non-activated coelomocytes is downregulated in response to bacteria and injury. based on the expression patterns in coelomocytes, spnfkb may be transcription activator for immune genes while spgatac may be a repressor (pancer et al., 1999). in embryos, sprunt expression is associated with proliferating cells (robertson et al., 2002) and consequently in coelomocytes it may be involved in proliferative responses to immune challenge. other lps-responsive ests in an effort to identify transcripts that appear in coelomocytes in response to lps challenge, an est study employed probes produced from subtractive suppressive hybridization using coelomocyte mrna from immunoquiescent sea urchins and the same sea urchins after lps challenge (nair et al., 2005). screens of arrayed coelomocyte cdna libraries identified ~6000 clones (of 92,160 clones in a library) representing putative lps-responsive genes (nair et al., 2005). est analysis of 1247 clones led to the identification of numerous novel genes expressed in s. purpuratus coelomocytes during lps challenge and included proteins that function in host defense, as cell surface receptors, signalling molecules, cytoskeletal and cytoskeleton modifying molecules, proteases, rna splicing, protein synthesis, protein processing, protein degradation, cell proliferation, and apoptosis. one of the largest categories of ests identified from both of the est studies (smith et al., 1996; nair et al., 2005) matched to cytoskeletal proteins including α and β-tubulin, dynein heavy chain, kinesin light chain, actin, gelsolin, fascin, and thymosin-β. additional matches include the mena neural variant protein, a receptor for activated protein kinase c (rack), integrin βc, protein tyrosine phosphatase receptor type f, protein tyrosine kinase 9-like protein, rho, rho-gdp dissociation inhibitor, cofilin, avena, and microtubule associated protein. this set of genes, which are upregulated in response to lps and encode both cytoskeletal proteins and cytoskeleton-regulating proteins, suggest that active and extensive remodelling of the cytoskeleton is a direct response of coelomocytes to immune challenge. this is in agreement with the initial identification of profilin up-regulation in coelomocytes responding to lps (smith et al., 1992). changes in nuclear activities were inferred from matches to proteins involved in dna transcription and mrna splicing. alternative splicing of defense-related transcripts has been observed for three complement components, spbf (terwilliger et al., 2004), spcrl and spcrs (multerer and smith, 2004), and the number of est matches with putative splicing function suggests that alternative splicing may be common. increases in messages encoding proteins involved in the synthesis, processing and degradation of proteins corresponds with coelomocyte responses to immune challenge. furthermore, matches to proteins that function in sorting (e.g. a p24 homologue which regulates vesicular traffic between the er and the golgi apparatus) and vesicular trafficking within the endosomal system (e.g. rab5interacting protein and the mannose-6-phosphate receptor homologues). indeed, previous studies have inferred that spc3 is synthesized and transported in cytoplasmic vesicles prior to secretion (clow et al., 2000; gross et al., 2000; smith, 2001). these results suggest that coelomocytes are involved in a significant level of protein production, transport and secretion beyond what is known about the complement components. est matches also indicate that coelomocytes express genes encoding proteins involved in stimulating cell proliferation (e.g. a polo-like kinase and allograft inflammatory factor), while other genes encode proteins that inhibit apoptosis (e.g. bax inhibitor-1). the activities of both these groups of proteins (i.e. proliferative and antiapoptotic) may serve to enhance coelomocyte numbers during immune challenges. such changes have been noted as increases in the numbers of cells expressing spc3 that appear in the coelomic fluid after immune challenge (clow et al., 2000). this suggests changes in cell numbers may be due to proliferation or reduced turnover rather than the release of emarginated coelomocytes into the coelomic fluid. aggressive clotting reactions are essential for protecting sea urchins from injuries and damage to the body wall because these animals have a ridged skeleton, or test, that cannot be contracted to close wounds. clotting of the coelomic fluid is a response to injuries and is a complex reaction involving both coelomocytes and coelomic fluid components. a recent study characterizing the clotting reaction in s. purpuratus showed that the coelomic fluid protein amassin is a prime mediator of the clotting reaction (hillier and vacquier, 2003) by employing disulfide bond formation; bertheussen and seljelid, 1978). this protein, which was also found to be expressed by lps-activated coelomocytes (nair et al., 2005), contains an olfactomedin domain and mediates the intercellular adhesion of coelomocytes during cellular clot formation. current analysis of the sea urchin genome (7/18/05 assembly) reveals four additional gene models with olfactomedin domains, although their functions are unknown (unpublished). although the cell surface receptor for amassin has not been identified, it may function in clotting by binding to integrins. it is noteworthy that a match to integrin βc was identified (nair et al., 2005), which was reported previously from sea urchin embryos (murray et al., 2000; burke et al., 2004). both the βc and βl integrins can be detected serologically on the surface of coelomocytes (r burke, personal communication), and a number of integrins have been identified in the sea urchin genome (unpublished). the 185/333 gene family message structure screens of the bacterially activated arrayed cdna library using subtracted probes showed that about 60 % of the positive clones matched to an unknown sequence that encoded a family of proteins (nair et al., 2005). re-screens showed that approximately 6.5 % of 35 fig. 6 full-length sequences from 81 cdna clones from the arrayed library were manually aligned and gaps (represented by horizontal lines) were inserted to optimize the alignment. the gaps separated blocks of sequence or elements, which are represented by colored boxes (numbered at the top). the figure shows a representative set of element patterns, which were established on the presence and type of element 15 (shown as varying sizes). an example of each group of cdna is represented here. group 1 is defined by element 15a (a2), group 2 by element 15b (b4), group 3 by element 15c (c1), group 4 by element 15d (d1), group 5 by element 15e (e2), and groups 6 (01) and 7 (not shown) do not have element 15. element 25 was divided into three sub-elements, 25a, b and c, based on the location of the stop codon (black vertical lines). the deduced protein shows little secondary structure. it is separated into a glycine-rich region (dotted horizontal line) and histidine-rich region (solid horizontal line). symbols indicate the presence of an rgd motif in element 7 (black star); conserved n-linked glycosylation sites (red circles) and o-link glycosylation sites (black circle); five types of repeats shown as numbered colored ovals (type 1 = red; type 2 = blue, type 3 = green; type 4 = purple; type 5 = yellow); secondary structure predictions (either á-helices or âstrands); short stretches of acidic amino acids (red vertical bars); histidine patches (purple vertical bars); and the five elements which are surrounded by putative cryptic splice signals. the scale bar is located at the lower right. (modified from terwilliger et al., 2006). the clones in the bacterially activated coelomocyte library were positive, compared to only 0.0087 % of the clones in the non-activated coelomocyte cdna library. this was in agreement with northern blots that revealed significant up-regulation in expression of this message in response to bacterial challenge (rast et al., 2000). originally identified as est333 (smith et al., 1996), later called dd185 (rast et al., 2000), and now referred to as 185/333 (nair et al., 2005), these transcripts are unusual because they display an elevated level of sequence diversity that is unexpected for an invertebrate. comparisons among 81 cdnas identified 67 different sequences encoding 64 different proteins (terwilliger et al., 2006) (fig. 6). the variability in the messages is based on the presence or absence of 25 blocks of sequence called elements, which resulted in 22 different element patterns (some of those patterns are shown in fig. 6) (terwilliger et al., 2006). besides the variations in element patterns, the 185/333 cdna sequences also show significant sequence diversity within the elements resulting from non-random single nucleotide polymorphisms (snps) and small insertions and deletions (indels). one effect of the extensive number of snps is to alter the position of the stop codon in element 25 (fig. 6). an initial analysis of sequence diversity for 42 unique ests and subsequent analysis of 81 full length cdnas found that synonymous/non-synonymous (dn/ds) ratios of the leader and first element are under positive selection for diversification, although most of the diversity is located within the first element and not the leader (nair et al., 2005; terwilliger et al., 2006). the 185/333 cdnas can be categorized into seven groups based on the presence or absence and length of element 15 (fig. 6), which is shared by most of the sequences, and has a large number of indels. notably, within these groups, the presence or absence of elements 13-25a (histidine-rich region and part of the terminal region) is identical, suggesting that certain suites of elements tend to appear together (terwilliger et al., 2006). analysis of the messages reveals elevated dn/ds ratios which suggest that the genes are undergoing selection for diversification, possibly as a result of pathogen pressure. initial analyses of 185/333 expression in individual sea urchins responding to bacterial and fungal molecular patterns show that the element patterns in messages present prior to challenge are generally different from those present after challenge (terwilliger et al., unpublished). furthermore, the sequence diversity of the messages present in coelomocytes after challenge is elevated compared to messages isolated prior to challenge. changes in the expression patterns of the various 185/333 transcripts in response to immune challenges suggests that expression 36 may be under some type of transcriptional or posttranscriptional control. the 185/333 proteins the 185/333 proteins are not similar to any known protein and showed little discernible secondary structure. all have a leader, all lacked cysteines, and have three distinct regions; a glycine-rich region, a histidine-rich region and a terminal region (fig. 6) (terwilliger et al., 2006). five types of repeats, 11 histidine patches, and six acidic patches are also present. a number of conserved sites for posttranslational modification are predicted throughout the deduced proteins, the most interesting of which is an rgd motif in element 7 suggesting that these proteins may bind to integrins. recent cytological analyses has shown that the 185/333 proteins are expressed by a subset of phagocytes and that they appear as diverse bands on western blots (brockton, et al.; unpublished). although the functions of the 185/333 proteins are not known, based on the timing of expression of the 185/333 genes in response to immune challenge (rast et al., 2000; nair et al., 2005), they may have an important immunological function. 185/333 gene structure and diversity the numerous element patterns initially identified from the transcripts suggested a significant level of alternative splicing (nair et al., 2005). this was thought to imply long primary transcripts from a very large gene that were spliced to produce all possible mature mrnas, a diversification mechanism similar to that found for dscam transcripts in drosophila (schmucker et al., 2000; watson et al., 2005). however, the two 185/333 genes identified in an early version of the sea urchin genome are not as predicted, and instead have two exons and a single, small intron (terwilliger et al., 2006). the first exon encodes the leader, while the second exon encodes the rest of the open reading frame, i.e., elements 1 through 25 (fig. 6). analysis of genomic dna by quantitative pcr suggests that the 185/333 gene family consisted of ~100 alleles, or 50 loci per genome (terwilliger et al., 2006). analysis of a later sea urchin genome assembly (7/18/05) shows two contigs with two or three 185/333 genes, which are spaced approximately 3 kb apart. based on the estimated number of genes per individual and the diversity levels of these genes, it is reasonable to conclude that the observed 185/333 transcript diversity may result from many small, closely linked genes that are members of a large family. this characteristic of clustered immune genes has been observed repeatedly in both animals and plants, and may be integral to gene diversification. sequences of genes cloned from two individuals show that element patterns mimic those found in the transcripts. the diversity observed from analyses of gene sequences corresponds to transcript diversity with respect to percent of unique nucleotide sequences, single nucleotide and amino acid polymorphisms, and diversity of sequences within sets of genes that have the same element pattern. these data suggest that the immune system of the sea urchin has an unknown mechanism of generating immune diversity in the 185/333 gene family. this is a shift from current paradigms, which assume that innate immunity is mediated by germline encoded proteins that have broad recognition specificities for identifying conserved molecular patterns in large classes of microbes. conclusions as we currently understand the sea urchin immune system, its most important functions appear to be opsonization and phagocytosis. the immune cells in the embryo, larva, and adult that mediate immune functions are amoeboid phagocytes. the adult coelomocytes are found in high concentrations in the coelomic fluid and are present in or on all tissues and organs in the animal. their mobility, phagocytosis and secretion functions are reflected in the large numbers of ests that match to cytoskeletal proteins and proteins involved in cytoskeletal modifications. opsonization to augment phagocytosis (or encapsulation) may be the general molecular response of sea urchins to foreign pathogens. a variety of types of opsonins are present in the sea urchin, including lectins, which bind oligosaccharides, srcrs, which bind many types of molecules including modified lipids, and the thioester-containing proteins of the complement system, which covalently bind to hydroxyl and amine groups. although the functions of the 185/333 proteins are not known, the expression of the genes in response to challenge from bacteria or lps plus their diversity supports the hypothesis that these proteins are somehow involved in coelomocyte interactions with microbes. the arrays of lectins, srcr proteins, 185/333 proteins, in addition to the expanded complement activation pathways that lead to opsonization, demonstrate the molecular diversity that is part of the sea urchin immune system. furthermore, the putative recognition system, as exemplified by the large numbers of tlrs and other llrcontaining proteins in the sea urchin genome, illustrates that both the detection and effector systems appear to be expanded and diverse. it is likely that diversification of innate immune systems in both invertebrates and plants may be driven by pathogen pressure. diversification of innate immune systems is a recent change in the previously accepted paradigm that innate immune systems are undiversified and static. the new paradigm results from recent data on sea urchins reviewed here and from analyses of other invertebrates. the importance of understanding the sea urchin system is based on the phylogenetic position of the echinoderms at the base of the deuterostome lineage and as a sister phylum to the chordates. determining how the sea urchin immune system functions and defining how it might be similar to other invertebrate and vertebrate systems will be of interest with respect to understanding the evolution of immune functions within the deuterostomes. the on-going analysis of the sea urchin genome is expected to provide additional surprises and insights into this invertebrate 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(lepidoptera: bombycidae) larvae to uvirradiation si faruki, pk kundu department of zoology, university of rajshahi, rajshahi, bangladesh accepted may 31, 2005 abstract the effects of uv-radiation on some commercially relevant traits of three instars viz. 1st, 2nd and 3rd of the two multivoltine strains, nistari-m and urboshi-1 of the silkworm, bombyx mori l. have been investigated. uv-rays reduced the weight of larvae, pupae and adults of both the strains and sexes of b. mori independently of the instar that has been treated. the cocoon weight, shell weight and shell ratios were also reduced due to uv-irradiation. increased larval mortality was recorded at all the doses of uvrays. key words: uv-radiation; bombyx mori l. growth; cocoon characters; larval mortality introduction in sericulture, the growth of various developmental stages of the mulberry silkworm, bombyx mori l. is of paramount importance because the quality of successful cocoon crop depends mostly on a healthy larval growth. radiation studies have been extensively carried out in different insects (calderon et al., 1985; mehta et al., 1990, 1991; islam et al., 1992; faruki and khan, 1993; sharma and dwivedi, 1997; hasan et al., 1998). in silkworms, few attempts have been made to find out the radiation sensitivity on different developmental stages through the use of chemical agents and ionizing radiations (mallik et al., 1968; park and hyun, 1968; subramany and reddy, 1982; tazima, 1983, 1984; ali and ali, 1998). it has been observed that radiosensitivity varies according to species, strain, individual and even at different developmental stages of the individual (tazima, 1978). dose dependent sensitivity of silkworm growth to different forms of ionizing radiations have also been reported by molnar et al. (1964), corresponding author: saiful islam faruki department of zoology, rajshahi university, rajshahi, 6205, bangladesh e-mail: faruki64@yahoo.com murakami and kondo (1964), shankarnarayanan (1982) and singh et al. (1990). moreover, gamma radiation has been used to identify the resistant and less resistant strains of silkworm (hirobe, 1974). the ultraviolet (uv) portion of the spectrum is widely used as germicide (bruce, 1975), in embryological-physiological studies (bodenstein, 1953) and for the surface disinfection of insect eggs from pathogens (guerra, et al. 1968). in bangladesh, some works have been conducted on the effect of gammarays on the eri-silkworm, samia cynthia ricini (rahman et al., 1982; khan and khan, 1991) and b. mori (rahman et al., 1983a, b). unfortunately, no investigation was done on the effect of ultraviolet radiation on the mulberry silkworm, b. mori. keeping in view the importance and feasibility of the use of uv-rays the present investigation was undertaken to evaluate the effect of uv-irradiation on commercially relevant aspects of b. mori. materials and methods the multivoltine strains of the silkworm, b. mori used in the present investigation were nistari-m and urboshi-1. newly-hatched larvae were brushed to wooden rearing trays (40 x 29 x 7.5 cm) and were reared on finely-chopped fresh, tender mulberry (morus 76 alba l.) leaves to get the 2nd and 3rd instar larvae . first instar larvae were exposed to uv-rays just after hatching from eggs, and the 2nd and 3rd instar larvae were irradiated just after completion of their moulting, i.e. no feeding was provided before irradiation. the larvae of all instars were irradiated with 254 nm wavelength of uv-rays at different durations (doses), e.g. 2, 4 and 8 min. a 15w germicidal lamp, ge15t8 measuring 20 x 4 cm was the source of uv radiation, emitting at a wavelength of 254 nm. for irradiation the test insects were kept in 15 cm diameter petri dishes placed on the surface apart 12 cm from the lamp. irradiated larvae were then reared on mulberry leaves in rearing trays up to pupation. a single batch of nonirradiated worms was simultaneously reared as controls on fresh mulberry leaves up to spinning. from the fourth instar onwards entire mulberry leaves were supplied to both irradiated and control groups. food was provided four times a day. three replications, each with 50 larvae, were made for each uv treatment and for controls. the rearing trays were kept in fine-netted cabinets. the weight of larvae was determined at maturity, i. e. one day before spinning. thirty larvae were taken randomly from each treatment and were individually weighed on an electric balance. mature larvae were transferred to bamboo-made mountages for spinning cocoons. after spinning and pupation the cocoons were harvested and stored according to their sexes. the sex was determined by cutting cocoons with a sharp blade and observing the external genitalia. cocoons were then retained for adult emergence. pupal and adult weight were individually recorded. the adult weight was determined after emergence but before coupling. the cocoon characters, i. e. whole cocoon and shell weight, and shell-ratio (%) were also noted. for each character and each treatment 30 males and 30 females were randomly selected. data of all the characters were subjected to analyses of variance. here, the variance ratio f was calculated from the ratio between treatment mean square and residual mean square and the value was compared with the tabulated value for significance. the differences between means were determined by the “student-newman-keuls (snk) test”. the mortality of b. mori larvae was observed up to pupation and data were corrected by abbott’s (1925) formula. all the experiments were conducted at a mean room temperature of 24 ± 2 °c. results and discussion the results on the effect of uv-rays on the weight of mature larvae, pupae and adults are shown in tables 1, 2 and 3. it was found that the weight of larvae decreased with increased radiation doses at all the instars of both the strains of b. mori (p < 0.001 for nistari and p < 0.05 for urboshi) (table 1). it was also observed that the effect of uv-rays was more pronounced at an early stage than an advanced stage. table 1 effect of uv-radiation on the weight (mg) of mature b. mori larvae (n = 30) instars 1st 2nd 3rd strains doses (min.) mean ± se mean ± se mean ± se f-ratio 0 (control) 1926.93 ± 15.03a 1926.93 ± 15.03a 1926.93 ± 15.03a 2 1530.86 ± 19.68b 1599.33 ± 21.74b 1670.00 ± 17.75b 4 1472.10 ± 15.63b 1703.16 ± 20.26b 1656.26 ± 24.75b nistari-m 8 1447.53 ± 20.97b 1584.43 ± 20.68b 1653.86 ± 20.57b (a) 23.91*** (b) 5.55* 0 (control) 1929.36 ± 20.92a 1929.36 ± 20.92a 1929.36 ± 20.92a 2 1735.73 ± 27.16b 1824.53 ± 50.29b 1917.30 ± 18.20a 4 1735.20 ± 45.49b 1726.30 ± 38.23bc 1916.70 ± 26.32a urboshi-1 8 1710.80 ± 37.26b 1693.96 ± 31.01c 1822.76 ± 18.67a (a) 6.92* (b) 6.25* (a) = between doses, (b) = between instars; * p < 0.05, *** p < 0.001 f = variance ratio. means followed by the same letter in each instar of each strain are not significantly different at p = 0.05 (snk test). 77 table 2. effect of uv-radiation on the weight (mg) of b. mori pupae (n = 30) instars 1st 2nd 3rd strains doses (min.) mean ± se mean ± se mean ± se f-ratio 0 (control) 808.26 ± 10.59a (914.60 ± 7.57k) 808.26 ± 10.59a (914.60 ± 7.57k) 808.26 ± 10.59a (914.60 ± 7.57k) 2 763.06 ± 9.28b (891.80 ± 6.40k) 745.66 ± 10.28b (895.86 ± 5.44k) 767.30 ± 8.56b (892.10 ± 6.33k) 4 690.73 ± 7.92c (799.26 ± 2.81l) 680.40 ± 6.48c (893.63 ± 4.46k) 732.50 ± 7.44bc (853.26 ± 7.39k) nistari-m 8 645.50 ± 11.33d (744.66 ± 8.17l) 607.23 ± 4.07d (901.10 ± 5.03k) 694.86 ± 6.87c (851.63 ± 8.65k) (a) 36.46*** (2.92ns) (b) 4.19ns (2.78ns) 0 (control) 810.13 ± 10.04a (875.26 ± 9.60k) 810.13 ± 10.04a (875.26 ± 9.60k) 810.13 ± 10.04a (875.26 ± 9.60k) 2 748.10 ± 11.30b (857.20 ±12.12k) 794.90 ± 8.77a (870.26 ± 10.78k) 800.86 ± 8.12a (858.60 ± 11.46k) 4 743.63 ± 10.35ab (849.06 ± 17.60k) 694.20 ± 5.61b (865.96 ± 8.05k) 794.33 ± 8.39a (863.50 ± 13.42k) urboshi-1 8 731.73 ± 8.17ab (783.80 ± 14.52l) 692.70 ± 4.36b (843.66 ± 8.25k) 722.90 ± 4.92b (847.86 ± 9.47k) (a) 6.07* (5.07*) (b) 1.44ns (2.24ns) (a) = between doses, (b) = between instars; * p < 0.05, *** p < 0.001 ns = not significant. data in parentheses indicate corresponding values in females. f = variance ratio. means followed by the same letter in each instar of each strain are not significantly different at p = 0.05 (snk test). there was a significant weight difference between the instars of both the strains (p < 0.05). in all the instars of nistari, uv-rays deleteriously reduced the weight of male pupae (p < 0.001) in comparison to controls but produced no effect on the weight of female pupae (table 2). the weight of male and female pupae of urboshi was significantly reduced (p < 0.05). similarly, the adult weight of both the strains and sexes were significantly reduced due to uv-irradiation (p < 0.01 for male and female of nistari, and p < 0.001 and p < 0.01 respectively for male and female of urboshi)(table 3). there was no significant difference regarding weight between the instars of both the sexes of pupae and adults of the two strains. lassota (1966), shigematsu and takeshita (1968) working with gamma-ray and coulon (1969) working with x-ray on b. mori reported that higher doses either on the eggs or the larvae decreased the body weight that corroborates with the present findings. similarly, khan and khan (1991) stated that the growth of the eri-silkworm, s. cynthia ricini was adversely affected when the eggs were irradiated with gamma rays. in the present investigation, significantly increased larval mortality was also recorded at all the instars and strains of b. mori due to uvirradiation (table 4). the cocoon weight of the two strains of b. mori was adversely affected when larvae of different instars were irradiated with uv-rays. the lighter cocoons were recorded at all the doses of uv-rays in both the strains and instars in comparison to controls (table 5). in nistari male cocoon weight was significantly (p < 0.001) reduced whereas both male and female cocoons of urboshi were severely affected (p < 0.05 and p < 0.01 respectively for male and female). the uv-rays produced no adverse effects on the shell weight of b. mori but except the male shells in 3rd instar of the strain nistari-m, the weight was reduced at all the doses of uv-rays in comparison to controls, which was not statistically significant (table 6). similarly, the shell ratios (%) was not affected except in the males of nistari where the shell ratios were significantly reduced (p < 0.001) due to uv-radiation (table 6). singh et al. (1990) also observed reduced cocoon and shell weight in b. mori due to gamma irradiation. similar result was observed by khan and khan (1991) using gamma irradiation against the eggs of s. cynthia ricini. 78 table 3. effect of uv-radiation on the weight (mg) of b. mori adults (n = 30) instars 1st 2nd 3rd strains doses (min.) mean ± se mean ± se mean ± se f-ratio 0 (control) 342.73 ± 4.20a (541.80 ± 5.77k) 342.73 ± 4.20a (541.80 ± 5.77k) 342.73 ± 4.20a (541.80 ± 5.77k) 2 326.80 ± 3.88a (522.30 ± 4.75k) 317.36 ± 4.82ab (557.66 ± 4.38k) 314.33 ± 3.20b (566.73 ± 3.01k) 4 290.83 ± 4.30b (495.53 ± 4.21l) 303.70 ± 2.54b (514.96 ± 3.13l) 308.93 ± 2.66b (551.66 ± 4.67k) nistari-m 8 263.93 ± 3.56c (480.60 ± 4.13l) 265.30 ± 4.91c (490.86 ± 4.77l) 303.63 ± 2.52b (512.93 ± 3.77l) (a) 14.24** (9.99**) (b) 0.97ns (6.22*) 0 (control) 343.50 ± 3.83a (700.06 ± 6.63k) 343.50 ± 3.83a (700.06 ± 6.63k) 343.50 ± 3.83a (700.06 ± 6.63k) 2 329.93 ± 4.72b (639.80 ± 3.93l) 331.36 ± 3.78b (634.16 ± 4.67l) 338.00 ± 3.08ab (640.16 ± 9.13l) 4 321.93 ± 3.09bc (546.76 ± 6.00m) 320.83 ± 3.03c (614.70 ± 4.50l) 330.96 ± 2.98b (602.40 ± 3.17lm) urboshi-1 8 318.53 ± 3.32c (489.10 ± 4.93m) 314.10 ± 2.99c (580.10 ± 5.95l) 310.83 ± 4.08c (574.03 ± 5.78m) (a) 26.18*** (18.56**) (b) 0.69ns (2.59ns) (a) = between doses, (b) = between instars; * p < 0.05, ** p < 0.01, *** p < 0.001 ns = not significant. data in parentheses indicate corresponding values in females. f = variance ratio. means followed by the same letter in each instar of each strain are not significantly different at p = 0.05 (snk test). table 4. effect of uv-radiation on the mortality of b. mori larvae corrected (%) mortality / instars f-ratio strains doses (min.) 1st 2nd 3rd 2 2.04 2.04 0.67 4 5.44 4.08 4.75 nistari-m 8 6.12 6.80 6.12 (a) 78.50*** (b) 0.85ns 2 2.71 8.16 2.71 4 3.39 3.39 4.75 urboshi-1 8 6.80 12.24 8.16 (a) 11.84** (b) 2.30ns control mortality of both the strains and all the instars = 2.00%, (a) = between doses, (b) =between instars, ** p < 0.01, *** p < 0.001; ns = not significant. f = variance ratio. 79 table 5. effect of uv-radiation on the cocoon weight (mg) of b. mori (n = 30) instars 1st 2nd 3rd strains doses (min.) mean ± se mean ± se mean ± se f-ratio 0 (control) 886.62 ± 10.73a (1004.93 ± 7.52k) 886.62 ± 10.73a (1004.93 ± 7.52k) 886.62 ± 10.73a (1004.93 ± 7.52k) 2 842.06 ± 9.59b (982.86 ± 6.54k) 823.26 ± 10.34b (982.36 ± 5.72k) 851.96 ± 8.48b (980.26 ± 6.52k) 4 766.49 ± 8.13c (879.86 ± 2.96m) 751.70 ± 7.46c (983.20 ± 4.40k) 815.53 ± 7.62c (941.20 ± 7.28k) nistari-m 8 718.56 ± 11.62d (820.30 ± 8.38m) 679.63 ± 4.12d (978.00 ± 4.96k) 744.02 ± 6.74d (937.80 ± 8.67k) (a) 60.19*** (3.25ns) (b) 5.52* (2.65ns) 0 (control) 955.40 ± 11.32a (1039.40 ± 10.41k) 955.40 ± 11.32a (1039.40 ± 10.41k) 955.40 ± 11.32a (1039.40 ± 10.41k) 2 871.36 ± 11.41a (1001.36 ± 12.62l) 930.63 ± 7.91a (1018.06 ± 12.81k) 931.86 ± 12.80a (1019.60 ± 9.01k) 4 881.56 ± 12.06a (1015.83 ± 17.02kl) 820.00 ± 5.55b (1007.42 ± 8.25kl) 807.26 ± 12.96b (1011.46 ± 13.50k) urboshi-1 8 867.03 ± 8.67a (938.36 ± 14.45m) 812.50 ± 4.28b (983.66 ± 6.04l) 836.40 ± 5.38b (984.36 ± 8.35l) (a) 9.25* (14.12**) (b) 0.20ns (1.49ns) (a) = between doses, (b) = between instars, * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant. data in parentheses indicate corresponding values in females. f = variance ratio. means followed by the same letter in each instar of each strain are not significantly different at p = 0.05 (snk test). 80 table 6. effect of uv-radiation on the shells of b. mori (n = 30) (a) = between doses, (b) = between instars, * p < 0.05, *** p < 0.001, ns = not significant. data in parentheses indicate corresponding values in females. f = variance ratio. shell weight / instars shell ratios (%) / instars 1st 2nd 3rd 1st 2nd 3rd strains doses (min.) mean ± se mean ± se mean ± se f-ratio mean ± se mean ± se mean ± se f-ratio 0 (control) 78.36 ± 1.37 (90.33 ± 1.01) 78.36 ± 1.37 (90.33 ± 1.01) 78.36 ± 1.37 (90.33 ± 1.01) 8.84 ± 0.18 (8.99 ± 0.12) 8.84 ± 0.18 (8.99 ± 0.12) 8.84 ± 0.18 (8.99 ± 0.12) 2 79.00 ± 1.22 (91.06 ± 1.13) 77.60 ± 0.61 (86.56 ± 1.08) 84.66 ± 1.44 (88.16 ± 1.41) 9.38 ± 0.16 (9.26 ± 0.12) 9.43 ± 0.14 (8.81 ± 0.10) 9.94 ± 0.19 (8.99 ± 0.19) 4 75.76 ± 0.81 (80.60 ± 0.78) 71.30 ± 0.65 (89.56 ± 0.42) 83.10 ± 1.11 (87.83 ± 1.07) 9.88 ± 0.12 (9.16 ± 0.08) 9.49 ± 0.11 (9.11 ± 0.06) 10.19 ± 0.14 (9.33 ± 0.13) nistarim 8 73.06 ± 1.42 (75.63 ± 1.46) 72.40 ± 0.53 (76.90 ± 0.61) 79.16 ± 1.18 (86.26 ± 1.29) (a) 2.64ns (4.22ns) (b) 6.98* (0.90ns) 10.18 ± 0.22 (9.22 ± 0.18) 10.65 ± 0.09 (7.86 ± 0.08) 10.64 ± 0.16 (9.20 ± 0.15) (a) 26.80*** (0.73 ns) (b) 2.57ns (2.03ns) 0 (control) 144.90 ± 2.67 (164.13 ± 3.12) 144.90 ± 2.67 (164.13 ± 3.12) 144.90 ± 2.67 (164.13 ± 3.12) 15.16 ± 0.23 (15.79 ± 0.30) 15.17 ± 0.23 (15.79 ± 0.30) 15.17 ± 0.23 (15.79 ± 0.30) 2 123.26 ± 2.61 (144.16 ± 2.50) 135.73 ± 0.69 (147.80 ± 1.52) 131.00 ± 3.81 (161.00 ± 2.60) 14.15 ± 0.33 (14.40 ± 0.26) 14.58 ± 0.13 (14.52 ± 0.22) 14.06 ± 0.32 (15.79 ± 0.22) 4 137.93 ± 3.37 (166.76 ± 2.58) 125.66 ± 1.46 (141.46 ± 1.03) 122.93 ± 3.10 (147.96 ± 2.77) 15.65 ± 0.33 (16.42 ± 0.37) 15.32 ± 0.20 (14.04 ± 0.15) 15.23 ± 0.27 (14.63 ± 0.28) urboshi1 8 135.30 ± 2.79 (154.60 ± 3.02) 119.86 ± 0.62 (140.00 ± 1.94) 113.50 ± 2.41 (136.50 ± 2.97) (a) 4.47ns (2.47ns) (b) 0.90ns (0.95ns) 15.60 ± 0.30 (16.48 ± 0.37) 14.75 ± 0.11 (14.23 ± 0.19) 13.57 ± 0.26 (13.87 ± 0.27) (a) 3.19ns (0.61ns) (b) 1.70ns (1.41ns) 81 hirobe (1974) stated that in silkworms, growth and other quantitative characters are changed by gammairradiation depending upon dose rate, total dosage, developmental stage, temperature, moisture and other environmental conditions. it has been demonstrated that the developmental stages of insects renew their cells and tissues, and a particular stage of these animals determine their radio-sensitivity to ionizing radiation (allotey, 1985). in the present investigation dose dependent sensitivity was observed in the strains of 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(eds), problems of threshold in chemical mutagenesis, the environmental mutagen society of japan, pp. 169-173, 1984 technical report isj 5: 192-215, 2008 issn 1824-307x technical report immune-neuroendocrine biology of invertebrates: a collection of methods l ballarin1*, m cammarata2*, f cima1*, a grimaldi3*, s lorenzon4*, d malagoli5*, e ottaviani5* 1department of biology, university of padova, padova, italy 2marine immunobiology laboratory, department of animal biology, university of palermo, italy 3department of structural and functional biology, university of insubria, varese, italy 4department of biological oceanography, national institute of oceanography and experimental geophysics, trieste, italy 5department of animal biology, university of modena and reggio emilia, modena, italy *the authors are inserted in alphabetical order accepted december 5, 2008 abstract in the last decade there has been a considerable increase of interest towards the elucidation of several aspects of invertebrate biology, including immunity and neuroendocrinology. however, due to the difficulties connected to the great variety of morphology and adaptations displayed by invertebrates, and also in consideration of the number of techniques that are applied in the various laboratories, research on invertebrates still suffers from hampering that have been substantially overcome in vertebrate models, especially in mammals. the aim of this technical report is to provide the reader a useful list of well-established morphological and morpho-functional protocols in order to facilitate the design and make more homogeneous the realization of experiments in the field of invertebrate immune-neuroendocrinology. key words: morphology; laboratory techniques; immunity; neuroendocrinology contents • introduction • collection of the hemolymph/immunocytes • morphological methods • morpho-functional methods • quantitative methods • concluding remarks • references introduction in the last decade the literature in the field of the invertebrate immune-neuroendocrine biology has been widely increasing and important contributions have allowed a better understanding of the mechanisms and the strategies of defenses set up from the species of different taxa. however, many ___________________________________________________________________________ corresponding author: enzo ottaviani department of animal biology university of modena and reggio emilia via campi 213/d, 41100 modena, italy e-mail: enzo.ottaviani@unimore.it problems remain unsolved in the field of comparative invertebrate immunology, such as the classification of circulating cells (ribeiro and bréhelin, 2006), the evolution of soluble mediators as the cytokines (gerber et al., 2007; malagoli and ottaviani, 2007), the presence of memory (bréhelin and roch, 2008) and the characterization of immune-related stem cells (bachère et al., 2004; söderhall et al., 2005) among others. in the insect drosophila melanogaster (lemaitre and hoffmann, 2007) and the nematode caenorhabditis elegans (alper et al., 2008) several steps have been taken towards a better comprehension of the molecular mechanisms of immune response, but comparative immunologists well know that two organisms, even if carefully and deeply characterized, cannot be considered as the paradigmatic representation of the million of species widespread along the protostomian and deuterostomian lineages of invertebrates. among comparative immunologists, it is commonly accepted that the difficulties in extending the results obtained in a given invertebrate species to others invertebrate taxa are not only the direct 192 consequence of the intrinsic difficulties connected to the great variety of morphology and adaptations displayed, but also to the variety of techniques that are applied and that often lead to different results in the various laboratories (hooper et al., 2007). indeed, the majority of the utilized techniques derive from those developed for investigations in vertebrate models and laboratories moving their first steps in the field of comparative immunology may encounter problems in adapting available protocols to their models. remarkably, molecular biology approaches are giving an important contribution in supporting/clarifying the data collected by mean of morphological and functional investigations. notwithstanding that, morphological and functional assays obviously remain the firstchoice approaches to collect those evidences that could eventually direct future molecular biologybased experiments. in this technical report we collected several well-established morphological and morphofunctional protocols used in the field of invertebrate immune-neuroendocrinology. this report has not the ambition to represent a complete guide for all the possible experiments that can be performed with invertebrate models. our aim is rather to provide the reader a useful guide in order to facilitate and unify the design of future studies. besides this, we also hope to offer a brief collection of methods that should be applied when studying invertebrate immune-neuroendocrinology. in particular, we will refer to protostomian and deuterostomian invertebrates, such as mollusca (planorbarius corneus, viviparus ater, lymnaea stagnalis, mytilus galloprovincialis and mytilus edulis), annelida (eisenia foetida and hirudo medicinalis), crustacea (astacus leptodactylus, homarus americanus, neprops norvegicus, munida rugosa, paguristes oculatus, palaemon elegans and squilla mantis), insecta (calliphora vomitoria and galleria mellonella, cell lines from estigmene acraea, lymantria dispar and mamestra brassicae) and tunicata (botryllus schlosseri, ciona intestinalis, phallusia mamillata, styela plicata). collection of the hemolymph mollusca in gastropods the hemolymph is obtained by exerting slight pressure on the animal’s foot and collected by a pasteur pipette. in bivalves, e.g., mytilus spp. the classical protocols indicate that hemolymph can be taken from the posterior adductor muscle using a 2 ml syringe (ottaviani, 1983; ottaviani et al., 1997b). recently, we have also proposed a protocol for m. galloprovincialis in which the hemolymph is collected from mussels directly by inserting a sterile syringe between the valves, just beneath the exit-point of the byssus. after insertion, hemolymph can be obtained by exerting a gentle aspiration. this method is useful when large amount of fluid is needed, but it is of extreme importance to check for the quality of the hemolymph immediately after the withdrawal, in order to ensure that neither the gonads nor the digestive gland have been touched or damaged by the needle (malagoli et al., 2007). annelida h. medicinalis immune cells, which are entrapped in a thick connective tissue, can be isolated and cultured utilizing the injection of matrigel matrix gel (mg) supplemented with different growth factor/cytokines selected among those that play a major role in invertebrate/vertebrate wound healing (grimaldi et al., 2008). mg is an extract of the murine engelbreth-holm-swarm (ehs) tumor grown in c57/b16 mice and produced as described by kleinman (1986). it is rich in basement membrane components (laminin, collagen iv, nidogen and perlecan) and it is a thermo-sensitive material liquid at 4 °c which polymerizes when warmed to room temperature (rt). leeches are injected with 300 μl of mg added either with 50 ng of monocyte chemoactractant protein-1 (mcp-1/ccl2, pepro tech, london, uk), with a pivotal role in the recruitment of monocytes and macrophages, or with 50 ng of vascular endothelial growth factor (vegf, pepro tech), playing a pivotal role in the recruitment of hematopoietic and endothelial precursor cells. for in vivo analysis, anesthetized leeches are dissected and polymerized matrigel pellets are removed and histologically, ultrastructurally examined (grimaldi et al., 2008). for in vitro analysis, each matrigel pellet is minced in small pieces using sterilized razor blades and plated in wells of 60 μm in diameter in dulbecco's modified eagle's medium (dmem, celbio, milan, italy) modified by dilution (1:4) to reach iso-osmolarity and supplemented with 1 % glutamine and 10 % fetal bovine serum (grimaldi et al., 2008). the coelomocyte collection from the earthworm e. foetida is performed putting the animals in a sterile plastic petri dish in cold phosphate buffered saline (pbs-1) (137 mm nacl, 2.7 mm kcl, 10 mm na2hpo4, 10 mm nah2po4, ph 7.3) diluted 1:1 with ethanol. after a few sec, the earthworms extrude coelomocytes vigorous though their dorsal pores. then the coelomocytes are transferred to plastic tubes and washed by centrifugation in pbs-1 without alcohol at 500xg for 5 min (cooper et al., 1995). crustacea hemolymph sample is taken from the pericardic sinus of shrimps (p. elegans), hermit crabs (p. oculatus), lobster (h. americanus), norway lobster (n. norvegicus), squat lobster (m. rugosa) crayfish (a. leptodactylus) and mantis shrimps (s. mantis). blood samples are taken from the coxal sinus of crab. in the smallest species 50 µl of hemolymph is collected using a sterile 1 ml syringe fitted with a 25g needle, in the biggest animals up to 1 ml of hemolymph is obtained, depending on of the size of specimens (lorenzon at al., 1999, 2007, 2008; giulianini et al., 2007). insecta in the fly c. vomitoria the hemolymph is taken from a small incision in the ptilinum at the front of the head. by softly squeezing of the abdomen and thorax, a drop of hemolymph is forced out of the incision and collected with a pipette (franchini et al., 1996). the hemolymph in the g. mellonella larvae is collected by piercing with a small needle in one of the first prolegs (wittwer et al., 1999). 193 tunicata in solitary ascidians the tunic is cleaned from epiphytes and sterilized with ethyl alcohol. in s. plicata incurrent siphon is incised and the exuding hemolymph is collected, while in c. intestinalis a syringe is inserted directly into the hearth and the hemolymph is collected. in both cases, hemolymph is put in sterile tubes containing a 5-fold excess of calcium/magnesium-free artificial sea water (asw) (9 mm kcl; 0.15 m nacl; 29 mm na2so4, nahco3, ph 7.4) with 10 mm edta (asw-edta) as anticoagulant (1:9 medium/hemolymph ratio), in ice. after centrifuging at 400xg for 10 min at 4 °c, the hemocytes are washed three times in sterile aswedta. in the colonial ascidian b. schlosseri, blood is collected with a glass micropipette after puncturing, with a fine tungsten needle, the tunic marginal vessel of colonies previously rinsed in 0.38 % nacitrate or 10 mm l-cysteine in filtered seawater (fsw), ph 7.5, to prevent hemocyte clotting. it is then centrifuged at 780xg for 10 min and the pellet is re-suspended in fsw at the final concentration of 6-8x106 cells/ml. the cell concentration is evaluated with a burker’s hemocytometer. sixty μl of hemocyte suspension are placed in the centre of culture chambers, made by gluing teflon rings (15 mm internal diameter, 1 mm thick) on siliconized glass slides (fig. 1a). washed coverslips are laid over the teflon rings, smeared with vaseline and gently pressed down to touch the drop of the cell suspension (ballarin et al., 1994). the culture slides fig. 1 hemocyte culture. a) schematic drawing of a culture chamber with a drop of cell suspension in the center a: upper view; b: side view. b) living b. schlosseri hemocytes adherent to coverslips. bar = 15 µm. fig. 2 cryosection of matrigel supplemented with vegf. the mg sponge is infiltrated by ovoidal and agranular hematopoietic precursor cells stained with may-grünwald and giemsa solution. are kept upside-down for 30 min at rt to allow the cells to settle and adhere to the coverslips (fig. 1b). morphological methods optical and electron microscopy-based methods for characterization of circulating hemocytes (immunocytes) mollusca in freshwater snails and marine bivalves, the immunocytes are obtained in hemolymph drops on glass or by cytocentrifugation (cytospin ii, shandon instruments, uk) of hemolymph samples onto a slide at 500-1000 rpm for 5-10 min. they are observed by phase-contrast microscope and stained with or without fixation for morphological observations (ottaviani, 1983, 1989; ottaviani et al., 1998b). annelida may-grünwald and giemsa differential staining (bio-optica, milan, italy) and the vital dye acetylated low density lipoprotein labeled with 1,1’-dioctadecyl3,3,3’,3’-tetramethyl-indo-carbocyanine perchlorate (dil-ac-ldl) (biomedical technologies inc., ma, usa) are a quick method to characterize leech immunocytes (grimaldi et al., 2008). may-grünwald and giemsa procedure in in vivo experiments mg implant is removed from the animal, embedded in polyfreeze tissue freezing medium (polysciences, eppelheim, germany) and immediately frozen in liquid nitrogen. cryosections (7 μm), obtained with a cryotome, are immerse in 100 % may-grünwald for 4 min and then directly transferred to 4 % giemsa (diluted in tap water) for 4 min. slides are then washed in tap water, air dried, mounted with eukitt mounting medium (electron microscopy science, washington, pa, usa) and subsequently observed under a light microscope (fig. 2). in in vivo experiments immunocytes of leech, extracted from mg and plated in wells, are allowed to air dry onto slides and then they are stained as described above (fig. 3). 194 fig. 3 may-grünwald and giemsa staining micrographs showing cultured hematopoietic precursor cells. dil-ac-ldl procedure in in vivo experiments, injection of 10 µl of 10 µg/ml dil-ac-ldl in pbs-2 buffer (138 mm nacl, 2.7 mm kcl, 4.3 mm na2hpo4, 1.5 mm kh2po4, ph 7.4) is performed at the level of the 80th superficial metamere of the leech, where the mg is subsequently inoculated. after 1 week the mg implant is removed from the animal and quickly frozen. cryosections are mounted in citifluor (citifluor ltd, london, uk) and observed on fluorescence microscope through a rhodamine filter set (excitation/emission filters 550/580 nm) to visualize the dil (fig. 4). in in vitro experiments, leech immunocytes are cultured in dmem containing 10 µg/ml dil-ac-ldl according to the manufacturer’s suggestions and tamaki et al. (2002). after 4 h at rt, cells are washed several times with probe-free medium and directly observed using an inverted-fluorescence microscope (fig. 5). nuclei are colored with 4'-6-diamidino-2phenylindole (dapi) (sigma, st louis, mo, usa) (excitation/emission filters 410/460 nm). utrastructural procedures mg pellets (see above) are fixed for 2 h in 0.1 m cacodylate buffer ph 7.2, containing 2 % glutaraldehyde. specimens are then washed in the same buffer and postfixed for 2 h with 1 % osmic acid in cacodylate buffer, ph 7.2. after standard serial ethanol dehydration, specimens are embedded in an epon-araldite 812 mixture. semithin sections (750 nm) are obtained with an ultratome, stained by conventional methods (crystal violet and basic fuchsin) according to moore et al. (1960), and observed under a light microscope. thin sections (80 nm) are stained by uranyl acetate and lead citrate and observed with a transmission electron microscope (tem) (fig. 6). crustacea to characterize the different classes of blood cells in a. leptodactylus, by means of light and electron microscopy, 200 µl of hemolymph (about 2x105 hemocytes counted by using a bürker’s hemocytometer) are drawn into a sterile plastic 1 ml syringe filled with an equal volume of fixative (2.5 % glutaraldehyde, 1 % paraformaldehyde, 7.5 % saturated picric acid solution in 0.1 m cacodylate buffer, ph 7.4) and, after a fixation for a minimum of 10 min, the hemocytes are pelleted in 1 ml of fixative by 10,300xg centrifugation for 10 min at 20 °c. the pellets obtained are then washed in 0.1 m cacodylate buffer ph 7.4 and post-fixed in 1 % osmium tetroxide in the same buffer, serially dehydrated in ethanol and embedded, via propylene oxide, in embed812/araldite (electron microscopy sciences, fort washington, pa, usa) or, without post-fixation, they are serially dehydrated in ethanol and embedded in lr-white (sigma). for light microscopy, sections (2 µm thick) are collected on slides, baked for 5 min at 80 °c and stained with 0.5 % toluidine blue in 0.1 % carbonate solution at ph 11.1 at the same temperature. for tem, silver/goldcoloured sections are stained with uranyl acetate and lead citrate and observed with a tem philips em 208. for light microscopy, selected areas were observed with an olympus bx50 microscope, and images are acquired with an olympus dp11 photo camera at a resolution of 1712x1368 pixels. for tem, negative plates are digitized with an epson photo perfection scanner at 1200 dpi (optical resolution) and saved as a tagged image format file. all measurements, statistical analyses and photocomposition are performed with image-pro plus 4.5 software (media cybernetics, silver spring, md, usa) (giulianini et al., 2007). insecta for the morphological studies, hemolymph from several c. vomitoria flies are pooled to a volume of 50-100 μl and cytocentrifuged on a slide using a cytocentrifuge cytospin ii (shandon instruments) running at 500 rpm for 5 min. subsequently the immunocytes are stained with may-grünwald and giemsa (franchini et al., 1996). fig. 4 cryosection of matrigel supplemented with vegf. the implanted mg is invaded by precursor cells and identified with dil-ac-ldl. nuclei are stained with dapi (in blue). 195 fig. 5 characterization of cultured immunocytes of leech by dil-ac-ldl uptake. in macrophages the fluorochrome-conjugated probe showed a spotted localization a), while a diffused cytoplasmic localization was evident in the hematopoietic precursor cells b). nuclei are stained with dapi (in blue). tunicata in b. schlosseri, several morphological protocols have been set up for both fixed or living hemocytes. however, it is important to underline the possibility to obtain sub-populations from a pool of circulating hemocytes. blood harvested from large colonies (of about 1000 zooids) is centrifuged at 780xg for 10 min. pelleted hemocytes are re-suspended in 1 ml of fsw (final concentration: 50x106 cells/ml) and layered over a ficoll discontinuous gradient obtained by dissolving ficoll 400 (pharmacia, canada) in fsw to final concentrations of 10, 14.5, 20 and 34 % (modified after michibata et al., 1987) and sequentially overlayering 2 ml of each solution into a 10 ml centrifuge tube. tubes are centrifuged at 400xg for 10 min at 4 °c. blood cell fractions are collected with a glass micropipette from the top of the tube, diluted in an equal volume of fsw, and centrifuged at 780xg for 10 min. pelleted cells are re-suspended in 300 µl of fsw and the concentration of hemocytes is estimated with a bürker’s hemocytometer. the fixation procedures for cytochemical staining of hemocytes (or hemocyte sub-population) is performed as follows. after adhesion of the hemocytes to the coverslips, the debris-containing fsw is discarded and cell monolayers are washed by dipping the coverslips (see above) repeatedly in a large volume (100 ml) of fsw. the best and simplest fixative mixture for cell morphology preservation is represented by a solution of 1 % glutaradehyde (fluka, buchs sg, switzerland) and 1 % saccharose in fsw at 4 °c for 30 min. cells are then washed in pbs-3 (1.37 m nacl, 0.03 m kcl, 0.015 m kh2po4, 0.065 m na2hpo4, ph 7.2) or in the buffers used for the cytochemical assays reported below. for b. schlosseri hemocyte identification and characterization, the following cytochemical staining methods can be used (cima et al., 2001; ballarin and cima, 2005): giemsa dye: fixed hemocytes adhering to coverslips are stained for 10 min with a 10 % giemsa (fluka) aqueous solution and then washed in distilled water and mounted in acquovitrex (carlo erba, milan, italy) on glass slides. with this dye, the nucleus appears blue and cytoplasm light blue or violet, due to metachromasia, under the light microscope. pappenheim’s panoptical stain: fixed hemocytes are stained for 3 min in may-grünwald’s dye (fluka). after washing in distilled water they are stained for 10 min with 5 % giemsa, washed again in distilled water and mounted in acquovitrex (c. erba) (mazzi, 1977; bancroft and gamble, 2002). with this technique, basophilic granules appear blue, neutrophils brown and acidophils dark pink (fig. 7a). ehrlich’s triacid mixture: this mixture is composed of 12 parts of saturated orange g aqueous solution, 8 parts of saturated acid fuchsin aqueous solution, 10 parts of saturated methyl green aqueous solution, 30 parts of distilled water, 18 parts of absolute ethanol and 5 parts of glycerin. fixed hemocytes are stained with this mixture for 15 min, washed in distilled water and mounted. basophilic granules appear light green, neutrophils violet and acidophils coppery red (mazzi, 1977; bancroft and gamble, 2002). toluidine blue staining: fixed hemocytes are stained for 10 s in a filtered aqueous solution of 0.5 % toluidine blue (fluka) and 0.5 % sodium tetraborate, to reveal the presence of mucosubstances and glucosaminoglycans which show pink-violet metachromasia. neutral red dye: after adhesion of hemocytes to coverslips, the fsw of the culture chambers is substituted with 60 µl of 8 mg/ml neutral red solution (merck, darmstadt, germany) in fsw. living hemocytes are directly observed. this dye specifically stains acid compartments (e.g., lysosomes or acid vacuolar contents) of living cells (mazzi, 1977; bancroft and gamble, 2002). sudan black for lipids: after adhesion of hemocytes, coverslips are dipped in 70 % ethanol for 30 s and stained with a saturated solution of sudan black (sigma) in 70 % ethanol for 15 min at 70 °c. cells are then rinsed in 70 % ethanol and washed in distilled water (mazzi, 1977; bancroft and gamble, 2002). black spots reveal the presence of lipids (fig. 7b). 196 periodic acid schiff (pas) reaction for polysaccharides: fixed hemocytes are incubated in 1 % periodic acid for 10 min, rinsed in tap water and stained with schiff’s reagent for 30 min at 37 °c. coverslips are then dipped in a solution of 0.6 % sodium metabisulfite in 0.02 m hcl for 6 min, washed in tap water for 10 min, and then rinsed in distilled water (mazzi, 1977; bancroft and gamble, 2002). positive sites appear primary red (fig. 7c). quinones: living hemocytes are incubated in a 2 mm solution of 3-methyl-2-benzothiazolinone hydrazone chloride (mbth) in fsw, containing 0.4 % dimethylformamide for 5 min, in the presence or in the absence (controls) of the po inhibitor 10 mm na-benzoate (winder and harris, 1991; ballarin et al., 1995). positivity is revealed by a marked red color (fig. 7d). dopa-containing proteins/quinoproteins: after the adhesion to coverslips, hemocytes are fixed as described above, washed in pbs-3 and incubated with a solution of 0.24 mm nitroblue tetrazolium (nb) (sigma) in 2 m potassium glycinate buffer (150 g/l glycine and koh 2 n to ph 10.0) and 20 mm sodium benzoate (flückiger et al., 1995). cells are then washed in pbs-3 and coverslips mounted with acquovitrex (c. erba). positive sites feature a dark blue color (fig. 7e). lectin cytochemistry fixed hemocytes are incubated for 30 min in pbs-3 containing 5 % powdered milk, washed three times for 10 min in pbs-3 and incubated for 60 min in a 50 µg/ml lectin solution in pbs-3 containing 0.1 m cacl2. both fitc-labeled and biotin-conjugated lectins can be used, e.g., ulex europaeus agglutinin-i (uea-i, specific for lfucose), datura stramonium lectin (dsl, specific for n-acetyl-β-d-glucosamine and n-acetyllactosamine), ricinus communis agglutinin-i (rca, recognizing β-d-galactosides), wheat germ agglutinin (wga, specific for n-acetyl-β-dglucosamine), helix pomatia agglutinin (hpa, recognizing n-acetyl-α-d-galactosamine), arachis hypogea agglutinin (pna, specific for galactosyl (β1,3) n-acetyl-galactosamine), concanavalin a (cona, recognising α-d-glucopyranosides and αd-mannopyranosides), vicia villosa agglutinin (vva, specific for n-acetyl-d-galactosamine), narcissus pseudonarcissus agglutinin (npa, specific for α-d-mannosyl carbohydrate residues). incubation with fitc-labeled lectins is followed by extensive washing in pbs-3, and coverslips are mounted with fluorsave (calbiochem, darmstadt, germany) on glass slides, and observed at a magnification of 1,250x with a fluorescence light microscope equipped with a filter block for fitc excitation (450-490 nm; emission at 525 nm). conversely, after incubation with biotin-conjugated lectins, hemocytes are washed in pbs-3, incubated for 30 min in avidin-biotin-peroxidase complex (abc) (vector lab., burlingame, ca, usa) in pbs-3, washed in pbs-3, incubated for 30 min in 0.5 mg/ml 3-3’ diaminobenzidine (dab) (sigma) in pbs-3 containing 0.04 % h2o2, mounted in acquovitrex (c. erba) and observed under microscope. fig. 6 tem micrographs. ultrastructural analysis of matrigel added with different cytokines. monocyte chemoactractant protein-1 (mcp-1/ccl2) a) selectively recruit cells characterized by cytoplasm filled with granules of diverse sizes while vascular endothelial growth factor (vegf) (b) recruits cells characterized by large nuclei with a scarce cytoplasm occupied by organelles and few small granules. 197 fig. 7 b. schlosseri hemocytes. a: fixed macrophage-lke cells (mlc) after pappenheim’s panoptical stain; b: living mlcs (arrowheads) stained with sudan black for lipids; c: fixed mlc positive for pas reaction; d: living morula cells treated with mbth to reveal quinones; e: fixed morula cell showing positivity at the reaction for dopacontaining proteins; f: actin cytoskeleton of fixed spreading phagocytes revealed with fitc-labeled phalloidin; g: tubulin cytoskeleton of fixed spreading phagocytes revealed, in immunofluorescence, with anti-tubulin antibodies; h: senescent living phagocyte with bleb after treatment with fluorescent annexin-v; i, j: fixed hyaline amoebocytes (spreading phagocyte; i) and mlc (j) showing tunel positivity for dna fragmentation; k: comet assay showing dna fragmentation in nuclei of senescent hemocytes; l: fixed hyaline amoebocyte showing positivity for βglucuronidase; m: fixed morula cell positive for phenoloxidase. bar = 10 µm. assay for cytosolic ca2+ sustained increases in cytosolic ca2+ are revealed as dark-blue precipitates by von kossa’s substitution method (callis and bone, 2002). glutaraldehyde-fixed monolayers are immersed in 5 % silver nitrate and exposed to ultraviolet light for 5 min. cells are then rinsed in distilled water, incubated for 2-4 min in a 5 % aqueous sodium thiosulfate solution, washed in distilled water, mounted in acquovitrex (c. erba), and observed under the light microscope. tem samples, represented by selected whole colonies or pellets of fixed hemocytes embedded in small pre-heated agar pieces, are fixed in a solution of 1.5 % glutaraldehyde in 0.2 m cacodylate buffer, ph 7.4, plus 1.6 % nacl for 2 h at 4 °c. specimens are then rinsed in cacodylate buffer containing 1.6 % nacl, post-fixed in 1.5 % oso4 in cacodylate buffer, dehydrated and embedded in epon. oneμm-thick sections are stained with toluidine blue and observed with light microscope. ultrathin sections, briefly stained with uranyl acetate and lead citrate, are examined under a tem at 75 kv. for enzymatic activity or antigen preservation, specimens are fixed by immersion in 4 % paraformaldehyde plus 0.1 % glutaraldehyde in saline buffer (sb) (0.2 m cacodylate buffer, ph 7.4, 1.7 % nacl and 1 % saccharose) for 2 h at 4 °c, and then washed three times in sb without postfixation in oso4. in the case of detection of enzymatic activity, a pre-embedding method is usually employed (cima et al., 2002a). specimens are pre-incubated in 0.1 % triton-x in sb for 10 min and then incubated overnight at rt in the reaction mixtures reported in the “cytoenzymatic assays” section. for each test, control specimens are prepared omitting the specific enzyme substrate. after incubation in the reaction and/or postincubation mixture, specimens are post-fixed in 1 % oso4 in 0.2 m cacodylate buffer, ph 7.4, dehydrated, and embedded in epon for sectioning. ultrathin sections are treated as reported above. in the case of immunocytochemistry, a postembedding method is applied. thin sections of fixed specimens, dehydrated and embedded in catalyzed lr white resin (polysciences, warrington, pa, usa) are collected on gold grids and processed for immunohistochemistry. after immersion in pbs-3 198 plus 10 % normal goat serum (vector lab., burlingame, ca, usa) for 10 min at rt, the grids are incubated in the primary antibody diluted in pbs-3 according to the manufacturer’s instructions overnight at 4 °c. they are then rinsed in pbs-3 and incubated in 10 nm gold-conjugated goat antirabbit igg (british biocell international, cardiff, uk), diluted 1:50, 1:100 in pbs-3 for 1 h at rt. in controls, the primary antibody is omitted. before observation with tem, the grids are also stained with uranyl acetate and lead citrate (fig. 8). scanning electron microscopy (sem) living hemocytes are left to adhere to poly-llysine-coated coverslips. they are then fixed for 30 min at 4 °c in the same fixative mixture for tem and post-fixed in 1 % oso4 in cacodylate buffer for 60 min. dehydration through a graded ethanol series is followed by critical-point drying in liquid co2 with absolute acetone as transitional fluid. cells are then sputtered with gold and observed with a sem. immunocytochemical procedures mollusca, annelida and insecta immunocytochemical assay is carried out on fixed or air-dried molluscan immunocytes in order to detect the presence of adrenocorticotropin hormone (acth) using a human acth polyclonal antibody (pab). after hemolymph cytocentrifugation, the immunocytes are air-dried, immersed in absolute ethanol followed to passages in 95 % ethanol (3 min each). endogenous peroxidase are inhibited by immersion in 0.3 % h202 in methanol for 15 min at rt, then the slides are immersed in running tap water for 10 min and rinsed in pbs-1 (3x5 min). incubate in normal goat serum (1:5) (vector lab.) for 30 min at rt; incubate in primary rabbit antihuman acth (1-24) pab (1:200) (dakopatts, denmark) overnight at 4 °c; wash in pbs-1 (3x10 min); incubate in secondary biotinylated anti-rabbit igg (1:200) (vector lab.) for 30 min at rt; wash in pbs-1 (3x10 min); incubate in abc (1:100) (vector lab.) in pbs-1 for 30 min at rt; wash in pbs-1 (3x10 min); incubate in a 0.025 % solution of dab tetrahydrochloride (sigma) in mcllvaine buffer at ph 5.5 containing 5 μl h202. stop the reaction by washing in tap water for 5 min and then rinse in distilled water; counterstain nuclei with hematoxylin; dehydrate through a graded ethanol series: 70, 80, 95 and 100 % (3 min each); clear in xylene and mount in eukitt (bio-optica, milan, italy). controls of immunocytochemical reaction are performed using the same procedure omitting the primary antibody or by pre-absorbing the primary antibody overnight at 4 °c with the homologous antigen (in excess) (ottaviani et al., 1990). the same immunocytochemical procedure is also applicable to the annelid e. foetida in studying the presence of cytokineand pomc-derived peptide-like molecules in the coelomocytes as well as for the immunocytes of the insect c. vomitoria (cooper et al., 1995; franchini et al., 1996). crustacea immunocytochemistry of neuroendocrine organs immunocytochemistry is performed on thick and semithin sections of the eyestalk of the test species to validate the specificity and cross-reactivity fig. 8 immunocytochemistry at tem: phagocyte with granular content labeled with colloidal gold. bar = 3 µm. of the purified antibody anti-nenchh (anti-n. norvegicus crustacean hyperglycaemic hormone) (giulianini et al., 2002). paraffin immunocytochemistry eyestalks dissected from n. norvegicus, a. leptodactylus, m. rugosa and s. mantis were fixed in bouin’s solution for 24 h, serially dehydrated in ethanol and embedded in paraffin. sections (7 μm thick) are collected on superfrost plus (bio-optica) slides and baked for 30 min at 50 °c. the slides are deparaffinized in xylene, hydrated, microwaved in 6 m urea at 800 w for 1 min, washed in ultra pure water and treated with tyramide amplification (nen, tsa-indirect kit, zavantem, belgium). endogenous peroxidases were blocked with 1 % h2o2 in pbs (sigma) for 15 min. slides are incubated in 10 % normal goat serum in 0.1 m tris-hcl, ph 7.5, 0.15 m nacl buffer containing 0.5 % tsa blocking reagent (tnb) for 1 h at rt. the sections are then incubated overnight at 4 °c with the primary antibody diluted 1:20,000 in tnb; concentrations of pre-immunization normal rabbit serum (nrs) as well as anti-gst (anti-glutathione-s-transferase) antibody are employed for parallel controls. after washing for 20 min in 0.1 m tris-hcl, ph 7.5, 0.15 m nacl buffer containing 0.05 % tween 20, they are incubate for 1 h in hrp-labelled goat anti-rabbit igg (nen) diluted 1:200 in tnb. afterwards, the slides are incubated in biotinyltyramide working solution for 10 min and in streptavidin-hrp diluted 1:100 in tnb for 30 min at rt. finally slides are developed in 50 mm tris-hcl buffer, ph 7.5, containing 0.05 % dab and 0.01 % h202 at dark for 20 min at 4 °c. sections are dehydrated and subsequently mounted in eukitt (bio-optica). resin immunocytochemistry eyestalks of n. norvegicus and p. elegans are fixed in 2 % glutaraldehyde in 0.1 m sodium cacodylate 199 and 0.4 m sucrose at ph 7.6 (and for n. norvegicus only post-fixed in 1 % oso4 in the same buffer), serially dehydrated in ethanol and embedded in lrwhite (sigma). one-μm-sections are collected on superfrost plus (bio-optica) slides and baked for 30 min at 80 °c. the slides are microwaved in 6 m urea at 800 w for 1 min, washed in ultra pure water, and only for n. norvegicus, deosmicated with 3 % h2o2 in pbs (sigma) for 15 min. slides are incubated in 10 % normal goat serum in 0.1 m trishcl, ph 8.3, 0.15 m nacl buffer (tris buffered saline, tbs-1) containing 1 % bovine serum albumin and 0.1 % tween 20 (tbb) for 30 min. sections are then incubated overnight at 4 °c with the primary antibody diluted 1:400 in tbb; concentrations of pre-immunization normal rabbit serum are employed for parallel controls. after washing for 20 min in tbs-1, the sections are treated with goat anti-rabbit igg labelled with 5 nm colloidal gold (british biocell international) diluted 1:200 in tbb. afterwards, the slides are washed in ultra pure water and developed with british biocell silver-enhancing kit (british biocell international) for 20 min at dark and mounted in eukitt (bio-optica). fluorescence-based morphological methods mollusca flurescence-based methods have been applied in molluscs especially to document cytoskeletal rearrangements following the exposure to chemotactic factors/cytokines (franchini and ottaviani, 1994; ottaviani et al., 2000). in the freshwater snail v. ater, acth is able to stimulate the motility of immunocytes. the withdrawn hemolymph plus acth are dropped onto a slide in which a small circle is delimited by a pvc adhesive strip. after 30 min of incubation in a humidified chamber, the supernatant is removed and the adhered immunocytes are left to air-dry for 30 min before the fixation in 1 % paraformaldehyde for 5 min at rt. after fixation, different procedures of permeabilization are followed on the basis of the target of the study. for the detection of actin and vinculin, the immunocyte membrane is permeabilized with an incubation in hepes-tritonx-100 for 5 min at rt, while for detection of microtubules, the permeabilization is achieved with a dehydration in cold absolute methanol (5 min at 20 °c), followed by cold acetone (5 min at -20 °c) and air drying. specific reactions are finally performed by incubating immunocytes for 30-60 min at 37 °c in a humidified chamber with either 2.5 μg/ml fluorescein isothiocyanate (fitc)-labeled phalloidin (a specific marker of f-actin) (sigma), or with 1-10 μg/ml mouse anti-vinculin mab (boeringher-mannheim, germany), or with rabbit anti-tubulin (chemicon). slides can be mounted with an ordinary aqueous mounting medium (e.g., 50 % pbs-1/glycerol) or with commercial anti-fading media. observations are performed under a fluorescence microscope (ex/em wavelengths 495/519 nm). in the bivalve m. galloprovincialis, the actin microfilament modifications in immunocytes exposed to 100 ng/ml human recombinant il-8 (pepro tech) have been analyzed by using a similar method. briefly, cell preparation is carried out as described above but fixation is performed with 4 % paraformaldehyde in pbs-1, ph 7.4, for 5 min at 4 °c. permeabilization is still achieved with a hepestriton x-100 solution (5 min at rt), then mussel immunocytes are incubated in a humidified chamber with 10 μg/ml fitc-labeled phalloidin at 37 °c for 30 min. finally, slides are again washed three times with pbs-1 and mounted in aqueous/anti-fading medium. insecta cytocentrifugation is the first-choice method for slide preparation when working with cell cultures growing in suspension. in the insect cell line iplbldfb, derived from the larval fat body of the lepidopteran l. dispar, it has been observed that cytocentrifugation has to be performed at a speed as low as possible, i.e., 200 rpm for 1 min. this is important since high speed cytocentrifugations can alter the morphology as well as the integrity of the cells and may alter results when studying cytoskeletal elements (malagoli et al., 2006). the cytoskeletal organization of iplb-ldfb cells has been evaluated by fitc-labelled phalloidin (sigma). after cytocentrifugation, the cells are fixed for 10 min at rt in 4 % paraformaldehyde, washed in pbs-1 for 5 min and incubated for 30 min at 37 °c in 30 nm fitc-labelled phalloidin in presence of 0.3 % triton x-100 in pbs-1. subsequently, nuclei are stained for 5 min at rt with 10 μm hoecsht 33342 and the cells are mounted in mowiol® (calbiochem). it should be remarked that fixation is a fundamental step not only for the preservation of cell morphology, but because it allows a tighter adhesion of cells to the slide. a similar cell preparation has been also applied for tunel assay (useful for detecting the presence of apoptotic nuclei) (malagoli et al., 2006). conversely, for the hoecsht 33342-propidium iodide (pi) staining, applied for the evaluation of cell membrane integrity, the cytocentrifugation is performed on unfixed cells after the incubation with hoecsht 33342 but before the incubation with pi. more in detail, iplb-ldfb cells are stained for 10 min at 26 °c with 10 μm hoecscht 33342 and subsequently cytocentrifuged. slides are then submerged at 26 °c in a 10 μm pi solution for 5 min and immediately observed (malagoli et al., 2005). the ex/em wavelengths for the cited fluorochromes are the following: 495/519 nm (fitc), 343/483 nm (hoecsht 33342), 536/617 nm (pi). tunicata some differences exist in fixation procedures for immunocytochemical protocols with respect to that described for characterization of circulating hemocytes of b. schlosseri. for antigen preservation in immunocytochemical assays, hemocytes are fixed in 4 % paraformaldehyde (serva, heidelberg, germany) plus 0.1 % glutaraldehyde in saline buffer (sb) for 30 min at 4 °c, and then washed in sb. fixed hemocytes are washed in pbs-3 plus 1 % sucrose and permeabilized with 0.1 % triton x100 (merck) in pbs-3 for 5 min. for the specific detection of f-actin, the monolayers are then incubated for 30 min at 25 °c in fitc-labeled phalloidin (sigma), 1 μg/ml in pbs-3. lastly, the coverslips are rinsed in 0.1 m carbonate buffer, ph 9.5, to amplify the fluorescent signal, mounted with fluorsave (calbiochem) on glass slides, and 200 observed at a magnification of 1,250x with a fluorescence light microscope equipped with a filter block for fitc excitation (450-490 nm; emission at 525 nm) (fig. 7f). to observe microtubules, hemocytes are preincubated in pbs-3 containing 3 % powdered milk and 0.5 % fetal calf serum for 30 min to block aspecific interactions of antibodies. coverslips are then incubated for 60 min in monoclonal anti-αtubulin antibody (sigma) (1:2,000 in pbs-3), washed in pbs-3 and incubated with 50 µg/ml fitc-labeled goat anti-mouse igg (sigma) in pbs3. slides are then mounted with fluorsave (calbiochem), as described above, and observed under the fluorescence microscope (fig. 7g). in addition to the analysis of the cytoskeleton, fluorescence-based methods have also been successfully applied to analyze apoptotic process of b. schlosseri hemocytes. we have analyzed both early-apoptosis and late-apoptosis markers, even if the boundary between these two phases is obviously not always clearly distinguishable. phosphatidilserine exposure (early apoptosis): living cells are exposed for 60 min to xenobiotics and then incubated with fitc-coupled annexin-v (annexin-v fluos roche diagnostics, indianapolis, in, usa), according to the manufacturer’s instructions, to detect the presence of phosphatidylserine in the outer leaflet of the plasma membrane of living cells, a marker of early apoptosis (martin et al., 1997). after 15 min, cells are observed under the fluorescence microscope, equipped with a filter block for fitc excitation, at the magnification of 1,250x. at least 300 cells/coverslip are counted to evaluate the apoptotic index, i.e., the percentage of fluorescent cells (fig. 7h). dna fragmentation (late apoptosis): to evaluate the occurrence of dna fragmentation, hemocytes ,exposed for 60 min to xenobiotics, are fixed for 30 min at 4 °c in 4 % paraformaldehyde plus 0.1 % glutaraldehyde in sb rinsed in pbs-3 and incubated for 30 min in 0.3 % h2o2 as blocking solution. then, cells are incubated in the permeabilization solution (0.1 % triton x-100 in 0.1 % na-citrate) for 2 min at 4 °c. samples are then rinsed twice with pbs-3 and incubated in the tunel reaction mixture (roche diagnostics) for 60 min at 37 °c according to the manufacturer’s instruction and immediately observed under the fluorescence microscope, equipped with a filter block for fitc excitation (abrams, 1997). the reaction product can also be longer preserved when, after this step, hemocytes are incubated with peroxidase-conjugated anti-fitc antibodies, stained with 0.05 % dab in pbs-3 containing 1.5 % h2o2, dehydrated, mounted with eukitt (bio-optica) and observed under the light microscope. the percentage of cells with fluorescent or stained (brown) nuclei (fig. 7i, j) is expressed as the dna fragmentation index. comet assay: this test for dna fragmentation is performed as described by singh et al. (1988) and modified by kamer and rinkevich (2002). ten µl hemocyte suspension, containing 104 cells are mixed with 90 µl of a 0.5 % solution of melted agarose (low-melting-point type), cast on a microscope slide pre-coated with 0.5 % regular agarose and followed, after solidification, by a layer of 0.5 % lowmelting-point agarose. slides are then immersed in lysis buffer (0.01 m tris-hcl, 2.5 m nacl, 0.1 m na2edta, 1 % triton x-100, 10 % dimethylsulfoxide, ph 10) for 2 h at 4 °c. the slides are placed on a horizontal gel electrophoresis unit filled with chilled electrophoretic buffer (0.3 m naoh, 1 mm na2edta) for 40 min, to denature dna. after electrophoresis (20 min at 20 v), the slides are washed in 0.4 m tris-hcl buffer (ph 7.5), blotted dry, stained with a 25 µg/ml solution of ethidium bromide, and observed under a fluorescence light microscope, with an excitation filter of 590 nm. the frequency of apoptosis is estimated by counting the percentage of cells showing nuclei transformed in typical apoptotic comets (fig. 7k) by counting at least 100 cells for each slide at 1,250x (bacsó et al., 2000). finally, a fluorescence-based immunocytochemical approach to identify stem hemocytes among a hemocyte population or subpopulation is here proposed. after adhesion to the coverslips, hemocytes are fixed for 30 min at 4 °c in 4 % paraformaldehyde plus 0.1 % glutaraldehyde in sb. they are washed in pbs-3, permeabilized with 0.1 % triton x-100 (merck) for 5 min, immersed for 30 min in a pbs-3 solution containing 10 % normal goat serum (vector lab.) to block aspecific reactions, and then washed again in pbs-3 for 5 min. cells are incubated in 50 µg/ml of anti-cd34 (calbiochem), anti-cd100 (alexis co, lausen, ch) or scf-r (sigma) mabs for 1 h at rt. cd34 is a highly glycosylated surface antigen of still unknown function, probably involved in cell adhesion (holyoake and alcorn, 1994); cd100 is also a transmembrane glycoprotein belonging to the igg superfamily, expressed by the majority of hemopoietic cells (hall et al., 1996); scf-r, receptor of stem cell growth factor (scf), also known as c-kit or cd117, seems to exert an essential role on vertebrate hemopoiesis (broudy, 1997). hemocytes are then washed in pbs-3 for 5 min and incubated for 30 min in 10 µg/ml fluoresceinated goat anti-mouse-ig antibody (sigma). lastly, they are washed for 5 min in pbs-3 and mounted in fluorsave (calbiochem). observations are carried out under a fluorescence light microscope equipped with a filter block for fitc excitation (ex/em wavelength 495/519 nm). in situ hybridization mollusca and insecta the following method has been successfully applied in molluscs (ottaviani et al., 1998c) as well as in the insect cell line crl-8003 (atcc 6538) from the cabbage moth m. brassicae (malagoli et al., 2002b). in the procedure reported for the detection of the expression of acth receptor-like mrna in molluscan immunocytes (fig. 9) plasmid pbluescript ii ks-phagemid containing about 3,000 bp bovine acth receptor cdna is used as probe (raikhinstein et al., 1994). the probe is labeled by incorporating digoxigenin (dig)-labeled deoxyuridine triphosphate (boehringer mannheim) by random primer dna-labeling, according to feinberg and vogelstein (1983). the reaction is stopped by adding 0.2 m edta, ph 8. the hybridization assay proceeds as follows: incubate unfixed and fixed immunocytes with pbs-1 plus glycine (0.7 %) and permeabilize with 0.3 % triton 201 fig. 9 in situ hybridization of the expression of acth receptor-like mrna in m. galloprovincialis immunocyte (a). negative control (b). bar = 10 μm. x-100 in pbs-1 for 15 min at rt; wash with pbs-1 and with a mixture containing 2x ssc (0.3 m nacl, 0.03 m sodium citrate, ph 7) for 10 min; incubate with pre-hybridization mixture (4x ssc), 40 % formamide and 1x denhardt’s solution for 1 h at rt; hybridization is performed by adding of a denaturated dig-labeled acth receptor cdna probe (50 μg/μl) to the pre-hybridization mixture overnight at 37 °c; rinse once in 2x ssc for 1 h at rt, once in 1x ssc for 30 min at rt, once 0.5x ssc for 30 min at 37 °c and once 0.5x ssc for 30 min at rt; incubate in a blocking solution [2 % normal sheep serum, 0.3 % triton x-100 in buffer 1 (100 mm tris-hcl, 150 mm nacl, ph 7.5)] for 30 min at rt; incubate with sheep anti-dig fab fragments conjugate to alkaline phosphatase diluted 1:500 in buffer 1 (boehringer mannheim) for 1 h at rt; wash twice in buffer 1 for 5 min and twice in buffer 3 (100 mm tris-hcl, 100 mm nacl, 50 mm mgcl2, ph 9.5) for 2 min at rt. the alkaline phosphatase activity is revealed by incubation in the following medium at rt: 45 μl nitro blue tetrazolium (75 mg/ml in dimethylformamide), 35 μl 5-bromo-4chloro-3-indolyl phosphate, toluidinium salt (50 mg/ml) (boehringer mannheim), 10 ml buffer 3, and 2.4 mg levamisole (sigma). the slides are mounted in glycerol diluted 50 % in te (10 mm tris-hcl, 1 mm edta, ph 8). controls are performed by mockhybridizing slides without probe. morpho-functional methods mollusca cell shape changes according to manske and bade (1994) cell motility is defined as cellular movements that do not result in translocation of the affected cell, while cell migration (chemotaxis) is a cell translocation over a clearly measurable distance as a consequence of the chemotactic activity exerted by a chemoattractant. cell shape changes can be easily evaluated by mean of a computer-assisted image analysis performed on digital pictures. when this procedure was proposed (schön et al., 1991) some specific tools were required. nowadays, the majority of laboratories has at least one digital camera installed on the microscope, while freeware softwares (e.g., imagej 1.32j, wayne rasband, national institute of health, usa, http://rsb.info.nih.gov/ij/) can be utilized to calculate the shape factor (sf), i.e., the circularity, of the cells. two are the true limiting factors in the application of this technique. the first is represented by the necessity to collect a reasonable amount of hemolymph (minimum 300 μl). the second is that the hemocytes have to remain functionally unaltered onto the slide for at least 30 min, after withdrawal. the hemolymph of m. galloprovincialis satisfies both of these requirements, thus it has been successfully utilized for cell shape analyses. the technique is based on the assumption that an inactive immunocyte has a round shape while, upon activation, it assumes a more elongated (ameboid) form. briefly, the sf is calculated as: ac/at = [lt/lc]2, where at is the area of an hypothetical circle with the same perimeter of the cell under measurement, lt is the perimeter of an hypothetical circle with the same area of the cell under measurement, and ac and lc are the actual area and perimeter, respectively, of the cell under measurement. the lower the sf value, the greater the perimeter of the cell with respect to its area, meaning that more the shape is ameboid. it descends that once a software able to calculate actual perimeter and area is available, it is possible to obtain the sf by relatively simple calculations. a b in cell shape change experiments, the hemolymph is collected form the mussel and 100 μl are spread onto the glass slide into a vaseline/rubber ring. the aim of the ring is to prevent the hemolymph from dropping out from the slide. a glass coverslip is then put over the ring, paying attention not to cover a small portion of the ring itself, so that it is possible to have access to the spread hemolymph in order to add molecules of interest, e.g., chemoattractants or signaling transduction pathway inhibitors (ottaviani et al., 1997b; malagoli et al., 2003). after 3-5 min for allowing immunocyte adherence, the operator has to select the cells (usually 5 for slide) that will be followed during the experiment. the photographs of these cells are taken at regular intervals (usually 5 min), and exposure to the light should be minimized. once the last photograph has been taken, it is strongly recommended to immediately perform the following controls. first, the cells that have been chosen must display a morphology similar to that of the majority of the other cells of the slide. if, for 202 example, the chosen cells are the only ones active in all the slide, the experiment should be discarded. second, in order to exclude any effect connected to the permanence of the slide under the microscope, it is important to immediately verify if a slide that was treated identically to that used for cell shape analysis but was not kept under the microscope, presents cells with a morphology almost overlapping to the first one. obviously, control slides in which the chemoattractant and/or the inhibitors are absent have also to be prepared and this explains why a relatively high amount of hemolymph is required for this method. the electronic images of the experiments can now be utilized for the actual evaluation of the sf, by following the edge of the cells with the pointer of the mouse and asking the software to directly evaluate the circularity or, as an alternative, the perimeter and the area of the chosen cells for each time point. chemotaxis the migration assay refers to the effect of human cytokines on molluscan immunocytes (ottaviani et al., 1995b). the collected hemolymph is centrifuged at 200xg for 15 min and the pellet resuspended in 1 ml of snail saline solution (sss) (na+ = 41.15 mm; k+ = 0.54 mm; ca2+ = 3.55 mm; mg2+ = 2.61 mm; ph 7.5; osmolarity = 109 ± 5 mos) (ottaviani, 1983) containing 0.1 % bovine serum albumin (bsa) (sigma). the test is carried out using 48-well microchemotaxis chambers (nucleopore, pleasanton, ca, usa), in which the upper and lower compartments are separated by a 5 μm pore, polycarbonate polyvinylpyrrolidone-free filter allowing the cell to migrate actively through the pores. fifty μl of immunocyte suspension is placed in the upper compartment, while in the lower one are placed different cytokines [concentrations: interleukin-1α (500, 50, 5 pg/ml), tumor necrosis factor (tnf)-α (100, 10, 1 u/ml)]. after 90 min incubation at 37 °c, the migrated immunocytes adhering to the distal part of the filter are fixed, stained, identified microscopically and counted using light microscopy (magnification 40x). data are expressed as migrate cells/field. controls are obtained measuring the immunocyte migration in the presence of sss. phagocytic tests mollusca a classical in vitro bacterial phagocytosis is reported in ottaviani et al. (1995b). one hundred μl of freshwater snail hemolymph, are placed on a slide in the form of a ring by applying an adhesive pvc strip, in which two holes (∅ 10 mm) are performed. the experiment is carried out by mixing the hemolymph with staphylococcus aureus (106108 bacteria/ml) in presence or in absence of different stimulators. the incubation is performed in humidified chamber and the phagocytic activity was tested after 40 min. the phagocytized bacteria is determined by immunocytochemical procedure (ottaviani, 1990) using a rabbit anti-s. aureus polyclonal antibody. the number of phagocytized bacteria is performed counting under the light microscopy (magnification 1,000x) 20 random immunocytes for each preparation. recently a different approach has been developed for the phagocytic test in the mussel m. galloprovincialis (malagoli et al., 2007). in this case, instead of bacteria, fluorescent micro-beads (diameter 1 μm) are used to assess phagocytic activity of immunocytes. after withdrawal, 100 μl of hemolymph (about 104 cells) are incubated at dark with 0.1 % v/v of 1 μm diameter green-fluorescent microspheres (fluospheres®, molecular probes, or, usa) in a 1.5 ml plastic tube and kept in gentle rotation for 30 min at rt. after incubation, immunocytes are placed on microscope slides and there left for a further 5 min to allow their adhesion. the number of phagocytic immunocytes out of 100 and the number of phagocytized microspheres from each phagocytic immunocyte are counted under a fluorescence microscope with the filter commonly used for fitc (ex/em wavelengths 495/519 nm). negative control, i.e., the discrimination between effectively phagocytized and non-specifically bound micro-beads, are performed by incubating the immunocytes with 1 % sodium azide alone for 5 min before the addition of the micro-beads. in this way it is possible to set a baseline percentage of phagocytic immunocytes that have to be taken into consideration when performing statistic calculations. insecta the in vitro bacterial phagocytosis is performed in 1.5 ml plastic tube, by adding s. aureus (108 bacteria/ml) to hemolymph collected from adult flies of c. vomitoria. the phagocytosis assay is determined after 30 min. the samples are cytocentrifuged, and the immunocytes are stained with 0.5 % toluidine blue. after staining, the cell phagocytic activity is recorded under the microscope (franchini et al., 1996). cytotoxicity the natural cytotoxicity is performed on freshwater molluscan immunocytes (franceschi et al., 1991) in a short term (4 h) 51cr release test following the classical protocol for human studies using the k562 cell line as a target (lozzio and lozzio, 1975; grimm and bonavida, 1979). target cells (t), are labeled overnight with na2 51cro4 solution (amersham int., amersham, uk) in complete medium. the effector (e)/t ratio varies from 100:1 to 6:1. the percentage of specific cytotoxicity is calculated according to the following formula: % s.c. = exp. rel.spontaneous rel. ________________________ max. rel. – spontaneous rel. x 100 s. c.= specific cytotoxicity exp. rel. = experimental release max. rel. = maximum release enzyme activity acid phosphatase activity has been examined in molluscan immunocytes (franchini and ottaviani, 1990). the activity is performed on fixed (1.5 % glutaraldehyde in 20 mm cacodylate buffer, ph 7.5) 203 and unfixed cytocentrifuged immunocytes. slides are incubated for 2 h at 37 °c in barka and anderson media (1962). after incubation, cells are treated with 1 % ammonium sulfate for 1 min and covered with acquovitrex (c. erba). flow cytometry the cytofluorimetric analysis has been applied to molluscan hemolymph in order to detect the different cell populations on the basis of physical characteristics, such as diameter, size, density, etc allowing to distinguish them on the basis of forward and side light scatters. a further phenotype characterization of immunocytes has been carried out using various antibodies, such as anti-humanacth pab and mab, anti-n-acetylmuramic acid pab and different mouse anti-human mabs recognizing different molecules (ottaviani and montagnani, 1989; franceschi et al., 1991; ottaviani et al., 1991). procedure using rabbit anti-humanacth pab and mouse anti-human acth mab: 400 μl of hemolymph are divided into two parts. one is incubated with 10 μl of anti-acth pab (dakopatts) or with mouse anti-acth mab (chemetron, milan, italy) for 20 min at 4 °c, while the second is taken as a negative control. the stained samples are then washed with cold pbs-1, incubated with 10 μl fitclabeled goat anti-rabbit igg (dakopatts) or with 10 μl phycoerythrin (pe)-labeled goat anti-mouse igg (becton-dickinson, san josè, ca, usa), washed with cold pbs-1 and analyzed with a flow cytometry (becton-dickinson). negative controls are prepared according to the above procedure, but omitting the anti-acth pab or anti-acth mab. samples are finally analyzed by using state of the art flow cytometry, taking into account the requirements for a precise setting of the fluorescence intensity and of the compensation, should one use two or more fluorochrome-conjugated antibodies to stain cells. tunicata trypan blue exclusion assay after cell adhesion, primary cultures of b. schlosseri hemocytes are exposed for 60 min to fsw containing xenobiotics at various concentrations. living hemocytes are then incubated for 5 min in a solution of 0.25 % trypan blue in fsw and observed under the light microscope at 1,250x. the percentage of stained hemocytes is estimated on a total count of at least 300 cells, and the concentration of xenobiotics able to cause mortality to 50 % of the hemocytes (lc50) is calculated according to the probit method (spss 11.0, spss corp., chicago, il, usa). adhesion assay since the capability of b. schlosseri hemocytes to adhere is fundamental for their role in immunity, adhesion assay can be usefully applied to acquire information on the effects of xenobiotics on this species. hemocytes are left to adhere for 60 min on clean coverslips in the presence of xenobiotics, at various concentrations. slides coated with 50 µg/ml poly-l-lysine are used as reference controls (100 % adhesion). after glutaraldehyde-fixation and staining for 5 min in 10 % giemsa solution, coverslips are mounted in acquovitrex (c. erba) and observed. in order to evaluate the ability of hemocytes to adhere to glass in the presence of xenobiotics, the total number of hemocytes in 10 optic fields at 1,250x is counted and expressed as the adhesion index, i.e., the ratio, expressed as percentage between the total hemocyte count in experimental slides and the cell count in similar conditions in poly-l-lysinecoated slides. cell spreading assay after 60 min-exposure to biocides at various concentrations, b. schlosseri hemocyte monolayers are fixed with glutaraldehyde, stained with giemsa solution and mounted in acquovitrex (c. erba). their morphology is then observed under the light microscope at 1,250x, and the cell-spreading index, i.e., the percentage of hemocytes with ameboid shape, is estimated after counting at least 300 cells per coverslip (cima and ballarin, 1999). in addition, computer-assisted image analysis (casting image nt, hp laboratories, palo alto, ca, usa) is performed to evaluate the sf, as defined above for cell shape change evaluation in molluscs. lower shape factors indicate larger perimeters with respect to the areas and, therefore, an increased ameboid shape. assay for reduced glutathione (gsh) content after treatment with xenobiotics, b. schlosseri hemocyte monolayers are washed in fsw, stained for 10 min at 37 °c in 40 mm chlorobimane (a fluorescent dye specific for gsh with λmax of 461 nm) (sigma) solution in fsw, obtained from a 20 mm stock solution in 95 % ethyl alcohol, and then rinsed in fsw (cookson et al., 1998). living cells are immediately observed under a fluorescence microscope equipped with an ultraviolet light filter block (ex/em 270-380/460 nm), at a magnification of 1,250x. positive sites appear fluorescent blue. the percentage of stained cells is then determined and expressed as the gsh index. morula cell (mc) degranulation assay fifty μl of b. schlosseri hemocyte suspension (15x106 cells/ml) are placed in the center of the culture chambers prepared as already described and left to adhere to washed coverslips for 30 min. after discarding the fsw, hemocytes are then incubated for 60 min at rt with 50 ml of autologous or heterologous blood plasma or with a suspension of escherichia coli (200x106 cells/ml), s. aureus (50x106 cells/ml), bacillus clausii spores (200x106 spores/ml) or ordinary baker’s yeast (saccharomyces cerevisiae) (50x106 cells/ml) in fsw, and washed in fsw (ballarin et al., 1998, 2005). the morphology of living mcs is then observed under a phase contrast microscope at a magnification of 1,200x (fig. 10). chemotactic assay migration of b. schlosseri hemocytes is analyzed using a 24-well chemotaxis chamber (transwell, costar, corning, ny, usa) with the lower and upper wells separated by 8-μm polycarbonate filters. the upper wells are filled with 100 μl of hemocyte suspension (5x105 cells) in fsw and 500 μl of fsw are added to the lower wells. solutions in upper and lower wells are allowed to equilibrate for 15 min, and then 20 μl of the lower solution are substituted with 20 μl of the putative chemoattractant at the concentration of 2 mg/ml 204 fig. 10 living morula cells incubated in the absence (a) or in the presence (b) of heterologous blood plasma. in this last case cells have degranulated and have released their vacuolar content. bar = 15 µm. (fsw in controls) (modified according to raftos et al., 1998). cells are allowed to migrate for 2 h at 18 °c. after incubation, the lower surface of the filters are flushed extensively with fsw, in order to detach adhering hemocytes and collect all the cells which have passed through the membrane. hemocytes in the lower wells are then collected in a 1.5 ml vial and centrifuged at 780xg for 10 min. the mean number of cells in 10 random optical fields, at a magnification of 1,250x, is used to define the migration stimulation index, i.e., the ratio between the mean number of migrated cells per field in experimental series and the mean number of migrated cells in controls. phagocytosis assay after adhesion, b. schlosseri hemocytes are incubated with 60 µl of a suspension in fsw of various test particles (ballarin et al., 1994) represented by living or autoclaved (15 min at 120 °c) cells of s. cerevisiae, sheep erythrocytes, zymosan (sigma), latex beads (1 and 3 µm diameter) (sigma), fluorescent e. coli cells (phagotest kit, orpegen, heidelberg, germany). the number of test particles is adjusted to a particle:hemocyte ratio of 10:1 after counting with a bürker’s hemocytometer. the best results are obtained with yeast suspensions in fsw since this type of target particles is both extensively engulfed by phagocytes and easily recognizable in phagosomes. cultures are kept upside-down for 5120 min at 25 °c and the uningested yeast is then removed by dipping repeatedly the coverslips in a large volume of fsw. viability, assessed by the trypan blue exclusion assay, exceeds 95 % after 2 h of incubation. when fluorescent test particles are used, uningested particles are quenched by quick immersion of coverslips in a solution of 2 mg/ml trypan blue and 2 mg/ml crystal violet in 0.02 m citrate buffer, ph 4.4 containing 33 mg/ml nacl. hemocyte monolayers are then fixed in 1 % glutaraldehyde and 1 % sucrose in fsw for 30 min at 4 °c and stained with 5 % giemsa for 5 min. the coverslips are mounted on glass slides with aquovitrex (c. erba) or fluorsave (calbiochem), the latter being used for assays with fluorescent test particles. at least 300 hemocytes are observed per slide under microscope, in ten optical fields, at the magnification of 1,250x, and, the phagocytic index, i.e., the percentage of hemocytes with ingested cells, is evaluated. the phagocytic index increases progressively with time reaching a plateau of 10-15 % after an incubation time of 60 min, which represents the optimum incubation time for this assay. in other experiments, at the end of the incubation time, the medium is collected and centrifuged to obtain a “conditioned” supernatant, which is used to detect enzymatic activity, reactive oxygen species, opsonins and cytokines released in the incubation medium (cima et al., 1996) and to study its effects on other phagocytosis assays (menin et al., 2005). finally, the phagocytosis assay can be also used for evaluating the immunosuppressant effects of xenobiotics added at various concentrations to the incubation medium containing the target particles. enzyme activity assays are carried out on glutaraldehyde-fixed hemocytes, previously exposed or not to xenobiotics. these methods can be applied either to investigations aimed at hemocyte characterization or to the study of xenobiotic effects on hemocyte functions. in controls, the specific enzyme substrate is omitted. after incubation in the reaction mixtures, monolayers are thoroughly washed in distilled water, coverslips mounted in acquovitrex (c. erba), and at least 300 cells per slide are counted to determine the fraction of positive hemocytes, expressed as the enzymatic index. acid phosphatase this is a lysosomal enzyme used as a marker of phagocytes. fixed hemocytes are washed in 0.1 m sodium acetate buffer, ph 5.2, for 10 min and incubated for 3 h at 37 °c in the reaction mixture made by 10 mg naphthol as-bi phosphate (sigma) in 400 μl dimethylformamide (dmf), 400 μl solution a (4 % new fuchsin (sigma), 2 % hcl in distilled water), 400 μl of a 4 % aqueous solution of nano2 solution b and 20 ml of 0.1 m sodium acetate buffer. positive sites appear red (lojda et al., 1979). alkaline phosphatase after fixation, hemocytes are washed in trishcl buffer 0.1 m, ph 9, for 10 min and incubated for 2 h at 37 °c in a reaction mixture similar to that used for acid phosphatase assay, but containing 20 ml of tris-hcl buffer instead of sodium acetate buffer (burstone, 1962). hemocytes are then washed in tris-hcl buffer for 10 min and mounted. positive sites stain red. β-glucuronidase it is another lysosomal hydrolytic enzyme, marker of phagocytes. fixed hemocytes, washed in 0.1 m sodium acetate buffer, ph 5.2, for 10 min, are incubated for 3 h at 37 °c in the reaction mixture made by 4 mg naphthol as-bi β-glucuronide (sigma) dissolved in 250 μl dmf, 400 μl solution a, 400 μl of aqueous solution of 4 % nano2 and 20 ml of sodium acetate buffer (lojda et al., 1979). positive sites stain red (fig. 7l). 205 5’-nucleotidase fixed hemocytes are washed for 10 min in tris–maleate buffer 0.2 m, ph 7.2 and incubated for 2 h at 37 °c in the following reaction mixture: 20 mg adenosine-5’-monophosphate (amp) (sigma), 22 ml distilled water, 20 ml tris-maleate buffer, 3 ml 2 % pbno3, 5 ml 2.5 % mgso4 (wachstein and meisel, 1957). hemocytes are then washed twice with distilled water and with ammonium sulfide solution (21 %) (fluka), diluted 1:100 in distilled water, for 2 min. they are then washed again with distilled water and mounted. positive sites stain black. acid esterase fixed hemocytes are washed for 10 min in phosphate-citric acid buffer 0.1 m, ph 5.5, and incubated for 16 h at 4 °c in the following reaction mixture: 3 mg naphthol acetate (sigma) dissolved in 500 µl acetone, 250 µl solution a, 250 µl solution b, 19 ml phosphate-citric acid buffer (lojda, 1977). hemocytes are then washed with distilled water and mounted. positive sites stain pinkish-brown. chloroacetylesterase hemocytes, washed in pbs-3 for 10 min, are incubated for 1 h at rt in the following reaction mixture: 6 mg naphthol chloroacetate (sigma) dissolved in 1 ml dmf and added to 19 ml pbs-3 containing 20 mg fast blue b (moloney et al., 1960). hemocytes are washed in pbs-3 for 10 min and mounted. positive sites stain blue. non-specific esterases non-specific esterases are reference hydrolytic enzymes, typical of phagocyte lysosomes. this assay was previously used to label the phagocytic line of b. schlosseri hemocytes (ballarin and cima, 2005) in acute toxicity assays with antifouling xenobiotics. hemocytes are washed in pbs-3 and incubated for 3 h at 4 °c in 100 ml of pbs-3 containing 2 ml of 1 % 1-napthyl acetate (sigma) as a substrate, previously dissolved in 1 ml acetone, 1 ml of hexazonium-p-rosaniline [500 µl of a solution of 0.4 g new fuchsin (c. erba) in 7.2 % hcl added to 500 µl of a 4 % solution of nano2 in distilled water] (davis et al., 1959). positive sites stain red. cytochrome-c-oxidase it is an enzyme of the mitochondrial respiratory chain, and its activity, related to mitochondrial functionality, is required for atp production. cells are washed in 0.1 m na-acetate buffer, ph 5.5, for 10 min, and incubated for 4 h at 37 °c in a solution of 0.2 % dab in 1 % mncl2 containing 0.01 % h2o2 (novikoff and goldfischer, 1969). positive sites stain brown. ca2+-atpase it is involved in the sequestration of ca2+ ions in intracellular stores. hemocytes are incubated for 20 min at 37 °c in 0.1 m na-barbital buffer, ph 9.0, containing 3 mm atp, 3.25 mm 2,4-dinitrophenol (dnp) and 0.1 m anhydrous cacl2. cells are then repeatedly washed in 1 % anhydrous cacl2, further rinsed in 1 % co(no3)2, immersed in 0.5 % ammonium sulfide for 1 min, and washed in distilled water (chayen et al., 1969). positive sites appear black. arylsulfatase hemocytes are washed in sodium acetate buffer 0.1 m, ph 5.2, for 10 min and incubated for 2 h at 37 °c in the following reaction mixture: 0.16 g p-nitrocatecholsulfate (sigma), 4 ml distilled water, 12 ml 0.1 m sodium acetate buffer, ph 5.5, 4 ml 8 % pbno3 (goldfischer, 1965). after incubation, hemocytes are washed with distilled water and then with 21 % ammonium sulfide solution (fluka) diluted 1:100 in distilled water, for 2 min. finally, they are washed with distilled water and mounted. positive sites stain brownish–black. peroxidase after fixation, hemocytes are washed in pbs-3 for 10 min and incubated for 2 h at 37 °c in the following reaction mixture (graham and karnovsky, 1966): 0.5 mg/ml dab in distilled water containing 0.02 % h2o2. hemocytes are then washed in distilled water and mounted. positive sites stain brown. phenoloxidase phenoloxidase is an oxidative enzyme with a cytotoxic activity involved in defense reactions in b. schlosseri, where it can be used as a marker for the cytotoxic line of blood cells. after fixation, hemocytes are incubated for 1 h in saturated dihydroxy-l-phenylalanine (l-dopa) solution in pbs3 (hose et al., 1987), washed in distilled water, and mounted in acquovitrex (c. erba). positive cells stain blackish-brown (fig. 7m). assay for lysozyme hemolymph is collected from the colonial marginal vessel with a fine micropipette and centrifuged at 780xg for 10 min. the supernatant, corresponding to the cell-free blood plasma, is immediately used for the lysozyme assay. pellets are resuspended in distilled water, sonicated for 5 min at 0 °c with a braün labsonic u sonifier at 50 % duty cycles, and centrifuged at 12,000xg for 15 min at 4 °c. supernatant, corresponding to the hemocyte lysate, is collected and immediately used for the lysozyme assay. one percent of methylene blue 0.13 % in distilled water is added to a 0.25 % suspension of micrococcus lysodeikticus (sigma) in 0.1 m phosphate/citrate buffer, ph 5.8, and incubated for 30 min at 25 °c to stain cells. seven ml of the above suspension are then added to 50 ml of 1.5 % melted agarose in 0.1 m phosphate/citrate buffer, ph 5.8, and 5 ml of the solution are poured into petri dishes. when the agarose solidifies, 5-mm wells are made and filled with 50 µl of hemocyte lysate. lysis diameter are observed after overnight incubation at 37 °c and compared with those obtained using known dilutions of lysozyme (5 and 10 mg/ml) from chicken egg white (sigma) in 0.1 m phosphate/citrate buffer, ph 5.8 (fig. 11a). protein concentration in hemocyte lysate and blood plasma is determined according to bradford (1976) using bsa as standard. hemagglutinating assay rabbit erythrocytes are washed three times by centrifugation at 500xg for 10 min in tris-buffered saline (tbs-2) (tris-hcl 50 mm, nacl 150 mm, ph 7.4) and incubated for 30 min at 37 °c in 0.1 mg/ml 206 trypsin in tbs-2 (ballarin et al., 1999). they are then washed again and resuspended in tbs-2 containing 0.2 % gelatin to get a 1 % solution (v:v). fifty µl of b. schlosseri blood plasma are serially diluted two-fold with tbs-2 in the wells of u-bottomed microtiter plates and an equal volume of 1 % rabbit erythrocyte suspension is added in each well. fsw is used in controls. tbs-2 containing 5 mm egta is also used to assess the ca2+-dependence of the reaction. in another experimental series blood plasma is incubated for 30 min with 1 % rabbit erythrocyte suspension in order to control the specificity of the interaction. the suspension is then centrifuged at 780xg for 10 min and the rabbit erythrocyte-absorbed supernatant is collected and used in the hemagglutination assay. plates are gently shaken, incubated for 1 h at 37 °c and then kept at 4 °c. the hemagglutination titre (ht), i.e., the reciprocal of the highest dilution giving positive hemagglutination is then evaluated (fig. 11b). fig. 11 a: lysis plaque of micrococcus lysodeikticus in agarose due to lysozyme activity; b: hemagglutination activity of blood plasma: absence and presence of agglutination in row a (control; fsw) and row b (hemagglutination titer = 128), respectively. agglutination activity is also evaluated in vitro by adding 50 µl of a 1 % trypsinized rabbit erythrocyte suspension in fsw to culture chambers. after 30 min of incubation at 25 °c, adhesion of erythrocytes is observable under the light microscope as rosette formation around some hemocyte types. in the solitary ascidian c. intestinalis erythrocytes from different species (i.e., rabbit, sheep, human abo group) were washed three times with tbs-2, 0.1 % (w:v) pig gelatin, suspended at 1 % in tbs-2 with or whitout 5 mm cacl2 ph 7.4, 0.1 % gelatin, and used in a microtitre plate hemagglutination assay (ha) in which 25 μl of serially diluted sample were mixed with an equal volume of erythrocyte suspension (fig. 12). serial dilutions (2-fold) of the tbs-2dialyzed and diluted serum (1:10) are performed with tbs-2 containing 0.1 % gelatin. the ht is evaluated after 1 h incubation (from 18 to 37 °c). also for c. intestinalis to increase the erythrocyte sensitivity to the hemagglutination assay, trypsintreated erythrocytes are prepared by resuspending an erythrocyte pellet (obtained from 1.0 ml blood) in 6 ml tbs-2 containing 300 μg trypsin (stock solution prepared in 10 mm hcl) (parrinello and canicattì, 1982). the reaction mixture is incubated at 37 °c for 15 min. the trypsin-treated erythrocytes are washed with tbs-2 and resuspended (1 %) in tbs-2. plaque-forming cell assay (pfc) a pfc assay using tunicate hemocyte effectors has been originally described by cunningham and szenberg (1968) for the human b cell/sheep red blood cells and subsequently modified for tunicate hemocytes (parrinello et al., 1996; cammarata et al., 1997). fifty µl of hemocytes suspension (1 x 106/ml) in marine solution (ms) (12 mm cacl2, 11 mm kcl, 26 mm mgcl2, 45 mm tris, 38 mm hcl, 0.45 m nacl, ph 7.4) are mixed with 50 µl of suspension of 5 % rabbit erythrocytes in ms. the reaction mixture is rapidly layered into the slide chamber by capillary action. the chamber has been constructed by placing three thin strips of doublestick tape placed between the borders and in the centre of a coverslip and another glass coverslip is then suspended onto the three pieces of tape forming a double chamber. after 15 min of incubation at 20 °c the cell mixture is examined under a phase contrast microscope. each slide chamber can hold just under 0.1 ml on either side of the tape (0.2 ml per slide) (fig. 13). phenoloxidase (po) activity determination of physical and chemical characteristics of lectins po activity is measured according to winder and harris (1991) using the reagent 3-methyl-2benzo-thiazolinone hydrazone (mbth) which reacts directly with dopaquinones. this method has the advantage of measuring the direct product of dihydroxyphenyl-l-alanine (l-dopa) oxidation since not all dopaquinones are converted to dopachromes which are measured when l-dopa only is used as substrate. briefly, 20 µl of hemolysate were incubated with 490 µl of pbs-3, 290 µl of 20.7 mm mbth in pbs-3 containing 4 % of dmf and 200 µl of l-dopa-saturated pbs-3. the reaction is read spectrophotometrically at 494 nm, each for 5 min. to examine divalent cation requirements for lectin binding, cacl2 or mgcl2 is added in the tbs-2 medium to obtain 5-10 mm final concentration. edta (1-10 mm) or egta (1-10 mm) are used to examine the effect of ca2+/mg2+ depletion on the hemoagglutinating activity. to examine the thermal stability of the protein, samples are incubated at different temperatures from 18 to 90 °c for 20 min and cooled down for 10 min on ice before the ha. susceptibility of the ha to freeze-thaw is examined by carrying out the ha on samples maintained at -20 °c for 2 months and thawed at rt. 207 serial dilution ct 2 4 8 16 32 64 128 256 512 1024 2048 1 2 cnt fig. 12 determination of hemoagglutinating titer. each well of the microtitre plate contained 25 μl of sample 1 and 2; 25 μl 1 % suspension of rabbit erythrocyte. the agglutinated erythrocytes form a carpet that cover completely the well, the last effective dilutions were indicated in blue (hemagglutination titer: sample 1=16; sample 2=512). in absence of agglutinin the cell form a button in the well. cnt= control in which sample has been substitute with tbs-2. carbohydrate specificity and chromatographic resin selection of lectins hemagglutinating activity is assayed with rabbit erythrocytes in the presence of saccharides as potential inhibitors of lectin binding. when the lectin activity has been detected in the hemolymph or extract of interest, the second step is to identify those carbohydrate ligands for which the lectin show the highest affinity for matrix selection in order to allow the lectin enrichments by affinity chromatography. inhibition experiments are carried out using decreasing concentrations (starting from 200 mm in tbs-2) of a large variety of monosaccharides that should include all four types of mäkelä’s classification (1957) plus sialic acid (i.e., l-fucose, d-arabinose d-galactose, d-glucose, dmannose, n-acetyl-galactosamine, and n-acetylglucosamine), oligosaccharides (i.e., maltose, lactose, d-raffinose, cellobiose), mannan and glycoprotein. the lectin solution is incubated with each one of the sugars at different concentrations for 60 min and the rabbit erythrocytes are then added. the agglutination titer in the presence of sugars are compared to the control where the sugars are substituted with tbs-2. the selected saccharide are coupled to the column support matrices by activation with cyanogen bromide or divinylsulfone. often other purification steps are required for an optimal lectin purity (i.e., size exclusion, ion exchange, hydrophobic chromatography). this additional protein purification step is an empirical process employing a large number of experiments, which often represent a limit especially for planning integrated project research. to overcome this limitation a plateor tube-based experiments could be used avoiding the preparation of numerous packed columns, the use of large volume of sample and test on chromatography systems. this parallel screening can facilitate the selection of media and development of optimal binding and elution conditions. for this test the media slurry can be incubated with the same volume of sample (i.e., hemolymph or extract of interest) and, after 1 h, centrifuged at 400xg for 5 min to remove not binding proteins. the binding activity can be detected analyzing the unbound proteins by electrophoresis or by centrifugation with the specific gel elution buffer for bound proteins recover. quantitative methods mollusca free amino acids (faa) the faa has been evaluated in molluscan hemolymph (ottaviani, 1984). the hemolymph is centrifuged at 3,000xg for 10 min and 100 μl of the supernatant are utilized to determine the faa using the amino acid analyzer liquimat iii (kontron, zurich, switzerland). radio-immuno assay (ria) test the following procedure has been utilized to quantify acth-like molecules in the molluscan serum and immunocytes (ottaviani et al., 1990). after hemolymph collection and the subsequently centrifugation at 600xg for 10 min, the serum is stored at -20 °c until use; the pellet must be resuspended in pbs-1, homogenized for 30 s and centrifuged at 1,500xg for 20 min at 4 °c before storing. the serum and pellet are freeze-dried, redissolved in pbs-1 and assayed via commercial ria kits for acth. high performance liquid chromatography (hplc) the hplc assay has been used in the determination of the biogenic amines (ba) in molluscan serum and immunocytes (ottaviani et al., 1992, 1993, 1994, 1995a, 1997a, 1998a). after centrifugation of the hemolymph, the ba are detected by hplc in the serum and pellet (immunocytes). the ba are extracted from the serum by using a clin-rep-catecholamine kit (recipe pharma vertriebs gmbh and co kg, münchen, germany) and the extracted sample is analyzed with a rapid and sample isocratic simultaneous determination of norepinephrine, epinephrine and dopamine. monoamine peaks are identified by comparing their retention times in the serum extracts with those of a standard solution. each ba is quantified using the internal standard 208 a b c figure 13 plaque forming cell assay. a: plaque of lysis observed by phase-contrast microscope. red arrowhead = plaque forming hemocytes capable of secreting lytic factors against rabbit erythrocytes; yellow arrows = erythrocyte ghosts; bar = 15 µm. b: low magnification observation, three plaques are marked in red; c: control of erythrocytes. (3,4-dihydroxybenzylamine, dhba) method with a correction factor. the pellet is washed with sss, homogenized with a fisher sonic dismembrator model 300 (globalspec inc., troy, ny, usa), centrifuged and the supernatant analyzed by direct injection into the chromatographic system. identification and calculation of the ba are performed by directly comparison areas of a standard calibration curve with those of the samples. the hplc system used consisted of a isocratic lc pump (mod. 250 perkin elmer, (waltham, ma, usa) equipped with a degasser (erc-3512-erma), an automatic injector (rheodyne 7125, 100 μl loop), an electrochemical detector (mod. 460 waters) and a data system pe nelson (mod. 1020 perkin elmer). spectrofluorimetry this method has been utilized to detect the neutral endopeptidase-24.11 (nep)-like activity in molluscan immunocytes (ottaviani and caselgrandi, 1997; caselgrandi et al., 2000). the collected hemolymph is centrifuged and the nep-like activity of the pellet is measured according to the deschodtlanckman et al. (1990) procedure. a maximum number of 700x103 immunocytes/ml have to be used for a correct evaluation since above this value enzymatic activity falls quickly in relation to a given number of cells. nep cleaves the substrate (sucala-ala-phe-amc) (sigma) to phe-amc. a fluorescent spectrometer (perkin elmer, mod. 204) at an excitation wavelength of 440 nm is utilized to detect the fluorescent product, free amc. the calibration curve is measured with pig nep enzyme. crustacea elisa to quantify the variation of chh (crustacean hyperglycaemic hormone) level in the eyestalks and hemolymph of p. elegans elisa test using an antibody against recombinant chh has been performed (lorenzon et al., 2004). in this method a group of 10 p. elegans is used for eyestalk ablation; animals are anaesthetized for 1 min on ice before ablation. the eyestalk was quickly frozen and the pigmented eyecup dissected. eyestalk homogenate is prepared from 20 eyestalks homogenized in 2 ml cold pbs (sigma) ph 8.0 and then centrifuged for 1 h at 930xg at 4 °c and the pellet is discarded. homogenates are quickly deep frozen at -20 °c and stored until required for study. hemolymph is withdrawn from groups of 10 animals, centrifuged for 1 min at 10,300xg at 4 °c and the supernatant is then stored at -20 °c. the standards are represented by a known concentration [from 1 to 0.001 µg in 100 µl of pbs (sigma)] of 6xhisnenchhwt (n. norvegicus chh wild type) recombinant protein (mettulio et al., 2004). one hundred µl of the samples are loaded onto a 96 microwells plate (costar) and incubated in duplicate overnight at 4 °c. the content of the wells is discarded and the wells are washed 4 times with 209 250 µl of pbs (sigma) + 0.1 % tween20, ph 7.4 (pbs-t); then filled with 100 µl of 3 % bsa (sigma) solution in pbs (sigma) ph 7.4 plus 5 % fetal calf serum (sigma) and left for 2 h at rt. the content is discarded and the plates are again washed 4 times as described above. one hundred µl of 1 µg/ml biotinylated anti-nenchh (anti n. norvegicus chh; giulianini et al., 2002) antibody is then added to each well and the plate is incubated for 3 h at 36 °c. after removal of the biotinylated antibody, plates are washed extensively with pbs-t, followed by the addition of 100 µl of streptavidin-peroxidase solution (sigma) diluted 1:5,000 and incubated for 1 h at rt. the plates are once again washed 4 times with pbs-t and developed with 2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid solution (sigma) in darkness for 1 h at rt (100 µl per well). the absorbance is measured in a multiwell plate reader (anthos 2020 version 1.1, krefeld, germany) at 405 nm. tunicata spectrophotometric assay for detoxifying enzymes glutathione s-transferase (gst) this enzyme plays a multiple role in xenobiotic metabolism, catalyzing the conjugation reactions of reduced glutathione (gsh) and electrophilic xenobiotics. gst acts as a mediator of xenobiotic detoxification and it has recently been successfully used as a biomarker, since it appears to be important in oxidative stress protection (cima et al., 2002b). gst activity is determined according to the method of habig et al. (1974) using 1-chloro-2,4dinitrobenzene (cdnb) (sigma) as substrate (λ = 340 nm, ε = 9.6 mm-1 cm –1) on hemolysates obtained after sonication at 4 °c at 50 % duty cycles for 5 min of hemocytes previously exposed in vitro to xenobiotics, then centrifuged and resuspended in 20 mm tris-hcl buffer, ph 7.5. the reaction is performed in a final volume of 1 ml containing 160 µl hemolysate or fsw in controls (blank), 700 µl of 100 mm k3po4, ph 7.0, 70 µl of 20 mm cdnb dissolved in absolute ethanol, 70 µl of 20 mm gsh (sigma). absorbances are read at 10 s intervals for 2 min, and enzyme activities, measured as decrease of substrate, are expressed as nmol/min/mg protein (mean ± sd) with the following formula: enz. act. = enz. act. = enzyme activity abs = absorbance glutathione peroxidase (gpx) it is an antioxidant enzyme which protects from the effects of reactive oxygen species through reduction of hydrogen peroxide (or organic peroxides) to water (or alcohols), respectively, requiring gsh consumption. it also protects phagocytes from the deleterious effects of reactive oxygen species produced during the respiratory burst. its activity is measured by the consumption of nadph monitored at 340 nm (ε = 6.22 mm-1 cm-1) using h2o2 or cumene hydroperoxide as substrates, for the se-dependent or the sum of se-dependent and se-independent forms, respectively. the rate of blank reaction is substracted from the total rate (gunzler and flohe, 1985). the reaction is performed in a final volume of 1 ml containing 500 µl of 0.1 m phosphate buffer, ph 7.0, 1 mm edta, 0.1 % nahco3, 100 µl of 2.4 u/ml glutathione reductase (sigma), 100 µl of 10 mm gsh, 100 µl hemolysate. the reaction is initiated by the addition of 100 µl of 0.03 % h2o2 in distilled water or 100 µl of 25 mm cumene hydroperoxide (sigma) in ethyl alcohol, respectively. absorbances are read at 10 s intervals for 2 min, and enzyme activities is expressed as nmol/min/mg protein (mean ± sd) and it is calculated with the formula reported above. protein concentration is measured according to bradford (1976) using bsa as standard. spectrophotometric assay for caspases whole blood from large colonies of b. schlosseri (≥ 10 systems) that have been previously blotted dry is collected and centrifuged at 780xg for 10 min. supernatants are discarded and the pellets resuspended in 100 μl of the lysis buffer from colorimetric activity assay kits for caspase-3 and -8 (chemicon). after a 10 min incubation, samples are centrifuged at 10,000xg for 10 min. supernatants, referred to as hemocyte lysates, are collected and their protein content evaluated according to bradford (1976). in the wells of a 96-well microtiter plate, 50 μl hemocyte lysate are incubated for 1 h at 37 °c with 10 μl of the specific colorimetric substrates n-acetyl-asp-glu-val-asp-p-nitroaniline or n-acetyl-ile-glu-thr-asp-p-nitroaniline, for caspase-3 and -8, respectively, according to the manufacturer’s instructions. the release of pnitroaniline is measured at 405 nm in a microplate reader. a p-nitroaniline reference curve is obtained by serial dilution of a 10 mm standard solution in dimethylsulfoxide. one unit of caspase activity is defined as the amount of enzyme able to cleave 1 nmole substrate per hour at 37 °c. the results are expressed as specific activities (u/mg protein). spectrophotometric assay for lysozyme activity lysozyme activity is quantified in cell lysate. in this case, hemolymph is collected from blood vessels without previous dipping in na-citrate, placed in 1.5 ml plastic tubes, and centrifuged at 780xg for 10 min. hemocytes are then incubated at 20 °c for 60 min in fsw and then centrifuged at 780xg for 10 min, re-suspended in 1 ml of distilled water, sonicated at 0 °c for 2 min at 50 % duty cycles, and centrifuged at 12,000xg for 15 min at 4 °c. supernatant, corresponding to cell lysate, is collected for the lysozyme assay. fifty μl of cell lysate, from both controls and treated hemocytes, are added to 950 μl of a 0.15 % suspension of m. lysodeikticus (sigma) in 66 mm phosphate buffer, ph 6.2, and the decrease in absorbance (da)/min is continuously recorded at 450 nm for 5 min at 20 °c, according to santarém et al. (1994). standard solutions containing 1, 2.5, 5 and 10 μg/ml lysozyme in phosphate buffer are prepared from crystalline hen egg white lysozyme (sigma). the average decrease in absorbance/min is determined for each enzyme solution and a standard curve of enzyme concentration versus da/min is drawn. one unit of lysozyme is defined as the amount of cell lysate equivalent to 1 μg of lysozyme, in the conditions (abssample – absblank) x final volume ______________________________ 1000 x ε x mg protein x ml substrate 210 described above. results are expressed as μg lysozyme/mg protein. protein concentration in cell lysate is quantified according to bradford (1976). assays for detection of reactive oxygen species superoxide anion in cytochemical assay, modified after song and hsieh (1994), yeast cells prepared for phagocytosis assay are resuspended in 0.3 % nitroblue tetrazolium (sigma) in fsw. sixty µl of this suspension are added to each culture chamber. after incubation for 60 min, hemocyte monolayers are fixed, washed and mounted as previously described for the phagocytosis assay. positive sites stain blue. yeast is omitted in negative controls, while 150 u superoxide dismutase (sigma) are added to the incubation medium as a control of specificity. the superoxide anion production is evaluated as percentage of hemocytes containing dark blue spots of precipitated formazan. in spectrophotometric assay, after incubation with 0.3 % nitroblue tetrazolium the hemocyte monolayers are washed and dipped in absolute methanol for 2 min before being air-dried. eighty µl of a solution of 2 m koh and dimethylsulfoxide (ratio 6:7) are added to the monolayers to dissolve formazan precipitates and recovered after 5 min. the absorbance at 620 nm is then read with a microplate reader. hydrogen peroxide collected hemocytes are centrifuged at 780xg for 15 min and resuspended in a freshly prepared hydrogen peroxide reaction mixture [200 µl of 1 % phenol red and 200 µl of 200 u/mg peroxidase (grade ii, roche) in 9.6 ml of fsw] (pick, 1986) containing yeast. at the end of the incubation period, suspensions are centrifuged at 780xg for 15 min, 100 µl of 1 n naoh are added to the supernatants and their absorbance at 620 nm is read with a microplate reader. hypochlorite ions washed hemocytes are resuspended in 1.87 % taurine (sigma) and yeast in fsw. after 60 min, 0.5 µl of a 50 mg/ml solution of catalase (sigma) are added to stop the reaction and suspensions are centrifuged at 780xg for 15 min. twenty mm ki is added to the supernatants and absorbance at 350 nm is read with a spectrophotometer (gressier et al., 1994). the thiol-containing antioxidant sodium 2mercaptoethane sulfonate (mesna; sigma) is added to the incubation medium at a concentration of 10 mm as a control for specificity. nitrite ions washed hemocytes are resuspended in yeastcontaining fsw and, at the end of the incubation period, they are centrifuged at 780xg for 15 min and 100 µl of supernatants are incubated for 10 min with 100 µl of griess reagent [equal volumes of 0.1 % naphtylethylenediamine (sigma) in distilled water and 1 % sulfanilamide (sigma) in 5 % h3po4]. the absorbance at 550 nm is then read with a microplate reader (shen et al., 1994). a precalibrated standard curve, with nano2 as standard, is used to calculate nitrite concentrations. yeast is omitted in controls. protein purification and analysis protein analysis by mean of electrophoresis and immunoblotting is usually applied in invertebrate models following protocols that are very similar to those realized in mammals. for instance, the common sds-page method (laemmli, 1970) followed by the blotting procedure (towbin, et al., 1979) has been successfully applied in mollusca (malagoli et al., 2003), insecta (malagoli et al., 2002a) and tunicata (gasparini et al., 2008). therefore, we report here only a method that has been applied to purify specific lectins from b. schlosseri hemolymph, since from protein purification onwards, the methods used do not significantly differ from invertebrate and mammalian models. affinity chromatography for β-galactosiderecognizing lectins various pools of 10-20 large (500-800) zooid colonies of b. schlosseri are homogenized in a homogenizer in 10 ml of a solution of 10 mm nabenzoate (sigma) in fsw, containing 0.1 mg/ml pepstatin and 1 mg/ml leupeptin (sigma). the homogenate is centrifuged at 2,000xg for 10 min at 4 °c and the supernatant is collected (average protein concentration: 2.5 mg/ml). affinity chromatography is carried out on acid-treated sepharose cl-6b (ge healthcare, chalfont st. giles, uk), previously equilibrated with pbs-3 according to parrinello and canicattì (1982). the column is washed with 1 m nacl, and absorbed proteins are eluted with a solution of 0.2 m dgalactose (sigma) in 1 m nacl. the flow rate is kept constant at 20 ml/h, and 2 ml fractions are collected, the absorbance of which is read at 280 nm. fractions corresponding to single absorbance peaks are pooled, dialyzed overnight against distilled water, vacuum-dried in a vacuum concentrator, and stored at -20 °c. protein content is evaluated according to bradford (1976). concluding remarks the majority of procedures developed for invertebrates and here reported find their counterpart in vertebrates, even if modifications to the canonic vertebrate protocols are needed. however, on the whole it emerges an important observation, i.e., the conservation in invertebrate and vertebrate species of the molecules involved in the immuneneuroendocrine interactions. this allows, for instance, that antibodies raised against components of vertebrate molecules can in most cases be used also for evidencing invertebrate molecules. however, nowadays, the immunoreactivity alone cannot be considered a sufficient indication to prove the existence of homologies between vertebrate and invertebrate molecules. the fundamental results collected by mean of careful morphological observations, as well as 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immune response; interleukin-2; nitric oxide; mollusc; mytilus galloprovincialis introduction molluscs constitute the widest phylum existing on earth after arthropods, with over 100,000 species present in all the ecosystems. many mollusc species, mainly marine, are very important from the economic point of view because of their use as human food resource. molluscs are also interesting because of their susceptibility to infection by parasites, bacteria, and viruses, which makes them transmitters of many diseases affecting different vertebrate species. finally, their ability to accumulate diverse toxins allows considering several mollusc species as excellent biosensors of the biologic quality of the ecosystems. unlike vertebrates, molluscs only have an ancestral defensive line, comparable to that known as immune innate system in vertebrates (medzhitov and janeway, 2000; plows et al., 2005), and constituted by a cellular component and the products that it generates (i.e. humoral component). ___________________________________________________________________________ corresponding author: juan ignacio ramos-martinez departamento de bioquímica y biología molecular, facultad de veterinaria, universidad de santiago de compostela, e-27002 lugo, spain e-mail: bniramos@lugo.usc.es the circulating cells, called hemocytes, are responsible for the phagocytosis, cytotoxic reactions, and the synthesis of the humoral factors, comprising antimicrobial peptides, agglutinins, lectins, cytokines, nitric oxide, etc. (ottaviani, 2006). due to their immune activity, hemocytes can be also referred to as immunocytes. although molluscan immunocytes show a wide heterogeneity, the cells involved in the defensive processes are similar to the macrophages from vertebrates. their morphological features include an irregular shape, a small nucleus in relation to cell size and presence of numerous granules (krupa et al., 1977; joky et al., 1983; ottaviani and franchini, 1988; ottaviani et al., 1998a; cao et al., 2003). moreover, molluscan hemolymph contains other hemocytes, roundish and with a bigger nucleus. in the particular case of mytilus galloprovincialis, both cell types seem to correspond to two maturation stages of the same cell (ottaviani et al., 1998a). as far as the humoral component is concerned, it promotes several responses involving cell shape changes, chemotaxis, phagocytosis, cytotoxicity, encapsulation, and neuroendocrine responses, thus suggesting the involvement of stress mediators in the immune response (for review see, ottaviani, 2006; ottaviani et al., 2007). 43 mailto:bniramos@lugo.usc.es in invertebrates, physiologic responses suggesting the presence of molecules with cytokinelike functions have been observed in molluscs, insects, annelids, echinoderms, and tunicates (ottaviani et al., 1995a, 2004; ottaviani, 2006). as for molluscs, cytokine-like molecules with immune and neuroendocrine functions have been detected in both marine and freshwater species. in this sense, heterologous cytokines provoking cell shape changes, chemotaxis or phagocytosis are also capable to induce the synthesis of biogenic amines (ba), nitric oxide (no) or oxygen radicals. these results suggest the existence of an ancestral immune-mobile brain that imitates the hypothalamicpituitary-adrenal axis (hpa) from vertebrates (ottaviani et al., 1993a; ottaviani, 2004, 2006). heterologous cytokines, growth factors, and cell differentiating factors increase epinephrine, norepinephrine and dopamine synthesis in molluscan hemocytes, both in those freshly extracted and those cultured for several days (franchini et al., 2000; cao et al., 2003, 2004b, 2007b). interleukin-2 (il-2) receptor in molluscan hemocytes early studies on the involvement of diverse cytokines in the activation of the immune response in molluscan hemocytes suggest the existence of a receptor with low specificity (ottaviani et al., 1994, 1995a, b). il-2 was one of the cytokines assayed, as it induces phagocytosis and provokes the strongest response in the synthesis of ba, no, etc (ottaviani et al., 1995a, b). studies on the interference of the inflammatory cytokine and corticotrophin-releasing hormone (crh) on ba production provided the initial clues about the possible presence of an il-2 receptor (ottaviani et al., 1994). subsequent studies on hemocytes of m. galloprovincialis allowed the detection of the three subunits constituting the il-2 receptor (barcia et al., 1999). flow cytometry confirmed the presence of a subunit of high affinity, termed il-2rα, and two of low affinity, termed il-2rβ and il-2rγ, which only one group of hemocytes expresses (barcia et al., 1999). also lipopolysaccharide (lps) induces the expression of the il-2 receptor, which can be explained as a collateral effect, similar to that detected in vertebrates, as lps might stimulate the hemocytes to synthesize molecules comparable to tnf-α, and the necrosis factor might in its turn induce il-2 the synthesis of molecules structurally/functionally related to il-2 (barcia et al., 1999). nitric oxide production in molluscs nitric oxide (no) is a gas that performs multiple biologic functions. among them outstands its character of intra and extracellular signal (moncada et al., 1991). in recent years, several studies have proved the presence of nitric oxide synthase (nos) and the involvement of no in cellular signaling in organisms dispersed through the whole animal kingdom (palumbo, 2006). the involvement of no in fig. 1 effect of different representative protein kinase inhibitors on the stimulation of no production by il-2. control: non-activated hemocytes. *compared with control p< 0.001. ** compared with il-2 p< 0.001. *** compared with il-2. (modified from novas et al., 2004). such processes as memory (korneev et al., 2005), bioluminescence (trimmer et al., 2001), sight (elphick et al., 1996), or cell proliferation (kuzin et al., 1996) was studied in invertebrates. particularly, in molluscs, no and the different forms of nos seem to be involved in the modulation of neuron activity (gelperin, 1994; moroz et al., 1996; hurst et al., 1999; stefano and ottaviani, 2002), metamorphosis (leise et al., 2004) and preferably in processes related to the immune defense (ottaviani, 2006). no may act directly, but it also reacts with free oxygen radicals, thus generating peroxinitrite (onoo-), a potent oxidizing agent whose action affects the peroxidation of the membrane lipids, the inhibition of tricarboxylic acid (tca) cycle, and the mitochondrial respiration (nappi and ottaviani, 2000). molluscan hemocytes remove bacteria by means of phagocytosis and no production. both processes are interdependent, although phagocytosis seems to happen before no synthesis. this means that the bacterial clumping takes place prior to no elimination. hemocyte incubation with sodium nitroprusside provokes bacterial clumping, and lps also display a similar action (ottaviani et al., 1993b; franchini et al., 1995; tafalla et al., 2002). in addition, incubation of hemocytes from the mussel m. galloprovincialis or the oyster crassostrea gigas with phorbol myristate acetate (pma) or laminarin leads to the synthesis of no and superoxide ions. these induce the synthesis of peroxinitrite, which has the cytotoxic properties commented above (arumugan et al., 2000, 44 fig. 2 no synthesis by haemocytes of mytilus galloprovincialis collected in winter and summer. a) the cells were cultured for 3 days in l-15 medium and then incubated for 24 h with the referred cytokines as expressed in novas et al., 2007c. the bars represent the standard deviation of six individual assays. results of a representative assay of each season. tumor necrosis factor-α (tnf-α); platelet-derived growth factor (pdgf); transforming growth factor-β1 (tgf-β1). 2001; gourdon et al., 2001; torreilles and romestand, 2001). these authors, however, discard lps as inducer of no synthesis. the existence of nos activity in molluscan hemocytes was proved in viviparus ater. the activity was stimulated by lps and was located in the soluble fraction of hemolymph (conte and ottaviani, 1995). the use of nos inhibitors promoted the canceling of the bacterial clumping induced by lps in the molluscan hemocytes of m. edulis and v. ater (ottaviani et al., 1993b). studies performed in hemocytes from freshwater molluscs show that the incubation with different cytokines induces a significant increase of no production (ottaviani et al., 1995a). similar works performed with hemocytes from marine molluscs offer similar results, with il-2 as the cytokine inducing the maximal response (ottaviani et al., 1995a). the results are similar, either if the hemocytes were freshly extracted or if they were subjected to culture for several days, and this was related to the capability of molluscan hemocytes to express the complete structure of the il-2 receptor (barcia et al., 1999; novas et al., 2004). during culturing, the hemocytes from m. galloprovincialis keep their ability to synthesize no induced by il-2. the results about the effect of lps on no production are different, maybe because of the use of lps of diverse origins lps does not induce the no synthesis in hemocytes of mytilus (arumugan et al., 2000, 2001; gourdon et al., 2001; torreilles and romestand, 2001), a different result to the obtained with cells of other models (conte and ottaviani, 1995). mytilus hemocytes respond to the presence of mammalian il-2 synthesizing ba and no, although the difference between the kinetics of no and ba synthesis (cao et al., 2004a, b; novas et al., 2004, 2007b) suggest different pathways of signal internalization. apparently, ba synthesis involves preferably the enzyme protein kinase c (p105) (cao et al., 2004a, 2007a), whereas the camp-dependent protein kinase (pka) plays a secondary role (cao et al., 2004a). both enzymes were purified from different tissues of m. galloprovincialis (mercado et al., 2002a, b, 2003; diaz-enrich et al., 2003; bardales et al., 2004), which led to the obtaining of specific antibodies that allowed learning more about the function of these enzymes in the action of il-2 on no production. as commented above, phorbol 12-myristate 13acetate (tpa) induces no synthesis, thus involving pkc in the process (arumugan et al., 2000, 2001; gourdon et al., 2001; torreilles and romestand, 2001). since il-2 has the same effect of tpa, all suggests that pkc is involved in the action induced by il-2. similar works on freshwater molluscs such as lymnaea stagnalis link pkc and erk (extracellular signal-regulated kinase) to the signaling mechanisms of the regulation of nos activity (wright et al., 2006). despite the involvement of pkc in mediating the signal borne by il-2, further experiments with specific pk inhibitors indicate that the signaling pathway by which il-2 induces no synthesis preferably involves pka. the action of inhibitors specific for these protein kinases suggests that the signal of the il-2-induced no synthesis preferably involves pka (fig. 1). the kinase activates a form of nos specific of mytilus hemocytes (novas et al., 2004), initially identified as a form of 130 kda and immunologically similar to inos from vertebrates (novas et al., 2004). the different kinetics of ba and no production that the cell shows in the presence of il-2 seems to have its base on the different protein kinase involved in each process. as it happens in many aspects of the physiology of marine molluscs (robledo et al., 1995; ramos-martinez et al., 1993), hemocyte immune response shows a seasonal variation in basal and il-2-induced no synthesis (novas et al., 2007c). fig. 3 il-2-induced production of no. control: basal non-stimulated hemocytes. il-2: hemocytes incubated with il-2. the pkc inhibitor bisindolylmaleimide (bsm) was added to the cells simultaneously with il-2. error bars are the sd of six assays as described in novas et al., 2007b. 45 fig. 4 schematic diagram illustrating the hypothetical implication of pka and pkc in the two seasonal pathways of hemocyte il-2-induced no synthesis. dotted lines represent the inhibiting effect of down-regulation (novas et al., 2007b). no basal production is higher during the summer months, whereas il-2-induced no synthesis by cells obtained in winter is 5-fold that of the cells obtained in summer (fig. 2). when studying the effect of pkc inhibitors on the seasonal variation of il-2-induced no production, pka appeared as the major activating agent of the mytilus constitutivenos (mc-nos) (novas et al., 2004). also, the results about no seasonal production in the presence of bisindolylmaleimide (bsm) suggest a participation of pkc that would be compatible with the hypothesis expressed in fig. 4 (novas et al., 2007b). pkc appears as a potent mc-nos inhibitor. in summer, and as a consequence of the presence of il-2, takes place pkc inhibition by downregulation, which implicates pka in keeping an activating action of no synthesis. the action described persists in winter, but in this season, a new nos-inducible enzyme was detected. this was termed mytilus winter-nos (mw-nos) and has similarities with the constitutive form of vertebrates susceptible of activation by pkc (fig. 4). pkc activating action on the new mw-nos inducible enzyme explains the result presented in fig. 3. concluding remarks in marine molluscs, there is a remarkable biologic paradox such as the fact that the widest mortalities occur in the seasons with most abundant food and when the immunologic potential is seems at its maximum (summer). apparently, the reason for the high mortality could be the increase of not only food but also parasites and pathogens occurring in phytoplankton blooms (robledo et al., 1995; lacoste et al., 2001; hernroth, 2003). when considering the basal no synthesis, the low production detected in winter is still compatible with a frame of reduced immune response in winter, hich may seem rather odd, considering the high degree of mortality registered in summer. indeed, this supposed immunodepression during wintertime is false, because an inflammatory phenomenon, and especially the increased production of molecules related to il-2 and to the activation of mw-nos trigger the mechanism that ensures the response, despite the low basal levels on no. fig. 5 seasonal variation of nitric oxide production by hemocytes of mytilus galloprovincialis lmk. in 2001 and 2002 the assays were performed during winter (w) and summer (s) (novas et al., 2007c). empty bars represent the basal production and solid bars represent no production of hemocytes incubated with 10 μg/ml il-2 for 24 h. error bars are the sd of 6 assays. 46 the results of the assay on no basal production and, mainly, hemocyte response against il-2 accounts for the utility of mytilus as biosensor, to judge by the consequences of the prestige oil spill in the immune response of the mollusc (ruizvillareal et al., 2006; soriano et al., 2006). as shown in fig. 5, no synthesis ability was stable in the years before the catastrophe; then it drops and does not recover until two years later. the process is especially dramatic if assayed no production by hemocytes treated with il-2. pollution inactivates no basal production, but it also cancels the no production mechanism induced by il-2 during the assay. hemocyte lack of response during assay time is parallel to other assays, such as the study of cell necrosis, apoptosis and others (novas et al., 2007a), and is compatible with a situation of immunodepression (laffon et al., 2006). finally, the test on il-2-induced no production by molluscan hemocytes may join other parameters assayed for the study of the effect of 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1995. ruiz-villarreal m, gonzález-pola c, diaz del rio g, lavin a, otero p, piedracoba s, et al. oceanographic conditions in north and northwest iberia and their influence on the prestige oil spill. mar. pollut. bull. 53: 220-38, 2006. soriano ja, viñas l, franco ma, gonzález jj, ortiz l, bayona jm, et al. spatial and temporal trends of petroleum hydrocarbons in wild mussels from the galician coast (nw spain) affected by the prestige oil spill sci. total environ. 370: 80-90, 2006. 48 http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22ruiz-villarreal%20m%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstractplusdrugs1 http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22gonz%c3%a1lez-pola%20c%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstractplusdrugs1 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http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22bayona%20jm%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstractplusdrugs1 stefano gb, ottaviani e. the biochemical substrate of nitric oxide signaling is present in primitive non-cognitive organisms. brain res. 924: 8289, 2002. tafalla c, novoa b, figueras a. production of nitric oxide by mussel (mytilus galloprovincialis) hemocytes and effect of exogenous nitric oxide on phagocytic functions. comp. biochem. physiol. 132b: 423-431, 2002. torreilles j, romestand b. in vitro production of peroxynitrite by haemocytes from marine bivalves: c-elisa determination of 3nitrotyrosine level in plasma proteins from mytilus galloprovincialis and crassotrea gigas. bmc immunol. 2: 1-5, 2001. trimmer ba, aprille jr, dudzinski dm, lagace cj, lewis sm, michel t, et al. nitric oxide and the control of firefly flashing. science 292: 24862488, 2001. wright b, lacchini ah, davies aj, walker aj. regulation of nitric oxide production in snail (lymnaea stagnalis) defence cells: a role for pkc and erk signalling pathways. biol. cell 98: 265-278, 2006. 49 interleukin-2 (il-2) receptor in molluscan hemocytes nitric oxide production in molluscs concluding remarks isj 4: xx-yy, 2007 isj 4: 92-94, 2007 issn 1824-307x technical report a full-length protocol to test hemolytic activity of palytoxin on human erythrocytes d malagoli department of animal biology, university of modena and reggio emilia, modena, italy accepted september 18, 2007 abstract the hemolytic assay protocols currently utilized to test the presence of the marine biotoxin palytoxin (ptx) are deeply analyzed. in some points, slight modifications and rearrangements have been realized, to obtain an exhaustive protocol suitable to test ptx activity on human erythrocytes. key words: palytoxin; hemolysis; method introduction palytoxin (ptx) is a non-protein toxin firstly isolated from the soft coral of the genus palythoa more than 30 years ago (moore and scheuer, 1971). ptx is considered one of the most toxic molecules occurring in nature (lenoir et al., 2004), and it has also recently been studied also as a skin tumor promoter (wattenberg, 2007). since marine animals display a certain degree of resistance to its action (gleibs and mebs, 1999), ptx can accumulates in the food chain therefore provoking severe, or even lethal, intoxications to humans (taniyama et al., 2003). even though ptx is not an hemolysin (habermann, 1989), its interaction with na+/k+ atpase on erythrocyte membranes (artigas and gadsby, 2003; hilgemann, 2003) provokes a delayed release of hemoglobin. this activity is the base of the hemolytic assay, proposed by bignami (1993) as a more sensitive alternative to enzymelinked immunoassay. bignami (1993) realized his protocol taking advantage from the already known hemolytic activity registered for ptx in mammalian erythrocytes (habermann et al., 1981). the text based its specificity on the capability of the glycoside ouabain to inhibit ptx activity (habermann and chhatwal, 1982; habermann, 1989). since then, several studies indirectly identified ptx in invertebrate (gleibs and mebs, 1999) and vertebrate (taniyama et al., 2001, 2003) extracts by mean of the hemolytic assay. however, some considerations have to introduced before analyzing the numerous report concerning the use of ptx in hemolytic assays. ___________________________________________________________________________ corresponding author: davide malagoli department of animal biology via campi, 213/d, 41100 modena italy e-mail: davide.malagoli@unimore.it different mammalian species exhibit a specific degree of sensitivity to ptx (habermann, 1989) and different modalities are utilized by the researcher for quantifying hemolytic activity of the samples, thus introducing slight modifications in the method described in each report (bignami, 1993; gleibs et al. 1995; gleibs and nebs, 1999; taniyama et al., 2001, 2003; lenoir et al., 2004; riobó et al., 2006). on these basis, it is difficult to obtain a complete and detailed protocol for evaluating the hemolytic activity of ptx, especially for human erythrocytes, from a single report. the aim of this technical report, is to present an exhaustive and detailed protocol to evaluate the hemolytic activity of ptx on human a+ erythrocytes and to confront the collected results with those already present in literature. materials and methods hemolysis assay human a+ whole blood was used within 24 h after bleeding and washed three times (gleibs et al., 1995) in 9 volumes of sterile 0.9 % nacl saline solution. after each washing, cells were pelleted by centrifugation at 150xg for 5 min and the supernatant was discarded. the final pellet was diluted 1:9 (v/v) in sterile 0.9 % nacl saline solution than 1:24 (v/v) in sterile dulbecco’s phosphate buffer saline (d-pbs), ph 7.0 (bignami, 1993) containing 0,5 mm boric acid and 1 mm calcium chloride (taniyama et al., 2001). red cell suspensions (1 ml of final volume) were incubated with an aqueous solution (taniyama et al., 2003) of ptx standard, from 10-3 to 103 ng/ml. the following times of incubations were tested: 4, 6, 8 and 24 h. however, since no differences were observed from samples incubated for 6 h or more, all the repetitions have been performed with incubation 92 fig. 1 dose-dependent hemolytic effects of ptx standard (blue) on human erythrocytes and their reversal in presence of 100 μm ouabain (purple). dose-ranging experiments were performed from 10-3 to 103 ng/ml of ptx. however, since for concentrations ≤ 100 ng/ml, no effects were observed, the 100 ng/ml concentration is represented as the first value considered. error bars represent sd. time of 6 h. incubation temperature was set at 37 °c (taniyama et al., 2001), and during the incubation the samples were occasionally (maximum once per h) resuspended by inversion. for negative controls, red blood cell suspension was added with 100 μm ouabain and incubated for 30 min at 37 °c, prior to the addition of ptx (gleibs et al., 1995). in order to avoid any possible differences due to a diverse manipulation, also samples that did not contain the glycoside ouabain were incubated for 30 min at 37 °c before the addition of ptx. total lysis of erythrocyte suspension was obtained by incubating the cells with 0,1 % v/v tween 20. in order to evaluate the degree of spontaneous lysis, also tubes containing exclusively the red blood cell suspension in d-pbs were set. for each concentration and control, the experiments were set in triplicate. a total of eight independent experiments have been performed. hemolysis evaluation after the incubation, the cell suspensions were centrifuged at 900xg for 10 min (taniyama et al., 2003) and the supernatant was carefully collected, by paying attention not to disturb the pellet. the absorbance at 405/540 nm of supernatant was measured with a “helios β" spectrophotometer (spectronic, unicam, cambridge, uk). the value of absorbance of erythrocytes maintained exclusively in d-pbs has been utilized to set the 0 value before reading the samples that contained ptx. hemolytic levels were expressed by percentage of hemolysis, calculated with the ratio between the value measured for each sample and that registered for the total hemolysis (taniyama et al., 2003). chemical reagents human erythrocytes were obtained from laboratorio medicina trasfusionale (policlinico, azienda ospedaliero universitaria, modena, italy), ptx standard was from wako chemicals (neuss, germany). a second ptx standard solution was generously gifted by centro di ricerche marine (cesenatico, fc, italy). all the other chemicals, including d-pbs and ouabain, came from sigmaaldrich (st louis, ca, usa). all the plastics used were sterile and cell-culture tested (sarstedt, nümbrecht, germany). results and discussion the applied protocol confirms that ptx is able to induce hemolysis in human erythrocytes (fig. 1), and the levels of the lysis are comparable with those observed by other groups using the same source for red blood cells (taniyama et al., 2001, 2003). it is worth noting that the value here presented indicated a discrepancy with those already published, since the effects of ptx were detectable only from 101 ng/ml, while at this value taniyama and collaborators found a clear activity of ptx (taniyama et al., 2001). in order to verify if present data could be due to an erroneous preparation of the ptx standard, experiments were repeated with a second ptx solution coming from centro di ricerche marine. however, data collected with this second standard substantially confirmed those collected with the first one. differently from this first result, there is a substantial agreement between data presented here and those collected by taniyama et al (2001) for what concerns the 93 maximum levels of hemolysis detectable in human erythrocytes. as it has been observed from a long time (habermann and chhatwal, 1982), ptx effects on human erythrocytes are completely reverted by the glycoside ouabain (taniyama et al., 2001). the results obtained by using 100 μm ouabain confirm this indication even if a small percentage of erythrolysis have been observed in the presence of the glycoside (fig. 1). also other concentrations have been suggested for ouabain (habermann and chhatwal 1982, taniyama et al., 2001), but that adopted here was the most effective and did not significantly influence the basal levels of erythrolysis. as far as data analysis is concerned, fig. 1 presents data collected by using the absorbance wavelength of 405 nm, following gleibs and collaborators (1995). different values of wavelength, such as 540 nm, have been utilized to evaluate the hemolysis of mouse erythrocytes (lenoir et al., 2004). however, even if also the latter wavelength has been utilized in the present research, the most repeatable data were obtained at 405 nm, after imposing to the spectrophotometer the value 0 for the samples maintained only in d-pbs. if some differences in the protocols used to test the hemolytic activity of ptx can be retrieved, probably the most notable ones concern the unity chosen to measure the levels of hemolysis. gleibs et al (1995), working on palythoa and other marine specimen, describe the hemolytic unit (hu) as the amount of material necessary to produce 50 % hemolysis in human erythrocytes within 4 h incubation ptx. a similar definition of the hu, without references to the time of incubation, is given also by lenoir et al (2004), that worked with mouse red cells. working with human erythrocytes, gleibs and mebs (1999) quantified one hemolytic unit (hu) in 0,4 μg of ptx, but due to the different sensitivity of mammalian erythocytes to ptx (habermann, 1989), this value should probably be referred only to human red cells. taniyama et al. (2001) quantify ptx effects with the percentage of hemolysis, calculated with the ratio between the value measured for a sample and that registered for the total hemolysis. the percentage is a pure number that can be applied independently from the source of the erythrocytes, therefore it has been chosen here to evaluate ptx activity from commercially available standards. however, since the percentage of hemolysis should be properly calculated as follows: [absorbance in test absorbance in dpbs]/[absorbance in 100 % hemolysis absorbance in d-pbs] (hubert et al., 1997), the value of absorbance in d-pbs has been utilized to set the 0 value on the spectrophotometer. only after this procedure, the ratio proposed by taniyama et al. 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice isj 6: s46-s57, 2009 issn 1824-307x review focusing on ciona intestinalis (tunicata) innate immune system. evolutionary implications n parrinello laboratory of marine immunobiology, department of animal biology, university of palermo, palermo, italy accepted march 13, 2009 abstract phylogenetic analyses based on molecular data provide compelling evidence that ascidians are of critical importance for studying chordate immune system evolution. the ciona intestinalis draft genome sequence allows searches for phylogenetic relationships, gene cloning and expression of immunorelevant molecules. acidians lack of the pivotal components of the vertebrate recombinatory adaptive immunity, i.e., mhc, tcrs and dimeric immunoglobulins. however, bioinformatic sequence analyses recognized genic elements indicating the essential features of the ig superfamily and ancestor proto-mhc genes, suggesting a primitive pre-duplication and pre-recombination status. c. intestinalis genes for individuality in the absence of mhc could encode diverse molecular markers, including a wide panel of complement factors that could be responsible for self-nonself discrimination. genome analysis reveals a number of innate immunity vertebrate-like genes which encode toll-like and virus receptors, complement pathways components and receptors, cd94/nk-receptor-like, lectins, tnf, il1-r, collagens. however, pure homology seeking for vertebrate-specific immunorelevant molecules is of limited value, and functional screening methods may be a more promising approach for tracing the immune system evolution. c. intestinalis, which displays acute and chronic inflammatory reactions, is a model organism for studying innate immunity genes expression and functions. key words: immunoevolution; genome; ciona intestinalis; ascidians; innate immunity; inflammatory response; gene expression evolutionary relevance of ascidian immunity studies ascidians (urochordata), including cosmopolitan compound and non-colonial species, occupy a key position in the phylogenetic line leading to the vertebrates (swalla et al., 2000; zeng and swalla, 2005; delsuc et al., 2006), therefore they have attained importance for immunity evolution studies recently promoted by available genome sequences (dehal et al., 2002b; satou et al. 2002; satoh, 2003; yokobori et al., 2003; kasahara et al., 2004; litman and cooper, 2007; ben-shlomo, 2008). bioinformatic approach and extensive in silico search of immunorelevant genes have been in part validated by expression patterns and biological properties of their products (davidson and swalla, 2002; nonaka and miyazawa, 2002; fujita, 2002; azumi et al., 2003; ___________________________________________________________________________ corresponding author: nicolò parrinello laboratory of marine immunobiology department of animal biology university of palermo via archirafi 18, 90123 palermo, italy e-mail: nicpar@unipa.it shida et al., 2003; terajima et al., 2003; fujita et al., 2004; du pasquier, 2004; kasahara et al., 2004; litman and cooper, 2007). botryllids provided of allorecognition reaction (de tomaso and weissman, 2004; de tomaso et al. 2005; ballarin , 2008; gasparini et al., 2008) and ciona intestinalis which displays acute inflammatory responses (parrinello, 1981; parrinello and patricolo, 1984; parrinello et al., 1984) are model organisms for studying chordate evolution. the whole genome of c. intestinalis has been sequenced and analyzed, genes have been annotated and some expression studies carried out, whereas adequate botryllid genome sequencing is lacking. adaptive immunity pivotal genes in vertebrates, the emergence of the adaptive immune system is linked to the acquisition of the enzyme machinery encoded by the recombination activating genes (rag) that provide to the rearrangement of immunoglobulin (ig) and t cell receptor (tcr) genes. both b and t cells carry s46 mailto:nicpar@unipa.it receptor molecules to recognize and respond to the antigens. b cell receptor is a prototype of the antibody, and tcr on the surface of t lymphocytes is responsible for recognizing antigens bound to major histocompatibility complex (mhc) molecules. these receptors are composed of two polypeptides of the ig superfamily (ig heavy h and light l chains; tcr α and β or γ and δ chains) with recognizable constant (c) and variable (v) domains. the c domain of c1-set characterizes only ig, tcr, and mhc class ii, class i and class i-related molecules, whereas the c2 set domain occurs in both vertebrates and invertebrates (see kasahara et al., 2004). the domain provided with variability is generated by somatic recombination of multiple elements scattered in the genic locus. in lymphocytes, rags cut dna at the recombinant signal sequence (rss) for mediating recombination of v(d)j regions through the joining gene segments. somatic hypermutation, rags and activation-induced cytidine deaminase (aid), regulators of secondary ig diversification, are crucial components of vertebrate adaptive immunity (manis et al., 2002; bransteitter et al., 2003) producing an unlimited repertoire of diversity that enables the cells to recognize and respond to unpredictable pathogens. analysis of c. intestinalis genome sequences did not reveal the pivotal genes and molecules for adaptive immunity, such as mhc genes, tcrs, or dimeric igs (dehal et al. 2002b; azumi et al., 2003; shida et al., 2003). nevertheless, bioinformatic sequence analyses have recognized two ig domaincontaining regions, key v regions, the essential feature of an ig superfamily vc1-like core trait, presumptive proto-mhc regions scattered throughout the genome, and three types of genes with receptor-like v-c architecture (du pasquier, 2004). in addition, there are indicators of v domains that could be targets for rag-mediated transposition. these genes belong to two families with recognizable homologs in vertebrates: the cortical thymocyte marker of xenopus/junctional adhesion molecules family (ci-ctx/jam) and the nectin ci-nec2 family. signal of ancestor proto-mhc genes can be drawn from the human mhc genome paralogy (kasahara, 2000). more than 100 genes are located in the human leukocyte complex (hla) and about 40 of them have paralogous copies on 1, 9 and 19 chromosomes. c. intestinalis genome contains a single copy gene with features of a precursor of multiple human mhc paralogous suggesting the existence of a pre-duplication region (bodmer, 1972; kasahara et al., 2004). chromosome duplications could be took place before the emergence of a common ancestor of vertebrates as indicated by mhc paralogous emergence. mhcbased allorecognition seems to be a unique feature of jawed vertebrates, and finding of variable lymphocyte receptors in agnates (pancer et al., 2004) indicate that different groups of molecules could be responsible for similar functions. accordingly, allorecognition machinery in urochordates has nothing in common with the mhcbased histocompatibility reaction of vertebrates (de tamaso et al., 2005; klein, 2006), and observations indicate that different ascidian species could have evolved their own independent allorecognition strategies. in example, the hystocompatibility fu/hc receptor of b. schlosseri has no direct homologs neither in vertebrates nor in c. intestinalis, indicating that self-nonself discrimination systems have branched off into a variety of unique and specialized systems during evolution. in the absence of vertebrate-like adaptive immunity, ciona‘s genes reflect a primitive preduplication and/or pre-recombination status leading to the genesis of mhc, tcr and ig (yu et al., 2005). moreover, it is generally accepted that c. intestinalis activating and inhibitory receptors of immunocytes have mhc-independent functions. presumptive molecular codes for sef-nonself discrimination in the absence of mhc although most animals are able to distinguish self from non-self (buss, 1987; chadwick-furman and rinkevich, 1994; cadavid et al., 2004), allorecognition molecular mechanisms do not seem of monophyletic origin and remain to establish if they evolved independently. different groups of invertebrates could have developed their own histocompatibility system and could express taxonspecific self-nonself determinants. ciona is a valuable model for deepening the question. it is hermaphrodite producing eggs and sperm simultaneously, but self-fertilization normally does not occur because the follicle cells accept only allogeneic sperm (rosati and de santis, 1978; pinto et al., 1995; murabe and hoshi, 2002). it can be assumed that a receptor, potentially involved in selfincompatibility, should have sequence and expression variable in oocytes from different individuals. based on this assumption, novel models have been proposed upon a c. intestinalis molecular code for individuality in the absence of mhc. to search for molecular markers of individuality, a suppression subtractive hybridisation approach has been used for comparing the somatic transcriptomes of two ciona individuals and for identifying individually variable cdnas (khalturin et al., 2005). the results show that two genes, cimeta2 and cis7, encode two classes of soluble proteins which exhibit high degree of interand intra-individual variability and contain multiple domains suitable for protein-protein interaction. both classes of individually variable proteins are coded in the genome by several gene copies organized in clusters. one class consists of secreted protein thrombospondin type 1-like domains (thrombospondin type 1 repeat, tsr superfamily), the second one consists of secreted proteins with multiple epidermal growth factor (egf)-like domains. although the functional significance is unknown, the authors suggest that these gene loci may participate in controlling non-self recognition. cis7 and cimeta2 isoforms with a high degree of aminoacid sequence variations are expressed in hemocytes and gametes which are mediators of recognition events. individuals have several genes and transcribe several mrnas coding for similar but s47 not identical proteins of each gene family. apparently each individual carries an unique repertoire (haplotype) in each locus, probably established by crossing over events during gamete maturation and/or fertilization. individuality may be encoded by the haplotype and the individual-specific combination of genes in the genome locus. this phenomenon resembles the variability of vertebrate killer cell ig-like receptors (kir), and fit with the predictions of self-nonself recognition molecules provided with individual variability, allorecognition and block of self-fertilization. to shed light on c. intestinalis specific receptors for allorecognition and self fertilization avoidance, kurn and colleagues (2007) subctracted gonadal cdna from three genetically unrelated c. intestinalis individuals by suppression subtractive hybridisation. individualspecific genes encoding variable transmembrane complement receptor-like protein (vcrl) have been identified. interestingly, they contain several complement controlling protein domains (scr/ccp). one of these genes reveals a high degree of inter-individual vcrl amino acid variation, and it is expressed in follicle cells and hemocytes. diverse vcrl1s, highly variable between individuals, show sequence similarity varying from 70 % to 93 %. each animal has its own version of vcrl1, therefore cells in different individuals are marked by non-overlapping receptors and corresponding ligands. intraindividual variants most likely are due to the two alleles of a single locus, and several alternative intracellular vcrl1 domains may be produced by alternative splicing. the cells within one individual are appropriately marked and will be referred to as “self” whereas any cell of genetically different individual will be distinguished as “non self”. in mammals, the complement components are not variable between individuals. in ciona, the number of genes encoding complement system components is greatly expanded compared to mammals and, together the vcrl high variability, allow to suppose that the ciona allorecognition reactions involve complementrelated receptors in a “missing self” mechanism. intriguingly, complement receptors are also expressed in human gametes and are involved in sperm/oocyte interaction (seya et al., 1999). the ciflrt transmembrane receptor gene expressed in hemocytes (kurn et al., 2007) was also analyzed. the corresponding protein consists of a signal peptide, nine leucine-rich repeats (lrrs, protein interaction modules), fibronectin type iii domain (mammalian fniii repeats on cell surface and extracellular) and a transmembrane domain (khalturin et al. 2005). the ciflrts from two individuals were amplified, sequenced and compared to sequences reported by khalturin and colleagues (2005). the distance between the ciflrt proteins of the four individuals is much smaller than that between vcrl1. the high degree of ccrl1 variation clearly exceeds the naturally occurring variations normally found in ciona genes. toll-like receptors the toll-like receptor (tlr) multigene family encodes recognition receptors of innate immune system conserved in invertebrates and vertebrates (kaisho and akira, 2000; medzhitov and janeway, 2000; imler and hoffmann, 2001; vasselson and detmers, 2002). tlrs recognize a variety of endogeneous and exogeneous ligands, they are pattern recognition receptors that bind molecules broadly shared by pathogens collectively referred to as pamps, many of which are conserved molecules essential for pathogen survival (zhong and kyriakis, 2007). c. intestinalis genome sequence analysis disclosed three distinct tlrs expressed genes and the corresponding signal transduction cascades (azumi et al., 2003; terajima et al., 2003; roach et al., 2005; shida et al., 2005). in invertebrates, tolllike receptors mediated antibacterial, antifungal and antiviral systems. recently, c. intestinalis inflammatory responses challenged by lps have been reported. the triggering mechanism presumably includes toll-like receptors (tlrs) which bind lps (a component of the pamps) initiating the inflammatory reactions. responses stimulated by lps including complement activation and products, galectincytokine-like and cd94-nk-c-type lectin-like receptor genes expression, and collagen synthesis have been reported (see below). genes for intracellular immunoreceptors, tyrosine-based inhibition motifs (ciitims) and tyrosine-based activation motifs (ciitams) may be responsible of the cell signaling pathways (azumi et al. 2003; shida et al., 2003). vertebrate itims and itams are short conserved sequences in the cytoplasmic tails of many immune cells inhibitory and activating receptors (including tcrs), respectively. virus receptors in c. intestinalis genome, homologs to vertebrate adhesion molecules members of membrane ig superfamily show ancestral features of antigen receptors (du pasquier, 2004). they include the junction adhesion molecule (reovirus receptor) and the cortical thymocyte marker of xenopus (adenovirus receptor) which are members of cictx/jam family, and the poliovirus receptor (pvr) members of the cinec family. pvr genes contain one distal v domain followed by two c domains, transmembrane and cytoplasmic segments. vertebrate pvrs are known to allow endocytosis of different viruses through the interaction with v domain. since in humans 4 paralogous groups exist, the ciona set of genes could correspond to a preduplication status. it has been suggested that the virus binding property of the members of this family were recruited in the vertebrate immune system following the introduction of the somatic rearrangement machinery. complement system the innate complement system evolved long s48 before the origin of vertebrate somatically rearranging antibodies. in vertebrates, there are about 30 complement protein components and three distinct pathways by which the complement system can be activated (nonaka and yashizaki, 2004a, b): 1. classical, antibody-mediated pathway; 2. lectin mediated pathway; 3. alternative pathway, triggered the pathogen surface. all the activation mechanisms converge to the c3 component proteolysis, and generate a same set of activation products. c3 is the central component, equipped with an unique intramolecular thioester bond which, upon activation, is exposed to the molecular surface and forms a covalent bond with invading microorganisms (lambris, 1990). c3 proteolysis enhances the phagocytosis (opsonin) of pathogens through some c3b fragment and the chemotaxis by the c3a-fragment which binds to macrophage and leucocyte receptors, furthermore c3b contribute in forming c5 convertase that activates c5-c9 late complement components leading to the formation of a cytolytic complex (membrane attack complex or mac). a large number of complement components are conserved between higher vertebrates and urochordates. in c. intestinalis, a wide and detailed architecture of the primitive complement system has been described (nonaka and yoshizaki, 2004a, b; fujita et al., 2004), and complement-like genes, most of which are transcriptionally active, indicate potential activity of lectin and alternative pathways. the ciona lectin mediated complement pathway has been revealed by genome sequence analysis, est, cloning and expression studies that identified nine mannose-binding lectins (cimbl), nine ficolins and four cimbl-associated serine proteases (cimasps c1r/c1s-like). the carbohydrate-binding collectin pathway (cimbls/ficolins as pattern recognition proteins), initiated by recognition of pamps and activated by cimasps, leads to cic4, cic2 and cic3 cleavage. several soluble components and receptors (four cimasps, cic3 and corresponding cicr3/cr4 receptors) have been recognized in the genome, and ests disclosed transcripts in hemocytes (fujita, 2002; azumi et al., 2003; fujia et al., 2004; wakoh et al., 2004). cimasps could exert trypsinlike activity, and the identified cimbl collectin genes encode proteins composed of the collagen and lectin-like domains. recently a cimbl has been cloned and sequenced, and its expression was promptly enhanced by lps (bonura et al., submitted). the deduced amino acid sequence (221 aa) showed a n-cysteine-rich terminal domain, a type 2 collagen domain, and a c-type mannose/glucose-specific crd. comparative analysis reveals 56.5 % similarity and 37.8 % identity with human mbl. marino and colleagues (2002) cloned two cic3-like genes (cic3-1 and cic3-2 cdna) from ciona hemocyte mrna. the deduced aminoacid sequences of both cic3 proteins show an overall similarity to the c3 molecules from vertebrate species, and exhibit a canonical processing site for αand β-chains, including a typical thioester site with the his residue required for nucleophilic activation. lps activates the complement, presumably via cimbl and cic3-1 proteolysis leading to the cic3a fragment which is a pro-inflammatory peptide akin to the vertebrate anaphylatoxin (pinto et al., 2003). the inflammatory challenge upregulates the cic3-1 expression in hemocytes and cic3-1a production. the recombinant cic3-1a exerts in vitro chemotactic effect on hemocytes interacting with a receptor molecule cic3ar coupled with gi protein and homologous to the mammalian receptor (melillo et al., 2006). an ancestor of the two cic3 seems to have diverged from a common ancestor of vertebrate c3/c4/c5 and has duplicated in two genes. accordingly, sequence phylogenetic analysis indicates that cimasps have diverged from the common ancestor of vertebrate masp/c1r/c1s. in addition, the genome presents two α2macroglobulin-like (ciα2m) genic elements (azumi et al., 2003). mammalian α2m is able to inactivate an enormous variety of proteinases, inhibits complement activation by masp and it is conceivable that it plays a regulatory role in mbpderived complement activation (terai et al., 1995). c3/c4/c5 and α2m form a molecular family different from other complement components and they do not show clear domain structure. in mammals, the factor b (bf) is the central serine esterase of the alternative pathway of complement activation, and in the active form (bb) is component of c3 convertase. in ciona three bflike genes, supported by cdna evidence, have also been identified and they, presumably responsible for the alternative pathway, encode predicted proteins with domain structures similar to the vertebrate bf/c2 gene family basic domain structure (fujita, 2002; azumi et al. 2003; fujita et al. 2004). bf and c2 are catalytic subunits of the c3 convertases of the alternative and vertebrate classic pathways respectively. the cibf genes are longer than those of jawed vertebrates, the deduced amino-acid sequences of cibf1-3 contain the usual domains of bf and, in addition, three extra domains at the n-terminus. similarly to vertebrate masp-2, masp-3 and c1r/c1s, cibf presents one additional short consensus repeat domain, and two low-density lipoprotein receptor (ldlr) domains. overall deduced amino-acid identity between cibf-1 and cibf -2 was 88 %, whereas cibf-3 showed 49 % identity to both cibf-1 and cibf-2. the cibf serine protease domain shared characteristic features with the complement components c1r/c1s, masp-2 and masp-3, and the active site appears to be of the agy codon type for the catalytic serine residue, and lacks of a histidine loop disulfide bridge (fumiko et al., 2005; yoshizaki et al., 2005). phylogenetic analysis indicates that cibf genes could be the result of duplication and gene conversion occurred after the divergence of the urochordate lineage from the vertebrate subphylum as also shown by genomic organization and intron/exon composition. presumably they have diverged from the common ancestor of vertebrate bf s49 and c2 before the divergence of bf and c2. these results indicate that complement genes have evolved through extensive exon shuffling events in the early stage of chordate evolution. exons 3 and 5 of the three cibf genes show an extremely high degree of nucleotide identity, indicating duplication and gene conversion, since its divergence from the vertebrate bf/c2 gene. the ciona genome analysis also allowed the identification of eleven presumptive genes with mac/perforin domain, nine of them exhibit domain structures similar to those of late complement components. although, activation mechanism and a functional linkage between cic3 and the possible lytic components have not been demonstrated, the lytic function is strongly suggested by the presence of the mac/perforin-like domain. combination of several domains assign the lytic components to the c6-c9 family. however, cic6 and cic7 lack the several c-terminal domains of the corresponding human components, and their functional link to cic3 remain unclear. finally, a group of 132 presumptive genes with complement control module (src domain) have been identified (azumi et al., 2003). in mammals, regulators for inhibiting undesirable complement activation against self cells are composed of repeat of short consensus repeats src domain. in brief, three functional units may be distinguished in activating cic3 and active factors production: 1. collectins, composed of collagen and lectin-like domains which recognize miscroorganisms, associated with serine proteases that presumably activate cic3 leading to a chemotactic product; 2. cibf and cic3, components of the vertebrate alternative pathway, and the activation product may have an opsonic activity; 3. presumptive cic6-c9 molecules forming the mac/perforin complex with cytolytic activity. cytokine-like molecules and receptors pleiotropic and multifunctional proinflammatory cytokines (tumour necrosis factor tnf, interleukins il1 and il6) play a pivotal role in innate immune responses, in cell proliferation, differentiation, apoptosis, and stimulation of the collagen synthesis in wound healing and tissue repair. in invertebrates, cytophilic humoral molecules with functional similarities to vertebrate cytokines have been reported (beck, 1998; beshin et al., 2001; ottaviani et al., 2008). in ascidians, cytokine-like molecules active in stimulating cell proliferation, phagocytosis and opsonisation, have been revealed by immunological and biochemical methods (see beshin et al., 2004). genome sequencing, cdna/est derived from c. intestinalis hemocytes, and identification of the corresponding genes in genome sequences (shida et al., 2003; terajima et al., 2003) revealed the existence of ciil1 receptor, ciil17 receptor genes and an ectodysplasin/tnf-like multigene family. the presumptive il6 gene was also identified. three interleukine-1-recepor-like (ciil-1r) genes present an extracellular ig and an intracellular tir domain which is the conserved toll/il-1 receptor (tir) domain of the two families of receptors (tong, 2005). this domain was first characterized due to the homology between the intracellular region of the mammalian il-1rs and the drosophila tlrs. like tlrs, il-1r signaling pathways are key mediators of the innate immune response to bacteria (lps), fungi, cytokines and growth factors. in mammals, the signalling pathways mediated by tlr, il-1r and tnfr share common components. the possibility exists that these signalling cascades are indeed functioning in c. intestinalis hemocytes. hemocyte citnfα gene expression is challenged by lps vertebrate tnfα is a component of a wide tnf family, it is a type ii transmembrane protein with an extracellular homotrimeric c-terminal domain. a membrane-bound form may be cleaved, and the mature cytokine may be released as soluble form by a variety of cell types including macrophages, monocytes, granulocytes, nk-cells. tnfs are promptly expressed, and regulate inflammatory reactions by interacting with other pro-inflammatory cytokines recruiting and activating inflammatory cells (arika et al., 1990). in the ciona genome, one citnf-like and three citnf-receptors–like genes have been identified (terajima et al., 2003). the c. intestinalis tnfα-like cdna (citnfα) has been cloned from the pharynx excised at 4 h after lps inoculation (parrinello et al., 2008). comparative analysis of the deduced aminoacid sequence discloses that the cloned citnfα-like clusters at a phylogenetic position close to vertebrate tnfα, whereas a considerable distance separates citnfα from drosophila melanogaster tnf-related eiger isoforms and earthworm ccf. like the vertebrate tnfα, citnfα is constitutively expressed in the hemocytes and it is promptly (4 h) increased by lps both in the pharynx after in vivo inoculation and in hemocytes challenged in vitro. western blot analysis with monoclonal antibodies specific for human recombinant tnfα, showed a cell bound form (43 kda) in hemocytes and a 15 kda soluble form in the serum suggesting the role of this cytokine in both local and systemic responses to inflammation. densitometry analysis of these bands confirms the gene upregulation. in particular the cell bound form is enhanced at 2 h post lps injection, whereas later (4 h pi.) the soluble form appears to be enhanced in the serum in accordance with the gene expression disclosed by real-time pcr analysis of the pharynx. the anticipated expression of the cellbound form in hemocytes challenged in vitro appears to be congruent with the presumed maturation process of the soluble one. similarly to vertebrates, different cell types can secrete the same cytokine. at 4 h after lps inoculation, amebocytes with large granules, contained in the pharynx vessels and in the connective tissue lining the tunic, as well as circulating hyaline amebocytes and granulocytes express the citnfα mrna as revealed by in situ s50 hybridization and immunohystochemistry with anti-human rtnfα monoclonal antibody. hemolymph galectins with il1α epitopes are modulated by lps the direct homologue of mammalian il-1 has not been found in the ciona genome. in mammals, il-1α and -β interleukines as well as galectins are pro-inflammatory molecules, furthermore many cytokines are bifunctional molecules containing a receptor-binding domain and an evolutionary conserved carbohydrate recognition domain (crd) that is typical of lectins. the carbohydrate binding is requested for the cytokine biological activity (beschin et al., 2004). in this regard, il1α and β can be considered as lectins (cebo et al., 2001, 2002) directly interacting and contributing to pathogen elimination via opsonization and/or leukocyte activation. recently, inducible galectin-like molecules with human recombinant il-1 epitopes and opsonic properties have been found. parrinello and colleagues (2007) have shown that ca2+independent cigalectin-like molecules, specific for d-galactose and d-galactosides, present human ril1α epitopes. the lps inoculation challenges a promptly (4 h) enhanced serum concentration of this lectin that has been related to the augmented serum opsonizing and hemagglutinating activities assayed with yeast and rabbit erythrocytes respectively. furthermore, human il-1 epitopes are involved in the opsonizing and hemagglutinating processes which are blocked by anti-human recombinant ilα monoclonal antibodies. the western blot pattern showed that, within the initial phase of the inflammatory response (4 h), several serum proteins (59, 37, 30, 23, 15 kda) cross-reacted with the antibody suggesting an oligomerization process of the opsonin/lectin. ciil-17 receptor hemocytes express an interleukine ciil-17r gene that encode a predicted polypeptide of 769 aminoacid residues (dehal et al., 2002b; shida et al., 2003). the c-terminal half of this protein shows homology to the cytoplasmic region of mammalian il-17r (27 % identity/40 % similarity). the central portion is rich in hydrophobic aminoacid residues presumably correspondent to a trans-membranous region, and 22 residues at the extreme n-terminal portion could be a signal peptide sequence. the nterminal portion, probably an extracellular region, shows no homology to the corresponding region of mammalian il-17r. il-17 is produced by mammalian t-lymphocytes whereas its receptor is expressed in a variety of cell types such as fibroblasts and stromal cells disclosing a wide spectrum of activity. a possible ligand of ciil17r has not been predicted. emergence of nk cells receptors natural killer (nk) cells are critical in the evolution of the innate immune system being active in discriminating and killing “normal” and virusinfected, tumor or allogeneic cells. in mammals, nk cells monitor mhc class i expression on target cells by means of inhibitory nk cell receptors (nkrs) (lanier, 2000; vivier et al., 2002). the nkrs transmit an inhibitory signal that cancels a program for cytotoxic action previously triggered by the target cell contact. nkrs belong to two distinct groups of molecules: ig-like receptors or c-type lectin receptors including human cd94 (boyington, 1999). c-type lectin receptors are type ii transmembrane glycoproteins with a c-type lectin domain (ctld) in the extracellular region that bind proteins in a ca2+independent manner rather than sugars. they are known to be a hallmark of surface markers for nk cells (biassoni et al., 2007). c-type lectin superfamily includes carbohydrate-binding proteins (lectins) involved in pathogen recognition and neutralization, leukocyte trafficking, phagocytosis, antigen uptake and processing, and apoptosis. the ca2+-dependent binding of their carbohydrate recognition domain (crd) characterized the ctdl. however, many c-type lectins included in the superfamily lack critical amino acid residues required for crd to bind carbohydrates (rogers and wong, 2003). in this respect it has been hypothesized that divergent evolution, acting on the ctld fold, has generated the lectin-like natural killer (nk) receptors that bind proteins in a ca2+independent manner, rather than sugars. hemocyte cicd94 gene expression modulated by lps is involved in phagocytosis c. intestinalis cd94 (cicd94-1) protein containing cictld is a homolog of the botryllus schlosseri (50/66 % identity/similarity) bscd94/nkr-p-1 molecule (khalturin et al., 2003), and human (30/46 % i/s) cd94. cicd94-1 has been cloned and sequenced, and hemocytes have been stimulated in vitro with lps (zucchetti et al., 2008). even though sequence homology situates the cicd94-1 molecule close to cd94, several features do not suggest that they are complete orthologs. cicd94-1 could be considered a c-type lectin which lacks ca2+-binding property and its carbohydraterecognition (mannose/galactose and related sugars) capacity, and it could be located along the evolutionary line leading to the nk receptors functionally related to the human cd94 which recognizes peptides in the groove of mhc class i molecules. on the other hand, the lack of mhc genes in c. intestinalis genome should indicate that cicd94 is functionally similar to the mice nk cells mhc-independent cd94 (iizuka et al., 2003; mcnerney et al., 2005). however the involvement of cicd94 in cytotoxic mechanism has not been shown, whereas it appears to be involved in phagocytosis of polystyrene latex beads by granular amebocytes inhibited in the presence of anticicd94-1 specific antibodies (zucchetti et al., 2008). no assays have been reported to disclose a cicd94-1 dependent cytotoxic activity by unilocular refractile granulocytes which are known to be cytotoxic (parrinello, 1996; parrinello et al., 1996). the cicd94-1 as receptor on phagocytes can be up-regulated by lps, presumably part of a s51 mechanism of self-nonself recognition (zucchetti et al., 2008). interestingly, granular amebocytes, together with compartment cells, are engaged in the production of cic3-1 and express cic3a-r following inoculation of lps. the presence in the genome of a cd94 homolog and molecules with inhibition (ciitim) and activation (ciitam) motifs reasonably sustain the activity of precursors of nk cells in ciona. the cicd94-1 protein has been found in about 20 % of the granular amebocytes of naïve ascidians suggesting the existence of a cell population that constitutively express cicd94-1 presumably acting as a self-nonself sentinel. altogether these results indicate that hemocytes are provided with a complex array of surface receptors and effector molecules, enabling them to be active in several immune responses. alternatively, distinct hemocyte populations originated from a same hemocyte type (lymphocytelike cells) could express distinct receptors and exert different activities following an inflammatory challenge. ca2+-dependent hemocyte cytotoxicity seems to be cicd94-independent apparently, a cicd94-independent a ca2+dependent cytotoxic activity of c. intestinalis hemocytes has been shown. the hemocyte type named unilocular refractile granulocytes (urg, the unique large granule occupies the cytoplasm), constitutively display cytotoxic activity and lyse rabbit erythrocytes and k562 tumour cell line. zucchetti and colleagues (2008) did not report any urg that express cicd94-1, furthermore immunocytochemical staining of percoll gradient separated hemocytes shows that, after a short incubation with lps, about 80 % granular amebocytes express the cicd94-1 protein. no signs of the receptor in hyaline amebocytes, that have been reported to be phagocytes (rowely, 1981), have been found. the receptor involved in hemocyte cytotoxic activity remain unknown. a plaque forming cell assay with rabbit erythrocytes showed that the cellkilling mechanism requires effector-target cells contacts for challenging the release in vitro of soluble cytolysins. the cytolysin is ca2+-dependent and is inhibited by sphingomyelin and carbohydrates (unpublished). the unique granule displays phenoloxidase activity, and urgs are components of the inflammatory reaction and densely populate the tunic matrix after lps inoculation (parrinello, 1996; parrinello et al., 1996; cammarata et al., 2008). although, any evidence exists on the involvement of cicd94-1-nkr in urg mediated cytotoxicity, in line with the observation that some c-type lectins bind carbohydrates in a ca2+independent manner (brown and gordon, 2001), carbohydrates could be still be potential ligands for cicd94-1, and the possibility exists that a coreceptor may be involved. since lectins have also been claimed as recognition molecules (quesenberry et al., 2003), presumably different self-nonself recognition molecules characterize ascidian hemocytes and tissues. cifacit-collagen expression as a component of the inflammatory reaction inflammation plays an important role in many processes, protecting organisms against pathogens, and also potentially promoting damage progression (henson, 2005). collagens are major structural components of extracellular matrix in tissues of vertebrates and invertebrates, involved in defence and reparative processes (singer and clark, 1999). in the mammalian acute inflammatory reaction, collagen fibres bundles are organized for tissue repair during the remodelling phase (nwomeh et al., 1998), moreover, the total collagens present in normal tissue, is increased from 2to 9-fold in the chronically inflamed tissue (narayanan et al., 1983). in this respect, activation of the innate immune response leads to the production of proinflammatory cytokines that can promote collagenolysis. collagen degradation, and collagen fragments modulate inflammation either augmenting or suppressing interleukine production from peripheral-blood cells (thomas et al., 2007). a family of non-fibrillar collagens, including type ix (facit) collagen, contain short triple helical domains, composed of three genetically distinct polypeptide-chains, interrupted by short non-helical domains (ricard-blum et al., 2005). this collagen type does not form fibrils, and interacts with fibrillar collagen (fibril-associated collagen of cartilage extracellular matrix) and with other extracellular matrix partners (eyre and wu, 2005). since type-ix collagen interacts with the cellular receptor integrins it may have an important function as mediator of cell adhesion to collagen fibrils (käpylä et al., 2004). a c. intestinalis type ix-like collagen cdna (citypeix-col 1α chain), with features of fibril associated collagens formed of interrupted triple helices (facit) has been cloned and sequenced (vizzini et al., 2002). the involvement of this collagen in the inflammatory response has been shown by real-time pcr analysis, ish assay and immunohistochemical methods. in addition flow cytometry with anti-ci-typeix-col 1α chain specific antibodies, showed a prompt (1-4h) and enhanced collagen expression in the circulating hemocytes treated in vitro with lps, and in epidermis cells after in vivo lps inoculation (vizzini et al., 2008). morula cells with large granules (morular feature) express this collagen revealing a fibroblast-like role. conclusions the barrel-shaped sea squirt c. intestinalis (non-colonial ascidian, tunicata) has its life cycle in shallow sea and ocean waters around the world. one day after an egg is fertilized, it develops into a swimming small tadpole that settles down and metamorphoses into an immobile adult. the settled adult feeds by siphoning seawater using a basketlike filter to capture particulate food and oxygen. despite the adult humble appearance, the tadpole larva, comprised of about 2,500 cells, is provided of notochord and dorsal neural tube revealing kinship s52 to vertebrates. the importance of tunicates as models for the vertebrate ancestor was recognized by kowalevsky, who first identified them as chordates, and they have played an important role in various evolution scenarios (see gee, 1996). due to the very simple ascidian body plan and an apparently increased body complexity of cephalochordates, ascidians were previously thought to be living form of the earliest chordate lineage. in the december 13, 2002, an issue of the journal science, an international consortium of researchers reported on the draft sequencing, assembly, and analysis of the c. intestinalis genome (dehal et al., 2002a). from then on, sequence comparison, cdnas, est and gene modulation and function studies provided new insights about the evolution of key vertebrate systems including immune system and development. recently, taking advantage of the genomes sequencing, delsuc and colleagues (2006) proposed that tunicates and not cephalochordates are the closest living relatives of vertebrates stimulating further research on chordates evolution. simplified form of vertebrate gene families were typically found in ciona, whereas the lancelet lineage diverged before the tunicates and vertebrates. genome sequences show that these relationships, reflected at the molecular level, indicates how similar systems and gene sets evolved in different ways from a common ancestor. the close relationship to vertebrates along with its compact genome (about 160 million base pairs, 1/20 the size of the human), makes this sea squirt an ideal model organism for studying chordate evolution. sequence analysis revealed that c. intestinalis genome contains about 16,000 genes, about 80 % of which are also present in humans and other vertebrates. however, the total number of ciona genes is only about half the number in vertebrates, presumably due to the fact that it has single copies of a large number of genes whereas they are present in multiple copies in vertebrates (francino, 2005). anyway, it can be retained that comparative analysis of genes and knowledge of the immunity genes evolution models are consistent with darwin’s 1871 suggestion that ascidians and vertebrates diverged from a common ancestor. increase in the extent of genome resources as well as understanding of transcription at both transcriptosome and spliceosome levels may unveil conserved features on the coding and non coding dna that sustain genetic stability or promote changes (litman and cooper, 2007) either they mutate away and disappear, or they evolve to perform other functions and advance in complexity. altogether adaptive and innate immune system, form a tremendous complex system to recognize non-self and provide protection from a wide variety of pathogens. the innate immune system is the most ancient of the two systems, and the adaptive immune system appeared more recently developing a high degree of complexity and interconnectivity. many components of the innate immune system in vertebrates can be reliably traced to urochordates. for example, genome analysis reveals a number of innate immunity vertebrate-like genes, including toll-like and virus receptor genes, complement pathways components and receptors, cd94/nkreceptor-like, lectins, tnf, il1-r, collagens. however, pure homology seeking for vertebratespecific immunorelevant molecules in invertebrates is of limited value, and functional screening methods may be a more promising approach. accordingly, the expression analysis of humoral and receptorial molecules in ciona’s tissues and hemocytes following a challenge indicate their involvement in ciona’s inflammatory response. there is no evidence of mhc orthologs, tcr, igs in urochordates and agnathans, and no evidence have been reported on the hundreds of key genes involved in vertebrate adaptive immunity. there is a lack of evidence for a gradual transition from the invertebrate innate immune system to the recombinatorial immune system of higher vertebrates (khalturin et al., 2004) and how the adaptive immune system emerged is still obscure. in the genome of c. intestinalis, genes that encode molecules with membrane receptor features have been found among many members of the ig superfamily. they contain the v, and c1-like domains typical of vertebrate antigen receptors and mhc class i and ii. the human homologs of these genes segregate in a single unit of four paralogous segments on chromosomes 1q, 3q, 11p, and 21q. in these regions there are several genes involved in the adaptive immune system, and mhc paralogs with some related members. presumably, an ancestral receptor emerged before the ragmediated rearrangement originated the ancestor of ig and tcr provided with a v domain, in which v and j regions were encoded by a single exon. it has been hypothesized that the simplest ancestral receptor could be a single chain made up of v and c domain, every one linked to a transmembrane segment and a short cytoplasmic tail for signaling (azumi et al., 2003; du pasquier, 2004; kasahara et al., 2004). the status of the urochordate genes reflects perhaps a primitive pre-duplication/prerecombination-activating gene (rag) stage that foreshadow the pathway leading to the genesis of the t-cell receptor (tcr) and antibodies (du pasquier et al., 2004). the absence of antigen-presenting molecules of the mhc-linked class i and ii types, raise questions on the ancestral chordates self-nonself self and allorecognition constituents. recently novel perspectives have been proposed upon a c. intestinalis molecular code for individuality in the absence of mhc (khalturin et al., 2005) as well as on the involvement of complement components. kurn and colleagues (2007) proposed that early during chordate phylogenesis the components of the complement system in addition to their role in pathogen elimination may be involved in allorecognition. so far the phylogenetic analysis of complement components indicates that gene expansion was generated by duplication events that occurred independently in the ascidian and vertebrate lineages. furthermore, since diverse lectin-crd repertoires in tunicates mediate broad recognition (quesenberry et al., 2003), molecular diversity in s53 non self recognition could be due to the glycome code. in addition, mounting evidence indicates that invertebrate immune-type receptors may undergo somatic diversification through elaborate rna processing mechanism (zhang et al., 2004; kalturin et al., 2005; watson et al., 2005; sadd and schimidhemplel, 2006). nowadays, much is known about the evolution of the immune system, but the details of its origin and ancestral genes functions remain to be elucidated (loker et al., 2004). obviously, we are expecting insight that will come by recognizing gene product functions and identifying the function of noncoding sequences of dna lying between the genes, which could regulate gene expression. finally, understanding the complex orchestration of ciona gene networks is crucial to much of biomedical research, while the comparative genomic studies are helping to determine the function and modulation of the genes and other dna regions in the human genome. acknowledgements this work was 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opsonizing ca2+-independent and β-galactoside specific lectins n parrinello, v arizza, m vazzana, m cammarata, ft giaramita, ml di bella, a vizzini, d parrinello marine immunobiology laboratory, department of animal biology, university of palermo, italy accepted june 14, 2007 abstract cytosolic lectins, ca2+-independent and β-galactoside-specific, were determined to be contained in hemocyte and pharynx lysate supernatants of ciona intestinalis, as revealed by hemagglutination assay with trypsinized rabbit erythrocytes. ca2+-independence and decreasing β-galactosides inhibitory capacity (tdg > lacnac ≥ lactose > galactose) have been considered properties typical of galectins. these lectins can be promptly released by hemocytes maintained in vitro suggesting their involvement in defense responses including inflammatory reactions. both cell lysate supernatants and hemocyte culture medium presented β-galactoside-inhibitable opsonizing activity versus yeast. although a percoll density gradient separation method showed that several hemocyte types contain and release β-galactoside-specific molecules, results suggest that hyaline and granular amoebocytes are the primary source of these molecules. key words: hemocyte lectins; hemagglutinins: β-galactosides; phagocytosis; opsonization; hemocytes; tunicates; ciona intestinalis introduction animal lectins, usually revealed by their hemagglutinating activity, are components of a wellconserved protein-carbohydrate recognition mechanism that function in a variety of biological systems (feizi, 2000; sharon and lis, 2003, 2004). functions include intraand extracellular transport processes, sensor branches of innate immunity and recognition of foreign glycans, induction and suppression of effector release, regulation of cellcell/cell-matrix adhesion or migration, positive/negative growth control with implication for differentiation and malignancy (kilpatrick, 2000, 2002; gobius et al., 2002; sharon and lis, 2003). matching glycan diversity with the lectin presence and various glycan epitopes identified as ligands, immune functions appear to be based on the sugar code. in vertebrates and invertebrates lectin families ___________________________________________________________________________ corresponding author: nicolò parrinello marine immunobiology laboratory department of animal biology university of palermo via archirafi 18 palermo, italy e-mail address: nicpar@unipa.it have been established (cooper and barondes, 1999; cooper 2002; kilpatrick, 2002; vasta et al., 1999, 2004a), and, among them, ca2+-independent soluble lectins, generally characterized for a typical carbohydrate recognition domain (s-crd) with affinity for β-galactosides (s-type/galectins), can be pro-inflammatory (brewer, 2002; rabinovich et al., 2002; vasta et al., 2004b). of critical importance for galectin characterization is the binding specificity of the basic unit of recognition as shown by the relative inhibitory efficiency of key oligosaccharides αlactose, n-acetyl-d-lactosamine (lac-nac) and thiodi-galactoside (tdg) (cooper and barondes, 1999; dodd and drickamer, 2001; sharon and lis, 2003). in invertebrates, the defence responses are mainly based on hemocyte types that release humoral factors, including lectins, lysins, clotting and antibacterial proteins (loker et al., 2004), or display cell-linked activities (parrinello, 1996; parrinello et al., 2003). cellular recognition has been attributed to a protein-carbohydrate molecular mechanism located at the cell surface. sugar-binding proteins have been found on hemocyte surface (amirante et al., 1978; vasta et al., 1984; parrinello and arizza, 1988) and they are present in the hemolymph of all mailto:nicpar@unipa.it 56 the examined species, probably involved in nonadaptive immune recognition. in many cases, an opsonic function has been demonstrated (cheng et al., 1984; renwrantz and stahmer, 1983). in ascidians, considered a key group in chordate phylogenesis (hori and osawa, 1987; field et al., 1988; swalla et al., 2000; zeng and swalla, 2005), multiple naturally occurring or inducible galectins have been found in cell-free hemolymph or bound to hemocyte surface (parrinello 1995; nair et al., 2001; vasta et al., 2001; green et al., 2003; quesemberry et al. 2003; vasta et al., 2004a). in the colonial ascidian botryllus schlosseri, ballarin et al. (1999, 2000) purified from the hemocyte lysate supernatant a dgalactose specific humoral opsonin with galectin properties. in styela clava a c-type humoral lectin with opsonin properties has been purified from the hemolymph (kelly et al., 1992). in addition, lectins could be released from hemocytes cultured in vitro (arizza et al., 1991; cammarata et al., 1993) and were contained and released from pharynx explants (raftos et al., 1990; arizza et al., 1991, 1997). previous papers reported that ciona intestinalis serum hemolymph contains ca2+-dependent (ctype) and ca2+-independent lectins (wright, 1974; parrinello and patricolo, 1975). a recent report showed that serum galectins could be enhanced in inflammatory responses (parrinello et al., 2007), whereas c-type lectins may be responsible of complement activation (pinto et al., 2003). in this respect c. intestinalis genome-wide analysis revealed that several c-type lectin and galectin genes have been annotated in the genome (hori and hosawa, 1987; cooper and barondes 1999; dehal et al., 2002). however, few data exist on the functional role and tissue distribution of lectins of this ascidian. to ascertain the lectin defence role, tissue localization compatible with internal defence are additional requirements to support the involvement of these molecules in immune protection. in this sense, circulating hemocytes assume particular interest as well as pharynx that represents the main route of pathogen entry. in c. intestinalis, several hemocyte types have been described (de leo, 1992), including stem cells, hyaline and granular amoebocytes, unilocular refractile granulocytes, signet ring cells, morula cells, small and large compartment cells. of these cell types, only hyaline and granular amoebocytes are capable of phagocytosis in vitro (rowley, 1981), and several granular and vacuolated hemocytes appeared to be mainly responsible of the inflammatory responses in vivo (parrinello, 1981; parrinello et al., 1984; parrinello and patricolo, 1984; parrinello et al., 1990). in the present paper we show that soluble lectins are contained in c. intestinalis hemocyte and pharynx lysate supernatants, as revealed by β-dgalactoside inhibition of rabbit erythrocytes agglutination. in addition, these lectins are promptly released by hemocytes in vitro. both cell lysate supernatants and hemocyte short-term culture medium presented opsonizing activity versus yeast, revealing the involvement of β-galactoside specific lectins as opsonins. a percoll density gradient separation method displayed that several hemocyte types, that have been shown to be involved in distinct phase of the inflammatory response, contain and release these lectins, whereas hyaline and granular amoebocytes, that posses in vitro phagocytic activity, appear to be rich in cytosolic lectins and are the main source of their release. materials and methods tunicates, hemolymph collection ascidians (7-10 cm long) were collected from mazara del vallo harbor (italy), held in refrigerated (18 °c) and aerated sea water (60 l aquaria) and fed every second day with a marine invertebrate filter feeding diet (kent marine inc. wi usa). the animals were blotted dry to remove any excess of seawater, and bled by removal of the tunic and puncture of the heart. to collect hemocytes, hemolymph was harvested into a fourfold excess of ice cold sterile artificial sea water without cacl2 and mgcl2 (fsw: 9 mm kcl, 29 mm na2so4, 2 mm nahco3, 0.5 m nacl, ph 7.4) containing 10 mm ethylenediaminetetracetic acid (fsw-edta) as an anticoagulant. after centrifugation at 850xg (10 min, 4 °c), pooled hemocytes (10 ascidians/experiment) were washed twice with fsw-edta and, finally, suspended in sterile fsw adjusted for osmolarity with the hemolymph (1,090 mosm kg-1). hemocyte mortality, estimated by trypan blue (0.05 % in fsw) exclusion test, was lower than 5 %. pharynx explants were surgically excised with sterile scissors and washed three times with sterilized fsw-edta. the same amount of tissue (about 1 gr) was used for every preparation. all media were sterilized through a 0.22 µm filter (millipore, millex). preparation of hemocyte and pharynx lysate supernatants (hls, phls) hemocytes from pooled hemolymph (15 ascidians for every preparation) were pelleted by centrifuging at 850 xg for 10 min at 4 °c. after two washings in fsw, hemocytes (10 x 106 cells/ml) were suspended in diluted ice-cold medium (1:5 in d.w.) to be sonicated at 4 °c for 60 seconds (branson, model b15, danbury, ct, usa). the cell lysate was spun (27,000 xg, 20 min, 4 °c), and the resulting supernatant (designed hls) was dialyzed against tbs (tris hcl 50 mm, nacl 0.15 m, ph 7.4), and used for the hemagglutination assay. pharynx explants (1 gr tissue) dried with filter paper, frozen at -80 °c and homogenized on ice, were sonicated at 4 °c for 60 seconds. after centrifuging at 27,000xg for 20 min at 4 °c, the supernatant (designed phls 0.6 – 0.7 mg/ml protein content) was extensively dialyzed against tbs. for hemagglutination and opsonization assays, samples were 100 times diluted. to examine the possible effect of cytosolic proteases, in previous experiments, a protease inhibitor cocktail (sigma, st. louis, usa) was added (0.1 % final concentration) into the medium just before hls preparation. 57 hemocyte culture and supernatant preparation details of the method have been reported elsewhere (cammarata et al., 1993). unfractionated or enriched hemocyte populations were suspended in sterile isosmotic artificial sea water (sw) (fsw containing 12 mm caci2.6 h2o and 26 mm mgci2.6 h2o). osmolarity was adjusted to 1090 mosm kg -1. hemocytes (3x106 in 200 µl medium) were put into each well of sterile flat-bottomed culture plates (nunc, denmark) and maintained at 4, 10, or 18 °c. in each experiment, cell-free medium from 10 cultures (designed hes) was pooled and dialyzed against tbs prior to be assayed. percentage of dead cells was evaluated with the trypan blue exclusion test. cell viability of hemocytes cultured for 1.5 h at 10 °c in sw was evaluated with neutral red vital stain (borenfreund and puerner, 1984). values lesser than 3 ± 0.5 % dead cells, and more than 97 ± 1.1 % viable cells were found. preparation of rabbit and sheep erythrocyte suspensions and hemagglutination assay rabbit erythrocytes (re) and sheep erythrocytes (se) were obtained from “istituto zooprofilattico della sicilia” (palermo). the erythrocyte pellet was washed with pbs (pbs: 6 mm kh2po4, 30 mm na2hpo4, 0,11 m nacl, ph 7.4) and centrifuged at 500xg for 10 min at 4 °c, then resuspended in tbs to obtain a 1 % suspension. as previously reported (parrinello and canicattì, 1982), hemagglutinating activity (designed ha) was determined in 96-well round bottom microtiter plates using tbs containing 0.1 % gelatin as a dilution medium (serial two-fold dilutions), and an equal volume of 1 % re or se in tbs. the microplate was incubated at 37 °c for 1 h, and 2 h at 4 °c. to increase the erythrocyte sensitivity to the hemagglutination assay, trypsin-treated erythrocytes (try-re, try-se) were prepared by suspending erythrocyte pellet, from 1.0 ml blood, into 6 ml tbs containing 300 µg trypsin (stock solution prepared in 10 mm hcl). the reaction mixture was incubated at 37 °c for 15 min, and, trypsinized erythrocytes, washed with tbs, were resuspended (1 %) in the same medium. to verify the role of ca2+, the hemagglutination assay was carried out in the presence of 20 mm edta or 10 mm cacl2. the titre of hemagglutinating activity (designed ht) was expressed as the reciprocal of the highest dilution giving unequivocal agglutination judged by eye or with a low power binocular microscope. the ht values, expressed as log2, were recorded as the average ± sd of 10 different assays. controls consisted of tbs samples in which serum was not added. to verify the role of ca2+, lysate supernatants were dialyzed against tbs in the presence of 20 mm edta or 10 mm cacl2, and erythrocytes were suspended in this medium for hemagglutination assay. formaldehyde-fixed rabbit erythrocytes (f-re) were prepared according to the csizmas’s method (1960), and suspended in tbs. yeast preparation, opsonization, and in vitro phagocytosis assay a saccharomyces cerevisiae (baker's yeast, type ii) suspension was prepared in distilled water at 0.25 % w/v (approx. 1x10 8 yeast cells/ml), autoclaved for 15 min, washed twice by centrifuging at 2,000xg (5 min, 4 °c), and finally incubated for 2 h at 20 °c with a solution of eosin-y at 0.05 % final concentration (cammarata and arizza, 1994). after repeated washing, yeast were suspended at 0.125 % final concentration in sterile calciumand magnesium-free sw (fsw), and used immediately. for opsonization, yeast were incubated with lysate supernatant (0.125 % w/v) for 1.5 h at 20 °c, washed in fsw (3 times), and finally suspended in the same medium at the initial concentration. after this treatment, yeast appeared to be agglutinated, forming small clumps, but they were easily resuspended by washing with fsw. to verify the role of divalent cations in opsoninyeast binding, opsonization was carried out in the presence of 20 mm edta. furthermore, the effect of added ca2+ or mg2+ was estimated with fsw containing 10 mm cacl2 or mgcl2. for the phagocytosis assay, 200 µl hemocyte suspension (1x10 6 cells in sw, indicated as he) was mixed with 100 µl of yeast preparation (10:1 yeast:hemocyte ratio), and incubated in 1 ml test plastic tubes with gentle stirring for 90 min at 20 °c. then, 50 µl of a quenching solution (2 mg/ml trypan blue, 2 mg/ml crystal violet in 0.02 citrate buffer, ph 4.4, containing 33 mg/ml nacl) was added. a drop of this suspension was smeared onto slides and examined under a light microscope equipped with a nomarsky differential interference contrast optic (diaplan, leica, wetzlar, germany). hemocytes (about 1000 for every assay, and at least 200/slide) were counted at 800x magnification. lysate supernatant opsonizing capacity was expressed as percent hemocytes showing ingested yeasts. results were compared to percent phagocytes in a reaction mixture in which hemocytes from ascidians were mixed with non-opsonized yeasts. the phagocytic index was calculated according to the following formula: total number of ingested yeasts/total number of counted phagocytes absorption with erythrocytes to absorb agglutinins or opsonins, fre or fse were used in the reaction mixture containing packed f-erythrocytes and lysate supernatant or culture medium (v/v). the mixture was incubated for 1 h at room temperature and overnight at 4 °c with occasional shaking, centrifuged at 800xg, and the supernatant assayed with try-re and try-se. to control the effect of experimental conditions on the hemagglutinating activity, no absorbed samples were treated as the absorbed ones. inhibition of hemagglutinating and opsonizing activities the supernatants (hls, phls, hes) were incubated for 60 min at 20 °c with decreasing sugar concentrations (starting from 100 mm final concentration), avoiding sample dilution. the 58 treated sample was then assayed for hemagglutinating activity. for inhibiting the opsonizing activity, yeasts were maintained for 1.5 h in lysate or culture supernatant preparations containing decreasing sugar concentrations, and then washed (2 times) with fsw before the phagocytosis assay. the last sugar concentration (mm) giving an unequivocal inhibitory activity was recorded. dgalactose, α-lactose, d-mannose, l-rhamnose, dglucose, l-fucose, n-acetyllactosamine (lacnac) and thio-digalactoside (tdg) were assayed. to examine the effect of sugar added to the yeast suspension, 25 µl of opsonizing serum containing 100 mm sugar was mixed on a slide with 25 µl yeast suspension and observed after 1.5 h incubation in a wet chamber. observations with a microscope equipped with nomarski differential interference contrast optics (leica) did not show any yeast clamps. to verify changes in yeast sensitivity to phagocyte, sugar treated yeasts were assayed, after washing, in a phagocytosis assay. identification and separation of the hemocytes through a percoll discontinuous density gradient the hemocytes were classified according to the most popular terminology (wright, 1981; de leo, 1992). in the hemolymph several hemocyte types have been recognized. lymphocyte-like cells are small stem cells. hyaline amoebocytes contain in their cytoplasm granules of uniform size; granular amoebocytes contain small or large granules; signet ring cells present a single large vacuole; compartment cells contain a variable number of large round and angular vacuoles distributed at the periphery of the cell; morula cells that, when allowed to stand, may assume a berry-like or morular appearance; unilocular refractile granulocyte (urg), characterized by an unique large granule that occupies the cytoplasm and appears to be refractile when observed under a light microscopy. the hemocyte populations were separated using the method described by parrinello et al. (1996). briefly, freshly collected hemocyte suspension (approximately 6x107/ml in 4 ml) diluted with fswedta was spun through a discontinuous gradient of equilibrated percoll (pharmacia fine chemicals uppsala, sweden) (dialyzed against hemocyte isosmotic fsw-edta). a gradient was performed with isosmotic medium to obtain decreasing densities (1.105, 1.098, 1.090, 1.079, 1.071, and 1.060 g/ml) into a 10 ml tube. the tube was centrifuged in a swing-out rotor (850xg, 15 min, 7 °c). bands of cells were gently removed by aspiration from the gradients and washed twice before suspension in fsw. the total population of hemocytes was portioned into six distinct, discrete bands (b1-b6). dead cells lower than 5 % were found, and viable cells were higher than 95 %. although each band was mainly enriched for certain hemocyte types, homogeneous populations could not be separated. for microscopy observations, the cells were removed from the gradients, washed twice in fsw. to check for the hemocyte types contained in each band, 200 µl of the cell suspension was layered on a slide soaked with the poly-l lysine, fixed (30 min) with 1 % saccharose and 1 % glutaraldeyde in fsw, and stained with hematoxilin-eosin (5 min). differential count of the hemocytes from each band was performed (at least 200 cells/slide). to prepare hls the correspondent bands from different percoll gradients were pooled to reach 1.0x107 cells/ml. protein content determination protein content was measured by the bradford method (1976). bovine serum albumin was used as standard. statistical analysis data were from five distinct experiments, and each assay repeated three times. hemagglutinin titres, recorded as log2 ± sd, were examined by the student t-test. differences were considered significant at p < 0.05. 2.9 chemicals unless otherwise reported, all chemicals were purchased from sigma. table 1 hemagglutinating activity of hemocyte lysate supernatant (hls), pharynx lysate supernatant (phls) and supernatant from cultured hemocytes (hes) cultured for 1.5 h in sw at 18 °c, assayed against trypsinized rabbit (try-re) or sheep erythrocytes (try-se), in the presence or absence of divalent cations hemagglutinating activity (log2 ± sd, n=5) hls phls hes medium try-re try-se try-re try-se try-re try-se tbs 5.2 ± 0.4 -4.8 ± 0.8 -3.8 ± 0.4 - tbs-10 mm cacl2 5.4 ± 0.5 -4.6 ± 0.8 -3.8 ± 0.8 - tbs-10 mm mgcl2 4.8 ± 0.8 -4.8 ± 0.4 -3.8 ± 1.0 - tbs-20 mm edta 4.8 ± 1.0 -4.4 ± 0.5 -3.6 ± 0.8 - sw 4.6 ± 0.8 -5.6 ± 0.5 -3.6 ± 0.8 - f-sw-edta 4.6 ± 0.8 -5.6 ± 0.8 -3.6 ± 0.5 - 59 results hls and phls agglutinated rabbit erythrocytes in the absence of ca2+ preliminary hemagglutination assays with re of five distinct hls (20-25 µg/ml protein content), and phls in 100 times diluted samples preparations (20-25 µg/ml) showed 3.0 ± 0.8 ht, whereas higher titres (ht: 5.0 ± 0.5 and 4.8 ± 0.5, respectively) were found with try-re as targets. consequently, trypsinized erythrocytes were used for the next agglutinin titration. no activity was found against se or try-se (table 1). the presence of a protease inhibitor cocktail in lysate preparations did not enhance the hemagglutinating titre compared to that of inhibitor-free samples (data not shown). the addition of 10 mm (final concentration) cacl2, mgcl2 or 20 mm edta into the medium did not affect the hemagglutinating activity of hls and phls samples (table 1). hemocytes release in vitro ca2+-independent hemagglutinins supernatant from hemocyte cultures (hes) agglutinated try-re but not try-se. the hemocytes cultured at 4 °c for 1.5 h released a low amount of lectins into the cell-free medium (ht: 2.2-2.4), whereas highest levels (ht: 3.6-3.8) were found at 10 °c (table 1). higher temperatures (up to 18 °c) did not significantly enhance the agglutinin release. the presence of 20 mm edta as well as the addition of cacl2 or mgcl2 in the hemagglutination medium (tbs) did not affect the activity vs both the erythrocyte types (table 1). to compare the activity of supernatants from unseparated hemocyte culture with hls, 15x106 hemocytes/ml were divided in two groups, one of the two was homogenized, and the other one was cultured (1.5 h, 10 °c). in four distinct experiments, the hlss presented the highest activity (ht: 6.7 ± 0.9) compared with the hemocyte culture supernatants (ht: 4.0 ± 0.15). protein content of unseparated hemocyte culture supernatants from twelve distinct experiments ranged fig. 1 a ciona intestinalis phagocyte with ingested eosin-y treated yeasts, as shown by nomarski contrast interference observation (a), or uv–light observation (b). y: eosin-y treated yeast. bar = 5 µm from 8 µg/ml to 20 µg/ml. a significant proportionality between protein content and hemagglutinin titre was not observed. the culture medium composition did not affect the agglutinin-release. in preliminary experiments the same hts were recorded by assaying supernatants from hemocytes cultured in m199 enriched medium or sw. therefore, in the next experiments hemocytes were cultured in sw. erythrocyte specificity absorption experiments with fre or fse showed that the hemagglutinating activity of hls and phls was lost after absorption with fre, whereas it was maintained after treatment of hls and phls with fse (ht: 4.6-5.6, respectively). the same effect was exerted by fre on hes. hls, phls and hes opsonize yeast table 2 shows that 15 % (mean value) of hemocytes spontaneously ingested non-opsonized yeast (fig. 1). when the targets were opsonized with hls, phls or hes, a greater number of hemocytes ingested yeast, and the percentage significantly increased up to 20-24 % (p < 0.05). the phagocytic index significantly increased as an effect of the opsonization. table 2 opsonizing activity, in the absence or presence of edta (20 mm), of hemocyte lysate supernatant (hls), pharynx lysate supernatant (phls), and supernatant from hemocytes (hes) cultured for 1.5 h in sw at 18 °c, assayed against yeast cells. percent (%) phagocytes with ingested opsonized yeasts were compared with % phagocytes assayed with non opsonized (sw) targets. values are expressed as mean percentage of hemocyte containing yeasts ± sd (n=5). phagocytic index = total number of ingested yeasts/total number of counted phagocytes. ** p < 0.01; ***p<0.001 10 mm edta yeasts treated with phagocytes (%) phagocytic index phagocytes (%) phagocytic index sw 15.3 ± 1.2 1.9 ± 0.2 14.6 ± 0.9 1.7 ± 0.3 hls 24.2 ± 3.2 (**) 2.6 ± 0.3 (**) 22.1 ± 2.3 (**) 2.9 ± 0.3 (**) phls 23.9 ± 1.2 (**) 2.4 ± 0.2 (**) 22.8 ± 1.4 (**) 2.6 ± 0.3 (**) hes 20.2 ± 2.1 (**) 3.3 ± 0.3 (***) 19.7 ± 1.8 (*) 3.2 ± 0.2 (***) 60 table 3 sugar inhibition of the hemagglutinating and the opsonizing activity of hemocyte lysate supernatant (hls), pharynx lysate supernatant (phls) and supernatant from hemocytes (hes) cultured for 1.5 h in sw at 18 °c, assayed against trypsinized rabbit erythrocytes and yeasts respectively. the lowest sugar concentration (mm) that abolished the hemagglutinating activity of the sample against rabbit erythrocytes, or giving significant inhibitory activity of yeast opsonization was recorded. 100 mm starting sugar concentration sugar inhibitory concentration (mm ± sd, n=5) hemagglutinating activity (1) opsonizing activity (2) compound hls phls hes hls phls hes d-galactose 21.6 ± 9,8 10.6 ± 4,6 5.25 ± 1.7 25.0 ± 5.6 25.0 ± 8.8 25.0 ± 6.3 α-lactose 21.6 ± 9,8 16.0 ± 0 12.5 ± 1.0 50.0 ± 12.9 50.0 ± 11.7 50.0 ± 11.3 lactulose 44.0 ± 19,0 27.3 ± 9,8 25.0 ± 1.5 50.0 ± 10.4 50.0 ± 7.5 25.0 ± 10.9 lacnac 25.0 ± 6.8 25.0 ± 4.8 1.5 ± 1.0 50.0 ± 11.7 25.0 ± 3.8 25.0 ± 3.2 tdg 1.5 ± 0.5 1.5 ± 0.5 0.5 ± 1.0 50.0 ± 10.1 25.0 ± 5.1 12.5 ± 9.7 l-fucose n.i. n.i. n.i. n.i. n.i. n.i. d-mannose n.i. n.i. n.i. 25.0 ± 3.7 25.0 ± 6.3 25.0 ± 8.7 d-glucose n.i n.i. n.i. n.i. n.i. n.i. nana n.i. n.i. n.i. n.i. n.i. n.i. nag n.i. n.i. n.i. n.i. n.i. n.i. laminarin (%) 0.006 ± 0.002 0.012 ± 0.04 0.015 ± 0.001 0.5 ± 0.09 0.05 ± 0.001 0.5 ± 0.05 (1) hemagglutination assay against trypsinized-rabbit erythrocytes. (2) supernatant opsonising activity vs yeast examined in a hemocyte phagocytosis assay. n.i.: no inhibition the ca2+-independence of the opsonizing activity was shown by treating yeast with hls, phls or hes in the presence of 20 mm edta, 10 mm cacl2, or mgcl2. no differences were observed as an effect of opsonization medium composition (table 2). finally, opsonins were absorbed by treating samples with packed fre whereas was unchanged after fse absorption. galactosides inhibit the hemagglutinating and opsonizing activities the sugar-lectin binding of both agglutinins and opsonins was shown by sugar inhibition assay. the hemagglutinating activity of hls and phls was abolished by d-galactose, α-lactose, lactulose, lacnac, at various mm concentrations (table 3). except lactulose, lower concentrations (ranging from 1.5 mm lacnac to 12.5 mm lactose) of these sugars inhibited hemocyte culture supernatant. tdg was the most effective saccharides in inhibiting hemagglutination activity of hls, phls and hes (ranging from 0.5 to 1.5 mm). in all cases, the hemagglutinating activity was not affected by 100 mm d-glucose, l-fucose, d-mannose (table 2). the above reported active sugars inhibited the opsonizing activity of hls, phls and hes (table 3) even if higher concentrations were needed, whereas mannose inhibited the yeast opsonization. the presence of high sugar concentration in the medium used for preparing yeast did not affect their sensitivity to phagocytes, and, after washing, they were phagocytosed as the untreated ones. hemagglutinating activity of hls and culture supernatants from hemocyte populations enriched through a percoll discontinuous density gradient the cells removed from density gradient separated bands were examined and identified for their morphology. differential count of the hemocytes from each band was performed (at least 200 cells/slide). b1 mainly contained hyaline amoebocytes (~ 76 %) and, to a lesser extent, stem cells (~ 9 %) and signet ring cells (~ 11 %); b2 was mainly enriched in hyaline amoebocytes (~57 %), but also contained lymphocyte-like cells (~ 5 %), granular amoebocytes (~ 20 %), signetring cells (~ 9 %); b3 consisted primarily of ~ 71 % granular amoebocytes, ~ 22 % hyaline amoebocytes and ~ 8 % signet ring cells; b4 was composed of granular amoebocytes (~ 45 %) and morula cells (~ 52 %); b5 contained morula cells (~ 59 %) and univacuolar refractile granulocytes (~ 41 %); finally, b6 was largely (~ 84 %) made up of morula cells. hemocyte types present in each band at very low percentage were not reported. 61 fig. 2 ciona intestinalis main hemocyte types in b2, b3 and b6 bands separated through a discontinous percoll density gradient. hyaline amoebocytes (ha), granulocytes (g); morula cells (mc). bar = 10 µm in fig. 2 hyaline amoebocytes, granular amoebocyte, univacuolar refringent granulocytes and morula cells contained in the separated bands are shown. to identify the hemocytes that contained antire lectins, hlss from percoll-gradient separated hemocytes were assayed for their activity. as shown in fig. 3, hls from enriched hemocyte populations, standardized at a same hemocyte number (10x106/ml), presented various levels of hemagglutinating activity revealing that b2-hls and b3-hls reached titres higher (ht: 8.2 and 5.9, respectively; p < 0.01) than hls from the whole hemocyte preparations (4.2 ht). lower levels were associated with b1 and b6 (ht: 4.0 and 3.6, respectively), whereas the lowest ones (ht: 1.4-2.0) were recorded in b4 and b5 hlss. to identify the lectin-releasing hemocytes, the density-gradient enriched hemocyte populations were cultured at 10 °c for three hours, and, then, cell-free medium from pools of each separated band was assayed. the hemagglutination titres of b2 cellfree culture medium, compared with the culture medium of the remaining bands or unseparated hemocytes (ht: 1.3), showed the highest hemagglutinating titre (ht: 3.4), whereas 1.7 ht was found for b1 (fig. 3). very low hts were recorded for b3-b6 cell free culture medium. discussion the serum hemolymph of c. intestinalis contains naturally occurring lectins that agglutinated rabbit and sheep erythrocytes (wright, 1974; parrinello and patricolo, 1975). in the present paper we show that hemocytes and pharynx lysate supernatants agglutinated rabbit erythrocytes, whereas they were inactive against sheep erythrocytes revealing a certain range of specificity in discriminating target membrane sugar components. accordingly, erythrocyte trypsinization of sheep erythrocytes that exposed glycosylated components of the membrane outer layer did not affect the hemagglutination of sheep erythrocytes whereas increased rabbit erythrocytes sensitiveness, and higher hemagglutinin titres were recorded. the anti-tryre agglutinin titres (ht: 4-5) revealed in the hls and phls were higher than those (ht: 1-3) reported by parrinello and patricolo (1975) for the hemolymph serum. hemocytes and pharynx tissues cannot be directly compared in their agglutinin titres, in fact pharynx blood vessels can contain various amounts of hemocytes. these agglutinins, are cytosolic being identified in water-soluble extracts from hemocyte and pharynx preparations. the sample preparation method, carried out on ice followed by a prompt separation of the supernatant at 4 °c, presumably avoided any effect of cytosolic proteases as shown by the unchanged hemagglutinin titres after the addition of an anti-protease inhibitor cocktail. hemagglutinins can be promptly released by hemocytes maintained viable in vitro for a short time (1-3 h). such a release was temperature-dependent as it decreased with low temperature and reached the highest values at 10-18 °c. we do not know if lectins are secreted or a non classical secretory mechanism may externalize lectins confined to cytoplasm as reported for some galectins (sato et al.,1993; sato and hughes, 1994). the anti-re agglutinins contained in hemocytes and pharynx as well as released by hemocytes are ca2+-independent, d-galactoside specific lectins that showed a relative inhibitory activity. tdg, an analog of lactose, and lacnac, an analog of lactose with a hydroxyl substituted with an acetamide group, were more active than lactose in inhibiting the agglutination. tdg, lacnac, lactose and galactose were effective at higher concentrations. a such lectin-binding affinity of β-galactosides (tdg > lacnac ≥ lactose > galactose) and the ca2+independence have been considered properties typical of galectins (vasta et al., 2004b). accordingly to the defence role, these βgalactoside-specific lectins showed opsonic properties vs yeast, and hemocytes could release them in vitro presumably as an effect of cell activation due to the experimental procedures. a low percentage of phagocytes internalized non opsonized yeasts, presumably due to mannose receptors on the phagocyte membrane, whereas the opsonization of the targets with supernatants from hemocyte and pharynx lysates, and hemocyte culture medium enable a significantly major number 62 fig. 3 ( ) hemagglutinating activity of hemocyte lysate supernatant from non fractioned hemocytes (nf), or enriched hemocyte populations separated through a discontinous percoll gradient as discrete bands b1, b2, b3, b4, b5 and b6. ( ) hemagglutinating activity of supernatant from cultured nf (1.5 h at 18 °c), or enriched hemocyte populations separated through a discontinous percoll gradient as discrete bands b1, b2, b3, b4, b5 and b6 and supernatant from 1.5 h hemocyte colture from cell populations enriched in bands b1-b6 through a discontinous percoll density gradient. inset: student t-test comparison between the various groups. *** p<0.001, **p<0.01, *p<0.05 of phagocytes to engulf opsonized targets, also showing an increased phagocytic index. the possibility exists that, as reported for mammalian galectin 10 (swaminthan et al., 1999), some crd variants could be contained in c. intestinalis β-galactosidespecific lectins that could interact with mannose. although pure hemocyte populations could not be separated, in an attempt of correlating lectin content and release with hemocyte types we examined cell populations separated on percoll discontinuous density gradient. lysate supernatants from the separated hemocyte bands, prepared from a constant cell number, presented a various degree of hemagglutination titres as compared with the unfractionated hemocytes. hemocytes from the separated bands appeared to be mixed cell populations therefore it is not possible to assign the content in cytosolic lectin molecules to discrete hemocytes. the highest hemagglutinating titres were found in b2-hls that mainly contained hyaline amoebocytes (~ 57 %) and a lower proportion of granulocytes (~ 20 %). likewise the high titre that characterized b1-hls, composed with 76 % hyaline amoebocytes in the absence of granular amoebocytes, emphasized the effect of the enrichment in hyaline amoebocytes as lectincontaining cells. on the other hand, also granular amoebocytes enriched in b3 (~ 71 % granular amoebocytes, and 22 % hyaline amoebocytes) could be responsible of the high titre registered in their lysate supernatant. finally, a low level was found in the lysate from morula cells enriched (85 %) in b6. cytosolic lectins appeared to be contained in several hemocyte types, supporting their role in multiple cell functions. in addition, the high content of lectins in inflammatory hemocytes, like hyaline and granular amoebocytes, suggested their involvement in inflammatory reactions. accordingly, a similar hemagglutin titre profile was observed by examining the activity of supernatant from b1-b6 hemocyte cultures even if the titres were lower than those observed in the hemocyte 63 lysate supernatants, probably due to a lesser number of hemocytes, as yielded from separated bands, cultured for a short time. the highest activity was found by culturing hemocytes from b1 and b2 mainly enriched in hyaline amoebocytes that have a role as phagocytes and presumably release lectins to be involved in opsonization. however, the low hemagglutinating activity of supernatants from enriched granular amoebocyte population (b3) does not exclude their involvement in lectin-dependent immune functions. in fact, the experimental conditions and the short time we used for preparing culture supernatants could affect the lectin release from this hemocyte type. further research is required to uncover the molecular structure of hemocyte β-galactosidespecific lectins and ascertain whether they belong to the galectin family as identified in the c. intestinalis genome. aknowledgements this work was supported by research grants from the italian ministry of education (prin 2004 4057000) to n parrinello co-funded by the university of palermo. tanks are also due to mr g miceli for collecting ascidians. references amirante ga, mazzalai fg. synthesis and localization of hemoagglutinins in hemocytes of the cockroach leucophaea maderae l. dev. comp. immunol. 2:735-40,1978. arizza v, cooper el, parrinello n. circulating hemocytes and pharyngeal explants of styela clava release hemagglutinin in vitro. j. mar. biotech. 5: 31-35, 1997. arizza v, parrinello n, schimmenti s. in vitro release of lectins by 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intestinalis to vertebrate erythrocytes. j. invertebr. pathol. 24: 29-36, 1974. wright, rk. in: ratcliffe na, rowley af (eds), urochordata, invertebrate blood cells, vol. 2, academic press, london, pp 565-625, 1981. zeng l, swalla bj. molecular phylogenesis of the protochordates: chordate evolution. can. j. zool. 83: 24-33, 2005. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=retrieve&dopt=abstractplus&list_uids=2243059&query_hl=16&itool=pubmed_docsum http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=search&itool=pubmed_abstractplus&term=%22vasta+gr%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=search&itool=pubmed_abstractplus&term=%22quesenberry+m%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=search&itool=pubmed_abstractplus&term=%22ahmed+h%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=search&itool=pubmed_abstractplus&term=%22o%27leary+n%22%5bauthor%5d 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice isj11x.pdf 1 isj 3: 1-3, 2006 issn 1824-307x short communication monitoring of the immune efficiency of mytilus galloprovincialis in adriatic sea mussel farms in 2005 d malagoli, l casarini, e ottaviani department of animal biology, university of modena and reggio emilia, modena, italy accepted january 13, 2006 abstract the monthly evaluation of the cytotoxicity of hemolymph from the mussel mytilus galloprovincialis revealed some variations in the percentage of cytotoxic animals during the year. cytotoxicity is confirmed to be a dynamic parameter that can be used as an indicator of immune efficiency and, therefore, of the state of health of the animals. key words: mytilus galloprovincialis; cytotoxicity assay; cytotoxic activity; adriatic sea mussel farms introduction the state of health of mytilus galloprovincialis mussels was evaluated in terms of cytotoxic activity in the hemolymph. it is well-known that molluscs, as all invertebrates, possess only innate or natural immunity and recognize and eliminate non-self material mainly through cellular and humoral components. among the latter, cytotoxicity is one of the most important immune functions (franceschi et al., 1991). this function has been well conserved during evolution and described both in invertebrates and vertebrates (ratcliffe et al., 1985; savary and lotzová, 1986). wittke and renwrantz (1984) have demonstrated that circulating cells (immunocytes) from mytilus edulis are able to produce cytotoxic substances, which lyse human erythrocytes. a cytotoxic protein complex has also been found in m. galloprovincialis hemolymph (hubert et al., 1997). recently, we have designed an easy-to-use and lowcost cytotoxicity test (malagoli and ottaviani, 2005) that has been used here to evaluate the cytotoxic activity of m. galloprovincialis collected each month during 2005 from adriatic sea mussel farms. corresponding author: enzo ottaviani department of animal biology via campi 213/d 41100 modena, italy e-mail: ottaviani.enzo@unimore.it materials and methods animals specimens of the mussel mytilus galloprovincialis were obtained each month from local fishermen in the cesenatico area (fc, italy) immediately after being caught. forty animals were sacrificed to collect hemolymph on the spot, while the remaining samples were taken to the department of animal biology (modena, italy) and maintained in the laboratory aquarium (artificial seawater, temperature 19 ± 1 °c, ph 8.0 ± 0.2, salinity 35 ± 2 psu) for at least 10 days before control experiments. no animals could be obtained in february because of adverse weather conditions. hemolymph preparation and cytotoxicity assay the detailed procedure for the cytotoxicity assay has been described elsewhere (malagoli and ottaviani, 2005). briefly, the hemolymph was collected from the posterior adductor muscle using a sterile syringe, filtered into sterile tubes with 0.2 µm filters, aliquoted, immediately frozen and maintained at –80 °c for at least 12 h before use. the cytotoxic activity was assessed by checking the cytolysis of human a positive erythrocytes. in order to eliminate damaged erythrocytes, the whole blood sample was washed at least three times in 9 vol. of sterile nacl 0.9 %, and the erythrocytes were then resuspended in sterile tbs (50 mm tris-hcl, 200 mm nacl, 10 mm cacl2, ph 8.5) at a final concentration of 2x109 cells/ml. 500 µl of filtered hemolymph were added 2 fig. 1 mussel cytotoxicity during 2005 in the cesenatico sea area. the red line represents the mean percentage. to 500 µl of erythrocyte suspension and incubated for 1h at 25 °c (hubert et al., 1997; malagoli and ottaviani, 2005). after incubation, samples were centrifuged at 3.000xg for 5 min at 4 °c, and the optical density (od) of supernatants was measured by evaluating the absorbance at 541 nm with a helios β spectrophotometer (spectronic unicam, cambidge, uk). it should be noted here that a threshold od level of 0,5 was fixed as the minimum for a positive test, while samples that clearly exceeded this value were considered positive without further measurement (malagoli and ottaviani, 2005). the experiment was repeated in duplicate two times for each animal. statistical analysis statistical analysis was performed using the χ2 test with p< 0.05 taken as significant. results and discussion the periodic evaluation of the cytotoxicity of hemolymph from the mussel m. galloprovincialis reveals variations in the percentage of cytotoxic animals during the year (fig. 1). the mean percentage of mussels positive to the cytotoxicity assay was around 53 %, but there were various peaks. it is interesting to note that the minimum and the maximum values were during the periods in which the water temperature reached its minimum and maximum levels, respectively, as indicated in the annual report on inshore water conditions of the italian region emilia-romagna in 2003. in a previous study, we observed that rapid changes in water temperature, salinity and ph resulted in a significant decrease in the mean cytotoxic activity. a rapid increase in water temperature resulted in a significant drop in the number of animals positive to the cytotoxicity assay (malagoli and ottaviani, 2005). conversely, the observations reported here seem to indicate a positive correlation between water temperature and the percentage of cytotoxic molluscs. however, no sudden fig. 2 comparison between mussel cytotoxicity immediately after being caught (a) or after a period of maintenance in the aquarium (b). the mean values of six independent experiments ± sd are shown. modifications in environmental parameters were seen in the present study. it should be underlined that the mean percentages of cytotoxic animals did not differ among mussels maintained in the laboratory aquarium for at least 10 days prior to control experiments (fig. 2). as far as the peak registered in september is concerned, it should be noted that the samples were collected after a period in which many animals had been died as a result of a loss of byssus. this pathology is still of uncertain origin, but a fungal infection has been observed to be involved in serious damage to the byssus apparatus (franchini et al., 2005). even if more detailed studies are needed to clarify the question, the large number of cytotoxic specimens in the survivor population could suggest a link between cytotoxic activity and the animal's ability to overcome epidemics. overall, cytotoxicity is confirmed as a dynamic parameter that can be used as an indicator of immune efficiency and, therefore, of the good state of health of the mussels. 0 10 20 30 40 50 60 70 80 90 100 jan feb mar apr may jun jul aug sep oct nov dec month % o f c y to to x ic a n im a ls % o f c y to to x ic a n im a ls month 0 10 20 30 40 50 60 70 80 90 100 % o f c y to to x ic a n im a ls a b % o f c y to to x ic a n im a ls 4 acknowledgement we are grateful to the centro di ricerche marine (cesenatico, fc, italy) for financial support for this study. the authors also wish to thank mr m marangoni who kindly provided the mussels and the centro trasfusionale policlinico (modena, italy) for providing the blood. references franceschi c, cossarizza a, ortolani c, monti d, ottaviani e. natural cytotoxicity in a freshwater polmonate mollusc: an unotorthodox comparative approach. adv. neuroimmunol. 1: 99–113, 1991. franchini a, malagoli d, ottaviani e. investigation of the loss of byssus in mytilus galloprovincialis from mussel farms in the adriatic sea. cell biol. int. 29: 857-860, 2005. hubert f, cooper el, roch ph. structure and differential target sensitivity of the stimulable cytotoxic complex from hemolymph of the mediterranean mussel mytilus galloprovincialis. biochim. biophys. acta 1361: 29–41, 1997. malagoli d, ottaviani e. cytotoxicity as a marker of mussel health status. j. mar. biol. ass. uk 85: 359-362, 2005. ratcliffe na, rowley af, fitzgerald sw, rhodes cp. invertebrate immunity: basic concepts and recent advances. int. rev. cytol. 97: 183–350, 1985. savary ca, lotzová e. phylogeny and ontogeny of nk cells. in: lotzová e, heberman rb (eds), immunobiology of natural killer cells, vol 1, fla: crc press, boca raton, pp 45–61, 1986. wittke m, renwrantz l. quantification of cytotoxic hemocytes of mytilus edulis using a cytotoxicity assay in agar. j. invertebr. pathol. 43: 248-253, 1984. 3 isj 6: 32-43, 2009 isj 6: 32-43, 2009 issn 1824-307x review signaling pathways in invertebrate immune and stress response r hatanaka, y sekine, t hayakawa, k takeda, h ichijo laboratory of cell signaling, graduate school of pharmaceutical sciences, strategic approach to drug discovery and development in pharmaceutical sciences, global center of excellence (gcoe) program, and core research for evolutional science and technology (crest), japan science and technology corporation, the university of tokyo, japan accepted march 4, 2009 abstract a wide variety of signaling pathways regulate immune and stress response in invertebrates and vertebrates. the fruit fly drosophila melanogaster and the nematode caenorhabditis elegans are extensively utilized model organisms for studies of such signaling pathways in invertebrates. intriguingly, major signaling pathways in immune response in drosophila and c. elegans, as represented by the toll and imd pathways and the dbl-1 and daf-2/daf-16 pathways, respectively, are different from each other. on the other hand, the mitogen-activated protein kinase (mapk) pathways function in common in these organisms not only in immune response but also in response to various abiotic stressors such as heat shock, ultraviolet (uv) irradiation, oxidative stress and osmotic shock. given that all of the above pathways are highly conserved and play diverse roles in vertebrates, particularly in mammals, drosophila and c. elegans are important invertebrate models that facilitate the elucidation of evolutionarily conserved mechanisms of immune and stress response. we therefore focus on signaling pathways that regulate immune and stress response in drosophila and c. elegans in this review. key words: innate immunity; stress; map kinase; toll; imd; daf-2 introduction to cope with pathogenic microorganisms, invertebrates rely solely on innate immunity because they do not have adaptive immunity. the well-characterized strategy against pathogens in invertebrate innate immunity is the induction of antimicrobial peptide (amp) genes, the regulatory mechanisms of which have been extensively explored using model organisms such as drosophila melanogaster and caenorhabditis elegans. in vertebrates, on the other hand, it has long been recognized that adaptive immunity is the main strategy used in the fight against various pathogens. however, a large body of recent evidence has demonstrated that innate immunity also plays a critical role in vertebrate immunity. therefore, in order to understand the highly complex immune systems in vertebrates, and in mammals in particular, it will be of great importance to elucidate the regulatory mechanisms of innate immunity in invertebrate models, ___________________________________________________________________________ corresponding author: hidenori ichijo laboratory of cell signaling graduate school of pharmaceutical sciences the university of tokyo, 7-3-1 hongo, bunkyo-ku tokyo 113-0033, japan e-mail: ichijo@mol.f.u-tokyo.ac.jp with a particular focus on signaling molecules that are conserved among species and involved in such mechanisms. invertebrate models also provide important information on the mechanisms that regulate the response to various abiotic stressors, such as heat shock, ultraviolet (uv) irradiation, oxidative stress and osmotic shock. the mitogen-activated protein kinase (mapk) pathways, especially those converging on two subgroups of stress-responsive mapks, jnk and p38, are the major players in a wide variety of response to these stressors (widmann et al., 1999; kyriakis and avruch, 2001). although these pathways are highly conserved from invertebrates to mammals and many physiological functions of these pathways have been revealed using mammalian models such as gene targeting mice, a great deal has also been revealed by studying these pathways as a primitive and efficient defense system in drosophila and c. elegans (stronach and perrimon, 1999; sakaguchi et al., 2004). in fact, the genetic evidence that the stress-responsive mapk pathways play pivotal roles not only in stress response but also in innate immunity was first revealed in analyses using these more rudimentary animals models (boutros et al., 2002; kim et al., 2002). 32 fig. 1 the toll pathway. cell wall components of gram-positive bacteria and fungi (lysine-type peptidoglycans and glucans, respectively) are recognized by pattern recognition proteins and activate protease cascades including such proteases as the serine protease grass. proteases secreted from fungi and gram-positive bacteria activate the serine protease persephone and its downstream protease cascade. these cascades finally activate spe, which cleaves pro-spätzle to form mature spätzle. upon the binding of spätzle to toll, dmmyd88, tube and pelle are recruited to toll. following the phosphorylation and degradation of cactus, dif and dorsal are released from cactus, translocate to the nucleus and induce amp genes such as drosomycin. in the first and second parts of this review, we focus on major signaling pathways in immune response in drosophila and c. elegans, as represented by the toll and imd pathways and the dbl-1 and daf-2/daf-16 pathways, respectively. in the last part we examine the functions of the stress-responsive mapk pathways as a common signaling system regulating immune and stress response in drosophila and c. elegans. signaling pathways in immune response in drosophila the toll and imd pathways have been extensively investigated as major signaling pathways in immune response in drosophila. these pathways are known to be functionally and molecularly conserved among other insects, such as the mosquitoes aedes aegypti and anopheles gambiae (christophides et al., 2002; waterhouse et al., 2007), the beetles tribolium castaneum (zou et al., 2007), the honey bees apis mellifera (evans et al., 2006) and the silkworms bombyx mori (tanaka et al., 2008), indicating that these pathways play central roles in immune response in insects. the toll pathway when flies are infected with gram-positive bacteria or fungi, amp gene expression is enhanced via the toll pathway in the fat body cells (lemaitre et al., 1996). the toll gene was first found to be required for the establishment of dorsal-ventral polarity in drosophila embryos (anderson et al., 1985). toll is a founder member of the highly conserved toll-like receptor (tlr) family, which is composed of type i transmembrane proteins with an ectodomain characterized by several repeats of leucine-rich motifs. although most members of this family recognize and directly bind to pathogen-associated molecular patterns (pamps) as host pattern recognition proteins, toll does not directly bind to pathogens or pamps but instead 33 binds to the mature form of the extracellular protein spätzle as an endogenous ligand (weber et al., 2003). upon infection with gram-positive bacteria, drosophila recognizes a bacterial cell wall component, lysine-type peptidoglycan (pgn), by means of pattern recognition proteins such as peptidoglycan-recognition protein (pgrp) short-form a (pgrp-sa), pgrp short-form d (pgrp-sd) and gram-negative binding protein 1 (gnbp1) in the hemolymph (werner et al., 2000; michel et al., 2001; gobert et al., 2003; bischoff et al., 2004) (fig. 1). results from genetic and biochemical analyses suggest that the ternary complex of all these proteins and the complex formed between pgrp-sa and gnbp1 differentially bind to pgns in a manner dependent on the bacterial strains from which pgns are purified (wang et al., 2008). these recognition proteins thereafter activate serine protease cascades, resulting in cleavage of pro-spätzle by the spätzle processing enzyme (spe) (jang et al., 2006). although the proteases that are engaged in spe activation have not been fully revealed, a recent study has clarified that the serine protease grass functions upstream of spe (el chamy et al., 2008). fungal infection also activates the toll pathway via protease cascades, and is dually detected by a pattern recognition protein gnbp3 and a serine protease persephone (psh) (gottar et al., 2006). fungal cell wall components such as β-(1,3)-glucans are recognized by gnbp3. proteases and chitinases secreted from fungi, which perforate the cuticle barrier and allow entry of fungi into the body cavity (clarkson and charnley, 1996), are sensed by psh. these recognition systems activate spe through protease cascades, leading to cleavage of pro-spätzle. recently, psh has also been shown to be required for sensing gram-positive bacterial proteases (el chamy et al., 2008). upon binding of spätzle to toll, dmmyd88, the drosophila ortholog of mammalian myd88, and the death domain proteins tube and pelle are recruited to the intracellular domain of toll, where they form a complex (horng and medzhitov, 2001; tauszig-delamasure et al., 2002). pelle is the drosophila ortholog of il-1 receptor-associated kinase (irak), while the counterpart of tube has not been found in mammals. this protein complex formation induces phosphorylation of cactus, the drosophila homolog of mammalian iκb (nicolas et al., 1998), although it has not yet been revealed whether the kinase pelle directly phosphorylates cactus. under unstimulated conditions, cactus retains and thus inhibits the nf-κb-like transcription factors dif and dorsal in the cytoplasm. once phosphorylated, cactus is degraded by the ubiquitin-proteasome system, and thereby dif and dorsal are released from cactus and translocate to the nucleus (ip et al., 1993), where they eventually induce the amp genes such as drosomycin (engström, 1999). the imd pathway another major immune control system in drosophila is the imd pathway (fig. 2). the imd gene was originally discovered in a survey of immune deficient mutant flies that exhibited lower viability than wild type flies with reduced expression of some amp genes in response to bacterial challenges (lemaitre et al., 1995). much as in the toll pathway, pgn recognition by pgrps is the initial step in the imd pathway. in this pathway, pgrp-lc recognizes diaminopimelic acid (dap)-type pgns, which are components of gram-negative bacteria and some gram-positive bacteria (choe et al., 2002; gottar et al., 2002; rämet et al., 2002). three alternatively spliced isoforms of pgrp-lc, lca, lcx and lcy, have been identified, and all of them are putative type ii transmembrane proteins with common n-terminal cytoplasmic and transmembrane domains but different extracellular domains (werner et al., 2003; kaneko et al., 2004). among these isoforms, pgrp-lca and pgrp-lcx have been shown to form the lca-lcx heterodimer and lcx-lcx homodimer, which function in the signal transduction induced by monomeric and polymeric pgns, respectively (mellroth et al., 2005). recent analyses have revealed that pgrp-le is another player in the recognition of dap-type pgns and functions synergistically with pgrp-lc (takehana et al., 2004; kaneko et al., 2006). consistent with the fact that pgrp-le possesses no predicted transmembrane domain or signal sequence, pgrp-le is detected in the cytosol and functions as an intracellular receptor for the monomeric dap-type pgn tracheal cytotoxin (tct). on the other hand, a version of pgrp-le (pgrp-lepg) containing only the pgrp domain that is conserved among all pgrps is found outside the cell and enhances pgrp-lc-mediated pgn recognition on the cell surface. these findings suggest that drosophila also employs conserved mechanisms for pamps recognition using both extracellular and intracellular receptors as in mammals (sansonetti, 2006). after the recognition of pgns, pgrp-lc and pgrp-le activate intracellular signaling. imd, a protein with a death domain similar to that of mammalian receptor interacting protein (rip), appears to be most proximal to the pgrps, since imd has been shown to physically interact with pgrp-lc and to a lesser extent with pgrp-le (georgel et al., 2001; choe et al., 2005; kaneko et al., 2006). although it remains elusive whether these interactions are indeed required for downstream signaling, imd subsequently binds to the drosophila fas-associated death domain-containing protein (dfadd). the imd-dfadd complex further recruits dredd, the drosophila ortholog of mammalian caspase-8, and elicits caspase activity of dredd (hu and yang, 2000; leulier et al., 2000; leulier et al., 2002; naitza et al., 2002). downstream of the formation of this active protein complex, drosophila tgf-β-activated kinase 1 (dtak1) is activated by a mechanism not fully understood but involving the drosophila homologs of human ubiquitin-conjugating enzymes ubc13 and uev1a (zhou et al., 2005). dtak1 then activates the drosophila iκb kinase (dmikk) complex (lu et al., 2001; vidal et al., 2001; silverman et al., 2003), which in turn conceivably directly phosphorylates the nf-κb-like transcription factor relish (silverman et al., 2000). relish possesses a dna-binding rel homology domain but 34 fig. 2 the imd pathway. upon the recognition of dap-type peptidoglycans of gram-negative bacteria by pattern recognition proteins, imd binds to dfadd and dredd, and the caspase activity of dredd is induced. dtak1 is also activated, and activated tak1 in turn activates the dmikk complex. subsequent dmikk-induced phosphorylation and dredd-mediated cleavage of relish generate the mature form of relish as a transcription factor, which translocates to the nucleus and induces amp genes such as diptericin. it is masked by an ankyrin repeats-containing iκb-like domain within the same molecule. phosphorylation of relish triggers endoproteolytic cleavage of the linker region between these two domains of relish, probably via the activation of dredd (stoven et al., 2003). finally, relish translocates to the nucleus and induces amp genes such as diptericin (cornwell and kirkpatrick, 2001). signaling pathways in the immune response in c. elegans whereas drosophila toll is a major player in immune response, as described above, the sole c. elegans toll homolog tol-1 has been assumed not to function in immune response. indeed, tol-1 deletion mutants have been reported not to exhibit altered sensitivity to various pathogens (pujol et al., 2001). recently, however, a possible role of tol-1 in combating bacteria has been proposed, because tol-1 mutants were shown to be sensitive to salmonella enterica and escherichia coli, and tol-1 was needed to prevent s. enterica from invading into the pharynx (tenor and aballay, 2008). however, the downstream signaling remains unknown mainly due to a lack of functionally conserved nf-κb-like factors in c. elegans. in this section, therefore, we focus on the dbl-1 and daf-2/daf-16 pathways as the major immune control systems in c. elegans. although these pathways are highly conserved from invertebrates to vertebrates, their direct immune functions have not been extensively investigated in other invertebrates than c. elegans, except for anopheles mosquitoes, in which these pathways have been shown to be critically involved in innate immunity to malaria parasite infection (lieber and luckhart, 2004; luckhart and riehle, 2007). the dbl-1 pathway in drosophila, induction of the amp genes upon infection is the most prominent mechanism of defense against pathogens, as described above. until recently, however, it was not understood whether such inducible anti-pathogen defense systems are also employed in c. elegans. mallo et al. clearly addressed this issue (mallo et al., 2002). by using a high-density cdna microarray, they showed that infection of c. elegans by serratia marcescens induced an upregulation of the expression of many genes–including those encoding lectins and lysozymes, which are known to be involved in immune response in other organisms, thereby demonstrating 35 fig. 3 functional interaction between the daf-2/daf-16 and jnk pathways. the daf-2/daf16 pathway starts with the binding of insulin-like peptides, which triggers activation of the pi3 kinase-like complex formed between age-1 and aap-1. age-1/aap-1 then catalyzes the conversion of pi-4,5-p2 (pip2) to pi-3,4,5-p3 (pip3). pdk-1 activated by pip3 phosphorylates and thus activates the akt-1–akt-2–sgk-1 kinase complex. finally, this complex phosphorylates and retains daf-16 in the cytoplasm. in contrast, jnk-1 promotes the translocation of daf-16 into the nucleus in response to environmental stressors, leading to the expression of numerous target genes to prevent damage to the cell. for the first time the existence of inducible antibacterial defenses in c. elegans. these amp genes in c. elegans have recently been shown to be preferentially expressed in the intestine, which is directly exposed to microbes (alper et al., 2007). intriguingly, some of the infection-inducible genes found in this study overlapped with genes under control of the dbl-1 signaling pathway (mochii et al., 1999). dbl-1 is a transforming growth factor (tgf)-β-related ligand that was first found in the context of body size regulation and male tail patterning (morita et al., 1999; suzuki et al., 1999), in accordance with the functions of the tgf-β-family members in body patterning in both invertebrates and vertebrates. consistent with these gene expression analyses, dbl-1 mutants have been shown to exhibit increased susceptibility to s. marcescens (mallo et al., 2002). recently, it has been demonstrated that neuronal expression of dbl-1 promotes expression of the caenacin family antimicrobial peptides in the epidermis probably in a paracrine manner after infection with the fungus drechmeria coniospora, suggesting the tissue-specific role of dbl-1 in immune response (zugasti and ewbank, 2009). although it remains to be clarified how the dbl-1 ligand is regulated in response to bacterial infection, it has been speculated that dbl-1 activates the following signaling pathway in its target cells (nicholas and hodgkin, 2004). in a manner analogous to the mammalian tgf-β receptor-smad system (kawabata and miyazono, 1999), sma-6 (type i receptor) and daf-4 (type ii receptor) form a heterodimer in response to dbl-1 stimulation, which induces phosphorylation of sma-2 and sma-3, the orthologs of the receptor-regulated smad proteins, mammalian smad1 and smad5, respectively. then sma-2 and sma-3 form a complex with sma-4, the ortholog of the mammalian common-mediator smad4, and translocate to the nucleus, leading to the expression of antimicrobial factors (savage et al., 1996; krishna et al., 1999). however, it has recently been shown that sma-3 alone, but neither sma-2 nor sma-4, is required for amp expression in response to d. coniospora infection, implying the existence of a non-canonical tgf-β signaling pathway in c. elegans (zugasti and ewbank, 2009). 36 fig. 4 the stress-responsive mapk pathways. the stress-responsive mapk pathways that converge on jnk and p38 in mammalian, drosophila and c. elegans are shown. each pathway consists of three classes of protein kinases: mapk, map2k and map3k. map3k phosphorylates and thereby activates map2k, and activated map2k in turn phosphorylates and activates mapk. the daf-2/daf-16 pathway daf-2 and daf-16 are c. elegans orthologs of the mammalian insulin/insulin-like growth factor (igf)-i receptor and the foxo family transcription factor, respectively (kimura et al., 1997; lin et al., 1997; ogg et al., 1997). the daf-2/daf-16 pathway has been investigated mainly from the viewpoint of metabolism, longevity, and dauer formation, which is an alternate larval stage that worms enter under unfavorable environmental conditions. the best-known phenotype of daf-2 mutants is the marked increase in life span, but they also exhibit resistance to pathogenic bacteria such as enterococcus faecalis, staphylococcus aureus, pseudomonas aeruginosa and bacillus subtilis (garsin et al., 2003). the daf-2/daf-16 pathway comprises signaling components highly homologous to those in well-characterized mammalian insulin/igf-i signaling. this pathway starts with the binding of insulin-like peptides to daf-2, which triggers the activation of age-1 and aap-1, the orthologs of the p110 catalytic and p55 regulatory subunits of mammalian pi3 kinase, respectively (morris et al., 1996; wolkow et al., 2002) (fig. 3). age-1/aap-1 then catalyzes the conversion of phosphatidylinositol-4,5-bisphosphate (pi-4,5-p2) to phosphatidyl-3,4,5-trisphosphate (pip3) (engelman et al., 2006). pip3 activates the 3-phosphoinositide-dependent kinase-1 homolog pdk-1 (paradis et al., 1999), which in turn phosphorylates and thus activates the akt-1–akt-2–sgk-1 kinase complex (hertweck et al., 2004). finally, this akt complex phosphorylates and retains daf-16 in the cytoplasm (paradis and ruvkun, 1998; lin et al., 2001). in the presence of daf-2 antagonists or perturbation of daf-2 signaling, daf-16 stays in the nucleus, which is a prerequisite for the induction of a wide variety of genes, including those regulating metabolism and cellular stress response (murphy et al., 2003). importantly, antimicrobial genes, such as the antibacterial lysozyme genes lys-7 and lys-8, which have been shown to be induced upon infection with s. marcescens (mallo et al., 2002), and the saposin-like gene spp-1, which has been shown to have antibacterial activity (bányai and patthy, 1998), are also the targets of daf-16. this finding suggests that insulin-like signaling in c. elegans counteracts the antibacterial activity through daf-16-dependent gene induction, and provides a reasonable explanation for the previous finding that daf-2 mutants exhibited increased resistance to pathogenic bacteria (garsin et al., 2003). consistent with these findings, p. aeruginosa was found to suppress daf-16-dependent immune response by activating the daf-2 pathway, probably through the induction of the insulin-like peptide ins-7 (evans et 37 al., 2008), further demonstrating the importance of the daf-2/daf-16 pathway in the immune response in c. elegans. in most literature, daf-16 regulation is mainly observed in the intestinal cells where it is feasible to detect nuclear translocation of daf-16, whereas regulation in other tissues remains to be elucidated. the mapk pathways the mapk pathways are signal transduction systems evolutionarily conserved in all eukaryotic organisms, and consist of three classes of protein kinases: mapk, mapk kinase (map2k) and map2k kinase (map3k) (widmann et al., 1999; kyriakis and avruch, 2001). map3k phosphorylates and thereby activates map2k, and activated map2k in turn phosphorylates and activates mapk. through these sequential biochemical events, the signals are amplified and diversified in a step-by-step manner (fig. 4). the stress-responsive mapk pathways that converge on the mapks, jnk and p38, are activated by various environmental stressors such as heat shock, uv irradiation, oxidative stress and osmotic shock. in response to these stressors, these pathways induce a wide variety of cellular responses such as dna repair, cell cycle arrest and apoptosis. these pathways are also activated in response to pathogen challenges and mediate immune responses. in this section, the roles of the stress-responsive mapk pathways in immune and stress response in drosophila and c. elegans are reviewed, although such roles have been assigned to highly conserved mapk pathways. the mapk pathways in immune response dtak1, which is the activator of the ikk complex in the drosophila imd pathway as mentioned above, is a member of the drosophila map3k family and activates the drosophila jnk (djnk) pathway (boutros et al., 2002; silverman et al., 2003). when lipopolysaccharides (lps), the principal cell wall components of gram-negative bacteria, are applied to adult flies and cultured cells, djnk is activated downstream of dtak1 and induces immune-related genes such as those encoding cytoskeletal regulators and pro-apoptotic signaling (sluss et al., 1996; boutros et al., 2002). however, it is still controversial whether the dtak1-djnk axis directly induces the amp genes (silverman et al., 2003; kallio et al., 2005; delaney et al., 2006). in addition to this transcriptional control, it has recently been proposed that the djnk pathway negatively controls the transcription of a group of genes mediated by the ikk-relish axis, another branch downstream of dtak1 (kim et al., 2005). conversely, the djnk pathway has been shown to be negatively regulated by relish-induced proteasomal degradation of dtak1 (park et al., 2004). this cross talk between two branches downstream of dtak1 appears to be critical to determine the duration, i.e., transient versus sustained, of gene induction in the immune response. thus, djnk is directly or indirectly involved in the control of a wide variety of genes, including the amp genes, and therefore is a critical regulator of immune response in drosophila. the drosophila p38 (dp38) pathway is also involved in immune response. among the three isoforms of dp38 identified so far-dp38a, dp38b and dp38c-dp38a and dp38b have mainly been examined in their role as mediators of immune and stress response in drosophila. treatment of drosophila cultured cells with the p38 inhibitor sb203580, which inhibits both dp38a and dp38b, has been shown to enhance lps-induced amp gene expression, while overexpression of dp38a reduced this expression in flies infected with bacteria (han et al., 1998b). however, the recent analysis of mutant flies deficient in dp38a has revealed that dp38a is not required for survival or for amp gene induction following infection with several bacteria (craig et al., 2004). nevertheless, the latter finding does not necessarily exclude the relevance of the dp38 pathway in immune response, because dp38b and/or dp38c may compensate for the deficiency of dp38a. consistent with this hypothesis, a possible immune function of dp38c was recently proposed. in response to bacterial septic injury, dp38c has been shown to mediate the induction of the dopa decarboxylase (ddc) gene. ddc catalyses the production of dopamine, which is metabolized to produce reactive quinones that kill invading microorganisms, and thus dp38c may upregulate quinones via the ddc gene (davis et al., 2008). in c. elegans, it remains unknown whether the jnk pathway is directly involved in innate immune response. on the other hand, the p38 ortholog pmk-1 has received much attention as a regulator in innate immunity. pmk-1 has been characterized as a mapk functioning downstream of the map2k sek-1 and the map3k nsy-1, orthologs of mammalian mkk3/mkk6 and ask1, respectively. nsy-1 and sek-1 were first identified as critical regulators of cell fate determination in a subset of olfactory neurons (sagasti et al., 2001; tanaka-hino et al., 2002). the nsy-1-sek-1-pmk-1 pathway was later found to play a crucial role in innate immune response (kim et al., 2002). loss-of-function mutation of or rnai against these genes clearly increases susceptibility to p. aeruginosa. however, mutation of unc-43, which encodes a ca2+/calmodulin-dependent protein kinase acting upstream of nsy-1 in cell fate determination in neurons (sagasti et al., 2001), did not result in enhanced susceptibility. instead, tir-1, the c. elegans ortholog of mammalian toll-interleukin 1 receptor (tir) domain protein sarm, appears to regulate the nsy-1-sek-1-pmk-1 pathway and to be required for survival and for the expression of amp genes following bacterial infection (couillault et al., 2004; liberati et al., 2004). moreover, tir-1, nsy-1 and sek-1 were found to physically interact with each other (tanaka-hino et al., 2002; chuang and bargmann, 2005). these findings suggest that tir-1 plays a pivotal role in innate immune response through activation of the nsy-1-sek-1-pmk-1 pathway. whereas the immune function of this pathway has been proposed to exert in the intestine (sakaguchi et al., 2004), this pathway has recently been shown to play a critical role in response to wounding as well as infection in the epidermis (pujol et al., 2008). 38 the mapk pathways in stress response apart from biotic stressors such as the bacterial components, various abiotic stressors also activate the jnk and p38 pathways. in drosophila, dp38 is activated by heat shock, uv irradiation, osmotic shock and oxidative stress in the cultured cells, and dp38a mutant flies are consistently susceptible to some of these stressors, indicating that dp38 confers stress resistance to flies (han et al., 1998a; han et al., 1998b; craig et al., 2004; zhuang et al., 2006). djnk is also activated by stressors similar to those that activate dp38 in the cultured cells (botella et al., 2001; chen et al., 2002; ryabinina et al., 2006). it has been shown using genetically engineered flies that djnk activity alleviates the toxic effects of reactive oxygen species (ros), probably through the induction of various categories of protective genes, thereby reducing the accumulation of oxidative damage and prolonging the lifespan of flies (wang et al., 2003). recently, it has been shown that djnk suppresses the insulin/igf-i signaling and activates dfoxo, the counterpart of c. elegans daf-16, in response to oxidative stress. djnk suppressed expression of insulin-like peptides in neuroendocrine cells in the brain, called the insulin-producing cells (ipcs), whereas djnk induced expression of target genes of dfoxo in the cells of peripheral tissues. this functional interaction between djnk and the insulin/igf-i signaling appears to contribute to the protective role of djnk against oxidative stress (wang et al., 2005). also in c. elegans, daf-16 is an important target molecule of jnk-1, an isoform among three jnk-like kinases in c. elegans (fig. 3). in response to environmental stressors such as heat shock, uv irradiation and excess ros, jnk-1 was found to directly interact with and phosphorylate daf-16 and promote the translocation of daf-16 into the nucleus, leading to the expression of numerous target genes to prevent damage from harmful stressors (oh et al., 2005; wolf et al., 2008). intriguingly, the jnk pathway acted in parallel with the daf-2 pathway to regulate lifespan, and both pathways converged on daf-16. moreover, in mammals, the nuclear translocation and transcriptional activation of foxo4, a member of the mammalian foxo family, in response to low levels of oxidative stress have been shown to be mediated through phosphorylation of foxo4 by jnk (essers et al., 2004). thus, this interaction between the jnk and insulin/igf-i pathways is functionally conserved among species and suggests that signaling pathways for stress response and lifespan are closely regulated by each other. heavy metals are well-studied stressors used in the analysis of stress response in c. elegans. mutants of the components of the jnk pathway have been shown to display elevated sensitivity to copper and cadmium, although the tissue(s) where the jnk pathway functions is unclear (koga et al., 2000; villanueva et al., 2001; mizuno et al., 2004). importantly, a daf-2 mutant and an age-1 mutant gained resistance to these metals, suggesting that the functional interaction between the jnk and daf-2 pathways is critical for the response to heavy metals as well (barsyte et al., 2001). perspectives and conclusions to take an overview of signaling pathways in invertebrate immune and stress response, we here focused on drosophila and c. elegans as suitable model organisms. however, we must note that a large body of evidence from other useful invertebrate models supports the important findings obtained using drosophila and c. elegans. as the present review has shown, these two organisms rely on different signaling pathways for their immune response, i.e., the toll and imd pathways versus the dbl-1 and daf-2/daf-16 pathways. this appears to depend at least in part on the existence or absence of the nf-κb-like factors and their regulatory systems. at the same time, the stress-responsive mapk pathways are utilized by both drosophila and c. elegans to regulate immune and stress response. taken together with the highly conserved functional interaction between the mapk and insulin/igf-i pathways, this suggests that the mapk pathways might play pivotal roles in integrating the 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2e7 2ismer-université du québec à rimouski, rimouski, 310 grande allée, rimouski, québec, canada, g5l 3a1 3environment canada, national water research institute, 867 lakeshore rd., po box 5050 burlington, ontario, canada, l7r 4a6 accepted october 27, 2009 abstract the purpose of this study was to examine the relationship between mitochondrial activity and gonad lipid stores in clams exposed to anthropogenic pollution at coldand warm-water sites. the balance between energy expenses and energy reserves was measured by mitochondrial electron transport (met) activity and lipid content in the gonad. the activity of malate dehydrogenase (mdh) was measured as an intermediary between energy production and the production of lipids in gonadal tissues. the results revealed that intertidal clam populations at warm-water sites under no source of pollution had less heavy metal content (ag, as, cr, hg and ni), lower mdh activity and temperaturedependent met than clams from cold-water sites. however, mdh activity measured at 6 oc was higher at the warm-water sites. lipid peroxidation in the gonad was higher in clams from the coldwater sites. the impacts of pollution differed among the study sites, clams from cold-water sites having increased mdh activity, temperature-dependent met activity, higher lipid content and dna strand breaks; clams from the warm-water sites had increased temperature-dependent mdh activity and lower gonadal lipid reserves. a multiple regression analysis revealed that gonad lipid reserves were positively correlated with mdh activity and negatively correlated with its temperature-dependent activity, suggesting that increased temperature sensitivity was negatively related to gonad energy reserves. the data show that pollution increases temperature sensitivity at the met level in clams in cold water, while temperature sensitivity in mdh activity was observed in clams from warm-water sites. discriminate function analysis revealed that pollution stress shows a tendency to be closer to clams adapted to warmer temperatures. in conclusion, pollution could increase mdh activity in cold-adapted clams which can lead to increased lipid stores in the gonad, oxidative stress and genotoxicity while pollution seems to increase the temperature dependence in met. in warm-adapted clams, temperature dependent mdh activity was higher by pollution with decreased lipid content in the gonad tissues which was independent of gonad maturation and size. key words: mitochondrial electron transport; malate dehydrogenase; gonad lipid; clam health introduction the st. lawrence river is subjected to various sources of chemical pollution that are historically linked to a variety of industrial (aluminum plants and pulp and paper mills), municipal effluents and agricultural runoffs from adjacent basins and harbors. clams are particularly at risk to these types of pollution since they are sessile, relatively long-lived (12-15 years for mya arenaria), and in close contact with sediments, which are well-known sinks for substances such as tributyltin (tbt) and industrial contaminants such as polycyclic aromatic hydrocarbons (pahs), polychlorinated biphenyls (pcbs) and heavy metals (gagnon et al., 2004; yang et al., 2006). the ecotoxicological impacts of pollution in this intertidal bivalve were extensively examined in the st. lawrence and one of its largest tributaries, the saguenay fjord (blaise et al., 2003; gagné et al., 2008a, b; pellerin et al., 2009). in these studies, it was revealed that the density of intertidal clam beds was closely related to immunocompetence (hemocyte concentration and phagocytic activity) and mitochondrial electron ___________________________________________________________________________ corresponding author: françois gagné fluvial ecosystem research section environment canada 105 mcgill st., montreal, quebec, canada h2y 2e7 e-mail:francois.gagne@ec.gc.ca 144 transport activity (met), a measure of respiration and energy expenditure. moreover, pollution increased clam temperature dependence or sensitivity to electron transport activity in mitochondria (gagné et al., 2008b). increased energy expenditure could occur at the cost of energy reserves in the form of lipids, sugars and proteins to a lesser extent (smolders et al., 2004). the balance between energy expenditure and reserves was introduced as the concept of cellular energy allocation, which proved a very predictive and sensitive endpoint of impacts at higher levels of biological effects such as survival, growth and reproduction in zebra mussels and daphnids (de coen and janssen, 2003). in a context of global warming and given the cumulative effects of environmental stressors, a recent study revealed that met activity was sensitive to temperature changes and pollution readily increased this temperature sensitivity (gagné et al., 2007). indeed, this study revealed that clams under pollution stress were more sensitive (i.e., spend more energy) to temperature increments than clams from lesscontaminated sites. to further investigate the interaction of pollution with thermal adaptation and stress, we undertook a spatial survey of intertidal clams at cold-water sites (st. lawrence estuary) and from areas of warmer water in the saguenay fjord. the balance between energy expenditure (met) and reserves in the gonad (gonad size and lipid content) was closely examined by tracking the transfer of precursors from the mitochondria to the cytoplasm to assist lipid synthesis. in cells, oxaloacetate is transferred in the cytosol to support lipid and amino acid synthesis through the malate shunt pathway, which involves nadh-dependent malate dehydrogenase (mdh). mdh catalyzes the reversible reduction of oxaloacetate to malate in the presence of the cofactor nadh in the mitochondria (stryer, 1995): oxaloacetate + nadh malate + nad+. malate diffuses out the mitochondria and is re-oxidized into oxaloacetate in the cytosol by a cytosolic mdh. during this process, nadh is formed which, in turn, can be used to support lipid synthesis. adaptation to cold or warm temperatures brings about changes in allelic mdh expression, selecting towards isoforms with various temperature dependence (fields et al., 2006). indeed, adaptation to colder temperatures was associated with lower enzyme affinity for nadh, but the activity was more sensitive to temperature. lower enzyme affinity for nadh will displace the reaction in favor of the oxidation of malate into oxaloacetate and nadh, hence is consistent with lipid production in organisms acclimated to colder temperatures. the production of oxaloacetate and malate will also depend on mitochondria respiration rate i.e. aerobic metabolism and the cytosolic activity of mdh. in another study, the affinity constant km for nadh was less sensitive to high temperatures in bivalve species adapted to warmer temperatures (l. scabra and l. gigantea) than in those adapted to colder temperatures such as l. scutum or l. pelta (dong and somero, 2009). this change in enzyme affinity was associated with a single amino acid substitution of glycine to serine at position 291 in cytosolic mdh, a change that favors hydrogen bonding and reduced conformational entropy, hence less affected to temperature variations. these studies suggest that warm/cold adaptation calls for changes in protein/enzyme kinetic activity and sensitivity to temperature changes. intertidal clams have to cope with air-temperature fluctuations during low tide which bring about shifts between anaerobic and aerobic metabolism and how clams handle these stresses in either coldor warm-water habitats is not fully understood. moreover, the interaction of pollution in these adaptation processes complicates the outcome of clam survival. there is a need to further study into how pollution stress and temperature interacts in feral clam populations. the purpose of this study was therefore to examine the impacts of pollution on the relationship between met, cytosolic mdh activity, lipid reserves in the gonad and the gonado-somatic index (gsi) of intertidal clam populations adapted to cold and warm water considering their relative distance from the shore (i.e., emersion time to air). moreover, toxic tissue damage was concurrently investigated by tracking changes in lipid peroxidation (lpo) and the formation of dna strand breaks. an attempt was made to relate the impacts of pollution on met and mdh activity, including their temperature-sensitive properties and gonad lipid reserves in clams obtained from coldand warm-water. materials and methods spatial survey and clam collection mya arenaria soft-shell clams were collected by hand on mud flats at morning low tide from two sites in the st. lawrence estuary and two sites in the saguenay fjord (fig. 1). sampling was made in later-half june of 2006 which corresponds to the pre-spawning period with gravid gametes and high gsi. clams from the former area thrive in the colder water of the st. lawrence (typically 3-6 oc compared to water temperatures of 5-12 oc at the fjord site) owing to the inputs of warmer river water draining nearby basins and adjacent tributaries (small streams and rivers, drainage of rainfall). both areas are influenced by tidal rhythms and they are interconnected and relatively close to each other; no significant ph and conductivity differences were observed for the surface waters at the site of collection. temperature measurements of the surface waters at the shore line were also determined during clam collection. among the st. lawrence estuary sites, baie sainte-catherine (bsc) is influenced by heavy traffic from sightseeing and whale-watching boats which is considered the cold temperature and polluted sites (ct+p); baiedu-moulin à baude (bau) was considered the coldtemperature reference site (ct) because of the absence of any direct source of pollution. as for the saguenay fjord sites, anse étienne site (ase), located on the south shore 20 km upstream of the estuary, is under no direct source of anthropogenic activity and was thus considered as the reference site for the fjord for the warm water temperature site (wt); the anse saint-jean (asj) site is exposed to the minimally treated (screening) urban wastewater of a population of about 2000 residents, located 40 145 fig. 1 clams were collected at four sites (identified by stars) in june 2006. the sites anse de saint-étienne (ase) and baie du moulin à baude (bau) were considered as the reference ct and wt sites respectively, while anse saint-jean (asj) and baie sainte-catherine (bsc) were the wt+p and ct+p sites respectively. clams from ase and asj sites were located in the warmer water temperatures of the saguenay river; clams from bau and bsc sites were located in the colder waters of the st. lawrence estuary. km upstream of the estuary and was considered the warm-temperature polluted site (wt-p). a total of 20 clams were collected at each site for the biomarker analyses. clam weights, soft tissue mass and shell length were determined on site and in the laboratory for gsi (gonad wet weight /soft tissues wet weight), condition factor (clam weight/shell length), and growth index (shell length/age ratio) determinations. age was determined by counting the number of major grooves on the shell. sex and gonad maturity were determined by microscopic analysis of gonad smears at 400x. parasitism was rarely observed but there were some occurrences of trematode infection; clams so infected were discarded. the clams retained for analysis were frozen under dry ice for transport to the laboratory and stored at -85 oc until the analyses. after thawing on ice, the visceral mass containing the gonad tissues were removed and homogenized with a teflon pestle tissue grinder apparatus in 25 mm hepes-naoh buffer, ph 7.4, containing 145 mm nacl, 10 µg/ml apoprotinin and 0.1 mm dithiothreitol at a 1:5 volume/volume ratio (five passes). samples (100 µl) of each homogenate were collected for total protein determinations (bradford, 1976), lipid peroxidation (lpo), total lipid content and dna damage. the remaining homogenate was centrifuged at 1,500xg (20 min at 2 oc) to remove cell debris and nuclei (pellet). the supernatant was centrifuged at 10,000xg (20 min at 2 oc) to isolate the mitochondria (pellet) for mitochondrial electron transport (met) activity, and the remaining supernatant was kept for the post-mitochrondrial malate deshydrogenase (mdh) activity evaluations. the mitochondria pellet was resuspended in 2 volumes of ice-cold homogenization buffer before measuring met activity during the same day. energy status cellular energy allocation was determined by measuring mitochondrial electron transport (met) activity and lipid content in gonad. met activity was determined in the mitochondrial fraction (10,000xg pellet) using the p-iodonitrotetrazolium dye method (king and packard, 1975; smolders et al., 2004). briefly, 100 µl of the supernatant was mixed with 100 mm tris-hcl, ph 8.5, containing 100 µm mgso4, 146 table 1 heavy metal content in m. arenaria clams and concordance analysis with the measured biomarkers metals bsc bau asj ase (µg/g) 0.58±0.25 0.14±0.02* 0.09±0.02 0.03±0.05 ag 0.91±0.05* 0.84±0.04 as 0.30±0.01 0.48±0.03 cd 0.06±0.01 0.04±0.02 0.09±0.01 0.11±0.01 7±1.4* 4±0.8 1.3 ±2* cr 4 ±0.9 1.3±0.26 1.5±0.03 cu 1.35±0.3 1.28±0.26 0.03±0.005* 0.03±0.003* hg 0.008±0.002 nd 4±0.8* ni 0.8±0.2 1.7±0.3 1.6±0.3 pb 0.04±0.003 0.06±0.1 0.09±0.02 0.065±0.01 0.12±0.05* sn 0.03±0.01 -- zn (x10) 1.1±0.2 1±0.2 1.7±0.1 1.3±0.3 sum 24.65 16.6 27.6 17.9 * indicates significance at p<0.05 0.1 % triton x-100 and 5 % polyvinylpyrrolidone for 1 min on ice. the reaction mixture was mixed with 1 mm and 0.2 mm nadh and nadph, respectively, on ice and then divided into two portions, one being equilibrated at 6 biomarkers of toxic effects toxic effects were determined by tracking lpo and dna strand breaks in gonad. lpo was determined in gonad homogenates using the thiobarbituric acid method (wills, 1987). standard solutions of tetramethoxypropane were used for calibration and fluorescence was measured at 520 nm excitation and 600 nm emission (chameleon-ii, multireader, bioscan, usa). given that the reagent could react with other aldehydes, results were expressed as µg of thiobarbituric acid reactants (tbars)/mg of homogenate protein. dna damage was determined by the alkaline precipitation assay developed by olive (1988), with fluorescence quantitation of dna strand breaks in the presence of detergents and an alkaline ph (bester et al., 1994). the assay principle is based on the potassiumdetergent (sds)-assisted precipitation of proteinlinked genomic dna, which leaves protein-free dna strand breaks in the supernatant. the number of dna strand breaks results from the dna repair of dna adducts and alkali-labile sites. the results were expressed as µg of dna/mg proteins. calibration was achieved with salmon sperm dna (sigma chemical company, usa). o oc and the other at 25 c. the reaction was initiated with the addition of 50 µl of 5 mm p-iodonitrotetrazolium for 30 min. absorbance readings were measured at 15-min intervals at 520 nm. the data were expressed as the loss of absorbance at 6 o oc and 25 c/30 min/mg total proteins in the corresponding supernatant. temperature-dependent met (mett) was determined and the rate difference in met at 25 oc and 4 oc was divided by the temperature change (25-6 o oc = 19 c) as determined previously (gagné et al., 2007). the data were expressed as the rate change (normalized against total proteins)/unit temperature in oc. gonad lipid levels were determined in gonad homogenates following the phosphovanillin method (frings et al., 1972). the detergent triton x-100 was used for calibration. the data were expressed as μg lipid equivalents/mg protein. malate dehydrogenase (mdh) activity in the mitochondrial fraction (s3) was determined by tracking the formation of nadh in the presence of 0.2 mm malate and nad+ o for 30 min at 20 c. the reaction buffer was 50 mm hepes-naoh, ph 7.4, containing 100 mm nacl and 0.1 mm edta. the appearance of nadh was measured by fluorescence (excitation 360 nm; emission 450 nm). temperature-dependent mdh was also determined as described with met data analysis a total of 20 clams were analyzed for each study site. the homogeneity of variances was determined using bartlett’s test. where the data proved heterogeneous, they were log-transformed. the data were subjected to a two-way anova, with gender and site of collection as the main factors. a discriminant analysis was performed to identify if the sites could be correctly classified using the biomarker t. the kinetic constants for mdh (km and vmax) were also determined at both temperatures using the classic lineweaver_burk plot method (stryer, 1995). 147 table 2 clam morphological characteristics clam weight/shell length growth index gonado-somatic index site age (shell length/age) saguenay fjord 10±0.31 0.49±0.022 6.04±0.25 0.12±0.01 ase (reference) asj 11.5±0.33 0.48±0.01 5.6±0.17 0.12±0.005 st. lawrence estuary b b b0.57±0.02 5.62±0.12 0.06±0.005bau (reference) 12±0.28 bsc 9.2±0.38 a,c a a,c a,c0.47±0.02 6.43±0.22 0.04±0.003 a significance at p<0.05 between the polluted site relative to the corresponding reference site b significant difference between the reference sites in coldand warm-water sectors c significant difference between polluted sites in coldand warm-water sectors test battery and which measurements contributed the most to site discrimination. correlation (pearson-moment) and multiple regression analyses were also performed. significance was set at p < 0.05 but marginal effects (0.1 < p < 0.05) were also provided. all statistical tests were performed using the statistica version 8 software package. results clams contamination and morphological characteristics the surface water ph, conductivity and temperature were measured in surface waters from the saguenay fjord and the st-lawrence estuary (fig. 1). clams collected at the st-lawrence area were exposed to colder surface water temperatures while those collected in the saguenay fjord were exposed to warmer surface water temperatures. indeed, the water temperature was significantly (p = 0.02) higher at the saguenay fjord (10 ± 4 oc) than the estuary (5 ± 1 oc) confirming the temperature differences between these two sectors. the ph and water conductivity did not significantly changed albeit a marginal (p = 0.07) increase in conductivity for the st-lawrence estuary sites. moreover, clams were analyzed for heavy metal content to confirm that they were indeed exposed to pollution stress (table 1). the data revealed that clams at the heavy-traffic harbor were more contaminated in arsenic (as), silver (ag), chromium (cr), mercury (hg), nickel (ni) and total tin (sn). clams exposed to urban wastewaters from a township of 2,000 inhabitants had the highest levels on ag with higher amounts of as, cr, and hg. these data corroborates previous findings (gagnon et al., 2004; yang et al., 2006) and confirms that the organisms were significantly more contaminated with some metals at least. the morphological characteristics of clams collected at two impacted sites, and their corresponding reference sites, were examined (table 2). globally, no significant changes in the sex ratio were observed throughout the study sites (kruskal-wallis rank anova, p = 0.51). the condition factor (clam weight/shell length) changed significantly according to site of collection. condition factor was significantly reduced at ct+p site compared to its corresponding reference ct site. the condition factor was significantly lower at wt than at ct reference sites. the growth index (shell length/age) significantly changed across the sites but gender was not significant. the growth index was significantly reduced at the ct+p sites. growth was significantly lower at wt than at ct sites. correlation analysis revealed that cf was significantly correlated with growth index (r = 0.26; p = 0.005) and shell length (r = 0.2; p = 0.03). gametogenic activity was determined by measuring the gsi. it was significantly affected by site location with no significant effects with clam’s gender. moreover, gsi increased significantly with clam age. the gsi was significantly reduced at site wt+p site and somewhat increased at site ct+p only when corrected for age differences. clams from the wt reference site had higher gsis than those from the ct reference site. a correlation analysis revealed that gsi was positively correlated with shell length (r = 0.19; p = 0.04), and age (r = 0.21; p = 0.03). energy status clam energy status in the gonad was determined by measuring temperature-dependent met at 25 and 6 o oc, mdh at 25 and 6 c), and gonad lipid content (figs 2a, b, c). met at 25 oc was significantly increased at ct+p site, with no change in mdh activity at the same temperature (fig. 2a). however, mdh activity at 25 oc was significantly higher at the wt reference site compared to the ct site. no significant differences in met activity at 25 oc were observed between the warmand cold-water sites. at colder temperatures (6 oc), there was no significant difference between the clean and polluted sites and both reference sites (fig. 2b). the temperature sensitivity of met activity (met ), but not mdh activity (mdh ), was readily t t 148 wt wt +p ct ct +p sites 0 1 2 3 4 5 6 7 m e t o r m d h a ct iv ity 25 o c met mdh a b a wt wt +p ct ct +p sites 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 m e t o r m d h a ct iv ity 6o c met mdh b wt wt+p ct ct+p sites 0,02 0,04 0,06 0,08 0,10 0,12 0,14 0,16 0,18 0,20 0,22 0,24 0,26 0,28 0,30 t em pe ra tu re -d ep en de nt m e t a nd m d h a ct iv ity d met/dt d mdh/dt c a b a wt wt+p ct ct+p site nu 2 4 6 8 10 12 14 16 18 li pi ds in g on ad (u g/ m g pr ot ei ns ) mean stand. error a ad fig. 2 temperature-dependent energy status in selected clam populations. the effects of clam length (size) and site location of m. arenaria clams are shown for met and mdh at 25 o oc (a), met and mdh at 6 c (b), temperature-dependent met and mdh (c) and gonad lipid reserves (d). met activity was expressed as the increase in dye reduction (520 nm)/min/mg proteins and mdh activity as the increase in nadh fluorescence/min/mg proteins. the letter ‘a’ indicates significance relative to each control sites (ase or bau) from the saguenay fjord and st. lawrence estuary. the letter ‘b’ denotes the significance between the cold-water and warm-water reference sites. increased at the ct+p site relative to the corresponding ct reference site (fig. 2c). mett but not mdht was significantly higher at the wt site compared to the ct site. lipid levels in gonad were significantly reduced at the wt+p site while they were increased at the ct+p site (fig. 2d). no significant change in lipid content was observed between the coldand warm-water reference sites (i.e., ct and wt). correlation analysis revealed that met at 25 oc was correlated with growth index (r = 0.3; p = 0.001), met at 6 oc (r = 0.59; p < 0.001), gonad lipids levels (r = -0.24; p = 0.011) and mett (r = 0.77; p < 0.001). met at 6 oc was significantly correlated with cf (r = -0.21; p = 0.02), growth index (r = -0.34; p < 0.001), mdh at 6 oc (r = -0.25; p = 0.01) and mett (r=0.53; p<0.001). mdh at 25 oc was significantly correlated with gsi (r=0.3; p=0.005) and mdht (r = 0.84; p < 0.001). mdh at 6 oc was significantly correlated with met at 6 o c (r = -0.25; p = 0.01), and mett (r = -0.40; p = 0.002). mdh was significantly correlated with gsi (r = 0.38; p = 0.003), cf (albeit marginally; r = 0.23; p = 0.08), gonad lipids (r = -0.7; p < 0.001) and mdh at 25 oc (r = 0.84; p < 0.001). because of the relationships between mdh activity and met (energy expenditure) and gonadal lipids (energy reserves), the kinetic properties of this enzyme complex were characterized at cold and warm temperatures (table 3). the enzyme has a higher affinity for nad+ than for malate, regardless of the water quality and thermal history of the site. the enzyme affinity at 25 oc for malate remains the same for both reference sites (wt and ct), but affinity increases in clams taken from the ct+p site. no changes were observed for nad+ when determined at 25 oc. however, at 6 oc, the enzyme affinity for both malate and nad+ increased at the ct site, whereas this was not found in clams at the ct+p site. temperature had more influence on the maximum rate of reaction (vmax) for malate and nad+. at cold temperatures (6 oc), vmax was increased at the ct+p site but this was lost at t 149 table 3 mdh* kinetic characteristics in m. arenaria clams enzyme turnover 25 enzyme turnover 6 temperature temperature vmax km vmax km sensitivity for enzyme affinity** sensitivity for reaction rate** substrate 6oc 6o o oc 25 c 25 c o oc c (vmax/km) (vmax/km) wt malate 9.7 0.2 28 0.6 0.02 0.02 18 0.4 0.19 0.11 1.3 0.4 nad 1.7 0.2 3 0.6 wt+p malate 0.02 0.07 17 0.06 7.8 0.52 25 0.6 0.6 0.12 0.5 0.2 nad 2.4 0.3 2.9 0.4 ct malate 5.4 0.2 27 0.5 0.02 0.03 22 0.3 0.58 1 1.1 0.6 nad 0.1 0.1 1.2 0.7 ct+p malate 8.2 0.5 12 0.4 0.03 0.06 4 -0.1 0.34 0.11 -1.1 0,2 nad 2.6 0.3 1.5 0.5 +* mdh activity: malate + nad oxaloacetate + nadh ** km or vmax at 25 oc km or vmax at 6 oc ohigher temperatures (25 c). the turnover number (vmax/km) at 6 oc was readily increased in clams from the ct+p site. a concordance analysis revealed that lipid gonad levels were significantly correlated with km and vmax, turnover number for nad+ at 6 oc (p < 0.001), km for nad + at 25 oc (p = 0.02), km and vmax, turnover number for malate at 6 oc (p < 0.001) and the turnover number at 25 oc (p < 0.001). the gsi was significantly correlated with km at 6 oc and 25 oc, vmax at 25 oc and temperature-dependent km and vmax for malate. for nad+, the gsi was significantly correlated with km and vmax at 25 oc (p = 0.02), turnover number at 6 oc (p = 0.02), temperature-dependent km and vmax (p = 0.02). it appears therefore that gonad lipids levels are more dependent on the respective activities at 6 oc (which is a function of clam immersion in cold water), while gsi is related more to temperature dependence, which is favored at the warm-water sites (and exposed to surface air temperature). the temperature-dependent mdh activity and mdh activity were correlated (as expected) with temperature-dependent vmax and vmax at 25 oc, respectively. tissue damage was investigated by measuring the level of dna strand breaks and lpo in gonad (figs 3a, b). lpo tended to be higher at the polluted sites but the increase was only significant for wt+p site (anova p < 0.01, least squares difference test). lpo at the ct site was significantly higher than the reference wt site in the saguenay fjord. the levels of dna strand breaks in the gonad were significantly higher at site wt+p site, reaching 1.9-fold the levels of the reference wt site. in an attempt to determine the interaction between pollution and energy production and transfers to lipid reserves and gonadal mass in gonad tissues, a discriminate function analysis was performed (fig. 4). the analysis revealed that sites were correctly classified in increasing order: ct+p site (68 %), wt site (68 %), wt+p site (70 %) and ct site (94 %). the following biomarkers were significantly correlated with the root 1 function of the x-axis, in descending order: gsi, gonad lipid, mdht and lpo in gonad. for the second function of the yaxis, the following biomarkers were significantly correlated, in descending order: mett, gonad lipid, condition factor and mdht. this indicates that temperature sensitivities of mdh and met activity are key variables of site discrimination. at the colder sites (ct and ct+p), the effects of pollution displaced the data towards the warmer site (ase). the effects of pollution at the warmer sites (ase, asj) displaced the data away from the colder sites, indicating that pollution has “warming” effects but not the reverse (i.e., no cooling effects). discussion increased met activity leads to increased activity in the malate shunt pathway, where malate is transferred to the cytosol and converted back to oxaloacetate by a cytosolic mdh. the process forms nadh, which can be used to support lipid synthesis. indeed, a multiple regression analysis revealed that lipid gonad levels were positively related with mdh activity but temperature dependence was negatively correlated with gonad lipid levels. in fact, lower-temperature dependence (from cold-adapted organisms) of mdh, but with increased temperature sensitivity for met, was related to increased lipid stores in gonad tissues. this suggests that increased mitochondrial energy production with a more temperature invariant mdh could favor the accumulation of lipids in gonad tissues. in turn, lipid synthesis calls for temperatureindependent mechanisms (hence, the production of lipid precursors is maintained at cold temperatures). mdh activity was shown to be less sensitive to high temperature changes in cold-adapted bivalves (fields 150 wt wt+p ct ct+p sites 0 5 10 15 20 25 30 35 40 li pi d pe ro xi da tio n (u g t b a r s /m g pr ot ei ns ) mean stand. error a b a 1000 1500 2000 2500 3000 3500 4000 4500 5000 d n a s tra nd b re ak s (u g d n a /m g pr ot ei ns ) mean stand. error b a wt wt+p ct ct+p sites fig. 3 tissue damage in m. arenaria clams collected at polluted sites. the levels of lpo (a) and dna damage (b) were measured in the gonad. the letter ‘a’ indicates significance at each reference site (ase or bau). et al., 2006). the present study corroborated the pollution-induced increase in temperature sensitivity at the warmer site (wt+p) than at the colder site (ct+p). increased sensitivity to temperature for mdh was associated with lower gonad lipids. it is noteworthy that these clams were fully ripped with no clear indication that spawning occurred. at the polluted cold-water site (ct+p), met activity was more sensitive to temperature than mdh activity. the relative distance from the shore differed from the various study sites indicating that the clams were exposed to different emersion times. however, the relative distance from the shore was not significantly related with neither mdh activity (25 in this study, increased met activity and lipid stores in gonad were observed at the ct+p site. in intertidal clams subject to large variations in temperature (i.e., eurythermal), oxygen radical formation was positively related with temperaturecontrolled respiration rates (state 3 and 4) and negatively correlated with mitochondrial coupling (abele et al., 2002). thus, wide temperature variations in clams from cold-adapted sites could lead to the formation of reactive oxygen species and lpo. however, no significant correlations were obtained with lpo in gonads and met activity at low and high temperatures, suggesting that antioxidant mechanisms such as superoxide dismutase, glutathione peroxidase and catalase were able to scavenge the oxygen radicals resulting from mitochondrial uncoupling. mitochondrial uncoupling from an increased temperature could be oc) nor with mdht. however, the influence from the distance from the shore should be considered since it was shown to have an important influence on oxidative stress in clams (gagné et al., 2009). 151 -4 -3 -2 -1 0 1 2 3 4 root 1 (gsi> lipid>mdht>lpo) -5 -4 -3 -2 -1 0 1 2 3 4 5 6 r oo t 2 (m e t t >l ip id e> c on d fa ct or >m d h t ) wt: 68 % ct+p: 62 % ct: 94 % wt+p: 70 % ct ct+p wt wt+p fig. 4 discriminate function analysis of clam morphological status, energy status and tissue damage. a discriminate function analysis revealed that the sites were correctly assigned at > 62 %. the site names shown in bold correspond to the centre of gravity of the discriminate function. the dotted arrows indicate departures from the reference sites to the corresponding polluted sites (within the cold-water or polluted-water sites. the result of changes in protein phosphorylation states in mitochondria of the clam mercenaria mercenaria (ulrich and marsh, 2009). the investigators identified three proteins that followed temperature-specific phosphorylation patterns and suggested that a suite of protein kinases and phosphatases regulate mitochondrial physiology in response to temperature increases. changes in protein phosphorylation in mitochondria show promise for use in investigating the interaction of temperature with pollution in the production of reactive oxygen species and oxidative stress. the data in the present study show that pollution could contribute to temperature sensitivity in mdh activity, which is related to lower lipid content in the gonad and dna strand breaks. this was consistent with discriminate function analysis of the biomarker data, which showed a shift in the centre of gravity of the discriminate functions of the polluted cold-water site toward the warmer sites (ase and asj). it is noteworthy that the principal biomarkers with the highest factorial weights were temperature sensitivity in mdh and met activities, gonad weight and lipid content. the physiological equivalency between low temperature dependence and cold adaptation observed here could perhaps favor the formation of dna strand breaks in the gonad, rendering the organisms more susceptible to genotoxic compounds. this is consistent with the observation that dna strand breaks were much higher at the polluted site in the cold-water sector (ct+p). however, this is difficult to ascertain at present since the genotoxicity could have been the result of relatively more contamination occurring at site ct+p relative to wt+p. in conclusion, clams from cold-water sites display less sensitivity to temperature but pollution increases temperature sensitivity in cytosolic mdh activity. temperaturedependent met at the polluted and cold-water site rises to levels identical to those of clams from the warmer sites, indicating that pollution alleviates cold-adaptation processes. although clams had lower gsis at the cold-water and polluted site, gonad lipid levels were higher at the polluted site than at the corresponding cold-water reference site indicating altered energy allocation towards gamete development. the impacts of pollution in clams from cold water site increased mdh activity at 25 oc and met temperature-dependence which would favor the formation of nadh in the cytosol for the production of lipids and steroidogenesis. the increase of mdh activity was related to a 2fold increase of the enzyme’s affinity constant towards malate at 25 oc while the affinity at 6 oc was somewhat lower. these changes were consistent with increased lipids stores at the ct-p site which were related with oxidative stress and genotoxicity. the impacts of pollution in clams from warm water sites seems to intervene rather at the mitochondria level (increased met activity) which was related to decreased lipid stores in the gonad and this was independent from changes in gsi or 152 gonad maturity in the clams. however, temperature-dependent mdh activity was higher at the polluted site. acknowledgements the authors are grateful for the technical assistance of s trépanier, b walker, s trottier and c boyko of the aquatic ecosystem protection research division, environment canada and to pascal rioux from uqar-ismer. this project was funded by environment canada under the st. lawrence action plan and by the nserc discovery grant attributed to j pellerin. references abele d, heise k, pörtner ho, puntarulo s. temperature-dependence of mitochondrial function and production of reactive oxygen species in the intertidal mud clam mya arenaria. j. exp. biol. 205: 1831-1841, 2002. bester mj, potgieter hc, vermaak wjh. cholate and ph reduce interference by sds in the determination of dna with hoescht. 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(ny) 11: 608618, 2009. wills ed. evaluation of lipid peroxidation in lipids and biological membranes. in: snell k, mullock b (eds), biochemical toxicology: a practical approach. irl press, washington, usa, pp 127-150, 1987. yang r, zhou q, jiang g. butyltin accumulation in the marine clam mya arenaria: an evaluation of its suitability for monitoring butyltin pollution. chemosphere 63: 1-8, 2006. 153 http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22abele%20d%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22heise%20k%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22p%c3%b6rtner%20ho%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22puntarulo%20s%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22dong%20y%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22somero%20gn%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22rudomin%20el%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22somero%20gn%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/pubmed/19698974?ordinalpos=1&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/19698974?ordinalpos=1&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/19698974?ordinalpos=1&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22gagnon%20f%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22tremblay%20t%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22rouette%20j%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22ulrich%20pn%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22marsh%20ag%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus javascript:al_get(this,%20'jour',%20'mar%20biotechnol%20(ny).'); arasite-host relationship: a lesson from a professional killer 41 isj 2: 41-53, 2005 issn 1824-307x review parasite-host relationship: a lesson from a professional killer mf brivio, m mastore, m pagani  dept of structural and functional biology (dbsf), university of insubria, varese, italy accepted april 18, 2005 abstract this paper outlines some of the main features of parasites immunoevasion/depression strategies. insects humoral and cellular responses are briefly discussed and correlated to the active and passive strategies of insects parasites, with particular emphasis on nematocomplexes used as biological insecticides. we have reviewed data on the interaction, at immunological level, of the parasite steinernema feltiae (rhabditidae) with the host model galleria mellonella (lepidoptera, pyralidae); the putative role of the parasite body-surface in active and passive evasion mechanisms has been evaluated and discussed. key words: insect; immunity; parasite; prrs-pamps; immunodepression; immunoevasion introduction invertebrates, particularly insects, act as vectors of important diseases such as malaria, chagas’ disease, sleeping sickness, filariases, dengue fever, yellow fever, etc. moreover many insect species, usually named insect pests, have a strong impact on the environment since they are phytophagous and harmful for both crops and urban areas. as a consequence of the widespread diffusion of insect species (reflecting the great success of this group) occupying almost all the habitats on earth, many insects live in environmental conditions infested by parasites and pathogens. insects can survive mainly because of the extreme efficacy of their immune system; any foreign parasite must then counteract these defenses to survive into its host. many parasites reproduce, develop and survive in invertebrate hosts that possess an immune system devoted to self-integrity and to discriminate self from not self. invertebrates lack finely tuned immunorecognition receptors but they possess instead useful patternrecognition molecules (prrs); these factors are able to interact specifically with a broad range of foreign corresponding author: maurizio f brivio dbsf, university of insubria, via jh dunant, 21100 varese, italy e-mail: maurizio.brivio@uninsubria.it  in memory of the late m pagani antigenic surface compounds (commonly named pamps and defined as pathogen-associated molecular patterns). pamps-prrs interaction is a key process among the discriminatory steps of innate immunity that usually precede the effectors-based mechanisms responsible for the elimination of not self (medzhitov, 2001; 2002; kanost et al., 2004 ). pamps consist of various compounds, including oligosaccharides, proteins, glycoproteins, lipids and distinct nucleic acid motifs that are unique to, and essential for, microorganism survival. an important feature of pamps is their strongly conserved structures, which are invariant among organisms of a given class (janeway and medzhitov, 2002; medzhitov and janeway, 2002). in insects, infections with different microorganisms or parasites selectively activate various defense reactions and effector-coding genes. the molecular basis of discrimination between different types of not self and the activation of immune responses is attributed to the specificity of prrs toward pamps, such as lps (lipopolysaccharide), pglc (peptidoglycan) or various glucans (medzhitov and janeway, 2002; dimopoulos, 2003). several proteins both in insect hemolymph or on hemocytes plasma membrane seem to function as prr, as they perform surveillance by binding to molecular patterns (hoffmann et al., 1999; hoffmann, 2003) (fig. 1). in galleria mellonella naïve larvae, two lpsbinding proteins, named lbp-1 (17.2 kd) and lbp-2 (26 kd), have been described by dunphy and halwani (1997); these humoral factors can be considered as prrs. these receptors bind the surface of bacteria and seem to act as detoxifiers, thus protecting 42 fig. 1 foreign stimula and receptors of insect immune system. perceiving of not-self in invertebrate seems to be modulated by the presence of pamps (pathogen-associated molecular patterns) which interact with free or membrane-bound receptors called prrs (pattern-recognizing receptors). these interactions lead to the activation of different cell-mediated and humoral effector immune processes. hemocytes from damage. both the lbps are specific for the lipid a portion of lps, in addition lbp-1 seem to act as an activator of galleria pro-phenoloxidase (propo) system. in the same year, wiesner and colleagues (1997) isolated and described a similar protein, called apolipophorin-iii (apolp-iii) of 17 kd of molecular mass; apolp-iii has been identified as an immune-stimulating molecule and is an exchangeable apolipoprotein, abundant in lepidopteran insects. the immune-stimulating capacity of apolp-iii came as a surprise, since this protein had been previously known to play a main role as lipid carrier in flying insects (niere et al., 1999). on insects hemocytes, cell receptors responsible of toll and imd pathways activation can be considered as the main prrs involved in cellular pamps sensing leading to antimicrobial peptides (amps) synthesis (de gregorio et al, 2002); their importance is not only restricted to the process of amps genes activation but, evolutionary, they also represent a further confirmation of the ancient origin of innate immunity, since they have been identified in several taxa from invertebrates to vertebrates (hoffmann and reichhart, 2002). after detection of not-self by pamps-prrs interactions, the recognition machinery can stimulate defensive humoral and cellular responses: among them, an important humoral defensive process is the melanization (humoral encapsulation) of foreign bodies. the melanization reaction, which is a common response to not self entry in invertebrates, especially arthropods, is due to the activity of an oxidoreductase called po. this enzyme, in the hemolymph, is a component of a complex system of proteases, proteases inhibitors (serpins) and humoral prrs, constituting the so-called propo-activating system (propo-as). propo-as is proposed to be a not self recognition system, because conversion of propo to the enzymatically active form can be induced by foreign pamps, particularly lipopolysaccharides and -1,3-glucans. propo-as, which is physiologically activated by invading micro-organisms or parasites, is a complex enzyme cascade in which the last active enzyme (phenoloxidase) can oxidize phenols into quinones, that in turn will convert into melanin autocatalytically (nappi et al., 2004). this system is a key element in the recognition of foreign bodies and in the production of opsonic factors; moreover, it is now considered to represent an integral component of the insect immune system (ashida, 1990; brivio et al., 1996; söderhall and cerenius, 1998; dimopoulous et al., 2001; cerenius and söderhall, 2004). several hemolymph prrs are involved in the propo system activation pathway: among them, -glucans-binding proteins (-gbp) and lps-binding proteins (lbps) seem to play a key role as receptors, triggering protease cascades that turn on prophenoloxidase enzymatic activity (jomori and natori, 1992; söderhall, 1999). besides, cell-mediated defenses are performed by cellular elements represented by several types of hemocytes that are commonly identified using morphological, histochemical and functional features (gupta, 1985, brehelin and zachary, 1986). hemocytes are immunoreactive cells playing a central role in maintaining host integrity; they are involved in various defense mechanisms such as phagocytosis, nodule formation, encapsulation, melanization, and synthesis of antimicrobial peptides (bulet et al., 1999; kanost et al., 2004). both cellular and humoral factors seem to be involved in the stimulation of cellular defenses, specifically in the early recognition and binding to pamps. several researches have demonstrated that humoral recognition receptors are also needed to stimulate hemocytes aggregation on the target surface during encapsulation processes (bulet et al., 1999, schmidt et al., 2001). the complex relationships between hosts and parasites can be clarified only considering the host 43 defense mechanisms and parasites evasion stategies altogether; the purpose of this paper is to outline some of the main parasite evasion strategies; in particular, the immunological interaction between entomopathogenic nematodes (steinernema feltiae) and the lepidopteran model insect g. mellonella will be described. the chosen examples are focused on insect hosts because of their economical and medical importance and considering their susceptibility to the symbiontic complex steinernema-xenorhabdus commercially available as biological insecticide. the convergence of parasitological and immunological studies also provides valuable knowledge in understanding the evolution of both parasitism and host immune system. parasite evasion strategies: general considerations parasites may successfully colonize their hosts by evading recognition, thus preventing immune defenses; circumvention of the host immune system can be achieved by molecular mimicry (or disguise) strategies or by colonization of young hosts, or host tissues, with low immunocompetence. alternatively (or concurrently) many parasites are able to depress either cell-mediated or humoral effectors mechanisms in a process usually called interference (lie and heyneman, 1976). as pointed out by götz and boman (1985), in order to survive, a parasite must reach an equilibrium with its host; a too efficient parasite may exterminate its hosts, whereas a too permissive parasite could have a low fitness and reproduction efficiency too low to guarantee its survival. in many cases, evolution and selection have finely-tuned host-parasite relationships leading to a long survival of parasitized invertebrate hosts; this process resulted in the production of vector species responsible of the transmission to human and animals of important diseases (richman and kafatos, 1995; ratcliffe and whitten, 2004). molecular mimicry is a strategy by which parasites became antigenically closely related to the host and thus avoid to evoke host immune responses. true molecular mimicry can be defined as the endogenous production of mimicking molecules that are usually exposed on the body-surface (or cell surface) of the parasite. despite the intuitive appeal of molecular mimicry as a mechanism of avoidance, few studies with any invertebrate parasites demonstrate a functionally protective effect of shared antigens (bayne et al., 1987; weston and kemp, 1993). however, antigens such as: -macroglobulin, immunoglobulin receptors, tropomyosin, mhc i and ii antigens, blood group glycolipids and oligosaccharides, related to both hosts and parasites have been identified and characterized (smithers et al., 1969; damian, 1991; vellupilai and harn, 1994). the molecular disguise, another form of mimicry, is described as the acquisition (sequestering) of molecular components from the host (ratcliffe et al., 1985; loker, 1994; strand and pech, 1995). in biomphalaria glabrata, several studies have demonstrated the ability of schistosoma mansoni to acquire host plasma proteins (e.g. hemoglobin, hemagglutinins, etc.) to form a coat of host factors (yoshino and bayne, 1983; dunn and yoshino, 1991; johnston and yoshino, 1996). more recently, kathirithamby and co-workers (2003) have described an alternative disguise mechanism carried out by a strepsiptera (stichotrema dallatorreanum) that is able to manipulate host (segestidea novaeguineae and s. d. defoliaria, orthoptera) epidermal tissues and wraps itself within it. this sort of bag acts thus as a camouflage for the endoparasite which is recognized as self by the host. the stage of development of the host is also essential in determining the outcome of parasitization (khafagi and hegazi, 2004). in general, early instars larvae of insects show a reduced immune activity often due to a lower hemocytes number or to a different array of cell populations (gardiner and strand, 2000; beetz et al., 2004). due to this, parasites penetrating young hosts can find a more favorable environment to overcome host defenses. finally, as a passive strategy, some parasites are able to colonize low-reactivity tissues of the host. a good example is represented by insect parasitoids that lay their eggs, with surgical precision, in nerve ganglia of their hosts into which the hemocytes do not normally circulate, so parasite embryos can develop unmolested within the host (götz and poinar, 1968; salt, 1971). as mentioned above, alternative active strategies are referred as interference; in this case, parasites show an aggressive suppression or alteration of the host immune system defenses. interference can be directed toward host humoral factors that are neutralized by the parasite or, as more commonly proposed, immunocompetent cells could be targeted instead (loker, 1994). another important aspect of host-parasite relationships is host humoral depression. with respect to this, propo system is one of the main target for many parasites or microorganisms; this is probably due to the need to neutralize its drastic and rapid effect when a host is in the presence of not self infections. many parasitic wasps inject maternal factors into the host’s hemocoel to suppress the host immune system and to ensure successful development of their progeny; asgari and colleagues (2003) isolated a 50 kd protein from cotesia rubecula (hymenoptera, braconidae) that blocked melanization in the hemolymph of its host pieris rapae (lepidoptera). the protein, named vn50, is a serine proteinase homolog containing an amino-terminal clip domain; recently the authors also demonstrated that vn50 is stable in the host hemolymph for at least 72 hrs after parasitization (zhang et al, 2004). using m. sexta as a model system, they found that vn50 efficiently downregulated propo system, by significantly reducing its proteolytic activation. this occurred without directly inhibiting and/or damaging the active phenoloxidase. according to the above description, we have obtained similar results in the tobacco budworm heliothis virescens (lepidoptera) larvae infected by toxoneuron nigriceps (hymenoptera). gregorio and ratcliffe (1991) demonstrated that the presence of tripanosoma rangeli in the hemolymph of two insects (rhodnius prolixus and 44 triatoma infestans) significantly reduced the level of propo activation. in their paper, the authors suggested that the susceptibility to tripanosoma infection of both the hosts is strongly dependent on the propo activation intensity. finally, in a recent study, we observed a drastic reduction of po activity in the hemolymph of g. mellonella induced by heat-killed entomoparasite nematodes (s. feltiae) or purified parasites cuticles (brivio et al., 2002). considering that cellular encapsulation is one of the most effective processes directed toward large parasites, it is reasonable to discuss interference mechanisms acting against host immunocompetent cells. with regard to this, parasites might reduce the recognition capability of hemocytes, by damaging surface prrs, by down-regulation of their synthesis, or simply rejecting active cells by means of noxious secretions or refractive body-surface; alternatively, direct damage of host hemocytes could be achieved. a great number of evidences suggest that parasitoid wasps inject factors suppressing host immune system; well-described suppressive factors are venom glands secretions (in braconid wasps), polydna virus and co-injected teratocytes cells (vinson, 1990; summers and dib-hajj, 1995). infection with wasps is known to affect host hemocytes (particularly plasmatocytes) ability to attach to substrates, to aggregate and to spread correctly. moreover, virus-containing calyx fluid from campoletis sonorensis induces a significant reduction in the number of circulating hemocytes, when injected in h. virescens larvae (davies and vinson, 1988). effects on host cells have been broadly described in insects infected (or in in vitro assays) with nematode symbiontic bacteria. the nematocomplex steinernema carpocapsae/xenorhabdus nematophilus seems to be responsible of almost two effects on lepidoptera hemocytes; ribeiro et al. (1999) demonstrated unsticking and cytotoxic effects of two factors released by nematocomplexes in in vitro experiments. however, from this paper is not clear if bacteria themselves, nematodes, or both, produced the above factors. an interesting aspect of the immunodepressive action of xenorhabdus was described by park et al. (2004) in m. sexta; inhibitor(s) released by live bacteria seems to block eicosanoid biosynthesis (that are crucial mediators of insects cellular defense reactions) by reducing phospholipase a2 intracellular activity. finally, processes such as the release of toxic factors from hemocytes, phagocytosis, encapsulation and nodulation are also probably to be targets of parasite-derived interference factors. however, it is reasonable to expect different parasites to adopt different strategies of immunoevasion and that any particular parasite species would employ a variety of evasive tactics. a short profile of the killer (entomopathogen nematocomplex) as described by nathan cobb (1915), nematodes are one of the most abundant type of animals on earth. nematodes, thanks to their small size, to the resistant cuticle and to the ability to adapt to severe environmental changes, have colonized a wide range of habitats including vertebrate and invertebrate bodies. nematodes may be free-living or parasitic; the latter are usually considered pests because they cause important diseases in animals, humans, and for their economic impact on many agricultural products. a small but significant number of parasitic nematodes, called entomopathogenic, are of considerable interest because they possess various features as biological control agents for pest insects (gaugler and kaya, 1990; georgis and manweiler, 1994; poinar, 1998). entomopathogenic nematodes must meet some criteria to be considered good candidates for an overall biological control: they should neutralize agriculture insect pests and, possibly, insect vectors responsible of human and animal diseases; practically, they should be able to kill, sterilize, or hamper the development of their insect targets. insect-parasitic nematodes that possess optimal features as bioinsecticides belong to the families steinernematidae and heterorhabditidae (nematoda, rhabditida). steinernematidae and heterorhabditidae are not closely related phylogenetically but, through convergent evolution, they share similar life histories (poinar, 1993); the main difference between them is the reproductive strategy (steinernematidae are gonochoric, heterorhabditidae are hermaphroditic). these families differ from other rhabditids by having a species-specific mutualistic relationship with bacteria of the genus xenorhabdus (enterobacteriaceae) (poinar, 1979; kaya and gaugler, 1993). x. nematophilus is associated with steinernematidae and photorhabdus luminescens with heterorhabditidae in a specie-specific manner (forst and nealson, 1996; forst et al., 1997; forst and clarke, 2001; silva et al., 2002). the symbiontic bacteria contribute to the mutualistic relationship actively, by killing insect host, by establishing and maintaining suitable conditions for nematode reproduction and by providing nutrients and microbial substances that inhibit growth of a wide range of microorganisms. at the same time the nematode acts as a vector for the symbiontic bacterium. the symbiosis is essential for the efficiency of the biocontrol and it enables nematodes to exploit a diverse array of insect hosts (dunphy and thurston, 1990). the basic life cycle of most entomopathogenic nematodes consists of several stages: an egg stage, four juvenile stages (l1, l2, l3, l4), and a complex adult stage that comprises l5 (early adult stage) and late adult (fig. 2). in general, nematodes moult four times during each life cycle with a moult occurring at the end of each larval stage. therefore, moults separate the first and second larval stages (l1 and l2), the second and third larval stages (l2 and l3), the third and fourth larval stages (l3 and l4) and also the fourth larval stages and immature adults (l4 and l5). the l5 grows up to the size limit of its new cuticle. the third juvenile stage (ij3) of nematodes is known as the “infective juvenile” or “dauer” stage and is the only free-living stage (womersley, 1993). the ij3 is capable to survive in the soil for extended periods until it is able to find a susceptible host; its function is to locate, attack, and infect an insect host (poinar, 1990; akhurst and dunphy, 1993). 45 fig. 2 a (l1) develops inside the egg, hatches (h), grows rapidly, then moults (m1) to l2. the second stage larva also shows a rapid growth followed by a second moult (m2) to third stage larva (l3), the infective juvenile stage 3 for many nematode species (also named ij3). this (l3) grows, then moults (m3) inside the host to a l4 larvae. the final larval stage grows and undertakes a final moult (m4) to an immature adult (l5). host infection consists of various steps (fig. 3); (a) the ij3 parasites find hosts by chemotaxis towards chemical concentration gradients of carbon dioxide, and/or host excretory products. infective juvenile stage enters the host through natural body openings (mouth, anus, spiracles), it reaches the hemocoel of the host, and later on (b) it releases bacterial spores by defecation or regurgitation. symbiontic bacteria live in a monoxenically area or in differentiated vesicles of the anterior part of the infective juvenile intestine modified as a bacterial chamber. after release, bacteria quickly multiply in the hemolymph (c); they are mainly responsible for the host mortality because they produce and release exoand endotoxins to which the insect succumbs by septicemia (d) within 24-48 h of infection; furthermore, bacteria secrete antibiotics that prevent multiplication of the other microflora (khandelwal and banerjee-bhatnagar, 2003). bacterial cells also express and release proteases and lipases that degrade insect host tissues to be utilized by the parasite as a food source. as reported by several authors (wouts, 1984, tanada and kaya, 1993), the parasite itself produces toxins that are lethal to the host, even without its associated bacteria but, in this case, it is unable to reproduce; moreover, without the nematode, bacteria cannot reach and invade the host hemocoel. after mating, the females lay the eggs that hatch as first-stage juveniles that moult successively to second, third and fourth-stage juveniles and then to males and females of the second generation (e). the adults mate and the eggs produced by these secondgeneration females hatch as first-stage juveniles that moult to the second stage. the adult nematodes produce hundreds of thousands of new juveniles. the late second stage juvenile ceases feeding, incorporates a small fresh group of bacteria in the bacterial chamber (f), and moults to the infective juvenile stage (ij3). when the host has been consumed, the infective juveniles (g) emerge from the exoskeleton of the host, move into the soil and begin the search for a new host (h). in nature, under ideal conditions, steinernematidae and heterorhabditidae emerge 6-11 and 12-14 days after the initial infection respectively (kaya and koppenhöfer, 1999). since entomopathogenic nematodes represent an alternative to chemicals for insect pest control, it is fundamental to understand the basis of the infectivity of nemato-bacterial complexes and the interaction with the insect host immune systems. although the immune depressive and lethal effects induced by bacteria (long-term infection phase) are well known (ffrenchconstant et al., 2000), the short-term infection phase, particularly the role of the parasite itself, are not clearly understood (brivio et al., 2002). gun and bullets: a lecture from the killer the efficacy of the nematode s. feltiae in killing its hosts is mainly attributable to the severe effects of its symbiontic bacteria (x. nematophilus) that, by means of multiple factors, cause the death of the host in the later phase of infection. symbiontic bacteria could be viewed as bullets of a gun (the parasite): as it is well known, after a murder the effect and characteristics of the bullets found on the crime scene are easily assessed; but detectives are in trouble when trying to investigate on the missing gun. given that, the bacterial bullets have been well studied; a lot of papers provide in depth descriptions of the immunodepressive effects of bacteria-released factors. host physiological disorder caused by the release and proliferation of the symbiontic bacteria have been investigated (chattopadhyay et al., 2004); particularly, these microorganisms seem to be able to arrange the environment (host’s body) in such a manner to allow the parasite to survive and spawn unmolested. x. nematophilus, upon release into the hemolymph of g. mellonella host, adhere to the surface of hemocytes, proliferate and damage the cells that became vacuolated, unable to adhere to surfaces and finally show positivity to trypan blue (dunphy and webster, 1984, 1986). at the same time, xenorhabdus synthesizes and releases antibiotic compounds within the insect hemocoel that suppress competing microorganisms; in this way they acquire the optimal condition to proliferate, allowing the parasites to complete its development (gaugler and kaya, 1990). a growing number of xenorhabdus-produced factors have been described; recently a cytotoxic pilin subunit (17 kd) of x. nematophilus has been isolated: the protein is expressed on the bacterial surface and also secreted in the extracellular medium; it binds to the surface of larval hemocytes and shows cytotoxic properties against immunocompetent cells of helicoverpa armigera, finally causing agglutination of the cells (khandelwal et al., 2004). ribeiro and colleagues (2003) reported the purification of a cytotoxin of 10 .7 kd of molecular weight from x. nematophilus, nam ed 46 fig. 3 life cycle of entomopathogenic nematodes. alpha-xenorhabdolysin (alphax) peptide; the plasma membrane of spodoptera littoralis hemocytes seems to be the main target of the peptide. alphax peptide induces an increase of monovalent cations permeability that is sensitive to potassium channel blockers, even on mammal macrophages or erythrocytes. as a consequence of alphax binding to the plasma membrane, several events occur intracellularly, such as selective vacuolation of the endoplasmic reticulum, cell swelling and cell death. in insects, bacterial infections usually evoke the activation of the toll/imd pathways that culminate in the synthesis of an array of antibacterial peptides; a well-known property of the parasite symbionts is to interfere with antibacterial responses. xenorhabdus affects antimicrobial activity of lepidoptera by means of two distinct released proteases; caldas and colleagues (2002) demonstrated that one of the two above mentioned proteases (protease ii) destroyed antibacterial activity in the hemolymph of insect larvae (g. mellonella and p. unipuncta) challenged with inoculated bacteria; particularly, the bacteriolytic activity of the inducible antibacterial peptide cecropin a was drastically reduced. furthermore, protease ii did not show toxicity to host hemocytes. finally, symbionts of entomopathogenic nematodes showed inhibitory effects on host propo activating system (yokoo et al., 1992; dunphy et al., 1998). the presence of a large number of studies on bacterial symbionts are mainly due to the economical interests that have led researchers to focus on bacteria-derived patentable molecules valuable in integrated pests management. on the other hand, in order to study the role of the parasite itself, it would be necessary to work with axenic nematodes; unfortunately, it is difficult to obtain nematodes completely deprived of their symbionts and, in this case, parasites might not be in a physiological condition, increasing the risk of obtaining artifacts. it is thus probably that experimental troubles and economical interests could be responsible for the scarcity of literature available on this topic. with the aim of clarifying the involvement of the parasite itself in the relationships with the host, at first we have carried out our experiments in a short period (0-30 min) following s. feltiae infection, when bacteria are not yet released into the g. mellonella hemolymph; besides, we carried out many studies utilizing the isolated body surface (i.e. cuticle and epicuticle) of the parasite. cuticles of steinernema have been obtained without bacterial contamination to a good level of purity (fig. 4) by developing a technique based on sonication, washes and sterilization of parasites. parasite immunoevasion strategies often involve the parasite body surface, which seems to play a key role in the interaction with the host environment (blaxter et al., 1992). nematodes moult several times throughout their developmental cycle, each time changing their body surface with the formation of a new cuticle (cox et al., 1981a, 1981b); although a common model of nematode cuticle has been proposed (maizels et al., 1993), single species may have significant differences in molecular organization and surface properties. this is particularly true for parasitic species (i.e. s. feltiae) that must interact with an unfavorable host environment. furthermore, parasitic nematodes may easily elaborate the composition and organization of the epicuticular external layer, depending upon the particular environment of each species (maizels et al., 1993). together with other surface and secreted molecules (politz and philipp, 1992), the cuticle of parasitic nematodes seems to be involved in immunoevasion and suppression of host’s defenses, as suggested also by akhurst and dunphy (1993); thus, it is likely that nematode body surface plays a crucial role in parasite success. the hypothesis of a key role of the body-surface of parasites was proposed early by vinson (1977); vinson suggested that in absence of active suppression mechanisms the prevention from encapsulation could be achieved by means of: a) the acquisition of a coat composed of host proteins (molecular disguise); b) the possession of heterophilic antigens; c) the presence of a non reactive bodysurface, or molecular mimicry. 47 in 1987 dunphy and webster presented preliminary evidences in favor of a possible role for the epicuticle layer of s. feltiae in cellular immunodepression. the paper pointed out interactions of the body surface of the entomopathogen with g. mellonella hemocytes and suggested its involvement in avoiding cellular encapsulation. the authors described a partial characterization of cuticle sugars by means of lectin specificity but, more interestingly, they assessed the role of the lipidic moiety of the epicuticle of s. feltiae. a simple assay based on lipase treatments determined that surface lipids played a role in escaping from hemocytes recognition; on this basis, they supposed that modifications of the lipidic surface resulted in a changed molecular architecture of the epicuticle, thus exposing discriminable antigens. primarily inspired by these suggestions, we have focused the research on the role of s. feltiae cuticle, with the aim to exclude any contribution from symbiontic bacteria and/or active secretions of the parasite. our preliminary observations (brivio et al., 2002) showed host propo system inhibition in g. mellonella larvae infected with heat-killed nematocomplexes; although these results suggested that factors released from the parasite were not responsible of propo inhibition, they did not completely exclude the involvement of bacteria. however, these data supported the attractive hypothesis that the parasite, after entry into the host hemocoel, exploits its body-surface to immunoevade and/or immunodepress host defenses. a strong confirmation of the above hypothesis came from the assays carried out with isolated cuticles. the suppression of the hemolymph phenoloxidase activity, observed after either in vivo cuticle injection or in vitro co-incubation (cuticles plus cell-free hemolymph), was comparable to that obtained in the experiments performed with killed whole parasites. the integrity of the molecular architecture of the cuticle seems to be essential to retain its immunodepressive properties, since chemical alterations of the structure result in a marked loss of inhibition of the host propo system. moreover, confirming dunphy’s suggestions, the main effect was observable after damage or removal of the lipidic layer obtained with lipase and methanol-chloroform treatments (fig. 5). these data confirmed the first assumption of a key role of the lipids in the host-parasite interaction, although no information concerning the mechanisms by which cuticular lipids may affect the activation of the propo system was provided (brivio et al., 2004). the process by means of which s. feltiae cuticle lipids showed inhibitory effects on the host propo system was further investigated hypothesizing that these molecules might interact with hemolymph factor involved in the activation pathway of host phenoloxidase. a set of experiments based on in vitro interaction of purified parasite cuticle with cell-free host hemolymph (fig. 6), demonstrated a specific binding property of the cuticle: particularly, the lipidic moiety interacts and sequesters three hemolymph proteins (17, 26, 35 kd), named hips (hostinteracting proteins), possibly involved in the propo activation cascade. concerning the identity and functions of hips, on the basis of preliminary characterization based on molecular mass and according to the literature (dettloff et al., 2001), we supposed that, firstly, the 17 kd hip could be identified as the insect lipid-carrier apolipophorin iii. besides reports on its well-known functions in the lipid metabolism (ryan and van der horst, 2000), exhaustive studies have been carried out on the involvement of this protein in immunological responses (wiesner et al., 1997; halwani and dunphy, 1999; zakarian et al., 2002). hip26 has a molecular weight comparable to that of lbp-2 (a lipopolysaccharide-binding protein) described by dunphy and halwani (1997) in g. mellonella. this protein shows a specific affinity for endotoxin lipid-a and seems to be involved both in hemocytes activation and propo system regulation. the third factor of 35 kd has a molecular weight similar to the protease-like molecule scolexin. insect scolexin is well characterized at molecular level, although its biological function is not yet understood (finnerty et al., 1999). the significant quantitative reduction of hips induced by the parasite is responsible for the blockage of the activation pathway of the propo system; when these components, eluted from the parasite body surface, are added during in vitro assays, the normal hemolymph phenoloxidase activity of the host is restored. furthermore, a function of the hips seems to be related to the activation of hemolymph serine proteases, given that their properties of reactivation (fig. 7) are lost if they are assayed in the presence of protease inhibitors. the precise reactivation mechanism of the propo system mediated by hips still has to be determined; to this goal we carried out far western blot experiments by which these proteins showed lps-binding properties (fig. 8). this affinity has been confirmed by their ability to bind to the wall of gram(-) bacteria (fig. 6, panel c, lane b). in addition, cuticle lipids seem to cross-react with anti-lps antibodies suggesting a structural correlation between the parasite lipids and the bacterial lps lipida domain (fig. 9). an intriguing hypothesis could be that surface lipids may act as pathogen-associated molecular patterns-like (pamps-like) but, in this case, their interaction with the host hips (comparable to prrs) would result in the removal of the latter from the insect hemolymph, thus preventing the activation of hemolymph serine proteases required for propo activation and melanotic encapsulation. the interference of the parasite with cell-mediated defenses of the host was investigated by ribeiro and co-workers (1999). the observed hemocyte damages were suggested to be related to factors released in the medium from nematocomplexes; these compounds (not identified) showed unsticking and cytotoxic effects on g. mellonella and m. unipunctata immunocompetent cells. the data presented seem to indicate an active interference process carried up by s. carpocapsae. moreover, the interaction between the parasite body-surface molecules and host hemolymph components could result in a coating effect of the nematode. this coat, composed of host self-proteins, could induce molecular disguise processes. to ascertain the above assumption we have performed various assays clearly showing that galleria hemocytes are unable to recognize the parasites as 48 fig. 4 a: longitudinal section of the parasite body (at tem level) showing the cuticle and epicuticular layer of s. feltiae; in b and c purified cuticular fragments (phase contrast microscopy). fig. 5 effects of the presence of s. feltiae whole individuals or isolated cuticles on propo activity of g. mellonella larvae. in the upper area is shown relative phenoloxidase activity as modulated by: l-n, living parasites; d-n, heat-killed parasites; cut, isolated cuticles; cut-lip, lipase-treated cuticles; cut-mc, methanol-chloroform-treated cuticles; extr-mc, lipidic extracts from cuticles. visualized below is the melanin production in microwells from the above assays. 49 fig. 6 to identify putative specific parasite-bound hips, high-salt elutions from parasite body surface (previously incubated with host hemolymph) were carried out. the supernatants of the elution were analyzed by sds–page (panel b). the parasites were thoroughly washed to eliminate all non-specific hemolymph proteins before elution. the electrophoretic pattern (panel b, lane “elu”) revealed the presence of four main bands with molecular masses of about 80, 35, 26, and 17 kd, respectively. when the assay was carried out with methanol–chloroform or lipasetreated parasites, the lower bands (35, 26, and 17 kd) disappeared (panel b, lanes “mc” and “lip”) and only a reduced amount of the 80 kd band was observed by sds–page. panel c: further interaction assays were carried out with hemolymph (<50 kd) incubated with gram(-) bacteria (e. cloacae). enterobacter was incubated with host hemolymph and 1m nacl eluted proteins were analyzed by sds–page (lane b). three main bands with molecular masses corresponding to the described hips were observed . as a control, high salt eluted bacteria (without prior hemolymph incubation) were analyzed (lane c). fig. 7 reactivation properties of hips directed against parasite-inhibited host propo system are shown (hips). hips activating properties were also assayed in the presence of protease inhibitors: under these conditions host propo system activity was not restored (hips-pi). fig. 8 far-western assay for lps-binding activity of hips. blotted hips were renatured in situ onto nitrocellulose sheets and incubated with lipopolysaccharides. anti-lps was used as primary antibody; the lps-binding to hips was revealed by anti-igg peroxidase-conjugated antibody and luminol. all hips (17, 26, and 35 kd) were positive to the assay (lane b); as a control, the assay was performed without hips in situ renaturation (lane a), without lps incubation (lane c1) or omitting the secondary antibody (lane c2). 50 fig. 9 cross-reactivity of parasite cuticle compounds with anti-lps antibodies. parasites were immunostained with the primary antibody and then with anti-igg tritc-conjugated; as shown, a strong signal is localized at the parasite surface (panel b). panel a shows light micrographs of parasite body section (arrowheads indicate cuticle and epicuticle zone). anti-lps western blot, carried out with samples from methanol–chloroform cuticle extracts (lane nem) shows a positive smeared band observable at the bottom of the gel. the assay was carried out also with bacterial lps, as a positive control (lane c). fig. 10 mimetic properties of s. feltiae. parasites (n1, n2, n3) or cuticles (c1, c2, c3) were incubated with cultured hemocytes to assay for escaping from host cell-mediated encapsulation. n1 and c1 micrographs show the lack of encapsulation of whole parasite and cuticle fragments. in n3 and c3 is seen the formation of capsules on lipase-treated parasites and cuticles. n2 and c2 show the healthy state of hemocytes that, even in the presence of s. feltiae or cuticles, are able to encapsulate beads. not self. the co-incubation experiments with parasites (or isolated cuticles) and abiotic materials provided further evidence that the host cells were healthy and capable of encapsulation (fig. 10). it can thus be supposed that this immunoevasion mechanism may be attributable to achievement of mimetic properties of the body-surface of steinernema rather than to the cells having been damaged by the parasite. finally, as observed in propo system inhibition assays carried out with pre-treated cuticle, upon removal of lipidic compounds parasites become unable to evade cellular encapsulation of the host. based on the data obtained from the relationships between s. feltiae and g. mellonella we propose a possible schematic model (fig. 11) describing some features of host-parasite (galleria-steinernema) immunological interaction. in our lab we have recently undertaken a study to 51 fig. 11 a scheme delineating some parasite immunoevasion/depression processes related to hemolymph prrs removal. ascertain the interference of the parasite with the inducible antibacterial response of g. mellonella; this topic is particularly relevant in the studied model since these parasites, besides escaping host immune defenses, seem to be able to down-regulate amps genes reducing the synthesis of molecules potentially harmful for their symbiontic bacteria (manuscript in preparation). concluding remarks our society increasingly demands alternatives to chemicals for managing insect pests and insect vectors of diseases; even though biological control by means of bioinsecticides holds great promises, no more than 1.5 % of commercial pesticides are represented by biologicals. researches directed towards a better knowledge of nematocomplexesinsects relationships could provide a valuable starting point for the improvement of integrated pest management techniques, with the aim of drastically reducing the use of chemicals. moreover, from studies of model insects immune system is clearly emerging a fascinating picture of the evolution of immune responses common to both invertebrates and vertebrates. acknowledgments we are grateful to magda de eguileor for tem micrograph, to michela curradi for the critical reading of the manuscript and to alberto vianelli for language checking and suggestions. references agaisse h, petersen um, boutros m, mathey-prevot b, perrimon n. signaling role of hemocytes in drosophila jak/stat-dependent response to septic injury. dev. cell 5: 441-50, 2003. akhurst, rj, dunphy gb. tripartite interactions between symbiotically associated entomopathogenic bacteria, nematodes, and their insect hosts. in: beckage ne, thompson sn, federici ba. 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endoparasitoid venom. insect biochem. mol. biol. 34: 477-483, 2004.1993. differential preference of coloured surface of tribolium castaneum (herbst) isj 3: 84-88, 2006 issn 1824-307x short communication differential preference of colored surface in tribolium castaneum (herbst) ams reza, s parween department of zoology, rajshahi university rajshahi 6205, bangladesh accepted july 28, 2006 ______________________________________________________________________________ abstract insects show color preferences mostly to those which resembles the color of foliage, flower or even their hosts. in the present study observations were made to determine vision towards different colored surfaces in young (second instar), and advanced (fourth instar) larvae, and adults of tribolium castaneum (herbst) (coleoptera: tenebrionidae). the larva and adult beetles showed significant color preferences when given a choice between white (control) and colored surfaces at 24and 48-h exposures. the second instar larvae were more attracted by yellow and pink than green surfaces. the fourth instar larvae did not show any significant preference between white and colored surfaces at 24-h exposure, but avoided red and pink surfaces (p>0.05) and had a marginal choice for black (p<0.05). the adults avoided green significantly at both exposure times and pink at 48-h exposure, but was moderately attracted by black (p<0.05) at both exposure times. key words: color preference; tribolium castaneum _____________________________________________________________________________________________________________________ introduction insect traps are used either for population sampling or for management of pest species. for collecting cropfield insects sticky traps and baited traps are used. the sticky traps are often made attractive by using pheromones in it, or by using different colore and shape traps (hoback et al., 1999). different insect families showed preferences for different trap colores. hoback et al. (1999) provided a list of insect families with their preferences for different color traps. members of the same family may prefer more than one color, for example in case of curculionidae (cross et al., 1976), even color preference may differ at species level (capinera and walmsley, 1978). lobdell et al. (2005) observed that the egg parasitoid trichogramma ostriniae showed differential responses to egg color or the hosts as well as the background color in a petri dish arena while searching for the host. ______________________________________________________________________________ corresponding author: a.m. saleh reza department of zoology, rajshahi university rajshahi 6205, bangladesh e-mail: salehbgd@yahoo.com studies on the cropland insects demonstrated the preferences of many insects for yellow color, as the peak color reflectance of plants is the yellow band at 50-560 nm, so this color may act as a super-normal foliage stimulant to herbivorous insects (prokopy and owens, 1983). sawflies showed least preference to dark colors like blue (anderbrant et al., 1989). it is reported that most hymenoptera have color receptors in the green, blue and ultra-violet range, with a few species also being able to perceive red (peitsch et al., 1992). the previous studies revealed that the insects are attracted to colors, which are close to their food color. numbers of attractant and repellent tests were carried for the stored product insect pests as part of their management program. most of these researches involved addition of different insecticides, hormones, pheromones, plant extracts, etc., to their food. however, color preference in stored product insects remains poorly studied. the objective of this study was to determine whether tribolium castaneum, the red flour beetle can differentiate colors? the differential preferences for colored surfaces were studied for young (2nd instar) and advance (4th instar) larvae and adults of t. castaneum. 84 mailto:salehbgd@yahoo.com materials and methods stages of tribolium castaneum used about 200 adults of t. castaneum (herbst) beetles were collected from the stock culture, which has been maintained in the stored product insect laboratory, department of zoology, rajshahi university, since 1985 without any loss of fitness. the adults were kept in a beaker and provided with 10 g of standard food (park and frank, 1948). after 24 h the subculture was sieved through 500 and 250 µm mesh to separate the adults and the eggs respectively from the food. the newly hatched larvae were collected from these eggs and provided with standard food and reared in beakers, covering the mouth with fine cloth to restrict escape of the larvae. a few similar subculture of the beetles were established during subsequent days. the subcultures were kept at 30 ± 1 °c in an incubator without controlling light and humidity. the food was changed after every three days to avoid contamination by the larvae (park, 1935). the second and fourth instar larvae were normally obtained on the third and ninth days respectively after hatching of the eggs (mondal, 1984). the adults emerged 21-22 days after hatching. in this experiment, 30 larvae of second or fourth instars, or adults of 48-h age were used for each color test. each of these experiments was replicated three times. table 1 distribution of larvae and adults of t. castaneum in different colored surfaces after 24 h exposure (n = 90) percentage distribution (number) life stage colored surface control (white) color χ2-values (df = 2) green 31.11 (28) 68.89 (62) 7.14* red 52.23 (47) 47.77 (43) 0.09 blue 35.55 (32) 64.45 (58) 4.18 chocolate 46.67 (42) 53.33 (48) 0.22 yellow 23.33 (21) 76.67 (69) 14.22*** pink 21.11 (19) 78.89 (71) 16.69*** second instar larvae black 47.77 (43) 52.33 (47) 0.11 green 56.66 (57) 43.34 (39) 0.89 red 50.00 (45) 50.00 (45) 0 blue 38.88 (35) 61.12 (55) 0.50 chocolate 35.55 (32) 64.45 (58) 4.18 yellow 48.88 (44) 51.12 (46) 0.02 pink 62.22 (56) 37.78 (34) 2.99 fourth instar larvae black 43.33 (39) 56.67 (51) 0.89 green 71.11 (64) 28.89 (26) 8.91* red 42.22 (38) 57.88 (52) 1.24 blue 40.00 (36) 60.00 (54) 2.00 chocolate 57.78 (52) 42.22 (38) 1.21 yellow 37.78 (34) 62.22 (56) 2.99 pink 57.78 (52) 42.22 (38) 1.21 adults black 31.11 (28) 68.89 (62) 7.14* *p<0.05; ***p<0.001 85 color choice test these tests were conducted in choice chamber. a choice chamber was made in glass petri dish of 9 cm diameter. a straight line was drawn through the middle of the petri dish. colored poster papers were used for the test, and white paper as the control. the papers of each color were first cut into a circle of 9 cm diameter, and then cut into two equal halves. at the outer surface of the petri dish color paper was pasted on one half and white paper was pasted on the other half. similarly, these choice chambers were made for each color. the color used in this experiment were green, red, blue, chocolate, yellow, pink and black. experimentation at the middle of the each choice chamber, 30 of either second or fourth instar larvae, or adults were released. the petri dish was covered with lid, and kept undisturbed at room temperature (20-22 °c). no food was given to them. after 24 h the number of larvae or adults at colored and white parts of the choice chamber were recorded, and the insects were kept undisturbed for another 24 h. similarly, the number of insects in colored and control halves were recorded after 48 h. table 2 distribution of larvae and adults of t. castaneum in different colored surfaces after 48 h exposure (n = 90) percentage distribution (number) life stage colored surface control (white) color χ2-values (df = 2) green 15.55 (14) 84.45 (76) 23.73*** red 34.44 (31) 65.56 (59) 4.48 blue 33.33 (30) 67.67 (60) 5.56 chocolate 61.11 (55) 38.89 (35) 2.47 yellow 24.44 (22) 75.56 (68) 13.07*** pink 17.78 (16) 82.22 (74) 20.76*** second instar larvae black 45.55 (41) 54.45 (49) 0.39 green 43.33 (39) 56.67 (51) 0.89 red 75.55 (68) 24.45 (22) 13.06*** blue 46.66 (42) 53.33 (48) 0.22 chocolate 56.66 (61) 43.34 (39) 0.89 yellow 43.33 (39) 56.67 (51) 0.89 pink 75.55 (68) 24.45 (22) 13.06*** fourth instar larvae black 28.89 (26) 71.11 (64) 8.91* green 81.11 (73) 18.89 (17) 19.36*** red 48.89 (44) 51.11 (46) 0.02 blue 33.33 (30) 66.67 (60) 5.56 chocolate 43.33 (39) 56.67 (51) 0.89 yellow 50.00 (45) 50.00 (45) 0 pink 67.78 (61) 32.22 (29) 6.32* adults black 28.89 (26) 71.11 (64) 8.91* *p<0.05; ***p<0.001 86 statistical analysis the distribution of larvae and adults on each colored surface was tested using χ2-test. anova was done to examine the effect of the exposure period and stages of t. castaneum on the choice of different colors. results and discussion there is difference for color preference among the larvae and adults of t. castaneum. the second instar larvae were mostly attracted to yellow and pink colors (p<0.001) at both 24and 48-h exposure (tables 1, 2). choice test for the fourth instar larvae showed no preference for any one of the tested colors at 24-h exposure (table 1). at 48-h exposure, the fourth instar larvae preferred the white color (control) more than red and pink (p<0.001) (table 2), and showed a little attraction towards black (p<0.05) (table 2). the adults showed no preference for colored or white surfaces except black (p<0.05) at both exposure periods (tables 1, 2). total avoidance to green surface was observed. during 48 h exposure the adults avoided pink whereas at 24-h exposure the distributions in white and pink colors were similar (tables 1, 2). the present results revealed that advance larvae and adults showed no preferences for colored surface. moreover, they avoided pink, red and green. white (control) was more preferred by the fourth instar larvae and the adults than the second instar larvae. table 3 preference or avoidance to colored surfaces by larvae and adults of t. castaneum preference/avoidance of beetles at two exposures 2nd instar larva 4th instar larva adults colors 24-h 48-h 24-h 48-h 24-h 48-h green mp sp nc nc ma sa red nc nc nc sa nc nc blue nc nc nc nc nc nc chocolate nc nc nc nc nc nc yellow sp sp nc nc nc nc pink sp sp nc sa nc mp black nc nc nc mp mp mp sp = strongly preferred (p<0.001); mp = moderately preferred (p<0.05); sa = strongly avoided; ma = moderately avoided; nc = no choice anova showed that there is no significant difference of color choice at different exposure periods (p>0.05, f= 5.58) by the larvae and the adults. the second instar larvae showed preference for green, yellow and pink colors, but had no significant choice for black. however, black was marginally preferred over white by the fourth instar larvae and adults (table 3). generally, a wide range of insects exhibit attraction towards yellow color (borror et al., 1989). the coleopteran families like chrysomelidae, coccinellidae, curculionidae and scarabaeidae showed preference for yellow traps (p<0.05) as stated by flemming et al. (1940), cross et al. (1976), dominick (1976) and dowell and cherry (1981). these beetles also showed choice for green and white traps. however, in the present investigation except young larvae, t. castaneum showed no choice for yellow color. an experiment on the farmland sawflies showed that all the collected species showed strong responses to colored traps, especially to yellow (barker et al., 1997). moreover, the hymenopteran egg parasitoids of the genus trichogramma showed preference for yellow and white, representing an adaptive preference for the egg color of the primary hosts (pak and de jong, 1987; lobdell et al., 2005). furthermore, romies et al. (1998) observed that naturally occurring trichogramma in sorghum fields in india were poorly attracted to yellow stick-traps as compared to white and green traps. khalil (1991) found that adult t. confusum attracted by red in a narrow space (45 ×1 cm) with a color area of 7×1 cm; while in a wide space (100×4 cm) having a color area of 16×4 cm, they were attracted by blue color. the author observed that sitophilous oryzae adults were attracted by green and black, and these responses were greatly affected by the exposure period. however, in the present experiment the adult t. castaneum no definite choice for either blue or red color was observed. normally, it is expected that there is no possibility of differential preference to colors between different species of an insect in the same habitat. however, different sawfly species showed significance difference between color choices (barker et al., 1997). in the present study no food was given in the choice chambers, which might be the reasons that the advanced larvae and the adults might be distributed evenly on white (control) and colored surfaces for the search of food at short exposure. moreover, they preferred dark colors, as a moderate choice for the black surface, for a site of refuge. the fourth instar larvae showed a strong avoidance to red and pink colors at 48-h post-exposure. the adults totally avoided green, and moderately pink. but the second instar larvae showed strong attraction to green and pink colors along with the yellow. hence, it can be said that the younger larvae are attracted to green, yellow and pink colors, and equally distributed on dark colors. longer exposure period provided to fourth instar larvae and adults gave them sufficient time for a good selection of a suitable color. color preference 87 may change during the insect’s life time. for example, the pre-reproductive adult anthomyiid flies were attracted to yellow colored traps, but reproductive flies were attracted to purple color (jenkins and roques, 1993); similarly tephritid flies are attracted to yellow color prior to maturation and red color during oviposition (kring, 1970). it can be concluded that for tribolium control in the grain stores, green, red and pink grain bags could be used to keep away the older larvae and adults, or the wall and surface of the stores may be painted with these colors. use of colored bags or colored surfaces may give better result in integrated pest management programs for the control of tribolium. acknowledgement the authors are thankful to professor dr md ataur rahman khan, department of zoology, rajshahi university, for his painstaking corrections of the manuscript and kind suggestions. references anderbrant o, lofquist j, jonssen j, marking e. effects of pheromone trap type, position and colour on the catch of the pine sawfly neodiprion sertifer (geoff.) (hym. diprionidae). j. appl. ent. 107: 365-369, 1989. barker am, sanbrooke kj, aebischer nj. the water trap colour preferences of farmland sawflies. ent. exp. et appl. 85: 83-86, 1997. borror dj, triplehorn ca, johnson nf. an introduction to the study of insects. saunders college publishing, philadelphia, pa, 1989. capinera jl, walmsley mr. visual responses of some sugarbeet insects to stick traps and water pan traps of various colours. j. econ. ent. 71: 926-927, 1978. cross wh, mitchell hc, hardee dd. boll weevils: response to light source and colours on traps. environ. ent. 5: 565571, 1976. dominick cb. collection of the tobacco flea beetle on coloured panels. j. econ. ent. 64: 1575, 1976. dowell rv, cherry rh. survey traps for parasitoids and coccinellid predators of the citrus black fly, aleurocanthus woglumi. ent. exp. et appl. 29: 356-362, 1981. flemming we, burgess de, maines ww. relation of colour to the effectiveness of japanese beetle traps. j. econ. ent. 33: 320-327, 1940. hoback ww, svatos tm, spomer sm, higley lg. trap colour and placement affects estimates of insects familylevel abundance and diversity in a nebraska salt marsh. ent. exp. et appl. 91: 393-402, 1999. jenkins mj, roques a. attractiveness of colour traps to strobilomyia spp. (diptera: anthomyiidae). env. ent. 22: 297-304, 1993. khalil me. the orientation responses of some coleopteraous adult, tribolium confusum (tenebrionidae) and sitophilus oryzae (curculionidae) to colours. j. egypt. ger. soc. zool. 005: 303-313, 1991. kring jb. red spheres and yellow panels combined to attract apple maggot flies. j. econ. ent. 63: 466-469, 1970 lobdell ce, yong tze-hei, hoffmann mp. host colour preferences and short-range searching behaviour of the egg parasitoid trichogramma ostriniae. ent. exp. et appl. 116: 127-134, 2005. mondal kamsh. effects of methyloquinine, aggregation pheromone and pirimophos-methyl on tribolium castaneum herbst larvae. ph.d. thesis, university of newcaste upon tyne, uk, 1984. pak ga, de jong ej. behavioural variations among strains of trichogramma spp. host recognition. netherlands j. zool. 37: 137-166, 1987. park t. studies on population physiological effects of conditioned flour upon tribolium confusum duv. and its population. physiol. zool. 8: 91-115, 1935. park t, frank mb. the fecundity and development of the flour beetles, tribolium confusum and tribolium castaneum at three constant temperatures. ecology 29: 368-375, 1948. peitsch d, fietz a, hertel h, desouza j, ventura, df, mendzel r. the spectral input systems of hymenopteran insects and their receptor-based colour vision. j. comp. physiol. 170a: 23-40, 1992. prokopy rj, owens ed. visual detection of plants by herbivorous insects. ann. review ent. 28: 337-364, 1983. romies j, shanowar tg, zebitz cpw. response of trichogramma egg parasitoids to coloured stick traps. biocontrol 43: 17-27, 1998. 88 ams reza, s parween department of zoology, rajshahi university rajshahi 6205, bangladesh accepted july 28, 2006 abstract materials and methods stages of tribolium castaneum used color choice test experimentation statistical analysis results and discussion adults acknowledgement seasonal variations in mu opiate receptor signaling in the nervous system of the blue mussel, mytilus edulis isj 7: 141-145, 2010 issn 1824-307x research report seasonal variations in mu opiate receptor signaling in the nervous system of the blue mussel, mytilus edulis: temperature controls physiological processes kj mantione, p cadet, f casares, w zhu, gb stefano neuroscience research institute, state university of new york at old westbury, ny 11568, usa accepted may 13, 2010 abstract it is anticipated that invertebrate processes will be subject to seasonal variations because of their poikilothermal characteristics. in the present study we determined if the morphine coupled nitric oxide (no) release, which is constitutive in nature, exhibits seasonal characteristics, which has previously been shown for catecholamine processes in the marine mollusc mytilus edulis. in this regard, morphine induced no release measured on a monthly basis for one year revealed a peak release value (39 ± 4 nm) during the late spring and early summer. the lowest no release occurred during the months of january (6.0 ± 0.5 nm) through march (6.5 ± 1.1 nm). the lowest sea surface temperatures (1.3 °c) were also recorded in these same three winter months in new york. relative mu opiate receptor gene expression was assessed by real time pcr during these seasons. the mrna expression reached a relative peak during the month of june and was at its lowest in february and march, further demonstrating the direct coupling of morphine with this receptor. we conclude that the temperature an animal is chronically exposed to serves to control cellular processes, i.e., opiate signaling. key words: nitric oxide; mu opiate receptor; morphine introduction through homeostasis, living organisms maintain their survival in the face of both internally and externally generated stimuli. this balance is constantly challenged and therefore the ability to overcome these normal perturbations is essential to survival and longevity (chrousos and gold, 1992; fricchione and stefano, 1994). in this regard, subjecting invertebrate animals to temperature changes has been shown to alter the ganglionic monoamine levels as well as affecting functionality of gill cilia in mytilus edulis (stefano et al., 1977a, 1977b; stefano and catapane, 1977b). additionally, opiate processes respond to various types of ___________________________________________________________________________ corresponding author: kirk j mantione neuroscience research institute suny college at old westbury p.o. box 210 old westbury, ny 11568-0210, usa e-mail: kjmantione@sunynri.org list of abbreviations: nitric oxide, no; phosphate buffered saline, pbs; s-nitroso-n-acetyl-dl-penicillamine, snap; constitutive nitric oxide synthase, cnos stressors in both vertebrates and invertebrates ( lee and spector, 1991; marrazzi et al., 1997; sonetti et al., 1999; zhu et al., 2001; cadet et al., 2002; guarna et al., 2002). we have previously demonstrated that endogenous morphine represents the terminal component of a successful stress response and that its actions are generally down regulating immune responses and metabolic rates (stefano et al., 2000). this down regulation occurs because of coupling of the mu opiate receptor to nitric oxide (no) release (cadet et al., 2003). our group has also demonstrated that opiate receptors and subsequent morphine induced no release can be profoundly impacted by temperature changes in m. edulis (cadet et al., 2002; mantione et al., 2003). furthermore, we have presented molecular evidence on the effect of rapid temperature changes on mu opiate receptor expression and morphine levels in this invertebrate’s nervous system (cadet et al., 2002). in cold stressed organisms, ganglionic mu opiate receptor decreases and morphine levels increase (cadet et al., 2002). in the present study, we investigated the seasonal variation in ganglionic mu opiate receptor expression in m. edulis. in addition, we performed a morphine induced no release assay to determine the functionality of the morphine signaling system in this model organism. 141 month jan febmarchapril may june july aug sept oct nov dec nm n itr ic o xi de 0 10 20 30 40 50 t em pe ra tu re o c 0 10 20 30 40 50 [no] sea surface temperature fig. 1 monthly measurements of sea surface temperature and morphine (1 µm) stimulated no release from mytilus edulis pedal ganglia (20 ganglia per assay, n = 4). paired t-tests revealed statistically significant differences between january or february or march and may or june or july (p = 0.002). material and methods animal collection and nitric oxide (no) determination mytilus edulis collected from shinnecock bay on long island were immediately transported to the laboratory in seawater for processing. the ambient seawater temperature was maintained using insulated coolers until dissection of animals. samples were collected monthly on the 15 day of the month and sea surface temperature was recorded. for no determination, approximately 20 pedal ganglia were placed in 1 ml phosphate buffered saline (pbs) at room temperature. no release from the tissues was immediately directly measured using a 200 μm flexible no-specific amperometric probe (world precision instruments, sarasota, fl) connected to a 4-channel biostat (esa, chelmsford, ma). the system was calibrated daily with s-nitroso-n-acetyl-dl-penicillamine (snap) in 0.1 m cu+2. the amperometric probe was allowed to equilibrate in pbs for at least 10 min prior to being transferred to the tube containing the tissue. morphine-stimulated no release was evaluated at a final concentration of 10-6 m. each experiment was repeated four times (4 groups of 20 ganglia for each month) along with a control (pbs only). a paired student’s t-test was performed to evaluate differences between selected months. mu opiate receptor expression pedal ganglia (15) were immediately processed after dissection. the ganglia were placed in 1.5 ml tubes and then washed with pbs (invitrogen, carlsbad, ca). total rna was isolated using the rneasy mini kit (qiagen, valencia, ca). ganglia were homogenized in 600 µl lysis buffer. the samples were then processed following the manufacturer’s instructions. in the final step, the rna was eluted with 50 µl of rnase-free water. this rna isolation process was repeated four times for each group of 15 ganglia. first-strand cdna synthesis was performed using random primers (invitrogen, carlsbad, ca). 1 µg of total rna was denatured at 95 °c and reverse transcribed at 40 °c for 1 h using superscript iii rnase h-rt (invitrogen, carlsbad, ca). ten microliters of the rt product was added to the pcr mix containing primers for the mu opiate receptor. primers and probes specific for the mu-opiate receptor gene (mor) were designed by the software primer express (applied biosystems, foster city, ca). the forward primer was 5’atgccagtgctcatcattac-3’ and the reverse primer sequence was 5’gatccttcgaagattcctgtcct-3’. the taqman probe was constructed with the 5’-reporter dye, 6carboxyfluorescein (fam), and a 3’-quencher dye, 6-carboxy-tetramethyl-rhodoamine (tamra). the probe sequence was 5’cgcctcaagagtgtccgcatgct-3’. the endogenous control gene, β-actin, was used to normalize the rt-pcr. the 2x universal master mix (applied biosystems) containing the pcr buffer, mgcl2, dntp’s, and the thermal stable amplitaq gold dna polymerase was used in the pcr reactions. in addition, 200 µm reverse and forward primers, 100 µm taqman probe, 3 µl of rt product and rnase/dnase-free water was added to the master mix to a final volume of 50 µl. the pcr reaction mixture was transferred to a microamp optical 96-well reaction plate and incubated at 95 °c for 10 min to activate the amplitaq gold dna polymerase and then run for 40 cycles at 95 °c for 30 s and 60 °c for 1 min on the applied biosystems geneamp 7500 sequence detection system(sds). each pcr was performed in triplicate. the pcr results were analyzed with the geneamp 7500 sds 142 month jan febmarchapril may june july aug sept oct nov dec r el at iv e g en e e xp re ss io n 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 fig. 2 relative mu opiate receptor gene expression in mytilus edulis pedal ganglia determined by real time pcr for each monthly sample (n = 4). paired t-tests revealed statistically significant differences between february or march and may or june or july (p = 0.004). software (applied biosystems). relative gene expression was calculated using the method of yoshikawa et al. (2001). standard curves were generated by serial dilution of the june cdna sample. r values were calculated and used to directly compare the monthly measurements. a paired student’s t-test was performed to evaluate differences between selected months. results morphine induced no release measured on a monthly basis for one year revealed a average peak value of 39 ± 4 nm, during the late spring and early summer (fig. 1). the lowest no release occurred during the months of january (6.0 ± 0.5nm) through march (6.5 ± 1.1nm) (fig. 1). the lowest sea surface temperatures (1.3 °c) were also recorded in these same three months (fig. 1). student’s t-tests revealed a statistically significant difference (p = 0.002) between the warm season high no values and cold season low no values. relative gene expression was assessed by real time pcr. the mrna expression reached a relative peak during the month of june and was at its lowest in february and march (fig. 2). student’s t-tests revealed a statistically significant difference (p = 0.004) between the warm season high r values (1.2 ± 0.10) and cold season low r values (0.46 ± 0.052). a regression analysis using mu opiate receptor expression as the independent variable and no release as the dependent variable showed a correlation between the measurements. the calculated r value was 0.729. discussion m. edulis neural tissues contain the typical biogenic amines, which includes dopamine (stefano et al., 1976). biogenic amines display variations in their ganglionic levels, which corresponds to the seasons and temperature, being high in warmer months and low in the winter months (stefano et al., 1977a, 1977b; stefano and catapane, 1977a, 1977b). interestingly, this same relationship occurs with opioid peptide expression along with their receptors (stefano et al., 1980; stefano and leung, 1986). as demonstrated in this report, this same phenomenon involves no release, which is coupled to opiate receptor activation, namely via μ3 ( liu and stefano, 1996; liu et al., 1996; magazine et al., 1996; stefano and scharrer, 1996; stefano et al., 1996). the coupling of catecholamine, no and morphinergic signaling has recently been reviewed (kream et al., 2009; stefano and kream, 2009; stefano et al., 2009; zhu and stefano, 2009). it is important to note that dopamine is a morphine precursor in this animal, which synthesizes endogenous morphine ( zhu et al., 2005; kream and stefano, 2006). the significance of this precursor status of dopamine emanates from previously noted reports in this document showing seasonal and temperature alterations of catecholamine levels, which can now be directly compared to morphinergic phenomena, including the ability of morphine to release constitutively derived no. we surmise that at colder “winter” temperatures all processes appear to be down regulated, including the homeostasis between an opiate receptor and its ligand levels, as currently demonstrated. this probably occurs because very cold temperatures influence all metabolic processes to decrease their activity levels, including those involved in various survival processes. this homeostasis mechanism occurs in all living organisms, including those with a pathogenic ability. thus, in both types of organisms processes providing a survival benefit are not required. this 143 probably extends into the energy metabolic processes found in mitochondrial-like structures where morphine exerts actions (kream and stefano, 2009). in conclusion, the therapeutic value of performing medical operations, maintaining food, etc., at very low temperatures probably arises from the ability of low temperatures to disassociate adaptive cellular processes, allowing for a rather universal down regulation, which depending on the organism has tremendous survival advantage. in the case of mytilus, if bacteria, viruses can’t survive or have a decreased infectious characteristic, why have a full functioning immune process with the activation of cytokines and opiate components at low temperature ( stefano and scharrer, 1994; stefano and salzet, 1999; stefano et al., 2000, 2008; stefano and kream, 2008). references cadet p, mantione kj, stefano gb. molecular identification and functional expression of mu3, a novel alternatively spliced variant of the human mu opiate receptor gene. j. immunol. 170: 5118-5123, 2003. cadet p, zhu w, mantione k, baggerman g, stefano gb. cold stress alters mytilus edulis pedal ganglia expression of μ opiate receptor transcripts determined by real-time rt-pcr and morphine levels. brain res. mol. brain res. 99: 26-33, 2002. chrousos gp, gold pw. the concepts of stress and stress system disorders: overview of physical and behavioral homeostasis. jama 267: 12441252, 1992. fricchione gl, stefano gb. the stress response and autoimmunoregulation. adv. neuroimmunol. 4: 13-28, 1994. guarna m, bianchi e, bartolini a, ghelardini c, galeotti n, bracci l, et al. endogenous morphine modulates acute thermonociception in mice. j. neurochem. 80: 271-277, 2002. kream rm, mantione kj, sheehan m, and stefano gb. morphine's chemical messenger status in animals. activitas nervosa superior rediviva 51: 2009. kream rm, stefano gb. de novo biosynthesis of morphine in animal cells: an evidence-based model. med. sci. monit. 12: ra207-ra219, 2006. kream rm, stefano gb. endogenous morphine and nitric oxide coupled regulation of mitochondrial processes. med. sci. monit. 15: ra263-ra268, 2009. lee cs, spector s. changes of endogenous morphine and codeine contents in the fasting rat. j. pharmacol. exp. ther. 257: 647-650, 1991. liu y, shenouda d, bilfinger tv, stefano ml, magazine hi, and stefano gb. morphine stimulates nitric oxide release from invertebrate microglia. brain res. 722: 125-131, 1996. liu y, stefano gb. hiv gp120 inhibits human and invertebrate immunocyte phagocytosis. chinese j. immunol. 12: 139-142, 1996. magazine hi, liu y, bilfinger tv, fricchione gl, stefano gb. morphine-induced conformational changes in human monocytes,granulocytes, and endothelial cells and in invertebrate immunocytes and microglia are mediated by nitric oxide. j. immunol. 156: 4845-4850, 1996. mantione k, hong r, im r, nam jh, simon m, cadet p, et al. effects of cold stress on morphine-induced nitric oxide production and mu-opiate receptor gene expression in mytilus edulis pedal ganglia. neuroendocrinol. lett. 24: 68-72, 2003. marrazzi ma, luby ed, kinzie j, munjal id, spector s. endogenous codeine and morphine in anorexia and bulimia nervosa. life sci. 60: 1741-1747, 1997. sonetti d, mola l, casares f, bianchi e, guarna m, stefano gb. endogenous morphine levels increase in molluscan neural and immune tissues after physical trauma. brain res. 835: 137-147, 1999. stefano gb, catapane ej. seasonal monoamine changes in the central nervous system of mytilus edulis. experientia 33: 1341-1342, 1977a. stefano gb, catapane ej. the effects of temperature acclimation on monoamine metabolism. j. pharmacol. exp. ther. 203: 449546, 1977b. stefano gb, catapane ej, aiello e. dopaminergic agents: influence on serotonin in the molluscan nervous system. science 194: 539541, 1976. stefano gb, catapane ej, stefano jm. temperature dependent ciliary rhythmicity in mytilus edulis and the effects of monoaminergic agents on its manifestation. biol. bull. 153: 618629, 1977a. stefano gb, goumon y, casares f, cadet p, fricchione gl, rialas c, et al. endogenous morphine. trends neurosci. 9: 436-442, 2000. stefano gb, hiripi l, catapane ej. the effects of short and long term temperature stress on serotonin, dopamine and norepinephrine concentrations in molluscan ganglia. j. thermal biol. 3: 79-83, 1977b. stefano gb, kream r. endogenous opiates, opioids, and immune function: evolutionary brokerage of defensive behaviors. semin. cancer biol. 18: 190-198, 2008. stefano gb, kream rm. dopamine, morphine, and nitric oxide: an evolutionary signaling triad. cns neurosci. ther. 2009 [epub ahead of print]. stefano gb, kream rm, mantione kj, sheehan m, cadet p, zhu w, et al. endogenous morphine/nitric oxide-coupled regulation of cellular physiology and gene expression: implications for cancer biology. semin. cancer biol. 18: 199-210, 2008. stefano gb, kream rm, zukin rs, catapane ej. seasonal variation of stereospecific enkephalin binding and dopamine responsiveness in mytilus edulis pedal ganglia. in: rozsa ks (ed), neurotransmitters in invertebrates, pergamon press, london, pp 453-459, 1980. stefano gb, leung mk. opioid aging and seasonal variations in invertebrate ganglia: evidence for an opioid compensatory mechanism. in: 144 stefano gb (ed), comparative opioid and related neuropeptide mechanisms, crc press, inc., boca raton, pp 233-242, 1986. stefano gb, salzet m. invertebrate opioid precursors: evolutionary conservation and the significance of enzymatic processing. int. rev. cytol. 187: 261-286, 1999. stefano gb, salzet m, ottaviani e. neuroimmune chemical messengers and their conservation during evolution. in: rinkevich b, matranga v. 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155-160, 2001. zhu w, mantione kj, shen l, cadet p, esch t, goumon y, et al. tyrosine and tyramine increase endogenous ganglionic morphine and dopamine levels in vitro and in vivo: cyp2d6 and tyrosine hydroxylase modulation demonstrates a dopamine coupling. med. sci. monit. 11: br397-br404, 2005. zhu w, stefano gb. comparative aspects of endogenous morphine synthesis and signaling in animals. ann. ny acad. sci. 1163: 330-339, 2009. 145 research report seasonal variations in mu opiate receptor signaling in the nervous system of the blue mussel, mytilus edulis: temperature controls physiological processes isj 3: 89-96, 2006 isj 3: 111-117, 2006 issn 1824-307x research report the effects of okadaic acid on enchytraeus crypticus (annelida: oligochaeta) a franchini, m marchetti department of animal biology, university of modena and reggio emilia, modena, italy accepted november 29, 2006 abstract we describe the morpho-functional effects of different concentrations of okadaic acid (oa) on specimens of enchytraeus crypticus. the results demonstrate that this experimental model is very sensitive to the treatment and presents timeand dose-related effects mainly involving an immune response associated with a reaction in the chloragogenous tissue. at the lower dose (100 nm), the main organs do not appear particularly affected except for a swelling of the coelomatic cavity and an increased number of circulating coelomocytes. at the higher dose (200 nm), the chloragogenous tissue extends in volume to occupy the body cavity almost completely, while the circulating amoebocytes and chloragocytic cells undergo conformational changes. at the highest oa dose (400 nm), there is a general cell suffering in the main animal organs. in control animals, the immunocytochemical reaction with anti-il-6 antibody is positive in neuron cell bodies and fibres from the ventral nerve cord and in circulating amoebocytes. following oa treatment, fewer immunoreactive cells are seen in the damaged nervous tissue, and the high number of recruited amoebocytes is also positive. key words: annelid; enchytraeus crypticus; okadaic acid; il-6-like molecules introduction okadaic acid (oa) is produced by toxigenic dinoflagellates from the dinophysis and prorocentrum genera and is involved in fish death, diarrhetic shellfish poisoning and, consequently, human intoxication (yasumoto et al., 1978, 1985). the molecular mechanisms responsible of the various biological actions attributed to this toxin involve the specific inhibition of serine/threonine protein phosphatases 1 and 2a (bialojan and takai, 1988) which are critical components in signalling cascades regulating a variety of cellular processes in eukariotic cells. in vivo studies using mice as experimental models have described the distribution and excretion of oa following oral administration, as well as the morpho-functional modifications of toxin target organs (edebo et al., 1988; ito and terao, 1994; ito et al., 2002; franchini et al., 2005). the small intestine was particularly affected, with an edema in the lamina propria of villi and desquamation ___________________________________________________________________________ corresponding author: antonella franchini department of animal biology university of modena and reggio emilia via campi 213/d, 41100 modena, italy e-mail: franchini.antonella@unimore.it of degenerated epithelium, while the liver accumulated a significant amount of toxin, but was not damaged (ito and terao, 1994; ito et al., 2002). the mouse thymus was also particularly affected, with a severe alteration in structural architecture and a significant depletion in lymphoid elements. the ability of oa to provoke both immunostimulation and systemic immunotoxicity has also been highlighted (franchini et al., 2005). oa is a well known inducer of proinflammatory responses (stanley et al., 2001) and a stimulator of inflammatory cytokine gene transcription in murine macrophages (tebo and hamilton, 1994). in the present investigation, the effects of oa on the structural organization of a species of enchytraeids are evaluated. the enchytraeids have been widely used for many years in ecotoxicological laboratory tests, in view of their keystone ecological status and the fact that they can serve as indicator organisms for chemical stress (didden and rombke, 2001). we have also examined the induced modifications for the presence and distribution of il6-like molecules. il-6 is a multifunctional cytokine able to modulate a variety of physiological events that play a main role in the immune system and inflammation. 111 mailto:franchini.antonella@unimore.it materials and methods adult specimens of the worm enchytraeus crypticus (annelida, oligochaeta, enchytraeidae) were grown on agar medium for 1 month before the experimental procedure. the animals were cultured in 90 mm petri dishes containing 1.1 % agar powder dissolved in distilled water and maintained at a temperature of 23 °c with a photoperiod of 8 h light and 16 h dark. the worms were fed with sterilized powdered rolled oats and water twice a week, checked daily and subcultured by transfer onto a new agar plate once every 10-14 days. fig. 1 longitudinal sections from e. crypticus controls (a, b, d) and specimens treated with 100 nm oa for 48 h (c) and 200 nm oa for 12 h (e, f) stained with mallory-azan stain (b, c, e, f) and gallocyanin-chrome alum (a, d). the chloragogenous tissue (a, b) from controls formed one or two layers of round vacuolated and basophilic cells (d) surrounding the intestine. after oa treatment, the tissue showed a higher number of cell layers and expanded into the coelomatic cavity (e). the toxin also induced an increase in the number of circulating coelomocytes (c, e). note the enlarged and rounded circulating chloragocytic cells (f). brain, b; ventral nervous cord, v; chloragogenous tissue, c; coelomocytes, arrowheads; intestine, i. bar = 10 μm 112 thirty groups of 5 worms each were placed in 30 mm petri dishes and treated as follows: 3 groups of control animals were fed normally at the beginning of the experiment, while 27 groups received powdered rolled oats mixed with a water solution of oa (calbiochem, usa) at different final concentrations (100, 200 and 400 nm) for 12, 24 or 48 h. treated and control specimens were then collected, immediately fixed in bouin’s mixture and embedded in agar/paraffin, as previously described (franchini et al., 2003). the following histological and histochemical stains were performed on 7 µm transversal or longitudinal paraffin serial sections: hematoxylin-eosin and mallory-azan stains for general morphology, and gallocyanin-chrome alum reaction for nucleic acids (bancroft and gamble, 2002). the immunocytochemical procedure was carried out on controls and the variously treated e. crypticus using goat anti-il-6 polyclonal antibody (r & d systems, usa) diluted 1:500. sections were incubated overnight at 4 °c in the primary antibody, fig. 2 longitudinal sections from e. crypticus treated with 200 nm oa for 48 h (a-c) and 400 nm oa for 48 h (df) stained with mallory-azan stain (d-f) and gallocyanin-chrome alum (a-c). brain, b; ventral nervous cord, v; chloragogenous tissue, c; clamped coelomocytes, arrows; intestine, i. bar = 10 μm. 113 and immunoreactivity was visualised by an immunoperoxidase technique using avidin-biotin peroxidase complex (hsu et al., 1981). controls of the immunocytochemical reactions were performed by substituting the primary antibodies with nonimmune sera and/or absorbing the antibody with homologous antigen. results examination of the histological sections revealed that e. crypticus is sensitive to oa treatment in a timeand dose-related manner. at the lower dose (100 nm), the main organs did not appear particularly affected, except for a swelling of the coelomatic cavity, where, in comparison to controls, an increased number of circulating coelomocytes was observed after 48 h (figs 1a-c). in e. crypticus, two main cell types were seen floating freely in the body cavity, a more numerous population of round or mostly elongated coelomocytes presenting a cytoplasm filled with large inclusions (oligochaete designed chloragocytic cells), and smaller cells devoid of inclusions with an irregular cytoplasm (oligochaete designed amoebocytes). in control worms the chloragogenous tissue presented one or two layers of large, vacuolated and basophilic cells surrounding the intestine (fig. 1d). at the higher oa dose (200 nm) and after 12 h of treatment, this tissue differed in morphology, extended in volume, and an increased number of cell layers was observed (fig. 1e). these cells were irregularly arranged and without cytoplasmic basophilia, while some were detached from the tissue and surrounded by amoebocytes that had undergone conformational changes into an active form. the circulating chloragocytic cells also changed morphology and became enlarged and rounded in shape (fig. 1f). after 48 h, the cells in the chloragogenous tissue appeared highly vacuolated and tended to break. they occupied the body cavity almost completely, so that the coelomocytes were concentrated in the prostomium, where the tissue was not present (figs 2a-c). the nervous system was also affected by a reduction in the nervous area, in particular in the ventral nerve cord extending through the entire animal length. at the highest oa dose (400 nm), a general cell suffering was found, i.e. the extended, irregularly shaped chloragogenous tissue cells embedded clumped coelomocytes (figs 2d, e), the epithelial cell layer of the medio-posterior intestine increased in surface area (fig. 2f), nephridia appeared disorganized in structure, and the ventral nerve cord comprising wrinkled nerve cells presented fewer neuronal cell bodies as a result of the invasion of expanding chloragogenous tissue (figs 3a, d). in control animals, the immunocytochemical reaction with anti-il-6 antibody was positive in the nervous system. in particular, the antibody labelled neuron cell bodies and fibres located in the ventral nerve cord (fig. 3a) and in the anterior ganglia near the mouth, but not brain nerve cells. one population of the circulating coelomocyte, i.e amoebocytes, was also positive (figs 3b, c). after oa treatment, fewer immunoreactive cells were seen in nervous tissue (fig. 3d) and the large number of recruited amoebocytes was positive, especially when seen in an active form with large and heterogeneous vacuola inside the cytoplasm (figs 3e-g). discussion the results from the present study demonstrate that the experimental model used is very sensitive to treatment with oa. the toxin induced timeand dose-related effects that mainly involved an immune response associated with a reaction in the chloragogenous tissue. the multi-functional cells in oligochaete chloragogenous tissue are known to be able to store glycogen, lipids and exogenous materials, such as pigments or metals, and have also been associated to immune defenses (needham, 1966; roots and johnston, 1966; prentø, 1979; cooper 1981; morgan 1981). in these cells, the chloragocytes, two cellular compartments have been implicated in metal trafficking: the cytoplasm organelles, called chloragosomes, undergo changes in autophagic derivatives, the debris vesicles. the subcellular modifications were found to be closely correlated with accumulated metal burdens in the tissue (morgan and turner, 2005). following oa treatment, the tissue increases its volume considerably and shows morphological changes in the cells. the earthworm’s chloragogenous tissue has some liver-like properties (laverack, 1963; prentø, 1987), and in e. crypticus, it may be involved in toxin accumulation and detoxification in a dose-related manner. the induction of a protein in response to toxin exposure, possibly related to detoxification reactions, has been demonstrated in the digestive gland of mytilus galloprovincialis (auriemma and battistella, 2004). oa also induces an inflammatory cell response, and the number of circulating coelomocytes, known to be responsible for the animal’s immune defence responses (valembois et al., 1982; vĕtvička and šima, 1998), increases both in the chloragocytic cell population, which originates in the chloragogenous tissue surrounding the intestine (fischer, 1993), and in the amoebocytes that change to an active form and show greater immunoreactivity to anti-il-6 antibody. oa is a well-known specific inhibitor of serine/threonine protein phosphatase 1 and 2a. these molecules have been found to act as endogenous regulators of inflammatory cell signalling (shanley et al., 2001), and to play a role in the control and stimulation of proinflammatory cytokine gene expression by murine mononuclear phagocytes and other cell types (falk et al., 1994; tebo and hamilton, 1994; wakiya and shibuya, 1999; feng et al., 2006). the enhanced inflammatory reaction is associated, through a protein phosphatase 2a-mediated mechanism, with the regulation of the c-jun nh2-terminal kinase, one of the major mitogen-activated protein kinase signalling pathways (shanley et al., 2001; avdi et al., 2002). it has also been suggested that the dynamic interplay between kinases and phosphatases modulates the activity of several proteins that are crucial in neuronal functions. oa provokes effects in the structure of the nervous system, while the protein hyperphoshorylation due 114 fig. 3 sections from e. crypticus controls (a-c) and specimens treated with 100 nm oa for 24 h (e), 200 nm oa for 24 h (f-h) and 400 nm oa for 48 h (d) immunostained with anti-il-6 polyclonal antibody and haematoxylin nuclear counterstaining. negative controls of the immunocytochemical reaction (h). ventral nervous cord, v; chloragogenous tissue, c; negative chloragocytic cells, arrows; positive amoebocytes, arrowheads. bar = 10 μm. 115 to the inhibition of phosphatases 1, 2a and possibly also calcineurin is known to induce neuronal stress and subsequent neurodegeneration (tapia et al., 1999; arias et al., 2002; ramirez-munguia et al., 2003). acknowledgement we thank prof. g pürschke (osnabruck university, germany) who kindly supplied the enchytraeus crypticus population and prof. e ottaviani (university of modena and reggio emilia, modena, italy) for critical reading of the manuscript. references arias c, montiel t, pena f, ferrera p, tapia r. okadaic acid induces epileptic seizures and hyperphosphorylation of the nr2b subunit of the nmda receptor in rat hippocampus in vivo. exp. neurol. 177: 284 -291, 2002. auriemma r, battistella s. biochemical and histological alterations of mytilus galloprovincialis digestive gland after exposure to okadaic acid and derivatives. inv. surv. j. 1: 66 -71, 2004. avdi nj, malcolm kc, nick ja, worthen gs. a role for protein phosphatase-2a in p38 mitogenactivated protein kinase-mediated regulation of the c-jun nh2-terminal kinase pathway in human neutrophils. j. 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nappi department of biology, loyola university chicago, chicago, illinois, usa accepted september 17, 2010 abstract various cellular innate immune responses protect invertebrates from attack by eukaryotic pathogens. in insects, assessments of the factor(s) causing, or contributing to, pathogen mortality have long considered as toxic components certain molecules associated with enzyme-mediated melanogenesis. in drosophila hosts, observations that have prompted additional or alternative considerations are those that document either the survival of certain endoparasitic wasps despite melanotic encapsulation, or the destruction of the parasite with no evidence of this type of host response. investigations of the production of some reactive intermediates of oxygen and nitrogen during infection provide a basis for proposing that these molecules constitute important elements of the immune arsenal of drosophila. studies of the target specificity of virulence factors injected by female wasps during infection that suppress the host immune response will likely facilitate identification of the toxic host molecules, and contribute to a more detailed understanding of the cellsignaling pathways that regulate their synthesis. key words: cellular innate immunity; reactive intermediates of oxygen; nitric oxide; melanization; encapsulation; drosophila introduction because of competition for some of the same limited metabolic resources, the outcome of the combative interaction between host and pathogen depends in large part on the effectiveness of apposing physiological, biochemical, and behavioral responses. in destroying pathogens, vertebrate hosts benefit from the collaborative interactions of two distinct, but not entirely separate, immune systems; adaptive and innate. the adaptive immune system produces an almost limitless repertoire of pathogen-specific responses, enabled in large part by considerable genetic plasticity that produces specific cell surface receptors, immunoglobulins, and cells possessing immune memory that rapidly initiate and enhance subsequent responses to the same antigen. insects and other invertebrates rely exclusively on innate immune responses, which many authors regard as the first line of defense. these responses are considered to be dependent on constitutive (i.e., germ-line encoded) and dedicated cell membrane-bound pattern recognition ___________________________________________________________________________ corresponding author: anthony j nappi department of biology, loyola university chicago, chicago, illinois, usa e-mail: anappi@luc.edu receptors with limited responsiveness to invariant molecular motifs of certain pathogens (pal and wu, 2009). some of the invertebrate innate immune effector responses elicited by prokaryotic infections include phagocytosis, hemolymph coagulation, the synthesis of pro-inflammatory cytokines, antimicrobial peptides, reactive intermediates of oxygen (roi) and nitrogen (rni), and stress-related proteins (nappi and vass, 2001; beutler, 2004; malagoli and ottaviani, 2007; malagoli et al., 2007, 2010; becker et al., 2010). eukaryotic pathogens succumb to an encapsulation response that is mediated in large part by macrophage-like blood cells (hemocytes). in insects and other arthropods, hemocyte-mediated encapsulations characteristically are accompanied by the synthesis of melanin, with intermediates such as quinones and semiquinones considered potentially toxic to invading pathogens (nappi and ottavani, 2000a; nappi and christensen, 2005; poirie et al., 2009) (fig. 1). in both adaptive and innate systems, the binding of foreign elements to cell-surface receptors leads to the activation of signal transduction pathways, the transcription of immune genes, and the generation of reactive cells and various toxic molecules. as counter strategies, some pathogens produce virulence factors that actively suppress immune responses, while others 198 fig. 1 overview of some toxic molecules manifested in the innate immune responses of various invertebrates. non-self recognition may involve plasma membrane receptors independently functioning, or cooperatively engaging non-self binding molecules in the host’s hemolymph. melanotic encapsulation, which is a common manifestation of the defense reaction made by arthropods infected with eukaryotic pathogens, involves activation of one or more of the following enzymes; dopa decarboxylase (ddc), dopachrome conversion enzyme (dce), phenylalanine hydroxylase (pah), and phenoloxidase (po). enzymes capable of generating reactive intermediates of oxygen (roi) and nitrogen (rni) include myeloperoxidase (myelo-px), nadph oxidase (nadph ox), nitric oxide synthase (nos), and superoxide dismutase (sod). melanogenic intermediates such as quinones and semiquinones can react with roi, rni and the active centers of certain metaloenzymes to contribute additional toxic molecules. passively avoid detection, either by molecular mimicry or by finding sanctuary within host tissues. the focus of this review concerns some unresolved aspects of the cellular innate immune responses of drosophila against certain endoparasitic wasps, including the nature of the toxic molecules generated during infection, as well as the mechanisms employed by pathogens to circumvent these potentially damaging molecules. drosophila-parasitic wasp interactions the availability of well-defined resistant and susceptible species and strains of drosophila, together with both virulent and avirulent lines of endoparasitic wasps (parasitoids), provide exceptionally good models for investigating not only the genetic and biochemical components of insect cellular innate immunity, but also the varied processes by which parasitoids deal with such reactions (fig. 2). leptopilina boulardi and l. heterotoma are two closely related wasp species that parasitize larvae of drosophila with varying degrees of success (vass and nappi, 2000; dubuffet et al., 2007, 2008, 2009). eggs of avirulent wasps characteristically provoke a rapid hemocytemediated melanotic encapsulation response when introduced into the hemocoel of resistant drosophila, 199 fig. 2 leptopilina boulardi and l. heterotoma parasitize larvae of drosophila. genetically resistant hosts exhibit a typical hemocyte-mediated melanotic encapsulation response. susceptible host have a faulty non-self recognition mechanism or fail to produce effective toxic responses. immune suppressive factors (isf) in the venom of virulent wasps block host cellular immune response. an atypical host response exhibited by d. paramelanica readily destroys l. heterotoma, but the response does not involve melanotic encapsulation. fig. 3 comparative hemocyte profiles illustrating involvement of these cells in the host response, and the immune suppressive effects manifested by virulent wasps. 200 fig 4 genetic complexity exhibited by the varying degrees of immune reactivity and parasite survival in different species and stains of drosophila and leptopilina (courtesy s dupas, m poirié, y carton, laboratoire evolution, génomes et spéciation, cnrs, gif-sur-yvette cedex, france). whereas the eggs of virulent wasps survive host defenses. melanin typically appears at the site of infection, generally just before or at about the same time hemocytes are observed adhering to the surface of the dead parasitoid. the drosophila encapsulation response involves the collaborative activities of three types of hemocyte: plasmatocytes, lamellocytes and crystal cells. comparative examinations of hemocyte profiles show significantly elevated numbers of hemocytes in resistant or immune competent hosts (fig 3). during infection, plasmatocytes and lamellocytes show precocious increases in numbers as they participate in the formation of the cellular 201 components of the capsule, while crystal cells, which represent an important source of melanin precursors, decline in number (nappi and streams, 1969). some of the immune-activated hemocytes participating in the encapsulation response appear to be recruited from those already in circulation at the time of infection, while others are mobilized from hematopoietic glands (i.e., lymph glands) (lanot et al., 2001; sorrentino et al., 2002; meister and lagueux, 2003; meister, 2004; carton et al., 2005, 2008; crozatier and meister, 2007; honti et al., 2010) and/or a subepidermal population of normally sessile cells ( krzemien et al., 2007; markus et al., 2009). the genetic complexity of the drosophila-wasp associations is illustrated by the varied outcomes of the combative interactions made by different species and strains of both host are parasite (fig 4). the gene for host resistance, which is associated with the second chromosome, is specific for each species of wasp. reciprocal chromosome exchange between resistant and susceptible host strains virtually completely reverses immune competence in each recipient. immune competence is later restored following reciprocal return of the chromosome (fig. 5). during oviposition, wasp venom also is introduced into the host hemocel. virus-like particles (i.e., immune suppressive factors, isf) in the venom protect the wasp egg from encapsulation, either by lysing host hemocytes, or interfering with essential transcriptional responses so as to abrogate or diminish host responses. unlike isf of virulent wasps, those present in the venom of avirulent species and strains exhibit little or no immune suppressive effect (labrosse et al., 2003; kohler et al., 2007). in experiments involving double infections, first by avirulent wasps followed by a virulent strain of l. boulard, isf from virulent parasitoids were found capable of also protecting avirulent wasps, provided the interval between infections is 12 hrs or less (fig. 6). also, the ability of leptopilina spp. to immune suppress depends on her egg-laying experience. during the latter part of the ovipositional period of l. boulardi, eggs introduced into d. melanogaster larvae are more susceptible to melanotic encapsulation than are eggs laid earlier (fig. 7). the decrease in immune suppression presumably correlates with a corresponding depletion of isf (vass and nappi, 1998). wasps with prior ovipositional experience not only lack or have a diminished capacity to immune suppress, but they also infect far fewer hosts than females with no prior ovipositional experience. if such ovipositional restraint retains eggs that would otherwise be encapsulated, selection pressure in host populations for evolving specific immune reactivity would be reduced. melanization and associated toxic molecules in immune reactive insects, melanin appears in many cases to occur concurrently with early capsule formation, an observation that has long been viewed as evidence that the proteinase cascade leading to activation of one or more enzymes involved in fig. 5 reciprocal exchange of resistant and non-resistant gene reverses the immune capacity of the recipient, which can be restored in subsequent exchanges (carton and nappi, 1997). 202 fig. 6 in superparasitized hosts, immune suppressive factors from virulent parasitoids also protect avirulent wasps provided the interval between infections is 12 hrs or less. fig. 7 the effects of prolonged oviposition on the diminishing capacity of isf from l. boulardi (vass and nappi, 1998) and l. heterotoma (streams, 1968) to suppress the immune response of d. melanogaster. wasp eggs introduced into hosts by females during the latter part of their ovipositional period are more susceptible to destruction than eggs laid earlier. the increase in parasitoid mortality is believed to result from a decline in isf. 203 catechol metabolism and pigment synthesis forms toxic molecules that target and destroy foreign organisms (nappi and vass, 2001; sugumaran, 2002; nappi and christensen, 2005; sideri et al., 2008; an et al., 2009; bidla et al., 2009; nappi et al., 2009). support for this proposal derives in large part from studies showing diminished immune responsiveness when components of the enzymeregulated melanin pathway are experimentally inhibited. reservations about such an interpretation concern the questionable specificity and excessive levels of agents injected into the host to inhibit and thereby demonstrate involvement of melanin intermediates in immune reactions. frequently overlooked in studies assessing the role of melanin in insect immunity is the initial enzyme-mediated reaction involving the hydroxylation of l-phenylalanine to l-tyrosine, a reaction catalyzed by phenylalanine hydroxylase (pah). ensuing oxidations of l-tyrosine and/or ldopa, which can be catalyzed either by phenoloxidase (po; terland et al., 2006), or peroxidase (per; kasraee, 2002; okun, 1996), generate dopaquinoine, a reactive intermediate essential for the formation of eumelanin, a brownish-black pigment, and, in the presence of sufficient levels of thio compounds, pheomelanin, a reddish-brown pigment. precursors of both pigments possess cytoprotective and cytotoxic properties, given their capacity to scavenge potentially toxic organic and inorganic cations and free-radical species, engage in metal-binding and sequestering responses, initiate redox reactions, cross-link proteins and mediate detoxification processes. to date, only eumelanin has been identified as the pigment type formed in the encapsulation response of d. melanogaster (nappi et al., 1992). following the formation of dopaquinone, a series of enzyme-regulated and/or spontaneous oxidoreductions occur yielding dopachrome and fig. 8 overview of the principal pathways involved in the formation of eumelanin and pheomelanin and some their reactive intermediates, including quinones and semiquinones. redox cycling and univalent transfers, which represent important mechanisms for generating cytotoxic molecules, also occur with dhi-derived indolequinone (iq) and indolesemiquine (not illustrated). insects apparently are incapable of forming dhica. 204 fig. 9 effects of injection of spn27a on the percentage of d. melanogaster hosts exhibiting a successful melanotic encapsulation of l. boulardi. enzymes involved in melanin synthesis include dopa decarboxylase (ddc), dopachrome conversion enzyme (dce), phenylalanine hydroxylase (pah), peroxidase (per), and phenoloxidase (po). additionally potentially cytotoxic eumelanin intermediates, including 5,6dihydroxyindole (dhi), 5,6-dihydroxyindole-2-carboxylic acid (dhica), and their respective indole quinones (iq, and iqca) (fig. 8). the dopa decarboxylase (ddc)-mediated pathway to dhi may be a principal route for production of pigment precursors in infected drosophila, as the melanotic encapsulation response against eggs of l. boulardi is severely compromised in temperature-sensitive ddcdeficient mutants (nappi et al., 1992) accordingly, it was recently shown that silencing the genes for ddc and dopachrome conversion enzyme (dce) significantly reduced melanization of foreign objects implanted in the mosquito anopheles gambiae (paskewitz and andreev, 2008). in the medfly ceratitis capitata, ddc-dependent pathways have been shown to regulate such immune functions as phagocytosis, nodulation and melanization by hemocytes (sideri et al., 2008). 205 fig. 10 l. boulardi isf diminishes the in vitro oxidations of two diphenol eumelanin precursors, dopamine and dhi. tyrosine and the diphenols dopa and dhica are not affected by isf. parasite suppression of host melanization melanization in insects is controlled by a cascade of serine proteases that ultimately activates prophenoloxidase (ppo) and leads to activated phenoloxidase (po) and pigment formation (tang et al., 2006, 2008; scherfer et al., 2008; tang, 2009). in drosophila, melanization induced by activated po is a tightly regulated reaction sequence (aggarwal and silverman, 2008; kan et al., 2008) involving at least three ppo isoforms, as well as serine protease inhibitors. two isoforms are expressed in crystal cells, the third is associated with lamellocytes (kan et al., 2008). an important regulating element in the cascade of proteolytic cleavages that converts ppo to po is the serine protease inhibitor serpin 27a (spn27a), which inhibits the terminal protease prophenoloxidase-activating enzyme (ppae) (de gregorio et al., 2002; nappi et al., 2005) (fig. 9). because of the critical role played by spn27a as a negative regulator of melanogenesis, the molecule and the signaling elements mediating its activity likely represent critically important factors in determining immune reactivity against leptopilina. this was established by experiments involving the introduction of spn27a into immune competent d. melanogaster larvae just before infection by l. boulardi. in these hosts, the ability to form melanotic capsules was significantly reduced. the specificity of action of spn27a establishes some of the components of the po-mediated pathway in the insect's defense response against l. boulardi (fig. 9). more recent comparative investigations using isf from virulent and avirulent wasps provide additional evidence that isf inhibits melanization in d. yakuba by affecting one or more steps in the cascade leading to po activation, but not po activity by itself (dubuffet et al., 2009). other experiments designed to determine if venom factors from l. boulardi targeted the principal oxidation pathways leading to synthesis of eumelanin, sensitive electrochemical detection methods showed that venom factors diminished the oxidations of the two diphenol eumelanin precursors, dopamine and dhi, while oxidations of the monophenol tyrosine, and two other related 206 fig. 11 comparative analyses of hemocytes and nitric oxide levels in infected and non-infected larvae of d. paramelanica, and the effects of introducing a nos inhibitor (ng-monomethyl-l-arginine on the fate of l. heterotoma (nappi et al., 2009). diphenols, dopa and dhica, were not significantly inhibited (kohler et al., 2007) (fig. 10). collectively, these related studies suggest that, in addition to targeting specific hemocytes, isf from leptopilina spp. specifically suppresses the oxidation pathways synthesizing certain pigment precursors, especially the decarboxylated pigment precursors derived from dhi. reactions lacking evidence of the involvement of melanization it is generally believed that at least some of the melanogenic enzymes and intermediate pigment products play a role in the defense reactions of insects (cerenius and soderhall, 2004; christensen et al., 2005; nappi et al., 2009), although this issue 207 still remains to be clarified (schnitger et al., 2007). reports that would appear to discredit or at least down-play the role of melanogenesis in insect immunity and prompt additional or alternative proposals are those that document parasite mortality prior to, or in the absence of, melanotic encapsulation (tardieu and rabasse, 1988; henter and via, 1995; vernick et al., 1995), and those that clearly show successful parasite development despite extensive melanotic responses (shin et al., 2003). in the d. paramelanica–l. heterotoma association, eggs of the endoparasite succumb with no evidence of blood cell-mediated encapsulation and no pigment reaction (fig. 2) (nappi and streams, 1970; carton et al., 2008). if melanization is not a universal feature of insect cellular immunity, the destruction of some pathogen must involve host molecules other than those associated with melanogenesis, and one would expect successful parasites to have evolved specific inhibition strategies that suppress or detoxify such potentially biochemically hostile reactions. although identity of the cytotoxic molecules remains unknown, attention has focused on roi and rni, given that elevated levels of some of these molecules have been found in immune responsive hosts (luckhart and li, 2001; foley and o'farrell, 2003; novas et al., 2004; whitten et al., 2007; molina-cruz et al., 2008), including those in which hemocyte-mediated melanotic encapsulation reactions are typically formed (nappi and vass, 1998, 2001a, b; nappi et al., 1995, 2000b), and in wasp-infected d. paramelanica where parasites are destroyed but no melanotic capsules are produced (carton et al., 1992). potentially damaging roi and rni can form during normal metabolism as a result of successive univalent reductions of molecular oxygen. initially, superoxide anion is produced, with subsequent electron transfers ultimately generating highly reactive and potentially cytotoxic molecules, including the hydroxyl radical (.oh-), peroxynitrite (onoo–) and hypochlorous acid (hocl-). interestingly, melanogenic intermediates may serve to promote or augment cytotoxic activity by reacting with certain transition metal ions, roi and rni (fig. 1). the univalent oxidations of redox active o-diphenols (qh2) such as l-dopa and dopamine by po and/or per can form semiquinones (.qh-) and quinones (q), which then can interact with roi and rni. reactions involving per and tyrosine can lead to the production of potentially injurious molecules, such as the tyrosyl radical, dityrosine, and tyrosine peroxide (fig. 1), without producing melanin. an important issue to consider is that the production of toxic molecules in response to infection must by a tightly regulated and localized reaction in order to avoid damage to nonspecific sites within the host's open circulatory system. the binding of the copper-containing po or the heme-containing per to pathogens would expose the metal active sites of these enzymes, a response that would facilitate their interaction with roi and rni and form .ohand other reactive molecules, and also serve to localize metal ionmediated cytotoxicity. because of its intrinsic coordination properties, copper can induce a more site-specific .ohcytotoxicity to bound ligands than can iron (berthon, 1993). recent studies (carton et al., 2009) support earlier reports that document the involvement of .no in mediating various toxic responses in drosophila and other invertebrates. nitric oxide is a well known signaling molecule associated with certain innate immune pathways. the radical serves an equally important role as a toxic effector molecule in eliminating pathogens (nappi and ottavani, 2000a; nappi et al., 2000b; luckhart and li, 2001; dimopoulos, 2003; han et al., 2009). in d. paramelanica where elevated levels of .no are produced almost immediately following infection by l. heterotoma, immune capacity is diminished when a specific nitric oxide synthase (nos) inhibitor is introduced in larvae prior to infection (fig. 11). these observations suggest .no is involved in the host immune response, either a critical signaling molecule in recruiting hemocytes to sites of infection, or as a component of the insect's arsenal of defense, given the capacity of the radical to readily react with various roi and rni. conclusions the associations between host and pathogen represent coevolved adaptations of great complexity. insects typically manifest a unique defense response against metazoan parasites that involves hemocyte-mediated melanotic encapsulation. the use of melanin for protection from foreign insult involves a multifaceted biochemistry and an equally complex genetic regulation. an equally fascinating component of insect host-parasitoid combative relationships is the ability of some wasp species and strains to develop unmolested within otherwise immune competent hosts. either such parasitoids evolve with passive immune evasion strategies that effectively preclude host detection, or with the capacity to actively combat and render ineffective host defenses. despite numerous descriptive accounts of non-self responses and associated cell-signaling molecules that summon blood cells to sites of infection, much remains to be learned about the identity of the killing molecules employed by insect hosts, knowledge of which 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dc, tandler b, aikawa m, miller lh. plasmodium gallinaceum: a refractory mechanism of ookinete killing in the mosquito, anopheles gambiae. exp. parasitol. 80: 583-595, 1995. whitten m, sun f, tew i, schaub g, soukou c, nappi a, et al. differential modulation of rhodnius prolixus nitric oxide activities following challenge with trypanosoma rangeli, t. cruzi and bacterial cell wall components. insect biochem. mol. biol. 37: 440-452, 2007. 210 comparative aspect of propo system in invertebrates isj 6: s67-s76, 2009 issn 1824-307x review the ascidian prophenoloxidase activating system m cammarata, n parrinello marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy accepted march 13, 2009 abstract phenoloxidases/tyrosinases initiate melanin synthesis in almost all organisms, and are involved in different biological activities such as the colour change of human hair and the browning or blackening of fruit skin etc. in many invertebrates, defence reactions are linked to phenoloxidase activity and/or melanization. contacts with foreign molecules are able to trigger the prophenoloxidase (propo) system that requires serine protease cleavage for activating the zymogen to phenoloxidase (po). it is generally accepted that the propo system is fully expressed in arthropods, and, recently, progress in the regulation of crustacean and insect propo activation steps have been achieved. after cells were stimulated by components of pathogen associated molecular pattern (pamp), propo activation takes place via zimogenic serine proteinase in turn activated by pamps followed by cascade, spatial and temporal control. the propo activating system plays a defensive role in arthropods, molluscs, annelids, ascidians and the cephalochordate branchiostoma belcheri. in the present paper, we report on ascidian propo system and related molecules, with particular focus on the biochemical, cellular and molecular aspects of the ciona intestinalis, propo system of circulating hemocytes from naïve ascidians as well as of body wall following lps inflammatory challenge. key words: ciona intestinalis; ascidians; propo; phenoloxidase; immune response; hemocytes phenoloxidases and related enzymes melanin is a pigment ubiquitous throughout the animal kingdom. in invertebrates melanization is related to phenoloxidase (po) and in part to tyrosinase, both are copper-dependent enzymes (monophenol, l-dopa; oxygen oxidoreductase; ec 1.14.18.1), that share similar active sites and catalyse the o-hydroxylation of monophenols (monophenoloxidase or cresolase activity) and the subsequent oxidation of the reaction products (odiphenols) to quinones. thus, substances forming copper ion complexes can be enzyme inhibitors (kahn, 1985; sugumaran et al., 1988). tyrosine is the natural substrate of tyrosinase, which exhibits a lag–phase during the tyrosine conversion ascribed to an autocatalytic mechanism due to the production of l-dopa in the initial phase of the reaction pathway (lerner, 1949). ________________________________________________________________________ corresponding author: matteo cammarata marine immunobiology laboratory department of animal biology university of palermo via archirafi, 90123 palermo, italy e-mail: camat@unipa.it vertebrate tyrosinases form dimers whereas pos, only found in invertebrates, form oligomers, from monomers to pentamers. although, both present two sites containing copper vary in their remaining sequence. in invertebrates, the prophenoloxidase (propo) is converted to po by proteolytic cleavage. the activation depends upon a cascade due to hemolymph proteases which are sensitive to peptidoglycans and lipopolysaccharides (lpss) or other bacterial carbohydrates or fungal β-glucans. in crayfish hemocytes propo is a 76 kda glycoprotein that, after activation by β-1.3-glucans, is cleaved by specific serine proteases to produce the 60 kda active po (aspan and söderhäll, 1991). a similar cascade has been reported in other invertebrates (beschin et al., 1998; parrinello et al., 2001; lunagonzales et al., 2003). molecular analysis of po and related protein pos can be sharply distinguished from tyrosinase and an independent evolution with short sequence traits conservation has been proposed. s67 fig. 1 a. the conserved domain architecture of c. intestinalis phenoloxidases performed by similarity searches of the ncbi entrez protein database (cdart). b. sequence alignments of c. intestinalis prophenoloxidases for comparison with arthropod pos, hemocyanins and various tyrosinases. the sequences shown are segments corresponding to the copper a and b binding sites of the hemocyanins. the homologous aa are shown in boldface, the three histidine residues (h) participating in the cu atom are shown in red. conversely, a close similarity between arthropod phenoloxidases and hemocyanin, an oxygen carrier protein, has been shown (söderhäll et al., 1996). both proteins present sequence similarity and contain two oxygen-binding sites that reversibly bind two copper atoms (decker and tuczek, 2000), moreover hemocyanin-like proteins can act as phenoloxidases (immesberger and burmester, 2004). like propo, hemocyanins can be activated in vitro by sds, trypsin and other effectors with denaturating property (decker et al., 2001; lee et al., 2004), or by chitin-binding antimicrobial peptides (nagai et al., 2001). this activation has been imputed to the cleavage of crayfish hemocyanin subunit 2 at the n-terminal part (lee et al., 2004). decker et al. (2004) described similar results on the tarantula hemocyanin, and hypothesized that after n-terminal cleavage the hemocyanin active site becomes accessible also for phenolic substances. propos from different arthropods have been cloned and sequenced (aspan et al., 1995; fujimoto et al., 1995; hall et al., 1995; kawabata et al., 1995). comparative sequence analysis (söderhäll et al., 1996), including several copper-containing proteins, showed that propos disclose sequence similarity to hemocyanins higher (49-59%) than to tyrosinases (3335 %) (parrinello et al., 2003). s68 table 1 possible genes involved in the c. intestinalis propo activated system cinpo-1 cinpo-2 peroxinectin-like gene cu-zn sod-like gene mrna aj7547813 aj7547814 predicted xp_002126285 predicted xp_002122526 protein complete; 794 aa partial at n-term; start atg absent. 774 aa (768 aa in jgi:279870) complete; 960 aa complete; 154 aa putative size (kda) 92.0 86.9 105.0 15.6 domain/binding sites cu binding sites cu binding sites peroxidase/cell binding site cu-zn reference burmester et al 2003 burmester et al 2003 present paper present paper immesberger and burmester (2004) cloned and sequenced two ciona intestinalis po cdnas (cinpo-1 and cinpo-2). cinpo-1 and cinpo-2, with predicted molecular masses of 92.0 kda and 86.9 kda respectively, showed 43.2 % identity and do not contain signal peptides indicating a non classical release. figure 1 shows the sequence alignment of c. intestinalis po sites compared to arthopod pos, hemocyanins and some tyrosinases, and displays a close similarity between pos and hemocyanins. a third putative cinpo showing high similarity to cinpo-2 had been identified by a search on jgi ciona intestinalis v2 as cinpo-3 (cammarata et al., 2008). however, after a further analysis cinpo-3 appeared to be a product of an uncorrect assembly of the sequences in the v2 genome version. in fact, a long stretch of undetermined nucleotides, at the scaffold end, explained the assumed high similarity of introns and esons as well as of the presumptive protein sequences. propo activating system the first defence line of the innate immunity includes po pathway products that participate in several responses such as melanization and encapsulation, cytotoxicity, phagocytosis, clotting, microbial killing and wound repair (söderhäll, 1982; cammarata et al., 1997; gillespie et al., 1997; huang et al., 2000; nagai and kawabata, 2000; nappi and ottaviani, 2000; cerenius and söderhäll, 2004; jiravanichpaisal et al., 2006; cerenius et al., 2008). it has been proposed that a molecular crosstalk takes place between the propo system and cellular defence responses which share activation by microbial products signals, such as coagulation and blood cell degranulation (lemaitre and hoffmann 2007; cerenius et al., 2008). crustacean granular and semigranular hemocytes contain propo, and, upon exposure to bacteria, they undergo degranulation in vitro leading to exocytosis (johansson and söderhäll, 1989b, 1999). in crayfish, pacifastacus leniusculus, the 76kda peroxinectin, purified from the hemocytes (johansson et al., 1995), mediates the attachment and spreading of hemocytes in vitro (johansson and söderhäll, 1988), and stimulates degranulation events when added to granular cells monolayer (johansson and söderhäll, 1989a, b). peroxinectinlike is a cell adhesion protein also detected in shrimp (poeneus monodon) hemocyte lysate supernatant (sritunyalucksana et al., 2001). sequence comparison shows that the shrimp protein is similar to crayfish peroxinectin (69 %) and to various peroxidases or putative peroxidases from invertebrates and vertebrates. since the biological effect of crayfish hemocyte peroxinectin is mimicked by the peptide gly-arg-glyasp-ser (grgds) (johansson and söderhäll, 1989c), the possibility exists that crayfish hemocyte integrin-like receptors recognize and bind rgd or kdg (rouslahti, 1996; holmblad et al., 1997). vertebrate integrins form a family of integral membrane proteins that act in cell-cell adhesion and as receptors in trans-membrane signalling (hines, 1992). peroxinectin may act as opsonin, and promote the adhesion of bacteria or other particles to the phagocyte surface, facilitating their subsequent ingestion by the cell. peroxonectin also binds cuzn-superoxide dismutase (cuznsod) at the surface of circulating hemocytes, and this interaction, facilitated by the close localization, modulates both the enzyme activities. the hydrogen peroxide, produced by the superoxide dismutases, can be substrate for the peroxinectin and antimicrobical substances (johansson et al., 1999). therefore, the cuznsod is involved in arthropod propo activating system. we carried out a bioinformatic analysis and, in table 1, show for the first time that the c. intestinalis predicted peroxinectin-like gene contains both the active site of the peroxidase and the cell binding site (gly-arg-gly-asp-ser, lkkgdr), moreover the deduced aminoacid complete sequence reveals 35 % identity with p. leniulusculus peroxinectin. in c. intestinalis genome, the presence of eleven alpha and five β chain integrin genes, suggest putative peroxinectin cell surface s69 table 2 properties and modulation of the ascidian phenoloxidases c. intestinalis s. plicata b.schlosseri p. mamillata h. roretzi references (1-7) (5,7,8) (9,10) (5,11) (12) treatment hls ths hls trypsin trypsin + sti -------------------- lps po inhibitors calcium effect no no no yes no no po containing cell types urg granular amebocytes morula morula compartment cell granular hemocytes nd po subunit mw 74 90 nd 80 70 62 method cloned hls isolated hls isolated biological activities cytotoxicity cytotoxicity non fusion reaction nd antimicrobial activity hemocyte lysate supernatant (hls); tunic homogenate supernatant (ths); unilocular refractile granulocytes(urg) (1) söderhäll and smith, 1992; (2) peddie and smith, 1993; (3) parrinello et al., 1995; (4) cammarata et al., 1996; (5) parrinello et al., 2003; (6) cammarata et al., 2008; (7) arizza et al., 1995; (8) cammarata et al., 1997; (9) ballarin et al., 1994; (10) ballarin et al., 2008; (11) cammarata et al., 1999; (12) hata et al., 1998 adhesion receptors (ewan et al., 2005). in table 1, we also report the sequence of a c. intestinalis putative gene cu-zn sod with 46 % identity to the predicted aminoacid sequence of the h. roretzi enzyme, obtained by using the aminoacid sequence of h. roretzi cu-znsod (abe et al., 1999) as a query in an aminoacid-based blast search (tblastn) versus the ncbi/genbank database. ascidian propo activating system and innate immunity ascidians occupy a key phylogenetic position in the evolutionary line leading to vertebrates (hori and osawa 1987; field et al., 1988; zeng and swalla 2005; delsuc et al., 2006), therefore both solitary and colonial ascidians are of interest in studying the evolution of defence mechanisms. phagocytosis, cytotoxicity, encapsulation and tissue damage (wright and cooper, 1983; parrinello and patricolo, 1984; parrinello et al., 1984, 2001, 2007; ballarin et al., 2008) in inflammatory responses, as well as in inflammatory events linked to allorecognition responses, have been shown (sabbadin, 1982; rinkevich, 1992; raftos et al., 1988). both share hemocytes degranulation in tissues of solitary ascidians (parrinello et al., 1984) and in the contact area of incompatible botryllid colonies (sabbadin, 1982; ballarin, 2008). differently than arthropod pos, which are monophenoloxidases, ascidian hemocyte pos are orthodiphenoloxidases. o-diphenol oxidase activity and phenolic compounds were at first identified by histochemical reaction (barrington and thorpe, 1968) in the tunic hemocytes suggesting a quinonetanning system involved in the production of tunic scleroprotein (chaga, 1980). although ascidian pos show the highest activity at 6-9 ph range (jackson et al., 1993; arizza et al., 1995; ballarin et al., 1994), they can differ in several biochemical properties. in botryllus schlosseri the ldopa oxydizing activity is enhanced by divalent cations (ballarin el al., 1994), whereas the po activity of other ascidian species does not appear to be ca2+ or mg2+-dependent (jackson et al., 1993; arizza et al., 1995; cammarata et al., 1999). in crustaceans, although calcium ions are requested, a high cation concentration exerts a suppressive effect. a further difference concerns the activating mechanism: β 1,3-glucans and oligosaccharides induce propo-activation in arthopods (söderhäll and smith, 1986; söderhäll, 1992) and in solitary ascidian c. intestinalis (jackson et al., 1993), whereas do not activate styela plicata (arizza et al., 1995) and b. schlosseri (ballarin et al., 1994) pos. ascidian orthodiphenoloxidases are copperdependent enzymes inhibitable by copper chelating substances (kahn, 1985; sugumaran et al., 1988). like in arthropods, the ascidian propo requires proteolytic cleavage for its activation. the level of po activity is significantly higher after incubation with serin-proteases, decreases as an effect of protease inhibitors, and it is activated by lps (table 2). smith and peddie (1992) and jackson et al. (1993) reported that the lps-sensitive protease activity, contained in the c. intestinalis hemocyte lysate supernatants, may be associated with in vivo prophenoloxidase activaction. benzamidine, soybean trypsin inhibitor (sti), phenylmethylsulphonyl fluoride, tosyl phenylalanyl s70 chloromethyl ketone, tosyl-l-lysin-chloromethyl ketone inhibited both protease and propo activation, this inhibitory activity is short lived and precedes hemocyte po activity (jackson and smith, 1993). at least three proteases are contained in solitary ascidians c. intestinalis, s. plicata (unpublished data) and phallusia mamillata hemocytes (guerrieri et al., 2000), and could regulate the otherwise uncontrolled protease activity. po containing hemocytes phenoloxidase participates in tunic formation (chaga, 1980), melanin production and non fusion reaction (ballarin et al., 1996), and an increased po activity can be found in c. intestinalis tissues inflamed by lps inoculation (parrinello et al., 2001). in c. intestinalis (smith and söderhäll, 1991) and b. schlosseri (ballarin et al., 1993) po oxidative activity of circulating hemocytes from naïve ascidians as well as hemocyte lysate supernatants has been revealed by l-dopa reaction. a similar activity has also been reported in ascidia mentula, ascidia virginea, ascidiella scabra, ascidiella aspersa, polycarpa pomaria, dendrodoa grossularia and morchellium argus (jackson et al., 1993). the specificity of the po reaction of s. plicata, p. mamillata and c. intestinalis hemocytes (arizza et al., 1995; parrinello et al., 2003) has been supported by tropolone, phenylthiourea and diethyltiocarbamate usually used as inhibitors (sugumaran et al., 1988; cadenas, 1989; kahn, 1995). the enzymatic assay of the hemocyte lysate and the cytochemical reaction of the hemocytes revealed a limited heterogeneity in the ascidian pocontaining cell types. po-activity as well as the possible substrates tunichrome and halocyaminereducing polyphenol, have mainly been identified in the hemocytes called "morula cells" (azumi et al., 1990; he et al., 1992; ballarin et al., 1996; parrinello et al., 2003). evidence of po positive “morula cells” have also been drawn by assaying hemocyte populations enriched through a density gradient separation of the hemolymph from c. intestinalis (jackson et al., 1993; parrinello et al., 2003), b. schosseri (ballarin et al., 1994) and s. plicata (arizza et al., 2005). this hemocyte-type is a round globular granulocyte (berry-like under the light microscope; 5.5-11.0 µm diameter) with large granules containing material of various electrondensity (de leo, 1992). light microscopy observations show large globular granules varying in number, shape and content feature. although smith and peddie (1992) suggested that c. intestinalis morula cells contain po, we found a weak reaction after the treatment with l-dopa-mbth. on the contrary, a strong po activity was found in the unilocular refractile granulocytes (urgs) identified in the hemolymph and characterized by a single popositive large granule that occupies the largest part of the cytoplasm (parrinello et al., 2003). in addition po-positive large granules were also found in granulocytes. although a defined differentiation line was not established, the possibility exists that granulocytes with large granules, urgs and morula cells may be components of a lineage characterized by a different state of the granule content including po activity level as well as hemocyte functions. accordingly, urgs exert po-dependent cytotoxic activity whereas morula cells do not show any cytotoxic activity against erythrocytes (parrinello et al., 1996) and tumour cell lines (peddie and smith, 1993). in p. mamillata, “hemocytes with large granules” show a low po activity whereas the morula cells do not show any positive reaction with l-dopa-mbth. the activity of the hemocyte lysate supernatant of enriched populations, obtained through a percoll density gradient, can be enhanced by trypsin that presumably activates the propo remaining after cell lysis. usually, hemocytes preparation and further treatments may cause the activation of a part of propo content by endogeneous proteases. in this ascidian, another hemocyte type named compartment cells characterized by few large vacuoles show spontaneous propo activation, and vacuoles appear to be po-positive. however, the lysate supernatant from enriched populations are less sensitive to trypsin proteolysis activation indicating that these cells may be more reactive to handling which can activate propo. proteolytic activation of propo is sustained by electrophoresis and l-dopa-mbth stain of trypsin-treated and untreated hemocytes with large granule lysate supernatants; the electropherograms show an increased mobility of the enzyme after proteolysis and, accordingly, reveal an additional protein fragment (parrinello et al., 2003). tunic pos and inflammatory response recently, the po activity of tunic homogenate supernatant (ths) from naïve c. intestinalis has been assayed (cammarata et al., 2008). as already reported upon the hemocyte lysate supernatant (hls), the po activity of ths is ca2+-independent, but, unlike hls (ph 6-9) requires a lower ph (7-8) and is more thermo-stable. the ths activity is lost after two days at 0 °c and after about one year at 80 °c, whereas the hls activity disappears after 2-3 h at 0 °c and 3-4 week at -80 °c. likewise the hlspropo, the treatment with exogenous trypsin enhances the activity and sti inhibits it, whereas a further difference resulted from a more effective activation due to lps acting at a lower concentration than that needed for activating hls-propo. in addition, since lps inoculation enhances the thspo activity of samples assayed in the absence of trypsin the possibility exists that in the tunic several serine proteases or different proteases could be involved in the activation phase (cammarata et al., 2008). to check for tunic matrix and tunic cells po activity in the inflammatory response, an in vitro enzyme assay of the inflamed tunic tissue excised at 24 or 48 h after lps inoculation has been performed. the po activity appears to be distributed throughout the tunic matrix, as well as inside the large granule of urgs and hemocytes with large granules. these observations are in accordance with the report on circulating hemocytes from naïve ascidians, whereas it is of interest that morula cells in the inflamed tunic disclose an evident po activity suggesting a distinct step of the cell lineage not found in circulating hemolymph from naïve ascidians s71 and presumably due to the lps challenge. microscope observations of wet inflamed tunic fragments after l-dopa-mbth treatment, showed both a strong po reaction and a high density of popositive hemocyte populations (fig. 2). since the deduced aminoacid sequence of a c. intestinalis cinpo-2 isoform has been reported (genbank accession n. aj547814) by immersberg and burmester (2004), a peptide (11-aa, efhndrrnrgf) has been selected through an antigen-prediction program, and anti-cinpo-2peptide specific antibodies has been raised in rabbits (cammarata et al., 2008). the immunohistochemistry reaction shows an intense dye of the inflammatory cells supporting that propo2 synthesis could be enhanced by lps inoculation (cammarata et al., 2008). the same assay revealed that, after lps inoculation, the enzyme is distributed in the tunic matrix, mainly in the outer layer, in addition anti-cinpo-2 antibodies also marked the pharynx vessel epithelia. immesberger and burmester (2004) reported that 86.9 kda is the predicted molecular size of cinpo-2, and this value fits with the 90kda revealed by the l-dopa-mbth reaction and immunoblotting analysis of ths from naïve ascidians (cammarata et al., 2008). after the lps inoculation, an additional 120 kda band reacts with l-dopa-mbth and anticinpo2 antibodies, whereas a 170 kda l-dopambth positive band does not react with the antibodies. presumably, this band could be considered as a dimeric form of cinpo-1 (92.0 kda) which cannot be identified by antibodies directed to a cinpo-2 peptide. anyway, an oligomerisation process may be responsible of cinpos higher in size (120 and 170 kda). a similar process may be taken in account for the differences observed in the thsand hls-po (parrinello et al. 2003) molecular sizes, and suggests that further analyses are needed. biological activity of po and related molecules in invertebrates, substances with cytotoxic activity include molecular derivates of oxygen and nitrogen, antimicrobial peptides, lectins and quinoid intermediates of melanin (parrinello 1996; nappi and ottaviani 2000). the oxygen reactive forms, such as superoxide anions, hydroxyl radicals, and hydrogen peroxide anions have been implicated as components of the cytotoxic mechanisms of vertebrates (cadenas, 1989) and invertebrates (bell and smith, 1993; anderson, 1994; valembois and lassegues, 1995; nappi and ottaviani 2000). the propensity of quinones for redox-cycling makes these eumelanin precursors potential sources of reactive forms of oxygen (riley, 1988; o'brien, 1991). indeed, it has been demonstrated (smit et al, 1996) that phenolic compounds, converted into toxic products by tyrosinase, exhibit cytotoxic activity towards human melanoma cells. we reported that the morula cells form s. plicata (cammarata et al., 1997) and urgs from c. intestinalis (parrinello et al., 1995) display podependent cytotoxic activity against tumour cell lines (unpublished data) and rabbit erythrocytes due to quinones which are known to be cytotoxic (pawelek and lerner, 1978; cotelle et al., 1991; fu et al., 1994; parrinello et al., 2003).the presence of quinones, possibly originated by po-driven tunichrome oxidation, has been reported in ascidians. differently, roi derived from the po pathway are cytotoxic factors in b. schosseri nonfusion alloreaction (ballarin et al., 2002, 2008). indeed, in vivo, quinones could be activated by enzymatic reduction as, under aerobic conditions, the semiquinone radical autoxidizes and forms superoxide anion radicals (nappi and vass, 1993). the superoxide anion, hydrogen peroxide and trace amounts of transitional-metal ions form a hydroxyl radical (fenton reaction), which is the most toxic of all oxygen products. there is only indirect evidence that, in c. intestinalis, propo-activation products have a stimulatory influence on hemocyte behaviour in vitro. in this ascidian, the crude hemocyte lysate supernatant, which contains active po and lpssensitive proteases, has a marked opsonic influence on the uptake of bacteria by phagocytic amoebocytes (smith and peddie, 1992). such opsonic potential match to the degree of the enzyme activity; in addition, po and proteases inhibition with benzamidine or sti precludes the opsonic effect, while pre-treatment of the lysate supernatants with lps produces a greatly elevated phagocytic response (smith and peddie, 1992). figure 3 shows a tentative model of the c. intestinalis propo activating system, based on present knowledge. finally, morula cells can release propo-activation products for tunic formation and regeneration in goniocarpa fig. 2 tunic fragment of c. intestinalis 24 h treated with l-dopa-mbth after the inoculation of lps. arrowhead indicates the po reaction at the injection site. bar (a) = 1 cm; (b) = 200 µm. s72 fig. 3 a model for the activation and modulation of the pro-po system in c. intestinalis, based on the following reference results: söderhäll and smith, 1992; peddie and smith, 1993; parrinello et al., 1995; cammarata et al., 1996, 1997; parrinello et al., 2003; cammarata et al., 2008. rustica, halocynthia aurantium (chaga, 1980) and b. schlosseri (zaniolo, 1981), and can be involved in the formation of clotting (wright, 1981), and in the encapsulation of foreign particles (anderson, 1971; parrinello, 1996). conclusions the melanogenetic pathway can be dependent on tyrosinase or phenoloxidase activity, the propo activating system appears to be a sophisticated system which represents an evolutionary independent defence mechanism characteristic of invertebrates. apparently protostomes and deuterostomes share propo and po-related factors and activities. phenoloxidase activity and propo-like components have been reported in the hemolymph of arthropods, annelids, molluscs, echinoderms and cephalocordata (roch et al., 1992; nappi and vass, 1993; beschin et al., 1998; luna-gonzales et al., 2003; pang et al., 2005). preliminary sequence analysis suggest that hemocyanin could be the evolutionary ancestor of po and a parallel molecular evolution cannot be excluded. taking in account the c. intestinalis inflammatory response challenged by lps, the propo system appears to be a component (cammarata et al., 2008) of a very complex reaction that presumably involves several interacting cell types and factors, namely inducible cytokine-like molecules (parrinello et al., 2007), cifacitcollagen (vizzini et al., 2007), and citnfα (parrinello et al., 2008). the lps or glucans activating effect could be associated with one or more serine proteases modulated by trypsin inhibitors, cu-znsod and peroxinectin-like molecules. therefore, lpsand glucan-binding proteins appear to be suitable candidates as recognition molecules central to several defence reactions. although some insight has been gained into the invertebrate propo system, host recognition proteins, biological activities, molecular interaction, and signal transduction pathways, little is known on the ascidian cascade. acknowledgements this work was supported by a miur (cofin2006 n. 2006059857) research grant. references abe y, ishikawa g, satoh h, azumi k, yokosawa h. primary structure and function of superoxide s73 dismutase from the ascidian halocynthia roretzi comp. biochem. physiol. 122b: 321-326, 1999. anderson rs. cellular responses to foreign bodies in the 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http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22raftos%20da%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22briscoe%20da%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22tait%20nn%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus report of meeting isj 4: 24-36, 2007 issn 1824-307x report of meeting viiith scientific meeting of the italian association for developmental and comparative immunology (iadci), 1 and 2 march 2007, area della ricerca, cnr, naples, italy organizers: u oreste, mr coscia institute of protein biochemistry, cnr, naples, italy session 1. chairman: e ottaviani, university of modena and reggio emilia, italy variomics of novel immunoglobulin-like transcripts in teleost fish rjm stet, e boumans, a oestergaard scottish fish immunology research centre, aberdeen university, aberdeen, uk the immune system uses a variety of rearranging and non-rearranging receptors to distinguish between self and non-self antigens. the rearranging receptors of the adaptive immune system such as the immunoglobulin proteins and t cell receptors have been studied in great detail in fish. it is hypothesized that the rearranging receptors, which form a subset of the immunoglobulin super family, arose from innate immune precursors that are not associated with somatic reorganization. non-rearranging receptors of the innate immune system, which recognise largely unknown ligands, collectively denoted as pathogen-associated molecular patterns, have only recently been described in fish. examples of these receptors are the toll-like receptors and novel immune-type receptors (nitrs). recently, highly polymorphic novel immunoglobulin-like transcripts (nilts) have been described in common carp, which encode leukocyte receptors composed of a single extra-cellular immunoglobulin domain, a stalk, transmembrane and cytoplasmic region. two receptors were isolated with opposing signal ability as indicated by the presence of either an immunoreceptor tyrosine-based activating (itam) or inhibitory (itim) motif. recently, we have extended these studies to salmonid fish. in atlantic salmon an est was retrieved with similarity to carp nilts. the complete sasa-nilt sequence was obtained by 3’ and 5’ race and was shown to encode the activating nilt receptor. we have analysed the presence of sasa-nilt receptors in two unrelated atlantic salmon individuals. this showed the presence of three framework sasa-nilt sequences that were present in both individuals. in addition to these framework sequences, each individual has its own set of polymorphic sasa-nilt receptors. the mechanisms that generate this receptor diversity are at present unknown. however, the haplotypic and allelic variation is reminiscent of the massive expansion and diversification of immunoglobulin-like loci encoded in the leukocyte receptor complex in chicken. self/nonself discrimination in ciliated protozoa: the molecular basis a vallesi, c alimenti, p luporini department of biology, university of camerino, camerino as also a recent review in cell on the "quality control in self/nonself discrimination" points out (boehm t. cell 125: 845-858, 2006), comparative studies of the mechanisms that avoid self-mating in more ancient eukaryotes are thought to be of key relevance for shedding light on the control of specificity in self/nonself discrimination, as well as on the evolutionary emergence of the antigen receptors in the adaptive immune system. these studies, however, traditionally drive most attention on the self-incompatibility of plants, self-sterility and allo-recognition of tunicates, mating types of fungi. scarce or no reference at all is made to ciliates. nevertheless, the ciliate highly multiple mating-type systems are providing insightful information not only on the molecular basis of self/nonself recognition in more ancient organisms, but also on the central question of how new receptor/ligand pairs are generated in complex recognition systems. this information essentially derives from: (i) nmr and crystallographic analyses (mostly carried 24 http://www.isj.unimo.it/articoli/isj093.pdf http://www.isj.unimo.it/articoli/isj093.pdf out in collaboration with the kurt wuthrich’s laboratory at the eth in zurich) of the threedimensional structures of a set of water-born protein signals (pheromones) produced by euplotes species (luporini et al. curr. pharm. des. 12: 30153024, 2006), and (ii) the determination of the splicing mechanism by which the same cell controls its own specific diffusible signal and the (autocrine) binding receptor of this signal (vallesi et al. eukaryot. cell 4: 1221-1227, 2005). a novel approach for studying hematopoietic cells in hirudo medicinalis a grimaldi, g tettamanti, c bianchi, g greco, r valvassori, m de eguileor department of structural and functional biology, university of insubria, varese, italy leeches have evolved a complex immune system that can recognize foreign antigens and can respond with a wide repertoire of reactions in relation to the non-self. surgical wounds or cytokine injection induce the formation of an extensive network of new vessels and the proliferation of hematopoietic precursors. these cells exert their functional role migrating through the extracellular matrix towards the stimulated areas. the different types of cells involved in leeches immune response have been characterized only in vivo, since its very difficult to isolate and culture these cells entrapped in the thick connective tissue. we establish an innovative system to isolate and culture specific population of leeches immune cells utilizing the injection of matrigel matrix gel added with different cytokines known to play a pivotal role in regulating proliferation, migration and differentiation of leech immune cells. after 48h from injection the matrigel sponge is infiltrated by cells. the infiltrated matrigel sponge dissected from leech is cultured in an appropriate medium. two types of cultured cells have been obtained depending on the added cytokines. after one week cells cultured in matrigel containing the vascular endothelial growth factor (vegf) differentiated in endothelial cells cd34+ while those cultured in matrigel containing the monocyte chemoattractant protein-1 (mcp-1) differentiated in macrophages cd14+. our method provides a controllable protocol for repetitive isolation and culture of precursors cells from a “parenchimatous” animal and it is an excellent tool to select different types of cell population. aging and il-6 immunoreactivity changes in the polychaete ophryotrocha labronica a franchini, e ottaviani department of animal biology, university of modena and reggio emilia, modena, italy the aging process is associated with dysregulation of the immune and inflammatory responses including modifications in the regulation and production of cytokines. il-6 is a pleiotropic proinflammatory cytokine thought to play a role in age physiology, even if its possible modulation by aging mechanisms has not been fully defined. the morpho-functional modifications and il-6 immunoreactivity during the aging process in a simple invertebrate model, the polychaete ophryotrocha labronica, are reported. the comparison between newly-hatched, juveniles (at 611 setigerous segments, max 2 weeks), young adult females (at 14 setigerous segments, about 3 weeks) and 3 month old females showed significant structural differences in the nervous and genital systems. a reduction in the nerve area with a substantial depletion in neurons of the central system was found. a decline in oocyte growth and maturation was observed at the gonad level, even if sexually mature o. labronica continued to produce egg mass until week 16 of their lives. the age induced morphological modifications were associated to a different distribution of il6-like molecules, that were detected in the central nervous system. a decreased number of reactive nerve cells and in particular in the anterior region of the brain of aged o. labronica was observed. session 2. chairman: l ballarin, university of padova, italy evolution of helical cytokines: a structural approach d malagoli, e ottaviani department of animal biology, university of modena and reggio emilia, modena, italy cytokines are small soluble factors retrieved in mammals and involved in several processes such as immunity and development. they are typically characterized by pleiotropicity, functional and receptor redundancy. in consideration of functional parallels between mammalian and invertebrate immunity, a lot of experiments have been dedicated to the unravelling of cytokine network in both protostomian and deuterostomian invertebrates. the presence of cytokine-like molecules has been evidenced by several morphological and functional investigations in different taxa of invertebrates, leading to the hypothesis that cytokines are molecules of ancient origin, present in metazoans before the division of protostomian and deuterostomian phyletic lines. however, all the recent molecular biology advances indicate that no sequence similarity can be retrieved between the known vertebrate cytokines and the whole genome of invertebrate species. on these basis, functional convergence has been proposed between 25 vertebrate and invertebrate cytokines. the functional convergence would be due to the lectinlike activity of vertebrate cytokines that can be retrieved also in some invertebrate molecules. in order to unravel this unsolved matter, we have adopted a new bioinformatics approach able to isolate proteins whose structure is comparable to that of mammalian helical cytokines from est and protein databases. through this method we have isolated a molecule from drosophila melanogaster databases that presents the structural characteristics of a helical cytokine (drosophila helical factor, dhf). functional experiments performed on third instars larvae and sl2 embryonic hemocyte cell line of d. melanogaster demonstrated that dhf expression was increased after different immune challenges. from the present findings, it emerges that the contradiction between the amount of morphological and functional evidences and the absence of any homology between mammalian and invertebrate cytokines, could be explained by evolution of cytokine genes thereby conserving specific protein structures rather than amino acid or nucleotide sequences. from the present findings, it emerges that the contradiction between the amount of morphological and functional evidences and the absence of any homology between mammalian and invertebrate cytokines, could be explained by evolution of cytokine genes thereby conserving specific protein structures rather than amino acid or nucleotide sequences. haemocytes of the cockle cerastoderma glaucum: cell types and involvement in immune responses v matozzo, mg marin department of biology, university of padua, padua, italy for the first time, morpho-functional characterisation of haemocytes from the cockle cerastoderma glaucum was performed to identify circulating cell types and to study their involvement in immune responses. haemocyte mean number was 5.5 (x105) cells/ml haemolymph (n=10). two main haemocyte types were found in haemolymph: granulocytes (85 %), about 10 µm in diameter and with evident cytoplasmic granules, and hyalinocytes (15%), 8 to 14 µm in diameter, with a few or no granules. most of the cytoplasmic granules stained in vivo with neutral red, indicating that they were lysosomes. on the basis of haemocyte staining properties, granulocytes and hyalinocytes were further classified as basophils and acidophils. acidophil hyalinocytes were the largest haemocyte type (about 14 µm in diameter) and had an eccentric nucleus and a large cytoplasmic vacuole. both granulocytes and hyalinocytes (except acidophils) were able to phagocytise yeast cells, although the basal phagocytic index was very low (about 2 %). it increased significantly (up to 26 %) after preincubation of yeast in cell-free haemolymph, suggesting that haemolymph has opsonising properties. haemocytes also produced superoxide anion. moreover, both granulocytes and hyalinocytes (except acidophils) were positive to some important hydrolytic and oxidative enzymes, such as acid phosphatase, non-specific esterase, acid esterase, and peroxidase. lysozyme-like activity was recorded in both cell-free haemolymph and haemocyte lysate, although enzyme activity in cell lysate was significantly higher. results indicate that haemocytes from c. glaucum are effective cells in immune responses. evidence for stem cell factor-induced proliferation/differentiation in bivalve hemocytes m betti*, b canonico°, c ciacci*, lc lorusso*, s papa°, l canesi^ *institute of physiological sciences, °cytometry and cytomorphology, university of urbino “carlo bo”, urbino, italy ^department of biology, university of genoa, genoa, italy bivalve hemocytes comprise both granular and agranular circulating cells that are capable of nonself recognition through lectins and chemotaxis, and, most of all, phagocytosis. as with vertebrate phagocytes, bivalve phagocytes are equipped with both oxidative and non-oxidative killing systems related to activities of lysosomal enzymes. in bivalves, the process of hematopoiesis is still unknown. the most generally accepted belief is that hemocytes may originate from connective tissue cells, although hemocyte proliferation at sites of inflammation has been demonstrated. stem cell factor (scf) is a member of hematopoietic cytokines, a group of glycoproteins that regulate the growth and differentiation of hematopoietic progenitor cells and functionally activate mature neutrophils or macrophages. in this work the possible effects of recombinant human scf on the hemocytes of the marine bivalve mytilus sp. were investigated. the in vitro effects of scf (50 ng/ml) on hemocyte functional parameters (lysosomal membrane stability-lms and lysozyme release-lr) were first evaluated. scf induced significant increase in lms and decrease in lr, this indicating a reduction in lysosomal membrane fusion processes. moreover, flow cytometry analysis showed that scf significantly affected both hemocyte number and cell cycle; in particular, increases in the number of both granular and agranular hemocytes were observed. the results obtained with heterologous sfc support the hypothesis that common pathways involved in modulating activity, differentiation and proliferation 26 of immune cells are shared by invertebrates and vertebrates. characterization of hemocytes from polistes dominulus (insecta, hymenoptera), target of the strepsipteran endoparasite xenos vesparum f manfredini*, e ottaviani°, r dallai* *department of evolutionary biology, university of siena, siena, italy °department of animal biology, university of modena and reggio emilia, modena, italy xenos vesparum (insecta, strepsiptera) is a macro-parasite of polistes dominulus, a primitively eusocial paper wasp. sem and tem observations after artificial infections allowed us to follow step by step the parasite development inside the hemocoel of the wasp. the host-seeking stage is the triungulin (free-living 1st instar larva) which is able to “softly” overcome the structural barriers of the larval wasp (cuticle and epidermis) without any traumatic reaction at the entry site. the parasite molts 48h later to a 2nd instar larva, which moves away from the 1st instar exuvium, molts twice more without ecdysis and pupates, if male, or develops into a neotenic female. some features result unusual: the encapsulation reaction involves the 1st instar exuvium (not the living parasite) and it starts only 48 h after host invasion; in addiction, no signs of melanization are visible. we suspect that x. vesparum inhibits host defense reactions during the early events of the infection and then the parasite seems to operate an elusion of p. dominulus immunity starting from the 2nd larval instar. we characterized the hemocytes present in the hemolymph of p. dominulus 3rd and 4th instar larvae (the main targets of triungulins) through morphological observations at tem, sem, light and phase-contrast microscopy; moreover we performed adhesion and phagocytosis functional tests and immunocytochemistry essays to check the presence of pomc-derived peptides and nep-like molecules. apart from the prohemocyte, the stem cell from which other hemocytes originate, two “types” or functional states are discernable, both of them adhering on glass slides and phagocytizing fluorescent beads, one type provided with structured and/or amorphous granules, the other one devoid of them. effect of cadmium exposure on phagocytosis and plaque lysis activity of paracentrotus lividus coelomocyte v arizza, f giaramita, g salerno, m vazzana, s basiricò, n parrinello marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy phagocytosis and plaque lysis activity (pla) of coelomocyte from paracentrotus lividus were examined after exposure to cadmium chloride (cdcl2·h2o), a potentially toxic metal salt, widely used in industry. p. lividus specimens were exposed at different cd concentration (100, 200, and 400 µg l-1) for 24 hours (sampled at 0, 6, 12 and 24 hrs) at 15°c, in tanks containing artificial sea water (asw) or injected with asw containing the metal at 50, 100 and 200 µg l-1 in to the coelomic cavity. the treatment without cd did not affect phagocytosis and pla, whereas treatment with cd, significantly lowered. this effect was dose and time dependent, presumably dependent on the cytotoxic effect of cadmium on coelomocyte as indicated by neutral uptake assay. session 3. chairman: l abelli, university of ferrara, italy invertebrate lectins present cytokine properties n parrinello, v arizza, m cammarata, m vazzana, a vizzini, d parrinello, ml di bella, m pergolizzi, f giaramita, m celi marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy the origin and evolution of the innate immunity, including cell-cell, cell-cytokines, cell-lectins, cellmatrix interactions, appear to be product of cells and genetic markers for self recognition that grow with molecules and mechanisms to identify and destroy non-self. lectins are components of a wellconserved protein-carbohydrate recognition system, the activity of most of them resides in a carbohydrate-recognition domain (crd). they present an ample repertoire and have been proposed to mediate cell-cell or cell-extracellular matrix interactions in developmental processes, cell adhesion, inflammation and metastasis. several immunomodulatory functions have been reported, among them mitogenic properties, opsonic properties, complement pathway activation and several immune responses. cytokines are the major regulators of the host defence processes and are involved in responses to exogenous and endogenous insults, tissue repair and recovery of homeostasis. many cytokines are bifunctional molecules having, beside a receptorbinding domain, a crd. the expression of the biological activity relies on the association between both domains. several reports have also shown that cytokine-crd can interact with various pathogens and presents the recognition site that contributes to pathogen elimination via opsonization and/or leukocyte activation. lectin-like activities of several cytokines, including il-1, have been described. our understanding of invertebrate cytokine-lectin 27 biological functions and evolution are lacking. in some studies, similarities at the physicochemical level of vertebrate cytokines and functional invertebrate analogues have also been described. among experimental approaches to identify cytokine-like molecules, antibodies neutralizing the activity of mammalian cytokines, have been used to screen for cross-reactivity with invertebrate factors in hemolymph. tunicates are a key group in chordate phylogenesis. in ascidian species, lectins are responsible for the in vitro opsonization, modulate cell proliferation activity, phagocytosis and complement activation, stimulate proliferation of mouse thymocytes and l-929 fibroblasts. recently we have shown that, in the serum from the lpschallenged ciona intestinalis, il1-like inducible components cross-reacted with anti-humanril1a antibodies. ciil1 with hemagglutinating properties, appears to be involved in sugar specific opsonization of yeast in an in vitro phagocytosis assay. therefore a putative structural model of the opsonin include both crd and il1 epitopes. we propose an evolutionary model in which multifunctional costituive/inducible lectins can express cytokine activity with crd responsible for pleiotropy and redundance, evolutionary conserved to guaranty a basic recogniton mechanism. several inflammatory factors have been hypothesized as responsible for this process including components known in mammal innate immunity (cytokines, complement components, collagens) and phenoloxidase activity. a putative evolutionary model in which lectins are represented as multifunctional inducible molecules (cytokine-like function) involved in adult defence mechanisms, could be hypothesized. the lectin crd, conserved in the evolution to guaranty a basic recognition mechanism, could explain lectin pleiotropy and redundancy as already suggested for mammalian cytokines. a novel rhamnose-binding lectin from the compound ascidian botryllus schlosseri l ballarin°, n franchi°, b spolaore*, f gasparini° °department of biology, university of padua, padua, italy *cribi, university of padua, padua, italy animal lectins play a fundamental role in invertebrate immunity, as they are involved in the recognition of microbial molecular patterns which, in turn, triggers various effector responses, such as opsonisation, encapsulation, activation of the propo activating system, phagocytosis. in a previous study, we purified by affinity chromatography and partially characterised a soluble ca2+-independent lectin, with specificity for b-galactosides, from the blood of the colonial ascidian botryllus schlosseri. the molecule can agglutinate rabbit erythrocytes, is secreted by haemocytes upon the recognition of foreign particles and behaves as an opsonins (ballarin et al., 1999, 2000). recently, we purified further this protein by rphplc, obtaining 4 lightly different peaks, likely isoforms of the same molecule. the mws estimated using mass spectrometry ranged between 10.7 and 11.1 kda. the lectin was digested with trypsin and tryptic fragments were sequenced by mass spectrometry. blast analysis of the main sequences obtained indicated a high degree of homology with rhamnose-binding proteins, a family of s-type lectins described in sea urchin and teleosts. the specificity for rhamnose (and the similar melibiose) was successively demonstrated in haemoagglutinating inhibition assays. we prepared a full length cdna library from botryllus colonies from which we obtained three full sequences of transcripts which, after blast analysis, resulted highly homologous to known genes for rhamnose-binding lectins. their putative aminoacid sequences contained our tryptic peptide sequences. serum lectins in fish innate immunity: molecular and functional aspects m cammarata*, g benenati*, g parisi*, d parrinello*, g salerno*, m vazzana*, a vizzini*, gr vasta°, n parrinello* *marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy °center of marine biotechnology, university of maryland, biotechnology institute, baltimore md, usa fucose-binding lectins (fbl) are present in tissues and fluids from invertebrates and vertebrates. the lectin repertoires in teleost fish are highly diversified and recently has been described the structure of the fucose-binding agglutinin that revealed a novel lectin fold (the “f-type” eel (anguilla anguilla) fold), which shared a unique fucose-binding sequence motif contained both in carbohydrate-binding proteins and unrelated proteins. in this report, we describe serum fbl from sea bass dicentrarchus labrax and sea bream sparus aurata. these lectins were purified, characterized, cloned and sequenced. studies on structural aspects, biological activity, tissue distribution as well as ontogenetic aspects were carried out. in addition, results on inflammatory response and opsonic activity against bacteria suggested that d. labrax fbl is involved in innate immunity. finally a new galactose binding lectin with agglutinating activity against bacteria purified from dicentrarchus labrax serum was also described and compared with fbl. 28 indifferentiating cells in the blood of the colonial ascidian botryllus schlosseri: a morphofunctional charaterisation f cima, l ballarin department of biology, university of padua, padua, italy colonies of the ascidian botryllus schlosseri undergo a periodic tissue renewal in the take-over stage of the colonial blastogenetic cycle, during which an extensive apoptosis occurs in the adult zooid tissues and the senescent cells are progressively removed by circulating phagocytes. the haemocytes which circulate in the common vascular system also die partly by apoptosis during this stage. these cells are replaced by new haemocytes, likely differentiating from stem cells. up to now, haemopoiesis was observed only in solitary ascidians in which haematopoietic noduli were described in the branchial wall. nothing is known on haemopoiesis in colonial species, in the blood circulation of which two cell types with the morphology of undifferentiated cells are recognizable: haemoblast and lymphocyte. we have studied the cytochemical and immunocytochemical properties of these haemocytes: results indicate the haemoblast as a pluripotent stem cell since it shows a basophilic nucleus labeled either with hoechst 33342 for euchromatin or anti-ki-67 and anti-pcna antibodies specific markers of nuclear proteins involved in cell proliferation and its plasma membrane is labeled by anti-cd34 and anti-cd100 antibodies, specific for haemopoietic cells in vertebrates. commercial antibodies for cytokine receptors, like interleukin 1 receptor i (il-1ri) and stem cell factor receptor (scf-r) label haemoblast plasma membrane, suggesting the presence of growth factor receptors. both lymphocytes and haemoblasts during the colonial cycle show a significant increase in concentration during the blastogenetic replacement. however, mitosis figures were rarely observed in circulating haemocytes. in vitro assays of haemocyte exposure to colchicine showed the presence of mitosis figures, which significantly increase after exposure to bacteria indicating a proliferating capability in blood circulation mainly as an immune response as observed in other invertebrates like molluscs. effects of different carbon dioxide concentrations on the adaptive immune system of cultured sea bass (dicentrarchus labrax) n romano^, e caccia^, t petochi°, s meloni^, l mastrolia^, g scapigliati^, l abelli*, g marino° ^department of environmental sciences, university of tuscia, viterbo, italy *department of biology and evolution, section of comparative anatomy, university of ferrara, ferrara, italy °icram, central institute for marine applied research, rome, italy low oxygen and high carbon dioxide concentrations could affect health and welfare of farmed fish. this study evaluated acute and chronic effects of different dissolved carbon dioxide concentration (2-45 co2 mg/l) on specific immune response of sea bass. fish were vaccinated against vibrio anguillarum before exposure to co2 and after 45 days were analysed for: a) the percentage of t and b lymphocytes in the leukocyte fraction of blood and head kidney (by flow cytometry using specific mabs dlt15 and dlig3 for t and b cells, respectively), b) proliferation capability of lymphocytes exposed to vibrio; c) the serum content of anti-vibrio ig by captured-elisa method and d) the agglutinating capacity of serum against vibrio bacteria. t and b lymphocytes significantly decreased (p<0.001) in fish maintained for 45 days at the highest co2 concentration respect to controls. the proliferation capability of head kidney lymphocytes was also significantly reduced in co2 treated fish. also anti-vibrio ig content decreased (50 %; p>0.001) in co2 exposed fish. non-immunised showed a lower vibrio-agglutination capability. these findings evidenced the strong effect of co2 on circulating lymphocytes and in their specific immune function and the sensitivity of farmed sea bass to carbon dioxide concentration higher than 40 mg/l. thymic morpho-functional changes after hypophysectomy and bursectomy in chicken embryos m aita*, n romano° *department of human physiology and pharmacology, faculty of medicine, university “la sapienza”, rome, italy °department of environmental sciences, university of tuscia, viterbo, italy experiments of hypophysectomy or bursectomy were performed in chicken embryos in order to give more information on the role of hypophysis and fabricius’ bursa in the thymus development. hypophysectomy was performed on chick embryos at 36-40 hr of incubation. the thymuses were collected on day 18 and tested for: 1) antithymostimulin (ts) immune reaction; 2) histoenzymatic activities (ldh, sdh, nadh, nadph, alfa-gpdh, ca2+-atp-ase). the total thymic size was reduced and anti-ts, sdh, atpase yielded negative reactions in the medullary epithelial cells. when hypophysectomized embryos received on day 12 a hypophyseal allograft from 18 day-old donor embryos, the thymic compartments improved and anti-ts immune reaction and enzymatic activities were partially recovered. bursectomy was performed at 68-72 hr of incubation. the thymuses were collected on day 17 and were tested for the pcna (proliferating cell 29 nuclear antigen) and cd3, cd8 and cd4 markers. a significant reduction of pcna-immunoreactive lymphocytes was observed in cortex (p<0,001) and a significant decrease of anti-cd3,-cd4,-cd8 lymphocytes was evidenced in medulla (p<0,01). these findings confirm, at one hand, the key role of the hypophysis in thymic ontogenic development and, on the other hand, that bursectomy interferes with a correct differentiation of thymocytes and that there is an interrelationship between thymus and bursa at least during embryonic life. session 4. chairman: n parrinello, university of palermo, italy evolution of the complement system: invertebrate animal models mr pinto stazione zoologica “anton dohrn”, naples, italy in the past decade, in the context of the renewed interest in innate immunity, the complement system has been investigated in increasing depth. one successful approach to analyzing the complement system has involved the study of its evolutionary origin. the search for complement components has been carried out in very divergent species, aided by the powerful tools provided by computational biology and the genome projects that are ongoing in many invertebrate species. a surprising result of these endeavours has been the finding that both c3, the key molecule of the complement system, and factor b have a very ancient origin. in cnidaria, these molecules seem to form a basic complement assembly that is able to opsonize bacteria through a primordial alternative pathway. carbohydrate-recognizing molecules, structural homologues of vertebrate ficolins, have been recruited by the complement system more recently in the protostomian lineage. high levels of complement complexity, comparable to those of mammals, have been reached in ascidians (urochordata), which experienced many gene duplication events specific to the urochordate lineage. as a result of this gene expansion, ascidians exhibit a complex complement system that operates through alternative and lectin pathways and exhibits proinflammatory and opsonic effector activities. expression pattern of c3 during ciona intestinalis embryo development d melillo*, r de santis*, s giacomelli*, jd lambris^, mr pinto* *stazione zoologica “anton dohrn”, naples, italy ^protein chemistry laboratory, university of pennsylvania, philadelphia, usa the identification of many complement components in ascidians indicates the presence of a complex complement system, comparable to that of mammals, activated via an alternative and an mbl-mediated pathways. c3 activation-dependent pro-inflammatory and opsonic effector activities have been demonstrated in this subphylum. in particular, c3, the central molecule of the system, is expressed in ciona blood granular amoebocytes and compartment cells, and the gene product is present in blood serum. while the immune defense mechanisms and molecules of adult ascidians have received some attention, no information is available on the immune surveillance, if any, during embryo development. to approach this topic, we have analyzed, in the present study, the spatial and temporal expression of c3 in ciona intestinalis embryo. following rt-pcr indications, we have carried out in situ hybridization experiments on developmental stages, from the unfertilized egg to the swimming larva. c3 shows an expression pattern restricted to mesenchyme cells and neural tissue cells. to extend these results western blot and immunochemical experiments have been also carried out. our preliminary data provide a first hint in defining the role of the c3 molecule, crucial in the innate immune response in the adults, during embryogenesis. cikll, the ascidian multipurpose c-type lectinlike receptor i zucchetti*, r marino*, mr pinto*, l du pasquier°, r de santis* *stazione zoologica “anton dohrn”, naples; italy °institute of zoology, university of basel, basel, switzerland c-type lectins, a family of diverse animal lectins characterized by a c-type lectin domain that serve for a broad range of biological processes, such as adhesion, endocytosis and pathogen recognition and neutralization, have been originally identified as carbohydrate-recognition molecules found in both invertebrates and vertebrates. the carbohydrate binding c-type lectin domains are part of a larger family of domains called c-type lectin-like domains (ctlds) that seem to have originated by a process of divergent evolution from a common ancestor. vertebrate cd94, one of the ctld-containing molecules characterized by a lack of ca2+-binding sites, and therefore, a putative lack of sugar-binding activity, is one of several vertebrate natural killer lymphocyte receptors. cd94, forming heterodimers with nkg2 family molecules, regulates cytotoxic activity of nk cells towards target cells by interacting specifically with the mhc class i molecules, thus representing a trait d’union between innate and adaptive immunity. 30 in order to provide further information on the evolution of c-type lectins, we have carried out an in depth study on cikll, a homolog of the bscd94/nkr-p1, a cd94-like gene identified in the colonial tunicate botryllus schlosseri, present in ciona intestinalis genome. cikll, showing intermediate structural features between a carbohydrate-binding protein and an nk cell receptor, is expressed in a blood cell type that is involved in the phagocytic activity during the immune response. furthermore, cikll is expressed in the larva and during early metamorphosis in structures related to the nervous system. these observations are in line with the current speculations on gene cooption in the course of evolution particularly between genes involved in immunity and those related to developmental processes of the nervous system. preliminary characterization of a c1q-like transcript from the ascidian ciona intestinalis s giacomelli, d melillo, r de santis, mr pinto stazione zoologica “anton dohrn”, naples, italy c1q is a subcomponent of the c1 enzyme complex that triggers the activation of the classical pathway of the complement system in the adaptive immune system of the mammalian species. c1q is known for its ability to bind antibodies as well as other ligands, including bacteria, viruses, parasites etc. c1q shows an hexameric assembly of three polypeptide chains, a, b and c chain. these chains possess the same topology, consisting in a collagen-like gly/pro-rich region, and a conserved c-terminal globular domain (c1q domain). the analysis of the urochordate ciona intestinalis genome has confirmed the absence of the pivotal genes for adaptive immunity in this species, which is phylogenetically at the basis of the vertebrate lineage. at the same time, this analysis has confirmed the presence of many complement genes, including a c1q domain-containing gene, cic1q-like, encoding a protein with the same modular organization of the mammalian c1q chains. to trace back to the invertebrates the origin of the classical activation pathway of the complement system, we have undertaken the molecular and functional characterization of the cic1q-like protein. we have verified the presence of cic1q-like mrna in ciona blood cells, by pcr analysis: pcr products have been cloned and sequenced. the 3’utr sequence has been determined on clones obtained from the 3’-race procedure. the c1q domain of the cic1q-like sequence has the 24-28 % of identity with the human orthologs. a phylogenetic tree generated by neighbour-joining method shows the relationship between the ciona c1q-domain and the other c1q-domain orthologs. the ascidian c1q could either act as a lectin, like the lamprey c1q, or interact with other unknown membrane bound receptor/s, as in the case of murine sign-r1. further investigations on cic1q-like expression and biological function may help to shed light on the origin and evolution of the complement system classical pathway and the c1q domain family. toll-like receptors in haemocytes of the colonial ascidian botryllus schlosseri: preliminary results a menin, l ballarin department of biology, university of padua, padua, italy toll-like receptors (tlrs) represent a wellknown family of pattern recognition receptors, expressed by immunocytes, the importance of which in non-self recognition was demonstrated in both vertebrates and invertebrates. in the colonial ascidian botryllus schlosseri, we used commercial anti-tlr2 and anti-tlr4 antibodies to inquire into the presence of toll-like receptors (tlr) in haemocytes lysates. after sdspage, the immunoblot analysis revealed single protein bands recognised by the two antibodies, of 34 kda and 32 kda for tlr2 and tlr4, respectively. immunocytochemical investigation on monolayers of fixed haemocytes, previously exposed to e. coli lps and yeast cells, revealed the expression of molecules recognised by tlr2 on activated phagocytes, whereas no labelling was observed with tlr4. we also studied the role of nf-kb in the signal transduction pathway related to phagocytosis. immunocytochemical analysis with anti-nf-kbp65 antibody revealed the labelling of the cytoplasm of untreated cells, whereas haemocytes exposed to yeast cells or bacillus clausii spores showed a marked staining of the phagocyte nucleus. the nfkb inhibitors na-pyrrolidinedithiocarbamate and parthenolide, at sublethal concentrations, significantly inhibits both the ingestion of yeast cells by botryllus phagocytes and the nuclear translocation of the activated factor. the same molecules have no effects on the morphology of haemocytes. on the whole, our data suggest that, in our species, tlr are involved in phagocytosis and act through the activation of nf-kb. signal transduction in phagocytosis of the colonial ascidian botryllus schlosseri: a preliminary approach a menin, e chemello, l ballarin department of biology, university of padua, padua, italy 31 in the course of our study on the role of immunocytes of the colonial ascidian botryllus schlosseri in immune responses, we began to investigate the signal transduction pathways involved in yeast cell phagocytosis. both calphostin c, a specific inhibitor of protein kinase c (pkc), and h-89, a specific inhibitor of protein kinase a (pka) significantly inhibit the increase in the phagocytic index. this indicates that both cyclic amp, which activates pka, and phospholipase c, which results in the production of ip3 and dag (the former mobilising ca2+ from intracellular stores, the latter activating pkc), are routinely required for phagocytosis. in addition, manumycin a, inhibiting ras activation, pd98059, inhibitor of erk activation, sp600125, preventing jnk activation, sb202190, inhibiting p38 kinase, significantly inhibit yeast phagocytosis by botryllus phagocytes. this suggest that the main map kinase pathways are involved in the ingestion of foreign cells. the frequency of phagocytes expressing molecules recognised by anti-pan ras antibody increase significantly when haemocytes were preincubated in the presence of foreign cells. activated haemocytes also express molecules recognised by anti-p-erk and anti-p38. therefore, a complex network of intersecting pathways is emerging and future research will aim to a better clarification of the main steps of signal transduction in ascidian phagocytosis. session 5. chairman: mr coscia, institute of protein biochemistry, cnr, naples, italy teleost immunoglobulins: genes and proteins u oreste institute of protein biochemistry, cnr, naples, italy the ability of teleosts to mount an antibodymediated immune response has been reported for the first time in the last century, at the beginning of the forties. serological methods, such as bacterial agglutination or hemolysis, provided the indirect evidence of the presence of antibodies in the teleost serum. the introduction of protein chemistry and immunochemistry methods in the sixties allowed the purification and characterization of the immunoglobulin (ig) molecules. the comparison with mammalian ig showed evidence of many peculiar features of teleost ig, such as isotype composition, polymeric assembly, lack of either secondary response or isotype switch. molecular biology tools have been introduced in fish immunology in 1989, when an ictalurus punctatus ig heavy chain (igh) cdna has been sequenced. afterwards, nucleotide sequences encoding ig genes have been obtained from more than 30 different teleost species. at present, data on ig gene structure, regulation, and expression, are also available. the antibody repertoire, the vh gene segment diversity, the occurrence of different igh and igl isotypes, the alternative splicing of primary igh transcripts have been deeply investigated in some model species. the ongoing genome and est sequencing of several teleost species, aided by computational biology, has enormously increased the knowledge of the immune gene organisation and functions: new igh isotypes, new igl gene segment organisation have been disclosed, allowing to draw a scheme of the vertebrate ig evolution. the knowledge on the most important molecule of vertebrate immunity has increased in the last decades at a very high rate thus prospecting fascinating results in the next future. molecular cloning, structural analysis and antigen-induced “in vivo” expression of interleukin-10 in sea bass (dicentrarchus labrax l.) f buonocore*, e randelli*, s bird°, cj secombes°, s costantini^, a facchiano^, s benedetti*, g scapigliati* *department of environmental sciences, university of tuscia, viterbo, italy °scottish fish immunology research centre, university of aberdeen, aberdeen, uk ^ institute of food science, cnr, avellino, italy interleukin-10 (il-10) is a regulatory cytokine mainly involved in the suppression or deactivation of immune responses and is produced by macrophages and by the t-helper cells (subset th2). recently il-10 has been discovered in the fugu genome and cloned in different fish species. here we describe the homology cloning of this cytokine in the mediterranean sea bass (dicentrarchus labrax l.) and investigate its structure and in vitro and in vivo expression upon various stimulants. the full-length il-10 cdna consists of 1015 bp and is translated in one reading frame to give the entire il-10 molecule containing 187 amino acids. a multiple alignment of the predicted translation of il-10 sea bass molecule with other known il-10 sequences showed the conservation of the fundamental features corresponding to il-10 molecules. a comparative 3d modelling using human il-10 as template showed that sea bass molecule is a symmetric homodimer, topologically similar to the structure of interferon-γ and about 70 % of the residues in each monomer assumes an α-helical conformation. expression analysis by real-time pcr was studied at a basal level in the main lymphatic tissues and after in vitro stimulation with lps and pha in the head kidney. moreover, an in vivo stimulation with the t-dependent antigen dnp human gamma 32 globulins (dnp-hgg), alone or in combination with an aluminium hydroxide emulsion, was performed. structural study of complex of mhc class i and co-receptor cd8 in sea bream by computational methods s costantini*, f buonocore°, e randelli°, g scapigliati°, am facchiano* *laboratory of bioinformatics and computational biology, institute of food science, cnr, avellino, italy °department of environmental sciences, university of tuscia, viterbo, italy comparative modelling represents the best predictive method for modelling the 3d structure of proteins. this method is applicable when the protein to be modelled is homologous to a protein whose 3d structure is known. the basis of this strategy is the observation that homologous proteins from different organisms can have low or high level of sequence identity, depending on the evolutive distance among them, but the 3d structure should be similar, being strongly related to the function played by that protein. on this basis, the 3d model of a protein can be created by similarity to the experimental model of known homologous protein. in this work we have applied the comparative modelling strategy to model mhc class i and homodimer cd8aa sequences in sea bream and the related complex. the three-dimensional models of the sea bream molecules and complexes were created by using human and mouse template models. as the sequence identities between the sea bream proteins and the homologous template models were about to 30 %, we used an accurate procedure to search for the best alignment of sequences, in order to improve the quality of the modelling results. the obtained models present a global structure similar to the reference proteins. comparing the complexes obtained for sea bream and the mammals ones, we have evidenced some differences both in structural and in energetic terms. cloning, expression and structural analysis of the mhc class ii β from sea bass dicentrarchus labrax e randelli*, f buonocore*, d casani*, rjm stet°, a facchiano^, s costantini^, g scapigliati* *department of environmental sciences, university of tuscia, viterbo, italy °scottish fish immunology research centre aberdeen university, aberdeen, uk ^institute of food science, cnr, avellino, italy major histocompatibility complex class ii (mhcii) molecules present foreign peptides to t cells of the cd4 subset, and are thus fundamental components of the adaptive immune system. the mhcii proteins belong to the immunoglobulin gene family, bind to lysosomally generated peptides and are expressed only by b cells and antigenpresenting cells. they consist in a heterodimer (α and β chain) having two extracellular domains, a short hydrophobic transmembrane section and a hydrophilic cytoplasmic domain. mhcii genes exhibit an extraordinary degree of allelic polymorphism and are likely candidates as gene markers associated with disease resistance. using degenerate primers corresponding to mhcii conserved regions of vertebrate sequences, we obtained an initial 190 bp product that, once sequenced and analysed by blastx search, corresponded to a mhcii-β gene fragment. from this fragment we designed specific primers that were used in 3’ and 5’ race pcr to complete the cdna sequence. an alignment was performed using available mhcii-β aminoacid sequences and a phylogenetic tree was generated with the putative aminoacid sequences lacking the signal peptide. moreover, two 3d mhcii sea bass models were obtained based on crystallographic mouse mhcii structures complexed with d10 t-cell antigen receptors and human cd4. finally, specific primers were used to analyse by real time-pcr the mhciiβ expression in kidney macrophages stimulated with different concentration of sea bass ril-1β and with lps for 4h and 24 h, which are known to modulate mhcii expression. molecular models of chionodraco hamatus igm transmembrane region s varriale*^, a merlino^, mr coscia*, l mazzarella^, u oreste* *institute of protein biochemistry, cnr, naples, italy ^department of chemistry, university of naples “federico ii”, naples, italy membrane-bound immunoglobulin m (igm) participates to the assembly of the b cell receptor (bcr). igm consists of two μ heavy chains crossing the cell membrane and two light chains. the μ chain region traversing the lipid bilayer (tm region) is highly conserved among species and contains a universal motif for antigen receptors that is important for bcr assembly and function. we analysed the tm region of igμ from the antarctic teleost chionodraco hamatus, belonging to the channichthyidae family. its membrane μ chain is particularly interesting because generated by an unusual mrna splicing mechanism. we determined the complete nucleotide sequence of c. hamatus membrane μ chain and analyzed the deduced amino acid sequence encoded by the tm exons. using different computational methods, we predicted the length and 33 the polarity of the α-helical region crossing the cell membrane, and build a molecular model of the c. hamatus μ chain tm region, using the h helix of the photosynthetic reaction center of rhodobacter sphaeroides as template. the stability of the model was investigated by molecular dynamics (md) simulations. models of a tm homodimer were also obtained by performing md simulations using two copies of the helix, at a 14-16 å distance between the centers of mass and in different orientations, as starting model. the obtained structures were related to the available experimental data collected on igm tm region of different species. identification of a new trematomus bernacchii immunoglobulin light chain isotype c de santi*, v morea^ mr coscia*, s giacomelli*, a tramontano°, u oreste* *institute of protein biochemistry, cnr, naples, italy ^ institute of molecular biology and pathology, cnr, rome, italy °department of biochemical sciences “a. rossi fanelli”, university of rome “la sapienza”, rome, italy immunoglobulin light chains (igl) have been sequenced in 20 different teleost species. in each species one, two or three different isotypes have been described. we have previously identified in the antarctic teleost trematomus bernacchii, two immunoglobulin light chain (igl) isotypes, referred to as trbel1 (distinguishable into two subgroups, trbel1a and trbel1b), and trbel3, based on comparison with the isotypes defined in other teleosts. in order to verify the presence of igl isotype 2 in t. bernacchii, a pcr approach was chosen. based on multiple alignment of igl2 sequences from different teleost species, two oligonucleotide primers, complementary to the most conserved part of the fr2 region (sense) and to the 3’ end of cl (antisense), were designed. the resulting pcr products were cloned into pgem-t easy vector and recombinant clones were isolated, sequenced, and found to belong to the trbel2 isotype. additional igl cdna clones were obtained by rt-pcr and 5’ race using isotype-specific primers. the percentages of identity of the total 30 clones confirmed their distribution in three isotypes, one of them distinguishable into two subisotypes. by multiple alignment of the cl domains, conserved positions and isotype-specific residues were identified. to compare the molecular structure of each isotype specific cl domain, molecular models were built. multiple alignment and phylogenetic tree of the cl sequences from t. bernacchii and other teleosts indicated that each t. bernacchii isotype fell into one of the three teleost isotype groups. session 6. chairman: l mastrolia, university of tuscia, viterbo, italy allelic polymorphism of the igµ exons in the antarctic teleost trematomus bernacchii mr coscia, r de feo, s giacomelli, u oreste institute of protein biochemistry, cnr, naples, italy in mammals, the immunoglobulin constant domains are relatively invariant, despite small amino acid differences are known to exist between products of the genes encoding iga, igm, and igg among the population. comparative studies in nonmammalian organisms have occasionally shown more than one igμ constant domain sequence, but they were usually attributed to gene duplication. to search for polymorphism in the antarctic teleost trematomus bernacchii igμ gene, total rna was extracted from the spleen of eight specimens caught in the same area. two specific pcr primers were designed to amplify the entire constant region. multiple alignment of the eight igμ sequences, revealed that at least 51 positions were polymorphic. the individuals analyzed were found to be heterozygous or homozygous for each polymorphic position as expressing one or two variants. the highest number of polymorphic positions was observed in a particular region. in fact 30 out of 51 nucleotide substitutions were found to fall within the “hinge” region which connects the ch2 and ch3 domains. this region not only displayed extensive nucleotide variation, but also length diversity; in fact several sequences were one amino acid shorter as resulting from the usage of a different splice acceptor site as demonstrated by the analysis of the genomic dna. polymorphism was observed also at some potential n-glycosylation sites. the ka/ks ratios of the polymorphic positions showed typical values higher than one, indicative of positive selection acting to polymorphic residues to favor amino acid replacements and maintain allelic polymorphism. comparison with other non-antarctic teleosts revealed that the high level of polymorphism in the “hinge” region is a peculiar feature of t. bernacchii. these results suggest that this property may have some biological significance, possibly related to modulating susceptibility/resistance to cleavage by bacterial or parasitic protease. secretory immunoglobulins in the skin of the antarctic teleost trematomus bernacchii c motta*, mr coscia^, a de santis*^, s tammaro*, u oreste^ *department of biological sciences, university of naples “federico ii”, naples, italy ^institute of protein biochemistry, cnr, naples, italy 34 the presence and localization of secretory immunoglobulins (ig) in the skin of the antarctic teleost trematomus bernacchii has been investigated by using specific antisera in situ and in western blots. analyses have indicated that l and h chains are present, that their molecular weights are similar to that of mucus ig and that they are localised in filamentous, but not in mucous cells. immuno-gold investigations have demonstrated that the ig are dispersed in the cytoplasm and concentrated at the level of the endoplasmic reticulum thus suggesting a local production. in situ hybridizations confirm the hypothesis since demonstrate that filamentous cells synthesize mrna for the ig h chain. hybridization also reveals that a significant synthesis of h chain mrna occurs in mucous cells indicating a selective activation of post-transcriptional control mechanisms in the different cell types forming the skin in trematomus. b cells in lymphoid tissues of the antarctic teleost trematomus bernacchii l abelli*, mr coscia°, f bertoni*, c caprera^, n romano^, u oreste° *department of biology and evolution, university of ferrara, ferrara, italy °institute of protein biochemistry, cnr, naples, italy ^department of environmental sciences, university of tuscia, viterbo, italy original data are presented on cytology and distribution of b cells in lymphoid tissues of the emerald rockcod (trematomus bernacchii, boulenger 1902), a bony fish living at sub-zero temperatures. b cells were identified by immunohistochemistry using antisera against homologous ig h and l chains. expression of membrane and secretory h chain transcripts and proteins was analised by rt-pcr and immunoblotting. according to general features of the teleost immune system, b cells were numerous in the head kidney and spleen, while were fewer in the gills and intestine. on the other hand, a striking finding was the concentration of ig+ cells in the thymus, clearly exceeding that found in all fish species studied so far. the differential distribution of membraneand cytoplasmic-ig+ cells (likely mature b cells, resting or stimulated) in the different vascular districts suggested a key role of head kidney and thymus in early b development, and of the spleen as the site of definitive maturation. in the gills, ig+ cells were scattered around the filament artery, in the pluristratified filament epithelium and around the outer marginal channel near the lamellar tip. in the intestine, ig+ cells constituted a minor leucocyte population, localised almost exclusively in the lamina propria and submucosa. an increasing gradient (towards the stomach) in the number of ig+ cells and eosinophilic granulocytes along the intestine suggested a higher inflammatory and immune responsiveness in the anterior segment, also mediated by hepatobiliary transport of immunoglobulins. localization of cd8α expressing t-cells in developing sea bass thymus: an hypothesis of positive selection s picchietti°, l guerra°, f buonocore°, am fausto°, m mazzini°, l abelli * °department of environmental sciences, university of tuscia, viterbo, italy *department of biology and evolution, section of comparative anatomy, university of ferrara, ferrara, italy these studies on thymus of the teleost dicentrarchus labrax (l.) focused on differentiation and positive selection of t cells, crucial steps in the development of a functional immune system in all jawed vertebrates. sea bass eggs, larvae (from day 2 until day 92 ph) and juveniles were analysed for developmental appearance of transcripts of two important genes in t cell function, cd8α and tcrβ. rt-pcrs detected tcrβ transcripts in larvae from 25 days ph. otherwise, cd8α expression was detected at day 51 ph when the thymus was well developed. in situ hybridization of cd8α  mrna identified thymocytes in the outer and lateral zones of the thymic paired glands. from day 75 ph on, the signal was mainly detected in the cortex and the corticomedullary junction. in one-year-old specimens cd8α and tcrβ expression patterns nearly overlapped, drawing a cortex-medulla demarcation in each thymic lobe. large cords of cd8α + tcrβ + cells lay in the medulla. a cd8α tcrβ+ subcapsular zone was evident near the septa coming from the inner connective capsule that delimited the thymus. no signal was found in subcapsular and cortical epithelial cells. the tcrβ and cd8α expression patterns demonstrated a compartmentalization of the thymus due to distinct localization of thymocytes at different developmental stages and suggested an hypothesis about positive selection in teleost thymus as described in mammals. localization of glucocorticoid receptor dlgr1 in tissues of teleost dicentrarchus labrax m vazzana, a vizzini, g salerno, ml di bella, n parrinello marine immunobiology laboratory, department of animal biology, university of palermo, palermo, italy cortisol is a glucocorticoid that affects a wide variety of biological responses, including immune 35 functions. virtually all tissues in the body are target organs and they can respond in different ways. the physiological response to cortisol is mediated through binding at two classes of intracellular receptors like mineral corticoids (mr) and glucocorticoids (gr) that act as ligand-dependant transcription factors. a cortisol receptor (dlgr1), (vizzini et al., 2007), from leukocytes of peritoneal cavity, previously cloned and sequenced, were employed for in situ hybridization and immunohistochemical assays. the experiments were performed on brain, head kidney, spleen, gills, intestine and hearth. the mrna and protein were expressed in the examined tissues. the wide expression and distribution of dlgr1 confirm the importance of the cortisol role in the maintenance of the homeostasis in the organisms. innate immune response of reared european sea bass dicentrarchus labrax to different environmental and husbandry conditions t petochi°, p di marco*, a priori*, mg finoia*, g marino* °icram, central institute for marine applied research, rome, italy *department of environmental sciences, university of tuscia, viterbo, italy innate immune parameters were evaluated as indicators of health and welfare in reared sea bass dicentrarchus labrax in relation to seasonal temperature changes, experimental carbon dioxide exposure (0-5, 15-20, 30-35 and 50-55 mg/l) for 45 days and different stocking densities (15, 30 and 45 kg/m3) for 35 days. complement activity (ach50) showed a seasonal trend, increasing during the summer and reaching maximum levels in september, with a positive correlation to high water temperature. significant increase in serum lysozyme was measured in late summer and autumn, but any evident correlation with water temperature was found. hypercapnia induced a transient decrease in ach50 activity within the first 24 hours of co2 exposure and in lysozyme levels in the first week of exposure. at the end of the experiment (45 days), there were no significant differences in lysozyme and ach50 among hypercapnic groups and controls and compared to initial levels. the respiratory burst activity in hypercapnic groups was significantly reduced compared to controls after 45 days of co2 exposure. high stocking density affected the ach50 activity in sea bass with a significant decrease in fish maintained at 30 and 45 kg/m3 for 35 days. lysozyme activity did not change in fish reared up to 45 kg/m3. results of present investigations indicate an influence of low water temperature, hypercapnia and high stocking density on innate immune response of sea bass that could be detrimental for health and welfare status. transferrins, nitric oxide and cytokines: complex responses to marek’s disease virus reinfection in a chicken lymphoblastoid cell line mf giardi, f giansanti, d botti department of basic and applied biology, university of l’aquila, l’aquila, italy in previous works, we have shown that a relevant antiviral activity against marek’s disease virus (mdv) is exterted by ovotransferrin (otrf), lactoferrin (lf) (giansanti et al. biochem. cell. biol. 80: 125-130, 2002) and two ovotransferrin derived peptides (giansanti et al. biochem. biophys. res. commun. 331: 69-73, 2005). in addition, we have also evidenced the production of otrf by a chicken lymphoblastoid cell line (mdcc-msb1) induced by mdv, following reinfection with mdv (giansanti et al. biochem. cell biol. in press). the aim of the present work is to verify if mechanisms other than tranferrins are involved in the defense against the above mentioned reinfection. for this reason, the possible role of no, il-8 and ifn-γ has been investigated. the data obtained indicate that no production by mdccmsb1 is strongly enhanced in the presence of transferrins and after the reinfection. the no production is completely inhibited by aminoguanidine (ag), an inhibitor of inos. it has also been observed that both cytokines stimulate the production of no, and that the maximum stimulatory effect was obtained in the presence of ifn-γ plus otrf or in the presence of il-8 plus lf. also in this case, ag was able to completely inhibit the production of no. 36 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice isj 4: xxx-yyy, 2007 isj 4: 112-118, 2007 issn 1824-307x review longevity genes across species: conservation versus evolvability s salvioli1,2,3, p tieri1,2, g castellani2,4, m capri1, c barbi1, a santoro1,2, serena altilia1,2, l invidia1,2, m pierini1,2, e bellavista1,2, d monti5, c franceschi1,2,3,6 1 department of experimental pathology, university of bologna, 40126 bologna, italy 2 interdepartmental center “l. galvani” (c.i.g.), university of bologna, 40126 bologna, italy 3 er-gentech laboratory, 44100 ferrara, italy 4 dimorfipa, university of bologna,, 40064 ozzano dell'emilia, italy 5 department of experimental pathology and oncology, university of florence, 50134 florence, italy 6 department of gerontological science, italian national research centre on aging (inrca), 60131 ancona, italy accepted november 11, 2007 abstract the search for longevity genes has greatly developed in recent years basing on the idea that a consistent part of longevity is determined by genetics. the ultimate goal of this research is to identify possible genetic determinants of human aging and longevity, but studies on humans are limited by a series of critical restrictions. for this reason, most of the studies in this field have been, and still are, performed on animal models, basing on the assumption that fundamental biological mechanisms are highly conserved throughout evolution and that, accordingly, extrapolation from model systems to humans is quite reasonable. indeed, many comparative data obtained on single genes or gene families fit with this assumption. however, it is also clear that, despite such a basic conservative scenario, major changes also occurred in evolution, particularly regarding biological regulatory processes and integration between and among pathways. this consideration raises the fundamental question of the transferability of the results obtained from model systems to humans. in this review, we discuss the differences between animal models and men regarding the genetics of aging and longevity, and the possible reasons that can explain such discrepancies, with a particular emphasis on the phenomena of conservation and evolvability of biological systems. finally we will suggest a possible strategy to identify putative longevity genes basing on their position inside conserved metabolic structures. key words: genetics of longevity; animal models; conservation; evolvability introduction in biological studies, the reductionist approach has been very successful, and the use of simple experimental models allowed the researcher to obtain an exceptional number of results. the most popular example of this approach is represented by the bacteriovorous nematode caenorhabditis elegans, a small worm composed of 959 cells in the adult hermaphrodite form. a defined subset of 131 cells undergoing programmed cell death has allowed to the identification of specific apoptosisrelated genes, the so called ced genes (hengartner ___________________________________________________________________________ corresponding author: claudio franceschi department of experimental pathology university of bologna via s. giacomo 12, 40126 bologna, italy e-mail: claudio.franceschi@unibo.it and horvitz, 1994). cloning of these c. elegans genes has revealed that nematodes and mammals share a common pathway for programmed cell death. the same reductionist approach has led to the identification of longevity genes in c. elegans since 1988 (friedman and johnson, 1988). in 1992, johnson and lithgow wrote: “if we are fortunate and aging processes exhibit evolutionary conservation, many exciting possibilities await”. at least in some cases, human homologs of invertebrate longevity genes have been found (in particular genes belonging to the igf-1 signalling pathway, see barbieri et al., 2003). such similarities are striking and suggest that the insulin/igf-i regulatory system arose early in evolution and that the fundamental mechanisms that control aging and longevity may be evolutionarily conserved from invertebrates (and even yeast) to mammals, not excluding humans. 112 table 1 comparison between animals and humans as experimental models for genetic studies on aging and longevity. *shorter life span and genetic inbreeding can be both advantageous and disadvantageous. for example, it is questioned whether the effect of the accumulation of mtdna mutations on aging can be seen in mice that live only two years at maximum (santoro et al., 2006). genetic inbreeding is generally advantageous, but it has hampered to discover the effects of mtdna haplogroups on longevity, that can only be seen in outbred populations as humans are (de benedictis et al., 1999). animal models humans advantages shorter life span* genetically inbred* possibility to use very high number of subjects controlled environment simple organisms (especially if invertebrates) subjects can be easily genetically manipulated many genes involved in longevity has been discovered only in human studies disadvantages shorter life span* genetically inbred* genetic and epigenetic differences with respect to humans different level of complexity with respect to humans long life span complex organisms no possibility of genetic manipulation large populationspecific genetic differences profound influence of the environmental background presence of cultural factors other than the relative simplicity of the organism to be studied, c. elegans has many other evident advantages over different experimental models: for example the short life-span, that allows the execution of experiments in short times (experiments that otherwise will be intolerably longer in animal species with a life span of many years, like primates). another advantage is the great number of animals that can be studied with a relatively small effort, and most importantly, the possibility to easily manipulate the genome of such animals, in order to obtain data on the role of particular genes in the aging process or in life span control. these and other pros and cons of invertebrates experimental models are listed in table 1. the main problem with these experimental models is that, for a series of reasons, we were not so fortunate as hoped by johnson and lithgow. indeed, many data obtained on animal models were not confirmed on humans or, on the contrary, some contributions to the genetics of longevity were discovered in humans and not always have been replicated in animals (franceschi et al., 2007). in this review we will briefly summarise some paradigmatic cases in which results found in animals have not been reproduced in men and vice versa, and we will discuss the possible reasons that can explain these differences, with a particular emphasis on two striking features of the conserved biological structures, i.e. conservation and evolvability. genetics of aging and longevity 1: the case of p66shc gene possible reason for the discrepancies between data obtained from animal models and humans can be the profound differences in the environment where experimental animals and humans live in. only recently it has been considered by researchers that the animals used for experiments live all their entire life in safe and pathogen-free environment. they do not have to cope with predators or pathogens, they do not suffer famine, they even do not have to struggle for food, nor to compete for reproduction. this setting assures basic requirement of scientific research, i.e. the possibility to replicate the results in different laboratories, but of course it is a strongly unnatural situation that may lead to confounding results, especially when studying the genetics of aging and longevity. an example of such situation is given by the discovery that the ablation of p66shc gene leads to an enhancement of longevity in mice (migliaccio et al., 1999). a fraction of p66shc has a mitochondrial localization where it can oxidise cytochrome c and give rise to the production of radical oxygen species (ros) (giorgio et al., 2005), and the localization to mitochondria seems to be regulated by pkc-β and pin-1 (pinton et al., 2006). thus, it seems that p66shc gene impinges upon aging and longevity by regulating the resistance to oxidative stress-mediated apoptosis (trinei et al., 2002). then, if p66shc is a bona fide 113 pro-aging gene, one should expect that long living animals do express this gene at low levels. to date, this hypothesis has not yet been tested. however, we studied p66shc expression in humans, and, unexpectedly, we found that centenarians have higher levels of p66shc with respect to young people (pandolfi et al., 2005). these findings lead to the conclusion that the effect on longevity of p66shc is either species-specific, or highly dependent on the environmental conditions. supposing that such effects are not species-specific, it has to be considered that all animal species including humans are evolved and still live in an environment that is profoundly different from that of a laboratory (for example it is very dirty from an immunological point of view), thus it can not be excluded that p66shc has important effects for the fitness of the organism in the wild that are not necessary in a cage. to say, it is possible that p66shc-/animals can have some disadvantages in survival with respect to wild type animals, and that such effects only become evident in the environment where genetic selection occurred. finally, it must be also considered that the ablation of p66shc gene can lead to an altered stress response that may be the real responsible for the increased life span observed in p66shc-/animals. genetics of aging and longevity 2: the case of mitochondrial dna the contribution to longevity of the inherited variants of mitochondrial dna (mtdna) has been neglected until studies performed on humans addressed the question. in particular, some haplogroups of the mtdna have been found to be more represented among centenarians and longliving subjects with respect to younger people (tanaka et al., 1998; de benedictis et al., 1999). this discovery would have never come from inbred animals which share the same mtdna molecule (mtdna does not recombine and it is inherited only from the mother). the involvement of mtdna inherited variants in longevity has not yet been confirmed in animals, but other very elegant experiments in mice have indicated that such variants can modulate functions that are likely important for survival, such as memory and learning (roubertoux et al., 2003). a possible positive role in longevity for specific somatic mutations of the mtdna has also been postulated basing on studies on humans (zhang et al., 2003; niemi et al., 2005; rose et al., 2007). the c150t mutation in the dloop of the mtdna molecule in particular has been found to be more represented in long-living people with respect to younger counterparts. such a mutation seems to cause an alternative origin of replication of the mtdna strands and would be thus advantageous for mtdna pool maintenance (zhang et al., 2003). more recently it has been reported that the tendency to accumulate somatic mutations at the mtdna seems to be genetically controlled, since centenarians and their offspring and nephews share the same levels of mtdna heteroplasmy at the level of d-loop, which differ from that of unrelated people (rose et al., 2007). these figures indicate that, despite their usefulness, animal models are unable to identify all the genetic determinants of human longevity, because of intrinsic limitations of the model, or for specific differences between animals and humans. the opposite situation is also possible, that is, results obtained in animal models that can not be reproduced in humans. for example, recent findings indicate that mtdna point mutations can accumulate in organs and tissues without affecting aging in mice (vermulst et al., 2007), but still the levels of mtdna mutations found in such a model (polgmut/+ mice) are much higher than that found in aged human colonic crypts (taylor et al., 2003), thus suggesting that the functional impact of mtdna mutations is likely different in mice and humans (khrapko and vijg, 2007). genetics of aging and longevity 3: population heterogeneity, heterozigosity and homozygosity a peculiar contribution to the study of the genetics of aging and longevity has come from observations on the levels of heterozygosity and homozygosity of genetic loci of interest. it is generally accepted that natural selection awards heterozygosity as a way to attain high levels of fitness during the reproductive period. nevertheless, it was not known whether the levels of heterozygosity were also fitting with longevity. by studying people of different ages including centenarians we observed that, contrary to what it was expected, the level of homozygosity of a locus in chromosome 1p35 was increased in centenarians with respect to young people (bonafè et al., 2001). in a following study, we observed that this locus, rich in alu sequences, contains a gene called ythdf2 that shows an increase in homozygosity in centenarians (cardelli et al., 2006). in this study we genotyped 412 participants of different ages, including 137 centenarians, and we confirmed the increased homozygosity in centenarians at this locus, and observed a concomitantly increased frequency of the most frequent allele and the corresponding homozygous genotype. remarkably, the same genotype was associated with increased ythdf2 messenger rna levels in immortalized lymphocytes. thus, these data suggest a possible role of this locus in human longevity, and more interesting, they suggest the counterintuitive concept that increased homozygosity can contribute to human longevity. these results has been obtained thanks to the study of a genetically heterogeneous population, as humans are. in animal models, different strains are used, but the animals of each strain are rather homogeneous from a genetic point of view, thus making this discovery almost impossible in such models. when these results will undergo replication in animal models, genetically heterogeneous animals must be used. the role of the environment 1: the antigenic load an important environmental factor impinging upon longevity is the antigenic load, i.e. the number and intensity of antigenic stimuli to which everybody is exposed during lifetime (de martinis et al., 2005). besides the life-threatening risks of the exposure to highly pathogenic microorganisms, it is becoming 114 evident that also the mild chronic exposure to antigenic stimuli (for example to viruses such as cytomegalovirus, cmv) for a period of time largely unpredicted by evolution profoundly affects the possibility to attain longevity (pawelec et al., 2006; vescovini et al., 2007). this life-long exposure to antigenic stimuli is one of the main causes of modifications occurring with age of the immune system, leading to the phenomenon known as immunosenescence and to an increase in inflammatory reactions that favours the onset of many age-associated diseases which do share an inflammatory pathogenic background, such as type ii diabetes, cardiovascular diseases, neurodegenerative diseases and many types of cancer. this agerelated increase in inflammatory markers has been termed “inflammaging” by our group (franceschi et al., 2000). the effect of such (acute or chronic) antigenic exposure is forcedly skipped out in most experimental models, where animals live in a pathogen-free environment. in particular, the role of chronic antigenic burden on longevity is extremely difficult to assess in animals living in a clean or even sterile environment. furthermore, the immune responses are clearly very important in order to attain longevity, and not only the immune system of a mammal is much more complicate than that of an invertebrate, but also the characteristics of immunosenescence, i.e. the age-related modifications of the immune responses, can be strikingly different among species (pawelec et al., 2002). such a confounding factor can overcome the effect of the gene of interest, thus leading the researcher to the conclusion that such a gene does not play a role in longevity. the role of the environment 2: the dietary restriction as mentioned at the beginning of this review, a striking difference between men and animal models regards metabolic regulatory processes. to date, the so called directionality theory distinguishes between species according to the ecological constraints they have to face within their ecological niche (braeckman et al., 2006). according to this theory, two groups of species are recognized: equilibrium species and opportunistic species. the first group is composed mainly by large mammals, like humans, that spent the most part of their evolutionary history in a stationary growth phase, with a limited but roughly constant amount of resources. the latter group includes many small mammals like rodents, insects and worms like c. elegans. these species are subject to fluctuations in size because of irregular availability of resources (periods of abundance and scarcity). it is very likely that species belonging to the two groups, despite the fact that they can share genes or even entire metabolic pathways, may have a different behaviour in terms of survival in face of environmental challenges. for example, the best known treatment that extends life span in animal models (mainly opportunistic species, according to the directionality theory) is dietary restriction (dr), i.e. the reduction in food intake. studies on c. elegans have showed that dr increases life span of such animals (klass, 1977; lakowski and hekimi, 1998; houthoofd et al., 2002). the same has been demonstrated for other “opportunistic” species that are classical animal experimental models, such as drosophila and mice, but contrasting results have been reported on long living mammals and humans (kayo et al., 2001; roth et al., 2004; everitt and le couteur, 2007). many data suggest that dr acts as a low intensity stressor and increases metabolic stability (masoro 1998, butov et al., 2001). equilibrium species, like humans, already have a strong metabolic stability, and thus dr would have a relatively small effect on such species (braekman et al., 2006; demetrius, 2006). from survival studies on overweight and obese people, it is estimated that long-term dr could add 3 to 13 years to human life expectancy (holloszy and fontana, 2007), quite far from the dramatic effects observed in worms and rodents, and in any case much lower than the effects of improved life-style conditions. one of the effects of dr is the reduction of the insulin/igf-1 signalling pathway (clancy et al., 2002), and, as mentioned, this pathway appears to be conserved throughout evolution from yeast to mammals. genetic studies on humans have indicated that some polymorphisms of genes involved in insulin/igf-1 pathway (igf-1r, pi3kcb) are associated with longevity (bonafè et al., 2003). these polymorphisms are correlated to low levels of igf-1, and this finding fits with the data obtained in experimental models, confirming that lower activity of the insulin/igf-1 pathway is a major determinant of longevity and that genes involved in such a pathway can be considered longevity genes. it is to note however that these and other genes, such as mtor and p66shc, increase longevity when they are (at least partially) turned off, and their ablation often produces dwarf animals with a series of defects such as obesity and decreased fertility (longo and finch, 2003). moreover, it has been reported that high levels of igf-1 and low levels of proinflammatory cytokines such as il-6 are beneficial for muscle mass maintenance and are associated with decreased risk of mortality in old people (barbieri et al., 2003). thus, there is a clear trade off between early life fitness and longevity, and every species is the result of a million-years evolution that tuned between these two contrasting goals, reaching a peculiar equilibrium for any species. hence, comparison between species in which this trade-off has led to a different equilibrium is risky. however, a series of possible pharmacological treatments have been envisaged to mimic the prolongevity effects of igf-1 deficiency escaping in the same time the detrimental side-effects (obesity, dwarfism, infertility, sarcopenia) of this deficiency (longo and finch, 2003). conservation and evolvability recent conceptualizations stressed the importance of robustness as one of the fundamental properties of living organisms, from cellular (stelling, 2004) to organismal level (kitano and oda, 2006; kitano, 2007), a characteristic acquired during evolution which allow them to be error-tolerant and to easily respond to external perturbations (jeong et 115 al., 2000). metabolic stability appears to be genetically determined and respond to the requisites of robustness. on the basis of available data obtained from different organisms, especially yeast and invertebrates, a “bow-tie” organizational architecture is likely to be a common feature of highly organized, robust systems (csete and doyle, 2002, 2004), which enables them to accommodate perturbations and fluctuations on many temporal and spatial scales. a bow-tie model is present when many inputs converge on, and are integrated by, few elements, and many different outputs come out as the product of the integration. the elements composing the core (“knot”) of the bow-tie in a biological system are “hub proteins” and the genes that encode them are likely to be in many cases robustness genes. such a structure has also an inner fragility, because few enzymes responsible for robustness can be easily hijacked by pathogenic microorganisms or used to amplify pathological processes (csete and doyle, 2002; kitano and oda, 2006; kitano, 2007). much of the core of a bow-tie is often conserved throughout evolution, but this conservation does not prevent, but rather facilitates the variability of the possible outputs. thus, a bowtie structure allows both robustness and evolvability (gerhart and kirschner, 1997; caporale, 2003). longevity could be intended as a consequence of the robustness of the animal system. indeed, it is conceivable that the outputs of a bow-tie structure include elements that ultimately control the life-span of the animal. if these elements can vary, as a feature of the bow-tie evolution, this variation may explain, at least in part, why knot genes, yet conserved, not always result to be involved in longevity on evolutionarily distant animals according to classical association or functional studies, thus casting some doubts on their role as real “longevity genes”. conclusions homo sapiens is one of the most long-living species among animals, but his longevity has had a dramatic increase in recent times, at least in western countries, due to profound modifications in lifestyle. thus, human longevity has two major components, whose effects are very difficult to distinguish, genetics and culture. this raises an insurmountable obstacle, since no animal species depends on culture as humans do. thereafter, results obtained on animals studies not only must be always confirmed on humans, but they are also not completely satisfactory for a series of reasons: these studies do not consider neither the genetic and epigenetic differences existing between men and animal species, even those much close to humans, nor the influence of culture on the duration of the life after the age of reproduction, i.e. after the period influenced by natural selection. as an example of specific genetic differences between man and animals, it can be mentioned the case of tp53 gene. this gene is crucial for a series of biological processes such as apoptosis, cell senescence, dna repair, and energy metabolism, and is considered a longevity gene in both animals and humans (donehower, 2005). in humans tp53 gene harbours a common functional polymorphism at codon 72 which alters the protein' functions (thomas et al., 1999; dumont et al., 2003; bonafè et al., 2004). this polymorphism has been and still is under investigation for its possible involvement in longevity (bonafè et al., 2002; van heemst et al., 2005), and such effects, if any, can be detected only in humans, because this polymorphism does not exist in chimpanzee, while in mice it seems to have no effects (phang and sabapathy, 2007). moreover, our data indicate that the effects of such a polymorphism become evident as the age of the studied subjects increases (bonafè et al., 2004; salvioli et al., 2005) thus suggesting that the role of such a gene does change with age. this could be a case of antagonistic pleiotropy, that is, a gene that has a positive effect for fitness at young age could turn to be detrimental later on, in a period of time not selected for reproductive fitness, or vice versa. the genetics of human longevity has thus been proposed to be described as a post-reproductive one (de benedictis and franceschi, 2006). it is at present unknown whether also laboratory animals have a post-reproductive genetics, and which similarities it has with the human one. in conclusion, animal models remain an irreplaceable tool to shed light into the genetics of aging and longevity, but the transferability of the results to humans is always an issue. as discussed all along this review, longevity genes are classically identified by their association with an extended life-span, but for a series of reasons this strategy is not always successful, and results can not always be reproduced in different experimental models. here we propose the idea that longevity genes could be identified by an alternative strategy. as mentioned, conservation and evolvability are inherent features of the bow-tie structures, that determine robustness. if robustness is important for longevity, it can be hypothesised that longevity genes should participate with a core position to a bow-tie metabolic structure. we propose that a new way to identify putative genetic determinants of longevity could be the conservation of their products in bow-tie structures all along evolution, rather than for their association with prolonged life-span. in this perspective, studies of comparative biology associated with a systems biology approach could be useful tools to get some insights also into the genetics of human longevity. acknowledgements this work was supported by eu (european union) grant “geha – genetics of healthy aging” fp6-503270; eu grant “proteomage” fp6518230; the prriitt program of the emilia-romagna region (and fondi strutturali obiettivo 2); italian ministry of health grant “progetto finalizzato <<studio delle differenze uomo-donna nei meccanismi patogenetici delle malattie cardiovascolari>>” to cf; italian ministry of university and research (miur) prin 2006 project to cf and dm (# 2006061707), and ss (# 2006063387); university of bologna grant “ricerca fondamentale orientata (rfo ex 60%) 2005”; roberto and cornelia pallotti legacy for cancer research grants to cf and ss. references barbieri m, bonafe m, franceschi c, paolisso g. insulin/igf-i-signaling pathway: an evolutionarily conserved mechanism of longevity from yeast to humans. am. j. physiol. 116 endocrinol. metab. 285: e1064-e1071, 2003. barbieri m, ferrucci l, ragno e, corsi a, bandinelli s, bonafe m, et al. chronic inflammation and the effect of igf-i on muscle strength and power in older persons. am. j. physiol. endocrinol. metab. 284: e481-487, 2003. bonafe m, barbi c, storci g, salvioli s, capri m, olivieri f, et al. what studies on human longevity tell us about the risk for 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in the human tp53 gene affects old age survival and cancer mortality. exp. gerontol. 40: 11-15, 2005. vermulst m, bielas jh, kujoth gc, ladiges wc, rabinovitch ps, prolla ta, et al. mitochondrial point mutations do not limit the natural lifespan of mice. nat. genet. 39: 540-543, 2007. vescovini r, biasini c, fagnoni ff, telera ar, zanlari l, pedrazzoni m, et al. massive load of functional effector cd4+ and cd8+ t cells against cytomegalovirus in very old subjects. j immunol. 179: 4283-4291, 2007. zhang j, asin-cayuela j, fish j, michikawa y, bonafe m, olivieri f, et al. strikingly higher frequency in centenarians and twins of mtdna mutation causing remodeling of replication origin in leukocytes. proc. natl. acad. sci. usa 100: 11161121, 2003. 118 isj 3: 89-96, 2006 isj 4: 10-12, 2007 issn 1824-307x short communication monitoring of the immune efficiency of mytilus galloprovincialis in adriatic sea mussel farms in 2006: regular changes of cytotoxicity during the year d malagoli, l casarini, e ottaviani department of animal biology, university of modena and reggio emilia, modena, italy accepted february 01, 2007 abstract by monitoring the course of hemolymph cytolytic activity in mytilus galloprovincialis during 2006, we have observed important fluctuations in the percentage of cytotoxic animals over the year. the changes seem to be correlated with seasonal variations in the temperature, but observations in mussels kept in aquaria indicated that this parameter is not the main cause of the fluctuations. data presented here suggest that normal levels of cytotoxicity can be predicted in a population for a specific period of the year, therefore confirming the value of this parameter in determining the immune efficiency of mussels at a given time. key words: mytilus galloprovincialis; cytotoxicity; immune efficiency __________________________________________________________________________________________ introduction the presence of hemolytic molecules in the hemolymph of molluscs has been reported on several occasions (wittke and renwrantz, 1984; merker and levine, 1986; hubert et al., 1997), but the natural target of these molecules has still not been clarified. it is conceivable that hemolyitic factors can be included in the humoral component of invertebrate innate immunity (hubert et al., 1997), but there is very little information about the relationship between hemolytic activity and immune efficiency in the mussel (malagoli and ottaviani, 2005). recently, the hemolytic activity of the bivalve mytilus galloprovincialis has been seen to be influenced by stressful and pathological conditions imposed either in laboratory aquaria (malagoli and ottaviani, 2005) or encountered in mussel farms (franchini et al., 2005). in order to take hemolytic activity as a valid parameter for evaluating whether the immune efficiency of mussels is compromised in particular circumstances, it is important to know how this activity changes during the year. this report provides data on fluctuations in hemolymph cytotoxic activity in mussels from the adriatic sea in italy during 2006. moreover, ___________________________________________________________________________ corresponding author: enzo ottaviani department of animal biology via campi 213/d 41100 modena, italy e-mail: ottaviani.enzo@unimore.it comparison of the present results with data collected in 2005 (malagoli et al., 2005) and with seasonal variations in water temperature suggest that hemolymph cytotoxicity is a parameter subjected to seasonally-regulated fluctuations. materials and methods animals specimens of the bivalve mollusc mytilus galloprovincialis were obtained monthly from local fishermen in the cesenatico area (fc, italy). after their collection, 40 animals were used to obtain the hemolymph immediately, while the remaining specimens were maintained in the laboratory aquaria in artificial seawater (temperature 16 ± 1 °c, ph 8.0 ± 0.2 and salinity 35 ± 1 psu). after 14 days, a further 40 mussels were sacrificed and the hemolymph withdrawn. hemolymph preparation and cytotoxicity assay the detailed procedure for the hemolysis assay is described elsewhere (malagoli and ottaviani, 2005). in short, the hemolymph was collected by gently aspirating with a sterile syringe inserted between the mussel valves and filtered into sterile tubes using 0.2 μm sterile filters. hemolytic activity was evaluated by checking the cytolysis of human a positive erythrocytes obtained after washing the whole blood at least three times in 9 vol. of sterile nacl 0.9 %. subsequently, the erythrocytes were 10 fig. 1 course of mussel cytotoxicity during 2005 and 2006 in the of cesenatico area. please note that the value for february 2005 is missing (malagoli et al., 2005). re-suspended in sterile tbs (50 mm tris-hcl, 200 mm nacl, 10 mm cacl2, ph 8.5) at a final concentration of 2x109 cells/ml. five hundred μl of filtered hemolymph were added to 500 μl of erythrocyte suspension and then incubated for 1 h at 25 °c (hubert et al., 1997; malagoli and ottaviani, 2005). after incubation, samples were centrifuged at 3000xg for 5 min at 4 °c, and the optical density (od) of the supernatants was evaluated by measuring absorbance at 541 nm with a helios β spectrophotometer (spectronic unicam, cambridge, uk). samples with an od above the fixed od threshold level of 0,5 where considered cytotoxic (malagoli and ottaviani, 2005). the experiments were repeated twice in duplicate for each animal. all chemical reagents came form sigma-aldrich (st louis, mo, usa). results and discussion the monthly evaluation of the hemolytic activity revealed significant fluctuations in the percentage of cytotoxic animals during the year (fig. 1). even if the mean percentage of cytotoxic bivalves is 45 %, two peaks were registered during the year: one at the end of the spring and the second at the end of the summer. interestingly, the trends in hemolyitic activity in the second half of 2005 and 2006 almost overlapped (fig. 1). since no peculiar situations were reported for the area in which the mussels were reared, the comparable results indicate that cytotoxicity in mussel populations is normally subject to fluctuations during the year. temperature can be considered the most common variable in the mussel farm area assessed in this study. we therefore compared the time course of water temperature with that of cytoxicity in the mussel population (fig. 2). the two variables seem to be correlated, since the peak in hemolytic activity in the hemolymph corresponded to the two periods in which the temperature either started to rise or to fall (fig. 2). however, the comparison of cytotoxicity between animals sacrificed immediately after collection and those kept for two weeks in aquaria at a constant temperature indicates that temperature cannot be the main parameter in determining cytotoxicity (fig. 3). for each month, the percentage fig. 2 comparison between hemolymph cytotoxic activity and temperature variations during 2005 (a) and 2006 (b). 11 fig. 3 comparison between cytotoxicity immediately after collection (white bars) or after a period of conservation in the aquarium at 17 °c (black bars). of cytotoxic mussels was almost identical in the two groups of animals. the essentially unmodified cytotoxic activity following conservation in the aquarium is in agreement with observations in 2005 (malagoli et al., 2005), demonstrating that even when mussels are maintained at a constant temperature different to that of the seawater, the level of cytotoxicity among population does not change significantly. in previous articles, we have reported that sudden changes in aquarium temperature can modify the number of animals displaying significant cytotoxic activity (malagoli and ottaviani, 2005), but we have also found that when sudden modifications in environmental parameters do not intervene, cytotoxic activity is a relatively stable immune function (malagoli et al., 2005). further studies are required to establish whether the component influencing the time course of cytotoxicity is mainly environmental or rather connected to the mussel life cycle. the mussel’s cytotoxic response to seasonal changes would represent a classical example of ecoimmunology. evolutionary ecologists assume that immunological defences must be minimized in terms of metabolic cost, because there must be a trade-off between maintaining a normal immune response and facing the significant changes in life conditions. from an evolutionary point of view, this balance plays a key role in species survival (lochmiller and deerenberg, 2000). concluding, mussel cytotoxicity is an activity that changes over the year with a regular time course, meaning that normal levels can be predicted for a given period. our observations support the idea of using hemolymph cytotoxicity as a useful parameter in evaluating the immune efficiency of mussels at a specific point in time (malagoli and ottaviani, 2005). acknowledgment we are grateful to centro di ricerche marine (cesenatico, fc, italy) for financial support for this work. the authors wish also to thank the centro trasfusionale policlinico (modena) for providing the blood, arpa-er for giving us the access to temperature measurements and mr m marangoni who kindly provided the mussels. references franchini a, malagoli d, ottaviani e. investigation of the loss of byssus in mytilus galloprovincialis from mussel farms in the adriatic sea. cell biol. int. 29: 857-860, 2005. hubert f, cooper el, roch p. structure and differential target sensitivity of the stimulable cytotoxic complex from hemolymph of the mediterranean mussel mytilus galloproíincialis. biochim. biophys. acta 1361: 29–41, 1997. lochmiller rl, deerenberg c. trade-offs in evolutionary immunology: just what is the cost of immunity? oikos 88: 87-98, 2000. malagoli d, ottaviani e. cytotoxicity as a marker of mussel health status j. mar. biol. ass. uk 85: 359-362, 2005. malagoli d, casarini l, ottaviani e. monitoring of the immune efficiency of mytilus galloprovincialis in adriatic sea mussel farms in 2005. inv. surv. j. 3: 1-3, 2006. merker mp, levine l. a protein from the marine mollusc aplysia californica that is hemolytic and stimulates arachidonic acid metabolism in cultured mammalian cells. toxicon 24: 451465, 1986. wittke m, renwrantz l. quantification of cytotoxic hemocytes of mytilus edulis using a cytotoxicity assay in agar. j. invertebr. pathol. 43: 248-253, 1984. 12 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice isj106.pdf isj 2: 124-131, 2005 issn 1824-307x research report wound repair in the marine worm sipunculus nudus (sipunculidae) g d’ancona lunetta institute of histology and embryology university of palermo, italy accepted september 14, 2005 abstract the cells and molecules involved in the wound healing of sipunculus nudus were studied. an incision, 5 mm in length, was cut longitudinally at a site opposite the anus and 10 mm from the introvert. the histological study performed at different times showed an involvement of both type i and type ii granulocytes in the process of healing. the former were capable of extracellular digestion and they were immunoreactive to anti-il-4, -il-10 and -epidermal growth factor (egf) antibodies (abs); the latter were involved in the synthesis of connective tissue from 24 h after the incision, thereby causing the initial closing of the wound. after 70 h, a continuous layer of type ii granulocytes was found on the sides of the wound where the future muscle tissue would be formed; many of these granulocytes had been partially degranulated. it was not possible to establish any existing relationship between the functions of type i granulocytes and their reactivity to anti-il-4, -il-10 and -egf abs. kew words: marine worm; sipunculus nudus; wound repair; cytokines introduction the process of wound healing has been the subject of intensive research, mainly in vertebrates (redd et al., 2004; harvey, 2005; whitney, 2005). with regard to invertebrates, kindred (1924) observed that the removal of a fragment of the body wall in echinoderms led to the healing of the wound, by the proliferation and infiltration of cells from the surrounding connective tissue. in asterias the aggregation of coelomocyte and of amoebocytes from adjacent tissue was seen to contribute to the wound healing (anderson, 1962, 1965). in the sea cucumber stichopus tremulus numerous morula cells, found in proximity of the incision, were supposed to play a significant role in healing wounds, and to be homologous with the vertebrate mastocytes (rollefsen, 1965). in contrast, in stichopus badionotus, after superficial cutaneous incisions cowden (1968) observed a rapid repair with complete fibrogenesis without the intervention of morula cells. corresponding author: d’ancona lunetta g istituto di istologia ed embriologia, dipartimento di biologia, università di palermo, viale delle scienze, 90123 palermo, italy e-mail: dancona@unipa.it the migration of epidermal and pigment cells from the periphery of the wound margin in thyone briareus caused re-epithelialization in the absence of evident mitotic activity, an morula cells seemed to be involved in this migration although their precise role is unknown (menton and eisen, 1974). fibroblast-like cells, pigment cells and numerous coelomocytes were the first cells to arrive at wound sites in the holothuria polii and the newly formed collagen fibres were synthesized by type ii spherula cells. cutaneous lesions performed after antigenic stimulation healed more slowly than controls since type ii spherula cells were also actively involved in the immune reaction by forming brown bodies in cooperation with the amoebocytes (d’ancona, unpublished). consequently, in this case, a lower number of type ii spherula cells are available for the synthesis of connective components (canicattì and d’ancona, 1989). recently, franchini and ottaviani (2000) have studied the effects of platelet-derived growth factor (pdgf-ab) and transforming growth factor (tgf-β)1 in the repair mechanism of wounds in the mollusc limax maximus. the main repair stages include an initial infiltration phase in which the hemocytes migrate and stratify at wound margins, actively phagocitize cell debris and damaged tissue and were immunoreactive to anti-il-1α, -il-8 and -tumor necrosis factor (tnf)-α antibodies (abs). this was followed by the formation of granulation tissue, the synthesis and depositing of 124 extracellular matrix components, such as fibronectin, collagen fibres and reticular fibres. finally, reepithelialization of the wound occurred. the exogenous administration of pdgf-ab and tgf-β1 stimulated the tissue healing process though a general acceleration of the activities involved. in the coelomic fluid of s. nudus two types of granulocytes (type i and type ii), are distinguishable on the basis of their morphology and chemical composition. type i granulocytes do not have a phagocytic capability, even though they contain lytic enzymes; type ii granulocytes show phagocytic activity. furthermore, haemerythrocytes, signet-ring cells, urna cells complexes, empty vesicles, vesicle fragments, laminar structures, aggregates of stem cells and brown bodies were also found (d’ancona et al., 2004). in the present paper the mechanism of wound repair in sipunculus nudus was studied. furthermore, it was also examined if cells reactive to anti-il-4, -il-10 and -egf abs were present and involved in the wound healing of the marine worm. materials and methods twenty adult specimens of sipunculus nudus were incised with a scalpel in the frontal part of the body, producing a wound in a site opposite to the anus and 10 mm from the introvert. the incision, 5 mm in length, was performed longitudinally and it involved the integument and the circular muscle structure below. the animals were sacrificed and fixed in bouin mixture at different times after the incision (3, 15, 18, 22, 24, 71 and 96 h) and unwounded specimens were used as controls. fragments including the wound and 2 mm of surrounding healthy tissue were included in paraffin and 7 µm thick sections were stained with gomori triple staining (the connective tissue resulted green and the type i granulocytes red) and alcian blue-pas (type ii granulocytes stained green and pink) (ganter and jòlles, 1969; mazzi, 1977). the immunocytochemical reactions were performed by incubating sections for 1 h at room temperature (rt) in mouse primary monoclonal abs (euroclon, celbio, italy), raised against il-4, il-10 and egf, diluted 1:100 in phosphate-buffered saline (pbs) (1.37 m nacl, 0.03 m kcl, 0.015 m kh2po4 and 0.065 m na2hpo4), rapidly washed in pbs, incubated for 30 min at rt in biotinylated goat anti-mouse immunoglobulins (kit dako cytomation, denmark), washed in pbs, incubated in streptavidin peroxidase conjugate for 30 min at rt. after washing, sections were stained for 15 min in the chromogen aminoethylcarbamate. sections incubated with normal non-immune rabbit serum were used as controls. results in physiological conditions the body walls of s. nudus consist of a cubic epithelium, secreting an outer cuticle, a derma, a layer of circular muscle, a layer of longitudinal muscle and the peritoneum. the derma contains fine fibres, connective cells and coelomatic, longitudinal canals, which communicate with each other and with the general coelom (hyman, 1959). as far as the wounded specimens are concerned, 3 h after the incision, the wound did not show any signs of healing, the layers of muscle and connective tissue were still damaged and the cuticle was missing. on the outer part of the wound, coelomocytes were present, most of which were acidophilic type i granulocytes (fig. 1). furthermore, type ii granulocytes and a few haemerythrocytes were observed. three h after the incision, an intense immunoreaction with anti-egf ab was observed in type i granulocytes, including those which were present in the coelomatic cavity (fig. 2). type ii granulocytes and haemerythrocytes were negative. no changes in the response were observed up to 15 h after the incision, but for an increase in exocytised acidophilic material. eighteen h after incision, new muscle fibres were not observed but fine connective fibres were evident. both partially degranulated type i granulocytes and type ii granulocytes were present, in addition to transparent spherical cells on the outer part and the sides of the wound. twenty-two h after incision, the wound had been externally closed up by fine collagen fibres and internally by numerous coelomocytes (haemerythrocytes, type i granulocytes and various type ii granulocytes). at this time, egf-like material was found in all type i granulocytes, while other coelomocytes were negative (fig. 3). twenty-four h after incision, the wound had closed: on the internal part of the wound, acidophilic type i granulocytes were present and the circular, muscle tissue was interrupted towards the central part of the wound. in its place there was newly synthesized connective tissue with numerous type i and type ii granulocytes nearby. type i granulocytes were highly reactive to the anti-il-4 ab, while type ii granulocytes, were less numerous and negative (figs 4, 5). in incisions of the entire animal wall, both the derma and muscle tissue were interrupted and the circular muscle fibres displayed rounded extremities, which were covered by connective tissue (fig. 6). the wound had been closed by various thin, connective lamina distributed over 2-3 layers at the extremities. each lamina contained type ii granulocytes and a few transparent cells (fig. 7). some type ii granulocytes start to loose their basophilic core, while others devoid of the basophilic granules, were amoeboid in shape with a flattened nucleus and were involved in collagen fibre production (fig. 8). twenty-four h after the incision, large spaces containing degranulating type i granulocytes were highlighted near the tissue on the internal part of the wound, and these degranulated cells flattened and formed a sort of barrier that trapped haemerythrocytes and type ii granulocytes (fig. 9). type i granulocytes located towards the coelomatic cavity were almost degranulated with a weak reaction to anti-il-10 ab. type ii granulocytes did not react with this ab, while degranulated material resulted highly immunopositive (fig. 10). ninety-six h after the incision, the walls of s. nudus had been reconstituted but the connective tissue was not always well compacted and the muscle fibres did not show their typical circular arrangement (fig. 11). in the lateral parts of the wound, the muscle fibres were interrupted and a great number of “spongy” type ii granulocytes, 125 fig. 1 histological section 3 h after incision, stained with gomori triple staining. note, on the outer part of the wound (op), coelomocytes (←) with acidophilic material and degranulated acidophilic material (*). the connective tissue and muscle tissue (**) are still damaged and the cuticle (c) is also missing. coelomatic cavity (cc). fig. 2 immunocytochemical reaction with anti-egf ab 3h after incision. positive type i granulocytes (g1); negative type ii granulocytes (g2) and haemerithrocytes (h) in the coelomatic cavity. fig. 3 immunocytochemical reaction with anti-egf ab 22 h after incision. positive type i granulocytes (g1) and degranulated material (dm); negative haemerithrocytes (h). outer part of the wound (op), cuticle (c). 126 fig. 4 immunocytochemical reaction with anti-il-4 ab 24 h after incision. positive type i granulocytes (g1) are present in the connetive tissue (ct). coelomatic cavity (cc); longitudinal muscle (lm), outer part of the wound (op). fig. 5 detail of fig. 4. type i granulocytes (g1) positive and type ii granulocytes (g2) negative to anti-il-4 ab. collagen fibres (cf). fig. 6 histological section 24 h after wound, stained with gomori triple staining. the incision concerned the entire thickness of the animal wall. the wound is closed by connective lamina (cl). longitudinal canal (lc); coelomocytes and acidophilic material (c); circular muscle fibres (cm); coelomatic cavity (cc); longitudinal muscle (lm). 127 fig. 7 detail of fig. 6. note longitudinal canal (lc); degranulated acidophilic material (*); coelomocytes (c); connective lamina distributed over 2-3 layers (cl); circular muscle fibres with rounded extremities (cm); coelomatic cavity (cc); longitudinal muscle (lm); outer part of the wound (op). fig. 8 detail of fig. 6. external surface of the wound. note type i granulocyte (g1); connective tissue (ct) where type ii granulocytes (g2a) displayed their basophilic core and type ii granulocytes shaped like a triangle (g2) are producing thin bundles of collagen fibrils (cf). fig. 9 histological section stained with gomori triple staining 24 h after a deep incision. note a barrier made up of flattened type i granulocytes (g1), which have trapped haemerythrocytes (h), type ii granulocytes (g2) and degranulated material (dm). 128 fig. 10 immunocytochemical reaction of section with anti-il-10 ab 24h after incision. partially degranulated type i granulocytes (g1) were moderately reactive whilst the degranulated material (dm) was highly reactive. negative type ii granulocyte (g2). note type i flattened granulocytes (fg1); connective tissue (ct); outer part of the wound (op); coelomatic cavity (cc). fig. 11 histological section 96 h after incision stained with gomori triple staining. the wound is partially healed. note cuticle (c); granulocytes; poorly compacted collagen fibrils (cf); circular muscle fibres (cmf) being formed; coelomic cavity (cc); longitudinal muscle (lm). fig. 12 detail of fig. 11 in the lateral parts of wound. note the interrupted muscle fibres (mf) and a great number of spongy type ii granulocytes (g2), separated by connective fibres (cf). 129 separated by connective material, formed a base onto which the muscle fibres were formed (fig. 12). elongated type i granulocytes, with varying amounts of acidophilic granules, were found between the forming muscle fibres. discussion the histological study performed 3-96 h after the incision in s. nudus revealed that type i and type ii granulocytes from the coelomatic cavity are responsible of the wound repair. type i granulocytes are the first cells to be activated and they degranulated thereby causing the transfer of their material to the surrounding area and, in particular, on type ii granulocytes. the contribution of the coelomocytes was not always the same. in most cases, the coelomocytes migrated from the coelomic cavity to the lesion zone, due to the probable presence of various chemotactic substances produced in situ (abercrombie, 1972; postletwaite, 1976). type i granulocytes contain lysosomal enzymes (matozzo et al., 2001; d’ancona et al., 2004) that could act in the digestion of cell debris and damaged tissue. the urna cell complexes present in the coelomatic liquid of s. nudus (d’ancona et al., 2004) and endowed with phagocytic activity (bang and bang, 1962) did not participate in wound repair process. unlike the hemocytes found in the wound repair of l. maximus (franchini and ottaviani, 2000), type i granulocytes do not take part directly by means of phagocytosis but they secrete enzymes into the extracellular environment. type i granulocytes also have an important hemostatic function. indeed, they move from the coelomatic cavity towards the wound in great amounts in order to create an aggregate that can prevent the passage of coelomatic material towards the outer and of pathogens towards the inner side of the body. these cells flattened after degranulation, stuck along the lower margins of the wound and form a lamina which entrap other cells, especially haemerythrocytes. many of these, in turn, flocked around the wound with the aim of supplying oxygen, which is necessary for metabolic activity (steins et al., 2001). rollefsen (1965) hypothesized that morula cells, present in the hydrovascular system and dermal connective tissue of s. tremulus, can be involved in the production of the intercellular substance of connective tissue, as they are both metachromatic and pas positive. morula cells may form fibres (endean, 1966) which are responsible for connective tissue growth and repair (smith, 1981). in other echinoderms polysaccharides and proteins are present in the granular inclusions of spherula cells (endean, 1958; hetzel, 1963; johnson, 1969; d’ancona and canicattì, 1990). type ii granulocytes in s. nudus showed the same cytological characteristics of the morula cells of s. tremulus and h. poli type i spherula cells (d’ancona and canicattì, 1990). they also have the same histochemical affinity of connective tissue and it can, therefore, be hypothesized that type ii granulocytes may be the main cell producers of connective material. the granulation tissue, typical of wound healing in vertebrates, was also found in l. maximus (franchini and ottaviani, 2000). in this mollusc the main cell type involved in the different phases of repair process is the hemocyte. indeed, this cell was immunoreactive to cytokines, was able to phagocytize cell debris and damaged tissue and showed fibroblastlike activity, as found by sminia et al. (1973) in l. stagnalis. in s. nudus numerous type i granulocytes are found everywhere and they continuously exocytise acidophilic material between other cells and muscle fibres. moreover these cells contain hydrolytic enzymes, able to remove cell debris and damaged tissue, and were also immunoreactive to egf-, il4 and il10 abs. in marine worm it can be supposed, as in vertebrates, that in the first hours after incision, an inflammatory response occurs, involving cellular proliferation of granulocytes (d’ancona et al., 2004). in mammals and some invertebrates, growth factors such as pdgf and tgf-β, play an important role in healing wounds as they are chemotactic and proliferative factors (seppa et al., 1982; deuel et al., 1982; senior et al., 1983; postlethwaite et al., 1987; wahl et al., 1987; clark et al., 1997; ottaviani et al., 1997; franchini and ottaviani, 2000). the presence of egf-, il-4and il-10-like material in s. nudus does not exclude the possibility that other cytokines or other factors, found in other invertebrates, could also be present in type i granulocytes, and that they could be directly involved in the process of wound healing. however, it is so far impossible to attribute a specific function to egf-, il-4and il-10-like material in the process of the healing of wounds in s. nudus. references abercrombie m. behavior of cells toward one another. in: montagna w, billingham re (eds), advances in biology of skin. v. wound healing, academic press inc, london, pp 95-112, 1972. anderson jm. studies on visceral regeneration in sea-stars. i. regeneration of pyloric caeca in henricia leviscula (stimpson). biol. bull. 122: 321-342, 1962. anderson jm. studies on visceral regeneration in sea-stars. ii. regeneration of pyloric caeca in asteriidae, with notes on the source of cells in regenerating organs. biol. bull. 128: 1-23, 1965. bang fb, bang bg. studies on sipunculid blood: immunological properties of coelomic fluid and morphology of “urn cell“. cahiers biol. mar. 3: 363-374, 1962. canicattì c, d’ancona g. cellul ar aspects of holothuria polii immune response. j. invert. pathol. 53: 152-158, 1989. clark ra, mccoy ga, folkvord jm, mcpherson jm. tgf-â 1 stimulates cultured human fibroblasts to proliferate and produce tissue-like fibroplasia: a fibronectin matrixdependent event. j. cell physiol. 170: 69-80, 1997. cowden rr. cytological and histochemical observations on connective tissue cells and cutaneous wound healing in the sea cucumber stichopus badionotus. j. invert. path. 10: 151-159, 1968. d’ancona g, canicattì c. the coelomocytes of holothuria polii (echinodermata). ii. cytochemical staining properties. bas. appl. histochem. 34: 209-218, 1990 d’ancona g, farina e, manione r. sipunculus nudus: particulate components of the coelomic fluid and its relationship with brown bodies. ital. j. zool. 71: 191-199, 2004. deuel tf, senior rm, huang js, griffin gl. chemotaxis of monocytes and neutrophils to platelet-derived growth factor. j. 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1987. whitney jd. overview: acute and chronic wounds. nurs. clin. north am. 40:191-205, 2005. 131 the fast evolutionary dynamics of ascidian mitochondrial genome: an exception to the general deuterostome evolutionary trend isj 6: s21-s28, 2009 issn 1824-307x review evolutionary mitogenomics of chordata: the strange case of ascidians and vertebrates c gissi, f griggio, f iannelli dipartimento di scienze biomolecolari e biotecnologie, università di milano, milano, italy accepted march 13, 2009 abstract the availability of almost one thousand complete mitochondrial genome (mtdna) sequences of chordates provides an almost unique opportunity to analyse the evolution of this genome in the phylum chordata, and to identify possible divergent evolutionary trends followed by the three chordate subphyla: vertebrata, cephalochordata and tunicata. here, we review some genome-level features of mtdna, such as genetic code, gene content, genome architecture and gene strand asymmetry, mostly focusing on differences existing between tunicates and remaining chordates. indeed, tunicate mtdnas show a surprisingly high variability in several genome-level features, even though the current tunicate taxon sampling is absolutely insufficient and is focused mainly on the class ascidiacea. on the contrary, a stabilization of the mtdna structural and evolutionary features is observed in both cephalochordates and vertebrates, where genome-level features are almost invariant. thus, different evolutionary dynamics, probably related to divergent functional constraints, have modelled the overall mtdna structure and organization of the three chordate subphyla. key words: mitochondrial genome; evolution; chordates; tunicates; ascidians introduction the mitochondrial genome (mtdna) is used both as a model system for studying genome evolution, and as a molecular marker to reconstruct animal phylogeny, both at high and low taxonomic levels (saccone et al., 1999; saccone et al., 2002). this dual interest in mtdna constitutes an advantage in phylogenetic studies, since the understanding of the evolutionary peculiarities of a given character greatly facilitates the evaluation of its reliability as a phylogenetic marker. for phylogenetic purposes, the sequences of one or more mitochondrial genes, or of the fast-evolving control region of vertebrates, are commonly analysed with traditional molecular evolutionary methods. besides the sequence itself, genome-level features have been also used for phylogenetic reconstructions: gene order, gene content, and changes in the genetic code are all examples of mt genome-level features, used to clarify controversial phylogenetic relationships, especially at high taxonomic levels (boore, 2006). ___________________________________________________________________________ corresponding author: carmela gissi dipartimento di scienze biomolecolari e biotecnologie università di milano via celoria 26, 20133 milano, italy e-mail: carmela.gissi@unimi.it to date, the entire mtdna sequence has been determined for 1206 metazoan species (gissi et al., 2008) but the taxon sampling is highly biased towards vertebrates and arthropods (genembl, september 2008). the taxon sampling of chordata is also quite heterogeneous, as it is almost exhaustive for vertebrates and cephalochordates (lancelets) but absolutely inadequate for tunicates, where sampling is almost exclusively derived from representatives of the class ascidiacea (fig. 1). in spite of the scarce data, it is evident that ascidian mtdnas possess several unusual features compared to other chordates, such as a fast nucleotide substitution rate (yokobori et al., 1999, 2005), gene orders that are extremely variable both within the class and compared to other metazoans (gissi et al., 2004; yokobori et al., 2005; iannelli et al., 2007a), and a variable number of trna genes (gissi and pesole, 2003; gissi et al., 2004; iannelli et al., 2007a). thus, several clues point to the existence of an almost opposite mtdna evolutionary trend between tunicate and remaining chordates, resulting in a strong variability of mt genome features in ascidians/tunicates versus a relative stability in vertebrates and cephalochordates (gissi et al., 2008). the “strange case” of ascidian mtdna is even more intriguing when we consider the controversies on the phylogenetic position of s21 mailto:carmela.gissi@unimi.it tunicates within chordates and the debated phylogenetic relationships among the three tunicate classes. traditional morphological data indicate that tunicates should constitute the basal chordate subphylum clustering cephalochordates and vertebrates in the euchordata group (rowe, 2004). however, recent molecular phylogenetic analyses based on a large number of nuclear genes strongly support a sister relationship between tunicates and vertebrates (olfactores clade) and a basal position of cephalochordates within chordates (bourlat et al., 2006; delsuc et al., 2006). in reality, the position of tunicates in molecular phylogenetic analyses is quite unstable and varies according to which genes or molecules (nuclear or mitochondrial dna) are used (zrzavy et al., 1998; cameron et al., 2000; winchell et al., 2002; oda et al., 2004; bourlat et al., 2006; delsuc et al., 2006). with regard to phylogenetic relationships within tunicata, the analyses of morphological and/or molecular characters have given rise to distinct phylogenetic hypotheses most of which suggest that larvacea is the sister group to the rest of tunicates, and that thaliacea is the sister group to phlebobranch ascidians, thus rendering ascidiacea a paraphyletic group (see references in stach and turbeville, 2002; zeng and swalla, 2005). in this review we will provide an overview of some structural and evolutionary peculiarities of the chordate mtdna, investigated through the analysis of a carefully revised dataset of 865 complete mtdnas (obtained from gissi et al., 2008). in particular, we will compare four genome-level features: genetic code, gene content, genome architecture and gene strand asymmetry, among the three subphyla of vertebrata, cephalochordata and tunicata, highlighting both differences and similarities. our synthesis should stimulate further comprehensive analyses of these and other mitochondrial genomic features within a phylogenetic framework. genetic code the three chordate subphyla use each their own modified mitochondrial genetic code (table 1), differing only in the meaning of agr codons. the cephalochordate genetic code is quite controversial, as the agr codons were initially assigned to glycine (spruyt et al., 1998) and then to serine (boore, 1999) based on the analysis of a single lancelet mtdna. further studies, carried out on a large number of mtdnas, and comparing the conservation within deuterostomes of amino acid sites with agr codons in lancelets, have confirmed the hypothesis that agr encodes serine (nohara et al., 2005a). it should be noted that the codon usage of lancelets is characterized by the absence or extremely low number of agg codons, which occur only 4 times in a total of 14 available complete mtdnas (see notes of table 1), thus the meaning of agg codon remains uncertain. these data leave the evolutionary history of the chordate mt genetic code an open question, particularly in the light of the recently reported basal phylogenetic position of lancelets compared to vertebrates and tunicates (bourlat et al., 2006; delsuc et al., 2006). in this fig. 1 number of available complete mitochondrial genomes of deuterostomes, corresponding to deuterostome mtdna dataset analysed in this review. the three taxonomic classes of tunicates are shown as a polytomy, due to phylogenetic controversies. deuterostome phylogeny is in accordance with bourlat et al. (2006). respect, it is interesting that the genetic codes of both vertebrates and tunicates are unique among metazoans, while the genetic code of lancelets is shared by many protostome and deuterostome phyla (i.e., annelida, arthropoda, mollusca, nematoda and xenoturbellida). thus, the data on genetic code seem to support the basal position of cephalochordates compared to remaining chordates. gene content within vertebrates, the most frequent mitochondrial gene content consists of 37 genes encoding for 13 protein subunits of the oxidative phosphorylation complexes, two ribosomal rnas, and 22 trnas necessary for the translational machinery located within mitochondria. among the 865 analysed chordates, there are no cases of rrna gene loss/acquisition, while only four vertebrate species lose/acquire one or two protein-coding genes (table 2). this is in accordance with observations carried out on metazoan mtdnas, where the number of proteincoding and rrna genes changes rarely (gissi et al., 2008). on the contrary, the trna gene content is quite variable between different metazoan phyla and, in general, it depends on the genetic code employed and the taxonomic group (gissi et al., 2008). according to the relaxed wobble rules in codon-anticodon recognition and to the genetic code observed (table 1), the mt translation machinery of chordata should require a minimum set of 22 trnas in vertebrates and lancelets, and 23 trnas in tunicates. in fact two trnas are needed to decode each of the six-fold degenerate amino acids (leucine and serine in all chordates, plus glycine in tunicates), while only one trna is needed to decode each remaining amino acid. this trna number, predicted on basis of the genetic code, holds for s22 table 1 mitochondrial genetic code of deuterostomes compared to the universal code. “codtab” indicates the number of the table containing the entire genetic code, available at the web site “http://www.ncbi.nlm.nih.gov/taxonomy/utils/wprintgc.cgi”. the genetic code of hemichordates is reported as codtab 9, although exceptions in the meaning of the aaa codon indicate that this is a new mt genetic code uga aua aga agg aaa codtab universal stop ile arg arg lys 1 mitochondrial chordata vertebrata trp met stop stop = 2 tunicata trp met gly gly = 13 cephalocordata a trp met ser ser/ absent = 5 hemichordata b trp = ser ser = new balanoglossus trp = ser absent absent 9 saccoglossus trp = ser ser = new echinodermata trp = ser ser asn 9 xenoturbellida trp met ser ser = 5 a: agg codons have been found only in epigonichthys maldivensis (2 occurrences) and in branchiostoma belcheri (1 occurrence in both the ab078191 and ab083383 mtdna sequences), while they are absent in the mtdna of remaining branchiostoma species and of asymmetron genus (7 and 4 mtdna sequences, respectively). b: the ac number of the two complete mtdna sequences of hemichordates are: af051097 (balanoglossus carnosus) and ay336131 (saccoglossus kowalevskii). vertebrates and lancelets but not for tunicates, whose most frequent (i.e. “standard”) trna gene number is 24 and includes an additional trna-met. in fact, all tunicates encode for the common trnamet with cat anticodon and for a trna-met with the unusual tat anticodon, probably acting as initiator and elongator trna-met, respectively (yokobori et al., 1999, 2003, 2005; gissi et al., 2004; iannelli et al., 2007a, 2007b). this additional trna-met(tat) gene is shared only with some mollusc bivalves, thus this feature has arisen independently in two distant animal lineages (gissi et al., 2008). variations from the standard trna gene content have been found less frequently in vertebrates and lancelets than in tunicates. as shown in table 2, only 1.6 % of the 849 vertebrate mtdnas analysed show loss/acquisition of trna genes. moreover, the loss of trna genes is an uncommon event compared to trna acquisition, as it has been found only in three of 849 vertebrates (table 2), suggesting that trna loss can not be easily compensated by an analogous function encoded by the nuclear genome. this may be due to differences in trna structure or difficulties in mitochondrial import of trnas. in addition, differences in trna content mainly concern amphibian species, which show acquisition of additional trna genes as results of duplication of mt regions and/or extensive gene order rearrangements (liu et al., 2005; mueller and boore, 2005; zhang et al., 2005; kurabayashi et al., 2006; san mauro et al., 2006). among lancelets, only branchiostoma floridae exhibits an additional trna gene (spruyt et al., 1998), although the modified cloverleaf structure and the presence of a non-canonical tct anticodon suggest that this is a trna-like secondary structure rather than a functional trna. finally, in tunicates the situation is extremely variable (see table 2): about half of the seven available tunicate mtdnas show differences from the expected trna gene number, moreover loss and acquisition of trna genes seem to be equally tolerated. although the tunicate taxon sampling is still insufficient to draw definitive conclusions, the current data suggest that the tunicate trna gene number could vary in a species-specific manner. genome architecture to compare the overall structure of mt genomes, we have introduced a new genomic feature, named genome “architecture” (ar). the genome ar takes into account both gene content and gene order, and it is defined as the order of the entire set of functional mt-encoded genes, including duplicated genes. therefore, two mtdnas with different architecture may show differences in gene content and/or gene order. the three chordate subphyla show different level of variability in genome ar, indeed genome ar is quite stable in vertebrates, moderately conserved in cephalochordates and highly variable in tunicates. in vertebrates, 80 % of the analysed species show the same genome ar as the human mtdna (hereafter named std, “standard”), while remaining species exhibit one of the 42 additional genome ars (table 3). in particular, the std architecture is shared by all eutherian and prototherian mammals, as well as by almost all fishes (except for few neopterygian species), and it is lost in all species belonging to metatheria, sauropsida (aves plus crocodylidae), and hyperoartia (table 3). in addition, non-standard ars are highly similar to the standard ar, as in most cases they can be converted into the standard ar by the translocation of few genes, most of which are located near to the mtdna replication origins (boore, 1999). s23 table 2 chordate taxa with a non-standard mitochondrial gene content (different from the expected number of 37 genes in vertebrates and lancelets; 39 genes in tunicates). the ac number of the corresponding mtdna sequences is also reported. the minus symbol indicates genes encoded by the minor strand. gsa: gene strand asymmetry, calculated as described in table 4 taxon (n° available mtdnas) ac number proteins trna gsa additional lost additional lost vertebrata (849) amphibia (82) polypedates megacephalus nc_006408 atp8, nad5 0.49 rhyacotriton variegatus nc_006331 trnt 0.53 plethodon elongatus nc_006335 trnt 0.53 mantella madagascariensis nc_007888 trnm 0.53 gegeneophis ramaswamii nc_006301 trnf 0.50 fejervarya limnocharis nc_005055 trnm 0.53 aneides hardii nc_006338 trnf, -trne, trnl(uur), -trnp 0.46 aves (73) diomedea melanophris a nc_007172 -nad6 trnt, -trnp, -trne 0.41 testudines (19) malacochersus tornieri b nc_007700 trnf 0.58 lepidosauria (44) cordylus warreni c nc_005962 trnt, -trnp 0.49 sphenodon punctatus nc_004815 nad5 trnk trnh, trnt 0.49 neopterygii (374) chionodraco rastrospinosus dq526431 nad6 trne 0.60 bathygadus antrodes nc_008222 trnl(uur) 0.53 cephalochordata (9) branchiostoma floridae y16474 trn(tct) d 0.51 tunicata (7) halocynthia roretzi ab024528 trnf 1 phallusia fumigata am292602 trni, trnx trnd 1 phallusia mammillata am292320 trnd 1 a: duplication of the region: +trnt-trnp-nad6-trne+cr, where cr corresponds to the control region (gibb et al., 2007) b: duplication of the region: +trnf+cr (parham et al., 2006), where cr corresponds to the control region c: tandem duplication of the region: +trnt-trnp (kumazawa, 2004) d: non-conventional trna with tct anticodon found in the b. floridae entry y16474 [first published as branchiostoma lanceolatum (spruyt et al., 1998), this sequence actually belongs to b. floridae (spruyt et al., 1998; nohara et al., 2005b)] in cephalochordates, the genome ar is slightly less conserved, as there are three different genome ars in 9 distinct species, and the most represented ar has been found in 55 % of the sampled species. finally, tunicates show an extreme ar variability, as each species has its own specific genome ar, with no ar shared by two or more mtdnas. this peculiarity appears even more surprising when considering that the seven sampled tunicates derive from a narrow taxonomic range and also include several congeneric ascidian species. additional mtdna sequences of ascidians produced in our laboratory (unpublished data) confirm this extreme ar variability. we have analysed the complete mtdna of ten additional ascidians, sampled both as closely (congeneric) and distantly related species, and no genome ar is present in more than one species. moreover, gene order variability at intra-genus level has been observed in all ascidian orders (aplousobranchiata, stolidobranchiata and phlebobranchiata). a comparative analysis among all chordates shows that no ar is shared by different subphyla when all genes are taken into account. on the contrary, the exclusion of trna genes makes the genome architectures (ar-trna) much more similar and sometimes identical between different subphyla. for example, the ar-trna of lancelets is identical or very similar to the standard vertebrate ar-trna depending on the genus (identical in branchiostoma plus epigonichthys; very similar in asymmetron). similarly, in vertebrates the exclusion of trna genes drastically reduces the number of different non-standard architectures (from 42 ar to 12 ar-trna; table 3) and increases the number of taxonomic groups showing a std architecture. in particular, excluding the trna genes, the std architecture is acquired by metatheria, crocodylidae and hyperoartia species, as well as by many lepidosaurians and amphibians (compare ar and ar-trna in table 3). these observations indicate that the extant genome ars of vertebrates and cephalochordates arose by modest rearrangements of the same ancestral ar, with modifications mainly concerning trna genes. s24 table 3 distribution of the standard (std) and non-standard (other) mtdna architecture (ar) between vertebrate taxonomic groups. ar: genome architecture calculated for all mt genes. ar(-trna): genome architecture calculated excluding the trna genes. std: standard ar, identical to the human mtdna architecture. nother : number of species showing a non-standard mtdna architecture. other_fishes: fish group including polypteridae, chondrostei, chondrichthyes, coelacanthiformes and dipnoi taxon mtdna n° n° ar n° ar(-trnas) std other a nother std other vertebrata 849 1 42 173 1 12 eutheria 197 1 1 prototheria 3 1 1 metatheria 22 1 22 1 testudines 19 1 2 2 1 1 lepidosauria 44 1 10 26 1 2 b aves 73 2 73 2 b crocodylidae 8 1 8 1 amphibia 82 1 16 26 1 4 b neopterygii 374 1 13 14 1 6 b other_fishes 23 1 1 hyperoartia 2 1 2 1 hyperotreti 2 1 1 a: four non-standard ar are shared by vertebrates belonging to different groups: ar-1 is shared by two neopterygians and two amphibians; ar-2 is shared by all metatherians and one amphibian; ar-3 is shared by one neopterygian and all crocodiles; ar-4 is shared by 72 birds and one lepidosaurian. b: the same non-standard genome ar(-trna) is shared by the following species: 72 aves, 2 neopterygii, 2 lepidosauria and 2 amphibia on the contrary, the genome architecture of tunicates does not show any similarity to that of remaining chordates. even the exclusion of trna genes, neither significantly reduces the number of different tunicate genome architectures (6 different ar-trna against 7 different ar when considering all genes) nor makes the tunicate mtdnas more similar in genome architecture to that of remaining chordates. these data remain valid also considering our enlarged and unpublished ascidian dataset. overall, these observations suggest a fast rate of mtdna rearrangements in the tunicate subphylum, with changes equally involving trna and other mitochondrial genes, while the strong similarity in ar-trna between vertebrates, cephalochordates, and remaining deuterostomes (excluding tunicates) (data not shown) supports the conservation of mtdna architecture during deuterostome diversification. gene strand distribution a peculiarity of metazoan mtdna is the asymmetric distribution of genes between the two strands, indeed a major and a minor strand are commonly recognized based on the number of encoded genes. this asymmetric gene distribution can be quantified using the gsa formula (gene strand asymmetry) (gissi et al., 2008), calculated as the absolute value of the difference in gene number between the two strands, divided by the total gene number. gsa values range from 0 to 1, with values close to zero indicating an almost equal number of genes encoded by the two strands, and values higher than 0.5 corresponding to the presence of at least 75 % of the genes on the major strand. the overall data on gene strand distribution in metazoans show a prevalence of high gsa values, while a symmetric gene distribution is very rare and it has been observed only in 17 phylogenetically distant species over 1206 complete metazoan mtdnas (gissi et al., 2008). among chordates, the gsa value is high and almost invariant within each subphylum. in vertebrates, the gsa is equal to 0.51 in almost all taxa, and the minor strand encodes only for one protein-coding gene, nad6, and 8 trna genes (table 4). the few exceptions (15 over 849 species) to this structure are related to inversion of a single trna gene (only two cases, table 4) or to changes in gene content (table 2). in the last cases, all additional duplicated genes are present on the same strand as the ancestral gene that gave rise to the duplication (table 2). in cephalochordates, the gsa ranges from 0.41 to 0.51, this modest variability being due to the different number of trnas encoded by the minor strand (from 8 to 10 trna genes, plus only one protein-coding gene, nad6 or nad5 depending on the species) (table 4). thus, vertebrates and lancelets exhibit a very similar gene strand asymmetry, as consequence of the similar genome architecture (see previous section), but they strongly differ in the tendency to switch the sense strand of a gene. indeed, lancelets show frequent gene inversions, both of large mt regions or single genes, while vertebrates show only two cases of gene inversion over a total of 849 analysed species (table 4). in particular, a strand switch of a 2.5 kb region including four genes (trnl(cun), nad5, trng, and nad6) has been found in the asymmetron genus compared to other lancelets and to the standard genome ar of vertebrates (nohara et al., 2005b; kon et al., 2007). s25 table 4 gene strand asymmetry (gsa) of chordate mtdnas, including data on number and name of genes encoded by the minor strand. for vertebrates, only species with the common mt gene content (37 genes) are reported in this table, and the minor strand genes are indicated as differences from the “standard” vertebrate situation. numbers in brackets refer to the number of species showing a given gsa taxon (n° species) gsa a minor strand genes n° trna cds vertebrata standard (833) 0.51 9 trnq, trna, trnn, trnc, trny, trns(ucn), trne, trnp nad6 neopterygii carapus bermudensis 0.46 10 standard plus trnm nad6 lepidosauria calotes versicolor 0.57 8 standard except trnp nad6 cephalochordata asymmetron inferum 0.46 10 trnn, trna, trnc, trny, trns(ucn), trng, trnl(cun), trne, trnp nad5 cephalochordata asymmetron lucayanum (3) 0.41 11 trna, trnc, trny, trnq, trnn, trns(ucn), trng, trnl(cun), trne, trnp nad5 cephalochordata branchiostoma (4) 0.51 9 trnq, trnn, trna, trnc, trny, trns(ucn), trne, trnp nad6 cephalochordata epigonichthys maldivensis 0.51 9 trnq, trnn, trna, trnc, trny, trns(ucn), trne, trnp nad6 tunicata (7) 1 0 a: the gsa is calculated as the absolute value of the difference in gene number between the two strands, divided by the total gene number. in addition, the inversion plus transposition of the trnq gene has been observed between asymmetron inferum and asymmetron lucayanus (kon et al., 2007). in vertebrates, the two observed cases of gene inversion concern a single trna gene (table 4) adjacent or translocated close to the control region (trnp and trnm, respectively), thus gene inversions have taken place near to the “hot spot” region of vertebrate genome rearrangements (boore, 1999). moreover, these rare events have occurred in two phylogenetically-distant species, a lizard and a fish: the inversion of trnp, found in the lizard calotes versicolor and shared by all south asian draconine agamids, appears incompatible with a trna remoulding process, and it has been explained by a homologous dna recombination helped by accidentally formed inverted repeats (amer and kumazawa, 2007); no hypothesis has yet been published to explain the translocation/inversion of trnm in the bone fish carapus bermudensis (see genembl accession number ap004404; (miya et al., 2003)). in general, gene inversion can not be explained by the tandem duplication/random gene loss model, the mechanism commonly invoked for mtdna rearrangements in metazoans, but can be easily explained by homologous or illegitimate recombination. thus, it has been proposed that the sporadic events of gene inversion observed in vertebrates are the result of rare dna recombination processes, while dna recombination is significantly high in the mtdna of cephalochordates, thus promoting frequent gene inversions in this subphylum (amer and kumazawa, 2007; kon et al., 2007). although the existence of recombination has been traditionally excluded in the mitochondria of metazoans, there is growing evidence for the occurrence of dna recombination in several taxonomic groups (lunt and hyman, 1997; hoarau et al., 2002; shao et al., 2005; tsaousis et al., 2005; guo et al., 2006) and for the presence of homologous enzymatic recombination activities in vertebrates (thyagarajan et al., 1996; kraytsberg et al., 2004). differently from other chordates, tunicates have a gsa equal to one, i.e. they show the most extreme gene strand asymmetry. thus, all genes of tunicates are encoded on the same strand, rendering the “minor” strand devoid of genes. this feature is shared by all tunicate species, including our unpublished dataset of ascidian mtdnas, making the gene strand asymmetry the only “invariant” mitochondrial feature currently found in tunicates. therefore, we can suppose that strong functional constraints have forced the tunicate mtdna to maintain all genes on the same strand, in spite of the numerous changes in genome ar and rearrangements of gene order: the overall transcription mechanism or processes regulating the level of transcription could be the most obvious functional constraints for the maintenance of all genes on the same strand. conclusions the data here summarized underline the existence of a significant variability between the three subphyla of chordata in several genome-level features. the genetic code exhibits a different specificity in each of the three subphyla, with cephalochordates retaining a genetic code more similar to that of remaining deuterostomes (table 1). similarities in genome architecture, i.e. in structural features such as gene content, gene order and gene strand distribution, cluster the vertebrates and cephalochordate subphyla to the exclusion of tunicates. in fact, tunicates are characterized by the s26 most extreme gene strand asymmetry, and a strong variability in genome ar, which is different between species, even when we analyse our unpublished dataset of ten additional ascidian species. on the contrary, cephalochordates are characterized by moderate rearrangements of genome ar, frequent gene inversions, and a gene order and gene strand asymmetry that can be easily traced back to that of vertebrates. finally, the genome ar of vertebrates is almost stable but not so invariant as previously reported: while gene inversions are very rare in all vertebrates, changes in gene content and gene order are quite frequent in amphibia and lepidosauria (see table 2 and table 3), and sporadic cases of genome rearrangements have been also observed in a few bony fishes, birds and turtles. thus, the initial dogma of a frozen genome ar in vertebrates has been broken by the identification of several deviations from the “standard” ar in some vertebrate groups (table 3). many of these nonstandard genome ars involve gene duplications or changes in the order of genes located close to the control region or surrounding the l-strand replication origin, suggesting that errors in the mtdna replication have given rise to these rearrangements. moreover, the trna genes are the most common gene category implicated in genome ar changes. although these differences in chordate mtdna evolutionary dynamics are already manifest, the extent of the surprising features of tunicate mtdna needs to be further confirmed through the analysis of a large number of ascidian mtdnas (our research field) and the sampling of the remaining tunicate classes of thaliacea and larvacea, for which mtdna sequences are almost absent. abbreviations cox1, cox2, cox3: cytochrome oxidase subunit i, ii, and iii protein genes; cob: cytochrome b gene; atp6, atp8: atp synthase subunit 6 and 8 genes; nad1, nad2, nad3, nad4, nad4l, nad5, nad6: nadh dehydrogenase subunit 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cladistics 14: 249-285, 1998. s28 genetic code gene content amphibia (82) nc_007172 -nad6 nc_007700 nc_005962 cephalochordata (9) tunicata (7) gene strand distribution conclusions isj 5: 103-xxx, 2008 isj 5: 103-109, 2008 issn 1824-307x visions and perspectives specificity, learning and memory in the innate immune response m brehélin1, p roch2 1 ecologie microbienne des insectes et interactions hôte-pathogène, université de montpellier 2, inra, france 2 ecosystèmes lagunaires, université de montpellier 2, cnrs, ifremer, france accepted july 29, 2008 summary immunity in invertebrates was for long analyzed in terms of the overall response; this resulted in misunderstandings concerning specificity and memory. recent reports of maternal transmission of immunity, and the discovery of the high diversity of receptors-effectors, have required the status of innate immunity to be reconsidered. there are few examples of obvious specificity towards some pathogens, but this cannot be generalized to all invertebrate species. the existence of memory is even more controversial. here, we suggest looking for immune memory by quantifying key molecular effectors (i) within single individuals following first and second exposures to a pathogen and (ii) in primed mother and her offspring. key words: innate immunity; invertebrates; vertebrates; drosophila introduction although invertebrates are able to develop immune reactions, it has long been assumed that their immune systems are non adaptive (not anticipatory) and respond identically to multiple challenges. in other words, it was believed that they lack specificity and memory, and this conviction was strongly supported by the fact that, unquestionably, invertebrates do not possess antibodies, or t or b cells. however, this dogma that adaptive (anticipatory) immunity is absent from invertebrates is now cracking, not because the long search for antibodies has succeeded, but because at least some invertebrates possess functional equivalents of the acquired responses of vertebrates (see kvell et al., 2007 for a review). since the work of carton et al. (1992), diverse examples of strongly specific immune responses in invertebrates against potential parasites or pathogens have been reported (little et al., 2003; pham et al., 2007). in addition, the wide diversity of receptors and effectors, including fibrinogen-related proteins (frep) in snail (adema et al., 1997),toll-like ___________________________________________________________________________ corresponding author: philippe roch equipe pathogènes et environnement cnrs-um2-ifremer ecolag université de montpellier 2 cc 093, place eugène bataillon f-34095 montpellier cedex 5, france e-mail: philippe.roch@univ-montp2.fr receptors (tlr) in drosophila (tauszig et al., 2000) and in sea urchin (hibino et al., 2006), the gene family 185/333 in sea urchin (buckley and smith, 2007), antimicrobial peptide (amp) in shrimps (padhi et al., 2007) and in mussels (padhi and verghese, 2008; pallavicini et al., 2008), the latest presumably generated by gene duplication and positive darwinian selection, argues in favour of the existence of a sharp specificity in some of the invertebrate immune responses. in most studies on invertebrates, and in the present paper as well, what was called “specificity” referred to the overall result of the immune reaction, whereas in mammal immunity studies, “specificity” referred to the process of antigen recognition. concerning memory, various studies concluded that previous experience of a pathogen can provide an individual invertebrate, or its descendant, with enhanced immunity (cooper and roch, 1986; kurtz and franz, 2003; little et al., 2003). this process of enhanced immunity following previous encounter, can be divided in two steps. in the first step (referring to "learning"), the host has met the pathogen and has learnt something from this meeting: this is a behavioural or dynamic feature. in the second step (referring to "memory") the host remembered what he has learnt: this is a physiological or static feature. learning can happen without memory (the lesson was lost) but memory cannot exist without previous learning. in other words, it is evident that memory is inseparable from 103 mailto:philippe.roch@univ-montp2.fr host d. melanogaster susceptible strain: s resistant strain: r virulent strain: v no capsule no capsule avirulent strain: av no capsule capsule p a r a s it o ïd l . b ou la rd i fig. 1 the cellular immune reaction of d. melanogaster against the parasitoïd l. boulardi is called a capsule. it is a multilayer sheath built by hemocytes around the parasitoïd egg which is killed. depending on the combination between susceptible (s) or resistant (r) strains of d. melanogaster and virulent (v) or avirulent (av) strains of l. boulardi, the capsule is built or not. the result of the interaction is predictable [adapted from carton and nappi (2001)]. learning and that "learning-memory" is different from specificity. curiously, this obviousness has been forgotten in some recently published papers (kurtz and franz, 2003). although the specificity of some immune responses in invertebrates has been reported as being much stronger than that generally allocated to innate immunity, there is still no direct evidence for memory in invertebrates. to this end, we suggest quantifying key molecular effectors to discriminate between what is related to transfer of effectors (see below) and what is indeed memory. what was tested when looking for specificity? studies with populations of drosophila melanogaster parasitized by the parasitoïd leptopilina boulardi demonstrated that the immune response was strain specific: some strains of the host fly can kill (encapsulate) some but not all strains of this parasitoïd species (fig. 1) (carton and nappi, 2001). similarly, different daphnia clones have been reported to be differently protected against diverse strains of the same pathogenic bacteria (little et al., 2003); thus, different individuals within the same species seemed to respond differently, and this corresponds to a level of specificity not far from that observed in vertebrate adaptive immunity. specificity is generally understood to be the recognition of a foreign body by the host (kurtz, 2005). for instance in d. melanogaster, both alternative splicing of peptidoglycan recognition protein (pgrp) can play a role in the immune response (werner et al., 2003) and also as many as 18,000 isoforms of the receptor down syndrome cell adhesion molecule (dscam) can be generated (watson et al., 2005). consequently, the observed specificity of d. melanogaster immune responses may be related to the selection of specific isoforms of receptors (agaisse, 2007), as also suggested for molluscs (zhang et al., 2004), or to synergism between receptors (schulenburg et al., 2007). in the d. melanogaster-l. boulardi model described by carton and nappi (2001) (fig. 1), the different steps of the defence reaction can be dissected because this system predicted the outcome of the immune response of selected host strains to particular parasitoïd strains. the immune reaction triggered in d. melanogaster larvae against parasitoïd eggs is called encapsulation and involves four main steps (russo et al., 1996, 2001): (i) an increase in the number of circulating hemocytes, (ii) differentiation of a particular hemocyte type, the lamellocytes, (iii) the activation of the phenoloxidase cascade and (iv) formation of the capsule by accumulation of lamellocytes. the egg must first be recognized as foreign for the reaction to occur and, as stated by several authors (carton et al., 1992; poirie et al., 2000; schmidt et al., 2001), the host-parasitoïd relationships in the d. melanogasterl. boulardi system resembles the "gene-for-gene" recognition model of plants. possibly, the absence of capsule formation results from non recognition of the parasitoïd egg by the host; however, various observations are inconsistent with this possibility. for instance, whether or not the capsule is formed (step iv), the first two steps, involving modifications of blood constituent, are in all cases completed (russo et al., 2001). in addition, when the same larvae of resistant d. melanogaster strain are parasitized both by a virulent l. boulardi strain (no capsule formation) and by an avirulent strain (capsule formation), both types of egg were protected from encapsulation (this reaction was termed "cross protection") (labrosse et al., 2003). if 104 only the avirulent strain were recognised, capsule formation would have been triggered; however, there was no capsule formation. finally, active inhibition of the drosophila defence response by the parasitoïd has been described (labrosse et al., 2003). all these data clearly demonstrated that in the four possible combinations of host-parasitoïd strains (fig. 1), the immune response was always triggered, but then actively inhibited by the parasitoïd in three of the combinations. whatever the combination, i.e. whatever the reaction outcome, parasitoïd eggs were always recognized as foreign bodies. therefore, the specificity of the reaction does not depend only on the recognition process, but also, and in this case mainly, on a putative depressive effect of the parasitoïd. note that in numerous studies performed on invertebrates, with the exception of some works in drosophila (lemaitre and hoffmann, 2007) and in molluscs (zhang et al., 2004), specificity refers to the overall result of the response and not only to the process that triggered the reaction. for instance, carton and nappi (2001) looked for the formation of a capsule around the parasitoïd egg, little et al. (2003) analyzed the fitness of daphnia after exposure to pathogen, kurtz and franz (2003) counted the percentages of copepods infected, moret and siva-jothy (2003) assessed the survival and global antibacterial activity of tenebrio, pham et al. (2007) tested for the protection of drosophila against pathogen; in all these studies what was called "immune specificity" was in fact the product of both (i) recognition (reviewed in du pasquier, 2005) and (ii) complex interactions between the host and the pathogen/parasite. putative supports of specificity there are clearly multiple targets for inhibiting factors in the d. melanogaster-l. boulardi system. once the potential parasite/pathogen has been recognized, signal pathways are triggered. these signal pathways involve various enzymes, especially kinases, phosphatases, hydrolases and gtpases. each one of these molecules is a potential target for immunosuppressive factors. for example, the entomopathogenic bacterium photorhabdus luminescens can prevent its phagocytosis by insect macrophages by inhibiting the activity of the rho and rac gtpases in these cells (brugirard-ricaud et al., 2005). indeed, there are tens or even hundreds of possible targets that could be exploited to abort an elicited defence reaction. each of the inhibitor/target complexes could display a very high specificity, as is characteristic of enzyme-substrate or enzymeinhibitor relationships. in addition, the putative inhibitor from l. boulardi and its putative target in d. melanogaster may be subject to mutations, which could affect the binding affinity. also, alternative splicing or other genetic diversification mechanisms (schulenburg et al., 2007) could greatly increase the efficiency of the combinations. specificity could also be improved by the combination of different inhibitors with different targets in the same hostparasite model. therefore, active inhibition could result in an overall immune response with stronger specificity than that expected from the recognition mechanisms alone. as a consequence, the dogma of the weak specificity of innate immunity led to misinterpretation of those invertebrate immune responses that display strong specificity. is there a need for immune memory in invertebrates? the first line of evidence involved graft transplantation assays and concluded that there was recognition of foreign antigens in annelids (cooper, 1969; valembois, 1963). it was suggested that so-called “anti-graft immunity” was mediated by particular leukocytes which are stimulated during rejection (cooper and roch, 1984; valembois and roch, 1977). anti-graft immunity can be transferred to naïve earthworms by transferring stem cells from a grafted individual, suggesting the existence of memory (roch, 1973). the earthworm’s anti-graft immunity was described as having three characteristics: accelerated rejection, weak specificity and short-term memory (less than 10 days). the same grafting technology applied to scleratinian coral (hildemann et al., 1977), the sea urchin lytechinus pictus (coffaro and hinegardner, 1977), and the marine sponge callyspongia diffusa (bigger et al., 1982) lead to similar conclusions. curiously, and despite repeated efforts in insects, a specific short-term memory has only been found in the american cockroach, periplaneta americana (karp and rheins, 1980; rheins et al., 1980; hartman and karp, 1989). in our example of the d. melanogaster-l. boulardi system (see above), the capsule formation process did not show any memory as the specific interaction is dependent on genetic and not phenotypic adaptation. however, there are an increasing number of reports of the existence of memory in invertebrate immune responses. even if the term memory was used by the authors, its short-term duration was destroying their efforts to demonstrate the existence of a true immune memory. immune memory is advantageous only if there is a chance of being exposed to a previously encountered pathogen; there is therefore a direct relationship with the lifespan of the species. most invertebrates will have died before a secondary exposure is likely, and long-lived invertebrates, such as the cephalopod mollusc nautilus, the horseshoe crab limulus polyphemus, and some insect species (up to 17 years for a north american cicada), seem more likely than for instance does a rotifer as candidates for immunological learning-memory. indeed, little and kraaijeveld (2004) suggested that the lifespan relative to the delay between exposures is probably important. even in short-lived invertebrates, several cycles of infections may occur according to the life cycle of the pathogen. additionally, the same individual may be repeatedly or persistently exposed to similar types of pathogen in their natural environment. consequently, specific immune memory might exist in short-lived as well as in long-lived invertebrates. 105 time intensity 1-m 1-o mothers offspring time intensity 1-m 1-o mothers offspring a b time intensity 1 2 time intensity 1 2 c d fig. 2 how to test the hypothesis of learning through transmission of memory from mothers to offspring (a-b), or within the same individual subjected to two different exposures to the same pathogen (c-d). evolutions of the intensity of the immune reaction according to time and reproduction (vertical bar) following a first exposure of the mothers to the pathogen (1-m), and a first exposure of offspring issued from the challenged mothers to the same pathogen (1-o). a: hypothesis of transmission of immunity by transfer of effectors leading to an increased response, i.e. no learning. b: hypothesis of learning after exposure of the mothers and transmission of the memory to offspring leading to an accelerated immune response. evolutions of the intensity of the immune reaction according to time in the same individual following a first exposure to the pathogen (1), and a second exposure to the same pathogen (2). c: hypothesis of the persistence of high concentration of immune effectors leading to an increased response after the second exposure, i.e. no learning. d: after the first challenge, the immune response return to baseline and the accelerated immune response observed after the second exposure is due to learning-memory resulting from the first exposure. the importance of the concept of immune learning the existence of long-lasting cells, known to be the support of memory in vertebrates, has never been fully demonstrated in invertebrates. however, in addition to graft rejection assays, there are several examples of a second response being modified by a previous encounter, suggesting the existence of memory derived from learning. in particular, experiments in shrimp, (huang and song, 1999) and daphnia (little et al., 2003) provide strong arguments in favour of immune learning and memory: in these studies, larvae were specifically protected against one pathogen after "vaccination" of their mothers. to explain their results, the authors suggested that mothers impregnated their eggs with immune peptides. it is also possible that there was a kind of learning and memory that allowed better protection. this is exactly what differentiates between innate and adaptive immunity and has led some authors to speculate on adaptive aspects of immune responses in invertebrates (flajnik and du pasquier, 2004; agaisse, 2007; kurtz, 2005; kvell et al., 2007; pham et al., 2007). invertebrates have no antibodies, or t and b cells, so there must be a 106 completely different system that supports learning and memory. before dissecting the molecular basis of such a putative system, we must be sure that the shrimp or daphnia (or other invertebrates) are indeed capable of immune learning. to achieve this goal, we suggested continuously measuring protection between two exposures to the same pathogen; surprisingly, reports of such studies are rare, if any. if the protection (expressed as the level of one particular effector, for instance) remained almost the same between the two exposures, the hypothesis of a transmission of immunity from mothers to eggs (by transfer of the effector) remains valid (fig. 2a). however, in contrast to the persistence of specific b cells in vertebrates, this cannot be called memory but only transfer of molecular effectors, as observed for antibodies between mothers and offspring through the placenta in mammals, for instance. in contrast, if there was a decrease in protection between mothers and offspring, followed by a large increase after exposure of the offspring (fig. 2b), this would argue for existence of immune learning and transmission of memory from mothers to offspring. however, this phenomenon has not been found in the adaptive immunity of vertebrates. the situation is somewhat different in the case of multiple exposures of one individual to different strains of a pathogen species. exposure of the copepod macrocyclops albidus to its tapeworm parasite, schistocephalus solidus, reduces the chances of re-infection of the same host by siblings of the infecting tapeworm but not by unrelated parasites (kurtz and franz, 2003); this clearly evidences immune specificity at the level of the strain, as observed in the d. melanogaster-l. boulardi system or in daphnia. but can we call this phenomenon memory? two different models may apply to these observations. if the protection (expressed as previously as the level of one particular effector) remained almost the same between the two exposures, the persistence of the molecular effector in itself could account for the improved protection at the second exposure (fig. 2c). however, if protection decreased after the first exposure, then was substantially accelerated on the second exposure, it would be good evidence for learning and memory induced by the first exposure (fig. 2d). to confirm that true memory is established, referring to a kind of “vaccination”, it would be relatively simple to test whether the level of protection just before the second challenge was returning to baseline. to do that, several prerequisites included at least established optimum breeding conditions and minimum knowledge of immune capacities of the species under investigation. existence of memory remains to be demonstrated when considering the global immune response generally (and not only the recognition process), it is evident that specificity is very accurate, at least in some invertebrates (carton et al., 1992; kurtz and franz, 2003; little et al., 2003; pham et al., 2007). the specificity of some innate immune responses is very probably also present in vertebrates where it might have been obscured by the strength of adaptive immunity. it seems likely, given the extreme diversity of invertebrate (and of vertebrate) species, that there is not one single universal system underlying specificity. we have shown that the inhibiting effect triggered by the parasitoïd could explain the strong specificity of d. melanogaster-l. boulardi relationships. the recently discovered extended genomic diversity of immune effectors (imler and bulet, 2005; padhi et al., 2007), and receptor pgrps (royet et al., 2005), dscam (watson et al., 2005) and tlrs (takeda et al., 2003), is still not understood but may also account for the specificity of innate immune responses. finally, the existence of a true memory remains to be clearly demonstrated in invertebrates. we agree with hauton and smith (2007) that before there can be a "theory", facts about invertebrate immune responses must be established for a wide range of taxa. but unlike these authors, we recommend establishing the existence of memory in an invertebrate species before looking for its biochemical and molecular supports, also suggested by little et al. 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(2005) extensive diversity of ig-superfamily proteins in the immune system of insects. science 309: 1874-1878, 2005. werner t, borge-renberg k, mellroth p, steiner h, hultmark d. functional diversity of the drosophila pgrp-lc gene cluster in the response to lipopolysaccharide and peptidoglycan. j. biol. chem. 278: 26319-26322, 2003. zhang sm, adema cm, kepler tb, loker es. diversification of ig superfamily genes in an invertebrate. science 305: 251-254, 2004. 109 cell death and the immune response of the sipunculid worm themiste petricola isj 7: 239-250, 2010 issn 1824-307x review cell death and the immune responses of the sipunculan worm themiste petricola ga blanco department of immunology, idehu-national research council (conicet), school of pharmacy and biochemistry, university of buenos aires (uba), buenos aires, argentina accepted october 25, 2010 abstract we have recently studied the role of cell death in the immune system of the sipunculan worm themiste petricola. typical biochemical and morphological changes of apoptosis were induced in celomocytes of these marine worms after in vitro exposure of cells to hydrogen peroxide. apoptosis was time and dose dependent, and required several hours to become apparent. surprisingly, in unexposed samples a subtype of granulocyte was observed to undergo homotypic aggregation, extensive cytoskeletal changes, and degranulation followed by cell death. this spontaneous response ending in cell death occurred in a divalent cation-dependent manner, served to entrap foreign particles, and was blocked by edta-containing saline solutions. even though the mode of granulocyte cell death shares some features with apoptosis, it appears to be a different form of programmed cell death since it occurs within minutes and does not produce single cell-derived apoptotic bodies but transforms itself into one or several syncytial masses with haemostatic and immune purposes. since numerous granulocyte types and multicellular masses involved in cellular immunity have been described in sipunculan worms, the review also discusses the potential influence of activation of granulocytes by sea water in expanding the variety of morphological types and multicellular structures identified through morphological studies among sipunculan species. key words: sipuncula; immune responses; hemostasis; cell death; celomocytes; apoptosis; coagulation introduction recent studies on celomocyte death in the sipunculan worm themiste petricola have introduced some new perspectives of cellular immune responses of these worms. the finding that a specific cell type of celomic granulocytes demonstrates rapid cell death as part of a well orchestrated response with hemostatic and immune ___________________________________________________________________________ corresponding author: guillermo a blanco catedra de inmunologia idehu uba conicet junin 956 4to piso, capital federal (1113), argentina e-mail: gblanco@ffyb.uba.ar list of abbreviations: lha, large hyaline amebocyte; lgl, large granular leukocyte; sgl, small granular leukocyte; pgrp peptidoglycan recognition protein; pgrp-s, pgrpsmall; fsc, forward light scatter; ssc, side light scatter; ps, phosphatidylserine; tunel, terminaldeoxynucleotidyl-transferase-mediated-dutp-nickend-labeling; dapi, 4',6-diamidino-2-phenylindole; jc-1, 5,5',6,6'-tetrachloro-1,1',3,3'tetraethylbenzimidazolylcarbocyanine iodide purposes (blanco, 2007; blanco et al., 2008; cavaliere et al., 2010) has implications to the interpretation of celomic cell morphological categories and cellular immune responses described hitherto in sipunculans. the first part of this review will present a brief summary of sipunculan celomic cells and cellular immune responses, as commonly described in previous studies conducted in different species of the phylum. then the main findings on a peculiar mode of rapid cell death of celomic granulocytes in t. petricola will be presented, and its implications to the immune system and hemostasis of sipunculans will be discussed. sipunculan body plan and the absence of a true circulatory system sipuncula is a phylum of unsegmented vermiform celomates with a plump trunk and a slender introvert that ends in a mouth encircled by tentacular outgrowths (stephen and edmonds, 1972; gibbs and cutler, 1987; rice, 1993). the body wall enclosing the celom cavity consists of several layers that include cuticle, epidermis, dermis, circular muscle layer, longitudinal muscle 239 layer, and peritoneum (fig. 1). the dermis varies from quite thick to very thin stratum and consists of a mesh of fine fibres containing connective-tissue cells, pigment cells, multinucleate bodies and leukocytes (hyman, 1959). a definite circulatory system is lacking in sipunculans and celomic fluid operates mechanically as a hydroskeleton. however, in some thick-walled species the dermis contains celomic canals or spaces communicating with each other and with the general celom (hyman, 1959).in larger species of the genus siphonosoma canals take the form of celomic diverticula that penetrate into the dermis as blind sacs of irregular shape, branched, and somewhat transversely arranged (hyman, 1959). the canals are lined by peritoneum and contain the same elements as the celomic fluid. thus celomic cells can reach the dermis and they can be found interspersed between a felt of fine fibres and connective-tissue cells (fig. 1). the main muscles of the interior are the retractors of the introvert. the contraction of the retractors causes the invagination of the introvert, which is extruded again by the general contraction of the circular layer of the body wall acting to compress the celomic fluid. this is the main factor causing celomic fluid flow (zuckerkandl, 1950). the tentacles consist of a separate lumen that contains the same elements as the celom, and although there are no openings into the celom the celomocytes seem able to penetrate into the system (maiorova and adrianov, 2005; adrianov et al., 2006). all sipunculans are dioecious and the sex cells are shed into the celom at an immature stage and complete their maturation while floating in the celomic fluid. they are voided by way of the nephridia, which act as gonoducts (hyman, 1959; adrianov et al., 2002). celomic cell types and cellular immune responses in sipunculans hemerythrocytes the celomic cavity contains a variety of dissimilar cell types. unfortunately, nomenclature of cell types is far from being standardized making it difficult to compare studies from different species. hemerythrocytes, the most abundant cells in the celomic fluid, are found in all species and confer the celomic fluid a pinkish tint due to the iron-containing substance hemerythrin (hyman, 1959; valembois and boiledieu, 1980; dybas, 1981a; rice, 1993; matozzo et al., 2001; lunetta, 2004; meyer and lieb, 2010). most authors agree in the morphological description of hemerythrocytes as nucleated biconvex disks, varying in size among different species, and often having one or more acid vacuoles (ochi, 1970; ochi and ohnishi, 1971). it has been suggested that they are of some assistance in ridding the celom of foreign particles since injected dyes are taken into these vacuoles (towle, 1975). other main cell types in the celom are leukocytes, multicellular structures and gametes (ochi, 1970). hyaline leukocytes leukocytes encompass a heterogenous group of cells mostly involved in cellular immune responses. main types of leukocytes, not necessarily all present fig. 1 the body wall of sipunculan worms consists of an external cuticle everywhere underlain by an epidermis with numerous glands (g). in some genera the dermis contains longitudinal canals (lc) communicating with the general celom. the bodywall musculature consists of outer circular (cm) and inner longitudinal layers (lm). a thin diagonal layer (dm) exists between the other two in some sipunculans but is infrequently mentioned (modified from hyman, 1959). in the same species, include hyaline cells with fine or no granules, and granulocytes with coarse granules that may be acidophilic, neutrophilic, or basophilic (marcou and volkonsky, 1933; hyman, 1959; matozzo et al., 2001). the presence of granules, extension of pseudopodia or an ameboid cell shape is often used as a morphological criterion to distinguish leukocytes from the most abundant biconcave disc-shaped hemerythrocytes. amebocytes are often referred to as phagocytic cells, with a hyaline cytoplasm having few or no granules and having one or several large lysosomic vacuoles often similar to that of hemerythrocytes (ochi and ohnishi, 1971). granular leukocytes most authors coincide in the occurrence of granulocytes among celomic leukocytes and their involvement in cellular immune responses (rice, 1993; matozzo et al., 2001; lunetta, 2004). they are often described as having numerous granules masking details of the cytoplasm and the nucleus (dybas, 1981b; matozzo et al., 2001; lunetta, 2004). the few studies that have quantified the relative abundance of granulocytes within celomic cells indicate a range between 5 % and 20 % (towle, 1975; matozzo et al., 2001; lunetta, 2004) more recently lunetta (2004) has introduced a classification that separates two broad categories of granulocytes from sipunculus nudus designated type i and type ii, and has provided differential characteristics between these two categories at the biochemical, morphological and functional level. this classification criterion may be applicable to most species of sipunculans and the categories proposed by lunetta (2004) will be recalled while discussing results obtained in t. petricola. granules 240 fig. 2 phagocytosis of zymosan particles (arrows) by large hyaline amebocyte (lha) stained with giemsa (a), and with acridine orange (b). in panels c and d degradation of particles was inhibited by alkalinizing the lysosomes with ammonium chloride. zymosan is engulfed but not degraded, thus the cytoplasm becomes packed with basophilic zymosan particles (c). note also that when lysosome vesicles are alkalinized acridine orange no longer stains them red-fluorescent (d). a sequence of nuclear apoptotic changes induced in lhas by exposure to hydrogen peroxide during 2 to 6 hours is shown in (e). cells were stained with acridine orange and ethidium bromide; green fluorescence indicates that cell membrane permeability is preserved (apoptotic live), while red fluorescence indicates loss of membrane permeability (apoptotic dead). exposure of phosphatydilserine during apoptosis of lhas and hemerythrocytes detected by annexin v-fitc and propidium iodide is shown in (f). red fluorescence indicates loss of membrane permeability. in panel (g) lhas and hemerythrocytes were stained with the potentiometric dye jc-1 showing normal mitochondrial membrane potential (orange fluorescence), while in (h) mitochondria became depolarized as indicated by shift from orange to green fluorescence. bar = 15 μm (modified from blanco et al., 2005). 241 from both categories of granulocytes have lytic enzymes including peroxidase, acid esterases, alkaline and acid phosphatases, and lipase (lunetta, 2004, 2005). even though type i granulocytes have lytic enzymes they do not contain any foreign material and do not show any phagocytic capability (lunetta, 2005). degranulation of type i granulocytes and production of extracellular acidophilic material has been noted by several authors (lunetta, 2004). in addition type i granulocytes often show thin cytoplasmatic filaments or filopodia, oriented in all directions. a wide range of morphological descriptions that may well be included in the type i category of granulocytes has been introduced by different authors. descriptions include elongated granulocytes, fusiform or curious shaped cells projecting pseudopodia, which often appear to join with one another (towle, 1975). yet dybas (1975) has reported a paired non-phagocytic granulocyte, occurring as a cell within a cell at a frequency of less than 0.5 % of total celomic cells. a recent study in phascolosoma esculenta reported granulocytes with condensed and clumped chromatin, syncytial granulocytes, cell complexes of granulocytes with podocytes, granulocytes forming pseudopodia and devoid of granules at the pseudopodia protrusion, and finally granulocytes having a large nucleus and often found in association with hemerythrocytes (ying et al., 2010). it should be noted that all of these cytological studies have been conducted in live or fixed preparations without concurrent assessment of cell viability prior to fixation. in addition it has been a common practice to use sea water as saline solution to dilute celomic fluid during cytological studies, either for supravital studies or prior to fixation of cells in suspensions. as it will be discussed further, non-phagocytic granulocytes may be activated in contact with sea water or saline solutions containing ca++ and show rapid cytoskeletal changes before dying within minutes. in contrast, type ii granulocytes of the classification proposed by lunetta (2004), are motile cells, actively phagocytic with lytic enzymes involved in intracellular digestion, and contain engulfed particulate material and lipids. both categories of granulocytes are reported to accumulate in the vicinity of foreign particles trapped within the celom together with masses of acidophilic material (lunetta, 2004). as will be discussed further type ii granulocytes of the category proposed by lunetta (2004) correspond to cells that do not lose viability as part of the cellular defence reactions but remain motile and actively phagocytic engulfing remnants of dying type i granulocytes and foreign particles as well. multicellular bodies various types of multicellular bodies are reported to occur in the celomic fluid of different species of sipunculans (hyman, 1959; rice, 1993). the main criterion to define these bodies has been the morphological identification of cells by light and electron microscopy. several authors have noted that the multicellular bodies include adhered cells and variable degrees of amorphous material that is thought to entrap particles and aid in adhesion. these masses, often of sizes from 30 to 100 μm, have received several names such as cell plates, giant multinucleate corpuscles or more recently brown bodies (lunetta, 2004). experimentally they have been induced by the injection of foreign particles into the celom and it has been considered a main cellular immune response of sipunculans (hyman, 1959; lunetta, 2004). brown bodies and other similar multicellular structures entrapping foreign bodies have been also referred to as capsules (tripplet et al., 1958), although there is not a histological resemblance to capsules occurring in other invertebrates such as insects and crustaceans. more recently lunetta (2004) described the structure of brown bodies induced by mammalian red blood cell injection as made of a core of acidophil material, probably derived from degranulation of type i granulocytes. the function of acidophilic material would be to cement the various parts of the brown body and external layers containing type i and type ii granulocytes (lunetta, 2004). granulocytes in brown bodies were described as misshaped, containing little cytoplasm, and having a large quantity of granulated material (lunetta, 2004). free urns the free urns, described only in some species of sipunculans, are cell complexes that swim freely within the celomic cavity and play an important role in the sipunculan immune defence by releasing sticky, mucoid tails that promptly trap invading pathogens (bang and bang, 1975; nicosia, 1979; bang and bang, 1980; nicosia and sowinski, 1995). urns are composed of two cells, a vesicle cell and a saucer-shaped ciliated cell, fitted together like an acorn into its cap (dybas, 1976). a smaller and variable population of cells, often referred to as thirdtype cells, is frequently found in close juxtaposition to the ciliated cell. lunetta (2004) showed that third type cells are adhering granulocytes that reacted positively with chloroacetate esterase and were enmeshed in acidophilic material. celomocyte cell death in t. petricola apoptosis in celomic cells apoptosis has been a main focus of research during the last 20 years and there is no doubt that it is a crucial process in adaptive and innate immunity of higher animals (kerr et al., 1972; opferman and korsmeyer, 2003; wang et al., 2003). apoptosis occurs during normal t and b cell ontogeny, central tolerance development, and downregulation of t and b cell responses (feig and peter, 2007). cell death in neutrophils during inflammation was classically thought to occur exclusively by passive necrosis due to release of lytic enzymes and damage by microbial products (sendo et al., 1996). however, recent studies have shown that neutrophils undergo apoptosis to ensure complete destruction of intracellular microbes, facilitate uptake by macrophages, and limit proinflammatory stimuli protecting bystander self cells (everett and mcfadden, 1999; fadok and chimini, 2001; fadok et al., 2001). 242 fig. 3 activation, shape changes and cytoplasmic disintegration in large granular leukocytes (lgl).a resting lgl is shown in (a). green fluorescence corresponds to intracellular ca++ level as indicated by ca++ probe fluo-4. early activated lgl emitting filopodia is shown in (b). note the increase in intracellular ca++ levels as indicated by green fluorescence. extensive shape changes observed at later time points after activation are shown in (c). nuclei were stained with dapi in a paraformaldehyde-fixed preparation in (c). a giemsa stained preparation is shown in (d) where cytoplasmic disintegration yields microparticles (mp), and isolated nuclei (n). microparticles are observed to be engulfed in a lha. in live preparation shown in (e) dapi stained only dna present in nuclear remnants and dead cells because the dye is non-permeant to live cells. note that dna-containing remnants were engulfed in lhas and sgls and remained in the cytoplasm, while nuclei were not stained indicating that the phagocytic cells were viable. in panels (f) and (g) a live preparation was stained with acridine orange showing acid granules in an early activated round lgl (right-bottom), and partial loss of granules in a late activated lgl with extensive pseudopodia formation (centre). bar = 15 μm (modified from blanco, 2007; blanco et al., 2008; cavaliere et al., 2010). the role of apoptosis in innate immunity has been also recognized in invertebrate animals where programmed cell destruction can aid in degradation of intracellular pathogens preventing spread of infection beyond the cell boundary (wang et al., 2003; russo and madec, 2007; wang et al., 2008; sokolova, 2009; kiss, 2010). yet some pathogens appear to have the ability to modulate the induction of apoptosis to their own advantage (sunila and labanca, 2003; wang et al., 2003; terahara and 243 takahashi, 2008). other studies have focused on cell death induced by ecotoxicants, since exposure to these substances may pose a threat to wildlife invertebrates by their ability to induce apoptosis in several cell types including immune cells (sokolova et al., 2004; cima et al., 2008; matozzo et al., 2008). all these findings have fostered the interest in characterizing apoptosis mechanism of immune cells in several invertebrate groups (podrabsky and krumschnabel, 2010). it should be pointed out that apoptosis is no longer a synonym of programmed cell death since other forms, including autophagic cell death and programmed necrosis, have an increasing relevance in immune response, amongst many other fields of study (sperandio et al., 2000; yorimitsu and klionsky, 2005; samara et al., 2008; kroemer et al., 2009). our first study on cell death in sipunculan celomocytes was focused on identifying standard indicators of apoptosis after exposure to hydrogen peroxide and evaluating to what extent hydrogen peroxide-induced cell death was similar to what had been described in other animals (blanco et al., 2005). that study was conducted on cell suspensions from celomic fluid containing hemeryhtrocytes and large hyaline amebocytes (lha) as their main constituents. both cell types have large acid vacuoles and particularly lha are actively phagocytic (blanco et al., 1995), and were shown to migrate in vitro toward gradients of endogenous or exogenous chemoattractants (cabrera et al., 2002). the lha vacuole has an extraordinary capacity to degrade engulfed material. when exposed to zymosan particles lha engulf the particles, route them to the vacuole and degrade it in such an efficient manner that only a scant number of particles can be observed in the cytoplasm (figs 2a, b). when acid degrading enzymes were inhibited by elevating the ph of the vacuole with ammonium chloride the cytoplasm became densely packed with particles since they were phagocytosed but not degraded (figs 2c, d) (blanco et al., 2005). exposure of this suspension of hemerythrocytes and lhas to hydrogen peroxide induced morphological and biochemical changes that are known to be present in apoptotic cells (blanco et al., 2005; galluzzi et al., 2009). changes at the nuclear level included chromatin condensation and fragmentation, nuclear membrane ripples, and dna degradation to oligonucleosome-sizes (fig. 2e) (blanco et al., 2005). extranuclear changes consisted of loss of mitochondrial membrane potential, cell volume decrease, exposure of phosphatidylserine (ps) on the outer membrane leaflet, and cell membrane blebbing (figs 2f-h) (blanco et al., 2005). activation and cell death in a subtype of granulocytes the celomic cell suspension used in our first study was particularly depleted of granular cells. the cell suspension was obtained by harvesting, washing, and incubating cells in saline solutions or culture media containing ca++ (blanco et al., 2005). when celomic fluid was stained with acridine orange and propidium iodide immediately after harvesting without washing and observed by fluorescence microscopy, we noted that a celomic cell type having acid granules was present in the samples (blanco, 2007). these cells, that we designated large granular leukocyte (lgl), were about 8 % of celomic cells, showed extensive morphological changes within minutes emitting filopodia and pseudopodia upon contact with glass surface (figs 3a-c), aggregated to each other, loss the acid granules and became dead as indicated by red fluorescence of the nuclei stained with propidium iodide (figs 3f, 3g, 4g-j). these granulocytes are similar in description to type i granulocyte category proposed by lunetta (2004). when celomic fluid was harvested in edta-containing solutions, lgls did not show morphological changes, preserved their round shape densely packed with granules and remained viable (blanco, 2007; blanco et al., 2008). moreover, when sea water was added to celomic fluid harvested in ca++ free saline preparations, lgls started to form aggregates and demonstrated shape changes, being more noticeable the larger the amount of sea water dispensed (blanco, 2007) (figs 4a-d). since all changes were blocked by edta we concluded that sea water and ca++ containing solution caused activation and aggregation of lgls in multicellular masses. a surprising finding was that although cell death of lgls occurred very rapidly, several apoptotic-like features were still observed including chromatin condensation and fragmentation, nuclear membrane ripples, loss of mitochondrial membrane potential and ps exposure (blanco, 2007; blanco et al., 2008). however, other typical apoptotic cytoplasmic changes were absent, including cell volume decrease, membrane blebbing and formation of apoptotic bodies. instead, the cytoplasm of lgls was often seen to disintegrate in small particles unless the cells remain in clumps, in which case the cytoplasmic remnants were kept as integral part of a syncytial mass (cavaliere et al., 2010) (figs 3d, 4ek). a suspected hemostatic role: isolating the cellular clot by the pullout method activation and aggregation of lgls was to some extent reminiscent of platelet aggregation and thrombus formation (george, 2000; heemskerk et al., 2002; harrison, 2005). both platelets and lgls show a series of prominent cytoskeletal changes after activation and continue to form a cell-cell aggregate, with exposure of ps residues in the outer leaflet of the plasma membrane and loss of mitochondrial membrane potential (pereira et al., 1999, 2002; li et al., 2000). the end product is in both cases an insoluble mass. however activation and aggregation systematically followed by death is not an accepted concept in platelet physiology due to its non-nucleated condition (perrotta et al., 2003). programmed cell death in platelets is still a concept that raises several concerns to most authors (wolf et al., 1999; zhang et al., 2007). however our finding in lgls was also reminiscent of cell death and disintegration of coagulocytes as occurs during arthropod coagulation (theopold et al., 2004). coagulocytes have an accessory role in arthropod clot formation 244 fig. 4 celomic fluid harvested in ca++ free saline solution is shown in (a). the effect of exposure to increasing concentrations of sea water (sw) is shown in (b) and (c). multiple lgl aggregates are observed as dark spots. in panel (d) lgl activation by 50 % sw was blocked by 6mm edta. panel (e) shows a large clot entrapping magnetic beads obtained in vitro and (f) shows a magnified image of (e) where beads are observed (arrows). in (g) and (h) a small clot entrapping beads (arrows) in vitro was stained with propidium iodide. in (i) and (j) a small clot entrapping magnetic beads (arrows) was stained with acridine orange. note loss of acid granules indicated by green fluorescence at the clot core and preservation at peripheral areas indicated by orange fluorescence. panel (k) shows small clots formed in vivo after injection of magnetic beads into the celom. no massive clots as shown in (e) were formed in vivo. these small clots entrapping beads coincide with morphological description of brown bodies (multicellular masses demonstrated to entrap particles injected in vivo in other species of sipunculans). bar = 100 μm in (a) through (e) and bar = 30 μm in (f) through (k) (modified from blanco, 2007; blanco et al., 2008). since extracellular strands formed by hemolymph coagulation proteins form the main clot structure (theopold et al., 2002; scherfer et al., 2004; bidla et al., 2005). even though cell-free celomic fluid does not form strands we speculated that sipunculans could yet form a clot mass, to some extent reminiscent of a platelet thrombus, by means of activation, adhesion and rapid death of nucleated cells. scherfer (2004) introduced a method to isolate clots from drosophila that was based on creating a clot over a suspension of magnetic beads and further separating the clot with a magnet (scherfer et al., 2004). using this so called pullout method we could isolate a massive clot ex vivo formed by lgls entrapping beads that could be further studied regarding several aspects of clot formation and structure (figs 4e-f). the hemostatic purpose of this cellular reaction was inferred upon the rapid formation of an insoluble macroscopic mass. however immediate entrapment of particles within the clot was also a strong evidence of being a first line immune response (blanco et al., 2008). 245 our first aim was to demonstrate that the reaction was indeed hemostatic. since a system of celomic canals allows sipunculan celomic cells to reach the dermis (fig. 1), we speculated that a body wall injury would create a sudden contact of celomic cells with sea water, causing lgls to activate, further aggregate, and demonstrate a systematic and rapid death to create a hemostatic plug. to prove this hypothesis we harvested celomic fluid and allow it to flow through a glass tube with an open end in contact with sea water. a massive clot was formed at the open end in contact with sea water which stopped the flow of the liquid column. when observed by light microscopy the clot was formed by aggregated lgls (cavaliere et al., 2010). the immune significance of cellular clotting and lgl death in t. petricola the immune significance of clot formation was made evident not only due to entrapment of magnetic beads but of several dissimilar biotic particles including bacteria, yeast and leukemic cells (cavaliere et al., 2010). by eliciting smaller clots over particle suspensions with controlled amounts of sea water we could observe that clots were formed by aggregating lgls that extended filopodia and adhered to each other creating a mesh where particles became entrapped (figs 4g-j). dna degradation as indicated by supravital propidium iodide staining of small clots was observed to occur quite fast at the core of lgl aggregates, while the peripheral layers tend to remain viable for a longer time (figs 4g, h). staining with lysosomotropic dyes like acridine orange showed that acid granules of lgls were released at the clot core, while most peripheral lgls showed intact granules indicating that there was a gradient of granule content release from the centre to the periphery (figs 4i, j). ps exposure as indicated by annexin v binding followed a similar gradient pattern from the centre to the periphery (blanco et al., 2008). dna degradation within the clot centre was further evidenced by the dna nickend labelling method (tunel) (cavaliere et al., 2010). thus we concluded that almost immediate entrapment of potential pathogens such as bacteria or fungi within the clot was followed by formation of a hostile environment within the clot core consisting of degradative enzymes derived from acid granules, and potentially from other enzymes activated as part of programmed cell death such as caspases, calpains and dnases (pasquet et al., 1996; shcherbina and remold-o'donnell, 1999; wolf et al., 1999). invertebrate cellular responses often utilize pattern-recognition receptors in the hemolymph or on the immune cell surface to identify pathogenassociated molecular patterns (kurata, 2010). members of the peptidoglycan recognition protein (pgrp) family recognize diverse bacteria-derived peptidoglycans and initiate appropriate immune reactions (kurata et al., 2006; goto and kurata, 2006). to further explore the immune significance of lgl clotting as a first line immune response we evaluated the presence of pgrp (blanco et al., 2008). using antibodies raised against different human recombinant pgrp we explored the expression of these pattern recognition proteins as indicated by immunofluorescence detection finding that lgls stained positive to anti pgrp-s (blanco et al., 2008). by harvesting celomic fluid in edta-containing saline solutions we were able to isolate resting lgls and evaluate its light dispersion properties through flow cytometry (blanco et al., 2008). these cells form a cluster of high side light scattering (ssc) due to the high granule content (fig. 5a). a high expression of pgrp-s was observed in resting lgls when stained with anti pgrp-s and evaluated by flow cytometry (blanco et al., 2008), since the pullout method allowed us to isolate clots entrapping magnetic beads we explored the presence of pgrp-s within these clot masses and also in clot supernatants noting that both the clot and the supernatant had high pgrp-s content (blanco et al., 2008). altogether these findings reinforce the role of clot formation as a first line immune cellular response with a role in pathogen-associated molecular pattern recognition. clot formation and the second line cellular immune response clot formation is not a single and isolated response in t. petricola but it is connected to a second line cellular immune response. light microscopic preparations of small clots were suggestive of two cell types involved in phagocytosis of lgl remnants, namely lhas and a second type of granulocyte that we designated small granular leukocyte (sgl) (fig. 3e). these granulocytes remained live as evidenced with viability probes, did not release granule content, and were often seen at the clot edges actively phagocytosing self remnants and particles shed from the clot (cavaliere et al., 2010). cytoskeletal arrangement of sgls was often suggestive of a polarized motile cell with uropodia and lamellipodia. thus these cells were coincident with type ii category of sipunculan granulocytes in the classification proposed by lunetta (2004). through flow cytometry sgls are found in the same cluster of resting lgls (fig. 5a). the cluster disappears when cell suspension is exposed to sea water due to activation, aggregation, and death of lgls, while many sgls may be washout with elicited lgl clots. thus sea water presence in harvested celomic cell suspensions that are further filtered through a small pore mesh, yields suspensions almost depleted of lgls and sgls (fig. 5b). in addition the cluster of non-clotting cells formed mainly by lhas and hemerythrocytes decreases its forward light side scatter properties most probably owing to cell shape changes associated with activation (fig. 5b). clot formation induces shedding of cytoplasmic remnants of a number of disintegrated lgls that are not completely retained at the clot. these cytoplasmic remnants can be stained and observed by flow cytometry (cavaliere et al., 2010). in addition, labelled cytoplasmic remnants were demonstrated to be phagocytosed by sgls, often at the clot neighbourhood (cavaliere et al., 2010). lhas were also detected to phagocytose these remnants although they appear to be loaded in the large lysosomic vacuoles and digested more 246 a b fig. 5 (a) flow cytometry dot plot of front (fsc) vs side (ssc) light scatter of celomic fluid harvested in edtacontaining saline solution. q1 quadrant shows resting lgls while q4 quadrant shows lhas, and hemerythrocytes that together encompass the most abundant cell types. (b) celomic fluid harvested and washed in ca++ or sea water-containing saline solutions becomes depleted of lgls as observed in q1 quadrant, and contains mainly lhas and hemeryhtrocytes observed in q4 quadrant. a decrease in fsc of this cluster is also observed as compared to (a). percentages were determined after running 80,000 celomic cells (modified from blanco et al., 2008). efficiently as occurs with most targets phagocytosed by these cells (figs 3d, e). dna remnants were similarly stained by dna labels and demonstrated to be engulfed by sgls and lhas (fig. 3e). in addition the tunel method demonstrated fragmentation of dna remnants that were engulfed by sgls and lhas (cavaliere et al., 2010). when clots were formed over suspensions of labelled bacteria the fate of these targets was similar to self remnants. bacteria appeared entrapped within the clot and sgls could be seen at the clot edges phagocytosing bacteria. lhas were also found to phagocytose bacteria although in areas not in much proximity to clot edges (cavaliere et al., 2010). clot formation in vivo and the origin of brown bodies since a large clot was formed in vitro by exposing the whole content of celomic fluid to a bead suspension, we expected that beads injected directly within the celom would elicit a similar massive clot in vivo. however injected beads recovered from the whole celomic fluid of an injected worm by the pullout method after 24 h showed several small clots of about 50 to 150 μm that entrapped magnetic beads (blanco, 2007). these small clots resembled brown bodies described in sipunculus nudus (lunetta, 2004) or similar multicellular masses described in other sipunculans (hyman, 1959; rice 1993). granulocytes could be barely recognized at the outer layers, while the core of multicellular masses was formed by an amorphous mass with entrapped beads interspersed (fig. 4k). these findings are in agreement with lunetta (2004) who proposed that formation of brown bodies, which is considered an immune response of sipunculans, would be somehow related to type i granulocytes since they were found in peripheral areas of brown bodies. we can now assert that amorphous material may not be derived from secretion of live cells but from activation, aggregation and active death of lgls as part of a cellular defence reaction. conclusion cell death of lgls is a central process of clot formation in t. petricola which involves both a hemostatic and an immune purpose. cell death is elicited following activation, occurs rapidly, and participates in a sequence of well orchestrated events. when evaluated in lgl aggregates, cell death occurs first at the clot core and later at the periphery. lgl death within the clot core provides a hostile environment to entrapped pathogens while containment at the periphery preserves neighbouring bystander cells and tissues. activation followed by cell death is responsible of several morphological changes in granulocytes. since granulocyte 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1824-307x research report vertebrate interleukins originated in invertebrates? s gerber, p cadet, m sheehan, gb stefano, kj mantione neuroscience research institute, state university of new york college at old westbury, old westbury, ny 11568-0210, usa accepted october 30, 2007 abstract previous studies have demonstrated that invertebrate immune and neural tissues contain mammalian-like cytokines, which activate specific cellular functions. therefore, it was of interest to attempt to identify these molecules via applied biosystems human genome survey arrays. the array was used to analyze the transcriptional profiles of mytilus edulis rna samples. the applied biosystems human genome survey array contains 31,700 60-mer oligonucleotides probes representing a set of 27, 868 individual human genes and more than 1,000 control probes. we show interleukin-like and tumor necrosis factor-like genes among other cytokine-like genes significantly expressed in this invertebrate tissue with a signal to noise value greater than 2. in morphine treated tissue additional cytokine genes were expressed. these cytokine-like genes are directly related to previously discovered molecules in invertebrates, suggesting that they first appeared earlier in evolution. key words: mussel; mytilus edulis; cytokines; microarray introduction mytilus edulis neural tissues contain both immuneand neural-like signaling molecules found in mammals (see stefano, 1982, 1990a, 1992). in regard to catecholamines, the neural tissues of mytilus contain both dopamine and norepinephrine (stefano, 1982, 1990b). in reference to cytokine-like molecules, interleukin (il)-1-, il-6and tumor necrosis factor (tnf)-like molecules have been identified via radio-immune assay in mytilus ganglia and immune tissues (hughes et al., 1990, 1991a; hughes and chin, 1994; scharrer et al., 1996). thus, invertebrate ganglia and immune tissues appear to have the potential to respond like mammalian tissues to these signaling molecules. concerning the signaling of these molecules, invertebrate immune tissues, i.e., immunocytes, ___________________________________________________________________________ corresponding author: kirk j mantione neuroscience research institute state university of new york college at old westbury old westbury, ny 11568-0210, usa e-mail: kjmantione@sunynri.org respond to il-1 and il-6 by undergoing conformational changes indicative of becoming activated, i.e., amoeboid, including stimulating mobility (hughes et al., 1990, 1991a,b; hughes and chin, 1994; scharrer et al., 1996). thus, given their presence and action in invertebrate physiological systems it was of great interest to determine if human microarray chips would also show that they are present given the many biochemical and pharmacological similarities. materials and methods mytilus edulis were harvested from the shores of long island sound at mattituck, new york during the month of march. animals were then transported to the laboratory in chilled seawater (4-10 °c). in the laboratory, they were maintained as previously described in detail (stefano et al., 1994). m. edulis pedal ganglia (20 per array) were dissected and kept on ice until needed. in order to determine the presence of 95 table 1 interleukin-like, tumor necrosis factor-like, and other cytokine-like genes that were significantly expressed as analyzed by the human genome survey microarray (applied biosystems) with a signal to noise value greater than 2 in the untreated mytilus edulis pedal ganglia tissue. tumor necrosis factor (tnf)-like molecules present tnfrsf11a tumor necrosis factor receptor superfamily, member 11a, activator of nfkb tnfrsf25; kiaa0720 tumor necrosis factor receptor superfamily, member 25; putative nfkb activating protein interleukins-like molecules present il16 interleukin 16 (lymphocyte chemoattractant factor) il7 interleukin 7 il31ra interleukin 31 receptor a il15 interleukin 15 il11ra interleukin 11 receptor, alpha il18bp interleukin 18 binding protein il23r interleukin-23 receptor additional cytokine-like molecules present chemokine and chemokine receptor activity ccl23 chemokine (c-c motif) ligand 23 ccrl2 chemokine (c-c motif) receptor-like 2 ccl1 chemokine (c-c motif) ligand 1 ccr9 chemokine (c-c motif) receptor 9 cxcr3 chemokine (c-x-c motif) receptor 3 ccl13 chemokine (c-c motif) ligand 13 ccl15; ccl14 chemokine (c-c motif) ligand 15; chemokine (c-c motif) ligand 14 ccl19 chemokine (c-c motif) ligand 19 ccl24 chemokine (c-c motif) ligand 24 mgc12815; ccl3l1; ccl3 chemokine (c-c motif) ligand 3-like, centromeric; chemokine (c-c motif) ligand 3-like 1; chemokine (c-c motif) ligand 3 ccr6 chemokine (c-c motif) receptor 6 cxcl12 chemokine (c-x-c motif) ligand 12 (stromal cell-derived factor 1) cell growth and growth factor activity gdf8 growth differentiation factor 8 gdf2 growth differentiation factor 2 ebaf endometrial bleeding associated factor (left-right determination, factor a; transforming growth factor beta superfamily) bmp1 bone morphogenetic protein 1 myh11 myosin, heavy polypeptide 11, smooth muscle cytokinesis and other cell-cycle activity cdk6 cyclin-dependent kinase 6 cdc23 cdc23 (cell division cycle 23, yeast, homolog) ccnd3 cyclin d3 cdc25a cell division cycle 25a ccnc cyclin c ccnl1 cyclin l1 cdc14a cdc14 cell division cycle 14 homolog a (s. cerevisiae) pard6a par-6 partitioning defective 6 homolog alpha (c.elegans) pard3 par-3 partitioning defective 3 homolog (c. elegans) prc1 protein regulator of cytokinesis 1 stat1 signal transducer and activator of transcription 1, 91kda nedd5 neural precursor cell expressed, developmentally down-regulated 5 3-sep septin 3 dock1 dedicator of cytokinesis 1 immune activity lif leukemia inhibitory factor (cholinergic differentiation factor) osm oncostatin m pf4 platelet factor 4 (chemokine (c-x-c motif) ligand 4) 96 tlr2 toll-like receptor 2 ifnk interferon, kappa other dapk1 death-associated protein kinase 1 lats1 lats, large tumor suppressor, homolog 1 (drosophila) pin1 protein (peptidyl-prolyl cis/trans isomerase) nima-interacting 1 pin1l protein (peptidyl-prolyl cis/trans isomerase) nima-interacting 1-like mobk1b mob1, mps one binder kinase activator-like 1b (yeast) sdfr1 stromal cell derived factor receptor 1 c17 cytokine-like protein c17 epor erythropoietin receptor csf2rb colony stimulating factor 2 receptor, beta, low-affinity (granulocyte-macrophage) acvr2b activin a receptor, type iib nrp1 neuropilin 1 pbef1 pre-b-cell colony enhancing factor 1 obrgrp; lepr leptin receptor gene-related protein; leptin receptor tlt4 trem-like transcript 4 loc392255 similar to growth differentiation factor 16 aforementioned molecules upon stimulation with a neuroimmune effecter using microarray, ganglia were incubated at 4 °c in filtered seawater or treated with 1 μm morphine for 18 h. applied biosystems expression array analysis applied biosystems human genome survey arrays were used to analyze the transcriptional profiles of rna samples. the applied biosystems human genome survey array contains 31,700 60mer oligonucleotides probes representing a set of 27, 868 individual human genes and more than 1,000 control probes. sequences used for microarray probe design are from curated transcripts from the celera genomics human genome database (www.celeradiscoverysystem.com), refseq transcripts that have been structurally curated from the locuslink (http://ncbi.nlm.nih.bov/locuslink/refseq.html) public database, high-quality cdna sequences from the mammalian gene collection (mgc) (http://mgc.nci.nih.gov) and transcripts that were experimentally validated at applied biosystems. total rna from 20 m. edulis pedal ganglia was isolated with the rneasy mini kit (qiagen, valencia, ca, usa). the tissue was lysed in 600 µl buffer rlt and homogenized by passing the lysate 5 times through a 20-gauge needle fitted to a 3 ml syringe. the samples were then processed following the manufacturer's detailed instructions. in the final step, the rna was eluted with 50 µl of rnase-free water by centrifugation for 1 min at 10,000 rpm. quality of the rna was analyzed using agilent 2100 bioanalyzer (agilent, santa clara, ca, usa) using the total rna nanochip according to manufacturer’s protocol. digoxigenin-utp labeled crna was generated and linearly amplified from 1 µg of total rna using applied biosystems chemiluminescent rt-ivt labeling kit v 2.0 and manufacturer's protocol. array hybridization, chemiluminescence detection, image acquisition and analysis were performed using applied biosystems chemiluminescence detection kit and applied biosystems 1700 chemiluminescent microarray analyzer following manufacturer's protocol. to each chip, 15 µg of labeled crna targets were hybridized at 55 °c for 19 h. ab1700 expression system software was used to extract assay signal, and assay signal to noise ratio values from the microarray images. to select expressed genes, the gene list was further filtered by removing genes with a signal to noise value less than two. results the previously discovered invertebrate cytokine-like molecules include tumor necrosis factor-like molecules as well as il-1-, il-2-, il-4-, il6and il-10-like molecules. table 1 demonstrates interleukin-like and tumor necrosis factor-like genes among other cytokine-like genes that were significantly expressed as analyzed by the human gene survey microarray (applied biosystems) with a signal to noise value greater than 2 in the untreated m. edulis pedal ganglia tissue. with a signal to noise value greater than 2, all genes expressed are thus considered to have a strong significant presence. tumor necrosis-like factors were present in the untreated tissue. additionally, several interleukin-like molecules also were present. in the morphine treated tissue, however, several additional genes were expressed (table 2). among these genes expressed was il-10, an interleukin-like molecule previously demonstrated in 97 http://www.celeradiscoverysystem.com/ http://ncbi.nlm.nih.bov/locuslink/refseq.html http://mgc.nci.nih.gov/ table 2 interleukin-like, tumor necrosis factor-like, and other cytokine-like genes that were significantly expressed in mytilus edulis pedal ganglia after morphine treatment. genes with a signal to noise ratio greater than 2 as analyzed by the human genome survey microarray (applied biosystems) were listed. tumor necrosis factor (tnf)-like molecules present tnfrsf10a tumor necrosis factor receptor superfamily, member 10a tnfsf12tnfsf13; tnfsf12; tnfsf13 tumor necrosis factor (ligand) superfamily, member 12-member 13; tumor necrosis factor (ligand) superfamily, member 12; tumor necrosis factor (ligand) superfamily, member 13 interleukin-like molecules present il17rb interleukin 17 receptor b il10 interleukin 10 il23a interleukin 23, alpha subunit p19 il5 interleukin 5 (colony-stimulating factor, eosinophil) il31ra interleukin 31 receptor a il18 interleukin 18 (interferon-gamma-inducing factor) il15ra interleukin 15 receptor, alpha additional cytokine-like molecules present cytokinesis and other cell-cycle activity cdc25b cell division cycle 25b anapc5 anaphase promoting complex subunit 5 cdk7 cyclin-dependent kinase 7 (mo15 homolog, xenopus laevis, cdk-activating kinase) cdk4 cyclin-dependent kinase 4 ropn1 ropporin, rhophilin associated protein 1 socs2 suppressor of cytokine signaling 2 pnutl1; gp1bb peanut-like 1 (drosophila); glycoprotein ib (platelet), beta polypeptide spag5 sperm associated antigen 5 anapc4 anaphase promoting complex subunit 4 cdc6 cdc6 cell division cycle 6 homolog (s. cerevisiae) ube2c ubiquitin-conjugating enzyme e2c dock3 dedicator of cytokinesis 3 other cntfr ciliary neurotrophic factor receptor asb9 ankyrin repeat and socs box-containing 9 prei3 preimplantation protein 3 stat2 signal transducer and activator of transcription 2, 113kda gab3 grb2-associated binding protein 3 obrgrp; lepr leptin receptor gene-related protein; leptin receptor asb10 ankyrin repeat and socs box-containing 10 crlf3 cytokine receptor-like factor 3 11-sep septin 11 asb1 ankyrin repeat and socs box-containing 1 ikbkb inhibitor of kappa light polypeptide gene enhancer in b-cells, kinase beta 98 table 3 additional interleukin-like genes expressed in presence of morphine as analyzed by the human genome survey microarray (applied biosystems) with a signal to noise ratio between 1 and 2. gene name gene ontology il1f10 interleukin 1 family member 10 (theta) immune response, interleukin-1 receptor antagonist activity, extracellular space il1f5 interleukin 1 family member 5, delta immune response; interleukin-1 receptor antagonist activity; extracellular space il1rl2 interleukin 1 receptor-like 2 interleukin-1, type i, activating receptor activity, transmembrane receptor activity, integral to membrane il1rl1 interleukin 1 receptor-like 1 signal transduction, transmembrane receptor activity; receptor signaling protein activity;interleukin-1 receptor activity il1f9 interleukin 1 family member 9 cell-cell signaling; immune response; response to pest/pathogen/parasite; interleukin-1 receptor antagonist activity; extracellular space il6st interleukin 6 signal transducer (gp 130, oncostatin m receptor) extracellular space; integral to membrane; protein binding; glycogen metabolic process; positive regulation of cell proliferation; regulation of notch signaling pathway; signal transduction il4 interleukin 4 cholesterol metabolism; regulation of isotype switching; cell proliferation; b-cell differentiation; cellular defense response; t-helper 2 type immune response; connective tissue growth factor biosynthesis; chemotaxis, interleukin4 receptor binding; extracellular space invertebrates (stefano et al., 1999) and tnf-like molecules different from those expressed in the untreated tissue. proinflammatory cytokines and tnf play a major role in inflammation response. the immunosuppressive effect of morphine treatment is demonstrated by a significant presence in expression of the anti-inflammatory il-10-like molecule. additionally, the significant presence of tnf receptor-like molecules indicates the down regulation of proinflammatory tnf-like molecules. the additional newly discovered cytokine-like molecules detected by microarray in both the untreated and morphine treated tissue provide researchers with a multitude of possible subjects for future investigation. given the logarithmic analysis supplied by the spotfire for functional genomics program (spotfire, somerville, maine), any positive signal to noise value indicates gene presence is in greater amounts than background noise. furthermore, the gene sequence of the human transcript on the microarray chip is not identical to the gene sequence of the corresponding m. edulis transcript. however, the array hybridization, as well as the washes, used the same stringency as human nucleic acid assays and we were still able to detect approximately 5000 genes. it is thus important to note the presence of any additional neuro-immune significant interleukinlike signal molecules that were detected upon morphine treatment of m. edulis pedal ganglia tissue (table 3). these interleukin-like genes are directly related to previously discovered interleukin-like molecules in invertebrates. discussion as noted earlier, invertebrate ganglia, immunocytes, and microglia contain il-1and il-6like signaling molecules (beck and habicht, 1986; hughes et al, 1990, 1991a; paemen et al, 1992; stefano, 1992; stefano et al, 1992; hughes and chin, 1994; scharrer et al, 1996). based on these findings, one can surmise that an interleukin-like molecule secreted from these invertebrate cells may have the ability to release dopamine from neurons. recently, sawada and colleagues, as well as others, demonstrated that mammalian il-1 and il-2 and -4 have the ability to alter invertebrate neural ion channels in a stereoselective manner, further strengthening the hypothesis that these immunocyte-derived molecules can alter neural activities as well as stimulate them ( sawada et al., 1991; szucs et al., 1992; franchini et al., 1996; rozsa et al., 1997; kletsas et al., 1998). in previous and current research, measures are taken to confirm gene expression including taqman probes (applied biosystems) and molecular methods including western blotting. the ability of microarray to corroborate with and/or confirm an expanse of previous research is demonstrated in this study of cytokine-like molecules found in m. edulis. given the comprehensive nature of a single microarray chip and the accuracy and precision of the data expressed by these chips, this research indicates that the use of microarray could be independently sufficient for determining gene expression. 99 in summary, it appears that immune-neural communication does occur in invertebrate neural tissues. certainly, the opposite has also been shown, i.e., that neuropeptides can alter and direct invertebrate immune actions (see stefano et al., 1996). this research has been able to confirm such previous findings using microarray technology. acknowledgments this work was supported in part by the following grants: nimh 47392, and ncmhd 001429. kj mantione is supported by the new york state empire innovation program. references beck g, habicht gs. isolation and characterization of a primitive interleukin-1-like protein from an invertebrate, asterias forbesi. proc. natl. acad. sci. usa 83: 7429-7433, 1986. franchini a, kletsas d, ottaviani e. immunocytochemical evidence of pdgfand tgf-β-like molecules in invertebrate and vertebrate immunocytes: an evolutionary approach. histochem. j. 28: 599-605, 1996. hughes tk, chin r. interactions of neuropeptides and cytokines. in: scharrer b, smith em, stefano gb (eds), neuropeptides and immunoregulation, springer-verlag, berlin, pp 101-119, 1994. hughes tk, chin r, smith em, leung mk, stefano gb. similarities of signal 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norepinephrine and octopamine: linking stress and immune function across phyla sa adamo department of psychology, dalhousie university, canada accepted february 8, 2008 abstract in species from three widely divergent phyla (arthropoda, mollusca and chordata) tyrosine derivatives (norepinephrine or octopamine) mediate a response to acute stress. part of this response is a change in immune function that results in a decrease in resistance to pathogens. this decrease in disease resistance appears maladaptive. however, if the connections between norepinephrine/octopamine and immune function were maladaptive, they should have been selected against. none of the four commonly proposed adaptive explanations for acute stress-induced changes in immune function fit the available data for species from all three phyla. however, this result is probably due to the lack of information about acute stress-induced immunosuppression in invertebrates and a lack of ecologically valid studies in vertebrates. understanding why immune function and disease resistance changes during acute stress will require greater comparative study. key words: immunocompetence; immunosuppression; insect; mollusc; vertebrate; adaptive benefits introduction when responding to danger, animals from across the animal kingdom alter their physiology in order to optimize it for the performance of flight-orfight behaviours (wingfield, 2003). this reaction is called the acute stress response. species from at least three diverse phyla (chordata, mollusca and arthropoda) coordinate their acute stress response using chemically similar derivatives of the amino acid tyrosine (ottaviani and franceschi, 1996). vertebrates (cooper et al., 2003) and molluscs (lacoste et al. 2001a) release norepinephrine (ne) during acute stress, while insects release norepinephrine’s chemical cousin, octopamine (o) (orchard et al., 1993). in both vertebrates (charmandari et al., 2005) and invertebrates (roeder, 2005) ne and oa mediate a range of stress related responses. most of these responses prepare the animal for extreme physical exertion (charmandari et al., 2005; roeder, 2005). however, the acute stress response also has complex, but largely immunosuppressive effects in a wide range of animals (adamo and parsons, 2006). the acute stress response can influence immune function ___________________________________________________________________________ corresponding author: shelley a adamo department of psychology dalhousie university halifax, ns, b3h 4j1, canada e-mail: sadamo@dal.ca because immune cells in vertebrates (e.g. webster et al., 2002; madden, 2003), molluscs (lacoste et al., 2001b) and insects (gole et al., 1982; orr et al., 1985) have receptors for ne or oa. the consistent connection between acute stress, ne (vertebrates and molluscs) or oa (insects) and immune function suggests that modulating immune function during acute stress serves an important adaptive function. in this paper, i use a comparative approach to examine four adaptive explanations for the existence of acute stress-induced immunosuppression. two tyrosine derivatives: norepinephrine and octopamine oa and ne are both derived from the amino acid tyrosine, although via different pathways (cooper et al., 2003). oa is synthesized from tyramine, while ne is synthesized from dopamine (fig. 1). oa and ne are identical in structure, except for the number of hydroxyl groups on the benzene ring (fig. 1). molluscs use both ne and oa as a signaling molecule (e.g. ne: sloley et al., 1990, lacoste et al., 2001a, b; oa vehovszky et al., 2005). insects, on the other hand, use oa, but not ne as a signaling molecule (roeder, 1999). vertebrates make extensive use of ne and its metabolite, epinephrine as signaling molecules (cooper et al., 12 mailto:sadamo@dal.ca fig. 1. biosynthetic pathways for norepinephrine and octopamine. adapted from cooper et al. (2003). 2003), but make little, if any, use of oa (roeder, 1999; pflüger and stevenson, 2005; farooqui, 2007). most invertebrate oa receptors have substantial sequence homology with vertebrate adrenergic (e.g. ne) receptors (evans and maqueira, 2005; roeder, 2005). also, oa receptors in invertebrates have similar pharmacological profiles to vertebrate adrenergic receptors (e.g. farooqui, 2007; evans and maqueira, 2005). moreover, pharmcological studies of invertebrate oa receptors demonstrate that they show some affinity for ne (evans and maqueira, 2005). for example, in the aquatic snail lymnaea stagnalis the cloned oa receptor has high affinity for oa, but also exhibits some affinity for ne (gerhardt et al., 1997). similarly, human alpha-adrenergic receptors (subtypes 2a, b, c) have high affinity for ne, but they also show some affinity for oa (gerhardt et al., 1997). the similarity between oa and ne receptors suggests that both had a common origin millions of years ago (pflüger and stevenson, 2005). oa and ne transporters also seem to have had a common origin (caveney et al., 2006). interestingly, molluscs appear to lack both an oa and an ne transporter, even though they contain both compounds. some insects also lack an oa transporter (e.g. drosophila) and must deactivate oa enzymatically (caveney et al., 2006). the chemical similarity between oa and ne, the similarity in the enzymes involved in their synthesis, and the similarities in the sequences of their receptor and transporter molecules support the argument that oa and ne pathways arose from the same ancestral pathway (caveney et al., 2006). both oa and ne play a role in stress adaptation, suggesting that this is an ancient conserved function for these compounds (gerhardt et al., 1997; roeder, 1999). norepinephrine, octopamine and acute stress molluscs (bivalves) react to stressful stimuli by contracting the large muscles that hold the shell closed (moore, 2006). this is the bivalve equivalent of flight-or-fight behaviour. it also results in an increase in ne in the hemolymph (lacoste et al., 2001a). ne is released by chromaffin-like cells in the oyster heart (lacoste et al., 2001a). in vertebrates, the sympathetic nervous system (sns) is activated in response to flight-or-fight situations and releases ne into immune organs (nance and sanders, 2007). all primary and secondary immune organs receive noradrenergic innervation, as do all body surfaces that are potential sites of microbial invasion (e.g. skin, gut or oral mucosa) (nance and sanders, 2007). ne also increases in concentration in the plasma (sachser, 1987; matt et al., 1997). therefore ne can reach the entire vertebrate immune system. in insects, oa is released as a neurohormone during flight-or-fight behaviours (orchard et al., 1997; pflüger and stevenson, 2005; roeder, 2005). oa is released into the periphery by dorsal unpaired medial cells (dum cells) (roeder, 2005). dum cells are considered to be the insect equivalent of the vertebrate sns based on their anatomy and pattern of innervation (evans and maqueira, 2005; roeder, 2005). therefore ne, or its chemical cousin oa, is released by a wide range of animals in response to acute stress. these compounds are widely disseminated allowing ne (charmandari et al., 2005) and oa (orchard et al, 1993; roeder, 2005) 13 to affect the immune system as well as mediating other stress responses. norepinephrine, octopamine and immune function immune cells release ne and it appears to have a paracrine-like function in both molluscs (ottaviani et al., 1993; ottaviani and franceschi, 1996; lacoste et al., 2001b) and vertebrates (flierl et al., 2007). therefore, the role of ne as an immunoregulator may be very ancient (ottaviani and franceschi, 1996). in molluscs, acute stress transiently suppresses immune function and increases susceptibility to bacterial infection (table 1). this increased susceptibility to disease is caused, at least in part, by the release of ne during acute stress. molluscan hemocytes contain receptors for ne (lacoste 2001b), and ne has negative effects on hemocyte function (table 1). injections of ne result in decreased bacterial clearance and increased mortality in oysters challenged with a bacterial pathogen (lacoste et al., 2001c). acute stress results in a transient decline in resistance to bacterial infection in insects as well (crickets, adamo and parsons, 2006). some of this decrease in disease resistance may be mediated by oa. injections of oa prior to a bacterial challenge results in increased mortality (adamo and parson, 2006). however, oa also has immunoenhancing effects (brey, 1994; table 2). it can even increase resistance to infection when the pathogen is coincubated with oa (baines et al., 1992; baines and downer, 1992). as in insects, acute stress in vertebrates results in a mix of immunosuppressive and immunoenhancing effects (dhabhar, 2002; ortega, 2003; gleeson et al., 2004; glaser and kiecoltglaser, 2005; nance and sanders, 2007; ortega et al., 2007). despite this complex mix of positive and negative effects, acute stress increases susceptibility to pathogens (e.g. cao and lawrence, 2002). one bout of intense exercise in mice (e.g. davis et al., 1997) or humans (gleeson et al., 2004) leads to an increased risk of disease and/or mortality in response to a pathogen challenge. ne appears to be causally involved in the increase in disease susceptibility after acute stress (e.g. kohut et al., 1998; cao et al., 2003; emeny et al., 2007). the complexity of the effects of ne on vertebrate immune function has prevented a clear adaptive explanation for these changes (sternberg, 2006). madden (2003), maestroni (2005), and kin and sanders (2006) suggest that these complex effects are a result of ne playing a role in maintaining immune function homeostasis. ne and oa may play a similar role in invertebrates. in molluscs and insects, ne or oa are present in the hemolymph of resting animals. although this could be because it is difficult to take blood from animals without stressing them, it may also indicate that oa and ne are chronically present in the hemolymph. oa has a half-life of 15 min or less in insect hemolymph (goosey and candy, 1982), and, therefore it should not be detectable unless it is constantly being released. a background level of oa or ne in non-stressed animals would be consistent with the hypothesis that these compounds help maintain normal immune function in invertebrates. however, if oa or ne helps maintain immune homeostasis in a variety of animals, why do the levels of oa and ne increase dramatically during acute stress? in other words, how does pushing the ‘immune thermostat’ towards an extreme end benefit animals during acute stress? adaptive function of ne and oa effects on immune function during acute stress in molluscs, insects and vertebrates, the acute stress response results in a brief period during which the animal’s ability to fight off infection is reduced (fig. 2, however see below). ne or oa appear to mediate some of this immunosuppression (fig. 2). this effect of ne or oa on the immune system appears to be maladaptive. increasing susceptibility to disease seems likely to reduce survival and reproductive success, and such a response should be selected against. as dhabhar (2002) has pointed out, during fighting or fleeing, animals run a real risk of injury and, therefore, exposure to pathogens. although it might make good adaptive sense to delay copulation, digestion, and egg laying until the predator has passed, the immune response may not be dispensable during flight-or-fight behaviours because of the increased risk of injury (dhabhar, 2002). nevertheless, the fact that animals from three different phyla exhibit the same apparently maladaptive response suggests that, despite the costs, it provides some benefit. below i review some of the suggestions as to why animals display acute stress-induced immunosuppression. these hypotheses are not mutually exclusive. in particular i explore whether there are any explanations that might fit the evidence from animals across all three phyla. table 1 effects of norepinephrine on molluscan immune functions immune functions effects references susceptibility to bacterial infection ↑ lacoste et al., 2001c phagocytosis at physiological concentrations ↓ lacoste et al., 2002a production of reactive oxygen species induced by interleukin-1 ↓ lacoste et al., 2001d apoptosis ↑ lacoste et al., 2002b 14 i focus on evidence obtained from whole animal studies. immune values taken in vitro are often different when measured in vivo (nance and sanders, 2007). more importantly, as kohut et al. (2005) comments, there is often a lack of association between declines in various immune functions and actual disease resistance (also see adamo, 2004). from an evolutionary perspective, it is the change in disease resistance that is important. 1. the ‘energy crisis’ hypothesis. one common hypothesis for the existence of acute stress-induced immunosuppression is that it allows animals to channel more energy into flight-or-fight behaviour (e.g. see råberg et al., 1998; segerstrom, 2007). however, it is unclear whether immunosuppression would save energy. for example, some mechanisms of immunosuppression, such as apoptosis, require an increase in energy expenditure (dhabhar, 2002). at present there is little direct evidence supporting the ‘energy-crisis’ hypothesis (adamo and parsons, 2006). 2. the ‘resource crunch’ hypothesis. animals make a number of physiological changes in order to make flight-or-fight possible (wingfield, 2003; charmandari et al., 2005). the ‘resource crunch’ hypothesis suggests that some of these changes will result in resources being shifted away from the immune system in order to optimize the flight-orfight response. this hypothesis differs from the ‘energy-crisis’ hypothesis because it is not energy per se that is limiting, but specific molecules that are required for both immunity and some other physiological function. the ‘resource crunch’ hypothesis explains, at least in part, acute stress-induced immunosuppression in insects. in crickets, conflicts between immune function and lipid transport can lead to acute stress-induced immunosuppression (adamo et al., 2008). crickets release oa during flight-or-fight behaviours (adamo et al., 1995). for about an hour after flying or fighting, crickets become more susceptible to bacterial infection (adamo and parsons, 2006). oa, either directly and/or indirectly, induces the mobilization of lipid from the fat body in order to fuel flight-or-fight behaviours (orchard et al., 1993). as lipid levels in the hemolymph increase, the protein apolipophorin iii (apolpiii) changes its confirmation, and combines with high density lipophorin (hdlp) to form low density lipophorin (ldlp) which has an increased lipid carrying capacity (see weers and ryan, 2006, for review). however, in the unlipidated form, apolpiii acts as an immune surveillance molecule (weers and ryan, 2006). once apolpiii becomes part of ldlp, it appears to loose that ability, resulting in a decline in immune surveillance. the decline in immune surveillance probably explains the increase in disease susceptibility after flying and fighting (adamo et al., 2008). therefore, in crickets, intense activity leads to transient immunosuppression because apolpiii is co-opted into lipid transport and becomes unavailable as an immune surveillance molecule. therefore, crickets become immunosuppressed during flight-or-fight even if they have abundant energy stores (adamo et al., 2008). the ability of oa to mobilize lipid explains why oa can produce immunosuppression when injected into crickets. injecting oa results in the release of lipid (woodring et al., 1989), which leads to a decline in the immune surveillance molecule apolpiii as it combines with hdlp to form ldlp (weers and ryan, 2006). but why does oa also have immunoenhancing effects (table 2)? i hypothesize that oa also works to maintain immune system function as some of the components of the immune system are being siphoned off into lipid transport. in other words, oa helps liberate lipid stores (needed to fuel flight-orfight behaviour) while simultaneously reconfiguring the immune system to maintain maximal function under the new physiological conditions. i predict that without the effects of oa on immune function, disease resistance would decline even more precipitously during flying or fighting in crickets. this hypothesis explains why oa can have both immunosuppressive and immunoenhancing effects. why do crickets not make enough apolpiii to support both immune surveillance and increased lipid transport? first, it would be energetically expensive to do so. apolpiii is already a very abundant protein in the hemolymph of many adult insects (weers and ryan, 2006). to produce more of this protein would decrease the energy available for reproduction and other activities. furthermore, as apolpiii concentrations increase, apolpiii may begin to bind to the animal’s own molecules, initiating an inappropriate immune response. such autoimmunity could be costly (e.g. sadd and sivajothy, 2006). therefore, shuttling apolpiii between immune surveillance and lipid transport may be the most adaptive response, even though it results in transient immunosuppression during flying or fighting. table 2 effects of octopamine on insect immune functions immune functions effects references susceptibility to bacterial infection ↑ adamo and parsons, 2006 phagocytosis ↑ baines et al., 1992 nodule formation (insect immune response) ↑ baines et al., 1992 hemocyte locomotion ↑ dielh-jones et al., 1996 hemocyte number at low (physiological) doses ↓ dunphy and downer, 1994 hemocyte number at higher (pharmacological) doses ↑ dunphy and downer, 1994 15 fig. 2. schematic outline of the connections between ne, oa, acute stress and immune function in different phyla. dum cells, dorsal unpaired median cells; ne, norepinephrine; oa, octopamine; sns, sympathetic nervous system. see text for references it is unclear whether a similar scenario can explain acute-stress induced immunosuppression in molluscs. in molluscs, the known effects of ne are all negative (table 1). however, there have been few studies on acute stress-induced immunosuppression in molluscs. more data are needed to assess whether molluscs suffer from a ‘resource crunch’ during acute stress. in vertebrates a number of molecules are shared between the immune system and other physiological systems. for example, lipid metabolism and immune function are also intertwined in vertebrates (e.g. van elzen et al., 2005). mammalian lipoproteins transport lipid (e.g. cholesterol), but they also participate in innate immunity (khovidhunkit et al., 2004). lipid carriers such as very low density lipoprotein (vldl) bind to and neutralize viruses and other pathogen products (khovidhunkit et al., 2004). during infection, vldl levels increase (khovidhunkit et al., 2004). however, after a single bout of intense exercise, the total concentration of vldl particles in the blood declines by 38 % in humans (børsheim et al., 1999). whether changes in mammalian lipoprotein concentrations during intense activity results in acute stress-induced immunosuppression remains unknown. some studies in mammals indirectly support the ‘resource crunch’ hypothesis. for example, despite the immunosuppressive effects of ne, mice that were stressed by minor surgery, and then exposed to infectious agents, were more likely to die from infection if they received ß-adrenoreceptor blockers (schmitz et al., 2007). in another study, mice given ß-adrenoreceptor blockers prior to intense exercise were more likely to die after a viral challenge compared with controls (kohut et al., 2005). these studies suggest that the effects of ne on immune function result in increased disease resistance when they occur within the context of an acute stress response. these results are consistent with the hypothesis that ne works to reconfigure the immune system in order to maintain immune function during a ‘resource crunch’. however, other studies have found that blocking ß1-adrenergic receptors decreased acute stress-induced immunosuppression (cao et al., 2003). emeny et al. (2007) found that mice lacking adrenoreceptors on their immune cells cleared a bacterial infection (listeria monocytogenes) more quickly after acute stress than mice with immune cells capable of responding to ne. however, the relationship between the speed with which an animal can clear bacteria from liver and spleen and its ability to survive an infection was not stated in these studies (cao and lawrence, 2002; cao et al., 2003; emeny et al., 2007). when determining the effects of various drugs on disease resistance, keil et al. (2001) used an ld10 dose of l. monocytogenes and measured the effect on mortality, not on bacterial clearance. in drosophila melanogaster, the ability to clear bacteria from the hemocoel does not correlate with the ability to survive an infection (corby-harris et al., 2007). 3. the ‘over excitation’ hypothesis. acute stress-induced immunosuppression may be beneficial because it prevents the immune system from becoming too active and harming the animal. during intense exercise, tissues such as muscle suffer minor damage, increasing the risk of inflammation and an autoimmune reaction (råberg et al., 1998). therefore, the immune system shifts towards a less inflammatory state, (i.e. a shift from th1 to th2 responses, elenkov and chrousos, 2006). this shift leads to a decrease in inflammation, but also an increased susceptibility to bacterial and viral pathogens. the cost of the increased risk of infection is thought to be less than an autoimmune reaction or damage from an overactive immune response. however, this key assumption remains untested. the ‘over-excitation’ hypothesis does raise the question as to why the prevention of ‘overexcitation’ occurs to the point that animals are left susceptible to bacterial infections during acute stress. it would be more adaptive to prevent autoimmunity and over-inflammation while maintaining normal anti-microbial defenses, unless there is some physiological constraint that makes this impossible. the ‘over-excitation’ hypothesis does fit the available data on molluscs (table 1), but is not supported by the insect data (table 2). in insects, immune cell activity appears to be up-regulated during acute stress (table 2). animals run the risk of having an over-active immune system during both an acute stress response and during an immune challenge. in vertebrates, an immune challenge also activates the acute stress response (elenkov and chrousos, 2006). this indirectly supports the ‘over-excitation’ hypothesis. however, insects release oa during an 16 immune challenge too (dunphy and downer, 1994), although its source is uncertain (adamo, 2005). given that oa appears to increase immune cell activity (table 2), these data do not support the ‘over-excitation’ hypothesis. immunologists should be wary of putting too much confidence in this hypothesis without more supporting data. 4. the ‘shift in focus’ hypothesis. this hypothesis suggests that during flight-or-fight animals are not immunosuppressed per se, but that they have shifted the focus of their immune effort from protecting against systemic invaders, to protecting against opportunistic organisms that might gain entry during wounding (dhabhar, 2002). dhabhar (2002) has shown that acute stress can increase a delayed-type hypersensitivity in rodents and that catecholamines (e.g. ne and epinephrine) play a role in this enhancement. this change could result in increased protection from wound infection (dhabhar, 2002). however, tests with real bacteria have mixed results. restraint stress produced a decrease in wound healing and a decrease in the ability mice to clear bacteria introduced into a wound (rojas et al., 2002). however, the duration of the stress (mice were restrained for 12 h at a time for 8 days) is more typical of a chronic than an acute stress. campisi et al. (2002) found that after a series of tail shocks given over 2 h, acutely stressed rats recovered more quickly than unstressed rats from a subcutaneous injection of a relatively benign bacterium (there was no mortality) (campisi et al., 2002). however, this experiment does not convincingly show that acutely stressed rats are less likely to develop infected wounds. the ‘shift in focus’ hypothesis does not appear to apply to insects. although oa enhances hemolymph clotting in some arthropods (e.g. battelle and kravitz, 1978), in insects, flight-or-fight behaviour results in an increase in infection after wounding (adamo and parsons, 2006). there is no evidence available from the molluscs. conclusions the involvement of ne (or oa) in mediating stress-induced changes in immune function may be ancient (ottaviani and franceschi, 1996). regardless of whether these connections have been conserved over millions of years or have evolved independently in multiple lineages, their existence in animals from different phyla suggests that there is strong selection pressure for a change in immune function in response to acute stress. none of the four suggested adaptive functions reviewed here explains acute stress-induced immunosuppression in all species. in part this is due to a lack of information about acute stress-induced immunosuppression in invertebrates. all of the information presented here rests on a handful of studies. but it is also because the key experiments are missing in vertebrate studies. for example, despite work on the ‘shift in focus’ hypothesis for more than a decade, whether acute stress actually decreases susceptibility to opportunistic wound infections under real world conditions remains unknown. the lack of a real world test of the ‘shift in focus’ hypothesis highlights a general lack of ecological context in these studies. for example, acute stress is typically produced using highly artificial stimuli, such as restraint stress or tail shock. these 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hyperglycaemia and hyperlipaemia in acheta domesticus. j. insect physiol. 35: 613-617, 1989. 19 abstract isj 7: 146-148, 2010 issn 1824-307x visions and perspectives cytokine network in invertebrates: the very next phase of comparative immunology d malagoli department of biology, university of modena and reggio emilia, modena, italy accepted may 20, 2010 abstract information on invertebrate cytokines has been growing impressively in the past five years. however, molecular characterization of newly discovered cytokines has not proportionally improved our understanding of the main reason underpinning their conservation among metazoans. one possible explanation is that cytokines have been conserved for the fundamental processes they control, but in mammals a single cytokine can hardly be considered as controller of complex reactions. in mammals, cytokines constitute a network of communication, and only this network can be considered the real controller of the effects that cytokines exert on immune or developmental functions. in all, the capability of constituting a network could represent the principal reason for cytokine evolution and conservation through diversification of metazoans. key words: cytokines; innate immunity; invertebrates; evolution introduction in mammals, cytokines are described as molecules responsible for the regulation of the maturation, proliferation, differentiation and survival of lymphocytes, macrophages and dendritic cells, and are further classified as lymphokines, monokines, interleukins and chemokines based on their origin and function (corbellini, 2010). among invertebrates (paraphiletic term but useful for the purposes of the manuscript), cytokines are present but much less characterized. no more than 6 years ago, the knowledge on invertebrate cytokines was limited and based on indirect evidence (ottaviani et al., 2004), and the very existence of possible counterparts of vertebrate cytokines was disputed (beschin et al., 2004). recent experiments have introduced dramatic changes in this scenario, so that it could be said now we are moving into the “second age” of invertebrate cytokines. the “second age” of invertebrate cytokines: state of art and future perspectives in the last three years, several reports on invertebrate immunity have used the terms “putative cytokine”, “cytokine-like” etc. (malagoli et al., 2007; parrinello et al., 2008; roberts et al., 2008; zhang et al., 2008; de zoysa et al., 2009, 2010; schikorski et ___________________________________________________________________________ corresponding author: davide malagoli department of biology, university of modena and reggio emilia, via campi 213/d, 41125 modena, italy e-mail: davide.malagoli@unimore.it al., 2009), but the factors that are commonly accepted as full-title invertebrate cytokines at present are spätzle (ferrandon et al., 2004) and unpaired (upd)-3 (agaisse et al., 2003) in drosophila melanogaster, insect chemotactic peptide (icp) in the moth pseudaletia separata (nakatogawa et al., 2009) and astakine-1 in pacifastacus leniusculus (söderhall et al., 2005). these cytokines do not present traits of similarity with vertebrate cytokines, but seem to be widespread in different invertebrate models (an et al., 2010; hsiao et al., 2010), and their receptor and the signalling pathway they elicit appear to be conserved among metazoans (malagoli et al., 2010). interestingly, while the research for cytokine in invertebrates have been prompted mainly by evolutionary-aimed studies (beschin et al., 2004; ottaviani et al., 2004), very few considerations on the evolution of cytokines have been made after the discovery of full-title cytokines. it may appear a paradox, but more speculations on the possible history of cytokines can be realized starting from molecules that at present appear to be candidate cytokines. indeed, some of those molecules, namely drosophila helical factor (dhf) (malagoli et al., 2007), ciona intestinalis tumor necrosis factor (citnf) (parrinello et al., 2008) and hirudo medicinalis endothelial monocyte-activating polypeptide ii (hmemap-ii) (schikorski et al., 2009) promise to give a significant contribution for the understanding of the evolution of cytokines. dhf, or more simply hf as indicated in flybase (www.flybase.org), is a molecule with a predicted structure typical of the vertebrate helical 146 cytokines (huising et al., 2006). the discovery of hf (malagoli et al., 2007) represented a significant contribution supporting the hypothesis that the molecular structure is a component of equal importance to gene and protein sequences in evolutionary studies (malagoli and ottaviani, 2007). citnf is another molecule in which sequence conservation is limited to key region of the molecule (parrinello et al., 2008), and the same is true for hmemap-ii (schikorski et al., 2009). in these respects, it has to be remarked that differently from hf, there is still no information about the activity of citnf and hmemap-ii in ciona and hirudo, respectively, because their discovery has not been followed by a detailed functional analysis, yet. functional assays, based for instance on the utilization of native and recombinant molecules, are however necessary in order to unravel if the structure/sequence conservation corresponds to similar function. however, even among the speculations on the evolution of cytokines, there is a fundamental aspect raised by the discovery of cytokines in invertebrates that has been neglected by comparative immunologists. in their efforts to isolate and characterize cytokines in invertebrates, researchers have been keeping their focus on the specific molecule under study. this led to very detailed and complete characterizations (nakatogawa et al., 2009), but some studies seemed almost concluded with the discovery and characterization themselves. the risk for the next future is to have a long list of new cytokines well-characterized in terms of molecular aspects, but very limited information on their implications for evolutionary biology. the presence of cytokines in organisms with quite different evolutionary histories make it difficult to understand the basis of their conservation. the most obvious explanation is that these molecules are ancient signals that, regulating reactions fundamental for survival and homeostasis maintenance, have been conserved in structure and function. but if we step back to the principle reference of comparative biologists, i.e., mammals and, above all, humans, we can observe that the term "cytokine" indicates a soluble factor that necessarily acts together with many other cytokines in order to determine an effect (corbellini, 2010). in mammals, the importance of cytokines relies principally on their capability of constituting molecular networks, and the functional meaning of a single cytokine is almost absent if we do not consider that molecule acting within a specific molecular context with many other players. accordingly, human immunologists have articulated numerous and complex proposals to describe the interactions between different systems and mediators, e.g., the bow-ties (ottaviani et al., 2008). molecules identified as cytokines have been conserved in diverging metazoan taxa for hundreds million years, and it should be asked if this is due to their capacity to interact and constitute molecular networks, or to function as single signal molecules. the object of natural selection have been cytokines or cytokine networks? admittedly, only in d. melanogaster more than a cytokine has been identified at present, but if we consider the amount of data collected in the past five years and the possibilities offered today by bioinformatic approaches and the high throughput technologies, the discovery of several other invertebrate cytokines appears just a matter of time. now the complete molecular characterization of single factors is a reality in several invertebrate models, the very next phase of comparative immunology must be the identification of networks of cytokines as occurs in vertebrate species. the research for cytokine networks in invertebrate is a topic that directly involve comparative immunologists and evolutionary biologists as well. even though there are several clues on the conservation of structure, receptors and signaling activities of invertebrate cytokines, if these molecules fail to constitute complex networks, should they still be called cytokines? references agaisse h, petersen um, boutros m, mathey-prevot b, perrimon n. signaling role of hemocytes in drosophila jak/stat-dependent response to septic injury. dev. cell 5: 441-450, 2003. an c, jiang h, kanost mr. proteolytic activation and function of the cytokine spätzle in the innate immune response of a lepidopteran insect, manduca sexta. febs j. 277:148-162, 2010. beschin a, bilej m, magez s, lucas r, de baetselier p. functional convergence of invertebrate and vertebrate cytokine-like molecules based on a similar lectin-like activity. prog. mol. subcell. biol. 34:145-163, 2004. corbellini g. immunology: a historical perspective. in: andrea grignolio (ed), immunology today: three historical perspectives under three theoretical horizons, bononia press university, bologna, italy, pp 35-52, 2010. de zoysa m, nikapitiya c, oh c, whang i, lee js, jung sj, et al. molecular evidence for the existence of lipopolysaccharide-induced tnfalpha factor (litaf) and rel/nf-kb pathways in disk abalone (haliotis discus discus). fish shellfish immunol. 28: 754-763, 2010. de zoysa m, jung s, lee j. first molluscan tnfalpha homologue of the tnf superfamily in disk abalone: molecular characterization and expression analysis. fish shellfish immunol. 26: 625-631, 2009. ferrandon d, imler jl, hoffmann ja. sensing infection in drosophila: toll and beyond. semin. immunol. 16: 43-53, 2004. hsiao cy, song yl. a long form of shrimp astakine transcript: molecular cloning, characterization and functional elucidation in promoting hematopoiesis. fish shellfish immunol. 28: 7786, 2010. huising mo, kruiswijk cp, flik g. phylogeny and evolution of class-i helical cytokines. j. endocrinol. 189: 1-25, 2006. malagoli d, conklin d, sacchi s, mandrioli m, ottaviani e. a putative helical cytokine functioning in innate immune signalling in drosophila melanogaster. biochim. biophys. acta 1770: 974-978, 2007. 147 malagoli d, sacchi s, ottaviani e. lectins and cytokines in celomatic invertebrates: two tales with the same end. inv. surv. j. 7: 1-10, 2010. malagoli d, ottaviani e. helical cytokines and invertebrate immunity: a new field of research. scand. j. immunol. 66: 484-485, 2007. nakatogawa s, oda y, kamiya m, kamijima t, aizawa t, clark kd, et al. a novel peptide mediates aggregation and migration of hemocytes from an insect. curr. biol. 19: 779785, 2009. ottaviani e, malagoli d, capri m, franceschi c. ecoimmunology: is there any room for the neuroendocrine system? bioessays 30: 868874, 2008. ottaviani e, malagoli d, franchini a. invertebrate humoral factors: cytokines as mediators of cell survival. prog. mol. subcell. biol. 34:1-25, 2004. parrinello n, vizzini a, arizza v, salerno g, parrinello d, cammarata m, et al. enhanced expression of a cloned and sequenced ciona intestinalis tnfalpha-like (citnf alpha) gene during the lps-induced inflammatory response. cell tissue res. 334: 305-317, 2008. roberts s, gueguen y, de lorgeril j, goetz f. rapid accumulation of an interleukin 17 homolog transcript in crassostrea gigas hemocytes following bacterial exposure. dev. comp. immunol. 32: 1099-1104, 2008. schikorski d, cuvillier-hot v, boidin-wichlacz c, slomianny c, salzet m, tasiemski a. deciphering the immune function and regulation by a tlr of the cytokine emapii in the lesioned central nervous system using a leech model. j. immunol. 183: 7119-7128, 2009. söderhäll i, kim ya, jiravanichpaisal p, lee sy, söderhäll k. an ancient role for a prokineticin domain in invertebrate hematopoiesis. j. immunol. 174: 6153-6160, 2005. zhang x, luan w, jin s, xiang j. a novel tumor necrosis factor ligand superfamily member (cstl) from ciona savignyi: molecular identification and expression analysis. dev. comp. immunol. 32: 1362-1373, 2008. 148 visions and perspectives isj 6: 1-6, 2009 issn 1824-307x visions and perspectives around the word stress: its biological and evolutive implications e ottaviani, d malagoli department of animal biology, university of modena and reggio emilia, modena, italy accepted january 7, 2009 abstract stress is a general adaptive reaction crucial for survival and basically positive that involves the neuroendocrine and the immune systems. in all bilaterian metazoans, the molecular mediators of the stress response, i.e., corticotrophin-releasing hormone, corticotrophin, catecholamines and glucocorticoids, have been preserved during evolution, even if the increased complexity of animals have corresponded to a more articulated stress response that, following the eco-immunology perspective, we speculate to be hierarchically organized along three levels. kew words: stressors; stress response; vertebrates; invertebrates; evolution eustress and distress, not simply “stress” among the general public, the word stress evokes a concept of negativity, which is maintained even among those that have, or should have, knowledge of biology. this situation becomes even more embarrassing when considering that the scientific concept of stress has had the good fortune to become very popular, but at the same time the misfortune to be insufficiently understood. moreover, the use of the term stress in the field of advertising has certainly not clarified its meaning. the present paper aims to provide a correct interpretation of the concept of stress, and especially to emphasize the importance of its positivity, i.e., the role played by stress response in the survival of all animal species on the earth and maintained during evolution. the phenomenon of stress was identified and conceptualized by hans selye who in 1936 published a paper, entitled: "a syndrome produced by different nocuous agents ". before describing the mechanisms of this phenomenon, we should underline some semantic details. stress is fundamentally characterized by two moments and aspects, i.e., the “stimulus” and the “response”. the word stress can indicate both, so creating a possible semantic ambiguity. selye (1978) suggested the word “stressors” (stressogenic ___________________________________________________________________________ corresponding author: enzo ottaviani department of animal biology via campi 213/d, 41100 modena, italy e-mail: enzo.ottaviani@unimore.it agent) to indicate the causal agent, while keeping the word “stress” and “stress response” (response to stress) to indicate the final outcome. moreover, according to selye (1978), the word “stress” has meaning only if related to specific biological situations. regarding the mechanisms of the response to stress, in mammals different organs belonging to the nervous and endocrine systems, such as the hypothalamus, the pituitary and adrenal glands, are involved (selye, 1978). the response triggers physiological processes that operate along two routes. the first is the nervous pathway involving the autonomic nervous system and the medullar portion of adrenal glands leading to the release of catecholamines (ca) (epinephrine and norepinephrine). these molecules provoke a very rapid response, inducing physiological changes, such as the degradation of glycogen to glucose and its increase in the blood, so improving the quality of the life. this situation is further improved with activation of the second track, the endocrine pathway, in which the cortex portion of adrenal glands is involved. schematically, the different stimuli that cause stress induce the release of the corticotrophin-releasing hormone (crh) by the hypothalamus. in turn, the crh provokes corticotrophin (acth) release from the pituitary. this hormone enters the bloodstream and binds specific receptors for acth present on the cells of the cortical portion of the adrenal glands and results in the release of steroid hormones such as glucorticoids (gc). these hormones (cortisol in humans and corticosterone in mice) have different 1 bilatera deuterostomia protostomia mollusca annelida arthropoda echinodermata urochordata cephalochordata vertebrata crh pomc-derived peptides gc ca ? ? ? ? ? ? ? ? fig. 1 presence of the molecules involved in the stress response in the most representative taxa of bilatera. effects; in particular they are involved in regulating the biosynthesis and release of ca. altogether, it emerges that the stress pathway involves molecules in the following order: crh, acth and ca. this complex mechanism that improves the quality of life by means of the release of ca, hormones and steroids is called “eustress” that means beneficial, positive stress. however, stress response must be of short duration. indeed, prolonged exposure to stressors leads to a sustained release of ca and cortisol associated with psychological, functional and pathological symptoms (including bleeding and ulcers) described by selye (1978). this overrun of the stress response is better defined as “distress” that means negative stress. the relationship between neuroendocrine and immune systems stressor and stress response, by one side, and antigens and immune response, on the other, have always been considered as two distinct phenomena, having been discovered and studied separately and, consequently, having become the topics of specific disciplines. however, this division is inconsistent with reality, and the distinction between stressor and antigen or stress and immune response, is to be considered only quantitative and semantic. this dualism was first overcome in experiments undertaken by hugo besedovsky and colleagues (1987). they showed that interleukin (il)-1, a classic mediator of the immune system, is able to activate the hypothalamus-pituitary-adrenal axis. this observation indicates that stressors that induce an immune response (bacteria, viruses, etc.) must also be inserted in the list of the stressogenic agents, suggesting that there is a deep correlation between the immune system and response to stress. edwin blalock and eric smith demonstrated that cells from the immune system, such as lymphocytes and macrophages may play a central role in the induction of stress (blalock and smith, 1985; blalock et al., 1985; blalock, 1989). indeed, lymphocytes and macrophages, well-known producers of cytokines, have also to be considered as neuroendocrine cells being able to synthesize a variety of hormones (i.e., classical molecules produced by the endocrine system) and neuropeptides (i.e., classical molecules produced by the nervous system). furthermore, lymphocytes and macrophages may, in turn, respond to hormones and neuropeptides produced by cells from the neuroendocrine system (blalock and smith, 1985; blalock et al., 1985; blalock, 1989). in summary, various levels of integration between the immune and neuroendocrine systems can be traced: • classical products from the immune system, i.e., cytokines, can act on cells from the neuroendocrine system, modifying the latter's functions; • immune stimuli and hypothalamic releasing factors induce immune cells to synthesize neuropeptides which, in turn, may influence the activity of the neuroendocrine system; • classical hormones and neurotransmitters bind to specific receptors on immune cells and modulate their activity; • cytokines and cytokine-like peptides that are potentially able to modulate immune cell activity are produced by cells from the nervous system. 2 these observations suggest that the three systems (immune, endocrine and nervous) should be considered as anatomically distinct components of a single integrated immuno-neuro-endocrine system involved in the maintenance of the body homeostasis, justifying the conclusion that the response to stress is essential for survival. accordingly, it should be underlined that this interplay between the immune and neuroendocrine systems is not restricted to mammals or other vertebrates, but can be retrieved also in invertebrates (ottaviani and franceschi, 1996), where the molecular cascade of stress response described in the previous paragraph has been observed in immune-competent cells. crh and acth crh has been isolated and characterized by hypothalamic extracts of sheep by vale’s group (1981). later searches showed the presence of crh also in not nervous tissue (seasholtz et al., 2002). a similar picture has been detected in cartilaginous and bony fish as well as in tetrapods, i.e., in all vertebrates (fig. 1) (sato and george, 1973; petrusz et al., 1983; waugh et al., 1985; panzica et al., 1986; roubos, 1997; lovejoy and balment, 1999; summers, 2001; engelsma et al., 2002; seasholtz et al., 2002; malagoli et al., 2004; huising et al., 2005). crh-like molecules were also found in the nervous system of different invertebrate taxa, such as molluscs (sonetti et al., 1986), annelids (rèmy et al., 1982) and insects (verhaert et al., 1984; malagoli et al., 2002), as well as in the immunocytes and hemolymph of molluscs (ottaviani et al., 1990). unfortunately, no data are at present available for echinoderms, urochordates and cephalochordates, that represent the most important invertebrate taxa sharing the deuterostomian lineage with vertebrates (fig. 1). acth is a small peptide enclosed within the pro-opiomelanocortin (pomc) precursor that was initially found in the human pituitary gland (phifer et al., 1974; eberle, 1988). subsequently, acth was also detected in mammalian extra-pituitary areas (ottaviani et al., 1997). as noted above for crh, acth-like molecules were also found in intraand extra-pituitary areas in other vertebrate taxa, namely fish, amphibians, reptiles and birds (fig. 1) (ottaviani et al., 1997; roubos, 1997; engelsma et al., 2002). also different invertebrate taxa (molluscs, annelids, insects, urochordates and cephalochordates) contain immunoreactive acth molecules (ottaviani et al., 1997). no data are available for echinoderms (fig. 1). gc, ca and cytokines in 1985, david norris identified the source of gc, in particular, of cortisol and corticosterone, in the cells of the adrenal cortex of mammals. nonmammalian vertebrates also produce gc (fig. 1) (summers, 2001; engelsma et al., 2002; wada, 2008), but the typical adrenal glands found in mammals are not present in these animals. fish present a group of cells homologue to adrenocortical and chromaffin mammalian tissue, and these two tissues are joined in various ways in tetrapods. the presence of gc-like molecules has also been detected in invertebrates, even if few studies are reported in literature. cortisol immunoreactive molecules were detected in immunocytes from molluscs using an immunocytochemical method (ottaviani et al., 1998), and cortisol and corticosterone have been recorded in the insect calliphora vicina by autoradiography (bidmon and stumpf, 1991). no further data are available for other invertebrate taxa. as far as the presence of ca is concerned, these molecules were detected in all vertebrates (leboulenger et al., 1984; korte et al., 1997; reid et al., 1998; summers, 2001; tsigos and chrousos, 2002). in invertebrates ca were found in molluscs (ottaviani and franceschi, 1996; lacoste et al., 2001; hooper et al., 2007; adamo, 2008), annelids (díaz-miranda et al., 1982; fleming, 1993), arthropods (murdock, 1971; klemm, 1983; adamo, 2008), echinoderms (huet and franquinet, 1981), urochordates (kimura et al., 2003) and cephalochordates (moret et al., 2004). finally, as for ca, cytokines have been observed in all vertebrate lineages (cohen and haynes, 1991; myers et al., 1992; abbas et al., 1994; scapigliati et al., 2000; engelsma et al., 2002; kaiser et al., 2004) and in some invertebrate taxa. with regard the latter, either cytokines or cytokinelike molecules were found in molluscs (ottaviani et al., 2004; de zoysa et al., in press), annelids (ottaviani et al., 2004), arthropods (morisato and anderson, 1994; agaisse et al., 2003; kauppila et al., 2003; söderhäll et al., 2005; ottaviani et al., 2004; lemaitre and hoffmann, 2007; malagoli et al., 2007) and urochordates (parrinello et al., 2008; zhang et al., 2008). a refined orchestra with the same players all the actors that play a role in the stress response must have appeared quite early in animals, since they can be retrieved in different bilaterian lineages (fig. 1). it should be underlined that the cascade of molecular events involved in the stress response is the same in all the bilaterians analyzed so far, i.e., crh, acth and ca. however, since invertebrates lack the organs usually related to vertebrate stress-response, i.e., hypothalamus, pituitary and adrenal glands, it remains to be established how invertebrate stress response can occur in such a simplified scenario. our experiments in molluscs let us to speculate that in less complex organisms the stress response involves the circulating and phagocytic immunocyte endowed with the same molecules that are released and act in the same order described above (ottaviani et al., 1997). however, if the primitive organization of stress response was restricted to single cells, how it came that it has been split up in different organs in vertebrates? in experiments in the catfish ameiurus nebulosus we have observed that fish exposed to lipopolysaccharide (lps) for 15 and 120 min showed an increase in procrh-like molecules in the brain after 15 min but not after 120 min, while the increase in procrh levels in the peripheral organs 3 such as the liver and head kidney persisted for the entire treatment. these findings suggest that stress response is hierarchicallyand time-regulated (malagoli et al., 2004). more precisely, the first and simpler level is the “cell” level by which circulating immunocytes and some cells in various organs, have maintained the capability to resume the stress response. the “cell” level can be taken to represent the persistence of the “ancestral” version of stress response in complex organisms. the second level is the “organ” level, representing a local stress response in which cells distributed within a whole organ are involved. in this case, other organs may not be interested by the stress response that is therefore managed by single components. finally, the third level is the “body” levels, involving different organs connected in a functional net, coordinating the entire system, as it is for the hypothalamuspituitary-adrenal gland axis (ottaviani et al., 1998). this level represent the most complex machinery in stress response, but not necessary its activity is overlapped to that of the other levels (malagoli et al., 2004). it may be surmized that during evolution of vertebrates, while circulating cells maintain their capability of promoting an immune-neuroendocrine response to stressor (“cell level”), some cells were specialized to respond to stressor within organs, thus constituting the “organ” level. the organization of a “system” or “body” level could derive form the constitution of a functional net between organs that were progressively specialized for the intertwined relations between increasingly complex nervous and endocrine systems. this concept of “hierarchy” is in agreement with the fundamental tenet of ecological immunology, i.e., to minimize the cost of biological responses (lochmiller and deerenberg, 2000). in this respect, the “organ” level described above represents a paradigmatic example. fish challenged with lps increased their expression of procrh-like molecules in the brain after 15 min but not after 120 min, while after 120 min the increase in procrh levels persisted in the liver and head kidney. in terms of energy expenditure, we can speculate that it is more convenient for the organism to face the stressor at first also with the “body” level, then, if the stressor does not change its intensity, the stress response is mainly transferred to the periphery and to the “organ” level, thus limiting the involvement of the central nervous system to just the first phase of the stress (malagoli et al., 2004; ottaviani et al., 2008). conclusions in their response to agents that are potentially able to alter their homeostasis and threaten their survival, living organisms exploit a complex and integrated mechanism involving the immuneneuroendocrine system and molecules that have been preserved during evolution, though differently located as a consequence of the increasing complexity of the organisms. stress is a general, adaptive reaction that is crucial for survival and basically positive. most of the negative effects reported in the literature derive from the general perception of stress and refer to 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antifungal activity; antimicrobial peptide; innate immunity; marine invertebrate introduction an increasing number of disease outbreaks have been recorded in marine invertebrates from viral, bacterial and fungal infections, which are largely influenced by environmental conditions, such as pollution and climate warming. perhaps the most well-known example is “coral breaching” which is partly caused by bacterial and fungal infections (mydlarz et al., 2006). immunological mechanisms in marine invertebrates are different from vertebrate immune system; they rely solely on innate immune systems that include both humoral and cellular responses, the former of which is performed by antimicrobial peptides contained in the blood and plasma. cellular immunity also involves antimicrobial peptides secreted into the hemolymph by hemocytes (tincu and taylor, 2004). naturally-occurring peptides are either synthesized by ribosomal machinery from 20 proteinogenic amino acids or by large enzymes and enzyme complexes called nonribosomal peptide synthases (mcintosh et al., 2009). antimicrobial peptides (amps) involved in marine invertebrate immunity are ribosomal peptides (gene-encoded peptides) and classified into: a) linear α-helical peptides, b) peptides with intramolecular disulfide _________________________________________________________________________ corresponding author: nobuhiro fusetani faculty of fisheries sciences hokkaido university, hakodate 041-8611, japan e-mail: anobu@fish.hokudai.ac.jp bridges, c) β-sheet and small proteins, and d) peptides with one or two predominant amino acids (bulet et al., 2004; tincu and taylor, 2004; hancock et al., 2006; jenssen et al., 2006). the majority of amps are amphiphilic and cationic, containing both hydrophilic and hydrophobic surfaces. they show antimicrobial activity by forming pores in microbial membranes or disrupting membranes (yeaman and yount, 2003; brogden, 2005; jenssen et al., 2006). a total of 1,518 amps are listed in the second version of antimicrobial peptide database, among which 442 peptides are antifungal (wang et al., 2009). nonribosomal peptides found in sponges, molluscs and tunicates are composed of unusual amino acids including d-amino acids and contain organic acids in addition to amino acids as cases of depsipeptides. they exhibit a wide range of biological activities, such as antimicrobial, cytotoxic, and enzyme inhibitory. a majority of them are considered to be derived either from microbial symbionts or cyanobacteria on which opisthobranch molluscs prey as mentioned later. surprisingly, a very limited number of peptides found in marine invertebrates have been reported to be antifungal; obviously much more peptides would be antifungal if tested. this review describes only the peptides reported to be antifungal. porifera sponges are the oldest metazoans and share a common ancestor with other metazoans. they 53 fig. 1 structure of discodermin a possess key molecules which are found in mammalian innate and adaptive immune systems (müller et al., 1999). even a marine sponge was reported to produce a perforin-like antibacterial protein (thankur et al., 2003). no antifungal peptides classified as amps have been identified in sponges, although antifungal cyclic peptides of ribosomal origin were reported from marine sponges as mentioned later. instead, sponges contain a large variety of bioactive compounds, including cytotoxc and antimicrobial (blunt et al., 2009), many of which are considered as microbial symbiont origin (piel, 2004). a number of bioactive unusual nonribosomal peptides were also isolated from sponges, some of which are antifungal (fusetani and matsunaga, 1993; matsunaga and fusetani, 2003; blunt et al., 2009). discodermin a (fig. 1), the first bioactive sponge peptide isolated from discodermia kiiensis, contains a large number of d-amino acids and such unusual amino acids as tert-leucine (t-leu), cysteinoic acid (cya), and sarcosine (sar) (fusetani, 1988; li et al., 1998). it possesses a wide range of bioactivities, including inhibition of various enzymes, antifungal (growth inhibition of candida albicans at 20 μg/disk). a number of its congeners were later isolated from various sponges (li et al., 1998). jaspamide (= jasplakinolide) (fig. 2), a cyclic depsipeptide comprising of such unsual amino acids as n-methyl-2-bromo-d-tryptophan (me-brtrp) and l-β-tyrosine (l-βtyr) isolated from fijian sponges of the genus jaspis, was fungicidal against c. albicans with both an mic and a minimal lethal concentration of 25 μg/ml (scott et al., 1988). it is known to promote actin polymerization (bubb et al., 1994). similar peptides have been reported from various sponges (li et al., 1998; molinski, 2004). marine sponges of the genus theonella are prolific in bioactive metabolites possessing unusual structures (bewley and faulkner, 1998). theonellamide f (fig. 3) is a bicyclic peptide isolated from a japanese theonella sp. containing several unusual amino acids, e.g. histidinoalanine, 3-methyl-p-bromophenylalanine, (2s,4r)-2-amino-4-hydroxyadipic acid (l-ahad), and (3s,4s,5e,7e)-3-amino-4-hydroxy-6-methyl-8 (p-bromophenyl)-5,7-octadienoic acid (aboa). it inhibited the growth of c. albicans with an mic 6.3 μg/ml (fusetani and matsunaga, 1993; li et al., 1998). a glycosylated peptide of the theonellamide family named theonegramide (fig. 4) with antifungal activity against c. albicans at 10 μg/disk was reported from t. swinhoei collected from palau (bewley and faulkner, 1996). fig. 2 structure of jaspamide 54 fig. 3 structure of theonellamide f fig. 4 structure of theonegramide cyclolithistide a (fig. 5) is another class of cyclic peptide isolated from a papua new guinean collection of t. swinhoei and contains unusual amino acids, including 4-chloroisoleucine (cl-ile), 2-amino-pentanoic acid (d-ape), and 4-amino-3,5-dihydroxyhexanoic acid (adha). it showed antifungal activity at 20 μg/disk (clark et al., 1998). microsclerodermin a (fig. 6), a highly unusual cyclic hexapeptide isolated from a palauan thenonella sp., contains new amino acids, e.g., (2s,3r,4s,5s,6s,11e)-3-amino-6-methyl-12-(p-me thoxyphenyl)2,4,5-trihydroxydodec-11-enoic acid (ammtd), (3r)-4-amino-3-hydroxylbutyric acid (gabob), and 3-hydroxy-4-amino-5-vinylpyrrolidone. it inhibited the growth of c. albicans at 1.5 μg/disk (bewley et al., 1994). several congeners have been reported from lithistid sponges (molinski, 2004). these peptides were suggested to be produced by a new δ-proteobacterium found in theonella sp. (bewley and faulkner, 1998). 55 fig. 5 structure of cyclolithistide a fig. 6 structure of microsclerodermin a callipeltin a (fig. 7) was originally isolated as an anti-hiv cyclodepsipeptide comprising of many unusual amino acids including (2r,3r,4s)-4-amino-7-guanidino-2,3-dihydroxyhep tanoic acid (agdhe) from a new caledonian lithistid sponge callipelta sp. it exhibited a 30 mm inhibitory zone at 100 μg/disk (zampella et al., 1996). more sponge nonribosomal peptides were reported to be antifungal (li et al., 1998; molinski 2004; blunt et al., 2009), but modes of antifungal activity of these nonribosomal peptides mentioned above have not been fully elucidated. antifungal ribosomal peptides have not been isolated from marine sponges, except for hymenamides, pro-rich cyclic heptapeptides isolated from hymeniacidon sp. collected in okinawa (kobayashi et al., 1993). hymenamide a (fig. 8) was antifungal against c. albicans with an mic 33 μg/ml as well as cytotoxic. cnidaria although antifungal activity has been detected in some gorgonian species, no antifungal peptides have been isolated. however, a cytotoxic pentapeptide named gymnangiamide (fig. 9) similar to dolastatin 10 (fig.10), a highly antifungal 56 fig. 7 structure of callipeltin a peptide isolated from the sea hare dolabella auricularia mentioned later was reported from the marine hydroid gymnangium regae, though no antifungal activity was described (milanowski et al., 2004). this peptide contains o-desmethyldolaproline (ddap), n-desmethyldolaisoleucine (ddil), l-threo-phenylserine (l-pser), and l-guanidinoserine (gser). perhaps this peptide was originated from symbiotic or sequestered cyanobacteria as the case of dolastatins. a 40-residue amp named aurelin isolated from the jellyfish aurelia aurita exhibited structural features of defensins and channel-blocking toxins of sea anemone origin, but no antifungal activity was reported (ovchinnikova et al., 2006). mollusca in molluscs, hemocytes are predominantly responsible for innate immune defense and release amps (bulet et al., 2004; tincu and taylor, 2004). amps have been reported from bivalves and opisthobrachs as shown in table 1; defensins were identified in hemocytes of the mussel mytilus galloprovincialis (mgd-1 and -2) (mitta et al., 1999b) and in the mantle tissue of the oyster crassostrea gigas (cg-def) (gueguen et al., 2006; gonzalez et al., 2007), respectively. mgds and cg-def inhibited growth of the fungus fusarium oxysporum with an mci value of 4.5-9 μm, respectively. the solution structure of mgp-1 obtained using 1h nmr spectroscopy consists of a helical part and two antiparallel β-strands. the cys-stabilized α-β motif is stabilized by 4 disulfide bridges (yang et al., 2000). similar antibacterial defensins a and b were isolated from hemocytes of m. edulis, but their antifungal activity has not been reported (charlet et al., 1996). defensin a and mgp-1 share some common properties in distribution of hydrophobic and hydrophilic side chains (yang et al, 2000). molluscan defensins are composed of a β-sheet and 3 disulfide bonds, which is similar to arthropod defensins. interestingly, an amp coined cg-prp (37-residue peptide) remarkably enhanced the antifungal activity of cg-def, although it is not antifungal (gueguen et al., 2009). fig. 8 structure of hymenamide a 57 fig. 9 structure of gymnangiamide fig. 10 structure of dolastatin 10 ap, a polyproline-type amp (47 redidues) isolated from the chilean scallop argopecten purpuratus, showed antifungal activity against f. oxisporum and saprolegnia parasitica with ic50 values of 2.1 and 0.85 μm, respectively (arenas et al., 2009). it is also highly antibacterial; perhaps ap enters in lipid bilayer to exhibit antifungal activity. a big defensin named aibd of the scallop a. irradians, similar to arthropod big defensins, has been cloned and expressed; the recombinant aibd (120 residues) was reportedly not only highly antibacterial, but also strongly fungicidal, though detailed fungicidal activity was not available (zhao et al., 2007). a novel argand cys-rich amp named myticin b (40 amino acids) (table 1) isolated from hemocytes of m. galloprovincialis showed antifungal activity against f. oxysporum with mic 5-10 μm (mitta et al., 1999a). interstingly, myticin a possessing a similar amino acid sequence to that of myticin b was not antifungal at 20 μm. mytilin b, a 34-residue amp containing 4 intramolecular disulfide bonds, purified from hemocytes of the same species exhibited antifungal activity against f. oxysporum with mic 0.7-1.4 μm (mitta et al., 2000). mytimycin, a novel antifungal cys-rich polypeptide of 6.2 kda that hindered the growth of fungi, was isolated and partially characterized from m. edulis (charlet et al., 1996). opisthobranch molluscs often sequester bioactive peptides from their prey organisms, especially cyanobacteria and seaweeds (cimino and ghiselin, 2001). dolastatins are highly cytotoxic linear peptides of nrps metabolites sequested from cyanobacteria of the genus lyngbya by the sea hare dolabella auricularia (garson, 2001), among which dolastatin 10, a highly unusual pentapeptide comprising of new amino acids, (2r,3r,4s)-dolaproline (dap), (3r,4s,5s)-dolaisoleucine (dil), l-dolapherine (doe), and l-dolavaline (dov), was shown to be highly antifungal against cryptococcus neoformans with mic 0.37 μg/ml, but not against other fungi (pettit et al., 1998). dolastatin 10 is a potent inhibitor of tubulin polymerization. similarly, kahalalide f (fig. 11), an unusual cyclic depsipeptide containing (z)-2-amino-2-dehydrobutyric acid (z-dhb) and l-ornithine (l-orn) accumulated by the hawaiian sacoglossan elysia rufescens from the green alga bryopsis pennata is highly cytotoxic as well as strongly antifungal against c. albicans, c. neoformans and aspergillus fumigatus with mic 5-10 μm (shilabin et al., 2007). kahalalide f is currently under phase ii clinical trials as anticancer drugs. dolabellin b2, a 33-residue amp (table 1) isolated from the body wall of the sea hare d. auricularia, is fungicidal against s. cerevisiae (ic50~25 μg/ml), while it is fungistatic agaist c. albicans (iijima et al., 2003). 58 fig. 11 structure of kahalalide f annelida and uchiura marine worms dwell in sediments, indicating the requirement of antimicrobial strategy for their survival. amps have been isolated from polychaete and echiuroid worms. arenicin-1 and -2 are 21-residue amps isolated from coelomoycytes of the polychaete arenicola marina (table 2) and show no structural similarity to any amps reported (ovchinnikova et al., 2004). arenicins are cationic peptides having two antiparallel β-strands and one disulfide bond between cys3 and cys20 (ovchinnikova et al., 2007). arenicin-1 showed antifungal activity against c. albicans, c. parasilosis, malasseria furfur, trichosporon beigelli and trichophyton rubrum with mics of 4.5-9 μm comparable to that of mellitin by disrupting fungal phospholipid membranes (park and lee, 2009). it is also antibacterial and hemolytic, which may be interpreted by its permeabilization of model membranes composed of phospholipids or lipopolysaccharides (andrä et al., 2009). perinerin, a 51 residue amp isolated from homogenates of the polychaete perinereis aibuhitensis, is a highly cationic, hydrophobic peptide that is not related to any known amps (pan et al., 2004). it was antifungal against paecilomyces heliothis with mics 12.5-25 μg/ml, in addition to bactericidal activity against gram-negative and –positive bacteria. some neuropeptides show potent antimicrobial activity, which is suggested to be involved in innate immunity (brogden et al., 2005). in fact, urechistachynins i and ii, five residue neuropeptides found in the echiuroid urechis unicinctus exhibit antibacterial and antifungal activities (mics 42 and 25 μm against c. albicans, respectively) (sung et al., 2008). it was suggested that the plasma membrane of fungi is structurally disrupted by urechistachynins. arthropoda amps play a major role in innate immunity of crustaceans and horseshoe crabs. penaeidins, a family of amps of 47-63 residues, were initially characterized from hemocytes of the shrimp litopenaeus vannamei (destoumieux et al., 1997) and later found to be expressed in all penaeid shrimps (bachère et al., 2000; destoumieux et al., 2000; cuthbertson et al., 2004: gueguen et al., 2006). they are composed of a pro-rich n-terminal domain, followed by a c-terminal domain stabilized by 3 intramolecular disulfide bonds, which is unique among amps (table 3). penaeidins exhibit not only antibacterial activity against gram-positive bacteria, but also antifungal activity against various filamentous fungi, but not against yeasts (e.g. mic 5-10 μm against f. oxysporum that is pathogenic to shrimps) (destoumieux et al., 1999; bachère et al., 2000). the solution structure of penaeidin 3 (litvan pen3-1) demonstrated that the surface of the cys-rich domain exhibits an amphipathic character required for antimicrobial properties (yang et al., 2003). a similar result was reported for penaeidin 4 (litset pen4-1) (cuthbertson et al., 2005). the penaeidin database, penbase (www.penbase.immunaqua.com), has been constructed; detailed information of 34 penaeidins is contained at moment (gueguen et al., 2006). they are classified into three subgroups based on amino acid sequences: penaeidin 2 (pen2), penaeidin 3 (pen3) and penaeidin 4 (pen4) (culthbertson et al., 2004). actually, penbase 59 http://www.penbase.immunaqua.com/ table 1 amino acid sequences of molluscan antifungal peptides mgd-1: gfgcpnnyqchrhcksipgrcggycggwhrlrctcyrcg mgd-2: gfgcpnnyachqhcksirgrcggycagwfrlrctcyrcg defensin a: gfgcpndypcksipgrxggycggxhrlrctcyr defensin b: gfgcpndypcksipgryggycggxhrlrctc cg-def: gfgcpgnqlkcnnhcksiscragycdaatlwlrctctdcngkk mytilin a: gcasrckakcagrrckgwasasfrgrcyckcfrc mytilin b: scasrckghcrarrcgyyvsvlyrgrcyckclrc myticinb: hphyctsyycskfcgtagcrrygcrnlhrgklcfclhcsrv ap: tympveegeyivnisyadqpkknspftakkqpgpkvdlsgvkaygpg dolabellanin b2: shqdcyealhkcmashskpfscsmkfhmclqqq each disulfide pair is highlighted in red, pink, green and light blue for mgds. cys residues are highlighted in red for the rest of peptides, while arg in violet for mytilins and myticin and pro in blue for ap. x in defensins is a not identified residue. contains more than 200 entries of penaeidins and the number is growing rapidly due to the active genomic and proteomic research. crustins are cationic, cys-rich antibacterial polypeptides of ca. 7-14 kd occurring in circulating hemocytes of crustaceans that contain a whey acidic protein (wap) domain in the c-terminus (smith et al., 2008). more than 50 crustin sequences have been reported from a variety of decapods and classified into 3 subgroups, type i to iii. no crustins had been reported to be antifungal until recently when crupc of 98 residues belonging to type ii and cruha1/2 of 90 residues (type i) were characterized from the red king crab paralithodes camtschaticus and the spider crab hyas araneus, respectively (sperstad et al., 2009). cruha1 inhibited the growth of s. cerevisiae with mic 12.5-25 μm. hemocytes of h. araneus also contain hyastatin, a gly-rich multi-domain polypeptide of 11.7 kd resembling type ii curstins (sperstad et al., 2009). it exhibited mics of 12.5 and 6.3-12.5 μm against s. cerevisiae and c. albicans, respectively. horseshoe crabs rely completely on innate immune system which is the first line of inducible host defense against bacterial, fungal and viral pathogens (iwanaga and lee, 2005). quite recently, an excellent review on the molecular basis for innate immune system of horseshoe crabs has been published in this journal (kawabata et al., 2009). the most well-studied species is tachypleaus tridentatus (japanese horseshoe crab); its hemolymph contains a variety of soluble defense molecules, e.g. hemocyanin, lectins/c-reactive proteins and α2-macroglobulin, in addition to granular hemocytes that comprise 99 % of total hemocytes. granular hemocytes consist of large and small granules which are sensitive to lipopolysaccharides (lps) of gram-negative bacteria and secret defense substances by stimulation of lps. small granules contain various amps, including tachyplesins, tachycitin, tachystatins and big defensin (table 4), while large granules release enzymes, lectins and proteins involved in hemolymph coagulation (iwanaga et al., 1998; 2005; kawabata et al., 2009). all amps isolated from horseshoe crabs showed binding activity to chitin, a primary target of the innate immune system. tachyplesins i and ii isolated from t. tridentatus are composed of 17 amino acids and contain 2 intramolecular disulfide bonds as shown in table 4 (nakamura et al., 1988; 60 table 2 amino acid sequences of antifungal peptides retrieved in worms arenicin 1: rwcvyayvrvrgvlvryrrcw arenicin 2: rwcvyayvrirgvlvryrrcw perinerin: fnklkqgsskrtcakcfrkimpsvhelderrrganrwaagfrkcvssicry urechistachykinin i: lrqsqfvgsr-nh2 urechistachykinin ii: aagmgffgar-nh2 table 3 amino acid sequences of penaeidins from the shrimp lytopenaeus vannamei litvan pen2-1 (pen2): eayrggytgpiprpppigrppfrpvcnacyrlsvsdarnccikfgscchlvkg litvan pen3-1 (pen3): qvykggytrpiprpppfvrplpggpigpyngcpvscrgisfsqrsccsrlgrcchvgkgysg litvan pen4-1 (pen4): hssgytrplpkpsrpifirpigcdvcygipsstarlccfrygdcchrg each disulfide pair is highlighted in red, green and light blue; pro residues are in pink. miyata et al., 1989). similar 18-residue amps named polyphemusins i and ii were isolated from the american horseshoe crab limulus polyphemus (miyata et al., 1989). these four amps showed similar antimicrobial activity against gram-negative and –positive bacteria as well as fungi (ic50 0.2 μg/ml against c. albicans)(osaki et al., 1999). a big defensin consisting of 79 amino acids was isolated from t. tridentatus hemocytes (saito et al., 1995). it is similar to rat defensins and showed potent antimicrobial activity against bacteria, but weak activity against fungi (ic50 20 μg/ml against c. albicans)(osaki et al., 1999). tachystatin a (44 residues), b (42) and c (41) isolated from t. tridentatus exhibited potent antifungal activity against c. albicans and p. pastoris with ic50 values of 0.9-3.0 and 0.1-0.3 μg/ml, respectively (osaki et al., 1999). tachystatins a and b showed sequence similarity to ω-agatoxin-iva of a funnel web venom, but tachystatin c, which is no significant sequence similarity to the former two, is similar to insecticidal spider neurotoxins. the solution structure of tachystatin a analyzed by nmr spectroscopy showed an amphiphilic folding found in membrane-interactive peptides (fujitani et al., 2002). tachystatins contain three disulfide bridges as shown in table 4. t. tridentatus hemocytes contain another amp named tachycitin which consists of 73 amino acids and five intramolecular disulfides linkages, but showed no significant sequence similarity to known amps (kawabata et al., 1996). it showed weak antimicrobial activity against antibacterial and fungi (ic50 52 μg/ml against c. albicans).. it should be noted that the apparent antimicrobial activity of horseshoe crab apms are significantly reduced under isotonic conditions (0.5 m nacl for horseshoe crabs) to those under hypotonic conditions (kawabata et al., 2009). echinodermata and urochordata starfishes and sea cucumbers are known to contain antifungal saponins named asterosaponins and holothurins, respectively (fusetani and kem, 2009). only a small number of works have been done on amps in echinoderms, although antibacterial activity of their coelomocytes were reported; cationic, defensin-like amps have been 61 table 4 amino acid sequences of antifungal peptides from horseshoe crabs tachyplesin i: kwcfrvcyrgicyrrcr-nh2 tachyplesin ii rwcfrvcyrgicyrkcr-nh2 polyphemusin i: rrwcfrvcyrgfcyrkcr-nh2 polyphemusin ii: rrwcfrvcykgfcyrkcr-nh2 tachystatin a: ysrcqlqgfncvvrsyglptipccrgltcrsyfpgstygrcqry tachystatinb1: yvsclfrgarcrvysgrsccfgyycrrdfpgsifgtcsrrnf tachystatin c: dydwslrgppkcatygqkcrtwsprnccwnlrckafrcrpr tachycitin: ylafrcgryspclddgpnvnlysccsfynchkclarlencpkglhynaylkvcdwpskagct each sulfide bond is highlighted in red, light blue and green table 5 amino acid sequences of antifungal peptides from tunicates clavanin a: vfqflgkiihhvgnfvhgfshvf-nh2 clavanin b: vfqflgriihhvgnfvhgfshvf-nh2 clavanin c: vfhllgkiihhvgnfvygfshvfnh2 clavanin d: afkllgriihhvgnfvhgfshvf-nh2 clavaspirin: flrfigsvihgighlvhhigval-nh2 recently reported from a sea urchin, but no information about its antifungal activity is available (li et al., 2008). tunicates (ascidians) often contain cytotoxic cyclic peptides, most of which are derived by nonribosaomal peptide synthases, but their antifungal activity have not been examined (blunt et al., 2009). an unusual diketopiperazine named etzionin (fig. 12) reported from a unidentified red sea tunicate inhibited the growth of c. albicans and aspergillus nidulans with mic of 3-12.5 μg/ml (hirsch et al., 1989). tunicates employ a prototype of vertebrate innate immune system for host defense; in fact, ciona intertinalis hemocytes are shown to express a number of host defense-related genes involved in innate immune systems (shida et al., 2003). halocyamines was the first ascidian-derived amps isolated from halocynthia roretzi which are post-translationally modified ribosomal peptides. halocyamine a (fig. 12) showed weak antifungal activity against cryptococcus neoformans with an mic of 100 μg/ml (azumi et al., 1990). styelin d also contains post-translationally modified residues but its antifungal activity is unknown (lehrer et al., 2003). clavanins a-d, α-helical amps of 23 residues identified in the solitary tunicate styela clava (table 5), showed antifungal activity against c. albicans with mic 5-20 μg/ml, in addition to antibacterial activity (lee et al., 1997). a his-rich, 23-residue apm named clavaspirin was identified in pharyngeal tissues of the same species (lee et al., 2001). a synthetic peptide of clavaspirin was antibacterial against gram-positive and –negative bacteria as well as antifungal against c. albicans 62 fig. 12 structure of etzionin and halocyamine a with mic ~5-10 μg/ml. it is highly hemolytic and cytotoxic; α-helical nature of the peptide may responsible for these activities. recently, search for amps using expressed sequence tag (est) database has been attempted for the tunicate ciona intestinalis on which the genome project was completed, which resulted in identification of gene families coding novel amps of α-helical types ( fedders and leippe, 2008; fedders et al., 2008). a synthetic peptide of c-terminal region consisting 24 amino acids of a putative amp coded ci-mam-a showed potent antifungal activity against c. albicans with mic 3.1 μm, in addition to potent antibacterial activity (fedders et al., 2008). importantly, its antibacterial activity was retained at high nacl concentrations (up to 450 mm). antimicrobial activity and mode of action of the intact peptide are interesting subjects. conclusion a large array of invertebrates live in marine environments harboring high concentrations of pathogenic microorganisms. amps are considered to be a major component of the innate immune defense on which marine invertebrates solely rely (tincu and taylor, 2004). therefore, it is surprising that amps have been explored for only limited phyla, mainly mollusca, arthropoda and urochordata. innate immune systems in marine invertebrates which are increasingly important, since emerging numbers of diseases reported for marine invertebrates partly due to seawater warming and water pollution. for better understanding of marine ecosystem, more knowledge should be accumulated for amps in a wide range of marine phyla. it is expected that more and more amps will be identified in a wide variety of marine invertebrates by 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and reggio emilia, modena, italy accepted july 9, 2007 abstract the immunocytochemical study performed on the annelid enchytraeus japonensis revealed the presence of immunoreactive heat shock protein (hsp)27 molecules in different areas of the body. positivity was observed in coelomocytes and in epithelial cells of the intestine wall. the exposition of the animals to 400 μt magnetic fields (50 hz) for 30 min provoked an increased immunoreactivity in some specimens, but after immunoblot experiments, no significant differences in the total content of hsp27-like molecules were found between exposed and non-exposed animals. key words: enchytraeus japonensis; hsp27; immunocytochemistry; extremely low frequency magnetic field introduction from the literature, it emerges that invertebrates are a suitable model to study the effects of extremely low frequency magnetic fields (elf-mf). after the pioneering work performed by goodman’s group (1976) on the slime mold physarum polycephalum, studies were subsequently carried out on the insects sciara coprophila and drosophila melanogaster, the molluscs cepea nemoralis and mytilus galloprovincialis and the nematode caenorhabditis elegans (kavaliers et al., 1991; goodman et al., 1995; junkersdorf et al., 2000; miyakawa et al., 2001; ottaviani et al., 2002; gobba et al., 2003; malagoli et al., 2003, 2004, 2006). mfs are able to influence a variety of biological systems (goodman et al, 1995; del re et al., 2006; malagoli et al., 2006; bernardini et al., 2007), and several investigations have focused on the alterations provoked by mfs on the membrane ion channels and on the expression of heat shock proteins (hsps). regarding ion channels in invertebrates, we have demonstrated that the exposure of the mussel m. galloprovincialis to 50 hz mfs ranging from 200 to 1000 μt disturbs the reactivity of circulating cells (immunocytes) towards ___________________________________________________________________________ corresponding author: davide malagoli department of animal biology via campi 213/d 41100 modena, italy e-mail: malagoli.davide@unimore.it n-formyl-meth-leu-phe (fmlp) by altering potassium and calcium channel permeability (ottaviani et al., 2002; gobba et al., 2003). also in the mollusc c. nemoralis, mfs provoke an alteration in calcium channel function (kavaliers et al., 1991). as other physiological stressors, mfs can provoke the expression of highly conserved genes coding for hsps (lindquist, 1986; goodman and blank, 1998). in the nematode c. elegans, exposure to mfs induces the expression of the hsp70 (goodman and blank, 1998) and the hsp16 genes (miyakawa et al., 2001), while an augmented expression of hsp70 and hsp90 was observed in the mussel m. galloprovincialis after repeated exposure to 400 and 600 μt mf (malagoli et al., 2004). in order to increase our knowledge of the possible effects of elf-mf on the small hsps in invertebrates, the present paper investigates the presence and expression of hsp27-like molecules in the annelid enchytraeus japonensis following exposure to a 400 μt intensity elf-mf. materials and methods animals prior to experimental procedures, adult samples of enchytraeus japonensis (annelida, oligochaeta, enchytraeidae) were grown on agar medium for 1 month, as previously described in detail for enchytraeus crypticus (franchini and marchetti, 2006). 82 mailto:malagoli.davide@unimore.it fig. 1 hsp27-like molecules in coelomocytes (arrows) (a) and in epithelial cells (arrows) of the intestine wall (b) of e. japonensis. nuclei are counterstained with hematoxylin. bar = 10 μm. exposure of animals to mfs each experiment was performed by placing 10 specimens for 2 h under a 50 hz mf generated by two pvc coated coils (10x10 cm, 1400 windings, 0.2 mm ∅ of copper wire) (igea, carpi, mo, italy) mounted horizontally 13 cm apart. the sinusoidal elf mf intensity was controlled by an electronic power source (california instruments, usa) connected to a computer and regulated by the “pgui32” ac source control program (california instruments, usa). elf mf intensity and the temperature between the coils were constantly checked using a “7010 gauss/teslameter” (f.w. bell, usa). mfs of 400 μt were generated. shamexposed animals were maintained under the coils for the same time as the treated specimens, but in the absence of any current. all experiments were performed at room temperature. after 4 h of recovery, some animals were immediately fixed in bouin’s mixture and embedded in agar/paraffin, following franchini and marchetti (2006), while others were sacrificed for western blot analysis. hematoxylin-eosin stain and immunocytochemical reactions were performed on 7 µm longitudinal sections. immunocytochemical procedure the immunocytochemical reaction was performed using avidin-biotin-peroxidase complex, as described in detail elsewhere (franchini and marchetti, 2006). anti-hsp27 polyclonal antibody (pab) (1:500) (santa cruz, ca, usa) was used as the primary antibody. negative control experiments were performed by either omitting the primary antibody or substituting it with non-immune serum. western blot analysis for hsp27 western blot analysis was performed on animals exposed to mfs and on sham-exposed animals (controls). immediately after the exposure to mfs, the animals were frozen at -80 °c for 30 min, then re-suspended in 95 μl of sample buffer (12.5 % 0.5 m tris-hcl ph 6.8, 10 % glycerol, 2 % sds, 0.5 % 2-mercaptoethanol, 0.025 % bromophenol blue) and boiled for 4 min at 1200 rpm in a thermomixer (eppendorf, germany). whole lysates were centrifuged at 13000xg at 4 °c for 30 min, the supernatant was collected, and the protein content quantified following bradford’s method (1976). protein extracts were separated by 12 % sds-polyacrylamide gel electrophoresis (laemmli, 1970) and electrophoretically transferred onto pvdf membranes (0.2-μm pore size). western blots were performed using anti-hsp27 pab (1:500) (santa cruz) and anti-β-tubulin monoclonal antibody (mab) (1:1000) (sigma, mo, usa) as primary antibodies. immunoreactive bands were visualized using a nbt/bcip detection system. densitometric analysis immunoblots were acquired using “gel doc xr” and digitally evaluated with the “quantity one” software (bio rad lab., milano, italy) and “imagej 1.32j” (wayne rasband, national institute of health, usa). statistical analysis statistical analysis of densitometric values was performed by anova. results and discussion the pot worm e. japonensis has normally been used as a model to study the mechanisms of annelid regeneration (myohara et al., 1999, 2006). in the present research, we sought to verify if the animal could also be used as an indicator for ecotoxicological stress, particularly in areas subjected to mf irradiation. previously, our and others laboratories have found interesting invertebrate models from different habitats that are very sensitive to mfs and hsp expression as a result of mf irradiation (lindquist, 1986; goodman and blank, 1998; miyakawa et al., 2001; malagoli et al., 2004). the majority of these investigations examined hsp70 and 90, which are typically related to stress-response (morimoto et al., 1997), but did not evaluate the effects of elf-mf on the small hsps, including hsp27. however, relationships between elf-mf and hsp27 could be of some interest in considering the role of these proteins in cytoskeletal dynamics (dalle donne et al., 2001; mounier and arrigo, 2002), as well as with regards to the suggested involvement of some cytoskeletal components in determining elf-mf effects (gartzke and lange, 2002). as far as the small hsps in 83 fig. 2 pot worm exposed for 2 h to 400 μt elf-mf shows an increased immunoreactivity to anti-hsp27 pab (b) with respect to control (a). nuclei are counterstained with hematoxylin. bar = 20 μm. invertebrates are concerned, miyakawa et al. (2001) demonstrated the presence of the hsp16 gene in c. elegans. working with d. melanogaster, tanguay’s group (2006) found that the family of small hsps is composed of 4 components: hsp22, 23, 26 and 27, localized in different cell compartments, i.e. hsp22 in the mitochondria, hsp23 and hsp26 in the cytosol and hsp27 in the nucleus. in e. japonensis, the hsp27-like molecules are distributed in the cytoplasm of coelomocytes and in epithelial cells of the intestine wall (fig. 1). even if in some specimens exposed to mfs, a higher immunoreactivity was detected compared to shamexposed animals (fig. 2), the hematoxylin-eosin staining did not reveal significant morphological modifications in the cells of treated animals. furthermore, western blot experiments performed on protein extracts of the whole animal failed to evidence any difference between exposed and control specimens (fig. 3). this findings are in agreement with previous data from the bivalve mollusc m. galloprovincialis. the exposure of mussels to elf-mfs in a range of 200-1000 μt induced intensity-correlated effects on immunocyte functionality. however, at 400 μt mfs only transient damage was observed, while the injury became permanent only after exposure to mf intensities ranging from 600 to 1000 t (ottaviani et al., 2002). moreover, hsp70 and 90 were not induced in mussel immunocytes at 400 μt after a single 30 min exposure (malagoli et al., 2004), a result that has also been confirmed by rt-pcr experiments (malagoli et al., 2006). as small hsps exert their cytoprotective role via antioxidant, antiapoptotic and actin-stabilizing properties during cell stress (ciocca et al., 1993; de franco et al., 2004; franklin et al., 2005), we can conclude that a single 2 h exposure to 400 μt elf-mf is not able to influence significantly cell integrity and hsp27 expression in the pot worm e. japonensis. this conclusion is based on western blot experiments performed on pooled animals. however, the immunocytochemical approach performed on single specimens revealed that some animals may be more sensitive to mf irradiation, fig. 3 western blot analysis of hsp-27 immunoreactivity in e. japonensis after 2 h exposure to 400 μt elf-mf (a). immunoreactivity towards βtubulin was adopted as loading control (b). mw = molecular weight standard; c = sham-exposed pot worms; t = exposed pot worms. displaying a high level of hsp27-like material after the elf-mf exposure. acknowledgements we thank prof. m myohara (developmental biology department, national institute of agrobiological sciences, tsukuba, ibaraki, japan) who kindly supplied the enchytraeus japonensis population and prof. e ottaviani (university of modena and reggio emilia, italy) for the critical 84 reading of the manuscript. this work was supported by an italian ministry of the university and the research (miur) grant to dm, and by the italian “istituto superiore per la prevenzione e la sicurezza del lavoro” (ispesl) and ministry of labour, grants 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5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 3 isj 6: 44-48, 2009 issn 1824-307x short communication seasonal changes in functional parameters of the hemolymph of mytilus galloprovincialis c ciaccia, r fabbrib, m bettia, p rochc, l canesib adisuan, dipartimento di scienze dell’uomo, dell’ambiente e della natura, urbino, italy bdipartimento di biologia, universita` di genova, italy cjru ecosystèmes lagunaires, cnrs-université de montpellier 2-ifremer, france accepted april 7, 2009 abstract in bivalves, many functional parameters show seasonal changes in relation to both abiotic (such as temperature and salinity) and biotic factors (such as gonad maturation, food availability). available data indicate that also immune parameters can show seasonal fluctuations in the marine mussel mytilus spp.. in this work we report data on hemocyte lysosomal membrane stability (lms) and phagocytic activity, as well as on soluble lysozyme activity, in the hemolymph of mussels (mytilus galloprovincialis) collected over a 24 month period in the adriatic sea (2006-2007). the results indicate that all the parameters measured show seasonal fluctuations over the year, with lysozyme activity showing the largest changes. lowest lms values were observed in early winter and early autumn, whereas maximal values of phagocytic activity were observed in winter and increasing serum lysozyme activities were recorded in autumn. the observed seasonal fluctuations are discussed in relation to both abiotic (temperature) and biotic (changes in endogenous modulators) factors. key words: mytilus; hemocytes; lysosomal membrane stability; phagocytosis; lysozyme; immune parameters; seasonal variation introduction bivalves (such are mussels, clams and oysters) possess both cellular and humoral defence mechanisms that co-operate to kill and eliminate invading bacteria (mitta et al., 2000; canesi et al., 2002). hemocytes are responsible for cell-mediated immunity through phagocytosis and various cytotoxic reactions, such as the release of lysosomal enzymes and antimicrobial peptides, and the production of oxygen metabolites (mitta et al., 2000, canesi et al., 2002). in the recent years, we have investigated the immune responses of mytilus galloprovincialis to different stimuli, from bacterial challenge to exposure to endogenous modulators and heterologous cytokines, in both in vitro and in vivo studies (canesi et al., 2001, 2003, 2004, 2005, 2006a, b; betti et al., 2006). although a number of assays can be utilized in order to evaluate the immune function in invertebrates (ballarin et al., 2008), the phagocytic activity is generally considered ______________________________________________________________________ corresponding author: laura canesi department of biology university of genoa corso europa 26, 16132, genoa, italy e-mail: laura.canesi@unige.it as one of the most important parameters, especially in the bivalve mytilus spp. where active phagocytes represent the main circulating cell type (carballal et al., 1998; ottaviani et al., 1998; wottoon et al., 2003). however, another hemocyte parameter, lysosomal membrane stability, evaluated in live hemocytes by the neutral red retention time (nrr) assay, has emerged as an extremely sensitive indicator not only of cellular stress due to environmental perturbations (lowe et al., 1995), but also of the functional status of immunocytes (hauton et al., 2001; pruzzo et al., 2005). in fact, the majority of mytilus hemocytes are endowed with an extremely developed lysosomal vacuolar system which is involved not only in digestion of engulfed foreign particles, but that also contains a number of hydrolases that are secreted for extracellular degradation of components from invading microorganisms, such as bacterial cell wall (canesi et al., 2002). in addition, lysosomal production of oxygen radicals has been demonstrated (winston et al., 1996). in mussels, lms response to a variety of extracellular stimuli has been long widely investigated in a number of studies so that recorded changes can be related to a different functional status of the cell; in particular, moderate lysosomal   44 mailto:laura.canesi@unige.it destabilisation indicates activation of lysosomal membrane fusion processes related to endo/exocytosis, whereas larger decreases in lms correspond to increasing cellular stress conditions that can lead to irreversible cellular damage and autophagy (moore et al., 2006). changes in both lms and phagocytosis are induced by immune stimuli, like bacterial challenge or exposure to cytokines (canesi et al., 2001, 2003; 2005; betti et al., 2006). moreover, we have identified the natural estrogen 17β-estradiol as an endogenous modulator of these parameters, as well as of lysosomal enzyme release and oxyradical production (canesi et al., 2004, 2006b). in bivalves, many functional parameters show seasonal changes in relation to both abiotic (such as temperature and salinity) and biotic factors (such as gonad maturation or food availability). these factors may affect also the immune function. in mytilus spp., only a few studies have been focused on seasonal variations in immune parameters (carballal et al., 1997, 1998; malagoli et al., 2006, 2007; novas et al., 2007). in this work, we report data on hemocyte lms and phagocytic activity, as well as on soluble lysozyme activity in mussels, mytilus galloprovincialis, collected over a 24 month period in the adriatic sea in 2006-2007. materials and methods chemicals all reagents were of analytical grade and were purchased by sigma (st. louis, mo). animals specimens of the bivalve mollusc mytilus galloprovincialis, collected in the cesenatico area (rn, italy) were purchased monthly from a local fishing company (sea, gabicce mare, pu) for two years (from january to december 2006 and 2007). analysis were carried out on individuals of 4-5 cm size. after collection, animals (30 mussels) were taken immediately to the laboratory where they were kept in an aquarium for 24 h in static tanks containing aerated artificial sea water (asw) (1 l/mussel), 36 % psu, at different temperatures (from 15 to 20 °c, depending on the sampling period to minimize the effect of laboratory conditions). hemolymph was sampled from the posterior adductor muscle using a sterile 1 ml syringe with an 18 g1/2” needle. with the needle removed, hemolymph was filtered through sterile gauze and pooled in 50 ml falcon tubes at 18°c. hemolymph samples from 8-10 mussels were pooled and utilised for subsequent analyses. hemolymph serum was obtained by centrifugation of whole hemolymph at 200xg and the supernatant was sterilised through a 0.22 µm pore size filter. all analyses were performed in quadruplicate. lysosomal membrane stability (lms) lms was evaluated by the nrr (neutral red retention time) assay as previously described (canesi et al., 2005) according to lowe et al. (1995). hemocyte monolayers on glass slides were incubated with 30 µl of a neutral red (nr) solution (final concentration 40 mg/ml from a stock solution of nr 40 µg/ml in dmso); after 15 min excess dye was washed out, 30 µl of asw was added, and slides were sealed with a coverslip. every 15 min, slides were examined under an optical microscope and the percentage of cells showing loss of the dye from lysosomes in each field was evaluated. for each time point 10 fields were randomly observed, each containing 8-10 cells. the end point of the assay was defined as the time at which 50 % of the cells showed sign of lysosomal leaking (the cytosol becoming red and the cells rounded). all incubations were carried out at 18 °c. phagocytosis assay phagocytosis of neutral red-stained zymosan was used to assess the phagocytic ability of hemocytes as previously described (canesi et al., 2006b) according to pipe et al. (1995). neutral redstained zymosan in 0.05m tris-hcl buffer (tbs), ph 7.8, containing 2 % nacl was added to each monolayer at a concentration of about 1:50 hemocytes:zymosan diluted in asw, and allowed to incubate for 30 and 60 min at 18 °c. monolayers were then washed three times with tbs, fixed with baker’s formol calcium (4 %, v/v, formaldehyde, 2 % nacl, 1 % calcium acetate) for 30 min and mounted in kaiser’s medium for microscopical examination with a vanox (olympus italy 1.2.1, mi) optical microscope. for each slide, the percentage of phagocytic hemocytes was calculated from a minimum of 200 cells. data are expressed as % of phagocytizing cells. serum lysozyme activity lysozyme activity in aliquots of serum was determined as previously described (pruzzo et al., 2005) following chu and la peyre (1989). briefly, lysozyme activity was determined as the ability to lyse a standard suspension of m. lysodeikticus (15 mg/100 ml in 66 mm phosphate buffer, ph 6.4) and measured as decrease in absorbance at 450 nm at room temperature. hen egg-white (hew) lysozyme was used to construct a standard curve and lysozyme activity was expressed as hew lysozyme equivalents/mg protein/ml. protein content was determined according to the lowry method using bovine serum albumin (bsa) as a standard. data analysis results are presented as the arithmetical mean ± sd of experiments carried out in quadruplicate. statistical analysis was performed by using the mann-whitney u-test with significance at p<0.05. results and discussion mussels were sampled for 24 months during 2006 and 2007. since no significant differences in the results obtained were recorded between the two years, only data from jan-dec 2007 are reported in fig. 1. figure 1a shows the results obtained for hemocyte lms: mean annual values of nr retention times were 117.75 ± 13.6 min. lowest lms (about 100 min) were recorded in winter (janfeb) and early autumn (sept-oct). with respect to these values, significantly higher lms were   45 recorded in spring, with a maximum in may (+39 %; p<0.05) and december + 31 %; p < 0.05). however, both minima and maxima did not significantly differ from mean annual values. data on hemocyte phagocytic activity are reported in fig. 1b as % of phagocytosing cells. mean values (%) were 53.87 ± 5.55. higher values were observed in winter, with a maximum in january (64 %), followed by a slow decrease in spring-early summer, that reached lowest values in june and september (-27 % and 26 %, respectively, with respect to january; p<0.05). however, as observed for lms data, also for phagocytosis neither minima nor maxima were significantly different from mean annual values. in figure 1c data on soluble lysozyme activity are reported. mean annual values were 147.25 ± 68 mu/mg protein. in this case, larger seasonal differences were observed, with lower values in late winter-early spring (less than 100 mu/mg protein from february to april, with a minimum in march of 84±15 mu/mg protein.). a large, progressive rise was observed from late summer to autumn (up to a +150 % increase in october and november with respect to july; p<0.05). maximal values recorded in october and november were also significantly different from mean annual values (+83 % and +100 %, respectively; p<0.05). taken together, the results indicate seasonal fluctuations in the parameters measured in the hemolymph of mussels from the adriatic sea. lowest lms values were observed in early winter and early autumn. on the other hand, maximal values of phagocytic activity were observed in winter and increasing serum lysozyme activities were recorded in autumn. these observations are in line with the fact that decreases in lms are associated with membrane fusion processes during both endo/phagocytosis and release of lysosomal enzymes by exocytosis. however, when data were analysed by the spearman rank correlation test, no significant correlation was observed among the different parameters measured (data not shown). the observed changes in hemolymph functional parameters can be related to both abiotic (such as temperature) and biotic factors (such as reproductive stage and also food availability). in the adriatic sea, fluctuations in hemolymph cytotoxicity of m. galloprovincialis were reported (malagoli et al., 2006, 2007), with two peaks at the end of spring and of summer. however, the temperature apparently was not the main parameter affecting cytotoxicity (malagoli et al., 2007). moreover, no significant correlation was found between changes in immune parameters recorded in the present work and environmental temperature (data not shown). although circulating hemocyte concentration did not show seasonal variations in m. galloprovincialis (carballal et al., 1998), the proportion of different hemocyte subpopulations endowed with different phagocytic activity due to different hemocyte maturation stages may change at different times of the year. since phagocytosis represents one of the main components of the innate immune response, lower phagocytic activities observed in this work during the summer may be fig. 1 seasonal trend of lysosomal membrane stability (lms) (a) and phagocytic activity (b) in hemocytes and of serum lysozyme activity (c) in hemolymph of mussels from adriatic sea. data, representing the mean ± sd of four replicates were analysed by the mann-whitney u test. a) * = p<0.05: may vs january and february; december vs september and october b) * = p<0.05: january vs june and september c) * = p<0.05: july vs october and november responsible for lower immune defence against invading micro-organisms during this period. moreover, the effect of seasonal changes in endogenous modulators (such as neuropeptides, cytokines and sex steroids) should be taken into account. in the hemocytes of m. galloprovincialis, physiological concentrations of the natural estrogen 17β-estradiol can stimulate immune parameters, including phagocytosis, both in vitro and in vivo (canesi et al., 2004, 2006b). in bivalves, 17βestradiol has a role in reproduction: seasonal changes in estrogen content have been observed in   46 different bivalve species in relation to gametogenesis, with increases during gonad maturation and decreases at spawning (osada et al., 2004; gauthier-clerc et al., 2006). however, in the present study, different extents of gonad ripening were observed in different individuals (males and females) throughout the year; although during acclimation in the laboratory a major gamete emission was observed in early spring, minor spawnings were also recorded at different times of the year. the absence of marked seasonal changes in gametogenesis may be partly related to a relatively constant food availability throughout the year in the adriatic sea. in m. galloprovincialis from the northern spanish coast, basal hemocyte no production showed lowest values in winter. however, only in mussels collected in winter il-2 greatly induced no production probably through an immunoreactive enos protein that was expressed only in this period by the hemocytes (novas et al., 2007). these data suggest that, in addition to fluctuations in basal values of immune parameters, changes in the inducibility of the immune response over the year should be considered. our data give a further insight on the seasonal inter-relationship among immune parameters in m. galloprovincialis. accumulation of this knowledge may greatly help understanding and predicting the immune response against potential pathogens, as well as the possible immunotoxic effects of exposure to environmental contaminants in an economically and ecologically relevant species of bivalves. acknowledgements this work was supported by the eu program imaquanim (food-ct-2005-007103) subcontract cnrs 009252. references ballarin l, cammarata m, cima f, grimaldi a, lorenzon s, malagoli d, et al. immuneneuroendocrine biology of invertebrates: a collection of methods. inv. surv. j. 5: 192-215, 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58-71, 2004. canesi l, betti m, ciacci c, lorusso lc, gallo g, pruzzo c. interactions between mytilus hemocytes and different strains of escherichia coli and vibrio cholerae o1 el tor: role of kinase-mediated signalling. cell. microbiol. 7: 667-674, 2005. canesi l, betti m, ciacci c, lorusso lc, pruzzo c, gallo g. cell signaling in the immune response of mussel hemocytes. inv. surv. j. 3: 40-49, 2006a. canesi l, ciacci c, lorusso lc, betti m, guarnieri t, tavolari s, et al. immunomodulation by 17βestradiol in bivalve hemocytes. am. j. physiol. integr. resp. comp. physiol. 29: r664-r673, 2006b. carballal mj, lopez c, azevedo c, villalba a. enzymes involved in defense functions of hemocytes of mussel mytilus galloprovincialis. j. invertebr. pathol. 70: 96-105, 1997. carballal mj, villalba a, lopez c. seasonal variation and effects of age, food availability, size, gonadal development, and parasitism on the hemogram of mytilus galloprovincialis. j. invert. pathol. 72: 304-312, 1998. chu fle, la peyre jf. effect of environmental factors and parasitism on hemolymph lysozyme and protein of american oysters (crassostrea virginica). j. invert. pathol. 54: 224-232, 1989. gauthier-clerc s, pellerin j, amiard jc. estradiol17β and testosteorne concentrations in male and female mya arenaria (mollusca bivalvia) during the reproductive cycle. gen. comp. endocrinol. 145: 133-139, 2006. hauton c, hawkins le, hutchinson s. response of hemocyte lysosomes to bacterial inoculation in the oysters ostrea edulis l. and crassostrea gigas (thunberg) and the scallop pecten maximus (l.). fish shellfish immunol. 11: 143153, 2001. lowe dm, fossato vu, depledge mh. contaminantinduced lysosomal membrane damage in blood cells of mussels mytilus galloprovincialis from the venice lagoon: an in vitro study. mar. ecol. prog. ser. 129: 89-196, 1995. malagoli d, casarini l, ottaviani e. monitoring of the immune efficiency of mytilus galloprovincialis in adriatic sea mussel farms in 2005. inv. surv. j. 3: 1-3, 2006. malagoli d, casarini l, ottaviani e.. monitoring of the immune efficiency of mytilus galloprovincialis in adriatic sea mussel farms in 2006: regular changes of cytotoxicity during the year. inv. surv. j. 4: 10-12, 2007. mitta g, vandenbulcke f, roch p. original involvement of antimicrobial peptides in mussel innate immunity. febs lett. 486: 185-190, 2000. moore mn, icarus allen j, mcveigh a. environmental prognostics: an integrated model supporting lysosomal stress responses as predictive biomarkers of animal health status. mar. environ. res. 61: 278-304, 2006.   47 http://www.isj.unimo.it/articoli/isj176.pdf http://www.isj.unimo.it/articoli/isj176.pdf http://www.isj.unimo.it/articoli/isj176.pdf http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids=16060858&query_hl=1 http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&dopt=abstract&list_uids=16060858&query_hl=1 http://www.ncbi.nlm.nih.gov/pubmed/16343609?ordinalpos=10&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvdocsum novas a, barcia r, ramos-martinez ji. nitric oxide production by haemocytes from mytilus galloprovincialis shows seasonal variations. fish shellfish immunol., 23: 886-891, 2007. osada m, tawarayama h, morii k. estrogen synthesis in relation to gonadal development of japanese scallop patipecten yeasoensis: gonadal profile and immunolocalization of p450 aromatase and estrogen. comp. biochem. physiol. 139b: 123-128, 2004. ottaviani e, franchini a, barbieri d, kletsas d. comparative and morphofunctional studies on mytilus galloprovincialis hemocytes: presence of two aging-related hemocyte stages. it. j. zool. 65: 349-354, 1998. pipe r, coles ja, farley sr. assays for measuring immune response in the mussel mytilus edulis. in: stolen j, fletcher tc, smith sa, zelikoff jt, kaattari sl, anderson, rs (eds), techniques in fish immunology: fish immunology technical communications, immunological and pathological techniques of aquatic invertebrates, vol. 4, sos publications, fair haven nj, pp 93-100, 1995. pruzzo c, gallo g, canesi l. persistence of vibrios in marine bivalves: the role of interactions with hemolymph components. environ. microbiol. 7: 761-772, 2005. winston gw, moore mn, kirchin ma, soverchia c. production of reactive oxygen species by hemocytes from the marine mussel mytilus edulis: lysosomal localization and effect of xenobiotics. comp. biochem. physiol. 113c: 221-229, 1996. wootton ec, dyrynda ea, ratcliffe n.a. bivalve immunity: comparison between the marine mussel (mytilus edulis), the edible cockle (cerastoderma edule) and the razor-shell (ensis siliqua). fish shellfish immunol. 15: 195210, 2003.   48 isj104.pdf 82 isj 2: 82-90, 2005 issn 1824-307x review mode of action of antimicrobial proteins, pore-forming toxins and biologically active peptides (hypothesis) o schmidt1, mm rahman1, g ma1, u theopold2, y sun3, m sarjan4, m fabbri5, h roberts1 1insect molecular biology, faculty of sciences (waite campus), university of adelaide, glen osmond, sa 5064, australia 2 department of molecular biology and functional genomics, stockholm university, s-10691 stockholm, sweden 3 the biotechnology research center, shanxi academy of agricultural sciences, 64 n.nongke, taiyuan, shanxi 030031, china 4 faculty of agriculture, university of mataram, lombok, indonesia 5 department of experimental oncology, european institute of oncology, i-20141 milan, italy accepted july 1, 2005 abstract antimicrobial peptides and pore-forming toxins are important effectors in innate immune defence reactions. but their mode of action, comprising the insertion into cholesterol-containing membranes is not known. here we explore the mechanical implications of pore-formation by extracellular protein assemblies that drive cellular uptake reactions by leverage-mediated (lm) processes, where oligomeric adhesion molecules bent membrane-receptors around ‘hinge’-like lipophorin particles. the interactions of antimicrobial peptides, pore-forming toxins and biologically active proteins with lmassemblies provide a new paradigm for the configurational specificity and sterical selectivity of biologically active peptides. key words: antimicrobial peptides; pore-forming toxins; peptide hormones; endocytosis; lipophorin; cholesterol; lipid rafts; channel formation; lectins introduction antimicrobial proteins are effectors of innate immunity with unique structural properties mediating mainly pore-forming activities in membranes (boman, 2000). since its discovery more than thirty years ago (boman et al., 1974; faye et al., 1975), peptides with antibacterial activities have been divided into a number of different categories (boman, 2003), including alpha-helical peptides, peptides with cysteine bonds and peptides enriched in one or more amino acids. a unique feature of anti-microbial peptides is their ability to permeate and disrupt target membranes (shai, 2002). corresponding author: otto schmidt insect molecular biology, faculty of sciences (waite campus), university of adelaide, glen osmond, sa 5064, australia e-mail: otto.schmidt@adelaide.edu.au antimicrobial peptides are believed to kill microorganisms via non-receptor-mediated mechanisms, although some peptides, such a nisin z bind to bacterial cell wall components (breuking et al., 1999). according to (shai, 2002) monomeric peptides that have random structures gain amphipathic structures and form oligomers in solution such that the hydrophobic regions are buried in the lumen of the oligomer and the hydrophilic regions are exposed to the solution. upon reaching the membrane the organization is reversed. the hydrophobic regions are exposed to the lipid constituents of the membrane, and the hydrophilic regions are either segregated in the lumen of the oligomer (if the peptide oligomerizes and inserts into the membrane via the ‘barrel’ mechanism (ehrenstein and lecar, 1977), or exposed to the solution (if the peptides lay on the surface of the membrane and insert via the ‘carpet’ mechanism (pouny and shai, 1992). two different mechanisms of peptide insertion have been implicated in related antimicrobial peptides (chen et al., 2003), where cecropin b molecules, (with one amphipathic and one hydrophilic á-helix) may be inserted into the 83 membrane in a concentration-dependent ‘barrel-stave’ mechanism, whereas synthetic cecropin b3 molecules (with two hydrophobic á-helices) may disrupt the membrane by a ‘carpet-like’ lysis mechanism. although many peptide properties have been described within the framework of the two models, a fundamental question remains: why are these peptides specific to bacterial membranes, but not damaging to most eukaryotic membranes? one argument is that the positive net charge of antibacterial peptides enables binding and permeation of negatively charged phospholipid membranes of bacteria but not to zwitterionic membranes, which are the major constituents of the outer leaflet of erythrocytes and other eukaryotic membranes (shai, 2002). but there are important exceptions, which suggest that it is not the charge alone, but that the secondary and tertiary structure of the peptide is also crucial for insertion into the membrane. this is apparent in some antimicrobial peptides that are active in bacteria and in mammalian cells, such as melittin, its hybrid cecropin a permutations and its diastereomeric analogs (merrifield et al., 1995b). it appears that neither the direction of the peptide bond, nor the turn of the helix in d-enantiomers (merrifield et al., 1995a) interferes with the membrane-lytic mechanism of the peptides (boman, 2003). this and other examples (staubitz et al., 2001) suggest that chiral and sequence-specific determinants are not required for membrane-disrupting activity, while target specificity is strongly influenced by the overall physico-chemical nature of the analogues (merrifield et al., 1995b; staubitz et al., 2001). apart from the observation that changes in a peptide’s sequence are more likely to destroy its activity to eukaryotic than to prokaryotic cells, there are no identifiable amino acid sequences that are responsible for the specificity (hancock and rozek, 2002). another argument put forward to explain peptide-specificities against bacterial membranes is that cholesterol, which is present in eukaryotic membranes, protects against the action of antibacterial peptides (boman, 2003). although this has been confirmed in artificial membrane systems, it raises the question of why a large portion of antibacterial peptides, such as defensins (hoffmann and reichart, 2002), dermaseptins (shai, 2002), cathelicidines (zanetti et al., 1997), pardaxin analogs (shai, 2002), and tachystatin (osaki et al., 1999) are also active against fungal membranes, which contain ergosterol. also difficult to understand in the context of current models are the non-lytic modes of action, such as the transfer of some peptides through the membrane into the underlying cytoplasm, where active peptides interfere with a diverse range of metabolic processes (hancock and rozek, 2002). in fact, some argue that the lytic action may not be the primary cause of death for bacteria (boman, 2003), since antibacterial peptides may have already irreversibly impaired the viability of bacteria before the apparent disruption of the membrane. membrane trafficking – an achilles’ heel? although most experiments are performed with artificial membrane systems comprising regularshaped vesicles, it is very likely that in vivo membrane disruptions by peptides are instigated during lipidbilayer disturbances, such as cellular processes involving extreme membrane curvature and membrane fusion. this is relevant for membrane trafficking in higher organisms, but also applies to prokaryotes during cell division. the in vitro studies mentioned above suggest that membrane lipid composition is crucial for susceptibility to peptide insertion. artificial membranes lacking cholesterol are more susceptible to peptide insertions than cholesterol-containing membranes (boman, 2003). furthermore, insertion of amphiphilic molecules forming nanotubes into cholesterol-free lipid bilayers has been modelled around hydrophobic-hydrophilic matching using molecular dynamics simulations (lopez et al., 2004). thus the presence of cholesterol and sphingolipids appear to be a major barrier for the insertion of peptides into the membranes. this may explain why bacteria are susceptible to most peptides, while animals and plants are not. in this context it is interesting to note that higher organisms with intensive membrane trafficking perform non-clathrin mediated uptake reactions in special membrane domains, called ‘lipid rafts’, which represent accumulations of cholesterol and sphingolipids (brown and london, 1998; ikonen, 2001). the fact that uptake reactions involve reorganisation of the membrane bilayer makes membrane trafficking vulnerable to peptide attacks. but if these membrane domains are perceived to protect against pore-forming toxins, why do many toxins interact with receptors in lipid rafts (kurzchalia, 2003; zhuang et al., 2002)? is it possible that poreforming toxins and channel-forming peptides exploit the protein machinery of cellular uptake reactions to achieve insertion into the membrane? pore-forming toxins an alternative mechanism of channel formation involves the membrane insertion of pore-forming toxins with â-barrel peptides (lesieur et al., 1997) by putative leverage-mediated (lm) uptake reactions (schmidt and theopold, 2004). in line with the model, receptors are assembled around ring-shaped lipoproteins, such as lipophorin or hexamerin. oligomeric adhesion molecules, such as lectins, cross-link receptors using membrane-distal mucindomains thereby bending receptors around hinge-like lipoproteins. in this model the configuration of the complex provides the leverage to curve the membrane and internalise receptors in a cluster of lm-complexes affecting cell shape changes (schmidt and theopold, 2004) and adhesive properties of the cell (schmidt and schreiber, in press). since many pore-forming toxins, such as crystal toxins from bacillus thuringiensis (bt-toxins), are lectins, the insertion into the membrane may be mediated by an lm-uptake reaction (schmidt and theopold, 2004). in the lm-scenario, the ring-shaped pore complex is formed before or during the assembly of receptors (fig. 1), which are bend around the hingelike lipoprotein by the oligomeric pore-forming complex (fig. 2). this is in contrast to the current assumption that bt-toxin molecules are inserted into the membrane as monomers by a receptor-mediated 84 fig. 1 schematic illustration of receptor-assemblies with the potential to provide configurational energy by leverage-mediated mechanisms. a) lipoproteins, such as lipophorin, contain ring-shaped proteins filled with lipids (canavoso et al., 2001), including phospholipids (green), cholesterol (blue) and diacylglycerols (yellow). lipophorin particles can bind to membrane-bound receptors to form lipophorin complexes (theopold and schmidt, 1997). alternatively, lectins may bind to glycolipids or glycoproteins on lipid particles to form lectinlipophorin complexes, before interacting with receptors. (glycodeterminants are indicated by black dots) b) formation of a receptor complex with the potential to create configurational energy by a leverage-mediated (lm) process. 1b 1a 85 fig. 2 schematic illustration of leverage-mediated uptake reactions. lm-uptake reactions are mediated by oligomeric adhesion molecules, such as lectins. a) assemblies, consisting of lipoproteins and multimeric lectins, interact with membrane-anchored molecules, such as membrane receptors, lipid anchored glycoproteins or glycolipids molecules. as a result of the lm-process the lipid particle is pushed into the underlying membrane thereby ‘unloading’ the lipophorin without the need to internalize the complex. note that multiple lm-complexes predict membrane domains enriched in particle-derived lipids, such as cholesterol that may alter the local composition and protect the membrane against antibacterial peptide attack. b) putative ‘shuttle’ mechanism: if the lipophorin particle changes shape in the process of lipid unloading, the resulting complex may not support a leverage-mediated uptake reaction and the complex unravels before internalization occurs. the unloaded lipophorin particles may then be released to become loaded on the gut membrane by an unknown process. 2a 2b 86 fig. 3 insertion of pore-forming toxins into the membrane by a putative lm-mechanism. a) lm-complex comprising oligomeric lectins containing amphipathic alpha-helices (red), which can engage in uptake reactions like other lectins. b) leverage-mediated uptake reactions may push the amphipathic peptide into the lipid layer opening a membrane gap to the cytoplasm. this allows ions and water to pass from the endosome into the cytoplasm, causing osmofragility in some lectins (pande et al., 1998) and pores in endotoxins. note that the poreforming peptides can be covalently attached to the oligomeric adhesion molecule (e.g. pore-forming toxin, such as bt-toxin). alternatively, the active peptide may be assembled with the lm-complex without being covalently attached to any components by its space-filling properties. in this case the active peptide may be toxic and form a pore, such as mellittin, or interfere with lm-functions by altering cellular processes and signalling functions as observed with biologically active peptides. 3a 3b 87 reaction and only assembled into pore-forming oligomeric complexes once inside the membrane bilayer (de maagd et al., 2001). however, it has been shown that some mature bt-toxins form tetrameric complexes when processed in vitro (ma et al., 2005), which would enable multiple interactions with receptors and lipoproteins before membrane insertion. another intriguing observation is that some plant lectins increase osmofragility (pande et al., 1998), which in itself is not damaging to membranes, but may provide clues for our understanding of how poreforming toxins are inserted into the membrane (schmidt and theopold, 2004). since many poreforming toxins are lectins, which recognise mucin-like glycoprotein-receptors (armstrong et al., 1996; kuwahara et al., 2000) and glycolipids (griffitts et al., 2005), such ring-shaped adhesion complexes could potentially be internalised by an lm-mechanism (schmidt and theopold, 2004), where the structural features of the complex predict a disruption of the lipid bilayer (fig. 2). in this context the structure of pore-forming toxins can be viewed as oligomeric lectins that have amphipathic peptides with antibacterial properties covalently attached. indeed, secondary structure predictions, helical wheel/net diagrams and molecular mechanics calculations of membrane-inserting peptides from the bt-toxin, form a strongly amphiphilic alpha-helix and show haemolytic activity in vitro comparable to that of bee venom peptide melittin (szabo et al., 1993). similar results were obtained with the isolated á4-loop-á5 hairpin from the bt-toxin, which showed that this peptide is extremely active compared to the isolated helices or their mixtures, indicating the complementary role of the two helices and the need for the loop for efficient insertion into membranes (gerber and shai, 2000). the concept that pore-forming toxins are oligomeric adhesion molecules with covalently attached antibacterial peptides is compatible with the idea that antibacterial peptides exploit lm-uptake reactions and are inserted into the membrane together with oligomeric adhesion molecules. a prediction of this model is that some antibacterial peptides can potentially overcome the cholesterol barrier of the membrane by assembling into the lm-machinery and become inserted into the membrane during lm-reactions. the fact that antibacterial peptides targeting eukaryotic cells are attached to oligomeric adhesion molecules, whereas peptides targeting prokaryotes are not, could indicate that peptides are effective in prokaryotes without the help of lm-mechanisms. alternatively, prokaryotic uptake mechanisms may be different (e.g. in the absence of cholesterol) and less selective against active peptides. lipid exchange in higher organisms cholesterol is transported between cells by a ring-shaped protein complex of apolipophorins that stabilize a mix of other hydrocarbons, such a phospholipids and diacylglycerols (dags). these lipid particles are in general internalised by cells via endocytosis reactions, except in insects, where lipophorin particles can act as a reusable shuttle by taking up lipid and delivering it to target tissues without internalization and degradation of the particle. for example, a single lipophorin particle, synthesized in the fat body, can take up dietary lipid at the midgut in the form of dag, diffuse through the hemolymph to the fat body, and deliver the dag for storage in the fat body without being internalised and degraded. this same particle can then return to the midgut surface and repeat the process (canavoso et al., 2001). how the lipophorin complex is able to release cholesterol and other lipids into the underlying cell membrane is not known. one of the possible implications of the lm-model is that an lm-uptake mechanism may provide the extracellular energy that is required to merge zwitterionic lipid layers. in this scenario, the lipid-loaded lipophorin complex is pushed into the membrane as receptors form linkages with an oligomeric adhesion molecule (fig. 2). while the nature and identity of receptors and adhesion molecules involved in lipophorin unloading is not known at this stage, recent observation suggest that lipophorin particles carry glycolipids and lipidanchored glycoproteins that can interact with lectins and membrane receptors. for example, when lectins were mixed with cell-free hemolymph from lepidopteran insects and lipophorin separated on density gradients, lectins were enriched in the lipophorin fractions (sarjan, 2002). conversely when lipophorin fractions where analysed on western blots with lectins a number of glycoproteins were found to be co-purified with lipophorin particles. one of these proteins, a ca 50 kda protein was identified (fabbri, 2003) and shown to belong to a group of chitinase-like molecules known as imaginal disc growth factors (idgfs) (asgari and schmidt, 2004) with possible lectin-like properties (homma et al., 1996; kawamura et al., 1999; li and aksoy, 2000) that are conserved in other invertebrates (akalal and nagle, 2001) and vertebrates (riazi et al., 2000). more recently, lipidanchored morphogens, such as wingless and hedgehog, have been found in association with drosophila lipophorin particles and shown to be morphogen carriers in the extracellular space of imaginal discs (panakova et al., 2005). it is therefore conceivable that lipophorin particles are pushed onto the membrane by functional lmcomplexes, and in the process may allow the lipid moieties to mix, releasing the lipids into the underlying membrane (fig. 2a). in this context a reversible shuttle function of lipophorin particles is possible if apolipophorin changes its conformation as a result of lipid depletion of the particle. in this case the hinge-like properties of lipophorin may be lost, disrupting the internalisation process and releasing the unloaded lipophorin (fig. 2b). while this is hypothetical, the importance of a lipophorin shuttle based on lm-mechanisms is that it makes specific predictions that can be experimentally tested. for example, mature crystal toxin from b. thuringiensis was co-purified with lipophorin particles on density gradients (fig. 4). since apolipophorin is not recognised by the toxin on western blots this implies that the toxin binds to other glycodeterminants, such as lipid-anchored glycoproteins (luo et al., 1999; panakova et al., 2005) and glycolipids (griffitts et al., 2005; hatakeyama et al., 1999; inoue et al., 2001; nedelkoska and benjamins, 1998; sandvig et al., 88 fig. 4 low density gradient of cell-free hemolymph (plasma) from galleria mellonella larvae mixed with mature bt-toxin (cry1ac). aliquots of fractions were analysed on western blots using anti-cry1ac antibodies. a 69 kda monomer was found predominantly in high density fractions (arrowhead) together with a minor 60 kda protein resulting from over-digestion of the pro-toxin. in addition, monomers were also found in low-density regions of the gradient between fractions 11 and 14, where apolipophorin i and ii subunits peak in addition to a minor peak between fractions 16 and 20. apart from monomers, oligomeric forms of the toxin, such as trimers and tetramers, are enriched in the lipophorin fractions (sarjan, 2002). it is not clear whether pre-existing oligomers preferentially bind to lipophorin particles or whether monomers are bound to particles and form oligomers on the particle as observed in other systems (park et al., 2005). 1989). although the identity of lectin-binding proteins from lipophorin particles in the gut lumen remains to be determined, this observation could suggest toxicity mechanisms at the gut lining involving lipophorinmediated lipid exchange or uptake. conversely, the fact that lipophorin (li et al., 2002) and other lipid carrying storage proteins (ma et al., 2005) are known to be involved in coagulation reactions could potentially be responsible for the observed aggregation reactions in the gut lumen (ma et al., 2005). the sequestration of toxin molecules in the gut lumen by immune-related coagulation reactions can also explain the observed tolerance to the toxin by immune induction (rahman et al., 2004; gunning et al., 2005; ma et al., 2005). configurational specificity if lm-uptake reactions are the driving force for the insertion of pore-forming complexes into membranes, the functioning of such multi-protein complexes will depend on the correct configurational compilation of the protein assembly. for example, only oligomeric adhesion molecules that are able to generate leverage by interacting with membrane receptors across a hinge-like protein will be able to curve the membrane (fig. 2a), a prerequisite for the insertion of amphipathic peptides into the membrane (fig. 3). in this context the role and specificity of antibacterial peptides that are inserted into cholesterol-containing membranes may depend on structural requirements, which allow the peptide to intercalate into gaps provided by the lm-assemblies comprising oligomeric adhesion molecules, membrane-receptors and hingelike proteins (fig. 3a), without damaging the functionality of the complex. this implies that the observed peptide-specificity may be based on spacefilling rather than protein-binding properties and that biologically active peptides are able to specifically interact with lm-assemblies without the need to bind to any individual proteins or receptors. the outcome of this interaction may be the formation of a damaging pore, but also non-toxic ion flux or alteration of the lmuptake process, which can modify cell behavior and signaling. in fact the 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acad. sci. 832: 147-162, 1997. zhuang m, oltean di, gomez i, pullikuth ak, soberon m, bravo a, gill ss. heliothis virescens and manduca sexta lipid rafts are involved in cry1a toxin binding to the midgut epithelium and subsequent pore formation. j. biol. chem. 277: 13863-13872, 2002. may 4, 2010 isj 7: 165-180, 2010 issn 1824-307x review role of α2-macroglobulin in the immune responses of invertebrates pb armstrong marine biological laboratory, 7 mbl street, woods hole, ma 02543, and department of molecular and cellular biology, university of california, davis, ca 95616, usa accepted june 30, 2010 abstract although proteases play essential roles in the lives of all organisms, they are also important agents of disease and pathogenesis in metazoans. most notably, proteases are essential virulence factors for a broad array of prokaryote and eukaryote parasites. one strategy used by the immune system of metazoans to defend against parasitic attack is to neutralize the toxins and essential virulence factors that allow the parasite to gain entry to the host and to survive and proliferate in the internal environment of the metazoan host. the particular defense system of interest to the present review is the system of endogenous protease inhibitors that operate to inactivate the secreted proteases utilized by invading parasites during the infection cycle within the host. protease inhibitors are of two broad classes, active-site inhibitors that bind to and inactivate the active sites of target proteases and the α2-macroglobulin class of inhibitors that operate as opsonins to bind and mark proteases in a manner that allows the subsequent endocytosis and intracellular proteolytic degradation of the α2-macroglobulin-protease complex. members of the α2-macroglobulin class of inhibitors interact with target proteases by the novel process of enfolding the protease into a pocket within the interior of the α2-macroglobulin molecule, which is followed by binding of the complex to the α2-macroglobulin receptor at the surfaces of macrophages and other endocytotic cells and its endocytosis and degradation. in contrast to the active-site protease inhibitors, each of which is specialized to interact with a small subset of all endopeptidases, the α2-macroglobulin inhibitors are remarkably promiscuous, binding proteases of all enzymatic classes and origins. this characteristic allows α2-macroglobulin to play an important role in immune defense because this one protein is capable of binding and neutralizing the diverse array of proteases that function as virulence factors of the diverse array of parasites out there in the environment of metazoa. key words: α2-macroglobulin; thiol ester protein family; protease inhibitor; innate immunity introduction an important barrier to the successful reproduction of metazoans is the disease and premature death that attends the attack by parasites. barriers to parasitic attack are the integuments, which limit the range of parasites that can gain entry to the internal milieu, and the immune system, which defends against parasites and their toxic products both at the surface and in the internal spaces. parasites may be unicellular or multicellular, prokaryote, eukaryote, or virus. in coelomate animals, the most important organ of the ___________________________________________________________________________ corresponding author: peter b armstrong department of molecular and cellular biology university of california, davis, ca 95616-8535, usa e-mail: pbarmstrong@ucdavis.edu 165 immune system is the blood, presumably because the blood has ready access to all parts of the body and is best prepared to concentrate defense effectors at sites of pathogen invasion. this review will be concerned with one of the important immune effector proteins found in the plasma of all major taxa of metazoans, α2-macroglobulin. α2-macroglobulin is an evolutionarily conserved element of the innate immune system whose bestcharacterized function is the clearance of active proteases from the tissue fluids. proteases have a diverse set of essential roles in the lives of eukaryotes, including protein digestion, the activation of peptide hormones and other effector proteins that are secreted as zymogens, the physiological turnover of intracellular proteins, the removal of effete extracellular proteins, and mailto:pbarmstrong@ucdavis.edu 166 remodeling of the extracellular matrix (lopez-otin and bond, 2008). however, in addition, proteases, whether of endogenous or exogenous origin, are capable of considerable mischief when free in the blood and tissue spaces. they are important agents in a variety of connective tissue diseases (perlmutter and pierce, 1989), contribute to neoplastic invasion and metastasis (deryugina and quigley, 2006; kessenbrock et al., 2010), and are important virulence factors contributing to the pathogenicity of prokaryotic and eucaryotic parasites (armstrong, 2006; mckerrow et al., 2006). in response to protease challenge, animals have evolved a diverse array of protease inhibitors (travis and salvesen, 1983). in mammals, approximately 3-5 % of the plasma proteins are protease inhibitors (laskowski and kato, 1980) and in the american horseshoe crab, limulus polyphemus, the hemolymph protease inhibitor, α2macroglobulin, is the third most abundant protein in the hemolymph (enghild et al., 1990). α2macroglobulin is one of the most abundant proteins of human plasma, at a concentration of 2-4 mg/ml (sottrup-jensen, 1989) and is the second-most abundant protein of the hemolymph of the cephalopod, sepia (vanhoorelbeke et al., 1993). α2macroglobulin, the subject of this review, is present in representatives of several metazoan phyla, including coelenterates (fujito et al., 2010) and representative species of the deuterostomia (echinodermata, tunicata, vertebrata), ecdysozoa (arthropoda, nematoda), and lophotrochozoa (mollusca) [for reviews see (armstrong and quigley, 1999; armstrong, 2006)]. a diverse variety of gramnegative eubacteria have acquired a gene encoding α2-macroglobulin from eukaryote associates (budd et al., 2004; doan and gettins, 2008). mechanisms for attack on proteases the protease inhibitors are of two fundamental classes, the active-site inhibitors, which bind to and inactivate the activate site of the targeted endopeptidase (huntington et al., 2000), and the α2macroglobulins, which react by a unique mechanism that involves the physical entrapment of the target protease within the folds of a molecule of α2macroglobulin (table 1). by binding to the active site of the protease, the active-site inhibitors destroy both its proteolytic activity against proteins and its ability to cleave the ester and amide bonds of low molecular mass artificial substrates. dedication for a particular active site means that the active-site inhibitors are restricted in their activity to one particular class, or even sub-class, of protease. by contrast, α2-macroglobulin shows a unique mechanism for interaction with target proteases which is initiated by its proteolytic cleavage at a defined motif that is constructed as an exposed and highly flexible stretch of 30 40 amino acids that presents a suite of peptide bonds that are inviting targets for most extant proteases (sottrup-jensen et al., 1989). this means that, whatever the protease, it will likely find a target for proteolytic attack at this “bait” region of the α2-macroglobulin molecule. this is the basis for the broadly reactive ability of α2-macroglobulin to bind proteases of diverse enzymatic reactivity and source. the forms of α2macroglobulin found in the tick (saravanan et al., 2003; buresova et al., 2009), horseshoe crab (husted et al., 2002), and carp (mutsuro et al., 2000) show multiple forms of the protein that differ by bait region sequence. presumably this functions to expand the list of proteases recognized by the α2macroglobulins of these species. proteolytic cleavage of the bait region is followed by the rapid re-folding of the α2-macroglobulin molecule so as to produce an enclosed interior pocket that now contains the target protease, entrapped within the folds of the α2-macroglobulin protein (starkey and barrett, 1973). protease entrapment disables the enzyme’s ability to proteolyze macromolecular substrates too large to penetrate the α2macroglobulin cage, but leaves intact the ability of the entrapped enzyme to hydrolyze low molecular mass substrates small enough to enter the α2macroglobulin cage and interact with the active site of the protease (starkey and barrett, 1973). one diagnostic feature to show that α2-macroglobulin is responsible for a protease inhibitory activity of an uncharacterized sample is the demonstration that the sample inhibits proteolytic but not amidolytic activity of an exogenously added protease. i am not aware of any other enzyme inhibitor that operates by this unique “trap” mechanism. the conformational change in the proteaseactivated α2-macroglobulin molecule exposes a domain at its carboxy terminus, the receptor-binding domain, that now targets the molecule, with its entrapped cargo of protease, for receptor-mediated endocytosis and proteolytic degradation by phagocytes and other cells that display the α2macroglobulin receptor at the cell surface (van leuven, 1984). in this fashion, α2-macroglobulin operates as an opsonin that delivers proteases of every enzymatic class to endocytotic cells of the innate immune system for internalization and destruction. several strands of evidence support this unusual scheme for the removal of potentially destructive proteolytic enzymes. active site protease inhibitors block both the proteolytic activity and the amidase and esterase activities against low molecular mass substrates. in contrast, proteases bound to α2-macroglobulin remain active against low molecular mass substrates while losing the ability to cleave peptide bonds of proteins (barrett and starkey, 1973; quigley and armstrong, 1983). this was interpreted to mean that the active site of α2-macroglobulin-bound proteases remained intact but was sterically blocked from reacting with macromolecular substrates. protein sequencing of α2-macroglobulins from mammals (sottrup-jensen et al., 1989) and a variety of invertebrates (iwaki et al., 1996; husted et al., 2002; saravanan et al., 2003) has identified the bait region as a defined stretch of approximately 30 amino acids that contains the protease-sensitive peptide bonds whose cleavage initiates the refolding of α2macroglobulin around the target protease. protein 167 table 1 comparison of α2-macroglobulin with active site protease inhibitors α2-macroglobulin active site inhibitors inhibits the proteolytic activity of proteases without inhibiting the hydrolysis of low molecular mass amide or ester substrates inhibits activity of target proteases against polypeptide and low molecular mass substrates reacts with endopeptidases of diverse catalytic mechanisms and substrate specificities reacts with a narrow spectrum of related proteases shields bound proteases from antibodies and high molecular mass active site inhibitors bound proteases remain reactive with antibodies presence of a unique internal reactive thiol ester group internal thiol ester is found only in proteins of the α2macroglobulin family and c3 families (the tep superfamily) sequencing has facilitated the molecular characterization of the receptor-recognition domain that is exposed by proteolytic cleavage (sottrupjensen et al., 1986; van leuven et al., 1986; enghild et al., 1989b; holtet et al., 1994). the molecular compaction responsible for the physical entrapment of a protease molecule was initially shown by the retarded elution of the proteasereacted form during gel filtration chromatography and by the increased electrophoretic mobility of the reacted form during non-denaturing polyacrylamide gel electrophoresis. this latter observation generated the term for the reacted and more compact form of the molecule as the “fast form” (barrett et al., 1979). the molecular compaction has also been demonstrated by direct electron microscopic comparison of α2-macroglobulin molecules before and after reaction with a protease (armstrong et al., 1991). the spectrum of proteases that interact with α2macroglobulin is unusually broad and includes proteases of all major enzymatic classes. although active-site protease inhibitors have been shown to function in immune defense (kanost, 1999), the promiscuous reactivity of α2-macroglobulin has adapted this single molecule to function as a particularly effective scavenger of the novel proteases introduced by parasites and pathogens. indeed, the α2-macroglobulins of vertebrates and invertebrates have been shown to react against the exoproteases of important parasites (freedman, 1991; araujo-jorge et al., 1992; fryer et al., 1996; srimal and armstrong, 1996; scharfstein, 2006). the broad spectrum of reactivity of α2macroglobulin contrasts with the much more restricted reactivity of the naturally-occurring activesite protease inhibitors and is another feature that can be used to demonstrate that an uncharacterized protease inhibitor is, indeed, α2-macroglobulin. interestingly, some microbial proteases are immune to certain versions of α2-macroglobulin. in some instances, the protease is too large to fit into the interior hydrophilic pocket of the reacted form of α2-macroglobulin [the collagenase of chlostridium perfringens has a molecular mass greater than 100 kda (abe et al., 1989)] but other non-reactive enzymes cleave peptide bonds not found in the bait region of certain versions of α2-macroglobulin. human α2-macroglobulin lacks the lysine residues in the bait region targeted by the lysyl endopeptidase of acromobacter lyticus and fails to react with that enzyme (ikai et al., 1999). a related example of this restriction is the relative insensitivity of the proteases of the limulus clotting system to limulus α2-macroglobulin, but with a sensitivity of those same proteases to human α2-macroglobulin (armstrong et al., 1984; iwaki et al., 1994). the limulus clotting protease specifically cleaves the clottable protein, coagulogen, at the carboxyl sides of two arg residues with the sequences leu-gly-arg and ser-gly-arg, which are conserved in coagulogens isolated from the four extant species of horseshoe crabs (iwanaga et al., 1992). limulus α2macroglobulin lacks the target sequence in the bait domain, but met-gly-arg is present in human α2macroglobulin, which may account for the differences in reactivity of these two forms of α2macroglobulin to limulus clotting enzyme. presumably, this is adaptive because blood clotting might be seriously compromised if the proteases necessary for the clotting reaction were rapidly inhibited by the principal protease inhibitor in the hemolymph. a similar situation may obtain in the hemolymph of the crayfish, pacifastacus, in which the proteases involved in activation of the prophenol oxidase system, a component of the systems involved in humoral immunity in crustaceans, are unaffected by the α2-macroglobulin found in the hemolymph of that organism (hergenhain et al., 1987). entrapment in the α2-macroglobulin cage both prevents the entrapped enzyme from accessing external proteins as substrates for proteolytic attack and protects the entrapped enzyme molecule from inactivation by macromolecular active site protease inhibitors. in other words, the protease molecule cannot get out of the α2-macroglobulin cage, but neither can macromolecular protein inhibitors get in. this has allowed the development of a specific and semi-quantitative assay for α2-macroglobulin, in which an uncharacterized sample is reacted first with trypsin, next with excess soybean trypsin inhibitor (sti, mr 21.5 kda), and then the residual trypsin activity is assayed with the low molecular mass amide substrate, bapna (na-benzoyl-dlarginine p-nitroanilide) (ganrot, 1966; armstrong et al., 1985). any trypsin not sequestered by α2macroglobulin is inactivated by sti and the only enzyme still able to hydrolyze bapna is that which is protected within the α2-macroglobulin cage. in the absence of α2-macroglobulin in the sample, degradation of bapna is zero. in the presence of increasing amounts of α2-macroglobulin, the hydrolysis of bapna is increased in a dosedependent fashion, indicative of increasing amounts of α2-macroglobulin-sequestered, and thus protected, trypsin. a positive reaction in a properly controlled sti-protection assay is a sure indicator for the presence of α2-macroglobulin in the sample, but the assay is subject to false negative responses that can occur if the sample also contains low molecular mass trypsin inhibitors small enough that they can enter the α2-macroglobulin cage and inactivate bound enzyme or if the α2-macroglobulin in the sample contacts proteases during sample collection and storage. because α2-macroglobulin is large, low molecular mass trypsin inhibitors can be removed by dialysis and premature reaction of α2macroglobulin can often be prevented by the inclusion of appropriate cocktails of low molecular mass protease inhibitors that can subsequently be removed by dialysis prior to assay with the stiprotection assay. we have seen now three diagnostic features of the α2-macroglobulin family of protease inhibitors: a broad reactive capacity against proteases of all classes, a unique molecular trap mechanism for interaction with target proteases, and a failure to inactivate the active site of the entrapped protease. a fourth diagnostic feature of the members of the α2-macroglobulin family of proteins is the presence of a reactive internal thiol ester bond linking cysteinyl and glutamyl residues, ⋅⋅⋅⋅g-c-g-e-q-n-m⋅⋅⋅⋅ * s____ c=o 168 which in limulus α2-macroglobulin are cys-999 and glx-1002 (iwaki et al., 1996). the thiol ester bond is cleaved when α2-macroglobulin experiences proteolytic attack on the bait domain, thereby exposing the cysteinyl thiol and the carbonyl of glutamic acid. this was seen by the exposure of a new titratable thiol group, the thiol of cys-999 in limulus α2-macroglobulin (armstrong and quigley, 1987). the newly-exposed carbonyl group of the glutamyl residue is highly reactive and can engage in isopeptide bonding with ε-amino groups of lysines of protein targets in the reaction environment (fig. 1). in human α2-macroglobulin, crosslinking of the thiol esterified glutamic acid is preferentially with the entrapped protease (sottrup-jensen et al., 1990b), whereas with α2-macroglobulin from the american horseshoe crab, limulus, the isopeptide bonds crosslink chains of α2-macroglobulin itself (dolmer et al., 1996). although the internal thiol ester of α2macroglobulin is stable in the intact protein, it will react with small primary amines such as ammonium and methylamine in the absence of proteolysis (fig. 1). an important diagnostic feature of α2macroglobulin is its sensitivity to methylamine. the contribution of α2-macroglobulin to the protease inhibitory activities of a biological sample is inactivated following methylamine treatment. protease clearance although α2-macroglobulin is usually thought of as a protease inhibitor, it might better be considered a protease-binding molecule whose principal function is to deliver its protease cargo to an endocytotic protease clearance pathway. in this context, unreacted α2-macroglobulin serves the recognition function and the protease-reacted fastform α2-macroglobulin serves the delivery function. the role of α2-macroglobulin in protease clearance is especially well illustrated in limulus because α2macroglobulin is the only protease inhibitor in the hemolymph (quigley and armstrong, 1983). clearance was studied by injecting fluoresceinlabeled proteins into the lumen of the heart and using a fluorometer to assay their concentration in blood drawn from the peripheral circulation at various times after injection. native proteins such as hemocyanin or native α2-macroglobulin (fig. 2b) require approximately 10 min to exit the heart and mix with the blood of the peripheral circulation, and then remain at constant concentration. in contrast, trypsin (fig. 2a) or the α2-macroglobulin-trypsin complex (fig. 2b) show rapid clearance from the hemolymph with a half-clearance time of 10 15 min. and maximal clearance at 20 25 min after dispersal from the injection site. clearance of trypsin depends on its catalytic activity because trypsin that has been inactivated by treatment with phenylmethylsulfonylfluoride (pmsf) is not cleared (fig. 2a). during the clearance stage, fluorescent trypsin is associated with a protein of high molecular mass. later, fluorescence reappears in the circulation as a form with mr < 10 kda (melchior et al., 1995). our interpretation of these observations is that the clearance of trypsin is mediated by binding to α2-macroglobulin and the protease-α2macroglobulin complex is then degraded to low molecular mass components that later reappear in the hemolymph. the blood cell appears to be involved in clearance and degradation because fig. 1 activation and cleavage of the internal thiol ester of α2-macroglobulin exposes a new thiol group on the cysteine and a reactive (-carbonyl on the glutamyl residue, which in limulus α2-macroglobulin are cys 999 and glx1002 . the reactive internal thiol ester of members of the α2-macroglobulin protein family is cleaved following proteolysis at the distantly-located protease bait region of the protein. thiol ester cleavage generates an activated (-carbonyl at the glutamyl residue and a free thiol at the cystenyl residue (top line of the diagram). the reactive glutamyl can form amide linkages with proteins (right arm of the diagram). the thiol ester can also react slowly with small primary amines, such as methylamine (left arm of diagram), even in the absence of proteolytic cleavage at the bait region. methylamine treatment eliminates many of the functional activities of α2-macroglobulin in parallel with its inactivation of the thiol ester. in general, sensitivity of a molecular function such as protease inhibition to treatment with methylamine is a useful test for the possibility that that function is dependent on the activity of a protein of the thiol ester protein family. fluoresceinated trypsin and fast-form α2macroglobulin associate with the blood cells at the very stages that these proteins are being cleared from the hemolymph (fig. 2b). the conformational change of protease-reacted α2-macroglobulin serves both to entrap the protease and to expose a previously cryptic domain at the carboxyl terminus, the receptor-binding domain (fig. 3) (sottrup-jensen et al., 1986; van leuven et al., 1986; enghild et al., 1989b; holtet et al., 1994), that is recognized by cell surface receptors, leading to the binding and endocytosis of the protease-α2macroglobulin complex (van leuven, 1984). this removes the protease from the circulation, thereby neutralizing its potentially damaging actions. the three major steps in protease recognition, capture, and delivery are mediated in part by three identifiable domains of the α2-macroglobulin molecule, the bait region, the thiol ester domain, and the receptor-recognition domain (fig. 3). presumably, there are additional domains that are under molecular strain in the unreacted form of α2macroglobulin and that are most actively involved in the active change in molecular shape following protease reaction and hydrolysis of the thiol ester, putative “hinge-domains”. in the absence of high resolution information of the structure of the native and fast-forms of the molecule, these have not been characterized. the best-characterized mammalian receptor for protease-reacted α2-macroglobulin (jensen et al., 1989; moestrup and gliemann, 1989; ashcom et al., 1990) has been identified as low density lipoprotein receptor-related protein-1 (lrp/α2m-r, a.k.a. lrp1, cd91) (kristensen et al., 1990; strickland et al., 1990). cd91 is synthesized as a 600 kda protein with a single transmembrane domain and is a member of the low density lipoprotein receptor family (for review see lillis et al., 2008). cd91, in addition to binding the α2-macroglobulin-protease complex, is also active as a receptor for a diverse variety of other exogenous ligands and plays an important regulatory role in a diverse array of important cellular processes (gonias et al., 2004; 169 fig. 2 clearance of proteases from the hemolymph of limulus is mediated by α2-macroglobulin. fluoresceinated proteins were injected into the lumen of the heart and their concentration in the peripheral hemolymph drawn from the leg joints was measured with a fluorometer. fluoresceinated protein reached the periphery by 5 10 min after injection. enzymatically active trypsin, but not trypsin inactivated by treatment with pmsf, was substantially cleared from the hemolymph by 30 min (fig. 1a). the fluorescence that appeared at later times was of low molecular mass, and presumably consisted of peptide digest products of the injected protein. fast-form, but not slow-form limulus α2-macroglobulin showed a similar pattern of clearance from the hemolymph. during the period of clearance of fast-form α2-macroglobulin from the hemolymph, fluorescence accumulated in the blood cells. this is interpreted to indicate that clearance of the introduced protease is mediated by its binding to α2-macroglobulin and that the blood cells participate in the uptake and clearance of the α2-macroglobulin-protease complex. may et al., 2007). of practical interest is its ability to bind two or more mols/mol of a 39 kda endogenous ligand, lrp receptor-associated protein (rap) (ashcom et al., 1990; herz et al., 1991). rap functions as a dedicated chaperone and regulator of members of the ldl receptor family of proteins (bu, 1998) and its high affinity binding makes rapconjugated sepharose an appropriate affinity matrix for purification of these proteins. to date, a receptor for protease-reacted α2macroglobulin has not been identified in an invertebrate. the family of proteins that includes vertebrate cd91 is, like α2-macroglobulin itself, an ancient family that was present prior to the great evolutionary radiation that established the divergent deuterostome and protostome invertebrate superphyla. representatives of the cd91 family have been found in modern representatives of lineages that diverged at the time of the precambrian radiation, notably in vertebrates, the nematode, caenorhabditis elegans (yochem and greenwald, 1993), and the arthropod, drosophila melanogaster (locus tag dmel cg33087). whether invertebrate orthologs of vertebrate cd91 operate as α2-macroglobulin receptors has not been decided. the limulus blood cell does contain a protein of very high molecular mass that, like cd91, binds rap (aimes et al., 1995) and human cd91 binds to protease-reacted limulus α2-macroglobulin (iwaki et al., 1996). although these observations hint at a possibility that the clearance pathway for protease-reacted limulus α2-macroglobulin involves an ortholog of cd91, they fall well short of any convincing demonstration of that prospect. phylogenetic distribution of the α2macroglobulins application of the criteria cited above has permitted the protein-based demonstration of α2macroglobulin in the plasma of lower vertebrates (starkey and barrett, 1982), arthropods, and molluscs (armstrong and quigley, 1999). the initial demonstration of α2-macroglobulin in an invertebrate occurred when james quigley and i stumbled upon the molecule while looking for fibrinolytic proteases active in the dissolution of blood clots in the american horseshoe crab, limulus polyphemus. we failed to find a fibrinolytic system, but we did find a hemolymph protease inhibitor that 170 fig. 3 domain structure of limulus α2-macroglobulin. based on the amino acid sequence of limulus α2macroglobulin (iwaki et al., 1996), it is possible to identify important functional domains by comparison with the already established domain structure of human α2-macroglobulin. the amino terminal end of limulus α2macroglobulin has a unique stretch identified as the c8 (domain, based on modest sequence identity with the human defense protein c8 (of the mammalian complement pathway. the bait region contains a sequence of amino acids that present target sites for nearly all proteases. proteolysis of the bait region initiates the compaction of α2-macroglobulin that is responsible for entrapment of the molecule of reacting protease. the thiol ester domain contains the unique thiol ester bond linking cysteine-999 and glutamic acid-1002. the receptor-binding domain at the carboxyl terminus of the polypeptide chain is exposed following molecular compaction and makes the protease-reacted form of α2-macroglobulin a target for binding to the cell surface α2-macroglobulin receptor. suppressed the action of serine, thiol, and metalloproteases against [14c]-casein, a protein substrate, but that failed to suppress hydrolysis of the amide substate bapna by trypsin. the antiprotease activity of hemolymph was eliminated if limulus hemolymph was treated with methylamine. this suggested the presence of α2-macroglobulin in the hemolymph of this invertebrate (quigley et al., 1982). our early analysis was facilitated by the absence of other trypsin inhibitors in limulus hemolymph (quigley and armstrong, 1983). subsequently, we showed that limulus hemolymph protected trypsin from inactivation by the macromolecular active-site inhibitor, soybean trypsin inhibitor (armstrong et al., 1985), that purified limulus α2-macroglobulin did indeed contain an internal thiol ester domain (armstrong and quigley, 1987), and that the protein from limulus shows significant sequence identity in key functional domains with human α2-macroglobulin (sottrupjensen et al., 1990a; iwaki et al., 1996). although overall sequence identity between human and limulus α2-macroglobulin is relatively low, the thiol ester domain shows significant sequence conservation, the receptor binding domains shows conservation of key basic amino acids, and the bait regions shows no sequence similarity. the strict conservation of the thiol ester domain of the various α2-macroglobulins has facilitated the molecular cloning of the genes for a variety of invertebrates from c-dna libraries. with the advent of complete genomic sequences for an everincreasing variety of species, data mining has revealed additional examples of genes encoding α2macroglobulin from these species. for example, the presence of genes for α2-macroglobulin in gramnegative bacteria was first demonstrated using the data-mining strategy (budd et al., 2004). these methods have identified both representatives of α2macroglobulin that, upon further investigation, have shown the unique suites of functional and structural characteristics of canonical α2-macroglobulin, and have also revealed novel related proteins, several of which await functional characterization. the α2-macroglobulin family includes the canonical α2-macroglobulins, a variety of similar protease-binding proteins, including pregnancyzone protein of humans, α1i3 of the rat, ovomacroglobulin of avian and reptile eggs, and proteins to be discussed below that were initially identified from the vertebrate complement system (sottrup-jensen, 1987). additionally, humans have two newly identified and incompletely characterized members of the family, cd109, which is linked to the cell surface by gpi-bonding (lin et al., 2002), and cpamd8, an acute-phase protein expressed by kidney, brain, and testis (li et al., 2004), and the settlement-inducing protein of the barnacle balanus amphitrite is an α2-macroglobulin (dreanno et al., 2006). the different α2-macroglobulins show differing orders of multimerization. human (jones et al., 1972; swenson and howard, 1979) and snail (bender and bayne, 1996) α2-macroglobulins are homotetramers, whereas many forms of the α2macroglobulins from a variety of lower vertebrates and arthropods are homodimers (starkey and barrett, 1982; feldman and pizzo, 1984; sand et al., 1985; feldman and pizzo, 1986; spycher et al., 1987; enghild et al., 1990; armstrong et al., 1991), and a few monomeric forms of α2-macroglobulin have been identified (enghild et al., 1989a). as indicated above, the molecular homologues of the canonical α2-macroglobulins have a wide phylogenetic distribution and share a defined suite of structural and functional characters (table 2). thiol ester protein family α2-macroglobulin is one of the founding members of the larger protein family, the thiol ester protein (tep) super-family (dodds and law, 1998). the thiol ester proteins share a similar domain 171 172 table 2 molecular and functional conservation of the α2-macroglobulins character specific attribute ref. 1. genetic identity overall peptide sequence identity 28-29 % identity of limulus and mammalian α2macroglobulins (sottrup-jensen et al., 1984; kan et al., 1985; gehring et al., 1987; van leuven et al., 1992; iwaki et al., 1996) bait region bounded by pxxc and exxr consensus sequences in limulus and mammalian α2macroglobulin (sottrup-jensen et al., 1989; iwaki et al., 1996) thiol ester domain >50 % identity for α2-macroglobulins of mammals, crustaceans, chelicerates, cephalopods, and gastropods (sottrup-jensen et al., 1984; spycher et al., 1987; hall et al., 1989; enghild et al., 1990; sottrup-jensen et al., 1990a; stocker et al., 1991; thogersen et al., 1992; bender and bayne, 1996; iwaki et al., 1996) receptor recognition domain conservation of two of the key lys residues of human α2-macroglobulin in limulus α2macroglobulin; of 73 residues conserved amongst the mammals, 45 are conserved in limulus α2macroglobulin (nielsen et al., 1996; iwaki et al., 1996) 2. biochemical homology susceptibility of bait region to proteolysis by proteases of all catalytic mechanisms reactivity of purified α2-macroglobulins of mammals, arthropods and molluscs to serine, cysteine, and metalloproteases (starkey and barrett, 1977; quigley and armstrong, 1985; hergenhahn and soderhall, 1985; enghild et al., 1990; thogersen et al., 1992; bender and bayne, 1996) molecular compaction; noncovalent trapping of proteases molecular compaction of protease-reacted α2macroglobulin of mammals and limulus (barrett and starkey, 1973; barrett et al., 1979; quigley et al., 1991; armstrong et al., 1991) covalent isopeptide bonding of thiol esterified glutamyl residue covalent bonding to trapped protease in mammalian α2-macroglobulin; covalent crosslinking to lys-254 of partner chain of α2macroglobulin dimer in limulus (sottrup-jensen et al., 1980; sottrup-jensen et al., 1990b; jacobsen and sottrup-jensen, 1993; dolmer et al., 1996) protease treatment exposes the recognition stretch for receptor-binding thiol ester-reacted limulus α2-macroglobulin binds the human α2-macroglobulin receptor (sottrup-jensen et al., 1986; van leuven et al., 1986; enghild et al., 1989b; iwaki et al., 1996) 3. functional similarity protease clearance mammals and limulus utilize α2-macroglobulin for the clearance of proteases from the internal milieu (feldman et al., 1985; davidsen et al., 1985; melchior et al., 1995) 173 architecture (janssen et al., 2005; doan and gettins, 2007), sequence similarity in key functional domains, and most, but not all, contain a stable internal thiol ester bond linking the cysteinyl and glutamyl residues of the thiol ester motif. members of this family that lack the internal thiol ester include complement component c5 (tack, 1983) and ovostatin from the albumin of the chicken egg (nagase et al., 1983) and drosophila mcr (ncbi accession number y11116). in all three proteins, the cys has has been substituted with another residue (nielsen and sottrup-jensen, 1993). but these proteins retain the other signature features that validate their assignment to the thiol ester protein family and ovostatin retains the ability to capture proteases by the molecular trap mechanism (nagase and harris, 1983). the taxonomy of the tep family is still a work in progress. one classification identifies two subfamilies, c3 and a2m (sottrup-jensen et al., 1985; fujito et al., 2010) named for their founding members, human complement component c3 and human α2-macroglobulin, respectively. a second scheme posits three distinct subfamilies, c3, a2m, and insect tep. members of the insect teps show sequence relatedness intermediate between the c3 and a2m subfamilies and functional similarity to members of the c3 subfamily. they also, like c3, the canonical member of the c3 subfamily, have a histidine residue (his 951 in drosophila tep1r; his 1,106 in human c3) in position to convert the reactive glx of the activated thiol ester to an intermediate that favors its subsequent covalent linkage to hydroxyl groups rather than showing the default reactivity with amines (dodds et al. 1966; law and dodds 1997). this residue is asp, asn, or ala in thiol ester proteins where the covalent reactivity of the thiol ester is with amines. subfamily membership is based on amino acid sequence and functional domain relatedness and is reflective of important functional differences between the members of the subfamilies. the best-characterized function of the a2m family members is protease binding, whereas the best-characterized function of the members of the c3 and insect tep proteins is covalent binding to the surfaces of foreign cells to mark them for immune destruction (dodds and sim, 1997; blandin et al., 2008). as will be described below, some members of the a2m subfamily, with well established protease-binding characters also, like members of the c3 and insect tep subfamilies, bind to the surfaces of foreign cells and facilitate their phagocytotic uptake. i am unaware of any reports of members of the c3 or insect tep proteins showing the unique protease capture abilities of the members of the a2m subfamily. the common ancestor of the tep family is presumably ancient because representatives of both the c3 and a2m subfamilies are found in cnidarians, which are basal metazoans. the sea anemone, haliplanella has two members of the c3 subfamily and two of the a2m subfamily (fujito et al., 2010). representatives of both families have been identified in vertebrates (sottrup-jensen et al., 1985), non-vertebrate chordates (nonaka et al., 1999), echinoderms (alsharif et al., 1998), and representatives of the ecdysozoans, the horseshoe crab (zhu et al., 2005; ariki et al., 2008), and the lophotrochozoans, a squid (castillo et al., 2009) and a clam (pradoalvarez et al., 2009). the insect tep subfamily has been reported only from insects and possibly nematodes (blandin and levashina, 2004), but not from other arthropods. it is important to note that the subdivision of the thiol ester proteins into just two or just three major subfamilies is not yet definitive. there are several tep proteins that may not fit within a twoor three-subfamily classification schemes (lin et al., 2002; li et al., 2004; dreanno et al., 2006). although the best-characterized function of the proteins of the a2m subfamily, both from vertebrates and invertebrates, is protease binding, biochemical pathways other than protease clearance have been identified. the α2macroglobulins also modulate cell proliferation and cell survival pathways (lamarre et al., 1991; shi et al., 2008), protease-independent pathways of the innate immune system (swarnakar et al., 2000; arnold et al., 2006; craig-barnes et al., 2010), antigen delivery in the operation of the adaptive immune system (bowers et al., 2009), and can operate as molecular chaparones (yerbury et al., 2009). as mentioned above, cd91, the canonical cell-surface receptor for protease-reacted α2-macroglobulin recognizes and binds a diverse suite of ligands and is an essential agent for a diverse array of important biological processes. the best-characterized function for members of the c3 subfamily is their covalent binding to the surfaces of foreign cells to target them for immune destruction, a function shared with members of the insect tep subfamily. an appealing example of this is the protein tep1, the mediator of an important pathway for the immune defense of the mosquito vector against the protozoan parasite, plasmodium falciparum, the agent of human malaria (reviewed in blandin et al., 2008). tep1 is a member of the thiol ester protein family from the anopheles mosquito. tep1 binds to bacteria and plasmodium cells and targets the cells for phagocytosis. the experimental elevation of the concentration of tep1 in the hemolymph of the mosquito reduces infection rates and experimental reduction increases susceptibility of the mosquito to infection (reviewed in volohonsky et al., 2010). different members of the insect teps show specialization of their targeting to the surfaces of different classes of microbes, for example, with drosophila mcr targeting candida albicans cells, drosophila tepiii targeting s. aureus, and drosophila tepii targeting e. coli (stroscheinstevenson et al., 2006). prospects for future research it is always presumptuous to predict the direction of research in any field of biology; the natural world holds many unexpected surprises and the ingenuity of biologists to identify and investigate those surprises seems without limit. that being said, some prospects for the future study of α2macroglobulin and of other members of the thiol ester protein superfamily are appropriate for a 174 review of this topic. a full characterization of the function(s) of the α2-macroglobulins in a diverse array of species will involve both the functional characterization of the molecule in vitro, which has been the principal topic of this review, and the elucidation of its function in vivo. what functions in the animal require α2-macroglobulin? the gene knock-out mouse has been available for a decade and a half and shows a surprisingly mild phenotype (umans et al., 1995; umans et al., 1999). interpretation is complicated by the artificially sanitary conditions of the life of the laboratory mouse. this mouse model will be particularly interesting when its sensitivity to an extended suite of proteasewielding pathogens is investigated (coutinho et al., 1999). in appropriate model invertebrates, rnai knock-down trials are possible and reduction of expression of various teps have been shown to significantly diminish host resistance to infection by some, but not all pathogens (buresova et al., 2009; volohonsky et al., 2010). one imagines refinements on this strategy where modified versions of α2macroglobulin are used to reconstitute function in α2-macroglobulin-depleted animals. in this context, the modified α2-macroglobulin constructs might feature bait regions with enhanced or restricted sensitivity to the various proteases of an appropriate pathogen (ikai et al., 1999). these could provide information on the roles of different proteases of the pathogen in the infection cycle and on the precise roles for α2-macroglobulin in immune defense. for example, might forms of α2-macroglobulin modified to include the appropriate novel cleavage sites for previously unrecognized microbial proteases now offer protection to the host from those bacteria? a related challenge to the functional characterization of the diverse members of the thiol ester protein family is the high resolution structural characterization of representative thiol ester protein family members for a detailed understanding of the relation of protein structure to the several functions of different members of this protein family. the goal of establishing a detailed understanding of the relations between protein structure and function is best exemplified by the characterization of human c3 (janssen et al., 2005) and an insect tep (baxter et al., 2007). the establishment of a high resolution structural characterization of α2-macroglobulin has proven elusive (jenner et al.,1998) but the insights provided by the characterization of the domain structure of c3 have been used to develop a model for human α2-macroglobulin (doan and gettins, 2007). one additional bonus of this line of research will be the provision of information for the refinement of the still-unsettled molecular taxonomy of the thiol ester protein superfamily. it will be interesting and illuminating to identify and characterize the clearance pathways for protease-reacted α2-macroglobulin in taxa other than mammals. to date, the sole experimental study is the investigation of the protease clearance pathway for the horseshoe crab (melchior et al., 1995) and this has implicated a cell-based clearance pathway that is selective for fast-form α2macroglobulin. initial evidence described above hints at a role for a role for a cell surface receptor with properties similar to mammalian cd91 in this pathway. although cd91 is the best-characterized cell surface receptor for mammalian α2macroglobulin, a second cell surface protein, grp78, has been shown to bind protease-reacted α2-macroglobulin with nm affinity (quinones et al., 2008; gonzalez-gronow et al., 2009). grp78 is a protein found in diverse eukaryotes and it will be interesting to discover if this or other undiscovered receptors operate to bind α2-macroglobulin in different metazoans. in mammals, both α2-macroglobulin and its receptor, cd91, have been shown to contribute to the operation of a number of physiological processes that are independent of protease clearance. it will be interesting to identify and characterize the possible diversity of functions of α2macroglobulin in invertebrates. a scattering of examples have already been identified. in invertebrates, α2-macroglobulin also regulates the activities of other immune effector proteins. in the shrimp, penaeusmonodon, α2-macroblobulin binds syntenin (tonganunt et al., 2005). syntenin is an acute-phase protein that is dramatically upregulated in shrimp infected with the white spot syndrome virus. in the horseshoe crab, limulus, protease-activated, but not native α2-macroglobulin binds and inactivates the cytolytic actions of limulin (armstrong et al., 1998; swarnakar et al., 2000). cytolytic destruction of foreign cells is a ubiquitous strategy of metazoan immune systems (canicatti, 1990; gabay, 1994). in mammals, cytolytic destruction of foreign cells is mediated in part by the multi-protein complement system (law and reid, 1995), whereas the hemolymph cytolytic pathway of limulus is mediated by the single protein limulin, a sialic acid-binding member of the pentraxin gene family (armstrong et al., 1996; harrington et al., 2008). the adaptive significance of the α2macroglobulin-limulin binding reaction has not been identified, but the pentraxins of limulus are multifunctional mediators of immunity, with potentially important roles in the inactivation of bacteral lipopolysaccharide (ng et al., 2004) and in the formation of hydrophilic pores across the lipopolysaccharide-rich outer membrane of gramnegative bacteria (harrington et al., 2009). it will be interesting to discover if the binding to proteasereacted α2-macroglobulin affects other functions of the limulus pentraxins or the pentraxins of other invertebrates. perhaps the reduced cytolytic activity of limulin complexed with α2-macroglobulin is correlated with augmentation of other immune functions of limulin. the recent identification of members of the c3 subfamily of tep proteins in diverse invertebrate taxa (zhu et al., 2005; fujito et al., 2010) and the insect tep subfamily in insects (blandin and levashina, 2004; blandin et al., 2008) have opened fertile fields for investigation. functional characterization of proteins of this class indicates that they operate much as c3 of the vertebrate complement system by their covalent binding to the surfaces of foreign cells to mark them for 175 subsequent immune destruction. it will be interesting to determine if this functional characterization is universal amongst metazoans or whether there is functional diversity for members of the c3 family from different taxa. for example are there instances where proteins of the c3 or insect tep sub-families function in a manner similar to the a2ms as protease binding proteins in addition to their canonical cell-binding functions or are there members of the a2m family that, in addition to the display of an ability to bind proteases, also function as opsonins that promote the phagocytosis of foreign cells that have become decorated with surface-associated α2-macroglobulin? for example, murine α2-macroglobulin binds to the surfaces of the pathogen, trypanosome cruzi (coutinho et al., 1997), and a form of α2-macroglobulin from a hard tick binds target bacteria and promotes their ingestion by phagocytes (buresova et al., 2009). conclusion it is the solemn duty of every animal to live to adulthood and to reproduce the species. survival requires efficient means to thwart the myriad invading pathogens that would compromise that survival. since many invertebrates regularly live to a considerable age, at least 20 years for limulus, 80 years for lobster, and 375 years for the ocean quahog, a mollusc (finch, 1990; philipp and abeke, 2010), while inhabiting highly septic environments, they have to possess efficient immune processes. these are largely of the innate class of immune systems, because invertebrates lack lymphocytes and the traditional rag-mediated adaptive immune system, which are restricted to the vertebrate lineage (marchalonis and schluter, 1990; agrawal et al., 1998). as we and others have shown, certain of these innate immune systems arose early in evolution and have been faithfully preserved in species of diverse lineage, and inhabiting very different environments and displaying very different life styles. for example both vertebrates and arthropods utilize the pentraxin (a.k.a., the c-reactive protein) and the tep families of proteins to protect from invading parasites. interest in immunity in invertebrates is driven by a basic curiosity in how species other than our own survives pathogenic attack and may have application to the development ___________________________________________________________________________ list of abbreviations: a2m, the α2-macroglobulin family of the tep protein superfamily; bapna, na-benzoyl-dl-arginine pnitroanilide; c3, the complement component 3 family of the tep protein superfamily; cd91, the canonical cell-surface receptor for fast-form α2macroglobulin and also known as lrp1, ldlreceptor-related protein/α2-macroglobulin-receptor; ma, methylamine, a small primary amine that reacts with the internal thiol ester of the teps; pmsf, phenylmethylsulfonylfluoride; rap, cd91associated protein, a 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molluscs is constituted by hemocytes and humoral factors that cooperate for the protection of the organism, triggering a wide range of immune responses. in molluscs, immune responses include phagocytosis, encapsulation, respiratory burst leading to reactive oxygen species (ros) production and nitric oxide (no) synthesis, release of antimicrobial molecules and the activation of phenoloxidase system. these responses are mediated firstly by a variety of hemocyte receptors binding to ligands that results to a cascade of signaling events. the processes of hemocytes adhesion to and migration through extracellular matrix (ecm) proteins play a crucial role in cell immunity. results suggest that cadmium and oxidants induce adhesion to and migration through ecm proteins in mytilus gallorovincialis hemocytes with the involvement of na+/h+ exchanger (nhe), phosphatidylinositol-3 kinase (pi-3k), protein kinase c (pkc), nadph oxidase, ros and no as well as with α2 integrin subunit. furthermore, the data so far suggests the involvement of additional signaling molecules such as mitogen-activated protein kinases (mapks), signal transducers and activators of transcription (stats), c-jun n-terminal kinase (jnk), extracellular signal-regulated kinase (erk), cyclic adenosine monophosphate (camp), responsive element binding protein (creb) and nuclear factor kappa b (nf-kb) in molluscs immunity. further research in mollusc immune system may lead to a more sufficient protection and to a better control of these economically important organisms. key words: mytilus galloprovincialis; immune response; adhesion; migration; integrin   introduction immune responses are highly complex including a variety of different cellular and molecular processes. the study of the immune processes in invertebrates is of great importance from ecological, economic and public health points of view (peteiro et al., 2007). furthermore, the study of the immune mechanisms in molluscs is also significant due to their susceptibility to infection by bacteria, viruses, and parasites that makes them transmitters of many diseases affecting different vertebrate species (barcia and ramos-martinez, 2008). however, the available data on the immune responses in invertebrates should be implemented (humphries and yoshino, 2003; tiscar and mosca, 2004; canesi et al., 2006; mydlarz et al., 2006; ottaviani, 2006; barcia and ramos-martinez, 2008). in this review we will refer to the phagocytic behavior of m.  galloprovincialis hemocytes. ___________________________________________________________________________ corresponding author: martha kaloyianni zoology department, aristotle university of thessaloniki 54124 thessaloniki, greece e-mail: kaloyian@bio.auth.gr immune system of molluscs among invertebrates, molluscs represent the widest phylum after arthropods. they are considered as excellent bio-indicators and they have been used intensively in research studies. the defense mechanisms of molluscs consist firstly of chemicophysical barriers (external skeletons, cockles, cuticles, mucus) that prevent host invasion and secondly of the circulating hemocytes and humoral factors that operate in co-ordination triggering a wide range of immune responses (renwrantz, 1990; rinkevich and muller, 1996; hine, 1999). according to mydlarz et al. (2006) the three essential components of innate immunity in invertebrates are: 1) phagocytosis which represents the cell-mediated immunity, 2) activation of humoral responses that result to opsonization, coagulation and melanization and 3) the production of humoral antimicrobial components. m. galloprovincialis immune system consists in at least four subtypes of hemocytes charged with different tasks in host defense: large granular (r1), large semigranular (r2), small semigranular   11 fig. 1 adhesion of mytilus galloprovincialis hemocytes to laminin, collagen iv and oxidized collagen iv after treatment with cadmium and inhibitors of nhe, pkc, pi-3k, nadph oxidase and no synthase. hemocytes were pre-incubated with the inhibitors cariporide (20 nm), go6976 (500 nm) and gf109203χ (10 μμ), wortmannin (50 nm), dpi (10 μm) and l-name (10 μμ) for 15 min at 20 0c, cdcl2 (5 μμ) was then added and the samples were incubated for 30 min at 20 0c. the results show the means of at least 4 experiments ± sd. the level of significance of the differences between the samples was calculated by anova with a student-newman-keuls post-hoc test (p<0.05). * indicates significant difference of the sample value with the control value. values that share a are significant different between each other. b,c,d indicate significant difference between each sample value with the respective control value (cd alone) (koutsogiannaki, 2008) (r3), and small hyaline (r4) hemocytes (garciagarcia et al., 2008). while large granular (r1), large semigranular (r2), and small semigranular (r3) cells are thought to be phagocytic, and capable of activating the respiratory burst, small hyalinocytes (r4) lack these two capabilities. nevertheless, all hemocyte subpopulations seem to be capable of nitric oxide (no) production (garcia-garcia et al., 2008). the presence of many hydrolytic enzymes in the large granules indicates their connection with lysosomes (pipe, 1990). ottaviani et al. (1998a) proposed just one type of immunocytes in different stages (young and old) in m. galloprovincialis supporting mix’s (1976) suggestion that hyalinocytes (agranular type) are a proliferative condition that after various stages mature into granulocytes. two of these four subtypes, large granular and large semigranular cells share common features with the mammalian professional phagocytes (garcia-garcia et al., 2008). furthermore, hemocytes secrete humoral factors that play a fundamental role in the innate immune responses in molluscs including lysosomal enzymes, agglutinins or lectins, cytokine-like molecules, bioactive peptides, no, and antimicrobial peptides (ottaviani, 2006). in addition to these, the defense mechanisms of mussels include the activation of phenoloxidase system (little et al., 2005). hemocytes are also involved in detoxification through accumulation of metallic and organic xenobiotics in their well developed lysosomal system (cajaraville et al., 1995). phagocytosis represents the main cellmediated immune response and is mediated by the hemocytes. it is comprised by different phases involving recognition, chemotactic migration, adhesion, ingestion, destruction and elimination of foreign cells (tiscar and mosca, 2004). we will focus on properties such as cell adhesion and cell migration through extracellular matrix proteins collagen iv, laminin-1 and on the signaling molecules that mediate these processes. cell adhesion, cell migration and extracellular matrix proteins among the immune responses in mussels, the processes of hemocyte adhesion to and migration through extracellular matrix play a crucial role in cell immunity. hemocyte adhesion is an initial step in phagocytosis of foreign particles (hynes and lander, 1992). cell-cell adhesion and cellsubstratum adhesion (e.g., to extracellular matrix) are critical for the development, maintenance and 12 fig. 2 migration of mytilus galloprovincialis hemocytes through collagen iv and oxidized collagen iv after treatment with cadmium and inhibitors of nhe, pkc, pi-3k, nadph oxidase and no synthase. hemocytes were pre-incubated with the inhibitors cariporide (20 nm), go6976 (500 nm) and gf109203χ (10 μμ) for 15 min at 20 0c, cdcl2 (5 μμ) was then added and the samples were incubated for 30 min at 20 0c. the results show the means of at least 4 experiments ± sd. the level of significance of the differences between the samples was calculated by anova with a student-newman-keuls post-hoc test (p<0.05). * indicates significant difference of the sample value with the control value. a,b indicate significant difference between each sample value with the respective control value (cd alone) (koutsogiannaki, 2008) function of multicellular organisms. moreover, hemocyte adhesion to different surfaces can result in important cellular behaviors such as parasitic encapsulation and hemocyte-mediated clotting responses (yoshino, 1998). hemocyte migration depends on directed cytoskeletal reorganization, ion transport membrane recycling by endocytosis and formation of focal adhesion sites with extracellular matrix. m. galloprovincialis hemocytes migration through extracellular matrix (ecm) proteins was reported after heavy metals (koutsogiannaki, 2008) and interleukin (il)8 affect (ottaviani, 2000). chemotaxis was also detected in immunocytes of the hard clam mercenaria mercenaria as a result of bacteria stimuli (fawcett and tripp, 1994). ecm plays a central role in the structure and maintenance of tissue architecture (adams and watt, 1993). it is now evident that ecm turnover is a critical step in tissue remodelling that accompanies many physiological as well as pathological processes in vertebrates, invertebrates and plants (massova et al., 1998). the macromolecules that are present in all extracellular matrices include collagen, proteoglycans and glycoproteins (mainly laminins). the collagens are a family of extracellular matrix proteins involved in cell adhesion, chemotaxis and migration, and the dynamic interplay between cells and collagens regulates tissue remodelling during growth, differentiation, morphogenesis and wound healing. cells encounter collagen in a number of different ways. cells may stably adhere to collagen in tissues and thus receive survival signals (e.g., dermal fibroblasts), migrate through the collagen-rich stroma as part of a normal morphogenic process (e.g., mammary gland branching) or in disease (e.g., tumour metastasis), or interact with collagen as a result of injury (e.g., homeostasis). interestingly, molluscan hemocytes have been reported to be involved in collagen synthesis and extracellular matrix deposition (serpentini et al., 2000). in accordance with these, studies in sections of integument from bivalve species suggest that molluscan integumental ecm contains collagens similar to type i, iv, v and vi collagens (corbetta et al., 2002). in addition, molluscan motoneurons adhere to laminin and type iv collagen (wildering et al, 1998). furthermore, results suggest that hemocytes after treatment with either cadmium or oxidants adhere to and migrate through collagen iv and oxidized collagen iv at a higher degree compared to control cells (koutsogiannaki, 2008) (figs 1-4). it is also suggested that m. galloprovincialis hemocytes adhere to collagen with the involvement of α2 integrin subunit (koutsogiannaki, 2008) (fig. 5) apart from collagens, laminins are also components of the extracellular matrix that determine the histoarchitecture and provide cells with biological information. the laminins are the major family of non collagenous heterodimeric glycoproteins that provide an integral part of the structural scaffolding in almost every tissue of an organism. it has been demonstrated that laminins are mainly involved in the organization of the basal membrane network and are also present in cellassociated extracellular matrices. they are involved in multiple physiological processes including cell proliferation, differentiation, migration, adhesion and survival. the laminins are found as trimeric proteins 13 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t2r-43cc954-g&_user=604493&_coverdate=03%2f31%2f2001&_rdoc=1&_fmt=full&_orig=search&_cdi=4925&_sort=d&_docanchor=&view=c&_acct=c000059656&_version=1&_urlversion=0&_userid=604493&md5=fc7fb64f38f647acc255c8becabaecbf#bib1 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t2r-43cc954-g&_user=604493&_coverdate=03%2f31%2f2001&_rdoc=1&_fmt=full&_orig=search&_cdi=4925&_sort=d&_docanchor=&view=c&_acct=c000059656&_version=1&_urlversion=0&_userid=604493&md5=fc7fb64f38f647acc255c8becabaecbf#bib1 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t2r-43cc954-g&_user=604493&_coverdate=03%2f31%2f2001&_rdoc=1&_fmt=full&_orig=search&_cdi=4925&_sort=d&_docanchor=&view=c&_acct=c000059656&_version=1&_urlversion=0&_userid=604493&md5=fc7fb64f38f647acc255c8becabaecbf#bib16 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t2r-43cc954-g&_user=604493&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=c000059656&_version=1&_urlversion=0&_userid=604493&md5=1c26a6c00a2ccf01e53e8c6ec9f5fcc8#bbib29 fig. 3 adhesion of mytilus galloprovincialis hemocytes to laminin, collagen iv and oxidized collagen iv after treatment with cadmium, oxidants and antioxidants. hemocytes were incubated with the oxidant rotenone (25 μμ) for 60 min at 20 0c or with the antioxidant nac for 15 hr at 20 0c. the results show the means of at least 4 experiments ± sd. the level of significance of the differences between the samples was calculated by anova with a student-newman-keuls post-hoc test (p<0.05). * indicates significant difference of the sample value with the control value. a,b,c indicate significant difference between each sample value with the respective control value (cd alone) (koutsogiannaki, 2008) which form a cross, giving a structure that can bind to other cell membrane and extracellular matrix molecules (timpl and brown, 1994; yurchenco and cheng, 1994; yurchenco and o'rear, 1994). it is reported that m. galloprovincialis hemocytes after treatment with either cadmium or oxidants adhere to the ecm protein laminin at a higher degree compared to control cells (koutsogiannaki, 2008) (figs1, 3). hemocyte receptors involved in immune responses the processes of adhesion and migration are mediated through hemocytes receptors interactions with binding groups. hemocyte receptors are grouped into several broad groups including lectins (or lectin-like receptors), integrins (or integrinrelated receptors) and growth factor/hormone/cytokine-like receptors (humphries and yoshimo, 2003). lectins, are glycoproteins which serve as recognition molecules by binding to non-self material through carbohydrate recognition sites (ottaviani, 2006). in addition, a unique family of proteins with cho-activity, referred to as fibrinogenrelated proteins or freps has been found to be induced in snails in response to infection (adema et al., 1997; leonard et al., 2001). moreover, selectinlike proteins has been referred to exist in molluscs. selectins constitute a family of cho-reactive membrane proteins that are present in endothelial cells, leukocytes and platelets in mammals. they are adhesion receptors involved in many processes such as leucocyte extravascular trafficking and inflammation (patel et al., 2002). it has also been demonstrated that receptors for platelet-derived growth factor (pdgf-alpha/β) and transforming growth factor β (tgf-β) are present in m. gallorovincialis hemocytes involved in many cellular functions such as phagocytosis and cell motility (ottaviani et al., 1997a; kletsas et al., 1998). moreover, receptors for bioactive peptides such as proopiomelanocortin (pomc) including β-endorphin, adrenocorticotrophic hormone (acth) and alpha-melanotropin receptors as well as insulin-like receptors have been found in molluscan hemocytes (stefano et al., 1989; duvax-miret et al., 1992; ottaviani et al., 1998b; sassi et al., 1998; lardans et al., 2001). furthermore, cytokine-like receptors have been found to be present in molluscan hemocytes as well. it has been shown that bioactive peptides and cytokines in invertebrates are related to cell shape changes and cell migration (hughes et al., 1990; ottaviani et al., 1995), induce no synthase (ottaviani et al., 1995) and increase phagocytic activity by activating the classical signal transduction pathways, i.e., protein kinase a, c and b (ottaviani et al., 1997b). among cytokines, interleukins which belong to chemotactic cytokines also referred as chemokines, are involved in acute inflammation. il-8 has been demonstrated to induce increased phagocytic activity and chemotactic response in m. galloprovincialis hemocytes (ottaviani et al., 2000). barcia et al. (1999) also detected the il-2 receptor to be present in m. galloprovincialis hemocytes. 14 http://www3.interscience.wiley.com/cgi-bin/fulltext/72503858/main.html,ftx_abs#bib269 http://www3.interscience.wiley.com/cgi-bin/fulltext/72503858/main.html,ftx_abs#bib269 fig. 4 migration of mytilus galloprovincialis hemocytes through collagen iv and oxidized collagen iv after treatment with cadmium, oxidants and antioxidants. hemocytes were incubated with the oxidant rotenone (25 μμ) for 60 min at 20 0c or with the antioxidant nac for 15 hr at 20 0c. the results show the means of at least 4 experiments ± sd. the level of significance of the differences between the samples was calculated by anova with a student-newman-keuls post-hoc test (p<0.05). * indicates significant difference of the sample value with the respective control value. a,b indicate significant difference between each sample value with the respective control value (cd alone) (koutsogiannaki, 2008) integrins comprehend a large family of cell surface receptors. in mammals there are integrins binding to laminin (α1β1, α2β1, α3β1, α6β1, α7,β1 and α6β1), integrins binding to collagen (α1β1, α2β1, α3β1, α10β1 and α11β1), integrins of leukocytes (αlβ2, αmβ2, αxβ2 and αdβ2) and integrins recognizing the rgd motif (α5β1, αvβ1, αvβ3, αvβ5, αvβ6, αvβ8 and αiibβ3) (heino et al., 2009) integrins function mainly as cell-matrix adhesion molecules and transducers of the signals between them (li et al., 2003). ecmintegrin interactions function in a bidirectional manner across cell membranes. as the extracellular domain of integrin receptors becomes occupied by ligand and cluster, the integrins set off a cascade of events termed “outside-in” signaling. in this regard integrins orchestrate multiple functions including proliferation, differentiation, gene expression, changes in intracellular ph and death (ross and borg, 2001). moreover, integrins interact with cytoskeleton regulating cell shape and cell migration. these interactions are mediated through binding of the cytoplasmatic domain of integrins to actin network and actin-binding proteins (ezrin, randixin, moesin) (hynes, 1992). this short cytoplasmatic domain serves also as a host of molecules such as kinases and small gtpases (ross and borg, 2001). integrins are presumed to be present in all the metazoan cells. in invertebrates the structures of integrins are well conserved and functions are correlated with adhesive processes and immune responses (tanzer, 2006). studies in molluscan neurons indicate that cells can attach to various substrates using both rgd-dependent and rgd-independent adhesion mechanisms suggesting that at least two different cell adhesion receptors, possibly belonging to the integrin family, are expressed in molluscan neurons (wildering et al., 1998). results have shown that α2 integrin subunit mediates the increased adhesion of m. gallorovincialis hemocytes to collagen and oxidized collagen induced by cadmium (koutsogiannaki, 2008) (fig. 5). in addition increased expression of α2 integrin subunit was observed after cadmium treatment in m. gallorovincialis hemocytes, which was due to na+/h+ exchanger (nhe), phosphatidylinositol-3 kinase (pi-3k), protein kinase c (pkc), nadph oxidase, reactive oxygen species (ros) and no involvement (koutsogiannaki, 2008) (fig. 6). among the adhesion receptors that have been also found in invertebrates are caderins and immunoglobulins (n-cam) as well as peroxinectin and psp1 peptide (plasmatocyte spreading peptide) (johansson, 1999). signaling molecules involved in immune responses the first step to initiate an immune response is the detection by hemocytes of foreign invaders and/or non-self cells, presumably through receptors associated with the surface membrane. signals generated by ligand binding are then transduced across the membrane resulting in a cascade of downstream chemical reactions, ultimately directing these signals to target organelles (e.g., nucleus, cytoskeleton) mediating the induction of appropriate cellular responses (heldin and purton, 1996). cells 15 fig. 5 adhesion of mytilus galloprovincialis hemocytes to collagen iv and oxidized collagen iv after treatment with the anti-alpha2 integrin subunit. hemocytes were incubated with the anti-alpha2 integrin subunit (cd49b) for 30 min at 20 0c. the results show the means of at least 4 experiments ± sd. the level of significance of the differences between the samples was calculated by anova with a student-newman-keuls post-hoc test (p<0.05). * indicates significant difference of the sample value with the respective control value. a indicates significant difference between each other (koutsogiannaki, 2008) mediating immunity are able to communicate with both their internal and external environments through well developed signaling pathways (hynes and zhao, 2000). receptor-ligand interactions result in the modulation of many cellular processes mediated by complex intracellular signal transduction pathways. in invertebrates little is known about these signaling pathways although the cumulative data implies that there is high homology with those of mammals (humphries and yoshino, 2003). studies concerning the induction of the immune system of m. galloprovincialis by various stimuli (bacteria, cytokines, hormones, environmental chemicals) suggest the involvement of p38 [(stress-activated p38 mitogen-activated protein kinase (mapk)], c-jun n-terminal kinase (jnk), extracellular signal-regulated kinase (erk), signal transducer and activator of transcription (stat)5, stat 3, nuclear factor kappa b (nf-kb), pkc, cyclic adenosine monophosphate (camp) dependent pka (camp/pka), pi-3 k, ros and no (ottaviani et al., 2000; canesi et al., 2006; cao et al., 2007; novas et al., 2007; barcia and ramosmartinez, 2008; garcia-garcia et al., 2008; malagoli et al., 2008). in addition, malagoli et al. (2007) reported that stressfull conditions in mussel hemocytes trigger increased phagocytic activity and/or modulation of their signal transduction pathways, mainly erk and map kinases. this flexibility suggests the possibility that accumulated substances exert different effects in diverse situations. on the other hand, garcia-garcia et al. (2008) suggest that the role of erk and pkc in phagocytosis regulation is less generalized due to differential stimulation of phagocytic receptors. in addition it has been suggested that different bacteria and bacterial strains can differently affect the host signaling pathways (zampini et al., 2003; canesi et al., 2005, 2006). ottaviani et al. (2000) suggested that il-8 triggers conformational changes, induces chemotaxis and increased phagocytic activity in m. galloprovincialis hemocytes through pka and pkc pathway followed by reorganization of the actin microfilaments. this study also suggests that pka signaling pathway could be more important than the pkc in mediating cell shape changes induced by il8. on the other hand, il-2 mediated biogenin amines (ba) synthesis involves preferably pkc whereas the camp dependent pka plays secondary role (cao et al., 2004, 2007). it has been found that camp activates nucleotide depended protein kinases in molluscs (macdonald and storey, 1999) and modulates phagocytic behavior of hemocyte (chen and bayne, 1995). results from our laboratory showed that treatment with 3-isobutyl-1methylxanthin (ibmx), that results in high camp cell content, didn’t significantly affect the processes of adhesion and migration of m. galloprovincialis hemocytes to extracellular matrix proteins laminin and collagen (koutsogiannaki, 2008). the role of camp in these processes warrants further investigation. no, different forms of nitric oxide synthase (nos) and ros represent some of the main immune mechanisms in invertebrates (pipe, 1992; anderson et al., 1992; gourdon et al., 2001; ottaviani, 2006; barcia and ramos-martinez, 2008). ros are produced through respiratory burst, which is a series of biochemical reactions leading to ros generation such as superoxide (o2-), hydrogen peroxide (h2o2) and hydroxyl radical (oh .) (cross 16 fig. 6 integrins expession of mytilus galloprovincialis hemocytes. hemocytes were incubated with the anti-alpha 2 integrin subunit for 10 min at 20 0c. the results show the means of at least 4 experiments ± sd. the level of significance of the differences between the samples was calculated by anova with a student-newman-keuls post-hoc test (p<0.05). * indicates significant difference of the sample value with the respective control value. a indicates significant difference between each sample value with cadmium value (koutsogiannaki, 2008) and segal, 2004). the activation of respiratory burst has been detected in hemocytes of many mollusc species including m. galloprovincialis (garcia-garcia et al., 2008). no is a highly cytotoxic and microbicidal molecule, that is responsible for the defense mechanisms mediated by macrophages in mammals. it is also capable of activating other leukocytes (armstrong, 2001). no synthesis has been demonstrated in many molluscs as well (ottaviani et al., 1993; arumugam et al., 2000; novas et al., 2004; stefano et al., 2004). it has been shown that no, o2and h2o2 are involved in the signaling pathway induced by cadmium leading to m. galloprovincialis hemocytes adhesion and migration through ecm proteins (koutsogiannaki, 2008). in addition, the use of oxidants caused increase in adhesion and migration of hemocytes through ecm proteins that was reversed in the presence of the antioxidant nac (koutsogiannaki, 2008) (figs 3, 4). the later observations confirm the fact that ros are implicated in immune responses of m. galloprovincialis hemocytes (koutsogiannaki, 2008). furthermore, the use of nos inhibitors resulted in elimination of the bacteria clumping induced by lipopolysaccharides (lps) in the molluscan hemocytes of m. edulis and v. alter (ottaviani et al., 1993). it has been demonstrated that metals can increase ros production in m. galloprovincialis hemocytes with the implication of pkc (kaloyianni et al., 2006). moreover, in mussel leukocytes no production seems to be mainly regulated by pi3-k, pkc and erk families (garciagarcia et al., 2008). according to the latter, erk and pkc regulate no production only in large semigranular hemocytes as a result of differential membrane phagocytic receptor stimulation. in addition, studies on lymnaea stagnalis relate pkc and erk to the signaling pathway that regulates no activity (wright et al., 2006). moreover, barcia and ramos-martinez (2008) showed that il-2 induces the synthesis of no in m. galloprovincialis hemocytes via activation mainly of the camp dependent pka and secondary of pkc. it has been also shown that pi-3k activation plays critical role in the immune responses of m. galloprovincialis against pathogens and environmental pollutants (canesi et al., 2002a-c). pi3k has central role in coordinating phagocytosis and is found to mediate production of ros, no synthesis and pkc activation in m. galloprovincialis hemocytes (chou et al. 1998; chen et al., 2003; garcia-garcia et al., 2008). in addition, there are studies that point out the role of pi3k in the signaling pathways involved in the interactions of cells with the extracellular matrix in invertebrates and in mammals (guan and chen, 1996; parson, 1996; wei et al., 1997; howe et al., 1998; koutsogiannaki, 2008; konstantinidis et al., 2009). it has been also reported that treatment with wortmannin (pi3-k inhibitor) caused inhibition of cell adhesion, migration, phagocytosis and reorganization of cytoskeleton in the colonial ascidian botryllus schlosseri (ballarin et al., 2002). similarly, it has been found that wortmannin effect 17 caused inhibition of hemocytes adhesion to and migration through ecm proteins (koutsogiannaki, 2008). finally, another signaling molecule that seems to be involved in immune processes is nhe. nhe plays a central role in intracellular ph regulation and homeostasis of cell volume and is also involved in many intracellular signaling pathways (dailianis and kaloyianni, 2004; dailianis et al., 2005; kaloyianni et al., 2005; koutsogiannaki et al., 2006). nhe activation is implicated in many other cell functions as cell survival and apoptosis (koliakos et al., 2008). it has been shown that treatment of m. galloprovincialis hemocytes with cadmium resulted in increased degree of hemocytes adhesion to and migration through laminin-1, collagen type iv and oxidized collagen type iv in relation to control cells, with the involvement of nhe and pkc (kaloyianni et al., 2006; koutsogiannaki, 2008). in addition, nhe’s implication in cell adhesion and cell migration is probably related to the fact that nhe is involved in focal anchoring sites together with focal adhesion kinase (fak) and proteins of the actin network (teline, vincoulin, paxiciline and others) through indirect connection with integrins (beningo et al., 2001; koliakos et al., 2001; webb et al., 2002; stock et al., 2005; kostidou et al., 2007) or cd44 (verfaillie et al., 1994). in 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a calcium ion concentration of 2 nm, the nos activity measured by citrulline formation was 27.1 ± 2.2 and 9.3 ± 0.8 pmol/min/106cell for soluble and particulate nos, respectively. the increase in free calcium ion concentration to 300 nm increases enzyme activity to 57.5 ± 4.1 and 23.5 ± 1.2 pmol/min/106cell, respectively. the 50 % activation of the calcium-dependent activity is 91 and 97 nm ca2+ for soluble and particulate enzymes. trifluoperazine, an inhibitor of the calmodulin-dependent enzyme, partially inhibits both activities. soluble nos is five times more sensitive than particulate nos. the behaviour of both activities with three nos inhibitors (7-nitroindazole, s-methylisothiourea sulphate, diphenyleneiodonium) is very similar, with ic50 values that are not significantly different. the calcium ion dependence of nos activities, in a range of free calcium ion variations, which are transiently observed in receptor-stimulated cells, suggests that nitric oxyde in v. ater immunocytes not only has a defensive role but also signalling relevance in crosstalking between immunocytes and other cells. key words: mollusc; viviparus ater; immunocytes; nitric oxide synthase; calcium ion dependence introduction immunocytes are the cells of the immune response in molluscs and other invertebrates against not-self materials. recognition, phagocytosis and killing of virus and bacteria are one of the most important functions of invertebrate immunocytes (ottaviani, 1992). in mammalian phagocytic cells, oxygen reactive species (ros), such as superoxide ions and hydrogen peroxide, hypochlorous acid and nitric oxide (no), are produced to kill phagocyted organisms. from no and superoxide ions, the more reactive peroxynitrite ion and other oxygen radicals are formed (beckman et al., 1990; porasuphatana et al., 2001; heales et al., 1999). the first evidence for no production and utilisation as a bactericidal agent by invertebrate mollusc immunocytes was reported by ottaviani et al. (1993). t h e n i t r i c o x i d e s y n t h a s e ( n o s ) a c t i v i t y o f corresponding author: davide tagliazucchi university of modena and reggio emilia, department of agricultural sciences, via kennedy 17, 42100 reggio emilia, italy e-mail: tagliazucchi.davide@unimore.it immunocytes of the fresh water snail viviparus ater has been partially characterised biochemically (conte and ottaviani, 1995). nos activity shows a partial dependence on calcium ions. it is also present in particulate fractions and is induced by lipopolysaccharides (lps) (conte and ottaviani, 1995). three main different forms of nos have been isolated from different cell types: neuronal nos (nnos), endothelial nos (enos) and inducible nos (inos) from macrophages (pollock et al., 1991; lamas et al., 1992; bredt et al., 1991; stuehr et al., 1991). the constitutive enos and nnos have an absolute requirement for calcium ions and calmodulin. enos is mainly, if not completely, membrane associated. the presence of covalentlybound myristoyl and palmitoyl residues on protein molecules have an important role in binding to membranes. nnos is mainly cytosolic, but may be bound to cell membranes. inos is cytosolic and is expressed upon immunological and inflammatory stimulation. this form is independent of calcium ions and calmodulin (cho et al., 1992; steven-truss and marletta, 1995). inos may produce toxic and lethal no concentrations. 55 the no produced by nnos and enos has a signalling role under the strict control of intracellular calcium ions. alternative spliced forms of nos have been demonstrated (silvagno et al., 1996; magee et al., 1996). the nos enzyme(s) from immunocytes of v. ater combine the properties of different noss (conte and ottaviani, 1995) and cannot be identified with any of the enzymes studied so far, in particular the inos of mammalian phagocytic cells. in the immunocytes of mytilus edulis, morphine induces a transient increase in intracellular calcium ions, followed by no release (nieto-fernandez et al., 1999) suggesting that the immunocytes of this invertebrate have nos with properties that are similar to those of v. ater. the aim of this study was the characterisation of calcium ion dependence of soluble and particulate nos of the snail v. ater. materials and methods reagents (6r)–5,6,7,8-tetrahydro-l-biopterin dihydrochloride was purchased from dr. b. schircks laboratories (jona, switzerland) and l-[2,3,4,5-3h]arginine monohydrochloride (58 ci/mmol) from amersham (buckinghamshire, england). the ion-exchange resin ag50wx-8 was supplied by bio-rad (milan, italy). calmodulin, glucose-6-phosphate dehydrogenase from yeast (ec 1.1.1.49) and glucose-6-phosphate were from serva (heidelberg, germany). 7-nitroindazole (7ni), s-methylisothiourea sulphate (smt), diphenyleneiodonium (dpi), and trifluoperazine (tfp) were from calbiochem (darmstadt, germany). nitrate reductase from aspergillus spp. (ec 1.6.6.2), lipopolysaccharide (lps) from escherichia coli, ngmonomethyl-l-arginine (l-nmma) and all other biochemicals were obtained from sigma (milan, italy). snails adult specimens of viviparus ater were collected from a freshwater canal near modena (italy), in spring and early summer. the animals were maintained at room temperature in de-chlorinated freshwater, for at least a week before the experiments. snail haemolymph was obtained by prodding the animal’s foot and collected with a pasteur pipette. immunocytes were obtained by centrifugation at 600 x g. the cells were washed twice with snail saline solution (ottaviani, 1983), counted and collected using centrifugation. determination of nos activity immunocytes were homogenised with ultra-turrax (ika-werk, germany) in 5 vols. of ice-cold solution, containing 320 mm sucrose, 50 mm tris, 1 mm edta, 1mm dithiothreitol (dtt), 1mm phenylmethylsulphonyl fluoride, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml antipain and 10 µg/ml bestatin, brought to ph 7.0 at 20°c with hcl. the homogenate was centrifuged at 20,000 x g for 30 min at 4 °c, and the supernatant was freed from low molecular mass compounds by sephadex g-25 chromatography (werner-felmayer et al., 1993). the protein fraction was eluted with a buffer containing 50 mm hepes, ph 7.4, 1 mm edta, 1 mm dtt and the above-mentioned protease inhibitors. nos activity was assayed by following the conversion of radiolabelled arginine to citrulline. standard reaction mixtures contained 50 mm hepes, ph 7.4, 0.5 mm edta, 1.4 mm cacl2, 1 mm mgcl2, 1 mm nadph, 1 mm dtt, 12 mm l-valine, 1 mm citrulline, a variable amount of l-arginine, 80,000-100,000 cpm of purified l-[2,3,4,5-3h]arginine monohydrochloride and 5-50 µl of sephadex g-25 eluate in a final volume of 100 µl. after 30 min incubation at 37 °c, [3h]citrulline was quantified by liquid scintillation counting, after separation from [3h]arginine by cation exchange (ag50wx-8) (bredt and snyder, 1989). the nos activity of the nonsoluble fraction was determined by washing the immunocyte pellet twice with pbs, centrifuging at 20,000 x g for 30 min at 4 °c and re-suspending with the buffer used for sephadex g-25 chromatography. nos activity was determined by calculating the difference between the [3h]citrulline produced in the presence and absence of 10 mm ng-monomethyl-larginine (l-nmma, an inhibitor of mammalian nos) in a standard reaction mixture. activity was calculated using the radiochemical method and expressed as pmol of [3h]citrulline formed/min/106 cells. when calcium ion dependence was investigated, variable amounts of cacl2 and egta were added to the reaction mixture. free calcium concentrations were calculated following fabiato (1988) and controlled by the fura 2 method, in accordance with mülsch et al. (1989). in some cases, incubations also contained variable concentrations of the nos inhibitors: 7-ni, smt, dpi or tfp. the results are expressed as means ± sd of at least three independent experiments performed in triplicate. proteins were determined in accordance with the modified lowry method (markwell et al, 1981), with serum albumin as standard. results the dependence of soluble and particulate nos activities on free calcium ion concentrations at ph 7.4 is reported in fig. 1. the soluble enzyme activity (upper line) at a free ca2+ concentration of 2 nm was 27.1 ± 2.2 pmol/min/106 cells and progressively increased to 57.5 ± 4.1 at 300 nm ca2+. the enzyme activity slightly decreased at higher calcium concentrations. the particulate nos activity (iower line), which is about 40 % of the soluble activity, showed similar calcium dependence. fifty percent activation is obtained at a 91 and 97 nm calcium ion concentration for soluble and particulate enzymes, respectively. calcium ions activated both soluble and particulate nos enzymes at concentrations that are physiologically observed during a transient calcium ion increase in cells stimulated by signalling molecules. the calmodulin dependence, evaluated by trifluoperazine inhibition of nos activity, is reported in fig. 2. trifluoperazine is a calmodulin antagonist that inhibits ca2+/calmodulin-dependent enzymes. the nos soluble activity (upper line), measured at a free ca2+ concentration of 300 nm, decreased with increasing concentration of tfp to 55 % of the 56 0 10 20 30 40 50 60 0 1 2 3 4 5 log [trifluoperazine] microm [3 h ] ci tr u lli n e p m o l/m in /1 0 6 c e lls particulate nos soluble nos fig. 1 dependence of soluble and particulate nos activity on free ca2+ concentration. each point is the mean ± sd of three independent experiments performed in triplicate. 0 10 20 30 40 50 60 0 1 2 3 4 5 6 7 log [ca2+] nm particulate nos soluble nos fig. 2 dependence of soluble and particulate nos activity on trifluoperazine concentration. each point is the mean ± sd of three independent experiments performed in triplicate. [3 h ] ci tr u lli n e p m o l/m in /1 0 6 c e lls [3 h ] ci tr u lli n e p m o l/m in /1 0 6 c e lls 57 table 1 ic50 values of some nos activity inhibitors. the inhibitors were included in standard reaction mixtures, containing 1 µm arginine and prepared as described in the text. the data are the means ± sd of three independent experiments performed in triplicate. soluble nos particulate nos tfp (µm) 74 ± 20 383 ± 92 7-ni (nm) 715 ± 134 648 ± 118 dpi (nm) 102 ± 29 130 ± 26 smt (nm) 877 ± 190 1051 ± 276 activity of the control at about 1 mm tfp. no further decrease in the enzyme activity was observed at higher tfp concentrations. the particulate bound activity (lower line) was inhibited by tfp at about a 5-times higher concentration than soluble nos (table 1) similarly to what we found with the carp enzyme (conte and ottaviani, 1998). table 1 reports the ic50 values of the three other inhibitors tested. no significant difference in the ic50 values was observed between the soluble and particulate enzymes. discussion we found that calcium and calmodulin dependence, evaluated by tfp inhibition, of nos activity from immunocytes of v. ater, measured by citrulline formation, is similar to those of the mutant ä 45 of the human enos enzyme (chen and wu, 2003). in this mutant, residues 594-606 and 614-645 of human enos are removed. these residues are not present in human and mouse macrophage inos. the ä 45 mutant is able to form no, measured by citrulline formation, in the absence of calmoduline or calcium ions, at 60% of the rate in their presence. it has been suggested by roman et al. (2000) that the ä 45 segments play a relevant role in ca2+/calmodulin dependence. the presence of these segments decreases or inhibits the electron flow from the reductase domain, which binds nadph, to the oxygenase domain, which binds arginine. ca2+/calmodulin, binding to the enos or nnos, remove inhibition and allow electron transfer from reductase to oxygenase. geller et al. (1993) reported the molecular cloning and expression in 293 embryonic kidney cells of nos induced by lps in human hepatocytes. the chelating agents edta and egta decreased the activity of this enzyme by 30 % but failed to obtain complete inhibition. trifluoperazine decreased activity by 50 %. the amino acid sequence reported by geller et al. (1993) appears to be the same as nos from human macrophages. whatever the molecular mechanism of partial calcium dependence of v. ater, nos raises several questions on its roles and regulation in immunocytes. nos has been demonstrated in the nervous system of several species of snail. no has neurotransmitterlike functions, for example, it is necessary for the transmission of sensory information to the central pattern generator for feeding behaviour (elphick et al., 1995) and the activation of buccal motor patterns (moroz et al., 1993). no is involved in neural transmission to intestinal muscles in helix lucorum but enteric release of no is blocked during snail dormancy (roszer et al., 2004). no has a role in developing the nervous system of the snail llyanassa obsoleta (thavaradhara and leise, 2001) and regulating the early embryonic behaviour in the snail helisoma trivolvis (cole et al., 2002). in all of the above examples, the constitutive calcium-dependent nnos is considered to be the enzyme that produces no. identification of nos enzymes has been carried out mainly by immunocytochemical methods, using antibodies to mammalian nnos. amino acid sequence studies show that neuronal nos from insects (drosophila melanogaster and schistocerca gregaria) and the snail lymnaea stagnalis, share 43-67 % identity with mammalian nnos (ogunshola et al., 1995; regulski and tully, 1995). it has been reported that v. ater soluble nos is inhibited by 70 % by mammalian nnos antibodies (conte and ottaviani, 1995). moroz et al. (1996) demonstrated constitutive calcium-independent nos activity in the nervous system of some molluscan species, which is largely associated with particulate fractions. using immunochemical investigations, xie et al. (2002) showed that the substrate arginine and the nos enzyme are localised in the separate, but adjacent, neurons of the snail helix pomatia. the product citrulline is observed in the neurons, which contain the nos enzyme, suggesting an unknown signalling pathway between neurons to maintain arginine and no homeostasis. the examples reported above show the complexity of the properties and regulation of nos activity in neuronal cells of snails, as well the expanding roles of no. the defense role of snail immunocyte nos has been demonstrated (ottaviani et al., 1993). during phagocytosis, these cells also produce oxygen species that may combine with no to form the more reactive peroxynitrite, increasing killing capacity. however, it seems that immunocytes are not only of immunological relevance, as they also synthesise signalling peptides (ottaviani et al., 1992). invertebrate immunocytes respond to multiple signal molecules that form the immunoregulatory network. cytokine-like factors affect immune functions such as cell motility, chemotaxis, phagocytosis and cytotoxicity in invertebrate immunocytes and are able to induce no (ottaviani et al., 2004). cytokinestimulated immunocytes in m. edulis modulate ganglionic no release, which later affects their activity level, demonstrating that ganglionic no is involved in down-regulating immunocyte activity (stefano et al., 2004). it has been demonstrated that m. edulis immunocytes have a µ3 morphine receptor, coupled to no release in an intracellular calciummediated manner (nieto-fernandez et al., 1999). no release may mediate morphine-induced changes in immunocyte conformation, lowering chemotactic activity, cellular velocity, and adherence (stefano et al., 1993). furthermore, naturally occurring cannabinoids may share the no-producing effector system with opiate alkaloids in these cells (stefano et al., 1996). not only can morphine-like and 58 cannabinoid substances be found in invertebrate immunocytes, there is also a 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characterization of the cytokine-induced macrophage nitric oxide synthase: an fad-and fmncontaining flavoprotein. proc. natl. acad. sci. usa 88: 7773-7777, 1991 thavaradhara k, leise em. localization of nitric oxide synthase-iike immunoreactivity in the developing nervous system of the snail llyanassa obsoleta. j. neurocytol. 30: 449-456, 2001. werner-felmayer g, werner er, fuchs d, hausen a, mayer b, reibnegger g, weiss g, wachter h. ca2+/calmodulin-dependent nitric oxide synthase activity in the human cervix carcinoma cell line me-180. biochem. j. 289: 357-361, 1993. xie m, hermann a, kerschbaum hh. complementary distribution of nadph-diaphorase and l-arginine in the snail nervous system. cell. tissue res. 307: 393-400, 2002. the hemocytes of polyandrocarpa mysakiensis and their role in defense reactions isj 6: 154-161, 2009 issn 1824-307x research report the hemocytes of polyandrocarpa mysakiensis: morphology and immune-related activities l ballarin1, k kawamura2 1department of biology, university of padua, padua, italy 2laboratory of cellular and molecular biotechnology, faculty of science, kochi university, japan accepted november 20, 2009 abstract a preliminary study of the hemocytes of developing buds of the compound ascidian polyandrocarpa misakiensis was carried out at the light microscope level for a better understanding of their biological role. similarly to other ascidians, p. misakiensis immunocytes are represented by phagocytes and morula cells. phagocytes include hyaline amoebocytes and round, giant phagocytes, the former the probable precursors of the latter. hyaline amoebocytes showed high macropinocytotic activity in the presence of bacteria, whereas yeast cells were ingested by phagocytosis. morula cells contain the enzyme phenoloxidase inside their vacuoles, probably stored as pro-enzyme, which is released upon the recognition of non-self. together with macrogranular leukocytes, morula cells were the most abundant hemocyte-types which stresses the importance of these cells in polyandrocarpa biology. macrogranular leukocytes are frequently found inside the vacuoles of phagocytes and were recognized by a polyclonal antibody raised against an opsonin purified from the colonial ascidian botryllus schlosseri, which suggests that a similar lectin can be involved in the interaction between these cells and phagocytes. key words: polyandrocarpa misakiensis; colonial ascidians; hemocytes; morphology; immunity introduction invertebrate chordates or protochordates are represented by cephalochordates and tunicates, the latter being the sister group of vertebrates (delsuc et al., 2006). the peculiar phylogenetic position of tunicates explains the increasing interest towards their biology, in particular, developmental biology and immunobiology. the majority of tunicates are represented by ascidians, sessile filter-feeding marine animals which include both solitary and colonial species. many types of hemocytes are found in ascidians. their morphology has been described by many authors (pérès, 1943; endean, 1955; sabbadin, 1955; andrew, 1961; overton, 1966; smith, 1970a, b; milanesi and burighel, 1978; scippa et al., 1982; burighel et al., 1983; schlumpberger et al., 1984; sawada et al., 1991, 1993; zhang et al., 1992; azumi et al., 1993; dansohkawa et al., 1995; arizza and parrinello, 2009) and various classification criteria have been ___________________________________________________________________________ corresponding author: loriano ballarin department of biology university of padua via u. bassi 58/b, 35100 padua, italy email: loriano.ballarin@unipd.it proposed (goodbody, 1974; wright, 1981; rowley et al., 1984; de leo, 1992; burighel and cloney, 1997). however, uncertainties still exist on their functions, mutual relationships and differentiation pathways. polyandrocarpa misakiensis (fig. 1) is a polystyelid compound ascidian, common along the coasts of the temperate regions of japan, which can reproduce asexually through continuous budding from parental zooids (kawamura and fujiwara, 1994; kawamura et al., 2008). the morphological, biochemical and molecular events occurring during bud differentiation and maturation have been widely studied (kawamura and fujiwara, 1994; hisata et al., 1998; kawamura and sugino, 1999; kamimura et al., 2000; matsumoto et al., 2001; sunanaga et al., 2007; kawamura et al., 2006, 2008). nevertheless, few data are available on polyandrocarpa hemocytes: their morphology has been studied at the electron microscope, but scanty data are available for light microscopy. in addition, the abundance of the different hemocyte types and their possible roles in immune defense have been little investigated. in order to fill this gap, we carried out a preliminary investigation aimed to a better characterization of polyandrocarpa hemocytes at the 154 fig. 1 colony of p. misakiensis. adult zooids (z) bear many buds (b). developing buds are indicated by asterisks. bar = 0.5 mm. light microscope, with particular reference to immunocytes, a well-defined class of circulating hemocytes responsible of both cellular and humoral (through their secretions) immune responses. results confirm the presence of phagocytes, able to quickly ingest foreign particles, through phagocytosis and macropinocytosis, and morula cells which, like many other compound ascidians, are probably involved in cytotoxic immune reactions. in addition, granular leukocytes, which are circulating trophocytes, are recognized by a polyclonal antibody, raised against a lectinic opsonin from botryllus schlosseri, which represents a good and specific marker for this cell type. materials and methods animals colonies of polyandrocarpa misakiensis were reared in the field, attached to glass plates, near the usa marine biological institute, kochi university, japan. when required, they were brought in the laboratory of cellular and molecular biotechnology of the faculty of science and kept at room temperature for few days before their use in a 20-l aerated aquarium filled with seawater. hemocyte collection and culture hemolymph was collected from developing buds (fig. 1) of colonies, previously immersed for few min in 0.38 % na-citrate in artificial seawater (asw), ph 7.5, in order to prevent cell clotting and then blotted dry. buds, already detached from the parent zooid, were punctured with a fine tungsten needle and hemolymph was collected with a glass micropipette and centrifuged at 700xg for 10 min at 4 °c; the pellet was re-suspended in asw to a final concentration of 106 cells/ml. fifty μl of hemocyte suspension were placed in the center of a glass coverslip, previously coated with poly-l-lysine (50 μg/ml), and left to adhere, for 60 min, at room temperature (rt) in a moist chamber. hemocytes were then observed under the light microscope or, alternatively, fixed in 1 % glutaraldehyde in asw containing 1 % sucrose, washed in phosphatebuffered saline (pbs: 8 g/l nacl, 0.2 g/l kcl, 0.2 g/l kh2po4, 1.15 g/l, ph 7.4) and stained for 5 min in 10 % giemsa’s solution. coverslips were then mounted on microscope slides with 80 % glycerol. hemocytes from at least 10 different colonies were observed and counted. blood plasma and hemocyte lysate preparation freshly collected hemolymph was centrifuged at 700xg for 10 min at 4 °c. the resulting supernatant was referred to as blood plasma (bp), whereas the pellet was re-suspended in an equal volume of distilled water, subjected to sonication for 20 s at 0 °c in a braun labsonic u sonifier at 50 % duty cycles and centrifuged at 10,000xg for 10 min in order to get hemocytes lysate (hl) as supernatant. phagocytosis and macropinocytosis assays after adhesion, hemocytes were incubated at rt, in a moist chamber, with 50 µl of a yeast (saccharomyces cerevisiae) suspension in asw (yeast/hemocyte ratio = 10:1) for 60 min, and the uningested yeast was then removed by dipping the coverslips repeatedly in a large volume of asw. living hemocytes were then observed under the light microscope. alternatively, monolayers were fixed and stained as described above before their observation. in another series of experiments, hemocytes were incubated for 60 min with a suspension of living escherichia coli in asw (109 cells/ml). 155 coverslips were then washed by repeated dipping in asw and cells were fixed and stained as described above before their observation under the light microscope. immunocytochemical analysis fixed hemocytes were incubated for 30 min in 1 % h2o2 to block endogenous peroxidase activity, washed in pbs, treated with 2 % powdered milk for 30 min and then incubated for 1 h with 50 μg/ml of purified polyclonal antibody raised against b. schlosseri rhamnose-binding lectin (bsrbl; ballarin et al., 2000; gasparini et al., 2008) in a moist chamber; pre-immune serum was used in controls. after washing in pbs containing 0.1 % tween 20, they were incubated for 30 min in 1 μg/ml of horseradish peroxidase-labelled mouse anti-rabbit secondary antibody (vector laboratories), washed in distilled water and treated with true blue (kpl), which stains positive sites blue, according to the manufacturer’s instructions. assay for phenoloxidase (po) activity on hemolymph, bp, hl and hemocyte incubation medium twenty μl of hemolymph, collected as described above, bp or hl were added to 180 μl of a saturated solution of dihydroxyphenyl-l-alanine (l-dopa) in pbs in the wells of flat bottomed, 96well microtiter plates and the time course of the reaction was read at 490 nm for 5 min with a biorad imark microplate reader. five mm na2so3, a po inhibitor (kong et al., 2000; cong et al., 2005), were added to the hemolymph in negative controls. one relative unit (ru) of po activity was defined as the increase in absorption of 0.001/min in the reaction mixture (söderhäll and smith, 1983). protein concentrations of the supernatants were determined according to lowry et al. (1951) and results were expressed as ru/mg protein. in another series of experiments, 100 μl of hemocytes suspension (106 cells/ml) were incubated for 60 min in asw or, alternatively in yeastcontaining asw (yeast:hemocyte ratio = 10:1). the supernatants were then collected by centrifugation at 700xg at 4 °c and assayed for po activity as described above. cytoenzymatic assay for po activity after fixation, hemocytes were incubated for 1h in saturated dihydroxy-l-phenylalanine (ldopa) solution in pbs in the presence or in the absence of 5 mm na2so3, washed in distilled water, and mounted in acquovitrex. positive cells converted l-dopa to dopachrome and stained blackish-brown. statistical analysis each experiments was repeated at least three times. po activity data were compared with the student’s t test. results the hemocytes of p. misakiensis the following main cell-types could be recognized: i) undifferentiated cells, 4-6 μm in diameter, with a high nucleus-cytoplasm ratio. they amounted to 4.4 ± 0.7 % of circulating cells (figs 2a, b); ii) hyaline amoebocytes, 6-12 μm in length, have a variable shape, homogeneous cytoplasm with various cytoplasmic protrusions (pseudopods), and a roundish nucleus (figs 2c, d). they represent 4.4 ± 0.4 % of the hemocytes; iii) round phagocytes, or macrophage-like cells, 8.1 ± 2.9 % of the hemocytes. they have a spheroidal shape, 10-15 µm in diameter; their cytoplasm can be stained metachromatically by giemsa’s dye, and shows one or few large vacuole(s) containing ingested material which occupy most of the cell volume (figs 2h-k); iv) microgranular leukocytes, 15.3 ± 3.5 % of the total hemocytes, 4-6 µm in diameter. they frequently show an amoeboid form and are characterized by the presence of small granules in their cytoplasm which frequently assume a metachromatic red color with giemsa’s dye (fig. 2e); v) macrogranular (granular) leukocytes, 10-15 µm in size, one of the most abundant circulating cell type in polyandrocarpa, representing 33.9 ± 3.1 % of the hemocytes. they are giant round cells with the nucleus usually found at the periphery of the cell and the cytoplasm filled with many granules, of variable size (up to 2-3 μm in diameter), which appear translucent in living cells and almost empty after fixation (figs 2i, l, m); vi) morula cells (mcs), 10-15 μm in diameter, 32.9 ± 4.4 % of the circulating cells. they are also round cells characterized by the presence of many vacuoles, variable in size, which appear yellowish in living cells and acquire a green color after aldehyde fixation (figs 2i, q) and vii) pigment cells,1.2 ± 0.2 % of the total hemocytes number, 10-15 µm in size. they are characterized by the presence of many small vacuoles filled with red pigment and a nucleus located at the periphery of the cell. their morphology is rarely preserved in fixed samples (fig. 2l). polyandrocarpa phagocytes behave differently in the presence of yeast and bacteria when hemocytes were incubated in the presence of yeast, phagocytes, mainly round cells, filled with yeast cells were frequently found after 60 min of incubation (figs 2h, k). no changes in mc morphology were observed. conversely, in the presence of bacteria most of hyaline amoebocytes showed heterogeneous macropinocytotic vesicles inside their cytoplasm which appeared empty under the light microscope (fig. 2f). most of the microbes resulted agglutinated outside the cells (fig. 2f) and in few cases they were visible inside phagocytes (fig. 2g). 156 fig. 2 hemocytes of p. misakiensis. a, b: living (a) and giemsa-stained (b) undifferentiated cells. c, d: living (c) and stained (d) hyaline amebocytes. e: microgranular leukocyte fixed and stained with giemsa. f: fixed and stained hyaline amebocyte after exposure to e. coli; many micropinocytotic vesicles (mv) are visible as well as agglutinated bacteria (arrowhead) outside the cell. g: fixed and stained hyaline amebocyte with ingested bacteria (arrowheads). h: living phagocytes with ingested yeast cells (arrows). i: living round phagocyte (ph), macrogranular leukocyte (ml) and morula cell (mc). j, k: fixed and stained round phagocytes with vacuoles containing ingested materials (yeast cells in k). l: living macrogranular leukocytes (ml) and pigment cell (p). m: fixed and stained macrogranular (ml) and microgranular (mi) leukocytes. n: fixed macrogranular leukocytes showing immunopositivity to anti-bsrbl antibody on their surface. o: round phagocyte having ingested a immunopositive to anti-bsrbl antibody (arrowhead). p: living macrogranular leukocyte (ml) and morula cells (mc). q: aldehyde-fixed (unstained) morula cells: vacuoles assume a green color. r: fixed hemocytes treated with l-dopa: stain for dopachrome production is evident in morula cells (mc) but not in macrogranular leukocytes (ml). bar = 5 µm. macrogranular leukocytes are recognized by the anti-bsrbl antibody the anti-bsrbl antibody recognized specifically the surface of macrogranular leukocytes (fig. 2n). in developing buds, these cells are frequently found inside phagocyte vacuoles and, in some cases, we could observe labeled cells inside round phagocytes (fig. 2o). po activity is located in mcs when the po activity of whole hemolymph (wh) and bp were compared, the former resulted more than three times higher than the latter, suggesting that the majority of the enzyme was present, in normal conditions, inside the hemocytes. this was confirmed by the higher enzyme activity (3 times that of the hemolymph) of hl. the addition of 157 table 1 po activity of whole hemolymph (wh), blood plasma (bp) and hemocyte lysate (hl) po source po activity (ru/mg protein) wh 1239.2 ± 53.1 wh + 5mm na2so3 0.7 ± 0.2 *** bp 374.3 ± 40.4 *** hl 3720.5 ± 99.3 *** *** p < 0.001 with respect to wh na2so3 to the hemolymph completely abolished the oxidation of l-dopa (table 1). after 60 min of incubation of hemocytes in asw, the po activity of the culture medium amounted to 29.8 ± 4.0 ru/mg protein. conversely, when blood cells were incubated in a suspension of yeast in asw, the resulting enzyme activity of the medium was significantly (p < 0.001) increased and reached the value of 63.3 ± 10.9 ru/mg protein. cytoenzymatic analysis in the presence of ldopa clearly showed that the only mcs, and no other cells type, were labeled in the presence of ldopa (fig. 2r). discussion colonies of the ascidian p. misakiensis continuously form new buds as outgrowths of the parental body which soon separate so that morphogenesis occurs without any contact with the parental organism. for these reasons, this species is considered an excellent model organism for the study of stem cell differentiation during asexual reproduction and regeneration (kawamura and fujiwara, 1994; hisata et al., 1998; kawamura and sugino, 1999; kamimura et al., 2000; matsumoto et al., 2001; sunanaga et al., 2007; kawamura et al., 2006, 2008). hemocytes have been claimed to take part in polyandrocarpa development to adulthood as both a source of undifferentiated cells (kawamura et al., 1991, 2008) and a reservoir of nutrients required for the completion of bud morphogenesis when the young individuals are not yet feeding (kawamura and nakauchi, 1986; kawamura et al., 1991, 1992). however, despite their importance, there are few data in the literature describing polyandrocarpa blood cells, their abundance and behavior (kawamura et al., 1992; sugino et al., 1993). in the present work, we carried out a light microscope morphological study on p. misakiensis hemocytes as a further contribution for better understanding the biological roles of these cells. undifferentiated hemocytes represent less than 5 % of the circulating cells, in agreement with what found in other compound ascidians (cima et al., 2001; ballarin et al., 2005). the presence of circulating undifferentiated cells, or hemoblasts, involved in asexual reproduction is a common feature of colonial ascidians. analogously to what described in stolonal budding of perophora (freeman, 1964) and in vascular budding of botryllid (oka and watanabe, 1957, 1959; sabbadin et al., 1975; rinkevich et al., 1995; rinkevich et al., 2007; voskoboynik et al., 2007), these cells exert a fundamental role in polyandrocarpa bud morphogenesis (kawamura and nakauchi, 1991; kawamura et al., 1991). hyaline amebocytes and round phagocytes have been previously included in the same category of hyaline leukocytes, involved in phagocytosis (sugino et al., 1993). indeed, like in botryllid ascidians, they are probably different morphs of a single phagocyte type which can actively move towards foreign cells or particles by ameboid progression and, upon the ingestion of non-self material, withdraws its cytoplasmic projections assuming a globular shape (cima et al., 2001; ballarin and cima, 2005). hyaline amebocytes of the compound ascidian botryllus schlosseri are capable of constitutive macropinocytosis (ballarin and burighel, 2006): the same process is probably responsible of the abundance of hollow vesicles observed in the cytoplasm of polyandrocarpa hyaline leukocytes (sugino et al., 1993). a clear macropinocytotic activity was observed in the presence of bacteria, in accordance with the general view of macropinocytosis as a process generally responsible of the ingestion of bacteria and necrotic material (krysko et al., 2003). the presence of agglutinated bacteria outside the cells suggests the release of agglutinins, likely lectins, by activated phagocytes. a similar agglutinating ability towards some bacterial strains have been recently demonstrated for bsrbl (unpublished data). conversely, no increase in macropinocytotic activity was observed when yeast cells were used as foreign particles: in this case, analogously to what described in other colonial ascidians (ballarin et al., 1994), phagocytosis occurred and phagocytes turned to large, round cells filled with yeastcontaining vacuoles. mcs represent one of the most abundant circulating hemocyte types. in botryllid ascidians, their frequency ranges from 20 to 60 % (ballarin, 2008) and they are important mediators of the response to non-self as both effectors of allorejection between contacting, genetically incompatible colonies (hirose et al., 1990; ballarin et al., 1995, 1998; rinkevich et al., 1998; shirae et 158 al., 1999, 2002; cima et al., 2004), and sites of synthesis of cytokine(s), able to influence phagocyte activity, in response to non-self recognition (ballarin et al., 2001; menin et al., 2005, menin and ballarin, 2008). in b. schlosseri, one of the first event consequent to the recognition of foreign molecules by mcs is their degranulation and the release of their vacuolar content, mainly po and its polyphenol substrates. like botryllid ascidians, in polyandrocarpa, po is contained inside hemocytes, probably as a precursor which can be readily activated by cell manipulation (ballarin et al., 1998), and mcs are the only cell type showing the enzyme activity. in addition, the enzyme can be released in the medium upon the recognition of foreign cells such as yeast cells. botryllid po is involved in the induction of cytotoxicity consequent to the recognition of non-self in both allorejection (ballarin et al., 1995, 1998; shirae et al., 2002) and responses towards foreign cells or particles (ballarin et al., 1998, 2005): as no allorecognition phenomena are known in polyandrocarpa, we can hypothesize that, in this species, mcs exert a general immunosurveillance role, being capable to induce cytotoxicity through the release of their vacuolar content, both po (present data) and peroxidase (kawamura et al., 1992), upon the recognition of non-self, even if no apparent signs of degranulation are visible in blood smears. macrogranular leukocytes constitute the other most common circulating cell-type in polyandrocarpa developing buds. their abundance suggests an important role for these cells and, indeed, there is a general agreement on the fact that they are trophocytes, involved in the uptake and storage of nutrients useful for bud morphogenesis (kawamura and nakauchi, 1986; kawamura et al., 1992); the alkaline phosphatase activity associated with their plasma membrane could be involved in this process (kawamura et al., 1992). this is in agreement with the observation that, in developing buds, they are frequently found inside phagocytes. the antibody raised against bsrbl recognized specifically the surface of macrogranular leukocytes suggesting the presence of molecules sharing some degree of similarity with bsrbl. a lectin with galactose specificity has already been demonstrated in these cells (kawamura et al., 1991). since the lectin from b. schlosseri has been demonstrated to exert an opsonic role through the coating of foreign cells before their phagocytosis and immunopositive macrogranular leukocytes are sometimes present in vacuoles of round phagocytes, we can hypothesize: i) that the molecule recognized by the anti-bsrbl antibody can have a role in the recognition of trophocytes by phagocytes and ii) that in polyandrocarpa asexual development, like in botryllus take-over (lauzon et al., 2002; cima et al., 2003, 2009; ballarin et al., 2008), phagocytosis is the main process which render available nutrients stored in trophocytes and dying cells, respectively. collectively, our results indicate or suggest some possible biological roles for polyandrocarpa hemocytes. however, many questions remain unanswered, such as the molecular mechanism of trophocyte recognition by phagocytes in order to render nutrients available to developing buds, or the role of mc and po in a species where allorecognition phenomena are not known, or the influence of mc on phagocyte activity. future research will help in clarifying the above points. acknowledgements this work was partially supported by a grant (fy2009) from the japan society for the promotion of science to one of us (lb) for working at kochi university. authors wish to thank the personnel at usa marine biological institute for technical help. references andrew w. phase microscope studies of living blood-cells of the tunicates under normal and experimental conditions, with a description of a new type of motile cell appendage. quart. j. microsc. sci. 102: 89-105, 1961. arizza v, parrinello d. inflammatory hemocytes in ciona intestinalis innate immune response. inv. surv. j. 6: s58-s66, 2009. azumi k, satoh n, yokosawa h. functional and structural characterization of hemocytes of the solitary ascidian, halocynthia roretzi. j. exp. zool. 265: 309-316, 1993. ballarin l. immunobiology of compound ascidians, with particular reference to botryllus schlosseri: state of art. inv. surv. j. 54-74, 2008. ballarin l, cima f. cytochemical properties of botryllus schlosseri haemocytes: indications for morpho-functional characterisation. eur. j. histochem. 49: 255-264, 2005. ballarin l, burighel p. rgd-containing molecules induce macropinocytosis in ascidian hyaline amoebocytes. j. invertebr. pathol. 91: 124-130, 2006. ballarin l, cima f, sabbadin a. phagocytosis in the colonial ascidian botryllus schlosseri .dev. comp. immunol. 18: 467-481, 1994. ballarin l, cima f, sabbadin a. morula cells and histocompatibility in the colonial ascidian botryllus schlosseri. zool. sci. 12: 757-764, 1995. ballarin l, cima f, sabbadin a. phenoloxidase and cytotoxicity in the compound ascidian botryllus schlosseri .dev. comp. immunol. 22: 479-492, 1998. ballarin l, tonello c, sabbadin a. humoral opsonin from the colonial ascidian botryllus schlosseri as a member of the galectin family. mar. biol. 136: 813-822, 2000. ballarin l, franchini a, ottaviani e, sabbadin a. morula cells as the major immunomodulatory hemocytes in ascidians: evidences from the colonial species botryllus schlosseri. biol. bull. 201: 59-64, 2001. ballarin l, menin a, franchi n, bertoloni g, cima f. morula cells and non-self recognition in the compound ascidian botryllus schlosseri. inv. surv. j. 2: 1-5, 2005. ballarin l, menin a, tallandini l, matozzo v, burighel p, basso g, et al. haemocytes and blastogenetic cycle in the colonial ascidian botryllus schlosseri: a matter of life and death. cell tissue res. 331: 555-564, 2008. 159 burighel p, cloney ra. urochordata: ascidiacea. in: harrison fw and ruppert ee (eds), microscopic anatomy of invertebrates, vol. 15, wiley-liss, new york, pp 221-347, 1997. burighel p, milanesi c, sabbadin a. blood cell ultrastructure of the ascidian botryllus schlosseri. ii. pigment cells. acta zool. 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italy accepted may 24, 2006 abstract in this work data on immune cell signallling in the circulating hemocytes of the edible bivalve, the mussel mytilus spp, are summarized. studies with different bacterial species and strains, heterologous cytokines and natural hormones, as well as with organic environmental chemicals, led to the identification of the role of conserved components of kinase-mediated transduction pathways, including cytosolic kinases (such as mapks and pkc) and kinase-activated transcription factors (such as stats, creb, nf-kb), in the immune response. from these data a general scenario emerged indicating that close similarities exist in the signalling pathways involved in cell mediated immunity in bivalve and mammalian immunocytes. in particular, the results indicate that both the extent and duration of activation of components of kinase-mediated cascades are crucial in determining the hemocyte response to extracellular stimuli. the identification of the basic mechanisms of immunity and its modulation in mussels can give important information for the possible utilization of these species as an invertebrate model for studies on innate immunity. moreover, the application of this knowledge to the understanding of the actual adaptive responses of bivalves when exposed to microorganisms in their natural environment can represent significant ecological, economical and public health-related interest. key words: mytilus; hemocytes; innate immunity; kinase-mediated cell signalling introduction innate immunity has recently received a renewed interest, in particular due to genetic and molecular evidence supporting the hypothesis that it represents an ancient and evolutionary conserved defense system (cooper et al., 2002, 2006). in particular, studies from the invertebrate model drosophila have been crucial in the identification of key molecular components (such as the toll-like receptors) of cell-mediated immunity also in vertebrates (miester and lagueux, 2003; nappi et al., 2004). however, invertebrates represent about 95 % of total species in animal kingdom and only for a few other invertebrate models information is becoming available on sequences coding for immune genes and genes corresponding author: laura canesi dipartimento di biologia, università di genova corso europa 26, 16132 genova, italy e-mail: laura.canesi@unige.it involved in immune cell signalling (gueguen et al., 2003; shida et al., 2003; venier et al., 2003; kim and ausuvel, 2005). therefore, for many invertebrate species, information on the mechanisms of activation of the innate immune response, and in particular on the signall transduction pathways involved, has been largely obtained by indirect means, in functional studies utilizing antibodies and pharmacological inhibitors directed towards the mammalian counterparts of signalling components. the scenario emerging from these studies indicates that a high degree of conservation occurs between the pathways involved in the activation of immunocytes from molluscan species (mainly gastropods and bivalves) and those of mammals. in bivalves (mussels, oysters, etc.) a few molecular studies have identified sequences potentially involved in immune signalling (escoubas et al., 1999; gueguen et al., 2003; montagnani et al., 2004). here we will summarize the results obtained on the signall transduction pathways involved in mediating the immune function of the edible marine 41 bivalve, the mussel mytilus galloprovincialis lamk. mussels are adapted to the natural exposure to a variety of microorganisms, and can accumulate in their tissues huge quantities of bacteria, mainly gram negative enterobacteria and vibrios, that can be pathogenic both to the mussels and to humans. although mussels are extremely simple organisms, they have a long life cycle (about 10 years) during which they are constantly exposed to microbes, and thus probably require rather complex homeostatic mechanisms to integrate both environmental and endogenous signalls. functional and molecular characterization of the basic mechanisms underlying the innate immune response in bivalves can give important comparative information on the immune response and on its modulation in this invertebrate group that can be applied to the understanding of the actual adaptive responses of these molluscs exposed to microorganisms in their natural environment. the immune system of bivalve mollusks the immune system of bivalves consists of the blood cells, the hemocytes, and of soluble hemolymph factors, that operate in a co-ordinated way to provide protection from invading micro-organisms (renwrantz, 1990; rinkevich and muller, 1996; hine, 1999). bivalve hemocytes are responsible for cell-mediated immunity through phagocytosis and various cytotoxic reactions (such as lysosomal enzyme and antimicrobial peptide release and oxyradical production); hemolymph serum contains humoral defense factors, such as soluble lectins, hydrolytic enzymes and antimicrobial peptides (canesi et al., 2002a; pruzzo et al., 2005) (fig. 1). bivalve hemocytes are extremely heterogeneous; classifications proposed for different bivalve species are reported elsewhere (fryer and bayne, 1996; ottaviani et al., 1998; hine 1999; wootton et al., 2003). in mytilus spp. granular hemocytes represent the dominant cell type in the hemolymph; they are characterised by a low nucleus/cytoplasm ratio, high phagocytic activity and capacity for oxyradical production (wottoon et al., 2003). survival of bacteria to the hemolymph microbicidal activity may depend on their different ability to attract phagocytes, to interact with opsonizing molecules, to bind to hemocyte surface, thus favouring or inhibiting the response of the host (canesi et al., 2002a; pruzzo et al., 2005). bacteria could evade hemocyte killing by avoiding phagocytosis, generally by inducing damage in the hemocyte, as well as by preventing the oxidative burst associated with the phagocytic process. finally, in analogy with host-pathogen interactions in mammalian models of infection (rosenberger and finlay, 2003), bacteria may undermine the hemocyte function through disregulation of the signalling pathways involved in hemocyte activation. cell signalling involved in the responses of mytilus hemocytes to bacterial challenge: the role of mapks and pkc in our first studies on the immune response of mytilus hemocytes to bacterial challenge we utilized an in vitro model of hemocyte monolayers incubated in the presence of the soluble hemolymph fraction (hemolymph serum) with e. coli mg155, a wild type (wt) strain carrying the type 1 fimbriae (canesi et al., 2001a). this strain was shown to adhere to and to be internalized by hemocytes through mannosesensitive interactions that were dependent on the presence of soluble serum components. the extent and time course of the hemocyte bactericidal were similar in vitro and in vivo (canesi et al., 2001a); therefore, the in vitro model was subsequently utilized for investigating the signall transduction pathways involved in the immune response. in mammalian systems, activation of a complex network of signalling pathways, that include proteinkinase and lipid-kinase driven cascades, is critical in innate immunity. in particular, one of these pathways, involving members of the highly conserved mitogen activated protein kinase (mapk) superfamily, plays a key role in both macrophage and neutrophil activation (caffrey et al., 1999). mapks are a family of serine-threonine kinases that are activated by phosphorylation in responses to different extracellular stimuli, with each member being activated by a distinct kinase cascade (caffrey et al., 1999). the extracellularly regulated mapks (erk mapks) are regulated by mitogens and growth factors; the stress-activated p38 and the c-jun n-terminal kinases (jnks) are activated by stress signalls such as uv, cytokines, heat and osmotic shock, endotoxin. a conserved role of mapk members in mediating the pleiotropic response to growth factors was demonstrated in mytilus cells (canesi et al., 2001b) cell signalling involved in the hemocyte response to bacterial challenge was first investigated by use of pharmacological inhibitors. hemocyte bactericidal activity (i.e. percentage of bacterial killing) towards wt e. coli mg155 was prevented by cell pre-treatment with specific inhibitors of the stress-activated p38 mapk and of pi-3kinase (phosphatidyl inositol 3 kinase), sb203580 and wortmannin, respectively. a significant decrease in the hemocyte response was also observed with inhibitors of enzymes involved in arachidonic acid production and metabolism, indicating a role for prostaglandins and leukotrienes in mediating the hemocyte response to bacterial challenge (canesi et al., 2002b). therefore, we first focused our attention on the involvement of mapks in hemocyte immune signalling. the presence and phosphorylation state (activation) of different mapk-like proteins were evaluated by sds-page electrophoresis and western blotting of hemocyte protein extracts with specific anti-phospho-antibodies directed against mammalian erk mapks and p38 and jnk mapks. the key role of mapks, in particular of the stressactivated mapks, in the immune response of mussel hemocytes towards the wt e. coli strain was confirmed by the rapid increase in phosphorylation of p38 and jnk mapks induced upon bacterial challenge in the presence of hemolymph serum; activation of erk mapks was also observed (canesi et al., 2002c). further information on the kinase-mediated pathways involved in the hemocyte response came from studies on the interactions with different bacterial species and strains; vibrio cholerae strains were utilized as a model of autoctonous marine 42 fig. 1 mechanisms involved in the bactericidal activity of bivalve hemolymph. vibrios that are particularly accumulated in mussel tissues, where they can persist after depuration processes in controlled waters (pruzzo et al., 2005). different strains of e. coli and v. cholerae incubated with hemocytes showed differences in adhesion and internalization (canesi et al., 2001a; zampini et al., 2003). these differences were associated with different sensitivities to the overall hemocyte bactericidal activity, with wt e. coli > mutant e. coli ≅ wt v. cholerae > mutant v. cholerae. we subsequently showed that different strains of e. coli and v. cholerae induced distinct patterns of mapk phosphorylation in mussel hemocytes within a narrow time range (5-60 min) (canesi et al., 2005a). although both wt and mutant e. coli strains induced an increase in the phosphorylation of the stressactivated p38 and jnk mapks, a different time course was observed in hemocytes incubated with the mutant e. coli strain compared to that observed with the wt e. coli: the increase in p38 mapk phosphorylation was delayed, and jnk phosphorylation was only transient. these differences may be ascribed to differences in adhesion between the two strains to hemocytes in the presence of serum; since the higher adhesion of the wild strain was due to serum opsonins (canesi et al., 2001a), different mechanisms in binding to hemocytes of the two strains may involve differential activation of receptors, receptor complexes and signalling pathways, this resulting in different patterns of mapk phosphorylation and leading to differential activation of the hemocyte response. interestingly, wt v. cholerae had little effect on mapk phosphorylation, in particular on the stressactivated mapks; on the other hand, this strain induced a rapid and large protein kinase c (pkc) phosphorylation. a distinct scenario was observed with the v. cholerae mutant, that, among the tested strains, showed the lowest sensitivity to hemocyte microbicidal activity (zampini et al., 2003). moreover, this strain induced severe cellular stress in the hemocytes, as indicated by the large destabilisation of lysosomal membranes, and the decrease in the phosphorylation state of all mapk members (erk, p38, jnks), as well as of pkc (canesi et al., 2005a). these data suggested that the inability of mussel hemocytes to mount an efficient defense response against the v. cholerae mutant may be related to down-regulation of the hemocyte signalling pathways, in particular through interruption of signalling cascades upstream of mapk activation, as previously described in mammalian host cells infected with pathogens (rosemberger and finlay, 2003). overall, the results demonstrated that, in bivalve hemocytes like in mammalian cells, different bacteria and bacterial strains can differently modulate the host signalling pathways. moreover, these studies indicated that not only the extent, but also the time course of bacteria-induced phosphorylation (activation) of different cytosolic kinases (mapks and pkc) is crucial in determining the hemocyte response. phosphorylation of transcription factors in mammalian immunocytes different stimuli can activate a number of cytosolic kinases, including mapks and pkc, leading to phosphorylation of different transcription factors that can modulate the expression of various genes involved in the immune response (su and karin, 1996; guha and mackman, 2001). among these, stat proteins (signall transducers and activators of transcription) are a family of transcription factors conserved in vertebrates and invertebrates unique in that they act both as signalling molecules and as transcription factors (decker and kovarik, 1999; horvath, 2000); moreover, they are the only transcription factor specifically activated by tyrosine phosphorylation (decker and kovarik, 1999; horvath, 2000). initially identified in interferon signalling (the jak/stat 43 pathway), stats have been recognised as essential components of both adaptive and innate immunity (stark et al., 1998). in particular, stat1 activation plays a critical role in the macrophage response against gram negative bacteria (ohmori and hamilton, 2001). in mussel hemocytes wt e. coli mg155 induced rapid and persistent tyrosine phosphorylation of immunoreactive stat1-, stat3-, and stat5-like proteins (canesi et al., 2003a); the time course of stat phosphorylation was consistent with that of the bactericidal activity, suggesting a physiological role for stat-like proteins in mediating the immune function. we have recently observed that also the wt v. cholerae strain induced significant tyrosine phosphorylation of stat1 and the results are reported in fig. 2a. interestingly, a distinct pattern of phosphorylation of stat1 was observed in response to different bacteria; v. cholerae rapidly induced a large but transient increase in stat1 phosphorylation, whereas the level of p-stat1 steadily increased with time in response to e. coli. we also investigated the effect of bacterial challenge on the phosphorylation state of the transcription factor creb (c-amp responsive element binding protein), whose activation is mediated by serine/threonine kinases. creb activation is an early response transcription factor whose role in inflammatory response has been well established; creb can be phosphorylated at ser133 by both pka and mapks; interactions between creb and other transcription factors such as stats can modulate transcriptional activity (decker and kovarik, 1999). a creb-like protein was identified in mussel hemocytes (canesi et al., 2005b). as shown in fig. 2b, wt e. coli induced a rapid and dramatic increase in creb phosphorylation; the level of p-creb peaked at 5 min after addition of bacteria (reaching about a maximal ten-fold increase with respect to controls) and remained high up to 60 min. also wt v. cholerae induced a significant creb phosphorylation; however, the effect was transient and much smaller than that observed with wt e. coli. in macrophages, differential activation of creb by different bacteria was shown to be dependent on differential activation of both pka and p38 mapk, with consequent effects on tumor necrosis factor α (tnf α) production (roach et al., 2005). these data further support the hypothesis that, like in mammalian macrophages, conserved kinase-mediated signalling cascades lead to phosphorylation of transcription factors, this possibly resulting in modulation of transcriptional responses in the hemocyte. moreover, the results further support the hypothesis that different bacteria have distinct effect on components of kinasemediated cell signalling, this resulting in differential activation of the immune response. cytokine signalling many studies indicate that host defense mechanisms in different invertebrate groups, including molluscs, can be modulated by a cytokine network as in vertebrates; however, most information relies on functional assays, using heterologous cytokines and antibodies directed towards vertebrate cytokines and their receptors (reviewed by beschin et al., 2003; ottaviani et al., 2004). although no molecular evidence for the presence of full length cytokine receptor or cytokine homologues has been provided in the genome of model invertebrates like drosophila melanogaster and caenorhabditis elegans, genes and corresponding proteins expressing domains found in vertebrate cytokines, cytokine receptors and components of cytokine signalling have been described in invertebrates (beschin et al., 2003). in mytilus spp, heterologous cytokines have been shown to modulate the activity of the hemocytes, and functional analogues of different cytokines and cytokine receptors have been described in this species (hughes et al., 1990, 1993; ottaviani et al., 1995, 2000; cao et al., 2004). in mammalian macrophages, complete activation results from stimulation with both bacterial products (lps) and the cytokine interferon γ (ifnγ) that act in concert to generate a maximal capacity to ingest and kill the microbes through activation of signalling pathways, including mapks and stats, leading to modulation of gene expression (su and karin, 1996, guha and mackman, 2001). we therefore tested the possibility that heterologous ifns may modulate mussel hemocyte signalling and function. we observed that short-term hemocyte pre-treatment with human recombinant ifnγ, but not with ifnα, significantly increased the bactericidal activity of mussel hemocytes towards e. coli (canesi et al., 2003a). ifnγ stimulated tyrosine phosphorylation of different stat-like members stat1, stat3 and stat5. in particular, ifnã lead to persistent phosphorylation of immunoreactive stat1; moreover, hemocyte pretreatment with ifnã, but not with ifnα, significantly increased stat1 phosphorylation induced by bacterial challenge with e. coli. ifnγ also affected the phosphorylation state of different mapks; in particular, activation of erk2 mapk and slow downregulation of stress-activate mapks were observed. the extent and time course of mapk phosphorylation induced by ifnγ were distinct from those elicited by either ifnα or bacterial challenge, again indicating a specificity of the hemocyte response to ifnγ. these results indicate that the hemocyte function can be modulated by heterologous cytokines and bacterial signals that act in concert through tyrosine kinasemediated transduction pathways involving both mapkand stat-like members. in particular, in mussel hemocytes, like in mammalian macrophages, both bacterial signals and ifnã converge on activation of stat1, a transcription factor that plays a critical role in the response towards gram negative bacteria. in previous studies, mussel hemocytes were shown to be responsive to heterologous tnfα and produce tnfα–like molecules in response to bacterial components (hughes et al., 1990, 1993; ottaviani et al., 1995).tnfα is a pleiotropic cytokine that plays a pivotal role in orchestrating innate immune responses, as well as in regulation of cell proliferation, differentiation and apoptosis in vertebrates (baud and karin, 2001). tnfα signalling involves binding to members of tnf receptor superfamily (tnfrs) and recruitment of a complex of adapter proteins; among these, tnf-receptorassociated factors (trafs) activate several 44 fig. 2 effects of bacterial challenge on the phosphorylation state of transcription factors of mussel hemocytes. changes in phosphorylation of stat1 (a) and creb (b) are shown in hemocytes incubated with wt e. coli (strain mg155) and v. cholerae (o1 el tor biotype strain n16961) in the presence of hemolymph serum for different periods of time. hemocyte protein extracts were subjected to 12 % and 10 % sds-page, respectively, followed by western blotting using polyclonal phosphospecific antibodies directed against phosphorylated stat1 (tyr701) and creb (ser133) as previously described (canesi et al., 2003a, 2005b). bands were stripped and reprobed with antibodies to the corresponding unphosphorylated stat1 and creb forms. data (mean±sd) of three independent experiments are expressed in % changes in p-stat1/stat1 and p-creb/creb with respect to controls. * = p�0.05, mann whitney u test. relative increases in band optical densities (arbitrary units) were normalised for the control band in each series. intracellular signall transduction pathways, in particular mapks and nf-kb, that lead to modulation of gene expression by different transcription factors (baud and karin, 2001). members of tnfα transduction machinery have been characterized in invertebrates; in drosophila a tnf superfamily ligand, eiger, has been identified, that triggers jnk-dependent apoptosis (cha et al., 2003). in drosophila, dtraf1 is essential for endogenous jnk activation, whereas dtraf2 is required for nf-kb signalling. when we first assayed hemocyte functional parameters in response to heterologous tnfα, distinct effects were observed in the presence and absence of hemolymph serum (betti et al., 2006). when added in the absence of serum (in asw-artificial sea water) tnfα induced cellular stress, as indicated by large lysosomal destabilization and decreased phagocytosis; on the other hand, in the presence of serum, tnfα did not affect lysosomal stability and significantly stimulated phagocytosis. tnfα induced rapid and large phosphorylation of the stress-activated p38 and jnk mapks, as well as of stat1; activation of p38 and jnks in mediating the effects of tnfá was confirmed by the use of specific mapk inhibitors. however, the effects on mapks and stat1 were persistent in asw but transient in serum, a difference that had not been previously observed with ifnã. flow cytometric analysis indicated that tnfα in the presence of serum induced transient phosphatidylserine exposure on the haemocyte surface, evaluated as annexin v binding; in asw, the cytokine resulted in a stable increase in the percentage of both annexin vand propidium iodide-positive cells, indicating possible apoptotic/necrotic processes. more recent data indicate tnfα can also affect nf-kb signalling in the hemocyte. transcription factors of the nf-kb/rel family play a pivotal role in the inflammatory and immune response (gosh et al., 1998). nf-kb/rel is composed of a set of structurally related and evolutionary conserved dna binding proteins: members of class i (p100 and 105 in mammals and relish in drosophila) and members of class ii rela/p65, c-rel, relb in mammals, dorsal, dif in drosophila and gambif in anopheles). a rel homolog, cg-rel, has been characterized in oysters (montagnani et al., 2004). p105 and p100 are activated by proteasomal degradation to p50 and p52 products that form dimeric complexes with rel proteins, which are then able to bind dna and regulate transcription. activation and nuclear translocation of nf-kb is modulated through both phosphorylation and ubiqutination induced by different stimuli (karin and ben-neriah, 2000). western blots of mussel hemocyte extracts with anti-nf-kb p105/p50 and anti-phospho-nf-kb-p65 antibodies showed the presence of immunoreactive protein bands corresponding to the p105 precursor and its cleavage product p50; moreover, a constitutively ser536 phosphorylated p65-like protein was identified (fig. 3). as shown in the figure, addition of tnfα to the hemocytes in the presence of serum did not apparently affect the level of p105 and p50; however, tnfá-induced a significant, rapid and transient increase in the level of phosphorylated p65. inducible phosphorylation of p65 at ser536 has been described in mammalian 45 fig. 3 effect of tnfα (200 nm) on the level and phosphorylation state of nf-kb components in mussel hemocytes. protein extracts from control and tnfα-treated hemocytes were subjected to 12 % sds-page followed by western blotting using polyclonal antibodies to p105 and p50 (a), or polyclonal phosphospecific antibodies to p65 (ser536) (b). bands were detected using enhanced chemioluminescence reagents. results are representative of three independent experiments. c = control. hmw and lmw protein standards. inset: densitometric analysis of blots (p-p65) from three independent experiments (mean±sd). * = p≤0.05, mann whitney u test. relative increases in band optical densities (arbitrary units) were normalised for the control band in each series. cells in response to tnf and lps, leading to increase in nf-kb transcriptional activity (sasaki et al., 2005). our data support the hypothesis that components of nf-kb signalling are present in mytilus hemocytes and that their activity may be modulated by heterologous tnfα. overall, the results indicate that tnfα can affect the function of bivalve haemocytes through conserved transduction pathways involving stress-activated mapks, stats, and components of nf-kb signalling and suggest that the haemocyte response to this cytokine is influenced by soluble hemolymph components. endogenous immunomodulators: 17ββ -estradiol signalling in bivalve molluscs, and mytilus in particular, a few endocrine and immune modulators have been identified so far (lacoste et al., 2001; stefano et al., 46 fig. 4 signalling pathways involved in the immune response of mytilus hemocytes. the main signalling components activated by both bacterial challenge and heterologous cytokines are reported. mapks = mitogen activated protein kinases; erk = extracellularly regulated kinase; p38 = stress-activated p38 mapk; jnk = cjun n-terminal kinase; pkc = protein kinase c; jak = janus activated kinase; stat = signall transducer and activators of transcription; creb =c-amp responsive element binding protein; nf-kb = nuclear factor kb; ros = reactive oxygen species. 2003a). estrogens have been identified in bivalves, where their role has been mainly investigated in the control of gametogenesis (osada et al., 2004; gauthierclerc et al., 2006). however, evidence has been provided that estrogen can represent an important signalling molecule involved in roles other than reproduction in these organisms. in neural tissues of mytilus spp., 17β-estradiol (e2) has been shown to downregulate ganglionic microglial cells after surgical insult; the effect was mediated by rapid induction of a ca2+-induced nitric oxide (no) release by nervous tissue and were antagonized by classical antiestrogens (stefano et al., 2003b). in mytilus, e2 was also shown to affect the digestive cells and circulating hemocytes, in particular at the level of the lysosomal function (moore et al., 1978; burlando et al., 2002). addition of e2 (in the low nm range) to hemocyte monolayers induced a moderate increase in cytosolic [ca2+], destabilisation of lysosomal membranes, morphological changes, hydrolytic enzyme release and stimulated the bactericidal activity towards e. coli (canesi et al., 2004a); all these effects were rapid, occurring from seconds to minutes from e2 addition and were prevented by the antiestrogen tamoxifen (canesi et al., 2004a). the effects of e2 were mediated by rapid activation of the stressactivated p38 mapk. moreover, in mussel hemocytes, e2 induced increased tyrosine phosphorylation of stat3and stat5-like members, as previously observed in mammalian cells. when the effects of e2 on components of the immune response were investigated in more detail, we observed that e2, in a narrow concentration range (5-25 nm), rapidly stimulated phagocytosis and oxyradical production; higher concentrations inhibited phagocytosis (canesi et al., 2006). e2induced oxidative burst was prevented by the nitric oxide synthase inhibitor l-nmma and by sod, indicating the involvement of both no and o2 -; no production was confirmed by nitrite accumulation. also these effects of e2 were prevented by the antiestrogen tamoxifen and by sb203580, further supporting a receptor-mediated event and involvement of the p38 mapk. e2-induced 47 stimulation of phagocytosis and oxidative burst were also prevented by the pkc inhibitor gf109203x, indicating a role also for pkc in mediating the effects of e2. in fact, e2 induced rapid and transient increases in the phosphorylation state of pkc, and in particular of a pkcα/βii-like isoform, as well as of the transcription factor creb. the results demonstrated that the signalling components that play a key role in the immune response of mussel hemocytes represent significant targets for the action of e2. the effects of e2 on the immune function were confirmed in vivo. for longer exposure times (6 and 24 hrs) in the hemocytes of e2-injected mussels lower concentrations resulted in immuno-stimulation, whereas higher concentrations had inhibitory effects (canesi et al., 2006). overall, the obtained data indicate that e2 signalling appears to be conserved from invertebrates to mammals. immune signalling as a target for environmental contaminants different environmental contaminants known as endocrine disrupting chemicals (edcs) (witorsch, 2002) have been shown to affect the hemocyte function through modulation of the signall transduction pathways involved in the activation of the immune response. first data were obtained with polychlorinated biphenyls (pcbs): different ortho-substituted, non coplanar pcb congeners were shown to affect the immune function through disregulation of mapk and stat signalling (canesi et al., 2003b). in particular, the di-orthosubstituted pcbs p47 (2,2’,4,4’-tetrachlorobiphenyl) and p153 (2,2’,4,4’,5,5’-hexachlorobiphenyl) were shown to rapidly increase the phosphorylation state of both mapks and stat members. among the tested congeners p47 showed the strongest immunotoxic effect, resulting in lysosomal destabilization, inhibition of e. coli-induced lysozyme release, decrease in the overall bactericidal activity; the effects were mainly due to a large and persistent phosphorylation of the stressactivated mapks p38 and jnks. other edcs, including synthetic estrogens, phytoestrogens and estrogenic chemicals that represent significant contaminants of the aquatic environment were shown to induce destabilization of lysosomal membranes in mussel hemocytes (canesi et al., 2004b). the effects were prevented by different kinase inhibitors, indicating that the effect of each compound was mediated by activation of different signalling components. when the effects of different edcs on the phosphorylation state of mapks and stats were evaluated, certain synthetic estrogens, like des (diethylstilbestrol) were shown to induce activation of p38 mapk and stats, with effects similar to those observed with the natural estrogen e2, although at higher concentrations; other estrogenic chemicals, such as the alkylphenols bisphenol a and nonylphenol, induced a large decrease in phosphorylation of p38 mapk and stat5. the effects of bpa on hemocyte function and signalling were confirmed in vivo, in the hemocytes of mussels sampled at different times postinjection with the compound (canesi et al., 2005b). the results showed that also in vivo, and at longer exposure times, bpa induced hemocyte lysosomal destabilization and decrease in phosphorylation state of p38 mapk, stat5, and creb, indicating down-regulation of cell signalling and possible immunodepression. another class of contaminants identified as potential hemocyte immunomodulators are certain brominated flame retardants (binmbaum and staskal, 2004). tbbpa (tetrabromobisphenol a) was shown to induce activation of erk, p38 and jnk mapks and pkc. these effects on cell signalling resulted in stimulation of the overall microbicidal activity through increase in phagocytosis and oxidative burst (canesi et al., 2005c). the results so far obtained indicate that different organic contaminants known as endocrine disruptors in vertebrate system can also act as immune disruptors in mussel hemocytes through disregulation of different components of kinase mediated cell signalling, resulting in distinct effects depending on the compound tested. conclusions the signalling pathways involved in the innate immune response show common features in bivalve hemocytes and mammalian immunocytes. from the results obtained so far, the main signalling components that play a role in the immune function of mytilus hemocytes can be summarized as shown in fig. 4. the results obtained with different stimuli (bacteria, cytokines, hormones, environmental chemicals) support the hypothesis that both the extent and duration of activation of components of kinase-mediated cascades are crucial in determining the hemocyte response to extracellular stimuli. in general, sustained but transient phosphorylation of both cytosolic kinases and transcription factors seems to be associated with efficient activation of the immune response; on the other hand, large and persistent activation of signalling components leads to cellular stress and irreversible damage. conversely, down-regulation of hemocyte signalling observed in response to certain bacteria of contaminants impairs hemocyte activation with different consequences ranging from immunodepression to citotoxicity. however, the study of immune signalling in bivalve molluscs, compared to that in other invertebrate models, is still at its infancy. the scheme depicted in fig. 4 does not include components whose involvement in mussel hemocytes has been suggested by indirect observations, such as the camp/protein kinase a (pka) pathway, pi-3 kinase, or enzymes involved in arachidonic acid metabolism. moreover, information immune-related receptors (copper, 2006), on signalling components upstream of mapk activation (caffrey, 1999), as well as on the role of the rho family gtpases, that are involved in many processes essential to the coordination of the complex machinery underlying innate immunity (bokoch, 2005), is still lacking. the identification of the basic mechanisms of immunity and its modulation in mussels can give important information for the possible utilization of this species as an useful invertebrate model for 48 studies on innate immunity. moreover, the application of this knowledge to comprehension of the actual adaptive responses of bivalves when exposed to microorganisms in their natural environment can represent significant interest not only from an ecological, but also a financial point of view. these studies can provide the basis for better understanding the reasons for the persistence of certain pathogenic agents in bivalve tissues, and possibly to prevent diseases in the molluscs themselves, with obvious advantages for the conservation of natural populations. the identification of the processes that influence the elimination of microorganisms by edible bivalves can also give useful information to devise strategies to ameliorate the depuration processes utilized in shellfish farming that are needed to lower the microbial load to levels acceptable for human consumption; these data can therefore be of interest for human health, since they contribute to prevent the risk of disease. a great effort is being dedicated by different groups at identifying immuneand signall transduction-related sequences in different molluscan species, 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issn 1824-307x review inflammatory hemocytes in ciona intestinalis innate immune response v arizza, d parrinello laboratory of marine immunobiology, department of animal biology, university of palermo, palermo, italy accepted march 13, 2009 abstract in the present paper an attempt is carried out to revise ciona intestinalis inflammatory hemocytes according to their morphology as formerly observed by light and electron microscopy, and taking in account recent reports on innate immunity gene expression. we also examine hemocyte morphofunctional aspects as derived from previous papers that refer to the tunic and body wall inflammatory responses challenged by corpusculate or soluble agents. lps inoculation into the body wall or treating hemocytes in vitro with lps have also been taken in account. lps inoculation stimulated the expression of citnfα, cifacit-αchain collagen, cic3a, cicd94 and enhanced phenoloxidase activity. these reports allow us to distinguish two main hemocyte types categories: 1. agranular hemocytes, including hemoblasts, circulating lymphocyte-like cells, hyaline amebocytes; 2. granular hemocytes including granulocytes with small granules, granulocytes with large granules, unilocular refractile granulocytes and morula cells. compartment cells and signet ring cells could be intermediate or terminal states presumably involved in releasing inflammatory factors or tunic matrix components. we suggest that the various hemocyte shapes, as shown by light and electron microscopy, could represent functional states as disclosed in inflamed tissues. although it cannot be excluded that a same cell expresses multiple activities, it is likely that several populations of a same cell type can exert distinct roles. key words: tunicate; innate immunity, inflammation, hemocyte, ciona intestinalis evolutionary relevance of ascidian immunology studies the developmental plan, the tadpole-like larva as well as molecular phylogenesis analysis support that tunicates are primitive members of the phylum chordata. ascidians (urochordata) occupy a critical position in the phylogenetic line leading to the vertebrates (swalla et al., 2000; zeng and swalla, 2005). recently delsuc et al. (2006) suggested that ascidians and not cephalochordates are the sister group of vertebrates, consequently urochordates have attained further importance for evolutionary immunology studies. ciona intestinalis is the representative species of the solitary ascidians generally retained a basic model for comparative biology research. the whole genome has been sequenced and several vertebrate homologous genes have been annotated (dehal et al., 2002; satou, 2002, 2003). bioinformatic ___________________________________________________________________________ corresponding author: vincenzo arizza department of animal biology university of palermo via archirafi, 18, 90123 palermo, italy e-mail: arizza@unipa.it approach and extensive in silico search have concerned immunorelevant molecules, gene expression patterns and some immune properties (davidson and swalla, 2002; fujita, 2002; nonaka and miyazawa, 2002; azumi et al., 2003; shida et al., 2003; terajima et al., 2003; du pasquier, 2004; fujita et al., 2004; kasahara et al., 2004). inflammatory reaction in the body wall of c. intestinalis, is a key evolutionary innate immunity model as well as can disclose hemocyte morpho functional aspects and pro-inflammatory products. soluble or particulate materials injected into the body wall are competent in challenging an inflammatory response including encapsulation and, in some cases, a tissue damage (parrinello, 1981; parrinello et al. 1984; parrinello and patricolo, 1984). in a variable time-course, these reactions can be visible through the transparent tunic, and microscopy observations show that in few hours the tunic matrix appears to be densely populated with hemocytes infiltrated through the epidermis(di bella and de leo, 2000), presumably coming from the pharynx and connective tissue close to the epidermis. hematogenic sites (crypts or nodules in the pharynx and emopoietic cells clusters) in the s58 pharynx and connective tissue (ermak, 1976) as well traits of proliferating epidermis have been shown (di bella and de leo, 2005). the nature of the used inflammatory agent as well as seasonal or environmental effects on the naïve ascidian populations could explain the variability in the timecourse and strength of the response. usually, in a few days, corpuscolate materials cause an intense heightened hemocyte populations density in the pharynx vessels, connective tissue, and tunic matrix where undergo differentiation, degranulation, necrosis, apoptosis, contributing in tunic matrix remodelling phase. the inflammatory cell-types show various shapes that could be related to activation and responding state, their products surround and isolate the host tunic containing the foreignness. in a significant number of individuals a degenerative process provokes a tunic wound which, successively, can be repaired (parrinello et al., 1977, 1984). although the timing sequence of the tunic reaction, after a second-set injection, is characterized by a heightened non-specific response, the chronic inflammation due to the first inoculation could maintain high hemocyte number and inflammatory factors level allowing an accelerate secondary response. therefore the presence of committed immunocompetent hemocytes may be excluded (parrinello et al., 1977). morphologically distinguishable hemocytes and their transitional forms, including cells that release their contents, have been observed. few stem cells, granular amebocytes and numerous hemocytes with large granules, signet ring cells and unilocular refractile granulocytes (urgs) have been found in the inflamed tunic. granulocytes degranulate, signet ring cells release their content, and numerous urgs express phenoloxidase (po) activity and release the active enzyme and/or pathway products. in the inflamed tunic matrix, a great amount of free granules and vacuoles, including lysosome particles, appear to be discharged by hemocytes. cellular debris and inflammatory molecules amplify the process also leading to tissue damage. after infection with e. coli, also circulating hemocytes promptly phagocytise the bacteria and excrete lysosome particles, while granular amebocytes liberate a lot of particles (liu et al., 2006). recently, we have examined c. intestinalis body wall inflammatory responses challenged by lipopolysaccharide (lps) (see below). lps is a component of the pathogen-associated molecular patterns (pamps). in organisms, ranging from invertebrates to humans, immune cells bear toll-like receptors (tlrs) that bind pamps (vasselon and detmers, 2002). three tlr distinct genes and the corresponding signal transduction cascades have been recognized in c. intestinalis draft genome (kimbrell and beutler, 2001; azumi et al. 2003; khalturin et al., 2004; roach et al., 2005). therefore it is presumable that lps-tlr binding induces responses including phagocytosis and release of inflammatory agents initiating the inflammatory response. ascidian hemocytes involved in immunity several approaches have been attempted to identify c. intestinalis hemocyte-types by using their morphological features under light or electron microscopy (rowley, 1981, 1982; de leo, 1992). in the last few years, interest in the mutual relationships between ascidians hemocyte types and products of innate immunity gene repertoire has led to a more clear-cut knowledge of these cells and their roles in immunity. this approach could also disclose that differentiation of activated cells yield to morpho-functional shapes of inflammatory hemocyte populations. a technique to classify c. intestinalis hemocytes is to look for the presence of granules, which allows to distinguish cells into two wide categories as agranulocytes (hemoblast, lymphocyte-like cells, hyaline amebocytes), or granulocytes (granulocytes with small or large granules, unilocular refractile granulocytes, morula cells). signet ring cells and compartment cells could be intermediate or final differentiation shapes following a challenge. variable frequency of circulating hemocyte types have been reported, presumably due to the variability within distinct ascidian populations as well as to seasonal factors and sea coastal environmental conditions. although transitional hemocytes have been identified in the hemolymph, they can be mainly found in inflamed tissues. hemoblasts and lymphocyte-like cells hemoblasts in hemopoietic nodules, are considered stem cells (about 3.0 5.0 µm) with a typical high nucleus/cytoplasm ratio (ermak, 1976, 1982). small hematogenic nodules are abundant in the pharyngeal wall and around the gut-loop; a few clusters also occur where the pharynx is attached to the body wall, under the endostyle and mesenteries. in the pharynx, hematogenic clusters are plentiful in transverse bars, spread in longitudinal bars, along the endostyle, and associated with the connective tissue. few hemoblasts with a lesser nucleus/cytoplasm ratio have been found in the circulating hemolymph (rowley, 1981; wright 1981; de leo, 1992). as shown by fine structure observations, the round nucleus contains condensed chromatine adherent to the nuclear envelope, and a characteristic prominent nucleolus. the cytoplasm presents free ribosomes and polyribosomes, mitochondria, occasional rough endoplasmic reticulum, rare golgi cisternae and a few lipid droplets. in different ascidian species, after an allogeneic challenge, circulating hemoblasts could proliferate and/or differentiate diverse hemocyte types (raftos and cooper, 1991). lymphocyte-like cells (llcs) (4.0-5.0 µm) in the hemolymph are similar to the circulating hemoblasts but present a lesser nucleus/cytoplasm ratio, the nucleus does not present a nucleolus, and the basophilic cytoplasm contains few small vesicles. light microscopy studies of unstained circulating cells do not allow a precise distinction between s59 http://en.wikipedia.org/wiki/pathogen-associated_molecular_pattern http://en.wikipedia.org/wiki/pathogen-associated_molecular_pattern http://en.wikipedia.org/wiki/granule_(cell_biology) http://en.wikipedia.org/wiki/granulocyte hemoblasts and llcs, and they are put on a pair with stem cells. frequently, structures recognized in the llcs presumably disclose initial differentiation steps, and some llcs wider in size are similar to small amebocytes containing numerous mitochondria and lysosome-like granules (warr et al,. 1977; fuke and fukumoto, 1993). various llcs frequency have been reported, presumably dependent on environmental conditions and the life cycle phase. frequency ranging from few cells up to about 20 % have been calculated (rowley 1981; wright 1981). recently liu et al. (2006) reported that some llcs can be marked by anti-cd34 monoclonal antibodies in agreement with the potential role of a pluripotent cell able to differentiate cell types. cd34 is a transmembrane protein expressed in mammalian hemopoietic cells (furukawa, 1998) and widely adopted as a marker of the human hemopoietic stem cells. although circulating hemoblasts were not distinguished, the llcs frequency slightly but significantly increases after in vivo bacterial (escherichia coli) challenge suggesting that they could proliferate in the circulatory system or in discrete body wall sites. llcs have been retained a primordial form of vertebrate lymphocyte (peddie and smith, 1995). although the enhanced proliferative activity may be related to their role in immunity, unequivocal evidences on c. intestinalis immunocompetent llcs in allorecognition have not been reported. in other ascidian species, llcs have been linked to non-self recognition and allograft rejection, also claimed as depositary of an adaptive histocompatibility-dependent cellular response including immunocompetent cell proliferation and differentiation of inflammatory hemocytes (raftos et al. 1987; raftos and cooper 1991). hyaline amebocytes in the hemolymph, hyaline amebocytes have been distinguished by electron microscopy as nonvacuolar or vacuolar hyaline amebocytes (rowley, 1982; de leo, 1992). in the inflammatory response, especially examined by light microscopy, immunocytochemistry and in situ hybridization methods, non-vacuolar and vacuolar amoebocytes cannot be distinguished, thus they are considered together as hyaline amoebocytes. electron microscopy observations show the cytoplasm with several vesicles of a variable size containing electron-lucid or a diffuse fibrous material, microtubules and microfilaments. vacuoles contain finely electron-dense granular inclusions, poorly developed endoplasmatic reticulum and golgi cisternae can also be observed. large and small vacuoles have been estimated as derived from expanded endoplasmic reticulum and golgi cisternae. morphologically some vesicles resemble the primary lysosomes of mammalian macrophages, while acpase positivity suggests phagolysosome formation (rowley, 1982). hyaline amebocytes are the most common cell type with phagocytic activity. within few minutes, they attach and, after the formation of pseudopodia, ingest formalinized sheep erythrocytes (more than 3/cell) (rowley, 1981), e. coli (liu et al., 2006) and yeast (personal observations), whereas they are not able to phagocytise polystyrene latex beads (zucchetti et al., 2008). after e. coli inoculation, secondary lysosomes can be formed and lysosomal enzyme particles secreted on the bacteria surface. part of the infected cells undergo cell death, either necrosis or apoptosis. electron microscopy observations show that the necrosed cells lose their membrane integrity, and broke completely. another portion of hemocytes which present integral membrane showed characteristics of apoptotic cells that promptly appear after in vivo infection. the phagocyte activity against yeast can be enhanced when the targets were opsonized with lectins (parrinello et al., 2007) suggesting that a lectin recognition mechanism characterizes these cells. hyaline amebocytes, with the above characters described for circulating cells, have not been observed in the inflamed tunic tissue, although the possibility exists that infiltrated cells undergo morpho-functional differentiation. in a recent paper parrinello et al. (2008) showed that, at 4 h after lps inoculation, circulating hyaline amoebocyte populations contain and presumably release the citnfα cytokine (cloned and sequenced) as shown by in situ hybridization analysis that identified the mrna mainly in the nucleus. granulocytes several circulating granulocytes have been described by light and electron microscopy, and distinguished by the granule size and abundance, shape and electron density of their content (de leo, 1992). in some cases, the granules contain a low electron-dense material and they have been referred as “vesicles” or “vacuoles”. rod-like and refractile granules have been observed upon phase contrast microscopy (rowley, 1981). light and electron microscopy showed that inflamed tissues including pharynx vessels, hemolymph and tunic were enriched with granulocytes containing granules of various shape, size, content density and fine structure organization. granular amebocytes with small granules following an inflammatory challenge numerous granular amebocytes promptly populate the tunic tissue, degranulate (parrinello et al., 1990) and release inflammatory factors. circulating granular amebocytes with small granules challenged in vitro and in vivo by e. coli (liu et al., 2006) lose their membrane integrity and degranulate, and electron microscopy shows small holes in the plasma membrane. after in vivo infection, apoptosis promptly appeared in the granular amebocytes, and massive density of necrotic hemocytes and degranulating granulocytes were also found in the inflamed tunic. in the inflamed tunic, cell functional states could be characterized by various electrondensity of the granular content that in some case appears heterogeneous in its fine structure. some granules present an electron-dense area surrounded with microtubules, whereas small s60 electron-transparent granules are acpase positive. rowley (1981) reports that these cells did not usually spread out to the same extent as the hyaline amebocytes and their cytoplasmatic extensions were often less evident. contrasting data have been reported on their phagocytic activity. although circulating granular amebocytes can fix bacteria, they do not phagocytised e. coli (liu et al., 2006), and it has been shown that after bacterial challenge the activated cells secrete in vitro numerous granules while typical apoptosis bodies emerge in ameboidic granulocytes. on the contrary several reports concern their phagocytic activity. smith and peddie (1992) showed that granular amebocytes collected from a percoll density band, ingest in vitro psychrobacter immobilis and their activity increases when the bacteria were pre-treated with hemocytelysate supernatant. however they were not able to distinguish between granular amebocytes and hyaline amebocytes both contained in the separated band. zucchetti et al. (2008) report that granular amebocytes are able to phagocytise polystyrene latex beads, and, after lps inoculation or in vitro treatment, these cells become more effective (up to 80 % granulocytes) in phagocytising the target. finally, rowley (1981) showed that these hemocytes promptly ingest formalinized sheep erythrocytes, showing indistinct ruffled membranes or spike-like pseudopodial extensions. the attachment of erythrocytes not always results in their internalization presumably due to differences in the recognition pattern. these various behaviours could be in accordance with granular amebocyte populations provided of different target specificity. lps inoculation puts in evidence that granular amebocytes, presumably distinct populations, are engaged in cic3a production and cicd94-1 expression. two c3-like genes, cic3-1 and cic3-2, from hemocyte total rna have been cloned and sequenced (marino et al., 2002). as in mammals, anaphylotoxin cic3a peptide is generated by proteolytic cleavage of the c3α-chain and it may exert proinflammatory activity including hemocyte recruitment (pinto et al., 2003). following lps inoculation, an increased number of these cells contain cic3a fragment with chemotactic activity (pinto et al., 2003), and also constitutively express a cic3a receptor (melillo et al., 2006) supporting the recruitment and activation of granular amebocytes and other effector cells in inflammation (c3ar positive circulating hyaline amebocytes identified by immunostaining could be granular amebocytes). inhibition experiments with the antibodies revealed that a cic3a-cic3ar binding is requested for exerting ci3a chemotactic activity on hyaline and granular amebocytes. a granular amebocyte population constitutively expresses cicd94-1 (homolog to vertebrate cd94 that marks nk cells), involved in the phagocytic activity, as a self-non-self recognition receptor with a c-type lectin domain exposed on the cell surface (zucchetti et al., 2008). after in vitro lps treatment 80% amebocytes express the cicd94 transcript. granulocytes characterized by the size and number of their granules they are large cells (ranging from 5.0 to 11.0 µm) with a cytoplasm, partially or almost entirely occupied by great granules containing materials of various density and refractile properties (rowley, 1981). the size and number of these granules can be very different (0.5-2.5 µm). granule number and size characterize granulocytes with many large granules, whereas other granulocytes contain a variable number of globules filled with material of low, moderate or high density. granules tend to fuse, and in the inflammatory reaction globular material appear to be released. an unique large granule occupies almost the whole cytoplasm and identify the unilocular refractile granulocytes. finally compartment and signet ring cells could be terminal hemocyte forms that release their content. the inflammatory response allows to distinguish the following hemocytes. granulocytes with large granules these granulocytes are abundant in the inflamed tunic, their inflammatory role is mainly indicated by phenoloxidase (po-2)-positive granules contained in the cytoplasm (parrinello et al., 2003). the enzyme was identified by dopa-mbth cytochemical reaction and anti-cipo-2 specific antibodies (immunoistochemistry) raised against a peptide designed from cloned and sequenced c. intestinalis po-2 (immesberger and burmester, 2004). the density of the po-positive granulocyte population as well as the enzyme activity increased in the tunic inflamed by lps inoculation, reaching the highest level within a few hours post injection (cammarata et al., 2008). in vitro assay of inflamed tissue and po assay of the tunic lysate supernatant supports that cells containing prophenoloxidase (propo) are activated. quinones, promptly (8 h) produced as an effect of cellular propo activation (proteolysis activated by lps), are distributed in the tunic matrix and presumably contribute to the inflammatory reaction. these granulocytes could have relationships with po-positive unilocular refractile granulocytes and morula cells. fine structure observations showed that numerous granulocytes with large granules undergo degranulation and granule contents (amorphous and granular) are released into the inflamed tunic matrix (de leo et al., 1992). unilocular refractile granulocytes (urgs) the cells present an unique large granule, filled with electrondense material, nearly occupies the whole cell and confines the nucleus at the periphery close to the cell membrane. this hemocyte-type has been identified in the hemolymph, the granule content appears to be refractile under phase contrast microscopy (rowley, 1981), provided with homogeneous fine granular content [electron microscopy (tem) observations] arranged in flocculent s61 structures or with loose or condensed fibrogranular material that forms masses aggregated into two or more foci often flowing together. the urgs are promptly involved in the tunic inflammatory reaction and are numerous in the inflamed tissue. the unique granule contains phenoloxidase (parrinello et al., 2003), cytochemical reaction with dopa-mbth shows a strong enzyme activity and the presence of cipo2 was revealed by specific antibodies. following lps injection (cammarata et al., 2008) numerous po-positive urgs occupy the tunic and presumably release products of po pathway. it is noteworthy that the circulating urgs exert cytotoxic activity, presumably cd94independent, when assayed in vitro with erythrocytes (parrinello et al., 1996). plaque forming cell assay demonstrated that cytotoxic molecules can be released following effector-target contacts (parrinello et al., 1996). an urg population from naïve ascidians expresses an antimicrobial peptide (cipap-a) that exerts a potent antimicrobial activity against a variety of bacteria and the yeast candida albicans (fedder and leippe, 2008). depending on the cell differentiation state, either the cytoplasm or the inclusion inside the large compartment contain cipap-a. although an assay on hemocytes from lps injected ascidians was not performed, it is reasonable that cipap-a can exert its antimicrobial activity in inflamed tissue injured by the injection procedure as well as by the inflammatory wound. multilocular granulocytes some large globular granules, tightly packed and close to the cell membrane, occupy the most cytoplasm. electron dense or electron transparent granule contents have also been used to distinguish two hemocyte types, morula cells and compartment cells respectively. morula cells they are spherical (8 16 µm), present variable number of tightly packed symmetrically arranged large globules (2 3 µm, max. 7). the nucleus, devoid of nucleolus, eccentrically located and compressed into an angular mass, is usually obscured by the globules. the globules are filled with high density homogeneous material, in some case the content appears electron-transparent with dense inclusion which may be granular or filamentous. it is peculiar that globules have a high refractive index (rowley, 1981). to one side of the nucleus there is a well-developed golgi apparatus containing irregularly shaped masses and an endoplasmic reticulum whose cisternae enclose dense granules (wright, 1981). cellular activity may be indicated by endoplasmic reticulum cisternae containing dense granules, and by irregularlyshaped masses in the golgi vesicles. when allowed to stand they assume a berry-like or morular appearance. presumably distinct morula cells populations could be distinguished. although phenoloxidase (dopa-mbth reaction and immunohistochemistry stain) was mainly found in granulocytes with numerous large granules and in urgs, it is noteworthy that, both in the hemolymph and in inflamed tissue (in vitro observations), some morula cells show globular granules with a faint po positivity. in addition these cells express (in situ hybridization) and contain (immunocytochemistry) citypeix-like (facit) collagen α-chain (parrinello et al., 2008). we cloned and sequenced a facit collagen (1α-chain) which is constitutively expressed in c. intestinalis tissues (vizzini et al., 2002). a prompt (4 h) expression of this collagen was shown by real-time pcr in the pharynx of lps inoculated ascidians as well as in circulating hemocytes challenged in vitro (flow cytometry analysis). collagens are major structural components of extracellular matrix in tissues of vertebrates and invertebrates, also involved in defence and reparative processes (singer and clark, 1999). in acute inflammatory reactions cellular, humoral, and molecular events are activated resulting in a regulated pattern of tissue repair with collagen fibres bundles organized during the remodelling (nwomeh et al., 1998). intermediate or fully developed hemocytes the possibility exists that compartment cells and signet ring cells may be intermediate or fully developed hemocytes. compartment cells spherical cells (8 12 µm) that present a variable number of large round globules containing electron transparent material with electron-dense granules of variable size on the inside. the nucleus contains no nucleolus and is centrally located, surrounded on one side by mitochondria and numerous profiles of dense rough endoplasmic reticulum and ribosomes (wright, 1981). rowley et al. (1984) report that compartment cells are usually far less common (5 % max.) than morula cells, moreover x-ray microanalysis results suggest interrelationship between these two cell types. we have found that, like morula cells they contain ci-facit collagen α-chain and are involved in tissue inflammatory response (vizzini et al., 2008). compartment cell-like cells releasing their globule content can be recognized in the inflamed tunic (de leo et al., 1992), suggesting they could be regarded as a fully developed hemocyte engaged in discharging inflammatory factors. in addition, they can express and contain inducible citnfα-chain in the cytoplasm lining the globules (parrinello et al., 2008). although both collagen chain and citnfα are constitutively expressed more numerous positive cells can be identified in the body wall after the lps challenge. the citnf enhanced time course expression is fast whereas that of the collagen increases after few days. finally, compartment cells express cic3a fragment (pinto et al., 2003) but not cic3a-receptor has been found (melillo et al., 2006). it is difficult to identify hemocyte types by using immunostaining method, and some cic3ar positive cells could be compartment cells. in additon, cic3ar positive granular amebocytes (presumably granulocytes with s62 table 1 features, activities and innate immunity genes expression of c. intestinalis inflammatory hemocytes observed in the inflamed tunic matrix or hemolymph after inoculation with lps. hemocyte drawings from light and electron microscopy observations hemoblast llc hematogenic tissue • steam cells circulating hemolymph • proliferation • cd34 positive • allograft reaction. liu et al., 2006; reddy et al., 1975; peddie et al., 1995 a gr an ul oc yt es circulantin hyaline amebocyte circulating hemolymph • express ci-tnf (lps) • fagocytosis fsrbc e. coli, saccharomices cerevisiae • necrosis/apoptosis. parrinello et al., 2008; rowley 1981; liu et al., 2006 granulocyte with small granule inflamed tunic • degranulation • cic3-1 expression • cic3a-r expression • cicd-94 expression circulating hemolymph • necrosis/apoptosis • phagocytosis: psicobacter immobilis, polystirene latex beads, fs rbc (not always), fix e. coli • degranulation following lps inoculation • cicd-94 expression. parrinello et al., 1990; liu et al., 2006; pinto et al., 2003; melillo et al., 2006; zucchetti et al., 2008; cammarata et al., 2008 granulocyte with large granules inflamed tunic • a few granules po-2 positive. cammarata et al., 2008 mc refractile globules inflamed tunic • same mcs contain po-2 positive globules • ci-facitα-chain collagen. vizzini et al., 2008 g ra nu lo cy te s urg unique refractile granulocyte inflamed tunic • po-positive unique large granule circulating hemolymph • po-positive unique large granule • cicd-94-independent cytotoxic activity (rbc, k562) • plaque forming cell • cipap-a expression against a variety of bacteria and candida albicans. zucchetti et al., 2008; parrinello et al., 1996; fedden and leippe, 2008; cammarata et al., 2008 compartment inflamed tunic (lps) • ci-facitα-chain collagen expression • ci-tnfα expression • ci-c3a • no ci-c3a-r. vizzini et al., 2008 parrinello et al., 2008 pinto et al., 2003 in te rm ed ia te a nd /o r te rm in al fo rm s signet ring cell inflamed tunic • encapsulation • release of electron transparent content. parrinello, 1981, 1990; de leo et al., 1992. s63 small granules) and some large round non ameboidic cells can be distinguished with difficulty from the negative cells identified as morula cells. signet ring cells these cells have been mainly described in the inflamed tunic (parrinello and patricolo, 1984; de leo et al., 1992), their frequency in the hemolymph may be very low (about 2 %), or they are absent. a large electron-transparent granule occupies almost entirely the cytoplasm and confines the nucleus at a peripheral site. electron microscopy observations show that these hemocytes (de leo et al., 1992) are very numerous in the inflamed tunic matrix where they discharge their granular or amorphous content as an effect of membrane dissolution. (parrinello 1981, 1990; de leo et al., 1992). the nature of the discharged granule materials is unknown, but they seem to contribute in encapsulation response. according to rowley (1981) presumably these cells represent the secretive state of challenged hemocytes. conclusions electron and light microscopy morphological features of c. intestinalis hemocytes allowed controversial classifications of the circulating cells. insights have been obtained by examining both cellular aspects of the inflammatory response and innate immunity genes expression (table 1). corpusculate and soluble inflammatory agents, bacteria and lps inoculation into the ascidian body wall, as well as in vitro challenge of circulating hemocytes, contribute to distinguish two main categories, agranulocytes (hemoblasts, lymphocytelike cells, hyaline amebocytes) and granulocytes (granulocytes with small granules, granulocytes with large granules, unilocular refractile granulocytes, morula cells). compartment cells and signet ring cells could be intermediate or fully developed hemocytes presumably involved in releasing inflammatory factors or tunic matrix components. gene expression studies, immunohistochemistry and immunocytochemistry assays disclose that hyaline amebocytes, mainly phagocytes, contain and presumably release citnfα-like cytokine, and show that granular amebocytes express cic3-1, cic3ar, cicd94 receptor and produce chemotactic cic3a supporting their inflammatory role. granulocytes with large granules show a weak po activity of a few granules, and could have relationships with po-positive unilocular refractile granulocytes and morula cells. urgs are the main components of the inflamed tissue where presumably release products of the po pathaway, whereas circulating urgs exert cytotoxic activity when assayed against erythrocytes and k562 tumor cell line. cytolysins cannot be related to the ca2+independent cicd94 nk-like receptor, they are released and exert ca2+-dependent activity. on the contrary, cicd94 nk-like receptor is involved in phagocytic activity. finally, an urg population from naïve ascidians expresses an antimicrobial peptide (cipap-a) with a potent antimicrobial activity. it is reasonable that cipap-a exerts its antimicrobial activity in inflamed tissue injured by the injection procedure and by the inflammatory wound. morula cells contain globules filled with high density homogeneous material showing high refractive index, and some of them are provided with faint po activity. in addition these cells express citypeix-like (facit) collagen α-chain following an lps challenge. some similarities suggest interrelationship between morula and compartment cells which contain a variable number of large round globules with electron transparent content. however, circulating compartment cells appear to be engaged in releasing inflammatory factors including citnfα-chain and cifacit α-chain, cic3a fragment. these hemocytes have been also found in inflamed tissues. finally signet ring cells could be a fully developed hemocytes discharging their granular or amorfous content as an effect of membrane dissolution, representing the secretive state of challenged hemocytes. we suggest that morpho-functional studies on challenged inflammatory hemocytes could contribute in establishing a more precise classification of the c. intestinalis hemocytes, taking in account that several populations of a same cell type can exert distinct roles. aknowledgments research on the c. intestinalis gene expression (citnf, cifacit collagen), po and cytotoxic activities of inflammatory hemocytes, as well as the present paper, have been supported by the princofin 2006 grant to prof n parrinello. references azumi k, de santis r, de tomaso a, rigoutsos i, yoshizaki f, pinto mr, et al. genomic analysis of immunity in a urochordate and the emergence of the vertebrate immune system: "waiting for godot”. immunogenetics 55: 570581, 2003. cammarata m, arizza v, cianciolo c, 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connective tissue forming large haemolymph sinuses isj 6: 125-137, 2009 issn 1824-307x review mechanisms of wound repair in crayfish x vafopoulou biology department, york university, 4700 keele street, toronto m3j 1p3, ontario, canada accepted september 8, 2009 abstract this review describes the complexity of events involved with repair to integumentary wounds and their regulation using the crayfish as a model system. injuries to integument precipitate a cascade of cellular events that lead to rapid healing of the wound, regeneration of damaged tissues and repair of the integument. the first step in this cascade is hemolymph clotting and subsequent melanization, events documented thoroughly elsewhere and not discussed here. wound healing and repair in crayfish involves the action of two physiological systems, the immune system and the neuroendocrine system regulating synthesis of the steroid molting hormones, ecdysteroids. injury promotes a swift rise in hemolymph ecdysteroids to a low, sustained plateau, followed by a premolt peak and molting. the plateau is essential for wound healing since its principal targets are the circulating cells of the immune system, the hemocytes, and healthy epidermal cells and fibrocytes. massive migration of these cells occurs under the wound and their concerted efforts under ecdysteroid control are paramount to wound healing and repair. these cells are likely engaged in physiological and biochemical activities that promote cell communication and cell to cell adhesion, removal of dead and harmful material and production of molecules essential to tissue regeneration. key words: crustacea; immune system; ecdysteroids; hemocytes; epidermis; regeneration introduction structural integrity in crustaceans is maintained by a rigid exoskeleton. the exoskeleton is also the main defense barrier against invasions by microbes and parasites. when this barrier is breached by mechanical injuries or erosion due to chemicals or bacteria, the animal becomes vulnerable to systemic infections. it is therefore vital for survival that injuries to integument are healed promptly and the damaged integument is repaired quickly. this review addresses events and mechanisms by which these responses are brought about. injury triggers a dramatic awakening of the machinery that directs restoration and repair of damaged tissues the details of which are discussed in the following section. wound healing is a complex and orderly process involving the coordinated behavior of different cell types. it relies heavily on rapid immune responses by the hemocytes which seal and protect the wound site and massive movements of cells which regenerate the damaged tissues. the initial event is coagulation of hemolymph components that ___________________________________________________________________________ corresponding author: xanthe vafopoulou biology department york university 4700 keele street, toronto m3j 1p3, ontario, canada e-mail: xanthev@yorku.ca seal off wounds temporarily and thus contain hemolymph loss and trap microbes (see for review lee and söderhäll, 2002; cerenius and söderhäll, 2004). ruptured hemocytes release clotting enzymes that coagulate hemolymph and enzymes that activate the pro-phenoloxidase (propo) cascade which leads to melanisation and ensures trapping of foreign and damaged cell material. additional immune responses are also triggered that include phagocytosis, cytotoxicity, nodule formation and encapsulation which kill invading microbes and remove cellular and matrix debris. concurrently with hemocyte recruitment, epidermis regeneration is triggered and when this process is completed the newly formed epidermal layer deposits a new cuticle that seals the wound permanently and restores the damaged integument to its original condition. the phenomena of wound healing and repair raise fundamental biological issues concerning the origin of cues that initiate the cascade of this process. cues may originate locally to act from one cell population to another or may be humoral in nature. for example the cues that trigger hemolymph coagulation are usually local. in the case of bacterial infection, local signals present on the surface of microbes initiate hemolymph clotting (see for review sritunyalucksana and söderhäll, 2000; theopold et al., 2002, 2004). by contrast, in 125 the case of physical wounding the cues are largely unknown. it is hypothesized that, as in vertebrates (e.g., gallucci and matzinger, 2001), unidentified “danger signals” are released from the damaged cells that trigger the clotting reaction. danger signals may include cellular components of damaged cells such as nucleotides, reactive oxygen intermediates or other intracellular proteins, which activate the hemolymph clotting cascade (review by theopold et al., 2002). another example of local signals derives from the study of limb regeneration in crabs following limb autotomy; regenerating limb buds release and respond to growth factor(s) that promote local growth (hopkins et al., 1979, 1999; hopkins, 2001). cues for the cascade of wound healing and repair may also be provided distantly, specifically from the central neuroendocrine system. the primary candidate for promoting tissue regeneration is the steroid molting hormones of crustaceans, ecdysteroids. for example, wound healing and repair depends on the presence of low levels of ecdysteroids following carapace injury in crabs (halcrow and steel, 1992) and crayfish (vafopoulou et al., 2007) or limb autotomy in crabs (mccarthy and skinner, 1977). freshwater crayfish provide an excellent model system to advance knowledge on the mechanisms of wound healing and repair. crayfish are of commercial and ecological importance. they are easy and inexpensive to rear in large numbers under laboratory conditions for experimentation in a simple aquatic environment. key components of the innate immunity system have been established in the crayfish (e.g., söderhäll et al., 1994; söderhäll and cerenius, 1998; wang et al., 2001a; lee and söderhäll, 2001, 2002) and much is known about the morphology and development of the animal (references in vogt, 2008). in the present review, we will discuss the current state of understanding of wound healing and tissue regeneration in the freshwater crayfish procambarus clarkii and the underlying mechanism of hormonal control that brings about these changes. wound healing and repair in crayfish we have found a direct physiological link between the immune system and the system that controls molting during wound healing and repair in the crayfish. specifically, we found in wounded animals immune responses affect the molting system and molting responses affect the immune system. interaction of these two systems is crucial in the ability and speed by which a crayfish heal wounds to the integument. the two physiological systems and their involvement in wound healing are described in details below. the first system to respond to injuries is the immune system. many aspects of the crustacean immune system have been elucidated in crayfish, making it a valuable animal to study the involvement of the immune system in wound healing. the immune system is centered on the circulating hemocytes and involves mechanisms to recognize and destroy non-self material and heal wounds. there are two components in innate immunity in crustaceans, the humoral and cellular components, both of which are activated upon immune challenges resulting from wounds to integument. cellular defenses include hemocyte-mediated responses such as phagocytosis, nodulation and encapsulation. humoral defences include antimicrobial peptides, coagulation and melanization of the hemolymph and production of reactive intermediates of oxygen and nitrite (see lee and söderhäll, 2002; lee et al., 2004). most of the bioactive peptides are produced by the hemocytes. among these bioactive compounds, the propoactivating system is the best studied and plays a pivotal role in innate immunity. in crayfish, propo and the enzymes responsible for its conversion to its active form phenoloxidase (po) reside as zymogens in the granules of granular and semigranular hemocytes, the contents of which are released by exocytosis into the hemolymph under challenges by pathogens or cell damage because of wounding (smith and söderhäll, 1991; wang et al., 2001a). this conversion ultimately results in the formation of melanin and the generation of a number of potent bioactive products, which assist phagocytosis, cell to cell adhesion and melanization (see söderhäll et al., 1994; söderhäll and cerenius, 1998; see for review lee and söderhäll, 2002; cerenius and söderhäll, 2004). propo has been cloned in crayfish (aspán et al., 1995). other important components of immune responses have also been extensively studied in crayfish. for example, components of the clotting and melanization system have been characterized such as a transglutaminase essential for hemolymph clotting (hall et al., 1999; wang et al., 2001b) and an inhibitory protein that halts melanization (söderhäll et al., 2009); antimicrobial peptides such as astacidin and crustin (jiravanichpaisal et al., 2007); hemocyanin which acts as a phenoloxidase in crayfish and a precursor to an antibacterial peptide (lee et al., 2004); peroxinectin, a multifunctional peptide critical for cell to cell adhesion (johansson and söderhäll, 1988), encapsulation (kobayashi et al., 1990) and degranulation (johansson and söderhäll, 1989); a pattern recognition peptide (lee and söderhäll, 2001; see for review sritunyalucksana and söderhäll, 2000; lee and söderhäll, 2002; cerenius and söderhäll, 2004). study of the cascade of events in wound healing depends on recognition of the morphological and functional characteristics of hemocytes. hemocytes are classified according to their morphology, cytochemistry and function (smith and söderhäll, 1983a; see for review johansson et al., 2000) and are recognized as hyaline, semigranular and granular hemocytes based on a classification system established by smith and söderhäll (1983a). based on this system, hemocytes have been characterized in several crayfish such as astacus astacus (smith and söderhäll, 1983a), procambarus zonangulus (cárdenas et al., 2000), pacifastacus leniusculus (wang et al., 2001a), procambarus clarkii (vafopoulou et al., 2007) and astacus leptodactylus (giulianini et al., 2007). in general, all three classes and subclasses of hemocytes, are recognized in all crustacean species (e.g., vázquez et al., 1997; johansson et al., 2000; sung and sun, 126 2002; battison et al., 2003; martin et al., 2003; liu et al., 2007; zhan et al., 2008). hemocytes have a defined division of labor in innate immunity. hyaline hemocytes are critical for hemolymph clotting (hall and söderhäll, 1994; wang et al., 2001a; reviews by theopold et al., 2002, 2004) and phagocytosis (smith and söderhäll, 1983b). the principal role of semigranular hemocytes is encapsulation (kobayashi et al., 1990) and cytotoxicity (söderhäll et al., 1985), whereas granular hemocytes are the primary source of the propo-activating system (johansson and söderhäll, 1985; see for review sritunyalucksana and söderhäll, 2000). the second system in crayfish that responds to injury is the neuroendocrine system that controls molting (vafopoulou et al., 2007). molting is the cyclical formation of a new exoskeleton and the shedding of the old one. growth and molting in crustaceans is regulated by ecdysteroids (such ecdysone and 20-hydroxyecdysone) the concentration of which displays a characteristic sequence of increases and decreases over a molt cycle (steel and vafopoulou, 1989). ecdysteroids are synthesized and secreted by the y-organs under the negative control of the neuropeptide molt-inhibiting hormone (mih). mih is synthesized by the x-organ in the brain and released from the sinus gland in the eyestalk (see for review webster, 1998). when present, it prevents the secretion of ecdysteroids from the y organ and holds crustaceans in intermolt (e.g., snyder and chang, 1986; see for review chang et al., 2001). new cuticle is secreted by the epidermis under the action of ecdysteroids; this period in the molt cycle is called ‘premolt’ and it culminates in shedding of the old exoskeleton (ecdysis) and hardening of the new one in the post-ecdysial period known as ‘postmolt’. during the remainder of a molt cycle, animals are devoid of ecdysteroids and not preparing to molt; this period is called ‘intermolt’. we have found that injuries to the crayfish integument during intermolt induce precocious production of ecdysteroids, measured by radioimmunoassay (ria), as shown in fig. 1 (details in vafopoulou et al., 2007). these injuries cause initially a small but significant elevation in the concentration of hemolymph ecdysteroids to a low, sustained plateau (10-12 days after injury) (fig. 1a, b; dark triangles). the plateau is then followed after about 2-3 weeks by a sharp increase to the peak values characteristic of premolt. ecdysis occurs at about 2 months later in crayfish. uninjured, healthy controls show only a slight increase in hemolymph ecdysteroid levels throughout the premolt period of wounded animals (fig. 1a, b; open triangles), and they never form a premolt peak. positive control animals which are subjected to removal of both eyestalks but no exocuticular injury exhibit no plateau phase but rather commence the steep premolt increase promptly after ablation (fig. 1a, b; open circles). bilateral eyestalk removal is known to precipitate an immediate premolt, because it removes the source of the neuropeptide moltinhibiting hormone (mih). therefore, both eyestalkless and wounded animals are induced to enter premolt but in wounded animals the premolt peak is delayed relative to eyestalkless animals by the duration of the plateau phase. fig. 1 induction of premolt and molt in intermolt crayfish determined by changes in hemolymph ecdysteroid titre using a ria. time zero indicates time of treatment. (a) bilateral eyestalk ablation (removal of molt inhibiting hormone) (positive control; open circles, orange line). premolt peak is reached at about 35 days and animals undergo ecdysis at about 50 days after treatment. dark triangles (light green line) represent wound to the integument. premolt peak is delayed compared to eyestalk ablated animals by about two weeks and animals undergo ecdysis at about 55 days after treatment. this delay is due to a plateau phase of low ecdysteroid level at days 10-12 after wounding. dark circles (brown line) represent integument wound plus bacteria infection. premolt peak is reached at about day 55 after treatment. a plateau phase (days 0-20) is further prolonged when compared to line b (dark triangles) by about a week. open triangles (dark green line) represent untreated, intact crayfish (negative control). these animals do not reach a premolt peak during the experiment and do not undergo ecdysis. (b) enlarged view of the plateau phase of ecdysteroid titre (days 0-15 underlined with a light blue line on the x-axis of panel (a). (modified from vafopoulou et al., 2007). 127 fig. 2 digital images of cells under the wound using confocal laser scanning microscopy and fluorescent immunohistochemistry on whole tissue mounts. tissues were stained with an antibody against ecr. ecdysteroidresponsive cells are revealed by ecr fluorescence which appears as yellow-green in the nuclei. cellular material and fibres of coagulated hemolymph without ecr are shown as olive green or red. optical sections are 1 μm thick. image a was taken immediately after wounding. images b-i were taken at 2 h after wounding. (a) hyaline hemocytes (short arrows) and strands of coagulated hemolymph fibres (long arrow). (b) aggregation of large number of ecdysone-responsive hyaline hemocytes (stack of 9 optical sections from a z-series). (c) granular hemocytes (arrows) interspersed among hyaline hemocytes (stack of 12 optical sections from a z-series). red, spherical cytoplasmic inclusions represent granules. (d-f) enlarged views of the three types of hemocytes in crayfish: (d) hyaline hemocytes; (e) semigranular hemocytes; arrow shows small size cytoplasmic granules; (f) granular hemocytes; arrow shows large size cytoplasmic granules. (g-i) high magnification of a single nucleus from hyaline hemocyte double-labelled with anti-ecr (g, green) and a fluorescent nucleic acid dye, propidium iodide (i, red) showing co-localization of ecr with chromatin fibres in the nucleus (h, shows merged image of g and f; co-localization is shown as yellow-green). this configuration of distribution of ecr fluorescence in the nuclei of ecdysone-responsive cells is suggested to represent the active state of ecr engaged in gene transcription (vafopoulou et al., 2005; vafopoulou and steel, 2006). bar = 10 μm. the plateau phase of ecdysteroids following injury is necessary for wound healing, epidermal regeneration and initiation of the process of repair of the wound on the integument. this plateau represents the main difference in the premolt processes between eyestalk ablated animals and wounded animals. successful restoration of the damaged integument during molting for wounded crayfish depends on previous regeneration of the damaged epidermis layer; a fully restored epidermis is required to secrete a new cuticle. during the plateau phase, we found the ecdysteroid receptor (ecr) localized in the nuclei of various types of cells involved in wound healing and repair using fluorescent antibodies to ecr, immunohistochemistry (ihc) and confocal laser scanning microscopy (fig. 2) (details in vafopoulou et al., 2007). cellular responses to ecdysteroids are mediated by ecr. ecr belongs to the nuclear receptor superfamily that includes receptors of hormones like estrogen, progesterone, thyroid hormone, vitamin d and others. ecr acts as ligand 128 fig. 3 confocal images of ecr-positive hemocytes with fillopodia (arrows) under the wound at 4 days after wounding. colors as in fig. 2. (a) hyaline hemocytes (modified from vafopoulou et al., 2007). (b) semigranular hemocytes. bar = 10 μm. inducible transcription factor (henrich, 2005). in crayfish, the presence of ecr was restricted in nuclei in association with chromatin (figs 2g-i). this property of ecr identifies these cells as specific targets of ecdysteroid action engaged in ecdysteroid-directed activities (vafopoulou et al., 2005, 2007). the morphology of uninjured tissue under the cuticle of intermolt crayfish reveals a single layer of columnar epithelium immediately under the cuticle and underneath a layer of loose connective tissue forming large hemolymph sinuses. fibrocytes and their long processes form these sinuses which are sparsely populated by small mostly hyaline hemocytes. these processes appear to provide a scaffolding structure the function of which is at the present unknown but it may be involved as a facilitator of cell movement and structural support under the integument. crayfish hyaline hemocytes are recognized as small, oval-shaped cells (about 10 μm in diameter) with a high nuclear to cytoplasmic (n:c) ratio and few, if any, small granules in the cytoplasm (fig. 2d). an immediate response to injury is coagulation of hemolymph under the damaged cuticle (fig. 2a, long arrow). the wound area is quickly populated by fibres of clotted hemolymph which protects and seals the wounded area probably by activation of the resident hemocytes (fig. 2a, short arrows). following an injury to integument, the morphology of the underlying tissue undergoes swift changes including rapid movements of various cell types. the integrated efforts of three classes of cells i.e., hemocytes, epidermal cells and fibrocytes, are required for proper wound healing and repair. by day 2 after wounding, some unknown cue (probably the release of a chemotactic factor from damaged cells?) triggers migration of large number of hemocytes into the wounded area. cell migration begins while the ecdysteroid level is still rising towards its plateau level. hemocytes rapidly infiltrate the area under the wound from the surrounded intact areas. masses of hyaline hemocytes form initially large aggregates and later a continuous sheath, embedded in a now thick disorganized matrix of strand-like coagulated hemolymph (fig. 2b). the rapid aggregation of hemocytes under the wound is to be expected since hyaline hemocytes are the primary cause of hemolymph clotting. the establishment of a hemocyte sheath now seals and protects the wound. a few semigranular and granular hemocytes also move under the wound and become dispersed among the hyaline hematocytes in the hemocyte sheath (fig. 2c, arrows). semigranular hemocytes are cells of about 20 μm in diameter and are characterized by a low n:c ratio. they carry a few small granules in their cytoplasm and their nucleus is slightly eccentrically located. (fig. 2e). granular hemocytes on the other hand are large cells (about 30 μm in diameter), have a slightly irregular shape and an eccentrically located nucleus. they are characterized by a low n:c ratio and the presence of numerous, large cytoplasmic granules (fig. 2f). fillopodia project from the cell bodies of various hemocytes at this time (fig. 3), presumably to facilitate movement under the wound. hemocyte movement under the wound coincides with the appearance of ecr in the nuclei of all hemocyte types (figs 2, 3) suggesting that hemocytes respond swiftly to slight elevation 129 fig. 4 confocal images of cells at 4 days after wounding. ecr-positive epidermal cells are shown in (a) and (c-f). colors as in fig. 2. (a) migrating epidermal cells under the wound originating from the border of the wound (modified from vafopoulou et al., 2007). (b) injured epidermis under the wound with clumped nuclear material (arrows) characteristic of apoptosis. these cells do not stain with anti-ecr. (c) epidermal cells begin to form a new epidermal sheath. (d) enlarged view of epidermal cells forming an epidermal layer. (e) migrating fibrocytes with elongated nuclei (modified from vafopoulou et al., 2007). bar = 10 μm. in ecdysteroid titre after injury and are therefore principal targets of ecdysteroid action. at this day after injury, the compacted cells of epidermis under the wound still have a normal appearance and they do not appear to carry ecr. around day 4 (beginning of ecdysteroid plateau) the epidermal cells under the wound have already become apoptotic as indicated by the presence of highly condensed chromatin in the nuclei (fig. 4b). these cells do not display ecr. a scab has also formed by this time above the sheath of hemocytes. the scab becomes quickly melanized, probably entrapping necrotic tissue. healthy epidermal cells around the periphery of the wound become aligned into rows pointing towards the wound site (fig. 4a). this arrangement of epidermal cells is indicative of cell migration from the edge of the wound towards its centre. as the ecdysteroid titre reaches the top of its plateau, the uninjured epidermal cells display abundant ecr in their nuclei (figs 4a, c, d), showing that they respond to ecdysteroid elevation. therefore, epidermal cells around the wound respond to ecdysteroids but only a few days later than the hemocytes and at relatively higher hormone concentrations. thus, injured and uninjured epidermal cells of the same animal respond differentially to ecdysteroids. these findings indicate that a primary response to the low ecdysone levels induced by injury is to activate epidermal cells to migrate across the injured area to restore the continuous sheath of epidermal cells. the migrating healthy epidermal cells make cell to cell contacts and are aligned in a single-cell layer (figs 4c, d), which becomes a complete sheath under the wound a few days later (see below). therefore, one of the early events in wound healing is cell to cell adhesion. if cell proliferation of epidermal cells takes 130 fig. 5 confocal images of granulocytes at 6 days after wounding with large cytoplasmic inclusions showing aggregation of these cells. these cells do not exhibit immunoreactivity with the ecr antiserum. (a) granulocyte containing enlarged granules and large cytoplasmic inclusions shown in red. (b) granulocyte with giant cytoplasmic inclusions (red). (c) aggregation of granulocytes with giant cytoplasmic inclusions. (d) a stack of 16 optical sections from a z-series shows a layer of hyaline hemocytes (small, round green nuclei) and underneath a layer of granulocytes with giant cytoplasmic inclusions (red). bar = 10 μm. place (and we suspect it does), it occurs not immediately under the wound but in healthy undamaged regions in the neighbourhood of the wound to produce the migrating sheets of epidermal cells that populate the wound area. along with the epidermal cells, fibrocytes, which are characterized by elongated nuclei, also migrate from the wound margin under the wound, probably to restore the scaffolding seen in intact, uninjured areas (fig. 4f). fibrocytes also develop ecr in their nuclei at this time indicating that they are also targets of ecdysteroid action (fig. 4f). at day 6 after injury, the area under the wound possesses abundant semigranular and granular hemocytes all of which display abundant nuclear ecr. many semigranular hemocytes possess conspicuous fillopodia. by this day, the aggregated hyaline hemocytes have formed a multilayered sheath. the appearance within 6 days following injury of ecr in the nuclei of cell types essential to wound healing and repair, such as epidermal cells, fibrocytes and all three types of hemocytes, shows unequivocally that all these cell types are targets of ecdysteroid action. however, the response to ecdysteroids is apparently elicited in each cell type by different hormone concentration. the gathering of granulocytes under the wound site is of particular interest because it indicates that copious amounts of po are probably needed locally for melanisation. indeed, concomitant to the rise of ecdysteroids to the plateau phase, the hemolymph po activity increases greatly (fig. 6, orange bar), and remains significantly higher during the plateau phase compared to those of intact animals (fig. 6, brown bar) and eyestalk-ablated crayfish (fig. 6, green bar). therefore wounding provoked the synthesis of high levels of po activity. the close correlation between the time of appearance of ecr in the hemocytes and the time of increase in po activity is suggestive of ecdysteroid control. this hypothesis is supported by the finding that ecdysone response elements are present in the flanking regions of the 131 fig. 6 changes in hemolymph po activity in crayfish during the plateau phase of the ecdysteroid titre shown in fig. 1. measurements were taken at time 0 (time of treatment) and then at days 2, 4, 6 and 10 after treatment. bars indicate measurements from 10 animals per treatment ± sem. brown bars show control, untreated animals. orange bars show animals with an integumentary wound. green bars show animals with bilateral eyestalk ablations. highly significant (p<0.01) increases in po activity were observed at days 2, 4 6 and 10 when animals with integumentary wounds were compared to controls. likewise, significant (p<0.05) increases in po activity were also measured at days 2, 4 and 6 between wounded and eyestalkless animals. enzyme responsible for catalyzing conversion of propo to po in the insect manduca sexta (zou et al., 2005) indicating direct control by ecdysteroids. further, injection of 20-hydroxyecdysone increased expression of propo mrna in hemocytes of honey bees (zufelato et al., 2004) and in a hemocyte cell line from mosquitoes (ahmed et al., 1999; müller et al., 1999). the phenomenon of increase of po activity following wounding in crayfish is also in agreement with observations in other crustaceans (mucklow and ebert, 2003; mucklow et al., 2004) and insects (nayar and knight, 1995). propo was found immunohistochemically to be distributed widely in the wound site in an insect suggesting that it acts locally and thus assists in the healing process (lai et al., 2002). therefore, it is plausible that the increase in po activity in injured crayfish indicates local participation of po essential for wound healing and repair. the close correlation between the time of appearance of ecr in the hemocytes and the time of increase in po activity suggests that this hemocyte function is probably directly influenced by ecdysteroids. at day 10 (end of plateau and beginning of premolt rise of ecdysteroids), a sheath of flattened and highly condensed granulocytes has been established between the necrotic tissue and the newly forming epidermis (fig. 5d) underneath the multilayer sheath of hyaline hemocytes (fig. 5d). the distinction between semigranular and granular hemocytes becomes unclear at this time because the cells become swollen with giant inclusions probably containing phagocytized and/or encapsulated material (figs 5a, b). ecr is present in the nuclei of all cell types, showing that they still respond to ecdysteroids. at this time, a pale ring around the melanized scab at the edges of the wound is seen under the dissecting microscope when the uppermost layer of the wound surface is peeled away, suggesting that deposition of repair cuticle had already started, as also reported in other crustaceans (dillaman and roer, 1980). around day 10, ecr ceases to be present in the nuclei of cells during the period of study of wound healing and repair. by day 15, after termination of the plateau phase, the titre commences its premolt rise and the epidermal cells have already become organised into a continuous sheet resulting from the coalescence of migrating epidermal cells which fully seals off the wound. at day 20, as the ecdysteroid titre continues to rise sharply to its premolt peak, the deposition of repair cuticle has become completed on the injured site. repair cuticle lacks epicuticle the characteristic outermost layer of arthropod cuticle; this is a phenomenon common to all crustaceans studied so far (halcrow, 1988). the requirement of a low level ecdysteroid plateau for wound healing and repair was highlighted in experiments where integumentary injury was combined with simultaneous injection of bacteria through 132 fig. 7 diagram showing the cascade of events of wound healing and repair in crayfish during the plateau phase of ecdysteroid titre, as summarised in the conclusion section of the text. red arrows indicate pathways where the triggering factors are known. white arrows indicate pathways where the triggering factors are speculated. the wound (vafopoulou et al., 2007). crayfish exposed to both injury and bacteria exhibited an extended ecdysteroid plateau and the consequent delay of the onset of the premolt peak when compared to injured animals (figs 1a, b). the presence of bacteria clearly taxes the resources of the immune system to a much greater extent and results in long delays in wound healing. therefore, the length of the hormonal plateau appears to be dependent on the extent of the immune challenges. thus, it is likely that the immune system functions as a modulator of the neuroendocrine system responsible for ecdysteroid synthesis. the dependence of tissue regeneration during 133 wound healing and repair on low levels of ecdysteroids can be generalized for decapod crustacean and probably for other arthropods. earlier studies in crabs suffering limb autotomy demonstrated that limb regeneration depends on low ecdysteroid levels (see for review skinner, 1985; hopkins, 2001) and results in ecr expression in whole limb regenerates (durica et al., 1996; 1999; 2002; chung et al., 1998). in insects, regeneration of imaginal discs also requires low ecdysteroid level and is inhibited by high (madhaven and schneiderman, 1969; kunieda et al., 1997). why are ecdysteroids needed for wound healing? the work with crayfish shows that hemocytes are principal targets of ecdysteroids and wounding results in elevation of po activity. these findings suggest that the nature of hemocyte responsiveness to ecdysteroids is activation of immune responses. this conclusion is supported by the fact that expression of several immune parameters in other decapod crustaceans is correlated with molt stage (cheng et al., 2003; liu et al., 2004, 2006; kuballa and elizur, 2008; ho et al., 2009; yeh et al., 2009) or with changes in ecdysteroid level following eyestalk ablation (sainzhernández et al., 2008). all this reinforces the hypothesis that immune responses in crustacea are regulated by ecdysteroids during wound healing. even though the area of ecdysteroid control of immune responses in crustaceans is largely unexplored, there is considerable evidence from insect systems supporting this hypothesis. hemocytes failed to respond to bacterial challenge in the absence of ecdysteroids (ahmed et al., 1999; müller et al., 1999). molt-dependent variations in immune parameters (yamamoto et al., 2001; zufelato et al., 2004; meylaers et al., 2006; eleftherianos et al., 2008) and positive correlations between ecdysteroids and immune responses have been observed in insects (meister and richards, 1996; dimarcq et al., 1997; dimopoulos et al., 1997; lee et al., 2002; sorrentino et al., 2002; korayem et al., 2004; aye et al., 2008; flatt et al., 2008). most interesting is the finding that ecdysteroid induction of antimicrobial peptides in the fruit fly drosophila melanogaster required the presence of ecr (flatt et al., 2008). therefore, it appears that the situation of ecdysteroid control of immune responses unites arthropods with vertebrates in which steroid hormones, their nuclear receptors and other members of the nuclear receptor family regulate adaptive and innate immunity (see for review flatt et al., 2008). another cellular aspect of ecdysteroid influence in wound healing in crayfish appears to be the massive migration of various cell types under a wound, notably hemocytes, epidermal cells and fibrocytes. again convincing supporting evidence derives from insect studies where it has been shown that cell migration during morphogenesis is regulated by ecdysteroids (hackney et al., 2007; jang et al., 2009; see for review montell, 2001). a less understood aspect of wound healing process is the cue(s) that activate the crayfish neuroendocrine system to synthesize and release ecdysteroids following an integumentary injury. we speculate that these cues may be supplied by the wound itself as is the case with limb regeneration in crabs. an inhibitory factor similar to molt-inhibiting hormone is released by the regenerating limb bud that inhibits proecdysial growth of limb buds and delays molting by acting on the central neuroendocrine system (see for review mykles, 2001; yu et al., 2002). conclusions in summary, integumentary wounds damage the underlying tissues and set in motion a cascade of events that lead to regeneration and repair of the damaged area. in the event of a wound, both the immune and neuroendocrine systems (ecdysteroid synthesis) are activated to repair the wound rapidly and prevent hemolymph and tissue loss and infection. a schematic summary of the events involved is shown in fig. 7. several triggering factors in this cascade are known (red arrows) and have been described above in detail, whereas the existence of many others is speculative (white arrows) and invites further research. in this scheme, a wound damages cells that lie under the cuticle and these cells leak a variety of cellular components which may trigger several reactions. they may act chemotactically and trigger cell migration towards and under the wound from neighboring healthy areas, e.g., hemocytes, epidermis and fibrocytes, and they may also activate synthesis of specific proteins by these cell types. or they may act on the neuroendocrine system and induce synthesis of ecdysteroids. likewise, the act of wounding itself may activate the neuroendocrine system by some unknown mechanism to initiate synthesis of ecdysteroids. factors released from damaged hemocytes are known to stimulate hemolymph clotting. migration of cells may also be triggered by ecdysteroids. ecdysteroids released into the hemolymph target primarily the immune system, e.g., the hemocytes, thereby appearing to regulate 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prophenoloxidase mrna. insect biochem. mol. biol. 34: 12571268, 2004. 137 39 isj 15: 39-51, 2018 issn 1824-307x research report the effects of cyanobacterial blooms on the immune system of elliptio complanata in urban and agricultural areas in the yamaska river watershed f gagné1*, m gélinas1, m fortier2, m fournier2 1acquatic contaminants research division, environment and climate change canada, 105 mcgill street, montreal, quebec, canada, h2y 2e7 2inrs – institut armand-frappier, 531 boulevard des prairies, laval, quebec, canada, h7v 1b7 accepted february 6, 2018 abstract the cumulative effects of cyanobacterial blooms at sites impacted by urban or agricultural activity, could be detrimental to local freshwater mussels. the purpose of this study was to examine and compare the resulting toxicity using an upstream–downstream approach at four sites in the yamaska river. mussels were caged and placed at a site where cyanobacterial blooms were present, a site that receives municipal effluent from a small city of 75,000 inhabitants, and a site that drains a large agricultural area—plus a reference site in lake saint-pierre (quebec, canada) for four months spanning the summer and fall (june to october). effects on immunocompetence were monitored by testing for hemocyte counts, viability, phagocytosis, production of nitric oxide (no), lysozyme (ly), reactive oxygen species (ros), thiol contents, and inflammation induced by cyclooxygenase (cox) activity and glutathione s-transferase (gst) activity. cyanobacterial blooms occurred at the target site only, reaching levels of 2.1 million cyanobacteria/l and 3 g/l of microcystin-lr in surface waters. although most of the immune parameters were affected at the urban, agricultural and cyanobacteria sites, ros and ly were the most responsive to cyanobacterial bloom, with a significantly greater response than the agriculture, urban and reference sites. the results also suggest that the effects of cyanobacterial blooms are spatially localized; they are not found at the downstream urban and agriculture sites in the yamaska river. key words: cyanobacteria, microcystins, hemocytes, immunocompetence, lysozymes, oxidative stress introduction the yamaska river is located in southeastern quebec (canada). it is 177 km long, covers a watershed of 4,784 km2 and originates from lake brome. the river ends in lake saint-pierre, which is part of the st. lawrence river. the watershed consists mainly of small townships (<75000 inhabitants) and supports intense agricultural activity. urban and agricultural pollutants including nutrients (total phosphorus and nitrogen) are released into the river, degrading its water quality. eutrophic conditions in aquatic environments can lead to blooms of pelagic and benthic cyanobacteria. for example, blooms of unicellular planktonic microcystis aeruginosa or filamentous anabaena, aphanizomenon or planktothrix can develop in lakes enriched with high nutrient loads. ___________________________________________________________________________ corresponding author: francois gagné acquatic contaminants research division environment and climate change canada 105 mcgill street, montreal, quebec, canada e-mail: francois.gagne@canada.ca m. aeruginosa produce toxic secondary metabolites called microcystins (mycst). these toxins are potent inhibitors of protein phosphatase 1 and 2a and cause liver damage and death to animals (jochimsen et al., 1998). the production of toxins is also associated with other unfavourable environmental conditions (e.g., hypoxia and high concentrations of suspended solids, organic matter and other associated pollutants), and the combined or cumulative effects can have profound effects on aquatic organisms. in freshwater environments, harmful algal blooms often occur in locales where bivalve molluscs are found. as sedentary filter-feeder organisms, mussels accumulate (cyano)bacteria, fungi, algae and suspended matter as sources of nourishment, thereby ingesting an important variety of environmental contaminants. thus, they may filter and accumulate large quantities of possibly toxic cyanobacteria. in a previous study, freshwater mussels (elliptio complanata) fed with anabeana flos-aqua in the laboratory for five days showed toxic effects, although the cyanobacteria were not 40 fig. 1 site location. the lake saint-pierre (lsp) site is part of the st. lawrence river and is located downstream from the other sites. the yam site is located 0.5 km from the junction of the yamaska river and lake saintpierre. lake boivin (boi) is within the municipality of granby. the choinière (cho) basin is northwest of granby. actively producing microcystins (gélinas et al., 2013). indeed, acetylcholinesterase activity and lipid peroxidation were higher in mussels fed with the cyanobacteria. although cyanobacteria are considered gram-negative, they contain unusual surface mucopeptides thick enough to react weakly with the gram stain (hoiczyk and hansel 2000). in addition, exposure of fish for 90 days to m. aeruginosa stimulated the immune system, including lysozyme activity, which is involved in the degradation of cell walls of gram-positive bacteria (das et al., 2013). in clams exposed to 100 µg/l microcystin-lr for 10 days, an increase in enzymes involved in oxidative stress, such as superoxide dismutase and catalase, was observed (pham et al., 2016). these responses were observed at tissue microcystin levels of 12.7 ± 2.4 µg/g dry weight, indicating that corbicula leana clams are relatively tolerant to these toxins. decreased glutathione stransferase (gst) activity was observed in dreissena polymorpha exposed to 10 µg/l and 50 µg/l microcystin-lr for 24 h and 7 days (burmester et al., 2012). the long-term and cumulative impacts of urban, agricultural and cyanobacteria-related pollution on organisms exposed to multiple contaminants are currently not well understood. for example, decreased gst activity in organisms exposed to cyanobacterial blooms could limit biotransformation and elimination of other organic pollutants to which they are exposed concurrently. increased levels of cyanobacteria in mussel digestive glands could have major impacts on the immune system. as a mussel filters its food, these harmful algae interact directly with the gills, digestive gland and other tissues during ingestion, allowing diffusion to the hemolymph throughout the bivalve’s open circulatory system. the internal defences against invading foreign bodies are mainly based upon non-specific innate immune system responses, such as phagocytosis and the production of humoral factors such as cytokines (auffret, 2005). hemocytes are responsible for phagocytosis and the production of humoral factors (cheng et al., 1996). these cells are not only found in the circulatory system, but also nest in interstitial tissues waiting to return to circulation following a stress signal. the release of hemocytes following an invasion by foreign bodies is a first response; phagocytosis and lysozyme (ly) production usually follow. hemocyte viability could be also affected following exposure to cyanobacteria extracts in crassostrea gasar oysters (queiroga et al., 2017). ly 41 is an enzyme involved in the degradation of cell walls of most gram-positive bacteria and cyanobacteria (which are gram-indeterminate), leading to lysis. this process is supported by phagocytocis, a further attempt to eradicate the pathogens/toxins in which the ingested bodies in phagosomes are destroyed by oxidative burst involving the production of nitric oxide (no) and hydrogen peroxide (peroxynitrite) as the main form of reactive oxygen species (ros). increased cyclooxygenase (cox) activity is associated with the production of pro-inflammatory precursors to assist the immune response (ottaviani 2004; gagné et al., 2005). recently, ros formation and antioxidant enzyme responses have been reported in organisms exposed to purified microcystins or microcystis extracts (wiegand and pflugmacher 2005; pietro et al., 2006; amado et al., 2010). ros, when improperly inactivated, can inflict damage on a wide range of cellular components (dna, proteins and lipids), and it is linked with the development of many pathological states. the observed resistance of mussels against cyanotoxins could also be attributed to increased biotransformation through gst activity and gsh, a main source of reduced thiols in cells (fernandes et al., 2009). the increase in gst activity coincided with peak levels of microcystin-lr in tissues, followed by a phase of decreasing activity after 8-day and 12-day depuration periods in toxin-free water. given that toxic marine algae could impair the immune system, we investigated whether blooms of m. aeruginosa could affect the immune system of a freshwater mussel, e. complanata, already multiply stressed by agricultural and urban activities in the yamaska river. this mussel is typical of the native fauna of the yamaska watershed and is representative of a sessile species that would endure blooms of m. aeruginosa in addition to other pollution inputs from agricultural and urban activities. the aim of the study was therefore to examine the effects of m. aeruginosa blooms on the immune system in caged mussels during the summer/fall months. we included sites contaminated by urban activities (municipal discharges) and agriculture during the same period to assess the contribution of other stressors to the health status of caged mussels. materials and methods study area sites were selected using an upstreamdownstream approach: the sites polluted by urban activity and algal blooms are located upstream in the yamaska river which discharges on the south shore of lake saint-pierre (lsp), which is part of the st. lawrence river (figure 1). three sites were located in the yamaska watershed, which supports agricultural activity and an urban area (granby; population circa 75,000). two sites were located upstream from granby: lake boivin (boi) and the choinière reservoir (cho). cho, the site located farthest upstream, is a 4.7 km2 reservoir built in 1972 that is prone to frequent cyanobacterial blooms (rolland et al., 2005). the boi site, which is a little farther downstream, was also formed by dams in the early 19th century. it receives the municipal effluents from the city of granby (aeration lagoons) and is also subject to cyanobacterial blooms. however, in this study, a cyanobacterial bloom occurred only at the cho site. the third site (yam) was in the yamaska river 3 km upstream from where it flows into lake saint-pierre, which drains more than 50 km of intensely farmed land. the fourth site was the reference site, lsp, located in lake saint-pierre near île plate which does not receive input from the yamaska river. mussel collection and caging exposure wild freshwater elliptio complanata mussels were collected in the first week of june 2011 in lake achigan (quebec, canada), which is not subjected to any direct sources of pollution. the animals were maintained in an aquarium at 15 °c under constant aeration, with a 16h-light/8h-dark photoperiod. they were fed daily with concentrates of phytoplankton (phytoplex®) and laboratory-cultured pseudokirchneriella subcapitata algae. mussels were then placed in two cylindrical nets (65×30 cm), with 30 mussels in each net (which includes extra mussels in case of unexpected mortality), and immersed at the four sites (lsp, yam, boi and cho) on june 20 and 21. the cages were secured with a cable and 10-kg cinderblocks. mussels were collected (10 randomly collected mussels per time) for analysis after one month (on july 30) and three months (september 30), placed in coolers at 4 ℃ and brought back to the laboratory for immediate immunocompetence assessments, as described below. water sampling and analysis water sampling was carried out monthly (in july, august, september and october). temperature, oxygen concentration, conductivity and ph were measured at 1 m depth in the water column with a ysi multiprobe. water samples were transferred into two 1 l polypropylene containers: a dark one for chlorophylla a (chl a) analysis and a clear one for the measurement of total phosphorus (tp), total nitrogen (tn), dissolved organic carbon (doc), ammonia (nh3) and nitrite-nitrate (no2-no3) (environment canada 2005). in the laboratory, chla water samples were filtered on gf/f whatman filters (0.7 µm pore size) that were kept frozen until analysis. chla pigments were extracted in cold 95% ethanol for 24 h and measured before and after acidification at 665 nm and 750 nm (spectronic genesys 5 spectrophotometer) (rice et al., 2012). tp and total dissolved phosphorus (tdp) were determined by acid digestion followed by colorimetry with ammonium molybdate (rice et al., 2012). tn was analyzed with a lachat continuous flow quick-chem 8000. no2-no3 was measured by reducing nitrate to nitrite in a cadmium column prior to colorimetry; nh3 was analyzed by colorimetry after the addition of sodium nitroprusside and phenate sodium. doc was oxidized to carbon dioxide by the addition of persulfate prior to infra-red detection (shimadzu tco-5000) (rice et al., 2012). the levels of mc-lr toxins in the water were determined using a commercial competitive immunoassay kit (enzo life sciences, usa). 42 hemolymph collection mussel hemolymph of 10 individuals per site and time of collection (july and end of september) was drawn (between 750 l and 1,000 l) from the posterior adductor muscle with a 3 ml syringe and a 23g needle. a portion of the hemolymph was set aside to measure hemocyte concentration and viability, reactive oxygen species (ros), nitric oxide production (no) and reduced thiols by flow cytometry. about 800 l was kept to measure lysozyme (ly), cyclooxygenase (cox) activities and nitric oxide (no) concentration. for ly, the hemolymph was centrifuged at 1,000 × g at 4 °c for 10 min, 100 µl of supernatant (plasma) was withdrawn for activity measurements, and the remaining pellet was re-suspended in 150 µl of phosphate buffered saline (pbs; pre-diluted 1/3 in water) for cox activity. total proteins in the cell-free hemolymph and hemocytes were determined by the protein-dye binding principle (bradford, 1976). flow cytometric analysis hemocyte counts and viability were evaluated by flow cytometry using a 3-colour guava easycyte plus cytometer with a laser emitting at 488 nm, using the supplied viacount kit (guava technologies, hayward, ca, usa). an aliquot of 20 μl of hemolymph was mixed with 80 µl of viacount solution after 10 min, in accordance with the supplier’s recommended procedure. the flow rate was set at 0.6 µl/sec, and 5,000 events were counted. the data were expressed as the number of cells/ml and percentage of live cells for hemocyte concentration and viability respectively. for phagocytosis activity, hemocytes were incubated with fluorescently labelled beads based on the protocol of brousseau et al., 2000. fluorescent latex beads (polysciences, pa, usa) were added to the cell suspensions at a 30:1 (beads:cell) ratio and incubated for 18 h at 15 oc in a humidified incubator. after the incubation period, the hemocyte suspension was re-suspended in prediluted pbs and layered over 4 ml of rpmi cell culture media (diluted 1/4 in water) supplemented with 3% bovine serum albumin (bsa) (sigma, ontario, canada). elimination of free or loosely bound beads was followed by centrifugation at 150 × g for 8 min at 4 °c. the cell pellet was then suspended in 0.5 ml of 0.5% formaldehyde and 0.2% sodium azide (sigma chemicals, canada) diluted in pbs (becton dickinson, ca, usa). cells were analyzed using flow cytometry, and at least 10,000 events were recorded for analysis. hemocyte populations were defined on the basis of their forward and right-angle scatter properties (fsc and ssc, respectively). results were analyzed with the cell quest pro software (becton dickinson). the percentage of cells (hemocytes) that engulfed at least one bead and at least three beads was measured in order to assess phagocytosis activity and efficiency, respectively (farcy et al., 2011). intracellular ros and reduced thiol were measured as previously described (brousseau et al., 2000). the probe dihydrochlorofluorescein diacetate (h2dcfda; cas 4091-99-0) was used to measure ros levels in hemocytes (collén and davison 1997). a 2 µm final concentration of h2dcfda was prepared and mixed with hemocytes and allowed to stand for 45 min in the dark. the presence of intracellular ros was determined by oxidation of h2dcfda dye and the dye is retained in viable cells by non-specific esterases (deacetylation of the diacetates into carboxylates). fluorescence was measured for 3,000 events using flow cytometry. for reduced thiol, the 5-chloromethylfluorescein diacetate probe (cmfda) was used. as the first step, non-specific thiol fluorescence background was blocked using n-ethylmaleimide (nem) as a negative control: hemocytes were treated with 100 µm nem for 10 min and washed in pre-diluted pbs. afterward, all hemocytes were stained with 5-µm cmfda and a total of 2,500 events were acquired by flow cytometry. immunological biomarkers the production of no was estimated by measuring the levels of nitrite concentration in the plasma (verdon et al., 1995). because no reacts readily with oxygen to produce nitrites and nitrates, nitrate reductase was added to convert nitrates into nitrites. following a 30 min pre-incubation step of 50 µl of hemolymph with 25 µl of nitrate reductase (80 units/l) and 25 µl of nadph (3 mm), the concentration of no was measured by adding 100 µl of griess reagent and reading the absorbance at 450 nm after 30 min. results are expressed as µmol of no/mg proteins. the activity of cox in hemocytes was measured by oxidation of the 2,7-dichlorofluorescein in the presence of arachidonic acid (gagné, 2014). a volume of 50 µl of the suspension (hemocytes and 1/4 pbs) was mixed with 150 µl of tris-hcl buffer 50 mm, ph 8.0, containing 0.05% tween 20, 0.1 µg/ml horseradish peroxidase, 25 µm arachidonic acid and 2 µm 2,7-dichlorofluorescein. fluorescence readings were taken at 485 nm for excitation and 520 nm for emission every 10 min for 30 min at 30 °c. a standard solution of fluorescein in pbs at 1 µm was used for instrument calibration. cox activity was expressed as fluorescein units/min/mg proteins. total protein concentration was determined using bovine serum albumin as standard, via the proteindye binding principle (bradford, 1976), with absorption at 595 nm measured by a microplate reader (powerwave, biotek). statistical analysis the biomarker responses were determined in n=10 mussels for each site and time of collection (season). the biomarkers were normalized to the lsp reference site for each season. the data was tested for normal distribution and homogeneity of variance using the shapiro–wilk and bartlett tests respectively. two-way factorial analysis of variance (anova) was used to test for the effects of sites (boi, choi, lsp, yam), seasons (summer and fall) and their interaction. intersite and between time comparisons were done using the least square difference test as the post-hoc test. correlation was examined using the pearson-moment procedure. significance was set at p<0.05. all statistical analyses were performed using statistica version 8. 43 table 1 change in physico-chemical characteristics of surface waters parameter turbidity oxygen (mg/l) chl a (mg/ml) blue-green algae (cells/ml) total n/nh3/no2 (mg/l) doc1 (mg/l) conductivity/ph s.cm-1 dissolved and total phosphates (mg/l) lsp j a s o 4.4 3.3 2.5 6.1 8.9 8.7 9.0 10.3 0.62 2.7 1.9 2.8 nd nd nd nd 0.545/0.04/0.18 0.475/0.04/0.17 0.54/0.05/0.19 0.58/0.04/0.22 3.5 3.5 3.4 3.4 260/8.5 276/8.6 272/8.3 271/8.2 0.18/0.01 0.18/0.01 0.02/0.01 0.03/0.01 yam j a s o 15.8 7.8 59.1 27.7 10.1 9.2 8.5 9.7 1.13 7,7 6.3 4.3 nd nd nd nd 0.48/0.02/0.05 0.69/0.02/0.03 0.54/0.05/0.2 1.9/0.05/1.3 3.2 3.3 7.5 7.5 280/8.3 286/8.7 289/8.1 332/8.3 0.03/0.008 0.03/0.01 0.19/0.07 0.14/0.06 cho j a s o 10.2 8.2 18.8 261 13.1 9.6 8.6 7.8 9.9 15.3 17.6 18 nd 613 2424 20900 0.06/0.001/0.02 0.68/0.02/0.02 0.75/0.08/1.2 0.75/0.01/0.02 5.2 5.4 6.8 6.8 129/9.2 129/8.6 122/7.5 130/6.9 0.02/0.01 0.03/0.01 0.04/0.01 0.04/0.01 boi j a s o 3.7 4.2 3.3 1.5 6.0 6.3 6.8 9.0 48 29.7 8.6 9 60 nd nd nd 0.1/0.02/0.02 1.1/0.03/0.02 0.84/0.02/0.02 0.84/0.02/0.03 9.5 9.5 11 11 156/7.4 161/7.8 180/7.2 218/8.0 0.22/0.1 0.17/0.1 0.11/0.09 0.11/0.09 1. dissolved organic carbon. results surface water chemistry during the first four weeks of the summer period, water temperature was stable at around 24 ℃. during the fall period, the mean water temperature was 17 °c and varied between 20 °c and 14 ℃. chl a measurement was higher at the boi site in the first summer month (july) but did not contain blue-green algae. chl a was higher at the cho site in fall, during the m. aeruginosa bloom (table 1). the number of blue-green algae increased considerably during the fall at cho site exclusively (table 1). cyanobacteria toxins were measured on every sampling date at all sites. no toxins were detected at any site in the summer, but during just one month in september at cho site. microcystin-lr equivalents were detected in filtered water (0.45 µm) taken from the water column (not the algae) at concentrations between 1.5 µg/l and 3 µg/l. immunocompetence responses for hemocyte density, two-way factorial anova revealed a positive interaction (f=32; p<0.001) between site and season (figure 2a). hemocyte density was significantly higher in the summer than in the fall: the mean cell density at the reference site, lsp, was 960 000 and 580 000 cells/ml in summer and fall respectively. in the summer, hemocyte density did not change significantly between sites. in the fall, hemocyte counts were significantly higher at the cho (5-fold) and boi (3.5-fold) sites than at the lsp and yam sites. the cho and boi levels were also significantly different from each other. hemocyte density was significantly correlated with ammonia (r=0.52; p<0.05), nitrates (r=-0.53; p<0.05) and ph (r=0.54; p<0.05). in respect to hemocyte metabolic activity (viability), based on general esterase activity, two-way factorial anova confirmed a significant interaction (f=15.7, p<0.001) between site and season (figure 2b). the initial metabolic activity (viability) index was 82% and 50% at the lsp site in summer and fall respectively. in the summer, hemocyte metabolic activity was significantly lower at the cho site (0.9-fold) compared to the lsp site. in the fall, hemocyte activity was significantly higher at the yam (1.2fold), boi (1.5-fold) and cho (1.45-fold) sites relative to the lsp site. hemocyte density was not significantly different between the cho and boi sites. hemocyte activity was significantly correlated with hemocyte density (r=0.39, p<0.05). phagocytosis activity and efficiency were also examined in mussels (figure 3). two-way factorial anova revealed a significant interaction (f=18.3, p<0.001) with site and season for phagocytosis activity. phagocytosis activity was significantly higher in the summer than in the fall (figure 3a). the initial activity was 41% and 14% at the lsp site for summer and fall respectively. in the summer, phagocytosis activity was significantly higher at the boi site (1.4-fold) compared to the lsp site. in the fall, phagocytosis activity at cho (2.3-fold), boi (2.1-fold) and yam (0.6-fold) were significantly 44 lsp yam boi cho sites 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 h e m o c y te d e n s it y (n o rm a liz e d t o r e fe re n c e s it e ) summer autumn a,b,c a,c a lsp yam boi cho sites 0,7 0,8 0,9 1,0 1,1 1,2 1,3 1,4 1,5 1,6 1,7 h e m o c y te v ia b il ty (n o rm a li z e d t o r e fe re n c e l s p s it e ) summer autumn a,c a,c a a b fig. 2 change in hemocyte density and esterase activity. mussels were caged at the sites in june and removed at the end of july and september. the letter a indicates a significant difference between the site and lsp (the reference site). the letter b indicates a significant difference from boi (impacted by urbanization but no bluegreen algae). the letter c indicates a significant difference from yam. different than at lsp. phagocytosis activity was significantly correlated with hemocyte density (r=0.55; p<0.001), viability (r=0.43; p<0.001), nitrates (r=-0.67; p<0.01), chl a (r=0.61; p=0.01) and water conductivity (r=-0.53; p<0.01). phagocytosis efficiency responded similarly to phagocytosis activity with a significant interaction (f=22.8, p<0.001) between site and time (figure 3b). the efficiency values were 27% and 5% at the lsp site in summer and fall respectively. in the case of ly activity, two-way factorial anova revealed a significant interaction (f=7.0, p<0.001) between site and season (figure 3c). ly activity was at 3.9 and 1.5 absorbance change/min/mg proteins at the lsp site in the summer and fall respectively. in the summer season, ly activity remained unchanged between sites. however, a significant increase in ly activity was observed at boi (6-fold) and cho (10-fold) compared to the lsp. there was also a significant difference in ly activity between the cho site (rural cyanobacterial bloom) and the boi (urban) site. correlation analysis revealed that ly activity was significantly correlated with blue-green cyanobacteria counts (r=0.50; p<0.05), ammonia (r=-0.52; p<0.05) and conductivity (r=-0.60; p=0.01). multiple regression analysis revealed a correlation 45 lsp yam boi cho sites 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 2,2 2,4 2,6 p h a g o c y to s is a c ti v it y (n o rm a liz e d t o r e fe re n c e l s p s it e ) summer autumn a a,c a,c a,c a lsp yam boi cho sites -0,5 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 p h a g o c y to s is e ffi c ie n c y (n o rm a liz e d to r e fe re n c e l s p s ite ) summer autumn b a,b,c a,b,c a lsp yam boi cho sites -2 0 2 4 6 8 10 12 14 l y s o z y m e a c tiv ity (n o rm a liz e d to r e fe re n c e l s p s ite ) summer autumn a,b,c a,c c fig. 3 change in phagocytosis and lysozyme activity in mussels exposed to multiple sources of pollution. mussels were caged at four sites from june to october. the letter a indicates a significant difference between the site and lsp (the reference site). the letter b indicates a significant difference from boi (impacted by urbanization but no blue-green algae). the letter c indicates a significant difference from yam. between lysozyme activity (dependent variable) and blue-green cell counts (β=0.50) with conductivity (β=-0.44) (r=0.80; p=0.002), suggesting that hemolymph ly activity increases with blue-green cell levels in water that has low conductivity. ros production following phagocytosis was measured, as well as the production of total nitrites in hemocytes (figure 4). for ros production, twoway factorial anova showed a significant interaction (f=12.6, p<0.001) between season and site (figure 4a). mean ros levels were 2,941 and 36 fluorescence units at the lsp site during the summer and fall seasons, respectively. in the summer, ros production did not differ significantly between sites. in the fall, ros production was significantly higher at the cho site only (20-fold) than at the yam and lsp sites. although the ros levels were high (5-fold) at boi, the difference was not significant owing to the variability. ros levels between cho and boi were significantly different. correlation analysis revealed that ros production was significantly correlated with hemocyte density (r=0.32; p=0.001), viability (r=0.42; p<0.001) and dissolved p/total p ratio (r=-0.68; p<0.01). for no production, two-way factorial anova revealed a significant interaction (f=4.6, p<0.01) between season and site (figure 4b). the mean no levels were 3.3 µg/mg and 0.1 µg/mg protein at the lsp site in the summer and fall respectively. in the summer, no production showed no significant difference between sites. in the fall, no production was significantly higher at cho (8-fold) compared to the lsp site. correlation analysis revealed that no level was significantly correlated with hemocyte density (r=0.37; p<0.001), phagocytosis efficiency (r=0.25; p=0.05) and dissolved p/total p ratio (r=0.49; p=0.05). cox activity was measured in hemocytes to assess inflammation in freshwater mussels (figure 4c). two-way factorial anova revealed a significant interaction (f=4.5, p<0.01) between site and season. cox activity was at 8,700 and 7,300 fluorescein units/min/mg proteins at the lsp site in the summer and fall respectively. in the summer, cox activity did not differ significantly by 46 lsp yam boi cho sites -5 0 5 10 15 20 25 30 h e m o c y te r o s le v e ls (n o rm a liz e d to l s p r e fe re n c e s ite ) summer autumn a,b,c a lsp yam boi cho sites -4 -2 0 2 4 6 8 10 12 n o p ro d u c ti o n (n o rm a li z e d t o r e fe re n c e l s p s it e ) summer autumn a,b,c b lsp yam boi cho sites 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 c o x a c ti v it y (n o rm a li z e d t o l s p r e fe re n c e s it e ) summer autumn a,cc c fig. 4 oxidative burst and inflammation in freshwater mussel hemolymph. mussels were caged at four sites during the summer and fall. the letter a indicates a significant difference between the site and lsp (the reference site). the letter b indicates a significant difference from boi (impacted by urbanization but no blue-green algae). the letter c indicates a significant difference from yam. 47 site. in the fall, cox activity was significantly lower at cho (0.6-fold) and boi (0.5-fold) compared to the reference site, lsp. correlation analysis revealed that cox activity was significantly correlated with hemocyte density (r=0.56; p<0.01), gst (r=0.26; p<0.01), thiol content (r=0.83; p<0.001) and lysozyme activity (r=-0.67; p<0.01). biotransformation activity was followed by reduced thiol content (for metals and ros inactivation) and gst (for organic pollutants) activity. for hemocyte thiol content, two-way factorial anova revealed a significant interaction (f=10.9, p<0.001) between site and season. the mean thiol content was 109 and 121 fluorescence units at the lsp site in the summer and fall respectively. in the summer, thiol content was significantly higher at cho (1.8 fold) and lower at yam (0.3-fold) compared to lsp (figure 5a). thiol content was significantly higher at the cho site than at the boi site. in the fall, thiol content was significantly lower at cho (0.2-fold) and boi (0.18fold) compared to yam and lsp. there was no significant difference between the boi and cho sites. thiol content was significantly correlated with ph (r=0.61; p=0.01) and oxygen content (r=0.51; p<0.05). lsp yam boi cho sites -0,5 0,0 0,5 1,0 1,5 2,0 2,5 h e m o c y te t h io l c o n te n t (n o rm a liz e d t o l s p r e fe re n c e s it e ) summer autumn a a,b,c c a a,b,ca,c lsp y am boi cho sites -0,2 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 g s t a c ti v it y (n o rm a liz e d t o l s p r e fe re n c e s it e ) summer autumn b a,b,c a,b,c a,c a,c fig. 5 biotransformation activity in hemocytes. mussels were caged at four sites in the yamaska river during the summer and fall. biotransformation activity was determined by total thiol content and gst activity. the letter a indicates a significant difference between the site and lsp (the reference site). the letter b indicates a significant difference from boi (impacted by urbanization but no blue-green algae). the letter c indicates a significant difference from yam. 48 for gst activity, two-way factorial anova revealed a significant difference for sites only (f=29.5, p<0.001) i.e. no seasonal effects and no interaction between season and sites. gst activity was globally similar in the summer and in the fall (figure 5b). the mean gst activity was 0.11 and 0.06 absorbance change/min/mg proteins at the lsp site in the summer and the fall. in the summer, gst activity significantly decreased at boi (0.45-fold) and cho (0.25-fold) compared to lsp. in the fall, the same pattern was observed but more intensely than in the summer, with 0.18-fold and 0.2-fold decreases at boi and cho respectively. there was no significant difference between the boi and cho sites by season. correlation analysis revealed that gst activity was significantly correlated with hemocyte density (r=0.23; p<0.05), phagocytosis efficiency (r=0.23; p<0.05), no production (r=0.42; p<0.001), doc (r=-0.66; p<0.01), conductivity (r=0.61; p=0.01) and dissolved p/total p ratio (r=-0.56; p<0.05). we examined the immunocompetence data to attempt to determine whether cyanobacterial blooms could produce specific, discernible effects in mussels. in respect to cyanobacterial blooms that occurred at the cho site only in the fall, we sought biomarkers that responded to the cho site and differed significantly from the urban pollution (boi), agriculture (yam) and reference (lsp) sites. we found that hemocyte density, phag efficiency, ros, no and ly activity satisfied the above criteria based on two-way factorial anova (table 2). moreover, ly activity was significantly correlated (r=0.67; p<0.01) with the levels of blue-green algae (cyanobacteria) in surface waters. multiple regression revealed that lysozyme activity was predicted (r=0.85) by blue-green algae (β =0.54; p<0.05), no3/total n (β=0.32; p<0.05) and conductivity (β=-0.42; p<0.05). table 2 identification of effects in caged freshwater mussels associated with cyanobacterial blooms biomarker difference before and after blooms at cho site difference from reference site lsp difference from urban pollution (boi) difference from agricultural pollution relationship with water parameters hemocyte density yes (+) yes(+) yes(+) yes(+) ph (r=0.5) hemocyte viability yes(+) yes(+) no yes(+) nil phagocytosis activity yes (-) yes (+) no yes (+) turbidity (r=-0.51) chl a (r=0.61) cond (r=-0.59) phagocytosis efficiency yes (+) yes (+) yes(+) yes (+) chl a (r=0.61) cond (r=-0.53) lysozyme yes (+) yes(+) yes(+) yes(+) cyanobacteria (r=0.66) cond (r=-0.63) ph (r=-0.60) ros yes(+) yes(+) yes(+) yes(+) pdissol/total p (r=-0.68) no yes(+) yes(+) yes(+) yes(+) cod (r=-0.58) pdissol/total p (r=-0.57) thiol content yes (-) yes (-) no yes (-) o2 (r=0.50) ph (r=0.61) gst activity no yes (-) no yes (-) cod (r=-0.66) cond (r=0.61) ph (r=0.5) pdissol/total p (r=-0.56) cox activity yes (-) yes (-) no yes (-) ph (r=0.67) the shaded rows indicate biomarkers that responded more strongly to cyanobacterial blooms than to urban and agricultural pollution. 49 discussion in nutrient (n and p)-rich environments, exponential growth (blooms) of cyanobacteria develop annually during summer and fall in lakes, reservoirs and slow-flowing rivers in temperate latitudes. however, it was found that total p levels alone were not always able to predict the presence of algal blooms (shimoda et al., 2016). multiple regression analysis showed that the occurrence of blue-green algae was significantly correlated with acidification of surface waters (ph, r=-0.52; p<0.05) but not with total phosphate and nitrogen levels. conditions in the yamaska river watershed are known to favour cyanobacterial blooms, which usually occur in late summer and fall (mddefp: http://www.mddelcc.gouv.qc.ca/eau/inter.htm). our two upstream sites, cho and boi, were located within the urban and agricultural area and one of them, cho, exhibited cyanobacterial bloom during the fall period, as confirmed by the presence of blue-green algae and microcystin-lr in surface waters and visual inspection (a thick green mat covered the water). given that mussel populations also thrive in the yamaska watershed, which drains both urban and agricultural areas, freshwater mussels are exposed to the cumulative input of urban and agricultural pollution and cyanobacterial blooms. the health consequences of this exposure are currently not well understood. gst activity in hemocytes was significantly depressed in both summer and fall at both the cho (algal blooms) and boi (municipal effluent) sites relative to lsp (the reference site), suggesting that not only cyanobacterial blooms but also other factors were at play in decreasing the activity. the decrease in gst activity could reflect a depletion of gsh stores from oxidative stress and biotransformation in mussels exposed long-term to pollution. this was corroborated by reduced thiol content in hemocytes in the fall (figure 3). interestingly, thiol content levels were significantly higher in the summer, which suggests that thiol stores were not fully depleted at this time. in a study with the freshwater clam diplodon chilensis patagonicus, clams were fed with a toxic strain of m. aeruginosa for 6 weeks, then oxidative stress and biotransformation activity were determined (sabati et al., 2011). antioxidant enzymes (superoxide dismutase and catalase), reduced gsh and gst activity were all significantly upregulated after 6 weeks. the reverse was found with decreased thiol content and gst in mussels caged at the cho site in the fall during cyanobacterial blooms. moreover, the ros increase was much greater than the increase in no production and was weakly correlated (r=0.19; p<0.05) with no, suggesting that oxidative stress involved other mechanisms than oxidative burst following phagocytosis. shrimps exposed for 70 days to cyanobacterial blooms had decreased hemocyte density and increased phagocytosis activity (gao et al., 2017). phagocytosis activity was increased at both the urban (boi) and cyanobacterial bloom (cho) sites, and the increase was stronger in the fall. however, both cho (cyanobacterial blooms) and boi (urban) showed increased phagocytosis activity and efficiency, although phagocytosis efficiency differed significantly between the cho and boi sites during the cyanobacterial bloom in the fall. nevertheless, the increase in phagocytosis activity and hemocyte density in mussels exposed to municipal effluents was reported elsewhere (farcy et al., 2011). interestingly, ly activity was not significantly increased by the effluents, indicating less sensitivity to human-generated sources of bacteria than to cyanobacterial blooms. microcystins from algal blooms could be toxic to clams (pham et al., 2016). exposure to crude extracts of cyanobacteria containing 400 µg/l of microcystin-l in clams for 10 days resulted in increased gst, superoxide dismutase and catalase in the gills and mantle, and inhibition of catalase and gst activities in the foot and other tissues. gst activity was also decreased at cho site, which contained cyanobacteria and microcystins at 3 µg/l but exposure was for much longer times than 10 days. gst activity was also reduced at boi, which receives urban discharges from the city of granby. no signs of cyanobacterial blooms were found there, indicating that other contaminants could also reduce the activity. in another study, freshwater clams (d. chilensis) were fed a toxic strain of m. aeruginosa for 6 weeks (sabatini et al., 2011). although neither protein nor lipid damage were observed, significant increases in gsh (thiol), superoxide dismutase, catalase and gst were observed, suggesting that antioxidant enzymes and cofactors were able to contain oxidative stress. this is in keeping with decreased inflammation (cox activity) in mussels caged at boi and cho. however, total thiol content was decreased at cho and boi indicating that other factors could have contributed to reduced thiol levels in the hemolymph. another possible explanation for the observed decreased in gst activity and the tolerance of bivalves to toxins is compensation mechanisms involving a multi-xenobiotic resistant p-glycoprotein (p-gp) pump (contardo-jara et al., 2008). the activity of the p-gp pump showed a significant increase up to 72 h in clams exposed to 100 µg/l microcystin-lr, indicating enhanced excretion of the toxin. this was accompanied by decreased gst and catalase activities after 72 h exposure times. hemocyte metabolic activity was greater when hemolymph was exposed in vitro to m. aeruginosa than when exposed to lyngbia wollei extracts (gélinas et al., 2014). this is consistent with increased hemocyte metabolic activity and density observed at the cho site, which was undergoing cyanobacterial blooms. however, hemocytes exposed to m. aeruginosa extracts had elevated ros levels and cox activity. a possible explanation is that protein thiol content was depleted by sustained exposure to contaminants including cyanotoxins and oxidative stress in mussels after four months. lower water temperatures in the fall might also have reduced the turnover rate of thiols and proteins in the hemolymph. no production, ly activity and ros levels appeared to be the most responsive and selective endpoints to cyanobacterial blooms when compared to urban and agriculture sites. indeed, ly activity http://www.mddelcc.gouv.qc.ca/eau/inter.htm 50 was maximal at the cyanobacterial bloom (cho) site, differing significantly from the urban (boi) and agriculture (yam) sites (table 2). ly activity was elevated at boi, but significantly less than at cho, compared to the reference lsp site, which makes sense given the occurrence of microorganisms in municipal effluents. increased hemolymph ly activity was also observed in hyriopsis cumingii mussels exposed to m. aeruginosa (hu et al., 2015). moreover, hypoxia was found to dampen the increase in ly activity. although oxygen content at cho (7.8 mg/l) was lower than at boi (9 mg/l), there was no significant correlation between ly activity and o2 levels in surface waters, within the range of o2 measured in the present study (6 mg/l to 13.1 mg/l). in conclusion, exposure to cyanobacterial blooms could contribute to contamination from urban and agricultural pollution in caged freshwater mussels. ros production and ly activity were the most responsive biomarkers for cyanobacterial blooms at the cho site and responded more strongly there than at the boi site, which receives municipal effluents from the city of granby. ros levels were not correlated with phagocytosis activity and were correlated only weakly with no levels, which suggests that there are other sources of ros from the immune response (oxidative burst), perhaps from mitochondria activity and biotransformation. the results support the hypothesis that cyanobacterial blooms exert additional stress on freshwater mussels by contributing to oxidative stress and to enhanced immunocompetence, as with other contaminants. acknowledgments this study was supported by the st. lawrence action plan of environment and climate change canada. the authors thank chantale andré and sophie trépanier for their assistance with mussel collection and cage preparation. references amado ll, monserrat jm. oxidative stress generation by microcystins 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https://www.ncbi.nlm.nih.gov/pubmed/?term=gao%20y%5bauthor%5d&cauthor=true&cauthor_uid=27266308 https://www.ncbi.nlm.nih.gov/pubmed/?term=dao%20ts%5bauthor%5d&cauthor=true&cauthor_uid=27266308 https://www.ncbi.nlm.nih.gov/pubmed/?term=utsumi%20m%5bauthor%5d&cauthor=true&cauthor_uid=27266308 https://www.ncbi.nlm.nih.gov/m/pubmed/?term=prieto%20ai%5bauthor%5d&sort=ac&from=/16455146/ac https://www.ncbi.nlm.nih.gov/m/pubmed/?term=jos%20a%5bauthor%5d&sort=ac&from=/16455146/ac https://www.ncbi.nlm.nih.gov/m/pubmed/?term=pichardo%20s%5bauthor%5d&sort=ac&from=/16455146/ac https://www.ncbi.nlm.nih.gov/m/pubmed/?term=moreno%20i%5bauthor%5d&sort=ac&from=/16455146/ac https://www.ncbi.nlm.nih.gov/m/pubmed/?term=came%c3%a1n%20am%5bauthor%5d&sort=ac&from=/16455146/ac https://www.ncbi.nlm.nih.gov/m/pubmed/?term=came%c3%a1n%20am%5bauthor%5d&sort=ac&from=/16455146/ac https://www.ncbi.nlm.nih.gov/pubmed/?term=queiroga%20fr%5bauthor%5d&cauthor=true&cauthor_uid=28407513 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https://www.ncbi.nlm.nih.gov/pubmed/?term=luquet%20cm%5bauthor%5d&cauthor=true&cauthor_uid=21477863 https://www.ncbi.nlm.nih.gov/pubmed/?term=san%20juli%c3%a1n%20m%5bauthor%5d&cauthor=true&cauthor_uid=21477863 https://www.ncbi.nlm.nih.gov/pubmed/?term=pirez%20m%5bauthor%5d&cauthor=true&cauthor_uid=21477863 https://www.ncbi.nlm.nih.gov/pubmed/?term=carmen%20r%c3%ados%20de%20molina%20md%5bauthor%5d&cauthor=true&cauthor_uid=21477863 https://www.ncbi.nlm.nih.gov/pubmed/21477863 https://www.ncbi.nlm.nih.gov/pubmed/?term=shimoda%20y%5bauthor%5d&cauthor=true&cauthor_uid=28073525 https://www.ncbi.nlm.nih.gov/pubmed/?term=watson%20sb%5bauthor%5d&cauthor=true&cauthor_uid=28073525 https://www.ncbi.nlm.nih.gov/pubmed/?term=palmer%20me%5bauthor%5d&cauthor=true&cauthor_uid=28073525 https://www.ncbi.nlm.nih.gov/pubmed/?term=koops%20ma%5bauthor%5d&cauthor=true&cauthor_uid=28073525 https://www.ncbi.nlm.nih.gov/pubmed/?term=mugalingam%20s%5bauthor%5d&cauthor=true&cauthor_uid=28073525 https://www.ncbi.nlm.nih.gov/pubmed/?term=morley%20a%5bauthor%5d&cauthor=true&cauthor_uid=28073525 https://www.ncbi.nlm.nih.gov/pubmed/28073525 https://www.ncbi.nlm.nih.gov/pubmed/28073525 http://www.sciencedirect.com/science/article/pii/s0003269785710792#! http://www.sciencedirect.com/science/article/pii/s0003269785710792#! http://www.sciencedirect.com/science/article/pii/s0003269785710792#! http://www.sciencedirect.com/science/journal/00032697 http://www.sciencedirect.com/science/journal/00032697/224/2 https://www.ncbi.nlm.nih.gov/pubmed/15737675 https://www.ncbi.nlm.nih.gov/pubmed/15737675 https://www.ncbi.nlm.nih.gov/pubmed/15737675 transplant rejection in m0lluscs isj 8: 15-20, 2011 issn 1824-307x minireview transplant rejection in terrestrial molluscs e furuta, k yamaguchi the research institute for comparative immunology, 1250-9-401, hasunuma, minuma-ku, saitama 337-0015, japan accepted december 20, 2010 abstract to know whether or not molluscs are capable of recognizing tissue allo-antigens, dorsal skin-allografts were exchanged between adult terrestrial slug, incilaria fruhstorferi. we succeeded for the first time in orthotopic transplantation of allografts and observed chronic rejection of allografts. cellular changes in the rejection process continued over for 40 days. two functional types of “effector” cells were recognized at the rejection site, but they were observed to be macrophages possessing perforin granules and phagocytosing damaged cells of the allograft. three days after transplantation, the perforin-positive cells were recognized only in the recipient tissue surrounding the allograft. five days after transplantation, these cells started to appear in the graft, while they disappeared from the host tissue. however, tunel-positive cells (apoptotic cells) were not observed throughout the graft-rejection process. electron microscopic examination of the graft tissue revealed autophagic degeneration of epithelial cells, mucous cells, pigment cells, fibroblasts, and muscle cells. these observations suggest that the slugs have the capability to recognize differences in cell-surface molecules between the allogeneic and recipient tissue, and that an allograft is chronically rejected due to a type of immunocyte (macrophage) that can induce perforin-dependent cell death. key words: allogeneic rejection; molluscs; orthotopic transplantation; autophagic cell death; perforin indroduction all metazoan animals need an internal defense system to protect themselves against foreign materials that succeed in getting past the external defense. it is well known that the internal defense mechanism in vertebrate consists of non-specific humoral and cellular factors, and specific cellular and humoral ones. vertebrates are able to respond against invading microorganisms first by non-specific element, such as lysozyme, complements, interferon, lysine, transferin and then by typical immune reactions of a clonal nature. moreover, macrophages are involved in part or all of those sequential defense processes both as phagocytes and antigen-presenting cells in some way and t and b-lymphocytes are engaged in only the later stage of specific defense reactions. however, invertebrates including the molluscs do not possess classical immune recognition molecules of vertebrates such as immunoglobulins, t-cells, mhc ___________________________________________________________________________ corresponding author: emiko furuta the research institute for comparative immunology 1250-9-401, hasunuma, minuma-ku saitama 337-0015, japan e-mail: furutaemk@nifty.com or antigen receptors. but they still manage to normally keep their internal body fluids sterile. they possess the capacity to distinguish not only between self and non-self, but also among non-self materials which differ in chemical properties (bayne, 1990; cooper et al., 1992). phagocytosis is considered to be the primary clearance mechanism in molluscs. incilaria fruhstorferi, the largest slug native of japan, possesses only macrophage, a kind of hemocyte, being able to recognize and phagocytose biotic and abiotic non-self materials (furuta et al., 1987; yamaguchi et al., 1988; furuta et al., 1990). the macrophage performs intracellular digestion of non-self materials. the destruction of tissue allografts in mammals is thought to be mediated primarily by cellular immune mechanisms, an interpretation supported by histological observations of the dense infiltration of recipient cells into the organs to be rejected. detailed and precise studies of the nature and the function of such cells are essential in order to clarify the mechanisms of transplant rejection. in mammals, the principal target of the immune response to allografts is the major histocompatibility complex (mhc, h-2 in mice). however, mhc molecules have not previously 15 fig. 1 procedure of allotransplantation of dorsal skin graft in the slug, incilaria fruhstorferi. a small piece of dorsal skin (2.5x4.5 mm in size and 0.1-0.2 mm in depth) was peeled off and placed on the recipient graft bed of other individual. been reported in invertebrates, and invertebrates possess neither antigen-specific lymphocytes nor any form of secreted immunoglobulins. yet, despite the lack of a highly developed specific immune response, those animals apparently have thrived for hundreds million years in a world full of pathogens. invertebrates possess several types of hemocytes and at least one of these functions as a phagocyte. phagocytes are known to play a crucial role in the internal defense system in invertebrates. in addition, a body of evidences suggests that invertebrate animals, especially molluscs, may possess the capability to recognize and reject allogeneic transplants. this is controversial. when digestive glands of helisoma duryinormale, the head/foot and digestive gland tissues, and the heart of biomphalaria glabrata were implanted into cephalopedal sinus of the other individuals, these heterotopic allografts had been encapsulated by hemocytes (cheng and galloway, 1970; jourdane and cheng, 1987; sullivan et al., 1992). on the other hand, when hemocyte producing organ was implanted into the hemocel, this kind of allograft not only survived without being encapsulated but hemopoietic activity was also still retained (sullivan, 1990). in lymnaea stagnalis, when vasa deferentia was implanted into the cephalopedal blood sinus, hemocytes had aggregated transiently at the cut surface at 24 h after implantation, and thereafter small groups of hemocytes were contact with the graft (sminia et al., 1974). sullivan et al. (1998) had found no conclusive evidence for chronic allograft rejection in b. glabrata regardless of tissue that was transplanted. the above results show that some grafts can often survive in a recipient for many months, whereas, others undergo varying degrees of hemocytic encapsulation and degenerative changes. these contradictory results may be due to the site, i.e., the heterotopic position. only two reports have been described so far on orthotopic transplantation in molluscs. according to röegener and renwrantz (1984), all small pieces of skin of the head-foot (autografts) of helix pomatia were destroyed within 6-9 days. in this case, the skin grafts may have been deficient in size for survival. however, alloand congeneric xeno-grafts of cerebral ganglia were tolerated in the mesocerebrum which were removed from helix aspersa (gomot and gomot, 1996). since results from mammals seem to indicate the presence of a barrier, considering the brain as privileged site for transplantation is still under discussion and this may apply in even to molluscs. in order to unravel these confused results mentioned above, orthotopic transplantation would be required. obstacles in performing orthotopic transplantation seem to lie in the technical difficulty of holding the donor tissue at the appropriate site in the recipient. we overcome this difficulty by a method recorded elsewhere (yamaguchi et al., 1999). we attempted to elucidate in our examination cytological change in both grafted and recipient tissue in response to orthotopic transplants of allogeneic skin using the terrestrial slug, i. fruhstorferi. 16 m acrophage n um bers /field 0 20 40 60 80 100 120 4 8 20 weeks after transplantation (a) autotransplantation 0 20 40 60 80 100 120 4 (b) allotransplantation 140 8 20 * * ; graft ; graft bed fig. 2 changes of macrophage numbers in autoand allografts, and their beds after transplantation. the number of macrophages phagocytosed cell debris in grafts and their beds were counted out at 10 visual fields randomly selected for each sample. in autotransplantation, the numbers at 20 weeks after transplantation significantly decreased in grafts and their beds comparing with them at 4 or 8 weeks. mean ± sem (n = 10). *p < 0.001 (student’s t-test) (yamaguchi et al., 1999). histology of the epithelial cells of the dorsal surface skin external surface of the dorsal skin epidermis is composed of a single layer of microvillous columnar cells which hold in place the covering of mucus and underneath epidermal cell layer, numerous pigment cells with many dendritic processes exist. the epidermis is supported by a mat of connective tissue through which run muscle fibers. five main cell types are distinguished in the epidermis: (1) microvillous cells, (2) round mucous cells (3) tubular mucous cells (4) channel cells and (5) ciliated cells. the epidermal cells closely cling to one another by zonula adherens in the apical region of cells. the presence of unicellular mucous glands is well established in the slug and the cells typically possess cell bodies located in the subepidermal connective tissue and secretary processes extending to the surface of the epidermis. macrophages are hardly observed in subepidermal connective tissue. furuta and shimozawa (1983), in an in vitro experiment, found that fibroblast of i. fruhstorferi differentiated into macrophage after injection of foreign materials, and the main macrophage producing site is in the cells that line the hemocel wall, which are derived from fibroblasts (furuta et al., 1994). tissue transplantation the terrestrial slug was selected as the donor and recipient for implantation, because it is easy to use, being not aquatic and possessing no shell. these features are favorable because the graft is not readily peeled off from the recipient body mechanically, as by a water stream or contact with a shell. transplants were made orthotopically: a piece of the dorsal skin (2.5x4.5 mm in size, 0.1~0.2 mm in depth) from a donor animal was placed on the cut surface at the corresponding site of the dorsal skin in the recipient (fig. 1). epidermis of the dorsal skin is arranged as simple columnar epithelial cells. although the body surface of the terrestrial slug is covered with only simple epithelium, the surface is prevented from evaporation and is protected from mechanical 17 fig. 3 perforin immunohistochemistry of an allografted recipient site of dorsal skin, 1 day (a) and 3 days (b) after transplantation. perforin-positive cells (arrowheads) are evident in the recipient connective tissue (ct) surrounding the graft 3 days after transplantation, but not 1day after transplantation as well as controlled immunohistochemistry. ep, epithelial cell; mu, mucous cell; pi, pigment cell. bar = 10 µm (furuta et al., 2006). injuries. the reason is that the surface skin of the slug is covered a large amount of mucus which is secreted by two mucous cell types whose necks reach the apical portion of the dorsal surface skin; these cells were present among the epithelial cells (yamaguchi et al.,1999). as the mucus rendered the physical attachment of the graft to the host graft bed difficult, secretion was inhibited by anesthetizing slugs on ice. on other hand, bacterial infection of the graft appears to be inhibited after transplantation by lectins in the mucus (furuta et al., 1995; yuasa et al., 1998). fate of autografts in transplant experiment of slugs, mucous cells, pigment cells and muscle fibers were provided as convenient criteria of graft viability. two weeks after transplantation, grafts had connected to host beds and various macrophages congregated around, particularly, underneath the graft sites and infiltrated into the graft matrix. numerous macrophages had already phagocytosed cells damaged by mechanical trauma. as a result of it, pigment cells, mucous cells and muscle fibers decreased in number in grafts and their beds. this phenomenon is seemed to heal wounds. until eight weeks after transplantation many macrophages observed in the graft site, whereas at four weeks after transplantation, grafted tissues such as muscle fibers and mucous cells began to regenerate slowly and the regeneration of these cells had been over twenty weeks after transplantation. at twenty weeks after transplantation, the macrophage numbers in autografts and their beds decreased significantly comparing with the numbers at four and eight weeks (p < 0.001, student’s t-test). by contrast, numerous macrophages were still present in allografts and their beds at eight and twenty weeks and revealed active phagocytosis (fig. 2). thus, the dorsal skin of host slug was completely repaired in autograft transplantation. fate of allografts the rejection of allografts in mammals is mainly mediated by cytotoxic t-lymphocytes, whereas no comparable immune cells have been described in invertebrates. the examination was undertaken to determine whether similar cytotoxic effector cells are present when allograft rejection occurs in the terrestrial slug i. fruhstorferi (yamaguchi et al., 1999; furuta et al., 2006). immunohistochemistry for perforin, detection of apoptosis by the tunel (tdt-mediated dutp-biotin nick-end labeling) method and electron microscopy were performed using both donor and recipient tissues. one day after transplantation, the grafted skin appeared normal, and both perforin-positive and tunel-positive cells were not recognized in the graft tissues (fig. 3a). three days after transplantation, perforin-positive cells (macrophages) were evident in the recipient tissue surrounding the graft (fig. 3b), but not present in the grafted tissue. five days after transplantation, perforin-positive cells were observed in the graft, but no these cells were seen any longer in the recipient tissue surrounding graft. beneath the epithelial layer, the cells composing the connective tissue of the graft (fibroblasts, muscle cells, pigment cells, nerve cells, etc.) began to appear shrunken, and chromatin-condensed nuclei (ccn, pro-ccn) were scattered throughout the tissue. autophagic 18 fig. 4 electron micrographs of the tissue of a dorsal skin allograft, 5 days after transplantation. columnar epithelial cells (ep) of the graft have become simple-squarmous type. autophagic or early degenerating cells are seen in the connective tissue of the graft. a) ep decreases in height and becomes simple-squarmous type. the cells composing the connective tissue of the graft begin to appear shrunken and contain chromatin-condensed nuclei (pro-ccn; p-ccn), and macrophages containing perforin-like granules (arrowheads) have infiltrated into the graft. bar = 5 µm. b) a mucous cell containing granules (mg) irregular in shape becomes autophagic and is surrounded by a macrophage (mp) containing several perforin-like granules (arrowheads). bar = 2 µm (furuta et al., 2006). vacuoles were observed in the cytoplasm of these cells (fig.4a, b). macrophages containing small perforin-like granules often appeared surrounding the cells with pccn (fig. 4b). the remnants of autophagic cell death were phagocytosed by macrophages that infiltrated into the grafted connective tissue. twelve days after transplantation, the constitutive cells of the connective tissue of the graft disappeared through phagocytosis by infiltrating macrophages. in the graft tissue, cell elements were gradually lost and cell-free space was formed at the site of cell disappearance. autophagic cell death was found to play an important role in tissue destruction. one-hundred forty days after transplantation, the grafted tissues were completely displaced by recipient tissues. from the above data, we concluded that terrestrial slugs (molluscs) have the capability of recognizing and rejecting allogeneic tissue transplants and that they possess perforin-like molecules that lead to a cytotoxic reaction for autophagic cell death in the rejection of allografts. the presence of a putative perforin in molluscs suggests that perforin may be an immune defense mechanism conserved across phylogenetic lineages. the existence of a perforin in terrestrial slugs implies a much earlier evolutionary origin of this molecule than has been previously thought. references bayne cj. phagocytosis and non-self recognition in invertebrates. bioscience, 40: 723-731, 1990. cheng tc, galloway pc. transplantation immunity in mollusks: the histocompatibility of helisoma duryinormale with allografts and xenografts. j. invertebr. pathol. 15: 177-192, 1970. cooper el, rinkevich g, valembois p. invertebrate immunity: another viewpoint. scand. j. immunol. 35: 247-266, 1992. furuta e, shimozawa a. primary culture of cells from the foot and mantle of the slug, incilaria fruhstorferi collinge. zool. mag. 92: 280-296, 1983. furuta e, yamaguchi k, shimozawa a. hemolymph cells and the platelet-like structures of the land slug, incilaria bilineata (gastropoda: pulmonata) anat. anz. 170: 99-109, 1990. furuta e, seo n, yamaguchi k. perforin-dependent cell death in skin allograft rejection of the terrestrial slug, incilaria fruhstorferi. zool. sci. 23: 1093-1100, 2006. 19 furuta e, takagi t, yamaguchi k, shimozawa a. incilaria mucus agglutinated human erythrocytes. j. exp. zool. 271: 340-347, 1995. furuta e, yamaguchi k, shimozawa a. blood cell-producing site in the land slug, incilaria fruhstorferi. acta. anat. nipponica 69: 751-764, 1994. furuta e, yamaguchi k, shimozawa a. phagocytosis by hemolymph cells of the land slug, incilaria fruhstorferi collinge (gastropoda: pulmonata). anat. anz. 163: 82-99, 1987. gomot a, gomot l. allogeneic and xenogeneic grafts in pumonate gastropod mollusks: fates of neural transplants. dev. comp. immunol. 20: 193-205, 1986. jourdane j, cheng tc. the two-phase recognition process allografts in a brazillian strain of biomphalaria glabrata. j. invertebr. pathol. 49: 145-158, 1987. rögener w, renwrantz l. destruction of autografts and wound healing in helix pomatia. zool. jb. physiol. 88: 515-527, 1984. sminia t, borghart-reinders te, van de linde aw. encapsulation of foreign materials experimentally introduced into the freshwater snail limnaea stagnalis. an electron microscopic and autoradiographic study. cell tissue res. 153: 307-326, 1974. sullivan jt, galvan ag, lares rr. comparison of several types of allografts in biomphalaria glabrata (mollusca: pulmonata). j. invertebr. pathol. 71: 1-8, 1998. sullivan jt. long-term survival of heterotopic allografts of amoebocyte producing organ in biomphalaria glabrata (mollusca: pulmonata). trans. am. microsc. soc. 109: 52-60, 1990. sullivan jt, andrew ja, currie rt. heterotopic heart transplantats in biomphalaria glabrata (mollusca: pulmonata): fate of allografts. trans. am. microsc. soc. 111: 1-15, 1992. yamaguchi k, furuta e, nakamura h. chronic skin allograft rejection in terrestrial slugs. zool. sci. 16: 485-495, 1999. yamaguchi k, furuta e, shimozawa a. morphological and functional studies on hemolymph cells of land slug, incilaria bilineata, in vitro and in vivo. in kuroda y, kurstak e, maramorosch k. (eds), invertebrate and fish tissue culture, springer-verlag, berlin, germany, pp 247-250, 1988. yuasa hj, furuta e, nakamura a, takagi t: (1998) cloning and sequencing of three c-type lectins from body surface mucus of the land slug, incilaria fruhstorferi. comp. biochem. physiol. 119b: 479-484, 1998. 20 research report isj 7: 89-106, 2010 issn 1824-307x research report the effect of oxidative stress on phagocytosis and apoptosis in the earthworm eisenia hortensis sl fuller-espie, t nacarelli, el blake, fm bearoff science department, cabrini college, 610 king of prussia road, radnor, pennsylvania 19087-3698, usa accepted february 17, 2010 abstract the effect of exogenous hydrogen peroxide (h202) on phagocytic function and apoptosis in coelomocytes from eisenia hortensis was investigated. treating coelomocytes with h202 (0.26 to 8.4 mm) evoked a significant increase in phagocytosis for one or more of the concentrations of h202 employed in 67 % of cases. using annexin v-fitc we show that h202 induced apoptosis of coelomocytes in vitro. we found that 100 % of viable coelomocyte populations exhibited significant increases in phosphatidylserine translocation for one or more of the concentrations of h202 tested (8.4 to 67.6 mm). using a fluorescent inhibitor of caspases, we revealed the presence of activated caspases observing increased caspase activity in 67 % of viable coelomocyte populations treated with 33.8mm h202, and in 100 % of cases treated with 67.6 mm h202. agarose gel electrophoresis and the tunel assay showed dna fragmentation in samples treated with 16.9 and 33.8 mm h202. in addition, endogenous h202 production during phagocytosis by hyaline amoebocytes was detected using a fluorogenic substrate. thus, free radicals not only appear to facilitate phagocytosis and are produced during phagocytosis, but they also promote an oxidative-stress-induced apoptosis that may play an important function in regulating innate immune responses in e. hortensis. key words: phagocytosis; hydrogen peroxide; annexin v; dna fragmentation; caspase, tunel assay introduction when the production of reactive oxygen species (ros) in microsomes, peroxisomes, mitochondria and the cytosol overwhelms a cell’s ability to either neutralize reactive intermediates or repair toxic effects from ros, a state of oxidative stress is initiated. toxic effects from ros include damage to nucleic acids (mutagenesis) and carbohydrates, lipid peroxidation of cellular membranes, and enzyme inactivation (imlay, 2003). oxidative stress is also linked to the aging process (larsen, 1993; helfand and rogina, 2003; rattan, 2006; csiszar et al., 2007). ros encompass a wide array of oxidants including hydrogen peroxide (h202, the focus of this study), superoxide anions, hydroxyl radicals, peroxyl radicals and organic hydroperoxides (dunford, 1987; coffey et al., 1995; panasenko et al., 2002). these oxidants are produced through various means including radiation, uncomplexed metals such as iron and copper, organic compounds such as quinones, uric acid and ___________________________________________________________________________ corresponding author: sl fuller-espie science department cabrini college 610 king of prussia road, radnor, pa 19087-3698, usa email: sfuller-espie@cabrini.edu homocysteines, certain classes of xenobiotics such as polycyclic aromatic hydrocarbons (pah) going through redox cycling, and thermal stress (diguilioi et al., 1989; livingstone et al., 1990; sundaram et al., 1990; livingstone et al., 1995; abele et al., 2001; tyagi et al., 2005; valko et al., 2006; strazzullo and puig, 2007; fato et al., 2008; pichaud et al., 2008; kell, 2009). they are also generated by intracellular enzymes including nadph oxidase, xanthine oxidase and cytochrome p450 (lewis, 2002; bedard and krause, 2007; jankov et al., 2008). paradoxically, ros not only exert damaging effects in cells, but they also afford protecive effects, for example during immune defense for phagocytosis where ros are toxic to phagocytized pathogens. another benefit of ros is their aility to participate in redox signaling (thannickal and fanburg, 2000; forman and torres, 2002; wang, 2009). because of the detrimental effects of ros on cellular components, it is imperative that organisms possess cellular antioxidant defense mechanisms for the detoxification of ros, often measured as the total oxyradical scavenging capacity (tosc), an important biomarker of oxidative stress (regoli, 2000; gorbi and regoli, 2003; dovzhenko et al., 2005). ros are neutralized by a variety of   89 mailto:sfuller-espie@cabrini.edu antioxidant processes aimed at stabilizing free radicals, terminating free radical reactions, and preventing the transfer of electrons from oxygen to organic molecules. one mechanism relies upon enzymatic detoxification of ros by catalase, glutathione peroxidase, superoxide dismutase, thioredoxin reductase, peroxiredoxins and sulfiredoxin (raes et al., 1994; nordberg and arnér, 2001; flohé et al., 2003; findlay et al., 2005). nonenzymatic antioxidants also provide antioxidant defenses and include a diverse array of molecules including antioxidant quenchers comprising cellular proteins (e.g. transferrin, ferritin, metallothionein, ceruplasmin and others) that chelate pro-oxidant minerals (cairo et al., 1995; kang et al., 2001; yamaji et al., 2004; laukens et al., 2009). in addition, glutathione, selenium, phytochemicals, vitamin e, vitamin c and provitamin a compounds (e.g. beta carotene) also provide protective antioxidant defenses (sies et al., 1992; loo, 2003; brenneisen et al., 2005; ghezzi, 2005). the primary goal of this study was to investigate the in vitro effects of oxidative stress on cellular activities in the immune cells (coelomocytes) of the earthworm eisenia hortensis (also known as the european nightcrawler) which reside in the coelomic cavity. investigations of innate immunity in earthworms have identified three distinct subpopulations of coelomocytes (leukocyte equivalents): hyaline amoebocytes (large coelomocytes), granular amoebocytes (small coelomocytes) and chloragocytes (eleocytes), most likely diverging developmentally from a common progenitor cell (prohemocyte), as suggested by hartenstein (2006). the immune functions of coelomocytes can be studied in vitro after harvesting the coelomic fluid, which is rich in coelomocytes, by extruding the coelomocytes through the dorsal pores of the body wall from experimentally-induced earthworms. the hyaline amoebocytes are the major phagocytic cells, the granular amoebocytes constitute the subpopulation exhibiting nk-like activity, and the eleocytes contain chloragosomes and do not participate in either phagocytic or nk-like activities, but they do secrete lytic substances (cooper, 1996; cossarizza et al., 1996; adamowicz and wojtaszek, 2001; engelmann et al., 2002; engelmann et al., 2005). differences in granularity and size between coelomocytes permits amoebocytes (hyaline and granular) and eleocytes to be distinguished using flow cytometry methodology employing forward light scatter (fsc) and side light scatter (ssc) measurements (cossarizza et al., 1996, 2005; engelmann et al., 2004; patel et al., 2007; fuller-espie et al., 2008). selective analysis of subpopulations is facilitated by specifying regions to identify particular subpopulations, and then gating on assigned regions, permitting the investigator to include only desired subpopulations and exclude irrelevant subpopulations from final analyses. light and fluorescent microscopy have been used by researchers to study immune functions in earthworms (adamowicz and wojtaszek, 2001; kalaç et al., 2002), however, these methods are more subjective than flow cytometry, and they impose restrictions on the number of cells included in analyses owing to time constraints. in contrast, flow cytometry is an objective, quantitative methodology that analyzes thousands of cells per second with the option of restricting analyses to predetermined subpopulations. this investigation focused specifically on phagocytic function and the induction of apoptosis in the amoebocytes of e. hortensis following exposure to exogenous h202. we evaluated the phagocytic uptake of escherichia coli using flow cytometry and found that in vitro exposure to h202 (0.26 8.4 mm) enhanced phagocytosis. using flow cytometric and agarose gel electrophoresis methodologies we also examined the effect of h202 on three events associated with apoptosis: 1) translocation of phosphatidylserine (ps) to the extracellular face of the plasma membrane using annexin-v binding; 2) caspase activation using a fluorescein-conjugated inhibitor of caspase activation (flica); and 3) dna fragmentation. we present data supporting an apoptotic-like cell death in coelomocytes of e. hortensis resulting from in vitro exposure to exogenous h202 (8.4 67.6 mm). finally, using the fluorogenic substrate dhr 123, a probe widely used to measure intracellular h202, we also show that h202 is generated in hyaline amoebocytes during phagocytosis of bacillus megaterium and pseudomonas stutzeri. materials and methods cell culture supplies and chemical reagents tissue culture plasticware was purchased from fisher scientific. phosphate buffered saline (pbs) was purchased from invitrogen. dulbecco’s modified eagle medium (dmem, invitrogen) was supplemented with either 10 % heat-inactivated fetal calf serum (invitrogen) or serum supreme (lonza biowhittaker), plus 100 μg ml-1 ampicillin (shelton scientific), 10 μg ml-1 kanamycin (shelton scientific), 10 μg ml-1 tetracycline, 5 μg ml-1 chloramphenicol (fluka biochemika), 1× penicillin, streptomycin and amphotericin b, 1× nonessential amino acids (invitrogen) and 1× l-glutamine (invitrogen) to comprise super dmem (sdmem). sdmem supplemented with serum supreme was used for all exogenous h202 assays while sdmem supplemented with fetal calf serum was used for endogenous h202 assays. earthworm husbandry eisenia hortensis (european nightcrawlers) was purchased from vermitechnology unlimited, orange lake, florida, usa, who imports e. hortensis from star food, holland, scherpenzeelseweg 95, 3772me barneveld, the netherlands. species identity was determined by the united states department of agriculture, usda permit #52262 (vermitechnology, personal communication). shortterm colonies were maintained at rt in the dark on moistened autoclaved pine woodchips sprinkled with single grain rice cereal or rice with bananas cereal (gerber) and covered with autoclaved, shredded and moistened paper towels. habitats were changed twice weekly. animals were euthanized by freezing at -20 oc.   90 extrusion of coelomocytes prior to experimentation, earthworms were first washed with distilled water on paper towels using a water bottle to remove wood chip fragments or food particles. they were then placed overnight on paper towels moistened with 2.5 μg ml-1 fungizone (fisher scientific) in 100 mm petri dishes to minimize fecal contamination during the extrusion process, and remove further any surface contaminants. to collect coelomocytes, earthworms were placed in either 100 mm petri dishes or in multichannel pipette reservoirs containing 3 ml bd facsflow sheath fluid (bd biosciences). the earthworms extruded their coelomocytes through their dorsal pores in response to this external stimulus without the need to use the alcohol extrusion method reported by others (engelmann et al., 2005). the coelomocytes were then transferred to 0.5 ml accumax (innovative cell technology) in 15 ml conical test tubes for a 5 min incubation period at rt to reduce aggregation of cells. finally, 5 ml pbs was added and the samples were centrifuged immediately at 150 x g, 5 min at 4 oc. after decanting the supernatant, the coelomocyte pellet was gently mixed by flicking the bottom of the centrifuge tube, and coelomocytes were resuspended in 0.5 ml sdmem. enumeration was carried out using a hemacytometer. only hyaline amoebocytes (large coelomocytes) and hyaline granulocytes (small coelomocytes) were included in the cell count; eleocytes were not counted but did factor into a quality score. samples with large numbers of eleocytes compared to large and small coelomocytes were not used in phagocytosis assays. samples were adjusted to 3.8 x 105 or 5 x 105 (phagocytosis and annexin v assays, see below) or 1 x 106 (caspase assays) coelomocytes ml-1 in sdmem. bacteria for phagocytosis assays e. coli/gfp: escherichia coli hb101 transformed with pglo (biorad) and expressing green fluorescent protein (gfp) were grown on tryptic soy agar containing 100 μg ml-1 ampicillin and 0.2 % (w/v) arabinose at 32 °c for 24 h. after washing the cells once in pbs, they were fixed chemically with 4 % (v/v) paraformaldehyde in pbs, 1 h at rt with periodic mixing, followed by three pbs washes. centrifugation was carried out at 3273xg for 5 min at 4 oc. the final cell pellet was resuspended in pbs, bacteria were enumerated using a hemacytometer, and then stored in the dark at 4 °c. bacillus megaterium and pseudomonas stutzeri (presque isle cultures) were grown overnight in tryptic soy broth at 37 oc in a shaking incubator. absorbance was measured using a spectrophotometer (600 nm) and compared to a standard curve to determine concentration. standard curves were generated by correlating absorbance with cell count using a hemacytometer. all bacteria were diluted in sdmem to obtain the desired multiplicity of infection (m.o.i.). phagocytosis: exogenous h202 pretreatment phagocytosis assays were carried out in sdmem. coelomocytes (50,000 per well) were pretreated with or without h202 (0 8.4 mm final concentration) 5 % co2, at 25 oc in 96-well, roundbottom plates in 200 μl sdmem. duplicate samples were used in every assay. following h202 pretreatment, cells were centrifuged (150xg) and washed once with pbs. finally, 200 μl e. coli/gfp was added to each well at a multiplicity of infection of 1000 bacteria:1 coelomocyte and incubated for 3 h at 30 °c. to control for non-specific binding of e. coli/gfp to the external surface of coelomocytes, 50 μm cytochalasin b (sigma aldrich) [an antibiotic that interferes with microfilament activity and thereby inhibits phagocytosis (axline and reaven, 1974)] was added to control wells 45 min before the addition of e.coli/gfp. following e. coli/gfp uptake, trypan blue (biowhittaker) was used at a final concentration of 0.02 % (w/v) for 30 min at rt in the dark, for quenching purposes to reduce background fluorescence (mosiman et al., 1997). the cells were transferred to flow cytometry tubes containing 100 μl facs flow buffer (bd biosciences), placed on ice in the dark, and run immediately on the flow cytometer. annexin v-fitc/pi assay recombinant human annexin v-fitc (invitrogen, annexinv01)) and propidium iodide (pi) (invitrogen, p3566) were used to detect ps translocation and to enable exclusion of dead cells from analyses. using a 96-well, round-bottom plate, 5 x 104 (assay 1) or 3.8 x 104 (assay 2) coelomocytes in 50 μl sdmem were added to appropriate experimental (h202-treated) and control (double negative autofluorescent background; single positive fitc; single positive pi; double positive annexin v/pi background) wells in triplicate. experimental wells received 50 μl of h202 (final concentrations of 67.6, 33.8, 16.9, and 8.45 mm). single positive fitc controls received 50 μl of h202 (270mm final). single positive pi controls received 50 μl saponin (0.01 % final). the plate was incubated 6 h, 25 oc, 5 % co2 before adding 100 μl pbs and centrifuging (5 min, 4 oc, 150xg). after removing the supernatant fraction, the wells were washed with 200 μl well-1 of pbs, and centrifuged again. the supernatant fraction was removed and the cells were resuspended in 200 μl sdmem containing 1× binding buffer (0.01m hepes, 0.14 mm nacl, 2.5mm cacl2) with or without annexin vfitc (3.75 μl well-1) and/or pi (0.5 μl well-1). the autofluorescent background control did not receive annexin v-fitc or pi. the single positive pi control did not receive annexin v-fitc. the single positive fitc control did not receive pi. all other samples received both annexin v-fitc and pi. samples were incubated for 5 min at rt and transferred to flow cytometry tubes containing 150 μl of facs flow sheath buffer containing 1× binding buffer. samples were kept on ice protected from light and analyzed immediately by flow cytometry. caspase assay caspase activation was measured using a vybrant® fam caspases assay kit (fitc) (invitrogen/molecular probes) according to the manufacturer’s instructions. this assay utilized a fluorescent inhibitor of caspases known as flica™   91 which detects activation of caspase enzymes in cells undergoing apoptosis. using a 96-well, vbottom plate, 1 x 105 coelomocytes in 0.1 ml sdmem were added to appropriate experimental (h202-treated) and control [untreated (0 mm) double negative autofluorescent background; single positive fitc; single positive pi; double positive caspase background] wells in duplicate. experimental wells received 50 μl h202 (final concentrations of 33.8 mm for ew f1-f3; 16.9 mm, 33.8 mm and 67.9 mm for ew f4-f6); single positive pi control wells received 50 μl 0.03 % saponin in sdmem; and single positive fitc control wells received 50 μl sdmem. all control and experimental samples were incubated for 6 h, 25 °c, 5 % co2. after centrifugation at 150xg (5 min, 4 oc), the supernatant fraction was removed and the wells were washed with 200 μl pbs. again the plate was centrifuged and the supernatant fraction was removed. untreated double-negative, autofluorescent control samples (fitc negative, pi negative) and single positive pi controls were resuspended in 100 μl of sdmem. untreated samples (caspase background) and h202treated samples were resuspended in 90 μl sdmem plus 10 μl of 10x flica reagent. single positive fitc control wells were resuspended in 50 μl sdmem, 10 μl of 10x flica reagent, and 40 μl of 10 % formaldehyde. the plate was incubated in the dark, 1 h, 25 °c, 5 % co2, with gentle mixing every 20 min before adding 100 μl of 1× washing buffer and centrifuging as above. after removing the supernatant fraction, the cells were washed twice with 200 μl well-1 of 1× wash buffer. following the last centrifugation and removal of the supernatant fraction, 200 μl well-1 of 1× wash buffer with or without pi was added to each well; double-negative and single positive fitc controls did not receive pi, all other samples received pi. samples were placed on ice protected from light and analyzed immediately by flow cytometry. cell volume measurements ew f4-f6 used in the caspase assay were also subjected to cell volume analysis using flow cytometry. forward scatter measurements of pi negative large coelomocytes treated in duplicate with 0, 16.9, 33.8 and 67.6 mm h202 were averaged and analyzed by student’s t test to determine if differences observed in forward light scatter measurements were statistically significant compared to controls. flow cytometry fluorescence was measured using fl-1 (fitc, gfp and rhodamine 123) and fl-2 (pi) detectors of a facscalibur flow cytometer (bd biosciences). autofluorescent controls were used to set voltages for forward scatter (fsc), side scatter (ssc), fl-1 and fl-2 during instrument set-up. single positive fitc and pi controls were used to adjust compensation settings (spectral overlap removal) for annexin v and caspase assays. listmode data was acquired and analyzed using cell quest (bd biosciences) and winlist5.0 (verity software house) software. only coelomocytes corresponding to the large coelomocyte population (phagocytosis assays) or large and small coelomocyte populations combined (annexin v and caspase assays), as determined by appropriate granularity and size, were gated for further analyses. dna fragmentation assay dna purification was carried out according to hermann et al. (1994) with some modifications. briefly, coelomocytes from ten individual earthworms were extruded and plated at 1.5 x 105 coelomocytes well -1 in 200 μl. for each treatment, 10 wells were used (1.5 x 106 coelomocytes per treatment from 10 individual earthworms), one well for each earthworm extruded, and each earthworm exposed to all treatments of the assay. h202 was added at 0, 8.4, 16.9 or 33.8 mm and then incubated at 30 oc, 5 % co2, 6 h. after the incubation period, the plate was centrifuged (150xg, 10 min, 4 oc), the supernatant was removed, and the cells were gently resuspended by vortexing. then 40 μl of lysis buffer (1% np-40, 20 mm edta, 50 mm tris-hcl, ph 7.5) was added to each well before pooling the 10 wells (10 individual earthworms) for each treatment group into a single microcentrifuge tube and centrifuging the lysate at 1600xg, 5 min to pellet debris. the supernatant was transferred to a new tube and the pellet was reextracted with 40 μl lysis buffer and respun. supernatants were combined for each treatment group, and adjusted to 1 % sds, 5 μg ml-1 rnase (fermentas life sciences) and incubated 2 h at 56oc before adding proteinase k (fisher, bp170050) (2.5 μg ml-1) and incubating 2 h at 37 oc. then 0.5 volume 7.5 mm ammonium acetate and 2.5 volume of absolute ethanol was added to precipitate the dna. dna pellets were collected by centrifugation (14,000xg), rinsed with ice cold 70 % ethanol and air dried. pellets were resuspended in 21 μl te (10 mm tris-cl, 1 mm edta, ph 8,0) at 37 oc, 5 min before adding 4 μl loading dye (6x blue/orange loading dye, promega, g1881) containing 1:100 sybr® safe gel stain (10,000x concentration in dmso, invitrogen). molecular weight markers (10 μl well-1) (exactgene, fisher bioreagents, bp257110) containing sybr® safe gel stain (2 μl well-1) and dna samples from each treatment group were electrophoresed in a 1.5 % agarose gel containing 1:10,000 sybr® safe gel stain in 1 x tbe (89 mm tris base, 89 mm boric acid, 2 mm edta, ph 8.3), 120v until first dye front was ~ 2 cm from bottom of gel. gels were photographed using a gel-documentation system (bio-rad). tunel assay a flow tacs apoptosis detection kit (trevigen, inc.) was used according to the manufacturer’s instruction except 150,000 cell ml-1 were used in 0.5 volume recommended, and optional pi was not included. coelomocytes were incubated in 0.2 ml sdmem for 12 h at 25oc, 5% co2 with or without h2o2 (33.8 mm) before washing and fixing the cells. flow cytometry measured fl-1 signals from gated amoebocytes. samples were run in duplicate and subjected to statistical analysis by student’s t test.   92 phagocytosis: endogenous h202 production values were statistically significant as exhibited as a p-value less than or equal to 0.05. all data is based on averages of either triplicate (exogenous h202/phagocytosis and annexin v assays) or duplicate (endogenous h202/phagocytosis, caspase and tunel assays) samples. bacteria were introduced to coelomocytes (50,000 per well in 96 v-bottom plate in duplicate) at a m.o.i. ranging from 10:1 to 1000:1. following 90 min incubation at 30 oc, dhr 123 (invitrogen, d632, 1 mm stock in dmso) was added (1 μm final, 1:1000). after 10 min incubation at rt, samples were placed on ice, protected from light, and run on the flow cytometer immediately. negative controls (no dhr 123) were incubated with 1:1000 dmso to control for carrier effect of dmso. fluorescence was measured using the fl-1 detector. results flow cytometry: exogenous phagocytosis assay figure 1 illustrates how our data was collected and analyzed to determine specific phagocytosis by hyaline amoebocytes from e. hortensis when using exogenous h202. the left panel of the top row is a dot plot representing a typical coelomocyte profile obtained on the flow cytometer when analyzing forward scatter (fsc) (abscissa) versus side scatter (ssc) (ordinate) properties of earthworm statistical analysis data analysis and graphs were generated using microsoft excel 2007. the student’s t-test assuming unequal variance was utilized with a 95 % confidence interval to determine if the experimental fig. 1 representative flow cytometry profile from phagocytosis assays. top row: left panel shows a typical coelomocyte profile of fsc (size) (abscissa) versus ssc (granularity) (ordinate) of extruded earthworm coelomocytes where r1 = hyaline amoebocytes, r2 = granular amoebocytes, and r3 = eleocytes; right panel shows fsc (abscissa) versus fl-1 (relative fluorescence intensity) (ordinate) of r1-gated, untreated coelomocytes cultured without e. coli/gfp. bottom row: fsc (abscissa) versus fl-1 (ordinate) of r1-gated coelomocytes cultured with e. coli/gfp without pretreatment (left panel) and with pretreatment (right panel) of h202. fsc = forward scatter; ssc = side scatter; fl-1 = relative fluorescence intensity of gfp; ur = upper right; lr = lower right.   93 fig. 2 phagocytosis is enhanced by pretreating earthworm coelomocytes with h202. asterisks indicate p ≤ 0.05. top row: earthworms ew-p1 – ew-p4 were pretreated with 0, 1.1, 2.1, 4.2 and 8.4 mm h202. middle and bottom rows: earthworms ew-p5 – ew-p12 were pretreated with 0, 0.26, 0.53, 1.1, 2,1, 4.2 and 8.4 mm h202. coelomocytes. r1 depicts large coelomocytes (hyaline amoebocytes), r2 depicts small coelomocytes (granular amoebocytes), and r3 depicts chloragocytes (eleocytes). for analysis purposes and the determination of percent specific phagocytosis, fsc (abscissa) versus fl-1 (ordinate) dot plots were gated on hyaline amoebocytes (r1), the phagocytic cell population. the fsc versus fl-1 dot plots were partitioned into quadrants moving the horizontal bar (left to right) such that all of the events fell within the two right quadrants. the vertical bar (up and down) for fl-1 was established based on the negative control population (i.e., autofluorescence). relative fluorescence intensity values for fl-1 delineate positive events (upper right quadrant ur) and negative events (lower right quadrant lr). the right panel of the top row shows fsc versus fl-1 for an untreated sample in the absence of e. coli/gfp, the negative control population. note that the vertical bar was placed above the majority of events. the left panel of the bottom row shows an untreated sample in the presence of e. coli/gfp, while the right panel of the bottom row shows an h202-treated sample in the presence of e. coli/gfp. note the shift of the coelomocyte population from the lr (negative events) to the ur (positive events) quadrants between these two dot plots as fluorescence intensity increases due to phagocytic uptake of e. coli/gfp when pretreated with h202. in this example percent positive events in ur increases from 7.54 to 13.23 % when coelomocytes were pretreated with h202. effects of exogenous h202 on phagocytosis having established the data analysis protocol, we studied the effect of h202 at concentrations ranging from 0.26 8.4 mm on the phagocytosis of e. coli/gfp by earthworm coelomocytes. percent specific phagocytosis of e. coli/gfp was determined by subtracting the percent positive events (ur) of negative controls (absence of e. coli/gfp) in fl-1 from each of the experimental samples (presence of e. coli/gfp with or without h202). the average of duplicates of controls (0 mm h202) versus h202treated samples were plotted and statistical significance was determined using the student’s t test. figure 2 displays the results obtained for   94 a b c d e fig. 3 representative flow cytometry profile of annexin v-fitc/pi assay using ew-a7 as an example for data collection. coelomocytes were pretreated with 0 (spontaneous apoptosis) (a), 8.4 (b), 16.9 (c), 33.8 (d) and 67.6 (e) mm h202. left hand column: fsc (abscissa) versus ssc (ordinate) of total, ungated coelomocytes population. region 1 (r1) depicts the amoebocytes population (hyaline and granular amoebocytes). middle column: fsc (abscissa) versus fl-2 (pi) (ordinate) of r1 gated amoebocytes (excluding eleocytes). region 6 (r6) depicts pinegative (fl-2 negative), viable amoebocyte population. right hand column: fl-1 (abscissa) versus cell number (ordinate) of amoebocytes gated on r1 and r6 (i.e. only viable amoebocytes that have not taken up pi). region 7 (r7) corresponds to annexin v negative amoebocytes while region 8 (r8) corresponds to annexin v positive (early apoptotic) amoebocytes. fsc = forward scatter; ssc = side scatter; fl-1 = relative fluorescence intensity of fitc, fl-2 = relative fluorescence intensity of pi.   95 table 1 percent early apoptotic cells binding annexin v-fitc in untreated and h202-treated samples. only cells residing in r1 and r6 were gated for annexin v-fitc analysis. r1 included hyaline and granular amoebocytes (not eleocytes) and r6 included pi-negative cells. background autofluorescence of untreated samples not receiving annexin v-fitc or pi was subtracted from all values for each indicated earthworm sample before averaging triplicate data and performing statistical analyses. results indicate data obtained from two assays performed by two independent researchers. percent positive values for annexin v-fitc (± sd) are shown for spontaneous apoptosis (0 mm h202) and two-fold serial dilutions from 67.6 to 8.4 mm h202. statistically significant values above spontaneous apoptosis levels are indicated as: * = p ≤ 0.05; ** = p ≤ 0.005; *** = p ≤ 0.0005 as determined by student’s t test. earthworm sample spontaneous apoptosis 0 mm 8.4 mm 16.9 mm 33.8 mm 67.6 mm ew-a1 46.23 (±2.42) 31.75 (±1.33) 42.27 (±0.95) 62.81 (±2.39)*** 82.86 (±1.48)*** ew-a2 54.68 (±2.07) 52.82 (±0.74) 64.91 (±2.17)** 66.70 (±1.10)** 81.72 (±1.40)*** ew-a3 31.76 (±1.67) 31.88 (±0.49) 39.51 (±1.13)** 49.99 (±1.26)*** 62.03 (±1.45)*** a ss ay 1 ew-a4 47.01 (±0.57) 36.99 (±1.22) 44.93 (±1.00) 49.78 (±5.04) 63.24 (±1.76)** ew-a5 63.11 (±4.80) 50.40 (±0.96) 58.90 (±1.22) 79.79 (±2.35)* 85.99 (±0.67)* ew-a6 72.68 (±1.68) 69.51 (±0.63) 70.75 (±1.30) 71.56 (±2.65) 80.43 (±2.40)** ew-a7 31.95 (±0.51) 59.35 (±1.48)*** 62.06 (±1.45)*** 67.56 (±2.45)** 75.95 (±2.04)*** a ss ay 2 ew-a8 38.75 (±2.25) 27.83 (±0.08) 27.25 (±1.23) 37.60 (±0.59) 46.88 (±1.12)* coelomocytes from 12 earthworms pretreated in vitro with h202 prior to phagocytosis. earthworms 1, 2, 3 and 4 (ew-p1-p4) were pretreated in the range of 1.1 8.4 mm h202, while ew-p5-p12 were pretreated in the range of 0.26 8.4 mm h202. eight of the 12 earthworms tested (67 %) exhibited statistically significant enhancement of phagocytosis to at least one of the concentrations employed. at doses above 8.4 mm, inhibitory effects on phagocytosis were observed (data not shown). flow cytometry detection of early apoptosis for the next two experiments, which were aimed at investigating the effects of h202 on ps translocation and caspase activation in amoebocytes of e. hortensis, it was important to be able to discriminate between necrotic/late apoptotic amoebocytes and amoebocytes undergoing early apoptosis to ensure that analyses were restricted to amoebocytes with intact plasma membranes. to do this, we utilized pi in addition to the fluoresceintagged reporters of ps translocation and caspase activation. pi exhibits a sufficiently large stokes shift compared to the fluorescein permitting simultaneous detection of fluorescein-labelled moieties and nuclear dna providing the appropriate optical filters and compensation adjustments for spectral overlap are utilized. in our case, we used a facscalibur flow cytometer which employes fl-1 for fluorescein detection and fl-2 for pi detection. pi is membrane impermeant and is thus excluded from viable cells, making this fluorescent counterstain an ideal marker for identifying necrotic/late apoptotic cells in a population when used together with a second fluorescent dye which has minimal spectral emission overlap. figure 3a illustrates the gating strategy used for the analysis of both the ps translocation and caspase activation experiments. first the coelomocytes were analyzed for fsc versus ssc (fig. 3a left panel) and region (r1) was set around the amoebocytes. next a dot plot of fsc versus fl2 gated on the r1 population was created and quadrants were established according to procedure described in fig. 1 (fig. 3a middle panel) positioning the vertical bar at a location that clearly delineated live from dead cells based on the saponin-treated, pi-stained single-positive control employed for compensation purposes. the quadrants corresponded to regions 2-5 (r2-r5). next another region (r6) was drawn around the pinegative cell population, serving to identify viable cells (this would include non-apoptotic, non-necrotic and early-apoptotic cells whose membranes were still intact). finally, a single parameter histogram measuring fluorescein (fl-1) was generated and gated on r1 (amoebocytes) and r6 (pi-negative cells). therefore, only events that satisfied both prerequisites of belonging to the amoebocytes pool (hyaline and granular) as well as being viable were quantified in the r1/r6 gated fl-1 histogram (fig. 3a right panel). this strategy permitted the exclusion of necrotic/late apoptotic cells from the final analysis.   96 table 2 relative fluorescence intensity (rfi) of early apoptotic, annexin v-fitc-positive cells in untreated and h202-treated samples. only cells residing in r1 and r6 were gated for annexin v analysis. r1 included hyaline and granular amoebocytes (not eleocytes) and r6 included pi-negative cells. triplicate data was averaged and subjected to statistical analyses. results indicate data obtained from two assays performed by two independent researchers. rfi values (geometric mean) above background detected by fl-1 (± sd) are shown for spontaneous apoptosis (0 mm h202) and two-fold serial dilutions from 67.6 to 8.4 mm h202. statistically significant values exceeding spontaneous apoptosis levels are indicated as: * = p ≤ 0.05; ** = p ≤ 0.005; *** = p ≤ 0.0005 as determined by student’s t test. earthworm sample spontaneous apoptosis 0 mm 8.4 mm 16.9 mm 33.8 mm 67.6 mm ew-a1 14.75 (±0.59) 10.65 (±0.42) 11.22 (±0.08) 14.03 (±0.66) 19.69 (±0.94)** ew-a2 11.99 (±0.16) 12.56 (±0.44) 12.58 (±0.02)* 13.91 (±0.21)*** 21.66 (±1.50)** ew-a3 15.12 (±0.57) 10.72 (±0.48) 11.70 (±0.47) 13.53 (±0.57) 14.90 (±0.41) a ss ay 1 ew-a4 12.79 (±0.12) 10.19 (±0.06) 11.20 (±0.12) 13.33 (±0.53) 18.02 (±0.39)** ew-a5 29.88 (±1.92) 17.81 (±1.46) 18.40 (±0.19) 30.45 (±3.50) 81.52 (±5.72)** ew-a6 25.55 (±2.74) 19.13 (±0.40) 22.40 (±0.70) 28.61 (±0.28) 45.16 (±1.65)** ew-a7 12.23 (±1.61) 14.46 (±0.24) 23.22 (±0.40)** 26.48 (±0.82)*** 31.84 (±0.65)*** a ss ay 2 ew-a8 10.31 (±0.18) 10.35 (±0.35) 11.72 (±0.75)* 13.58 (±0.56)** 19.37 (±2.04)* ps translocation: annexin v-fitc binding cells undergoing early apoptosis can be easily identified using annexin v, an anticoagulant protein that exhibits a high degree of specificity for ps, and when conjugated to a reporter molecule, such as fluorescein isothiocyanate (fitc), can be used an indicator of early apoptosis. ps is a phospholipid that is normally retained on the inner leaflet of the plasma membrane, however, in cells undergoing apoptosis, it is translocated to the outer leaflet and is exposed on the surface of the cell. once exposed on the surface, it is accessible to annexin v binding. early apoptotic cells maintain the integrity of their plasma membrane and are thus impermeable to pi. we conducted experiments to determine the effect of h202 on ps translocation. we treated coelomocytes with h202 in the range of 0 to 67.6 mm and after washing incubated them annexin vfitc and pi. figure 3 illustrates a representative set of flow cytometry obtained for earthworm 7 (ewa7). as the concentration of h202 increased, the degree of ps translocation also increased (right panels). the left hand panels illustrate that h202 affected the morphology of the coelomocytes; as h202 concentration increased, granularity (ssc) decreased (note tightening of ssc signal on ordinate). the middle panels shows that h202 induced cell necrosis/late apoptosis at 33.8 mm and above. in two separate assays performed by two independent researchers on separate days, eight earthworms were analyzed. tables 1-3 report the overall findings of these experiments. table 1 shows % annexin v-fitc binding of viable amoebocytes; 100 % (8/8), 63 % (5/8), 38 % (3/8) and 12.5 % (1/8) exhibited statistically significant ps translocation when exposed to 67.6, 33.8, 16.9 and 8.4 mm h202, respectively. note that untreated amoebocytes exhibit signs of spontaneous apoptosis, perhaps attributed to a stimulus delivered during isolation and/or in vitro culturing. table 2 illustrates the increase in relative fluorescence intensity (rfi) of annexin v-fitc compared to baseline controls; 88 % (7/8), 38 % (3/8), and 38 % (3/8) revealed statistically significant increases in annexin v-fitc levels on the cell surface of viable amoebocytes following exposure to 67.6, 33.8 and 16.9 mm h202, respectively. there was no significant increase in rfi in any of the earthworms when exposed to 8.4 mm h202. table 3 reveals that necrosis/late apoptosis correlates with increasing concentration of h202 but only at the highest concentrations used; 100 % (8/8) and 63 % (5/8) of samples had statistically significant increases of necrosis/cell death compared to controls when exposed to 67.6 and 33.8 mm h202, respectively. no significant difference in necrosis/cell death was observed at concentrations of 16.9 and 8.4 mm h202. caspase activation in coelomocytes to detect the events associated with signal transduction in cells undergoing apoptosis, we measured the activation of caspases in coelomocytes exposed to h202 by using a reporter reagent called fam-vad-fmk flica where 1) fam is the carboxyfluorescein group which fluoresces and is detectable in the fl-1 detector of the flow cyometer, 2) vad is the three amino acid (valine, alanine, aspartic acid) generic probe that binds to most caspases (including caspases -1, -3, -4, -5, -6, -7, -8, and -9) and 3) fmk is the fluoromethyl ketone moiety which anchors the fam-vad-fmk reagent   97 table 3 percent late apoptotic/necrotic cells in untreated and h202-treated samples. only cells residing in r1 were gated for pi analysis. r1 included hyaline and granular amoebocytes (not eleocytes). triplicate data was averaged and subjected to statistical analyses. results indicate data obtained from two assays performed by two independent researchers. percent pi-positive cells detected by fl-2 (± sd) are shown for spontaneous apoptosis (0 mm h202) and two-fold serial dilutions from 67.6 to 8.4 mm h202. statistically significant pi values exceeding spontaneous apoptosis levels are indicated as: * = p ≤ 0.05; ** = p ≤ 0.005; *** = p ≤ 0.0005 as determined by student’s t test. earthworm sample spontaneous apoptosis 0 mm 8.4 mm 16.9 mm 33.8 mm 67.6 mm ew-a1 24.55 (±1.19) 18.47 (±2.07) 25.93 (±1.00) 39.12 (±1.57)*** 58.95 (±1.07)*** ew-a2 14.03 (±2.71) 10.79 (±0.39) 11.43 (±0.40) 12.48 (±1.13) 49.93 (±1.80)*** ew-a3 25.03 (±2.09) 18.79 (±0.63) 19.70 (±0.72) 22.85 (±0.20) 44.87 (±0.44)** a ss ay 1 ew-a4 24.85 (±0.14) 19.43 (±1.80) 20.90 (±1.01) 29.75 (±10.7) 45.75 (±2.89)** ew-a5 50.61 (±1.86) 34.53 (±1.39) 39.47 (±2.29) 54.49 (±2.26)* 72.34 (±1.07)*** ew-a6 35.93 (±2.79) 34.84 (±1.62) 35.96 (±2.17) 43.69 (±2.22)* 52.78 (±3.94)** ew-a7 15.48 (±2.05) 11.34 (±1.15) 16.11 (±1.15) 21.15 (±1.47)* 45.75 (±1.71)*** a ss ay 2 ew-a8 20.17 (±3.34) 20.76 (±0.31) 23.69 (±4.15) 29.33 (±1.34)* 57.27 (±1.23)*** to activated caspases in the cell via a covalent cysteine linkage. unbound fam-vad-fmk reagent is washed from the cell while bound reagent is anchored in the cell and fluoresces during flow cytometry, hence enabling the detection of activated caspases in cells undergoing the initial stages of apoptosis. we tested six earthworms in two separate assays. figure 4 shows the results of these assays where coelomocytes were pretreated with 0 (a), 16.9 (b), 33.8 (c) and 67.6 (d) mm h202. the same strategy for excluding necrotic/late apoptotic cells described in fig. 3 was used in these experiments, i.e., relevant cells used for analysis were obtained by gating r1 (amoebocytes) and r6 (pi-negative) positive coelomocytes (fig. 4). a marked increase in necrosis/cell death was observed between 33.8 and 67.6 mm h202 (fig. 4cd). again we observed changes in cellular morphology (as detected by fsc versus ssc profiles) as the concentration of h202 increased, particularly at 67.6 mm (fig. 4, middle panels). statistically significant increases in caspase activation were detected in 100 % (3/3) and 67 % (4/6) of samples exposed to 67.6 and 33.8 mm h202, respectively (fig. 5). morphological changes in coelomocytes ew f4-f6 were subjected to further analysis to determine if morphological changes associated with a decrease in cell volume, another hallmark of cells undergoing apoptosis, was occurring in response to h202 treatment. treated and untreated coelomocytes incubated with fam-vad-fmk flica and pi (as described in caspase assay above) were gated to select for pi-negative (viable and early apoptotic), large coelomocytes. gated cells were analyzed in a single-parameter histogram measuring forward light scatter (fsc). duplicate samples were averaged and p values were determined by student’s t test. figure 6 shows a decrease in cell volume; in all cases except one (16.9 mm h202 for ewf5), decreases in cell volume of h202-treated samples were statistically significant (p < 0.05) compared to untreated samples. the difference in cell volume was much more pronounced in the large coelomocytes (fig. 6) than in the small coelomocytes (data not shown). these results show that in earthworms undergoing biochemical changes (caspase activation), morphological alterations (cell volume) are also occurring. dna fragmentation another hallmark of apoptosis is the fragmentation of dna which occurs when an endonuclease (caspase-activated deoxyribonuclease, cad) is activated as a consequence of programmed cell death induction. genomic dna is cleaved at sites between nucleosomes at ~ 200 base pairs (bp) intervals (nagata, 2000). in this study, total coelomocytes (including amoebocytes and eleocytes) were pretreated with h202 prior to the purification and electrophoresis of dna. in the first assay (fig. 7, right hand gel), h202 was used at 0 and 33.8 mm. dna fragmentation consistent with apoptosis was   98 fig. 4 representative flow cytometry profile of caspase (flica/pi) assay using ew-f6 as an example for data collection. coelomocytes were pretreated with 0 (spontaneous apoptosis) (a), 16.9 (b), 33.8 (c), and 67.6 (d) mm h202. left hand column: fsc (abscissa) versus ssc (ordinate) of total, ungated coelomocyte population. region 1 (r1) depicts the amoebocyte population (hyaline and granular amoebocytes). middle column: fsc (abscissa) versus fl-2 (pi) (ordinate) of r1 gated amoebocytes (excluding eleocytes). region 6 (r6) depicts pi-negative (fl-2 negative), viable amoebocyte population. right hand column: fl-1 (abscissa) versus cell number (ordinate) of amoebocytes gated on r1 and r6 (i.e. only viable amoebocytes that have not taken up pi). region 7 (r7) corresponds to fluorescein negative amoebocytes while region 8 (r8) corresponds to fluorescein positive (early apoptotic) amoebocytes. fsc = forward scatter; ssc = side scatter; fl-1 = relative fluorescence intensity of fluorescein, fl-2 = relative fluorescence intensity of pi.   99 fig. 5 caspase activation in untreated and h202-treated coelomocytes. top row: percent caspase positive amoebocytes of ew-f1-f3 treated with 0 mm (spontaneous apoptosis) or 33.8 mm h202. bottom row: percent caspase positive amoebocytes of ew-f4-f6 treated with 0, 16.9, 33.8 and 67.6 mm h202. asterisks denote p ≤ 0.05 compared to control. observed compared to the untreated control. in a second assay (fig. 7, two left hand gels) h202 was used at 0, 33.8, 16.9 and 8.4 mm. laddering was observed only when coelomocytes were exposed to 33.8 and 16.9 mm h202. these results indicate that initiation of the stereotypical internucleosomal degradation of genomic dna characteristic of apoptosis is occurring in earthworm coelomocytes in response to oxidative stress. tunel assay a terminal dutp nick-end labeling (tunel) method was used to enzymatically verify dna fragmentation in h2o2-treated coelomocytes. this method involved labeling the 3’-hydroxyl dna ends generated during dna fragmentation using a terminal deoxynucleotidyl transferase (tdt) and biotin-labeled utp. labeled dna was detected by incubating with fitc-conjugated streptavidin and subjecting the samples to flow cytometric analysis. amoebocytes identified on forward versus side scatter dot plots were gated for fl-1 (fitc) analysis. four earthworms were treated with 33.8 mm h2o2, however, only three exhibited any demonstrable difference in labeling compared to untreated controls(ew t1, ew t2 & ew t4); two of these (ew t1 & ew t2) were statistically significant (p < 0.006). figure 8 shows the results from the three responding earthworms. note the increase in relative fluorescence intensity compared to untreated controls. a nuclease control was included for verification purposes (data not shown). these results complement the agarose gel electrophoresis results and illustrate that flow cytometry combined with the tunel assay is a useful and rapid method to detect dna fragmentation in earthworm coelomocytes. h202 production during phagocytosis our final investigation was aimed at determining whether hyaline amoebocytes of e. hortensis undergo a respiratory burst and produce endogenous h202 upon phagocytosis of the soil bacteria p. stutzeri (gram negative) and b. megaterium (gram positive). we used dhr 123, a fluorogenic molecular probe (excitation 505 nm; emission 534 nm) to detect the generation of h202. dhr 123 is oxidized by h202 and is converted consequently to it fluorescent derivative rhodamine 123, which can be measured intracellularly by the flow cytometer. flow cytometry analysis involved: 1) setting a region around the hyaline amoebocytes (as described in fig. 1, top row, left hand dot plot); 2) generating a fl-1, single-parameter histogram gated on this region; and 3) establishing a boundary (markers) to separate negative events from positive events (as described in fig. 4, right hand histograms). the boundary was established based   100 fig. 6 cell volume changes in untreated and h202-treated coelomocytes. ew f4-f6 samples from the caspase assay were gated on pi-negative events falling in the region corresponding to the large coelomocyte population. h202 concentration (abscissa) versus geometric mean of forward side scatter (ordinate) are plotted for ew f4 (diamonds), ew f5 (squares) and ew f6 (triangles). with the exception of ew f5 at 16.9 mm, p values < 0.05 compared to controls (untreated) were obtained. on the negative control sample which was incubated in the absence of dhr 123 (but with an equivalent amount of dmso compared to experimental samples) to control for autofluorescence. any events exceeding the fl-1 boundary marker were considered positive (percent positive hyaline amoebocytes). eight earthworms were extruded and three (ew 2, 7 and 8) were selected for the phagocytosis assay based on the quality of the coelomocytes (i.e., higher proportion of amoebocytes compared to eleocytes and adequate cell numbers). figure 9 shows the results of these three earthworms and illustrates that dhr 123 is oxidized to rhodamine 123 during phagocytosis of p. stutzeri and b. megaterium. statistical significance was determined based on comparison between control (dhr 123 only) and experimental (bacteria plus dhr 123) samples. p. stutzeri generated statistically significant results in 100 % and 67 % of cases at a m.o.i. of 100:1 and 10:1, respectively. b. megaterium generated statistically significant results in 67 % of cases at a m.o.i. of 100:1 and 10:1. a second assay employing p. stutzeri at a m.o.i. of 1000:1 revealed enhanced h2o2 production in 67% of cases (4 out of 6 earthworms) compared to controls (data not shown). discussion this study demonstrates that exogenous h202 (0.26 8.4 mm) caused an increase in phagocytosis by e. hortensis; 67 % of earthworms tested exhibited statistically significant enhancement of percent specific phagocytosis for one or more of the concentrations of h202 used. bejarano et al. (2007), gamaley et al. (1994) and takeda et al. (1998) examined the effects of oxygen free radicals in cultured human neutrophils, murine macrophages and rat ameboid microglia, respectively, and found that exposure to exogenous h202 also caused an increase in phagocytic function. it is interesting to propose that in our model h202 may be acting as a second messenger involved in evoking a significant elevation of phagocytic function. we are interested in determining if calcium mobilization from agonistsensitive intracellular stores or influx across the plasma membrane accompanies h202-enhanced phagocytosis, an effect reported by others (redondo et al., 2004; bejarano et al., 2007). the use of a calcium quelator (e.g., dimethyl bapta) would help to reveal the role, if any, that calcium mobilization plays in phagocytosis in earthworms, and to facilitate a better understanding of the physiological role of oxygen free radicals in calcium homeostasis. we also plan to examine the effect of pre-treating earthworm amoebocytes with catalase, an enzyme facilitating h202 decomposition into water and oxygen, prior to the addition of exogenous h202 in phagocytosis assays. in parallel with the invertebrate studies of blanco et al. (2005) which examined the effects of h202 on coelomocytes of themiste petricola (the sipunculan marine worm), we also revealed that h202 induced apoptosis-like cell death in coelomocytes of e. hortensis. using pi, we were able to discriminate between viable coelomocytes (nonapoptotic and early apoptotic) and nonviable coelomocytes (necrotic/late apoptotic); pi-positive cells were excluded from flow cytometric analyses due to dna labeling. note that eleocytes were not included in   101 fig. 7 dna fragmentation of h202-treated coelomocytes. these three gels represent two independent dna fragmentation assays where earthworm coelomocytes were treated with h202 for 6 h and dna was isolated and electrophoresed in 1.5 % agarose gels in tbe buffer. lanes m (molecular weight markers in bp are indicated for the nine distinct bands of the 100 bp ladder), lanes c (untreated cells, 0 mm h202), lanes 1 (33.8 mm h202), lane 2 (16.9 mm h202), and lane 3 (8.4 mm h202). the two left-hand gels are from the same assay and same population of coelomocytes. the right-hand gel is from a separate assay with a different population of coelomocytes. these analyses owing to relatively high level of autofluorescence exhibited by this subpopulation. we observed ps translocation and caspase activation when amoebocytes were treated with h202 in the range of 8.4 67.6 mm and 16.9 67.6 mm, respectively, under the conditions specified. it is interesting to note that relatively high concentrations of h202 were required to induce apoptosis, suggestive of high tosc in earthworm coelomocytes. we also observed dose-dependent changes in fsc when amoebocytes were pretreated with h202, a phenomenon consistent with apoptotic volume decrease (avd) (maeno et al., 2000; okada and maeno, 2001) when cells undergoing apoptosis experience cell shrinkage and subsequent cell fragmentation (apoptotic bodies). exposure of ps on the surface of infected coelomocytes may be one mechanism by which phagocytes recognize and remove cellular reservoirs of pathogens during innate immune responses. fadok et al. (1992), for example, showed that murine macrophages rapidly recognize and remove apoptotic lymphocytes following exposure of ps on the outer leaflet of the plasma membrane of apoptotic cells, thereby prevent potential tissue damage from lysis of these cells in vivo. caspases are highly conserved and have been identified in invertebrates; for example the ced-3 protein of the nematode caehorhabditis elegans was the first to be characterized. the gene encoding ced-3 is known to exhibit homology to murine and human caspase-1 (formerly known as interleukin-1 beta converting enzyme) (yuan et al., 1993). several caspases known to participate in apoptosis have also been identified in drosophila melanogaster fig. 8 tunel analysis of h202-treated coelomocytes. coelomocytes treated with 0 mm h2o2 (filled graph line) (control) or 33.8 mm h2o2 (unfilled graph line) were chemically fixed, and the fragmented dna ends were labeled with biotinconjugated utp and stained with streptavidin fitc. the gated amoebocyte population was analyzed for fl-1 (fitc) fluorescence intensity (abscissa) versus cell number (ordinate). ew t1 and ew t2 exhibited statistical significance (p < 0.006) between untreated and treated amoebocytes.   102 fig. 9 endogenous h202 is produced during phagocytosis. the percent hyaline amoebocytes oxidizing dhr 123 to rhodamine 123 for each treatment is shown (percent positive hyaline amebocyes). phagocytosis assays using p. stutzeri and b. megaterium at m.o.i. of 100:1 and 10: 1 compared to control (dhr 123) shows statistically significant (p ≤ 0.05) increases in rhodamine 123 production (*) for ew 2, 7 and 8. (reviewed in boyce et al., 2004). in addition, the crystal structure of sf-caspase-1 from spodoptera frugiperda has been resolved and its overall fold found to be exceedingly similar to active caspases from humans (forsyth et al., 2004). using a fluorescent inhibitor of poly-caspases, our results show that h202 induces caspase activation in earthworm amoebocytes. it would be worthwhile to try to dissect the caspase signaling pathway in e. hortensis using inhibitors known to target caspases with specificity for particular caspase subfamilies (matsura et al., 1999). we also evaluated oligonucleosomal dna fragmentation using agarose gel electrophoresis to resolve dna fragments. a dose-dependent laddering pattern characteristic of apoptotic cells was observed when coelomocytes were treated with 16.9 and 33.8 mm, but not 8.4 mm h202. perhaps a longer incubation period would have resulted in dna laddering at the lower concentration. the presence of discrete ~200 bp dna fragments is consistent with apoptosis and not necrosis as the latter would result in random dna fragmentation causing smears on an agarose gel rather than discrete repetitive oligonucleosomal fragments generated by endonuclease cleavage between nucleosomes (walker et al., 1988). in addition, we conducted tunel assays in conjunction with flow cytometry which provided enzymatic verification of dna fragmentation in h2o2-treated coelomocytes, and a more rapid procedure for detecting dna fragmentation in apoptotic cells. we are also interested in examining mitochondrial transmembrane potential using a mitochondriaspecific probe such as jc-1 which incorporates into mitochondria and undergoes a shift in fluorescence emission spectra during membrane depolarization (cossarizza et al., 1995). finally, our results show that endogenous h202 is produced by hyaline amoebocytes during phagocytosis of gram positive (b. megaterium) and gram negative (p. stutzeri) bacteria at m.o.i. ranging from 10:1 to 1000:1. 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et al. hydrogen peroxide enhances phagocytic activity of ameboid microglia. neurosci. lett. 240: 5-8, 1998. thannickal fj, fanburg bl. reactive oxygen species in cell signaling. am. j. phsiol. lung cell. mol. physiol. 279: l1005-l1028, 2000. tyagi n, sedoris kc, steed m, ovechkin av, moshal ks, tyagi sc. mechanisms of homocysteine-induced oxidative stress. am. j. physiol. heart circ. physiol. 289: h2649h2656, 2005. valko m, rhodes, cj, moncol j, izakovic m, mazur m. free radicals, metals and antioxidants in oxidative stress-induced cancer. chem. biol. interact. 160: 1-40, 2006. walker ni, harmon bv, gobé gc, kerr jf. patterns of cell death. methods achiev. exp. pathol. 13: 18-54, 1988. wang g. nadph oxidase and reactive oxygen species as signaling molecules in carcinogenesis. front. med. china 3: 1-7, 2009. yamaji y, nakazato y, oshima n, hayashi m, saruta t. oxidative stress induced by iron released from transferrin in low ph peritoneal dialysis solution. nephrol. dial. transplant. 19: 2592-2597, 2004. yuan j, shaham, s, ledoux s, ellis hm, horvitz hr. the c. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme. cell 75: 641-652, 1993.   106 http://www.ncbi.nlm.nih.gov/pubmed?term=%22gob%c3%a9%20gc%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22kerr%20jf%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract isj 8: 56-58, 2011 isj 8: 56-58, 2011 issn 1824-307x visions and perspectives stress-based modulation of the immune response in molluscan hemocytes: a tworeceptor model r barcia, ji ramos-martínez department of biochemistry and molecular biology, school of veterinary, university of santiago de compostela, campus de lugo, lugo, spain accepted march 23, 2011 abstract in molluscs, hemocytes perform the molecular mechanisms related to immunity. these cells have the ability to respond to the different varieties of stress by modulating their responses. the stressors may be bacterial toxins, cytokines or growth factors, and even physical agents such as changes in temperature or oxygen partial pressure. in the first place, hemocytes synthesise catecholamines, which, in turn, modify the immune response in terms of phagocytosis or nitric oxide synthesis. according to studies on the hemocytes of the mussel mytilus galloprovincialis, we propose a model for a sequential action where the il-2 receptor and its wide agonist specificity play an important role. also, α and β-adrenergic receptors suggest the functioning of a return-to-hemocyte mechanism. the model is proposed taking into account the possible relationship between the pathways mediated by campactivated protein kinase and protein kinase c in hemocytes. key words: molluscs; mytilus galloprovincialis; stress; endocrinology; immunology   introduction in molluscs the immune response is performed by specialized cells termed hemocytes. because molluscs lack an adaptative immune system, the response against toxic agents of diverse origin is performed through an ancestral process that was preserved along the evolution until vertebrates and ___________________________________________________________________________ corresponding author: juan ignacio ramos-martínez department of biochemistry and molecular biology school of veterinary university of santiago de compostela campus de lugo, 2702 lugo, spain e-mail: juanignacio.ramos@usc.es list of abbreviations: acth: adrenocorticotropic hormone; crh: corticotrophin-releasing hormone; erk: extracellular signal-regulated kinase; ifn: interferon; il-2: interleukin-2; (il-2r: il-2 receptor; jnk: c-jun n-terminal kinase; lps: lipopolysaccharide; mapk: mitogen-activated protein kinase; no: nitric oxide; pdgf: plateletderived growth factor; pka: camp-activated protein kinase; pkc: protein kinase c; ros: oxygen radicals; stat: signal transducers and activators of transcription protein; tnf: tumor necrosis factor. is known as innate immunity (medzitov and janeway, 1997). up to date, the system has been admitted to involve a rigid response; however, new data regarding the structures of the recognition elements suggest a certain selective ability, which allows guessing the elaboration of a possible molecular memory (brehélin and roch, 2008; ottaviani, 2011). the immune response of molluscan hemocytes is modulated by stress in a way apparently similar to that described in vertebrates as “hypothalamushypophysis-adrenal axis” (hpa axis), constituting a proto-response to stress centered on these cells, which can be qualified as an “immune-mobile brain” (ottaviani et al. 1993). the receptor-based modulation of molluscan immunity when a foreign element enters the organism, the immune response increases the phagocytic activity, among other actions. phagocytosis develops in several stages, and one of them is the synthesis of ros and no, leading to the generation of peroxynitrite. when molluscan hemocytes are incubated with agonists so different in structure as growth factors (pdgf, tnf), peptidic hormones (acth, crh), interleukins (il-2), or even bacterial toxins (lps), catecholamine and no synthesis is induced (ottaviani and franceschi, 1996). the   56 mailto:juanignacio.ramos@usc.es http://en.wikipedia.org/wiki/corticotropin-releasing_hormone fig. 1 hypothesis on the stress-based molecular mechanisms modulating the immune response in hemocytes of mytilus galloprovincialis lmk. nitric oxide synthase (nos). other abbreviations, in the text. the red lines suggest non demonstrated pathways. effects are detectable, both in freshly extracted hemocytes (therefore, stressed because of extraction procedure) and in mytilus hemocytes stabilized after three days in culture (cao et al., 2004, 2007a). depending on the agonist assayed, differences in the expression of the subunits forming the il-2 receptor were detected, mainly of the α subunit (cao et al., 2004, 2007a). this agrees with the hypothesis about the presence of a unique ancestral receptor with wide specificity (ottaviani and franceschi, 1996). on the other hand, the same agonists also provoke remarkable increases in catecholamine production, which evidencing a stress on these cells (cao et al., 2004, 2007a, b). the catecholamines that the hemocytes or other cells secrete might take a return-to-hemocyte pathway, or else, act directly as suggested by the presence of α and β adrenergic receptors in crassostrea gigas (lacoste et al., 2001). the signals generated by catecholamines are internalized through pka and pkc, as observed in crassostrea hemocytes (lacoste et al., 2001, 2002). also, the signals of lps and other bacterial factors involve the activation of stress kinases, such as p38-mapk, jnk o erk (canesi et al., 2002, betti et al., 2006, ciacci et al., 2010). at the same time, pka and pkc mediate no synthesis (barcia and ramos-martínez, 2008, gonzalez-riopedre et al., 2009). a common intermediation of some protein kinases related to stress (mapk) in phagocytosis and in synthesis of thermal shock proteins has been reported in mytilus hemocytes incubated with toxins and bacteria (canesi et al., 2002; gaitanaki et al., 2004; kefaloyianni et al., 2005), and mapk is known to mediate processes activated through g protein receptors. therefore, there it seems to be a network between the different protein elements involved in the internalization of the stress processes and those evidencing infective or inflammatory actions. canesi and colleagues also reported that the incubation of mytilus hemocytes with the macrophage activator ifn-γ induced stat phosphorylation, which proves the convergence towards mapk-like phosphorylation mediators (canesi et al., 2003). this result leaves open the possibility of the existence in hemocytes of two types of receptor for immune response activating agonists. one of them with wide specificity, similarly to il-2r of vertebrates, and another one similar to the tyrosine kinase receptors, which would internalize stat activating signals. in addition, at least other two adrenergic receptors should operate the modulation of the catecholamine-generated response. the differences in the timing of signal internalization account for the operability of the different types of receptors and suggests a possible consecutive connection of their actions. in this sense, il-2 and lps increase catecholamine synthesis quickly (30 60 min) and then, a return to basal values is detected with longer incubation times (cao et al., 2004, 2007a, b; gonzalezriopedre et al., 2009). on the contrary, no synthesis requires a more prolonged incubation with the agonists (novas et al., 2004). the results obtained suggest that il-2 and lps make pkc activity to increase rapidly (barcia and ramosmartínez, 2008; gonzalez-riopedre et al., 2009). catecholamine secretion and later binding to   57 adrenergic receptors may trigger late no synthesis (24 h). this hypothesis would justify the early pka and pkc increase induced by il-2 or lps (barcia and ramos-martínez. 2008; gonzalez-riopedre et al., 2009). the reduction of no synthesis detected at 30 min of cell incubation with il-2 or lps would confirm an early kinase action (novas et al., 2004). this ensemble of actions can be summarized in the hypothesis shown in figure 1. the timing of successive actions of cytokines must play an important role in the activation of the different mapks. in this sense, and taking into account the implication of mapks in the synthesis of thermal shock proteins (cellura et al., 2006; gourgou et al., 2010), the biphasic expression of the hsp70 when incubating the cells with il-2 or lps confirms the hypothesis unfolded in the present text (gonzalez-riopedre et al., 2007) and opens new perspectives in the study of these cells and their function in cell signalling in molluscan neuroimmunology. references barcia r, ramos-martínez ji. effects of interleukin2 on nitric oxide production in molluscan innate immunity. inv. surv. j. 5: 43-49, 2008. betti m, ciacci c, lorusso lc, canonico b, falcioni t, gallo g, et al. effects of tumour necrosis factor alpha (tnf alpha) on mytilus haemocytes: role of stress-activated mitogenactivated protein kinases (mapks). biol. cell 98: 233-244, 2006. brehélin m, roch p. specificity, learning and memory in the innate immune response. inv. surv. j. 5: 103-109, 2008. canesi l, betti m, ciacci c, scarpato a, citterio b, pruzzo c, et al. signalling pathways involved in the physiological response of mussel hemocytes to bacterial challenge: the role of stress-activated p38 map kinases. dev. comp. immunol. 26: 325-334, 2002. canesi, l., betti, m, ciacci, c, citterio b, pruzzo c, gallo, g. tyrosine kinase-mediated cell signalling in the activation of mytilus hemocytes: possible role of stat-like proteins. biol. cell 95: 603-613, 2003. cao a, novas a, ramos-martínez ji, barcia r. seasonal variations in haemocytes response in the mussel mytilus galloprovincialis lmk. aquaculture 263: 310-319, 2007a. cao a, ramos-martínez ji, barcia r. in vitro effects of lps, il-2, pdgf and crf on haemocytes of mytilus galloprovincialis lmk. fish shellfish immunol. 16: 215-225, 2004. cao a, ramos-martínez ji, barcia r. in haemocytes from mytilus galloprovincialis lmk., treatment with corticotrophin of growth factors conditions catecholamine release. int. immunopharmacol. 7: 1395-1402, 2007b. cellura c, toubiana m, parrinello n, roch p. hsp70 gene expression in mytilus galoprovincialis hemocytes is triggered by moderate heat shock and vibrio angilarum, but not by v. splendidus or micrococcus lysodeikticus. dev. comp. immunol. 30: 984997, 2006. ciacci c, betti m, canonico b, citterio b, roch p, canesi l. specificity on anti-vibrio immune response through p38 mapk and pkc activation in the hemocytes of the mussel mytilus galloprovincialis. j. invertebr. pathol. 105: 49-55, 2010. gaitanaki c, kefaloyianni e, marmari a, beis i. various stressors rapidly activate the p38mapk signaling pathway in mytilus galloprovincialis (lam.) mol. cell biochem. 260: 119-127, 2004. gonzalez-riopedre m, novás a, dobaño e, ramosmartínez ji, barcia r. effect of termal stress on protein expression in the mussel mytilus galloprovincialis lmk. comp. biochem. physiol. 147b: 531-540, 2007. gonzalez-riopedre m, barcia r, ramos-martínez ji. implication of pkc isozymes in the release of biogenic amines by mussel hemocytes: effect of pdgf, il-2 and lps. j. exp. zool. 311a: 727-734, 2009. gougou e, aggeli ik, beis i, gaitanaki c. hyperthermia-induced hsp70 and mt20 transcritional upregulation are mediated by p38mapk and jnks in mytilus galloprovincialis (lamarck); a prosurvival response. j. exp. biol. 213: 347-357, 2010. kefaloyianni e, gourgou e, ferle v, kotsakis, e, gaitanaki c, beis i. acute thermal stress and various heavy metals induce tissue-specific pro or anti-apoptotic evetnts via the p38-mapk signal transduction pathway in mytilus galloprovincialis (lam.). j. exp. biol. 208: 44274436, 2005. lacoste a, de cian mc, cueff a, poulet sa. noradrenaline and alpha-adrenergic signalling induce the hsp70 gene promoter in mollusk immune cells. j. cell sci. 114: 3557-3564, 2001. lacoste a, malham sk, gelèbart f, cueff a, poulet sa. stress-induced immune changes in the oyster crassotrea gigas. dev. comp. immunol. 26: 1-9, 2002. medzhitov r, janeway cajr. innate immunity: the virtues of a nonclonal system of recognition. cell 91: 295-298, 1997. novas a, cao a, barcia r, ramos-martínez ji. nitric oxide release by hemocytes of the mussel mytilus galloprovincialis lmk kwas provoked by interleukin-2 but not by lipopolysaccharide. int. j. biochem. cell biol. 36: 390-394, 2004. ottaviani e. is the distinction between innate and adaptative immunity in invertebrates still as clear-cut as throught?. ital. j. zool., 2011 [in press]. ottaviani e, caselgrandi e, franchini a, franceschi c. crf provokes the release of norepinephrine by hemocytes of viviparus ater (gastropoda, prosobranchia): further evidence in favour of the evolutionary hypothesis of the mobile immune-brain. biochem. biophys. res. commun. 193: 446-452, 1993. ottaviani e, franceschi c. the neurobiology of stress from invertebrates to man. progr. neurobiol. 48: 421-440, 1996.   58 http://www.ncbi.nlm.nih.gov/pubmed/14720463 http://www.ncbi.nlm.nih.gov/pubmed/14720463 http://www.ncbi.nlm.nih.gov/pubmed/14720463 http://www.ncbi.nlm.nih.gov/pubmed/8503933 http://www.ncbi.nlm.nih.gov/pubmed/8503933 http://www.ncbi.nlm.nih.gov/pubmed/8503933 http://www.ncbi.nlm.nih.gov/pubmed/8503933 http://www.ncbi.nlm.nih.gov/pubmed/8503933 isj 5: xx-yy, 2008 isj 5: 54-74, 2008 issn 1824-307x review immunobiology of compound ascidians, with particular reference to botryllus schlosseri: state of art l ballarin department of biology, university of padua, padua, italy accepted may 29, 2008 abstract the phylogenetic position of invertebrate chordates closely related to vertebrates explains the increasing interest towards tunicate immunobiology. most of the tunicates are ascidians which, like all other invertebrates, rely only on innate immunity for their defense. compound ascidians differ from solitary species for the presence of colony specificity, i.e. the ability for intraspecific non-self recognition. the immunobiology of compound ascidians has been particularly studied in botryllus schlosseri, which is an emerging model organism for this kind of studies. in b. schlosseri and related species, immunocytes are represented by phagocytes and cytotoxic morula cells, the former able to ingest foreign cell and particles, the latter representing the effectors of the inflammatory reaction which follows the contact between genetically incompatible colonies. activated phagocytes release lectins with opsonic activity and are involved in the clearance of apoptotic cells during the colonial generational change. morula cells recognize the presence of foreign molecules as well as allogeneic soluble factors diffusing from an alien colony and as a consequence they: i) release cytokines in the medium which have chemotactic activity and activate phagocytes; ii) degranulate and release phenoloxidase which induces necrotic cell death by oxidative stress. a better knowledge of botryllus genome will allow a deeper insight into open problems in immunobiology of compound ascidians. key words: colonial ascidians; botryllus; immunobiology; immunocytes; allorecognition; phagocytosis introduction deuterostomes of the phylum chordata feature: i) the presence of a notochord, permanent or temporary, which prevents shortening of the body when longitudinal muscles contract; ii) a dorsal nerve cord, in the form of a hollow tube, enlarged to some extent at the front end to form a brain; iii) a ventral digestive tract, which expands anteriorly to form a pharynx, provided with gill slits or pharyngeal pouches and a ventral gland secreting iodoproteins (endostyle or thyroid). both the notochord and the neural tube extend to the tail, the muscular postanal part of the body. tunicates and cephalochordates, collectively named protochordates, represent the invertebrate relatives of vertebrata (schubert et al., 2006), the major chordate subphylum with nearly 50,000 species. unlike vertebrates, which radiated on lands, ___________________________________________________________________________ corresponding author: loriano ballarin department of biology, university of padua, via u. bassi 58/b, 35100 padova, italy email: loriano.ballarin@unipd.it freshwater and seawater and are active predators and/or grazers, protochordates are marine filterfeeding animals, most with a sedentary life-style. tunicates (or urochordates) owe their name to the tunic or test, the peculiar tissue which embeds the larval and adult body. although of epidermal origin, it resembles connective tissue in having an amorphous matrix in which tunicine fibres, similar to cellulose in composition, and interspersed ameboid cells are found. the results of a recent phylogenetic study, based on analysis of many nuclear genes, suggest that tunicates are the closest living relatives of vertebrates (delsuc et al., 2006), thus increasing interest towards these organisms. the majority of tunicates are represented by ascidians or sea squirts, sessile animals widespread all over the world with approximately 3,000 species, both solitary and colonial. today, the genome of some solitary ascidian species (ciona intestinalis, ciona savignyi, halocynthia roretzi) has been fully or partially sequenced (dehal et al., 2002; yokobori et al., 2003) which renders these organisms important 54 fig. 1 a: dorsal view of a b. schlosseri colony; b: colonial system showing buds and budlets; c: colony at generational change or take-over; d: schematic representation of the main stages of b. schlosseri colonial blastogenetic cycle. days from the beginning of the cycle are in brackets. a: ampullae; b: buds; bl: budlet; cs: cloacal siphon; os: oral siphon; s: system; t: tunic; tv: tunic vessel; z: zooids; bar = 1 mm. models in studying the molecular control of embryogenesis and differentiation (nishida, 2002a, b; oda-ishii et al., 2005; passamaneck and di gregorio, 2005; satoh and levine, 2005; dufour et al., 2006). immunity in ascidians the evolutionary relationships of tunicates with vertebrates explain the increasing interest in the immunobiology of ascidians. unlike vertebrates and like all invertebrates, tunicates rely only on innate immunity, characterized by neither somatic recombination nor long-term immune memory, low discriminative power, and a limited array of effector responses. nevertheless, precursors of vertebrate immune components have been described in ascidians, e.g. proteins of the lectin pathway of the complement activation system (nonaka et al., 1999; nair et al., 2000; nonaka, 2001; sekine et al., 2001; raftos et al., 2002; pinto et al., 2003), orthologues of the c-type lectin-like receptor cd94 (khalturin et al., 2003; zucchetti et al., 2007) and other genes involved in innate immunity (azumi et al., 2003; shida et al., 2003; oren et al., 2007) the immunobiology of solitary ascidians has been studied in the past, mostly in relation with inflammation, cytotoxicity, encapsulation, phagocytosis and allorecognition (parrinello et al., 1977, 1984, 1993; parrinello and patricolo, 1984; raftos et al., 1987a, b; raftos, 1991; peddie and smith, 1993, 1994; ohtake et al., 1994; cammarata et al., 1995; dan-sohkawa et al., 1995; lipari et al., 1995). this kind of research has been improved by the availability of the genomes of ciona and halocynthia (azumi et al., 2003; shida et al., 2003), which allowed to study the molecular control of immune responses. a review on the immunity of solitary ascidians is presented elsewhere in this journal. although less well studied at the molecular level, compound ascidians have some advantages with respect to solitary species. for instance, several developmental pathways (embryogenesis, blastogenesis, and regeneration) leading to adult, filter-feeding zooids may be compared in the same 55 organism and at various levels (morphological, biochemical, molecular). immunity in compound ascidians growing interest in the defense reactions of compound ascidians has been stimulated by the presence of colony specificity, i.e. the ability for intraspecific non-self recognition (allorecognition), and consequent attempts to understand the biological basis of the phenomenon, which frequently results in an inflammatory response (see below). most research has been carried out in botryllid ascidians, mainly in the cosmopolitan species botryllus schlosseri, which is emerging as a model organism for studying immunobiology (manni et al., 2007): i) it is easily found in the field; ii) it can grow and reproduce in laboratory conditions; iii) colonies are embedded in a soft and transparent tunic which allows direct observation of biological processes; iv) large colonies can be cut into clonal fragments which are able to grow and reproduce, so that subclones of the same colony can be used in control and experimental series; v) colonies undergo recurrent generation changes, with diffuse cell death by natural apoptosis in zooid tissues and efferocytosis by circulating phagocytes; vi) colonies have a well-defined tunic circulatory system and hemocytes can easily be collected with glass micropipettes after the tunic vessels have been punctured with a fine tungsten needle. this review, therefore, focuses mainly on this model organism as an important reference species. when available, various data from studies on other compound ascidians are reported. the colony of b. schlosseri three blastogenetic generations are usually present in a colony of b. schlosseri (fig. 1a): i) adult, filter-feeding zooids, 1.5-2 mm in length, are grouped in star-shaped systems of 10-12 individuals and oral siphons are located in the anterior part of each zooid (i.e. towards the periphery of the system), whereas the central cloacal siphon connects the cloacal chamber, into which individual atrial siphons open, with the exterior; ii) primary palleal buds on zooids are able to replace the parental generation, and iii) secondary buds (budlets) on buds grow to buds and, lastly, to zooids (fig. 1b). budding occurs continuously, in an orderly and synchronized way, as well as the cyclical change of adult generations or take-over (fig. 1c). one of the first report of budding and generational changes in botryllus was that of spallanzani in 1784 (in gibin, 1997). after that, botryllus blastogenesis was described by several researchers (metschnikow, 1869; della valle, 1882; oka, 1892; hjort, 1893; pizon, 1893; ärnbäck-christie-linde, 1923; berrill, 1941a, b; watterson, 1945; sabbadin, 1955a; milkman, 1967; izzard, 1973). the time interval between one generation change and the next is called a blastogenetic cycle: its length is temperature-dependent and lasts one week at 19 °c (fig. 1d). at take-over, old zooids close their siphons, cease filtering, contract (fig. 1c), undergo massive, diffuse apoptosis of their tissues (in parallel with some necrosis in the digestive tube), and are gradually resorbed by either wandering professional or fixed occasional phagocytes (burighel and schiavinato, 1984; lauzon et al., 1993, cima et al., 2003). they are replaced by primary buds, which open their siphons 24–36 h after the beginning of take-over; in the meantime, secondary buds become primary buds and give rise to a new budlet generation (sabbadin, 1955a, 1958; manni et al., 2007). therefore, the lifespan of a zooid, from its appearance as a budlet primordium to its resorption at take-over, covers approximately three weeks. as colonies cannot feed until the new adult generation opens its siphons, they rely on recycling of the components of dying zooids, which are used for growth of the developing buds (sabbadin, 1956; lauzon et al., 2002). each colony is a clone, as it derives from a founder oozoid, which is the outcome of the metamorphosis of a tadpole-like, swimming larva (fig. 2a) deriving from sexual reproduction. oozoids (fig. 2b) bear a palleal bud on their right side, which matures into a filter-feeding blastozooids (fig. 2c), replacing the parental zooid and producing two or more palleal buds on each side of the body, able to produce budlets and grow to mature blastozooids (sabbadin, 1969). zooids, buds and budlets are embedded in a common tunic and interconnected by fig. 2 swimming larva (a), oozooid (b) and first blastozooids (c) of b. schlosseri. a: ampullae; b: bud; bl: budlet; o: oozooid; z: blastozooids. bar = 0.5 mm. 56 a network of vessels of epidermal origin crossing the tunic and joined to a colonial marginal vessel which runs along the contour of the colony and gives rise to many sausage-like blind endings, known as ampullae, where blood cells are stored (brunetti and burighel, 1969; fig. 3). since adult zooids are cyclically resorbed and replaced by their growing buds, a healthy colony can be formed of hundreds or thousands of zooids and buds, synchronized in their development by the common vascular system. the development of buds and zooids in botryllus is highly coordinated (berrill, 1941a, b; watanabe, 1953; sabbadin, 1955a; milkman, 1967) so that the developmental stages of a colony during the blastogenetic cycle can be univocally defined by the developmental stages of its blastogenetic generations. stages more than one day from the preceding or the following take-over are called midcycle stages (lauzon et al., 1992). immunocytes of compound ascidians invertebrate immunocytes represent the key component of immunity, responsible for cellmediated immune reactions and, through their secretions, of most humoral responses. they are wandering cells, active in immunosurveillance which, in ascidians, represent a well-defined class of circulating hemocytes (ballarin and cima, 2005). like all other ascidian species, many types of circulating hemocytes are found in colonial ascidians. the morphology of both living and fixed cells has been widely described by many authors (pérès, 1943; sabbadin, 1955b; andrew, 1961; schlumpberger et al., 1984; cima et al., 2001; ballarin and cima, 2005), as well as their ultrastructure (overton, 1966; fujimoto and watanabe, 1976; milanesi and burighel, 1978; burighel et al., 1983; hirose and mukai, 1992; sugino et al., 1993; cima et al., 2001; hirose et al., 2003) and various attempts have been made to find unifying classification criteria (e.g. de leo, 1992; burighel and cloney, 1997). however, doubts still exist about their functions, mutual relationships, and differentiation pathways. in colonial botryllid ascidians, especially in the species botryllus schlosseri, hemocytes have been particularly investigated for their role in immunity (ballarin et al., 1993, 1995; cima et al., 1996, 2001; ballarin and cima, 2005). in this species, circulating hemocytes are grouped into three main categories: i) undifferentiated cells; ii) immunocytes; iii) storage cells (pigment cells and nephrocytes). immunocytes are represented by cytotoxic morula cells (mcs) and phagocytes, the latter including hyaline amebocytes and macrophage-like cells. morula cells mcs represent the most abundant circulating hemocyte type in botryllid ascidians, their frequency ranging from 20 to 60 %. they have a diameter of 10-15 µm, contain many vacuoles of uniform size, approximately 2 μm in diameter (fig. 4a), and, after treatment with aldehydes, assume a yellowish color and shrink (fig. 4b), thus conferring the typical fig. 3 schematic drawing of the tunic circulatory system of a b. schlosseri colony (modified after bunetti and burighel, 1969). a: ampullae; b: bud; z: zooid; t: tunic. berry-like morphology on fixed cells (sabbadin, 1955b; ballarin et al., 1993; ballarin and cima, 2005). the content of mc vacuoles has reducing properties, as demonstrated by its capability to reduce osmium, and shows positivity for peroxidase, phenoloxidase (po) and arylsulfatase (ballarin and cima, 2005). in addition, it can be stained with eosin (shirae et al., 2002), gives positive cytochemical reactions for polyphenols, quinones and dopacontaining proteins (ballarin et al., 1995; ballarin and cima, 2005) and is recognized by antibodies raised against tunichromes of solitary ascidians (ballarin, 2008). the precursors of mcs are probably granular amebocytes, with which they share similar cytochemical properties and enzymatic content (ballarin and cima, 2005). mcs are abundant inside the lumen of the ampullae of the colonial growing edge (cima et al., 2006a), have specific homing sites such as the lacunae inside the tentacles of the oral siphon (rinkevich et al., 1998; rinkevich, 2005), and their frequency inside the contacting ampullae increases abruptly in early stages of the non-fusion reaction (see below). mcs can sense foreign molecules and, on recognizing them, degranulate (fig. 4c) and release their vacuolar content (ballarin et al., 1995, 1998, 2005) consequently inducing cytotoxicity in neighbouring cells. this effect is directly related to the presence in the medium of active po, as demonstrated by the significantly lower cytotoxicity with respect to controls in the presence of po inhibitors such as sodium benzoate, phenylthiourea and dimethyldithiocarbamate (ballarin et al., 1998, 2005). b. schlosseri po has been purified and partially characterized: the monomeric form has a molecular 57 weight of 80 kda and can polymerize to larger complexes (frizzo et al., 1999). using a polyclonal antibody against the purified protein, it was possible to confirm the enzyme location inside mc vacuoles (frizzo et al., 2000). the observation that serine protease inhibitors can decrease po activity suggests that the enzyme is stored, at least partly, as a proenzyme (pro-po) which, as in arthropods, is converted to active po through the removal of a short peptide by serine proteases (söderhäll and cerenius, 1998). polyphenol substrata are probably represented by tunichromes, small peptides containing dopa or topa (oltz et al., 1987; smith et al., 1991), present inside ascidian mcs (dorsett et al., 1987; azumi et al., 1990; ballarin et al., 1995; taylor et al., 1995;. bayer et al., 1997). they are probably present in a masked form inside mc vacuoles, with sulfates, also revealed in mcs, bound to the aromatic ring, and made available to po by the action of the enzyme arylsulfatase which detaches sulfates from phenols (ballarin et al., 1995). phagocytes in all metazoans, phagocytes can recognize and ingest foreign cells or particles entering the organism, thus ensuring their clearance. in b. schlosseri, circulating professional phagocytes are represented by hyaline amebocytes (fig. 4d) and macrophage-like cells (fig. 4e), which represent two diverse morphologies of the same hemocyte type (sabbadin, 1955b; ballarin et al., 1993). the former represent cells active in phagocytosis which, upon ingestion, withdraw their pseudopods and turn to the globular morphology of macrophage-like cells (ballarin et al., 1993, 1994; ballarin and cima, 2005). the two hemocyte types have similar cytochemical properties and common contents of lysosomal and fig. 4 b. schlosseri hemocytes. a, c: living mcs incubated in the absence (a) or in the presence (c) of foreign molecules or cells (from ballarin et al., 2005); b: aldehyde-fixed mcs; d, e: fixed b. schlosseri phagocytes (from menin and ballarin, 2006). d: hyanine amebocyte; e: macrophage-like cell. bar = 10 µm. antioxidant enzymes (such as phosphatases, 5’nucleotidase, β-glucuronidase and esterases), share the same surface carbohydrates, as demonstrated by their labelling with narcissus pseudonarcissus agglutinin, and are immunopositive to anti-cd39 antibody (ballarin and cima, 2005). similar results were obtained in botrylloides leachi (cima et al., 2001). the release of acid phosphatase in the medium has been observed in in vitro phagocytosis by b. schlosseri hemocytes (cima et al., 1996), due to leakage of the enzyme by phagocytes during phagosome formation (davies and bonney, 1980). tunic and external immunocytes the ascidian tunic contains numerous cells deriving from the hemocytes which leave the circulation and colonize the test matrix (burighel and cloney, 1997). a common tunic cell type is represented by spreading cells or amebocytes which closely resemble circulating hyaline amebocytes. in b. schlosseri three distinct types of cells were recognized: fusiform, fibrocytic and vacuolated cells, the latter deriving from morula cells migrated through the epidermis into the tunic (zaniolo, 1981). a role in immune defense has been postulated for some of these cells, confirmed by the observation that, in both solitary and colonial ascidians, they have phagocytic activity (smith, 1970; hirose et al. 1994a; burighel and cloney, 1997). in addition, as demonstrated in solitary ascidians, hemocytes can infiltrate the tunic in response to the presence of non-self cells or particles, where they contribute to form a capsule (parrinello and patricolo, 1984). in botryllid ascidians, massive infiltration of immunocytes into the tunic is associated with the non-fusion reaction between contacting, genetically incompatible colonies (see below). in the colonial ascidian b. schlosseri, we recently reported the presence of amebocytes, completely exposed to seawater, attached to the tunic of siphonal regions. these cells share many cytochemical features with circulating phagocytes, respond to the same lectins and antibodies used as markers of the phagocytic cell line, and can also engulf carmine particles. all these results suggest that these cells guard the entrances to the branchial and atrial chambers, and trigger a systemic defense response toward non-self particles or cells (cima et al., 2006b). non-self recognition phagocytosis invertebrate phagocytes recognize non-self molecular patterns through a series of poorly known receptors. in b. schlosseri, indirect evidence suggests the involvement of a mannose receptor in yeast recognition (ballarin et al., 1994); in addition, yeast-matched hemocytes express molecules cross-reacting with antibodies raised against mammalian toll-like receptor (tlr)-2 and -4, located in phagocytes (menin and ballarin, 2007), which suggests the involvement of tlrs in the recognition of foreign cells. vertebrate phagocytosis implies a zipper-like mechanism involving a close adhesion, mediated by 58 integrins, of phagocyte projections with the surface of foreign particles or cells (griffin et al., 1975a, b) in b. schlosseri, the interaction with yeast cells requires the activation of phagocyte integrins after recognition of the arg-gly-asp (rgd) motif on the surface of target particles, for both pseudopod formation and cell spreading, as suggested by the fact that the soluble antagonist tetrapeptide arg-glyasp-ser (rgds) significantly inhibits both yeast phagocytosis and cell spreading and disrupts cytoskeletal organization (ballarin et al., 2002a). in b. schlosseri, the occurrence of the respiratory burst after the interaction of phagocytes with non-self particles has been clearly documented in the case of yeast phagocytosis, in which an increase in the production of cytotoxic superoxide anions, peroxides and hypochlorite by phagocytes has been reported (ballarin et al., 1994; cima et al., 1996). an increase of nitrite in the culture medium was also observed when hemocytes were incubated with yeast cells (cima et al., 1996), suggesting that, as in vertebrates (hibbs et al., 1987), the activation of inducible nitric oxide synthase (nos) in botryllus phagocytes occurs on recognition of foreign cells and, as a consequence, nitric oxide (no) with microbicidal activity is produced. this hypothesis may be strengthened by the immunocytochemical assay on phagocytes with anti-nos antibodies. non-self recognition by b. schlosseri phagocytes triggers various signal transduction pathways. the observation that h89 and calphostin, inhibitors of protein kinase a (pka) and protein kinase c (pkc), respectively, can decrease the ingestion of yeast cells by phagocytes (menin and ballarin, 2006), suggests the involvement, in phagocytosis, of transduction pathways activated by camp and diacylglycerol, respectively, and controlled by trimeric g proteins (gomperts et al., 2002). pkc activation follows activation, by trimeric g proteins, of a membrane-associated phospholipase c which produces inositol triphosphate (ip3) and diacylglycerol (dag), the former increasing the cytosolic ca2+ concentration, the latter acting on pkc. a transient rise in cytosolic ca2+ concentration is required for ingestion to occur, as demonstrated by the inhibition of phagocytosis in the presence of edta or when hemocytes and target cells are incubated in the presence of either thimerosal, which depletes intracellular calcium stores, or inhibitors of the calmodulin-dependent ca2+-atpase, such as thapsigargin, pimozide or tributiltin (tbt) chloride, which cause a sustained increase in cytosolic ca2+ concentration (cima et al., 1995; ballarin et al., 1997; cima and ballarin, 2000). manumicin, an inhibitor of monomeric g proteins, decreases the fraction of in vitro phagocytosing cells, indicating the involvement of these proteins, also suggested by the expression, revealed by immunohistochemical and immunoblot analysis, of molecules recognized by anti-pan-ras antibodies in yeast-matched phagocytes (menin et al., 2007). we recently demonstrated that various mapk inhibitors, such as pd98059 (erk pathway), sp600125 (jnk pathway) and sb202190 (p38 pathway) can decrease the frequency of yeast-ingesting hemocytes, and an increase in the expression of various activated mapks, such as p38, erk, sapk/jnk, was observed in immunoblot analysis of yeast-matched hemocyte lysates (menin et al., 2007). phosphatidylinositol-3-kinase (pi3k) is also involved in phagocytosis, and its role seems to be related to the cytoskeletal re-organization required for pseudopod formation (ballarin et al., 2002a). immunopositivity to anti-nf-kb antibodies is located in the cytoplasm of unstimulated phagocytes, whereas it migrates into the nucleus of yeastactivated cells (ballarin, 2008) suggesting the involvement of this transcription factor in phagocytosis. efferocytosis, i.e. phagocytosis of apoptotic cells (de cathelineau and henson, 2003; gardai et al., 2005), occurs massively during take-over and is performed by professional, circulating phagocytes massively recruited in the senescent tissues (fig. 5a), and by occasional phagocytes such as cells of the digestive epithelium (tiozzo et al., 2006). during the generational change, there is a significant increase in the frequency of circulating macrophage-like cells with respect to mid-cycle stages (fig. 5b), paralleled by a decrease in the fraction of circulating hyaline amebocytes (fig. 5c) (cima et al., 2003; ballarin et al., 2008), matching the hypothesis that the two hemocyte types represent different stages of the same cell. as a result of intense phagocytosis, there is a significant increase in the fraction of hemocytes showing positivity for acid phosphatase which, as stated above, is a phagocyte marker, and in both the activity of acid phosphatase and the concentration of peroxides in the blood plasma (cima et al., 1996). botryllus phagocytes recognize phosphatidylserine on the surface of effete cells and corpses, as soluble phospho-l-serine can inhibit their ingestion. in addition, clearance of senescent cells requires the expression of molecules recognized by antimammalian-cd34 antibodies on the phagocyte surface, the expression of which increases at takeover (cima et al., 2003). phagocytes of b. schlosseri are also capable of constitutive macropinocytosis, a process generally responsible of the ingestion of bacteria and necrotic material (krysko et al., 2003), at sites of membrane ruffling along their leading edge. this activity is enhanced by the presence, on the substrate, of molecules containing the rgd motif, such as fibronectin or fibrinogen. this suggests that, as in mammals (meier et al., 2002), integrins can also regulate macropinocytosis. the increase in fluidphase endocytosis is associated with a rise in both oxygen consumption, as indicated by the higher production of reactive oxygen species, and increased cytochrome oxidase activity, related to the synthesis of atp (ballarin and burighel, 2006). allorecognition as stressed by buss (1987), clonal organisms are characterized by colony specificity or allorecognition, i.e. the ability for intraspecific nonself recognition. colony specificity of botryllid ascidians has been known since the pioneer observations by bancroft (1903). it was further investigated in japanese species (oka and watanabe, 1957a, 1960; oka, 1970; mukai and watanabe, 1974) and in b. schlosseri (karakashian 59 fig. 5 a: tissues of senescent zooid during take-over. infiltrated macrophage-like cells having ingested apoptotic cells and corpses are marked by asterisks. bar = 15 µm. b, c: frequencies of circulating macrophage-like cells (mlc) and hyaline amebocytes (ha) in 4 colonies, at mid-cycle (red bars) and take-over (yellow bars). significant differences between mid-cycle and take-over in the same colony are marked by asterisks. *p < 0.05; **p < 0.01; ***p < 0.001. and milkman, 1967; sabbadin, 1962). colony specificity manifests itself as either fusion or nonfusion of genetically compatible or incompatible colonies, respectively, contacting each other at their growing edges. the phenomenon has been studied in 14 species of botryllid ascidians (sabbadin, 1962, 1982; tanaka, 1975; taneda et al., 1985; rinkevich, 1992, 2005; saito et al., 1994; hirose, 2003), particularly in the japanese species botryllus primigenus and the cosmopolitan species b. schlosseri, where the outcome of the contact is genetically controlled by a highly polymorphic fu/hc gene, the alleles of which are co-dominantly expressed (oka and watanabe, 1957a, 1960; sabbadin, 1962; oka, 1970). in b. schlosseri, the fu/hc locus also controls the vascularization and the completion of development of secondary buds grafted from a colony in the tunic of an alien colony, which occur only if the two colonies are fusible (sabbadin, 1982; sabbadin et al., 1991). the great majority of botryllid colonies in nature are heterozygous at the fu/hc locus and fusion occurs when at least one allele is shared by the two colonies, otherwise non-fusion is observed. fusion implies the formation of a chimeric colony, with anastomosis of tunic and circulation (katow and watanabe, 1980; zaniolo et al., 2006); non-fusion requires a partial fusion of the facing tunics with the local disappearance of the limiting cuticles (tanaka and watanabe, 1973; taneda et al., 1985; sabbadin et al., 1992; saito et al., 1994; hirose, 2003; rinkevich, 2005; zaniolo et al., 2006) and is usually characterized by the appearance of a series of cytotoxic foci (rejection reaction), called points of rejection, along the contact border (oka and watanabe, 1957a, 1960; sabbadin, 1962; oka, 1970; rinkevich, 1992, 2005). the growth of contacting ampullae to unusually large size (megaloampullae) during allorecognition has been reported in botrylloides leachi (rinkevich et al., 60 1994; zaniolo et al., 2006). as a consequence of the non-fusion reaction, a change in vectorial growth of the contacting colonies (retreat growth) occurs (rinkevich and weissman, 1988). both cells and soluble factors are involved in botryllus allorecognition. in b. primigenus and b. schlosseri, the partners of a chimeric colony separated after a fusion of at least four days show altered fusibility (mukai, 1967; sabbadin and astorri, 1988). in b. schlosseri, sabbadin and astorri (1988) demonstrated that this alteration is related with the persistence, in a colony, of cells from the partner with which it was previously fused. tanaka (1973, 1975), working with colonies of b. primigenus of defined genotype at the fu/hc locus, showed that fusion conferred to the chimera ac-bc the ability to reject a colony bd when contacting the bc partner. the fusibility of b. primigenus colonies was altered by the previous exposure to x-rays which reduces circulating undifferentiated cells, stressing the role of blood cells in allorecognition (taneda and watanabe, 1982c). non-fusion reaction is irreversible after the fusion of the tunic cuticles occurred, even if one colony is removed after the contact, suggesting the involvement of diffusible (circulating) humoral factors from one colony to the other through the fused tunics (tanaka, 1975; watanabe and taneda, 1982). this is supported by the observation that, in early stages of non-fusion reaction, an increase in permeability of the ampullar epithelium occurs (taneda and watanabe, 1982a) and that the reaction can be mimicked by the injection of whole blood and blood plasma from a colony into the vessels of a genetically incompatible host (taneda and watanabe, 1982b; saito and watanabe, 1984). the observation of a simultaneous occurrence of fusion and non-fusion reactions between contacting colonies fits the hypothesis of the recognition of soluble histocompatibility factors diffusing from one colony by effector hemocytes of the facing partner (taneda, 1985). humoral factors involved in the non-fusion reaction have been partially characterized from blood plasma of botrylloides simodensis: they are resistant to dyalisis heat-labile and require divalent cations for their activity (saito and watanabe, 1984). in b. primigenus and b. schlosseri, the non-fusion reaction shares many characteristics with vertebrate inflammation, such as cell recruitment by chemotaxis, extravasation, cell degranulation, and induction of cytotoxicity. as stated before, it is preceded by partial fusion of the contacting tunics, in front of the facing marginal ampullae, which allows the diffusion of soluble factors from one colony to the next. this event induces selective crowding of mcs inside the ampullae of the growing edge, their migration through the epithelium of the ampullar tips into the tunic, and their final degranulation, with the consequent release of their vacuolar content (taneda and watanabe, 1982a; sabbadin et al., 1992; saito et al., 1994; rinkevich et al., 1998; cima et al., 2006c), in particular, the enzyme po and its polyphenol substrata, which contribute to inducing the observed cytotoxicity (fig. 6; ballarin et al., 1995, 1998; cima et al., 2004, 2006c). analogous behavior, although restricted to a very limited region in front of the facing ampullae (subcuticular rejection), has been described in b. simodensis, botrylloides fuscus and botrylloides violaceus (hirose et al., 1988, 1990, 1997; shirae et al., 2002) and b. leachi (zaniolo et al., 2006; ballarin and zaniolo, 2007). in b. schlosseri, mc degranulation and the induction of cytotoxicity may be mimicked in vitro by exposing hemocytes to the blood plasma of incompatible colonies (ballarin et al., 1995; cima et al., 2006c) and prevented by the above-reported po inhibitors (ballarin et al., 1998). cytotoxicity is related to the induction of oxidative stress, as indicated by the reduction of nitroblue tetrazolium when added to incompatible blood plasma in in vitro assays, and the observation that scavengers of reactive oxygen species, such as superoxide dismutase, catalase and sorbitol, can suppress cell death both in vitro, when hemocytes are incubated with incompatible blood plasma, and in vivo, in colony allorecognition, although they have no effects on mc degranulation or po activity (ballarin et al., 1998, 2002b). the role of no in the induction of cell death is suggested by the in vitro production of nitrite ions when hemocytes are exposed to nonself molecules and the decrease of in vitro cytotoxicity in the presence of the nos inhibitor nωnitro-l-arginine methyl ester (cima et al., 2004). the necrotic masses at rejection points share several chemical and cytochemical properties with mc content (ballarin et al., 1995), thus strengthening the assumption that mcs infiltrated into the tunic play a major role in their formation. various properties of the pigment deposited at rejection points are also indicative of its melanic nature (ballarin et al., 1995), and melanins are the end-product of po activity in metazoans (waite, 1992). despite the well-defined role of mcs as effectors of non-fusion reactions in most botryllid ascidians, even phagocytes take part in rejection reactions: i) in b. schlosseri, fusion between allogeneic colonies, sharing only one allele at the fu/hc locus, usually leads to the resorption of one of the partners during take-over of one of the blastogenetic cycles of the chimeric colony following fusion (rinkevich and weissman, 1987, 1992; sabbadin and astorri, 1988; rinkevich, 2002, 2005). colony resorption, therefore, mimicks zooid resorption at take-over, implying the involvement of phagocytes in the elimination of tissues of the “losing” partner; ii) a more direct role of phagocytes in allorejection has been described in botryllus scalaris, which is the only botryllid species reported so far in which phagocytes and not mcs are involved in allorecognition between contacting, incompatible colonies. in this case, the rejection reaction starts after fusion of the ampullae of the facing growing edges and the beginning of blood exchange through the fused vessels. phagocytes crowd inside the fused ampullae and stimulate the aggregation of hemocytes into large clusters which are finally encapsulated by other phagocytes. in this way, hemocyte clusters plug the lumen of the fused ampullae and blood flow is interrupted in a few 61 fig. 6 schematic representation of the rejection reaction in b. schlosseri. for sake of simplicity, the main steps are indicated only on the left colony. 1: fusion of the contacting tunics after the local disappearance of the cuticles; 2: diffusion of histocompatible factor(s) through the fused tunics; 3: recognition of alien factors by mcs inside the tips of ampullae; 4: release of cytokines by activated mcs enhancing recruitment; 5: extravasation of mc and their degranulation in the tunic; 6: release of po; 7: cytotoxicity and melanin formation at points of rejection. modified after hirose (2003). minutes. differently from other botryllid species studied so far, no signs of selective recruitment or degranulation of mcs were observed (shirae et al., 1999). when incompatible colonies of botryllid ascidians are artificially brought into contact at their cut surfaces (cut surface allorecognition assay), an intense rejection reaction is observed in ovoviviparous species (rinkevich, 1992, 2005; saito et al., 1994; hirose, 2003; zaniolo et al., 2006; ballarin and zaniolo, 2007). however, fusion of tunics and blood vessels (surgical fusion) always occurs in the case of the viviparous species studied so far, suggesting that the hemocytes of these species have lost the ability for allorecognition, which persists in tunic cells (saito et al., 1994; hirose et al., 1994b; okuyama et al., 2002; hirose, 2003; rinkevich, 2005). this may be in relation with the necessity to prevent the immune system from attacking the brooded embryos which shares only one allele at the fu/hc locus with the mother colony and might undergo rejection/resorption as they are exposed to the circulation for more than a week (hirose, 2003). in partial confirmation of the above statement, the po activity of the hemolysate of viviparous species is much lower than that of ovoviviparous ones (shirae and saito, 2000; okuyama et al., 2002). humoral factors lectins it is well-known that ascidian hemolymph contains lectins, often revealed by their 62 hemagglutinating activity (hence the name hemagglutinins), with various carbohydrate specificities (vasta et al., 1982; coombe et al., 1984a; parrinello, 1995). a role in immune defense has been postulated for some of them, but clear involvement in cell proliferation after non-self recognition or in the modulation of phagocytosis has been demonstrated in a few cases (coombe et al., 1984b; kelly et al., 1992; pearce et al., 2001). in compound ascidians, the presence of soluble agglutinins has been reported in various species of the genera amaroucium, aplidium, botrylloides, botryllus, didemnum, diplosoma, clavelina and polyandrocarpa (vasta et al., 1982, 1986; coombe et al., 1984a; suzuki et al., 1990; parrinello, 1995). in b. schlosseri, we demonstrated that yeastactivated phagocytes can synthesize and release, through apocrine secretion, lectins with specificity for β-galactosides, which can agglutinate rabbit erythrocytes and yeast cells (ballarin et al., 1999; fig. 7) and which were previously thought to be members of the galectin family on the basis of their ca2+-independence (ballarin et al., 2000). polyclonal antibodies against these molecules recognize the surface of erythrocyte or yeast cells clumped by exposure to affinity-purified lectins (ballarin et al., 1999). these lectins can improve yeast phagocytosis by acting as opsonins and promoting interactions between target cells and phagocytes (ballarin et al., 1999, 2000). recently, in a full-length cdna library from botryllus colonies, we identified five transcripts homologous to known rhamnose-binding lectins (rbls). their predicted amino acid sequences exactly match the sequences of the tryptic fragments previously obtained from the above agglutinins purified by affinity chromatography. they probably represent different isoforms of a novel rbl, called b. schlosseri rbl (bsrbl), with a molecular weight of approximately 11 kda. they contain the eight cysteines which characterize rbls, form four disulfide bonds, and have a single carbohydrate recognition domain; through non-covalent interactions they can form multivalent complexes able to act as bridges between target particles and the phagocyte surface and enhance phagocytosis (gasparini et al., 2008). antiviral, antimicrobial and antitumoral factors various bioactive molecules, with antiviral or cytotoxic activities against microbial or tumoral cells have been described in compound ascidians. most of these compounds were isolated from didemnid ascidians. ulithiacyclamide and ulicyclamide are cyclic peptides from lissoclinum patella showing antineoplastic and antiviral activity (ireland and sheuer, 1980; ireland et al., 1982; wasylyk et al., 1983). in the same species, a lactone, named lissoclinolide, with antibacterial and antitumoral activity has been isolated (davidson and ireland, 1990; richardson and ireland, 2004). lissoclibadins are cytotoxic and antimicrobial alkaloids from the indonesian lissoclinum cf. badium (nakazawa et al., 2007). didemnins and eudistomins are antiviral molecules isolated from whole extracts of colonies of trididemnum sp. and eudistoma olivaceum, respectively. the former are cyclic peptides, able to inhibit the replication of various rna and dna viruses, fig. 7 agglutination of rabbit erythrocytes (a) and yeast cells (c) in the presence of purified bsrbl. immunostaining with polyclonal anti-bsrbl antibodies reveal the presence of the lectin on the surface of clumped erythrocytes (b) or yeast cells (d). bar = 10 µm. which also exert antitumor activity (rinehart et al., 1981a, b), the latter are β-carboline derivatives containing bromine, active against herpex simplex virus (rinehart et al., 1984). lepadins d, e and f are decahydroquinoline derivatives from didemnum sp. with antiplasmodial and antitrypanosomal activity (wright et al., 2002). anticancer hydroquinones derivatives with cytotoxic properties have been described in aplidium californicum (howard and clarkson, 1979; cotelle et al., 1991). in the mediterranean aplidium albicans, the cytotoxic peptide aplidin induces perturbations of the cell cycle and apoptosis of human leukaemia cells (erba et al., 2002). another antitumoral peptide, vitilevuamide, isolated from didemnum cuculiferum and polysyncraton lithostrotum, was able to inhibit tubulin polymerization required for the organization of the mitotic spindle (edler et al., 2002). metabolites inducing apoptosis of human cancer cells were recently isolated from the japanese ascidian diplosoma virens (ogi et al., 2008). the enzyme po, as stated above, is also involved in preventing microbial infections through its cytotoxic activity and b. schlosseri mcs degranulate and release cytotoxic active po in response to the recognition of yeast cells or bacterial spores (ballarin et al., 2005). cytokines the presence of soluble immunomodulatory molecules, cross-reacting with antibodies raised against mammalian il-1, has been reported in ascidians since the pioneering work by beck et al. (1989). b. schlosseri and a member of the genus didemnum were among the tunicate species investigated, showing the presence of lymphocyte activation factors, the activity of which was neutralized by anti-il-1 antibodies. 63 in b. schlosseri, mcs are the main source of molecules recognized by antibodies raised against the mammalian pro-inflammatory cytokines il-1-α and tnf-α: when hemocytes are exposed to nonself molecules, such as mannan or incompatible blood plasma, or particles, such as yeast cells or microbial spores, mcs acquire immunopositivity to the above antibodies (ballarin et al., 2001, 2005). immunopositive mcs are also observed inside the facing ampullae of contacting incompatible colonies (cima et al., 2004) in early stages of the non-fusion reaction. in b. leachi, mcs are recognized by anti-il-1-α antibodies, whereas positivity to anti-tnf-α antibodies has been unexpectedly observed in another type of hemocytes which, according to cima et al. (2001), were classified as granular cells (ballarin and zaniolo, 2007). in b. schlosseri, the above antibodies inhibit both the rise in cell death observed when hemocytes are incubated with incompatible blood plasma (cima et al., 2004) and mc chemotaxis (cima et al., 2006c), suggesting that the recognized molecules stimulate both the recruitment of mcs inside the tips of facing ampullae and their degranulation with the consequent release of po (fig. 7). the supernatants from cultures of b. schlosseri hemocytes, matched with non-self particles such as yeast cells or zymosan (conditioned media) enhance the phagocytosis of yeast cells (menin et al., 2005). these effects were abolished by the addition of anti-il-1-α or antitnf-α antibodies, suggesting that molecules recognized by the above antibodies are involved in immunomodulation and may be considered as cytokines in the broad sense of the term. using cell fractionation by density gradient centrifugation in ficoll 400, we obtained four hemocyte bands, with different immunocyte distribution, from which we prepared four different conditioned media, one for each band. in this way, we could demonstrate that the enhancing effect on phagocytosis was present in conditioned media derived from hemocyte bands enriched in mcs, but that it was not present in conditioned media from bands rich in phagocytes (fig. 8), in agreement with previous results indicating mcs as the source of molecules crossreacting with anti-mammalian cytokine antibodies (menin et al., 2005). immunoblot analysis of the supernatant from zymosan-matched hemocytes showed a 60 kda band cross-reacting with both anti-il-1-α and antitnf-α. in addition, a 37 kda band, recognized by anti-bsrbl antibodies, was detected in the above conditioned media, and the presence of the lectins was confirmed by hemagglutinating assay (menin and ballarin, 2008). all the above results fit a scenario in which mcs are the main organism sentinel cells which sense non-self molecules, being recruited as a consequence of the recognition and, on the basis of the nature of the foreign molecules, they are able to trigger a cytotoxic response or stimulate phagocytes to ingest foreign cells and release agglutinins which potentiate their activity. immunity and xenobiotics b. schlosseri is one of the most diffuse encrusting organisms in the lagoon of venice, where it characterizes the relative climax of the ecological succession of hard-substratum macrobenthos (cima et al., 2006c). as colonial ascidians have been reported to be very susceptible to antifoulants (henderson, 1986), we studied the effects of short-term exposure of b. schlosseri hemocytes to biocides such as organotin compounds (tributyltin, triphenyltin) and new organic compounds used in antifouling paint formulations after the ban on tin-based antifoulants, on cell functions in order to reveal the cellular targets of these molecules (cima et al., 1995, 1997, 1998, 2008; cima and ballarin, 1999, 2000, 2004). organotin compounds alter the morphology of phagocytes, their capability to ingest target yeast cells, and to induce the respiratory burst in a dosedependent manner, and these changes are related to disruption of cytoskeletal components (cima and ballarin, 2000, 2008; cima et al., 1995, 1997, 1998). tributyltin can interact with calmodulin and alter the activity of calmodulin-dependent ca2+-atpase (cima et al., 2002a) or react directly with cytosolic thiols (cima and ballarin, 2004). as a consequence, they alter cytosolic calcium and thiol homeostasis which cause the observed alterations in cell morphology (cima et al., 1995, 1997), inhibition of hydrolytic, detoxifying and mitochondrial enzymes (cima et al., 2002b), and eventually lead to apoptosis (cima and ballarin, 1999), probably due to the severe oxidative stress following the reduction of cytosolic thiols (cima et al., 2004). among the new antifoulants, we assayed seaninetm (4,5 dichloro-2-n-octyl-4-isothiazoline-3-one), chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile), diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) and tcms pyridine (2,3,5,6-tetrachloro-4(methylsulfonyl)pyridine). both sea-ninetm and chlorothalonil have a negative effect on the phagocyte cytoskeleton, altering cell morphology and severely hindering phagocytosis. both compounds decrease intracellular reduced glutathione, inducing oxidative stress which is probably the cause of the observed cell death by apoptosis (cima et al., 2008), and perturb the mitochondrial respiratory chain, whereas only sea-ninetm disrupts cytosolic calcium homeostasis (cima et al., 2008). diuron and tcms pyridine inhibit phagocytosis and cell spreading in a dose-dependent manner, suggesting cytoskeletal alteration, and can induce apoptosis at the higher concentrations assayed (100 and 20 μm, for diuron and tcms pyridine, respectively). the two biocides did not have any negative effect on esterase or cytochrome-c oxidase activities, or on the homeostasis of cytosolic calcium (menin et al., 2008). conclusions and perspectives today, the study of ascidian immunobiology is a topical subject and it is a widespread opinion among scientists that protochordates can greatly 64 fig. 8 density gradient centrifugation of b. schlosseri hemocytes. a: enriched fractions of cells obtained after centrifugation in discontinuous gradient of ficoll 400. b: concentration of mcs (yellow bars) and phagocytes (brown bars) in each fraction. c: percentage of phagocytizing cells in hemocyte cultures exposed to a suspension of yeast cells in seawater (control) or in the supernatants from cultures of hemocytes of each band obtained after density gradient centrifugation; significant differences with respect to the control are marked by asterisks. *p < 0.05; **p < 0.01. modified after menin et al. (2005). contribute to answering the still unresolved problems of the origin of the highly sophisticated and complex vertebrate immune system. among compound ascidians, b. schlosseri is diffuse and studied worldwide, and represents an optimal animal model for immunobiological studies, despite the size of its zooids, thanks to good knowledge of the biology of its immunocytes (manni et al., 2007). in addition, this kind of study has been frequently motivated by interest in understanding the cellular basis of allorecognition. as in other invertebrates, immune responses in ascidians rely mainly on circulating immunocytes which act as the effector arms of immunity, and can ingest or kill non-self cells or release opsonins and immunomodulatory molecules which enhance or reinforce cellular responses. as reported, two distinct immunocyte differentiation lines are present in botryllid ascidians: phagocytes and cytotoxic, phenoloxidase-containing morula cells. nevertheless, despite the abundance of data gathered in the last two decades, unlike solitary ascidians of the genus ciona and halocyntyhia (dehal et al., 2002; yokobori et al., 2003), compound ascidians and b. schlosseri in particular still lack an adequate molecular approach to the study of their genomes. few genes other than the bsrbl genes reported above (gasparini et al., 2008), which might be involved in non-self recognition, have been isolated. examples are a cd94 orthologue of the vertebrate cd94 receptor on nk cells, expressed on a subset of b. schlosseri blood cells, probably phagocytes, mainly in the contacting ampullae (khalturin et al., 2003), and genes for putative homologue of c-type lectin (pancer et al., 1997), human eb1 (pancer et al., 1996a), fk506-binding protein (pancer et al., 1993), vertebrate receptor for antigens (pancer et al., 1996b) and hsp70 (fagan and weissman, 1996). recently, a subtraction cdna library of contacting, genetically incompatible colonies of b. schlosseri allowed the identification of more than 100 genes differentially expressed and related to immunity (oren et al., 2007). for deeper insight into the immunobiology of compound ascidians, better knowledge of the genome of these organisms, at least of botryllus, on which many of the studies have been concentrated, is required. in addition, there are still some open questions regarding basic biological processes related to immunity, which represent good challenges for future research on compound ascidians. some of them are listed below. origin and differentiation hemocytes (and immunocytes) ascidian hemocytes derive from the embryonic mesenchyme (cowden, 1968; sala, 1973; sabbadin et al., 1999). undifferentiated cells are known as hemoblasts and are characterized by high nucleocytoplasmic ratio, a well-defined nucleolus and a basophilic cytoplasm whereas the term lymphocyte has been used either as a synonymous of hemoblast or to indicate a cell type thought to represent an immature differentiating hemocyte (pérès, 1943; sabbadin, 1955b; ermak, 1976; kawamura et al., 1988). however, the real nature of the lymphocyte and the differentiation pathways of hemocytes are not clear and require further investigations. it is known that, in b. schlosseri, almost 30 % of hemocytes die by apoptosis at take-over and are replaced by new hemoblasts entering the circulation 65 at the same stage of the blastogenetic cycle (ballarin et al., 2008). however, their origin is still unknown as no structures comparable to the branchial hemopoietic nodules, reported by ermak (1977) in solitary species, have been described in compound ascidians. in certain experimental conditions, mitosis figures have been observed in circulating hemocytes of (cima and ballarin, 2007). undifferentiated blood cells are directly involved in asexual reproduction by stolonal (freeman, 1964) or vascular budding (oka and watanabe, 1957b). the latter occurs spontaneously in b. primigenus (oka and watanabe, 1957b) and in dormant colonies of b. leachi (burighel et al., 1976) and can lead to the re-building of a whole colony from small colony fragments, in botrylloides violaceus (oka and watanabe, 1959) and b. leachi (rinkevich et al., 2007), or from the colonial matrix deprived of zooids and buds, in b. schlosseri (milkman, 1967; sabbadin et al., 1975). both blood and epithelial cells can be cultured in vitro in appropriate conditions, and this represents an interesting tool in studying cell differentiation (sala, 1973; rinkevich and rabinowitz, 1993, 1994; rabinowitz and rinkevich, 2003). allorecognition: old problems, new questions i) some japanese authors, in comparing the non-fusion reactions of various botryllid ascidians, hypothesized the key role of tunic cells and the epithelium of the facing ampullae in allorecognition (taneda et al., 1985; saito et al., 1994; hirose, 2003). the fu/hc gene of b. schlosseri has recently been characterized (de tomaso et al., 2005), as well as that of the putative receptor involved in allorecognition (nyholm et al., 2006). the receptor is expressed in facing ampullae: intense labelling is observed in the epithelium of the ampullar tips but not in mcs (nyholm et al., 2006). in addition, the epithelium of the ampullar tips express high levels of bs cadherin in early stages of the non-fusion reaction (rosner et al., 2007). indeed, the ampullar epithelium plays a fundamental role in botryllus allorecognition in allowing the diffusion of non-self factors from the alien colony into the ampullar lumen, where they can alert mcs. during the non-fusion reaction, it is highly fenestrated and increases its permeability in both b. primigenus and b. schlosseri (taneda et al., 1982a; sabbadin et al., 1992), which suggests that it perceives signals which are absent in the fusion reaction. as all these events precede mc activation, represent prerequisites for the following inflammatory reaction and are worthy of study. ii) in botryllid ascidians, in addition to allorecognition, the fu/hc gene also controls the recognition between sperm and egg. it has been reported that the sperm of the japanese species botryllus primigenus cannot fertilize eggs when it shares a fu/hc allele with the diploid, maternallyderived, egg envelope (oka, 1970; saito et al., 1994). this limitation also seems to exist in some populations of b. schlosseri (scofield et al., 1982) but not in others (sabbadin, 1971, 1982; grosberg 1987). however, at least in the population from the lagoon of venice, high levels of inbreeding depression have been reported (sabbadin, 1971). this offers the possibility of investigating relationships among allorecognition, fertilization and inbreeding depression, and their implications in the ecology of natural populations. iii) as stated before, in laboratory conditions, resorption of one partner in the chimera usually follows colony fusion (rinkevich and weissman, 1987; rinkevich, 2002, 2005). in natural populations, multichimeras form as the result of kin recognition, controlled by the fu/hc gene, allowing larvae to settle near parental of genetically compatible adult colonies with which, once metamorphosed, they can fuse (grosberg and quinn, 1986; grosberg, 1987; ben-shlomo et al., 2008). this natural multichimerism gives colonies greater fitness in terms of faster growth rate, maximum size reached, better competition for the substrate, earlier achievement of sexual maturity and increased genetic diversity (sabbadin, 1994; rinkevich and shapira, 1999; paz and rinkevich, 2002; de tomaso, 2006). in addition, stem cells can be maintained and proliferate for a long time within a chimeric colony, although the corresponding soma has been resorbed (sabbadin and zaniolo, 1979; sabbadin and astorri, 1988; stoner et al., 1999; laird et al., 2005; de tomaso, 2006). according to sabbadin (1994) chimerism has the important role of maintaining the genetic structure of a population allowing the preservation of the genomes of fusible colonies, even in the case of their resorption by a partner, and seems to be diffuse in colonies collected from the field (ben-shlomo et al., 2008). for this reason, botryllus is an interesting model for the study of the evolution of microchimerism (rinkevich, 2001), which seems to be responsible for various human pathologies (adams and nelson, 2004), and its relationships with the immune system. apoptosis and efferocytosis at take-over despite the abundance of in vitro model systems, mainly represented by selected cell lines, there is an increasing need for reliable models for in vivo investigations of the biological role of apoptosis in organisms. with its spontaneous and recurrent apoptosis of zooid tissues at the end of each blastogenetic cycle, b. schlosseri is an interesting model organism for the study of apoptosis and its genetic control. two genes changing their expression during take-over have already been described (lauzon et al., 1996), but we do not know the nature of the cyclical signal inducing weekly cell death. in addition, phagocytes have been reported to play an important role in regulating the clearance of senescent cells, the extent of growth of buds and their budding activity (voskoboynik et al., 2004), but the molecular mechanisms underlying phagocyte recruitment in senescent tissues and recognition of effete cells are still largely unclear. tunicate cytokines: what is their relationship with their vertebrate counterparts? vertebrate cytokines are immunomodulatory proteins secreted by activated immunocytes after the recognition of foreign molecules, and they take part in several immunological processes such as inflammation, apoptosis, clearance of effete cells 66 and corpses, cytotoxicity and phagocytosis. in addition, they guarantee fine cooperation between sentinel cells, which recognize non-self molecules, and effector cells, which can mount active responses towards foreign cells or particles, in order to ensure the health and survival of individuals (abbas et al., 2000). in tunicates, molecules recognized by antibodies raised against the mammalian proinflammatory cytokines il-1 have been reported in various ascidian species (beck et al., 1989; raftos et al., 1991, 1992; ballarin et al., 2001, cima et al., 2004; parrinello et al., 2007). they have been partially characterized and their molecular weights range between 12 and 59 kda, as resolved by sdspage and gel chromatography (beck et al., 1989; raftos et al., 1992; parrinello et al., 2007). it is common opinion that invertebrate cytokines share no homologies with their vertebrate counterparts (beck, 1998; beschin et al., 2001, 2004) and this may explain the absence of orthologues of vertebrate pro-inflammatory cytokine genes in the genome of ciona instestinalis (azumi et al., 2003). however, it has been reported that various vertebrate cytokines have a lectin domain and can bind carbohydrates (cebo et al., 2002; beschin et al., 2004) and this probably represents the 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dev. comp. immunol. 6: 665-673, 1982c. 74 review immunobiology of compound ascidians, with particular reference to botryllus schlosseri: state of art immunity in ascidians immunity in compound ascidians immunocytes of compound ascidians non-self recognition phagocytosis humoral factors cytokines immunity and xenobiotics acknowledgements references isj 6: xx-yy, 2009 isj 6: 59-77, 2009 issn 1824-307x review the lipopolysaccharide-activated innate immune response network of the horseshoe crab s kawabata, t koshiba, t shibata department of biology, faculty of sciences, kyushu university, fukuoka 812-8581, japan accepted may 20, 2009 abstract primary stimulation of the horseshoe crab innate immune system by bacterial lipopolysaccharide (lps) activates a network of responses to ensure host defense against invading pathogens. granular hemocytes selectively respond to lps via a g protein-dependent exocytic pathway that critically depends on the proteolytic activity of the lps-responsive coagulation factor c. in response to stimulation by lps, the hemocyte secretes transglutaminase (tgase) and several kinds of defense molecules, such as coagulation factors, lectins, antimicrobial peptides, and protein substrates for tgase. lps-induced hemocyte exocytosis is enhanced by a feedback mechanism in which the antimicrobial peptide tachyplesin serves as an endogenous mediator. the coagulation cascade triggered by lps or β-1,3-d-glucans results in the formation of coagulin fibrils that are subsequently stabilized by tgase-dependent cross-linking. a cuticle-derived chitin-binding protein additionally forms a tgase-stabilized mesh at sites of injury. invading pathogens are agglutinated by both hemocyteand plasma-derived lectins. in addition, the proclotting enzyme and tachyplesin functionally convert hemocyanin to phenoloxidase. in the plasma, coagulation factor c acts an lps-sensitive complement c3 convertase on the surface of gram-negative bacteria. in this manner, lps-induced hemocyte exocytosis leads not only to coagulation but also activates a sophisticated innate immune response network that coordinately effects pathogen recognition, prophenoloxidase activation, pathogen clearance, and tgase-dependent wound healing. key words: horseshoe crab; lipopolysaccharide; innate immunity; hemolymph coagulation; transglutaminase; complement c3 introduction innate immunity, which defends the host against infectious pathogens, is an ancient and ubiquitous immune system in both vertebrates and invertebrates. each species employs a variety of environment-specific adaptations to ensure host defense, whereas a generalized recognition strategy against invading pathogens underlies the innate immune reaction. the innate immune system recognizes broadly conserved microbial cell wall components known as pathogen-associated molecular patterns (pamps), such as lipopolysaccharides (lps) of gram-negative bacteria, peptidoglycans of gram-positive bacteria, and β-1,3-d-glucans of fungi via pattern-recognition ___________________________________________________________________________ corresponding author: shun-ichiro kawabata 6-10-1 hakozaki higashi-ku, department of biology faculty of sciences, kyushu university fukuoka 812-8581, japan e-mail: skawascb@kyudai.jp proteins (janeway, 1989; hoffmann, 2003; akira et al., 2006). innate immune systems in invertebrates consist of pathways that promote recognition of pathogen-associated macromolecules, hemolymph coagulation, phenoloxidase-mediated melanization, cell agglutination, antimicrobial activity, and phagocytosis (nappi et al., 2004; theopold et al., 2004; iwanaga and lee, 2005; kurata et al., 2006; nakanishi and shiratsuchi, 2006). the horseshoe crab belongs to the class merostomata and is phylogenetically more closely related to arachnoidea than it is to crustacea. fossils of horseshoe crabs, such as mesolimulus walchi and limulus coffini, have been found in deposits from the paleozoic era to the cenozoic era in europe and north america (størmer, 1952). extant horseshoe crabs comprise four species, limulus polyphemus, tachypleus tridentatus, t. gigas, and carcinoscorpius rotundicauda, each having a distinct geographic distribution; l. polyphemus is distributed along the east coast of north america, 59 fig. 1 lps-activated innate immune response network of the horseshoe crab. granular hemocytes selectively respond to lps via a g protein-dependent exocytic pathway that critically depends on the proteolytic activity of the lps-responsive coagulation factor c. in response to stimulation by lps, the hemocyte secretes several kinds of defense molecules, such as coagulation factors, lectins, antimicrobial peptides, and protein substrates for tgase involved in protein cross-linking. lps-induced hemocyte exocytosis is enhanced by a feedback mechanism in which the antimicrobial peptide tachyplesin serves as an endogenous mediator. the coagulation cascade triggered by lps or β-1,3-d-glucans results in the formation of coagulin fibrils that are subsequently stabilized by tgase-dependent cross-linking with stablin and proxin. a cuticle-derived chitin-binding protein caraxin additionally forms a tgase-stabilized mesh at sites of injury. invading pathogens are agglutinated by both hemocyteand plasma-derived tachylectins and crp. in addition, the proclotting enzyme and tachyplesin functionally convert hemocyanin to phenoloxidase. in the plasma, coagulation factor c also acts an lps-sensitive complement c3 convertase on the surface of gram-negative bacteria. an immunocompetent cell with phagocytotic activity against gram-negative bacteria has not been identified in the horseshoe crab and the complement-dependent clearance system of invading pathogens remains to be examined. po, phenoloxidase; plc, phospholipase c; pip2, phosphatidylinositol-4,5-biphosphate; ip3, inositol-1,4,5-triphosphate; er, endoplasmic reticulum. and the other three species are mainly distributed throughout southeast asia. in japan, t. tridentatus inhabits coastal areas of the northern part of kyushu island as well as the inland sea. t. tridentatus has proven to be a suitable model system for the investigation of arthropod immunity, since, in addition to having a sophisticated innate immune system, it is relatively long-lived; the embryo molts four times within the fertilized egg, and after hatching it molts every year over 15 years to become a mature adult (sekiguchi et al., 1988). here we review our current knowledge of horseshoe crab innate immunity at the molecular level with an emphasis on the importance of hemocytes and hemolymph plasma. lps-induced hemocyte exocytosis and its endogenous amplification system in t. tridentatus, granular hemocytes, as determined by morphological classification, constitute 99 % of all hemocytes, and play a key role in the innate immune system (iwanaga et al., 1998; iwanaga, 2002; kawabata and tsuda, 2002). horseshoe crab hemocytes respond selectively to lps but not to other pamps, such as β-1,3-d-glucans and peptidoglycans (ariki et al., 2004). a variety of defense molecules are stored in the secretory granules of the hemocyte; large granules contain serine protease zymogens for hemolymph coagulation (factor c, factor g, factor b, 60 fig. 2 factor c is a membrane associated lps sensor on hemocytes. hemocytes were treated without (left panel) and with (right panels) fitc-labeled lps (green), and stained with a monoclonal antibody (2c12) against factor c. for the detection, cy3-conjugated anti-mouse secondary antibody was used (red). arrowheads indicate lps co-localized with membrane bound factor c. bar = 10 μm. and the proclotting enzyme), the clottable protein coagulogen, serine protease inhibitors (serpins), lectins, and substrates for transglutaminase (tgase), whereas small granules contains antimicrobial peptides. in response to stimulation by lps, these defense molecules are rapidly secreted by the hemocyte (fig. 1). factor c is a unique lps-responsive serine protease zymogen that is stored in the large granules of hemocytes and acts as an lps sensor to potentiate hemocyte exocytosis. upon activation by lps, factor c initiates hemocyte exocytosis via a g-protein-dependent exocytic pathway that is dependent upon the proteolytic activity of factor c. in this respect, the activation of hemocytes by factor c is analogous to the thrombin-thrombin receptor (the protease-activated g protein-coupled receptor, par) signaling axis in mammalian platelets (ariki et al., 2004). hemocyte exocytosis can be quantitatively assayed by elisa using an antibody against a granular component such as coagulogen in the presence of 50 mm mg2+ and 10 mm ca2+, equivalent to the concentrations of these cations in hemolymph plasma. exclusion of divalent cations from the assay buffer inhibits exocytosis even at high concentrations of lps. moreover, in the absence of lps, hemocyte exocytosis can be induced by synthetic hexapeptides corresponding to the tethered ligands of mammalian pars, supporting the notion of a par-like receptor on the hemocyte surface. immunofluorescence microscopy of the hemocyte using an anti-factor c antibody detects factor c, which is localized in a punctate distribution on the hemocyte surface (kurata et al., 2006; koshiba et al., 2007) (fig. 2). when hemocytes are incubated with fitc-labeled lps, the factor c antigen co-localizes with fitc-lps accumulated on the cell surface (koshiba et al., 2007). the accumulation of fitc-lps is not observed on hemocytes fixed with formaldehyde, as would be expected with chemical modification and inactivation of cell surface proteins. these results also suggest that factor c is a membrane-bound lps sensor on the hemocyte surface. factor c has no obvious transmembrane domain within in its sequence, whereas it strongly interacts with acidic phospholipids (nakamura et al., 1988a). surface plasmon resonance analyses indicate that factor c interacts with phosphatidylserine (kd = 2.5×10 -9 m) and phosphatidylinositol (kd = 4.7×10 -9 m) as well as with cholesterol (kd = 1.4×10 -9 m) (ariki et al., 2004). the interaction of factor c with lps (kd = 7.6×10 -10 m) is 61 62 competitively inhibited by the addition of the acidic phospholipids. in contrast, cholesterol does not inhibit the interaction of factor c with lps, suggesting that factor c interacts with cholesterol through a binding site that is distinct from that for lps, and raising the possibility that factor c may be localized on cholesterol-rich microdomains or lipid rafts on the hemocyte membrane. the horseshoe crab hemocyte has an endogenous positive feedback mechanism for lps-induced hemocyte exocytosis (ozaki et al., 2005). the hemolymph contains hemocytes at ~106 cells/ml. lps-induced hemocyte exocytosis is highly dependent on the cell density, namely, an increase in cell density from 0.05×106 to 0.8×106 cells/ml results in a 106-fold change in the apparent lps sensitivity (from 10-7 to 10-13 g/ml of lps), suggesting the presence of feedback mechanism for secretion via an unknown secretagogue secreted from hemocytes in response to the stimulation by lps. interestingly, tachyplesin in the exocytosed fluid acts as a secondary secretagogue, thereby dramatically enhancing the sensitivity of the hemocyte to lps. tachyplesin (17 residues) is a potent antimicrobial peptide and one of the most abundant components stored in the hemocyte (nakamura et al., 1988b; shigenaga et al., 1990). the effective concentration of tachyplesin required for hemocyte exocytosis ranges from 5 to 10 μm, indicating that a high concentration of tachyplesin is required to act as an effective endogenous secretagogue. tachyplesin has structural properties in common with mastoparan, a basic tetradecapeptide from wasp venom. mastoparan interacts directly with g proteins without direct stimulation of the upstream receptor, and induces exocytosis in the mast cell (higashijima et al., 1988). consistent with these findings, mastoparan is able to induce hemocyte exocytosis in t. tridentatus (ariki et al., 2004). moreover, tachyplesin binds to bovine g protein an equilibrium dissociation constant (kd) of 8.8×10 -7 m (ozaki et al., 2005). these data suggest that tachyplesin interacts with g-proteins in the hemocyte in a manner similar to that of mastoparan. in addition, tachyplesin has an ability to bind to hemocyanin (nagai et al., 2001), the major protein in the hemolymph plasma, suggesting that hemocyanin may serve as a sink for tachyplesin released from hemocytes, thereby spatially restricting its hemocyte-stimulating effect to the site of infection. on the other hand, insect and mammals conserve a signaling pathway of the innate immune system through cell-surface receptors called tolls and toll-like receptors (hoffmann, 2003; akira et al., 2006). a toll-like receptor (ttoll) has been identified in t. tridentatus, which is most closely related to drosophila toll in both domain architecture and overall length (inamori et al., 2000, 2004). the two receptors show a significant sequence identity between their tir domains (39 %). interestingly, spätzle, a protein ligand for drosophila toll, shows significant structurally similarity to horseshoe crab coagulogen (see "hemolymph coagulation"). moreover, ttoll is nonspecifically expressed in all tissues examined (inamori et al., 2004), suggesting that ttoll does not act as an lps receptor on granular hemocytes. in addition, nf-κb and iκb homologues (crnf-κb and criκb) have been identified in the horseshoe crab c. rotundicauda (wang et al., 2006). gram-negative bacteria infection causes degradation of criκb and nuclear translocation of crnf-κb, leading to up-regulation of immune-related gene expression, including nitric oxide synthase and factor c, indicating that the nf-κb/iκb signaling cascade remains well conserved from horseshoe crabs to mammals, playing a fundamental role in regulating the expression of critical immune defense molecules. hemolymph coagulation the hemocyte releases coagulation factors by lps-induced exocytosis, leading to the activation of the proteolytic coagulation cascade (fig. 1). factor c secreted from hemocytes is autocatalytically activated in the presence of gram-negative bacteria or lps. the resulting activated factor c activates coagulation factor b, which in turn converts the proclotting enzyme into the clotting enzyme. the clotting enzyme then promotes the proteolytic conversion of coagulogen to coagulin, which spontaneously forms an insoluble polymer. alternatively, activated factor g in the presence of β-1,3-d-glucans triggers the activation of the proclotting enzyme to the clotting enzyme. in this manner, factor c and factor g independently serve to couple the recognition of lps and β-1,3-d-glucans, respectively, to the formation of a physical barrier at the site of microbial invasion. the vertebrate coagulation system acts locally on the phospholipid surface in cooperation with ca2+ at the site of vascular injury. in an analogous fashion, hemolymph coagulation in the horseshoe is restricted to the surfaces of invading pathogens, such as gram-negative bacteria and fungi. this mechanistic similarity between the coagulation cascades of vertebrates and horseshoe crabs may lead to the erroneous assumption of a common evolutionary origin (fig. 3). in fact, fibrinogen homologues of the horseshoe crab, named tachylectins-5a and -5b, act as non-self recognizing proteins rather than as target proteins of the coagulation cascade (gokudan et al., 1999). also coagulogen has no structural similarity or evolutionary relatedness to fibrinogen (bergner et al., 1996). a protease cascade in drosophila has been well characterized as the morphogenetic cascade for determining embryonic dorsal-ventral polarity, leading to the production of the toll ligand spätzle (belvin and anderson, 1996). the drosophila toll pathway additionally controls resistance to fungal and gram-positive bacterial infections (ferrandon et al., 2007). spätzle, belongs to nerve growth factor family and possesses a structural similarity to horseshoe crab coagulogen (smith and delotto, 1992; bergner et al., 1996; bergner et al., 1997). in addition, a clip-like domain located in the n-terminal region of horseshoe crab coagulation factor b and the proclotting enzyme, originally identified in the proclotting enzyme as a disulfide-knotted domain, fig. 3 comparison of proteolytic cascades between horseshoe crab hemolymph coagulation, mammalian blood coagulation, and drosophila toll pathway. the serine protease zymogens are indicated by asterisks. homologous proteins are connected by ladders. has been identified in the proteins snake and easter of the drosophila toll pathway (muta et al., 1990, 1993). the structural similarity between coagulogen to spätzle, as well as that between the serine protease zymogens participating in the two cascades, suggests that the two functionally distinct cascades may have a common evolutionary origin (fig. 3) (krem and di cera, 2002; kawabata et al., 2003). the coagulation cascade in the horseshoe crab is regulated by three types of serpins that form stable 1:1 covalent complexes with target coagulation proteases (fig. 1); serpins-1, -2, and -3 inhibit activated factor c, the clotting enzyme, and activated factor g, respectively (miura et al., 1994, 1995; lal agarwala et al., 1996). all three serpins are stored in the large granules of hemocytes and are secreted upon hemocyte exocytosis in response to stimulation by lps. these serpins appear to prevent diffusion of the activated forms of coagulation factors by scavenging activated proteases that escape into the hemolymph from the surface of microbes at the site of injury, and thereby prevent unnecessary clot formation. horseshoe crab serpins are more closely related to mammalian serpins than they are to insect serpins. for example, serpin-1 shows higher sequence identities to human plasminogen activator inhibitor (40 %) and human neutrophil elastase inhibitor (39 %) than to an elastase inhibitor from manduca sexta (29 %) and silkworm antichymotrypsin (27 %) (miura et al., 1994). mammalian serpin-protease complexes are hypothesized to be rapidly cleared through a cell-surface receptor, which recognizes a hydrophobic consensus sequence that is selectively exposed on the complexed forms of serpins; for instance, phe-val-phe-leu-met in the c-terminal region of α1-antitrypsin (joslin et al., 1991). this consensus sequence is conserved in horseshoe crab serpins in the corresponding region (ex. phe-val-phe-phe-ile for serpin-1), suggesting that a similar clearance mechanism exists in horseshoe crabs. lps recognition by factor c factor c is a multidomain glycoprotein with an apparent molecular mass of 120 kda. in addition to a typical serine protease domain at the c-terminus, factor c contains a cys-rich region, an epidermal factor (egf)-like domain, five complement control protein (ccp) modules, a c-type lectin domain, and an lccl module (derived from a conserved domain of limulus factor c, coch-5b2, and lg11) (muta et al., 1991; trexler et al., 2000) (fig. 4a). structure-function analyses reveal that the lps-binding site in factor c is present in the n-terminal cys-rich region of the molecule and contains a tripeptide sequence (-arg36-trp37-arg38-) consisting of an aromatic residue flanked by two basic residues (koshiba et al., 2007). a recombinant version of the cys-rich/egf fragment (positions 1-116) with paired substitutions of arg36/arg38 to 63 fig. 4 schematic domain structure of horseshoe crab innate immune proteins. (a) domain structure of factor c. egf, epidermal growth factor; ccp, complement control protein; lccl, limulus factor c, coch-5b2, and lg11. (b) domain structure of factor g. z1 and z2, xylanase z-like modules. (c) domain structures of tachylectins. tl, tachylectin. (d) domain structure of ttc3. cub, complement-urchin-bone. glu36/glu38 (r36e/r38e mutant) is incapable of binding to lps. moreover, a mutant protein with substitution of trp37 to ala37 also lacks the ability to bind lps. these data indicate the essential nature of the tripeptide motif for lps recognition by factor c. the cys-rich/egf fragment interacts with lps with kd = 2.0×10 -10 m, which is similar to that of the wild-type protein (shibata et al., unpublished data). this tripeptide motif is conserved in horseshoe crab anti-lps factor (aketagawa et al., 1986) and several mammalian lps-recognizing proteins such as lps-binding protein (schumann et al., 1990) and bactericidal/permeability-increasing protein (marra et al., 1990) (fig. 5a). anti-lps factor (102 residues) has been identified as an inhibitor for lps-mediated hemolymph coagulation. anti-lps factor is stored in the large granule of hemocytes, and shows sequence similarity to the α-lactoalbumin/lysozyme family. the crystal structure shows that anti-lps factor has a single domain consisting of three α-helices packed against a four-stranded β-sheet to form a wedge-shaped molecule with a striking charge distribution and amphipathicity (hoess et al., 1993). the binding site for lps likely encompasses the extended amphiphilic loop with the tripeptide motif (-lys43-trp44-lys45-) (pristovsek et al., 2005) (fig. 5b). in addition to its physical association with factor c, lps promotes its autocatalytic activation upon binding to its cys-rich region. in contrast to wild-type factor c, the r36e/r38e variant is incapable of autoactivation in the presence of lps (fig. 6) (koshiba et al., unpublished data). β-1,3-d-glucan recognition by factor g factor g is another pattern-recognition protein in the coagulation cascade that acts as a sensitive and specific sensor for β-1,3-d-glucans. in other arthropods, such as crustaceans and insects, the recognition of β-1,3-d-glucans also triggers a serine protease cascade, leading to the activation of prophenoloxidase, a key enzyme in the melanization of pathogens and damaged tissues (cerenius and söderhäll, 2004; kanost et al., 2004; vetvicka and sima, 2004). in vertebrates, the recognition of β-1,3-d-glucans by dectin-1, a c-type lectin family member, potentiates the production of cytokines and antifungal reactive oxygen species by dendritic cells and macrophages (brown, 2006). factor g is a heterodimeric serine protease zymogen composed of two non-covalently associated subunits, α and β (seki et al., 1994) (fig. 4b). the β subunit contains a serine protease domain, 64 fig. 5 a model of bacterial recognition by membrane associated factor c via n-terminal cys-rich region. (a) the n-terminal sequence of factor c containing cys-rich and egf-like regions (r1-g116) is shown. the tripeptide motifs in the cys-rich region are highlighted. hlbp, human lps-binding protein; hbpi, human bacterial permeability-increasing protein; hmd-2, human md-2; lalf, limulus anti-lps factor; and rcap18, rabbit cationic antimicrobial protein. (b) the recognition of lps on gram-negative bacteria via membrane-associated factor c initiates the horseshoe crab innate immune response. the modeled 3d structure of the lalf-lps complex is shown in the right panel (pristovsek et al., 2005) and this model may resemble that of the complex between n-terminal elements of factor c and lps. and the α subunit acts as a pattern-recognition subunit. the α subunit comprises three types of non-catalytic glycosidase-like modules: a single β-1,3-d-glucanase a1-like module, three tandem xylanase a-like modules, and two tandem xylanase z-like modules. of the three types of glycosidase-like modules, the two xylanase z-like modules (z1 and z2) have been identified as independent binding sites for β-1,3-d-glucans (takaki et al., 2002). this observation, taken together with the high degree of sequence identity between z1 and z2 (91 %), suggests that duplicated binding sites for β-1,3-d-glucans may increase avidity to allow stable and specific recognition of pathogens. 65 both z1 and z2 show significant sequence similarity to a carbohydrate-binding module of endoglucanase 5a from the aerobic soil bacterium cellvibrio mixtus (45 % sequence identity). endoglucanase 5a from c. mixtus contains an n-terminal catalytic domain and two tandem repeats of non-catalytic family 6 carbohydrate-binding modules, cmcbm6-1 and cmcbm6-2 (fontes et al., 1998). our recent structural studies of recombinant z2 domain by nuclear magnetic resonance spectroscopy clearly indicate that the ligand-binding site in z2 is located in a cleft on a β-sheet in a predicted β-sandwich structure, which is superimposed onto cleft b in cmcbm6-2 (ueda et al., unpublished data). pattern recognition for β-1,3-d-glucans by factor g may be accomplished by a carbohydrate-binding cleft that is evolutionally conserved among invertebrates and bacteria. a crystal structure of the extracellular domain of mouse fig. 6 the r36/38e mutant of factor c abolishes the autocatalytic activation of factor c by gram-negative bacteria. (a) the wild-type factor c was specifically activated by gram-negative bacteria (e. coli b) but not by gram-positive bacteria (s. aureus) and fungi (pichia. pastoris), whereas the r36/38e mutant was not converted into the active form by e. coli b. the wild-type and the mutant factor c contained a myc-epitope tag at their c-terminal ends and were identified by western blotting with the anti-myc monoclonal antibody (9e10). the zymogen form and the active form (the light chain containing the protease domain) of factor c are indicated by the arrows a and b, respectively. (b) the time course for the autocatalytic activation of the wild-type and mutant factor c in the presence of e. coli b. dectin-1 has been determined (brown et al., 2007), and the putative ligand-binding site of dectin-1 exhibits no structural similarity to that of the predicted structure of z2. the “sliding” of factor g molecules on β-1,3-d-glucans seems be essential to increase the frequency of collision between factor g molecules and resultant autocatalytic activation (takaki et al., 2002). an insoluble β-1,3-d-glucan (curdlan) activates factor g at a minimum concentration of 0.01 ng/ml, whereas a 1000-fold higher concentration of a soluble β-1,3-d-glucan (laminarin) is required for activation (tanaka et al., 1991). β-1,3-d-glucans have specific molecular structures, and high molecular weight β-1,3-d-glucans have higher ordered structures such as triple helices (bohn and bemiller, 1995). therefore, to induce the autoactivation efficiently, the dual binding of the repeating modules may occur in the case of the interaction of factor g with curdlan but not with laminarin. the importance of dual binding of repeating modules to β-1,3-d-glucans for the autoactivation of factor g remains to be examined. pamp recognition by tachylectins given the complexity and diversity of pathogen-associated carbohydrate moieties and the essential nature of specific carbohydrate recognition in innate immunity, it has been proposed that a third biochemical alphabet, the sugar code, is indispensable (gabius et al., 2002). in t. tridentatus, four types of lectins have been identified in the large granule of hemocytes, including tachylectin-1 (saito et al., 1995a), tachylectin-2 (okino et al., 1995), tachylectin-3 (saito et al., 1997), tachylectin-4 (inamori et al., 1999), all of which are secreted upon lps-induced hemocyte exocytosis (fig. 1). although each tachylectin recognizes pamps, their carbohydrate ligand specificities differ. tachylectin-1 interacts with 2-keto-3-deoxyoctonate on the surface of gram-negative bacteria, whereas tachylectin-2 binds to glcnac or galnac and recognizes lipoteichoic acids of gram-positive bacteria. in contrast, tachylectin-3 specifically recognizes a certain sugar moiety on o-antigens of s-type lps from several specific gram-negative bacteria, such as escherichia coli o111:b4. tachylectin-4 also recognizes the o-antigen of e. coli o111:b4 and shows the ligand specificity for colitose (3-deoxy-l-fucose), a unique sugar present in the o-antigen. in addition, an isoprotein of tachylectin-1, named tachylectin-p, has also been found in the perivitelline space of the egg (nagai et al., 1999). the crystal structure of tachylectin-2 clearly points out the importance of multivalency for achieving high specificity and high affinity (beisel et al., 1999). tachylectin-2 (236 residues) belongs to the so-called wd-protein family, characterized by the repetitive sequence of 40-60 residues containing trp and asp residues (neer et al., 1994). tachylectin-2 contains five tandem wd-repeats of 47 residues and adopts a five-bladed β-propeller structure with five equivalent glcnac/galnac-binding site (fig. 7a). each propeller blade has an independent binding site for the ligand. the specific recognition by tachylectin-2 is reinforced by the short distance between the individual binding sites (25 å), according 66 fig. 7 crystal structures of tachylectins-2 and -5a. (a) the 5-fold β-propeller structure of tachylectin-2 in complex with glcnac. (b) oligomeric arrangement of tachylectin-5a subunits around the 4-fold crystallographic axis. to the pentagonal geometry, required to interact with pamps. tachylectin-1 (221 residues) and tachylectin-3 (123 residues), despite lacking significant overall sequence similarity to tachylectin-2, also consist of wd repeats, with six-wd-repeats for tachyectin-1 and two-wd-repeats for tachylectin-3, suggesting β-propeller structures analogous to that of tachylectin-2. ultracentrifugation analysis shows that tachylectin-3 is present in dimer in solution. in contrast, tachylectin-4 (232 residues) is an oligomeric glycoprotein of 470 kda and is homologous to the n-terminal domain of xenopus pentraxin-1 (fig. 4c). the function of the n-terminal domain of pentraxin-1 is unknown. hemolymph plasma also contains several lectins, such as isoforms of tachylectin-1 (chiou et al., 2000; chen et al., 2001) and three types of c-reactive proteins (crps), all of which exhibit functional and structural diversity (iwaki et al., 1999). human crp is an acute-phase reactant, its concentration increasing rapidly in response to stress, injury or infection, and it belongs to a protein family of pentraxin (osmand et al., 1977; tennent and pepys, 1994). horseshoe crab crp is a predominant lps-binding protein and is up-regulated at transcript levels by pseudomonas infection, suggesting the importance of horseshoe crab crp as a conserved molecule for pathogen recognition (ng et al., 2004). crp from the horseshoe crab l. polyphemus forms extended fibrilar structures that encapsulate liposomes in the presence of ca2+ (harrington et al., 2009). furthermore, the membranes of limulus crp-treated bacteria exhibit significantly different mechano-elastic properties than those of untreated bacteria, suggesting the protein's role as a primary defense molecule, acting in the entrapment and killing of potential pathogens. in addition, tachylectins-5a and -5b, with binding specificity for acetyl-group have been identified in hemolymph plasma (gokudan et al., 1999). the overall sequence identity between tachylectins-5a (269 residues) and -5b (289 residues) is 45 %. about two thirds of the total hemagglutinating activity in the hemolymph plasma can be attributed to tachylectins-5 a and -5b, which strongly agglutinate all types of human erythrocytes at a minimal concentration of 0.004 μg/ml, and also agglutinate both gram-negative and gram-positive bacteria. the concentration of tachylectins-5a or -5b in the plasma is at least 10 μg/ml, suggesting that they play an important role in the recognition of invading pathogens at the forefront of the innate immune system. tachylectins-5a and -5b show significant sequence similarity to the c-terminal globular domain of the γ-chain of vertebrate fibrinogens and fibrinogen-related proteins, with pairwise identities ranging from 35 to 51 % and highest for the fibrinogen-like domain of human ficolin. ficolin is composed of an n-terminal cys-containing oligomerization segment followed by a collagen-like domain and the c-terminal fibrinogen-like domain, forming an overall structure that resembles a bundle-of-tulips (matsushita and fujita, 2001; fujita, 2002). in plasma, ficolin exists as a complex with specific serine proteases, the mannose-binding lectin-associated serine proteases, through the collagen-like domain, leading to complement activation after the recognition of invading pathogens. an analogous collagenous domain is absent in the corresponding regions of tachylecins-5a and 5b. 67 68 the crystal structure of tachylecitn-5a is readily superimposed onto that of the c-terminal polymerization domain of the γ-chain of fibrinogen (kairies et al., 2001) (fig. 7b). there is a dramatic structural similarity between these proteins, not only with respect to the overall topology, but also within their ca2+-binding sites. during the final stage of mammalian blood coagulation, thrombin cleaves the n-terminal portion of the fibrinogen α-chain, and the newly created n-terminal sequence containing gly-pro-arg-prois recognized by the polymerization pocket on the γ-chain to form fibrin protofibrils (pratt et al, 1997; spraggon et al., 1997). the polymerization pocket within the γ-chain structurally corresponds to the acetyl group-binding site of tachylectin-5a, a finding that highlights the evolutionary connection between hemostasis and non-self recognition. tachylectin-5a is present in oligomer in hemolymph plasma, and its propeller-like arrangement is evident by electron microscopy (gokudan et al., 1999). given that tachylectins -2 and -5 exhibit virtually no sideor main-chain conformational changes upon ligand binding, it is likely that the polyvalent nature of these molecules underlies their avidity by allowing them to recognize specific densities or clustering patterns ligands on the surface of pathogens. roles of antimicrobial peptides in innate immunity in arthropods, antimicrobial peptides are widely recognized to be very important for host defense against, and killing of, invasive microbes. however, under isotonic conditions (0.5 m nacl concentration for horseshoe crabs), the apparent antimicrobial activities of horseshoe crab antimicrobial peptides are dramatically reduced to those under hypotonic conditions. therefore, the antimicrobial peptides of horseshoe crabs may act as endogenous mediators and regulators in the innate immune system by enhancing such processes as hemocyte exocytosis and conversion of hemocyanin to phenoloxidase (described below). in t. tridentatus, the small granules of hemocytes contain several kinds of cysteine-rich peptides with antimicrobial activities, including tachyplesin (nakamura et al., 1988b), tachystatins (osaki et al., 1999), big defensin (saito et al., 1995b; kawabata et al., 1997), and tachycitin (kawabata et al., 1996). the ic50 values for these antimicrobial peptides under hypotonic conditions are listed in table 1 (osaki et al., 1999). all of the antimicrobial peptides identified in the horseshoe crab have an affinity for chitin, primary target of the innate immune system. tachyplesin significantly inhibits the growth of gram-negative and gram-positive bacteria and fungi (table 1). tachyplesin forms a rigid hairpin loop constrained by two disulfide bridges and adopts the conformation comprising an antiparallel β-sheet connected to a β-turn (kawano et al., 1990; laederach et al., 2002; mizuguchi et al., 2005) (fig. 8a). when viewed as a planar surface, six basic residues are localized on one face, and six hydrophobic residues are distributed on the opposing face. this amphiphilic structure is presumed table 1 antimicrobial activities of horseshoe crab antimicrobial peptides -------------------------------------------------------------------- e. coli s. aureus c. albicans -------------------------------------------------------------------- ic50 (μg/ml) tachyplesin 2.5 0.3 0.2 tachystatin a 25 4.2 3.0 tachystatin b no inhibition 7.4 3.0 tachystatin c 1.2 0.8 0.9 big defensin 2.5 2.5 20 tachycitin 33 56 52 -------------------------------------------------------------------- to be closely associated with its antimicrobial activity. tachystatins a (44 residues), b (42 residues), and c (41 residues) also exhibit a broad spectrum of antimicrobial activity (table 1). of the tachystatins, tachystatin c shows the most potent antimicrobial activity, and tachystatin c, but not tachystatins a and b, additionally exhibits hemolytic activity against sheep erythrocytes. furthermore, tachystatin c has strong cell lysis activity against budding yeast. tachystatins a and b have 42 % sequence identity. tachystatin c contains a disulfide motif analogous to that found in tachystatin a, but otherwise shows very low sequence similarity to tachystatin a. tachystatin a is homologous to ω-agatoxin-iva of funnel web spider venom, a potent blocker of voltage-dependent ca2+ channels (mintz et al., 1992). despite this similarity, tachystatin a exhibits no blocking activity toward p-type ca2+ channel in rat purkinje cells. tachystatin a consists of a cysteine-stabilized triple-stranded β-sheet and shows an amphiphilicity commonly observed in membrane-interactive peptides (fujitani et al., 2002) (fig. 8b). tachystatin a shares structural similarity with ω-agatoxin-iva. interestingly, ω-agatoxin-iva also has antimicrobial and chitin-binding activities. the three dimensional structure of tachystatin a shows no structural homology with well known chitin-binding motifs, suggesting that tachystatin a belongs to a new family of chitin-binding peptides. as expected, tachystatin b shows a significant three dimensional structural similarity to tachystatin a (42 % sequence identity between tachystatins a and b ) (fujitani et al., 2007) (fig. 8c). big defensin has potent antimicrobial activities against both gram-negative and gram-positive bacteria, but not against fungi (table 1). big defensin (79 residues) is distinct from the mammalian defensins with respect to molecular size (29-34 residues for the latter). it is proteolytically cleaved by trypsin into two domains, the hydrophobic n-terminal domain and a c-terminal domain that is homologous to β-defensins. interestingly, the n-terminal domain possesses a more potent antimicrobial activity against gram-positive bacteria than the c-terminal domain. in contrast, the c-terminal domain displays more fig. 8 nmr structures of antimicrobial peptides of the horseshoe crab. (a) ball-and-stick model of tachyplesin. (b) ribbon representation of the energy-minimized average structure of tachystatin a. (c) ribbon representation of the energy-minimized average structure of tachystatin b. (d) ribbon representation of the energy-minimized average structure of big defensin. (e) ribbon representation of the energy-minimized average structure of tachycitin. potent antimicrobial activity than the n-terminal domain against gram-negative bacteria. these data suggest a new sub-class within the defensin family that possesses two discrete functional domains with different antimicrobial activities. it is noteworthy that tachylectins-5a and -5b enhance the antimicrobial activity of big defensin against gram-positive bacteria (gokudan et al., 1999). structurally, big defensin represents a new class within the defensin family; the c-terminal domain adopts a β-defensin structure, whereas the n-terminal domain forms a unique globular conformation (kouno et al., 2008) (fig. 8d). interestingly, the hydrophobic n-terminal domain, but not the c-terminal domain, undergoes a conformational change in micelle solution, which may be associated with the antimicrobial activity against gram-positive bacteria. 69 tachycitin (73 residues) contains five disulfide bridges and has no significant sequence similarity to known antimicrobial peptides. the antimicrobial activity of tachycitin is not strong by itself (table 1). however, tachycitin synergistically enhances the antimicrobial activity of big defensin as demonstrated by a 50-fold reduction in the ic50 value of big defensin against gram-negative bacteria when a small amount of tachycitin is present. the structure of tachycitin is largely divided into the n-terminal domain and the c-terminal domain (suetake et al., 2000) (fig. 8e). in the c-terminal domain, tachycitin forms a hairpin loop connecting a two-stranded β-sheet, a structural motif shared by the chitin-binding site of hevein, an antifungal peptide from the rubber tree hevea brasiliensis (broekaert et al., 1990). in hevein, the aromatic side chains of the two trp residues in this loop directly interact with chitin-derived oligosaccharides. the side chain of tyr and val residues in the corresponding loop of tachycitin are structurally analogous to these two trp residues in hevein. tachyplesin also contains the similar hairpin loop that connects a two-stranded β-sheet. the hydrophobic residues clustered on the one face of their β-hairpin loops possibly participate in chitin-binding sites. roles of tgase-dependent protein cross-linking in the innate immune system in mammals, the blood coagulation cascade culminates in the proteolytic conversion of soluble fibrinogen into insoluble fibrin. the resulting fibrin fibrils are further stabilized by intermolecular ε-(γ-glutamyl) lysine cross-linking by the plasma fig. 9 stablin as a component of the clotting mesh. (a and b) co-localization of stablin with coagulin fibrils. hemocytes were treated with 1 mg/ml lps (salmonella minnesota r595) for 1 h and stained with a monoclonal antibody against coagulogen (a) and a polyclonal antibody against stablin (b). as secondary antibodies, alexa fluor® 488 goat anti-mouse immunoglobulin and rhodamine-conjugated swine anti-rabbit immunoglobulins were used. bar = 10 μm. (c and d) bacterial immobilization by the clotting mesh. hemocytes were treated with e. coli expressing enhanced green fluorescence protein for 1 h, stained with the monoclonal antibody against coagulogen (c), and detected by a cy3-conjugated anti-mouse secondary antibody. e. coli was identified by the fluorescence of enhanced green fluorescence protein (d). bar = 10 μm. (e and f) involvement of stablin in the formation of the clotting mesh. hemocytes were preincubated with 0.1 mg/ml of an antibody against stablin (f) or without the antibody (e). hemocytes were treated with e. coli for 1 h, stained with the monoclonal antibody against coagulogen, and detected by the cy3-conjugated secondary antibody. bar = 100 μm. tgase, factor xiiia (davie et al., 1991). in crustaceans, hemolymph coagulation directly depends on the tgase-mediated cross-linking of a specific clotting protein (a vitellogenin-related protein) without prior proteolytic cleavage (doolittle and riley, 1990; hall et al., 1999; theopold et al., 2004). in horseshoe crabs, hemolymph coagulation cascade triggered by lps or β-1,3-d-glucans promotes in the conversion of coagulogen to coagulin. in the resulting coagulin homopolymers, coagulin monomers associate non-covalently in a head-to-tail manner (bergner et al., 1996; kawasaki et al., 2000). horseshoe crab tgase is not present in hemolymph plasma. it is ordinarily restricted to cytoplasm of the hemocyte and secreted via an unknown mechanism in response to stimulation by lps (tokunaga et al., 1993a, 1993b; osaki et al., 2002). although horseshoe crab tgase neither catalyzes monodansylcadaverine incorporation into coagulin nor cross-links coagulin intermolecularly, it cross-links coagulin to other hemocyte-derived proteins, such as the proline-rich protein, proxin (osaki et al., 2002) and the cysteine-rich protein stablin (matsuda et al., 2007a), resulting coagulin fibrils with enhanced stability. an anti-stablin antibody strongly inhibits the proper formation of the clotting fibrils. proxin and stablin have been detected in the large granules of hemocytes and are secreted by lps-induced exocytosis. in the absence of tgase, proxin non-covalently interacts with coagulin but not coagulogen. moreover, proxin exhibits the specific interaction with stablin (kd = 4.0×10 -9 m). in contrast, stablin interacts with lps and lipoteichoic acids and exhibits bacterial agglutinating activity against both gram-negative and gram-positive bacteria. consequently, stablin co-localizes with coagulin fibrils that are cross-linked with proxin, effectively trapping bacteria (fig. 9). in addition, stablin binds to chitin, a major component of the arthropod cuticle (kd = 1.5×10 -8 m). these data suggest that proxin and stablin promote not only the formation of the stable clotting fibrils but also the immobilization of invading microbes at injured sites. horseshoe crab tgase additionally cross-links carapace-derived chitin-binding proteins, known as caraxins, that are specifically localized to the sub-cuticular epidermis (matsuda et al., 2007b). one of these homologues, caraxin-1, comprises 135 amino acid residues and consists of nand c-terminal heptad repeats that flank a central domain consisting of a pentapeptide tandem repeat structure. recombinant caraxin-1 exists as an oligomer (~20-mer) in solution, and these oligomers are cross-linked by tgase to form an elaborate mesh 70 fig. 10 scanning electron microscopy of caraxin mesh. (a and c) caraxin-1 was incubated with tgase at 37 °c for 16 h. also, hemocytes were stimulated with 1μg/ml lps for 1 h (b and d). both samples were fixed in 4 % paraformaldehyde for 1 h. bars were 20 μm (a and c) and 2 μm (b and d). of honeycomb structures that is distinguishable, by electron microscopy, from the clotting mesh triggered by lps (fig. 10). the α-helical structures of the nand c-terminal domains of caraxin-1 are essential for proper mesh formation. horseshoe crab hemocytes are actively motile, and one of the principal functions of the hemocyte is to seal scars in the cuticle. this function is fulfilled in part by the adherence of hemocytes to injured sites (armstrong, 1985). the wound repair process of l. polyphemus was observed for 180 days by making a cut (1×40×3 mm) on the carapace (bursey, 1977). according to these observations, a coagulation plug is formed within 10 min, and the coagulum is then infiltrated by hemocytes to form a cellular plug within 24 h. the sub-cuticular epithelial cells begin to migrate into the wound after 15 days, and the epithelial cells span the wound between the cut ends of the exoskeleton by day 30, and then probably secrete cuticular components to complete the wound repair process. at the initial stage of this wound repair process sufficient quantities of tgase may be secreted from hemocytes recruited to the site of injury, and immediately activated by ca2+ in hemolymph plasma. at the same time caraxin may be secreted from the sub-cuticular epithelial cells. the resulting cross-linked clotting fibrils and caraxin mesh may function to seal the wound to stop bleeding, serve as a barrier to the entry of pathogens into the interior of the animal via the wound, and operate as a transient extracellular matrix for the migration of epithelial cells that facilitate wound healing (fig. 1). the complement system the vertebrate complement system consists of three activation pathways: the classical, lectin, and alternative pathways (klein and horejsi, 1997; fujita, 2002). the classical and the lectin pathways are serine protease cascades initiated by the recognition of microbes by antibodies and lectins, respectively. although the alternative pathway can be activated spontaneously, it is potently activated upon microbial infection. activation of each of the three pathways results in generation of a proteolytic complex known as the c3 convertase (c4bc2a in the classical and lectin pathways, or c3bbb in the alternative pathway) that plays a central role in promoting downstream responses. the c3 convertase cleaves c3 to generate c3a, a peptide also known as anaphylatoxin that attracts inflammatory cells to sites of infection. the c3 convertase concomitantly generates c3b, a large polypeptide that covalently 71 72 attaches to microbial surfaces and promotes c3 receptor-dependent phagocytosis by leukocytes as well as the formation of the membrane attack complex. vertebrate complement factors d (df) and b (bf) are involved in the activation of the alternative pathway. df is a single-chain serine protease circulating in the blood as an active protease that cleaves bf, resulting in the formation of c3bbb. although several homologs of vertebrate complement factors are known to be present in ascidians, cyclostomes, and echinoderms, the functional analog of df that triggers the alternative pathway has not been identified in protostomes (endo et al., 2006; dodds and matsushita, 2007). the complement-related protein, α2-macroglobulin, has previously been identified in horseshoe crabs (iwaki et al., 1996). recently, a homolog of complement component c3 has been identified in the horseshoe crab c. rotundicauda (crc3), indicating the presence of a complement system capable of promoting the phagocytosis of invading microbes in protostomes (zhu et al., 2005). in a phylogenetic tree of reactive thioester-containing proteins from vertebrates and invertebrates, crc3 shows the highest similarity to c3 sequences from lower deuterostomes and forms a clade with amphioxus c3, cnidaria c3-like protein, and sea urchin c3. however, despite the identification of the key complement component in protostomes, the molecular mechanism underlying c3 activation has remained unknown. in an effort to elucidate the mechanism of c3 activation in horseshoe crabs, we have isolated and characterized an ortholog of c3 (ttc3) from t. tridentatus (ariki et al., 2008). ttc3 consists of 1,716 residues with an overall domain structure that is identical to vertebrate c3, including α2-macroglobulin domains, complement-urchin-bone (cub) domains, a thioester-containing domain, an anaphylatoxin domain, and a c345c domain (fig. 4d). a thioester motif (-cys-gly-glu-gln-) at residues 1004 to 1007 and a catalytic his at the position 1116 are conserved in the deduced sequence. the sequence identity between ttc3 and crc3 (98 %) is considerably higher than that between coagulogens from the two species (90 %) (srimal et al., 1985). ttc3 forms a disulfide-linked three-chain structure (α-, β-, and γ-chains) in hemolymph plasma, unlike vertebrate c3 which is present in a two-chain form. the horseshoe crab complement system promotes the deposition of ttc3b on the surface of gram-negative or gram-positive bacteria in hemolymph plasma. however, evaluation of the ability of pamps to promote the proteolytic conversion of ttc3 to ttc3b revealed that lps, but not zymosan, peptidoglycan, or laminarin, strongly induces this conversion, highlighting the selective response of the complement system to lps stimulation. factor c stored in hemocytes has been identified as an lps sensor and an initiator of hemolymph coagulation in the horseshoe crab innate immune system. as would be expected from these findings, an anti-factor c antibody inhibits both the proteolytic conversion of ttc3 and the deposition of ttc3b on the surface of gram-negative bacteria in hemolymph plasma. moreover, activated factor c present on the surface of gram-negative bacteria directly catalyzes the proteolytic conversion of the purified ttc3, thereby promoting ttc3 deposition. these data indicate that factor c acts as an lps-sensitive c3 convertase on the surface of invading gram-negative bacteria in the initial phase of horseshoe crab complement activation (fig. 1). in the alternative pathway of the vertebrate complement system, the interaction between c3b and bb is essential to form c3 convertase (c3bbb). a homolog of complement bf has been identified in c. rotundicauda (zhu et al., 2005). although physiological function of this bf homolog in the horseshoe crab complement system remains unknown, it is likely that it may be responsible for the formation of the second c3 convertase. ttc3 binds to factor c at kd = 4.9×10 -8m (ariki et al., 2008). ttc3 is present at a concentration of at least 300 μg/ml in hemolymph plasma, whereas the amount of factor c in hemolymph plasma is very low (~10 μg/ml). the relatively high concentration of ttc3 and its high affinity to factor c suggest that factor c exists in a complex with ttc3 in hemolymph plasma, and that formation of this complex is a prerequisite for the immediate activation of ttc3 by factor c on the surface of gram-negative bacteria. factor c antigen has been shown to be present all the tissues examined by western blotting. in contrast, factor g and the proclotting enzyme are not detectable in hemolymph plasma. in contrast to these findings, the anti-factor c antibody exhibits no effect on the deposition of ttc3b on staphylococcus aureus, suggesting the presence of a factor c-independent pathway to initiate the opsonization of gram-positive bacteria (ariki et al., 2008). pamps or complement factors in hemolymph plasma required for the deposition of ttc3b on gram-positive bacteria remain to be examined. factor c and complement bf from c. rotundicauda interact with plasma-derived lectins, including galactose-binding protein, carcinolectin-5, and c-reactive protein (saux et al., 2008). also in t. tridentatus, hemocytes and hemolymph plasma contain several lectins responsible for microbial agglutination (kawabata and tsuda, 2002). therefore, the protease-lectin complexes on the surface of gram-positive bacteria may enhance the deposition of ttc3b. although the complement-dependent clearance system of invading pathogens in horseshoe crabs remains to be examined, phagocytosis of gram-positive bacteria by hemocytes both in vivo and in vitro is inhibited by protease inhibitors, raising the possibility that the proteolytic dependence of opsonization by c3b may underlie phagocytosis by hemocytes (zhu et al., 2005). roles of hemocyanin in the innate immune system in crustaceans and insects, the prophenoloxidase activation system is an important part of the innate immunity, where it acts to detect and kill invading pathogens as well as to synthesize melanin for wound healing and encapsulation of 73 pathogens (cerenius and söderhäll, 2004; nappi et al., 2004). arthropod prophenoloxidases are known to be non-enzymatically activated by treatment with detergents, lipids, or organic solvents (ashida and yamasaki, 1990). in the horseshoe crab as well, the induction of phenoloxidase activity in hemolymph plasma is evident upon similar treatment (nellaiappan and sugumaran, 1996), yet prophenoloxidase has not been identified in horseshoe crabs. arthropod prophenoloxidases have been sequenced, and show significant homology to hemocyanins (aspan et al., 1995; fujimoto et al., 1995; kawabata et al., 1995). both prophenoloxidase and hemocyanin contain two functional copper-binding sites capable of reversibly binding an oxygen molecule (solomon et al., 1996). moreover, the origin of arthropod hemocyanins appears to be an ancient prophenoloxidase-like protein (burmester and scheller, 1996). under physiological conditions, arthropod prophenoloxidases require a proteolytic cleavage for activation by a specific protease (aspan et al., 1995). interestingly, tarantula hemocyanin is found to express phenoloxidase activity after limited proteolysis with trypsin or chymotrypsin (decker and rimke, 1998). in horseshoe crabs, the clotting enzyme or activated coagulation factor b efficiently produces phenoloxidase activity in hemolymph plasma, whereas activated factor c, activated factor g, or trypsin do not (nagai et al., 2000). the phenoloxidase activity in hemolymph plasma disappears upon removal of hemocyanin by ultracentrifugation, suggesting that hemocyanin is involved in the prophenoloxidase activation system in horseshoe crabs. horseshoe crab hemocyanin is composed of six subunits (α, β, γ, δ, ε, and ζ), each of which have a molecular mass of 70 kda and, in their purified forms, independently express phenoloxidase activity. the clotting enzyme converts the α-subunit of hemocyanin to phenoloxidase in a dose-dependent manner, and the resulting phenoloxidase activity reaches a plateau at a 1:1 molar ratio. the proteolytic cleavage of the hemocyanin subunit is not required for the functional conversion, and the zymogen forms of the coagulation proteases, the proclotting enzyme and coagulation factor b, are effective activators. a common structural feature of these two coagulation factors is the presence of an n-terminal clip domain (muta et al., 1990, 1993). homologous clip domains are present in the n-terminal regions of insect prophenoloxidase-activating enzymes (jiang et al., 1998; lee et al., 1998; satoh et al., 1999). therefore, the clip domain of the proclotting enzyme or coagulation factor b may promote the interaction of these factors with hemocyanin effect its functional conversion to an active phenoloxidase. horseshoe crab hemocyanin is also converted to phenoloxidase by treatment with amphiphilic substances such as sds and phosphatidylethanolamine (nagai and kawabata, 2000). consistent with the amphiphilic nature of the antimicrobial peptides secreted from the horseshoe crab hemocyte, tachyplesin interacts with the α-subunit of hemocyanin (kd = 3.4×10 -6 m) and induces its intrinsic phenoloxidase activity. the phenoloxidase activity induced by tachyplesin is inhibited by phenylthiourea, a typical inhibitor of phenoloxidase. although tachyplesin is the most effective activator of hemocyanin, other hemocyte-derived antimicrobial peptides such as tachystatins and tachycitin significantly induce its phenoloxidase activity. mutation of tachyplesin at trp2 or tyr8 and try13 on its hydrophobic face, but not mutation of basic residues on its cationic face, significantly impair its interaction with the α-subunit, implicating the hydrophobic face of tachyplesin in the functional conversion of hemocyanin to a phenoloxidase. horseshoe crab antimicrobial peptides are all capable of binding to chitin, a cell wall component of fungi and also the major structural component of the arthropod cuticle. the antimicrobial peptides likely recognize chitin exposed at sites of injury as well as on the surface of invading microbes. in addition, hemocyanin binds to tachyplesin-coated chitin. although horseshoe crab hemocyanin present at high concentration in hemolymph plasma (~70 mg/ml) and acts as an oxygen carrier under normal physiological conditions, it may be selectively converted to phenoloxidase by the antimicrobial peptides. we hypothesize that the chitin coated with antimicrobial peptides may serve as a scaffold for the binding of hemocyanin and that the resulting phenoloxidase activity acts as trigger for wound healing in the cuticle (fig. 1). in the crayfish pacifastacus leniusculus, an antibacterial peptide astacidin 1 has been identified (lee et al., 2003). interestingly, astacidin 1 is released from the c-terminal part of hemocyanin by a cysteine-like protease and is up-regulated by injection of lps or β-1,3-d-glucans, indicating that hemocyanin acts as a multifunctional protein in the innate immune system. recently, horseshoe crab hemocyanin and 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patthy l. eur. j. biochem. the lccl module. 267: 5751-5757, 2000. vetvicka v, sima p. β-glucan in invertebrates. inv. surv. j. 1: 60-65, 2004. wang xw, tan ns, ho b, ding jl. evidence for the ancient origin of the nf-b/ib cascade: its archaic role in pathogen infection and immunity. proc. natl. acad. sci. usa 103: 4204-4209, 2006. zhu y, thangamani s, ho b, ding jl. the ancient origin of the complement system. embo j. 24: 382-394 , 2005. isj isj 6: s29-s34, 2009 issn 1824-307x review solitary ascidians embryos (chordata, tunicata) as model organisms for testing coastal pollutant toxicity g zega, r pennati, s candiani, m pestarino, f de bernardi department of biology, university of milan, milan, italy accepted march 13, 2009 abstract marine coastal communities are daily exposed to several chemical compounds commonly used in agriculture and industrial activities. therefore, toxicological studies evaluating the effects of these compounds on marine organisms are of primary importance for marine environment preservation. different model organisms are used to perform toxicity tests with potential pollutants, under laboratory conditions. in last decades, solitary ascidians have been selected as valuable model organisms to run bioassays with embryos and larvae. in fact, by in vitro fertilization, it is easy to obtain thousands of embryos, rapidly developing and therefore allowing a fast screen of pollutant toxicity. the aim of this review was to summarize results from toxicity tests, run with heavy metals, organometal and organic compounds, on solitary ascidian development and settlement to evidence that these animals offer several advantages as models to perform these kind of studies. first of all, they have a sensitiveness directly comparable to that of other marine model organisms. moreover, the effects of toxicants on exposed embryos and larvae could be studied using different approaches, from ultrastructure to genetic analysis. finally, since ascidians are chordates morphological and gene expression analyses could provide data for comparative studies with vertebrates. key words: heavy-metals; antifoulants; pesticides; development; tunicates; ascidians introduction marine environment pollution is a concrete risk along densely populated coastal regions, where urban and industrial development could facilitate the dispersal of several chemical agents. therefore, marine coastal ecosystems could be endangered by pollutants, such as heavy metals, pesticides and antifoulants that could be easily detected in the water column or in the sediment of harbours and estuaries (castillo et al., 2006; antizar-ladislao, 2008; bellas et al., 2008). these areas, often very rich in nutrients, host filter-feeders communities encompassing bivalves, serpulids and ascidians. marine mussels have been selected early for the study of coastal pollution impact on marine life. more recently, ascidians have been selected as potential model organisms for testing pollutants toxicity as they offer several advantages for these studies (mansueto et al., 1993; cooper et al., 1995; cima et al., 1996, 2008; bellas et al., 2003). ___________________________________________________________________________ correspondig author: fiorenza de bernardi department of biology university of milan via celoria 26, 20133 milan, italy e-mail: fiorenza.debernardi@unimi.it solitary ascidians (chordata, tunicata) are marine benthic filter-feeders that occur in dense populations along eutrophic coastal habitats, and therefore they could be easily sampled. they are hermaphrodite organisms that reproduce sexually by the simultaneous emission of eggs and sperm. fertilized eggs develop in the water column in about a day into a planktonic tadpole larva that shows some chordate characters, a dorsal hollow neural tube and a notochord flanked by muscle cells. adult solitary ascidians of ciona, phallusia and styela genus are world wide distributed and fertile almost all year round. gametes can be easily obtained by gonoduct dissection and, from in vitro fertilization, it is possible to obtain thousands of synchronously dividing embryos . under laboratory conditions, development is completed in about 16-24 hours in a range of decreasing temperature from 22 to 16°c. for these characteristics, solitary ascidians are valuable and reliable organisms to run toxicity tests on gametes and embryos, for the high number of specimen easy available every time and the rapid development. in last decades, several studies have been made to test the effect of different pollutants on ascidian development that is evaluating the percentage of s29 mailto:fiorenza.debernardi@unimi.it table 1 list of compounds whose toxicity has been tested on solitary ascidians embryos to determine median effective (ec50) concentration on development and settlement. when available the environmental concentration is also listed in bold, together with its reference compounds chemical classification ec50 (µm) embryos ec50 (µm) larval settlement environmental concentration (µm) hg heavy metal 0.22 0.39 0.002 bellas et al., 2004; ospar commission, 2000 cu heavy metal 0.58 1.61 5.67 ″ cd heavy metal 6.42 6.7 0.23 ″ cr heavy metal 226 289 ″ tbt tributyltin organometallic anti-foulant 0.02 0.01 bellas et al., 2005 zinc pyrithione (zpt) zinc 1-oxidopyridin-1-ium-2thiolate organometallic bactericide, antifoulant 0.23 0.11 bellas, 2005 lindane 1,2,3,4,5,6hexachlorocyclohexane organochloride insecticide 15.20 0.004 bellas et al., 2005; ospar commission, 2000 chlorpyrifos diethoxy-sulfanylidene-(3,5,6trichloropyridin-2yl)oxyphosphorane organophosphorus pesticide 15.70 ″ diuron 3-(3,4-dichlorophenyl)-1,1dimethylurea urea derived herbicide 17.80 ″ chlorothalonil 2,4,5,6-tetrachlorobenzene-1,3dicarbonitrile organochloride fungicide, antifoulant 0.12 0.16 0.005 bellas, 2006 sea-nine 211 (kathon 930) 4,5-dichloro-2-octyl-1,2-thiazol-3one organochloride anti-foulant 0.37 0.15 0.013 ″ ″ dichlofluanid n-(dichloro-fluoromethyl)sulfanyln-(dimethylsulfamoyl)aniline organochloride anti-foulant 0.85 0.39 0.017 ″ ″ tolylfluanid n-(dichloro-fluoromethyl)sulfanyln-(dimethylsulfamoyl)-4methylaniline organochloride anti-foulant 0.62 0.28 ″ irgarol 1051 n-tert-butyl-n'-cyclopropyl-6methylsulfanyl-1,3,5-triazine-2,4diamine anti-foulant 8.34 >25.60 0.016 ″ ″ imazalil 1-[2-(2,4-dichlorophenyl)-2-prop2-enoxyethyl]imidazole organochloride triazoleimidazole fungicide 0.67 0.47 pennati et al., 2006; fao, 2001 triadimefon 1-(4-chlorophenoxy)-3,3-dimethyl1-(1,2,4-triazol-1-yl)butan-2-one organochloride triazole fungicide 29.56 ″ fluconazole 2-(2,4-difluorophenyl)-1,3bis(1,2,4-triazol-1-yl)propan-2-ol organofluoride triazole fungicide 74.70 groppelli et al., 2007 s30 http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=mesh&dopt=full&list_uids=67080469 normal larvae hatching from different treatments. in fact, larvae have a simple body plan that allows the rapid screening of malformed specimen. the tadpole larva body is formed by a trunk and a tail. the trunk bears main sensory organs: the three adhesive papillae or palps, situated at its anterior end, and one or two pigmented organs, situated in the sensory vesicle. the palps contain sensory neurons, through which the larva is able to choose a substratum where to settle, and mucus secreting cells to perform permanent attachment (groppelli et al., 2003, pennati et al., 2007). commonly, larvae possess two pigmented organs, the ocellus and the otolith respectively a photoand gravity-receptor, but some larvae could have only one, usually called the photolith, as it perceives both kind of stimuli. the sensory vesicle is the anterior portion of the central nervous system that continues towards the posterior end as a dorsal hollow tube, divided in three portions, the neck, the visceral ganglion and the tail nerve cord. the neck, devoid of neurons, connects the sensory vesicle to the visceral ganglion. this latter contains neurons with ascending projections and motor neurons with descending projections to tail muscle cells (imai and meinertzhagen, 2007). interestingly, tunicates are considered the vertebrate sister group (delsuc et al., 2006) and their embryos and larvae share basic homologies with vertebrates also at the level of the expression of developmental regulatory genes (meinertzaghen et al., 2004). in particular, the availability of ciona intestinalis genome sequences (dehal et al., 2002) could favour the study of toxicant effects on gene expression. for example, a chip for cdna microarray analysis has been developed to investigate gene expression profiles in tbt (table 1) exposed ascidians (azumi et al., 2004). in this review, results from toxicity tests run with heavy metals, organometals and organic compounds, such as pesticides and anti-foulants, on ascidian development will be reported. this overview has the aim of evidencing how it is possible to take advantage of solitary ascidians to perform toxicity studies, with several approaches. effects of heavy metals, organometallic and organic compounds on ascidian development and settlement among pollutants, heavy metals and organometallic compounds showed the highest toxic effects on ascidian development and settlement. exposure to hg, cu, cd and cr of c. intestinalis embryos for 20 h severely reduced percentage of hatching of normal larvae and of settlement. the ec50 (median effective concentration that determines larval malformation) values of hg, cu and cd were very low indicating that these metals could effectively impair development and consequently larval attachment (table 1). moreover, c. intestinalis sensitiveness to such pollutants resulted comparable to what previously reported for other marine organisms commonly used in toxicity test, such as the bivalve mytilus galloprovincialis and the sea-urchin paracentrotus lividus (bellas et al., 2004). in the group of tested organic compounds, organo-metallic ones resulted the most toxic for ascidians such as styela plicata and c. intestinalis. micromolar doses of organotin compounds (tbt, tpt, tcht), blocked development of s. plicata embryos in a stage-dependent manner. in fact, earliest developmental stages, 2-4 cells to gastrula, were more sensitive (cima et al., 1996) (table 2). the ultrastructural analysis of 1h exposed embryos of different stages revealed the presence of electron-dense precipitates in mithocondria, whose membrane were severely damaged. moreover, blastomere shape and adhesion were also affected most probably because organotin compounds could interfere with cytoskeletal proteins. similarly, c. intestinalis embryos exposed from neurula stage for 1 h showed malformed and disorganized blastomeres, lacking cytoskeletal elements. as a consequence, neurulation was blocked (dolcemascolo et al., 2005) (table 2). the effect of tbt was studied also on late developmental stage of c. intestinalis. pre-hatching and swimming larvae exposed for 1 h to 0.1µm tbt showed severe tail malformations. muscle cells had an abnormal distribution along the tail and irregularly shaped nuclei. moreover, the ultrastructure of sarcomeres and muscle mitochondria appeared completely compromised (gianguzza et al., 1996) (table 2). when c. intestinalis embryos were exposed to tbt throughout development (about 20h) development was blocked and ec50 was 0.022µm (bellas et al., 2005). another potent organometallic anti-foulant, zinc pyritione (zpt) showed similar effect on c. intestinalis development and settlement (table 1) (bellas, 2005). pesticides and anti-foulants are the last group of compounds whose toxicity was investigated on ascidian development (table 1). these substances have a broad-spectrum activity and their action on ascidian embryos were studied mainly evaluating dose-depending effects on development. for each compound the ec50 value was calculated (table 1). for some organic pesticides and anti-foulants, such as lindane, chlorpyrifos, diuron, irgarol 1051, triadimenfon and fluconazole, ec50 values were quite high in terms of toxicity, corresponding to micro-molar concentrations. organochloride antifoulants (chlorothalonil, sea-nine 211, dichlofluanid, tolylfluanid) instead resulted the most toxic substances, for their very low ec50 values. moreover, among fungicides, imazalil, that contains two chlorine atoms, showed a similar toxicity for ascidian embryos (bellas et al., 2005; bellas, 2006; pennati et al., 2006; groppelli et al., 2007). effects of the triazole fungicide was also evaluated in terms of teratogenicity as these substances induced specific malformations whose severity was dosedependent. therefore, larval phenotypes obtained after triazole exposure throughout development were classified using a dissection microscope and further characterized by means of histology and immunohistochemistry experiments. triazole exposed larvae showed typical malformation: the trunk appeared shortened, the palps were fused or not completely differentiated, and the sensory vesicle was reduced with displaced pigmented organs (fig. 1a, b). moreover, the anterior nervous s31 fig. 1 control larvae (a, c, e) and larvae developed from embryos exposed to 5 µm imazalil (b, d, f) of the solitary ascidian ciona intestinalis. control (a) and malformed larva (b) showing the typical imazalil induced phenotype. immunohistochemical localization of β-tubulin in control (c, e) and malformed (d, f) larvae, whose anterior nervous network appeared disorganized. bars = 100 µm. network was compromised, as evidenced by immunolocalization of β-tubulin (fig. 1c-f). in these studies, the teratogenic action of triazoles on ascidian development was directly compared with what known on vertebrate embryos, where these fungicides typically affect differentiation of the anterior structures, interfering with retinoic acid catabolism. the authors found evidences that also in ascidians the observed phenotypes could be due to an alteration of retinoic acid signalling (pennati et al., 2006; groppelli et al., 2007). conclusions coastal pollution could stress marine communities determining a decrease in biodiversity for the disappearing of more sensitive species (castillo et al., 2006; bellas et al., 2008). marine benthic invertebrates are easily exposed to toxic compounds commonly used in agriculture, industrial and harbour activities, and have been selected as model organisms to evaluate effects of these substances on life processes. some of the listed inorganic and organic compounds were proved to impair ascidian development at very low doses, ranging from nano to micro-molar concentrations. even if the average environmental concentration of these compounds is lower than their ec50 values on ascidian development (table 1), we believe that the chance of endangering coastal populations of sessile tunicates is a realistic risk. in fact, given the wide production of pesticides (tilman et al., 2001), the possibility of local accumulation by accidental spills must be also considered. similarly, the extensive uses of anti-foulants favour their accumulation in harbours shallow waters (bellas, 2006). moreover, among the substances considered in this review, cu, tbt and imazalil were detected in water or soils in concentrations directly comparable to their ec50 values on ascidian development (table 1). from the conspicuous studies reviewed here, it is clear that, among benthic coastal invertebrates, solitary ascidians are valuable organisms to be considered among models to test toxicity of potential or known pollutants on their development and settlement ability. in fact, it is possible to run toxicity tests on a high number of embryos and to rapidly (1 day) screen the effects. solitary ascidians embryos and larvae can be used in these laboratory studies with different approaches, from exposure bio-assays of different developmental stages to morphological analysis at different levels (ultrastructure, histology, immunohistochemistry). results summarized here evidenced that several common pollutants strongly impaired ascidians development and consequently their dispersal and recruitment phases. s32 table 2 effects of different compounds on the development and/or morphology of larvae of three different ascidian species effective concentrations (µm) developmental stage exposure time (h) target organs effects styela plicata tbt 0.1/1 tpt 0.1 tcht 0.1 2-4 cells, morula, gastrula 1 mitochondria, cytoskeleton block of cleavage cima et al., 1996 ciona intestinalis pre-hatching larvae tbt 0.1 swimming larvae 1 tail muscle cells, mitochondria, cytoskeleton block of hatching/swimming gianguzza et al., 1996 tbt 0.1/10 neurula 1 mitochondria, cytoskeleton block of neurulation dolcemascolo et al., 2005 phallusia mammillata imazalil 5 triadimefon 125 2 cells 10 palps, central nervous system pennati et al., 2006 fluconazole 125 2 cells 18 palps, central nervous system groppelli et al., 2007 recently, 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http://www.myendnoteweb.com/endnoteweb/2.3/release/endnoteweb.html?selectedfolderid=0&searchitem=cima,%20f.&searchfield=all&authorflag=1& http://www.myendnoteweb.com/endnoteweb/2.3/release/endnoteweb.html?selectedfolderid=0&searchitem=bragadin,%20m.&searchfield=all&authorflag=1& http://www.myendnoteweb.com/endnoteweb/2.3/release/endnoteweb.html?selectedfolderid=0&searchitem=ballarin,%20l.&searchfield=all&authorflag=1& http://www.myendnoteweb.com/endnoteweb/2.3/release/endnoteweb.html?selectedfolderid=0&searchitem=cooper,%20e.%20l.&searchfield=all&authorflag=1& http://www.myendnoteweb.com/endnoteweb/2.3/release/endnoteweb.html?selectedfolderid=0&searchitem=arizza,%20v.&searchfield=all&authorflag=1& http://www.myendnoteweb.com/endnoteweb/2.3/release/endnoteweb.html?selectedfolderid=0&searchitem=cammarata,%20m.&searchfield=all&authorflag=1& http://www.myendnoteweb.com/endnoteweb/2.3/release/endnoteweb.html?selectedfolderid=0&searchitem=pellerito,%20l.&searchfield=all&authorflag=1& http://www.myendnoteweb.com/endnoteweb/2.3/release/endnoteweb.html?selectedfolderid=0&searchitem=parrinello,%20n.&searchfield=all&authorflag=1& phagocyotsis of apoptotic cells in invertebrates isj 3: 89-96, 2006 issn 1824-307x minireview mechanisms and roles of phagocytosis in drosophila and caenorhabditis elegans y nakanishi, a shiratsuchi graduate school of medical science, kanazawa university, kanazawa, ishikawa 920-1192, japan accepted september 19, 2006 abstract our understanding of the humoral immune response in both vertebrates and invertebrates has dramatically deepened in the past decade. in contrast, many of the mechanisms and roles of the cellular immune response remain to be elucidated. phagocytosis is at the center of the cellular responses in both innate and adaptive immunity. targets of phagocytosis are either invading microbes or altered self, that is, own cells that have become dispensable or harmful. the selective recognition and engulfment of target cells by phagocytes are achieved through the specific binding of receptors of phagocytes to ligands present on the surface of the target cells. however, these phagocytosis receptors and ligands are still being identified. the fundamental mechanism of phagocytosis appears to be the same in vertebrates and invertebrates, but whether or not genes are evolutionally conserved has yet to be determined. key words: apoptosis; caenorhabditis elegans; drosophila; innate immunity; microbial pathogen; phagocyte; phagocytosis introduction innate immunity is defined as a type of immune responses in which only products of genes already present in germ lines play roles, in contrast to adaptive immunity that uses both germ line genes and genes acquired by rearrangement of existing genes during development (janeway and medzhitov, 2002). simpler organisms like invertebrate animals have only innate immunity, while organisms more complex than jawed fish possess both innate and adaptive immune systems. innate immunity was once thought to be a prototype of the “more sophisticated” adaptive immunity, but it is now apparent that the two systems play individual roles and cooperate to protect against invaders and endogenous insults (hoebe et al., 2004). furthermore, the boundary between the two immune systems is becoming obscure (flajnik and pasquier, 2004). given that either form of immunity consists of humoral and cellular responses, there are two types of humoral and cellular reactions in vertebrates and only one type in invertebrates. ___________________________________________________________________________ corresponding author: yoshinobu nakanishi graduate school of medical science, kanazawa university, shizenken, kakuma-machi, kanazawa, ishikawa 920-1192, japan e-mail: nakanaka@kenroku.kanazawa-u.ac.jp this raises the possibility that the roles of innate immunity in vertebrates and invertebrates are somewhat distinct. if so, a better way to fully understand innate immunity would be to compare the mechanisms and roles of immune responses between vertebrates and invertebrates. previous studies have revealed that at least the mechanistic part of the innate immune response is well conserved between vertebrates and invertebrates although the players are not exactly the same (iwanaga, 2002; brennan and anderson, 2004). while the mechanisms and consequences of the humoral innate immune response have been intensively investigated particularly in insects and mammals (janeway and medzhitov, 2002), many issues still remain to be solved before we can gain a good understanding of the cellular response. provided that the fundamental mechanisms of the cellular innate immune response are almost the same in vertebrates and invertebrates, a smarter way to address these issues is to begin with the analysis of genetically tractable invertebrate animals such as drosophila and caenorhabditis elegans (mylonakis and aballay, 2005). in this minireview, we summarize what is known of phagocytosis, an event at the center of cellular responses, in drosophila and c. elegans, in an attempt to gain a deeper understanding of this phenomenon. 89 mailto:nakanaka@kenroku.kanazawa-u.ac.jp phagocytosis at a glance phagocytosis, a phenomenon whereby cells are engulfed and digested by other cells (aderem and underhill, 1999), is at the center of the cellular immune response in both innate and adaptive immunity; other cellular responses include cell-mediated killing in vertebrates, and cell-mediated killing and encapsulation in invertebrate animals. the cells in charge of phagocytosis are called phagocytes and consist of various types. phagocytes are classified into two groups, professional and amateur cells. professional phagocytes, macrophages as a representative, are full-time executors. in contrast, amateur phagocytes, which exert functions other than phagocytosis most of the time, exhibit phagocytic activity only when it is needed. there is another way to classify phagocytes; that is, phagocytes that circulate through the body and are responsible for phagocytosis in various places, and others that are localized to certain places and engaged in phagocytosis only there. the phagocytes of invertebrate animals have not been intensively characterized compared with those of vertebrates. in many invertebrates circulatory cells (either coelomocytes or hemocytes) exist, several types of which act as professional phagocytes. in the fruit fly drosophila, plasmatocytes are such phagocytes and responsible for the phagocytic elimination of invaders and altered self in many areas of the body (meister and lagueux, 2003). glia and some ectodermal cells also act as phagocytes in drosophila. in contrast, there are no mobile phagocytes in c. elegans; the cells that simply neighbor target cells seem to carry out phagocytosis (gravato-nobre and hodgkin, 2005). phagocytes selectively recognize and engulf cells that are foreign to our body or own cells that have become dispensable. the foreign cells are invading microbes, and the altered self includes cells that have become structurally and/or functionally spent, unwanted, aged, or harmful. removal of the former targets is accomplished to eliminate microbial pathogens that may cause infectious diseases, while that of the latter is necessary for morphogenesis, establishment of tissue functions, and maintenance of tissue homeostasis, i.e. tissue renewal, avoidance of excessive cellular actions, and extermination of pathogenic or noxious materials (stuart and ezekowitz, 2005). failure in the expeditious removal of such “unwanted” cells impairs normal development as well as increases the risk of infectious diseases, inflammation, or autoimmunity. phagocytosis is induced when receptors present on the surface of phagocytes are activated by target cells (aderem and underhill, 1999) (fig. 1). upon the binding of target cells, the intracellular portion of phagocytosis receptors activates a signaling pathway, which in most cases leads to rearrangement of the actin cytoskeleton. as a result, the plasma membrane of phagocytes locally extends and surrounds the target, which is then ingested as membrane vesicles called phagosomes. there is another mode of phagocytosis whereby target cells appear to “sink” into phagocytes without any extension of the plasma membrane. it is presumed fig. 1 recognition and engulfment of target cells by phagocytes. phagocytosis receptors of phagocytes bind to phagocytosis markers present at the surface of target cells. marker-bound receptors activate a signaling pathway that leads to reorganization of the actin cytoskeleton. portions of the plasma membrane of phagocytes then extend and surround targets. finally, target cells are incorporated into phagocytes as phagosomes. that the mode of engulfment varies depending on which receptors are responsible for the induction of phagocytosis as well as the size and shape of target cells (champion and mitragotri, 2006). target selectivity in phagocytosis is achieved through specific molecular recognition between phagocytosis receptors residing at the surface of phagocytes and their ligands or phagocytosis markers on the surface of target cells. the phagocytosis markers are either constituents of the surface of target cells or soluble molecules in body fluid that bind to the targets. the latter are called opsonins, being represented by immunoglobulin and complement of vertebrates that presumably do not exist in invertebrates: it is unclear if opsonin-dependent phagocytosis occurs in invertebrate animals (see below). cell surface constituents specific for microbes are exemplified by a group of molecules called the pathogen-associated molecular patterns that are recognized by a group of receptors of immune cells, the pattern recognition receptor (janeway, 2001). however, it is presumed that pattern recognition receptors and pathogen-associated molecular patterns, which are responsible for the induction of humoral innate immune responses, do not serve, at least in a direct way, as receptors and ligands, respectively, in phagocytosis. mammalian receptors responsible for the phagocytosis of microbes have been characterized, including mannose receptors, fc receptors, and complement receptors (aderem and underhill, 1999; taylor et al., 2005). mannose receptors directly recognize components of the bacterial cell wall, but fc receptors and complement receptors bind to the opsonins immunoglobulin and complement, respectively. several membrane proteins have recently been proposed to be receptors responsible for the phagocytosis of bacteria by phagocytes of drosophila, but the corresponding bacterial ligands have yet to be identified (see below). altered own cells are often induced to undergo apoptosis, a physiologic mode of cell death (wyllie et al., 1980; ellis et al., 1991), and become susceptible to phagocytosis (savill et al., 1993; savill and fadok, 2000). apoptotic cells 90 express phagocytosis ligands that do not exist at the surface of viable cells. these ligands are either endogenous molecules that move to the cell surface or pre-existing molecules at the surface whose structure or distribution changes during apoptosis (lauber et al., 2004). the apoptosis-dependent structural reorganization of the cell surface has been well studied with mammalian cells, and the membrane phospholipid phosphatidylserine is the best-characterized ligand for phagocytosis receptors (fadok et al., 1998; schlegel and williamson, 2001). apoptosis-dependent expression of phosphatidylserine at the cell surface is also observed in cells of drosophila and c. elegans. however, it is unclear whether phosphatidylserine serves as a phagocytosis ligand for apoptotic cells to be recognized by phagocytes of drosophila and c. elegans. to date, no molecule has been identified as a marker for phagocytosis of altered self in drosophila and c. elegans targets are incorporated into phagocytes as membrane vesicles called phagosomes, which are surrounded by the plasma membrane of phagocytes (aderem and underhill, 1999). the main fate of engulfed target cells is decomposition and digestion. phagosomes are processed so that the engulfed targets are killed and degraded mainly by reactive species such as reactive oxygen species and reactive nitrogen species (halliwell, 2006) and lysosomal enzymes, respectively (fig. 2). a key step in the production of reactive oxygen species is the activation of an enzyme called nadph oxidase (underhill and ozinsky, 2002; geiszt and leto, 2004). a prerequisite of the lysosomal degradation of engulfed target cells is the fusion of phagosomes with lysosomes that provide various enzymes for degrading components of engulfed cells. these processes are collectively called phagosome maturation and seem to be under a strict regulation. fig. 2 fate of engulfed cells in phagocytes. engulfed cells are killed and digested by a reactive oxygen-mediated mechanism and lysosomal enzymes, respectively. both reactions occur during structural and functional changes of phagosomes. contents of engulfed cells are sometimes used as antigens for the activation of t lymphocytes and thus for the induction of adaptive immunity. mhc, major histocompatibility complex. on the other hand, engulfed targets are sometimes not completely degraded but subjected to partial digestion. this occurs in a particular type of phagocyte, i.e. antigen-presenting cells such as dendritic cells in vertebrate animals, and processed components of engulfed cells are expressed at the surface together with the major histocompatibility complex for the activation of t lymphocytes (ackerman and cresswell, 2004). microbes, some types of bacteria in particular, possess the ability to resist the actions of phagocytes at various steps (ernst, 2000; coombes et al., 2004). yersinia inhibits phagocytosis itself through the actions of their own proteins that are delivered to phagocytes via the type iii secretion system. salmonella produce, after engulfment, proteins that inhibit the activation of nadph oxidase in phagosomes. listeria sneaks out of phagosomes by disrupting phagosome membranes using their own proteins. other bacteria including leginonella and chlamydia, and the protozoa leishmania also inhibit phagosome maturation. phagocytes of the host organisms counterattack such microbes, in at least two ways as follows. bacteria that have come out of phagosomes are surrounded again by membranes through a process called autophagy (shintani and klionsky, 2004), and phagocytes invaded by long-living microbes are often induced to undergo apoptosis and engulfed together with the microbes by other phagocytes. all these phenomena have been observed with mammalian phagocytes, and whether or not the same is true for phagocytes of invertebrate animals remains to be determined. phagocytosis of microbes in drosophila and c. elegans phagocytosis of bacteria by drosophila phagocytes in drosophila, most humoral immune responses are accomplished by cells of the fat body, a tissue equivalent to the mammalian liver, while blood cells called hemocytes are responsible for most cellular events (hoffmann and reichhart, 2002; mylonakis and aballay, 2005). there exist three cell lineages of drosophila hemocytes, which emerge at different stages of development and participate in particular types of cellular immune responses (meister and lagueux, 2003). two lineages, the plasmatocyte and the crystal cell, differentiate during the second half of embryogenesis. plasmatocytes account for no less than 90 % of circulating hemocytes and act primarily as phagocytes. therefore, these cells are considered to be responsible for the phagocytic elimination of invading microbes and altered self in drosophila. crystal cells are seemingly involved in humoral melanization mediated by phenoloxidase, but their role is not fully understood. the third hemocyte lineage emerges at the larval stage, and these cells called lamellocytes are responsible for the encapsulation of microbes, a cellular response often followed by phagocytosis. differently from mammalian phagocytes such as neutrophils and macrophages, how plasmatocytes act to phagocytose target cells has not been intensively studied. even the identity of phagocytosis receptors and corresponding microbial ligands has been elusive. it is presumed that most phagocytosis 91 receptors of drosophila phagocytes directly recognize molecules present at the surface of target microbes and apoptotic cells, because no molecules present in the hemolymph have been shown in vivo to serve as opsonin. several proteins have been proposed to be phagocytosis receptors (table 1) (cherry and silverman, 2006), although their ligands presumably present at the surface of microbes remain to be identified. the first receptor reported is known as a pattern recognition receptor. in drosophila, a family of proteins called peptidoglycan recognition proteins (pgrps) serves as pattern recognition receptors (brennan and anderson, 2004), as do toll-like receptors in mammals (akira et al., 2006). pgrp-lc, not pgrp-la or -ld, has been identified as a protein, a decrease in the expression of which leads to a decrease in the level of phagocytosis of gram-negative escherichia coli, but not gram-positive staphylococcus aureus, by s2 cells (rämet et al., 2002), a cell line established from hemocytes of drosophila embryos. this approach, namely, a genome-wide screen with rna interference-mediated inhibition of gene expression in phagocytes, was adopted by other investigators, and a group of proteins resembling a human protein called cd36 have been spotlighted. cd36 belongs to the class b scavenger receptor family (sr-b) that is responsible for the control of serum cholesterol levels as well as the removal of denatured serum proteins in mammals (peiser and gordon, 2001). besides these actions, two sr-b proteins, sr-bi of mammals (shiratsuchi et al., 1999) and croquemort of drosophila (franc et al., 1996), have been shown to be involved in the phagocytosis of apoptotic cells. the genome-wide screen revealed that the drosophila sr-b proteins peste and croquemort serve as receptors for the phagocytosis of bacteria by s2 cells. peste targets mycobacteria and lysteria but not e. coli and s. aureus (agaisse et al., 2005), but this specificity seems to change when peste is expressed in mammalian cells (philips et al., 2005). on the other hand, s. aureus is a preferred target for croquemort in phagocytosis by s2 cells (stuart et al., 2005). in addition, sr-ci, a class c scavenger receptor, of drosophila seems to have some role in the phagocytosis of bacteria (rämet et al., 2001; philips et al., 2005). all the aforementioned proteins remain as candidate phagocytosis receptors at present, because their role in vivo in the phagocytic removal of bacteria is yet to be shown. ezekowitz and colleagues extended their rna interference screen with s2 cells, in which the transcription factor serpent was found to be important for the phagocytosis of bacteria (rämet et al., 2002). they searched for gene products whose expression is regulated by serpent and examined their role in the phagocytosis of bacteria by s2 cells. eventually one protein named eater was found to bind to and engulf both e. coli and s. aureus (kocks et al., 2005). eater is a single-path membrane protein with epidermal growth factor (egf)-like repeats in its extracellular portion, and is expressed primarily in plasmatocytes. hemocytes prepared from mutant flies lacking the expression of eater showed a decreased level of the phagocytosis of both e. coli and s. aureus. furthermore, the phagocytosis of those bacteria injected into the adult mutant flies was significantly impaired. the final candidate for a phagocytosis receptor of drosophila is quite unique in that it possesses an immunoglobulin-like structure and is expressed as over one thousand isoforms through alternative splicing in hemocytes and the fat body (watson et al., 2005). this family of proteins, named dscam for immunoglobulin-superfamily receptor down syndrome cell adhesion molecule, bind to e. coli, and larval hemocytes prepared from mutant flies with a reduced level of the expression of dscam showed less activity to phagocytose e. coli than table 1 candidate receptors for the phagocytosis of bacteria by drosophila phagocytes receptor name domains e. coli s. aureus mycobacterium other targets in vivo evidence references___ pgrp-lc peptidoglycan binding yes no nd1 m.luteus2 nd2 rämet et al., 2002 peste scavenger receptor3 no no yes listeria nd philips et al., 2005 croquemort scavenger receptor3 no yes nd nd stuart et al., 2005 sr-ci scavenger receptor4 yes5 nd yes5 nd philips et al., 2005 eater egf-like repeat yes yes nd yes6 kocks et al., 2005 dscam immunoglobulin-like yes nd nd yes7 watson et al., 2005 1. not determined; 2. only viability of flies lacking pgrp-lc expression upon infection with bacteria was examined; 3. class b scavenger receptor family; 4. class c scavenger receptor family; 5. extent of contribution is small; 6. levels of phagocytosis of bacteria by larval hemocytes of flies lacking eater expression are reduced. levels of phagocytosis of bacteria injected into adult mutant flies are reduced; 7. levels of phagocytosis of bacteria by larval hemocytes of flies lacking dscam expression are reduced. 92 those from wild-type flies. in addition, its soluble form is present in the hemolymph. these findings evoke the possibility that dscam serves not only as a receptor but also as an immunoglobulin-like opsonin in the phagocytosis of bacteria. more recently, a family of secreted proteins, called teps for thioester-containing proteins, which serve as opsonins to mediate phagocytosis of microbes by s2 cells, was reported (stroschein-stevenson et al., 2006). of 6 teps tepii, tepiii, and tepvi have been suggested to be involved in the phagocytosis of e. coli, s. aureus, and candida albicans, respectively, though in vivo confirmation is required. teps resemble the complement c3, and the presence of other complement-like proteins has also been noted though their action as opsonins remains to be shown (lagueux et al., 2000; kocks et al., 2003). phagocytosis of bacteria by c. elegans phagocytes the nematode c. elegans maintained in laboratories propagates when fed with e. coli, and the lifespan of this worm is altered when the food source is changed to other bacteria. this suggests that c. elegans is immune to microbial pathogens. the study of innate immunity in c. elegans has begun with the analysis of the humoral immune response, and its mechanism and role have been shown to be basically the same as those in drosophila and mammals (gravato-nobre and hodgkin, 2005; kim and ausubel, 2005; mylonakis and aballay, 2005). infection with some types of bacteria causes apoptosis in germ lines, and a mutant line of the worm lacking the expression of ced-3 and ced-4 is more sensitive to infection. this means that c. elegans uses the apoptotic pathway for innate immune responses against invading microbes. in contrast, the role of cellular responses in the protection of the worm from infectious diseases has not yet been settled. there exists a type of cell that serves as a phagocyte in c. elegans, but the importance of the phagocytic elimination of pathogenic microbes is expected to be small. the phagocytosis of invading microbes by c. elegans phagocytes is inefficient, simply because those phagocytes are not mobile. further studies are needed to clarify the mechanisms and roles of the phagocytosis of microbes by c. elegans phagocytes. phagocytosis of apoptotic cells in drosophila and c. elegans phagocytosis of apoptotic cells by c. elegans phagocytes although the contribution of phagocytosis to defense against the invasion of pathogenic microbes is unclear, apoptotic cells are definitely eliminated by phagocytosis in c. elegans. there are no circulating “professional” phagocytes in this worm, and cells that neighbor dying cells are in charge of phagocytosis. the pioneer work done by horvitz and coworkers has revealed the existence of a set of genes responsible for the induction, execution, and regulation of programmed cell death or apoptosis in c. elegans (ellis et al., 1991; lettre and hengartner, 2006). such genes include those that play roles at the final stage of apoptosis, i.e. the engulfment and degradation of apoptotic cells (gumienny and hengartner, 2001; fig. 3 signaling pathways for the induction of phagocytosis of apoptotic cells. two partly overlapping signaling pathways for the induction of phagocytosis of apoptotic cells, which were genetically identified in c. elegans and are considered to be conserved beyond species, are schematically presented. shown in the parentheses are names of the drosophila counterparts of the c. elegans proteins. reddien and horvitz, 2004; mangahas and zhou, 2005). seemingly there are two partly overlapping signaling pathways, which involve signal mediators conserved beyond species, for the induction of the phagocytosis of apoptotic cells by c. elegans phagocytes (lettre and hengartner, 2006) (fig. 3), although a different opinion was recently provided (yu et al., 2006). the onset of engulfment should be the activation of receptors residing at the surface of phagocytes. this occurs most likely by the binding of marker molecules of phagocytosis present on the surface of target apoptotic cells. presumably there are two sets of phagocytosis receptor and ligand, but only one receptor has been identified to date. a single-path membrane protein named ced-1 has been genetically discovered and shown to serve as a phagocytosis receptor (zhou et al., 2001). there are counterparts of ced-1 in drosophila and human, which are respectively called draper (freeman et al., 2003) and megf10 (callebaut et al., 2003). draper seems to be responsible for the phagocytic removal of apoptotic cells by drosophila phagocytes (freeman et al., 2003; manaka et al., 2004) (see below), but whether or not megf10 plays roles in the clearance of apoptotic cells by mammalian phagocytes remains to be determined. ced-1 contains several structural domains, including egf-like repeats, in the extracellular region and two segments containing tyrosine residues, which are candidate domains for protein-protein interaction, in the intracellular region. the former domain could serve as a site for the binding of an as-yet unidentified phagocytosis ligand, and the latter could be, most likely after phosphorylation of the tyrosine residues, a site for the assembly of downstream signal mediators such as ced-6 (mangahas and zhou, 2005). there has been no information regarding the identity of the other phagocytosis receptor. in contrast to the fact that many molecules have been proposed to be phagocytosis markers in mammals (lauber et al., 2004), no molecules have 93 been shown to be ligands for phagocytosis receptors of c. elegans. c. elegans cells seem to express phosphatidylserine at their surface during apoptosis, but it is not known if externalized phosphatidylserine serves as a phagocytosis marker. the involvement of the c. elegans homologue of the mammalian phosphatidylserine receptor in the phagocytosis of apoptotic cells was reported, but the role for the mammalian protein itself as a phosphatidylserine-recognizing phagocytosis receptor is now doubted. it will be necessary to adopt an experimental strategy other than genetics for the identification of the other phagocytosis receptor and a couple of ligands, but the c. elegans system where mobile phagocytes and suitable cell lines are unavailable does not appear to be suitable for a rapid solution of these issues. phagocytosis of apoptotic cells by drosophila phagocytes in contrast to studies with c. elegans (see above) and mammals (lauber et al., 2004), mechanisms of the phagocytosis of apoptotic cells in drosophila have not been intensely investigated. three membrane proteins have so far been proposed as receptors responsible for the phagocytosis of apoptotic cells by drosophila phagocytes, but no molecules have been identified as phagocytosis markers presumably present on the surface of apoptotic cells. franc and coworkers were the first to identify a receptor responsible for the phagocytosis of apoptotic cells by drosophila phagocytes (franc et al., 1996, 1999). they searched for members of the c-type lectin family in larvae at the third instar stage and found a protein that belongs not to the c-type lectin family but to the sr-b family. this protein, named croquemort, standing for “catcher of death”, is a membrane protein (singleor double-path) and expressed in hemocytes (plasmatocytes/lamellocytes) of embryos and larvae. analyses of flies with a chromosomal deletion including the croquemort locus revealed that croquemort is responsible at least in part for the phagocytosis of apoptotic cells, but not of bacteria, in drosophila embryos, though the latter conclusion recently became controversial (stuart et al., 2005). it is still unknown what molecule at the surface of apoptotic cells croquemort binds to, and as to whether this receptor is contained in either one of the two signaling pathways (see fig. 3) is not clear. another molecule that has been shown to act as a receptor for the phagocytosis of apoptotic cells is draper, the drosophila homologue of the c. elegans phagocytosis receptor ced-1. draper, a single-path membrane protein with egf-like repeats, appears to serve as a receptor for the phagocytic elimination of apoptotic cells by both hemocytes and glia (freeman et al., 2003; manaka et al., 2004). moreover, draper is involved in the removal of axons by glia during metamorphosis for remodeling of the neural network and recovery from injury (awasaki et al., 2006; hoopfer et al., 2006; macdonald et al., 2006). a ligand(s) for draper remains to be identified for both apoptotic cells and degenerating axons, but phosphatidylserine is not likely to be the one (manaka et al., 2004). the externalization of phosphatidylserine occurs also in drosophila cells during apoptosis, but whether or not phosphatidylserine serves as a phagocytosis marker remains to be determined. the third candidate for a drosophila phagocytosis receptor is a protein named six microns under (simu) that is expressed in hemocytes and glia (kurant et al., 2006). the overall structure of simu resembles that of draper; both are single-path membrane proteins containing egf-like repeats in the extracellular region. a decrease in the expression level of simu in a hemocyte-derived cell line as well as in embryos leads to an increase in the number of apoptotic cells. two structurally similar phagocytosis receptors, draper and simu, are co-expressed in hemocytes and glia, and how they cooperate with each other needs to be investigated. perspectives in animals having both innate and adaptive immunity, cooperation between the two systems is necessary to maximize immune responses. for invertebrate animals lacking adaptive immunity, innate immune responses, either humoral or cellular, are more important than those in animals with both types of immunity. it is thus speculated that the role and mode of action of innate immune responses in invertebrate animals are somewhat different from those in vertebrate animals. phagocytosis is at the center of cellular immune responses, and thus clarification of its mechanisms and consequences in invertebrate animals should lead to a better understanding of immunity in general. there are many issues to be solved in order to achieve a full understanding of innate immunity in invertebrate animals. first, it is not known how these animals die upon being infected with some types of microbes. in mammals, septic shock is considered a consequence of excessive host responses to invading microbes, which are mostly mediated by proteins called cytokines secreted from immune cells. is this also true for invertebrates? probably the answer is yes, because cytokine-like proteins are secreted from drosophila hemocytes when insults such as infections with microbes occur (agaisse et al., 2003). the secreted proteins move via the hemolymph and stimulate the fat body, a drosophila tissue equivalent to the mammalian liver, to produce stress proteins. there could thus be septic shock in invertebrate animals, at least in drosophila. taking into consideration that drosophila produce immunoglobulin-like proteins (watson et al., 2005) and complement-like proteins (lagueux et al., 2000; kocks et al., 2003; stroschein-stevenson et al., 2006), the architecture and operation of immunity do not seem to differ between invertebrate and vertebrate animals. it needs to be confirmed in vivo if these proteins act as opsonins to mediate the phagocytosis of microbes and altered self by drosophila phagocytes. conversely, it is necessary to determine to what extent the opsonin-independent phagocytosis of microbes and microbe-infected cells contributes to the immune response against pathogenic microbes in vertebrate animals. another question to be answered is how invertebrates protect themselves against microbes other than bacteria, such as viruses and protozoa. this issue is also important for humans, because arthropods such as 94 insects can act as a vector for parasitic protozoa that cause severe infectious diseases. it is totally unclear how immune surveillance against invading plasmodia, the parasite responsible for malaria, is accomplished in mosquitoes. a study on innate immune responses to viral infections has just started with drosophila (cherry and silverman, 2006). the final general question is whether or not the contents of engulfed cells, microbes or altered self, are used to evoke further immune reactions in invertebrate animals. in mammals, cell contents are sometimes processed and presented as a complex with the major histocompatibility complex at the surface of specialized immune cells, antigen-presenting cells, for the activation of t lymphocytes (ackerman and cresswell, 2004). this seems unlikely to occur in invertebrates lacking adaptive immunity, but the presence of immunoglobulin-like molecules in drosophila catches our imagination. leaving the above-mentioned questions for a future task, issues to be immediately addressed are: 1) to identify marker molecules that exist at the surface of bacteria and are bound by phagocytosis receptors of drosophila phagocytes; 2) to identify a molecule that exists at the surface of apoptotic cells and is bound by c. elegans ced-1/drosophila draper; and 3) to identify the second phagocytosis receptor, after ced-1/draper, and its ligand for the phagocytosis of apoptotic cells by phagocytes of c. elegans and drosophila. acknowledgements we thank an anonymous reviewer for helping us to improve the manuscript. our studies 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mediates cell corpse engulfment in c. elegans. cell 104: 43–56, 2001. 96 mechanisms and roles of phagocytosis in drosophila and caenorhabditis elegans y nakanishi, a shiratsuchi graduate school of medical science, kanazawa university, kanazawa, ishikawa 920-1192, japan abstract introduction phagocytosis at a glance phagocytosis of microbes in drosophila and c. elegans phagocytosis of bacteria by drosophila phagocytes those from wild-type flies. in addition, its soluble form is present in the hemolymph. these findings evoke the possibility that dscam serves not only as a receptor but also as an immunoglobulin-like opsonin in the phagocytosis of bacteria. more recently, a family of secreted proteins, called teps for thioester-containing proteins, which serve as opsonins to mediate phagocytosis of microbes by s2 cells, was reported (stroschein-stevenson et al., 2006). of 6 teps tepii, tepiii, and tepvi have been suggested to be involved in the phagocytosis of e. coli, s. aureus, and candida albicans, respectively, though in vivo confirmation is required. teps resemble the complement c3, and the presence of other complement-like proteins has also been noted though their action as opsonins remains to be shown (lagueux et al., 2000; kocks et al., 2003). phagocytosis of bacteria by c. elegans phagocytes phagocytosis of apoptotic cells in drosophila and c. elegans phagocytosis of apoptotic cells by c. elegans phagocytes phagocytosis of apoptotic cells by drosophila phagocytes perspectives references tributiltyn-induced effects on mapk signaling in ascidian embryos isj 6: s87-s94, 2009 issn 1824-307x research report effects of tributyltin chloride in ascidian embryos: modulation of kinase-mediated signalling pathways f damiani, m gianguzza, g dolcemascolo dipartimento di biopatologia e metodologie biomediche, sez. di biologia e genetica, università di palermo, palermo, italy accepted march 13, 2009 abstract we studied the effects of various tbt concentrations by assaying the activity of erk 1/2 (p44/42) and phospho-erk1/2 (phospho-p44/42), proteins with a key role in ascidian development, and tyrosine kinase-dependent pathway. the effects of this xenobiotic and the role of some signalling mechanisms on ascidian embryos were examined by using western immunoblotting. the tyrosine phosphorylation pattern in the ascidians ciona intestinalis and phallusia mammillata development was examined and different levels of protein phosphorylation were found as a response to tbt at µm range. to determine whether another key signalling pathway was activated, the effects of tbt on the phosphorylation state of a component of tyrosine kinase-mediated signal transduction mapk, erk 1/2 (p44/42) were evaluated. embryos of ciona intestinalis exposed to 0.1, 0.25 and 0.5 µm tbt showed a slight decrement in the level of phosphorylated erk, while a remarkable decrement in level of phopshorylated erk were observed at higher tbt concentrations (0.5 µm to 10 µm). these data indicated that exposures to tbt induced changes in the total pattern of phosphotyrosine and in the phosphorylation levels of erk 1/2 but there were no changes on the overall level of total erk in ascidian embryos. key words: tributyltin-induced effect; tyrosine kinase signalling; mapk; erk (p44/42); ascidian embryos introduction among the class of organotin compounds, tributyltin (tbt) is well known. organotin compounds have many applications, which include use in pvc, as catalyst in chemical reactions, agricultural pesticides, glass coatings and food packaging materials (forsyth et al., 1993; ohno et al., 2002) and antifungal treatments for textile polymers (allsopp et al., 2000, 2001). in particular, tbt has been used in marine antifouling paints leading to the widespread distribution of tbt and its breakdown products in the marine sediments and biota (elgethun et al., 2000; connelly et al., 2001; de brito et al., 2002; lee et al., 2005). high levels of tbt dissolved in fresh and seawater impair reproduction, by inhibiting embryogenesis and larval development, in a variety of marine organisms (coelho ___________________________________________________________________________ corresponding author: francesca damiani dipartimento di biopatologia e metodologie biomediche sez. di biologia e genetica università di palermo via divisi 83, 90133 palermo, italy e-mail: francescadamiani@unipa.it et al., 2006; beiras et al., 2008). in invertebrates, symptoms of such a tbt exposure includes the development of male sexual characteristics as a penis and vas deferens by female (imposex) (miller et al., 1999; ten hallers-tjabbes et al., 2003; santos et al., 2004). tbt effects have been studied in many marine organisms including ascidians, in which immunotoxic effects such as decreasing of phagocytic activity and phenoloxidase activity have been observed (cooper et al., 1995; cima et al., 1998; cima and ballarin, 2000; tujula et al., 2001; arizza et al., 1995; cima et al., 1995). ascidians are an useful model for developmental biology, and they are also sensitive bio-indicators of environment degradation. previous light and electron microscope studies on ciona intestinalis embryos and larvae incubated in tbt chloride solutions showed morphological and fine structural changes in the embryos and larvae. in particular, changes in cytomembranes and mitochondria and anomalous blastomere arrangement during gastrulation were found (mansueto et al., 1993; gianguzza et al., 1996; dolcemascolo et al., 2005). s87 mailto:francescadamiani@unipa.it in ascidian embryos, a fibroblast growth factor (fgf)-like signal has been proposed to be involved in induction of notochord and mesoderm formation (nakatani and nishida, 1994; kim and nishida, 1999). a main pathway is a protein kinase transduction pathway, which include ras, raf, mek and mitogen-activated protein kinase (mapk). kim and nishida (2001) suggested that a mek-mapk signalling cascade is widely involved in embryonic induction in ascidians. mapks are serine/threonine kinases that transduce signals from the plasma-membrane to the nucleus (garrington and johnson, 1999; cobb and goldsmith, 2000) and they play a critical role in controlling cell survival, proliferation, and differentiation (chang and karin, 2001). three major subfamilies have been characterized, including the extracellular signal-regulated kinases (erks), the cjun n-terminal kinases (jnks), also known as stress-activated protein kinases (sapks) and the p38-mapks (kyriakis et al., 1996; seger et al., 1995; cohen, 1997). mapks have also a key role in responding to a wide variety of environmental stresses (storey and storey, 2001). canesi et al. (2004a) have shown that in hemocytes of the mussel mitylus galloprovincialis, 17β-estrediol (e2) induced shape changes, lysosomial membrane destabilization and release of hydrolytic enzymes by activation of the stress-activated p38 mapk and signal transducers and activators of transcription (stat). among signalling mechanisms, tyrosine kinasedependent pathways are triggered by citokines, growth factors and hormones and they are implicated in cell signalling, cell growth, differentiation and apoptosis (fischer, 1999). different stressors such as heavy metals, prooxidants and pollutants are known to stimulate tyrosine kinase signalling (rahman et al., 1993; nakashima et al., 1994; katano et al., 1995; burlando et al., 2006). therefore the possibility exists that both signal transduction pathways (tyrosine phosphorylation and mapk) could be used to study signalling mechanisms in organisms under enviromental stress. in the present paper, the effects of the tbt treatments on cell signalling, variation of protein tyrosine phosphorylation levels and erk module in embryos of ciona intestinalis and phallusia mammillata embryos were examined by the immunoblotting method. materials and methods animals adult specimens of ciona intestinalis and phallusia mammillata were collected from the gulf of palermo and termini imerese harbour (palermo). they were maintained in sea water and kept in aerated aquaria (200 liters). ascidians (about 100 for every experiments set) were held at 18-20 °c for no more than 7 days before use and were daily fed with various food types including freeze-dried rotifers, green unicellular algae and artificial diet. tbt chloride solutions concentrated stock solution of tributyltin (iv) chloride (schering bergkamen, germany) was obtained by dissolving the compound at 10 mm concentration in dimethylsulfoxide (dmso). working solutions were obtained by further diluitions of the stock in millipore-filtered seawater (fsw). these solutions were diluited at 0.1, 0.5, 1, 10, 50 and 100 µm final concentrations. experimental procedure in each experiment female and male gametes were removed from gonoducts and transferred into agar-coated syracuse dishes containing filtered sea water at 22 °c for cross-fertilization. ten min after fertilization, sperm excess was removed and sea water renewed. gastrulae were separated to form the following groups: (1) control a (ctrl): c. intestinalis or p. mammillata gastrulae in fsw (2) control b: c. intestinalis gastrulae in 0.1 % dmso (3) c. intestinalis gastrulae treated for 60 min with 0.1 µm, 0.5µm, 1µm, 10 µm tbt chloride solution (4) p. mammillata gastrulae treated for 60 min with 0.5µm, 1µm, 10 µm, 50 µm, 100 µm tbt chloride solution the final dmso concentration chosen to make up the experimental solution was 0.1 %; this represents a no-toxic concentration which is less than the one reported by bellas et al. (2005); in fact the controls treated with this concentration of dmso did not show significant differences with controls in fsw. electrophoresis and western blotting the levels of tyrosine phosphorylation and phosphorylated erk in whole cell extracts from ascidian embryos were determined using specific antibodies. different lots of ascidian embryos were incubated with tbt solutions for 60 min. then the sample were centrifugated and lysed in buffer (300 mm nacl, 50 mm tris-hcl ph 7.6, 0.1 % triton, 1 % protease inhibitor cocktail (sigma-aldrich), 4 mm edta, 2 mm sodium orthovanadate, 10 mm sodium pyrophosphate and 100 mm sodium fluoride) on ice for 120 min. the lysates were centrifuged at high speed (10.000xg) for 15 min and an aliquot of the supernatant was assayed to determine protein concentration by the bradford method (bradford, 1976). equal amounts of proteins (30 µg) were separated by 12 % sds-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (pvdf) membrane (immobilon-p, millipore, billerica, ma, usa) in 0.1m 3-(cyclo-hexylamino)-1propanesulfonic acid (caps, sigma-aldrich), ph 11; 10 % methanol, at 170 ma for 45 min. membranes were stained with ponceau s, incubated in block solution (3 % albumin from bovine serum, 10 % fetal bovine serum in phosphate buffer), and probed overnight with anti-phosphotyrosine antibody s88 fig. 1 effects of tbt chloride on protein tyrosine phosphorylation in c. intestinalis gastrulae. protein extracts from ascidian embryos were subjected to 12 % sds-page followed by western blot using anti-phosphotyrosine antibody. (a): representative blot obtained from embryos controls and tbt treatment (0.10.250.5 μm); (c): representative blot obtained from embryos control and tbt treatment (0.5110 μm). (b) and (d): densitometric analysis of phosphotyrosine bands; data plotted on bar charts represent the mean intensities (±sd, n = 3) obtained from all the bands of each lane. statistical evaluation of means were carried out using the student’s t test and statistically significant differences as compared to the control are indicated by asterisks. *p < 0.05, **p < 0.01, ***p< 0.001. (py20; santa cruz biotechnology, ca, usa). after 5 washes with washing solution (1x phosphate buffer, 0.1 % tween-20) the membranes were incubated with alkaline phosphatase-conjugated secondary antibody (promega corporation madison, usa). then the membranes were washed with alkaline phosphatase buffer (0.1 m tris hcl, 0.1 m nacl, 5 mm mgcl2, ph 9.5) and proteins were detected with 5-bromo-4chloro-3-indolyl phosphate/nitroblue tetrazolium liquid substrate system (bcip/nbt liquid substrate system sigma, saint louis, ms, usa). for p44/42 map kinase (erk 1/2) and phospho-p44/42 map kinase (p-erk 1/2) protein level evaluation, the samples were separated by sds-polyacrylamide gel electrophoresis, transferred to pvdf membrane and then the membranes were probed overnight with specific antibodies against erk 1/2 (cell signalling technology, beverly, ma, usa) and perk 1/2 (cell signalling technology), respectively. protein corresponding to erk 1/2 and p-erk 1/2 were indentified by using the detection protocol mentionated earlier. data analysis data from desitometric analyses of western blots are means±sd of three independent experiments. statistical evaluation of the data was performed with the student’s t test and p< 0.05 was assumed to be statistically significant. results effect of tbt chloride on tyrosine phosphorylation proteins pathway tyrosine phosphorylation as marker for signal transduction was examined. western blot analyses with a monoclonal antibody that specifically recognizes phosphorylated tyrosine residues allowed to detect different levels of phosphorylation in various proteins obtained by treating different embryos of c. intestinalis and p. mammillata with tbt. s89 fig. 2 effects of tbt chloride on protein tyrosine phosphorylation in p. mammillata gastrulae. protein extracts from ascidian embryos were subjected to 12 % sds-page followed by western blot using anti-phosphotyrosine antibody. (a): representative western blot showing variations in protein phosphorylation of p. mammillata gastrulae after tbt treatment (0.511050100 μm). (b): densitometric analysis of phosphotyrosine bands; data plotted on bar charts represent the mean intensities (±sd, n= 3) obtained from all the bands of each lane. statistical evaluation of means were carried out using the student’s t test and statistically significant differences as compared to the control are indicated by asterisks. **p < 0.01, ***p < 0.001. exposure of c. intestinalis embryos at gastrula stage to 0.1µm, 0.25µm or 0.5 µm tbt for 60 min induced only a slight increase of phosphorylated proteins around 116 kda and 180 kda. (figs 1a, b). when embryos of c. intestinalis were treated with 0.5 µm, 1 µm and 10 µm tbt solution, they showed an increase of phosphotyrosine levels, reaching, at 10 µm tbt exposure, a significant variation of about 1-fold over the control (p<0.001) (figs 1c, d). in particular, phosphorylated proteins of 36 kda, 48 kda, 58 kda, 90 kda and 180 kda were mainly identified. densitometry analysis values of the protein patterns were presented as mean density of the total bands in each sds-page lane. the same experiments were carried out in p. mammillata gastrula stage by using a 0.5-100 µm tbt. at 10 µm tbt an increase of phosphorylation level of proteins with molecular size nearby 48 kda and 55 kda was found. the bands nearby 36 kda, showed a slight increase at 0.5 µm e 1 µm and then decrease until the control levels at 10 µm (fig. 2a). because variations of the densitometric analysis were evaluated as a mean of the total pattern of tyrosine protein phosphorylation, the decrease of a single band gives an unimportant contribution respect to the density increase of the other proteins. as shown in fig. 2b, the highest increase in tyrosine phosphorylation was found at 10 µm tbt (0.18-fold above the control; p< 0.001); exposure of embryos to 50 and 100 µm tbt resulted in a remarkable decrement in tyrosine phosphorylation levels of total pattern (0.6and 0.7fold beneath the control respectively; p < 0.01). effect of tbt chloride on p44/42 and phospho p44/42 levels the erk module responds primarily to growth factors and mitogens and stimulates transcriptional responses in the nucleus. generally, activation of an erk signalling pathway has a role in mediating cell division, migration and survival. to determine whether erk 1/2 signalling pathway was activated in response to tbt treatments, whole embryos extracts were prepared and immunoblotted with antibodies specifically recognizing the phosphorylated and active forms of erk 1/2 (p44 and p42). at first a putative protein was identified in ciona genomic database (blast genomic database and swiss prot, embl) showing about 90 % of sequence similarity with mammalian erk1 and erk2. exposure of c. intestinalis embryos to 0.1 µm, 0.25 µm, and 0.5 µm tbt induced only a slight decrease in the level of phosphorylated erk (fig. 3a, top panel); at 0.5 µm of tbt exposure the average decrement was 0,4-fold beneath the control (p< 0.05) (fig. 3b), whereas the overall p44/42 levels remained did not change as shown by representative experiment in fig. 3a (bottom panel). as shown in fig. 4 a (top panel), when higher tbt concentration, from 0.5 µm to 10 µm, a remarkable dose-dependent erk phosphorylation inhibition was evident, and an average decrease of 0.85-fold beneath the control at 10 µm tbt (p < 0.001) (fig. 4b) was reached. there were no changes about endogenous levels of total erk due to tbt treatment (fig. 4a, bottom panel). high concentrations of tbt inhibit erk signalling as shown by above mentioned results. s90 fig. 3 effects of tbt chloride on erk 1/2 (p44/42) and phospho-erk1/2 (phosphop44/42) in c. intestinalis gastrulae. (a, top panel): representative experiment of phopho-erk 1/2 levels obtained from embryos of control (ctrl) and embryos treated with tbt (0.10.250.5 μm). (a, bottom panel): representative experiment of erk 1/2 levels at the same concentration above-mentionated. (b): densitometric analysis of phospho-p44/42 bands. results are means for three independent experiments (mean±sd). statistical evaluation of means were carried out using the student’s t test and statistically significant differences (p < 0.05) as compared to the control are indicated by an asterisk. discussion ascidians represent an intriguing candidate experimental system for studying the effects of environmental stress. in fact, these organisms are able to survival in a wide range of marine pollutions and some species are abundant in highly transformed and altered environments such as harbors and industrial areas. for this reason, numerous studies have outlined the importance of this group as pollution bio-indicators (papadopouolu et al., 1972; papadopouolu and kanias, 1977; bell et al., 1982; galletly et al., 2007). among the various signal transduction pathways involved in response to environmental stress, both tyrosine kinase signalling and mapks appear to play a significant role (burlando et al., 2006; schaffer and weber, 1999; widmann et al., 1999; kyriakis and avruch, 2001). to further elucidate molecular mechanisms affected by tbt exposure we studied the two signal transduction pathways above mentioned. they have an important role in the response of marine organisms to pollutant, and are considered as biomarkers. different stressors are known to stimulate tyrosine kinase activities and this could explain a wide spectrum of effects produced by pollutants on different organisms. at the beginning, we examined the effects of tbt exposure on tyrosine kinase signalling in ascidian embryos by western immunoblotting. to evaluate whether the effects of the xenobiotic might be associated with a change in tyrosine phosphorylation levels, embryos of c. intestinalis at gastrula stage, were exposed for 60 min to µm range of tbt solution. the treatments with 1 µm and 10 µm tbt, showed an enhanced phosphorylation level for proteins ranging from 36 and about 180 kda. the fold increase in phosphotyrosine levels, reached a significant variation of about 1-fold over the control (p < 0.001) at 10 µm tbt, compared to the control. in p. mammillata embryos at gastrula stage, the exposure to 10 µm tbt, induced a significant increase in phosphotyrosine levels (p< 0.001). at higher tbt concentrations (50 µm and 100 µm) a remarkable decrease in the phosphorylated protein pattern was observed. the possibility exists that such a decrease can be linked to apoptotic or necrotic events caused by high tbt concentrations. taking in account total pattern, the highest phosphorylation values were found after treatment with 10 µm tbt. it has been suggested that distinct upstream mechanisms exist leading to apoptosis and necrosis by different concentrations of an organotin compounds (gunasekar et al., 2001; jurkiewicz et al., 2004). although further research are needed to elucidate the role of tyrosine kinase signalling and mechanism of tbt-induced apoptosis, the here reported results suggest that protein tyrosine phosphorylation may represent a key element in the signal transduction of ascidian embyos exposed to marine pollution. in this respect these embryos could be used for detecting the involvement of cell signalling in organisms exposed to pollutant or stressors. mapk cascades are important amplifying modules that can transduce stress signals into cellular responses (kultz and avila, 2001; poonam et al., 2002; ranganna et al., 2002). studies on the characterization and function of mapk modules have been carried out by using mammalian models and non mammalian experimental systems, for i.e. xenopus laevis, drosophila melanoganster and caenorhabditis elegans (widmann et al., 1999), and recently mussel hemocytes or isolated digestive gland cells (canesi et al., 2001, 2002). in the present study the stimulation of mapk signal transduction pathways by tbt treatment was examined in ascidian embryos. in particular, we observed the response of proteins tyrosine phosphorylation and erk 1/2 signalling pathway to tbt exposure. the erk signalling pathway responds mainly to growth factors and mitogens, stimulating s91 fig. 4 effects of tbt chloride on erk 1/2 (p44/42) and phospho-erk1/2 (phosphop44/42) in c. intestinalis gastrulae. (a, top panel): representative experiment of phopho-erk 1/2 levels obtained from embryos of control (ctrl) and embryos treated with tbt (0.51and 10 μm). (a, bottom panel): representative experiment of erk 1/2 levels at the same concentration above-mentionated. (b): densitometric analysis of phospho-p44/42 bands. results are means for three independent experiments (mean ±sd). statistical evaluation of means were carried out using the student’s t test and statistically significant differences as compared to the control are indicated by asterisks. *p < 0.05, **p < 0.01, ***p < 0.001. transcriptional responses and its activation has a role in mediating cell division, migration and survival (garrington and johnson, 1999; cobb and goldsmith, 2000). as regards the role of erk 1/2 signalling pathway, the results of our studies revealed that 0.1 µm, 0.25 µm and 0.5 µm tbt induced only a slight decrement in the level of phopshorylated erk, while the levels of total erk were unvaried. higher tbt concentration up to 10 µm leads to a remarkable decrement in the level of phopshorylated erk, but no effects on endogenous levels of total erk were found. these results indicate that these tbt exposures caused an inhibition in the phosphorylation of erk 1/2 while not changing their overall levels. it could be suggested that tbt, a strong toxic agent, could interfere with normal mechanisms of signal transduction involved in ascidians embryo development. there are indications that the mapk signalling cascade, mediated via the erk family, plays a role in the onset of apoptosis during the embryogenesis (kling et al., 2002; yao et al., 2003) and that mapk plays a pro-apoptotic role during the ascidian metamorphosis. in fact, chambon et al. (2002) have demonstrated that the activation of the c. intestinalis mapk/erk (ci-erk) in tail cells precedes the wave of apoptosis, suggesting that the phosphorylated form of ci-erk transduces the death-activating signal in tail tissues during metamorphosis, whereas the inhibition of ci-erk blocks metamorphosis. moreover it has been proposed that mitogenactivated protein kinase (mapk) signalling might be involved in the regulation of hsp expression in blue mussels (anestis et al., 2007). batel et al. 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2002. widmann c, gibson s, jarpe mb, johnson gl. mitogen-activated protein kinase: conservation of a three kinase module from yeast to human. physiol. rev. 79:143-180, 1999. s94 19 isj 15: 19-30, 2018 issn 1824-307x research report sequence features, expression profiles and biochemical characteristics of a sigma class glutathione s-transferase gene (aigstσ) from bay scallop argopecten irradians m wang1, 3, b wang1, m liu1, k jiang1, l wang1, 2 1key laboratory of experimental marine biology, institute of oceanology, chinese academy of sciences, qingdao 266071, china 2laboratory for marine biology and biotechnology, qingdao national laboratory for marine science and technology, qingdao 266237, china 3research platform for marine molecular biotechnology, qingdao national laboratory for marine science and technology, qingdao 266237, china accepted december 5, 2017 abstract glutathione s-transferases (gsts) are a class of enzymes that facilitate the detoxification of xenobiotics and also play important roles in innate immunity. in the present study, a novel sigma class gst gene (designated as aigstσ) was cloned from the bay scallop argopecten irradians via rapid amplification of cdna ends (race) technique. the complete cdna sequence of aigstσ consisted of a 5’ untranslated regions (utr) of 48 bp, a 3’ utr of 113 bp with a poly a tail and an open reading frame (orf) of 618 bp. the orf encoded a polypeptide of 205 amino acid residues with a calculated molecular mass of approximately 23.11 kda and a theoretical isoelectric point of 5.354. the deduced amino acid sequence of aigstσ contained a gst_n domain and a gst_c domain, and exhibited high similarity with other reported sigma class gsts. in the phylogenetic tree, aigstσ was located in the sigma class gsts sub-branch. the aigstσ mrna transcripts were constitutively expressed in the tissues of hemocytes, muscle, mantle, gill, hepatopancreas and gonad, with the highest expression level in hemocytes, and the mrna expression levels of aigstσ were significantly up-regulated in hemocytes after various pathogen associated molecular patterns (pamps) stimulation. the purified recombinant aigstσ protein exhibited catalytic activity against the common substrate 1-chloro-2, 4-dinitrobenzene (cdnb) with low thermal stability and narrow optimum ph spectrum. all these results indicated that aigstσ was a fragile but efficient antioxidant enzyme and was potentially involved in the innate immune responses of scallop. key words: argopecten irradians; glutathione s-transferase; innate immunity introduction the innate immunity is almost the solo defence mechanism for invertebrates that protects hosts against microbial invaders (song et al., 2015). in the innate immune defence mechanism, hemocytes can phagocytize and kill the microbial pathogens (lu et al., 2013; chen et al., 2014; wang et al., 2014). when the host is attacked by microbial invaders, phagocytosis is activated with high oxygen consumption named the respiratory burst and followed by mass reactive oxygen intermediates (roi) and reactive oxygen species (ros) production ___________________________________________________________________________ corresponding author: lei w ang key laboratory of experimental marine biology institute of oceanology chinese academy of sciences qingdao 266071, china e-mail: wanglei@qdio.ac.cn (shao et al., 2017). therefore, organisms employ the antioxidant system to maintain roi and ros at the normal physiological levels (zhang et al., 2017a, b). as an essential kind of antioxidant enzymes, glutathione s-transferases (gsts, ec 2.5.1.18) are a superfamily of multifunctional phase ii enzymes primarily catalyzing reduced glutathione to both endogenous and exogenous electrophiles (sheehan et al., 2001). gsts have been identified from the cytosol, mitochondria and microsomes of all the prokaryotic and eukaryotic organisms that have been studied (raza et al., 2002). generally, based on their primary and tertiary structures, substrate and inhibitor specificity, and immunological cross reactivity, gsts could be grouped into at least fifteen classes, which were termed as alpha (α), beta (β), delta (δ), epsilon (ε), kappa (κ), lambda (λ), mu (μ), omega (ω), phi (φ), pi (π), sigma (σ), tau (τ), theta (θ), zeta (ζ) and rho (ρ) (wilce and parker, 1994). 20 table 1 primers used in the present study primer sequence (5`-3`) brief information adaptor primer ggccacgcgtcgactagtac anchor primer for 3` race adaptor primer-oligo (dt) ggccacgcgtcgactagtact17vn olido (dt) for cdna synthetize aiactin-qrt-f caaacagcagcctcctcgtcat internal control for real-time pcr aiactin-qrt-r ctgggcacctgaacctttcgtt internal control for real-time pcr aigstσ-cds-f atgccttcctacaaacttatctac gene specific primer for cds aigstσ-cds-r ttagatcacgctctcgggacgcga gene specific primer for cds aigstσ-qrt-f ctgatccgtctcgctttcgct gene specific primer for real-time pcr aigstσ-qrt-r gctgtttcccgtccacttcca gene specific primer for real-time pcr aigstσ-race-f1 cccaaatttgccgaaatc gene specific primer for race aigstσ-race-f2 agttgaacccagattgtttgaagg gene specific primer for race m13-47 cgccagggttttcccagtcacgac vector primer for sequencing rv-m gagcggataacaatttcacacagg vector primer for sequencing t7 acatccactttgcctttctc vector primer for sequencing t7-ter tgctagttattgctcagcgg vector primer for sequencing among all the gsts classes, sigma class gst (gstσ) comprises one of the largest gst subfamilies identified from invertebrates to vertebrates, which was believed to evolve from ancestral gst genes and exhibit high levels of enzymatic activity toward the common substrate 1-chloro-2, 4-dinitrobenzene (cdnb) (flanagan and smythe, 2011). recently, several sigma class gsts were identified and investigated in marine invertebrates (boutet et al., 2004; lee et al., 2007; wan et al., 2008; ren et al., 2009; li et al., 2012; umasuthan et al., 2012; yang et al., 2012; zhang et al., 2012a, b; wang et al., 2013a; li et al., 2015). among these sigma class gsts, the abgstsigma gene from abalone haliotis diversicolor was significantly expressed in the hemocytes, gill, mantle and digestive gland of bacteria-challenged abalone (ren et al., 2009). bacterial challenge could significantly up-regulate the mrna expression of both vpgst-1 and vpgst-2 from manila clam ruditapes (venerupis) philippinarum (li et al., 2012). the mrna expression level of sggst-s1 in hemocytes was significantly up-regulated after razor clam solen grandis was stimulated by peptidoglycan (pgn) or β-1, 3-glucan (glucan) (yang et al., 2012). while after bacterial challenge, the mrna expression levels of sigma class gsts in hemocytes were all significantly higher than those of the control group in mussels mytilus galloprovincialis (wang et al., 2013a). all these research achievements revealed that sigma class gsts from marine invertebrates were functional diversity and might not only serve as an antioxidant enzyme involving in the detoxification but also play important roles in the modulation of innate immune responses. bay scallop argopecten irradians was introduced from usa in 1982 and has become one of the most important aquaculture species in china, due to its high economic value, fast growth rate and adaptation ability to different regions for aquaculture (li et al., 2007). and a. irradians was also considered as an attractive model to study immunology because of its relatively simple innate immune system and its propensity to undergo various manipulations, which allows researchers to study the effects of both biological and non-biological factors on the innate immune responses (matozzo, 2016). by now, several antioxidant enzyme genes have been identified and characterized in a. irradians, such as metallothionein (mt) (wang et al., 2009), peroxiredoxin (prx) (li et al., 2011) and superoxide dismutase (sod) (bao et al., 2008, 2009a, b, 2010), however, no information about gst genes was available in bay scallop till now. to bridge this gap, the main objectives of the present study were (1) to clone the full-length cdna of sigma class gst from a. irradians (designated as aigstσ), (2) to investigate the tissue distribution of aigstσ mrna transcripts and their temporal expression after different pathogen associated molecular patterns (pamps) stimulation, and (3) to validate the activities of recombinant aigstσ protein under different treatments. materials and methods scallops, immune stimulation and sample collection the bay scallops used in the present study were obtained from a local farm in qingdao, china, and all the experiments were conducted in accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health (nih). the study protocol and all the experimental design were conducted with approval from experimental animal ethics committee of institute of oceanology, chinese academy of sciences. approximately 200 scallops with an average 50 mm in shell length were employed for the pamps stimulation treatment. the scallops were randomly divided into 6 groups and each group contained about 30 40 individuals. the scallops were received an injection of 50 μl phosphate buffered saline (pbs, 0.14 mol l-1 sodium chloride, 3 mmol l-1 potassium chloride, 8 mmol l-1 disodium hydrogen phosphate dodecahydrate, 1.5 mmol l-1 21 table 2 information of gst proteins used in phylogenetic analysis class species accession number omega chlamys farreri adf32018 craassostrea gigas xp_011429380 danio rerio np_001002621 haliotis discus discus abo26600 haliotis madaka alu63761 perna viridis agn03944 sigma argopecten irradians ang56313 chlamys farreri acf25904 chlamys farreri adf32019 hyriopsis cumingii agu68336 pinctada fucata jas04242 ruditapes philippinarum aew46325 rho chlamys farreri acf25903 cyprinus carpio bas29983 ruditapes philippinarum aew46331 sebastes schlegelii anw83217 siniperca chuatsi aci32418 solea senegalensis bag12568 zeta chlamys farreri add82544 cyprinus carpio bas29981 oplegnathus fasciatus ady80028 xenopus laevis xp_018084636 microsomal chlamys farreri adf45336 gallus gallus np_001129022 microtus ochrogaster xp_005364596 osmerus mordax aco10098 sinonovacula constricta alc77324 xenopus tropicalis np_001011245 potassium phosphate monobasic, ph 7.4), lipopolysaccharides from escherichia coli 0111:b4 (lps, l2630, sigma-aldrich, usa, 0.5 mg ml-1 in pbs), pgn from staphylococcus aureus (77140, sigma-aldrich, usa, 0.5 mg ml-1 in pbs), glucan from baker’s yeast saccharomyces cerevisiae (g5011, sigma-aldrich, usa, 0.5 mg ml-1 in pbs) or polyinosinic-polycytidylic acid (poly ic, p1530, sigma-aldrich, usa, 0.5 mg ml-1 in pbs), respectively. the injected scallops were returned to seawater tanks immediately and five individuals were randomly sampled from each stimulated and unstimulated group at 3, 6, 12, 24 and 48 h post injection. the hemolymphs were collected and centrifuged at 800 g, 4 °c for 10 min to harvest the hemocytes for rna preparation. hemocytes, muscle, mantle, gill, hepatopancreas and gonad from five untreated scallops were collected to determine mrna transcripts of aigstσ. rna isolation and cdna synthesis total rna was isolated from the hemocytes of scallops with rnaiso plus reagent (9108, takara, japan). the first-strand synthesis was carried out using the dnaseⅰ (rq1, m6101, promega, usa) treated raw rna as template and adaptor primer-oligo (dt) as primer (table 1). the reaction were performed at 42 °c for 1 h, terminated by heating at 95 °c for 5 min, and then stored at -80 °c till use. est analysis and cloning of full-length aigstσ cdna an est (ai_f00346) from bay scallop cdna library in national center for biotechnology information (ncbi) homologous to previously identified sigma class gst genes was selected for further cloning the cdna of aigstσ. two gene-specific primers, aigstσ-race-f1/f2 (table 22 1), were designed to clone the 3’ sequence of aigstσ cdna by rapid amplification of cdna ends (race) technique. and the coding sequence (cds) of aigstσ was amplified and confirmed using another two gene-specific primers, aigstσ-cds-f/r. all pcr amplification was performed in an a300 fast thermal cycler (longgene, china), and the pcr products were purified using monarch dna gel extraction kit (t1020s, neb, usa) and cloned into the pmd18-t simple vector (d103a, takara, japan). after being transformed into the competent cells escherichia coli strain dh5α (cb101, tiangen, china), the positive recombinants were identified via anti-ampicillin selection and verified by pcr screening using m13-47 and rv-m primers (table 1). three of the positive clones were sequenced using a prism 3730xl automated sequencer (thermo fisher scientific, usa). bioinformatical analysis of cdna and protein sequences the protein sequences information for homologous and phylogenetic analysis was listed in table 2. the search for protein sequence similarity was conducted with blastp 2.6.0. the deduced protein sequences were analyzed by the editseq module in lasergene program suite 14.0.0.88. the function domains were predicted using simple modular architecture research tool (smart) 7.0. multiple sequence alignments were performed with clustal omega 1.2.4 and visualized by multiple alignment show module in sequence manipulation suite 2.0. a neighbor-joining (nj) phylogenic tree was constructed with mega 7.0.26. to derive confidence value for the phylogeny analysis, bootstrap trials were replicated 1,000 times. expression patterns analysis via quantitative real-time pcr the mrna transcripts of aigstσ in different tissues or their temporal expression patterns in hemocytes of scallops stimulated with various pamps were investigated by quantitative real-time pcr (qrt-pcr). all qrt-pcr reactions were performed with the sybr premix extaq (tli rnaseh plus) (rr420, takara, japan) using 100 ng cdna template in a linegene k fqd-48a (a4) fluorescence quantitative pcr detection system (bioer, china). all the primers using in qrt-pcr were listed in table 1. the mrna expression levels of aigstσ were normalized to those of β-actin for each sample. the relative mrna expression levels of aigstσ were generated using comparative ct method (2-δδct method) (schmittgen and livak, 2008). all the data were subjected to one-way analysis of variance (anova) followed by a multiple comparison using costat 6.400, and the p values less than 0.05 were considered statistically significant. recombinant and purification of aigstσ in e. coli the cds of aigstσ was amplified using two gene-specific primers, aigstσ-cds-f/r (table 1), and ligated to the expression vector peasy-blunt e1 (ce111, transgen, china). the recombinant plasmid, peasy-blunt e1/aigstσ, was isolated by monarch plasmid miniprep kit (t1010s, neb, usa) and then transformed into e. coli strain bl21 (de3) (cd601, transgen, china). the positive transformants, e. coli bl21 (de3)/peasy-blunt e1/aigstσ, were incubated in artmedia protein expression auto-inducing medium (cp101, transgen, china) containing 100 mg l-1 ampicillin (gg101, transgen, china) at 28 °c with shaking at 220 rpm for 24 h. the recombinant protein (designated as raigstσ) was purified using a his-tag protein purification kit (p2226, beyotime, china) under natural condition. the resultant protein was separated by 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) and visualized with protein stains h (c510041, sangon, china). analysis of enzymatic activity of raigstσ the specific activities of raigstσ were measured as described in previous reports (habig et al., 1974 wan et al., 2008; umasuthan et al., 2012). briefly, the reaction was carried out in a 1 ml mixture containing 100 mm pbs, 10 mm gsh (s0073, beyotime, china), and an appropriate amount of raigstσ. the enzyme mixture was incubated at 25 °c for 5 min before the reaction was initiated by adding 1 mm cdnb (703318, cayman chemical, usa) and absorbance was monitored for 5 min at 340 nm while the reaction was maintained at 25 °c. the changes in absorbance per minute were converted into amounts of substrate conjugated per min per mg enzyme by using the molar extinction coefficient for cdnb ε340 = 9.6 mm-1 cm-1. to characterize the raigstσ, enzymatic activity was evaluated at different temperature and ph. to determine the optimal temperature, protein samples were treated at 10 °c intervals between 10 °c and 90 °c for 1 h. to investigate the optimal ph, protein samples were treated between ph 3.5 and 10.5 at 1.0 ph intervals using different buffers for 1 h. acetate, phosphate and glycine-naoh buffers were used to obtain the ph ranges of 3.5-5.5, 6.5-7.5 and 8.5-10.5, respectively, according to previously reports (wang et al., 2013b, 2015). results sequence features of aigstσ a sigma class gst gene, aigstσ, was identified from the bay scallop est database, and its full-length cdna sequence was obtained via race technique and deposited into genbank under the accession number ku301768. the full-length cdna sequence of aigstσ comprised 779 bp, containing a 5’ untranslated regions (utr) of 48 bp, a 3’ utr of 113 bp with a poly a tail and an open reading frame (orf) of 618 bp. the orf encoded a polypeptide of 205 amino acid residues with a calculated molecular mass of approximately 23.11 kda and a theoretical isoelectric point of 5.354. no signal peptide was revealed in the deduced amino acid sequence of aigstσ by signalp program. a gst_n domain (from y4 to r73) and a gst_c domain (from i92 to n190) were found in the deduced amino acid sequence of aigstσ (fig. 1). 23 fig. 1 nucleotide and deduced amino acid sequences of aigstσ. the nucleotides and amino acids were numbered along the left margin. the function domain was in shade. the asterisks indicated the stop codon. two single typical polyadenylation signal sequences (aataaa aataaa) was underlined. phylogenetic ananlysis of aigstσ the deduced protein sequence of aigstσ exhibited high similarity with other previously identified sigma class gsts, such as 78 % identity with that of sigma class gst 2 from chlamys farreri (adf32019). the nj phylogenetic tree based on protein sequences from multiple gst genes was positioned separately into five main branches, and aigstσ were clustered with sigma class gst 2 from c. farreri and located in the sigma class gsts sub-branch (fig. 2). tissue distribution of aigstσ mrna transcripts the qrt-pcr technique was employed to detect the distribution of aigstσ mrna transcripts in different tissues with β-actin gene as internal control (fig. 3). the highest mrna expression level of aigstσ was found in hemocytes, which was 21.30-fold (p < 0.05) of that in muscle, while that in hepatopancreas was 13.28-fold (p < 0.05) of that in muscle. expression profiles of aigstσ mrna transcripts the temporal mrna expression profiles of aigstσ in hemocytes after various pamps stimulation were also examined via qrt-pcr (fig. 4a-d). the mrna transcripts of aigstσ all increased for the first time at 3-6 h and reached the peak at 12 h post different pamps stimulation. the mrna transcripts of aigstσ significantly increased at 3 h after lps stimulation (2.79-fold compared with the origin level, p < 0.05, fig. 4a), with the highest level observed at 12 h (17.74-fold, p < 0.05, fig. 4a). the mrna expression level of aigstσ was up-regulated at 6 h post pgn stimulation (3.13-fold, p < 0.05, fig. 4b) and then up-regulated to the highest level at 12 h (6.82-fold, p < 0.05, fig. 4b), and finally down-regulated to the normal level at 48 h. in the glucan stimulation group, after a significant increase at 3 h post stimulation (3.18-fold, p < 0.05, fig. 4c), the mrna transcripts of aigstσ increased to the peak at 12 h (12.15-fold, p < 0.05, fig. 4c), and finally decreased to the original level at 48 h. 24 fig. 2 consensus neighbor-joining phylogenetic tree based on the amino acid sequences of gsts from different organisms. the evolutionary history was inferred using the neighbor-joining method. the bootstrap consensus tree inferred from 1,000 replicates was taken to represent the evolutionary history of the taxa analyzed. all positions containing gaps and missing data were eliminated. the numbers at the forks indicated the bootstrap value. the sequence information has been listed in table 2. the mrna transcripts of aigstσ significantly increased at 6 h post poly ic stimulation (6.71-fold, p < 0.05, fig. 4d), reached the peak at 12 h (9.07-fold, p < 0.05, fig. 4d), and then decreased to the normal level at 24 h. in the normal group, no significant change of aigstσ mrna expression level was observed during the whole experiment, while after pbs injection, a slight but significant increase was observed at 6 h (2.93-fold, p < 0.05, fig. 4a-d). purification of recombinant aigstσ protein to investigate the potential activities of aigstσ, the recombinant plasmid peasyblunt e1/aigstσ was transformed into e. coli strain bl21 (de3). after auto-induction, the whole-cell lysate was separated by sds-page, and a distinct band of raigstσ was revealed (fig. 5). biochemical characteristics of recombinant aigstσ protein according to the method previously described, the activity of raigstσ was measured for five times, and raigstσ exhibited detectable activity towards cdnb, which was 3.28 ± 0.03 μmol min-1 mg-1. to investigate the stability of aigstσ, the enzymatic activities of raigstσ were measured at different ph and temperature. for the optimal ph assay, raigstσ could maintain more than 50 % of its activity at a ph range from 7.5 to 9.5, but lost more than 60 % of its activity when the ph was lower than 6.5 or at 10.5 (fig. 6a). while when the temperature increased from 10 °c to 20 °c, raigstσ exhibited stable enzymatic activities, but lost more than 60 % of its enzymatic activity over 30 °c and almost devitalized at 50 °c (fig. 6b). 25 fig. 3 tissue distribution of aigstσ mrna transcripts detected by qrt-pcr. the β-actin gene was used as an internal control to calibrate the cdna template for each sample. the mrna expression level of aigstσ in hemocytes, muscle, mantle, gill, hepatopancreas and gonad of five adult scallops was normalized to that of muscle. vertical bars represented mean ± sd (n = 5), and bars with different characters indicated significantly different (p < 0.05). discussion sigma class gsts are a large sub-family of gsts (flanagan and smythe, 2011), and accumulating research achievements revealed that sigma class gsts from marine invertebrates were functional diversity and might not only serve as an antioxidant enzyme involving in the detoxification but also play important roles in the modulation of innate immune responses (boutet et al., 2004; lee et al., 2007; wan et al., 2008; ren et al., 2009; li et al., 2012; yang et al., 2012; umasuthan et al., 2012; zhang et al., 2012a, b; wang et al., 2013a; li et al., 2015). in the present study, the full-length cdna sequence of aigstσ was obtained from bay scallop a. irradians. the deduced polypeptide of aigstσ consisted of 205 amino acids, and its calculated molecular weight was 23.11 kda, which was very close to gsts of vertebrate and invertebrate. the amino acid sequence of aigstσ shared as high as 78 % identity with the previously identified sigma class gst 2 from c. farreri. in the phylogenetic tree, aigstσ was located in the sigma class gsts sub-branch. its sequence characteristics, high similarity with other known sigma class gsts and the phylogenetic relationship collectively suggested that aigstσ is a novel member of invertebrate sigma class gst family and may have similar function with sigma class gsts from other marine invertebrates. sigma class gst acts as the principal scavenger of xenobiotics (flanagan and smythe, 2011), and it was reported to be ubiquitously distributed in multiple tissues in marine invertebrates (boutet et al., 2004; lee et al., 2007; wan et al., 2008; ren et al., 2009; li et al., 2012; yang et al., 2012; umasuthan et al., 2012; zhang et al., 2012a, b; wang et al., 2013a; li et al., 2015). in the present study, the tissue distribution of aigstσ mrna transcripts was detected by qrt-pcr to investigate its possible function, and the ubiquity of aigstσ transcripts indicated that it could be involved in many important physiological processes of scallops. similar to the observation in sigma class gsts from m. galloprovincialis, s. grandis and v. philippinarum (yang et al., 2012; zhang et al., 2012a; wang et al., 2013a), the highest mrna expression level of aigstσ was observed in hemocytes, followed by hepatopancreas. the variable tissue distribution of aigstσ mrna transcripts was speculated to be related with tissue dependent oxidative load. the hemocytes have been considered to play pivotal roles 26 fig. 4 temporal mrna expression profiles of aigstσ detected by qrt-pcr in hemocytes at 3, 6, 12, 24 and 48 h post different pamps stimulation (a: lps, b: pgn, c: glucan, d: poly ic). the β-actin gene was used as an internal control to calibrate the cdna template for each sample. each values was shown as mean ± sd (n = 5), and bars with different characters indicated significantly different (p < 0.05). in the innate immune response in invertebrates mainly via phagocytosis, which was usually companied with oxidative stress, while the hepatopancreas is regarded as the main organ where multiple oxidative reactions and antioxidant defenses occur with high metabolic activity (song et al., 2015). additionally, hemocytes and hepatopancreas were also considered as the main immune related organs in scallops (song et al., 2015), the high mrna expression level of aigstσ in these two organs indicated that it could be involved in the innate immunity of scallop. it has been reported that sigma class gsts could rapidly respond to various foreign particles or invading microbes in mrna levels. for examples, a sigma class gst gene from h. diversicolor could be significantly induced in the hemocytes, gill, mantle and digestive gland of bacteria-challenged abalone (ren et al., 2009). bacterial challenge could significantly induce the mrna expression of two sigma class gsts from v. philippinarum (li et al., 2012). the mrna expression of a sigma class gst in hemocytes was significantly up-regulated after razor clam was stimulated by pgn or glucan (yang et al., 2012). while after bacterial challenge, the mrna expression levels of sigma class gsts in hemocytes were all significantly up-regulated in m. galloprovincialis (wang et al., 2013a). in the present study, the mrna transcripts of aigstσ could be significantly induced by the stimulation of four typical pamps, confirming the hypothesis that it could be involved in the innate immune response of scallops. additionally, a slight but significant increase of aigstσ mrna transcripts was also observed at 6 h after pbs injection, indicating aigstσ might be also involved in the responses to injury in scallop. to further investigate the potential role of aigstσ in bay scallop, the catalytic activity of its recombinant protein was determined in vitro using 27 fig. 5 sds-page analysis of the raigstσ protein in e. coli strain bl21 (de3). line m was the unstained protein marker (26610 thermo fisher scientific, usa). line u was the supernatant of non-induced bacteria lysate. line i was the supernatant of auto-induced bacteria lysate. line p was the purified recombinant protein. cdnb as substrate. in a previous research, the recombinant hdgsts1 and hdgsts2 proteins in h. discus discus exhibited catalytic activities of 0.17 ± 0.01μmol min-1 mg-1 and 1.06 ± 0.02 μmol min-1 mg-1, respectively, with relatively broad optimum ph spectrum and temperature range (wan et al., 2008). while rrpgstσ from r. philippinarum demonstrated a high catalytic ability toward cdnb of 4.64 ± 0.17 μmol-1 min-1 mg-1, but xhibited narrow optimal ph spectrum and temperature range (umasuthan et al., 2012). similarly, in the present study, aigstσ exhibited a high enzymatic activity of 3.28 ± 0.03 μmol min-1 mg-1, but lost more than 60% of its activity when the ph was lower than 6.5 or when the temperature was over 30 °c. it has been reported that both sea surface temperature rise and ocean acidification affect survival and reproduction of marine organisms negatively, including scallop (zhang et al., 2014; lagos et al., 2016). so, the lower active stability of raigstσ, especially susceptible to low ph or high temperature, might provide valuable insights into a possible mechanism of large scale mortalities of cultured bay scallops in summer. in conclusion, the full-length cdna encoding a sigma class gst was identified from bay scallop a. irradians. it was constitutively expressed in all the tested tissues, including hemocytes, muscle, mantle, gill, hepatopancreas and gonad, and the mrna expression levels of aigstσ were all up-regulated in hemocytes after various pamps stimulaton. the purified raigstσ protein exhibited relatively high catalytic activity against cdnb with low thermal stability and narrow optimum spectrum of ph. all these results indicated that it was a fragile but efficient antioxidant enzyme and was potentially involved in the innate immune responses of scallop. this study would enrich the understanding of the scallop innate immunity. 28 fig. 6 the enzymatic activities of raigstσ under different treatment (a: ph, b: temperature). each values was shown as mean ± sd (n = 5), and bars with different characters were significantly different (p < 0.05). 29 acknowledgement this research was supported by the key research program of the chinese academy of sciences (kfzd-sw-106). we would like to thank all the three expert reviewers for their constructive suggestions and enlightening comments during the revision. references bao yb, li l, wu q, zhang gf. cloning, characterization, and expression analysis of extracellular copper/zinc superoxide dismutase gene from bay scallop 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79-87, 2017a. zhang z, lv zm, wei zx, li ch, shao yn, zhang ww, et al. microsomal glutathione transferase 2 modulates ltc4 synthesis and ros production in apostichopus japonicus. mol. immunol. 91: 114-122. 2017b. isj 5: 179-yyy, 2008 issn 1824-307x isj 5: 190-191, 2008 issn 1824-307x erratum to: lipase and invertase activities in midgut and salivary glands of chilo suppressalis (walker) (lepidoptera, pyralidae), rice striped stem borer [5: 180-189, 2008] a zibaee, ar bandani, s ramzi plant protection department, faculty of agriculture, university of tehran, karaj 31584, iran in the above article table 2 was reproduced incorrectly, the table and caption should have appeared as below: table 2 relative activity of c. suppressalis invertase towards different compounds compounds concentration (mmol/l) relative activity (midgut) relative activity (salivary gland) control 100 100 nacl 5 83.33 152.63* 10 63.88* 121.05* 20 41.66* 94.7* 40 10* 52.63* cacl2 5 25.83* 78.94* 10 66.66* 131.57* 20 113.88 157.89* 40 125* 194.73* kcl 5 161.11* 273.68* 10 105.55 252.63* 20 69.44* 194.73* 40 50* 89.47* mgcl2 5 97.22 168.42* 10 61.11* 152.63* 20 44.44* 84.21* 190 40 21.38* 27.36* edta 0.5 144.44* 168.42* 1 116.66 131.57* 2 97.22* 105.28 4 52.77* 73.68* sds 2 111.11 173.68* 4 91.66 136.84* 6 61.11* 121.05 8 38.88* 73.68* urea 1000 83.33* 173.68* 2000 63.88* 147.36* 4000 50* 115.78 5000 33.33* 73.68* 6000 25* 47.36* plant extract 10 % 82* 91* 15 % 64* 74* 25% 21* 52* *p < 0.05 vs control ___________________________________________________________________________ corresponding author: ali reza bandani plant protection department college of agriculture and natural resources university of tehran, karaj, 31584 iran e-mail: abandani@ut.ac.ir 191 isj111.pdf 4 isj 3: 4-17, 2006 issn 1824-307x review proteomics and insect immunity l shi, sm paskewitz department of entomology, university of wisconsin, madison, wisconsin, usa accepted january 24, 2006 abstract insect innate immunity is both a model for vertebrate immunity as well as a key system that impacts medically important pathogens that are transmitted by insects. recent developments in proteomics and protein identification techniques combined with the completion of genome sequences for anopheles gambiae and drosophila melanogaster provided the tools for examining insect immunity at a new level of molecular detail. application of proteomics to insect immunity resulted in predictions of new roles in immunity for proteins already known in other contexts (e.g. ferritin, transferrin, chi-lectins) and helped to target specific members of multi-gene families that respond to different pathogens (e.g. serine proteases, thioester proteins). in addition, proteomics studies verify that post-translational modifications play a key role in insect immunity since many of the identified proteins are modified in some way. these studies complement recent work on insect transcriptomes and provide new directions for further investigation of innate immunity. key words: phagocytosis; antimicrobial peptides; melanization; drosophila melanogaster; anopheles gambiae; 2dpage; hemolymph introduction innate immunity refers to the first-line host defense against the early phases of microbial infection and is an evolutionarily ancient defense mechanism. insects and vertebrates display considerable overlap in the intracellular signaling pathways that regulate innate immune responses (salzet, 2001; giot et al., 2003; hultmark, 2003) and in some of the effector mechanisms used against microbes (e.g. phagocytosis, fluid lysozymes). thus, discoveries made through research in the fruit fly, drosophila melanogaster may be applicable to innate immunity in humans (fallon et al., 2001). corresponding author: susan m paskewitz department of entomology, 237 russell labs, 1630 linden drive, university of wisconsin, madison, wisconsin 53706, usa email: paskewit@entomology.wisc.edu the study of innate immunity in insects has also garnered increasing attention because of the role of many insects in transmission of human disease agents. understanding how the insect immune system interacts with pathogens may contribute to development of new strategies to block transmission of disease agents (christophides, 2005). for example, cecropin is a protein originally identified for its antibacterial activity in lepidopteran insects but eventually shown to reduce malaria parasite development (gwadz et al., 1989). as a result, transgenic mosquitoes were developed to overexpress cecropin in the midgut, resulting in significant decreases in the number of developing malaria parasites following infection (kim et al., 2004). the sequencing of the genomes coupled with est projects for two dipteran species, d. melanogaster and the african malaria mosquito, anopheles gambiae, provided new opportunities for studying immunity. new genes with candidate immune functions were quickly identified (christophides et al., 2002) and microarrays were applied to survey transcriptome changes after bacterial, fungal or parasite infections (degregorio et al., 5 2001; irving et al., 2001; dimopoulos et al., 2002; roxstrom-lindquist et al., 2004). these studies have been fruitful in identifying a set of genes that can be tested for functional involvement in insect immune responses. however, mrna-based approaches suffer some limitations. first, they can be misleading in estimating how much protein is present. although changes in mrna levels sometimes accurately serve as surrogates for changes in the respective protein levels, several studies have shown that this is not the case about 40-50 % of the time (gygy et al., 1999; ideker et al., 2001). a specific example of this type of problem can be seen in mosquito immunity where the antimicrobial peptide defensin displayed high levels of transcript that did not correlate with detectable peptide (bartholomay et al., 2004). second, mrna analyses tell us nothing about whether the encoded proteins are active or not as the functions of proteins are often regulated by post-translational modification. for example, several proteolytic cascades are involved in insect responses to pathogens, with cleavage of a series of proteins necessary for activation of the end product. these functional modifications cannot be directly determined from dna sequence information or mrna levels. third, mrna cannot be used to profile changes in secreted proteins that occur in the hemolymph when the body cavity is invaded by pathogens or to identify components of extracellular reactions such as melanization or coagulation. thus, a complementary approach for investigating immunity in greater detail is to focus on the proteins themselves. proteomics is a tool for detecting changes in protein expression and modification in whole organisms and in specific cells, tissues, and fluids. this review will focus on the methods and applications of proteomics to insect immunity. methodologies definition of proteomics the term “proteome" was coined in 1994 and defined as the entire protein complement expressed by a sample. proteomics encompasses a broad set of disciplines aimed at understanding and monitoring proteins. this includes work correlating genetic sequence with three-dimensional protein structure and 3d structure with protein function, development of protein separation and protein profiling techniques, and investigation of protein-protein interactions. recent studies of insect immunity concentrate on “profiling/expression" and “functional" proteomics (table 1). profiling or expression proteomics focuses on the description of the whole proteome in a given tissue, body fluid, cell type, or organelle, and differential measurement of protein expression levels in samples collected under different conditions (choudhary and grant, 2004). functional proteomics includes research approaches that directly analyze a subset of proteins, such as a family of sequenceor function-related proteins (kocks et al., 2003), as well as those that characterize the protein’s biological functions, proteinprotein or protein-dna/rna interactions, or posttranslational modifications (cai et al., 2004). the tools of proteomics have been developing over the past three decades, but it was not until mass spectrometry began to be used for the identification of proteins in complex mixtures that the field really started to take off (karas and hillenkamp, 1988; fenn et al., 1989). a summary of methodologies used for insect immunity studies will be presented next, with notes concerning limitations of the various procedures. protein separation all proteomic technologies rely on the ability to separate a complex mixture so that individual proteins are more easily processed with other techniques. twodimensional gel electrophoresis (2-de) is still the most widely used protein separation technology for insect immunity (o’farrell 1975; table 1; fig. 1). in this approach, proteins are separated in the first dimension by isoelectric focusing using immobilized ph gradient strips. then, these proteins are again separated, this time by molecular weight in standard polyacrylamide gel electrophoresis, resulting in a 2-dimensional display of proteins. the advantage of this method is that a large number (3,000 to 10,000) of proteins can be visually separated and those spots exhibiting changes between treatments can then be singled out for further exploration. electrophoresis is followed by excision of specific spots, digestion, and protein identification (fig. 1). a number of problems have to be confronted when using 2-de as a protein separation and expression profiling technique. for example, proteins present at low concentrations, those of very high or very low molecular weight, or membrane proteins are generally not detected on the gels. detection of some of these proteins can be improved by pre-separation fractionation and processing of tissue and cell extracts or by altering the conditions of the 2-de (fig. 1b; chevallet et al., 1998), but pre-separation requires additional manipulation to make quantification possible. insect tissues such as the fat body that have high lipid contents may also require pre-separation fractionation (e.g. stadler and hales, 2002). perhaps most frustrating, the reproducibility of 2-de experiments can be poor, requiring many replicates to ensure confidence in the results. however, several tools have reduced some of the variability associated with 2-de. immobilized ph gradients are commercially available and replace the unstandardized tube gels used in older protocols. 2ddige (see below) provides internal standards and reduces the non-biological variability associated with standard 2-de. companies have introduced software that greatly facilitates 2-d gel image analysis. such programs generally automate the alignment of spots on one gel with corresponding spots on another, facilitating analysis even when gel distortions occur. an alternative to 2-de that is likely to become more widely used in the future is mudpit (multidimensional protein identification technology), which couples twodimensional chromatography of peptides in mass 5 table 1. summary of proteomics studies in insect immunity insect samples treatment(s) methods ref profiling proteomics drosophila melanogaster 3th instar larval hemolymph lps, m.luteus, s. cerevisiae 2d-dige vierstraete et al., 2005 3th instar larval hemolymph no 2de, malditof vierstraete et al., 2003 3th instar larval hemolymph m. luteus, s. cerevisiae, lps. 2d-dige, ms vierstraete et al., 2004a, b 3th instar larval hemolymph m. luteus, e. coli, b. bassiana 2de, malditof levy et al., 2004a, b 3th instar larval hemolymph d. pneumoniae, n. catarrhalis, s. aureus, k. pneumoniae, h. influenza, s. pyogenes 2de, malditof guedes et al., 2005 3th instar larval hemolymph hemolymph clot 2de, malditof karlsson et al., 2004 mbn-2 cell line lps 2de, malditof loseva and engstrom, 2004 anopheles gambiae adult 4a rr and l3-5 strains, hemolymph bacteria (m. luteus and e. coli) sephadex beads 2de, ms paskewitz and shi, 2005 chun et al., 2000 adult g3 strain female salivary gland blood meal sds-page, lc-ms/ms kalume et al., 2005 g3 strain male and female midgut sugar-fed or blood meal 2de prevot et al., 2003 anopheles stephensi female midgut from mosquitoes of different susceptibility to p. falciparum sugar-fed or blood meal 2de prevot et al., 1998 aedes aegypti larval tissues v. culicis 2de, malditof biron et al, 2005 fat body eclosion or blood meal 2de shih and fallon, 2001 rkf, lvp, rlvp strain female thoracic tissue sucrose meal, blood meal or b. malayi sds-page, 2de wattam and christensen, 1992 bombyx mori 5th instar larvae hemolymph, midgut and fatbody lps 2de, malditof wang et al., 2004 functional proteomics drosophila melanogaster drosophila s2 cell line dcg-o4 sds-page ms kocks et al., 2003 anopheles gambiae adult female midgut, 4a-3a cell line purified p. berghei ookinetes bind annexins maldi-tof, 2de kotsyfakis et al., 2005 spectrometry-compatible solutions directly to tandem mass spectrometry (2d-lc-ms/ms), allowing for the identification of proteins from highly complex mixtures (fig. 1c). a useful aspect of this technique is the ability to analyze proteins when the amount of starting material is too small for 2-de. this technique has been used by levy et al. (2004b) to investigate small peptide (1-11 kda) profiles in hemolymph from immune challenged and naïve drosophila adults and by florens et al. (2002) to investigate mosquito-stage proteins of the malaria parasite, plasmodium falciparum. protein modification almost all proteins are modified following translation. specialized methods have been developed to study phosporylation (phosphoproteomics; salih, 2005) and glycosylation (glycoproteomics; hirabayashi et al., 2002) but these have not yet been applied to studies of insect immunity. however, changes due to glycosylation and protein spot was quantified by its staining intensity. in this approach, protein mixtures are often prepared from two different samples and resolved by separate 2d gels for subsequent comparison of protein expression or changes in protein modification. gels can be compared by eye but are now usually digitized and analyzed using imaging software. for reliable densitometric analysis, image acquisition needs to be done by calibrated gel scanners with a wide dynamic range. several software packages that can analyze this input exist and have greatly facilitated spot quantification and comparison. 6 5 fig. 1 strategies for proteome analysis. (a) analysis of whole proteomes by two-dimensional gel electrophoresis (2dpage). in this approach, a protein extract is prepared from two different samples and the proteins are resolved by 2d gel electrophoresis. proteins that differ by some variable then are selected for identification by mass spectrometry (ms). although this method allows for the selection of relevant proteins, few low-copy proteins can be identified. (b) analysis of whole proteomes by ms. in this approach, all cellular proteins are converted to peptides. the peptides are resolved by liquid chromatography and analyzed by ms. this method allows for the identification of low-abundance proteins but, since there is no selection for relevant proteins, all proteins must be analyzed by ms. (c) analysis of sub-proteomes. in this approach, a protein extract is separated into individual sub-proteomes by fractionation or specialized affinity chromatography and proteins are resolved by 1d or 2d-page. this allows for the enrichment of low-copy proteins and their selection for further analysis (graves and haystead, 2003). 7 5 protein quantification traditionally, visualization of spots in a 2d gel was by coomassie or silver staining. proteomics analysis utilizing 2-de protein separation is frequently criticized as being lowthroughput, in part due to the time-consuming process of image analysis that is necessary to determine differential protein expression. this process can be laborious due to gel-to-gel variations that confound the analysis process. through the use of fluorescent dyes to label protein samples prior to 2-de, the dige (difference gel electrophoresis) technique allows multiple samples to be co-separated and visualized on one 2d gel (tonge et al., 2001; fig. 2). the protein extracts, for example one control and one treated, are labeled with different fluorescent dyes (e.g. cy2, cy3), then combined and separated by 2-de. in this example, two images of the gel would be captured – using the cy2 and cy3 excitation wavelengths. the images are then merged, and differences between them can be determined using image analysis software. the method minimizes the gelto-gel variation implicit in 2-de but cannot eliminate variation due to differences in the extraction or labeling steps. the dyes are reported to produce a linear response to protein concentration over five orders of magnitude, have enhanced sensitivity in comparison with other commonly used protein stains, and are compatible with ms analysis. the 2d-dige method was used successfully by veirstraete and colleagues (2004a,b; 2005) to profile changes in the larval hemolymph of d. melanogaster following immune challenge. protein identification protein identification is now an essential part of almost every proteomics experiment. some of the studies of insect immunity incorporated n-terminal sequencing or sequencing of proteolytic fragments from spots by edman degradation to generate sequences for database searches (chun et al., 2000). currently, however, the most commonly used identification approach is ms. ms can rapidly, and with high sensitivity, determine masses and structures of peptides. software is available to use the two different types of data generated by mass spectrometers to search sequence databases. protein identification using peptide mass fingerprinting is an effective technique when studying organisms with completed genomes. using the programs mascot (www.matrixsciences.com), profound (http://prowl. rockefeller.edu/) and peptldent (www.expasy.org/tools/ peptident.html), one can analyze the peptide mass profiles produced by ms. findmod (www.expasy.org/ tools/findmod/) is used to find modifications for analysis of unmatched peptide masses. a second method for protein identification is based on the use of sequence data created by tandem mass spectrometers. this information can be used to search databases of translated protein sequences as well as nucleotide databases such as expressed sequence tag (est) sequences. the ability to search nucleotide databases is an advantage when analyzing data obtained from organisms whose genomes are not yet completed, but for which a large amount of expressed gene sequence is available. data analysis to make the most of the wealth of proteomics data being produced around the world, it is important to establish standards for storing and reporting proteomic data enable comparisons across platforms and research groups. the universal protein resource, or uniprot, (http://www.pir.uniprot.org/) was recently established by nih as a centralized database of protein information such as function, classification and cross-reference. uniprot combines the resources from the major annotated protein databases swissprot and trembl from the european bioinformatics institute (ebi) and the swiss institite for bioinformatics (sib) as well as the protein sequence database (psd) from the protein information resource (pir) (http://pir.georgetown.edu/) (apweiler et al., 2004). for drosophila melanogaster or anopheles gambiae, gene identity and predicted protein function are found at the flybase report (http://flybase.bio.indiana.edu/) or anobase (www.anobase.org/anobase/genes/ano-xcel) (loseva and engstrom, 2004; ribeiro et al., 2004). application of proteomics to the study of insect immunity mechanisms of insect immunity in this section, we will briefly introduce proteins that have known or likely functions in insect immunity to place them in context when discussing the results of proteomics studies. insect innate immunity is based on the recognition of microbial molecules, such as lps, peptidoglycans, or β-1,3-glucans, by specific receptors with the subsequent activation of immune effector responses. non-microbial surfaces (e.g. sephadex beads) also elicit responses. proteins that are considered to have a recognition or opsonizing function include gnbp, tep and pgrp. recognition leads to activation of cellular and/or humoral effector mechanisms. these include phagocytosis by hemocytes, encapsulation or nodulation of pathogens by hemocytes, activation of proteolytic cascades leading to localized melanization and hemolymph clotting, and synthesis of a battery of amps. the latter process can occur in most insect tissues including the fat body, hemocytes, respiratory system, cuticular and midgut epithelia, malpighian tubules, and male and female genital tracts (tzou et al., 2000). the activation of amp synthesis is the best described of these effector processes. in drosophila, there are several groups of amps that act mainly against either gram positive bacteria, gram negative bacteria or fungi. two distinct signaling pathways, toll and imd (immune deficiency), control their expression. the toll pathway is activated by an extracellular proteolytic cascade, where serine proteases are important. serine proteases can be regulated by inhibitors, including serpins and kunitz types. 8 4 fig. 2 schematic representation of the 2d-dige (differential in gel electrophoresis) method. two samples for comparison are individually labeled with distinct fluorochromes. the two samples are mixed and separated on the same 2d gel to reduced gel-to-gel variation. the resulting gel is imaged twice using the two different wavelengths for the two fluors. image analysis software is used to detect spots in each image, overlay gels, and quantify differences. 9 4 2002) and, with a few exceptions, specific targets have not been identified for them. some members of a subgroup called the clip-domain serine proteases, as well as their inhibitors, are important in activating the toll pathway and in localized melanization responses. mosquitoes are known to melanize malaria parasites, nematode worms, yeast, microsporidial spores, bacteria and sephadex beads. the melanin pathway depends on the activity of phenoloxidase, which exists in a zymogen form prior to cleavage by an activating serine protease. phenoloxidases and serine proteases are also involved in hemolymph clotting (karlsson et al., 2004). interestingly, a serpin mutant suggests that toll activation is linked to the melanization pathway in drosophila (green et al., 2000; ligoxygakis et al., 2002). expression profiling proteomics protein expression profiling of insect immune responses has been initiated for several insect species and tissues (table 1) and many differentially expressed proteins have been identified by ms. most of these studies have been carried out in d. melanogaster (uttenweiler-joseph et al. 1998; guedes et al., 2003; sabatier et al. 2003; vierstraete et al. 2003, 2004a,b, 2005; engstrom et al., 2004; levy et al., 2004a,b; guedes et al., 2005). other taxa where immunity has been investigated by proteomic methods include the mosquitoes, a. gambiae (chun et al. 2000; paskewitz and shi 2005) and aedes aegypti (wattam and christensen, 1992; biron et al., 2005), the silkworm, bombyx mori (wang et al., 2004), and the locust, odaleus australis (stadler and hales 2002). overall, we are lacking in genomic and proteomic information for hemimetabolous insects as well as broad coverage of the holometabolous orders. there are significant and extensive methodological differences between the above mentioned proteomics studies, including types of challenge agents (single species or mixes of living bacteria, lps, bacterial lysates, yeast, filamentous fungi, microsporidia, picorna-like virus, sephadex beads) as well as the method of introduction of the agent (feeding, external exposure, injection), the length of incubation time after exposure, the tissue examined (hemolymph, hemocyte-like cells in culture, fat body, midgut, thorax, whole insect larvae), the method of tissue collection, and the stage of the insect (larvae vs adult). standardizing these aspects would provide better ability to compare innate immunity across taxonomic groups. nevertheless, taken together these studies identify some patterns in proteins that are affected by immune challenges and provide a framework for future investigations. hemolymph profiles hemolymph is a critical immune fluid in insects. it contains hemocytes and is a transport tissue for effector molecules like the fat body-produced amps. most proteomic data are from this fluid. several studies provide reference maps of identified hemolymph proteins that are not immune responsive but may be useful for other studies (guedes et al., 2003; vierstraete et al., 2003; karlsson et al., 2004; paskewitz and shi, 2005). the hemolymph reference maps contain constitutively expressed proteins from several groups: storage and transport proteins, metabolic proteins, cytoskeletal proteins, defense/immune proteins, antioxidant and stress proteins, and novel proteins. there are relatively few proteins in common between some of the drosophila studies, reflecting the methodological issues described above. many of the identified proteins in the reference maps of drosophila hemolymph are intracellular metabolic proteins while more than half of those identified in a. gambiae are related to immunity and appear to be secreted proteins. cellular proteins occur in these samples because hemocytes and other contaminants are generally not removed (but see karlsson et al., 2004 for serum versus plasma maps). in a. gambiae, fat body can be a major hemolymph contaminant, so care must be taken in attributing changes in cellular proteins to the hemocytes. in addition, some of the hemocyte types are known to be quite labile (e.g. crystal cells in drosophila, oenocytoids in anopheles) meaning that they break down and quickly release their contents when their environment changes. we attributed finding phenoloxidases in hemolymph and plasma to this feature, since oenocytoids produce pos and they do not contain signal peptides (paskewitz and shi 2005). other cellular proteins that are abundant in labile hemocytes might also exhibit this pattern. cellular protein profiles may also be affected by changes in hemocyte behavior after infection. microbial challenge can result in mobilization of sessile hemocytes, so the relative cellular content of immune-challenged versus unchallenged hemolymph may not be equal. some considerations as to controls controls for immune-induced hemolymph samples must be carefully chosen since the manner of introduction of the immune challenge is often traumatic. pricking an insect with a needle dipped in a pellet of bacteria is the most common challenge, although some studies use feeding or external exposure to simulate more natural conditions. we found that several of the proteins we had labeled as “wound-induced” proteins in a. gambiae were probably a specific result of damage to the thoracic musculature during aseptic wounding, rather than a generalized response to damaging the cuticle that would include hemolymph clotting and wound healing. comparison with mosquitoes wounded in the abdomen did not produce the same group of proteins and the thoracic wound samples contained some proteins of obvious muscle origin (paskewitz and shi, 2005). damage to tissue in vertebrates can also lead to the release of cellular proteins into circulation (alleyne et al., 2001; renz et al., 2001) if defined numbers of microbes or aliquots of surface molecules are to be injected, it is critical to use a sterile solution of the suspension buffer for the controls. vierstraete et al. (2004a) report that they injected lps in a solution that contained 1 % ethanol but did not use ethanol in the controls. this may explain the fact that the strongest induction they observed was for alcohol dehydrogenase in the lps-injected samples. 10 5 fig. 3 two dimensional gel profiles of anopheles gambiae hemolymph incubated in vitro in the absence (a) or presence (b) of heat-killed bacteria (m. luteus and e. coli). black arrows indicate the chi-lectins br1 and br2 in the bacterially-induced sample. red arrows indicate some of the other proteins altered after exposure to bacteria in this sample. guedes et al. (2005) tried to overcome the problems of injection by feeding bacterial lysates to larvae. they combined lysates from six different bacterial species and incorporated them into the feeding medium. this procedure might result in relatively low activation of the immune system, since only the digestive and exoskeletal systems would be directly exposed to bacterial products and bacteria are a normal part of the larval feeding environment. the controls for this experiment were not exposed to a change in their normal feeding medium. it is possible that a change in food quality could induce stress or cause metabolic shifts in an insect as it does in vertebrate animals. indeed, most of the induced proteins identified were metabolic or stress-related. amp production could be a useful marker for verifying immune induction under these conditions. peptidomic studies of hemolymph most proteomic studies are not designed to capture low molecular weight proteins. unfortunately, many of the amps fall into this category. uttenweiler-joseph et al. (1998) and levy et al. (2004a) report results of using hplc and maldi-tof for analysis of bacteriallyinduced peptides (1-15 kda) in adult drosophila hemolymph. of the 28 induced peptides that were characterized, ten were amps exhibiting posttranslational modifications and one was a kunitz type serine protease inhibitor; many of the others are hypothesized to have a chemokine function (levy et al., 2004a). additional kunitz type inhibitors were described in the reference maps of larval hemolymph (vierstraete et al., 2003). the targets of these inhibitors are not known. similar methodology has been applied to peptides induced in the hemolymph of a. aegypti after bacterial challenge (lowenberger, 2001). several amps (defensins and cecropin) were identified as well as novel peptides. the identification of a large number of peptides with unknown functions in drosophila and aedes indicates that much remains to be done to fully characterize even this narrow aspect of immunity. proteomic studies of hemolymph approximately 130 larger proteins that are described as immune-induced have been documented in larval or adult hemolymph from d. melanogaster by 2de and protein identification methods (levy et al. 2004a, b; vierstraete et al. 2004a,b; guedes et al. 2005). in general, the studies identify the following groups of proteins as regulated by the immune treatments: i) a b 11 5 immune-responsive proteins that have functional roles that are somewhat or well defined; ii) immuneresponsive proteins that have hypothesized functions that can be tested; iii) cellular proteins that are involved in stress responses; iv) proteins that appear following injury; v) metabolic proteins; and vi) proteins that are not similar to any other proteins in databases and as yet have no hypothesized functions. catalogs of these proteins can be found in the studies listed above and not all of these groups will be discussed in detail in this review. here we will first consider some of the patterns suggested by these studies. an example of a 2d-page separation of hemolymph proteins is provided in fig. 3. in studies of adult hemolymph following bacterial challenge, a similar number of proteins were affected in drosophila and anopheles. for example, 50 of 350 (14 %) silver-stained spots were upor down-regulated in drosophila (levy et al., 2004a, b) while 14 of 280 (5 %) silver-stained spots were upregulated in a. gambiae (paskewitz and shi, 2005). by comparison, a much larger number of spots were said to be specifically regulated in adult fruit flies by 72 h after fungal exposure (levy et al., 2004a, b). comparison of a subset of 42 proteins regulated following fungal exposure showed that twelve of the proteins were also affected by bacterial infection. three of the twelve were upregulated following both types of challenge (αamylase distal; hsp20-like chaperone; fructose 1,6, bisphosphate aldolase) while nine were regulated oppositely (propo-ae cg16705; dnase ii cg7780; aldehyde dehydrogenase; glyceraldehyde 3 phsophate dehydrogenase; enolase; cathepsin l; transferrin; ferritin; obp99c). in larval drosophila hemolymph, proteins that were immune-regulated were compared at 25 min after injection of micrococcus luteus or saccharomyces cerevisiae. thirteen were upregulated with either challenge agent. three additional proteins were regulated only following saccharomyces and two were regulated only following micrococcus inoculation. some proteins were present as multiple spots, and these were often differentially regulated between challenges. for example, several different ferritin heavy chain spots were identified, each upregulated after a different type of challenge agent (vierstraete et al., 2004b). in drosophila larval hemolymph, 131 of approximately 289 (45 %) silver-stained spots were altered following exposure to a diet that included bacterial lysates (guedes et al., 2005). the majority of the 71 proteins that were identified were cellular proteins involved in metabolism and stress responses. since only 20 % of the immune-regulated proteins in a. gambiae and 14 % of those in drosophila adult hemolymph were identified, we don’t yet have a complete picture of the changes that are occurring at the protein level at any time after infection of adults. however, one study did identify 94 % of the proteins found to be upregulated in larval fruit fly hemolymph by 25 min after immune challenge (vierstraete et al., 2004b). this study provides a good baseline for examining the overall profile of the types of immune proteins that are quickly secreted or processed following infection, and that would not be detectable by transcript analysis. teps are complement-like proteins characterized by a conserved thioester motif that enables covalent binding to target surfaces. these proteins appear to be fundamental to a number of immune processes in insects. reverse genetics clearly demonstrate that a. gambiae tep1 regulates phagocytosis of bacteria and killing of malaria parasites in this mosquito (levashina et al., 2001; blandin et al., 2004). among the proteins that increase in drosophila hemolymph upon infection are other members of the family, including tep2 following m. luteus or lps injection and tep4 after beauvaria bassiana exposure (levy et al., 2004b; vierstraete et al., 2004a,b). tep2 was especially sensitive to m. luteus injection; this was the strongest upregulation seen in this study of larval proteins. the rapid appearance of tep2 (within 25 min) means that other recognition processes occur to trigger its release or cleavage in the hemolymph. all of the tep proteins identified in the proteomics studies were smaller than predicted based on gene sequences and probably represent cleaved forms. the only other recognition/opsonizing protein identified by proteomics is a protein related to gram negative binding proteins. this was the most strongly upregulated protein seen in samples taken following fungal infection of drosophila adults (gnbp3; levy et al., 2004b). serine proteases and serine protease inhibitors play important roles in modulating and amplifying the toll signaling and melanization activation pathways. four different clip-domain serine proteases were identified following fungal/yeast infection in larval or adult drosophila. one, cg5390, was found within 25 min of challenge with saccharomyces cerevisiae but not after m. luteus or lps inoculation (vierstraete et al. 2004b). three others (cg1102, cg16705 and cg9372) were induced 72 h after b. bassiana exposure (levy et al., 2004a, b). two clip-domain serine proteases were identified in the a. gambiae proteomic studies but neither was reliably immune-induced (chun et al., 2000; paskewitz and shi, 2005). finally, three other serine proteases (not clip domain sps) were upregulated in hemolymph when high doses of bacterial lysates were fed to drosophila larvae (guedes et al., 2005). the significance of this observation is not clear but some serine proteases have direct antibacterial activity (tsuji et al., 1998). inhibitors of serine proteases called serpins are also important in immunomodulation. two serpins (srpn2 and srpn15) were identified in a. gambiae, both as constitutively expressed proteins (paskewitz and shi, 2005). two drosophila serpins (cg1857, cg6687) were significantly upregulated in adult flies on fungal but not bacterial infection. one of them (nec, cg1857) had previously been shown to regulate the induction of the toll pathway through inhibition of the activation of spaetzle, the toll ligand (levy et al., 2004a, b). a serpin was also found in hemolymph of injected silkworm larvae at 24 h post inoculation (wang et al., 2004). serpins were not found in the studies of the fruitfly larval hemolymph taken at 25 min after challenge (vierstraete 12 6 et al., 2004a,b). in addition to recognition factors and immunomodulators, some of the known effector proteins have also been identified. as noted above, many of the amps are small molecules and need to be examined by special methods. these studies have shown that posttranslational modifications are frequent and biochemical analysis indicates that the modifications affect the antimicrobial activity of the peptides. phenoloxidases are another effector category. in a. gambiae there are nine po genes, while drosophila has only three. the contribution of the individual po gene products to immunity has not yet been examined. proteomics pinpointed the po6 protein as strongly upregulated at one timepoint (24 h) following bacterial injection in a. gambiae (paskewitz and shi, 2005). one of the drosophila pos was down-regulated upon feeding of bacterial lysates. both of these observations could be reconciled by considering processing of ppo. that is, the down-regulated spot might represent ppo and the upregulated spot might be activated po6. po2 was identified as a constitutive protein in a. gambiae hemolymph that was not altered on bacterial injection. the proteomics studies pointed to post-translational modifications of proteins involved in iron metabolism as a potentially fruitful area for investigation. paskewitz and shi (2005) found decreases in both ferritin subunits at 6 and 24 h after a bacterial challenge. levy et al. (2004b) reported that ferritin was down-regulated in fungally challenged adults but unchanged after bacterial infection, although a more complex pattern is indicated in levy et al. (2004a). vierstraete et al. (2004b) found a large increase in the amount of a spot corresponding to the ferritin heavy chain homologue in larval hemolymph 25 minutes after m. luteus challenge. the spot is described as “shifted” indicating that post-translational modifications occurred. additional information on multiple forms of the light and heavy chains of ferritin can be found in levy et al. (2004a) and vierstraete et al. (2004b). the biology of ferritin in relation to immunity is not at all clear but it might serve to sequester iron from invading microorganisms (yoshiga et al., 1997). tsf is an iron transport protein that also occurs in the hemolymph. the tsf protein was found to be upregulated upon treatment of mosquito cells with bacteria (yoshiga et al., 1997) and during encapsulation of filarial worms in a. aegypti (beerntsen et al., 1994). proteomics studies also identified differences in tsf during fungal infections (levy et al., 2004a). in addition to an increase in tsf production, fungal infection induced proteolytic cleavage of tsf. in vertebrates tsf fragments have been linked to immunity, acting directly as antimicrobial peptides or as inducers of nitric oxide production by macrophages. a role in sequestering iron is also possible (yoshiga et al., 1997). another group of proteins ripe for investigation belong to a group called chi-lectins. we identified two of these proteins (br1 and br2) by proteomic analysis of a. gambiae as they were very strongly induced by bacterial infection (shi and paskewitz, 2004). the two new spots represented c-terminal peptides and we found that both proteins are converted to smaller forms in hemolymph in vivo or in vitro on exposure to bacteria. we could identify this processing as early as 5 min after incubation of hemolymph with bacteria in vitro. a related protein, drosophila ds47 behaves similarly (shi and paskewitz, 2004). br2 and ds47 also can be processed on exposure to peptidoglycan alone but not lps (shi and paskewitz, 2004). other members of this group are called imaginal disc growth factor proteins (idgfs) in drosophila, because of a role in stimulating proliferation of an imaginal disc cell line in conjunction with insulin (kawamura et al., 1999). vierstraete and colleagues (2004b) reported that two spots identified as chain a of idgf2 were significantly regulated by 25 min after either yeast or bacterial challenge, while ds47 was down-regulated after bacteria only. levy et al. (2004a) found that spots corresponding to idgf2 and idgf3 were down-regulated after fungal or bacterial infections, respectively. again, differences in direction of regulation likely reflect processing, with down-regulated spots corresponding to the full-length proteins and upregulated spots representing the processed forms. this group of proteins is particularly interesting because vertebrates have members of the family that are also immune responsive but not yet well-understood (houston et al., 2003). some act as growth factors while others promote migration of immune cells (owhashi et al., 2000; chang et al., 2001; hung et al., 2002; recklies et al., 2002). a related molecule in manduca sexta, haip, inhibits aggregation of hemocytes in vitro (kanost et al., 1994). finally, the proteomics studies have identified several types of proteins that belong to categories not previously known to have immune functions. for example, an odorant binding protein (obp99c) was upregulated after fungal infection of drosophila adults (levy et al., 2004a). odorant binding proteins (obp) bind small hydrophobic molecules and function in chemoreception. obps are found in the antennae but some members of the group appear in the hemolymph (paskewitz and shi, 2005). two pherokines, proteins related to the obp family, were also induced by viral or bacterial infections (sabatier et al., 2003). another group of proteins that was found in to be immuneinduced in both larval and adult drosophila contains members with a phosphatidylethanolamine binding domain. the peb family is widespread but little is known about the function of this group. one possibility is in coordinating regulation of the various signaling pathways but see levy et al. (2004a) for other possible functions. hemocytes (blood cells) hemocytes play important roles in immunity in all insects but there is a great deal of variation in cell types and number between taxa. in general, an insect will have several different types of hemocytes, each of which has a specialized function. some hemocytes are capable of phagocytosis of foreign targets, including latex beads, bacteria, and malaria sporozoites. larger targets can elicit encapsulation responses, where layers of hemocytes adhere to the target surface and enclose it. 13 7 this process resembles some types of granuloma formation in vertebrates. hemocytes also produce some of the effector molecules for humoral immunity, including components of the po cascade and some of the amps. cellular responses were examined in many of the hemolymph studies, since neither hemocytes nor other cellular contaminants were separated from the plasma component. cellular responses were also examined by using a drosophila cell line called mbn-2 for analysis of cytoplasmic and nuclear proteins that changed following exposure to lps (loseva and engstrom, 2004). the mbn-2 line is hemocyte-like and retains its ability to phagocytose bacteria and the signaling systems necessary to activate the genes coding for amps. in this proteomic profiling study, 24 intracellular proteins were identified as regulated (up or down) or modified in response to immune challenge. at 30 min following lps administration, proteins that regulate post-translational modifications and traffic over the nuclear membrane were up-regulated including lamin dm (lam), nuclear porin p62 (nup62), calmodulin (cam), and the receptor of activated protein kinase c1 (rack1). after 6 h of lps treatment, proteins that are directly involved in effecting an immune response proteins were differentially regulated. these included actin-binding/cytoskeletal remodeling proteins and lysosomal proteases (cathepsin l, d, k), (loseva and engstrom, 2004). examination of immune-challenged hemolymph resulted in the identification of a few of the same proteins as well as others likely to be involved in the same processes (vierstraete et al., 2004a,b; guedes et al., 2005). changes in cytoskeletal proteins and their regulators are likely markers of activation of cells in preparation for phagocytosis. lysosomal cathepsins are also related to the phagocytic function since they localize to the phagolysosome in mammals and in drosophila. cathepsin d (cg1548) was found in seven isoforms and lps treatment of cells resulted in the disappearance of four of these (loseva and engstrom, 2004) while lps treatment of larvae resulted in upregulation of a cathepsin d spot (vierstraete et al., 2004b). cathepsins were further characterized in drosophila s2 cells using a functional proteomics technique whereby these enzymes are labeled covalently in an activity dependent manner. chemical tagging allowed the investigators to follow increases in cathepsin activity within the phagolysosome after phagocytosis of latex beads (kocks et al., 2003). in addition to the direct immune responses described above, proteomic studies reveal that infection causes shifts in homeostasis that alter proteins involved in metabolic and redox processes. oxidative stress results when the production of reactive oxygen species exceeds the ability of the animal to remove them. both transcript and proteome analyses indicate that oxidative stress occurs in insects during infection. members of the peroxiredoxin family (cg12405 and cg1633) were regulated in mbn-2 cells (loseva and engstrom, 2004) and in drosophila hemolymph following bacterial and fungal challenge (vierstraete et al., 2004b) or after feeding on bacterial lysates (guedes et al. 2005). glutathione s-transferase was also upregulated in all cases of infection and may have a protective role against oxidative stress (vierstraete et al., 2004b; guedes et al., 2005). two forms of gst were also identified as upregulated in a. gambiae following wounding (paskewitz and shi, 2005) and gst activity increased in hemolymph sampled following immune challenge (data not shown). a large number of cellular proteins involved in carbohydrate and amino acid metabolism were also described as immune-responsive in hemolymph and cell line studies. possible involvement of these proteins in responses to oxidative damage or in shifts in energy/biosynthetic pathways is discussed in the relevant articles (levy et al., 2004a; vierstraete et al., 2004b; guedes et al., 2005). other tissues because of their important role as developmental sites for vector-borne disease agents, mosquito midguts, salivary glands, and thoracic tissues are also potential targets for studying immune responses to parasites. work is in progress on identification of midgut proteins that are altered on malaria parasite infection. other proteomic studies of the mosquito midgut have been undertaken on strains of anopheles stephensi that exhibited different susceptibility to the human parasite, plasmodium falciparum (prevot et al., 1998). whether susceptibility is governed by immune factors is not known but 29 differences in spot patterns were identified following blood feeding in the susceptible line. additional work was done to describe spots that differed between male and female mosquitoes and following blood feeding (prevot et al., 2003). nematode worms such as brugia malayi occupy a different developmental location in mosquitoes, traveling from the midgut to the thoracic musculature. wattam and christensen (1992) compared different strains of a. aegypti and reported that thoracic muscle of refractory strains (rlvp and rkf) produced seven polypeptides in response to a blood meal, whereas a susceptible strain (lvp) did not exhibit this pattern. subsequent work identified mosquito transferrin as a protein altered in this tissue upon worm infection (yoshiga et al., 1997). the proteome of a. aegypti larvae was examined following infection by a microsporidian parasite, vavraia culicis (biron et al., 2005). samples contained the head, thorax and part of the abdomen of larvae that were sampled at either 5 or 15 days following infection. fifteen proteins that were upregulated following infection were identified by peptide mass fingerprinting. among others, these included heat shock protein co-chaperone cdc37, proteins involved in protein biosynthesis, an obp, a nitrophorin, and a gst. additional proteins that were suppressed included proteins involved in ornithine metabolism, nitric-oxide synthase, gsts, and an obp. future perspectives as an important complement to genomics, proteo 14 8 mics allows for the examination of the entire complement of proteins in an organism, tissue, or celltype. current proteomics technologies not only identify protein expression, but also post-translation modifications and interactions of immune response proteins in insect. successful protein profiling with identifications has only recently been applied to insect immunity and only the two dipterans with completed genomes have been used as models. clearly, many milestones have yet to be reached. in the future, increased use of lc-ms/ms will increase the sensitivity, resolution, dynamic range and throughput of proteomics. future studies will take advantage of the availability of new genomes and better identification technologies so that we will be able to compare proteomic results across taxonomic groups and life stages, leading to advances in our understanding of the evolutionary history of innate immunity. within model organisms, reverse genetics will allow us to inactivate key immune regulators through rna interference and to examine the effects on proteome profiles. identification of post-translational modifications suggests experiments designed to identify activators or changes in efficacy of the modified molecules. protein-protein interactions and subcellular locations will provide more precise information about the functions of the unknown proteins that are induced by infection. in short, we predict satisfying application of the methods of proteomics to questions of insect immunity on all fronts within the coming decades. abbreviations 2-de: 2 dimensional electrophoresis; 2d-dige 2: dimensional differential in gel electrophoresis; amp: antimicrobial peptide; br1: bacterially responsive protein 1; br2: bacterially responsive protein 2; est: expressed sequence tags; gnbp: gram negative binding protein; gst: glutathione s transferase; hplc: high performance liquid chromatography; hsp20: heat shock protein 20; idgf: imaginal disc growth factor; imd: immune deficiency protein; lps: lipopolysaccharide; ms: mass spectrometry; maldi-tof: matrix-assisted laser desorption/ionization time-of-flight; nec: necrotic; obp: odorant binding protein; peb: phosphatidylethanolamine binding protein; pgrp: peptidoglycan recognition protein; ppo: prophenoloxidase; po: phenoloxidase; srpn: serpin; 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refractoriness in aedes aegypti. proc. natl. acad. sci. usa 89: 6502-6505, 1992. yoshiga t, hernandez vp, fallon am, law jh. mosquito transferrin, an acute-phase protein that is up-regulated upon infection. proc. natl. acad. sci. usa 94: 1233712342, 1997. 17 56 isj 14: 56-62, 2017 issn 1824-307x research report gene expression of hsp90 and hsp70 in four silkworm hybrids (bombyx mori l.) in response to severe thermal shock sf mousavi1, sh hosseini moghaddam2, n ghavi hossein-zadeh1, sz mirhosseini2 1department of animal science, faculty of agricultural sciences, university of guilan, rasht, iran 2department of animal science and department of sericulture, faculty of agricultural sciences, university of guilan, rasht, iran accepted march 6, 2017 abstract usually silkworm egg producers provide several silkworm hybrids with different qualities of productivity and viability traits. this study was conducted to compare the expression of hsp70 and hsp90 genes among different silkworm hybrids. in the fourth day of fifth larval instar of two japanese maternal parents (103×104 and 107×110) and two chinese maternal parents (110×107 and 104×103), silkworm larval fat body was sampled from heat shock (45 °c for 35 min) and non-heat shock groups. sampling was done at 0, 2, 4 and 24 h after heat shock. gene expression of hsp70 and hsp90 (target genes) were measured by qrt-pcr using rpl27a as reference gene. the prolonged heat shock (8 h at 39 °c) was utilized to examine the thermal tolerance of larvae in comparison with control group (24°c). the results showed both hsp70 and hsp90 have been up-regulated in the treated larvae. the hybrids with japanese female parents (107×110 and 103×104) were more sensitive than two others; moreover in these hybrids, hsp90 and hsp70 were expressed significantly more than others after heat exposure. the maximum expression was occurred in the time zero, then a decreasing trend was observed over time. comparison of larval mortality among four hybrids revealed that (103×104) and (110×107) hybrids had the highest and the lowest mortality rate. key words: gene expression; heat shock proteins; real-time pcr; silkworm hybrids; thermotolerance introduction sericulture has an important role in village economy in countries like iran where sustainable rural development is very important. high sensitivity of commercial hybrids to high temperature is the main barrier in its expansion in hot and dry climates. therefore, factors affecting silkworm tolerance should be considered in the silkworm breeding and introduction of new hybrids (hosseini moghaddam, 2005). moreover, substantial studies have been carried out by researcher to evaluate the effect of heat stress on various organisms due to probability of sudden and rapid climate change when the earth is getting warmer. when insects expose to extreme temperature, they respond to this temperature change by arising heat shock proteins (hsps) as molecular chaperones to protect of protein folding process. hsps family is divided according to their molecular __________________________________________ corresponding author: seyed hossein hosseini moghaddam department of sericulture, faculty of agriculture sciences university of guilan, rasht, iran e-mail: hosseini@guilan.ac.ir weight, function and sequence homology into several groups including hsp100, hsp90, hsp70, hsp60, hsp40, small hsp (shsps) and hsp10(zhao and jones, 2012). among these hsps, high molecular weight hsp70s as a housekeeping molecule has different responsibilities in insects such as developmental processes and fecundity (jensen et al., 2014), development (huang et al., 2009), diapause (rinehart et al., 2007), longevity (choi et al., 2014), and metamorphosis (zheng et al., 2010). hsp70s genes were up-regulated in response to cold or heat stress in a variety of insects, including coleoptera (mahroof et al., 2005), diptera (gray, 2013) and lepidoptera (jiang et al., 2012; choi et al., 2014; shen et al., 2014). in a study on flash fly (sarcophaga crassipalpis) in response to hypoxia, hsp90 showed little response, however, hsp70 was the most responsive and increased several hundred fold in the cells (michaud et al., 2011). results of previous studies on the chilo suppressalis, liriomyza trifolii, and pteromalus puparum showed that hsp70 can be related to thermotolerance and survivability (cui et al., 2010; zheng et al., 2010). in drosophila melanogaster, mailto:hosseini@guilan.ac.ir 57 family of hsp70’s could not make thermotolerance against severe thermal shock (bettencourt et al., 2008). the well-defined role of hsps in acquired thermotolerance in the silkworm and other insects is not clearly known yet (manjunatha et al., 2010). hsp90 that forms about 1 2 % of cellular proteins is one of the most abundant proteins in the living cells. when insects are under normal conditions, the hsp90 expressed at low levels (wegele et al., 2004). hsp90 acted as a protective against thermal stress in some insects including plutella xylostella and liriomyza huidobrensis, (sonoda et al., 2006; huang and kang, 2007). zhang et al. (2009) reported that among different tissues of silkworm, sometimes the hsp90 gene expression was up-regulated and sometimes was down-regulated. the commercial silkworms are usually twoway cross hybrids (direct and reciprocal crosses) which are synthetized by two parents including japanese (e.g. 103, 107) and chinese (e.g. 104, 110) strains. according to previous report (hosseini moghaddam, 2005) the productivity traits (cocoon weight and cocoon shell weight) of japanese maternal parents (103×104 and 107×110) were higher than chinese maternal parents (110×107 and 104×103) and conversely for viability traits. the purpose of this research is the comparison of gene expression of two high molecular weight hsps in four commercial silkworm hybrids (including two maternal chinese and two maternal japanese parents), besides the relationship of gene expression with thermal tolerance of silkworm larvae. materials and methods silkworm rearing, thermal treatment and sampling two iranian silkworm hybrids: 110×107 and 104×103 (first parent is female and a chinese cocoon shape strain) and their reciprocal 103×104 and 107×110 (first parent is female and a japanese cocoon shape strain) were reared in the iran silk research center. the 103 and 104 silkworm strains are originally from japan and the strains 107 and 110 were isolated from a korean hybrid under fao/undp tcp project (1992-1997). fifth instar larvae were transferred to the silkworm laboratory, faculty of agricultural sciences, university of guilan to continue rearing and heat shock treatments. sampling was done from fat body. this tissue is under larval skin that can easily affected by heat exposure. fat body as a site for energy storage tissue in the silkworm, synthetize many biological proteins like heat shock proteins that these substances are expected to be active in the fat body (kajiwara et al., 2006). while all process was performed on ice, the fat body free of muscle was collected in the fourth day of fifth larval instar from both heat shock (45 °c for 35 min) and non-heat shock larvae at 0, 2, 4 and 24 h after heat treatment. before collection of fat body, the surface of dissected larva was washed by icecold insect physiological salt solution (0.7 % nacl) (chavadi et al., 2006). three independent fat body samples were mixed together to minimize variation and to get enough amount of samples. they were store immediately at -80 °c for further use. rna extraction, cdna synthesis and real time-pcr rna extraction procedure was carried out by trizol reagent (invitrogen, usa) based on the supplier’s instructions. the quality and quantity of extracted rna was evaluted using nanodrop-2000 spectrophotometer (thermo scientific) and the quality of agarose gel electrophoresis bands. dnase i treatment (takara, japan) was applied to avoid possible contamination of genomic dna. the cdna of all samples were made using cdna synthesis kit (thermo scientific) based on manufacturer protocol. specific primers of hsp70 and hsp90 were designed using online software primer 3 (table 1). real time-pcr was carried out using maxima sybr green/rox qpcr master mix (thermo scientific). data was normalized using livak (2001) formula (2-δδct). a factorial experiment (4×4) with completely randomized design was utilized to evaluate the effects of different hybrids (110×107, 104×103, 103×104, 107×110) and the times after heat shock (0, 2, 4, 24 h), as fixed effects, and their interactions on gene expression data. statistical analysis of data was performed using the glm procedure of sas 9.0. least squares means were used to identify the main effects. table 1 specific primer sequences for real-time pcr gene ncbi reference primer sequence pcr product length annealing temperature rpl27a nm_001044057 right : tgacaggttgtttggggag left: cagacgaggctgaagtatgc 144 60 hsp70 nm_001043931.1 right: gtgcttcatgtcctgctgaa left: tcgccttgaaccctaacaac 100 60 hsp90 nm_001043411.1 right: aggccttcgaacttcacctt left: atggcaagacccttgtatcg 103 54.7 58 table 2 least-squares means of hsp70 and hsp90 gene expression in the studied silkworm hybrids at four times after thermal exposure expression of hsp90 expression of hsp70 times hybrids 2183.49a 1549.54a 0 107×110 595.96b 646.83b 2 107×110 83.54c 1.002e 4 107×110 1.85c 0.01e 24 107×110 32.78c 277.47c 0 110×107 7.26c 7.51e 2 110×107 0.13c 0.06e 4 110×107 0.04c 0.007e 24 110×107 278.11a 1598.42a 0 103×104 49.90c 26.07 d 2 103×104 2.15c 21.11 d 4 103×104 0.43c 0.09 e 24 103×104 26.99c 97.57 d 0 104×103 20.87c 85.62 d 2 104×103 10.06c 2.28 e 4 104×103 0.01c 80.86* 0.07 e 27.78* 24 104×103 se a-e different superscripts within a column indicate significant differences (p < 0.0001) * standard error for all least squares means of gene expression evaluation of silkworm viability and thermal tolerance in order to measure thermotolerance of different silkworm hybrids, larvae were exposed to 39 °c for 8 h (9 am to 5 pm) from second to seventh day of fifth instar as long-term heat stress; in other word six days from one day after forth molting to one day before larval cocooning. while larval mortality was recorded daily, mortality rate was measured after two days (lo1) and also at the end of heat shock period (lo2). because, it is expected that sensitive genotypes will respond more quickly to the initial thermal shock and die; therefore, the first time measurement of mortality rate (lo1) was considered as a thermotolerance criterion. in the same time, larval mortality was recorded for nonheat stress groups. lo2 is not a good criterion to rank the silkworm hybrids; since long-term heat exposure may cause to be killed most of larvae in each tray. average larval mortality in response to heat shock was reported as log-transformed values to assure normality. a factorial experiment (4×2) with completely randomized design was utilized to evaluate the effects of different silkworm hybrids (110×107, 104×103, 103×104, 107×110) and heat exposure (with or without thermal shock), as fixed effects, and their interactions on larval mortality (lo1 and lo2). statistical analysis of data was carried out using the glm procedure of sas 9.0. least squares means were used for identifying the main effects. results the results showed that gene expression of hsp90 and hsp70 in hybrids with japanese maternal parents) (103×104 and 107×110) were significantly (p < 0.0001) higher than hybrids with chinese maternal parents (104×103 and 110×107). table 2 shows that the fluctuation of hsp expression for both genes is almost the same. in other word, the expression of hsp90 was in good accordance with hsp70. least-squares means of hsp70 and hsp90 gene expression (table 2) indicated that there was a high significant difference(p < 0.0001) in the hsp70 and hsp90 expression between two reciprocal hybrids (110×107 vs. 107×110 and 103×104 vs. 104×103) immediately after heat exposure (fig. 1). expression of both genes immediately after heat shock (time zero) was significantly higher than other times (p < 0.0001) in all genotypes, afterwards, gradually decreased to the lowest level after 24 h (table 2). in this time down-regulation was observed in almost all genotypes, in fact, hsp70 and hsp90 expression reduced over time. four h after thermal exposure, while hsp70 expression in the three hybrids declined but still 103×104 had higher expression. the hybrid 104×103 had significantly lower expression among hybrids (p < 0.0001) which proposed that this hybrid need less hsp70 and hsp90 in thermal shock conditions. in another experiment a prolonged heat shock (8 h at 39 ◦c for six days) was implemented to examine the thermotolerance of larvae. the mortality rate (in %) was considered as a measure of thermotolerance. mortality of all treatment groups was higher than control groups. the hybrid 110×107 and 103×104 had the lowest and the highest mortality rates, respectively. in fact, 103×104 as a sensitive hybrid had the highest losses and 110×107 hybrid as a resistant hybrid had the lowest 59 fig. 1 hsp70 and hsp90 gene expression immediately after heat exposure (time zero) in the studied silkworm hybrids losses (fig. 2). the same result was obtained control group; however differences was not significant (table 3). 110×107 and 107×110, two silkworm hybrids that their parents were originally from korea, were more resistant than hybrids 103×104 and 104×103 with japanese parents. among four genotypes, the sensitive hybrids (103×104 and 107×110) had the highest gene expression immediately after heat exposure (zero time) implying that these genotypes need more hsp70 and hsp90 under severe thermal conditions. in fact, hybrids with japanese maternal parents (107×110 and 103×104) were sensitive and hybrids with chinese maternal parents (110×107 and 104×103) were relatively resistant. therefore, sensitive hybrids immediately after heat exposure need both hsp70 and hsp90 proteins to reduce thermal effects. discussion a large difference was observed between the genotypes in terms of hsp expression. generally, the results showed that the hybrids with japanese maternal parents had more hsps expression than chinese maternal parents. there are some reports on genetic differences of hsp70 or hsp90 among silkworm genotypes such as comparison of nistari and jingsong (hosseini moghaddam et al., 2008), nistari and nb4d2 (velu et al., 2008), c. nichi, pure mysore and nb4d2 strains (joy and gopinathan, 1995) and pure mysore and nb4d2 (sosalegowda et al., 2010). velu et al. (2008) reported little differences in hsp70 expression between two resistant and sensitive strains on agarose gel (semiquantitative) after a mild heat shock (41 °c for one hour) but li et al.)2011(using qrt-pcr method showed that expression of hsp70 in jingsong (a thermosensitive commercial strains) was significantly more than nistari breed )a thermotolerant multivoltine breed in tropical region). it means that thermo-sensitive breed induced strongly hsp70 mrna after heat shock treatments. this result was consistent to our study which among four genotypes, the sensitive hybrids (103×104 and 107×110), as japanese maternal parents, had the highest gene expression. garbuz et al. (2002) reported that among drosophila species and strains originating from different climatic zones, some of species and strains exhibited positive correlation between hsp70 expression and thermotolerance and for others negative correlation. table 3 average larval mortality in the four silkworm hybrids for both thermal treatment and control groups hybrids lo 1* lo 2 107 ×110 (treatment) 0.70 ± 0.07c 1.84 ± 0.02a 107×110 (control) 0.13 ± 0.02d 0.93 ± 0.01d 110 ×107 (treatment) 0.50± 0.08cd 1.66 ± 0.01b 110×107 (control) 0.09d 0.57 ± 0.005e 103×104 (treatment) 1.66± 0.07a 2.02± 0.003a 103×104 (control) 0.34 ± 0.02d 1.23 ± 0.07c 104 ×103 (treatment) 1.19± 0.03b 1.91± 0.005a 104 ×103 (control) 0.09d 0.7 ± 0.04d a-e different superscripts within a column indicate significant differences (p < 0.0001) * average larval mortality is log-transformed values (lo1: first period of measuring mortality; lo2: second period of measuring mortality) 60 fig. 2 larval mortality percentage by treatment and control groups in the studied silkworm hybrids (lo1: first period of measuring mortality; lo2: second period of measuring mortality; average larval mortality is log-transformed values) in the current study hsp70 and hsp90 expression was up-regulated in the fat body of silkworm larvae after severe (45 °c) but non-lethal heat shock. . li et al. (2011) observed higher expression of hsp70 in fat body rather than testis and ovary in a thermosensitive strain of silkworm. velu et al. (2008) reported mid gut and fat body tissues had higher hsp70 expression than the cuticle and silk gland tissues. keshan et al. (2014) studied hsp90 expression in various tissues of silkworm and indicated that a higher hsp90 expression in all examined larval tissues after mild (39 °c) and mild to severe (42 °c) heat treatments. the highest expression was observed just immediately after heat exposure (time zero) in all silkworm hybrids. to our knowledge, this is the first study where the differences of hsp gene expression were evaluated for reciprocal crosses in the silkworm. in the silkworm, high thermotolerance in fifth instar larvae reflects its adaptation to high temperature (manjunatha et al., 2010). a large difference was also observed between the genotypes in terms of heat tolerance. the hybrids with japanese maternal parents were more sensitive than the chinese ones, which was in line with other researches (vasudha et al., 2006; firdose and reddy, 2009). hsieh et al. (1995) by comparing chinese and japanese maternal parents demonstrated that feng, a chinese strain, was the most tolerant strain followed by japanese races, kuo and j-09, while another chinese strain, c-54 was most susceptible. in different studies, different genotypes had different reactions to the heat stress. this diversity among silkworm varieties is related to their genetic background and related silkworm breeding plan. our results showed that this kind of differences could be detected in molecular levels. further investigations of these differences can help us to understand the mechanisms of protecting cells against environmental stresses and also identify variation of hsps genes expression among susceptible/ tolerant strains and hybrids of silkworm. conclusion the results of this study showed that both hsp70 and hsp90 have been up-regulated in the treated larvae. the hybrids with japanese female parents (107×110 and 103×104) were more thermosensitive than two others, moreover hsp90 and hsp70 were also significantly expressed in these two hybrids after heat shock. comparison of larval mortality among four hybrids revealed that the hybrid 103×104 had the highest mortality rate and 110×107 had the lowest mortality rate. high expression of hsps in thermo-sensitive hybrids showed importance of hsps in survivability. generally, it can be concluded that the expression patterns of high molecular weight hsp can 61 determine the various levels of thermotolerance in the different silkworm genotypes. acknowledgement we are grateful to the iran silk research center (guilan province) for cooperation in silkworm rearing. this work was supported by biotechnology committee of university of guilan under their block grant. references bettencourt br, hogan cc, nimali m, drohan bw. inducible and constitutive heat shock gene expression responds to modification of hsp70 copy number in drosophila melanogaster but does not compensate for loss of thermotolerance in hsp70 null flies. 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identification of two hsp90 genes from the marine crab, portunus trituberculatus and their specific expression profiles under different environmental conditions. comp. biochem. physiol. 150c: 465-473, 2009. zhao l, jones wa. expression of heat shock protein genes in insect stress responses. inv. surv. j. 9: 93-101, 2012. zheng ww, yang dt, wang jx, song qs, gilbert li, zhao xf. hsc70 binds to ultra-spiracle resulting in the up-regulation of 20hydroxyecdsone-responsive genes in helicoverpa armigera. mol. cell. endocrinol. 315: 282-291, 2010. isj 7: 211-220, 2010     211 isj 7: 211-220, 2010 issn 1824-307x review echinoderm immunity f ramírez-gómez1, je garcía-arrarás2 1department of biology, university of massachusetts dartmouth, 285 old westport road, north dartmouth, ma 02747, usa 2department of biology, university of puerto rico, p.o. box 23360, upr station, río piedras, san juan, pr 00931-3360, usa accepted september 27, 2010 abstract echinoderms are exclusively marine animals that, after the chordates, represent the second largest group of deuterostomes. their diverse species composition and singular ecological niches provide at the same time challenges and rewards when studying the broad range of responses that make up their immune mechanisms. two types of responses comprise the immune system of echinoderms: a cellular response and a humoral one. cell-based immunity is carried by the celomocytes, a morphologically heterogeneous population of free roaming cells that are capable of recognizing and neutralizing pathogens. celomocytes present diverse morphologies and functions, which include phagocytosis, encapsulation, clotting, cytotoxicity, wound healing among others. humoral immunity is mediated by a wide variety of secreted compounds that can be found in the celomic fluid and play important roles in defense against infection. compounds such as lectins, agglutinins, perforins, complement and some cytokines make up some of the humoral responses of echinoderms. recent advances in the field of molecular biology, genomics and transcriptomics have allowed for the discovery of new immune genes and their products. these discoveries have expanded our knowledge of echinoderm immunity and are setting up the stage for future experiments to better understand the evolution of the immune mechanisms of deuterostomes. key words: comparative immunology; echinoderm; immunity; celomocytes; genes introduction the phylum echinodermata is a very diverse group of marine animals that have sparked the interests of scientists for over a century. significant discoveries have been made using echinoderms in the areas of cell biology, developmental biology and immunology. five classes comprise the phylum: asteroidea (sea stars or starfish), crinoidea (crinoids or feather stars), ophiuroidea (brittle stars), echinoidea (sea urchins and sand dollars) and holothuroidea (sea cucumbers or holothurians). even though research has been done on all echinoderm classes, one group excels as the favorite of scientists: the echinoids. thus, sea urchins have become one of the classical animal models and have been particularly exploited in studies of fertilization and developmental biology. similarly, in the field of echinoderm immunology, sea urchins comprise the group that has been most ___________________________________________________________________________ corresponding author: francisco j ramirez-gomez university of massachusetts dartmouth, 285 old westport road, north dartmouth, ma 02747-2300, usa e-mail: framirez@umassd.edu extensively studied (smith et al., 2006). furthermore, the availability of the genome sequence for the purple sea urchin (strongylocentrotus purpuratus) has allowed for in depth studies into the genetic aspects of its immune response (hibino et al., 2006; rast et al., 2006). this trend has helped advance the field and at present, echinoderms are catching the attention of comparative immunologists. however, due to the inherent diversity of the echinoderm phylum, general assumptions cannot be easily established and what is true for one specific class may not apply to others. interest in echinoderm immunobiology also originates form the aquaculture field. although little known in the western hemisphere, holothurian and echinoid cultures are an important economic activity in asia. with an increasing demand for sea urchin roe and trepang (a generic name for sea cucumbers), commercial culture venues have increased in order to maintain the demands for these organisms. with increase in aquacultures one observes an increase in diseases, mainly infections, and therefore an increase interest in understanding how the organisms protect themselves from mailto:framirez@umassd.edu     212 pathogenic threat. the present review will attempt to summarize the latest published research work on the echinoderm immune system with a special emphasis on non-echinoid groups. the review focuses on the different immune components and mechanisms present in the phyla and highlights how rich, diverse and complex this group of animals can be. general aspects of echinoderm immunity in terms of their immune systems, echinoderms display the same basic types of responses that most multicellular (including vertebrates) animals do. they can recognize self from non-self and, if a foreign material (e.g., microorganism/pathogen) enters the body cavity, they can readily neutralize it and dispose of it (yui and bayne, 1983; dybas and fankboner, 1986; jans et al., 1996; glinski and jarosz, 2000). additionally, echinoderms possess very good wound-healing capabilities, a key feature that also plays an important role in one of the best known characteristics of the group: regeneration of lost body parts. these defense mechanisms are mediated by cellular and humoral responses, with several homologous and analogous components found in other invertebrates and vertebrates alike. in fact, it is their key position in the evolutionary tree, being invertebrate deuterostomes (thus sharing a common evolutionary branch with vertebrates) that makes the study of their immune system a very interesting and exciting field. therefore, this advantageous phylogenetic position allows for comparisons between immune mechanisms that have been well studied in vertebrates with those of their echinoderm counterparts. thus, echinoderms can provide important information on the evolution of the immune response. as in many other systems, echinoderm immune responses can be divided between cellular and humoral responses. cellular responses are mediated by the celomocytes, which are free roaming cells that occupy the celomic cavity but can also infiltrate tissues and organs. these cells circulate in the celomic fluid and exert the vast majority of immune functions. on the other hand, humoral responses are defined by the broad variety of molecules present in the celomic fluid. these molecules are capable of recognizing and neutralizing foreign material, promoting cell migration and agglutination and also playing roles in wound healing ( ryoyama, 1973; kanungo, 1982; canicatti et al., 1992; smith and davidson, 1992). cellular components celomocytes are a very abundant and diverse cell types that are present in all echinoderms. these cells are heterogeneous in morphology, size, relative abundance and function, which make a single standard classification for all echinoderms a difficult task. extensive research has been done during the past century on the morphological aspects of celomocytes. comprehensive reports on the celomocyte types of different echinoderms classes have also been published (kindred, 1924; boolootian and giese, 1958, 1959; boolootian, 1962; endean, 1966). these studies clearly show the wide variety of cell morphologies present in the echinoderm celomic fluid. however, the absence of a standard reference among groups and particularly, differences in terminology and even specimen preparation, contribute to the existing heterogeneity. nonetheless, some types of celomocytes can be found in all classes, while others have been considered to be specific to certain classes. these cell types are summarized in table 1 along with the particular functions that have been ascribed to certain cells. the distribution of these cell types is highly variable among species and also even at the individual level. for example, in some sea star species the vast majority (> 90 %) of celomocyte types are amebocytes, while other cell types seem to be exclusive of certain groups (e.g., holothurian crystal cells). table 2 summarizes the general distribution of celomocytes in three echinoderm classes (echinoids, holothuroids and asteroids) and how they differ depending on the group and the species. this cell distribution is also very dynamic, changing in accordance to the physiological or immune state of the animal. for example, in the sea star asterias rubens specific sub-populations of amoebocytes increase in number after injection of gram-positive bacteria while other sub-groups remain unchanged (coteur et al., 2002). our studies with the sea cucumber holothuria glaberrima have shown that the total number of celomocytes remains unchanged after challenges with diverse pathogen associated molecular patterns (pamps). however, the distribution of particular sub-types changes after immuno-stimulation, e.g., lymphocytes numbers diminish, while phagocytes increase (ramirezgomez et al., 2010). from all the celomocyte types, probably the one that is present in all the echinoderm classes is the phagocyte/amebocyte type. this cell ranges in size from 3 to 20 μm and its main characteristic is its ability to phagocytize other cells or foreign particles (endean, 1966). other roles have been attributed to phagocytes, most of them immune related, demonstrating that this cell type is the main effector of the echinoderm immune system. in fact, the discovery of these cells in the sea star, back in the late 1800’s by russian zoologist ilya metchnikoff gave rise to the field of cellular immunity, for which he was awarded the nobel prize in 1908 (metchnikoff, 1891). amebocyte roles include: graft rejection, chemotaxis, reactive oxygen species production, encapsulation, cytotoxicity, immune gene expression, agglutination and clotting reactions (gross et al., 1999, 2000; beck et al., 2001; coteur et al., 2001; lin et al., 2001; hillier and vacquier, 2003; clow et al., 2004; matranga et al., 2005; sun et al., 2008). several authors subcategorize phagocytes according to their size and morphology, but since these classifications are not the same for all echinoderms, some sub-types may overlap or on the other hand can be rendered as a different cell type altogether. lymphocytes are another cell type that might be present in all echinoderms (endean, 1966), but it is most frequently found in holothurians and some sea stars     213 table 1 summary of celomocyte types reported for echinoderm classes. e: echinoidea, h: holothuroidea, a: asteroidea, c: crinoidea, o: ophiuroidea. cell type present in class role reference discoidal cell e, h polygonal cell e small phagocyte e, h amebocytes /phagocytes e, h, a, c, o phagocytosis, clotting, encapsulation, chemotaxis, opsonisation, graft rejection (coteur et al., 2002; de faria and da silva, 2008; eliseikina and magarlamov, 2002; endean, 1966; matranga et al. 2005; ramirez-gomez et al., 2010; smith et al., 2006) colored spherule e, h, c antibacterial activity (de faria and da silva, 2008; endean, 1966; smith et al., 2006) colorless spherule e, h, a, c, o antibacterial, inflammation, wound healing, ecm remodeling (coteur et al., 2002; de faria and da silva, 2008; eliseikina and magarlamov, 2002; endean, 1966; garcia-arraras et al., 2006; ramirez-gomez et al., 2010; smith et al., 2006) lymphocyte e, h, a progenitor cells (coteur et al., 2002; eliseikina and magarlamov, 2002; endean, 1966; ramirez-gomez et al., 2010; xing et al., 2008) vibratile e, h, a, o celomic fluid movement, clotting (de faria and da silva, 2008; eliseikina and magarlamov, 2002; endean, 1966; matranga et al., 2005; pinsino et al., 2008; ramirez-gomez et al., 2010; smith et al., 2006; xing et al., 2008) crystal cells h osmoregulation (eliseikina and magarlamov, 2002; endean, 1966; ramirezgomez et al., 2010; xing et al., 2008) hemocytes h, a, o oxygen transport (eliseikina and magarlamov, 2002; endean, 1966; pinsino et al., 2008) (smith, 1981). these are small cells (4-6 μm), with a large nucleus and a thin layer of cytoplasm whose only common characteristic with their vertebrate namesakes is their morphology. lymphocytes are regarded as progenitor cells and may be the precursors of other celomocyte types (xing et al., 2008; ramirez-gomez et al., 2010). they can show phagocytic capabilities but this may reflect an intermediate state of maturity before becoming phagocytes (ramirez-gomez et al., 2010). spherule cells (spherulocytes) are present mostly in echinoids and holothuroids (endean, 1966; eliseikina and magarlamov, 2002; smith et al., 2006; de faria and da silva, 2008; xing et al., 2008; ramirez-gomez et al., 2010) and in at least one sea star species (penn, 1979). they are characterized by the presence of vesicles in their cytoplasm, some containing pigment (red, yellow, green, brown) other being colorless. spherulocytes range in sizes from 8 to 20 μm and their distribution varies substantially between species. they have been associated with antibacterial activity (johnson, 1969; service and wardlaw, 1984; haug et al., 2002), inflammatory responses (pagliara and canicatti, 1993), extracellular matrix remodeling (garcia-arraras et al., 2006), and wound healing (san miguel-ruiz and garcia-arraras, 2007). another cell type present in echinoids and holothuroids are the vibratile cells. these are cells whose size ranges from 6 to 20 μm and are highly motile due to the presence of a flagellum. their distribution varies accordingly to the species and their function is still not completely determined. they have been associated with clotting reactions (bertheussen and seijelid, 1978) and are also thought to be involved in the movement of the celomic fluid (xing et al., 2008). crystal cells seem to be exclusive of holothurians, these cells display a very regular geometric morphology (rhomboidal or hexagonal) and present a crystal inclusion within their cytoplasm (endean, 1966). their role is still not well defined, but it is likely that they play osmoregulatory roles (xing et al., 2008).     214 table 2 summary of celomocyte distribution in the celomic fluid in three echinoderm classes and six different species. l.var: lytechinus variegatus; s. purp: strongylocentrotus purpuratus; e. luc: echinometra lucunter; h. glab: holothuria glaberrima; a. jap: apostichopus japonicus; a. rub: asterias rubens. cell type echinoidea holothuroidea asteroidea l. var (borges et al., 2005) s. purp (smith et al., 2006) e. luc (de faria and da silva, 2008) h. glab (ramirezgomez et al., 2010) a. jap (xing et al., 2008) a. rub (coteur et al., 2002) lymphocytes n.f. .n.f n.f. 60 % 59 % n.f. phagocytes/ amebocytes > 60 % 40-80 % 77 % 30 % 17 % 80-95 % colored spherules < 40 % 7-40 % 1 % n.f. n.f. n.f. colorless spherules + 3-25 % 3 % 5 % 23 % n.f. vibratile cells + 11-20 % 19 % < 1 % n.a. n.f. n.f., not found; n.a., not accounted. n.f., not found. + spherules and vibratile cells were accounted together. interestingly, echinoderm cellular immunology has escaped the classical characterization of their components by phenotyping (identification of cellspecific epitopes expressed on cell membranes) as vertebrate lymphocytes do. the lack of definite surface markers for celomocytes has helped maintain the confusion in distinguishing between specific cell types and sub-types, limiting it to just morphological observations. however, this trend is slowly changing, as demonstrated by studies with sea urchin celomocytes, in which a sub-population of phagocytes was defined by nk cell surface markers and characterized by their cytotoxic properties (lin et al., 2001). furthermore, monoclonal antibodies were generated against these cells showing a successful identification of a specific cytotoxic phagocyte sub-type (lin et al., 2007). our research group has also identified subgroups of sea cucumber celomocytes using monoclonal antibodies, each sub-population showing distinct characteristics and different responses to immunostimulation (ramirez-gomez et al., 2010). similarly, a recent study in the sea cucumber apostichopus japonicus, has also led to the development of a monoclonal antibody that specifically recognizes spherulocytes. initial characterization of the antigen being recognized by this antibody resulted in the identification of a 136 kda protein according to western blotting (li et al., 2010). however, what is still missing is the full characterization of the antigens these antibodies are recognizing (protein sequences and cloning). future experiments where cell markers are used to describe celomocyte populations and compare these populations among the different echinoderm classes should be the basis for a clear and universal classification of echinoderm celomocytes. celomocyte origin the origin of celomocytes is still a matter of debate. two theories have been proposed to address this issue: one involving specific organs or tissues as the source of celomocytes while the other points at the celomocytes themselves as selfreplicating cells (bossche and jangoux, 1976; matranga et al., 2005). potential cytopoietic organs include the axial organ, tiedemann bodies, polian vesicles, connective tissue and the celomic epithelium (endean, 1966). the latter has received particular attention in studies involving the sea star a. rubens, showing the epithelial origin of sea star celomocytes (bossche and jangoux, 1976; holm et al., 2008). even though no direct evidence have been proposed for a self-replicating population of celomocytes, the idea of a circulating stem cell has not been ruled out and if anything has become more attractive in view of recent findings of stem cells in other metazoans (handberg-thorsager et al., 2008; watanabe et al., 2009; funayama, 2010). it must be stated that the evidence to ascertain the origin of celomocytes is far from definitive, and would not be acceptable by modern scientific standards. thus, it is necessary for scientists to use modern methodologies to verify the celomocyte origins proposed by past investigators. humoral components echinoderms present a wide rich variety of secreted immune molecules. they have been the subjects of extensive research, even to the point of potential medical applications (kelly, 2005). as mentioned before, the humoral components present in celomic fluid of echinoderms are capable of     215 recognizing foreign matter, neutralizing or destroying pathogens, inducing or enhancing cellular responses (opsonization) and helping during wound healing. a well-known group of recognition molecules are the lectins, which recognize carbohydrate moieties on the surface of host cells (self) and of bacteria and fungi (non-self). several lectins have been identified from the celomic fluid of echinoderms, where they play important roles in opsonization, lytic cytotoxicity, clot formation and wound repair (gross et al., 1999). different lectins with specific recognition abilities have been found in asteroids (kamiya et al., 1992), and holothuroids (matsui et al., 1994; gowda et al., 2008a, b). echinoidin, a c-type lectin (calcium-dependent) identified in a sea urchin also possess an rgd sequence, suggesting an additional role in cell-tocell adhesion (giga et al., 1987; ozeki et al., 1991). moreover, the c-type lectin cel-iii from the sea cucumber cucumaria miniata, which possess a strong hemolytic activity have been transgenically expressed in mosquitoes and shown to successfully impair malaria parasite development (yoshida et al., 2007). other humoral factors include hemolysins, that interact with plasma membranes and form holes in the membrane of cells causing lysis of target cells (canicatti, 1990, 1991). hemolysins have been identified in sea stars (leonard et al., 1990), sea urchins (ryoyama, 1973; stabili et al., 1992) and sea cucumbers (canicatti and parrinello, 1985). agglutinins are another type of humoral factor that play roles in cell aggregation, encapsulation and clotting and have also been studied in wound repair. they have been found in echinoids (ryoyama, 1973; canicatti et al., 1992), the sea star asteria pectinifera (kamiya et al., 1992) and in the sea cucumber holothuria polii (canicatti and parrinello, 1985). in vertebrates a well-known group of effector molecules are the cytokines, which play a wide variety of roles in the immune response. in echinoderms, homologues of cytokines have also been identified. the first glance at an echinoderm cytokine came from the sea star a. forbesi, in which a humoral factor named the sea star factor was isolated and found to possess cytokine-like properties (prendergast and suzuki, 1970; prendergast and liu, 1976; kerlin et al., 1994). furthermore, interleukin-like molecules were identified in the sea star, e.g., a protein with il-1 activity and an il-6 like molecule ( beck et al., 1989, 1993; beck and habicht, 1991a, 1991b, 1996). however, none of these findings resulted in a definite identification of the cytokine factor or the cloning of the corresponding gene(s). the issue appears to be complicated since no il-1 homologues were found in the sea urchin genome. however, other members of the cytokine network (mostly pro-inflammatory) have indeed been found, e.g., tnf and il-17 (hibino et al., 2006). another important humoral factor present in echinoderms is the complement protein family. most of the components of the alternative and lectin pathways have been identified in the sea urchins, being the purple sea urchin the first invertebrate in which a complement system was identified (smith et al., 1996; smith, 2001). initial evidence gathered from sea urchins, sea cucumbers and sea stars hinted at the presence of a complement system in echinoderms (kaplan and bertheussen, 1977; parrinello et al., 1979; bertheussen 1981a, 1981b, 1982, 1983; bertheussen and seljelid, 1982), but they were mostly from complement-derived or dependent activity and no definite identification of a complement protein was achieved. smith and colleagues (1996, 1998) successfully identified the first echinoderm (and invertebrate) homologue of the c3 component and later another complement protein was found (factor b) (smith et al., 1998). a recent publication reported the finding of a c3 complement homologue in the sea star a. rubens, whose expression is also induced by lps stimulation (mogilenko et al., 2010). additional components of the system have been identified from the sea urchin genome, suggesting that echinoderms possess a complement pathway mostly directed towards opsonization, since the components of canonical terminal pathway could not be found (hibino et al., 2006). molecular studies and the genomic era the vast majority of molecular studies have been done in echinoids, particularly the purple sea urchin s. purpuratus. a broad number of immune genes have been identified from the sea urchin since the early 1990’s and pinnacled with the publication of the s. purpuratus genome (sodergren et al., 2006). an in depth analysis of the immune repertoire contained within the sea urchin genome can be found in the publications of hibino et al. (2006) and rast et al. (2006). however, other species of echinoderms have also been the subject of molecular studies in order to better understand their immune responses. these studies altogether benefit greatly the advancement of the field, providing further insights into the genetic and molecular aspects of echinoderm immunity. echinoderm molecular immunogenetics has evolved in parallel with the technologies available for its study. starting with gene-by-gene approaches, in which single genes were analyzed at a time and their immune roles determined. an example of this is the case of the profilin gene, an actin binding and cytoskeletal modification protein, expressed in celomocytes and up-regulated after injury and lps injections (smith et al., 1992, 994, 1995). then, when sequencing technologies became accessible, high-throughput sequencing projects were launched, mostly screening cdna libraries. in the late 1990’s a survey of a cdna library from lps-activated celomocytes provided the first glimpses of the immune repertoire of an echinoderm (smith et al., 1996). several interesting findings were made in this study on sea urchins, beginning with the discovery of an echinoderm complement and a collection of putative immune effector genes that set the basis for future comparative studies between echinoderm species. our research group has been dwelling into the molecular immune aspects of holothurians since the     216 year 2000, when a homologue of the acute phase response protein serum amyloid a (saa) was identified for the first time in an invertebrate (santiago et al., 2000). its expression was found mostly in intestinal tissues during the regeneration process but also after immune stimulation with lps. saa mrna was found to be overexpressed following an immune challenge not only in the intestine (santiago-cardona et al., 2003) but also in celomocytes (ramirez-gomez et al., 2008). additionally, a series of immune-related genes were identified in the holothurian from intestinal cdna libraries. this identification was mostly done by sequence comparison with other immune genes present in other organisms and whose immune role was clearly defined. the expression of these holothurian immune genes was corroborated in celomocytes to determine if they were part of the gene repertoire of these cells. in addition their expression was analized after an lps challenge (ramirez-gomez et al., 2008). among these genes we found a c-type lectin, ferritin, cathepsin, toposome and an alpha 2 macroglobulin domain (a2m)-containing protein. one sequence of particular interest was a homologue of the dd104 protein from the sea urchin, which is up-regulated in celomocytes after injury and infection (rast et al., 2000) with the holothurian dd104 following a similar pattern but with higher expression levels in celomocytes after lps injection. recently, analysis of the expression of immune-related genes has also been done in embryos and larvae of the sea cucumber, a. japonicus. nine genes were studied: six of them (heat shock proteins -70, -90 and -gp96; thymosin-beta, ferritin and dd104) showed no changes upon lps challenge while the remaining three (mannan-binding c-type lectin, lysozyme and serine proteinase inhibitor) were found to be upregulated upon challenge (yang et al., 2010). the advent of array technologies that allow for studies on the expression of multiple genes at the same time, have opened the door for the identification of potential novel genes. many of these genes were missed in previous approaches probably due to their lack of homology to known genes. this approach was first carried out with the sea urchin, comparing immune-stimulated and immunoquiescent animals. an unexpected diversity of genes was found to be differentially expressed and, more interestingly, a set of novel genes, the 185/333 family of proteins, were then identified (nair et al., 2005). this family of genes represents a highly variable set of proteins that are involved in the immune response of the sea urchin (reviewed in ghosh et al., 2010). we have also used immune activation and microarray technologies to compare lps-injected sea cucumbers with seawater-injected controls and thus, identify immune-responsive genes in the holothurians. we have found 50 unique sequences differentially expressed after lps stimulation. the vast majority of these sequences showed no known homologies in the databases (ramirez-gomez et al., 2009). ongoing efforts are being done to further characterize these unknown genes. by complete sequencing of the mrnas we expect to find similarities and/or conserved domains that will provide either proper identification, or the characterization of novel holothurian lpsresponsive genes. an interesting case derived from our microarray study, is the echinoderm mayor yolk protein (myp) and its closely related protein, toposome. in our microarray, the holothurian myp gene was one of the top genes that showed differentially expression following lps injection. echinoderm myp was initially identified as an unconventional iron-binding vitellogenic protein making up to 50 % of the protein content of the sea urchin celomic fluid (brooks and wessel, 2002). it is synthesized in the digestive tract and also binds zinc ions (unuma et al., 2007, 2009). a possible immune role for this protein had been suggested due to its affinity for iron, making it an excellent bacteriostatic agent. our results from the holothurian microarray, have shown that myp mrna is up-regulated after lps stimulation in the digestive tract but its expression remains unchanged in celomocytes (ramirez-gomez et al., 2009). nonetheless, anti-myp labeling is found in phagocytic lymphocytes (ramirez-gomez et al., 2010). the toposome protein (which is closely related to myp), functions as an adhesion protein in the sea urchin embryo (cervello and matranga, 1989; scaturro et al., 1998; noll et al., 2007), but is also related to stress and injury respones (cervello et al., 1994; matranga et al., 2005; pinsino et al., 2007). we have found toposome mrna to be expressed in h. glaberrima celomocytes at relatively high levels that remain unchanged after lps stimulation (ramirez-gomez et al., 2008) as well as in intestinal tissues, in which its levels remained unchanged also (ramirez-gomez et al., 2009). these results show that both myp and toposome are indeed associated with the immune response but also suggest additional roles that might not be part of the traditional functions associated with celomocytes but might be associated with the immune functions of the digestive tract. now that we have entered the genomic era, further advances are expected as genome sequencing technologies become faster and more economically accessible. the s. purpuratus genome represents a cornerstone in echinoderm research that can be used to compare findings from other echinoderm species. however, as presented here, the great diversity of the animals within the echinoderm phylum suggest that having the genome of only one member of only one echinoderm class will not be enough to understand the echinoderm immune system. take for example one of the most diverse set of genes found in the sea urchin genome: the nlr gene family (nucleotide-binding domain, leucine-rich repeat containing proteins). these genes encode cytoplasmic pattern recognition proteins, which in humans are represented by about 20 genes (inohara and nunez, 2003). however, in the sea urchin 203 nlr predicted genes can be found. similar to the vertebrate counterparts, the major site of expression of the sea urchin nlrs is the gut (hibino et al., 2006). nonetheless, we were not able to identify sequences for this gene family in any of our holothurian intestinal cdna libraries nor in our intestinal microarray studies. this may reflect key     217 differences in gene repertoires between these two species related to their phylogenetic divergence. this variety of gene repertoires may also be attributed to differences in habitat and developmental history, and to differences in the microbe flora that challenges the organisms. these differences will eventually shape the type of immune responses that organisms react to. therefore, we still need more information on immune-related genes present in other species from as many different groups as possible in order to have a better understanding of the molecular events that are involved with the echinoderm immune response. concluding remarks echinoderm immunity is a challenging yet promising field to study. the large diversity of echinoderm species, with different internal organs (most of them with little physiological information as to their functions) and different lifestyles make it difficult to identify those tissues or cells that might be playing an immune role. moreover, different species might be responding to different immune challenges not usually associated with other animal groups (think about the fact that echinoderms occupy large 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obtained from stem cells isolated from b. mori larvae cultured in grace’s medium supplemented with 20-hydroxyecdysone (20-he) and α-arylphorin. after three weeks, up to 60 % of the cultured cells were differentiated into columnar and goblet cells, the two predominant cell types in the midgut epithelium. these cells presented in vitro the same shape, morphology and polarity recorded in vivo, even if their dimensions were slightly reduced. columnar cells displayed a well developed cytoskeletal arrangement, with actin filaments highly organized within the thick brush border and distributed in faint filaments in the cell cytoplasm. microtubules formed a substantial net just beneath the brush border and ran longitudinally from the apical to the basal pole of the cell. cultured cells homogenates displayed aminopeptidase n and alkaline phosphatase activity, proving that these two enzymes, involved in vivo in the intermediate and final digestion, are expressed also in vitro. the columnar cells differentiated in culture were able to internalize two model proteins with quite different transport rates. key words: bombyx mori larval midgut; stem cells; columnar cells in culture; cytoskeletal scaffolding; digestive enzymes; protein uptake introduction the lepidopteran midgut is formed by a folded epithelial cells monolayer, separated from underlying muscles and tracheae by a thin basal membrane and composed by three main cell types, goblet, columnar, stem cells (cioffi, 1979; baldwin and hakim, 1991), and by fewer endocrine cells. goblet cells have a peculiar shape, with a basally located nucleus and a cavity lined by an apical plasma membrane forming numerous microvilli, where a v-h+-atpase pump (wieczorek et al., 1989) and a k+/2h+ antiport (azuma et al., 1995) are expressed. the combined activity of these two transporters generates the high electrical voltage, the active secretion of k+ and the extreme luminal ___________________________________________________________________________ corresponding author morena casartelli dipartimento di biologia università degli studi di milano via celoria 26, 20133 milano, italy e-mail: morena.casartelli@unimi.it alkalinisation typical of the lepidopteran midgut epitehelium. columnar cells have an almost cylindrical shape with a central nucleus, an apical thick brush border and deep infoldings of the basal plasma membrane (cioffi, 1979; baldwin and hakim, 1991). this cell type is involved in nutrient digestion (terra and ferreira, 2005) and absorption (giordana et al., 1982, 1998). the small stem cells, roughly conicalor spindle-shaped with a large nucleus, are located at the base of the epithelium (turbeck, 1974; baldwin and hakim, 1991): they are the only cell type that undergoes mitosis (loeb and hakim, 1996) and their proliferation initiates in proximity of the moults (baldwin and hakim, 1991). in the last decade primary cultures of mature cells from several lepidopteran species were obtained successfully from the isolated midgut stem cells maintained in a culture medium integrated with 20hydroxyecdisone and α-arylphorin, a subunit isolated from a perivisceral fat body extract of manduca sexta pupae (sadrud-din et al., 1994, 119 1996; loeb and hakim, 1996; blackburn et al., 2004). differentiation to goblet and columnar cells requires the presence of factors released in the medium by the actively developing cell culture (sadrud-din et al., 1996). more recently, some of the peptides that, acting like the mammalian growth factors, induce cell differentiation have been identified (loeb et al., 1999; goto et al., 2001; loeb and jaffe, 2002). we have recently shown that bombyx mori larval midgut isolated and perfused in vitro can transport two selected proteins unaltered from the lumen to the haemolymph side of the epithelium by transcytosis (casartelli et al., 2005, 2007). the availability of single mature columnar cells in culture may represent the best tool to study in detail the multiple steps involved in this complex pathway. we have therefore standardised a primary culture of larval midgut cells from b. mori and examined some morphological features of the isolated cells grown and differentiated in culture. we also investigated if they expressed some of the digestive enzymes detected in vivo and if they could internalize two different proteins, albumin labelled with fluorescein isothiocyanate (fitc-albumin) and green fluorescent protein (gfp). materials and methods experimental animals bombyx mori eggs and the artificial diet (cappellozza et al., 2005) were provided by crainstitute for sericulture (padova, italy). larvae were reared under controlled conditions (25 ± 1 °c, 65-70 % rh, 12l:12d photoperiod). histological analysis of iv instar midgut epithelium during pre-moult, moult and v instar feeding period silkworms at the above indicated stages of development were anaesthetized with co2. the midgut was explanted, deprived of the peritrophic membrane and malpighian tubules and fixed in pycric acid, formaldehyde and glutaraldehyde (pafg) at room temperature for 2 h and then at 4 °c over-night according to ermak and eakin (1976). the samples were then washed in 0.1 m cacodylate buffer (ph 7.4), postfixed in 1 % osmium tetroxide in the same buffer for 2 h, washed in distilled water and left for 2 h in 2 % uranyl acetate. after dehydratation in a graded ethanol series, samples were embedded in epon resin and the polymerisation was performed at 60 °c for 48 h. semithin sections were cut with a reichert ultracut e microtome and observed at the light microscope (axiovert 200m equipped with axiocam hrm, zeiss, germany). preparation of midgut cells cultures larvae of b. mori at the end on the iv instar, just before the iv moult, were anaesthetized with co2 and surface-sterilized by consecutive immersions, lasting approximately 2 min each, in the following solutions: 10 % (v/v) detergent (pharma soap medical); 2 % (v/v) p-hydroxybenzoic acid methyl ester (sigma), prepared from a stock solution of 15 % p-hydroxybenzoic acid methyl ester (w/v) in 95 % ethanol; 0.1 % (v/v) sodium hypochlorite. silkworms were cut between the second and the third pair of thoracic legs and behind the third pair of abdominal appendages to exclude the foregut and the hindgut, and the peritrophic membrane along with the enclosed intestinal contents was removed. the central part of the larva was transferred to a petri dish containing a sterile physiological solution composed of 47 mm kcl, 20.5 mm mgcl2, 20 mm mgso4, 5.3 mm k2hpo4, 5.6 mm kh2po4, 1 mm cacl2, 75 mm sucrose at ph 7, 0.2 % (v/v) gentamicin (50 mg/ml, sigma), 0.01 % (v/v) antibiotic-antimycotic solution 1x (sigma). to this solution was added 0.003 ‰ (v/v) sodium hypochlorite. the ventral cuticle was cut longitudinally and the midgut, deprived of muscles and silk glands, was isolated. dissected midguts from 8-10 animals were cut along the longitudinal axis and rinsed twice (10 min for each rinse) in the above mentioned sterile physiological solution added with 0.003 ‰ (v/v) sodium hypochlorite, then again twice (for 10 min each) in the sterile physiological solution. midguts were pooled into a strainer (100 μm mesh size), placed in a petri dish containing few ml of the latter solution and left under mild agitation for 1 h. in these conditions, the loosely attached stem cells migrated away from the tissue. the tissue within the sieve was discarded and the free cells in the filtrate were collected and pelletted by gentle centrifugation at 400xg for 5 min. cells were then resuspended in growth medium, composed by a mixture of 67.4 % grace’s insect medium (gibco), 11.2 % 100 mm koh, 6.7 % fetal bovine serum (gibco), 0.5 % vitamins mix (composed by, in mg/100ml: 300 riboflavin, 150 pyridoxine hydrochloride, 150 thiamine hydrochloride, 150 folic acid, 600 nicotinic acid, 600 pantothenic acid, 12 biotin, 1.2 vitamin bb12), 0.018 ‰ antibiotic-antimycotic solution 1x (sigma), 0.1 % gentamicine (50 mg/ml, sigma). cultured cells were supplemented with 6x10 m 20hydroxyecdysone (sigma) and 100 ng/ml αarylphorin (purified according to blackburn et al., 2004 in insect bio-control laboratory, usda, beltsville, md, usa), kindly donated by prof rs hakim, howard university, washington, dc, usa. all the solutions used were routinely sterilized by filtration (nalgene, 0.2 μm pore size) prior to use. three ml of the cell suspension in growth medium were distributed in the wells (35 mm in diameter) of six well plates. cultures were incubated at 25 °c. one ml of medium from each well was routinely replaced with 1 ml of fresh medium once a week. -8 viability of cells in culture, recognition and count of stem cells, differentiating cells and mature cells along six weeks cell viability was checked with the trypan blue test in the initial stem cell culture and every seven days: viable cells excluded the dye, whereas dead cells became blue. an aliquot of the cell culture was removed, the cells were centrifuged for 5 min at 400xg and then resuspended in a known amount of the physiological solution (see above). an aliquot of the suspension was mixed with 0.4 % (w/v) trypan blue (sigma) (2:1). after 2 min, viable and dead cells were counted under the inverted microscope using a haemocytometer slide (burker). viable cells 120 were then classified in four different categories (stem, differentiating, columnar and goblet cells) on the basis of their morphological features and counted every seven days for six weeks. differences between the four categories along this experimental period were tested by student’s t test. immunodetection of microtubules in cultured columnar cells three weeks old cultured cells were pelletted at 400xg for 5 min and resuspended in the suitable volume of physiological solution (see above). cells were then fixed and permeabilized for 5 min with ice-cold methanol. after 3 rinses in pbs, cells were incubated for 15 min in pbs containing 1 % bovine serum albumin (bsa) and for 1 h with the anti-αtubulin mouse igg (sigma), diluted 1:500 in pbs with 1 % bsa. the cells were then washed 3 times in pbs plus 1 % bsa and incubated for 1 h with alexa fluor 488-conjugated anti-mouse igg antibody raised in donkey (molecular probes) diluted 1:1000 in pbs plus 1 % bsa. after 3 rinses in pbs, the samples were mounted in dabco (sigma)-mowiol (calbiochem), covered with a cover-slip and examined with a clsm tcs sp2 aobs (leica microsystems heidelberg gmbh, germany) equipped with an argon ion laser (458, 476, 488, 496 or 514 nm excitation), two hene lasers (543, 594 and 633 nm excitation) and tunable emission wavelength collection. a 63x leica oil immersion plan apo (na1,4) objective and a 2x zoom were used for all the experiments. detection of actin filaments in cultured columnar cells three weeks old cultured cells were pelletted at 400xg for 5 min and resuspended in the appropriate volume of physiological solution (see above). cells were then incubated for 10 min in 4 % paraformaldehyde in pbs, washed 3 times in pbs and permeabilized for 4 min with 0.1 % triton-x100 in pbs. the cells were washed 3 times in pbs and then incubated for 20 min with 4.3 μg/ml tritcphalloidin. after 3 rinses in pbs, the samples where mounted in dabco (sigma)-mowiol (calbiochem), covered with a cover-slip and examined with a confocal microscope (see above). enzymes assay three weeks old midgut cells in culture were pelletted by gentle centrifugation at 400xg for 5 min and resuspended in a small amount of physiological solution (see above). after three washes, the pellet was resuspended in a buffer solution (100 mm mannitol, 10 mm hepes-tris at ph 7.2) and lysated in the eppendorf vial with a motor for pellet pestle (sigma). protein concentration in the lysate was determined according to bradford (1976) with bsa as standard. all enzymatic assays were conducted under conditions in which products formation depended linearly on enzyme concentration. aminopeptidase n and alkaline phosphatase activities were determined at 25 °c by measuring the release of p-nitroaniline from l-leucine-pnitroanilide in 40 mm tris-hcl at ph 7.5 or of pnitrophenol from p-nitrophenylphosphate in 1 m trishcl at ph 8, respectively. enzymes activities were determined in triplicate or quadruplicate in a pharmacia biotech ultrospec 3000 spectrophotometer. proteins internalization in cultured columnar cells three weeks old cultured cells were pelletted by gentle centrifugation at 400xg for 5 min and resuspended in a small volume of physiological solution (see above). cells were incubated at 25 °c for 2 h in the presence of 1.4 μm fitc-albumin (sigma) or 1.5 μm gfp (vector etc). at the end of the incubation, the cells were washed three times with the physiological solution, fixed for 10 min with 4 % paraformaldehyde in pbs and then rinsed three times in pbs. cells were mounted in dabco (sigma)-mowiol (calbiochem) and examined with a confocal microscope (see above). results stem cells differentiation in culture and morphology of midgut cells in vivo and in vitro to isolate the largest possible number of stem cells from b. mori midgut, we performed an histological analysis of the tissue in three different instances of the larval development. in fig. 1 is shown the midgut epithelium dissected from larvae immediately before the iv moult (a), during the iv moult (b) or in the middle of the v instar during the feeding period (c). in the period just before the last larval-larval moult, the stem cells are located in numerous nidi at the base of the epithelium (fig. 1a). during the moult (fig. 1b), the stem cells proliferate and then differentiate, each of the newly developed cell inserting between the mature cells of the iv instar epithelium. in the v instar, during the feeding period (fig. 1c), very few single stem cells are visible, far less numerous than in the pre-moult period. therefore, the largest number of stem cells can be isolated from b. mori larval midguts in the period just preceding the iv moult. to monitor cell development in culture with time, we followed the growth, differentiation and percentage distribution of each cell type for 42 days (figs 2, 3), by identifying the different cell types every 7 days on the basis of their morphological features. stem cells were round, with a diameter of 4-8 μm (fig. 2a) and some of them could be observed in mitosis (fig. 2b). cells in an early stage of differentiation were round, with long tenuous membrane projections and numerous granules in the cytoplasm (fig. 2c): as suggested by sadruddin et al. (1996), these spherical cells with uniformly distributed microvilli appears to correspond to the initial phase of differentiation of columnar cells, that in a more advanced phase were triangular in shape (fig. 2d), differing from adult ones for their small dimension (10-25 μm). young columnar (fig. 2e) and goblet (fig. 2g) cells had the same shape of the respective mature cells (figs 2f, h) but their dimensions were smaller (between 25-30 μm). mature columnar cells were characterized by a well developed apical membrane with numerous microvilli, a centrally placed nucleus and a cylindrical or cubical shape (fig. 2f), while mature goblet cells were flask-like and presented the typical wide cavity, the apical valve and a basally located 121 fig. 1 semithin sections of the midgut epithelium of bombyx mori larvae dissected immediately before the iv moult (a), during the moult (b) and in the v instar feeding period (c). stem cells nidi (a), proliferating cells (b) and a single stem cell (c) are indicated by arrows. bars = 10 μm. nucleus (fig. 2h). both these cells showed most of the apparent morphological features seen in vivo but they were never as tall as those of the original epithelium (60 to 80 μm, fig. 1). as shown in fig. 3, in the filtrate collected after a mild agitation of the pre-moult midgut tissue, the stem cells represented the 87.4 ± 3.6 % (3 determinations) of the total viable cells present in the medium. after six days in culture, the reduction of stem cells was accompanied by an increase of differentiating, columnar and goblet cells. at the end of the following 7 days, the percentage of stem cells were further decreased, while differentiating cells were the most represented cell type. from the third week on, less than 10 % were stem cells and the remaining cells were represented by percentage values not statistically different (ranging between 27.6 ± 3.4 % (3 determinations) and 41.4 ± 5.3 % (3 determinations)) of columnar, goblet and differentiating cells. all along the experimental period here considered, viable cells were 79.2 ± 5.1 % (21 determinations) of the total cells present in the culture. the columnar cells used for the experiments reported in this paper came from three weeks-old cultures, although the cells maintained the same functional properties till day 42 (data not shown). organisation of the cytoskeleton in cultured columnar cells we examined the distribution of microtubules and actin filaments in columnar cells. as shown in figure 4 b, a large number of microtubules ran parallel to the apical cell surface, forming a dense network just below the apical microvilli. deeper down the cell, numerous bundles of microtubules are oriented longitudinally along the basal-apical axis of the cell. phalloidin stained conspicuously actin filaments within the brush border microvilli of columnar cells both in the initial phase of differentiation (fig. 5a) and in the mature phase (fig. 5b). in the latter figure a number of filaments running from the basolateral membrane deep into the cytoplasm were also detectable. enzymes activity columnar cells in vivo are responsible for the production of the different classes of enzymes involved in the digestion of ingested nutrients (terra and ferreira, 2005). we investigated if the activity of two enzymes currently used as marker enzymes of the apical membrane of midgut columnar cells, i.e. leucine-aminopeptidase n (apn) and alkaline phosphatase (alp), could be detected in the lysate of three weeks old cell cultures. although after 20 days in culture columnar cells represented only the 28.9 ± 2.7 % (3 determinations) of all the cells in culture (fig. 3), an activity of both enzymes could be measured: apn and alp specific activities (mu/mg of protein) were 890 ± 88 (5 determinations) and 131 ± 12 (4 determinations) respectively. proteins absorption in cultured columnar cells we have shown that fitc-albumin is transported across the lepidopteran larval midgut in vitro by transcytosis (casartelli et al., 2005), entering the cell through the apical membrane and being released in the haemocoel across the basolateral membrane. at variance, the green fluorescent protein gfp fed in vivo to the hemipteran lygus hesperus reached without degradation the haemocoel following apparently a paracellular pathway across the junctional complex (habibi et al., 2002). we investigated if mature columnar cells in culture were able to internalize these two proteins, by incubating the cells for 2 h in the presence of 1.4 μm fitc-albumin or 1.5 μm gfp. the fluorescent proteins taken up by columnar cells after 2 h of incubation were detected by confocal laser microscopy: while columnar cells internalized abundantly fitc-albumin (fig. 6a), gfp could not be detected inside the cells (fig. 6b). intracellular fitc-albumin was never diffused uniformly into the cytoplasm but was always localized in vesicular structures clearly visible as spots. 122 fig. 2 morphology of cultured midgut cells. brightfield images (acquired with confocal laser scanning microscope) of fixed cells: stem cell (a); stem cell in mitosis (b); differentiating cells (c, d); columnar (e) and goblet (g) cells in an early phase of differentiation; and fully differentiated (f, h). bars = 5 μm (a, b); 10 μm (c-h). discussion detailed information on the mechanisms involved in peptide and protein absorption by the insect gut can have a considerable impact on the development of new delivery strategies for orally administered insecticidal proteins targeting haemocoelic receptors. proteins are absorbed by the insect midgut in vivo (fishman et al., 1984; modespacher et al., 1986; zlotkin et al., 1992; benyakir and shochat, 1996; powell et al., 1998; habibi et al., 2002; kurahashi et al., 2005), and recent studies performed in vitro in b. mori larval midgut showed that two selected proteins crossed unaltered the intestinal barrier by transcytosis (casartelli et al., 2005, 2007). we considered that midgut columnar cells in primary culture could be a powerful tool to identify the different steps involved in this composite transcellular pathway. sadrud-din et al. (1994, 1996) obtained primary cultures of midgut cells from stem cells isolated from m. sexta larval epithelium, identifying the conditions in vitro in which the stem cells could survive, multiply and differentiate. stem cells from the insect midgut can be easily removed from the tissue because they are not linked to the other cells by junctions and their proliferation in vitro can be induced by ecdysone or 20-hydroxyecdysone (smagghe et al., 2005) and by fat body extracts or its derivates (hakim et al., 2007), while differentiation is stimulated by various factors produced by the mature and differentiating cells in the culture (loeb et al., 2004). following the same method, we isolated the stem cells from the midguts of pre-moult iv instar b. mori larvae and obtained their proliferation and differentiation in culture. we analysed the evolution of the culture by counting the total number of cells, testing their viability and determining the different cell types among the living cells every seven days for 6 weeks (fig. 3). the initial culture was almost exclusively composed of stem cells (87.4 ± 3.6 %, 3 determinations) but six days later all the cell types shown in figure 2 were already present. the progressive drop in stem cells observed in the following 21 days was compensated by the parallel increase in differentiating cells and in young and mature columnar and goblet cells. from the third week on, these three cell types represented about 30 % each of total living cells. the morphology of the different kind of cells shown in figure 2 is in large agreement with that reported by sadrud-din et al. (1996). it is worth noting that, following a drastic decrease after 27 days, stem cells fig. 3 percentage of the various viable cell types in culture in the different days since stem cells isolation. each bar represents the mean ± se of three different determinations. 123 fig. 4 brightfield (a) and confocal laser scanning micrographs (maximum projection) (b) of a typical cultured columnar cell in which is visible microtubules organisation. immunolocalisation of microtubules was performed with an anti-α-tubulin primary antibody visualised with an alexa fluor 488conjugated secondary antibody. bars =10 μm. number increases progressively in the subsequent two weeks, suggesting an enhanced production by the cell culture of specific proliferation factors, once reached a steady state. although our present report of the cell culture properties is referred only to six weeks (fig. 3), the culture maintains almost unaltered the characteristics shown here for up to three months (data not shown). the integrity of the cytoskeleton is fundamental for the maintenance of cell shape and polarity and for the correct localization of membrane proteins in the apical and basal domains of the cell surface. efficient targeting of vesicles loaded with the specific proteins, from the golgi apparatus to the correct membrane domain, requires an intact microtubule organization (reviewed by yeaman et al., 1999). it has been shown in enterocytes and other polarized epithelia that disruption of microtubule architecture by colchicine or nocodazole leads to a preferential alteration of the delivery to the apical rather than to the basolatelal membrane. in rat intestinal epithelium, the microtubules organizing center(s), identified with anti γ-tubulin antibodies, is/are located as a band close to the sub-apical space near the terminal web, from which the fast growing positive ends of microtubules grow towards the basolateral membrane, forming bundles of apical-basal filaments along the cell axis, while the negative ends are apically located (waschke and drenckhahn, 2000). in the subapical-space microtubules still run in parallel and only few are oriented obliquely or perpendicularly, but they never cross the terminal web (waschke and drenckhahn, 2000). microtubules distribution from the basal to the apical pole in b. mori midgut cells largely follows this scheme but, at variance with mammalian cells, in the insect they form a well developed felt just under the brush border (fig. 4b). this particular structure could be related to the lack in insect columnar intestinal cells of a terminal web organized as that of mammals (hull and staehelin, 1979; bonfanti et al, 1992; gibson and perrimon, 2003). as a matter of fact, in caco-2 cells monolayer, an intestinal model epithelium, in which a complete terminal web presumably does never fully differentiate, microtubules in the apical cytoplasm have a network-like arrangement (waschke and drenckhahn, 2000). the disposition of actin cytoskeleton in the mature cell shown in fig. 5b follows that classically described for polarized columnar cells (yeaman et al., 1999): it is highly organized within the apical microvilli and some filaments running from the basolateral membrane deep into the cytoplasm were also visible. well structured actin filaments were also observable in columnar cells in an earlier phase of differentiation (fig. 5a). as reported in the results, some of the differentiating cells were round shaped with microvilli uniformly distributed all over the surface (fig. 2c). sadrud-din et al. (1996) suggested that these cells corresponded to the initial phase of differentiation of columnar cells. the cell reported in fig. 5a, a representative of columnar cells in a more advanced stage, suggests that the differentiation of the basolateral membrane, at least from a morphological point of view, is a delayed process. the midgut epithelium performs in vivo the intermediate and final digestion of nutrients (terra and ferreira, 2005). two enzymes involved in this process highly abundant in the midgut epithelium are aminopeptidase n (apn) and alkaline phosphatase (alp). different isoforms of apn were characterized in the insect midgut (terra and ferreira, 2005), where they play a critical role in the final digestion of polypeptides by catalyzing the hydrolysis of amino acid residues at the n-terminal end. alkaline phosphatases, also present in the midgut epithelium as different isoforms (ferreira and fig. 5 brightfield and confocal laser scanning micrographs (maximum projection) of typical cultured columnar cells, in the initial phase of differentiation (a) and in the mature phase (b), in which is visible microfilaments organisation. actin filaments were visualised with tritc-phalloidin. bars = 10 μm. 124 fig. 6 brightfield and confocal laser scanning micrographs of a single optical section in a middle cell focal plane (where the nucleus was clearly evident) of cultured midgut columnar cells incubated for 2 h in the presence of 1.4 μm fitc-albumin (a) or 1.5 μm gfp (b). in the figure typical columnar cells are reported. bars = 10 μm. terra, 2005), are responsible for the removal of the phosphate groups from various phosphorylated substrates, an important step to allow the subsequent absorption of the molecule. both enzymes are expressed by columnar cells in culture, since their activity could be measured in the cell lysate. finally, we tested the ability to take up proteins from the medium by mature columnar cells. fitcalbumin, a protein actively absorbed by transcytosis by b. mori midgut epithelium in vitro (casartelli et al., 2005), was already detected inside the cell after 20 min (not shown) and was uniformly distributed as numerous spots after 2 h (fig. 6a). conversely, gfp, a protein that was supposed to reach the haemocel in the hemipteran lygus hesperus following the paracellular pathway (habibi et al., 2002), was not found inside columnar cells after 2 h (fig. 6b), intracellular traces of the protein becoming apparent only after 16 h (not shown). it is therefore feasible that the appearance of this protein in the haemocoel in vivo could be due to its passive leakage through the paracellular route rather than by an active process like transcytosis. in conclusion, b. mori columnar cells, differentiated from stem cells in vitro, maintained in culture the typical morphological features of the epithelium in vivo and were able to perform at least some of the normal absorptive and digestive functions. of particular interest for us was the ability of the cells to act selectively as regards the uptake of two model proteins. the very rapid internalization by the cell of fitc-albumin designates this protein as an excellent tool to assess the specific mechanisms involved in endocytosis and to finely characterize the multiple sequence of intracellular events involved in transcytosis. acknowledgements this work was supported by the italian ministry of education university and research (cofin 2004, project no. 2004077251; cofin 2006, project no. 20060794417). we are indebted to prof. rs hakim for his support and advise on the preparation of midgut cell cultures. we also thank dr r marotta e dr c di benedetto for their suggestions in the preparation of the histological samples. references azuma m, harvey wr, wieczorek h. stoichiometry of k+/h+ antiport helps to explain extracellular ph 11 in a model epithelium. febs lett. 361: 153-156, 1995. baldwin km, hakim rs. growth and differentiation of the larval midgut epithelium 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hyperglycemic stress response in crustacea s lorenzon brain center, department of biology, university of trieste, italy accepted september 27, 2005 abstract blood glucose level in crustaceans is controlled by the crustacean hyperglycemic hormone (chh), released from the eyestalk neuroendocrine centres both under physiological and environmental stress conditions. hyperglycemia is a typical response of many aquatic animals to pollutants and stress and, in crustaceans, increased circulating chh and hyperglycemia are reported to result from exposure to several environmental stressors. biogenic amines and enkephalin have been found to mediate the release of several neurohormones from crustacean neuroendocrine tissue and a model of the controlling network is proposed. key words: crustacea; glucose; crustacean hyperglycemic hormone (chh); stress response; neuroendocrine control introduction hyperglycemia is a typical response of many aquatic animals to harmful physical and chemical environmental changes. in crustaceans increased circulating crustacean hyperglycemic hormone (chh) titres and hyperglycemia are reported to occur following exposure to several environmental stressors (durand et al., 2000; lorenzon et al., 1997; 2002; santos et al., 2001) in intact but not in eyestalkless animals (fig.1), suggesting a chh mediated response (fingerman et al., 1981; reddy and bhagyalakshimi, 1994; reddy et al., 1996; lorenzon et al., 2000, 2004a). toxicity induced by a pollutant is the result of interaction of the compound or one of its metabolites, with the biochemical events involved in the homeostatic control of a physiological process (brouwer et al., 1990). physiological processes are mostly coordinated by hormones. anthropogenic chemicals can alter the hormonal (endocrine) systems of wildlife and the corresponding author: simonetta lorenzon brain center, department of biology, university of trieste, via giorgieri 7, i-34127 trieste, italy e-mail: lorenzon@units.it effects of organic and inorganic contaminants on functions regulated by hormones in crustaceans are being investigated with increased frequency because several of these phenomena could be used as biomarkers of environmental contamination. heavy metals and organic compounds have been found to negatively affect hormonally-regulated functions, such as reproduction, molting, blood glucose level and pigmentary effectors in crustaceans (fingerman et al., 1998; depledge and billinghurst, 1999). therefore, biosentinel parameters and “early warning” of toxicity can be identified by looking for alterations in endocrine patterns (fingerman et al., 1996). neurosecretory structures in the eyestalk are the most important components of the neuroendocrine system of the stalk-eyed crustaceans. the hemolymph glucose concentration is mainly controlled by the chh synthesized within the x-organ (xo) and released from the sinus gland (sg) complex in the eyestalk (abramowitz et al., 1944; fingerman, 1987). biogenic amines and enkephalin (l/m-enk) control the release of neurohormones from the crustacean neuroendocrine tissue. serotonin (5-ht) is involved in regulating important aspects of behaviour and a variety of systemic physiological functions. 5-ht has long been known (bauchau and mengeot, 1966) to have a potent hyperglycemic effect in several crustacean species (lorenzon et al., 1999, 2004b; lee et al., 132 2000; komali et al., 2005;), while dopamine (da) and enkephalin showed conflicting results in different species (sarojini et al., 1995; lorenzon et al., 1999, 2004b; zou et al., 2003; komali et al., 2005). the crustacean hyperglycemic hormone (chh) multiple forms of the chh represent one member of an eyestalk neuropeptide family (bocking et al, 2001), that includes the moult inhibiting hormone (mih) and the gonad inhibiting hormone (gih): the chh/mih/gih family. these neuropeptides, synthesized in the xo, a cluster of neuron perikarya located in the medulla terminalis of the eyestalk, are transported to and stored in the axon terminals forming a neurohemal organ named sg and released by exocytosis into the hemolymph (fig. 2). the main function of chh is the regulation of hemolymph sugar level: chhs are also involved in other functions such as reproduction (de kleijn et al., 1998; de kleijn and van herp, 1998), molting (chung et al., 1999; webster et al., 2000), lipid metabolism (santos et al., 1997), stress response (lorenzon et al., 1997; 2002;chang et al., 1999; durand et al., 2000; santos et al., 2001) and hydromineral regulation (spanings-pierrot et al., 2000; serrano et al., 2003). on the basis of the primary structure, the chh/mih/gih family can be divided into two subfamilies (de kleijn et al., 1995; lacombe et al., 1999): the chh sub-family characterized by the chh precursor-related peptide (cprp) and the mih/gih sub-family without cprp. the prepropeptide chh consists of a signal peptide, cprp and a peptide with 72–74 amino acids. usually, the mature peptide has an amidated carboxyl terminus (de kleijn and van herp, 1998; lacombe et al., 1999), which is important in conferring hyperglycemic activity in penaeus japonicus as evidenced by bioassay of recombinant peptide (katayama et al., 2003). in several crustacean species, different isoforms of chh exist. in the american lobster homarus americanus, chh-a (8.583 da) and chh-b (8.638 da) have been found, with different actions during the female biannual reproductive cycle (de kleijn et al., 1995). role of biogenic amines and enkephalin in blood glucose regulation neurotransmitters such as 5-ht, da and l/m-enk play a fundamental role in hormone modulation (fingerman et al., 1994) and at the same time their level and functions can be altered by pollutants (amiard-triquet et al., 1986, reddy et al., 1997). 5-ht is well known as a neurotransmitter in crustaceans on several grounds, and its levels have been measured in the nervous system and hemolymph of various crustacean species (elofsson et al., 1982; laxmyr, 1984; kulkarni and fingerman 1992), thus suggesting a possible role as a neurohormone (rodriguez-soza et al., 1997). in crustaceans 5ht is linked with discrete circuits that control movements of the foregut, escape behaviour, locomotion and posture as well as with higher-order behaviours such as aggression (sosa et al., 2004). in addition 5-ht levels are sensitive to environmental stress. 5-ht has long been known to have a potent hyperglycemic effect in several crustacean species (bauchau and mengeot, 1966; keller and beyer 1968; lüschen et al., 1993; kuo et al., 1995; santos et al., 2001). in our laboratory (lorenzon et al., 1999, 2004b) we have demonstrated that 5-ht elevates blood glucose in palaemon elegans, astacus leptodactylus and squilla mantis. however no such effects were found in eyestalkless individuals of these species, suggesting the involvement of the eyestalk hormone chh in the hyperglycemic response. in all the species injection of the antagonist, ketanserin and cph (cyproheptadine, 5-ht1 receptor inhibitor) were able to inhibit the hyperglycemic effect of 5-ht. 5-ht1 like receptors seemed to be more likely involved in mediating 5-ht action, as cph was a more effective antagonist than ketanserin (5-ht2 receptor inhibitor and also putative da antagonist). these data agree with those by lee et al. (2000) in procambarus clarkii suggesting that 5-ht induced hyperglycemia is mediated by 5-ht1 and 5-ht2 like receptors. using elisa very recently we have demonstrated in p. elegans that injection of 5-ht induced a rapid and massive release of chh from the eyestalk into the hemolymph followed by hyperglycemia. on the contrary da did not significantly affect chh release and hyperglycemia (lorenzon et al. 2005). da and enkephalins showed conflicting results in different species (table 1). injection of da induced marked decrease in blood glucose levels in p. elegans and s. mantis (lorenzon et al., 1999, 2004b). on the other hand injection of the da receptor blocker inhibits the effects on blood glucose, apparently allowing the release of chh. these findings are in contrast with those by lüschen et al., (1993) for carcinus maenas, kuo et al. (1995) for penaeus monodon and komali et al. (2005) for macrobrachium malcolmsonii where da induced hyperglycemia in intact animals. as for enkephalins, l/m-enk elicited hypoglycemic response in intact s. mantis but not in eyestalkless individuals (lorenzon et al. 2004a). these results confirm those of jaros (1990), lüschen et al. (1991), rothe et al. (1991) and sarojini et al. (1995) who reported that l/m-enk induced hypoglycemia in uca pugilator, c. maenas and p. clarkii respectively. on the other hand l-enk induced hyperglycemic response in intact but not in eyestalkless a. leptodactylus (lorenzon et al. 2004). these observations are consistent with our previous findings in p. elegans (lorenzon et al., 1999) and also with recent reports on oziotelphusa senex senex (reddy and basha, 2001), on the mud crab scylla serrata (reddy and kishori, 2001) and in the two prawns, penaeus indicus and metapenaeus monocerus (kishori et al., 2001). in s. mantis injection of the opioid antagonist naloxone reversed the inhibitory effect on blood glucose of l-enk while in a. leptodactylus an additive effect on hyperglycemia was recorded (lorenzon et al., 2004b). all these results corroborate the commonly held view that 5-ht, is a potent hyperglycemic effector and exerts its effect through chh release from the 133 fig.1 stress response in crustacea. medulla terminalis xo-sinus gland complex (mtxosg), mediated by modulation of electrical activity of xo cells (saenz et al., 1997). a detailed reconstruction of the underlying neural circuitry suffers from lack of precise identification of neurosecretory cell types, contrasting results of electrophysiological evidence and discrepancies due to interspecific differences (glowik et al., 1997; saenz et al., 1997). finally 5-ht appears to provide a major control mechanism for glucose mobilization whereas da and l/m-enk act as modulators whose plasticity in use or actions varied among even closely related species. stress response stress induced by changes in environmental parameters, emersion, handling and transport during commercial processes requires homeostatic regulation that brings about behavioural and physiological alterations in aquatic animals. hemolymph glucose concentration can change significantly with altered physiological and environmental conditions. exposure to air during commercial transport and hypoxia are reported to induce hyperglycemia in many crustacean species like the spiny lobster, jasus edwardsii (morris and oliver 1999; speed et al., 2001), the crab, eriocheir sinensis (zou et al., 1996), the spider crab, maia squinado (durand et al., 2000) and the norway lobster, nephrops norvegicus (spicer et al., 1990). moreover hyperglycemia is reported in the giant prawn, macrobrachium rosenbergii as a response to cold shock (kuo and yang, 1999). blood glucose level increased in p. elegans and other crustacean species after injection of lipopolysaccharide (lps) and the hyperglycemic effect, is likely mediated by the chh since it does not occur in eyestalkless animals. it is dose-related and dependent on the different gram negative bacterial lps (lorenzon et al., 1997, 2002). heavy metals like cd, hg, and cu cause hyperglycemia in the freshwater prawn, macrobrachium kistenensis, the crab, barytelphusa canicularis (nagabhushanam and kulkarni, 1981, machele et al., 1989) and s. serrata (reddy and bhagyalakshmi, 1994). moreover, cdcl2 induces hyperglycemia in intact crayfish p. clarkii, but not in the absence of the eyestalks, suggesting a chh mediated response (reddy et al., 1996). our studies (lorenzon et al., 2000) on the effect of heavy metals on blood glucose levels in p. elegans showed that the intermediate sublethal concentrations of hg, cd and pb produced significant hyperglycemic responses while the highest concentrations elicited no hyperglycemia in 24 h. in contrast, animals exposed to cu and zn showed hyperglycemia even at high concentrations. this difference in response could be explained on the basis of the physiological roles these two essential 134 fig. 2 general organization of neuroendocrine tissues in the eyestalk of crustaceans. elements play in crustaceans, and consequent tolerance adaptations, as opposed to the toxic, xenobiotic heavy metals cd, hg and pb. on the other hand both groups of heavy metals failed to elicit a hyperglycemic responses in eyestalk ablated animals suggesting the involvement of mtxo-sg hormones, most likely chh. however, in spite of the richness of information regarding variations in blood glucose levels following stress, much less is known about the stress-induced variation in chh levels in the sinus gland and in the hemolymph. in the crayfish orconectes limosus subjected to hypoxia, blood chh titers raise within 15 min (keller and orth, 1990). in cancer pagurus emersion induced an increase in the hemolymph chh after 4 h (webster, 1996). using elisa chang et al. (1998) observed variation in the blood chh in homarus americanus following exposure to various environmental stresses. emersion was found to be a potent stimulator of blood chh while temperature and salinity variations were less effective. moreover an increase in water temperature increased blood chh in c. pagurus and p. clarckii (wilcockson et al. 2002; zou et al., 2003). in c. maenas it has been shown that the concentration of the chh in the hemolymph increases dramatically during molting from 1-5 fmol 100µl-1 in the intermolt up to 150-200 fmol 100µl-1 during ecdysis (chung et al., 1999). variation in the hemolymph chh titer were also observed in n. norvegicus infected by the parasitic dinoflagellate hematodinium sp. (stentiford et al., 2001). using elisa and bioassay tests we have recently demonstrated the relationship between an environmental stressor and the release of chh from the eyestalk into the hemolymph and the hyperglycemic response in the shrimp, p. elegans (lorenzon et al., 2004a). moreover with this work we validated the use of a cross reactive antibody, antinenchh, to assess chh level in the eyestalk and hemolymph of p. elegans. with the help of standard immunocytochemistry the antibody had previously been tested for recognition of chh in the eyestalks of different species belonging to systematic groups increasingly remote in the phylogenetic tree: the decapods a. leptodactylus, n. norvegicus, p. elegans, munida rugosa and the stomatopod s. mantis (giulianini et al., 2002). finally we have quantified the variations in the hemolymph chh after a challenge with different stressors. in p. elegans exposure to copper induced a dose-related rapid and massive release of chh from the eyestalk into the hemolymph at the higher, lethal concentration while a gradual and reduced discharge was observed at the lower concentration (fig. 3). the relationship between exposure to a toxicant and release of the chh was confirmed by variation in blood glucose with a dose related hyperglycemia that peaked 2 h after exposure to copper (fig. 4). animals exposed to sublethal concentrations of hg showed similar quantitative and time course relations between toxicant, release of chh from the eyestalk, increment of hormone level in the hemolymph and subsequent hyperglycemia as already described for copper contamination. interestingly, however, the highest, lethal concentration induced the release of chh from the eyestalk into the hemolymph but was not followed by a significant variation in blood glucose (figs 5, 6). this situation could be related to the high toxicity of hg which may interfere with the finer mechanisms that regulate hyperglycemic response. it is neither due to synaptic blockage of the superimposed neuronal release network (lorenzon et al., 1999) nor limited release of circulating chh as high levels of chh are discharged from the sg into the hemolymph. it is not due to inhibition of peripheral receptors on glycogenolytic target organs: indeed native sg homogenate injected into eyestalkless shrimps exposed to lethal concentration of hg for 3 h is still able to cause hyperglycemia (lorenzon et al., 2000). high concentrations of hg, instead, may change the functionality of the prepro-chh processed during secretory steps and due to its ability to bind cysteines six of which represent a highly conserved feature of the peptide structure (lacombe et al., 1999) hg might alter the active configuration of the peptide, as seen in other systems (rodgers et al., 2001), but not its immunoreactivity. moreover hg is known to impair osmoregulatory mechanisms in the crab, eriocheir sinensis (péqueux et al., 1996); and inhibit acetylcholinesterase activity in p. clarkii (devi and fingerman, 1995). the altered response in p. elegans exposed to high concentrations of hg may also be related to physiological modifications induced by hg at a different systemic level (lorenzon et al., 2004a). cu contamination induced variations of 5-ht of the eyestalk and hemolymph of p. elegans (lorenzon et al., 2005). the release of 5-ht from the eyestalk appears to be very rapid and dose dependent. in the hemolymph 5-ht peak occurs after 30 min and again the concentration of circulating 5ht is dose dependent. after 1 h the level of 5-ht slowly decreases to the basal level (fig. 7). 135 0,5 1 2 3 control 0 1 2 3 4 5 6 7 8 9 10 a ch h ( p m o l s g e -1 ) time (h) cu ++ 0.1 mg l -1 cu ++ 5 mg l -1 0,5 1 2 3 control 0 1 2 3 4 5 6 7 8 9 10 11 12 b ch h ( p m o l m l -1 ) time (h) cu ++ 0.1 mgl -1 cu ++ 5 mgl -1 fig. 3 time course of chh in the eyestalk homogenates (a) and in the hemolymph (b) of p. elegans after exposure to different concentrations of cu++ and in relation to untreated controls. values are expressed as means ± sd (n=4 repeated measures). 0,5 1 2 3 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 2,2 2,4 2,6 2,8 3,0 3,2 3,4 in cr e m e n t o f g ly ce m ia time (h) cu ++ 0.1 mg l -1 cu ++ 5 mg l -1 control fig. 4 time course of glycemia in the hemolymph of p. elegans after exposure to different concentrations of cu++ and in relation to untreated controls. values of increment given as: [(experimental value)/ (value displayed by the same animal at 0 h)]–1, are expressed means ± sd (n=10 repeated measures). the release of 5-ht from the eyestalk into the hemolymph after cu exposure precedes in its time course the release of chh, confirming its role as neurotransmitter acting on chh neuroendocrine cells. the rapid and massive release of 5-ht from the eyestalk of individual species following exposure to cu might have induced release of the chh resulting in hyperglycemia in intact but not in eyestalkless animals. lastly contamination with different doses of lps, a bacterial thermostable endotoxin from e. coli, confirms the dose-related and convergent chain of events that leads to hyperglycemia. this suggests that blood glucose elevation is a general-purpose response to stressors and is likely to perform a protective role (lorenzon et al., 2004a). conclusion in spite of the vastness of information on hyperglcemic stress response in crustacea, there still exist many questions. in the scheme presented in figure 8 a possible model of the controlling network is proposed. stressors have been demonstrated to release the chh and 5-ht from the eyestalk leading to an increase in their hemolymph concentrations. 5-ht exerts a positive influence inducing the release of chh from the sg into the blood. the chh then acts upon the target organs to release more than normal level glucose resulting in hyperglycemia. the da 136 0,5 1 2 3 control 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 a ch h ( pm ol s g -1 ) time (h) hg ++ 0.1 mg l -1 hg ++ 0.5 mg l -1 hg ++ 5 mg l -1 control 0,5 1 2 3 control 0 1 2 3 4 5 6 7 8 9 10 11 12 btime (h) ch h ( p m o l m l -1 ) hg ++ 0.1 mg l -1 hg ++ 0.5 mg l -1 hg ++ 5 mg l -1 control fig. 5 time course of chh in the eyestalk homogenates (a) and in the hemolymph (b) of p. elegans after exposure to three different concentrations of hg++ and in relation to untreated control. values are expressed means ± sd (n=4 repeated measures). 0,5 1 2 3 5 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 in cr e m e n t o f g ly ce m ia time (h) hg ++ 0.1 mg l -1 hg ++ 0.5 mg l -1 hg ++ 5 mg l -1 control fig. 6 time course of glycemia in the hemolymph of p. elegans after exposure to three different concentrations of hg++ and in relation to untreated controls. values of increment given as: [(experimental value)/ (value displayed by the same animal at 0 h)]–1, are expressed means ± sd (n=10 repeated measures). receptor blocker, spiperone, inhibited the hypoglycemic action of da and was found not to affect the ability of l/m-enk to produce hypoglycemia. on the other hand, naloxone blocked the action of both l/m-enk and da, thereby allowing the release of chh (sarojini et al., 1995, lorenzon et al., 1999). apparently da and l-enk produced hypoglycemia by inhibiting chh release. these results suggest that in the chain of neurons terminating at the neuroendocrine cells that secrete chh, dopaminergic neurons precede enkephalinergic neurons. we also suggest a role for the hemocytes in the hyperglycemic stress response as stressors affect both the total (thc) and the differential haemocyte count and that exocytosis of chh granules from the eyestalk neuroendocrine cells can be elicited either by an early release from hemocytes of cytokines and/or other circulating messengers like 5-ht. moreover lps treated eyestalkless animals undergo less haemocytopenia than intact individuals. this suggests that previous chh release and hyperglycemia can cause a decrease in thc, which eventually exerts a protective function (lorenzon et al., 2002). in summary it may be said that indicators of stress responses are useful in assessing the shortterm well-being or long-term health status of an animal (fossi et al., 1997; paterson and spanoghe, 1997) and, such indicators have received considerable attention in commercially important species of decapod crustaceans (paterson 137 0 0,5 1 2 3 control 0 5 10 15 20 25 30 35 40 a hemolymph cu ++ 5 mg l -1 cu ++ 0.1 mg l -1 time(h) 5 -h t ( n g m l -1 ) 0 0,5 1 2 3 control 0 1 2 3 4 5 6 7 8 9 10 11 12 b eyestalk 5 -h t ( n g m l -1 ) time (h) cu ++ 5 mg l -1 cu ++ 0.1 mg l -1 fig. 7 time course (0.5-3 h) of 5-ht in the hemolymph (a) and in the eyestalk (b) of p. elegans after exposure to different concentrations of cu++ and in relation to untreated controls. values are expressed means ± sd (n=4 repeated measures). fig. 8 possible model of hyperglycemic stress response controlling network. in the scheme: continuous arrow=demonstrated effect, dotted arrow= hypothesized effect, red arrow= stimulation, blue arrow= inhibition, green arrow=release. 138 and spanoghe, 1997; chang et al., 1999). a number of researchers have suggested different methods for quantifying the stress responses in crustaceans; which include the measurement of different hemocyte types in the hemolymph (jussila et al., 1997 lorenzon et al., 1999, 2001), and the physiological, biochemical (paterson and spanoghe, 1997; stentiford et al.. 1999), and molecular changes in the tissues and the hemolymph (fossi et al., 1997). thus variations in the hemolymph glucose concentration in the hemolymph and of the chh level in relation to stressors could be used as a tool to monitor a variety of stress responses. acknowledgements the author is grateful to prof. ea ferrero and dr. pg giulianini for useful discussion and comments on the manuscript. the constructive comments of anonymous referees are kindly acknowledged. this work was supported by 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105-109, 1996. 141 visions and perspectives isj 9: 89-92, 2012 issn 1824-307x visions and perspectives contribution of invertebrate models to aging and longevity studies e ottaviani1, c franceschi 2, 3 1 department of biology, university of modena and reggio emilia, modena, italy 2 department of experimental pathology, university of bologna, bologna, italy 3 cig-interdepartmental center "l galvani", university of bologna, bologna, italy accepted may 28, 2012 abstract this paper summarizes pros and cons of the invertebrate models involved in aging and longevity. the worm caenorhabditis elegans and the fruit fly drosophila melanogaster are the two models that have given the major contributions on this topic. furthermore, we also discuss the possible contribution of recent theories on aging and inflammation to understand the complex phenotype of aged soma, from invertebrates to humans. key words: aging; longevity; invertebrates   introduction according to kirkwood (1985) animal species keep a balance between energy investments in maintenance, growth and repair on one hand and reproductive activity on the other hand, and this balance is related to aging. despite the great variability present among the animal kingdom, invertebrates and vertebrates use a common pool of highly conserved molecules combinatorially assembled under the constrain of selection for fitness (ottaviani et al., 1997, 2007; franceschi et al., 2000). we surmized that same or similar molecules found in invertebrates are present in vertebrates and in these higher forms of life their function remains basically similar. however, nature has apparently made new uses of these old molecules, while at the same time evolving towards more complex and centralized functions and organs (ottaviani et al., 1991). the molecular mechanisms able to affect aging and longevity are also involved in the capability of the organisms to cope with a variety of stressors (franceschi et al., 2000). furthermore, a prediction of these arguments is that the processes that extend lifespan and longevity in invertebrates will have a counterpart in vertebrates, including humans. however, one of the major differences between invertebrates and vertebrates is the appearance of the acquired immune response, characterized by high levels of specificity and memory. likely, the ___________________________________________________________________________ corresponding author: enzo ottaviani department of biology university of modena and reggio emilia via campi 231/d, 41125 modena, italy e-mail: enzo.ottaviani@unimore.it immune system is, at a higher level of biological organization and complexity, the counterpart of the anti-stress response network identified in invertebrates as the major determinant of survival. in this context, invertebrates respond to stressors utilizing the same basis set of molecules found in vertebrates, but in the last, the stress response becomes more specialized and specific, involving an evolutionary well maintained network of responses (ottaviani and franceschi, 1996). the study of the mechanisms that underlie aging and longevity was conducted primarily in invertebrates without forgetting yeast, while human studies were considered secondary and only recently they have assumed considerable importance. with regards the human model, we have proposed the inflamm-aging theory, that represents the major characteristic of the aging process (franceschi et al., 2000). indeed, the ability to cope with a variety of stressors and the concomitant progressive increase in the proinflammatory status is considered a major cause related to a continuous, lifelong antigenic load and stress. in this paper the main invertebrate species used as models for the study of aging and longevity are reported. the invertebrate models used in aging and longevity studies the worm caenorhabditis elegans and the fruit fly drosophila melanogaster are the two models that have given the major contributions to the knowledge of the molecular mechanisms underpinning aging and longevity. it should be noted that in 1978, 451     89 references were listed on this topic for d. melanogaster (van heukelem, 1978). c. elegans is important for the study of aging as in this metazoan it was shown for the first time that single-gene mutations are able of extending maximum lifespan. in particular, the mutations of 4 genes age-1, daf-2, spe-26 and clk-1 induce a life extension of more than 40 %. the overexpression of tkr-1 in transgenic worms increases the longevity 40 100 % and confers increased resistance to heat and ultraviolet irradiation (see for review lithgow, 1996; murakami and johnson, 1998). the effects on c. elegans longevity and resistance to infection were also observed in worms fed with lactobacilli and bifidobacteria (ikeda et al., 2007; komura et al., 2010). with regards d. melanogaster, spencer et al. (2003) found a group of gene called “aging genes” (methuselah, indy, inr, chico, superoxide dismutase) that extend the fly lifespan by up to 85 %. further experiments using axenic cultures and antibiotic treatment showed that the presence of bacteria during the first week of adult life of flies enhanced longevity by 30 35 %. conversely, the presence of bacteria in the last stage of life caused a slight decrease (brummel et al., 2004). the addition of escherichia coli to the diet significantly prolonged the fly longevity in the two oregon r d. melanogaster strains, selected for different longevities: a short-life with an average adult lifespan of 10 days and a long-life standard r strain with an average adult lifespan of 50 days. furthermore, it has also been observed modifications on the structure and the histochemical reactivity of the fat body. the increased survival was associated with a great amount of glycogen accumulated in fat body cells from both strains. in aged control animals, fed with standard diet, lipid droplets were seen to be stored in fat body of shortlived, but not long-lived flies (franchini et al., 2012). for what the increased survival is concerned, it is important to mention that recently blagosklonny and hall (2009) suggest a new view, i.e., that "excessive growth is driving for aging", involving basic shared molecular pathways and processes such as the evolutionary conserved tor pathway. tor, the target of the antifungal drug rapamycin, has been described from yeast saccharomyces cerevisiae to higher eukaryotes, and its decreased activity has been found to slow aging in yeast, c. elegans and d. melanogaster (katewa and kapahi, 2011; mccormick et al., 2011). a different approach in the study of aging and longevity is represented by the medfly populations of ceratitis capitata in the wild (carey et al., 2008), where unlike the laboratory animals, it is not possible to control the experimental conditions. in this study, a new method for estimating age structure in insect populations was proposed, and it revealed that the major modifications were found in field populations, demonstrating that middle-aged individuals are common in the wild, and revealing the extraordinary lifespan of wild-caught insects. according to abel et al. (2009), bivalves are another excellent model to study aging since several parameters can be evaluated. for instance, the shell can provide information for determining the individual age and, at the same time, provides information on changes in environmental conditions that could influence the life of animals. it was also noted how different molluscan lifestyles regulate the balance between ros production and antioxidant defense, playing an important role in the determination the maximum lifespan. buick and ivany (2004) suggested that one of the processes in extending bivalve lifespan from high latitudes could be the seasonal limitations of light and food availability. however, other valuable model for the aging study are potentially available among marine invertebrates characterized by the ''negligible senescence", i.e., animals that do not show an increase in mortality rate or a decrease in fertility, physiological function or disease resistance with age (finch and austad, 2001). in this context, a list is reported by bodnar (2009). a new theoretical scenario within an evolutionary perspective, the theoretical field of aging has been dominated by few major theories, such as the mutation accumulation theory (medawar, 1952), the antagonistic pleiotropy theory (williams, 1957) and the disposable soma theory (kirkwood, 1977). in this paper we would like to discuss some of the implications of the more recent and above-mentioned conceptualization of blagosklonny and hall (2009). this quasiprogrammed hypothesis prompted us in extending the inflamm-aging theory to invertebrates. indeed, inflammation is an ancestral and highly conserved process which plays a fundamental physiological role for survival from invertebrates to homo sapiens. in a more general perspective, the age-related inflammatory process, which develops and increases with aging, owing to a lifelong persistent antigenic load as well as to other stimuli (accumulation of senescent cells), might be interpreted as a quasi-programmed extension to later life of the physiological tendency to activate inflammation and tissue repair crucial for survival in young age. accordingly, we not only predict a major role of the gut microbiota in life extension in the invertebrates (ottaviani et al., 2011), but we also suggest that inflamm-aging, and the underpinning molecular mechanisms and pathways, will also play a prominent role in invertebrate models of life extension. this extension to invertebrates of the inflamm-aging theory can be complemented by the recently advanced hypothesis of metaflammation (hummasti and hotamisligil, 2010). this conceptualization suggests that an excess intake of nutrients is the driving force triggering the inflammatory status characteristic of obesity and metabolic diseases, such as metabolic syndrome and diabetes in humans and mice. thus, at least two inflammatory process, inflamm-aging and metaflammation, have been identified, which likely affect aging, age-related diseases and longevity. we surmize that a theoretical merging of inflammaging and metaflammation could be useful to understand, at least in part, a variety of major topics in the aging field, such as the negative effects of excess nutrient and the positive effects of caloric restriction, the positive effects of inflammation for     90 table 1 major advantages and disadvantages of invertebrate model systems in comparison to humans _____________________________________________________________ advantages • the relative simplicity of their systems • the rapid generation time • the short life-span • the small size • standardization of the environmental conditions, including diet, temperature, among others • easy possibility to perform genetic studies disadvantages • inbreeding • artificial environment, including diet, temperature, among others • altered cycle of day and night • less complex immune system • scarce knowledge of the gut microbiota • scarce knowledge on mitochondrial dna • scarce knowledge of the pathology _____________________________________________________________ survival against infectious agents and the negative effects of inflamm-aging, among others. concluding remarks in our perspective the take home message of the this paper can be summarized in the table 1 where we list the major advantages and disadvantages of invertebrate model systems in comparison to humans, within a "balanced" view which appreciate what and how the different models, including humans can contribute for a better understanding of the highly complex phenotype of aged soma. references abele d, brey t, philipp e. bivalve models of aging and the determination of molluscan lifespans. exp. gerontol. 44: 307-315, 2009. blagosklonny mv, hall mn growth and aging: a common molecular mechanism. aging (albany ny) 1: 357-362, 2009. bodnar ag. marine invertebrates as models for aging research. exp. gerontol. 44: 477-484, 2009. brummel t, ching a, seroude l, simon af, benzer s. drosophila 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mccormick ma, tsai sy, kennedy bk. tor and ageing: a complex pathway for a complex process. philos. trans. r. soc. lond. b biol. sci. 366: 17-27, 2011. medawar pb. an unsolved problem of biology, hk lewis & co., london, 1952. murakami s, johnson te. life extension and stress resistance in caenorhabditis elegans modulated by the tkr-1 gene. curr. biol. 8: 1091-1094, 1998. ottaviani e, caselgrandi e, bondi m, cossarizza a, monti d, franceschi c. the "immuno-mobile brain": evolutionary evidence. adv. neuroimmunol. 1: 27-39, 1991. ottaviani e, franceschi c. the neuroimmunology of the stress response from invertebrates to man. prog. neurobiol. 48: 421-440, 1996. ottaviani e, franceschi f. the invertebrate phagocytic immunocyte: clues to a common evolution of immune and neuroendocrine systems. immunol. today 18: 169-174, 1997. ottaviani e, malagoli d, franceschi c. common evolutionary origin of the immune and neuroendocrine systems: from morphological and functional evidences to in silico approaches. trends immunol. 28: 497-502, 2007. ottaviani e, ventura n, mandrioli m, candela m, franchini a, franceschi c. gut microbiota as a candidate for lifespan extension: an ecological/evolutionary perspective targeted on living organisms as metaorganisms. biogerontology 12: 599-609, 2011. spencer cc, howell ce, wright ar, promislow de. testing an 'aging gene' in long-lived drosophila strains: increased longevity depends on sex and genetic background. aging cell 2: 123-130, 2003. van heukelem wf. aging in lower animals, in: behnke ja, finch ce, moment b (eds), the biology of aging, plenum press, new york, pp 115-130, 1978. williams gc. pleiotropy, natural selection, and the evolution of scenescence. evolution 11: 398411, 1957.     92 microsoft word isj-2010-222     251 isj 7: 251-261, 2010 issn 1824-307x research report effects of bacillus thuringiensis var. kurstaki and medicinal plants on hyphantria cunea drury (lepidoptera: arctiidae) i zibaee1, ar bandani1, jj sendi2, r talaei-hassanloei1, b kouchaki2 1department of plant protection, agricultural and natural resources campus, university of tehran, karaj 31584, iran 2department of plant protection, college of agriculture, university of guilan, rasht, 41635-1314, iran accepted november 2, 2010 abstract the fall armyworm, hyphantria cunea drury (lepidoptera: arctiidae) is an insect native to north america that was recently introduced into iran resulting in severe damage to trees and agricultural production. an experiment was conducted to examine potential effects of medicinal plants, artemisia annua and lavandula stoechas and the insect pathogenic bacterium bacillus thuringiensis var. kurstaki on activities of digestive enzymes (α-amylase, αand β-glucosidase, lipase and proteases) and lactate dehydrogenase (ldh) in h. cunea by using two hosts, mulberry and sycamore. results showed that b. thuringiensis var. kurstaki and plant extracts when administered orally, affected the digestive enzyme profiles of h. cunea. combined effect of b. thuringiensis, a. annua and l. stoechas extracts on mulberry decreased the activities of digestive enzymes in a dose-related manner, except for β-glucosidase and lipase. when larvae were treated by different concentrations of the mentioned insecticides, ldh activity increased i.e. the higher activity was obtained by b. thurengiensis alone and b. thurengiensis and l. stoechas extracts together. the least activity was observed in the case of l. stoechas extracts alone on both hosts. physiological analysis would be particularly informative when using combination of biopesticides to enhance the efficiency of a safe management process. key words: hyphantria cunea; bacillus thuringiensis var. kurstaki; artemisia annua extract; lavandula stoechas extract; digestive enzymes; lactate dehydrogenase introduction the fall armyworm, hyphantria cunea drury (lepidoptera: arctiidae) is an insect native in north america that is presently distributed in many areas in the northern hemisphere (warren and tadic, 1970) and new zealand (kean and kumarasinghe, 2007). it has been introduced to different areas of europe and asia (li et al., 2001). since, 2002, h. cunea established itself in northern areas of iran, causing severe damage to trees. it is a multivoltine pest feeding on leaves of trees and hibernates as a pupa in soil around the damaged trees. research has been conducted to look for natural plant protection compounds such as botanical insecticides, antifeedants and microorganisms such as fungi and bacteria. bacillus thuringiensis is a gram-positive, soil dwelling bacterium which is commonly used as a ___________________________________________________________________________ corresponding author: idin zibaee department of plant protection, agricultural and natural resources campus, university of tehran, kardj, 31584, iran e-mail: izibaee@gmx.com pesticide. the genus artemisia is a member of a large plant family asteraceae (compositae) encompassing more than 300 different species of this diverse genus (shekari et al., 2008). the species a. annua, known as sweet worm wood, grows widely in europe and america and is grown in china, turkey, vietnam, afghanistan and australia (bhakuni et al., 2001; shekari et al., 2008). several isolated compounds from this species have shown antimalarial, antibacterial, antiinflamatory, plant growth regulatory and cytotoxicity (antitumor) activities (akhtar and isman, 2004). lavandula stoechas (french lavender), lamiaceae, occurs naturally in the mediterranean region and is a perennial shrub that grows to 30-100 cm tall. it was declared a toxic weed and has potential for use as a botanical insecticide. reduced efficacy of synthetic insecticides has been highlighted in the last two decades. the first alternative was b. thuringiensis berliner (bt), but the increasing number of reports on the resistance to bt led also to choose other biological insecticides such as those coming from plants (senthil nathana et al., 2006). several studies have investigated the     252 fig. 1 mortality probit of the bacillus thurengiensis, artemisia annua and lavandula stoechas in the presence of two hosts on the larvae of hyphantaria cunea. combination of bt with baculoviruses, however other combinations should be explored (senthil nathana et al., 2006). it is clear that botanical insecticides and microbes such as b. thuringiensis affect insect physiology in different ways including decrease of digestive enzyme activities. hence, in this paper research was conducted to examine potential effects of two botanical insecticides and bacterial toxin on activities of digestive enzymes and lactate dehydrogenase (ldh) in the h. cunea in the presence of two hosts, mulberry, an important tree in orchards, and sycamore, an important tree in urban areas, in order to find more suitable ways to decrease its population and damage.     253 materials and methods insects first instar larvae of hyphantria cunea were collected from the field and reared separately on mulberry and sycamore to reach 4th instar larvae in the laboratory at 27 ± 2 °c under a 14 h light:10 h dark photoperiod. these larvae were used to initiate the experiments. preparation of bacillus thurengiensis var. kurstaki and plant extracts a stock suspension of bacillus thuringiensis var kurstaki (109 spore/ml) was provided by giah company (iran) and a serial concentration prepared using distilled water. medicinal plant (artemisia annua and lavandula stoechas) leaves were collected, washed with distilled water and dried at room temperature in the shade. methanolic extraction was carried out according to the procedure described by shekari et al. (2008). briefly, 30 g of dried leaves were stirred with 300 ml of 85 % methanol in a flask, left for 48 h at 4 °c, then filtered through whatman no.4 filter paper. the solvent was removed by vacuum in a rotary evaporator and the dark green residue was dissolved in 10 ml acetone and used as a starting stock solution. further dilutions with either acetone or distilled water were used to prepare different concentrations. bioassay bioassays were performed with first instar larvae of h. cunea using 102, 104 and 106 spores/ml of bt on mulberry and 104, 106 and 108 spore/ml on sycamore. concentrations of 0.09, 0.22 and 0.42 % of a. annua on mulberry and concentrations of 0.13, 0.28 and 0.48 % 0n sycamore were used. concentrations of 0.02, 0.11 and 0.32 of l. stoechas on mulberry and concentrations of 0.13, 0.38 and 0.79 on sycamore were used. control leaves were treated with distilled water. for each treatment 30 larvae in three replicates were used in all the experiments and whole experiments were replicated twice. during the experiments, the larvae of each experimental condition were kept separately. the effective concentration (lc50) was calculated after 24 h using probit analysis (finney, 1971). fresh leaves were sprayed with different concentrations of the bt, a. annua, and l. stoechas and allowed to air dry. control leaves were treated with methanol alone. first instar larvae were starved for 4 h and then fed on leaves treated with the different concentrations of btk, a. annua, and l. stoechas. the uneaten leaves were removed every 24 h and the larvae were fed fresh treated leaves until larvae reached to fourth instar when the biochemical experiments were initiated. sample preparation for enzymatic assay enzyme samples from the midguts of fourth instar larvae were prepared based on zibaee and bandani (2009). briefly, larvae were randomly selected and their midguts were removed by dissection under a stereo microscope in ice-cold saline buffer (6 μmol/l nacl). the midgut was separated from the insect body, rinsed in-cold saline buffer, placed in a pre-cooled homogenizer and ground in 1 ml of universal buffer containing succinate (5 mm), glycine (2 mm) and 2morpholinoethanensulfonic acid (ph 7.2). the homogenates from both preparations were separately transferred to the 1.5 ml centrifuge tubes and centrifuged at 15000 rpm for 20 min at 4 °c. the supernatants were pooled and stored at -20 °c for subsequent analyses (zibaee and bandani, 2009). digestive enzyme assays α-amylase activity α-amylase activity was assayed by the dinitrosalicylic acid (dns) procedure (bernfeld, 1955), using 1% soluble starch (merck, darmstadt, germany) as substrate. twenty microliters of the enzyme were incubated for 30 min at 35 °c with 500 μl universal buffer and 40 μl soluble starch. the reaction was stopped by addition of 100 μl dns and heating in boiling water for 10 min. dns is a color reagent hence, the reducing groups released from starch by α-amylase action were measured by the reduction of dns. the boiling water stops the αamylase activity and catalyzes the reaction between dns and the reducing groups of starch. absorbance was then read at 540 nm. one unit of α-amylase activity was defined as the amount of enzyme required to produce 1 mg maltose in 30 min at 35 °c. a blank sample without substrate with αamylase extract and a negative control containing no α-amylase extract with substrate were run simultaneously. all assays were performed in duplicate and each assay was repeated at least three times. αand β-glucosidase activity for solubilization of membrane hydrolyses (α, β-glocusidases) in triton x-100, membrane preparations were exposed to triton x-100 for 20 h at 40 ˚c, in a ratio of 10 mg of triton x-100/mg of protein, before being centrifuged at 15,000 rpm for 30 min. no sediment was visible after the centrifugation of this supernatant at 10,000 rpm for 60 min. the activity of the enzymes remains unchanged, at -20c, for periods of at least a month (ferreira and terra, 1983). the α, β-glucosidases activity was assayed by incubating 50 μl of enzyme solution with 75 μl of p-nitrophenyl-α-dglucopyranoside (pnαg) (5 mm), p-nitrophenyl-β-d glucopyranoside (pnβg) (5 mm) and 125 μl of 100 mm universal buffer (ph 5.0) at 37 °c for 10 min. the reaction were stopped by adding 2 ml of sodium carbonate (1 m) and read at 450 nm (ferreira and terra, 1983). lipase activity the enzyme assays were carried out as described by tsujita et al. (1989). thirty µl of midgut tissue extracts, 0.5 ml of universal buffer solution (1m) (ph 7.2), and 100 µl of p-nitrophenyl butyrate (50 mm), as substrate, were incorporated, mixed thoroughly and incubated at 37 °c. after 1 min, 100 µl distilled water was added to each tube (control and experimental samples) and absorbance was read at 405 nm. one unit of enzyme release 1.0     254 table 1 toxicity of bacillus thurengiensis, artemisia annua and lavandula stoechas extracts on the larvae hyphantaria cunea concentration1 bacillus thurengiensis artemisia annua lavandula stoechas mulberry sycamore mulberry sycamore mulberry sycamore ld10 95% confidence interval2 102 101-104 104 103-105 0.09 0.03-0.13 0.13 0.07-0.18 0.02 0.001-0.06 0.13 0.05-0.20 ld30 95 % confidence interval 104 103-106 106 105-107 0.22 0.16-0.28 0.28 0.22-0.35 0.11 0.03-0.18 0.38 0.27-0.51 ld50 95 % confidence interval 106 106-108 108 107-1010 0.42 0.33-0.63 0.48 0.38-0.71 0.32 0.20-0.49 0.79 0.20-0.57 l90 95 % confidence interval 109 108-1011 1012 1010-1014 1.93 1.06-8.14 1.76 1.05-5.48 4.12 1.69-5.46 4.77 2.28-8.17 slope±se 0.34-0.073 0.25-0.056 1.95±0.50 2.29±0.54 1.15±0.35 1.64±0.40 x2 (df) 4.70 4.44 2.46 2.37 2.83 0.82 p-value 0.48 1.14 0.72 0.70 0.16 0.64 1concentration of bacillus thurengiensis is spore/ml and plant extracts are percentage 2confidence limits have been calculated with 95 % confidence nanomole (10-9 mole) of p-nitrophenol per minute at ph 7.2 at 37 °c using p-nitrophenyl butyrate as substrate. the negative control tube was placed in a boiling water bath for 15 min to destroy the enzyme activity and then cooled prior to be added with the substrate. protease activity general protease activity of adult midguts was determined using azocasein as substrate (elpidina et al., 2001). the reaction mixture was 80 µl of 2 % azocasein solution in 40 mm universal buffer of specified ph and 30 µl enzyme. the reaction mixture was incubated at 37 °c for 60 min. proteolysis was stopped by addition of 300 µl of 10 % trichloroacetic acid (tca). appropriate blanks in which tca was added first to the substrate were prepared for each assay. precipitation was achieved by cooling at 4 °c for 120 min and the reaction mixture was centrifuged at 16,000 rpm for 10 min. an equal volume of 1 n naoh was added to the supernatant and the absorbance was recorded at 440 nm. lactate dehydrogenase (ldh) assay for evaluating lactate dehydrogenase (ldh), the king’s (1965) method was used. to standardize volumes, 0.2 ml nad+ solution was added to the test tubes and 0.2 ml of water was added to control test tubes, each containing 1 ml of the buffered substrate. 0.01 ml of the sample was also added to the test tubes. test tube samples were incubated for exactly 15 min at 37 ˚c and then arrested by adding 1 ml of color reagent (2,4-dinitrophenyl hydrazine) to each tube and the incubation continued for an additional 15 min. after the contents were cooled to room temperature, 10 ml of 0.4 n naoh was added to each tube to make the solutions strongly alkaline. at exactly 60 s after the addition of alkali to each tube, the intensity of color was measured at 440 nm. protein determination protein concentrations were measured according to the method of bradford (1976), using bovine serum albumin (bio-rad, münchen, germany) as a standard. statistical analysis the mortality and lethal concentration were obtained by using probit analysis (robertson et al., 2007) and polo-pc software (leora, 1987). in this case, significant differences among the concentrations were recorded when 95 % confidence intervals (ci) did not overlap. other data were compared by one-way analysis of variance (anova) followed by tukey's studentisized test when significant differences were found at p = 0.05 (sas, 1997). differences among samples were considered statistically significant (p < 0.05).     255 results dose-response relationships the results showed that plant extracts and bacterial toxins produced a dose response in the insect species on both host species (fig. 1, table 1). the ld50 values of b. thurengiensis were significantly different in sycamore and mulberry (table 1). in the case of a. annua extracts, no significant difference was observed between sycamore and mulberry, whereas extracts from l. stoechas produced effects similar to those observed for b. thurengiensis (table 1). effect of btk and plant extracts on digestive enzymes results showed that b. thuringiensis and plant extract affected the digestive enzymatic profiles of h. cunea at several concentrations by using oral ingestion treatment in the presence of two hosts (tables 2-6). when larvae fed on leaves treated by btk, activity of all digestive enzymes was decreased and showed a dose-related status (table 2). a. annua treatment decreased digestive enzyme activities in larvae feed on both mulberry and sycamore in a dos-related manner (table 3). treatment of leaves by l. stoechas demonstrated a slightly decrease on digestive enzymes except for protease and lipase. however, the effect of l. stoechas extracts on enzyme activities on sycamore was more with regard to mulberry (table 4). combined effect of b. thuringiensis and a. annua on mulberry showed that digestive enzyme activities decreased except for β-glucosidase and lipase (table 5). similar results were found in the case of b. thuringiensis and l. stoechas (table 6). effect of btk and plant extracts on lactate dehydrogenase activity table 7 shows effect of b. thuringiensis, a. annua and l. stoechas on ldh activity of h. cunea. results demonstrated that ldh activity increased by treating different concentrations of insecticides on larvae and the higher activity was obtained by b. thuringiensis alone and b. thuringiensis and l. stoechas together. the least activity was observed in the case of l. stoechas alone. similar results found when sycamore was used as host. discussion crude botanical components for various purposes were well known in traditional cultures for centuries (schmutter, 1990). their extracts and active ingredients are a good choice for different investigations including pest management tactics. here we observed that the treatment of b. thuringiensis, a. annua and l. stoechas separately as well as the combined effect of bacteria and plant extracts exerted a significant effect on h. cunea digestive enzyme and ldh when spread onto two plant hosts, namely mulberry and sycamore. several studies have shown that feeding is necessary for the stimulation of digestive enzyme activities (sibley, 1981; broadway and duffey, 1988). results demonstrated that sublethal doses of these biopesticides individually decreased digestive enzyme activities such as α-amylase, αand βglucosidase, lipase and protease and increased ldh activity. higher enzyme activities in the midgut of control insects are most probably due to consumption and utilization of large quantities of food (senthil-nathan et al., 2006). imbalance in enzyme-substrate complex and inhibition of peristaltic movement of the gut (hori, 1969) might have inhibited the enzyme activities in the treated insects (zibaee and bandani, 2009). it is clear that exposure of diet to botanical insecticides has significant effects on several enzyme activities found in the late instar larvae of h. cunea. botanical insecticides may interfere with the production of certain types of proteins (smirle et al., 1996; senthil-nathan et al., 2006). in the case of decreasing activity of digestive enzymes due to b. thuringiensis treatment, this bacterium causes damage to the epithelial cells of the midgut through crystalline parasporal bodies, which release the active toxin after digestion by serine proteases under the alkaline conditions in the intestinal fluid (senthil-nathan et al., 2006). therefore one would expect that such damage to the midgut would cause a decrease in digestive enzyme activities (eguchi et al., 1972; mathavan et al., 1989; smirle et al., 1996; senthil nathan et al., 2006). α-amylase is an endo-digestive enzyme that catalyzes the breakdown of 1:4-α-glucosidase bonds in polysaccharides and converts starches into maltose (disaccharide) and glycogen into glucose. in the many studies, pesticides including b. thuringiensis, synthetic chemicals and botanical components significantly decreased the activity of αamylase in the midgut of different insects (senthilnathan et al., 2006; shekari et al., 2008; zibaee et al., 2008; zibaee and bandani, 2009). saleem and shakoori (1987) showed that sublethal concentrations of pyrethroids decreased the α-amylase activity in larval gut of the beetle tribolium castaneum herbst (coleoptera: tenebrionidae). lee et al. (1994) showed that some igrs decreased the activity level of α-amylase and esterase in the treated larvae. ascher and ishaaya (2004) showed that the activity level of this enzyme increased 30 % in s. littoralis boisd (lepidoptera: noctuidae) treated with phentine acetate compared with control. senthilnathan et al. (2006) found that b. thuringiensis decreased the activity level of this enzyme; the activity was much lower when bacterial spores and botanical components were combined. zibaee et al. (2008) showed that along with elevation of spraying times, the activity level of α-amylase would sharply decrease in chilo suppressalis walker (lepidoptera: crambidae) larvae. shekari et al. (2008) demonstrated that α-amylase activity level decreased 24 h after treatment and sharply increased at 48 h after treatment with a. annua extract of the elm leaf beetle. similar results were found when adults of eurygaster integriceps puton (heteroptera: scutelleridae) fed on grain and water contained a. annua extract (zibaee and bandani, 2009). in this study we found that b. thuringiensis, a. annua and l. lavandula, individually, decreased activity level of α-amylase and the highest inhibitions were obtained when larvae had been fed on sycamore and combined exposures were made.     256 table 2 effect of bacillus thurengiensis on the activity of different enzymes (µmol/min/mg protein) of hyphantaria cunea larvae in the presence of two different hosts treatment1 α-amylase α-glucosidase β-glucosidase protease lipase mulberry sycamore mulberry sycamore mulberry sycamore mulberry sycamore mulberry sycamore control 2.44±0.20a 1.85±0.08a 2.72±0.34a 2.32±0.98a 2.91±1.35a 2.64±0.66a 3.54±0.00a 2.14±0.004a 4.00±0.008a 3.44±0.004a ld10 2.07±0.06ab 1.56±0.07ab 2.26±0.21a 1.62±0.40ab 3.74±0.36a 2.62±0.31a 3.08±0.001a 2.01±0.001ab 2.80±0.003b 2.8±0.004b ld30 1.69±0.06b 1.53±0.03b 1.60±0.32b 1.21±0.33b 2.84±0.12ab 1.77±0.60b 2.18±0.003b 1.13±0.00b 2.14±0.003c 2.02±0.002c ld50 1.15±0.14c 1.30±0.06b 1.58±0.44b 0.63±0.30c 2.45±0.30b 1.21±0.50b 0.71±0.001c 0.77±0.001c 1.49±0.003c 1.55±0.002c 1concentration of is b. thurengiensis spore/ml. ld10, ld30 and ld50 are 10 2, 104 and 106 on mulberry and 104, 106 and 108 on sycamore. 2means ± sem followed by the same letters indicate no significant difference (p < 0.05) according to the tukey test. table 3 effect of artemisia annua extract on the activity of different enzymes (µmol/min/mg protein) of hyphantaria cunea larvae in the presence of two different hosts treatment1 α-amylase α-glucosidase β-glucosidase protease lipase mulberry sycamore mulberry sycamore mulberry sycamore mulberry sycamore mulberry sycamore control 1.87±0.09a 1.75±0.28a 2.05±0.54a 1.90±0.4a 3.88±1.03a 2.67±0.28a 3.80±0.00a 2.36±0.00a 3.43±0.00a 3.34±0.00a ld10 1.44±0.05b 1.69±0.05a 1.39±0.10b 1.55±0.27b 2.48±0.07c 2.50±0.39b 3.16±0.00ab 1.62±0.00ab 2.79±0.00b 2.61±0.00ab ld30 1.13±0.00c 1.19±0.04ab 1.24±0.28b ±0.950.09c 1.39±0.14c 1.55±0.21c 1.88±0.00b 0.80±0.00b 2.00±0.00c 2.01±0.00b ld50 0.84±0.06c 0.99±0.03b 0.52±0.19c 0.14±0.08d 0.28±0.49d 1.25±0.31d 0.76±0.00c 0.50±0.00c 1.47±0.00d 0.56±0.00c 1concentrations of plant extract are 0.09, 0.22 and 0.42 on mulberry and 0.13, 0.28 and 0.48 on sycamore as ld10, ld30 and ld50. 2means ± sem followed by the same letters indicate no significant difference (p < 0.05) according to the tukey test.     257 table 4 effect of lavandula stoechas extract on the activity of different enzymes (µmol/min/mg protein) of hyphantaria cunea larvae in the presence of two different hosts treatment1 α-amylase α-glucosidase β-glucosidase protease lipase mulberry sycamore mulberry sycamore mulberry sycamore mulberry sycamore mulberry sycamore control 2.08±0.01a 1.97±0.03a 2.20±0.20a 1.49±0.91a 2.89±0.33a 2.61±0.21a 3.64±0.00a 3.51±0.00a 3.25±0.00a 2.77±0.02a ld10 2.05±0.03a 1.61±0.02b 1.77±0.65b 1.62±0.23 2.42±0.68a 2.46±0.48a 3.65±0.00a 3.43±0.00a 3.18±0.00a 2.49±0.00a ld30 1.89±0.03b 1.38±0.08b 2.37±0.74a 1.53±0.30a 2.71±0.12a 1.79±0.70a 3.42±0.00a 3.20±0.00a 2.91±0.00a 2.42±0.00a ld50 1.71±0.02b 1.11±0.05c 2.15±0.75a 1.54±0.34a 2.45±0.23a 1.41±0.23b 3.39±0.00a 3.25±0.00a 2.69±0.00a 2.38±0.00a 1concentrations of plant extract are 0.02, 0.11 and 0.32 on mulberry and 0.13, 0.38 and 0.79 on sycamore as ld10, ld30 and ld50. 2means ± sem followed by the same letters indicate no significant difference (p < 0.05) according to the tukey test. table 5 combined effect of bacillus thurengiensis and artemisia annua extract on the activity of different enzymes (µmol/min/mg protein) of hyphantaria cunea larvae on mulberry as the host treatment1 α-amylase α-glucosidase β-glucosidase protease lipase control 2.01±0.01a 1.99±0.021 3.32±0.80a 3.93±0.00a 3.16±0.00a ld10 2.00±0.018a 1.52±0.016b 2.89±0.34a 3.93±0.00a 3.14±0.00a ld30 1.72±0.03ab 1.45±0.08b 2.96±0.87a 2.82±0.00ab 3.14±0.00a ld50 0.86±0.06c 0.69±0.32b 2.61±0.87a 0.91±0.00b 3.07±0.00a 1each ld value shows b. thurengiensis + plant extract concentration as ld10: 10 2+ 0.09, ld30: 10 4+0.22, ld50: 10 6+0.42. 2.means ± sem followed by the same letters indicate no significant difference (p < 0.05) according to the tukey test.     258 table 6 combined effect of bacillus thurengiensis and lavandula stoechas extract on the activity of different enzymes (µmol/min/mg protein) of hyphantaria cunea larvae in the presence of mulberry leaves as the host treatment1 α-amylase α-glucosidase β-glucosidase protease lipase control 1.93±0.03a 1.92±0.10a 2.99±0.79a 3.35±0.00a 3.30±0.00a ld10 1.75±0.11ab 1.77±0.30a 2.76±1.01a 1.05±0.00b 2.50±0.00b ld30 1.24±±0.19b 1.34±0.14ab 3.12±0.75a 0.33±0.00b 1.87±0.00b ld50 0.38±0.13c 1.20±1.08b 2.52±1.00b 0.14±0.00b 1.03±0.00c 1each ld value shows b. thurengiensis + plant extract concentration as ld10: 10 2+ 0.02, ld30: 10 4+0.11, ld50: 10 6+0.32. 2means ± sem followed by the same letters indicate no significant difference (p < 0.05) according to the tukey test. table 7 effect of bacillus thurengiensis, artemisia annua and lavandula stoechas extract on lactate dehydrogenase (µmol/min/mg protein) of hyphantaria cunea larvae on mulberry and sycamore as the host treatment1 bacillus thurengiensis artemisia annua lavandula stoechas b.t. + artemisia annua b.t. + lavandula stoechas mulberry sycamore mulberry sycamore mulberry sycamore mulberry sycamore mulberry sycamore control 0.17±0.05d 0.21±0.10d 0.19±0.08c 0.18±0.03c 0.23±0.07c 0.19±0.07b 0.20±0.04c 0.25±0.09d 0.23±0.031d 0.17±0.06c ld10 0.26±0.08c 0.35±0.08c 0.21±0.09c 0.22±0.01c 0.26±0.06b 0.21±0.03b 0.27±0.06b 0.38±0.03c 0.40±0.08c 0.34±0.02b ld30 0.50±0.10b 0.49±0.08b 0.35±0.07b 0.45±0.05b 0.27±0.07b 0.29±0.06ab 0.53±0.1bc 0.67±0.09b 0.61±0.19b 0.39±0.04b ld50 0.71±0.14a 0.95±0.02a 0.57±0.07a 0.81±0.04a 0.54±0.10a 0.48±0.07a 0.67±0.17a 0.97±0.12a 0.96±0.21a 0.86±0.09a 1each ld value shows b. thurengiensis (b.t.) and plant extract alone and b.t. + plant extract concentration together in the presence of mulberry leaves as the host. 2means ± sem followed by the same letters indicate no significant difference (p < 0.05) according to the tukey test.     259 the glycosidases catalyze the hydrolysis of terminal, non-reducing 1, 4-linked α-d-glucose residues with release of α-d-glucose. study of glucosidase in herbivorous insects is important not only for understanding digestion biochemistry but also for developing insect pest management strategies. plants produce a wide variety of allelochemicals which act as defensive compounds. these include alkaloids, cyanogenic and triterpenoid glycosides, phenols, flavenoids and nonprotein amino acids (hsiao, 1985). among these allomones, glycosides seem to play an important role in host plant resistance to insects. for example, tomatine, an alkaloid glycoside and rutin (quercetin 3-rutinoside) are involved in the resistance of tomato to the tomato fruitworm, heliothis zea fabricius (lepidoptera: noctuidae), by acting as feeding deterrents (pratviel-sosa et al., 1986). dimboa is another glycoside which is present in young corn tissues. the toxic action of these glycosides is due to their corresponding aglycones liberated by the action of β-glucosidase. in the current study, treatment of h. cunea larvae with sublethal concentrations of different biopesticides showed a reduction in the activity level of αand βglucosidases. the increase of plant extract concentrations on sycamore corresponded to a reduced enzymatic activity in larvae. this may be due to a drop in the consumption rates and leveling off or decline in food conversion efficiencies (zibaee and bandani, 2009). these decreasing activities were reported in other insects. hemmingi and lindroth (1999, 2000) studying effect of phenolic components in gypsy moth (lepidoptera, lymantriidae) and forest tent caterpillar (lepidoptera, lasiocampidae) demonstrated that glucosidase activities declined for both insect species when reared on diets with phenolic glycosides in addition to decreasing growth and increasing developmental time. zibaee and bandani (2009) found that a. annua extract significantly decreased activity of αand βglucosidases on e. integriceps adults so that the lowest activity was obtained at 25 % concentration of plant extract treatment of adults. lipases (triacylglycerol acylhydrolase; ec 3.1.1.3), which catalyses the hydrolysis of fatty acid ester bonds, are widely distributed among animals, plants and microorganisms (zibaee et al., 2009). it was found that b. thuringiensis and plant extracts decreased the activity level of lipase but in the case of l. stoechas no significant differences were observed. senthil nathan et al. (2006) showed that treating cnaphalocrocis medinalis (guenee) (lepidoptera: pyralidae), the rice leaffolder, by btk, nske, and vnle (azadirachtin and neem components) sharply decreased the activity level of lipase in the midgut. proteases hydrolyze proteins to amino acids classified as endopeptidases (ec 3.4.21-24) and exopeptidases (ec 3.2.4.11-19) based on their catalytic mechanism (pascual-ruiz et al., 2009). it was observed that biopesticides significantly decreased activity of protease in the midgut of h. cunea larvae especially on sycamore but no significant differences were obtained in the case of l. stoechas on both hosts. because proteases are necessary for activation of b. thuringiensis protoxin to active toxin, this biopesticide could be a logical choice for control of caterpillars due to high ph value and suitable activity of proteases in the their midgut. zibaee and bandani (2009) found that a. annua extract significantly decreased the activity of protease on e. integriceps adults so that the lowest activity were obtained when 25 % concentration of plant extract was used on adults. physiological conditions of h. cunea affects the activity of the tested enzymes and reflects the absorption, digestion, and transport of nutrients in the midgut. b. thurengiensis damages the epithelial cells of the midgut through crystalline parasporal bodies, so decreasing levels of digestive enzymes. this results in reduced phosphorous liberation for energy metabolism, decreased rate of metabolism and decreased rate of metabolite transport, maybe due to the direct effects on enzyme regulation and synthesis. ldh is an important glycolytic enzyme being present virtually in all tissues (kaplan and pesce, 1996). it is also involved in carbohydrate metabolism and has been used as an indicative criterion of exposure to chemical stress (wu and lam, 1997; diamantino et al., 2001) and as an index of anaerobic metabolism (chamberlin and king, 1998). activity level of ldh in culex after treatment with ddt, malathion and cyfluthrin decreased 58.88 %, 33.33 % and 66.66 %, respectively (arshad et al., 2002). senthil-nathan and kalviani (2005) showed that feeding of spodoptera litura on ricinus communis treated with azadirachtin and nucleopolyhedrovirus decreases the amount of this enzyme in midgut that demonstrates low nutritional efficiency of the larvae. similar results were also observed on effectiveness of melia azedarach on rice leaffolder (senthilnathan, 2006). results in our current study showed that reduction of digestive enzyme activities due to using different bio-pesticides on larvae fed on sycamore were higher than those of mulberry. in addition, induction of ldh activity on larvae fed on sycamore was higher than that of mulberry. there may be different reasons for these differences. first of all, sycamore trees have been planted extensively around the city but mulberry orchards exist in the limited areas. hence, sycamore trees are more available to larvae than mulberry. secondly, almost all mulberry trees in the area have been modified genetically for silkworm rearing and have less plant secondary metabolites than sycamore. this matter is being observed more obviously on activity of digestive enzymes in control. but, treatment of sycamore leaves by biopesticides has a synergistic relationship with secondary metabolites existing in the plant tissue and caused more reduction in the digestive enzyme activities. as a conclusion, a. annua and l. stoechas extracts had significant effects on the larvae of h. cunea so that they act synergistically with b. thurengiensis var. kurstaki toxin, causing reduction of digestive enzyme activity and elevation of ldh activity. h. cunea is a widely distributed pest which causes severe damages to trees in orchards. hence, widely spraying by common synthetic     260 insecticide has high environmental risks specially on human therefore, studies on biopesticides and their combinations are necessary specially for physiological effect to decrease the population density of the pest. physiological analysis would be particularly informative to get insight into which combinations of biopesticides enhance the efficiency of a safe management process. acknowledgment this study was supported by a university of tehran grant. we thank m allahyari for assistance. references abudulai m, shepard bm, mitchell pl. parasitism and predation on eggs of leptoglossus phyllopus (l.) 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(asteracea) on the digestive enzymatic profiles and the cellular immune reactions of the sunn pest, eurygaster integriceps (heteroptera: scutellaridae), against beauveria bassiana. bull. entomol. res. 100: 185-196, 2010. zibaee a, bandani ar, ramzi s. lipase and invertase activities in midgut and salivary glands of chilo suppressalis (walker) (lepidoptera, pyralidae), rice striped stem borer. inv. surv. j. 5: 180-189, 2009. zibaee a, jalali sendi j, etebari k, alinia f, ghadamyari m. the effect of diazinon on some biochemical characteristics of chilo suppressalis walker (lepidoptera: pyralidae), rice striped stem borer. munis. entomol. zool. 3: 255-264, 2008. isj 5: 20-xx, 2008 isj 5: 20-29, 2008 issn 1824-307x research report a study on biochemical differences among five different groups of rice striped stem borer chilo suppressalis walker (lepidoptera: pyralidae) a zibaee1, jj sendi1, f alinia2, k etebari3 1department of plant protection, faculty of agriculture, the university of guilan, rasht 41635-1314, iran 2rice research institute of iran (rrii), rasht 41635-1658, iran 3department of sericulture, faculty of natural resources, the university of guilan, somehe sara, iran accepted february 21, 2008 abstract identification of biodiversity in different rice striped stem borer (chilo supprressalis) populations is very important to adopt suitable integrated pest management procedures. larvae were collected from five different regions in north of iran including gourabzarmikh (go), sheikhmahaleh (sh), rasht (ra), amol (am) and babol (ba). activity levels of five enzymes including alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and alpha-amylase were evaluated in 4th instar larvae. in addition, five non-enzymatic compounds such as glucose, cholesterol, total protein, uric acid and urea were also measured. amount of measured compounds showed significant differences in all groups except for alanine aminotransferase and aspartate aminotransferase. hierarchical agglomerative clustering under upgma model demonstrated that ba population had the most genetic distance and was separated from other groups. in the second group, go population had the most genetic distance from others and two groups of ra and sh had the least genetic distances. key words: rice stripped stem borer; hierarchical agglomerative clustering; biochemical characteristics introduction the rice striped stem borer (chilo supprressalis) is a cosmopolitan and destructive pest in rice fields of the world (khanjani, 2006). this pest was introduced to iran in 1973 and has been widely distributed in all rice fields of iran. its distribution is random and aggregative with 2-3 generations per year (saeb and gramy, 2000). in north of iran, this pest has been distributed in all areas and its density is more than economic injury level (dezfoulian and moustofipoor, 1972). in 1995, it has been reported from other provinces of iran such as isfahan, shiraz, eilam and khozestan. severe damages have been reported from these areas (moghaddas and saiiad-nasiri, 1995). the chemical control especially organophosphorous compounds has been a common practice for more ___________________________________________________________________________ corresponding author: jalal jalali sendi department of plant protection faculty of agriculture the university of guilan rasht 41635-1314, iran e-mail: jjalali2001@yahoo.com than three decades (khosroshahi et al., 1979). however, other methods such as cultural practices and biological control with trichogramma spp., have been incorporated. in recent years, control of c. suppressalis has been concentrated on using resistance varieties and pheromone traps. saeb and mohammad-salehi (1998) studied 78 different varieties and showed that binam variety with 15 % white head was the most resistant one. saeb (1999) studying on different germplasts of rice showed that khazar variety was resistant to first generation of rice striped stem borer and susceptible to second generation. saeb (2002a) suggested that using pheromone traps including z-13, octadecenal, z-11, hexadecenal and z-9, hexadecenal was a usefull practice for rice striped stem borer control in north of iran. using different markers to determine the intraspecific biodiversity and better understanding of genetic polymorphisms has always been within the range of researchers' interests (chatterjee and data, 1992; eguchi, 1995; etebari and matindoost, 2004b; etebari et al., 2005). "biochemical marker" is a term used for some biochemical compounds, 20 which are able to demonstrate the differences between two species or different biotypes of the same species (stoikova et al., 1998). bartelett (1989) used these biochemical markers for identification and presence of biotypes in different species of heliothis. loxdale and brookes (1990) used the same markers to identify the biotypes of blackberry grain aphid (sitobiom avenae) in southeast england. there are many biochemical markers in insects which explicit differences among various individuals in the same population. the measurement of αamylase and invertase could divide the silkworm populations in two classes, one group with two generations and high silk production and the other with several generations and low production (chatterjee and data, 1992). chatterjee et al. (1993) reported that there is a significant correlation between some biochemical parameters of hemolymph and midgut fluid of the silkworm larvae of which the most important compounds are: amylase, invertase and alkalin phosphatase. the amount of these compounds in larval hemolymph depends on different factors such as the type of food, environmental conditions, genetics and etc. enzymes with respect to their genetic structure are less changeable than other biochemical compounds in larval hemolymph. generally for this aspect, different qualitative enzymatic analyses or isoenzymes are being utilized (etebari et al., 2005). in this study, larvae were collected from five different regions in north of iran including gourabzarmikh, sheikhmahaleh, rasht, amol and babol. activity levels of five enzymes including alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and alpha-amylase and non-enzymatic coumponds such as total protein, glucose, cholesterol, urea and uric acid were evaluated in 4th instar larvae of rice striped stem borer. materials and methods insects the larvae of chilo suppressalis were collected from five different sites of rice fields including amol (am), babol (ba) in mazandaran province and sheikhmahaleh (sh), gouramzarmikh (go) and experimental blocks of rice research institute of iran, rasht (ra) in guilan province (300 larvae from each location) and reared on dorfac variety of rice, at 25 ± 1 ºc temperature, 70 ± 5 % rh and 16l:8d. thirthy larvae of fourth instar from each site were randomly selected and used in biochemical experiments with 3 replications. sample preparation and biochemical analysis whole larval body was homogenized in fluid nitrogen and samples of each region were diluted with phosphate buffer in weight to volume proportion and centrifuged for 10 min in 10,000 rpm. the supernatant was transferred to new tubes and was preserved at -20 ºc until the onset of the experiments. protein was measured based on biuret's method by utilizing a total protein assay kit (biochem co, iran). in this method, proteins makes a complex purplish blue with an alkaline copper solution, which its absorption value at 540 nm has a direct relation to the amount of whole body protein. to measure total cholesterol, richmonds's (1971) method was performed. the principles of this method are based on hydrolysis of cholesterol esters by cholesterol oxidase, cholesterol esterase and peroxidase. glucose was analyzed as a method described by siegert (1987). alanine aminotrasferase (alt) and aspartate aminotransferase (ast) were measured using thomas' (1998) procedure. method of mihara et al. (1988) was used to analyze alkaline phosphatase (alp) and p-nitrophenylphosphate is used as a substrate and light absorption was evaluated at 400 nm. uric acid contents were determined using uricase as described by valovage and brooks (1979); this enzyme produces a purplish color which has a direct correlation (at 500 nm) with uric acid concentration. urea was measured with urease gdh kit (biochem. co, iran). in this method, ammonia ion is produced by urease enzyme and second reaction was catalyzed by glutamate dehydrogenase. finally, reducing absorption rate was calculated at 340 nm. for evaluating lactate dehydrogenase (ldh), king’s method (1965) was used. based on this method, the catalytic potential of the enzyme in conversion of lactate to pyrouvat and simultaneously the reduction of nad+ to nadh is considered. alpha-amylase was measured using kondo et al (1988) method. in this method, cnpg3 substrate is used in which 2chloro-4-nitrophenol has been bound to maltoriose. cnpg3 is hydrolysed by alpha-amylase and its concentration is determined at 405 nm. statistical and clustering methodology all data were analyzed using sas software and tukey’s studentized range (hsd) test in a complete randomized design (sas, 1997). hierarchical agglomerative clustering was done using ntsys software, employing the method of average linkage between groups (romesburg, 1984) under upgma (unweighted pair-group method sing arithmetic average). the clustering was based on the squared euclidean distance. the average linkage between two groups are considered as the average of distance among all pairs of cases with one number from each group. hierarchical clustering analysis was carried out by considering all ten biochemical parameters. results the quantitative differences of analyzed compounds activity levels and the amount of biochemical compounds in 4th instar larvae from five groups of rice stripped stem borer have been represented in figs 1 and 2. in all larval groups, significant differences among enzyme activity levels and amount of non-enzymatic compounds were observed, except for ast and alt. activity of ast in different groups was fluctuating between 1,420 to 21 fig. 1 changes of nonenzymatic macromolecules in five populations of rice stem borer. amol (am), babol (ba), rasht (ra), sheikhmahale (sh) and gourabzarmikh (go). 2,850 iu/l (table 1). the minimum value of this enzyme was measured in larvae of am and maximum value was in larvae of ra. the activity level of alt was less than ast and the minimum and maximum value of it was observed in ra and sh, respectively. however, these enzymes were not significantly different among various groups of larvae (fig. 1). the amount of alp had a significant difference in all populations (table 1). the highest amount of this enzyme (1,006 iu/l) was measured in am population and the lowest amount (261 iu/l) was evaluated in go population. activity levels of alphaamylase and ldh also showed significant differences in various populations of rice stripped stem borer. the maximum value of alpha-amylase and ldh were 40 iu/l, 1,040 iu/l in go and 21iu/l, 249 iu/l in sh, respectively. the measurement of five non-enzymatic compounds including total protein, cholesterol, uric 22 fig. 2 changes of enzymatic macromolecules in five populations of rice stem borer. amol (am), babol (ba), rasht (ra), sheikhmahale (sh) and gourabzarmikh (go). acid, urea and glucose in the larvae of various regions demonstrated significant differences (fig. 2). the highest value of protein (2.6 g/dl) was measured in am and the lowest value was evaluated in ra. the highest and the lowest amount of urea were 7 mg/dl and 3 mg/dl, which were observed in ba and am larvae, respectively. the uric acid amount had the maximum value in go population (3.4 mg/dl) and the minimum value in ba population (104 mg/dl). the highest and the lowest value of cholesterol were observes in go and ba larvae, which were measured 35 mg/dl and 16 mg/dl, respectively. finally, the amount of glucose was the maximum in ba population (341 mg/dl) and minimum in am population (131 mg/dl). hierarchical agglomerative clustering table 2 shows genetic distances in five groups of larvae based on enzymatic activity levels. as it shows, genetic distance between ba and sh larvae 23 table 1 enzyme activity and non-enzymatic compounds amount of 4th instar larvae of stripped stem borer biochemical compounds no. range mean f value c. v. aspartate aminotrasferase (iu/l) 30 1,420-2,580 1,973.79 1.90 13.28 alanine aminotransferase (iu/l) 30 1,600-2460 2,414.82 0.87 114.90 alkaline phosphatase (iu/l) 30 261-926 633.20 89.83 10.48 alpha-amylase (iu/l) 30 21-40 33.13 13.02 10.11 lactate dehydrogenase (iu/l) 30 249-1040 616.06 187.43 7.88 total protein (g/dl) 30 1-2.6 1.56 5.78 16.60 cholestrol (mg/dl) 30 16-35 22.68 10.25 15.93 glucose (mg/dl) 30 131-341 261.75 32.9 8.39 urea (mg/dl) 30 3-7 4.51 4.39 19.18 uric acid (mg/dl) 30 1.4-5 2.48 7.36 23.69 table 2 genetic distance of five groups of rice striped stem borer based on enzyme activity levels populations go sh ra ba am gourabzarmikh (go) 0.0000 sheikhmahale (sh) 12.864 0.0000 rasht (ra) 6.1312 9.0180 0.0000 baboul (ba) 12.785 15.186 10.314 0.0000 amoul (am) 13.149 6.0702 7.5441 6.9349 0.0000 table 3 genetic distance among five different populations of rice striped stem borer based on non-enzymatic compounds populations go sh ra ba am gourabzarmikh (go) 0.0000 sheikhmahale (sh) 5.8316 0.0000 rasht (ra) 6.8027 79.599 0.0000 baboul (ba) 20.219 9.0972 8.1452 0.0000 amoul (am) 16.682 6.4529 5.9860 18.100 0.0000 table 4 genetic distance among five different populations of rice striped stem borer based on all biochemical parameters populations go sh ra ba am gourabzarmikh (go) 0.0000 sheikhmahale (sh) 10.8316 0.0000 rasht (ra) 6.1027 7.599 0.0000 baboul (ba) 18.269 12.100 8.1552 0.0000 amoul (am) 12.722 8.2548 6.6560 14.1020 0.0000 was maximum (15.86) and the most similarity was measured 6.1312 between ra and go larvae. on the basis of these data, hierarchical clustering was divided into two groups, ba population was in one part and the rest of the populations were placed in another cluster. in current group, go population was separated from others (fig. 3). genetic distances in five groups of larvae based on non-enzymatic compounds are shown in table 3. the highest value, observed between ba and go groups, was 20.219, while the least value was between sh and go groups, 5.8316. hierarchical clustering on the basis of non-enzymatic compounds is similar to enzyme activity figure (fig. 4). 24 fig. 3 hierarchical cluster of five populations of rice striped stem borer based on enzymatic characteristics. amol (am), babol (ba), rasht (ra), sheikhmahale (sh) and gourabzarmikh (go). in the table 4, genetic distance of groups on the basis of both enzyme levels and non-enzymatic compounds are shown. on the basis of these data, genetic distance of ba and go larvae with 34.904 was the highest and the least genetic distance was between ra and sh groups. the nearest group to them was ra and go whose genetic distance was 12.933. figure 5 demonstrates hierarchical analysis among these five groups on the basis of all biochemical parameters. on the basis of this dendrogram and a transaction in 50 %, larvae were divided into two distinct clusters; ba was in one part and the rest were placed in another part. in the second group, go population was separated from others. sh and ra population had the least distance. discussion in this study, the activities of two aminotransferases presented in the larval body were evaluated. the aminotransferases are important components of amino acid catabolism and they are mainly involved in transferring an amino group from one amino acid to another keto acid. the ast and alt serve as a strategic linkage between the carbohydrates and protein metabolism that are known to be altered during various physiological and pathological conditions (etebari et al., 2005). horie and nakamura (1986) figured out that activity of alt in the silk gland of silkworm larvae was much and demonstrated it to be more than the midgut and fat body, while maximum activity of ast was reported from the fat body. therefore, activity levels of these enzymes are different in various tissues. scaraffia et al. (2005) showed that when females of aedes aegypti ate a blood meal, activity level of these enzymes increased in fat body and midgut. researches have shown that isoenzyme pattern of ast is easily able to differentiate between the two species of stem borers chilo sp. (kioko et al., 1995). in this study, the amount of these enzymes didn’t demonstrate a significant difference among various groups. etebari et al. (2005) showed that the activity levels of aminotransferases have a significant difference among eight groups of silkworm. several factors are effective on amount of these enzymes. the increase in temperature causes the enhancement of alt and ast activity (reddy and benchmain, 1992), the diet and type of food also have high impact on activity levels of enzymes (gogoi and yadav, 1995). for this reason, rearing conditions such as variety of rice, temperature, relative humidity and etc were uniform for each population in this study. the sampling time and biochemical analysis were also similar for all populations so that results had the least side effects. but as it was said, no significant differences were measured among different populations. in the present study, a significant difference between activity levels of alp were observed in various groups. the alp is a set of hydrolytic enzymes that hydrolyze phosphomonoesters under the alkaline condition (miao, 1988). the production of this enzyme has a clear relationship with feeding behavior. in addition, activity of it depends on larval status, absorption, digestion and transportation of nutrients in midgut (eguchi and iwamoto, 1975; yoshitake et al., 1966). toxic chemicals in food decrease nutrition efficiency and alp activity. nathan et al. (2005) showed that treatment of rice plants with neem limonoids and melia azedarach extracts decreased the activity level of alp in cnaphalcrocis 25 fig. 4 hierarchical cluster of five populations of rice striped stem borer based on nonenzymatic characteristics. amol (am), babol (ba), rasht (ra), sheikhmahale (sh) and gourabzarmikh (go). medinalis. they also showed that feeding of spodoptera litura on ricinus communis treated with azadirachtin decreased the amount of this enzyme in midgut. present results demonstrated that, because of suitable conditions, am group had been widely distributed, but go group was vice versa. the larvae of am, ba and ra regions have been sprayed with diazinon for more than 30 years. because of alp role in hydrolyzing phosphomonoesters, it could be concluded that there were some degrees of resistance in these populations. saeb (2002b) reported no chemical resistance in his field collected specimens however, we found that rice striped stem borer in ra, ba, am and sh had resistance ratio of 12.88, 12.81, 8.8 and 4.4 to diazinon, respectively in 2006 (unpublished data). ldh is an important glycolytic enzyme being present in virtually all tissues (kaplan and pesce, 1996); it is also involved in carbohydrate metabolism and has been used as an indicative criterion of exposure to chemical stress (wu and lam, 1997; diamantino, amadeu and soaresa, 2001). and it is used as an index of anaerobic metabolism (chamberlin and king, 1998). in this study, activity level of ldh in five groups of larvae showed a significant difference. nathan et al. (2005) showed that feeding of s. litura on r. communis treated with azadirachtin and nucleopolyhedrovirus decreased the amount of this enzyme in midgut that demonstrated low nutritional efficiency of the larvae. similar results were also observed on effectiveness of m. azedarach on rice leaffolder (nathan, 2006). kim et al. (2002) showed that feeding on different varieties of mulberry affect the amount of ldh and cholesterol in longicorn beetle. therefore, different factors such as feeding, growth stages and even type of tissues affect on quantitative changes of this enzyme in insect bodies. also, smith and collier (2001) showed that activity level of ldh among different population of orthopsyche embriata and acanthophlebia cruentata have no significant differences. in current study, all populations are affected by diazinon except for go larvae. because of stress caused by this chemical, the amount of ldh in go population was the highest among others. alpha-amylase is one of the midgut enzymes that is involved in starch and other carbohydrates metabolism. the activity level of this enzyme depends on feeding diet is different. in insects feeding on wool this enzyme is in the lowest amount while in phytophagous insects, it is the highest, especially in clethrophagous insects (chapman, 1998). in this study, amount of this enzyme was significantly different among various groups of larvae. go population had the maximum level of alpha-amylase that showed suitable feeding habitat. hirano and ishi (1961) showed that starch in rice stem was very suitable for nutrition of rice striped stem borer which higher amount of alpha-amylase in go population confirmed this idea. because in go area those varieties were planted that was more susceptible to rice stem borer than those in ba, hence, type of planted variety caused differences among these five different groups. 26 fig. 5 hierarchical cluster of five populations of rice striped stem borer based on all biochemical parameters. amol (am), babol (ba), rasht (ra), sheikhmahale (sh) and gourabzarmikh (go). considerable differences were observed in the amount of five non-enzymatic compounds, including glucose, cholesterol, urea, uric acid and protein. on the basis of glucose amount, ba group was differentiated from others. etebari et al. (2005) showed that amount of glucose differentiates line 104, x2 and 107 from others in silkworm. etebari and matindoost (2004b) reported when the feeding activity was appropriate, glucose and cholesterol of the silkworm hemolymph increased and in these larvae a considerable improvement was observed in production characteristics. in contrast, when the feeding of larvae was interrupted, the amount of this compound severely decreased (etebari and matindoost, 2004a). several activities of insects depend on carbohydrates metabolism. the amount of glucose demonstrates the available sugar for cells that could represent the metabolism of carbohydrates (satake et al., 2000). the quality of consumed food and starvation affect the hemolymph sugar (etebari and matindoost, 2004b). satake et al. (2000) observed when the fifth instar larvae were under starvation, the glucose of hemolymph decreased immediately. etebari (2002) showed that the type of food could affect the amount of cholesterol in larval hemolymph. therefore, in the present study, larvae of each group were feed upon the same rice variety and the enhancement of cholesterol and glucose level could be relative to the efficiency of absorption system for each group of larvae. etebari and matindoost (2004b) demonstrated that adding vitamin b3 increased the amount of protein and cholesterol in silkworm hemolymph. in current study, enhancement of protein and cholesterol in ra and am larvae showed appropriate status of feeding. mosavi (1979) showed that there was a direct relationship between larval weight and fertilization in adults. the weight of insects depends on amount of carbohydrates, proteins, lipids and other substances. in this study, the weight of go larvae was 10-14 mg and ba larvae was 9-11 mg and amount of total protein, cholesterol, urea, uric acid and glucose were the highest in go population. these data showed that approximate digestibility (ad) of this population was better than ba population. on the basis of hierarchical clustering in different groups of rice stripped stem borer, all the studied populations were divided into two groups, ba in one and the rest in another group. this showed that ba group based on biochemical markers was different from others. in the second cluster, go population has been separated from others and sh and ra groups have the least genetic distance on the basis of biochemical parameters. chattarijee and data (1992) utilized the biochemical markers to classify 54 silkworm strains with different geographical origins. they also obtained similar results on some strains with different origin in one group and also strains with the same origin in different groups. identification of different biotypes in rice stripped stem borer is the most important for adoption 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice gene function and cellular pathways in higher vertebrates, including humans, have increasingly shown to be highly conserved thr isj 6: 7-14, 2009 issn 1824-307x review immunorecognition and immunoreceptors in the cnidaria sr dunn center for marine studies, university of queensland, australia accepted january 29, 2009 abstract recent studies that are focused on cnidarians as model systems for cell biology are offering key insight into the complexities of higher metazoan biology. the innate immune system of these basal invertebrates is one of the cellular processes that until recently, was undescribed. the knowledge regarding both innate immunity and other cellular processes in cnidarians is far from complete. however, the evidence acquired so far, suggests highly conserved components of these cellular processes are more closely related to vertebrate homologues than more complex, but divergent invertebrate model systems. this review examines the immunorecognition and receptors that have been identified within the cnidarians so far. the complement of receptors and pathways already identified indicates that these basal invertebrates are far from “simple” in the array of methods they possess for dealing with potential invading microbes and pathogens. key words: cnidaria; immunity; symbiosis; pathogen; symbiodinium; pamp; prr introduction gene function and cellular pathways in higher vertebrates, including humans, have increasingly shown to be highly conserved through metazoan evolution from the discovery of homologues in basal metazoans, such as sponges and cnidarians (kortschak et al., 2003; kuo et al., 2004; kusserow et al., 2005; dunn et al., 2006; hemmerich et al., 2007). the recent use of cnidarians as a metazoan model system (for review see weis et al., 2008), has brought about a wealth of new information that is unravelling the cellular processes involved in symbiosis, immunity regulation, cell death and organism longevity. there are, of course, considerable differences between the lower and higher metazoans: cnidarians lack a vertebrate-like adaptive immune system in so far as immunological memory is absent. furthermore, whilst amoebocytes have been shown to play a role in cnidarian cell defenses (olano and bigger 2000; mullen et al., 2004; mydlarz et al., 2008), specialization of cnidarian cells into components of an adaptive immune system is not evident. the ability of cnidarians to distinguish between self and non-self has previously been shown to occur in anthozoans and hydrozoans (frank et al., ___________________________________________________________________________ corresponding author: simon r dunn center for marine studies university of queensland st lucia, brisbane qld 4072, australia e-mail: s.dunn@cms.uq.edu.au 2001; rinkevich 2004; bosch, 2008). the capacity of allorecognition may vary across and between classes, colonies, such as anthopleura sp. (class: anthozoa) (lubbock, 1980) and solitary individuals, such as hydra sp. (class: hydrozoa) (kuznetsov and bosch, 2003). the molecular triggers that lead to allorecognition in cnidarians may utilize the innate immune response, however at present, the cellular mechanisms remain unknown and beyond the scope of this review. this review focuses upon the complexity of cnidarian immunorecognition. a cellular process that was once thought of as ‘basic’ in these simple metazoans is in fact complemented by an array highly conserved defenses, which offer key insight into the foundation of the higher metazoan innate immunity. one of the first lines of defense of the invertebrate immune system against potential invasive microbes are the pattern recognition receptors (prr’s). the host prr’s detect and bind to highly conserved components of microbe cell walls, such as proteins, lipids, carbohydrates and lipoteichoic acids (gram-positive bacteria) or lipopolysaccharides (lps; gram-negative bacteria), which form a recognisable matrix or pattern, known as pathogen-associated molecular patterns (pamp) or microbe-associated molecular patterns (mamps) (murphy et al., 2008). the activation of signal responses following the binding of host prr’s to pamp’s is rapid and can operate in three ways: firstly, to stimulate microbe ingestion through phagocytosis and enzymatic degradation. secondly, 7 through chemotactic directives for molecules to move to sites of infection, and thirdly, the induction of effector molecules that leads to a cell signal cascade and ultimately an immune response (murphy et al., 2008). at present, the complement of cnidarian prr’s appears to be diverse across classes, although the limited number of descriptive immunoreceptor studies still hinders a full evaluation and requires much more in-depth research. recent extensive cnidarian expressed sequence tag (est) and genome sequencing projects have highlighted the broad range of highly conserved biological processes within all metazoans that are also found in cnidarians, including the innate immune system (miller et al., 2007). across animal evolution, there has also been significant gene loss, and cnidarians are no exception. gene loss and duplication has occurred across all of the cnidarian classes, and suggests that the hydrozoa are divergent from basal anthozoa, and the scyphozoa from hydrozoa (miller et al., 2007; bosch, 2008). several key domains and conserved components of pathways associated with innate immune receptors that have already been identified are; 1) toll-like receptors (tlr’s) containing leucine rich repeats (lrr’s), which recognise pamps, 2) components similar to that of the complement cascade, and 3) lectins (for review see hemmerich et al., 2007; miller et al., 2007; kvennefors et al., 2008). toll-like and lrr receptors toll and toll-like receptors (tlr) are part of a metazoan receptor superfamily that all share a toll interleukin-1 receptor domain (tir). in the cnidarians, tlr-domain conserved proteins vary in number and structure. in the hydrozoan, hydra magnipapillata, there are four tlr-domain proteins described so far. two of which, hmmyd88-1 and hymyd88-2 are related to the downstream myd88, each displaying an additional characteristic death domain. the two remaining hydra tir-domain proteins, hytrr-1 and hytrr2 have a typical transmembrane and short extracellular scaffold, and are likely to be pathway initiator receptors. yet unusually, they lack the typical lrr-domains, and may be devoid of canonical structure (miller et al., 2007). however, this structural variation may not be uncommon or necessary for the same function (sun jin and lee, 2008). recent work by bosch et al. (in press) has revealed additional tlr-related lrr domain proteins in hydra may function synergistically with hytrr-1 and hytrr-2 in pamp recognition, indicating multiple receptor responses to an immune challenge operate in the cnidarians. the anthozoa, in comparison to hydrozoans, have a broader selection of tlr-domain proteins reflecting their basal phylogeny in the group. the estuarine sea anemone, nematostella vectensis have at least 5 tlr-domain proteins from the predicted structures. one of the tlr-domain proteins, nvmyd88 has a similar structure to the hmmyd88-1 and 2 and is thought to function in the same manner to induce expression of immune response genes through activation of the transcription factor, nfkb. a second tlr-domain protein, nvtlr-1, has multiple lrr domains and both carboxy and amino-terminal flanking cysteine rich motifs, which is characteristic of an ancestral domain structure (miller et al., 2007). the remaining identified predicted tlr-domain protein structures all contain immunoglobulin (ig) domains. the ig domains, which may function in cell-cell recognition, form a distinct clade away from the higher vertebrate structures. in comparison to other anthozoan sequences so far screened, n. vectensis has the highest complement of tlrs. at present, only one tlr-domain protein has been identified in the corals, acropora palmata and acropora millepora. both of the coral tlr-domains were similar to the n. vectensis ig-domain tlr, nvil1r1. however, no extracellular domains were detected in the coral receptors (miller et al., 2007). in addition to the tlr-domain structures described by (miller et al., 2007), downstream components of the toll/tlr pathways were also described from the est/ genome analysis, such as those associated with the c-jun n-terminal kinases (jnk)/mitogenactivated protein kinase (mapk) pathway and nfkb transcription that can lead to cell death. the depth of the known pathway homology indicates that it is not just toll/tlr receptors that are highly conserved, but a complete representative prr immune response pathway. antimicrobial peptides and metabolites an important contribution to the cnidarian immune response is the array of anti-microbial peptides. peptides are small signalling molecules that control a variety of processes, such as development, muscle contraction and the control of target gene expression (for review see bosch and fujisawa, 2001). antimicrobial peptides secreted by microbes within the mucus of corals are known to be a potent inter-specific microbial regulator of the epiphytic coral microbial community (ritchie, 2006). the composition of the cnidarian microbial community structure is important to host health (ritchie 2006; fraune and bosch, 2007), yet the roles of host anti-microbial peptides in host immunity until recently were unknown. host peptides have a stable structure that allows translocation to different areas around the cnidarian diploblastic body structure via the interepithelial space or mesoglea (fraune and bosch, 2007). the antimicrobial properties of peptides such as, aurelin, from the scyphozoan, aurelia aurita may focus activity on removing invasive gram-positive or gram-negative bacteria (ovchinnikova et al., 2006). host peptides may play a role in regulating associated microbial community populations to benefit the host, such as observed in the hydrozoan, hydra olgactis (fraune and bosch, 2007). in hydra, the induction of antimicrobial peptides is mediated by the interaction of a lrr domain protein with a tir-domain protein lacking lrr’s. the antimicrobial peptides from hydra, such as hydramacin-1 (jung et al., 2009), which is upregulated in the presence of lps and the peptide, pereculin-1, upregulated in the presence of lps and flagellin, are important components of the 8 microbe and stress host response. these particular peptides have been shown to have important therapeutic qualities as potent antibiotics against drug resistant human pathogens (bosch et al., in press). the initial descriptions of the antimicrobial peptide structures indicate that they are unique, but also, as is the case with aurelin, have similarity to defensins and k+-channel blocking toxins (ovchinnikova et al., 2006). a current rapidly expanding area of research is that of biodiscovery or bioprospecting. one target of this research are peptides, the other are secondary metabolites and their important roles in cellular homeostasis and defense against predation, parasites and disease (newman and hill, 2006; dunlap et al., 2007). the roles of many peptides and secondary metabolites as part of the cnidarian innate immune system is now being explored and have been shown to function as antioxidants and as antimicrobial compounds (mydlarz and jacobs 2004, shapo et al., 2007). however, the pathways associated with the synthesis and receptor mediation of these metabolites remain unknown. complement and lectins the complement signalling cascade is a major part of the vertebrate innate immune system, whereby microbes and foreign cells that are detected undergo opsonisation, phagocytosis and lysis. complement is composed of four pathways. three of the pathways are involved in activation and contain a thiolester c3 component, which leads to the fourth membrane attack complex (mac) lytic pathway (murphy et al., 2008). in vertebrates the primary function of c3, c4 and other members of the alpha-2-macroglobulin (a2m) paralogous gene family is opsonisation of microbes or immune complexes (armstrong and quigley, 1999). there are complement or precursors of the complement pathways, in the form of c3-like thiolester-containing proteins (tep) that predate the protostome-deuterostome split identified in cnidarians (dishaw et al., 2005; miller et al., 2007). the first c3-like, tep cdna to be identified from the gorgonian coral, swiftia exserta, had high conservation to vertebrate c3. there was an overall similarity with mammalian c3, c4 and c5 sequences, with a characteristic anaphylatoxin region which is absent in other a2m proteins, and cleavage sites of vertebrate c4 (dishaw et al., 2005). this basal, multiple ‘attribute’ domain protein, which may predate a later divergence into a larger protein family with individual domains and specific function, has previously been suggested to be a feature of cnidarians, such as caspases and bcl-2 family members of the apoptotic pathway (dunn et al., 2006). a similar c3-like protein has also identified in the anthozoans, acropora and nematostella, and although the counterpart in hydra was absent, hydra was shown to contain an a2m-like protein. it is interesting to note that all forms were restricted to the endoderm/gastrodermal tissues respectively (miller et al., 2007). although they have different structures, all forms may play a role in opsonisation fig. 1 a laser confocal microscopy image of the surface of a cultured symbiodinum sp. labelled with fitc-labelled concanavalin-a lectin (green), which binds specifically to glycans, including α-mannose. (fc: 100 μg/ml). sample prepared by d logan and s davy, university of wellington, new zealand in accordance with wood-charlson et al. (2006). image taken by srd. key: red = chlorophyll autofluorescence. bar = 5 μm. and therefore, their presence in cells associated with food particle /microbe selection and uptake is not surprising. in addition, miller et al. (2007) described a suite of predicted proteins with a membrane attack complex (mac) and perforin domains associated with the final phase of the complement cascade, indicating that multiple components from different stages of the complement cascade pathways exist in the cnidarians. activation of complement-like pathways and opsonization through the formation of a lectinbinding complex has been indicated in cnidarians during the onset of the complement-like cascade. the lectin-binding complex may also be of particular relevance in the onset and specificity of the prolific symbiosis with the dinoflagellate, symbiodinium sp., found in many cnidarians. the role of lectins in cell surface recognition of potential pathogens or symbionts is the important first step in cnidarian cell surface recognition. in previous studies using glycosides and concanavalin-a lectin to bind and mask the surface sugars of symbiodinium sp. (fig. 1) prior to infection of aposymbiotic hosts, have shown differential uptake and onset of symbioses (jimbo et al., 2000; lin et al., 2000; koike et al., 2004; wood-charlson et al., 2006). at present, only one cnidarian lectin has been characterised, millectin from the scleractinian anthozoan, a. millepora (kvennefors et al., 2008). millectin has sequence homology to the lectin domain of a range of c-type lectins and is a relative of both the collectins, mannose binding lectin (mbl) and the surfactant, sp-a. in addition, millectin has the unusual characteristic of having extensive variability in the substrate binding region, indicating 9 a potential broad range of pamp’s that may be recognized and bound by millectin/s, and in part, may be due to a dual role in pathogen/potential symbiont recognition of this receptor (kvennefors et al., 2008). cnidarian cells detect previously ‘undetectable’ or persistent invading microbes are only now being identified and may also operate to remove dysfunctional symbionts under stress (for review see weis, 2008). two key components of this intracellular innate immune repertoire can lead to cell death activation: firstly, the upregulation of nitric oxide (no) in the anthozoan, aiptasia pallida in response to lps, and hyperthermic stress (perez and weis, 2006). although inducible nitric oxide synthase is still yet to be identified in cnidarians, nitric oxide up-regulation has been shown to play an active role in the removal of symbiont symbiodinium sp. under symbiosis stress through cell death pathway activation (trapido-rosenthal et al., 2001; perez and weis, 2006). intracellular immune receptors so far, this review has covered receptors associated with intercellular-mediated innate immune recognition, which act as a first line of defense and gateway to phagocytosis and entry into the host. cnidarian innate immunorecognition is not limited to the cell surface or just the ‘gate’ into the cell. the cnidarian cell is well equipped with intracellular receptors and pathways that act as a second line of defense to recognise and remove the “trojan horse” that has managed to slip through the first line of defense and is now inside the walls. this multilevel approach to microbe recognition for removal of the microbe/pathogen and/or retention of the symbiotic ‘rent payer’ is key to the onset and specificity of symbiosis known as the ‘winnowing process’ that was first described in the squid, euprymna scolpes and vibrio fischeri bacterium, symbiosis (nyholm and mcfall-ngai, 2004). secondly, expression of a member of the cd36 family, the scavenger receptor sr-b1, is upregulated in the symbiotic anthozoan, anthopleura elegantissima compared to aposymbiotic individuals, (rodriguez-lanetty et al., 2006). cd36 family members including srb-1, act in host defense through the lipid metabolism. members of the cd36 family are known to be manipulated by invasive pathogens to gain entry into the host cell, (stafford et al., 2002) and may be controlled through bridging molecules, such as thrombospondin-1, c1q collectins and β2 glycoproteins (for review see májai, 2006). although est’s to homologs of a number of bridging molecules have been identified in a.millepora (meyer et al., 2007), a direct link between either lipids or bridging molecules and cd36 in host defense is yet to be shown in cnidarians. one of the intracellular recognition immune response found in cnidarians is the increased enzyme driven production of melanin (petes et al., 2003; mydlarz et al., 2008; palmer et al., 2008). the phenoloxidase (po) cascade that leads to melanin production, is known to play an active associated role with phagocytic aggregation and microbe/pathogen removal in many other invertebrates (johansson and söderhäll, 1996). initiation of the po cascade through pro-form cleavage in other invertebrate systems may vary according to substrate, suggesting a functioning role in recognition. in arthropods, activation occurs through contact with specific polysaccharides such as β-1,3-glucan (β13g), lps and peptidoglycan, in urochordates the inducers are lps and β13g, and in echinoderms only β13g has been shown to activate the cascade (johansson and söderhäll, 1996). in cnidarians, differential po activity was shown by experimental substrate addition to samples of both a healthy and compromised branching acroporid coral species, and to a lesser degree in the massive porites spp. (palmer et al., 2008). however, although previous studies have detected increased melanin within coral tissues (petes et al., 2003; mydlarz et al., 2008; palmer et al., 2008), it is important to note that similar host pigmentation occurs in response to a multitude of different stimuli (roff et al., 2008), and quite normally is often more visible in areas of healthy new growth or in areas of varying symbiotic dinoflagellate density. therefore, defining and attributing a cause to the different areas of host pigmentation is important to resolving the extent to which the po-melanin pathway is activated and associated with inflammation and immunity across a broad spectrum of cnidarian hosts. in all metazoans the destruction or removal of microbes/pathogens through host cell apoptosis, autophagy or induced microbe programmed cell death (pcd) can occur at initial contact stage to prevent infection, or at an intracellular level to mitigate damage (james and green, 2004). the initiation of cnidarian host apoptosis, autophagy and / or in situ symbiont pcd /necrosis, is known to occur during development (cikala et al., 1999), allorecognition (seipp et al., 2001; kuznetsov and bosch, 2003), in response to disease (ainsworth et al., 2007), in response to hyperthermic oxidative stress (dunn et al., 2002, 2004, 2007; franklin et al., 2004; richier et al., 2006), and as a postphagocytic removal mechanism of symbiodinium sp. during the onset of symbiosis (dunn and weis, 2009). the success of symbiosis in cnidarians appears to stem from the ability to restrain or prevent autophagy/apoptotic cell death pathways (rodriguez-lanetty et al., 2006; dunn et al., 2007), and corresponds with similar cell death inhibition in other invertebrate symbioses (pannebakker et al., 2007). intracellular parasites (the “trojan horse”) of vertebrates host cells, such as leishmania donovani and mycobacterium tuberculosis, also avoid immuno-detection by retarding apoptotic and autophagic pathways (for reviews see dermine et al., 2000; koul, 2004; gutierrez et al., 2004). the pathogenic control over these pathway occurs by manipulating ca2+ signalling, phosphoinositide metabolism, phosphatidylinositol 3-kinase signalling the intracellular immune receptors by which 10 fig. 2 a schematic representation of the innate immunoreceptors and associated protein domains, and peptides found in cnidarians that are prosed to contribute to the innate immune response. some receptors are part of the primary response at the cell surface leading to increased secretion of peptides and lectins or to a secondary intracellular response, gene expression and pathway activation. key to abbreviations are reported in table 1. cascade and inhibiting pro-cell death molecular triggers (fratti et al., 2001; chua et al., 2004; koul et al., 2004; hilbi 2006). whether the cnidariandinoflagellate symbiosis is controlled by the host, or if there is a symbiont directed manipulation of host immunity and associated cell-death pathways at present remains unresolved. in conclusion, there is now strong evidence that cnidarian innate immunorecognition is far from simple and members of all classes display a diverse armoury of innate immune receptors that operate at both an inter-cellular and intra-cellular level. the interaction between tlr-domain and lrr domain proteins and the production of peptides (possibly with the addition of secondary metabolites) is complemented with lectin secretion that may interact with a c3-like complement phagocytic signalling cascade (fig. 2). this multiple attribute defense system leading to an immune response, offers a key insight into the basal function of more complex innate immune pathways found in vertebrates. there is no doubt that the discovery of homologous, highly conserved cell pathways, and their functioning molecular components within cnidarians, will continue to promote members of the phylum as an ideal model system for the study of not only innate immunity, but much broader areas of higher metazoan cell biology. table 1 different classes of receptors retrieved in cnidarians and their conserved domains abbreviation domain name lrr leucine rich repeat tmd trans membrane domain tir (toll-like) toll interleukin-1 receptor ig immunoglobulin a2m alpha-2-macroglobulin sr-b1 scavenger receptor b1 cell surface receptor signalling pro-po prophenoloxidase tir/ death tir (as above) /death domain po phenoloxidase no nitric oxide intracellular signalling ros reactive oxygen species 11 acknowledgements i would like to thank c kvennefors, m pernice and s dove for their comments and views, and t bosch for the communications provided in advance of publications. references ainsworth td, kvennefors ec, blackall ll, fine m, hoegh-guldberg o. disease and cell death in white syndrome of acroporid corals on the great barrier reef. mar. biol. 151: 19-29, 2007. armstrong pb, quigley jp. alpha-2 macroglobulin: an evolutionary conserved arm of the innate immune system dev. comp. immunol. 23: 375390, 1999. bosch tcg. the path less explored: innate immune reactions in cnidarians. in: heine h (ed), innate immunity of plants, animals, and humans (vol. 21), springer-verlag, berlin, pp 27-42, 2008. bosch tcg, augustin r, anton-erxleben f, fraune s, hemmerich g, zill h, et al. uncovering the evolutionary history of innate immunity: the simple metazoan hydra uses epithelial cells for host defence. dev. comp. immunol. 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http://www.bio.utexas.edu/research/matz%5flab/matzlab/454.html http://www.bio.utexas.edu/research/matz%5flab/matzlab/454.html weis vm. cellular mechanisms of cnidarian bleaching: stress causes the collapse of symbiosis. j. exp. biol. 211: 3059-3066, 2008. wood-charlson em, hollingsworth ll, krupp da, weis vm. lectin/glycan interactions play a role in recognition in a coral/dinoflagellate symbiosis. cell. microbiol. 8: 1985-1993, 2006. 14 ii scientific meeting of the italian ascidiologists, 30 june – 1 july 2008, department of isj 6: s1-s2, 2009 issn 1824-307x editorial ii° scientific meeting of the italian ascidiologists: dedicated to professor giuseppe reverberi n parrinello department of animal biology, university of palermo, palermo, italy accepted march 13, 2009 periodically the italian ascidiologists are engaged in a meeting on ascidian biology. the 2008 meeting has been dedicated to the memory of prof giuseppe reverberi who, in the second half of the past century, was devoted to study of ascidian developmental biology. he held (1948-1971) the chair of zoology at the university of palermo (italy) where, with valid collaborators, started and strengthened the main scientific route leading to significant improvements, internationally appreciated, in embryology with an experimental approach. he dedicated his life to research and was a master for numerous scholar generations who attained the frame of the scientific method and achieved keenness for research in animal biology. a lot of scholars and scientists, inside and outside his schooling place, appreciated his rigor in research and the valuable teaching method that also improved their human and scientific personality. prof f de bernardi (university of milan, italy), ascidian embryologist, validly retraced the reverberi scientific activity that still nowadays is a milestone in ascidian development study. the meeting broadened diverse fields of ascidian biology and appreciated lectures and oral communications have been presented. due to their phylogenetic key position, ascidians have attained importance for evolutionary studies. the genome of some solitary species (ciona intestinalis, ciona savignyi, halocynthia roretzi) has been fully or partially sequenced, analyzed and annotated and can be validated by gene expression patterns for several specific biological properties and activities. the first session was rich in reports on development and morphogenesis including differentiation of adult sensory organs and larval papillae, developmental expression and gene organization of synapses, distribution of larval neural phenotypes, meiotic progression and fertilization, musculature differentiation and gene expression, evolution of anterior hox regulatory elements, larval metamorphic and juvenile phases, role of nitric oxide ___________________________________________________________________________ corresponding author: nicolò parrinello department of animal biology university of palermo via archirafi 18, 90123 palermo, italy e-mail: nicpar@unipa.it during the development, pigment organ formation during the embryogenesis, angiogenetic mechanism in the colonial circulatory system, and the effects of a xenobiotic on development and metamorphosis. the next session on phylogeny and microevolution concerned the homologies between chordate invertebrates and vertebrates as revealed by the neurophysiology of swimming tadpoles, the fast evolutionary dynamics of mitochondrial genome including new data and forward genetic that unveil and characterize two ciona cryptic species, and the phylogenetic conservation of csf-related genes. since this ascidian is a cosmopolitan representative model, cryptic species identification is of great relevance for research and comparative analysis of results involving several laboratories. at the second day, after a plenary lecture on stem cells and chimerism in colonial ascidians (a voskoboynik, stanford university, usa), the lectures were mainly devoted to immunity and inflammatory response. bioinformatic results, disclosed est and extensive in silico searches have concerned immunorelevant molecules, gene expression patterns and some specific immune characteristics. phenoloxidases, variable domaincontaining chitin-binding proteins (vcbps) genes and their expression, anaphylotoxin and specific receptors, immunomodulatory factors, hemocyte cytotoxic activity, collectin and cytokine-like cloning and genes expression, hemocytes provided with acetylcholinesterasis, were the topics of the reports. finally, some communications concerned with environmental stressors effects on embryos and hemocytes. several italian research groups, that esteemed the prof reverberi research, attended the meeting. besides the ascidiologists from the university of palermo who organized the event, scientists from the universities of padua, genoa, milan, bari, from the stazione zoologica “anton dohrn” of naples, and from palermo cnr, contributed in the success of the scientific happening. in particular, we were honoured for the interest and presence of the prof. armando sabbadin emeritus at the padua university and a founder of the research field on colonial ascidian allorecogniton. moreover prof e ottaviani, president in charge of the italian association of developmental and comparative immunology and editor in chief of the open access s1 journal “invertebrate survival journal”, was an appreciated guest. the abstracts of the scientific contributions have been published in inv. surv. j. 5: 83-96, 2008. acknowledgements we are grateful to prof g silvestri rector of the university of palermo, and to the department of animal biology of the university of palermo for their helpful support and sponsorship. thanks are due to prof e ottaviani, editor in chief, for his contribution in publishing the inv. surv. j. special issue. miur-prin cofin 2006-2008 grant to n parrinello also contributed to the meeting success. s2 61 isj 15: 61-65, 2018 issn 1824-307x visions and perspectives going beyond a static picture: the apple snail pomacea canaliculata can tell us the life history of molluscan hemocytes d malagoli department of life sciences, university of modena and reggio emilia, italy accepted february 27, 2018 abstract more than 40 years of studies on molluscan immunity have revealed a complex and dynamic immune system endowed with multifunctional circulating cells, i.e., hemocytes that are regulated by diverse signaling molecules. however, very little is known about the dynamic processes that drive hemocyte proliferation, differentiation, maturation, and senescence. evidence reported here highlights how the apple snail pomacea canaliculata is an extremely promising research organism that will provide answers to the numerous questions regarding the life-history of molluscan and lophotrochozoan hemocytes. key words: ampullariidae; gastropoda; hemocyte maturation; immunity; invertebrate hematopoiesis; pomacea canaliculata metazoans base their survival and reproductive success on the simultaneous functioning of different organ systems. while many organ systems have a well-defined location, clear anatomical borders, and a recognized physiological role, the immune system does not have a fixed anatomy, and its primary function is maintaining the equilibrium between the organism and all the microorganisms that live in or around it (bosch, 2014; bachère et al., 2015). this structure and function make it extremely difficult to identify and dissect the many activities that the immune system plays during homeostasis. most researchers have focused on mechanisms that are activated when the organism is challenged by pathogens or stressful events (malagoli et al., 2017). since the 1980s, numerous scientists have studied the immune system of invertebrates. they were principally attracted by its relative simplicity and the lack of components related to acquired immunity, such as lymphocytes and immunoglobulins. the main rationale for these analyses was to both clarify the functional basis of the innate component of the human immune system and to improve our understanding of the many pathologies resulting from the malfunctioning of the innate immune system (notarangelo et al., 2009). surprisingly, the invertebrate immune system has been revealed to be much more complex than ___________________________________________________________________________ corresponding author: davide malagoli department of life sciences university of modena and reggio emilia via campi 213/d, 41125, modena italy e-mail: davide.malagoli@unimore.it expected, changing experimental approaches and revising our current knowledge of the comparative immunology field. one of the main changes that has occurred in the last decade was the publication of numerous papers that revealed the high degree of complexity in the invertebrate immune system, in addition to the presence of highly variable molecules (armitage and brites, 2016; doolittle, 2016; oren et al., 2016). the involvement of these molecules in the immune response of invertebrates has yet to be understood, but it is undeniable that these molecules are strikingly similar to vertebrate antibodies (immunoglobulins), which are characterized by hypervariable regions. among invertebrates, the most important and commonly studied model is drosophila melanogaster (buchon et al., 2014). however, several other research organisms are available and well-studied, mainly because of their economic relevance (such as crustaceans and bivalves [smith et al., 2016; bachère et al., 2015]) or their importance as vectors of parasitic diseases (e.g., mosquitoes and some gastropods) (choi et al., 2012; adema et al., 2017). over the past few years, our research group has used a molluscan gastropod, pomacea canaliculata, as a research organism. this organism has great potential in the comparative immunology field because it gathers together many point of interest mentioned above, and many biological features make it easy to maintain in the lab and work with (fig. 1a). p. canaliculata (aka, the golden apple snail), is indexed among the most invasive species in the world (www.issg.org/worst100_species.html), and its fast and vast spread raised concerns for several reasons. in economic terms, p. canaliculata is a 62 fig. 1 a) the apple snail p. canaliculata laying eggs outside the water onto the wall of the tank. the eggs are bright pink because of the neurotoxic perivitellin fluid contained inside the egg shell. every egg clutch consists of hundreds of eggs. b) p. canaliculata hemocytes. the hemolymph has been collected and cytocentrifuged onto a slide. hemocytes have been stained with diff quik kit. a small blast-like cell (high nuclear/cytoplasmic ratio, white arrow), many large hyalinocytes (agranular blue cytoplasm), and a large granulocyte (granular cytoplasm, black arrows) are present. bar in a = 2 cm; bar in b = 20 um. voracious grazer of crops, and this problem is particularly relevant in asia (gilioli et al., 2017; lei et al., 2017) where this species was introduced as source of food. unfortunately, p. canaliculata was demonstrated to be potentially neurotoxic (sun et al., 2010) and not edible, and, thanks to the small number of predators, it freely eats and ultimately kills young rice plants (horgan, 2018; http://www.knowledgebank.irri.org/step-by-stepproduction/growth/pests-and-diseases/goldenapple-snails). aware of its impact on the environment and economy, both the eu and some states in the usa implemented tight regulations that forbid circulation and commercialization of the freshwater snails belonging to the genus pomacea (commission implementing decision, 2012; united states department of agriculture, animal and plant health inspection service [usda_aphis]; lei et al., 2017). pomacea and its relationship to the environment have also been studied, suggesting that this snail is a potential bio-indicator as a result of its ability to accumulate heavy metals (hoang et al., 2011). apple snails are also interesting from a biomedical perspective because they can regenerate complex organs de novo, such as the eyes and the tentacles (important tactile and chemosensory organs). recent studies have highlighted common aspects and mechanisms shared between adult regeneration and embryonic development (accorsi et al., 2017b). advances in the understanding of both regeneration and immune responses are especially interesting, and in the past few years, many researchers have been trying to elucidate the deep relationships between regeneration abilities and characteristics of the immune system by comparing different vertebrate and invertebrate model systems with varying regenerative abilities and types of immune systems (godwin et al., 2017; neves et al., 2016; tasiemski and salzet, 2017). the adaptability to different external conditions, the immune-tolerance towards a parasite (song et al., 2016), and the astonishing regenerative capacity of p. canaliculata are possible thanks to the role played by a common and fundamental component that is the immune system. as a consequence, the study of the immune system of p. canaliculata becomes of wide interest. the characterization of the cellular component of p. canaliculata immune system, aka the circulating hemocytes, demonstrated that p. canaliculata is not significantly different from other gastropods, since small, blastlike cells (aka pro-hemocytes) and larger hemocytes have been identified (accorsi et al., 2013; smith et al., 2016). among the larger hemocytes, hyalinocytes (agranular) and granulocytes (granular cytoplasm) were distinguished, with the former endowed with phagocytic activity (fig. 1b). direct observation of the organ structures and the comparison with other gastropod anatomical descriptions allow us to define the pericardial fluid a plausible candidate for the hematopoietic tissue. this tissue has a gel-like texture in younger animals, and it is more fluid in older adults. however, in both cases, it fills the pericardial 63 chamber in which the heart and the ampulla are housed (accorsi et al., 2014). after performing experiments involving repeated hemolymph withdrawal and immunostaining with a mitotic marker, we confirmed the presence of dividing cells in the pericardial fluid, and we hypothesized the involvement of the ampulla as a hemocyte reservoir. the tight functional and anatomical connections between the hemolymph, the heart, the ampulla and the pericardial fluid intrigued us, prompting us to perform further studies on hematopoiesis in the apple snail. real-time pcr experiments demonstrated the expression of a prokineticin-like protein in the apple snail pericardial fluid, supporting our model of hemocyte replication in this freshwater snail (accorsi et al., 2017a). these significant advances represent a solid foundation for studying the dynamic nature of the immune system of mollusks, particularly with respect to hemocyte turnover, maturation, and senescence. despite the characterization of hemocytes in mollusks (smith et al., 2016) and the identification and localization of a hematopoietic tissue/organ (accorsi et al., 2014; smith et al., 2016), hemocytes maturation still remains to be described, traced, and mechanistically understood. which anatomical sites are involved in this process? what are the stages of hemocyte differentiation and maturation? when does an hemocyte start to express the receptors that characterize it as a functional immune cell and when it becomes an old and no more functional cell? can this process be influenced by external events, such as environmental cues or pathogen exposure? (fig. 2). fig. 2 schematic representation of the hypothesis described in this paper about the turnover and maturation of p. canaliculata hemocytes. the hematopoietic cells (pink cells) proliferate in the hematopoietic organ(s) where they probably both self-renew and give rise to a population of immature hemocytes (orange cells) that differentiate. the differentiating cells mature into the hemocyte stage (green cells), which are functional until they become senescent hemocytes (grey cells) and eventually die. this process has not been carefully dissected in any mollusc. the hypothesis presented here suggests that mature and senescent hemocytes that are already functional can interact with maturing cells (blue cells), influencing their maturation process. this mature/maturing hemocyte interaction would drive the maturation of the new hemocytes towards specific and already encountered targets, enriching for a pool of cells active against the immune challenges that they are likely to face. besides hemocyte/hemocyte interactions, i hypothesize that also the environmental cues can influence the hematopoietic cell replication rate and hemocyte differentiation process, influencing the compositon of circulating cells on the basis of changes in environmental stimuli. this is, to the best of my knowledge, a new and dynamic perspective for considering the main cellular component of the molluscan immune system, consisting of the circulating hemocytes. the hemocytes have a short lifespan and constant turnover and this might provide to p. canaliculata its ability to overcome both persistent and new immune challenges during their long lifespan. 64 in accordance with observations performed also in other molluscan models (ottaviani et al., 2013; tascedda et al., 2015; malagoli and ottaviani, 2010), the main hypothesis of my laboratory is that the immune system of p. canaliculata is constantly refined on the basis of the immune stimulations encountered during an individual organism’s lifetime. this would also explain the remarkable capacity of these animals to quickly adapt and begin reproducing in new environments (accorsi, personal communication; gilioli et al., 2017; lei et al., 2017). to answer these and many other questions regarding the biology of p. canaliculata, several tools are now available thanks to the efforts of several laboratories around the world. recently, a few organ-specific transcriptomes have been published (yang et al., 2017; zhou et al., 2016), and the sánchez alvarado group at the stowers institute for medical research (kansas city, mo, usa) is developing an extensive database that includes organ-specific transcriptomes of many adult organs and the transcriptomes of many embryonic stages (accorsi, personal communication). proteomic studies have also been performed, which identified the presence of a neurotoxin in the perivitellin fluid of their eggs that likely evolved as defense against predators, expanding the scientific community’s interest in this model (sun et al., 2010, 2012; mu et al., 2017). at present, more than one research group is sequencing the genome of p. canaliculata in order to obtain a reliable genome database. the first assembly of the genome will soon be published and made available to the community working on apple snails (accorsi, personal communication; zhou et al., 2016; guo et al., 2018). the availability of these databases will provide our community the opportunity to efficiently search for genes, transcripts, and proteins of interest for both descriptive and functional studies (malagoli et al., 2011; tascedda et al., 2015; malagoli et al., 2017). altogether, this increasing body of evidence and the exponential growth and refining of molecular databases will allow a deeper understanding of the biology of p. canaliculata such that a wider set of experiments can be performed. while the data collected on p. canaliculata are increasing, as well as the number of papers published per year on this snail, it is important to emphasize how frequent species misidentification can be among apple snails. in this field, light has been shed by hayes et al. (2009), who described different apple snail species and their phylogenetic relationships, providing a detailed list of approaches to correctly identify each species (hayes et al., 2009, 2015; guo et al., 2018). the evidence recapitulated in this vision and perspectives show that p. canaliculata is a versatile model for several studies in the biological field and single it out among mollusks as one of the best candidate to study the process of replication, maturation, and senescence of immune-competent cells in lophotrochozoans. acknowledgments the author wishes to thank drs. a. accorsi and a. sánchez alvarado (stowers institute for medical research, kansas city, mo, usa) for helpful discussion and essential input. the author would also like to thank dr. s elliott for the careful linguistic revision of the manuscript. references accorsi a, benatti s, ross e, nasi m, malagoli d. a prokineticin-like protein responds to immune challenges in the gastropod pest pomacea canaliculata. dev. 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https://www.aphis.usda.gov/aphis/ourfocus/plan thealth/import-information/permits/regulatedorganism-and-soilpermits/sa_snails_slugs/ct_snails_slugs. yang l, cheng ty, zhao fy. comparative profiling of hepatopancreas transcriptomes in satiated and starving pomacea canaliculata. bmc genet. 18: 18, 2017. zhou x, chen y, zhu s, xu h, liu y, chen l. the complete mitochondrial genome of pomacea canaliculata (gastropoda: ampullariidae). mitochondrial dna 27a: 884-885, 2016. 32 isj 14: 32-43, 2017 issn 1824-307x research report effect of heavy metals on four different earthworm’s species specific autofluorescing eleocytes a chatterjee, r thilagaraj, m gobi department of biotechnology, school of bioengineering, srm university potheri 603203, tamilnadu, india accepted january 11, 2017 abstract earthworm is key bio-indicator of soil milieu to assess heavy metal contaminations. the celomic fluid of earthworm plays a significant role in the storage of riboflavin within the celomic cavity thereby maintaining its homeostasis. so the measurement of these autofluorescent ‘self-marking’ eleocytes will give more information about chloragocyte derived cells and species. the present study is to investigate the percentage of autofluorescing cells, riboflavin content, elemental and heavy analysis of immunologically significant eleocytes. the present study is focused on four different earthworms namely lampito mauritii, octochaetona serrata, eudrilus eugeniae and eisenia fetida, to characterize immune factors such as cellular and riboflavin content exposed to various heavy metal concentrations under laboratory conditions. four different earthworms were exposed to three different concentrations (63.5, 112.4 and 207.2 µg/ml) of heavy metals (cu, cd, and pb) over 96 h. the celomic fluid were subjected for facs analysis to find out the percentage difference of autofluorescent eleocytes cells between control and exposed species. the discrepancies between riboflavin content of control and heavy metal exposed worms were analyzed by spectrofluorescence. the low and negligible percentage of autofluorescent cells were recorded in e. eugeniae and l. mauritii but large numbers of autofluorescent cells were recorded in e. fetida and o. serrata. the experimental results show that riboflavin content and autofluroscence cells of the heavy metal exposed worms displays significant decrease in the celomic fluid. the present study clearly demonstrated that investigated species possess the significant population of celomocytes, but they differ considerably in the number of cells per body mass and by species. this non-invasive technique proves stable cellular biomarkers in earthworm for toxicological and biological soil monitoring studies. key words: earthworms; autofluorescence; heavy metals; riboflavin; facs introduction earthworms are ubiquitous and used as a model organism for many eco-toxicological studies. the interaction between earthworm and soil leads to increase in soil fertility and mobilization of heavy metals from soil to terrestrial ecosystem. the homeostasis properties of earthworm play a vital role/ significant role in maintaining the elemental concentration in the coelomic fluid. when the heavy metal concentration increases in the soil, it affects the homeostasis of the earthworm which leads to the excretion of heavy metals in the celomic fluid. the riboflavin storage is universal in earthworm ___________________________________________________________________________ corresponding author: muthukaruppan gobi department of biotechnology school of bioengineering srm university potheri 603203 tamilnadu, india e-mail: gobicc@gmail.com species, as it was detected both in attached chloragocytes forming the chloragogen tissue and in chloragocyte-derived eleocytes (mazur et al., 2011). according to (ottaviani, 2011), the celomocytes of all earthworm species contain amebocytes. immune system parameters may be used as a sensitive sublethal endpoint to assess the toxicity of atmospheric deposition to earthworms. the earthworm immune system is composed mainly of celomocytes, i.e., cells found within the fluids in the worm celomic cavity (stein et al., 1977). it has been demonstrated that many chemicals, including various trace elements, can adversely affect the immune system (fournier et al., 2000). the immunodeficiency of exposed species is interpreted as an indication of toxic effects of environmental contaminants (dales and kalac, 1992; fournier et al., 2000). the celomic fluid exhibits “molecules of many biological functions” (antimicrobial peptides and lysozymes) in annelids which provides effective 33 protection mechanisms against invaders. the composition of celomocytes in earthworm are species-specific, particularly in regard to autofluorescent eleocytes (koziol et al., 2006, plytycz et al., 2006). the chloragocyte and riboflavin inclusion in the earthworm are species specific and depends on the soil metal quality in lumbricidae (verdrengh and tarkowski, 2005; iwanaga et al., 2007). there was no previous report for celomocytes community of lampito, octochaetona and eudrilus worms. the aim of this work was to characterize the selected immune factors like cellular and riboflavin content. it was of interest to address the differences between immunological features of these four species, because although they share many similarities, their natural environment varies considerably. therefore, it was expected to discover some discrepancies between the immunological features of the four species. the differences of cellular levels and riboflavin content of control and heavy metal exposed worms were analyzed by facs and spectrofluorescence. furthermore, the variations of elements were quantitively measured by aas. materials and methods collection and identification of earthworm four different species of earthworms lampito mauritii, octochaetona serrata,(two anecics worms) eudrilus eugeniae and eisenia fetida (two epigeic)were mass cultured at our vermiary (400 800 lux light) and then these earthworms were identified based on the morphological characters. gut cleaning collected earthworms were washed with distilled water after which 50 ml jars were filled with 30 ml of 1.5 % agar gel prepared with deionised water. after getting cooled and solidified in the jars, the gel was taken out and cut into small pieces. after which earthworms were transferred separately into jars and kept for 96hrs at room temperature (400 800 lux light) to remove all the soil from their gut (pokarzherskii et al., 2000). harvesting of celomic fluid after removing the gut contents, diverse species of earthworms were cranked for 30 sec with a 4.5 v electric current to expel celomic fluid with suspended celomocytes through the dorsal pores, as described previously (plytycz et al., 2006). briefly, earthworms were placed individually in petri dishes containing 3ml of extrusion fluid (phosphatebuffered saline, pbs, supplemented with 2.5 g/l ethylenediamine tetraacetic acid, edta). freshly prepared 2 ml suspensions were used for spectrofluorimetry and flow cytometry analysis. further, celomic fluid was collected by above said method without pbs and edta. cell counting and viability cells viability were assessed by trypan blue exclusion test, mixing an equal volume of celomic fluid and 0.4 % trypan blue solution. cell viability should always exceed 90 %. collected celomic fluid was smeared on a clean glass slide, to observe the autofluorescence of celomic fluid cells. flow cytometric measurements and analysis a thin smear was prepared on a clean glass slide using collected celomic fluids and examined under olympus inverted fluorescence microscope. samples of celomocytes were analyzed with a bd facscalibur flow cytometer system. during scientific experiments, 10,000 threshold events per worm sample were collected and analyzed by their forward scatter (fs) (for cell size) and sideward scatter (ss) (cell complexity) properties. fluorescence fl1-h (emission 530 nm; excitation 488 nm) was recorded and resulting data were analyzed using winmdi 2.8 software, by producing dot plots of cell size versus fl1 autofluorescence. table 1 shows the species-specific characteristics of celomocytes in four earthworm species species n bw (g) tc (x 105) tc/bw (x 105/g) % afc afc/ bw (x 105/g) eisenia fetida 4 0.16 ± 0.00 3.4 ± 0.4 21.2 55.1 ± 0.07 344.3 lampito mauritii 4 0.54 ± 0.02 3.06 ± 0.3 5.6 18.8 ± 1.25 34.8 octochaetona serrata 4 0.51 ± 0.01 4.4 ±0.23 8.6 42.4 ± 0.97 83.1 eudrilus eugeniae 4 1.2 ± 0.07 1.6± 0.3 1.3 0.18 ± 0.09 0.15 means ± sd; n = number of individuals; bw = body weight; tc = total celomocytes; tc/bw = celomocytes /gram of body weight; % afc = percentage of autofluorescent celomocytes; afc/bw = percentage of autofluorescent celomocytes/gram body weight. 34 estimation of riboflavin by spectrofluorometer spectrofluorometric analyses were performed using the celomocyte lysate. different species of the earthworm celomocyte suspension lysates with 2 % triton. the supernatant was collected, and it was subjected to spectrofluorometric analyses with standard riboflavin (himedia laboratories). excitation spectra were recorded at 300 and 520 nm (excitation at λ = 525 nm) while emission spectra were recorded at 380 and 700 nm using excitation at λ = 370 nm. the spectrofluorometric signatures of unbound riboflavin were characterized by two maxima (at 370 nm and 450 nm) in the excitation spectrum, and an emission spectrum maximum at 525 nm. arbitrary units (au) of fluorescence were recorded using microsoft excel v. 2007. metal exposure metal chlorides (cu, cd, pb) were dissolved in double distilled water to a final 1mm metal concentration being equivalent of 63.5, 112.4 and 207.2 µg/ml respectively. dissolved metal chlorides were incorporated in 1.5 % agar gel, after cooling and solidifying this gel in the jars, it was taken out and cut into small pieces. the earthworms were then transferred to jars containing agar pieces and kept for 96 h and these agar pieces were made possible to consume easily by the earthworm. one group served as a control, and other three groups were exposed to a concentration of 63.5, 112.4 and 207.2 µg/ml, respectively. each level was tested in three replicates using ten animals. heavy metal analysis the concentration of heavy metals was analyzed in acid digested samples of earthworm by atomic absorption spectrometer as described by aqac (1999). heavy metal bioavailability to earthworm evaluated both in terms of relative toxicity and bioaccumulation factor (baf) (saxe et al., 2001). the baf was calculated as per equation; baf = [metal] earthworm/[metal] agar, where [metal] earthworm represents the total metal concentration of earthworm (mg kg), and [metal] agar represents the total metal concentration of sludge (mg kg-1). statistical analysis results were expressed as mean ± standard deviations. further data were analyzed using the one-way anova with post hoc tukey’s test. the level of statistical significance was set at p ˂ 0.05. results celomocytes the celomycytes of four different earthworms were calculated as per its body weight as shown in table 1. the low and negligible percentage of autofluorescent cells were recorded in e. eugeniae and l. mauritii. the high rate and the large numbers of autofluorescent cells were recorded in e. fetida and o. serrata. the celomocytes number per gram of body weight was found to be maximum in e. fetida (4.6x105/g), and it was recorded as 2.2x105/g in o. serrata, but it was 1.27x105/g, 3.0x105/g in l. mauritii and e. eugeniae respectively. it indicates the absence of a simple correlation between the body size/ weight and the number of celomocytes inhabiting the celomic cavity. flow cytometric analysis of eleocyte fluorescence microscopic observation of celomocytes from representative adults of four different species of earthworm revealed autofluorescent eleocytes and some adherent amebocytes which are devoid of autofluorescence. moreover, flow cytometry and fluorescent microscopy revealed the significant proportion of eleocytes in octochaetanea and e. fetida, while autofluorescent cells were very lower in lampito and eudrilus. flow cytometric analysis showed the high proportion of amebocytes in lampito and eudrilus. figure 1 shows the percentage of granular fig. 1 percentage of granular and eleocytes in four different species of earthworm at flow cytometric analysis. mean ± sd; significant p < 0.01. 35 figs 2 (a, b) riboflavin derived fluorescence spectra of celomocyte lysates from four different species of earthworm, au = arbitrary units of fluorescence intensity. mean ± sd; significant p < 0.01. and eleocytes of four different earthworms species. the forward scatter is an indicator of particle size (cell volume), whereas side scatter is an indicator of particle granularity (internal complexity) in the flow cytometer dot plot analysis. both measurements are obtained uniformly in the absence of fluorochrome molecule. the fluorochrome molecule is bound to the particles and get excited in to higher energy state and followed by the release of energy as a photon of light at a longer wavelength (lower energy state) which is known as stokes shift. density plots of the cell size versus cell complexity show that all the four different species of earthworm possess two distinct celomocyte populations i.e., agranular amebocytes and eleocytes. density plots of ssc-h versus fl1-h show that the latter exhibit very distinct fl1-h autofluorescence, arbitrarily assessed as that more intensive than 102 units on x-axis. the percentage of eleocytes are differ from species to species, and the ratio of eleocytes is directly proportional to granular cells. riboflavin content measurement by spectrofluorometry all earthworms e. fetida, e. eugeniae, l. maruitii and o. serrata possess riboflavin in celomocytes lysates. figure 2a shows emission spectra of four different species of earthworm celomic fluid and the comparison of the emission spectra of evocative samples of 1x105 celomocytes. the riboflavin content in the eleocytes is proportional to the peak fluorescence at 525 nm. a) b) 36 figs 3 (a, b, c, d) riboflavin-derived fluorescence spectra of control and heavy metal treated worm’s celomocyte lysates. au = arbitrary units of fluorescence intensity. emission spectra (kex = 370 nm) with peaks at 525 nm. the peaks at 525 nm is evident in each of them, albeit of different height in the order of e. fetida>> lampito spp>> octochatenea spp. >> eudrilus spp. it confirms, the richness of the riboflavin content in the eleocytes of eisenia, lampito and octochatenea inhabiting the unpolluted soil. the amount of riboflavin is proportional to the intensity of the emission band measured at 525 nm (expressed in arbitrary units, au). when compared to the control, the intenstity of riboflavin fluorescence found to be drastically reduced in eudrilus celomocytes. figures 3a, b, c, d displays the comparative riboflavin emission spectra of celomic fluids of four different species of earthworms. the experimental results show that that the emission spectrum occurs very high in e. fetida followed by lampito and octochaeta. the fluorescence spectrum observed from e. eugeniae is almost nonexistent, compared to control. the fluorescence emission spectrum of fluorophore characterizes the electron distribution of the molecule in the ground state. thus, it helps to identify the structure or the nature of the emitting molecule. a) b) c) d) 37 fig. 4 analysis of celomocytes of four different earthworms after 3 days exposure to heavy metal: a) comparative total riboflavin content; b) biomass of four different species of earthworm. mean ± sd; significant p < 0.01. figures 4a, b shows body weights of four different species were unaffected by the experiment days, and the mean biomass of four different worms was slightly decreased. one-way anova revealed that significant effect of metals on total number of celomocytes f value = 181.4; p-value = 0.000; p < 0.01), percentage of eleocytes set up by flow cytometry (p < 0.01) and eleocytes number (p < 0.01) and also riboflavin content in arbitrary units (au) established by spectrofluorometry (p < 0.01). results of post hoc t-tukey’s test revealed that all investigated parameters coelomocytes, eleocytes and riboflavin were consistently lowest in celomocytes from exposed worms. quantitative analysis of celomocytes flow cytometer analysis offers excellent information on the nature of the cells present in the earthworm celomic fluid and allows the identification of different cells structure which is capable of interacting with metal ions. all the earthworm was retaining two distinct population i.e., granular amebocytes (green) and autofluorescent eleocytes (red). predisposed eleocytes population were observed in the right of 102 units (figs 5 a, b, c, d) than in control. the autofluorescent intensity increases from left to right indicating a more intensive population of eleocytes in control than in heavy metal treated worms. figures 6, 7 shows the autofluorescent eleocytes of octochaetona was more biased towards the right than in control. but the eleocyte population is influenced toward left in heavy metal exposed worms indicating a reduction in the percentage of eleocytes. the dot plot intensity of granulocytes is more in control than in exposed worms. similarly, flow cytometry of lampito confirmed the presence of amebocytes and eleocytes, the latter exhibiting strong autofluorescence. the earthworms from control possessed a significantly higher percentage of autofluorescence value than their counterparts from the metalliferous soil. dot plot analysis of eudrilus revealed a significant reduction in the amoebocytes population in the exposed worms when compared to control. a) b) 38 control exposed figs 5 (a, b, c, d) analysis by flow cytometry in respect of percentages of celomocytes extruded from four different species of earthworms a) eisenia b) octochaeta c) lampito d) eudrilus. a) b) d) c) 39 fig. 6 influence of heavy metals on eleocytes in four different species of earthworm. mean ± sd; significant p < 0.01. figures 6, 7 depicts the influence of heavy metals on eleocytes and granulocytes in four different species of earthworm. heavy metal could, of course, have affected the riboflavin content, granulocytes and eleocytes percentage and viability of cells, which in turn could have affected the growth and reproduction indirectly. the present study also proves, cells variability depends on species-specific, and the individual differences in riboflavin content may reflect species-specific, nutritional preferences and vitamin b12 availability of the local resources. further, present study concludes that metal contamination of the soil caused their accumulation in the earthworm body including their coelomocytes which in turn affects their health and viability. figure 8 depicts the accumulated heavy metal contents of four different species of earthworm. the cr concentration was 0.36 ± 0.01, 0.34 ± 0.15, 0.27 ± 0.02, 0.23 ± 0.01 and cd concentration was 0.76 ± 0.02, 0.56 ± 0.02, 0.63 ± 0.15, 0.0.47 ± 0.20 in eisenia, lampito, octocheta and eudrilus respectively. their bioaccumlation factor (baf) values were 0.135, 0.168, 0.14 and 0.11. so the present study reveals that earthworm subjected to heavy metal (cd, cr and cu) exposure exhibited a higher accumulation of the heavy metals in the coelomic fluid. fig. 7 influence of heavy metals on granulocytes in four different species of earthworm. mean ± sd; significant p < 0.01. 40 fig. 8 bioaccumulation and baf of heavy metals in earthworm celomic fluid. mean ± sd; significant p < 0.01. discussion in the present study, celomocytes are collected from celomic fluid retrieved non-invasively from four different species of earthworm by electrostimulation was analyzed by spectrofluorimetry and flow cytometry. an astonishing divergence was detected in the four earthworm species with autofluorescent cells (eleocytes), and high number of celomocytes per body weight. plytycz et al. (2006) concluded that amebocytes with phagocytic properties are present in all species, but the presence of autofluorescent eleocytes, derived from the chloragogenous tissue, is species specific. as koziol et al. (2006) showed, the partial source of fluorescence in eleocytes is riboflavin, which is involved in immune responses in vertebrates (verdrengh and tarkowski, 2005), and is also considered responsible for balancing between earthworms and microorganisms present in soil (homa et al., 2010). content and composition of celomocytes, as well as riboflavin content, may vary not only between species but also within them depending on the presence of stress factors such as heavy metals (plytycz and morgan, 2011). in the present study, percentages of autofluorescent eleocytes were relatively stable, as well as the amounts of riboflavin measured in celomocyte lysates samples (plytycz et al., 2012). it is well established that riboflavin is essential for the proper functioning of the innate immunity of both animals (powers, 2003; verdrengh and tarkowski, 2005) and plants (dong and beer, 2000; asai et al., 2010; yoshioka et al., 2011), as well as being a signaling molecule in bacterial quorum sensing (rajamani et al., 2008; atkinson and williams 2009). riboflavin is considered as the chemoattractant for immunocytes in earthworms (plytycz et al., 2011) that putatively mobilizes the defense response during a disrupted balance of host and microbes/parasites induced by toxic factors (plytycz et al., 2006). the present work clearly showed that there is no correlation between body weight and riboflavin content. furthermore, a maximum decrease of riboflavin was recorded in the e. eugeniae than other species while exposed to heavy metals. and also it was observed that the physical nature of the celomic fluid of the four different species shows remarkably different in color. the celomic fluid of the e. eugeniae is color less where as other three species are brilliant yellow in color due to the presence phosphorescence pigments. this adaptation might be connected with very efficient riboflavin metabolism, because its amount in celomocytes of eisenia sp. is much higher than in another earthworm species (plytycz et al., 2006). from experimental results, it was observed that the body weight and the coelomic fluid does not correlate with respect to immunological defense point of view. fluorescence data shown in the present study indicates clearly that e. fetida, l. maruitii, o. serreta and e. eugeniae, display different molecular composition in their celomic fluid and thus have different metabolisms. therefore, interpretations of toxicological and immunological studies obtained on earthworms could be significantly different depending upon the different and the species interactions. the purpose of studying this parameter was to determine if the contaminant exposure reduced the cell defense system regarding cell survival. eisenia lampito octo eudrilus 0.168 0.135 0.14 0.11 41 many studies concerned the effects of environmental pollution, including heavy metals on earthworm immune functions mediated by celomocytes (scott-fordsmand and week, 2000; kurek et al., 2002). fugere et al. (1996) found that inhibition of the phagocytic activity of coelomocytes exposed to the heavy metal solution. plytycz et al. (2009) discovered that riboflavin content was reduced in the eleocytes of worms transferred from unpolluted to the metal-polluted soil. recent findings indicate that the riboflavin/lipofuscin balance may be a sensitive indicator of soil pollution intensity (cygal et al., 2007). according to plytycz et al. (2009), states that the heavy metal pollution strongly influences the riboflavin content in the immune competent cells of d. rubidus. the reduction of riboflavin content in the eleocytes cells leads to decrease in the fluorescence signal (riboflavin quenching) due to heavy metal binding. indeed, earthworms living in stress conditions are obligated to manage their energy, because they are constantly exposed to the high cost of maintaining homeostasis. these results are concordant with previous in vitro observations (sauve´ et al., 1998) showing that the immune function of celomocytes exposed to trace elements is impaired before the onset of cell death (brousseau, 1997). heavy metal accumulation depends on the exposure duration whereas the accumulation of cu, cd and cr is dependent upon the metabolic turnover (gobi, 2015). the use of celomic fluid for elemental analysis in earthworm offers many advantages on the commonly used whole-body measure, particularly for in field application. 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helminth infections k nava-castro1, s muñiz-hernández2, r hernández-bello3, j morales-montor3 1departamento de inmunología e infectología perinatal, instituto nacional de perinatología, secretaría de salud, méxico, d.f. cp 11000. méxico 2subdirección de investigación básica, instituto nacional de cancerología, secretaría de salud, méxico, d.f. cp 14080. méxico 3departamento de inmunología, instituto de investigaciones biomédicas, universidad nacional autónoma de méxico, ap 70228, méxico, d.f. 04510, méxico accepted july 25, 2011 abstract the physiological interactions during the course of the immune response to helminthes are complex. as our understanding of the neuroendocrine system grows, it has become increasingly clear that this complex network of neurotransmitters, hormones, and cytokines plays an important role in mediating immunity, in general, but in the case of helminthes this interaction among different systems is crucial. helminthes present a complex relationship in the host’s physiological systems, with neuro and hormonally dependent host factors such as sex, age, and the host physiological status correlated with parasite success. on top of the effect that this particular type of parasites may have on the invaded host, recent experimental evidence suggest that helminth parasites not only actively evade immune response, but are also able to exploit the hormonal microenvironment within their host to favor their establishment, growth and reproduction. the close interaction of the worm with the host’s homeostatic systems, the molecules produced by them, and the activation of immune mediated mechanisms to eliminate it, activate a complex neuroendocrine network, that produces strong behavioral changes in the infected host. understanding how the host’s neuroendocrine system can under certain circumstances favor the establishment of a parasitic infection opens interesting perspectives into the host parasite relationship field. this review focuses on the host-parasite neuroendocrine network activated by parasite worm infections. key words: neuroendocrine network; helminthes; worm; immunity; endocrine host-parasite relationship introduction the interaction of the nervous, endocrine and immune systems is crucial in the maintenance of homeostasis in vertebrates, and is absolutely vital in mammals. the capacity of the immune system to discriminate between self and non-self is based on a wide spectrum of specificity expressed by the immune system cells. this feature of the immune system implies that it can perceive an internal image of the organism’s components and react to the distortions of this image (such as transformed cells of the self). the immune response, as a homeostatic response under physiological control, ___________________________________________________________________________ corresponding author: jorge morales-montor departamento de inmunología instituto de investigaciones biomédicas u.n.a.m., ap 70228, méxico d.f. 04510 e-mail: jmontor66@biomedicas.unam.mx contributes to maintain the integrity of the body cells and tissues. hormones and neurotransmitters present in the immune cell microenvironment can restrict its autonomy, probably by acting on the receptors of these neuroendocrine factors. efficient communication among these three systems implies the existence of afferent and efferent pathways, constituting a complex feedback system. the alterations of this network trigger pathologies that involve its components (bottasso and moralesmontor, 2009; perez et al., 2009). in recent years, information on the multiple functions of the immune system has expanded remarkably. one of these functions has been biological adaptation through pathogens, such as helminthes, and its elimination from the organism. immune functions, in turn, require delicate control of the involved cells, which allow adaptation of the organism to the different physiological and 143 mailto:jmontor66@biomedicas.unam.mx pathological situations that it will face along life. to meet this end, interaction with the nervous and endocrine systems of the organism is necessary. this interaction is constant and makes the merged functioning of the three systems possible. this communication involves common messengers and receptors, simultaneously participating in a complex feedback system. alterations in communication among the three systems, lead to different pathologies. this is the case with the neuropsychiatric disorders that cause immunosuppression, such as depression (blume et al., 2011), and immune disorders that cause endocrinological problems such as hashimoto’s thyroiditis (tomer and huber, 2009) and diabetes mellitus type i (lehuen et al., 2010), both examples of the functional interaction between the immune and the neuroendocrine systems (wilder, 1995). numerous experimental data show that, as with other body cells, the cells of the immune system are influenced by the neuroendocrine system, which displays various control levels, from metabolism to cell division, regulated by hormones and neurotransmitters (jacobs et al., 2010; muñoz-cruz et al., 2011). the immune response is possibly the only physiological phenomenon in which the amplification of the response is based on cell proliferation and the specific transformation of its components. this process requires metabolic changes and growth factors, which make the immune response dependent on neuroendocrine control (fig. 1). it is well known that cd4+ t cells play an important role on the adaptive immunity against pathogens as well as on autoimmune diseases. also they are a crucial key for immunological memory. the activation phase of non-differentiated cd4+ t lymphocytes is determined by the specific recognition of antigenic determinants, which appear in the context of major histocompatibility complex class ii molecules (mhc-ii) and are expressed on professional antigen-presenting cells, such as macrophages, b or dendritic cells (zhu et al., 2010). the specificity of the immune response determined by cd4+ t lymphocytes is modulated by selective expansion of the clones capable of recognizing these antigenic determinants and, thus, of differentiating into helper cells (th1, th2, th17 and tregs) which contribute to the protection of the organism against infectious diseases (zhu et al., 2010). the classification of th1 and th2 was based on the specific pattern of cytokines they produce: th1 lymphocytes produce cytokines such as interleukin 2 (il-2), gamma interferon (ifn-γ) and the tumor necrosis factor alpha (tnf-α) are mainly involved in protection against intracellular pathogens through cell-mediated immunity, macrophage activation, and also in delayed hypersensitivity (cox et al., 2011). on the other hand, th2 lymphocytes produce interleukins il-4, il-5, il-6, il-10 and il-13, and regulate the humoral immune response through the proliferation of b lymphocytes and the change of the specific antibody isotype, beside promoting eosinophil and mastocyte differentiation (reviewed in wan and bramson (2001)). th17 lymphocytes, the third effector population of cd4 t cell, are characterized by producing il-17, il-21 and il-22 principally (nurieva et al., 2007). the t regulatory cells (tregs) are implicated on the control and regulation of particular subsets of cd4 t cells (zhu et al., 2010). however, both hormones and neurotransmitters have influence on immune cells, since they affect the production of several cytokines, and various products of the immune response, both th1 and th2, have a regulatory effect on the neuroendocrine system (fig. 1). the host-parasite neuroimmunoendocrine network the relationship between parasites (p), particularly helminthes, and their hosts (h), implies biochemical co-evolution and communication between their complex physiological and metabolic systems among themselves and with the environment, at all levels of biological organization (derijk and berkenbosch, 1991; grossman et al., 1991). hormones regulate a variety of cellular and physiological functions of organisms such as growth, reproduction and differentiation. hormones and immune actors are prominent in h-p relationships (klein, 2004). the comparatively sophisticated immune systems of vertebrates add complexity to h-p interactions. mammals sense and react with their innate and acquired immunological systems to the presence of a parasite and the parasite is also sensitive and reactive to the host’s immune systems effectors. host’s hormones are also involved in the modulation of the immune system’s protective or pathogenic functions and also on the parasite’s metabolism and reproduction (escobedo et al., 2005). host’s adrenal hormones are well known as immune modulators (loria et al., 1996), whilst sex steroids (estradiol, progesterone and testosterone) are recognized to also significantly affect the immune system’s functions (hughes and randolph, 2001; roberts et al., 2001). more recently, the ability of hormones to affect the immunological response directed against pathogenic agents, particularly helminthes, has gained attention (bottasso and morales-montor, 2009). this is clearly evident during various parasitic diseases including malaria (cernetich et al., 2006), schistosomiasis (morales-montor et al., 2008), toxoplasmosis (henriquez et al., 2009), cysticercosis (larralde et al., 1995), trypanosomiasis (brazão et al., 2009), leishmaniasis (snider et al., 2009), where strong hormonal regulation of the immune response has been described. however, other factors than the immunoendocrine response affect the course of a parasitic infection. a striking example of exploitation of host molecules is the ability of a number of parasites to use host-synthesized cytokines as indirect growth factors for the parasite (damian, 1997). the case of helminthes helminthes are estimated to include 18,000 to 24,000 species, and are divided into two subclasses 144 fig. 1 proposed neuroimmunological interactions that occur in higher vertebrates. in physiological conditions there is a crosstalk between neurological and immune systems of the host. external stimuli, such as infections, results in a th1/th2 systemic cytokine production of the immune response. in addition to, central nervous system (cns) is able to actively induce cytokines expression, which may affect the cns function. (touassem et al., 1992). nearly all trematodes are parasites of molluscs and vertebrates. the smaller aspidogastrea, comprising about 100 species, are obligated parasites of molluscs and may also infect turtles and fishes, including cartilaginous fishes (rosas-valdez and leon, 2011). the digenea, which constitute the majority of trematode diversity, are obligate parasites of both molluscs and vertebrates, but rarely occur in cartilaginous fishes. one-quarter of a billion people are infected with parasitic trematode worms worldwide (razomendivil and perez-ponce de leon, 2011). diseaseassociated symptoms occur in 120 million people, and 20 million people suffer from severe morbidity (cribb et al., 2002). cestoda is the class of parasitic flatworms, commonly called tapeworms that live in the digestive tract of vertebrates as adults and often in the bodies of various animals as juveniles (olson and caira, 1999). there are two subclasses in class cestoda, the cestodaria and the eucestoda. by far the most common and widespread are the eucestoda, with only a few species of unusual worms in subclass cestodaria. the cyclophyllideans are the most important to humans because they infect people and livestock (hoberg, et al., 1999). two important tapeworms are the pork tapeworm taenia solium, and the beef tapeworm t. saginata (levron et al., 2010). taennids, particularly taenia solium (causal agent of porcine cysticercosis and human neurocysticercosis) and taenia crassiceps (causal agent of murine cysticercosis) are highly evolved parasites that have developed diverse mechanisms of survival within the host that facilitate their establishment (hoberg, 2006). these mechanisms can be roughly grouped into two types. the first is evasion of the immune response by molecular mimicry or by inactivating effector immune processes (i.e., complement inhibition) (ludin et al., 2011). in the second mechanism, the parasite exploits the host system to its benefit in its establishment, growth or reproduction (long and boots, 2011). this exploitation mechanism provides 145 parasites with a dual benefit: first, obtaining amino acids for metabolism, and second preventing the surface-bound antibody from interfering with cytotoxic cells interacting with the parasite (long et al., 2011). effect of steroid hormones on helminth infections in last years, research has proved the influence of sex hormones in the immune system regulation (arteaga et al., 2002), and the idea of a neuroimmunoendocrine network was released. since then, investigations focused in the role of these hormones and their possible mechanism to intervene in the host susceptibility or resistance have grown (klein, 2000). it is well-known that males of vertebrate species tend to exhibit higher rates of parasites than females, and sex-associated hormones may influence immunocompetence. thus, sex hormones are hypothezised to lead to this bias (hoby et al., 2006). in this point, females have also been shown to have higher susceptibility to many parasitic infections, a finding particularly striking in helminth infections such as those produced by taenia solium (morales-montor et al., 2004) and trichinella spiralis (hernandez-bello et al., 2011). there are enough evidence about corticoids and their influence in the regulation of the immune response involved in parasitic infections (aly et al., 2010). however, there is recent data about their direct influence on the growth of the parasite, without an immune regulation. for instance, in a moderately resistant strain of mice, cysts of e. multilocularis developed into hydatid cysts in cortisone-treated mice (barnard et al., 1998). cortisone treatment significantly increased the average number of cysts, the average area of each cyst, and the total surface area occupied by cysts when compared with the untreated mice. collectively, the cysts in the treated mice occupied more of the surface area of the liver but less of the same area in the untreated mice (barnard et al., 1998). treatment of a e. multilocularis resistant mouse strain with cortisone drastically increased both the number of cysts and the average size of each cyst if the treatment occurred early in the infection. consequently, this treatment increases the susceptibility of mice to primary infections with e. multilocularis. based on these results, it could not be determined whether the increased susceptibility results from physiological or immunological effects caused by the cortisone treatment (hildreth et al., 2003). 146 these results are in agreement with studies of the nematode heligmosomoides polygyrus. in this infection, peripheral immune responsiveness in male laboratory mice was reduced by infection with the parasite. responsiveness was also lower among high-rankers or aggressive males regardless of infection status. reduced responsiveness on infected animals and high rankers was associated with elevated serum corticosterone concentration among high-ranking males (perkins et al., 2008). although glucocorticoids have a stimulatory effect on the initial cell proliferation phase of t lymphocytes, and thus some elevation might have been expected on this account, the change in corticosterone concentration during the infection phase was the best hormone-measure predictor of eventual worm burden. the negative relationship between the immune status and high corticosteroids levels is more in keeping with the later impact of glucocorticoids on the secretion of th2 cytokines and thus depression of the th2 arm of the immune response. this is consistent with effects of glucocorticoids in prolonging intestinal nematode infections, increasing the susceptibility of rodent hosts to h. polygyrus and depressing the expression of acquired resistance to h. polygyrus. there was no testosterone-dependent increase in parasite burden among high rankers in this experiment, perhaps because resistance to the parasite relies on a different emphasis on the th1 and th2 arms of the acquired immune response (barnard et al., 1998). schistosomiasis is another example in which steroids play an important role in the host susceptibility (kurtis et al., 2006). the disproportionately high intensity and prevalence of schistosome infection in children, compared with adults, has been documented for decades, so understanding the mechanisms of this naturally occurring protection may guide efforts to develop a vaccine for schistosomiasis (kurtis et al., 2006). then, the importance of the hormonal changes during pubertal development, including increases in the levels of the adrenal hormones dhea-s (dehydroepiandrosterone sulfate) and dhea (dehydroepiandrosterone) may have part of responsibility for the dramatic reduction in age susceptibility to schistosome infection. another evidences that points out to the relationship between increasing pubertal development, dhea-s levels, and resistance to schistosome infection are: (1) in mice, exogenous administration of dhea-s leads to decreased schistosome worm burdens after challenge infection, thus dehydroepiandrosterone sulfate treatment of mice modulates infection with schistosoma mansoni; (2) dhea-s kills larval and adult parasites in culture at physiologic concentrations; (3) in another 2 cross-sectional studies increased dhea-s levels are associated with decreased intensity of infection in humans; and (4) increased dhea-s levels are associated with decreased intensity of re-infection after treatment with praziquantel (pzq) (kurtis et al., 2006). dhea-s is known to have potent immunomodulatory activities, including upregulation of th2-driven antibody isotypes and down-regulation of pro-inflammatory cytokines. finally dhea-s could mediate resistance through a direct anti-parasite effect. but also via innate immune mechanisms, such as host skin thickness or fat deposition, then capitalizing on these mechanisms for vaccine development will be difficult. however, if dhea-s mediates resistance via enhancement of acquired protective immune responses, then vaccine strategies designed to induce and augment these protective acquired immune responses, including hormonal adjuvants, may be promising (kurtis et al., 2006) (fig. 2). fig. 2 regulation of cytokine production in th1 and th2 lymphocytes by dhea. the differentiation from th0 towards th1 or th2 is simetric, each one controls a unique type of immune response and increases the development of cells of the same subclass while suppress the expansion and effector functions of the other subtype. dhea can control th1/th2 balance by inducing the development of one subtype and inhibit the other subtype. dhea effects could depend on concentration, having a dual effect. also, dhea has been demonstrated to have direct helminticidal effects on different parasites, without mediation of immune response. finally, the prediction of male biased parasitism was tested in free ranging chamois (rupicapra rupicapra), which are infested intensely by gastrointestinal and lung helminthes. male chamois had a higher output of gastrointestinal eggs and lungworm larvae when compared to females. male biased parasitism originating in sex related hormone levels was confirmed for the elevated output of lungworm larvae, but not for the gastrointestinal nematodes. the faecal output of lungworm larvae was significantly correlated with androgen and cortisol metabolite levels. the immunosuppressant effects of these hormones may explain the greater susceptibility of males to infection by parasites and developing disease. the stress of the rutting season with elevated glucocorticoid levels is hypothesized to reduce humoral antibodies, and to enhance larval output of nematodes in males. however, it should be considered that the subset samples were collected predominantly around the period when androgen levels between sexes differ most significantly. in contrast to the output of lungworm larvae, the male bias in quantitative gastrointestinal nematode output was not significantly correlated with sex differences in steroid levels (hoby et al., 2006). for instance, a strong negative correlation was found between sex and adrenal steroid hormone circulating levels and some proinflammatory cytokines in microfilaremic women. plasma samples from amicrofilaremic women contained higher concentrations of testosterone and estradiol than those from microfilaremic ones. testosterone was also negatively correlated with il6 and estradiol with ifn-γ. the fact that cortisol concentrations were not elevated in women with filariasis may be related to the chronic nature of the disease (mavoungou, et al., 2005). another research that focused on the immuneendocrine system relationship in an helminth infection, is a study designed in rodents infected with strongiloides ratti, in which a sex-related differences in host susceptibility was previously known. in this infection, male mice were more susceptible to s. ratti and the difference was seen in migrating larvae. it has been shown that natural immunity against migrating larvae of strongyloides ratti is regulated by macrophages. in the small intestine, host mast cells were related to adult worm expulsion (watanabe et al., 1999). according to this study, the sex-related difference are clearly mediated by testosterone during the migration of larvae, suggesting that testosterone renders mice 147 susceptible to migrating larvae by modulating their natural defense mechanisms (watanabe et al., 1999). so, steroid hormones produced by the adrenal cortex, such as dhea-s and cortisol, influence the intensity of the immune response during s. mansoni infections and have been implicated among the most important host factors controlling the onset, establishment, and pathogenesis of schistosomiasis. these hormones inhibit oviposition by s. mansoni both in vitro and in vivo. in vivo, the increased numbers of worms, larger number of eggs and more vigorous hepatic granulomas can be related to the lack of circulating glucocorticoids, whose presence in some way ameliorates the inflammatory immune response in the liver. the effect of adrenalectomy produced high levels of infection and more severe pathology, a fact that can be related to the well-known glucocorticoid antiinflammatory effect. low levels of cortisol could promote vigorous granuloma formation and the production of cytokines necessary for schistosome reproduction, such as tnf-α. the immunosuppresive effects of hydrocortisone and dexamethasone are counteracted by dhea, suggesting a tightly controlled balance in the secretion of these hormones to regulate the inflammatory response. an intriguing question is how the lack of adrenal hormones affects the infection in the parasitized host. host derived candidates that could possibly be affected by adrenalectomy are the interleukins (il’s), which are known to be altered during schistosomiasis. potential endocrinological-immunological mediators of this process are il-1, il-6, tnf-α and macrophage migratory inhibitory factor, all known regulated by adrenal steroids. the changes produced in the infected adrenalectomized host could thus be cytokine mediated. further work could elucidate the mechanism by which adrenalectomy induces changes in the immune function during disease progression and could establish causal links, if indeed they exist (morales-montor et al., 2004). trichuris muris infection is an ideal model for defining t-cell-driven immunity, and also provides essential insights that may impact on potential helminth therapies currently in development. the female-associated hormone 17-β estradiol (e2) significantly enhanced the generation of a th2 response in vitro (hepworth et al., 2010); however, this stimulatory effect was found to be dispensable for the generation of immunity to trichuris in the gender-biased il-4 ko mouse model (hepworth, et al., 2010). these mice are compromised in their ability to generate an efficient th2 response, necessary for parasite expulsion, as they lack il-4 a key th2 polarizing cytokine. in addition, female il-4 ko balb/c mice mount an unusually delayed th2 response, associated with t-cell and accessory nk cell-derived il-13 and an associated decrease in the levels of the pro-inflammatory cytokines tnf-α and il-6, which combine to mediate worm expulsion (hepworth et al., 2010). conversely, male littermates are unable to expel the parasite and retain high adult worm burdens, a phenotype found to be dependent on il-18. in contrast, the maleassociated hormone dihydrotestosterone (dht), significantly inhibited the t-cell stimulatory capacity of dc and directly suppressed the immune response of male il-4 ko mice, with worm expulsion restored following castration (hepworth et al., 2010). this finding was associated to a dramatically reduced il-18 mrna expression, suggesting that androgens may act via this cytokine to suppress th2 immunity to trichuris muris infection (hepworth et al., 2010). behavioral changes in the infected host several behavioral changes that are induced by infections with parasites have been described. for instance, there are sexual changes in body morphology as well as sex-related behavioral changes in crabs when parasitized with a rhizocefalan, through mechanisms that are still obscure but could involve changes in the hormonal pattern of the host (cited in larralde et al., 1995). taenia taeniformis is also known to alter reproduction in rats by interfering with sex-steroids (lin et al., 1990). perhaps the most studied helminth that induces strong hormonal, and behavioral changes is the helminth parasite taenia crassiceps. male mice infected with t. crassiceps show remarkable changes in sexual behavior, characterized by a complete loss of the ejaculation response early at the infection (six weeks), followed by a gradual decrease in the number of mounts and intromissions, and their latencies increased, until none of the parasitized mice showed any sexual response toward female mice (morales et al., 1996). moreover, it was demonstrated that alterations in sexual behavior were due to the change in the normal production of sex-steroids by the mouse, since the testosterone or dihydrotestosterone restitution of infected male mice, showed a complete restoration of their sexual behavior (morales-montor et al., 2002). since c-fos and progesterone receptor (pr), both are key estradiolregulated genes involved in the regulation of sexual behavior, we studied possible changes of c-fos and pr expression in the central nervous system (cns) of infected male mice. indeed, c-fos and pr expression oscillated with time of infection and to different magnitudes in hypothalamus, brain cortex and preoptic area but neither in other areas of the brain nor in several other organs of the host (morales-montor et al., 1999, 2004). furthermore, infection disrupts the dominantsubordinate status (gourbal et al., 2002). in infected male mice strong perturbations in territorial behavior and aggressiveness were found. in addition, during confrontation between naive infected and healthy mice, infected animals more often assumed a subordinate status than healthy ones. the effects of the infection by t. crassiceps were more likely to prevent adult male mice from becoming behaviorally dominant than to reverse existing dominance relationships (gourbal et al., 2002). significant cns changes in c-fos, and progesterone receptor (pr) expression during infection signifies the brain senses the infection 148 fig. 3 chart of the proposed host-parasite neuroimmunoendocrine network. sex steroids may act directly upon the parasite whereby progesterone (p4), and oestradiol (e2) favor its reproduction. also, sex steroids act upon parasite via the immune system, e2 favoring a permissive th2 response that inhibits the restrictive th1. the brain of the host shows changes in pr, that could be the result of the high oestrogens levels that reveal that it senses and may react to the infection. also, the outcome of the feminization process is shown, that directly affects the sexual behavior of the infected male mice, through out the binding of sex steroids and/or neurosteroids to pr. the arrows show the interconnection among all system components. (+) positive stimulation; (-) negative stimulation. episode and may be involved in the ensuing behavioral changes of the infected mice, as well as, through its connectivity, extend the effects of infection to other physiological systems under its influence. that these changes in cns are beneficial to the host or parasite remains speculative (morales-montor et al., 1999, 2004). one could argue that feminization of male hosts favors the parasite by allowing its reproduction, however it is equally arguable to consider feminization of the male host as deleterious to the parasite’s completion of its cycle since there is a reduction of male exposure to its predators, the definitive hosts. other similar mutually conflicting statements may be elaborated with the above premises, the true ones remain to be identified and could perhaps vary with each different host-parasite relationship (fig. 3). not only male mice are behaviorally affected by cysticercosis, female mice also suffer perturbations in their sexual behavior, i.e., receptivity to the male, as well as disruption of the estrous cycle (arteaga et al., 2010). concluding remarks until some years ago, the immune system was perceived as a system isolated from other body systems. the present review makes evident that the immune and neuroendocrine systems share numerous ligands and receptors, which results in a constant bidirectional communication. in fact, it has been postulated that an important function of the immune system is to serve as a sensory organ for cognitive stimuli that pass unnoticed to the nervous system, as could be infectious agents, such as parasites. our present proposal is to reintegrate an important system to the physiological context of the whole organism. this will doubtlessly lead to an improved understanding of physiology, and generate changes in modern medical practice. for further understanding of the bidirectional communication process between the immune and neuroendocrine systems, it will be necessary to continue the search for ligands and receptors common to both systems, and to examine in depth 149 the similarities and differences in their functional regulation. in addition, it will also constitute a challenge for physiologists to integrate this information to the context of the whole organism during helminth infections. on the other hand, new information about immunoneuroendocrine interactions in infected hosts will help us to design novel therapies for the treatment and diagnosis of human diseases of apparently immune or endocrine origin. we have documented here that a complex interactive network involving the immune, endocrine and nervous systems, is in control of the parasite growth, reproduction and establishment. if such complex a management of the parasite loads, as that we shown here between different hosts and worms, extends to other parasite diseases of mammals, as current research seems to indicate in a number of helminthes infections, their means of exploration, fuller understandings and forms of control must be reviewed and approached with designs matching in complexity and plasticity that of the infections. the evidence presented above illustrates the complexity and importance of neuroimmunoendocrine interactions during cysticercosis and provides clues to the many other possible mechanisms of parasite establishment, growth and reproduction in an immunocompetent host. further, strong neuroimmunoendocrine interactions may have implications in the control of transmission and treatment of this parasitic disease in porcines and humans. in practical importance, the complexity of the cysticerci-host relationship suggests that all physiological factors (i.e., sex, age) should be taken into account in the design of vaccines and new drugs. acknowledgements financial support was provided by grant # in 214011-3 from programa de apoyo a proyectos de innovación tecnológica, dirección general de asuntos del personal académico, (papiit, dgapa), 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purpose of this study was to investigate metal contamination in tissues and changes in metallothioneins (mt) in respect to its redox status in mya arenaria clams collected at three polluted sites. the phosphorylation state of mt was also investigated to determine whether this state is changed in clams collected at heavy-metal contaminated site and its involvement in cytoprotective signaling during stress contamination. the results show that clams collected at least one of the three polluted sites presented significantly higher concentrations of silver (ag), arsenic (as), cobalt (co), copper (cu), mercury (hg), nickel (ni), tin (sn) and lead (pb) in tissues. in the visceral tissue, total mt levels and the reduced, metal-binding form of the protein were significantly induced at the sites. the phosphorylation of mt and mitochondrial activity, as determined by electron transport and cytochrome c oxidase activities, were also significantly reduced at the contaminated sites. reduced phosphate levels in mt were negatively correlated with total mt levels, suggesting that decreased phosphorylation was involved in kinase-mediated signaling during cellular stress and could possibly alter the protein’s affinity to confer cytoprotection against heavy metal contamination. these preliminary investigations revealed that the phosphorylation state could change in polluted environment and provide some clues on the modulation of binding affinities during heavy-metal and oxidative stress in clams. key words: phosphorylated metallothioneins; mitochondrial electron transport activity; cytochrome c oxidase; heavy metals; mya arenaria introduction metallothioneins (mt) are low-molecular-weight (6–7 kda) metal-binding proteins. they are ubiquitous in life forms, from bacteria to mammals, as well as being rich in cysteine (25–30%) and heatstable owing to a lack of aromatic amino acids and hydrophobic regions (klaassen et al., 1999). these characteristics make them efficient transitional metal-binding proteins that are capable of binding ag(i), au(i), cd (ii), co(ii), cu(ii), hg(ii), pb(ii) and zn(ii). moreover, it was demonstrated that mt not only binds metals but also has the capacity to sequester reactive oxygen in the oyster (andersen et al., 1999) and nitrogen species such as superoxide anion and nitric oxide (atif et al., 2006). ___________________________________________________________________________ corresponding author: françois gagné fluvial ecosystem research section environment canada 105 mcgill st., montreal, quebec, canada h2y 2e7 e-mail: francois.gagne@ec.gc.ca upon reacting with these radicals, the metal thiolate clusters of mt oxidize, liberating metals from the protein (kang, 2006; gagné et al., 2008). hence, mt exists in both a reduced metal-binding form and in an oxidized metal-releasing form in the cytoplasm of cells. in contaminated mya arenaria populations exhibiting lower clam-bed density, growth and increased mean age values, marked responses in mt, oxidative stress and gonad size were observed (blaise et al., 2003). the same was also found in freshwater mussels, where cd concentrations in the high-molecular-weight protein fraction, where mt intervenes to reduce the metal content of these fractions, was the biomarker response that was most frequently and strongly correlated with the population variables (perceval et al., 2004). the mt biomarker is therefore considered a relevant biomarker of stress and has predictive value at higher levels of biological organization. recent studies suggest that ser/thr protein kinases (protein kinase c, or pkc) can phosphorylate 22 fig. 1 clams were collected at four sites (identified by stars) in june 2007. the site baie du moulin à baude (bau-r) was considered as the reference site; anse saint-jean (asj-e), baie sainte-catherine (bsc-t) and baie éternité (be-m) were the pollution-impacted sites. mt in neuronal cells (aras et al., 2009). pkc is a family of enzymes that are involved in controlling the function of other proteins through the phosphorylation of hydroxyl groups of aliphatic ser and thr amino acids. indeed, zn-induced metalresponsive element activation was significantly decreased in neuronal cells expressing a recombinant mt-1 devoid of its phosphorylation site, while cells expressing normal mt-1 had enhanced expression (aras et al., 2009). this suggests that pkc signaling could influence the phosphorylation of mt, which could, in turn, change its affinity for zn. however, it was recognized that the phosphorylation of proteins causes them to be degraded by the atpdependent ubiquitin/proteasome pathway. these target proteins become substrates for particular e3 ubiquitin ligases only when they are phosphorylated. it is interesting to observe that ubiquitin is activated by the formation of a thioester bond between the cterminal carboxyl group of ubiquitin and a (metal sensitive?) cys residue of the e1 enzyme, a process requiring atp as an energy source. at present, the function of mt phosphorylation is unclear despite its apparent involvement in pkc signaling and possible ubiquitinylation proteasome tagging. in another study, mt was seemingly involved in protein phosphorylation signaling: the re-activation of cysteine proteinase like caspases or other phosphatases. to show this, mt-iii prevented the activation of caspase-3 and -9 and the release of mitochondrial cytochrome c to the cytoplasm in neuronal cells (kim et al., 2009). mt-iii also increased the activation of akt, a ser/thr protein kinase, the phosphorylation (and degradation) of ikappa b, which can activate either an inflammatory or immune response, a cell survival response or cellular proliferation. these studies reveal that the phosphorylation state of mt might provide some clues about its involvement in cell signaling during cellular stress. this study represents the first attempt to track changes in mt-phosphate levels in m. arenaria clam populations obtained at polluted sites. the levels of total phosphate bound to mt and the proportion of oxidized/reduced mt collected at three sites contaminated by heavy metals were determined. the implication of mitochrondrial respiration (metabolic activity) was also examined by tracking mitochondrial electron transport and cytochrome c oxidase activities, the terminal enzyme 23 http://en.wikipedia.org/wiki/ubiquitin http://en.wikipedia.org/wiki/proteasome http://en.wikipedia.org/wiki/ubiquitin fig. 2 change in mt characteristics in clams from the st. lawrence estuary and saguenay fjord. clams were analyzed for mt in the visceral mass tissues. the total and phosphate-bound mt (a) and the redox status of mt (b) were determined. before the formation of atp, which is the main source of reactive-oxygen species formation in cells. an attempt was made to examine the influence of mt phosphate level, total mt and redox status on clam’s condition status. materials and methods spatial survey and clam collection soft-shell clams (mya arenaria) were handcollected at low tide in the morning from three pollution-impacted sites and one reference (i.e., under no direct source of pollution) site located in the saguenay fjord and the st. lawrence estuary (figure 1). in the saguenay fjord, the anse-saintjean site is located 40 km upstream from the estuary and receives the primary-treated (screened) effluent of about 2,000 residents (asj-e). the baie éternité site, which is located 15 km farther upstream, is historically recognized to be contaminated by heavy metals (be-m) (blaise et al., 2002). in the st. lawrence estuary, the baie saintecatherine site was chosen because it is heavily impacted by local commercial and pleasure-boat traffic from sightseeing and whale-watching operations, and has displayed a history of contamination by organotin (bsc-t) compounds and heavy metals (gagné et al., 2005). the baie du moulin à baude site is located 3 km downstream along the north shore of the st. lawrence estuary; it was selected as the reference site because of the absence of any direct source of pollution (bau-r). a total number of 30 clams were collected from clam beds at each site during early morning low tide. the clams were processed for biomarker analysis. clam age was estimated by the number of major grooves on the shell. clam weights and maximum longitudinal shell length were determined. the soft and gonad tissues were dissected out at 4 oc to determine the gonadosomatic index (gsi: wet weight of gonad/wet weight of soft tissues), condition factor (cf: clam weight/shell length) and growth index (gi: shell length/age). gender was determined by microscopic examination of gonad smears at 400 times magnification. parasitism in gonad tissues was seldom observed; any infected clams were discarded when present. the clam tissue samples were then frozen using dry ice for transportation to the laboratory and stored at -85 oc until analysis. after thawing on ice, the visceral tissues containing the gonad were crushed with a teflon pestle tissue grinder and homogenized in a 50 mm hepes-naoh buffer, ph 7.4, containing 150 mm nacl, 10 µg/ml apoprotinin and 0.5 mm dithiothreitol at a 1:5 volume/volume ratio (five passes). the dithiothreitol concentration was selected to stabilize the homogenates against mt oxidation during tissue processing (minkel et al., 1980). samples (100 µl) of each homogenate were collected for total protein determinations (bradford, 1976). the remaining homogenates were centrifuged at 1,500 x g (20 min at 2 oc) and the supernatant was centrifuged at 10,000 x g for 20 min at 2 oc to isolate the mitochondria in pellet. the mitochondria were resuspended in two volumes of the homogenization buffer. the remaining supernatant was centrifuged at 15,000 x g (20 min at 2 oc) for mt characterization, as described below. all biomarkers were normalized with total protein levels in the corresponding fractions. heavy metals and metalloids (i.e., ag, al, as, cd, co, cr, cu, hg, ni, pb, sn, and zn) were analyzed in whole soft tissues (three pools of ten individuals per site) according to standard methods of the national laboratory for environmental testing (nlet, 1994). the data were expressed as ng of metals or metalloids/g of dry weight. metallothionein characterization the levels of total, oxidized and reduced mt were determined using a modified version of the spectrophotometric method of viarengo et al. (1997), as described elsewhere (gagné et al., 2008). the original methodology uses a series of 24 solvent fractionation steps to isolate the mt fraction from high-molecular-weight proteins and peptides such as glutathione. the modification includes a step for the complete reduction of mt (with the highly potent reducer tris[2carboxyethyl]phosphine) in the s15 fraction, which was stabilized at the homogenization step using a small amount of dithiothreitol, which was not sufficient to further reduce mt in the homogenates (minkel et al., 1980). the s15 samples were divided between two tubes for total mt and metallic (reduced) mt evaluations. for the former, 200 µl of the s15 fraction was pre-treated with 50 µl of 50 mm tris(2-carboxyethyl)phosphine (sigma chemical company, mo, usa) for 30 min before the addition of an acidic ethanol/chloroform solution and subsequent ethanol fractionation steps to obtain the total level (reduced and oxidized) of mt. for the metal-binding (reduced) mt form, 200 µl of the s15 fraction was mixed with 50 µl of water alone with no reduction step. a sample of the mt pellet was set aside for total phosphate determinations according to the phosphomolybdate methodology of stanton (1968) after treating the mt pellet in 1 m naoh for 30 min to liberate inorganic phosphate. the data were expressed as µmol of thiol (gsh) equivalents/mg total protein for total and reduced mt or as µg phosphates/mg total proteins in the s15 fraction for phosphate mt. the oxidized fraction of mt was calculated as follows: mtoxidized = mttotal (phosphine treated) mtmetallic. analysis of the fractionated mt samples by high-resolution polyacrylamide gel electrophoresis with coomassie blue staining revealed a major band around 10 kda that is characteristic of mt, with no contaminating high-molecular-weight protein bands apparent. mitochondrial activity energy expenditures were determined by measuring mitochondrial electron transport (met) activity (smolders et al., 2004). met activity was determined in the mitochondrial fraction (resuspended 10,000 x g pellet) using the piodonitrotetrazolium dye method (king and packard, 1975; smolders et al., 2004). briefly, 100 µl of the resuspended mitochondrial pellet was mixed with 100 mm tris-hcl, ph 8.5, containing 100 µm mgso4, 0.1% triton x-100 and 5% polyvinylpyrrolidone for 1 min on ice. the reaction mixture was mixed with 1 mm and 0.2 mm nadh and nadph, respectively, on ice. the reaction was initiated with the addition of 50 µl of 5 mm piodonitrotetrazolium for 30 min at 20 oc. absorbance readings were measured at 15-min intervals at 520 nm. the data were expressed as the loss of absorbance at 20 oc/30 min/mg total proteins in the mitochondria. cytochrome c oxidase (ccox) was determined by a spectrophotometric cytochrome c oxidation assay (sakai et al., 1988). data analysis a total of 12 clams were analyzed for each site. the homogeneity of variances was determined using bartlett’s test. where the data proved to be heterogeneous they were log-transformed. the data were subjected to an analysis of variance using the benferroni t test for comparison of the biomarker data against the reference site (i.e., bau-r). a pearson product-moment analysis was performed to determine if there were any correlations within the metal and biomarker of effects data. factorial and discriminant function analyses were also performed to examine the interrelatedness among the data, the biomarkers and the metal loads in tissues in order to discriminate among and to examine the sites. significance was set at p<0.05. all statistical tests were performed using statistica software (version 8). results based on the investigation on the primary sequences of mt from various species in public medline database (pubmed), the sequences reveal the substantial presence of serine (ser) and threonine (thr), which can be potential aliphatic phosphorylation sites (table 1). the proportion of {ser+thr} amino acids represents about 17% of the amino acids in various species, ranging from protozoans to mammals. in bivalves, the proportion of {ser+thr} represents between 14% (m. edulis) to 18% (dreissena polymorpha) of the amino acids of the mt protein. the highest proportion was found in rainbow trout (oncorhynchus mykiss) mt-1 with 23% of the amino acids as either ser or thr residues. the proportion of {ser+thr} residues was significantly correlated with the proportion of cys (r=0.78; p<0.01) and independent of the total number of amino acids of mt. moreover, ser and thr residues were often located in the vicinity of cys residues, frequently forming -cys-ser-cysor –cysthr-cyssequences. this suggests that mt could undergo phosphorylation and perhaps influence the protein’s capacity to bind heavy metals and/or reactive oxygen species, which is well-established in mts. the investigation of mt sequences also revealed that these proteins contains lysine residues which are possible ubiquitinylation sites. indeed, mt typically has 10% lysine residues, which suggests possible ubiquitinylation sites (table 1). the proportion of lysine appears to be independent (i.e., not correlated) of the proportion of ser+thr, of cys and of the total number of amino acids in the various species of mt. clams were harvested at one reference and one polluted site in the st. lawrence estuary (baur and bsc-t, respectively) and two sites located upstream in the saguenay fjord (asj-e and be-m, respectively). in an attempt to characterize the quality of each site, total heavy-metal contents were determined in m. arenaria clams (table 2). the data revealed that as and co were significantly increased at the bsc-t sites while cu was elevated at be-m sites (p<0.05 level). cr was significantly higher at the asj-e and be-m sites. ni loads in the clam tissues were significantly higher at site asj-e. pb was significantly higher at all sites relative to the reference site bau-r. mercury, pb and sn were significantly higher at the bsc-t site, already known to be contaminated by organotin compounds (viglino et al., 2006). zn was significantly reduced at this site. a correlation analysis revealed that ag was significantly correlated with cd (r=0.67; p<0.001), cu (r=0.68; p<0.001) and hg (r=0.89; p<0.001). tissue 25 table 1. characteristics of mt in different species. organism species proportion of ser and thr amino acids1 proportion of lysine residues2 proportion of cysteine (%) total amino acids protist tetrahymena pyriformis 16 14 29 107 mussels mytilus galloprovincialis 15 7 26 72 psychotria viridis 16 5 28 75 mytilus edulis 14 10 25 73 clams dreissena polymorpha 18 8 28 73 musca lusoria 14 12 28 76 gastropods helix pomatia 16 12 24 67 nematodes caenorhabditis elegans 11 13 25 63 fish oncorhynchus mykiss 23 12 31 61 perca fluviatilis 20 10 33 60 human homo sapiens 20 11 31 61 mean±sd 16.6±3.4 10.4±2.7 28±3 71±13 1 potential phosphorylation sites as estimated by the percentage of number (ser+thr)*100/total number of amino acids of mt. 2 potential ubiquitinylation sites as calculated by the percentage of lysine residues: number lys (*100)/total number of amino acids of mt. al content was significantly correlated with as (r=0.44; p<0.05), pb (r=0.4; p=0.05) and sn (r=0.83; p<0.001). tissue as levels were significantly correlated with co (r=0.61; p=0.001), cr (r=0.51; p=0.01), cu (r=0.52; p<0.01) and pb (r=0.66; p<0.001). cd levels in tissues were significantly correlated with cr (r=0.53; p<0.01), cu (r=0.53; p<0.01), zn (r=0.72; p<0.001) and hg (r=0.66; p<0.001). co tissue levels were significantly correlated with pb (r=0.78; p<0.001) only. cr levels were significantly correlated with cu (r=0.47; p<0.05) and zn (r=0.47; p<0.05). tissue cu levels were significantly correlated with sn (r=-0.44; p<0.05), zn (r=0.66; p<0.001) and hg (r=0.63; p=0.001). tissue sn levels were significantly correlated with hg (r=-0.50; p=0.01). zn tissue levels were significantly correlated with hg (r=0.44; p<0.05). it is noteworthy that most correlations were positive, with the exception of sn levels in tissue, which were negatively correlated with metals such as ag, cu and hg. clam condition factor (cf) varied significantly across all sites (anova p<0.001). the cf was significantly reduced (1.3-fold) at the bsc-t and asj-e (1.5-fold) sites. no significant change was observed in cf at the be-m site (table 3). the gi was significantly reduced by 1.2 at only one site (bsc-t). gsi also significantly affected (anova p<0.001) at one site and was significantly increased 1.4-fold at the be-m site. a correlation analysis revealed that cf and gsi were significantly correlated (r=0.31; p<0.05). correlations between biomarkers of effects were included in table 4 while correlations with the heavy metal tissue loadings were cited in the text. clam cf was significantly correlated with tissue ag (r=0.51; p=0.01), cu (r=0.51; p=0.01) and hg (r=0.57; p<0.05). the growth index was negatively correlated with tissue co (r=-0.42; p<0.05) and sn (r=-0.4; p=0.05) levels and positively correlated with tissue cu (r=0.48; p<0.05) and zn (r=0.50; p=0.01) levels. the gsi was not correlated with any of the metal burdens in tissue. the levels of mt in the clam gonad/visceral mass were determined (fig. 2a and b). the results revealed that the total levels of mt were significantly affected at the polluted sites (fig. 2a). total mt levels decreased at bsc-t while they were significantly elevated at the be-m site. at the asj-e site, only a marginal increase was observed (p=0.07). the phosphate levels of the mt fraction were significantly reduced at the bsc-t and be-m sites, while a marginal decrease was found at site asj-e (p=0.06). a correlation analysis revealed that total mt was negatively correlated with the amount of phosphate bound to mt (r=-0.69; p<0.001) and positively so with as (r=0.50; p=0.05), cu (r=0.83; p<0.001), zn (r=0.79; p<0.001) and hg (r=0.49; p=0.07). the phosphate associated with mt levels was negatively correlated with as (r=0.65; p<0.01), co (r=-0.61; p<0.01), cu (r=-0.50; p<0.05) and pb (r=-0.64; p<0.01). the redox status of mt was also appraised (fig. 2b). the proportion of the reduced and metal-binding forms of mt rose significantly at all three polluted sites. the proportion of reduced metal-binding mt was significantly correlated with phosphorylated mt (r=0.43; p=0.05) and cr (r=0.47; p<0.05). this indicates that mt phosphorylation follows the formation of oxidized (metal-releasing) mt at the expense of the metal-binding (reduced) fraction of mt. 26 table 2. heavy-metal content of m. arenaria clams from the st. lawrence estuary and saguenay fjord. metals (µg/g) bau-r bsc-t asj-e be-m ag 0.212±0.02 0.044±0.006 0.122±0.022 0.36±0.14* al 91±13 122±24 96±6 109±5 as 0.63±0.04 0.83±0.02* 0.77±0.001 0.96±0.1* cd 0.08±0.008 0.06±0.003 0.09±0.002 0.094±0.009 co 0.115±0.02 0.2±0.01* 0.12±0.009 0.18±0.006* cr 0.4±0.09 0.48±0.08 0.8±0.05* 0.74±0.05* cu 1.2±0.05 0.7±0.06* 1.16±0.07 1.65±0.2* ni 0.37±0.04 0.39±0.05 0.51±0.03* 0.46±0.04 pb 0.03±0.001 0.06±0.005* 0.04±0.002 0.05±0.002* sn 0.02±0.02 0.117±0.02 0.043±0.02 0.03±0.02 zn 14±1 10±1* 14±1 15±0.5 hg 0.05±0.005 0.027±0.004* 0.03±0.001 0.064±0.01* * indicates statistical significance from the reference site at p<0.05 (anova and least-square-difference tests). table 3. values of morphometric parameters at four study sites. site bau-r bsc-t asj-e be-m parameter condition factor (cf) 0.67±0.02 0.54±0.01* 0.44±0.01* 0.70±0.02 growth index 9.3±0.3 7.6±0.1* 9.2±0.3 8.8±0.3 gsi 0.08±0.01 0.09±0.01 0.080±0.003 0.110±0.004* sex ratio (1= all males; 2= all females) 1.5±0.1 1.67±0.14 1.75±0.10 1.6±0.2 * indicates significance at p<0.05 level. the influence of metabolic activity and oxidative stress on the expression of mt was determined by following mitochondrial electron transport activity (a measure related to oxygen production in mitochondria) and ccox, the last enzyme complex involved in electron transport activity (fig. 3a and 3b). met activity was readily reduced at the asj-e (2.8-fold) and be-m (2.3-fold) sites. met activity was significantly correlated with phosphates bound to mt (r=0.58; p<0.01), co (r=0.51; p<0.05), cr (r=0.64; p<0.01), cu (r=-0.54, p<0.05) and marginally correlated with ni (r=-0,43; p=0.09). the activity of ccox was also affected in mitochondria (fig. 3b). its activity was significantly decreased at the bsc-t (1.6-fold) and be-m (2.2-fold) sites, significantly correlated with phosphorylated mt (r=0.50; p=0.05) and marginally so with redox mt (r=0.41; p=0.07 for oxidized mt). ccox was not significantly related to any of the measured metals in tissues. the various tissue biomarker and metal loads were analyzed using factorial and discriminant function analyses to identify the major biomarkers that could explain most of the data responses and identify site-specific characteristics (fig. 4a and 4b). a factorial analysis revealed that most of the variance (60%) was explained by three factors. the biomarkers with the highest factorial weights were ag, cu, hg, pb, sn, zn, cf and total mt. these metals formed a cluster close to total mt, cf, growth and gsi (fig. 4a), indicating that these metals were statistically related to diminished clam condition and altered gonad development. phosphorylated mt was located at the opposite side of this cluster and was closely located with ccox activity and met endpoints. a discriminant function analysis revealed that all sites were well discriminated at 100% accuracy (fig. 4b). the first root function was able to discriminate between sites be-m and asj-e, with the following biomarkers having the highest factorial weights: cf, mt-p and total mt. the second root function well discriminated the reference site bau-r from the other sites, with the following biomarkers having the highest factorial weights: cf, mt-p and tissue pb levels. these analyses revealed that the phosphorylation state of mt is an important factor relating to clam morphological status and health condition. 27 table 4 correlation analyses of the measured endpoints. total mt oxidized mt reduced mt mt-p met ccox gsi growth cf total mt 0.25 -0.25 -0.68 -0.17 -0.33 0.51 0.22 0.67 p>0.1 p>0.1 p=0.001 p>0.1 p=0.1 p<0.01 p>0.1 p<0.001 oxidized mt 0.40 0.15 0.44 -0.14 0.04 0.42 p=0.05 p>0.1 p<0.05 p>0.1 p>0.1 p<0.05 reduced mt -0.40 -0.15 -0.44 0.14 -0.05 -0.42 p=0.05 p>0.1 p<0.05 p>0.1 p>0.1 p<0.05 mt-p 0.58 0.50 -0.56 0.05 -0.43 p<0.01 p<0.05 p=0.001 p>0.1 p=0.01 met -0.04 -0.1 -0.19 0.1 p>0.1 p>0.1 p>0.1 p>0.1 ccox -0.41 0.08 -0.32 p<0.05 p>0.1 p=0.09 gsi 0.09 0.31 p>0.1 p<0.05 growth 0.11 p>0.1 discussion the phosphorylation state of mt in clam tissues appears to be related to the catabolism of mt in cells. our data support this hypothesis since mtphosphate was negatively related with the total amount of mt in cells. the increase in mt phosphate was also related to the proportion of oxidized mt in cells. since mitochondria are the principal source of reactive oxygen species in cells (abele et al., 2002), we examined their activity by tracking electron transport and ccox activities. the data revealed that mt-phosphate was closely related to changes in both met and ccox activity in tissues, suggesting that 1) mt phosphorylation is coupled in some way with mitochondrial electron transport activity and/or 2) mt is oxidized by increased mitochondrial respiration rates and then phosphorylated for removal, perhaps through the ubiquitin/proteasome pathway. the latter hypothesis was consistent with the observation that mtphosphate was negatively related to total mt levels and positively related to oxidized mt, met and ccox activities. protein phosphorylation is a prerequisite for tagging proteasome degradation and ubiquitin requires lysine residues for binding to the phosphorylated proteins which are found in various mt sequences of different species (table 1). however, this would have to be verified by more specific experiments (i.e., finding ubiquitylated mt in bivalve tissues by western blot analysis using antiubiquitin antibodies). it is noteworthy that the decrease in mt-phosphate was associated with clams with lower cfs and altered gonad weights. clams contaminated by metals from polluted sites had decreased phosphate bound to mt with a concomitant elevation in the metal-binding form of mt and total mt levels. this indicates that clams from metal-contaminated sites increase the levels of mt for metal sequestration, perhaps at the expense of oxygen radical scavenging. this was corroborated by a previous study in which the metalbinding form of mt was more closely associated to oxidative stress and tissue damage than was the oxidized form of mt (gagné et al., 2008). perhaps this explains, at least in part, why met and ccox activity tended to be lower at the polluted sites. moreover, it has long been established that phosphorylation plays an important role in controlling mitochondrial metabolism, where a delicate balance between electron transport for respiration and protection against the production of reactive oxygen species is required (foster et al., 2009; sokolova et al., 2005). met and related enzyme complexes involved in respiration (ccox) would release reactive oxygen species that are less sequestered by mt and thus likely to do damage. this was consistent with increased lipid peroxidation in the gonadal homogenates of clams at the polluted sites in this study (results not shown). it is noteworthy that sn in tissues were negatively related to ag, cu and hg in clams. according to the factorial analysis, sn tissue loadings were relatively close to the mt-phosphate and ccox cluster. a possible explanation for would be that sn competes with ag and hg to cu (and zn) binding sites. moreover, organotin compounds are well-known to have a high affinity for hemoproteins which could decouple electron flow in mitochondria (simionatto et al., 1984). to the best of our knowledge, this is the first report of the modulation of mt phosphorylation in clam populations from polluted sites as determined by increased heavy-metal tissue loadings. ser and thr protein kinases are also ubiquitous in bivalves (dailianis and kaloyianni, 2004). the external face of 28 fig. 3 mitochondrial electron transport activity in selected clam populations. mitochondrial electron transport (a) and cytochrome c oxidase (b) activities were measured in isolated mitochondria from feral clam populations. asterisks (*) indicate significance at p<0.05 the outer mitochondrial membranes is filled with various intracellular receptors and serves as a site for signals implicated in steroidogenesis, cytoprotection and energy demands, where pkcinteracting proteins are found (smith et al., 2006; poole et al., 2004). this family of protein kinases is also involved in zn signaling in neuronal cells, which confers tolerance towards zn2+ and suggests that pkc acts directly on the intracellular source of zn and the expression of zn-regulated genes such as mt (aras et al., 2009). in mt, a protein kinase c phosphorylation site was identified at serine 32 regardless of the species examined. moreover, mt phosphorylation appears to modulate the zn-binding efficiency of mt, as shown by the reduction in zn2+induced metal-responsive element in cells expressing a mutated mt that is devoid of a phosphorylation site. this was explained by the phosphate-associated mt, which lowers the binding affinity for zn and favoring the subsequent release of zn2+ for the activation of metal-responsive elements and the production of de novo zndependent genes such as mt. our data revealed an inverse relationship between phosphate-bound mt and total mt/metal-binding mt, which suggests that decreased mt phosphorylation might have intervened in the mt forms in handling metal homeostasis in clams from metal-contaminated sites. in another study, copper-induced mt-1 transcription was regulated by pkc and a metal transcription factor (mattie and freedman, 2004). the role of mt in the maintenance of redox homeostatis appears to recall other protein phosphatases such as protein kinase b, leading to ikappa b degradation which, in turn, activates the expression of nf-kappab activity during the inflammatory response and oxidative stress (kim et al., 2009). the inhibition of several intracellular protein kinases such as protein kinase a and c, mapk and calmodulin kinase-ii eliminated the neuroprotective effect of mt, highlighting the interplay between metal-binding capacity, oxidative stress and protein phosphorylation (asmussen et al., 2009). in mytilus galloprovincialis, cd and zn caused an increase in superoxide anion production, with cd being more potent (2.2-fold) than zn (1.5fold) (koutsogiannaki et al., 2006). in addition, the metal effect was reversed in the presence of calphostin, a pkc inhibitor, and an amiloride analogue that blocks na/k-exchanges. conversely, cd induced pyruvate kinase activity and a rise in intracellular ph was augmented by phorbol esters, a potent activator of pkc (dailianis and kaloyianni, 2004). this is in agreement with the increase in metal affinity in less phosphorylated-mt hypothesis, which confers cytoprotection to cd and zn but perhaps at the expense of radical oxygen scavenging. taken together, these studies reveal a hormone-like effect of divalent metals such as cd and zn at the β-adrenergic and pkc signal transduction pathways in m. galloprovincialis. however, gsi was not correlated with any of the biomarkers in this study suggesting perhaps that hormonal signaling involved in gametogenesis (i.e., steroids) had no important effects on the phosphorylation state of mt. we should bear in mind that these sites were not only contaminated by heavy metals but with organic contaminants such as polyaromatic hydrocarbons which could also produce oxidative stress and perhaps influence the phosphorylation state of mts. more research is required to better understand the physiological role of mt phosphorylation in the handling of heavy metals and oxygen radicals in bivalves. in conclusion, m. arenaria clams from heavy metalcontaminated sites show increased total and metalbinding mt with decreased met and ccox activities. total mt and the metal-binding (reduced) form of mt were closely related with levels of ag, cu, hg and zn in tissue. moreover, mt phosphorylation was also reduced at the contaminated sites (less-phosphorylated mt would have a stronger metal-binding capacity), suggesting 29 http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22dailianis%20s%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22kaloyianni%20m%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus fig. 4 factorial and discriminant function analyses of the biomarker responses. the factorial analysis was performed with the principal component extraction procedure (a). the discriminant function analysis was also performed to examine the capacity of the biomarkers used in this study to identify the sites (b). the involvement of pkc signaling pathways that can change the metal-binding affinity of mt towards exposure to various metals. the measurement of phosphates bound to the mt fraction might provide some clues on the hormone-like effects of metals and the modulation of binding affinities towards metal contamination of the environment. acknowledgements the authors thank sophie trépanier for her technical support with the biomarker analyses. this work was funded by environment canada 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press, washington dc, usa, pp 127–150, 1987. 31 http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22freedman%20jh%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus caenorhabditis elegans minireview isj 9: 58-63, 2012 issn 1824-307x minireview someone like it hot? effects of global warming on insect immunity and microbiota m mandrioli department of biology, university of modena and reggio emilia, modena, italy accepted march 14, 2012 abstract global warming represents a substantial challenge on a broad range of organisms with diverse life-history traits and geographical distributions. up till now several studies correlated global warming to changes in body mass, growth rate or fat content, whereas the effects on immune function and microbiota composition remained almost unexplored. on the contrary, some pioneering studies are showing that increased temperature may influence the insect immune function and the microbiota composition, making global warming in a pivotal position influencing insect survival and adaptation to a warming planet. key words: global warming; immunity; microbiota composition; thermal tolerance; symbionts       58 living in a warming planet temperature is considered one of the most important ecological factors for ectothermic organisms and the ability to tolerate temperature fluctuations is essential for individual survival (overgaard and sørensen, 2008). consequently, global warming may pose a substantial challenge on many natural systems and in particular for tropical ectotherms, living close to their upper critical thermal limits, making them particularly vulnerable to global warming (sala et al., 2000; thomas et al., 2004). insects are among the groups of organisms most likely to be affected by climatic changes because climate has direct influences on their development, reproduction and survival (bale et al., 2002; savage et al., 2004; frazier et al., 2006; menéndez, 2007). nevertheless, insects have short generation times and high reproductive rates, so that they can respond quicker to climate change than long-lived organisms, such as plants and vertebrates (bale et al., 2002; menéndez, 2007). warming can therefore potentially affect several aspects of insect life-cycle and ecology, and potential responses could include changes in phenological patterns and habitat selection and the expansion/contraction of geographic and altitudinal ranges (bale et al., 2002; menéndez, 2007; berg et al., 2010). ___________________________________________________________________________ corresponding author: mauro mandrioli department of biology, university of modena and reggio emilia, via campi 213/d, 41125 modena, italy e-mail: mauro.mandrioli@unimo.it global average surface temperature increased by about 0.6 ºc during the past century, and the third ipcc report predicts that temperatures will continue to rise during the next century, with increases of up to 5.8 ºc by the year 2100 (houghton et al., 2001). the study of how these human-induced changes in climate may affect biodiversity has attracted a vast research effort during the last two decades in order to study the ecological impacts of current warming on a broad range of organisms with diverse life-history traits and geographical distributions (menéndez, 2007). due to the importance of insects for human health and activities (such as agriculture), several studies focused their attention on insects showing that in temperate regions, climate warming is predicted to benefit many insect species since less severe winter months could results in higher overwintering survival and increase in the population sizes (bale et al., 2002; botkin et al., 2007). moreover, climate warming could lengthen the growing season resulting in increased growth, reproduction rate and number of generations per year. the effects could be hampered in cool climate zones, where climate warming could increase insect fitness by bringing them closer to their physiological optima (bale et al., 2002; botkin et al., 2007). is therefore global warming always beneficial for insects? what could happen to tropical insects that already live close to their optimal temperature? can climate warming decrease their fitness by exceeding the physiological optima? aphids, for instance, are not always able to adapt physiologically to high temperatures since they are already living close to their upper temperature limit for survival (neve et al., 2009; hazel et al., 2010). as reported by chiu et al. (2012), global warming seems to affect the fitness of the aphid myzus varians altering several physiological functions, including immunity, development and reproduction. in particular, more than 90% of m. varians nymphs reached adulthood in the temperature regimes with a daily mean temperature of 28.8 and 30.0 °c, whereas no nymphs reached adulthood at 32.5 °c. at the same time, the mean fecundity of aphids reared at 28.8 °c was greater than that for aphids reared at 30.0 °c, showing that even a small increase in mean temperature from 28.8 to 30.0 °c could cause a decline in the fitness of m. varians so that aphid populations could go extinct locally and populations will not rebound even when temperatures become favourable in the fall (chiu et al., 2012).     59 up till now several studies correlated global warming to changes in some aspects, such as body mass, growth rate or fat content as fitness-related parameters (bale et al., 2002; botkin et al., 2007), whereas the effects on immune function and microbiota composition remained almost unexplored. in view of this assumption the present paper has been focussed mainly on the effects of warming on insect immune function and microbiota composition, parameters that could play important roles in insect survival and adaptation to a warming planet. insect immunity and global warming insects present an immune system endowed with only innate immune components consisting of cellular and humoral factors (mandrioli et al., 2003; nappi et al., 2004; malagoli et al., 2007, 2010). cellmediated immunity includes phagocytosis and encapsulation, exerted by specific cell types (ballarin et al., 2008), while humoral mediators comprise several factors among which the antimicrobial peptides (amps) and the components of the pro-phenoloxidase (pro-po) cascade have been the most elucidated (bulet and stöcklin, 2005). it is important to observe that cellular and humoral components have not to be considered as separate elements, because several findings indicated that secreted factors are fundamental for clotting and pathogen recognition and engulfment (bulet and stöcklin, 2005). the maintenance and deployment of an efficient immune response may shift away resources from other functions (such as reproduction), so that immune function results from physiological trade-offs in insects (bonneaud et al., 2003; schmid-hempel, 2003, 2005; rolff et al., 2004). in view of its cost, the immune response is influenced therefore by both biotic and abiotic factors (such as food availability and temperature) (le moullac and haffner, 2000; mydlarz et al., 2006; de block and stoks, 2008; karl et al., 2011). according to these assumptions, the predicted increase in thermal stress due to global warming (diffenbaugh et al., 2005, 2007) is likely to induce cascading effects on other functions such as the fig. 1 global warming can positively influence immunity (by enhancing the activity of some molecules), reproduction (favouring increased egg deposition, faster grow and over-wintering survival) and resource availability (for instance by making faster grow plants). however, global warming also induces heat stress that has costs that could reduce the resources available for reproduction and immune responses. immune response, thus further reducing the individual fitness and favouring the distribution and prevalence of infectious diseases (lafferty, 2009; travers et al., 2009). in order to verify this hypothesis, karl et al. (2011) investigated in the tropical butterfly bicyclus anynana the effects of temperature changes on fitness-related adult traits (such as body mass and fat content) and on phenoloxidase (po) activity and hemocyte numbers that are two key parameters for evaluating the immune function at both cellular and humoral levels. interestingly, results on body mass and fat content suggest that global warming could be beneficial, whereas haemocyte numbers and po activity decreased at increasing temperatures (karl et al., 2011) supporting the hypothesis that immune parameters were negatively affected by global warming. at higher temperatures, insects may therefore increase their rate of growth and reproduction, but may become more susceptible to diseases, leading to reduced lifespan and possibly reduced fitness in the field (figure 1). at the same time, karl et al. (2011) observed that the decrease in po activity with increasing temperature was more evident in food-deprived individuals, in respect to butterflies having access to food showing a trade-off relating energy shortage and immune response. even if the molecular mechanism at the basis of this change in the po activity has not been studied, a direct link between the expression of heat shock proteins and a decrease in immune parameters could be     60 suggested. interestingly, two mediators of the response to stress (norepinephrine and glucocorticoids) are known in mammals to act as immune-suppressors thus decreasing disease resistance (sternberg, 2006). the study of the trade-off between immune response and thermal tolerance is absolutely relevant in medicine and agriculture since if both immune function and reproduction are simultaneously enhanced, then climate change will result in the reproduction of more insects that will be more resistant to diseases. as a consequence, we will have more pest crop insects in our fields with more damages for agriculture, together with larger numbers of insects relevant for the transmission of human diseases. on the contrary, if higher temperatures induce or exacerbate trade-offs between reproduction and immune function, in order to face the global warming insects have to define if put their resources mainly on reproduction or on the immune systems. a possible reply to this question can be obtained from studies performed in the cricket gryllus texensis, where empirical evidence suggested that warmer temperatures lead to a decline in some immune functions (e.g. melanisation) (suwanchaichanda and paskewitz, 1998; adamo and lovett, 2004). in particular, it emerged that g. texensis may become more susceptible to some pathogens at higher temperatures suggesting that reproductive rate and immune function are not simultaneously enhanced at higher temperatures. a further confirmation of the presence of a strict trade-off between immune response, reproduction and temperature has been observed by an interesting set of data published by adamo and lovett in 2004. in particular they reported that elevated temperatures resulted in increased egg laying, faster egg development and greater mass gain in gryllus texensis. in the same experiments a reduction in the resistance to the gram-positive bacterium bacillus cereus was observed both above or below the average field temperature (26°c) suggesting that increased temperatures induce trade-offs between reproduction and disease resistance for some species–pathogen interactions (adamo and lovett, 2004). interestingly, these results also explain the choice of ecological niches by g. texensis that prefers temperatures lower than those corresponding to the optimal reproductive output, but that assure the presence of an efficient immune response. the ecological immunity could suffer global warming bacteria commonly interact with insects in intimate associations known as symbioses, where symbionts increase host fitness (for a review see russell and moran, 2006). several evidences suggested that symbiotic bacteria present in the insect gut resulted to be involved not only in the degradation of specific substances in the food (brummel et al., 2004), but also in other complex interactions protecting the host from invasion by pathogenic microorganisms (a process known as “colonization resistance”) and modulating the insect immune system (dillon and charnley, 1996; ryu et al., 2008). microbiota seems therefore to act in insects (and actually not only in insects) as a sort of ecological immunity or extended immune system being able of affecting the efficiency of the host immune system and limiting the accumulation of pathobionts (ottaviani et al., 2012). according to this proposal, germ-free locusts died prematurely due to infection of pathogens, such as pseudomonas aeruginosa, penicillium spp. and bacillus subtilis (charnley et al., 1995; dillon and charnley, 1996, 2005), suggesting that they are more susceptible to infection than normal insects and that the gut microbiota exerted a protective function out-competing potentially harmful organisms (dillon et al., 2005). the involvement of microbiota in the host immune protection can vary during insect life, since the composition of the bacterial community that populates the insect gut is not stable, but can change during lifespan due to variation in the nutritional composition of the food and the aging process (deveale et al., 2004). in a recent paper, chiu et al. (2012) reported a low survival of nymphs of the aphid m. varians at high temperatures as a consequence of the elimination of endosymbionts, such as buchnera (as previously suggested by ohtaka and ishikawa, 1991). this effect probably results from a temperature-mediated decrease in aphid endosymbionts, which synthesize amino acids essential for their insect hosts (chen et al., 2009). in the last years, different roles have been suggested for symbionts other than the synthesis of amino acids only (russell and moran, 2006). buchnera might, for instance, play a key role in aphid thermal tolerance since endosymbionts code for heat shock proteins, which deter degradation of host protein secondary structure (dunbar et al., 2007). secondary endosymbionts, such as serratia simbiotica, play a similar role in the thermal tolerance of their host strengthening the ability of aphids to evolve further adaptations to overcome the impacts of warming (russell and moran, 2006). buchnera are at least partly able to survive at high temperatures because of constitutive expression of genes that are normally up-regulated in response to heat and aphids could be able to thrive under temperatures as high as 35°c in the laboratory (dunbar et al., 2007). surprisingly, a single nucleotide deletion in the buchnera ibpa gene encoding for a small heat-shock protein virtually eliminates the transcriptional response of ibpa to heat stress and lowers its expression even at cool or moderate temperatures (dunbar et al., 2007). in the presence of this mutant allele, a short heat exposure in juveniles has strong effects on aphids that produce few or no progeny and contain almost no buchnera, in contrast to aphids bearing symbionts without the deletion. the ibpa mutated allele has appreciable frequencies in field populations supporting the view that lowering of ibpa expression improves host fitness under some conditions (dunbar et al., 2007).     61 as previously suggested, the response to stress (including thermal stress) is part of a large trade-off that relates stress response to reproduction and immunity. this mutation by switching off the response to heat stimuli could favor aphid reproduction and immunity. however, the prolonged permanence of aphids at high temperatures (for instance in hot summer with daily mean temperature of 32.5 °c) results in the elimination of buchnera reducing not only the thermal tolerance of aphids, but also their fecundity since the lack of endosymbionts results in a lost synthesis of amino acids essential for the hosts (chiu et al., 2012). according to these results, global warming could be difficultly faced by aphids in tropical regions due to buchnera symbiont depletion. interestingly, in the presence of low density of primary symbionts, secondary symbionts (such as hamiltonella defensa, s. symbiotica, regiella insecticola) could be more present affecting not only the aphid thermal tolerance to high temperatures, but also their symbiont-based immune response (poirié and coustau, 2011). the effects of global warming on the composition of aphid microbiota are of particular interest since, as recently reviewed by poirié and coustau (2011), the immune deficiency (imd) signalling pathway was apparently non functional in aphids and no genes coding for peptidoglycan recognition proteins (pgrps) and several wellconserved antimicrobial peptides, such as defensins and cecropins, have been predicted in the pea aphid acyrthosiphon pisum genome (gerardo et al., 2010), making the microbiota-based immunity essential to protect the host against natural enemies (poirié and coustau, 2011). global warming: threat or opportunity? global warming is a well-studied phenomenon referring to the current temperature of earth’s atmosphere and oceans and it is responsible for climate-driven habitat changes that could influence insect survival and distribution. according to some proposals, the predicted increase in the earth temperature could benefit many insect species since they will face less severe winter months resulting in higher over-wintering survival and increase in the population sizes. moreover, climate warming could lengthen the growing season resulting in increased growth, reproduction rate and number of generations per year. even if these hypotheses are intriguing, the scenario could be more complex in view of the existence of different trade-offs that balance the cost of reproduction and stress and immune responses. as suggested by some pioneering studies, global warming could have detrimental effects not only on insect immune system, but also on the composition of their microbiota making insects more vulnerable to pathogens. the occurrence of increasing temperature could therefore exacerbate the tradeoffs between reproduction and immune function, making few resources available for disease resistance. the study of trade-offs related to global warming has an important value not only from a biological point of view, 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2009. global warming: threat or opportunity? s isj 8: 179-189, 2011 issn 1824-307x review how gene expression profiles disclose vital processes and immune responses in mytilus spp. s domeneghetti1*, c manfrin2*, l varotto1, u rosani1, m gerdol2, g de moro2, a pallavicini2, p venier1 1department of biology, university of padua, padua, italy 2department of life sciences, university of trieste, trieste, italy *equal contribution accepted september 08, 2011 abstract gene expression studies largely support the understanding of gene-environment interactions in humans and other living organisms but the lack of genomic and genetic information often complicates the analysis of functional responses in non-traditional model species. nevertheless, the fast advancement of dna microarray and sequencing technologies now makes global gene expression analysis possible in virtually any species of interest. as regards the mytilus genus, tens of thousands expressed sequence tags (ests) are currently available for m. californianus and m. galloprovincialis, and dna microarrays have been developed. among them, immunochip 1.0 specifically includes 1,820 probes of genes centrally involved or modulated in the innate immune responses of the mediterranean mussel. this review recalls peculiarities and applications of the existing mussel dna microarrays and finally summarizes facts concerning a variety of transcript sequences likely involved in the mussel immunity. beside dna microarrays, next generation sequencing (ngs) technologies now offer new and broader research perspectives, from the whole transcriptome coverage to the mytilus genome sequencing. key words: mytilus; dna microarray; innate immunity; ests; antimicrobial peptides; c1q introduction global gene expression analyses in organisms selected to represent a given ecosystem currently support ecotoxicological investigations and create a conceptual bridge between the early organism responses and late population changes (steinberg et al., 2008). the animal response to a variety of detrimental conditions usually starts with alarm signals followed by adjustment reactions aimed to neutralize the physiological unbalance, and may end up in a general decline of vital processes ultimately marked by disease and death. depending on the stress type and exposure intensity, the expression of definite sets of genes makes available specific proteins and other molecules in cells and tissues. appeared in the 1990s, the dna microarray technology enables the simultaneous expression ___________________________________________________________________________ corresponding author: paola venier department of biology university of padua via u. bassi, 58/b, 35121 padua, italy e-mail: paola.venier@unipd.it measure of thousands of genes represented in the microarray platform by unambiguous polynucleotide probes (schena et al., 1995; lockhart et al., 1996). the gene expression profiles emerging from suitable sampled cells or tissues can provide a dynamic view of biological processes and allow the correct sorting of different functional states. based on the availability of sequence data, dna microarrays can be used to solve a variety of biological questions: from the identification of molecular markers pathognomonic of disease and transcriptional signatures of various stress factors to the understanding of complex phenomena such as the epigenome in normality and disease (martínsubero and esteller, 2011). specific microarray platforms and advanced deep sequencing technologies now support studies on the cellular functions of micrornas and their role in human diseases (thomas et al., 2010). leading research institutions are currently using both the mrna and mirna expression profiling to examine the genomic responses to environmental stresses (nct). central to the toxicogenomics studies is the concept of ‘phenotypic anchoring’ which recalls the 179 fig. 1 number of pubmed publications including the terms "dna-microarray" (blue line) or "mytilus" (purple line) from 1995 to 2010. has the dna microarray revolution reached its peak? importance to correlate the observed gene expression changes to adverse effects defined by conventional parameters of toxicity and pathology. in the controlled vocabulary of the natl. library of medicine, the term ‘dna microarray’ is indexed under the following category which indicates the large application range of such innovative technology (mesh): oligonucleotide array sequence analysisthe hybridization of a nucleic acid sample to a very large set of oligonucleotide probes, which are attached to a solid support, to determine sequence or to detect variations in a gene sequence or expression or for gene mapping. relevant to the gene expression profiling research area is gene expression omnibus, a public repository that archives and freely distributes microarray, next-generation sequencing, and other forms of high-throughput functional genomics data submitted by the scientific community (geo). to fulfil the current standards (minimum information about a microarray experiment) the contents submitted to geo should include the following: raw hybridization data; normalized data from which the main experimental findings can be outlined; description of the tested samples and whole experimental design, with details on the biological and technical replicates; identity and location of all probes and controls of the microarray platform, with external reference in the case of commercial arrays; concise but precise description of laboratory and data processing protocols related to the experiment under submission. according to the aims of the microarray gene expression data society, dating back to the late ‘90s, the compliance to the miame standards should assure the data comparability among different platforms and testing protocols while supporting common work criteria and the reduction of random data variation (rogers and cambrosio, 2007). based on the comparative data analysis, the guidelines for standardization and reporting have been further refined (chen et al., 2007; shi et al., 2008). at present, geo contains as much as 9,000 platform records which can be accessed and browsed in full detail. figure 1 illustrates the annual increase of pubmed records including the term "dnamicroarray" or "mytilus" (subject heading or title/abstract) and suggests that pioneering technologies open the way to new ideas more than an unconventional model organisms. in fact, the gene expression profiling field has substantially diversified: specialized equipments and various related software make today the dna microarrays powerful tools for the study of gene sequence, structure and expression, particularly for the best known model organisms. nonetheless, one must remember that transcription is just one step in gene expression, and post transcriptional events referred to maturation of the primary transcript, rna editing and rna silencing as well as various modifications of the translation products overall influence the final amounts and activity of cellular proteins. mytilus dna microarrays: preparation strategy and applications six geo records refer to mussel dna microarrays at july 2011. mytarray 1.0 (geo platform gpl1799, oct 2006) is composed by 1,712 cdna probes, univocally tagging the 3’-end region of transcripts from the main tissues of adult mussels (mytilus galloprovincialis) and 46 unrelated cdna control probes, all printed in duplicate and twice per slide (1.7 k mussel probes per array, 7.0 k total probes per slide). the probes were designed in the 3’-utr, 180 fig. 2 work diagram referred to the competitive hybridization of two dye-swap-labelled samples on a cdna microarray with two-channel detection of the fluorescence signals (modified from gibson and muse, 2004). one among the least conserved gene regions, so that competition of different mrnas from genes with similar coding sequence and cross-hybridization to the same microarray probe should be minimal. also, the probe size of 400 800 bp is expected to ensure comparable efficiency in the amplification and spotting of the cdna inserts as well as uniform hybridization kinetics (venier et al., 2006). mytarray 1.0 was first used to investigate the specificity of gene transcription in mussel tissues with different functional role and the transcriptional profiles of mussels treated with chemical mixtures or living wild in different sites of the venice lagoon (venier et al., 2006; geo series gse2176, gse2183 and gse2184). sample pairs combined according to dye-swap labelling (reference and test samples labelled with cy3/cy5 cyanine dyes in alternate combinations) were competitively hybridized on the two equal arrays of cdnas spotted on the same slide (fig. 2). gills, digestive 181 gland, tissues involved in contraction/motility (foot, adductor muscles, ligaments) and reproduction (gonads and mantle) displayed specific transcriptional footprints, as expected. the results obtained in mussels treated with mixtures of inorganic metal salts or persistent organic chemicals guided the interpretation of the gene expression profiles of mussels living in the inner industrial canals or at the lagoon border open to the sea (this exercise yielded a provisional list of contamination marker probes). in this study, the evident transcriptional down-regulation detected in the reproductive tissues was consistent with the depleted status of the mussel gonads whereas the greatest variety and abundance of transcripts was found in the digestive gland. additional analysis of these expression data is reported elsewhere (pantzartzi et al., 2010). the same platform was then used to evaluate in a time-course study the gene expression changes in the digestive gland of mussels exposed to okadaic acid (oa) via food contamination for five weeks (manfrin et al., 2010; geo series gse14885). one relevant purpose of the study was the identification of molecular biomarkers which could enable an easy and rapid detection of the diarrhoeic shellfish poisoning biotoxins in marketable mussel stocks, i.e., novel reliable assays complementing the existing diagnostic methods. an unsaturated loop design, combining control and treated samples with different dye-labelling for the competitive hybridization on mytarray 1.0, was adopted to take into account all the time points and the biological replicates, with some combinations only inferred (kerr and churchill, 2001). a considerable number of transcriptional changes was detected in the oa-exposed mussels, with a prevalence of up-regulated probes at 3 days and a subsequent progressive increase of down-regulated probes (from 58 % over-expressed to 76 % underexpressed genes, respectively detected at day 3 and day 35). the biphasic time-related trend of response observed in this study recalls the changes occurring in the mussel digestive gland along different phases of the mussel reaction to the experimental stimulus, from the early acute response to the late overall unbalance of the functional processes. many candidate markers are now under study to evaluate their predictive value in the diagnosis of biotoxin-contaminated mussels. mytarray 1.1 (gpl102699, march 2010) contains the same cdna probes of mytarray 1.0 in a slightly modified platform geometry. it has been used to study the gene expression profiles of m. galloprovincialis with monthly samplings for one year, hence taking into account seasonal differences which are known to influence metabolism rates and gonad development among other vital functions (banni et al., 2011; geo series gse22915, gse23049gse23051). mussels were collected from an anthropized and industrialized lagoon of the southern mediterranean sea (ben said et al., 2009) and competitive hybridizations were performed with dye-swap-labelled samples (dual colour analysis). following a loop design with 3-4 biological replicates and parallel histological evaluation of the gonad status, the authors could analyze the transcriptional profiles of digestive gland tissue of female mussels collected during 12 months, and those of digestive gland and mantle tissues from male and female individuals representing all four gonad maturation stages. in the examined annual period, the transcriptional profiles globally highlighted the higher expression of genes associated to mussel nutrition and digestion in mayaugust compared to the other months, and trends for gonad transcripts consistent with the reproductive mussel status. the same cdna platform contributed to the toxicological evaluation of a neonicotinoid insecticide mixture (dondero et al., 2010), an organophosphate compound (canesi et al., 2011) and to the integrated measure of the functional mussel responses in the estuarine tamar region in uk (shaw et al., 2011). the hofmann_ucsb_mytilus_2.5k_v1.0 record (gpl5795, mar 2008) describes a platform of nearly 2500 spotted cdnas of mytilus californianus consisting of both unsequenced and sequenced clones referring to gill and muscle of environmentally challenged mussels. the related geo series gse8935 include data on latitudinal gene expression changes. five biological replicates from four populations of californian mussels were compared to a common reference sample in dual colour analysis (dye-swap labelling). the hms/somerolab-mytilus-105k array-v1.0 (gpl9676, jun 2010) and hms/somero-mytilus105k agilent-v1.0 salinity stress (gpl11156, jan 2011) are two successive versions of a platform composed by oligomer probes in-situ synthesized by agilent technologies (santa clara, ca, usa). these microarrays include probes of both m. californianus and m. galloprovincialis, and are intended for homologous and heterologous gene expression profiling. the processing and assembling of about 26,000 ests from m. californianus (gracey et al., 2008) and 3,984 ests from m. galloprovincialis (venier et al., 2003) resulted in a total of 12,961 and 1,688 transcript clusters or singletons, respectively. long (60-mer) oligoprobes were designed against the m. californianus series and the resulting 43,969 total unique probes (2.6 probes per transcript sequence) were analyzed through blast searches against the m. galloprovincialis series to support selection and design of related probes (556 probe pairs matching transcripts of both species, with a mean number of 4.6 divergent nucleotide bases per probe). a total of 44,524 unique probes were duplicated or triplicated randomly to fill a microarray of 105,000 elements (105 k probes). these two platforms have been used to investigate the transcriptional responses to thermal and osmotic stresses in m. californianus, m. trossulus and m. galloprovincialis (evans and somero, 2010; lockwood et al., 2010; lockwood and somero, 2011). to control the effects of sequence mismatches in the case of m. galloprovincialis probes included in the gpl9676 platform, only probes experimentally confirmed in the hybridization of 84 samples of both m. galloprovincialis and m. trossulus were used in the related data analysis. following a large set of 182 hybridization experiments and stringent quality control, misleading probes were removed from the dataset and the second platform version (gpl11156/agilent 019153) was generated. in the central and southern coasts of california, m. galloprovincialis has largely displaced the native congener, m. trossulus, and such evidence could be explained by species differences in physiological traits related to the adaptation to warm habitats. to investigate the hypothesis, gene expression profiling was performed on gill rna from mussels subjected to acute heat-stress (geo series gse19031). a total of 1,531 probes, out of 4,488 different genes represented on the microarray and recognizing mrnas of both species, showed temperaturedependent expression changes highly similar in the two congeners whereas 96 probes denoting oxidative stress, proteolysis, energy metabolism, ion transport, cell signalling, and cytoskeleton reorganization outlined species-specific responses to the heat-stress. among them, the one encoding the small heat shock protein 24 was highly induced in the mediterranean mussel and showed only a small change in m. trossulus. six biological replicates per mussel group were included in this study which exemplifies the use of a cross-species microarray as well as heterologous and homologous hybridization. according to the authors and published literature, m. trossulus and m. galloprovincialis are approximately 7.6 million years divergent from m. californianus, and only 3.5 million years divergent from each other: in other words, the heterologous hybridization of target sequences from m. trossulus should occur on microarray probes from m. galloprovincialis without inherent sequence bias and should provide a reliable comparison of their transcriptional responses. though debated, prudent evaluations of the sequence divergence by in silico approaches and phylogenetic data could expand the use of cross-species hybridization as a compromise solution for investigating gene expression in species with unsequenced genomes (costa et al., 2010; nazar et al., 2010; ptitsyn et al., 2010). gene expression profiling was also performed on gill rna from mussels subjected to salinity stress (geo series gse25111). a total of 117 probes, out of 6,777 genes represented on the microarray, showed significant changes similar between m. californianus and m. galloprovincialis whereas 12 probes, denoting mrna splicing, polyamine synthesis, exocytosis, translation, cell adhesion, and cell signaling, outlined species-specific responses. the study was based on alexafluor-labelling (555 and 647 fluorescence dyes) of amplified rna, pooled reference samples, six biological replicates, and competitive hybridization in agreement to the recommended agilent protocols. in addition to the overall stringent processing of the fluorescence signals, the heterologous hybridization design suggested the elimination of data from probes with low signal intensity (signal intensity < 150 % of the local background and hybridized spot diameter < 30 % of the nominal spot diameter). the work performed at the a. gracey’s and g.n. somero’s laboratories (university of southern california -los angeles, ca, u.s.a. and stanford university -palo alto, ca, u.s.a., respectively) on mytilus (geo series gse19031 and gse25111) and other species is facing the fundamental aspects of the organism adaptation to fluctuating environments and global climate changes, and gene expression profiling has been essential to their findings. for instance, the study of gene-expression changes in the californian mussels at different phases in the tidal cycle revealed at least four distinct physiological states, corresponding to metabolism and respiration phase, cell-division phase, and two stress-response signatures linked to moderate and severe heat-stress events. the metabolism and cell-division phases appeared to be functionally linked and anti-correlated in time whereas magnitude and timing of the above states resulted to be influenced by the microhabitat conditions according to the vertical position on the shore (gracey et al., 2008). based on comparative physiology, a recent paper offers an overview on the expected consequences of global climate changes (somero, 2011). finally, the mussel immunochip 1.0 (gpl10758, april 2011) is a spotted oligonucleotide platform consisting of four-replicated 1820 oligomer probes plus unrelated controls prepared at cribi for the purposes of a recent european project (imaquanim). oligomers of 57 bases average length were designed at short distance from the 3’ end of transcript sequences selected previously in mytibase, the interactive knowledgebase of m. galloprovincialis which includes most of the ests publicly available for this species (venier et al., 2009). based on multiple criteria, the subset of transcripts selected from mytibase as putatively immune-related molecules should denote central “players” of the mussel innate immunity or genes whose expression is modulated during the mussel responses to immunostimulation (venier et al., 2011). in the platform description, the probe id is hyperlinked to the relative mytibase record: for instance the probe mgo_07346 relates to mgc07346, a mussel transcript featured by the protein domain ipr000098-interleukin 10 and yet functionally unknown. the performance of immunochip 1.0 was tested with hemolymph samples collected at 3 and 48 h from vibriochallenged mussels (geo series gse23535) according to competitive hybridization of dye-swap labelled amplified rna samples. in agreement with the above descriptions, figure 3 provides an updated summary of the nucleotide and protein sequences publicly available at july 2011 and highlights the importance of est sequencing for the preparation of new dna microarrays. more about the molecular “players” of the innate immunity and the immune responses of m. galloprovincialis is reported in the following paragraph. how much can simple sequences tell us about the mussel immune responses? taking advantage of the continuous increase of the nucleotide and amino acid sequences in the public databases, the current methods of bioinformatics can extract instructive data from 183 fig. 3 number of sequence records available for selected mollusc species at the natl center for biotechnology information at july 2011. dna, rna and protein sequences refer to biomphalaria glabrata (gastropoda) and bivalves belonging to the veneroida, unionoida, mytiloida, ostreoida, pectinoida and pterioida orders. simple sequences: from the analysis of various gene/transcript regions to the evaluation of protein/peptide structure and to the comparative analysis of evolutionary differences across the tree of life. this procedural approach complements and integrates the data derived from long-standing disciplines such as measures of structural changes and protein amounts/activity, among others. the overall analysis of 18,788 high-quality ests rationally organized in 7,112 independent clusters or singletons (mytibase transcript collection) highlighted some particularly abundant transcript groups: namely, transcripts featured by a complement component c1q-like domain, antimicrobial peptide (amp) precursors of all four families known in the mediterranean mussel and many heterogeneous lectins including fibrinogenrelated molecules (venier et al., 2011). to explain the abundance of immune-related molecules in mytibase it is important to remember that such collection has been prepared by 16 primary (5 from hemocytes) and 1 normalized cdna libraries from mussels subjected to various challenges, for instance mussels immune stimulated with preparations of gram positive and gram negative cells and viral-like molecules. searches by protein domain revealed a total of 168 different mytibase transcripts containing the c1q signature ipr001073, almost invariably associated with the overlapping tnf-like ipr008983 motif. curiously, the c1q domaincontaining proteins predicted from the transcript sequences, display a short n-terminal signal peptide and a c-terminal globular domain but no central collagen-like repeats which are instead typical of vertebrate c1q domain-containing proteins. according to the current literature, these mussel proteins could represent secreted globular receptors, components of ancient complement pathways expected to mediate pathogen recognition and lysis (dodds and matsushita, 2007). the modularity and versatility of binding mediated by the globular c1q domain explain the variety of roles currently attributed to this still expanding family of proteins, and also supports their involvement in pathogen pattern recognition (carland and gerwick, 2010). the abundance and variety of mussel c1q domain-containing transcripts are consistent with this view. one among these transcripts, named mgc1q, resulted to be expressed at detectable levels in the main tissues of naïve adult mussels, with the hemocytes showing the highest expression levels, and from 2 h post-fertilization up to 3 months later. the mgc1q expression was significantly modulated after mussel infection with gram positive or gram 184 negative bacteria, data which confirm mgc1q as an immune-related gene. the striking molecular diversity of mgc1q was confirmed at both the dna and cdna levels, hence posing mechanistic questions on the origin of such variation (gestal et al., 2010). experimental findings and sequence analyses support the hypothesis of gene duplication, functional diversification and positive selection of many c1qdc variants in selected taxa, including the mussel lineage (gerdol et al., 2011). defensins, mytilins, myticins and mytimycins are cationic antimicrobial peptides stabilized by 4 intrachain disulphide bonds (6 in mytimycin) in a typical 3-d motif (yeaman and yount, 2007). a remarkable diversity of a new group of myticins, with specific variant profiles detectable in single mussels, was reported in m. galloprovincialis (pallavicini et al., 2008; costa et al., 2009). following the discovery of the myticin-c variants, their molecular diversity and evolution has been further discussed (padhi and verghese, 2008) and the most recent findings indicate myticin c as a chemotactic molecule with antiviral activity and immunoregulatory properties (balseiro et al., 2011). just one singleton and other four similar sequences denote the antifungal amp mytimycin in mytibase (rare transcript). mytimycin is composed by 54 aminoacids (6.2 6.3 kda, 12 cysteines) and two main precursor variants, both featured by a signal peptide and a c-terminal extension, are expressed in mussels from different european regions (sonthi et al., 2011). the presence of a calcium binding (ef hand) motif in the c-terminal extension suggests further characterization of such unusual amp. the "effector" role of the mussel antimicrobial peptides (amps) is confirmed in many experimental studies and a comprehensive review have been recently provided (li et al., 2011). whether these effectors can modulate the mussel immune responses with mechanisms other than membrane disruption, as reported for mammalian amps, it is not clear. based on deep amplicon sequencing, the sequence diversity of mussel amps is now under study in natural mussel populations from different geographical regions and in mussels challenged with bacterial cells. lectins are a rather heterogeneous protein family comprising 8 to 15 subgroups, depending on the scientist’s view (dodd and drickamer, 2001). lectins typically possess carbohydrate binding domains and participate in many cell processes. similarly to the mammalian c1q, the c-terminal fibrinogen-like domain ipr002181 of ficolins forms a tulip-like structure able to bind the carbohydrate residues of foreign and apoptotic cells (with consequent opsonization, phagocytosis and cell clearance) or triggering the proteolytic complement cascade and pathogen lysis. fibrinogen-related lectin proteins (freps) are expressed also in mussels (venier et al., 2011) and are codified by at least 2 (m. edulis) 4 (m. californianus) and 7 genes (m. galloprovincialis) (gorbushin and iakovleva, 2011). these molecules can be regarded as immune pattern-recognition receptors and their involvement in the native immunity is supported by the evidence of species-specific expansion of freps in the snail biomphalaria glabrata and the mosquito anopheles gambiae (waterhouse et al., 2007; zhang et al., 2008). in mussel, freps are significantly up-regulated after bacterial infection or pamp treatment, and display opsonizing activity similar to that of mammalian ficolins; moreover, the different sets of frep sequences detected among and within individuals further emphasize the great complexity of the invertebrate immune systems (romero et al., 2011). other lectin-like sequences expressed in mussels are commented in venier et al. (2011). the cases reported above are a few examples of the many classes of transcripts specifically expressed or modulated during the mussel response to potential pathogens. considering in a dynamic view the behaviour of one cell population only, the versatile mussel hemocytes, one can imagine that almost all cellular processes could be influenced by the contact with pathogen-associated molecular patterns: from the cytoskeleton remodelling supportive of chemotaxis, migration and phagocytosis to the intracellular signalling possibly shaping the inflammatory response and finely tuned expression of many regulatory and effector genes. cross-talking signalling pathways have been traced in mussel and the mytibase collection includes transcripts denoting the regulatory cytokine mif (migration inhibiting factor) and cytokine-related molecules, consistent with the idea of an invertebrate cytokine network (malagoli, 2010). the recent definition of a species-specific immunochip aims to the experimental validation of a selected subset of transcripts: a synopsis of the main gene expression changes detected in mussels at 3 and 48 h after challenge with live bacterial cells is reported in fig. 4. the general amp downregulation observed in this particular laboratory treatment was confirmed by quantitative pcr data and is discussed also in li et al. (2010). concluding remarks est sequencing and dna microarrays have substantially improved the identification of genes expressed in the mytilus species. compared to the first est collection and the related cdna microarray, mytibase includes an interesting variety of immune-related molecules which can be further characterized with traditional and innovative approaches as exemplified by romero et al. (2011). nonetheless, in the mytibase collection about half of the mussel transcripts are still unknown, devoid of functional annotation. hence, much work remains to be done both in silico and in laboratory to provide a comprehensive view of the global gene transcription in mussels, particularly the part of the transcriptome mediating the response to potential invaders (immunome). undoubtedly, the application of the available mussel dna microarray platforms can further reveal expression trends of different gene categories and identify useful markers of functional state, if not global molecular signatures useful to disentangle the complex mussel physiology. depending on the study design and on the type of microarray platform, independent validation of the expression data can be accomplished by quantitative pcr or with other 185 fig. 4 main transcriptional changes detected in mussels at 3 h and 48 h from the injection of live vibrio cells (modified from venier et al., 2011). only relevant molecular "players" represented in the immunochip of m. galloprovincialis are reported (framed). in each frame, the detected expression trends are indicated in red, green and yellow (upand down-regulation and not homogeneous trends, respectively). annotations based only on protein domains are reported in brackets. overall, the figure draws the attention to a number of mussel genes, still not characterized, whose expression is modulated in response to immune stimulation. abbreviation list (fig. 4): aif: allograft inflammatory factor apaf1: apoptotic peptidase activating factor 1 bcl2: baculoviral apoptosis regulator 2 c1-c5: complement component 1-5 calr: calreticulin casp: caspase cd63/limp: tetraspanin-7 (lysosome membrane protein) clr: c-type lectin receptor clr: c-type lectin receptor damps: damage-associated molecular patterns fadd: fas (tnfrsf)-associated via death domain fnbp1: formin-binding protein 1 grp 78/94: glucose-regulated protein 78/94 hsc70: heat shock cognate 70 hsp70/90: heast shock protein 70/90 iap: inhibitor of apoptosis proteins; ikbα: inhibitor of nuclear factor kappa-b kinase alpha ikk: inhibitor of nuclear factor kappa-b kinase complex il: interleukin irak4: interleukin receptor-associated kinase 4 jak: janus kinase klhl: kelch-like protein ldlr: low-density lipoprotein receptor litaf: lps-induced tnfalpha factor lps: lipopolysaccharide mapks: mitogen-activated protein kinases mbl: mannose binding lectin mgd1/2: mytilus galloprovincialis defensin 1 /2 mif: migration inhibitory factor mnk: map kinase-interacting serine/threonine-protein kinase mr1: mannose receptor 1 myd88: myeloid differentiation primary response gene 88 nalps: natch, lrr, and pyr containing proteins nfkb: nuclear factor of kappa light polypeptide gene enhancer in b-cells nlr: nod-like receptor nod: nucleotide binding oligomerization domain p13k: phosphatidylinositol-4,5-bisphosphate 3-kinase pac2: proteasome assembly chaperone 2 pamps: pathogen associated molecular patterns pgrp: peptidoglycan recognition protein pi31: proteasome inhibitor pi31 subunit pim: proto-oncogene serine/threonine-protein kinase pim prr: pathogen recognition receptors rab: ras-related gtp-binding protein rip: receptor-interacting serine-threonine kinase ros: reactive oxygen species sec22: vesicle transport protein sec22 sod: superoxide dismutase stat: signal transducer and activator of transcription protein srcr: scavenger receptor cysteine-rich protein precursor tak: mitogen activated protein kinase kinase timp3: tissue inhibitors of metalloproteinase 3 tnf: tumour necrosis factor tnfr: tumour necrosis factor receptor traf6: tnf receptor-associated factor 6 ub: ubiquitin ubr5: ubiquitin protein ligase e3 (component n-recognin 5) α2: proteasome subunit alpha type 2 β4, β5: proteasome subunit beta type 4/5 186 experimental measures. all the steps of the dna microarray testing could be used to strengthen the final data interpretation, from the microarray preparation strategy to the stringency of the hybridization reaction to the algorithms applied to data processing. the maintenance of the physical collection of the cdnas, i.e., recombinant bacterial clones, is a prerequisite for the use of spotted cdna microarrays (for instance, the current use of mytarray 1.0 slides, printed at the cribi facility depends on long work performed at the department of biology, university of padua). such work is not more affordable as long as the clustered ests increase in number, and external commercial services or deep sequencing become an attractive alternative. as a matter of fact, next-generation sequencing (ngs) technologies are now complementing and challenging the dna microarrays as alternative tools for genome analysis and transcriptome sequencing (hurd and nelson, 2009; morozova et al., 2009). for instance, the so called 454 pyrosequencing has been already applied to the study of tissue-specific expression patterns in m. galloprovincialis (craft et al., 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vertebrate macrophage; immune and neuroendocrine responses; evolution introduction the historical term “serfdom” indicates the low socio-economic status of several peasants during feudalism. as a paradox, it may be stated that a very low level in the functioning of immune system has been attributed for a long time to the vertebrate macrophage and, for extension, also to invertebrate phagocytic immune cells. indeed, expecially among vertebrate immunolgists’ attitude, it appears that these cells do not have the "royal" position occupied in the immune system by the lymphocytes. despite this obsolete point of view, the invertebrate immunocyte/vertebrate macrophage plays a crucial role in immune and neuroendocrine responses in multicellular organisms, and its importance has been neglected for a long time even though it is obviously the pivotal immune cell in forms of life endowed of only innate immunity, as invertebrates. beside this wrong opinion, also the concept that the invertebrate recognition is gross and far less sophisticated than vertebrates was quite diffused before the discovery of molecules such as dscam, 185/333 and frep (watson et al., 2005; bowden et al., 2007; ghosh et al., 2010; romero et al., 2011). before the advances of the last decade in invertebrate immunity, several vertebrate immunologists underestimated the power of invertebrate immune system, since their scientific interest was mainly focused on mammalian models. despite the difficulty of debunking this old concept ___________________________________________________________________________ corresponding author: enzo ottaviani department of life sciences university of modena and reggio emilia via campi 213/d,41125 modena, italy e-mail: enzo.ottaviani@unimore.it among human immunologists, every day new evidence emerge about the role played by phagocytic immune cells. the root of the misconception may be linked to the attitude of the two scientists who formulated the two theories of the immunity, the cellular (elie metchnikoff) and humoral (paul ehrlich). not surprisingly, both of them thought that one component was more significant than the other, but the humoral theory for a long time has played a dominant role as giving responses to unanswered questions on the immunological mechanisms and related diseases. in this minireview we recapitulate the findings that justify why the pahogytic immune cells should be considered the major players of the immune system. phagocyte: morpho-functional characteristics in mammals, macrophage appears to derive from a common progenitor in the bone marrow which gives rise to a separate lineage. all the professional phagocytic cells in humans derive from circulating monocytes and acquire new morphological and physiological characteristics according to the organs and microenvironments in which they settle. this unitarian origin is uncertain for circulating and tissue phagocytes in invertebrates, so we proposed the term “immunocyte” to indicate circulating cells endowed of phagocytic activity, irrespective of their developmental origin (ottaviani, 2011). in our view, the most general characteristic shared by immunocytes and macrophages throughout metazoans is the ability to be activated by not-self material and to react through the release 134 mailto:enzo.ottaviani@unimore.it fig. 1. leech body wall after injection with lps. a) semi-thin section of leech body wall. the adjacent fields of muscle fibers (m) usually in close contact are separated by numerous migrating cells (arrowhead). b) thin section of leech body wall. under the epithelium, among the muscle fibers (m) forming the muscular sac, migrating cells (arrowheads), irregularly shaped (their dimensions vary due to emission of pseudopodia) are recognizable. c, d) cryosections, indirect immunofluorescence staining of migrating cells using anti-d14 (c) and anti-cd68 (d). cd14 and cd68 are typically expressed by macrophages in a wide variety of tissues. e, f) tem. macrophage-like cells moving in the connective tissue show ruffled surface and projections. g) cryosection. cathepsin b (arrowhead), indicated by dark brown signal, is localized in macrophage-like cells migrating among muscle fibers (m). h) sem. area of lesion where macrophage-like cell, showing thin pseudopodia (arrowhead), bridges the epithelial edges. i) semi-thin section of lesioned leech body wall. macrophage-like cells are involved in the obstruction of the wound (arrowheads). migrating cells form a wedge-shaped cluster between muscle fibers (m). bars: a: 25 μm; b: 10 μm; e: 2 μm; f: 5 μm; h: 20 nm. 135 a variety of biologically active molecules, such as cytokines, nitric oxide, reactive oxygen species, hydrolytic enzymes, adrenocorticotropic hormone (acth), β-endorphin and corticotropin-releasing hormone (crh) that affect the immune and the neuroendocrine responses (ottaviani and franceschi, 1997; ottaviani et al., 1998, 2004, 2007). as far as the immune role of the reported molecules is concerned, it has been found that mammalian cytokines, acth peptide fragments and crh are able to influence the chemotactic and phagocytic activity of invertebrate immunocytes and vertebrate macrophages (ottaviani et al., 1990, 1992, 1994, 1997, 2004; genedani et al., 1994). these findings revealed that the pro-chemotactic and prophagocytic effects of the above reported mammalian molecules on invertebrate immunocytes are not straightforward being specie-specific and dose-correlated. the effects on phagocytosis are more uniform than those registered on chemotaxis, making the general assumption that chemotaxis and phagocytosis are strictly correlated, not valid for all invertebrates. the discrepancies observed for the effects of different heterologous peptides on diverse invertebrate immune functions may be attributed to several factors, not excluded the possibility that the immunocyte responds to the same stimulus on the basis of contingency (ottaviani et al., 1997, 2004; malagoli and ottaviani, 2010a). as far as the neuroendocrine responses are concerned, it has been demonstrated that the principle mediators of stress response in invertebrates are fundamentally comparable to those known in vertebrates and have been conserved during evolution (malagoli et al., 2011). we have found that molecules similar to the mediators of mammalian stress, crh, acth, biogenic amines, glucocorticoids and cytokines, are present in and/or released by invertebrate immunocyte (ottaviani and franceschi, 1996; ottaviani et al., 1998; malagoli et al., 2007). it is to underline that even though in invertebrates the molecules are those that participate also in vertebrate stress response, in invertebrates the framework is simpler. in vertebrates, various organs are involved, while all the molecules determining invertebrate stress response are harboured into the immunocyte. in other words, the prototypical response in invertebrates appears to be concentrated into a single, multifunctional cell, representing the best example of an immuneneuroendocrine cell observed so far. during animal diversification, four fundamental evolutionary events that have influenced the formation of the increasingly complex vertebrate immune system, can be recognized. the first evolutionary event is represented by the formation of multicellular organisms, not so much the formation of colonies where the cells are more or less identical to one another, but the association of cells that differ and are able to perform different integrated functions. this series of events is observed for the first time in porifera. the second event is the appearance of a body cavity, i.e., a place in which the circulating cells may be retrieved. the third evolutionary event, is represented by deuterostomia (the blastopore becomes the anus) characteristic of some invertebrate (e.g., echinoderms) and the vertebrate embryos. the fourth event coincides with the transition from the aquatic to the terrestrial area by tetrapode vertebrates. by examining the different species included in the four evolutionary events reported, the key role played by the phagocyte emerges (see turner, 1994). it should be underlined that in animals without a digestive tract, such as porifera and coelenterata, the phagocyte plays a double role i.e., defense and nutrition. hildemann et al. (1979) identified in the immune system of the sponge callyspongia diffusa (porifera), the three functional components as minimal criteria for immunological competence: cytotoxicity, specificity and memory. from invertebrates to vertebrates immune cells are capable of sophisticated performances. as far as immune and neuroendocrine functions are concerned, it is amazing to observe how phagocyting immune cells perform functions ranging from chemotaxis, phagocytosis, encaspsulation, transplantation, wound healing, cytotoxic activity to the stress response (turner, 1994; ottaviani et al., 1997; de eguileor et al., 2000a, b; grimaldi et al., 2004). on the whole, it emerges that phagocytic immune cells are endowed of a vastly dinamic morphology and, beside molluscs (ottaviani, 2011), an interesting example is also represented by hirudo immunocytes (de eguileor et al.,1999, 2000a, b). in leeches, the responses to surgical wounds, grafts or lps injection are similar to those obtained in vertebrates and involve a sequence of events triggered by inflammatory reactions (de eguileor et al., 2003, tettamanti et al., 2003a, b). after immune stimulation a large number of cells migrate through the extracellular matrix from the inner regions of the body close to the gut, towards the superficial body area (figs 1a, b). these migrating cells have been characterized by morphological, histochemical, and immunohistochemical methods. macrophage-like cells, nk-cells and granulocytes are the cell types more represented. in particular several migrating cells involved in leech defense system, display features and behaviour observed also in invertebrate and vertebrate activated macrophages being cd25+, cd14+, cd61+, cd68+, cd11b+ and cd11c+ (de eguileor et al., 2000a). furthermore, immunocytes modify their morphology in relation to the time elapsed after the immune stimulation. during the phase of migration immunocytes change their shape showing ruffled borders (figs 1a-g) and supply enzymes (cathepsin b) to degrade components of extracellular matrix outside the cells, ensuring the movement in the connective tissue (fig. 1g) (grimaldi et al., 2004). in lesioned leeches immunocytes are the first cells that are also involved in closing the wound by using pseudopodia to bridge the epithelial edges (fig. 1h). subsequently additional immunocytes togheter with granulocytes and nk cells complete the obstruction (fig. 1i) (de eguileor et al., 2000a). during wound healing the principal function of immunocytes is phagocitosis. indeed, in their cytoplasm large phagocytic vacuoles containing citoplasmic debris or bacteria or any kind of foreign antigen are always visible (fig. 1e) (de eguileor et al., 2000a). 136 concluding remarks overall, we sustain for the circulating and phagocytic cells the part of principal actor not only during inflammatory response and immunity, as originally suggested by mechnikoff (tauber, 2003), but also during stress response, at least in invertebrates. in this scenario, an unitarian view emerges in which phagocytic immune cells, the most ancestral defense cell of the body, continues to play a fundamental role throughout evolution in the most complex phenomena responsible for body homeostasis, i.e., immunity, inflammation and stress response (ottaviani and franceschi, 1997; ottaviani et al., 2007). however, without minimizing the role played by the cells of acquired immunity, i.e., the vertebrate lymphocytes, they have a more restricted number of functions requiring the involvement of components of innate immunity to be completed. furthermore, the lymphocytes play a key role in immunological memory, an immune function crucial for homeothermic vertebrates, while for the coldblooded ones it seems less relevant (malagoli and ottaviani, 2010b). in this context, the bony fish have their lymphocytes suppressed at low temperatures and, in this situation, found in phagocytic cells the main actors of the immune defense (bly et 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obsoleta isj 7: 67-78, 2010 issn 1824-307x research report fate and distribution of pyrene in ilyanassa obsoleta exposed through the diet s erskine1, dg beach2,3, c rouleau4, j hellou2,3,5 1department of biology, dalhousie university, halifax, ns, canada 2department of chemistry, dalhousie university, halifax, ns, canada 3bedford institute of oceanography, fisheries and oceans canada, ns, canada 4institut maurice lamontagne, fisheries and oceans canada, québec, canada 5department of oceanography, dalhousie university, ns, canada accepted february 2, 2010 abstract the ability of eastern mud snails to bioaccumulate and biotransform a model polycyclic aromatic hydrocarbon (pah), pyrene (py), was investigated. the contaminant was added to fish at levels ranging from 2 to 5,000 ng/animal fed 20 mg/each and fed to snails. a linear relationship (p=0.03) was observed between the level of py and the sum of metabolites consisting predominantly of 1hydroxypyrene and pyrene-1-sulfate detected in soft tissues of snails. in healthy animals, more py than metabolites was detected, with more biotransformation relative to the parent compound apparent at lower levels of exposure. in ten snails fed together, the body burden of py-related compounds displayed 49% variability as well as a similar mean and median. one snail within that group had five times more metabolites than py and was retracted inside the shell, indicating that the animal was stressed. radio-labelled py was detected in the largest proportion in the kidney of the animals. three snails that died during the exposure had also greater than five times more py metabolites relative to live counterparts. this study is unique in the links that it establishes between stress and the balance of fates of anthropogenic chemicals in biota. key words: body burden; bioaccumulation; biotransformation; pyrene; dose-response; health introduction the bioavailability and bioaccessibility of chemicals labelled as priority pollutants is an area of research that generates continual interest (thorsen et al., 2004; johnsen et al., 2006). such interest results from population expansion that has enhanced the production of these anthropogenic compounds and from a heightened awareness of the associated deleterious effects. an assessment of the presence of contaminants in the abiotic environment provides a means of comparing the actual state of various locations with published guidelines for an acceptable or ideal background state (chapman et al., 1987; ospar, 2009). the latter publication states that “the ultimate aim of achieving concentrations in the marine environment near background values for naturally occurring substances and close to zero for man-made synthetic substances”. questions about the potential impact of exposure on organisms’ health may then be asked. the investigation of the fate of organic ___________________________________________________________________________ corresponding author: jocelyne hellou department of chemistry dalhousie university halifax, ns, canada e-mail: jocelyne.hellou@mar.dfo-mpo.gc.ca compounds in biota reflects the uptake and potential risk caused by exposure to contaminants which bioaccumulate and biotransform. this body burden information can provide the means to link the presence of anthropogenic chemicals to the probability of generating toxic effects (meador et al., 2008). the effects of exposure to contaminants on the health of organisms can cover a broad range of end points. the challenge is to interpret the implications from simple chemical and biochemical levels of organisation, to the more complex of populations, ecosystems and communities (hinton, 1993). contaminants’ mechanism of action differs with dose, where lethality represents an acute endpoint, and does not readily reflect the outcome of exposures. this reality raises the need for additional studies of effects. a balanced assessment based on the “weight of evidence” approach can be achieved (chapman, 2007) by combining knowledge generated in laboratory investigations with the results of field observations. environmental studies aim to discover the state of a site and its inhabitants relative to other or to past situations (myers, et al., 2003; leung et al., 2005). the goal of canada’s oceans strategy is “to ensure healthy and prosperous oceans for the benefit of current and   67 mailto:jocelyne.hellou@mar.dfo-mpo.gc.ca   68 future generations of canadians”. our research has pursued the further development of tools which are the basis of efforts to monitor and assess sediment quality. polycyclic aromatic hydrocarbons (pah) are recognized priority pollutants and pyrene (py) is a commonly abundant pah found in air, freshwater, rain, snow, seawater, sediments and soil (garrigues, narbonne, lafaurie, ribera, lemaire, raoux et al., 1993; lane, leithead, baroi, lee, graham, 2008; lima, farrington, reddy, 2005; williams, meares, brooks, watts, lemieux, 1994). it mainly derives from combustion sources, such as the burning of coal, wood, food and garbage, or the presence of industries involving aluminum smelters, coke ovens, carbon or graphite-electrodes, as well as from natural events such as forest fires and volcanic eruptions. this tetracyclic molecule is always present in complex mixtures with numerous pah and various additional chemicals, especially if sampling is near sewage effluents (soclo et al., 2000; hellou et al., 2002). the physical-chemical properties of py lead to a relatively long residence time in organisms during depuration (hellou and leonard, 2004; hellou, et al., 2009b). in the environment, py has a slower degradation rate than smaller pah, and in sediments, its concentration correlates to that of other large pyrogenic pah associated with mutagenicity and carcinogenicity. these characteristics, along with the availability of a couple of metabolite standards, contribute to choosing py as a model pah for inter-species comparisons of fate (grainger et al., 2005; santella et al., 1994). in the aquatic environment, pah with an octanol water partition coefficient (log kow representing the amount in octanol/amount in water) values of <3.0 reside preferentially in water (mckim, 1994). those with a log kow of 3.0 to 6.0 partition between water and particles, with an increasing tendency to associate with the latter with increasing log kow (mackay et al., 1992). particle-bound pah with log kow >6.0 will be available from lipophilic material such as food. since py has a log kow of 5.2 under steady state conditions, it will be found in small proportion in water and more so in dietary items of the aquatic food web. once taken up by biota, this reactive chemical can undergo a one or two step enzymatic oxidation to 1-hydoxypyrene (pyoh) and it can further conjugate with a biogenic moiety (hellou and leonard 2004; pesch et al., 2007). metabolites represent markers of exposure and effects (perera , et al., 2005). the purpose of biotransformation is to produce more polar and easily eliminated derivatives and represents a biochemical defense mechanism. however intermediate pah products formed during biotransformation such as quinones are toxic (zielinska-park et al., 2004). livingstone (1998) described four types of studies where the role of biotransformation by invertebrates and vertebrates would be of interest, such as in research regarding animal ecology and evolution, in modelling, as biomarkers and in toxicity tests. since less is known about the ability of gastropods to biotransform reactive molecules, our research recently centered on two species of offshore large snails, neptunea lyrata and buccinum undatum, a poisonous and an edible species, respectively (beach et al., 2010, in press; beach and hellou, 2010, in press). an analytical approach was developed for the extraction, separation and identification of bioaccumulation and biotransformation products present in soft tissues of invertebrates exposed to py and pyoh (beach et al., 2009). the accumulation and transformation of these two chemicals yielded up to eight phase i (oxidation) and phase ii (conjugation) metabolites. a smaller readily available related species, the eastern mud snail, ilyanassa obsoleta, is seen at low tide on estuarine beaches where it occupies a variety of habitat surfaces, including fine grain sediments, marsh grasses, pilings or rocks. this mollusc is reported to feed on lettuce, spinach, shrimp or fish when held in tanks, where animals can be easily maintained for experimentation. these snails are found in densities reaching thousands of individuals/m2 (cranford, 1988). they tolerate highly variable weather conditions, surviving temperatures of 40-45 °c for up to 30-90 min and a salinity of 0‰ for more than two days. being surface deposit feeders, as grazers they ingest microalgae and diatoms as well as carrion, where small particles are enriched in lipophilic contaminants. because of their abundance, ilyanassa obsoleta could be used to monitor the availability of contaminants in field, mesocosm or laboratory experiments. the behavioural response of this species towards contaminated sediments and seawater was previously examined in our laboratory (hellou et al., 2009a; marklevitz et al., 2008a, b). in the present study, our goal was to study the fate of py in snails exposed to food containing a range of py levels. the behaviour of the animals was also noted in conjunction to exposure in order to examine potential differences between the behavioural response and fate. the long-term goal of our research aims to support studies evaluating the richness and abundance of fish species from an integrated ecosystem management perspective. such research would also help to link toxicity and contaminants when assessing environmental quality with triad studies (chapman, 1987; 2007) combining toxicity tests, chemical analyses and examining the benthic community. materials and methods care and use of ilyanassa obsoleta snails were collected in summer-early fall from an inter-tidal mud flat in hantsport, nova scotia, canada. sediment and snails were obtained by scraping the upper 5-10 mm surface of the beach and they were transported to the laboratory in halffilled buckets with a couple of cm of additional seawater. upon arrival (1 h drive), animals and sediments were separated. sediments were placed in bags and frozen for future use. our previous work had determined that the grain size of the mud was very fine <50 μm. animals were housed in 1 m2 tanks with flowing seawater maintained at a regulated temperature 15 ± 2 ˚c, with aeration and some sediment (<1cm) placed on the bottom. snails were fed lettuce every other week, sediment was added every month and some was removed to maintain a depth of <1cm in the tank. a smaller 7 l aquaria covered with plastic wrap with pinholes was   69 placed in the holding tank to maintain snails deprived of food for 2.5 days prior to the beginning of experimentation. to select animals for exposures performed in the fall, snails measuring 17-21 mm were removed from the tank, placed in a second one with seawater to determine whether they would take an upright position. they were then distributed randomly among the required 1 l beakers, placing ten per beaker. animals that did not emerge from the shell and begin moving within a few minutes were replaced with active ones. beakers were covered with plastic wrap and punctured with pinholes. behavior of snails all chemical exposures were performed at 1920 °c and snails were counted for their position, on non-immersed glass or in seawater in the 1l beakers containing 800 ml of seawater. experiments were performed over a single season, fall, because of changes previously observed with time. to examine behaviour relative to food preference, exposures were done in the same size beakers with 200 ml of seawater. animals were observed for being upright; in a reversed position lying on their shell with soft tissue extended, “distressed”; or completely within the shell with the operculum covering the soft tissue “retracted” (harry and aldrich, 1963). exposure to spiked food food preference was examined by comparing the consumption of algae, soft tissue of shrimp and filets of whitefish. when fish was offered, the material disappeared more quickly and snails remained on the offered food for a shorter period of time than with the other items. this behavior would indicate less potential loss of contaminant. snails were then fed small pieces of the latter, and it was estimated that after 2.5 days without food, each snail would consume on average 20 mg of fish. therefore, the 200 mg of food required for 10 snails was spiked evenly with <100 μl of an acetone solution while placed in a glass dish, the solvent was evaporated for 10 min and the food was introduced in the beaker and pulled into small pieces with tweezers. in all exposures, animals began to feed readily and consumed most of the food within 15 min, with no traces of fish visible after <60 min. spiking levels were of 100, 1,000, 10,000 and 250,000 ng/g of py in food and represent a dose of 2, 20, 200 and 5,000 ng of py per snail in each beaker. dissection in most cases, because of chemical analyses and the trace levels used in the exposures, animals from one beaker were pooled for analyses, except for any that died during the study, and except at the highest exposure level where each of ten individuals were analyzed separately. for the lipid and chemical extractions, whole animals were frozen until ready for processing. the material was then thawed, the operculum removed and the retractor muscle severed using pointed tweezers. in most cases the snail could then be pulled intact from the shell. when this was not possible, a small screw clamp was used to carefully crack open the shell. prior to weighing, any parts of the shell remaining on the soft tissue were removed. it was important to weigh the snails soon after removing the shells because the moisture evaporated readily and the wet weight became measurably less within minutes of removing the shell. dissection of snails revealed a soft tissue weight of 300-600 mg (wet), with nearly equal weights of muscle and visceral mass. these parts were not divided when processing samples, but only for preliminary physiological determinations. lipid and moisture content lipid content was determined by air-drying tissue in the fume hood overnight with a subsample placed at 70 ˚c in order to confirm the moisture in the air-dried tissue. moisture represents the difference in weight before and after oven drying, expressed as a percentage of wet weight. portions of 200-500 mg of air-dried tissue were weighed into centrifuge tubes and crushed using a manual homogenizing pestle. lipids were extracted using three 10 ml portions of a solution of 1:1 hexane:dichloromethane. this latter solvent was also used to rinse residues off equipment to prevent the loss of material. for each extraction, the tissue and solvent were mixed by vortexing for 30 s and then sonicated for 3 min. after mixing, samples were centrifuged at 2000 rpm for 5-8 min, and the supernatant collected before adding the next aliquot of solvent. combined extract was treated with ovendried sodium sulphate (60-80 °c, continuously), the solution was filtered after 5 min and it was evaporated to dryness. the residue was then transferred to a pre-weighed vial with repetitive rinses of dichloromethane. further evaporation using nitrogen and gravimetric determination of the lipid content was expressed as percent of dry tissue. analysis of soft tissue soft tissue was placed in a pre-weighed aluminum dish using ten animals from each exposure level that were extracted together. the detailed method has been published by beach et al. (2009) and consists of a stepwise methanol extraction repeated four times. ten snails provided 3.9 to 4.7 g of wet tissue. however, smaller samples such as three dead animals of 0.230 g total mass were extracted separately from the remaining ones. the tissue was spiked with 51 ng of 2hydroxyfluorene (floh) using 100 µl of a 0.51 µg/ml solution in acetone and left for 10 min to allow the solvent to evaporate. this phenolic compound represented a surrogate standard to determine the loss of material during processing. tissue was transferred to a centrifuge tube and the petri dish rinsed into a centrifuge tube with 10ml dichloromethane and then 10ml methanol. scoops of dry sodium sulphate were added to ensure that the tissue would deposit in the tube. the mixture was homogenized using a polytron blender, then vortexed for 30 s and sonicated for 3 min. instruments were rinsed with methanol and scraped into the tube to minimize the loss of material. the mixture was centrifuged at 2000 rpm for 5-8 min and the supernatant transferred to a round-bottomed flask. the extraction step was repeated three more times: twice with 10ml methanol and once with 10ml dichloromethane and the extract evaporated using a rotary evaporator at 34-42 ˚c. the flask was carefully rinsed with three 1 ml portions of methanol, blown down under nitrogen,   70 then rinsed again with 2 ml dichloromethane and blown down again. the residue was suspended in 2 ml methanol and filtered through glass wool into another calibrated test tube, rinsing with more methanol. the solvent was evaporated and the residue weighed. a measured amount of methanol was added to give the desired final volume depending on the concentration of py. a subsample of the liquid was transferred to a 2 ml amber hplc vial using a syringe with a filter tip (waters millex hv 4.5 µm) to remove any remaining particles. the extract was analyzed by highperformance liquid chromatography (hplc) with fluorescence detection, and diluted and re-analyzed as necessary to give a concentration within the range of the calibration curves. quality assurance/quality control snail tissue spiked with py, pyoh, pyrene-1sulfate (pyos) and pyrene-1-glucuronide (pyoglca), as well as floh as surrogate standard, were extracted as described above and detailed in beach et al. (2009). a snail matrix spiked with 10100 ng of each of the available standards and processed to examine the efficiency of the approach yielded generally >70% for py and pyoh, while the amount of the more polar compounds was always underestimated. each processed sample was spiked with floh to examine variations in the processing. floh recoveries were above 60% and reaching up to 96%. the concentrations of analytes were therefore adjusted for losses. concentrations are expressed in terms of available standards and there is a need for isotopically labeled standards that could be used to examine the recoveries of the more polar compounds during the work up of each sample. exposure to 14c labeled py and sample preparation a spiking solution was prepared by mixing 22 µl of a 100 µci/ml 14c labeled py in methanol with 35 µl (of the 992 µg/ml of py). the solution was blown dry with a gentle stream of n2 and dissolved in 100 µl of acetone. this amount was applied dropwise to 200 mg of whitefish and allowed to dry for 10 min. the food was given to ten illyanassa held in a 1 l beaker as described above and animals sampled 72 h later. the operculum was removed from live shellfish, they were then cracked with a screw clamp and the soft tissue was removed. each of the animals was slowly lowered into liquid nitrogen with a spoon and held there for 1-2 min. the frozen animal was transferred with forceps to a 50 ml beaker containing 40 ml of carboxymethylcellulose solution (26 g/l). larger forceps were then used to dip the beaker halfway into liquid nitrogen and hold it there until the gel became thoroughly white (5-10 min). as the freezing procedure began, a micro-spatula was used to prevent the tissue from floating to the gel’s surface. finally, the beakers were placed in ziplock bags and stored in the freezer until they were shipped to another location. snails were then processed as described by frouin, et al. (2007) with more than 30 slices generated for each snail, where images were further examined for radiolabeling. results and discussion lipid content the morphometrics and the amount of neutral lipids measured in snails are presented in order to enable comparison by other researchers in future endeavors (table 1). mature animals were chosen according to shell length within a restricted size range having 7% variability (standard deviation/mean). the soft tissue weights of the visceral mass and muscle displayed a wider range of results with 22 and 26% variability, respectively. mean lipid content was 5.8% and varied by 47% over that period. the fish used to feed the snails contained a similar amount of lipid, 5.8%. the mean moisture content of snails and fish was also similar, 83 and 80%, respectively. py food exposure the food preference of the snails was determined by comparing their attraction to, and the time taken to deplete offered items, i.e., algae, shrimp and fish. experiments determined that animals not fed over a period of 2.5 days would be readily attracted to and consume fish more rapidly than other substances. this preliminary conditioning was previously needed in experiments which examined the behavior of snails placed in tanks containing two choices of sediments (marklevitz et al., 2008a, b). this standardization prior to exposure helped to interpret the avoidance/preference response of snails relative to spiked seawater or to single spiked sediment (hellou et al., 2009a). under these holding conditions, an average mature snail readily ingested 20 mg of fish within a few minutes. table 1 morphometric data of some of the snails (n=22) sampled between september and december. shell shell visceral muscle index moisture lipid length width mass mass v/m content content (mm) (mm) (mg) (mg) % % mean 481 19 11 261 1.1 83 5.8 sd 139 1 74 58 0.2 4 2.7 minimum 204 17 9 156 0.6 78 3.4 maximum 861 21 11 431 1.6 87 10.5 fig. 1 body burden of ten snails exposed to the highest level of pyrene (py) spiked in food and representing 5,000 ng per snail for a total of 50,000 ng in 200 mg of fish or 250,000 ng/g fish. behavior towards spiked food behavior was examined because avoidance represents a stress response towards chemical or physical disturbances like spillage of oil or changes in temperature and wind conditions. interest in behavioral research using fish and invertebrates has recently broadened (roudez et al., 2008; robinson, 2009). the stress stages that can develop in snails have been described by harry and aldrich (1963). they consist initially of an attraction-or-avoidance reaction. thereafter animals may be observed to appear “distressed”, soft tissue protruding as they lie on their shells, and eventually to have completely “retracted” soft tissue within their shells. these stages reflect the quality of the snails’ environment and have been detected in exposures to contaminated environments (burris et al., 1990; marklevitz et al., 2008a, b; hellou et al., 2009a). animals were observed during feeding and at intervals during the three-day experiments. none of the offered food was avoided. some variability was detected with time, but without a statistically significant difference in stress (better than 5% level) between exposure levels. in contrast to this behavior towards spiked food, animals exposed to harbor sediments and to sediment spiked with a mixture of seven abundant pah avoided that matrix (marklevitz et al., 2008b). although py has been associated with a range of toxic and therapeutic endpoints (long et al., 1995; law and klungsoyr, 2000; jensen and sverdrup, 2003; clément et al., 2005; culotta et al., 2007) stress was not observed during feeding. variability in contaminants’ uptake: prior to analyzing pools of soft tissue from snails exposed to low levels of py, it was important to determine the distribution in the uptake that would be detected in ten snails exposed together to spiked food. animals exposed to an expected, calculated 5,000 ng of py/animal were therefore analyzed individually. this group of snails was of a restricted mean mass 0.53 g (+0.09 g). assuming that the spiked food was homogenous in py content, since the solvent spike was added uniformly to the food and with great care, then the lowest and highest consumed amount of food interpreted from the body burden consisting of summed py and derivatives was 20 and 170% (fig. 1). the mean and median of the body burden were nearly equal (4,800 + 2,300 and 5,100 ng equivalents of py/animal) and displayed a normal distribution with five animals with concentrations above and below the mean. the inter-individual variability of 49% likely reflects the competition between snails or a snail’s need for food. this investigation provided the standard deviation to be expected with the analysis of pooled tissue of snails exposed to lower levels of py that could not be analyzed individually due to the available instrumentation. concentrations differing by less than 50% between exposure groups would be deemed similar. a high variability has been observed when analyzing animals exposed in the laboratory to spiked food. for example, a wide standard deviation has been reported in studies involving terrestrial and aquatic invertebrates (stroomberg et al., 2004; dam et al., 2006; granberg and forbes, 2006). rates of feeding, excretion and metabolism differ between members of a species even when individuals of a restricted size range are chosen in an experiment in order to minimize the morphometric differences associated with age. fingerprint of uptake the fingerprint of the extracts demonstrated the complexity of the biotransformation process taking place in this species (fig. 2). the proposed structures of the metabolites are based on earlier studies performed in our laboratory and involving larger gastropods, n. lyrata and b. undatum (beach, et al., in press, 2010; beach and hellou, in press, 2010). the presently detected metabolites were identified by comparing the chromatograms of the   71 fig. 2 high performance liquid chromatography (hplc) fluorescence chromatograms displayed on two scales to highlight the fine fingerprint, with pyrene (py) derivatives detected in tissue extracts, with 1-hydroxypyrene (pyoh), fluorenol (floh) representing the surrogate standard, two isomers of pyrene diol monosulfate (pydoms) are followed by two isomers of pyrene diol disulfate (pydods). chromatogram a represents a sample that is more concentrated and injected from a smaller volume of solvent (2 ml), while b is more dilute and injected from a larger volume of solvent (20 ml). larger and smaller species that were injected on the hplc using a common protocol, i.e., same extraction method, guard column and column, solvent gradient, as well as temperature, with injections performed on the same day. the identity of the peaks was previously determined (beach, et al., 2009; beach and hellou, in press, 2010) in part by comparing the retention times and fluorescence spectra of detected peaks to available standards, by performing enzymatic hydrolyses and especially by using a hyphenated chromatography-spectroscopy technique (lc-ms) on an extract fractionated by solid phase extraction. at the highest exposure level, py represented between 61 and 93% of the sum of compounds. the next two more detectable derivatives were pyoh and pyos, followed by two isomers of disulfate derivatives representing between 0.2 and 1.2% of the sum of compounds. two additional diols present as monosulfates were detected at lower levels. pyglca was detected at trace levels. animals with more bioaccumulated py also produced more metabolites, with a significant linear regression between these values (p=0.005, r2=0.6623, with n=9, fig. 3). the exception was for one snail with 1,500 ng of py detected along with 965 ng of metabolites (as py equivalents), where pyoh and pyos were of equivalent proportion. the regression would predict about five times less metabolites or nearly 200 ng. this animal’s behavior differed from that of the others as its body was extended in a manner that is symptomatic of stress. the sum of py and metabolites extracted from this group of snails represented 96% of the amount spiked in food. this result confirmed that the experimental setup was optimal because most of   72 fig. 3 a. linear regression obtained from the analysis of nine healthy individual snails at the higher pyrene exposure, with the star representing the tenth outlier snail. b. linear regression obtained from the analysis of healthy individual or pooled snails (including those in fig. a). the food was consumed. little discharge was apparent in the beakers over the 72 h exposure, reflecting retention of the food or re-ingestion of eliminated material. this has also been observed by beach and hellou (in press, 2010) exposing larger marine gastropods and by dindal and wurzingerref (1971) studying terrestrial snails. it highlights the importance of the recycling of contaminants in the environment with potential trophic transfer by animals consuming detritus. lower levels of exposure three lower exposure levels offering the same amount of white fish with 2, 20 and 200 ng of py per animal were performed. the control and 2 ng group displayed close body burdens (fig. 4). since body burden could be expressed in various units, results obtained for reference animals highlight these differences for an average animal weighing 0.5 g (fig. 4). these body burdens indicate that a similar amount of py was present in the dietary intake of control snails ingesting small particles and animals from the low dietary exposure. in extracts from control snails and the lowest exposure, nearly half, 45-55% of the anthropogenic material, detected in an animal was present as parent py (fig. 4). in extracts from snails exposed to 20 and 200 ng/animal, py represented 65 and 80% of the body burden, respectively, with pyos and pyoh representing the abundant metabolites, and pyos displaying twice the concentration of pyoh. therefore, more biotransformation took place at the lower exposure levels. in healthy animals, tissue extracts contained from 21 to 76% of the py spiked on food. recovery rates of py equivalents were 76 and 96% in the lowest and highest exposures, respectively. therefore, in the middle exposure, either the spike remained in uneaten minute food particles, or py leached out prior to food consumption. the present species handles low levels of xenobiotics through efficient biotransformation better than higher levels (fig. 3). dead animals on a per animal basis, seven times more pyoh and pyos were detected in three dead animals than in surviving animals from the 200ng/snail exposure (fig. 4). these two metabolites were 30% more abundant as py (0.56 vs 0.45 nmol/g), while in healthy animals metabolites represented 20% of the detected py (0.08 vs 0.36 nmol/g). in this mid exposure, metabolites were produced in a somewhat similar opposite ratio as in the distressed animal at the highest food exposure. in the 5,000 ng/animal exposure, healthy animals had metabolites representing 12% of the parent py, while the stressed snail had metabolites representing 60% of the level of py (11 vs 17 nmol/g animal in the one animal compared to a mean of 5.1 vs 42 nmol/g animal in the others). therefore, at least five times more metabolites were produced in animals experiencing toxicity. whether this effect is due to specific derivatives such as quinones (zielinskaet al., 2004) remains to be investigated.   73 fig. 4 a. mean concentrations of pyrene (py) derivatives expressed in different units and detected in reference snails, levels are reported for 1-hydroxypyrene (pyoh), pyrenesulfate (pyos) and pyreneneglucuronide (pyglca). b. concentration of py derivatives detected in animals exposed to various concentrations of pyrene spiked in fish. ref stands for reference, 2, 20 and 200 is the amount of spiked py (ng) expected to be taken up per animal. comparing fate to that in other species the fate of this combustion-derived contaminant has been investigated in the bile of many finfish species from freshwater and marine environments. since the gall bladder bile represents a distinct relatively easy matrix to handle, many studies have examined metabolites in this liquid (solbakken et al., 1982; escartin and porta, 1999). in terrestrial vertebrates, the non-invasive urine analysis has been used most frequently (grainger et al., 2005; walker et al., 2006) with glucuronide and sulfate conjugates detected. interest in the uptake kinetics of pah, in terms of concomitant bioaccumulation and biotransformation, has been relatively restricted. some of the initial studies performed more than two decades ago demonstrated differences in the ability of finfish and shellfish to biotranform vs bioaccumulate pah (varanasi et al., 1985; varanasi et al., 1986; mcelroy, 1990). for example, only one of two species of amphipods was capable of transforming py (reichert et al., 1985). some of the early studies used labeled material to determine the soluble nature of labeled extracts and enzymatic hydrolysis to further pursue the structure of conjugates (collier et al., 1978; solbakken et al., 1979; cravedi and tulliez, 1982; solbakken et al., 1978; mcelroy, 1990). these investigations pursued a balance of fates and the publication by mcelroy (1990) is an excellent example. concern with invertebrates has expanded over the last ten years. a diversity of metabolites has been discovered (eickoff et al., 2003; stromberg, et al., 2004; lee, 2005;) and reviewed in beach et al. (2009). briefly, monohydroxy and dihydroxy pyrene, phase i metabolites, along with glucuronide, sulfate, malonate, glucoside phase ii products, have been identified in tissues of isopod, clam, crab and whelk. the largest number of identified py related compounds was detected in a large gastropod, b. undatum (beach et al., 2010, in press). there are few studies of fates and effects that discuss the presence of metabolites relative to effects (dam et al., 2006). the present observations are unique.   74 fig. 5 example of two images of radio labelled snail tissue with colour associated with the intensity of the labelling and highlighting identified organs. 14[c] labeled pyrene distribution since animals were analyzed whole, an additional exposure to the highest level of py was performed using 14[c] labeled py in food, with identical experimental conditions as used for unlabelled py. the interest was to discover the tissue-specific distribution of py after a three day exposure. as observed with whelks (beach et al., 2010, in press), the most labeling was apparent in the nephridium/kidney (fig. 5). a much lower proportion of labeling was detected in the rest of the digestive tract, particularly in the esophagus, as the images indicate. there was no evidence of labeling in the reproductive system, most likely because of the length of exposure. this species of snails reproduces during may-july under our climate conditions. conclusions and perspectives a linear relationship (p= 0.03) was observed between the level of py accumulated and transformed in soft snail tissues. results demonstrate the complex metabolic capacity of i. obsoleta exposed to realistic levels of food contamination. animals starved for 2.5 days prior to exposure did not avoid py-spiked food. they also did not display significant levels of stress over the following three days, except for one and three snails offered a mean of 5,000 and 200 ng of py per food portion, respectively. these animals displayed more than five times the amount of metabolites detected in soft tissues of healthy members of the respective groups. the difference in the fates of py points to the importance of pursuing the multi-faceted fates of pah to understand the chemical-biological link between fates and effects. with more balanced research, the development of environmental assessment criteria (ospar, 2009) would benefit from the type of research described herein. the eastern mud snails could be used as model organisms for the production and comparison of aromatic metabolites difficult to purchase commercially. the potential presence of a specific py metabolite that would be associated with the   75   76 observed stress needs to be pursued. more investigations should also be performed in order to investigate the link between the fate of other contaminants and stress in various animals. acknowledgements: funding was provided by the national science and engineering research council of canada (jh), by the bedford institute of oceanography (jh) and l’institut maurice lamontagne (cr), of the department of fisheries and oceans. we would like to thank other members of the hellou’s group who helped to collect and maintain snails in our fisheries laboratory and those in the rouleau‘s group who performed the screening of radio-labeled tissue. we also acknowledge the support of dalhousie university to the students (se and dgb) who are co-authors in this publication. references angerer j, mannschreck c, gündel j. biological monitoring and biochemical effect monitoring of exposure to polycyclic aromatic hydrocarbons. int. arch. occup. environ. health 70: 365-377, 1997. beach dg, hellou j. bioaccumulation and biotransformation of 1-hydroxypyrene by the marine whelk neptunea lyrata. int j environ anal chem. 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calf thymus dna and hela s3 cells produced by bacterial quinone metabolites of fluoranthene and pyrene. carcinogenesis 25: 1727-1733, 2004. care and use of ilyanassa obsoleta isj 8: xx-xx, 2011 isj 8: 247-255, 2011 issn 1824-307x review the evolutionary ecology of aphids' immunity m poirié1,2,3, c coustau1,2,3 1from the evolution and specificity of multitrophic interactions (esim), umr 1301 "biotic interactions and plant health" (ibsv), institut national de la recherche agronomique, inra paca, sophia antipolis, france 2 umr 6243, centre national de la recherche scientifique, cnrs, france 3 université nice sophia antipolis, ufr sciences, france accepted december 19, 2011 abstract aphids comprise 4,400 species that live in close interactions with their host-plants, the parasitoid wasps and fungi they encounter, as well as several bacteria including buchnera aphidicola, an obligatory, nutrient-providing symbiont. aphids also interact with a cohort of facultative secondary symbionts that strongly interfere with their major life history traits such as host-plant specialization, heat tolerance and resistance to natural enemies. here, we present some evolutionary and ecologically-relevant aspects of these interactions, focusing on aphid defenses to parasitism, and considering aphids either as "extended organisms" comprising aphid and symbionts' genomes, or as "single-genome" organisms whose immune components are still poorly known. we highlight the complexity of predicting evolution of aphid immune resistance in the field, due to variable selection pressures, short-term costs, and cross-talk between symbionts. finally, we present perspectives to strongly improve our understanding of the "aphid-symbiont-bacteriophage" meta-organism defenses and to elucidate the interactions between immunity, pathogenicity and symbiosis. key words: aphid; immune defenses; symbionts; parasitoids; extended phenotype, ecological immunity introduction all organisms have to maintain homeostasis and ensure growth and reproduction in changing abiotic and biotic environments. there is no doubt that one of the most challenging environmental condition is the presence of a large diversity of pathogens and parasites, and that the main host defense relies on the immune system. furthermore, the existence of a continuum from pathogenic to beneficial microorganisms is now largely admitted and current research focuses on the immune system as a main factor in the establishment and maintenance of mutualist/symbiotic interactions (slack et al., 2009; login et al., 2011). investigation of the complex interactions between immunity, pathogenicity and symbiosis mostly relies on insect models that are numerous and diversified. indeed, it is roughly estimated that more than 70 % of species host one or more bacterial symbionts (hurst and darby, 2009). ___________________________________________________________________________ corresponding author: marylène poirié evolution and specificity of multitrophic interactions (esim) umr 1301 "biotic interactions and plant health" (ibsv) institut national de la recherche agronomique inra paca, 400 route des chappes, sophia antipolis, 06903, france e-mail:marylene.poirie@sophia.inra.fr the complex pathways of innate immunity have been at least partly deciphered in model species, allowing comparative analyses to be performed. of course, as immunity is a major fitness-related trait, interest is also given to variation of some immune components in relation with biological characteristics such as the developmental stage, the morph, the sex, or the occurrence of a previous immune challenge, as well as with physical environmental characteristics, such as the temperature. evolutionary important features as the existence of trade-offs between constitutive or induced immunity and, for instance, survival or reproduction, are also considered, and the evolution of the level of specificity of the immune response is largely discussed (sadd and schmidt-hempel, 2009; schulenburg et al., 2009). however, data on all these aspects are scarce in homopteran insects and notably in one of the most representative groups, the aphids. aphids are remarkable organisms at the evolutionary and ecological level that have adapted to drastic nutritional and ecological constraints thanks to specific life-history traits and complex polymorphisms. firstly, their life-cycle is characterized by a succession of sexual and asexual morphs, dependent on the environmental 247 mailto:francedominique.colinet@sophia.inra.fr conditions. their impressive ability to proliferate thus largely relies on clonal multiplication while the resulting lack of genetic diversity is mainly compensated by a high phenotypic plasticity. dissemination and colonization of new host plants is ensured by the production of winged individuals when resources become scarce (le ralec et al., 2010). secondly, the biology of aphids is characterized by multiple inter-specific interactions. they are sapfeeding insects that establish a durable interaction with their host plant, managing to avoid or control the plant defenses, and manipulating the plant physiology to ensure the compatibility of the interaction (giordanengo et al., 2010). adaptation to the restricted phloem sieve diet is ensured by an obligate (primary) symbiosis with the bacteria buchnera aphidicola which is essential in providing the missing nutrients (amino acids) (brinza et al., 2009). aphids can also carry secondary, facultative symbionts that are not required for survival but can be mutualistic in affecting positively various life history traits such as suitability to the plant host, heat tolerance, or protection against natural enemies (montlor et al., 2002; oliver et al., 2010). finally, like all living organisms, aphids are attacked by natural enemies such as pathogenic fungi and parasitoid wasps. the immunity of aphids is of particularly interest as it likely affects the network of inter-specific interactions, therefore playing a central role in their ecology and evolution (fig. 1). understanding the functioning and evolutionary ecology of aphid immune defenses is therefore of central importance for future development of control strategies involving pathogenic agents or targeting the aphid-symbionts equilibrium. however, data in this area remain scarce. here, we present a brief overview of our current knowledge, mainly focusing on the pea aphid a. pisum whose genome has been sequenced, and which represents a good example of aphids' functioning as a meta-organism. we then discuss the evolutionary and ecologically-relevant traits of aphid biology that should be explored in the next future in relation to immune findings. the aphid complex biology aphid diversity and plasticity aphids belong to the aphidoidea and the phylloxeroidea super families of hemiptera and they comprises about 4,400 species (blackman and eastop, 1994). among these, about 250 species are agricultural pests, mainly because they vehicle and transmit plant viruses. most aphid species are found in temperate regions but some have adapted to tropical environments (dixon et al., 1986) or even extreme climates such as sub-antarctic (hullé et al., 2003), resulting in a world-wide distribution. aphids can feed on virtually all plant families, the majority of species being specialized to a single host plant, while some have a broad host-plant range (pecoud et al., 2010). aphid speciation and diversification is thought to be widely driven by their specific adaptation to host plants (pecoud et al., 2010). aphid life cycles involve a succession of sexual and asexual morphs. in the simplest cycles, such as that of the pea aphid acyrthosiphon pisum, a single sexual generation occurring in autumn alternates with several parthenogenetic generations where each female produces hundreds of viviparous offspring (helle, 1987). changes in sexual fate and reproductive mode are condition-dependent and they illustrate the aphid extraordinary developmental plasticity in response to environmental cues. altogether, whether variability of a given trait of aphids results from an existing genetic diversity among clones, as evidenced for their adaptation to the host plant, or from high phenotypic plasticity, is sometimes difficult to establish. aphid multiple inter-specific interactions one of the major characteristics of nearly all aphids is their adaptation to plant feeding through association with the obligatory nutrient-providing bacterial symbiont buchnera aphidicola. this gramnegative proteobacterium has co-evolved with aphids for 160-280 millions years (moran and baumann, 1994; wilson et al., 2010). bacteria are located only in specialized cells, the bacteriocytes, and they are transmitted vertically to the embryos. buchnera has a dramatically reduced genome (<1mb), typical of well-integrated obligatory intracellular endosymbionts, where genetic and metabolic redundancy has been minimized (gil et al., 2002; toft and andersson, 2010). it has been estimated that nearly 10 % of the coding capacity is devoted to biosynthesis of 10 essential amino acids that are lacking in the aphid phloem sap diet (wilson et al., 2010). while metabolic interactions between aphids and buchnera have been extensively studied (hansen and moran, 2011), the role of the aphid immune system in the establishment and maintenance of this mutualistic interaction remains to be examined. aphids are attacked by various enemies, notably parasitoid wasps. primary parasitoids of aphids belong to two specialized taxa, the subfamily aphidiinae (hymenoptera: braconidae) and the genus aphelinus (hymenoptera: aphelinidae). female wasps lay eggs in different developmental stages of aphids, from larvae to adults. by the time the parasitoid larvae is fully developed, the aphid dies and its cuticle hardens to form a so-called "mummy" from which an adult wasp will emerge (le ralec et al., 2010). aphids are also infected by various fungi, which generally induce death within a few days (butt et al., 1990). differences in aphid susceptibility/resistance to parasitoids or pathogens have been reported in the field (henter and via, 1995) but the underlying mechanisms are still largely unknown. finally, aphids also interact with bacterial secondary endosymbionts (oliver et al., 2010). they are facultative and found free in the hemolymph as well as within various cell types including bacteriocytes (oliver et al., 2010). interestingly, secondary symbionts can impact important fitnessrelated traits, further complicating the evolutionary ecology of aphids. for instance, serratia symbiotica has a beneficial effect on a. pisum reproduction and viability under heat stress (montllor et al., 2002), thus providing a functional explanation to the previous observations that its frequency reached 80 248 fig. 1 aphid’s immunity is likely involved in interactions with the host plant, the primary and secondary symbionts, as well as the pathogens or parasitoids. % in hot places (oliver et al., 2010). another reported symbiont effect is the change in aphid color. a recent study indeed evidenced that the presence of rickettsiella induces a body color change from red to green (tsuchida et al., 2010). such a modification is likely to affect prey-predatorparasite interactions since ladybird beetles preferentially consume red aphids while parasitoids are more attracted by green ones. finally, a largely affected fitness-related trait is adaptation to the host plant. in particular, results from several independent studies revealed a complex association between infection by regiella insecticola, the aphid genotype, and the host plant use (ferrari et al., 2007). host-plant specialization of aphids can also be directly affected, as infection by r. insecticola would improve aphids' fitness specifically on clover (tsuchida et al., 2004). most fitness-related traits and interactions of aphids with other species can therefore be diversely affected by the presence of symbionts, whether alone or in combination (oliver et al., 2006). symbiont transmission in contrast to b. aphidicola, secondary symbionts are generally transmitted vertically. however, occasional horizontal transmission has been reported. for instance, one secondary symbiont was shown to be possibly transmitted through artificial diet, and its presence was reported in aphid honeydew as well as siphuncular fluid samples (darby and douglas, 2003). the lateral transfer of symbionts may not only generate exchanges between otherwise independent clonal lines but also allow a much quicker spread of symbionts among populations. most interestingly, symbiont transmission was also reported to differ between the parthenogenetic and sexual reproduction stages. first, the maternal transfer of symbionts appeared to be far more imperfect during sexual reproduction than during parthenogenesis, which might be a source of uninfected aphids (moran and dunbar, 2006). besides, paternal transfer of symbionts could lead either to infection of previously non-infected aphids, to double infections, or to replacement of the maternal symbiont (moran and dunbar, 2006). the occurrence of paternal transfer of symbionts likely impacts their population dynamics, notably because of the possible establishment of double infections. new combinations of symbionts might indeed confer new characteristics to the host, as well as generate synergistic or antagonistic interactions. in addition, recombination events and phage gene exchanges might occur, representing a source for rapid evolutionary changes. the evolutionary ecology of aphids immune interactions one major difficulty in understanding how ecological factors, either biotic or abiotic, shape the evolution of the immune system is probably that this question that defines ecological immunology (schulenburg, 2009) is at the interface between different scientific areas. as secondary symbionts are main components of the biotic environment of aphids and strongly influence their resistance to pathogens, the study of aphid defenses belongs naturally to ecological immunity. the use of classical tools to estimate overall defenses (survival to pathogens, encapsulation ability, hemocyte numbers, phagocytic activity, phenol oxidase activity, antimicrobial activity, quantitative pcr on 249 immune-relevant genes, etc.) under various biotic and abiotic conditions is thus a major approach to explain and predict the complex interactions between symbiosis and immunity in the aphid model. an important feature at this time is also the definition of the "organism" to be considered. aphids can indeed be perceived either as species whose immune phenotype is mainly determined by their own genome, or as "extended organisms"1 (in the sense of dawkins' extended phenotype) or metaorganisms, whose defenses may result from the intricate effect of different genomes, including that of symbionts. aphids as "extended organisms" though the aphid meta-organism was reported to interact with host-plants, parasitoids and pathogenic fungi, studies have mainly focused on the "resistance to parasitoids" phenotype, and more specifically on the resistance associated with secondary symbionts. clonal resistance to braconid parasitoids has been described in populations of a. pisum (hufbauer and via, 1999; ferrari et al., 2001), myzus persicae (von burg et al., 2008) and aphis fabae (vorburger et al., 2009), although it is quite rare. in a resistant aphid, failure of the parasitoid can occur either at an early stage when the egg fails to develop or at the larval stage (li et al., 2002), and it is the "larval stage" resistance that is largely influenced by secondary symbionts. in order to understand how symbionts increase aphids' resistance, several studies have experimentally manipulated the symbiotic associations, either by suppressing symbionts using antibiotics treatment, or by introducing a new symbiont thanks to microinjection. for instance, aphidius ervi parasitism success on a. pisum was shown to be reduced by 42 % and 23 % in aphid lines harboring h. defensa and s. symbiotica respectively (oliver et al., 2003). when aphids were experimentally super-infected with both symbionts, the reduction in parasitism success reached 60 %, a benefit that correlated with a marked decrease in fecundity (oliver et al., 2006). h. defensa was also reported to provide resistance to a. ervi against aphidius eadyi (ferrari et al., 2004; oliver et al., 2009), and more recently to aphis fabae against lysiphlebus fabarum (vorburger et al., 2009). it is noteworthy that different strains of h. defensa confer various levels of protection against a. ervi (oliver et al., 2005). strikingly, however, a toxin-encoding bacteriophage, apse (a. pisum secondary endosymbiont), was demonstrated to be required, and likely responsible, for the protective phenotype (oliver et al., 2009). more recently, an independent study evidenced that a. pisum clones infected with both h. defensa and the newly discovered symbiont paxs displayed a high resistance to a. ervi (guay et al., 2009). another symbiont, regiella insecticola, was previously known to confer resistance to a fungal pathogen (scarborough et al., 2005) but not to parasitoids. however, unlike other strains of this bacterium, a specific isolate from myzus persicae provides a protection against the wasp aphidius colemani (vorburger et al., 2010). altogether, these data suggests that the ability to protect the host against natural enemies may evolve readily in different endosymbiotic bacteria, maybe in relation with occurrence of genetic exchanges and gene transfer among symbionts or phages in double-infected hosts (see above). to date, the only described mechanism explaining the symbiont-associated protection is a direct effect, involving the use of bacteriophage' toxins. however, bacterial toxins might be used as well, since most symbionts, including hamiltonella have retained virulence-associated genes in their genomes (degnan et al., 2009). alternatively, symbionts may act indirectly, through an existing host system, and, for instance, positively affect the host immunity. the evolution of symbiont-associated resistance in populations depends on the selection pressures induced by parasitism rates, on the costs associated with the presence of a given symbiont and of the cross-talk between the symbiotic companions in case of multi-infection (oliver et al., 2006). interestingly, the symbiont-associated cost may itself vary. most facultative symbionts have detrimental effects on their host fitness under sexinducing conditions (simon et al., 2011), and the estimated cost on aphis fabae longevity associated with hamiltonella depends on genotype×genotype interactions between the host and the symbiont (vorburger and gouskov, 2011). the selective advantage provided by symbionts can also vary. for instance, the resistance associated with hamiltonella's bacteriophage evolves quickly due to repeated losses of the phage under laboratory conditions, probably because of an imperfect vertical transmission (oliver et al., 2009). resistance to a. ervi in the presence of h. defensa can also change from complete protection to high susceptibility depending on the temperature (bensadia et al., 2006). finally, parasitoids exposed to h. defensa-harboring resistant clones rapidly gain virulence over time (dion et al., 2011), so that resistance is overcome, but they also experience a reduction in fitness. the case of hamiltonella well illustrates the prediction that symbiont-associated resistance may be less stable than genetic resistance (hurst and darby, 2009). indeed, the rate of vertical transmission is not always 100 %, so that bacteria can be lost, and the presence of symbionts is possibly costly, at least energetically. the complex pattern of selective advantages and disadvantages may then explain the large fluctuations of hamiltonella frequency and aphid resistance to parasitoids reported in the field. also, it may facilitate the acquisition/evolution of new resistanceassociated secondary symbionts (maybe explaining the observed resistance conferred by paxs in association with hamiltonella, guay et al., 2009). 1 the term "extended-species" we initially choose do not strictly apply to the aphid model since the host and the symbionts, including buchnera, are considered as different taxonomic units. however, we have clear examples from the long-term evolution that an intricate symbiosis can ultimately lead to the formation of a unique taxonomic entity. 250 http://www.ncbi.nlm.nih.gov/pubmed?term=%22vorburger%20c%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22gouskov%20a%22%5bauthor%5d aphids as a "single genome" species vorburger et al. (2008) has suggested that aphid parasitoids may be confronted with two lines of defense: the "innate defences” and the “acquired defences” provided by secondary endosymbionts, which likely differ in their effectiveness and specificity. significant clonal variation in resistance was indeed observed in several studies, which suggest the existence of an aphid innate resistance. for example, susceptibility of the pea aphid to a. ervi was shown to vary among clones of a single population (henter and via, 1995). although occurrence of such a "genetic" variation suggested a potential for resistance to evolve in response to selection, the average resistance remained unchanged between aphids from this population collected early or late in the summer and exposed meanwhile to a high parasitism rate (henter and via, 1995). the authors hypothesized that the lack of response to selection was due to trade-offs between resistance and other fitness-related traits. significant clonal variation and co-variation in resistance of a. pisum to two parasitoid wasps and to a pathogenic fungi was also reported, without evidence of a trade-off between resistance and fecundity (ferrari et al., 2001). on the contrary, myzus persicae effectiveness to survive a. colemani attacks was correlated with a loss of fecundity in individuals surviving the attack (vorburger et al., 2008). in other words, clones that were more resistant to the parasitoid experienced a higher loss in fecundity when attacked. such a trade-off may impair selection for resistance in natural populations and participate to the maintenance of genetic variation for resistance (vorburger et al., 2008). finally, recent work from dion et al. (2011) also demonstrated a large clonal variation in resistance to a. ervi in the absence of secondary symbionts. importantly, caution must be taken in concluding on the genetic basis of a variation in resistance since the absence of secondary symbionts has not always been assessed or accurately demonstrated. when tested, the presence of symbionts was assessed through pcr analysis based on known sequences, while novel aphid secondary symbionts are regularly described (guay et al., 2009; tsushida et al., 2010). although these studies nevertheless highlight natural variation in aphid ability to fight pathogens, the mechanisms underlying this variation are totally unknown and the involvement of the immune system has not been investigated. understanding resistance to parasitoids in aphids and their evolution thus requires a thorough study of aphids' own immune defences, as well as of the parasitoid strategies selected to avoid or circumvent all aphid defense categories. the aphid immune system: what do we know? intriguingly, neither the physiology nor the molecular biology of the immune defenses of aphids have ever attracted attention. possible reasons for that are the small size of most aphid species. also, insect immunity was primary studied on diptera and lepidoptera that are submitted to frequent bacterial challenges, while aphids belong to the homoptera and were mainly described as interacting with parasitoid wasps. ecological immunologists often estimate the immune response level by counting the number of immune cells, and measuring the phenol oxidase (po) activity. however, there are very few available descriptions of immune cells in aphids, the most detailed being by far the one of boiteau and perron (1976), which described six hemocyte categories in macrosiphon euphorbiae: prohemocytes, oenocytoids, plasmatocytes, granulocytes, spherulocytes and wax cells. surprisingly, the first data on a. pisum (laughton et al., 2011), reported only three morphologically distinct types of hemocytes: prohemocytes, granulocytes that may phagocyte bacteria, and oenocytoids. more accurate, thorough analyses, including ultra-structural description of the cells, and description of their adhesion profiles, are strongly needed to perform functional analyses. comparison of aphid hemocyte numbers from different morphs and under different environmental conditions nevertheless remains a complicated task, due to the low cell number and the quantity of debris and symbionts in the hemolymph. regarding the phenoloxidase pathway, detailed annotation work of a. pisum genome suggests that it exists in the pea aphid (gerardo et al., 2010), and a constitutive phenoloxidase activity can be measured (m poirié, personal data). whether or not it differs between morphs and can be activated by a pathogen challenge remains to be established. the question of the immune molecular processes underlying the biotic interactions of aphids is then far from being elucidated. most information comes from the recent sequencing of the first aphid genome (a. pisum) by the international aphid genomics consortium (iagc) (iagc, 2010) that has raised novel evolutionarily and functionally-relevant questions. for instance, a total of approximately 34,000 genes were predicted, which is nearly twice as described for other insect sequenced genomes belonging to diptera, coleoptera and hymenoptera (iagc, 2010; tagu et al., 2010). this is at least partly explained by the existence of many gene duplications (tagu et al., 2010). in a first search for immune-related genes in a. pisum genome, gerardo and collaborators (2010) identified key elements of the toll and janus kinase/signal transducer (jak/stat) pathways, as well as corresponding recognition and effector genes. surprisingly, however, the immune deficiency (imd) signaling pathway was apparently non functional, with some of the genes missing, and no peptidoglycan recognition proteins (pgrps) were found. in addition, well-conserved antimicrobial peptides such as defensins and cecropins could not be predicted (gerardo et al., 2010). experimental analyses were designed to characterize immune response through the isolation of rna transcripts from immune-challenged pea aphids but they uncovered few immune-related products. these data and the low expression levels of some characterized aphid immune genes suggested a low overall antibacterial immune response (altincicek, 2008; gerardo, 2010) in agreement with aphid susceptibility to experimental bacterial infection (grenier, 2006; altincicek, 2011). 251 fig. 2 studies on aphid immune-related traits should be performed in a sequential manner aimed at understanding the respective influence of aphid genotype (level 1), of the presence of secondary symbionts (level 2), and possibly the presence of phages in secondary symbionts (level 3). the parasitoid effect should be tested in combination with all these levels and the temperature effect will likely have to be considered as well. note that secondary symbionts may belong to different species or may represent different strains of a given species, therefore complexifying the approach. different evolutionary hypotheses have been proposed to explain this surprising result. for instance, aphid increased investment in reproduction following infection, or symbiontmediated host protection might "compensate" for the "deficient" immunity. this latter hypothesis implies of course that symbionts do not act indirectly through manipulation of host immunity. also, the reduced antibacterial defense was suggested to be an adaptation for the symbiosis with the bacterium buchnera aphidicola, that is known to elicit an immune response in drosophila s2 cells (douglas et al., 2011). this selection to "accommodate" the bacterial partner could have also ended in a reduced antibacterial defense specific to the bacteriome as reported in a weewil species (anselme et al., 2008) given that buchnera cells are rarely encountered outside the bacteriome. a main concern in answering the question of a “deficient” or a “different” immune system in aphids is the lack of information both on genes potentially involved in the anti-parasitoid response, and on occurrence of resistance to bacterial pathogens. besides, it is possible that a substantial part of the aphid immune genes escaped annotation due either to assembly problems or to biases, since gene prediction and identification strictly rely on similarities with genes previously described in other models. the "deficient immunity" hypothesis thus remains first to be tested accurately, taking into account other elements of the immune response such as the signaling pathways involved in cellular responses (including mapk pathways), the receptors involved in phagocytosis ability, or the reactive oxygen species-mediated defenses. future directions aphid’s life history traits, including immune performances, must be viewed as extended phenotypes (dawkins, 1989) resulting not only from the expression of the aphid genome itself, but also from the expression of genes from their bacterial symbionts and eventually from the bacterial phages. in many insects, including drosophila, bacterial symbionts can indeed positively or negatively impact host defenses against pathogens and even participate in the formation of the immune system (xie et al., 2010; weiss et al., 2011). characterization of immune traits thus have to be performed in aphids naturally or artificially deprived of secondary symbionts. this will allow subsequent comparison of different genetic backgrounds and different morphs, under different conditions (fig. 2). in a second step, it will be possible to compare immune components between genetically identical lines without secondary symbionts or with a single secondary symbiont, or different strains of this symbiont, with or without associated phages (fig. 2). this will provide essential information on how the aphid immune system and the symbionts interfere with each other, depending of the symbiont strain or species. strikingly, understanding the immune ecology of aphids as meta-organisms will also require addressing the important question of the multiple-infections and of the impact of abiotic conditions. in the field, future studies aimed at characterizing immune processes or at examining an immune-related trait, such as the ability to fight infection by a particular pathogen or parasitoid, should be carefully designed to control or characterize the extended genotype (fig. 2). the diagnostic of the presence of microorganisms by observatory methods (such as microscopy, immunelabeling or pcr) is restricted to the known symbiont species. however, the rapid progression of genomesequencing methods and facilities should allow characterization of the metagenome of aphid clones in a close future, opening the way to comparative 252 genomics of clones presenting different immunerelated traits (i.e., resistance/susceptibility to a pathogen). applied to human gut, microbial metagenomics recently revealed more than 1,000 prevalent bacterial species in a single cohort of 124 individuals, each individual hosting at least 160 of such species (qin et al., 2010), whether commensal or potentially pathogenic. this commensal microbiota is now known to shape the host immune system. aphids host a comparatively much smaller number of bacteria but they have highly intricate relationships with most of them, then appearing as good models for deciphering the interactions between immunity, pathogenicity, and symbiosis. they are also important models to address the central question of how to use our increasing knowledge on the symbiont-mediated modification of essential life-history traits for improving human and plant health. acknowledgments we are thankful to jc simon, jl gatti and a schmitz for fruitful discussions as well as to an anonymous reviewer for comments and suggestions to improve the 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successful animals in view of their great adaptability to a wide range of terrestrial niches. symbiotic bacteria gave a precious contribution to such a success playing crucial roles in different contexts such as nutrition, development, reproduction, immunity, defense against natural enemies and speciation. recently, the study of symbiosis furnished precious data not only on insect evolution, but also on the mechanisms involved in the bacterial host choice giving us new perspectives to study this process that was poorly understood up to date through the study of pathogenic interactions. key words: symbiosis; host choice; symbiont genome degeneration; insect-bacteria interaction introduction insects are undoubtedly one of the most successful animal group in nature in view of the high number of species and the high number of individuals observed in insect population. the success of insects is due to their remarkable adaptability to a vast array of terrestrial habitats, including those that are strongly limited or imbalanced in nutrients and to their ability to face pathogens (such as bacteria). nevertheless, insect success is also due to the collaboration with bacteria in term of symbiosis since bacteria play crucial roles in the biology and life cycle of most insects species, affecting nutrition, development, reproduction, immunity, defense against natural enemies and speciation (buchner, 1965; moran and baumann, 2000; moran, 2001; moran, 2006).  up to date several papers faced the relationship between bacteria and insects in term of insect defense so that most of the attention has been put on insect pathogens and antimicrobial peptides synthesized by insects or onto other strategies that they set up to avoid bacterial infection (mandrioli et al., 2003; brivio et al., 2005; schmidt et al., 2005; brown and hancock, 2006; lemaitre and hoffmann, 2007; lazzaro, 2008; müller et al., 2008). ___________________________________________________________________________ corresponding author: mauro mandrioli department of animal biology university of modena and reggio emilia via campi 213/d, 41100 modena, italy e-mail: mauro.mandrioli@unimo.it however, in the last years the interaction between insects and bacteria has been studied with particular attention to symbiosis and, interestingly, the study of mutualism and symbiosis is furnishing several intriguing evidences about the host choice giving us new data about this process that has been poorly understood up to date through the study of pathogenic interactions (mandel et al., 2009). symbioses are categorized according to the extent of dependence between the host and the symbionts, which generally depends on evolutionary antiquity of the symbiosis (moran and baumann, 2000; moran, 2001). while obligate primary symbionts are essential for the host survival and/or reproduction, secondary are facultative and thought to be of more recent acquisition, even though they can contribute to the fitness of the host, e.g., conferring resistance to parasites (moran and baumann, 2000; moran, 2001). most primary symbionts are vertically transmitted to the progeny with a process starting at early stages of oogenesis or embryogenesis. vertical transmission is common also in secondary symbionts, but they can also colonize novel hosts through horizontal transmission among host individuals belonging to the same or different species (dale and moran, 2006). sequencing of bacterial genomes is facilitating our understanding of the relationships between insects and their symbionts bringing to a better comprehension of the genome interdependence that occurs between host and bacteria (zientz et al.,     98 fig. 1 whole mount in situ hybridization: analysis of the distribution of fitc-labeled (green) asaia bacteria in anopheles gambiae salivary glands stained with propidium iodide (red) observed by confocal microscopy. bar = 100 μm (a). magnification of a portion of the left salivary gland showing the presence of asaia in the gland duct. bar = 100 μm (b). 2001; feldhaar and gross, 2009). in particular, symbiosis results in a genome reduction in endosymbiotic bacterial lineages that loose preferentially genes involved in catabolic pathways, since these functions may be played by the insect metabolism (zientz et al., 2001; feldhaar and gross, 2009). genome reduction may also affect the anabolic pathways if symbionts succeed in recruiting metabolic precursors from the host cell metabolisms bringing to a further rationalization of the symbiont’s genome (andersson and kurland, 1998; andresson and andersson, 1999; goebel and gross, 2001; moran and mira, 2001). interestingly, genome degeneration could be a key aspect not only in the study of symbiont genome evolution, but in the understanding of the host choice, since genome degeneration can not affect genes that are essential for the interaction with the host, neither genes that serve to avoid the exposition of bacteria to the host’s immune system. therefore, the occurrence of smaller genomes can make symbionts perfect experimental models to test the role of different genes in the host-bacteria interaction. examples include three genomes of buchnera aphidicola strains from different aphid hosts, two of candidatus blochmannia species from ants, one of wigglesworthia glossinidia from tsetse flies, and one each of candidatus baumannia cicadellinicola and candidatus sulcia muelleri from leafhoppers (dale and moran, 2006; mccutcheon and moran, 2007). their genomes are below one megabase in size and are known to encode as few as 500 genes. an extreme case is that of carsonella ruddii, a primary symbiont of psillids, that has a genome of 160 kb, the smallest bacterial genome described so far (nakabachi et al., 2006). in contrast, escherichia coli and other free-living relatives in this group have genomes of about four to five megabases encoding some 5000 genes. despite these advances, however, the mechanisms by which host-symbiont specificity develops in animal-bacterial interactions are not clear. many animals, including humans, are born devoid of symbionts and must recruit their microbiota from the environment and the process by which hosts and symbionts find each other to initiate a mutualism must be sensitive enough to identify the correct partner even when the symbiont is a minority constituent of the microbial community, and specific enough to exclude interlopers from gaining access to the host (mandel et al., 2009). the species specificity is also poorly understood for pathogenic interactions and at present is very difficult to explain why similar congeneric bacteria have distinct host ranges as reported, for example, in salmonella and brucella species (edwards et al., 2002; rajashekara et al., 2004). attempts to understand the molecular basis of host specificity have been unsuccessful in many pathogen-host animal interactions, including humans. salmonella enterica serovar typhi, for instance, can infect humans only, whereas serovar typhimurium has a broad range of hosts that includes mice, although the genomes of these two strains are over 97 % identical (edwards et al., 2002). similarly, different brucella species share over 98 % identity across 90 % of their genes, but exhibit strict host specificity (rajashekara et al., 2004). in contrast, the study of mutualism is providing insights into how specificity develops. for     99     100 instance, works from many laboratories has established nitrogen-fixing, nodulating rhizobia as the best-understood system for the development and evolution of host specificity in plant-associated bacteria (long, 2001). a strong confirmation of the hypothesis that symbionts may favour our understanding of the mechanisms involved in host choice better than pathogens has been recently published by mandel and colleagues (2009) showing that a single regulatory gene is sufficient to alter host range in an animal-bacterial mutualism, suggesting that the same could be true in the host-pathogen interaction. despite the relevance of this paper, however, the fundamental biological questions on how animalbacterial partnerships are established is still difficult to access and it is still impossible to define when bacteria passed the thin line that separates patogenicity and mutualism/symbiosis (gilmore and ferretti, 2003). in insects, some good candidates for taking a glance into the mechanisms involved in host choice are already present and in particular bacteria of the genus asaia could be perfect experimental models since they are cultivable in vitro (that is not a common feature for symbionts), can be manipulated at a chromosomal level in order to obtain stable transgenic strains and can be used for study of colonization of the insect body (favia et al., 2007). asaia belong to the group of the acetic acid bacteria that can be identified in virtue of their ability to oxidize ethanol into acetic acid even if asaia differentiates because it does not (or weakly) oxidize ethanol to acetic acid. besides tropical plants, where it was originally isolated (malimas et al., 2008), asaia has thus far been found associated to the insects scaphoideus titanus, the leafhopper vector of the phytoplasma causing flavescence dorée, a severe disease of grapevine (marzorati et al., 2006), and three mosquito vectors of malaria, anopheles stephensi, anopheles maculipennis and anopheles gambiae. in particular asaia has been found stably associated with larvae and adults of a. stephensi, dominating the microbiota of the mosquito (favia et al., 2007). the distribution of asaia in the body of a. stephensi has been investigated by the use of a strain, previously isolated from the mosquito, after genetic modification to express a green fluorescent protein (gfp). the gfp-tagged strain efficiently colonized the gut, salivary glands, and male and female reproductive organs. it is noteworthy that asaia, after assumption with a sugar-based diet by females, was detected in the gut and then in the salivary glands of the insect (fig. 1), crucial organs for the development of the cycle of the malaria parasites plasmodium spp. (favia et al., 2007). by using fluorescent strains it was shown that in a. stephensi, asaia is vertically transmitted from the mother to offspring (favia et al., 2007), but also undergoes paternal transmission to the progeny, by the way of venereal transfer from male to female during mating (damiani et al., 2008). the efficient capacity of asaia of colonizing adults and larvae of a. stephensi and the discovery of this bacterium in other insect vectors (i.e., other anopheles species and scaphoideus titanus) rise the question of whether this bacterium can crosscolonize different insect hosts. the reply to this question could be very useful not only in order to better understand asaia biology, but also to verify which asaia genes may be involved in the host choice that is necessary for establishing a symbiotic interaction. finally, it is important to underline that the investigation of the basis of host-symbiont interaction could be very useful also from an applicative point of view since asaia represents a promising bacterial species for the development of asaia-based symbiotic control approaches to block parasite transmission by insect vectors (favia et al., 2008). the symbiotic control approach would utilize bacteria capable of colonizing the insect body to produce effector molecules (natural or transgenic in the paratransgenic models) that kill or inhibit the causative agent of the disease or interfere with the survival of parasitic insects (beard et al., 2001). considering the localization in the insect body, the capability of colonizing very different hosts the culturability and the genetic transformability, asaia may be also accounted as potential interesting agents for natural or paratransgenic symbiotic control opening new perspectives also from an applicative point of view. acknowledgements this work is supported by the grant "far" from the university of modena and reggio emilia (mm). references andersson jo, andersson sg. insights into the evolutionary process of genome degradation. curr. opin. genet. dev. 9: 664-671, 1999. andersson sg, kurland cg. reductive evolution of resident genomes. trends microbiol. 6: 263268, 1998. beard cb, dotson em, pennington pm, eichler s, cordon-rosales sc, durvasula sr. bacterial symbiosis and paratransgenic control of vectorborne chagas disease. int. j. parasitol. 31: 621-627, 2001. brivio mf, mastore, m, pagani m. parasite-host relationship: a lesson from a professional killer. inv. surv. j. 2: 41-53, 2005. brown kl, hancock re. cationic host defense (antimicrobial) peptides. curr. opin. immunol. 18: 24-30, 2006. buchner p. endosymbiosis of animals with plant microorganisms. wiley interscience, new york, 1965. dale c, moran, na. molecular interaction between bacterial symbionts and their 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comparative whole-genome hybridization reveals genomic islands in brucella species. j. bacteriol. 186: 5040-5051, 2004. schmidt o, rahman mm, ma g, theopold u, sun y, sarjan m, et al. mode of action of antimicrobial proteins, pore-forming toxins and biologically active peptides (hypothesis). inv. surv. j. 2: 8290, 2005. mauro mandrioli acknowledgements isj 9: yyy-xxx, 2012 isj 9: 163-168, 2012 issn 1824-307x minireview utilization of a silkworm model for understanding host-pathogen interactions c kaito, h yoshikai, k sekimizu graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan accepted september 27, 2012 abstract studies of the interactions between humans and pathogenic microorganisms require adequate representative animal infection models. further, the availability of invertebrate models overcomes the ethical and financial issues of studying vertebrate materials. insects have an innate immune system that is conserved in mammals. the recent utilization of silkworms as an animal infection model led to the identification of novel virulence genes of human pathogenic microorganisms and novel innate immune factors in the silkworm. the silkworm infection model is effective for identifying and evaluating novel factors involved in host-pathogen interactions. key words: insect model; innate immune factor; bacteria; fungi; virulence factor advantages of the silkworm as an animal infection model invertebrate animals possess an innate immune system, but lack an acquired immune system. many aspects of the innate immune system of invertebrate animals are conserved in mammals. for example, cationic antimicrobial peptides and toll receptors recognizing pathogens are found in both invertebrate animals and mammals (okada and natori, 1983; hoffmann, 1995; natori, 2010). therefore, studies using invertebrate animals can be performed to develop a better understanding of the host-pathogen interactions in mammals without the ethical and financial issues (seabra and bhogal, 2009). silkworms are larvae of the moth bombyx mori, a lepidopteran species (fig. 1). silkworms form cocoons where they develop into pupae. humans have used these cocoons as raw materials for silk for over 5000 years (goldsmith et al., 2005). bombyx mori is the only domesticated insect species, and the silkworm cannot survive in the natural world, probably due to their ineffective locomotion. in contrast to wild insects, silkworms can barely bite human fingers or escape from a breeding cage. silkworms typically consume mulberry leaves, but an artificial diet for silkworms has also been established and is commercially available. thus, rearing silkworms in the laboratory is easy. studies of host-pathogen interactions require quantitative evaluation of the virulence properties of ___________________________________________________________________________ corresponding author: chikara kaito graduate school of pharmaceutical sciences the university of tokyo 7-3-1, hongo, bunkyo-ku, tokyo, 113-0033, japan e-mail: kaito@mol.f.u-tokyo.ac.jp pathogenic microorganisms. to evaluate pathogenic virulence quantitatively, injection of a precise amount of the pathogen solution into model animals is essential. the large body size of the fifth instar silkworm (~ 5 cm) allows for the injection of a very precise amount of the pathogen solution into the silkworm hemolymph using a tuberculin syringe equipped with a 27-gauge needle (kaito and sekimizu, 2007), whereas injection of a precise sample amount is more difficult in small body-sized invertebrates such as drosophila melanogaster and caenorhabditis elegans. injection of human pathogenic bacteria such as staphylococcus aureus and pseudomonas aeruginosa into the silkworm hemolymph kills the silkworm (kaito et al., 2002). s. aureus injected into silkworms proliferates in the hemolymph. the lethal effects of s. aureus injection in silkworms are blocked by the injection of antibiotics. these observations suggest that the lethal effects of s. aureus in silkworms require bacterial proliferation (kaito et al., 2002). the silkworm-s. aureus infection model allows for the identification of biologic molecules involved in the ability of s. aureus to escape various innate immune factors of the silkworm and to proliferate in the silkworm hemolymph. importantly, infection experiments using silkworms can be performed at 37 ˚c, the temperature at which most human pathogenic microorganisms exhibit high virulence properties (kaito et al., 2011). genetic and biochemical analyses of silkworms are essential for identifying biologic molecules of silkworms that are involved in host-pathogen interactions. the bombyx mori genome project was recently completed and genome data are now 163 fig. 1 5th instar larvae of bombyx mori. tuberculin syringe equipped with a 27-gauge needle is shown above the silkworm. available on line (shimomura et al., 2009). in addition, construction of transgenic silkworms is established (tomita, et al., 2003). for biochemical analysis, biologic molecules from crude silkworm biomaterials must first be purified and identified. a fifth instar silkworm weighs around 2 grams, and thus an adequate amount of silkworm biomaterial can easily be prepared for purifying biologic molecules. identification of bacterial and fungal virulence factors using silkworms s. aureus is a pathogenic gram-positive bacterium present in the noses of 30 % of healthy individuals. to identify novel virulence factors of s. aureus, 100 hypothetical genes that are conserved among bacteria were disrupted and examined for lethal activity against silkworms. gene-disrupted mutants of three novel genes, named cvfa, cvfb, and cvfc (conserved virulence factor a, b, and c), exhibited attenuated lethality in silkworms (kaito et al., 2005) (table 1). these gene-disrupted mutants also showed attenuated virulence in mice, indicating that these genes contribute to the virulence of s. aureus not only in insects but also in mammals (kaito et al., 2005; matsumoto et al., 2007; marincola et al., 2012). streptococcus pyogenes is a human pathogenic gram-positive bacterium that causes various diseases, including adenoiditis and necrotizing fasciitis. the cvfa gene is also required for the lethality of s. pyogenes in silkworms and mice, and it is involved in the expression of various genes in s. pyogenes (kaito et al., 2005; kang et al., 2010; kang et al., 2012) (table 1). the cvfa gene is required for hemolysin production in both s. aureus and s. pyogenes. cvfa protein is a cyclic phosphodiesterase that cleaves a 2’,3’-cyclic phosphodiester linkage at the 3’-terminal nucleotide of rna (kaito et al., 2005; nagata et al., 2008). the cvfb gene contributes to s. aureus hemolysin production via a virulence regulatory gene, agr (matsumoto et al., 2007). crystal structure analysis revealed that cvfb has a novel l-shaped structure comprising three s1 rna binding domains and a winged-helix domain (matsumoto et al., 2010). the cvfc gene contributes to s. aureus resistance to detergents via the expression of thymidylate synthetase (ikuo et al., 2010). these novel virulence factors are conserved in many human pathogenic bacteria and their molecular functions are different from those of other well-known virulence factors. to determine whether s. aureus virulence factors against mammals contribute to s. aureus lethality in silkworms, s. aureus gene-disrupted mutants of hemolysins, cell wall proteins, and virulence regulators were examined for their attenuated lethality against silkworms (miyazaki et al., 2012) (table 1). the results demonstrated that s. aureus hemolysins are not required for virulence in silkworms. in contrast, several cell wall proteins and virulence regulators are required for s. aureus lethality in silkworms. thus, although not all s. aureus virulence factors against mammals can be evaluated in silkworms, silkworms are useful for evaluating the effects of s. aureus cell wall proteins and virulence regulators. that is, interactions between the host animal and s. aureus cell wall proteins or between the host animal and s. aureus virulence regulators are conserved among invertebrates and vertebrates. the silkworm model is also applicable for evaluating virulence factors of gram-negative human pathogenic bacteria. enterohemorrhagic escherichia coli (ehec) is a human pathogen that causes encephalopathy and nephropathy. ehec o157:h7 produces shiga toxins that are toxic to mammalian cells. the ehec gene-deleted mutant of shiga toxin exhibits attenuated virulence in a mouse infection model (eaton et al., 2008), but not in a silkworm model (miyashita et al., 2012). in contrast, the ehec gene-deleted mutant of lipopolysaccharide (lps) o-antigen synthase showed attenuated lethality in both silkworms and mice (miyashita et al., 2012) (table 1). the lps o-antigen mutant of ehec is sensitive to both silkworm and porcine antimicrobial factors (miyashita et al., 2012). therefore, lps o-antigen is required for the lethal effects of ehec in silkworms and mice via conferring resistance against innate immune factors of insects and mammals. a transposon mutant library of serratia marcescens, a 164 table 1 summary of biologic molecules identified in the silkworm infection model pathogenic microorganism gene category function references gram-positive bacteria staphylococcus aureus cvfa regulator 2', 3'-cyclic phosphodiesterase (kaito et al., 2005) cvfb regulator rna binding protein (matsumoto, et al., 2010) cvfc regulator conributing to detergent resistance (ikuo et al., 2010) sarz regulator transcription factor (kaito et al., 2006) agr regulator transcription factor and regulatory rna (miyazaki et al., 2012) saers regulator a two-component regulatory system (miyazaki et al., 2012) arlrs regulator a two-component regulatory system (miyazaki et al., 2012) srta cell wall protein anchoring proteins to cell wall (miyazaki et al., 2012) clfb cell wall protein binding mammalian cytokeratins (miyazaki et al., 2012) fnbb cell wall protein binding mammalian fibronectin (miyazaki et al., 2012) sdrc cell wall protein adherence to mammalian epithelial cells (miyazaki et al., 2012) streptococcus pyogenes cvfa regulator 2', 3'-cyclic phosphodiesterase (kaito et al., 2005) gram-negative bacteria enterohemorrhagic escherichia coli rfbe lipopolysaccharide lipopolysaccharide o-antigen synthesis (miyashita et al., 2012) waal lipopolysaccharide lipopolysaccharide o-antigen ligation (miyashita et al., 2012) serratia marcescens weca lipopolysaccharide lipopolysaccharide o-antigen synthesis (ishii et al., 2012) flhd flagella flagella synthesis (ishii et al., 2012) flir flagella flagella synthesis (ishii et al., 2012) pseudomonas aeruginosa toxa toxin exotoxin a (chieda et al., 2011) exos toxin type iii effector protein (okuda et al., 2010) sodm stress response manganese-superoxide dismutase (iiyama et al., 2007) sodb stress response iron-superoxide dismutase (iiyama et al., 2007) fungi cryptococcus neoformans gpa1 regulator g-protein alpha subunit (matsumoto et al., 2012) pka1 regulator catalytic subunit of protein kinase a (matsumoto et al., 2012) cna1 regulator catalytic subunit of calcineurin (matsumoto et al., 2012) candida albicans cmp1 regulator protein phosphatase (hanaoka et al., 2008) yvh1 regulator protein phosphatase (hanaoka et al., 2008) sit4 regulator protein phosphatase (hanaoka et al., 2008) ptc1 regulator protein phosphatase (hanaoka et al., 2008) candida glabrata cyb2p metabolism lactate dehydrogenase (ueno et al., 2011) host animal silkworms apolp-ii/i virulence inhibitor suppressing s. aureus hemolysin production (hanada et al., 2011) pp cytokine inducing innate immune responses (ishii, et al., 2010) silkworm hybrid (kinshu × showa) was used in studies of p. aeruginosa (iiyama et al., 2007; chieda et al., 2011). silkworm hybrid (hu • yo × tukuba • ne) was used in other studies. 165 human pathogenic gram-negative bacterium, was screened for its attenuated lethality in silkworms, leading to the identification of lps o-antigen synthase as the factor required for silkworm lethality (ishii et al., 2012). exotoxin a, a type iii effector protein exos, and superoxide dismutase of p. aeruginosa, which are virulence factors in mammals, are also required for killing silkworms (iiyama et al., 2007; okuda et al., 2010; chieda et al., 2011) (table 1). in contrast, p. aeruginosa pyocyanin, which is a virulence factor in mammals, is not required for killing silkworms (chieda et al., 2008). many factors in gram-negative bacteria are required for virulence in both silkworms and mammals, although some factors are specifically required for virulence in mammals. several virulence factors of human pathogenic fungi, including cryptococcus neoformans, candida glabrata, and candida albicans, were identified by infecting silkworms with gene-deletion mutants (hanaoka et al., 2008; ueno et al., 2011; matsumoto et al., 2012). gene-deletion mutants of the virulence factors of c. neoformans and c. albicans in mammals showed attenuated virulence in silkworms (hanaoka et al., 2008; matsumoto et al., 2012) (table 1). cyb2p of c. glabrata and ptc2 of c. albicans have been identified as virulence factors in silkworms and these genes are also required for virulence in mice (hanaoka et al., 2008; ueno et al., 2011) (table 1). these results suggest that human pathogen virulence factors of gram-positive bacteria, gram-negative bacteria, and fungi can be identified and evaluated in a silkworm model by infecting silkworms with gene-disrupted mutants. identification of innate immune factors in silkworms injection of s. aureus hemolysins into silkworms kills silkworms (hossain et al., 2006). in contrast, s. aureus hemolysin gene-deleted mutants did not exhibit attenuated killing ability against silkworms (miyazaki et al., 2012). these findings suggest that silkworm hemolymph contains a factor that inhibits s. aureus hemolysin production. a lipid carrier protein, apolipophorin (apolp), purified from silkworm hemolymph shows inhibitory activity against s. aureus hemolysin production (hanada et al., 2011) (table 1). the addition of apolp to s. aureus culture decreases the expression of saers, which is a positive regulator of s. aureus hemolysin genes. injection of anti-apolp antibodies into silkworms sensitizes silkworms against s. aureus. these findings suggest that apolp inactivates s. aureus saers and decreases hemolysin expression, leading to silkworm resistance against s. aureus. mammalian mucin also inhibits s. aureus hemolysin production, indicating that resistance to infection by the inhibition of hemolysin production is conserved among insects and mammals. most innate immune factors contribute to infection resistance by killing pathogenic microorganisms. novel innate immune factors that do not inhibit bacterial proliferation and inhibit bacterial virulence are not well understood. in addition to apolp, apolipoprotein b in mammalian blood and hydrogen peroxide produced by macrophages inhibit s. aureus virulence (rothfork et al., 2004; peterson et al., 2008). apolp is the first invertebrate biologic molecule found to inhibit bacterial virulence. silkworm hemolymph contains a cytokine-like peptide named paralytic peptide (pp) (ishii et al., 2008) (table 1). pp is synthesized as an inactive precursor and constitutively exists in silkworm hemolymph. bacterial peptidoglycans and fungal glucans induce reactive oxygen species (ros) from silkworm hemocytes and ros activate serine protease. the activated serine protease digests the pp precursor to produce matured pp. the matured pp activates humoral and cellular immune responses, including phagocytosis by silkworm hemocytes, phosphorylation of p38 mitogen-activated protein kinase, and production of antimicrobial peptides (ishii et al., 2010). because injection of the anti-pp antibody into silkworms sensitizes silkworms against s. aureus (ishii et al., 2008), pp contributes to silkworm resistance against s. aureus. pp was originally identified as a biologic molecule that induces muscle contraction in silkworms (ha et al., 1999). the biologic significance of the muscle-contracting activity of pp in the innate immune system is unknown. concluding remarks this minireview describes biologic molecules identified in the silkworm infection model. in most cases, the biologic molecules identified in the silkworm infection model are involved in mammalian host-pathogen interactions. utilization of a multitude of silkworms allows for quantitative evaluation of the virulence of many gene-disrupted mutants of pathogenic microorganisms. the silkworm infection model will be a powerful tool to further our understanding of host-pathogen interactions. references chieda y, iiyama k, lee jm, kusakabe t, yasunaga-aoki c, shimizu s. inactivation of pyocyanin synthesis genes has no effect on the virulence of pseudomonas 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row, mardyke, cork, ireland 2departamento de bioquímica y biología molecular, university of cordoba, campus de rabanales, córdoba 14071, spain accepted september 3, 2008 abstract bivalves are of major importance in aquatic ecology, aquaculture, are widely used as sentinel species in environmental toxicology and show remarkable plasticity to molecular oxygen. excess reactive oxygen species (ros) arising from molecular oxygen can cause oxidative stress and this is also a consequence of exposure to many common environmental pollutants. indices of oxidative stress have therefore found favor as biomarkers of exposure and effect in environmental toxicology. however, there is a growing body of literature on the use of discovery-led proteomics methods to detect oxidative stress in bivalves. this is because proteins absorb up to 70 % of ros leading to complication of the proteome. this article explores the background to these developments and assesses the practice and future potential of proteomics in the study of oxidative stress in bivalves. key words: bivalve; oxidative stress; mussel; clam; ecotoxicology introduction molecular oxygen (o2) first accumulated on earth ~ 2.3 billion years ago due to the appearance of photosynthesis. the redox characteristics of the earth’s atmosphere fundamentally altered from reducing to strongly oxidizing and living cells, with reducing internal environments, for the first time needed to expend considerable energy to survive the surrounding oxidizing environment. oxygen is paradoxical in that it is on the one hand essential for the most efficient form of energy metabolism; aerobic metabolism. on the other hand, it is a potential chemical threat because it can lead to formation of reactive oxygen species (ros) (halliwell and gutteridge, 2007; winterbourn, 2008). these include species such as h2o2, the hydroxyl and superoxide radicals (fig. 1) which are naturally formed in living cells especially in subcellular organelles such as the mitochondrion and endoplasmic reticulum. cells developed elaborate ___________________________________________________________________________ corresponding author: david sheehan proteomics research group, dept. biochemistry, university college cork, lee maltings, prospect row, mardyke, cork, ireland e-mail: d.sheehan@ucc.ie strategies to cope with ros as part of adaptation to their changed redox situation including antioxidant enzyme activities (e.g., catalases), small antioxidant molecules (e.g., glutathione, gsh; vitamin e) and redoxins (e.g., thioredoxins). under normal circumstances, these defenses cope well with the levels of ros produced and cells exist in a state of redox homeostasis. however, in certain circumstances, the balancing act between ros production and antioxidant defences tilts in favor of build-up of ros in the cell leading to a state of oxidative stress (halliwell and gutteridge, 2007). bivalves (informally including mussels, clams, oysters and scallops) comprise animals of the class bivalvia in the phylum mollusca which first appeared late in the cambrian explosion (~ 530 million years ago) and eventually came to dominate over brachiopods in the palaezoic era (gould and calloway, 1980). they are characterized by a shell which is divided from front to back into left and right valves, have adapted their gills as filter-feeding organs and often have a well-developed byssus apparatus allowing them to attach to rocky substrates (bayne, 1976). the precise reason for the evolutionary success of bivalves is a matter for conjecture (gould and calloway, 1980; miller, 1998), but it is evident that they have shown considerable resilience and now occupy niches in a 110 mailto:d.sheehan@ucc.ie fig. 1 oxidative stress arises when there is an imbalance between production and neutralisation of ros. these can arise from endogenous enzyme mechanisms or from exogenous sources such as metals and pahs. oxidative stress can result in modification of biomolecules, especially proteins and lead to toxicity. wide range of aquatic environments. they are often the major macrofauna on rocky substrates of littoral, shallow sub-littoral and deep-sea vents (bayne, 1976; lutz and kennish, 1993). their filter-feeding habit adds greatly to their ecological significance in that bivalves are important calcium and carbon accumulators, they link primary producers (bacteria and phytoplankton) with higher organisms in aquatic food-chains and are responsible in tidal zones for filtration of the water body (newell, 2004). since most human interaction with the aquatic environment is concentrated along rivers, coastlines and estuaries (halpern et al., 2008), bivalves have importance in addition to their evolutionary and ecological significance. they are an important foodsource for human populations and can be cultured for food and other reasons (naylor et al., 2000; newell, 2004). bivalves have genomes generally comparable in size to the human genome yet they are poorly represented in dna sequence databases. for this reason, there is growing interest in bivalve genomics as a means of elucidating evolutionary, genetic and toxicological relationships (mckillen et al., 2005; tanguy et al., 2008). because of their sedentary lifestyle, filterfeeding habit, abundance, ease of identification, tolerance to pollution and wide geographical distribution bivalves have found particular applications as sentinel species in environmental toxicology (ecotoxicology) (bayne, 1979; goldberg and bertine, 2000). this has led to a considerable body of research focusing on using levels of particular biomolecules (biomarkers) to reveal effects of environmental pollutants and give insights to the pollution status of specific sampling sites (cajaraville et al., 2000; handy et al., 2003; depledge and galloway, 2005; galloway, 2006). since many environmental pollutants such as heavy metals, polyaromatic hydrocarbons (pahs), polychlorinated biphenyls (pcbs) and nanomaterials are known to be strongly pro-oxidant, much of this research has focused on biomarkers reflecting oxidative stress (viarengo et al., 1991; lópez-barea and pueyo, 1998; lesser, 2006; valavanidis et al., 2006). it has increasingly become clear that such studies also need to allow for effects due to non-pollution variables such as seasonality, nutritional status and the normal redox variations inherent in the bivalve lifestyle (power and sheehan, 1996; manduzio et al., 2004). more recently, interest has grown in extending this essentially hypothesis-driven biomarker approach to a discovery-driven approach in which highthroughput proteomics techniques are applied to bivalves to identify novel biomarkers for exposure to environmental pollution. this review explores how bivalves cope with ros under normal circumstances, describes the biomarker approach to study of oxidative stress and reviews the technology and opportunities offered by proteomics in this important area of research. bottlenecks to exploitation of this experimental approach are briefly described. reactive oxygen species and oxidative stress in aerobic metabolism, molecular oxygen is eventually reduced to water. in this process, a 111 variety of oxygen derivatives, collectively called ros, are naturally produced. these include the superoxide anion radical (o2• -), hydrogen peroxide (h2o2), peroxyl radicals (roo•), nitrogen oxide (no•) and hydroxyl radical (ho•) (lesser, 2006; winterbourne, 2008). some ros contain unpaired electrons (i.e. they are free radicals) whilst others are non-radical species (e.g., h2o2) (fig.1). ros can also be formed in response to a wide range of exogenous agents including radiation (x-ray, gamma, uv, or visible light in the presence of a sensitizer), metal ions, solvents, particulate matter, nitrogen oxides, ozone etc. (davies, 2005). the ho• is the most important ros in biology with an oxidation rate constant for protein components comparable to the rate of diffusion ~ 108-10 m-1 s-1 (davies, 2005; winterbourn, 2008). due to its high reactivity ho• is quite non-specific in its targets for oxidation, whereas ros with lower rate constants react more specifically (davies, 2005; winterbourn, 2008). ros are generally removed rapidly by antioxidant mechanisms as they can affect major cellular components including lipids, proteins, carbohydrates and dna and can ultimately lead to cell death (fig. 1). ros can cause serious toxicity because they are capable of interacting rapidly and efficiently with important biomolecular targets (davies, 2005; valavanidis et al., 2006; winterbourne, 2008). the most toxic ros is ho• which can attack biological membranes in a diffusion-controlled fashion, initiating free radicalmediated chain reactions (davies, 2005; lesser, 2006; winterbourn, 2008). ros capable of diffusion across biological membranes (e.g., h2o2) can enter into numerous other reactions and so cause effects at a distance from their site of formation. the extent of damage which ros can generate is dependent on a number of factors including: the concentration of target, the rate constant for reaction of oxidant with the target, the location of the target when compared to the site of oxidant formation, the occurrence of secondary events, the occurrence of oxidant-scavenging reactions and repair reactions (davies, 2005; winterbourn, 2008). oxidative stress in bivalves: what is “normal”? oxidative stress refers to a state where there is an imbalance between the generation and neutralisation of ros by antioxidants, caused by excessive production of ros, loss of antioxidant defenses or both (halliwell and gutteridge, 2007). several aspects of bivalve biology make oxidative stress of especial significance. bivalves can be exposed to relatively high levels of pro-oxidants as a consequence of their filter-feeding habit. common environmental pollutants known to be pro-oxidants such as pcbs, pahs, heavy metals and organochlorines are bioconcentrated within bivalves leading to oxidative stress (viarengo et al., 1991; rodríguez-ariza et al., 1992, 1993, 2003; cheung et al., 2001; downs et al., 2002; rodríguez-ortega et al., 2002; valavanidis et al., 2006). moreover, intertidal animals exposed to air during the tidal cycle face particular challenges. these only survive dehydration because of the integrity of the seal between the two halves of their shell. however, this seal also cuts off access to oxygen from the animals’ surroundings and oxygen inside the shell is rapidly depleted (stachowitz et al., 2007). depending on local conditions, individual animals may be exposed to air for a comparatively short time-period (at the lower end of the intertidal zone) or for up to 7 h in every 12 (at the highest end of the intertidal zone). when exposed to air, animals soon experience hypoxia which can turn to anoxia in the more extreme circumstances of the highest part of the intertidal. this is a significant cause of mortality and individual animals can physiologically adapt to this challenge (widdows et al., 1979; altieri, 2006; stachowitz et al., 2007). normal aerobic metabolism is very quickly depressed with glycolytic fermentation progressively increasing (widdows et al., 1979; widdows and shick, 1985). on reimmersion, the bivalve opens its shell and experiences a rapid increase in oxygen level. simultaneously, oxidative metabolism resumes (widdows and shick, 1985) resulting in a burst of ros similar to the reperfusion-ischemia injury of mammals (grace, 1994). thus, as a routine part of their life-cycle, bivalves must have sufficient plasticity to cope with relatively large fluctuations in oxygen levels which can be further modulated by factors such as seasonality, pollution exposure and nutritional status (viarengo et al., 1991; rodríguezariza et al., 1992, 1993, 2003; power and sheehan, 1996; manduzio et al., 2004; lesser, 2006). moreover, recent research (primarily in mammalian systems) suggests that, even in sub-stress scenarios, redox modifications may be an important aspect of normal cell signalling capable of triggering apoptosis (biswas et al., 2006; ying et al., 2007; oktyabrsky and smirnova, 2007; poole and nelson, 2008). while outside the scope of this review, bivalve species will no doubt be a fruitful area for future research into this important role of ros. oxidative stress in bivalves: the biomarker approach bivalves have found especial favor as “sentinels” of environmental pollution and have come to be widely used in pollution surveillance programmes (bayne, 1979; goldberg and bertine, 2000). biomarkers are indices of the presence of chemical pollutants in particular environmental contexts (cajaraville et al., 2000; handy et al., 2003; depledge and galloway, 2005; galloway, 2006). as has been pointed out (power and sheehan, 1996; manduzio et al., 2004) effects of environmental pollutants are to some extent dependent on biological (seasonal, nutritional and reproductive status) and environmental variables (e.g., temperature, exposure to sunlight). bivalves have evolved robust biochemical and physiological defenses against chemical pollution and the varying levels of oxygen exposure to which they are subject (widdows et al., 1979; widdows and shick, 1985). they have an extensive battery of antioxidant defenses (winston, 1991; valavanidis et al., 2006) and variations in these were found to be useful in environmental monitoring (rodríguez-ariza et al., 1992, 1993, 2003; sole et al., 1996; regoli et al., 2002; rodríguez-ortega et al., 2002; manduzio et 112 al., 2004). other important biomarkers include glutathione transferases (fitzpatrick et al., 1995; lyons et al., 2003), heat shock proteins (sanders, 1993; lyons et al., 2003), dna damage (steinert, 1999), ubiquitination (hofmann and somero, 1995; buckley et al., 2001) , acetylcholine esterase, and metallothioneins (vergani et al., 2005; lehtonen et al., 2006). investigation of these biomarkers was largely prompted by analogy with mammalian and/or in vitro toxicology and was therefore hypothesisdriven in that these biomarkers would be expected a priori to vary in response to chemical pollution. notwithstanding this expectation, there have been some surprising instances where bivalve biomarkers show significant differences either in biochemical properties or toxicological response when compared to those from higher species (livingstone, 1998; vergani et al., 2005; sole and livingstone, 2005). a significant body of literature has now grown up on biomarkers of oxidative stress in bivalves as pollution indices. proteomics approaches the biomarker approach depends heavily on study of single variables which could either be serendipitously observed to occur in nature or predicted as likely effects of particular pollutants. proteomics offers an alternative discovery-based approach for biomarker discovery of targets not necessarily predicted a priori. proteomic technologies each type of biological sample (organism, tissue, cell) expresses a characteristic subset of the proteins encoded in its genome. this protein population is further complicated by a range of possible post-translational modifications such as oxidation (mann and jensen, 2003; spickett et al., 2006; sheehan, 2006). the proteome is defined as the complement of proteins expressed in a given biological system under a defined set of conditions. in contrast with the genome, the proteome is highly dynamic, changing with the type of cell and in response to variables such as nutritional or pollution status. since chemical pollutants can alter the profile of proteins in the sample by altering protein structure (e.g., by post-translational modification; ptm) or by changing expression level of specific proteins (protein expression signature; pes) proteomics provides a potentially highly-sensitive means of detecting effects as well as offering potential insights to toxicity mechanisms (heijne et al., 2005). this approach has the potential to reveal changes in the level/status of specific proteins against a background of unchanged proteins using high-throughput experimental platforms (reviewed in sheehan, 2007) such as two dimensional electrophoresis (2de) (gőrg et al., 2004) and mass spectrometry (ms) (aebersold and mann, 2003). while these approaches will be especially emphasized here, emerging proteomic technologies include protein microarrays (cretich et al., 2006) and the yeast two hybrid system (zhu et al., 2003). sample origin and preparation are key variables in environmental proteomics since, as well as studying whole cell extracts prepared from plants or animals, selected sub-proteomes can be studied such as those prepared by affinity selection (lee and lee, 2004) and subcellular organelles (kislinger et al., 2006). moreover, when working with genetically illdefined animals such as bivalves (as opposed to inbred laboratory strains), it is necessary to have sufficient replicates to allow for inter-animal variation (biological replicates). in addition, multiple analyses are required to allow for experimental variation (analytical replicates). these are important facets of experimental design in environmental toxicology studies with bivalves (karp et al., 2005; karp and lilley, 2006; monsinjon et al., 2006). 2de 2de consists of sequential separation of proteins based first on isoelectric focusing (ief) followed by orthogonal sds page (o’farrell, 1975). originally, a stable ph gradient was established in a polyacrylamide gel by including local buffering agents (ampholytes) when casting the gel. proteins would migrate along the ph gradient until arriving at their isoelectric point (pi, the ph at which the protein has no net charge) and then stop moving. in this way proteins focus at their pi and the protein population separates in the rod gel into specific bands. in practice, it was technically demanding to achieve reproducible ph gradients in this system and there was a tendency for the entire gradient itself to migrate towards the cathode (“cathodic drift”; lognonne, 1994). immobilized ph gradients (ipgs) were later developed in which ampholytes were covalently immobilized along plastic strips (görg et al., 2000). after ief, the focused strip is exposed to sds page sample buffer, laid across the top of an sds page gel and electrophoresed. when stained with coomassie or silver stain (rabilloud, 1992), individual proteins become visible as spots which are usually sharplydefined (fig. 2). since few proteins have identical pi and relative molecular mass (mr), especially highresolution separations are achievable in which pairs of samples (e.g., test and control) can be compared. by increasing the size of the gel, up to thousands of spots may be evident but, even in the smallest gels, several hundred spots may be resolved. separations are captured and analyzed by image analysis software (corzett et al., 2006). independent labeling of lysines in test and control proteomes with up to three different fluorescent labels followed by their mixing and separation on a single gel allows comparison of tests and control samples without gel-to-gel variation, a technique called difference gel electrophoresis (dige) (gade et al., 2003). for the purposes of the present review, it should be noted that 2de separations can also be interrogated to identify proteins targeted by specific redox lesions, thus revealing redox-related differences not detectable by pes alone (sheehan, 2006). mass spectrometry (ms) methods chemical species with a given mass to charge ratio (m/z) describe a unique trajectory in an electromagnetic field in vacuo (aebersold and mann, 2003, domon and aebersold, 2006a). if the charge of all chemical species in a sample is identical, a mixture can be separated based on m 113 fig. 2 protein expression signatures (pes) of mytilus edulis. whole body proteomes separated by 2de from animals exposed to (a, b) arachlor 1248, (c, d) copper or (e, f) lowered salinity. these are composite gels (eight gels, four animals per treatment) and spots highlighted as being up-regulated (relative to controls) in at least 75 % of gels are highlighted with boxes (a, c, e). spots down-regulated in 75 % of gels are highlighted as circles (b, d, f). from shepard jl, olsson b, tedengren m, bradely bp. protein expression signatures identified in mytilus edulis exposed to pcbs, copper and salinity stress. mar. environ. res. 50: 337-340, 2000. alone yielding a very accurate mass estimate of the order ± 0.1 %. this allows identification of proteins and peptides which are composed of wellunderstood structural components of known m. in proteomics, ms analyzes differences between proteomes at the level of individual proteins or is used to identify proteins from tryptic digests of spots in two dimensional gel separations. protein identification is achieved either by peptide mass mapping (or fingerprinting) (thiede et al., 2003) or by peptide sequencing (wu et al., 2006). in both methods proteins are digested into a population of peptides with trypsin (or another protease) and these peptides are identified by comparison with predicted m values from sequence databases. rapid interrogation/analysis of sequence databases is made possible with powerful bioinformatics programs (wu et al., 2006; palagi et al., 2006; domon and aebersold, 2006b). two common ionization methods are used for ms of proteins/peptides: matrix-assisted laser desorption ionisation (maldi) (tanaka et al., 1988) and electrospray ionization (esi) (fenn et al., 1990). maldi is most commonly used in conjunction with a “time-of-flight” (tof) detector so this platform is called maldi-tof (fig. 3). sample is mixed with a “matrix” of a material capable of absorbing some of the laser energy used to ionize the protein or peptide into the gas phase (e.g., sinapinnic acid) and then placed on a target for laser-induced ionization. surface enhanced laser desorption ionization (seldi) is a modification of maldi increasingly used in biomarker discovery and ecotoxicology in which sub-proteomes are selected for categories of proteins on chips functionalized with selective chemical groups such as ion exchange (merchant and weinberger, 2000; poon, 2007) (fig. 4). esi ms achieves ionization of intact proteins by progressively drying droplets containing sample (fenn et al., 1990). when the droplet becomes sufficiently small, the charges on ions now highly concentrated within the droplet repel each other so strongly that a coulombic explosion occurs ejecting structurally intact ions into the gas phase for separation/detection. this produces extremely accurate m determinations with a minimum of structural breakdown. tandem ms (also called ms/ms or ms2) uses two ms sectors separated by a collision cell (aebersold and mann, 2003, 2006a; wu et al., 2006) (fig. 5). a tryptic digest is first separated in one ms giving a unique m for each peptide. the peptide then passes through the collision cell where it fragments systematically giving a “ladder” of daughter ions which are separated on the second ms based on m. since each fragment has a unique m and is composed of a limited number of chemical building blocks, the ladder can be read as a de novo amino acid sequence which can be used to screen sequence databases identifying the protein giving 114 fig. 3 outline of maldi-tof. sample is mixed with matrix (e.g., sinapinic acid) on a target. a laser beam impacts on this imparting sufficient energy to peptides or proteins to propel them through the tof analyser. this estimates the time required for each peptide type to reach the detector which depends on momentum (μ). from this time of flight, an accurate m/z value can be calculated. matching masses of tryptic peptides to masses predicted from sequence databases identifies proteins of origin by pmf. rise to the original digest (palagi et al., 2006; aebersold and mann, 2006b). this dependence on sequence databases means that protein identification by this method in species such as bivalves can be problematic. in such cases, identification may instead need to be based on recognizing sequence homology rather than identity representing a major limitation in identifying target proteins in ecotoxicology (dowling and sheehan, 2006; mcdonagh and sheehan, 2007). highresolution steps such as capillary liquid chromatography (lc) or capillary electrophoresis ce can precede tandem ms giving “hyphenated” techniques; lc-tandem ms (powell et al., 2006) and ce-tandem ms (hu et al., 2005) facilitating extremely high resolution analysis of complex mixtures. in “shotgun sequencing”, a tryptic digest of the entire proteome is separated using two dimensional lc (e.g., ion exchange followed by reversed phase) followed by sequencing in tandem ms (mcdonald and yates, 2002; peng et al., 2003) (fig. 6). the very large amounts of ms data generated by such experiments raise important questions for experimental design and statistical analysis which have recently been reviewed (domon and aebersold 2006b; nesvizhskii et al., 2007). proteomic studies in bivalves environmental toxicology has recently begun to embrace proteomic technologies as a set of techniques suitable for understanding biological responses to environmental stress (dowling and sheehan, 2007; monsinjon and knigge, 2007; nesatyy and suter, 2007). as is usual with a new field, the approach has been at first tentative with an early emphasis, for example, on identifying pes rather than specific proteins. however, increasingly sophisticated proteomics technologies such as dige and seldi-tof have gradually been introduced in important landmark studies. this work has revealed a number of points of difficulty some of which are further explored near the end of this article. for example, when studying bivalves significant care must be taken when selecting the study organism with respect to size, sensitivity to stress, availability and previously published data. moreover, tissues from different organisms may not necessarily be comparable for physiological reasons: mussels are filter feeders while clams are generally sediment feeders. therefore, proteomic responses in gill from both species might be very different. notwithstanding these practical limitations, proteomics is now increasingly accepted as a valid means of detecting often subtle effects of environmental pollutants. while not all of the following studies refer explicitly to oxidative stress, they often feature model compounds thought to function as pro-oxidants such as metals and hydrocarbons. in an early application of 2de to bivalves, shepard et al. (2000) defined a pes for mussels exposed to copper or high salinity. sets of proteins induced or repressed were identified as a sort of fingerprint for stress. although this approach allowed 115 (b) fig. 4 (a) outline of seldi-tof. multiple protein samples can be added to proteinchip® arrays which select proteins based on ion exchange, reversed phase or affinity interactions with groups immobilised on the array. use of 8-well or 96-well arrays allows scaling to number of samples required with high throughput. non-specificallybound proteins are removed by washing and matrix (e.g., sinapinnic acid) is added. a laser beam propels proteins through a tof analyser. the output may be represented as a plot of intensity versus m/z or as a synthetic “gel view” (i.e. how the sample might appear if separated by 1d sds page). (b) selection of results of seldi-tof from knigge et al. (2004) (reproduced with permission). a protein with m/z = 3268 was up-regulated in m. edulis digestive gland proteomes from animals in sites contaminated with pah (hog; n = 45) in comparison with reference animals (for; n = 39). from knigge t, monsinjon t, andersen ok. surface-enhanced laser desorption/ionization-time of flight-mass spectrometry approach to biomarker discovery in blue mussels(mytilus edulis) exposed to polyaromatic hydrocarbons and heavy metals under field conditions. proteomics 4: 2722-2727, 2004. copyright wiley-vch verlag gmbh & co. kgaa. reproduced with permission. 116 fig. 5 lc-tandem ms. proteins in 2de spots are digested with trypsin to form tryptic peptides. these are separated by capillary hplc followed by m/z determination in first ms sector. peptides are then fragmented in a collision cell and fragments are analysed in a second ms sector. because proteins/peptides fragment in predictable ways, the series of fragments can be interpreted as a sequence. sequences of several tryptic peptides are searched in sequence databases to give high-confidence identification. distinction between two samples without any protein identification, it revealed little about underlying mechanisms of toxicity. the first bivalve study combining 2de with ms identification was in the clam chamaelea gallina, exposed to four model pollutants (rodriguez-ortega et al., 2003). the 15 most dramatically altered protein spots were excised and analysed by maldi-tof ms, of which 4 proteins were identified. however, these proteins were cytoskeletal in origin which may point to high relative abundance, prevalence of cytoskeletal proteins in sequence databases or a cytoskeletonrelated oxidative stress response (mirabelli et al., 1989). this approach was later extended to another clam species, scrobicularia plana, sampled from sites of varying pollution status leading to identification of site-specific pes, identification of sequence tags for 16 proteins of increased abundance and unambiguous identification of more abundant hypoxanthine guanine phosphoribosyl transferase and glyceraldehyde 3-phosphate dehydrogenase (romero-ruiz et al., 2006). as a further development manduzio et al. (2004), identified 19 protein spots in gills of mussels exposed to crude oil. again, some cytoskeletal proteins were identified but also some antioxidant and metabolic enzymes such as gst, heavy metal binding proteins and aldolase. the authors also reported that, although a number of spots produced good maldi-tof spectra, they did not yield a significant score for peptide mapping and hence identification. this highlights the difficulty in identifying bivalve proteins when they are so poorly represented in sequence databases, a recurring theme in bivalve proteomics (dowling and sheehan, 2007; monsinjon and knigge, 2007). knigge et al. (2004) applied a seldi-tof approach for biomarker discovery to mussels exposed to pahs and heavy metals. seldi-tof should be capable of giving a proteomic profile of changes in cells under stress conditions as well as being well suited for low abundance and small proteins. however, a major problem encountered in this study was high variability amongst individual samples. these authors went on to combine protein array technology with seldi-tof of mussel serum from animals exposed to oil either on its own or spiked with alkylphenols (bjørnstad et al., 2006). a total of 83 mass peaks were perturbed in the spiked samples while only 49 were altered by oil alone. as with earlier studies, individual proteins were not explicitly identified but the mass patterns could be used as a fingerprint of effect. these studies demonstrate the power of seldi-tof for detection of effects when processing relatively large sets of individual complex protein samples although, again, analysis of such datasets is not without its difficulties (monsinjon et al., 2006). dige technology was applied to digestive gland peroxisome-enriched fractions of m. edulis exposed under controlled conditions to model pollutants (apraiz et al., 2006; mi et al., 2007). these workers reported significant changes between groups and that pes identified could be used to assess oil exposure in marine pollution. a major advantage of this approach is that upor down-regulated spots are readily identifiable on the same gel, thus improving identification by spot matching, reducing the number of gel replicates needed and raising the confidence levels for detection and quantification. dige also overcomes some of the shortcomings of traditionally stained gels which are susceptible to 117 fig. 6 shotgun proteomics. this involves tryptic digestion of whole proteomes with high-resolution separation and identification technologies. in this schematic, tryptic peptides are first passed through 2d lc (ion exchange first with each peptide peak further separated by reversed phase) then each resultant 2d lc peak is passed to tandem ms. high-speed algorithms identify peptide sequences and thus allow identification of proteins in the starting proteome. up to hundreds of proteins are identifiable in a single experiment. variation in spot patterns, staining intensity and which have a low linear range (nesatyy and suter, 2007). oxidative stress can cause a range of reversible and irreversible modifications to proteins and their side chains. some of these can lead to protein inactivation, some are protective of the protein’s structural integrity and some may be viewed as sensing changes in the cell’s redox status (davies, 2005; sheehan, 2006). protein side chains can be irreversibly modified to aldehyde or ketone groups in a process termed carbonylation. protein carbonylation can lead to protein aggregation, inactivation and degradation (levine et al., 1990; davies, 2005). the 2de proteomic approach can be combined with immunoblotting to investigate the carbonylation response in individual tissues in response to pollutants known to induce oxidative stress in both mussels and clams (mcdonagh et al., 2005; dowling et al., 2006; mcdonagh and sheehan, 2006). these studies revealed that levels of carbonylated proteins increased in general with oxidative stress although there was a tissue-specific response between organisms and the response was also pollutant-specific. damaged proteins are removed from cells by proteolysis, mainly via the ubiquitin proteasome pathway (upp) (marques et al., 2004). the upp is responsible for selective digestion of many short lived intracellular regulatory proteins or abnormal cytosolic or nuclear proteins (marques et al., 2004). changes in ubiquitination levels have been studied in bivalves by immunoblotting combined with 1de in response to temperature and season (hofmann and somero, 1995; et al., 2001buckley ) and in m. trossulus exposed to exxon valdez oil (downs et al., 2002). this approach has been extended to 2de, for analysing mussels and clams exposed to a variety of oxidative stressors (mcdonagh and sheehan, 2006; chora et al., 2008; tedesco et al., 2008). somewhat surprizingly, the ubiquitination pes is distinct from that for carbonylation suggesting that carbonylated proteins are degraded in a uppindependent manner. irreversible modifications are most likely indices of protein damage, whereas reversible modifications such as glutathionylation and the formation of inter or intradisulphide bonds can play a regulatory role both in sub-stress scenarios (biswas et al., 2006; ying et al., 2007; oktyabrsky and smirnova, 2007; poole and nelson, 2008) and in the organism's response to oxidative stress (stadtman and levine, 2000). gill tissues of m. edulis give a greater increase in glutathionylation in response to h2o2 treatment than digestive gland (mcdonagh et al., 2005). gills of mussels exposed to h2o2 and menadione, as well as baltic mussels sampled from a salinity gradient and exposed to pollutants were analysed using a diagonal electrophoresis technique to reveal formation of interand intrachain disulphides in proteins (mcdonagh et al., 2006; mcdonagh and sheehan, 2007, 2008; prevodnik et al., 2007). in this technique proteins are initially run in a non-denaturing primary dimension, the gel lane is then excised, proteins reduced in-gel and the lane is then placed orthogonally over a second dimension gel. proteins not involved in inter-or intrachain disulphides fall along a diagonal while those involved in interchain disulphides will fall below the diagonal and those involved in intrachain disulphides will fall above the diagonal. it was found that actin and protein disulphide isomerase are involved in a number of disulfide bonds. as many proteins form disulphides in response to oxidative 118 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t4g-4kc2j80-1&_user=723077&_coverdate=10%2f12%2f2006&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=c000040418&_version=1&_urlversion=0&_userid=723077&md5=c91d2d4772622558acf759b7e3d9b794#bib37 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t4g-4kc2j80-1&_user=723077&_coverdate=10%2f12%2f2006&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=c000040418&_version=1&_urlversion=0&_userid=723077&md5=c91d2d4772622558acf759b7e3d9b794#bib37 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t4g-4kc2j80-1&_user=723077&_coverdate=10%2f12%2f2006&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=c000040418&_version=1&_urlversion=0&_userid=723077&md5=c91d2d4772622558acf759b7e3d9b794#bib37 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t4g-4kc2j80-1&_user=723077&_coverdate=10%2f12%2f2006&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=c000040418&_version=1&_urlversion=0&_userid=723077&md5=c91d2d4772622558acf759b7e3d9b794#bib22 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t4g-4kc2j80-1&_user=723077&_coverdate=10%2f12%2f2006&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=c000040418&_version=1&_urlversion=0&_userid=723077&md5=c91d2d4772622558acf759b7e3d9b794#bib22 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t4g-4kc2j80-1&_user=723077&_coverdate=10%2f12%2f2006&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=c000040418&_version=1&_urlversion=0&_userid=723077&md5=c91d2d4772622558acf759b7e3d9b794#bib5 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t4g-4kc2j80-1&_user=723077&_coverdate=10%2f12%2f2006&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=c000040418&_version=1&_urlversion=0&_userid=723077&md5=c91d2d4772622558acf759b7e3d9b794#bib13 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6v7h-4jnf06t-5&_user=723077&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=c000040418&_version=1&_urlversion=0&_userid=723077&md5=941b07af22dd0735abd170e9a61d0951#bib10 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6v7h-4jnf06t-5&_user=723077&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=c000040418&_version=1&_urlversion=0&_userid=723077&md5=941b07af22dd0735abd170e9a61d0951#bib10 stress, the method of baty et al. (2005) was adapted to analyse changes in thiol status within the proteome. in this approach, free thiols are initially blocked with n-ethylmaleimide, disulphides reduced by dtt and resulting free thiols labelled with the thiolreactive fluorescent compound, 5iodoacetamidofluorescein (5-iaf). this enabled identification of a number of menadione-responsive proteins in the gills of m. edulis by lc-ms/ms (mcdonagh and sheehan, 2007, 2008). another technique that uses an affinity column to pre-select for a subproteome in combination with 2de has also been used in the clam tapes semidecussatus, where a gsh-agarose column was used and tissuespecific expression of pi class gst’s was discovered (dowling et al., 2006). these studies take a sub-proteome approach to oxidative stress in bivalves considerably simplifying subsequent analysis. conclusions and perspectives recent years have seen development of new proteomic techniques, many of which have yet to be fully embraced by environmental toxicology. some of these techniques such as “shotgun proteomics” necessitate fully-covered genome sequences which currently precludes many bivalves. others, such as blue native gel electrophoresis have especial promise for studies of oxidative stress as this technique resolves protein-protein complexes in systems such as mitochondria (krause, 2006). in the coming years, significantly more sequence data are expected to become available (mckillen et al., 2005; dupont et al., 2007; tanguy et al., 2008). this is especially important as “gel-free” techniques overcome many of the well-known shortcomings of 2de such as the need for detailed image analysis, misidentification due to co-migration of proteins, under/over-selection of some proteins due to their physical properties or position in the cell (e.g., membrane proteins, are not suitable for 2de separations) (görg et al., 2004). one of proteomic’s ultimate goals is to unearth networks of interactions to understand a particular biological state as well as to identify and quantify involvement of individual proteins (domon and aebersold, 2006a). relative quantification between protein samples is a crucial aspect of analysis and 2de, when combined with image analysis, allows relative spot comparisons to be made between gels. however, numerous gel replicates and careful consideration of total amount of protein on gels is necessary. as the dynamic range of many protein stains is limited, relative quantification can be difficult, although the newer fluorescent stains and dyes have been a significant improvement (nesatyy and suter, 2008). some of the newer approaches for quantification include, isobaric tags for relative and absolute quantification (itraq) allowing relative quantification between tandem mass spectra and use of tags of varying mass that label n-terminus and side-chains of all peptides. samples are then pooled and analysed by ms/ms. isotope coded affinity tag (icat) reagents that are cysteinespecific offer options especially relevant to redox proteomics. cysteines are one of the most rarely used amino acids but are often highly-conserved and involved in the regulation of important cellular processes. icat can provide information about the oxidative state of individual proteins (leichert et al., 2008). many proteomic studies of oxidative stress in bivalves have so far been performed under laboratory-controlled conditions, where bivalves are subjected to high levels of contaminants or stressors. this is understandable as proteomics is relatively new to environmental monitoring, and researchers often use artificially high exposures to see if there is measurable response. the next step is to develop proteomic techniques to the point that they can reveal differences in the field at environmentally-relevant pollutant concentrations. a major issue to be considered in environmental proteomics studies so far has been the repeated identification of multifunctional proteins such as those of the cytoskeleton. an analysis of 15 environmental proteomics studies indicated that actin, tubulin or myosin were identified in almost 50 % of studies (monsinjon and knigge, 2007). whether this is due to the high abundance of these proteins, that they are highly conserved across species or that it is a genuine stress-response remains to be elucidated. the presence of high abundance proteins could mask the oxidative stress response of low abundance proteins that are modified to greatest degree. when initially designing a proteomics experiment it should be kept in mind what the overall objectives of the study are, as with some careful initial planning the maximum information may be gleaned from results. a major bottleneck in proteomic studies is how to analyse the final data. if the question posed is to look for changes in a particular protein, a univariate statistical analysis such as the student’s t-test may be sufficient, while a multivariate analysis is more appropriate for looking at changes in patterns of expression of proteins (karp and lilley, 2007). these issues should be kept in mind as they will affect the number of biological and analytical replicates required. the issue of whether to pool samples from a number of individuals in a proteomic study has been a subject of much debate and has been extensively reviewed elsewhere (monsinjon and knigge, 2007; karp and lilley, 2007). briefly, pooling of samples may reduce biological variation and enhance statistical performance which, in a 2de approach that is laborious and timeconsuming, has obvious advantages. on the other hand, one aberrant sample could negatively impact on the quality of a particular pool and any phenotypic data is impossible to distinguish. subpooling has been suggested as an alternative approach but again some considerations include that the pool should be large enough to reflect the average population and that subpooling creates a hierarchical structure within the data (karp et al., 2005). a 3-year european research project beep (biological effects of environmental pollution in marine ecosystems) published in a special edition of aquatic toxicology evaluated the use of biomarkers determined in marine organisms as a means of assessment of chemical contamination (pampanin et al., 2006). this report included a 119 number of proteomic studies including 2de and seldi-tof, and highlighted the need for multivariate statistical methods for interpretation of complex datasets. in general, careful initial planning and outlining of overall objectives can save time over the course of the study. together with the increasing availability of complete gene and protein databases we should see further developments in the field of environmental monitoring of bivalves as a response to oxidative stress (mckillen et al., 2005; dupont et al., 2007). acknowledgements our laboratory is supported by the irish research council for science engineering and technology. references aebersold r, mann m. 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253-278, 2006. ying j, clavreul n, sethuraman m, adachi t, cohen ra. thiol oxidation in signalling and response to stress: detection and quantification of physiological and pathophysiological thiol modifications. free radic. biol. med. 43: 10991108, 2007. zhu h, bilgin m, snyder m. proteomics. annu. rev. biochem. 72: 783-812, 2003. 123 isj 5: xx-yy, 2008 isj 5: 75-82, 2008 issn 1824-307x review antimicrobial peptides in annelids a tasiemski laboratoire de neuroimmunologie des annélides (lna), cnrs fre2933, «groupe signaux de danger, voies de signalisation et effecteurs», université de lille1, france accepted june 11, 2008 abstract gene encoded antimicrobial peptides (amps) are widely distributed among living organisms including plants, invertebrates and vertebrates. they constitute important effectors of the innate immune response by exerting multiple roles as mediators of inflammation with impact on epithelial and inflammatory cells influencing diverse processes such as cytokine release, cell proliferation, angiogenesis, wound healing, chemotaxis and immune induction. in invertebrates, most of the data describe the characterization and/or the function of amps in the numerically and economically most representative group which are arthropods. annelids are among the first coelomates and are therefore of special phylogenetic interest. compared to other invertebrate groups, data on annelid’s immunity reveal heavier emphasis on the cellular than on the humoral response suggesting that immune defense of annelids seems to be principally developed as cellular immunity.this paper gives an overview of the variety of amps identified in the three classes of annelids, i.e. polychaetes, oligochaetes and achaetes. their functions, when they have been studied, in the humoral or cellular response of annelids are also mentioned. key words: antimicrobial peptides; annelids; lophotrochozoan; immunity introduction numerous studies on the effectors of the innate immune system have demonstrated the contribution of amp to the host defense (zasloff, 2002). antibiotic peptides are small molecules. basically on their structural features, five major classes were defined (bulet et al., 2004): 1) linear α helical peptides without cysteines (the prototype of this family are the cecropins); 2) loop forming peptides containing a unique disulfide bond (mainly isolated from amphibian skin); 3) open-ended cyclic cysteine-rich peptides (among which defensins are the most widespread); 4) linear peptides containing a high proportion of one or two amino acids, e.g. indolicidin; 5) peptides derived from larger molecules known to exert multiple functions. in spite of great primary structure diversity, the majority of documented amps are characterized by a preponderance of cationic and hydrophobic amino acids. this amphipathic structure allows them to ___________________________________________________________________________ corresponding author: aurélie tasiemski laboratoire de neuroimmunologie des annélides (lna), cnrs fre2933, «groupe signaux de danger, voies de signalisation et effecteurs» université de lille1, france e-mail: aurelie.tasiemski@univ-lille1.fr interact with bacterial membrane. however, brogden et al. (1998) have evidenced in ovines that some peptides with anionic properties can also exert antimicrobial activities. amps appear to be essential anti-infectious factors that have been conserved during evolution. meanwhile, their implications in immune processes are different according to species, cells and tissues. the involvement of amps in natural resistance to infection is sustained by their strategic location in phagocytes, in body fluids and at epithelial level, i.e. at interfaces between organisms and its environment. this action is strengthened by the rapid induction of such amp genes in bacteriachallenged plants or animals (zasloff, 1992). annelids belonging to the group of lophotrochozoans are primitive coelomates that possess specially developed cellular immunity against pathogens including phagocytosis, encapsulation and spontaneous cytotoxicity of coelomocytes against allogenic or xenogenic cells (salzet et al., 2006). they have also developed an important humoral immunity that is based on antimicrobial, hemolytic and clotting properties of their body fluid. the present review summarizes the function of different amps that adaptation has taken during the course of evolution of the three 75 mailto:aurelie.tasiemski@univ-lille1.fr classes of annelids, i.e. polychaetes, oligochaetes and achaetes. polychaetes the large majority of polychaetes is restricted to the marine environment. they are considered as the most primitive annelids, based on morphology, physiology and development. to date, amps have been studied in three species of polychaetes, arenicola marina (ovchinnikova et al., 2004), nereis diversicolor (tasiemski et al., 2007) and perinereis aibuhitensis (pan et al., 2004). they all live in estuary sediments which are rich in microorganisms but also of toxic agents resulting from pollution. their abundance in this type of environment suggests these worms have developed efficient detoxification and immunodefense strategies. in the asian clamworm p. aibuhitensis, a cationic amp named perinerin was isolated and partially characterized by pan et al.(2004) from the homogenate of adults. this peptide does not show any similarities with other described amps. perinerin consists of 51 amino acids, including 4 cysteine residues presumably implicated in two disulfide bridges. no cdna cloning was performed so no information is available about the precursor sequence of the mature form. antimicrobial assays performed with the native perinerin evidenced an activity directed against a large spectrum of microorganisms including fungi, gram positive and gram negative bacteria at physiological concentrations. a rapid bactericidal activity was evidenced towards gram positive bacteria: indeed, when incubated with a culture of bacillus megaterium during exponential phase, perinerin killed all the bacteria in less than three minutes, suggesting a pore forming activity. the cellular localization as well as the physiological role of perinerin in the anti-infectious response of p. aibuhitensis has not been yet elucidated. the authors suggest that the peptide should be constitutively expressed since perinerin was purified from unchallenged worms. however, there are no indications that immunization of p. aibuhitensis by bacterial challenge has modified the synthesis site and/or the concentration of native perinerin. still in 2004, two novel 21 amino acids amps, namely arenicin-1 and arenicin-2, were isolated and fully characterized from the coelomocytes of the lugworm, a. marina (ovchinnikova et al., 2004). each isoform possesses two cysteine residues implicated in one disulfide bond. amps containing only one disulfide bond have been isolated from frog skin earlier but their cysteine bridged loop is smaller than the one of arenicin (bulet et al., 2004). recent publications showed that arenicin folds into a two stranded antiparallel β-sheet which are twisted to expose an amphipatic surface (ovchinnikova et al., 2007; andra et al., 2008). other amps such as the tachyplesin found in the horseshoe crab adopt this conformation although the β-sheet of arenicin is not caged into two disulfide bonds (tamamura et al., 1993). both isoforms were shown to be active against fungi, gram positive and gram negative bacteria. antimicrobial activities of arenicin-1 and 2 were absolutely equal. as arenicin is amphipatic and rich in hydrophobic and arginine, andra et al. (2008) hypothesized that the killing of the bacteria implicated a disruption of the membrane. by applying an elegant approach, they demonstrated that incubation of gram negative bacteria with arenicin leads to a rapid membrane permeabilization accompanied by the intercalation of the amp peptide into the lipid bilayer and the release of cytoplasmic material. these active forms are processed from a larger precursor containing a predicted signal peptide followed by a long prodomain. the presence of a signal peptide suggests that the peptides can be secreted thought a conventional pathway (ovchinnikova et al., 2004). data related to arenicin have been more focused on its structure and mode of action than on its immune function in a. marina. the isolation of arenicins from coelomocytes lets presume that arenicin may play function in the cellular immunity of l. rubellus in a way comparable to what has been described for hedistin in n. diversicolor (tasiemski et al., 2007). hedistin was identified from the coelomocytes of the sandworm n. diversicolor. like perinerin and arenicin, this amp showed no obvious similarities with other known peptides. hedistin is active against a large spectrum of gram positive bacteria. interestingly, when tested with different gram negative microorganisms, hedistin is active especially on marine bacteria v. alginolyticus which is a causative agent of episodes of mass mortality of larvae of bivalves in commercial hatcheries. this could be attributable to the capacity of vibrio to extensively degrade native cuticle collagen of nereis. vibrial collagenase would help bacteria entrance into the worm body, making the mechanical defense barrier of the cuticle inefficient against vibrio invasion. thus, hedistin synthesis would follow from the adaptation of nereis immune defense towards bacteria of its environment. no cytotoxicity of either hedistin forms was observed against nereis cœlomocytes. hedistin is a linear peptide containing bromotryptophan residues in its amino acid sequence. although many antimicrobial peptides have been characterized in marine metazoans, very few are reported to contain bromotryptophan residues (taylor et al., 2000). bromination of tryptophan which is described as a result of post translational modifications seems to be typical of organisms living in sea water like tunicates (craig et al., 1997; jimenez et al., 1997). for example, mammalian cathelicidins, a family of amps, do not contain bromotryptophan while hagfish cathelicidins do (shinnar, butler et al., 2003; uzzell et al., 2003). bromination of tryptophan residues does not seem to play a role in the antibacterial activity of hedistin. in addition to bromotryptophan, the hedistin primary structure includes a c-terminal (ct) amidation. the presence of a ct amide increases the net cationic charge and consequently the electrostatic attraction to target membrane like the negative charged bacteria membrane. this suggests that c-terminal amidation of hedistin might be implicated in its bactericidal properties. moreover, as for the hagfish cathelicidins, the cterminal amidation and the unusual amino acid bromotryptophan could make hedistin a poorer substrate for endogenous proteolytic enzyme by 76 providing resistance to c-terminal exopeptidase and to proteolysis for steric reasons respectively (uzzell et al., 2003). such protease resistance could extend the lifetimes of hedistin in vivo sustaining antimicrobial activity. the hedistin gene is strongly and exclusively expressed in cœlomocytes evenly distributed in the whole cœlomic cavity. these are referred as the type 3 granulocytes also called nk-like cells because of their natural cytotoxicity (porchethennere et al., 1992). even if the level of transcript does not increase after bacteria challenge, “hedistin containing cœlomocytes” appeared to accumulate around infection sites where the presence of bacterial motifs triggers the release of the active peptide into the local environment. these results are reminiscent of those reported for marine organisms of different taxa like the shrimp penaeus vannamei (bachere et al., 2004), the mussel mytilus galloprovincialis (mitta et al., 1999) and the horseshoe crab tachyplesus tridentatus (iwanaga, 2002). in m. galloprovincialis, microbial challenge provokes the release of the antimicrobial peptide mgd1 from the hemocytes into the plasma. in t. tridentatus, bacterial stimulation triggers the degranulation and the release of different immune molecules among them amps oligochaetes oligochaetes have a larger repartition area than polychaetes: they can be terrestrial, semior fully aquatic in freshwater or, more rarely, in seawater. they live in an environment such as water, soil and manure containing abundant microorganisms that are ingested during feeding. members of the amp family have been identified in the following earthworms: lumbricus rubellus, pheretima tshiliensis and eisenia foetida. the first amp evidenced in oligochaetes and in annelids in general, was isolated and characterized from a homogenate of l. rubellus (cho et al., 1998). lumbricin-1 is a proline-rich antimicrobial peptide of 62 amino acids showing antimicrobial activity in vitro against fungi, gram positive and gram negative bacteria without hemolytic activity. a 29-amino acid peptide, named lumbricin-1 (6-34), which was derived from residues 6-34 of lumbricin-1, showed marginally stronger antimicrobial activity than authentic lumbricin-1. many proline-rich amps were discovered from various vertebrates and invertebrates species (bulet et al., 2004). even if they possess the common characteristics of a high content of proline and a highly positive charge, their mode of action and their antimicrobial spectra are quite different from one to another. for example, apidaecin has been shown to act through a non pore forming mechanism involving stereospecificity whereas pr39 blocks protein and dna synthesis in cells ( casteels and tempst, 1994; gaczynska et al., 2003). the mode of action of lumbricin-1 has not been yet described. concerning its physiological role, lumbricin-1 is processed from a precursor containing a signal peptide directly followed by the active molecule suggesting that this amp can be found in the extracellular environment. northern blot data showed that lumbricin-1 gene is constitutively expressed and is not inducible upon bacterial infection(cho et al., 1998). moreover, lumbricin-1 transcripts were detected in unchallenged six month old adults but not in one week old young adult and in eggs suggesting that the transition to the adult stage might be inducer of lumbricin-1 gene expression. that would be interesting to check whether the expression of the gene encoding lumbricin-1 is not induced in bacterially challenged young or eggs. in 2003, a cdna encoding a molecule presenting high percentage homology with lumbricin-1 was described in the asian worm p. tschiliensis (wang et al., 2003). despite the great similarity with lumbricin i, this putative antimicrobial peptide, so called pp-1, lacks an obvious signal peptide. however, immunohistochemical studies showed that pp-1 was immunodetected in the mucus covering the animal and not into the epidermis cells. the extracellular presence of peptides devoid of peptide signals was also described for lysenin which was purified from the celomic fluid of e. foetida (sekizawa et al., 1997) and more recently for hm-lumbricin, an amp similar to lumbricin-1 that was purified in the medicinal leech hirudo medicinalis (schikorski et al., 2008). further investigations on this uncommon mechanism of secretion have been proposed to be performed by wang et al. (2003). in the tiger worm e. foetida, three very short antibiotic peptides, f-1, f-2 and oep3121, have been isolated and identified ( zhang et al., 2002; liu et al., 2004). these are described as exhibiting an activity against gram positive, gram negative bacteria and fungi. all of three are composed of only five amino acids that do not present primary structural homology with other known amps. to date, there is no information in the literature about the nature of their eventual precursors or their production site. achaetes achaetes, also called leeches, live in an environment similar to that of their near relatives, oligochaetes. both are clitellates and in contrast to polychaetes do not present a larval stage during their development. amps have been studied in two species of leeches: the rhynchobdellid leech theromyzon tessulatum and the gnathobdellid leech h. medicinalis. these are blood suckers of vertebrates suggesting that they could have developed an immune response adapted to microorganisms possibly pathogens of mammals. t. tessulatum is an ectoparasite of aquatic birds. its life cycle was arbitrarily subdivided in stages (these are not larval stages) defined by taking, as indicators, the three blood meals. the third stage which corresponds to the gametogenesis phase is characterized by an important water uptake making the collect of the body fluid easy. for this reason, t. tessulatum constitutes a convenient model for studying the antimicrobial response which takes place at the systemic level of the leech. three amps were isolated and fully characterized from the body fluid of t. tessulatum. these are theromacin, a cysteine rich amp exhibiting bactericidal activities, theromyzin an anionic peptide with bacteriostatic properties (tasiemski et al., 2004) and peptide b an 77 anionic peptide matured from a neuropeptide precursor, proenkephalin a (pea) (tasiemski et al., 2000). they all present an activity directed against gram positive bacteria. several types of amps containing cysteine residues have been described in multiple organisms. in invertebrates, most of them share the disulfide array of the insect/arthropod defensin (bulet et al., 2004). in addition to having ten cysteine residues instead of six, theromacin does not harbor this consensus sequence. besides, it appeared that the peptide has no significant similarity with other known peptides. so theromacin is a novel cysteine rich amp. majority of amps including members of the cysteine rich family possess a global positive charge allowing their interaction with the negatively charged bacterial membrane. theromyzin and peptide b, in contrast to theromacin, are anionic molecules. in vertebrates, amps with anionic properties were evidenced in the human and the sheep lung (brogden et al., 1998). amps are anionic because of homopolymeric regions of aspartate, and require zinc as a cofactor for bactericidal activity (melino et al., 1999). histatins, a family of histidin rich amps found in human saliva, also need the presence of zinc ions for bactericidal activities. circular dichroism studies showed that the antimicrobial activities of histatin-5 require a conformational change that results from the interaction of the peptide with both zinc ions and negatively charged membranes (brewer and lajoie, 2000). the abundance of histidine residues at the n-terminal part of theromyzin could argue in favor of some common structures between the leech antibacterial peptide and histatins. based on these data, we can hypothesize that the active part of theromyzin might be reduced to the n-terminal part enriched in histidine and aspartate residues and that theromyzin could require a cofactor for bactericidal activity. theromacin and theromyzin genes are exclusively expressed in large fat cells (lfc) evenly distributed in the leech. their transcriptional level is enhanced after bacteria challenge evidencing a regulation of the leech amp similar to that of the insect antimicrobial peptides genes. indeed, in the fruit fly, genes encoding antibiotic peptides are rapidly induced following a septic injury (hoffmann, 2003). the similarity between the antibacterial response of the leech and those of holometabolous insects is also supported by the functional resemblance between the leech lfc and the insect fat body which possess the common capacity to produce egg-yolk proteins (baert et al., 1991). moreover, no difference in gene expression was observed after gram positive or gram negative injection suggesting that the antibacterial response of theromyzon is aspecific. this non-specificity has also been assumed in drosophila until the work of lemaitre et al. (1997) demonstrated that the humoral antimicrobial response of the fruit fly discriminates between various classes of microorganisms and mounts a response that is adapted to the infection. this suggested that in a more natural modality of infection, also the leech could adapt its antimicrobial response, what was recently confirmed (schikorski et al., 2008). as for lumbricin-1 in l. rubellus, physiological events occurring during gametogenesis phase appeared to be inducers of the theromacin and theromyzin gene expression in t. tessulatum. this indicates that several hormonal factors implicated in the sexual maturation may participate in the induction of genes encoded amps in annelids as described in drosophila by meister and richards (1996). the peptide sequences deduced from the theromacin and theromyzin genes contain putative signal peptides, indicating that mature peptides correspond to secreted molecules. the massive production of theromacin and theromyzin is immediately followed by a rapid release of the peptides into the celomic fluid after septic injury. thus theromacin and theromyzin may play their antimicrobial activities through a systemic action. moreover the presence of these peptides in the intestinal epithelial cells and at the epidermis level suggests that leech antimicrobial peptides could also play a role in epithelial defense. the localization of antibiotic molecules in gastrointestinal tract has also been reported in insects and in vertebrates where they provide a rapid local immune response against exogenous pathogens brought in during feeding (zasloff, 2002). as for pp1, theromacin and theromyzin were detected in the mucous that covers the animal. that reminds the local defensive response reported in frogs in which antibacterial peptides secreted in the mucous prevent bacteria colonization and/or subsequent infection (zasloff, 1992). in addition, since a mucous membrane covers the eggs after laying, we hypothesized a protective role of leech theromacin and theromyzin against bacteria during eggs development. peptide b is not produced by the lfc although it was also isolated from the body fluid of the leech. its precursor, pea, was immunodetected into circulating coelomocytes suggesting that peptide b could be released from these cells. in contrast to theromacin and theromyzin, the production of peptide b seems to be more regulated at the translational level by the enzymes implicated in the pea processing than at the transcriptional level. consequently, t. tessulatum is an original invertebrate model which has developed two modes of fighting infections by amps: (i) storage of antibacterial peptide derived from pea and release of the peptide into the celomic fluid after immune challenge (ii) induction after septic injury of gene coding for more classical amps, mainly in lfc, and rapid release into the body fluid of the antibiotic peptides. unpublished data collected by our group evidence that the same amps participate to the systemic antimicrobial response of the medicinal leech h. medicinalis. this leech presents celomic cavities filled by cells constituting the bothryoidal tissue, making the collect of the body fluid impossible. in fact, the medicinal leech has been extensively used as a model organism for cellular analyses of nervous system function (blackshaw and 78 table 1 summary of the amps characterized in anellids nicholls, 1995; emes et al., 2003). of particular significance, the medicinal leech has been used to study repair of the nervous system at the level of identified nerve cells, an approach that is currently difficult or not possible with more complex nervous systems. the nerve cord of the leech can be easily removed from the animal and maintained in culture for weeks in the absence of peripheral immune system components and blood cells that might infiltrate the nerve cord after injury. that allows focusing the studies on the intrinsic immune response developed by the leech nervous system. unlike mammals, the leech central nerovous system (cns) has a demonstrated capacity to repair itself after injury and to restore function (blackshaw et al., 1997; burrell et al., 2003). the process of regeneration is accompanied by a rapid activation of microglial cells leading to their accumulation at the lesion site where they phagocytize damaged tissue. interestingly, we recently evidenced that the leech nerve cord used a common panel of proteins to initiate an antimicrobial response and regrowth program. indeed, we have demonstrated that microbial challenge promoted the regenerative process of the injured cns of the medicinal leech by inducing the synthesis of amps in neurons and microglia (schikorski et al., 2008). two newly characterized amps, hm-lumbricin and neuromacin have been shown to be produced by microglial cells and by neurons themselves in response to cns injury. neuromacin is a relative of theromacin. theromacin possesses a longer c-terminal domain than neuromacin, which probably results in two different conformations, that could determine different biological activities for the two peptides. theromacin and neuromacin present a differential tissue expression. theromacin is expressed in the peripheral lfc, whereas neuromacin expression is restricted to the nervous system. curiously, only neuromacin-like molecules have been found in other invertebrates, such as molluscs and other annelids (mitta et al., 2005; moroz et al., 2006). thus far, the larger theromacin appears to be unique to leeches. neuromacin, like theromacin, displayed bactericidal activity against gram-positive bacteria. the importance of neuromacin and hm-lumbricin in the antiinfectious immunity of the leech cns is emphasized by the presence of their transcripts in neuronal cells and by the fact that their gene expression is upregulated by some microbial components. a difference in neuromacin and hmlumbricin gene expression was observed after infection with different microbial components, suggesting that the antibacterial response of the medicinal leech cns is specific to the antigens presented. this was not expected, given that in the leech t. tessulatum our previous work demonstrated the non-specificity of the humoral antimicrobial response to infection. this ability to discriminate pathogens might be relevant to the use, in the experiments presented here, of bacteria naturally living in the environment of the leech. intriguingly, a neuromacin-like peptide reported as theromacin-like has recently been detected by sequencing cdna libraries from the cns of the mollusc aplysia californica, but the roles and the production sites of the peptide were not detailed (moroz et al., 2006). in caenorhabditis elegans, several genes encoding neuropeptide-like proteins named nlp-29, nlp-31 and nlp-33, the sequences of which were deduced from an in silico analysis of an est library, have been shown to be induced in the hypodermis by fungal infection. origin organism name primary structure production site polychaetes perinereis aibuhitensis perinerin cysteine-rich undetermined arenicola marina arenicins-1, 2 loop forming peptides coelomocytes nereis diversicolor hedistin linear peptide containing bromo-tryptophans coelomocytes (nk cells-like) oligochaetes lumbricus rubellus lumbricin-1 linear peptide, proline-rich undetermined pheretima tschiliensis pp-1 linear peptide, proline-rich tegument eisenia foetida oep3121 short linear peptide undetermined f-1, f-2 short linear peptides undetermined achaetes theromyzon tessulatum, peptide b peptide derived from a larger precursor coelomocytes hirudo medinalis theromacin cysteine-rich large fat cells (lfc) theromyzin linear, anionic lfc hm-lumbricin and tt-lumbricin linear peptide, proline-rich lfc, neuron and microglia neuromacin cysteine-rich neuron and microglia 79 interestingly, the chemically synthesized nlp-31 exhibited antifungal activity but in contrast to most of the nlp family members, nlp-29, nlp-31 and nlp33 were not detected in neurons using gfp reporter genes (couillault et al., 2004). in addition to manifesting antibacterial properties, neuromacin and hm-lumbricin exert impressive regenerative effects on the leech cns. in vertebrates, one study provided evidence for the positive effects of an antimicrobial peptide on the restoration of the functions of a lesioned peripheric nerve. indeed, the addition of neutrophil defensin np-1 on the lesioned sciatic nerve in rats lead to increase the rate of growth of regenerative nerve fibers by 30 % (nozdrachev et al., 2006). so these data are the first evidencing the participation of an amps produced by the nervous system itself in the regeneration process of the cns. conclusions this review presents the large variety of amps that can be found in annelids since the five groups of amps evoked in introduction are all represented (table 1). amps produced by polychaetes seem to be distinct from those produced by leeches and oligochaetes. for examples, h. medicinalis and l. rubellus produce common amps, such as lumbricin and theromacin (found in the lumbricus est database) which are different from those synthesized by polychaetes. neither hedistin nor arenicin was found in the leech. this suggests that for the selection of amps retained by an organism, the environment where it lives might be relevant. it is interesting to remark that, at present, no defensins have been isolated from annelids although more than 70 different invertebrate defensins have been identified in molluscs, nematods and arthropods. defensins which are considered as the most widespread family of invertebrate amps have not been found neither in the genome of the leech helobdella robusta (http://genome.jgi-psf.org/helro1/helro1.home.html) nor in the expressed sequence tag (est) libraries of lumbricus terrestris, e. foetida (http://www.earthworms.org) and h. medicinalis. 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bacterial symbiont; symbiosis; hologenome; holobiont introduction aphids (hemiptera: aphididae) are ancient insects, whose fossils go back to the triassic, about 220-210 myr ago (grimaldi and engel, 2005), that have conquered most of the biomes, including the arctic and subarctic regions and infest a huge range of plants (loxdale, 2008a). aphids reproduce primarily by apomictic parthenogenesis, a form of reproduction whereby adult females give birth to a female progeny without any male fertilization (soumalainen et al., 1987; loxdale, 2008a). several authors frequently suggested that no genetic recombination occurs in the parthenogenetic generations (soumalainen et al., 1987; spence and blackman, 1998), so that it has been assumed that the aphid offspring represents a genetically identical clone of aphids (dixon, 1989). in 1977, dan janzen argued that clonal lineages of aphids could be considered as “evolutionary individuals” with the ability to exploit resources over a wide geographical area. these multiple individuals thereby have a competitive edge over single organisms that lack the capacity to ___________________________________________________________________________ corresponding author: mauro mandrioli department of life sciences university of modena and reggio emilia via campi 213/d, 41125 modena, italy e-mail: mauro.mandrioli@unimore.it propagate parthenogenetically (apomictically). aphids can reproduce quickly and it has been calculated that under ideal conditions (absence of predators, parasites, pathogens and benign climatic conditions, especially including optimal temperatures of 20 -25 °c), a single asexual female could in theory produce 7.6x1028 offspring in a single growing season (with a generation given as 7 days, 50 offspring per female and 18 generations a year) (harrington, 1994). however, whether they can maintain a long-term genetic fidelity, if at all, and how long so-called clones persist unchanged either in the laboratory or in the field is still a contentious issue (loxdale, 2008a, b, 2009). actually, aphid lineages within a same species have been found to differ for colour (loxdale, 2008b), size (jenkins, 1991), intrinsic rate of increase (jenkins, 1991), ovariole number (dixon, 1989), reproductive modes (loxdale, 2008b), ability to transfer pathogenic plant viruses (terradot et al., 1999) and susceptibility/resistance to predators, parasites, pathogens and pesticides (losey et al., 1997; devonshire et al., 1999; loxdale, 2008b). these findings strongly suggest that clonality in aphids has been overestimated prompting an evaluation of the true nature and reality of the concept of clone (loxdale, 2008a, b). in the last years, a growing amount of molecular evidences suggested that aphid asexual lineages are not true clones, since they can rapidly 1   fig. 1 different microorganisms can be harboured in diverse aphid organs. the primary symbiont b. aphidicola is generally hosted in the bacteriocytes (b), specialized fat body cells located near the aphid gut (g). a highly diversified bacterial community has been identified in the aphid gut since differences in the microbiome have been observed not only comparing diverse species, but also making a comparison of different populations of the same species, but feeding on different plants. mutate and this variation is selectable and may affect some phenotypic traits, such as the host choice (loxdale, 2008a, b; martens et al., 2009), so that the real nature of clone in aphids is not simply semantics. indeed, the presence of genetic differences among clones could be very important for aphid evolution since several studies showed that cryptic sympatric speciation occurred in a wide range of aphid species (loxdale, 2008a, b), including evidences of rapid chromosomal changes affecting speciation events in the aphids rhopalosiphum maidis and myzus persicae (blackman, 1987; brown and blackman, 1988; monti et al., 2012). 2   starting from the hypothesis of the clonal reproduction of aphids, some authors referred to aphids as a single genome species suggesting not only that each aphid presents a clear correspondence one genome-one organism, but also accepting the idea that the aphid phenotype relies on the nuclear genome only (as recently revised in loxdale, 2008a, b). in view of their impact in agriculture (in particular for virus transmission), aphids need to be controlled by pesticides and/or using biological control agents. however, in the absence of a thorough understating of the genetics of aphid populations/clones, the identification of transmissible and adaptative variations could make biological and chemical controls not really effective (loxdale, 2008b). same nuclear genome, different microbiome? aphids have close associations with various lineages of microorganisms and most of them may harbour, for instance, the obligate mutualist (usually called primary symbiont) buchnera aphidicola (russell and moran, 2006). in addition to b. aphidicola, other maternally transmitted intracellular bacteria, such as rickettsia sp. (α-proteobacteria), spiroplasma sp. (mollicutes) and various γproteobacterial microbes (including hamiltonella defensa, regiella insecticola, serratia symbiotica and arsenophonus sp.), are harboured in aphids constituting their microbiome (chen et al., 1996; fukatsu et al., 2000, 2001; gomez-valero et al., 2004) (fig. 1). aphid secondary symbionts are often shared between divergent lineages and they seem to undergo both vertical and horizontal transfer among matrilines within and between species (russell and moran, 2006). this transmission is due to the ability of the secondary symbionts to overcome host immune responses and invade various types of host cells, including germ cells (russell and moran, 2006). several data suggested that symbiotic bacteria are involved in different traits of the aphid biology, including resistance to parasitoid wasps (oliver et al., 2003), tolerance to heat stress (montllor et al., 2002) and changes in the host plant range (tsuchida et al., 2004). moreover, as also showed in other insects, symbionts may modulate other complex interactions protecting the host from the invasion by pathogenic microorganisms (a process known as “colonization resistance”) and modulating the aphid immune system (russell and moran, 2006; poirié and coustau, 2011). microbiome seems therefore to act in aphids (and also in taxa other than insects) as a sort of ecological immunity or extended immune system being able of affecting 3   the efficiency of the host immune system and limiting the accumulation of pathobionts. the role of the aphid microbiome could be particularly relevant since, as recently reviewed, the immune deficiency (imd) signalling pathway was apparently non functional in aphids (poirié and coustau, 2011). moreover, no genes coding for peptidoglycan recognition proteins (pgrps) and several wellconserved antimicrobial peptides, such as defensins and cecropins, have been predicted in the pea aphid acyrthosiphon pisum genome (gerardo et al., 2010; poirié and coustau, 2011), making the microbiota-based immunity essential to protect aphids against natural enemies (poirié and coustau, 2011). different roles have been suggested for symbionts other than the synthesis of amino acids (douglas et al., 1998). buchnera might, for instance, play a key role in aphid thermal tolerance. thermal tolerance of the primary endosymbiont buchnera is attributed to genes coding for heat shock proteins, which deter degradation of protein secondary structure (shigenobu et al., 2000). interestingly, in the presence of low density of primary symbionts, secondary symbionts (such as h. defensa, s. symbiotica and r. insecticola) could be more numerous affecting the aphid thermal tolerance (oliver et al., 2010). for instance, s. symbiotica has a beneficial effect on a. pisum reproduction and viability under heat stress (montllor et al., 2002), thus providing a functional explanation to the previous observations that its frequency reached 80 % in hot places (oliver et al., 2010). secondary symbionts may change aphid colour, as showed for rickettsiella that induces a body colour change from red to green affecting prey-predator interactions, since ladybird beetles preferentially consume red aphids, whereas parasitoids are more attracted by green ones (tsuchida et al., 2010). secondary symbiont can also affect the adaptation to the host plant as assessed for infections by r. insecticola that could improve aphids' fitness specifically on trifolium plants (tsuchida et al., 2004). lastly, resistance to parasitoids is associated with secondary symbionts, as showed by several studies where the symbiotic associations have been experimentally manipulated either by suppressing symbionts using antibiotics treatment or by introducing a new symbiont through bacterial microinjection. according to literature data, aphidius ervi parasitism success on a. pisum is lower in aphid lines harbouring h. defensa and s. symbiotica respectively (oliver et al., 2003, 2005, 2006; ferrari et al., 2004; vorburger et al., 2009; poirié and coustau, 2011). on the whole, secondary symbionts play different roles, but are they stable within an aphid species? can they vary among populations? according to sandström et al. (2001) the associations with secondary symbionts are quite variable, suggesting a rather labile relationship between aphid species and their secondary symbionts. therefore, in contrast to the stable association between aphids and their primary symbionts (moran et al., 1993; clark et al., 2000), secondary symbionts can be lost due to infidelity of the vertical transmission or gained by horizontal transmissions. for instance, an a. pisum laboratory clone that lost two secondary symbiont types has been described in literature (sandström et al., 2001), together with the identification in the field of aphid populations hosting multiple or single secondary symbionts (sandström et al., 2001). moreover, a differential resistance to braconid parasitoids has been described in populations of a. pisum (hufbauer and via, 1999; ferrari et al., 2001), m. persicae (von burg et al., 2008) and aphis fabae (vorburger et al., 2009), suggesting that a differential composition of the microbiome strongly affect the survival and reproduction of aphids. if we therefore accept that a large community of bacteria may invade aphids and that they are transmitted both vertically and horizontally within and among aphid lineages and that different populations may have different microbiomes, can we surmise without any doubt that each aphid within a clone harbours the same microbiome? in view of the horizontal transfer of symbionts and their beneficial effects on the reproductive success of parthenogenetic females, a single gain of a secondary symbiont can have beneficial effects on the aphid carriers influencing their fitness. at this regards, gehrer and vorburger (2013) demonstrated a previously undescribed route of horizontal transmission consisting in the parasitoidmediated transfer of endosymbionts among aphid clones by sequentially stabbing infected and uninfected aphids. the wasp’s ovipositor appears to act as a ‘dirty needle’ that can inoculate previously uninfected aphids. if the recipient aphid resists the parasitoid and survives the attack, this can result in a new, heritable infection. considering that many aphid parasitoids use multiple hosts, it is likely that they can transfer symbionts not just within, but also between, aphid species (gehrer and vorburger, 2013). due to their relevant roles in aphid biology, the presence/absence of a secondary symbiont may have an important phenotypic effect so that aphid microbial symbionts form a true second genome with its own genetic inheritance (moran, 2007; gilbert, 2011). furthermore, symbiotic bacteria provide a selectable allelic variation (thermotolerance, colour and parasitoid resistance) that enables aphids to persist under different environmental conditions so that mutations occurring in the symbiont genomes may affect aphid fitness and evolution (dunbar et al., 2007; tsuchida et al., 2010). aphids as holobionts more than a hundred years of biological research demonstrated the importance of microorganisms in the health and disease of higher organisms, including humans (ottaviani et al., 2011). as a result of the recent development of culture-free molecular techniques, it is now accepted that in many cases the number of symbiotic microorganisms and their combined genetic information far exceed that of their hosts so that for each gene in our genome, we host about http://rstb.royalsocietypublishing.org/content/365/1540/671.full http://rstb.royalsocietypublishing.org/content/365/1540/671.full http://www.plosbiology.org/article/info%3adoi%2f10.1371%2fjournal.pbio.0050096 http://www.plosbiology.org/article/info%3adoi%2f10.1371%2fjournal.pbio.0050096 4   100 genes belonging to human bacterial symbionts (the human microbiome project consortium, 2012). in view of this new view of the symbiotic interactions, zilber-rosenberg and rosenberg suggested the hologenome theory of evolution defining hologenome as the sum of the genetic information of the host and its microbiome (rosenberg et al., 2007; zilber-rosenberg and rosenberg, 2008). as a consequence of the hologenome theory of evolution, each organism should be viewed as an holobiont including the host and its symbiotic microbiome and the holobiont is the true unit of selection in evolution in place of the single host as individual separated from its symbionts (rosenberg et al., 2007; zilber-rosenberg and rosenberg, 2008). at this regards, it has to be underlined that relatively rapid variation in the diverse microbial symbionts can have an important role in the adaptation and evolution of holobionts identifying them as dynamic entities in which a vast amount of the genetic information and variability is contributed by microorganisms. in view of this assumption, the evolution of holobionts can occur by changes in the host genome and/or in any of the hosted microbial genome, and relies on the cooperation between the genomes within the holobiont, as much as on competition with other holobionts. similarly, genetic variation can arise from changes in either the host or the symbiont genomes. variation in host genomes occurs during sexual reproduction, chromosome rearrangements and ultimately by mutations, but the same processes occur in microbial symbionts with the noteworthy difference that in haploid bacteria recombination occurs also by conjugation, transduction and dna transformation among different species (rosenberg et al., 2007; zilber-rosenberg and rosenberg, 2008). concluding remarks contrarily to rare recombinations and mutations of the host genome, changes in the genetic information related to symbionts can occur quickly by microbial amplification, acquisition of novel bacterial strains and horizontal gene transfer between different species (including gene transfer from the symbiont to the host genome). in particular, the microbial amplification is the most rapid and easy mechanism to achieve variations in holobionts, since it involves changes in the relative numbers of the diverse types of associated microorganisms that can occur as a result of changing temperatures, nutrient availability, exposure to xenobiotics or other environmental factors (moran, 2007; rosenberg et al., 2007; zilber-rosenberg and rosenberg, 2008; gilbert, 2011). the main advantage for hosts is therefore that they can survive, multiply and gain the time necessary for their genome to evolve using the genetic information available in their symbionts (moran, 2007; gilbert et al., 2012). as a whole, although dan janzen’s (1977) original concept was fascinating and not implausible thirty years ago, the use of the one-genome/oneorganism paradigm of classical genetics has been eclipsed by recent studies on symbiosis suggesting a revision of our approach to the aphid biology and evolution. aphid clones cannot be considered as evolutionary individuals in any sense of the term neither the idea of aphids as a single genome species should be further considered in the light of the existence of the aphid hologenome. on the whole, also taking into account the peculiar structure of their immune system, aphids should be regarded as highly plastic organisms (probably more than other insects), whose evolution has been shaped by their symbionts. references caspi-fluger a, inbar m, mozes-daube n, katzir n, portnoy v, belausov e, et al. horizontal transmission of the insect symbiont rickettsia is plant-mediated. proc. r. soc. b. 279: 17911796, 2011. chen dq, campbell bc, purcell ah. a new rickettsia from a herbivorous insect, the pea aphid acyrthosiphon pisum (harris). curr. microbiol. 33: 123-128, 1996. devonshire al, field lm, foster sp, moores gd, williamson ms, et al. the evolution of insecticide resistance in the peach–potato aphid, myzus persicae. in: denholm i, pickett ja, devonshire al (eds), insecticide resistance: from mechanisms to management. wallingford, oxon, cabi publishing, pp 1-9, 1999. dixon afg. parthenogenetic reproduction and the rate of increase in aphids. in: minks a, harrewijn p (eds), aphids, their biology, natural enemies and control. vol. a, elsevier, the netherlands, pp. 269-287, 1989. douglas ae. nutritional interactions in insectmicrobial symbioses: aphids and their symbiotic bacteria buchnera. annu. rev. entomol. 43: 1737, 1998. ferrari j, muller cb, kraaijveld ar, godfray hcj. clonal variation and covariation in aphid resistance to parasitoids and pathogen. evolution 9: 1805-1814, 2001. fukatsu t, nikoh n, kawai r, koga r. the secondary endosymbiotic bacterium of the pea aphid acyrthosiphon pisum (insecta: homoptera). appl. environ. microbiol. 66: 27482758, 2000. fukatsu t, tsuchida t, nikoh n, koga r. spiroplasma symbiont of the pea aphid, acyrthosiphon pisum (insecta: homoptera). appl. environ. microbiol. 67: 1284-1291, 2001. gehrer l, vorburger c. parasitoids as vectors of facultative bacterial endosymbionts in aphids. biol. lett. 2013 [in press]. gerardo nm, altincicek b, anselme c, atamian h, barribeau sm. immunity and other defenses in pea aphids, acyrthosiphon pisum. genome biol. 11: r21, 2010. gilbert sf. symbionts as genetic sources of hereditable variation. in: gissis sb, jablonka e. 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genotypic variation and the role of defensive endosymbionts in an all parthenogenetic host-parasitoid interaction. evolution 63: 1439-1450, 2009. zilber-rosenberg i, rosenberg e. role of microorganisms in the evolution of animals and plants: the hologenome theory of evolution. fems microbiol. rev. 32: 723-735, 2008. isj 9: yyy-xxx, 2012 isj 9: 200-206, 2012 issn 1824-307x minireview the immune role of the arthropod exoskeleton y moret, j moreau équipe écologie évolutive, umr cnrs 6282 biogéosciences, université de bourgogne, 6 boulevard gabriel f21000 dijon, france accepted november 17, 2012 abstract the exoskeleton or cuticle of arthropods is an important feature that contributes to their great success in colonising numerous habitats on earth. it has numerous functions among which to provide protection against parasites. whereas often regarded as a simple physical barrier to the outside world, the immune protection of the cuticle is slightly more complex than that. here, we provide an overview of the cuticle defensive traits against parasites and examine their variation as a response to parasitism. it appears that the cuticle is an efficient line of defense, which includes physical, biochemical and physiological defensive components that are potentially subject to genetic and plastic variation in response to parasitism. it also appears that the cuticle defense systems are relatively understudied despite it may determine for large part the success of parasitic attacks. key words: invertebrates; immunity; cuticle defense; parasite; ecological immunology introduction arthropods include insects, spiders, centipedes, shrimp, and crayfish, and make up the most abundant phylum in the animal kingdom. a key feature that contributed to the phenomenal success of this taxon is their cuticle. the cuticle, corresponds to the relatively thin, but tough and flexible, layer of non-cellular material that cover the whole external body surface, as well as respiratory organs, the anterior and posterior portion of the digestive tract, and reproductive ducts (wigglesworth, 1957). the cuticle is often viewed as the “skin” of arthropods but it has many other functions. it provides physical support and protection of internal organs against chemical and mechanical damages, and serves as skeleton (exoskeleton) to which muscles are attached for locomotion. the cuticle also functions to limit the entry or loss of water, and form an efficient barrier protecting against invasion by eukaryotic parasites and infection by microorganisms. given the propensity of many arthropods to live in microbial-rich environments, each wounding event is likely to be accompanied by opportunistic infections (siva-jothy et al., 2005). furthermore, even in the most aseptic habitats, the outer surface of arthropod cuticle probably harbours ___________________________________________________________________________ corresponding author: yannick moret équipe écologie évolutive umr cnrs 6282 biogéosciences université de bourgogne 6 boulevard gabriel, f-21000 dijon, france email: yannick.moret@u-bourgogne.fr a diverse and abundant community of parasites (brey et al., 1986). since parasites decrease host fitness, arthropods evolved a suite of mechanisms of defense acting at different levels in the sequences of the parasitic infection to reduce the probability and impact of parasite infection (schmid-hempel and ebert, 2003; siva-jothy et al., 2005; duneau et al., 2011). these defense components include behavioural, physical and physiological mechanisms and the cuticle is one of these sequential defense mechanisms. the first line of defense involves behavioural mechanisms to avoid or remove parasites (cade and wyatt, 1984; cade, 1991; bischoff, 2003; and see kurtz et al., 2002; thomas and blandford, 2003). once an invader has overcome behavioural defenses and breached the protection of the exoskeleton, he is then exposed to the last line of defense that is the innate immune system or haemocoelic internal defenses. from these three lines of defense, boundary defense provided by the cuticle is probably the less known and the most poorly studied mechanism of resistance against parasites in arthropods. yet, the cuticle contributes greatly to successful defense against most parasites aiming to colonize the haemocoel. the success of the cuticle in this duty is well supported by the increase vulnerability of moulting arthropods to opportunistic parasites entering into the haemocoel with sometime dramatic survival consequences (le moullac et al., 1997; morado et al., 1999). a number of cuticular traits such as the chemical composition, structural 200 architecture and thickness reported to be involved in disease resistance, exhibit a great range of variation among and within species. they may also vary within an individual’s lifetime, depending on environmental cues directly or indirectly related to the threat of disease. therefore, it seems that the cuticle of arthropods is subject to modifications, which enhance its ability to act as physical or chemical barrier to penetration by parasites when the threat of disease is high (wilson et al., 2001). whereas moulting is a critical period of vulnerability to parasites, under certain circumstances, it may serve as a defensive mechanism reducing the negative effects of wounding, epibionts or parasites from the shell (duneau and ebert, 2012). in this respect, moulting could be included as part of the cuticle defense system, especially if the moulting event is affected by the presence of parasites in the environment (moret et al., 2010; but see duneau and ebert, 2012). here, we provide an overview of the cuticle defensive traits against parasites and examine their variation as a result of parasitism selection pressure. in addition, we further discuss the role of moulting as part of the cuticle defense system based on its interaction with parasite infection success. structure and formation of the cuticle of arthropods a typical arthropod integument is a layered structure mainly composed of three main regions (fig. 1). the innermost region is the basal membrane (or basal lamina), a thin connective tissue layer attached to the above epidermis and separating the integument from the haemocoel. the epidermis is a more or less continuous monolayer of epidermal cells responsible for the production and secretion of various materials for the above cuticle, which is the extracellular outer layer of the integument that gives most of its rigidity and colour (nation, 2002). the cuticle further stratifies into subsequent layers (fig. 1), typically with a thin waxy epicuticle covering a thicker procuticle consisting of protein, lipid, and chitin cross-linked to a varying degree to provide elasticity and hardness. in crustaceans and myriapods (and in few insects see leschen and cutler, 1994), the above cuticular regions are calcified by the addition of a substantial amount of calcium carbonate, leading to great mechanical strength. the epicuticle is the main water-proof barrier. this thin layer is unlikely to provide mechanical protection but may harbour antibacterial and/or cytolytic activity (harrington et al., 2008). in contrast, the procuticle is the bulk of the cuticle, which provides resistance to mechanical loads and is the next barrier to infection, mainly because of its thickness and architecture. in some part of the body the procuticle differentiates into a hard, outer exocuticle and a soft, inner endocuticle. differentiation of the exocuticle results from the sclerotisation of proteins that occurs shortly after each moult (see below). the formation of new cuticle comprises a succession of events that occurs during moulting. the rigidity of the cuticle restricts growth, so arthropods replace it periodically by moulting or shedding the old cuticle after growing a new one (fig. 2). moulting cycles occur nearly continuously until the animal reaches full size and are controlled by hormones (wigglesworth, 1957). the initial phase of moulting, or pre-ecdysis, is induced by an increased level of ecdysone, the arthropod-moulting hormone. ecdysone stimulates the growth of the epidermis and the release of moulting fluid containing enzymes that digests the endocuticle and thus detaches the old cuticle. this is also the phase when the new deposition of the new cuticle is initiated. when this stage is complete, it is followed by ecdysis during which the remnant of the old cuticle is shed through the uptake of air or water from the environment, causing the exoskeleton to rupture. at this point, the soft new cuticle is wrinkled and the new endocuticle has not yet formed. during postecdysis, the animal continues to pump itself up to expand the new cuticle, then hardens the new exocuticle and eliminates the excess air or water. by the end of this phase the new endocuticle has formed. hardening of the new exocuticle results from a biochemical process known as sclerotization during which the proteins of the outer procuticle are covalently bound to each other. sclerotisation is allowed by the presence, beneath the epicuticle, of enzymes including phenoloxidases such as laccase and tyrosinase. these enzymes catalyse the synthesis of quinones that polymerize to form melanin deposits in interaction with the cuticular proteins and chitin, to crosslink and harden them (andersen et al., 1996; terwilliger, 1999; andersen, 2010). in crustaceans and some other arthropods (see above), hardening of the cuticle also results from the process of mineralization, which involves the deposits of calcium salts in all layers of the cuticle except the outer layer of the epicuticle that calcify after ecdysis (stevenson, 1985). physical component of the cuticle defense intuitively, the protective function of the cuticle of arthropods relies on boundary defense, which consists of a tough and flexible integument covering the animal surface. this protection even extends to the digestive system, where a protective cuticular membrane called the peritrophic membrane, covers the midgut. (peters, 1992). despite this physical barrier, parasites, can invade directly through the exoskeleton. parasites that penetrate the cuticle are mainly bacteria, fungi and parasitoids. for instance, the entomopathogenic fungi such as metarhizium anisopliae and beauvaria bassiana, or bacteria responsible for the shell disease syndrome (e.g., vibrio sp., which induces characteristic black-spot lesions on the exoskeleton of marine crustaceans) use a combination of physical and enzymatic processes, such as chitinase and protease, to breach the cuticle (charnley and st. leger, 1991; vogan et al., 2001; freimoser et al., 2003, 2005; cho et al., 2006). resistance to these microbial infections resides mainly in cuticular thickness, the degree of cross-linking within the cuticular laminae 201 fig. 1 a schematic figure of the arthropod integument, showing the different structural layers of cuticle. and the degree of sclerotisation in the cuticle. in this respect, melanin deposits in the exocuticle and the epicuticle are likely to have an important role to increase the immune protection of the cuticle. as a polymer, melanin may strengthen the cuticle and so improves its ability to act as a physical barrier to the penetration of parasites (st. leger et al., 1988; hajek and st. leger, 1994). furthermore, melanin is toxic to microorganisms and has potent antimicrobial activity (e.g. söderhäll and ajaxon, 1982; montefiori and zhou, 1991; ourth and renis, 1993; sidibe et al., 1996; ishikawa et al., 2000), perhaps by binding a range of proteins (doering et al., 1999) and inhibiting lytic enzymes produced by microorganisms, including proteases and chitinases (kuo and alexander, 1967; bull, 1970). melanisation of the cuticle largely occurs shortly after moult. thus moult represents a critical period during which melanisation of the cuticle might be plastically altered from one intermoult period to the other, depending on the perceived risk of infection by the animal. examples of such plastic changes in the degree of melanisation of the cuticle are illustrated by the literature on density dependent polyphenism of the cuticular colour in insects (wilson, 2005). parasite transmission is assumed to be positively density-dependent (anderson and may, 1979), and the increased density of conspecific may represent a reliable cue for increased threat of diseases (wilson and reeson, 1998; mccallum et al., 2001). in response to this cue, individual insects exhibit a stronger melanisation of the cuticle during the next moult, which is associated with increased parasite resistance and immune function (kunimi and yamada, 1990; reeson et al., 1998; barnes and siva-jothy, 2000; wilson et al., 2001; cotter et al., 2004a, b). for instance, melanic caterpillars of two lepidopteran species, spodoptera exempta and spodoptera littoralis, were respectively found more resistant to the ectoparasitoid wasp, euplectrus laphygmae, and the entomopathogenic fungus, b. bassiana, than non-melanic caterpillars (wilson et al., 2001). similarly, the mealworm beetle, tenebrio molitor, shows density dependent polyphenism of adult cuticular colour. in this insect, the degree of cuticular melanization is a strong indicator of resistance to the entomopathogenic fungus, m. anisopliae, with darker beetles being more resistant than lighter ones (barnes and siva-jothy, 2000). thus, cuticular melanisation could be considered as an immune parameter in its own right (wilson et al., 2001). biochemical component of the cuticle defense if cuticular melanisation occurs mainly during the process of moulting, it could also be induced in response to a mechanical scratch or by microbial invasion. this defense response results from the presence of phenoloxidase zymogens, which are produced by haemocytes and transported to the cuticle across the epidermis (ashida and brey, 1995). this process is often used for wound healing and results in the formation of dark melanised plugs around the damage zone of the cuticle (plaistow et al., 2003; fig. 3). yet, whether phenoloxidase enzymes are present in the cuticle for the purpose of improving the physical structure of the integument or to afford defense against infection is unclear (siva-jothy et al., 2005). nevertheless, the activity of these enzymes is directed towards parasites in the cuticle since fungal germ tubes are melanised as they pass through the cuticle before they entered the haemocoel (golkar et al., 1993). inducible biochemical defense also involves the production of antibacterial peptides by epidermal cells, such as cecropins, which are transported in the vicinity of the microbial challenge to abraded cuticle (brey et al., 1993). hence, in addition to its obvious physical characteristics, the cuticle of arthropods also provides an active biochemical barrier. moulting as a component of cuticle defense growth and development of the arthropods involve a series of moults during which the old cuticle is digested while a new cuticle is formed and the remnant is discarded (fig. 2; see above). in addition to allow growth of the animal, moulting may 202 fig. 2 gammarus pulex (crustacea: amphipoda) shortly after ecdysis on the right side with its old cuticle on the left side. photo by f vogelweith. serve as a defensive mechanism reducing the negative effects of wounding or parasites from the shell. in nature, wounds can be extremely prevalent and form the main points of entry for parasites (plaistow et al., 2003). although wounds are rapidly healed by melanotic plugs (fig. 3), they may be less protective than a new cuticle. wounds favour the development of shell disease in crustaceans, resulting from the spread of chynolytic microorganisms in the cuticle (vogan et al., 2008). recent data show that moulting soon after parasite exposure helps to remove parasites attached to the cuticle and reduces the likelihood of successful infection. for instance, in the cotton aphid, aphis gossypii, exposed to the entomopathogen fungus, lecanicillium attenuatum, moulting removes conidia attached on the cuticle before their germ tubes penetrated the insect haemolymph (kim and robert, 2012). similarly, in the crustacean daphnia magna, moulting helps to remove up to 30 % of the spores of the castrating bacterium, pasteuria ramosa, attached on the oesophageal cuticle and reduces greatly the infection success of the parasite when it occurs within 12 h after exposure (duneau and ebert, 2012). these results show that moulting is an effective mechanism of resistance against parasites attached on the cuticle. however, they also stress the point that moulting, in order to be really beneficial, has to occur rapidly after the parasite is attached to the cuticle (before the parasite penetrates the host). it seems then that the benefit of moulting could be improved if it was combined with the ability for the host to perform precocious moulting in response to wounding or parasite attachment. unfortunately, available data on this matter are scarce and not conclusive. the only study that examined specifically whether hosts exposed to parasite could shorten time to ecdysis is the one by duneau and ebert (2012) who found no significant difference in moult interval between experimentally exposed and unexposed d. magna to spores of p. ramosa. however, there are indirect evidences that suggest different outcomes in other systems. indeed, laufer et al. (2005) found that levels of the moulting hormone (ecdysone, which initiate the process of moulting) are much higher in shell diseased lobsters than in unaffected ones. unfortunately, moult cycling was not measured in this study. maybe another indirect evidence is the one provided by the study of roth and kurtz (2008) on the red flour beetle, tribolium castaneum. the authors found that larvae wounded with a needle, either sterile or mucked with heat-killed bacteria, reduce larval development time compare to unmanipulated larvae. rapid growth allows moulting events to occur more frequently. in addition, in species such as t. castenum, the number of larval moults could be highly variable ranging from 8 to 20. one could hypothesise that in a parasite rich environment, larvae should perform more moults than in a parasite poor environment. however, such a response to parasitism should be costly because it 203 should lead to the development of smaller adults that usually exhibit a low investment to reproduction (thornhill and alcock, 1983). this hypothesis remains to be tested. whether parasite may speed up or not its occurrence, moulting, by preventing infection, might select parasite for higher parasite penetration speed (duneau and ebert, 2012) or to develop means that prevent or slowdown moulting (kamimura et al., 2012). if this is true, there is therefore room for an arm-race between moulting defence and parasite infectivity. on the other hand, when shedding the old cuticle, the animal exposes a new, soft and untanned cuticle to the outside world. this may represent a critical period of vulnerability to parasites entering the host through the cuticle until the properties of the integument are fully reestablished (le moullac et al., 1997; morado et al., 1999). however, recent findings show that antimicrobial peptides can be prophylactically produced during this period of vulnerability (an et al., 2012). the presence of parasites in the environment is temporally and spatially variable, resulting in periods or sites of variable risk of infection. therefore, arthropods may gain a survival advantage by adjusting moult timing to correspond to the lowest risks of infection. consistent with this hypothesis, the crustacean, gammarus pulex, was found to exhibit temporal adjustments of its moult cycling in response to elevated risks of infection by postponing ecdysis by several days when the individuals are exposed to ‘micro-organismenriched’ water (moret et al., 2010). concluding remarks this minireview intended to provide the reader with an overview of the defensive traits of the cuticle of arthropods against parasites in order to highlight its important role in the immune protection of these animals. the defense line that confers the cuticle of arthropods is probably less complex but also fairly understudied compare to haemocoelic defense systems (see lemaître and hoffmann, 2007 for a review). consequently, its involvement in resistance to parasitic infections is probably often underestimated. yet, the outcome of parasitic attacks depends to a large extent on cuticular defenses. furthermore, beyond the acknowledgement of its great protective efficacy, the immune role of the exoskeleton of arthropods cannot be simply regarded as an inert and invariable physical barrier to the outside world. indeed, defense components of the exoskeleton also comprise inducible biochemical processes and ecdysis in addition of the physical structure of the cuticle. all these traits are potentially subject to variation depending on level of the parasitic selective pressure. furthermore, variation could also occur plastically within an individual lifetime in response to environmental cues presaging changes in the threat of disease. from a functional as well as ecological point of view, investigation about the causes and consequences of variation of cuticular defenses should provide significant insight into the evolution of immune defence in a larger picture. fig. 3 larvae of the european grape berry moth, eupoecilia ambiguella (lepidoptera: tortricidae) exhibiting melanic plugs on sites of wounding of the cuticle. photo by f vogelweith. acknowledgements we thank maf gillingham for valuable comments on the manuscript and f vogelweith for kindly providing the illustrative pictures. support was provided by the cnrs and the anr (anr-07-jcjc0134 and anr-08-jcjc-0006). references an s, dong s, wang q, li s, gilbert li, stanley d, et al. insect neuropeptide bursicon homodimers induce innate immune and stress genes during molting by activating the nf-κb transcription factor relish. plos one 7(3): e34510. doi:10.1371/journal.pone.0034510, 2012. anderson rm, may rm. population biology of infectious diseases: part i. nature 280: 361367, 1979. andersen so. insect cuticular sclerotization: a review. insect biochem. mol. biol. 40: 166-178, 2010. andersen so, peter mg, roepstorff p. cuticular sclerotization in insects. comp. biochem. physiol. 113b: 689-705, 1996. 204 ashida m, brey pt. role of the integument in insect defense: pro-phenol oxidase cascade in the cuticular matrix. proc. natl. acad. sci. usa 92: 10698-10702, 1995. barnes a., siva-jothy mt. density-dependent prophylaxis in the mealworm beetle tenebrio molitor l. 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50-57, 2013 issn 1824-307x research report heat stress induces ros production and histone phosphorylation in celomocytes of eisenia hortensis ra tumminello, sl fuller-espie science department, cabrini college, 610 king of prussia road, radnor, pennsylvania 19087-3698, usa accepted june 18, 2013 abstract the effect of heat stress on celomocytes (leukocytes) from eisenia hortensis was investigated by measuring the production of reactive oxygen species (ros). after culturing celomocytes at temperatures ranging from 4 °c (control) to 44 °c for 3-16 h, ros levels were measured using a flow cytometric method employing dihydrorhodamine 123 (dhr123) for ros detection and 7aminoactinomycin d (7-aad) as a viability stain. reproducibly we observed significant (p < 0.05) increases in ros production and decreases in cell viability at temperatures of 28 °c and above. we then examined the effect of heat stress on histone phosphorylation employing antibodies specific for γh2ax as an indicator of histone modification. celomocytes were incubated at temperatures ranging between 20 °c to 35 °c for 16 h and antibodies specific for phosphorylated serines in h2ax histones were employed through flow cytometric analysis. comparing controls to heat-stressed samples using three separate assays reproducibly confirmed significant h2ax phosphorylation (p < 0.05). collectively, these results emphasize the importance of selecting appropriate temperatures for rearing invertebrates in laboratory-based habitats and for culturing invertebrate cells when conducting in vitro assays in order to minimize oxidative stress. the possible cellular effects of heat stress in soil ecosystems associated with global warming events is also considered. key words: reactive oxygen species (ros); γh2ax; histone phosphorylation; heat stress; in vitro cell culture   introduction reactive oxygen species (ros) are highly reactive molecules used during innate host defense mechanisms to eradicate pathogens and initiate programmed cell death signal cascades (bolwell, 1996; forman and torres, 2002; pastori, 2002; reth, 2002). ros are produced by phagocytes such as neutrophils and macrophages and are a universal indicator of oxidative cellular stress (finkel, 2001; fuller-espie et al., 2010). the formation of ros from ground state oxygen takes place through a series of reductions provided by either energy transfers or by electron transfer reactions. this series of reactions leads to the formation of superoxide radical ions, which are reduced to hydrogen peroxide and then reduced to a hydroxyl radical through a series of electron transfers (klotz, 2002; apel and hirt, 2004). ros is a natural byproduct of metabolism, but when an excess ___________________________________________________________________________ corresponding author: sheryl l. fuller-espie science department cabrini college 610 king of prussia road, radnor, pa 19087-3698, usa e-mail: sfuller-espie@cabrini.edu of these highly radical molecules accumulate during times of stress, the ability of the cell to neutralize intermediates or repair their toxic effects is overwhelmed leading to damage of cellular membranes, dna, neighboring cells and tissues through the induction of inflammatory events (duval et al., 2003; fuller-espie et al.,2010). ros resulting in dna damage have been shown to be produced through a variety of environmental stimuli including ionizing radiation, chemical mutagens, as well as heat (beckman and ames, 1998; bruskov et al., 2002). the effect of heat stress on organisms has been studied and confirmed to increase the production of ros, possibly by interfering with electron transport assemblies of the inner mitochondrial membrane and may be a factor resulting in disruption of biological molecules such as dna, proteins, and lipids (ando et al., 1997; bruskov et al., 2002; mujahid et al., 2005). invertebrate heat-induced ros is a concern for laboratories frequently using invertebrate experimental organisms for in vitro assays. in addition, organisms not reared at appropriate temperatures in laboratory-based habitats may generate ros leading to perturbation   50 of results observed in both in vivo and in vitro studies. in our lab, various in vitro cellular assays are conducted routinely including innate immune response assays to investigate pathogen-associated molecular patterns (pamps) and xenobiotics, natural killer cell-like activity assays, and phagocytosis assays (patel et al., 2007; fullerespie, 2010; nacarelli and fuller-espie, 2011; fuller-espie et al., 2011). the primary goal of this in vitro study was to investigate the effect of heat stress on the celomocytes of eisenia hortensis (also known as the european nightcrawler) and to determine the temperature threshold which triggers increased generation of ros for optimal in vitro studies. to determine the onset of heat-induced cellular damage, ros production was detected through a range of temperatures along with analyzing cellular viability and h2ax histone phosphorylation. the generation of dna damage was observed through double-stranded breaks which were identified by phosphorylated histone protein h2ax at the flank of the double-strand break at ser-139 at the cterminus (shimohara et al.,2008). phosphorylation is mediated by dna-dependent protein kinases and the resulting phosphorylated form of h2ax is referred to as gamma-h2ax (γh2ax) (rogakou et al., 1999). h2ax is unique and is highly conserved between eukaryotes (madigan et al., 2002). furthermore, it has been reported that antibodies that specifically bind phosphorylated h2ax in mammals also recognize radiation-induced proteins in muntiacus muntjac, xenopus laevis and drosophila melanogaster (rogakou et al., 1999). this observed conservation between various organisms made the h2ax protein an ideal target for working with e. hortensis. earthworm celomocytes can be studied in vitro through experimental extrusion through the dorsal pores of the celomic cavity where the celomocytes are located. three distinct subpopulations of celomocytes exist which are believed to derive from a single progenitor cell (prohemocyte): phagocytic hyaline amebocytes (large celomocytes), granular natural killer-like amebocytes (small celomocytes), and eleocytes which contain chloragosomes which are used to secrete lytic substances (engelmann et al., 2004; hartenstein, 2006; fuller-espie et al., 2008). post extrusion, cells are analyzed with flow cytometry allowing simultaneous measurements of multiple characteristics of a single cell at a rapid rate. flow cytometry measures relative size (forward scatter), the granularity or internal complexity (side scatter) (allowing the differentiation between subpopulations), and also fluorescence associated with cellular subpopulations. in our experiments relative fluorescence intensity using fluorochromes was measured by the instrument to determine: 1) cellular viability using 7-aminoactinomycin d (7aad) which only binds to double-stranded nucleic acid and fluoresces upon entry into a non-viable, membrane-compromised cell; 2) ros production upon oxidation of the reporter molecule dihydrorhodamine 123 (dhr 123); and 3) histone phosphorylation using antibodies specific for γh2ax and fluorescein isothiocyanate(fitc)-conjugated secondary antibodies (rabinovitch et al., 1986; nicoletti et al., 1991; huang and darzynkiewicz, 2006). materials and methods reagents general laboratory reagents and plastic ware were purchased from fisher scientific unless otherwise noted. cell culture all cell culture reagents and phosphate-buffered saline (pbs) were purchased from invitrogen unless otherwise noted. dulbecco’s modified eagle medium (dmem) was supplemented with 10% serum supreme (lonza bio whittaker), 100 μg/ml ampicillin (shelton scientific), 10 μg/ml kanamycin (shelton scientific), 10 μg/ml tetracycline, 5 μg/ml chloramphenicol (fluka biochemika), 1x penicillin/streptomycin/amphotericin b (gibco), 1x nonessential amino acids (gibco) and 1x lglutamine (gibco) to comprise super dmem (sdmem). all assays were conducted in vitro post celomotycte extrusion. the 3 h incubation experiments were conducted in water baths in test tubes. the 16 h incubations were conducted in 5% co2 incubators in 96-well v-bottom plates. earthworm husbandry e. hortensis were purchased from vermitechnology unlimited, orange lake, florida, usa, who import e. hortensis from star food, holland, scherpenzeelseweg 95, 3772me barneveld, the netherlands. species identity was determined by the united states department of agriculture, usda permit #52262 (vermitechnology, personal communication). short-term colonies were maintained at room temperature (rt) in the dark on autoclaved pine wood chips and shredded paper moistened with water and single grain rice cereal or rice with bananas cereal (gerber) until use. bedding and food were changed twice weekly. extrusion of celomocytes prior to experimentation, earthworms were chosen based on their color and activity; earthworms with healthy deep coloration, lacking yellow appearance, and with high activity were placed overnight on moist paper towels saturated with 2.5 μg/ml fungizone (fisher scientific) to reduce the level of fecal material and other surface contaminants. to collect celomocytes from an earthworm, earthworms were extruded according to bearoff and fuller-espie (2011). briefly, earthworms were placed in a plastic autoclaved trough containing 3 ml of facs flow sheath fluid (bd biosciences) used as an extrusion buffer. the celomocytes were then transferred to 0.5 ml accumax (innovative cell technology) for a 5 min incubation period at rt. then the cells were washed with 5 ml pbs prior to centrifugation at 150 xg for 5 min at 4 °c. celomocytes were resuspended in 1 ml or 0.5 ml sdmem and enumerated using a hemocytometer. only large celomocytes (lc) and small celomocytes (sc) were included in the cell count; eleocytes (which are highly autofluorescent) were not counted but did   51 fig. 1 representative flow cytometry profiles for ros and viability assays. (a) depicts a typical celomocyte profile produced post flow cytometric acquisition of fsc (size) (abscissa) versus ssc (granularity) (ordinate) of celomocytes where r1 = granular amebocytes, which were the desired celomocyte subpopulation for this study. (b) shows fsc (abscissa) versus fl-3 fluorescence (ordinate) of r1-gated celomocytes to distinguish viable from non-viable cells. r2 depicts non-viable 7-aad positive cells (fl-3 positive) and r3 depicts viable 7-aad negative cells (fl-3 negative). (c and d) show fl-1 (abscissa) versus cell number (ordinate) of amebocytes gated on both r1 and r3. control (c) and heat-treated (d) samples are shown comparing temperatures 20 °c to 35 °c, respectively. fsc = forward scatter; ssc = side scatter; fl-1 = relative fluorescence intensity of dhr123; fl-3 = relative fluorescence intensity of 7-aad. factor into a quality score. samples with large numbers of eleocytes compared to lc and sc were not used in assays. for preliminary ros production and cellular viability assays, individual earthworms (n = 3) were selected and celomocytes were extruded and analyzed in duplicate. during later ros production and cellular viability assays, as well as γh2ax assays, selected celomocytes from approximately seven earthworms were batched and assays were performed in duplicate or triplicate as indicated. detection of cell viability and ros the viability dye 7-aminoactinomycin d (7-aad; bd biosciences) was employed for two purposes: 1) to enable the elimination of dead cells from our ros analyses; and 2) to determine percent viable cells. for positive controls, celomocytes (105 cells in 0.2 ml) were pretreated with saponin (0.1%) at 25 °c. 7aad fluorescence was measured using the fl-3 detector. cell viability was measured after establishing a threshold to distinguish live versus dead cells using saponin-treated celomocytes as the dead cell indicator (see fig. 1). the higher the percent of cells entering the fl-3 positive region (r2) of the saponin control, the lower the percent cell viability. ros production was measured in viable celomocytes by gating on small celomocytes that were 7-aad negative using dihydrorhodamine 123 (dhr 123; invitrogen, 1 μm in sdmem) which was converted to rhodamine 123 upon oxidation. for positive controls, celomocytes (105 cells in 0.2 ml) were pretreated with h o2 2 (67.6 mm), or cdcl2 (500 µm) (data not shown). rhodamine 123 fluorescence was measured using the fl-1 detector. fixation of celomocytes for γh2ax analysis celomocytes from 7 earthworms were pooled (batched) and used at 2-2.5 x 105 in 0.2 ml and incubated at temperatures ranging from 20 °c – 35 °c in three separate assays in duplicate or triplicate. following heat treatment, celomocytes were fixed according to huang and darzynkiewicz (2006) with   52 minor modification. celomocytes were centrifuged (150 xg, 5 min, 4 °c) and treated with 0.2 ml icecold 1% methanol-free formaldehyde. following a 15 min incubation celomocytes were centrifuged and then resuspended in ice-cold 70% ethanol. after fixation (up to one week at -20 °c) the celomocytes were centrifuged, the ethanol was removed, and cells were washed twice in 200 µl 1% bovine serum albumin, 0.2% v/v triton x-100 in pbs (bsa-t-pbs) before adding blocking serum. immunofluorescence γh2ax antibodies detection of γh2ax using flow cytometric analysis was performed according to huang and darzynkiewicz (2006) with minor modification. postfixation and prior to primary antibody incubations, cells were incubated in 10% blocking serum (mouse serum sc-45051) for 20 min at rt. γh2ax was detected using primary rabbit polyclonal anti-γh2ax antibody specific for the phosphorylated serine 129 (sc-101696). in addition isotype-matched control normal rabbit igg (sc-3888) antibody was used to ensure antibody specificity. secondary mouse antirabbit igg fitc-conjugated antibody (sc-2359) was used. all antibodies and sera were ordered from santa cruz biotechnology, inc. and used at the concentrations recommended by the supplier. flow cytometric analyses flow cytometry was carried out using a facscalibur flow cytometer (bd biosciences). listmode data was acquired and analyzed using cell quest pro software. fig. 1 illustrates the approach used to analyze ros production. first, a two-parameter dot plot depicting forward scatter (fsc) versus side scatter (ssc) was created (fig. 1a). region 1 (r1) was placed around the granular amebocytes. next, a two parameter dot plot gated on the r1 subpopulation measured forward scatter versus 7-aad relative fluorescence intensity (fl-3), and two additional regions were placed on the cellular events. r2 represents the 7-aad-positive population (non-viable cells), and r3 represents the 7-aad-negative population (viable cells) (fig. 1b). after exposure to heat, ros production in live granular amebocytes was measured using the fluorogenic substrate dhr 123, a probe widely used to measure intracellular h202. in order to detect ros production a single parameter histogram gated on both r1 and r3 population measured rhodamine 123 (fl-1). therefore, the final analysis was restricted to live granular amebocytes. fig. 1 shows representative histograms comparing a 20 °c control (fig. 1c) versus a 35 °c heat-stressed sample (fig. 1d). marker 1 (m1) was set at approximately the median (~50%) of the control histogram as a baseline for comparison. events were considered positive if they exhibited a higher relative fluorescence intensity than the median fluorescence value obtained in the control cells. figure 1c shows the 20 °c control sample with a value of 50.91% compared to the 35 °c heatstressed sample shown in figure 1d which has a value of 89.79%. preliminary samples were run in duplicate (figs 2a and 2b) whereas later assays were run in triplicate (figs 2c and 2d) and average values were graphed showing standard deviation. statistical analysis all graphs and data analysis were created and processed using microsoft excel 2007. student’s ttest paired two samples for means was used with a 95% confidence interval to determine statistical significance. results flow cytometric detection of ros production and viability healthy celomocytes were identified through the use of the viability dye 7-aad which enters only membrane-compromised cells and upon binding to double-stranded nucleic acid fluoresces, thereby indicating non-viability. therefore, 7-aad negative cells exhibiting no fluorescence could be preferentially selected for final analysis of rhodamine 123 (fl-1 detector) by creating regions and gating on desired subpopulations. cells undergoing late apoptotic or necrotic pathways are considered non-viable (rabinovitch et al., 1986; bearoff and fuller-espie, 2011). in order to identify viable cells a two parameter dot plot gated on the r1 subpopulation measured forward scatter versus 7-aad relative fluorescence intensity (fl-3) and consisted of two additional regions (r2 and r3) which were utilized to determine the effects of heat stress on cellular viability (fig. 1b). as a starting point, a wide range of incubation temperatures (4 °c 44 °c) was employed in duplicate to examine ros production and viability. figure 2a illustrates the ros results while figure 2b shows the viability results for this temperature range. in this experiment, the 4 °c sample was the control to which the others were compared, and a 3 h incubation period was used. this lead to further experiments that expanded the temperature range to represent their natural habitat more closely (20 °c – 35 °c) and also increased the duration of incubation to 16 h. three additional assays were performed using either individual earthworms in duplicate or triplicate (data not shown), or batches of earthworms (7 per batch) in triplicate. figure 2c shows ros production and figure 2d shows viability, both representative of data acquired for all three assays. here the 20 °c sample was the control to which the others were compared. statistically significant (p ≤ 0.05) results were obtained for viability assays at temperatures of 25 °c and higher, and for ros assays at temperatures of 30 °c and higher for the majority of batches examined. therefore, these results clearly show increased cell death and ros production as temperatures increase above 25 °c or 30 °c, respectively. flow cytometric detection of h2ax phosphorylation figure 3a illustrates the specificity of the antiγh2ax antibody that was used in this study and the degree of nonspecific binding of antibodies to celomocytes. celomocyte samples incubated at 35°c and subsequently fixed were stained with either an isotype-matched control antibody (left curve) or the anti-γh2ax antibody (right curve). this result shows that the anti-γh2ax antibody binds to celomocytes with specificity. figure 3b depicts a   53 fig. 2 heat-stressed granular amebocytes exhibit increased ros production and decreased cell viability. (a and c) are gated on both r1 and r3 (see fig. 1) and incubation temperature (abscissa) versus the percent increase in ros production above the median for the untreated control (ordinate) (a control = 4 °c; c control = 20 °c) are shown. (b and d) indicate the percent of all cells in r1 that are also included in r3 (see fig. 1) showing incubation temperature (abscissa) versus the percent of viable cells post-treatment (ordinate). each bar color corresponds to an individual earthworm analyzed in duplicate (a and b) or the average of different batches of earthworms (n=7) analyzed in triplicate (c and d). * = p ≤ 0.05; ** = p ≤ 0.005; *** = p ≤ 0.0005; **** = p ≤ 0.00005. error bars signify standard deviation. histogram overlay comparing a control sample (25 °c) to a heat-treated sample (35 °c) measuring h2ax phosphorylation. it shows the positioning of the m1 marker set at the approximate median of the control sample. figures 3c and 3d are representative of results obtained from a total of three assays. the assay depicted in figure 3c was conducted in duplicate at incubation temperatures of 20 °c, 25 °c, 30 °c, or 35 °c, and the control was the 20 °c sample. the assay depicted in figure 3d was conducted in triplicate at incubation temperatures of 25 °c, 30 °c, 32.5 °c, or 35 °c and the control was the 25 °c sample. these results indicate that statistically significant (p ≤ 0.05) heatinduced h2ax phosphorylation is taking place in celomocytes at 30 °c and above compared to controls. discussion this study demonstrates that heat stress (28 °c and above) causes ros production, decreases cell viability, and results in the phosphorylation of the h2ax histone in e. hortensis and therefore appropriate temperatures for rearing organisms and conducting cell culture assays must be established and implemented. avoiding heat-induced stress will serve to minimize undesired background when conducting in vitro cellular assays. although the mechanism of interaction between ros and γh2ax is not well understood, it has been suggested that ros production may be induced by the accumulation of γh2ax during dna damage response (ddr) which results in γh2ax regulation of nox1-mediated ros generation post-dna damage (kang et al., 2012). γh2ax has been called “the histone guardian of the genome” and seems to play an intricate role in ddr as the “marker of dna breaks” signaling the need for dna repair survival or apoptosis (fernadez-capetillo et al., 2004; cook et al., 2009). although a homolog of p53 has not been identified in e. hortensis, it is still of interest to note that a relationship between γh2ax-induced ros generation seems to play a role in upstream signals that trigger p53 activation (bragado et al., 2007; liu et al., 2008). although this study focused on heat stress in the earthworm, it is interesting to note that recently the effect of gene expression in earthworms in response to cold stress conditions was investigated leading to the construction of a coldresponse regulatory-gene network (kim et al., 2012).   54 fig. 3 heat stress induces phosphorylation of h2ax. (a) shows representative fl-1 (abscissa) versus cell number (ordinate) on a histogram overlay between a control sample incubated with isotype-matched antibody (left curve) and a sample incubated with anti-γh2ax antibody (right curve), both heat-treated at 35 °c utilizing the m1 marker method to determine antibody specificity as previously stated. (b) shows representative fl-1 (abscissa) versus cell number (ordinate) on a histogram overlay between a control sample (25 °c) (not bolded) and a heat-treated sample (35 °c) (bolded), both incubated with anti-γh2ax antibody, and indicates the placement of the m1 marker method stated previously. (c) and (d) show incubation temperature (abscissa) versus the percent increase in h2ax phosphorylation (ordinate) above the median at which the m1 marker is set for the untreated control at 20 °c (c) or 25 °c for (d) in two separate assays. fl-1 = relative fluorescence intensity of fitc. each bar represents a batch of earthworms (n=7). * = p ≤ 0.05, ** = p ≤ 0.005. ros-induced dna damage causes apoptosis through a variety of intermediates, and could therefore disrupt cellular physiology if a temperature threshold is exceeded (circu and aw, 2010; hahm et al., 2011; matés and sánchez-jiménez, 2012). in the future we would like to conduct cell cycle analysis to determine at what temperature the celomocytes are reaching an apoptotic threshold perhaps mediated by a p53-like homolog, and determine if these cells can mediate a ddr-like mechanism. in addition to its role in host defense mechanisms, ros has been shown to be involved in many other cellular processes including proliferation, growth arrest, as well as the initiation of apoptosis (liu et al., 2008). it would be of interest to determine if ros participate in these cellular processes in the earthworm. at this stage it is unclear whether heat stress in earthworm celomocytes induces ros production that culminates in ddr or vice versa. it is clear from the results of our study that there is a correlation between increased ros production and cell death as invertebrate celomocytes are subjected to heat stress. therefore, as ros begin to accumulate they have a negative effect on cellular viability and physiological processes. evidence indicates that ros-mediated damage predisposes complex organisms to cancer (pelicano et al., 2004). although cancer has not been observed in the earthworm, ros-mediated damage can result in impaired physiological function through cellular damage of dna, proteins, lipids and other macromolecules (rowe et al., 2008). it is possible that heat-induced ros can be generated in other organisms that share the soil environment through heat stress. through our flow cytometric analysis of ros production, cell viability and histone phosphorylation we show that the earthworm has the potential to be used as a bioindicator to monitor heat-related environmental stressors such as climate change. global warming has the potential to compromise soil ecosystems in the next thirty to sixty years by influencing biotic and abiotic factors, and may mediate habitat displacement of soil-dwelling invertebrates by reducing the resources available for immune defense mechanisms and reproduction (houle et al., 2012; mandrioli, 2012).   55 acknowledgments this work was funded by the pennsylvania academy of science, beta beta beta biological honor society and the cabrini college science department research fund. references ando mk, katagiri s, yamamoto k, wakamatsu i, kawahara s, asanuma m, et al. age related effects of heat stress on protective enzymes for peroxides and microsomal 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analysis in bombyx mori nucleopolyhedrovirus-infected silkworms z nie, p lü*, x chen, q wang, x meng, s lu, x dong, k chen* 1institute of life sciences, jiangsu university, zhenjiang, 212013, jiangsu, p. r. china * these authors contributed equally to this work. accepted march 29, 2017 abstract bombyx mori nucleopolyhedrovirus (bmnpv) is the most serious viral disease in silkworms. to investigate the mechanisms of the immune responses of b. mori to a bmnpv infection, a suitable reference gene (rg) is necessary for normalizing data when studying the expression of genes in bmnpv-infected silkworms or cells. thus, quantitative real-time pcr polymerase chain reaction was used to compare the stability of expression of nine potential rgs, including the actin a3, translation initiation factor 3 (tif-3), tif-a4, glyceraldehyde-3-phosphate dehydrogenase (gapdh), 18s rna, 28s rna, tata-binding protein (tbp), ribosomal protein l3 (rpl3), and α-tubulin genes, in silkworms infected with bmnpv. the results were analyzed by bestkeeper, genorm, and normfinder software. overall, α-tubulin exhibited the most stable gene expression in bmnpv-infected silkworms, and this was verified by western blotting of the α-tubulin protein. moreover, we detected the expression of some genes involved in the immune signaling pathways of silkworms after bmnpv infection using α-tubulin as an internal rg. key words: bombyx mori; reference genes; bmnpv; qpcr; immune genes introduction the domesticated silkworm (bombyx mori) is a lepidopteran model insect and an important economic insect for silk production. sericulture is a principal source of income for farmers in many developing countries (jiang and xia, 2014). china has the most prominent sericulture history of any country. however, the disease caused by b. mori nucleopolyhedrovirus (bmnpv) is the most serious viral infectious disease of silkworms, and it is difficult to control, which has resulted in 60 % decreases in silk production in major sericultural areas worldwide. silkworms have an efficient and potent innate immune system to discriminate and eliminate invading pathogens and parasites. there are four signaling pathways that mediate immune responses ___________________________________________________________________ ________ corresponding authors: keping chen institute of life sciences jiangsu university zhenjiang, 212013, jiangsu, p. r. china e-mail: kpchen@ujs.edu.cn peng lü institute of life sciences jiangsu university zhenjiang, 212013, jiangsu, p. r. china e-mail: penglu@ujs.edu.cn against different pathogens, including the kinase/signal transducer and activator of transcription janus (jak-stat) signaling pathway, the toll signaling pathway, and the immunodeficiency (imd) signaling pathway. moreover, most silkworm strains are highly susceptible to bmnpv, while only a few are resistant (chen et al., 2003). therefore, it is important to screen resistance genes and elucidate the immune mechanisms of silkworms against bmnpv. however, the mechanisms underlying the immune response to bmnpv are still unknown. many studies have examined the immune responses of silkworms after bmnpv infection. however, they have generated conflicting results. for example, some studies indicated that the toll pathway is activated after bmnpv infection (yang et al., 2013), whereas others did not (liu et al., 2015). while the jak-stat pathway of silkworms is activated after silkworms challenged with bmnpv (liu et al., 2015), overexpression of bmstat in bmn cells does not enhance anti-bmnpv activity (zhang, 2011). we believe that the use of different reference genes (rgs) by different laboratories may account for these discrepancies. gene expression analysis has become a hot topic in many research fields. quantitative methods of analyzing gene expression at the transcriptional mailto:kpchen@ujs.edu.cn 95 table 1 candidate rgs and their primers for qpcr analysis level, such as quantitate polymerase chain reaction (qpcr), rna blotting, rnase protection analysis, and gene chip technology, as well as quantitative methods that analyze expression at the protein level, such as western blotting, all require the calculation of a rg to target gene expression ratio to obtain reliable results. in this study, we screened nine candidate rgs (actin a3, translation initiation factor 3 (tif-3), tif-a4, glyceraldehyde-3-phosphate dehydrogenase (gapdh), 18s rna (18s), 28s rna (28s), tata-binding protein (tbp), ribosomal protein l3 (rpl3), and α-tubulin) using qpcr, and we analyzed the data with bestkeeper, genorm, and normfinder software. the results suggested that α-tubulin is the most appropriate rg for gene expression analyses when challenging silkworms with bmnpv. moreover, we detected the expression of some genes involved in the immune signaling pathways of silkworms after bmnpv infection using α-tubulin as an internal rg. materials and methods silkworm strains and virus the bmnpv-resistant silkworm strain nb (median lethal dose [ld50] = 2.5×10 8 polyhedral inclusion bodies [pibs]/larva), the bmnpv-susceptible silkworm strain 306 (ld50 = 3.4×105 pibs/larva), and the bc8 strain, which is also bmnpv-resistant and nearly isogenic to the nb strain, were used in this study. the near-isogenic (bc8) line was prepared in accordance with the method (chen et al., 2003). these strains were preserved in our laboratory, and newly exuviated fifth-instar larvae were used for these experiments. bmnpv was propagated in silkworm strain 306, and the occlusion bodies (obs) of bmnpv were isolated and purified from the infected b. mori larvae. the numbers of obtained obs and cells were examined using a hemocytometer under light microscope. (peng et al., 2013). virus inoculation and midgut collection all larvae were infected orally with 5 µl of the bmnpv virus at a concentration of 2×108 pibs/ml. then, the midgut was collected for rna extraction at 0, 12, 24, 48, and 96 h post-infection, washed quickly using phosphate-buffered saline, and stored in an eppendorf tube containing sample protector for rna/dna (takara, dalian, china). the tube was immersed in liquid nitrogen and then stored at −70 ℃ for further use. for comparison, a control group was set up for each silkworm strain without the virus treatment. rna extraction and cdna synthesis total rna was extracted using the trizol reagent (invitrogen, carlsbad, ca, usa). rna was reverse-transcribed from 3 g of total rna using the moloney murine leukemia virus reverse transcriptase (vazyme, nanjing, china) according to the manufacturer’s instructions. primer name sequence(5’-3’) length (bp) actin a3 f:cggaatcgtcactaactggg r:gcgggcgtgttgaatgt 173 tif-a4 f: gaatggaccctgggacactt r: ctgactgggcttgagcgata 186 gapdh f: tgttgagggcttgatgac r: accttacccacagctttg 150 α-tubulin f: ctccctcctccataccct r: atcaactaccagccaccc 186 28srna f:cccagtgctctgaatgtcaac r:agatagggacagtgggaatctc 150 18srna f: cgatccgccgacgttactaca r: gtccgggcctggtgagattt 201 tbp f:ggttgtgcctgggactgt r:cactcacccgaagttttcc 210 rpl3 f:gaagatgatccgctactgt r:tatcctttgcccttggtg 232 tif3 f:agatgacggggagcttgatggt r:gagggcggaatgtacttgttgc 200 96 table 2 bombyx mori genes and their primers for qpcr analysis primer name sequence (5’-3’) length bmtoll9-1 f:atgagtcagtggtgccagttc r:atcagatagtggagggtcgtt 133 bmmyd88 f:ttatacctagcgatggacctgat r:cttattgctacactggtggatgg 139 bmstat f:gccgagatgctggacgaca r:tccgccaaccaacagacga 200 bmjun f:cgcttccaaatgtagacgacg r:acctgctcctttagcctgtgc 140 bmimd f:acgaagaagttatcattgaggaa r:ttatggttgttagggtcaggttt 169 vector construction and qpcr cdnas of the nine rgs were amplified by pcr and individually cloned into the pmd18-t vector (takara, dalina, china). the positive recombinant plasmids were used as standard quantitative templates to construct standard curves for absolute transcript quantifications. the templates were diluted to different concentrations for qpcr. qpcr was performed using a 7300 fast system (applied biosystems, foster city, ca, usa) with the sybr green master mix kit (vazyme, nanjing, china), according to the manufacturer’s instructions. the primers used for qpcr are listed in tables 1 and 2. the reaction volume was 20 µl with 2 µl diluted cdna, 10 µl 2×sybr master mix, and 200 nm of each primer. all samples were amplified in triplicates and three biological replicates were performed. the ct values and the corresponding numerical value were imported into microsoft excel and used for further analysis. calculation of the stability of expression of the rgs the stability analysis was dependent on the qpcr quantification using the standard curves, and the data were analyzed with the bestkeeper, genorm, and normfinder tools. the relative expression of immune genes was calculated with the δδct value. first, the δct for each sample of the virus-infected and control groups was calculated. second, the maximal differences between the values were calculated as the δδct. the concrete data analysis strategies were described in results. western blotting the midgut of silkworms were collected and mixed with 5×loading buffer and boiled for 5 min. the protein samples were separated on sds-page, transferred on to a nitrocellulose membrane and incubated with mouse α-tubulin (1:2,000; proteintech, china), then, the membrane was further incubated with hrp labeled goat anti-mouse igg (1:20,000; proteintech, china). the protein bands were identified by exposure to the clarity western ecl substrate (bio-rad, usa). results qpcr analysis of candidate rgs after bmnpv infection we used actin a3, tif-3, tif-a4, gapdh, 18s, 28s, tbp, rpl3, and α-tubulin as candidate rgs. qpcr primers were designed based on the gene sequences in the national center for biotechnology information, and pcr results showed that all the qpcr primers exhibited a high degree of specificity (fig. 1). to assess the expression stabilities of the nine candidate rgs in bmnpv-infected samples, we individually cloned them into the pmd18-t vector. positive recombinant plasmids were used as templates to construct standard curves for absolute transcript quantifications. because of the various behaviors of the candidate rgs, we evaluated their expression stability in bmnpv-infected silkworms. to identify the optimal rg, we used the bestkeeper, genorm, and normfinder data analysis software tools, which are used most frequently for analyzing the stability of rgs (vandesompele et al., 2002; pfaffl et al., 2004; mallona et al., 2010). table 3 analysis of the candidate reference genes in view of the stability values estimated by normfinder gene name rank stability value actin-a3 1 0.009 tif-3 2 0.055 α-tubulin 3 0.081 28srna 4 0.101 tif-a4 5 0.104 rpl3 6 0.105 18srna 7 0.117 gapdh 8 0.131 tbp 9 0.349 97 table 4 bestkeeper analysis results of the candidate reference genes normfinder analysis normfinder is one of the visual basic application tools for microsoft excel. it is an add-in for microsoft excel; namely, the normfinder function is added directly to the microsoft excel software package. for this algorithm, more stable genes have lower stability values (vandesompele et al., 2002). additionally, normfinder can estimate intraand inter-group variations as well. the normfinder results are shown in table 3. actin-a3, tif-3, and α-tubulin were estimated to be the most stable rgs, with stability values of 0.009, 0.055, and 0.081 respectively, while the least stable gene was tbp, with a stability value of 0.349. genorm analysis genorm software is also a visual basic application tool for microsoft excel. it identifies the most stable reference genes from a given sample and determines the gene expression normalization factors according to the geometric mean values of candidate genes. the parameter employed by genorm to measure the stability of candidate genes is the average expression stability (m) value. the m value is calculated according to the average pairwise variation among all detected genes. a lower m value indicates higher stability of gene expression (bustin et al., 2009). the genorm results are shown in figure 2. for the bmnpv-infected samples, tif-4a and α-tubulin were the most stable rgs, while tbp was the least stable rg. bestkeeper analysis bestkeeper is an excel-based spreadsheet software application. different from the above two tools, bestkeeper can analyze raw ct values, without any conversion (tang et al., 2015). when the original ct values were imported, the descriptive statistics of each candidate gene were computed. the bestkeeper results are shown in table 4. the fig. 1 identification of primer specificity for qpcr amplification by pcr. 1.0 % agarose gel electrophoresis displayed the pcr products of each primer pair. m: marker; line1: actin-a3; line2: tif-a4; line3: gapdh; line4: α-tubulin; line5: 28srna; line7: 18srna; line8: tbp; line9: rpl3; line10: tif3. gene name rank r p-value sd(±cp) α-tubulin 1 0.95 0.001 1.271 actin-a3 2 0.93 0.001 0.802 28srna 3 0.92 0.001 1.179 rpl3 4 0.92 0.001 1.708 tif-a4 5 0.91 0.002 1.160 gapdh 6 0.89 0.003 1.288 tif3 7 0.88 0.004 0.724 18srna 8 0.82 0.012 1.617 tbp 9 0.20 0.628 3.036 98 fig. 2 average expression stability (𝑀) values of the candidate genes. the average expression stability (𝑀) values are acquired through the stepwise exclusion of the least stable reference gene. starting from the least stable gene at the left, the genes are ranked according to the ascending expression stability, ending with the two most stable genes at the right. fig. 3 protein expression of α-tubulin in midgut of silkworm after bmnpv infected and no-infected. the black columns represent control group and the gray columns represent treatment group. control represents no viral infection. 99 fig. 4 the expression levels of bmtoll9-1 in midguts 306, bc8 and nb infected with bmnpv at different time points. a: 306; b: bc8; c: nb. fig. 5 the expression levels of bmmyd88 in midguts 306, bc8 and nb infected with bmnpv at different time points. a: 306; b: bc8; c: nb. fig. 6 the expression levels of bmstat in midguts 306, bc8 and nb infected with bmnpv at different time points. a: 306; b: bc8; c: nb fig. 7 the expression levels of bmjun in midguts 306, bc8 and nb infected with bmnpv at different time points. a: 306; b: bc8; c: nb 100 fig. 8 the expression levels of bmimd in midguts 306, bc8 and nb infected with bmnpv at different time points. a: 306; b: bc8; c: nb results indicated that α-tubulin was the optimal rg, while tbp was the least stable rg. in brief, the bestkeeper result were almost identical to those obtained using normfinder and genorm. protein expression level of α-tubulin to verify the stability of α-tubulin expression in the midgut of silkworms after bmnpv infection, we examined its expression at the protein level. western blotting results showed that the expression of α-tubulin did not differ significantly between the control and treatment groups (fig. 3). thus, we selected α-tubulin as the optimal rg for further validation. expression of immune genes in silkworm strains 306, nb, and bc8 infected with bmnpv the transcription level of bmtoll9-1 in the bmnpv-infected group increased steadily and significantly from 0 h to 96 h. in the 306 strain (which is susceptible to bmnpv), the highest transcription level was reached at 48 h, and then it began to decrease, which was probably because the virus replication rate decreased after this time point (fig. 4a). when the bmnpv-resistant nb and bc8 strains were infected with bmnpv, the expression levels of bmtoll9-1 increased notably in the virus-treated groups (figs 4b, c). the highest expression levels were reached at 24 h. the bmmyd88 transcription level of the bmnpv-infected group decreased compared with that of the control group in strain 306 (fig. 5a), but it increased sharply during the first 24 h and remained at a relatively high level in the bmnpv-infected nb and bc8 strains (figs 5b, c). the data indicate that virus invasion can stimulate the expression of bmtoll9-1 and bmmyd88 early in the nb and bc8 strains, and relatively late in strain 306. we observed a slight decrease of bmmyd88 expression in the virus-treated silkworms after 96 h, which may have contributed to the decreased virus replication rate, similar to the case of bmtoll9-1. in contrast to bmtoll9-1, the transcription level of bmstat mostly slightly decreased in the bmnpv-infected 306 strain at 24 h (fig. 6a), while it increased briefly at 48 h. however, its level increased slightly in the bmnpv-resistant nb and bc8 strains infected with bmnpv at 24 h (figs 6b, c). this differed substantially from the bmtoll9-1 and bmmyd88 genes in the 306, nb, and bc8 strains, whose expression increased significantly. bmtoll9-1 and bmmyd88 belong to the toll signaling pathway, while bmstat belongs to the jak-stat signaling pathway, which indicates that the toll signaling pathway can be activated rapidly after virus invasion, while virus invasion only has a small effect on the jak-stat signaling pathway. the transcription level of bmjun decreased and remained below the starting level in the untreated 306 strain (fig. 7a), while it increased notably after bmnpv treatment. for the nb and bc8 strains, bmjun expression also always remained below the starting level; it is interesting that it decreased notably after virus treatment (figs 7b, c). these results suggest that bmnpv can upregulate gene expression in a bmnpv-susceptible strain, while downregulating expression in bmnpv-resistant strains. the transcription level of bmimd was mostly unchanged in the 306 control group (fig. 8a), and it decreased in the nb and bc8 control groups (fig. 8b). it remained steady in the virus-treated 306 strain and increased briefly and then decreased in the virus-treated nb and bc8 strains (fig. 8c). the data indicate that virus invasion can stimulate the expression of bmimd temporarily in virus-resistant strains. discussion bombyx mori is a lepidopteran model insect, and it is an important economic insect for silk production. antiviral studies in silkworms are essential to improve sericulture production and control insect pests (jiang and xia, 2014; zhang et al., 2014a). therefore, to understand silkworms at the molecular level and to determine the specific mechanisms of bmnpv resistance, the selection of stable rgs for gene expression normalization in silkworms is imperative. in previous studies, different traditional rgs (such as actin-3 (gao et al., 2014b; wang et al., 2014, 2015; kolliopoulou et al., 2015), tif-3 (zhou, et al., 2013), gapdh (bao et al., 2009), tif-4a (also known as sw22934) (jiang et al., 2012a, b, 2013b), and 18s) were used as internal rgs in bmnpv-infected silkworms. although (guo et al., 2014) selected an internal rg for an expression analysis, using relative expression ananlysis method, when challenging silkworms with bmnpv, bmcpv, and bmbdv, it is necessary to select a more optimal rg when silkworms are infected only with bmnpv. thus, in the present study, the bestkeeper, genorm, and normfinder tools were employed to calculate 101 the stabilities of nine candidate rgs in b. mori after bmnpv infection. the results suggested that α-tubulin is a suitable rg for gene expression analyses after bmnpv infection in silkworms. using this rg, the transcriptional changes of five immune genes related to virus infections were investigated systematically by qpcr. bmnpv can initiate viral infections in the silkworm midgut; thus, the midgut was collected and analyzed, and a time-course analysis was used to examine the transcriptional changes. when α-tubulin was used as the rg after bmnpv infection, the transcription levels of bmtoll9-1 and bmjun were upregulated in the silkworm strain 306, whereas there were no significant increases, and even slight decreases, of bmmyd88, bmstat, and bmimd expression in the bmnpv-infected 306 group, which is consistent with previous conclusions, except for bmstat. the use of α-tubulin as a rg in the previous study may explain why the expression of bmstat was upregulated when silkworms were infected with bmnpv and bmbdv; however, overexpression of bmstat could not obviously enhance the anti-bmnpv activity of the silkworms. it is known that baculovirus can alter cellular filamentous actin and affect the actin expression level during viral infection and replication (volkman, 2007); thus, actin is not a suitable rg for qpcr analysis in bmnpv infection experiments. moreover, the toll and jak-stat pathways should be further analyzed to determine whether they are activated in silkworms after bmnpv infection, because we did not observe any significant changes in the expression of bmmyd88 of the toll pathway and bmstat of the jak-stat after bmnpv infection, although we demonstrated that bmtoll9-1 and bmjun were upregulated. in addition, we identified a highly bmnpv-resistant strain, nb, and constructed a bmnpv-resistant near-isogenic line, bc8. our results showed that the expression of bmtoll9-1, bmmyd88, bmjun, bmstat, and bmimd were upregulated significantly in both bmnpv-resistant strains, indicating that both the toll and jak-stat innate immune pathways may be activated in the bmnpv-resistant nb strains. it is interesting to note that the jnk pathway, also named the stress-activated protein kinase pathway (sapk), is essential for providing a cellular response to extracellular changes such as ultraviolet and reactive oxygen species-induced dna damage, mechanical stress, and osmolality changes (neganova et al., 2016). many researchers have reported that the jnk/sapk signaling pathway can be activated by stress responses, but few indicated that virus invasion can stimulate this pathway. in this study, we found that bmnpv can stimulate the upregulation of the bmjun gene in a viral-susceptible strain, while bmjun expression was downregulated in virus resistant strains. all these results are deserving of further study. acknowledgement this work was supported by the project of the priority academic program development of jiangsu higher education institutions, the national natural science foundation of china (no. 31572467, 31570150 and 31602008), the national science foundation of jiangsu province (bk20150495, bk20140539)，the start-up research funding of jiangsu university for distinguished scholars (15jdg055), china postdoctoral science foundation (2015m571701), and postgraduate research and innovation project of jiangsu province (no. cxlx12-0671). reference bao yy, tang xd, lv zy, wang xy, tian ch, xu yp, et al. gene expression profiling of resistant and susceptible bombyx mori strains reveals nucleopolyhedrovirus-associated variations in host 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y-tube olfactometer isj 11: 4-10, 2014 issn 1824-307x research report heat and desiccation tolerances of heterorhabditis bacteriophora strains and relationships between their tolerances and some bioecological characteristics tc ulu, ia susurluk uludag university, agriculture faculty, plant protection department, 16059 nilüfer, bursa, turkey accepted december 16, 2013 abstract heat tolerances, desiccation tolerances, and effectiveness of 10 heterorhabditis bacteriophora strains isolated from different climatic regions in turkey were analyzed in laboratory conditions. all strains were exposed to heat and desiccation conditions to determine their tolerance levels, and different doses of the strains were applied to the host larva to detect infection capabilities. correlations between heat and desiccation tolerances as well as effectiveness of all strains were investigated. moreover, relationships between the tolerances and geographic origins were examined. the results showed that there was no correlation between desiccation tolerance and effectiveness as well as between heat and desiccation tolerances. however, a significant correlation was found between heat tolerance and effectiveness. furthermore, there was a correlation between heat tolerances and origins, but no correlation existed between desiccation tolerances and origins. key words: desiccation; efficacy; heterorhabditis bacteriophora; heat; tolerance introduction entomopathogenic nematodes (epns), which belong to the families steinernematidae and heterorhabditidae, have been used to control a wide range of soil-borne insect pests (ehlers, 1996). control of soil-dwelling insect pest larvae with chemical insecticides is limited because insecticides are rapidly decomposed or adsorbed in the soil. thus, insecticides cannot effectively reach target insect larvae. however, controlling the host larvae with epns may be more effective than chemicals because cruiser epns can reach their target hosts in soil up to a 50 cm soil depth (susurluk, 2008b). epns are safe for non-targets and the environment (boemare et al., 1996; ehlers, 2003), and they can be mass produced in liquid culture (lunau et al., 1993; ehlers et al., 1998; strauch and ehlers, 1998; ehlers, 2001) for widespread commercial use. in soil, infective juveniles (ijs), a free-living stage of epns, penetrate insect hosts through natural openings (mouth, anus, and spiracles) or directly through the cuticle (poinar, 1979). after penetration, ijs release their symbiotic bacteria into the hemocoel (photorhabdus spp. for heterorhabditis ___________________________________________________________________________ corresponding author: ismail alper susurluk uludag university agriculture faculty plant protection department 16059 nilüfer, bursa, turkey e-mail: susurluk@uludag.edu.tr spp. and xenorhabdus spp. for steinernema spp.), and they kill insect hosts through septicaemia within 36 48 h and convert cadavers into biomass, which is a suitable condition for feeding and reproduction of epns (poinar, 1975; brown and gaugler, 1997; susurluk et al., 2001; adams and nguyen, 2002; susurluk, 2008a). ijs are resistant to severe environmental conditions for a long time. thus, ijs can persist in soil without any insect host for up to 22 months (susurluk and ehlers, 2008). furthermore, ijs can resist shear stress, and they can be applied with standard pesticide sprayers or irrigation systems (georgis, 1990; wright et al., 2005). in addition to these advantages, heat and desiccation are two major stress factors in large scale field applications, and both stress factors cause a short shelf life (strauch et al., 2000). in general, temperatures below 0 °c and above 40 °c are lethal to most ijs. however, the negative effect of temperature depends on exposure time (koppenhöfer, 2000). use of strains tolerant to heat and desiccation increases the success of their control on target insect pests in outdoor applications. however, there are few studies on heat and desiccation tolerance of epns. several studies have shown that genes controlling heat and desiccation characters have high heritability for heterorhabditis bacteriophora (poinar, 1976) (rhabditida: heterorhabditidae) (glazer et al., 1991; strauch et al., 2004; ehlers et al., 2005; mukuka et 4 fig. 1 the stars on the map of turkey indicate the geographic origins where h. bacteriophora strains used in the study were isolated. al., 2010b, c). in the present study, 10 h. bacteriophora strains isolated from different climatic regions in turkey were used because h. bacteriophora strains have high heritability of both stress factors and have variable heat and desiccation tolerances depending on geographic regions (mukuka et al., 2010d). the main objective of this study was to determine heat and desiccation tolerance levels of 10 different turkish strains of h. bacteriophora and to detect their relationships between effectiveness of the strains and tolerances to both stress factors. moreover, relationships between tolerances to both stresses and the highest average annual temperatures and average annual precipitation levels for geographic origins over 35 years were examined in the present study. material and methods heterorhabditis bacteriophora strains in the present study, heterorhabditis bacteriophora strains were used due to high variability in tolerances among strains (mukuka et al., 2010d). one-week-old strains were used in this experiment. all strains were identified by a pcrrflp molecular technique (unpublished data). the strains and their geographical origins are described in figure 1. the strains were cultured using the last instar of galleria mellonella (lepidoptera: pyralidae) as described by kaya and stock (1997) and were stored at 4 °c. determination of heat tolerance the following temperatures were used in the present study: 32, 34, 36, 38, 40 and 42 °c. the heat tolerance tests were carried out in 24-well plates (each well had a 1.4 cm diameter and 3 cm3 volume). before the experiment, the strain cultures stored at 4 °c were adapted to room temperature (20 22 °c) for 2 h. a total of 500 ijs were transferred into one well filled with 500 µl of distilled water, and the plates were sealed with parafilm. the strains were then exposed to the adjusted temperature for 2 h (mukuka et al., 2010d). after exposure to heat, the strains were adapted to room table 1 strains, mt50 values, mt10 values, geographic origins (cities) and highest average annual temperatures of the origins strains mt50 mt10 geographic origins highest average annual temperature (°c)* hb 10 39.27 42.58 adana 36.45 hsu 40.75 43.69 şanlıurfa 34.95 hb 6 40.50 44.06 antalya 34.67 hiz 40.90 44.00 i̇zmir 34.40 hb 13 38.62 41.31 yalova 34.38 h-101 39.31 42.34 samsun 33.17 hb 17 40.46 43.78 kırklareli 31.11 han 39.12 41.91 ankara 29.76 hb 876 38.60 41.94 çanakkale 29.48 hb 11 38.00 40.58 erzurum 23.97 *mean values of the highest average annual temperatures from 1976 to 2011 for each origin. 5 table 2 strains, lc50 values, lc90 values, geographic origins (cities) and average annual precipitation levels of the origins strains lc50 lc90 geographic origins average annual precipitation levels (kg/m2)* hb 6 49.06 69.94 antalya 90.92 hb 13 39.54 53.06 yalova 62.69 hiz 43.21 57.82 i̇zmir 58.48 h-101 34.67 49.48 samsun 57.80 hb 10 43.04 55.84 adana 55.08 hb 876 48.99 69.53 çanakkale 50.46 hb 17 46.65 64.73 kırklareli 46.31 hsu 42.04 54.61 şanlıurfa 36.85 hb 11 42.42 54.71 erzurum 33.87 han 43.02 55.94 ankara 33.51 *mean values of the average annual rainfall from 1976 to 2011 for each origin. temperature for 24 h. following the adaptation period, dead and living individuals of each strain were counted under a stereomicroscope, and mortality ratios were detected at each used temperature. the results were expressed as mean temperature tolerated by 50 % of the population (mt50) and mean temperature tolerated by only 10 % (mt10) of the strains. the experiment was replicated five times. determination of desiccation tolerance polyethylene glycol (peg; hoch2-ch2-(o-ch2ch2)(n-1)-oh) was used as a desiccator in the desiccation experiment at the following peg concentrations: 10, 20, 30, 40, 50, 60, 70 and 80 %. the desiccation tolerance tests were performed in 24-well plates. a total of 500 ijs from the culture flask filled with ringer’s solution (laboratory standard containing 9 g of nacl, 0.42 g of kcl, 0.37 g of cacl2 x 2h2o, 0.2 g of nahco3 and water to a final volume of 1000 ml) was added into one well filled with 500 µl of adjusted peg concentration, and the plates were then sealed with parafilm. the strains were then exposed to various peg concentrations for 24 h at 25 °c. after the incubation period, the strains were washed with distilled water and stored for an additional 24 h at 25 °c for rehydration. dead and alive individuals were counted for each strain at each peg concentration under a stereomicroscope, and mortality ratios were calculated for each peg concentration. effects of the peg concentration on the strains were described as lethal concentration (lc50 and lc90). the experiment was replicated five times. effectiveness of the strains infectivity experiments were conducted in 24well plates using last instar larvae of tenebrio molitor (coleoptera: tenebrionidae), which are less sensitive than g. mellonella larvae and are commonly used in epn effectiveness tests. the use of t. molitor larvae allows a more accurate infectivity of epns on insect larvae to be determined (koppenhöfer et al., 1995; aydın and susurluk, 2005). one t. molitor larva was placed at the bottom of each well followed by the addition of sand (particle size of 300-400 µm) with a water content of 10 % (susurluk et al., 2001). the doses of 2, 5, 10, 20, 50 and 75 ijs/t. molitor larva were applied to the sand in the each well. only the dose of 75 ijs per larva was used for the hb 6 and hsu strains. for each dose, 20 larvae were inoculated with ijs of the different strains, and the plates were sealed with parafilm and kept at 25 °c for 3 days. three days after inoculation, dead and alive larvae were counted. the dead larvae were dissected to verify the presence of nematodes. eventually, infectivity capabilities of the strains were represented by ld50 and ld90 values for each strain. the experiment was replicated three times. statistical analyses the mt50, mt10, lc50, lc90, ld50 and ld90 values were calculated by probit analysis using the biostat® 2010 program. in the correlation analyses, heat tolerances, effectiveness and desiccation tolerances were indicated as the mean of mt50 and mt10, ld50 and ld90, lc50 and lc90, respectively. correlations between heat tolerances and infectivity as well as between desiccation tolerances and infectivity of the strains were analyzed by pearson’s correlation coefficient at a 5% confidence level test using jmp® 7.0 software. results determination of heat tolerance the three most tolerant strains to heat were hiz from i̇zmir, hb 6 from antalya and hsu from şanlıurfa. the three most susceptible strains to heat were hb 11 from erzurum, hb 13 from yalova and hb 876 from çanakkale. the highest average annual temperatures in the geographic origins of the strains and heat toleration levels as indicated by mt50 and mt10 are shown in table 1. determination of desiccation tolerance the most tolerant strains to desiccation were hb 6 from antalya, hb 876 from çanakkale and hb 17 from kırklareli. importantly, these regions had higher annual average precipitation levels than other regions examined in the present study. the 6 table 3 ld50 and ld90 values of the strains on t. molitor larvae strains ld50 confidence interval (ld50) ld90 confidence interval (ld90) hb 17 4.98 2.08-15.93 27.56 11.92-49.00 hb 13 2.48 -10.02-12.42 21.93 4.20-42.15 hb 876 0.58 -10.99-9.33 21.43 8.79-38.67 hb 6 5.20 2.40-12.04 28.89 11.32-44.17 hb 10 5.42 2.30-8.03 26.56 19.95-33.30 hb 11 4.50 -3.19-9.95 23.47 12.19-39.25 h-101 0.25 -14.45-12.20 19.68 3.83-40.93 han 3.35 -6.44-11.39 23.22 7.58-41.59 hsu 5.27 -4.63-13.23 31.20 13.88-51.66 hiz 5.06 -2.83-12.22 24.26 9.00-43.01 lowest tolerant strains to desiccation were han from ankara, hb 13 from yalova and hsu from şanlıurfa. similarly, these regions did not have the highest precipitation levels among the studied regions. the average annual precipitation levels in the geographic origins of the strains and desiccation tolerances as indicated by lc50 and lc90 values are shown in table 2. effectiveness of the strains mortalities of all strains reached 100 % at the dose of 50 ijs, except for the hb 6 and hsu strains. however, these two strains caused 100 % mortalities at the dose of 75 ijs. infectivity of the strains at all studied doses were indicated by ld50 and ld90 values (table 3). the most effective strain was h-101 from samsun, and the strain with the lowest infection capability was hsu from şanlıurfa. correlations there was a statistically significant correlation between heat tolerances and effectiveness (y (mt) = 37.29 + 0.27x (ld); r = 0.64; p = 0.045) as well as between heat tolerances and origins (y (mt) = 35.14 + 0.18x (highest average annual temperature); r = 0.61; p = 0.048). based on mt values and the highest average annual temperatures in geographic origins, these results showed that heat tolerant strains had lower infectivity capabilities. in contrast, no statistically significant correlation was detected between desiccation tolerances of the strains and their effectiveness (y (lc) = 41.52 + 0.66x (ld); r = 0.32; p = 0.373). similar results were also found between heat and desiccation tolerances (y (mt) = 37.90 + 0.06x (lc); r = 0.31; p = 0.377) as well as between desiccation tolerances and origins (y (lc) = 45.05 + 0.11x (average annual precipitation); r = 0.34; p = 0.334). moreover, no significant relationship between the highest average annual temperatures and average annual precipitation levels of the origins was found y (highest average annual temperature) = 26.17 + 0.12x (average annual precipitation); r = 0.54; p = 0.101) (fig. 2). discussion the most negative effects on survival of epns are heat and desiccation in field applications. to detect h. bacteriophora strains that are more tolerant to heat and desiccation, 10 h. bacteriophora strains from different regions in turkey were examined for tolerance against these stress factors. the obtained results were compared with average annual precipitation levels and highest average annual temperatures of the strain origins. moreover, the relationship between effectiveness of the strains and their tolerances were investigated in the present study. the present study showed that strain heat tolerances were correlated with their geographic origins. these results were in accordance with the highest average annual temperatures at the geographic origins of the strains. similarly, mukuka et al. (2010d) reported a correlation between the mt50 and mt10 values of heat-adapted and nonadapted populations of h. bacteriophora, h. megidis and h. indica, and they also reported the mean annual temperatures at the origins, except for the mt50 of non-adapted populations. when only h. bacteriophora was considered in mukuka et al. (2010d), a significant correlation was found only for the mt10 of the adapted population. however, their result does not agree with the present results. importantly, the averages of mt50 and mt10 were used in the present study instead of using both mt values individually as was done in the study by mukuka et al. (2010d). mukuka et al. (2010d) also indicated that the influence of the strain origins on their tolerance might be less important because the soil temperatures (except for the top of the soil) have much lower variability than air temperatures. moreover, grewal et al. (1994) indicated that each nematode species has a well known thermal niche where it is not affected by climatic situations. however, mukuka et al. (2010d) suggested that the correlation analysis with the highest temperature recorded in the origins might be better to understand the relationship. thus, the results in the present study may be more objective than the results presented by mukuka et al. (2010d) due to the use of the highest temperatures of the origins. if the highest temperatures of the origins were used in the correlation analyses performed by mukuka et al. (2010d), the correlation for h. bacteriophora would have been significant. another result of the present study suggested that heat tolerant strains have lower effect capabilities 7 fig. 2 correlations between heat tolerances and effectiveness (a), heat tolerances and origins (b), desiccation tolerances and effectiveness (c), heat and desiccation tolerances (d) and desiccation tolerances and the origins (e). because a significant relationship was detected between heat tolerance and effect capabilities. it is known that high temperatures above 30 °c have an adverse effect on epn survival, pathogenicity and longevity (zervos et al., 1991; grewal et al., 1994; glazer, 2002; somasekhar et al., 2002; hirao and ehlers, 2009). symbiotic bacteria have a crucial role on infectivity of an epn. extreme temperatures (> 40 °c) can kill the bacteria instantly (ehlers et al., 2000), but adaptation to high temperatures for long time periods may reduce the number of symbiotic bacteria cells and, thus, reduce infectivity, which has also been indicated by mukuka et al. (2010a). thus, the potential reduction of symbiotic bacteria may explain why heat tolerant strains had less infectivity capabilities in the present study. in contrast, due to no correlation between the desiccation tolerances and effectiveness of the strains, the desiccation tolerant strains did not have less infectivity. this conclusion was not applicable to the heat tolerant strains. moreover, no correlation between the desiccation tolerance and average annual precipitation of the origins was detected in the present study. however, any studies regarding this relationship have been published. likewise, no correlation was detected between heat and desiccation tolerances, which can be explained by the lack of correlation between the highest average annual temperatures and average annual precipitation levels of the origins in this study. thus, the warmest origin might not be the most arid place (tables 1, 2). this study is the first record of detecting heat and desiccation tolerances of domestic h. bacteriophora strains in turkey. further studies on 8 other biological features (e.g., reproduction, penetration, and longevity) of tolerant strains of h. bacteriophora should be performed to detect strains that are better fitted for use in field applications. acknowledgements this study was financially supported by the scientific and technological research council of turkey (tubitak; project number: tovag 110o161). moreover, we would like to thank yasemin kongu, a msc student, for technical support. references adams bj, nguyen kb. taxonomy and systematics. in: gaugler r (ed.), entomopathogenic nematology, cabi, oxon, new york, pp 1-34, 2002. aydın h, susurluk a. competitive abilities of the entomopathogenic nematodes steinernema feltiae and heterorhabditis bacteriophora in the same host at different temperature. turk. j. biol. 29: 35-39, 2005. boemare ne, laumond c, mauleon h. the entomopathogenic nematode-bacterium complex: biology, life cycle and vertebrate safety. 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abstract the immune system in molluscs, as well as in other invertebrates, is endowed with an innate immune response which, however, shows in its recognition processes both molecular mechanisms and effectors similar to those employed by vertebrates in eliminating pathogens and parasites. furthermore, as in vertebrates, invertebrates also present a profound correlation between immune and neuroendocrine responses. in this review, the players in the immune and neuroendocrine systems have been examined, with particular references to gastropods and bivalves. key words: molluscs; immune and neuroendocrine responses introduction in the immune system, two categories of immune response are distinguishable, innate or natural and adaptive or acquired. invertebrates only possess the innate response, which, however, shows in its recognition processes both molecular mechanisms and effectors similar to those employed by vertebrates in eliminating pathogens and parasites (hoffman et al., 1999; medzhitov and janeway, 2000; plows et al., 2005). furthermore, as in vertebrates, invertebrates also present a profound correlation between immune and neuroendocrine responses (ottaviani and franceschi, 1997). pioneering studies were performed in mammals by blalock and co-workers (blalock, 1984; blalock and smith, 1985; weigent and blalock, 1987, 1989), who have demonstrated that the immune and neuroendocrine systems share common pools of molecules and cooperate in coping with dangerous internal and external agents in order to maintain body homeostasis. basically, the levels of integration between the two systems can be summarized as follows: i) the use of cytokines and neuropeptides for communication between immune and neuroendocrine systems, i.e. the same signal molecules used to communicate within each system; corresponding author: enzo ottaviani department of animal biology, university of modena and reggio emilia, via campi 213/d, 41100 modena, italy e-mail: ottaviani.enzo@unimore.it i) the use of the same cell, the lymphocyte, to perform simultaneous immune and neuroendocrine responses. this central cell in the immune system has many characteristics of a neuroendocrine cell, e.g. receptors for both hypothalamic releasing factors and neuroendocrine peptides, production of neuroendocrine hormones and cytokines. thus the distinction between “hormones”, “neurotransmitters” and “cytokines” becomes open to discussion, as the same molecule can be included in each group depending on the target that is involved. for instance, interleukin (il)-1 is described as a cytokine when it mediates the interaction between macrophages and t lymphocytes, but it may be considered a neurotransmitter when acts on hypothalamic neurons in inducing fever. in both cases, we have a scenario in which a molecule produced by a cell provokes effects on other close or distant cells, in other words the typical action of a hormone. the distinction, then, would appear to an old conception rather than indicating true functional differences. the sharing of the same mediators by the immune and neuroendocrine systems suggests that nature has followed the same general strategy in the construction of these systems. given this, we have surmized from an evolutionary point of view that both systems have a common origin in which the invertebrate phagocytic immunocyte plays a pivotal role (ottaviani and franceschi, 1996, 1997; ottaviani et al., 1997a). this review presents the actors in the immune and neuroendocrine systems and their respective performances in the molluscs, in particular in the gastropods and bivalves. 51 as all invertebrates endowed with a coelomic cavity, molluscs are able to recognize and discriminate between self and not-self principally by means of the cellular and humoral components of the hemolymph. however, other structures, for example the skin and body wall, as well as phagocytic cells located in various tissues are also involved in defense. in gastropods, cells with phagocytic activity are scattered in the connective tissue (lymnaea stagnalis; sminia et al., 1979) and in organs such as the digestive gland. these cells represent a fixed phagocyte system in helix pomatia (reade, 1968), while in planorbarius corneus they are distributed throughout the entire gland (ottaviani, 1990). in h. pomatia, antigen-trapping cells have been described in blood sinus and kidney (renwrantz et al., 1981). it has been reported that while digestible particles are degraded within the immunocytes in the bivalve crassostrea virginica (tripp, 1958a, b, 1960; feng, 1959, 1965), the indigestible particles are eliminated via the migration of particle-laden phagocytes across epithelial borders (tripp, 1960; feng, 1965). cellular component as already reported in insects (ottaviani, 2005), the classification of the types of immunocytes present in the hemolymph remains an unresolved problem also in molluscs. the cells derive from circulating immunocytes lacking true hemopoietic organs (sminia, 1981; ottaviani, 1983). in gastropods, and in particular in various species of planorbids and lymnaea palustris (kinoti, 1971; lie et al., 1975; rachford, 1976; jeong et al., 1983; ottaviani, 1988a), a hemocyte-producing organ (hpo) lying between the mantle cavity and the pericardium has been identified (figs 1-3). immunoblasts transformed into immunocytes migrate into the hpo sinus. while no hemopoietic organs have been found in bivalves, it is generally accepted that the immunocytes may originate from connective tissue cells (cheng, 1981). together with the number of types of immunocyte, their naming also causes difficulty. nevertheless, it can be said that the majority of gastropods is endowed with two types of immunocytes. the two types are described in p. corneus as spreading and round hemocytes (sh, rh) (figs 4-7) (ottaviani, 1983; ottaviani and franchini, 1988). the sh show ultrastructural similarities with the spreading amoebocytes of l. stagnalis (stang-voss, 1970; sminia, 1972), the granulocytes of bulinus guernei (krupa et al., 1977) and the granulocytes of biomphalaria glabrata (harris, 1975; joky et al., 1983). despite their different names, these cells show the same morphology and functions, in particular phagocytosis. the p. corneus rh is comparable only with the round amoebocytes of l. stagnalis (sminia, 1972, 1981). cytofluorimetric analysis have revealed that both sh and rh react with several anti-human monoclonal antibodies, including those directed against epitopes typical of mammalian natural killer (nk) cells and cell-adhesion molecules (table 1) (franceschi et al., 1991). with regards bivalves, cheng (1981) in his review proposed dividing the blood cells simply into granular (granulocytes) and agranular (hyalinocytes) types. however, another specialized figs 1-3 1) hemocyte-producing organ (hpo) in p. corneus (arrow). m, mantle cavity; p, pericardial cavity. bar = 50 µm; 2) immunoblasts (b) scattered in the stroma and immunocyte (h) in the blood sinus (s) of the hpo. bar = 10 µm; fig 3) mitotic division in the circulating immunocyte. bar = 5 µm (from ottaviani, 1988a). type of immunocyte (the serous cell) involved in excretion has been described. in contrast, mix (1976) suggested that hyalinocytes are a proliferative condition that after various stages mature into granulocytes. in mytilus galloprovincialis, only one cell type in two different stages (young or old) has been proposed (ottaviani et al., 1998a) (figs 8-13). these findings support mix’s model, but in m. galloprovincialis both young and old immunocytes have the same functions, i.e. phagocytosis and the expression of common signal molecules such as cd5, cd11b and cd16, while the differences in cytology and number seem to be only a consequence of animal aging. it is clear that the classification system is far from unified, as further witnessed by the paper by hine (1999), who 52 figs 4-7 immunocytes of p. corneus. light microscopy: 4) spreading (sh); 5), round (rh) bar = 10 µm. electron microscopy: 6) sh (x18.600); 7) rh (x11.000) (from ottaviani and franchini, 1988) (reprinted with permission). suggested a new and more complex scheme than cheng's (1981) cell-type division. humoral component humoral factors play a fundamental role in the innate immune responses in molluscs. agglutinins or lectins, bioactive peptides, cytokine-like molecules, nitric oxide (no), lysozyme and other lysosomal enzymes, anti-microbial peptides and others have been described. agglutinins or lectins invertebrates possess humoral components with agglutinating capacity. agglutinins, or lectins, are glycoproteins consisting of more than one subunit, which function as recognition molecules (ractliffe et al., 1985; olafsen, 1986). they may act as opsonins by binding to the non-self material via their carbohydrate recognition sites. however, the binding between the ligand and the phagocytic surface is not been cleared yet. the presence of natural agglutinins or lectins has been documented both in gastropods and bivalves (ractliffe et al., 1985; olafsen, 1986, 1996). in gastropods, lectins have been found in helix aspersa (prowse and tait, 1969; hammarström, 1974), l. stagnalis (van der knaap et al., 1983) and b. glabrata (bretting et al., 1983). in p. corneus, natural and induced bacterial agglutinins were isolated by affinity chromatography on sephadex gel g 150 (ottaviani and tarugi, 1986, 1989), and sh are involved in the agglutinin synthesis (ottaviani, 1988b). natural agglutinin is a glycoprotein with a molecular weight (mw) of 130 kda, while the induced form shows a mw of about 330-350 kda. it is interesting to note that the carbohydrate component of the natural agglutinin contains n-acetylmuramic acid, typical of prokariotes, and not sialic acid as expected for eukariotes (ottaviani et al., 1990a). in bivalves, lectins have been shown in crassostrea virginica (tripp, 1966), in mytilus edulis (renwrantz and stahmer, 1983) and in c. gigas (olafsen et al., 1992). the latter presents lectins able to agglutinate horse and human erythrocytes and bacteria such as vibrio anguilarum. increased lectin activity was observed after exposing c. gigas to v. anguillarum for 6 h. bioactive peptides using a variety of techniques (immunocytochemistry, cytofluorimetric analysis, ria test and in situ hybridization), phagocytic immunocytes have been shown to contain a variety of bioactive peptides. in particular, pro-opiomelanocortin (pomc)-mrna and the related immunoreactive peptides, adrenocorticotropin hormone (acth) and β-endorphin, have been detected in the gastropods p. corneus and 53 figs 8-13 immunocytes of m. galloprovincialis. 8) proimmunocyte (a), type i (b) and type ii (c) immunocytes of an adult specimen. bar = 10 µm; 9-13) different stage of immunocyte maturation from proimmunocyte to type i and type ii in young specimens: 9) the cytoplasm of the proimmunocyte (a) gradually increases and the nucleus changes from a round through a reniform (b); 10) to a polymorphic shape; 11) vacuola then appear in the cytoplasm; 12) the cell becomes irregularly shaped (a) and the latter cell type, considered type i, becomes rich in cytoplasmic inclusions (b) that are typical of type ii; 13) intermediate forms resembling type ii, with a polymorphic nucleus are also found. bar = 10 µm (from ottaviani et al., 1998a) (reprinted with permission). figs 14-17 expression of pomc-mrna (14) and presence of acth (15) in sh (a) and not in rh (b) of p. corneus; expression of pomc-mrna (16) and presence of acth (17) in m. galloproviancialis immunocytes. bar = 10 µm. 54 table 1 cytofluorimetric analysis of p. corneus immunocytes by using mouse anti-human monoclonal antibodies (mab) ____________________________________________________________________________________________________ mab anti-: sh rh cd 1a, cd16, cd26, cd29, cd56 + + cd5, cd34, cd45ra, cd54, cd61, cd71 + cd2, cd3, cd4, cd7, cd8, cd11a, cd11b cd11c, cd13, cd18, cd19, cd20, cd21, cd22, cd23, cd25, cd33, cd38, cd43, cd45ro, cd57, hla-dr, αβtcr, γδtcr ____________________________________________________________________________________________________ sh, spreading immunocytes; rh, round immunocytes (modified from franceschi et al., 1991) table 2 presence of cytokine-like molecules in molluscs species refs __________________________________________________________________________________________ planorbarius corneus il-1α,β, il-2, il-6, tnf-α, ottaviani et al. (1993b), pdgf-ab, tgf-β1 franchini et al. (1996) viviparus ater il-1α,β ,il-2, il-6, tnf-α, ottaviani et al. (1993b), pdgf-ab, tgf-β1 franchini et al. (1996) biomphalaria glabrata il-1, tnf-α granath et al. (1994), owe-missi-oukem-boyer et al. (1994) viviparus contectus pdgf-ab, tgf-β1 franchini et al. (1996) lymnaea stagnalis pdgf-ab, tgf-β1, egf, franchini et al. (1996), neurotrophic factor hermann et al. (2000), fainzilber et al. (1996) mytilus edulis il-1α,β, il-6, tnf-α hughes et al. (1990, 1991, 1992), stefano et al. (1991), paeman et al. (1992) mytilus galloprovincialis il-8, pdgf-ab, tgf-β1 franchini et al. (1996), ottaviani et al. (2000) crassostrea gigas tgf-β lelong et al. (2000) __________________________________________________________________________________________ viviparus ater and in the mussel m. galloprovincialis (figs 14-17) (ottaviani et al., 1990b, 1995a; franchini et al., 1994). furthermore, the acth receptor-like mrna has also been detected in the mussel (ottaviani et al., 1998c). the acth, β-endorphin and corticotropin-releasing hormone (crh) in the immunocytes and cell-free hemolymph were quantified by ria test (ottaviani et al., 1990b). with regards crh receptors, it has been reported that m. galloprovincialis immunocytes express molecules homologous to human mrnas of the two receptor subtypes (crh-r1 and crh-r2) (figs 18, 19) (malagoli et al., 2000). finally, a further 14 immunoreactive peptides, including bombesin, cck-8, neurotensin, oxytocin, substance p and vasopressin, have been detected (ottaviani and cossarizza, 1990). cytokine-like molecules cytokines are soluble factors involved in the immune responses mediating the interactions between different 55 cell types. however, it has been found that these molecules have a wide range of action as primary mediators of a variety of physiological functions even in non-immune environments such as the neuroendocrine system. cytokine immunoreactive molecules have been detected in different tissues of various invertebrate species, including molluscs (ottaviani et al., 2004) (table 2). using different technical approaches, these molecules have been detected in immunocytes, in the hemolymph, in eggs, in embryos, in larvae, in neurons and in glial cells from gastropods and bivalves (hughes et al., 1990, 1991, 1992; stefano et al., 1991; paeman et al., 1992; ottaviani et al., 1993b, 2000; granath et al., 1994; owe-missi-oukem-boyer et al., 1994; fainzilber et al., 1996; franchini et al., 1996; lelong et al., 2000). furthermore, platelet-derived growth factor (pdgf) receptor-αand -βand transforming growth factor (tgf)-β receptor (type ii)-like molecules have been detected on the plasma membranes of immunocytes from the mussel m. galloprovincialis (kletsas et al., 1998). nitric oxide synthase (nos) studies on nos in invertebrates have been mainly concentrated on the nervous system, and molluscs have been extensively used for investigations of the mechanisms involved in intercellular communication (stefano and ottaviani, 2002). with regards the immune system, biochemical, histochemical and immunocytochemical procedures demonstrated the presence of nos and related immune functions in the immunocytes of v. ater (conte and ottaviani, 1995; franchini et al., 1995). these findings are also supported by production of no by immunocytes from m. edulis and v. ater (ottaviani et al., 1993b). nos was also induced by injection of different cytokines, such as il-1α, il-2 and tumour necrosis factor (tnf)α, in the foot of a mollusc (ottaviani et al., 1995b). anti-microbial peptides most of the data on anti-microbial peptides regards insects (bulet et al., 1999), while molluscs have been reviewed only recently (mitta et al., 2000). in m. edulis two defensins (a and b) containing six cysteines and mytilinis (a and b isoforms) have been found (charlet et al., 1996). m. galloprovincialis presents a defensin-like molecule, named mgd1, containing 8 cysteines (hubert et al., 1996; mitta et al., 1999), while a second isoform, mgd2, has been identified from immunocyte mrna (mitta et al., 1999). furthermore, mytilinis (b, c, d, g1 isoforms) and myticins (a isoform in the plasma, a and b isoforms in immunocytes) have also been reported (mitta et al., 1999, 2000). immune-neuroendocrine interactions the present section reports how the immune and neuroendocrine responses are intermixed and interconnected in forming an unitarian network to neutralize threatening agents. with regards the immune response, cell shape changes, chemotaxis, phagocytosis, cytotoxicity, encapsulation, transplantation and wound healing will be examined, while for the neuroendocrine responses, the stress response is highlighted. cell shape changes (the expression of cell motility), chemotaxis (the expression of cell migration) and phagocytosis are the main ancestral mechanisms used by all organisms to eliminate non-self material (manke and bade, 1994). using computer-assisted microscopic analysis, it has been shown that bioactive peptides, such as acth and crh (sassi et al., 1998; malagoli et al., 2000), and cytokines, e.g. pdgf, tgfβ and il-8 (figs 20, 21) (kletsas et al., 1998; ottaviani et al., 2000), provoke changes in the cellular shape of mussel immunocytes. the extracellular signals are transduced by activating the classical pathways, i.e. protein kinase a, c and b. furthermore, bioactive peptides, cytokines, endorphins and crh have a chemoattractant effect on molluscan immunocytes (ottaviani et al., 1997a, b). in particular, acth, βendorphin and their related fragments exert different chemoattractant activity (ottaviani et al., 1997a). in this context, the first quantitative study on chemotaxis was performed by schmid (1975) on immunocytes from the snail viviparus malleatus that migrate toward staphylococcus aureus. chemotaxis has also been observed in the immunocytes of the hard clam mercenaria mercenaria in the presence of bacterial products (fawcett and tripp, 1994). figs 18, 19 expression in m. galloproviancialis immunocytes of human crh receptor subtypes: crhr1(18) and crh-r2-mrnas (19). bar = 10 µm (modified from malagoli et al., 2000). 56 figs 20, 21 immunocyte cell shape changes in m. galloprovincialis after incubation with il-8: actin microfilament modifications (21). control (20). bar = 10 µm (modified from ottaviani et al., 2000). phagocytosis, the last step in the response to nonself particulate material, is also well documented in molluscs (fig. 22) (cheng, 1981; sminia, 1981; bayne, 1983). here, it should be underlined that acth and its fragments, crh and cytokines, such as il-1α, il-2, tnf-α, il-8, pdgf and tgf-β all increase phagocytic activity (ottaviani et al., 1994, 1995b, 2004). as far as the relation between the nos system and immunocyte phagocytic activity is concerned, it has been observed that the no system is able to provoke bacterial clumping and killing, but is not an alternative to phagocytosis, as both are fundamental in bacterial elimination by immunocytes (ottaviani et al., 1993b; franchini et al., 1995). on the whole, these findings suggest that: i. a direct correlation between chemotaxis and phagocytosis does not exist; ii. not only peptides with a complete aminoacid sequence, but also peptide fragments of 4 or 5 aminoacids are able to stimulate or inhibit immune functions, as seen with small peptides and mammalian immune functions (werner et al., 1986); iii. a close correlation exists between molluscan immune functions and peptides typical of the neuroendocrine system. despite the lack of an adaptive immune response, invertebrates are able to cope with pathogen microorganisms present in their environment. accordingly, primitive but very efficient forms of immunity can be predicted, and cytotoxicity is one of fig. 22 m. galloprovincialis immunocytes phagocitizing bacteria (arrows). bar = 10 µm. these. in gastropods, bayne et al. (1980) have found that the immunocytes of b. glabrata have a cytotoxic effect on schistosoma mansoni sporocysts. decker et al. (1981) have demonstrated that in different marine invertebrates, including molluscs, cytotoxic activity seems to be independent of prior antigen exposure. in p. corneus, only one type of immunocyte, rh, was able to exert cytotoxic activity on k562 cells in a short-term (4 h) 51cr release essay used to evaluate human nk cell activity. this suggests that a nk-like cytotoxic activity is present in molluscs. furthermore, the nk-like activity was severely reduced after 18 h incubation at 24 ºc with il -2 (franceschi et al., 1991). in the bivalve m. edulis, the immunocytes are able to produce cytotoxic substances that cause lysis of human erythrocytes (wittke and renwrantz, 1984). the cytotoxic substances, which do not require a preceding contact with target cells (leippe and renwrantz, 1988), were purified, found to possess a mw of 72 kda and act even without free ca2+ (lieppe, 1989). cytotoxicity molecules may be important in the encapsulation reactions against parasites. for detailed information of the responses of gastropods and bivalves against metazoan, protozoan and fungal parasites, see the interesting chapter in bayne (1983). the ultrastructural studies of capsule formation in p. corneus have shown that both sh and rh are involved (ottaviani et al., 1991a), and the process can be correlated with the encapsulation process observed in b. glabrata using electron microscopy (harris, 1975) or enzymatic markers such as acid phosphatase (cheng and garrabrant, 1977). the granulocyte corresponding to the sh of p. corneus (ottaviani and franchini, 1988) is one of the two cell types present in b. glabrata (cheng, 1975) responsible for both phagocytosis and the encapsulation process (cheng and garrabrant, 1977). the other p. corneus cell type, the rh, is the first to reach the graft and behaves in a functionally equivalent manner to b. glabrata hyalinocytes (cheng and garrabrant, 1977). the p. corneus capsule 57 figs 23-26 ultrastructural aspect of the p. corneus graft capsule (96 h): rh (23); sh (24); fibroblast-like cells (25, 26). bar = 0.5 µm (modified from ottaviani et al., 1991a). figs 27-32 allograft (ganglia) implant in p. corneus after 96 h (27, 28): the fibres were green with the gabe and martoja-pierson trichromic stain (29); 192 h after implant (30); xenograft implant after 96 h (31) and 192 h (32), f, foot; c, capsule; n, new blood vessel; g, ganglia. 27, 30, 32) bar = 100 µm; 28) bar = 30 µm; 29) bar = 10 µm (modified from ottaviani and vergine, 1990). 58 figs 33-36 autograft (tentacle) implant in p. corneus after 24 h (33); 48 h (34); 96 (35) and 168 h (36). f, foot; t, tentacle, h, immunocytes; dt, degenerative tentacle. 33, 34) bar = 100 µm; 35, 36) bar = 30 µm (modified from ottaviani and vergine, 1990). contains immunocytes and fibroblast-like cells (figs 23-26), while in helisoma duryi normale, epithelioid cells and multinucleate macrophages have been observed (cheng and garrabrant, 1970). in l. stagnalis, fibroblast-like cells have been shown to be transformed blood cells (sminia et al., 1974), and a similar phenomenon probably also occurs in p. corneus. indeed, bearing in mind that n-acetylmuramic acid is a sh marker (ottaviani and montagnani, 1989), and that both collagen fibres and sh are positive to the corresponding polyclonal antibody, fibres may be formed by the sh that can so be considered fibroblastlike cells (ottaviani et al., 1991a). studies on cellular encapsulation have shown that the extension and the speed of the process is related to the degree of compatibility between the host and the encapsulated parasite from a foreign body (sminia, 1981). in this respect, transplantation experiments are a good tool to investigate the degree of discrimination of the molluscan recognition system. however, the various gastropod models have not given a uniform response. australorbis glabratus (tripp, 1961) and l. stagnalis (sminia et al., 1974) are able to discriminate between autografts and xenografts, but fail to recognize allografts. also in b. glabrata, several alloimplats are not rejected (sullivan et al., 1998). in contrast, h. duryi normale (cheng and garrabrant, 1970) and p. corneus (ottaviani and vergine, 1990) recognize allografts. in p. corneus, cerebral ganglia from samples of the same (allografts) or different (xenografts) species were implanted in the foot. for the autograft experiments a removed tentacle was used. the histological observations 72 h after alloimplant showed that the immunocytes were stratified, flattened and infiltrated in the graft tissue. after 96 h, the graft was completely encapsulated (fig. 27). the capsule showed immunocytes, thin fibril bundles loosely dispersed in an amorphous substance and the presence of new blood vessels (fig. 28), as witnessed by the presence of an angiogenesis process. the amorphous substance and the fibrillar component were positive to bromophenol blue, pas and alcian blue ph 2.5 reactions. the fibres were green with the gabe and martoja-pierson trichromic stain (fig. 29). from 168 h onwards, the capsule decreased in thickness. the implanted ganglia showed suffering and after 192 h almost all the structure had been destroyed (fig. 30). the ganglia xenoimplant in the foot of p. corneus was encapsulated after 48 h, and the capsule showed maximum thickness after 96 h (fig. 31), diminishing drastically after 192 h (fig. 32). the autograft experiments have revealed that the immunocytes migrated towards the implanted tentacle (fig. 33), and that after 48 h they were not longer observed (fig. 34). later at 96 h, the autograft seemed to integrate into the host tissue (fig. 35). at 168 h, the degenerative process was observed in the graft (fig. 36). the non-survival of the autograft can have several explanations, including blood supply, the nature and the number of cells, tissue mitotic activity after vascolarization, the capacity of the graft to stimulate host fibroblasts and so on. this experiment has shown that p. corneus seems to have a specific immunorecognition system able to discriminate between the different grafts. in bivalves, allotoimplants were performed by inserting small portions of living mantle from c. gigas into a slit in the connective tissue near the palps of another animal (des voigne and sparks, 1969). the findings have indicated that some alloimplants are rejected, while others seem to remain normal during the duration of the experiment. xenoimplants, performed by placing mya arenaria mantle orthotopically on m. californianus mantle (bayne et al., 1979), have shown that the secondand 59 figs 37-40 wound healing in the gastropod l. maximus. 37). a large number of immunocytes (arrow) stratify at the wound margins; 38, 39) a well-developed granulation tissue (g) with hematic lacunae (arrows); 40) the repairing wound is covered by cubic epithelial cells (arrows). bar = 10 µm (modified from franchini and ottaviani, 2000). are rejected more rapidly than the first-set, while there is a more localized host response to the third-set implant. however, the authors have claimed that the response is not specific. wound healing is a complex process involving different biological events to restore tissue integrity and functions. this process is characterized by inflammation, new tissue formation and tissue remodelling. experiments performed on the gastropod limax maximus (franchini and ottaviani, 2000), in which longitudinal incisions were made through the skin of the latero-dorsal part of the body, have shown that wound repair is first characterized by an infiltration phase, during which the immunocytes are recruited, accumulated and activated in the injured area (fig. 37). flattened immunocytes are stratified at wound margins and actively phagocytize cell debris and damaged tissue surrounding the area. the inflammation response is also observed in other molluscs (cheng and garrabrant, 1970; sminia, 1981; ottaviani and vergine, 1990). as in mammals, the second step in the process involves the formation of granulation tissue, in which several small blood lacunae are formed and a provisional matrix is synthesized and deposited by immunocytes presenting fibroblast-like activity (figs 38, 39). finally, there is a wound re-epithelialization phase (fig. 40). exogenous administration of pdgf-ab and tgf-β1 stimulates the tissue healing process by accelerating all the activities involved. wound healing experiments in c. gigas have shown a similar chronology. initially, the leukocytes infiltrate, elongate and arrange themselves parallel to the direction of the lesion. just when the cells have filled the lesion, the scar is formed and subsequently substituted by fusiform leukocytes that create a pseudoepithelium at the body surface. the oyster healing process is closely to that observed in annelids (des voigne and sparks, 1968). the wound healing observed in the freshwater mussel anodonta oregonensis have shown an immediate inflammation response, followed by the repair process, as seen in most vertebrates and other invertebrates. however, there is no pronounced cellular response in this mussel (pauley and heaton, 1969). memory-type response the presence of some kind of memory in invertebrates is still debated. the evidence in favour of the existence of a memory-type response in lower and advanced invertebrates has been proposed by various authors (cooper, 1969, 1976; hostetter and cooper, 1973; karp and hildemann, 1976; hildemann et al., 1977, 1979a,b) and by karp and rheins (1980). in the present review, a further example of a memory-type response in an invertebrate (p. corneus) is presented. according to hildemann et al. (1979), three functional components must be identified as minimal criteria for immunological competence, i.e. cytotoxicity, specificity and memory. data in favour of the presence of cytotoxicity and specificity have been reported before, while results supporting memory in p. corneus have been obtained from humoral and cellular experiments as well as bacterial clearance studies (ottaviani et al., 1986; ottaviani, 1992). after injecting the bacteria s. aureus and escherichia coli repeatedly into the foot of the mollusc, specific and aspecific agglutinins which were undetectable before injection were observed. 60 specific agglutinins observed in direct-agglutination tests showed an increased titre after the second injection. aspecific agglutinins evaluated in crossagglutination tests showed no changes in titre after the second injection (ottaviani, 1992). the in vitro bacterial phagocytosis experiments have shown a higher bacterial elimination across the entire time-range considered (30, 60, 90, 120, 150 min) in the snails that had already contacted the bacteria to be phagocytized. differences between control and sham-injected control snails were not observed (ottaviani, 1992). the bacterial clearance investigations have revealed that after the second (14 days) and third (73 days) bacterial injections, clearance rates are faster and clearance patterns markedly different than after the first injection (ottaviani et al., 1986). on the basis of the above findings, some considerations can be advanced. according to klein (1989), the evolutionary key opening the door to the vertebrate immune system should not be sought in the invertebrate immune system, since the vertebrates are not the crowing phase of invertebrate evolution. looking at the problem from another point of view, i.e. considering the dna as the possible prime mover of evolution, this statement is not completely true. the dna is conservative, but all forms of life are the expression of casual combinations of dna sequences. the resulting products, that are the expression of selective advantage, appear to be re-proposed every time that new forms derive from dna. a typical example is represented by molecules and functions of the immune system, such as bioactive peptides, cd, phagocytosis, nk activity and others. in this respect, it is useful to examine the immune mechanisms present and conserved in lower forms in order to understand the complexity of higher life. furthermore, as every form of life survives, it must possess an immune system sufficient to its needs and, as suggested by hildemann (1974), it is reasonable to recognize different levels of immunity. therefore, observations on the structural simplicity of invertebrates and the consequent lack of a true immune system with an anamnestic-accelerated secondary response (klein, 1989) are quite inadequate, as invertebrates possess immunological competence. however, bearing in mind that invertebrate memory is not based on clonal selection, it would be better to use the term “invertebrate memory”, rather than “immunological memory”. stress response pomc-derived peptides and crh are the central mediators of stress response. in mammals, the main circuit involves a hypothalamic-pituitary-adrenal gland axis. crh produced in the hypothalamic paraventricular nuclei controls the pituitary secretion of acth that, in turn, stimulates the synthesis and the release of adrenocorticosteroids. together with neural activation, glucocorticoids regulate catecholamine biosynthesis in adrenal medulla. the question of the evolution of this apparently very complex type of response that involves several distant organs and a variety of cell types has been resolved in invertebrates by a simplified scheme, in which the basic mechanisms are well conserved. indeed, the phagocytic immunocytes contain the key mediator molecules and the series follows the same order and pattern, i.e. crh à acth à biogenic amines (ba) (ottaviani and franceschi, 1996). in molluscs, in addition to this acth-mediated pathway, another more direct crh à ba pathway appears to exist. as far as ba are concerned, epinephrine, norepinephrine, dopamine have been found in molluscan brain, hemolymph and immunocytes using a hplc procedure (ottaviani and franceschi, 1996). furthermore, enzymes of the ba biosynthesis and cortisol have been reported in the immunocytes by immunocytochemical procedure (ottaviani et al., 1993a, 1998b; ottaviani and franceschi, 1996). studies on stress have also been performed by stefano’s group. the immunocytes of m. edulis produce and reacte to opioid peptides (stefano et al., 1990) and the cellular response to stress observed is the same as that found in mammalian immunocytes. similarly to vertebrates, cytokines are also involved in molluscan stress responses. indeed it has been observed that il-2, il-1α, and β, tnf-α and β, pdgfab and tgf-β1 induce ba release from immunocytes (ottaviani et al., 2004). all these events occur in the immunocyte, a cell type that has been proposed as an “immune-mobile brain”, capable of both immune and neuroendocrine responses (ottaviani et al., 1991a, 1993a). concluding remarks in conclusion, the findings reported in this review support the following considerations and speculations: • despite their apparent simplicity, molluscs, as all celomatic invertebrates, are capable of very sophisticated performances regarding immune and neuroendocrine functions; 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assay in agar. j. invertebr. pathol. 43: 248-253, 1984. xi meeting italian association of developmental and comparative immunobiology (iadci) isj 7: 107-118, 2010 issn 1824-307x report of meeting xith scientific meeting of the italian association of developmental and comparative immunobiology (iadci), 24 26 february 2010, department of animal biology, university of modena and reggio emilia, modena, italy organizers: e ottaviani, d malagoli department of animal biology, university of modena and reggio emilia, modena, italy session 1. chairman: e ottaviani, university of modena and reggio emilia, modena, italy mussel hemocytes: the role of pkc isozymes in the innate immune response r barcia, m gonzalez-riopedre, ji ramosmartínez department of biochemistry and molecular biology, university of santiago de compostela, campus of lugo, lugo, spain the ancestral relationship between immune response and stress has evolved from a common cellular space such as the hemocyte of invertebrates to the implication of diverse endocrine elements (hpa axis) in vertebrates. in marine molluscs, the hemocytes shoot the immune response by means of molecular and physical stimuli that are redundant and pleiotropic. it is well known that different molecules as toxins, cytokines or growth factors induce responses apparently similar, but differing in power, seasonality etc., which proves that every signal has an internal regulating ability. the subjects of this study were two protein kinases c isolated from mytilus galloprovincialis, namely p60 and p105. our goal was to investigate their implication in the synthesis of catecholamines, as stress markers, and nitric oxide, associated to the respiratory burst and to phagocytosis, in mussel hemocytes. cultured mytilus hemocytes were treated with different agonist and then catecholamine synthesis was monitored. the results obtained were compatible with the modifications that pkcs undergo. il-2 or lps activate catecholamine synthesis by the implication of both isoforms of pkc. for its part, pdgf activates dopamine and norepinefrine synthesis through p60, while modifying the balance between p105 active and inactive forms by inhibiting epinefrine synthesis. also, hemocyte response varied seasonally when activated both by lps and by il-2. mytilus hemocytes treated with lps did not show nitric oxide synthesis. on the contrary, il-2 provoked a remarkable no production which involved preferably the camp-dependent protein kinase (pka). the use of pkc inhibitors, the study of the seasonal variations, and the detection of an inducible nos isoform, prove that pkc acts upon the constitutive nos throughout the year preferably as an inhibitor. in winter, the maximal no production detected is the result of p105 activating action on a new inducible nos. these results prove that the different actions are in accordance with regulating processes through cell signaling mechanisms. on the contrary, they do not support the existence of a specific receptor for each modulator or inducer, thus confirming the hypothesis of the presence of an ancestral common receptor. responses of mytilus galloprovincialis hemocytes to heat killed vibrio splendidus lgp32: role of p38 mapk and pkc c ciacci1, b citterio2, m betti1, b canonico1, p roch3, l canesi4 1disuan, university of urbino “carlo bo”, urbino, italy 2department of biomolecular sciences, university of urbino “carlo bo”, urbino, italy 3 jru ecosystèmes lagunaires, cnrs-university of montpellier 2, france 4department of biology, university of genoa, genoa, italy mytilus hemocytes have been recently demonstrated to show differential functional responses to injection of different vibrio species. in particular, in mytilus galloprovincialis hemocytes, differences were observed in responses to v. splendidus lgp32, a strain associated with oyster larvae and juvenile stage mortalities, and to v. anguillarum. in this work, the in vitro effects of heat-killed v. splendidus lgp32 on mytilus hemocytes and the mechanisms involved were investigated. the results were compared with those obtained with v. anguillarum (atcc 19264). hemocyte adhesion, lysosomal membrane stability, lysozyme release, oxidative burst and no production were evaluated, as well as the phosphorylation of the stress 107 activated p38 mapk and pkc, that play a key role in the hemocyte response to bacterial challenge. v. splendidus did not affect total hemocyte adhesion, but decreased adhesion of large granulocytes, induced lysosomal membrane destabilization, rapid and persistent lysozyme release, stimulation of oxidative burst and no production. these effects were associated with rapid and persistent activation of p38 mapk and pkc. in contrast, v. anguillarum had no effect on oxidative burst, and induced significantly lower lysozyme release and phosphorylation of p38 mapk and pkc with respect to v. splendidus. these data indicate the existence of specific interactions between v. splendidus lgp32 and mussel hemocytes and suggest that this vibrio strain might affect host bivalve cells through disregulation of immune signaling. overall, the results support the hypothesis that responses of bivalve hemocytes to different bacterial stimuli may also depend on the cell subtype, thus leading to differential activation of different kinases. effects of vibrio challenge on digestive gland biomarkers and antioxidant gene expression in mytilus galloprovincialis c barmo1, r fabbri1, c ciacci2, l. vergani1, p roch3, g gallo1, l canesi1 1department of biology, university of genoa, genoa, italy 2disuan, university of urbino “carlo bo”, urbino, italy 3jru ecosystèmes lagunaires, cnrs-university of montpellier 2, france in bivalves, functional and molecular responses to bacterial infection have been largely characterized in the immune cells, the hemocytes. although soft tissues are endowed with bacteriostatic activity, responses at the tissue level, where bacterial infection may cause stressful conditions, have been not specifically investigated. in this work, the effects of heat-killed vibrio species, v. splendidus lgp32 and v. anguillarum (atcc19264), in the digestive gland of mytilus galloprovincialis were investigated. mussels were injected with either vibrio and tissues sampled at 3, 6 and 24 h post injection (p.i.). lysosomal biomarkers (lysosomal membrane stability-lms and lipofuscin accumulation), activities of antioxidant enzymes (catalase and glutathione transferasegst) were evaluated, as well as expression of antioxidant molecules (catalase, gst-π and metallothioneins mt10 and mt20) by quantitative rt-pcr were evaluated. both v. splendidus and v. anguillarum significantly affected all parameters measured, to a different extent at different times p.i.. both vibrios induced similar effects on lms and antioxidant enzyme activities. in contrast, v. splendidus induced a general up-regulation of antioxidant gene expression, whereas v. anguillarum did not. the lack of this response was reflected in stronger tissue oxidative stress conditions in mussels challenged with v. anguillarum, as indicated by higher lipofuscin accumulation observed at longer times p.i.. the results indicate that mytilus digestive gland can mount an efficient antioxidant response towards v. splendidus lgp32, a strain pathogenic to oyster juveniles and larvae. overall, lysosomal and oxidative stress biomarkers could be usefully applied in order to monitor early changes in the health status of bivalves induced by bacterial infection. differential response of mytilus galloprovincialis hemocytes to in vivo and in vitro bacteria challenge p pagliara1, p cito1, p roch2 1department of biological and environmental sciences and technology, university of salento, lecce, italy 2jru ecosystèmes lagunaires, cnrs-university of montpellier 2, france marine bivalves are filter-feeding organisms able to accumulate a large number of bacteria. the massive presence of bacteria does not affect their survival due to a very efficient immune response. in mussels, hyalinocytes and granulocytes are responsible for cell-mediate immunity. granulocytes are generally considered to play a prominent role in such defense mechanisms. in this study the response of mytilus galloprovincialis hemocytes to in vivo and in vitro challenges with two bacteria (vibrio anguillarum and micrococcus lysodeikticus) was evaluated. direct injection of live bacteria into the hemolymph induced different hemocyte responses. in particular, the presence of v. anguillarum caused morphological changes in hyalinocytes that resulted in apoptosis or in modified cytoplasm containing a large number of vacuoles, while the majority of granulocytes showed a necrotic aspect. nevertheless some granulocytes resulted rounded with a strongly condensed nucleus. on the other hand, m. lysodeikticus induced morphological changes associable to apoptotic process, in both cell types. also in vitro, bacteria were able to differentially modulate the hemocyte response, being m. lysodeikticus able to induce chromatin condensation and rounding of cells and v. anguillarum inducing necrosis. so, the in vivo or in vitro conditions differently affected the bacteria action, resulting v. anguillarum unable in vitro to induce apoptotic morphology in hemocytes. about morphology of apoptotic hemocytes, we evidenced the ability of bacteria to induce a rounding of cells and chromatin condensation, while not relevant nuclear fragmentation, generally considered to be the last step of apoptotic process. effects of different environmental vibrio strains on immune responses of mytilus hemocytes c ciacci1, a manti1, b canonico1, w baffone2, r campana2, l canesi3 1disuan, university of urbino “carlo bo”, urbino, italy 2department of biomolecular sciences, university of urbino “carlo bo”, urbino, italy 108 3department of biology, university of genoa, genoa, italy vibrios are gram (-) autochthonous bacteria of the marine environment, where they occur both free in water and associated with living organism and organic and inorganic surfaces, thus representing a considerable part of heterotrophic bacterial populations. vibrios are also considered an important cause of human food-borne illnesses associated with the consumption of seafood, including bivalves. although in bivalve immunocytes responses to bacterial challenge have been widely investigated, few reports are available on responses of mytilus hemocytes to environmental vibrio isolates. in this work, the effects of in vitro challenge of mussel hemocytes with v. parahaemolyticus 80 (isolated from mussel samples of the conero coast), and v. alginolyticus 1513 (isolated from marine plankton), were compared with those of available reference strains, v. parahaemolyticus atcc 43996 and v. vulnificus 509. the results indicate differential responses to different vibrios in terms of lysosomal membrane destabilization and activation of immune parameters (lysozyme release, oxidative burst and no production). v. parahaemolyticus strains, and in particular the v. parahaemolyticus 80 isolate, producing the virulence factor tdh (thermostable direct hemolysin), induced stronger lysosomal destabilization and smaller activation of immune responses in comparison to other vibrios. flow cytometry analysis showed that different vibrios did not affect total hemocyte counts (thc), but induced changes in hemocyte subpopulations. these results support the hypothesis that mytilus hemocytes represent a sensitive target for the action of different vibrio species and strains and indicate that these invertebrate cells can be utilized for elucidating the mechanisms for pathogen action also in vertebrates. changes in hemocyte subpopulations and immunoreactivity to anti-integrins and progenitors antibodies of mytilus galloprovincialis after bacterial challenge b canonico1, c ciacci1 , m arcangeletti1, m betti1, b citterio2, p roch3, s papa1, l canesi4 1disuan, university of urbino “carlo bo”, urbino, italy 2department of biomolecular sciences, university of urbino “carlo bo”, urbino, italy 3jru ecosystèmes lagunaires, cnrs-university of montpellier 2, france 4department of biology, university of genoa, genoa, italy the involvement of circulating hemocytes as the principal cellular effector mediating molluscan immune responses is well established. microbial invasion poses an immediate threat to survival, and a vigorous defense response ensues an effort to clear the pathogen from the host. overall, experimental evidence suggests that molluscan immune responses rely on molecules that share homology with those of vertebrate systems. in this work, flow cytometric techniques and anti-human mouse monoclonal antibodies (mabs) were utilized to monitor changes in hemocyte subpopulations of mytilus galloprovincialis, after bacterial challenge, in vivo and in vitro. we focused on total hemocyte count (thc), fsc and ssc physical characteristics, positivity to anti cd34, anti cd117 and anti cd11b antibodies. these reagents in humans recognized stem and progenitor cells (cd117 and cd34) and neutrophils/monocytes (cd11b). we applied cytometric internal controls to monitor cell autofluorescence, avoid artifacts due to formaldehyde fixation. the results show that 6h after bacterial challenge, a decrease in large granulocyte subpopulation occurs, particularly with vibrio splendidus. furthermore, we found a different expression of the antigens investigated in hyalinocytes, small and large granulocytes. inoculation of v. splendidus, v. anguillarum and micrococcus lysodeitkicus seems to produce a rearrangement in the distribution of these antigens. these in vivo data are also substantiated by the adhesion test after in vitro bacterial challenge. this study confirm the existence of heterogeneity among circulating hemocytes, the presence of differentially engaged cell populations during bacterial challenge and a kind of proliferation/differentiation pathway differently primed by various bacteria. functional differential hemocytes behaviour in the clearance of bacteria and humoral defense factors variability in mytilus galloprovincialis mg parisi1, m cammarata , h li , m toubiana , n 1 2 2 parrinello , p roch1 2 1department of animal biology, university of palermo, palermo, italy 2jru ecosystèmes lagunaires, cnrs-university of montpellier 2, france bivalve molluscs are constantly exposed to various pathogen agents. to survive in the aquatic environment, they have developed active cellular and humoral immune responses. in this study, flow cytometry was used to identify three hemocytes sub-populations in m. galloprovincialis: hyalinocytes, small and large granulocytes. when bacteria of vibrio and micrococcus genus were injected into mussel circulation, proportions of the three cells categories varied differentially and increment of living intrahemocyte bacteria number suggested intense phagocytosis. lysozyme gene expression study during bacterial infection and in vivo heat shock and cold temperature treatment showed that hemocyte populations were also capable of discriminating between stress factors and pathogenic species. finally sequence diversity in antimicrobial peptides (amp) from mediterranean mussels were found. in addition, mytilin b mrna appeared to be translated into propeptide and his mature form obtained inside hemocyte granules. polymorphism observed at specific locations indicated negative selection pressure on signal and mature peptide domains. however, a positive selection pressure for coohterminus domain can be suggested. studies are in progress to explain the relationship between 109 different environment conditions and mussel amps polymorphism. variability of antimicrobial peptides in mytilus galloprovincialis u rosani1, l varotto1, a rossi1, b novoa2, p roch3, a figueras2, p venier1 1department of biology, university of padua, padua, italy. 2instituto de investigaciones marinas (csic), vigo, spain 3jru ecosystèmes lagunaires, cnrs-university of montpellier 2, france in mytilus spp. four different antimicrobial peptide (amp) families have been described. amps are produced both constitutively and in response to vaious stimuli, and show complex expression patterns in the mussel hemocytes. in fact, amps precursors can represent 25-40 % of the whole hemocyte transcriptome and a remarkable sequence variability, with unique profiles in individual mussels, has been reported for myticin c. aiming to investigate the natural variability of amp transcripts in farmed mussels we adopted a high throughput sequencing approach which allows the detection of rare sequence variants. primers were carefully designed to cover almost all the known sequence variants (cds) of mytilin, myticin, defensin and mytimycin. following pcr amplification, the tagged amplification products were sequenced with a genome sequencer flx™ system. at first glance, the variability of the amp transcript precursors is comparable to that reported for the myticin c and mainly represented by single nucleotide substitutions (sequence variability of the mature peptides is substantially reduced). specific patterns of variation will be compared with appropriate statistical tests and the overall analysis will also include the comparison between different geographical regions. session 2. chairman: l ballarin, university of padua, padua, italy cytokines do exist in invertebrates!...now what? d malagoli department of animal biology, university of modena and reggio emilia, modena, italy cytokines are soluble mediators of a relatively small molecular weight mainly produced by the cells of the immune or neuroendocrine systems during immune response. cytokines have been described principally in mammals, even though during 80’s and 90’s several authors have reported the presence of cytokine-like molecules in invertebrates. the existence of cytokines in protostomians was only confirmed in the last decade, when molecular biology and functional studies lead to the discovery of spätzle and unpaired (upd)-3 in d. melanogaster, hemocyte chemotactic peptide (hcp) in the moth pseudaletia separata and astakine 1 in the freshwater crayfish pacifastacus leniusculus. these findings have finally demonstrated that cytokines exist in invertebrates and probably several other factors will be discovered in the next future. however, the fundamental questions pertaining the evolution of cytokines (i.e., which is their origin and how their differentiation have proceeded) remain unanswered. the analysis of cytokine genes in mammals have demonstrated the extreme variability of the cytokine sequences, especially of interleukins. this makes the study of cytokine evolution a very difficult task, that requires specific algorithms to find potentially conserved molecules and full molecular and structural characterization as a final step. in this perspective, molecules such as drosophila helical factor (dhf) may prove of help in describing the evolution of one of the major classes of immunerelated molecules, but further studies are required before we gain the knowledge necessary to trace a draft of a phylogeny tree for cytokines. implication of an il-16 homologous in microglial cells recruitment after cns injury in the medicinal leech hirudo medicinalis j vizioli, f croq, a garçon-bocquet, m tahtouh, p-e sautière, c van camp, m salzet, j pestel, c lefebvre laboratory of annelids neuroimmunology, cnrs fre 2933, university of lille1, cité scientifique bat. sn3 59655 villeneuve d’ascq, france the medicinal leech hirudo medicinalis can totally repair its central nervous system (cns) after injury. this invertebrate model offers unique opportunities to study the molecular and cellular basis of the cns repair processes. when the leech cns is injured, microglial cells migrate and accumulate at the site of lesion, this phenomenon is essential for the usual sprouting of injured axons. we recently isolated a novel molecule, named hmil-16, having homologies with human interleukin-16 (il-16) active form. hmil-16 has a chemotactic activity on leech microglial cells similar to that observed using a gradient of human il-16. the pre-incubation of microglial cells either with an anti-human il-16 or with anti-hmil-16 antibodies highly reduced the leech conditioned mediummediated microglia migration. moreover, hmil-16 was demonstrated to promote human cd4+ t cells migration. using either antibody against human il16, an il-16 antagonist peptide or soluble cd4, human cd4+ t cells migration promoted by hm-il16 was reduced. in leech, immunohistochemistry studies on cns suggests that hmil-16 protein present in the neurons is rapidly transported and stored along the axonal processes to promote the recruitment of microglial cells close to injured axons. to our knowledge, this is the first time that an interleukin-16 has been found in cns invertebrate with an associated biological function. a homologous of interleukine-16 is involved in leech wound healing a grimaldi1, c bianchi1, g greco1, g tettamanti1, r valvassori1, m de eguileor1, a garçonbocquet2, c lefebvre2, j vizioli2 110 1department of biotechnology and molecular sciences, university of insubria, varese, italy 2laboratory of annelids neuroimmunology, cnrs fre 2933, university of lille1, cité scientifique bat. sn3 59655 villeneuve d’ascq, france several reports in these recent years have highlighted that cytokines and their signalling pathways have been highly preserved during evolution. we have already described the remarkable conservation of these molecules and their related function in leeches as well. recently a new cytokine has been identified in the leech hirudo medicinalis. this molecule is homolog to human interleukin-16 (il-16) an it is involved in the central nervous system repair. since in vertebrates il-16 plays an important role in innate immune responses and is a major chemotactic signal for cd4+ cells, we focused our study on the possible role of hmil-16 in the regulation of inflammation and wound healing processes in leech as well. in particular we investigated on the expression of hmil-16 in the two tissue of leech mainly involved in these processes: the botryoidal tissue, involved in the myelo/erythroid and hematopoietic cell production and in the angiogenic activity, and the vasofibrous tissues, responsible in the formation of the pseudoblastema during wound healing processes. we found that hmil-6 injection in the leech body wall induces the proliferation and migration, towards the stimulated area, of the vasofibrous tissue cells. this type of cells highly express both hmil-16 and its receptor cd4+. the chemoattractant activity of hmil-16 for leech immune cells cd4+ was validated by using the innovative technique of matrigel biopolymer supplemented with hmil16 and injected in leech body wall. immune response of anopheles stephensi immunocytes to asaia infection m mandrioli department of animal biology, university of modena and reggio emilia, modena, italy the acetic acid bacteria belonging to the genus asaia have been recently identified in midgut, salivary glands and reproductive organs of the asian malaria vector anopheles stephensi. asaia has been proven to be easily cultivable and transformable, and modified strains of asaia expressing green fluorescent protein (gfp) or red fluorescent protein (dsred), efficiently colonize recipient mosquitoes. asaia is horizontally transmitted to members of mosquito populations by co-feeding and mating and by maternal and paternal vertical transmission routes. even if the transmission routes are quite well established, at present it is not clear how asaia can move from the gut to the salivary glands and the reproductive organs without being killed by the host immune system. at this aim, immunocytes have been isolated from a. stephensi adult mosquitoes and maintained in culture in order to test their response to asaia. in particular the effect of asaia on the transcriptional levels of cecropin, defensin and gambicin genes has been evaluated by rt-pcr. moreover, phagocytosis tests have been performed in order to verify if asaia could activate this process. on the basis of the results, the symbiont asaia induces an immune response that is similar to that observed after escherichia coli induction, but asaia is not phagocytized at the contrary of what happens with e. coli. it could be therefore very intriguing to verify if asaia is killed by the antimicrobial peptides whose transcription is increased in response to asaia infection or if asaia is resistant to these peptides. proliferation and differentiation of mussel hemocytes are conserved from molluscs to mammals m betti1, c ciacci1, p gobbi1, b canonico1, s papa1, l canesi2 1disuan, university of urbino “carlo bo”, urbino 2department of biology, university of genoa, italy stem cell factor (scf) is a cytokine that binds to the c-kit receptor (cd117). scf can exist both as a transmembrane protein and a soluble protein. this cytokine plays an important role in hematopoiesis, spermatogenesis, and melanogenesis. soluble and transmembrane scf bind to c-kit and are biologically active. both forms of scf is produced by fibroblasts and endothelial cells. soluble scf has a molecular weight is 18.5 kda and forms a dimer. scf plays an important role in the hematopoiesis during embryonic development. scf plays a role in the regulation of hematopoietic stem cells (hscs): scf has been shown to increase the survival of hscs in vitro and contributes to the self renewal and maintenance of hscs in vivo. scf binds to the c-kit receptor (cd 117), a receptor tyrosine kinase. c-kit is expressed in hscs, mast cells, melanocytes, and germ cells. it is also expressed in hematopoietic progenitor cells including erythroblasts, myeloblasts, and megakaryocytes. scf binding to c-kit causes the receptor to homodimerize and auto-phosphorylate at tyrosine residues. the activation of c-kit leads to the activation of multiple signaling cascades, including the ras/erk, pi3-kinase, src kinase, and jak/stat pathways. flow cytometry analysis of control hemocytes of mytilus sp. showed immunoreactivity towards cd34 and cd90 antibody utilised has a markers of hscs. scf-treated mussel hemocytes showed increase in phagocytic activity, reduction in lysosomal membrane fusion processes and increase expression of cyclin a and d. ultrastructural morphological studies in colcemidetreated hemocytes confirmed that the mussel cells are able to enter the mitotic phase of the cell cycle. confocal microscopy analysis of control and scftreated hemocytes showed that these effects of scf involved the activation of c-kit tyrosine kinase-like receptors. new insight into the genetic basis of the highmultiple mating type systems of the modern species of the ciliate euplotes a vallesi1, c alimenti1, s federici2, g di giuseppe2, f dini2, p luporini1 111 1department of enviromental and natural sciences, university of camerino, camerino, italy 2department of biology, university of pisa, pisa, italy the high-multiple (“open”) systems of mating types (mt) that control self/non-self recognition in the most recently evolved complex of euplotes species are traditionally considered to be genetically determined by series of single-locus alleles designated as mat-1, mat-2, mat-3, and so forth. these genes are inherited and expressed accordingly to a mendelian mechanism of serial dominance (i.e., mat-1 > mat-2 > mat-3 and so forth). therefore, the heterozygous condition (e. g., mat-1/mat-2) would mimic the corresponding dominant condition (i. e., mat-1/mat-1), both gene combinations expressing the same phenotype mt-i due to the production of only one chemical signal (pheromone) specified by the dominant gene mat-1. working on a paradigmatic modern species, e. crassus, we first characterized the amino acid sequence of a pheromone (designated as ec-ph1) purified from the culture filtrates of the strain l2d. based on the knowledge of this sequence, we synthesized oligos for pcr-cloning the ec-ph1 gene from the strain l2d, as well as other pheromone genes from other e. crassus strains. we obtained evidence that the ec-ph1 gene: (i) coexists in the strain l2d with a second pheromone gene (ec-ph2) which is structurally divergent from the ec-ph1 gene and equally expressed; (ii) is present and coexpressed also in other e. crassus strains in combination with other homologous (allelic) pheromone genes (ec-ph3, ec-ph4, and so forth). these observations thus imply that in the modern species of euplotes, as is the case in the ancient ones, the multiple series of mat genes are regulated by relationships of co-dominance rather than of serial dominance. crucial insights to understand the functional differences between the two models await a definitive characterization of the expression and activity of the ec-ph1 pheromone gene in multiple e. crassus strains. an unusual chordate metallothionein gene in ciona intestinalis genome: structure and expression studies n franchi, m del favero, e piccinni, l ballarin department of biology, university of padua, padua, italy metallothioneins (mts) are able to bind essential and non-essential heavy metal ions, thus controlling cellular homeostasis and detossification. in addition, they act as scavengers for reactive oxygen species (ros), thanks to their abundant thiols groups. they have a role also in the regulation of inflammatory responses through the modulation of immunomodulatory humoral components. chordata represents the major phylum of deuterostomes, including about 45,000 species distributed in three subphyla: tunicata (urochordata), cephalochordata and vertebrata. invertebrate chordata, about 3 % of the total chordate species, are collectively named protochordata. unfortunately, no mt genes have been annotated so far in protochordates. in order to allow a comparison with the vertebrate mts we undertook a search for mt genes in the genome of the solitary tunicate ciona intestinalis. we were able to find a mt gene (cimt1), which represents the first mt gene identified in tunicates. its expression is limited to hemocytes and modulated by cd, zn and cu. the deduced protein is only 39 amino acids in length with no typical α and β domains. however the sequence shows that this protein shares the usual percentage (≥ 30) of cys residues arranged in typical conserved motifs reported for vertebrates. further insight on ciona intestinalis prophenoloxidase system activated during the lps induced inflammatory response m cammarata, v mangano, mr trapani, v arizza, a vizzini, d parrinello, m vazzana, m pergolizzi, n parrinello department of animal biology, university of palermo, palermo, italy in invertebrates, activated phenoloxidases participates in the melanization and is involved in different biological activities including the immune responses. a contact with foreign agents activates, through a serine protease pathway, challenges the prophenoloxidase system (propo) producing the active form of the enzyme (po). in ciona intestinalis, the po is a o-diphenoloxidase contained in hemocytes. in the present work we report on the propo system and related molecules in c. intestinalis tunic during the lps induced inflammatory responses with particular attention to the biochemical, cellular and molecular aspects. following an inflammatory stimulus numerous hemocytes infiltrate the inflamed tissue, degranulate contributing to inflammatory events and capsule formation. the tunic inflammatory cells containing phenoloxidases were identified by microscopic observations, tunic explants were assayed for the enzymatic reaction, and immunohistochemistry performed with specific antibodies. hplc purification of po extracted from the tunic supported the high molecular weight of native phenoloxidase as well as the enhancement in its activity following treatment with trypsin. for the first time a stable po form was purified from ascidians allowing further functional studies. analysis of separated fractions showed that the po activity after lps injections is due to modulation of two components different in size. the possibility exist that, as already reported in other invertebrates, the enzyme activation event was due to multiple subunits molecular association. finally, the genes for the po1, and po2, peroxinectin and cu-zn superoxide dismutase, were identified in silico, the presence in hemocytes demonstrated by pcr mrna amplification, and the reaction product sequenced. why animals invented endogenous morphine before plants? d sonetti1, b okere1, e bianchi2 1department of animal biology, university of modena and reggio emilia, modena, italy 112 2department of neurosciences, section of pharmacology, university of siena, siena, italy morphine, the most experimented alkaloid in human history, is commonly thought to derive from poppy plant, papaver somniferum. its biosynthetic pathway includes several enzymatic steps starting from tyrosine and passing through dopamine as intermediate. the presence and origin of endogenous morphine even in animal tissues is now well documented. its role as communication molecule has been mainly demonstrated in nervous and immune systems both in vertebrates and invertebrates where a similar biosynthetic pathway has been postulated. in previous studies, an evolutionary linearity was suggested indicating the origin of morphine in plants than, following the same pathway, in invertebrates in which the origin of cathecolamines might start from the intermediate dopamine with production of nor-epinephrine and finally in the vertebrates where the final step is the production of epinephrine. recently, some data indicate that morphinergic neurons should start the pathway directly from dopamine even if they do not produce catecholamines by themselves. we now hypothesize that morphinergic neurons import from extracellular space dopamine to produce morphine and preliminary data indicate the possible presence of dopamine transporters on morphinergic neurons. in the light of our results we now question the previous evolutionary hypothesis even because a look to the appearance of angiospermae (to which belongs the genus papaver) put them in a geological period in which not only the most of invertebrates were already present but also evolved vertebrate like reptiles were too. endogenous morphine, thus, was “invented” by animals as important signaling molecule and the discovering of this narcotic in plants by man and its use (and abuse) was just a secondary fact. session 3. chairman: n parrinello, university of palermo, palermo, italy invertebrate immunity: what remains to be learned about the detection and destruction of non-self aj nappi department of biology, loyola university chicago, chicago, il 60525, usa invertebrate host-parasite interactions have been the subject of numerous investigations involving physiological, biochemical, molecular, ecological, and behavioral components. the dynamic and varied competing interactions of several species of drosophila and their endoparasitic wasps have been helpful in our attempts to analyze and compare whole genome microarray data with transcriptional responses. genetic manipulations of virulent and avirulent parasitoids with both resistant and susceptible hosts have provided limited insight into the nature of the killing molecules, some understanding of the immune signaling pathways employed, and possibly some likely targets of immune suppression by invading pathogens. our inability to directly link altered gene expression with translational events that are indisputably involved in pathogen destruction or immune suppression represents an enduring challenge for future studies. cellular and biochemical responses in experimentally-stressed crabs (carcinus aestuarii): effects of bacterial challenge, leg ablation and starvation v matozzo, c gallo, mg marin department of biology, university of padua, padua, italy in the first experiment, crabs (carcinus aestuarii) were challenged in vivo with micrococcus lysodeikticus, and hemolymph was collected after 24 h. in the second experiment, crabs were subjected to leg ablation, and hemolymph was collected after 1, 3 and 7 days. in the third experiment, crabs did not receive food for 7 days, and hemolymph was then collected. total hemocyte count (thc), cell proliferation, phenoloxidase (po) activity in both hemocyte lysate (hl) and cell-free hemolymph (cfh), and cfh glucose levels were evaluated. in all the experiments, the above responses were measured at the same time in both treated and control crabs and then compared. no significant variation in thc was observed between bacteria-injected crabs and controls, whereas cell proliferation and glucose levels increased significantly in the former when compared to the latter. no significant variation in po activity was observed between bacteria-injected crabs and controls. one-day after leg ablation, significantly increased thc was found in de-clawed crabs. in declawed crabs, cell proliferation was significantly higher after 7 days, whereas glucose and cfh po activity significantly increased after 1 and 3 days. starvation caused a statistically significant increase in thc after 7 days, whereas no significant differences in cell proliferation were observed in starved and control crabs. glucose levels in hemolymph and po activity in hl significantly increased in starved organisms with respect to controls. overall, results obtained demonstrated that stress conditions induced in c. aestuarii can alter both cellular and biochemical responses investigated, suggesting differing immunomodulation pathways depending on the type of stress undergone. crayfish hemocyte type classification via sr infrared microspectroscopy s lorenzon1, pg giulianini2, a mosco2, l vaccari3 1department of biological oceanography, national institute of oceanography and experimental geophysics, trieste, italy 2department of life science, university of trieste, trieste, italy 3elettra synchrotron light laboratory, area science park, trieste, italy 113 the circulating hemocytes of crustaceans play a central role in innate immunity. three hemocyte types are commonly described in crustaceans: hyaline hemocytes (hh), small granule hemocytes (sgh), and large granule hemocytes (lgh). recently, we have identified in the freshwater crayfish astacus leptodactylus a fourth type, medium diameter granule hemocytes (mgh), that represents about 4 % of total circulating hemocytes. hh are involved in hemolymph clotting, while the granular cells contribute to the humoral immune response through the release of immune factors and phagocytosis. the traditional morphological crustacean hemocyte classification is based essentially on thresholds of granule number and size and on nucleus to cell ratio using phase contrast or brightfield light microscopy and transmission electron microscopy. in this work we used an original approach to crustacean hemocyte classification based on synchrotron radiation (sr) infrared microspectroscopy (irms). irms is an absorption spectroscopy, well established as a sensitive bioanalytical tool for the characterization of the chemical composition of the tested samples. the analysis gives new information on the spatial distribution of different cellular macromolecule constituents (proteins, lipids, nucleic acids and carbohydrates), holding also to a possible clarification of the origin (single or different cell lines) of the circulating hemocytes. a response of rhynchophorous ferrugineus (coleoptera: curculionidae) larval hemocytes to bacillus thuringiensis b manachini, v arizza, d parrinello, n parrinello department of animal biology, university of palermo, palermo, italy rhynchophorous ferrugineus olivier larvae are a pest of palm trees who are still difficult to combat with both chemical and biological control. the bacterium bacillus thuringiensis (bt) is a pathogen of many insect species and is actively used in biocontrol. little is known on the interaction of pathogens with the defense responses of these insects. insect circulating hemocytes are primarily responsible for the immune defense against parasites and pathogens. here, we report on the response of r. ferrugineus (5th-instar nymphs) circulating hemocytes following bt spore ingestion and vegetative form inoculation. in the hemolymph, plasmatocytes, granulocytes, prohemocytes, oenocytes and spherulocytes were identified. after ingestion of a sub lethal dose (lc50), of commercial product based on spores of bt, rpw larvae after bt treatment lose 30 % in weight and had a decrement in the total number of circulating hemocytes. particularly there was a reduction in the plasmatocytes which also lost their typical spindleshape. many specimens of vegetative forms of bt were found in rpw larvae fed with bt spores. the vegetative form as been reported as involved in insect septicemia process. the decrease in the hemocyte number was also found following bt (vegetative form)-inoculation. the percent plasmatocytes was drastically reduced. however further research are necessary to clarify the role of plasmatocytes in the larvae and their interaction with bt. anti biofilm activity of paracentrotus lividus coelomocytes v arizza1, d schillaci2, s molin3, n parrinello1 1department of animal biology, university of palermo, palermo, italy 2department of medicinal chemistry and technology, university of palermo, palermo, italy 3department of systems biology, technical university of denmark, lyngby, denmark defense system of marine invertebrates is based solely on the innate immune system that includes both humoral and cellular responses. antimicrobial peptides (amps) are a major component of the humoral immunity, and they display broad antimicrobial activities with remarkable specificity for prokaryotes. amps have been found to exert an antimicrobial activity also against human pathogens. they are characterized by a small size due to 10-50 amino acids provided with positive or amphipathic charges. in this work, we isolated a peptide fraction from the coelomocyte supernatant of sea-urchins paracentrotus lividus (5cc) showing a mass not exceeding 5 kda. this fraction displayed in vitro a wide spectrum of antimicrobial activity against all strains of human pathogens bacteria tested, such as staphylococcus aureus atcc 29213, atcc 25923, atcc 43866, staphylococcus epidermidis dsm3269, 1457, escherichia coli atcc 25922, pseudomonas aeruginosa atcc 9027, candida albicans atcc 10231 and candida tropicalis atcc 13813. the minimum inhibitory concentration (mic) of 5-cc varies from 253.7 to 15.8 mg/ml-1. in addition, as shown through a confocal microscope, the 5-cc also inhibited staphylococcus biofilm. to characterize the amp contained in the peptide fraction, the 5-cc was analysed with a rphplc/nesi-msms. three main peptides (parcentrin i, ii and iii) disclosing 1251.7, 2088.1 and 2292.2 daltons molecular size were identified. the msms analysis showed that these peptides include the aminoacid sequences 9-19, 12-31 and 24-41 of ß-thymosin from p. lividus (an aj439718) respectively. ß-thymosin is an antibacterial peptide contained in vertebrates platelets. research in progress will disclose the amp expression by hemocytes and tissues, moreover in vivo and in vitro modulation by micro-organisms will be examined. finally, an amp named “paracentrin” could be candidate as anti-human biofilm pathogens as well as antifouling substances. the heparin-histamine system in the phagocytic line of a tunicate: an ancient cell system equivalent to vertebrate mast cells? f cima department of biology, university of padua, padua, italy 114 in the compound ascidian botryllus schlosseri, sentinel-cells were observed in the oral siphon where they play an immunosurvellance role in the opening of the pharynx. their morphology, histochemical characteristics and ability to engulf test-particles are typical of hyaline amebocytes of the blood phagocytic line. histochemical and immunohistochemical studies at light and electron microscope reveal that, like in vertebrate mast cells, heparin and histamine co-localize inside the granules of this cell type and exposure to compound 48/80, a specific degranulating agent of vertebrate mast cells, leads to cell degranulation, suggesting that polyfunctional cells separated functions and competences among various specialized cell types of the innate immunity throughout the chordate evolution. heparin and histamine were found in the temporary “plug” of colloidal matter that closed the oral siphon after 15 min exposure to bacterial spores in seawater, resulting by degranulation of the sentinel-cells. the main physiological functions of these substances is discussed. heparin might be involved in the releasing activity of proteases, antimicrobial peptides, histamine, cytokines and growth factors. histamine might be involved in the modulation of the ciliary beat in the pharynx. in short-term branchial cultures, exogenous histamine significantly increases the ciliary beat frequency and the presence of h2 receptors was indirectly demonstrated by means of the specific antagonist ranitidine suggesting a model of clearance similar to the mucociliary transport in the vertebrate respiratory tract. inflamed adult pharynx tissues and swimming larva of ciona intestinalis share citnfαproducing cells n parrinello, a vizzini, g salerno, ma sanfratello, m cammarata, v arizza, m vazzana, d parrinello department of animal biology, university of palermo, palermo, italy in situ hybridization and immunohistochemistry analyses showed that the ciona intestinalis citnfα, previously cloned and sequenced, is both a component of the inflammatory pharynx response to lipopolisaccharide and is involved in swimming larva. specific antibodies against a citnfα peptide identified a 43 kda cell-bound form and observations of pharynx histological sections (at 4h and 8h p.i.), and in naïve and sham ascidians, disclosed the tissue response. granulocytes with large granules and compartment/morula cells were citnfα-producing inflammatory hemocytes; the vessel endothelium was also involved in the response. hemocyte nodules in the vessel lumen or associated with the endothelium showed the involvement of citnfα in producing lymphocyte-like cells in differentiating inflammatory hemocytes. finally, larva histological sections and whole mount preparations revealed that citnfα was expressed by trunk mesenchymal, preoral lobe, and tunic cells, suggesting mesenchyme immigration events and an ontogenetic role. the hemocytes of polyandrocarpa misakiensis, with particular reference to immunocytes l ballarin1, k kawamura2 1department of biology, university of padua, padua, italy 2laboratory of cellular and molecular biotechnology, faculty of science, kochi university, kochi, japan polyandrocarpa misakiensis is a polystyelid compound ascidian, common along the coasts of the temperate regions of japan, which can reproduce asexually through continuous budding from parental zooids. the hemolymph of this species contains the following main cell-types: undifferentiated cells, phagocytes, including both hyaline amebocytes and macrophage-like cells, microgranular leukocytes, macrogranular leukocytes, morula cells and pigment cells. immunocytes are represented by phagocytes and morula cells. phagocytes are capable of constitutive macropinocytosis, can release lectins and ingest macrogranular leukocytes in order to provide nutrients required for growth of developing buds. like other compound ascidians, polyandrocarpa morula cells (mcs) represent one of the most abundant circulating hemocyte types. they share the presence of dopa-oxidase activity inside their vacuoles, ascribable to the presence of phenoloxidase, a key enzyme in invertebrate immune responses, which is released in extracellular compartment upon the recognition of foreign cells or molecules thus inducing a cytotoxic response. this enzyme can be considered, in all respects, a differentiation marker of morula cells as no other cell types show similar enzyme activity. recently, we were able to clone a cdna sequence of 1985 bp, representing most of the po transcript. it shows homology with arthropod hemocyanins and, through in situ hybridization, we demonstrated that mc are the only cells expressing the corresponding mrna. natural apoptosis during the blastogenetic cycle of the colonial ascidian botryllus schlosseri f schiavon, l manni, m del favero, l ballarin department of biology, university of padua, padua, italy colonies of the compound ascidian botryllus schlosseri undergo regular generation changes during which adult zooids are progressively resorbed and replaced by growing buds. the generation change, or take-over, is characterized by massive cell death by apoptosis, changes in the expression of surface molecules by senescent cells of zooid tissues and recruitment of circulating phagocytes in zooid tissues which assure the complete clearing of the dying cells. the entire process lasts 24-36 h at 20 °c and has been subdivided, on the basis of the degree of contraction of old zooids, in four substages. it has an antero-posterior progression, at least in the digestive tube, as trace of apoptosis can be found in the epidermis, peribranchial epithelium, and 115 heart in the late take-over, whereas they are easily found in the branchial basket after 2-4 h from the beginning of the generation change. during the take-over, massive recruitment of phagocytes, which ingest senescent cells, occurs: this suggests the release of diffusible chemoattracting factors by effete cells, able to attract phagocytes toward them. we are going to investigate the expression of genes related to apoptosis, such as iap (inhibitor of apoptosis) and hsp70 during the various phases of the colonial blastogenetic cycle. session 4. chairman: u oreste cnr, institute of protein biochemistry, naples, italy the chick embryo as a hatching model for molecular biology of development and functional genomics studies v zappavigna department of animal biology, university of modena and reggio emilia, modena, italy the chick embryo has since long represented an ideal animal model for the study of development in vertebrates. it's easy accessibility for in ovo manipulation and it's relatively inexpensive handling have always represented an added bonus with respect to more conventional rodent animal models. in recent years, the possibility to perform gene expression analysis and gainand loss-of-function experiments, together with the completion of the first draft of the sequence of its genome, has made the chick one of the most versatile experimental systems available. past and novel applications of the chick embryo model will be discussed, along with possible future developments of this effective in vivo system for fundamental and biomedical research. effects of marine toxins on xenopus laevis early development a franchini, d malagoli, e ottaviani department of animal biology, university of modena and reggio emilia, modena, italy okadaic acid (oa) is a lipophilic compound produced by several marine dinoflagellates, almost exclusively accumulated in mussel digestive gland. the consumption of contaminated animals provokes a syndrome in humans known as diarrheic shellfish poisoning. palytoxin (ptx) is a large, water soluble polyalcohol found in a variety of marine organisms ranging from dinoflagellates to fish, implicated in seafood poisoning and classified as neurotoxin. our experiments were performed by using the frog embryo teratogenesis assay-xenopus (fetax) protocol, and x. laevis embryos at early gastrula stage were treated with different toxin concentrations (0.1, 1 and 10 nm for oa and 0.37, 37 and 370 nm for ptx) for 5 days. the bioassay showed that both toxins affected xenopus development in terms of mortality, delayed growth and embryo malformation. in particular significant mortality rates were observed with ptx higher dose and the initial sample population decreased by about 80 % at assay end. the observation of surviving larvae showed a marked tail folding. further histological and histochemical studies revealed disorders to the nervous system (the most sensitive tissue) and to the tail skeletal musculature, while alterations also involved the main visceral organs. molecular biology-based experiments assessed the expression of four genes (siamois, engrailed-2, bmp4, and myf5) involved in the early events of xenopus development (stages 11-47) and showed that ptx induces an increase in expression levels in all genes, while the response to oa is stagedependent, with the embryonic development stages more sensitive than the larval stages. the deregulated gene expression patterns may account for fetax and histological data. regenerative capacity in xenopus laevis tadpole tail e bertolotti, a franchini department of animal biology, university of modena and reggio emilia, modena, italy the ability to regenerate lost tissue and organs varies among animal species, tissue and life cycle stages and the nature of repair responses has been related to the dynamic, reciprocal interactions among several signalling molecules and the cell types present and activated at the wound site. amphibians, and in particular xenopus laevis, provide excellent models to examine cellular and molecular mechanisms involved in the progressive loss of regenerative potential. in this context, x. laevis tadpoles in different regenerative competence stages (st 50 and st 55) were studied after tail partial amputation. the histochemical results showed similar sequences of repair events, i.e., the epithelial wound closure, the formation of a neural ampulla and a bullet-shaped mass of cells at the cut end of the notochord surrounded by mesenchymal-like cells, but they were by means different morphological patterns. moreover differences were in a delayed full tail reconstitution in st 55, and in the extent of inflammatory responses and angiogenesis at the wound tail stumps. immunoreactivity to antibodies for critical healing immune mediators, such as inducible nitric oxide (no) synthase, was also found in a greater number of cells, mainly leukocytes, from the early healing phase in older tadpoles. on the whole, our data indicate an important involvement of the immune cells and induction of no synthesis in wound microenvironment in modulating the degree of immune responses and repair quality outcomes that may be at least partly related to the gradual decline of tail regeneration efficiency during tadpole development. bathyraja eatonii immunoglobulin heavy chains v avagliano, mr coscia, s varriale, e chianelli, e cocca, u oreste cnr, institute of protein biochemistry, naples, italy 116 chondroichthyes immunoglobulins (ig) have been extensively studied in sharks; in skates investigations remain scarce and fragmentary. only two heavy chain isotypes igm and igw (previously called igx) have been reported in leucoraja erinacea and raja eglanteria; cdna encoding the igm membrane-bound immunoglobulin heavy chain has been sequenced only in leucoraja ericacea. to focus on rajdae igs we chose a cold adapted species, bathyraja eatonii which lives in antarctic seas at sub-zero temperature. we prepared mrna from the spleen of an adult individual caught near the usa palmer station. rtpcr experiments were performed with two oligonucleotides designed on the rajdae sequences available: the first, used as sense primer, in the fr3 region of the variable domain, the second, used as anti-sense primer at the end of the ch4 domain, including the stop signal. the pcr products were analysed on agarose gel and found to be homogeniously 1400-nt long. they were cloned in the psc-a vector and 11 positive clones were sequenced. the sequences shared on average 97.3 % nucleotide identity. by shannon entropy analysis, the highest position diversity was found in the cdr3 region, whose length varied between 8 and 12 amino acid residues. a distance tree was built by the neighbor-joining method and two indipendent branches, defining two igm subisotypes, were obtained. based on comparison of the b. eatonii amino acid sequence with that of l. erinacea we found one additional cystein residue in the ch3 domain and same number of glycosilation sites. evidence for local production of immunoglobulins in the skin of antarctic teleosts e chianelli1, c motta2, s giacomelli3, mr coscia1, s varriale1,4, v avagliano1, u oreste1 1cnr, institute of protein biochemistry, naples, italy 2department of evolutive and comparative biology, university of naples “federico ii”, naples, italy 3zoological station “anton dohrn”, naples, italy 4department of chemistry, university of naples “federico ii”, naples, italy the skin of teleost fishes has an important immunological role against pathogens, similarly to other tissues such as the gastrointestinal, respiratory, and genitourinary tracts. it has been reported that skin and gut mucus of teleost fishes contain tetrameric igm, that are produced independently of the systemic antibodies, thus indicating the existence of a mucosal immune system in teleosts. secretory immunoglobulins (ig) similar in size to serum ig, have been purified from the skin mucus of the antarctic teleost trematomus bernacchii. lymphocytes that produce ig have been localized in the skin tissue by immunohistochemistry, using specific antisera, and by in situ hybridization. skin sections were analyzed to verify the expression and synthesis of igm and localize the cells involved. by in situ hybridization using an antisense probe, the expression of both l and h chain genes was demonstrated in occasional lymphocytes located close to the basal membrane as well as dispersed in the superficial stratum. by contrast, filamentous cells and goblets cells showed no specific labelling. no signal was detected in any of the above mentioned cells when hybridization was carried out using a sense probe. moreover, rt-pcr experiments performed using specific primers revealed the presence of mature transcripts encoding the secretory ig in the skin of t. bernacchii and that of the membranebound form in the case of chionodraco hamatus. the ζ-ζ cd3 component of the tcr complex in lipid bilayers s varriale1,2, a merlino2, v avagliano1, mr coscia1, e chianelli1, l mazzarella2, u oreste1 1cnr, institute of protein biochemistry, naples , italy 2department of chemistry, university of naples "federico ii", naples, italy the human t-lymphocytes receptor complex consists of the antigenic peptide binding subunit, the heterodimer α-β, and the transducing subunit cd3, the latter resulting from the assembly of three dimers: γ-δ, γ-ε and ζ-ζ. while the structure of the extracellular portions of α-β, γ-δ and γ-ε has been determined, that of the regions traversing the cell membrane (tm) are unresolved with the exception of ζ-ζ. in fact by nmr spectroscopy in water, the structure of ζ-ζ tm region has been recently proposed. the present work has been aimed at analysing the human homodimer tm structure in lipid bilayers by molecular dynamic simulations. we considered two types of lipid bilayer models: the palmitoyl-oleoyl-phosphatidylcholine (popc) which better resembles the cell membrane in lipid composition, and the popc/cholesterol/palmitoyl-sphyngomyelin (1:1:1) which resembles the raft membrane microdomains, thought to be the sites of the signal transducing machinery. the simulations were performed using the gromacs package for a total time longer than 25 ns. each simulation showed the formation of a stable homodimer with high conservation of the secondary structure. the model in raft had an ahelix structure more extended (for each chain: 6.317.0 å in raft and 12.3-27.4 å in popc); a more compact packing (the distance between the centers of mass of the two helices was 7.9 å in raft and 9.7 å in popc). our results suggest that during the translocation of the tcr complex in the raft membrane domains, the ζ-ζ dimer assumes a specific conformation probably necessary to the correct signal transduction. molecular and structural characterisation of a macrophage migration inhibitory factor from sea bass (dicentrarchus labrax) e randelli1, f buonocore1, am facchiano2 , a pallavicini3, m modonut3, g scapigliati1 117 1department of environmental sciences, tuscia university, viterbo, italy 2cnr, institute of food sciences, avellino, italy 3department of life science, university of trieste, trieste, italy the macrophage migration inhibitory factor (mif) is a cytokine mainly produced by t lymphocytes and macrophages in response to inflammatory stimuli. mif is also implicated in a wide range of biological activities, related to hormone-like and enzymatic functions. we report here the identification, from a thymus cdna library, of a cdna encoding a mif molecule from sea bass (dicentrarchus labrax), the transcription levels, and the 3d structure. sea bass mif cdna consists of 609 bp and encodes for a putative protein of 115 amino acids. real time pcr analyses revealed that mif is constitutively expressed in various tissues and organs, with the highest mrna level observed in thymus. mif expression was induced after 4 hours in vitro stimulation of hk leukocytes with lps and decreased after 24 h. the predicted 3d model of sea bass mif is comparable with human model and has been used to verify the presence of structural requirements for its known biological activities in mammals. our results will raise the possibility of investigating more in detail the involvement of mif in sea bass immune responses and add new insight on the evolution and biological activities of this important cytokine present both in vertebrates and invertebrates. immunomodulatory effects of aloe arborescens ethanolic extract on saf-1 cell line c bernini1, c belardinelli1, e ovidi1, d ceccarelli1, f buonocore1, l abelli2, g scapigliati1, am fausto1, s picchietti1 1department of environmental sciences, tuscia university, viterbo, italy 2department of biology and evolution, section comparative anatomy, ferrara university, ferrara, italy modulation of cytokine secretion may offer novel approaches in the treatment of a variety of fish diseases. the use of herbal extracts can represent a new strategy for the modulation of cytokine expression. deeply studied cytokines are interferons (ifns), which may induce vertebrate cells into an antiviral state. the in vitro research performed in this study demonstrated that an ethanolic extract (1.2 mg/ml) of aloe arborescens, a plant that is widely used in korea as ingredient of health food and cosmetics, significantly increased after 48 h of treatment the expression level of the ifn type i in saf-1 (sparus aurata fibroblast cell line) cells stimulated with poly i:c. moreover, the treatment significantly upregulated (after 48 h) the expression levels of mhc class i-α and mx, a protein endowed with antiviral properties, compared with control cells. this work provides a new perspective for the use of medicinal plants in fish to prevent viral diseases. further studies are in progress to characterize the active principles of the plant extract and to establish feeding protocols for food additives. 118 a franchini, d malagoli, e ottaviani isj 6: 1-6, 2009 isj 6: 15-20, 2009 issn 1824-307x research report presence of a low molecular weight lectin in the coelomic fluid of the sea urchin paracentrotus lividus f dragoa, d malagolib, fm pezzinoc, v d’ursoa, f sammartanoa adepartment of animal biology, university of catania, catania, italy bdepartment of animal biology, university of modena and reggio emilia, modena, italy cdepartment of biomedical sciences, university of catania, catania, italy accepted february 1, 2009 abstract a low molecular weight (mw) lectin (paracentrotus lividus small lectin, plsl) has been found in the sea urchin, paracentrotus lividus. after gel electrophoresis under denaturing conditions, plsl exhibits a mw of 13 kda, while its hemagglutinating activity is ca2+-independent and inhibited by dglucose, l-rhamnose, d-arabinose, l-fucose and n-acetyl-d-glucosamine. electrophoretic analysis of the coleomic fluid of p. lividus reveals that the presence of plsl increases following immune challenge with bacteria, whereas it is annulled as a consequence of osmotic stress. interestingly, two other putative inducible hemagglutinins of an approximate mw of 11 and 32 kda were retrieved in concomitance with the stress-promoted disappearance of plsl. key words: paracentrotus lividus; sea urchin; lectin; immunity; stress introduction lectins are carbohydrate binding proteins other than immunoglobulins that display no enzymatic activity towards the recognized sugars (loris, 2002). lectins have been retrieved in almost all forms of life, and animal lectins, though fulfilling a variety of functions, have been implicated in defense against pathogens, immune regulation and prevention of autoimmunity (kilpatrick, 2002). in marine invertebrates, lectins have been thought to participate in immune response by inducing bacterial agglutination or by acting as opsonins to enhanced phagocytosis by coelomocytes (yui and bayne, 1983; bayne, 1990). an extensive discussion regarding the physiological significance of lectins in both vertebrates and invertebrates has been reported (turner, 1994; kilpatrick, 2002). in the case of invertebrate humoral lectins, it has been postulated more than twenty years ago that one of their physiological functions is distinction of self from non-self in cooperation with cellular and humoral defense processes (renwrantz, 1986). a large number of lectins has been isolated and characterized from deuterostomian and protostomian ___________________________________________________________________________ corresponding author: francesca sammartano department of animal biology university of catania via androne 81, 95124 catania, italy e-mail: sammarta@unict.it invertebrates, such as tunicates (vasta et al., 1986; belogortseva et al., 1998), insects (racliffe et al., 1985; murdock and shade, 2002), crustaceans (beisel et al., 1999) and molluscs (racliffe et al., 1985; bulgakov, 2004; ottaviani, 2006). as far as the presence of lectins in echinoderms is concerned, lectins have been found in the sea urchins anthocidaris crassispina (giga et al., 1985, 1987) and paracentrotus lividus (canicatti et al., 1992). in the latter species, the reported authors described the occurrence of a natural c-type lectin with a molecular weight (mw) of more than 200 kda in the coelomic fluid. in the present study, a low mw lectin was purified from the coelomic fluid of the sea urchin p. lividus by ion-exchange chromatography on deaesephadex. this molecule has a mw of 13 kda and displays a ca2+-independent hemagglutinating activity. materials and methods animals adult specimens of the sea urchin paracentrotus lividus lamarck 1816 (echinodermata) were collected along the coast of catania (sicily, italy) and transferred to the laboratory in aerated tanks with sea water (salinity: 35 psu, temperature range: 15°-18 °c) and immediately utilized as controls or for either bacterial challenge or osmotic-stress. 15 bacteria injection escherichia coli (tb1) (invitrogen) was reconstituted in lb broth (luria-bertani) (sambrook, 1989) and grown overnight under constant agitation at room temperature (rt) as indicated by the supplier. the bacteria density in lb was quantified at the spectrophotometer by evaluating the optical absorbance at 550 nm. a 500 μl suspension of e. coli (3x106 living bacteria/ml) was injected into the perivisceral coelom through the peristomal membrane. the coelomic fluid taken from each specimen was examined 3 h and 24 h after injection. in order to discriminate between the effects of the injection and that of the bacterial challenge, controls for these experiments were performed by injecting an identical volume of sterile lb broth. osmotic stress the sea urchins were placed in aquaria containing sea water mixed with distilled water (70:30) (salinity 24.5 psu). sea water was wellaerated and maintained at a temperature varying between 15 °c and 18 °c. the sea urchins were collected 3 (short exposure) and 12 h (long exposure) after osmotic stress. the health of sea urchins was checked visually by monitoring if they tended to crawl up on the sides of the aquaria, as unhealthy animals usually remain on the bottom. furthermore, healthy animals eat algal food avidly. coelomic fluid collection the coelomic fluid was obtained from untreated, bacterial-challenged and osmoticstressed animals by cutting a slit into the peristomal membrane and allowed to clot for 1 h at 0 °c and clotted proteins were discarded. the resulting supernatant (serum) was dialyzed against 50 mm tris-hcl (ph 7.5) and stored at 0 °c or at -80 °c. purification of hemagglutinin the hemagglutinin was purified according to giga (1985), with minor modifications. briefly, the dialyzed serum was applied to a deae-sephadex (sigma, st. louis, mo, usa) column equilibrated with 50 mm tris-hcl (ph 7.5). the column was washed with a serially increasing concentration of nacl (0.5 m, 0.6 m and 0.7 m) diluted in 50 mm tris-hcl (ph 7.5). protein concentration was determined in all fractions according to the bca protein assay reagent (bicinchoninic acid) (pierce biotechnology, rockford, ll, usa) using bovine serum albumin (bsa, ranging from 0.003 mg/ml to 0.125 mg/ml) as a standard with both spectrophotometry nd1000 (nanodrop technologies, usa) and microplate reader opsys mr™ (thermolab system franklin,na, usa). after protein measurement, the hemagglutinating activity (ha) against human erythrocytes was analyzed in all fractions. only those fractions displaying ha were retained for the experiments described below. gel electrophoresis density-gradient sds-page electrophoresis (4-12 %) was carried out in denaturing conditions according to laemmli (1970). before loading, the protein sample was denatured at 100 °c for 2 min in a solution of 1 % sds laemmli buffer. after protein separation (approximately 10 μg/lane), the gel was silver-stained following, with slight modification, the protocol of mortz et al. (2001). the multimark-multicolored standard (1x) (invitrogen, carlsbad, ca, usa) was used as standard mw. assay of ha with whole and trypsinized erythrocytes ha assay of the dialyzed serum was performed as indicated by giga et al. (1985). samples of human blood groups a, b and 0, were washed three times by centrifugation at 500xg for 10 min in tris buffered saline (tbs: 50 mm tris-hcl, 150 mm nacl, fraction number (1 ml/tube) fig. 1 pattern of column chromatography of the whole coelomic fluid of p. lividus, displaying the fractions (from fraction 2 to fraction 18) with hemagglutinating activity. 16 table 1 effect of different sugars on hemagglutining activity of p. lividus hemagglutinin sugar 50 mm 100 mm 200 mm d-glucose + sucrose d-lactose d-melibiose l-rhamnose ++ d-sorbitol d-arabinose ++ l-fucose + d-mannose d-galactose n-acetyl-d-galactosamine n-acetyl-d-glucosamine + +, partial inhibition of the hemagglutinating activity ++, total inhibition of the hemagglutinating activity -, no hemagglutination ph 8.0). ha assays were carried out on u-bottom microtiter plates (corning incorporated, corning, ny, usa). serial dilutions (from 1:2 to 1:10) of the serum were made in 25 µl of test buffer (50 mm tris-hcl, 150 mm nacl and 1 mm cacl2, ph 7.5). twenty-five μl of a suspension of 3 % human erythrocytes in test buffer were then added to each well. after gently mixing, the microplates were allowed to stand for 1 h at rt and macroscopic agglutination was recorded. the maximum dilution causing hemagglutination was referred to as the hemagglutination titer. the ha assays were also performed with trypsinized erythrocytes, since it has been reported that proteolytic treatment increases the agglutinating activity. the removal of surface proteins gives the lectins greater accessibility to the receptors (kapáček et al., 1993). the preparation of the erythrocytes followed the same procedure described above, but before tests, the red blood cells were incubated with 0.1 mg/ml trypsin in tbs for 30 min at 37 °c and then washed three times in tbs. hemagglutination inhibition assay to assess their effects on the agglutination of trypsinized erythrocytes, the following sugars were assayed: d-sorbitol, d-glucose, d-galactose, dmannose, n-acetyl-d-glucosamine, n-acetyl-dgalactosamine, d-galactose, l-fucose, sucrose, d-lactose, d-melibiose, l-rhamnose and darabinose. all sugars were purchased from sigma (except l-fucose and n-acetyl-d-glucosamine, which was obtained from united states biochemical corporation, cleveland, oh, usa). sugars were added to tbs to yield 0.4 m storage solutions. a total of 25 µl of hemagglutinating serum fractions were then added to an equal volume of various dilutions (50 mm, 100 mm, 200 mm) of carbohydrates in the wells of u-bottomed microtiter plates and incubated for 30 min at 37 °c. erythrocyte suspension was then added, and after a further 60 min of incubation at 37 °c, the lowest carbohydrate concentrations able to inhibit agglutination were recorded. the dependence of agglutinating activity on divalent cations was assessed by performing the hemagglutination assay in the presence of 1 and 2 mm egta or by adding 10 mm cacl2. results purification of hemagglutinin and evaluation of its ha the different eluate fractions collected were tested with erythrocytes to highlight their agglutinatining capacity (fig. 1). the ha of coelomic fluid from p. lividus was assayed against human erytrocytes a, b and 0, and no differences referable to the human blood groups were observed. the analysis of the protein content of the agglutinating fractions by means of sds-page revealed that ha was invariably related to the presence of a protein with an electrophoretic mobility corresponding to a mw of 13 kda (fig. 2). effect of divalent cations on ha experiments performed with trypsinized erythrocytes treated with egta and mixed with hemagglutinating fractions demonstrated that egta cannot influence ha activity. moreover, the addition of ca2+ to the serum did not affect the hemagglutination. effect of sugars on ha ha against trypsinized human erythrocytes was inhibited by the presence of l-rhamnose and darabinose. also d-glucose, l-fucose and n-acetyld-glucosamine showed weak inhibition. conversely, the oligosaccharides, sucrose, dlactose, d-melibiose, d-sorbitol, d-mannose, nacetyl-d-galactosamine and d-galactose, did not inhibit the ha (table 1). 17 fig. 2 sds-page of hemagglutinating fractions. lane b) lectin in controls; lane c) 3 h after bacterial injection; lane d) 12 h after injection. lane a) mw marker. bands observed in treated sea urchins and not retrievable in controls were the followings: a= 34 kda; b= 32 kda; c= 21 kda; d= 18 kda; e= 16 kda; f= 29 kda; g= 11 kda. effect of bacterial injection three h after the injection of the bacterial suspension, sds-page showed a band at 13 kda of higher intensity than controls (fig. 2). in the same agglutinating fractions, bands at approximately 16, 18, 21, 32 and 34 kda were also evident, although with a lower intensity than the 13 kda band. surprisingly, 12 h after the injection, the band at 13 kda was no longer visible, while the samples maintained the ha and displayed bands at 11, 29, 32 and 34 kda (fig. 2). osmotic stress after a short exposure (3 h) to osmotic stress, all the hemagglutinating fractions showed six bands at a mw of approximately 11, 14, 26, 28 and 32 kda after sds-page. the highest intensity was seen at bands 11, 14 and 32 kda (fig. 3). it should be noted that the 13 kda band typical of unstressed samples was not present in the hemagglutinating fractions. also, after long exposure (12 h) to osmotic stress, the sds-page analysis of the hemagglutinating fraction revealed the absence of the 13 kda band retrieved in the controls. on the other hand, two bands of approximately 11 and 32 kda were visible. discussion the present investigation shows that in the coelomic fluid of the sea urchin p. lividus a 13 kda band is present in all the hemagglutinating fractions. this observation let us to hypothesize that the band corresponds to a lectin able to agglutinate human red blood cells. the carbohydrate recognition domain of the protein can bind d-glucose, lrhamnose, d-arabinose, l-fucose and n-acetyl-dglucosamine that, in turn, are able to inhibit hemagglutination. it is well-known that erythroagglutination by hemagglutinins is generally inhibited by monosaccarides, which presumably are part of or closely related to saccaride receptor sites on the erythrocyte surface (giga et al., 1985). we named the newly discovered lectin plsl (paracentrotus lividus small lectin). the proposed name is based on the observation that plsl is smaller than the other lectin found in p. lividus, which has a mw of more than 200 kda (canicattì et al., 1992). such a difference in the molecular size of lectins belonging to the same organism is not new, at least not in echinoderms. indeed, in the sea cucumber, stichopus japonicus, lectins of 400, 60, 15 and 13 kda have been reported (hatakeyama et al., 1993; matsui et al., 1994). four lectins with an estimated mw of 27, 35, 45 and 68 kda have been retrieved in cucumaria echinata (hatakeyama et al., 1994), whereas only one 44 kda lectin was found in cucumaria japonica (bulgakov et al., 2000). in other echinoderms, lectins with a mw ranging from 220 kda in the sea cucumber holothuria polii (canicattì and rizzo, 1991) and starfish asterina pectibifera (kamiya et al., 1992) to 182 kda in the sea cucumber holothuria scabra (gowda et al., 2008), have been recorded. on the whole, these findings indicate that the echinodermal lectins are a very heterogeneous group, at least with regards molecular size, and therefore deserve a more detailed biochemical characterization. while the ha of plsl is ca2+-independent, most echinodermal lectins are ca2+-dependent (hatakeyama et al., 1993, 1994; himeshima et al., 1994; matsui et al., 1994; hatakeyama et al., 1995; bulgakov et al., 2000), and this is also true for the other lectin found in p. lividus (canicattì et al., 1992). interestingly, from a. crassispina eggs it has been identified a ca2+-independent lectin named suel, whose mw is 11.5 kda and 23 kda under reducing and non-reducing conditions, respectively (ozeki et al., 1991). suel is suggested to be important for sea urchin development, but at present no definitive conclusions have been reported in this sense (ozeki et al., 1995). fig. 3 sds-page of hemagglutinating fractions. lane b) lectin in controls; lane c) 3 h after osmotic stress; lane d) 12 h after osmotic stress. lane a) mw marker. bands observed in treated sea urchins and not retrievable in controls were the followings: a= 32 kda; b= 28 kda; c= 26 kda; d= 16 kda; e= 14 kda; f= 11 kda. 18 as far as the role of plsl as a humoral component of the immune system of p. lividus is concerned, our data show the increase of the density of the13 kda band 3 h after the injection with e. coli. unexpectedly, plsl is no longer visible 12 h after the injection, even if the collected fractions maintain their hemagglutinating capacity. similarly, in the osmotic stress assays, plsl is no longer visible either 3 or 12 h after injection, but the coelomic fluid of the sea urchin continues to agglutinate red blood cells. an increased ha has been observed as a consequence of bacterial challenge in the sea cucumber h. scabra, and it has been suggested that this finding is related to an increased expression in lectins (gowda et al., 2008). moreover, a relationship between lectins and bacterial clearance has been proposed (gowda et al., 2008), indicating that data collected at different time points after injection of bacteria could present some discrepancies. an interesting outcome of our experiments is that even if the band corresponding to plsl is no longer visible under most of the stressful conditions tested, the ha of coleomic fluid is maintained. when comparing the electrophoretic profiles of hemagglutinating fractions collected 12 h after bacterial injection or osmotic stress, it seems reasonable to impute the ha to the 11 and/or 32 kda bands. further studies are needed to verify if these proteins correspond to two new lectins, with slower synthesis, especially because it cannot be excluded that the 11 and 32 kda bands represent post-traslational modifications in plsl. acknoledgements the authors wish to thank prof e ottaviani (university of modena and reggio emilia) for the helpful indications given during the course of this study and prof l ballarin (university of padua) for the suggestions related to sugar tests. references bayne cj. phagocytosis and non-self recognition in invertebrates. bioscience 40: 723-731, 1990. beisel hg, kawabata si, iwanaga s, huber r, bode w. tachylectin-2: crystal structure of a specific glcnac/galnac-binding lectin involved in the innate immunity host defense of the japanese horseshoe crab tachypleus tridentatus. embo j. 18: 2313-2322, 1999. belogortseva ni, molchanova vi, kurika av, skobun as, glazkova ve. isolation and characterization of new galnac/gal specific lectin from the sea mussel crenomytilus grayanus. comp. biochem. physiol. 119c: 4550, 1998. bulgakov aa, park k, choi k, 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laboratory press, cold spring harbor, ny, 1989. turner rj. immunology: a comparative approach. john wiles & sons ltd, chichester, england, 1994. vasta gr, marchalonis jj, decker jm. binding and mitogenic properties of a galactosyl-specific lectin from the tunicate didemnum candidum for murine thymocytes and splenocytes. j. immunol. 137: 3216-3233, 1986. yui m, bayne c. echinoderm immunology: bacterial clearance by the sea urchin strongylocentrotus purpuratus. biol. bull. 165: 473-85, 1983. 20 review isj 8: 59-69, 2011 issn 1824-307x review regulation of the innate immune responses in the silkworm, bombyx mori h tanaka1, m yamakawa2, 3 1insect mimetics research unit, national institute of agrobiological sciences, 1-2 owashi, tsukuba, ibaraki 305-8634, japan 2division of insect sciences, national institute of agrobiological sciences, 1-2 owashi, tsukuba, ibaraki 305-8634, japan 3graduate school of life and environmental sciences, university of tsukuba, tsukuba, ibaraki 305-8572, japan accepted april 18, 2011 abstract insects possess an effective innate immune system against foreign microorganisms. innate immunity of insects is divided into two major reaction types: humoral and cellular reactions. humoral reactions involve soluble proteins in the hemolymph such as phenoloxidase, antimicrobial proteins (amps), lysozymes, and lectins, whereas hemocytes mediate cellular reactions such as phagocytosis, encapsulation and nodule formation. in bombyx mori, six different families of amps have been identified: cecropin, attacin, lebocin, moricin, gloverin, and defensin. one lysozyme and three lysozyme-like proteins, one of which is involved in elimination of invading pathogens, are also found in the silkworm. both lysine-containing peptidoglycan (lys-pgn) and meso-diaminopimelic acid containing peptidoglycan (dap-pgn) trigger expression of amp genes, probably through the toll and imd pathways, respectively. dap-pgn has stronger elicitor activity than lys-pgn in b. mori because of the difference in transcriptional activity between bmrelishes and bmrels, which are effectors of the imd and toll pathways, respectively. furthermore, two recognition proteins and a serine protease are involved in activation of prophenoloxidase for melanization, and several c-type lectins, which participated in cellular reactions, were identified in b. mori. moreover, a paralytic peptide was reported to play important roles in silkworm immunity. recent development of transgenic technologies and silkworm genome information are expected to accelerate silkworm immunity studies. key words: innate immunity; bombyx mori; antimicrobial peptides; melanization; cellular immunity; paralytic peptide introduction insects first appeared on the earth approximately 350 400 million years ago; today, they account for more than 70 % of the animal species on the earth (mayhew, 2007). evolution of an effective innate immune system is one reason for the prosperity of insects on the earth. they have a primitive innate immunity system, but lack acquired immunity for antibody production or immunological memory similar to that present in vertebrates (brennan and anderson, 2004; pinheiro and ellar, 2006; lemaitre and hoffmann, 2007). insects have developed this powerful innate immune system against invading microorganisms during their evolution. however, they belong to diverse families ___________________________________________________________________________ corresponding author: hiromitsu tanaka insect mimetics research unit national institute of agrobiological sciences 1-2 owashi, tsukuba, ibaraki 305-8634, japan e-mail: htanaka1@affrc.go.jp with different life cycles and feeding methods. thus, recognition and elimination systems against invading pathogens may also vary among them. at present, insect immunity studies using the common vinegar fly, drosophila melanogaster, are the most advanced, and a large amount of data has been accumulated about drosophila immunity (brennan and anderson, 2004; cherry and silverman, 2006; ferrandon et al., 2007; lemaitre and hoffmann, 2007; pal and wu, 2009). however, data from d. melanogaster may not always be applicable to other insect species owing to their diversity. the silkworm bombyx mori has been domesticated for sericulture in the past 5,000 years. from the beginning of the nineteenth century, the silkworm has been used for basic science studies, such as genetics, physiology, and pathology, because of its large body size, its importance in sericulture, easy rearing, and a large number of described mutants (willis, et al., 1995). now, the technology to construct a transgenic silkworm has 59 been developed for use in functional analysis of unknown gene products and to produce recombinant proteins (yamao et al., 1999; tamura et al., 2000; tomita et al., 2003). furthermore, b. mori is the first lepidopteran insect to have an almost complete genome sequence documented (the international silkworm genome consortium, 2008). therefore, the silkworm is very useful for scientific studies as a model for lepidoptera, which includes the most disruptive agricultural pests. by the 1980s, studies using b. mori also led to the insect immunity research, such as analysis of the activation mechanism for melanization, and analysis of the morphology of hemocytes in response to invading pathogens. although silkworm immunity studies, particularly on the molecular level, are not as advanced as those of the common vinegar fly and mosquitoes, increasing studies on silkworm immunity using silkworm genome information and transgenic technology are being published. in addition, silkworm immunity mechanisms different from those of d. melanogaster have been reported. in this article, we will review research on the immune responses of b. mori. some excellent review articles are available on innate immunity of the lepidopteran, manduca sexta (kanost et al., 2004; ragan et al., 2009; kanost et al., 2009). humoral immunity and regulation of its response insect immunity is divided into two major response types: humoral and cellular defense responses. humoral responses involve phenoloxidase (po) and immune proteins such as antimicrobial proteins (amps), lysozyme, and lectins. in response to microbial infection, amps and lysozyme are rapidly produced primarily in the fat body (fb) and hemocytes, and subsequently secreted into the hemolymph to eliminate invading pathogens. in addition, melanization induced by po, and lectins are implicated in segregation of invaded microorganisms in the hemolymph. antimicrobial proteins and lysozyme in b. mori, six different families of amps have been identified: cecropin (morishima et al., 1990; taniai et al., 1992; kato et al., 1993; yamano et al., 1994; kim et al., 1998; yang et al., 1999; cheng et al., 2006; hong et al., 2008), attacin (sugiyama et al., 1995), lebocin (hara and yamakawa, 1995a; chowdhury et al., 1995; furukawa et al., 1997), moricin (hara and yamakawa, 1995b; furukawa et al., 1999), gloverin (cheng et al., 2006; kaneko et al., 2007; kawaoka et al., 2008), and defensin (kaneko et al., 2008; wen et al., 2009) (table 1). cecropins, which were originally isolated from the giant moth hyalophora cecropia (steiner et al., 1981), consist of approximately 40 amino acids, and kill both gram-positive and -negative bacteria by forming ion channels in the bacterial membrane (christensen et al., 1988). they were discovered as a family of 13 genes including two cecropin a, six cecropin b, one cecropin c, one cecropin d, one cecropin e, and two enbocin; eleven of these genes form a cluster on the same chromosome (tanaka et al., 2008). attacin is a glycine-rich protein with a molecular mass of approximately 20 kda (sugiyama et al., 1995). in escherichia coli, treatment with attacin from h. cecropia led to specific inhibition of the synthesis of several outer membrane proteins, such as ompc, ompf, ompa, and lamb, and subsequent elevation of its outer membrane permeability (carlsson et al., 1991). therefore, bombyx attacin also appears to suppress the growth of gram-negative bacteria by inhibiting the production of the bacterial outer membrane proteins. silkworms possess two copies of attacin, located in tandem on chromosome 6 (tanaka et al., 2008). lebocin, a proline-rich glycosylated peptide, consists of 32 amino acid residues. glycosylation of threonine 15 with n-acetylgalactosamine and galactose or solely with n-acetylgalactosamine is involved in its antibacterial activity (hara and yamakawa, 1995a). moricin is a highly basic peptide 60 consisting of 42 amino acid residues; it shows antibacterial activity against both gram-positive and -negative bacteria by attacking the bacterial membrane (hara and yamakawa, 1995b). lebocin and moricin genes are present as single genes (tanaka et al., 2008). gloverin is a glycine-rich protein. b. mori has 4 different isoforms named gloverin 1-4 (kaneko et al., 2007; kawaoka et al., 2008). all gloverins are reported to exhibit antibacterial activity against gram-negative bacteria by using the recombinant proteins, even though their activity was weak (kawaoka et al., 2008). defensins are basic peptides consisting of 30 40 amino acids with six cysteine residues to form three pairs of disulfide bonds. in b. mori, two defensins, designated defensin a and defensin b, have been identified, even though their antimicrobial activity has not yet been confirmed (kaneko et al., 2008; wen et al., 2009). lysozyme catalyzes the cleavage of the β-1,4-glycosidic linkage between the n-acetylglucosamine and n-acetylmuramic acids of peptidoglycan (pgn), a major component of the bacterial cell wall. a lysozyme from b. mori shows higher antimicrobial activity against gram-positive bacteria than against gram-negative bacteria similar to that of the lysozyme from other organisms (lee and brey, 1995). recently, three lysozyme-like proteins (llps) were reported to exist in b. mori (gandhe et al., 2007) (table 1). all of llps lack catalytic amino acid residues for muramidase activity, but llp1 inhibits the growth of e. coli and micrococcus luteus, probably by an unknown mechanism different from that of lysozyme (gandhe et al., 2007). the function of other two llps has not yet been elucidated. amp genes are rapidly expressed in response to microbial infection in specific tissues, mainly in fb and hemocytes but not in the posterior silk gland (psg) (yamano et al., 1994; sugiyama et al., 1995; furukawa et al., 1997; kaneko et al., 2007). however, the transcription of amp genes was observed in an in vitro transcription system, using not only fb nuclear extract but also psg nuclear extract (tanaka et al., 2005a). comparison of nucleosomal structure near the promoter regions of amp genes indicated clear nucleosomal arrangements in the psg but not in the fb, suggesting that regulation of tissue-specific expression of amp genes occurs at least at the chromatin level (tanaka et al., 2005a). the induction mechanism of amp genes has been extensively studied in d. melanogaster, and two distinct pathways, toll and immune deficiency (imd) pathways, play important roles in the activation of amp genes (pinheiro and ellar, 2006; ferrandon et al., 2007; lemaitre and hoffmann, 2007; aggarwal and silverman, 2008; valanne et al., 2011). the toll pathway is known to be stimulated by lysine-containing pgn (lys-type pgn), a cell wall component of many gram-positive bacteria with the complex of pgn recognition protein sa (pgrp-sa) and pgrp-sd, and gram-negative binding protein 1 (gnbp1) (govert et al., 2003; pili-floury et al., 2004; bischoff et al., 2004). this recognition activates the extra-cellular serine protease signaling cascade, and leads to cleavage of pro-spätzle. the binding of spätzle to the receptor toll leads to the translocation of nf-κb transcription factors, dif and dorsal, into the nuclei to activate target amp genes such as drosomycin. imd pathway is stimulated by meso-diaminopimelic acid containing pgn (dap-type pgn) from gram-negative bacteria and subclass of gram-positive bacteria such as the bacillus species, along with other pgn recognition proteins, pgrp-lc and pgrp-le (gottar et al., 2002; rämet et al., 2002; takehana et al., 2002; kaneko et al., 2005). initiation of the intracellular signal transduction cascade by pgrp-lc results in the cleavage of an nf-κb factor, relish, and subsequent activation of expression of amp genes such as diptericin. in contrast, the signal transduction pathways involved in the up-regulation of amp genes have not been well studied in b. mori. in addition, recognition factors that trigger expression of amp genes have not yet been identified. recently, comparative genome analysis indicated that the silkworm also possesses both toll and imd pathways, since almost all orthologous genes encoding these intracellular components exist in the silkworm genome (tanaka et al., 2008) (fig. 1). however, 1:1 orthologous genes of drosophila pgrps involved in the recognition of pgns and subsequent activation of both the pathways were not found in the silkworm genome by bootstrap analysis, although twelve silkworm pgrp genes were identified (tanaka et al., 2008). we neither identified the 1:1 orthologous genes of serine proteases involved in extra-cellular toll signaling such as grass, spirit, and persephone (tanaka et al., 2008). these data suggest that the recognition factors and serine proteases that activate intracellular toll signaling are not well evolutionally conserved between b. mori and d. melanogaster. although 13 or 14 genes have been identified, the toll receptor involved in the toll pathway in b. mori has not yet been identified (cheng et al., 2008; tanaka et al., 2008). bootstrap analysis also failed to identify an orthologous gene of drosophila toll in b. mori. however, bmtoll9-1 is speculated to activate the toll pathway because drosophila toll9, an ortholog of bmtoll9-1, is known to constitutively activate the toll pathway. in fact, the expression of bmtoll9-1 is elevated in response to bacterial and fungal infection (wu et al., 2010). furthermore, bombyx mori spätzle-1 (bmspz-1), a putative ligand for the toll receptor, was also cloned in b. mori (wang et al., 2007). the bmspz-1 generated by proteolytic processing of its precursor, pro-bmspz-1 up-regulates transcription of amp genes, suggesting that bmspz-1 has a similar function as the drosophila spätzle. we found that dap-type pgn induces expression of amp genes more strongly than lys-type pgn in the fb of silkworm larvae (tanaka et al., 2009b). we further confirmed that bacteria possessing dap-type pgn elicited stronger expression of amp genes than those bacteria that possess lys-type pgn; this suggests that the apparent bacterial difference in the influence on the induction levels of these genes depends on the type of pgn (tanaka et al., 2009b). transgenic knockdown analysis in silkworm showed that the dorsal and relish orthologs, bmrels and bmrelishes, respectively, participated in the activation 61 fig. 1 factors involved in toll and imd pathway in d. melanogaster, and b. mori orthologs corresponding to these factors. the factors represented by red letters indicate that b. mori also has 1:1 ortholog of genes encoding these factors. the factors represented by black letters indicate that b. mori appears to not have 1:1 ortholog of them. black boxes indicate proteins involved in recognition, red boxes indicate proteins involved in the extra-cellular toll pathway, blue boxes indicate transcription factors, and yellow boxes indicate proteins involved in the intracellular pathways. spirit does not appear to cleave spe directly, although it is located up-stream of spz. psh and grass also do not appear to cleave spirit directly. pgrp; peptidoglycan recognition protein, gnbp; gram-negative bacteria-binding protein, nec; necrotic, psh; persephone, spe; spätzle-precessing enzyme, spz; spätzle, ecsit; evolutionarily conserved intermediate in toll pathways, traf2; tumor necrosis factor receptor-associated factor 2, fadd; fas associated protein with death domain, iap2; inhibitor of apoptosis 2, tak1; transforming growth factor-activated kinase 1, tab2; tak1 binding protein 2, ikkβ; iκb kinase β, ikkγ; iκb kinase γ, hem; hemipterous, jnk; c-jun nh2-terminal kinase. of expression of amp genes triggered by gram-positive and -negative bacteria, respectively (tanaka et al., 2005b; tanaka et al., 2007; see below in detail). this suggests that toll and imd pathways are also activated by lys-type and dap-type pgns, respectively, in b. mori. these results and suggestions lead us to further speculate that expression of amp genes in silkworm is up-regulated by both pathways, but that the activation of these genes by the toll pathway is lower than that by the imd pathway. we demonstrated two mechanisms for this differential activation (tanaka et al., 2009b). one is that the quantity of bmrelishes, effector of the imd pathway, is greater than that of bmrels, effector of the toll pathway. the other is that bmrelishes have a greater ability to enhance the promoter activity of amp genes than bmrels, and this difference is at least because of the difference in the binding affinity of the rel family proteins to the target sites. regulation of promoter activity of amp genes has been analyzed relatively well in b. mori. κb elements, which are known as regulatory elements of amp genes in other insects, are also conserved in the 5′ up-stream regions of silkworm amp genes (yamakawa and tanaka, 1999; cheng et al., 2006). rel family transcription factors, bmrels and bmrelishes binding to these sequences have already been cloned from b. mori (tanaka et al., 2005b; tanaka et al., 2007). a single bmrel gene produces two alternative splicing isoforms, bmrela and bmrelb. the two rel proteins bmrela and bmrelb have identical amino acid sequences with a rel homology domain (rhd), except that bmrela possesses extra 52 amino acids at the n-terminus. these rel proteins appeared to have different functions: bmrelb activates cecropin b1, attacin, enbocin2, gloverin2, and groverin4 more strongly than bmrela, whereas bmrela activates lebocin4, a polymorphic gene of lebocin, more strongly than bmrelb. the fact that a minor structural change in a rel protein can induce considerably different activation of amp genes has been reported only in b. mori, suggests that b. mori has a novel regulatory mechanism for expression of amp genes. bmrelish also produces two alternative splicing products, bmrelish1 and bmrelish2. bmrelish1 has a rhd at the n-terminus, and ankyrin repeats at the 62 c-terminus. bmrelish1 is assumed to be activated by endoproteolytic cleavage and subsequent removal of the c-terminal ankyrin repeats as seen in drosophila relish. bmrelish2 lacks a putative transcriptional activation domain and an ankyrin repeat; bmrelish2 seems to be a dominant negative factor against bmrelish1 (tanaka et al., 2007). the function of bmrelish2 in vivo remains unknown because the amount of bmrelish2 mrna in the b. mori larval fb is extremely low compared to that of bmrelish1 mrna. another nucleotide sequence, the “cattt/a” motif located on the 5′ region of cecropinb1 and attacin is also reported to be involved in promoter activation by crude lipopolysaccharide (lps), in which pgn is probably contaminated (taniai and tomita, 2000; tanaka et al., 2005a). further electro mobility shift assay revealed that the binding factors are contained in the fb nuclear extract (taniai and tomita, 2000; tanaka et al., 2005a), but they have not yet been identified. gata motif, which has been reported to play an important role in expression of amp genes in d. melanogaster, seems to have no effect on promoter activity in b. mori (taniai and tomita, 2000). unlike pgn, lps, which is a major cell surface component of gram-negative bacteria, does not activate the toll or imd pathway in d. melanogaster. on the contrary, we found that lps elicits expression of amp genes in the fb of b. mori larvae using highly purified lps, although the expression level is lower than that elicited by crude lps and pgn (tanaka et al., 2009a). this signal transduction pathway for transcriptional activation of amp genes by lps remains unknown. some other molecules have also been shown to elicit the synthesis of immune-related proteins in b. mori. eicosanoids were shown to mediate induction of amp genes in the fb of silkworm by treatment with eicosanoid biosynthesis inhibitors (morishima et al., 1997). in addition, nitric oxide (no) produced by b. mori nitric oxide synthase seems to be involved in a intracellular signaling to induce expression of amp genes, since the gene expression of cecropin b is induced in the fb of b. mori larvae injected with no donors (imamura et al., 2002). furthermore, tian et al. (2010) reported that juvenile hormone (jh) up-regulated expression of amp genes, whereas 20-hydroxyecdysone (20e) suppressed it in the fb of silkworm larvae. they further reported that the 20e receptor complex, ecdysone receptor, and ultraspiracle were involved in the inhibition of amp gene expression by 20e. the regulation of amp gene expression by these two hormones in b. mori is contrary to that observed in d. melanogaster, in which 20e induces the expression of amp genes and jh inhibits 20e-dependent up-regulation of amp genes (flatt et al., 2008). further analysis will be necessary to clarify the molecular mechanism of jh and 20e-mediated transcriptional regulation of amp genes in b. mori and d. melanogaster. melanization invasion of microorganisms into the hemocoel triggers rapid synthesis of polymeric melanin from phenolic substances to encapsulate invading foreign organisms (cerenius and söderhäll, 2004). a key enzyme involved in melanization is po. more than 40 years ago, ashida et al. were the first to demonstrate that silkworm po exists as a zymogen, propo (ashida and ohnishi, 1967), and then it was found that infection of pathogens leads to its proteolytic activation of propo through a stepwise process called the propo cascade, composed of several serine proteases in the silkworm (ashida et al., 1974; katsumi et al., 1995; johansson and söderhäll, 1996). ashida et al. also identified in 1983 that pgn and β-1,3-glucan were elicitors to activate the silkworm propo cascade (ashida et al., 1983). subsequently, they purified for the first time a pgrp and a β-1,3-glucan recognition protein (βgrp) from larval hemolymph as proteins to recognize pgn and β-1,3-glucan, respectively (ochiai and ashida, 1988; yoshida et al., 1996). by now, the activated propo cascade is known to cleave propo activating enzyme (proppae) to active ppae, which is required for conversion from propo to po (satoh et al., 1999). an unknown serine protease to process proppae also converts probaeease to baeease, whose target substrate in silkworm is not yet known (katsumi et al., 1995). recently, lipid a, a component of lps, has been reported as an elicitor to activate melanization (kaneko et al., 2005). in another lepidopteran, manduca sexta, two branches of the propo cascade/system have been identified (kanost et al., 2009; cerenius et al., 2010). one consists of the serine proteases, hemolymph protein 6 (hp6) and propo activating proteinase 1 (pap1), and the other consists of hp14, hp21, pap2 and pap3. the serine protease homolog (sph) 1 and 2 are also reported to be involved in the cleavage of propo. a genome-wide analysis of silkworm showed that silkworm has at least 15 clip domain serine protease genes (bmclip1 to 15). among them, bmclip1 and bmclip2 correspond to ppae and baee, respectively (tanaka et al., 2008). bootstrap analysis demonstrated that bmclip8 and bmclip11 (equal to bmsph1) are definitive 1:1 orthologs of pap1, and sph1, respectively, but none has a 1:1 ortholog relationship with pap2, pap3, hp6, hp14, hp21 or sph2. this comparative genomic analysis suggests that the propo cascade between b. mori and m. sexta is not well conserved, although the two insects are lepidoptera. production of reactive oxygen species (ros) is also reported in the course of the melanization reaction for antimicrobial defense (nappi, et al., 2009). recently, experiments of injecting pgn from porphyromanas gingivalis into silkworm larvae demonstrated that overproduction of ros generated during the melanization reaction has lethal effects on the silkworm (ishii et al., 2010). cellular immunity and regulation of its response hemocytes play an important role in host cellular defense mechanisms such as phagocytosis, encapsulation, and nodule formation (lavine and strand, 2002; lemaitre and hoffmann, 2007). silkworm hemocytes are classified into five types based on their morphology and function: granulocytes, plasmatocytes, oenocytoids, prohemocytes and spherulocytes (akai and sato, 1973, 1976; nakahara et al., 2009). among them, 63 granulocytes and plasmatocytes, shared similarity with mammalian neutrophils and macrophage, respectively are involved in cellular immunity (wago, 1980). additionally, oenocytoids produce propo in response to microbial infection (iwama and ashida, 1986). phagocytosis is the engulfing of particles smaller than own cells such as bacteria, viruses, and latex beads by hemocytes, and it proceeds in three phases: attachment, filopodial elongation, and actual internalization by the veil-like membrane processes (wago, 1982, 1983). in b. mori, granulocytes are primarily involved in phagocytosis (wago, 1982, 1983). larger foreign materials such as wasp eggs or larvae are encapsulated by granulocytes in cooperation with plasmatocytes (encapsulation) (sato et al., 1976). in addition, granulocytes and plasmatocytes can surround and isolate aggregated foreign materials when many materials are incorporated into the hemocoel by septic infection (nodule formation) (ratcliffe and gagen, 1977; koizumi et al., 1999; sakamoto et al., 2011). in nodule formation, melanization occurs in the nodule matrix, which consists of aggregated bacteria and hemocytes (koizumi et al., 1999; sakamoto et al., 2011). recently, sakamoto et al. (2011) demonstrated that the precursor of sph1 from b. mori (bmsph1) was cleaved to an active species in the nodule but not in the plasma in bacteria-injected silkworm larvae. additionally, they found that anti-bmsph1 inhibited melanization in nodules. these results suggest that bmsph1 is involved in melanization in nodules but not in the hemolymph. in contrast, m. sexta sph-1, a putative ortholog of bmsph1 appears to regulate the initiation of melanization in the hemolymph (yu et al., 2003). more detailed analysis is needed to elucidate the different functions of sph-1 between b. mori and m. sexta. the recognition of invading pathogens by pattern recognition receptors elicits cellular immunity responses. in d. melanogaster, several kinds of receptors have been identified, such as the pgrps (kurata, 2004; aggrawal and silverman 2007), eater (ertürk-hasdemir and silverman, 2005), nimrod family proteins (kurucz et al., 2007), scavenger-receptor family proteins (rämet et al., 2001), and an immunoglobulin superfamily domain protein, dscam (watson et al., 2005). soluble proteins, thioester-containing proteins (blandin and levashina, 2004) and c-type lectins (ao et al., 2007) are also known to act as opsonins, which promote the cellular immunity reactions. a genome-wide analysis revealed that the gene families encoding these recognition receptors and soluble proteins also exist in the silkworm genome except for eater, which seems to be present only in dipteran insects (tanaka et al., 2008). however, receptors that elicit cellular immunity reactions have not yet been identified in b. mori except for c-type lectins, such as the b. mori lps-binding protein (bmlbp) (koizumi et al., 1997, 1999) and b. mori multiple saccharide-binding protein (bmmbp) (watanabe et al., 2006). both bmlbp and bmmbp are investigated to be involved in the elimination of invading pathogens from the hemolymph. bmlbp recognizes gram-negative bacteria through lps, whereas bmmbp recognizes gram-positive bacteria and yeast through lipoteichoic acid and mannose, respectively. most recently, other c-type lectins—b. mori low-expression lectin 1 (bmlel1) and bmlel2—have been reported to bind to rough and smooth strains of gram-negative bacteria, respectively, indicating that they also act as recognition molecules to induce immunity reactions (takase et al., 2009). hemolin, which belongs to the immunoglobulin superfamily, is also a soluble recognition molecule involved in humoral and cellular immunity (sun et al., 1990; ladendorff and kanost 1991; eleftherianos et al., 2007). hemolin is detected exclusively in lepidopterans, including b. mori, suggesting that the gene has evolved as a lineage-specific gene for lepidoptera (schmidt et al., 1993; tanaka et al., 2008). immune response by paralytic peptide a cytokine-like factor, b. mori paralytic peptide (bmpp), which was purified from the silkworm hemolymph (ha et al., 1999), belongs to the enf peptide family. this family shares a similar amino acid sequence particularly at the c-terminal region, and is named after the n-terminal-conserved three amino acids (glu-asn-phe) (strand et al., 2000). enf family proteins are synthesized as inactive precursors and are activated by a serine protease cleavage. mature enf family proteins, including bmpp, have been reported to show multiple effects, such as induction of morphological changes of plasmatocytes, inhibition of larval growth, promotion of cell growth, and local muscle contraction (ha et al., 1999; sasagawa et al., 2001; miura et al., 2002; nakahara et al., 2003). recently, ishii et al. (2008) reported that pgn and β-glucan-dependent ros production in hemocytes stimulates the activation of serine proteases that lead to proteolytic activation of bmpp in the hemolymph. they also showed that bmpp activation enhances the immunity against bacterial infection, and that the enhancing host immunity against invading pathogens by active bmpp is because of the activation of hemocytic phagocytosis and up-regulation of expression of amp genes through activation of p38 map kinase signaling (ishii et al., 2010). nevertheless, it still remains unclear whether the serine proteases leading to proteolytic activation of bmpp are the same as those involved in the activation of the toll pathway or propo cascade, and whether the p38 map kinase signaling pathway is implicated with the toll and imd pathways. it would be interesting to know whether these phenomena specifically occur in silkworm (or lepidopteran insects) or not. perspective in this review, we have described the regulation of the innate immune response in the silkworm, b. mori (fig. 2). although a recognition factor to activate the immune systems was first identified in b. mori, recent silkworm immunity studies, particularly at the molecular level, are less advanced than those on the common vinegar fly and mosquitoes. recently, the entire genome analysis of b. mori was almost completed (the international silkworm genome consortium, 2008), and a subsequent 64 fig. 2 overview of innate immune responses in b. mori. pgrp; peptidoglycan recognition protein, βgrp; β glucan recognition protein, ppae; prophenol oxidase activating enzyme, po; phenol oxidase, pp; paralytic peptide. genome-wide analysis of immune-related genes was performed (tanaka et al., 2008). this analysis revealed that the factors involved in intracellular signal transduction pathways are well conserved between silkworm and non-lepidopteran insects, whereas the recognition proteins and effectors are structurally diverse among them. as for genes encoding recognition proteins and effectors, a dynamic lineage-specific gene evolution probably occurred in lepidoptera to adapt the silkworm or lepidopteran insects to the pathogens that preferentially infect them. unfortunately, the function of most genes encoding recognition proteins in b. mori is unknown. nonetheless, they can be elucidated by the development of functional analyses such as rna interference (rnai) and overproduction of proteins using silkworm cell lines and/or transgenic silkworms. dna-based rnai in cultured silkworm cells is now available (isobe et al., 2002; fujita et al., 2009; tanaka et al., 2009c). in addition, the established gal4/uas system allows a target gene or hairpin rna corresponding to a target gene to be expressed in appropriate tissues and developmental stages in b. mori (imamura et al., 2003; tatematsu et al., 2010; kobayashi et al., 2011). moreover, the following two methods for gene targeting mutagenesis have been established, homologous recombination using autographa californica nucleopolyhedrovirus (npv) (yamao et al., 1999) and zinc-finger nuclease (takasu et al., 2010). further studies on the functional analyses of immune-related genes using these techniques are expected in the near future. although we did not describe it in this review, it is also important to study immune systems against viruses. in contrast to studies of antiviral immunity against rna viruses, which are proceeded mainly in diptera (lemaitre and hoffmann, 2007; kemp et al., 2009; sabin et al., 2010), antiviral immunity against dna viruses is poorly understood in insects. the silkworm is surely a good model for studies of immunity against dna viruses because of the thorough characterizations of the npv, an enveloped dna virus that infects silkworms (yao et al., 2006). recently, we identified up-regulated and down-regulated host genes from a silkworm cell line in response to b. mori npv infection (sagisaka et al., 2010). we believe that elucidating 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is required for the induced transcription of antimicrobial peptide genes in lepidopteran insects. dev. comp. immunol. 31: 1002-1012, 2007. yu xq, jiang h, wang y, kanost mr. nonproteolytic serine proteinase homologs are involved in prophenoloxidase activation in the tobacco hornworm, manduca sexta. insect biochem. mol. biol. 33: 197-208, 2003. 69 research report isj 11: 66-72, 2014 issn 1824-307x research report determination of lipase activity in the larval midgut of bacterocera oleae gmelin (diptera: tephritidae) s delkash-roudsari1, a zibaee1, mr abbacimozhdehi2 1department of plant protection, faculty of agricultural sciences, university of guilan, rasht 41476-1314, iran 2agricultural and natural resources center, rasht, iran accepted february 7, 2014 abstract in the current study, digestive lipase activity was determined and characterized in the third larval instars of olive fly, bactericera oleae as the first time in dipteran order. by using two sample fractions, it was found that the enzyme had higher activity in membrane-bound fraction than that of soluble fraction. optimal ph of soluble lipase was found to be 4 and 6 but membrane-bound lipase showed ph 4 as optimal value. optimal temperatures for soluble and membrane-bound lipase were obtained to be 35 and 50 °c, respectively. activities of digestive soluble and membrane-bound lipases decreased by using various monoand di-valent ions. since, fruits of olive are full of various oils, digestive lipases of b. oleae larvae have critical role in their digestion. so, these enzymes might be a good target for developing inhibitors and resistant varieties. key words: bacterocera oleae; digestive lipase; characterization   introduction bacterocera oleae (diptera: tephritidae) is the most destructive pest of olive around the world (richard et al., 2003). the insect have been introduced to north of iran as a serious pest since 2004 (jafari and rezaee, 2005; mirrahimi et al., 2008). females lay their eggs in fruits especially large green ones, larvae feed on the fruit pulp causing severe loss and feasibility of pathogen entrances to fruits. pest control tactics depend on chemical treatments against adults, biological control and physical control such as sticky and pheromone traps (laskowski and kato, 1980). in insects, lipids are involved in several physiological functions like moulting during larval and adult development (kawooya and law, 1988), reproduction (majumder and sengupta, 1979), energy supplement during starvation (cheeseman, 1976; ziegler, 1991) and immunity. lipases [ec 3.1.1.3] are the enzymes that hydrolyze the esteric bonds at the interface between insoluble substrate and water. the enzymes are synthesized by animals, plants, fungi and bacteria (jaeger et al., 1999; gupta et al., 2004; grillo et al., 2007). digestive ___________________________________________________________________________ corresponding author: arash zibaee faculty of agricultural sciences university of guilan, rasht iran, 416351314 e-mail: arash.zibaee@gmx.com lipases of insects are divided into triacylglycerol lipases (tag-lipases), alkaline and acid phosphatases as well as phospholipases (terra and ferreira, 2012). majority of ingested lipids by insects are the storage lipids (triglycerids) that must be digested to diand monoglycerids in midgut (zibaee et al., 2008). during digestion process, triacylglyceride lipases hydrolyze certain positions of triacylglycerols while different phospholipases act on different ester bonds within phospholipids (zibae e et al., 2008). digestive lipases has been studied in a few insects such as rhodnius prolixus l. (lepidoptera: reduviidae) (grillo et al., 2007), chilo suppressalis (lepidoptera: crambidae) (zibaee et al., 2008), naranga aenescens (lepidoptera: noctuidae) (zibaee and fazeli-dinan, 2012), pieris brassicae (lepidoptera: pieridae) (zibaee, 2012) and andrallus spinidens (hemiptera: pentatomidae) (zibaee et al., 2012). although lipases have not been well studied like other digestive enzymes but the enzymes have critical role in digestion process of insects. moreover, characterization of lipases is mandatory to find inhibitors against lipases. on the other hand, inhibitors do cause severe reduction in growth and development and even mortality due to the importance of long chain unsaturated fatty acids in essential dietary components. this could be discovered from host plants that insect feeding causes lower activity of digestive lipases. moreover, biochemical behavior of the lipases must be elucidated before any exposure 66 fig. 1 activity level of soluble and membrane-bound lipases in 3th instar larvae of olive fruit fly. to inhibitors. hence, a biochemical characterization of the digestive lipase from b. oleae has been made via evaluating ph and temperature, effects of different ions and specific inhibitors. this is the first report of digestive lipases in a dipteran. materials and methods insect rearing larvae of bacterocera oleae were reared on the olive fruits of arbequina variety in containers of 20×12 cm under laboratory conditions of 25 ± 1 °c, 70 % of relative humidity and 16l:8d of photoperiod. rearing containers were daily checked and cleaned to remove any contaminations. when the larvae reached to 3rd larval instars, they were randomly selected and used in biochemical experiments. sample preparation third larval instars were separated from olive fruits and dissected under a stereomicroscope in ice cold saline solution (nacl, 10 mm). whole guts were homogenized in distilled water by a glass homogenizer, centrifuged at 25000xg for 20 min at 4 °c and supernatants were used in lipase assay (zibaee, 2012). membrane preparations were exposed from pellets of centrifuged digestive tract (see above) to triton x-100 for 20 h at 4 °c, in a ratio of 10 mg triton x-100 per mg of protein. samples were centrifuged at 25000xg for 20 min. activity of the enzyme remains unchanged at -20 °c for at least a month (zibaee, 2012). lipase assay the enzyme assay was carried out as described by tsujita et al. (1989). five microliter of larval gut extract and 15 μl of p-nitrophenyl-butyrate (pnpb, 27 mm) as substrate were added into 40 μl of universal buffer (20 mm, ph 7), mixed thoroughly and incubated at 30 °c. for negative control, samples were placed in a boiling water bath for 15 min to destroy the enzymatic activity.finally, absorbance was read after 10 min at 492 nm. one unit of enzyme released 1.0 nmol of p-nitrophenol per min at ph 7.2 and 37 °c when p-nitrophenyl butyrate was used as substrate. effect of ph and temperature on the activity and the stability of the enzyme effects of temperature and ph on lipase activity were examined by using p-nitrophenol-butyrate as substrate in various ph and temperature values. optimal ph was determined using universal buffer (20 mm) with ph set at 3 12. the effect of temperature on the enzyme activity was determined by incubating the reaction mixture at 20, 25, 30, 35, 40, 45, 50 and 60 °c. the procedures for both ph and temperature assays were quite similar to mentioned lipase assay (see above). effect of monoand di-valent cations on lipase activity different concentrations of cations (1, 3 and 5 mm) were assayed to find their effects on lipase activity in b. oleae larvae. used cations were ca2+, cu2+, fe2+, k+, mg2+, na+ and zn2+. briefly, 5 µl of a solution containing each concentration of ions and 5 67 a) b) fig. 2 effect of ph on the activities of soluble and membrane-bound lipases extracted from the digestive system of b.oleae larvae. statistical deifferences have been shown by various letters (tukey’s test, p ≤ 0.05). µl of enzyme were pre-incubated for 10 min at ph 7 and room temperature. the pre-incubated mixture was added to a solution including 30 µl of universal buffer and 10 µl of substrate (pnpb, 27 mm). other steps were carried out as mentioned earlier. effect of specific chelating agent on lipase activity the effects of enzyme inhibitors on lipase activity were studied using different concentrations (2, 4, 6, 8 and 10 mm) of ethylene glycolbis (βaminoethylether) n,n, n′, n′-tetraacetic acid (egta), triethylenetetramine hexa acetic acid (ttha), phenylmethylsulfonyl fluoride (pmsf), diethyldithiocarbamate (dtc), and ethylenediaminetetraacetic acid (edta). the enzyme (5 µl) was pre-incubated with inhibitors for 10 min at ph 7 and room temperature. the preincubated mixture was added to a solution including 10 µl of substrate. other steps were carried out as mentioned earlier. protein determination protein concentrations were assayed according to the method described by lowry et al. (1951). 68 a) b) fig. 3 effect of temperature on the activities of soluble and membrane-bound lipases extracted from the digestive system of b.oleae larvae. statistical deifferences have been shown by various letters (tukey’s test, p ≤ 0.05). statistical analysis the data were compared by one-way analysis of variance (anova) followed by tukey’s test when significant differences were found at p ≤ 0.05 using sas program (sas, 1997). results and discussion lipases have important roles in digestion of lipids by insects and intermediary metabolism including lipid storage and mobilization (horne et al., 2009). our results clearly demonstrated presence of a digestive lipase in the gut of b. oleae. on the other hands, it could be inferred that alimentary canal of larval olive fruit fly has ability to digest lipid by the action of soluble and membrane-bound lipases.in the current study, lipase activity was observed in both soluble and membrane-bound fractions by higher activity of membrane-bound fraction (fig. 1) althopugh, majority of studies have been determined 69 in soluble content of the midgut. since digestive enzyme secretion is a secretagogue process, digestive enzymes are presence as enzymatic vesicle inside of epithelial cells during starvation (klowden, 2007). during ingestion, these vesicles are released into midgut lumen. hence, higher activity of membrane-bound lipase could be attributed to the higher amounts of enzyme as vesicles inside the epithelial cells. temperature and ph are the two factors that affect biochemical reactions via various approaches table 1 effect of monoand divalent cations on soluble lipase activity of b.oleae compound concentration specific activity c 1.857±0481 a ca2+ 1 3 5 c 0.312±0.064 b 0.122±0.093 b 0.047±0.037 b 1.857±0481 a cu2+ 1 3 5 c 0.442±0.034 b 0.289±0.75 b 0.537±0.074 b 1.857±0481 a fe2+ 1 3 5 c 1.088±0.194 b 1.598±0.119 ab 1.714±0.167 ab 1.857±0481 a k+ 1 3 5 c 0.741±0.134 ab 0.224±0.081 b 0.047±0.013 b 1.857±0481 a mg2+ 1 3 5 8.913±0.649 b 2.415±0.350 b 1.592±1.354 b c 1.857±0481 a na+ 1 3 5 c 0.340±0.018 b 0.115±0.013 b 1.769±0.426 ab 1.857±0481 a zn2+ 1 3 5 1.585±0.204 a 0.122±0.031 b 0.394±0.013 b *statistical differences have been shown by various letters (tukey’s test, p ≤ 0.05). table 2 effect of monoand divalent cations on membrane-bound lipase activity of b. oleae *statistical differences have been shown by various letters (tukey’s test, p ≤ 0.05). like substrate and enzyme stability, their combination, tertiary structure of the enzyme and etc. both of these factors provide optimal conditions to more adjustment of enzyme leading to better affinity and velocity during enzymatic reactions (zibaee et al., 2012). it was found that optimal ph of the digestive solubleand membrane-bound lipases in the gut of b. oleae larvae were obtained 4 and 6 for soluble lipase and 6 for membrane-bound one (fig. 2). meanwhile, optimal temperatures for soluble and membrane lipases were observed at 50 compound concentration specific activity c 6.800±0.322 a ca2+ 1 3 5 c 0.628±0.081 b 0.052±0.028 b 0.231±0.087 b 6.800±0.322 a cu2+ 1 3 5 c 0.304±0.066 b 1175±62.915 b 0.555±0.277 b 6.800±0.322 a fe2+ 1 3 5 c 0.463±0.176 b 1.283±0.162 b 0.972±0.251 b 6.800±0.322 a k+ 1 3 5 c 0.807±0.184 b 142.719±719 b 140.778±12.165 b 6.800±0.322 a mg2+ 1 3 5 3.380±0.587 b 1.574±0.767 b 1.316±0.324 b c 6.800±0.322 a na+ 1 3 5 c 0.469±0.017 b 0.205±0.102 b 0.608±0.283 b 6.800±0.322 a zn2+ 1 3 5 1.223±0.160 b 0.205±0.102 b 0.641±0.509 b 70 °c and 35 °c, respectively (fig. 3). grillo et al. (2007) reported that digestive lipase from the midgut of rhodnius prolixus (hemiptera: reduviidae) had maximal activity in ph 7-7.5. zibaee et al. (2008) found the optimal ph and temperature of 10 and 37 40 °c in larvae of chilo suppressalis. zibaee (2012) found optimal ph and temperature of digestive lipasein p. brassicae as ph 11 and temperature of 30 °c. the optimal ph of 10 and temperature of 35 40 ◦c observed for a lipase from larvae of n. aenescens (zibaee and fazeli-dinan, 2012). the optimal ph for tag-lipase activity was obtained at ph 9 and temperature 40 °c for a. spinidens (zibaee et al., 2012). the optimal temperature for activity of an enzyme reflects temperature in which organism is living in it. also, activity of enzymes increase along with elevation of temperature up to optimal value then it decrease because the hydrogen bonds in enzyme structure break in extreme temperature and disrupt threedimensional structure leading to enzyme denaturation (zeng and cohen, 2000). ions are one of the significant components in active sites of enzymes. ions can take and release electrons; affect electrophiles and nucleophiles, increase efficiency of enzyme-substrate complex and stability of enzymes (zibaee, 2012). in the present study, different concentrations of ions and chelating agents caused various effects on lipase activity in b oleae larvae (tables 1 4). activities of the digestive soluble and membrane-bound lipases in the gut of b. oleae larvae were decreased by using all ions (tables 1, 2). the effects of several chelating agent and pmsf were examined to find their possible effects on digestive solubleand membrane-bound lipase activities (tables 3, 4). all the chelating agents except for ttha in soluble lipase had no inhibitory effects on the enzymatic activity but pmsf sharply decreased enzymatic activity in both fractions (tables 3, 4). applebaum 1985) reported that ca2+ ion increased lipase activity table 3 effect of specific inhibitors on soluble lipase activity of b. oleae compound concentration specific activity c 0.217±0.013 a dtc 10 0.442±0.114 a pmsf c 10 1.857±0.481 a 0.421±0.118 b egta c 10 925±0.156 b 1.809±0.119 a edta c 10 0.217±0.013 a 0.442±0.114 a ttha c 10 1.687±0.203 a 0.591±0.142 b *statistical differences have been shown by various letters (tukey’s test, p ≤ 0.05). table 4 effect of specific inhibitors on membranebound lipase activity of b. oleae compound concentration specific activity c 0.555±0.052 b dtc 10 1.223±0.152 a pmsf c 10 6.800±0.322 a 1.706±0.558 b egta c 10 1.574±0.220 a 1.488±0.045 a edta c 10 0.840±0.262 a 1.303±0.304 a ttha c 10 1.580±0.077 a 0.077±0.0.056 a *statistical differences have been shown by various letters (tukey’s test, p ≤ 0.05). of callosobruchus chinensis. zibaee (2012) found that mg2+, na+, edta and ttha significantly affect digestive lipase activity in p. brassicae. grillo et al. (2007) found that ca2+ increase activity of lipases in r. prolixus. similar results were found in case of c. suppressalis and n. aenescens (grillo et al., 2007; zibaee et al., 2008; zibaee and fazeli-dinan, 2012). ca2+, mg2+, k+, na+, mn+, pmsf and egta showed significant effects on lipase activity in a. spinidens (zibaee et al., 2012). although digestive lipases of insects have been less studied but they have a great potential to be inhibited like amylases and proteases. this property could be used to develop resistant varieties in sustainable agricultural system. there are many reports on inhibition of vertebrate pancreatic lipases by secondary metabolites of plants but relevant studies on insect digestive lipases are few (markwick et al., 2011). senthil-nathan et al. (2006) and zibaee and bandani (2010) reported that feeding of eurygaster integriceps (hemiptera: scutelleridae) and cnaphalocrocis medinalis on the diets containing botanical extracts lead to significant inhibition of digestive lipases. also, markwick et al. (2011) demonstrated effects of tetrahydrolipstatin (thl), on neonate epiphyas postvittana (lepidoptera, tortricidae) larvae by feeding on control artificial diets and diets containing one of three concentrations of thl (0.011 %, 0.037 % and 0.11 %). the authors reported significant decrease in growth, pupation and time to pupation in comparison with control. although reports on digestive lipases of insects revealed promising results but it is necessary to carried out more studies to confirm that digestive lipases of insects could be a new control for insect pest or not. lipase inhibitors do cause severe reduction in growth and development and even mortality due to the importance of long chain unsaturated fatty acids being essential dietary components. hence, a plant breeding program might 71 be adopted to increase levels of naturally occurring lipase inhibitors. the current study was the first one to characterize a digestive lipase of dipteran larvae. this is a basic study that will continue to develop inhibitors leading to an efficient control of b. oelae. references applebaum sw. biochemistry of digestion. in: kerkut ga, gilbert ll (eds), comprehensive insect physiology, biochemistry and pharmacology, vol. 4, regulation, digestion, excretion, pergamon press, oxford, pp 279307, 1985. cheeseman p, jutsum ar, goldsworthy gj. quantitative studies on the release of locust adipokinetic hormone. physiol. entomol. 1: 115121, 1976. grillo la, majerowicz d, gondim kc. lipid metabolism in rhodnius prolixus (hemiptera: reduviidae): role of a midgut triacylglycerollipase. insect biochem. mol. biol. 37: 579-588, 2007. gupta r, gupta n, rathi p.bacterial lipases: an overview of production, purification and biochemical properties. appl. microbiol. biotechnol. 64: 763-781, 2004. horne i, haritos vs, oakeshott jg. comparative and functional genomics of lipases in holometabolous insects. insect biochem. mol. biol. 39: 547-567, 2009. jaeger ke, dijkstra bw, reetz mt. bacterial biocatalysts: molecular biology, threedimensional structures, and biotechnological applications of lipases. ann. rev. microbiol. 53: 315-351, 1999. jafari y, rezaee v. first report of import olive fly to country. new entomol. soc. iran. 6: 1-22, 2004 [in persian]. kawooya jt, law jh.role of lipophorin in lipid transport to the insect egg. j. biol. chem. 263: 8748-8753, 1988. klowden mj. physiological systems in insects, 2nd ed., elsevier, new york, ny, 2007. laskowski jr, kato m. protein inhibitors of proteinases. ann. rev. biochem. 49: 593-626, 1980. lowry oh, rosebrough nj, farr al, randall rj. protein measurement with the folin phenol reagent. j. biol. chem. 193: 265-275, 1951. majumder uk, sengupta a. triglyceride composition of chrysalis oil, an insect lipid.j. american. oil chem. soc. 56: 620-623, 1979. markwick np, poulton j, mcghie tk, wohlers mw, christeller jt. the effects of the broadspecificity lipase inhibitor, tetrahydrolipstatin, on the growth, development and survival of the larvae of epiphyas postvittana (walker) (tortricidae, lepidoptera). j. insect physiol. 57: 1643-1650, 2011. mirrahimi s, khalaghani j, nouri h. effect of time, direction and site of sampling on olive infestation by olive fruit fly. proceedings of 18th iranian plant protection, 2008. richard r, phillips pa, stewart-leslie js ibbett gs. olive fruit fly population in central and southern california. 57. doi: 10.3733/ca.v057n04p122, 2003. sas institute.sas/stat user’s guide for personal computers. sas institute, cary, nc, 1997. senthil-nathan s, chung pg, murugan k.combined effect of biopesticides on the digestive enzymatic profiles of cnaphalocrocis medinalis (guenee) (the rice leaffolder) (insecta: lepidoptera: pyralidae). ecotox. environ. safe. 64: 382-389, 2006. tan-kristanto a. characterisation of lipase genes in helicoverpa armigera. department of genetics. the university of melbourne, 2006. terra wr, ferriera c. biochemistry of digestion. in: gilbert li (ed.), insect molecular biology and biochemistry, elsevier, pp 365-418, 2012. tsujita t, ninomiya h, okuda h. p-nitrophenyl butyrate hydrolyzing activity of hormonesensitive lipase from bovine adipose tissue. j. lipid res. 30: 997-1004, 1989. zeng f, cohen ac. partial characterization of αamylase in the salivary glands of lygus hesperus and l. lineolaris. comp. biochem. physiol. 126b: 9-16, 2000. zeng f, zhu y, cohen ac. molecular cloning and partial characterization of a trypsin-like protein in salivary gland of lygus hesperus (hemiptera: miridae). insect biochem. mol. biol. 32: 455464, 2002. zibaee a. a digestive lipase of pieris brassicae l. (lepidoptera: pieridae): purification, characterization and host plants effects. arch. insect biochem. physiol. 81: 1-19, 2012. zibaee a, bandani ar.effects of artemisia annua l. (asteracea) on digestive enzymes profiles and cellular immune reactions of sunn pest, eurygaster integriceps (heteroptera: scutellaridae), against beauvaria bassiana. bull. entomol. res. 100: 185-196, 2010. zibaee a, bandani ar, ramzi s. lipase and invertase activities in midgut and salivary glands of chilo suppressalis (walker) (lepidoptera: pyralidae), rice striped stem borer. inv. surv. j. 5: 180-189, 2008. zibaee a, fazeli-dinan m. purification and characterization of a digestive lipase in naranga aenescens moore (lepidoptera: noctuidae). soaj 1: 38-54, 2012. zibaee a, hoda h, fazeli-dinan m. a tag-lipase activity in the salivary secretions of a predaceous bug, andrallus spinidens fabricius (hemiptera: pentatomidae).trends entomol. 9: 48-57, 2012. ziegler r. changes in lipid and carbohydrate metabolism during starvation in adult manduca sexta. j. comp. physiol. 161: 125-131, 1991. 72 review isj 8: 21-32, 2011 issn 1824-307x review the complement c3 protein family in invertebrates m nonaka department of biological sciences, graduate school of science, the university of tokyo, 7-3-1 hongo, bunkyo-ku, tokyo 113-0033, japan accepted january 11, 2011 abstract complement c3 plays a pivotal role in the innate immune system of mammals as the central component of the complement system essential for its activation mechanism and effecter function. c3 has a unique intra-chain thioester bond that is shared by some complement and non-complement proteins forming a thioester protein (tep) family. phylogenetic analysis of tep family genes of vertebrates and invertebrates revealed that the tep family is divided into two subfamilies, the c3 subfamily and the alpha-2-macroglobulin (a2m) subfamily. the establishment of the tep genes and differentiation of them into the c3 and a2m subfamilies occurred prior to the divergence of cnidaria and bilateria, in a common ancestor of eumetazoa more than 600 mya. since then the a2m subfamily has been retained by all metazoan lineages analyzed thus far. in contrast, the c3 subfamily has been retained only by deuterostomes and some protostomes, and has been lost in multiple protostome lineages. although the direct functional analysis of the most invertebrate teps is still to be performed, conservation of the basic domain structure and functionally important residues for each molecule suggests that the basic function is also conserved. functional analyses performed on a few invertebrate c3 support this conclusion. the gene duplication events that generated c4 and c5 from c3 occurred in a common ancestor of jawed vertebrates, indicating that invertebrate and cyclostome c3s represent the pre-duplication state. in addition to c3, complement bf and masp involved in the activation of c3 are also identified in cnidaria and some invertebrates, indicating that the complement system is one of the most ancient innate immune systems of eumetazoa. key words: thioester-containing protein (tep); α-2 macroglobulin (a2m); evolution; cnidaria introduction the human complement system is composed of about 30 plasma and cell surface proteins and has three physiological functions, host defense against infection, interface between innate and adaptive immunity and disposal of immune complex and apoptotic cells (volanakis, 1998; walport, 2001). since the latter two functions are intimately connected with the canonical adaptive immune system unique to the jawed vertebrates (kasahara et al., 1997), the original function of the complement system, found in both vertebrates and invertebrates, is believed to be the host defense against infection. in the human complement system, this function is attained by three major effector mechanisms, opsonization, induction of inflammation by chemotaxis and activation of leucocytes, and lysis of bacteria and cells. ___________________________________________________________________________ corresponding author: masaru nonaka department of biological sciences graduate school of science, university of tokyo 7-3-1 hongo, bunkyo-ku, tokyo 113-0033, japan e-mail: mnonaka@biol.s.u-tokyo.ac.jp there are three activation pathways for the mammalian complement system (volanakis, 1998). the classical pathway, termed so because it was discovered first among three pathways, is initiated by binding of c1 to antigen-bound antibody, and results in the formation of the classical pathway c3 convertase composed of c4 and c2, which is responsible for the proteolytic activation of the central component c3. the next found alternative pathway consists of factor d (df), factor b (bf) and c3, and the alternative pathway c3 convertase is composed of c3 itself and bf (pangburn and muller-eberhard, 1984). the activation mechanism of the alternative pathway is still not totally clear, although regulatory factors such as factor h and properdin are reported to play a critical role in initiation of the pathway. the lastly found lectin pathway is initiated by recognition of pamp (pathogen-associated molecular pattern) by the lectins, mbl (mannose-binding lectin) and ficolin, and the lectin-associated serine proteases, masp (mbl-associated serine protease) activate c4 and c2, merging to the classical pathway (matsushita and fujita, 1992). 21 fig. 1 distribution of the complement genes with characteristic domain structure in invertebrate deuterostome, protostome and cnidaria. most key components of the complement system possess unique domain structure, and are classified into five mosaic protein families, c3, bf, masp, c6 and if families. the presence of these family genes is schematically shown for the representative species of vertebrate (human, h. sapiens), invertebrate deuterostome (sea squirt, c. intestinalis), protostome (horseshoe crab, c. rotundicauda) and cnidaria (sea anemone, n. vectensis). since the genome sequence information is not yet available for c. rotundicauda, the absence of the masp, c6 and if family member is still tentative. the question mark near the c6 family members of c. intestinalis indicates that these molecules are probably not involved in the sea squirt complement system in spite of close structural similarity to mammalian c6 family members (see text). abbreviations of domain names are: mg, macroglobulin; ana, anaphylatoxin; cub/tep, cub domain inserted with thioester region; c345c, c-terminal of c3, c4 and c5; ccp, complement control protein; vwa, von willebrand factor type a; sp, serine protease; cub, c1r, c1s, uegf, and bone morphogenetic protein; egf, epidermal growth factor-like; tsp, thrombospondin type 1 repeats; mac/p, membrane-attack complex/perforin; ldl, low-density lipoprotein receptor domain class a; fim, factor i/membrane attack complex; and sr, scavenger receptor cys-rich. upon activation by c3 convertases, c3 is cleaved into the larger c3b and the smaller c3a fragments. c3b has ability to bind covalently to acceptor molecules on cell surfaces via ester or amide linkages (law et al., 1979), and cell-bound c3b is recognized by cr1 (complement receptor 1) on phagocytic cells, resulting in opsonic function (ehlenberger and nussenzweig, 1977). c3b can also react with c4b or c3b of the c3 convertases, leading to loss of c3 convertase activity and acquisition of c5 convertase activity (takata et al., 1987; kinoshita et al., 1988). like c3 convertase, c5 convertase cleaves c5 into c5b and c5a. c5b initiates the assembly of the mac (membrane attack complex) by sequential binding of c6, c7, c8 and c9. during this assembly process, hydrophobic domains of the participating proteins become exposed on the surface of the complex, and the complex becomes gradually inserted into the lipid bilayer and eventually forms a transmembrane channel (podack et al., 1981), leading to killing of the susceptible cells. c3a, c5a and the equivalent peptide derived from c4, c4a are termed as anaphylatoxin, since these peptides have prominent pro-inflammatory activity to induce chemotaxis and degranulation of leukocytes (hugli, 1984). c3, c4 and c5 are structurally homologous genes (wetsel et al., 1987), arose from a common ancestor by gene duplications occurred in the early stage of vertebrate evolution (nonaka et al., 1984). they are similar in size (~200kda) and are composed of α and β subunits (c3 and c5) or α, β and γ subunits (c4). during complement activation, all three proteins are proteolytically cleaved at near the n-terminal end of α chain, liberating the c3a, c4a and c5a anaphylatoxins. c3 and c4 have a unique intrachain thioester bond formed between cys and gln in α chain, which is hidden inside of the molecule (janatova and tack, 1981; levine and dodds, 1990). upon structural change induced by proteolytic activation of these molecules, the highly reactive thioester bond is exposed at the molecular surface, and can form an ester bond with a hydroxyl group or amide bond with an amino group on the target surface. this covalent tagging of the foreign particles seems to be the central function of the complement system, enabling the following elimination or killing of the pathogens. 22 fig. 2 phylogenetic tree of animals. out of more than 30 animal phyla, only those relevant to this review are shown with photographs of a belonging species. taxonomic groups higher than phylum are shown in bold, whereas lower than phylum are shown in italic. the 3-dimensional structure analysis of human c3 revealed the presence of the unpredicted macroglobulin (mg) domain, which repeats eight times and constitute the core of the tep family proteins (janssen et al., 2005). most functional sites of human c3 are present in the ana, ted and c345c domains (see fig.1 for the abbreviation and location of the domains) inserted within or between these eight mg domains. further analysis of the 3-dimensional structure of c3b indicated that the dramatic conformational change actually occurs when c3 is proteolytically activated into c3b as predicted from biochemical analyses (janssen et al., 2006). this review provides an overview of our current knowledge on the c3 genes or proteins of invertebrates, and will try to reconstitute the evolution of the complement system. although there is no logical basis of an argument whether the common ancestral gene for c3, c4 and c5 should be called c3, c4 or c5, here i refer it as c3 since the role of c3 in the mammalian complement system is so pivotal that it is difficult to imagine a complement system without c3. animal phylogeny figure 2 shows the phylogenetic tree of the animal phyla relevant to this review based mainly on the recent multigene molecular phylogenetic analyses (philippe et al., 2005; dunn et al., 2008). multi-cellular animals are divided into porifera (sponges) without typical germ layers and eumetazoa with typical germ layers. eumetazoa is further divided into cnidaria (sea anemone, hydra etc) possessing two germ layers and bilateria possessing three germ layers as well as left-right symmetric body. bilateria has two major groups, protostomia and deuterostomia. protostomia is further divided into ecdysozoa and lophotrochozoa, each containing several phyla. deuterostomia has four phyla, xenoturbellida, hemichordata, echinodermata and chordata. vertebrata is one of the three subphyla of chordata. invertebrate, all animals except for vertebrates, is thus not a proper taxonomic group. from the viewpoint of the complement evolution also, it is difficult to classify the invertebrate and vertebrate complement systems. rather, the complement system of cyclostomes (agnatha), the most basal extant vertebrates, shows more similarity to the invertebrate complement system than to the jawed vertebrate complement system. this is also true for the c3 family proteins as will be discussed in the following. origin of the tep family genes the thioester-containing protein (tep) family members possess the unique intrachain thioester bond originally found in the human protease inhibitor, alpha-2-macroglobulin (a2m) and complement c3 (dodds and law, 1998). although some members such as complement c5 (wetsel et al., 1987) and certain insect tep (lagueux et al., 2000) secondary lost the thioester bond, they are identified as members of this family based on an overall sequence homology. seven members of this family are encoded in the human genome: c3, c4, c5, a2m (sottrup-jensen et al., 1985), pregnancy zone protein (pzp) (sottrup-jensen et al., 1984), cd109 (lin et al. 2002) and the complement 3 and pzp-like a2m domain-containing 8 (cpamd8) (li et al., 2004). c3, c4 and c5 are complement components derived from a c3-like common ancestor by gene duplications in the early stage of jawed vertebrate evolution (nonaka and takahashi, 1992; terado et al., 2003). thus, whereas all c3, c4 and c5 are present in sharks (terado et al., 2003; graham et al., 2009) and higher vertebrates, only c3 has been identified from lamprey (nonaka and takahashi, 1992) and hagfish (ishiguro et al., 1992). although c4 shows a close structural and functional similarity to c3, c5 lacks the thioester bond and plays a function that has diverged markedly from those of c3 23 and c4 in the human complement system (lambris et al., 1998). a2m is a serum protease inhibitor, inhibiting the diverse array of proteases by trapping proteases inside of the molecule rather than binding to the active site (armstrong, 2010). the domain structure of a2m is essentially the same with that of c3 except that a2m has a bait domain instead of the ana domain and lacks the c345c domain (janssen et al., 2005). pzp is a major pregnancy-associated plasma protein, with similar structure and function to a2m (sottrup-jensen et al., 1984). cd109, unlike other members of the tep family, is a gpi-linked glycoprotein originally found on endothelial cells, platelets and activated t-cells (lin et al., 2002). cd109 suppresses transforming growth factor (tgf)-β signaling in human keratinocytes by binding to tgf-β receptor i (finnson et al., 2006), and a high level of cd109 expression is detected in squamous cell carcinomas of the esophagus, lung, uterus and oral cavity (hagiwara et al., 2008). however, biochemical details of the cd109 function are still unknown except that the involvement of furin in processing is reported recently (hagiwara et al., 2010). cpamd8, also termed kiaa1283, has a kazal-type serine proteinase inhibitor-like domain at the c-terminus and is expressed mainly in the kidney, brain and testis, although its function is poorly characterized (li et al., 2004). no tep gene is present in the published genome information of a sponge, amphimedon queenslandica and a choanoflagellate, monosiga brevicollis (king et al., 2008; kimura et al., 2009; srivastava et al., 2010). thus, it is suggested that the tep gene arose in the eumetazoa lineage. comprehensive cloning of tep genes of a cnidarian, a sea anemone, haliplanella lineate resulted in identification of four tep genes (fujito et al., 2010). the genome analysis performed in another sea anemone species, nematostella vectensis, also identified the same set of tep genes (kimura et al., 2009), indicating that anthozoan cnidaria has these four tep genes. phylogenetic analysis of the four identified cnidarian tep genes and various tep genes of many eumetazoa resulted in a nj tree shown in fig. 3. although this is an unrooted tree, the root most probably resides at the branch marked by the black triangle, since genes of various animals from cnidaria to vertebrata are present on both sides of this branch indicating that this branch represents an ancient diversifying event, and the ana and c345c domains are present in all members on one side of this branch but none of the members on the other side. thus, the tep gene family is divided by this branch into two subfamilies: the a2m subfamily including a2m, cd109, cpamd8 and insect teps, and the c3 subfamily including complement c3, c4 and c5 (fig. 3). insect teps were originally found in drosophila melanogaster, and six members, tep1-tep6, have been identified in this species (lagueux et al., 2000). drosophila teps contain a hypervariable region at the position corresponding to the bait region of a2m and the ana domain of c3. tep2, tep3 and tep6 bind to escherichia coli, staphylococcus aureus and candida albicans, respectively, and promote their phagocytosis by cultured s2 cells (stroschein-stevenson et al., 2006). in a mosquito, anopheles gambiaeare, the tep1 gene was shown to promote phagocytosis of some gram-negative bacteria (levashina et al., 2001), and to bind to the surface of plasmodium ookinetes and promote their lysis and melanization (blandin et al., 2004). therefore, the insect tep genes were referred to as complement-like genes. however, the phylogenetic analysis clearly indicates that they belong to the a2m subfamily (fig. 3), suggesting that the reported functional similarity of these insect teps and mammalian c3 was caused by convergent molecular evolution. two of the four cnidarian tep genes belonged to the a2m subfamily, showing a close similarity to human a2m and cd109, respectively, and thus were termed halia2m and halicd109 (fujito et al., 2010). the other two genes belonged to the c3 subfamily, and were termed halic3-1 and halic3-2 (fujito et al., 2010). cnidarian teps retained the basic domain structure and functionally important residues for each molecule, and their mrna were detected at different parts of the sea anemone body. thus a strong signal for halic3-1 was detected in the endoderm of tentacles, halia2m was detected in endoderm of the mesentery as strong granular signals, and halicd109 showed strong granular expressions in ectoderm of tentacles and in endoderm of the mesentery (fujito et al., 2010). different expression pattern implies functional differentiation among these three genes. therefore it is suggested that gene duplication and subsequent functional differentiation among c3, a2m and cd109 were very ancient events predating the divergence of the cnidaria and bilateria more than 600 mya. in contrast, the genome of hydra magnipapillata, belonging to hydrozoa, contained only a2m subfamily member (miller et al., 2007). these results indicate that the creation of the tep genes and subsequent gene duplication and functional differentiation into c3, a2m and cd109 have occurred in a relatively short evolutionary period after the divergence of sponges and before the divergence of cnidaria from the bilateria lineage, and that the c3 subfamily was lost by some cnidaria lineages. since both c3 and a2m subfamily members of the tep family are present in cnidaria and bilateria, it is not clear which subfamily arose first. the recent genome analysis in placozoa (srivastava et al., 2008) identified at least two tep genes, and both of them belong to the a2m subfamily, one at the basal position of a2m cluster and the other in the cd109 cluster (fig. 3). although the phylogenetic relationship among porifera, placozoa, cnidaria and bilateria is not conclusively resolved (philippe et al., 2009; schierwater et al., 2009), if placozoa diverged prior to the divergence between cnidaria and bilateria, it is suggested that the a2m subfamily is more ancient than the c3 subfamily. structural features of the common ancestor of the c3 and a2m subfamilies can be deduced from the comparison of the human and sea anemone tep structures (fig. 4). comparison of four human teps and three sea anemone teps showed that the signal peptide and the thioester domain are present in all members, suggesting that these features were present in the common ancestor of the c3 and a2m subfamilies. in addition, the α−β processing site and the his residue 24 fig. 3 phylogenetic tree of tep genes. the tree was constructed based on the alignment of the full length amino acid sequences of the tep family genes, using the neighbor-joining method. gaps were not excluded. bootstrap percentages of more than 50 with 1000 replicates are given. accession numbers of the used sequences and scientific names of animals are; human (homo sapiens) c3, c4, c5, a2m, cd109, cpamd8, and pzp (np_000055, p0c0l4, aaa51925, p01023, np_598000, np_056507 and caa38255), squid (euprymna scolope) c3 (acf04700), sea urchin (strongylocentrotus purpuratus) c3 and cpamd8 (np_999686 and xp_785018), sea squirt (ciona intestinalis) c3-1, c3-2, cd109, and cpamd8 (np_001027684, cac85958, np_001027688 and xp_002124325), lamprey (lethenteron japonicum) c3 and a2m (q00685 and baa02762), hagfish (eptatretus burgeri) c3 and cd109 (p98094 and bad12264), horseshoe crab 1 (cainoscorpius rotundicauda) c3 (aaq08323), horseshoe crab 2 (tachypleus tridentatus) c3 and a2m (bah02276 and baa19844), amphioxus (branchiostoma floridae) c3-1, c3-2, cd109 and cpamd8 (aam18874, xp_002612866, xp_002586872 and xp_002612485), coral 1 (swiftia exserta) c3 (aan86548), coral 2 (acropora millepora) c3 (abk78771), sea anemone (haliplanella lineate) c3-1, c3-2, a2m, and cd109 (ab481383, ab481384, ab481385 and ab481386), fruit fly (drosophila melanogaster) tep1 (np_523578), mosquito (anopheles gambiae) tep (aag00600), nematode (caenorhabditis elegans) cd109 (np_493614), clam (hyriopsis cumingii) a2m (abj89824), clam (venerupis decussatus) c3 (fj392025), ticks (ornithodoros moubata) a2m (aan10129), snail (euphaedusa tau) cd109 (bae44110), sea cucumber (apostichopus japonicus) c3 (adn97000), acorn worm (saccoglossus kowalevskii) c3 (xp_002732077), placozoa (trichoplax adhaerens) cd109 and tep (xp_002111588 and xp_002111589). 25 which catalyze the cleavage of the thioester bond are present in most members, although they are missing from human a2m. thus, the common ancestor molecule likely had these characters, showing a closer similarity to human c3 than to human a2m. for the position for the ana or bait domains and the c-terminus where c345c, kazal and gpi attachment domains appear, it is difficult to deduce the ancestral state, since none of them is in majority. therefore, the common ancestor of the c3 and a2m subfamilies most probably was, secreted protein synthesized with the signal peptide, two subunit (α and β) chain protein, and endowed with the thioester bond with the catalytic histidine. cnidarian c3 and complement system in addition to c3-1 and c3-2 genes identified from sea anemone species, haliplanella lineate (fujito et al., 2010) and nematostella vectensis (kimura et al., 2009), the c3 genes have been reported from two coral species, swifta exserta (dishaw et al., 2005) and acropora millepora (miller et al., 2007). the basic domain structure of the c3 family and the signal peptide for secretion were completely conserved in these cnidarian c3 proteins (table 1). the conservation of the c3a anaphylatoxin region, the thioester site (gcgeq), and the catalytic his residue for cleavage of thioester suggest that the cnidaria c3 proteins retain the inflammatory and opsonic functions. the c345c domain was also identified and five cys residues characteristic to this domain are perfectly conserved. since the mammalian c345c domain of c3 is involved in the interaction with the vwa domain of bf, in the alternative pathway c3 convertase, c3bbb (rooijakkers et al., 2009; torreira et al. 2009), presence of this domain in cnidarian c3 proteins imply that the cnidarian c3 proteins are capable to form c3 convertase with bf. the conservation of the α/β chains processing site ‘rxxr’ suggested that cnidarian c3 proteins are processed into the two-subunit chain structure. however, both of cys residues involved in the disulfide linkage between the α and β chains of mammalian c3 (dolmer and sottrup-jensen, 1993) were substituted into another residue, and there is no other pair of cys residues, which is possibly involved in the inter-chain linkage. the most unique feature of cnidarian c3s is the presence of about 50 residue-long, highly lys/arg-rich insertion in the mg8 domain. although insertion into the mg8 domain was also found in the horshoe crab c3 (zhu et al., 2005), ascidian c3 (nonaka et al., 1999; marino et al., 2002), lamprey c3 (nonaka and takahashi, 1992) and vertebrate c4, the lys/arg content of cnidarian c3 is extremely high (~60 %), likely providing a unique, extremely positive charge to this region of cnidarian c3. the α/γ chain-processing motif ‘rxxr’ has been reported in the jawed vertebrate c4, lamprey c3, and horseshoe crab c3 at this region. it is possible, therefore, that the lys/arg-rich insertion was present in the common ancestor of c3 subfamily proteins, and the α/γ chain-processing motif is its remnant. in addition to two c3, two bf and one masp genes were identified in the draft genome sequence of nematostella vectensis (kimura et al., 2009). in contrast, no c6 and factor i (if) family genes were identified. the deduced primary structures of the cnidarian bf and masp shared the unique domain structures and most functionally critical amino acid residues with their mammalian counterparts, suggesting the conservation of basic biochemical functions throughout the metazoan evolution. in situ hybridization analysis indicated that all five cnidarian complement genes are co-expressed at the tentacles, the pharynx and the mesentery in an endoderm-specific manner (kimura et al., 2009). these results indicated that the multi-component complement system composed of at least c3, bf and masp was established in a common ancestor of cnidaria and bilateria more than 600 mya to protect coelenterons, the primitive gut cavity with putative circulatory functions. protostome c3 and complement system the presence of c3 in cnidaria indicated that the c3 gene has been established prior to the divergence of cnidaria and bilateria. however, the firstly-elucidated protostome genomes of drosophila melanogaster (adams et al., 2000) and caenorhabditis elegans (consortium 1998) did not contain the c3 gene. moreover, our attempt to rt-pcr amplify tep cdnas using universal primers compatible with both the c3 and a2m subfamily members in four lophotrochozoa species, planocera multitentaculata (platyhelminthes), siphonosoma cumanense (sipuncula), hesione reticulata (annelida) and euphaedusa tau (mollusca), resulted in isolation of only a2m family cdna, suggesting the absence of c3 in these species (kim, fujito and nonaka, unpublished data). on the other hand, the c3 subfamily members have been reported from ecdysozoan horseshoe crab, carcinoscorpius rotundicauda and tachypleus tridentatus (zhu et al., 2005; ariki et al., 2008) and lophotrochozoan squid, euprymna scolope (castillo et al., 2009) and clam, hyriopsis cumingii (prado-alvarez et al., 2009). these results indicate that whereas a2m has been retained by all protostomes, c3 has been lost secondarily multiple times during the protostome evolution. the deduced primary structure of these protostome c3 proteins did not show any large insertion or deletion compared to vertebrate c3, and retained all primary structure motifs reported to have functional importance, such as the thioester, anaphylatoxin, and α−β processing regions (table 1). for squid and clam, the composition of the complement system is still unknown except that the bf-like gene was identified from clam (prado-alvarez et al., 2009). however, the active center ser is substituted into ile in this bf-like molecule, making it unlikely that this molecule functions as the catalytic subunit of a c3 convertase. in contrast, horseshoe crab c3 is the best-analyzed c3 of invertebrates at the biochemical level. molecular cloning of the c3 has been reported from two distantly-related horseshoe crab species, carcinoscorpius rotundicauda (cr) (zhu et al., 2005) and tachypleus tridentatus (tt) (ariki et al., 2008). interestingly, the primary structures of crc3 and ttc3 show quite high degree of identity (fig. 3), in spite of a remote phylogenetic relationship of these two species. crc3 26 table 1 structural features of invertebrate c3s showed binding activity to staphylococcus aureus and other bacteria and this binding activity was inhibited by hydroxylamine, suggesting the involvement of the thioester bond of crc3 in binding. in addition, divalent cation-dependent induction of trypsin-like proteolytic activity in horseshoe crab plasma was observed, although it is still to be demonstrated directly if this activity is due to crbf, and if crbf directly activates crc3 (zhu et al., 2005). for ttc3, factor c originally identified as an lps-sensitive initiator of hemolymph coagulation stored within hemocytes was identified as an activating enzyme (ariki et al., 2008). thus, upon invasion of gram-negative bacteria, an lps-responsive factor c plays the central role in the initiation of the horseshoe crab complement activation. however, factor c seems not to be involved in ttc3 binding to s. aureus, suggesting the presence of other c3 activating enzymes in horseshoe crab, possibly the c3 convertase comprising bf. in support of this view, direct binding of factor c and bf to prrs was reported in c. rotundicauda (le saux et al., 2008). the apparent functional substitution by factor c for masp suggest a significant deviation of the horseshoe crab complement system from the other complement systems. in this context, the reported primary structure of horseshoe crab bf shows curious characteristics (zhu et al., 2005). the serine protease domains of mammalian bf and c2 are known to be unique in the following points (volanakis and arlaud, 1998). firstly, although they cleave peptide bonds following a positively-charged amino acid residue, they lack asp189 (chymotrypsinogen numbering) which in trypsin positions the positively charged p1 residue. instead, asp226 is responsible for substrate specificity of human bf (jing et al., 2000). secondary, they lack the highly conserved and functionally important n-terminal sequences of serine proteases. thus, following activation their serine α−β ana thioester catalytic his α−γ haliplanella lineate 1 rkkr cc,c,c,cc#1 gcgeq h kr-rich haliplanella lineate 2 ? ?,cc gcgeq h kr-rich swifta exserta rkrr cc,c,c,cc gcgeq h kr-rich acropora millepora rkkr cc,c,c,cc gcgeq h kr-rich euprymna scolopes rykr cc,c,cc gcgeq h venerupis decussatus rkrr c,cc,c,c,c gcgeq h rkkr carcinoscorpius rotundicauda rkkr cc,c,c,cc gcgeq h lr-rich tachypleus tridentatus rkkr cc,c,c,cc gcgeq h lr-rich apostichopus japonicus rrrr c,c,cc gcgeq h dr-rich strongylocentrotus purpuratus rrkr c,c gcgeq h saccoglossus kowalevskii #2 cc gcgeq h dr-rich branchiostoma belcheri 1 #2 c,cc gcgeq h edsr-rich branchiostoma belcheri 2 #2 c,cc gcgeq h edsr-rich ciona intestinalis 1 rkkr c,c gcgeq h ciona intestinalis 2 rnkr c,c gcgeq h eptatretus burgeri rrkr cc,c,c,cc gcgeq h lethenteron japonicum rkpr cc,c,c,cc gcgeq h rrrr homo sapiens c3 rrrr cc,c,c,cc gcgeq h homo sapiens c4 rkkr cc,c,c,cc gcgeq h rrrr homo sapiens c5 rprr cc,c,c,c,cc gsaea p #1distrition of the cys residues in the ana domain is shown. "," represents multiple amino acid residues. #2blank indicates the absence of characteristic residues. however, the absence of the α−β processing signal and some cys residues in the ana domain of these three c3s could be due to inaccurate gene prediction. 27 fig. 4 structural features of human and sea anemone tep molecules. structural features of human (h. sapiens) and sea anemone (h. lineate) tep molecules are compared, and the structure of the common ancestor of the tep molecules is deduced based on maximum parsimony principle. question marks in the ancestor molecule indicate that there are multiple candidates with equal probability. protease domains remain attached to a vwa domain. comparison of amino acid sequences of the serine protease domain of bfs from various animals shown in figure 5 suggests that both structural specializations occurred in a common ancestor of jawed vertebrates (terado et al., 2001). thus, both structural specializations seem to have occurred simultaneously leading to a drastic change in structure and activation mechanism of bf. the horseshoe crab bf is exceptional in that it retains the original specificity-determining asp189 without retaining the conserved n-terminal sequence of the serine protease domain. thus the activation mechanism of the horseshoe crab bf, and whole complement system also, could be a highly deviated one, although the details are still to be clarified. deuterostome c3 and complement system c3 sequence of invertebrate deuterostome has been reported from sea squirt (urochordata) (nonaka et al., 1999; raftos et al., 2002; azumi et al., 2003; pinto et al., 2003), amphioxus (cephalochordata) (suzuki et al., 2002; holland and gibson-brown, 2003), acorn worm (hemichordata) (xp_002732077), sea urchin (al-sharif et al., 1998) and sea cucumber (echinodermata) (adn97000) thus far. since gene duplications among c3, c4 and c5, and subsequent functional differentiation among them likely occurred at the early stage of jawed vertebrate evolution (nonaka and takahashi, 1992; nonaka and kimura, 2006), these c3 genes together with cyclostome c3 genes are considered to represent the ancestral state before the gene duplications. all these invertebrate deuterostome c3 proteins retain the thioester site, the his residue which catalyzes the cleavage of the thioester bond, and the α−β processing site. in contrast, the cys residues in the ana domain and the leu-x-arg sequence at the cleavage site of the c3 convertase are poorly conserved by these c3 proteins (table 1). in addition to c3, bf gene has been identified from sea urchin (smith et al., 1998), and several complement genes have been identified from sea squirt, mainly from two species, halocynthia roretzi and ciona intestinalis. those genes are; bf (azumi et al., 2003; yoshizaki et al., 2005), masps (ji et al., 1997; azumi et al., 2003), mannan-binding lectin (mbl) (bonura et al., 2009), ficolin (kenjo et al., 2001), complement receptor 3 (cr3) alpha (miyazawa et al., 2001) and beta (miyazawa et al., 2001) and glucose binding lectin (gbl) lacking the collagen domain as a possible functional substitute for mbl (sekine et al., 2001). thus, the sea squirt complement system is the best-analyzed invertebrate complement system from the viewpoint of the component genes. for the functional aspect, opsonic function of the sea squirt complement system has been demonstrated in h. roretzi, where c3, ficolin and gbl proteins isolated from the body fluid work together as opsonin. moreover, the c3a fragment was shown to have a chemotactic activity (pinto et al., 2003; raftos et al. 2003), indicating that the role of the complement system in inflammation is also conserved between mammals and urochordates. 28 fig. 5 evolution of bf primary structure. the primary structures of bf and c2 of various animals were compared at two regions of the serine protease domain. the upper panel: the region around the active center ser corresponding to 656th-746th residues of human bf. the active center ser is marked by #. the asp residue at the s1 pocket essential for the trypsin-like specificity is shown in red. the lower panel: the n-terminal region of the serine protease domain orresponding to 478 th 495 th residues of human bf. the possible proteolytic activation site is shown in red. in contrast, the third activity of the mammalian complement system, cytolytic activity, has not been recognized in the urochordate complement system. evolution of the complement system figure 1 summarize the distribution of the complement genes with the characteristic domain structure in the representative species of cnidaria (n. vectensis), protostomia (c. rotundicauda), invertebrate deuterostomia (c. intestinalis) and vertebrata (homo sapiens). c3 arose in a common ancestor of cnidaria and bilateria more than 600 mya from an ancestral tep gene with unknown function. bf and masp seem to be generated at the same time, establishing a primitive complement system consisting of c3, bf and masp. conservation of the c3 c345c domain and the bf vwa domain suggests that they are able to assemble to form the alternative pathway c3 convertase, c3bbb. masp is likely an initiating enzyme to induce c3bbb formation, and covalent binding of c3b to the surface of foreign molecules most probably enhanced phagocytosis. in addition to this opsonic function, the conservation of the ana domain between cnidaria and vertebrata c3 suggests that the primitive complement system also had another function to induce inflammation. by unknown reason, the complement system has been lost multiple times in the protostome lineages. even in some protostome species which retains c3, masp has not been identified, and bfs has highly deviated structure, either proteolitically inactive or lacking the conserved n-terminal sequence of the serine protease domain. thus, the activation mechanism of protostome complement system should be very different from that of cnidaria or vertebrata. interestingly, some insect species lacking the c3 gene have tep molecules functioning as opsonin like vertebrate c3. however, molecular phylogenetic analyses indicate that these insect teps belong to the a2m subfamily, suggesting that the similar functions were attained by convergent evolution. in contrast, the complement system of invertebrate deuterostome represented by c. intestinalis shows much closer similarity to the mammalian complement system. however it lacks if, the main component of the regulatory mechanism. in addition, although there are several c6-like genes with the membrane attack complex/perforin (macp) domain in the c. intestinalis genome, all of them lack the c-terminal short consensus repeat (scr) and factor i/membrane attack complex (fim) domains reported to be essential for interaction with other complement components. thus, it is unlikely that these c6-like molecules are integrated in the urochordate complement system. these results indicate that the lytic 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noctuidae) sk mirhaghparast, a zibaee, j hajizadeh department of plant protection, faculty of agricultural sciences, university of guilan, rashtiran, 416351314 accepted october 27, 2013 abstract in the current study, fifth larval instars of spodoptera littoralis were injected by spores of beauveria bassiana and metarhizium anisopliae to find their effects on cellular immunity and enzymes involved in intermediary metabolism. the highest numbers of plasmatocytes were observed 6 hours post-injection for both entomopathogenic fungi although there were the slight significant differences between time intervals of 3-12 hours. the highest numbers of granulocytes were observed 6 hours post-injection for both fungi although slight statistical differences were found by injecting spores of b. bassiana after 3-6 hours. injection of larvae by b. bassiana spores caused the highest number of nodules 12 hours postinjection but spores of m. anisopliae caused the highest number of nodules after 1-6 hours of postinjection. the highest activity of phenoloxidase was obtained 6-12 hours post-injection by b. bassiana spores while the highest enzymatic activity was found 12 hours after injection by m. anisopliae spores. in case of assayed enzymes including, alanine aminotransferase, aspartate aminotransferase, δglutamyl transferase, acid phosphatase, alkaline phosphatase and lactate dehydrogenase, the highest activities were observed 6-12 hours post-injection by fungal spores. the results demonstrated that the highest physiological phenomena like immune responses and intermediary metabolisms were occurred 12 hours post-injection by entomopathogenic fungi. determination of these processes could be helpful to improve quality of entomopathogenic fungi and their efficiency to decrease population outbreaks of pests. key words: entomopathogenic fungus, spodoptera littoralis, immunity, intermediary metabolism   introduction the entomopathogenic fungi are the promising agents that are used against insect pests for several decades. these organisms include taxa of several fungal groups like hypocreales of ascomycota that beauveria bassiana and metarhizium anisopliae are the two most recognized species (vincent et al., 2007). b. bassiana and m. anisopliae grow naturally throughout the world and acts as parasites of many arthropod species causing white and green muscardine diseases due to the color of their spores (vincent et al., 2007). besides entomopathogenic fungi caused natural mortality on insects, these agents are environmentally safe so there is a worldwide interest of their using and improvement for biological control of insects. when a spore adheres ___________________________________________________________________________ corresponding author: arash zibaee department of plant protection, faculty of agricultural sciences university of guilan, rasht, 41635-1314, iran e-mail: arash.zibaee@gmx.com to cuticle of insects, a germ tube is generated and pass through the integument by mechanical and enzymatic (e.g. chitinases, proteases, lipases and etc) processe. when it reaches to the hemocoel, it produces blastospores which are the final pathogenic parts for host infection (vincent et al., 2007). hemolymph of insects is a medium for several physiological processes like immune responses and intermediary metabolism. when an invader enters hemocoel of insects, hemocytes are engaged to remove non-self-target by phagocytosis, nodule formation, encapsulation, synthesis of antimicrobial peptides and reactive metabolites (beckage, 2008). intermediary metabolism consists of various pathways in which ingested and stored nutrients such as carbohydrates, lipids and proteins are processed to produce energy via their degradation or synthesis. in details, locked up energy in the nutrients is released by several biochemical reactions like glycolysis, β-oxidation of lipids, citric   110 a) b) fig. 1 effects of b. bassiana and m. anisopliae injections on number of plasmatocytes (a) and granulocytes (b) in s. littoralis larvae. statistical differences have been shown by various letters (p≤0.05). acid cycle, electron transport system, transaminations and etc (nation, 2008). spodoptera littoralis boisduval (lepidoptera: noctuidae) known as african cotton leaf-worm or mediterranean brocadeis is one of the most destructive agricultural pests in subtropical and tropical regions that causes severe damages of plants belonging to 44 different families including grasses, legumes, crucifers and deciduous fruit trees by highly economic importance (abdelmegeed, 1975). larvae intensively fed on leaves and sometimes fruits of crops. the pest could be active nine months of a year and complete a generation within 30 days (gharib, 1979). although cultural procedure, sanitation and chemical control are used to decrease population outbreaks of the pest but it annually causes several damages worldwide. in addition to activation of immune responses, microbial infections could alter intermediary metabolism of insects by affecting activity of involved enzymes and detoxifying ones although majority of studies have been concentrated on evaluation of general esterases and phosphatases   111 a) b) fig. 2 effects of b. bassiana and m. anisopliae injections on nodule formation in s. littoralis larvae (a). an image of observed nodule (b). statistical differences have been shown by various letters (p≤0.05). (sokolova and sundukov, 1999; xia et al., 2000; xia et al., 2001). since b. bassiana and m. anisopliae are the two main entomopathogenic fungi, their interaction with immune system and intermediary metabolism of s. littoralis could be useful to improve efficiency of these microbial agents. hence, objectives of the current studies are (i) determination of larval cellular responses to b. bassiana and m. anisopliae, and (ii) determination of changes in intermediary metabolism by measurements of involved enzymes. materials and methods insect rearing larvae were collected from strawberry field in pirbazar, rasht (north of iran) and reared on the same leaves at 25±1 °c, 70% of relative humidity   112 fig. 3 effects of b. bassiana and m. anisopliae injections on phenoloxidase activity in s. littoralis larvae. statistical differences have been shown by various letters (p≤0.05). and 16l:8d of photoperiod (the larvae was identified by dr. jalil hajizade, professor of insect taxonomy in our department). fifth larval instars were used to carry out the experiments. entomopathogenic fungi culture b. bassiana (isolate b3 isolated from fashand soil-iran) and m. anisopliae (isolated from rice fields) were cultured at 25 ± 1 °c on sabouraud dextrose agar (merck co., germany) (ph = 5.6) amended with 1% yeast extract. after 14 days, conidia were washed off with a 0.01% aqueous solution of tween 80 (sigma aldrich co., austria) and different concentrations of spores were prepared. effect of fungal spore on hemocyte numbers   113 to determine possible changes of hemocyte numbers followed by treatment of entomopathogenic fungi, fifth larval instars were injected laterally into the latest segment of thorax with 1 μl of a 105 spores/ml concentration of mentioned fungal isolates. hemolymph was collected at intervals of 1, 3, 6, 12, 24 and 48 hours after injection. samples of hemolymph were bled into 1 ml of ice–cold anticoagulant buffer in 1.5 ml plastic tubes. the tubes were gently inverted 5 to 7 times to facilitate mixing, and both total and different hemocyte numbers were counted using an improved neubauer hemocytometer (chemkind co. china). for each treatment, 6 larvae were used and the experiment had five replicates (n=30, n=5). effect of fungal spores on nodulation injections were carried out as described in the previous section to find number of formed nodules in response to spores of different entomopathogenic fungi. number of nodules was calculated at intervals of 1, 3, 6, 12, 24 and 48 hours post-injection. injected larvae were chilled on ice, hemolymph was gathered in a capillary tube, and then 200 μl of samples in three replicates were poured in a hemocytometer and nodules were counted. effects of fungal spores on phenoloxidase activity (po) after injecting of larvae with fungal spores, hemolymph was collected at mentioned intervals. a hemocyte lysate supernatant was prepared after injections based on leonard et al. (1985). collected hemolymph from larvae was mixed with anticoagulant buffer and centrifuged at 13,000 rpm for 5 min; the supernatant was discarded and the pellet washed gently twice with a phosphate buffer (0.02 m, ph = 7.1). cells were homogenized in 200 µl of phosphate buffer centrifuged at 13,000 rpm for 15 min, and the hemocyte lysate supernatant was used in po assays. samples (10 µl) were preincubated with phosphate buffer at 30 °c for 3 min before the addition of 20 µl of 10 mm aqueous solution of l-dihydroxyphenylalanin (sighma-aldrich co., usa) as substrate. the mixture was incubated for an additional five min at 30 °c and po activity was measured at 495 nm. one unit of po activity a) b) c) fig. 4 effects of b. bassiana and m. anisopliae injections on alt (a), ast (b) and δ-gt (c) activities in s. littoralis larvae. statistical differences have been shown by various letters (p≤0.05).   114   115 represents the amount of enzyme required to produce an increase in absorbance of 0.01 min−1 (dularay & lackie, 1985). activity in treated assays was compared with that of controls (n=3). sample preparation hemolymph samples gathred from larvae in each time intervals were poured in 1.5 ml tubes containing 100 µl of anticoagulant solution, centrifuges and supernatant was used as enzymatic source of intermediary metabolism. estimation of aspartate (ec 2.6.1.1) and alanine aminotransferases (ec 2.6.1.1) alanine aminotrasferase (alt) and aspartate aminotransferase (ast) were measured using thomas' (1998) procedure. this assay was done by ast and alt kit (biochem co, iran). on this basis, solution 1 and 2 were mixed (4:1). then, samples were added and absorption was read at 340 nm. estimation of δ-glutamyl transferase the method described by tate and meister (1985) was used to assay activity of �-glutamyl transferase. in a kit by ziest-chem. co. (tehran, iran), reagent a was incubated with samples for 5 min, then reagent b was added and absorbance was read at 405 nm. assay of estimation of acid (ec 3.1.3.2) and alkaline phosphatases (ec 3.1.3.1) the enzyme assays were carried out as described by bessey et al. (1946). the buffered substrate (phosphate buffer, 0.02 m, ph 7.2) was incubated with samples for 30 min. alkali were added to stop the reaction and adjust the ph for the determination of concentration of the product formed. the spectral absorbance of p-nitrophenolate was maximal at 310 nm. the molar absorbance of pnitrophenolate at 400 nm is about double that of pnitrophenyl phosphate at 310 nm. on converting the p-nitrophenolate into p-nitrophenol by acidification, the absorption maximum is shifted to about 320 nm with no detectable absorption at 400 nm. estimation of lactate dehydrogenase (ec 1.1.1.27) for evaluating lactate dehydrogenase (ldh), the king (1965) method was used. to standardize volumes, 0.2 ml nad+ solution was added to the test tubes and 0.2 ml of water was added to control test tubes, each containing 1 ml of the buffered substrate. the sample containing 0.01 ml was also added to the test tubes. test tube samples were incubated for exactly 15 min at 37°c and then arrested by adding 1 ml of color reagent (2,4 dinitrophenyl hydrazine) to each tube and the incubation continued for an additional 15 min. after cooling at room temperature, 10 ml of 0.4n naoh was added to each tube to make the solutions strongly alkaline. at exactly 60 s after the addition of alkali to each tube, the intensity of color was measured at 440 nm. protein assay protein concentrations were assayed according to the method described by lowry et al. (1951). statistical analysis all data were compared by one-way analysis of variance (anova) followed by tukey's studentisized test when significant differences were found at p ≤ 0.05 and marked in tables with letters. results and discussion it was found that injection of s. littoralis larvae by b. bassiana and m. anisopliae spores significantly affected cellular immunity and phenoloxidase activity at various time intervals. the highest numbers of plasmatocytes were observed 6 hours post-injection for both injected spores of the entomopathogenic fungi although there were the slight significant differences between time intervals of 6-24 hours (f= 7.65, pr>f: 0.0019; f=14.87, pr>f: 0.0001) (figure 1a). the highest numbers of granulocytes were observed 6 hours post-injection for b. bassiana and 3-6 hours for m. anisopliae (f= 5.31, pr>f: 0.0084; f=56.26, pr>f: 0.0001) (figure 1b). meanwhile, injection of b. bassiana spores caused higher numbers of these hemocytes in the larvae and overall numbers of plasmatocytes were higher than that of granulocytes (fig. 1a,b). several studies have been reported fluctuation of hemocyte numbers in the immune challenged insects such as melanoplus sanguinipes fabricius (orthoptera: acrididae), schistocerca gregaria l. (orthoptera: acrididae), periplaneta americana l. (blattaria: blattidae) spodoptera exigua hubner (lepidoptera: noctuidae), galleria mellonella l. (lep., pyralidae), reticulitermes flavipes kollar (isoptera: rhinotermitidae), oxya japonica thunberg (orthoptera: acrididae), eurygaster integriceps puton (hemiptera: scutelleridae), (bidochka and khachatourians, 1987; gunnarsson, 1988; hung and boucias, 1992; sewify and hashem, 2001; chouvenc et al., 2009; anggraeni et al., 2011; zibaee et al., 2011). these fluctuations could be attributed to some factors like; taking part of hemocytes in nodule formation after injection, cytotoxic effect of fungal secondary metabolite on hemocytes and composition of spore surface mainly hydrophobin proteins. meanwhile, differences in numbers of plasmatocytes and granulocytes followed injection by b. bassiana and m. anisopliae could be attributed to different properties of fungal spores in production of secondary metabolites and composition of spore surface. nodule formation is one of the major cellular responses of insects against pathogens since it is considered to be the last defensive line (chouvenc et al., 2009). injection of larvae by b. bassiana spores caused the highest number of nodules 12 hours post-injection (f= 7.52, pr>f: 0.0013) but spores of m. anisopliae caused the highest number of nodules 3-6 hours (f= 11.23, pr>f: 0.0052) although spores of b. bassiana caused higher number of nodules versus m. anisopliae (fig. 2). these findings are correspondence with the higher number of plasmatocytes and granulocytes after 3 hours of injection. hence it could be concluded that higher production of plasmatocytes and granulocytes is due to involvements of these hemocytes in nodule formation. a) b) fig. 5 effects of b. bassiana and m. anisopliae injections on acp and alp activities in s. littoralis larvae. statistical differences have been shown by various letters (p≤0.05). the highest activity of phenoloxidase was obtained 6-12 hours post-injection by b. bassiana spores (f= 214.9, pr>f: 0.0001) while the highest enzymatic activity was found 12 hours after injection by m. anisopliae spores (f= 12.2, pr>f: 0.0043) (fig. 3). overall activity of the enzymes in the larvae injected by m. anisopliae spores was higher than that of b. bassiana spores (fig. 3). phenoloxidases are activated upon wounding or infection as part of the innate immune response (kanost and gorman 2008). the enzymes have two biochemical functions in hydroxylation of tyrosine to form dihydroxyphenylalanine and oxidizing o-diphenols to form quinones (gorman et al., 2007). after forthcoming reactions, quinones changes to form melanin, which is deposited on the surface of encapsulated parasites, hemocyte nodules, and wound sites (kanost & gorman 2008). results of the current study are attributed to melanin deposition to complete of nodule formation process.   116 fig. 6 effects of b. bassiana and m. anisopliae injections on ldh activity in s. littoralis larvae. statistical differences have been shown by various letters (p≤0.05). melanin deposition prevents absorption of nutrients to kill parasites due to starvation (chen and chen, 1995). also, formation of cytotoxic reactive oxygen and nitrogen intermediates during melanin synthesis causes to kill invading organisms (nappi and christensen, 2005). alanin amino transferase (alt) and aspartate aminotransferase (ast) are the enzymes that are involved in transamination process of various tissues (nation, 2008). these enzymes catalyze conversion of alanine, aspartate and α-ketoglutarate to oxaloacetate and glutamate. any changes in activities of alt and ast are due to existence of a physiological challenge in body such as microorganism infections, damage to some tissues or being a toxic material (giboney et al., 2005). meanwhile, �-glutamyl transferase is another aminotransferase. also, it plays a key role in the gamma-glutamyl cycle, a pathway for the synthesis and degradation of glutathione and drug and xenobiotic detoxification (tate and meister, 1985). in the current study, the highest activities of two aminotransferase were observed 6-12 hours post injection (figure 4a,b; f= 12.24, pr>f: 0.0043; f=12.09, pr>f: 0.0042). δ-glutanyl transferase (δgt) showed the highest activity 3-12 hours post injection (figure 4c; f= 12.24, pr>f: 0.0042; f=26.32, pr>f: 0.0003). although activity of δgt had no significant differences in the larvae injected by b. bassiana and m. anisopiae spores but the larvae treated by b. bassiana had higher activity of alt and larvae treated by m. anisopliae had higher activity of ast (fig. 4). these results implies that immune challenge of larvae by entomopathogenic fungi cause a protein shortage due to differentiation of hemocytes and detoxifying of fungal secondary metabolites. so, enzymes involve in transamination get the higher activity to increase availability of amino acids for physiological processes. these observations are similar to insects treated by chemical insecticides (ender et al., 2005; etebari et al., 2005; zibaee et al., 2008; zibaee et al., 2011). acid (acp) and alkaline phosphatases (alp) are the hydrolytic enzymes responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids in alkaline and acidic conditions, respectively under name of dephosphorylation (zibaee et al., 2011). also, these enzymes are involved in lipid hydrolysis in several tissues like midgut, hemolymph and fat bodies (zibaee et al., 2011). overall activities of acp and alp in the larvae injected by m. anisopliae were higher than those by b. bassiana (fig. 5) but both enzymes showed the highest activity 12 hours post injection (fig. 5a,b; f= 19.38, pr>f: 0.0012; f=31.09, pr>f: 0.0003). activity elevations of these enzymes in immune challenged s. littoralis could be due to energy demands for compensatory mechanisms like treated insects by chemical insecticides. lactate dehydrogenase catalyzes the interconversion of pyruvate and lactate with concomitant inter-conversion of nadh and nad+ in glycolysis cycle (kaplan and pesce, 1996). shortage in oxygen or tissue breakdown elevates levels of ldh considering as a medicinal biomarker (kaplan and pesce, 1996). in the current study, the highest activity of ldh was observed 12 hours post-injection although there was a slight statistical difference between intervals 6-12 hours (fig. 6; f= 26.36,   117   118 pr>f: 0.0001; f=43.27, pr>f: 0.0001). now, it has been determined that entomopathogenic fungi produce toxic secondary metabolites to disable immune system of insects. these materials kill hemocytes and intervene in phagocytosis or nodule formation against parasites (zibaee et al., 2011). so, this could be one of the reasons for elevation of ldh activity in immune challenged larvae of s. littoralis. another reason could be energy demands for removing parasite from hemolymph by elevation of glycolysis rate leading to conversion of pyruvate to lactate. results of the current study clearly revealed different effects of b. bassiana and m. anisopliae spores on cellular immunity and phenoloxidase activity in the fifth larval instars of s. littoralis. these differences were due to surface properties of spores and their capability in production of secondary metabolites which are toxic on hemocytes and other tissues. moreover, findings on changes of enzymatic activity involved in intermediary metabolism support results of previous studies on effect of fungal spores and secondary metabolite on chemical composition of insect hemolymph (madziara-borusiewiez and kucera, 1978; sujak et al., 1978; kol’chevskaya and kol’chevskii, 1988; shiotsuki and kato, 1996; sokolova and sundukov, 1999; xia et al., 2000, 2001; serebrov et al., 2006; zibaee et al., 2009). meanwhile, it was obtained that 6-12 hours postinjection the highest physiological phenomena were observed in both immune responses and intermediary metabolisms. hence, it could be concluded that pathogenicity of an entomopathogenic fungi may occur via overcoming on immune responses and discrepancies of intermediary metabolisms. these finding show significant role of these agents against agricultural pests and might be used to improve their quality and efficiency in future. references abdel-megeed mi. field observations on the vertical distribution of the cotton leafworm, spodoptera littoralis on cotton plants. ang. entomol. 78: 597-62, 1975. anggraeni t, melanie p, putra re. cellular and humoral immune defenses of oxya japonica (orthoptera: acrididae) to entomopathogenic fungi metarhizium anisopliae. entomol. res. 41: 1–6, 2011. beckage ne. insect immunology. academic press. 348 pp, 2008. bessey oa, lowry oh, brock mj. a method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. j. biol. chem. 164: 321–329, 1946. bidochka mj, khachatourians gg. hemocytic defense response to the entomopathogenic fungus beauveria bassiana in the migratory grasshopper melanoplus saguenipens. entomol. experim. app. 45: 151–156, 1987. chen cc, chen cs. brugia pahangi: effects of melanization on the uptake of nutrients by microfilariae in vitro. experim. parasitol. 81: 72– 78, 1995. chouvenc t, su ny, robert a. cellular encapsulation in the eastern subterranean termite, reticulitermes flavipes (isoptera), against infection by the entomopathogenic fungus metarhizium anisopliae. j. invert. pathol. 101: 234–241, 2009. dularay b, lackie am. haemocytic encapsulation and the prophenoloxidaseactivaion pathway in the locust schistocerca gregaria. j. insect. physiol. 15: 827-834, 1985. el-hawary fm, abd el-salam ame. laboratory bioassay of some entomopathogenic fungi on spodoptera littoralis (boisd.) and agrotis ipsilon (hufn.) larvae (lepidoptera: noctuidae). egypt. acad. j. biol. sci. 2: 14, 2009. ender i, ferah a, kemal b, ahmet g. biochemial stress indicators of greater wax moth, galleria mellonella exposure to organophosphorus insecticides. j. econ. entomol. 98: 358–366, 2005. etebari k, bizhannia ar, sorati r, matindoost l. biochemical changes in haemolymph of silkworm larvae due to pyriproxyfen residue. pestic. biochem. physiol. 88: 14–19, 2007. gharib a. rahe pest in khozestan. j. pest. plant. pathol. 47: 161-178, 1979. giboney pt. mildly elevated liver transaminase levels in the asymptomatic patient. am. fam. physic. 71: 1105–1110, 2005. gorman mj, an c, kanost mr. characterization of tyrosine hydroxylase from manduca sexta. insect. biochem. mole. biol. 37: 1327-1337, 2007. hung sm, boucuas dg. influence of beauveria bassiana on cellular defense response of the beet army worm, spodoptera exigua. j. invert. pathol. 60: 152–158, 1992. kanost mr, gorman mj. phenoloxidases in insect immunity. in: insect immunology, by beckage, n. e. academic press. page 69-96, 2008. kaplan la, pesce aj. clinical chemistry – theory analysis and correlation, mosby-year book, mo, 1996. king j. the dehydrogenases or oxidoreductases. lactate dehydrogenase, in: practical clinical enzymology, van nostrand d, london, pp. 8393, 1965. leonard c, kenneth s, ratcliffe na. studies on prophenoloxidase and protease activity of blaberua craniifer haemocytes. insect. biochem. 15: 803-810, 1985. lowry oh, rosenbrough nj, farr ll, randall rj. protein measurement with the folin phenol reagent. j. biol. chem. 193: 265-75, 1951. madziara-borusiewiez k, kucera m. enzyme changes in galleria mellonella caused by an unknown pathogen from the larvae of acantholyda nemoralis (hymenoptera, pamphiliidae). acta. entomol. bohemoslica. 75: 353-356, 1978. nappi aj, christensen bm. melanogenesis and associated cytotoxic reactions: applications to insect innate immunity. insect. biochem. mole. biol. 35: 443–459, 2005. nation jl. insect physiology and biochemistry. 2nd edition. crc press. new york. 544 pp, 2008.   119 serebrov vv, gerber on, malyarchuk aa, martemyanov vv, alekseev aa, glupov vv. effect of entomopathogenic fungi on detoxification enzyme activity in greater wax moth galleria mellonella l. 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a high proportion of earthworms (60 %) exhibited inhibitory effects. additionally, the collapse in membrane potential and ps translocation were found to strongly correlate (r > 0.5) in 89 % of cases when induced by cadmium and copper. thus, heavy metals appear to induce death in celomocytes of e. hortensis through apoptosis by means of caspase dependent pathways. key words: cadmium and copper; jc-1; annexin v-fitc; mitochondrial membrane depolarization; phosphatidylserine translocation; caspases   introduction programmed cell death (pcd) is a universal cellular process in which the induction of a tightly regulated signal cascade ultimately leads to the destruction of the cell. it serves multiple roles such as facilitating death of the mother cell during spore formation in the bacterium bacillus subtilis, removal of tissue during fetal development, and elimination of infected tissues in both plants and animals (haanen and vermes, 1996; lewis, 2000; collazo and chacón, 2006; kinchen, 2010; harrison et al., 2011). in multicellular organisms, apoptosis is the mode of pcd characterized by many morphological changes such as membrane blebbing, nuclear dna fragmentation, loss of mitochondrial membrane potential (∆ψm), and phosphatidylserine (ps) translocation across the plasma membrane (ly et al., 2003; kiss, 2010). many of these apoptotic events are initiated through caspases which are cysteine proteases that initiate signal cascades by ___________________________________________________________________________ corresponding author: sheryl l. fuller-espie science department, cabrini college 610 king of prussia road, radnor, pennsylvania 190873698, usa e-mail: sfuller-espie@cabrini.edu cleaving at aspartic acid residues (shimizu and pommier, 1997). these signal cascades can be initiated in times of cellular stress by many factors such reactive oxygen species (ros) generation, exogenous h2o2 exposure, cytokine stimulation, and exposure to environmental pollutants such as heavy metals and cytotoxic organic compounds like camptothecin (shimizu and pommier, 1997; blanco et al., 2005; belyaeva et al., 2008; kim et al., 2008; fuller-espie et al., 2010). heavy metals such as cadmium and copper are increasingly becoming an environmental concern due to the improper disposal of electronic devices such as cell phones and batteries in which they are used. toxicity associated with accumulation of cadmium and copper in soil has been shown in both plant and earthworm (ew) models leading to decreased root growth, mortality, and dna damage due to increased production of 8-oxoguanine (an, 2004; arnold et al., 2004; hirano and tamae, 2010). ews are well suited for assessing soil quality since they bioaccumulate these metals and have relativity long life spans at an average of 4.25 years (morgan and morgan, 1988; mulder et al., 2007; hirano and tamae, 2010).   98 mailto:sfuller-espie@cabrini.edu fig. 1 representative flow cytometry profile for all assays. (a) shows a typical celomocyte profile of fsc (size) (abscissa) versus ssc (granularity) (ordinate) of extruded ew celomocytes where r1 = hyaline and granular amebocytes and r3 = eleocytes. (b) shows fsc (abscissa) versus fl-2/3 fluorescence (ordinate) of r1 gated celomocytes. r2 depicts pi-negative (fl-2 negative) or 7-aad negative (fl-3 negative) viable amebocyte populations. left dot plot indicates untreated celomocytes while right dot plot indicates cadmium-treated celomocytes. percentages in upper right and lower right quadrants are shown corresponding to dead and alive celomocytes, respectively. (c) depicts fl-1 (abscissa) versus fl-2 (ordinate) of amebocytes gated on r1 and r2 and stained with 7-aad plus jc-1 in ∆ψm assays. therefore, gated events represent only viable amebocytes that have not taken up 7-aad. events in lower right quadrant set at the median fl-1 fluorescence value of the untreated control were counted as positive. percentages in lower right quadrant are indicated for untreated (left dot plot) and cadmium-treated (right dot plot) celomocytes. (d) depicts fl-1 (abscissa) versus cell number (ordinate) of amebocytes gated on r1 and r2. events in the m1 marker set at the median fl-1 fluorescence value of the untreated control were counted as positive for ∆ψm (jc-1) and ps (annexin v fitc) assays. shown are ∆ψm results for untreated (left), valinomycin-treated (middle) and cadmium-treated (right) celomocytes. fsc = forward scatter; ssc = side scatter; fl-1 = relative fluorescence intensity of fitc and monomeric jc-1, fl-2 = relative fluorescence intensity of pi (ps assays) or j-aggregates (∆ψm assays), fl-3 = relative fluorescence intensity of 7-aad.   99 the primary goal of this in vitro study was to investigate the effects of the heavy metals cadmium and copper on the early apoptotic events of involving ∆ψm and ps translocation in the immune cells (celomocytes) of the ew eisenia hortensis (also known as the european nightcrawler). mitochondrial membrane potential (δψm) is the product of stored energy for the mitochondrial respiratory chain maintained by a balance of ions such as sodium and potassium within the mitochondrion. this potential difference normally exists at -180 to -200 mv and is necessary for transport of precursor proteins into the mitochondrion (martin et al., 1991; cossarizza et al., 1995). during early apoptosis the mitochondrial membrane becomes depolarized leading to increased permeabilization and the release of mitochondrial contents which, in addition to cytochrome c, contains a second mitochondria-derived activator of caspases (smacs) that initiate apoptosis through the deactivation of inhibitor of apoptosis (iap) proteins (fesik and shi, 2001). it has been shown that mitochondria are target organelles for heavy metals and can induce the apoptosis signal cascade (belyaeva et al., 2008). ps translocation from the inner to outer leaflet of the cellular membrane is also associated with early apoptosis   100 table 1 loss of ∆ψm in response to cadmium and copper exposure ∆ψm  cd50  cd125  cd250  cd500  cu50  cu125  cu250  cu500  ew‐j1  ‐  t  t*  ‐  ‐  t  t  ‐  ew‐j2  ‐  t  t  ‐  ‐  t  t  ‐  ew‐j3  ‐  t*  t*  t*  ‐  t*  t*  t*  ew‐j4  ‐  t  t  t*  ‐  t  t  t  ew‐j5  ‐  t  t*  t*  ‐  t  t  t  ew‐j6  ‐  ‐  ‐  t*  ‐  ‐  ‐  t  ew‐j7  ‐  ‐  ‐  t  ‐  ‐  ‐  t  ew‐j8  ‐  ‐  ‐  t  ‐  ‐  ‐  t  ew‐j9  ‐  ‐  ‐  t*  ‐  ‐  ‐  t*  ew‐j10  ‐  ‐  ‐  t  ‐  ‐  ‐  t  ew‐j11  ‐  ‐  ‐  t*  ‐  ‐  ‐  t  ew‐j12  ‐  ‐  ‐  t*  ‐  ‐  ‐  t  ew‐j13  ‐  ‐  ‐  t*  ‐  ‐  ‐  t  ew‐j14  ‐  ‐  ‐  t  ‐  ‐  ‐  t  ew‐j15  ‐  ‐  ‐  t  ‐  ‐  ‐  t  ew‐j16  ‐  ‐  ‐  t*  ‐  ‐  ‐  t*  ew‐j17  ‐  ‐  ‐  t*  ‐  ‐  ‐  t  ew‐j18  ‐  ‐  ‐  t*  ‐  ‐  ‐  t*  ew‐j19  ‐  ‐  ‐  t  ‐  ‐  ‐  t  ew‐j20  ‐  ‐  ‐  t*  ‐  ‐  ‐  t  ‐  20%  60%  66.7%  ‐  20%  20%  22.2%  % response of ew‐j1‐20  (0/0)  (1/5)  (3/5)  (12/18)  (0/0)  (1/5)  (1/5)  (4/18)  ew‐ja1  ‐  ‐  t*  t*  ‐  ‐  t*  t*  ew‐ja2  ‐  ‐  t  t  ‐  ‐  t*  t*  ew‐ja3  ‐  ‐  t  t*  ‐  ‐  t*  t*  ew‐ja4  t*  ‐  ‐  t*  t  ‐  ‐  t*  ew‐ja5  t  ‐  ‐  t*  t  ‐  ‐  t*  ew‐ja6  t  ‐  ‐  t*  t  ‐  ‐  t*  ew‐ja7  t*  ‐  ‐  t*  t  ‐  ‐  t*  ew‐ja8  t  ‐  ‐  t*  t  ‐  ‐  t  ew‐ja9  t*  ‐  ‐  t  t  ‐  ‐  t*  50%  ‐  33%  77.8%  0%  ‐  100%  88.9%  % response of ew‐ja1‐9  (3/6)  (0/0)  (1/3)  (7/9)  (0/6)  (0/0)  (3/3)  (8/9)  50%  20%  50%  70.4%  0%  20%  50%  44.4%  % response  (3/6)  (1/5)  (4/8)  (19/27)  (0/6)  (1/5)  (4/8)  (12/27)  total  58.7%  37%  response  (27/46)  (17/46)  shown is a summary of all ews treated with cadmium and copper at 50 µm, 125 µm, 250 µm, and 500 µm. statistically significant increases (p ≤ 0.05) were greater for cadmium at all concentrations compared to copper with total responses of 58.7 % and 37 % respectively. = not tested, t = tested, t* = tested and statistically significant.   101 and is brought about by the oxidation of these phospholipids with caspase-3 involvement (kagan et al., 2000; mandal et al., 2002). the externalization of ps serves as a signal for engulfment of apoptotic cells by phagocytes (fadeel and xue, 2009). ew celomocytes reside in the celomic cavity and consist of three distinct subpopulations which are most likely derived from a common progenitor cell (prohemocyte): hyaline amebocytes (large celomocytes), granular amebocytes (small celomocytes), and chloragocytes (eleocytes) (hartenstein, 2006). functionally, the hyaline amebocytes are phagocytic, the granular amebocytes exhibit nk-like activity, and the eleocytes contain chloragosomes which are used to secrete lytic substances (cossarizza et al., 1995, 1996; fuller-espie et al., 2010). these celomocytes can be studied in vitro by experimentally inducing extrusion of the celomocyte rich celomic fluid through the dorsal pores. once collected, these cells can be analyzed through flow cytometry where amebocytes (hyaline and granular) and eleocytes can be differentiated based on their size (forward scatter) and granularity (side scatter) (cossarizza et al., 2005). using flow cytometry, early apoptotic cells can be distinguished with dna binding viability dyes such as propidium iodide (pi) and 7aminoactinomycin d (7-aad) that only penetrate late apoptotic and necrotic cells (rabinovitch et al., 1986; nicoletti et al., 1991). additionally, changes in ∆ψm and ps translocation can be detected through flow cytometry with the fluorescent compounds jc-1 and annexin v-fitc respectively (salvioli et al., 1997; van engeland et al., 1998; fuller-espie et al., 2010). materials and methods cell culture supplies and chemical reagents tissue culture plasticware was purchased from fisher scientific. phosphate buffered saline (pbs) was purchased from invitrogen. dulbecco’s modified eagle medium (dmem, invitrogen) was supplemented with serum supreme (lonza biowhittaker), plus 100μg ml-1 ampicillin (shelton scientific), 10μg ml-1 kanamycin (shelton scientific), 10μg ml-1 tetracycline, 5μg ml-1 chloramphenicol (fluka biochemika), 1×penicillin, streptomycin and amphotericin b (invitrogen), 1×nonessential amino acids (invitrogen), 1×l-glutamine (invitrogen) and 1×hepes (invitrogen) to comprise super dmem (sdmem). copper (ii) chloride (cucl2, 221783100g) and cadmium chloride (cdcl2, 655198-5g) were purchased from sigma-aldrich and stock solutions were prepared in deionized water and sterilized by filtration. ew husbandry eisenia hortensis (european nightcrawlers) were purchased from vermitechnology unlimited, orange lake, florida, usa, who imports e. hortensis from star food, holland, scherpenzeelseweg 95, 3772me barneveld, the netherlands. species identity was determined by the united states department of agriculture, usda permit #52262 (vermitechnology, personal communication). short-term colonies were maintained at rt in the dark on moistened autoclaved pine woodchips sprinkled with single grain rice cereal or rice with bananas cereal (gerber) and covered with autoclaved, shredded, and moistened paper towels. habitats were changed twice weekly. animals were euthanized by freezing at -20 ºc. extrusion of celomocytes prior to experimentation, ews were first washed with distilled water on paper towels using a water bottle to remove wood chip fragments and food particles. they were then placed overnight on paper towels moistened with 2.5 μg ml-1 fungizone (fisher scientific) in 100mm petri dishes to minimize fecal contamination during the extrusion process, and remove further any surface contaminants. to collect celomocytes, ews were placed in multichannel pipette reservoirs containing 3 ml bd facsflow sheath fluid (bd biosciences). the ews extruded their celomocytes through their dorsal pores in response to this external stimulus without the need to use the alcohol extrusion method reported by others (engelmann et al., 2005). the celomocytes were then transferred to 0.5 ml accumax (innovative cell technology) in 15 ml conical test tubes for a 5 min incubation period at rt to reduce aggregation of cells. finally, 6.5 ml pbs was added and the samples were centrifuged immediately at 150xg, 7 min at 4 ºc. after decanting the supernatant, the celomocyte pellet was gently mixed by flicking the bottom of the centrifuge tube, and celomocytes were resuspended in 1 ml sdmem. enumeration was carried out using a hemacytometer. only large celomocytes (hyaline amebocytes) and small celomocytes (granular amebocytes) were included in the cell count; chloragocytes (eleocytes) were not counted but did factor into a quality score. samples with large numbers of chloragocytes compared to large and small celomocytes were not used in assays due to their high background autofluorescence. samples were adjusted to 1 x 106 celomocytes ml-1 in sdmem. ew coding ews were coded according to the particular in vitro assays in which their celomocytes were used. ews used in detection of changes to ∆ψm with jc-1 were labeled with a j and numbered (ew-j1 through ew-j20). ews used in phosphatidylserine translocation and caspase assays detected with annexin v-fitc were labeled with ac and numbered (ew-ac1 through ew-ac10). lastly, ews used in the correlation assays were labeled with ja and numbered (ew-ja1 through ew-ja9). ∆ψm assays changes in ∆ψm were measured with the lipophilic cationic fluorescent compound jc-1 (5,5',6,6’-tetrachloro-1,1’,3,3’tetraethylbenzimidazol-dazolcarbocyanine iodide) (anaspec, 88060). jc-1 penetrates the plasma membrane readily entering the cytosol where it binds to intact mitochondrial membranes with large ∆ψm and forms j-aggregates which emit orange at   102 590 nm when excited. during membrane depolarization these j-aggregates dissociate into the monomeric form which emit a green color at 527 nm (salvioli et al., 1997). using a bd facscalibur flow cytometer with a 488 nm excitation argon laser, celomocytes undergoing membrane depolarization were identified by increases in green fluorescence compared to the untreated control. 7aminoactinomycin d (7-aad) (bd pharmingen, 559925) was also used to exclude dead cells from analyses. using a 96-well v-bottom plate, 1x 105 celomocytes in 0.1 ml sdmem were added to experimental (cadmium and copper treated) and control [untreated (0 µm) double negative autofluorescent background; single positive jc-1; single negative jc-1, single positive 7-aad] wells. experimental wells received final concentration of 0 µm, 125 µm, 250 µm, or 500 µm of cadmium or copper, 5µg ml-1 jc-1, and 2.5 µl 7-aad. single positive jc-1 control wells received 20 µm valinomycin, single negative jc-1 control wells received 0 µm valinomycin, and single positive 7aad control wells received 0.01 % saponin (acros 41923-1000). experimental and control wells were incubated with medium, heavy metals, or valinomycin for 20 h at 25 ºc, 5 % co2. after incubation the cells were centrifuged at 150xg (5 min, 4 ºc) and washed once with 100 μl pbs. cells in experimental wells were then resuspended in sdmem containing jc-1 and 7-aad; control wells were resuspended in sdmem containing either jc1 or 7-aad/saponin. cells were then incubated 15 min at rt in the dark, transferred to flow cytometry tubes on ice, and analyzed immediately by flow cytometry. the final outcome measure is based on the overall proportion of responder ews to at least one of the concentrations of cadmium or copper employed. phosphatidylserine translocation and caspase assays translocation of phosphatidylserine (ps) from the plasma membrane inner leaflet to the outer leaflet was detected with fluorescein isothiocyanate (fitc)-conjugated annexin v (invitrogen, annexinv01). upon translocation, ps is bound by the annexin v component and the fitc component emits at 518 nm detectable in the fl-1 channel. similar to 7-aad, propidium iodide (pi) (invitrogen, p3566) also binds nucleic acids and was used to identify and exclude membrane-compromised cells of the late apoptotic and necrotic varieties. upon excitation, pi emits at 617 nm which is detectable in the fl-2 channel. the tripeptide caspase inhibitor nbenzyloxycarbonyl-val-ala-asp-fluoromethyketone (z-vad-fmk) (bd pharmingen, 550377), which binds to many caspases but principally inhibits caspases 1 and 3 (yang et al., 2004), was used to test the involvement of caspase activity on ps translocation. z-vad-fmk enters the cell as an omethyl ester and is converted to the active form within the cell by esterases (shimizu and pommier, 1997). once active, the peptide irreversibly binds to and inhibits caspase function through its valinealanine-aspartic acid (vad) sequence. the aspartic acid at the p1 position of this peptide is specifically involved in this interaction where it alkylates the cysteine residue in the active site (sarin, 1996). this peptide contains a fluoromethyl ketone (fmk) group responsible for anchoring to the caspase via a cysteine linkage and whose effects are controlled for with the dipeptide n-benzyloxycarbonyl-phe-alafluoromethylketone (z-fa-fmk) (bd pharmingen, 550411) which serves as a negative control (braun et al., 1999). this peptide has a phenylalaninealanine (fa) sequence which lacks the aspartic acid at the p1 position and therefore does not inhibit caspase activity in the cell. using a 96-well v-bottom plate, 1x105 celomocytes in 0.1ml sdmem were added to experimental [cadmium-treated in 0.3 % dmso (carrier control), cadmiumand z-vad-fmk-treated, and cadmiumand z-fa-fmk-treated] and control [untreated (0 µm) double negative autofluorescent background; single positive annexin v-fitc; single positive pi] wells. experimental wells received final concentrations of 0 µm, 62.5 µm, 125 µm, 250 µm, or 500 µm of cadmium, 40 µm z-vad-fmk or z-fafmk, 2.5 µl annexin v-fitc, and 2.5 µl pi. single positive annexin v-fitc control wells received 67.6 mm h2o2 (fischer h325-500) and single positive pi control wells received 0.01 % saponin. experimental and control wells were incubated with the cadmium and caspase inhibitor/negative caspase inhibitor control/dmso carrier control for 20 h at 25 ºc, 5 % co2. after incubation the cells were centrifuged at 150xg (5 min, 4 ºc) and washed with 100 μl pbs. cells in experimental wells were then resuspended in sdmem, annexin v-fitc, and pi; control wells were resuspended in sdmem and treated with either annexin v-fitc/h2o2 or pi/saponin to a final volume of 100 μl. cells were then incubated 15 min at rt in the dark, transferred to flow cytometry tubes on ice and analyzed immediately by flow cytometry. correlation assays the correlation between ongoing mitochondrial membrane depolarization and ps translocation was determined by conducting assays in which cells from individual ews were assessed for both conditions independently. these assays were conducted simultaneously in separate v-bottom plates using the same protocols mentioned above without the addition of caspase inhibitors/negative inhibitor controls/dmso carrier control. experimental wells received final concentration of 0 µm, 50 µm, 250 µm, or 500 µm cadmium or copper. flow cytometry analysis fluorescence was measured using fl-1 (annexin v-fitc and jc-1), fl-2 (jc-1 and pi), and fl-3 (7-aad) detectors of a facscalibur flow cytometer (bd biosciences). autofluorescent controls were used to set voltages for forward scatter (fsc), side scatter (ssc), fl-1, fl-2, and fl-3 during instrument set-up. singly positive annexin v-fitc and pi controls were used to adjust compensation settings (spectral overlap removal) for ps assays and singly positive jc-1 and 7-aad for ∆ψm assays. listmode data was acquired and analyzed using cell quest pro (bd biosciences)   103 software. only celomocytes corresponding to the large celomocytes (hyaline amebocytes) and small celomocyte (granular amebocytes) populations combined, as determined by appropriate size (fsc) (abscissa) and granularity (ssc) (ordinate), were gated for further analyses in region 1(r1) (fig. 1a). viable cells were only counted for analysis by gating on fl-2 (pi) or fl-3(7-aad) negative events for ps and ∆ψm, respectively, in region 2(r2) (fig. 1b). changes in ∆ψm or ps were assessed by placing a marker (m1) on a fl-1 histogram gated on r1 and r2 at the median fluorescence value for untreated 0μm samples (fig. 1d). this marker was set based on the untreated 0μm dmso carrier control for analysis of ps translocation assays. samples considered positive are those exhibiting a greater percentage of events occurring above the median fluorescence value measured in the untreated cells in the fl-1 channel. statistical analysis data analysis and graphs were generated using microsoft excel 2007. the student’s t-test paired two samples for means was utilized with a 95 % confidence interval to determine if the experimental values were statistically significant as exhibited by a p-value less than or equal to 0.05. only events exhibiting statistically significant increases above background are reported for ∆ψm and correlation assays; only events exhibiting statistically significant decreases in z-vad-fmk treated groups compared to z-fa-fmk treated groups are counted as positive events for ps caspase assays. linear correlation between ∆ψm and ps was determined by calculating pearson product-moment correlation coefficients (r). degree of correlation was dependent by the magnitude of positive r-values with values from 0.5 to 1.0 considered strong correlations. all data is based on averages of triplicate samples except ew-j1 and ew-j2 which were performed in duplicate. results flow cytometric detection of changes in ∆ψm early apoptotic celomocytes were identified with the viability dye 7-aminoactinomycin d (7-aad) which only penetrates membrane-compromised cells and binds to nucleic acids. upon excitation, membrane-compromised cells, such as those that are late apoptotic and necrotic, will emit at 650 nm which is detectable in the fl-3 channel (rabinovitch et al., 1986). using three-color analysis, these events were excluded so that only live and early apoptotic celomocytes could be evaluated for changes in ∆ψm. figure 1 illustrates how data was collected and analyzed to detect alterations in ∆ψm in response to varying concentrations of cadmium and copper. shown in fig. 1d is the typical profile observed for the untreated negative control (left), valinomycin-treated positive control (middle), and heavy metal-treated sample (right). twenty-nine ews in total were assayed for alterations in ∆ψm at cadmium or copper concentrations of 50 µm, 125 µm, 250 µm, and 500 µm as summarized in table 1 where 6 ews were assessed at 50 µm, 5 ews at 125 µm, 8 ews at 250 table 2 caspase involvement for ps translocation in response to cadmium exposure ps cd62.5 cd125 cd250 cd500 ew-ac1 t t ew-ac2 t* t ew-ac3 t t ew-ac4 t* t ew-ac5 t* t ew-ac6 t t* ew-ac7 t t* ew-ac8 t t ew-ac9 t t* ew-ac10 t t 0% 60% 60% 0% % response (0/5) (3/5) (3/5) (0/5) ews were treated at cadmium concentrations of 62.5 µm, 125 µm, 250 µm, and 500 µm. statistically significant decreases (p ≤ 0.05) in z-vad-fmk treated celomocytes compared to the z-fa-fmk negative caspase control were exhibited only at concentrations of 125 µm and 250 µm in 60 % of ews. = not tested, t = tested, t* = tested and statistically significant. µm, and 27 ews at 500 µm of cadmium or copper. response rates for cadmium were varied but were recorded above the 50 % marker region for all concentrations other than 125 µm with a total response of 58.7 % for all ews at all concentrations. responses for copper were generally lower with a maximum response of 50 % for 250 µm. total response for copper among all ews for all concentrations was lower than cadmium at only 37 %. interference of phosphatidylserine translocation with caspase inhibitors flow cytometric data was collected and analyzed similarly to ∆ψm shown in fig. 1. ten ews in total were assayed for inhibition of cadmiuminduced ps translocation: five (ew-ac1-5) at concentrations of 62.5 μm and 125 μm, and another five (ew-ac6-10) at the higher concentrations of 250 μm and 500 μm (table 2). representative data for 2 ews (ew-ac6 and ew-ac7) at 125μm is shown in fig. 2 where cadmium treatment with dmso and z-fa-fmk produced significant increases compared to untreated samples and cadmium treated z-vad-fmk samples were inhibited compared to untreated samples. when treated at concentrations of 125 μm and 250 μm, significant inhibition of ps translocation was observed in 60 % of cases compared to the z-fa-fmk negative control, but was not observed at the lowest (62.5 μm) fig. 2 representative interference of ps translocation using caspase inhibitor. the percent viable hyaline and granular amebocytes exhibiting ps translocation above the 50 % m1 marker for each treatment is shown for two ews (ew-ac6 and ew-ac7). for each ew the left set of bars is the untreated 0 µm cadmium control and the right set of bars with borders is 125 µm cadmium treated. for each set the first bar is the dmso carrier control, the second is treated with the negative control of caspase inhibition (z-fa-fmk), and the third is treated with the general caspase inhibitor z-vad-fmk. statistically significant decreases  (*, p ≤ 0.05) compared to the negative caspase control are shown for both ews at 125 µm. error bars indicate standard deviation. or highest (500 μm) concentrations as summarized in table 2. correlation of changes in ∆ψm and phosphatidylserine translocation   104 since mitochondrial membrane depolarization and ps translocation are both hallmarks of early apoptosis, there was an interest in the existence of correlation between these two events. these separate events were evaluated for nine ews in two separate assays: one using copper and cadmium concentrations of 250 μm and 500 μm for a two-fold difference, and another using 50 μm and 500 μm for a ten-fold difference (fig. 3). these events were detected to some degree at all concentrations used except ∆ψm at a copper concentration of 50 μm (table 3). a very high degree of correlation was found with eight ews correlating strongly and mean r value of 0.71. results for ews ew-ja7 through ew-ja9 are shown in fig. 3. note the strong correlation between events and concentrations for ew-ja7 where there is a decreasing effect moving from 500 μm to 50 μm and from cadmium to copper. the only ew that did not correlate strongly was ewja9 where at 500 μm copper ps translocation increased but ∆ψm decreased. discussion this study demonstrates that the heavy metals cadmium (50 500 µm) and copper (125-500 µm) induce the early apoptotic event of mitochondrial membrane depolarization in celomocytes of the ew e. hortensis. it was found that cadmium induced an equal, if not greater, response at every concentration measured with a total response rate of 58.7 % compared to copper at 37 %. copper required a higher concentration than cadmium (125 µm vs. 50 µm) to induce membrane depolarization. the decrease of membrane potential has also been reported for both metals in rat hepatocytes and for cadmium in human intestinal cells (martel et al., 1990; pourahmad, 2000; bolduc et al., 2004). pourahmad (2000) also reported a similar effect where cadmium induced depolarization at lower concentrations than copper (20 vs. 50 µm). although fig. 3 loss of ∆ψm and ps translocation are correlated. in the assay using 50 µm and 500 µm cdcl2, the percent viable hyaline and granular amoebocytes exhibiting loss of ∆ψm (a) and ps translocation (b) above the 50 % m1 marker for each treatment is shown for 3 ews. statistically significant increases (*, p ≤ 0.05) compared to the untreated control are indicated. the correlation coefficient (r) is indicated for each ew where ew-ja7 and ewja8 are strongly correlated whereas ew-ja9 is not. error bars indicate standard deviation. concentrations needed for a response in celomocytes of e. hortensis were higher than those needed in rat hepatocytes, ew cells may be adapted to higher concentrations since they bioaccumulate these metals from the naturally metalliferous soils they live in (morgan and morgan, 1988). this tolerance may be attributed to heavy metal binding proteins such as those for cadmium demonstrated in eisenia foetida and copper in the annelid eudistylia vancouveri (young and roesijadi, 1983; suzuki et al, 1980). it would be interesting to investigate the presence of similar proteins in our e. hortensis model to confirm this. additionally, the caspase dependence of cadmium on ps translocation was also investigated. in regards to the events of early apoptosis, mitochondrial membrane depolarization is one of the earliest apoptotic events with caspase independent pathways and typically precedes ps translocation (bortner and cidlowski, 1999; fabbri et al., 2006). translocation of ps has been shown to be dependent upon caspase-3 which is inhibited by zvad-fmk (marianski et al., 2002). little is known of caspase involvement in ews although exogenous h2o2 has been shown to activate caspases in the e. hortensis model (fuller-espie et al., 2010). results of this study show that caspases also play a role in the translocation of ps induced by cadmium in celomocytes of e. hortensis as measured with annexin v-fitc. it was shown that caspase involvement only occurred within a narrow range of 125-250 µm. it is possible that z-vad-fmk is not active at concentrations lower than this since low doses of cadmium (5-20 µm) have been shown to have inhibitory effects as well (yuan et al., 2000). at the higher concentration it is possible that cadmium is subverting caspase dependent pathways through oxidative induction of ps translocation and is therefore unaffected by z-vad as has been shown in human lung cells (shih et al., 2003). it would be interesting to study these oxidative effects in parallel to ps translocation with a fluorescent detector of ros such as dihydrorhodamine123 or dihydroethidium (kagan et al., 2002; fuller-espie et al., 2010). lastly, the relation between mitochondrial membrane depolarization and ps translocation was also investigated in celomocytes of e. hortensis exposed to cadmium and copper. it was found that these events correlated strongly (r > 0.5) in 89 % of   105   106 table 3 correlation between loss of ∆ψm and ps translocation cd50 cd250 cd500 cu50 cu250 cu500 ∆ψm and ps ∆ψm ps ∆ψm ps ∆ψm ps ∆ψm ps ∆ψm ps ∆ψm ps r correlation ew-ja1 t* t* t* t* t* t* t* t* 0.80 strong ew-ja2 t t* t t* t* t* t* t* 0.69 strong ew-ja3 t t t* t* t* t* t* t* 0.84 strong ew-ja4 t* t* t* t* t t t* t* 0.69 strong ew-ja5 t t t* t* t t t* t* 0.71 strong ew-ja6 t t t* t* t t t* t* 0.97 strong ew-ja7 t* t* t* t* t t t* t 0.98 strong ew-ja8 t t* t* t* t t* t t* 0.87 strong ew-ja9 t* t* t t* t t t* t -0.14 none 50% 67% 33% 67% 78% 100% 0% 17% 100% 100% 89% 78% mean 89% % response (3/6) (4/6) (1/3) (2/3) (7/9) (9/9) (0/6) (1/6) (3/3) (3/3) (8/9) (7/9) 0.71 (8/9) ews were treated in two separate assays at cadmium and copper concentrations of 50 µm and 500 µm for a tenfold difference, or 250 µm and 500 µm for a two-fold difference. loss of ∆ψm and ps translocation were strongly correlated (r > 0.5) in 89 % of cases with an average r value of 0.71. statistically significant increases (p ≤ 0.05) were present for both events and both heavy metals. = not tested, t = tested, t* = tested and statistically significant. cases (8 of 9 ews) with an average r value of 0.71. these data suggest that loss of membrane potential is not a pre-requisite for externalization of ps since in many cases ps translocation was detected while depolarization of the mitochondrial membrane was not such as in ew-ja2 for cadmium at 250 µm and 500 µm. the use of an inhibitor of mitochondrial membrane depolarization and cytochrome c release like ursodeoxycholic acid would help determine the involvement of loss of ∆ψm on ps translocation (rodrigues et al., 1999). these data also show that heavy metal toxicity is inducing these early apoptotic events after the same exposure time (20 h) which could further be studied to determine if 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inhibitors directed towards caspase-1 and -3 are less effective than pan caspase inhibition in preventing renal proximal tubular cell apoptosis. nephron exp. nephrol. 96: 39-51, 2004. young j, roesijadi g. reparatory adaptation to copper-induced injury and occurrence of a copper-binding protein in the polychaete, eudistylia vancouveri. mar. pollut. bull. 14: 3032,1983. yuan c, kadiiska m, achanzar w, mason r, waalkes m. possible role of caspase-3 inhibition in cadmium-induced blockage of apoptosis. toxicol. appl. pharmacol. 164: 3219, 2000. review isj 7: 262-284, 2010 issn 1824-307x review antimicrobial peptides in crustaceans rd rosa, ma barracco laboratory of immunology applied to aquaculture, departamento de biologia celular, embriologia e genética, universidade federal de santa catarina, 88040-900 florianópolis, brazil accepted november 9, 2010 abstract crustaceans are a large and diverse invertebrate animal group that mounts a complex and efficient innate immune response against a variety of microorganisms. the crustacean immune system is primarily related to cellular responses and the production and release of important immune effectors into the hemolymph. antimicrobial proteins and/or peptides (amps) are key components of innate immunity and are widespread in nature, from bacteria to vertebrate animals. in crustaceans, 15 distinct amp families are currently recognized, although the great majority (14 families) comes from members of the order decapoda. crustacean amps are generally cationic, gene-encoded molecules that are mainly produced by circulating immune-competent cells (hemocytes) or are derived from unrelated proteins primarily involved in other biological functions. in this review, we tentatively classified the crustacean amps into four main groups based on their amino acid composition, structural features and multi-functionality. we also attempted to summarize the current knowledge on their implication both in an efficient response to microbial infections and in crustacean survival. key words: antimicrobial peptides; innate immunity; crustacean defense; crustaceans; decapods introduction crustaceans compose a large, ancient and diverse animal group that includes many wellknown, commercially exploited members, such as shrimp, crab, crayfish and lobster. together with insects, they are by far the most numerous, diverse and widespread animals on earth. crustaceans are primarily marine organisms. they are the most abundant animals inhabiting the world oceans, but there are also freshwater, terrestrial and semiterrestrial species. during their long-standing existence, crustaceans have confronted a broad variety of challenges to their self-integrity because their natural habitats are typically overloaded with infectious organisms, such as viruses, bacteria, fungi and other parasites. their evolutionary success confirms the efficient strategies they use for survival in such a potentially hostile and microbeenriched environment. part of this coexisting microbial biota is beneficial and can establish advantageous associations with the crustacean in a commensal or mutualistic manner, whereas another ___________________________________________________________________________ corresponding author: margherita a barracco laboratory of immunology applied to aquaculture, departamento de biologia celular, embriologia e genética, universidade federal de santa catarina, pob 476, 88040-900 florianópolis, brazil e-mail: barracco@mbox1.ufsc.br part of this biota is potentially harmful. indeed, crustaceans can harbor specific microbial communities in their surface and internal epithelia that have important roles in nutrition and defense (gil-turnes et al., 1989). in normal conditions, crustaceans maintain a healthy state and keep infections under control. externally, they are covered by a hard, rigid exoskeleton that functions as an efficient physicochemical barrier against mechanical injury and microbe invasion. their gastrointestinal tract, another important route for pathogen invasion, is also protected almost entirely by chitinous membranes. this cuticular coat, in combination with an acid environment rich in digestive enzymes, is able to inactivate and degrade most viruses and bacteria (jiravanichpaisal et al., 2006). however, once the cuticle barriers are disrupted, pathogenic and/or opportunist microorganisms can penetrate into the hemocoel and thus activate the internal immune defenses of the crustacean. crustaceans lack the complex and highly specific adaptive immune system of vertebrates, which is based on lymphocytes, immunoglobulins and immunological memory. their internal defenses rely only on innate immune responses that are relatively less specific, but are fast and efficient defenses against microbes. the innate immune system of crustaceans is primarily related to their 262 mailto:barracco@mbox1.ufsc.br blood or hemolymph and is comprised of cellular and humoral responses. humoral defenses include pattern-recognition receptors/proteins that recognize pathogen-associated molecular patterns (pamps), the production of toxic oxygen and nitrogen metabolites, complex enzymatic cascades leading to melanization, clotting proteins and antimicrobial peptides. conversely, cellular immune responses are mediated by circulating blood cells or hemocytes (hyaline and granular hemocytes) that are capable of neutralizing and/or eliminating pathogens by phagocytosing microbes and/or trapping them in hemocyte aggregates or nodules or by encapsulating larger microorganisms and parasites (see review of jiravanichpaisal et al., 2006). antimicrobial peptides or proteins (amps) are one of the major components of the innate immune defense and are ubiquitously found in all kingdoms from bacteria to mammals, including fungi and plants. amps are primarily known as “natural antibiotics” because of their rapid and efficient antimicrobial effects against a broad range of microorganisms, including gram-positive and gramnegative bacteria, yeast, filamentous fungi and, to a lesser extent, protozoans and enveloped viruses (bulet et al., 2004; yount et al., 2006; guaní-guerra et al., 2010). recent evidence has shown that in addition to their antimicrobial activity, amps may encompass a number of other diverse biological roles and are, in fact, multifunctional molecules. it was demonstrated that these peptides have antitumor effects, mitogenic activity and, most importantly, participate in immunoregulatory mechanisms by modulating signal transduction and cytokine production and/or release (kamysz et al., 2003; bowdish et al., 2005; brown and hancock, 2006; yount et al., 2006; easton et al., 2009; lai and gallo, 2009; guaní-guerra et al., 2010). thus far, more than 1,500 amps sequences have been identified and are accessible on databases (http://aps.unmc.edu/ap/main.php) or in journal publications (see for review thomas et al., 2010). amps are classically described as small cationic, amphipathic, gene-encoded molecules (<10 kda, 15-100 amino acids) that differ considerably in amino acid sequence and structural conformation. they are commonly found in the blood or epithelial (mucosal) surfaces that are most exposed to microorganisms. more recently, other groups of antimicrobial molecules have been identified that do not fit into this classical definition. these are anionic peptides, proteins larger than 10 kda and multifunctional proteins that contain antimicrobial sub-domains that are cleaved under certain conditions and generate fragments that resemble classical antimicrobial peptides (brogden, 2005; yount et al., 2006). the amp mode of action is basically determined by structural conformation, cationic charge and amphipathicity. it is generally agreed that amps predominantly act by disrupting the membrane integrity of the cell target. the cationic portion of the peptide is first attracted to the negatively-charged bacterial and fungal cell walls and/or membranes, and following this first electrostatic interaction, the peptide inserts into and permeabilizes the microbial cell membranes through its hydrophobic portion. the microorganisms are then destroyed via membrane destabilization and/or pore formation (brogden, 2005; yount et al., 2006). the detailed mechanism of pore formation (barrel-stave, toroidal and carpet-like models) has been described in detail elsewhere (brogden, 2005; salditt et al., 2006). beyond this direct interaction with microbial membranes, a growing body of evidence suggests that amps may have additional mechanisms to inactivate pathogens. numerous recent studies have documented that amps can be translocated into the cytoplasm of the microorganism where they act on specific intracellular targets. once inside, the peptides interfere with several essential metabolic functions, such as protein, nucleic acid and cell wall syntheses, leading to bacterial cell death (kamysz et al., 2003; brogden, 2005; yount et al., 2006; hale and hancock, 2007; nicolas, 2009). moreover, it appears that many amps may be multifunctional microbicides, acting simultaneously at the cell membrane and internal sites (yount et al., 2006). different families of amps have been identified in crustaceans. for economic reasons, the best characterized peptides come from cultivated species, such as marine shrimp. the cultivation of penaeid shrimp is a potent industry worldwide, which generates significant profits for the producing countries. this industry is consistently threatened by devastating disease outbreaks caused by infectious agents that usually lead to massive losses in shrimp production. to overcome this constraint, the study of shrimp immune system, including the antimicrobial peptides, is essential in order to better comprehend their internal defenses and open new perspectives for developing novel strategies to prevent and control infections in aquaculture. besides marine shrimp, amps have been also identified and characterized in a number of other crustaceans, primarily decapods, including marine crabs and lobsters, freshwater crayfishes and prawns and also terrestrial species, such as isopods. the amps of penaeid shrimp have been very recently reviewed in great detail (zhao and wang, 2008; tassanakajon et al., 2010). in this review, we discuss the current information on the different antimicrobial peptide families identified to date in all crustacean groups. special emphasis is given to their structure, biological properties and involvement in host defense and survival. we also draw attention to the fact that crustaceans possess a significant number of single-gene encoded amps that are composed of distinct domains and are larger molecules (>10 kda) than the classic antimicrobial peptides in other organisms. crustacean amps: structure, classification and biological properties the earliest studies on isolation and characterization of amps in crustaceans date back to mid-1990s. the first isolated amps, including bac-like, crustins and penaeidins, were purified from the hemolymph of crab and shrimp by biochemical approaches. since then, novel amp families have been identified in crustaceans using 263 http://aps.unmc.edu/ap/main.php table 1 summarized characteristics of crustacean amps amps molecular mass (kda) charge crustacean order first descriptions single-domain linear α-helical amps and peptides enriched in certain amino acids bac-like 6.5 cationic decapoda (crab) schnapp et al., 1996 callinectin 3.7 cationic decapoda (crab) khoo et al., 1999 astacidin 2 1.8 cationic decapoda (crayfish) jiravanichpaisal et al., 2007a armadillidin 5.2 cationic isopoda (woodlouse) herbinière et al., 2005 homarin 4-6 cationic decapoda (lobster) battison et al., 2008 single-domain peptides containing cysteine residues engaged in disulfide bonds defensin 6.7-7.1 cationic decapoda (lobster) pisuttharachai et al., 2009b anti-lps factor 7-11 cationic decapoda (various) gross et al., 2001 supungul et al., 2002 scygonadin 10.8-11.4 anionic decapoda (crab) huang et al., 2006 multi-domain or chimeric amps penaeidin 5.5-6.6 cationic decapoda (penaeid shrimp) destoumieux et al., 1997 crustin 7-14 cationic decapoda amphipoda relf et al., 1999 hyastatin 11.7 cationic decapoda (crab) sperstad et al., 2009b arasin 4.3-4.8 cationic decapoda (crab) stensvåg et al., 2008 stylicin 8.9 anionic decapoda (penaeid shrimp) rolland et al., 2010 unconventional amps histones and derived fragments 11-15 cationic decapoda (penaeid shrimp) patat et al., 2004 hemocyanin-derived peptides 2.7-8.3 (shrimp) 1.9 (crayfish) anionic (shrimp) cationic (crayfish) decapoda (shrimp, crayfish) destoumieux-garzón et al., 2001 lee et al., 2003 different molecular approaches. at the present time, 15 amp families or single peptides sharing common molecular features with the currently known amp families have been recognized in crustaceans. all crustacean amp families display antimicrobial activities against a number of specific microorganisms (with the exception of two putative β-defensin-like peptides identified in a spiny lobster species by a genomic approach). however, the high diversity of the variants (subgroups and isoforms) found in most crustacean amp classes can presumably confer a broad spectrum of activity to a single amp family. in terms of structure, crustacean amps can be primarily defined as small, cationic, amphipathic molecules that are encoded by a single gene, as commonly described in other organisms. however, more recently, this definition was expanded to include less common amps, such as anionic peptides, multifunctional proteins that are primarily implicated in other cellular functions and proteinderived fragments that display antimicrobial activities. interestingly, an important number of gene-encoded amps in crustaceans are composed of different structural domains. each of these domains possess singular features found in other amp groups, such as the overrepresentation of specific amino acids or the presence of cysteine residues that form disulfide bonds. it has been proposed that these chimeric peptides could act as multifunctional proteins in different physiological systems in addition to their role in innate immunity (bachère et al., 2004). based on amino acid composition and structure, we tentatively clustered the families of antimicrobial peptides found in crustacean species into four main groups: (1) single-domain linear αhelical amps and peptides enriched in certain amino acids, (2) single-domain peptides containing cysteine residues engaged in disulfide bonds, (3) multi-domain or chimeric amps and (4) unconventional amps including multifunctional proteins and protein-derived fragments that exhibit antimicrobial functions (table 1). these groups 264 encompass the major characteristics of crustacean amps, although using this classification, some families could be categorized into more than one group. single-domain linear α-helical amps and peptides enriched in certain amino acids linear amphipathic peptides comprise a large group of amps that lack cysteine residues engaged in disulfide linkages. these peptides are usually unfolded in solution, but can adopt an amphipathic α-helical structure in the presence of lipid bilayers (brogden, 2005). they are largely found in many vertebrate and invertebrate species, displaying a broad spectrum of antimicrobial activities against bacteria, fungi and protozoa. additionally, some linear peptides are enriched with a high proportion of select residues, particularly arginine, proline, glycine, tryptophan or histidine (tossi and sandri, 2002). to date in crustaceans, five linear amps have been described, including homarin, armadillidin and three proline/arginine-rich peptides (bac-like, callinectin and astacidin 2). proline/arginine-rich peptides historically, the first antimicrobial peptide characterized in crustaceans was a 6.5-kda proline/arginine-rich (or prp-rich) cationic peptide isolated from the hemocytes of carcinus maenas (schnapp et al., 1996). partial sequence analysis of the amino (n)-terminal region revealed that this peptide contains three repetitions of the motif prp (proline-arginine-proline), which are usually found in other insect antimicrobial peptides (bulet and stöcklin, 2005) (fig. 1a). moreover, due to significant amino acid similarities to bactenecin-7, a cathelicidin antimicrobial peptide from bovine neutrophils (frank et al., 1990), this amp is also known as bac-like. similar to the bovine amp, the 6.5-kda peptide was active against both grampositive (micrococcus luteus) and gram-negative (psychrobacter immobilis) bacteria in inhibition zone assays. interestingly, it also shares high sequence similarities with the prp-rich domain of penaeidins (see section 3.1), an amp family found exclusively in penaeid shrimp. unfortunately, this first-identified crustacean amp was not further characterized, and it remains unclear if its full-length sequence contains only prp-rich motifs or if it includes additional unidentified molecular domains. new information on its complete amino acid sequence will reveal if baclike is related to the penaeidin family or if it is a distinct class of single domain antimicrobial peptides. a few years later, another prp-rich peptide, named callinectin, was isolated from the hemocytes of the blue crab callinectes sapidus (khoo et al., 1999). in contrast to bac-like, the partially characterized n-terminal sequence of callinectin showed no significant homology with any other known antimicrobial peptide (fig. 1a). this novel cationic peptide of 3.7 kda was only tested against escherichia coli d31, and information on its fulllength sequence and spectrum of antimicrobial activity are currently lacking. similar to bac-like, it is uncertain if callinectin is a genuine single-domain prp-rich amp. another peptide belonging to the prp-rich family was isolated from the hemocytes of the freshwater crayfish pacifastacus leniusculus and was fully characterized (fig. 1a). this original peptide, named astacidin 2, is composed of 14 amino acids and displays strong antimicrobial activity against both gram-positive (minimal inhibitory concentration [mic] of about 5.5-10.3 µm: bacillus sp., staphylococcus aureus and m. luteus) and gram-negative bacteria (mic of about 0.5-4.3 µm: shigella flexneri, proteus vulgaris, e. coli and pseudomonas aeruginosa) (jiravanichpaisal et al., 2007a). unlike astacidin 1, a crayfish antimicrobial peptide derived from the carboxyl (c)-terminus of hemocyanin (see section 4.2), astacidin 2 is a geneencoded molecule that is synthesized as a prepropeptide and then processed at both nand cterminal regions. this highly cationic peptide of 1.8 kda (isoelectric point or pi of 11.8) is constitutively expressed in crayfish hemocytes and shows significant homology to metalnikowin-1 from the insect palomena prasina (chernysh et al., 1996). recently, an astacidin 2 homolog was also identified in the crayfish procambarus clarkii (shi et al., 2009). armadillidin: a glycine-rich peptide antimicrobial peptides rich in glycine residues have been isolated from a variety of arthropod species, including insects and spiders (bulet et al., 1991; lorenzini et al., 2003). in crustaceans, the first glycine-rich antimicrobial peptide, named armadillidin, was purified from the hemocytes of the terrestrial isopod armadillidium vulgare (herbinière et al., 2005). armadillidin is a linear cationic peptide (pi of 12.1) that is characterized by the presence of a high content of glycine residues (about 47 %). this peptide displays antibacterial activity against the gram-positive bacteria bacillus megaterium (mic: 0.5 µm). the mature peptide (5.2 kda) is amidated at the c-terminus and encloses a unique five-fold repeated motif gggfh(r/s) (fig. 1b). this glycine-rich peptide is the unique crustacean amp currently characterized in a non-decapod species. interestingly, armadillidin was found only in the hemocytes of the woodlouse a. vulgare, but not in other isopod species such as oniscus asellus, porcellio dilatatus petiti and armadillo officinalis (herbinière et al., 2005). homarin: a linear α-helical peptide homarin (also designated as cationic antimicrobial peptide or cap) is a short linear peptide isolated from the hemocytes of the american lobster homarus americanus and does not contain any specific abundant amino acid residues (battison et al., 2008). this cationic peptide shares evident amino acid similarities with temporins (mangoni, 2006) (fig. 1c). temporins are small amphipathic α-helical amps from amphibian skin that exhibit antimicrobial activity against grampositive bacteria (simmaco et al., 1996). in contrast, two synthetic homarin variants (scap-1.1 and scap-1.4) were shown to be effective against a vibrio sp. isolated from the lobster intestinal tract and also exhibited protozoacidal activity against an 265 fig. 1 primary structure of crustacean single-domain linear α-helical amps and peptides enriched in certain amino acids. (a) the proline and arginine residues are shadowed with a black background (where x is any amino acid residue). (b) the fivefold repeated motif gggfh(r/s) is shown in bold and underlined. (c) the amino acid sequence of the linear α-helical homarin, which is identical to the amphibian temporins, is represented in bold type. the asterisks indicate the non-yet fully characterized crustacean amps. important ciliate pathogen for lobsters and crabs (battison et al., 2008). single-domain peptides containing cysteine residues engaged in disulfide bonds this group of single domain amps is characterized by the presence of pairs of cysteine residues that are capable of forming intramolecular disulfide bridges. the number of cysteine residues (generally 2 to 12) and their binding result in the formation of cyclic or open-ended cyclic stabilized peptides. cysteine-containing amps are found in many species, from bacteria to eukaryotes, including fungi, plants and both vertebrate and invertebrate animals (tossi and sandri, 2002). this amp group includes thanatin, tachyplesins, gomesin, protegrins and the well-known defensins (plant-, invertebrate-, α-, βand θ-defensins) (for review, see bulet et al., 2004). in decapod crustaceans, only three cysteine-containing amps have been characterized (defensins, antilipopolysaccharide factors and scygonadins), although a significant number of cysteine-containing domains are also present in crustacean multidomain antimicrobial peptides. defensins the defensin family appears to be the best characterized and the most widespread family of antimicrobial peptides, occurring in most plants and invertebrate and vertebrate animals (bulet et al., 2004). curiously, in crustaceans, defensin members were only very recently identified in the japanese spiny lobster panulirus japonicus using an expressed sequence tag (est) approach (pisuttharachai et al., 2009a). in this species, two different isoforms, pjd1 (7.1 kda) and pjd2 (6.7 kda), were detected in various tissues through reverse transcription-polymerase chain reaction (rt-pcr), including hemocytes, heart, gills and hepatopancreas (pisuttharachai et al., 2009b). both isoforms possess an n-terminal signal peptide and a c-terminal defensin-like domain. interestingly, the cysteine pattern found in p. japonicus defensins (cx4-8-c-x3-5-c-x9-13-c-x4-7-c-c) is distinct from those present in invertebrate defensins, but identical to that of β-defensins found in vertebrates (taylor et al., 2008) (fig. 2a). unfortunately, to date, defensins are merely considered to be “putative amps” in crustaceans because their spectrum of antimicrobial activity has not yet been determined. anti-lipopolysaccharide factors anti-lipopolysaccharide factors (anti-lps factors or alfs) were first purified from the hemolymph cells (amebocytes) of two marine chelicerate arthropods, the horseshoe crabs limulus polyphemus and tachypleus tridentatus (tanaka et al., 1982). limulus and tachypleus antilps factors (lalf and talf) have been initially identified as potent inhibitors of lps-mediated hemolymph coagulation. the coagulation system of horseshoe crabs can be activated by two distinct pathways (triggered by lps or 1,3,-β-d-glucan) that mediate the activation of the proclotting enzyme, 266 resulting in the formation of hemolymph clots, a defense reaction against microbial invasion (kawabata et al., 2009). however, in addition to inhibiting the lps-mediated coagulation pathway, the horseshoe crab anti-lps factor also exhibits a strong antibacterial activity against r-types of gramnegative bacteria, such as salmonella minnesota r595 (morita et al., 1985), thus acting as a multifunctional protein. structurally, both lalf and talf are large cationic peptides of approximately 100 amino acids that contain two conserved cysteine residues and a hydrophobic n-terminal region (aketagawa et al., 1986; muta et al., 1987). a cluster of positively-charged residues is comprised between the two cysteine residues that form the disulfide-bond loop, which has been defined as the lps-binding domain (hoess et al., 1993). horseshoe crab anti-lps factors are stored in the large granules of amebocytes, although no signal sequence has been recognized in the cdna sequence of talf (wang et al., 2002). in crustaceans, homologues to horseshoe crab alfs were first identified from the hemocytes of the penaeid shrimp species litopenaeus setiferus (gross et al., 2001) and penaeus monodon (supungul et al., 2002) using an est approach. to date, the genes encoding alfs have been only identified in decapod crustaceans, such as penaeid shrimps (tassanakajon et al., 2010), freshwater prawns (rosa et al., 2008; lu et al., 2009), crayfish (liu et al., 2006), lobster (beale et al., 2008) and crabs (imjongjirak et al., 2007; li et al., 2008; yedery and reddy, 2009a; yue et al., 2010). these peptides are part of a very well-characterized family of crustacean amps composed of different subgroups and variants that are either encoded by distinct genes or generated by alternative mrna splicing (tharntada et al., 2008). in the tiger shrimp p. monodon, the most expressed variants, alfpm2 and alfpm3, are transcribed by different genes with distinct genomic organization, designated as group a and b genes (fig. 2b). a genomic structure similar to the shrimp group a gene was also found in the crabs scylla paramamosain and eriocheir sinensis (imjongjirak et al., 2007; li et al., 2008). these peptides are constitutively expressed in circulating granular hemocytes, although cisregulatory elements, which are involved in transcriptional regulation, were also recognized in their gene promoters (tharntada et al., 2008). alf sequences from decapod crustaceans are encoded as precursor molecules. these precursors are composed of a leader sequence followed by a large mature peptide (of about 11 kda) containing a highly hydrophobic n-terminal region and the two characteristic cysteine residues. recently, the threedimensional structure of shrimp alf heterologously expressed in yeast cells (ralfpm3) was resolved by nuclear magnetic resonance (yang et al., 2009) (fig. 2b). the alf structure consists of three αhelices (one at the n-terminus and two at the cterminus) packed against a four-stranded β-sheet, as found in limulids. based on amino acid sequence alignments, the alf family was tentatively classified into three main groups: alf cluster i, ii and iii (zhao and wang, 2008). later, another classification was proposed, but considered only penaeid shrimp alfs: groups i, ii and iii alfs (tassanakajon et al., 2010). although an increasing number of studies have focused on the characterization of this amp family, a consensus nomenclature and classification regarding structural features and phylogenetic relationships among different crustacean species and limulids are not still accessible. notably, alfs exhibit a potent (mic < 6.25 µm) and broad spectrum of antimicrobial activity against a large number of both gram-positive and gramnegative bacteria, including several opportunistic/pathogenic vibrio species, fungi and human enveloped virus (somboonwiwat et al., 2005; carriel-gomes et al., 2007; yedery and reddy, 2009a). interestingly, ralfpm3 showed a bactericidal effect against a large number of bacterial strains, including shrimp pathogens (mic < 1.56 µm) (somboonwiwat et al., 2005). moreover, it was very recently shown that ralfpm3 also has the ability to bind to both negatively-charged lipopolysaccharide (lps) and lipoteichoic acid (lta), the major cell wall components of gramnegative and gram-positive bacteria, respectively (somboonwiwat et al., 2008). synthetic peptides corresponding to the alf putative lps-binding domain of the kuruma prawn marsupenaeus japonicus displayed an efficient lps neutralizing activity in vitro (nagoshi et al., 2006), suggesting that crustacean alfs are also multifunctional proteins. interestingly, in their recent review, smith et al. (2010) do not classify alfs as conventional amps, but as binding proteins. this classification is due to their ability to bind to lps (lipid a portion) (somboonwiwat et al., 2008) in addition to their strong antimicrobial property. moreover, the authors also point out that horseshoe crab alfs are not segregated into the same granular population (small granules) as the classic antimicrobial peptides (tachyplesin, polyphemusin, tachycitin and tachystatin), but into the large amebocyte granule population together with the components of the coagulation system (iwanaga and lee, 2005). this separate compartmentalization apparently suggests that limulid alfs are primarily implicated in the regulation of the coagulation cascade. however, in crustaceans, the coagulation process is distinct from that of horseshoe crabs and is more similar to the insects (theopold et al., 2004), and alfs do not seem to take part in this process. scygonadins in contrast to the cationic hemocyte-produced amps, scygonadin is an anionic (pi of 6.09) sexspecific large antimicrobial peptide of 10.8 kda that was originally purified from the seminal plasma of the mud crab scylla serrata (huang et al., 2006). the scygonadin gene is composed of three exons and two introns, and its expression is restricted to the ejaculatory duct of adult males (wang et al., 2007). the purified peptide only showed antibacterial activity against the gram-positive bacteria m. luteus (huang et al., 2006). later, a scygonadin homolog of 11.4 kda, named ssap (for s. serrata antimicrobial protein), was purified from granular hemocytes of the same crab species 267 fig. 2 crustacean single-domain peptides containing cysteine residues engaged in disulfide bonds. (a) amino acid sequence alignment between two defensins isoforms (pjd1 and pjd2) from the japanese spiny lobster panulirus japonicus. (b) amino acid sequence comparison between two alf variants encoded by distinct genes (alfpm2 and alfpm3) from the shrimp penaeus monodon and the three-dimensional structure of alfpm3. (c) amino acid sequence alignment between the male-specific peptide scygonadin and the non-gender specific scylla serrata antimicrobial protein (ssap). identical residues are marked with an asterisk (*) and the cysteine residues are shadowed with a black background. (yedery and reddy, 2009b) (fig. 2c). both anionic scygonadin and ssap (pi of 5.7) contain two cysteine residues that are arranged as in alfs. however, a phylogenetic relationship between both anionic peptides from s. serrata and alfs was not clearly evidenced. in contrast to scygonadin, ssap is expressed in multiple tissues (as determined by rt-pcr and northern and western blot analyses) of both male and female crabs (yedery and reddy, 2009b). ssap displayed antibacterial activity mainly against gram-positive bacteria (mic of about 7.5-30 µm: m. luteus, streptococcus pyogenes and s. aureus), but not against yeast and filamentous fungi (yedery and reddy, 2009b; peng et al., 2010). multi-domain or chimeric amps antimicrobial molecules with at least two distinct domains comprise a remarkable group of crustacean amps. each domain may exhibit particular characteristics of classical single-domain amps, such as prpor cysteine-rich peptides. multi-domain amps are not common in all living kingdoms, and apart from crustaceans, they have been identified in insects and arachnids (wicker et al., 1990; saito et al., 1995). in some cases, chimeric constitution is essential for establishing cationicity and amphipathicity. a cationic domain can be committed in electrostatic interactions with negatively-charged microbial walls, while a hydrophobic domain can be responsible for membrane destabilization. the presence of different structural arrangements in a single molecule can also provide multifunctional and/or synergic properties in addition to its antimicrobial activities. additional biological functions have been shown for the crustacean multi-domain penaeidins, crustins and hyastatin as discussed hereafter. 268 penaeidins penaeidins are unquestionably the most wellcharacterized family of antimicrobial peptides described in crustaceans so far and have been the subject of many review articles (bachère et al., 2000, 2004; destoumieux et al., 2000a; cuthbertson et al., 2008; tassanakajon et al., 2010). they are chimeric cationic peptides composed of an unstructured n-terminal prp-rich domain and a cterminal region containing six cysteine residues that are engaged in three intramolecular disulfide bridges (destoumieux et al., 1997; yang et al., 2003; cuthbertson et al., 2005) (fig. 3a). interestingly, the n-terminal domain shares high sequence similarities with prp-enriched peptides, in particular with crab bac-like (schnapp et al., 1996). on the other hand, the c-terminal cysteinerich domain does not correspond to any other “cysteine motifs” previously described in cysteinecontaining amps. penaeidins were originally isolated from the hemolymph of the pacific white shrimp litopenaeus vannamei (destoumieux et al., 1997) and appear to be ubiquitous only in the family penaeidae. all penaeidin precursors comprise a highly conserved signal peptide followed by a cationic mature peptide (5.48-6.62 kda) with a calculated isoelectric point above 9 (bachère et al., 2004). because the signal peptide is cleaved, the mature peptides can be posttranslationally processed by the formation of a pyroglutamic acid in the n-terminus and/or by a cterminal amidation involving the elimination of a glycine residue (destoumieux et al., 1997, 2000a). based on amino acid sequence comparisons and the position of some precise residues in both the n and c-terminal regions, four distinct subgroups of penaeidins have been classified: pen2, pen3, pen4 and pen5 (cuthbertson et al., 2002; gueguen et al., 2006; kang et al., 2007) (fig. 3a). the subgroup pen1, which was initially purified from shrimp hemolymph, was later classified as a pen2 variant (cuthbertson et al., 2002). each penaeidin subgroup possesses a characteristic amino acid signature and common biochemical features. gueguen et al. (2006) have proposed and developed a specific nomenclature as well as a specialized database for the penaeidin family (penbase, available at http://www.penbase.immunaqua.com). the genomic structure of the penaeidin genes is variable according to each subgroup. both pen2 and pen4 genes from l. vannamei (o’leary and gross, 2006), the pen3 gene from p. monodon (chiou et al., 2007) and the pen5 gene from the fleshy prawn fenneropenaeus chinensis (kang et al., 2007) are encoded by two exons interrupted by one intron. in all of these cases, a single intron of variable length divides the prpand cysteine-rich domains. conversely, the pen3 gene from l. vannamei lacks this typical intron sequence (o’leary and gross, 2006). interestingly, in two species of litopenaeus, transcripts of pen2, pen3 and pen4 were all found to be expressed in a single individual (cuthbertson et al., 2002). in naïve (unchallenged) shrimp, penaeidin precursors are constitutively expressed, processed and addressed to cytoplasmic granules of both granular and semigranular hemocytes (destoumieux et al., 2000b). the production of penaeidins is restricted to granule-containing hemocytes that can be free in the hemolymph or infiltrating shrimp tissues (bachère et al., 2004). interestingly, no pen2 and pen4 members were described in asiatic shrimps, while the pen5 subgroup seems to be absent in “occidental” species (tassanakajon et al., 2010). the spectrum of the antimicrobial activity of penaeidins has been studied in detail using native peptides purified from shrimp hemolymph, synthetic peptides and recombinant variants produced in heterologous expression systems (destoumieux et al., 1997, 1999; cuthbertson et al., 2004, 2005, 2006; li et al., 2005; kang et al., 2007). these peptides have been shown to be particularly effective against gram-positive bacteria (mic of about 0.3-2.5 µm: aerococcus, micrococcus, bacillus, staphylococcus) and filamentous fungi (mic of about 1.25-2.5 µm: fusarium, nectria, alternaria, neurospora, botritys, penicillium), but poorly or not active against gram-negative bacteria and marine vibrios (with the exception of fenchi pen5, which appears to be active against klebsiella pneumoniae). moreover, recent studies have confirmed that penaeidin subgroups can display distinct antimicrobial activities (for review, see cuthbertson et al., 2008). interestingly, besides their antimicrobial properties, penaeidins are also able to bind to chitin. a conserved chitin-binding motif is recognized in the cysteine-rich domain, whereas the prp-rich domain is preferentially involved with the antimicrobial activities (destoumieux et al., 2000a; cuthbertson et al., 2004). according to destoumieux et al. (2000b), the chitin-binding motif of penaeidins could have additional functions, such as the ability to bind to shrimp carapace upon injury and participate in wound healing and/or molting processes. crustins crustins are generally defined as multi-domain cationic antibacterial polypeptides (7-14 kda) containing one whey acidic protein (wap) domain at the c-terminus (smith et al., 2008). the first identified crustin member is an 11.5-kda protein purified from the granular hemocytes of the shore crab c. maenas that exhibits specific activity towards gram-positive marine or salt-tolerant bacteria (relf et al., 1999). the term crustin was later proposed by bartlett et al. (2002) to describe transcripts found in two penaeid shrimp species (l. vannamei and l. setiferus) with high sequence similarity to the crab 11.5-kda protein, which was later designated carcinin (brockton et al., 2007). since the first characterization of crustin from c. maenas, over 50 crustins and crustin-like sequences have been identified in numerous crustacean species, including crayfish, shrimps, freshwater prawns, crabs and lobsters, and also in non-decapod crustaceans, such as amphipods, (through est-based approaches) (smith et al., 2008). in terms of structure, all known crustin precursors possess a leader sequence at the nterminus and a c-terminal region containing a wap domain. “wap” is a general family of proteins usually found in the whey fraction of mammalian 269 milk that contains eight conserved cysteine residues in a conserved arrangement, forming a single four disulfide core (4dsc). this molecular motif seems to exert a protease inhibitory activity, in addition to other biological functions (ranganathan et al., 1999). it is well established that protease inhibitors can play an essential role in crustacean immunity, such as inhibiting microbial proteases or regulating immune-protease cascades (cerenius and söderhäll, 2004). based on their structural features, smith et al. (2008) categorized crustins into three main types: types i, ii and iii (fig. 3b). type i crustins comprise the members most related to carcinin and possess a cysteine-rich region of variable length between the leader sequence and the wap domain. this type of crustins is mainly present in crabs (relf et al., 1999; sperstad et al., 2009a; imjongjirak et al., 2009; mu et al., 2010; yue et al., 2010), lobsters (stoss et al., 2004; hauton et al., 2006; christie et al., 2007; pisuttharachai et al., 2009c), crayfish (jiravanichpaisal et al., 2007a; shi et al., 2009), shrimp (sun et al., 2010) and freshwater prawn (dai et al., 2009). on the other hand, type ii crustins are characterized by the presence of a hydrophobic region containing an overrepresentation of glycine residues upstream of the cysteine-rich and wap domains found in type i crustins. this group is frequently documented in penaeid shrimps (bartlett et al., 2002; rattanachai et al., 2004; supungul et al., 2004; de lorgeril et al., 2005; rosa et al., 2007; zhang et al., 2007; antony et al., 2010) and crayfish (jiravanichpaisal et al., 2007a). conversely, type iii crustins possess a short prp-rich region between the leader sequence and the single wap domain, but do not contain the characteristic cysteine-rich domain present in both type i and ii crustins nor the glycine region motif. these peptides are usually known as single-whey domain (swd) proteins, chelonianin-like proteins or antileukoprotease-like proteins and have been found in shrimp and crayfish species (jiménez-vega et al., 2004; amparyup et al., 2008a; jia et al., 2008; du et al., 2010). interestingly, in penaeids, proteins with two 4dsc motifs have also been recently identified and thus named double wap domain (dwd)-containing proteins (jiménez-vega et al., 2007; chen et al., 2008; du et al., 2009). these dwd proteins display protease inhibitory activity against bacterial proteases (du et al., 2009), but they are not defined as crustins because they have more than one wap domain in their structure (smith et al., 2008). protease inhibitory and antibacterial activities seem to be related to a particular structure of the wap domain. protease inhibitory activity is generally associated with the presence of a methionine residue adjacent to the second cysteine in the 4dsc. this residue is substituted by cationic or hydrophobic amino acids in wap-containing proteins with antibacterial activity (hagiwara et al., 2003). despite the presence of the wap domain in all crustin groups, antiprotease activities have only been reported for type iii crustins (amparyup et al., 2008a; jia et al., 2008). this antiprotease capacity could be important to inactivate microbial proteases during infection and/or regulate endogenous protease cascades, such as the propo system that leads to melanization. thus, type iii crustins seem to be multifunctional immune proteins that combine both antimicrobial and antiprotease properties (amparyup et al., 2008a; jia et al., 2008). some antimicrobial studies have revealed that all crustin groups are mainly active against grampositive bacteria (mic<8 µm). susceptible bacteria include the gram-positive strains of the genera micrococcus, aerococcus, planococcus, staphylococcus, streptococcus, corynebacterium and bacillus (relf et al., 1999; zhang et al., 2007; supungul et al., 2008; imjongjirak et al., 2009; sperstad et al., 2009a). conversely, a type ii crustin from p. monodon (crustin-likepm) showed strong antibacterial activity against both grampositive and gram-negative bacteria (mic < 5 µm), including the crustacean opportunist/pathogen vibrio harveyi (amparyup et al., 2008b). the antimicrobial activity of crustins appears to be related to the tertiary structure of the 4dsc (tightly constrained by three disulfide bonds and containing a small α-helix). zhang et al. (2007) have shown that the crustin-like cshfc from f. chinensis, which lacks the authentic wap domain, did not affect any bacteria tested, in contrast to the wap-containing type ii crustin crufc, which inhibited the growth of gram-positive bacteria in vitro. interestingly, another classification for crustins was also proposed by zhao and wang (2008). these authors consider that the ‘crustin signature’ should not be restricted to the sole presence of a wap domain at the c-terminal region of the molecule. in their classification, the crustin domain comprises the arrangement of the 12 conserved cysteine residues found in type i and ii crustins, which includes the wap domain (eight cysteines). accordingly, the swd (type iii crustins) and dwdcontaining proteins, which only contain wap domains (one for swd and two for dwd), cannot be considered as authentic crustin molecules. however, as mentioned above, shrimp swds combine antiprotease and antimicrobial properties. in addition, regarding the spacing of the cysteine residues within the crustin domain, zhao and wang (2008) proposed that shrimp crustins could be recognized as either crustin i or crustin ii. although crustin antimicrobial peptides have been extensively reviewed and categorized into distinct types, a consensus nomenclature and classification as established for penaeidins (gueguen et al., 2006) has not yet been proposed. in this review, we adopted the nomenclature used by smith et al. (2008) to facilitate comparisons between the different crustin subgroups already reported in different crustaceans. the genomic organization of crustins is distinct among the different groups. the genes of two type i crustins are encoded by four exons interrupted by three introns (brockton et al., 2007; imjongjirak et al., 2009), in contrast to the swdpm2 gene from p. monodon, which belongs to the type iii crustin group and contains three exons and two introns (amparyup et al., 2008a). furthermore, two crustin variants from the shrimp p. monodon are encoded by different genes; crustinpm5 contains four exons separated by three introns, and crustin-likepm contains only two exons and one intron (amparyup 270 et al., 2008b; vatanavicharn et al., 2009). expression of most crustin-encoding genes has mainly been found in circulating hemocytes, although some reports point to the possibility of expression in other tissues (as determined by rtpcr), such as heart, ovary and intestines (smith et al., 2008). surprisingly, through rt-pcr analysis, transcripts of different crustin types, such as crustinpm5, plcrustin2, fc-crus 3 and pet-15, were essentially identified in the epipodite, hematopoietic tissue, ovary and olfactory organ, respectively (stoss et al., 2003; jiravanichpaisal et al., 2007a; vatanavicharn et al., 2009; sun et al., 2010). recent findings from our laboratory indicate that the type ii crustin crusfpau from the pink shrimp farfantepenaeus paulensis (rosa et al., 2007) is constitutively produced and stored in the granules of some populations of the shrimp granular hemocytes. moreover, monospecific polyclonal antibodies raised against the recombinant rcrusfpau were able to cross-react with corresponding crustins in the hemocyte granules of other penaeids (l. vannamei and litopenaeus schmitti), freshwater prawn (macrobrachium potiuna) and crab (c. sapidus), suggesting that these antimicrobial peptides are produced and stored in the hemocyte granules, as described for penaeidins and alfs (unpublished data). hyastatin hyastatin is a novel multi-domain antimicrobial peptide that was purified and characterized from the hemocytes of the small spider crab hyas araneus (sperstad et al., 2009b). the mature cationic molecule (pi of 9.84) is a large polypeptide of 11.7 kda that is composed of a noteworthy glycine-rich n-terminal domain, a short prp-containing portion and a c-terminal region with six cysteine residues (fig. 3c). interestingly, the glycine-rich domain consists of about 27 % of glycine residues and is very similar to that of type ii crustins. on the other hand, both the prpand cysteine-containing domains are comparable to those of penaeidins. interestingly, the arrangement of the six cysteine residues of hyastatin is identical to the cysteine pattern found in all penaeidin groups (gueguen et al., 2006). moreover, hyastatin has chitin-binding abilities as described for penaeidins (destoumieux et al., 2000b). recently, gene sequences encoding hyastatin peptides were also discovered in est libraries of other crab species, such as c. sapidus, e. sinensis, cancer magister, portunus trituberculatus and celuca pugilator. to date, information about gene arrangement and organization is not yet available for this peptide. hyastatin was assayed against a reduced number of microorganism species but showed a broad spectrum of activity. it is capable to inhibit the growth of yeast (mic of about 6.3-12.5 µm: saccharomyces cerevisiae and candida albicans), gram-positive bacteria (one species: corynebacterium glutamicum mic of 0.4 µm) and gram-negative bacteria (one species: e. coli mic of 12.5 µm), thus differing from penaeidins and crustins that possess a more restricted antimicrobial activity to gram-positive bacteria. curiously, the antimicrobial activity of hyastatin appears to be related to the cysteine-rich domain (sperstad et al., 2009b) rather than to the prp-rich domain as presumed for penaeidins (cuthbertson et al., 2004). moreover, the chitin-binding property of hyastatin is linked to its n-terminal region (prpand glycine-rich domains) instead of to the cysteine-rich domain as in penaeidins (sperstad et al., 2009b). therefore, even though hyastatin and penaeidins share sequence and structural similarities, the functional properties of their molecular domains appear to be distinct. arasins like hyastatin, arasins are cationic chimeric peptides (pi ~11) isolated from the hemocytes of h. araneus (stensvåg et al., 2008). these peptides contain a leader sequence of 25 amino acids followed by a linear prp-rich n-terminal region and a c-terminal portion containing four cysteine residues engaged in two disulfide linkages (fig. 3d). the n-terminal prp motif is similar to that of bactenecin-7, metalnikowin-1 and of crustacean prp-containing amps, such as bac-like and astacidin 2 (schnapp et al., 1996; jiravanichpaisal et al., 2007a). furthermore, the four cysteine residues from the c-terminal region are arranged similarly to vertebrate protegrins (capone et al., 2010). like hyastatin, arasin 1 (4.3 kda) was only tested against a few microorganisms species and exhibited activity against both gram-positive (c. glutamicum mic of 0.8 µm) and gram-negative (listonella anguillarum and e. coli mic of 6.3-12.5 µm) bacteria in vitro. arasin 2 was only identified through sequencing of a hemocyte cdna library, and its spectrum of antimicrobial activity has not yet been determined. like most other crustacean antimicrobial peptides, transcripts of both arasin forms were mainly detected in circulating hemocytes (stensvåg et al., 2008). unfortunately, information about arasin encoding-genes is not currently available. stylicins similar to scygonadins, stylicins are anionic peptides with a theoretical pi of 5. they are composed of 82 amino acids (8.9 kda) and are characterized by a proline-rich n-terminal region and a c-terminal portion containing 13 cysteine residues (rolland et al., 2010) (fig. 3e). this family of antimicrobial peptides shows homology to mouse cryptdin and was first evidenced in the shrimp litopenaeus stylirostris that was able to survive an infection of the pathogenic vibrio penaeicida (de lorgeril et al., 2005). these peptides were also identified in est libraries of two other penaeid species, l. vannamei and p. monodon. ls-stylicin1 from l. stylirostris is encoded by two exons interrupted by one intron, and its expression is strictly limited to the hemocytes. ls-stylicin1 did not display significant activity against either gram-positive or gramnegative bacteria, but exhibited strong antifungal effect on fusarium oxysporum. interestingly, lsstylicin1 showed a potent lps-binding activity (comparable to ralfpm3) and was able to agglutinate v. penaeicida in vitro in a lectin-like manner (rolland et al., 2010). 271 unconventional amps: multifunctional proteins and protein fragments with antimicrobial activities this last group of crustacean amps is composed of multifunctional proteins that primarily serve other functions and protein fragments that display antimicrobial activity and are generated by the processing of larger proteins. these unconventional amps, so called by smith et al. (2010), possess important molecular elements found in the structure of classical amps, such as charge, hydrophobicity and/or amphipathicity (brogden, 2005), and have been isolated and characterized from many different invertebrate and vertebrate species. they include whole proteins, such as histones, ribosomal proteins and mammalian milk proteins, and peptide fragments derived from large precursors with no evident antimicrobial properties, such as hemoglobin, lactoferrin and hemocyanin (bulet et al., 2004). in crustaceans, antimicrobial activities were reported for histones and histone fragments and also for peptides derived from the crustacean oxygen carrier hemocyanin. histones and derived fragments histones are major protein components of chromatin that are directly implicated in dna packing and the regulation of gene expression. they are cationic proteins, highly conserved in all eukaryotic cells that might also be involved in antimicrobial defense (cho et al., 2009). antimicrobial activity has been reported for different types of histones (h1, h2a, h2b, h3 and h4) in many vertebrate and invertebrate species, confirming their multifunctional properties (kashima, 1991; park et al., 1996, 1998; richards et al., 2001). their high content of cationic residues and amphipathic secondary structure could be responsible for their antimicrobial property. in fish and amphibians, histones and histone-derived fragments with antimicrobial activity have been found in skin secretions and mucus (park et al., 1998; robinette et al., 1998; fernandes et al., 2002, 2004). the antimicrobial property of histones seems to be related to their capacity of destabilizing bacterial membranes and not to their ability of forming stable pores (fernandes et al., 2002). however, the mechanisms by which histones are recruited to sites outside the cell nucleus and the regulation of histone production in response to microbial infections is far from being clear. in crustaceans, histones and histone-derived fragments displaying antimicrobial activities were recently identified in the shrimp l. vannamei (patat et al., 2004). using biochemical approaches, high levels of the four core histone proteins (h2a, h2b, h3 and h4) were detected in circulating hemocytes. both full length histone h2a and its n-terminal fragment, which is similar to buforin i and parasin amps, were strongly active (mic < 4.5 µm) against m. luteus and two strains of bacillus. likewise, a chromatographic fraction containing both h2b/h4 histones inhibited m. luteus in liquid growth inhibition assays. moreover, a shrimp h1-derived peptide was also found to be active against m. luteus (patat et al., 2004). hemocyanin-derived peptides hemocyanin is a copper-containing oxygen transport protein in crustaceans that is produced in the hepatopancreas and then released into the plasma. hemocyanin is the most abundant protein in the crustacean hemolymph, representing more than 95 % of the total protein in the plasma. arthropod hemocyanins are organized as multiples of hexamers, and each hexamer contains monomers of about 75 kda (van holde et al., 2001). in addition to its role as an oxygen carrier, hemocyanin appears as a multifunctional protein since it is also involved in osmoregulation, protein storage and some immune reactions (decker and jaenicke, 2004). in chelicerates, the n-terminal region of hemocyanin was suggested to have phenoloxidase activity after proteolytic cleavage (decker and rimke, 1998; nagai and kawabata, 2000). the prophenoloxidase system (or propo system) involves a complex cascade in which phenol compounds are oxidized and many toxic molecules (quinones and reactive oxygen intermediates) are generated in response to a microbial infection (cerenius and söderhäll, 2004; cerenius et al., 2008). in crustaceans, some antimicrobial peptides derived from the c-terminal part of hemocyanin were isolated and characterized from the plasma of the penaeids l. vannamei (pvhct) and l. stylirostris (pshct1, pshct2) (destoumieux-garzón et al., 2001) and the crayfish p. leniusculus (astacidin 1) (lee et al., 2003). the three peptides derived from shrimp hemocyanin exhibited strong antifungal activity against many filamentous strains (mic of 3.15-12.5 µm) (destoumieux-garzón et al., 2001). by contrast, astacidin 1 from crayfish was active against gram-positive (mic of 1.9-12.8 µm: bacillus sp. and m. luteus) and gram-negative bacteria (mic of 15 µm: s. flexneri and e. coli). all three shrimp hemocyanin-derived peptides are anionic molecules with molecular masses of 2.7 kda (pvhct), 7.9 kda (pshct1) and 8.3 kda (pshct2) and are induced or activated in the plasma of animals stimulated by microbial injection (destoumieux-garzón et al., 2001). these peptides are generated from the c-terminal region of hemocyanin, which lacks the copper-binding site. in the crayfish p. leniusculus, astacidin 1 (about 1.9 kda) appears to be released from the c-terminus of hemocyanin by a cysteine-like protease, and its production is enhanced by injection of lps or glucan into the crayfish (lee et al., 2003). involvement of amps in crustacean host defense and survival evidence of the implication of amps in innate host defenses has been reported in multiple species across different taxa, including crustaceans (hancock and scott, 2000; pazgier et al., 2006; de lorgeril et al., 2008; conlon, 2010; smith et al., 2010). in most decapod species, especially in penaeid shrimp, the modulation of gene expression of some amps in response to microbial challenge 272 has been extensively studied (muñoz et al., 2002, 2004; sunpugul et al., 2004; jiravanichpaisal et al., 2007a; robalino et al., 2007; de la vega et al., 2008; sperstad et al., 2010). interestingly, the expression pattern of the well-characterized crustacean amps differs according to the amp family and/or the crustacean species. alf transcripts in the circulating hemocytes of p. monodon (alfpm3), f. chinensis and l. stylirostris increase in abundance in the first hours after a vibrio challenge (supungul et al., 2004; liu et al., 2005; de lorgeril et al., 2008). in contrast, the mrna concentration of shrimp crustins, penaeidins and stylicin significantly decrease at this stage of infection (muñoz et al., 2004; supungul et al., 2004; de lorgeril et al., 2008). furthermore, the expression of these last amps returns to basal levels 24-72 h post-infection, followed by a subsequent remarkable increase when compared to unchallenged animals (muñoz et al., 2004; de lorgeril et al., 2008). conversely, the in vitro gene expression of three amps (arasin-1, hyastatin and a type i crustin) in primary hemocyte cultures of the crab h. araneus were not affected neither by gram-positive nor by gram-negative bacteria stimulation (sperstad et al., 2010). however, these observations cannot be extended to an in vivo situation, in which the conditions are considerably different. interestingly, in the crayfish p. leniusculus, the modulation of crustin expression appears to vary according to the crustin type. the expression of plcrustin1 (type i crustin) was increased in circulating hemocytes and hematopoietic tissue after stimulation with both gram-positive and gram-negative bacteria, while plcrustin3 (type ii crustin) expression was only induced after injection with a non-pathogenic gramnegative bacteria (acinetobacter sp.). in contrast, the expression of plcrustin2 (another crayfish type i crustin) and astacidin 2 were not modulated by microbial challenge (jiravanichpaisal et al., 2007a). according to bachère et al. (2004), two main phases are recognized in shrimp (l. vannamei) immune response to a vibrio infection, based on penaeidin expression: phase i (local reaction) and phase ii (systemic reaction). during the first phase (12 h post-infection), there is a decrease in the mrna abundance of penaeidins due to the migration of penaeidin-producing hemocytes to the sites of infection. in response to a presumed chemotactic effect, these hemocyte populations release large amounts of penaeidins in the infected tissues. the second phase occurs at about 48 h post-infection and is characterized by the activation of hematopoiesis and the appearance of penaeidinexpressing hemocytes in both hemolymph and shrimp tissues. a similar observation was also observed in alf kinetics in p. monodon after a v. harveyi injection (somboonwiwat et al., 2008). recently, the in vivo role of some crustacean amps was investigated in shrimp and crayfish using rna interference (liu et al., 2006; de la vega et al., 2008; shockey et al., 2009; tharntada et al., 2009). a significant increase in mortality after silencing crustin gene transcripts was observed in l. vannamei infected with the gram-negative pathogen v. penaeicida, but not with the virulent fungus f. oxysporum, as compared to crustin-expressing animals (shockey et al., 2009). these results are curious because crustins do not seem to have an effect on marine vibrios, according to in vitro assays (see section 3.2). on the other hand, in the same shrimp species, alf was shown to be involved in both bacterial and fungal infections (de la vega et al., 2008). in addition to bacterial infections, crustaceans are also attacked by different classes of viruses. to date, viral infections are the most serious constraint to shrimp farming worldwide, particularly the disease caused by the white spot syndrome virus (wssv). in the crayfish p. leniusculus, injection of alfdsrna resulted in an increased expression of the wssv envelope protein vp28 in hematopoietic tissue cell cultures, indicating that this molecule is involved in antiviral defense (liu et al., 2006). moreover, preincubation of wssv with ralfpm3 (from p. monodon) reduced virus propagation in both crayfish cell cultures (hematopoietic tissue) and infected p. monodon shrimp (tharntada et al., 2009). conversely, the alf member lvalf1 does not seem to be responsible for direct virus protection in the shrimp l. vannamei (de la vega et al., 2008). these results, together with differences in alf antimicrobial activities according to species, suggest that this amp family could be implicated differentially in the immune responses of each crustacean group. over the past few years a significant number of large-scale est sequencing projects have successfully identified many immune genes, such as antimicrobial peptide family genes, in important cultivated shrimp species (gross et al., 2001; rojtinnakorn et al., 2002; supungul et al., 2002; tassanakajon et al., 2006; dong and xiang, 2007). currently, over 170,000 sequences are available in the genbank database from various tissues of different shrimp species (for review, see robalino et al., 2009). in addition, the analysis of these est libraries has confirmed that hemocytes are the main site for amp synthesis. sequences encoding amps in l. vannamei and l. setiferus comprise about 20 % of all sequenced transcripts in hemocyte cdna libraries (gross et al., 2001). in this context, it has been shown that penaeidins, crustins and alfs appear to be the most highly expressed amps in shrimp hemocytes (gross et al., 2001; supungul et al., 2002; dong and xiang, 2007). furthermore, in specific est libraries of p. monodon, some amps were found to be up-regulated after both wssv and v. harveyi challenge (tassanakajon et al., 2006). the developmental expression of the three most well-characterized crustacean amps was studied in detail in penaeid shrimp and crayfish. significant mrna expression levels of penaeidins, crustins and alfs were detected in the early developmental stages (nauplius, zoea and mysis) of l. vannamei, p. monodon and f. chinensis (muñoz et al., 2003; chiou et al., 2007; jiravanichpaisal et al., 2007b; sun et al., 2010; tassanakajon et al., 2010). interestingly, two type ii crustins from f. chinensis (fc-crus 1 and fc-crus 2) are expressed in several shrimp developmental stages, while fccrus 3 (the sole type i crustin member found in penaeid shrimp to date) is found only in the ovaries 273 fig. 3 crustacean multi-domain or chimeric amp families. (a) sequence comparison of different penaeidin subgroups (pen2 to pen5) and the three-dimensional structure of litvan pen3-1 from litopenaeus vannamei. (b) a not-to-scale schematic illustration of the three crustins types (type i to iii crustin). (c) schematic comparison (notto-scale) of hyastatin, penaeidins and type ii crustins. (d) amino acid sequence alignment between the two arasins from the spider crab hyas araneus. (e) schematic representation (not-to-scale) of stylicin from the blue shrimp litopenaeus stylirostris. in the amino acid alignments, the proline/arginine residues and conserved cysteines are highlighted in grey and black boxes, respectively. 274 (via rt-pcr) of adult animals (sun et al., 2010). conversely, in the middle stage of crayfish embryo development, the expression of type i crustins (plcrustin1 and plcrustin2) is inferior to that of the type ii crustin plcrustin3 (zhang et al., 2010). taken together, these results suggest that some families of amps could be preferentially produced during distinct periods of the crustacean life cycle and highlight their importance during ontogenesis. recently, genes potentially associated with shrimp survival capacity were identified in l. stylirostris using the suppression subtractive hybridization (ssh) strategy (de lorgeril et al., 2005). among the genes differentially expressed in the circulating hemocytes of naïve versus vibrioinfected shrimp, many were immune-related genes, including some crustacean amp families. it was shown that the mrna abundance of penaeidins (litsty pen2 and litsty pen3), crustins and stylicin (previously called cysteine-rich peptide or cryptdinlike) before a v. penaeicida infection could predict shrimp survival. these results evidenced for the first time a relationship between the gene expression profile and the capacity of shrimp to survive an infection (de lorgeril et al., 2008). additionally, it was shown that shrimp amps could display different expression kinetics after microbial stimulation. it is noteworthy that one single animal can concomitantly express multiple amp families, including many different subgroups and isoforms, and that this can confer a broad spectrum of antimicrobial responses to this animal. unfortunately, at the present moment it is not known if different crustacean amps co-localize in the same vesicles of granular hemocytes or if each peptide family is segregated in different granules or cellular subpopulations. it would be also interesting to elucidate if crustacean amps co-localize with other immune effectors, such as the proteins of the propo system and lysosomal degrading enzymes in the hemocyte granules, as shown in detail in limulids (iwanaga, 2002; iwanaga and lee, 2005). other considerations on crustacean amps the knowledge acquired over the last two decades on the identification and characterization of antimicrobial peptides in crustaceans has revealed their essential role in the immune response and also in the capacity of these animals to survive infection. however, from the identified amp families, only a few members have been tested against crustacean pathogens, as marine vibrios that may cause severe infections to these animals (table 2). from these, solely the alfs displayed a consistent and potent effect against a variety of vibrio species, including strains that are pathogenic to crustaceans (somboonwiwat et al., 2005). in addition, alfs are also implicated in crustacean antiviral defense against wssv (liu et al., 2006). these properties make the alfs a particularly interesting antiinfective amp family within crustaceans. to date, alf members have been identified in several decapod species, but their occurrence and distribution in other crustacean orders remain unknown. curiously, the complete genomic sequencing of the water flea daphnia pulex (a basal crustacean lineage: class branchiopoda, order cladocera) did not reveal the presence of genes encoding alfs or any other currently known amp family identified in crustaceans (mctaggart et al., 2009). these findings are intriguing because gene sequences encoding alfs are present in ancient chelicerates (horseshoe crabs) as well as in decapods (derived crustacean lineage), which are obviously more phylogenetically related to branchiopods than to chelicerates. therefore, it remains unclear if alf sequences were lost in d. pulex or if they have never been present. with respect to crustins, est sequences containing a putative wap domain with characteristics of the crustin wap domain were identified in the marine branchiopod artemia franciscana and the copepod calanus finmarchius (smith et al., 2008). however, in other molecular aspects, these partial sequences did not share significant similarities with the well-characterized crustin family. conversely, two est sequences were identified in the amphipod gammarus pulex (a derived crustacean lineage), including many features of type ii crustins, such as a glycine-rich region similar to those of shrimp and a cysteine-rich region containing the wap domain (smith et al., 2008). the authors suggest that although incomplete, these sequences are strong candidates for being true members of the crustin family. therefore, crustins appear to be the unique amp family characterized in decapods that is also present in non-decapod species (at least in amphipods) to date. it would be interesting to elucidate if certain amp families are widely distributed across crustaceans, as it appears to be the case for crustins, while others have a more restricted distribution, such as penaeidins, which are only present in penaeid shrimp. another important point concerns the in vitro assessment of the activity of the different amp families of crustaceans (table 2). in most studies, the physiological conditions of the animals are not taken into account, particularly of marine crustaceans (the most well-studied group in terms of amps), which have high salt concentrations in their body fluids. it is well known that amps lose their effect at high salinity due to alterations of their charge and structural conformation (lee et al., 1997; löfgren et al., 2009). it is therefore essential to confirm whether the amps of marine animals are stable under salt conditions to better comprehend their effect in vivo. indeed, it was shown that the penaeidin variant litvan pen3-1 lost its activity against m. luteus at a salinity above 0.9 % (marine salinity is about 3.5 %). conversely, the antimicrobial property of the alf ralfpm3 was not significantly affected by high nacl concentration (löfgren et al., 2009), and this is another interesting feature of this molecule. in contrast, carcinin (type i crustin) requires high salt concentrations for its activity (relf et al., 1999). the great majority of amps was isolated and/or identified from crustacean hemocytes. it was shown that penaeidins, alfs and type ii crustins from shrimp are synthesized and stored in the granules of granular hemocytes. however, it was also reported that some amp families could be also expressed in 275 table 2 antimicrobial properties of crustacean amps bacteria fungi amp families gram-positive gram-negative filamentous yeast bac-like nt nt callinectin nt 1 nt nt astacidin 2 nt nt armadillidin 1 na nt nt homarin na nt nt defensin nt nt nt nt alf na scygonadin na na na penaeidin subgroup 2 (pen2) na na subgroup 3 (pen3) na subgroup 4 (pen4) na subgroup 5 (pen5) nt crustin2 type i na na type ii nt type iii hyastatin 1 1 na 1 arasin 1 1 nt nt stylicin na na histones and derived fragments 1 nt nt nt hemocyanin-derived peptides astacidin 1 nt nt pvhct/pshct1/pshct2 na na nt minimal inhibitory concentration (mic) values: up to 10 µm; 10-20 µm; 20-40 µm. na: not active (>40 µm). nt: not tested. 1amps tested against a reduced number of microorganisms. 2crustin classification proposed by smith et al. (2008). other crustacean tissues, such as the defensins of the spiny lobster p. japonicus (pisuttharachai et al., 2009b) and certain crustin types (smith et al., 2008). however, it happens that most of these results are based on rt-pcr and northern blot analyses whose results can be misleading. crustaceans possess an open circulatory system, and the circulating blood cells can infiltrate most of their tissues. consequently, the presence of amp transcripts in different tissues can actually originate from infiltrated hemocytes. indeed, the expression of penaeidins was initially identified in a wide range of tissues in l. vannamei using northern blot analysis (destoumieux et al., 2000b). however, after in situ hybridization assays it was revealed that penaeidins were only expressed in shrimp granular hemocytes (muñoz et al., 2002). similar results were also obtained for the alfs of f. chinensis and p. monodon (liu et al., 2005; soombowiwat et al., 2008). hence, it is of prime importance to determine the gene expression sites of the other crustacean amp families using more appropriate technical approaches (such as in situ hybridization and immunohistochemistry) to avoid misinterpretations. 276 fig. 4 schematic illustration of crustacean cellular and humoral immune-reactions after microbial challenge. hh = hyaline hemocytes, gh = granular hemocytes another relevant aspect that remains to be clarified concerns the co-localization of the different amp families within the crustacean hemocytes. to date, it is unclear if the different amp classes and/or isoforms are selectively or randomly segregated into different granules of distinct granular hemocyte populations. as previously mentioned, this specific subcellular compartmentalization was elegantly demonstrated in horseshoe crabs, in which the classic amps are stored within small granules, while alf is segregated into large granules in association with the components of the coagulation cascade (iwanaga and lee, 2005), suggesting its primary role in horseshoe crab coagulation. in crustaceans, nothing is currently known about the co-localization of the different amp families within immune cells. a 277 better elucidation of their storage in the same or different granules could help to predict their behavior during infections. it is generally assumed that crustacean amps are promptly released into the hemolymph from the granules upon cell stimulation by invading pathogens (jiravanichpaisal et al., 2006), as it occurs in limulids (iwanaga and lee, 2005). accordingly, crustacean amps should combat and control infections extracellularly in a systemic response. however, these molecules could also exert their activity intracellularly within phagosomes. interestingly, a significant number of crustacean amps are composed of distinct domains (penaeidins, crustins, hyastatin, arasins and stylicins). this structural feature contrasts with the amps commonly described in other organisms, even the phylogenetically related groups, such as limulids (iwanaga et al., 2005) and insects (bulet and stöcklin, 2005), which mainly possess small single-domain peptides. it would be interesting to elucidate when and how the genes encoding these multi-domain proteins have been formed and rearranged during crustacean evolution. the implication of the different domains in different immune or physiological functions in crustaceans is uncertain. perhaps the biological role of these proteins is primarily a reflex of their overall structural conformation (forming cationic amphipathic structures) and not of their independent intramolecular domains. however, the multifunctional role of some crustacean amp is better documented. for example, the chitin-binding activity of penaeidin and hyastatin presumably participates in wound healing and molting (destoumieux et al., 2000a; sperstad et al., 2009b), the lps-binding properties of alfs and stylicins and the protease inhibition activity of type iii crustins block pathogen proteases and/or regulate endogenous protease cascades. concerning the unconventional amps, the antimicrobial activity of histones and histone-derived fragments was indeed an expected phenomenon in crustaceans because the antimicrobial property of these highly conserved proteins was already reported from fish to mammals. in shrimp, patat et al. (2004) hypothesized that these proteins are stored in hemocyte granules and released into the hemolymph during microbial infection, together with other classical amps. however, the presence of histones in granules is unexpected because their transcripts do not possess a signal sequence to direct them to cytoplasmic granules. expectedly, they contain nuclear localization signals within their n-terminal domain for translocation into the nuclear compartment. consequently, their storage in hemocyte granules and their presumed release into the hemolymph in response to infection remain to be demonstrated. finally, maybe the most important aspect regarding crustacean amps concerns the complementary and synergistic antibiotic roles that these molecules probably encompass. it is reasonable to assume that the multiple classes of variants and isoforms of crustacean amps, which are concurrently present in the hemocytes of decapods, should act in a cooperative and complementary manner in order to circumvent infections and ensure host survival (fig. 4). to date, synergistic experiments have only been reported for l. vannamei penaeidins. however, no synergistic effects were observed between litvan pen2-1 and litvan pen3-1 (destoumieux et al., 1999). also, in addition to amps, the immune cells of crustaceans produce several other well-known immune effectors, such as lysozyme and other degrading enzymes and the components of the propo system, to control infections. smith et al. (2010) suggest that lysozyme, a widely distributed antimicrobial enzyme that cleaves the carbohydrate portion of the peptidoglycan from the bacterial cell wall, potential plays a synergistic role with the common antimicrobial peptides. after disrupting the bacterial cell wall, lysozyme could facilitate the access of classic amps to permeabilize the bacterial membrane. therefore, lysozyme could simultaneously serve as a direct effector and synergistic agent for enhancing amp activity. concluding remarks in conclusion, as illustrated above, both in vitro and in vivo properties of amps confirm that they are indeed essential components of the crustacean immune system. unfortunately, the vast majority of the current knowledge refers only to the order decapoda and is far from representing the entire and highly diverse group of crustaceans. this is clearly evidenced by the apparent absence of gene sequences encoding for hitherto described amps in the complete genome of the branchiopod d. pulex. perhaps a more refined analysis of its genome could reveal the presence of gene sequences with similarity to some amp domains that have already been characterized in decapods. moreover, in view of the exceptional diversity of crustaceans, it is expected that novel unknown amp families with singular molecular structures and interesting biological properties are waiting to be discovered. on the other hand, concerning the already identified crustacean amps, it would be interesting to fill in gaps of missing knowledge, such as their precise mode of action, in vivo activity, co-localization with other amp members and especially their synergistic effect with other amp families and/or other immunologic effectors also present in the immune cells of crustaceans. this information would certainly significantly contribute to a more comprehensive and integrated vision of the essential role that these molecules exert in crustacean immunity. references aketagawa j, miyata t, ohtsubo s, nakamura t, morita t, hayashida h, et al. primary structure of limulus anticoagulant anti-lipopolysaccharide factor. j. biol. chem. 261: 7357-7365, 1986. amparyup p, donpudsa s, tassanakajon a. shrimp single wap domain (swd)-containing protein exhibits proteinase inhibitory and antimicrobial activities. dev. comp. immunol. 32: 1497-1509, 2008a. amparyup p, kondo h, hirono i, aoki t, tassanakajon a. molecular cloning, genomic 278 organization and recombinant expression of a crustin-like antimicrobial peptide from black tiger shrimp penaeus monodon. mol. immunol. 45: 1085-1093, 2008b. antony sp, bright singh is, philip r. molecular characterization of a crustin-like, putative antimicrobial peptide, fi-crustin, from the indian white shrimp, fenneropenaeus indicus. fish shellfish immunol. 28: 216-220, 2010. bachère e, destoumieux d, bulet p. penaeidins, antimicrobial peptides of shrimp: a comparison with other effectors of innate immunity. aquaculture 191: 71-88, 2000. bachère e, gueguen y, gonzalez m, de lorgeril j, garnier j, romestand b. insights into the antimicrobial defense of marine invertebrates: the penaeid shrimps and the oyster crassostrea gigas. immunol. rev. 198: 149-168, 2004. bartlett tc, cuthbertson bj, shepard ef, chapman rw, gross ps, warr gw. crustins, homologues of an 11.5-kda antibacterial peptide, from two species of penaeid shrimp, litopenaeus vannamei and litopenaeus setiferus. mar. biotechnol. 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shrimp. inv. surv. j. 5: 162-179, 2008. 284 review antimicrobial peptides in crustaceans purification and characterization of phenoloxidase from the hemolymph of hyphantria cunea (lepidoptera isj 9: 64-71, 2012 issn 1824-307x research report purification and characterization of phenoloxidase from the hemolymph of hyphantria cunea (lepidoptera: arctiidae) m ajamhassani1, jj sendi1, mj farsi2, a zibaee1 1department of plant protection, college of agriculture, university of guilan-rasht, 41635-1314, iran 2department of plant protection, research institute of forest and range land, tehran, 13185-116, iran accepted may 4, 2012 abstract phenoloxidase (po) is a key factor in insect immunity. on invasion of microorganisms and pathogens, prophenoloxidase changes to its active form to po. this study investigated purification biochemical properties of po from the hemolymph of 5th instar larvae of hyphantria cunea (lepidoptera). the purification fold was determined as 9.67 with a recovery of 0.12 and a specific activity of 23.28 u/mg protein identified. kinetic parameters of purified po from the insect h. cunea were determined. the michaelis constant (km) and the maximal velocity (vmax) were 4.08 and 12.98 µmol/min/mg protein, respectively. the optimal ph and temperature of the enzyme for oxidation of ldopa were 10.0 and 35 ºc, respectively. the ions zn2+, cu2+, k+ and na+ significantly increased the enzyme activity and synthetic inhibitors such as diethyldithiocarbamate (detc) significantly decreased it. finally, it was found that purified po had a molecular mass of 33 kda. this study demonstrated some po properties and its inhibitory effects demonstrating that it can be employed as useful methods for developing novel insecticides to replace traditionally used ones. key words: purification; phenoloxidase; hyphantria cunea; enzyme property introduction insect immunity consists of both humoral and cellular defensive reactions (gillespie et al., 1997; lavine and strand, 2002). cellular immunity includes phagocytosis, encapsulation and nodule formation (lavine and strand, 2002). but antimicrobial peptides and the phenoloxidase (po) system both have major roles in humoral defense. po is a key factor of insect immunity, having important roles in the processes of coagulation, melanization and wound healing. in arthropods po is synthesized as inactive zymogen and prophenoloxidase is then activated in the presence of serine proteinases. this phenomenon occurs when pathogen agents and parasitoids enter the hemocel (söderhäll and cerenius, 1998). po causes modifying tyrosine to form dihydroxyphenylalanine, and they oxidize odiphenols to quinones to form melanin for nodulation and encapsulation. melanotic encapsulation and nodulation play important roles in ___________________________________________________________________________ corresponding author: jalal j sendi department of plant protection college of agriculture, university of guilan-rasht 41635-1314, iran e-mail: jjalali@guilan.ac.ir innate immune response against large microorganisms (ling and yu, 2005). insects have both types of phenoloxidases, namely laccase-type enzymes (ec 1.10.3.2) and tyrosinase-like enzymes. laccase-type change oxidize oor pdiphenols to quinones, this function is fundamental in sclerotization and tanning of the cuticle (dittmer et al., 2004; arakane et al., 2005). also, those enzymes in the hemolymph that have tyrosinaselike activity can hydroxylate tyrosine (ec 1.14.18.1) and oxidize o-diphenols to quinones (ec 1.10.3.1) (gorman et al., 2007a). the function of propo in arthropods’ immunity and regulation of its activating system have been discussed in other researches (ashida and brey, 1997; cerenius, 1998; sugumaran, 2002; cerenius and söderhäll, 2004; kanost et al., 2004; christensen et al., 2005; nappi and christensen, 2005). po activity has been investigated in other insects such as manduca sexta (lepidoptera: sphingidae), (hall et al., 1995), drosophila melanogaster (diptera: drosophilidae) (sezaki et al., 2001), apis mellifera (hymenoptera: apidae) (zufelato et al., 2004), pieris rapae (lepidoptera: pieridae) (xue et al., 2006), heliothis virescens (lepidoptera: noctuidae) (shelbi et al., 2006), ostrinia furnacalis (lepidoptera: pyralidae) (feng et 64 mailto:jjalali@guilan.ac.ir fig. 1 column chromatography of po from h. cunea. profiles corresponding to 490 nm absorbance and enzymatic activity of collected fractions are shown. a) sepharyl g-100 gel-filtration of po after ammonium sulfate (30 % and 70 %) treatment. po was applied to a sepharyl g-100 column and eluted with 25 mm tris-hcl buffer (ph 8). fractions 17 24 contained the highest enzymatic activity on 10 mm l-dopa and collected for next steps. b) cm-sepharose ion-exchange chromatography of the gel-filtrated po from h. cunea. sepharyl g-100 runoff fractions were applied to a cm-sepharose column and eluted with a linear gradient (1, 3 and 5 m ) nacl in 25 mm tris-hcl buffer (ph 8). fractions 10 14 contained the highest enzymatic activity on 10 mm l-dopa and used for continuing the experiments. al., 2008), and eurygaster integriceps (hemiptera: scutelleridae) (zibaee et al., 2011). several studies have investigated purification and characterization of different insect pos (durrant et al., 1993; gillespie et al., 1997; chase et al., 2000). hyphantria cunea or fall web worm (lepidoptera: arctiidae) is a polyphagous insect found throughout the world. it was first reported in iran in 2002 in the caspian forests (guilan province, northern iran). a large population of h. cunea has been established during the last few years and the insect is one of the most harmful pests in the region. this insect has a broad range of hosts from different forest trees, fruit trees and ornamentals to annual crops and weeds. the preferred hosts include morus, maple, platanus, oak, poplar, elm, fagus, willow and alder (yarmand et al., 2009). there is a need for an advanced program to keep this pest under control. the use of bacillus thuringiensis has been worked out as an entomopathogenic agent decreasing the population of larvae of h. cunea in the field. furthermore, pilot experiments have shown that beauveria bassiana can affect its survival and longevity (unpublished data). ajamhassani and colleagues evaluated decreases in total hemocytes, nodulation and po activity of h. cunea against different isolations of the fungus b. bassiana (unpublished data). but there appears to be very little known research on po in this species. therefore, it is essential to obtain more information on the physicochemical properties of h. cunea po. the purpose of this study was to purify po from the plasma of h. cunea, and to examine its biochemical properties. 65 table 1 purification of the po from the hemolymph of h. cunea. purification steps total activity (u) total protein (mg) specific activity (u/mg) recovery (%) purification fold crude extract 0.36825397 0.153667 2.40688889 100 1 ammonium sulfate 30 % 0.26243386 0.017667 15.4372941 72.22 6.413796 ammonium sulfate 70 % 0.23915344 0.016333 14.9470625 63.88 6.210117 sepharyl g-100 0.08465608 0.004 21.164 0.23 8.793094 cm-sepharose 0.04656085 0.002 23.2805 0.12 9.672445 material and methods insects eggs of hyphantria cunea drury were collected from the forests of guilan province and reared on mulberry leaves in laboratory conditions (26 ± 1 °c, 80 % rh and 14 h light:10 h dark). two-day-old 5th instar larvae were used in this study. collection of hemolymph hemolymph samples (10 larvae, 200 μl hemolymph) were collected from severed third prolegs of fifth instar larvae. the hemolymph was immediately diluted with an anticoagulant solution (0.01 m ethylenediamine tetraacetic acid, 0.1 m glucose, 0.062 m nacl, and 0.026 m citric acid, ph 4.6) (azambuja et al., 1991). the hemolymph was diluted with a phosphate buffer (ph 7, 10 mm). po preparation the collected hemolymph (35 µl of hemolymph and 15 µl of anticoagulant solution) was mixed with the phosphate buffer and centrifuged at 6,000 g for 30 min. supernatant was mixed with 25 g ammonium sulfate and centrifuged at 6000 g for 30 min. pellets were mixed with tris hcl 25 m m (ph 8). this crude extract was used to assess po. samples were pre-incubated with the buffer (trishcl, ph 7) at 30 °c for 30 min before the addition of 50 ml of 10 mm aqueous solution of substrate ldihydroxyphenylalanine (l-dopa). the mixture was incubated for 5 min at 30 °c and po activity (one unit of po activity represents the amount of enzyme required to produce an increase in od490 of 0.01 per min) was measured in a spectrophotometer at 490 nm (dularay and lackie, 1985). assays were done in triplicates. purification of po purification of the po extracted from h. cunea hemolymph was done according to a three-step procedure described by pang et al. (2005) at 30 ◦c: a) ammonium sulfate treatment. the samples were subjected to ammonium sulfate precipitation using concentrations of 30 % and 70 %. then two different levels of ammonium sulfate treatments were collected by centrifugation at 10,000 g and the pellets obtained in each treatment were suspended in a minimal volume of 100 mm tris-hcl, ph 8.0. to change samples from the first ammonium sulfate precipitation to the second one, the 30 % pellet was discarded, the supernatant precipitated with 70 % ammonium sulfate and the pellet was used for the next step; b) sepharyl g-100 gel filtration. the ammonium sulfate fractions were subjected to gel filtration using a sepharyl g-100 column (2 cm×100 cm) equilibrated with 25 mm tris-hcl ph 8.0 containing 0.05 % (v/v) triton x-100. fractions of 5 ml were collected at a flow rate of 20 ml/h with the same buffer. protein content and po activity were measured and fractions showing po activities were pooled; c) cm-sepharose separation. fractions with fig. 2 double reciprocal plot to show the kinetic parameters of the purified po from the hemolyph of h. cunea l-dopa (10 mm) was used as substrate. (1/vmax = intercept on the 1/v0 ordinate, −1/km = intercept on the negative side of the 1/[s] abscissa). 66 po activity were applied to a cm-sepharose column (3 cm×30 cm) equilibrated with 25 mm tris-hcl buffer ph 6.0. after washing the column with the same buffer, bound proteins were eluted with a linear gradient of nacl (1, 3 and 5 m) in the equilibrating buffer. fractions (5 ml each were collected at a flow rate of 1.0 ml/min. fractions with po activities were pooled and stored at -20 °c for further analysis. kinetic parameters (vmax and km) of po to measure the kinetic parameters of po different concentrations of l-dopa (1, 3, 5, 7 and 10 mm) were mixed with 20 μl of enzyme solution and read at 490 nm. michaelis constant (km) and maximal velocity (vmax) was estimated by sigmaplot software version 11 and the results of km and vmax were the means ± se of three replicates (n = 3) for each concentration. effects of ph and temperature on the enzyme activity the effects of temperature and ph on po activity were examined using 10 mm solution of ldopa as a substrate. optimal ph was determined using 25 mm tris-hcl buffer at a range of 4 12. the effect of temperature on po activity was determined by incubating the reaction mixture at temperatures of 20, 25, 30, 35, 40, 50 and 60 °c for 30 min (liu et al., 2006), followed by measurements of activity. effects of ions and enzyme inhibitors on protease activity the effect of various ions (0.5, 3 and 5 mm) on enzyme activity was investigated using cacl2, cucl, znso4, mgcl2, kcl and nacl. the activity of the enzyme in the absence of added ions was considered as 100 %. the effect of enzyme inhibitors on po activity was studied using ethylenediaminetetraacetic acid (edta, 10mm), diethyldithiocarbamate, n, n, n′, n′-tetraacetic acid (egta, 10 mm), sodiumdodecylsulfate (sds, 10 mm), phenylthiourea (10 mm), diethyldithiocarbamate (detc, 10 mm) and phenylmethylsulfonyl fluoride (pmsf, 10 mm). the purified enzyme was pre-incubated with the inhibitors for 30 min at 35 °c and ph 8, and enzyme activity was determined with l-dopa as a substrate. in all experiments, the activity of the enzyme without the addition of inhibitors was considered as 100 percent. polyacrylamide gel electrophoresis sds polyacrylamide gel electrophoresis (denaturing sds-page) was used to determine the purity and molecular mass of the enzyme as described by laemmli (1970) using a 4 % (w/v) stacking gel and a 10 % (w/v) separating gel. the molecular mass of the enzyme was estimated using the following standards: β-galactosidase (116 kda), bovine serum albumin (66.2 kda), ovalbumin (45 kda), lactate dehydrogenase (35.5 kda), restricting endonuclease bsp 981 (25 kda), β-lactoglobulin (18.4 kda) an lysozyme (14.4 kda). after sdspage, proteins on the polyacrylamide gel were stained with 0.2 % coomassie brilliant blue r-250. fig. 3 effect of ph (a) and temperature (b) on the activity of the hemolymph-derived po in h. cunea. different letters show significant differences among values (tukey's test, p < 0.05). protein determination protein concentrations were measured according to the method of bradford (1976), using bovine serum albumin (bio-rad) as a standard. statistical analysis data were compared by the one-way analysis of variance (anova) followed by tukey's studentisized test and significant differences were considered at p < 0.05 (sas, 1997). significant differences were marked in figures and tables. results purification of po po from the hemolymph of h. cunea was purified and the results are presented in fig. 1 and table 1. in the first step, samples were precipitated with ammonium sulfate in concentrations of 30 % and 70 %. in the second step, the 70 % ammonium sulfate fraction was eluted by gel filtration using sephadex g-100 column followed by ion exchange chromatography on cm-sepharose column. about 10 ml of the ammonium sulfate-precipitated po was loaded onto a sepharyl g-100 gel-filtration column (fig. 1a). fractions 17 24 with high enzymatic activities using 10 mm l-dopa were collected and pooled. the pooled fractions contained 0.004 mg/ml protein and the total enzymatic activity was 67 table 2 effect of different compounds (ions as activators and organic molecules as inhibitors) on the po activity of h. cunea. compounds concentration (mm) percentage of po activity (%) control 100 cu+ 0.5 106.19 3 129.64 5 202.65* zn2+ 0.5 215.04* 3 217.69* 5 273.45* ca2+ 0.5 27.43 3 26.54 5 9.73 mg2+ 0.5 24.77 3 12.38 5 12.38 k+ 0.5 29.20 3 123 5 199.11* na+ 0.5 38.93 3 42.47 5 136.28 edta 10 72.94 egta 10 83.52 detc 10 2.35* pmsf 10 22.35* phenylthiourea 10 56.47* the activity of the po was determined by incubating the enzyme in the presence of various compounds for 30 min at 30 °c and ph 8. asterisks show the significant differences among treatments and control (tukey's test, p < 0.05). 0.23 u. the pooled fractions from sepharyl g-100 gel-filtration were loaded onto an ion-exchange column (fig. 1b). fractions 10-14 with high enzymatic activities were eluted at a nacl concentrations 1,3 and 5 m, and were pooled. the 3 ml pooled fraction had a protein concentration of 0.002 mg/ml and a total enzymatic activity of 0.046 u. the final purification step achieved 9.67-fold purity with a recovery of 0.12 % and a specific activity of 23.28 u/mg proteins (table 1). kinetic analyses using lineweaver-burk plots showed that vmax of purified po was 12.98 u/mg proteins with a km of 10 mm (fig. 2). effect of ph and temperature on po activity the effect of ph on po activity of h. cunea hemolymph was analyzed by ph range of 4 12. activity reached a maximum value at ph 10 with ldopa substrate. the activity decreased more than 50 % when other ph levels were used. at 35 ºc, po showed maximum activity. at 10 ºc below this maximum value, i.e., at 25 and 20 ºc, the activity decreased to approximately 60 %. there was a decrease recorded of approximately 100% when the level of activity was above that of the temperature tested (fig. 3). effect of ions and inhibitors the effects of various ions (0.5, 3 and 5 mm) on activity of the purified enzyme were studied at ph 10 and 35 °c (table 2). experiments were carried out at 25 30 °c because this was considered suitable for growth and development of h. cunea in the forest. different concentrations of cu+ and zn2+ increased po activity, the highest effect correlated to high concentrations of these ions (202.65 % and 273.65 %) respectively, compared with the control (table 2). also, the highest concentration of k+ and na+ had more effect on activity of the purified enzyme (199.11 % and 136.28 %) whereas additions of mg2+ and ca+ decreased the activity of the enzyme (table 2), in other experiments, respectively. enzyme activity was measured in the presence of different enzyme inhibitors (table 2). results indicated that some inhibitors including detc (10 mm), pmsf (10 mm) and phenylthiourea (10 mm) decreased enzyme activity to 2.35 %, 22.35 % and 56,47%, respectively (table 2). sds-page sds-page showed a single major protein band at molecular mass 33 kda compared with a large smear of proteins in the crude extract (fig. 4). 68 discussion in this study we characterized a po enzyme in the hemolymph of h. cunea. the native enzyme was estimated as 33 kda by gel filtration in sepharose and sds-page. these data are compatible with the purified enzyme from sarcophaga bullata (chase et al., 2000), hyalophora cecropia (anderson et al., 1989), locusta migratoria (cherqui et al., 1998) and e. integriceps (zibaee et al., 2011) that a single isoform has been characterized from them. it has been reported that different isoforms of po have been detected in several insects. for example, there are two isoforms in g. mellonella (kopácek et al., 1995) and bombyx mori (yasuhara et al., 1995), six in the mosquito anopheles gambiae (müller et al., 1999) and three in the fruit fly drosophila melanogaster (fujimoto et al., 1993). however, it is essential to have more detailed investigations on the structure of po. the physiological significance of po isoforms in the above mentioned insects still remains to be studied (feng et al., 2008). analysis by lineweaver-burk plots identified the kinetic parameters of the enzyme, vmax and km using l-dopa as the substrate measured 12.98 µmol/min/mg protein and 4.08 mm, respectively. km was higher than that of other insects, such as the h. virescens with 2.25 mmol (lockey and ourth, 1992), spodoptera littoralis with 1.35 mmol (lee and anstee, 1995), a. mellifera with 0.17 mmol (zufelato et al., 2004), housefly (musca domestica) pupae with 3.93 mmol, blowfly (sarcophaga bullata) pupae with 1.54 mmol (wang et al., 2004), p. rapae with 0.8 mmol (xue et al., 2006), o. furnacalis with 0.92 mmol (feng et al., 2008) but lower than e. integriceps with10 mmol (zibaee et al., 2011). catalytic efficiency was calculated by vmax and km in the presence of l-dopa. although l-dopa has traditionally been used as a substrate for characterization of pos from arthropods in general (durrant et al.,1993; kopácek et al.,1995; lee and anstee, 1995; brivio et al.,1996; cherqui et al., 1996), data in this study showed a low affinity of h. cunea po for l-dopa . the po binding affinity was significantly affected by the nature of the active part of po. however, differences in substrate-protein contact points or differences in the size of the substrate-binding pocket can affect po binding affinity in different insects (feng et al., 2008). it would appear that sometimes insect po prefers other substrates. the ph that permitted the higher h. cunea po activity, i.e., 10, was different from those obtained for this enzyme from the other species that were studied, for example, ph 7.5 for g. mellonella (dunphy, 1991), 7.4 and 7.5 for larval and pupal po from m. domestica (hara, et al., 1993), ph 7.0 7.5 for s. littoralis (lee and anstee, 1995), ph 6.5 for a. mellifera (zufelato et al., 2004), ph 7.0 for p. rapae (xue et al., 2006) and ph 6.0 for e. integriceps (zibaee et al., 2011). however, some reported optimum ph values were higher, such as ph 8.0 for po from l. dispar (dunphy, 1991), and ph 9.0 for this activity in h. virescense (lockey and orth, 1992), suggesting distinct properties for po from different sources. fig. 4 sds-page of the purified po from h. cunea. left to right; mm: molecular mass markers, 1: crude extract, 2: ammonium sulphate 30 %, 3: ammonium sulphate 70 %, 4: sepharyl g-100 chromatography and 5: cm-sepharose ion exchange chromatography. the optimal temperature was 35 40 ºc for the l-dopa catalysis reaction with po activity. for several species, po showed maximum activity at 30 45 º c. such as l. migratoria (30 – 35 ºc) (cerqui et al., 1996), e. integriceps (30 35 ºc) (zibaee et al., 2011) and h. virescens (45 ºc) (lockey and orth, 1992). also, the present result showed that po activity reached approximately 0 when the temperature was 50 60º c as the purified po was not extremely heat stable and in most cases was partially or totally destroyed after short exposure to temperature above 50 or 60 ºc. several metal ions (0.5, 3.5 mm) were tested with po of h. cunea showing that all concentrations of zn2+ and cu2+ and high concentration (5 mm) of k+ and na+ increased po activity, whereas mg2+ and ca2+ decreased enzyme activity. zn2+ increased po activity 2 2.5 fold. copper is the center of the po structure and causes high activity in the presence of cu2+. this phenomenon was verified using specific chelating agents of cu2+ and zn2+. this result is similar to other studies (anderson et al., 1989; feng et al., 2008; zibaee et al., 2011). ca2+ -modulating po activity enhancement has been reported for a large number of insects, e.g., b. mori (ashida et al., 1983), schistocerca gregaria (dularay and lackie, 1985), blaberus craniifer (leonard et al., 1985), l. migratoria (brehelin et al., 1989), l. dispar and g. mellonella (dunphy, 1991), but in this research ca2+ decreased po activity. lockey and orth (1992) reported ca2+ was not required for po activity in h. virescens. some metal ions can significantly modify the structure of po (li et al., 2000) that leads to increased or decreased 69 po activity. also, the structure of po could be reversed by salt concentration. however, in the presence of high concentrations of k+ and na+ po activity increased in d. melanogaster (sezaki et al., 2001) that was also likely in this study. among inhibitors, detc significantly decreased po activity (p < 0.05), detc is a specific chelator for copper. inhibition of h. cunea po by dtc was similar to that found in pos of h. virescens (lockey and qurth, 1992), aedes aegypti, anopheles quadrimaculatus (nayar and bradley, 1994) and other arthropods such as limulus polyphemus (nellaiappan and sugumaran, 1996), o. furnacalis (feng et al., 2008) and e. integriceps (zibaee et al., 2011). pmsf as an inhibitor affects po activity in h. cunea. it also abolished po activity in venom and plasma of parasitoid nasonia vitripenni (abt et al., 2007). in contrast, however edta and phenylthiourea are important inhibitors of po activity in insects, it showed no inhibitory effect on h. cunea. it seems these differences are correlated to the molecular structure of the enzyme. po has crucial roles in insect development and immunity (xue et al., 2006; gorman et al., 2007a). hence it should be possible to control insect pests by inhibiting this enzyme. this could provide a basic tool for the development of new insecticides to replace others being used that are environmentally threatening. for example xue et al. 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receptors: an overview from invertebrates to vertebrates mr coscia, s giacomelli, u oreste institute of protein biochemistry, cnr, naples, italy accepted november 10, 2011 abstract toll-like receptors (tlrs) are membrane glycoproteins consisting of an ectodomain, encompassing tandem lrrs (leucine-rich repeats), a membrane spanning segment and a globular cytoplasmic toll/interleukin-1 receptor (tir) domain. they detect microbes on the basis of conserved pathogen-associated molecular patterns (pamps). tlrs share molecular architecture and common ancestors with arthropod toll molecules, which show a dual role, in the dorsoventral patterning of the embryo, and in the immune response to fungal infections in the adult. during the metazoan evolution toll/tlrs modified the number and the arrangment of lrrs (protostomeand vertebrate-type), the localization of the loops interacting with different ligands, the ability to reside in the cellular or endosomal membrane and the complexity of the signaling pathway. the evolutionary mechanisms of toll/tlr gene diversification included gene duplication, retrotranscription, high gene expansion rate within species, and alternative splicing of the transcripts. the aim of this review is to supply a schematic representation of a very complex, but still, fascinating story. key words: toll-like receptor; signaling; pathogens recognition receptors; tir domain; leucine-rich repeat; metazoan evolution foreground there is a growing interest in toll-like receptors (tlrs), as demonstrated by the 2011 nobel prize in medicine awarded to ba beutler and ja hoffmann for their studies on tlr role in physiology and pathology. ___________________________________________________________________________ corresponding author: umberto oreste institute of protein biochemistry, cnr via pietro castellino 111, 80131, napoli e-mail: u.oreste@ibp.cnr.it the founding member of the tlr family was identified as a protein involved in dorsoventral patterning of drosophila melanogaster embryos (anderson et al., 1985); later, it was shown also to play a role in responding to fungal infection of the adults in the same species (lemaitre et al., 1996). a human homologue of the drosophila toll protein was identified as activator of adaptive immunity (medzhitov et al., 1997). at present, tlrs are the most extensively studied pathogens recognition receptors (prr) in list of abbreviations: ctlrr: c-terminal lrr; gnbp: gram negative binding proteins; hklp: heat-killed legionella pneumophila; hmm: hidden markov model; iκb: nf-κb inhibitor; ikk: iκb kinase; il1r: interleukin-1 receptor; imd: immune deficiency; inf: interferon; irak: interleukin-1 receptor-associated kinase; lps: lipopolysaccharide; lrr: leucine-rich repeat; lta: lymphotoxin-alpha; mal (synonym of tirap): myd88 adaptor-like; mekk: mytogenactivated protein kinase kinase kinase, also known as map3k; mpd: muramyl dipeptide; myd88: myeloid differentiation factor 88; nf-κb: nuclear factor κ light chain enhancer of b cells; ntlrr: n-terminal lrr; orf open-reading frame; pamp: pathogen-associated molecular patterns; pgrp: peptidoglycan recognition protein; pik-1: pelle and il1r associated kinase; png: peptidoglycan; prr: pathogens recognition receptors; ticam: toll-like receptor adaptor molecule; tir: toll/interleukin-1 receptor; tirap (synonym of mal): tir containing adaptor molecule; tlr: toll-like receptor; tnf: tumor necrosis factor; tollip: toll interacting protein; tram: trif related adaptor molecule, also known as tcam; trif: tir domain containing adaptor inducing interferon-β; sarm: sterile αand armadillo-motif-containing protein; slip: lps-interacting protein; vlr: variable lymphocyte receptor; utr: untraslated region; wssv: white spot syndrome virus 210 fig. 1 phylogenetic relationships of the reported taxa. the tree was built according to the lineages reported by ncbi taxonomy database (http://www.ncbi.nlm.nih.gov/taxonomy). divergence times, expressed in mya, are indicated only at the nodes analyzed by hedges et al. (2004) using molecular clock methods on all available eukaryote protein sequences. 211 http://www.ncbi.nlm.nih.gov/taxonomy both vertebrate and invertebrate species. several authors focus on tlrs role (pasare and medzhitov, 2005; iwasaki and medzhitov, 2010), structure (gay and gangloff, 2007; jin and lee, 2008; botos et al., 2011), signaling (barton and medzhitov, 2003; gay et al., 2011), and evolution (leulier and lamaitre, 2008; werling et al., 2008; satake and sasaki, 2010; wu et al., 2011). due to the huge amounts of data present in literature, a brief excursus is here presented on all phyla in which genes encoding tlrs or their homologues have been identified (fig. 1). we will focus special attention on tlr molecules from species that lie on the boundary between the vertebrates and invertebrates to provide a comprehensive guide to the evolution of these molecules at the emergence of the adaptive immune system. all species analyzed in the present review are listed in table 1. introduction the tlr family mediates sensing of microbial pathogens (beutler, 2004). for a brief description of the molecular structure, we shall remind that tlrs are type i integral membrane glycoproteins and different members of the family detect microbes on the basis of conserved pathogen-associated molecular patterns (pamps). they consist of an ectodomain, a membrane spanning segment and a globular cytoplasmic toll/interleukin-1 receptor (tir) domain (fig. 2). the ectodomain is arranged in a horseshoe structure (fig. 2), encompassing 19 27 tandem lrrs (leucine-rich repeats). each lrr contains a conserved 11-residue segment, being the consensus sequence lxxlxlxxnxl, where x can be any amino acid, l is a hydrophobic residue (leucine, valine, isoleucine or phenylalanine) and n can be asparagine or cysteine (kobe and kajava, 2001). each repeat consists of a β-strand and an αhelix connected by loops. two regions, each containing several cysteines, flank the lrrs, ntlrr (n-terminal lrr) and ctlrr (c-terminal lrr). a tir domain (fig. 2) is present in the cytoplasmic region; it has an α β fold consisting of a central five-stranded parallel β-sheet surrounded by five α-helices. the same fold occurs also in the adaptor proteins myd88 (myeloid differentiation factor 88), mal (myd88 adaptor-like), trif (tir domain containing adaptor inducing interferon-β) tram (trif related adaptor molecule) and sarm (sterile αand armadillo-motif-containing protein) (o’neill and bowie, 2007). the adaptors associate with each other or with tlrs by the respective tir domains (dunne et al., 2003). the receptor complex recruits, in succession, iraks (interleukin-1 receptor-associated kinases), traf6 (tnf receptor-associated factor 6), ikk, which phosphorylates the nfκb inhibitor iκb, which activates the transcription (fig. 3). tlr signaling is also tuned by micrornas, which target 3’utr of transcripts that encode signaling components (o’neill et al., 2011). the signaling induces production of pro-inflammatory cytokines such as interleukins, interferon, tnf, responsible for direct innate response and for triggering adaptive immune cells. table 1 metazoan species whose toll-tlr molecules have been considered in the present study amphimedon queenslandica porifera suberites domuncula acropora millepora acropora palmata hydra magnipapillata montastraea faveolata cnidaria nematostella vectensis nematodes caenorhabditis elegans platyhelminthes schmidtea mediterranea capitella capitata helobdella robusta annellida hirudo medicinalis chlamys farreri crassostrea gigas euprymna scolopes mollusca mya arenaria tachypleus tridentatus merostomata carcinoscorpius rotundicauda aedes aegypti anopheles gambiae apis mellifera drosophila melanogaster insecta tribolium castaneum fenneropenaeus chinensis litopaeneus vannamei marsupenaeus japonicus crustacea penaeus monodon echinodermata strongylocentrotus purpuratus branchiostoma belcheri cephalocordata branchiostoma floridae boltenia villosa ciona intestinalis tunicata oikopleura dioica hyperoartia lethenteron camitschaticum callorhincus milii chondrichthyes chiloscyllium griseum cynoglossus semilaevis cyprinus carpio danio rerio gobiocypris rarus ictalurus punctatus oncorhynchus mykiss paralichthys olivaceus pseudosciaena crocea salmo salar teleostei takifugu rubripes xenopus laevis amphybia xenopus tropicalis reptiles anolis carolinensis accipiter cooperii amazona albifrons carpodacus mexicanum dromaius novaehollandiae falco naumanni gallus gallus oceanodroma leucorhpoa picoides pubescens aves taeniopygia guttata 212 fig. 2 tlr molecule. on the right side the molecular structure of the mammalian tlr. at the top the ectodomain of mouse tlr2 (pdb id: 3a7c), at the bottom the human tir domain (pdb id: 1fyx). the majority of tlrs associate to form homodimers. however, there are some exceptions, one is represented by tlr2 that dimerizes with tlr1, tlr6 (takeuchi et al., 2002), or tlr10 (govindaraj et al., 2010) to provide different pamp specificity. c-terminal ectodomains dimerize while n-termini are oriented in opposite directions. this architecture is crucial for both the ligand binding and the assembly with the adaptors. tlr functions are well known in mammals and all knowledge accumulated so far is based on interpretation of the general role tlrs play in nonmammalian species. some tlrs are expressed on the cell membrane and their ectodomain recognizes extracellular pamps, while others are expressed in endosomes to detect internalized pamps. tlrs are expressed not only in immune cells (dendritic cells, monocytes, macrophages, b lymphocytes) but also in non-immune cells, including fibroblasts, endothelial cells, adipocytes, epithelial cells and glial cells. the tlr ligands can be categorized into lipid, protein and nucleic acid components. tlr ligand specificity is due to different modes of lrrs assembly. moreover, ligandinteracting residues have been demonstrated to be present on both the concave and the convex side of the horseshoe (jin and lee, 2008). so far, at least 23 vertebrate tlrs have been identified, based on amino acid similarity, genomic structure, and ligand properties. they can be grouped into six major families (roach et al., 2005) as reported in table 2. porifera porifera represents the most ancient metazoan phylum showing nucleotide sequences reminiscent of tlr. two species, suberites domuncula (wiens et al., 2005, 2007) and amphimedon queenslandica (gauthier, 2010), belonging to this phylum, have been investigated in an attempt to trace back the evolutionary origin of tlr. both are demosponges. 213 the muller group has identified in s. domuncula several proteins resembling some toll/tlr-pathway components, including a lps-interacting protein (slip) whose predicted ectodomain lacks lrrs; it dimerizes and bind a myd88 homologue but it appears significantly atypical in that it lacks a clear death domain (wiens et al., 2005). as sponge myd88 and slip are co-immunoprecipitated by the reciprocal antibodies, they clearly can interact in vitro. subsequently, the same group has reported the cloning of three major elements of the sponge innate immune response: tlr-like, irak and caspase, highly homologous with vertebrate orthologs. in particular, a tir domain, highly homologous to mammalian tir, is present in the tlr-like cytoplasmic region. in the ectodomain no clear lrr has been detected (wiens et al., 2007). it has been hypothesized that s. domuncula tlr corresponds to a short splice variant of a longer transcript as reported in vertebrate tlrs (iwami et al., 2000; wells et al., 2006). the availability of the whole-genome sequence of the sponge a. queenslandica has allowed the identification of two related receptors, amqigtirs, which comprise at least three extracellular il1r-like immunoglobulin domains and an intracellular tir domain. the remainder of the tlr/il1r pathway is mostly conserved and includes genes known to interact with tlrs and il1rs in bilaterians, such as tollip (toll interacting protein) and myd88 (gauthier et al., 2010). however, the ambiguous status of the sponge myd88 related protein means that it is unclear whether sponges have a canonical toll/tlr signaling pathway. because a. queenslandica and basal eumetazoans encode similar proteins with extracellular il1r-like ig domains and an intracellular tlr-like tir domain, it can be suggested that a similar receptor existed in the last common metazoan ancestor. this implicates that in eumetazoans the genes encoding tir domains with tlr features and those encoding lrr-containing domains were combined together yielding the ancestral gene of the canonical toll/tlr family. this is in line with the extensive exon-shuffling event occurred in metazoan and eumetazoan lineages (srivastava, 2010). cnidaria the phylum cnidaria provides crucial insights into the early evolution of animals because it is the likely sister group of the superphylum bilateria. although the literature on cnidarian immunity is wide, the distinction between historecognition and host-response/disease is unclear in this group of primitive organisms (rinkevich, 2011). results on cnidaria molecules resembling tlrs have been reviewed by hemmrich et al. (2007) and by dunn (2009). data collected in five different cnidarian species are reviewed below. the species investigated are as follows: hydra magnipapillata belonging to hydrozoa; nematostella vectensis, acropora millepora, acropora palmata and montastraea faveolata belonging to anthozoa. fig. 3 tlr signaling pathway in mammals. extensive searching the hydra predicted protein collection, using the available hidden markov models (hmms) has not succeeded in identifying proteins having the canonical toll/tlr structure. on the other hand, four tir domain-containing proteins have been found. two of them show sequence features related to myd88 including the typical death domain. the two other hydra tir domaincontaining proteins show relatively short ectodomains lacking lrrs. these proteins are known as hytrr-1 and hytrr-2 and are expressed on the epithelial cells (miller et al., 2007; augustin et al., 2010). by functional studies it has been demonstrated that they are capable of mounting an immune response through a nonconventional signaling pathway (bosch et al., 2009). sequence comparison has provided further evidence that these tir-domain sequences cluster with tir domains of other animal tlrs, rather than with intracellular tir domain adaptors, suggesting that they are tlr-related molecules (zheng et al., 2005). in addition, no nf-κb homologues have been identified in hydra (miller et al., 2007). instead, different data have been obtained in anthozoan species. a tlr gene (nvtlr-1) is present in the genome of the starlet sea anemone, nematostella vectensis (putnam et al., 2007; sullivan et al., 2007), a basal cnidarian, but not in the genomes of other anthozoans, such as the coral species acropora millepora and montastraea faveolata (schwarz et al., 2008). using hmm-based 214 search methods five tir-containing proteins have been identified in n. vectensis (miller et al., 2007). nvtlr-1 is clearly related to members of the toll/tlr family. the typical tlr architecture consists of only a c-terminal cys-rich motif flanking the lrrs proximal to the membrane, instead n. vectensis nvtlr-1 is predicted to contain the ntlrr and an additional ctlrr within the lrrs. moreover, a phylogenetic analysis of tir-containing proteins has grouped the nvtlr-1 tir with its fly and human counterparts. interestingly, three more n. vectensis tir-containing proteins contain multiple (two or three) immunoglobulin domains, resembling the structure of mammalian il-1rs. in addition, a single myd88 homologue (nvmyd88) and several kinases involved in the toll-tlr signaling have been identified (sullivan et al., 2007). searching the datasets of nucleotide sequences from a. palmata and a. millepora has shown that the respective tirs are very similar to those contained in the nematostella il-1r-like proteins (miller et al., 2007). finally, characterization of m. faveolata est dataset has led to identification of partial sequences of genes involved in immune response such as mapk, nf-κb, and tir-containing proteins (schwarz et al., 2008). nematodes and platyhelminthes nematodes are bilaterian animals belonging to pseudocoelomata and are very abundant on earth. analysis of the caenorhabditis elegans genome identified a gene encoding a tlr (tol-1), other genes encoding proteins involved in toll-tlr signaling pathway (trf-1, pik-1, iκb) and one more tir-containing protein (tir-1), which is homologous to human sarm1. however, homologues of myd88 or nf-κb have not been found in the c. elegans genome (irazoqui et al., 2010). the predicted tol-1 ectodomain contains 22 lrrs, with one interspersed ntlrr and two ctlrrs, followed by a putative transmembrane region and a cytosolic tir domain (pujol et al., 2001). this architecture is similar to that of nvtlr1, but the number of lrrs is larger in the nematode species than in cnidarian species. tol-1 is preferentially expressed in the nervous system of adult animals. the developmental role of tol-1 in embryogenesis has been ascertained by analysis of mutans, but there is no evidence for a role in resistance to a number of pathogens. since myd88 and nf-κb are absent and the only tir-containing protein seems to be not involved in immune signaling, other signaling pathways, like p38 mitogen-activated protein kinase (mapk) cascade probably assume immune functions (kim et al., 2002). these additional immune pathways might have evolved in primitive metazoans and have been maintained throughout metazoan evolution, functioning in concert with tlr pathways. although very important because of infecting millions of people, plathelminthes remain scarcely investigated at molecular level. an important contribution comes from the work of sanchez et al. (2002), which have characterized about 3,000 nonredundant cdna from a clonal line of the planarian table 2 vertebrate tlr families. letters preceeding some tlrs are as: x, xenopus; t, teleost; c, chicken; m, mouse. the remaining tlrs are shared by all vertebrate species family 1 tlr1, tlr2, tlr6, tlr10, xtlr14, ttlr14, t tlr18, ctlr15, 3 tlr3 4 tlr4 5 tlr5 7 tlr7, tlr8, tlr9 11 mtlr11, mtlr12, mtlr13, ctlr21, xtlr21, xtlr22, 21 ttlr13, ttlr19, ttlr20, ttlr21, ttlr22, ttlr23 schidtea mediterranea. in this library a single 620-nt long mrna sequence (ay066289) was found to be similar to mammalian tlr4. because of the low value of similarity, this datum is scarcely useful. annelida annelida belongs to protostoma. the leech helobdella robusta and the polychaete capitella capitata (davidson et al., 2008) have been analyzed for the presence of tlrs: 16 tlr contigs were detected in the genome of h. robusta and 105 in c. capitata. h. robusta tlr sequences do not seem to be orthologous to those of c. capitata; the majority of c. capitata tlr sequences are very similar to each other and seem to result from a recent gene duplication event. both species show a basic tlr domain structure (extracellular lrrs, transmembrane segment, tir domain), but h. robusta tlrs present the extracellular lrr clusters with tandem ctlrr and nlrr (protostome-type) whereas all c. capitata, but one, tlrs present a structure similar to mammalian tlrs (vertebrate-type). in c. capitata, but not in h. robusta genome, the putative orthologs of tlr signaling pathway proteins (mekk, iκb e and nf-κb) have also been identified whereas myd88 and traf homologues have been found in both genomes. recently, a cdna library of hirudo medicinalis has been analyzed (cuvillier-hot et al., 2011) and the complete sequence of hmtlr1 has been determined; the ectodomain presents 6 lrrs preceded by one ntlrr. hmtlr1 shows great homologies with mus musculus and monodelphis domestica tlr13, while no significant homology with molecules identified in other annelids has been noticed. the transcripts are preferentially expressed in neurons as well as in microglial cells and the protein, similarly to mouse tlr3, has been localized in endosomal compartment of neuronal cells. to explore the biological functions of hmtlr1, gene expression has been quantified during the regeneration process and following microbial challenges. by regeneration tests, a differentiation role of hmtlr1 gene has been excluded; on the 215 other hand, after h. medicinalis exposition to different microbes, the levels of hmtlr1 transcripts have been observed to differently increase, demonstrating the capability of distinguishing microbial components (cuvillier-hot et al., 2011). mollusca from an evolutionary point of view mollusks are placed between the two traditional model organisms d. melanogaster, in which toll protein and toll signaling pathway have been identified for the first time, and c. elegans, in which little evidence for the existence of tlr signaling pathway have been accumulated so far. the molluscan species investigated in this context are the bivalvian chlamys farreri, mya arenaria, crassostrea gigas, and the cephalopod euprymna scolopes. in c. farreri most of the toll-tlr signaling pathway components have been demonstrated to occur: cftoll-1 (qiu et al., 2007a), cfmyd88 (qiu et al., 2007b), cftraf6 (qiu et al., 2009), cfnfκb and cfiκb, indicating the possibility of the presence of a toll-tlr signaling pathway in mollusks. the sequence features of these five key genes involved in tlr signaling pathway in scallop c. farreri have been characterized (wang et al., 2011a). the expression levels of cftlr, cfmyd88 (limei et al., 2007), cftraf6 (limei et al., 2009), cfiκb and cfnfκb increase after lps stimulation and decrease after rnai suppression. an interaction between recombinant cftlr-tir domain and recombinant cfmyd88 has been proved by elisa assay. zhang et al. (2011) have cloned the tlr gene in c. gigas (cgtoll-1) and have found its expression to be affected by bacterial challenge. a phylogenetic analysis of the molecules involved in toll-tlr signaling indicates that c. gigas downstream genes iκb (escoubas et al., 1999; montagnani et al., 2008) and rel (montagnani et al., 2004) cluster with protostome orthologs, while upstream genes, myd88, traf6 (gueguen et al., 2003; roberts et al., 2009) and irak cluster with deuterostome orthologs. cgtoll-1 is a typical single-pass transmembrane protein including signal peptide, 19 lrrs containing an interspersed ntlrr, a transmembrane domain and a tir domain. moreover, among 20 potential n-linked glycosylation sites in cgtoll-1, nine are anchored at the convex surface of lrrs accounting for a high degree of putative n-linked glycosylation in this region (zhang et al., 2011). although widely distributed, cgtoll-1 is preferentially expressed in hemolymph; this feature resembles that of cftoll (qiu et al., 2007a). furthermore, it has been reported that cgtoll-1 upregulates the expression of myd88 in c. gigas (tirape et al., 2007). haemocytes have been demonstrated to have a crucial role in mollusk defense system, and a high expression level of cgtoll-1 in these cells suggests the key role of cgtoll-1 in c. gigas immune response. studies performed on immune gene expression levels in mya arenaria haemocytes, have revealed that genes encoding homologues of tlr2 and irak4 are significantly regulated following in vivo infection with vibrio splendidus (mateo et al., 2010). finally, goodson et al. (2005) have described seven component of the toll-tlr pathway by screening an est library from the juvenile light organ of the cephalopod e. scolopes. e. scolopes tlr architecture is consistent with that of mammals and drosophila. merostomata merostomata is an arthropod class including the order xiphosura and the extinct order eurypterida. a tlr, named ttoll, has been identified in the haemocytes of the horseshoe crab tachypleus tridentatus (family limulidae). the architecture and length of ttoll are similar to those of drosophila toll1 (inamori et al., 2000; 2004). ttoll consists of 22-25 residue-long lrrs flanked by cysrich motives and containing ntlrr and ctlrr; a putative transmembrane domain is placed near the ectodomain and a tir domain is present in the cterminal cytoplasmic region. twenty potential nlinked glycosylation sites are localized in the ectodomain. ttoll and drosophila toll1 ectodomains share 23 % identity, whereas ttoll tir shows 39 % sequence identity with drosophila toll1 and 31 % with human tlr2 and tlr4. the structural relationship with drosophila toll1 and the absence of insertions in the lrrs suggest that, similarly to drosophila toll1, ttoll does not function as pamp-binding receptor, and that its real ligand might be a protein resembling drosophila späetzle (kurata et al., 2006). coagulogen, the final target of the coagulation cascade of horseshoe crabs, whose structure is similar to späetzle, could be the candidate molecule. its cleaved form, coagulin, could promote ttoll dimerization and, in turn, the activation of intracellular signaling. in another merotostome species, carcinoscorpius rotundicauda, the tlr adaptor sarm has been identified and shown to share many signature motives with vertebrate and invertebrate sarmss (belinda et al., 2008). insecta insecta is an arthropod class with more than a million of species, and includes the most diverse group of animals. studies performed on the model species drosophila melanogaster, has opened the way for knowledge of fundamental mechanisms of embryo development and immune response in insects. at present toll-like nucleotide sequences have been determined in 24 different insect species. after toll identification (anderson et al., 1985), additional toll family members (toll2-9) have been recognized in the d. melanogaster genome (hoffmann, 2003; valanne et al., 2011). gradually, the dual function in embriogenesis and immune response has been ascertained (ferrandon et al., 2007). all tolls, but toll9, contain 1 4 additional cys-rich motives interspersed in the lrr region (imler and hoffmann, 2001). tolls do not bind any pamps, however require accessory proteins. persephone or pgrps and gnbps are the molecules recognizing fungi or gram-positive 216 bacteria, respectively (gobert et al., 2003; pal and wu, 2009). in drosophila molecules unrelated to tolls, called imd (immune deficiency) mediate the resistence to gram-negative bacteria (lemaitre et al., 1995; de gregorio et al., 2002). persephone, which is a protease, directly cleaves a protein called späetzle, which is able to bind toll and activate the signaling cascade. pgrps, and gnbps activate proteolytic cascades, finally cleaving späetzle. tolls have been demonstrated to dimerize (hu et al., 2004) and the molecular structure of the tollspäetzle complex has been investigated by molecular modeling and electron microscopy; the complex shows a stoichiometry and architecture totally unrelated to that of tlr-ligand complexes, being the toll binding sites at the n-terminal end of each monomer (gangloff et al., 2008). adaptor proteins, including myd88, are involved in the signaling mechanism; however, antimicrobial peptides rather than cytokines are the immune defense molecules induced by the signaling. toll genes have also been searched in sequenced genomes of other species that belong to the class insecta. the genomes of five insect species revealed the presence of a different number of toll encoding genes: 5 in apis mellifera (evans et al., 2006), 9 in tribolium castaneum (zou et al., 2007), 11 including two pseudogenes, in bombyx mori (cheng et al., 2008), 10 in anopheles gambiae (cristophides et al., 2002), 12 in aedes aegypti (waterhouse et al., 2007). a comparison of the insect tolls suggests a species-specific diversification process. crustacea crustacea is another arthropod class that is distinct from insecta by the possession of twoparted limbs. tlrs have been found in four crustacean species, all belonging to the family penaeidae. lvtoll1, the first crustacean toll identified in litopenaeus vannamei, (yang et al., 2007), has a typical protostome-like tlr structure with an ectodomain composed by 16 lrrs, a transmembrane domain and an intracellular tir domain. a sequence comparison of lvtoll tir with that of insect toll shows high similarities, between 54.3 and 59.9 %. other two novel tlrs have been identified more recently in the same species (wang et al., 2011b); lvtoll2 and lvtoll3 share 43.2 % and 25.4 % identity with lvtoll1, respectively. the cellular localization of the three lvtolls is different: lvtoll1 and lvtoll3 are present in both membrane and cytoplasm, while lvtoll2 is restricted to the cytoplasm. they are constitutively expressed in many tissues including gill, stomach, intestine, nerve, muscle, pyloric caecum, spermary, and epidermis. upon challenges with vibrio alginolyticus and wssv, the three lvtolls show a different response: lvtoll1 is up regulated with both challenges; lvtoll2 is up regulated only with wssv challenge; finally, lvtoll3 is up regulated with both challenges as lvtoll1, but at different times. they also may be involved in phagocytosis (wang et al., 2011b). a partial sequence of a similar toll-related gene has also been identified in a penaeus monodon gill cdna library. it is expressed, with no significant differences, in gut, gill and hepatopancreas haemocytes and compound eye (arts et al., 2007). in 2008 tlrs have been described in two additional crustacean species, fenneropenaeus chinensis (yang et al., 2008) and marsupenaeus japonicus (mekata et al., 2008). fctoll shows a structure and expression pattern similar to those of the previously identified shrimp tolls. mjtoll shows high identity (96.9 %) with pmtoll and low identity with other crustacean homologues (59.0 %). the mjtoll gene is constitutively expressed in the same tissues as lvtoll1 and its expression is increased by peptidoglycans. echinodermata echinodermata is a phylum of deuterostomes that separated from chordata about 600 mya (ayala et al., 1998). a survey of the genome of the purple sea urchin strongylocentrotus purpuratus (family strongylocentrotidae), a member of the phylum echinodermata, has revealed the presence of a large number (4 5 % of the identified genes) of vertebrate immune gene homologues (hibino et al., 2006; rast et al., 2006; buckley and smith, 2007; buckley et al., 2008). 222 toll-like receptor gene models have been identified; these tlrs, are grouped into two categories, a greatly expanded multigene family, including 211 genes comprised of seven subfamilies, and a very small set of 11 divergent genes. the expanded tlr genes are intronless and code for proteins with a vertebrate-type structure; three of the small set have a typical protostome-like structure and their tir domains exhibit a protostome-like sequence and a shorter c-terminal β-strand. protostome-like sequences have not been identified in other deuterostomes, suggesting that they were present in the common ancestor of modern bilateria and then were lost in vertebrate lineage (rast et al., 2006). the remaining five tlr genes of the small group code for an unusual short ectodomain and seems to have affinities with the protostome-type genes (hibino et al., 2006). the majorirty of the sea urchin tlr genes are more similar to each other than to those of other animals: this suggests that a tlr gene expansion occurred in s. purpuratus. the majority of the differences among these genes fall in the ectodomain probably accounting for the diversification of immune recognition specificity. differences consist of: individual amino acid substitutions, small insertion/deletions, insertions of long sequences between or within lrr motives or insertions of additional lrrs. in protostomes long insertions are less frequent than in vertebrates in which they modify ligand specificity (bell et al., 2003). the hypervariability is confined to particular lrrs. the presence of recently duplicated genes, the occurrence of many pseudogenes (25 30 %) and the regionalized hypervariability suggest that the sea urchin tlr genes undergo a dynamic 217 evolution characterized by a high gene turnover rate (rast et al., 2006). expanded tlr genes are absent or weakly expressed in embryos prior to the end of gastrulation whereas their expression increases in early pluteus. protostome-like tlr genes are absent in embryos whereas are predominatly expressed in coelomocytes and in tube feet in the adult (hibino et al., 2006). in addition, the survey of s. purpuratus genome has identified 26 genes coding for potential tlr adaptor proteins: a myd88 ortholog and three more genes with a myd88-like domain, an orthologue of sarm, 14 sarm-related genes, and 7 genes encoding cytoplasmic tir domain proteins. the expansion of tlr genes has occurred in parallel with a modest expansion of tlr adaptor signaling protein genes. the presence of homologues of tlr signal transduction proteins suggests that the engagement of tlrs may lead to the activation of nf-κb, already known since isolated and characterized in s. purpuratus (pancer et al., 1999). cephalochordata the phylum chordata comprises cephalochordates (amphioxus), urochordates (tunicates), and vertebrates. these groups diverged from a common ancestor during or prior to the cambrian explosion (holland et al., 2008). by recent phylogenetic studies cephalochordates have been recognized as the basal group of the phylum chordata, since vertebrates and urochordates have diverged later (bourlat et al., 2006; delsuc et al., 2006). this indicates that cephalochordates represent the oldest still existing lineage of the phylum chordata. sequencing the genome of the cephalochordate branchiostoma floridae has allowed a better understanding of the basal chordate evolution. a recent insight into b. floridae genome has revealed that its tlr system possesses an unprecedented degree of arrangement since including an expanded vertebrate-type family, consisting at least of 36 tlrs, a protostome-type group of 12 elements and 40 tir-containing adaptors. rapid tandem gene duplication has been suggested to be the mechanism generating the majority of b. floridae vertebrate-type tlrs (huang et al., 2008). based on phylogenetic analysis of the tir domains of amphioxus and vertebrate tlrs, surprisingly protostome-type b. floridae tlrs has been shown to cluster with the vertebrate tlr4 lineage; on the other hand, 33 variable-type b. floridae tlrs show a paraphyletic relationship with vertebrate tlr11 lineage, 19 of them comprising a distinct clade designated as sc75. in this clade there are two pseudogenes and 12 intronless genes, probably generated by retrotranscription events. in the expanded group, the tir domain is highly conserved (identity higher than 85%), whereas the ectodomain is more variable. the occurrence of positively selected positions has been demonstrated in lrrs. the evolution of sc75 clade resembles that of the s. purputatus variable-type tlrs (huang et al., 2008). the search for elements of the toll-tlr signaling pathway in the amphioxus genome has also been carried out: 4 myd88-like, 10 sarm-like, one tirap-like and one ticam2-like gene have been identified, whereas no homologue of ticam1 has been detected. a single tlr gene, bbttlr1, which is inserted into an intron in the reverse orientation, has been identified, cloned and characterized in the chinese amphioxus branchiostoma belcheri tsingtauense (yuan et al., 2009). the genomic region containing bbttlr1 has also been demonstrated to be highly polymorphic. its structure is of vertebrate-type showing one ntlrr, 22 lrrs and one ctlrr in the ectodomain, a transmembrane domain, and a tir domain in the cytoplasmic region. bbttlr1 shares 77 % identity with bftlr1. most of the bbtlr1 lrrs show the canonical lrr motif except lrr6 and 7; two large insertions are present next to lrr3 and 10; in addition, 10 putative nglycosilation sites have been detected. bbttlr1 expression is detectable in the villi of the gut epithelial cells, midgut diverticulum, in the connective tissues and coelome cells: it is predominantly expressed in certain regions that represent the frontline of host defense. it was also demonstrated that bbttrl1 is a surface receptor expressed on cell membrane. the expression of its transcript can be greatly upregulated by lps and gram-negative bacteria (vibrio vulnificus) and scarcely by lta, pgn and gram-positive bacteria (staphylococcus aureus), and is unaffected by glucans and poly i:c. a myd88 homologue, bbtmyd88, has been characterized and its involvement in nf-κb activation has been demonstrated (yuan et al., 2009). tunicata urochordates are considered the invertebrate group more closely related to vertebrates. the presence of tlr-like molecules in urochordates has been investigated in two ascidian species boltenia villosa (family piuridae) (davidson and swalla, 2002), and ciona intestinalis (family cionidae) (azumi et al., 2003; sasaki et al., 2009; nonaka and satake, 2011) and in one appendicularian species, oikopleura dioica (family oikopleuridae) (denoeud et al., 2010) davidson and swalla (2002) have isolated in a b. villosa cdna library a gene, bvlrr, resambling d. melanogaster toll and showing distinct peaks of expression during larval or post-larval development. azumi et al. (2003) by screening the draft genome sequence of c. intestinalis have identified only 3 tlr gene models, homologous to tlr4, 6, and 7, respectively, and several genes involved in the tlr signaling, including myd88, irak, traf, iκb, and nfκb. sasaki et al. (2009) have investigated the structures, localization, ligand recognition, activity and cytokine production of two tlrs of c. intestinalis, citlr1 and citlr2. both deduced protein sequences show the typical tlr architecture consisting of an intracellular tir domain, a transmembrane domain, and multiple 218 extracellular lrrs. in particular, citlr1 exhibits 7 putative lrrs and only one ctlrr (vertebratetype), while citlr2 displays 13 lrrs and three ctlrrs, which are features found in the protostome-type tlr. the overall amino acid sequence of citlrs shares no significant sequence homology with human tlrs: tir domains of citlr1 and citlr2 are more similar to human tlr4 and tlr6, and the overall sequences are most homologous to human tlr7 and tlr8, respectively. in juveniles citlr1 and citlr2 genes are expressed intensively in stomach, intestine and in haemocytes. in the adult both genes are equally expressed in the stomach; in anterior and middle intestine citlr1 expression predominates over that of citlr2. unlike mammalian tlrs, which have been found to be exclusively either on plasma membrane or in endosomes, citlr1 and citlr2 present both localizations, even if citlr2 is more intensively expressed in endosomes than on cell membrane. citlr1 and citlr2 interact with multiple pamps, which are differentially recognized by vertebrate tlrs; citlr1 induces a response to zymosan, while citlr2 elicits a prominent dosedependent response to poly i:c, (specific ligand for human tlr3), to heat-killed legionella pneumophila (hklp) (specific ligand for human tlr2) and flagellin (specific ligand for human tlr5). it is ascertained that citlrs, like vertebrate tlrs, directly recognize their pamps, without requiring association of additional specific proteins. notably, both citlr show equipotent nf-κb activation in response to the same ligand. finally, up regulation of tnf-α in response to citlr ligands has been demonstrated to occur in stomach and intestine. also in the pelagic appendicularian o. dioica the genome has been surveyed for pathogen sensors (denoeud et al., 2010): only one tlr-like protein has been identified and search for myd88, sarm, tirap and ticam has been unsuccessful. hyperoartia hyperoartia is a class of jawless fish (agnathans), that diverged from gnathostomes about 520 mya (hedges et al., 2004). in the surviving jawless fish which are the lowest class of vertebrates including lamprey and hagfish, an exclusive adaptive immune system comprises variable lymphocyte receptors (vlrs) containing lrr subunits (pancer et al., 2004). two tlrs, named latlr14a and latlr14b, were initially identified in lampreta japonica (synonym: lethenteron camitschaticum, petromyzontidae) by pcr-based cloning using primers designed on sequences of tlr2 from various species (ishii et al., 2007). both latlrs contain 8 lrrs, a transmembrane region, and a cytoplasmic tir domain. the two latlrs show 56% homology with each other, and the tirs are similar to those of the human tlr2 subfamily, probabily orthologs of fish tlr14. an 85-kda protein has been identified in a human hek293 transfectant by using in immunoblotting a polyclonal ab specific for latlr14b. facs, histochemical, and confocal analyses have shown that latlr14b is expressed in the cells, preferentially in gills, gut, and leukocytes. further investigation is required to determine whether the lamprey tlrs are localized on macrophages/monocytes. these cells should be different from lamprey lymphocytes, which have been shown to be vlr-positive cells. it has been demonstrated that by artificial dimerization of latlr14b, nf-κb, as well as inf-β promoter can be activated; however the pattern of pamps recognition by these latlrs remains unknown. more recently, advances in whole genome sequencing and annotation have allowed the identification of 16 genes predicted to encode tlrs from the latest petromyzon marinus (synonym: lethenteron camitschaticum) genome database (pre-ensemble lamprey genome browser) and ncbi trace archive. it should be reminded that the predicted protein sequences of the lamprey tlrs and their respective tir domains have been subjected to comparative analyses using the ncbi non-redundant protein database and blastp search. the repertoire of predicted lamprey tlrs has been determined and phylogenetic analyses indicate that the repertoire consists of both fishand mammalian-type tlrs (kasamatsu et al., 2010). at present, three types of tlrs belonging to the tlr2 subfamily have been found in the lamprey, which correspond to tlr24 (pmtlr2a-d), tlr14 (pmtlr14a-c), and the ortholog of jawed vertebrate tlr14 (tlr14d), forming clearly distinct clusters in a phylogenetic tree (kasamatsu et al., 2010). similarly, two tlr7/8 and three tlr21 genes have been identified in the lamprey genome, able to recognize foreign rna molecules and unmethylated cpg dnas, respectively. the tlr2 subfamily, tlr3, tlr5, tlr7/8, and tlr21/22 are conserved in the lamprey and teleosts, suggesting that lampreys and jawed vertebrates share the same tlr family with both mammalianand fish-type tlrs. since both types may have arisen together with vertebrates, they may represent the origin of the tlr repertoire in vertebrates. finally by the genome analysis, four other proteins have been identified as tlr adaptor-like proteins since they are similar to myd88, ticam or sarm. chondrichthyes data about chondrichthyan tlr are surprisingly scarce. the only datum we found is a nucleotide sequence of chiloscyllium griseum (family hemiscylliidae), 270-nt long, which has been registered in genbank as tlr2 under the accession number jf792813. blast search has shown a high homology of c. griseum tlr2 with mammalian tlr2a (e value: 3e-84); a smart analysis of the deduced amino acid sequence has revealed the occurrence of a partial tir domain. the partial sequence length does not allow to speculate about chondrichthyan tlr. in callorhinchus milii genome two gene models encoding ticam and one tirap, all supposed to be tlr signaling components, have been identified (wu et al., 2011). 219 teleostei the nucleotide sequences assigned to tlr genes have been determined in 31 teleost species belonging to 8 different families (table 3). data available at present have been obtained by 7 sequenced genome or transcriptomes (danio rerio, takifugu rubripes, tetraodon nigroviridis, oryzias latipes and gasterosteus aculeatus, oncorhynchus mykiss, and salmo salar). the determined sequences can be attributed to 16 different tlr types, 8 out of 16 being teleost specific. with respect to family 1, which includes mammalian tlr1, 2, 6, and 10 (roach et al. 2005), orthologs of mammalian tlr6 and tlr10 are absent but a further member of the same family, tlr14, is present and shares some features with tlr1, 6 and 10. both families 3 and 7 (including tlr7, 8 and 9) share teleost and mammalian orthologs. concerning the family 4, the majority of teleost species, but the most anciently diverged otocephala taxon, has lost tlr4; and whenever present, as in d. rerio and gobiocyris rarus, it does not recognize the mammalian agonist, lps (sepulcre et al., 2009). the family 21 comprises the other members specific for the teleost lineage (tlr13, 19, 20, 21, 22 and 23) (palti, 2011). in addition, paralogous or duplicated tlr genes, probably resulting from the third or fourth round of the whole genome duplication event, have been identified in d. rerio (jault et al., 2004; meijer et al., 2004), o. mykiss (palti, 2010), cyprinus carpio (kongchum et al., 2011). a unique feature of teleost tlrs is the presence, in addition to tlr5, of a soluble tlr5 molecule (tlr5s), which lacks the transmenbrane and tir domains in o. mykiss (tsujita et al., 2004, 2006) and t. rubripes (oshiumi et al., 2003) genomes. in o. mykiss tlr5s has been demonstrated to possess an adjuvant role in the response to bacteria via physical binding to flagellin (tsujita et al., 2006). soluble forms of tlrs are usually absent in mammalian genomes and those previously identified (iwami et al., 2000; lebouder et al. 2003) are probably generated by alternative splicing. tlr5s has also been identified in transcripts of s. salar (tsoi et al., 2006) and ictalurus punctatus (baoprasertkul et al., 2007a). its expression pattern generally differs from that of the membrane form and is increased upon infection of catfish specimens with edwardsiella ictaluri (bilodeau and waldbieser, 2005). functional data have been obtained in many teleost species. in o. mykiss increased tlr3 trascriptional activity was demonstrated after poly (i:c) or infectious hematopoietic virus treatments (rodriguez et al., 2005). in the same species areomonas salmonicida induced higher expression of tlr22 (rebl et al., 2007) and vaccine adjuvant r848 amplified tlr7 expression (palti et al. , 2010). exposure of i. punctatus to the gram-negative bacterium edwardsiella ictaluri activated the tlr3 and ticam production (baopraserkul et al., 2006), and modestely down-regulated tlr2 (baopraserkul et al., 2007b). the ortholog molecules involved in the tlr signaling cascades have also been identified in teleosts (takano et al., 2010). in the genome of d. rerio myd88, mal, ticam, trif and sarm have been identified (jault et al. 2004; meijer et al. 2004). the functionality of teleost myd88 has been confirmed by infecting d. rerio with the pathogen salmonella enterica (van der sar et al., 2006) or paralichthys olivaceus with ewardsiella tarda (takano et al., 2006). additional myd88 sequences are known from pseudosciaena crocea, cynoglossus semilaevis, s. salar (rebl et al., 2010). takano et al. (2010) discussed the presence of other elements (tirap, ticam, sarm, irak, ikk, traf) of the teleost tlr signaling pathway. nf-κb proteins have been characterized in d. rerio (phelan et al., 2005) and p. olivaceus (yazawa et al., 2005) demonstrating that teleost nf-κb has a critical role in the transcription occurring downstream of signaling cascade. amphybia the developmental and immunological studies on xenopus laevis and xenopus (silurana) tropicalis have been reviewed by robert and otha (2009). the presence in x. laevis of a signaling pathway primed by a spätzle/toll in dorsoventral patterning has been demonstrated by armstrong et al. (1998) and a mammalian myd88 homologue has been cloned (prothmann et al., 2000). more recently the availability of x. tropicalis genome database has provided a useful tool to deeply investigate the tlr pattern composition. in the draft genome, roach (2005) has identified several tlr genes. subsequently, ishii et al. (2007) have searched the last genome version (jgi 4.1) for tlr by blastp analysis using the tir domain of t. rubripes tlrs and have found 19 proteins. nineteen out of 23 complete sequences have shown high scores of identity with mammalian tlrs; the remaining 4 have been annotated as x. tropicalis myd88 molecules. tlr2 and 6 are duplicated. an ortholog for each teleost tlr has been found, except for t. rubripes tlr23; among tlrs present in mammals but absent in teleosts, orthologs of mammalian tlr6, 12 and 13, have been found, while the search for tlr4, 10 and 11 has failed. a particular attention has been focused on tlr4 whose presence in teleosts is controversial; its gene seems to be buried in a dna region where prediction tools indicate no exons. the size and number of the lrr of each x. tropicalis tlr are similar to those of the human tlr counterpart. moreover the same authors have demonstrated that x. tropicalis tlrs are costitutively expressed in the tadpole as well as in the adult (ishii et al., 2007). sauropsida the phylum sauropsida includes reptiles and birds. while tlr (or presumptive tlr) sequences from 17 different bird species are present in databanks, the search for reptile sequences unveiled only one species, anolis carolinensis. sequences of this species have been annotated as molecules resembling mammalian tlr2, 3, 4, 5, 6, 7 and 13. at present there are not articles in the literature that describe these genes. 220 on the contrary, a lot of papers deal with bird tlrs. it should be reminded that avian immune system differs from that of mammals in some aspects. it presents different immune organs, such as the bursa of fabricius, it lacks organized lymph nodes, neutrophils and eosinophils, but have heterophils. birds have also a different repertoire of cytokines, chemokines, defensins and integrins (kaiser, 2007). knowledge of the avian immunology has considerably expanded by the assembly of the gallus gallus genome (consortium, 2004). bird genome analysis has been extended to a species diverged from g. gallus lineage about 100 mya, the zebra finch (taeniopygia guttata, family passeriformes) in an attempt to obtain a more complete definition of the tlr types (brownlie and allan, 2011). the existence of ten avian tlrs have been confirmed by several authors (smith et al., 2004; yilmaz et al., 2005; boyd et al., 2007; temperley et al., 2008; alcaide and edwards, 2011). avian tlr1 and tlr2 are duplicated and also tlr7 exists in two copies in t. guttata. five out of ten avian tlrs (tlr2a, 2b, 4, 5, and 7) have clear mammalian orthologs. tlr15, belonging to the vertebrate family 1, appears to be specific within the avian species whereas tlr21 is related to teleost and amphybian tlr21. alcaide and edward (2011) have conducted the characterization of tlrs in seven distant nonmodel species falco naumanni, (falconidae), carpodacus mexicanum (fringillidae), oceanodroma leucorhoa (hydrobatidae), accipiter cooperii (accipitridae), amazona albifrons (psittacidae), picoides pubescens (picidae), and dromaius novaehollandiae (casuariidae). by using tests of selection, these authors have found that avian tlrs are dominated by purifying selection, but patterns of positive selection acts on specific amino acid residues; many of positively selected positions can be mapped to putative ligand-binding regions, suggesting that variations are linked to speciesspecific differences in pamps recognition. a systems biology approach has been used to identify orthologous relationships between human and g. gallus tlrs and tlr-interacting molecules: annotations of chicken sequences were generated both manually and by computer, using a human reactome knowledgebase, previously integrated by tlr signaling genes. in particular 33 out of 38 proteins of the human tlr3 pathway were annotated integrating manual and computational analysis (gillespie et al., 2011). mammalia it is extremely complex to summarize current knowledge on mammalian tlrs and this is not the main objective of this review. we will limit to indicate only several recent reviews covering parts of the topic that appear to be more important. casanova et al. (2011) reviewed the role of both tlrs and il1rs in host defense from multiple points of view. evolutionary genetic studies have shown that human intracellular tlrs (tlr3, 7, 8, and 9) evolved under stronger negative selection than surface-expressed tlrs (barreiro et al., 2009). epidemiological studies have revealed the association between infection diseases and variants in tlr genes. clinical investigations have demonstrated that rare mutations affecting the tlr3-trif pathway underlie herpes simplex encephalitis (zhang et al., 2007). also in domestical animals variation in amino acid sequence in tlr ectodomains could be associated with infectious diseases (huenishi et al, 2011; jungi et al., 2011). on the other hand, the viral evolution to escape the tlr recognition by manipulating their genomic content is a fascinating area that deserves more attention (barton, 2007). concluding commentaries in the last decade the number of genes encoding tlr or tlr-like molecules, identified from different species is extremely increased. however no tlr signaling pathways have been completely clarified in any animal lower than d. melanogaster. the evolutionary analysis of the available tlr sequences allows identification of orthologous relationships only in neighboring species; indeed sequences from phylogenetically distant species never occur in the same clade. this suggests that each lineage evolved independently by gene duplication. the evolution of tlr architecture seems to be shaped by structural conservation and divergence. in the cytoplasmic region the tir domain was highly conserved allowing to use in separate lineages, basically common signaling mechanisms and analogous regulatory modules. on the other hand, the ectodomain underwent significant variations. in more recently diverged species, the most relevant novelty was the loss of the additional cysteine clusters intermingled with the lrrs (protostometype). the number of lrrs appears to increase and their sequences seem to converge in the “typical motif”. different ligand-binding sites were shaped in the ectodomain through molecular evolution: while drosophila toll dimer accommodates two ligand molecules on the n-terminal region of each monomer, all vertebrate tlrs interact with a single ligand molecule and both monomers cooperate in the binding. furthermore a specific feature of the vertebrate tlr evolution is the ability to sense pamps directly, without using a cytokine intermediate as the insect spätzle. by reviewing the presence of tlrs in different species it appears clear that evolution used tlr molecules for different functions, ranging from immune response to developmental signaling and cell adhesion. while studies at genomic level have been performed on a large number of species covering the majority of the most important phyla, unfortunately at present investigations on the tlr functions are limited to a few species, particularly in the protostome lineage. this hampers the definition of 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http://www.ncbi.nlm.nih.gov/pubmed?term= http://www.ncbi.nlm.nih.gov/pubmed?term= http://www.ncbi.nlm.nih.gov/pubmed?term= http://www.ncbi.nlm.nih.gov/pubmed?term=a%20toll%20receptor%20from%20chinese%20shrimp%20fenneropenaeus%20chinensis%20is%20responsive%20to%20vibrio%20anguillarum%20infection%20changjian%20yang http://www.ncbi.nlm.nih.gov/pubmed?term=a%20toll%20receptor%20from%20chinese%20shrimp%20fenneropenaeus%20chinensis%20is%20responsive%20to%20vibrio%20anguillarum%20infection%20changjian%20yang http://www.ncbi.nlm.nih.gov/pubmed?term=%2522zou%20z%2522%255bauthor%255d http://www.ncbi.nlm.nih.gov/pubmed?term=%2522evans%20jd%2522%255bauthor%255d http://www.ncbi.nlm.nih.gov/pubmed?term=%2522lu%20z%2522%255bauthor%255d http://www.ncbi.nlm.nih.gov/pubmed?term=%2522zhao%20p%2522%255bauthor%255d http://www.ncbi.nlm.nih.gov/pubmed?term=%2522williams%20m%2522%255bauthor%255d http://www.ncbi.nlm.nih.gov/pubmed?term=%2522sumathipala%20n%2522%255bauthor%255d http://www.ncbi.nlm.nih.gov/pubmed?term=zou%20comparative%20genomic%20analysis%20of%20the%20tribolium the characteristic of immune parameters in zhikong scallop chlamys farreri and bay scallop argopecten irradians isj 11: 39-46, 2014 issn 1824-307x research report granulocytes of sea anemone actinia equina (linnaeus, 1758) body fluid contain and release cytolysins forming plaques of lysis mg parisi, mr trapani, m cammarata marine immunobiology laboratory, department of biological, chemical, pharmaceutical science and technology, university of palermo, palermo, italy accepted january 20, 2014 abstract the cnidaria phylum includes organisms that are among the most poisonous animals. the exact composition of cnidarian bioactive molecules is not known in detail, but little is known on the cells that produce the toxins. here we have shown that the presence of cytolysins is not exclusive of nematocysts. a plaque-forming assay was carried out with cell populations extracted from the percoled body fluid showed for the first time that anthozoan granulocytes are able to form plaque of lysis. we have partitioned the total population of free cells into three distinct discrete bands by discontinuous percoll gradient, and we have identified six small different types cells: morular granulocytes; cells with large or small peripherical granules, granulocytes with irregular shape containing blue and red granules, cells showing one fine red granule of uniform size and, finally, cells with elongated shape and small dispersed granules. cell lysate of each cellular band resulted cytolytic toward different erythrocytes types. sds page analysis of the lysate cell fraction showed a predominant of 20 kda that corresponds to the weight of the cytolytic equinatoxin. the nature of equinatoxins-related activity was demonstrated by inhibition experiments using bovine sphingomyelin. key words: cytolysin; actinia equina; granulocytes; plaque of lysis; sphingomyelin introduction cnidarians are the rich source of bioactive molecules like antimicrobial peptides, cytolytic proteins and neurotoxins. sea anemones (actininaria, cnidaria) are benthic sessile species that depend on their toxic venom for survival, providing for defense and predation. among last decades three classes of toxins, 20 kda pore-forming cytolytic hemolysins (kem, 1988; anderluh and macek, 2002, bakrac and anderluh, 2010), 3-5 kda sodium channel toxins (kem, 1988; norton, 1991) and 3.5 6.5 kda potassium channel toxins (castaneda et al., 1995; schweitz et al., 1995; cotton et al., 1997; gendeh et al., 1997; diochot et al., 1998; minagawa et al., 1998), have been isolated and well characterized from a great number of sea anemones. the strongest actinia equina cytolysins, equinatoxins (eqii), a pore forming protein of the actinoporin family of approximately 20 kda (shon et ___________________________________________________________________________ corresponding author matteo cammarata department of biological, chemical pharmaceutical science and technology marine immunobiology laboratory university of palermo via archirafi 18, palermo, italy e-mail: matteo.cammarata@unipa.it al., 2008; kristan et al., 2009; bakrac and anderluh, 2010) have been extensively studied for their chemical and pharmacological properties. it exhibits a wide range of pharmacological activities, including platelet aggregation (teng et al., 1988), cardiotoxicity (sket et al., 1974), cytotoxicity (batista et al., 1990) and an ability to cause pulmonary edema (lafranconi et al., 1984). at a cellular level, its most important outcome is the formation of pores in lipid membranes thank to cytolytic and cytotoxic effects on different types of cells. cytolysins may have several isoforms and in actinia equina five sequences of equinatoxins have been found to show a multigenic family (anderluh et al., 1999). regarding eqii, protein and sequence analysis (simpson et al., 1990; belmonte et al., 1994; anderluh et al., 1996), pores formation (mavek et al., 1994; bonev et al., 2003), crystallographic and solution structure resolution (zhang et al., 2000; athanasiadis et al., 2001; hinds et al, 2002) have been investigated. despite these studies at the molecular and structural level, is not yet known if other cells, in addition to the nematocysts, contain or release cytolysins, what is their localization and characterization. 39 as others invertebrates, a. equina possess various morphological categories of amebocytes, such as phagocytes and non granular type (hutton and smith, 1996). research on cnidarian venom and toxins focused on the nematocysts, recognized as the venomous apparatus in sea anemones and jellyfish. it is widely accepted that the venom of cnidarians is produced in the nematocytes and is injected via the nematocysts upon encounter (kass-simon and scappaticci, 2002). some studies have shown that the nematocyst-derived fractions were often less toxic than whole tentacle extracts, suggesting the possibility that toxins may also reside in non-nematocyst compartments (xiao et al., 2009). the origin of neurotoxin and cytolytic substances remained unanswered despite the knowledge on their storage in nematocysts. thus, for very few toxins has been shown that the origin of formation is the nematocysts (lotan et al., 1996) and it was not known the cell compartment involved in the toxins production. moran et al., (2009; 2013) reported that toxins are held also in ectodermal gland cells. in anemonia viridis neurotoxins are associated with both nematocytes and ectodermal gland cells. honma et al. (2008) described gigantoxins derived from unknown organelles other than nematocysts. the aim of this research is to characterize actinia equina body fluid percoled cell types and their cytotoxic activity, the presence of equinatoxin or other cytolytic molecules and the ability to lyse foreign cells. materials and methods collection of the sea anemone actinia equina the sea anemone a. equina (linnaeus, 1758) was collected from a typical rocky shore of the intertidal zone of capo gallo coast (palermo italy). specimens were maintained and powered in a closed-circuit aquarium at 20 °c with aerated sea water (150 l aquaria) and fed every second day with a marine invertebrate filter feeding diet (kent marine inc. wi usa) until experimental use. cell extraction free cells were extracted from the body fluid percolate and mesenteric filaments by making an incision into the center of the pedal disc and excising the filaments into a sterile 15-ml plastic centrifuge tube (hutton and smirh, 1996). the filaments and liquid of internal cavity were mixed with free sea water fsw (nacl 0.5 m, kcl 8 mm, na2so4 30 mm, nahco3 2mm) with 10 mm edta (ethylendiamminotetraacetic acid) as an anticoagulant and vigorously pipetted to dissociate the cell. free cell were separated from internal liquid-fsw edta by centrifuging (800×g) for 10 min at 4 °c, immediately suspended in ice cold fsw-edta and counted in a neubauer chamber. after observation under a light microscope material was immediately used or stored at −20 °c. preparation of percoll gradient according to cammarata et al. (1993), a 75.0, 55.0, 37.5 % discontinuous gradient of percoll (pharmacia, uppsala, sweden) in fsw with 10 mm edta and 10 mm cysteine, was formed in 6 ml round-bottom polyethylene tubes (du pont de nemours & co. inc. instrument product, newton, ct, usa). briefly, freshly collected cell suspension (approximately 4 ml of 6x107/ml) diluted with fsw-edta was spun through a discontinuous gradient of equilibrated percoll. the tube was centrifuged in a swing-out rotor (850xg, 30 min, 4 °c). bands of cells were gently removed by aspiration from the gradients and washed twice before suspension in fsw. for microscopy observations, the cells were removed with a pipette from the gradients and washed twice in fsw before being deposited on a glass slide. also each cellular band was removed gently and, after being washed with fsw, was employed for the lysis plaques assay. plaque-forming cell assay (pfc) a pfc assay has been originally described by cunningham and szenberg (1968) for the human b cell/sheep red blood cells and subsequently modified for invertebrate blood cells (parrinello et al., 1996; cammarata et al, 1997; ballarin et al., 2008). fifty μl of hemocytes suspension (1x106/ml) in marine solution (ms:12 mm cacl2, 11 mm kcl, 26 mm mgcl2, 45 mm tris, 38 mm hcl, 0.45 m nacl, ph 7.4) are mixed with 10 μl of suspension of 5 % rabbit erythrocytes in ms. the reaction mixture is rapidly layered into the slide chamber by capillary action. the chamber has been manufactured by placing three thin strips of double-stick tape placed between the borders and in the center of a coverslip and another glass coverslip is then suspended onto the three pieces of tape forming a double chamber. each slide chamber can accommodate just under 0.1 ml on either side of the tape (0.2 ml per slide). after 15 min of incubation at 20 °c the cell mixture was examined under a phase contrast microscope. the control was prepared with 10 μl of erythrocytes and 50 μl of ms. cell staining for the cells characterization 100 μl of cell suspension in fsw-edta containing cysteine were placed on a glass slide treated with poly-l-lysine (sigma diagnostics inc.) and left for 30 min at room temperature. the cells were fixed with lavdowsky fixative (2.5 ml of 37 % formaldehyde, 1 ml acetic acid, 12.5 ml 96 % ethanol, 10 ml distilled water) for 30 minutes. after washing with pbs (0.1 m nacl; 0.02 m kcl; kh2po4 0.01 m; na2hpo4 0.06 m) cells suspension was treated with toluidine blu stain 0.1 % in pbs. after two times washing with pbs, the slide was closed with a solution of pbs and 20 % glycerol and observed under a microscope. cell cytotoxic assay the rabbit erythrocytes (rbc) obtained by istituto zooprofilattico della sicilia in alsever solution (0.42 % nacl; 0.08 % sodium citrate dihydrate, citric acid monohydrate 0.045 %, 2.05 % d-glucose ph 7.2) were washed three times with pbse (kh2po4 6mm; na2hpo4 0.11 m; nacl 30 mm; ph 7.4) and centrifuged at 1800 rpm for 10 min at 4 °c. the supernatant was removed by gentle aspiration and the above process was repeated two times. the 40 erythrocytes were finally resuspended in tbs gel (tris-hcl 50 mm; nacl 0.15 m; ph 7.4, gelatin 0.1 %) to make a 1 % final concentration.to perform cytotoxic assay 500 μl of hemocyte suspensions (1x106) unfractionated cells in ms or 500 μl of cellular band lysate was mixed with an equal volume of freshly prepared sheep, bovine, pig or rabbit erythrocyte suspensions (8x106 cells) in ms. hemocyte counts were determined in the final volume of the reaction mixture. the mixture was incubated with continuous and moderate shaking at 25 °c for 1 h. at the end of the incubation, the supernatant was separated and the amount of the released hemoglobin (hb) was estimated by reading the absorbance at 541 nm in a spectrophotometer. the degree of hemolysis was determined according to the equation: measured release spontaneous release percent hemolysis = ------------------------------------------x 100 complete release spontaneous release complete hemolysis was obtained by preparing an erythrocyte suspension in distilled water. control erythrocyte suspensions were also prepared in the same medium and incubated as reaction mixtures: spontaneous hemoglobin release never exceeded 5 % of the total release. for each experiment three samples were assayed. cell lysate from the percoll cell bands each cell fraction recovered from the percoll gradient was used to carry out cell release and extracts for rabbit erythrocytes cytotoxic assay. to get the released cell product, the cell fractions (10x106 cells) were removed from percoll gradient, washed in ms and left in this solution (1 ml) for one hour. then the suspension taken out was centrifuged at 800xg, 4 °c for 10 min and the supernatant was and used for the lysis assay. the cell from each fraction (1.2x106 cells) were washed and resuspended in tbs and placed in polycarbonate tubes to be sonicated at 4 °c for 60 s (branson, model b15, danbury, ct, usa). the cell lysate was centrifuged at 27,000 g for 20 min at 4 °c and the resulting hemocyte lysate supernatant was used for the assay. sphingomyelin (sigma-chemicals) was dissolved in ms to obtain 0.25 and 250 ng/ml concentrations in reaction mixture. due to insolubility stock solution sphingomyelin was briefly sonicated (vibra cell sonics & materials, inc. dambury). sodium dodecyl sulfate polyacrylamide gel electrophoresis (sdspage) lysed and released cell products were analyzed by 10 % sds-page according to the method of laemmli (1970) under reducing conditions (5 % mercaptoethanol). after electrophoresis, protein bands were stained with coomassie brilliant blue r250 (sigma). relative molecular weights of the protein bands were determined by using molecular weight markers: albumin, bovine serum (66.0 kda), ovalbumin (45.0 kda), glyceraldehyde-3phosphate dehydrogenase (36.0 kda), carbonic anhydrase (29.0 kda), trypsinogen (24.0 kda), and alpha lactalbumin (14.2 kda). results separation on a percoll discontinuous density gradient and characterization of a. equina percoled body fluid cells the percoled body fluid of a. equina cell suspension was obtained and separated into the percoll gradient. the total population of hemocytes was separated into three distinct discrete bands (b1-b3). the cells removed from density gradient separated bands were examined and identified for their morphology. differential count of the hemocytes from each set was performed (at least 200 cells/slide). six small types of different cells have been identified, as reported in figure 1: granulocytes (a) that assume a morular appearance, considered as globular cells with a variable vacuoles stained with toluidine blue 0.1 % in pbs; b type cells have a globular shape 8and contain a variable number of large fig. 1 characterization of a. equina cell types present in percoled body fluid separated by percoll gradient. cell differential distribution (%) in bands obtained from a discontinuous percoll density gradient (b1, b2, b3) and in the unfractionated cells (un). identification of cell types (a-f) was carried out with toluidine blue 0.1 % in pbs stain. bar = 10 μm. 41 fig. 2 a. equina cells cytotoxic activity against mammals erythrocytes. typical lytic activity of a. equina cell assayed in vitro against different erythrocytes at various effecter/target ratios. cell lysates are serially diluted twofold and incubated with an equal volume of 1 % rabbit erythrocyte suspension for 1 h at 37 °c. after centrifugation the absorbance of the supernatants was determined at 545 nm. the value were at least the mean of three experiments, each performed in duplicate. large granules distributed at the periphery of cells; c type cells are distinguished by a granular inclusions and a blue less staining than in other classes; d cells are known as granular cells with irregular shape that contain blue and red granules with variable dimensions; cells of category e contain in their cytoplasm one fine red granule of uniform size; f type cells with very large nuclei appear with round or elongated shape and small granules dispersed in the peripheral regions of the cell bodies. gradient centrifugation allows separating cell debris in the one band located on the top of the percoll tube (fig. 1) where no whole cell has been identified through microscopic observation. as indicated in figure 1, b1 band mainly contained cells of b and c category, respectively 23 % and 42 % and, to a lesser extent, granulocytes (d) and round small cells with a red nucleus (e). b2 band consisted primarily of ~40 % granular cells (d), ~28 % granulocytes, an equal percentage of c and e categories and ~9 % of cells with large nuclei of f category. instead, this last type of cells is predominant in the b3 followed by the presence of b and d type granulocytes with percentage of 26 %. cytotoxic activity at various effector:target cell ratios toxic activity of effector cells has been clarified by examining the effector:target cell ratio. as reported in figure 2, the curve of hemolysis in vitro to rabbit, sheep, bovine and pig erythrocytes is similar in shape but higher towards rabbit erythrocytes. the highest value of the undiluted sample (t) decreases until the last dilution of 1:512, although it showed an increase at the dilutions between 1:32 and 1:64 for all the used targets. plaque of lysis are induced by granulocytes a plaque-forming assay was carried out with cell populations separated into three bands by percoll gradient to investigate which cell type was responsible for the cytotoxic activity and plaque formation. for the first time, anthozoan granulocytes that form plaque of lysis are shown (fig. 3). in three distinct experiments, by using rabbit erythrocytes, plaques constituted 10 20 % of the effector cells. on the contrary, plaques were not found when sheep erythrocytes were used as a target (fig. 3 cnt). fig. 3 plaque lysis assay of cells from separated bands from a percoll gradient. plaque of lysis of a. equina cells fractionated in b1, b2 and b3 bands after separation on percoll density gradient were observed by phase-contrast microscope. figure 3a shows a plaque of lysis formed by cells isolated from the band b1. two typical plaques of lysis against rabbit erythrocytes with the effector cell at the center of the plate are shown. arrowed indicate the ghosts of erythrocytes. bar = 20 μm. inset a is a magnification of the cell (type d) responsible for cytotoxicity. bar = 60 μm. figure 3b shows a plaque of lysis from the band b2 cell. bar = 20 μm. cnt: monolayer of rabbit erythrocytes. bar = 20 μm. in figure 3c small plaques from b3 are shown. 42 plaques were observed when cells present in b1 and b2 fraction were placed in contact with the erythrocyte suspension in a cunningham szenberg chamber (figs 3a, b), in which a clear granulocyte is present at the center as an effector releasing cell. in the band that contains cells debris the lysis process is very fast and takes place immediately after contact between the cells and the erythrocytes used. cells of the b3 (fig. 3c) not showed plaques of lysis although in the bands lysate cytotoxicity has indicated a moderate lytic activity. the addition of sphingomyelin at 250 and 25 µg/ml inhibited the plaque formation, whereas there was no effect on cell or erythrocyte suspensions used as controls. cell bands are cytolytic towards various erythrocyte targets and lysis is inhibited by sphingomyelin a. equina cells lysate was assayed for its hemolytic activity (clc) using the rabbit erythrocytes. all the data were obtained using aliquots of the same cell population bands lysate. the results were reported in figure 4a. the samples caused 75 % and 88 % hemolysis respectively in band of cells debris and in the cell fraction of b1. cell component from the density percoll gradient of bands 2 and 3, b2 and b3, show a 65 % and 57 % of clc, respectively. percentage of clc is clearly higher in the unfractionated samples (94 %). with the aim of examining the cytolytic mechanism of an enriched population of effector cells, the interaction between erythrocytes membrane lipids and lysins was examined by inhibition experiments. rabbit erythrocytes have been utilized for the values of the hemolysis ratio where the higher respect to another target. as shown in figure 4b, sphingomyelin inhibits the cytotoxic activity of unfractionated, band1 and 2 cells at 250 μg/ml and at various e/t ratios. percentage of actinia equina cell mortality is always less than 4 % in the presence of sphingomyelin. the figure 5 reported the sds page stained with coomassie brilliant blue that showed a pattern of total protein in the lysate cell fraction. many bands were recorded with different molecular weights ranging from 66 to 14.2 kda. the predominant band is the one with an apparent molecular weight of 20 kda that corresponds to the equinatoxin mw, a more intense band is visible in b1 and b2 and in unfractionated sample. fig. 4 cell lysate cytotoxicity from a percoll gradient cell band and sphingomyelin inhibition. a) percentage of hemolysis towards rabbit erythrocytes of a. equina cell lysate from bands b1, b2, b3, unfractionated cell and debris. b) inhibitory effect of sphingomyelin on cytotoxicity towards re at the concentration of 250 µg/ml. vertical bars represented the mean ± se (n = 3). significant differences across control were indicated with an asterisk at p < 0.05 and two at p < 0.01. 43 fig. 5 electrophoretic analysis of a. equina cell lysates. sds-page gel of a. equina cell lysate from b1, b2 and b3 bands obtained from percoll density gradient stained with coomassie blue. lane 1 3: electrophoretical profile of b1, b2 and b3 cell bands. lane 4: protein molecular weight standards. lane 5: unfractionated cell lysate. discussion anthozoans are important modulators of marine habitats in benthic communities. from a. equina, one of the conspicuous species of intertidal rocky shore areas, have been isolated many bioactive molecules (frazão et al., 2012) but few is known about source of these. in this work morphology characterization of a. equina cell from percolated animal body fluid after percoll gradient density separation has been carried out. previous histological studies indicated anthozoa as very simply organized with a body built up of sheets of tissue, above the mesoglea, and on whose surface is present an epithelium (gadelha et al., 2012). here, six cells categories distributed into three cell bands have been characterized. for the first time we showed that a. equina cells able to form plaque of lysis versus sheep erythrocytes. cytotoxic activity was different for the different cell bands depending from the cell types composition. cells from debris band were not recognized even though there is a strong cytotoxic activity, probably due to lytic factors rapidly released after the contact with the targets. most likely the cell responsible for lytic activity are the granulocytes in b1 and b2 as seen from low magnification of a cell of b1 band regardless of the percentage of cells calculated for each fraction (fig. 3a). it is not excluded combination of lytic factor present in the sample to determine the cytotoxic results. the component capable of mediating the cytotoxic response has been investigated analyzing supernatant obtained by cell release or cell lysate. sds page analysis of cell lysate component showed similar electrophoretic profile. the predominant band is the one with a molecular weight of 20 kda, molecular mass characteristic of numerous toxins of type ii inhibited by sphingomyelin. these cytolysins are also called actinoporins due to their ability to hold the membrane phospholipids domains of the host organism, oligomerizing and forming cation selective pores (kem, 1988). they belong to the unique family of the α-pore-forming toxins (pfts) (monastyrnaya et al., 2010). the most representative of cytolytic anthozoan actinoporins is equinatoxin (macek and lebez, 1988; anderluh and macek, 2002), a mixture of five isoforms of which equinotoxin ii is the most abundant one, exclusively found in sea anemones (anderluh et al., 2009). studies about the cytotoxicity activity of living sea anemones or isolated toxins showed that 20 kda cytolysins are able to destroying the tissues of not symbiotic fishes (anderluh and macek, 2002). one of the hallmarks of actinoporins is their sphingomyelin specificity, as they efficiently make pores in lipid membranes containing this lipid (kristan et al., 2009). therefore, here we wanted to assess the interaction between erythrocytes membrane lipids and lysins by inhibition experiments carried out using rabbit erythrocytes. sphingomyelin inhibits the cytotoxic activity at 25 and 250 μg/ml and at various e/t ratios particularly in band 2. in higher metazoans the defense responses are mainly based on hemocyte types that release humoral factors (iwanaga and lee, 2005; loker et al., 2004) or display cell-linked activities (parrinello, 1996; parrinello et al., 2003). cellular recognition has been attributed to proteins that are located on the cell surface. the ability of cnidarians to distinguish between self and non-self has previously been shown to occur in anthozoans and hydrozoans (rinkevich 2004; bosch, 2008). the anti-erythrocyte cytotoxic activity examined in this paper resides in different cell bands, able to recognize erythrocyte target and appear dependent from equinatoxin presence. many studies focused interest to the employment of cytolysins as model proteins to study protein-lipid membrane interaction (anderluh and macek, 2002) and to study the eradication of tumour cells and parasites (tejuca et al., 2009), cardio-stimulating, dermatonecrotic and antihistamine properties (klyshko et al., 2004). equinatoxin ii has been also been studied as an alternative permeabilizing agent that lyses the limiting membrane of plasmodium falciparum 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toxicity and tolerance to isolation and purification procedures. toxicon 53: 146-152, 2009. 46 moran y, gordon d, gurevitz m. sea anemone toxins affecting voltage-gated sodium channels--molecular and evolutionary features. toxicon 54: 1089-1101, 2009. hypothetical photosensory structure in ciliated protozoan, blepharisma isj 11: 213-218, 2014 issn 1824-307x research report identification of camp-dependent phosphorylated proteins involved in the formation of environment-resistant resting cysts by the terrestrial ciliate colpoda cucullus y sogame1, k kojima2, t takeshita2, e kinoshita3, t matsuoka1 1department of biological science, faculty of science, kochi university, kochi 780-8520, japan 2department of microbiology and immunology, shinshu university school of medicine, 3-1-1 asahi, matsumoto, nagano 390-8621, japan 3department of functional molecular science, institute of biomedical & health sciences, hiroshima university, kasumi 1-2-3, hiroshima 734-8553, japan accepted june 26, 2014 abstract in the terrestrial ciliate colpoda cucullus, an elevation of the intracellular camp concentration was reported to be involved in environment-resistant resting cyst formation. in the present study, camp-dependently phosphorylated proteins of encystment-induced c. cucullus were isolated with phos-tag agarose phosphate-affinity beads and subsequent sds-page. in a liquid chromatography/tandem mass spectrometry analysis of these phosphoproteins, 27-, 37and 43-kda proteins (p27, p37 and p43) were identified as rieske iron-sulfur protein, histone h4 (hyperacetylated form), and actin, respectively. key words: environment-resistant cyst; colpoda; encystment; protein phosphorylation; camp introduction the terrestrial protists, inhabitants of temporary puddles, form resting cysts when they detect approaching hostile environmental conditions. they excyst when conditions favorable to survive and to proliferate are regained. in general, the resting cysts of terrestrial protists are resistant to extreme environments. for example, dried cysts of the terrestrial ciliate colpoda cucullus have been reported to survive up to about 120 °c (taylor and strickland, 1936). the resting cyst of this species is also resistant to environmental stresses such as desiccation, freezing and even extreme lower ph (1 m hcl) (maeda et al., 2005; sogame et al., 2011b). for better understandings of the mechanisms underlying the cellular morphogenesis and acquisition of tolerance during the encystment of c. cucullus, we studied this species from morphological and molecular perspectives, as follows. the cyst formation (encystment) process involves remarkable cellular morphogenetic transformation as follows (funatani et al., 2010): some mitochondria are fragmented within 1 h after the onset of encystment induction and then digested ___________________________________________________________________________ corresponding author tatsuomi matsuoka department of biological science faculty of science kochi university, 780-8520, japan e-mail: tmatsuok@kochi-u.ac.jp by autophagy. net-like globules called lepidosomes (foissner et al., 2011) are formed inside the intracellular vacuoles, to be expelled within 1 2 h after the onset of encystment induction, and subsequently the ectocyst (an outermost layer of cyst wall) is formed. in this stage (2 3 h after the onset of encystment induction), small-sized chromatin granules extruded from the macronucleus are digested by autophagy, and the mitochondrial membrane potential begins to disappear (sogame et al., 2014). ectocyst formation is followed by the formation of several layers of endocyst between the ectocyst and plasma membrane for several days. within 1 2 weeks, the structures characterizing vegetative cells such as the ciliary apparatus are disintegrated, and electron-lucent granules that may be reserve granules accumulate in the center of the mature resting cyst. encystment of c. cucullus can be induced by the overpopulation of vegetative cells in the presence of ca2+ (yamaoka et al. 2004; matsuoka et al., 2009). intracellular signaling pathways leading to the encystment of c. cucullus are triggered by an increase in the intracellular ca2+ concentration (sogame and matsuoka, 2013), which is promoted by cell-to-cell mechanical stimulation in the presence of external ca2+ (matsuoka et al., 2009). in these signaling pathways, the intracellular camp concentration is elevated (matsuoka et al., 2009; sogame et al., 2011a, 2011c), as are the 213 a) b) fig. 1 camp-dependent protein phosphorylation detected by biotinylated phos-tag/ecl assays (a),phosphoproteins isolated with phos-tag agarose phosphate-affinity beads in encystment-induced c. cucullus (b). (a) ‘cbb’: blots stained with cbb after the biotinylated phos-tag/ecl detection. ‘p-tag’: protein phosphorylation detected by biotinylated phos-tag/ecl assays. protein phosphorylation and encystment was induced by c. cucullus vegetative cells being suspended for 1 h at a low cell density (2,000 cells/ml) in 1 mm tris-hcl (ph 7.2) containing 10 µm camp-am (fig. 1a, ‘camp-am’). as a control (a, ‘none’), the cells were suspended in 1 mm tris-hcl (ph 7.2) without camp-am at a low cell density (2,000 cells/ml); (b) isolation of phosphoproteins of encystment-induced c. cucullus (encystment induction by ca2+/overpopulation) with phos-tag agarose phosphate-affinity beads and subsequent sds-page/western blotting. cbb: blots stained with cbb after the biotinylated phos-tag/ecl detection (‘p-tag’). ‘p-tag’: biotinylated phos-tag/ecl detection of phosphoproteins, isolated with phos-tag agarose phosphate-affinity beads. phosphorylation levels of several proteins (sogame et al., 2012a), and followed by the alteration of protein expression (sogame et al., 2012b, 2014). among encystment-dependently phosphorylated proteins, some proteins (33-, 37-, 43-, 47and 49-kda proteins) have been reported to be camp-dependently phosphorylated (sogame et al., 2011c). in the present study, in addition to these proteins, several phosphoproteins were found to be also phosphorylated in camp-dependent manner. the purpose of the present study is to identify camp-dependently phosphorylated proteins in the early phase of encystment of c. cucullus by a liquid chromatography/tandem mass spectrometry (lc-ms/ms) analysis. materials and methods cell culture and encystment induction colpoda cucullus (nag-1 strain) collected from the soil surface in kochi prefecture, japan was cultured in a 0.05 % (w/v) infusion of dried wheat leaves, which was periodically inoculated with a non-pathogenic strain (6081) of klebsiella pneumoniae. the bacteria were cultured on agar plates containing 1.5 % (w/v) agar (wako pure chemical industries, osaka, japan), 0.5 % (w/v) polypeptone (nihon pharmaceutical co., tokyo), 1 % (w/v) beef extract (becton dickinson, lincoln park, ny), and 0.5 % (w/v) nacl. the vegetative cells of c. cucullus cultured for 1 2 days were collected by 214 centrifugation (1,500×g for 2 min) and resuspended in 1 mm tris-hcl (ph 7.2). in order to induce protein phosphorylation and encystment, the cells collected again by centrifugation (1,500×g for 2 min) were suspended at a low cell density (2,000 cells/ml) in 1 mm tris-hcl (ph 7.2) containing 10 µm adenosine 3’, 5’-cyclic monophosphate acetoxymethyl ester (camp-am) (encystment induction by camp), or suspended at a high cell density (50,000 cells/ml) in 1 mm tris-hcl (ph 7.2) containing 1 mm cacl2 (encystment induction by ca2+/overpopulation). as a control of encystment induction by camp, the colpoda cells were suspended in 1 mm tris-hcl (ph 7.2) containing 0.1 % dimethyl sulfoxide (dmso) at a low cell density (2,000 cells/ml). chemicals adenosine 3’, 5’-cyclic monophosphate acetoxymethyl ester (camp-am) was purchased from sigma-aldrich (st. louis, mo). camp-am was dissolved in dmso to give 10 mm stock solution. prior to the assays, the stock solution was diluted 1,000 times to produce 10 µm camp-am solution containing 0.1% dmso. phenylmethylsulfonyl fluoride (pmsf) was purchased from boehringer-mannheim (gaithersburg, md), aprotinin from sigma-aldrich, leupeptin and pepstatin from peptide institute inc. (osaka, japan), sodium fluoride (naf) from wako pure chemical industries (osaka, japan), and sodium orthovanadate from sigma-aldrich. pmsf and pepstatin were dissolved in dmso to give 1 m and1 mg/ml stock solutions, respectively. for use, these stock solutions were diluted 1,000 times to produce solutions with final concentrations of 1 mm and 1 µg/ml, respectively, and containing 0.1 % dmso. leupeptin, aprotinin and sodium orthovanadate (na3vo4) were dissolved in pure water to produce 1 mg/ml, 1 mg/ml and 1 m stock solutions, respectively. prior to the assays, they were diluted 1,000 times to produce final concentrations of 1 µg/ml, 1 µg/ml and 1 mm solutions, respectively. naf was dissolved in pure water to give a 200 mm stock solution, and diluted 200 times to produce a solution with a final concentration of 1 mm. sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) we performed the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) essentially according to laemmli’s method (laemmli, 1970). the colpoda cells were solubilized in the sds-sample buffer containing 30 mm tris-hcl (ph 6.8), 1 % (w/v) sds, 5 % (v/v) 2-mercaptoethanol and 10% glycerol, and then boiled for 3 min. the total proteins (approx. 50 mg) corresponding to 5,000 cells in each lane were electrophoresed on a 10 % (fig. 1a) or 12.5 % (fig. 1b) gel at 150 v. western blotting and biotinylated phos-tag /ecl assay electrophoresed proteins were transferred to an immobilon-p transfer membrane (millipore, bedford, ma) for 3 h at 350 ma in a transfer buffer (ph 11.0) containing 10 mm 3-(cyclohexylamino)-1-propanesulfonic acid (caps) and 10 % methanol, transferred for 60 min at 100 ma using a semi-dry blotting system (hoefer te70, amersham, tokyo) with three different types of blotting solutions containing 20 % methanol each (solution a, 300 mm tris; solution b, 25 mm tris; solution c, 25 mm tris-borate buffer [ph 9.5]). for the phos-tag (phosphate-binding tag molecule) detection of phosphorylated proteins, we prepared a complex consisting of biotin-pendant phosphate-binding tag molecule (phos-tag) (zn2+-phos-tag™ btl-104; available at http:www.phos-tag.com) and horseradish peroxidase (hrp)-conjugated streptavidin (ge healthcare bio-sciences, buckinghamshire, uk).phosphorylated proteins were detected according to the method reported by kinoshita et al. (2006). in this assay, the phosphoproteins on the transfer membranes were detected by an enhanced chemiluminescence (ecl) detection system (ge healthcare) (exposed to hyperfilm and developed at 20 °c). thereafter, the blots were stained for 1 min with 0.1 % cbb r250, 40 % (v/v) methanol, 1% glacial acetic acid solution, and then destained in 50 % (v/v) methanol. imagej analysis theoptical density of each lane (indicated by the framework) of cbb-stained gels (fig. 1a, left) was determined by an imagej analysis. each phos-tag detected band (fig. 1a, right) was also analyzed by imagej, and the ratios of theoptical density of each corresponding band are indicated in the parentheses. isolation of phosphoproteins with phos-tag agarose phosphate-affinity beads for the isolation of phosphoproteins using phos-tag agarose phosphate-affinity beads, the colpoda cells were solubilized for 2 h on ice in 50 mm tris-hcl (ph 7.4) containing 2.5 % (w/v) sodium deoxycholate, 2 % (v/v) nonidet p-40 (np-40), 1 mm n, n, n’, n’-ethylenediaminetetraacetic acid (edta), 0.15 m nacl, protease inhibitors (1 mm pmsf, 1 µg/ml aprotinin, 1 µg/ ml leupeptin and 1 µg/ml pepstatin) and phosphatase inhibitors (1 mm na3vo4 and 1 mm naf). the isolation of phosphoproteins using phos-tag agarose phosphate-affinity beads and sds-page was performed according to the methods reported by kinoshita-kikuta et al. (2009). liquid chromatography tandem mass spectrometry (lc-ms/ms) prior to the lc-ms/ms analysis, the phosphoproteins trapped on phos-tag agarose phosphate-affinity beads were separated by sds-page and then electroblotted to an immobilon-p transfer membrane (fig. 1b, left lane). the blots used in the phos-tag detection assays were incubated in 1 n aqueous nh3 for 15 min three times to remove the biotinylated phos-tag complex (nakanishi et al., 2007). protein bands (fig. 1b, left lane) visualized by cbb staining were cut out and then subjected to reduction with 40 mm dtt (dithiothreitol) for 1 h at 37 °c and alkylation with 100 mm iodoacetic acid for 20 min at room temperature to generate the carboxymethylation of cysteine residues. to block the non-specific binding of protease, membrane pieces were treated in 100 mm 215 table 1 novel colpoda cucullus proteins isolated with phosphate-affinity beads and tentatively identified by lc-ms/ms whose phosphorylation level is enhanced by encystment induction protein name bands obtained by sds-page sequences of exactly matched peptides partially matched peptides (2) accession no. (organisms) sequence coverage (%) actin p43 agfagddapr, mpgimvgmdqk, dsyvgdeaqsk, lteaplnpk, eltalapstmk gi 157093087 (karlodinium micrum) 14 (52aa/376aa) histone h4 (1) p37 isgliyeetr, tlygfgg gi 294888716 (perkinsus marinus) 17 (17aa/103aa) rieske iron-sulfur protein p27 lvedk, pgnfgdhidfk hlvedkptffvtssr, vnidnwfdenr, lyamgvigr, eenelpsntlldk, evilsdagnt gi 146164447 (tetrahymena thermophila) 26 (69aa/269aa) hypothetical protein p21 lydpntfyehgdnpafk gtaseeelk nwddflqrdck pvgshgitk gi 146142959 (tetrahymena thermophila) 22 (46aa/210aa) (1)hyperacetylated form (2)de novo sequences. the residues matched with those in sequences predicted by peaks online 5.3 are underlined acetic acid containing 0.5 % polyvinylpyrrolidone (pvp40) for 30 min at 37 °c. on-membrane digestion of proteins was performed in 10 ml of 30 mm tris-hcl (ph 8.5) containing 10 % acetonitrile and 1 pmol trypsin (sigma-aldrich) for 18 h at 37 °c. peptides produced by tryptic digestion were separated by a 0% 40% linear gradient with acetonitrile and analyzed with an ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (uplc qtof) system (xevo®, waters, milford, ma). the raw data were processed with proteinlynx global server 2.4 software (waters). proteins were identified by searching against the alveolata protein sequences registered in ncbi entrez protein records using peaks online 5.3 (bioinformatics solutions inc., waterloo, on, canada). results and discussion when the cells were suspended at a low cell density in 1 mm tris-hcl (ph 7.2) containing 10 µm camp-am, the phosphorylation level was evidently elevated (greater than about twofold) in 21-, 22-, 24-, 26-, 27-, 31-, 33-, 37-, and 43-kda proteins (p21 p43) (fig. 1a, ‘p-tag’), indicating that these proteins may be phosphorylated through camp-dependent kinase (protein kinase a; pka). the ratios indicated in the parentheses in fig. 1a (‘p-tag’) are the ratio of the optical density between each band in each lane (‘none’ and ‘camp-am’), which reflects the degree of protein phosphorylation. in this case, the total protein content contained in each lane was equivalent, because the imagej analysis of the cbb-stained western blots (fig. 1a, ‘cbb’) showed the equivalent optical density between the two lanes. among the camp-dependently phosphorylated proteins, p24 has already been identified to be ribosomal s5 protein, and p31 and p33 to be ribosomal p0 protein (sogame et al., 2012a). the phosphorylated proteins contained in encystment-induced cells (1 h after the onset of encystment induction) which were isolated with phos-tag agarose phosphate-affinity beads, separated by sds-page, and the western blots (fig. 1b, ‘cbb’) were analyzed by biotinylated phos-tag/ecl (fig. 1b, ‘p-tag’) prior to cbb staining. cbb-stained protein bands (p21, p27, p37, and p43) 216 on the transfer membrane whose phosphorylation level was evidently elevated in camp-dependent manner, were analyzed by lc-ms/ms, followed by a database search. in the present assays, the amino acid sequences of protease-digested fragments of p21, p27, p37 and p43 completely and/or partially coincided with the sequences of the tetrahymena thermophila hypothetical protein, rieske iron-sulfur protein (risp) of t. thermophila, histone h4 (hyperacetylated form; http://datasheets.scbt.com/sc-34264.pdf) of perkinsus marinus, and actin of karlodinium micrum, respectively (table 1).the hypothetical protein (p21) failed to be identified, because no proteins highly homologous to the t. thermophila hypothetical protein were found. risp has been suggested to have functions other than a core polypeptide of complex iii of a mitochondrial electron transport chain. that is, risp may form part of the mitochondrial permeability transition pore complex, and the phosphorylation and dephosphorylation of this protein may be involved in the regulation of the mitochondrial permeability transition (he and lemasters, 2005) which reduces mitochondrial membrane potential. in encystment-induced c. cucullus, the mitochondrial membrane potential begins to disappear 2 3 h after the onset of encystment induction (sogame et al., 2014). the encystment-dependent phosphorylation of a colpoda risp (p27) may be involved in the disappearance of the mitochondrial membrane potential through the regulation of the mitochondrial permeability transition. the tails of the histone proteins including histone h4 can be epigenetically modified by acetylation, methylation, or phosphorylation. it is known that histone phosphorylation is involved in diverse nuclear events such as dna damage repair, transcription regulation, and apoptosis-induced chromatin condensation (rossetto et al., 2012). in the early stage of colpoda encystment (2 3 h after the onset of encystment induction), chromatin condensation occurs in a macronucleus, and subsequently many condensed chromatin granules are extruded from the macronucleus to be digested by autophagy (funatani et al., 2010). the camp-dependent phosphorylation of colpoda histone h4 (p37) in encystment-induced c. cucullus may contribute to the chromatin condensation of the macronucleus. the phosphorylated form of g-actin has no polymerizing activity (sonobe et al. 1986; furuhashi et al., 1998), suggesting that the camp-dependent phosphorylation of colpoda actin (p43) in encystment-induced c. cucullus may be involved in the disintegration of actin filaments of the vegetative cell structure. acknowledgements this research was supported by a sasagawa scientific research grant (#24-407) from the japan science society and by a research fellowship from the japan society for the promotion of science for young scientists (#13j08784). references foissner w, stoeck t, agatha s, dunthorn m. intraclass evolution and classification of the colpodea (ciliophora). j. euk. microbiol. 58: 397-415, 2011. funatani r, kida a, watoh t, matsuoka t. morphological events during resting cyst formation (encystment) in the ciliated protozoan colpoda cucullus. protistology 6: 204-217, 2010. furuhashi k, ishigami m, suzuki m, titani k. dry stress-induced phosphorylation of physarum actin. biochem. biophys. res. commun. 242: 653-658, 1998. he l, lemasters jj. dephosphorylation of the rieske iron-sulfur protein after induction of the mitochondrial permeability transition. biochem. biophys. res. commun. 334: 829-837, 2005. kinoshita e, kinoshita-kikuta e, takiyama k, koike t. phosphate-binding tag: a new tool to visualized phosphorylated proteins. mol. cell. proteomics 5: 749-757, 2006. kinoshita-kikuta e, kinoshita e, koike t. phos-tag beads as an immunoblotting enhancer for selective detection of phosphoproteins in cell lysates. anal. biochem. 389: 83-85, 2009. laemmli uk. cleavage of structural proteins during the assembly of the head of bacteriophage t4. nature 227: 680-685, 1970. maeda h, akematsu t, fukui r, matsuoka t. studies on the resting cyst of ciliated protozoan colpoda cucullus: resistance to temperature and additional inducing factors for en-or excystment. j. protozool. res. 15: 7-13, 2005. matsuoka t, kondoh a, sabashi k, nagano n, akematsu t, kida a, iino r. role of ca2+ and camp in a cell signaling pathway for resting cyst formation of ciliated protozoan colpoda cucullus. protistology 6: 103-110, 2009. nakanishi t, ando e, furuta m, kinoshita e, kinoshita-kikuta e, koike t, et al. identification on membrane and characterization of phosphoproteins using an alkoxide-bridged dinuclear metal complex as a phosphate-binding tag molecule. j. biomol. tech. 18: 278-286, 2007. rossetto d, avvakumov n, côté j. histone phosphorylation. a chromatin modification involved in diverse nuclear events. epigenetics 7: 1098-1108, 2012. sogame y, asami h, kinoshita e, matsuoka t. possible involvement of camp and protein phosphorylation in the cell signaling pathway for resting cyst formation of ciliated protozoan colpoda cucullus. acta protozool. 50: 71-79, 2011a. sogame y, kida a, matsuoka t. possible involvement of endocyst in tolerance of the resting cyst of colpoda cucullus against hcl. afr. j. microbiol. res. 5: 4316-4320, 2011b. sogame y, kinoshita e, matsuoka t. ca2+-dependent in vivo protein phosphorylation and encystment induction in the ciliated protozoan colpoda cucullus. eur. j. protistol. 47: 208-213, 2011c. sogame y, kojima k, takeshita t, fujiwara s, miyata s, kinoshita e, et al. protein phosphorylation in encystment-induced colpoda cucullus: localization and identification of phosphoproteins. fems microbiol. lett. 331: 128-135, 2012a. 217 sogame y, kojima k, takeshita t, kinoshita e, matsuoka t. ef-1 α and mitochondrial atp synthase β chain: alteration of their expression in encystment-induced colpoda cucullus. j. euk. microbiol. 59: 401-406, 2012b. sogame y, kojima k, takeshita t, kinoshita e, matsuoka t. identification of differentially expressed water-insoluble proteins in the encystment process of colpoda cucullus by two-dimensional electrophoresis and lc-ms/ms analysis. j. euk. microbiol. 61: 51-60, 2014. sogame y, matsuoka t. evaluation of intracellular ca2+ concentration by fura 2 ratiometry in encystment-induced colpoda cucullus. acta protozool. 52: 51-54, 2013. sonobe s, takahashi s, hatano s, kuroda k. phosphorylation of amoeba g-actin and its effect on actin polymerization. j. biol. chem. 261: 14837-14843, 1986. taylor cv, strickland agr. effects of high vacua and extreme temperatures on the cysts of colpoda cucullus. physiol. zool. 9: 15-26, 1936. yamaoka m, watoh t, matsuoka t. effects of salt concentration and bacteria on encystment induction in ciliated protozoan colpoda sp. acta protozool. 43: 93-98, 2004. 218 dopa-containing proteins in the compound ascidian botryllus schlosseri isj 9: 1-6, 2012 issn 1824-307x minireview ascidian cytotoxic cells: state of the art and research perspectives l ballarin department of biology, university of padua, padua, italy accepted january 11, 2012 abstract ascidian cytotoxic cells are multivacuolated cells, variable in morphology, abundantly represented in the circulation, playing important roles in ascidian immunosurveillance. upon the recognition of foreign molecules, they are selectively recruited to the infection site where they release the content of their vacuoles. their cytotoxic activity closely linked to the activity of the enzyme phenoloxidase (po), a copper-containing enzyme widely distributed in invertebrates, contained inside their vacuoles together with its polyphenol substrata. recent molecular data indicate that ascidian po shares similarities with arthropod propo but, unlike the latter, do not require enzymatic cleavage by extracellular serine proteinases for their activity. possible ways of ascidian po activation are discussed. key words: tunicates; ascidians; cytotoxic cells; phenoloxidase introduction in recent years, the interest towards invertebrate immunity has considerably raised driven by comparative, evolutionary and ecological studies. despite their relying only on innate immunity, invertebrates are capable of complex cellmediated and humoral responses able to guarantee the survival of individuals and, consequently, of the species. two main immunocyte types are shared by invertebrates: professional phagocytes and cytotoxic cells which coordinate their efforts to cope with potentially pathogenic microbes having entered the organism. this paper will focus on ascidian cytotoxic cells, whose importance in immune surveillance, although still not completely clear, is progressively emerging thanks to the efforts of relatively few research groups focusing on a limited number of species. ascidians: a renewed phylogenetic interest since the publication of the paper by delsuc et al. in 2006, changing their phylogenetic position within the phylum chordata to the rank of vertebrate sister group, tunicates have experienced a period of scientific renown testified by the great increase in the interest of scientists towards this group of marine, filter-feeding organisms. ascidians represent ___________________________________________________________________________ corresponding author: loriano ballarin department of biology, university of padua, via ugo bassi 58/b, 35100 padua, italy e-mail: loriano.ballarin@unipd.it the best known and richest in species class of tunicates. embryos give rise to free swimming tadpole-like larvae with a real notochord in their muscular tail, ventral to the neural tube which are replaced, at metamorphosis, by sessile, barrelshaped adults sharing, beyond the external tunic, a fissured pharynx, which occupies most of the volume of the organism, and a ventral endostyle able to secrete the mucous net useful in removing suspended particles from seawater passing through the pharyngeal slits (ballarin and burighel, 2002). adult ascidians also have a well-defined tubular heart and an open circulatory system with hemolymph flowing within lacunae and sinuses inside internal tissues and, in most species, entering the tunic inside vessels derived from the epidermis. hemocytes or blood cells represent an important constituent of the hemolymph, as they are involved in a variety of important biological processes such as wound repair, nutrient mobilization and transport, asexual reproduction and regeneration, waste accumulation, tunic synthesis, allorecognition and, last but not least, immune responses (goodbody, 1974; wright, 1981). the ascidian professional killer cells and their poorly known history as invertebrates, ascidians rely only on innate immunity to cope with potentially pathogenic microbes or molecules having entered their organisms and their immune responses are mainly based on the activity of circulating immunocytes which include professional phagocytes, able to 1 mailto:loriano.ballarin@unipd.it secrete a variety of immune-relevant molecules (fuke and fukumoto, 1993; ballarin, 2008; franchi et al., 2011), and a peculiar cytotoxic hemocyte-type which exert its biocidal activity through the enzymatic oxidation of polyphenol substrata and the induction of molecular damage consequent to the production of quinones and highly reactive oxygen species (ros) (cammarata et al., 1997; ballarin and cima, 2005). in most cases, these cells, which can collectively be called phenoloxidase (po)-containing cells (pocc), assume a typical berry-like shape which justifies the name “morula cells” (mcs) used to indicate them and usually constitute one of the most abundant circulating haemocyte type (sometimes representing more than 50 % of the hemocytes). mcs are characterized by large diameters (10-15 μm) and cytoplasms filled by many vacuoles where the enzyme po resides (chaga, 1980; smith and peddie, 1992; arizza et al., 1995, cammarata et al., 1997; frizzo et al., 2000; shirae and saito, 2000; shirae et al., 2002; parrinello et al., 2003; cammarata et al., 2008). until the end of the 1980s, poccs were simply one of the numerous circulating hemocyte types reported in scientific papers, whose function was not well defined (wright, 1981). they were considered too differentiated to be able to exert any important biological function and represented the research subject of relatively few investigators interested to the unusual acidic content of their vacuoles, which did not share any apparent relationship with the lysosomal machinery, and to their supposed ability to store metals such as vanadium (in most cases) and iron, hence the old names of vanadocytes and ferrocytes with which these cells were also known (endean, 1960; overton, 1966; smith, 1970; pirie and bell, 1984; milanesi and burighel, 1978). this assumption resulted not true in the case of vanadium and the term vanadocytes today applies to cells different from mcs (michibata et al., 1990; nette et al., 1999; yamaguchi et al., 2006). in 1980, in a paper written in russian (which, unfortunately, limited its diffusion), chaga reported, for the first time, that ascidian mcs have po activity, a fundamental step towards the comprehension on the cytotoxic role of poccs, but ten additional years were required for the first clear demonstration of the involvement of mcs in the induction of toxicity and the decisive advancement was made using botryllid ascidians as model organisms and one of the outcome of allorecognition, i.e., the rejection (nonfusion) reaction between contacting, genetically incompatible colonies of the same species resulting in the formation of a series of pigmented necrotic spots along the borders of the facing colonies, as the reference phenomenon (hirose et al., 1990). although known for many years, botryllid allorecognition was studied mainly in terms of morphology and formal genetics (oka and watanabe, 1957, 1960; sabbadin, 1962; mukai and watanabe, 1974; taneda et al., 1985) and papers dealing with the importance of hemocytes as mediators of the reaction appeared only from 1970s (tanaka and watanabe, 1973; taneda et al., 1982a, b); some observations suggesting a role of mcs in the rejection reaction were published in 1980s (taneda et al., 1982a; scofield and nagashima, 1983). the paper by hirose et al. (1990) was the first histological analysis of the rejection reaction which unequivocally indicated mcs played a decisive role in the considered phenomenon. today, we know that, during this reaction, mcs are selectively recruited from the general circulation to the lumen of the peripheral ampullae (the blind, sausage-like termini of marginal vascular vessels) facing the alien colony, then they leave the ampullar tips, cross the ampullar epithelium and enter the tunic where they degranulate and release their vacuolar content which, as reported below, is responsible of the induction of the observed cytotoxicity, a series of events sharing many features with vertebrate inflammation (sabbadin et al., 1992). in b. schlosseri, where the nonfusion reaction has been particularly studied, mcs are activated by the recognition of soluble factors diffusing from the alien colony and express chemotactic molecules recognized by antibodies raised against mammalian proinflammatory cytokines, such as il1α and tnfα, responsible of their selective recruitment inside the ampullar lumen and migration to the tunic (cima et al., 2006; ballarin, 2008). the selective recruitment of poccs and their role in inflammatory and cytotoxic reactions consequent to graft transplantation or injection of nonself molecules was confirmed also in solitary ascidians (cammarata et al., 1995, 1997, 2008; parrinello, 1996; parrinello et al., 1996). the cytotoxic role of poccs, suggested by research on colonial and solitary ascidians, helped to insert in the same frame apparently dissimilar phenomena such as botryllid allorecognition, graft rejection and inflammatory reactions in solitary ascidians, contact reaction in mixed cultures of solitary species hemocytes, all induced by nonself recognition and involving poccs as effector cells (parrinello, 1996). today, we can consider these cells as a sort of immunological sentinel able to recognize foreign molecules and quickly respond with the induction of cytotoxicity (ballarin et al., 2001, 2005). phenoloxidase (po): the ascidian molecular weapon against nonself pos are copper-containing enzymes, widely distributed in invertebrates, with orthodiphenoloxidase (catecholase) activity, able to convert phenolic substrata to quinones which, then, polymerize to form melanin. they exert a role in immune responses related to the induction of cytotoxicity, once released by specific hemocytes, through the production of ros and semiquinones which can either induce oxidative stress or rapidly react with biomolecules altering their functionality. quinones and melanin themselves are toxic: the former can undergo oxidation and generate reactive oxygen species (nappi and vass, 1993; nappi and ottaviani, 2000) or react with -sh groups on biomolecules (kato et al., 1986), whereas the deposition of the latter in nodules of non-self material enveloped by host immunocytes contributes to the formation of the so-called brown bodies which prevent the survival of foreign 2 organisms in their interior (nappi and ottaviani, 2000). the role of po in ascidian cytotoxicity has been clearly demonstrated in both solitary and colonial species with the use of specific inhibitors in appropriate in vitro assays (akita and hoshi, 1995; cammarata et al., 1997, 2008; ballarin et al., 1998, 2005; hata et al., 1998; parrinello et al., 2003). in b. schlosseri, during the rejection reaction, cytotoxicity is the consequence of a severe oxidative stress consequent to the depletion of reduced intracellular thiols (ballarin et al., 2002). the involvement of the enzyme in the nonfusion reaction of other colonial species was also demonstrated (shirae and saito, 2000; shirae et al., 2002). cytochemical analyses indicate that mc vacuoles also contain polyphenols, probably the po natural substrata (cammarata et al., 1997; frizzo et al., 2000; ballarin and cima, 2005). ascidian po: certainties and uncertainties the regulation of po activity and its role in immune defense has been particularly studied in crustaceans, where the enzyme is usually stored, as inactive zymogen (propo), inside the granules of po-containing hemocytes, the morphology of which varies greatly among the different species (aspán et al., 1995; kawabata et al., 1995). the activation of propo to po requires the degranulation of pocontaining cells, the release of the zymogen in the extracellular milieu and its conversion to active po by extracellular serine proteases which are the last components of a finely regulated series of events, collectively called the propo activating system, triggered by the recognition of foreign molecules on the surface of microbial cells (cerenius and söderhäll, 2004; cerenius et al., 2008). recent molecular investigations indicate that the primary, secondary and tertiary sequences of ascidian po show many similarities with those of arthropod hemocyanins and propos, including the organization in three domains and the conservation of the histidines at both the copper-binding sites (immesberger and burmester, 2004). similarly to arthropod pos, having a molecular weight ranging around 70 80 kda (decker et al., 2007), the molecular weight of ascidian pos ranges between 60 and 90 kda (hata e et al., 1998; frizzo et al., 1999; parrinello et al. 2003; cammarata et al., 2008): these values agree molecular data predicting a molecular weight of 92 and 87 kda for ciona intestinalis pos (immesberger and burmester, 1994). however, unlike arthropod pos, which assemble in vivo to form hexamers (decker et al., 2007), electrophoretic data indicate that ascidian po monomers interact each other to form dimers (frizzo et al., 1999; parrinello et al., 2003; cammarata et al., 2008). in addition, despite the absence of incontrovertible data, there is a general consensus that, like arthropods, a propo activating system is present in all the invertebrates species in which po activity has been demonstrated (smith and söderhäll, 1991; cerenius and söderhäll, 2004), ascidians included (smith and söderhäll, 1991; jackson et al., 1993; arizza et al., 1995; ballarin et al., 1998; shirae and saito, 2000; shirae et al., 2002; parrinello et al., 2003; cammarata et al., 2008). however, some doubts that this holds true also for ascidians come from the observation that the activation by exogenous serine proteases is never required for detecting the activity of ascidian po in both hemocyte monolayers and hemolymph or hemocyte lysates (smith and söderhäll, 1991; jackson et al., 1993; arizza et al., 1995; ballarin et al., 1994, 1998; ballarin and cima, 2005; parrinello et al., 2003; cammarata et al., 2008). moreover, po-positive mcs are easily observed in histoenzymatic assays on paraffin sections of colonial ascidians (frizzo et al., 2000; shirae et al., 2002), implying that either the enzyme is constitutively active, at least in part, inside mc vacuoles or it can be easily activated during hemocyte collection or colony manipulation. the reported increase in enzyme activity observed after treatment with exogenous serine proteinases such as trypsin and chymotrypsin (smith and söderhäll, 1991; ballarin et al., 1994, 1998; arizza et al., 1995; cammarata et al., 2003, 2008; parrinello et al., 2003) which also leads to an increase in electrophoretic mobility of the enzyme (parrinello et al., 2003), interpreted as evidences in favor of the presence of a propo in ascidians, can be the result of unspecific effects of the exogenous proteinases which, acting on some cleavage sites and through the removal of enzyme fragment(s), decrease the protein molecular weight, with the consequent increase of its mobility during electrophoresis, and render the copper-binding sites more accessible to the substrate which results in the observed increment of enzyme activity. since, as reported above, also the polyphenol substrata are contained inside the vacuoles of poccs (cammarata et al., 1997; ballarin and cima, 2005), a control of the enzyme activity inside pocc vacuoles is required which can be achieved in at least two possible ways: i) the inhibition of the enzyme inside pocc vacuoles, and ii) the masking of the polyphenol substrata through chemical modifications. conclusions and perspectives the two possibilities suggested above are supported by the fact that it is known that pocc vacuoles contain reducing compounds such as thiols (ballarin et al., 1995) and tunichromes (bruening et al., 1986; oltz et al., 1988; martoja et al., 1994) having inhibitory effects on po (ballarin et al., 1998; hata et al., 1998). tunichromes, in turn, are polyphenolic compounds (bruening et al., 1986; oltz et al., 1988) and, therefore, good candidates as natural substrata of po. the modulation of po activity by polyphenol substrata is an interesting point worth of deeper investigations. as for the second point, it is known that mcs contain sulfur in the form of bound sulfates inside their vacuoles (bell et al., 1982; scippa et al.,1985; frank et al., 1987; ballarin et al., 1995). the presence of the enzyme arylsulfatase inside botryllus mc vacuoles (ballarin et al., 1993) suggests that sulfates may be bound to the aromatic rings of polyphenols. sulfates exert inhibitory effect on ascidian po (hata et al., 1998) 3 and the action of the enzyme may be important in detaching them thus rendering phenols available to po. in conclusion, the importance of ascidian poccs in immune responses is, today, definitively established; however, the regulation of the activity of po, their main cytotoxic tool, and the relationships between polyphenols and po remain controversial and require future investigations for a better clarification of these points. acknowledgements thanks are due to the italian miur for supporting the research. references akita n, hoshi m. 1995. hemocytes release phenoloxidase upon contact reaction, an allogeneic interaction, in the ascidian halocynthia roretzi. cell struct. funct. 20: 8187. arizza v, cammarata m, tomasino mc, parrinello n. 1995. phenoloxidase characterization in vacuolar hemocytes from the solitary ascidian styela plicata. j. invertebr. pathol. 66: 297-302. aspán a, huang ts, cerenius l, söderhäll k. 1995. cdna cloning of prophenoloxidase from the freshwater crayfish pacifastacus leniusculus and its activation. proc natl acad sci usa 92: 939–943. ballarin l. 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abundantly expressed in insects are important modulators of insect survival. the expression of different hsp genes are induced and modulated in insects in response to environmental inputs including abiotic stresses such as heat shock, ultraviolet radiation, chemical pesticides, as well as biotic stresses such as viruses, bacteria, fungi and other insects. this minireview will provide useful information related to the expression of hsp genes in response to abiotic and biotic stressors as well as developmental regulation and modulation of hsp genes involved with insect survival. key words: heat shock protein (hsp); gene expression; abiotic stress; biotic stress introduction insects respond to elevated temperature and to a variety of chemical and physical stresses by a rapid increase in the synthesis of a set of conserved polypeptides collectively referred to as heat shock proteins (hsps). hsps, named according to their molecular weight, such as hsp100, hsp90, hsp70, hsp60, hsp40, small hsp (shsp) and hsp10, are a class of functionally related proteins involved in the folding and unfolding of other proteins. ritossa first reported that heat and the metabolic uncoupler dinitrophenol induced a characteristic pattern of puffing in salivary gland chromosomes in the fruit fly, drosophila busckii (ritossa, 1962, 1963). this discovery eventually led to the identification of hsps which were represented by these puffs. increased synthesis of selected proteins in the cells of drosophila following stresses such as heat shock was first reported in 1974 (tissieres et al., 1974). an enormous literature has now accumulated that describes a wide variety of events in a cell’s response to a wide array of biotic and abiotic sources of stress in a variety of insects (lindquist, 1981; schlesinger, 1990; wu, 1995; garcia et al., 2002, lakhotia, 2011). ___________________________________________________________________________ corresponding authors: liming zhao, walker a jones biological control of pests research unit national biological control laboratory agricultural research service united states department of agriculture p.o. box 67, stoneville, ms 38776, usa e-mails: liming.zhao@ars.usda.gov, walker.jones@ars.usda.gov hsps are found in practically all living organisms, from bacteria to humans (nevins, 1982; wu et al., 1985; walter et al., 1989; marrs et al., 1993; zhang et al., 1998; kanagasabai et al., 2011). more than 7,000 related hsps papers have been published in various journals. table 1 lists hsp genes expressed by insects in response to environmental stresses that have been published during past three decades. this minireview is focused on the expression of hsp genes in response to abiotic and biotic stressors as well as developmental regulation. abiotic stress responses insects respond to elevated temperatures and to chemical and other stresses by an increase in the synthesis of hsps. hsps appear to serve a significant role in the insect’s responses to abiotic stressors such as elevated temperature (garcia et al., 2003, huang et al., 2007; wang et al., 2008; kostal and tollarova-borovanska, 2009; zhao et al., 2009, 2010a), ultraviolet radiation (rangel et al., 2008, nguyen et al., 2009), drought and dehydration (xu et al., 2010, cornette and kikawada 2011), anhydrobiosis (lopez-martinez et al., 2009, gusev et al., 2010, cornette and kikawada, 2011), chemical (planello et al., 2008, 2011), metal (shu et al., 2010; zhao et al., 2010b), nutrient (benoit et al., 2011), injury or adaptation (colinet et al., 2009; kostal and tollarova-borovanska, 2009), hypoxia (michaud et al., 2011) and double stranded rna (benoit et al., 2009, kostal and tollarovaborovanska, 2009; lu and wan, 2011). 93 http://en.wikipedia.org/wiki/protein http://en.wikipedia.org/wiki/dinitrophenol mailto:liming.zhao@ars.usda.gov mailto:walker.jones@ars.usda.gov http://en.wikipedia.org/wiki/bacteria http://en.wikipedia.org/wiki/human 94 table 1 examples of heat shock protein gene expression in insects species heat shock proteins references aedes aegypti hsp26, hsp83, hsp70, hsc70 zhao et al., 2009 aedes aegypti hsp26, hsp83, hsp70 zhao et al., 2010a aedes aegypti, arthropods hsp70 benoit et al., 2011 aedes aegypti, anopheles gambiae, culex pipiens hsp70, hsp90 benoit et al., 2009 apis mellifera hsp70 elekonich, 2009 belgica antarctica shsp*, hsp70, hsp90 lopez-martinez et al., 2008; lopez-martinez et al., 2009 belgica antarctica shsp, hsp70, hsp90 rinehart et al., 2006 bemisia tabaci hsps** mahadav et al., 2009 bemisia tabaci hsp23, hsp70, hsp90 lu and wan, 2011 bombyx mori hsp40, hsp70, hsp90, hsc70*** hong et al., 2010 bombyx mori hsp20.4, hsp40, hsp70, hsp90 ponnuvel et al.,2010 bombyx mori hsps, hsp83, hsp70, hsp 90, hsp 84, hsp62, hsp60, hsp52, hsp33 sosalegowda et al., 2010 calanus finmarchicus hsp21, hsp22, p26, hsp90, hsp70 aruda et al., 2011 chironomus ramosus hsp70 datkhile et al., 2010 chironomus riparius hsp40, hsp90 park and kwak, 2008 chironomus riparius hsp70 planello et al., 2008 chironomus riparius hsp70, hsc70 planello et al., 2011 chortoicetes terminifera hsp40, hsc70, hsp90, hsp20.5, hsp20.6, hsp20.7 chapuis et al., 2011 culex quinquefasciatus hsp70 zhao et al.,2010b drosophila melanogaster hsps lindquist, 1981 drosophila shsps, hsp22 berger et al.,1985 drosophila melanogaster hsp23, hsp27 dubrovsky et al., 1994 drosophila hsp70 zhang and odenwald, 1995 drosophila melanogaster hsp70, hsp26 lohe et al., 1995 drosophila melanogaster hsp23 dubrovsky et al., 1996 drosophila melanogaster hsp22, hsp23, hsp26, hsp27, hsp40, hsp60, hsp67ba, hsp68, hsp70aa, hsc70-1, hsp83 colinet et al., 2009 drosophila hsp90 pflanz and hoch, 2000 drosophila melanogaster hsps takahashi et al., 2011 galleria mellonella hsp90 wojda and jakubowicz, 2007 lepinotus reticulates, liposcelis entomophila hsp70, hsp23, hsp27 guedes et al., 2008 liriomyza huidobrensis hsp90, hsp70, hsp60, hsp40, hsp 20 huang et al., 2007 manduca sexta hsp70/hsc70 rybczynski and gilbert, 1995 penaeus monodon hsp21, hsp70, hsp90 rungrassamee et al., 2010 plodia interpunctella shsp, hsc70, hsp90 shim et al., 2008 polypedilum vanderplanki hsps cornette et al., 2010 polypedilum vanderplanki hsp90, hsp70, hsc70, hsp60, hsp20, hsp23 gusev et al., 2011 pteromalus puparum hsc70 wang et al., 2008 pyrrhocoris apterus hsp70, hsc70 kostal and tollarova-borovanska, 2009 sarcophaga crassipalpis hsp90, hsp70, hsp60, hsp40, shsps michaud et al., 2011 sarcophaga crassipalpis hsps, hsp23, hsp70, hsp90 rinehart et al.,2007 sarcophaga crassipalpis hsp23, hsp70, hsp90 hayward et al., 2005 spodoptera frugiperda hsp70, hsp70 lyupina et al., 2010 spodoptera litura hsp70, hsp90 shu et al., 2010 stratiomys singularior hsp70, hsp68 garbuz et al., 2011 steinernema carpocapsae hsps hao et al., 2009 tribolium castaneum hsp83 xu et al., 2010 * small heat shock proteins. ** heat shock protein family. ***heat shock protein chaperones and modulation of hsp genes involved with insect survival. 95 temperature hsps can protect cells and organisms from thermal damage. in the red flour beetle, tribolium castaneum, the expression of the hsp83 gene could be induced with heat stress at 40 ºc for 1 h in teneral and mature beetles (xu et al., 2010). high temperature can alter gene expression including hsps and other genes in a vector mosquito population using suppression subtractive hybridization (zhao et al., 2009). aeahsp26 and aeahsp83 are important markers of stress and may function as critical proteins to protect and enhance survival of aedes aegypti larvae and pupae (zhao et al., 2010a). different sequential thermal shocks can trigger different mechanisms of cellular protection against stress in the cone-nose bug, panstrongylus megistus, allowing the insect to adapt to different ecosystems (garcia et al., 2002). pretreating insects with a mild heat stressor can induce expression of hsp genes and result in protection from subsequent stresses. this phenomenon has been termed "rapid heat hardening" and is apparently caused by the resolubilization of proteins that were denatured during the stressing episode (huang et al., 2007; manwell and heikkila, 2007; de crecy et al., 2009; elekonich 2009; mahadav et al., 2009; rangel et al., 2010). mild heat hardening improves thermotolerance of the pea leafminer, liriomyza huidobrensis, which significantly increased the expression of mrna levels of hsp70 and hsp20 but at the cost of impairment of fecundity (huang et al., 2007). the induced expression of mrna may play an important role in balancing the functional tradeoff of thermal protection and reproductive impairment (huang et al., 2007). hsps also play important roles of the recovery phase for repairing chilling injuries (colinet et al., 2009). it has been demonstrated that expressed levels of hsp genes of males and females are different in the silverleaf whitefly, bemisia tabaci (lu and wan, 2011). the survival rate of females fed dsrna significantly decreased following exposure to 44 ºc for 1 h, but male survival rate was not significantly affected (lu and wan, 2011). their study also revealed that the optimum mrna expression of hsp genes in females promoted a higher survival rate under heat shock conditions; hsp23 and hsp70 played a key role in heat tolerance in females but not in males, and hsp90 showed no significant role in heat tolerance in either females or males (lu and wan, 2011). antarctic flightless midge, belgica antarctica, has adapted in the antarctica’s terrestrial environment, whose larvae survived the lengthy austral winter to complete their two years life cycle (rinehart et al., 2006). in survival strategies, larvae b. antarctica constitutively upregulates its heat shock proteins and maintain a high inherent tolerance to temperature stress (rinehart et al., 2006). the midge larvae have adopted the unusual strategy of expressing hsps continuously, possibly to facilitate proper protein folding in a cold habitat to enhance thermotolerance (rinehart et al., 2006). ultraviolet radiation solar radiation can be important sources of abiotic stress for herbivorous insects living in close association with plants. greater homeostatic capabilities as revealed at the proteomic level could explain the higher tolerance of the alate morph of the aphid, macrosiphum euphorbiae, to environmental stress and its more stable performance and fitness (nguyen et al., 2009). a tropical species of radiation-tolerant midge, chironomus ramosus, has been shown to express elevated levels of hsp70 mrna and proteins in salivary gland cells of larvae immediately after gamma radiation exposure (datkhile et al., 2011). the expressed hsp70 might be one of the gamma radiation-induced stress proteins required during the early stages of radiation stress management in aquatic midge larvae (datkhile et al., 2011). ultraviolet radiation can affect cross protection. elevated tolerance to uv radiation and heat-shock may be induced in conidia produced by fungi exposed to sublethal stresses other than heat or uv radiation during mycelial growth (rangel et al., 2008). drought dehydration and anhydrobiosis some insects are able to survive the loss of almost all their body water content, entering a latent state known as anhydrobiosis. hsp genes were identified as important up-regulated genes for anhydrobiosis in the sleeping chironomid, polypedilum vanderplanki (cornette et al., 2011). expression of the hsps mrna in response to dehydration in the a. aegypti, anopheles gambiae and culex pipiens is different, and knock-down expressions of the transcripts using rnai have revealed potential functions of the hsps in maintenance of water balance in these mosquito species (benoit et al., 2009). interestingly, it has been demonstrated that in the flesh fly, sarcophaga crassipalpis, expression levels for most of the hsp genes were significantly up-regulated during hypoxia, suggesting an important role for hsp genes in responding to low oxygen environments (michaud et al., 2011). the molecular responses of dehydration, rehydration and overhydration were investigated in larvae of the antarctic midge, b. antarctica (lopezmartinez et al., 2009). using suppression subtractive hybridization, heat shock proteins (shsp, hsp70, hsp90) were found the most responsive to changes in the hydration state in all genes examined (lopez-martinez et al., 2009). the authors speculated that the midge larvae are thus responding quickly to water loss and gain by expressing genes that encode hsps and other proteins contributing to maintenance of proper protein function, protection and overall cell homeostasis during times of osmotic flux, a challenge that is particularly acute in the antarctic environment (lopez-martinez et al., 2009). chemicals and metals expression of hsp70 and other hsp genes was significantly induced after exposure of oriental leafworm moths, spodoptera litura, to zinc. the 96 results also showed that the induced response of s. litura hsp90 to zinc was more sensitive than that of hsp70, whereas the inhibited response of hsp70 was much stronger than that of hsp90 (shu et al., 2010). magnesium is crucial for baculovirus transmission in culex nigripalpus and culex quinquefasciatus larvae. target transcripts up/downregulated by magnesium included hsp70. magnesium can alter gene transcription in a vector mosquito population, and understanding this process can provide insight into the mechanistic role of magnesium in baculovirus transmission (zhao et al., 2010b). chromosomal responses to heat and heavy metal shocks were studied in the trichogen polytene chromosomes of the australian sheep blowfly, lucilia cuprina (joshi and tiwari, 2000). arsenate and mercury, two of the most common toxic environmental chemical pollutants, also induced almost the same set of puffs, suggesting that a common set of gene loci encoding heat shock proteins is responsive to diverse environmental stresses (joshi and tiwari, 2000). the function of hsps and other genes has been recently studied using dsrna interference (rnai) knock down techniques (benoit et al., 2009; kostal and tollarova-borovanska, 2009; papaconstantinou et al., 2010). in addition, heat treatment can be used as a control tactic against stored-product insects such as the psocid, liposcelis entomophila, a major concern in stored grain (guedes et al., 2008). biotic stress responses biotic stress mainly refers to the stress that occurs as a result of damage to plants and animals by other living organisms such as bacteria, viruses, fungi, parasites, beneficial and harmful insects, weeds, and cultivated or native plants. recently, many reports have shown that hsp genes are induced by virus, bacteria, fungi, and insects to confer protection against stressors (selkirk et al., 1987; wojda and jakubowicz, 2007; mahadav et al., 2009; hong et al., 2010; lyupina et al., 2010; rungrassamee et al., 2010; ying and feng, 2011). high population densities are involved in resistance to other ecologically relevant types of stresses (chapuis et al., 2011). parasites stressor-induced tissue damage is involved in various diseases. parasites are undoubtedly a biotic factor that produces stress. parasitoid virulence and host resistance are complex interactions depending on metabolic rate and cellular activity. among other factors, natural control of variably susceptible host populations by aphid parasitoids is more likely at moderate to high temperatures (bensadia et al., 2006). serratia symbiotica is a facultative symbiont of pea aphids (acyrthosiphon pisum) that provides tolerance to heat stress. although s. symbiotica has a major influence on its host's metabolism and resistance to heat, it induces little change in gene expression in its host (burke and moran, 2011). envenomization by the ectoparasitoid, bracon hebetor, on the expression of shsp,hsc70 and hsp90 in the lepidopteran host, the indian meal moth, plodia interpunctella, suggested that upregulation of hsp genes may produce potent factors that have important roles in the mechanism of host-parasitoid relationships (shim et al., 2008). steinernema carpocapsae is an insect-parasitic nematode widely used in pest control programs. hsp genes have been detected in the cdna library of the parasitic phase of s. carpocapsae, and has provided useful information for the study of the parasitic mechanisms exhibited by this parasitoid (hao et al., 2009). cross-protection cross-protection occurred in the honeycomb moth, galleria mellonella, when larvae were exposed to mild heat-shock at 38 ºc, showing an enhanced humoral immune response after microbial infection in comparison to infected animals grown at 28 ºc, and was correlated with the changes in hsp90 protein and increased level of 55kda protein, suggesting hsp90 may play a significant role in converging pathways involved in insect immune response and heat-shock (wojda and jakubowicz, 2007). the whitefly b. tabaci causes tremendous losses to agriculture by direct feeding on plants and by vectoring several families of plant viruses (mahadav et al., 2009). using dna markers and biological characteristics, the b. tabaci species complex was shown to have over 10 genetic biotypes, including the most dominant and damaging b and q biotypes, which differ considerably in fecundity, host range, insecticide resistance, virus vectoring ability and in the symbiotic bacteria they harbor (mahadav et al., 2009). exposing b biotype whiteflies to heat stress changed its gene expression, suggesting that these clear-cut differences between biotype response are due to differences in adaptation of one biotype over another and are partly responsible for observed changes in the local and global distribution of both biotypes (mahadav et al., 2009). pathogens baculovirus expression systems are broadly used for recombinant protein production in lepidopteran cells or larvae. in transgenic silkworms using its heat-shock proteins, the expression levels of the transgenes were found to be under the control of a hsp-promoter driven by a specific activator (hong et al., 2010). it has been shown that the his-tagged baculovirus expression system featuring the chaperone effect hsp70 and hop70 of transgenic silkworms increased the yield of soluble and functional foreign gene products (hong et al., 2010). another study showed that baculoviruses serve as a stress factor that can activate both death-inducing and cytoprotective pathways in infected cells (lyupina et al., 2010). the infection potentiated the response to heat shock by boosting the hsp/hsc70s content in infected cells several-fold http://en.wikipedia.org/wiki/stress_(biological) http://en.wikipedia.org/wiki/native_plant 97 in comparison with uninfected cells (lyupina et al., 2010). addition of a known inhibitor of inducible hsps decreased the rate of viral dna synthesis in infected cells and markedly suppressed the release of budded viruses, indicating the importance of the heat shock response for baculovirus replication (lyupina et al., 2010). there is speculation that an immune response rather than tolerance to these proteins allows, not only for an immediately protection from infection by a variety of pathogens, but also for immune surveillance, an activity of the immune system that eliminates abnormal and damaged cells (schlesinger, 1990). a sudden increase in temperature results in heat shock stress of cultured shrimp such as the giant tiger prawn, penaeus monodon. under heat shock conditions, only hsp90 was induced in all tissues of p. monodon when compared to its untreated level (rungrassamee et al., 2010). the expression levels of hsp70 and hsp90 in p. monodon were significantly increased after a 3-h exposure to the marine bacterium, vibrio harveyi (johnson and shunk) baumann, where the hsp21 transcript was induced later after a 24-h exposure, suggesting putative roles and involvement of hsp genes as a part of an immune response against v. harveyi (rungrassamee et al., 2010). developmental regulation and mutants development hsp genes are developmentally regulated in the different insects. diapause, the dormancy common to overwintering insects, evokes a unique pattern of gene expression. most hsp genes are up-regulated, which appears to be common to diapause in species representing diverse insect orders including diptera, lepidoptera, coleoptera and hymenoptera as well as in diapause that occur in different developmental stages including embryos, larvae, pupae and adult stages (rinehart et al., 2007). one study of the multivoltine silkworm b. mori provides an overview of the differential expression levels of metabolic enzyme and hsp genes in non-diapause and diapause-induced eggs within 48 h after oviposition, confirming the major role of in early embryogenesis (ponnuvel et al., 2010). it has been demonstrated that up-regulation of hsp genes during diapause is a major factor contributing to cold-hardiness of overwintering insects (rinehart et al., 2007). research on the flesh fly, sarcophaga crassipalpis, has shown hsp23 and hsp70 are strongly up-regulated during pupal diapause (rinehart and denlinger, 2000). expression patterns of hsp genes and other genes associated with pupal diapause were reported in s. crassipalpis (hayward et al., 2005). expression of hsp90 gene was downregulated two days after pupariation, while hsp23 and hsp70 transcripts were up-regulated just after the start of diapause, 5 days after pupariation (hayward et al., 2005). although both cold and heat shock evoked elevated expression, the response of hsp90 to heat shock and cold shock remained intact during diapause, which indicates differential regulation of hsp genes during diapause and in response to thermal injury inflicted on diapausing pupae (rinehart and denlinger, 2000). however, expression of most of the hsp genes examined did not vary in response to diapause, perhaps because the diapause of calanus finmarchicus, a key component of marine food webs, is not associated with extreme environmental conditions (aruda et al., 2011). during the early stages of drosophila development, the heat-shock response cannot be induced (fang et al., 2001). it is thought that the adverse effects on cell cycle and cell growth brought about by hsp70 induction must outweigh the beneficial aspects of hsp70 induction in the early embryo (fang et al., 2001). early drosophila embryos are refractory to heat shock as a result of dhsf nuclear exclusion (fang et al., 2001). however, the late embryo can respond to heat shock (fang et al., 2001). steroid hormone in cultured drosophila cells, northern blot analysis showed that shsp genes can be induced by high temperature shock and by exposure to physiological doses of the insect molting hormone ecdysterone (berger et al., 1985). the ecdysone causes dramatic changes in the genetic programs leading to the pupation of d. melanogaster, and regulates developmental changes in transcription and chromatin structure of four small hsp genes (dubrovsky et al., 1996). it has been shown that the ecdysone response element is necessary but not sufficient for full developmental expression of hsp23 in the late third instar and that there is another regulatory element (dubrovsky et al., 1996). in the tobacco hornworm manduca sexta, ecdysteroids coordinate molting and metamorphosis of insects. ecdysteroids are produced by the prothoracic glands under the acute control of the brain neuropeptide prothoracicotropic hormone, which upregulated hsc70 synthesis both translational and transcriptional levels (rybczynski and gilbert, 1995). mutants mutations can be an important means for insects to adapt to various environmental stresses and to survive in new environments. mutants from drosophila had little effect on two measures commonly used to assess heat tolerance, heatknockdown time and heat hardening ability, suggesting that more subtle heat-related fitness components need to be examined for the effects of these mutations (johnson et al., 2011). the quantitative trait locus found in the drosophila melanogaster genome encompassed hsps and 19 heat-responsive genes, suggesting that they were strong candidates for triggering heat resistance, emphasizing the advantages of genome-wide deficiency screening using isogenic deficiency libraries (takahashi et al., 2011). during oogenesis in mutant females of the fruit fly, drosophila virilis, following heat stress, there is an increase in early vitellogenic oocyte degradation and some http://en.wikipedia.org/wiki/charles_johnson http://en.wikipedia.org/w/index.php?title=constantin_auguste_napol%c3%a9on_baumann&action=edit&redlink=1 98 degradation of late-forming egg chambers (gruntenko et al., 2003). gruntenko and his colleague showed that 20-hydroxyecdysone levels change following heat stress in mutant females of d. virilis (gruntenko et al., 2003). other mutants of the mnn1 gene of d. melanogaster are hypersensitive to several stressors and display increased genome instability when subjected to conditions such as heat shock, generally regarded as non-genotoxic (papaconstantinou et al., 2010). menin, a widespread regulator of heat shock gene expression and a critical factor in the maintenance of genome integrity, also links the stress response to the control of genome stability in d. melanogaster (papaconstantinou et al., 2010). conclusions hsp genes are induced and modulated in insects in response to environmental factors including abiotic and biotic stresses. hsp genes are also developmentally regulated, which is important for insects to survive and adapt to their environments. the very widespread occurrence of hsp activity in insects will have a significant bearing on insect adaptability as our climate changes. it may be likely that via hsp activity, many pest and beneficial species will be able to adapt to global warming more than previously thought. changes in environmental conditions can rapidly shift allele frequencies in populations of species linked to evolutionary responses to pollution, global warming and other changes (hoffmann and willi, 2008). new technologies such as microarray, suppression subtractive hybridization and quantitative real-time pcr advances promise to accelerate the development of genetic methods including the genomic function for monitoring insects’ adaptation to environmental change in several ways. hsps may play an important role in biodiversity. many exotic invasive species displace native fauna, while others have little or no effect. the mechanisms for the differences often elude explanation. it is likely that hsps could be playing a role in competition for space and resources. if hsps prove to have a more widespread effect on resistance to parasites and diseases, future methods might be developed that allows increased susceptibility of pests and increased resistance to host defense by natural enemies, which would provide a 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stress. j. med. entomol. 47: 367-375, 2010a zhao l, pridgeon j, becnel jj, clark gg, linthicum kl. identification of genes differentially expressed during heat shock treatment in aedes aegypti. j. med. entomol. 46: 490-495, 2009. zhao l, becnel jj, clark gg, linthicum kj, chen j, jin x. identification and expression profile of multiple genes in response to magnesium exposure in culex quinquefasciatus larvae. j. med. entomol. 47: 1053-1061, 2010b. 129 isj 14: 129-139, 2017 issn 1824-307x research report study of the toxicity of bacillus cereus on silkworm (bombyx mori) and the relevant proteome x dong1, p lü1, w cao2, c zhang1, f zhu1, x meng1, z nie1, s lu1, k chen1 1institute of life sciences, jiangsu university, zhenjiang, 212013 jiangsu, china 2the forth people’s hospital of zhenjiang, jiangsu, china accepted april 11, 2017 abstract twenty-six strains of bacteria were obtained by random sampling previously in our lab. the most pathogenic strains were identified as bacillus cereus through toxicity assessment of silkworm. silkworm larvae were killed within 12 h of injection of b. cereus. the changes in cytoplasm and cell membrane caused by the infection were observed by histopathological examination; these were obvious at 6 10 h after injection of the bacteria, indicating its acute pathogenicity. the differential protein spots of silkworms in the treatment group and control group were detected by two-dimensional electrophoresis. five kinds of silkworm protein were identified in fat body tissue; of these, four proteins were up-regulated and one was down-regulated. thirteen silkworm proteins were detected in midgut tissues; of these, six proteins were up-regulated and expression of seven proteins was down-regulated. after biological analysis, a 14-3-3zeta protein was expressed in both tissues, and several other up-regulated proteins were found to be involved in the anti-inflammatory, immune, phagocytotic and metabolic pathways of the body, indicating that the host had an emergency response to b. cereus injection. the down-regulated proteins were associated with cell biofilm stability, signal transduction and detoxification, respectively. the protein expression was verified by fluorescence quantitative pcr. the results showed that the expression of proteins was consistent with the changes in gene expression. key words: bombyx mori; bacillus cereus; tissue slice; proteome; qpcr introduction bacillus cereus, a gram-positive bacterium, is an aerobic or facultative anaerobic type. according to the whole gene information by referring to genome sequencing data ， b. thuringiensis, b. anthracis and b. cereus all belong to the b. cereus community (rasko et al., 2005). in recent years, b. cereus has been widely used as an insecticide in agricultural production. it can be made by fermentation, but also by direct extraction of the physiologically active substances from b. cereus (guan et al., 2007; ruiu et al., 2015). bacillus cereus contains a variety of virulence factors, and the level of virulence factors in the plasmid is variable (frenzel et al., 2012). non-toxic b. cereus can be used as a drug to improve the intestinal microenvironment, while cereulides, diarrhea toxins hbl, enterotoxin nhe and cytotoxin cytk secreted by ___________________________________________________________________________ corresponding author: keping chen institute of life sciences jiangsu university zhenjiang, 212013 jiangsu, china e-mail: kpchen@ujs.edu.cn toxin-like baculopsis are the main pathogenic virulence factors involved in food poisoning (agata et al., 1996; lindbäck et al., 2004; maria-elisabeth et al., 2016). one study found that vomiting symptoms occur 0.5 6 h after intake of small, circulating and thermo-stable b. cereus. diarrhea symptoms are caused by single or multiple heat-labile enterotoxins in gastrointestinal epithelial cells after 8 16 h of incubation (didier et al., 2016). since 2008, china public health incident report management information system data showed that the number of incidents and the number of victims associated with b. cereus were in the top three among all food poisoning events (chu et al., 2012). hospitals can experience bacillus cereus outbreaks. it has been reported that b. cereus isolated from hospitals is an important pathogen (sasahara et al., 2011) that can cause a variety of clinical infections, such as eye infections (endophthalmitis) (veysseyre et al., 2015), pulmonary infections (pneumonia) (bottone et al., 2010), bacteremia (catheter-related bloodstream infection) (hilliard et al., 2003; inoue et al., 2010), gas gangrene-like infections (bottone et al., 2010), skin and soft tissue infections (cellulitis) (bottone et ../../xinxinxin/desktop/kpchen@ujs.edu.cn 130 fig. 1 toxicity test of bacillus cereus. all physiological lesions of silkworm fifth instar larvae were observed at 4, 10, 12, 24 h. a) pathology of bombyx mori 4 h after injection; b) physiological phenotype of the silkworms 10 h after injection; c) physiological phenotype of the silkworms 12 h after injection; d) physiological phenotype 24 h after injection al., 2010), endocarditis, bone and joint infections (osteomyelitis), urinary tract infections (pyelonephritis) and central nervous system infections (meningitis and brain abscess) (gaur et al., 2001). therefore, evaluation of b. cereus toxicity becomes very important. at present, the silkworm genome has been sequenced and the genetic background is clear. it has a complete set of organs and tissues including brain, heart, liver, kidney, gastrointestinal, nerve, muscle and others that keep the body function properly. therefore, it has become popular as a model animal in replace of mammals in various studies, particularly in toxicity assessment. kaito et al. (2011) isolated 122 strains of bacteria from seafood and injected into silkworm, respectively. it was found that 40 % of the strains were fatal to silkworm, 10 of them were highly pathogenic (kaito et al., 2011). sekimizu et al. (2012) injected staphylococcus aureus and pseudomonas aeruginosa into the silkworm hemolymph and killed the silkworm larvae. hamamoto et al. use silkworm to assess the therapeutic effect of antibiotics and found that the effective dose of antibiotics (ed50) and toxic lethal dose (ld50) were consistent in mice and silkworm (hamamoto et al., 2004). uchida found nosokomycin a, b, c, d and other new antimicrobial substances by silkworm screening (uchida et al., 2010). ogata used diabetes silkworm model to identify hypoglycemic substances separated from food and finally confirmed their effectiveness with mammals (ogata et al., 2008). twenty-six strains of bacteria were isolated from a local hospital in zhenjiang city (jiangsu province, china) by random sampling, and the most toxic strain were identified as b. cereus. in this work, we use silkworm to evaluate the toxicity of b. cereus and investigated proteomic changes in silkworm body in an attempt to find bacteria-responsible proteins. the pathological changes were observed by tissue sections, and the midgut and fat body proteome of silkworm were analyzed. this work set as an example of using silkworm as a model to evaluate toxicity of environmental pathogenic microorganisms and provided theoretical basis for the pathogenicity of the microorganisms at the molecular level, not only for silkworm but also for other insects. materials and methods strains the bacteria strains were kindly provided by the fourth people’s hospital of zhenjiang city in jiangsu province. b. cereus was obtained from the ophthalmology ward. stock cultures were maintained with 20 % glycerol. fresh cultures were obtained by transferring an aliquot to tubes containing lb culture medium (1 % yeast extract, 0.5 % trytone, 1 % nacl, ph 7.0) and incubated overnight (10 12 h) at 37 °c. virulence assay the virulence of the strain was tested according to the silkworm physiology phenotype. silkworm larvae were injected with 5 µl of the bacterial suspension (incubated overnight) at the internode membrane into the hemolymph using a germ-free trace syringe; pressure was applied for 10 sec to stop the bleeding; and an alcohol cotton ball was used to wipe around the wound. the control group was injected with 0.6 % nacl into the hemolymph. after infection, the silkworms were kept at room temperature (25 °c) with normal feeding. on a regular basis (every 2 h), the larvae were checked and the number of deaths recorded. protein extraction silkworm tissue protein was ground into a powder, with the addition of abrasive slurry buffer w1, w2 and dtt, for three minutes. the homogenate was centrifuged (4 °c, 15,000xg for 90 min) to remove the precipitate, the supernatant was harvested and the same volume of tris-phenol was added. the sample was centrifuged to separate the phenol and precipitate (4 °c, 15,000×g for 20 min), the phenol collected, three volumes of ammonium acetate-methanol was added and the sample left to stand overnight. on the second day, the homogenate was centrifuged (4 °c, 15,000×g for 20 min) to remove the supernatant. the homogenate was then centrifuged (76,000×g for 90 min) to remove the tissue debris and the protein fraction was used for further analysis. the clear liquid was decanted and the precipitate washed with cold acetone (0.05 % dtt) until no yellow color remained; a thermostatic vacuum drier was used to obtain the protein, which was stored at -70c. 131 fig. 2 the histopathological changes of fifth instar larval midgut tissue under microscopic examination (10×). infected with bacillus cereus and uninfected silkworm midgut tissue, respectively, was made into paraffin sections. the nucleus was stained by hematoxylin to give a blue-violet color; cytoplasm was stained pink; a) microscopic observation of midgut tissue cells 2 h after infection; control group (0.6 % saline, above), treatment group (bacterial suspension, below); b) microscopic examination of midgut tissue 4 h after infection; c) microscopic observation of midgut tissue cells 6 h after infection. the cells began to show pathological changes; d) microscopic observation of midgut tissue 8 h after infection; e) the midgut tissue cells 10 h after infection. cells show significant pathological changes. histopathological examination histological analysis was performed as described previously. the larvae were injected with bacteria, and then the posterior midgut of b. cereus-infected and control larvae was injected with normal saline. the tissues were fixed in pbs 4 % paraformaldehyde overnight at 4 °c. tissue specimens were subjected to conventional dehydration followed by paraffin embedding and sectioning (4 μm thick). the slides were deparaffinized with dimethylbenzene and rehydrated through an ethanol series. hematoxylin and eosin (he) staining was performed to examine the pathological changes of the midgut. sections of the midgut were observed using a leica dfc280 light microscope and analyzed using the leica q win plus v3 image analysis system (leica micros imaging solutions ltd.; cambridge, uk). two-dimensional (2d) gel electrophoresis the mixed, labeled protein samples were diluted with lysis buffer to 120 μl. isoelectric focusing (ief) was done using an ipgphor focusing system (bio-rad, hercules, ca, usa) according to the manufacturer’s instructions. ipg strips (linear ph 4 7 gradient; 17 cm) were run at 20 °c. the protein sample (1.4 mg) was first rehydrated in buffer using active rehydration (13 h at 50 v) in a total volume of 400 l, after which, ief was done with a voltage gradient of 250 v (0.5 h), 1000 v (1 h) and 8000 v (5 h), and then continued for a total of 60 kvh at 10 kv. the focused strip was equilibrated for 15 min with equilibration solution (6m urea, 0.375 m tris-hcl, ph 8.8, 20 % (v/v) glycerol, 2 % (w/v) sds and 0.002 % (w/v) bromophenol blue) containing 2% (w/v) dtt and for another 15 min with the same solution containing 2.5 % (w/v) iodoacetamide. sds-page was done using 12 % gels at 30 ma (constant) until the dye front reached the bottom of the gel. the gel was stained with 0.1 % coomassie brilliant blue g-250 (bio-safetm, bio-rad) and photographed with a digital single lens reflex camera (nikon d5000) and a standard lens (af 50 mm f/1.4d). protein identification by mass spectrometry in total, 306 protein spots from the midgut and 433 protein spots from the fat body detected in the 2d gel by coomassie brilliant blue staining were manually excised, transferred to eppendorf tubes and then destained, reduced, alkylated and digested with trypsin. after digestion, the protein peptides were extracted twice in 0.5% trifluoroacetic acid (tfa) and 2.5 % tfa/50 % acetonitrile. a 1-µl sample was then spotted onto an mtp anchor chip board (bruker, billerica, ma, usa) and analyzed with a maldi-tof mass spectrometer. peptide mass fingerprints of 1,000 4,000 da were obtained. standard peptides were used as external standards. the peak value of the trypsin peptide and matrix were used as internal parameters. bioinformatics analysis open reading frames were identified by using an in-house program based on ‘getorf’ from emboss. gene annotation was done by blastp searching against the swiss-prot and genbank databases with an e value cutoff of 1e 3. to identify the proteins, the ms fingerprints were screened 132 against the ncbinr and swiss-prot sequence databases with the search engine mascot. unidentified proteins were searched in a local database constructed specifically for this purpose. fixed modification was set to carbamidomethyl (c) and variable modification was set to be oxidation (m). the mass tolerance was set as 0.2 to 0.8 da. the species were set as chordata and a ci score > 62 was considered to be a positive match. the resulting protein sequences were aligned with interpro scan to obtain the gene ontology (go) identifications and the collected information was then analyzed using wego. rna extraction and quantitative rt-pcr for real-time pcr experiments, total rna was isolated from the tissues of silkworm (midgut and fat body). the reaction used 0.2 µl upstream and downstream primers (10 tendency/l); 2 µl template; 6.8 µl ddh2o, in 20 µl total system. fluorescence quantitative pcr reaction conditions were as follows: 95 c 5 min; 95 c 10 s; 60 c 30 s; 95 c 15 s; 60 c 60 s; 95 c 15 s, repeated for 45 cycles. three repetitions were used in each group. results virulence assay of the bacillus cereus strain all five instar larvae were injected beneath the skin, and all died within 12 h after injection of bacillus cereus bacterial fluid. figure 1 shows the physiological phenotypic changes at 6 h, 10 h, and 24 h after the infection of larvae with bacterial liquid. the results showed that the bacteria had strong lethality to silkworm. after bacterial infection, the larvae started to eat less, moved slower, exhibited vomiting and diarrhea and the body began to swell (fig. 1a); part of the larval head became green, and all stopped eating at 10 hours (fig. 1b). twelve hours later, the silkworm body blackened slowly and became soft and some larval limbs become stiff after the body swelled; all larvae died (fig. 1c). after 1 day of observation, the larvae body became black, followed by the outflow of black liquid (fig. 1d). histological study of the larval midgut cells infected with bacillus cereus the midgut tissue paraffin sections were made after b. cereus infection of the silkworms and microscopic examination followed he staining. we compared the pathological changes in midgut cells after 2 10 h in the control group and the treatment group. during this period, no obvious lesions were observed in the nucleus, but pathological changes of the midgut cells were observed (figs 2a, b). six hours later, compared with the control group, the infected midgut cell folded structure was loose, the cell wall was destroyed and the columnar cells became smaller (figs 2c, d, e). 2d gel electrophoresis of the proteome of the fat body pdquest 7.1 two-dimensional image analysis software was used for the analysis; a total of 306 protein points were detected, as seen in figure 3a. the software automatically identifies the protein spots on the map, and then automatically identifies the points for manual editing; some points not recognized by the software can be added and some points misrecognized can be deleted. the statistical difference value for the anova was set as ≤0.05, and the expression of protein variation ratio was set fig. 3 two-dimensional electrophoretic (2-de) map of bombyx mori fat body protein. a) 0.6 % saline control group; b) proteins in treatment group after injection with bacillus cereus. numbers indicate the coding of differentially expressed proteins. 133 table 1 proteins identified by maldi-tof mass spectrometry and mascot spot no. protein name protein id species pia/mwb p value fold change seq cov (%)c score 1 endoribonuclease homolog gi|512922547 bombyx mori 5.70/39943 3.50e-07 0.4 19% 88 2 low molecular 30 kda lipoprotein gi|827538302 bombyx mori 5.98/30367 2.00e-02 5.4 54% 161 3 proteasome subunit beta type-6 gi|512892807 bombyx mori 5.89/24430 4.50e-03 3.2 50% 139 4 proteasome subunit alpha 3 gi|114051245 bombyx mori 5.27/28354 2.20e-10 3.9 42% 137 5 14-3-3 protein zeta gi|114050901 bombyx mori 4.90/28266 1.10e-06 4.6 49% 150 a isoelectric point b molecular weight c sequence coverage as ≥1.8, giving 21 different protein spots. finally, five differentially expressed proteins were obtained. sample handling and mass spectrometry identification were carried out; one stage and two stage mass spectrometry were analyzed, and five silkworm protein spots were detected (table 1). protein spot no.1 is the protein whose expression was down-regulated after inoculation of bacteria, protein spots nos. 2, 3, 4, and 5 represent up-regulated protein expression after inoculation of bacteria (fig. 3b). 2d gel electrophor esis of the proteome of the midgut in total, 433 protein spots (fig. 4a) were detected by pdquest 7.1 two-dimensional image analysis software. finally, 26 differential protein spots were obtained. a primary mass spectrometry and secondary mass spectrometry were performed to detect 13 silkworm protein spots (table 2). protein numbers 2, 4, 5, 6, 8, 12, and 13 were proteins whose expression was down-regulated, and the protein spots 1, 3, 7, 9, 10, and 11 were up-regulated after inoculation with bacteria. the results showed that a total of seven proteins were down-regulated after inoculation, and six proteins were up-regulated after inoculation (fig. 4b). the pdquest 7.1 two-dimensional image analysis software was used for analysis, and a total of 433 protein points were detected, as can be seen in figure 4a. the software automatically identifies the protein spots on the map, and then automatically identifies the points for manual editing; finally, 13 differentially expressed proteins were obtained. sample handling and mass spectrometry identification were carried out; after the two stage mass spectrometry results were analyzed, 13 silkworm protein spots were detected (table 2). protein spots nos. 2, 4, 5, 6, 8, 12, and 13 were down-regulated after inoculation of bacteria, and nos.1, 3, 7, 9, 10, and 11 represent the up-regulated proteins after inoculation of bacteria (fig. 4b). verification of observed differential protein levels at the gene expression level using qpcr on fat body and midgut the infected and uninfected fat bodies and midgut tissues from the fifth instar larval silkworms at different time points were selected and identified by the template cdna. the differential proteins up-regulated in the two tissues were verified by fluorescence quantitative pcr (fig. 5). the results showed that proteasome (no. 3), proteasome (no. 4), and 14-3-3 protein zeta (no. 5) were significantly up-regulated at 6 h in fat tissue compared with the control group, which is consistent with the electrophoresis results. lipoprotein (30 kda, no. 2)was slightly down-regulated at 6 h, and there was a difference with the results of electrophoresis. in the midgut tissue, beta-tublin (no. 1)，glutamine synthetase (no. 9), tubulin (no. 3), fumarylacetoacetate hydrolase (fah, no.7), 14-3-3 protein zeta (no.10) were significantly up-regulated at 6 h, which is consistent with the protein level. the gene expression level of proteasome (no.11) was different from the protein level at 6 h. we speculate that the detection sensitivity of the fluorescence quantitative pcr at the gene level is relatively high, and the detection of protein has many interfering factors, resulting in this difference. the results are shown below. 134 fig. 4 two-dimensional electrophoretic (2-de) map of bombyx mori midgut protein. a) control group (0.6 % saline); b) proteins in the treatment group after injection with bacillus cereus. numbers indicate the coding of differentially expressed proteins discussion our results showed that hypodermic inoculation of bacteria into bombyx mori can result in strong pathogenicity. symptoms were observed on silkworm similar to those seen on human patients, including vomiting and diarrhea. in addition, silkworm stopped eating with body became stiff and blackened. its head became green, followed by outflow of black liquid. our experiment successfully demonstrated that silkworm can be used as a model organism for evaluating the toxicity of pathogenic microorganisms. the results indicate that the bacteria strain b. cereus may have strong toxicity to human as well, suggesting that public places like hospitals should pay particular attention to b. cereus infection in wounds. studies have indicated that enterotoxin h1yii can be secreted by b. cereus and that it can be integrated with the cell membrane without receptors (sinev et al., 1993; budarina et al., 1994; andreeva et al., 2006), thus infecting the host via adhesion. the histopathological result showed that the pathological changes in the cytoplasm and cell membrane can be seen 2 h after infection of b. moris, but there was no change in the nucleus, which implies that the bacteria mainly work on the cell membrane and have acute pathogenicity. according to the result of 2d electrophoresis, there is a great difference in the level of expression shown by five kinds of b. moris after injection of bacteria into the fat body tissue. the down-regulated protein was endoribonuclease (no. 1), and the up-regulated proteins were lipoprotein (no. 2) 30 kda, proteasome (no. 3), proteasome (no. 4), and 14-3-3 protein (no. 5) (table 1). proteasome (no. 3, no. 4) is involved in many key cellular functions and plays an important role in the cellullar immune response, regulation of periodicity, cell growth and apoptosis, cell signal transduction, and ion channels in insects. the extracellular protein is mainly degraded via lysosomes (yang et al., 2015). once b. moris has been infected by b. cereus, the hosts must digest the exotic protein because it is not necessary for their bodies. the up-regulation of the proteasome is the result of adaptation to such change. the 14-3-3 protein zeta is a critical apoptosis inhibition factor that can inhibit apoptosis at various levels through different mechanisms; for instance, by combining with bad, it can prevent the integration of bad and anti-apoptotic factors bcl-2 and bcl-xl, thus inhibiting apoptosis (subramanian et al., 2001). meanwhile, 14-3-3zeta protein is also an important adaptor protein in the signal transduction pathway (chen et al., 2002). analysis of the pathway has verified that the protein is correlated to the mapk pathway, and 14-3-3zeta can facilitate the activation of the erk/mapk signal transduction pathway and adjust the cellular growth, differentiation, stress adaptation to the environment, inflammatory reaction and many other important cell physiological and pathological processes through the enhancement of rkip phosphorylation (dabbous et al., 2011; huan et al., 2012). for b. moris injected with b. cereus, the bacterial solution is toxic and may cause inflammation and an emergency reaction of their bodies, thereby leading to the rise in protein expression. other research implies that the probability of having glioma will increase significantly owing to the up-regulated expression of 14-3-3zeta protein, which can increase the probability of apoptosis by nearly 8 times (niemantsverdriet et al., 2008). in recent years, research has shown that 14-3-3 zeta is relevant to diseases including diabetic 135 table 2 proteins identified by maldi-tof mass spectrometry and mascot spot no. protein name protein id species pia/mwb p value fold change seq cov (%) c sco re 1 beta-tubulin gi|112983318 bombyx mori 5.53/50638 4.50e-10 2.8 61% 286 2 vacuolar atp synthase catalytic subunit a gi|148298878 bombyx mori 5.27/88558 2.00e-02 0.7 53% 311 3 tubulin alpha chain gi|1729841 bombyx mori 4.96/50558 1.90e-02 1.7 40% 103 4 uncharacterized protein loc101744771 gi|512936517 bombyx mori 5.64/49702 3.30e-02 0.6 18% 84 5 xaa-pro dipeptidase isoform x1 gi|512915099 bombyx mori 6.36/61701 1.90e-02 0.9 17% 87 6 ehdomain-containing protein 3 isoform x2 gi|512911643 bombyx mori 6.74/60980 5.40e-02 0.8 36% 162 7 fumarylacetoacetate hydrolase isoform b gi|87248329 bombyx mori 6.68/70059 1.90e-02 2.7 3% 67 8 proteasome subunit alpha gi|512891246 bombyx mori 5.61/30776 1.80e-06 0.8 9% 66 9 glutamine synthetase 2 cytoplasmic gi|512921350 bombyx mori 6.64/51608 2.10e-02 2.8 6% 78 10 14-3-3 protein zeta gi|114050901 bombyx mori 5.90/38266 2.70e-02 4.3 47% 124 11 proteasome subunit alpha type-4 gi|827562552 bombyx mori 6.09/26776 3.30e-02 3.7 8% 63 12 proteasome alpha 3 subunit gi|114051245 bombyx mori 5.59/28354 1.60e-04 0.3 24% 93 13 annexin ix isoform c gi|162952017 bombyx mori 5.00/36068 1.90e-02 0.6 45% 166 aisoelectric point; bmolecular weight; csequence coverage nephropathy, depression, and senile dementia. however, the related mechanisms have not yet been fully demonstrated. the excessive expression of 14-3-3zeta protein in breast cancer and liver cancer is a potential marker in the diagnosis of tumors (luo et al., 2016). the lipoprotein of low molecular weight is a unique protein that exists in the fat body of b. moris, it plays a critical role in inhibiting programmed cell death and immunologic defense of b. moris during embryogenesis (van et al., 1993). in 2004, kim et al. expressed the 30k protein of b. moris in sf9 cells by making use of the baculovirus expression system; they found that the survival rate of sf9 cells expressing the 30k protein was significantly higher than that of the control group (yu et al., 2013). after infection with b. cereus, b. moris may show an immune response. endoribonuclease is of great importance to the survival ability and nervous system activity of b. moris. research has shown that the flight performance, life and nervous system of drosophila are greatly affected by cg3303 protein 136 fig. 5 fluorescence quantitative polymerase chain reaction (qpcr) for fifth instar larval silkworm fat body (fig. 5a) and midgut (fig. 5b). the arrow indicates the differential protein spots. the protein spots were obtained following treatment with saline (control group) or bacillus cereus. “black” indicates the differential protein fluorescence quantitative pcr results of cdna template from the control group of silkworm tissues, and “light” represents the results of the silkworm pcr in the treatment group. the experiments were repeated three times. statistically significant differences (mean ± s.d, p <0.05) were detected when the results were compared with the control group. a) b) 137 (laneve et al., 2017). in b. moris infected with b. cereus, body degradation ability and nerve conduction can be affected. this result is similar to the findings of the research on drosophila. the protein go analysis of the differentially expressed proteins showed that proteins in the fat body tissue are much involved in biological processes and that metabolic processes account for a large proportion of the biological processes. in the midgut tissue, 13 proteins of b. moris show significant differences in the level of expression after injection with the bacteria, when compared with the control group. the expression of seven proteins was down-regulated after injection with the bacterial solution, and that of six proteins was up-regulated under the same conditions (fig. 4). the down-regulated proteins were atp synthase (no. 2), an uncharacterized protein (no. 4), dipeptidase (no. 5), ehdomain-containing protein (no. 6), proteasome (no. 12), proteasome (no. 8), and annexin (no. 13). the up-regulated proteins were beta-tubulin (no. 1), fumarylacetoacetate hydrolase (no. 7), glutamine synthetase (no. 9), tubulin (no. 3), 14-3-3 protein (no. 10), and proteasome (no. 11) (table 1). beta-tubulin (1) and tubulin (3) are two proteins related to phagocytosis, which is one of the most basic defense mechanisms in the body of insects. the literature has shown that phagocytosis can be stimulated once the host is invaded by some viruses or bacteria (klerk et al., 2017). as an immune response of bodies, the protein expression quantity will be up-regulated and the phagocytosis will be enhanced. fumarylacetoacetate hydrolase (7)-fah is an enzyme that plays a key role in degradation in the paf pathway (phaneuf et al., 1995). it involves in the degradation of tyrosine and has the metabolic detoxification capability in vivo, so as to clear accumulated toxic metabolites in the blood or tissues and prevent damage to organs like the liver and kidneys (li et al., 2017). according to a review of the literature, hydrolase of b. moris can be activated in cases of body trauma, external induction or metamorphosis, thereby improving the activity of clusterin, which exists in vivo (xu et al., 2012). glutamine synthetase (no. 9) can catalyze the reaction of glutamic acid in vivo to generate glutamine, and it is a key enzyme in the metabolism of glutamic acid. the enzyme is involved in the maintenance of normal neurotransmission as well as the repair of damaged nerves. the heteromorphosis of this enzyme will result in various neurocognitive diseases. research has indicated that glutamine synthetase is indirectly involved in immune defense to resist infection with pathogenic microorganisms (zong-jun et al., 2011). the up-regulated expression of both proteins suggests that there is emergency response when b. moris is infected by bacteria. protein 14-3-3zeta (no.10) was detected in the fat body tissue of b. moris, and it plays a critical role in the immune response. therefore, this protein may be used as a molecular index in future research and be applied to the research of human bacterial infection. among the down-regulated proteins, annexin (no. 13) is a calcium-dependent phospholipid binding protein, which participates in various activities relevant to biological membrane structure and functions, thus functioning in the stability of the biological membrane, formation of phagocytic vacuoles, and ion transport. it can become integrated with a protein named s100a10 and facilitate membrane fusion (shyu et al., 2017). the down-regulation of protein expression directly affected the stability of the biological membrane structure of the midgut cells of b. moris, which is consistent with the observation of the tissue slices. in addition, the cell phagocytosis and signal transduction of b. moris as well as their lifespan can be influenced. the ef-hand domain pair structural domain of ef domain-containing protein (no. 6) is mainly involved in the integration of metal ions. it is related to the increase of calcium ion binding protein. the down-regulation of this protein may have an impact on the cell membrane bioelectric potential and nerve conduction in b. moris, thus further affecting the signal pathways of b. moris as well as their growth (lewit et al., 2014). based on the go analysis results for the differentially expressed proteins, many proteins are related to biological processes and cellular elements. regarding the latter category, there are many proteins related to cytoplasmic elements. b. cereus can cause a series of obvious pathological changes in silkworm and serious destruction of midgut cells, affecting a variety of silkworm metabolic pathways and signal transduction pathways, with strong acute lethality. these proteins with significant expression level is vital to study the toxicity of b. cereus. the genes can be used as the early warning genes, which may be the root causing silkworm death and can be important to the development of antimicrobial agents and biological control of sericulture. acknowledgement this research was supported by the project of the priority academic program development of jiangsu higher education institutions, the national natural science foundation of china (no. 31572467). references 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regulation of insect innate immune by ubiquitin-proteasome system. j. environ. entomol. 37: 634-644, 2015. yu w, ying h, tong f, zhang c, quan y, zhang yz. protective effect of the silkworm protein 30kc6 on human vascular endothelial cells damaged by oxidized low density lipoprotein (ox-ldl). plos one 8: e68746, 2013. zong-jun du, liu y, jiang j. relationship between glutamine and immune defense functions of aquatic animals: a review. fisheries sci. 12: 616-619, 2011. https://www.ncbi.nlm.nih.gov/pubmed/?term=veysseyre+f+and+2015 microsoft word isj310 isj 10: 151-161, 2013 issn 1824-307x research report reversible inhibition of reproduction during regeneration of cerebral ganglia and celomocytes in the earthworm dendrobaena veneta j okrzesik1, n kachamakova-trojanowska2, a jozkowicz2, aj morgan3, b plytycz1 1department of evolutionary immunology, institute of zoology, jagiellonian university, krakow, poland 2department of medical biotechnology, faculty of biochemistry, biophysics, and biotechnology, jagiellonian university, krakow, poland 3cardiff school of biosciences, main building, cardiff university, cardiff cf10 3us, wales, uk accepted november 25, 2013 abstract earthworms may be subjected to mechanical/chemical stimuli and/or sub-lethal predator attacks leading to the extrusion of celomocytes and/or loss of body parts; thus, regeneration of cells, tissues and organs has adaptive value. the aim of present study on the lumbricid earthworm dendrobaena veneta was to determine the interactive effects of celomocytes and the brain on the regeneration of either system after experimental depletion or extirpation, and to assess the effects of such treatments on reproductive performance. decerebration was achieved either by amputating the first six anterior segments, or by surgery; celomocyte depletion was achieved by a standard electro-stimulation procedure. celomocytes (amebocyte and eleocytes, respectively) were counted by hemocytometry, and riboflavin content in celomocyte lysates measured by spectrofluorimetry. the main findings were: (i) d. veneta regenerated anatomically intact brain, including neurosecretory cells, within 10 18 weeks after its removal plus celomocyte depletion (i.e. dual treatment); (ii) amoebocyte counts recovered to control levels by 10 weeks after extrusion treatment alone, but were still lower (60 %) than in controls at 18 weeks after dual treatment; eleocyte recovery after electro-stimulation alone was slow, reaching control levels only after 18 weeks, and was further retarded (31 % of controls at 18 weeks) by brain extirpation; (iii) riboflavin content was lower than controls only in the dual-treatment worms at 5 weeks; riboflavin content relative to eleocyte numbers was initially higher than controls in both treatment groups; this index was restored to control levels by 18 weeks in the electro-stimulation only treatment whilst recovery was somewhat retarded in the brain-extirpated group; (iv) celomocyte depletion treatment alone slightly impaired reproductive output, whilst brain removal had pronounced and protracted inhibitory effects. the observations engender the hypothesis that brain-derived neurosecretions and immune-competent celomocytes act in tandem to modulate neural regeneration and reproduction. key words: dendrobaena veneta; immune-neuroendocrine interactions; celomocytes; riboflavin; cocoon production; regeneration introduction interest in earthworms as metazoan models for studying fundamental aspects of tissue regeneration date back well over a century to the series of seminal publications by the nobel prize-winning geneticist thomas hunt morgan (morgan, 1897). tissue regeneration, or the restoration of lost body parts as it has been defined (bely and nyberg, 2009), ___________________________________________________________________________ corresponding author: barbara plytycz department of evolutionary immunology institute of zoology jagiellonian university gronostajowa 9, pl 30-387 krakow, poland e-mail: barbara.plytycz@uj.edu.pl whilst commonplace within members of the annelida, is extensively variable within the phylum with some enchytraeid oligochaetes able to regenerate complete organisms from fragments comprised of a few segments (takeo et al., 2010), other taxa amongst the oligochetes and polychetes possessing different degrees of ability to regenerate anterior and posterior segments, with posterior regeneration particularly prevalent, and hirudinea apparently incapable of any segment replacement (bely, 2006). earthworms are anatomically more intricate than other favoured model organisms, such as sponges, hydra and flatworms, since they are metamerically-segmented coelomates with a closed circulation and a well-developed nervous system. 151 fig. 1 the regeneration of surgically-extirpated cerebral ganglion in d. veneta. (a) whole cerebral ganglion (cg) experimentally removed through a small dorsal incision at time 0; (b) micrograph of a transverse section through the anterior segments of d. veneta fixed 18 weeks after cg extirpation; the plane of the section, stained with paraldehyde fuchsin, corresponded with the position of the fully regenerated cerebral ganglion (cg); spg subpharyngeal ganglion. (c) a magnified view of the regenerated cerebral ganglion (cg) depicted in fig. 1b; note the distinct cluster of neurosecretory cells (nsc). bars = 1 mm. earthworms are relatively easy to culture and manipulate in the laboratory, and knowledge of particular aspects of their physiology and molecular genetics is substantial and burgeoning (stürzenbaum et al., 2009). thus, they provide several advantages in terms of understanding the mechanisms underlying the regeneration of tissues and organs in a complex, albeit accessible, organism (cho et al., 2009). neurosecretions released by the elements of the ventral nerve cord and cerebral ganglion (‘brain’) are evidently involved, either directly or indirectly, in regulating not only earthworm tissue regeneration but also other vital functions such as reproduction and electrolyte balance (alonso-bedate and sequeros, 1985). the remarkable ability of the earthworm to regenerate transected or ablated giant axons (birse and bittner, 1981), and relatively quickly (70 to 80 days) and completely the cerebral ganglion, has drawn the attention of neuroscientists seeking convenient models for studies on neuronal regeneration (lubics et al., 2002; csoknya et al., 2003). during the early phases of cephalic regeneration, when the earthworm lacks a brain and is unable to consume food, there is evidence of gross regression of developing gametes (jamieson, 1981). free-floating celomocytes migrate early to the locus of wounding in earthworms, and are involved in wound plug formation and subsequent tissue remodelling by virtue of their phagocytic activities and delivery of nutrients and growth-stimulating molecules (jamieson, 1981; varhalmi et al., 2008). a number of neuropeptides have been immunolocalised within the celomocytes of the earthworm eisenia fetida (wilhelm et al., 2006). the observation (somogyi et al., 2009) that pituitary adenylate cyclase-activating polypeptide (pacap)like proteins not only occurs in earthworm celomocytes but it is enriched in these immunecompetent cells located in the vicinity of regenerating tissues is particularly noteworthy. pacap in vertebrates protects cells within the central nervous system against stress-induced apoptosis (vaudry et al., 2002), thus providing support for the notion (somogyi et al., 2009) of a functional link between the earthworm nervous system and celomocyte-mediated regenerative activities. indeed, there is now a substantial body of evidence supporting the general principle of intimate interactions between the neuroendocrine and innate defense systems in invertebrates and vertebrates (cohen and kinney, 2007). 152 samuel et al. (2012) recently detected autofluorescent cells in the regeneration blastema of the earthworm eudrilus eugeniae, and that riboflavin (vitamin b2) was the main source of the cytoplasmic fluorescence. moreover, these authors also observed that injected riboflavin promoted regeneration in a dose-dependent mode. whilst it is plausible that earthworms may contain more than one type of riboflavin-containing autofluorescent cells, it has been conclusively demonstrated that one cohort of celomocytes, the eleocytes (wandering chloragocytes), are particularly riboflavin-rich in some lumbricid species such as compost-inhabiting e. fetida and dendrobaena veneta (koziol et al., 2006; cygal et al., 2007; plytycz and morgan, 2011; sulik et al., 2012; rorat et al., 2013). it is significant that earthworms possess the temperature-dependent capacity to regenerate their grossly depleted celomocyte community after experimental extrusion, with amoebocyte numbers recovering within a few weeks whilst eleocyte numbers were fully recovered much later, depending on the ambient temperature (homa et al., 2008; klimek et al., 2012). these reported differences in restoration kinetics between the two morphologically and functionally distinct celomocyte types is entirely consistent with the differences in proliferative behaviour of amebocytes and eleocytes after wounding and tissue grafting (parry, 1976). both the loss of celomocytes and body segments may occur under natural conditions due to mechanical stimuli, autotomy of toxinor waste product-laden tissues, or sub-lethal predation (bely and nyberg, 2009), thus the regeneration of cells and tissues in earthworms has potential adaptive value. just as there has been a long-term interest in tissue regeneration in earthworms, there has also been considerable research activity centered on the impact of environmental toxicants on earthworm celomocytes (plytycz et al., 2007; plytycz and morgan, 2011) whose momentum continues apace into the nanotechnology age (hayashi and engelmann, 2013). despite these efforts much remains to be known and understood about the interactions within the neural-immune axis in the context of cell proliferation and regeneration. therefore, the primary aim of the present study was to determine the effect of brain extirpation on the temporal recovery of amebocyte and eleocyte populations in d. veneta after electro-stimulated expulsion. these changes, including the riboflavin content of eleocytes, were tracked in relation to the morphology of the regenerating brain. a subsidiary aim was to monitor the effects of brain removal and regeneration on reproductive output in terms of cocoon and hatchling counts, given that neurosecretions probably regulate restorative growth and reproduction at some level in earthworms (alonso-bedate and sequeros, 1985). materials and methods earthworms adult specimens of d. veneta from a commercial supplier (ekargo, krepa slupska) were reared at the institute of zoology, jagiellonian university, krakow under controlled laboratory conditions (16 19 °c; 12:12 ld, 25 % humidity). they were kept in plastic boxes with perforated lids in the soil from a commercial supplier (ppuh biovita, tenczynek, poland). the worms were fed ad libitum on boiled nettle (urtica dioica) leaves and the commercial soil of proper humidity was exchanged at 4 6 week intervals to avoid its intoxication by metabolic products. experimental scheme earthworms of similar body weights (0.7 0.9 g) were selected from the stock culture and divided into control and experimental groups. within each experimental series the number of worms and soil content in the control and ‘treatment’ boxes were the same, i.e., 10 worms per box with 400 g of soil in preliminary experiments, and 15 worms per box with 500 g of soil in three experimental series of the main experiment. control groups consisted of untreated worms (group ‘c’). worms whose celomocytes were extruded by mild electrostimulation (4.5 v, 30 sec), as described below, were designated the ‘v’ treatment group. pilot experiments in a pilot experiment, groups of electrostimulated worms had either 6 anterior or 6 posterior body segments amputated by transection; these treatment groups were designated ‘va’ and ‘vp’, respectively. therefore the exploratory experiment comprised of treatment groups c, v, va and vp, each maintained in a separate box, with 10 individual worms per box. this experiment was informative, but in the case of the critical va treatment it was not possible to distinguish the effects of neurosecretory disturbance from those of interrupted ability to consume food. for this reason, a second more refined experiment was conducted where the cerebral ganglion as a discrete entity was removed. main experiments in the main experiment, the ‘new’ treatment was the surgical extirpation of the cerebral ganglion in worms that had previously been electro-stimulated, and designated group ‘ve’. therefore the main experiment comprised of c, v, and ve treatment groups of worms kept in separate boxes, 15 worms per box. these experiments were repeated three times. at the start of main experiments (i.e., on day 0) 15 earthworms from each of the treatment groups were placed on moist filter paper on the surface of fresh soil to recover from any experimental manipulation they had experienced, and after recovery they burrowed into the soil. at weeks 5, 10 and 18 post treatment, 5 worms from each of the c, v, and ve treatment boxes were weighed and subjected to the extrusion of celomocytes by electric shock followed by removal and fixation of the first 6 segments for the histological evaluation of cerebral ganglion regeneration (see below). body segments amputation and cerebral ganglia extirpation our attempts to anaesthetise earthworms either by immersion in co2-containing cold water, or cooling 153 fig. 2 cocoon production rates (expressed as number of cocoons per individual worm per week) by d. veneta during the 6-week maintenance period after the initiation of treatment. group c = untreated control worms; group v = worms whose coelomocytes were extruded at t0 by electro-stimulation alone; group va = worms treated by electro-stimulation followed by amputation of first 6 anterior (cerebral) segments at t0; group vp = worms treated by electro-stimulation followed by amputation of the 6 terminal (caudal) segments at t0. in each case the treatment groups comprised of 10 worms. them on a cold plate with or without exposure to chloroform, resulted in uncontrolled partial celomocyte extrusion which would obviously confound the celomocyte restoration observations. therefore for present purposes controlled celomocyte extrusion was induced by the electrostimulation. electro-stimulation conveniently caused a temporary state of immobilisation that facilitated effective brain extirpation by transection or surgery without any form of anaesthesia. in the va and vp treatment groups, celomocyte extrusion was immediately followed by transection of either the 6 anterior (va) or 6 posterior (vp) body segments. in the ve treatment, celomocyte extrusion was followed by cerebral ganglion extirpation by cutting the circumpharyngeal connectives through a small dorsal incision in the 3rd segment; surgery was performed under a dissecting microscope. the removed cerebral ganglia were fixed in 4 % formalin. at the end of each experiment, cerebral ganglia of some representative worms from each treatment group were removed and fixed in 4 % formalin for a gross anatomical observation; in some representative cases the first 6 anterior segments were removed and fixed in bouin's solution, dehydrated in a graded series of ethyl alcohol, rinsed in benzene, and embedded in paraffin wax according to the siekierska (2002) protocol. serial wax sections (6 μm thick) were mounted on glass slides and stained with paraldehyde fuchsin for neurosecretion (cameron and steel, 1959). cocoon and juvenile counts cocoons were manually collected and counted every time the soils in the treatment boxes were replaced with fresh soil. the cocoons were cultured further at 16 19 oc either in the soil or individually on moist filter paper in the wells of 96-well plate until hatching to determine their viabilities as expressed by hatchling counts. celomocyte extrusion earthworms were stimulated for 30 sec with a 4.5 v electric current to expel coelomic fluid with suspended celomocytes through the dorsal pores as described previously (plytycz et al., 2006). in summary, the earthworms were placed individually in petri dishes containing 3 ml of extrusion fluid (phosphate-buffered saline, pbs, containing 2.5 g/l ethylenediamine tetra-acetic acid, edta (sigmaaldrich), to prevent cell clumping). freshly prepared 2 ml suspensions were used for spectrofluorimetry, and the remaining sample from each worm was fixed in 2 % formalin for flow cytometry and for celomocyte counting in a haemocytometer. spectrofluorimetry the spectrofluorimetric measurements were performed on celomocyte suspension-lysates (2 % triton; sigma-aldrich) using a perkin-elmer spectrofluorimeter ls50b. excitation spectra were recorded between 300 520 nm (λ = 525 nm), while emission spectra were recorded between 380 700 nm (λ = 370 nm). the spectrofluorimetric signatures of unbound riboflavin are two excitation maxima (370 nm and 450 nm) and one emission maximum (525 nm). arbitrary units (au) of fluorescence at 525 nm emission wavelength were recorded using microsoft excel v. 2007. statistics recorded parameter values for earthworms and their celomocytes were expressed both as direct means and as fold-differences in relation to the 154 http://www.sciencedirect.com/science/article/pii/s0269749103001726#bib9 values for the appropriate control groups. data were expressed as x ± se and analysed by anova with post-hock tukey’s test; p < 0.05 was established as the level of significance. results cerebral ganglion regeneration figure 1 shows the gross morphology of a freshly removed cerebral ganglion (fig. 1a). a micrograph shows a transverse histological section through the 3rd anterior segment of an earthworm that had been surgically depleted of suprapharyngeal (cerebral) ganglion 18 weeks earlier (fig. 1b), and a micrograph depicting the paraldehyde fuchsin-stained neurosecretory cells of a regenerated cerebral ganglion at 18 weeks postsurgery (fig. 1c). it is evident that d. veneta can regenerate the morphology and (apparently) the function of its cerebral ganglion within 18 weeks of extirpation. cocoon and hatchling (juvenile) counts the pilot experiment revealed that the cumulative numbers of cocoons laid during the first 6 post-operative weeks by the c, v, va, and vp treatment groups of worms, 10 animal per group, were 47, 40, 5, and 57, respectively, i.e., 0.8, 0.7, 0.1, and 0.9 cocoons per worm per week, respectively (fig. 2). celomocyte loss by electrostimulation only slightly impaired reproductive output (group v). in contrast, the removal of anterior segments containing the suprapharyngeal and subpharyngeal ganglia, as well as the mouth and pharynx, almost completely inhibited cocoon production (group va); whilst removal of posterior segments (group vp) appeared to stimulate cocoon production during the early regeneration phase. cocoon production by worms in each treatment group of the representative series of the main experiment in relation to intact controls is presented in figure 3. compared with controls (c), cocoon production rate in the celomocyte-deprived worms (v group) was lowered to 72 % during the first 5 post-operative weeks, then increased to 83 % of the control level between weeks 6 and 7, and then attained values (ranging from 90 % to 110 %) similar to controls at four time intervals up to 18 weeks. in contrast, in the brain-extirpated worms (ve group) cocoon production was almost completely abolished during the first 5 postoperative weeks; between weeks 6 and 7 it reached a level equivalent to19 % of the control, and then oscillated to some extent between 31 % and 52 % of the control level between weeks 8 and 18 (fig. 3). figure 4 shows the numbers of cocoons and hatchlings produced by worm per week in the c, v, and ve groups from the representative series of the main experiment. in c and ve groups the numbers of hatchlings were consistently equal to or slightly higher than cocoon numbers, indicating that most cocoons contained one embryo. in contrast, in the ve group, only 3 cocoons and no hatchling were produced up to the 5th week after surgery; the numbers of juveniles were much lower than the numbers of cocoons produced during subsequent postoperative weeks (fig. 4). the reproductive performance by worms from three experimental series of the main experiment in relation to intact controls is presented in fig. 5. compared with controls (c), cocoon production rate in the celomocyte-deprived worms (v group) were lowered to 75 % during the first 5 post-operative weeks, and then attained values similar to controls at 10 and 18 weeks (103 % and 95 %, respectively). in contrast, in the brain-extirpated worms (ve group) cocoon production was almost completely abolished during the first 5 post-operative weeks (till 5 % of the control), and then reached a level equivalent to 23 % and 30 % of the control at 10 and 18 postoperative weeks, being still significantly lower than those in the c and v groups (fig. 5a). numbers of juveniles attained similar values in c and v groups at 5, 10 and 18 post-operative weeks. in contrast, in the ve group, none juveniles hatched from cocoons produced up to the 5th week after surgery. the numbers of juveniles were consistently very low throughout experimental period, as they reached only 5 % and 15 % of the control of by the 10th and 18th postoperative weeks (fig. 5b). thus, at 18 weeks after celomocyte retrieval and brain extirpation the reproductive performance of ve worms was still only ~15 % of the observed outputs in c and v worms (fig. 5b). earthworm body weights the mean body weights of worms in the v and ve treatment groups within the main experiment did not differ significantly from control values at postoperative weeks 5, 10, and 18 (data not shown). amebocyte and eleocyte counts the number of amoebocytes (an) in the celomocyte extrusion-only treatment group (v) was reduced to 49 % at 5 weeks after electrostimulation, but recovered to the control level by 10 weeks. amoebocyte numbers in the worms treated with both electro-stimulation and brain extirpation (ve group) were significantly lower than in the nonmanipulated control worms even 18 weeks after surgery; the values at post-operative weeks 5, 10, and 18 were 21 %, 45 %, and 62 %, respectively (fig. 6a). compared with amebocytes, the post-treatment recovery of eleocytes was slower. eleocyte numbers (en) in the v group reached those in the control worms only after 18 weeks, with levels of 40 % and 70 % relative to controls at post-treatment weeks 5 and 10. recovery of eleocytes was even slower in the brain-extirpated group (ve), with numbers equivalent to 18 %, 30 %, and 31 % of controls 5, 10, and 18 weeks, respectively, after electrostimulation and surgery (fig. 6b). riboflavin content riboflavin content, measured by spectrofluorimetry in celomocyte lysates, was considered to be proportional to the peak of emission at 525 nm (rf), and expressed as values relative to the corresponding control values at given post-treatment intervals (fig. 6c). riboflavin content was also adjusted to account for eleocyte counts (rf/en), and again expressed in relation to the control value (fig. 6d). 155 fig. 3 cocoon production by treated groups of d. veneta relative to untreated controls. cocoons were counted at different time intervals up to 18 weeks after the initiation of the treatments. the data are presented as a ratio of the measured number of cocoons per individual worm per week in a given treatment group to the same parameter in control worms during the same time interval. group c = untreated control worms; group v = worms whose coelomocytes were extruded at t0 by electro-stimulation alone; group ve = worms treated by electro-stimulation followed by surgical extirpation/removal of the cerebral ganglion (brain) at t0. data from the representative experimental series. despite the significantly lower eleocyte numbers relative to controls at 5 and 10 weeks in earthworms subjected to experimental celomocyte extrusion (v), and at 5, 10, and 18 weeks in earthworms subjected to celomocyte extrusion and brain extirpation (ve) (fig. 6b), the riboflavin content in celomocyte lysates was significantly lower only in the ve group at 5 weeks post-treatment (fig. 6c). however, the amount of riboflavin as a function of eleocyte counts (rf/en) was considerably higher (up to ~x3) than control values in the ve earthworm group at 5, 10, and 18 weeks post treatment, but with an apparently steady downward trend toward values observed in untreated animals (fig. 6d). the mean rf/en value in celomocyte extrusion-only worms (v treatment group) was significantly higher (~x3) than in control worms at 5 weeks posttreatment, but by week 10 was not significantly different from the control mean value, an observation substantiated by the v versus c comparison at 18 weeks (fig. 6d). at 18 weeks post-treatment, the significantly higher mean rf/en value observed in the brain-extirpated group (ve) compared with celomocyte extrusion-only treatment group (v) implied that surgery retarded recovery (fig. 6d). discussion the annelid central nervous system (cns) is a highly differentiated neuroendocrine structure which produces neurohormones and neurotransmitters (e.g., takahama et al., 1998; hartenstein 2006; wilhelm et al., 2006; herbert et al., 2009). in earthworms the cns is comprised of the ventral nerve cord (vnc) consisting of segmentally repeated ganglia joined across the midline by comissures and longitudinally by connectives. all vnc ganglia are uniform in anatomical organization except for the first vnc ganglia; these are fused to form the suboesophageal ganglion which is connected by paired circumpharyngeal connectives to a dorsal cerebral ganglion, loosely referred to as the ‘brain’. the time-course of regeneration of extirpated cerebral ganglia has been described in three lumbricid earthworm species: the brain was observed to have regained its original morphological fidelity within 6 weeks in lumbricus terrestris (aros and vigh, 1962), within 5 6 weeks in allolobophora chlorotica (koritsanszky and hartwig, 1974), and within 10 weeks in e. fetida (lubics et al., 2002). in the present study, d. veneta reformed a histologically complete brain, including previously described neurosecretory cells (siekierska, 2003), within 18 weeks after surgical extirpation combined with the additional restorative burden of replacing celomocytes extruded by electro-stimulation. this apparent delay in brain regeneration observed in the dual treatment leads us to postulate a bi-directional relationship between the neural and immune system of earthworms. it has long been recognized that neurosecretions promote the regeneration of, for example, amputated caudal segments, and that the activities of immunocompetent celomocytes (bilej et al., 2011) at the locus of regeneration are crucial and maybe functionally orchestrated by neurosecretions (somogyi et al., 2009). on the other hand, the regeneration of the brain itself, whilst largely dependent by the suboesophageal ganglion via the connectives (lubics et al., 2002) and possibly aided by neurotrophin-like molecules (davoli et al., 2002), appears also to be nurtured by 156 fig. 4 reproductive outputs of control and treated earthworm groups measured at 5, 10 and 18 weeks after the initiation of treatment (cf. fig. 3). data expressed as number of cocoons or juvenile worms per adult worm in a given treatment group per week. c = untreated control worms; group v = worms whose coelomocytes were extruded at t0 by electro-stimulation alone; group ve = worms treated by electro-stimulation followed by surgical extirpation/removal of the cerebral ganglion (brain) at t0. data from the representative experimental series. celomocytes. it is plausible that members of the celomocyte community deliver nutrients, signaling molecules, growth factors, and vitamins, as well as providing various protective roles in the vicinity of neural damage and repair (jamieson, 1981; varhalmi et al., 2008). whilst it is imprudent to attempt a consolidation of this notion because of the relatively meager state of knowledge about the composition and function of celomocytes, it is pertinent to note that the functional intimacy of the neural and immune systems has been more thoroughly expounded in another taxon of clitellate annelids, the leeches, where the term “neuroimmune system” is accepted in the lexicon (salzet and macagno, 2009). expulsion of celomocyte-containing coelomic fluid may be induced by various chemical and physical stressors, such as thermal, chemical, and mechanical stimuli, including sub-lethal predator attacks. stress-inducing stimuli can ultimately reconfigure the neuroendocrine-immune network at molecular, cellular, and physiological levels (adamo, 2012). the indiscriminate loss of a significant proportion of the free-floating celomocytes of d. veneta in the present study evidently modified the process of brain regeneration. it is presently unclear what roles the main celomocyte types, amebocytes and eleocytes, play in space and time during regeneration. klimek et al. (2012) reported that the amebocyte count in d. veneta returned to control levels within 4 weeks after experimental extrusion in otherwise intact worms. this recovery rate is rather faster than observed in the present study, but both studies agree that the recovery of eleocyte counts lags significantly behind that of amoebocytes. this is not surprising given the different life cycles of the two cell types. parry (1975) found that ‘free’ amebocytes are mitotically active; eleocytes, on the other hand, are detached mature chloragocytes (klimek et al., 2012) with consequential higher replenishment inertia. the compositional distinctiveness of amoebocytes (somogyi et al., 2009) is also reflected in their functional capacities as displayed, for example, by the selective uptake of metallo-nanoparticles (hayashi and engelman, 2013). thus, it is tempting to conclude that amoebocytes play more prominent roles than eleocytes in brain regeneration, particularly during the early phases, because eleocyte counts are still very low at 18 weeks post-treatment at a time when the brain has fully recovered its morphological integrity. however, our observations indicate that cell counts alone may be misleading. eleocytes, but not amebocytes, exhibit autofluorescence, a signal confined to their intracellular ‘chloragosome’ granules (plytycz et al., 2007) and derived primarily from riboflavin (vitamin b2) (koziol et al., 2006; cygal et al., 2007; sulik et al., 2012). riboflavin (vitamin b2) plays an important role in immunity of animals (verdrengh and tarkowski, 2005) and plants (zhang et al., 2009). the recent findings by samuel et al. (2012) that 157 fig. 5 reproductive outputs of control and treated earthworm groups measured at 5, 10 and 18 weeks after the initiation of treatment (cf. fig. 3). data expressed as number of cocoons or juvenile worms per adult worm in a given treatment group per week. c = untreated control worms; group v = worms whose coelomocytes were extruded at t0 by electro-stimulation alone; group ve = worms treated by electro-stimulation followed by surgical extirpation/removal of the cerebral ganglion (brain) at t0. all values relative to those measured in respective control samples considered to be 1. data were combined from three different replicated experiments. data presented as x ± se (n = 5 15). at a given time interval post initiation of treatment, columns with the same lower-case letter were not statistically (mann-whitney) different at p < 0.05. riboflavin augments regeneration of amputated earthworm body segments are very instructive; riboflavin not only promoted blastema growth but, when co-injected with antibiotics, it blocked the inhibitory effects of the antibiotics on blastema growth. it is a curious observation in our study that, in the presence of a brain, the riboflavin content of celomocyte lysate reached the control level by 5 weeks post-extrusion even though the eleocyte count was still significantly below control at this point. even in brain-extirpated worms the riboflavin content of the lysate was similar to controls by 10 weeks after treatment, although the neural disruption significantly retarded eleocyte count recovery. interpreting the disparity between the rapid restoration of riboflavin status and the lag in riboflavin-storing cell (eleocyte) counts is difficult. it is evident that the cohort of chloragocyte-derived eleocytes detached in response to electrostimulated extrusion possesses higher riboflavin content, but what is the source of the ‘extra’ vitamin? one possibility is that stress-mediated neuro-immune reconfiguration (adamo, 2012) is involved. this could entail the trafficking of riboflavin 158 fig. 6 regeneration of coelomocyte system after 5 (5w), 10 (10w), and 18 weeks (18w) after experimental treatments: (a) amebocyte counts (an); (b) eleocyte counts (en); (c) amount of riboflavin (rf) in celomocyte lysated measured by spectrofluorimetry; (d) the relative amount of riboflavin in coelomocyte lysates expressed as a function of eleocyte counts (rf/en); all values relative to those measured in respective control samples considered to be 1. c = untreated control worms; group v = worms whose coelomocytes were extruded at t0 by electro-stimulation alone; group ve = worms treated by electro-stimulation followed by surgical extirpation/removal of the cerebral ganglion (brain) at t0. data were combined from three different replicated experiments. data presented as x ± se, n = 5. at a given time interval post initiation of treatment, columns with the same lower-case letter were not statistically (mann-whitney) different at p < 0.05. from relatively immature attached chloragocytes to more mature chloragocytes that are about to be released into the coelom to become eleocytes. an alternative hypothesis is that bacterial and fungal gut endo-symbionts, the main source of riboflavin for earthworms according to sulik et al. (2012), are somehow regulated by factors involved in the neuroimmune system linked to regeneration pathways. these hypotheses warrant robust investigations. however, whatever the source of the riboflavin, it is evident that riboflavin content is restored sufficiently rapidly after electro-stimulated celomocyte depletion in otherwise intact worms, and in worms subjected to celomocyte depletion after brain extirpation, to potentially play a role in brain regeneration analogous to that described by samuel et al. (2012) in the regeneration of amputated segments. cocoon production is inhibited both in starved earthworms and in earthworms restoring experimentally extruded celomocytes (polanek et al., 2006). these observations were putatively confirmed in the present study, where caudal amputation did not impair cocoon production, whilst cerebral amputation of segments containing mouth, pharynx, and cerebral and subpharyngeal ganglia completely inhibited reproductive output for several weeks after transection. however, the transection of anterior segments precludes distinguishing the effects of interrupted feeding ability from the effects of removing a key source of neurosecretions as well as the nutritional cost of regenerating vital organs. in our main experiment, reproduction was completely inhibited in worms with surgically extirpated brains and subsequently recovered slowly during the 18 week post-operative period, but had not fully restored to control levels even when the brain was apparently fully regenerated. there is some evidence that both restorative growth and reproduction in earthworms are controlled by neurosecretions (alonso-bedate and sequeros, 1985). moreover, an oxytocin-related neuropeptide, annetocin, eliciting stereotypical egg-laying behaviour patterns (oumi et al., 1996), is secreted by the cerebral ganglia (takama et al., 1998) with receptors located within urine-forming nephridial tubules in the clitellum region (kawada et al., 2004). sound microscopic evidence has been published indicating that brain removal causes immediate and profound structural disruption within the gonads of the earthworm d. veneta, but that the damage is largely repaired at 20 days after decerebration (siekierska, 2002, 2007). these observations 159 combined with our findings suggest that the attenuated suppression of reproductive output in worms with surgically excised brains and depleted celomocytes is at least partly, directly or otherwise, a consequence of the experimental manipulation of the immune-competent circulating cells. in conclusion, the present study provides some evidence that the neurosecretory brain of earthworms and the celomocytes act in concert to regulate fundamentally important functions such as tissue regeneration and reproduction. over a century after the pioneering tissue regeneration work of morgan and others, the detailed investigations of the molecular-genetic and physiological basis of these interactions within the “neuroimmune system” of terrestrial oligochetes is long overdue. acknowledgments we thank the doctor m klimek for a valuable technical help. this work was supported by k/zds/003252 and b/nz4/01640 (k/pbo/000178). references adamo sa. the effects of the stress response on immune function in invertebrates: an evolutionary perspective on an ancient connection. horm. behav. 62: 324-330, 2012. alonso-bedate m, sequeros e. suggested regulatory mechanisms for caudal regeneration in allolobophora molleri 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oxytocin-related peptide, and identification and ultrastructural characteristics of the annetocin-secretory cells in the oligochaete earthworm eisenia foetida. zool. sci. 15: 381-388, 1998. takeo m, yoshida-noro c, tochinai s. functional analysis of grimp, a novel gene required for cell proliferation at an initial stage of regeneration in enchytraeus japonensis (enchytraeidae, oligochaeta). int. j. dev. biol. 54: 151-160, 2010. varhalmi e, somogyi i, kiszler g, nemeth j, reglodi d, lubics a, et al. expression of pacap-like compounds during the caudal regeneration of the earthworm eisenia fetida. j. mol. neurosci. 36: 166-174, 2008. vaudry d, pamantung tf, basille m, rousselle c, fournier a, vaudry h, et al. beauvillain jc, gonzalez bj. pacap protects cerebellar granule neurons against oxidative stressinduced apoptosis. eur. j. neurosci. 15: 14511460, 2002. verdrengh m, tarkowski a. riboflavin in innate and acquired immune responses. inflammation res. 54: 390-393, 2005. wilhelm m, koza a, engelmann p, nemeth p, scokya m. evidence for the presence of thyroid-stimulating hormone, thyroglobulin and their receptors in eisenia fetida: a multilevel hormonal interface between the nervous system and the peripheral tissues. cell tissue res. 324: 535-436, 2006. zhang s, yang x, sun m, sun f, deng s, dong h. riboflavin-induced priming for pathogen defense in arabidopsis thaliana. j. integr. plant biol. 51: 167-174, 2009. 161 http://www.ncbi.nlm.nih.gov/pubmed?term=beryl%20vedha%20y%5bauthor%5d&cauthor=true&cauthor_uid=22150027 http://www.ncbi.nlm.nih.gov/pubmed?term=amutha%20k%5bauthor%5d&cauthor=true&cauthor_uid=22150027 http://www.ncbi.nlm.nih.gov/pubmed?term=amutha%20k%5bauthor%5d&cauthor=true&cauthor_uid=22150027 http://www.ncbi.nlm.nih.gov/pubmed?term=dinesh%20sm%5bauthor%5d&cauthor=true&cauthor_uid=22150027 http://www.ncbi.nlm.nih.gov/pubmed?term=jackson%20durairaj%20sc%5bauthor%5d&cauthor=true&cauthor_uid=22150027 http://www.ncbi.nlm.nih.gov/pubmed?term=elaiya%20raja%20s%5bauthor%5d&cauthor=true&cauthor_uid=22150027 http://www.ncbi.nlm.nih.gov/pubmed?term=elaiya%20raja%20s%5bauthor%5d&cauthor=true&cauthor_uid=22150027 spectrofluorimetry isj 5: 134-xxx, 2008 isj 5: 135-161, 2008 issn 1824-307x review hsp expression in bivalves e fabbri, p valbonesi, s franzellitti interdepartment centre for environmental science research, university of bologna, campus of ravenna, italy accepted september 26, 2008 abstract one of the molecular responses which mostly contribute to the physiological plasticity of bivalves is the heat shock response mediated by heat shock proteins (hsp). variations of hsp response were observed under environmental conditions, correlated with differences in environmental temperature and degree of heterogeneity across geographic thermal gradients and through time. laboratory experiments characterized the expressions of different protein isoforms and coding genes, which are induced by heat as a prototypical stimulus. nevertheless, other physical and chemical factors significantly induce hsp gene and protein expressions in bivalves, that can be different depending on tissues and the nature of the insult. multiple alignments of the deduced amino acid sequences indicated that the bivalve hsp70 proteins share common structural and evolutionary features with the mammalian hsp70, while some appear to be exclusive. the rate at which new findings are made regarding the bivalve hsp response is still increasing. however, some major questions remain unanswered. among them, the possibility that the bivalve hsp response is related to cell signalling pathways and acts as a component of the acute systemic response to stress is also discussed in this review. key words: bivalves; hsp; hsp expression; hsp phylogeny; hsp sequence; stress response introduction living systems have evolved a variety of strategies to respond to external or internal environmental challenges. while these responses are often behavioural or metabolic, a powerful mechanism widely employed to maintain cellular homeostasis under stress is the adjustment of gene expression. due to their immobility, sessile organisms in particular rely on this physiological plasticity to adapt to environmental insults and colonize rapidly fluctuating habitats. bivalves living in intertidal zones are one of the best examples of ectotherms surviving highly stressful conditions, facing exposure to natural changes in temperature, salinity, and oxygen availability exacerbated by the extensive presence of pollutants and anthropogenic disturbances in general (hofmann, 1999). these same ___________________________________________________________________________ corresponding author: elena fabbri interdepartment centre for environmental science research university of bologna, campus of ravenna via s. alberto 163, 48100 ravenna, italy e-mail: elena.fabbri@unibo.it organisms living at their physiological stress limit will be affected by the continuous rise in temperature predicted under climate change scenarios, and only those endowed with sufficient defence mechanisms will be able to survive. the aim of this review is to outline the current state of our knowledge of one of the molecular responses which most contribute to the physiological plasticity of bivalves, i.e., the heat shock response mediated by heat shock proteins (hsp), examined at the level of gene and protein expressions. we also discuss structural features of the hsp70 and hsp90 multigene families in bivalves, and their phylogenetic relationships. traditionally the heat shock response has been considered an intracellular phenomenon with little or no association with intra/extracellular signalling. in this review we want to draw attention to the fact that the hsp response is related to cell signalling pathways, and as it is a component of the acute systemic response to stress, it is integrated with the physiological stress response. these relationships are presently under active investigation in mammals, and initial findings suggest that they may also apply to bivalves (lacoste et al., 2001a). 135 mailto:elena.fabbri@unibo.it the terminology used is in accordance with the following criteria: capital letters are used to refer to whole hsp or the whole family (e.g., hsp70). noncapital letters are used to refer to a specific protein from a family (e.g., hsp70, hsp72, etc.). italics are used for genes (i.e., hsp70, hsp72, etc.). the integrated stress response the definition of stress and stressors has a long and controversial history. in this review, however, we will adopt the definition used by wendelaar bonga (1997) that stress is a condition in which dynamic equilibrium of animal organisms, called homeostasis, is threatened or disturbed by intrinsic or extrinsic stimuli, commonly defined as stressors. these responses typically involve all levels of animal organization and are referred to as the “integrated stress response” (wendelaar bonga, 1997). in this light, stress acquires a less negative connotation, in so far as it indicates all the forces or stimuli in the environment, internal or external, that can induce changes and adaptations in the organism to help it better fit its environment. however, if the stress or the stress factors persist without physiological adaptation, illness or even death may occur. many hormones are involved in the mammalian integrated stress response, but the dominant role of catecholamines (ca) and glucocorticoids in this response is generally recognized (wendelaar bonga, 1997). these hormones are the primary messengers of the two major routes through which the brain coordinates the stress response: the brain-sympathetic-adrenal medulla axis and the brain-pituitary-adrenal axis. their main functions involve stimulation of oxygen uptake and transfer, mobilization of energy substrates, reallocation of energy away from growth and reproduction. past studies on molluscs revealed remarkable structural, functional and biochemical parallelisms with vertebrates. numerous studies actually identified in mussels and other molluscs neuroendocrine and nervous system functions analogous to the hypothalamic-pituitary system in vertebrates (stefano et al., 2002). in general the elements at the basis of the response and their triggering are similar to those of vertebrates, but take place in a different and simpler scenario (ottaviani and franceschi, 1996). most mechanisms are represented in one single multifunctional cell, the hemocyte (malagoli et al., 2000). as the cell component of bivalve hemolymph, this cell is involved in several functions, including respiration and nutrition, immune response, and stress response (ottaviani and franceschi, 1996). ca together with β-endorphin, α-msh, and acth are the most important mediators of stress response in bivalves, and some molecules similar to cortisol were identified (ottaviani et al., 1998a, 1999). although the mechanisms controlling the ca release in invertebrates have not been fully elucidated, the production of crh by nerve cells probably induces the release of acth-like molecules, which in turn trigger the release of ca, mainly noradrenaline and dopamine. in oysters, the release of ca starts from neurosecretory cells which resemble the vertebrate chromaffin cells (lacoste et al., 2001b). there is clear evidence that, under stress conditions, the crh-acth modulated axis also determines the activation of bivalve hemocytes, which possess crh and acth receptors (ottaviani et al., 1998b; malagoli et al., 2000). it has also been suggested that that the platelet derived growth factor (pdgf) and transforming growth factor (tgf-β) exert a direct control on ca release, thus playing an important role in stress response in these organisms (ottaviani et al., 1998a, 2001) relationship between integrated and cell response to stress in mammals, circulating levels of prolactin and glucocorticoids are increased by thermal stress and are associated with the modification of intracellular heat shock by enhancing expression of hsp genes (vijayan et al., 2003). prostaglandins are also associated with thermal stress and are known to induce hsp synthesis (collier et al., 2006). mechanical disturbances and changes in temperature or salinity were shown to increase circulating levels of noradrenaline and dopamine in oysters. the response was rapid and transient, according to the duration of the stress exposure (lacoste et al., 2001c). the ca response to acute stimulation took place in a few minutes, raising the circulating levels of noradrenaline and dopamine (lacoste et al., 2001c). further experiments clearly demonstrated that treatments with adrenergic compounds induced the hsp70 gene promoter in oyster hemocytes, indicating that the integrated response to stress is related to the heat shock response elaborated by cells. specifically, the response was stimulated through α-adrenoceptor mediated pathways, involving plc and pkc activation (lacoste et al., 2001a). treatments with ca also induced high viability in hemocytes exposed to severe heat stress, indicating that αadrenergic stimulation leads to cell thermotolerance through hsp overexpression (lacoste et al., 2001a). although this relationship has not been further elucidated, it represents one of the most interesting issues to be investigated in this field. the cell response to stress cellular exposure to stress factors induces a number of anomalies in cellular function, including a general inhibition of protein synthesis, alterations in protein structure and function, cytoskeleton rearrangements, shifts in metabolism, alterations in cell membrane dynamics and fluidity, etc. these anomalies trigger major changes in gene transcription and protein synthesis, known as the cell response to stress mediated by hsp, metallothioneins, antioxidant enzymes, etc. the timing and success of these changes ultimately determines cell survival and acclimation or cell death. therefore, cells have developed strategies that allow the identification of, and the reaction to, stress conditions. although in oysters it has been demonstrated that hsp is induced by ca (lacoste et al., 2001a) these proteins are also naturally induced at stress levels lower than those required 136 for activation of the integrated stress response. the overexpression of hsp in the absence of an integrated stress response involving the whole organism reflects the fact that the expression of these proteins can be regulated at the cellular or tissue level, not only at the organism level. as a general pattern, a transcription factor family known as heat shock factor (hsf) is the first responder during the onset of elevated cell temperature. the physiological importance of hsf is exemplified by its evolutionary conservation, from unicellular organisms to mammals (pirkkala et al., 2001). amongst other hsf, hsf1 is primarily responsible for induction of hsp gene expression after heat shock (pirkkala et al., 2001). the current model of hsf1 transcriptional activity indicates that nonstressed cells contain folded hsf1 monomers bound to hsp within the cytoplasm. upon heat stress, the hsp dissociate from hsf1 monomers, which then unfold and bind to two other hsf1 monomers to form a trimer which then translocates to the nucleus. once in the nucleus, the trimer binds promoters containing heat shock elements (hse) to trigger heat shock gene transcription. although hsf1 was traditionally associated with regulation of hsp, recent evidence now links it to regulation of mammalian carbohydrate metabolism, transport, cytoskeleton, and ubiquitination during heat shock (page et al., 2006). the heat shock proteins hsp are evolutionarily ancient and highly conserved intracellular molecular chaperones constituting several multigene superfamilies (barral et al., 2004). they are present in all the different subcellular compartments of all cell types from prokaryotes to eukaryotes. the initial nomenclature for hsp was based on the apparent molecular weight of each single protein (i.e., hsp70, 72, 73, etc.) and they were grouped according to their nearest size (e.g., the hsp70 family). some proteins are referred to by different names, as they were thought to be different when they were discovered and, for historical reasons, these names have been preserved (see vos et al., 2008 for the most recent classification of human hsp). hsp can also be grouped into constitutive and inducible isoforms. proteins reported as hsc (heat shock cognate) represent the constitutive forms; these are always expressed under physiological conditions and serve as molecular chaperones. the inducible forms, properly referred to as hsp, are synthesised by cells under stressful conditions and display a cytoprotective role. further proteins constitutively present but also induced by stress have been reported in most bivalves (piano et al., 2002). molecular chaperones appear to be essential for protein folding or trafficking, and for regulated proteolysis in cells (hendrick and hartl, 1995; fink, 1999). most of the information we have on chaperone functioning relies on studies on bacteria, where proteins are on average smaller and less complex than the corresponding proteins of eukaryotes (hartl and hayer-hartl, 2002). however main research on human chaperones was performed, also due to the correlation between protein misfolding and major diseases (barral et al., 2004). to become functionally active, newly synthesized proteins must fold into unique threedimensional conformations, based on the information encoded in their amino acid sequences. although many proteins can fold to their native state spontaneously in vitro, the involvement of high efficiency molecular chaperones is required in vivo. a further role of molecular chaperones is to prevent protein misfolding and aggregation, anomalous reactions that would otherwise impair cell functioning. the properties of the peptide bond confer a high degree of conformational flexibility to the protein backbone, while the amino acid side chains allow a large number of mostly non-covalent interactions. thus the protein chain can theoretically adopt an enormous number of different conformations, and generally only one of these corresponds to its native state, sufficiently stable and biologically active under physiological conditions. these non-native states often expose hydrophobic residues and tend to self-associate into disordered aggregates, driven by hydrophobic forces and interchain hydrogen bonding. chaperones promote the re-folding process through cycles of substrate binding and release, generally regulated by atpase activity and various co-factors. more recent reports showed that in addition to protein folding and refolding, chaperones are also involved in several other physiological processes in mammals, including signal transduction, apoptosis, immune response, etc. (van noort, 2008). members of the hsp70 family are the most highly conserved molecular chaperones. hsp70 found in prokaryotic and eukaryotic cells are able to recognize exposed hydrophobic amino acid side chains within an accessible polypeptide backbone, features that are generally found in non-native proteins. all hsp70 functions are performed through an atp-regulated cycle of substrate binding and release in the presence of different cofactors, including dnaj-proteins or the eukaryotic equivalent hsp40. biochemical studies of hsc70 fragments generated using recombinant dna technology have led to mapping and characterization of the domains. the 44-kda fragment (amino acid residues 1-386) from the n-terminus has been characterized by xray crystallography and contains the atpase domain. the 18-kda fragment contains the peptidebinding domain that binds unfolded and folded peptides (wang et al., 1993). the 44and 18-kda fragments are the same as those making up hsp70. the 10 kda c-terminal of hsp70 differs by 26 amino acid residues relative to hsc70 and is 6 amino acids shorter (leung and hightower, 1997). members of the 90 kda heat-shock protein (hsp90) family act downstream of the hsp70/hsp40-chaperone system, and play an important role in conformational protein regulation and cell signalling. they are highly conserved, essential proteins found in all organisms from bacteria to humans. hsp90 are the most abundant chaperones in the cell, representing 1 2% of cellular proteins, making it one of the most abundant proteins even in the absence of stress. hsp90 137 http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22hartl%20fu%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22hayer-hartl%20m%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus act at the core of a network of protein complexes, with over a dozen known cofactors, some of which link it to other multi-protein complexes such as the ubiquitin proteasome system and the hsp70 system. unlike hsp70, hsp90 has several identified specific interactions, for example, with cytoskeleton elements, signal transduction proteins including steroid hormone receptors, and protein kinases (fink, 1999). moreover, it plays a regulatory role in inducing conformational changes in folded, native-like substrate proteins, leading to their activation or stabilization (wandinger et al., 2008) hsp60 are large, oligomeric, ring-shaped proteins known as chaperonins, present in all biological compartments with the exception of the endoplasmic reticulum. this family includes two groups of proteins on the basis of sequence homology: groel-like proteins, present in bacteria, mitochondria and chloroplasts, and the tcp-1 (cct or tric) family, found in archaea and the eukaryotic cytosol. hsp60 play an essential role in all cells, assisting a large variety of newly synthesized and newly translocated proteins to reach their native forms by binding them and facilitating their folding. the folding-active state is reached by conformational changes, induced by the action of atp binding and, for the organellar/bacterial chaperonins, also by the binding of a lid-like co-chaperonin, (cpn10) (groes in e. coli) (bukau and horwich, 1998). other hsp distributed among organisms are the small heat shock proteins (shsp). this family consists of 12 to 43 kda proteins, which assemble into large multimeric structures, present in the cytosol, nucleus, and mitochondria. these proteins act as atp-independent chaperones and many of them are produced only under stress conditions. one of the best studied shsp is hsp27 (also denoted hspb1, hsp28, and hsp25 in murine cells). hsp27 protect cells against oxidative stress by counteracting the accumulation of proteolysisresistant large aggregates of oxidized proteins (lipofuscin) which interfere with proteasome activity and are extremely deleterious to the cell. hsp27 is an early target for phosphorylation, which occurs rapidly following exposure to various stresses, but also in unstressed cells upon stimulation by serum or a variety of mitogens, cytokines, and inducers of differentiation (fink, 1999). the heat shock proteins in bivalves aquatic environments are often highly dynamic and provide a wide variety of stress factors for individuals living there. bivalves may be subject to a variety of sources of stress, including temperature and salinity fluctuations, oxygen availability, different quantities and quality of food, presence of predators or competitors, presence of toxic natural compounds and contaminants. moreover, they are always in contact with microbes and their immune system is alerted to avoid an accumulation of invading pathogens. aquaculture introduces additional forms of stress, including mechanical stress during handling and transportation (lacoste et al., 2001c). the heat shock response entails the rapid synthesis of hsp, which have been ubiquitously detected in all bivalves studied so far. the absence of hsp response was reported in an antarctic fish (hofmann et al., 2000) and an antarctic ciliate (la terza et al., 2001); however, the antarctic clam laternula elliptica does rely on hsp to counteract the effects of thermal stress (park et al., 2007). although the name “hsp” derives from stress due to temperature elevation (ritossa, 1962), a number of factors have actually been shown to induce this response in bivalves and will be discussed below. nevertheless, hsp are also constitutively expressed and play a role in bivalves under physiological conditions. hsp70 is the best investigated family, and most of the following discussion will focus on these proteins. mitochondrial hsp60 induction was proposed as an early warning of adverse physiological effects in bivalves, as it is enhanced in mussels (sanders et al., 1992; sanders and martin, 1993; snyder et al., 2001), oysters (ivanina et al., 2008a), and clams (franco et al., 2006) exposed to stress stimuli. dowling et al. (2006) observed no effect of pesticides on hsp60 expression in gill, mantle and digestive gland of ruditapes decussatus, shedding some doubts as to the role of hsp60 as biomarkers of stress. in bivalves, hsp90 have received less attention than other hsp. it is true that the behaviour of hsp90 expression is often similar to that of hsp70 in response to cadmium exposure (choi et al., 2008), prolonged heat stress (snyder et al., 2001; anestis et al., 2007), or low electromagnetic fields (malagoli et al., 2004), but a tissue specific and species-specific expression of hsp90 in bivalves is underlined by several authors (lyons et al., 2003; dowling et al., 2006; ivanina et al., 2008a). other hsp have been poorly studied or not studied at all in bivalves. however, they will be considered in this review, where data are available. the hsp70 multigene family in bivalves along with the extensive characterization of the hsp70 response at the functional level, the bivalve hsp70 multigene family was also investigated at the gene level, and the availability of nucleotide and deduced amino acid sequences has increased noticeably during the last few years (table 1). marine mussels and oysters are amongst the bivalves most often used to study the heat shock response, and studies on hsp70 gene regulation in these organisms provide important insights into the molecular basis of the response to environmental challenges. sequences encoding multiple hsp70 isoforms are reported mainly for the mediterranean mussel mytilus galloprovincialis, and for the ostreidae ostrea edulis and crassostrea gigas (table 1). for these species, different full-length sequences encoding both hsc70 and hsp70 proteins are available, and detailed characterizations of their functional features and gene expression profiles have been carried out (boutet et al., 2003a, b; piano et al., 2004, 2005; franzellitti and fabbri, 2005, 2006; kourtidis et al., 2006; cellura et al., 2006, 138 http://www.ncbi.nlm.nih.gov/pubmed/11385587?ordinalpos=2&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvdocsum table 1 the bivalve hsp70 protein sequences available in genbank species (abbreviation) gene genebank ac numb reference crassostrea ariakensis (cariak) hsc70 aao41703 unpublished crassostrea columbiensis (ccol) hsp70 abc02062 unpublished crassostrea gigas (cgig) hsc71 bad15287 unpublished hsc72 aad31042 (gourdon et al., 2000) hsp70 cac83009 (boutet et al., 2003b) hsp70 bad15286 unpublished grp78 bad15288 (yokoyama et al., 2006) grp94 baf63637 unpublished hsp68 bad15285 unpublished hsc70 cac83683 (boutet et al., 2003b) crassostrea virginica (cvir) hsp70 cab89802 (rathinam et al., 2000) ostrea edulis (oed) hsc70 cac83684 (boutet et al., 2003a) hsp70 aam46635 (piano et al., 2005) hsp70 aam46634 (piano et al., 2005) hsp70 cac83010 (boutet et al., 2003a) saccostrea palmula (sacp) hsp70 abc02063 unpublished mytilus galloprovincialis (mg) hsc71 cah04109 (kourtidis et al., 2006) hsc70 cah04110 (kourtidis et al., 2006; kourtidis and scouras, 2005) hsc70 aba61049 (franzellitti and fabbri, 2005) hsp70 aaw52766 (cellura et al., 2006) hsp70 aba61046 (franzellitti and fabbri, 2005) hsp70 aba61047 (franzellitti and fabbri, 2005) hsp70b bad99027 (toyohara et al., 2005) hsp70a bad99026 (toyohara et al., 2005) hsp70 cah04108 (kourtidis et al., 2006) hsp70 cae51348 (kourtidis et al., 2006) hsp70 cah04106 (kourtidis et al., 2006) hsp70 cah04107 (kourtidis et al., 2006) mytilus edulis (med) hsc70 aad48065 (luedeking and koehler, 2002) bathymodiolus azoricus (baz) hsp70 caj40877 unpublished perna viridis (pvir) hsp71 abj98722 unpublished hsc71 abq11278 unpublished diplodon chilensis (dpic) hsp70 abw06851 unpublished chlamys farreri (cfar) hsp70 aao38780 unpublished chlamys farreri (cfar) hsp70 abe77386 unpublished argopecten irradians (airr) hsp70 aas17723 (song et al., 2006) mizuhopecten yessoensis (myess) hsp70 aas17724 (song et al., 2006) pinctada fucata (pfuct) hsp70 abj97378 unpublished pteria penguin (ppeng) hsp70 abj97377 unpublished laternula elliptica (lellipt) hsp70a cal25331 (clark et al., 2008) hsp70b cal25333 (clark et al., 2008) hsc70 cal25332 (clark et al., 2008) grp78 cal25334 (clark et al., 2008) hsp70 abm92345 (park et al., 2007) venerupis decussates (vdec) hsp70 acb38005 unpublished dreissena polymorpha (dpol) hsp70 abp88104 unpublished 139 2007). other full-length or partial sequences have been obtained for crassostrea virginica, crassostrea ariakensis, crassostrea columbiensis, and saccostrea palmula (table 1). the scallops argopecten irradians and chlamys farreri have received recent attention due their extensive employment in aquaculture (song et al., 2006). other bivalves have been studied as representatives of peculiar environments; for example an hsp70 cdna sequence was recently obtained for the antarctic clam l. elliptica (park et al., 2007). a cdna sequence encoding the 78 kda glucose regulated (grp78) protein was cloned from c. gigas (yokoyama et al., 2006). a further partial sequence from c. gigas putatively encoding a 94 kda grp is also available (unpublished, table 1). to our knowledge these are the only representatives of the grp members of the hsp70 family obtained from a bivalve species. the multiple alignment of the full-length hsp70 amino acid sequences of bivalves with their human homologues (fig. 1) indicates that they share common structural and evolutionary features (piano et al., 2005; kourtidis et al., 2006). in particular, they display the canonical conserved domain structure of the hsp70 consisting of: i) a cleavable signal sequence at the n-terminus, which characterizes the grp78 proteins (yokoyama et al., 2006); ii) an atpase domain; iii) a peptide binding domain; iv) a g/p-rich c-terminal domain region, which enables the proteins to bind co-chaperones and other hsp (daugaard et al., 2007), and contains intracellular localization signal sequences (fig. 1). further important structural features, such as the presence of three hsp70 family signatures idlgttys, ifdlgggtfdvsil, and vvlvggdtripkiqk (gupta and singh, 1994), were also highly conserved (fig. 1). nevertheless, some structural features appear to be exclusive to the bivalve hsp70. the hsc70 from c. gigas and o. edulis possess an extra nqsq tetrapeptide within the atpase domain (fig. 2). this sequence is also found in other oyster hsc70 (kourtidis et al., 2006), and appears to be unique for ostreidae. it should be noted that the nqsq tetrapeptide encodes a putative glycosylation domain (laursen et al., 1997), so that its correlation with peculiar functional and expression features of the ostreidae hsc70 deserves further investigation. the inducible hsp70 gene products from mussel and oyster possess an extra serine (s) residue within the conserved atpase domain (fig. 2). as previously reported (kourtidis et al., 2006), this serine residue is shared by the inducible hsp70 of all invertebrate species studied so far. however, no specific role has yet been ascribed to this residue. as shown in fig. 3, the o. edulis hsc70 display a large amino acid deletion of about 60 residues encompassing the end of the peptidebinding domain and a part of the c-terminal domain. a similar deletion was also found in other ostreidae hsp70 (fig. 3, kourtidis et al., 2006), which also share a reduced molecular weight. this deletion could be responsible for a change in the functional role of these 65 kda-proteins. in fact, two cognate variants in humans (hsc54; tsukahara et al., 2000) and rats (hsc49; yamada et al., 1999) bearing a similar deletion were found to act as negative competitors towards the normal hsc70. as previously reported in mammals (demand et al., 1998; fuertes et al., 2004), a low homology within the c-terminal domain is also observed in mollusc hsp70, particularly between hsp70 and hsc70 (piano et al., 2005; fig. 3). this domain is thought to be involved in the interaction with cochaperones of the dnaj class and in the regulation of the substrate binding kinetics and affinity (fuertes et al., 2004). interestingly, in this domain the bivalve hsc70 possess a large insertion compared with the inducible hsp70. less extensive sequence variations are also identified in the same region when comparing mammalian hsp70 and hsc70. they might be partially responsible for the functional differences between inducible and constitutive hsp70 (fuertes et al., 2004). this could also be true for the bivalve proteins, since the hsc70 sequence insertion contains two repeats of the tetrapeptide ggmp (fig. 3), an important element mediating cofactor binding to the hsp molecule (demand et al., 1998). since no extensive biochemical characterization of the bivalve hsp70 and hsc70 genes is available, at present we do not know the extent to which such a structural variation affects their expression profiles as hypothesized in vertebrates (demand et al., 1998; fuertes et al., 2004). the c-terminal region contains a consensus motif that enables hsp70 to be localized into specific cellular compartments where these proteins perform different functions, including protein folding (endoplasmic reticulum), translocation to organelles (mitochondria), and stress response (cytosol/nucleus) (de maio, 1999). the localization motif specific for the cytosolic proteins is gp(t/k)(v/i)ee(v/m)d (boorstein et al., 1994; demand et al., 1998). this sequence is found in the bivalve hsp70 sequences analyzed thus far, indicating that most of the hsp70 gene products obtained for bivalves encode cytosolic proteins. only one full-length protein sequence was obtained for an endoplasmic reticulum (er) hsp70 protein, i.e., the grp78 protein from c. gigas (table 1); no records are currently available for the mitochondrial hsp70. the oyster grp78 sequence displayed the characteristic er localization motif lys-asp-glu-leu (kdel), and shared a high sequence homology with grp78 homologues from fish and other vertebrates (fig. 1; yokoyama et al., 2006). the phylogenetic analysis of several molluscan, mammalian, and fish cytosolic hsp70 reveals that the bivalve genes can be divided into two groups (fig. 4), one containing the inducible (bivalve hsp70), and the other containing the cognate genes (bivalve hsc70). the branching pattern is in agreement with the generally accepted molluscan classification, differentiating bivalvia from gastropoda (kourtidis et al., 2006). some hsp70 sequences obtained from c. gigas (genbank ac. numb cac83009; boutet et al., 2003b), o. edulis (genbank ac. numb cac83010; boutet et al., 2003a), m. galloprovincialis (genbank 140 atpase domain peptide bindingdomain c-terminal domain localization signal h2n cooh signal sequence (grp78 only) human hsc70(bc019816) mskgp--avgidlgttyscvgvfqhgkveiiandqgnrttpsyvaft-dter 80 cgig_hsc71_(bad15287) ...paqq.i......................................-.... mg_hsc71_(cah04109) .a.tg-p.i......................................-.... human hsp70(p17066) .qaprel.................q.r...l................-.... cgig_hsp70_(bad15286) .aska-p.i.......f..............................-.... mg_hsp70_(bad99027) .agkg-p.i...................d..................-.... human grp78(np_005338) m-klslvaamllllsaaraeeed-----kkedvgtv...............kn.r...........i........peg.. cgig_grp78_(bad15288) mrkllflglaillvswsradddegekkkdkesvgtvi..............kn.r...........i........a.n.. human hsc70(bc019816) ligdaaknqvamnptntvfdakrligrrfddavvqsdmkhwpfmvvndagrpkvqveykg-etksfypeevssmvltkmk 160 cgig_hsc71_(bad15287) .v............n..i.........k.n..s..........t.i.q.sk.mik.....-.e.t.sa........n... mg_hsc71_(cah04109) ..............v............k....t..........t.....sk..it.d...-...t.f...i.....v... human hsp70(p17066) .v.....s.a.l..h............k.a.tt..........r..seg.k...r.c.r.-.d.t.....i.....s... cgig_hsp70_(bad15286) .............an..i.........k.n.ds..........t.i..g.k..le..f.n-.k.r.t...i......... mg_hsp70_(bad99027) .v.........l.a...i.........n.s.st....i.....k.i.sg.k..l.a.h..-...t.t...i.....v... human grp78(np_005338) .........lts..e............twn.ps..q.i.fl..k..ekktk.yi..dig.gq..t.a...i.a....... cgig_grp78_(bad15288) .........lts..e..i..v......tw..ks..k.iqyy..k.i.kn.k.his..as.-.e.v.a.....a...g..r human hsc70(bc019816) eiaeaylgktvtnavvtvpayfndsqrqatkdagtiaglnvlriineptaaaiaygldkkvg----aernvlifdlgggt 240 cgig_hsc71_(bad15287) .t........in........................s.........................nqsqg............. mg_hsc71_(cah04109) .t.......l.n.s.i....................s.m.......................----g............. human hsp70(p17066) .t......qp.kh..i..................a.......................rrga----g............. cgig_hsp70_(bad15286) .t......q..rd..........na..e......v.........v.......l......nis----g.k........... mg_hsp70_(bad99027) .t......qk..d..i...........l......f.................l......nls----g.k........... human grp78(np_005338) .t.......k..h...........a................m.................re.-----.k.i.v....... cgig_grp78_(bad15288) ....gf...kin...i........a................m..................e.-----.k.i.v....... human hsc70(bc019816) fdvsiltiedg-ifevkstagdthlggedfdnrmvnhfiaefkrkhkkdisenkravrrlrtacerakrtlssstqasie 320 cgig_hsc71_(bad15287) ...........-.......s...................q..................................s..... mg_hsc71_(cah04109) ...........-.......s...................q......................................v. human hsp70(p17066) ....v.s.da.-v....a...............l....me..r...g..l.g....l....................tl. cgig_hsp70_(bad15286) ........de.s....r.....................vq.....yn....k.n.sl.................se.n.. mg_hsp70_(bad99027) ........de.sl...r.....................vn.....cg....g.n..l........k.........e.nv. human grp78(np_005338) ....l...dn.-v...va.n...........q.vme...kly.k.tg..vrkdn...qk..rev.k...a...qh..r.. cgig_grp78_(bad15288) ....l...dn.-v...va.n...........q.vme...kly.k.kg...rkdn...qk..rev.k...a...qh..k.. human hsc70(bc019816) idslyegidfytsitrarfeelnadlfrgtldpvekalrdakldksqihdivlvggstripkiqkllqdffngkelnksi 400 cgig_hsc71_(bad15287) ....f.........................me.............a.................................. mg_hsc71_(cah04109) ....f..v......................me.............aav.e.............................. human hsp70(p17066) ....f..v..............cs....s..e.............a....v...........v................. cgig_hsp70_(bad15286) ....f..m...sk........mc........e..........m...k..ev..............m....mg........ mg_hsp70_(bad99027) ....f..t....k.........cs....t..e..............s.qe...............m.se.m........v human grp78(np_005338) .e.f...e..setl...k.....m....s.mk..q.v.e.sd.k..d.de..............q.vke......psrg. cgig_grp78_(bad15288) .e..fd.e..setl.........m....s.mk..kqv.e..d.k.ee.de............v.q.vk.......p.rgv human hsc70(bc019816) npdeavaygaavqaailsgdksenvqdlllldvtplslgietaggvmtvlikrnttiptkqtqtfttysdnqpgvliqvy 480 cgig_hsc71_(bad15287) .......................e........................n............................... mg_hsc71_(cah04109) .......................e........................a............................... human hsp70(p17066) ...............v.m...c.k.........a.....l........t..q..a....................f.... cgig_hsp70_(bad15286) .................k....daik.v..v.................kive..ak....as.............s...f mg_hsp70_(bad99027) ....................r.dtik.v..v..a.............ak..d...k....as.i.........a.s...f human grp78(np_005338) ..............gv....--qdtg..v....c..t.....v.....k..p...vv...ks.i.s.a.....t.t.k.. cgig_grp78_(bad15288) ..............gv...e--.dtg.......n..tm....v.....k..p...v....ks.i.s.aa....t.t...f human hsc70(bc019816) egeramtkdnnllgkfeltgippaprgvpqievtfdidangilnvsavdkstgkenkititndkgrlskediermvqeae 560 cgig_hsc71_(bad15287) .....................................................................de.d...n... mg_hsc71_(cah04109) ......................................................................e.....nd.. human hsp70(p17066) ..............r...s........................s.t.t.r....a...............ev....h... cgig_hsp70_(bad15286) ...........k..t...n............d.e.............k......s..............a......n... mg_hsp70_(bad99027) ...........h....d............k...e.......l.....k.q.s.nsk.......r........d...sd.. human grp78(np_005338) ....pl....h...t.d..................e..v....r.t.e..g..nk........qn..tp.e.....nd.. cgig_grp78_(bad15288) ....p.....h.....d..................e..v....k.t.e..g..tk.h.v.q..nn...p......ind.. human hsc70(bc019816) kykaedekqrdkvssknslesyafnmkatvedek-lqgkindedkqkildkcneiinwldknqtaekeefehqqkelekv 640 cgig_hsc71_(bad15287) ...q......eriaa..g.........s..d...-.kd..seg..kt.....e...k.m.q..l.d......k.....g. mg_hsc71_(cah04109) .........k.rita.......s....q......-.kd..ses..ke.m...d...k...a.nl........k.....g. human hsp70(p17066) q......a...r.aa.....ahv.hv.gslqe.s-.rd..pe..rr.mq...r.vla..eh..l.....y...kr...qi cgig_hsp70_(bad15286) t..e..d...qriaar.q....v.tv.qaa..t---gd.lqs...et.srv.s.tvs...n.al..vd.y.fkl..vq.. mg_hsp70_(bad99027) ...e.....tqrit.r.q..n.i.sv.qaig.s---gd.lstq..ddlgka.e.slk...n.sl...d.yddkm...q.i human grp78(np_005338) .fae..k.lkeridtr.e.....ysl.nqig.kek.g..lss...etmekave.k.e..esh.d.di.d.kakk....ei cgig_grp78_(bad15288) .fadd.k.vke..ea..e.....ysl.nqig.kek.g..ls....kt.eeavd.k.k.mes.ad..v.dlka.k....ei human hsc70(bc019816) cnpiitklyqsaggmpg-gmp---------ggfpgggappsggass-gptieevd696 cgig_hsc71_(bad15287) ..........as..a..g...ggmpnfg--..a......gg.sgg--........mg_hsc71_(cah04109) ..............a..g...----nfgga..a...apgsg.tgg.g........m human hsp70(p17066) .r..fsr..ggp.--vp-------------..ssc.tqarq.dp.t-..i.....cgig_hsp70_(bad15286) .s..ma..h-----------------------qn.stgn.gpas..q...v..m.mg_hsp70_(bad99027) .t.vms..hg--------------------.aqn.qsnste.ys..n...v....human grp78(np_005338) vq...s...g----------------------sa.--p..t.eedt--aekd.l-cgig_grp78_(bad15288) vq..m.....----------------------ga..ap....eega--dekd.l-atpase domain peptide binding domain c-terminal domain signal sequence a b 141 fig. 1 predicted amino acid sequences of the bivalve hsp70 family proteins and their domain structures. (a) cartoon showing a linear representation of the general domain structure for the hsp70 protein family. (b) multiple alignment of deduced amino acid sequences of bivalve hsp70, hsc70, and grp78 with the human homologoues showing the position of the conserved domains. sequences from c. gigas (cgig) and m. galloprovincialis (mg) are used as representatives for the bivalve hsp70 and hsc70 sequences. the grp78 sequence from c. gigas is the representative for the endoplasmic reticulum hsp70 of bivalves (yokoyama et al., 2006). genbank accession numbers are given in brackets. identical amino acid residues are indicated by dots. gaps (indicated by dashes) were added to improve the alignment. dashed squares indicate the three hsp70 family signatures idlgttys, ifdlgggtfdvsil, and vvlvggdtripkiqk. a grey square indicates the cterminal localization signal. the alignment was performed with the mega4 software (www.megasoftware.net). ac. numb aaw52766; kourtidis et al., 2006), and from the pectinidae c. farreri (unpublished genbank ac. numb aao38780 and abe77386), a. irradians (genbank ac. numb aas17723; song et al., 2006), and mizuhopecten yessoensis (genbank ac. numb aas17724; song et al., 2006), were originally referred to as inducible hsp70 proteins, although according to the phylogenetic analysis they belong to the bivalve hsc70 cluster (fig. 4). this finding is further supported by the occurrence of structural characteristics of the cognate gene products in all the above protein sequences (piano et al., 2005; kourtidis et al., 2006; figs 2, 3). however, since no data regarding their expression pattern are available, we cannot exclude a different functional classification. also the hsp68 gene product from c. gigas (table 1) has an uncertain phylogenetic position. this sequence is highly differentiated, and lacks the c-terminal eevd tetrapeptide. we can speculate that it represents a pseudogene or is truncated because of sequencing errors. the phylogenetic analysis also indicated that bivalve hsp70 and hsc70 gene sequences are more closely related to other bivalve hsp70 and hsc70, respectively, than to each other. this is a common feature of the hsp70 multigene family, and similar inter-specific homology between hsp70/hsc70 members was also observed in fish and mammals (ohta, 1994; yamashita et al., 2004). it is consistent with the occurrence of hsp70 gene duplication events during evolution, suggesting that inducible and cognate genes undergo divergent evolution. this can explain why inducible and cognate genes, apart from having different expression patterns, also perform related but different functions. in fact, divergent evolution predominates when different functions that have to be maintained are acquired (ohta and nei, 1994). the presence of multiple copies of the heatinducible hsp70 gene products (table 1) is in agreement with the occurrence of gene duplication events, indicating that the evolutionary model proposed for vertebrates (yamashita et al., 2004) could also be applied for the bivalve hsp70. in fact, a recent phylogenetic reconstruction of hsp70 evolution in bivalves indicated the occurrence of multiple duplication events in the hsp70 family, and also suggested a possible scenario for the hsp70 evolution in which three successive duplication events occurred (kourtidis et al., 2006). the hsp90 gene family in bivalves apart from the hsp70 family, there is very little information currently available on gene identification in bivalves for other hsp subfamilies. however, some progress has been made very recently on the cloning and characterization of the 90 kda members of the family. to our knowledge, three full-length protein sequences are currently available, from the pacific oyster c. gigas (choi et al., 2008), the scallops c. farreri (gao et al., 2007) and a. irradians (gao et al., 2008). the multiple alignment indicates that these bivalve hsp90 display a high degree of sequence homology with human hsp90, and contain conserved structural features typical of these proteins. in particular, they possess five signal peptides that are referred to as signatures for the hsp90 family (gupta, 1995; gao et al., 2007), and the meevd consensus sequence at the c-terminus, which characterizes the cytosolic hsp90 proteins (fig. 5). two different cytosolic hsp90 genes have been identified in vertebrates, i.e., hsp90-α and hsp90-β; they are different in so far as the hsp90-β isoform lacks the glutamine-rich sequence (qtqdq) at the n-terminus, a site of phosphorylation by a dsdna-dependent kinase (lees-miller and anderson, 1989). all three bivalve sequences obtained so far lack this qtqdq sequence (fig. 5), so that they display a greater similarity with the vertebrate hsp90-β. until now only genes encoding the hsp90-β isoform have been reported in invertebrates, except for anopheles albimanus, which contains both isogenes (benedict et al., 1996); however, sequencing information is too scarce to exclude the occurrence of a hsp90-α gene in bivalves. the bivalve hsp response to thermal stress field observations variations of hsp expression in nature are correlated with differences in average environmental temperature and degree of environmental heterogeneity across largeand small-scale geographic thermal gradients and through time (hofmann and somero, 1995; roberts et al., 1997; chapple et al., 1998; dahlhoff and rank, 2000; dahlhoff et al., 2001; helmuth and hofmann, 2001). the observed variations encompass a suite of traits including expression of different hsp isoforms, i.e., ubiquitin, 142 cariak_hsc70_(aao41703) aygldkkvgnqsqgernvlifdlgggtfdvsiltiedg-ifevksts cgig_hsc71_(bad15287) ......................................-........ cgig_hsc70_(cac83683) ......................................-........ oed_hsc70_(cac83684) ..........................a...........-........ mg_hsc71_(cah04109) .........----.........................-........ pvir_hsc71_(abq11278) .......at----.........................-........ cfar_hsp70_(abe77386) .........----.........................-........ cfar_hsp70_(aao38780) .........----.........................-........ airr_hsp70_(aas17723) .........----t.k......................-........ myess_hsp70_(aas17724) .........----.........................-........ pfuct_hsp70_(abj97378) .........----.........................-........ ppeng_hsp70_(abj97377) .........----.........................-........ lellipt_hsp70_(abm92345) .........----..................v......-........ cgig_hsp68_(bad15285) .f..e.nii----..k..mvy..............de.sv...l..a cgig_hsp70_(cac83009) ......................................-........ cgig_hsp70_(bad15286) ......nis----..k...................de.s....r..a cgig_hsp70_(aad31042) ......................................-........ oed_hsp70_(cac83010) ......................................-........ oed_hsp70_(aam46634) ......nis----..k...................de.s....l..a oed_hsp70_(aam46635) ......nis----..k...................de.s....r..a cvir_hsp70_(cab89802) ......nis----.dk...................de.s....r..a mg_hsp70_(aaw52766) .........----......................k..-........ mg_hsp70_(bad99027) ......nls----..k...................de.sl...r..a mg_hsp70_(bad99026) ......nls----..k...................de.sl...r..a mg_hsp70_(cah04106) ......nls----..k...................de.sl...r..a mg_hsp70_(cah04107) ......nls----..k...................de.sl...r..a mg_hsp70_(cah04108) ......nls----..k...................de.sl...r..a mg_hsp70_(cae51348) ......nls----..k...................de.sl...r..a med_hsc70_(aad48065) .........----.........................-........ pvir_hsp71_(abj98722) .......at----.........................-........ dipc_hsp70_(abw06851) .........----.........................-.......a vdec_hsp70_(acb38005) ...f............de.s.......a fig 2 multiple sequence alignment of several bivalve hsc70 and hsp70 proteins in the variable region of the atpase domain. grey squares indicated the insertion of the glycosilation (nqsq) motif in the ostreidae, and of the serine (s) residue in the bivalve inducible hsp70 gene products. bivalve specie abbreviations are given in table 1. genbank accession numbers are given in brackets. identical amino acid residues are indicated by dots. gaps (indicated by dashes) were added to improve the alignment. the alignment was performed with the mega4 software (www.megasoftware.net). constitutive, or inducible isoforms, variations in endogenous levels of hsp, and different threshold temperature at which the hsp gene expression is activated. one concept at the basis of this physiological feature is that the function of most hsp requires atp, and so are costly for organisms. considering the energetic costs associated with hsp activity, natural selection appears to have worked towards optimizing the cost/benefit ratio of stress proteins, so that variations in hsp production occur as a function of environmental conditions. however, seasonal acclimatization involves other physiological adaptations, including the different utilization of metabolic pathways, modification of protein turnover, change in membrane composition, etc., so that hsp seasonality appears as only one of the multiple aspects of a complex strategy that allows life and reproduction in a particular habitat (hofmann and somero, 1996; chapple et al., 1998; feder and hofmann, 1999). a number of fundamental experiments carried out in the past (hofmann and somero, 1996; chapple et al., 1998; feder and hofmann, 1999; hofmann, 1999) provided some crucial evidence that is still at the basis of our understanding of hsp expression. there were observed to be seasonal differences in the endogenous hsp70 levels, which were higher in summer than in winter-acclimatized mytilus trossulus (hofmann and somero, 1995). similarly, in mytilus californianus collected from the field, hsp70 levels were higher in summer than in winter (roberts et al., 1997). in the same intertidal mussels, the threshold temperature of hsp induction showed seasonal differences, rising to higher temperatures in summer. amongst intertidal mussels, differences in latitude distribution were correlated with 143 cariak_hsc70_(aao41703) nvsavdkstgkenkititndkgrlskdeidrmvneaekykqedekqreriaaksglesyafnmkstvddeklkdkisegd cgig_hsc71_(bad15287) .....................................................n.......................... cgig_hsc70_(cac83683) .....................----------------------------------------------------------oed_hsc70_(cac83684) .....................----------------------------------------------------------mg_hsc71_(cah04109) ..........................e..e....d.....a.....kd..t..ns....s....q..e..........s. pvir_hsc71_(abq11278) ..........................e..e....d.....d.....kd..g..ns............e..........d. cfar_hsp70_(abe77386) ............d...........t.e..e....d...h.a..dv..n.vs..n.......r....ae.d........e. cfar_hsp70_(aao38780) ..........................e..e....d.....a..dv..n.vs..n.......q....ae.d........e. airr_hsp70_(aas17723) .............................e....d.....a..dv..s.vs..na......q.....e.dn..s....d. myess_hsp70_(aas17724) ..........................e..e....d.....s..dt..n.vss.n.......q....ae.d........e. pfuct_hsp70_(abj97378) ..q.t...........................ls............kd..t..ns..........i.e........e... ppeng_hsp70_(abj97377) ..............................k.lsd...........kd..g..n.............e.........dt. lellipt_hsp70_(abm92345) ....a......................d......d.....n.....kn..q..ns....s.......e..........e. cgig_hsp68_(bad15285) ..t.r.rd...s.q..vsk..--..p..lns.k.k.m..qe...legq.veh.nh....lifvqkcaqfa--eealddre cgig_hsp70_(cac83009) h....................----------------------------------------------------------cgig_hsp70_(bad15286) ....k......s..............ad.e.......t..e..d...q....rnq....v.tv.qaae.t--g..lqse. cgig_hsp70_(aad31042) .....................................................n.......................... oed_hsp70_(cac83010) .....................----------------------------------------------------------oed_hsp70_(aam46634) ....k......s.h............ad.e...sd..r..e..d...q..ggrnq....v.sv.qvteen--g..lqse. oed_hsp70_(aam46635) ....k......s.r............ad.e..........e..d...q..g.rnq....v.sa.qateen--g..lqse. cvir_hsp70_(cab89802) ....k......s............t.ad.e.......r..e..d...q....rnq....v.tv.qateen--g..lqge. mhg_hsp70_(aaw52766) ..........................e..e....d.....a.....kd..t..ns....s....q..e..........s. mg_hsp70_(bad99027) ....k.q.s.nsk.......r.....ed.....sd.....e.....tq..tsrnq..n.i.sv.qaig.s--g..l.tq. mg_hsp70_(bad99026) ....k.q.s.nsk.......r.....ed.....sd.....e.....tq..tsrnq..n.i.sv.qaig.s--g..l.tq. mg_hsp70_(cah04106) ....k.q.s.nsk.......r.....ed.....sd.....e.....tq..tsrnq..n.i.sv.qaig.s--g..l.tq. mg_hsp70_(cah04107) ....k.q.s.nsk.......r.....ed.....sd.....e.....tq..tsrnq..n.i.sv.qaig.s--g..l.tq. mg_hsp70_(cah04108) ....k.q.s.nsk.......r.....ed.....sd.....e.....tq..tsrnq..n.i.sv.qaig.s--g..l.tq. mg_hsp70_(cae51348) ....k.q.s.nsk.......r.....ed.....sd.....e.....tq..tsrnq..n.i.sv.qaig.s--g..l.tq. cariak_hsc70_(aao41703) kktildkceeiikwmdqnqladkeefehkqkelegvcnpiitklyqasggapgggmpg--gmpn--fgggapggg-apgg cgig_hsc71_(bad15287) ..........................................................--....--.........-.... cgig_hsc70_(cac83683) -.........................................................--....--.........-.... oed_hsc70_(cac83684) -.........................................................--....--.........-.... mg_hsc71_(cah04109) ..e.m...d.....l.a.n..e........................sa.........nf-.gag----.apg.apgsg.t pvir_hsc71_(abq11278) ..v.m...d.....l.a.t..e.....d......kt...........a......aggmp-.gmp--nf...g.ptgga.s cfar_hsp70_(abe77386) ....a...s.v.s.l.a....e............a.....v.....ga......mpg----gmpggmp.----.adgast cfar_hsp70_(aao38780) ....t...s.v.s.l.a....e............a.....v.....ga......mpg.mp.gmpggmp.----.adgast airr_hsp70_(aas17723) ....v...s.v.t.l.a....e.d.y....................ga...g.mpggmp-.gmpggmp..m...adsqst myess_hsp70_(aas17724) ....s...s.v.a.l.a....e............ai....v.....ga......mpg.mp.gmpggmp..m...adgast pfuct_hsp70_(abj97378) .nk.ke..d.....l.t....e.....d......ke...........a......a---p-.gmpp-nf...a-p.ggse. ppeng_hsp70_(abj97377) ..q.t...d.....l.a....e...y.d......................ga.ap-----.gmp--nf...a-p.g..da lellipt_hsp70_(abm92345) ..i.....ndv.t.l.a....et....qq..d..ka....v....gga..p--.....--...gg-..a....e.a.... cgig_hsp68_(bad15285) lqslsll.nktfs.l.h.sg.alf.l.l.l..vqvf.s...e.iqn..k------------------------------cgig_hsp70_(cac83009) -..........n..............................................--....--.........-.... cgig_hsp70_(bad15286) .e..srv.s.tvs.l.n.a..evd.y.f.l..vqk..s..ma..h---qngst.npgp---------------------a cgig_hsp70_(aad31042) ..........................................................--....--.........-.... oed_hsp70_(cac83010) -.........................................................--....--....dk...-.... oed_hsp70_(aam46634) .e..ssm.s.tls.l.n.a..eid.y.f.l..vqk..s..ma..h---qngcsenpnf---------------------. oed_hsp70_(aam46635) .e..skv.n.tls.l.n.a..eid.y.f.l..vqk..s..ma..h---qngss.npgh---------------------s cvir_hsp70_(cab89802) re.vsrv.s.tvs.l.n.a..evd.y.f.l..vqk..s..ma..h---qngss.nsgh---------------------a mhg_hsp70_(aaw52766) ..e.m...d.....l.a.n..e........................sa.........nf-.gag----.apg.apg.g.t mg_hsp70_(bad99027) .ddlgka...sl..l.n.s..e.d.ydd.m...qki.t.vms..hggaqngqsnste.---------------------y mg_hsp70_(bad99026) .edlgka...sl..l.n.s..e...ydd.m....k..t.vms..hggaqng.ssstg.---------------------h mg_hsp70_(cah04106) .ddlgka...sl..l.n.s..e...ydd.m...qki.t.vms..hggaqngqsnstg.---------------------q mg_hsp70_(cah04107) .ddlgka...sl..l.n.s..e.d.ydd.m...qki.t.vms..hggaqngqsnste.---------------------y mg_hsp70_(cah04108) .ddlgka...sl..l.n.s..e.d.ydd.m...qk..t.vms..hggaqngqsnstg.---------------------q mg_hsp70_(cae51348) .ddlgka...sl..l.n.s..e.d.ydd.m...qki.t.vms..hggaqngqsnstg.---------------------y cariak_hsc70_(aao41703) gsgggptieevd cgig_hsc71_(bad15287) ............ cgig_hsc70_(cac83683) ............ oed_hsc70_(cac83684) ............ mg_hsc71_(cah04109) .gs......... pvir_hsc71_(abq11278) .gs......... cfar_hsp70_(abe77386) .g.......... cfar_hsp70_(aao38780) .g.......... airr_hsp70_(aas17723) .gs......... myess_hsp70_(aas17724) .gs......... pfuct_hsp70_(abj97378) ..s......... ppeng_hsp70_(abj97377) .t.......... lellipt_hsp70_(abm92345) ............ cgig_hsp68_(bad15285) -----------cgig_hsp70_(cac83009) ............ cgig_hsp70_(bad15286) s.sq...v..m. cgig_hsp70_(aad31042) ............ oed_hsp70_(cac83010) ............ oed_hsp70_(aam46634) d.nq-------oed_hsp70_(aam46635) ...q-------cvir_hsp70_(cab89802) s..q...v..m. mhg_hsp70_(aaw52766) .gs......... mg_hsp70_(bad99027) s.sn...v.... mg_hsp70_(bad99026) n.sn...v.... mg_hsp70_(cah04106) stsn...v.... mg_hsp70_(cah04107) s.sn...v.... mg_hsp70_(cah04108) stsn...v.... mg_hsp70_(cae51348) s.sn...v.... 144 fig 3 multiple sequence alignment of several bivalve hsc70 and hsp70 proteins in the variable region of the cterminal domain. a grey square indicates the characteristic gg[a,m]p repeats characterizing the hsc70 gene products. a solid-line square indicates the 60 amino acid deletion showed by four ostreidae gene products. a dashed-line square indicates the truncated c-terminal region of the c. gigas hsp68 gene product. bivalve species abbreviations are given in table 1. genbank accession numbers are given in brackets. identical amino acid residues are indicated by dots. gaps (indicated by dashes) were added to improve the alignment. the alignment was performed with the mega4 software (www.megasoftware.net). interspecies difference in thermal sensitivity. m. trossulus, the northern species along the pacific coast of usa, appeared more sensitive to heat stress than the congener m. galloprovincialis, living at a southern latitude (hofmann and somero, 1996). both species were acclimated at 13 °c and analysed for some parameters related to thermal sensitivity. higher levels of hsp70 were found in m. trossulus than in m. galloprovincialis, indicating that a greater degree of protein denaturation took place in the former. moreover, higher levels of ubiquitinated proteins were measured in m. trossulus, indicating a higher protein degradation, which was consistent with the lower protein stability of organisms living at more northern latitudes (hofmann and somero, 1996). c. gigas showed significant increases in constitutively expressed hsp70 in summer as compared to winter. consistently, increases in thermal limits and threshold for stress-inducible isoforms were observed (hamdoun et al., 2003) the expression of hsp is under the control of specific transcription factors (scharf et al., 1998), and there is evidence that this regulation differs among organisms, according to their life history and adaptation capacity (hofmann and somero, 1995). in nature, organisms continuously experience changes in the physico-chemical conditions of the environment, on a day or seasonal scale. a wellstudied case is that of intertidal molluscs, which undergo regimes of immersion and emersion and are exposed to a series of abiotic stresses. during midday low tide, mussels may experience an increase of up to 25 °c in body temperature within 8 h accompanied by a strong induction of hsp70 synthesis (hofmann and somero, 1995). the hsp70 synthesis in m. trossulus was compared with that in m. galloprovincialis, a species which is phylogenetically and ecologically similar, although living at a lower latitude and higher temperatures. a comparison of the intensity of the hsp70 expression following thermal stress in individuals from these two species, either collected from the natural environment or maintained in aquaria, showed that it was significantly higher in m. trossulus (about 8 times the basal level) than in m. galloprovincialis (1.5 times). moreover, the northern species showed an increase of different hsp70 isoforms and greater levels of damaged proteins as a consequence of similar temperature changes (hofmann, 1999). interestingly, hsp expression differed for mussels living at different heights in the intertidal zone; specimens of m. californianus occurring in locations farther up the shore displayed higher hsp70 levels than mussels collected from the lower portion of the mussel bed (roberts et al., 1997). these considerations are supported by further results, including the very recent observations on m. californianus analysed along a vertical stress gradient, from the low intertidal lowstress zone to the high intertidal high-stress zone (petes et al., 2008). high-edge mussels developed an hsp response modulated by acute stress during a single tide, and chronic stress from repeated exposure at low tide. furthermore, the total hsp70 produced was always significantly higher in the high-edge mussels. a negative correlation was found in these mussels between hsp synthesis and reproductive potential. maintenance of hsp response is costly for organisms, and this may negatively influence a diversified energy allocation. according to the above studies, the temperature of acclimatization or acclimation influenced the set-point of the “cellular thermometer” and changed the temperature required to induce hsp synthesis in mussels. therefore the hypothesis proposed by several authors (hofmann and somero, 1995, 1996; chapple et al., 1998; hofmann, 1999; buckley et al., 2001) that environmentally-induced protein damage plays a role in setting the limits of species distribution is rather convincing. as antarctic animal species have evolved under a cold and thermally stable environment, it seemed plausible that the inability of heat shock to induce the hsp response was due to the loss of the regulation pathway in the hsp gene expression during their evolutionary history (hofmann et al., 2000; la terza et al., 2001). however, the antarctic clam l. elliptica showed the constitutive expression of hsp70-mrna levels in gills and digestive glands at their living temperature of 1 °c, and levels were enhanced by exposure to 10 °c. the transcript levels were significantly higher than in controls after 6 h, increasing further up to 12 24 h, and declining thereafter (park et al., 2007). gills were more responsive to stress than digestive glands, developing a faster and more marked response. this ability would appear to be of great importance as we move towards global warming scenarios, when temperature will be a major factor affecting the growth and survival of antarctic species. interesting persective is provided by studies on deep-sea mussels. bathymodiolus childressi is adapted to a cold, thermally stable environment, it is exposed to a variety of stressors, including hydrocarbons, low oxygen levels, high salinity and hydrogen sulphide. interestingly, this species possesses a high thermal tolerance, although it does not express an inducible hsp70 protein. high constitutive levels of hsp70 are indeed present that probably remediate protein damage from the above 145 fig d. bivalves hsp70 gasteropods hsp70 mammals hsp70 fish hsp70 fish hsc70 mammals hsc70 molluscs hsp70 molluscs hsc70bivalves hsc70 mg hsp70 (bad99027) bivalves hsc70 gasteropods hsc70 mg hsp70 (cae51348) mg hsp70 (bad99026) mg hsp70 (cah04106) mg hsp70 (cah04107) mg hsp70 (cah04108) cvir hsp70 (cab89802) cgig hsp70 (bad15286) oed hsp70 (aam46634) oed hsp70 (aam46635) has hsp70 (abr15462) bglab hsp70 (aab95297) bglab hsp70 (aab99911) h. sapiens hsp70b(x51757) h. sapiens hsp70 1a(aah18740) b. taurus hsp70(np776769) m. musculus hsp70(aah04714) d. rerio hsp70(aah56709) o. tshawytscha hsp70(q91233) o. mykiss hsp70(bab72233) o. mykiss hsc70(p08108) x. maculatus hsc70(bab72169) d. rerio hsc70(aaq97970) b. taurus hsc70(p19120) r. norvergicus hsc70(np077327) h. sapiens hsc70 (bc019816) ppeng hsp70 (abj97377) airr hsp70 (aas17723) myess hsp70 (aas17724) cfar hsp70 (aao38780) cfar hsp70 (abe77386) hdish hsp70 (abc54952) htub hsp70 (cak95236) baz hsp70 (caj40877) mg hsc71 (cah04109) pvir hsc71 (abq11278) mg hsp70 (aaw52766) pfuct hsp70 (abj97378) lellipt hsp70 (abm92345) cgig hsc70 (cac83683) oed hsc70 (cac83684) oed hsp70 (cac83010) cariak hsc70 (aao41703) cgig hsp70 (cac83009) cgig hsc71 (bad15287) cgig hsp70 (aad31042) ssa1(hhbya1) 100 99 99 94 77 68 89 60 83 74 66 65 62 58 52 54 0.01 fig. 4 phylogenetic relationship among hsp70 deduced amino acid sequences of mammals, fish, and bivalves. the tree was constructed by the neighbour joining algorithm using the mega 4 software (www.megasoftware.net). bootstrap confidence values for the sequence groupings are indicated in the tree (n = 1000 replicates). ssa1 from s. cerevisae was used as outgroup. bivalve species abbreviations are given in table 1. gasteropod specie abbreviations: bglab, biomphalaria glabrata; has, haliotis asinina; hdish, h. discus hannai; htub, h. tubulata. genbank accession numbers are given in brackets. 146 stressors and confer tolerance against thermal stress (berger and young, 2006). differently, bathymodiolus azoricus inhabits the harsh hydrothermal vent environment, being thus exposed to elevated temperature and pressure, heavy metals, sulphide and radionuclides. the expression patterns of the hsp70 levels in b. azoricus show tissue-specific and cellular localization differences. a constitutive hsp70 was detected in mantle and in gills. two additional hsp70 isoforms were found in this latter tissue, one of which was stress-inducible, pointing to a crucial role played by these stress proteins throughout the life of the vent mussel in its naturally hostile environment (pruski and dixon, 2007). laboratory observations all bivalves studied to date showed hsp70 overexpression in response to thermal stress, whether studied in the natural environment or in laboratory conditions. within the wide range of ostreidae distribution, water temperature may occasionally exceed 40 °c, evoking a strong synthesis of inducible hsp, as demonstrated in haemocytes of c. virginica (tirard et al., 1995). gills of c. gigas acclimated at 12 °c and exposed to 37 °c for 1 h showed an increase in the constitutive isoforms hsp72 and 77, together with the induction of a newly synthetized 69 kda isoform (clegg et al., 1998). in o. edulis we recognized two hsp isoforms of about 72 and 77 kda in both gills and mantle of animals maintained in control conditions, while expression of a 69 kda isoform increased after 1 h of exposure to heat shock at different temperatures. the hsp69 protein was newly induced after exposure at temperatures ≥32 °c. the maximum expression measured after 3 h of post-stress recovery was detected at 35 °c, while individuals exposed to 38 °c showed low, if any, expression of hsp69 (piano et al., 2002). further experiments showed that in oysters exposed for 1 h to 38 °c the hsp69 mrna transcription actually was performed, but after a striking delay, as it was significant after 24 h of post stress recovery (piano et al., 2004). we argued that the high temperature partially compromises the biochemical machinery at the basis of the hsp response, and that this delayed response may be related to the high mortality of o. edulis observed at this temperature (piano et al., 2002). the maximum expression of hsp69 caused by 1 h of heat shock at 35 °c was developed after 24 and 48 h of post-stress recovery in the gills and mantle, respectively, and the protein was still clearly detectable in both tissues 7 days after the heat challenge, persisting in the gills for up to 14 days. high levels of hsp70 were also expressed in tissues of c. gigas for up to 14 days after hs, during which period the animals were able to tolerate an otherwise lethal heat shock (clegg et al., 1998). expression of hsp72 and hsp77 in the gills and mantle of o. edulis remained easily detectable at the end of the 14 day period. this confirms the constitutive role of these proteins. the hsp69 expression was never detected in the digestive glands of oysters exposed to thermal stress. the expression of hsp69 in gills and mantle of c. gigas was induced at temperatures ≥38 °c, which interestingly corresponded to the apparent half-lethal temperature stated for o. edulis, and densitometric analysis indicated that the maximum was reached at 40 °c in both tissues. in agreement with previous reports (clegg et al., 1998), 44 °c was found to be the minimum lethal temperature for c. gigas. according to the models for the transcriptional activation of hsp genes, denatured proteins are the trigger for the enhancement of hsp synthesis, and it has been suggested that induction of the hsp response mirrors the thermal stability of cell proteins (dietz and somero, 1992). in this context, the biochemical machinery of o. edulis might have a higher susceptibility to heat than that of c. gigas. an alternative suggestion is that heat directly activates a single transcriptional factor, hsf1, which trimerizes and binds to specific regions in the promoter of hsp genes (zhong et al., 1998). in this case, the hypothesis is that the two oysters might differ as to the thermal sensitivity of the promoter or the hsf. as a matter of fact, c. gigas is more resistant to stress stimuli than are other oyster species (tirard et al., 1995), and one could speculate that a higher threshold of stress sensitivity contributes to the ability of c. gigas to colonize new habitats in competition with autochthonous species. the expression of newly synthesised hsp70 isoforms, like the hsp69 in oysters, is not a common feature in bivalves exposed to thermal stress. the clams tapes philippinarum and scapharca inaequivalvis exposed to thermal stress for 1 h in the range of 30 40 °c did not show the expression of merely inducible isoforms, although hsp proteins already present in control conditions were significantly over-expressed by heat (piano et al., 2004). moreover, reports from different laboratories on different species indicate that also mussels exposed to thermal stress display a strong overexpression of apparently two hsp70 isoforms already present in control conditions without the synthesis of new isoforms (hofmann and somero, 1995, 1996; piano et al., 2004). however, due to a better resolution or more probably to the different specificity of the antibody used, snyder et al. (2001) reported three distinct hsp70 bands of 67, 70, and 74 kda. in digestive glands of the mussel m. galloprovincialis. the hsp67 was poorly expressed and not over-expressed by heat. after animal exposure to 28 °c for 1 h, hsp70 and hsp74 declined within 2 h, whereas they increased after 15 h of post stress recovery at 15 °c. this feature may explain why no increase of hsp70 was shown in the digestive glands of mussels exposed to heat shock and allowed to recover for up to 6 h (piano et al., 2004). table 2 summarizes the patterns of hsp70 bands detected by western blotting in several stress conditions, analysed in different organisms and tissues through different antibodies. the occurrence of stress-inducible hsp70 mrna or the related proteins under unstressed conditions is consistent with the ability of mussels and some clams to thrive in transitional environments, where significant fluctuations in physical and chemical parameters may 147 hsapiens_hsp90a_(np_005339) --mpeetqtqdqpmeeeevetfafqaeiaqlmsliintfysnkeiflrelisnssdaldkiryesltdpskldsgkelhi 80 hsapiens_hsp890b1_(np_031381) --....vhhg-----......................................a........................k. cgig_hsp90_(abs18268) --...pehm-----..g....................................a......................d.e. cfar_hsp90_(aar11781) mpe..gqam-----.dg.........g..........................c........................e. airr_hsp90_(abs50431) --...nqam-----.dgd...................................c......................d.e. hsapiens_hsp90a_(np_005339) nlipnkqdrtltivdtgigmtkadlinnlgtiaksgtkafmealqagadismigqfgvgfysaylvaekvtvitkhndde 160 hsapiens_hsp890b1_(np_031381) di...p.e....l.........................................................v......... cgig_hsp90_(abs18268) riv.d.esk....m...........v.........................................dr.v.e....... cfar_hsp90_(aar11781) kiv...d.n..s.m...........v.......r.................................dr.v.e..n.... airr_hsp90_(abs50431) kiv...d.n....m...........v.......r.................................d..v.e..n.... hsapiens_hsp90a_(np_005339) qyawessaggsftvrtdtgepmgrgtkvilhlkedqteyleerrikeivkkhsqfigypitlfvekerdkevsddeaeek 240 hsapiens_hsp890b1_(np_031381) ...............a.h...i......................v..v..............yl....e..i.......e cgig_hsp90_(abs18268) ..i...........k.csenti.....it.f................v............k.l.............e..e cfar_hsp90_(aar11781) h.i............sg-dgsfil..rit..m....a.....kkv...............k.q......v......e..e airr_hsp90_(abs50431) h.i............sg-dgsfn....it..m....a.....kkv...............k.q......v......e..e hsapiens_hsp90a_(np_005339) edkeeekekeekesedkpeiedvgsdeeeekkdgdkkkkkkikekyidqeelnktkpiwtrnpdditneeygefyksltn 320 hsapiens_hsp890b1_(np_031381) kg---...e.d.dd.e..k.........ddsgkdk...t............................q............ cgig_hsp90_(abs18268) kk--..dk-a.eke....kv..ld-ed..ddskskd..........ted..................q............ cfar_hsp90_(aar11781) kk--..dkda..sed...kv..lddeddd.d-kskd.......g...ed..................q............ airr_hsp90_(abs50431) kk--..dkda..nede..kv..lddeddddddkskd..........med..................q............ hsapiens_hsp90a_(np_005339) dwedhlavkhfsvegqlefrallfvprrapfdlfenrkkknniklyvrrvfimdnceelipeylnfirgvvdsedlplni 400 hsapiens_hsp890b1_(np_031381) ........................i...........k.................s.d....................... cgig_hsp90_(abs18268) ...r-----p.gc...........i.....l.....k.............................a............. cfar_hsp90_(aar11781) ....................................k...................n.v.......v............. airr_hsp90_(abs50431) ....................................k...................d.v.......v............. hsapiens_hsp90a_(np_005339) sremlqqskilkvirknlvkkclelftelaedkenykkfyeqfskniklgihedsqnrkklsellryytsasgdemvslk 480 hsapiens_hsp890b1_(np_031381) .................i........s..............a....l........t..rr.......h..q.....t..s cgig_hsp90_(abs18268) ......................i..ied.t...d.........a..l........t.....adf....s.q.....t... cfar_hsp90_(aar11781) ......................m...ddi..............a..l.......tt....iadf...h..q.....t.f. airr_hsp90_(abs50431) ......................m...ddi..............a..l.......tt....iadf...h..q.....t.f. hsapiens_hsp90a_(np_005339) dyctrmkenqkhiyyitgetkdqvansafverlrkhgleviymiepideycvqqlkefegktlvsvtkeglelpedeeek 560 hsapiens_hsp890b1_(np_031381) e.vs....t..s.......s.e..........v..r.f..v..t..............d..s.................. cgig_hsp90_(abs18268) ..vs.......s.......srev.qs......vk.r.m.....vd.....a......yd..p..n..............r cfar_hsp90_(aar11781) e.vs.......s.......srev.qs.....nvk.r.i.....vd.....a......y...................... airr_hsp90_(abs50431) e.vs.......s.......srev.qs.....nvk.r.i.....vd.....a......yd..................... hsapiens_hsp90a_(np_005339) kkqeekktkfenlckimkdilekkvekvvvsnrlvtspccivtstygwtanmerimkaqalrdnstmgymaakkhleinp 640 hsapiens_hsp890b1_(np_031381) ..m..s.a.......l..e..d......ti.....s..................................m......... cgig_hsp90_(abs18268) .rf..aeaey.g...v.....d......................q...s..............s................ cfar_hsp90_(aar11781) .rf..ataey.g...vv.e..d......t...............q...s..............s....c........... airr_hsp90_(abs50431) .rf..ataay.g...vi.e..d......t...............q...s..............s................ hsapiens_hsp90a_(np_005339) dhsiietlrqkaeadkndksvkdlvillyetallssgfsledpqthanriyrmiklglgideddptaddtsaavteempp 720 hsapiens_hsp890b1_(np_031381) ..p.v..............a.....v..f.................s................eva.eepn...pd.i.. cgig_hsp90_(abs18268) .....ks.kd...............m..f..s..a......e.g...s..h............e--tpe.qep...d... cfar_hsp90_(aar11781) ..a..ks.ke..gl...........l..f..sm.a......e.g......h..........d..sg.pe..denv..p.. airr_hsp90_(abs50431) ..a..ks.ke..t............l..f..sm.a......e.g......h..........d..ag..n.-ees...... hsapiens_hsp90a_(np_005339) legd-ddtsrmeevd 735 hsapiens_hsp890b1_(np_031381) ....-e.a....... cgig_hsp90_(abs18268) ....e..a....... cfar_hsp90_(aar11781) ....e..a....... airr_hsp90_(abs50431) ....e..a....... fig. 5 multiple alignment of deduced amino acid sequences of bivalve hsp90 with the human homologues (hsp90α and hsp90β) showing the position of conserved domains. the hsp90 sequences available for the bivalves were from c. gigas (cgig), c. farreri (cfar) and a. irradians (airr). genbank accession numbers are given in brackets. identical amino acid residues are indicated by dots. gaps (indicated by dashes) were added to improve the alignment. a solid-line square indicates the glutamine-rich qtqdq consensus sequence; a grey square indicates the hsp90 family signatures. a dashed-line square indicates the c-terminal localization signal. the alignment was performed with the mega4 software (www.megasoftware.net). occur. to minimize the effects of these environmental stressors, these animals may elaborate a molecular strategy where inducible hsp70 isoforms are physiologically expressed at low levels, their synthesis promptly increasing as the animal experiences adverse environmental change. we believe that the relatively rapid induction of the heat shock response by means of the inducible hsp70 gene product is another component of the molecular adaptation of mussels to transitional environments, providing an effective tool to cope with rapidly changing environmental conditions. this hypothesis is corroborated by the occurrence of analogous mechanisms in several mussel species (minier et al., 2000; buckley et al., 2001) and aquatic vertebrates exposed to fluctuating environments, including the teleosts fundulus heteroclitus (koban et al., 1991) and sparus sarba (deane and woo, 2005). further and more detailed observations have recently been made of the hsp response in different tissues of animals exposed to thermal stress. in the 148 table 2 an overview of hsp70 detected in different bivalve species by western blotting species tissue hsp70 primary antibody immunogen reference m. galloprovincialis m, pam 2 bands mouse mab clone brm-22 (h-5147) sigma-aldrich bovine brain hsp70 (anestis et al., 2007) m. trossulus g 2 bands rat mab clone 7.10 (ma3-001) affinity bioreagents human hsp70 (437-479 aa) (buckley et al., 2001) g, m 3 bands m. edulis pam 2 bands mouse mab clone 5a5 (ma3-007) affinity bioreagents human hsp70 (122-264 aa) (chapple et al., 1997, 1998) p. perna g 2 bands rabbit pab (spa-811) stressgen human hsp70 (franco et al., 2006) f no bands g 1 band h 2 bands pam 1 band m. galloprovincialis m 1 band mouse mab clone n27f3-4 calbiochem human hsp70/hsc70 (from hela cells) (gonzalez-riopedre et al., 2007) m. galloprovincialis g 2 bands mouse mab clone brm-22 (h-5147) sigma-aldrich bovine brain hsp70 (hamer et al., 2004) m. trossulus g up to 4 bands rat mab (clone 7.10) dr susan lindquist hsp70/hsc70 (hofmann and somero, 1995) m. trossulus g up to 3 bands m. galloprovincialis g up to 3 bands rat mab clone 7.10 (ma3-001) affinity bioreagents human hsp70 (437-479 aa) (hofmann and somero, 1996) m. galloprovincialis g, m 1 band rabbit pab cell signalling technology human hsp70 (kefaloyianni et al., 2005) m. galloprovincialis h 1 band goat pab santacruz human hsp70 (c-terminus) (malagoli et al. 2004, 2006) m. galloprovincialis dg 2 bands mouse mab clone brm-22 (h-5147) sigma-aldrich bovine brain hsp70 (minier et al., 2000) m. edulis g, m 2 bands g 3 bands (1 merely inducible) b. azoricus m 1 band rabbit pab (spa-812) stressgen human hsp70 (hsp72) (pruski and dixon, 2007) m. edulis g 1 band mouse mab clone 3a3 (ma3-006) affinity bioreagents recombinant human hsp70 (over expressed in e. coli.) (radlowska and pempkowiak, 2002) m. californianus g up to 4 bands (1 merely inducible) rat mab (clone 7.10) dr. susan lindquist hsp70/hsc70 (roberts et al., 1997) g 3 bands (2 merely inducibles) m. edulis m 1 band rabbit pab (s. ullrich, according to ehrhart et al., 1988) human hsp70 (23 aa at the c-terminus) (sanders and martin, 1993; sanders et al., 1994) d. polymorpha soft tissues 2 bands mouse mab clone brm-22 (h-5147) sigma-aldrich bovine brain hsp70 (singer et al., 2005) m. edulis g 3 bands (1 merely inducible) mouse mab clone 5a5 (ma3-007) affinity bioreagents human hsp70 (122-264 aa) (smerdon et al., 1995) m. galloprovincialis dg 3 bands mouse mab (spa-822) stressgen chicken hsp70/hsp90 complex (snyder et al., 2001) m. galloprovincialis m 3 bands (1 merely inducible) mouse mab clone brm-22 (h-5147) sigma-aldrich bovine brain hsp70 (toyohara et al., 2005) c. gigas g 3 bands (1 merely inducible) rat mab clone 7.10 (ma3-001) affinity bioreagents human hsp70 (437-479 aa) (clegg et al., 1998) c. gigas h 2 bands (1 merely inducible) mouse mab clone 3a3 (ma3-006) affinity bioreagents recombinant human hsp70 (over expressed in e. coli.) (lacoste et al., 2001a) c. gigas g 3 bands (1 merely inducible) rat mab clone 7.10 (ma3-001) affinity bioreagents human hsp70 (437-479 aa) (hamdoun et al., 2003) dg 2 bands o. edulis g, m 3 bands (1 merely inducible) c. gigas g 3 bands (1 merely inducible) rat mab clone 7.10 (ma3-001) affinity bioreagents human hsp70 (437-479 aa) (piano et al., 2002, 2004) c. virginica h 2 bands mouse mab clone 3a3 (ma3-006) affinity bioreagents recombinant human hsp70 (over expressed in e. coli.) (tirard et al., 1995) r. decussatus dg, g, m 1 band rabbit pab (386035) calbiochem recombinant human hsp70 (hsp72) (dowling et al., 2006) s. inaequivalvis g 1 band t. philippinarum g 1 band rat mab clone 7.10 (ma3-001) affinity bioreagents human hsp70 (437-479 aa) (piano et al., 2004) key abbreviations: dg, digestive gland; f, foot muscle; g, gills; h, hemocytes; m, mantle; pam, posterior adductor muscle 149 mussel m. galloprovincialis, all the tissues examined developed a different response to heat shock. considering only the results obtained by western blotting, since these were more comparable to other studies where mammalian anti-hsp antibodies were used, gonzalez-riopedre et al. (2007) observed that mussels acclimated at 20 °c displayed different proteins in different tissues: hsp70 was detected in the mantle, in the adductor posterior muscle and in the hemocytes; hsp60 was present only in the mantle and in the gills. the posterior muscle displayed only hsp70, while neither hsp70 nor hsp60 was present in basal conditions in the foot muscle. however, hsp70 was no longer detected in the mantle from individuals exposed for 5 or 45 min to 45 °c, while levels of hsp60 remained stable. in the adductor posterior muscle hsp70 was overexpressed after 5 min, and was not detected after 45 min of heat shock at 45 °c. in the foot muscle, neither hsp70 nor hsp60 levels were expressed after heat shock. hsp70 was never detected in the gills, while hsp60 was expressed in a significant manner. interestingly, both hsp70 and hsp60 were present in haemocytes withdrawn from stressed animals, although apparently only hsp60 expression was affected by heat; in contrast, haemocytes withdrawn from unstressed animals and cultured for three days at 20 °c did not show basal levels of either hsp70 or hsp60. the two protein isoforms remained absent after 5 min exposure, whereas they were detected after 45 min exposure at 45 °c. a different hsp response in the different tissues was highlighted by different laboratories; for example, we observed (see above) that oyster mantle and gills developed a strong hsp response to thermal stress, while no effect was seen in the digestive glands (piano et al., 2004). however, if we compare the above results with previous findings on mussels, some main discrepancies appear, in particular regarding the hsp70 expression in gills. in fact, hsp70 constitutive expression has been documented in gills of all mussel species studied so far (e.g. hofmann, 1999; piano et al., 2004), significantly increasing after heat shock. the disappearance of both hsp70 and hsp60 isoforms in the mantle of individuals exposed to 45 °c for 5 or 45 min does not fully agree with previous data either. although such factors as seasonality, feeding, reproductive states etc. may interfere with the protein expression, in the light of previous observations it is worth noting that gonzalesriopedre et al. (2007) assessed the hsp response immediately after heat shock, which lasted 5 or 45 min. in other laboratory experiments, a period of post-stress recovery was adopted to let the organisms develop the response (piano et al., 2002, 2004), or the exposure period lasted longer. some hours of treatment and/or of post-stress recovery had in fact been considered necessary in previous experiments to allow the response to develop completely (e.g., hofmann and somero, 1995; piano et al., 2002; franzellitti and fabbri, 2005). the delayed protein expression is also supported by data on gene expression, which show that mrna transcripts reach maximum levels after 3 h of poststress recovery (franzellitti and fabbri, 2005), declining thereafter. similar observations were made on other bivalves, including oysters and clams (piano et al., 2004; park et al., 2007). interestingly in this connection is also the transient reduction of hsp70 transcript levels observed at 1 h of recovery in m. galloprovincialis (franzellitti and fabbri, 2005) as well as in o. edulis exposed to 35 °c for 1 h (piano et al., 2004). this feature is consistent with the peculiar regulation of rna metabolism during the heat shock response (yost et al., 1990) and with the onset of several and probably time-delayed mechanisms underlying hsp70 gene expression control (de maio, 1999). in general, the different responses obtained in the same mussel tissues may be partly attributable to the different times allowed for the hsp response to develop. the disappearance of hsp after 45 min exposure to 45 °c appears to relate to the phenomenon observed in oysters (piano et al., 2002), where the animals’ exposure to 38 °c, a temperature lethal for most organisms, led to a strong reduction of hsp70 expression levels. concurrently, the mrna transcripts were absent and were once more expressed only after 24 h after the heat shock (piano et al., 2004). mussel hemocyte cells withdrawn from stressed mussels displayed basal levels of hsp and a significant hsp response to heat (gonzalesriopedre et al., 2007), apparently involving different protein isoforms. only one study focused on hemocytes withdrawn from control mussels, cultured for three days at 20 °c and subsequently exposed to thermal stress (gonzales-riopedre et al., 2007). while these hemocytes did not display hsp60 or hsp70 at 20 °c or after 5 min of exposure at 45 °c, protein bands were detected after 45 min exposure at 45 °c. on the basis of this single report, we can speculate that cultured cells lose the physiological modulation performed by neuroendocrine factors, which may influence the hsp pattern both in basal as well as in stressed conditions (lacoste et al., 2001a), thus leading to substantial differences between the two cell populations. in general, in line with what is reported by hofmann (1999), new evidence confirms that hsp overexpression is a ubiquitous molecular mechanism for coping with stress, but animals show individual responses with different thresholds of sensitivity and tissue specificity. some of these differences have been analysed in the same individuals exposed to different conditions, and account for physiological differences in thermal stress response. the bivalve hsp response to non-thermal stress although the term hsp specifically refers to heat shock, the accumulation of these proteins is not increased only by heat. a large body of evidence indicates that there is a variety of stimuli that result in an increase in their concentrations. the question arises of how different harmful stimuli can provoke such a similar effect. a first explanation was provided by hightower (1991), who noticed that many factors responsible for the hsp response acted in vitro as protein denaturating agents, i.e., 150 substances altering the tridimensional configuration of proteins, thus provoking the loss of biological functionality. different chemical substances directly or indirectly affect cell proteins, e.g., by oxidation of thiol groups and disulfide-bonds, thus destabilizing the protein structure. this occurs for example in the case of heavy metals, free radicals, and pesticides (feder and hofmann, 1999; gonzales-riopedre et al., 2007; farcy et al., 2007). organic compounds including alcohols, phenols, and solvents in general can also affect protein integrity by interacting with the hydrophobic domains, normally located within the hydrophobic core (ait-aissa et al., 2000). further evidence showed that radiation must be included in the list of hsp inducers (malagoli et al., 2004). because of the great diversity in toxic factors, it can be hypothesised that the hsp response is triggered by multiple mechanisms; however, proteotoxicity remains at the moment the sole common factor at the cellular level. we discuss here some of the numerous observations on hsp response induced by non-thermal factors in bivalves. effect of non essential metals of the substances able to induce hsp overexpression, heavy metals have attracted major attention. chemicals may have toxic effects at the cell and tissue level and, above a certain threshold, also elicit an integrated stress response. considerable experimental evidence obtained in vitro and in vivo testifies to the ability of cadmium to increase hsp levels in humans (polla et al., 1995; valbonesi et al., 2008), rats (curtis et al., 1996), fish (hansen et al., 2007), sponges (schroder et al., 1999), drosophila (courgeon et al., 1984) etc. no role for cd in the metabolism of living organisms is yet known, and the metal is extremely hazardous to animal life. recent studies implicated cd in the elevated metabolic demand and concomitant impairment of atp production, reduced aerobic capacity and oxidative stress in oysters (cherkasov et al., 2007; ivanina et al., 2008a). the short term effect of cd exposure induced a dose-dependent increase of metallothioneins, hsp60, and hsp70, but not hsp90 synthesis, in c. virginica (ivanina et al., 2008a). within 4 h of exposure to 10 2000 μm cd, a great difference was observed between the tissues analysed, with the hsp expression much greater in gills than in digestive glands. interestingly, this was inversely related to the metallothionein induction, which was greater in oyster digestive glands than in gills (ivanina et al., 2008a). when other evidence on the activation of antioxidant systems was also taken into account, it was concluded that the hsp response is a secondary line of cellular defence significantly activated in gills, when inducible metallothioneins and gsh appear to be insufficient to fully prevent damage due to cd exposure (ivanina et al., 2008a). however, longer animal exposures to 4 μm cd indicated that metallothioneins (at 3, 7 and 15 days) and hsp70 (at 3 and 15 days) can be simultaneously immunolocalized in both gills and digestive glands of c. gigas (moraga et al., 2005). in agreement with previous reports (boutet et al., 2003b) it emerged clearly that hsp70 levels decreased at 7 days. several hypotheses have been made regarding the inhibition of hsp expression by metals or degradation temporarily exceeding the synthesis rate. further considerations emerging from data on constitutive and inducible hsp70 gene expression in mussels exposed to hg2+ or cr6+ (franzellitti and fabbri, 2005) will be discussed below. cd also induced hsp70 overexpression in o. edulis exposed to the metal (100 500 μg/l) for 7 days (piano et al., 2004). at these experimental conditions, cd elicited significant increases of both metallothioneins and hsp70 in gills and digestive glands. the response to heat shock in oysters (clegg et al., 1998; piano et al., 2002) led to a strong induction of hsp69. interestingly, cd induced the expression of hsp69 in gills and also in digestive glands of o. edulis where hsp69 was not induced by heat (piano et al., 2004). this would suggest that the hsp69 mrna transcription is differently regulated by the two stress factors at least in digestive glands. cd affects the expression of hsp90, as was well demonstrated in c. gigas (choi et al., 2008). hsp90 mrna levels increased doseand timedependently, up to a 40 fold expression, after 7 days of treatment at 0.1 ppm cd. expression was significantly reduced after 11 days of exposure, probably due to a decrease in metabolic capacity of the organisms or to oxidative stress generated by prolonged exposure to cd. the response was similar in gills and digestive glands, and indicated that, in bivalves, hsp90 may be induced to maintain homeostasis and protect the cells against xenobiotics. in general few studies are available on the hsp90 response to chemical stressors, and point to a substantial similarity to the hsp70 response. studies carried out at the gene expression level made it possible to clearly distinguish between differently time-modulated hsp responses to heavy metals, and also to establish a differential hsp70 expression in bivalves exposed to different stress stimuli (franzellitti and fabbri, 2005, 2006). time course experiments showed that the inducible hsp70 and the constitutive hsc70 gene expressions were differently modulated during exposure to hg2+. the abundance of hsp70 transcript increased during the early response to hg2+ (8 24 h) and the basal level was recovered within 6 days, while hsc70 expression showed a biphasic response with a reduction at 1 day and an induction at 6 days of treatment. this pattern may be partially related to the peculiar structural, functional, and regulatory features of heat shock genes. in fact, in accordance with their role as stress-responsive genes, hsp70 genes are typically intron-less (gunther and walter, 1994), while the hsc70 coding region is interrupted by several introns. nevertheless, we also hypothesize that differential expression of the two genes is related to a specific mechanism of short and long-term cell protection. the peculiar hsp70/hsc70 expression profile after prolonged hg2+ exposure was confirmed by data from cr6+ exposure. after a 1 week treatment, induction of hsc70 and inhibition of hsp70 expression were observed. in more detail, the mghsp70 expression increased immediately after a 1 h heat shock or after an 8 h heavy metal 151 exposure, while hsc70 was either unmodified or inhibited. hsc70 induction occurred only after a longer-term exposure to hg2+or cr6+, at a time when hsp70 expression had returned to basal levels. franzellitti and fabbri (2006) also showed that the chemical form of the contamination could dramatically affect the toxic potential of metals and, as a consequence, the magnitude of the cellular response to its exposure. ch3hg +, probably by virtue of its higher hydrophobicity, was more effective in evoking a cytoprotective response than hg2+. in particular, the response to ch3hg + exposure was achieved through the strong, stable up-regulation of the constitutive transcript hsc70, while the inducible counterpart hsp70 was progressively down-regulated. this is in contrast with the expression profiles following hg2+ exposure described above, and suggests that the higher toxicity of ch3hg + requires the onset of the chronic, rather than the acute, response pathway. a low density microarray approach was applied to evaluate alterations of gene expression profiles after mussel exposure to hg2+ (dondero et al., 2006). the set of genes included hsp27 and hsp70. the expression of none of them was modified after m. galloprovincialis exposure to 750 nm hg2+ for 6 days, although an alteration in hsp27 expression was expected on the basis of mammalian responses (lavoie et al., 1995). as to hsp70, the oligoprobes were designed in a region of high homology amongst inducible and constitutive hsp70, so that the microarray analysis could not discriminate between the expression of the two genes. these data might reflect the lack of inducible hsp70 overexpression at 6 days of exposure to hg2+ observed in m. galloprovincialis by franzellitti and fabbri (2005); the sum of hsp70 and hsc70 at this stage brought about a protein expression level similar to control values. platinum group elements (pge) have been released in the last few decades mainly as a consequence of anthropogenic activities. major sources are hospital releases, because of pt-based anticancer drugs, and overall car emissions due to the use of pge in catalytic converters. soluble and particle-bound pge are biologically available to living organisms (ravindra et al., 2004). the few data available on the ability of pge to induce hsp overexpression (singer et al., 2005) are nevertheless interesting. zebra mussels, dreissena polimorpha, exposed for up to 10 weeks to 500 μg/l rh, pd and pt, bioaccumulated the metals and showed a significant overexpression of 70 kda proteins. a 19-fold increase compared to the control levels was obtained with pt and rh, and a 25-fold increase with pd. in parallel experiments mussels were exposed to cd or pb, and an increase in hsp70 expression of about 6 and 12 fold respectively was obtained. although further data on pge are not available, the clear induction of hsp70 expression suggests that these metals produced strong proteotoxic effects. effect of essential metals some data are available on the hsp response after bivalve exposure to essential metals, namely copper and zinc. it is well-known that prolonged exposure to cu or zn produces toxic effects and decreases animal fecundity, hatchability and reproduction (lock and janssen, 2003), although these metals have a physiological role (madsen and gitlin, 2007). m. edulis exposed for 7 days to increasing cu concentrations (30-100 μg/l) showed an increased expression of hsp60 and hsp70 in the mantle, and to a greater extent in the gills (sanders et al., 1994). in particular, the highest cu concentration induced the ex novo synthesis of two protein isoforms, with the appearance of three protein bands in the related immunoblotting. radlowska and pempkowiak (2002), evaluated on the same organisms the effects of cd and cu administered alone or in combination resembling the most common condition in nature. these experiments revealed that mixtures had a synergistic effect on hsp70 expression with respect to the single contaminants. moraga et al. (2005) demonstrated the greater effect of a combination of cu and cd on c. gigas hsp70 immunolabelling. in contrast, the combination of cd and zn induced a significant increase of hsp70 expression in gills and digestive glands of o. edulis, an increase which, however, was smaller than the effect of cd alone used at the same concentration (piano et al., 2004). zn per se did not change the hsp70 expression. the lower effect of the combination could be ascribed to a lower water filtering rate by the animal exposed to high levels of contaminants, or to competition between the two metals for cellular uptake mechanisms. there are examples of the reduction of cd assimilation and uptake after perna viridis preexposure to zn (blackmore and wang, 2002). the absence of any effect of zn on hsp70 expression has also recently been confirmed in tissues of brown mussels, perna perna (franco et al., 2006). the different parameters analysed clearly indicated that animals exposed to 10, 30 and 100 μm zn for 48 h were subjected to oxidative stress; in this context, hsp60 proteins were significantly overexpressed while hsp70 levels remained unchanged (franco et al., 2006). the overall responses to the essential metals cu and zn show once again that different species respond differently and that often mixtures of metals induce synergistic effects. although the small number of reports does not allow a more detailed discussion, we may posit that other cytoprotective responses are elicited in cells by excesses of cu and zn, namely metallothioneins and antioxidant enzymes. therefore, the different extent of the hsp response may be related to the different induction of the other cytoprotective responses activated. effect of organic compounds besides heavy metals, other environmental contaminants were examined for their ability to stimulate the hsp response in bivalves. the mussel m. galloprovincialis exposed to xenobiotics (phenobarbital, heptaclor and pentachlorophenol) or to a mixture of hydrocarbon degradation products showed increases in hsp expression, although to different extents (snyder et al., 2001). in mussel digestive glands a doseand time-dependent increase of hsp67, hsp70 and hsp74 isoforms was observed after exposure to degraded oil. c. gigas 152 were exposed to the same mixture of hydrocarbons reported by snyder (2001), and significant increases in both mrna and protein levels were observed after 7 days of exposure, reaching a maximum at 15 days, and declining thereafter (boutet et al., 2004). the effect of oil mixtures on mussel gene expression was recently assessed (dondero et al., 2006). application of a low-density microarray produced the observation that 8 days of exposure to 0.5 ppm of a crude oil mixture (north sea oil) caused a negative modulation of several gene expressions in mussel digestive glands, including that of hsp70 genes. as only a few isolated studies have been performed on the effects of hydrocarbons on hsp response, with different concentrations and times of exposure, we are not yet ready to draw a picture of the cytoprotective role played by hsp towards these compounds. chlorinated organic compounds are lipophilic chemicals widespread in the environment, the bestknown of them being ddt. although banned they are highly persistent, and are therefore still responsible for inducing clear endocrine disruptive effects (binelli et al., 2004). the ddt metabolite dde (p,p’-dichlorodipehnyldichloroethylene) was assessed for its potential effects on stress response and protein carbonylation in the clam r. decussatus (dowling et al., 2006). hsp60 and hsp70 isoforms were present in gills, mantle and digestive glands of control animals, while hsp90 was constitutively present only in gills. neither hsp60 nor hsp70 proteins were over-expressed by dde exposure in gills and mantle. similarly, dde did not cause hsp90 overexpression in gills. instead, a newly synthesised band of 90 kda appeared in the mantle. carbonylation in response to ros generation after dde exposure was evident in digestive glands and mantle, while not observed in gills. thus the lack of hsp overexpression in gills was well related with the lack of a dde effect on the proteome. the strong induction of hsp90 in the mantle may reflect the need for cytoprotective proteins in this tissue where ros were mainly generated. not in line with these results is the lack of induction or overexpression of hsp in digestive glands. this reflects once more the tissue-specific nature of hsp expression in bivalves often highlighted in this review. effect of hypoxia quantification of hsp70 revealed an increased protein expression in tissues of c. gigas exposed to hypoxia from 0 to 21 days; instead, hsp70 transcription was up regulated after 17 days, but down regulated after 21 days of exposure (david et al., 2005). these inconsistent results let to the conclusion that the hsp response to hypoxia in oysters remains relatively unknown. clams were vulnerable to moderate hypoxia which caused high mortality (joyner-matos et al., 2006). however, the stimulus did not trigger any change in shsp, hsp60 or hsp70. the response elaborated by clams towards hypoxia or hyperoxia was influenced by seasonality. exposure to the stressors for more than 24 h was lethal. clams collected in spring showed an increased stress protein response, decreased levels of lipid peroxidation and increased survival compared to those collected in fall (joyner-matos et al., 2006). a number of factors besides temperature vary between seasons, including availability of nutrients, reproductive status, and growth cycle. we must consider the possibility that all of them may influence the animal’s response to stress factors. effect of electromagnetic fields low frequency electromagnetic fields (emf) may affect several physiological functions in cells and tissues (funk and monsees, 2006), and elicit the hsp response. according to goodman and blank (1998) the control of hsp70 induction by low frequency electromagnetic fields takes place at the level of gene transcription and is mediated at the level of two peculiar nucleotide sequences (nctctn) located on the hsp70 promoter gene. haemocytes from mussels exposed for 30 min at fifty hertz emf at 300 or 400 μt intensity showed a delayed response to chemotactic factors, and at 600 μt their motility was suppressed (malagoli et al., 2003). the different physiological alterations concomitant with this effect included the hsp response, elicited in hemocytes of mussels exposed to emf (malagoli et al., 2004). while at 300 μt no modification of the hsp pattern was observed, at 400 μt and 600 μt overexpression of both hsp70 and hsp90 was elicited, and increased with multiple exposure and with the duration of exposure. the triggering of hsp expression indicates that emf represent a physical stressor to which the mussel reacts. given the few studies carried out on this topic, we are far from understanding the action mechanisms through which emf may induce the hsp response in bivalves. further evidence of emf effects on different invertebrate parameters may provide useful clues for understanding the phenomenon in more complex organisms. at present little is known about the action mechanism of emf in mammals either, and it is the object of major debate. effect of pathogens various pathogens induce hsp overexpression in infected tissues. examples are the increase in hsp70 levels in human macrophages exposed to staphylococcus aureus, erhytrocytes infected by escherichia coli and glial cells attacked by mycobacteria (kantengwa and polla, 1993), and hsp60 increases in neutrophils infected by s. aureus (zheng et al., 2004). c. virginica infected by perkinsus marinus showed the overexpression of hsp69 (encomio and chu, 2007). oysters preexposed to a sublethal thermal stress (40 °c for 1 h) and subsequently challenged with p. marinus improved their survival, indicating that hsp overexpression by heat provides tolerance to a further stress stimulus, namely p. marinus. stress factor interactions a large body of evidence indicates that a mild heat shock provides greater resistance to hyperthermia, anoxia, heavy metals, hydrogen peroxide, etc (de maio, 1999). oyster specimens (c. gigas) cultivated at 12 °c did not survive 1 h exposure to 44 °c, which is therefore the lethal temperature for these organisms (clegg et al., 153 1998). however, c. gigas exposed for 1 h to 37 °c survived the subsequent treatment of 1 h to 44 °c to which they were subjected one week later. such a tolerance phenomenon is present in primitive organisms such as bacteria through more complex organisms up to mammals, leading to the hypothesis that it is indeed a primitive mechanism of cellular defence. a mild heat shock protects cell processes such as transcription, splicing, trafficking etc. from further exposure to different type of stress (yost et al., 1990; parsell and lindquist, 1993; de maio, 1995) probably because the hsp levels are already higher, so that the defence mechanisms can be activated faster. unfortunately this issue has not received much attention in the latest studies on bivalves, and a comparison amongst species or stressors is difficult to perform. consistently, the molecular mechanisms at its basis are far from being understood. hsp response and cell signaling interestingly, and in a manner related to the above topics, a difference was observed between the temperature inducing an increase in protein levels and the temperature at which gene expression is triggered. while the threshold temperature for hsp70 induction varied according to the thermal history of two groups of m. trossulus, there were no variations between the endogenous levels of the constitutive hsp70 isoform and of hsf1. moreover, the activation temperature of hsf1 found in m. trossulus was not identical to the threshold temperature for hsp70 synthesis in the congeneric m. californianus. as described above, transactivation of heat shock genes is mediated by the interaction of hsf1 and hse (wu, 1995). hsf1 is normally located in the cytosol, linked to hsp70/90 and other proteins; it is released by hsp70 in response to stress and translocates into the nucleus. it appears that hsf1 is released by hsp70 in response to small increases in temperature, and remains presumably inactive on the promoter until a higher temperature is reached. consistently, a quantitative change in levels of hsf1 is not necessary to tune the heat-shock response during acclimatization. rather, the controlling steps underlying the “adjustment of the thermostat” probably occur after hsf1 has bound the promoter. these might involve further cell proteins and also signalling pathways, including the mitogen-activated protein kinases (mapk) signalling cascade which is responsible for hsf1 phosphorylation events preceding gene transactivation (buckley et al., 2001). different mapk may be involved in this mechanism; in fact, a marked increase in the levels of the phosphorylated form of p38-mapk and c-jun n-terminal kinase (jnk, also known as stress activated protein kinase, sapk) in tissues from m. galloprovincialis exposed to temperatures beyond 24 °c has been reported (anestis et al., 2007). the increased phosphorylation of kinases paralleled increased hsp expression, strongly supporting involvement of mapk signalling cascade in the induction of hsp genes in the tissues of m. galloprovincialis during thermal stress. in these experiments it was shown that m. galloprovincialis cannot survive sea water temperatures beyond 26 °c over extended periods of time, and that the mortality of mussels increased drastically during warming to 30 °c. consistently with further evidence, it was established that the increase in the mortality of mussels during exposure to temperatures higher than 24 °c might be attributed to a reduced ability to assimilate food and associated energy, before the direct consequences due to heat. however, acclimation up to 28 °c induced overexpression of hsp70 and hsp90 and concomitant phosphorylation of sapk and p38mapk. although the cause-effect relationship between mapk and hsp has not been proven in bivalves, the involvement of mapk signalling in hsp expression may account for the regulation of hsp expression by integrated factors, besides protein damage. in agreement with the above evidence are also data reported by malagoli et al. (2004), who observed the parallel induction of p38mapk and hsp70/90 by electromagnetic fields. moreover, in gills of m. galloprovincialis, stimuli that are known to trigger the hsp response also induce activation of mapk pathway. cu, zn and cd induced p38-mapk activation with maximum levels reached within 1 h. hypothermia (4 °c) induced a moderate kinase phosphorylation (maximised at 30·min), whereas hyperthermia (30 °c) induced rapid p38-mapk phosphorylation that remained considerably above basal levels for at least 2 h (kefaloyianni et al., 2005). in fact, besides hsp overexpression, which takes place in a few hours, another evolutionarily conserved response to heat shock develops in minutes and leads to activation of the major signalling transduction pathways involving jnk and p38-mapk (dorion et al., 1999). the mechanisms of activation and the roles of these pathways during heat shock have been the subject of several studies, with no clear conclusions (dorion and landry, 2002). recent findings revealed that hsp in mammals can regulate both the signalling and the execution of major cell death pathways (reviewed in jäättelä, 1999; beere and green, 2001). consequently, hsp play a primary role in the resistance to a variety of toxic agents and situations that do not necessarily involve protein denaturation. overexpression of hsp70 can inhibit jnk activation by various stimuli, including heat shock, uv light, and h2o2 through a mechanism involving the direct binding of hsp70 to jnk or an hsp70-mediated protection of a jnk phosphatase from heat denaturation (park et al., 2001). the inhibition of jnk activity could therefore be a factor of acquired thermotolerance. in contrast, the activation of the p38-mapk pathway leads to the phosphorylation of hsp27 (huot et al., 1995), an event that is generally assumed to be protective. phosphorylation of hsp27 is catalyzed by mapk-2, a serine-protein kinase itself activated by phosphorylation by p38-mapk (rouse et al., 1994; huot et al., 1995). this recent progress in the field of cellular stress underlines a new concept that cell response to stress stimuli mediated by hsp is not only the consequence of protein damage. it results from the integration of stress and evolutionarily conserved and interconnected physiological mechanisms, i.e., the activation of hsp response and of mapk 154 cascades. in fact, hsp regulate mapk activation and mapk regulate hsp activation and activities in mammals (dai et al., 2000). much remains to be studied in bivalves regarding this issue. although not dealing with mapk-hsp interaction, elucidation on the role of mapk pathways in mussels was recently provided by kefaloyianni et al. (2005), anestis et al. (2007) and by the extensive work of canesi et al. (2005, 2006a, 2006b). hsp as molecular biomarkers as detailed above, the induction of several hsp isoforms by environmental contaminants is widely documented. for a number of years hsp have been considered as potential biomarkers to be included in biomonitoring programmes (nadeau et al., 2001; snyder et al., 2001; radlowska and pempkowiak, 2002). the implication of these proteins within the main mechanisms of cellular protection would render them good markers of the stress status of an organism. the advantages are that they give information about general conditions of health and provide early warnings of intoxication, before complex functions are compromised. although in some cases hsp may show a specific pattern of response to selected stimuli, they do not usually provide specific information on the type of stress factor present in the environment. however, this application of hsp requires a certain caution, and their use as biomarkers should be based on previous studies on the biochemical and physiological features of the analysed animal. in fact, hsp are differently expressed in different tissues of the same organism; they are also subject to seasonal and physiological variations e.g. related to temperature, oxygen availability and salinity (minier et al., 2000, bodin et al. 2004, hamer et al. 2004). the main difficulty in using biomarkers in a monitoring program is the interference of natural environmental factors with the biological responses. although the hsp response is one of the fastest and most sensitive, we know that it is also subject to seasonality. these drawbacks have led to criticism in some reports of hsp being used as biomarkers (pyza et al., 1997; bierkens, 2000). we believe that one main obstacle to this use is the methodology needed to assess the hsp response, requiring the use of the western blotting technique for protein assessment and of the pcr, qpcr or microarray for gene expression profiling. if we agree that biomarkers should be clear and repetitive responses measured through relatively simple and cheap methodologies (viarengo et al., 2007), hsp cannot meet these requirements at the moment. nevertheless, including hsp assessment in a battery of biomarkers can add useful information and sometimes point the way to further studies. future challenges there was a perception that the major patterns of hsp expression in eukaryotes were becoming so obvious that additional descriptive work was difficult to justify (feder and hofmann, 1999). however, the rate at which new findings are being made is still increasing, and major advances have occurred in the last few years. some major questions remain unanswered, while new aspects have emerged in mammals which have not yet been taken into consideration in bivalves. only one detailed study has been addressed to the evaluation of hsp in aging bivalves (ivanina et al., 2008b). the issue deserves more attention, both as an approach to the physiology of aging in bivalves, and also because knowledge of ancestral mechanisms may provide clues for understanding the complex aging phenomenon in humans. the oyster c. virginica and the clam mercenaria mercenaria were studied at different ages from 7 months to 4 years. mitochondrial hsp60 significantly decreased with age, suggesting an age-related decline in mitochondrial chaperone protection. however, the possibility that other protective mechanisms are increased by senescence in mitochondria was not evaluated. different trends in the hsp70 and hsp90 expressions were developed by the two bivalves at different ages (ivanina et al., 2008b). hsp90 levels in c. virginica increased progressively, while hsp70 levels did not change. hsp70 levels increased with age in m. mercenaria, while hsp90 decreased. this non-uniformity also appeared in non-bivalve organisms from drosophila to humans (wheeler et al., 1995; colotti et al., 2005), so that at present we cannot answer the question of whether chaperone systems compensate for aging-related proteotoxicity or not. the relationship established in mammals and more recently in mussels (kefaloyianni et al., 2005; anestis et al., 2007) between the mapk signalling cascade and the induction of hsp genes during thermal stress is rather intriguing, since it links the hsp response to intracellular signalling. moreover, is well known that mapk cascades are activated by stress factors and also by physiological factors, which could therefore modulate hsp expression. on the other hand, catecholamines induced hsp gene expression in bivalves (lacoste et al., 2001a). from this initial evidence it is clear that the hsp response must be regarded as an integrated phenomenon, with intracellular and systemic components. well-related to the above observations is the fact that extracellular hsp70 are emerging as important mediators of intercellular signalling and transport in mammals (calderwood et al., 2007). the release of these proteins from cells is triggered by physical trauma, stress, and exposure to immunological stimuli. stress protein release occurs both through physiological secretion mechanisms and during cell death by necrosis. after release into the extracellular fluid, hsp enter the bloodstream and possess the ability to act at distant sites in the body, then bind to the surfaces of adjacent cells and initiate signal transduction cascades as well as the transport of antigenic peptides (van noort, 2008). many of the effects of extracellular stress proteins are mediated through cell surface receptors located on neurons, immune cells, blood vessels, etc. to the best of our knowledge it appears that the phenomenon of hsp release to the extracellular environment has not yet been tackled in bivalves. considering the roles played by bivalve haemocytes in stress response, immunity and metabolism, 155 investigations in this field are strongly recommended. we are fully aware that the challenges we have chosen to outline here represent just a few of the many topics deserving of wider exploration. however, the emergence of major new roles for hsp points the way forward to a fruitful line of future study. from a broader perspective, apart from providing further basic information, we hope that the new synergistic approaches will help to elucidate the integration between the hsp response and cell signalling at the cellular level, as well as the role of hsp as a cellular component of the integrated stress response. acknowledgement the authors are grateful to dr. a piano, who set up the first studies on hsp in prof. fabbri’s laboratory during her phd, and allowed us to continue this work on solid basis of knowledge. in this review we focused mainly on bivalve hsp. however, it was impossible to acknowledge all the work that has led to our current knowledge on the hsp response. we apologise for any omissions and invite readers to find further information in the excellent reviews and articles cited herein. reference ait-aissa s, porcher j, 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hsp90 chaperone machinery. j. biol. chem. 283: 18473-18477, 2008. zhong m, orosz a, wu c. direct sensing of heat and oxidation by drosophila heat shock transcription factor. mol. cell. 2: 101-108, 1998. 161 minireview isj 9: 207-211, 2012 issn 1824-307x minireview the immuneregulator role of neprilysin (nep) in invertebrates e ottaviani1, d malagoli1, a grimaldi2, m de eguileor2 1department of life sciences, university of modena and reggio emilia, via campi 213/d,41125 modena, italy 2department of biotechnology and life science, university of insubria, via j. h. dunant 3, 21100 varese, italy accepted november 17, 2012 abstract neprilysin (nep) represents an important enzyme in both vertebrates and invertebrates. in the present report we have focused our attention to invertebrates. in particular, a structure related to cd10/nep as well as its activity in different tissues, such as immunocytes, nervous tissue and muscle of various species were detected. moreover, the role played by the enzyme in the interactions between host and parasite has also been reported. the findings indicate that nep immunoregulation is a wellbalanced process that, with appropriate physiological and homeostatic responses to challenges, allows the survival and well-being of the species. key words: neprilysin (nep); invertebrates; immunoregulation   introduction neutral endopeptidase, nep (ec 3.4.24.11) now referred to as neprilysin is a type ii integral membrane protein with a mw of 93 kda, consists of a short nh2-terminal cytoplasmic domain of 27 amino acids, a trasmembrane region of 22 hydrophobic residues, and a large extracellular domain of about 700 residues that contains zinc in the active center (fig. 1). it cleaves substrates on the amino side of hydrophobic amino acids (kerr et al., 1974a, b; gafford et al., 1983; turner and tanzawa, 1997; turner et al., 2001). the nep gene exists in a single copy, extents more than 80 kb, is composed of 24 exons and is highly conserved in mammalian species (d'adamio et al., 1989). furthermore, nep was shown to be identical to cd10, a tumor-associated cluster differentiation antigen expressed on the surfce of neutrophils and some lymphoid progenitors, and also known as the common acute lymphoblastic leukemia antigen (calla) (letarte et al., 1988). neprilysin (nep) the nep has been reported in vertebrates and invertebrates. with regards vertebrates, the majority of the data refer mammals. in particular, from the ___________________________________________________________________________ corresponding author: enzo ottaviani department of biotechnology and life sciences university of modena and reggio emilia via campi 213/d, 41125 modena, italy e-mail: enzo.ottaviani@unimore.it literature it emerges that this enzyme is mainly localized in the human, rat, rabbit and pig kidney (kerr and kenny, 1974a, b; mumford et al., 1981; gafford et al., 1983; edwards et al., 1999), but also in human fibroblasts (lorkowski et al., 1987; kletsas et al., 1998), human genital tract (erdös and skidgel, 1989), rat brain (back and gorenstein, 1989), human blood cells (connelly et al.,1993), rat nerve ending membranes (vandenbulcke et al., 1994), mammalian membrane (turner and tanzawa, 1997), mouse mesangial cells (ebihara et al., 2003), and so on. as far as invertebrates are concerned, a structure related to cd10/nep as well as its activity were detected in the molluscan immunocytes of mytilus edulis (shipp et al., 1990), planorbarius corneus, viviparus ater (ottaviani and caselgrandi, 1997) and mytilus galloprovincialis (caselgrandi et al., 2000), and in the central nervous system of aplysia californica (zappulla et al., 1999). with refer other groups, data were reported on the hemocytes of the insect heliothis virescens (grimaldi et al., 2012), neural membranes from the locust schistocerca gregaria (isaac, 1988), from the nematode ascaris suum muscle (sajid and isaac, 1995) and from the head parts of the leech theromyzon tessulatum (laurent and salzet, 1995). genomic studies revealed that caenorhabditis elegans and drosophila melanogaster contain 22 and 24 nep-like genes (turner et al., 2001). moreover, further investigations in d. melanogaster have reported that the nep2 gene codes for a secreted endopeptidase with a highly restricted 207   mailto:enzo.ottaviani@unimore.it pattern of expression, and this protein for its localization seems involved in renal function and in spermatogenesis (thomas et al., 2005). we detected the presence of nep and its activity by using different approaches. in the first case, the cytofluorimetric analysis of m. galloprovincialis immunocytes revealed that the cells were positive to cd10 in a range from 8 to 10 % (fig. 2) (caselgrandi et al., 2000), while in granulocytes of the insect h. virescens the presence of the enzyme was detected by immunocytochemical (fig. 3) and western blot analysis (grimaldi et al., 2012). for the determination of the nep activity, we used a spectrofluorimetric procedure (ottaviani and caselgrandi, 1997) and the shape factor (sf) protocol (ottaviani et al., 1997; sassi et al., 1998; caselgrandi et al., 2000). this last procedure is based on the capacity of nep to cleave biological peptides (duvaux-miret et al., 1992; otttaviani and ceselgrandi, 1997) and cytokines (pierart et al., 1988; casey et al., 1993; caselgrandi et al., 2000) molecules that, in turn, activate the cell motility of immunocytes (scharrer and stefano, 1994; sassi et al., 1998; caselgrandi et al., 2000; ottaviani et al., 2004). the enzymatic activity of nep was confirmed by phosphoramidon, a potent inhibitor of nep (fulcher et al., 1982). the induced changes in cell shape, from a round form (inactive) to an ameboid form (active), were recorded by measuring the cellular area and the perimeter allowing the evaluataion of the sf (fig. 4). the sf formula of the american innovision analysis system software package (san diego, ca) was used to express changes mathematically, as described in detail elsewhere (schön et al., 1991). the evaluation of the sf has been described previously (sassi et al., 1998). briefly, this method is based on the use of a computer-assisted microscopic image analysis. a vaseline ring on a microscopy slide delimited a chamber within which 100 μl of hemolymph were placed (fig. 5), as described in detail in a previous paper (ottaviani et al., 1997). functions of neprilysin (nep) this endopeptidase plays a regulatory role on the peptides that are involved in the physiological mechanisms of mammalian nervous, cardiovascular and immune systems (turner et al., 2001). in the present paper we will focus our attention on its role in the invertebrate immune system. at a glance observation of literature it emerges that the enzymatic degradation of nep induces a downregulation according to the following scheme: a first stimulus (for instance biopeptides, cytokines) activating an immunocyte upregulates nep. consequently immunocyte response to a second stimulus, that serves as nep substrate, is downregulated (fricchione and stefano, 1994). the activation of invertebrate immunocytes was found to be suppressed by adrenocorticotropin hormone (acth) and alpha-melanocyte-stimulating hormone (α-msh). the effect of acth is largely due to its conversion to α-msh by immunocyte fig. 1 schematic representation of nep. associated nep. this is the topic point, since αmsh inhibits adherence and locomotory activity of polymorphonuclear leukocytes (pmn), monocytes and invertebrate immunocytes. it should be underlined that while α-msh acts rapidly (min) on the cells, acth requires much more time (h) in order to act, and this is due its processing into αmsh (smith et al., 1992). fig. 2 cytofluorimetric analysis of m. galloprovincialis immunocytes. (a) control, (b) staning with anti-cd10 mab. fl1 = fluorescence 1 channel; fl2 = fluorescence 2 channel. from caselgrandi et al., 2000 (reprinted with permission). 208   fig. 3 immunocytochemical evidence of nep in the in larva hemocytes of the insect h. virescens. bar = 15 μm. 209   fig. 4 phase-contrast photographs of m. galloprovincialis immunocytes. (a) control, (b) activated immunocytes 5 min after the addition of 10-8 m acth (1-24). bar = 10 μm. sf = 0.87 sf = 0.58 parasites use a similar mechanism for immunosuppresion involving the proopiomelanocortin-derived peptides released from the parasite schistosoma mansoni (duvaux-miret et al., 1992). s. mansoni may escape immune reactions from its vertebrate (man) by using signal molecules common to both host and parasite. in the experiments of coincubation of adult worms with human pmn or snail immunocytes has been detected the presence of α-msh in the medium suggesting that α-msh results from the conversion of the parasite acth by nep. nep has also been detected in the t. nigriceps/h. virescens parasitic model. during the parasitization of the insect h. virescens larva by another insect toxoneuron nigriceps, the host activates a series of humoral and cellular defenses in which plasmatocytes and granulocytes are the circulating immunocytes (grimaldi et al., 2012). the granulocytes activated by parasitization produce fig. 5 microscopy slide for cell activity assay. s = substance to test. large amount of amyloid fibrils to package melanin. the stimulation in response to parasitic attack involves a cross-talk between the immune and neuroendocrine systems with the activation of stress-sensoring circuits to produce and release molecules, such as acth (responsible of the autocrine/paracrine activation of cells), α-msh (resulting in activation of melanin production), nep and the overproduction of reactive oxygen species. in this contex nep present on the cell surface plays an important role in controlling the acth/α-msh loop modulation. the same enzyme after exocitosis of amyloid fibrils massively hydrolyzes amyloid fibrils poured in circulating fluid and this cleavage prevents the unnecessary accumulation in hemolymph of amyloid resistant material. conclusive remaks the majority of the findings reported in the present paper on nep have been observed in parallel also in man (duvaux-miret et al., 1992; smith et al.,1992; scharrer and stefano, 1994) suggesting that: 1) nep activity is present on blood cells, immunocytes, peripheral fluids and hemolymph; 2) nep is a highly important factor in controlling the response of immunocytes in invertebrates and blood cells in man to the influence of biologically active substances; 3) the presence and the activity of nep in invertebrates and man substantiate the importance of this intercellular regulatory mechanism of communication. in summary the nep immunoregulation is a well-balanced process that, with appropriate physiological and homeostatic responses to challenges, allows the survival and well-being of the species. references back sa, gorenstein c. histochemical visualization of neutral endopeptidase-24.11 (enkephalinase) activity in rat brain: cellular localization and codistribution with enkephalins in the globus pallidus. j. neurosci. 9: 4439-4455,1989. caselgrandi e, kletsas d, ottaviani e. neutral 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sciences, yantai 264003, pr china 2yantai oceanic environmental monitoring central station of soa, yantai 264006, pr china 3china agriculture university (yantai), yantai 264670, pr china accepted may 16, 2014 abstract glutathione peroxidases (gpxs) are key enzymes in the antioxidant defense system of living organisms, and protect organisms against oxidative stresses. in this study, the full-length cdna sequences encoding cytosolic gpx (mgcgpx) and phospholipid-hydroperoxide gpx (mggpx4) were identified from mytilus galloprovincialis. the mussels were exposed to 0, 1, 10, and 100 μg/l cadmium and arsenate for 30 days. the mrna transcripts of these two genes and total gpx activity were examined in the gills and digestive gland after contaminants exposure. the mussels exposed to cadmium and arsenate responded mainly by down-regulating mgcgpx and mggpx4 mrna transcription in gills and up-regulating transcription in digestive gland. however, total gpx activities increased following cadmium exposure but decreased after arsenate stress in both tissues. these results suggest that mgcgpx and mggpx4 perhaps play an important role in maintaining cellular redox homeostasis and protecting m. galloprovincialis against cadmium and arsenate toxicity. it can also be inferred that these genes have the potential to be used as molecular biomarkers for assessing cellular stress and toxicity of contaminants in this mussel. key words: cytosolic gpx; phospholipid-hydroperoxide gpx; arsenic; cadmium; bivalve; antioxidant enzyme introduction in recent years, a great number of contaminants including metals, petroleum oils and organic pollutants have been discharged into the bohai sea, a territorial sea in north china (ma et al., 2001). among these contaminants, metal contaminants have been recognized as the major pollutants in coastal areas. cadmium (cd) has been found in high concentrations in marine environments of the bohai sea (meng et al., 2004; meng et al., 2008; wang et al., 2009). in addition, arsenic (as) pollution also has been of great concern because a total arsenic concentration was up to 440 μg l-1 in some polluted estuarine areas of the bohai sea (meng et al., 2004; wu et al., 2013). ___________________________________________________________________________ corresponding authors: huifeng wu jianmin zhao key laboratory of coastal zone environment processes and ecological remediation yantai institute of coastal zone research chinese academy of sciences yantai 264003, pr china e-mail: hfwu@yic.ac.cn. e-mail: jmzhao@yic.ac.cn the pollutants including metal contaminants constitute a potential threat to marine organisms (winston and di giulio, 1991). the toxic damage induced by metals and arsenic is generally associated with excessive formation of reactive oxygen species (ros), which cause oxidative modification of the major cellular macromolecules (leonard et al., 2004; ventura-lima et al., 2011). both cd and arsenic have been shown to be toxic to marine bivalves and affect their physiological status by causing oxidative stress (dovzhenko et al., 2005; wang et al., 2010; chakraborty et al., 2010). excessive ros production has been found to oxidize and damage cell membrane, proteins, and nucleic acids (hensley et al., 2000). in marine animals, the oxidative stress can be counteracted by non-enzymatic antioxidant and enzymatic antioxidant systems (fernández et al., 2010). the key components of these enzymatic antioxidant systems include glutathione peroxidase (gpx), catalase (cat) and superoxide dismutase (sod) (apel and hirt, 2004). gpx (ec 1.11.1.9 and ec 1.11.1.12) can 149 mailto:jmzhao@yic.ac.cn catalyze the reduction of h2o2 or organic hydroperoxides to water or corresponding alcohols using reduced glutathione (gsh) (margis, 2008). they are generally classified into two subgroups as selenium-dependent gpx and non-selenium gpx, based on the presence of selenocysteine (sec) encoded by a stop codon tga (arthur, 2000). at present, eight gpx isozymes have been characterized in mammals, including: cytosolic gpx (gpx1), gastrointestinal gpx (gpx2), plasma gpx (gpx3), phospholipid hydroperoxide gpx (gpx4), epididymal gpx (gpx5), olfactory epithelium gpx (gpx6), endoplasmic reticulum phospholipid hydroperoxide gpxs (gpx7 and gpx8) (herbette et al., 2007; nguyen et al., 2011). in contrast to other tetrameric gpxs, gpx4 is monomeric and membrane-associated, especially in membrane fractions. gpx4 is unique in its substrate specificity because it can interact with lipophilic substrates, including the peroxidized phospholipids and cholesterol, and reduce these hydroperoxides to hydroxide compounds (thomas et al., 1990). they mainly participate in the repair of disrupted bio-membranes, which has been considered as the main defense strategy against oxidative membrane damage (bae et al., 2009). the role of cytosolic selenium-dependent gpxs in host immune and antioxidant defense system has been well documented in many mollusks, such as unio tumidus (doyen et al., 2006), crassostrea gigas (jo et al., 2008), chlamys farreri (mu et al., 2010), meretrix meretrix (wang et al., 2011a), venerupis philippinarum (zhang et al., 2011; zhang et al., 2012) and haliotis discus hannai (wu et al., 2010). however, gpx4s have been less characterized and their roles in antioxidant system are less studied in aquatic animals. until recently, only a few gpx4 genes have been cloned in aquatic organisms such as hydra (dashe et al., 2006), midge (nair et al., 2012) and several fishes (wang et al., 2012; pacitti et al., 2013). to our knowledge, no gpx4 gene has been cloned from mollusks although gpx4 activity was observed in the mussel perna perna (almeida et al., 2005). the mussel mytilus galloprovincialis, extensively dispersed in the bohai sea, has a high adaptive ability to environments and occupies a broad range of habitat types. they are apt to be exposed to pollutants and suffer from oxidative stress in esutarine and coastal area of the bohai sea. in china, this mussel has been used as a biomonitoring organism to assess the marine environment. therefore, it is necessary to study how the antioxidant system of the mussel responds to environmental stress. to investigate the possible role of gpxs in protecting marine mussel from oxidative damage, two gpx genes were characterized and their transcriptional responses to sub-chronic cadmium and arsenic exposures were studied. in addition, the possibility of these genes used as biomarkers was also discussed. materials and methods animal culture adult animals (shell length: 5.0 7.0 cm) were purchased from a local mussel culture farm and acclimatized in aerated seawater (33 psu) at 20 °c for 7 days before commencement of the exposure experiment. during the acclimatization period, the mussels were fed with phaeodactylum tricornutum and platymonas helgolandica, and the seawater was completely renewed once every 24 h. contaminant exposure after the acclimatization, the mussels were divided into 21 groups and cultured in polyethylene tanks with 20 l seawater, each containing 20 individuals. the exposure experiments were performed using analytical grade salts of cdcl2·2.5h2o and na2haso4·12h2o (guoyao, shanghai, china). stock solutions were prepared in deionized water at a concentration of 1 g l-1, which was high enough to prevent weighing errors and salinity change. the exposure experiment for each contaminant included three concentrations: 1, 10 and 100 μg l-1 (contaminant concentration, not salt concentrations). there were 3 replicates (tanks) per treatment and the other three untreated tanks were employed as the control groups. the mussels were fed as described above and the culture medium was renewed once every 24 h. the gills and digestive glands of four individuals from each tank were randomly sampled for gene expression and antioxidant enzyme activity analysis after 30 days of exposure respectively. all the samples were quickly frozen in liquid nitrogen and then transferred to a -80 °c refrigerator till use. rna isolation, cdna synthesis and cloning of mussel gpx genes total rna was extracted from gills using the trizol reagent (invitrogen, usa) following the supplier’s protocol. the extracted rna was then treated with rq1 rnase-free dnase (promega, usa) to remove dna contamination. single-stranded cdnas were synthesized from one microgram of the total rna using m-mlv reverse transcriptase (promega, usa) according to the manufacturer’s instructions. blast analysis of all expressed sequence tag (est) sequences from a cdna library of m. gallopronvicialis (unpublished) revealed that two ests were highly similar to the previously identified gpx genes, respectively. the 3′ ends of mgcgpx and mggpx4 were obtained by rapid amplification of cdna ends (race) using the smarttm race cdna amplification kit (clontech, usa) according to the manufacturer’s manual. two race primers were designed according to these ests and they were included in table 1. the pcr products were purified using agarose gel electrophoresis, cloned into the pmd-18t simple vector (takara, japan) and then sequenced in both directions by the chinese national human genome center (sinogenomax, beijing, china). sequence analysis the mgcgpx and mggpx4 cdna sequences were analyzed using the blastx search program (http://www.ncbi.nlm.nih.gov/blast/). the deduced amino acid sequences were analyzed using the expasy server (http://www.expasy.org/tools/). multiple alignments were performed with the clustalw program (http://www.ebi.ac.uk/clustalw/). a 150 table 1 primers used in this study gene name forward primer (5'-3') reverse primer (5’-3’) description mgcgpx ggaaaatggaaacggtgaag tatgggaacctgtgacaagaac 3’ race mggpx4 aggagcctggaactgaagc gttctggggatgtcaacctg 3’ race β-actin gctatccaggccgtactct gcggtggttgtgaatgag qrt-pcr α-tubulin gaccacccataccaccctt ctccgtgagatcgacattc qrt-pcr 18s rrna tcggattggtgagactggat tgctgccttccttggatgt qrt-pcr 28s rrna ccgagaccgaggatttgcc accgattcgccactgaccc qrt-pcr ubiquitin c atcaacagcgtctcatct gctcaacttctagcgtaat qrt-pcr ef1 gctggtatctcatctaacg cttcactgtatggtggttc qrt-pcr gapdh agggtccaatgaagggtg ttaagagcgatgccagct qrt-pcr ddx catcagaagaaggtggct aacagttggtcgtagggt qrt-pcr mgcgpx aatgcccttgagcatgttcg tgaaactaacagcatcgtcgc qrt-pcr mggpx4 agtcaggagcctggaactga tgcctccttgtttgtgtttg qrt-pcr phylogenetic tree was constructed with mega4.1 software using the neighbor-joining (nj) method based on the alignment (tamura et al., 2007). bootstrap analysis was used with 1000 replicates to test the relative support for the branches produced by the nj analysis. the selenocysteine insertion sequence (secis) elements were searched using the secisearch 2.19 program (http://genome.unl.edu./secisearch.html). quantitative real time pcr (qrt-pcr) analysis of mgcgpx and mggpx4 expression in order to select appropriate internal standards for qrt-pcr analysis of gene expression in mussel exposed to cadmium and arsenic, the expressions of eight housekeeping genes, i.e., β-actin, α-tubulin, elongation factor 1-α (ef1), 18s rrna, 28s rrna, ubiquitin c, dead-box rna helicase (ddx) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) were determined. the data were analyzed with genorm to calculate the expression stability (m values) of potential reference genes required for accurate normalization (v values) (dang and sun, 2011). hemocytes, gills, digestive gland, adductor muscle, mantle and gonad of six untreated individuals were sampled to investigate the tissue-specific distribution of mgcgpx and mggpx4 transcripts. the tissues of digestive gland and gills were selected to analyze temporal expression profile of mgcgpx and mggpx4 challenged by cd and asv, respectively. total rna extraction and cdna synthesis were performed as described above. qrt-pcr was carried out in an abi 7500 real-time detection system by using the sybr exscript qrt-pcr kit (takara) as described before (wang et al., 2013). the pcr amplification was carried out in a total volume of 50 μl, containing 25 μl of 2 × sybr green pcr master mix, 20 μl of the diluted cdna, 1 μl of each of primers (10 μmol l-1), and 3 μl of depc-treated water. the thermal profile for qpcr was 50 °c for 2 min, 95 °c for 10 min followed by 40 cycles of 95 °c for 15 s and 60 °c for 1 min. all reactions were run in triplicate. dissociation curve analysis of amplicon was performed at the end of each pcr reaction to confirm that only one pcr product was amplified and detected. the expression level of mgcgpx and mggpx4 was analyzed using 2−δδct method (livak and schmittgen, 2001). the qrt-pcr primers of mgcgpx and mggpx4 are shown in table 1. measurement of total gpx activity in digestive gland and gills for assays on total gpx activity, the tissues of digestive gland and gills from six mussels were weighed and homogenized individually in 10 % (w/v) 50 mm tris-hcl buffer (ph 7.5) using a tissue grinder (ultra turrax ika t10 basic, germany) held in an ice-water cooled bath. the homogenate was centrifuged at 10,000×g (4 ℃) for 30 min (eppendorf centrifuge 5804r, germany). then the supernatant was collected and kept at 4 °c until used for further analysis. the total protein concentrations were determined according to the method described by bradford (1976). total gpx activity was measured as described before (wang et al., 2010). all measurements were carried out in a magellan plate reader (tecan, switzerland). statistical analysis spss 16.0 software (spss inc., usa) was used for statistical analysis. the data were checked for homogeneity of variance before analysis. the data were analyzed by one-way analysis of variance (one-way anova), followed by a tukey's post hoc test. a probability level of p < 0.05 was considered significant. 151 http://genome.unl.edu./secisearch.html a) b) fig. 1 the complete nucleotide and deduced amino acid sequences of mgcgpx (a) and mggpx4 (b). the asterisk (*) indicates the stop codon. the start and stop codons are included in a box. the selenocysteine (u) is shaded. the polyadenylation signal is underlined in bold font and the predicted secis element is underlined in italic font. results sequence analysis two nucleotide sequences of 972 bp and 889 bp representing the complete cdna sequence of mgcgpx and mggpx4 were obtained by overlapping est and the amplified fragments. full-length cdnas of mgcgpx and mggpx4 were deposited in genbank under the accession numbers of hq891311 and jq866922, respectively. the deduced amino acid sequences of mgcgpx and mggpx4 were shown below and the corresponding nucleotide acid sequence in figure 1. the complete sequence of mgcgpx cdna 152 a) b) fig. 2 multiple sequence alignments of mgcgpx (a) and mggpx4 (b) with other gpx orthologs deposited in genbank. the black shadow region indicates positions where all sequences share the same amino acid residue. gaps are indicated by dashes to improve the alignment. the gpx signature motif 2 elements and active site motifs are marked by frame, respectively. the catalytically important residues are indicated by asterisks. the genbank accession numbers of gpx sequences are shown in table 2. 153 fig. 3 phylogenetic tree constructed by neighbour-joining method based on the sequences of gpxs from different animals. numbers at the forks indicate the bootstrap values (in %) out of 1,000 replicates. the sequences used to construct phylogeny trees of gpxs are shown in table 2. table 2 sequences used for multiple sequence alignments and phylogenetic analysis gene species common name accession numbers gpx1 homo sapiens human np_000572.2 gpx1 equus caballus horse np_001159951.1 gpx1 bos taurus cattle np_776501.1 gpx1 danio rerio zebra fish np_001007282.2 gpx2 homo sapiens human np_002074.2 gpx2 rattus norvegicus brown rat np_899653.2 gpx3 homo sapiens human aap50261.1 gpx4 rhipicephalus microplus tick aba62390.1 gpx4 hydra vulgaris hydroid abc25027.1 gpx4 xenopus laevis african clawed frog np_001165213.1 gpx4 salmo salar atlantic salmon ach86324.1 gpx4 gallus gallus chicken aam18080.2 gpx4 mus musculus house mouse baa22780.1 gpx4 homo sapiens human caa50793.1 gpx4 mytilus galloprovincialis mediterranean mussel jq866922 gpx5 homo sapiens human caa06463.1 gpx6 homo sapiens human aay68223.1 gpx mizuhopecten yessoensis yesso scallop adq92353.1 gpx hyriopsis cumingii triangle shell mussel acy72387.1 gpx sepiella maindroni cuttlefish aek48346.1 gpx pinctada fucata pearl oyster adc35417.1 gpx mytilus galloprovincialis mediterranean mussel hq891311 154 a) b) fig. 4 tissue-specific mrna expression of mgcgpx (a) and mggpx4 (b) determined by quantitative real-time pcr. each bar represented mean ± se (n = 4). encoded a polypeptide of 199 amino acids with a predicted molecular weight of 22.97 kda and a theoretical isoelectric point (pi) of 9.11. smart program analysis revealed that mgcgpx contained a typical gshpx domain ranging from phe15 to arg128. the full-length cdna of mggpx4 encoded a polypeptide of 170 amino acids with a predicted molecular weight of 19.48 kda and a theoretical pi of 8.62. smart program analysis demonstrated that mggpx4 also contained a gshpx domain ranging from ile14 to lys123. signalp program analysis revealed that no putative signal peptide existed in the deduced amino acids of mgcgpx and mggpx4. in addition, a 98 bp and a 93 bp secis element were identified in the 3’ untranslated region (utr) of mgcgpx and mggpx4 cdnas, respectively. both the secis elements were predicted to form a stem-loop secondary structure and belonged to the form 2 secis element. multiple alignments and phylogenetic analysis multiple alignments revealed that the characteristic sec (u48) residue, catalytically essential residues (q82, w160), the typical gpx signature 2 motif 72lgfpcnqf79 and the conserved active site motif 160wnfekf165 were well conserved in mgcgpx and other selected mollusk gpx sequences (fig. 2a). similarly, mggpx4 also contained the characteristic residues (u46, q82 and w136), the conserved 72lgfpcnqf79 motif and 136wnftkf141 motif in its sequence (fig. 2b). to evaluate the molecular evolutionary relationship of mgcgpx and mggpx4 against other gpxs, the amino acid sequences of 20 representative gpxs were selected to construct the phylogenetic tree (fig. 3). the phylogenetic tree of gpxs included three major clades: the gpx1 and gpx2 (including gpxs from mollusks) clade; the gpx3, gpx5 and gpx6 clade; and the gpx4 clade. 155 a) b) fig. 5 temporal mrna expression profiles of mgcgpx (a) and mggpx4 (b) mrna in gill and digestive gland after cd-exposure measured by quantitative real-time pcr. each bar represented mean ± se (n = 4). significance across control was indicated with an asterisk at p < 0.05. mgcgpx firstly clustered with gpxs from the yesso scallop mizuhopecten yessoensis and pearl oyster pinctada fucata, then formed a mollusc subgroup with gpxs from the triangle shell mussel hyriopsis cumingii and the cuttlefish sepiella maindroni, and it further grouped with typical gpx1s and gpx2s from vertebrates. in the gpx4 clade, mggpx4 firstly grouped with gpx4s from the tick rhipicephalus microplus and the hydroid hydra vulgaris and then clustered with gpx4s from other vertebrates including fish, amphibian, bird and mammals. the phylogenetic analysis indicated that mgcgpx was derived from a common ancestor with most mollusc gpx family and mggpx4 was a typical member of gpx4 family. tissue-specific distribution of mgcgpx and mggpx4transcripts genorm identified β-actin as the most stable gene with a v2/3 value 0.139 less than the proposed genorm cutoff value of 0.15, which was then followed by ubiquitin c, α-tubulin, gapdh, ddx and ef1. therefore, β-actin was used as the internal control for gene expression normalization. the tissue-specific distribution patterns of mgcgpx and mggpx4 mrna were investigated by qrt-pcr with β-actin as the internal control. for mgcgpx, mggpx4 and β-actin genes, there was only one peak at the corresponding melting temperature in the dissociation curve analysis, indicating that the pcr was specifically amplified (data not shown). the transcripts of mgcgpx and mggpx4 were detected in all the examined tissues, including digestive gland, gills, hemocytes, adductor muscle, mantle and gonad. the mgcgpx transcript was mainly detected in the tissue of hemcoytes, digestive gland and gills, moderately expressed in adductor muscle and gonad (fig. 4a). a similar tissue-specific distribution pattern was also observed for mggpx4 transcript with the highest expression level in hemocytes, moderate levels in gills and digestive gland, least level in mantle (fig. 4b). 156 a) b) fig. 6 temporal mrna expression profiles of mgcgpx (a) and mggpx4 (b) mrna in gill and digestive gland after asv-exposure measured by quantitative real-time pcr. each bar represented mean ± se (n = 4). significance across control was indicated with an asterisk at p < 0.05. transcriptional response of mgcgpx and mggpx4 in gills and digestive gland exposed to cd after sub-chronic cd exposure, the transcript level of mgcgpx was significantly down-regulated in gills at relative high concentrations (10 μg l-1 and 100 μg l-1), whereas the transcriptional levels of mgcgpx increased significantly in digestive gland at all exposure concentrations compared to the control (p < 0.05) (fig. 5a). however, a significantly higher transcriptional up-regulation of mggpx4 in digestive gland was only observed in the mussels exposed to 100 μg l-1 cd. in gills, the mrna expression of mggpx4 was also significantly inhibited at the highest concentration (100 μg l-1) compared to that of the control (p < 0.05) (fig. 5b). transcriptional response of mgcgpx and mggpx4 in gills and digestive gland exposed to asv in gills, the transcription of mgcgpx was significantly inhibited at all exposure concentrations compared to the control. however, the mrna expression level of mgcgpx in digestive gland had no significant change at all concentrations compared to the control (p < 0.05) (fig. 6a). similarly, the transcription level of mggpx4 in gills significantly decreased at all concentration. the mrna expression level of mggpx4 was significantly higher in the digestive gland of mussels treated with higher concentrations (10 μg l-1 and 100 μg l-1) compared to the control (p < 0.05) (fig. 6b). alteration of total gpx activities in gills and digestive gland after cd and asv exposure a significant decrease in gpx activity was observed in gills of the mussels exposed to 10 μg l-1 and 100 μg l-1 cd compared to the control (p < 0.05). in digestive gland, the gpx activity increased significantly at all exposure concentrations (fig. 7a). the modulation in transcript level of gpx coincided with the change in total gpx activity following cd 157 a) b) fig. 7 alteration of total gpx activities in gill and digestive gland after cd (a) and asv (b) exposure. each bar represented mean ± se (n = 4). significance across control was indicated with an asterisk at p < 0.05. exposure. after sub-chronic exposure, 1 μg l-1 and 10 μg l-1 asv significantly inhibited gpx activity in gills (p < 0.05). arsenate also inhibited gpx activity in digestive gland, and a statistically significant effect was observed in the 100 μg l-1 treated group (fig. 7b). there was no obvious relation between gene expression and enzymatic activity of gpx following asv exposure in this mussel. discussion glutathione peroxidase enzymes are involved in the elimination of ros produced in physiological and pathological processes. their differential expression as compared to normal expression pattern can indicate exposure to cellular stress and adverse cellular effects (dash et al., 2006). thus, gpxs have been proposed as biomarkers for the monitoring of physiological stress and toxicity induced by environmental toxicants in different aquatic organisms such as mollusks and fishes (cossu et al., 1997; sayeed et al., 2003; almeida et al., 2004; zhang et al., 2004; valavanidis et al., 2006). in some field and laboratory studies, the gpx activities in m. galloprovincialis have been already examined to assess the toxic effects of contaminants in marine environment (cravo et al., 2009; maria et al., 2009; chatziargyriou and dailianis, 2010; gomes et al., 2012; nahrgang et al., 2013). however, no gpx genes have been characterized from this ecotoxicologically important species so far, although the mrna expression of mgcgpx has been examined in mussels exposed to chemical mixtures (giuliani et al., 2013). in this study, typical mollusc gpx and gpx4 genes were cloned and characterized in m. galloprovincialis. both amino acid sequences contained a selenocysteine encoded by a terminator codon tga, suggesting that they belonged to the family of selenocysteine-containing proteins. multiple sequence alignment showed that both sequences contained the conserved gpx signature motif 2 and active site motif, which strongly implied that they were new members of the 158 selenium-dependent gpx family. phylogenetic analysis demonstrated that mgcgpx was similar to vertebrate gpx1s and gpx2s, indicating it might be a gpx1 or gpx2 isoform. furthermore, the mollusc gpx branch was in the base of gpx1 and gpx2 branch, suggesting a parallel evolution among most mollusc gpxs and vertebrate gpx1/gpx2 (fu et al., 2012). tissue-specific distribution patterns of mgcgpx and mggpx4 transcripts indicated that both transcripts were mainly expressed in hemocytes, gills and digestive gland. in bivalves, ros production had been shown to be involved in the immune defense and detoxification process of hemocytes (gomez-mendikute and cajaraville, 2003). the comparably high expression levels of mgcgpx and mggpx4 may be associated with the high oxidative stress occurring in hemocytes. the enrichment of mgcgpx and mggpx4 transcripts in digestive gland was probably due to the fact that digestive gland was the major metabolic site for pollutants detoxification in molluscs (livingstone, 1991). the high transcript level of gpx in digestive gland was also reported in scallops and clams (mu et al., 2010; wang et al., 2011a; zhang et al., 2011). the gill is continuously exposed to environmental stress factors such as metallic ion, salinity, toxic substance and pathogen. therefore, the transcripts of mgcgpx and mggpx4 might be highly transcribed in this tissue. similar results were also found in abalone, oyster and clam (de zoysa et al., 2008; jo et al., 2008; zhang et al., 2011, 2012). cadmium and arsenic are prooxidant contaminants and exposure to them is often associated with antioxidant response in aquatic organisms (winston and di giulio, 1991; ventura-lima et al., 2011). recently, the change of gpx at transcriptional level has been used to evaluate the antioxidant response to contaminant stress in molluscs (jo et al., 2008; bigot et al., 2011, zhang et al., 2011; wang et al., 2012). several studies have demonstrated that mollusc gpx activity can be modulated upon cadmium exposure (chandran et al., 2005; legeay et al., 2005; wang et al., 2010; wang et al., 2011b). however, the antioxidant response to arsenic exposure was less studied in molluscs (chakraborty et al., 2010). in this report, a dose-response study was carried out to elucidate gene expression profiles of mgcgpx and mggpx4 upon contaminants exposure. in the gill tissues, the expression levels of both transcripts decreased in a dose-dependent manner after cd exposure, although a significant decrease was only observed in relative high concentration. after asv exposure, both transcripts of mgcgpx and mggpx4 were significantly down-regulated at all concentrations in gills, indicating that both these genes responded sensitively to arsenic exposure even at the environmentally relevant concentration. however, another study reported that gpx transcript in gills of oyster c. gigas increased as the cd concentration (10, 50 and 100 μg l-1) and exposure time (11 day) increased. it was deduced that short term cadmium exposure might stimulate the mrna expression of gpx to counteract the antioxidant stress in oyster (jo et al., 2008). however, sub-chronic contaminant exposure might cause strong oxidative stress in gills which are usually constantly contacted with pollutants. the stress could dramatically lower the metabolic capacity and result in a decrease in mrna expression of antioxidant enzymes (zhang et al., 2004, 2011). additionally, the tissue structure damage in gills of bivalve might result in the inhibition of gpx mrna expression after asv exposure (chakraborty et al., 2010). it has also been reported that the mrna expression of gpx4 was inhibited in fish olfactory tissue which was frequently contact with contaminant. in common carp, the mrna expression of gpx4b was greatly down-regulated following cd (10 mg l-1) exposure for 21 d (hermesz and ferencz, 2009). in coho salmon, both low (3.7 μg l-1) and high (347 μg l-1) level cd exposures for 24 48 h significantly decreased gpx4a mrna expression in olfactory (wang et al., 2012). in addition, negative correlations were observed between the mrna expression levels of mgcgpx and mggpx4 in gills and the concentration of contaminant exposure, suggesting that the mrna expression of these genes might be used as potential biomarkers for assessing the toxicity associated with contaminant exposures. several previous studies also proposed the use of gpx4 transcript level in aquatic animals as biomarker for monitoring environmental pollution (dash et al., 2006; nair et al., 2012). in digestive gland, cd but not asv exposure modulated the mrna expression of mgcgpx at all concentrations, suggesting that mgcgpx was perhaps involved in the detoxification process of cd in digestive gland. similarly, the up-regulation of gpx mrna expression had also been reported in digestive gland of v. philippinarum after cd exposure (zhang et al., 2011). however, few studies have investigated the possible role of bivalve gpx4 in the detoxification process. it was reported that gpx4 activity could be stimulated by cd (200 μg l-1) stress in digestive gland, indicating a protective role of gpx4 against lipid peroxidation in p. perna (almeida et al., 2004). no information is available on the expression pattern of gpx4 in response to pollutant in mollusc so far. in the present study, the transcription of mggpx4 was up-regulated in mussels exposed to both cd and asv at the high concentration (100 μg l-1). these results indicated that the transcript of mggpx4 was modulated to protect the mussel against oxidative stress in the digestive glands of m. galloprovincialis. similarly, the expression of gpx4 mrna was also induced after cd (49.2 mg l-1) and asv (24.0 mg l-1) exposure for 6 h in hydra h. vulgaris (dash et al., 2006). in the aquatic midge c. riparius, the expression of gpx4 mrna was found to be modulated upon exposure to different concentrations and time durations of cd (nair et al., 2012). in this study, total gpx activity was also assayed to determine the correlation between the gene expression and enzymatic activity. it was found that the increase in transcript level of mgcgpx and mggpx4 mainly coincided with the increase of total gpx activity following cd exposure. however, the change of total gpx activity was different with the mrna expression pattern of mgcgpx and mggpx4 in digestive gland after asv exposure. a different 159 sensitivity towards the inducers at the transcription level, mrna processing, or transport and protein stability may lead to discrepancies between transcript level and enzyme activity (okey, 1990). moreover, mgcgpx and mggpx4 contributed only partially to the total enzymatic activity of gpx in this mussel. the inhibition of gpx activity by arsenate might be ascribed to the fact that asv can form complexes with gsh to facilitate the excretion and biotransformation processes of this element (aposhian et al., 2004). therefore, asv might have adverse impact on the content of gsh in cells to affect the total gpx activity. in summary, we have cloned two gpx genes (mgcgpx and mggpx4) in m. galloprovincialis. both the encoded gpx proteins had the conserved selenocysteine residue, catalytic sites and signature motifs. the constitutive transcripts of these genes were detected in six tissues examined. the modulation of mgcgpx and mggpx4 transcript were involved in the antioxidant responses against pollutants challenge. this work will contribute to a better understanding of detoxification systems in marine bivalves. moreover, the expression of mgcgpx and mggpx4 mrna can be considered as potential molecular biomarkers for assessing contaminant toxicity and aquatic environmental quality. acknowledgements this work was supported by grants from national natural science foundation of china (no. 41206105), key deployment program of chinese academy of sciences (kzzd-ew-14-03) and 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from manila clam venerupis philippinarum. aquac. res. 43: 1176-1183, 2012. 162 insect digestive enzymes as a target for pest control" isj 8: 190-198, 2011 issn 1824-307x review insect digestive enzymes as a target for pest control ml rodrigues macedo1, m das graças machado freire2 1departamento de tecnologia de alimentos e saúde pública, centro de ciências biológicas e da saúde, universidade federal de mato grosso do sul, campo grande 79070-900, ms, brazil 2laboratório de química e biomoléculas, centro de pesquisas, institutos superiores do censa, campos dos goytacazes 28010-970, rj, brazil accepted october 4, 2011 abstract the continual need to increase food production necessitates the development and application of novel biotechnologies to enable the provision of improved crop varieties in a timely and cost-effective way. plants and herbivores have been co-evolving for thousands of years, and as a result, plants have defence mechanisms that offer protection against many herbivores/predators. plant proteinase inhibitors (pis), which play a potent defensive role against predators and pathogens, are natural, defense-related proteins often present in seeds and induced in certain plant tissues by herbivory or wounding. this review describes the main classes of proteinase inhibitors and proteinases, their distribution and localization, general properties, and their main functions. possible applications utilities for the pi and proteolytic enzymes in plant biotechnology have been reviewed. key words: proteinase inhibitor; herbivory; plant; biotechnology introduction losses of agricultural production due to pests and diseases have been estimated at 37 % in europe and worldwide. most damage is caused by arthropods and the methods available today for protecting plant crops against insect predation are heavily dependent on environmentally-aggressive chemicals and that have been estimated to reduce losses by only about 7 % (oerke et al., 1994). this fact justifies the necessity for the research and development of alternative approaches to this problem (carlini and fatima, 2002). plant defenses against insect herbivores are mediated, in part, by enzymes that impair digestive processes in the insect gut. little is known about the evolutionary origins of these enzymes, their distribution in the plant kingdom, or the mechanisms by which they act in the protease-rich environment of the animal digestive tract (chen et al., 2007).the transgenic expression of insecticidal proteins such as α-amylase and protease inhibitors is also being ___________________________________________________________________________ corresponding author: maria lígia r macedo departamento de tecnologia de alimentos e saúde pública centro de ciências biológicas e da saúde universidade federal de mato grosso do sul campo grande 79070-900, ms, brazil e-mail address: bioplant@terra.com.br evaluated as a potential protective strategy against insects (schuler et al., 1998). digestive proteinases of insects the proteinases are a major group of hydrolytic enzymes in insects and are involved in digestive processes, proenzyme activation, liberation of physiologically active peptides, complement activation, and inflammation processes amongst others (neurath, 1984). the proteinases are classified according to their mechanism of catalysis: (1) serine proteinases; (2) cysteine proteinases; (3) aspartic proteinases, and (4) metalloproteinases (bode and huber, 1992). for an efficient management of pest control through proteinase inhibitor transgenes, it is imperative to know the type of enzymes present in the gut of insects and pests. the two major proteinase classes in the digestive systems of phytophagous insects are the serine and cysteine proteinases (haq et al., 2004). murdock et al. (1987) carried out an elaborate study of the midgut enzymes of various pests belonging to coleoptera, while srinivasan et al. (2008) have reported on the midgut enzymes of various pests belonging to lepidoptera. serine proteases are known to dominate the larval gut environment and contribute to about 95 % of the 190 mailto:bioplant@terra.com.br total digestive activity in lepidoptera, whereas the coleopteran species have a wider range of dominant gut proteinases. cysteine proteinases cysteine proteinases, endopeptidyl hydrolases with a cysteine residue in their active center are usually identified based on the effect of their active site inhibitors (iodoacetate, iodoacetamide and e64) and activation of the enzymes by thiol compounds (grudkowska and zagdańska, 2004). in insects, the cysteine proteinases are utilized in the digestive processes (rawlings and barrett, 1993), but are found in several other tissues, indicating that they may also play other roles (matsumoto et al.,1997). studies on the ph dependence of cysteine proteinase activity in the crude extract of insect larvae have indicated that this activity was generally in the alkaline range (bode and huber, 1992; oliveira et al., 2003). the papain family contains peptidases with a wide variety of activities, including endopeptidases with broad specificity (such as papain), endopeptidases with very narrow specificity (such as glycyl endopeptidases), aminopeptidases, dipeptidyl-peptidase, and peptidases with both endopeptidase and exopeptidase activities (such as cathepsins b and h). there are also family members that show no catalytic activity (dubey et al., 2007). the three-dimensional structure of papain, a representative member of papain-like cysteine proteases (kamphuis et al., 1984), has been elucidated (fig. 1), as well as other members. all papain-like cysteine proteases share similar sequences (berti and storer, 1995) and have similar 3-dimensional structures. the structural data provides strong evidence that these proteinases all arose from a common ancestor (dubey et al., 2007). proteinaceous inhibitors of cysteine proteinases are subdivided into three families (stefin, cystatin and kininogen) based on their sequence homology, the presence and position of intrachain disulfide bonds, and the molecular mass of the protein (turk and bode, 1991). serine proteinases serine proteinases are widely distributed in nearly all animals and microorganisms (joanitti et al., 2006). in higher organisms, nearly 2 % of genes code for these enzymes (barrette-ng et al., 2003). being essentially indispensable to the maintenance and survival of their host organism, serine proteases play key roles in many biological processes. serine proteases are classically categorized by their substrate specificity, notably by whether the residue at p1: trypsin-like (lys/arg preferred at p1), chymotrypsin-like (large hydrophobic residues such as phe/tyr/leu at p1), or elastase-like (small hydrophobic residues such as ala/val at p1) (revised by tyndall et al., 2005). serine proteases are a class of proteolytic enzymes whose central catalytic machinery is composed of three invariant residues, an aspartic acid, a histidine and a uniquely fig. 1 molscript diagram of the final model of papain, in complex with ccpi (cowpea cystatin proteinase inhibitor). the papain portion (below) is shown as a gray cα trace and the cystatin (above) as a ribbon diagram. side chains of cystatin residues interacting with the enzyme are drawn in a ball-and-stick representation and labeled. the orientation was chosen to clearly display the role of the inhibitor n-terminus, leading to partial concealment of residue glu 18 (aguiar et al., 2006). reactive serine, the latter giving rise to their name, the “catalytic triad” (fig. 2). the asp-his-ser triad can be found in at least four different structural contexts (hedstrom, 2002). these four clans of serine proteases are typified by chymotrypsin, subtilisin, carboxypeptidase y, and clp protease. the three serine proteases of the chymotrypsin-like clan that have been studied in greatest detail are chymotrypsin, trypsin, and elastase. all three of these enzymes are similar in configuration, as shown by their x-ray structures (figs 2, 3). more recently, serine proteases with novel catalytic triads and dyads have been discovered, including ser-his-glu, ser-lys/his, hisser-his, and n-terminal ser (hedstrom, 2002). proteinase inhibitors inhibitor proteins have been found for each of the four mechanistic classes of proteinases and a large number of proteinase inhibitors are directed towards serineand cysteine proteinases (barrett et al., 1987; turk and bode, 1991). in contrast, only a few of these inhibitors are known for asparticand metalo-proteinases (jouanin et al., 1998; oliveira et al., 2003). plant proteins that inhibit various types of enzymes from a wide range of organisms have 191 http://en.wikipedia.org/wiki/chymotrypsin http://en.wikipedia.org/wiki/trypsin http://en.wikipedia.org/wiki/elastase http://en.wikipedia.org/wiki/x-ray_structure been extensively studied. proteinase inhibitors (pis) comprise one of the most abundant classes of proteins in plants. most storage organs such as seeds and tubers contain 1 to 10 % of their total proteins as pis, which inhibit different types of enzymes (ryan et al., 1981). inhibitors bind tightly to the enzyme’s active site in a substrate-like manner, resulting in a stable complex unlike that of the weak complexes between enzyme-substrate and enzyme-product, which dissociate in a short spantime (oliva et al., 2010). the function of the inhibitors is to control proteolysis within cells, organelles or fluids when limited proteolysis is important for the biochemical or physiological process. a large number of transgenic plants have been developed with pis that confer resistance to different families of insects. serine proteinases inhibitors have been the subject of more research than any other class of proteinase inhibitors and are effective against the serine proteinases in the gut of many insect families, particularly lepidoptera. the role of pis against herbivory was hypothesized due to the abundance of these proteins and the lack of activity against endogenous proteins. extensive studies have shown that pis are induced as components of many defense cascades under various stress-prone conditions, such as insect attack and mechanical wounding (ryan, 1990) fig. 2 crystal structure of bovine chymotrypsin. the catalytic residues are shown as yellow sticks. rendered from pdb 1cbw. cystatin the name cystatin was first used by barrett (1981) to describe an inhibitor that had been discovered and partially characterized from chicken egg-white of papain, ficin and other related cysteine (sen and whitaker, 1973). when other protein inhibitors of cysteine proteinases were characterized and their amino acid sequences determined, it became apparent that these are related to chicken cystatin and, thus, are members of the cystatin superfamily (barrett et al., 1986). tertiary structures were determined (fig. 1) (aguiar et al., 2006). some of these show homology with serine proteinase inhibitors, such as the potato tuber cysteine proteinase inhibitor that belongs to the kunitz-type trypsin inhibitor family, and do not contain the conserved region (gln-x-val-y-gly) that characterizes the cystatin superfamily (carlini and grossi-de-sá, 2002). cystatins, similarly to other competitive protease inhibitors, form a tight complex with the active site of target proteases to cause inhibition and interfere with dietary protein digestive functions in herbivorous organisms (arai et al., 2002). scientific articles have been published reporting on the role of phytocystatins in the control of cys protease activities in plants, or their potential as potent inhibitors of cys proteases in biological systems of practical interest. plant cystatins are now known to be involved in a large variety of physiological processes, ranging from the control of endogenous proteolysis in reproductive and vegetative organs to the inhibition of digestive, extracellular cys proteases of herbivore arthropods, parasitic nematodes and microbial pathogens (revised by benchabane et al., 2010). phytocystatins plant cystatins or phytocystatins are the second most studied class of inhibitors and have been identified and characterized from several plants, including cowpea, potato, cabbage, ragweed, carrot, papaya, apple fruit, avocado, chestnut, and job’s tears, among others. cystatins have also been isolated from seeds of a wide range of crop plants. these crop plants include those of the sunflower, rice, wheat, maize, soybean, sugarcane, etc. (revised by haq et al., 2004). serine proteinase inhibitors protease inhibitors (pis), which are ubiquitous in nature, are a group of prime pest-control candidates, with highly proven inhibitory activities against insect pests and the ability to suppress the enzymatic activity of phytopathogenic micro organisms and nematodes. the possible role of pis in plant protection was investigated as early as 1947 by mickel and standish and the first transgenic the phytocystatins (5 87 kda) present characteristics found in the cystatin subfamilies i and ii ( arai et al., 2002). most phytocystatins have a molecular mass in the 12 16 kda range and are devoid of disulphide bonds and of putative glycosylation sites. several cysteine proteinase inhibitors have been identified and their primary and 192 a2 a1 b1 fig. 3 x-ray crystallographic structure of serine proteinases. (a1) trypsin structure and (a2) structure catalytic triad in detail (pdb id: 2ptc); (b) elastase (pdbi id: 1gvk) tobacco plant expressing pis was first reported in 1987 (hilder et al., 1987). plant proteins that inhibit various types of enzymes from a wide range of organisms have been extensively studied. proteinase inhibitors (pis) comprise one of the most abundant classes of proteins in plants. most storage organs such as seeds and tubers contain 1 to 10 % of their total proteins as pis, which inhibit different types of enzymes (ryan et al., 1981). inhibitors bind tightly to the enzyme’s active site in a substrate-like manner, resulting in a stable complex unlike that of the weak complexes between enzyme-substrate and enzyme-product, which dissociate in a short spantime (oliva et al., 2010). the function of the inhibitors is to control proteolysis within cells, organelles or fluids when limited proteolysis is important for the biochemical or physiological process. one of the recent developments in the field of plant genetic engineering is the manipulation of plants for disease and insect resistance. in an effort to develop insect-resistant crops, the role of plantderived pis was recognized early on. by transferring a single defensive gene from one plant to another either with its own promoter or with constitutive 193 promoters, genetically modified plants can be readily obtained. a large number of transgenic plants have been developed with pis that confer resistance to different families of insects. serine proteinases inhibitors have been the subject of more research than any other class of proteinase inhibitors and are effective against the serine proteinases in the gut of many insect families, particularly lepidoptera. the role of pis against herbivory was hypothesized due to the abundance of these proteins and the lack of activity against endogenous proteins. extensive studies have shown that pis are induced as components of many defense cascades under various stress-prone conditions, such as insect attack and mechanical wounding (ryan, 1990) the size of plant proteinase inhibitor (pi) proteins ranges from 4 to 85 kda, with a great proportion being small proteins of only 8 20 kda. their amino acid composition is enriched in cysteine residues that are significant in the formation disulfide bridges and in conferring stability to heat, ph changes, and proteolysis (revised by chye et al., 2006). the serine proteinase inhibitors are found in plants including the kunitz (soybean trypsin inhibitor) family, the bowman-birk (soybean proteinase inhibitor) family, potato i inhibitor family, potato ii inhibitor family, barley trypsin inhibitor family, and squash inhibitor family (norton, 1991). bowman-birk type inhibitors are small polypeptides (8 kda), typically found in legume seeds. they are double-headed, binding simultaneously and independently to two separate proteinase molecules, such as trypsin and chymotrypsin (bode and huber, 1992). plant kunitz inhibitors are widely distributed in plants and are mainly concentrated in leguminous seeds of the taxonomic subfamilies mimosoideae, caesalpinioideae and papilionoideae. the structural pattern of most plant kunitz inhibitors is a single polypeptide chain of approximately 20 kda with two disulfide bonds (cys39-cys86 and cys136-cys145) and a single reactive site (oliva et al., 2010). some plant serine proteinase inhibitors are bifunctional molecules and are able to inhibit trypsins as well as α-amylase (strobl et al., 1995). recently, two kunitz inhibitors from prosopis juliflora and adenanthera pavonina have been shown to possess potent cysteineinhibitor activity (franco et al., 2002; macedo et al., 2004). developing insect resistant transgenic plants expressing proteinase inhibitors during the past decade, fundamental changes have taken place in the field of plant molecular biology. among the large number of new technologies that are available, commercial interest has focused on the ability of plants to integrate and express foreign genes and to produce recombinant proteins in bulk quantities at a relatively low cost (franken et al.,1997). new inhibitors against predatory insects with the potential for use in plantgenetic engineering to develop transgenic resistant plants have been characterized (ussuf et al., 2001). the main function of plant cysteine protease inhibitors is thought to be for plant defense.the defensive role of plant cystatins may be due to their inhibitory activities towards the digestive enzymes of insects, their larvae and other proteases involved in some vital processes. several other transgenic plants expressing cysteine protease inhibitors have been shown to be effective against phytophagous insects. (mosolov and valueva, 2005; dubey et al., 2007). the expression of cystatins in transgenic plants to increase host-plant resistance has only been marginally successful. for example, transgenic potatos expressing rice cystatin inhibited larval growth and exhibited mortality of the colorado potato beetle (lecardonnel et al., 1999). however, growth compensation and faster development of the same species feeding on potato foliage expressing rice cystatin has also been observed (cloutier et al., 2000). in two varieties of transgenic poplar, expressing cysteine proteinase inhibitors from rice (leple et al., 1995) and arabidopsis (delledonne et al., 2001), substantial levels of resistance to two chrysomelid beetle species were achieved. in an artificial diet, soyacystatin n, a soybean cysteine proteinase inhibitor, inhibited both western corn rootworm gut proteolysis and larval growth (kiowa et al., 2000). apparently, one or more cathepsin l-like cysteine proteinases of the papain superfamily, present in the rootworm gut, are the targets of this inhibitor (revised by fabrick et al., 2002). the presence of inhibitory domains in serine proteinase inhibitor prompted us to question their physiological functions. it was thought that many plant serine proteinase inhibitor proteins do not have endogenous functions against plant proteases, but show specificities for animal or microbial enzymes. as such, these inhibitor proteins could be applied to combat invasion by pests or pathogens due to their action on foreign proteolytic enzymes. its actions on insect gut proteases were nonetheless experimentally demonstrated using artificial diets and in vitro inhibition assays on insect gut proteases (revised by chye et al., 2006; macedo et al., 2004; ramos et al., 2008). the cpti gene isolated from the cowpea plant (vigna unguiculata) has been extensively used in the generation of insect resistant plants. this is the first plant-originated insect resistance gene to be successfully transferred into other plants species (hilder et al., 1987). cpti is a member of the bowman-birk superfamily of protease inhibitors and possesses the insecticidal properties against the insect groups of lepidoptera, coleoptera and orthoptera (gatehouse et al., 1997). cowpea trypsin inhibitor gene cpti has also been introduced into brassica oleracea var. capitata cultivars yingchun and jingfeng (fang et al. 1997). the transformed plants showed resistance to pieris rapae in laboratory tests. transgenic tobacco expressing high levels of kunitz type of trypsin inhibitor from soybean demonstrated resistance helicoverpa virescens (sharma et al., 2000). pis of the potato inhibitor i and ii family (pin1 and pin2) are the best characterized plant serine pis in terms of their molecular properties (sin and chye, 2004). the heterologous expression of pin1 and pin2 proteins confers insect resistance in transgenic plants. pin1 and pin2 inhibitor target the digestive serine proteinases trypsin and 194 chymotrypsin, the major enzymes contributing to protein digestion in the gut of lepidopteran larvae. jonhson et al. (1989) expressed pin 1 and ii inhibitors in transgenic tobacco plants. the authors shown that the growth of manduca sexta larvae (tobacco hornworms) feeding on leaves of transgenic plants containing inhibitor ii was significantly retarded, compared to growth of larvae fed untransformed leaves. however, the presence of tomato inhibitor i protein, a potent inhibitor of chymotrypsin but a weak inhibitor of trypsin, in transgenic tobacco leaves had little effect on the growth of the larvae. currently, the genes of more than 14 proteins, proteinase inhibitors, are expressed in various cultured plants. the majority of transgenic plants containing proteinase inhibitor genes are characterized by increased resistance to insects and some other pests. apparently, the most promising are plants containing the genes of proteinase inhibitor in combination with genes of other proteins. it can be assumed that in these cases proteinase inhibitors not only act by themselves, but also protect other recombinant proteins from the destructive action of plant proteinases (valueva and mosolov, 2004). adaptive strategies from insects to proteinase inhibitors when ingested protease inhibitors (pi) block protease activity and increase insect mortality by restricting the availability of essential amino acids. mechanisms of insect resistance to pis include the upregulation of enzymes that degrade the pis (yang et al., 2009), the induction of enzymes that resist inactivation by pis (broadway, 1996), and overproduction of enzymes to maintain normal levels of gut proteolysis (brioschi et al., 2007). some insects exhibit an amazing flexibility in adapting to various host plants by altering the specificities of their gut proteases in response to qualitative changes in dietary protein content and when the existing proteases are ineffective and/or inefficient for digestion (gatehouse et al., 1997). studies on insect responses to the dietary incorporation of plant-derived pis have indicated a biphasic response characterized by an initial upregulation of all digestive protease specificities, which precedes a simultaneous downregulation of pi-sensitive proteases and upregulation of piinsensitive proteases (bown et al., 2004). a similar response can be expected with a change in host plant. although it is clear that insects are able to express a variety of proteinases in response to pi exposure, the mechanism of enzyme induction is unknown. brito et al. (2001) found that heliothis virescens larvae vary their complement of trypsin activities when fed control or inhibitor-containing diet. data indicated that newly-synthesized trypsins have altered substrate specificities, probably reflecting different interactions with substrates and plant inhibitors. volpicella et al. (2003) showed that helicoverpa zea larvae express two different trypsins depending on whether larvae were fed control or inhibitor-containing diets. these enzymes differ in some residues predicted to be involved with inhibitor interactions (lopes et al., 2004). other strategies involve the overexpression of proteases from alternative functional classes following inhibitor ingestion (rivard et al., 2004), the degradation of inhibitor with non-target insensitive proteases (zhusalzman et al., 2003), and a reallocation of cellular resources towards inhibitor-induced compensatory processes (liu et al., 2004). it is now well recognized that protease/ inhibitor interactions in plant-insect systems are the result of a long coevolutionary process triggering the continuous diversification of (insect) proteolytic and (plant) protease inhibitory functions (kiggundu et al., 2006; benchabane et al., 2010) mechanism of action in insect guts the mode of action of pis at the tissue level in insect guts is extremely selective and different types of pis have a different mechanism of action. the activity of pis is due to their capacity to form stable complexes with target proteases, blocking, altering or preventing access to the enzyme active site. pis with activity against serine proteases, the most widespread in nature, act as a potential substrate for proteases. residues forming the scissile peptide bond are indicated as p1-p1’ and are generally located on an external loop of the protein, interacting with proteases. the p1 residue determines the specific type of serine protease inhibited. other residues around the reactive site also play a role in determining the strength of the pienzyme interaction (fan and guo-jiang, 1997). the possible role of pis in plant protection was envisaged as early on as 1947 when mickel and standish observed that the larvae of certain insects were unable to develop normally on soybean products (haq et al., 2004). reese (1983) had proposed a simple hypothesis that growth rates were reduced due to reduced rates of proteolysis, which was later dismissed when broadway and duffey examined the physiological effects of pis in the gut protease activity in insects. these authors suggested that a feedback mechanism led to the hyperproduction of proteinases to compensate for the loss of activity, which in turn led to the depletion of essential amino acids and finally resulted in retarded growth rates. in a study conducted by marchetti et al. (2000), it was observed that larvae fed on transgenic plants expressing a kunitz inhibitor gradually lost their turgor and became shrunken; hence it appears that food avoidance also has a dramatic effect on the water balance of the feeding larvae (revised by lawrence et al., 2002). ramos et al. (2009) suggested that the toxic effect of the protease inhibitors induces the insect to eliminate its digestive enzymes in feces, complicating its digestion. in contrast, some insects, such as spodoptera littoralis (lepidopteran), can overcome the deleterious effects of protease inhibitors by synthesizing different proteases that are insensitive to particular inhibitors (paulillo et al., 2000; brito et al., 2001; de leo et al., 2001; volpicella et al., 2003). hence, the exact mechanism of action of pis, at the tissue level of insects, is not well described (carlini et al., 2002; amirhusin et al., 2007). 195 future trends the aim of this literature review was to highlight the ability of some proteins, including pis, as resistant factors against some important insect pests to reduce the massive use of chemical compounds. these proteins have demonstrated direct insecticidal activity on a wide range of insect pests and have the potential for expression in transgenic crops, conferring insect resistance to plants. a considerable amount of transgenic plants expressing the genes for serine and cysteine proteinases have been obtained over two decades of research (valueva et al., 2004). since the discovery that economically important insect pests, namely lepidoptera, diptera and coleoptera, use serine and cysteine proteinases in their digestive system to degrade proteins in ingested food, efforts have been directed at defining genes encoding pis that are active against these mechanistic classes of proteases for developing transgenic plants (habib and fazili, 2007). in a number of cases, the degree of plant protection (assessed by the level of their damage or the effects on the insects) was as high as 50%. however, this value is still lower than those obtained for plants harboring the genes of bt toxins (95% or higher) (gatehouse, 2008). the main reason consists of the rapid adaptation of the digestive tract of phytophagous insects to the effects of the inhibitors, which occur due to the genetic diversity of proteolytic enzymes. further refinement of the method requires new, more efficient proteinase inhibitors to be identified (or those already known or modified, including by constructing hybrid proteins) (revised by mosolov and valueva, 2008). the insect midgut reportedly contains centimes different proteases (bown et al., 1997). these are differentially regulated and cannot all be inhibited by a plant’s pis (broadway, 1996). with the development of transgenic, insectand pestresistant crop varieties, the proteinase inhibitor genes can make a promising contribution towards maximizing yields and minimizing losses due to insects and pests. we can anticipate a number of promising possibilities for pest control through insecticidal genes. all need to be explored and prudently tapped for their implementation in integrated pest management programs (revised by fan and wu, 2005) acknowledgements this work was supported by fundect (fundação de apoio ao desenvolvimento do ensino, ciência e tecnologia do estado de mato grosso do sul) and cnpq (conselho nacional de desenvolvimento científico e tecnológico). we gratefully acknowledge dr n coran (unicamp) for carefully reading the manuscript and assistance in language revision. references aguiar jm, franco ol, rigden dj, bloch-jr c, monteiro acs, flores vmk, et al. proteins: structure, function, and bioinformatics. biochem. mol. biol. 63: 662-670, 2006. amirhusin b, shade re, koiwa h, hasegawa pm, bressan ra, murdock ll, et al. protease inhibitors from several classes work synergistically against callosobruchus maculatus. j. insect physiol. 53: 734-740, 2007. arai s, matsumoto i, emori y, abe k. plant seed cystatins and their target enzymes of endogenous and exogenous origin. j. agric. food chem. 50: 6612-6617, 2002. barrett aj, fritz h, grubb a, isemura s, järvinen m, katunuma n, et al. nomenclature and classification of the proteins homologous with the cysteine-proteinase inhibitor 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(bioorganicheskaya khimiya), 29: 499-504, 2004. ramos vs, freire mgm, parra jrp, macedo mlr. regulatory effects of an inhibitor from plathymenia foliolosa seeds on the larval development of anagasta kuehniella (lepidoptera). comp. biochem. physiol. 152: 255-261, 2009. volpicella m, ceci lr, cordewener j, america t, gallerani r, bode w, et al. properties of purified gut trypsin from helicoverpa zea, adapted to proteinase inhibitors. eur. j. biochem. 270: 10-19, 2003. ramos vs, silva gs, freire mgm, parra jrp, macedo mlr. purification and characterization of a trypsin inhibitor from plathymenia foliolosa seeds. j. agric. food chem. 10: 11348-11355, 2008. yang l, fang z, dicke m, van loon jj, jongsma ma. the diamondback moth, plutella xylostella, specifically inactivates mustard trypsin inhibitor 2 (mti2) to overcome host plant defence. insect biochem. mol. biol. 39: 55-61, 2009. rawlings nd, barrett aj. evolutionary families of peptidases. biochem. j. 290: 205-218, 1993. zhu-salzman k, koiwa h, salzman ra, shade re, ahn je. cowpea bruchid callosobruchus maculatus uses a three-component strategy to overcome a plant defensive cysteine protease inhibitor. insect mol. biol. 12: 321-330, 2003. reese, jc. in: p.a. hedlin (ed), plant resistance to insects, am. chem. soc., washington dc, 231244, 116, 1983. 198 http://www.ncbi.nlm.nih.gov/pubmed/4782525## http://www.ncbi.nlm.nih.gov/pubmed/4782525## acknowledgements dubey vk, pande m, singh, bk, jagannadham, mv. papain-like proteases: applications of their inhibitors. african j. biotechnol. 9: 1077-1086, 2007. fabrick j, behnke c, czapla t, bala k, rao ag, kramer kj, et al. . effects of a potato cysteine proteinase inhibitor on midgut proteolytic enzyme activity and growth of the southern corn rootworm, diabrotica undecimpunctata howardi (coleoptera: chrysomelidae). insect biochem. mol. biol. 32: 405-415, 2002. johnson r, narvaez j, an g, ryan c. expression of proteinase inhibitors i and ii in transgenic tobacco plants: effects on natural defense against manduca sexta larvae. proc. natl. acad. sci. usa 86: 9871-9875, 1989. visions and perspectives isj 8: 153-161, 2011 issn 1824-307x visions and perspectives the ‘immunology trap’ of anthozoans b rinkevich national institute of oceanography, tel shikmona, p.o. box 8030, haifa 31080, israel accepted august 08, 2011 abstract organisms commonly respond to infectious agents via effector arms of immune systems. however, whereas innate immunity in vertebrates has been intensely investigated, we still strive to understand how cnidarians’ immunity operates, consulting literature that is rife with unsubstantiated statements. here i contend that the striking superficial similarities with regard to some vertebrate genes promote the false notion that considers vertebrate’s and cnidarian’s immunities as homologous. this is enhanced by intermingling allorecognition with host-parasite interactions and by synthetic comparisons of anthozoans-vertebrates putative immune genes. as complex as it is, cnidarian’s historecognition is probably not associated with host-parasite/disease responses and studies on anthozoan host-parasite interactions are not yet supported by underlying mechanisms. therefore, i demarcate allorecognition from other aspects of anthozoan immunity and discuss the lack of research studies on the anecdotally recorded anthozoan phagocytes. further attention is given to the roles of ‘non-immunological defenses’, stand-alone defense mechanisms that respond to environmental assaults independently of immunity, also mistakenly regarded as revealing immune properties. because defining immunity in the anthozoa remains deficient, reflecting the needs for improved distinction between historecognition and host-response/disease disciplines, it is required to establish an accepted synthesis for what immunity in cnidarians is or is not, and to evaluate changes in immunocompetence through quantitative approaches. following the current state-of-the-art on cnidarian immunity, six counsels for re-evaluating immune criteria are offered. key words: allorecognition, cnidaria, coral, disease, innate immunity, non-immunological defenses   scientia vincere tenebras (conquering darkness by science) the prevalence of diseases in reef organisms, many of which are highly virulent (weil et al., 2006; mydlarz et al., 2010; reed et al., 2010) has stimulated scientific discussion on its causes and corals’ immune mediated mechanisms (mydlarz et al., 2006, 2009, 2010; reed et al., 2010), all based on the known abilities of reef organisms to display discriminatory tissue reactions to foreign grafts (allorecognition; particularly corals; rinkevich, 2004, 2011). in addition, corals exhibit a suite of effector mechanisms to rid themselves of sediment, settling organisms (including pathogens), on top of cellular (phagocytic cells; bigger and olano, 1993; olano and bigger, 2000; mydlarz et al., 2008) and biochemical/antimicrobial properties, usually with broad spectrum of antimicrobial activities (jensen, ___________________________________________________________________________ corresponding author: buki rinkevich national institute of oceanography, tel shikmona p.o. box 8030, haifa 31080, israel e-mail: buki@ocean.org.il 1996; koh, 1997; kim et al., 2000a, b; petes et al., 2003; ritchie, 2006; mydlarz and harvell, 2007; couch et al., 2008; gochfeld and aeby, 2008; kvennefors et al., 2008; palmer et al., 2008; dunn, 2009; mydlarz et al., 2009). recent studies have also scanned anthozoans genomes to elucidate immune pathways and immune gene families (miller et al., 2007; anderson and gilchrist, 2008; hayes et al., 2010; oren et al., 2010; polato et al., 2010). i contend that these studies, cumulatively, have led to the vague impression that we know what immunity in the anthozoa is. this is not the situation. whilst the vertebrate innate immunity has been the subject of intense investigation, revealing to a great extent an overwhelming complex system (e.g., du pasquier, 2005; ellis et al., 2011), the research on anthozoan immunology suffers from documentation paucity and a lack of an accepted synthesis of what innate immunity is or is not (loker et al., 2004; rinkevich, 2011). also, the synthetic comparisons of cnidarians genes with seemingly counterpart vertebrate   153 immune genes carried very limited valid information on the nature of anthozoan immunity. i further claim that the frequent use of buzz words (e.g., immunological ‘tool kit’; miller et al., 2007) and the tendency to mix allorecognition with host-parasite interactions (e.g., mydlarz et al., 2006) have been erroneously practiced as delivering evidence for cnidarian innate immunity, or have been inappropriately applied to describe immunological responses of reef corals, such as to climate change deliverables (e.g., mydlarz et al., 2009, 2010; reed et al., 2010). here, i further contend that addressing anthozoan immunity by means of deduced genomic sequences or gene homology comparisons amidst vague descriptions of the underlying mechanisms (without having a basic knowledge on the nature of anthozoan immunity) is an inadequate approach, leading to invalid conclusions and rendering this discipline imprecise and elusive. this essay accentuates the fact that we still strive to understand what immunity in the cnidaria is and how cnidarian immunity operates, and that until these are achieved any untenable conclusion could lead to erroneous assumptions. cnidarian immunityhistorecognition anthozoans are sessile organisms that cannot move away from points of settlement (or exhibit very restricted movement capabilities), sometimes living in densely populated communities, in environments that are laden with infectious agents. dense populations also lead to allogeneic and xenogeneic encounters with other permanently attached-tohard-surfaces organisms, which settle in close proximity. the literature attests that anthozoans (as all cnidarians) do not harbor specialized immune cells, wandering discriminatory cells or circulatory systems. however, they exhibit surprisingly complex sets of allorecognition and xenorecognition phenomena, exemplified by extreme allotypic diversity, wide range of effector arms, unconfounded immunological specificity, quasiimmunological memory, allogeneic maturation, fusion events that lead to chimerism, and episodes associated with ‘ecological immunity’, e.g., intraspecific and interspecific competitions (reviewed in lang and chornesky, 1990; leddy and green, 1991; rinkevich 1996a, b, 1999, 2004, 2011). the effector mechanisms that are used by the anthozoans during allogeneic/xenogeneic challenges are enormously complex. the list includes contact avoidance through chemical sensing, allelopathy, barrier formation, tissue and skeletal overgrowths, development of sweeper tentacles, employment of mesenterial filaments, creation of pseudofusions, morphological resorption of chimeric individuals, bleaching, retarded growth rates, transitory fusions, nematocyst firing, developing of delayed responses, necrosis formation, tissue growth without calcification, attraction of motile phagocytic cells, retreat growths, allogeneic reversals and more (details and reviews in hildemann et al., 1979; bak et al., 1982; rinkevich and loya, 1983; hidaka, 1985; sauer et al., 1986; rinkevich and weissman, 1987; chornesky, 1989; lang and chornesky, 1990; romano, 1990; leddy and green, 1991; salter-cid and bigger, 1991; alino et al., 1992; rinkevich et al., 1993, 1994; tanner, 1993, 1997; chadwickfurman and rinkevich, 1994; ding et al., 1994; frank and rinkevich, 1994, 2001; jokiel and bigger, 1994; frank et al., 1995, 1996, 1997; bruno and witman, 1996; rinkevich 1996a, 2004, 2011; van veghel et al., 1996; griffith, 1997; hidaka et al., 1997; abelson and loya, 1999; peach and hoeghguldberg, 1999; aerts, 2000; olano and bigger, 2000; rinkevich and sakai, 2001; barki et al., 2002; lapid et al., 2004; nozawa and loya, 2005; lapid and chadwick, 2006; amar and rinkevich, 2008, 2010). however, many of the phenomenological outcomes of cnidarian historecognition offer little in terms of the cellular and molecular constituents that lead to the morphological outcomes. as complex as they are, these historecognition attributes for rejecting alien tissues are probably not associated with host-parasitic and disease responses in the cnidarians (rinkevich, 2011). moreover, based on our current knowledge, most discussions on anthozoan host parasitic interactions (see below) are weak and flawed because they are not yet supported by any elucidated underlying mechanism. cnidarian immunityphagocytosis and associates as i specified above, the scientific propensity that combines cnidarian historecognition phenomena with host-parasitic/disease events has emerged as a serious obstacle in elucidating the nature of cnidarian (mainly reef corals) immunology (rinkevich, 2011) and its cellular components. therefore, it is not surprising to find in the literature discussions, merging the concept of invertebrates’ immunity with vague, generalized immunological phrases (like ‘the invertebrate immune system is based on self/nonself recognition and cellular and humoral processes’ [mydlarz et al., 2006]), or immunological properties, under a unified ‘immunological umbrella’. one such example for ill-chosen practice in the research of anthozoan immunity is the phenomenon of phagocytosis and its associated molecular cascades. indeed, the predominant mechanism of innate immunity in excluding parasitic/infectious forms involves phagocytosis by immune cells. however, the literature on anthozoan immunity, while documenting a wide repertoire of allogeneic phenomena (hildemann et al., 1977,1979; leddy and green, 1991; rinkevich 1996a, 2004, 2011), does not detail any clearly mounted defensive response on the cellular level, not any evidence for phagocytosis response, nor any cell type that specifically disables infectious agents, or targets direct elimination of infected cells. this argument is further illuminated by a recent study on acropora pathogenesis (work and aeby, 2011) that has employed histological observations on coral lesions. indeed, very few studies (neither one was performed on hermatypic corals) have documented the participation of motile phagocytic cells, epithelial cells, and amebocytes (no specialized phagocytic cells) in cnidarian’s biological phenomena, mostly in   154 wound healing scenarios (mezaros and bigger, 1999; olano and bigger, 2000), but also in response to general stressors (mydlarz et al., 2008). in a more detailed study on a sea anemone (hutton and smith, 1996), phagocytosis by amebocytes was found to be inefficient, as only about 40 % of the cells were observed to ingest bacteria in vitro in over 45 min. these amebocytes, however, showed some antibacterial properties, primarily, when cells were lyzed (hutton and smith, 1996). in association with phagocytosis, cnidarians also possess some hemolytic polypeptids in celentric fluids (meinardi et al., 1994), peroxidase activities (mydlarz and harvell, 2007), antifungal and antibacterial lipid metabolites (koh, 1997; kim et al., 2000a; dunn, 2009), members of the complement system (dishaw et al., 2005; miller et al., 2007; dunn, 2009; kimura et al., 2009), and lectins (kvennefors et al., 2008) which, again, without any direct verification, are conjectured to be involved in the animals’ immune reactions to pathogenic and parasitic insults. this specifically applies to the documentation on phenoloxidaseactivating pathways in anthozoa (petes et al., 2003; mydlarz and harvell, 2007; palmer et al., 2008). while activating this melanin synthesis cascade is widely documented in invertebrates immunity, it has not been confirmed yet whether the anthozoans pathway resides in the cellular free compartments (e.g., celentric fluids), within either type of specialized phagocytes or in any other enigmatic cellular compartment (e.g., ‘granular epidermal cells’; palmer et al., 2008) and if it is an effector arm of the anthozoan immune defense or just a common response to localized or general environmental stress, used as a barrier forming device (petes et al., 2003; mydlarz et al., 2009). to my knowledge, there is no detailed research study on anthozoan phagocytosis pathways (or associated molecular cascades) and no attempt has been launched to identify specific disease-borne responses on the cellular level. furthermore, nothing is known on how cnidarians’ phagocytes recognize a pathogen (e.g., via the use of lectins; pipe, 1990). amebocytes (but not phagocytosis) were anecdotally recorded in diseased anthozoan tissues (e.g., ellner et al., 2007) but more information, such as systemic increase in their numbers (mydlarz et al., 2008), apparent cell infiltration or cellular proliferation, are needed to address the current, immunologically critical questions. on the other hand, other possible cellular and humoral immune functions may go unnoticed if phagocytosis continues to be targeted as the major valuable end point for innate immunity. the issue of the effector cells (including the alleged roles of phagocytes) and associated molecular cascades in cnidarian immunity, therefore, remain untested and offer no resolution in elucidating disease and hostparasitic interactions. immunity, environmental stressors and global changes another mistaken research approach tries to use selected components of invertebrates’ immune systems as the proxy for overall immunocompetence; thus an a-priory set of immune dysfunction factors and associated conclusions become entirely reliant on the immunocompetence proxy parameters, leading to erroneous conclusions (ellis et al., 2011). this flawed approach has also been expressed in studies on cnidarian immunity, such as the attempt to link anthozoans immunity with global change impacts (mydlarz et al., 2009, 2010), without addressing any actual or quantitative change in the overall immunity as a response to pathogenic or environmental challenges. unsustainable statements, like those claiming that acroporid and pocilloporid corals are more susceptible to diseases ‘due to low investment in immunity’, or the use of jargon like ‘overall immunocompetence’ (mydlarz et al., 2010) ‘spatial immunodynamics’ (ellner et al., 2007) and ‘factors that shape the immune physiology of colonies’ (couch et al., 2008) further convey the wrong impression that we are well acquainted with cnidarian and coral immunity. this is not the case. some authors (e.g., lesser et al., 2007) have also suggested that with rare exceptions, coral diseases should be considered as opportunistic infections (syndromes), secondary to exposure to physiological insults such as elevated temperature that result in uncontrolled growth of bacteria normally benign and non-pathogenic. therefore, cnidarians’ disease prevalence may be or may not be plausibly associated with global change impacts. hence, any argument on the cnidarians’ tight connection between immunity and environmental stress necessitates a solid validation, quantification and optimization, as generalized for other cases (viney et al., 2005). various studies in other organisms have elucidated the interactions and impacts of nonimmunological ‘defenses’ (see below) on environmental insults, host parasitic interactions and disease prevalence. relevant examples for nonimmunological ‘defenses’ are impacts of ingested plant material on the resistance of insects to their parasitic forms (cory and hoover, 2006) and the possibility that, at least, part of the worldwide recorded shell disease syndrome in crustaceans is not the resultant of causative agents but a disruptive chitin recycling (vogan et al., 2008). the same may apply to documented responses of corals to elevated water temperature, such as the enhanced expression of heat shock proteins, or the elevation in intracellular calcium (fang et al., 1997). therefore, with regard to coral diseases and syndromes (the later probably best characterizes coral diseases, as in the vast majority of cases, no single causative agent has been found as associated with prevalent phenomena), the data supporting the connections between cnidarian immunity and global change impacts is awkward, based largely on anecdotal observations that had been generalized to predict anthozoan immunity. it hampers our ability to evaluate the genuine impacts of environmental stressors and global changes on anthozoans immunocompetence. in the same way, the possible roles of the yet enigmatic cnidarian immunity in the animals’ resistance/susceptibility to infectious agents following, for example, bleaching events have yet to be explained.   155 the sensitiveness of cnidarians’ immune system components to environmental perturbation is another unsolved enigma. this issue was extensively studied in some marine invertebrates, revealing, as an example, that changes in phagocytic activity can serve as sensitive parameter to environmental insult and to anthropogenicallyinduced stressors (ellis et al., 2011). the cnidarian arena remains, however, deficient in spite of documentation revealing seasonal and site variations, across a geographic region, in disease prevalence, antimicrobial and enzyme properties (e.g., ritchie, 2006; toledo-hernández et al., 2007; couch et al., 2008). in the same way, very little has been comprehended on the crux of the apparent coral bleaching in relation to immunity. one ‘non immunological’ possibility (see also below) is that ‘corals that have undergone bleaching become more vulnerable to pathogens because the protective contributions of their zooxanthellae have been lost’ (loker et al., 2004). in drosophila, abiotic conditions (such as elevated temperature) directly affect susceptibility to parasites regardless of the functionality of its immune systems (linder et al., 2008), a phenomenon that may well be comparable to the cnidarians’ increased disease prevalence following elevated seawater temperatures. similarly, the variations in resistance of pea aphids attacked by parasitoid wasps are related to the impacts of the facultative bacterial symbionts, not the host genotype’s immunity (oliver et al., 2005). stressinduced diseases in corals also recall the phenomenon of the environmentally inflicted stressinduced senescence, a premature senescence induced by various stressors in the absence of telomere loss or dysfunction (reviewed in kuilman et al., 2011). understanding how the cnidarian immune system responds to environmental challenges and how it reflects seasonal variability (e.g., duchemin et al., 2007) are of primary importance. however, dealing with global change impacts on immunity (without validating what is cnidarian immunity or what are the cnidarian immune characteristics), and overlooking the roles of environmental, nonimmunological factors in corals’ susceptibility to diseases, may lead to wrong conclusions. non-immunological ‘defenses’ and how should anthozoan immunity be defined? while organisms do respond to infectious agents via the effector arms of their immune systems, recent studies have revealed the importance of ‘non-immunological defenses’, standalone defense mechanisms that operate autonomous to immune system machineries (but interact with immune systems) and contribute to the organism ability to withstand the impacts of infectious agents (reviewed in parker et al., 2011). examples of non-immunological defenses include behavior (e.g., the hygienic behavior in honey bees that limits diseases and individual host susceptibility; wilson-rich et al., 2009), fecundity compensation (petes et al., 2003), physiological properties, anorexia, symbiont mediating immunity, and social immune mechanisms (parker et al., 2011). some further illustrations for very effective non-immunological defenses are (1) the contribution of feeding regimen among daphnia clones to the variation recorded in the animals’ susceptibility to fungi (hall et al., 2010), and (2) the secretion of a special thick mucus layer, normally expressed in non intestinal mucosa, in the intestine of mammals resistant to parasite infection, lowering the viability of gut-dwelling nematode worms (hasnain et al., 2011). likewise, cnidarian ‘chemical warfare’ against microbes (koh, 1997), cytotoxicity of the secreted mucus (ding et al., 1994), the expression of chitinolytic enzymes (douglas et al., 2007), and the emancipating of non-specific antifungal and antimicrobial compounds (jensen, 1996; kim et al., 2000a, b; ritchie, 2006; gochfeld and aeby, 2008) should all be considered as responses associated with non-immunological defenses (lesser et al., 2007; parker et al., 2011), unless proven otherwise (being an integral participant of immunity, part of the effector arm). this could also apply to the vast majority of melanization phenomena, as recorded in the cnidarians (petes et al., 2003; mydlarz and harvell, 2007; palmer et al., 2008). the arguments presented here stand for all cnidarians, including hydrozoans, but for clarity and the lack of space this assay concentrates on the anthozoa. here i wish to highlight, again, the claim that anthozoan immunity, including recognition elements and effector arms, is poorly understood and that the term ‘anthozoan immunity’ (and associated versions) is wrongly used in a broad sense, ignoring the fact that the effector arms used by one group of organisms (e.g., vertebrates) are probably different from their parallel in other taxa (e.g., corals; loker et al., 2004). special consideration should be given to the demonstrated wide range of interspecific and intraspecific differences in responses to any single biological/environmental assault, even to different levels of a single stressor, or to stressors generated by a single cause or in a combination of several sources (ellis et al., 2011). indeed, progress has recently been made in expounding the molecular details of cnidarians genomes, revealing, by the use of bioinformatics, homologous sequences to the vertebrate immune genes (miller et al., 2007; anderson and gilchrist, 2008; hayes et al., 2010; oren et al., 2010; polato et al., 2010). however, the striking superficial similarities offered with regard to some genes and processes in the cnidarians in general and anthozoan in particular, are based on the wrong notion that considers vertebrate immunity and cnidarian immunity as homologous (stemming from the rationale that the early appearance of host defense indicates that same immune constituents are shared by most multicellular organisms; a sort of anthropocentrism). conclusionsin rerum natura (in the nature of things) cnidarians, as many other invertebrates (rinkevich, 1999), may employ alternative means to generate immunity, making this discipline highly complex. in a similar fashion, it has been   156 questioned whether the roles of toll (a family of proteins that triggers innate immunity) in drosophila host resistance are comparable to the roles of tolllike receptors in mammalian immunity (trinchieri and sher, 2007). on the other hand, bioinformatics approaches and genome screenings, without ‘forcing’ vertebrate immunological notions onto cnidarian immunity, can be used as powerful tools in the research. as immune function in vertebrates is one of the biological attributes enriched with genes under positive or balancing selection (e.g., fumagalli et al., 2009; barreiro and quintana-murci, 2010), the two evolutionary forces underlying adaptation, employing bioinformatics approaches on proposed cnidarians ‘host-pathogen interaction genes’ that reveal signatures of adaptation, may emerged as the ultimate tool in the research. this is further highlighted by the vertebrate/invertebrate outcomes that innate immunity systems act in a semi-specific way by recognizing pathogenassociated molecular patterns (pamps), which are essential and conserved components of pathogen entities. although the literature on cnidarian immunity is rife with unsubstantiated statements and conclusions, it is deficient with regard to what is immunity in this group of primitive organisms (rinkevich, 2011). more challenging is the outcome that at least some invertebrates possess functional equivalents of the acquired responses of vertebrates (reviewed in kvell et al., 2007). while this adds to the foreseen complexity of cnidarian immunity, as specified above, except for the phenomenon of allorecognition where much research has been done (hildemann et al., 1979; leddy and green, 1991; rinkevich 1996a, 2004, 2011), we are still limited in our understanding of what is cnidarian immunity in general, and do not fundamentally grasp yet coral immunity, in particular. recent approaches and research attempts that have delved into the molecular level (trying to infer analogous from the vertebrate arena, before exploring the full repertoire of the invertebrates morphological and cellular mechanisms) run the risk of overlooking the real phenomenological outcomes, and neglect the possible new immunity avenues explored by the cnidaria (little et al., 2005) by falling into the ‘homology trap’ (klein, 1997). it is also dangerous to tightly connect other phenomena, like those associating coral tumors (domart-coulonet al., 2006) with coral immunity (palmer et al., 2008; mydlarz et al., 2010). the only real phenomenological homology between marine invertebrates and vertebrate immunities is probably allorecognition, marked by the explicit notion that the mechanisms underlying them are similar only in the general paradigm of self/nonself recognition (rinkevich, 2011). the effector arms and expression pathways, all evolving in harmony for orchestrating the immunocompetence in allorecognition and infections/disease responses, are probably disparate, thus conclusions for the nature of each component of innate immunity can be reached only through controlled experiments. clearly, the incomplete understanding of anthozoan immunocompetence hampers our ability to study immune related responses. immunity in invertebrates was for long analyzed in terms of the overall response, resulting in misunderstandings concerning its biological properties (brehélin and roch, 2008). to overcome such a difficulty, hildemann et al. (1977, 1979) have proposed a minimal set of criteria to test invertebrates’ immunity, a major step in the research since it put forth, for the first time, a defined set of criteria required for use of a term. this led to discussions and rebuttals for validity, exclusion or inclusion of immunological criteria in experimental outcomes, or what is required to meet those criteria. however, even after more than three decades of research on coral biology, very little is known about coral immunology, even though much work exists on historecognition reactions. in this regards, researchers have attempted to provide some empirical evidence of what that immune system may possess (i.e., gene products) as well as some preliminary, even anecdotal, evidence as to how that system may function (e.g., phagocytosis), all without much success to reveal the nature of cnidarian immunity. i argue that the extent to which anthozoan ‘immunity’ (but not historecognition) is modulated or modified by parasitic forms, environmentally laden microbes, or environmental insults cannot be inferred from the current literature. therefore reconsidering the approach for ‘criteria’ (hildemann et al., 1977, 1979) in the research of cnidarian immunity/diseases/host-parasitic interactions may clear up the way for understanding the impacts of environmental insults on anthozoan immunocompetence. the current state-of-the-art reveals that we still do not really know what immunity in the cnidaria is. hence, before statements on cnidarian immunity can be made (like the roles of immunocompetence in coral diseases, impacts of environmental stress on coral immunity), we need an improved distinction between historecognition, host-response and disease disciplines in the cnidaria. then, the research on anthozoans immunity needs (a) to establish, with high standards of scientific scrutiny, an accepted synthesis of what immunity in this group of organisms is or is not (e.g., theoretically, as long as pathogens are correctly identified, there is no need for the fine detection of all non-self versus self), (b) to recognize that the ability of corals to ward off opportunistic infections and the capacity for highly regulated allorecognition might have evolved from different origins under differing evolutionary pressures. unless proven otherwise, both phenomena should be considered independently, thus any scientific outcome to be assigned to either immunity route, not a-priori shared by both, (c) to evaluate the changes in immunocompetence following virulent/environmental assaults through quantitative approaches, such as the ‘clearance efficiency’ assay, phagocytic index, cellular reactive oxygen intermediate production, bactericidal activity of cells and other assays (ellis et al., 2011), (d) to perform ‘’experimental immunization assays’, for elucidating the properties of cnidarians immunity. such an approach may test the likelihood that   157 infection can impose changes in the activity of certain highly defined immune functions (e.g., nonspecific immunological priming). for example, moret and siva-jothy (2003) showed that injection of bacterial cell wall components increased the resistance of insects against a fungus, up regulating of a generic immune response, also showing that an induced response can occur without specificity, (e) to clarify the roles and importance of ‘nonimmunological defenses’ (like mucus shedding in corals) in cnidarian immunosurveillance, and (f) to employ bioinformatics approaches and genome screenings not only as comparative tools. molecular biology approaches should be exercised to better design functional experiments of cnidarian hostpathogen interactions and immunosurveillance (as successfully employed on cnidarian historecognition) and to analyze the footprint of adaptive selection signatures in the innate immune mechanisms. since the ‘immunology trap’ is not unique to the cnidaria, above six major suggestions for re-evaluating immune criteria may 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they rely on plant-sourced diets and intestinal bacteria for their supplies. a unique feature of earthworm ‘hepatocyte-like’ chloragocytes and chloragocyte-derived eleocytes floating in celomic cavity is the storage of riboflavin within intracellular granules. the hypothesis was that vegetarian food-deprivation or antibiotic/antifungal treatment inhibits riboflavin accumulation in eleocytes of eisenia andrei. the 7-week starvation inhibited worm body weight gain and worm reproduction but had insignificant effects on celomocytes, both amoebocytes and eleocytes, and eleocyte riboflavin accumulation. the 1 week or 3 week antibiotic exposure had insignificant effects on worm coelomocytes and riboflavin content. thus, a vegetarian diet and intestinal bacteria are not the exclusive or perhaps even the main sources of eleocyte riboflavin. the role of endosymbionts in earthworm flavonoid economy warrants targeted investigation. moreover, the possibility of horizontal transfer of riboflavin biosynthesis genes from bacteria/fungi to earthworm genomes cannot be neglected. key words: eisenia andrei; food deprivation; antibiotic treatment; celomocytes; riboflavin introduction the earthworm immune system is very efficient (bilej et al., 2011). the immunocompetent cells (celomocytes) include amebocytes, which are classical immunocytes according to nomenclature proposed by ottaviani (2011), and species-specific chloragogen tissue-derived free chloragocytes (eleocytes). eleocytes (detached chloragocytes), but not amebocytes, exhibit autofluorescence (cholewa et al., 2006) that is evidently restricted to chloragosomal vesicles (plytycz et al., 2007). autofluorescent self-marking, and the simplicity of non-invasive retrieval of celomocyte suspensions, make eleocytes ideal subjects for flow cytometry analysis (e.g., cholewa et al., 2006; plytycz and morgan, 2011). spectrofluorimetry has revealed that riboflavin (vitamin b2) is a prominent fluorophore contributing to eleocyte autofluorescence (koziol et al., 2006; cygal et al., 2007). the percentage of autofluorescent eleocytes among celomocytes and ___________________________________________________________________________ corresponding author: barbara plytycz department of evolutionary immunobiology, institute of zoology jagiellonian university, krakow, poland e-mail: barbara.plytycz@uj.edu.pl the amount of riboflavin stored within eleocytes are species-specific, with some species possessing a high proportion of fluid-suspended eleocytes whilst other species have very few such eleocytes in their celom (plytycz et al, 2006). moreover, intrinsic edaphic variables (plytycz and morgan, 2011; plytycz et al., 2011), including metal contamination (e.g., kwadrans et al., 2008; homa et al., 2010; piotrowska et al., 2010; podolak et al., 2011), can modulate eleocyte counts and riboflavin content. lumbricid worms can expel celomocytes through their dorsal pores when irritated in natural environments (e.g., by predators) or experimentally by physicochemical stimuli such as a mild electric shock (roch, 1979), ultrasound (hendawi et al., 2004), or 5 % ethanol (cooper at al., 1995). experimental manipulations do not affect worm viability, and their immune system gradually recovers after electro-stimulation (olchawa et al., 2003; polanek et al., 2011). riboflavin is not stored exclusively in eleocytes. recent observations indicate that the attached chloragocytes of lumbricid earthworms, whether they have high or very low numbers of chloragocyte-derived eleocytes floating freely in the celomic fluid, contain significant riboflavin levels (mazur et al., 2011). this finding 169 mailto:barbara.plytycz@uj.edu.pl fig. 1 schematic representation of two independent experiments designed to investigate the effects of (a) food deprivation and (b) antibiotic/antifungal treatment, respectively, on riboflavin content in the eleocytes of eisenia andrei. in experiment a, worms were either fed ad libitum (f treatment) or unfed (u treatment). in experiment b, worms were exposed either to water-soaked filter paper (controls: c treatments) or to filter paper soaked with antibiotic/antifungal cocktail cefuroxime/fluconazole (a treatments). at the end of 7-week (experiment a) or 1week and 3-week (experiment b) experimental periods, celomic fluid was expelled by electro-stimulation (x-es) and analysed. indicates that riboflavin storage plays an important role in earthworm biology. riboflavin is synthesized by plants and many microorganisms (bacher et al., 2000). animals lack the riboflavin biosynthesis machinery (fassbinder et al., 2000). consequently, the main sources of the vitamin for earthworms are highly likely to be a plant-based detritivorous diet, intestinal microflora, and from other endosymbionts. for example, there is direct evidence for insects (nakabachi and ishikawa, 1999) and nematodes (taylor et al., 2012) being provided with riboflavin by their endosymbiotic bacteria. the main aim of the present work was to find the primary source of the riboflavin accumulated in the eleocytes of the epigeic, composting, earthworm species eisenia andrei. in the first series of experiments the worms were food-deprived (u unfed) while in the second series they were treated with an antibiotic/antifungal cocktail (a). the working hypothesis was that eleocyte riboflavin accumulation would be decreased in celomocyte lysates by worm starvation or by reducing the gut flora, respectively. it turned out, however, that the effects of such treatments were statistically insignificant, and therefore other putative sources of riboflavin accumulated in eleocytes are discussed. materials and methods earthworms adult eisenia andrei (oligochaeta; lumbricidae), field-sampled in manure and compost heap in the sadecki mountains (southern poland), were kept under controlled conditions (16 ± 1 °c; 12:12 ld) in the laboratory. the worms were kept in plastic boxes with perforated lids and the moisture level was checked weekly. groups of worms with similar individual body weights (c. 0.5 -0.6 g) were used. experimental design (fig. 1) worms were subjected to celomocyte extrusion at the end of 7-week experiments on effects of starvation or after 1-week or 3-week experiments on effects of antibacterial/antifungal factors. food deprivation (fig. 1a) worms with similar body weights (appr. 0.6 g) (2 boxes, 8 worms per box) were maintained on 170 fresh commercial soil (ppuh biovita, tenczynek). in the first box worms were fed (‘f’), i.e., provided ad libitum with a mixed diet comprised of dried/boiled nettle (urtica dioica) and dandelion (taraxacum officinale) leaves, and in the second the worms were deprived of food (i.e., unfed, ‘u’). after 7 weeks on the contrasting dietary regimes, all of the worms were weighed, their celomocytecontaining celomic fluid was extruded and analysed, and the egg capsules (cocoons) in each box were counted. the results were compared and statistically analysed. antibacterial and antifungal treatment (fig. 1b) earthworms were exposed dermally to solution of antibacterial/antifungal agents since such a procedure was very efficient in studies on effects of heavy metal solutions on worm celomocytes (e.g., see olchawa et al. 2006; plytycz et al. 2011). worms of similar body weight (appr. 0.5 g), 10 worms per group, were kept individually in plastic tubes on a substrate comprised entirely of dailyexchanged shredded soft filter paper (5.7 g per tubes) soaked either with water (i.e., controls: ‘c’ groups) or with an antibiotic/antifungal cocktail (‘a’ groups). the antibiotic used was cefuroxime (zinacef 750 inj., glaxo), a second generation cephalosporin. the antifungal agent was fluconazole (diflucan 2 mg/ml inj., pfizer), a triazole antifungal drug. these agents were diluted and combined to give concentrations of 10,000 mg kg-1 of cefuroxime and 200 mg kg-1 of fluconazole per in the filter paper. after one week and 3 weeks, 5 individual worms from each of the treatments (‘c’ and ‘a’, respectively) were retrieved, weighed, and their celomocytes extruded and analysed. comparisons and statistical analysis were performed between ‘c’/‘a’ groups after 1 week, and 3 weeks, separately. celomocyte extrusion the earthworms were stimulated for 1 min. with an electric current (4.5 v) to expel celomic fluid with suspended celomocytes through the dorsal pores. briefly, the weighed earthworms were individually placed in petri dishes containing 3 ml of extrusion fluid (phosphate-buffered saline, pbs, supplemented with 2.5 g/l ethylenediamine tetraacetic acid, edta), and 2 ml samples of the extruded celomocyte suspensions were used for spectrofluorimetric analysis; 1 ml was fixed in 2 % formalin and used for cell counting in hemocytometer and flow cytometry. soil-derived bacteria soil samples were prepared according to modified procedure used previously (wieczorekolchawa et al., 2003). in short, sample of air dried commercial soil (2 g) used in present experiments was shaken in 10 ml of sterile pbs on a laboratory shaker type 358 s (elpan, poland). after 1 h sedimentation, 1 ml of supernatant was added to 9 ml sterile bacterial broth (biomed, warszawa, poland). after overnight incubation at room temperature, suspension of soil-derived bacteria was used for testing the efficiency of antibacterial/antifungal agents. effects of cefuroxime and fluconazole on soilderived bacteria ten μl of bacteria suspensions were added to 90 μl of bacterial broth in 96-well flat-bottomed plates (falcon), supplemented either with cefuroxime (c) (final concentration 10,000 mg/l), or fluconazole (f) (final concentration 200 mg/l), or cefuroxime/fluconazole cocktail (cf) at the same concentrations, or with an equivalent volume of pbs (controls); wells filled with broth supplemented with c, f, c/f, pbs, but without addition of soil-derived bacteria served as controls of sterility. 8 wells were used per each treatment. plates were incubated for 20 h at room temperature. the viability of bacteria in the various treatment groups was assessed by mtt reduction test, according to modified method described by wieczorek-olchawa et al. (2003). the yellow mtt (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl-tetrazolium bromide; sigma) is reduced to blue formazan by dehydrogenases of living bacteria. in practice, 10 μl of mtt (from 5 mg/ml working solution) was added to each well and incubated for 15 min in darkness. absorbance was measured at 570 nm on an expertplus (asyshitech gmbh, austria) spectrometer. flow cytometric measurement and analysis samples of celomocytes were analysed with a facscalibur flow cytometer (bd biosciences). during analytical experiments, 10,000 thresholded events per worm sample were collected and analysed on the basis of their forward scatter (fs) (for cell size) and sideward scatter (ss) (cell complexity) properties. fluorescence fl1-h (emission 530 nm; excitation 488 nm) was recorded. the resulting files were analysed using winmdi 2.8 software (joe trotter, http://facs.scripps.edu), by producing dot plots of cell size versus fl1 autofluorescence. spectrofluorimetry and analysis spectrofluorometric measurements were performed on 2 ml riboflavin (sigma-aldrich) solution as a standard and on celomocytesuspension lysates (lysed with 2 % triton; sigmaaldrich) using an ls50b perkin-elmer spectrofluorometer. emission spectra of riboflavin were recorded in the 460 680 nm range (lambda at 370 nm), while excitation spectra were recorded in the 300 500 nm range (lambda at 525 nm). the spectrofluorimetric signatures of unbound riboflavin are characterised by two maxima (at 370 nm and 450 nm) in the excitation spectrum, and a maximum at 525 nm in the emission spectrum. riboflavin standard curve was prepared using serial dilution of pure riboflavin (sigma-aldrich). the emission value at 525 nm in each particular celomocyte lysates was converted to amount of riboflavin (in μg) according to the standard curve, as described previously (plytycz et al., 2006). to verify species identification, in randomly selected samples spectra of fluorophore specific for e. andrei were recorded according to the albani et al. (2003) protocol; emission spectra were recorded in the 340 480 nm range (lambda at 320 nm), while excitation spectra were recorded in the 260 360 nm 171 http://facs.scripps.edu/ fig. 2 fluorescence spectra of celomocyte lysates from eisenia andrei. top row: spectra of fluorophore specific for e. andrei: a emission (mem lambda at 320 nm; peak at 370 380 nm); b excitation (mex lambda at 380 nm; peak at 314 320 nm). bottom row: spectra of riboflavin: c emission (rem lambda at 370 nm; peak at 520 525 nm); d excitation (rex lambda at 525 nm; peaks at 370 nm and 450 nm). range (lambda at 380 nm). the spectrofluorimetric signature of e. andrei is characterised by a maximum at 314 nm in the excitation spectrum, and a maximum at 370 380 nm in the emission spectrum. arbitrary units (au) of fluorescence were recorded using microsoft excel v. 97. statistical analysis the results were expressed as means ± standard errors. differences between the means were determined with the mann-whitney u-test (statgraphics plus 5.0), with the level of significance established at p < 0.05. results species identification (fig. 2) worms sampled in the sadecki mountains and reared in the institute of zoology in krakow were identified as e. andrei on the basis of the uniformly reddish body coloration. celomic fluid lysates of worms from the present experiments contained fluorescence spectra similar to those characteristic for the fluorophore described by albani et al. (2003) as a presumptive diagnostic molecular marker for e. andrei (fig. 2, top), and riboflavin-specific fluorescence spectra (fig. 2, bottom). cocoon production cocoon production was inhibited in unfed worms compared with their fed counterparts (17 and 37 cocoons, respectively, produced in the 7 week treatment period, corresponding to 0.30 and 0.66 cocoons/worm/week). cocoons were absent in worms kept for 3 weeks on filters soaked with water or antibiotic/antifungal cocktail. effects of antibiotic/antifungal agents on soil-derived bacteria/fungi (fig. 3) mtt reduction assay revealed the high amount of soil-derived bacteria multiplied in bacterial broth during overnight incubation. the amounts of bacteria in the control wells filled only with bacterial broth was high. similarly high amounts of bacteria were detected in wells supplemented with antifungal agent, fluconazole (f wells). in a sharp contrast, living bacteria were absent in broth supplemented with antibacterial cefuroxime (c), and in the cocktail of these two agents (cf), as optical densities read at 570 nm were very low and almost identical to those of bacteria-free wells supplemented with pbs (controls of sterility), or with f, c, or cf (fig. 3). effects of food deprivation on coelomocytes (fig. 4) at the end of 7-week experiments body weights were statistically significantly lower in unfed worms than in their fed ad libitum counterparts (fig. 5). numbers of celomocytes (cn), among them both amebocytes (an) and eleocytes (en), were similar in fed and unfed worms, both those counted in extruded fluid and after adjustment for reduced body mass (cn/bw, an/bw, en/bw). percentages of eleocytes (e) and riboflavin content in celomocyte lysates (r) were unaffected by 7-week 172 fig. 3 viability of soil-derived bacteria measured by mtt reduction test after overnight in vitro incubation in the control bacterial broth or that supplemented with antifungal fluconazole (f), or antibacterial cefuroxime (c) or with cocktail of these two agents (c/f). solid and empty bars samples with and without addition of soil-derived bacteria, respectively. od optical density read at 570 nm. data are presented as means ± se; n = 8 wells per treatment. food deprivation, riboflavin amount being even higher in unfed worms after adjustment for reduced body mass (rf/bw) or eleocyte number (rf/en), but the increase was statistically insignificant (fig. 4). effects of antibiotics on worm celomocytes (fig 5) one-week dermal exposure to soft filter papers soaked with water or cefuroxime/fuconazole (cf) aqueous solution had no adverse effects on worm viability, while worms exposed for 3 weeks to antibiotic/antifungal agents showed impaired mobility thus experiments were terminated. autopsy revealed presence of ingested pieces of filters in worm intestines indicating that animals were penetrated by antibacterial/antifungal cocktail both via the derma and through the digestive tract. at the end of 1-week experiments body weights were similar in worms maintained on a substrate of filter paper as the only nutritional source, and moistened either with water (c control group) or antibiotic/fungicide cefuroxime/fuconazole (cf) aqueous solution (a group) (fig. 5a). body weights (bw) were unaffected during 1-week cf exposure, while the percentages of eleocytes (e %) tended to be increased. in the worms exposed to antibiotics/fungicides (a groups), also numbers of celomocytes, among them amebocytes and eleocytes tended to be increased. amount of riboflavin was unaffected, both that measured in celomocyte lysates and after adjustment for body mass (rf and rf/bw, respectively), but that concerning riboflavin adjusted to eleocytes number (rf/en) was lower in cells from worms exposed to antibacterial/antifungal cocktail than that in water exposed worms, but the difference was statistically insignificant (fig. 5a). at the end of 3-week experiments (fig. 5b) body weights (bw) and percentages of eleocytes (e %) among coelomocytes were similar in worms maintained on the waterand c/f-soaked filter papers as the only nutritional source (fig. 5b). in the worms exposed to antibiotics/fungicides, numbers of amebocytes tended to be increased (cn), eleocytes (en) were unaffected, while amount of riboflavin tended to be reduced, both total and adjusted for body mass (rf and rf/bw, respectively), with differences being statistically insignificant, but that concerning riboflavin adjusted to eleocytes number (rf/en) was higher in the control worms that that from the a (cf-treated) group, the difference being close to significance (p = 0.06) (fig. 5b). discussion it is well established that riboflavin is important for the proper functioning of the innate immunity of both animals (e.g., powers 2003; verdrengh and tarkowski, 2005) and plants (dong and beer, 2000; asai et al., 2010; yoshioka et al., 2011), as well as being a signalling molecule in bacterial quorum sensing (rajamani et al., 2008; atkinson and williams, 2009). in earthworms riboflavin acts as a chemoattractant for celomocytes what may facilitate the targeted recruitment of immune-competent celomocytes to the site of pathogen invasion (mazur et al., 2011). therefore, accumulation of riboflavin in chloragosomes of chlogagocytes and chloragocytederived eleocytes is probably of adaptive value for earthworm species, and eleocyte riboflavin stores are mobilized mainly in a case of pathogen invasion to support immune functions (plytycz and morgan, 2011). 173 fig. 4 effects of 7-week food deprivation on adult e. andrei. worm groups fed ad libitum on nettle and dandelion leaves were designated ‘f’ (open bars), and unfed groups were designated ‘u’ (solid bars). body weights (bw), percentages of eleocytes (e, %), celomocyte numbers (nc), among them amoebocyte numbers (an) and eleocyte numbers (en), and body weight-adjusted (cn/bw; an/bw; en/bw); total riboflavin content (rf) in extruded celomocytes, body weight-adjusted riboflavin content (rf/bw), and riboflavin content adjusted to extruded eleocyte numbers (rf/en). data are presented as means ± se; n = 8 worms per group. lack of statistically significant differences according to mann-whitney u-test between f and u treatments (p > 0.05). our working hypothesis was that riboflavin stores in eleocytes are exhausted in food-deprived worms and especially in those fed only on filter papers and exposed dermally/orally to solution of antibacterial and antifungal agents. contrary to our expectations, these experimental manipulations had no or little effects on riboflavin status in eleocytes of e. andrei. in the first part of the present study we revealed that the riboflavin contents in eleocyte-rich celomocyte lysates were very similar in e. andrei fed ad libitum on nettle and dandelion leaves and in worms deprived of plant-derived food. dietary restriction over a period of a few weeks has previously (piotrowska et al., 2010; polanek et al., 2011) been observed to have a little or no effect on the riboflavin status of circulating immunecompetent cells in other earthworm species. consequently, we excluded such a possibility that the main sources of the vitamin for earthworms is a plant-based detritivorous diet. these observations may be interpreted in a number of ways. first, it is feasible that if riboflavin is not consumed to counteract a pathogen challenge during a given period, then the vitamin budget could be maintained by recycling directly or indirectly from senescent eleocytes to newly formed eleocytes. second, earthworms may possess enough riboflavin stored internally elsewhere, for example in the chloragogenous tissue, to maintain optimal levels in 174 fig. 5 effects of 1 week (fig. 5a) or 3-weeks (fig. 5b) antibacterial/antifungal treatments on adult e. andrei. worms were kept on filter paper soaked either with water (control, c, open bars) or with a cocktail of antibiotic/antifungal agents (a, solid bars). body weights (bw), percentages of eleocytes (e, %), celomocyte numbers (nc), among them amebocyte numbers (an) and eleocyte numbers (en), and body weight-adjusted (cn/bw; an/bw; en/bw); total riboflavin content (rf) in extruded celomocytes, body weight-adjusted riboflavin content (rf/bw), and riboflavin content adjusted to extruded eleocyte numbers (rf/en). data are presented as means ± se; n = 5 worms per group. lack of statistically significant differences according to mann-whitney u-test between c and a treatments (p > 0.05). the eleocytes stores over the time period. third, during a period of dietary restriction riboflavin may be replenished from endosymbiont source or sources, possibly driven by upregulation through metabolic feedback. it is not possible to infer from present knowledge which of these possibilities is the most likely. measuring systemic riboflavin-binding protein, and the immuno-localisation of its membrane-bound counterpart (foraker et al., 2003), would be helpful in this regard. in order to check if the main source of earthworm riboflavin is intestinal microflora the second part of experiments was performed. in an attempt to reduce if not eliminate the gut flora (thakuria et al., 2008) earthworms were maintained for a period of several weeks on a filter paper substrate spiked with water (control) or a cocktail of antibiotic and antifungal agents. post mortem revealed that worm intestines were filled with papers, what strongly supports that cefuroxime/fluconazole cocktail, which successfully killed soil bacteria, might eliminate the worm gut flora. contrary to our expectations, the riboflavin accumulation within the eleocytes was only slightly reduced in the antibiotic-exposed worms fed only with antibiotic-soaked filter paper. it is worth to notice that such dermal exposure to filter papers soaked with metal chlorides was very efficient in studies on effects of heavy metals on coelomocytes of several earthworm species, putatively by disruption of balance between coelom-invading pathogens and worm immune system (e.g., wieczorek-olchawa at al., 2003; olchawa et al., 2006; plytycz et al., 2011). it is not known whether the antibiotic is trafficked through the earthworm body and has a negative impact on endosymbionts such as verminephrobacter (e.g., lund et al., 2010a, b). however, investigations on insects provide concrete evidence that bacterial endosymbionts do synthesise and supply riboflavin to their hosts (nakabachi and ishikawa, 1999). more recently, studies have shown that the depleted genomes of the bacterial endosymbionts of certain insect species have retained ancestral genes encoding essential amino acids and components of the riboflavin-synthesis pathway (moran et al., 2003). in an exquisite example of co-evolution, it has been 175 observed that different endosymbiont species within a given insect host species retain complimentary components of common and essential metabolic pathways (mccutcheon et al., 2009). it is not implausible that endosymbionts play similar nutritive roles in earthworms, but there is no direct evidence for the phenomenon. the fact that verminephrobacter eiseniae is vertically inherited via the cocoons of eisenia fetida (davidson et al., 2008) does, however, raise the intriguing possibility that the riboflavin requirements of the developing embryo are supplied directly by this or other endosymbiont species within the egg capsule. another possible mechanism for keeping a relatively constant riboflavin content in earthworm celomocytes would be the ‘insertion’ of riboflavin biosynthesis genes into the earthworm genome by horizontal dna transfer from microbes. this is not as inplausible as it may appear because such lateral transfer of fungal carotenoid genes into arthropods is well documented phenomenon (moran and jarvik, 2010; 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et al. the vitamin riboflavin and its derivative lumichrome activate the lasr bacterial quorum-sensing receptor. mol. plant microbe interact. 21: 1184-1192, 2008. roch p. protein analysis of earthworm coelomic fluid: i. polymorphic system of natural hemolysin of eisenia fetida andrei. dev. comp. immunol. 3: 599-608, 1979. segerstrom sc. stress, energy, and immunity: an ecological view. curr. dir. psychol.sci. 16:.326330, 2007. taylor m, mediannikov o, raoult d, greub g. endosymbiotic bacteria associated with nematodes, ticks nd amoebae. fems immunol. med. microbiol. 64: 21-31, 2012. thakuria d, schmidt o, liliensiek ak, egan d, doohan fm. field preservation and dna extraction methods for intestinal microbial diversity analysis in earthworms. j. microbiol. methods 76: 226-233, 2008. verdrengh m, tarkowski a. riboflavin in innate and acquired immune responses. inflamm. res. 54, 390-393, 2005. wieczorek-olchawa e, niklinska m, miedzobrodzki j, plytycz b. effects of temperature and soil pollution on the presence of bacteria, coelomocytes and brown bodies in coelomic fluid of dendrobaena veneta. pedobiologia 47: 702-709, 2003. yoshioka h, mase k, yoshioka m, kobayashi m, asai s. regulatory mechanisms of nitric oxide and reactive oxygen species generation and their role in plant immunity. nitric oxide 25: 216-221, 2011. 177 http://www.ncbi.nlm.nih.gov/pubmed/18645630 http://www.ncbi.nlm.nih.gov/pubmed/19038293 http://www.ncbi.nlm.nih.gov/pubmed/19038293 http://www.ncbi.nlm.nih.gov/pubmed/19038293 1department of evolutionary immunobiology, institute of zoology, jagiellonian university, krakow, poland this work was supported by k/zds/001955 from the jagiellonian university. sessioni: isj 10: 15-28, 2013 issn 1824-307x report of meeting xivth scientific meeting of the italian association of developmental and comparative immunobiology (iadci), 14 16 february 2013, department of biological chemical pharmaceutical science and technology, university of palermo, palermo, italy organizers: n parrinello, v arizza, m cammarata, mg parisi, m vazzana, a vizzini department of biological chemical pharmaceutical science and technology, university of palermo, palermo, italy plenary lecture from signal transduction to gene expression in innate immunity: learning from the mussel model p roch, m toubiana ecologie des systèmes marins et côtiers (ecosym), université montpellier 2-cnrs, montpellier, france antimicrobial peptides (amp) are certainly one of the major effectors of the anti-infectious innate immunity. they are present in all the living creatures, including bacteria and plants. they are of multiple amino acid sequences and characteristics. in addition, several amp families are simultaneously present in the same animal, not only in immune circulating cells, but also in various epithelia. complete molecular cascades of signal transduction from pathogen recognition to amp activity are known in several vertebrates, but only in drosophila (ecdysozoa), although still in debate. the present lecture will present the few we know on the mussel mytilus (lophotrochozoa). first, biochemical analysis revealed the existence of particular amps, grouped in several families, including several isoforms, with different antibacterial capabilities. synthetic fragments of peptides have been developed for applied biotechnology against bacteria and virus infections. second, were molecular biology approaches, which extended the diversity of amp mrnas, including within the same mussel, suggesting a complex system of molecular effectors. at that time, we realized how naïve was the idea of a primitive simple innate immune defense. third, expression regulations and specificities have been investigated following challenges with bacteria, fungi, and physical stresses. no strict correlation has been yet established between structure and function of amps in mussel. last, focus was on recognition-signal transduction steps by analogy with drosophila in which the toll-nf-κb pathway controlled the synthesis of amps. blast of sequences was less successful than looking for conserved domains of partners within est databases. thus, illumina read assemblies have completed identified sequences and revealed new ones. regulation of gene expression following challenges gave some light on the existence of the cascade in lophotrochozoa. in conclusion, mussels possess some of the nf-κb pathway intermediates. but most of them remain to be identified and the functionality of such complex system in the course of real infection is still pending. present lecture resulted from data obtained in collaboration with colleagues and students from the italian universities of padua, trieste, palermo, and the csic-vigo from spain. session 1. chairmen: n parrinello, university of palermo, palermo, italy; e ottaviani, university of modena and reggio emilia, modena, italy; l abelli, university of ferrara, ferrara, italy; l ballarin, university of padua, padua, italy immunity evolution. responses and mechanisms evolution of the intracellular transport mechanisms in eukaryotes: ciliates and mammals use the same translocation and nuclear localization signals a candelori1, m montani2, a amici2, p luporini1, a vallesi1 1department of environmental and natural sciences, university of camerino, camerino, italy 2department of biosciences and biotechnology, university of camerino, camerino italy in the ciliate e. raikovi, self/non-self recognition phenomena are controlled by cell type-specific, water-borne signal proteins (pheromones) by their binding to target cell-surface receptors. the downstream signal transduction pathway activated by the pheromone-receptor interactions of self type (that promote the vegetative, mitogenic cell growth) involves the phosphorylation of a nuclear protein kinase, designated er-mapk1, which is structurally similar to the "intestinal-cell kinase" and "male germ cell-associated kinase" described in mammals. to identify the sequence segments responsible for ermapk1 nuclear localization, mouse fibroblasts were   15 transfected with plasmids containing the reporter gene for the "green-fluorescent protein" (gfp) associated to different fragments of the er-mapk1 coding sequence. by expressing gfp-tagged protein constructs in mammalian cells, in the cterminal domain of er-mapk1 it was effectively possible to identify an arg/lys-rich motif that is required for the nuclear entry of gfp-fused constructs. these results provide evidence that distant related organisms such as ciliates and mammals use the same molecular language for the nuclear translocation and localization of proteins, thus suggesting that this language arose early in the evolution of the eukaryotic cell. cloning and expression of methionine sulfoxide reductase genes in the ciliated protozoan tetrahymena thermophila d ferro1, f cattalini1, o coppellotti1, r bakiu2, e piccinni1, g santovito1 1department of biology, university of padua, padua, italy 2department of animal production, university of tirana, tirana, albania accumulative post-translational modification to proteins, mediated by the action of reactive oxygen species (ros), is thought to be one of the major causes of aging and age-related diseases. thus, mechanisms have been evolved to prevent or reverse these protein modifications. while most protein damage by ros is irreversible, methionine oxidation to proteins can be reversed by the methionine sulfoxide reductase (msr) system, which includes msra (that repairs methioninesulfoxide senantiomer) and msrb (that repairs the methioninesulfoxide r-enantiomer). the action of the msr system may prevent irreversible protein damage (e.g. protein carbonylation), contribute to cellular antioxidant resistance resulting in life span extension of the organism. moreover, many work demonstrated that methionine oxidations in both inhibitor of kappa b-alpha and ca2+/calmodulinregulated phosphatase calcineurin may alter their functions and consequently affect transcription levels mediated by nfat and nfκb especially in tlymphocytes of the immune system. with the aim to explore this problem we projected some experiments using the ciliated protozoan t. thermophila, as model organism, characterizing the genes codifying for msr. total rna has been purified from t. thermophila cells (sb210 strain) cultured in ppyg medium and the cells were harvested after three days during exponential growth. the primers for the amplification of msr cdnas were designed after cross analyses between ncbi and t. thermophila genome databases. the obtained data seem to indicate that only one of the four annotated mrs genes is constitutively expressed. the nucleotide and amino acid sequences of all genes were compared with orthologous of other organisms and used for phylogenetic analyses. time-course of gene expression was analyzed by rt-pcr, using the same primers employed for cloning and sequencing, in t. thermophila cells grown in normal condition and after exposure of cu2+, used as pro-oxidant. the injection of lps induces epigenetic changes in pomacea canaliculata neurons a accorsi1, g rigillo1, d malagoli1, jmc blom2, f tascedda1, e ottaviani1 1department of life sciences, university of modena and reggio emilia, modena, italy 2department of medical and surgical sciences for children and adults, university of modena and reggio emilia, modena, italy epigenetic changes allow to modify gene expression on the basis of cell necessities and environmental stimuli. recent experiments suggested that resilience or vulnerability to stressful events is sustained by specific changes in gene expression that can affect the behavioral and molecular consequences of stress. in the present study, we have observed by means of immunocytochemical and western blot analyses that the injection of escherichia coliderived lps (o55:b5) in the foot of the apple snail pomacea canaliculata (gastropoda: ampullariidae) promotes the phospho-acetylation of histones, a phenomenon usually associated with changes in transcriptional patterns. more in detail, lps injection provoked an increase in immunoreactivity of antiphospho (ser10)-acetyl (lys14)-histone h3 pab in neurons of the pedal ganglia after 2 and 6 hours. in pedal ganglion neurons, the cytoplasmic immunoreactivity of the stress-related crh-like and acth-like factors is also enhanced after lps injection. western blot analysis showed also the increase of the transcriptional factor c-fos after the immune challenge. in mammals, the expression of c-fos is promoted by intravenous administration of crh or exposure to restraint stress. moreover, several researches have demonstrated a link between immune challenges and activation of the stress axis in vertebrates and invertebrates. the morphological and molecular data collected in snail neurons allow to surmize that acetylation of lysin residues in histone h3 may play a role in regulating the expression of molecules involved in the stress response of molluscs, as it has been suggested for mammals. lps challenge regulates gene expression and tissue localization of a ciona intestinalis gene through an alternative polyadenylation mechanism a vizzini1, a bonura2, d parrinello1, ma sanfratello1, v longo2, p colombo2 1department of biological chemical pharmaceutical science and technology, university of palermo, palermo, italy 2institute of biomedicine and molecular immunology “alberto monroy” of the national research council, palermo, italy a subtractive hybridization strategy for the identification of differentially expressed genes was performed between lps-challenged and naïve ciona intestinalis. this strategy allowed the   16 characterization of two transcripts (ci8short and ci8long) generated by the use of two alternative polyadenylation (apa) sites. the ci8long transcript contains a protein domain with relevant homology to several components of the receptor transporting protein (rtp) family not present in the ci8short mrna. by means of real time pcr, the ci8short and ci8long transcripts showed a different pattern of gene expression with the ci8short mrna being strongly activated after lps injection in the pharynx. in situ hybridization analysis demonstrated that the activation of the apa site also influenced the tissue localization of the ci8short transcript. this analysis showed that the ci8long mrna was expressed in hemocytes meanwhile the ci8short mrna was highly transcribed also in vessel endothelial cells and in the epithelium of pharynx stigmata. these findings demonstrated that regulation of gene expression based on different polyadenylation sites is an ancestral powerful strategy influencing both the level of expression and tissue distribution of alternative transcripts. exosomes and epigenetic information e capelli1, v curti1, s rossi2 1department of earth and environmental sciences (dista)-animal biology, università di pavia, pavia, italy 2medicina vi, irccs-policlinico s. matteo, pavia, italy the epigenetic changes play an important role in the differentiation of cells and tissues. among epigenetic factors, the micro rnas (mirnas) have assumed a considerable interest, thanks to the availability of new technologies that make these molecules easier to study. not long ago, non-coding genomic sequences were considered “junk”. today we know that many transcripts play a regulatory role by itself. non-coding rnas (ncrnas) can interact with dna, rna, proteins and engage in different structural and functional activities; they have an important role in nuclear and trascriptional organization, in post-trascriptional and epigenetic processes. mirnas are endogenous ncrnas of about 22 nucleotides (bartel, 2004) that negatively regulate gene expression by acting as inhibitors of the translation process and by determining the degradation of the mrna target. mirnas are carried by exosomes in biological fluids so they can be considered as circulating molecular messenger that regulate mrnas. exosomes are particles of 30200nm in diameter, derived from multi-vesicular bodies (mbv), delimited by a lipid bilayer containing a wide range of proteins and nucleic acids (particularly mrna and mirna) (martins et al., 2012). exosomes contain distinct signatures of mirnas that are characteristic of the cell from which they are derived and operate as signaling platforms that can influence cells destiny. we studied the diffusion of mirnas in exosomes derived from serum of patients with hepatocellular carcinoma (hcc) to relate the mirnas identified in tumor tissues with those ones identified in serum. we tested in particular the presence of mirnas characterizing hepatocellular carcinoma in exosomes isolated from serum and their expression level. our data are in agreement with the opinion that exosomes could represent the means of transport of metastasizing information. mirnas have a great potential as diagnostic and prognostic biomarkers and exosomes could be used as drug delivery system. molecular cloning, characterization and expression analysis of peroxinectin from ciona intestinalis ma sanfratello, a vizzini, m cammarata, d parrinello, v mangano, n parrinello department of biological chemical pharmaceutical science and technology, university of palermo, palermo, italy ascidians occupy a key phylogenetic position and are retained the sister group of vertebrates. peroxinectins, included into the peroxidasecyclooxygenase gene superfamily, function as hemoperoxidase and cell adhesion factor and could be involved in invertebrate immune reaction like po activating system. in this study, the ascidian (ciona intestinalis) peroxinectin gene (cipxt) and its expression during the inflammatory response have been examined. lps was inoculated into the ascidians, total rna was extracted from pharynx and cdna produced. the cipxt full-length cdna and the deduced amino acid sequence contain a peroxidase domain and an integrin binding motif (lys-gly-asp). sequence analysis showed high similarity of cipxt to echinoderms peroxinectins and mammalian myeloperoxidase (mpo); eosinophil peroxidase (epo); thyroid peroxidase (tpo). homology modelling process showed the expected molecular structure. cipxt, a new member of the peroxidase-cyclooxygenase gene superfamily, is very close to the chordate group and appears to be the out-group of mammalian mpo, epo and tpo clades. the cipxt molecular structure model resulted superimposable to the human myeloperoxidase. since the c. intestinalis pharynx is involved in the inflammatory response to lps (parrinello et al., 2010; vizzini et al., 2012; giacomelli et al., 2012) tissue expression of the cipxt gene was examined by real-time pcr analysis and in situ hybridization. cipxt gene expression is upregulated by lps inoculation suggesting it is involved in c. intestinalis inflammatory response. the cipxt riboprobe was found in vessel haemocytes, in the zones 7-8-9 of the endostyle (a special pharynx organs with a functional homology to the vertebrate thyroid gland) and in stigmata. the present report supports the role of the ascidians’ pharynx in the innate immunity. in vitro effect of leptin on rainbow trout oncorhynchus mykiss leucocytes g mariano1, r stilo1,2, j terrazzano3,4, e coccia1, f russo1, p vito1,2, e varricchio1, m paolucci1 1dipartimento di scienze biologiche, geologiche ed ambientali, university of sannio, benevento, italy 2biogem consortium, ariano irpino, avellino, italy 3dipartimento di scienze, university of basilicata,   17 potenza, italy 4department of biology and cellular and molecular pathology, university of naples federico ii, naples, italy leptin is an adipocyte-derived hormone discovered in 1994 (zhang et al., 1994). the finding that the leptin concentration circulating in the plasma is proportional to the body adiposity led to the theory that leptin acts as an “adipostat”, a humoral signal carrying information regarding energy reserves (maffei et al., 1995). in addition to the regulation of appetite and body weight, leptin has been reported to play a role in a different range of physiological functions (bjorbaek and kahn, 2004; la cava and matarese, 2004). leptin three dimensional structure is similar to that of a cytokine consisting of a four α-helix bundle motif which is common to the il-6 family of cytokines (zhang et al.,1997). despite the fact that numerous studies carried out in mammals point out at leptin role in the modulation of immune function evidence of immunomodulatory effect of leptin in fish is still unknown. trout recombinant leptin (rt-lep) has been produced and tested for its biological activity to trigger cellular pathways usually active in the cells of mammals immune system in in vitro incubations with blood leucocytes of the rainbow trout oncorhynchus mykiss. nfκb and map kinase activation were assessed by immunoblotting with a phospho (anti pikbα; anti pjnk; anti pp38; anti perk) specific antibody. rt-lep caused a decrease in the superoxide anion production in trout blood and head kidney leucocytes showing that this hormone plays in teleosts pleiotropic actions as in mammals, however, its actions not always conforms to the picture emerging for mammals. putative rhamnose-binding lectin in the solitary ascidian ciona intestinalis n franchi1, ma sanfratello1, d parrinello1, m vazzana1, ml di bella1, m cammarata1, l ballarin2, n parrinello1 1department of biological chemical pharmaceutical science and technology, university of palermo, palermo, italy 2department of biology, university of padua, padua, italy lectins are sugar-binding proteins involved in cell-cell interaction and the recognition of carbohydrate-containing molecules. they act as humoral factors in non-self recognition, are a key component of the innate immune systems of many metazoans and are involved in phagocyte activation through their opsonizing activity. in recent years, a new lectin family, the rhamnose-binding lectin (rbl) family, has been described and its members can modulate the inflammatory response in fish (watanabe et al., 2009) as well as in various invertebrates, including the colonial tunicate botryllus schlosseri. recently, we succeeded in purifying, by affinity chromatography using a rhamnose column, a putative rbl, from the hemolymph of the solitary ascidian ciona intestinalis. the molecule is ca2+independent and promptly (within 4h) inducible, after lps inoculation. the eluted fractions, when examined by 15 % sds-page under reducing condition, showed four bands with apparent molecular masses of 65, 54, 30 and 19 kda. the agglutinating activity of the isolated fraction was demonstrated using trypsinized rabbit erythrocytes and it was inhibited by glycosides such as rhamnose, galactose and lactose. moreover, by immunocytochemical analysis using antibodies produced against b. schlosseri rbl and in situ hybridization with a riboprobe for the annotated c. intestinalis rbl, we have observed a positive signal in hyaline and granular amoebocytes and in the endothelium of the pharynx vessels. antarctic teleost antibodies: evolution, structure, function u oreste institute of protein biochemistry, cnr, naples, italy over the past 55 million years, the temperature of the southern ocean has undergone a progressive reduction from about 15 °c to the present-day -1.86 °c. in consequence, many temperate fish species became extinct and one group, the notothenioidei, succeeded in adapting to the environmental conditions, and were diversified in situ. eight notothenioid families, including five that are predominantly antarctic, encompass a total of 44 genera and 129 species, 101 antarctic and 28 non-antarctic. evolution under constant cold conditions was also accompanied by significant gene duplication suggesting that the genomes of notothenioids are evolutionarily dynamic, thus contributing to the overall success of the group. due to the plasticity of its gene locus, immunoglobulin (ig) is an ideal candidate to contribute to the characterization of the evolutionary modifications undergone by the notothenioid genome. investigations conducted during many years, on 23 different notothenioid species allowed us to obtain an overall view of their ig genes evolution. we purified and characterized ig molecules, studied the ig epato-biliary transport and the secretion into the skin mucus, demonstrated the antibody response to nematode parasites, investigated the evolution of the heavy chain (igh) gene locus, inspected the diversity of the variable gene segments, identified three different ig light chain (igl) isotypes, mapped the igh locus on the chromosome of several species, examined the alternative splicing of the igh primary transcript and looked at the molecular structure of the igh transmembrane segment. the achieved results convinced us that the antarctic teleost igs are very fascinating molecules. t cells and t cell receptor gamma chain of european sea bass dicentrarchus labrax (l.) g scapigliati1, p boudinot2, e randelli1, c marozzi1, f buonocore1 1department dibaf, university of tuscia, viterbo, italy   18 2virologie et immunologie moléculaires, inra, jouyen-josas, france the sea bass is a marine fish species for which much information on its immune system is available, and results obtained from recent investigations on this species can be reassumed as follows. mucosal tissues are preferential sites for transcription of the t cell-related genes trb, trg, cd3, cd4, cd8a, mhcii, as well as are the sites where the highest number of t cells has been measured by mabs against t cells (dlt15), cd45 receptor (dlt22), and where cells expressing in situ mrna for cd4, cd8a, mhcii are present. interestingly, the rag-1 gene is highly expressed in the intestine, suggesting a possible extrathymic somatic rearrangement of tr and ig in lymphocytes. leukocytes from intestine and spleen display a spontaneous cytotoxic activity against allogenic and xenogenic cell targets, which was abolished when t cells were depleted by mab dlt15, suggesting presence of a ctl activity. in agreement, dlt15-enriched leukocytes from intestine and spleen showed an higher expression of trb and cd8a compared to negative dlt15cells. splenocytes respond better than kidney leukocytes to lectin stimulation, and when induced to proliferate by pha and cona, a significant increase in the number of t cells and of cd45bearing cells was measured. we recently cloned in sea bass cdnas coding for cellular receptors cd45 and cd83, and observed that the transcription of these gene was modulated in vitro and in vivo by lps, cona, pha, il-1, poly i:c, and betanodavirus. a peculiarity of γδ t cells is that they may recognize unconventional without the need of mhci or mhcii presentation, and we cloned full-length cdna sequences of sea bass trg, and trg-cdr3 length spectratyping revealed the structure of the junction diversity in the thymus and intestine of juveniles. trg expression can be modulated in vivo in the intestine by betanodavirus infection, and in kidney cells by poly i:c stimulation. high basal trg mrna expression levels were found in thymus and intestine, that were up-regulated in head kidney and intestinal leukocytes after in vitro stimulation by poly i:c. an in vivo infection of juveniles with betanodavirus caused an up-regulation of trγ expression levels in the head kidney and a downregulation in intestine. our data can be of importance for studies on mucosal immunity of teleost fish. the vertebrate allograft inflammatory factor-1 (aif-1) homologous in hirudo medicinalis is involved in wound healing process t schorn1, l pulze1, f drago2, m de eguileor1, r valvassori1, j vizioli2, a grimaldi1 1department of biotechnology and life science, university of insubria, varese, italy 2lsmbfa ea4550-equipe activation microgliale, université lille 1, villeneuve d’ascq, france analysis of an est library from medicinal leech cns reveals the presence of a gene, named hmaif1, showing a high homology with vertebrate aif-1. this factor is a 17 kda cytokine-inducible calciumbinding protein that in vertebrates plays an important role in allografts immune response and vasculopathy. since its expression is mainly limited to the monocyte/macrophage lineage, it was recently suggested that it could play a key role during inflammatory responses, allograft rejection, as well as in the activation of macrophages. to clarify this point we have focused our research on the possible role of aif-1 during the inflammatory response after injury in the leech hirudo medicinalis (annelida, irudinea). this invertebrate is an excellent animal model since the responses evoked during inflammation and tissue repair are clear and easily detectable and have a striking similarity with vertebrate responses. our preliminary data show that hmaif-1 is constitutively expressed in unlesioned leeches, but dramatically increase 24 h after a lesion. immunohistochemical experiments, using an anti aif-1 polyclonal antibody, shows that hmaif-1 is present in spread, cd68+ / cd45+ macrophage-like cells. a few days after chirurgical wound of the body wall, the amount of these immunopositive cells increases at the lesion site. in conclusion here we propose that in leech hmaif-1 factor is involved in inflammation events like its vertebrate counterparts. cellular and humoral components of innate immunity in squilla mantis (crustacea, stomatopoda): a preliminary approach l ballarin department of biology, university of padua, padua, italy the morphology and enzyme content of circulating cells of the mantis shrimp squilla mantis from the north adriatic sea, together with their ability to phagocytize foreign cells, were studied for deeper insights into the function of crustacean hemocytes in immune responses. the enzyme content and the agglutinating and hemolytic activities of cell-free hemolymph were also assayed. three hemocyte types, i.e., hyalinocytes, semigranulocytes and granulocytes, were distinguished, according to cell and nuclear morphology and the presence of cytoplasmic granules, in agreement with previous reports. all of them share the same patterns of enzyme activities and are recognized by the same lectins. spreading cells (hyalinocytes and semigranulocytes) can ingest foreign cells; granules of semigranular and granular cells have similar cytochemical properties. injection of micrococcus luteus into the heart sinus results in an increase in the frequency of hyaline cells and a decrease in the frequency of granulocytes. after 24 h from the injection, a decrease in the number of phagocytizing hyalinocytes, and a general decrease in the frequency of acid phosphatase-positive cells was reported. the above results suggest the existence of a single differentiation pathway for squilla haemocytes with the three hemocyte morphs as different stages of cell differentiation. results also indicate that squilla hemolymph performs immunosurveillance, through rapid changes in hemocyte distribution,   19 increase of antimicrobial and antioxidant enzymes and secretion of lectins stimulating agglutination, phagocytosis and encapsulation. characterization of hemocytes and plasmatic prophenoloxidase from adults of pterostichus melas italicus dejean, 1828 (coleoptera, carabidae) a giglio1, p brandmayr1, e perrotta1, pg giulianini2 1department of biology, ecology and earth sciences, university of calabria, cosenza, italy 2department of life sciences, brain centre, university of trieste, trieste, italy carabid beetles are among the most important groups of beneficial arthropods in the agroecosystem food chain where they are predators of many pests (including aphids, lepidopterans, slugs and diptera). previous studies have been shown that they are good models to investigate the negative effects of agrochemical used in agricultural management practices on natural enemies of insect pests. in ecological immunology, variation on immune capacity of insects is an early warning, highly sensitive biomarker to monitor the sub-lethal effect of toxicants introduced into environment as a result of industrial or agricultural activity. however, morpho-functional data about immune system of carabid are absent in spite of their ecological relevance. in this study, we have investigated the immune function of pterostichus melas italicus (dejean, 1828) for their use in eco-toxicological monitoring. this species is a generalist predator, eurytopic and thermophilous and it is very common in calabrian (south italy) agroecosystems (i.e. olive grove) and acts as a predator against insect pests (i.e., bactrocera oleae). tests performed on adult involve a general screen of immune function and include: characterization of circulating hemocytes, phagocytosis in vivo and phenoloxidase activity. the cellular population has been characterized by light and electron microscopy analysis and four morphotypes of circulating hemocytes were found: prohemocytes (1.1 ± 0.35 %), plasmatocytes (76.13 ± 7.00 %), granulocytes (13.40 ± 5.84 %), oenocytoids (1.93 ± 0.96 %). the phagocytosis assays were performed in vivo by injection of 0.9 µm carboxylate-modified polystyrene latex beads in order to identify the hemocyte types involved in phagocytosis. after non-self challenge treatment, specimens showed a decrease of granulocyte and prohemocyte percentages (6.13 ± 3.36 % and 0.80 ± 0.41 % respectively) and a non-specific immune response involving phagocytosis performed by plasmatocytes (42.50 ± 3.39 %). moreover the plasmatic phenoloxidase (po) activity has been evaluated spectrophotometrically recording the formation of dopachrome by a non-enzimatic reaction from dl-dopa substrate and it was expressed as absorbance units at 492 nm/μl of hemolymph. the po level was low in unstimulated specimens (0.0521 ± 0.0026 a492/µl at 30’; n = 20), while we detected an increase of po activity (0.0816 ± 0.0153 a492/µl at 30’; n = 26) after the activation of the prophenoloxidase (propo) into po with methanol. effects of steinernema carpocapsae (nematoda: steinernematidae) on biological parameters of larvae responses of rhynchophorus ferrugineus (coleoptera: curculionidae b manachini, d schillaci, v arizza department of biological chemical pharmaceutical science and technology, university of palermo, palermo, italy rhynchophorus ferrugineus (coleoptera: curculionidae) known as the red palm weevil (rpw) is becoming more and more of a problem in italy, and especially in sicily, where it is well adapted. the infestations are mainly in the urban areas, and for that reason, chemical control is not advisable. data from literature show that entomopathogenic nematodes (epn) particularly steirnenema carpocapsae, have a quite successfully control of rpw. however, results coming from the laboratories are often in contrast with each other and no data are available on precise doses and s. carpocapsae seems not be able to reproduce itself in the host. the effect of epn on the rpw immune system is totally unknown. different dosages of s. carpocapsae and varying durations of exposure were assessed. larval mortality showed a positive linear correlation with both nematode dosage and the duration of exposure. median lethal dose (dl50) and the median lethal time (tl50) were calculated for older larvae. the number of nematodes that gained access to the haemocoel of larvae was always low, but increased with dosage and exposure time. epn had also a detrimental effect on larval weight. in this paper we also investigate in vivo and in vitro effects of administered s. carpocapsae on the phagocytic responses of r. ferrugineus later-instar larval haemocytes. after a few hours, the nematodes were measured in the hemolymph of the insect and it appeared that the immune system was not activated by the presence of these foreign bodies. the nematodes suddenly moulted in the hemolymph totally undisturbed by the hemocytes but they were unable to complete the life cycle and to reproduce. after 24 hours, the number of the hemocytes (thc) recorded in the larvae treated with s. carpocapsae was dramatically inferior compared to the thc found in the control larvae. the study of the interaction between epn and rpw could be crucial understanding the mode of action of epn in the different instars and the reason for the response to different doses. we also evaluated the defence ability of rpw humoral and cellular immunity system in vitro against the bacterium xenorhabdus nematophila associated with epn, through the minimum inhibitory concentrations (mics) assay. to our knowledge, this is the first time that such assay has been used to evaluate the ability of insect immune system against entomopathogenic bacterium. comparison among the responses of the greater   20 wax moth, galleria mellonella and red palm weevil rhynchophorus ferrugineus to the entomopathogenic nematodes, steinernema carpocapsae v orlando, d russo, d schillaci, v arizza, b manachini department of biological chemical pharmaceutical science and technology, university of palermo, palermo, italy the entomopathogenic nematode-bacterium complex of steinernema-xenorhabdus has high potential as lethal biological control agent against many insect pest species. the red palm weevil (rpw) rhynchophorus ferrugineus (coleoptera: curculionidae) is an important worldwide palm trees pest. this insect is a quarantined pest, accidentally introduced in sicily in 2005. the pest is killed by steirnenema carpocapsae, but nematodes are unable to reproduce in the rpw larvae. this research try to understand the reasons of the inability of s. carpocapsae to complete its life cycle in the host comparing what happens in one of the most suitable host, the greater wax moth, galleria mellonella (lepidoptera: pyralidae), for this nematode species. g. mellonella is a pest of beehives; the larvae feed on wax and do considerable damage to the wax and to honey production in italy. the lethal doses were comparable if pondered at the weight of the hosts. in both g. mellonella and r. ferrugineus there were no encapsulation or melanisation responses against s. carpocapsae. however s. carpocapsae successfully complete its life cycle in g. mellonella producing thousands of offspring while just few moults were recorded in rpw larvae and not male and female were found in the rpw larvae cadavers. xenorhabdus nematophila is a gram-negative member of the family enterobacteriaceae that lives in a symbiotic association with s. carpocapsae in a highly effective symbiosis of pathogens. we found that rpw was unable to defence its self against the bacterium. through viable plate counts we measure the quantity in terms of cfu (colony forming units)/ml of x. nematophila in the g. mellonella and in the r. ferrugineus hemolymph. the results show that the quantity of x. nematophila was higher in g. mellonella than in rpw already after 24 h from bioassay. the presented combination of nematode species, the two host species and the bacterium studied in terms of reproduction of the complex nematode-bacterium, is unique to this study. time-related changes of hemocyte morphology in the gastropod pomacea canaliculata d malagoli1, a accorsi1, l bucci2, m de eguileor3, e ottaviani1 1department of life sciences, university of modena and reggio emilia, modena, italy 2department of experimental, diagnostic and specialty medicine, university of bologna, bologna, italy 3department of biotechnology and life sciences, university of insubria, varese, italy the freshwater gastropod pomacea canaliculata is an established model for ecotoxicological and parasitological researches, and a promising model for the analysis of the hemocytemediated immune and stress reactions. in this study, flow cytometry allowed the categorization of p. canaliculata hemocytes into two populations, i.e., small and large hemocytes. histochemical staining demonstrated that small hemocytes are round cells, with agranular and acidophilic cytoplasm. large hemocyte population displays a more articulated morphology, with either agranular or granular cells. agranular cells may have acidophilic or basophilic cytoplasm. cytoplasmic granules are acidic. this frame is characteristic of just-withdrawn hemolymph. from 20 up to 40 min after hemolymph collection, changes in large hemocytes have been observed by flow cytometry and light microscopy. morphological observations showed the presence of large hemocytes with vacuolated cytoplasm, and the increase of the percentage of large granular cells. in order to test the basic immune-related functions of p. canaliculata hemocytes, adhesion and phagocytosis assays were performed. p. canaliculata hemocytes rarely adhere on a glass surface as individual cells, and they rather clump into loosely adherent aggregates. a spontaneous phagocytic activity of p. canaliculata hemocytes has been demonstrated by transmission electron microscopy. phagocytosis assay confirmed these observations and indicated that more than 30 % of circulating hemocytes phagocitize heat-inactivated escherichia coli but no phagocytizing small hemocytes were observed. concluding, p. canaliculata hemocytes may be divided into two populations showing distinct morphological and functional features as well as specific dynamic changes after hemolymph withdrawal. differential interactions between mytilus hemolymph components and vibrio aestuarianus and vibrio splendidus: in vitro and in vivo studies c ciacci1, r cuppini1, t balbi2, r fabbri2, a smerilli2, e pezzati2, c grande2, l vezzulli2, c pruzzo2, l canesi2 1disteva, dipartimento di scienze della terra, della vita e dell’ambiente, university of urbino ‘carlo bo’, urbino, italy 2distav, dipartimento di scienze della terra, dell’ambiente e della vita, university of genoa, genoa, italy marine bivalves are filter-feeding invertebrates that can accumulate large numbers of bacteria in their tissues, in particular vibrio species particularly abundant in coastal waters. persistence of different bacteria in bivalve tissues largely depends on their sensitivity to the bactericidal activity of mussel hemolymph, resulting from complex interactions between bacteria and circulating hemocytes. host– pathogen interactions have been increasingly investigated in different bivalves, with the aim of   21 understanding the pathogenesis of diseases in species susceptible to infection by certain vibrio spp. and strains. v. splendidus strains have been associated with the ‘summer mortalities’ syndrome of juvenile oysters. on the other hand, the mussel mytilus galloprovincialis is considered to be particularly resistant to vibrio infection. in this work, in order to explore the susceptibility of mussels to different vibrio species in controlled conditions, the interactions between mytilus hemolymph components and v. aestuarianus and v. splendidus were investigated in vitro and in vivo. in vitro, the bactericidal activity of whole hemolymph towards different vibrios, the capacity of bacteria to adhere to hemocyte monolayers both in the presence and in the absence of different sugars, as well as their effects on hemocyte lysosomal membrane stability (lms) were evaluated. the results were compared with those obtained in oyster (crassostrea gigas) hemocytes. the results indicate distinct interactions between mussel and oyster hemocytes and different vibrios. mussels were also injected with v. aestuarianus and v. splendidus and different endpoints (hemolymph bactericidal activity, bacterial concentration in mussel tissues, hemocyte lms and serum lysozyme activity) were evaluated at 6, 24 and 96 h p.i. moreover, lms was evaluated in mussel digestive gland as a biomarker of general stress. the results indicate clear differences in the interactions between mussel hemolymph and different vibrio species, with v. splendidus inducing more stressful conditions in the host and showing higher resistance to hemolymph bactericidal activity compared to v. aestuarianus. however, the effects of v. splendidus were transient, thus confirming the high resistance of mytilus to bacterial pathogens. characterization of haemolytic activity of coelomocytes of holothuria tubulosa m vazzana, v arizza, m celi, d russo, n parrinello department of biological chemical pharmaceutical science and technology, university of palermo, palermo, italy phylogenetic analysis recognizes echinoderms as a key group of deuterostomes, therefore the species in this group are useful for the study of the evolution of innate immunity responses. in addition, this marine invertebrate lives in coastal and estuarine waters that are directly exposed to potentially pathogenic microorganisms and stressful anthropogenic factors. coelomocyte populations seem to be essential to immune-defence functions such as phagocytosis, roi production, cytotoxicity, synthesis and release of antimicrobial substances including lectin, cytokine, c3-like expression, prophenoloxidase activity and capsule formation. holothurians’ coelomocyte populations contain several coelomocyte types, including phagocytes, and can form brown bodies in response to multicellular parasites. holothuria tubulosa coelomic fluid contains three main coelomocyte categories: amoebocytes, spherulocytes and progenitor cells. the amoebocytes represent about 30 % of the total population, and in living cell preparations exhibit two distinct forms: petaloid and philopodial; spherule cells represent the numerically dominant cell type (about 60 %). progenitor cells are present in a lesser amount (about 20 %); they are similar to lymphocytes and show a nucleus that is typically prominent with a thin rim of cytoplasm. in the present report we show that coelomocytes of holothuria tubulosa are able to exert cytotoxic activity against different cellular targets such as rabbit or sheep erythrocytes and the human erythromyeloid leukaemia-derived cell line k562. moreover, the unseparated coelomocyte supernatant lysate (cls) exerts a lytic activity, even in the absence of calcium, against the same targets. analysis of the coelomocyte lysate by overlay assays with sheep and rabbit erythrocytes on page without sds showed a protein pattern composed by two main hemolytic bands with different electrophoretic mobility. the one with low mobility (i) showed calcium independent haemolytic activity while high mobility band (ii) showed calcium dependent activity. the two bands were eluted from the gel and analyzed by sds-page and stained with silver stain; band i could be separated in three components of different sizes (52, 42 and 41 kda), and band ii had a size of 43 kda. spherule cells seemed to be the effector cells, as shown by a plaque lysis assay. further studies are in progress to identify the lytic proteins and the lytic mechanism at work on both a molecular and cellular level. cloning and expression of glutathione peroxidase genes in the chordate invertebrate ciona intestinalis d ferro1, r bakiu2, n franchi1, l ballarin1, g santovito1 1department of biology, university of padua, padua, italy 2department of animal production, university of tirana, tirana, albania ascidians represent interesting models from an evolutionary and ecotoxicology point of view, because of their large distribution in temperate sea and their phylogenetic position of invertebrate chordates. immune responses imply an increase in oxygen consumption with a consequent risk of oxidative stress. with the aim to study the components of the antioxidant defense system in the solitary ascidian ciona intestinalis, we have characterized the genes codifying for two glutathione peroxidases (gpx), metalloenzymes that catalyze the reduction of hydrogen peroxide or organic hydroperoxides to water or corresponding alcohols, using reduced glutathione (gsh) as an electron donor. in the genebank database five gpx-like sequences from c. intestinalis are present, but only two of those genes demonstrated an effective transcription after cloning and sequencing analyses. the respective proteins, named ci-gpx7 and ci-gpxb, show a good level of sequence conservation with metazoan orthologs, especially for residues that are important for the catalytic activity.   22 in the 3’-utr region of ci-gpxb cdna we have found a typical secis-element, confirming that this protein may be a selenium gpx. phylogenetic reconstruction, performed with bayesian methods using metazoan gpxs, indicate that ci-gpxb emerges in the tree with the tetrameric gpxs, confirming as previously hypothesized. preliminary data, obtained by homology modeling, confirmed the tetrameric structure and show that the gpxb is similar to gpx3 from homo sapiens, a selenium protein. thus, we propose to name this gene cigpx3. as expected, ci-gpx7 clusterized with other gpx7s. the transcription of both these genes, measured by rt-sqpcr, resulted inducible by cd, cu and zn, which have different effects. the peroxidase activity decreases in the cell-free extract from specimens treated with each considered metals, probably in relation to metal-induction of gsh biosynthesis, as indicated by the presence of positive correlation by the time-dependent cd accumulation an increase of ros and gsh production. the data presented here improved our knowledge about the evolution of the antioxidant system in metazoans and emphasize the importance of a complex regulation for the antioxidant system, including the transcription of cigpx7 and ci-gpx3 genes, which can create an efficient detoxification pathway allowing c. intestinalis to survive in the presence of metals in the environment. session 2. chairman: g scapigliati, university of tuscia, viterbo, italy environmental stress responses amyloidogenesis as stress response: winner or loser process? m de eguileor1, r girardello1, a grimaldi1, d malagoli2, l pulze1, g tettamanti1, e ottaviani2, r valvassori1 1department of biotechnology and molecular sciences, university of insubria, varese, italy 2department of life sciences, university of modena and reggio emilia, modena, italy contacts with foreign molecules from bacteria (lps), fungi (pma), parasites, and chemicals can provoke serious stress stimuli able in turn to induce protective responses. the defense answers, due to non-self recognition, belong to acquired immunity, or to innate immunity. in the last case any stress event sets off a mix of responses from immune and neuroimmune systems. animals generally show fundamental biological principles revealing conserved regulation of the involved processes. this is also true for cellular stress responses, a complex and dynamic process of restoring cellular homeostasis characterized by the same specific stages. we have previously shown that cellular stress conditions promote in different animal models (invertebrates and vertebrates) the same massive morphological and physiological modifications (fowler et al., 2006; grimaldi et al., 2012). any kind of insult mimicking a stress condition (sundry chemical, immune, neuroendocrine and inflammatory) provokes, always and in any type of cell/tissue, detectable series of events that start with ros over expression, acth axis activation, and cytokine such as il-18 production. these general overexpressions sustain a massive amyloid fibril synthesis that provides a resistant scaffold in turn driving melanin deposition. (falabella et al., 2011; grimaldi et al., 2012). now we show that the amyloid production is not only linked to precursor melanin activation and synthesis but it is fundamentally important to guaranty a state of cellular redox equilibrium due to regulation of ros presence, i.e., amyloid fibrils production could be considered as a basic cellular compensatory response endeavouring to attenuate oxidative stress in different cell types. moreover, the relationship between amyloidogenesis and stressors allows to surmize a new background of information on the effects of stress. molecular and physiological characterization of in vivo sulfamethoxazole response in procambarus clarkii a nicosia1, m celi2, m vazzana2, a damiano1, n parrinello2, f d’agostino1, g avellone2, s indelicato2,3, s mazzola1, a cuttitta1 1istituto per l’ambiente marino costiero uos capo granitola cnr, trapani, italy 2department of biological chemical pharmaceutical science and technology, university of palermo, palermo, italy 3centro grandi apparecchiature, university of palermo, palermo, italy sulfamethoxazole (smz) is one of the most widely sulfonamides employed to treat human urinary infections, in veterinary practice and aquaculture. smz, acting as broad spectrum antibiotic microbial drug, blocks the folic acid metabolism. because of smz widespread use considerable amount is indeed expected to be introduced into the environment. the smz derived cytotoxicity is mediated by an arylamine bioactivation to the arylhydroxylamine metabolites (s-noh) of smz and it is associated to the generation of reactive oxygen species (ros). there has been very limited information relating to the toxic potential of smz at cellular and molecular level particularly in aquatic or non-target organisms. in the present study, the red swamp crayfish (procambarus clarkii), because of its tolerance to extreme environmental conditions and resistance to diseases, was used as a model organism to profile the molecular and physiological response to smz. haemato-immunological parameters such as glucose serum levels and total haemocyte count (thc) were alterated as compared to controls; moreover a significative increase in hsp70 serum level was detected for the first time. variation at transcriptional levels of proinflammatory genes (cox 1 and cox 2), antioxidant enzymes (gst and mnsod), stress response and fenton reaction inhibitor genes (hsp70, mt and ft) were evaluated and alteration in the canonical gene expression pattern emerged. considering the above exposed results, specific mechanisms involved in maintaining physiological homeostasis and adaptation in response to perturbation   23 are suggested. physiological and agonistic behavioural response of procambarus clarkii to an acoustic stimulus m celi1, f filiciotto2, d parrinello1, g buscaino2, ma damiano1, a cuttitta2, s d’angelo3 s mazzola2, m vazzana1 1department of biological chemical pharmaceutical science and technology, university of palermo, palermo, italy 2istituto per l’ambiente marino costiero uos capo granitola cnr, trapani, italy 3wwf italia, mazara del vallo, trapani, italy the impact of human activity on acquatic habitats can produce adaptive alterations and other significant changes in animals. in recent years, many studies have been carried out with the aim of evaluating the effects of anthropogenic acoustic disturbance on marine and freshwater organisms, thus increasing the awareness of the damage done to animals exposed to human related underwater sounds. this study examined the effects of an acoustic stimulus on the agonistic behaviour and on haemolymphatic parameters of the red swamp crayfish procambarus clarkii. the experiment was conducted in a tank equipped with a video recording system using 6 groups (3 control and 3 test groups) of five adult crayfish (30 specimens in total). after one hour of habituation, the behaviour of the crayfish was monitored for two hours. during the second hour, the animals in the test groups were exposed to a linear sweep (frequency range 0.1 25 khz; peak amplitude 148 dbrms re 1 μpa at 12 khz) acoustic stimulus for 30 minutes. exposure to the noise produced significant variations in haematoimmunological parameters as well as a reduction in agonistic behaviour. in particular, the acoustic stimulus induced a decrease in the natural aggressive activity (number of fights and tail flip events) and a significant increase in haemolymph glucose levels and on total serum protein concentration. also, the number of circulating total haemocytes, of the stressed crayfish decreased by approximately 50 % relative to the initial. a different pattern was observed for the differential haemocytes count. in tested crayfish, a significant increase in hyaline cell number (from 20 % to 59 %, p <0.005) was accompanied by significant decreases in the relative proportions of granular and semigranular cells relative to the values determined for the control group. furthermore we show for the first time that acoustic stimuli induce hsp70 overexpression in p. clarkii haemocytes as expression of a stress status. bacillus thuringiensis treatment modulate the hsp70 expression in larva and adult brain of rhynchophorus ferrugineus d russo, m celi, v arizza, m vazzana, b manachini department of biological chemical pharmaceutical science and technology, university of palermo, palermo, italy to study the pathogen-host relationship, we used the model of the entomopathogenic bacterium bacillus thuringiensis (bt) and rhynchophorus ferrugineus, a quarantine pest that attacks palm trees. in particular, we focused on the bt stressinduced infections. we studied the effect of bt on larval and adult growth, and on the expression of the heat shock proteins (hsps), rapidly synthesized in the cell after exposure to stress including pathogens. bt has negative effects on larval and adult growth, on total hemocytes counts and on the hemocyte type. hsp70 was evaluated in the supernatant of the brain lysate obtained from larvae and adults fed with sublethal doses of bt. hsp70 expression was modulated in time (3, 6, 12 and 24 h) in response to bt ingestion, highlighting that bt is a stress factor for the r. ferrugineus. further investigation is needed to understand the possible correlation between the reduction of hemocytes and hsp70 modulation. the potential costs of the different bioassay used will be compared. the toxic pesticide lindane affects the immunological competence of the sea urchin paracentrotus lividus l stabili1,2, ra cavallo2, p pagliara1 1dipartimento di scienze e tecnologie biologiche e ambientali, university of salento, lecce, italy 2istituto per l’ambiente marino costiero uos capo granitola cnr, trapani, italy pollution of marine environment has become an international problem. this issue is of particular interest in coastal areas usually subjected to a variety of pressures related to the introduction of high nutrient loads, hazardous chemicals and pathogens affecting marine organisms health the presence of pollutants may affect survival, growth, reproduction, metabolism and immunity of marine invertebrates. the scant available studies demonstrated the effect of environmental perturbations on echinoderm immunological competence. in this framework, in previous works, we evaluated the effect of a high zinc concentration on several immunological parameters of the sea urchin paracentrotus lividus as well as the seastar marthasterias glacialis by comparing zinc treated and untreated specimens. the observed modifications in echinoderm immunological competence led us to conclude that they may give an early indication of disease susceptibility thus suggesting to consider the examined defence mechanisms as potential biological indicators of pollution. to generalize our results other xenobiotics effects have to be examined thus, in the present study, we analyzed the effects of the toxic pesticide lindane on the immunological parameters of the sea urchin p. lividus. in particular we evaluated the effect of this xenobiotic on celomocytes haemolytic and lysozyme-like activites as well as antibacterial activity on vibrio alginolyticus. in addition, we examined changes in coelomocytes composition and morphology. the inhibitory action of this pollutant on the immunological defence of the sea urchin was evidenced thus, taken all together, our   24 results lead to hypothesize the feasibility of using sea urchin coelomocytes and some humoral parameters as novel biosensors of environmental stress useful for sea urchins disease surveillance and environmental health assessment. ambient noise of aquaculture systems: impact on sparus aurata welfare f filiciotto1, vm giacalone1, f fazio2, g buffa1, g piccione2, v maccarrone1, v di stefano1, s mazzola1, g buscaino1 1istituto per l’ambiente marino costiero uos capo granitola cnr, trapani, italy 2department of experimental science and applied biotechnology, university of messina, messina, italy the present study evaluated the impact of onshore and offshore aquaculture systems’ ambient noise on the welfare of gilthead sea bream juveniles (sparus aurata) through primary, secondary and tertiary stress responses. some biochemical (cortisol and glucose) and haematological (white blood cell count, red blood cell count, haematocrit value and haemoglobin concentration) indexes of stress and the growth performances of fish were measured. the experiment lasted 120 days during which two different playlists of acoustic stimuli were projected inside six experimental tanks (each condition was replicated in three tanks): offshore aquaculture noise condition that recreated the typical acoustic field in proximity of an offshore sea cage for fish farming using a random sequence of quite sea background and boat noises and onshore aquaculture noise that represented the acoustic field inside an onshore open concrete tank for fish farming. the other three tanks were used as a control condition without acoustic projection. the weights and lengths of fish exposed to offshore aquaculture noise were higher than the specimens in the control and onshore aquaculture groups. moreover, higher levels of serum cortisol, glucose, red blood cell count, haematocrit value and haemoglobin content and lower levels of white blood cells were recorded in fish groups from the control and onshore treatments. these results allow us to hypothesise that offshore aquaculture noise and the sea soundscape in particular positively influence growth performance and could reduce stress and improve the welfare of the sea bream. engineered nanoparticles of titanium dioxide (tio2) in a fish species (dicentrarchus labrax l.): uptake and biological effects c bernini1, s picchietti1, f buonocore1, ar taddei2, g scapigliati1 1department for innovation in biological, agro-food and forest systems (dibaf), university of tuscia, viterbo, italy 2interdipartmental centre of electron microscopy (cime), university of tuscia, viterbo, italy the general aim of this work was to investigate if the engineered nanoparticles of tio2 (np-tio2) have access to marine species used as food for humans, and monitor if the nanoparticles may affect their physiology. np-tio2 is widely used in various fields, such as products for personal use, cosmetics and sunscreens and its use is about two million tons a year. the toxicity to humans is well documented but there is a lack of data concerning the effects on animals and its presence in the marine environment, in particular with regard species widely used for human food and therefore considered potential carriers of np-tio2. in addition, it is known that nptio2 may bind dangerous contaminants present in traces in marine water such as cadmium (cd), and hence allow magnification of these poisons in trophic chains. the present work tried to assess how the engineered np-tio2 may be incorporated in a fish model species and evaluated the biological effects in a sea bass cell line (dlec). the uptake of nptio2 has been examined by transmission and scanning electron microscopy, this latter coupled with energy dispersive x-ray spectroscopy (edx) for particle element detection. the effects of controlled exposure of np-tio2 and np-tio2-cd to dlec have been studied evaluating different quantitative parameters related to metabolic functions, such as intracellular atp concentration and cellular viability. furthermore, we studied the effects of np-tio2 on the expression of target genes associated with innate immune defences (mx, il-8, cox-2, tgfbeta) by real-time pcr. finally, considering that there are some evidences that pre-irradiation of np-tio2 with uv light can promote increased production of free radicals and general toxicity, we used uv irradiation to also investigate the cytotoxic potential of photoactivated np-tio2 on dlec cells. our study supports the notion that nanoparticles can enter fish cells quickly and easily. in addition np-tio2 were non toxic, contrary to what is demonstrated, by cell viability measurements, for the photo-activated np-tio2. these speculations warrant further studies because of the important implications for environmental and human health risk assessment and preventive actions to limit exposure. session 3. chairmen: u oreste, ibp cnr, naples, italy; v arizza, university of palermo, palermo, italy antimicrobial peptides de novo discovery of antimicrobial peptides from invertebrate transcriptomes a pallavicini1, m gerdol1, g leoni1, c manfrin1, g de moro1, v torboli1, u rosani2, s domeneghetti2, l varotto2, s battistella1, m scocchi1, p venier2, p giulianini1 1department of life sciences, university of trieste, trieste, italy 2department of biology, university of padua, padua, italy the innate defense systems of aquatic invertebrates include antimicrobial peptides (amps),   25 usually small, positively charged molecules effective against a broad range of pathogens. several amp families have been widely studies and described in a many different phyla (e.g. defensins) many others represent genusor even species-specific acquisitions. the methodological advances achieved in the last decade now allow a large scale analysis of entire genomes and transcriptomes of non-model animals. bioinformatics can be used for the development of in silico tools aimed at the mining of large sequence databases and identification of both amps belonging to known families and novel candidates satisfying specific user-defined requirements. here we present a bioinformatic pipeline for the whole-transcriptome scale mining of sequences encoding peptides with a potential antimicrobial activity. based on the known chemical-physical properties of these bioactive peptides, we developed a perl script which permits to filter a target sequence file based on user-defined parameters, including sequence length, isoelectric point, amino acid composition or the presence of specific amino acid patterns. all these searches can be performed on windows of variable length to deal with the peptide precursors. to date, linear amps rich in specific amino acid residues have not yet been described in the bivalve mytilus galloprovincialis. about 37,500 putative peptides (ranging from 40 to 120 aa) were translated from the 110k coatings that make up the digestive gland transcriptome of the mediterranean mussel. out of these, 932 seem to be secreted. within this reduced dataset, 14 present at least 30 % content in lys/arg or pro in a sliding window of 30 residues. four of them also show an extremely basic pi (>10). moreover, 63 peptides are cysteinerich, bearing at least 3 disulfide bridges within 30 amino acids. as a positive control, several known cys-rich mussel amps were included among these mined sequences. this result indicate that this approach may be successfully applied to de novo transcriptome assemblies of non-model marine and freshwater invertebrates. in this respect we are approaching with this pipeline the transcriptome of the crayfish astacus leptodactylus, and we will also take advantage of the wealth of public data available for non model invertebrate organisms. characterization of the salmonid cathelicidins and of their biological activities m furlan1, g cannone1, p venier2, u rosani2, p irato2, a pallavicini1, r gennaro1, m scocchi1 1department of life sciences, university of trieste, trieste, italy 2department of biology, university of padua, padua, italy cathelicidins are a family of cationic antimicrobial peptides (amps) and are an important component of the innate immune response. two members of this family have recently been identified in salmonids and other fishes. analysis of the cath-1 and cath-2 salmonid cathelicidin gene sequences showed a protein organization with a characteristic conserved cathelin-like n-terminal domain and a varied glycine/serine-rich c-terminal domains corresponding to the active peptides. in this study we characterized the antimicrobial activity spectrum, the mode of action and tissue expression of salmonid cathelicidins. different peptide fragments representing specific c-terminal regions of cath1 and/or cath2 of rainbow trout (oncorhynchus mykiss), brook trout (salvelinus fontinalis), grayling (thymallus thymallus) and brown trout (salmo trutta fario) have been chemically synthesized and their antimicrobial activity evaluated against standard bacterial strains and some fish pathogens. most peptides showed a medium-dependent antimicrobial activity with mic values ranging from 4 µm to 64 µm. killing kinetics, membrane permeabilization assays and hemolytic assays indicated that these peptides rapidly kill bacteria by permeabilization of their cell membranes, and at the same time show very low toxicity against erythrocytes. to detect cath-1 in trout tissues and to study its processing a polyclonal antibody was raised against a complete recombinant cath-1 protein derived from o. mykiss spleen cdna. western blot analysis, revealed that cath-1 is abundantly expressed in spleen and head kidney tissues of trout. experiments are ongoing to determine eventual cath-1 post translational modifications. to evaluate gene expression and tissue distribution of cath-1 and cath-2, an in vivo experiment was performed infecting o. mykiss samples with the salmon pathogen yersinia ruckeri and tissues have been collected at different times and analyzed by real-time pcr. preliminary data evidenced a high induction expression level 24 hours post-infection in spleen (average induction i.e. 10and 180-fold for cath1 and cath2 respectively), head-kidney and in intestine 48 hours after the challenge. results will contribute to a comparative understanding of the functions of cathelicidins in the vertebrates. amps and biotechnology application for new generation of medical devices l inguglia1, d schillaci2, m leto1, c mazzarella2, a vizzini2, d parrinello2, mg cusimano2, ma sanfratello2, m cacioppo1, v arizza2 1biotechnology laboratory general medical supplies s.r.l. calatafimi, segesta, trapani, italy 2department of biological chemical pharmaceutical science and technology, university of palermo, palermo, italy while a variety of gram-positive and negative bacteria as well as fungi have been involved as causative organisms in foreign body-related infections (fbris), staphylococci, particularly staphylococcus epidermidis and other coagulasenegative staphylococci (cons) account for the majority of infections, both of temporarily inserted and of permanently implanted material. normally, these bacteria are found as normal inhabitants of human skin and mucous membranes. however, in the appropriate clinical setting, specifically when there is a possible infection of a medical device, cons may be associated with considerable hospital   26 expenditures, morbidity and also an increased mortality rate. (von eiff et al., 2002). antimicrobial peptides (amps), principle effectors in the innate immunity, are molecules highlighted as potential candidates to produce new antibiotics, able to fight hard-to-treat infections. the presence of peptides with antimicrobial and antibiofilm activity was discovered in the sea urchin coelomocytes of paracentrotus lividus (schillaci et al., 2010). based on these results, we are studying the possibility to develop a new orthopedic device, able to prevent infections, coated with one of these amps. individual variability and gene expression specificity of the mussel antifungal mytimycin (mytm) mg parisi1, f cantet2,3, m toubiana2, m sonthi2,4, mr trapani1, m cammarata1, n parrinello1, p roch2 1department of biological chemical pharmaceutical science and technology, university of palermo, palermo, italy 2ecosym, université montpellier 2-cnrs, montpellier, france 3cpbs, univiversité montpellier 1-cnrs, montpellier, france 4faculty of marine technology, burapha university, chonburi, thailand four antimicrobial peptides (amp) families have been identified and characterized in the mediterranean mussel, mytilus galloprovincialis: defensin, mytilin, myticin and the antifungal mytimycin (mytm). they are synthesized mainly by hemocytes. nucleotide sequences were reported as different from one mussel to another. here, we reported on mytm gene expression and specificity of induction comparing the responses after challenges. q-pcr showed that mytm gene expression in circulating hemocytes was dosedependent. specificity of the mytm gene regulation has been investigated measuring the expression kinetics of mytilin and myticin genes in fungi injected mussels. in vivo challenge with yeasts and bacteria did not increase the expression of mytm gene as measured by q-pcr in hemocytes. on the opposite, injection of spores from the filamentous fungus, fusarium oxysporum, resulted in a rapid and significant up-regulation of mytm gene expression at 9 h post injection. the lack of stimulation suggested the existence of two different signal transduction pathways, one activated by bacteria and yeast, the other delayed and triggered by filamentous fungi. the second response to f. oxysporum challenge was significantly lower than the first one, suggesting a less efficient response more than a better protection and arguing against memory. then, research focused on the expression of mytm gene in individual m. galloprovincialis, before and after f. oxysporum challenge by q-pcr quantifying mytm mrna and in situ hybridization (ish). only some mussels reacted to the injection by increasing the expression of mytm gene. differences in mrna quantification values might result from variable numbers of circulating hemocytes expressing mytm gene. thus mytm transcript levels have been quantified from mrna extracted from the entire posterior adductor muscle 9 h after fungi injection. variability of mytm gene expression measured in hemocytes was not due to the sampling process, but to the fact that not all the mussels reacted to the challenge with f. oxysporum. cytology of mytm-expressing hemocytes showed that only granulocytes were labeled, suggesting trafficking of mytm mrna. gene expression increased in only some of the injected mussels and no correlation has been found between q-pcr quantification and number of labeled hemocytes as observed in ish. in conclusion we report here different behavior of individual mussels towards the same challenge. inflammatory-like reaction following bacterial injection and antimicrobial peptide isolation from anemonia sulcata (cnidaria) mr trapani1,2, d parrinello1, ma sanfratello1, g benenati1, mg parisi1, p roch2, m cammarata1 1department of biological chemical pharmaceutical science and technology, university of palermo, palermo, italy 2ecosym, université montpellier 2-cnrs, montpellier, france the diversity in the body plan, life and habitat of anthozoa raises crucial questions related to immunity. inflammation represents a rapid and efficient elimination mechanism of damaged tissue and microbes, and eventually the restoration of tissue functionality. we demonstrate here the presence of an inflammatory-like response in anemonia sulcata after injection of rabbit erythrocytes or bacteria escherichia coli, vibrio proteolyticus, vibrio alginolyticus and shigella putrefaciens, in the upper part of the basal disk. the reactions occurring mainly with e. coli are observed at 24 h post-injection showed the presence of a swelling in the ascending column of the body and in the basal disk. the reaction developed until to the appearance of a brown capsule. we performed histological analysis of injection sites to characterize the cells and tissues involved in the inflammatory response. in addition we analyzed the crude extract of a. sulcata body and tentacles by acid extraction, acid-urea page, hplc purifications and antibacterial assays. the purification procedure consisted of first step on sep pak c8 vac column followed by several hplc runs on c18 (interchrom up5odb-25qs) using different buffers and running conditions. among the several peaks, two showing the strongest antimicrobial activity was pooled and lyophilized. these fractions named as-amp1 and 2 show antibacterial activity against micrococcus lysodeikticus evaluated by the minimum inhibitory concentration (mic) after 16h incubation at 37°c and minimum bactericidal concentration assays counting the colony-forming-units in dilutions lowers than mic. further analyses are in progress for obtaining the primary sequence of as-apms.   27 components of hemocyte extracts from marine invertebrates exert antimicrobial activity ma damiano1,2, v arizza1, m cammarata1, m celi1, mg parisi1, d parrinello1, d russo1, ma sanfratello1, mr trapani1, m vazzana1, a occhialini2 1department of biological chemical pharmaceutical science and technology, university of palermo, palermo, italy 2umr 5236, centre d’études d’agents pathogènes et biotechnologie pour la santé, montpellier, france during the last decade, several biologically active peptides, compounds from 12 to 50 amino acids, have been isolated from a wide range of organisms including mammals, insects, plants and bacteria. these molecules are part of the innate immunity of organisms at all levels phylogenetic, also they have a broad spectrum of action against viruses, bacteria, fungi and protozoa pathogens and are able to destroy microorganisms and/or inhibiting their growth (boman, 2000; tincu and taylor, 2004). present in blood cells and plasma (taylor et al., 1997), these molecules show a wide range of mechanisms of action, mainly correlated with the destruction of the microbial cell wall. an in vitro study about the inhibitory effect (antimicrobial) of samples isolated from different species of marine invertebrates (actinia equina, pelagia noctiluca, procambarus clarkii, cancer pagurus, paracentrotus lividus), on the growth of pathogenic microorganisms or commensals of man, was carried out. in a representative collection of bacterial strains (escherichia coli, pseudomonas aeruginosa, enterococcus faecalis, staphylococcus aureus, bacillus subtilis, corynebacterium glutamicum, candida albicans, brucella abortus and brucella suis) assays chosen to evaluate the antimicrobial activity were: minimum concentration inhibitory (mic) and minimum bactericidal concentration (mbc). in parallel, in order to establish whether the action of extracts from hemocytes is specifically antimicrobial, was estimated their cytotoxic effect on cells of the type j774-a1. antibiotics, ampicillin and polymyxin b, were used as a control. the results indicate that cellular estracts from actinia equina, pelagia noctiluca, procambarus clarkii, cancer pagurus and paracentrotus lividus at the concentrations tested, have aspecific inhibitory effect and/or bactericidal action. the extract from actinia equina also showed a dose-dependent cytotoxic activity towards target cells. based on the obtained results and the current literature, the active fractions will be purified and the mechanism of action will be outlined.   28 isj 11: 331-336, 2014 isj 11: 331-336, 2014 issn 1824-307x research report physico-chemical characterization of a natural agglutinin from the hemolymph of a millipede thyropygus descriptus srmr basil-rose1, mh ravindranath2, srpd mercy1 1department of zoology, holy cross college, nagercoil-629 004, india 2terasaki foundation laboratory, los angeles, ca, usa accepted november 12, 2014 abstract natural hemagglutinins with specific affinity for the glycocalyx of rabbit erythrocytes is identified in the hemolymph of the millipedes, thyropygus descriptus, xenobolus acuticonus, arthrosphaera disticta and a. craspedota. of the tested species, maximum hemagglutinability is observed in the hemolymph of the millipede thyropygus descriptus. further characterization of the hemolymph agglutinin of thyropygus descriptus showed optimum agglutinability at ph 6.5 and temperatures 30 35 ºc. starvation up to 20 days had no influence on the hemagglutinability of the hemolymph. agglutinability was impervious by change in diet, and inclusion of diverse concentrations of cations or chelators in the buffer. the agglutinin, though agglutinates rabbit, rat and human a erythrocytes, when pre-adsorbed with erythrocytes of a particular species, loses its ability to agglutinate erythrocytes of any species suggesting the presence of a single agglutinin in the hemolymph. in general, the agglutinating activity of the agglutinin is inhibited by the glycoproteins porcine stomach mucin, lactoferrin, bovine submaxillary mucin, transferrin, fetuin and the sugars n-acetyl galactosamine, nacetyl lactosamine, lactose, galactose and n-acetyl neuraminic acid. the sialic acid specificity of the agglutinin is revealed by the reduction in hemagglutination activity when treated with the desialylated rabbit erythrocytes. key words: millipede; thyropygus descriptus; agglutinin; hemolymph; erythrocytes; hemagglutination assay; hemagglutination inhibition assay introduction lectins, the sugar binding proteins are found in a wide range of organisms including viruses, bacteria, fungi, plants and animals (sharon, 2008). they react with sugars in glycolipids, glycoproteins, or oligosaccharides and agglutinate erythrocytes via cell surface glycoproteins and glycolipids (stromberg et al.,1991; sharon, 2008). their specificity is usually defined in terms of a monosaccharide(s) or simple oligosaccharides that inhibit lectin-induced agglutination (sharon and lis, 1972; goldstein et al.,1980). an agglutinin may recognize a part of a sugar (shimizu et al.,1977), a whole sugar (bretting and kabat, 1976), their glycosidic linkages (koch et al., 1982) or a sequence of sugars (kobiler and mirelman, 1980; mauchamp, 1982). among protostomian invertebrates that are incapable of synthesizing sialic acids, molluscan ___________________________________________________________________________ corresponding author: sr. basil rose holy cross college nagercoil tamilnadu-629 004, india email: basilrosemr@gmail.com and arthropodan agglutinins recognize a unique kind of sugar called sialic acids (mullainadhan et al., 1984). the type of sialic acids and the glycosidic linkages with adjacent sugar in an oligosaccharide differ among pathogenic bacteria (ravindranath and cooper, 1984) and human cancer (ravindranath et al.,1985b; higashi et al., 1985; kawai et al., 1991). therefore, lectins specifically recognizing various sialic acids and their carbohydrate binding patterns can be used as tools for identifying various sialyl epitopes on pathogens or in biopsy of malignant tumors. a search for such sialic acid specific lectin is made in a millipede, thyropygus descriptus, a diplopodan representative of the super class myriapoda, a new group for lectin study. to develop strategies for affinity purification we have studied the physico-chemical properties of the agglutinin. material and methods materials millipedes thyropygus descriptus, xenobolus acuticonus, arthrosphaera disticta and a. craspedota used in this investigation were collected from the forest region of kanyakumari (anayadi and 331 table 1 survey of hemagglutinins in the hemolymph of millipedes ha titer erythrocytes t. descriptus (n = 25) x. acuticonus (n = 25) a. disticta (n = 25) a. craspedota (n = 25) rabbit 2048 32 512 1024 rat 128 0 256 128 human a 64 0 512 128 human b 0 0 512 128 human o 0 0 32 16 ox 0 0 0 0 horse 0 0 0 0 pig 0 0 0 0 mouse 0 0 0 0 kodayar) and tirunelveli (kalacaud and thalayanai) districts, tamil nadu, india. hemolymph collection the arthropodial membrane in between the collum and the adjacent segment was punctured after cleaning the area with wet cotton. the exuding hemolymph was collected in 15 ml polypropylene tubes kept on ice and stored in refrigerator. erythrocyte collection blood from different mammals was collected by venipuncture of the ear (rabbit), fore arm (human a, b, o and horse), cardiac puncture (mouse and rat) and from the slaughter house (ox and pig) directly in modified alsevier's medium (ph 6.1) containing sodium citrate (30 mm), sodium chloride (77 mm), glucose (114 mm), neomycin sulphate (100 μg/ml) and chloramphenicol (330 μg/ml) at a ratio of 2:8. erythrocytes were suspended and washed three times by centrifugation at 4,000g for 5 min with ten volumes of tris-buffered saline (tbs) ph 6.5 (trishcl 50 mm, nacl 100 mm, cacl2 10 mm) and resuspended in the same as 1.5 % suspension. hemagglutination (ha) assay the ha activity of the hemolymph agglutinin was assayed by measuring its ability to agglutinate erythrocytes. ha assays were performed at 30 °c by serial dilution of the hemolymph (25 µl) with tbs (25 µl) and mixing with 25 µl of 1.5 % erythrocyte suspension. ha titer was determined by the visual estimation of erythrocyte agglutination on microtiter plates 60 min after adding the cells. the ha titer (the units of agglutinin activity) is the reciprocal of the highest dilution of the sample that gave agglutination. to develop strategies for affinity purification, ha assay was also performed with high agglutinating rabbit erythrocytes at different ph, temperature and using buffer with different concentrations of cations calcium, magnesium and manganese and chelators, edta and egta. table 2 effect of moulting on the biochemical factors and ha titer of the hemolymph of the millipede of thyropygus descriptus parameters analysed premoult (n = 15) freshmoult (n = 10) postmoult (n = 10) intermoult (n = 25) volume (ml/animal) 2.568±1.229 2.967±1.09 2.78±1. 922 2. 65±1.27 water content (mg%) 96.887±1.1 98.35±1.218 97.232±1. 29 97.335±1.209 calcium (mm) 12.983±0.045 13.101±0.321 12.503±0.245 12.801±0.504 protein (µg/25µl) 0.430±0.031 0.432±0. 5 0.441±0.053 0.461±0.035 ha titer 1024 4096 2048 2048 332 fig. 1 effect of ph, temperature and starvation on hemagglutination assay of the hemolymph agglutinin of the millipede thyropygus descriptus to study the effect of ph on ha titer, the hemolymph sample was mixed with tbs at specific ph (5 9) at 1:1 ratio and incubated for 1 h and serially diluted in microtiter plates having tbs of same ph before adding erythrocyte suspension. to study the effect of temperature on ha titer, the hemolymph sample was incubated for 1 h at specific temperature (10 50 °c) and used for ha assay. to study the effect of cations and chelators on ha titer, the hemolymph sample incubated for 1 h in equal volume of tbs containing specific concentration (0.01,0.1,1.0 and 10 mm) of cations (calcium, magnesium and manganese) and chelators (edta and egta) was used for ha assay. in addition, ha was also carried out in the hemolymph obtained from animals fed with different foods, subjected to starvation and various stages of moult cycle after determining the volume, protein, calcium and water content of the hemolymph. cross-adsorption test packed erythrocytes (rabbit/rat/human a) were prepared by repeated washing of erythrocytes in 0.9 % saline by centrifugation at 4,000g for 5 min until we get a clear pellet. hemolymph was mixed with equal volume of packed rabbit/rat/human a erythrocytes and incubated for 18 h at 4 °c with occasional shaking. after centrifugation, the supernatant was analyzed for ha. hemagglutination inhibition (hai) assay hemolymph (25 µl) diluted to sub agglutination concentration (dilution at which hemolymph was able to provide 2 wells ha) was added to each well containing 25 µl of known concentration of serially diluted inhibitors (glycoproteins, mono and oligosaccharides). after incubation for 1 h, 25 µl of 1.5 % rabbit erythrocyte suspension was added. the hai titer is reported as the reciprocal of the highest dilution of inhibitors giving complete inhibition of agglutination after 60 min. protease treatment rabbit erythrocytes washed with tbs by centrifugation at 4,000g were mixed with equal volumes of mg/ml of trypsin and chymotrypsin and 0.25 mg/ml of pronase and incubated for 4 h at 37 °c. the treated cells were pelleted by low speed centrifugation in tbs-bsa and used for ha assay. sialidase treatment a reaction mixture containing 10 % washed rabbit erythrocytes in tbs-bsa (ph 6.5) and 140 mu sialidase of c. perfringens (sigma-type x) was incubated at 37 °c for 4 h. sialidase treated cells were washed with tbs-bsa three times, pelleted by low speed centrifugation and used for ha assay. 333 results ha activity of hemolymph the hemolymph of all the millipedes t. descriptus, x. acuticonus, a. disticta and a. craspedota agglutinated erythrocytes with diverse specificity. all the tested species agglutinated rabbit erythrocytes much higher than the other red blood cells tested (table 1). among the species assayed, maximum agglutinability was observed in the hemolymph of the millipede t. descriptus. hence further characterizations were restricted to the hemolymph of the millipede, t. descriptus. hemolymph had maximum agglutinin activity at ph 6.5 (fig.1a) and temperature 30 35 °c (fig. 1b). calcium, magnesium, manganese, edta and egta at all the concentrations (0.01, 0.1, 1.0, 10.0 mm) did not have any influence on ha. maximum ha was observed in the freshmoult stages than in the pre and post and intermoult stages (table 2). although varieties of food fed to the animals did not alter the ha, prolonged starvation significantly reduced the ha of the hemolymph (fig. 1c). ha activity after adsorption with different erythrocytes when t. descriptus hemolymph adsorbed to rabbit, rat and human a erythrocytes was used for ha assay with rabbit, rat and human a erythrocytes, it failed to agglutinate the erythrocytes of any other species except rabbit erythrocytes (table 3). table 3 cross adsorption assay of the hemolymph agglutinin of the millipede thyropygus descriptus erythrocyte adsorbed (n = 10) ha titer rabbit rat human a none 2048 128 64 rabbit 0 0 0 human a 16 (0) 0 0 rat 16 (0) 0 0 values in parenthesis refer to ha titers after second and subsequent adsorptions. inhibitors of ha n-acetyl lactosamine (lacnac), n-acetyl galactosamine (galnac) and lactose effectively inhibited the hemagglutinating activity. though sialidase treatment reduced the agglutinability, free sialic acid was a weak inhibitor of the hemolymph agglutinin. among glycoproteins, porcine stomach mucin (psm) and lactoferrin inhibited the hemolymph agglutinin strongly (tables 4). table 4 hemagglutination inhibition (hai) of the hemolymph agglutinin of the millipede thyropygus descriptus inhibition by sugars inhibitors (n = 5) hai titer min. con. req. for hai (mm) inhibitory potency (%) n-acetyl lactosamine 64 1.062 100 n-acetyl galactosamine 64 1.062 100 lactose 64 1.062 100 galactose 16 6.25 25 n-acetyl neuraminic acid 8 12.5 12.5 inhibition by glycoproteins inhibitors hai titer min. con. req. for hai (µg/ml) inhibitory potency (%) porcine stomach mucin 2048 4.88 100 lactoferrin 512 19.53 25 bovine submaxillary mucin 8 1250 0.39 transferrin 8 1250 0.39 fetuin 4 2500 0.195 thyroglobulin 0 334 table 5 effect of enzymatic cleavage of rabbit erythrocytes on hemagglutination assay of the hemolymph agglutinin of the millipede thyropygus descriptus enzymes used site of enzyme action ha titer none 2048 neuraminidase c. perfringens type x (140 mu) neuacα2,3gal;neuac α2,6gal; neuacα2,8gal; 512 trypsin (1 mg/ml) arg-, lys32768 chymotrypsin (1 mg/ml) tyr-trp-phe-leu32768 pronase (0.25 mg/ml) all peptide links 65536 sugars such as n-acetyl glucosamine, glucose, maltose, mannose, xylose, fructose and arabinose and glycoproteins such as bovine and porcine thyroglobulin did not inhibit ha at concentration 50 mm and 2.5 mg/ml respectively. ha activity of hemolymph after enzymatic alteration of erythrocytes ha activity got reduced when tested with neuraminidase (sialidase) treated rabbit erythrocytes and increased when tested with protease treated rabbit erythrocytes (table 5). discussion the hemolymph of all the four millipedes t. descriptus, x. acuticonus, a. disticta and a. craspedota recognized rabbit erythrocytes with great specificity. the ability of the millipede agglutinin to agglutinate rabbit erythrocytes argues for the specific recognition of the sugars constituting the glycocalyx of these erythrocytes, which serve as receptors to ligands as in the eukaryotic cells (hakomori, 1973). it has been found that different animal species have characteristic receptor determinants on their erythrocyte surface (yamakawa and suzuki, 1953) and interspecies variations (yasue et al., 1978). reduction in ha following starvation beyond 20 days and stability of ha after feeding different types of food reveals the presence of a natural agglutinin. identification of maximum hemagglutinability in the freshmoult stage of the millipede suggest defense role of this lectin in protecting the animals from foreign invaders and in the development of the millipede. as the exoskeleton of the freshmoult animals is extremely soft it may remain susceptible to the attack of any pathogen. hence the presence of high amount of agglutinin in the hemolymph could be a defense mechanism to evade microbial attack. like agglutinins from many other species (miller et al., 1972), the t. descriptus agglutinin is also sensitive to ph and temperature. the loss of biological activity of the agglutinin with increased temperature could be related to destabilization of sporadic weak interactions of tertiary structure responsible for native conformation of lectin (singh and saxena, 2013). ha assay with different concentrations of cations such as calcium, magnesium and manganese and chelators such as egta and edta suggests that the lectin is calcium independent. the removal of ha following adsorption of the hemolymph to rabbit erythrocytes suggest the presence of a single hemagglutinin as reported in c. antennarius (ravindranath et al., 1985a), scylla serrata (mercy and ravindranath, 1992, 1993) and paratelphusa jaquemontii (maghil et al., 2003). however, the hemolymph when adsorbed to rat and human a erythrocytes continued to agglutinate rabbit erythrocytes suggesting the presence of remnants of agglutinability in the hemolymph capable of recognizing rabbit erythrocytes even after repeated adsorptions. this is supported by the serological studies which show that activity to one type of erythrocyte can be adsorbed by that type of erythrocytes, leaving residual agglutinating activity to other type of erythrocytes (noguchi, 1903). the rise in ha activity following treatment of erythrocytes with proteases may be due to the removal of certain proteins which mask the glycocalyx of the rabbit erythrocytes that are specifically recognized by the millipede hemolymph agglutinin. the potent inhibitor of the agglutinin is psm, a glycoprotein rich in galnac residues. accordingly, galnac also inhibits the agglutinating activity at 1.5 mm concentration, but the ability of the hemolymph to agglutinate rabbit erythrocytes and its reduction following desialylation of erythrocytes argues for the affinity of the agglutinin to sialic acids. among the sialic acid content of psm, 90 % exist as nglycolylneuraminic acid and 10 % as n-acetyl neuraminic acid and traces as n-o-acetyl neuraminic acid. high ha with rabbit erythrocytes containing neugc (bhavananthan et al., 1964) and inhibition by psm containing 90 % neugc (schoop and faillard, 1967) accounts for neugc specificity, which can be confirmed only after affinity purification of the lectin. conclusion in spite of various information, the exact specificity of the agglutinin based on sugar specificity can be clearly stated only upon purification. this study provides the physico335 chemical requirements of the hemolymph agglutinin for affinity purification. the presence of sialic acid binding agglutinins in arthropods such as t. descriptus that are incapable of synthesizing sialic acids suggest that these agglutinins may be involved in the innate immunity of these organisms. references bhavanandan vp, buddecke e, carubell r, gottschalk a. the complete enzymatic degradation of glycopeptides containing o-seryl and o-threonyl linked carbohydrate. biochem. biophys. res. commun. 16: 353-361, 1964. bretting h, kabat, ea. purification and characterization of agglutinins from the sponge axinella polypoides and a study of their combining sites. biochemistry 15: 3228-3236, 1976. goldstein ij, hughes rc, monsigny m, osawa t, sharon n. what should be called a lectin? nature 285: 66, 1980. hakomori si. glycolipids of tumor cell membrane. adv. cancer. res. 18: 265-315, 1973. higashi h, hirabayashi y, fukui y, naiki m, matsumoto m, ueda s, et al. characterization of n-glycolylneuraminic acid containing gangliosides as tumor associated hanganutziudeicher antigen in human colon cancer. cancer res. 45: 3796-3802, 1985. kawai t, kato a, higashi h, kato s, naiki m. quantitative determination of nglycolylneuraminic acid expression in human cancerous tissues and avian lymphoma cell lines as tumor-associated sialic acid by gas chromatography mass spectrometry, cancer res. 51: 1242-1247, 1991. kobiler d, mirelman d. lectin activity in entamoeba histolytica trophozoites. infect. immun. 29: 221225, 1980. koch om, lee ck, uhlenbruck g. cerianthinlectins: a new group of agglutinins from cerianthus membranaceus. immunobiology 163: 53-62, 1982. maghil d, mercy pd, bai rn, suriya js. purification and characterization of a sialic acid specific lectin from the hemolymph of the freshwater crab paratelphusa jaquemontii. eur. j. biochem. 270: 4348-4355, 2003. mauchamp b. purification of an n-acetyl-dglucosamine specific lectin (p.b.a.) from epidermal cell membranes of pieris brassicae. l. biochimie 64: 1001-1008, 1982. mercy sr. pd, ravindranath mh. an agglutinin with unique specificity for n-glycolylsialic acid residues of thyroglobulin in the hemolymph of a marine crab scylla serrata (forskal). experientia 48: 498-500, 1992. mercy sr. pd, ravindranath mh. purification and characterization of n-glycolylneuraminic acid specific lectin from scylla serrata. eur. j. biochem. 215: 697-704, 1993. miller h, ballback s, tauley gb, krassner sm. a preliminary physico-chemical characterization of an agglutinin found in the hemolymph of the cray fish procambarus clarkii. j. invertebr. pathol. 19: 83-93, 1972. mullainadhan p, ravindranath mh, wright rk, cooper el. crustacean defense strategies 1, molecular weight dependent clearance of dyes in the mud crab scylla serrata (forskal), (portunidae:brachyura). dev.comp. immunol. 8: 41-50, 1984. noguchi, h. on the multiplicity of the serum hemagglutinin of cold blooded animals. zentr. bakt. abt. 34: 2865, 1903. ravindranath mh, cooper el. crab lectins: receptor specificity and biomedical applications. progr. clin. biol. res. 157: 83-96, 1984. ravindranath mh, higa hh, cooper el, paulson jc. purification and characterization of an o-acetyl sialic acid specific lectin from a marine crab cancer antennarius. j. biol. chem. 260: 88508856, 1985a. ravindranath mh, paulson jc. irie rf. human melanoma antigen o-acetylated ganglioside gd3 is recognized by cancer antennarius. j. biol. chem. 260: 8838-8845, 1985b. schoop hj, faillard h. contribution to the biosynthesis of the glycolyl group of n glycolylneuraminic acid. hoppe seylers z. physiol. chem. 348: 1518-1524, 1967. sharon n, lis h. cell-agglutinating and sugarspecific proteins. science 177: 949-959, 1972. sharon n. lectins: past, present and future biochem. soci. trans. 36: 1457-1460, 2008. shimizu s, ito m. niwa m. lectins in the hemolymph of japanese horseshoe crab, tachypleus tridentatus. biochem. biophys. acta 500: 71-79, 1977. singh ap, saxena kd. effect of temperature, ph and denaturing agents on biological activity of mcj lectin. chem. sci. trans. 2: 1508-1512, 2013. stromberg p, nyholmt g, paschert i, normark s. saccharide orientation at the cell surface affects glycolipid receptor function, proc. natl. acad. sci. usa 88: 9340-9344, 1991. yamakawa t, suzuki, s. the chemistry of lipids of post hemolytic residue or stroma of erythrocytes. iv. distribution of lipid hexamine and lipid hemataminic acid in red blood corpuscles of various species of animals. j. biochem. 40: 7-10, 1953. yasue s, honda s, miyagawa s, inoue j, hasegava a, yamakava t. difference in the form of sialic acid in red blood cells glycolipids of different breeds of dogs. j. biochem. 83: 1101-1107, 1978. 336 a new cell line established from silkworm embryo is highly susceptible to the nucleopolyhedrovirus infection isj 12: 13-18, 2015 issn 1824-307x research report establishment and characterization of a new embryonic cell line from the silkworm, bombyx mori m xu#, j tan#, x wang, x zhong, h cui state key laboratory of silkworm genome biology, southwest university, chongqing 400716, china #equal contribution accepted december 18, 2014 abstract insect cell lines are widely used for basic and applied research in the fields of insect pathology, genetics, and molecular biology. in the present study, a new continuous cell line designated bme-swu3 was established from blastokinesis-stage embryos of the silkworm bombyx mori (furong strain). the primary culture was initially performed using grace’s medium supplemented with 20 % foetal bovine serum (fbs) at a constant temperature of 27 °c. the dominant cell type was round and spindle-shaped. thus far, this cell line has been cultured continuously for 60 passages. the cell doubling time was approximately 3.0 days. the ssr profile of bme-swu3 differs from those of the silkworm bme and bmn-swu1 cell lines and from those of the spodoptera frugiperda cell line sf9 and the drosophila cell line s2. however, the ssr profiles among the various passages of bme-swu3 were stable and identical. this new cell line was highly susceptible to bombyx mori nucleopolyhedrovirus (bmnpv). semi-quantitative rt-pcr indicated that the tissue-specific gene expression patterns were completely distinct from those of bme and bmn-swu1. key words: embryonic cell line; bme-swu3; dna fingerprinting; bmnpv infection; bombyx mori   introduction as an experimental tool, cell lines offer great advantages due to their easy handling and amenability to manipulation. ever since thomas grace and shangyin gao established cell lines from antheraea eucalypti and bombyx mori, respectively, the development of insect cell lines has progressed rapidly (grace, 1962; gaw et al., 1958). indeed, approximately 800 types of continuous cell lines have been developed from over 100 insect species, including coleoptera, hymenoptera, orthoptera, homoptera, and hemiptera, with the majority derived from lepidopteron and diptera (lynn, 1999, 2002; zhang et al., 2007; shao et al., 2008). insect cell lines have been widely applied to produce certain virus species for use as biopesticides and also can be used as a tool to generate recombinant proteins. in addition, certain lines are used to investigate specific virus pathogenic mechanisms in basic research (blissard, 1996). several types of insect cell lines have been widely used to date. the s2 (schneider 2) cell line, ___________________________________________________________________________ corresponding author: hongjuan cui state key laboratory of silkworm genome biology southwest university chongqing 400716, china e-mail: hongjuan.cui@gmail.com derived from late embryonic-stage drosophila melanogaster, is one of the most commonly used cell lines (schneider, 1972). the lepidopteron cell line sf9 (spodoptera frugiperda 9) is commonly used and was isolated from the sf21 (spodoptera frugiperda 21) cell line, which was originally established from ovarian tissue (vaughn et al., 1977). cell lines established from embryonic, larval, pupal or adult stages have become useful tools for basic and applied research. over 30 cell lines have been established from the silkworm, and most of them are derived from the embryonic or ovarian tissues of larvae or pupae (lynn, 2001; pan et al., 2007; khurad et al., 2006, 2009). for example, bme, reported in 2007, was established from silkworm embryonic tissue of the reversion phase (pan et al., 2007). bmn-swu1 was established from the ovarian tissue of 3-day-old fourth-instar silkworm larvae of the 21 872nlw strain (pan et al., 2010). as the most commonly used silkworm cell lines were established decades ago, new practical and affordable cell lines are needed to facilitate the study of silkworm functional genomics. in this report, we present a new embryonic cell line, bme-swu3, which was isolated from blastokinesis-stage embryos of the silkworm bombyx mori (lepidoptera: bombycidae). its distinct 13 fig. 1 cell morphology of the bme-swu3 cell line established from silkworm embryos. (a) and (b) show cells cultured for 3 days and 6 months, respectively. (c) and (d) show cells at the 8th and 30th passages, respectively. scale bars = 100 µm. ssr dna and mrna expression profiles indicate that bme-swu3 is a novel cell line and distinct from the bme, bmn-swu1, sf9 or s2 cell lines, which are either from the silkworm or other insect species. due to its high susceptibility to viral infection, bme-swu3 can be used to study the bmnpv baculovirus expression system and the mechanism of virus replication. materials and methods insects all experiments were conducted with embryos of the furong strain, which is a pure strain that is widely used in research. eggs from the silkworm gene bank of southwest university were incubated at 28 °c with a 16:8 h light:dark photoperiod for 3 days and then collected immediately after they entered the blastokinesis stage. primary cultures the eggs were sterilised by submersion in 70 % ethyl alcohol for 3 min before processing using previously described procedures (pan et al., 2007). briefly, the embryos were cut into small pieces and dissociated with 0.25 % trypsin for 5 min; this reaction was stopped by adding fbs (invitrogen). the pellets were collected, centrifuged at 100g for 5 min, then suspended in 2 ml grace’s medium (ph 6.8) supplemented with 20 % fbs. the suspension was placed into a 25-cm2 culture flask and cultured at 28 °c. fresh medium was added after the majority of the embryonic tissues were attached, and half of the medium was replenished every two weeks until the culture was split. fig. 2 bme-swu3 cell line growth curve. cell growth was assessed using the cck-8 cell proliferation assay. for each time point, 5 wells were examined. data are presented as the means ± sd. 14 fig. 3 ssr-pcr dna profile analysis. (a), (b), and (c) show the results of ssr-pcr using primers s0205, s0518, and s2618, respectively. lanes 1 4 represent the pcr results of bme-swu3 cells at the 7th, 9th, 19th, and 34th passages, and lanes 5-8 represent the pcr results for the bme, bmn-swu1, s2, and sf9 cell lines, respectively. subculture and morphological observation the cells kept split and were subcultured in half year after the original culture. briefly, the cells were suspended using a sterile pasteur pipette and centrifuged at 100g for 5 min; the cell pellets were resuspended in 5 ml medium containing 15 % fbs and then transferred into a new culture flask. the culture medium was replaced every 5 days. the fbs concentration was decreased from 20 % to 15 % at the 10th passage and from 15 % to 10 % at the 20th passage. the cells were observed under a nikon te2000 inverted phase-contrast microscope. growth analysis growth curves were determined at the 30th passage. a sample of 8,000 cells in 200 μl medium was seeded into a 96-well plate. the cell number was counted every 24 h using cell counting kit-8 (cck-8) (beyotime), and the growth curve was plotted as od values at 450 nm. fingerprinting analysis dna was extracted and purified from bme-swu3, bme, bmn-swu1, s2, and sf9 cells using the easypure genomic dna kit (transgene biotechnology, inc.). ten pairs of ssr primers (kind gifts from prof fangyin dai in southwest university) were employed (miao et al, 2005): s0105, s0201, s0205, s0317, s0411, s0518, s0709, s0804, s0818, s2502, s2503, s2612, and s2618. pcr amplification was performed in reaction volumes of 15 μl containing 1x pcr buffer, 2.5 mm mgcl2, 0.2 mm of each dntp, 0.6 µm of ssr primers, 0.4 unit of taq polymerase (takara), and 20 ng of template dna. the pcr conditions were as follows: 94 °c for 30 sec, 63 °c for 1 min, and 72 °c for 1 min; 15 cycles of 94 °c for 30 sec, 62.5 °c for 1 min, and 72 °c for 1 min, with the temperature lowered 0.5 °c per cycle; followed by 25 cycles of 94 °c for 30 sec, 56 °c for 1 min, and 72 °c for 1 min. the pcr products were analysed by 12 % polyacrylamide gel electrophoresis (page). gel images were captured using a bio-rad gel documentation system. reverse transcription-pcr analysis total rna from bme-swu3, bme, and bmn-swu1 cells was isolated using trizol (invitrogen). cdna was synthesised with 2 µg total rna using reverse transcriptase and the supplied solutions (promega). pcr was performed as follows: 94 °c for 3 min; 35 cycles of 94 °c for 30 sec, 55 °c for 30 sec, and 72 °c for 1 min; and 72 °c for 10 min. the products were analysed by 1 % agarose gel electrophoresis, and gel images were captured using a bio-rad gel documentation system. the sequences of the primers used in the rt-pcr experiments are as follows: vasa-f, 5' ggaggaggcgatagaaatg 3'; vasa-r, 5' atgatacacgattcctttcca 3'; lsp-f, 5' tatcaccactgccgattacaa 3'; lsp-r, 5' gcttagcgggcttcagg 3'; fibl-f, 5' tttttggtattactcgtcgct 3'; fibl-r, 5' 15 tcactgctggctaagattgc 3'; apn-f, 5' caaacaatggcgttatcactt 3'; apn-r, 5' tcctaaactgtctccatttctga 3'; rpl3-f, 5' cggtgttgttggatacattgag 3'; and rpl3, 5' gctcatcctgccatttcttact 3'. virus infection bme-swu3 cells or bmn-swu1 cells were seeded into 96-well plates at 8×103 cells/well and infected with recombinant bmnpv expressing egfp (enhanced green fluorescent protein) under the bmnpv 39k promoter (v39kprm-egfp) at an moi (multiplicity of infection) of 20. after 3 days of infection, the titres were determined by tcid50 analysis based on egfp as described (o'reilly, 1993). results establishment of the bme-swu3 cell line after 6 days of incubation, the cells had migrated from the embryonic tissue sections. the cell number gradually increased, and some cells were de-attached when the cell confluence reached 80 %. the suspended cells were harvested and then re-cultured in 3 ml grace’s medium (ph 6.8) supplemented with 20 % fbs. the bme-swu3 line was successfully subcultured after another 6 months of culturing. the interval of subculture for the first five passages was 30 days at a ratio of 1:2, then became 10 days. the morphology of the majority of cells was round and smaller than the cells of the bme cell line, with a spindle shape that became round at 80 % confluence (fig. 1). to date, the cells were successfully subcultured for 60 passages at an interval of 5 days. cell growth analysis cell growth was assessed using the cck-8 cell proliferation assay when the cells reached the 35th passage (fig. 2). the growth curve of bme-swu3 cells expressed as od values showed an obvious “s” shape. the population doubling time was approximately 3 days, which was calculated using the formula provided by hayflick (1973). dna profiling dna fingerprinting is a valuable and reliable technique for cell line identification (mcintosh et al., 1996). we conducted pcr with 13 primers to analyse the dna profiles of the bme-swu3 cell line in comparison with those of the bme, bmn-swu1, sf9 or s2 cell lines. pcr using 10 primers generated clear dna fragments in the silkworm cell lines. because the ssr primers used are specific for b. mori, only 4 of them were able to generate dna fragments in the s2 cell line, with 7 in the sf9 cell line. however, the dna banding patterns of the s2 and sf9 cell lines were highly distinct from those of the silkworm cell lines. the dna fingerprinting profiles using s0205 (fig. 3a) and s0518 (fig. 3b) indicated no difference among the bme-swu3, bme, and bmn-swu1 cells. in contrast, the dna fingerprinting profiles with the s2618 primer (fig. 3c) and another 7 primers were significantly different among the bme-swu3, bme, and bmn-swu1 cell lines (fig. 3b). however, no polymorphism was detected between the various passages of the bme-swu3 cell line. all of these results confirmed that the bme-swu3 line is genetically closer to b. mori cell lines than to cells from other insect species. fig. 4 bme-swu3 and bmn-swu1 cells were infected with recombinant bmnpv for 72 h. the infected cells were indicated by egfp signals (green). scale bars = 200 µm. 16 fig. 5 semi-quantitative rt-pcr of bme-swu3, bme, and bmn-swu1 cells. (a) apn4, l-fib, vasa, and lsp were analysed using semi-quantitative pcr. rpl3 was included as a control. (b) quantitative analysis of the mrna expression level. viral susceptibility bme-swu3 and bmn-swu1 cells were infected with recombinant bmnpv for 72 h, and egfp signals were used to monitor viral infection via fluorescence microscopy (fig. 4). according to the formula described by o’reilly (o'reilly, 1993), the virus titres in the bme-swu3 and bmn-swu1 cells were 1.756×108 tcid50/ml and 2.338×108 tcid50/ml, respectively. bmn-swu1 cells have been reported to be highly susceptible to bmnpv (bombyx mori nucleopolyhedrovirus), and the similar tcid50 values indicate that bme-swu3 is also highly susceptible to bmnpv. tissue-specific gene expression patterns in bme-swu3, bme and bmn-swu1 cells apn4 (aminopeptidase n4), l-fib (l-fibroin), vasa, and lsp (larval serum protein) are specifically expressed in the midgut, silk gland, gonad, and fat body, respectively, in silkworm larvae (hua et al., 1998; tang et al., 2003; tomita et al., 2003; nakao et al., 2006). thus, we chose these four tissue-specific genes to characterize the bme-swu3 cell line. rt-pcr data indicated that the bme-swu3 mrna expression patterns were significantly different from those of bme or bmn-swu1 cells (fig. 5). the bme-swu3 cells were negative for apn4, l-fib, and lsp, but positive for vasa, confirming that bme-swu3 cells are derived from the germ line rather than from other tissues, such as the midgut, silk gland or fat body. discussion in this study, we established and characterised a new embryonic cell line, bme-swu3, from the silkworm b. mori. this is the first cell line cultured from the furong strain, which is a pure strain that has been commonly applied in many research fields. bme-swu3 was initiated from the third-day embryos, the prophase blastokinesis stage, which stage belongs to gastrula and also had the totipotency capability. it’s different from bme which was generated from the fourth-day embryos, which stage belongs to the reversion phase. in this stage, ectoderm, endoderm and mesoblast formed consecutively, all of the organs began differentiate and develop. as we expected, the tissue-specific gene expression patterns in bme-swu3 were different from bme or bmn-swu1. derived from different stage, and owned different genetic background, and different gene transcriptional regulation, and that would provide diversity for silkworm basic research. in addition, the ssr profile of bme-swu3 was compared with those of the current silkworm cell lines (bme-swu1 and bmn-swu1) and another two other insect cell lines (sf9 and s2) at the dna level, confirming that bme-swu3 was derived from the silkworm b. mori and is distinct from other cell lines. moreover, sf9 cell line is derived from spodoptera frugiperda, and bme-swu3 is from b.mori, both of them belong to lepidopteron, but s2 17 cell line is from the diptera drosophila melanogaster, the expression profile is totally different from the b. mori. as the same lepidopteron resource, the sf9 cell line expression profile showed some similarity with bme-swu3, both of them shared a clear band with the s2618 primer. these results confirmed that bme-swu3 cell line is genetically closer to sf 9 cell line rather than s2 cell line. more important, bme-swu3 has high susceptibility to bmnpv, and it could be used in research of the mechanism about infection, replication, assembly and release of bmnpv and utilised as a foreign gene-expressing system to produce useful recombinant proteins with bmnpv-derived expression vectors (e.g., bac-to-bac baculovirus expression system). in summary, bme-swu3 is a brand new cell line derived from silkworm, and it’s very practical and affordable, and it could be widely used in insect gene functions research and application. acknowledgments this research was supported by national basic research program of china (no. 2012cb114603), research fund for the doctoral program of higher education of china(20130182110003), the natural science foundation of chongqing (cstc2013jcyjy0007), supported by fundamental research funds for the central universities (swu111014). we would like to thank dr. fangyin dai from southwest university of china for providing the ssr primers and the furong embryos. references blissard gw. baculovirus--insect cell interactions. cytotechnology 20: 73-93, 1996. gaw zy, liu nt, zia tu. tissue culture methods for cultivation of virus grasserie. acta virol. 3 (suppl.): 55-60, 1959. grace td. establishment of four strains of cells from insect tissues grown in vitro. nature 195: 788-789, 1962. hayflick l. subculturing human diploid fibroblast cultures, academic press, new york, 1973. hua g, tsukamoto k, taguchi r, tomita m, miyajima s, ikezawa h. characterization of aminopeptidase n from the brush border membrane of the larvae midgut of silkworm, bombyx. mori as a zinc enzyme. biochim. biophys. acta 1383: 301-310, 1998. khurad am, kanginakudru s, qureshi so, rathod mk, rai mm, nagaraju j. a new bombyx mori larval ovarian cell line highly 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tettamantia, r valvassoria, m de eguileora adepartment of biotechnology and life sciences, university of insubria, varese, italy buniversitätsklinikum, freiburg neuroonkologie, breisacher straße 64, 79106 freiburg, germany accepted february 18, 2013 abstract in some leeches like hirudo medicinalis, any kind of stimulation (surgical wound or growth factor injection) provokes the botryoidal tissue response. this peculiar tissue, localized in the loose connective tissue between gut and body wall, is formed by granular botryoidal cells and flattened endothelial-like cells. under stimulation, the botryoidal tissue changes its shape to form new capillaries. in mammals, the molecular regulation of the angiogenic phenotype requires coordinated input from a number of signalling molecules: among them the gtpase ras is one of the major actor. in our current study, we determine whether ras activation alone would be sufficient to drive vessels formation from leech botryoidal tissue. our findings indicate that assembly and disassembly of actin filaments regulated by ras protein is involved in morphological modification of botryoidal tissue cells during leech angiogenic process. key words: leech; ras; cytoskeleton; angiogenesis introduction hirudo medicinalis shows a simple anatomical organization: a muscular-cutaneous sac, practically avascular (de eguileor et al., 2001a, 2004), containing several organs embedded in a loose connective tissue. in hirudidae between the gut and the body wall, a peculiar tissue, the botryoidal tissue is localized. this multifunctional tissue, formed by clusters of roundish granular botryoidal cells, and small, flattened, endothelial-like cells, displaying myelo/erythroid and storage functions (sawyer, 1986), is involved in angiogenesis (de eguileor et al., 2001b). we have previously demonstrated that both surgical and biochemical stimuli (i.e. injection of cytokines such as vascular endothelial cell growth factor (vegf), basic fibroblast growth factor (bfgf) or granulocyte macrophage colony-stimulating (gm-csf) evoke in botryoidal tissue cells, angiogenic signalling pathways analogous to those observed in vertebrates (grimaldi et al. 2006; tettamanti et al., 2003a; tettamanti et al., 2003b, 2006). immediately after stimuli, botryoidal tissue changes its shape from a solid cord of cells to a tubular, pre-vascular structure through a dehiscence ___________________________________________________________________________ corresponding author: annalisa grimaldi department of biotechnology and life sciences university of insubria via j. h. dunant 3, 21100 varese, italy e-mail: annalisa.grimaldi@uninsubria.it process. in this contest, the remodelling is characterized by marked cellular changes: thinning, flattening and tapering of these cells allow the definition of lumen, and the increase in diameter and length of new capillary. the molecular control of the complex angiogenic phenotype requires coordinated input from a number of signalling molecules. in mammalian, one of basic regulators of the angiogenic response is ras. this gtpase, due to its involvement in endothelial cell motility (sosnowski et al., 1993; fox et al., 1994), plays an important role in several systems including bfgfmediated wound closure and pro-angiogenic response to vegf. its activation is also required for the proliferation, migration, and branching morphogenesis of vascular endothelial cells (meadows et al., 2001, 2004). ras protein controls signal transduction pathways (carpenter 2000; kranenburg and moolenaar, 2001) leading to gene expression changes and cell motility through the map/erk kinase cascade. (ehrhardt et al., 2002). the focus of the present work is to demonstrate that as in vertebrates (joneson et al., 1996; leblanc et al., 1998; meadows et al., 2001; kranenburg, 2004; meadows et al., 2004; serban et al., 2008) ras plays a key role in the leech angiogenic process by remodelling actin cytoskeleton even in absence of vegf, generally considered the principal regulator of angiogenesis. 7 fig. 1 a-j morphological and immunocytochemical analysis of control and vegf stimulated botryoidal tissue cells. semi-thin (a, c) and thin (b, d-f) sections of control and vegf stimulated cultured botryoidal tissue. the control tissue is formed by clusters of botryoidal cells with a granule-filled cytoplasm (arrowheads in a and b) and endothelial cells (arrows in a and b). after 6 h from vegf administration, large cavities (lv in c and d) lined by granulated botryoidal cells (arrowheads in b and c) and by flattened endothelial-like cells (arrows in c, d) are visible. bundles of actin filaments are visible at tem in the cytoplasm of vegf stimulated endothelial cells (arrowheads in f), but not in the control untreated endothelial cells (e). in both vegf stimulated (g, h) and vegf+anti-ras (i, j) treated botryoidal tissue, the diffuse distribution of actin, lacking a specific organization in bundles is detected by immunofluorescence using a specific monoclonal antibody anti-beta actin (g, i), while factin (h, j) is stained using -conjugated phalloidin. the formation of actin filaments is highly reduced in vegf+anti-ras treated endothelial cells (j). bars in a, c, g-j: 100 μm; bar in b: 4 μm; bar in d:10 μm; bars in e, f: 200 nm. 8 material and methods animals and treatments leeches (hirudo medicinalis, annelida, hirudinea, hirudidae from ricarimpex, eysines, france) were kept in water at 19 20 °c in aerated tanks. animals were fed weekly with calf blood. before each experiment, leeches were anaesthetized with a 10 % ethanol solution. cell culture leeches were longitudinally dissected on the dorsal side using sterilized razor blades and botryoidal tissue cells, localized between the muscular body wall and the gut, were harvested from the animals using sterilized forceps. cells were then transferred to a single well of 24 well plates in 400 ml of tissue culture medium containing dmem medium (celbio, milan, italy) modified by dilution (1:4) to reach iso-osmolality and supplemented with 1% glutamine,10 % fetal bovine serum and 1 % antibiotic antimycotic solution (sigma, st. louis, mo) as already described (grimaldi et al., 2008, 2009). cells were maintained at 20 °c for 24 h before to be differently processed. cell culture and vegf administration to evaluate the effect of vegf165 on botryoidal tissue cells shape modification cells were plated in 24-multiwell plates in medium alone or in medium supplemented with 100 ng/ml of vegf165. all cultures were performed in quadruplicate and scored at 6 in vitro using an inverted olympus microscope (olympus, tokyo, japan). data were recorded with a ds-5m-l1 digital camera system (nikon, tokyo, japan). cell culture and transformation assays plasmids (kindly gifted by renata zippel, university of milano, italy) utilized for botryoidal cells transfection were: pc3mycras61, coding for a constitutively activated form of ras (glu61 substituted with leu) and pegfp-ci (coding for a green fluorescence protein). dna transfection of botryoidal tissue cells were performed using calcium phosphate precipitation (calcium phosphate kit, sigma). cells were kept in a 1xhepes-buffered saline solution ph 7.05 (sigma) containing 8.4 μl cacl2 (2,5m), 2,5 μg of pegfp-ci and 4.5 μg of pc3mycras61. control experiments were performed using a 1xhepes-buffered saline solution ph 7.05 containing only 8.4 μl cacl2 (2,5m) and 7 μg of pegfp-ci. after 20 h the medium containing dna were removed and cells were treated for 2 minutes with a 50 % glycerol in 1xhepes-buffered saline solution ph 7.05 to increase efficiency of transfection. cells were then plated in culture medium and analyzed after 3 days. transfection efficiency was visualized by using a fluorescence microscope olympus (excitation/emission filters 490/525 nm for fluorescein isothiocyanate-fitc). inhibition of ras signalling in ras transformed cells an antibody anti-ras and the synthetic inhibitor of the map kinase pathway pd 098059, (2′-amino3′methoxyflavone, alexis, bingham, u.k.) were used to block ras activity (alessi et al., 1995). for antibody-mediated neutralization experiments, 1 μg of the polyclonal antibody anti-rabbit ras (stressgen biotechnologies corporation, victoria, bc, canada), was added to the medium in which gfp/ras61 co-transfected cells or vegf stimulated cells were cultured. pd 098059 was dissolved in dmso to give a final concentration of 37 μm and stored at -80 °c. gfp/ras61 transformed cells were then cultured in a medium containing pd 098059 to a final concentration of 100 μm. cells plated in both modified culture medium were analyzed after 3 days. inhibition of actin bundles formation cytochalasin d (goddette and frieden, 1987), that has the ability to bind actin filaments, was use to block polymerization and the elongation of actin. gfp or gfp/ras61 transformed cells were incubated with culture medium containing 5 μg/ml of cytochalasin d (sigma) and analyzed after 3 days. optical and electron microscopy botryoidal tissue cells were fixed for 2 h in 0.1 m cacodylate buffer ph 7.2, containing 2 % glutaraldehyde. specimens were then washed in the same buffer and postfixed for 2 hrs with 1 % osmic acid in cacodylate buffer, ph 7.2. after standard serial ethanol dehydration, specimens were embedded in an epon-araldite 812 mixture. sections were obtained with a reichert ultracut s ultratome (leica, wien, austria). semithin sections were stained by conventional methods (crystal violet and basic fuchsin) according to moore et al. (1960), and subsequently observed under a light microscope (olympus). data were recorded with a ds-5m-l1 digital camera system (nikon). images were combined with adobe photoshop (adobe systems, inc.). thin sections were stained by uranyl acetate and lead citrate and observed with a jeol 1010 ex electron microscope (jeol, tokyo, japan). data were recorded with a morada digital camera system (olympus). immunofluorescence staining cultured cells were fixed with 4 % paraformaldehyde in phosphate-buffer (pbs) for 30 min at 4 °c. after washing in pbs cells were preincubated for 30 min in a blocking solution (pbs, 2 % bovine serum albumin (bsa), tween 0.1 %). cells were then incubated for 1 h at 37 °c with the primary antibodies mouse anti-beta actin (sigma), to visualize g-actin, and rabbit anti-ras (stressgen biotechnologies corp.), diluted 1:100 in blocking solution. the washed specimens were incubated for 1 h at room temperature with the appropriate secondary antibody cy3 conjugated (1:200) (jackson, immuno research laboratories, west grove, pa, usa). samples were directly examined with a fluorescence microscope olympus or embedded in polyfreeze tissue freezing medium (polysciences, eppelheim, germany), immediately frozen in liquid nitrogen. and cut with a leica cm 1850. the staining was visualized using excitation/emission filters 550/580 nm for cy3. data were recorded with a ds-5m-l1 digital camera system (nikon). images were combined with adobe photoshop (adobe systems, inc.). in control samples, primary antibodies were omitted and cells, 9 fig. 2 a-l ectopic expression of ras61 enhances a dehiscent process in botroyidal tissue cells. the morphology of gfp transformed botryoidal cells in growth medium (a, b) and sectioned (e) is similar to that observed in control untreated cells. botryoidal tissue cells maintain a rope shape. in cultured (c, d) and sectioned (f) gfp/ras61 transformed cells morphological changes are observed. botryoidal tissue cells show an elongated phenotype (c, d) and undergo a dehiscence process lining a new cavity (lv in f). the expression of ras was detected by immunofluorescence in both gfp (g-i) and gfp/ras61 (j-l) transformed cells. after ras61 transfection, the expression of ras protein (in red in h, i, k, l) is mainly localized in the elongated and flattened endothelial cells (j-l). the gfp marker indicates infected cells (green in b, d, h, i. k, l). bars in a-l: 25 μm. 10 pre-incubated for 30 min with pbs/bsa, were incubated only with the secondary antibodies. to visualize f-actin filaments, cells were treated for 10 min at 4 °c with a permeabilizing solution (hepes 20 mm, ph 7.4, 300 mm sucrose, 50 mm nacl, 3 mm mgcl2, 0.5 % triton x-100), then washed with pbs buffer and finally incubated 1 h at 37 °c with tetramethylrhodamine (tritc)-labeled phalloidin (sigma) (diluted 50 μg/ml) in pbs buffer. results morphological analysis of cultured botryoidal tissue cells cultured botryoidal tissue, extracted from untreated control leech, is formed by clustered cells mostly packed in cord or lining practically virtual spaces (figs 1a, b). these cells (de eguileor et al., 2001) belong to different categories due to their size and to the presence of large amount of granules in the cytoplasm: large roundish granular botryoidal and small flattened endothelial-like cells are easily recognizable (figs 1a, b). effects of vegf in vitro the vegf administration (figs 1c, d) is responsible of botryoidal tissue cells changes. a dehiscence process led the compact cell cords to gradually line new cavities. during early phases of angiogenesis (i.e., during the formation of prevascular lacunae), botryoidal cells acquired a semilunar shape (figs 1c, d) while small, flattened and elongated endothelial-like cells become visible interposed among botryoidal ones (figs 1c, d). unlike control, vegf stimulated endothelial-like cells showed bundles of cytoskeletal filaments (figs 1e, f). characterization of cultured botryoidal tissue cells cytoskeleton after vegf administration, botryoidal tissue cells, showing an elongated phenotype, displayed a weak expression of anti-beta actin that localize diffuse g-actin, and a strong tritc-conjugated phalloidin staining f-actin (i.e., detecting filament bundles) (figs 1g, h). in contrast vegf/anti-ras treated cells showed a high positivity for the betaalpha actin antibody while a low signal was detectable for f-actin staining (figs 1i, j). effect of ras61 on botryoidal tissue cells botryoidal tissue cells have been transformed both with gfp and ras61 (constitutively activated ras protein) in order to: a) define the effects of ras activation on botryoidal tissue phenotypes; b) to determine whether ras activation was sufficient to cause actin filaments polymerization, driving angiogenic responses without the synergistic vegf induction. botryoidal tissue cells co-expressing ras61 and gfp showed transformed phenotype (figs 2a-f). cells expressing only gfp maintained a cord shape (figs 2 a, b, e) while the ectopic expression of ras61 promoted in vitro the elongation and the formation of new vessels, (figs 2 c, d, f). the ectopic expression of ras61 in both gfp and gfp/ras61 co-transfected cells was evaluated by using an antibody anti-ras. in the control gfp transfected cells, ras was low expressed at the periphery of the botryoidal tissue cells organized in clusters (figs 2g-i). in contrast, in ras61 transformed cells, ras signalling was strong and it was mainly localized in the flattened endothelial cells lining the new lumen of vessel (figs 2j-l). paralleling, cells expressing only gfp showed a strong peripheral localization of g actin (fig. 3a), and a weak rhodamine-phalloidin positivity indicating that there were very few actin filaments (fig. 3b) as also ultrastructurally validated (fig. 3c). in gfp/ras61 co-transfected cells (figs 3d-f) the presence of f-actin (confirmed by staining with tritc-phalloidin), mainly located at level of endothelial like cells, was preponderant in respect to g-actin (confirmed by staining with anti-beta actin). the presence of filament bundles was verified by ultrastructural analysis (figs 3f). therefore, we assessed the role of ras signalling in endothelial cells cytoskeleton reorganization by inhibition of ras signalling in ras transformed cells. the effect of ras was abolished when an antibody anti-ras or the selective map kinase kinase 1 (mek1) inhibitor, pd 098059, were administrated in vitro (figs 4 a, b). a similar result was obtained adding to the culture medium the inhibitor of actin polymerization cytochalasin d (fig. 4c). all three different treatments inhibited the formation of f-actin, as shown by tritc-phalloidin staining (figs 4 d-f) blocking the consequent modification of the botryoidal tissue shape. discussion reorganization of the actin cytoskeleton, composed of actin filaments and many specialized actin-binding proteins (stossel, 1993; small, 1994; zigmond, 1996; takai et al., 2001), has a key role in many cellular functions such as cytokinesis, change of cell shape, cell motility and cell adhesion. the modulations of actin scaffold is due to the tightly regulated polymerization and depolymerisation of actin filaments and numerous studies have identified in gtpase ras intervention the clou event leading to remodelling of the actin cytoskeleton (joneson et al. 1996; serban et al., 2008). in vertebrates ras activation is sufficient to induce many of the requisite phenotypes for angiogenesis by inducing profound morphological changes of the vascular endothelium (xu, et al., 1998), however discordant opinions are about the importance in assigning the different factors involvement. in fact it is known that ras signalling, even if its mode of action is unclear, is capable of driving an angiogenic switch in the phenotype of primary endothelial cells in the absence of angiogenic factors (meadows et al., 2004). in addition some evidences have accumulated that both the classical extracellular activated kinase erk1/2 mitogen activated protein (map) kinase pathway and the phosphoinositide 3kinase (pi 3-kinase) pathway can contribute to alterations in the actin cytoskeleton (castro barros and marshall, 2005). to assess the role of ras/map/erk signalling on cells shape modification we used, as an in vitro approach, the botryoidal tissue of h. medicinalis. this tissue (made of endothelial and botryoidal cells) 11 fig. 3 a-f analysis of cytoskeleton in sectioned gfp and gfp/ras61 transformed cells. distribution of diffuse and polymerized actin in transformed cells is assessed respectively with an anti-beta actin antibody (a, b) and by staining with -phalloidin (d, e). gfp transformed cells contain more diffuse actin (red in a) than polymerized factin (red in b), as also showed by electron microscopy image (c). in gfp/ras61 tranformed cells a downregulation of g-actin isoform (red in d) and a strong signal for f-actin (red in e) is observed. bundles of actin filaments are visible at tem (arrowhead in f). the gfp marker indicates infected cells (green in a, b, d, e). lv: lacunae vessel; n: nucleus. bars in a, b, d, e: 10 μm; bar in c: 400 nm; bar in f: 200 nm. 12 can be considered an useful tool to evaluate ras effects on cell shape modification since it, during angiogenesis in response to vegf administration, undergoes to a radical remodelling leading to prevascular/vascular lumen formation characterized by marked cellular changes (i.e., by flattening, lengthening and stretching especially of endotheliallike cells (de eguileor et al., 2001b). an interesting outcome of our investigation showed that constitutively activated protein ras61 expression in stably transfected cells causes modification of actin cytoskeleton with vessels cavities formation also in total absence of the angiogenic factors vegf, whereas cells expressing only gfp show the typical solid cord structure of unstimulated tissue. moreover the angiogenic phenotype in both gfp/ras61 transformed cells and vegf stimulated cells was inhibited by using an antibody anti-ras. immunodetection and ultrastructural analysis of cytoskeleton alterations of both vegf stimulated and ras61-transformed botryoidal tissue cells show the presence of oriented bundles of polymerized filaments in all botryoidal tissue cells. these cells with an elongated and flattened shape are characterized by actin filament bundles and in their cytoplasm the presence of polymerized f-actin (rhodamine-phalloidin positivity) is predominant in respect to g actin (anti-beta globular actin positivity). these data are validated by treatment of gfp/ras61 co-transfected botryoidal tissue cells with cytochalasin d, an inhibitor of f-actin polymerization (goddette and frieden, 1987), leading to disappearance of actin bundles (decrease in fluorescence intensity of phalloidin). on the contrary, the diffuse distribution of g-actin, challenges the hypothesis of the relationship between down-regulation of actin polymerization and morphological changes of botryoidal tissue cells. we demonstrate also that map kinase activity is essential to the maintenance of the ras-induced phenotype, utilizing a potent inhibitor, the pd 098059 that reduces the basal map kinase activity in ras-transformed botryoidal cells that are unable to form vessels. summarizing: i) ras is the most involved factor in actin cytoskeleton reorganization of botryoidal tissue cells; ii) the cytoskeletal reorganization of botryoidal tissue cells lead to new vessel formation; iii) ras/map/erk signalling on actin filaments polymerization and their spatial arrangements are directly involved in the morphological modification of botryoidal tissue cells. fig. 4 a-f effect of anti-ras; pd 098059 and cytochalasin d on ras61 activated cells. a-c: semithin sections of ras-61 transformed botryoidal tissue cells. the anti-ras antibody (a), the map kinase kinase 1 (mek1) inhibitor pd09805 (b) and the inhibitor of actin polymerization cytochalasin d (c) inhibit ras induced vessel formation. df: cryosections of gfp/ras-61 co-transformed botryoidal tissue cells treated with anti-ras antibody (d), the map kinase kinase 1 (mek1) inhibitor pd09805 (e) and the inhibitor of actin polymerization cytochalasin d (f). a low signal for f-actin is detected with tritc-conjugated phalloidin (red in d-f). bars in a, b, d, e: 10 μm; bars in c, f: 5 μm. 13 acknowledgments this work was supported by fondi di ateneo per la ricerca (far) and centro grandi attrezzature (cga) of the university of insubria. references alessi dr, cuenda a, cohen p, dudley dt, saltiel ar. j. biol. chem. 270: 27489, 1995. castro barros j, marshall cj. activation of either erk1/2 or erk5 map kinase pathways can lead to disruption of the actin cytoskeleton. j. cell sci. 118: 1663-1671, 2005. carpenter g, the egf receptor: a nexus for trafficking and signaling, bioessays 22: 697707, 2000. de eguileor m, grimaldi a, tettamanti g, ferrarese r, congiu t, protasoni m, et al. hirudo medicinalis: a new model for testing activators and inhibitors of angiogenesis. angiogenesis 4: 299-312, 2001a. de eguileor m, grimaldi a, tettamanti g, congiu t, protasoni m, reguzzoni m, et al. ultrastructure and functional versatility of hyrudinean botryoidal tissue. tissue cell 33: 332-341, 2001b. de eguileor m, tettamanti g, grimaldi a, perletti g, congiu t, rinaldi l, et al. hirudo medicinalis: avascular tissues for clear-cut angiogenesis studies? curr. pharm. des. 10: 1979-1988, 2004. ehrhardt a, ehrhardt gr, guo x, schrader jw. ras and relatives--job sharing and networking keep an old family together. exp. hematol. 30: 10891106, 2002. fox pl, sa g, dobrowolski sf, stacey dw. the regulation of endothelial cell motility by p21 ras.oncogene 9: 3519-3526, 1994. goddette d. w.; frieden c. actin polymerization the mechanism of action of cytochalasin d. j. biol. chem. 261: 15974-15980, 1987. grimaldi a, tettamanti g, perletti g, valvassori r, de eguileor m. hematopoietic cell formation in leech wound healing. curr. pharm. des. 12: 3033-3041, 2006. joneson t, white ma, wigler mh, bar-sagi d. stimulation of membrane ruffling and map kinase activation by distinct effectors of ras. science 271: 810-812, 1996. kranenburg o, moolenaar wh. ras-map kinase signaling by lysophosphatidic acid and other g protein-coupled receptor agonists, oncogene 20: 1540-1546, 2001. kranenburg o, mfbg gebbink, ee voest. stimulation of angiogenesis by ras proteins. biochim. biophys. acta 1654: 23-37, 2004. leblanc v, tocque b, delumeau i. ras-gap controls rho-mediated cytoskeletal reorganization through its sh3 domain. mol. cell. biol. vol. 18, 9: 5567-5578, 1998. meadows kn, bryant p, pumiglia k. vascular endothelial growth factor induction of the angiogenic phenotype requires ras activation. j. biol. chem. 276: 49289-49298, 2001. meadows kn, bryant p, vincent pa, pumiglia km. activated ras induces a proangiogenic phenotype in primary endothelial cells. oncogene 23: 192-200, 2004. moore rd, mumaw v, shomberg md. optical microscopy of ultrathin tissue sections. j. ultrastruct. res. 4: 113-116, 1960. sawyer rt. leech biology and behaviour: anatomy, physiology and behaviour, oxford science publications, oxford, 1986. serban s, leng j, cheresh d. h-ras regulates angiogenesis and vascular permeability by activation of distinct downstream effectors. circ. res. 102:1350-1358, 2008. small l jv. lamellipodia architecture: actin filament turnover and the lateral flow of actin filaments during motility. semin. cell biol. 5: 157–163, 1994. sosnowski rg, feldman s, feramisco jr. interference with endogenous ras function inhibits cellular responses to wounding. j. cell biol. 121: 113-119, 1993. stossel tp. on the crawling of animal cells. science 260: 1086-1094, 1993. takai y, sasaki t, matozaki t. small gtp-binding proteins. physiol. rev. 81: 154-208, 2001. tettamanti g, grimaldi a, ferrarese r, palazzi m, perletti g, valvassori r, et al. leech responses to tissue transplantation. tissue cell 35: 199212, 2003a. tettamanti g, grimaldi a, valvassori r, rinaldi l, de eguileor m. vascular endothelial growth factor is involved in neoangiogenesis in hirudo medicinalis (annelida, hirudinea). cytokine 22: 168-179, 2003b. tettamanti g, malagoli d, benelli r, albini a, grimaldi a, perletti g, et al. growth factors and chemokines: a comparative functional approach between invertebrates and vertebrates. curr. med. chem. 13: 2737-2750, 2006. weyman cm, ramocki mb, taparowsky ej, wolfman a. distinct signaling pathways regulate transformation and inhibition of skeletal muscle differentiation by oncogenic ras. oncogene 6: 697-704, 1997. xu xs, vanderziel c, bennett cf, monia bp. a role for c-raf kinase and ha-ras in cytokinemediated induction of cell adhesion molecules. j. biol. chem. vol. 273, 50: 33230-33238, 1998. zigmond sh. signal transduction and actin filament organization. curr. opin. cell biol. 8: 66-73, 1996. 14 visions and perspectives isj 8: 1-4, 2011 issn 1824-307x visions and perspectives immunocyte: the invertebrate counterpart of the vertebrate macrophage e ottaviani department of biology, university of modena and reggio emilia, modena, italy accepted december 15, 2010 abstract the circulating phagocytic immune cell is considered to be the main effector of the invertebrate defense system, involved in both immune and neuroendocrine responses, showing the functional characteristics of vertebrate macrophage. various names have been used to define this cell in different taxa i.e., hemocyte, celomocyte, amebocyte, plasmatocyte, etc. however, regardless of the terminology, these cells perform the same immune function, and possess very similar morphology. for these reasons, it is suggested that the general term immunocyte be used to describe these cells in invertebrates. key words: immunocyte; invertebrates; immune system introduction elie metchnikoff was the first to suggest an evolutionary mechanism devoted to protecting organisms. he demonstrated the phagocytic role of certain cells of the freshwater crustacean daphnia magna infected by the parasitic fungus monospora bicuspidata (metchnikoff, 1884), and described in vertebrates two phagocytic cell types, microphages (polymorphonuclear leucocytes) and the mononuclear macrophages (metchnikoff, 1901). the discovery of phagocytic cells in humans has diverted attention of the cellular mechanisms present in invertebrates to those found in vertebrates. however, in the last few decades several papers and reviews have been dedicated to phagocytic cells in invertebrates. different names have been used to describe invertebrate immune cells that exhibit phagocytic activity. regrettably, this has promoted more confusion than clarification of cell function. there currently is no information on the homology of phagocytic cells in different taxa, and the use of different names for cells that perform the same activities does not enhance understanding among investigators. the present contribution attempts to promote the acceptance and usage of the general name immunocyte to the specific cells in different invertebrates that perform phagocytic activity. such acceptance would hopefully benefit especially those researches that report cross-taxa information. ___________________________________________________________________________ corresponding author: enzo ottaviani department of biology university of modena and reggio emilia via campi 213/d, 41125 modena, italy e-mail: enzo.ottaviani@unimore.it anatomycal background of the immune system in non-vertebrate models the presence of a body cavity can be used as a phylogenic parameter for classifying different groups of metazoans. acelomate animals lack a body cavity (barrington, 1967; barnes et al., 1988). pseudocelomates have a pseudocel, a cavity lacking in own walls arising directly from the blastocelic cavity filled of fluid. celomates that have a true cavity, called celom, that develops within the endomesoderm, covered on its outer surface by the somatic mesoderm and on inner surface by the splanchnic mesoderm. the body cavity of celomates is filled of fluid and is lined by an epithelium. an open circulatory system is present in molluscs and arthropods, in which a fluid is contained in the dominant body cavity or hemocel. the fluid, usually called hemolymph, can bathe the organs directly and there is no distinction between blood and interstitial fluid. conversely, annelids, cephalopods and echinoderms possess a closed system characterized by the presence of blood vessels (brusca and brusca, 2003). it should be noted that different circulatory systems are not linked to the taxon, consequently they are not good parameters from a phylogenetic point of view. currently, the relationships among circulating cells and open or closed circulatory systems are unknown. invertebrate immune cells it is well-documented that the immune cells represent the cellular component of invertebrate immune systems (ratcliffe and rowley, 1981). 1 however, there is a general problem in defining the number of circulating immune cells present in the hemolymph, which represents a subject of great debate. there are several reasons for this unsolved problem, including different methods employed in examining invertebrate immune cells, the lacking of an hemopoietic organ in numerous models, and the inability to document cellular maturation. in this context, an initial morphological examination of adult mytilus galloprovincialis revealed the presence of two cell types in the circulating hemolymph. however, more detailed studies such as functional tests, cytochemical and enzymatic reactions and cytofluorimetric analysis revealed the presence of a single cell type in two different stages, both of which possessed phagocytic activity (ottaviani et al., 1998). using light and electronic microscopy two cell types have been described in m. edulis and m. galloprovincialis (rasmussen et al., 1985; renwrantz, 1990; noël et al., 1993; cajaraville and pal, 1995). however, using monoclonal antibodies, sub-populations of a single cell type were identified in m. edulis (dyrynda et al., 1997). in synthesizing the different interpretations of circulating immune cells in bivalves, cheng (1981) concluded that only two cell types are present: hyalinocytes and granulocyes. conversely, mix (1976) suggested that hyalinocytes are an intermediate proliferative stage that matures to become granulocytes. another notable example is the classification of immune cells in insects. rowley and ratcliffe (1981) reported the presence of the following cell types in the hemolymph: prohemocytes, plasmatocytes, granular cells, cystocytes, spherule cells and oenocytoids. while brehélin and zachary (1986) proposed another classification of insect blood cells where nine cell types are described: prohemocytes, plasmatocytes, oenocytoids, spherule cells, thrombocytoids and four types of granular hemocytes. three basic cell types are observed in the hemolymph of adult insect calliphora vomitoria: prohemocytes, plasmatocytes and granular cells (franchini et al., 1996). in drosophila melanogaster plasmatocytes, lamellocytes and crystal cells have been described as cells derived from progenitors originated in the larval lymph gland (nappi et al., 2004). as previously noted, the lack of an hematopoietic organ presents a problem in classifying blood cell types. however, even when this organ is present problems occur. indeed the different species of planorbids and lymnaea palustris (kinoti, 1971; lie et al., 1975; rachford, 1976; jeong et al., 1983; ottaviani, 1988) in which a hemopoietic organ is present, it is insufficient to produce the quantity of immune cells that need to the animals, and the majority of the cells comes from the hemolymph. in annelids, echinoderms and tunicates, a terminology for immune cells includes celomocytes, amebocytes among others (smith et al., 2006; ballarin, 2008; lefebvre et al., 2008; arizza and parrinello, 2009; vetvicka and sima, 2009). in the leech theromyzon tessulatum three distinct celomic cell populations are reported: the chloragocytes which were initially defined as large celomocytes, the granular amebocytes and small celomic cells (lefebvre et al., 2008). in general, the various annelid cell types are usually characterized as amebocytes, elocytes, erythrocytes and hemocytes in polychaeta, celomocytes, amebocytes, vascular lymphocytes, elocytes and macrophages in oligochaeta and amebocytes and chloragocytes in hirudinea (vetvicka and sima, 2009). in the sea urchin strongylocentrotus purpuratus are described the following celomocyte types: type 1 (discoidal phagocyte), type 2 (polygonal phagocyte), small phagocyte, red spherule cell, colorless spherule cell and vibratile cell (smith et al., 2006). in colonial botryllid ascidians and in particular in the species botryllus schlosseri the circulating immune cells are grouped into three main categories: undifferentiated cells, immunocytes, and storage cells (pigment cells and nephrocytes). immunocytes are represented by cytotoxic morula cells and phagocytes, the latter including hyaline amebocytes and macrophage-like cells (ballarin, 2008). in the solitary ascidian ciona intestinalis various cell types are reported: agranular hemocytes, including hemoblasts, circulating lymphocyte-like cells, hyaline amebocytes; granular hemocytes including granulocytes with small granules, granulocytes with large granules, unilocular refractile granulocytes and morula cells (arizza and parrinello, 2009). invertebrate phagocytic immune cells as noted above, in different taxa numerous cell types are described and various functions are assigned. although these cells play a fundamental role in immunity, they unfortunately are designated with different names, and no information is available on their homology. in the mollusc planorbarius corneus the phagocytic cell has been called spreading hemocyte (ottaviani, 1983; ottaviani and franchini, 1988). these cells show the same function of the spreading amebocytes of lymnaea stagnalis (stang-voss, 1970; sminia, 1972), of the granulocytes of bulinus guernei (krupa et al., 1977) and the granulocytes of biomphalaria glabrata (harris, 1975; joky et al., 1983). in various insects, the plasmatocytes represent cells with the typical functions of a macrophage, i.e., glass adhesion with the emission of pseudopodia allowing amoeboid movement, phagocytic capacity, encapsulation, nodule formation and wound repair (rowley and ratcliffe, 1981; brehélin and zachary, 1986; franchini et al., 1996). however, in lepidoptera granulocytes are considered the phagocytic cells, whereas plasmatocytes are larger and adhesive cells that cooperate in encapsulation (nakatogawa et al., 2009) the ameboid celomocytes are very important cells of the immune system of annelids. they are involved in non-self recognition, transplantation reaction, cytotoxicity, encapsulation, endocytosis and enzymatic digestion of engulfed material. in 2 addition, they actively participate in regenerative processes and wound healing (vetvicka and sima, 2009). smith et al. (2006) in their interesting review report that in echinoderms the phagocytes represent the majority of celomocytes involved in graft rejection, chemotaxis, phagocytosis, encapsulation, immune gene expression, agglutination and clotting reactions. in b. schlosseri, the circulating professional phagocytes are considered hyaline amebocytes and macrophage-like cells, which represent two diverse morphologies of the same hemocyte type (sabbadin, 1955; ballarin et al., 1993). also in c. intestinalis hyaline amebocytes are the most common cell type with phagocytic activity (arizza and parrinello, 2009). in conclusion, on the basis of the reported observations, i suggest, from a conceptual point of view, to adopt the general term of immunocyte for the invertebrate cells endowed of characteristic functions of vertebrate macrophage. in this way one immediately understands the kind of cell described, regardless of the taxonomic group involved. acknowledgements the author wish to thank dr aj nappi, emeritus professor (department of biology, loyola university, ca, usa) for the critical reading. references arizza v, parrinello d. inflammatory hemocytes in ciona intestinalis innate immune response. inv. surv. j. 6: s58-s66, 2009. ballarin l, cima f, sabbadin a. histoenzymatic staining and characterization of the colonial ascidian botryllus schlosseri hemocytes. boll. zool. 60: 19-24, 1993. ballarin l. immunobiology of compound ascidians, with particular reference to botryllus schlosseri: state of art. inv. surv. j. 5: 54-74, 2008. barnes, rsk, calow, p, olive, pjw, golding, dw. the invertebrates: a new synthesis. blackell scientific publications, oxford, 1988. barrington, ejw. invertebrate structure and function. thomas nelson and sons ltd, australia, 1967. brusca rc, brusca gj. invertebrates. 2nd ed. sinauer associates, inc., publishers, sunderland, ma, usa, 2003. brehélin m, zachary d. insect haemocytes: a new classification to rule out the controversy. in: brehélin m (ed), 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(pulmonata). z. zellforsch. 107: 142-156, 1970. vetvicka v, sima p. origins and functions of annelide immune cells: the concise survey. inv. surv. j. 6: 138-143, 2009. 4 invertebrate immune cells invertebrate phagocytic immune cells research report isj 11: 257-272, 2014 issn 1824-307x research report influence of sericin in alleviating the hydrogen peroxide induced oxidative stress in silkworm bombyx mori: role of the amino acids as micheal, m subramanyam department of life science, bangalore university, bangalore 560 056, india accepted september 15, 2014 abstract sericin is an important peptide derived from silk fibre spun by the silkworm bombyx mori and has various biological activities. the aim of the present study was to characterize the major constituents of sericin that are providing cytoprotective effect against hydrogen peroxide-induced cell damage in midgut epithelial cells and hemocytes of silkworm. extracted sericin was subjected to lcms analysis for amino acid composition. isolated cells of midgut and hemocytes were incubated with sericin or with mixture of serine and aspartic acid prior to suboptimal concentration of hydrogen peroxide treatment. sericin as well as amino acid mixture reduced the activity of antioxidant enzymes triggered by hydrogen peroxide, inhibited oxidative derivatives such as protein carbonyl and malondialdehyde and increased antioxidant capacity in both the cells studied. furthermore, sericin and amino acid mixture significantly decreased intracellular reactive oxygen species as assessed by fluorescent detection. these results suggest that major constituent amino acids of sericin defend midgut epithelial cells and hemocytes against oxidative damage by scavenging reactive oxygen species rather than activating antioxidant enzyme system thereby inhibiting cell damage. key words: amino acids; antioxidant enzymes; bombyx mori; oxidative stress; reactive oxygen species; sericin   introduction reactive oxygen species (ros) are produced during normal cellular metabolism or are derived from exogenous sources, and play an important role in cellular homeostasis. exogenous sources, including prooxidant allelochemicals, severely affect herbivorous insects during host interactions and stress such as starvation may contribute to endogenous sources of ros accumulation. unchecked or increased levels of ros can cause severe damage to various cellular compartments, including dna, protein and lipids, thereby causing oxidative injury. oxidative injury to the cell membrane results in structural changes and increased permeability to ions and fluids (nagasaka et al., 2004). nonetheless, insects have evolved a complex antioxidant mechanism to overcome the toxic effects of ros (krisnhnan and kodrik, 2006). antioxidant defence is primarily contributed by antioxidant enzymes (aoes) such as peroxidase (pox, e.c. 1.11.1.7), superoxide dismutase (sod, e.c. 1.15.1.1) and catalase (cat, e.c. 1. 11. 1.6) ___________________________________________________________________________ corresponding author: muthangi subramanyam department of life science bangalore university bangalore 560 056, india e-mail: asuba@vsnl.net (felton and summer, 1995). glutathione peroxidase (gpx) reduces h2o2 and hydroperoxides, thereby scavenging oxidative radicals in cell membranes, whereas sod converts o2 to molecular o2 and h2o2 (maiorino et al., 2003; imlay, 2008). h2o2 is subsequently scavenged by cat, resulting in the production of water and molecular oxygen (kashiwagi et al., 1997). several naturally occurring macromolecules (balsano and alisi, 2009) and amino acids have been found to be effective as antioxidants (wu et al., 2003a; atmaca, 2004; liu et al., 2004; movahedian et al., 2006; selvaraju and subbashinidevi, 2011). among the macromolecular proteins that combat oxidative stress (elisa et al., 2008; medina-navarro et al., 2010), the silk protein sericin role as an antioxidant is commendable because of the presence of polyphenols and flavanoids (devi et al., 2011; prasong, 2011). sericin is an active biomolecule with several implications as a therapeutic agent (khudaiderdier, 1997; aramwit and sangcakul, 2007; li et al., 2008) and component of cosmetics (padamwar and pawar 2004; patel and modasiya, 2011). silk protein filament has two protein fractions, fibroin, a fibrous component and glue-like sericin that holds the fibroin components together and often discarded as waste product in silk industry. sericin exhibits 257   mailto:asuba@vsnl.net fig. 1 (a) lcms spectra of sericin showing prominent amino acids. (b) spectra of the major amino acids serine (a), aspartic acid (b), glycine (c), threonine (d) and glutamic acid (e). the x-axis is retention time and the y-axis is intensity in cps. (c) prominent amino acid composition of sericin protein in b. mori. several pharmacological effects, such as enhancing digestion and cryoprotection. dietary sericin reduces lipids and ameliorates glucose tolerance in rats fed a high fat diet and also acts as an anticoagulant upon sulphonation (kato et al., 1998; sasaki et al., 2000; tsujimoto et al., 2001; okazaki et al., 2010). sericin is specifically synthesized in the middle silk gland of the silkworm b. mori. it is a polypeptide with 18 amino acids, most of which have strong polar side groups such as hydroxyl, carbonyl and amino groups (wu et al., 2007) and is especially rich in serine (~32 %) (kwang et al., 2003). in general, amino acids found in proteins have the potential to interact with free radicals if the energy of the radicals insult is high (elias et al., 2008) and antioxidant activity of proteins in radical mediated oxidation reactions may be due to their ability to act as radical trapping devices (ostdal et al., 2002). the silkworm bombyx mori is a monophagus insect known worldwide because of the lustrous silk it produces in the final instar of the larval stage. one of the reason for the decline in silk production and the survivability of highly domesticated mulberry silkworm is due to food contaminated with pesticides (vyjayanthi and subramanyam, 2002a, b). in an earlier study, we demonstrated a transient increase in antioxidant defence mechanisms in silkworms stressed with short-term exposure to low temperature, hypoxia and viral infection (micheal and subramanyam, 2013). the purpose of the present study was to assess the effect of sericin on 258   fig. 2 malondialdehyde levels in silkworm b. mori midgut epithelial cells (a) and hemocytes (b) treated with hydrogen peroxide and pre-incubated with sericin or amino acid mixture prior to hydrogen peroxide treatment. data are shown as mean ± se (n = 6); p < 0.05 was considered significant. values between the treatments are represented by lower case letters (a, b) and between the instars are represented by upper case letters (a, b). those not sharing the same letters are significant. hydrogen peroxide-induced oxidative stress in the midgut epithelial cells and hemocytes of silkworm and to explore the possible components of antioxidant activity that alleviate oxidative injury caused by transient exposure to hydrogen peroxide. we have demonstrated that the major amino acids of sericin acts as scavenger of radical oxygen species in hydrogen peroxide induced oxidative stress. materials and methods chemicals thiobarbituric acid (tba), glutathione reductase, horseradish peroxidase and dinitrophenylhydrazine (dnph) were purchased from sigma-aldrich, (st. louis, mo, usa). h2dcfda was purchased from molecular probes (eugene, usa). reduced glutathione (gsh), nadph, t-butylhydroperoxide, 259   fig. 3 protein carbonyl levels in silkworm b. mori midgut epithelial cells (a) and hemocytes (b) treated with hydrogen peroxide and pre-incubated with sericin or amino acid mixture prior to hydrogen peroxide treatment. data are shown as mean ± se (n = 6); p < 0.05 was considered significant. values between the treatments are represented by lower case letters (a, b) between the instars are represented by upper case letters (a, b). those not sharing the same letters are significant. hydrogen peroxide (h2o2), triton x-100, epinephrine, sodium dodecyl sulphate (sds), acetic acid, butanol, pyridine, tetra methoxy propane (tmp) and 2,4,6-tripyridyl-s-triazine (tptz) were purchased from sisco research laboratory (mumbai, india). l-aspartic acid and lserine from spectochem (mumbai, india). insects and experimental design the present study was approved by the institutional animal ethics committee (iaec) of bangalore university, bangalore, india. second instar larvae were procured from the kunigal seed area, karnataka, india and were maintained in the laboratory throughout the larval stages and were fed 260   fig. 4 sod activity in midgut epithelial cells (a) and hemocytes (b) of iv and v instar of the silkworm b. mori treated with hydrogen peroxide and pre-incubated with sericin or amino acid mixture prior to hydrogen peroxide treatment. data are shown as mean ± se (n = 6); p < 0.05 was considered significant. values between the treatments are represented by lower case letters (a, b) between the instars are represented by upper case letters (a, b). those not sharing the same letters are significant. ad libitum with m5 variety mulberry leaves (vyjayanthi and subramanyam, 2002a, b). the uniformly grown healthy larvae of iv and v instars were used in all experiments and were maintained at 24 25 oc with a relative humidity of 70 75 %. midgut epithelial cells were isolated by micro dissection and 1 % collagenase treatment. dissociated cells were washed repeatedly with cold phosphate buffer and were allowed to stand in cold phosphate buffer of ph 7.4 for further usage. hemolymph was collected in pre-cooled 2 ml vials containing 5 mg thiourea by gentle incision on the caudal horn of the larvae. hemocytes were separated by centrifuging the diluted hemolymph at 3,000 rpm for 10 min in cold. cold phosphate buffer of ph 7.4 was used for the tissue homogenate preparation and for the separation of hemocytes. isolated cells were exposed to h2o2 (20 μm) for 10 min to induce oxidative stress. to evaluate the antioxidant nature of sericin, isolated cells were incubated with 28 mg/ml sericin for 10 min at rt prior to treatment with h2o2. the isolated cells were 261   fig. 5 cat activity in midgut epithelial cells (a) and hemocytes (b) of iv and v instar of the silkworm b. mori treated with hydrogen peroxide and pre-incubated with sericin or amino acid mixture prior to hydrogen peroxide treatment. data are shown as mean ± se (n = 6); p < 0.05 was considered significant. values between the treatments are represented by lower case letters (a, b) between the instars are represented by upper case letters (a, b). those not sharing the same letters are significant. also incubated with 0.5 mm each of l-serine and laspartic acid for 10 min at room temperature prior to treatment with h2o2 to access the role of amino acids as antioxidants. extraction of sericin sericin was extracted from silk cocoon according to (wu et al., 2007) with slight modification. briefly, multi-voltine cocoons were boiled for several hours in distilled water to extract sericin. the water extract was condensed further by evaporating the water at 50 ºc, and the concentrate was spray dried and collected as a powder. the powder was dissolved in distilled water in a 10:1 ratio (w/v), and was chilled overnight at 4 ºc. pure chilled ethanol was added to the sericin solution with constant stirring to obtain a final ethanol concentration of 75 % (w/v). the obtained mixture was then kept at -20 ºc overnight, 262   fig. 6 gpx-like activity in midgut epithelial cells (a) and hemocytes (b) of iv and v instar of the silkworm b. mori treated with hydrogen peroxide and pre-incubated with sericin or amino acid mixture prior to hydrogen peroxide treatment. data are shown as mean ± se (n = 6); p < 0.05 was considered significant. values between the treatments are represented by lower case letters (a, b) between the instars are represented by upper case letters (a, b). those not sharing the same letters are significant. followed by centrifugation at 3500 rpm for 20 min. alcohol evaporation was performed at 40 ºc and the samples were lyophilized and stored until use. lc-ms analysis of sericin protein lc-ms analysis was performed using an api 3000 lc-ms system fitted with a turbo ion spray source and a quadrupole mass spectrometer (perkin elmer sciex, thornton, canada). the instrument was operated in positive ion mode with a spray voltage of 5500 v and a source temperature of 475 ºc using a phenomenex column (2540x6.6 mm) with methanol:water (3:1) as the mobile phase. data were analysed with analyst software version 1.4.2. lipid peroxidation level malondialdehyde (mda), a product of lpo, was determined as described by ohkawa et al. (1979). in brief, 200 µl of cell homogenates were added to 8.1 % sds, vortexed and incubated for 10 min. 375 µl of 20 % acetic acid and 0.6 % thiobarbituric acid 263   were added to the reaction mixture and placed in a boiling water bath for 60 min. the samples were allowed to cool and 1.25 ml of a butanol:pyridine mixture (15:1, v/v) was added and centrifuged at 640 g for 5 min. absorbance was measured at 532 nm using 1,3,3-tetramethoxy propane (tmp) as the standard. the mda concentration was expressed as nmol/mg protein. protein carbonyl level protein carbonyl (prc) was measured according to the method of uchida and stadtman (1993). 0.1 % dnph in 2 n hcl was added to 800 µl of cell homogenate. samples were kept in the dark for 1 h. the protein was precipitated with 20 % trichloroacetic acid and centrifuged. the pellets were washed thrice with ethanol and ethyl acetate (1:1, v/v) and were dissolved in 2 ml of 8 m guanidine hydrochloride, and centrifuged. the supernatant was used to measure the absorbance at 365 nm and the prc level was calculated using a molar absorption coefficient of 22,000 m-1 cm-1. the results were expressed as µm/mg protein. superoxide dismutase (sod, e.c. 1.15.1.1) activity sod activity was measured according to misra and fridovich (1972) with slight modifications. briefly, 100 µl of a 5 % cell homogenate was added to 880 µl of carbonate buffer (0.5 m, ph 10.2). 20 µl of epinephrine (30 mm in 0.05 % acetic acid) were added to the mixture and measured spectrophotometrically (model: genova mk3, jenway, uk) at 480 nm for 4 min. sod activity was measured as the amount of enzyme that inhibits oxidation of epinephrine by 50 %, which is equal to 1 unit. catalase (cat, e.c. 1.11.1.6) activity catalase was determined by the method of aebi (1984). briefly, 100 µl enzyme samples with 10 µl of absolute alcohol were incubated for 30 min at 0 oc followed by the addition of 10 µl triton x-100. an aliquot of 50 µl was placed in 1.25 ml of 0.066 m h2o2 in phosphate buffer and the decrease in absorbance was measured at 240 nm for 60 s in a spectrophotometer. an extinction coefficient of 43.6 m cm-1 was used to determine enzyme activity and was expressed as one µmole of h2o2 degraded/min/mg protein. glutathione peroxidase-like enzyme (gpx, e. c. 1.11.1.9) activity gpx-like enzyme activity was analysed by the method of flohe and gunzler (1984). 50 µl of 0.1 m phosphate buffer (ph 7.0), 100 µl of the enzyme sample, 100µl glutathione reductase (0.24 units) and 100 µl of 10 mm gsh were mixed. the mixture was pre incubated for 10 min at 37 oc followed by the addition of 100 µl of 1.5 mm nadph in 0.1 % nahco3. 50 µl of 12 mm t-butylhydroperoxide was added to monitor the hydrogen peroxide independent concentration of nadph for 3 min. the overall reaction was started by adding 100 µl of prewarmed h2o2, and the decrease in absorption at 340 nm was monitored for 5 min. gpx-like activity was expressed as µm nadph oxidized/min/mg protein. antioxidant capacity antioxidant capacity was analysed by the modified method of benzie and strain (1996) using ascorbic acid as the standard. frap reagent was prepared fresh from acetate buffer (ph 3.6). 10 mm tptz diluted with 40 mm hcl and 20 mm ferric chloride solution at a ratio of 10:1:1 (v/v), respectively, were warmed to 37 c prior to use. 100 µl of cell homogenate and 3 ml of the frap reagent were vortexed and absorbance was measured at 593 nm at 0 min. the samples were incubated at 37 c and absorbance was recorded after 30 min. the antioxidant capacity of the cell homogenate was expressed in µm fe /100 mg cell mass. o o 2+ fluorescent microscopy studies to assess the intracellular ros levels in isolated midgut epithelial cells and hemocytes, the cells were loaded with an ros-sensitive indicator, cm-h2dcfda of 20 µm for 10 min at rt. the excess stain was washed off with insect ringer’s solution. in cells, esterase cleaves cm-h2dcfda to release cm-h2dcfh, which is converted to the fluorescent product cm-h2dcf when exposed to ros (xie et al., 1999). cm-h2dcfda was excited at 520 nm and the emitted light was collected at 570 nm using a fluorescent microscope (olympus ix 71, japan). ros was quantified using image pro express version 6.3 software from stored images. statistical analysis data are shown as the mean ± sd of six observations. changes between the groups were analysed by manova and further tested by the bonferroni post-hoc test using statistical package for social sciences (spss) software (huberty and olejnik, 2006) and p < 0.05 was considered significant. statistically significant data are presented in the text. results composition of silk protein sericin a flow rate of 0.3 ml/min through the phenomenex column was used in the lc-ms studies to provide information on the retention times and the endogenous concentrations of various amino acids in the silk protein sericin. the retention times from the samples were compared with synthetic standards, as shown in figures 1a and b. among the amino acids, serine was the most abundant, followed by glycine, aspartic acid, threonine and glutamic acid (fig. 1c). oxidative stress markers incubation with 20 µm hydrogen peroxide significantly increased mda, a product of lipid peroxidation, in both midgut epithelial cells and hemocytes irrespective of the instars studied. however, the mda level was significantly higher in iv instar larvae when compared to v instar larvae. incubation with sericin or a combination of serine and aspartic acid for 10 min prior to hydrogen peroxide treatment inhibited hydrogen peroxideinduced lipid peroxidation in both midgut epithelial cells and hemocytes (figs 2a, b).a significant increase in protein carbonyl content (a product of 264   table 1 antioxidant capacity of sericin and the effect of sericin and amino acid mixture to induce antioxidant capacity in midgut epithelial cells and hemocytes of iv and v instar silkworm b. mori treated with hydrogen peroxide midgut epithelial cells (µm fe / 100mg cell mass)2+ hemocytes (µm fe / 100mg cell mass)2+nature of the treatment iv v iv v control 13.6 a ±0.4 11.8a ±0.9 15.2a ±0.3 14.9a ±0.3 incubation with h2o2 8.6a ±0.6 7.2a ±0.5 14.5a ±0.4 12.7a ±0.5 incubation with sericin prior to h2o2 23.8b ±0.4 17.2b ±0.7 25.3b ±0.4 28.8b ±0.4 incubation with amino acids prior to h2o2 36.5 b ±0.9 17.4 b ±0.8 24.8 b ±0.8 23.3 b ±0.9 frap value of sericin 35.8 c (µm fe / 100mg sample)2+ ±0.2 data are means ± se (n = 6); p < 0.05 was considered significant. values between the treatments are represented in lower cases (a, b, c). those not sharing the same letters are significant. protein oxidation), as a result oxidative stress, was evident following treatment with hydrogen peroxide in both in midgut epithelial cells and hemocytes (figs 3a, b). prior incubation with sericin and amino acid mixture significantly inhibited protein oxidation in both types of cells. antioxidant enzymes incubation of isolated midgut epithelial cells and hemocytes with 20 µm hydrogen peroxide significantly increased sod activity in both instars, but more so in iv instar over v instar silkworms irrespective of the treatment. incubation with sericin and serine, aspartic acid mixture prior to hydrogen peroxide treatment inhibited the hydrogen peroxideinduced increase in enzymatic activity. sod activity in midgut epithelial cells was significantly higher than in hemocytes per assay (figs 4a, b). catalase hydrolyses hydrogen peroxide into h2o and o2. to assess the role of sericin as a probable antioxidant, isolated midgut epithelial cells and hemocytes were incubated with 20 µm hydrogen peroxide with or without prior treatment with sericin. cat activity was found to be significantly higher in iv instar larvae than in v instar larvae of both tissues studied. incubation with hydrogen peroxide significantly increased cat activity in midgut epithelial cells as well as in hemocytes. cells treated with sericin or mixture of aspartic acid and serine before the induction of oxidative stress did not elicit a rise in cat activity under stress (figs 5a, b). a significant increase in gpx-like activity was noted in the context of oxidative stress in both midgut epithelial cells and hemocytes. prior treatment with sericin or the mixture of aspartic acid and serine prevented the oxidative stress-induced increase in enzymatic activity in both tissues (figs 6a, b). the instar-dependent decrease in antioxidant enzymes such as sod and cat was not observed for the gpx-like enzyme. antioxidant capacity of silk protein and a mixture of serine and aspartic acid the antioxidant capacity of the silk protein sericin was determined by a frap assay using ascorbic acid as the standard. the residual antioxidant capacity was found to be 13.6 and 11.8 µm fe /100 mg cell mass2+ in midgut epithelial cells, while in hemocytes it was found to be 14.5 and 14.9 µm fe /100 mg cell mass2+ in iv and v instar silkworms, respectively. cells incubated with sericin and also with combination of serine/aspartic acid prior to hydrogen peroxide treatment showed significantly increased antioxidant capacity by 0.72 fold and 0.45 fold in midgut epithelial cells, while in hemocytes the increase was 0.74 fold and 1.6 fold in iv and v instar silkworms, respectively (table 1). hydrogen peroxide-induced increase in reactive oxygen species h2dcfda staining was used to study the h2o2induced change in reactive oxygen species in midgut epithelial cells and hemocytes under a fluorescent microscope. under phase contrast, blebbing of the plasma membrane and swelling of the cells were observed in cells treated with h2o2 at concentrations above 20 µm. in contrast, pre incubation with 28 ng/ml sericin or with mixture of 0,5 mm serine and aspartic acid inhibited h2o2induced morphological changes in both types of cells studied (figs 7, 8). h2dcfda stained cells revealed a higher intensity of fluorescence, indicating an increase in ros upon exposure to 20 µm h2o2 (supplementary figs s1, s2). 265   fig. 7 effect of sericin and amino acid mixture on the hydrogen peroxide-induced increase in reactive oxygen species in the midgut epithelial cells of the silkworm b. mori. (a, b) normal midgut epithelial cells, (c, d) hydrogen peroxide treated cells, (e, f) cells incubated with sericin prior to hydrogen peroxide treatment and (g, h) cells incubated with amino acid mixture prior to hydrogen peroxide treatment. discussion hydrogen peroxide is membrane permeable, diffusible, less reactive and longer-lived than oh.or o2 .(stone and yang, 2006). the physiological range of the intracellular hydrogen peroxide concentration appears to be remarkably conserved in different forms of life (meller, 2000). notably, among the ros, hydrogen peroxide is the only species that is generated by several specific enzymes, which suggests that the intracellular concentration of hydrogen peroxide is tightly regulated and may serve several specific cellular functions. it potentiates antioxidant mechanisms; however, if produced in excess, it has deleterious consequences (veal et al., 2007). it has been reported to be less effective as an intracellular signalling molecule when added exogenously than endogenously produced hydrogen peroxide (choi et al., 2005). in the present study, 20 µm hydrogen peroxide, when applied exogenously, caused close to 50 % mortality in midgut epithelial cells and hemocytes with a concomitant increase in ros, as reflected in the fluorescent microscopy study. hydrogen peroxide can cause oxidative modifications in proteins (stadtman, 1992; dalledonne et al., 2002) and peroxidise unsaturated lipids in the cell membrane (fridovich, 1978). in our experiments, exposure of midgut epithelial cells and hemocytes to 20 µm hydrogen peroxide significantly increased the level of lipid peroxidation products, i.e., mda; prior treatment with sericin and with a mixture of amino acids was found to inhibit hydrogen peroxide-mediated effects in the cell membrane. sericin was found to suppress lipid peroxidation in rat brain homogenates (kato et al., 1998) and several amino acids have the property of reducing the lipid peroxidation (movahedian et al., 2006; selvaraju and subbashinidevi, 2011). in our present study a mixture of serine and aspartic acid have similar effect of reducing the lipid peroxidation in midgut epithelial cells and hemocytes of silkworm. however, there has been no evidence of a role of sericin as an inhibitor of protein oxidation, as protein thiols were found to inhibit protein hydroperoxide formation (platt and gieseg, 2003). the current study clearly indicates that sericin functions as an inhibitor of protein oxidation. up-regulation of antioxidant enzymes (aoes) affords protection against ros (fulda et al., 2010). in current study, exogenous treatment with h2o2 resulted in a significant increase in the antioxidant enzymes sod, cat and gpx in the midgut epithelial cells and hemocytes of the silkworm prior to the treatment with sericin or with combination of serine and asparatic acid. such an increase in sod (tsai et al., 2011), cat (dash et al., 2008a) and gpx (caldinin et al., 1998) upon exposure to hydrogen peroxide was observed in fibroblasts. an increase in sod, cat and gpx were reported in silkworms infected with bm npv and reared on an artificial diet containing cecl3 (li et al., 2011). the increased aoes were explained by the fact that cecl3 acts as scavenger of hydroxyl radicals in pathogenic states. also, treatment with 4methylumbelliferone, a model drug, up-regulated the activities of cat and gpx against oxidative stress in fat body cells of the silkworm (fang et al., 2014). our 266   fig. 8 effect of sericin and amino acid mixture on the hydrogen peroxide-induced increase in reactive oxygen species in hemocytes of the silkworm b. mori. (a f) normal hemocytes, (g l) hydrogen peroxide treated hemocytes, (m r) hemocytes incubated with sericin prior to hydrogen peroxide treatment and (s x) hemocytes incubated with amino acid mixture prior to hydrogen peroxide treatment. results show a significant increase in aoes upon hydrogen peroxide treatment. a possible explanation for increased aoes seen in h2o2 treated cells, but not in cells pre-treated with sericin and amino acids, is that the increased antioxidant defence reduces more of the generated ros, such that the ros signal is not generated or rosmediated damage may not occur. in our study, the instar-dependent increase in antioxidant enzymes such as sod and cat was not observed for gpx. similar variations in the antioxidant system as an ontogenic effect has been reported in the beetle tenebrio molitor (gulevsky et al., 2006a, b). in the present study prior treatment with sericin and a mixture of amino acids reduced the aoe activities that were evoked by hydrogen peroxide exposure 267   alone. contradictory findings have been reported regarding the effect of sericin and amino acids on aoes. sericin elevated sod, cat and gpx activity in rat livers treated with alcohol (li et al., 2008, selvaraju and subhashinidevi, 2011), while sericin had no effect on sod in fibroblasts (dash et al., 2008a). it is possible that sericin and amino acids may participate in detoxifying harmful radicals rather than potentiating the activity of aoes, thereby ameliorating oxidative stress-induced cell damage. the ability of a compound to reduce fe3+ and fe2+ serves as an important indicator of its potential antioxidant power (benzine and strain, 1996; yen et al., 1999). in the present study, treatment with 20 µm h2o2 failed to evoke a change in antioxidant capacity in midgut epithelial cells and hemocytes. perhaps, the elevated antioxidant status upon oxidative stress might have been utilized by the cells to overcome the radicals in the initial period. nonetheless, prior incubation with sericin and also with combination of serine and asparatic acid significantly increased the frap values in both tissues subjected to oxidative stress. exposure to hydrogen peroxide in excess of 20 µm induced changes such as swelling and blebbing in both tissues studied. similar changes in morphology were also observed in fibroblasts (dash et al., 2008a) upon hydrogen peroxide treatment. furthermore, an increased hydrogen peroxide concentration was reported in keratinocytes irradiated with uvb (dash et al., 2008b). changes in the intracellular concentration of peroxyl radicals were evident from our fluorescent microscopy studies. the cells labelled with h2dcfda showed a higher intensity upon treatment with hydrogen peroxide, and prior incubation with sericin or with amino acids obliterated the accumulation of peroxyl radicals induced by hydrogen peroxide. our observations are in concurrence with earlier work of fan et al. (2009). it was evident from our study that hydrogen peroxide induces peroxide radical generation and sericin and mixture of serine and aspartic acid may act as a scavenger of free radicals. the ability of proteins to scavenge free radicals has been reported in several systems (kong and xiong, 2006; sakanaka and tachibana, 2006; elias et al., 2008). the results of our study substantiate earlier studies and provide evidence for free radical scavenging by sericin and lcms studies clearly indicate that sericin is rich in serine, followed by aspartic acid, glycine, threonine and glutamine. evidence for protective role of amino acids against oxidative stress is well documented in several experimental models (patterson et al., 2003; movahedian et al., 2006; selvaraju and subbashinidevi, 2011). in the present study a mixture of serine and aspartic acid 0,5 mm each significantly scavenged ros thereby prevented protein oxidation and lipid peroxidation without altering the antioxidant enzymes. serine acts as an antioxidant (maralani et al., 2012), efficient nucleophilic reagent and particularly play a vital role in enzyme catalyst (anderson et al., 1961). whereas, aspartic acid is known as an antioxidant (liu et al., 2004) and also undergo transamination to function directly or indirectly as an antioxidant. although l-serine or l aspartic acid alone scavenged ros to certain extent, a mixture of the amino acids significantly decreased the ros. perhaps synergistic effect of serine and aspartic acid may be responsible for the scavenging effects however, such synergistic effects were not found on antioxidation among the compounds used in combination (wu et al., 2003b). our present result further support antioxidant properties of l-serine (kitazawa et al., 2005) and laspartic acid (chen and nawar, 1991; wang and xiong, 2005). earlier studies on antioxidant property of sericin were appertained to phenol compound (prasong, 2011) and flavonoids (devi et al., 2011). however, our results on a mixture of serine, aspartic acid have clearly indicated the scavenging of ros by these amino acids without altering the antioxidant enzymes. here we report for the first time that the antioxidant property of sericin as scavenger of free radicals rather than enhancing the activities of aoe’s. we suggest that fortification of silkworm diet with a mixture of serine and aspartic acid can alleviate the effects of ros thereby larvae can with stand environmental born stressors to certain extent. although, the mechanism of protection offered is through scavenging of free radicals by major constituent hydroxyl amino acids, suboptimal hydrogen peroxide also helped to enhance the endogenous antioxidant machinery as defense against initial ros signaling or ros damage. acknowledgments this work was supported in part by the promotion of university research and scientific excellence (purse), department of science and technology, new delhi (no.sr/s9/z-23/2010138). fellowship offered by 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systems. j. sci. food agric. 79: 1213-1217, 1999. 270   http://pubs.acs.org/action/dosearch?action=search&author=wang%2c+l+l&qssearcharea=author http://pubs.acs.org/action/dosearch?action=search&author=xiong%2c+y+l&qssearcharea=author fig. s1 effect of sericin and amino acid mixture on the hydrogen peroxide induced increase in reactive oxygen species in hemocytes (a) prohemocytes, (b) plasmocytes and (c) granular hemocytes of the silkworm b. mori. data are shown as mean ± se (n = 3); p < 0.05 was considered significant. values between the stressors are represented by lower case letters (a, b). those not sharing the same letters are significant. 271   fig. s2 effect of sericin and amino acid mixture on the hydrogen peroxide induced increase in reactive oxygen species in midgut epithelial cells of the silkworm b. mori. data are shown as mean ± se (n = 3); p < 0.05 was considered significant. values between the stressors are represented by lower case letters (a, b). those not sharing the same letters are significant. 272   maiorino m, scapin m, ursini f, biasolo m, bosello v, flohe l. distinct promoters determine alternative transcription of gpx-4 into phospholipid-hydroperoxide glutathione peroxidase variants. j. biol. chem. 278: 34286-34290, 2003. maralani mn, movahedian a, javanmard shh. antioxidant and cytoprotective effects of l-serine on human endothelial cells. res. pharm. sci. 7: 209-215, 2012. medina-navarro r, duran-reye g, diaz-flores m, vilar-rojas c. protein antioxidant response to the stress and the relationship between molecular structure and antioxidant function. plos one 5: e8971, 2010. minireview isj 11: 73-78, 2014 issn 1824-307x minireview the main actors involved in extending the invertebrate life span e ottaviani, a franchini, m mandrioli department of life sciences, university of modena and reggio emilia, modena, italy accepted february 3, 2014 abstract classical invertebrate models, i.e., drosophila melanogaster and caenorhabditis elegans, have provided the keys to understand the life span regulation. in the present paper we summarize the mechanisms involved in this process with particular emphasis on the role of the fly fat body. it is interesting to note that pathways which lead to an extension of life span are highly conserved in animals so that “longevity pathways” identified in invertebrates provide templates for the identification of genes and drugs that regulate longevity and diseases also in other animals, including mammals. key words: drosophila melanogaster; insulin/igf-1-like pathway; fat body; gut microbiota; longevity   introduction aging is a well-conserved process during evolution that involves different actors including dietary restriction (dr), fat body and/or adipose tissue and insulin/insulin growth factor-1 (igf-1)-like pathway (klöting and blüher, 2005). the regulation of life span in different organisms starts from glucose (as observed in yeasts) or insulin/igf-1-like (as described in worms and flies). the increase of growth and mortality occurs through the downregulation of antioxidant enzymes and heat shock proteins, together with the reduction of the accumulation of glycogen and/or fat. vice versa, the decrease of these pathways prolonged the life span by simulating the dr (see for review, katic and kahn, 2005). insulin/igf-1-like pathway and dr the insulin/igf-1-like (iis) pathway probably plays a central role in the evolution of multicellularity (skorokhod et al., 1999). it is involved in several processes, including growth and longevity and a reduced activity of the pathway extends life span (partridge and gems, 2002; tatar et al., 2003; kenyon, 2005). the regulation of growth and size in drosophila melanogaster requires the following components: the insulin/igf-1 receptor inr (insulin-like receptor), the inr substrate chico, the pi3k, the pi3k target pkb (also known as akt1) and dfoxo, the fly forkhead transcription factor ___________________________________________________________________________ corresponding author: enzo ottaviani department of life sciences university of modena and reggio emilia via campi 213/d, 41125 modena, italy e-mail: enzo.ottaviani@unimore.it phosphorylated and inactivated in response to iis (weinkove and leevers, 2000) (fig. 1). studies performed in invertebrates, such as d. melanogaster, caenorhabditis elegans and trechus angusticollis, demonstrated that a dr was able to extend life span at the expense of the fecundity (chippindale et al., 1993; partridge et al., 2005; heestand et al., 2013). however, it is still unclear if the observed increase in life span is due to a specific nutrient or dietary (tatar, 2011) and the trade-off between longevity and fecundity related to dr is not always observed (heestand et al., 2013). in flies the extension of life span by dr involves the rapamycin (tor) signaling pathway (kapahi et al., 2004), and the increase of triacylglycerols (tag) (bohni et al., 1999; zhang et al., 2000). furthermore, dr intervenes in the fatty acid metabolism, a process required for the life span extension (katewa et al., 2012). fat body fat body is an important source of energy that is stored as glycogen and tag (leopold and perrimon, 2007; arrese and soulages, 2010). tag are the core of the so-called lipid particles, also known as lipid droplets or lipid bodies (ottaviani et al., 2011a). at a phylogenetic level, tag are present in yeasts, such as saccharomyces cerevisae (zweytick et al., 2000; daum et al., 2007) and candida parapsilosis (neugnot et al., 2002), the nematode c. elegans (watts, 2009), the molluscs ifremeria nautilei (saito and hashimoto, 2010) and haliotis fulgens (nelson, 2002), the insect d. melanogaster (grönke et al., 2005), the sea star asterias rubens (allen, 1998), the sea urchin 73 echinus esculentus (allen, 1998) and the sea cucumber holothuria forskali (allen, 1998). it has been reported that the reduction of adipose tissue influences the extension of the life span in different invertebrates (hwangbo et al., 2004; kenyon, 2005; klöting and blüher, 2005). for instance, the reduction in fat mass in d. melanogaster provokes an overexpression of dfoxo with the consequent extension of the life span. 74 recent findings showed that the addition of escherichia coli to the diet of d. melanogaster females significantly increase the longevity of both the two strains examined [a short-life strain (bloomington drosophila stock center (fbst0006971) with an average adult life span of 10 days and a long-life standard lived r strain with an average adult life span of 50 days] (franchini et al., 2012). in the short-life flies the lengthening of lifespan was particularly evident at days 7 and 9 when the survived flies grown in presence of bacteria were four and three times more numerous than controls. moreover, 5 % of flies fed with e. coli were still alive at day 11, whereas controls were all dead. in the long-life strain, an extension of the life span was also observed: at days 45 and 48 the percentages of survived flies were three and five times higher in the bacteria fed samples than in controls and about 20 25 % of flies grown in presence of e. coli was still alive while controls were all dead (between the 49th and 51st day). the comparison of structural and histochemical observations from flies fed with different diets, demonstrated that the presence of e. coli induced modifications in the fat body. this organ was characterized by a loose tissue of both layers of cells close to the integument and different sized lobes surrounding the internal organs in thorax and abdomen cavities. the most abundant cell type consisted of large polygonal cells containing different amounts of stored materials. at day 4, when no difference in survival of females from shortlife strain was found, the morphology and histochemical reactivity of fat body from samples fed by e. coli were similar to those of most of the controls. it was well developed with the main cell type rich in glycogen and few empty unstained vacuola of different size (fig. 2a). however, the network of loose perivisceral lobes of cells poor in glycogen and rich in lipid droplets, observed in some control flies, were not detected in bacteria fed samples. at day 9, the fat body of control survived flies appeared reduced in its perivisceral lobes that were constituted by vacuolated cells mainly storing lipid droplets (fig. 2b). in contrast, fat bodies from most flies grown in the presence of bacteria were formed by well-developed islets of cells, full of glycogen (fig. 2c). in some flies, the perivisceral cell aggregates contained lipid droplets (fig. 2d). the fat body in control and bacteria fed samples from the long-life strain, similar to that of short lived flies, did not show relevant differences in structure and histochemical reactivity when no difference in survival was detected. in contrast, when the longevity was significatively extended, an higher percentage of controls contained reduced glycogen stores in comparison with bacteria fed flies (figs 2e, f). the fat body cells were pas-negative in 85 % of control survived flies against a 55 % of bacteria fed flies at day 48. in contrast to the short lived strain, no lipid droplets have seen to accumulate in empty vacuolated cells in the course of fly aging. gut microbiota and insulin signaling pathway in recent years, the presence of a reduced microbiota (less than 30 species) made d. melanogaster an intriguing model to understand the principles that govern host-microbiota interactions (kostic et al., 2013). indeed, drosophila represents an experimentally tractable system to discover the molecular underpinnings the host-commensal interactions also in other insects, including those that act as vectors of infectious diseases or are of importance to agriculture (douglas et al., 2011). in other cases, the implications of drosophilamicrobiota interaction allowed to uncover broader concepts of mutualism that are conserved among higher-order organisms (kostic et al., 2013). fig. 1. regulative pathway of life span in flies. a reduction of the insulin/igf-1pathway activates a cascade resulting in dfoxo overexpression and an extended longevity. inr: insulin-like receptor. chico: inr substrate. pi3k: phosphatidylinositol-3 kinase. akt/pkb: serine/threonine kinase b. dfoxo: fly homologue of the mammalian forkhead (foxo) family of transcription factors. fig. 2 longitudinal sections of the fat body from short(a-d) and long (e, f) life drosophila females fed with standard diet (controls) and in the presence of e. coli (pas/hematoxylin, a-c, e, f; hematoxylin-eosin, d). at day 4, when no survival differences were found between controls and bacteria fed flies, the structure and histochemical reactivity of fat body were similar (a). at day 9, in control survived flies the perivisceral lobes were reduced and the vacuolated cells mainly stored lipid droplets (b). in contrast, in most flies grown with addition of bacteria to the diet, well developed islets of cells were full of glycogen (c) while in some females the cells of perivisceral aggregates contained lipid droplets (d). the fat body from 45 day old long-life control females, with strongly reduced glycogen stores (e), is compared with that from bacteria fed flies (f). bars: 50 μm (a-c, e, f); 10 μm (d). according to previous studies, the microbiota regulates the accumulation of fat by promoting the storage of tag in the adipocytes (bäckhed et al., 2004), and regulates the life span in flies (seung et al. 2011). interestingly, the fly commensal bacterium acetobacter pomorum modulates the iis expression in the fat body affecting host homeostatic programs controlling developmental rate, body size, energy metabolism and intestinal stem cell activity. this result is due to the ability of a. pomorum to induce the activation of the pi3k suggesting that this commensal bacterium also have an effect on the fly life span (shin et al., 2011). the possible regulative role of a. pomorum in fly life span is not surprising taking into account that the digestive tract of many insect species harbours several bacteria that perform different beneficial functions to their host and may affect host longevity 75 fig. 3. the circuitry of longevity in flies. tag: triacylglycerols. dr: dietary restriction. (ottaviani et al., 2011b). for instance, ceratitis capitata life span was extended after feeding with enterobacteriaceae due to direct effects of these symbionts on medfly metabolism and development (behar et al., 2008). similarly, non-virulent strains of wolbachia can extend drosophila life span (fry and rand, 2002) and experiments performed using axenic cultures and antibiotic treatment revealed that exposure to bacteria during the first week of adult life increased longevity by 30 35 % in flies (pletcher et al., 2002; seroude et al., 2002; brummel et al., 2004). a second set of intriguing data have been published by storelli et al. (2011) evidencing a reduced insulin signaling in germ-free drosophila, while the addition of the commensal bacterium lactobacillus plantarum is sufficient on its own to restore the natural drosophila microbiota growthpromoting effect. according to the published data, l. plantarum exerts its benefit by acting genetically upstream of the tor-dependent host nutrient sensing system controlling hormonal growth signaling. the key implication of this study, together with data of shin et al. (2011), is that different bacterial products, derived from taxonomically divergent bacteria, can affect insulin signaling in drosophila. as a whole, these data on drosophila open some intriguing questions, since as suggested by douglas (2011), multiple bacterial products may interact (competitively, additively and/or synergistically) with the drosophila insulin signaling networks, so that the “standard” setpoint of the fly insulin signaling could be a titration between the high and low preferred setpoints of the bacteria and fly respectively (douglas, 2011). interestingly, if the intrinsic set point of flies is calibrated constitutively to account for bacterial manipulation (as is likely because bacteria are always present in naturally occurring drosophila), then the signaling would be depressed in the germ-free flies, which lack the manipulative up-regulation by the bacteria (douglas, 2011). the insulin signaling may be therefore the result of an evolutionary “agreement” between drosophila and its microbiota, explicable not only in the context of fly ecology, but also in terms of the long evolutionary history of fly-microbiota interactions. bacterial intervention in animal signaling networks can be considered as part of how the resident microbiota keeps flies healthy and also mediate the life span extension (douglas 2011). conclusive remarks the data here reported show that several components, such as insulin pathways, dr, fat body and gut microbiota are deeply interconnected in insect aging and interact for extending life span (fig. 3). the life history of each animal is therefore a trade-off resulting from the complex evolutionary history of each species that should face different kinds of competitively, additively and/or synergistically interactions. this scenario opens an intriguing perspective for human health and aging since if our life span has been defined by an heterogeneous set of interactions among our genome, diet, microbiota and environment, we 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http://www.ncbi.nlm.nih.gov/pubmed/16125891 78 storelli g, defaye a, erkosar b, hols p, royet j, leulier f. lactobacillus plantarum promotes drosophila systemic growth by modulating hormonal signals through tor-dependent nutrient sensing. cell metab. 14: 403-414, 2011. tatar m. the plate half-full: status of research on the mechanisms of dietary restriction in drosophila melanogaster. exp. gerontol. 46: 363-368, 2011. tatar m, bartke a, antebi a. the endocrine regulation of aging by insulin-like signals. science 299: 1346-1351, 2003. watts jl. fat synthesis and adiposity regulation in caenorhabditis elegans, trends endocrinol. metab. 20: 58-65, 2009. weinkove d, leevers s. the genetic control of organ growth: insight from drosophila. curr. opin. genet. dev. 10: 75-80, 2000. zhang h, stallock jp, ng jc, reinhard c, neufeld tp. regulation of cellular growth by the drosophila target of rapamycin dtor. genes dev. 14: 2712-2724, 2000. zweytick d, athenstaedt k, daum g. intracellular lipid particles of eukaryotic cells. biochim. biophys. acta 1469: 101-120, 2000.   vision and perspectives isj 8: 227-230, 2011 issn 1824-307x visions and perspectives the central role of immunity in the symbiotic event referred as parasitism m de eguileor1, e ottaviani2 1department of biotechnology and life science, university of insubria, varese, italy 2 department of biology, university of modena and reggio emilia, modena, italy accepted november 25, 2011 abstract several papers have been published on the communications between species, including hostparasite and predator-prey interactions. here we stress the crucial role of immune system in symbiosis and parasitism. in particular, it appears that during the coevolution between any interacting populations the immune system was selected accordingly to a flexible strategy in order to adapt itself to the needs of the homeostasis, thus allowing the evolution of symbiotic relationships. key words: immune system; symbiotic interactions introduction accordingly to a traditional view, parasite benefits at the expenses of the host. however even if this is the more diffused situation, cases are reported in which the host alone or both the host and the parasite, survive. what determines the outcome among the existing alternatives? likely intrinsic and extrinsic factors are involved. the first concerns the characteristics of host and parasite, while the latter may include the environment, the ecological niches in which the interactions take place, etc. moreover, the relationships parasite-host also are subjected to a basic principle of ecoimmunology, i.e., to minimize the energy costs of immune responses (lochmiller and deerenberg, 2000; ottaviani et al., 2008). to explain the evolutive advantage represented by the complex and expensive immune system different models have been proposed, including the trade-off theory. according to holt and polis (1997), trade-off theory predicts the coexistence of competition for resources in dynamic populations in which a direct predator-prey interactions occurs allowing a trade-off (transition of energy) which then leads to a partitioning of the ecological niches. in this framework, the worst competitor for the same available resources can find a second source of energy in the best competitor, becoming for instance a predator or a parasite or a symbiont. in symbiotic interactions, the role of the immune tolerance have to be considered. edwards (2009) ___________________________________________________________________________ corresponding author: enzo ottaviani department of biology university of modena and reggio emilia via campi 213/d, 41125 modena, italy e-mail: enzo.ottaviani@unimore.it suggested that tolerance may be involved in promoting the evolution of mutualism or in its maintenance. also, tolerance may supply a pathway for autonomy and breakdown of mutualism. another concept helping us to explain the energy sustainability of immune responses in a host/parasite system is allostasis, that is the process of maintaining stability through deep and transient alterations involving numerous systems (nervous, circulatory, endocrine systems, etc.) (korte et al., 2005). bearing in mind that immune system is devoted to maintain the integrity of an organism through the recognition of self from not-self, a symbiont/parasite must either be recognized as own by the host or escape the host immunosurveillance, for instance by inhibiting the host immune system. in both cases, immune system is a central player. one important point is the role of immune system in defining the demarcation between self and not-self, because harmful and nutritious not-self may be very similar (ulvestad, 2009). another important point is that immune tolerance is an important evolutive mechanism that also influences the outcome of the parasitism (edwards, 2009; ulvestad, 2009). indeed, the evolution of the multicellular organisms must have been based on mechanisms resembling the immune tolerance that allows multicellularity where the cells that make up a given organism may not be identical to each other (ulvestad, 2009). finally, potential symbionts that can not modulate the host immune system can be “hidden” within specialized structures such as endosymbionts within bacteryocytes (baumann et al., 2000). among the most extreme and intimate examples there is the leech symbionts usually harbored in the cytoplasm of mycetocytes that in turn are detected in various 227 mailto:enzo.ottaviani@unimore.it tissues as the epidermis, salivary glands, gut where help the digestion of the blood meal, providing essential nutrients and prevent colonization by other potentially harmful microorganisms (graf et al., 2006). examples of strategies adopted in the interaction between host and parasite/symbiont for a parasite is fundamental to escape the host immune system. in this context it has been demonstrated that the parasitic trematode schistosoma mansoni is able to elude the immunosurveillance of the host, the mollusc biomphalaria glabrata (duvaux-miret et al., 1992). the release of adrenocorticotropic hormone by the immunocytes of the parasite is converted by neutral endopeptidase 24.11 to α-melanocyte-stimulating hormone, a molecule that inhibits the adherence and locomotory activity of b. glabrata immunocytes as it has also been observed for human polymorphonuclear cells and monocytes. fig. 1 light microscopy. semithin section of the body of the larva (l) surrounded by a thick “serosa” (evidenced area, harrowheads). fig. 2 tem image of “serosa” lining the parasitoid larva (l) surface. serosal cells (s) are coated by thick fibrillar basal membrane (arrowheads) (bar = 0.5 μm). a complex approach is used in another host/parasitoid system, i.e., heliothis virescens/toxoneuron nigriceps where the parasitoid wasp t. nigriceps injects in the host, eggs and maternal fluids (venom, calyx fluid with polydnaviruses and the ovarian proteins) provoking severe damage to the immune and neuroendocrine systems of h. virescens larva. during the early phase of parasitization it has been demonstrated that humoral host prophenoloxidase system is rapidly and temporarily switched off and this neutralization is paralleled with a depression of host cellular defense (ferrarese et al., 2005; falabella et al., 2011). in addition the parasitoid shows the concurrent presence of active and passive immunoevasive strategies in order to better insure the survival of the progeny. t. nigriceps, from the embryo stage up to the moult of first-instars larva, is protected by a persisting extra-embryonic membrane, the “larval serosa” (figs 1-3). this complex structure fulfills different functions contributing both to the immune evasion, acting as a barrier for macromolecules and to the nutritional exploitation being able to hydrolyze and absorb nutrients (grimaldi et al., 2006). when the developing parasitoid looses this own protection, it completes the development adopting a molecular mimicking strategy sequestering host hemolymph components close to its body surface (ratcliffe et al., 1985; strand and peck, 1995; brivio et al., 2010). a different modality resulting in the same effect is the unique manipulation adopted from several strepsiptera that for avoiding host immune responses complete their grow in a “bag” derived from the host epidermal tissue. stichotrema dallatorreanum wraps itself with segestidea defoliaria defoliaria tissue; thus, due to this camouflage, the endoparasite is recognized as self. this strategy has been reported as a good example 228 of host/parasitoid coevolution (kathirithamby et al., 2003; kathirithamby, 2008). a complete different strategy is documented by the endosymbiotic prokaryotes. endosymbiosis is common in insects, with more than 10 % of insect species that depend on intracellular bacteria for their development and survival (baumann et al., 2000). the endosymbiotic bacteria are transmitted maternally and during the embryogenesis reach specialized cells called bacteriocytes (fig. 4), cells that derive from the hemocyte line, the plasmatocytes (sacchi et al., 1989; sacchi, 2004; heddi et al., 2005). sometime the bacteriocytes form a specific organ, the bacteriome, an outgrowth of the insect’s gut (anselme et al., 2006). it has been found that aphid bacteriocyte expresses three transcription factors: dll, en, and ubx or abd-a. these transcription factors play important roles during later stages of insect development (braendle et al., 2003). furthermore, it has been found a relationship between bacterial virulence and host immune defense, indeed an overexpression of pgrp gene family is detected in the bacteriome tissue of the host (haddi et al., 2005; anselme et al., 2006), as well as the induction of antibacterial peptide genes outside of bacteriome (anselme et al., 2008). last but not least, the studies on the bacterium wolbachia pipientis must be remembered. drosophila melanogaster is protected from rna viruses when infected by the intracellular bacterium w. pipientis (hedges et al., 2008). in particular, the antiviral activity of w. pipeintis is exerted in two different ways: i) the bacterium interferes with the virus infection cycle provoking a delay in the virus accumulation resulting in a host resistance to virus infection; ii) w. pipeintis infection protects the flies increasing the host tolerance to virus infection (osborne et al., 2009). in this context, eberl (2010) coined the term of “superorganism” to define the new functional entity composed by host and symbiotic microbiota where they crosstalk with the immune system in order to maintain the homeostasis of the “superorganism”. fig. 3 tem image of “serosa”. in the detail serosal cell (s) shows pressed microvilli (arrowheads) and a coat of thick basal membranes (bm) (bar = 1 μm) (courtesy dr. grimaldi a, department of biotechnology and life science, university of insubria, varese, italy). n b b fig. 4 tem image of a bacteriocyte from the fat body of the insect blattella germanica (n, nucleus; b, bacteria) (bar = 1.2 μm) (courtesy prof. sacchi l, department of animal biology, university of pavia, pavia, italy). conclusive remarks on the whole we can stress the following points: i) coevolution can occur between any interacting populations, for instance between prey-predator, host-pathogen, etc. this event is very important because this association provokes selective pressures of one on the other participants resulting in different effects on their fitness inducing benefits; ii) the immune system is crucial for the success of the symbiotic interactions. furthermore, it emerges an apparent paradox because the defense system recognize not-symbiotic bacteria, while, likely the gut local immune response, avoid a permanent systemic response to the commensal bacteria (anselme et al., 2008); iii) the immune system is not a killer, but a system that adapts itself to the needs of the 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y, wäckers f. molecular and cellular profiles of insect bacteriocytes: mutualism and harm at the initial evolutionary step of symbiogenesis. cell microbiol. 7: 293-305, 2005. strand mr, pech ll. immunological basis for compatibility in parasitoid-host relationship. annu. rev. entomol. 40: 31-56, 1995. ulvestad e. cooperation and conflict in hostmicrobe relations. apmis 117: 311-322, 2009. hedges lm, brownlie jc, o’neill sl, johnson kn. wolbachia and virus protection in insects. science 322: 702, 2008. 230 falabella p, riviello l, pascale m, di lelio r, tettamanti g, grimaldi a, et al. functional amyloids in insect immune response. insect biochem. mol. biol. 2011[in press]. summary 309 isj 11: 309-318, 2014 issn 1824-307x research report the genetic diversity and differentiation of shrimp fenneropenaeus chinensis in the yellow sea revealed by polymorphism in control region of mitochondrial dna l wang a , j yang a , m sun a,b , c yang a , z cui a , in k jang c , l song a a key laboratory of experimental marine biology, institute of oceanology, chinese academy of sciences, qingdao 266071, china b university of chinese academy of sciences, beijing 100049, china c west sea mariculture research center, national fisheries research & development institute, taean, chungnam 357945, south korea accepted october 29, 2014 abstract chinese white shrimp fenneropenaeus chinensis is a commercially important species in northern china and korea. in the present study, the genetic diversity of five populations collected from qingdao (qd), rizhao (rz) of china, and narodo island (kn), taean (kt), yeongguang (ky) of korea in the yellow sea was investigated using the mitochondrial control region (cr). the length of the amplified partial mitochondrial control region (mtcr) ranged from 600 to 622 bp, and the sequence variations were distributed among 13 polymorphic sites. the pattern of nucleotide substitution was biased in favour of transitions over transversions in variable sites, including 12 transitions (si, 4 a↔g and 8 t↔c changes) and only one was transversion (sv, 1 t↔g changes). altogether, 24 unique haplotypes were identified from five populations in yellow sea. the overall haplotype diversity and nucleotide diversity were 0.368 0.421 and 0.052 0.079, respectively, and the lowest genetic diversity was found in qd population. there was no differentiation between the two chinese populations (fst = 0.039). within the korean populations, there was a slight differentiation (fst = 0.075, p < 0.05) between kn and kt. the relative bigger differentiation was shown between rz and kn population (fst = 0.170, p < 0.05). the relative further genetic distance was shown between rz and kn population as well as between qd and kn population, while the relative closer genetic distance was shown between kt and ky, and between kt and rz population. the low variability in the mitochondrial control region among f. chinensis in the yellow sea indicated the low genetic diversity in comparison to other shrimp species. the results suggested a slight population differentiation among f. chinensis populations. such information will assist in sustainable use, management, and conservation of the species. key words: fenneropenaeus chinensis; control region; genetic diversity; population genetics; polymorphism introduction the chinese shrimp fenneropenaeus chinensis, mostly distributed in the yellow sea, bohai sea in china and the west coast of the korean peninsula (liu, 1959, 1990). it has been playing an economically important role in the fishing and farming industries in northern china (ye, 1984, 1994; xin, 1999). the chinese shrimp aquaculture industry suffered a severe mortality problem caused by shrimp white spot syndrome virus in the middle of 1990s, and fishing production also decreased ___________________________________________________________________________ corresponding author: linsheng song institute of oceanology chinese academy of sciences 7 nanhai rd., qingdao 266071, china e-mail: lshsong@ms.qdio.ac.cn significantly (liu, 2003; guo, 2006). shrimp larvae have been released annually into the yellow sea over the last two decades to replenish the decreasing shrimp stocks in china. however, such large scale restocking of hatchery raised larvae and the escape of farmed individuals can significantly affect the genetic structure of shrimp population in yellow sea. f. chinensis wild stocks are further threatened by overfishing, viral epizootics, and habitat contamination. a better understanding of population structure is important to the effective fisheries management and conservation of genetic resources in exploited marine organisms (rolda’n et al., 2000). recent researches on the genetic variation within f. chinensis have utilized new analytical and technical tools that provide high-resolution genetic information. mailto:lshsong@ms.qdio.ac.cn 310 table 1 shrimp fenneropenaeus chinensis populations sampled for present study sampling locality abbrev. sample for sequencing average wet weight (g) narodo island, korea kn 23 94.37 yeongguang, korea ky 25 60.25 taean, korea kt 20 70.75 qingdao, china qd 12 26.13 rizhao, china rz 20 46.45 two geographic populations of f. chinensis had previously been defined, one along the coast of northern china and the other on the west coast of the korean peninsula (kim, 1973; deng et al., 1983, 1990; zhuang et al., 2001). recently, a new population of f. chinensis was found near the jeju island in southern korea, called the southern coast population of the korean peninsula (liu et al., 2004, 2006). its spawning location and migration routes are different from the others. most previous researches revealed the very low genetic diversity of f. chinensis in yellow sea and bohai sea (liu et al., 2000a b; qiu et al., 2001; ma et al., 2004; meng et al., 2004; cui et al., 2007) using various approaches such as radom amplified polymorphic dna (rapd) and microsatellite dna. the sequence and structure of mitochondrial genomes (mtgenomes) are gaining increasing popularity in higher-level phylogenetic analysis because of their ability to provide better resolution for relationships than single or multi-gene analysis and the relative ease of sequencing of the entire genome, which can provide information on phylogenetic relationships and on the genetic structure of populations and patterns of gene flow (cameron et al., 2004). this information may derive from gene order, the sequences of individual genes, restriction fragment length polymorphism (rflp) analysis of mtdna, or the sequences of complete genomes. although there are various reports on the genetic diversity of f. chinensis and population differentiation by using different dna markers, their resolutions are not sufficient to discriminate the less genetically differentiated populations. the mitochondrial 16s rrna and cytochrome oxidase i (coi) genes were often used in elucidating population structure of penaeid shrimps over a broad geographic range (klinbunga et al., 2001; tsoi et al., 2007), but they were found less useful in identifying genetic difference over smaller geographic scales (tsoi et al., 2007), as in the case of f. chinensis. in the present study, the population structure of shrimp (f. chinensis) in the yellow sea was inferred using highly variable control region of mitochondrial dna to provide more information on the genetic diversity of f. chinensis for the sustainable utilization of its wild stocks, the management of fisheries and the health of ecosystem in the yellow sea in terms of genetic diversity in exploited stocks. materials and methods sample collection and dna extraction shrimps f. chinensis were collected from the following five localities (table 1, fig. 1): taean (abbreviated to kt, chungnam-do,), narodo island (kn, jeollanam-do), and yeongguang (ky, jeollanam-do) from the korean peninsula in the yellow sea; and qingdao (qd, shandong), rizhao (rz, shandong) from the coast of northern china in the yellow sea. the specimens were kept at -20 °c until analysis. total genomic dna was isolated from approximately 20 50 mg of pleopod tissue that was minced and digested at 55 °c for 3 h in 500 μl of extraction buffer (10mm tris/hcl at ph 8.0, 50 mm fig. 1 purple triangles indicate sampling locations of five shrimp populations: taean (abbreviated to kt, chungnam-do), narodo island (kn, jeollanam-do), and yeongguang (ky, jeollanam-do) from the korean peninsula in the yellow sea; and qingdao (qd, shandong), rizhao (rz, shandong) from the coast of northern china in the yellow sea (referenced from wang et al., 2006). 311 edta, 1 % sodium dodecyl sulfate and 100 μg ml −1 proteinase k). the mixture was then extracted twice with phenol, phenol/chloroform (1:1) and once with chloroform/isoamyl alcohol (24:1), followed by ethanol precipitation. the samples were washed with 70 % ethanol, air-dried, and re-dissolved in distilled water. the detailed sampling location, sampling size and other information were shown in table 1 and figure 1. sequencing of partial control region a total of 100 shrimps were employed for amplification of the 5’ segment of mitochondrial cr using the primers 12s (5’-aag aac cag cta gga taa aac ttt-3’) and pcr-1r (5’-gat caa aga aca ttc ttt aac tac-3’) (chu et al., 2003). the reaction mixture (20 ml) contained ~100 ng of dna template, 0.2 mm of each primer, 1×thermophilic dna polymerase buffer, and 2.5 u taq dna polymerase (qiagen). the thermal cycling profile for amplification was one cycle of 3 min at 94 °c, 35 cycles of 40 s at 94 °c, 40 s at 48 °c, and 30 s at 72 °c, and a final extension at 72 °c for 3 min. single bands of the predicted size were obtained from pcr. prior to sequencing, pcr products were purified using either the qiaquick pcr purification kit or gel purification kit according to the manufacturer’s instructions (qiagen). sequences of purified pcr products were obtained from both directions using the same primers for pcr. sequence assembly and annotation sequences from both strands in each specimen were aligned with clustal x1.81 (thompson et al., 1997) and individual consensus sequences were retrieved with both alignment and manual check. contig sequences were checked for ambiguous base calls and only non-ambiguous regions were used for annotation. the partial control region sequences of the f. chinensis were compared with those submitted to genbank, and blast database searches were performed to make sure correct target sequences amplified. software computation molecular diversity indices such as number of haplotypes, polymorphic sites, transitions, transversions and indels were obtained using arlequin (ver. 2.000, schneider et al., 2000). haplotype diversity (h), nucleotide diversity (π), and their corresponding variances were calculated following nei (1987) as implemented in arlequin. the amounts of genetic variability partitioned within and among populations were accessed by an analysis of molecular variance (amova; excoffier et al., 1992). significance of pairwise population comparison was tested by 20,000 permutations. organization of the amova tests was in a hierarchical manner and 1,000 permutation procedures were used to construct null distributions and to test the significance of variance components (guo and thompson, 1992). amova and bootstrap analysis with 5,000 replicates were performed in arlequin. the inclusive tamura-nei (trn) (tamura and nei, 1993) model was used to calculate the genetic pairwise distances between haplotypes. the isolation-by-distance effects on population genetic structure were estimated by pairwise fst statistics (wright, 1951, 1965). the significance (5 % level) of the fst was tested by 1,000 permutations for each pairwise comparison. phylogenetic trees of the haplotypes were constructed using mega 4.0 (tamura et al., 2007). the neighbour-joining (nj) algorithm (saitou and nei, 1987) was implemented to construct a phylogenetic tree from the maximum likelihood (ml) distances estimated under the selected models. relationships between haplotypes were also determined with the kimura two-parameter distance model by using the neighbour-joining method in mega 4.0. bootstrap analysis with 1,000 replicates was used to evaluate reliability of phylogenetic relationships (felsenstein, 1985). results sequence variation pcr products from the mitochondrial control region of 100 individuals were obtained with the primers 12s and pcr-1r, which ranged from 600 to 622 bp (including primers). the nucleotide composition of this fragment consisted of 9.55% cytosine, 45.06 % thymine, 37.04 % adenine, and 8.35 % guanine. of the partial mitochondrial control region (mtcr) sequences, the sequence variations were distributed among 13 polymorphic sites (table 2). the pattern of nucleotide substitution was biased in favour of transitions over transversions in variable sites, including 12 transitions (si, 4 a↔g and 8 t↔c changes) and only one was transversion (sv, 1 t↔g changes) (table 2). altogether, 24 unique haplotypes were identified from five populations in yellow sea (table 2). haplotype ky12 was the most common and was observed in all samples, and its frequency was 24 % in the total samples. another two haplotypes (kn02, qd12) occurred frequently in five populations, and kn02 showed higher frequency in korean population than that in chinese population. haplotype rz08, rz11 and rz16 were shared by the qd and rz samples from the chinese coast. the distribution of ten mtcr haplotypes was restricted to only one population. and they were sample-specific haplotypes. none of the haplotypes was shared only among the populations from the region of korean coast (kn, ky and kt). the population pairs and the numbers of haplotypes shared between the populations from chinese and korean coast were as follows: kn and qd populations (five); kn and rz populations (five); kt and qd populations (six); kt and rz populations (four); ky and rz populations (four); ky and qd populations (six). haplotype frequencies of cr fragment and their distributions in the five samples are shown in table 2. genetic diversity and population structure the number of haplotypes, haplotype diversity (h), and nucleotide diversity (π) for each population were listed in table 3. the genetic variation level was low, whether measured as haplotype diversity (0.368 0.421) or nucleotide diversity (0.052 0.079). the lowest genetic diversity was found in qd population from qingdao. and kn population from 312 table 2 variable nucleotide positions defining the mtdna control region haplotype from each of five shrimp population sampled (sequence identity to reference sequence in top row kn02), haplotype frequencies in 5 shrimp populations and total number of individuals of each haplotype (n). haplotype variable sites and positions populations 7 1 7 7 3 1 5 3 3 4 3 6 4 3 8 6 4 0 0 4 1 3 4 2 0 4 2 1 4 2 2 4 4 2 5 6 2 kn ky kt qd rz n kn 01 a t t g t t t t t a t a c 1 2 1 2 0 6 kn02 a g t g c t t t c a t a t 5 3 3 2 3 16 kn13 a g c g c t t t c a t a t 1 0 0 0 0 1 kn17 a t t a c t t t t a t g t 1 0 0 0 0 1 kn19 a g t g c t c t t a t a t 1 0 0 0 1 2 ky02 a t c g t t t t t a t a c 0 1 0 0 0 1 ky06 a t t g t t t t t a t a t 0 1 0 0 0 1 ky12 g g c g c t t t c a t a t 9 8 4 2 1 24 ky20 a g t g c t t t t a t a t 1 1 0 0 0 2 ky21 g g c g c t t t c a c a t 0 1 0 0 0 1 kt12 a t t g c t t t t a t a t 1 0 1 0 0 2 kt13 a t c g c t t t t a c a t 0 0 1 0 0 1 kt19 a g t g c c t t t g t a t 1 0 1 0 0 2 kt22 a t t a c t t t t g t a t 0 1 1 0 4 6 kt23 a g t g c t t t c a c a t 0 0 1 0 0 1 qd12 a t t g t t t t t a c a c 1 3 2 1 4 11 qd25 g g c g c t t t c a t g t 1 0 0 2 1 4 qd30 a t t g c t t c t a t a t 0 1 2 1 0 4 qd37 a t t g c c c t t a t a t 0 3 3 1 0 7 rz01 a t c g c t t t t a t a t 0 0 0 0 1 1 rz05 a g t g c t t t c a t g t 0 0 0 1 1 2 rz08 a t t g t t t t t a c g c 0 0 0 0 1 1 rz11 a t t g c t t c t a c a t 0 0 0 0 2 2 rz16 a t t a c t t t t g t g t 0 0 0 0 1 1 total 12 transition, 1 transversion 23 25 20 12 20 narodo island, korea also showed lower genetic variation level than rz (rizhao) population in china, and other kt and ky populations from the korean side. when the samples were pooled into different regional groupings, amova analysis showed that more than 93 % of the total molecular variance was distributed within populations (table 4). the fst values for korean (kt, kn, ky) and chinese groups (rz, qd) was 0.044 and the fst value for north (qd, kt) and south groupings (rz, kn, ky) was 0.021 therefore indicated no significant differentiation. however, the fst value obtained when comparing the southern korean population (kn) with the rest (qd, kt, rz, ky) was 0.069, and the fst value obtained when comparing kn, qd with the rest (kt, rz, ky) was 0.058 (table 4), indicating a slight population differentiation throughout the range of f. chinensis populations in yellow sea. 313 genetic differentiation between populations genetic differentiation among shrimp populations was assessed using fst pairwise comparisons. in the 10 possible comparisons, four of the pairwise fst estimates were negative (table 5), indicating that the variation within samples was greater than variation between samples. there was no differentiation between the two chinese populations (fst = 0.039). within the korean populations, there was a slight differentiation (fst = 0.075) between kn and kt (p < 0.05). and the kn and rz population showed a severe differentiation (p < 0.05) (fst = 0.170). the relative further genetic distance was shown between rz and kn population as well as between qd and kn population, while the relative closer genetic distance was shown between kt and ky, and between kt and rz population (table 6, fig. 2). the relationship among all the 24 haplotypes was shown in figure 3. all the 24 haplotypes were clustered with two separate groups. three haplotypes (ky12, kn02 and qd12) were found in all samples, while ten mtcr haplotypes were restricted to one population. eleven mtcr haplotypes were shared in any two of the shrimp populations. table 3 summary of molecular diversity for f. chinensis, h, haplotype diversity, π, nucleotide diversity population no. of haplotype h π kn 5 0.372 0.052 ky 5 0.397 0.073 kt 5 0.421 0.078 qd 4 0.368 0.052 rz 5 0.416 0.078 discussion in general, the genetic diversity of f. chinensis in the yellow sea appears to have diminished in the past years. the heterozygosity has decreased from table 4 the amova analysis of shrimp populations in different groups. source of variation sum of squares variance components percentage variation p values fst group (ky, kt, kn) and group (qd, rz) between groups 4.118 0.02726 1.41 0.00000 0.044 between populations within groups 8.999 0.05753 2.98 0.00000 within populations 175.392 1.84624 95.61 0.00933 total 188.510 1.93103 group (qd, kt) and group (rz, kn, ky) between groups 0.786 -0.07335 -3.89 0.00000 0.021 between populations within groups 12.332 0.11294 5.99 0.00000 within populations 175.392 1.84624 97.90 0.00387 total 188.510 1.88583 group (qd, kt, rz, ky) and group (kn) between groups 6.394 0.11517 5.81 0.00000 0.069 between populations within groups 6.723 0.02092 1.06 0.00000 within populations 175.392 1.84624 93.13 0.01153 total 188.510 1.98233 group (kt, rz, ky) and group (qd, kn) between groups 6.329 0.08972 4.70 0.00000 0.058 between populations within groups 6.702 0.02230 1.17 0.00000 within populations 170.669 1.79652 94.13 0.01045 total 183.700 1.90854 314 table 5 pairwise difference among shrimp populations (the significance (5 % level) of the fst was tested by 1,000 permutations for each pairwise comparison) population kn ky kt qd rz kn 0.00000 ky 0.03065 0.00000 kt 0.07510* -0.01863 0.00000 qd -0.00094 -0.03762 -0.01306 0.00000 rz 0.17016* 0.04899 0.00763 0.03887 0.00000 1995 to 1998 based on allozyme analysis, and the percentage of polymorphism loci and heterozygosity has also decreased from 1997 to 2001 based on rapd analysis. an investigation of the genetic variation within this species will provide useful information that can be used to manage shrimp stocks and protect genetic variation in this species. the control region in mtdna is a non-coding dna area, which is the most polymorphic region of mtdna genome. of the partial mitochondrial control region (mtcr) sequences identified in the present study, sequence variations were distributed among 13 polymorphic sites across five populations. the pattern of nucleotide substitution in f. chinensis was biased in favour of transitions over transversions in variable sites, consistent with the bias in favour of transitions characteristics of the control region in other animal species (brown et al., 1982; kocher et al., 1989; ram´ırez-mac´ıas et al., 2007). similar to the results of others (shi, 1999; song, 1999; wang, 2001; liu et al., 2004; cui et al., 2007), the present results indicated that the genetic diversity of f. chinensis was low, and maybe the lowest among penaeus species studied so far (hualkasin et al., 2003; mcmillen-jackson and bert, 2003, 2004; tzeng et al., 2004; table 3). the low genetic diversity found in the population kn from narodo island near the jeju island in korea might indicate southern coast population of the korean peninsula to be somewhat different from populations in other areas (liu et al., 2004). as different methods were used in the prior studies, it was difficult to draw a conclusion whether there was a decrease or increase in genetic diversity compared with the current study. even though the same mtcr was used in the present study and in an earlier one (cui et al., 2007), the data were not comparable as the mtcr information was derived differently from restriction fragment length polymorphism (rflp) analysis and the sequencing of partial control region. though the estimated mutation rate was different when using different mtdna markers, the level of genetic variation in chinese shrimp was still low. the lower diversity of chinese qingdao population in comparison with the rz, ky and kt populations, suggested that qd population had undergone a recent population bottleneck or founder event and might has lost some specific alleles. the reduced genetic diversity of f. chinensis in china appeared to be due to the use of a small pool of broodstock during decades of extensive prawn farming and release of the excess postlarvae on a large-scale for stock enhancement (zhuang et al., 2001). integration of genetic diversity data from the present study and previous research indicated that the f. chinensis populations bore low genetic diversity in contrast to other penaeus species, likely due to the reduction of effective population size arising from habitat instability during sea-level variations. because the extent of genetic variation is closely related to the evolution of organisms, food supply and the ability to withstand adverse environment, the low genetic diversity not only challenges the future existence of f. chinensis, but is also one of the reasons why f. chinensis has low resistance against disease and adverse environments. table 6 genetic distances (tamura-nei method) between five populations of f. chinensis from control region sequences population qd rz kt ky kn qd - rz 0.007 - kt 0.006 0.005 - ky 0.007 0.007 0.005 - kn 0.011 0.015 0.008 0.009 - 315 in the present study, amova analysis showed most of the total molecular variance was distributed within populations at different hierarchical levels. all the conventional population fst statistics showed significant difference between the population kn and the other four populations as a north group. within the korean populations, there was a slight differentiation between kn and kt. and the kn and rz population showed a severe differentiation. there was no differentiation between the two chinese populations from the yellow sea. these results were consistent with the opinions that there were two geographic populations of f. chinensis in the yellow sea and the bohai sea from recapture data (deng et al., 1990), and the variation among korea and chinese populations was larger than the variation within populations, indicating the possible differentiation among the two populations (shi et al., 1999). in addition, meng et al. (2004) found over three quarters of variation occurred within samples from the yellow sea and the bohai sea, and this differentiation had taken place to some extent among f. chinensis. the high fst statistics between the population kn and the other four populations as a north group indicated the differentiation between them. although the sampling area and the sampling season were not so strict, (the sampling date for kn, ky, kt was april, may, june in 2008, respectively, and the rz and qd were sampled in august 2008), the results from the present study favoured the definition of a new f. chinensis population near southern korea suggested by a previous study with a different spawning location and migration route from the others (liu et al., 2004). fig. 2 neighbour-joining tree of five populations for shrimp f. chinensis. cui et al. (2007) pointed out that there was little genetic differentiation among f. chinensis populations because of extensive gene flow. in the present study, three haplotypes (ky12, kn02 and qd12) were found in all samples, indicating a common source of origin for these populations and eleven mtcr haplotypes were shared in any two of the shrimp populations suggesting that gene flow was likely to have occurred between samples as a result of the long migrations and wide fig. 3 neighbour-joining tree of mtdna control region haplotypes of shrimp f. chinensis. 316 dispersal of these shrimps. instances of uniform marine populations are regarded to be due either to non-equilibrium populations or to a truly high degree of larval dispersal (palumbi, 2003). however, ten mtcr haplotypes were restricted to one population suggesting there were some barriers between populations, and this was consistant with the previous study by meng et al. (2009) which suggested that the persistent reproductive isolation had likely contributed to the genetic differentiation among geographic populations of f. chinensis in the yellow and bohai sea. with regard to the origin/formation and genetic background of kn were still unknown, different opinions about the genetic differentiation of f. chinensis nevertheless remain. in any case, it is undeniable that low genetic divergence will lead to germplasm depression characterized by slow growth rate, reduced productivity and disease susceptibility (zhang et al. 2002). the mtdna variations of diversity in this species are the legacy of historical events. the current study favoured the previous result from liu et al. (2004), in which a new population of f. chinensis was found around the jeju island near southern korea. given the differences in spawning, mating, migrating time and over-wintering places, f. chinensis may have three geographic populations: the yellow and bohai sea (yb) coast population, the western korean peninsula (kw) coast population, and the southern coast of korean peninsula (ks). f. chinensis is also found in small quantities near the shengsi and zhoushan archipelago in the north part of the east china sea and the mouth of the pear river in the south china sea (liu and zhong, 1988). however the amount of f. chinensis fished in the east china sea and the south china sea was very low and usually mixed with fenneropenaeus penicillatus and other penaeus species (liu et al., 1959, 1988). unlike those individuals in the bohai sea and the yellow sea, which usually migrate twice a year for reproduction and over-wintering during their life cycles , individuals of f. chinensis in the south china sea only move within a small area and show no long distance migration (liu and zhong,1988). this unique behaviour of f. chinensis in the bohai sea and the yellow sea was presumed to have resulted from gradual adaptation during the process of evolution. given the highly migratory nature of f. chinensis in bohai sea and yellow sea, and the lowly migratory nature of f. chinensis in east china sea and south china sea, some tagging studies centred on the spawning location and migration routes for f. chinensis from these areas are crucial to fill important gaps on f. chinensis biology, and particularly migration patterns. acknowledgments the authors would like to thank m walton for kindly read this manuscript, and all labmates for their help with critical steps in the laboratory and their comments on the draft of the manuscript. the work described in this paper was fully supported by united nations development programme (reducing environmental stress in the yellow sea large 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1994. ye, cc. the prawn in bohai sea and their fishery. penaeid shrimp-their biology and management, news books limited farham., surry, england, 49-60, 1984. zhang xc, liang yb, liu ry, wang lj, yang bo. the genetic diversity of reared population of bay scallop argopecten irradians (lamarck). acta. oceanol. sin. 24: 107-113, 2002 (in chinese). http://dlib.cnki.net/kns50/navi/bridge.aspx?linktype=baselink&dbcode=cjfd&tablename=cjfdbaseinfo&field=baseid&value=sckx&navilink=%e6%b0%b4%e4%ba%a7%e5%ad%a6%e6%8a%a5 http://dlib.cnki.net/kns50/navi/bridge.aspx?linktype=baselink&dbcode=cjfd&tablename=cjfdbaseinfo&field=baseid&value=ssdb&navilink=%e4%b8%8a%e6%b5%b7%e6%b0%b4%e4%ba%a7%e5%a4%a7%e5%ad%a6%e5%ad%a6%e6%8a%a5 http://222.195.226.79/kns50/navi/bridge.aspx?linktype=baselink&dbcode=cjfd&tablename=cjfdbaseinfo&field=baseid&value=hyfz&navilink=%e6%b5%b7%e6%b4%8b%e4%b8%8e%e6%b9%96%e6%b2%bc http://222.195.226.79/kns50/navi/bridge.aspx?linktype=baselink&dbcode=cjfd&tablename=cjfdbaseinfo&field=baseid&value=hyfz&navilink=%e6%b5%b7%e6%b4%8b%e4%b8%8e%e6%b9%96%e6%b2%bc http://dlib.cnki.net/kns50/navi/bridge.aspx?linktype=baselink&dbcode=cjfd&tablename=cjfdbaseinfo&field=baseid&value=hysc&navilink=%e6%b5%b7%e6%b4%8b%e6%b0%b4%e4%ba%a7%e7%a0%94%e7%a9%b6 http://dlib.cnki.net/kns50/navi/bridge.aspx?linktype=baselink&dbcode=cjfd&tablename=cjfdbaseinfo&field=baseid&value=hysc&navilink=%e6%b5%b7%e6%b4%8b%e6%b0%b4%e4%ba%a7%e7%a0%94%e7%a9%b6 http://www.medsci.cn/sci/journal.asp?id=e4aa111 http://www.medsci.cn/sci/journal.asp?id=e4aa111 318 zhuang zm, shi t, kong j, liu p, liu zh, meng xh, et al. genetic diversity in penaeus chinensis shrimp as revealed by rapd technique. prog. nat. sci. 11: 432-438, 2001 (in chinese). title: isj 11: 87-102, 2014 issn 1824-307x research report sipunculan celomocytes increase the resistance to h2o2-induced cell death under hypoxia t lombardoa, dm peraltaa, l kornblihttb , ga blancoa alaboratorio deinmunotoxicología (laito), idehu-conicet, hospital de clínicas, josé de san martín, universidad de buenos aires (uba), buenos aires, argentina bservicio de hematología, hospital de clínicas, josé de san martín (uba), buenos aires, argentina accepted march 4, 2014 abstract themiste petricola is a marine intertidal endolithic worm that experiences transient hypoxia within its habitat, owing to natural sediment movements or increased organic enrichment. we characterized and quantified the cytotoxic effect of h2o2 in celomocytes of the sipunculan themiste petricola under normoxia and hypoxia (o2 < 0.1 %) through the median effect method. the 50 % cell death h2o2 dose at 24 h (ec50) under normoxia was 1.5 mm. the range ec10-ec90 was 0.6 mm 3.9 mm. the fraction of cells having collapsed mitochondrial membrane potential (mmp) was increased dosedependently after 3 h exposure with 24 h cytotoxic doses of h2o2 from ec10 to ec90. the 24 h cytotoxic dose inducing 50 % of cells with collapsed mmp at 3 h was 3.67 mm. intracellular superoxide anion production was increased dose-dependently, while reduced glutathione was decreased dosedependently at 3 h with h2o2 from ec10 to ec90. exposure to 24 h hypoxia did not cause cell death but induced intracellular acidification. the 24 h ec50 of h2o2 under hypoxia was increased to 4.7 mm while the range ec10-ec90 was increased to 0.9 mm 25.1 mm. we conclude that hypoxia induces anaerobic metabolism and increases tolerance to h2o2-induced cell death in celomocytes of themiste petricola preserving the immune functions and providing an advantage to survive under low oxygen tension. key words: sipunculans; hypoxia; hydrogen peroxide; ros; cell death; median effect; celomocytes; polychaetes; marine worms introduction sipuncula is a phylum of unsegmented marine celomate worms closely related to the polychaete annelids (cutler 1994; kristof et al., 2008; schulze and rice 2009). themiste petricola is an endolithic intertidal sipunculan species found within sedimentary rocks of variable grades of cohesion (amor et al., 1991). most of sipunculan taxa came into existence in paleozoic and mesozoic times, when oxygen reached near-modern values and this has been suggested to explain why animals radiated so dramatically beginning at about 540 million years (payne et al. 2011; sperling et al., 2013). while intertidal environments may be temporarily deprived of accessible oxygen, sipunculans are oxyconformants and may reduce ___________________________________________________________________________ corresponding author: guillermo a. blanco laboratorio de inmunotoxicología (laito) idehu conicet uba junin 956 4to piso capital federal (1113), argentina e-mail: gblanco@ffyb.uba.ar metabolic rate and energy demand as part of an adaptive response to hypoxia. the oxygen consumption of sipunculus nudus conforms to the ambient oxygen tension (portner et al., 1985) and the same is true for themiste cymodoceae and phascolopsis gouldi (edmonds 1957). however sipunculans can also revert to anaerobic pathways and live without oxygen for several days, producing lactic acid as one of the end products of metabolism (edmonds, 1957). sipunculans have a celomic cavity filled with abundant red cells known as hemerythrocytes carrying the respiratory pigment hemerythrin and leukocytes mainly involved in innate immunity. collectively these cells are also named celomocytes (cutler, 1994; blanco, 2010). sipunculans are devoid of a circulatory and a respiratory system, but movements of the celomic fluid through activities of the animal and circulation in the tentacular system accomplish the functions of circulation and respiration (cavaliere et al., 2010). in ordinary conditions of sipunculan life, the hemerythrin is nearly completely saturated with oxygen, and hence 87 is well adapted to the needs of the animal.(hyman, 1959; meyer and lieb, 2010). oxidative stress is defined as an imbalance between oxidants and antioxidants which results in an excess of oxidative species (ros) that leads to disruption in signalling, redox control, and molecular damage (sheehan and mcdonagh, 2008). when pro-oxidants and antioxidants are in a steady state, ros are involved in intra and intercellular communication, and when the equilibrium is shifted towards pro-oxidants the damage to intracellular constituents ensues (comhair and erzurum, 2002; speakman and selman, 2011). environmental pollutants including heavy metals, polycyclic aromatic hydrocarbons, and polychlorinated biphenyls have the potential to cause oxidative stress in aquatic organisms through ros mechanisms (sheehan and mcdonagh, 2008). aquatic organisms can uptake pollutants that cause oxidative stress damage from sediments and food sources (valavanidis et al., 2006). marine invertebrates, especially bivalve mollusks, have been used extensively as sensitive bioindicators for aquatic pollutants associated with ros generation (chora et al., 2008; mcdonagh and sheehan, 2008). hypoxia is a known source of oxidative stress in vertebrate cells (lushchak and bagnyukova, 2007). for example, hypoxia caused a dose-related increase in ros production in cardiac myocytes (duranteau et al., 1998) and mammalian cell lines (chandel et al., 1998). in animals with closed circulatory system hypoxia-induced ros facilitates a rapid microvascular inflammatory response characterized by enhanced leukocyte-endothelial adherence and emigration, which increases vascular permeability (wood et al., 1999; peng et al., 2003). additionally ros may be involved in defence against pathogens during phagocytosis (oxidative burst) (fuller-espie et al., 2010). even though hypoxia causes increased ros several antioxidant mechanisms are also increased together with metabolic changes to increase anaerobic respiration (lushchak and bagnyukova, 2007). mitochondrial respiration, where oxygen is the final acceptor of electrons, is the main source of ros, and superoxide anion (o2 -), hydroxyl radical and hydrogen peroxide (h2o2) are produced even under normal metabolism. superoxides are relatively unstable, with a half-life of only milliseconds, and do not easily cross cell membranes although it may cause damage to amino acids or loss of protein function (valko et al., 2007). the most harmful result of excessive intracellular ros production is cell death. most often cell death is initiated in the mitochondria where ros concentration may be five to ten times higher than the cytosol (cadenas and davies, 2000). peroxidation of mitochondrial lipids compromises the normal function of the electron transport chain (etc) leading to lower mitochondrial membrane potential (mmp) and altered regulation of ca2+. this in turn may initiate programmed cell death (zhang et al., 1990). although h2o2 is not a free radical, it is an extremely harmful ros because it acts as an intermediate in hydroxyl radical producing reactions, such as fenton’s reaction table 1 cytotoxic doses of h2o2 in celomocytes (*) n is the number of cells used to determine the fraction of dead cells per sample; n is the number of samples used for regression analysis. (koppenol, 2001). h2o2 has a long half-life and is able to cross several lipid layers and react with transition metals and some hemoproteins (miller et al., 2010). it can also induce dna damage, chromosomal alterations, and oxidize sulfhydryl compounds (cantoni et al., 1989). when h2o2 is added experimentally at increasing doses, an active programmed cell death is elicited while higher doses will induce passive necrosis (jiang et al., 2013). in vertebrate cells active cell death by excess oxidative damage caused by h2o2 is initiated at the mitochondria (luo et al., 2010; palomba et al., 1999). this is similar to the cytotoxic mechanism of several xenobiotics including heavy metals and pesticides that target the mitochondria and cause abnormal function of the etc with increased ros production, decreased mmp and initiation of cell death (wang et al., 2012). we have previously shown that h2o2 induces apoptotic-like cell death in celomocytes of t. petricola (blanco et al., 2005). however the celomocyte population includes cell types such as hemerythrocytes and leukocytes that may exhibit differential sensitivity over a range of h2o2 doses. in this study we assessed the whole range of cytotoxic effects of h2o2 in celomocytes by the median effect method, which is basically a log-transformed regression method where parameters of a sigmoid dose-response curve are estimated (chou, 2011; lombardo et al., 2012, 2011). we also characterized the early effects caused by h2o2 exposure including mitochondrial damage, ros production and anti-oxidative response in normoxia, and we further evaluated the cytotoxic effect of h2o2 in celomocytes under hypoxia. 88 materials and methods worms adult themiste petricola were collected from crevices in intertidal rocks at santa elena beach on the coast of argentina (34°s latitude) and maintained in plastic boxes with frequently renewed filtered sea water with 32 parts per thousand (ppt) salinity at 18 °c. the salinity of the sea water that the subject worms were originally found in was 32 ppt. celomocyte suspensions were prepared as indicated previously (blanco et al., 2005). briefly, celomic fluid was harvested by incision of the body wall with a sterile surgical blade, allowing the fluid to drip into a 15 ml tube. germinal cells were excluded with sterile filters of 30 µm mesh. the suspension was washed two times by centrifugation in a saline solution made of 26g/l nacl (890 mosm; 0.444 m nacl) with 5 % sea water (v/v). addition of 5 % sea water depletes celomic fluid from large and small granular leukocytes by forming a small but macroscopic clot (blanco, 2010), while washing in 0.4 m nacl alone preserves both types of granular cells. the cell suspension was washed once more in rpmi-1640 (invitrogen, argentina) supplemented with l-glutamine, 17g/l nacl, and sodium bicarbonate ph 7.4 (900 mosm/kg). all subsequent references to rpmi correspond to this modified culture medium. reagents and in vitro culture of celomocytes fresh stock solutions of h2o2 were prepared for each experiment. fluorescein-diacetate (fda), dihydroethidine (he), tetramethylrhodamine-ester (tmre), 5-chloromethylfluorescein (5cmf), 4´,6´diamidine-2-phenylindol (dapi) and propidium iodide (pi) were purchased to invitrogen. the stock solutions of he and pi were prepared in dmso and the rest in phosphate buffer saline (pbs, 295 mosm). assessment of cell death celomocytes harvested from five to ten worms as described above were suspended in rpmi and decanted in 24-well culture plates 1.0x106 per well in 0.5 ml. serial dilutions of h2o2 in 0.5 ml rpmi were added in triplicate. rpmi (0.5 ml per well) was added as untreated control in triplicate. experiments were repeated widening, narrowing or shifting the dose ranges and intervals between doses until a suitable scheme was found to cover the entire effect range (i.e., from no cytotoxic effect to maximal cytotoxic effect). this was necessary because the median effect method requires experimental values of cytotoxicity to cover the entire range of effects from no effect (negative control) to 100 % effect (maximum positive effect control) with sufficient intermediate values to obtain statistically precise estimates of the slope (m) and intercept (– m . log[dm]) parameters in a log-linear regression (see description of the median effect method in the section below). the plates were incubated for 24 h at 18 °c. to identify dead and live cells samples were incubated in 1 μm fda in rpmi for 15 min, washed three times in nacl 0.4m by centrifugation at 300xg during 5 min at rt, transferred to flow cytometry tubes, stained with 2 μm pi for 5 min in 1ml nacl 0.4m, and run in a partec pas iii flow cytometer equipped with a 100 w uv-mercury lamp source light and a 20 mw 488 argon laser (partec, gmbh, münster, germany). a total of 20,000 events were analyzed per sample tube containing 1 x 106 cells in 1 ml of 0.4m nacl. each sample tube corresponded to cells harvested from each well of a 24-well plate. the fda and pi fluorescence were collected through a 535/15 nm and 680/15 bandpath filter respectively. quadrant analysis was done with flomax software (partec, germany) and winmdi 2.8 (scripps research institute, la jolla, ca, usa). median-effect and combination index analysis of cytotoxicity live vs. dead discrimination at the single cell level through flow cytometry allowed us to use a quantal dose-response model where fa was the fraction of dead cells (obtained from the percentage of cells in upper quadrants of flow cytometry dot plots) while fu was the fraction of live cells and fu = (1-fa) (lombardo et al., 2012). we further created a median-effect plot as log (d) vs. log (fa/fu), where d was the dose of h2o2 in each experimental sample point (each well of a 24-well culture plate). by linear regression we obtained the slope and the intercept estimates of the equation log(fa/fu) = m . log(d) – m . log(dm) to further derive the estimate of the median effect dose (dm). with the slope m and the median effect dose dm we derived the median effect formula for h2o2: d=dm (fa/(1-fa))1/m with this formula we could further estimate the dose d of h2o2 that induces cytotoxicity in a fraction fa of cells in a 24 h incubation experiment. thus we refer to the fa value as cytotoxic effect level (ec), and we denote the estimated dose dm that kills 50 % of cells (fa = 0.5) in a 24 h assay as ec50 %. in the same way a dose d that according to the formula is estimated to kill 30 % of cells (fa = 0.3) in a 24 h assay is referred to as ec30 and so on for any arbitrary cytotoxic effect level. the same method was used to determine the dose d of h2o2 that causes the collapse of mitochondrial membrane potential in a 3 h assay, and that we refer to as ed. in this case the fraction fa was derived from the percentage of cells with complete collapse of the mmp, which in turn was obtained from histograms of each of the replicates. further details of the median effect method can be found in (chou, 2011; lombardo et al., 2011, 2012). calculations were performed with the software calcusyn (biosoft, uk). the se of ec% values were calculated with calcusyn software according to the following formula (lombardo et al., 2012): se(d) = 1/2 . {10 [log(d)+se(log(d)]-10 [log(d)-se(log(d)]} where: se(log (d)) = {log(d). [se(b)/log(fa/(1-fa)b]2+[se(m)/m]2+2[-(log(d))1/2. se(m)/se(b)]. se(b)/b. se(m)/m}1/2, and b = -m . log(dm), dm=ec50, and d=ec% for each particular effect level. 89 assessment under hypoxia to assess the effect of h2o2 under hypoxia the culture plates were prepared as described above and were further placed in a mic-101 chamber (bilrups rothemberg, usa), connected to a dual flow meter that supplied a gas mixture containing 95 % n and 5 % co2. under such conditions the chamber is designed to keep o2 concentrations below 0.1 % when flushed for at list 10 min. evaluation of reduced glutathione (gsh) intracellular content after exposing celomocytes to h2o2 for the appropriate time they were washed by centrifugation at 300xg during 5 min at rt in 1 ml of 0.4m nacl to avoid the interference of thiol groups that could be present in the rpmi medium, and were further incubated in 0.3 μm 5cmf in 1 ml of 0.4m nacl during 20 min at rt. this probe becomes fluorescent in live cells and then binds covalently to gsh with high specificity (sarkar et al., 2009). cells were washed once more at 300xg during 5 min at rt in 1 ml of 0.4m nacl and then pi was added at a final cc of 2 μm in 0.4 m nacl to obtain an additional and independent parameter of cell death. assessment of o2 production and statistical analysis he, a probe that is oxidized to ethidium by intracellular o2 (chen et al., 2009) was prepared as a 5 mm stock solution in dmso and stored at -70 °c. a 500 μm working solution was prepared by diluting 1/100 the stock solution in 0.4 m nacl. after exposure to h2o2 in 3 h assays cells were washed and incubated during 20 min at rt with he 2 μm final concentration. cells were washed again by centrifugation at 300xg during 5 min at rt in 1 ml of 0.4m nacl and further analyzed by flow cytometry. fluorescence of a positive control made by exposing a sample of cells to 50 μm carbonyl cyanide m-chlorophenyl hydrazone (cccp; sigmaaldrich) in 1 ml of 0.4m nacl 10 min prior to adding the he probe, and basal fluorescence of nontreated samples were used as reference to compare changes induced by h2o2 treatment. assessment of mitochondrial membrane potential (mmp) celomocytes were treated with h2o2 during different time intervals at a fixed ec50 dose or at a fixed time interval of 3 h at increasing doses of h2o2 in 24-well culture plates (1x106 per well in 1 ml rpmi) at 18 °c. each dose or time point was assessed in triplicate. the cells from each well were further transferred to microcentrifuge tubes and centrifuged at 300xg at rt during 5 min. the supernatant was discarded and the cells were suspended 1 ml of nacl 0.4 m with the potentiometric probe tmre 0.05 μm final cc during 30 min. a positive control was included by treating a sample of cells (1x106 in 1 ml of 0.4 m nacl) during 10 min with the mitochondrial electron transport chain uncoupler cccp (50 μm final cc). this treatment, which was applied prior to labelling with tmre, causes complete collapse of mmp and serves as a positive control of depolarizing effect. the untreated samples with normal mmp served as negative controls or baseline of depolarizing effect. after labelling cells with the probe tmre cells were washed by centrifugation at 300xg at rt in 1 ml 0.4 m nacl. the cells were finally suspended in 1 ml nacl 0.4 m, transferred to flow cytometry tubes and analyzed by flow cytometry. a total of 20,000 events were acquired from each of the 24 tubes. assessment of phagocytosis zymosan particles (sigma) were suspended at 1 mg/ml in 0.4 m nacl and labelled with dapi 10 μg/ml final cc at rt during 1 h. this suspension was washed three times by centrifugation at 900xg in 0.4 m nacl during 5 min at rt. celomocytes were incubated in 24-well culture plates with these labelled particles at three time intervals (1 h to 3 h) and two concentrations (250 μg/ml and 500 μg/ml). to assess production of o2 during phagocytosis the samples were further incubated with he as described in the correspondent section above. a sample of celomocytes was exposed to ec90 h2o2 (3.9 mm) for 18 h at 18 °c to obtain a preparation of cells undergoing cell death. these cells were labelled with 10 μg/ml dapi in rpmi. a separate sample of untreated cells was suspended at 1x106/ml in rpmi with 1 μm fda and incubated for 15 min at 18 °c. these cells were washed three times in rpmi by centrifugation at 300xg during 5 min at rt and decanted into a 24-well culture plate at 1x106 per well in 0.5 ml. the dapi-labelled cells were added at 1x106 per well in 0.5 ml and the coculture was incubated for another 4h to allow live fda-stained cells to phagocytose dapi-stained dead cells. after incubation the samples were transferred to flow cytometry tubes then analyzed with dual 360 nm uv and 488 nm blue excited flow cytometry. additional controls included fda-stained cells without dapi-stained cells, dapi-stained cells without fda-stained cells, dapi-labelled zymosan particles and unlabelled zymosan particles. a total of 20,000 events were acquired from each flow cytometry tube containing more than 1x106 cells in 1 ml. statistical analysis flow cytometry histograms or bivariate plots were analyzed using multiparametric gating with the software winmdi 2.8 (scripps research institute, la jolla, ca, usa). the frequency values of cell death were obtained from quadrant analysis of fda vs. pi plots in triplicates (20,000 cells acquired per sample) for each of the doses tested. the frequency values of cells with complete collapse of mmp were obtained from tmre fluorescence histograms in triplicates (20,000 cells acquired per sample) for each of the doses tested. these data were further used in a log-transformed regression as indicated in the correspondent section above. for statistical analysis of fluorescence intensity the original logfluorescence data were exported with winmdi software to graph pad prism 4.0 software (graphpad, san diego, usa). we applied the nonparametric kruskal-wallis test for differences between fluorescence medians among all treatments followed by dunn´s post hoc test to evaluate differences of the medians between treated and non-treated samples (lombardo et al., 90 fig. 1 assessment of dose-dependent h2o2-induced cell death after 24 h incubation at 18 °c. a) changes in the forward (fsc) and side (ssc) light dispersion of celomocytes exposed at increasing doses of h2o2. the red arrow indicates the progression of fsc-ssc changes during h2o2-induced cell death ("death pathway"). each density plot corresponds to a representative single sample tube from triplicates for each dose (20,000 acquired per tube). b) discrimination between live and dead cells by metabolic activity (fda) and membrane damage (pi); the amount of dead cells increased dose-dependently with h2o2 exposure. live cells were detected as fda positive and pi negative. each density plot corresponds to a single sample tube from triplicates for each dose (20,000 acquired per tube) c) calculation of ec50 of h2o2 applying a regression of log-transformed data and using the median effect equation (left). the slope and the intercept obtained were used to calculate the ec50 and the parameters of the dose-effect curve (right). red dots correspond to experimental values. the ec% values estimated from the median effect equation (together with the estimated se for each ec% dose) and further used in this study are shown in table 1. see materials and methods section for details on the median effect equation. 91 fig. 2 evaluation of mmp after exposure of celomocytes to h2o2 . a) effect of ec50 h2o2 at increasing time intervals (up to 6 h). representative histograms of cells labelled with the potentiometric probe tmre and assessed at increasing time points are shown. m1 refers to the region of cells having a positive mmp while m2 refers to the region of cells with collapsed mmp. the histogram labelled 0h-cccp corresponds to a positive control of complete collapse of mmp made of untreated cells exposed to the mitochondrial etc uncoupler cccp and used to accurately define the m2 and m1 regions. the histogram labelled 0h is a negative control of complete collapse of mmp and is a baseline corresponding to the normal mmp of untreated cells b) bar graph showing that the percentage of ec50 h2o2-treated cells with collapsed mmp (cells in m2) increased with time length up to 3 h when it achieved a plateau (scale on left axis). dots correspond to the mean tmre value (from triplicates) of cells that still kept a positive mmp (cells in m1 region) and the scale is shown on the right axis. error bars around dots or grey bars correspond to the mean value ± sd of triplicates. c) representative histograms of cells treated with increasing doses of h2o2 and evaluated after 3 h for tmre fluorescence. insets show fsc-ssc density plots to appreciate the shape changes after 3 h exposure. d) bar graph showing that the amount of cells with collapsed mmp increased with increasing doses of h2o2 (left axis). in addition those cells that still kept a positive mmp showed a progressively decreased mean value of tmre fluorescence (black dots with scale on right axis). error line bars around dots or grey striped bars correspond to the mean value ± sd from triplicates. for the actual h2o2 concentration of ec% values see table1. e) calculation of the h2o2 dose that causes mmp collapse at 3 h in 50 % of cells (ed50) applying a linear regression of log-transformed data and the median effect equation. a plot of the complete dose-effect equation (blue line) together with experimental values (red dots) is shown. see materials and methods section for additional details on the median effect equation and the calculation method. 92 2011). box-whisker plots were obtained with graphpad prism 4.0 as part of the exploratory data analysis to further visualize the h2o2-induced changes in fluorescence intensity frequency distribution. correlation analysis between gsh content and superoxide anion production was conducted by calculation of the pearsons´s r correlation coefficient with graphpad prism 4.0 software. nonlinear regression was applied to a hyperbolic function of the type y = k/x+b representing the inverse relationship between gsh and superoxide anion using graph pad prism 4.0 software. results dose-related h2o2-induced cell death in sipunculan celomocytes celomocytes exposed to h2o2 over a range 0.25 mm to 5.0 mm during 24 h showed a doserelated increase in the amount of dead cells as determined by fda and pi labelling and flow cytometry assessment. increasing doses of h2o2 also caused changes in cell shape as indicated by forward and side light dispersion in flow cytometry bivariate plots (figs 1a, b). at low effect doses within the range 0.25 mm to 1.0 mm h2o2 we observed an increase in the side scatter light parameter (ssc) of cells but at the same time only a slight increase in the fraction of dead cells which remained below 11 %. however at doses above 1.0 mm h2o2 both the fraction of dead cells and the ssc were increased while the forward light scatter (fsc) was decreased (figs 1a, b). this profile became clearer when experimental values were used to calculate the h2o2 dose causing death in 50 % of cells (ec50 = 1.52 mm, se = 0.10) and to obtain a sigmoid dose-effect curve (fig. 1c). the linear increase in cell death was observed between 1.0 mm and 2.0 mm (fig. 1c). we presumed that the increase in ssc without increase in cell death at low h2o2 doses could be due to the fact that phagocytes, which may have a higher oxidative stress tolerance, could be engulfing dead cells (or even cells undergoing programmed cell death) that were sensitive to lower doses of h2o2. thus we explored this possibility in a section below. as expected, the sigmoid shape and the steepness of the linear segment showed that there was a range of susceptibilities to h2o2-induced cell death among celomocytes, where some cells may be killed with 1.0 mm h2o2 while others may tolerate more than 2.0 mm h2o2. the actual h2o2 concentrations for ec% values derived from the median effect equation and used in the study are show in table 1. assessment of mitochondrial damage caused by h2o2 exposure we further evaluated mmp through the potentiometric probe tmre at several time points after exposing cells to the 24 h ec50 h2o2 (1.5 mm). the proportion of cells with collapsed mmp increased linearly up to 3 h and remained constant afterwards (figs 2a, b). in addition those cells that still kept a positive mmp maintained stable and high polarization as indicated by a steady value of the mean fluorescence of tmre. we next evaluated the mmp at 3 h post-exposure at several doses of h2o2 ranging from ec0 to ec98. we observed a dosedependent increase in the amount of cells with collapsed mmp and also a progressive decrease in mmp among those cells that still kept a positive mmp (figs 2c, d). we next calculated the dose of h2o2 that causes the complete collapse of mmp in 50 % of cells (ed50 = 3.67 mm) by the method of the median effect equation, and we further obtained the parameters of a curve describing the relationship of h2o2 dose to mmp collapse at 3 h (fig. 2e). thus, by comparing this curve with the one in figure 1c, the fraction of cells with collapsed mmp at 3 h can be used as a biomarker to forecast the amount of cell death that will be observed at 24 h. mitochondrial damage causes an increase in oxidative stress the direct consequence of mitochondrial damage originated in the pulse of h2o2 is the failure of etc and the production of ros. the production of o2 was increased dose-dependently at 3 h in agreement with increased mitochondrial failure (figs 3a, b). to evaluate if celomocytes attempted to reduce the impact of increased ros production by means of an antioxidant response we measured the amount of intracellular gsh by the fluorescent probe 5cmf at 3 h, since gsh is one of the most ubiquitous and efficient cellular antioxidants. as expected, increasing doses of h2o2 caused a progressive decrease in the amount of gsh detected (figs 4a, b). the amount of gsh detected was inversely correlated to the amount of intracellular o2 , and also inversely correlated to the amount of cells showing complete collapse of mmp (figs 4c, d). phagocytosis may contribute to eliminate potentially harmful damaged self cells we evaluated the ability of celomocytes to phagocytose dapi-stained zymosan particles. as shown in the left panel of figure 5a cells unexposed to dapi-stained zymosan had no blue fluorescence because no engulfment of fluorescent particles occurred. this sample represented a negative control or baseline of blue autofluorescence for comparison with cells exposed to dapi-stained zymosan particles. in the right panel of figure 5a cells that engulfed dapi-stained zymosan particles appear above the baseline of blue autofluorescence. the fact that fsc of the zymosan-exposed cells had a similar distribution as untreated cells denotes that these cells had acquired the blue fluorescence by engulfing the already labelled dapi-stained zymosan particles. as shown in figure 5b when celomocytes were exposed to dapi-stained zymosan the highest amount of intracellular particles was detected after 1 h and decreased progressively at 2 h and 3 h. in addition at the 1 h time point the phagocytosis of dapi-stained zymosan particles was coincident with an increase in o2 production as detected by the simultaneous presence of dapi-stained zymosan particles (blue fluorescence) and increased he fluorescence by flow cytometry (fig. 5c). therefore, 93 fig. 3 assessment of intracellular o2production after 3 h incubation at 18 °c with increasing doses of h2o2. a) overlaid histograms of cells treated with increasing doses of h2o2 and evaluated after 3 h for he fluorescence indicating the amount intracellular o2 production. b) box whisker plots of the samples shown in a. data from 20,000 events from each sample were exported to the software graphpad prism 4.0 for further statistical analysis. median values were compared by the non-parametric kruskal-wallis test with significance p < 0.001 and median values of h2o2-treated samples and the cccp control were compared to untreated cells using dunn´s post test for median differences. ***: p < 0.001 for difference with median of untreated cells in dunn´s post test. whiskers represent ± 1.5 inter-quartile distance (iqd) (le meur et al., 2007). among celomocytes exposed to h2o2 the phagocytes could be the more resistant ones and could also be responsible for eliminating the more sensitive cells that undergo cell death at the lower doses. to provide some evidence to this hypothesis we exposed cells to h2o2 at a high dose (ec90) during 18 h. we then labelled these cells with dapi, and we further incubated these dead cells during 4h more with live cells that were previously labelled with fda to make them green fluorescent. as shown in fig. 6a live and dead cells had completely different fsc vs. ssc profile. we then identified the amount of live cells that had engulfed dead cells, the amount of cells that were alive but had not engulfed dead cells and the amount of dead cells that had not been engulfed and remained free (figs 6b, c). this experiment proved that phagocytes can actively engulf dead cells and in fact it could be possible that they can also engulf cells that are still alive but undergoing a death program as a mean to eliminate a potential source of ros and tissue damage. as shown in fig. 6c those live cells that actively engulfed dead cells can exhibit an increase in ssc as was observed in figure 1a. h2o2-induced cell death is decreased under hypoxia when celomocytes were exposed for 24 h to less than 0.1 % o2 but in the absence of h2o2, they survived without any change in cell viability. however the culture media shifted to a strong yellow color indicating a rapid acidification of the extracellular media (data not shown), while the fda 94 fig. 4 assessment of intracellular gsh level after 3 h incubation at 18 °c with increasing doses of h2o2. a) representative histograms of cells treated with increasing doses of h2o2 and evaluated after 3 h with 5cmf to measure the level of intracellular gsh. insets show fsc-ssc density plots of the same sample to appreciate the shape changes induced after 3h exposure. b) bar graph showing that intracellular gsh decreases with increasing doses of h2o2 when evaluated after 3h. error bars correspond to the mean value ± sd from triplicates. c) correlation analysis of intracellular gsh content vs. intracellular o2 production. pearson´s correlation coefficient r was -0.67 with significance p < 0.05. the nonlinear regression curve representing the inverse relationship corresponds to a hyperbolic function of the type y = k/x+b (r2 = 0.64). d) correlation analysis of gsh content vs. amount of cells with collapsed mmp having a pearson´s correlation coefficient of -0.80 with p < 0.001. the nonlinear regression curve also corresponds to a hyperbolic function of the type y = k/x+b (r2 = 0.75). fluorescence intensity of hypoxic celomocytes was reduced indicating that the cytoplasm had also become acid (figs 7a, b). the reduction of intensity was about 100 times and the reason why fluorescence decreases is that fda light absorption at 488 nm is reduced depending on how low the intracellular ph is (geisow, 1984; martin and lindqvist, 1975). this property has been used to measure intracellular acidification with fda in nerve cells under intense glycolisis and high production of lactic acid (khodorov et al., 1994). the ec50 of h2o2 under hypoxia was increased to 4.72 ± 0.31 mm (mean ± se estimated from linear regression of log-transformed data using the median effect equation), and the dose effect curve was deviated to the right indicating that cells became more tolerant to oxidative stress as compared to the same doses applied under normoxia (figs 7c e). the ec% values for other cytotoxic effect levels under hypoxia are shown in table 1. of notice, in samples treated with h2o2 under hypoxia the fda fluorescence was decreased but to a less extent than untreated samples (about 10 times reduction compared to 100 times reduction in untreated samples under hypoxia; figs 7b d). this acidification under severe hypoxia is known as "pasteur effect" and is due to enhanced anaerobic glycolisis with overproduction of lactic acid. thus, even though hypoxia may trigger antioxidant defences, h2o2 may also compromise the efficiency 95 fig. 5 phagocytosis and oxidative burst in celomocytes exposed to zymosan. a) phagocytosis of dapi-stained zymosan particles by celomocytes. the left panel shows a negative control of phagocytosis where only baseline levels of blue fluorescence are present. in the right panel those cells that engulfed zymosan particles became blue-fluorescent (z-dapi y axis) and appear above the baseline of blue autofluorescence. the percentage of celomocytes above this value indicates the percentage of phagocytic cells free non-phagocytosed particles are excluded due to their low fsc signal. b) phagocytosis of dapi-stained zymosan particles by celomocytes evaluated at three time points and two concentrations of target particles. bars indicate the mean value ± sd of cells that engulfed fluorescent zymosan particles from triplicates. c) the median red fluorescence of he indicating o2 production among cells that engulfed dapi-stained zymosan particles (blue fluorescent) was computed as an indicator of the amount of intracellular oxidative burst during phagocytosis. data from 20,000 events from each sample were exported to the software graphpad prism 4.0 for further statistical analysis. the box-whisker plot corresponds to cells exposed to 500μg/ml dapi-stained zymosan and evaluated after 1, 2 and 3 h, with he. cccp corresponds to the positive control for o2 production. treatments were compared with the non-parametric kruskal-wallis test with significance p < 0.001 and medians further compared by dunn´s post test. ***: p < 0.001 and **: p < 0.01 for differences with median of untreated cells in dunn´s post test. whiskers represent ± 1.5 iqd (le meur et al., 2007). 96 fig. 6 phagocytosis of dead celomocytes by live celomocytes. a) density plots showing fsc and ssc in live untreated cells (left) and changes occurring in dead cells (right) after 18 h culture with ec90 h2o2 at 18 °c. b) the suspension of dead cells was labelled with dapi, which is a nonpermeant dye excluded by live cells similarly to pi but has blue fluorescence, while the suspension of live cells was stained with fda (green fluorescence of fda indicates metabolic activity of viable cells) and then they were coincubated for 4 h at 18 °c. the left panel shows the fsc vs. ssc distribution of coincubated cells. the right panel shows the bivariate distribution of fda vs. dapi fluorescence that allows distinction of three clusters located in three separate regions r1, r2, and r3. region r1 corresponds to live cells that did not engulfed dapi-stained dead cells and thus remain fda single positive. region r2 corresponds to dapi-stained cells that were not engulfed. region r3 corresponds to live cells (fda positive) that have engulfed dapi-stained dead cells and became blue fluorescent in addition to being green fluorescent (i.e., double positive). c) backgating of the three regions identified in b) applied to show the individual ssc characteristics of each group. live cells that did not engulf dead cells have no change in ssc (r1, left panel). dead cells that were not engulfed by live cells had increased ssc (r2, mid panel). the majority of live cells that engulfed dapi-stained dead cells had no change in ssc (85.5 %) but a fraction of them showed increased ssc (14.5 %) corresponding to the most active phagocytic cells. 97 of anaerobic glycolisis. in addition the range ec10ec90 was increased from [0.58 mm 3.94 mm] under normoxia to [0.89 mm 25.0 mm] under hypoxia (table1) and the steepness of the linear part of the dose-response curve was decreased. the slope of the log regression was 2.32 under normoxia (fig. 1c left panel) and was reduced to 1.30 under hypoxia (fig. 7e left panel). this indicates that the variability in the susceptibilities to h2o2-induced cell death among celomocytes was also increased under hypoxia. discussion in this study we characterized the dose-effect relationship of h2o2-induced cell death evaluated at 24 h in sipunculan celomocytes under normoxia and hypoxia and we also characterized the dose-effect profile of some early indicators of oxidative damage and antioxidant response. marine coastal pollution often involves increased levels of heavy metals and pesticides that negatively impact the benthic communities. at the cellular level these pollutants often increase oxidative stress, causing altered cellular functions and eventually cell death. at the same time tidal flat habitats are transiently exposed to hypoxic conditions resulting from organic enrichment, algal bloom decomposition, and sediment movements in estuarine mudflats and litoral tidal flats (lim et al., 2006; neira et al., 2006). in fact, low-oxygen conditions are important in structuring the diversity and abundance of benthic communities (van colen et al., 2010). our model of h2o2-induced cell death resembles a variety of cell injury situations caused by agents targeting the mitochondria. many xenobiotics including several heavy metals and some pesticides target the mitochondria and cause increased intracellular production of ros (bagchi et al., 2002). as shown above, a single pulse of h2o2 caused cell death at 24 h in a dose-dependent manner. however, the sigmoid shape of the curve indicated that a linear dose-dependent increase in cell death is present only at doses above ec30 corresponding to 1.0 mm h2o2. in addition the smooth slope of this linear phase of the dose-effect curve indicated that celomocytes were heterogeneous in their susceptibility to h2o2induced cell death. the dose range, another indicator of variable susceptibility, was 0.6 mm-3.9 mm for ec10-ec90 indicating that some celomocytes may require up to 7 times more h2o2 than others to be killed. the level of mitochondrial damage caused by the pulse of h2o2, as determined by the amount of cells with collapsed mmp, became stabilized after 3 h. thus we explored the effect of a range of doses causing a known level of cell death at 24 h over some biomarkers of oxidative damage assessed at 3h post-exposure. both the amount of cells with collapsed mmp and intracellular o2 production were increased after 3 h exposure in a dose dependent manner consistent with the idea that an increase in mitochondrial damage will cause an increase in intracellular ros level. gsh is the most abundant intracellular thiol and plays an essential role in maintaining the intracellular redox environment (meister and anderson, 1983). the h2o2 dose-dependent decrease in gsh at 3 h indicated a widespread oxidation of thiols associated with increased intracellular ros in an attempt to reduce the impact of oxidative damage. the negative correlations gsh level vs. o2 , and gsh vs. cells with collapsed mmp confirmed this interpretation. interestingly, a decrease in cellular gsh concentration has been reported to be an early event in the apoptotic cascade induced by death receptor activation, mitochondrial apoptotic signalling, drug exposure and oxidative stress (circu and aw, 2008). thus, o2 production, the amount of cells with collapsed mmp and the reduction in gsh can all be used as early biomarkers of effect anticipating the amount of cell death caused by oxidative damage at 24h and could be of potential ecotoxicological usefulness (da silva et al., 1998). the most abundant cells in the celomic fluid are hemerythrocytes which are involved in o2 transport (above 75 %), while phagocytic cells with a diameter close to that of hemerythrocytes are the second largest cell type representing up to 25 % of celomocytes. this percentage figures apply to suspensions of celomocytes prepared under the conditions used in this study that exclude gonadal cells and two granular cell types designated large and small granular leukocytes (for a detailed description and discussion of cell types and relative proportions found in t. petricola and its comparison to other species of sipuncula see (blanco, 2010). we showed that phagocytes produced o2 after engulfment of zymosan, and that o2 level peaked at 1 h but decreased afterwards providing evidence of the occurrence of oxidative burst (chang et al., 2006; xian et al., 2010). thus phagocytes should be well equipped to tolerate transient increases of intracellular ros and may be more resistant to h2o2 than hemerythrocytes. the sigmoid shape of the cytotoxic h2o2 dose-effect curve showed that at lower doses the rate of change in cell death effect was low. we also observed that even though cell death rate was low in these samples, the net number of cells was decreased. we also noted that ssc was increased even with the lower doses and that mmp dose effect curve was less sigmoid and more linear in shape at low doses. thus we speculated that phagocytes could be engulfing the more susceptible celomocytes that were killed at lower doses masking the true rate of dead cells. as shown in figure 6, healthy phagocytes were able to engulf h2o2-treated cells causing them to increase their own ssc signal. uptake of dead or dying cells should be important in the study of cell death in mixed populations where phagocytes may completely eliminate apoptotic cells before they can be detected by regular methods such as fluorescent annexin-v labelling. paradoxically uptake of dead cells will be even more evident at low cytotoxic effect doses where the apoptotic phenotype is supposed to be more readily detected. in fact the finding of engulfed dead cells by resident macrophages has been underscored as a robust apoptotic indicator in tissues (kroemer et al., 2009). exposure of phosphatidylserine is known as an "eat me" signal in eukaryotic cells and the phagocytosis of 98 fig. 7 comparative effects of h2o2 under hypoxia and normoxia. a) density plots showing fsc vs. ssc parameters (left) and fda vs. pi (right) of untreated cells cultured for 24 h under normoxia at 18 °c. fda fluorescence ranges between 100 and 1000 over a log scale. b) a similar set of density plots of untreated cells cultured for 24 h at 18 °c under hypoxia. fda fluorescence is decreased to values around 10 in a log scale owing to strong ph decrease (khodorov et al., 1994). however viability remains the same as cells cultured in normoxia, as indicated by the complete exclusion of the pi probe (right plot). c) density plots of cells treated with ec50 h2o2 under normoxia. fda fluorescence ranges between 100 and 1,000 in a log scale. d) density plots of cells treated with ec50 h2o2 under hypoxia. viability remains high and fda fluorescence is decreased although to a lesser extent than untreated cells shown in b). e) calculation of ec50 h2o2 in cells cultured under hypoxia applying a regression of log data and using the median effect equation (left). the slope and the intercept obtained were used to calculate the ec50 (mean ± se = 4.72 mm ± 0.31; n = 24) and the parameters of the dose-effect curve under hypoxia and compared to the curve obtained under normoxia (right). red dots correspond to experimental values. 99 apoptotic cells may also contribute to eliminate the release of potentially harmful material including ros production and intracellular pathogens (birge and ucker, 2008; krysko and vandenabeele, 2008). we have already demonstrated in a previous work that h2o2 induces apoptotic cell death using several classical morphological and biochemical indicators of apoptotic death phenotype such as chromatin condensation, nuclear membrane rippling, cell membrane blebbing, dna degradation to multiples of oligonucleosomal size, mitochondrial membrane depolarization and exposure of phosphatidylserine in the outer leaflet of the cell membrane (blanco et al., 2005). in the present study we did not address the question of whether h2o2 used at a fixed dose induces apoptosis or alternative forms of cell death but instead we evaluated how sensitive or resistant were celomocytes to h2o2 under hypoxia as compared to normoxia. although apoptotic indicators are good measures of cell death phenotype they are not necessarily good measures of cell death rates to calculate ec50 dose. many apoptotic indicators require the cells to be killed by fixation and even apoptotic cells detected by annexin v are often live as indicated by exclusion of pi. the amount of cells with complete collapse of mmp is even considered an early indicator of apoptosis and as shown in this study it does not provide a measure of cell death (kroemer et al., 2009). however, since the ec50 dose is also the minimal dose that kills at least 50 % of cells, it is not surprising that the death phenotype at the ec50 dose is often apoptosis or alternative forms of programmed cell death rather than passive necrosis. hypoxia causes increased ros production and damage of lipids and proteins (prabhakar et al., 2007; solaini et al., 2010). response to hypoxia is characterized by adaptive changes in the circulatory and hemopoyetic systems and includes angiogenesis, vasodilation, and increased erythropoiesis, while expression of several enzymes and transporters are modified to enhance uptake and breakdown of glucose through anaerobic glycolysis. since hypoxia increases ros it may be anticipated that h2o2 will be more toxic under hypoxia. however, the assays conducted under hypoxia showed that the cytotoxic effect of h2o2 was diminished. the ec50 was increased from 1.5 mm to 4.7 mm. in addition, the range ec10-ec90 (0.6 mm 3.9 mm under normoxia) was expanded to 0.9 mm 25.1 mm under hypoxia indicating that variability was also increased and that the tolerance increase was exceedingly high in some particular cells. studies in other eukaryotes have demonstrated that hypoxia-induced mitochondrial ros is part of the mechanism of o2 sensing (chandel et al., 2000), and that antioxidant mechanisms are activated in concert with metabolic changes due to the activation of hypoxia inducing factor and upregulation of several genes (gorr et al., 2010). thus the net result could be an increased tolerance to oxidative damage as we have observed in our study in sipunculans. the strong acidification of the celomocyte cytoplasm after 24 h hypoxia without showing any change in cell viability is consistent with enhanced anaerobic metabolism and production of lactic acid. as said before sipunculans are oxyconformants but prolonged hypoxia ends up in anerobiosis with increased production of lactic acid. interestingly, it has been suggested that the body wall with longitudinal and 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anaerobic mitochondria; opines; mytilus edulis; mollusc   introduction mitochondria represent an endosymbiont model of complex organelle development driven by evolutionary modification of permanently enslaved primordial purple non-sulphur bacteria (gray et al., 1999). from a teleological perspective, endosymbiotic enhancement of eukaryotic cellular energy requirements indicates a convergence of metabolic processes within the mitochondrial matrix for optimal synthesis of atp from adp and inorganic phosphate. bacterial and mitochondrial atp synthases (f-atpases) require a defined membrane ___________________________________________________________________________ corresponding author: george b stefano neuroscience research institute state university of new york college at old westbury old westbury, ny, usa e-mail: gstefano@sunynri.org potential to achieve transductive transmembrane proton-motive force across the inner membrane linked to high efficiency of atp production (stefano et al., 2012). this necessitates an evolutionarily driven retrofit of the bacterial plasma membrane into the inner mitochondrial membrane. the protonmotive force is functionally coupled via mechanical transductive events within discrete protein subunits localized to the transmembrane domains of fatpases and involves sequential protonation and deprotonation of glutamate side-chains of cytochrome c-subunits within functional pores. evolutionary pressure is predicted to provide an existential advantage to the host eukaryotic cell at this primal level of energy production (stefano et al., 2012). recent elegant work has confirmed this key contention by demonstrating an enhanced efficiency of 2.7 vs. 3.3 5 protons per synthesized atp molecule by eukaryotic vs. prokaryotic f-atpases, 22 mailto:gstefano@sunynri.org fig. 1 anaerobic production and re-oxidation of the opine, octopine, in mitylus edulis. pyruvate is condensed with the amino acid, arginine, to produce octopine. the reaction is catalyzed by octopine dehydrogenase (od). opines are stored until oxygen becomes available to reverse this reaction and produce pyruvate for the krebs cycle. respectively (watt et al., 2010). mechanistically, endosymbiosis has apparently resulted in seamless coupling of cytochrome c oxidase (cox) to f-atpase for maximal atp production in respiring mitochondria, thereby effecting essential partitioning of glycolytic and tca cycle metabolic processes within discrete cellular domains. cox is an inner mitochondrial multisubunit enzyme complex expressed and assembled as a mosaic from nuclear and mitochondrial genomes. a recent review presents the case for cox as a key regulator of mitochondrial atp production (pierron et al., 2012). the authors propose that the evolutionarily driven addition of nuclear-encoded cox subunits provides the host eukaryotic cell with high order control over the ancestral activity of cox subunits encoded by mtdna genes in the face of fluctuating mitochondrial oxygen tensions and potentially dangerous reactive oxygen species. anaerobic respiration in invertebrates the intertidal habitat of the marine mussel, m. edulis, poses unique metabolic challenges to the survival of the species. during low tide, the mussel must close its valves to avoid water loss and therefore experiences hypoxic conditions. m. edulis has evolved to cope with hypoxia by switching to anaerobic respiration (de zwaan et al., 1976; connor et al., 2012). this strategy is not only employed by mussels; it has also been observed in other marine invertebrates (hochachka et al., 1977), numerous other eukaryotes [see review (muller et al., 2012)], plants (igamberdiev et al., 2009; shingaki-wells et al., 2014), and of course in prokaryotes. to effectively mediate anaerobic metabolic demands, m. edulis synthesizes and utilizes amino acid adducts termed opines as chemically defined energy reserves (de zwaan et al., 1976). opines were first discovered in the mollusc, octopus (morizawa, 1927), notably the prototypic compound octopine, the enzymatically derived condensation product of arginine and pyruvate. in addition to the utilization of opines as anaerobic metabolic intermediates by invertebrate organisms, opines were also discovered and characterized as metabolic intermediates in plant parasites, specifically crown gall tumors (holsters et al., 1978; guyon et al., 1980; toothman, 1982; dessaux et al., 1993). synthesized opines are effectively stored until oxygen levels are sufficient to resume aerobic respiration followed by enzymatic oxidation to release pyruvate as an essential tca cycle substrate (grieshaber et al., 1994) (fig. 1). the amino acids used in the biosynthesis of opines are alanine, arginine or glycine (fields et al., 1980, siegmund et al., 1983; grieshaber et al., 1994). the enzyme required for production of octopine from arginine and pyruvate has recently been isolated and purified (vazquez-dorado et al., 2011). presumably, this strategy evolved to maintain osmolality and to produce a by-product less acidic than lactate (ballantyne, 2004). this metabolic pathway, like glycolysis, only produces 2 atp per mole of glucose. anaerobically functioning mitochondria in invertebrates in recent times the dynamic nature of the mitochondrion has been observed to exhibit biochemical and functional variation, including the capacity for anaerobic respiration (muller et al., 2012). in this regard, m. edulis has been well studied (doeller et al., 2001; connor et al., 2012). when a prolonged period of hypoxia leads to anoxia 23 fig. 2 (adapted from (muller et al., 2012) anaerobic and aerobic metabolic pathways within the cytoplasm and mitochondria of the mussel, mytilus edulis. glucose can be converted to phosphoenolpyruvate and to pyruvate. pyruvate can be used in the mitochondrial krebs cycle or condensed with amino acids to produce opines. phosphoenolpyruvate can be converted to malate before being simultaneously reduced to fumarate and oxidized to pyruvate (malate dismutation). pyruvate can be used in the krebs cycle. the fumarate is further reduced by fumarate reductase (fr) and rhodoquinone (rq) to succinate. succinate is then transformed into propionate as an end product. in the mussel, an additional metabolic pathway is employed instead of opine production (woo et al., 2011). malate dismutation contains the favored reactions and malate’s reduction to fumarate, via a reaction that is essentially part of the krebs cycle running in reverse, leads to the production of succinate (muller et al., 2012). m. edulis utilizes fumarate reductase and rhodoquinone to reduce fumarate to succinate (tielens et al., 2002). succinate is further metabolized to propionate resulting in approximately 5 atp (tielens et al., 2002) (fig. 2). this process allows the organism to survive at a lower energy state while yielding more atp than can be achieved by glycolysis alone. furthermore, in this state of lower energy production there are less free radicals generated, offering a degree of protection while in this physiological state (rivera-ingraham et al., 2013). interestingly, each tissue type in the mussel responds differently to hypoxia as a result of mitochondrial functional differences in gene expression. the gills, digestive glands, mantle, and adductor muscle have been shown to respond to hypoxia by switching to anaerobic respiration, (ibarguren et al.,1989; lushchak et al., 1997; bacchiocchi et al., 2000, doeller et al., 2001; diazenrich et al., 2002). in the case of gill ciliated epithelium, which is most important for the survival of the individual, the metabolic process is kept on. this can be surmised by the fact that the gill cilia are densely packed with mitochondria (paparo, 1972). the ciliated gill epithelium of m. edulis has been studied not only for its ciliary activity but for its innervation as well (paparo, 1972; stefano et al., 1975, 1976). this epithelium is innervated via serotoninergic and dopaminergic neurons, providing for cilio-excitation and cilio-inhibition, respectively. clearly, this necessitates greater energy requirements, which may be difficult at intertidal intervals. we surmise this difficulty is overcome by way of nervous system integration of the tissue, exerting specific and rapid responses to respiratory and waste needs carried out by the ciliated epithelium (stefano, 1990; stefano et al., 1991). anaerobically functioning mitochondria and cancer biology a careful review of the biomedical literature indicates functional similarities between anaerobic mitochondrial subtypes in m. edulis and crown gall tissue and metabolic processes in human tumors. cancer cells utilize anaerobic energy metabolism under hypoxic, anoxic and even during normoxic conditions (gonzalez et al., 2012; amoedo et al., 2013; witkiewicz et al., 2013; chen et al., 2014). it has been suggested that carcinogenic processes might target normal mitochondrial functioning and cause a disruption of the krebs cycle and electron 24 transport enzymes (gonzalez et al., 2012). it has been recently proposed that normative mitochondrial function in non-proliferating cells affects relatively high cytosolic atp/adp ratios resulting in functional inhibition of aerobic glycolysis (maldonado et al., 2014). in contrast, the bioenergetics of the “warburg” effect that has been extensively linked to the metabolic phenotype of numerous cancer cell types is characterized by enhanced aerobic glycolysis and suppression of aerobic mitochondrial metabolism (gonzalez et al., 2012; amoedo et al., 2013; witkiewicz et al., 2013; chen et al., 2014). furthermore, aerobic respiration in proliferating cells leads to deleterious production of free radicals that can damage dna and proteins. accordingly, free radical damage is proposed to exacerbate compromised mitochondrial functioning thereby diminishing the existential viability of cancer cells. along these lines, davila and zamarano (2013) posit that cancer can be viewed as a cell that has phenotypically reverted to the last common eukaryotic ancestor of the host cell. they surmise that a cancer cell is functioning as a facultative anaerobic microbe with unlimited replication potential (davila and zamarano, 2013). interestingly, anaerobic mitochondria in gill cilia of m. edulis have evolved to utilize the phenotype of a facultative anaerobe (doeller et al., 1993, 2001). mytilus mitochondrial dna, trna and a link to cancer for over a decade, an ostensibly unresolved issue relating to essential genes expressed by mitochondrial dna (mtdna) from m. edulis and related species of marine mussels is the absence of a traditionally defined gene encoding subunit 8 of the mitochondrial atp synthase complex (atp8) (boore, et al., 2004; breton et al., 2010; smietanka et al., 2010). the protein expressed by the atp8 gene has been established as an integral component of the atp synthase stator stalk in yeast and all metazoan phyla and is essential for coupled atp production within the mitochondrial matrix. recently, two laboratories have independently defined an open reading frame (orf) corresponding to a never before annotated atp8 variant in the mtdna of several mytilus species and have speculated that evolutionary resolution of mtdna contributions by both male and female underlies its novel representation within the mitochondrial genome (breton et al., 2010; smietanka et al., 2010). a very recent publication reinforces the functional role of atp8 mtdna gene expression in the process of carcinogenesis (grzybowska-szatkowska et al., 2014). five identified mutations and polymorphisms of the atp8 gene were identified in tissues obtained from breast cancer patients, thereby supporting the contention that functional modification/impairment of an essential subunit of the mitochondrial atp synthase complex represents causative factor in carcinogenesis. another interesting characteristic of mytilus mitochondrial genome is the presence of an additional novel methionyl trna. its uau anticodon makes it unique among taxa (hoffmann et al.,1992; boore et al., 2004). the presence of this additional trna raises questions in regard to potential similarities with tumor cells since these tend to exhibit elevated levels of initiator methionyl trna expression (kanduc, 1997; kanduc et al., 1997; marshall et al., 2008; pavon-eternod et al., 2009; zhou et al., 2009). it has been postulated that altering the trna expression profile in cells might influence the regulation of translation of growth factors, proto-oncogenes and other proteins involved in cell cycle (kanduc, 1997; marshall et al., 2008; kolitz et al., 2009; pavon-eternod et al., 2013). in particular, it has been demonstrated that increasing the levels of initiator trnamet caused a concomitant elevation of other trna molecules, resulting in increased metabolic activity and cell proliferation (pavon-eternod et al., 2013). accordingly, after partial hepatectomy, levels of initiator trnamet increase in rat hepatocytes compared to those of elongator trnamet during cellcycle progression (kanduc, 1997). similar trnamet pattern shift was observed in human colorectal and gastro-intestinal tumors (kanduc et al.,1997). moreover, in embryonic fibroblasts from mice, overexpression of initiator trna resulted in induction of tumorigenesis (marshall et al., 2008), and in breast cancer and multiple myeloma cell lines initiator trna levels were also found to be elevated (pavon-eternod et al., 2009; zhou et al., 2009). mytilus as a model to study cancer in humans, the v-ki-ras2 kirsten rat sarcoma viral oncogene homolog (kras) gene encodes a small gtpase involved in key regulatory signaling cascades (franks et al., 1987) and in tumorigenesis (chetty et al., 2013). amplification of kras gene expression and/or oncogenic activating gain-offunction kras mutations have been functionally linked to enhanced growth, survival, and metastasis of major classes of human tumor types included in small-cell lung cancer (minuti et al., 2013) colorectal cancer (brand et al., 2012), pancreatic cancer (di magliano et al., 2013, fang et al., 2013), and intrahepatic cholangiocarcinoma (robertson et al., 2013). of equivalent importance, dysregulation of the cellular epidermal growth factor receptor (egfr) signaling pathway has been demonstrated to be critically important in promoting tumor growth, survival, and metastasis in human tumors (goffin et al., 2013) and development of several frontline anticancer therapeutic agents have attempted to achieve efficacious selective targeting of the oncogenic egfr signaling pathway (kohler et al., 2013). recent studies indicate that kras tumorigenicity is functionally linked to the “warburg” phenotype favoring a high rate of aerobic glycolysis and anaerobic mitochondrial function (weinberg et al., 2010). this establishes the facultative anaerobic mitochondrion in m. edulis tissues as a potentially valuable high resolution model system for the development of novel anticancer therapeutic agents. conclusions this review documents the phenomenon and existence of anaerobically functioning mitochondria in m. edulis as a model for invertebrate energy generating systems. in this regard, this mechanism 25 is used to benefit the organisms when large amounts of energy translocation are not present. it is clear mytilus may use this pathway to survive when an abundant source of oxygen is not present e.g., intertidal periodicity. accordingly, if mitochondria represents evolutionary defined endosymbiont organelles, they have retained part of the anaerobic process associated with bacteria. this dynamic capacity would have survival value under hypoxic environmental conditions. in part, we surmise, that dysfunctional mitochondria in cancer cells may have their origin in the early evolution of eukaryotic cells by retaining this information and/or processes to implement this phenomenon in times of stress. however, in metastatic processes this pathway may emerge due to poor chemical messenger regulation. importantly, mytilus may yet be another invertebrate that can be used as a model system because of its broad scope of energy balance and dynamic capacity to adapt. acknowledgements this work, in part, was supported by mitogenetics, llc. 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glycoproteins, freely dissolved in the hemolymph, of many arthropods and mollusks isj 10: 120-127, 2013 issn 1824-307x review antiviral activity of hemocyanins p dolashka , w voelter1 2 1institute of organic chemistry with centre of phytochemistry, bas, sofia, bulgaria 2interfacultary institute of biochemistry, university of tϋbingen, hoppe-seyler-strasse 4, d-72076 tϋbingen, germany. accepted october 11, 2013 abstract hemocyanins are giant biological macromolecules acting as oxygen-transporting glycoproteins. most of them are respiratory proteins of arthropods and mollusks, but besides they also exhibit protecting effects against bacterial, fungal and viral invasions. as discovered by 2-dge proteomics analyses, several proteins including hemocyanins of hemocytes from virus-infected arthropods increased upon infection, confirming hemocyanin’s role as part of the organism’s defence system. based on the structural analyses of molluscan hcs it is suggested that the carbohydrate chains of the glycoproteins seem to interact with surface-exposed amino acid or carbohydrate residues of the viruses through van der waals interactions. key words: functional units; hemocyanin; mollusks; arthropods; viruses introduction the persistence of the virus in the organism leads to the development of lymphoproliferative disease, the formation of various carcinomas and to the affection of the peripheral and central nervous system. antiviral drugs may be divided into acyclic nucleoside analogues (aciclovir, ganciclovir, penciclovir), acyclic nucleotide analogues (cidofovir and adefovir), and substances of natural origin (de clercq, 2004). most of the submitted drugs are potent inhibitors of viral reproduction, but not all of them are promising for clinical application, since they are very different in terms of toxicity. recently it was found that hemocyanins (hcs), the oxygen-transporting glycoproteins of many arthropods and mollusks, could be also potential inhibitors of some virus infections (chongsatja et al., 2007; flegel et al., 2011). these giant glycoproteins which differ in molecular mass, structure, carbohydrate content, and monosaccharide composition have recently received increasing interest due to their significant immunostimulatory, antitumor, and antiviral properties (dolashkaangelova et al., 2009; dolashka et al., 2010; mičetić et al., 2010; de smet et al., 2011; rehm et al., 2012; markl et al., 2013). both species, molluscan ___________________________________________________________________________ corresponding author: pavlina dolashka institute of organic chemistry with centre of phytochemistry bas, sofia, bulgaria e-mail: pda54@abv.bg; dolashka@orgchm.bas.bg and arthropodan hcs, contain copper-containing active sites in which the cu(i,i) is oxidized to the cu(ii,ii) state, thus accounting for their distinctive deep blue color. the biosinthesis of hemocyanins from e.g. concholepas concholepas (cch) takes place in the hepatopancreas (manubens et al., 2010) but megathura crenulata’s hcs originate from rhogocytes (martin et al., 2011). investigations on several molluscan hemocyanins, e.g. from haliotis tuberculata (hth, abalon) (markl et al., 2001); helix lucorum (hlh, garden snail), rapana venosa (rvh, black sea murex) (dolashka-angelova et al., 2003, 2009, 2011; iliev et al., 2008) or c. concholepas (cch, loco) from the pacific chilean coast (moltedo et al., 2006, arancibia et al., 2012) demonstrated their remarkable immunostimulatory properties in experimental animal model and clinical studies. moreover, hemocyanins have been extensively used as carriers to generate antibodies against diverse hapten molecules and peptides to induce antigen-specific cd8+ and cd4+ t cell responses (minozzi et al., 2007; arancibia et al., 2012). recently, a vaccine potential of oncomelania hupensis hc against schistosoma japonicum parasite was also established (guo et al., 2011). probably, due to their high carbohydrate content and specific monosaccharide composition, hcs were also found to be active against viruses (dolashka-angelova et al., 2009, 2010). proteomic analysis of differentially-expressed proteins in arthropodan penaeus vannamei hemocytes upon 120 http://www.sciencedirect.com/science/article/pii/s1570963913000836## mailto:pda54@abv.bg mailto:dolashka@orgchm.bas.bg fig. 1 pseudoatomic model of emperor scorpion hemocyanin used for molecular replacement (jaenicke et al., 2012). taura syndrome virus (tsv) infection showed increased expression of hemocyanin (rattanarojpong et al., 2009). the efficacy, against white spot syndrome virus (wssv) and singapore grouper iridovirus (sgiv) is also assigned to a protein of the arthropod penaeus monodon (zhang et al., 2004). antiviral properties of molluscan hcs were reported for the first time discribing the inhibition effect of hemocyanins from gastropod r. venosa (rvh) against respiratory syncytial virus (rsv) and herpes simplex virus type-1, strain vic (hsv-1) (dolashka et al., 2010; dolashka-angelova et al., 2011; nesterova et al., 2011). as this property seems to be associated with the glycosylation of functional units, the oligosaccharide structures of r. venosa and related molluscan and arthropodan hemocyanins were determined using different analytical physycochemical thechniques, (sandra et al., 2007; dolashka-angelova et al., 2009; dolashka et al., 2010). in this short review, current knownledge about structural and biochemical aspects and eventual potential future clinical applications of arthropodan and molluscan hemocyanins as antiviral drugs is presented, and biochemical mechanisms causing the antiviral activity are suggested. arthropodan hemocyanins structure of arthropodan hemocyanins hemocyanins evolved early in the arthropod stem lineage from phenoloxidases, o2 -consuming enzymes involved in the melanin pathway. they form hexamers or oligo-hexamers of identical or related subunits with a molecular mass of about 75 kda (fig. 1) (dolashka-angelova et al., 2001; mičetić et al., 2010; jaenicke et al., 2012). each subunit contains three domains and o2-binding is mediated by two cu+ ions which are coordinated by six histidine residues ("type iii” copper binding site). subunit interactions within the multisubunit hemocyanin complex lead to diverse allosteric effects such as the highest cooperativity for oxygen binding found in nature. based on biochemical, immunochemical and molecular phylogenetic analyses, distinct hemocyanin subunit types have been identified in chelicerata, myriapoda, crustacea and hexapoda (huang et al., 2008; rehm et al., 2012). the crystal structure of a native hemocyanin oligomer larger than a hexameric substructure was published for the first time for the 24-meric hemocyanin (mw = 1.8 mda) from emperor scorpion (pandinus imperator) (fig. 1) (jaenicke et al., 2012). antiviral activity of arthropodan hemocyanins several reports on antiviral effects of arthropodan hcs appeared in the literature, e.g. hcs from shrimp penaeus japonicus (pjh) and p. vannamei (pvh), against white spot syndrome virus (wssv), taura syndrome virus (tsv), yellow head virus (yhv) (lei et al., 2008; chongsatja et al., 2007; rattanarojpong et al., 2007; nesterova et al., 2011). hcs-based antiviral therapies are also reported (lin, 2005; balzarini et al., 2007). one of these viruses, the white spot syndrome virus (wssv) caused billions of dollars of losses for 121 http://www.ncbi.nlm.nih.gov/pubmed?term=jaenicke%20e%5bauthor%5d&cauthor=true&cauthor_uid=22403673 http://www.ncbi.nlm.nih.gov/pubmed?term=jaenicke%20e%5bauthor%5d&cauthor=true&cauthor_uid=22403673 http://www.ncbi.nlm.nih.gov/pubmed?term=jaenicke%20e%5bauthor%5d&cauthor=true&cauthor_uid=22403673 fig. 2 quaternary structure of molluscan hemocyanins: a) side view of didecamer; b) top view of decamer; c) structural subunits and functional units; d) electron microscopic picture of the native molecule of molluscan hemocyanin (lieb et al. 2008). shrimp farmers. to reduce shrimp production losses, stimulated since 2000 a plethora of research programmes to test shrimp’s responses against viral pathogens at the molecular level. humoral responses, binding studies between shrimp and viral structural proteins including intracellular responses, viral persistence co-interactions as well as viral sequence determination of the shrimp genome were subject of these investigations. based on these results, a novel practical method for improved disease control was developed (flegel et al., 2011). studies on shrimp p. japonicus (pjh) showed that its hemocyanin could delay the infection of white spot syndrome virus (wssv) in vivo and its subunits have different behavior in anti-wssv defense (table 1). during wssv infection a strong induced one also cloned subunits, pjhcl was observed in contrast to subunit pj-hcy. these findings suggest a possible discrepancy between the two subunits in shrimp innate immunity (lei et al., 2008). to understand the molecular responses of crustacean hemocytes to virus infection, a twodimensional electrophoresis (2-de) proteomics approach was applied to determine altered proteins in hemocytes of p. vannamei during taura syndrome virus (tsv) and yellow head virus (yhv) infections (rattanarojpong et al., 2007; chongsatja et al., 2007). proteomic analysis of p. vannamei hemocytes (pvh) upon yhv and tsv infection show the differentially-expressed proteins from the hemocytes. in the proteomic analysis of gills from yellow head virus-infected p. vannamei, 13 spots with up-regulated protein expression levels and five spots with down-regulated levels were identified. lc-nano-esi-ms/ms indicated that the up-regulated proteins included enzymes in the glycolytic pathway, the tricarboxylic acid cycle and amino acid metabolism. the other upregulated proteins were arginine kinase, imaginal disk growth factor (idgf) and a ras-like gtp protein. these results provide preliminary data, however, it was not able to assign specific yhv-response status to any of the upregulated or down-regulated genes identified after yhv challenge (rattanarojpong et al., 2007). proteomic analysis was also applied to explain the antiviral effect of p. vannamei hemocyanin (pvh) against taura syndrome virus (tsv) (chongsatja et al., 2007). at 24 h post infection of pvh with taura syndrome virus (tsv), quantitative 122 table 1 antiviral activity of several molluscan and arthropodan hemocyanins against different viruses hemocyanins viruses efficacy references arthropodan hcs p. japonicus (pjh), white spot syndrome virus (wssv) lei et al., 2008 pjhcy, pjhcl wssv + lei et al., 2008 p. vannamei (pvh), taura syndrome virus (tsv) + chongsatja et al., 2007 pvh yellow head virus (yhv) + rattanarojpong et al., 2007 p. monodon singapore grouper iridovirus (sgiv) zhang et al. 2004 molluscan hcs rapana venosa rvh, rvh1 and rvh2 polio virus type 1(lsc-2ab) dolashka-angelova 2009 rvh, rvh1 and rvh2 respiratory syncytial virus (rsv) dolashka-angelova 2009 rvh, rvh1 and rvh2 coxsackie virus b1 (cv-b1) dolashka-angelova 2009 gglycosylated rvh-b rsv dolashka-angelova 2009 nonglycosylated rvh-b rsv dolashka-angelova 2009 gglycosylated rvh-c rsv + dolashka-angelova 2009 nonglycosylated rvh-c rsv dolashka-angelova 2009 rvh, rvh1 and rvh2 epstein barr virus velkova et al., 2009 helix vulgaris (hvh) epstein barr virus zagorodnya et al., 2011 rvh, rvh1 and rvh2 hsv-1 nesterova et al., 2010 helix vulgaris (hvh) hsv-1 nesterova et al., 2010 rvh2-e hsv-1 + nesterova et al., 2010 intensity analysis and nano-lc-esi-ms/ms revealed 8 proteins that were significantly upregulated, whereas 5 proteins were significantly down-regulated in the infected shrimps. all of these proteins (hemocyanin, catalase, carboxylesterase, transglutaminase, and glutathione transferase) play important roles in host defense, signal transduction, carbohydrate metabolism, cellular structure and erstress response. however, hemocyanin, is one of these up-regulated proteins indicating a relation between this protein and the organism’s defence system. to get more insight into the molecular mechanism of the immune response of p. vannamei during taura syndrome virus (tsv) infection, expression and functional proteomics studies were performed on its hemocyanin, which is a major abundant protein in shrimp hemocytes. twodimensional electrophoresis revealed up-regulation of several c-terminal fragments of hemocyanin, whereas the n-terminal fragments were downregulated during tsv infection. 2-d western blot analysis showed that the c-terminal hemocyanin fragments had more acidic isoelectric points (pi), whereas the n-terminal fragments had less acidic pis. by motif scanning an important motif was discovered in the c-terminal hemocyanin, the erk d-domain, of required for activation of erk1/2 effector kinase, as a kinase-binding site at val527 in the hemocyanins c-terminus, whereas no functional domain was found in the n-terminus. coimmunoprecipitation confirmed the interaction between the c-terminal hemocyanin and erk1/2 which was also up-regulated during tsv infection. these findings demonstrate for the first time that the erk1/2 signaling pathway may play an important role in molecular immune response of p. vannamei upon tsv infection through its interaction with the c-terminal hemocyanin (havanapan et al., 2009). molluscan hemocyanins structure of molluscan hemocyanins molluscan hemocyanins (hcs) are giant biological macromolecules acting as oxygentransporting glycoproteins. they have recently received particular interest due to their immunostimulatory and antitumoral properties. most of them are glycoproteins with molecular mass around 9000 kda organized as decamers or didecamers (fig. 2) (lieb et al. 2008; markl, 2013). the native hemocyanins contain one or two structural subunits with molecular masses around 350 450 kda. seven or eighth functional units with molecular masses of 50 kda organize one structural subunits (fig. 2) (dolashka-angelova et al., 2003a; lieb et al. 2008; de smet et al., 2011). molluscan hemocyanins possess certain inhibitory properties against tumor cells and viruses (iliev et al., 2008; toshkova et al., 2009; dolashka et al., 2011), as also found for the molluscan hemocyanins r. venosa (rvh) and h. lucorum (hlh) against different viruses (dolashka-angelova et al., 2009, 2010; nesterova et al., 2010 2011; zagorodnya et al., 2011; velkova et al., 2011). antiviral activity of molluscan hemocyanins the antiviral effect of rvh and its isoforms was studied against polio virus type 1 (lsc-2ab), coxsackie virus b1 (cv-b1), respiratory syncytial virus (rsv) and epstein barr virus (ebv). after treatment of the viruses with the native rvh, the non-glycosylated and glycosylated functional units rvh-b and rvh-c, the quantitative virucidal suspension test did not show any effect against polio virus type 1 (lsc-2ab), coxsackie virus b1 (cv-b1). also no antiviral effects of native rvh against rsv was observed. however, the results presented in table 1 show that only the glycosylated 123 fig. 3 3d model of βchlh-g created by using the swiss pdb viewer and the model of functional unit “g” from octopus dofleini (odh-g) hemocyanin. glycans and the putative glycosilated sites n125 and n245 are represent as balls (kostadinova et al., 2013). fu, rvh-c, possesses some antiviral effect against the replication of rsv, at both viral doses tested (dolashka-angelova et al., 2009). in contrast, nonglycosylated fu rvh-b does not reveal any antiviral activity against the replication of rsv, poliovirus type 1 (lsc-2ab) and cv-b1. this is the first report of the fact that molluscan hemocyanin functional units with specific glycosylation possess antiviral activity (dolashka-angelova et al., 2009). the properties of native hemocyanins from r. venosa and helix vulgaris (hvh) snails and their structural subunits rvh1, rvh2, hvh1, and hvh2 have been studied in vitro as substances with feasible antiebv activity (velkova et al., 2009). epstein barr virus (family herpesveridae) is one of the etiologic agents that cause burkitt's lymphoma and nasopharyngeal carcinoma, but drugs for antiebv treatment are limited. there are few approaches for the treatment of this viral infection, namely, the use of effective antiviral chemotherapy, serotherapy or seroprevention which supposes the use of specific human immunoglobulins and vaccines (pagano and gershburg, 2005). the influence of r. venosa hemocyanin on viability and proliferative activity of lymphoblastoid cells was characterized by cytomorphological and colorimetric methods and an antiviral effect was observed after treatment with fus rvh1-a and rvh2-e (nesterova et al., 2010). antiviral effects on r. venosa and h. vulgaris (hvh) hemocyanins, their structural subunits, the glycosylated functional unit rvh2-e and the nonglycosylated unit rvh2-c were also investigated against hsv virus type 1: only the glycosylated fu rvh2-e exhibited an antiviral activity, which probably is due to its high carbohydrate content and specific monosaccharide composition (zagorodnya et al., 2011; velkova et al., 2011). based on the obtained results, we suggested for the first time that the complete native rvh molecule lacks any antiviral activity because the carbohydrate chains are buried in between the structural subunits of the global protein and therefore, are unable to interact with the viral surface glycoproteins. to support this hypothesis, we found that two putative glycosylated sites in fus βchlh-g are exposed on the surface of the molecule (fig. 3) (kostadinova et al., 2013) and that fu rvh1-a, exhibits antiviral activity and bears two nglycosidic resirues attached to asn262 and asn401. 124 fig. 4 3d model of functional unit rvh2-e. amino acid residues included in the loops are shown in blue and green (dolashka et al., 2010) glycosylation of hemocyanins the relevance of specific glycosylation patterns as a differentiating factor for immunostimulatory properties has already been raised in studies on the hemocyanin isoforms klh1 and klh2 of m. crenulata and r. venosa, (geyer et al., 2005; sandra et. al., 2007). molluscan hemocyanin genes contain several potential n-glycosylation sites, and some of those have been already effectively demonstrated to be glycosylated using a series of analytical strategies and maldi-tof-ms and tandem mass spectrometry (lieb et al. 2008; dolashka-angelova et al., 2009; dolashka et al., 2010; de smet et al., 2011). the identification of the oligosaccharide structure of several molluscan hemocyanins revealed a highly heterogeneous mixture of glycans of the compositions hex0–9 hexnac2–4 hex0–3 pent0–3 fuc0–3. a novel type of n-glycan with an internal fucose residue connecting one galnac(b1-2) and one hexuronic acid, was detected which also occurs in subunits rvh1 and rvh2 (sandra et. al., 2007; dolashkaangelova et al., 2009; dolashka et al., 2010). based on the obtained information about carbohydrate structure of hemocyanins the antiviral properties of hemocyanins against herpes simplex virus type 1, was suggested. one potential site for n-glycosylation in fu rvh2-e at asn-127 was shown to be glycosylated and this carbohydrate chain is likely to interact with specific regions of glycoproteins of hsv, through van der waals interactions in general or with certain amino acid residues in particular. as is shown in figure 4 several groups of these residues can be identified on the surface of rvh2-e which may interact with a carbohydrate chains of glycoprotein gp120 of hsv. the surface of glycoprotein gp120 was very well analysed (sander et al., 2002). development and identification of polyclonal antibodies against viruses hemocyanins are commonly used to develop polyclonal antibodies e.g. also against predicted b cell epitopes in hiv-1 accessory protein vpr. the synthesized b-cell peptide epitopes were subsequently conjugated with keyhole limpet hemocyanin (klh) and then used to immunize rabbits. the antibody titers and specifities were determined by elisa, western-blotting, and immunoprecipitation analyses, respectively. two b 125 cell epitopes of vpr were successfully predicted by bio-informatical methods and polyclonal antibodies against those peptides could be successfully prepared (sun et al., 2012). klh was used to analyse the protective potential of polyclonal igg antibodies specific to the ectodomain (em2) of m2 protein of influenza a virus (iav) against lethal influenza infection of mice. two fractions, klh alone and the ectodomain em2, conjugated to klh, were administered with freund's adjuvant intraperitoneally (i.p.) to balb/c mice. analysis of the preparation of anti-klh-em2 iggs by elisa revealed that it contained about 25% of antiem2 iggs and 75% of anti-klh iggs and after infection with 3 ld50 iav, a 100% survival of mice was observed with 320 µg anti-em2iggs (király et al., 2011). recently, molluscan hemocyanins receive increasing interest in vaccine development. several carrier proteins were used to prepare vaccines against highly variable hiv-1. two synthesized peptides, jy1 (v3 region of hiv-1 gp120, subtype d) and jy1-map8, were coupled to carrier proteins such as klh, hepatitis b surface antigen (hbsag) and meningococcal p64k protein and used for immunization in mice. it was found that jy1-map8 conjugates were more immunogenic than jy1-map8 alone. furthermore, conjugates to hbsag and klh were more immunogenic than those to p64k. the analysis showed that conjugate-based immunogens are more prompt to elicit immunogenicity and crossreactivity and hemocyanins are promising candidates the development of hiv vaccines (cruz et al., 2009). we found that hemocyanins are a new class of natural antiviral compounds against different viruses. however, arthropodan hemocyanins are active against those own viruses of arthropods, such as wssv and tsv, while the molluscan hemocyanins are active against viruses of human, such as rsv and ebv. maybe the mechanisms of antiviral activity of both species are different and therefore, we consider them as an appropriate for further investigation as antiviral agents. acknowledgement this work was supported by deutsche forschungsgemeinstchaft (dfg-ste 1819/51/2012), germany, bulgarian ministry of education, projects tk01-496/2009, vu-l-310/2007,daad17/2007 and “young researchers” dmu 03/26, grant №bg051po001-3.3.06-0025, financed by the european social fund and operational programme human resources development (2007 – 2013) and co-financed by bulgarian ministry of education, youth and science and fund for scientific research-flanders (fwo-vlaanderen, project vs.011.06n). references arancibia s, salazar f, inés becker m. hemocyanins in the immunotherapy of superficial bladder cancer – from basic science to robotic surgery. review 221-242, 2012. balzarini j. targeting the glycans of glycoproteins: a novel paradigm for antiviral 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http://www.scirus.com/srsapp/sciruslink?src=sd&url=http%3a%2f%2fwww.sciencedirect.com%2fscience%3f_ob%3dgatewayurl%26_origin%3dsciencesearch%26_method%3dcitationsearch%26_piikey%3ds0166354211001860%26_version%3d1%26_returnurl%3dhttp%253a%252f%252fwww.scirus.com%252fsrsapp%252fsearch%253fq%253ddolashka%2526t%253dall%2526sort%253d1%2526p%253d0%2526drill%253dyes%26md5%3d5b474872b3b0aa85456e4c1ca7752cd1 jaenicke e, pairet b, hartmann h, decker h. crystallization and preliminary analysis of crystals of the 24-meric hemocyanin of the emperor scorpion (pandinus imperator). plos one.7: 3, e32548, 2012. isj 9: 139-xxx, 2012   isj 9: 139-152, 2012 issn 1824-307x review oenocytes in insects gf martins1, jm ramalho-ortigão2 1departamento de biologia geral, universidade federal de viçosa (dbg/ufv). campus universitário, viçosa, minas gerais, brazil. cep 36570-000 2department of entomology, kansas state university (ksu), manhattan, kansas, usa, 66506 accepted august 17, 2012 abstract oenocytes are insect cells responsible for lipid processing and detoxification. of ectodermic origin, they are found in close association with the insect epidermis, or fat body cells, or both depending on the insect species and developmental stage. they are easily distinguishable either by staining or by their ability to form cell clusters lined by a basal lamina, which makes it possible to isolate them from other cells. the most noticeable characteristic of the oenocytes ultrastructure is the presence of a welldeveloped smooth endoplasmic reticulum that can fill almost entire cell cytoplasm that for a long time was suggestive of lipid processing capacity. this capacity was confirmed lately through the usage of genetic, molecular and biochemistry approaches and other functions are also addressed to these cells, such as cuticular hydrocarbons and pheromones synthesis and detoxification. additionally, oenocytes are considered analogous to mammalian hepatocytes based on their gene expression profiles and cell functions. in spite of the current knowledge about oenocytes, much about their protein expression profile remains unknown. in this review we provide a general overview of the state of the art related to oenocytes studies and certain morphological and biochemical aspects of such cells crucial for insect survival. key words: insects; oenocytes; oenocyte ultrastructure; oenocyte metabolism   introduction oenocytes are polyploid insect cells of ectodermic origin and are usually found in close association with the epidermis or fat body cells, or both depending on the insect species and developmental stages (locke, 1969; dorn and romer, 1976; hartenstein et al., 1992). although oenocytes have been known for over a century (landois, 1865; koschevnikov, 1900; imms, 1907; vickery, 1915) only recently their role in lipid metabolism and detoxification has been confirmed. according to snodgrass (1935) the term oenocyte refers to the usual pale amber color of the cells (oeno or oinos means wine in greek), however oenocytes may also display colorations that vary from brown, to yellow, to green, or red, and sometimes are even colorless. koschevnikov (1900) was one of the first to speculate on the possible role played by oenocytes (wrongly thought to be “urinary ___________________________________________________________________________ corresponding author: gustavo ferreira martins departamento de biologia geral universidade federal de viçosa (dbg/ufv) campus universitário, viçosa, minas gerais, brazil. cep 36570-000 e-mail: gmartins@ufv.br cells”), however, until the early 2000’s oenocytes were considered one of the least studied cells of invertebrates (gould et al., 2001). more recently, aspects of oenocytes differentiation (elstob et al., 2001; burns et al., 2012), gene expression (lycett et al., 2006; gutierrez et al., 2007; martins et al., 2011a), biochemistry (wicker-thomas et al., 2009) and physiological roles (knauf et al., 2002; gutierrez et al., 2007) have been investigated in details. arthropods utilize different strategies against the harmful effects of waste metabolites. such strategies may include metabolites excretion by feces and urine, and also neutralizing and storing them in the fat body. lycett et al. (2006) demonstrated that oenocytes play an important role in detoxification, protecting the organism against toxic and potentially lethal compounds such as insecticides. further, through the synthesis of hydrocarbons present on the outer surface of the insect cuticle (reviewed by lockey, 1988), oenocytes have a role in preventing water loss (fan et al., 2003), and have been shown to participate in the intraspecific communication as chemical signals (wicker-thomas et al., 2009). oenocytes are crucial for insect survival as indicated by their many key roles. here we provide 139     fig. 1 histological sections showing the location and organization of oenocytes in insects. hematoxylin and eosin staining of oenocytes (o) located underneath the abdominal epidermis (e) in brontocoris tabidus (heteroptera; pentatomidae) (5th instar nymph) is shown in a. toluidine blue staining of oenocytes (o) of toxorhynchites theobaldi (diptera; culicidae) 4th larva (b), where clustered oenocytes are attached to the epidermis (e) and in close association with the fat body (f); and newly-emerged adult female (c), where oenocytes are seen as clustered or single cells scattered between trophocytes in the periphery or inside the fat body (f). arrowoenocyte nucleus; ccuticle; hhemolymph. bar = 10 µm. 140     a comprehensive review on the roles played by oenocytes in insects, and details of these cells’ location, general morphology, ultrastructure, biochemistry and gene expression. oenocytes location and types the location of the oenocytes within insect body varies depending on the species or developmental stage. they can be found either associated with the epidermis (figs 1a-b), scattered between abdominal fat body cells (fig. 1c), or both within the same individual, as summarized in table 1. when associated with the abdominal fat body cells, oenocytes are organized as either single or clustered cells within the same insect. in aedes aegypti (culicidae) larvae, they are associated with the epidermis and located among the parietal fat body cells (wigglesworth, 1942). this also is the case for toxorhynchites theobaldi (culicidae) larvae (martins et al., unpublished data) (fig. 1b). in adults, however, the oenocytes appear in the periphery of the fat body, or inside fat body lobes either as single or clustered cells in a manner similar to what is observed in other haematophagous mosquitoes (martins et al., 2011b, c) (fig. 1c). according to fan et al. (2003), oenocytes that are arranged in discrete clusters within the hemocel are readily accessible for experimentation, and most investigations have concentrated on insect species whose oenocytes display such arrangement. moreover, the presence of oenocytes cell clusters facilitates their dissection and the establishment of in vitro culture (krupp and levine 2010; martins et al., 2011a, d). previously it has also been shown that in larvae of the fruit fly drosophila melanogaster, oenocytes form groups of four to nine cells attached to the lateral epidermis of each body segment. in addition, d. melanogaster oenocytes do not divide following eclosion of the larvae and simply grow in size (reviewed by elstob et al., 2001; gould et al., 2001). in larvae of anopheles maculipennis (culicidae) oenocytes are separated in large and small. large oenocytes are segmentally arranged in clusters, and are present in each of the first seven abdominal segments. they are absent from abdominal segments eight and nine, and from the thorax. in each of the larval abdominal segments of a. maculipennis oenocytes are located ventro-laterally and consist of two pairs of vary large cells on both sides of abdomen. small oenocytes on the other hand are numerous and have no defined arrangement although they are sometimes found in pairs.the small oenocytes are present just beneath the epidermis in the vicinity of each of the clusters formed by the large oenocytes. they are located anteriorly with respect to the large oenocytes but can be found along the base of each segment on either side of the nerve cord. small oenocytes are generally observed in the first eight abdominal segments and occasionally in the last segment (imms, 1907). in adults of apis mellifera (apidae), different populations of oenocytes also can be distinguished according to their localization and size. in newlyemerged queens, the small oenocytes are parietal and present in small groups immersed in the fat body while larger oenocytes appear as isolated cells scattered in the perivisceral fat body. in older queens only one population of oenocytes is present in the parietal fat body, and located mainly ventrally (hepburn et al., 1991). for the worker bees, the number of oenocytes varies according to age and body segment, with younger individuals having fewer oenocytes than the older workers, and there are fewer oenocytes in their head as compared to the abdomen. in addition, head oenocytes are larger than those in the abdomen, with bigger cellular and nuclear volumes (ruvolo and cruz-landim, 1993). the association between fat body cells and oenocytes persists throughout all developmental stages of the locust schistocerca gregaria. in this species, oenocytes are more numerous in the subepidermal region, and are intimately associated with trophocytes (coupland, 1975). in the beetles tenebrio molitor and tenebrio obscurus, adult insects display oenocytes arranged in grape-like clusters along the length of the dorsal surface of the lateral longitudinal trachea of the abdomen, but may also extend upwards along the dorsal trachea in the region of the spiracles. these clusters, made of a variable number of individual cells and with diameters ranging from 96 to 170 µm, lie within among the fat body and can be easily recognized by their brown color (roth, 1942). in nymphs and adults of heteropterans, oenocytes are located between the basal membrane and the epidermis and are clearly distinct from the fat body cells, as observed in the soldier bug brontocoris tabidus (pentatomidae) nymph (martins et al., unpublished data) (fig. 1a). in kissing bugs, such as rhodnius prolixus and triatoma infestans (triatominae), oenocytes are located in tergites and sternites in the abdomen, with sizes ranging from 10 to 100 µm, depending on the developmental stage [juárez and fernández (2007) and references therein]. wigglesworth (1933) observed that at the time of blood-feeding of r. prolixus nymphs there are two types of oenocytes: one large solitary and more or less lobulated with condensed chromatin near the center of the nucleus, and another small rounded oenocyte considered to be a new generation of cells. these newly generated cells can be differentiated as they are almost always in pairs and are sometimes linked by an unbroken strand of cytoplasm with the condensed chromatin more evenly distributed over the nuclear membrane. in adult a. aegypti, oenocytes are located on the periphery of the fat body and between trophocytes of the abdominal fat body. each rounded or oval cell is surrounded by a thin, finely granular basal lamina (tadkowski et al., 1977; martins et al., 2011b). similarly, in the stingless bee melipona quadrifasciata numerous oenocytes occur among the fat body cells and they have small nucleus and an acidophilic cytoplasm (paes-deoliveira and cruz-landim, 2006). the greatest polymorphism observed in insect oenocytes has been described in calpodes etlhius (lepidoptera), and that also seem to be the case for 141     table 1 distribution and location of oenocytes according to insect species, order and developmental stage __________________________________________________________________________________________ insect species order (common name) stage oenocytes location and organization reference monomorium pharaonis hymenoptera (pharaoh ant) associated to abdominal trophocytes in the parietal fat body jensen and børgesen, 2000 cyphomyrmex rimosus mycetarotes parallelus acromyrmex disciger atta laevigata hymenoptera (leaf-cutting ants) associated to trophocytes in the parietal and perivisceral fat bodies roma et al., 2006, 2008 pachycondyla villosa distributed among the trophocytes; right underneath the epidermis; isolated or in cluster of three to five cells, in both the thorax and abdomen, and more abundant in the latter, near the intersegmentary muscles zara and caetano, 2004 pachycondyla striata hymenoptera (panther ants) adult distributed among the trophocytes thiele and camargomathias, 2003 apis mellifera hymenoptera (honey bee) adult below epidermis in close association with the fat bodies hepburn et al., 1991; ruvolo and cruz-landim, 1993 musca domestica diptera (house fly) near the periphery of the abdominal segments and attached singly or in groups to the tracheae studinger and willig, 1975 dacus tryoni diptera (queensland fruit fly) larvae are located between the epidermis and the fat body evans, 1967 larvae arranged in lateral and subepidermal clusters of, on average, 6 cells per abdominal hemisegment elstob et al., 2001; gutierrez et al., 2007 drosophila melanogaster diptera (fruit fly) adult subcuticular abdominal cells found in segmentally repeated rows that form crescent-shaped strands on the tergites and small clusters on the sternites ferveur et al., 1997; billeter et al., 2009 adult pupa martins et al., 2011b, c aedes aegypti diptera (yelow fever mosquito) larva wigglesworth, 942 1clustered or single cells scattered between trophocytes in the parietal fat body aedes albopictus, aedes fluviatilis culex quinquefasciatus anopheles aquasalis anopheles darlingi adult martins et al., 2011c anopheles maculipennis diptera (hematophagous mosquitos) larvae located ventro-laterally, consisting of two pairs of large cells on both sides of abdomen imms, 1907 larva clustered in the parietal fat body toxorhynchites theobaldi diptera (mosquito hawk) adult clustered or single cells scattered between trophocytes in the parietal fat body martins et al., unpublished 142     brontocoris tabidus heteroptera (soldier bug) fourth instar nymph rhodnius prolixus triatoma infestans heteroptera (kissing bugs) nymph and adult attached to epidermis juárez and fernández, 2007; wigglesworth, 1933 oncopeltus fasciatus heteroptera (milkweed bug) embryo spread between trophocytes dorn and romer, 1976 echidnophaga oshanini aphaniptera (flea) associated to trophocytes in the parietal fat body vashchenok, 1966 blattella germanica blattaria (german cockroach) adult located beneath the epidermis, close to the basal lamina fan et al., 2003 leucophaea maderae blattaria (madeira cockroach) embryo included in the epidermis, associated to dermal glandular cells rinterknecht, 1985 lipeurus lawrensis tropicalis phthiraptera (lice) nymph and adult attached to epidermis, single or in cluster of two-six cells saxena and agarwal, 1980 adult in close association with the fat body coupland, 1975 schistocerca gregaria orthoptera (desert locust) nymph parietal abdominal fat body in association with trophocytes and urate cells diehl, 1973 bombyx mori lepidoptera (silkworm) metamerical groups on both sides of the abdomen associated with the spiracles vickery, 1915 calpodes ethlius lepidoptera (brazilian skipper) larvae close to specialized wax glands and subepidermal locke, 1969 tenebrio molitor tenebrioobscuros coleoptera (darkling beetles) adult in close association with the fat bodies and tracheal system roth, 1942 attagenus megatoma coleoptera (black carpet beetle) larva and adult in each of abdominal segments and in the secondand third adult thoracic segments; in cluster of five to eight cells associated with dorsoventral muscles toward the anterior of each ofthese segments; often associated with fat bodies and trachea dunkel and mallory, 1968 __________________________________________________________________________________________ other hesperids. also, three types of oenocytes can be recognized in the larvae according to location and morphology (locke, 1969): permanent oenocytes, which are located below the wax glands and remain enlarged throughout the intermoult period; segmentally arranged oenocytes, which only enlarge during the moulting cycle; and smaller subepidermal oenocytes, which appear just before pupation and occur in large numbers. although the role(s) of these three types of oenocytes is yet to be determined, they are likely to be distinct (locke, 1969). oenocytes in other arthropods in addition to insects, oenocytes have been described in chelicerata (romer and gnatzy, 1981), miriapoda (fontanetti et al., 2004), and crustacea (symonová and smrž, 2009). however, these reports are few and mostly descriptive. in adult opilionid (arachnida), oenocytes were described in the legs (romer and gnatzy, 1981), wedged between the bases of the epidermal cells and their cytoplasm. like their counterparts in insects, arachnida oenocytes also are 143     distinguishable by the presence of well-developed smooth endoplasmic reticulum (ser) (see below). another notable characteristic is the presence of many autophagic vacuoles and the conspicuous infoldings of the basal plasma membrane (romer and gnatzy, 1981). oenocytes were reported associated with the fat body cells of rhinocricus padbergi (diplopoda, spirobolida) adults (camargomathias and fontanetti, 2000; fontanetti et al., 2004; souza et al., 2011) and they are very similar to the insect oenocytes in terms of location, staining properties and ultrastructure that will be discussed further. in crustacea, oenocytes were described in juveniles and adults of ostracodes where they are present in large numbers in the body cavity and in the appendages of juveniles but less frequent in adults. their shape has been described as irregularly oval, measuring approximately two-three µm in diameter (symonová and smrž, 2009). oenocytes changes during post-embryonic development oenocytes arise during embryonic and postembryonic development and the differentiation of their ectodermic precursors have been studied in several insect species (lawrence and johnston, 1982; rinterknecht and matz, 1983; hartenstein et al., 1992; burns et al., 2012). oenocytes formation and differentiation also occur during insect postembryonic development, however, it is restricted to metamorphosis and ecdysis in holoand hemimetabolous insects, respectively (wigglesworth, 1933; evans, 1967). during metamorphosis, oenocytes have been shown to arise via de novo mechanisms. in dacus tryoni (diptera, trypetidae), larval oenocytes dissociate and eventually disintegrate simultaneously with the larval fat body cells during metamorphosis. at the beginning of pupation, segmentally arranged clusters of small cells lie among the large cells of the larval epidermis. between the second and fourth days following pupation, the smaller cells invade the surrounding epidermis, displacing it, and forming the adult epidermis. in this process, some of the adult cells migrate from the clusters into the body cavity (becoming free cells). by the sixth day of the pupal stage, these free cells have increased in size and are observed scattered under the entire surface of the abdominal integument. in addition, at this point the free cells become multinucleated and during the next few days will continue to enlarge. from the time they first appear, the adult oenocytes gradually increase in size until emergence of the adult insect (evans, 1967). it has been shown that oenocytes obtained from the last instar larvae of the cabbage armyworm mamestra brassicae (lepidoptera) can differentiate in vitro. moreover, they also are capable of affecting differentiation of the wing imaginal discs when in culture in the presence of the prothoracic gland and in media containing oenocytes and α-ecdysone. however, the wing discs display greater development than when cultured with α-ecdysone alone (agui, 1974). table 2 oenocyte size, number, and proportion according to abdominal volume in drosophila melanogaster (johson and butterworth, 1985). age (days) oenocytes number oenocyte size (µm2) oenocytes as a percentage of abdominal volume (%) female 0-1 6-7 56 7,138 5,855 4,819 315 475 658 1.7 1.4 1.5 male 0-1 6-7 56 4,224 6,602 5,271 257 715 665 1.1 3.2 3.3 morphometric analyses showed that in d. melanogaster the size and number of oenocytes vary greatly during aging of the adult insect and according to sex (johnson and butterworth, 1985) (table 2). interestingly, for the aging d. melanogaster male, an increase in cell size (nearly three times the size observed in newly emerged) translates in the oenocytes representing almost three percent of the abdomen of the fruit fly (table 2). morphologic and morphometric aspects of a. mellifera oenocytes were also studied in queens and workers in different adult ages (ruvolo and cruz-landim, 1993, 1995). for instance, size increase of 10.18 % and 7.47 % were noticed in a. mellifera workers oenocytesin the 5th and 7th days post emergence, respectively (ruvolo and cruzlandim, 1995). after r. prolixus nymph blood-feeding, whereas the small oenocytes grow rapidly, acquiring pseudopodia, old large cells break down completely. nine days post blood meal the oenocytes gradually reduce their size, and this trend continues for a few days after the next moulting as the new cuticle is being formed. oenocytes do not change until the next blood meal followed by a new moulting cycle. thus, in r. prolixus a new generation of oenocytes arises at each moulting cycle from undifferentiated cells in the epidermis with the oenocytes from the previous instar persisting into this new stage of development (wigglesworth, 1933). oenocytes hystochemistryand cytochemistry one of the main features used to identify oenocytes in histological sections is their acidophilic cytoplasm by using routine laboratory staining such as hematoxylin and eosin and toluidine blue (figs 1a-c). additionally, different cell staining methods including the bromophenol blue and millon reaction for proteins, periodic acid schiff (pas) for neutral sugars, feulgen reaction for dna, sudan black (sb), oil-red and osmium (oso4) impregnation for lipids and pyronin for rna (locke, 1969; coupland, 1975; zara and caetano, 2004; roma et al., 2006, gutierrez et al., 2007; roma et al., 2008; martins et al., 2011b) have been extensively applied to the 144     study of oenocytes in several insects, helping to reveal their distinguishable staining properties. these properties include the oenocytes’ cytoplasm positivity for proteins and lipids, and non-positivity for polysaccharides as discussed below. in addition, streptavidin also has been used to help track the fate of oenocytes during embryonic development and the differentiation of its ectodermic precursor in in the red floor beetle tribolium castaneum (burns et al., 2012). in oenocytes of adult eusocial ant workers such as the basal attini cyphomyrmex rimosus and mycetarotes parallelus and the derived acromyrmex disciger and atta laevigata strong reaction to bromophenol blue indicated the presence of large amounts of proteins in these cells (zara and caetano, 2004; roma et al., 2006). oenocytes of the parietal and perivisceral fat bodies of c. rimosus and m. parallelus are characterized by small and very electrondense protein granules distributed throughout their cytoplasm. on the other hand, lipids with large electrondense granules are commonly found and basic proteins are represented by granules in oenocytes of all species (roma et al., 2008). these studies also demonstrated that oenocytes are weakly positive for the pas test (zara and caetano, 2004; roma et al., 2006, 2008). histochemical tests for detection of lipids also evidenced the presence of positive cytoplasmic inclusions that contain unsaturated lipids that are more abundant in oenocytes of derived ants than basal ones (roma et al., 2008). additional details of oenocytes organization and staining profile in ants can be found elsewhere (roma et al., 2010). in t. molitor larvae, oenocytes impregnated with oso4 displayed different levels of lipid staining, which was also reported for the staining of esterases with naphthyl-acetate. in addition, oenocytes in t. molitor pupal and adult stages contain lipases as demonstrated by the presence of brownish crystals as the end product of this enzyme activity (leadsulfide) (romer, 1980). in the adult honeybee a. mellifera ruvolo and cruz-landim (1993) showed that the staining pattern of oenocytes varied according to the caste. for example, the abdominal oenocytes of queens are less acidophilic and showed weak reaction to sb than those of workers. these authors pointed that the differences between the oenocytes of queens and workers indicate that these cells may have different functions in the two castes. in 12-day old bees oenocytes showed a very strong reaction to sb and the intensity of staining is reduced gradually between 17and 29-day old bees. treatment with nile blue also indicated that these cells contain acid lipids that could be participating in wax synthesis. acid phosphatase (ap) was also observed in oenocytes in all workers and queens of a. mellifera adults, with queens displaying comparatively larger amounts of the enzyme. moreover, in workers, the levels of ap are higher in fiveand 29-day old bees. for queens, the presence of ap within oenocytes throughout their life is consistent with continued intracellular digestion activity in oenocytes, as ap is frequently associated with lysosomes (ruvolo and cruz-landim, 1993). worker honeybees are sensitive to earth’s magnetic field. in a provocative study, kuterbach et al. (1982) indicated that oenocytes in this caste of adult bees contain magnetite (fe3o4) as numerous electron-opaque, iron containing granules in the cytoplasm and are concentrated in the ventral abdomen under each segmental ganglion in the adult foraging worker. in light of the high iron content of these cells, these investigators suggested that in honey bees these oenocytes are involved in insect orientation. our studies on oenocytes of a. aegypti adult females indicated that, histochemically, these cells display a pas-negative cytoplasm, which also was strongly and uniformly stained by bromophenol blue and oso4 (martins et al., 2011b). however, the staining intensity changed depending on the diet. for example, in blood-fed mosquitoes the oenocytes are weakly stained in comparison to newly-emerged and sugar-fed individuals. oenocytes are positive for lipid staining and negative for pas reaction indicating that polysaccharides content is not as pronounced as the amount of proteins and lipids. similar results also were found for several mosquito species including aedes albopictus, aedes fluviatilis, culex quinquefasciatus, anopheles aquasalis and anopheles darlingi. in these insects the oenocytes also have uniformly stained cytoplasm (using gomori’s trichrome) confirming the high protein content in these cells (martins et al., 2011c). the cytoplasm of oenocytes has also been shown to be poor of glycogen in several insects. this is the case in a. aegypti (tadkowski et al., 1977; martins et al., 2011b) and in at least four ant species (zara and caetano 2004; roma et al., 2006, 2008). for the cricket gryllus bimaculatus (orthoptera) no glycogen is observed within the first 25 h of the first instar (romer, 1974). however, glycogen can be detected as pas-positive spots reaching nearly 3 µm in diameter within the cells of the cricket as larvae development proceeds. in crickets shortly before moulting glycogen rosettes have completely disappeared. these observations suggest that glycogen function as energy source for the increasing cell metabolism towards the end of the g. bimaculatus first instar (romer, 1974). it seems that the presence of large amount of glycogen is not common in insect oenocytes and generally is restricted to early developmental stages as an energy supply. in agreement with these observations, following apolysis of l. maderae oenocytes still display clusters of glycogen that remain until soon after the first larval epicuticle deposition (rinterknecht, 1985). an exception to the availability of glycogen in insect oenocytes described above is dacus tryoni (diptera, trypetidae). oenocytes in adults of this species display many electron-dense granules, likely glycogen, throughout their cytoplasm (evans, 1967). regarding ribosomes, romer (1974) used pyronine staining during the molting cycle of g. bimaculatus nymphs to assess oenocytes profile. under light microscopy, oenocytes display a patchy staining and under the transmission electron microscope (tem) few ribosomes are found in oenocytes from immediately post-hatch larvae and 145     this condition changes 12 h post moulting. especially around the nucleus, many extended polysomes accumulate ribosomes and the number of ribosomes becomes maximal mainly in the regions of rough endoplasmic reticulum (rer) 35 to 55 h post moult in g. bimaculatus. oenocytes ultrastructure one of the most striking albeit not surprising characteristics of insect oenocytes is the presence of a well-developed ser. the oenocyte ser sometimes occupies almost all of the cell’s cytoplasm (martins et al., 2011d), resembling steroidogenic cells (rinterknecht and matz, 1983), and this is in accordance with their function in lipid synthesis and processing and detoxification (evans, 1967; locke, 1969; clark and dahm, 1973; martins et al., 2011b). the oenocyte’s ser consists of a net of ramified tubules that in the case of g. bimaculatus nymphs can vary in size from 170 to 340 å depending on age (romer, 1974). the development of such prominent ser may start during the insect’s early developmental stages (i.e. embryo) (dorn and romer, 1976; rinterknecht and matz, 1983) and can also occur during the entire larval, pupal and adult stages (locke, 1969; romer, 1974; stoppie et al., 1981; martins et al., 2011b, d). ser-associated organelles such as the multivesicular bodies, the peroxisome-like organelles, and the microbodies described in the insect oenocytes are discussed below. in comparison to the ser, the rer and golgi complex are not prominent in oenocytes. in general, the rer is formed by short stacks that are restricted to certain areas of the cytoplasm (wigglesworth, 1933) or limited to the perinuclear region (tadkowski et al., 1977; martins et al., 2011b, d). these subcellular characteristics are not only observed in adult oenoyctes but are also seen in oenocytes during early insect embryogenesis (dorn and romer, 1976). changes in the ultrastructure of oenocytes were studied considering embryonic (dorn and romer, 1976; rinterknecht, 1985) and postembryonic developments, including hemiand holometabolous insects (wigglesworth, 1933; evans, 1967; locke, 1969; romer, 1974; dorn and romer, 1976). in general, oenocytes are always separated from the fat body cells and other tissues by a basal lamina (stoppie et al., 1981) and another notable characteristic is the abundant number of mitochondria scattered throughout their cytoplasm (romer, 1974; clark and dahm, 1973; sohal, 1973). during the early embryogenesis the oenocyte cell population is heterogeneous. rinterknecht and matz (1983) reported an entire scale of intermediate levels of differentiation in l. maderae. however, it has not been possible to establish whether this heterogeneity reflects the existence of a plurality of cell functions, or whether this is related to the progress in terms of differentiation of a unique cell population. when individual pleuropodial cuticle deposition occurs there is a rapid increase in the number of differentiating oenocytes of l. maderae embryo. at the time of dorsal closure, the deposition of the epicuticle of the embryonic cuticle is preceded by the ser proliferation and by the differentiation of a new oenocyte generation (rinterknecht and matz, 1983). just before general apolysis and during its occurence, the oenocytes display the welldeveloped ser. the oenocyte population then regresses after epicuticle deposition of the first larval cuticle. just prior to the cuticulin layer of the embryonic cuticle is observed, another wave of oenocyte differentiation takes place. in this case, oenocyte differentiation is marked by a rapid biogenesis of the cell membrane and was correlated with the ectodermal coating, the titer of the ecdysone and the differentiation of the prothoracic gland (rinterknecht, 1985). in musca domestica (diptera) oenocytes change according to age in adults and in contrast to other insect which display mononucleated oenocytes, here oenocytes either have one or two nuclei (clark and dahm, 1973; sohal, 1973). curiously, no difference in the appearance of oenocytes between adult males or females has been described for m. domestica (clark and dahm, 1973). differently to four-day-old flies, in older flies (over 30 days), oenocytes undergo a variety of degenerative alterations that may include reduction in the quantity of elements of the ser, a reduction in the matricial density and the number of cristae in mitochondria and the vacuolization of several areas of the cytoplasm. another prominent feature of the degenerating oenocytes of m. domestica adults is the presence of lysosomes (sohal, 1973). in glossina austeni (diptera) adult females, oenocytes exhibit cyclical changes during the postemergence growth and during subsequent cycles of pregnancy. in newly emerged flies, the cytoplasm of the oenocytes contains a few deeply staining inclusions, which become more prominent during the period between emergence and ovulation and remain large during the first cycle of pregnancy (tobe et al., 1973). in one-two-day-old sarcophaga bullata (diptera) adults, the ser is already very dense with many small vesicles. during vitellogenesis of this species, the oenocytes display invaginations of the plasma membrane and high electron dense granules in their cytoplasm that accumulate following completion of vitellogenesis. in the post-vitellogenesis period, the mitochondria become more spherical and the typical parallel arrangement of the cristae disappears. in spite of all the changes observed, it is still not clear what role oenocytes play during the process of adult vitellogenesis of s. bullata (stoppie et al., 1981). oenocytes of the o. fasciatus embryo, which has just differentiated, have a basal membrane already formed and the mitochondria are relatively large, but not as numerous. after migration of the oenocytes into the fat body, pronounced changes in cellular structure occur with the ser occupying almost the whole cytoplasm. vacuoles only occur in embryos and adults, not in larvae, however, microbodies are numerous in the fifth larvae. here, anchorage of oenocytes to fat body cells by desmosomes is occasionally seen in the embryo, but not in larvae or adults of o. fasciatus (dorn and romer, 1976). in a. aegypti adults, oenocytes have a single, large, round, centrally-located nucleus with a 146     prominent nucleolus and chromatin appears in irregular granular clumps, especially around the edge of the nucleus (tadkowski et al., 1977; martins et al., 2011b, d). yet in the mosquito, the oenocytes ultrastructure changes significantly form pupae to adults. in pupae the ser occupies the large extensions of the cytoplasm, while in adults ser are restricted to some areas (for details see martins et al., 2011b, d). a close correlation is also to be observed between the moulting cycle and the differentiation of golgi complex in oenocytes of g. bimaculatus nymphs. immediately after hatching, several golgi complexes are found and certain areas show coated vesicles. as rer increases, the number of golgi complex decreases, but increases again close to the end of the moult. some mitochondria show a circular arrangement of the cristae and they appear in cells showing pronounced lytic activity close to moulting (romer, 1974). fifty four hours after molting of g. bimaculatus, oenocytes cytoplasmic membrane has infoldings, reaching almost to the nucleus (romer, 1974). these canaliculli are observed around the cell’s periphery in a. aegypti, sometimes with delicate membranous and/or fine amorphous flocculent material (tadkowski et al., 1977; martins et al., 2011b, d). they are more developed in a. aegypti blood-fed females in comparison to newly-emerged ones (martins et al., 2011b). in c. ethlius (lepidoptera) larvae, these plasma membrane invaginations are described as a reticular system (locke, 1969; jackson and locke, 1989), however it does not seems to occur in other insects such as g. bimaculatus (romer, 1974). the peroxisome-like organelles are closely associated with ser and scattered throughtout the cytoplasm of l. maderae embryo (rinterknecht, 1985) and in nymphs of g. bimaculatus the ser is associated to microbodies that shows no electrondense content, and reaches a diameter of up to 0.5 µm and transitions from tubules to vesicles are also found. these microbodies show peroxidase positivity demonstrated by benzidine oxidation (romer, 1974). other intriguing structures are found in the oenocytes cytoplasm of insect larvae and adults. they correspond to clefts and may extend through the entire cell and extend for considerable distances and are interpreted as lipid deposits (romer, 1974). clark and dahm (1973) reported that exposure of m. domestica adults to phenobarbital, led to the formation of membranelike scrolls at 48 and 72 h. these membrane-like scrolls resemble to the clefts described in oenocytes of other insects (locke, 1969; romer, 1974) and probably are made of lipids or lipoproteins related to the ser for export, as suggested for c. ethlius larvae (locke, 1969). the isolation and degeneration of parts of the oenocyte cytoplasm takes place by the formation of autophagic vacuoles that include parts of the ser and mitochondria. the autophagosomes dynamics in the first and second instar of g. bimaculatus and c. ethlius larvae are discussed in details by romer (1974) and locke (1969), respectively. oenocytes function, biochemistry and gene expression in spite of studies pointing to oenocytes as lipid processing cells (gutierrez et al., 2007; martins et al., 2011a), their role is not limited only to this function. oenocytes have been shown to participate in homeostasis (e.g., detoxification of xenobiotics) (clark and dahm, 1973; lycett et al., 2006), in the synthesis of several long chain hydrocarbon sex pheromones (wicker-thomas et al., 2009) and other cuticle components (fan et al., 2003), and innate immunity (martins et al., 2011a). oenocytes also play a role in the differentiation of neurons during d. melanogaster embryogenesis through the secretion of semaphorin (sema2a), a peptide that drives axon elongation (bates and whitington, 2007). other studies have indicated that oenocytes produce lipids related to the impermeabilization of the insect’s body (reviewed by lockey, 1988). for instance, in r. prolixus nymphs, oenocytes are associated with epidermis through cell prolongations in which lipid transport from oenocytes to epidermal cells is thought to occur (wigglesworth, 1988), and in c. ethlius larvae, oenocytes are located near epidermal wax glands, suggesting that they participate in the synthesis of wax precursors (locke, 1969). gutierrez et al. (2007) suggested that the oenocytes’ analogous liver function in storage of sugar and in lipid processing appear to be divided between the fat body trophocytes and oenocytes in d. melanogaster larvae. the lipid mobilization during starvation of fruit fly larvae led to a rise in lipid droplets within oenocytes, which maintained a low level of lipids in the hemolymph. this is similar to what happens with the adipose tissue and the liver in human during steatosis or adipose degeneration. however, unlike the mammalian liver, d. melanogaster oenocytes are distributed in discreet paired clusters along the larval body wall allowing the cells to be in intimate contact with the hemolymph where nutrients circulate (reviewed by bharucha, 2009). it has been shown that oenocytes participate directly during courtship behavior in d. melanogaster adults by means of the hydrocarbons they secrete, and in the female fruit flies these cells are the primary organ for communicating species and sex identity to males (ferveur et al., 1997; billeter et al., 2009). through an elegant set of experiments, billeter et al. (2009) generated an oenocyte gal4 driver derived from the regulatory sequence of one of the desat1 promoters expressed specifically in oenocytes of adult females. ablation of adult oenocytes in males by inducing expression of the pro-apoptotic gene hind suggested that the oenocyte-less (oe-) males display normal courtship behavior towards wild-type females, but in a fashion considered less intense than control males. however, wild-type females are less receptive to oe males than control males (billeter et al., 2009). the oenocytes ablation induced an unnatural behavior in males that have vigorous courtship by each other (ferveur et al., 1997). surprisingly, oefemales, i.e., lacking hydrocarbons, are more attractive than those 147     with a normal hydrocarbon profile to wild males, suggesting that female hydrocarbons normally act to slow down male mating attempts. billeter et al. (2009) also tested the behavior of males from other species towards d. melanogaster or females. they showed that males of other drosophila species (such as d. simulans, d. yakuba and d. erecta) engaged in courtship of the d. melanogaster oe females, but exhibit limited or no courtship towards control. these data indicated that oenocytes and their hydrocarbon products are important components of the reproductive isolation barrier. the synthesis of pheromone in the adult of d. melanogaster has been demonstrated to be a result of desaturase activity expressed mainly in the oenocytes. wicker-thomas et al. (2009) used rnai to knock down a desaturase gene (desat1) in oenocytes resulting in 96 % decrease in unsaturated hydrocarbons in adult males and 78 % in females. inactivation of the female specific desatf gene (responsible for diene formation), resulted in a dramatic loss of pheromones (98 %) combined witha two-fold increase in monoenes. another gene whose expression is prominent in the fat body, in oenocytes, and in midgut tissues of d. melanogaster is indy (for “i’m not dead yet”) (knauf et al., 2002). oenocytes are supposedly related to fruit fly longevity and, interestingly, a reduction in indy proteins levels dramatically extended the life span in fruit flies without sacrificing their fertility or physical activity (knauf et al., 2002). also, the expression of catalase in d. melanogasteris mostly confined to these same tissues indicated above, starting from the embryonic development and extending into adult stages, and the expression levels rise according to age (klichko et al., 2004). oenocytes seem to be an important site for the biosynthesis of hemoproteins in insects. in the d. melanogaster embryo, a site for porphyrin biosynthesis, and expression of the enzyme δaminolevulinate synthase (alas) is specifically detected in oenocytes suggesting a role of these cells in hemoproteins biosynthesis (ruiz de mena et al., 1999). our group previously investigated the transcriptome of oenocytes isolated from a. aegypti pupae (martins et al., 2011a). our data indicated that eight percent of the transcripts in these oenocytes coded for the hemoprotein cytochrome p450. this enzyme participates in the metabolism of different molecules such as sterols, steroid hormones, and several lipids, and also in a variety of physiological roles ranging from development, to feeding and growth, to resistance to pesticides and tolerance to plant toxins [martins et al. (2011a) and papers therein]. accordingly, p450 expressed in oenocytes likely participate in steroid metabolism as previously suggested by romer et al. (1974) demonstrating that oenocytes isolated from t. molitor larvae synthesize αand β-ecdysone from 414c-cholesterol precursors in vitro. in addition, approximately four percent of the transcripts in the oenocytes from a. aegypti pupae encoded other types of detoxification proteins, such as alcohol dehydrogenase, catalase, and a nadph cytochrome p450 reductase (cpr) (martins et al., 2011a). in anopheles gambiae, oenocytes are one of the major sites of xenobiotic metabolism in the adult mosquito (lycett et al., 2006). in both a. gambiae and d. melanogaster, oenocytes express high levels of nadph cytochrome p450 reductase (cpr) that is required for cytochrome p450s involved in metabolic insecticide resistance, and rnai mediated knockdown of cpr in oenocytes led to enhanced sensitivity to permethrin (lycett et al., 2006). aedes aegypti oenocytes also express 23 transcripts that code for lipid-metabolizing proteins, such as fatty acid synthase (fas), elongase and estradiol dehydrogenase. these enzymes are related to integument hydrocarbon synthesis, such as long-chain fatty acids, and pheromone synthesis [martins et al. (2011a) and papers therein]. such findings are in agreement with results obtained by fan et al. (2003) showing that oenocytes are responsible for hydrocarbon synthesis in b. germanica. interestingly, a small fraction of a. aegypti transcripts (1.9 %) coding for proteins such as lysozymes and serine proteases that likely participate in mechanisms of innate immune responses, is suggestive of a role for oenocytes in insect immunity. hydrocarbon synthesis by oenocytes depends on age (hepburn et al., 1991). in this case, honeybee (a. mellifera) workers displayed an increase in saturated c25 and c27 hydrocarbons with a concomitant decrease in the c33 molecules present in oenocytes as they become older. in addition, the hydrocarbon and fatty acid profiles of isolated oenocytes were also found in newly synthesized wax, suggesting that oenocytes are the likely source of hydrocarbons for this cuticular component (hepburn et al., 1991). additional studies using labeled molecules to investigate the metabolism in oenocytes were performed by diehl (1973, 1975), tobe and davey (1974), and romer (1980). for example, using h3tyrosine and -leucine injected in g. austeni females tobe and davey (1974) demonstrated that the incorporation of these molecules by oenocytes is age dependent, and is highest at eclosion and just after larviposition. in other insect models, such as in fifth-instar nymphs of s. gregaria, oenocytes synthesize and secret hydrocarbons, mainly cuticular lipids, from 14c-acetate and several other lipids of unknown nature (diehl, 1973, 1975). also, incorporation of 14c-labeled cholesterol following abdominal injections of m. domestica larvae indicated a strong labeling of certain portions of the fat body especially within oenocytes suggesting that these cells play a central role in the metabolism and/or storage of sterols and they possibly take part in abdominal ecdysone biosynthesis (studinger and willig, 1975). oenocytes infection little is known about the ability of pathogens to infect insect oenocytes. generally speaking, question such as the type of pathogens that are able to invade these cells, or how invasion and establishment of infection proceeds (in case of those pathogens shown able to infect), as well as changes or responses to infection by oenocytes, have never been fully addressed. nevertheless, it is 148     known that microsporidia fungi are able to infect mosquito oenocytes, including larvae, pupae and imago, and that these infections occur transovarially (kellen et al., 1965, 1967; hall, 1985; sweeney et al., 1988). among the different fungi species that were shown to infect oenocytes (of mosquitoes) we include those in the genera thelohania, amblyospora, nosema, stempellia, parathelohania and plistophora (kellen et al., 1967; hall, 1985; sweeney et al., 1988; garcia et al., 1993). infection of oenocytes can sometimes be recognized, depending of the mosquito species or the invading fungi, as the oenocytes hypertrophied as the parasites multiply, and infections are fatal to the insect (kellen et al., 1965). however, in females of culex salinarius (diptera) infected by amblyospora sp., parasites do not multiply significantly until late pupal or early adult stages at which time they gradually fill the oenocytes (hall, 1985). hall (1985) also reported that in healthy mosquitoes, the larval oenocytes break down during the pupal and early adult stages, but in infected individuals they persist, undergoing fusion during the pupal stage to form syncytia containing two to seven nuclei per syncytial oenocyte. during this period, the infected oenocytes break loose from the fat body and begin circulate throughout the hemocel, mainly in the head and particularly the thorax in the vicinity of the foregut (hall, 1985). infected hypertrophied oenocytes of anopheles pseudopunctipennis franciscanus adults adhered closely to the gut and harbored various stages in the life cycle of nosema chapmani. in larvae and adults of culex tarsalis infected by nosema lunatum, oenocytes were especially evident in adult females, where they occasionally attached to ovaries and fat body (kellen et al., 1967). amblyospora sp. in c. salinarius exhibited two distinct developmental stages, one in each host sex. in females, the entire life cycle was restricted to host oenocytes where multiplication of diplokarya occurred during merogony (andreadis, 1978). infections were initiated when small binucleated sporoplasms infected the developing eggs within the female host and were subsequently transferred to the next generation when the eggs were laid. within embryonated eggs and newly hatched larvae of both sexes small, oval, diplokaryotic stages invaded host oenocytes and underwent an initial multiplicative phase (merogony) where they divided mitotically to produce more diplokarya. diplokarya subsequently broke out of the oenocytes and invaded trophocytes where they multiplied repeatedly (andreadis,1978). in anopheles quadrimaculatus, diplokaryotic meronts of parathelohania anophelis (microspora: amblyosporidae) were observed within oenocytes of third-instar female larvae. parasites containing six to 40 nuclei were also noted in oenocytes from the second and third abdominal segments of female mosquitoes 24 h after emergence. from larvae to newly emerged adults, parasites multiplied within the oenocytes restricted to the second abdominal segment and in the anterior of the third abdominal segment. the development of the spore took place inside large groups of oenocytes in the interior of the abdomen during days three to five after emergence and before the female took a blood meal (garcia et al., 1993). the infection of oenocytes by viruses has been poorly studied, and little data are available. nevertheless, it has been reported that old adults of d. melanogaster oenocytes were supposedly infected by unknown virus-like particles (philpott et al., 1969), whereas dengue virus serotype 2 was able to infected oenocytes isolated from a. aegypti in culture (guedes and pimenta, 2009). oenocytes isolation and culture until recently, very few studies involving isolated insect oenocytes were reported. today, with the improvement of dissection and harvesting techniques in vitro, oenocyte cultures can be established opening new avenues of research focused on the role of these cells in insect metabolism. the ability to isolate and maintain oenocytes in vitro led to the first studies demonstrating the role of these cells in the metabolism of ecdsteroids. this was accomplished by romer et al. (1974) using oenocytes isolated from the abdomen of t. molitor larvae that demonstrated that these cells are able to synthesize αand β-ecdysone from 14c-labeled cholesterol. oenocytes were also successfully isolated from b. germanica and found to represent different cell types (fan et al., 2003). using a mild enzymatic treatment these authors obtained oenocytes in suspension after digesting their basal lamina. from this highly oenocyte-enriched cell suspension they demonstrated that oenocytes can produce hydrocarbons. although obtaining isolated oenocytes can be tricky due to the nature of how these cells are distributed in the insect and how they might associate with other tissues, oenocytes in the mosquito pupa are easily separated and collected (martins et al., 2011a, d). additionally, a detailed demonstration on how to identify and collect oenocyte clusters from d. melanogaster adults can be found at krupp and levine (2010). studies involving the primary cultivation of insect oenocytes are rare and one example is the work of martins et al. (2011d). maintaining viable in culture for up to two months they described details about the morphology of these cells combining various microscopic approaches. these cells were attached to the glass substrate and were visualized as single or clustered cells that maintain main cytoplasmic characteristics found in freshly isolated cells, such as the general chromatin organization and the cytoplasm filled with ser. however, there is a decrease in the mitochondria number and size in the cultured cells. during this cultivation period some cells keep their basal lamina and do not divided and this result was expected since oenocytes are highly differentiated and specialized cells. the absence of proliferation occurs also in oenocytes from d. melanogaster embryo’s in vitro. however, in this case, oenocytes are capable to aggregate after dissociation (lesseps, 1965), what not happens with pupal oenocytes of a. aegypti (martins et al., 2011d). 149     clearly, methods to isolate and maintain oenocytes in vitro can contribute towards studies aimed at understanding the metabolism of such cell type, perhaps providing novel strategies for insect control. further, the long-term survival of viable oenocytes in primary culture also provides a tool for investigating their interactions with pathogens (martins et al., 2011d). conclusions and perspectives this review was intended to provide the reader with a general perspective of oenocyte function by outlining some of the key findings related to oenocyte’s location within insects, histochemistry, biochemistry, and gene expression. with the use of advanced molecular tools it has become clear that insect oenocytes play a wider role during insect metabolism than lipid processing. the identification of proteins (or of transcripts) expressed within oenocytes demonstrated that these cells also participate in detoxification. in addition, little is known about the role(s) of different oenocyte populations during insect post-embryonic development, including larval and imaginal stages. gene ablation targeting d. melanogater oenocytes in larval stages indicate that these cells can be considered analogous to mammalian hepatocytes (see gutierrez et al., 2007). on the other hand, in adult fruit flies, oenocytes play key role in the sexual behavior by means the synthesis of hydrocarbons (ferveuret al., 1997; billeter et al., 2009). however, such oenocyte functions in d. melanogaster have not yet been confirmed for other insects. thus, in our view, the ability to harvest and maintain oenocytes in culture can positively impact future studies focused on oenocytes metabolism, and gene and protein expressions, and assist in identifying other potential roles in these cells so crucial for insect survival. references agui n. joint action of prothoracic glands and oenocytes on the cultivated wing discs of the cabbage armyworm, mamestra brassicae l. in vitro (lepidoptera: noctuidae). appl. ent. zool. 9: 256-260, 1974. andreadistg. life cycle and epidemiology of aniblyospora sp. 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(2010) concluded that immunological parameters, such as phenoloxidase activity, provide good indicator of coral immunity and underpin linkages between the susceptibility of corals to disease. immunity refers to the ability of an organism to resist infection with the nonspecific and immediate innate immune pathways providing the first line of internal defense. a key component of invertebrate innate immunity is the presence and activation of the melanin-synthesis pathway in response to invasion by foreign organisms or physical injury (rinkevich, 2004). melanin pathway activity, as ___________________________________________________________________________ corresponding author: analia fernández gimenez instituto de investigaciones marinas y costeras facultad de ciencias exactas y naturales universidad nacional de mar del plata funes 3350. 7600, mar del plata, argentina e mail: fgimenez@mdp.edu.ar indicated by levels of the activating enzyme phenoloxidase, has been documented in scleractinian corals, gorgonians and true soft corals from the caribbean and indo-pacific (palmer et al., 2008, 2011; mydlarz et al., 2009; mydlarz and palmer, 2011). furthermore, melanin is a redoxactive pigment and therefore has the potential not only to be cytotoxic and kill pathogens, but also to scavenge oxygen radicals that may be harmful to the host. oxygen radical scavengers and enzymatic antioxidants are important during infection, as host responses frequently induce oxidative stress conditions (palmer et al., 2011). several studies have linked cnidarian peroxidase activity with antioxidant potential (hawkridge et al., 2000; olano and bigger, 2000) and oxidation of fatty acid hydroperoxides (koljak et al., 1997). mydlarz and harvell (2007) argued that enzymatically driven resistance measures, such as peroxidase activity, are important in the early responses of the sea fan gorgonia ventalina to a fungal pathogen aspergillus sydowii. as immunity determines, the ability of an organism to resist and eliminate infection and to recover from injury, it can be used as a predictor of compromised health susceptibility. the immune defenses increase fitness by promoting survival, through disease resistance and the maintenance of tissue integrity. the study of marine invertebrate 192 mailto:fgimenez@mdp.edu.ar ecological immunity is advancing very quickly and critical signaling pathways and cytotoxic responses are being elucidated. the presence and relative activities of phenoloxidase and antioxidants, such as, peroxidase were investigated in several coral species, mainly scleractinian (mydlarz and harvell, 2006). the presence of immune responses within sea anemone species have received little or virtually no attention, but their biology suggests that diseaseresisting defenses would be adaptive. hutton and smith (1996) investigated the antimicrobial defenses of actinia equina and hawkridge et al. (2000) determined the subcellular distribution of antioxidant enzymes in the temperate sea anemone anemonia viridis. according to the scarce knowledge on this topic related with sea anemones we decided to investigate the phenoloxidase and peroxidase activities in ectoderm, endoderm and tentacles of actiniarians aulactinia marplatensis and bunodosoma zamponii. these species are the most common species in the rocky intertidal zone of mar del plata (38º 05’ s-57º 32’w). they are found mainly attached to hard quartzitic substrate and the taxonomical status of both species was studied by acuña et al. (2007) and braga gomes et al. (2012), respectively. many aspects of their biology and ecology were also studied, like those related with reproduction (zamponi and excoffon, 1986; excoffon and zamponi, 1991, 1997), population ecology (acuña and zamponi, 1995a, 1996a, 1998), feeding (acuña and zamponi, 1995b; 1996b; acuña, 1997; acuña et al., 1999), as well as particular topics like analyses of the cnidae (acuña and zamponi, 1997) and mycosporine-like amino acid content (arbeloa et al., 2010). however other aspects, like immunological, remain unknown. this study, represents the first record toward specific immune information about the mentioned sea anemone species of argentina, and thus permits prediction of the potential effects of environmental factors on immune response. materials and methods sample collection specimens of the sea anemones aulactinia marplatensis and bunodosoma zamponii were obtained from the intertidal zone of the rocky area with a quartzitic substrate in punta cantera, mar del plata (38° 05’s and 57° 38’w). the individuals, all around the same size (30 mm in basal diameter), were caught in december 2012 in the same area for both species during the low tide, but covered by water. the organisms, n = 10 for each species, were maintained at room temperature in an aquarium with decanted and aerated sea water and they were sampled one day after collection. for both anemone species, tissue of epidermis, endodermis (gastrodermis) and tentacles, were removed and homogenized with a 50 mm phosphate buffer at ph 7.8 on ice. samples were then centrifuged for 5 min at 4,000 rpm and 4 °c, avoiding the mucus layer and the supernatant (protein extract) was carefully removed and stored at -20 °c. soluble protein in protein extract was measured by the method described by bradford (1976), using chicken egg white albumin as the standard. phenoloxidase and peroxidase activities were determined according to palmer et al. (2011). phenoloxidase was assay using 50 µl of protein extract; 100 µl of phosphate buffer (50 mm ph 7.8) and 50 µl double distilled water pyrogen free were incubated for 20 min at room temperature, then 50 µl l-dopa (3 mg ml-1) (aldrich, 333786) was added and after 10 min 350 µl of cacodylate buffer (200 mm ph7.4) was addedand absorbance at 490 nm was recorded (shimadzu uv-2102 pc, uv-visible scanning spectrophotometer). two control treatments were used, without l-dopa or without protein extract. peroxidase activity was determined using 60 µl of protein extract, 210 µl of phosphate buffer (10 mm ph 6) and 240 µl of pyrogallol (sigma p0381) with 150 µl hydrogen peroxide 1.6 volumes to activate the assay, after 3 min the absorbance was recorded at 470 nm. control treatment containing 60 µl of protein extract and 600 µl of phosphate buffer (10 mm ph 6) was done. phenoloxidase and peroxidase activities were expressed as the change in absorbance per mg protein (abs mg protein-1). all assays were run by triplicate. soluble protein content and enzymatic activity were analyzed with anova after testing normality and homogeneity of variances. significant differences were considered at p < 0.05. when significant differences were found, a tukey-kramer multiple comparison test was performed to locate these differences. analysis were made using ncss 8 software. results and discussion the two sea anemone species used in this study, a. marplatensis and b. zamponii, demonstrated differing levels of constituent immunity, as indicated by the immune parameter activities. soluble protein did not differ significantly among species and tissues, with levels between 15.8 and 20.2 mg ml-1 for a. marplatensis and 17.0 and 21.8 mg ml-1 for b. zamponii. phenoloxidase (po) activity was observed in all tissues evaluated, having the ectoderm of b. zamponii the highest po activity at 0.025 abs 470 nm mg protein-1. mean po activities of endoderm and tentacles for both species and ectoderm of a. marplatensis were significantly lower than b. zamponii's ectoderm (figs 1a, c). palmer et al. (2010) demonstrated that po activity was present in different coral families, such as, euphylidae, acroporidae, pocilloporidae, alcyonacae, merulinidae, faviidae, mussidae, fungiidae, poritidae and oculinidae; and varied significantly among them. phenoloxidase is the activating enzyme of the melanin-synthesis pathway, a key component of invertebrate immunity and the melanin-synthesis which provides cytotoxic defense, a protective barrier and structural support (palmer et al., 2011). for anthozoans, melanization was the first documented within a sea fan, as a barrier against a fungal infection (petes et al., 2003; mullen et al., 2004) described the amebocytes involved. in the same species, aggregations of amebocytes were documented around fungal infections, and their granular content was confirmed to be melanin 193 fig. 1 (a, b, c, d). aulactinia marplatensis versus bunodosoma zamponii tissue comparisons for mean (± s.e.) phenoloxidase and peroxidase activities, ect = ectoderm, end = endoderm, tent = tentacles. (mydlarz et al., 2008). tucker et al. (2011) demonstrated that amebocytes were commonly encountered in the mesoglea, in the thick fibril free zone under the epidermis of anemone nematostella vectensis, while melanin-containing granular cells were located predominantly in the epidermis in 15 scleractinian species by palmer et al. (2010), this observation could be explain the highest po activity registered in ectoderm of b. zamponii in the current study. invertebrates with low po activity are more susceptible to disease and similarly, scleractinian corals are more susceptible to bleaching and disease, as recently documented in a wide range of coral families (palmer et al., 2011). differences in residual po activity among coral families indicate physiological disparities that may have implications at an ecological scale in terms of disease resistance, with families having higher po activity being more able to resist infection. this prediction is consistent with correlations found between po activity and immunocompetence for numerous invertebrates (palmer et al., 2010). according to previous information, we may be suggesting that b. zamponii is more able to resist infection, however additional research is necessary. the super-family of peroxidase enzymes contains many isoforms which partake in a variety of metabolic functions. in animal, peroxidase enzymes (pe) are involved in disease resistance and stress responses. changes in peroxidase levels can signify immunomodulation due to contaminants and other environmental stressors (mydlarz and harvell, 2007). these authors examined the inducibility of coral peroxidases by experimentally exposing corals to fungal pathogen and found that enzyme activity was induced after an incubation period and they also hypothesize that gorgonia ventalina utilizes the peroxidases as an integral component in disease resistance pathways. palmer et al. (2011) 194 evidenced peroxidase activity in bleached and healthy colonies of acropora millepora and proposed that this enzyme is potentially important for mitigate the effect of oxidative stress. dikens and shick (1984) established that peroxidase activity in the sea anemone anthopleura elegantissima was highest in the tentacles and oral disc. a more detailed localisation of this enzyme was attemped by hawkridge et al. (2000) who localised the antioxidant enzymes in granulated vesicles, accumulation bodies of endosymbiotic algae and all forms of cnida in the temperate sea anemone anemonia viridis and tropical coral goniopora stokesi, both species considered abundant in their respective habitats. in the current study, mean peroxidase activity was approximately equivalent (~0.06 abs 470 nm mg protein-1) for all tissues of a. marplatensis and tentacles of b. zamponii, and significantly higher than ectoderm and endoderm of b. zamponii (~0.03 abs 470 nm mg protein-1) (figs 1b, d). phenoloxidase and peroxidase activities are commonly associated with the mechanisms of innate immunity in invertebrates, and were confirmed for the first time in the studied actiniarians. the high production of phenoloxidase observed in b. zamponii would provide a continual level of resistance to infection and this species to be less susceptible to stress and disease, compared to a. marplatensis. however the latter has a possible additional defense, the attached cover of gravel on its column that could constitute a physical barrier to pathogens. this cover is common on the column of intertidal sea anemones with verrucae, and can support a small but rich community of invertebrates and algae (barcellini, 2011). thus, via collaborative efforts between ecologists, immunologists, cell biologists, and physiologists, we may expand the current understanding of innate immunity in naturally occurring ecologically important species. with greater understanding of the connections between environment and organismal immunity, we can make predictions about the effects of changing climate and environment on immunocompetence and disease outbreaks (mydlarz et al., 2006). acknowledgements this research was financially supported by the mar del plata national university (argentina) grant no. 585-12. we are also grateful to lic. yamila rodriguez for her suggestions for the draft of this manuscript. references acuña fh. 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issn 1824-307x research report immune reactions of the lesser mulberry pyralid, glyphodes pyloalis walker (lepidoptera: pyralidae) to the entomopathogenic fungus, beauveria bassiana (bals.criv.) vuill and two developmental hormones r khosravi1, jj sendi1, a zibaee1, ma shokrgozar2 1department of plant protection, faculty of agricultural sciences, university of guilan, rasht 41635-1314, iran 2pasteur institute of iran (ipi), no. 69, pasteur ave, tehran, 1316943551, iran accepted december 27, 2013 abstarct effects of beauveria bassiana (bals.-criv.) spores on immune functions of glyphodes pyloalis walker larvae were studied. total and differential hemocyte counts revealed that infection by b. bassiana caused a dramatic change in hemocyte number. in vivo and in vitro studies demonstrated that increase in time exposure to fungal pathogen resulted in elevated phagocytic activity. nodules were formed in response to spore injection and their numbers were maximal 6 h post injection. the phenoloxidase (po) activity in treated larvae changed significantly 3 and 6 h after spore injection compared with the control larvae. the reduction in po activity was observed 12 and 24 h post injection. effect of two developmental hormones, juvenile hormone (jh) and ecdysone on cellular immune response of g. pyloalis were also evaluated. larval treatment with jh prior to b. bassiana spore injection reduced nodulation while ecdysone enhanced it. these results demonstrated that ecdysone and jh play an important regulatory role in the immune response in the studied insect. key words: hemocyte; nodulation; juvenile hormone (jh); ecdysone; cellular immunity   introduction entomopathogens are important regulatory factors of insect populations. at present, several species of entomopathogenic fungi are used as biocontrol agents in insect pests (bogus et al., 2007). the entomopathogenic fungus, beauveria bassiana (bals.-criv.) shows great potential for the control of a broad range of insect species. this fungus has been developed for use as a biological pesticide (ansari and butt, 2012). the process of infection begins with attachment of the conidia to the cuticle. the fungus then germinates, grows and penetrates the integument. in addition to being able to reach the cuticle barrier, this pathogen possesses the ability to replicate in the insect hemocel (xiong et al., 2013). upon entering the hemocel, fungal cells interact with insect hemocytes. they then, induce peptides and proteins that mediate humoral immunity (borges et al., 2008). insects defend themselves from infection by a variety of potential pathogens including; bacteria, ___________________________________________________________________________ corresponding author: roya khosravi department of plant protection faculty of agriculture university of guilan rasht 41635-1314, iran e-mail: khosravi.roya@yahoo.com fungi and parasites in natural habitats. they have therefore evolved efficient host-defense mechanisms to survive (yamauchi, 2001). the immunity system in insects consists of cellular and humoral defense responses. humoral defenses refer to antimicrobial peptides, cell adhesion molecules, lysozyme, lectins, and the prophenoloxidase (propo) (hoffmann, 2003; kanost et al., 2004). cellular defenses include responses such as phagocytosis, encapsulation, and clotting that with hemocytes acting as important mediators of such processes (lavine and strand, 2002). several morphologically distinct hemocyte cell types work together in the immune reactions. cellular reaction include phagocytosis in which individual hemocyte ingest large particles that enter the hemocel from the environment. this process is essential for host defense in higher eukaryotes against infectious microorganisms and for the elimination of apoptic cells generated during development. this is regarded as the main cellular reaction of innate immunity in vertebrates and invertebrates (borges et al., 2008). foreign particles are identified by phagocytic cells via recognition by a series of receptors on cell membranes that bind to pathogen-associated molecules (rosales, 2011). two types of hemocytes involved in phgocytosis are the granulocytes and the plasmatocytes. however, 11   http://en.wikipedia.org/wiki/giuseppe_gabriel_balsamo-crivelli http://en.wikipedia.org/wiki/giuseppe_gabriel_balsamo-crivelli http://en.wikipedia.org/wiki/jean_paul_vuillemin mailto:khosravi.roya@yahoo.com fig. 1 total hemocyte count (cells ×104/ml) of g. pyloalis infected with b. bassiana spores. *means ± se followed by the asterisk indicate significant differences versus control (p < 0.05) according to the tukey’s test. their contribution in this process varies between insect species (moushumi et al., 2008). after entering the hemocel, micro-aggregations of hemocytes (granulocytes and plasmatocytes) are initiated on microorganisms. this process is initiated by changing of circulating hemocytes from nonadhesive to adhesive cells that are able to bind to microorganisms (lavine and strand, 2002). these micro-aggregation will eventually lead to the formation of nodules. in the later stages of nodule formation, melanization takes place within the nodules. finally, foreign particles almost die. several factors such as asphyxiation, production of toxic quinones or hydroquinones via the propo cascade and antibacterial peptides have been proposed to function as killing agents (nappi and christensen, 2005). phenoloxidases (pos) are vital enzymes involved in a number of crucial processes, such as defense, wound healing, coagulation, sclerotization, and melanization (bogus et al., 2007). pos are present as inactive precursor, the propo in insect hemolymph and are activated in response to wounding or infection as a part of the innate immune response (cerenius et al., 2008). these enzymes are similar to mammalian tyrosinases in their ability to use reactive sites containing copper atoms to catalyze two types of reactions that require molecular oxygen as a substrate. the cytotoxic quinones that are produced during oxidation are placed over the surface of large foreign materials in order to form melanized layers, which help to kill the encapsulated fungi, bacteria or parasites (ling et al., 2005). table 1 number of hemocyte types from control and treated larvae of g. pyloalis with b.bassiana at different times after the immune challenge. control treatment 1 h 3h 6h 12 h 24 h 1 h 3 h 6 h 12 h 24 h pr 20.28±2.02 17±2.9 18±1.89 18.6±2.03 15.6±1.69 20.2±1.76 17.4±1.74 19.6±1.44 15±1.49 18±1.89 pl 107.6±3.36 124.6±3.59 112.4±2.85 119±2.07 123±3.8 185.2±3.64* 214±1.81* 183±3.22* 99.8±4.41* 86±4.84* gr 52.4±2.4 57.2±1.06 65±3.48 62±2.47 68.2±2.26 67±3.61* 110±3.96* 72±3.02* 49±4.13* 41±3.42* sp 7.2±0.91 8.6±1.28 11.2±1.28 10±1.25 9±1.1 7.6±1.06 9±1.31 9±1.0* 8.2±1.21* 8.2±0.91 oe 10.6±1.39 9.8±2.12 10±1.23 9.8±1.21 8.6±0.94 13.4±1.29* 12±1.45 11.2±1.43 13±1.49* 9.6±1.23 pr= prohemocyte; pl= plasmatocyte; gr= granulocyte; sp= spherulocyte; oe= oenocytoid 12   fig. 2 in vivo and in vitro phagocytosis of b. bassiana spores by plasmatocytes and granulocytes. endocrine factors are involved in the immune system and mediate immune signals. the eicosanoids (stanley, 2000), biogenic amines (baines et al., 1992) adipokinetic hormone (goldsworthy et al., 2003), juvenile hormone (jh) and 20-hydroxyecdysone (20e) (franssens et al., 2006) change insect immune response to pathogenic agents. inhibition of hemocyte encapsulation response was observed in tenebrio molitor after injection of jh (rantala et al., 2003). the 20e promotes nodule formation of neobelliera bullata in response to injection of laminarin, the components of bacterial cell wall (franssens et al., 2006). the lesser mulberry pyralid, glyphodes pyloalis (lepidoptera: pyralidae) is a specialist insect on mulberry, and is widely distributed throughout asia and the northern province of iran. this pest has caused severe damage to mulberry plantations in northern iran and has posed a serious concern to silkworm growers. fifth instar larvae feed on the whole leaf until only the ribs remain (khosravi and jalali sendi, 2010). populations of most pests are usually regulated by density dependent factors involving pathogens and parasites. thus, understanding the interactions of pest with their pathogens and parasites and hence, the cellular defense responses are needed for developing best methods of pests' control. hemocytes perform certain vital activities in insects, and thus hematological studies are fundamental in the field of insect physiology (el-aziz and awad, 2010). an important necessity in the study of the fungal pathogen host interaction is the ability to detect mechanisms that are used by pathogens to overcome the insect’s immune defense system. table 2 thc and number of plasmatocytes and granulocytes (×104 cell/ml) of g. pyloalis larvae at 3, 6 and 12 h after jh treatment prior to b. bassiana spore injection. thc number of plasmatocytes number of granulocytes 3 h 6 h 12 h 3 h 6 h 12 h 3 h 6 h 12 h k 347.67±4.21a 337.67± 4.78a 292.33± 3.53a 215± 6.13a 241.33± 3.01a 239.66± 2.22a 85.66± 2.74a 87.3±3. 16a 84.00± 1.62a a 323.67±3.6a 278.00± 4.6b 251.67± 3.64b 220±2. 08a 181.00± 3.04c 166.00± 1.58c 68.33± 2.24b 69.66± 2.42b 69.33± 2.21b b 332.47±3.09a 287.33± 4.3b 256.33± 3.53b 224±2. 95a 193.00± 2.56bc 173.33± 2.12c 69.66± 2.24b 64.3± 1.74b 67.00± 2.46b c 330.00±3.92a 307.00± 2.79ab 283.33± 3.96ab 213.6± 1.87a 191.33± 2.42bc 213.33± 2.58ab 73.00± 2.64ab 75.00± 1.89ab 66.4±1. 44b d 327.43±2.38a 309.67± 3.53ab 276.33± 2.53ab 224.5± 2.88a 201.00± 3.14b 198.00± 3.08bc 75.65± 1.87ab 74.66± 2.85b 74.32± 2.17ab k = control; a = jh 0.5 mg/ml; b = jh 0.25 mg/ml, c = jh 0.125 mg/ml; d = jh 0.062 mg/ml. means ± se within the same column followed by the same letter are not significantly different (p ≤ 0.05 tukey test). 13   this study was undertaken to investigate the effects of b. bassiana isolate fashand on cellular immune responses and the po activity of g. pyloalis. secondly, in order to understand the role of insect hormones in immune responses, the effects of jh and ecdysone, two key insect hormones on immune reactions of this pest were evaluated. materials and methods insects rearing larvae of g. pyloalis were collected from mulberry orchards (kenmuchi var.) in rasht (37°16′51″n 49°34′59″e), north of iran. they were maintained in the laboratory in a rearing chamber of constant temperature (25 ± 1 ˚c), relative humidity (75 ± 5 %) and photoperiod (16:8 h light:dark). larvae were placed in plastic jars 10×20 cm and were daily provided with fresh mulberry leaves (kenmuchi var.). on adult emergence, they were transferred to 18×7 cm transparent jars and were provided with fresh leaves for egg laying and cotton wool soaked in 10 % honey for feeding. beauveria bassiana culture b. bassiana isolate fashand was grown in sterile petri dishes containing potato dextrose agar (pda) and were incubated at 25 ± 1 ˚c in complete darkness. spores were harvested from pda plates with a sterile scalpel after 14 days. the final concentration was adjusted to 1×105 spores/ml using a hemocytometer in distilled water containing 0.01 % of tween 20. injection of insects with spores fifth instar larvae of g. pyloalis were immobilized on ice for 5 min and were surface sterilized with 70 % ethanol. this experiment was replicated three times and ten insects were used in each replicate. after injection with 1×105 spores/ml fig. 3 induction of nodule formation in g. pyloalis larvae with 2 μlb. bassiana spores injection. (2 µl) by a 10 µl hamilton syringe, the larvae were transferred to rearing jars provided with fresh mulberry leaves, to follow the course of the assay for further observation. the control larvae were injected with distilled water containing 0.01 % of tween 20 (2 µl) alone. effect of fungal spores of b. bassiana on hemocyte number to determine, whether injection of spores of b. bassiana could cause any changes in the levels of total and differential hemocyte numbers, the fifth instar larvae were injected with 2 µl of 1×105 spores/ml concentration between the second and third prolegs. similarly, the controls were injected with 2 µl of sterile distilled water containing 0.01 % fig. 4 effect of b. bassiana spores on nodule formation in fifth instar larvae of g. pylalis. *means ± se followed by the asterisk indicate significant differences versus control (p < 0.05) according to the tukey’s test. 14   http://tools.wmflabs.org/geohack/geohack.php?pagename=rasht&params=37_16_51_n_49_34_59_e_type:city_region:ir fig. 5 effect of b. bassiana spores on phenoloxidase (po) activity in fifth instar larvae of g. pylalis. *means ± se followed by the asterisk indicate significant differences versus control (p < 0.05) according to the tukey’s test of tween 20. hemolymph was collected by cutting one of the prologs 1, 3, 6, 12, and 24 h after injection from chilled, surface sterilized (70 % ethanol) larvae. this experiment was replicated three times and ten insects were used in each replicate. samples of hemolymph from each larva were diluted 5-fold with a cold anticoagulant buffer (0.098 m naoh, 0.186 m nacl, 0.017 m edta and 0.041 m citric acid, ph 4.5). then, the total and differential hemocyte numbers were counted on an improved neubauer hemocytometer for each treatment (el-aziz and awad, 2010). labeling of b. bassiana spores the spores of b. bassiana for labeling were obtained from the 10 14 day culture on pda medium. the spores were re-suspended in 10 ml of phosphate buffered saline (pbs) (0.13 m nacl, 2.68 mm kcl, 8.1 mm na2hpo4 and 1.47 mm kh2po4, ph 7.4, autoclaved), were then washed and resuspended in a sterile co3-hco3 buffer at ph 9.4 (9.5 ml 0.2 m na2co3 was mixed with 41.5 ml 0.2 m nahco3). the solution was made up to 200 ml and was labeled by mixing this solution with 1mg of fitc (fluorescein isothiocyanate, sigma) on a shaker for 30 min at room temperature in complete darkness (rohloff et al., 1994).the spores were rinsed by phosphate buffered saline four times, pelletized, and the pellets from the last wash were re-suspended in 1 ml of grace’s insect medium (gim, gibco) and stored at -20 ˚c until needed. phagocytosis assay the phagocytic activity of hemocytes was assessed using the fitc-labeled b. bassiana spores. larvae were injected with 2 µl of 1×105 fitclabeled b. bassiana spores/ml and phagocytic activity of hemocytes was investigated 15, 30, and 60 min post injection. this experiment was replicated three times and ten insects were used in each replicate. the body surface of each larva was sterilized with 70 % ethanol before extracting the blood. the larvae were bled by cutting one of the prolegs. the hemolymph was then collected into ice-cold anticoagulant solution and poured over microscopic slide. the slide was incubated in a moist dark chamber at room temperature for 5min with 10 µl of trypan blue solution (2 mg/ml) in order to quench spores that were not ingested. for the in vitro phagocytosis assays, 10 µl of fitc-labeled b. bassiana spores were mixed with 10 µl of freshly collected hemolymph on a microscopic slide and were then incubated in a moist dark chamber at room temperature for 60 and 120 min. then, 10 µl of trypan blue solution was added and the mixture was incubated for another 5 min. in both the tests the phagocytic activity was determined by counting hemocytes with or without ingested spores under a fluorescence microscope (leica, wetzlar, germany). ten photo-frames per microscope slide were counted and the average was calculated. each experiment was repeated for 3 times (tseng et al., 2008). phagocytosic activity was calculated as the number of phagocytosing cells×(number of total cells)-1×100. effect of fungal spore on nodulation injections were carried out as mentioned above. the number of nodules formed at 1, 3, 6, 12, and 24 h post injection were determined. hemolymph was collected from each larva, then samples in 5 replicates were poured into a hemocytometer, and the number of nodules was counted (franssens et al., 2006). assay for po activity po activity was measured according to the procedure of catalan et al. (2012) with slight 15   modification. in order to test the effect of b. bassiana spores on the po system in g. pyloalis larvae, 10 µl of hemolymph was diluted with 90 µl of ice-cold sterile phosphate buffered saline and then were vortexed. samples were frozen at -20 ˚c for 48 h. for assay of po activity, l-dopa was used as a substrate (wilson et al., 2001; cotter et al., 2004). then samples were centrifuged at 5,000g at 4 ˚c for 5 min. fifty microliters of hemolymphbuffer supernatant was mixed with 150 µl of ldopa (10 mm). po activity was measured (in 3 replicates) at 490 nm for 30 min in 5 min intervals using a microplate reader (awareness technology inc, florida, usa). protein determination the method of bradford (1976) was used for determining total protein, using bovine serum albumin (bio-rad, munchen, germany) as the standard. effect of ecdysone and juvenile hormone (jh) on immune responses ecdysone was dissolved in ringer’s solution (0.123 m nacl, 1.53 m cacl2, 4.96 m kcl, ph 7.4) at a concentration of 5 mg/ml. initially, preliminary tests were performed to find the effective dose ranges of ecdysone and jh on the development of g. pyloalis. then the stock solution of ecdysone was diluted with ringer to a concentration of 0.5, 0.25, 0.125, and 0.062 mg/ml. jh was dissolved in acetone at 5, 2.5, 1.25 and 0.625 mg/ml and 2 µl of this solution was topically applied onto the metathoracic tergum of each larva. four hours after topical application of jh on metathoracic tergum or injection of ecdysone, a suspension of b. bassiana spores (1×105 spores/ml) was prepared and 2 µl of which was injected to the larva by a hamilton syringe. then, after 3, 6, and 12 h total and differentiated hemocyte number and nodules were counted. control specimens were either first topically treated with 2µl of acetone or were first injected with ringer’s solution and then with b. bassiana spores. three replicates were used for each concentration (n = 30) and totally 120 insects were used for immunological assays. statistical analysis all data obtained from the experiments were subjected to analysis of variance (anova) (p < 0.05). means were compared by tukey’s studentized range test, accepting significant differences at p ≤ 0.05 (sas institute, 1997). results effect of b. bassiana spores on hemocyte number, phagocytosis and nodulation total number of circulating hemocytes in lesser mulberry pyralid, g. pyloalis fifth instar larvae exhibited major changes after b. bassiana spore injection (fig. 1). a significant increase in total hemocyte count (thc) was recorded 3 and 6 h post injection (365 and 481×104 cell/ml respectively), while in control thc was recorded only 295 and 305×104 cell/ml, respectively (p ≤ 0.05). as shown in table 1, significant changes in the profile of five hemocyte types were observed in various intervals post-injection of b. bassiana spore compared with the control. the number of plasmatocytes and granulocytes increased 1, 3, and 6 h postinjection, and then decreased after 12 and 24 h. fungal infection generally increases the number of plasmatocytes in first interval after the inoculation. the data revealed that the numbers of oenocytoids were increased significantly after 1 and 12 h pos-injection compared with the control. the number of prohemocytes did not change significantly but the number of spherulocytes decreased 6 and 12 h post injection of b. bassiana spores. hemocytes of g. pyloalis showed a basic phagocytosis activity against b. bassiana spores. results of this study showed that plasmatocytes and granulocytes of g. pyloalis have an important role in phagocytosis of foreign particles. the most phagocytic activity was observed 30 and 60 min after injection of spores. the phagocytic potential of g. pyloalis was higher in vivo than in vitro (fig. 2). nodule formation (fig. 3) in g. pyloalis larvae was significant after injection of b. bassiana spores. most of the nodules occurred 3, 6 and 12 h after inoculation of fungi. the highest number of nodules could be observed 6 h post injection, and then decreased 24 h post-injection (fig. 4). effect of b. bassiana spores on phenoloxidase activity when fifth instar larvae of g. pyloalis were injected with b. bassiana spores (1×105), the po system was activated during intervals after inoculation (fig. 5). the highest po activity was observed 3 and 6 h after the injection, and then decreasing after 12 and 24 h but not significant statistically. effect of exogenous ecdysone and jh on immune responses in g. pyloalis quantitative analysis of thc of insects treated with exogenous jh is shown in table 2. by increasing the concentration of exogenous jh from 0.625 to 5 mg/ml a dose-dependent decrease in total number of hemocytes was observed compared with the control. plasmatocyte numbers sharply decreased along with increase in the concentrations. the number of granulocytes decreased at higher concentrations compared with the control. the changes of total hemocyte count in the fifth instar g. pyloalis larvae were affected by injection of exogenous ecdysone. as shown in table 3, total hemocyte count significantly increased with time and concentration. higher concentrations of exogenous ecdysone seemed to play a strong facilitating role in promoting the increase in granulocyte numbers. treatment of larvae with exogenous jh significantly inhibited the nodule formation in larvae injected with b. bassiana spores (fig. 6a). while, increasing concentrations of exogenous ecdysone (0.5 and 0.25 mg/ml), enhanced nodulation 3 and 6 h after b. bassiana injection (fig. 6b). 16   a) b) fig. 6 influence of different concentrations of jh (a) and ecdysone (b) on nodule formation of g. pyloalis larvae at 3, 6 and 12 h after injection with b. bassiana spores. data of each treatment at any time were compared with control at the same time. discussion to understand the role of hemocytes and their involvement in the defense reactions of lesser mulberry pyralid larvae a pathogenic agent was used. it was found that, total hemocyte count (thc) first increased during infection, then declined 12 and 24 h post-injection. as the infection progressed the thc and more specifically the granulocyte number were reduced. similar reduction in circulating hemocytes was reported for spodoptera exigua infected by b. bassiana (hung and boucias, 1992). previous studies by other researchers showed a major effect of pathogenesis on thc in insects (moushumi et al., 2008; ajamhassani et al., 2013). bandani (2008) reported no significant reduction in the total hemocyte count (thc) when galleria mellonella larvae were injected with tolypocladium cylindrosporum spores until 24 hours post injection. however, thc was suppressed 48 h post-treatment of larvae with spores. meshrif et al. (2011) also indicated that population of s. littoralis hemocytes 48 h post injection of b. bassiana spores were significantly lower than the control, but 72 h post injection they were increased. the decline in thc in the later stages of infection could partly be attributed to the hemocyte aggregation (nodule formation), induced by soluble wall components released from circulating fungus (gillespie et al., 2000). furthermore, cytotoxic fungal metabolites may play a key role in the reduction of hemocyte numbers. the differential hemocyte count (dhc) showed an initial increase in plasmatocytes (pls) and granulocytes (grs) during infection, followed by a decline in these cell numbers. this result was in accordance to other reports (gunnarsson, 1987; pech and strand, 1995; gillespie et al., 2000; meshrif et al., 2011). the total granulocyte and plasmotocyte numbers also increased 12 h after treatment with metarhizium anisopliae in oxya japonica (anggraeni and putra, 2011). pls and grs are known to give out cytoplasmic processes in retaliation to any invading foreign material (sharma et al., 2008). 17   table 3 thc and number of plasmatocytes and granulocytes (×104 cell/ml) of g. pyloalis larvae at 3, 6 and 12 h after ecdysone treatment prior to b. bassiana spore injection thc number of plasmatocytes number of granulocytes 3 h 6 h 12 h 3 h 6 h 12 h 3 h 6 h 12 h k 341.33± 4.26a 326.00± 3.88c 292.23± 3.53c 215.00± 4.13ab 241.3± 3.01b 239.66± 2.22ab 79.00± 2.74b 87.26± 3.16c 84± 1.62c a 373.64± 3.17a 423.0± 3.18a 386.00± 3.43a 257.22± 3.53a 304.43± 2.60a 254.33± 4.26a 108.00± 3.08a 143.66± 3.16a 110.4± 3.6b b 362± 4.28a 392.65± 3.45ab 354.3± 4.32ab 232± 2.56ab 270 ±3.48b 240± 3.47ab 80.3± 2.76b 131± 2.92ab 124.33± 1.72a c 358.33± 6.36a 363.2± 6.62bc 323.67± 6.23bc 221± 5.46ab 236.12± 4.98b 259± 3.48a 106± 3.6a 119.42± 4.09b 120± 1.44ab d 334.43± 2.73a 337.5± 3.17bc 329.± 2.49bc 207.35± 4.93b 243.67± 5.36b 221.27± 3.08b 91.2± 3.6ab 110.1± 3.53b 123.3± 2.24a k = control; a = ecdysone 0.5 mg/ml; b = ecdysone 0.25 mg/ml, c = ecdysone 0.125 mg/ml; d = ecdysone 0.062 mg/ml. means ± se within the same column followed by the same letter are not significantly different (p ≤ 0.05 tukey test). in the current study we have demonstrated that oenocytoides' (oe) number changed in response to b. bassiana spores. changes in the numbers of oes following fungal injection may be attributed to the beginning of humoral activity or the active transformation of grs into sps and oes as suggested by gupta (1985). the decrease of pls and grs can be attributed principally to their involvement in nodule formation and partially to programmed cell death induced by toxins secreted by the growing fungi. the decline in grs observed may be due to their involvement in the latter stages of nodule formation, as has been reported in other insects (gotz and boman, 1985; pech and strand, 1995; gillespie et al., 1997). a significant increase in the percentage of grs was observed in latex bead-treated insects 60 and 120 min after the inoculation. the percentage of oenocytoides significantly varied in response to staphylococcus aureus infection, with an initial increase at 30 min followed by accentuated decrease 60 and 120 min post-inoculation (borges et al., 2008). in the process of phagocytosis cells recognize, bind and ingest relatively large elements and protect insects. several authors have indicated that pls are the major cell type involved in phagocytosis (ratcliffe et al., 1985; anggraeni and ratcliffe, 1991; rohloff et al., 1994). immune reactions of g. pyloalis against b. bassiana showed that pls and grs are the main factors in phagocytosis and nodulation. in this study, most of phagocytotic activity occurred 30 and 60 min after injection. this study showed that nodules are formed in response to injection of b. bassiana spores. the maximal number of nodules were observed 3, 6 and 12 h post-injection, but declined later. similar results were recorded by other researchers (gillespie et al., 2000; meshrif et al., 2011; ajamhassani et al., 2013). it is generally agreed that the formation of nodules is initiated by degranulation of grs, and the contents released act as an opsonin in the employment of other hemocytes, mainly pls (ratcliffe et al., 1985). contact of grs with the fungus, or the release of β-1, 3 glucan of other soluble material from the fungus could be the cause of this initial degranulation (gillespie et al., 2000). the subsequent decline in nodule numbers may be due to their running out of circulation and attaching to the body wall when they attain a stable size (brookman et al., 1989). it is likely that the immunosuppressive effects of fungal metabolites might have a role in their decline. the dark color of the nodules demonstrates the synthesis of melanin and at least a localized activation of propo, which is indicative of their formation by hemocytes (lavin and strand, 2002). po is responsible for the activation of melanogenesis in invertebrates. the main role of po in melanogenesis is to convert phenols to quinones which subsequently polymerize to form melanin (gonzález-santoyo and cόrdoba-aguilar, 2011). furthermore, recent research has documented po as an important tool used against several pathogens (cerenius and söderhäll, 2004). wounding or infection activates po system as part of immune response (kanost and gorman, 2008). in the present study we observed high level of po activity in hemolymph of larvae injected with b. bassiana spores 3, 6 and 12 h post-injection. gillespie et al. (2000) demonstrated that a topical application of conidia from m. anisopliae var. acridum led to significant elevation of propo in the hemolymph. we investigated the effect of different concentrations of jh and ecdysone on immune parameters including total hemocyte counts and nodule formation. we found that high concentrations of jh reduced total hemocyte number. also both plasmatocyte and granulocyte numbers sharply 18   decreased in a time and concentration-dependent manner. zibaee et al. (2012) demonstrated that pyriproxyfen (pyridine-based pesticide with jh mimicking activity) caused significant reduction in thc, plasmatocyte and granulocyte population in eurygaster integriceps adults. kim et al. (2008) showed a dose response relationship for 20e on the number of plasmatocytes in spodoptera exigua. treatment of g. pyloalis larvae with jh caused reduction in the ability of the larvae to form nodules in response to injection of b. bassiana. similar results have been reported by rantala et al. (2003) and zibaee et al. (2012). we have also assessed the influence of ecdysone on total hemocyte count and nodulation. it was observed that ecdysone stimulated nodule formation in a dose dependent manner. when larvae of n. bullata were treated with 20e prior to laminarin (a storage glucan) injection increased the nodulation response in a dose-dependent manner. contrary to ecdysone treated larvae with jh or juvenile hormone analogs (jha), fenoxycarb and pyriproxyfen, significantly reduced the formation of nodules in response to laminarin (franssens et al., 2006). the phagocytic activity of plasmatocytes in larvae of n. bullata was enhanced after 20e injection (lanot et al., 2001). it was also shown that 20e signaling was needed for drosophila lymph gland growth and hematopoiesis, both of which are required for pathogen encapsulation (sorrentino et al., 2002). in the grey flesh fly larvae n. bullata, 20e promotes cell-mediated nodulation response (franssens et al., 2006). in the tobacco hornworm manduca sexta jh acts as inhibitor for po synthesis and thus cuticular melanization did not occur (hiruma and riddiford, 1988). similarly in the mealworm beetle t. molitor, jh reduced immune parameters such as po levels and encapsulation (rolff and siva-jothy, 2002; rantala et al., 2003). from these results, it is well established that 20e positively regulate the innate immune system of insects, while jh works as an immuno-suppressor (flatt et al., 2005). flatt and kawecki (2007) showed that jh and 20e have hostile effects on the induction of antimicrobial peptide genes in fruit fly, drosophila melanogaster. the present study demonstrated that the entomopathogenic fungus, b. bassiana has properties that allow for its successful replication in lesser mulberry pyralid and strongly affect the immune responses of this pest. the understanding of interaction between entomopathogenic fungus and the insect is an important step in fungal disease propagation. further, understanding fungal induced immune response and the identification of fungal virulence factors and their targets may provide significant methods for development of efficient mycoinsecticides in biological control of dangerous pests. jh and ecdysone have significant effect on immune response of g. pyloalis to b. bassiana spores. it can be stated that the results of this experiment suggests jh or its analogues as an immunosuppressive agent and may be used in a control program prior to use of fungal pathogens while, ecdyson facilitates effective immune reactions. acknowledgements the authors express their sincere gratitude to university of guilan for financial support and professor k slama of institute of entomology, c budejovice, czech repuplic for generous supply of ecdysone and jh. references ajamhassani m, sendi jj, zibaee a, askary h, farsi mj. immunoliogical responses of hyphantria cunea (drury) (lepidoptera: arctiidae) to entomopathogenic fungi, beauveria bassiana (bals.-criy) and isaria farinosae (holmsk.) fr. j. 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wilson k, cotter s, reeson a, pell j. melanism and disease resistance in insects. ecol. lett. 4: 637649, 2001. xiong q, xie y, zhu y, xue j, li j, fan r. morphological and ultrastructural characterization of carposina sasakii larvae (lepidoptera: carposinidae) infected by beauveria bassiana (ascomycota: hypocreales: clavicipitaceae). micron 44: 303-311, 2013. yamauchi h. two novel insect defensins from larvae of the cupreous chafer, anomala cuprea: purification, amino acid sequences and antibacterial activity. insect biochem. mol. biol. 32: 75-84, 2001. zibaee a, bandani ar, malagoli d. methoxyfenozide and pyriproxifen alter the cellular immune reactions of eurygaster integriceps puton (hemiptera: scutelleridae) against beauveria bassiana. pestic. biochem. physiol. 102: 30-37, 2012. 21   meshrif ws, rohlfs m, hegazi ma, barakat em, seif ai, shehata mg. interactions of spodoptera littoralis haemocytes following injection with the entomopathogenic fungi: beauveria bassiana and nomuraea rileyi. j. egypt. soc. parasitol. 41: 699-714, 2011. combined effects of temperature and salinity on functional responses of haemocytes and survival in air of the clam ruditapes philippinarum isj 8: 70-77, 2011 issn 1824-307x review bivalve immune responses and climate changes: is there a relationship? v matozzo, mg marin department of biology, university of padua, padua, italy accepted may 2, 2011 abstract global climate changes (gccs) are predicted to occur in the next hundred years through increases in temperature, water acidification and changes in seawater salinity. increasing atmospheric co2 concentrations are considered to be the main responsible for gccs. climate changes can pose risks for aquatic ecosystems, mainly for marine coastal areas that are ecologically and economically important. in this context, increasing effort has been addressed to the evaluation of effects of variations in abiotic factors, such as temperature, salinity and ph, on biological responses of marine organisms, as deviation from the optimum for these factors may result in deleterious consequences for the physiological performance of animals. in a climate change scenario, the present review focuses on the effects of variations in some important environmental factors mainly temperature, salinity and ph on immune parameters of bivalves. key words: climate changes; bivalves; immune parameters; temperature; salinity; acidification introduction global climate changes (gccs) are predicted to occur in the next hundred years through increases in temperature, water acidification and changes in seawater salinity (ipcc, 2007). metaphorically speaking, we are cooking our planet (fig. 1). it is commonly accepted that increases in anthropogenic emissions of carbon dioxide (co2) in the atmosphere are the main responsible for gccs. indeed, increased co2 levels are postulated to affect average air and ocean temperatures, to cause widespread melting of snow and ice and to rise average sea level (ipcc, 2007). pre-industrial atmospheric co2 levels were approximately 280 ppm; at present, co2 levels are increased to over 380 ppm, mostly as a result of anthropogenic activities (feely et al., 2004). the capability of oceans to uptake co2 may influence seawater carbonate chemistry, with a consequent decrease in ph values, concentration of carbonate ions and the related calcium carbonate (caco3) saturation state of seawater (orr et al., 2005). nowadays, it is assumed that seawater acidification due to the continued release of co2 into the atmosphere has already caused a reduction in ocean ph values of about 0.1 units with respect to the pre-industrial levels, and reductions from 0.3 to 0.5 ph units are ___________________________________________________________________________ corresponding author valerio matozzo department of biology university of padua via ugo bassi, 58/b, 35131 padua, italy e-mail: matozzo@bio.unipd.it predicted within the end of 21st century (caldeira and wickett, 2005; raven et al., 2005; ipcc, 2007). increasing atmosphere co2 levels may cause a ph reduction of 0.7 units by 2300 (caldeira and wickett, 2003). with regard to temperature, it is demonstrated that mean global temperature has increased by about 0.7 °c in the last century, and further increases are expected during the present century (ipcc 2007; mann et al., 2008). indeed, global temperature is hypothesised to increase of about 1.8 to 4.0 °c by the end of the 21st century (ipcc, 2007). in any case, warming is expected to occur heterogeneously, with land warming faster than oceans, high latitudes warming faster than mid-latitudes, and winters warming more than summers (ipcc, 2007). there is also concern about future alterations in seawater salinity values, mainly in estuarine and coastal areas (booij, 2005; kay et al., 2006). indeed, global warming should be associated with changes in the hydrological cycle at a large scale: increases in precipitations could occur at high latitudes and near the tropics, whereas decreases could be recorded in sub-tropical and mid-latitude regions (fenoglio et al., 2010). as a consequence, many areas of our planet will be subjected to increases either in drought or in flooding. in particular, frequent flood events are hypothesised to lead to prolonged and frequency-increased periods of reduced salinity, in estuarine areas in particular (bussell et al., 2008). in a gcc scenario, increasing concern is addressed to the well-being of living organisms. 70 indeed, it is suggested that gccs could affect population dynamics and distribution of livestock parasites, causing increases in disease incidence and production loss (morgan and wall, 2009). generally, when an organism is subject to stressful conditions it can cope with stress modifying its physiological, biochemical and behavioural responses. at immunological level, responses comprise a complex network of specific and nonspecific humoral and cell-mediated components. in this context, information concerning direct effects of gccs on animal immune functions are not available in the literature, to our knowledge at least. conversely, data about the effects of individual environmental factors on immune parameters are abundant. taking into account that it is not possible to summarize all information available, in the present review we have summarized results of the most recent studies concerning the influence of environmental factors temperature, salinity and ph in particular on immune parameters of molluscs, discussing them in a possible scenario of gccs. effects of temperature on mollusc immune parameters environmental factors including temperature, salinity, oxygen, food availability, and contaminants can affect immune parameters of molluscs (fisher, 1988). in particular, temperature is one of the most studied abiotic factors being able to affect many physiological processes in animals, mainly ectotherms. numerous studies have demonstrated that temperature can influence markedly immune responses of molluscs, even if temperature effects differ among species. we have recently demonstrated that high temperatures affect some important functional responses of hemocytes in the clam chamelea gallina (monari et al., 2007). in that study, clams were kept for 7 days at 20, 25 and 30 °c and total hemocyte count (thc), phagocytosis, lysozyme activity (in both hemocyte lysate and cell-free hemolymph), activity and expression of the antioxidant enzyme superoxide dismutase (sod) (in both hemocyte lysate and cell-free hemolymph) were measured. the highest temperature increased significantly thc in c. gallina (fig. 2). data concerning effects of temperature on thc in bivalves are contradictory. for example, hemocyte number of mytilus galloprovincialis was positively correlated with water temperature, the lowest values being found in winter and the highest in summer (carballal et al., 1998). conversely, in the same mussel species, increased temperature (exposure to 25 °c for 24 h) did not exert any significant effect on the number of circulating immunocytes (malagoli et al., 2007). in the oyster, crassostrea virginica, thc was lowest in july and august, when the highest water temperatures were recorded (fisher and oliver, 1996). the effects of acute temperature challenge (from 17 °c to 11, 23 and 28 °c for 72 h) on thc were also evaluated in the scallop, chlamys farreri (chen et al., 2007). in that study, just after 1h of acute temperature stress, thc increased significantly from 3.7×107 cells ml−1 to an average of 5.3×107 cells ml−1 in scallops transferred to 11, 23 fig. 1 a metaphorical image of global climate changes. and 28 °c. after 72h, thc of scallops held at 11 °c remained significantly higher than those of the other treatment groups. significantly higher thc values were also found in clams (ruditapes philippinarum) kept at the highest temperature (21 °c) (paillard et al., 2004). monari et al. (2007) have hypothesised that the increased number of hemocytes found in c. gallina kept at 30 °c could be a consequence of a mobilisation of cells from tissues to hemolymph, in order to respond to bacteria. indeed, although bacteria were not inoculated in clams, a great number of bacteria surrounding hemocytes were observed in hemocyte cultures from 30 °c-exposed animals (monari et al., 2007). likewise, inoculation of both marteilia refringens and vibrio tapetis caused a significant increase in circulating hemocyte number in mytilus galloprovincialis (carballal et al., 1998) and r. philippinarum (oubella et al., 1994), respectively. in a recent study, perrigault et al. (2011) have investigated the effects of temperature on defence factors in the clam mercenaria mercenaria. clams were maintained at 13, 21 and 27 °c for 4 months, and cellular and humoral defence parameters were assessed after 2 and 4 months. thc exhibited variations according to temperature value and sampling time: thc increased significantly after 2 months at 27 °c, but it decreased significantly after 4 months at the same temperature (perrigault et al., 2011). 71 fig. 2 effects of temperature on thc, expressed as number of hemocytes (x106)/ml hemolymph, in chamelea gallina. values are means ± sem (n = 3). inset: significance of comparisons between experimental groups. *p<0.05, n.s.: not significant (from: monari et al., 2007). monari et al. (2007) have also observed a significant inhibition of phagocytic activity in clams, c. gallina, kept at 30 °c. likewise, maintenance of c. virginica at 28 °c for 7 days caused a significant decrease in phagocytic activity (hégaret et al., 2003). in the same species, a significant increase in hemocyte phagocytic activity was recorded in oysters held at 20 °c for 68 days, compared to those held at 10 °c, but activity reduced in oysters kept at 25 °c (chu and la peyre, 1993). in m. galloprovincialis, carballal et al. (1997) demonstrated that the in vitro capability of hemocytes to engulf foreign particles was lower at 10 °c than at 20 °c and 30 °c. in the same species, malagoli et al. (2007) observed that higher temperature (25 °c, 24 h) did not significantly influence the percentage of phagocytic immunocytes. malham et al. (2009) have recently demonstrated that oysters (crassostrea gigas) held at 12 °c had significantly higher phagocytic hemocytes than oysters held at 21 °c. in an in vitro study, incubation of hemocytes at 40, 50 and 60 °c for 4 h caused cell mortality, while percentages of aminopeptidaseand esterase-positive cells were significantly lower after hemocyte incubation at 50 and 60 °c (gagnaire et al., 2006). in the same study, incubation of oysters for 4 h at 60 °c caused a significant decrease in both hemocyte phagocytic activity and percentage of esterase-positive cells. the authors suggested that the temperatureinduced decrease in enzymatic and phagocytic activities of c. gigas hemocytes was mainly due to increased cell mortality (gagnaire et al., 2006). although those temperatures were particularly high, it should be highlighted that oysters face high temperatures (40 °c) during the summer period in marennes-oleron bay (charente-maritime, france), where the study was performed (gagnaire et al., 2006). in c. farreri, the percentage of phagocytic hemocytes was significantly lower in scallops kept at 28 °c than in those placed at 11 °c and 23 °c after 1-h stress application, and significantly decreased over the whole stress application, from 13.9 % initially to 6.3 % at the end of the experiment (chen et al., 2007). recently, higher phagocytic activity was observed after 2 and 4 months in m. mercenaria held at 21 °c, as compared to levels recorded at 13 °c and 27 °c (perrigault et al., 2011). it has been demonstrated that temperature can affect other important immune functions of bivalves. in c. gallina, significantly increased lysozyme activity was observed in hemocyte lysate from clams kept at 25 °c, and in cell-free hemolymph from animals held at 20 and 30 °c (monari et al., 2007). a relationship between lysozyme activity in hemocyte lysate and cell-free hemolymph was observed at 20 °c, enzyme activity being significantly reduced in hemocytes and increased in cell-free hemolymph. this was probably due to the high phagocytic activity of hemocytes from clams held at 20 °c. interestingly, animals maintained at the highest temperature (30 °c) also showed increased cell-free hemolymph lysozyme activity, 72 fig. 3 hemocyte mortality percentage of oysters after an in vitro 2 h or 18 h incubation period at different salinities. values are mean of four replicates. bars represent standard deviation. ***p<0.001 (from gagnaire et al., 2006). even if their phagocytic activity was low. clams kept at 30 °c might have been induced to release lysozyme in order to respond to the high number of bacteria in the hemolymph. in the surf clam, mactra veneriformis, maintenance of animals at 10 °c decreased thc, lysozyme activity and neutral red retention nrr times, but increased the phagocytic activity. in the same study, the highest temperature tested (30 °c) significantly increased thc, whereas it decreased phagocytic activity, lysozyme activity and nrr times (yu et al., 2009). the cytotoxicity of m. galloprovincialis hemolymph (evaluated by the cytolysis of human a-positive erythrocytes) was significantly reduced after maintenance of animals at 25 °c for 96 h (cytotoxic activity reduced significantly from 75 % in controls to 16 % in stressed animals) (malagoli and ottaviani, 2005). that study demonstrated that high temperature reduced cytotoxic responses in mussels, making them more vulnerable to pathogen aggression. in m. mercenaria, unstimulated reactive oxygen species (ros) production was also significantly affected by temperature, with higher levels at 13 °c with respect to 21 and 27 °c, at both sampling times (2 and 4 months) (perrigault et al., 2011). in the same study, higher lysozyme activity was observed after 4 months in clams maintained at 13 °c as compared to those held at 21 °c and 27 °c. a significant decrease in hemocyte mn-sod and cu/zn-sod activities was also recorded with increasing temperature (monari et al., 2007). in cell-free hemolymph, the highest mn-sod activity was recorded at 30 °c, whereas the cu/zn-sod activity showed no significant changes in clams maintained at the three temperatures tested (20, 25 and 30 °c). all these studies demonstrate that temperature (high temperatures, in particular) influences strongly immune parameters of bivalves. in a scenario of a possible global warming, information available on temperature-induced immunemodulation in invertebrates could provide essential knowledge useful to define early warning systems based on immunomarkers. effects of salinity on mollusc immune parameters as stated above, gccs can also occur through changes in seawater salinity. warmer temperatures could increase seawater evaporation and reduce rainfall, concentrating salt in the water. on the other hand, warming could form areas of heavy tropical rainfall, with consequent decreases in seawater salinity, mainly along the coasts. salinity can influence several metabolic and physiological parameters in aquatic organisms. salinity was also shown to influence immune parameters in molluscs. results from both laboratory and field studies have demonstrated a relationship between variations in salinity levels and infection in bivalves (gauthier et al., 1990; chu et al., 1993; reid et al., 2003). in oysters (ostrea edulis) kept for 7 days at differing salinity levels (32, 25 and 16 psu), the highest salinity promoted the growth of the opportunistic bacterial pathogen listonella anguillarum, increased the number of large granulocytes, and decreased the concentration of the microbicidal agent, hydrogen peroxide, in the hemolymph (hauton et al., 2000). oysters (o. edulis) acclimated at 32, 28 and 25 psu showed a neutral red dye retention time in the lysosomal compartment longer than oysters kept at low salinities (16 and 19 psu), suggesting a reduced cell membrane stability (hauton et al., 1998). in a recent in vitro study, a reduction in salinity levels (6.5, 3 and 0 psu) induced high hemocyte mortality (fig. 3), whereas one day in hyposalinity conditions (15 psu) significantly reduced phagocytic activity in c. gigas 73 (gagnaire et al., 2006). in the abalone haliotis diversicolor, cheng et al. (2004) found low hemocyte numbers at low salinities, and low hemocyte phagocytic capability at both reduced and elevated salinities. reid et al. (2003) found significantly increased thc values with increasing salinity from 20 psu (control) to 40 psu in r. philippinarum. conversely, in clams (c. gallina) kept at 28 psu, thc significantly increased with respect to animals kept at 34 and 40 psu, whereas higher phagocytic activity was recorded in hemocytes from clams kept at 34 psu, with respect to those kept at 28 and 40 psu (matozzo et al., 2007). in m. galloprovincialis, the number of circulating immunocytes increased significantly in mussels exposed for 24 h to 40 psu salinity, whereas the percentage of phagocytic immunocytes did not change significantly (malagoli et al., 2007). bussell et al. (2008) have demonstrated that mussels (m. edulis) kept for two days at reduced salinity (16 psu) had a significant reduction in the number of hemocytes, percentage of eosinophils and the percentage of phagocytic activity, with respect to mussels kept at environmental salinity (32 psu). in that study, the authors suggested that alterations in immune parameters due to low salinity were a consequence of reduced movement of hemocytes caused by the osmotic stress or enhanced infiltration of hemocytes into the connective tissue of differing organs. in a recent study, the effects of hypo-saline conditions on immune parameters of the akoya pearl oyster, pinctada imbricata, have been evaluated (kuchel et al., 2010). both phagocytosis and phenoloxidase activity decreased significantly when oysters were exposed to low salinity (25 psu), whereas thc significantly increased. also in this case, the authors hypothesised that the reduction in phagocytic activity was a consequence of either osmotic stress or increased infiltration of hemocytes into connective tissue and organs (kuchel et al., 2010). in the same study, the frequency of circulating granulocytes was significantly higher in oysters kept in hypo-saline conditions, than in control oysters. in addition, total protein content of hemolymph increased significantly when oysters were subject to low salinity (kuchel et al., 2010). after 96 h, low salinity (25 psu) also reduced hemolymph cytotoxicity in mussels, m. galloprovincialis, with respect to controls (35 psu), and only 12 % of the animals showed cytotoxic activity (malagoli and ottaviani, 2005). results here summarised suggest that bivalves experiencing changes in salinity have altered immunesurveillance, and become more susceptible to infection/diseases. combined effects of temperature and salinity on mollusc immune parameters in aquatic environments, differing stressors act jointly. as a consequence, effects of stressors should be evaluated in combination. however, very few studies have investigated the combined effects of environmental parameters on bivalve immune responses, to our knowledge at least. in a recent survey, the combined effects of various temperatures (5, 15, and 30 °c) and salinities (18, 28, and 38 psu) on hemocyte functionality of the clam r. philippinarum were evaluated (munari et al., 2011). in that study, both the extreme temperature and salinity values were chosen considering their occurrence in lagoon shallow waters where clams live. effects of the resulting 9 experimental conditions on thc, neutral red uptake (nru, indicative of hemocyte pinocytotic capability), hemolymph protein concentration, and lysozyme activity in both hemocyte lysate and cell-free hemolymph were studied. temperature influenced significantly thc and nru, whereas salinity and temperature/salinity interaction affected nru only. temperature and salinity did not affect significantly hemocyte lysate and cell-free hemolymph lysozyme activity, as well as hemolymph total protein content. overall results suggested a better physiological condition for animals kept at 15 °c temperature and 18 psu salinity. effects of acidification on mollusc immune parameters a possible consequence of increased atmospheric co2 levels is ocean acidification. indeed, almost one half of the anthropogenically produced co2 should be taken up by the oceans, with consequent increases in hydrogen ion and in carbonic acid and bicarbonate ion concentrations, whereas concentration of carbonate ions should reduce (raven et al., 2005; bibby et al., 2008). it is well known that calcium-carbonate minerals (e.g., calcite and aragonite) constitute the shells of many aquatic species, such as bivalves. as a consequence, reduction in ph values could have biological implications for such animals, both wild and cultivated, that require carbonate to form their shells or skeletons (wikfors and krome, 2009; range et al., 2011). some studies have demonstrated the role of mantle cells and circulating hemocytes in shell deposition in molluscs. in particular, mount et al. (2004) reported that mantleepithelial cells create a protein matrix, while hemocytes deposit calcium-carbonate crystals during shell growth of oysters (c. virginica). due to the functional role of hemocytes in shell deposition, efforts should be addressed to the evaluation of acidification effects on these important circulating cells. in addition, the involvement of mollusc hemocytes in immune responses is well known. therefore, it can be hypothesised that water acidification can also compromise the immunosurveillance status of animals. however, only few studies have investigated the effects of acidification on mollusc immunosurveillance. malagoli and ottaviani (2005) investigated the effects of low ph keeping m. galloprovincialis at 7.3 ph for 96 h. applied stress reduced significantly hemolymph cytotoxicity, with respect to controls (ph = 8.0), suggesting that changes in the water physical conditions can reduce hemolymph cytotoxicity in mussels, making them more vulnerable to pathogens. in a recent study, effects of hypercapnia on the immune response of m. edulis were investigated by exposing mussels for 32 days to acidified (by co2) sea water (ph 7.7, 7.5 74 fig. 4 number of phagocytosed zymosan by hemocytes of mussels (mytilus edulis) exposed for 32 days to differing ph values (from: bibby et al., 2008). and 6.7; control: ph 7.8) (bibby et al., 2008). in that study, although phagocytic activity increased significantly during the exposure period at all ph values tested, phagocytosis was significantly lower in mussels kept in acidified sea water at the end of the exposure (fig. 4). acidified sea water did not have significant effects on the other immune parameters measured (superoxide anion production, total and differential cell counts). the authors stated that although their study did not clarify exactly the mechanisms of actions of reduced ph, one possible explanation of the results obtained was the dissolution of the mussel shell, resulting in elevated levels of ca2+ in the hemolymph. in turn, increased hemolymph ca2+ levels affected cellular metabolism, function and signalling pathways (bibby et al., 2008). in another study, adults of the oyster saccostrea glomerata were exposed to a seawater matrix of varying salinity, sulphuric acid and al3+ for 48 h, and the expression of 7 genes involved in immune responses were evaluated by quantitative reverse-transcription polymerase chain reaction (green and barnes, 2010). sulphuric acid was used to reproduce acidification conditions, as this acid is naturally produced by oxidation of acid sulphate soils and released into water ecosystems after rains. in that study, a reduction in salinity from 35 to 15 psu caused a 1.7-fold down-regulation in the expression of peroxiredoxin 6 gene. however, changes in salinity, sulfuric acid and al3+ concentrations did not affect the expression of other target genes. the effects of salinity, sulfuric acid and al3+ on the capability of immune genes to respond to bacteria were also evaluated by exposing oysters to the seawater matrix for 40 h, followed by injection with heat-killed vibrio alginolyticus. a combination of reduced salinity and v. alginolyticus injection caused a down-regulation of peroxiredoxin 6 and c1q-like protein, whereas exposure to sulfuric acid and al3+ had no significant effects on the immune gene responses. on the basis of the results obtained, the authors suggested that reduction in salinity, and not acidification, was the main factor affecting immunesurveillance in oysters (green and barnes, 2010). conclusions information summarized in the present review demonstrate, once again, that changes in abiotic environmetal factors influence strongly mollusc immune parameters. however, most of the studies performed up to here have investigated the effects of individual environmental factors, whereas surveys aimed at evaluating the combined effects of differing abiotic factors are poor. as a consequence, taking into account that gcc could occur in the next decades through contemporaneous changes in various environmental factors, efforts should be addressed to the evaluation of the combined effects of abiotic factors on immune responses of animals. this approach could be essential in understanding thoroughly the possible relationship between environmental conditions and immunemodulation mechanisms in invertebrates, and in highlighting the susceptibility of each species to infections. considering that invertebrates represent about 95 % of living species and play important roles in ecosystems (aquatic in particular), the knowledge of the mechanisms involved in immunemodulation will be crucial to develop management and conservation programs in future gcc scenarios. references bibby r, widdicombe s, parry h, spicer j, pipe r. effects of ocean acidification on the immune response of the blue mussel mytilus edulis. aquat. biol. 2: 67-74, 2008. booij mj. impact of climate change on river flooding assessed 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j. exp. mar. biol. ecol. 396: 177184, 2011. raven j, caldeira k, elderfield h, hoegh-guldberg o, liss p, riebesell u, et al. ocean acidification due to increasing atmospheric carbon dioxide. policy document. the royal society, london, 2005. reid hi, soudant p, lambert c, paillard c, birkbeck th. salinity effects on immune parameters of ruditapes philippinarum challenged with vibrio tapetis. dis. aquat. org. 56: 249-258, 2003. wikfors gh, krome c. ocean acidification and molluscan hemocytes: basis and rationale for experimental studies. j. shellfish res. 28: 658659, 2009. yu jh, song jh, choi mc, park sw. effects of water temperature change on immune function in surf clams, mactra veneriformis (bivalvia: mactridae). j. invertebr. pathol. 102: 30-35, 2009. 77 abstract introduction corresponding author conclusions a new in vivo assay of coagulation in invertebrates isj 9: 178-183, 2012 issn 1824-307x technical report a new in vivo capillary assay of coagulation in invertebrates c bajzek, ms dushay biology department, illinois institute of technology, chicago, il, usa accepted october 11, 2012 abstract greater understanding of insect clotting requires better tests that can be performed in whole, living insects. we found we could collect hemolymph bleeding from wounded drosophila larvae in microcapillary tubes. the capillary assay showed a difference in the amount of bleeding between feeding and wandering stage third instar larvae, and performed well with clotting mutations. this new in vivo coagulation assay will be helpful for studies of coagulation in drosophila and other invertebrates. key words: drosophila; hemolymph; hemolectin; hemocytes; phenoloxidase introduction coagulation prevents excessive fluid loss and contributes to immunity and wound healing in invertebrates (theopold and dushay, 2007; dushay, 2009; cerenius and söderhäll, 2011; loof et al., 2011). we are focused on the fruit fly, drosophila melanogaster, with its extensive molecular genetic advantages. goto et al. (2001) first identified hemolectin (hml) as a gene expressed in hemocytes, and then showed its involvement in coagulation by increased bleeding in hml rnai knockdown larvae (goto et al., 2003). larval clotting factors, including hemolectin, were later identified and confirmed with ex vivo clotting assays (karlsson et al., 2004; scherfer et al. 2004). we then sought to study coagulation in vivo. although loss of hml completely blocked coagulation as measured by bead aggregation and spaghetti assay (karlsson et al., 2004; scherfer et al., 2004), unstaged hmlf03374 larvae survived wounding as well as controls (lesch et al., 2007). when larvae were wounded in the feeding stage of the third instar with fewer circulating hemocytes to participate in clotting, hmlf03374 lshowed a small, but significant reduction in survival (chang et al., 2012). however, the great difference between the strong effect of hmlf03374 l on ex vivo assays of coagulation and its small effect on wound survival revealed the need for another in vivo assay of coagulation. we have now developed a capillary assay that ___________________________________________________________________________ corresponding author: mitchell s. dushay biology department, illinois institute of technology 3101 s dearborn st, chicago il 6016, usa e-mail: mdushay@iit.edu allows quick and quantitative measure of how much wounded larvae bleed as the clot and other hemostatic mechanisms work inside the animals. thus, the assay indirectly measures coagulation inside living larvae. as expected, this assay demonstrates that larvae bleed more when wounded in the feeding stage than in the wandering stage of the third instar, and it shows a greater effect of hmlf03374 than was shown by wound survival. here, we describe the first uses of this assay, which we believe will be a welcome tool for further studies on coagulation in drosophila and other insect and invertebrate species. materials and methods fly stocks with the exceptions noted below, all fly stocks were obtained from the bloomington drosophila stock center. the hmlf03374 strain used was the outcrossed strain described in (chang et al., 2012). flies were kept on mashed potato, sugar, yeast medium. larvae were staged by culturing on medium with 0.5 % bromophenol blue (fletcher and thummel, 1995). feeding stage third instar larvae had visibly blue guts, while wandering stage larvae that had stopped eating had paler guts. capillary assay feeding stage third instar larvae were selected from vials of blue fly food as described in chang et al. (2012). larvae were washed in deionized water, gently dried for a few seconds on tissue paper, and placed on plastic petri dishes. these clean dry larvae did not move very much, so they were easily manipulated. each larva was rolled onto its side and 178 fig. 1 feeding stage larvae were selected, washed in deionized water, and gently dried. larvae were wounded in the lateral caudal-most region with a fine tungsten needle. a 1 µl microcapillary tube was then used to collect the hemolymph as the animal bled. finally, the amount of hemolymph in the tube was measured. held with a paintbrush while a fine tungsten needle was used to wound the larva in the lateral caudal region, not striking the gut. immediately after wounding, the needle was removed and a 1µl microcapillary tube (drummond scientific number 1000-0010) was placed over the wound to collect the hemolymph as larvae bled. some larvae moved at this point, but it was not difficult to hold the capillary over the wound and follow the larva. hemolymph sometimes pooled around larvae, and this too was collected with the capillary tube. the capillary tube was held over the wound until the larvae stopped bleeding. this took less than 20 sec., and we could see when hemolymph stopped moving up the capillary tube and no more hemolymph pooled around the larvae. the amount of hemolymph in the tubes was measured in mm. a schematic drawing of the method is shown in figure 1. to be clear; coagulation was not monitored in the capillary tubes. rather, the tubes were used to measure how much hemolymph bled from wounded larvae. results we found we could collect hemolymph bled from wounded larvae with microcapillary tubes as described in materials and methods and shown schematically in figure 1. we first tested the assay using wild type larvae wounded in feeding or wandering stages of the third instar. more hemocytes circulate in the wandering stage than in feeding stage larvae, so coagulation is likely to be greater and/or faster in the wandering stage. capillary assay results confirmed that larvae wounded in the wandering stage bleed less than when wounded in the feeding stage. (fig. 2). then we tested whether the capillary assay would provide a larger signal for effects of the hmlf03374 mutation than displayed by wound survival. as shown in figure 3, hmlf03374 mutant larvae bled significantly more than controls, and this difference was of greater magnitude than the slight difference found in fig. 2 capillary assay results for feeding and wandering stages of third instar larvae. wandering stage larvae bled significantly less (p < 0.01 by student's t-test). error bars indicate standard deviation, and n = 30 for each group. 179 fig. 3 capillary assay results for hmlf03374 and w1118 wildtype. as expected for a strong coagulation mutant, more hemolymph was collected from hmlf03374 (p < 0.01 by student's t-test). error bars indicate standard deviation, and n = 30 for each group. wound survival (chang et al., 2012). these results confirmed the capillary assay as a valid and useful measure of coagulation. next, we tested the effects of removing hemocytes completely. in drosophila, apoptosis can be induced by the expression of grim (chen et al., 1996). we employed the uas gal4 system (brand and perrimon, 1993) and drove the expression of grim in hemocytes by setting crosses to generate hml∆gal4uas::gfp>>uas::grim larvae (charroux and royet, 2009). gfp-expressing hemocytes fluoresce in hml∆gal4uas::gfp larvae (fig. 4a). in contrast, this fluorescence is not visible in larvae bearing both hml∆gal4 and uas::grim, indicating the loss of hemocytes by apoptosis. capillary assays performed on these larvae showed increased bleeding comparable to hmlf03374 mutant larvae (fig. 4b). the bleeding defect of larvae lacking hemocytes reinforced the value of the capillary assay for measuring coagulation in vivo. finally, we tested bc larvae lacking phenoloxidase. this enzyme is known for its role in invertebrate immune defense (cerenius et al. 2008), although the importance of phenoloxidase in drosophila immune defense has been contested (leclerc et al., 2006). the mature clot is melanized by phenoloxidase, and the enzyme is thought to play a late role in coagulation (bidla et al., 2007), as well as in subsequent wound healing (galko and krasnow, 2004). while loss of phenoloxidase in bc mutant larvae reduced wound survival (chang et al., 2012), its effect on bleeding specifically was not tested at that time. the capillary assay showed that bc mutant larvae bled more than controls, but much less than hml mutant larvae (fig 5). discussion in vivo studies are needed to learn more about coagulation in drosophila and other insects. wound survival has been tried, but it is not specific to coagulation, as survival is also affected by other still-poorly understood hemostatic processes, immune defense, and wound healing. in addition, the high wound survival rate of strong clotting mutant hmlf03374 showed survival alone was a poor measure of coagulation. we developed a capillary assay to better monitor coagulation in vivo. this assay showed that drosophila larvae bleed more after wounding in the feeding stage of the third instar than in the wandering stage, in line with the change in numbers of hemocytes circulating in the different larval stages. the capillary assay also showed greater magnitude effects of hmlf03374 on bleeding, more consistent with tests of coagulation ex vivo than its slight effect on wound survival. hemolymph coagulation requires both humoral and cellular factors. we tested hml∆gal4 uas::gfp>>uas::grim larvae lacking hemocytes to measure the limits of the capillary bleeding assay. we do not argue that all hemocytes were completely removed from these larvae: only the loss of hmlexpressing cells as demonstrated by absence of gfp fluorescence. regardless, the similar amounts of bleeding by these larvae and hmlf03374 mutants is 180 a) b) fig. 4 a) green fluorescent protein-expressing hemocytes can be seen in hmlδgal4uas::gfp larvae (top). apoptosis was induced by expressing the grim gene using the binary gal4 uas system. the hemocytes were killed and the fluorescence lost in larvae expressing the grim cell death gene in hmlδgal4 uasgfp>>uas::grim larvae (bottom). b) capillary assay results for larvae expressing hmlδgal4 uasgfp>>uas::grim. the mean of the capillary measurements of w1118 was set to 1, and the capillary values collected for the other genotypes were set relative to this. the expression of the grim cell death gene led to the death of hemocytes and an increase in bleeding. the values for hmlδgal4 uasgfp>>uas::grim and hmlf03374 were statistically indistinguishable. the differences between the controls, w1118, hmlδgal4 uasgfp, and uas::grim, were also not statistically significant. the hmlδgal4 uasgfp>> uas::grim and hmlf03374 larvae bled significantly more than w1118 (p < 0.01 by student’s t-test), indicated by asterisks. error bars indicate standard deviation, and n = 30 for each group. 181 fig. 5 capillary assay results for bc larvae lacking phenoloxidase. bc larvae bled significantly more than controls (p < 0.05 by student’s t-test), but less than hmlf03374 mutants, whose greater difference from controls (p < 0.01 by student’s t-test) is indicated by double asterisks. consistent with hemolectin being a major hemocyte coagulation factor, since loss of hemocytes did not markedly increase bleeding compared to loss of hml. finally, the capillary bleeding assay showed that wounded bc larvae bled more than wildtype, but less than hmlf03374 mutants. this too is consistent with current models of drosophila coagulation, and suggests that much of the effect of bc on wound survival is due to loss of phenoloxidase and defects in plug formation and wound healing, similar to the effects of comparable phenoloxidase blocking mutation lz15 (galko and krasnow, 2004). the capillary assay has advantages over wound survival as a means to measure coagulation in vivo. the capillary assay yields results in one day, unlike the wounding assay, which takes twice as long. also, whereas wound survival may be affected by a variety of processes that may add to or obscure the effects of mutations on clotting, capillary assay results are more specific to coagulation and hemostasis. this was highlighted by our study of hmlf03374 mutants. this mutation that abolished coagulation in ex vivo assays only reduced larval wound survival by 5 %. in contrast, the capillary assay showed that hmlf03374 mutant larvae bled twice as much as wildtype. the capillary assay thus provides a robust in vivo measure of coagulation that is more specific, easier, and faster to perform than wound survival. this assay should be easy to adapt to other small invertebrates. acknowledgements we thank the bloomington drosophila stock center for fly stocks. flybase provided important information used in this work. the authors are grateful to iit for support. cb was also supported by the iit csl summer undergraduate research fellowship. references bidla g, dushay ms, theopold u. crystal cell 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coagulation and immunity in drosophila melanogaster. dev. comp. immunol. 31: 1255-1263, 2007. loof tg, schmidt o, herwald h ,theopold u. coagulation systems of invertebrates and vertebrates and their roles in innate immunity: the same side of two coins? j. innate immunity 3: 34-40, 2011. scherfer c, karlsson c, loseva o, bidla g, goto a, havemann j, et al. isolation and characterization of hemolymph clotting factors in drosophila melanogaster by a novel pull-out assay. curr. biol. 14: 625-629, 2004. theopold u, dushay ms. mechanisms of drosophila immunity an immune system at work. curr. immunol. rev. 3: 276-288, 2007. 183 research report isj 9: 72-81, 2012 issn 1824-307x research report biochemical characterization of α-amylases from gut and hemolymph of rhynchophorus ferrugineus olivieri (col.: curculionidae) and their inhibition by extracts from the legumes vigna radiata l. and phaseolus vulgaris l. n saberi riseh, m ghadamyari department of plant protection, faculty of agricultural science, university of guilan, rasht, iran accepted may 8, 2012 abstract α-amylase inhibitors represent an important tool in engineering crop plants against insect pests. for achieving this goal, it is necessary to find the nature of α-amylases and their properties for possible use in a pest management procedure. because rhynchophorus ferrugineus olivieri is a devastating pest of palm trees in the southeast of iran, we attempted to characterize α-amylases from larval gut and hemolymph, and to study their interaction with inhibitors extracted from the common bean and the green mung bean. the optimal phs for gut and hemolymph α-amylases were 4 5 and 5 6, respectively. also, high gut amylolytic activity was found at temperatures of 40 – 50 °c. the highest and lowest specific α-amylase activities were detected in the guts of last instar and adult males, and in the hemolymph of last instar, respectively. as calculated from lineweaver-burk plots, the km values for gut and hemolymph α-amylases of the last instar were 0.54 and 2.15 %, respectively, when glycogen was used as the substrate. also, when starch was used as the substrate, the km values for gut and hemolymph α-amylases were 1.37 and 0.15 %, respectively. zymogram pattern in the native gel revealed that r. ferrugineus gut and hemolymph α-amylases had two isoforms. α-amylase inhibitors partially purified from vigna radiata l. and phaseolus vulgaris l. by ionic exchange deae cellulose column, inhibited the r. ferrugineus gut α-amylase activity by 19 ± 0.64 % and 11.56 ± 0.69 %, respectively. key words: rhynchophorus ferrugineus; gut and hemolymph α-amylases; common bean; green mung bean; inhibitors   introduction α-amylases (α-1,4-glucan-4-glucanohydrolases; e.c. 3.2.1.1) constitute a family of hydrolyses that cleave α-d-(1,4)-glucan linkages in starch components, glycogen and various other related carbohydrates. α-amylases are digestive enzymes that play an essential role in starch digestion and are consequently involved in energy production in insects (pelegrini et al., 2006). many studies have focused on the characterization of the digestive αamylases of economically important insect pests including rhyzopertha dominica (baker, 1991), tenebrio molitor (buonocore et al., 1976), zabrotes subfasciatus (pelegrini et al., 2006), naranga aenescens l. (asadi et al., 2010), xanthogaleruca luteola mull. (sharifi et al., 2011) and eurygaster ___________________________________________________________________________ corresponding author: mohammad ghadamyari department of plant protection faculty of agricultural science university of guilan, rasht, iran e-mail: ghadamyari@guilan.ac.ir integriceps (kazzazi et al., 2005). insect control strategies interfering with αamylases, and thus food digestion, are known to reduce insect survival and growth, and, for this reason, many studies have focused on characterizing the effects of digestive α-amylase inhibitors on insect α-amylases. disruption of carbohydrate digestion by transformation of plant genomes with α-amylase inhibitors represents an alternative approach to the control of insect pests. α-amylase inhibitors naturally occur in many plants and are particularly abundant in cereals and legumes, as part of natural defense mechanisms against herbivores insects (carlini and grossi-desá, 2002; franco et al., 2002). many insecticidal proteins and molecules of plant origin such as lectins, and α -amylase and protease inhibitors can retard insect survival, growth and development when ingested (boulter, 1993; ussuf et al., 2001). when the α-amylase inhibitor gene from the common bean was expressed in transgenic peas, seeds became resistant to the infestation of bruchid 72    mailto:ghadamyari@guilan.ac.ir fig. 1 different parts (v1, v2 and v3) of the gut in the larvae of r. ferrugineus. weevils (shade et al., 1994; shroeder et al., 1995). the α-amylase inhibitor markedly inhibited the αamylase activity in the larval midgut of the weevils (ryan, 1990; ishimoto and chrispeels, 1996). the red palm weevil, rhynchophorus ferrugineus is widely considered to be the most damaging insect pest of palms in the world. this pest is usually attracted from unhealthy palm trees, but they will often attack healthy palms too. r. ferrugineus larvae feed within the apical growing point of the palms, causing extensive damage to palm tissues and to the structure of the palm trunk. good sanitation practices are needed to prevent the red palm weevil spreading from infested palms (murphy and briscoe, 1999). because of the concealed nature of the larvae, effective methods for the management of the red palm weevils have been difficult to develop. current methods recommended for the management of r. ferrugineus have focused on integrated pest management (ipm) strategies involving surveillance, pheromone lures, cultural control methods and chemical treatments (murphy and briscoe, 1999). insecticides are probably the most common control tools used against the red palm weevil in iran, and can be applied in a variety of ways for r. ferrugineus suppression including applications as dusts, and/or liquid sprays. trunk injections or soil applications of systemic insecticides that move inside the palm poisoning weevil larvae and adults may also be effective (murphy and briscoe, 1999). however, because of the many problems associated with the use of synthetic insecticides in table 1 the mean α-amylase specific activity (±se) in guts of adults and last instars and hemolymph of last instars of r. ferrugineus. activity of α-amylase (µmol/min/mg protein) tissue 3.16 ± 0.022av1 segment of larval gut 2.21 ± 0.033bv2 segment of larval gut 1.32 ± 0.0055cv3 segment of larval gut 2.06 ± 0.011badult female whole gut 3.05 ± 0.03aadult male whole gut 2.98 ± 0.03alarval gut 0.27 ± 0.003dlast instars hemolymph different letters indicate that the activity of enzymes in different tissue are significantly different from each other by tukey’s test (p < 0.05). 73    fig. 2 the effect of ph on hemolymph α-amylase activity in larvae of r. ferrugineus. integrated pest management approaches, the use of chemicals against this pest presents some difficulties. therefore, the use of genetic engineering to produce pest resistant transgenic plants offers an alternative strategy for the control of this pest. due to the significant damage caused by the red palm weevil, we attempted to characterize its gut and hemolymph α-amylases and to study its inhibition by inhibitors extracted from the common bean and the green mung bean. materials and methods insect the insect was collected from the palm trees in the sistan and baluchestan province of iran. the final instars larvae and male and female adults were randomly selected for measuring the enzyme activity. enzyme sample preparation final instars larvae were immobilized on ice and dissected under a stereomicroscope in ice-cold saline buffer (10 µm nacl, ph = 7). whole parts of the gut (experimentally divided into v1, 2 and 3 segments) were removed from the body and thoroughly rinsed in ice-cold distilled water. each digestive system was homogenized in a known volume of cold double-distilled water using a handheld glass homogenizer before measuring the optimum ph. also, the sample for measuring specific activity and effect of temperature on enzyme activity were prepared in buffer (optimum ph specific for each enzyme). the homogenates were centrifuged at 13,000×g for 15 min at 4 °c and supernatants were used for enzyme activity assays. also the hemolymph was collected from larvae. small incisions were made in the soft cuticle anterior to the second thoracic segment of larvae and the hemolymph was collected with a 75 μl glass capillary tube. chemicals 3,5-dinitrosalicylic acid (dns), triton x-100 and edta (ethylenediaminetetra acetic acid) were purchased from sigma (st. louis, mo, usa). deae (diethylaminoethyl) cellulose was obtained from bio-rad laboratories ltd. (uk). all other chemicals (reagent grade) were from merck (merck, darmstadt, germany). determination of α-amylase activity and protein concentration α-amylase activity was determined at room temperature in 20 mm acetate-citrate (in optimum ph for each tissue). supernatant (10 µl) was added to a tube containing 40 µl of the buffer and 50 µl of 1 % (w/v) starch. the concentration of reducing sugars obtained from the catalyzed reaction for 30 min was measured by the dns method according to 74    fig. 3 the effect of ph on gut α-amylase activity in larvae of r. ferrugineus. bernfeld (1955). absorbance was measured at 545 nm with a microplate reder model stat fax® 3200 (awareness technology inc.). one unit of αamylase is defined as the amount of enzyme that liberates 1.0 μmol of reducing sugar/min with maltose as a standard. protein concentration was determined by the bradford's method (1976) with bovine serum albumin (bsa) as standard. effects of ph and temperature on α-amylase activity the ph profiles of the α-amylase activity were determined at room temperature in a mixed buffer containing phosphate, glycine and acetate (25 mm of each) adjusted to various phs (ph from 2 to 12) by adding hcl or naoh for acidic and basic ph values, respectively. before determining activity, the reaction mixtures were incubated at different phs at room temperature for 5 min. the activity of αamylase was determined by incubating the reaction mixtures in optimal ph value (20 mm acetate-citrate) at different temperatures ranging from 20 to 60 °c with 10 °c intervals. polyacrylamide gel electrophoresis and zymogram analysis non-denaturing polyacrylamide gel electrophoresis (page) (8 %) was carried out as described by davis (1964) and electrophoresis was performed applying a 100 v electric field at 4 °c. afterward, the gel was incubated in 2.5 % (v/v) triton x-100 for 30 min at room temperature with gentle agitation. then, the gel was rinsed with distilled water and washed in 25 mm tris-hcl (ph 7.4). the washed gel was incubated in fresh acetate buffer (ph 5) containing 1 % (w/v) soluble starch at 30 ºc for 60 min. after being washed with distilled water, the gel was subjected to staining with lugol solution (i2 1.3 % and ki 3 %) at room temperature until the appearance of clear zones in protein bands with α-amylase activity against a dark blue background. kinetic parameters of α-amylases catalytic activities of the enzymes were investigated at different concentrations of starch and glycogen over the range 0.05 1.5 % and 0.1 3 % (w/v) in 20 mm acetate (ph 5), respectively. the michaelis-menten constant (km) was estimated from the lineweaver-burk plots. purification of v. radiata and p. vulgaris α-amylase inhibitors from seeds seeds were ground into flour and extracted with 0.15 m nacl with continuous stirring for 1 h at 4 °c. the materials were then centrifuged at 6,000×g at 4 °c for 30 min. supernatants were submitted to 80 °c and centrifuged at 6,000×g for 15 min. the supernatant was then submitted to fractionation with ammonium sulfate (with 80 % saturation). after dialysis against a 20 mm tris-hcl, ph 7, buffer, the 75    fig. 4 the effect of temperature on gut αamylase activity of last instar larvae rhynchophorus ferrugineus. the relative activities were based on the ratio of the activity obtained at a temperature to the maximum activity obtained at that range and expressed as a percentage. different letters indicate that the activity of enzymes in different temperature is significantly different from each other by tukey’s test (p < 0.05). fractions were applied to an ionic exchange deae cellulose column equilibrated with 20 mm tris-hcl buffer (ph 7.0), with a flow rate of 0.5 ml/min. the column was eluted with a linear nacl gradient of 0 0.5 m at the flow rate of 0.5 ml/min. the absorbance of the effluent was monitored at 280 nm in a biophotometer plus (eppendorf, germany) amylase inhibition assay ten µl of the enzyme sample (protein content = 45 µg) was pre-incubated with 10 µl of inhibitor and 30 µl of acetate buffer (ph 5) for 30 min at 37 °c; then the same procedure was applied to the amylase and its activity was determined by measuring absorbance at 540 nm. experiments were performed in four replicates. statistical analysis the data were compared by one-way analysis of variance (anova) followed by tukey’s test when significant differences were found at p = 0.05 using sas program (sas institute, 1997). results α-amylase activity the activity of α-amylases was characterized in crude extracts of r. ferrugineus. the results showed that there were significant differences between the α-amylase activities in tissues and that the highest and the lowest activities were detected in the guts of last instars and adult males, and in the hemolymph, respectively. the results showed that there were significant differences between the amylases activities in different parts of the digestive system and the highest activity was detected in the part v1 (fig. 1, table 1). the effect of ph and temperature on enzyme activity the influence of ph on the gut and hemolymph α-amylase activity is shown in the figs 2 and 3. hemolymph α-amylase was most active at ph 6, while α-amylase extracted from digestive systems showed highest activity at ph 5. the ph activity profile of hemolymph α-amylase was distributed along a very broad ph range (3 7) and the enzyme activity retained more than 55 % of its maximal activity in the ph range of 3 to 8. as is shown in fig. 4, the optimal temperature for gut α-amylase was 40 50 °c. the substrate was hydrolyzed at a broad range of temperatures (20 60 °c). zymogram analysis of α-amylase at different ph the crude r. ferrugineus larval extracts were analyzed by native page. after α-amylase activity staining, two isoforms of α-amylase were clearly detected in the digestive system of the last instars. however, as depicted in fig. 5, the light intensity of bands representing α-amylase activity was lower 76    with a relative mobility (rm) of 0.72 instead of 0.92. zymogram of gut α-amylase from larval r. ferrugineus at different phs is shown in fig. 6. αamylase activity was observed at phs 3, 4, 5, 6, 8 and 9. maximum in-gel α-amylase activity was determined at ph 5, which is consistent with the results from our tube assays (fig. 6). kinetic parameters of α-amylases results showed that kinetic behavior of r. ferrugineus α-amylase toward starch and glycogen was significantly different. as calculated from lineweaver-burk plots, when starch and glycogen were used as substrates, the km values for hemolymph α-amylase were 0.1665 and 2.159 %, respectively. also, when starch and glycogen were used as substrates, the km values for gut α-amylase from last instars were 1.375 and 0.541 %, respectively (table 2). effects of p. vulgaris and v. radiata α-amylase inhibitors on r. ferrugineus α-amylase activity ammonium sulfate fraction of inhibitors was further fractionated on an ionic exchange deae cellulose column. the profile of p. vulgaris inhibitors showed three major peaks and three minor peaks (fig. 7). also, the chromatogram of v. radiata inhibitors showed three major peaks and 6 minor peaks. assay of peaks revealed that one peak of p. vulgaris inhibited the r. ferrugineus gut α-amylase by 11.56 ± 0.69 %, while the others did not. in the chromatogram obtained from v. radiate the peak number 17 had the highest inhibitory effect on αamylase activity. discussion this study has clearly demonstrated α-amylase activity in the digestive system and hemolymph of the red palm weevil's last instars, and male and female adults. the specific activity of gut α-amylase derived from adult males was found to be higher than that of females and hemolymph of last instars (table 1). sharifi et al. (2011) showed that the specific activity of gut α-amylase from last instars of x. luteola was 1.46-fold higher than that of adults. reports of differences in digestive enzyme activities between male and female insects are contradictory. in numerous insects, females have additional enzymes along the digestive tract with respects to males, apparently to meet the metabolic demands of egg production. however, in some insect species, males show higher enzyme activity than females. mandal et al. (1981) showed that the protease activity in schizodactylus monstrosus was higher in adult males compared to females and larval stages. investigation of midgut trypsin and chymotrypsin specific activities in adult castes of four polister species showed that chymotrypsin activity in males of p. mericus, p. fuscatus and p. exclamans were 1.97-, 2.78and 1.13fold higher than in females, respectively. also, trypsin activity in males of p. mericus and p. fuscatus were 2and 2.65fold higher than in females, respectively (kayes, 1978). α-amylase is present in all regions of the alimentary canal of the last larval instar of r. ferrugineus (table 1). our results show a significant difference in the fig. 5 zymogram analysis of α-amylase activity from larval r. ferrugineus. a, gut; b, hemolymph. activity of α-amylase in v1, v2 and v3 gut regions of last larval instar (fig. 1, table 1). the rank order, from the highest to the lowest α-amylase activity was v1>v2>v3. the alimentary canal of the pistachio green stink bug, brachynema germari kolenati (hemiptera: pentatomidae) was divided into four distinct regions (v1, v2, v3 and v4) by ramzi and hosseininaveh (2010) and activities of αamylase and αand β-glucosidases were measured in these parts. their results showed that the highest α-amylase and αand β-glucosidase activities were observed in v3, whereas the lowest activities were measured in v4. also, sharifi et al. (2011) divided the alimentary canal of elm leaf beetle into three distinct divisions and their results showed that the αamylase activity in the midgut of last instars was 3.125and 4.16-fold higher than that in foregut and hindgut, respectively. our results show that the highest activities of gut and hemolymph α-amylase are in 4-5 and 5-6 ph ranges, respectively (figs 2, 3). α-amylase in r. ferrugineus was active in acidic ph, which is consistent with the optimal neutral to slightly acidic conditions reported for other coleopteran species (baker, 1983; terra et al., 1996). optimal ph values for α-amylases in larvae of several coleopterans range from 4 to 5.8 (baker, 1983). α-amylase in h. postica larvae have an acidic optimal ph (phs 3 6) (vatanparast and hoseininaveh, 2010). also, αamylase extracted from hypothenemus hampei has an optimal activity at ph 5 (valencia-jimenez et al., 2000). the optimal ph values were found at ph 5.2 77    fig. 6 zymogram analysis of gut α-amylase activity from larvae of r. ferrugineus at different phs. as shown, the α-amylase activity is affected by ph variations. for callosobruchus chinensis (podoler and applebaum, 1971) and ph 5.4 for tribolium castaneum (applebaum and konijn 1965). however, in some coleopterans such as trogoderma granarium (dermestidae), optimum α-amylase activity mostly occurs at alkaline ph range (e.g., 6 9) (hosseininaveh et al., 2007). sharifi et al. (2011) showed that in the case of larval x. luteola, the optimum ph for gut α-amylase activity was 5. asadi et al. (2010) showed that the optimum phs for αamylases midgut, salivary and hemolymph αamylases of naranga aenescens were 8, 9 and 9, respectively. the maximum amylase activity at phs 5 6 observed in the hemolymph of r. ferrugineus, which is consistent with physiological ph of the hemolymph as reported for most insects. also, the optimum ph 6.8 was reported for hemolymph amylase in bombyx mori (abraham et al., 1992). activity of digestive α-amylase in the larval gut of r. ferrugineus was highest at 40 50°c. optimum temperature for h. posticae α-amylase has been reported as 35°c (vatanparast and hosseininaveh, 2010). the activity of α-amylase was also characterized by zymogram analysis after native page which allowed visualization of the enzyme activity in situ. the results indicated two isoforms of α-amylase in crude gut and hemolymph of the last instar with the same electrophoretic pattern (fig. 5). however, as depicted in fig. 5, intensity of both bands showing α-amylase activity in hemolymph were less than that in the gut, which is correlated well with α-amylase activities in crude extracts. our results from a zymogram analysis of αamylase activity at different phs f show two bands in the extracts from the larval digestive system of r. ferrugineus. no α-amylase activity was observed at ph 2. maximum in-gel α-amylase activity was determined at ph 5 based on the intensity of bands (fig. 6). in other coleopteran insects, the gut αamylases of x. luteola (sharifi et al., 2011) and osphranteria coerulescens redt. (saberi riseh and ghadamyari, unpublished results) has present just one isoform. the number of α-amylases identified in different insect species varied from 1 to 8 isoforms e.g., helicoverpa armigera, spodoptera litura, callosobruchus chinensis and carcyra cephalonica exhibited more than five isoforms whereas sitophilus oryzae and tribolium castaneum possess only one isoform (sivakumar et al., 2006). wisessing et al. (2008) showed that callosobruchus table 2 km values of α-amylases from gut and hemolymph of larval r. ferrugineus on starch and glycogen. substrate tissue glycogen starch gut 0.541±0.08 1.375±0.02 hemolymph 2.159±0.01 0.1665±0.005 78    a b fig. 7 retained fraction obtained from ionic exchange deae cellulose chromatography, equilibrated with 20 mm tris-hcl buffer, ph 7.0, with a flow rate of 0.5 ml/min. dashed line represents 0 0.5 m nacl linear gradient. a) and b) chromatograms for inhibitors extracted from p. vulgaris and v. radiata, respectively. maculatus α-amylase had one isoform with a molecular weight of 50 kda. two α-amylase isoforms in the gut extracts of two species of sitophilus, including s. zeamais and s. granarius (baker,1983), prostephanus truncatus (mendiolaolaya et al., 2000) and hypothenemus hampei (valencia-jimenez et al., 2000) were reported. v. radiata and p. vulgaris seeds seem to contain a number of r. ferrugineus α-amylase inhibitors that can be separated by ion-exchange chromatography. these proteins show α-amylase inhibitory activity against r. ferrugineus gut αamylases. our results confirmed that α-amylase inhibitors, purified from v. radiata and p. vulgaris by ionic exchange deae cellulose column, exert inhibitory activity on r. ferrugineus gut α-amylase. the peak number 37 (nacl concentration 0.3 mm; protein concentration = 0.93 mg/ml) from p. vulgaris and peak number 17 (nacl concentration 0.1 mm; protein concentration = 1.17 mg/ml) from v. radiata 79    inhibited α-amylase activity by 11.56 ± 0.69 % and 19.8 ± 0.64 %, respectively. plant α-amylase inhibitors show a great potential as tools to engineer the resistance of crop plants against pests. the in vivo effect of the α-amylase inhibitor on mortality of c. maculatus showed that the α-amylase inhibitor purified from v. radiata acts on c. maculatus during the developmental stage, by reducing carbohydrate digestion necessary for growth and development, rather than during the egg laying/hatching stage (wisessing et al., 2010). also, engkagul et al. (2004) reported that crude protein extracts from the kamphaengsaen 1 variety of mung bean (kps1) inhibited the c. maculatus α-amylase, which in turn prevents growth and development of insect larvae infesting seeds. these α-amylase inhibitors, are attractive candidates for seed weevil biocontrol, and have been purified and characterized from different varieties of common bean including the white kidney bean (yamaguchi, 1991), red kidney bean and black kidney bean (lajolo and finardi-filho, 1985). in conclusion, protein inhibitors active against different types of hydrolytic enzymes are widely distributed in plants. these inhibitors play a protective role against insect attack. the transgenic expression of insecticidal proteins such as αamylase inhibitors is being evaluated as a potential protective strategy against insects (schuler et al., 1998). our results show that the inhibitor extracted from v. radiata seeds has a more potent inhibitory activity against r. ferrugineus gut α-amylase 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protease inhibitors in plants. genes for improving defense against insects and 80    http://www.ncbi.nlm.nih.gov/pubmed?term=%22mandal%20s%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22roy%20s%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22chaudhuri%20dk%22%5bauthor%5d pathogens. ann. rev. phytopathol. 28: 25-45, 1990. schroeder he, gollash s, moore a. bean αamylase inhibitor confers resistance to the pea weevil (bruchus pisorum) in transgenic peas (pisum sativum l.). plant physiol. 107: 12331239, 1995. schuler th, poppy gm, kerry br, denholm i. environmental risk assessment of transgene products using honey bee (apis mellifera) larvae. trends biotechnol. 16: 168-175, 1998. shade re, schroeder re, poueyo jj, tabe lm, murdock li, higgins tjv, et al. transgenic pea seeds expressing the α-amylase inhibitor of the common bean are resistant to bruchid beetles. nat. biotechnol. 12: 793-796, 1994. sharifi m, ghadamyari m, mahdavi m, saeedi f. biochemical characterization of digestive carbohydrases from xanthogaleruca luteola and inhibition of its α-amylase by inhibitors extracted from the common bean. arch. biol. sci. 63: 705-716, 2011. sivakumar s, mohan m, franco o, thayumanavan b. inhibition of insect pest α-amylases by little and winger millet inhibitors. pestic. biochem. physiol. 85: 155-160, 2006. terra wr, ferreira c, jordao bp, dillon rj. digestive enzymes. in: lehane mj, billingsley pf (eds), biology of the insect midgut, chapman & hall, london, pp 153-194, 1996. ussuf kk, laxmi nh, mita r. protease inhibitors: plant derived genes of insecticidal protein for developing insect resistant transgenic plants. curr. sci. 80: 847-853, 2001. valencia-jimenez a, bustillo ae, ossa ga, chrispeels mj. α-amylases of the coffee berry borer (hypothenemus hampei) and their inhibition by two plant amylase inhibitors. insect biochem. mol. biol. 30: 207-213, 2000. vatanparast m, hosseininaveh v. digestive amylase and pectinase activity in the larvae of alfalfa weevil, hypera postica (coleoptera: curculionidae). entomol. res. 40: 328-335, 2010. wisessing a, engkagula a, wongpiyasatida a, chuwongkomon k. purification and characterization of callosobruchus maculatus kasetsart α-amylase. j. natur. sci. 42: 240244, 2008. wisessing a, engkagul a, wongpiyasatid a, choowongkomon k. biochemical characterization of the α-amylase inhibitor in mung beans and its application in inhibiting the growth of callosobruchus maculates. j. agric. food chem. 58: 2131-2137, 2010. yamaguchi h. isolation and characterization of the subunits of phaseolus vulgaris α-amylase inhibitor. j. biochem. 110: 785-789, 1991. 81    our results show that the highest activities of gut and hemolymph α-amylase are in 4-5 and 5-6 ph ranges, respectively (figs 2, 3). α-amylase in r. ferrugineus was active in acidic ph, which is consistent with the optimal neutral to slightly acidic conditions reported for other coleopteran species (baker, 1983; terra et al., 1996). optimal ph values for α-amylases in larvae of several coleopterans range from 4 to 5.8 (baker, 1983). α-amylase in h. postica larvae have an acidic optimal ph (phs 3 6) (vatanparast and hoseininaveh, 2010). also, α-amylase extracted from hypothenemus hampei has an optimal activity at ph 5 (valencia-jimenez et al., 2000). the optimal ph values were found at ph 5.2 for callosobruchus chinensis (podoler and applebaum, 1971) and ph 5.4 for tribolium castaneum (applebaum and konijn 1965). however, in some coleopterans such as trogoderma granarium (dermestidae), optimum α-amylase activity mostly occurs at alkaline ph range (e.g., 6 9) (hosseininaveh et al., 2007). sharifi et al. (2011) showed that in the case of larval x. luteola, the optimum ph for gut α-amylase activity was 5. asadi et al. (2010) showed that the optimum phs for α-amylases midgut, salivary and hemolymph α-amylases of naranga aenescens were 8, 9 and 9, respectively. the maximum amylase activity at phs 5 6 observed in the hemolymph of r. ferrugineus, which is consistent with physiological ph of the hemolymph as reported for most insects. also, the optimum ph 6.8 was reported for hemolymph amylase in bombyx mori (abraham et al., 1992). activity of digestive α-amylase in the larval gut of r. ferrugineus was highest at 40 50°c. optimum temperature for h. posticae α-amylase has been reported as 35°c (vatanparast and hosseininaveh, 2010). review isj 10: 58-68, 2013 issn 1824-307x review apolipophorins and insects immune response a zdybicka-barabas, m cytryńska department of immunobiology, institute of biology and biochemistry, faculty of biology and biotechnology, maria curie-skłodowska university, 19 akademicka st., 20-033 lublin, poland accepted july 30, 2013 abstract insect lipoproteins, called lipophorins, are non-covalent assemblies of lipids and proteins serving as lipid transport vehicles. the protein moiety of lipophorin comprises two glycosylated apolipoproteins, apolipophorin i (apolp-i) and apolipophorin ii (apolp-ii), constantly present in a lipophorin particle, and an exchangeable protein, apolipophorin iii (apolp-iii). apolp-iii is an abundant protein occurring in hemolymph in lipid-free and lipid-bound state and playing an important role in lipid transport and insect innate immunity. in immune response apolp-iii serves as a pattern recognition molecule. it binds and detoxifies microbial cell wall components, i.e., lipopolysaccharide, lipoteichoic acid, and β-1,3-glucan. apolp-iii activates expression of antimicrobial peptides and proteins, stimulates their antimicrobial activity, participates in regulation of the phenoloxidase system and in hemolymph clotting. in addition, the protein is involved in cellular immune response, influencing hemocyte adhesion, phagocytosis and nodule formation, and in gut immunity. although apolp-iii is the best studied apolipophorin in insect immunity so far, a literature review suggests that all the three apolipoproteins, apolp-i, apolp-ii and apolp-iii, function together in a coordinated defense against pathogens. key words: lipophorin; apolipophorin iii; insect immunity; galleria mellonella introduction insect lipoproteins, called lipophorins, are wellstudied complexes of multifunctional molecules. these non-covalent assemblies of lipids and proteins serve as lipid transport vehicles. lipophorins have a structure similar to mammalian lipoproteins. they possess a hydrophobic core composed of nonpolar lipid constituents, surrounded by a monolayer of amphiphilic phospholipids (pl) and apolipoproteins. the protein moiety of lipophorin comprises two glycosylated apolipoproteins, arising from a common precursor preproapolipophorin, i.e. apolipophorin i (apolp-i, ca 240 kda) and apolipophorin ii (apolp-ii, ca 80kda) which are present in a 1:1 molar ratio in lipophorin particles (ryan et al., 1984; weers et al., 1993; blacklock and ryan, 1994; ryan and van der horst, 2000; marinotti et al., 2006). the fat body, a functional analog of mammalian ___________________________________________________________________________ corresponding author: małgorzata cytryńska department of immunobiology institute of biology and biochemistry faculty of biology and biotechnology maria curie-skłodowska university 19 akademicka st., 20-033 lublin, poland e-mail: cytryna@poczta.umcs.lublin.pl liver, is the site of apolp-i, apolp-ii and lipid synthesis as well as lipid storage, and assembling of lipoprotein particles. lipophorins are released as high or very high density lipoproteins into the hemolymph, depending on the insect species (prasad et al., 1986; venkatesh et al., 1987; de capuro and de bianchi, 1990; weers et al., 1992; van heusden et al., 1998). one of the major roles of lipoprotein is lipid transport during larval development and long-distance flight of insects. adipokinetic hormones (akhs), which are stored in the secretory granules of neuroendocrine cells (corpus cardiaca), are released during flight activity and are involved in lipid mobilization (beenakkers et al., 1985; van der horst, 2003). akhs trigger conversion of the fat body triacylglycerol (tag) stores into diacylglycerol (dag) by a specific lipase. the insect lipids are then released as dag and, after being assembled with apolp-i and apolp-ii, form high-density lipophorins (hdlp). two proteins, apolp-i and apolp-ii, are constantly present in a lipophorin particle, whereas the third protein, apolipophorin iii (apolp-iii), is an exchangeable molecule in these complexes. association of dag and apolp-iii with hdlp converts them to lowdensity lipophorins (ldlp), which serve as transport vehicles for lipids in hemolymph to a target tissue. 58 fig. 1 main functions of apolipophorin iii in insects. (*) denotes processes in which lipophorin particles or apolpi/ii are involved from 14 to 16 molecules of apolp-iii can be associated with the ldlp surface (kawooya et al., 1984, 1986; wells et al., 1987; van der horst et al., 1991). after release of a lipid load, apolp-iii dissociates from the complex and together with hdlp can be reused for another cycle of dag transport (weers et al., 1992; blacklock and ryan, 1994; soulages et al., 1995; ryan and van der horst, 2000; niere et al., 2001). among apolipophorins, especially apolp-iii is considered to be an important factor of insect immunity. the review summarizes available data on apolp-iii involvement in insect immune response (fig. 1). apolipophorin iii and lipid interactions apolp-iii was detected in different insect tissues, e.g., eggs, fat body, hemocytes, and hemolymph. this protein was found in hemolymph plasma of larvae, pupae, adults as well as in the molting fluid. it is a water-soluble and heat-stable protein of molecular mass 17-20 kda depending on the insect species (table 1). apolp-iiis contain no cysteine, some of them are glycosylated in orthoptera species, but other, e.g. in lepidoptera species, lack this modification (kawooya et al., 1984; chino and yazawa, 1986; chung and ourth, 2002). the studies on l. migratoria and g. mellonella have shown that the protein can occur as isoforms, differing in pi values (chino and yazawa, 1986; van der horst et al., 1991; wiesner et al., 1997; zdybicka-barabas and cytryńska, 2011). the apolp-iii molecule is formed by a bundle of five antiparallel amphipathic α-helices organized in an up-and-down topology, which are connected by short hinged loop regions (breiter et al., 1991). this bundle motif is a stable arrangement of the protein, which allows it to exist in hemolymph. a majority of residues in the molecule interior are hydrophobic, while hydrophilic residues are exposed to the aqueous environment of hemolymph. although the degree of amino acid sequence identity of apolpiiis from evolutionally divergent species is low, the three-dimensional structure of these proteins in their lipid-free state shows a striking similarity in molecular architecture, which is connected with the physiological functions of apolp-iii. physiologically, the protein occurs in a lipid-free or lipid-bound state that readily converts from one to the other depending on the metabolic setting. the low intrinsic stability of the helix bundle in the lipid-free state probably facilitates interaction with lipid surfaces. during the formation of the complex between apolp-iii and lipid, considerable conformational changes in the protein were observed, i.e., helix-helix interactions were replaced by helix-lipid interactions in the lipid-bound open conformation (wientzek et al., 1994; raussens et al., 1995). the lipid-bound state is the active form of 59 table 1 properties of apolipophorin iii of selected insect species molecular mass (a,b) (da) number of residues pi (a,b)/ isoforms reference lepidoptera acherontia atropos 17247 b 20 kdaa 161 surholt et al., 1988 bombyx mori 18420 18378b 164 8.04 b yamauchi et al., 2000 diatraea grandiosella 17964b 165 6.8a burks et al., 1992 galleria mellonella 18075.5 a 18075b 163 6.38b 6.5a 5.9a 6.1a weise et al., 1998 zdybicka-barabas and cytryńska, 2011 heliothis virescens 18 kda a 17965.9a chung and ourth, 2002 hyalophora cecropia 18 kdaa telfer et al., 1991 hyphantria cunea 18.3 kda a 18344b 165 6.23 b kim et al., 2004 manduca sexta 18364 18380b 166 5.88 b kawooya et al., 1984 spodoptera exigua 16523b 149 6.25b rizwan-ul-haq et al., 2011 spodoptera litura 18.3 kdaa 166 kim et al., 1998 thitarodes pui 18606a 171 5.61b sun et al., 2012 orthoptera acheta domesticus 17248 b 17.2 kdaa 161 smith et al., 1994 strobel et al., 1990 locusta migratoria 20488a (glc) 17583a (non-glc) 17327b 162-164 5.35a /5.43a 5.10ba/5.11a 4.98a van der horst et al., 1984; 1991 weers et al., 2000 chino and yazawa, 1986 coleoptera derobrachus geminatus 18039 b 18 kdaa 165 smith et al., 1994 diptera anopheles gambiae 19247b 170 4.82b gupta et al., 2010 a molecular mass and isoelectric point (pi) determined empirically; b theoretical molecular mass and pi; glc – glycosylated; non-glc – non-glycosylated the protein and occurs when apolp-iii associates with lipid-enriched lipophorins. it has been demonstrated that interaction of apolp-iii with model phospholipid vesicles, composed of dimyristoylphosphatidylcholine (dmpc), transforms them into discoidal particles surrounded by apolp-iii (weers et al., 1999, 2000; weers and ryan, 2003; vasques et al., 2009; narayanaswami et al., 2010; wan et al., 2011). the interaction of the protein with membrane lipid bilayers does not affect their permeability and occurs via polar and/or electrostatic forces at the bilayer surface without penetration of the hydrophobic core of the bilayer (zhang et al., 1993; sahoo et al., 1998). the packing defects in the phospholipid bilayer generate sites of apolp-iii binding, which was reported for various dmpc and sphingomyelin (sm) vesicles (chiu et al., 2009). it is known that lipid binding involves the hydrophobic surface of the helix bundle. upon lipid binding, apolp-iii molecule undergoes conformational changes and its hydrophobic interior is exposed to the lipid environment, which allows hydrophobic amino acid side-chains to gain direct access to the lipid surface (wientzek et al., 1994; garda et al., 2002; sahoo et al., 2002). a plausible model of the interactions of apolp-iii with model phospholipid particles is based on reorientation in the bundle of α-helices, i.e., three 60 of them move away from the two others (breiter et al., 1991; ryan and van der horst, 2000; van der horst et al., 2001, 2002). it has been suggested that the lipid binding is initiated at one end of the helix bundle. different models have been proposed for description of the initial binding steps. one model has been suggested for l. migratoria apolp-iii by breiter et al. (1991), where directed helix-lipid contact is made possible by conformational opening of the bundle involving ‘hinged’ loops connecting helices 2 and 3 and helices 4 and 5. the resulting exposure of its interior permits formation of a stable interaction with hydrophobic patches on lipophorin particles which appear as a function of dag enrichment. in turn, kawooya et al. (1986) have proposed that m. sexta apolp-iii recognizes potential lipid surface binding sites via one of its end and then breiter et al. (1991) have shown that loop a connecting helices 1 and 2 and loop c between helices 3 and 4 play this role. furthermore, in the structure of apolp-iii of l. migratoria, m. sexta, and thitarodes pui short additional helices (called 4’ and 3’) located at one end of the α-helix bundle are present. they adapt an orientation nearly perpendicular to the long axis of the bundle (wang et al., 1997; narayanaswami et al., 1999; fan et al., 2003; sun et al., 2012). because they are exposed to the solvent, it has been suggested that these short helices play a critical role in initiating the lipid binding with apolp-iii. it is believed that repositioning of the first and last helices in the molecule is an essential step in the binding process of apolp-iii, facilitating further opening of the hydrophobic helix bundle interior and separation of all helices from each other, which allows the protein to spread out on the lipid surface (narayanaswami et al., 1996; garda et al., 2002; sahoo et al., 2002). it has been demonstrated that apolp-iii of m. sexta, l. migratoria and g. mellonella were unable to bind lipid surfaces when helix 1 and helix 5 were tethered by a disulfide bond (garda et al., 2002; sahoo et al., 2002; leon et al., 2006a). based on spectroscopic analyses, raussens et al. (1995) inferred that the helices of m. sexta apolp-iii in the lipid-bound state are oriented perpendicularly to fatty acyl chains. apolp-iii shares similarities in the structure and in the mechanism of lipid binding with the n-terminal domain of human apolipoprotein e (apoe) and apolipoprotein a-i (apoa-i). the structure of the 22 kda n-terminal domain in apoe3 in the lipid-free state comprises four amphipathic α-helices with buried hydrophobic residues and exposed hydrophilic residues (aggerbeck et al., 1988; wilson et al., 1991). this domain can alter its conformation in a manner similar to apolp-iii on the lipoprotein particle. like the human apolipoproteins, apolp-iii forms discoidal complexes with phospholipid liposomes in which extended helices of the protein are wrapped around nanodiscs (saito et al., 2004; hatters et al., 2006; davidson et al., 2007; narayanaswami et al., 2010). involvement of apolipophorin iii in insects immunity the level of apolp-iii in hemolymph of immunechallenged insects, e.g., g. mellonella, heliothis virescens, plutella xylostella, undergoes changes, depending on the insect species and the pathogen/parasite, which indicates participation of apolp-iii in immune response against microbial infections (chung and ourth, 2002; song et al., 2008; zdybicka-barabas and cytryńska, 2011). it has been postulated that during an early step of insect immune response an interaction of apolp-iii with lipids occurs and that apolp-iii in the lipidbound state is involved in insect immunity (wiesner et al., 1997; dettloff et al., 2001a,b). a relationship between the lipid transport and immune function of apolp-iii has been shown in crickets. it appeared that the two processes compete for the protein, as after flight, the crickets became less able to fight bacterial infection (adamo et al., 2008). recognition of non-self proper recognition of invading pathogens by the immune system is a necessary prerequisite for activation and mounting of effective humoral as well as cellular immune response. it has been documented that apolp-iii binds microbial cell wall components, such as gram-negative bacteria lipopolysaccharide (lps), gram-positive bacteria lipoteichoic acids (lta), and fungal β-1,3-glucan (dunphy and halwani, 1997; halwani et al., 2000; pratt and weers, 2004; whitten et al., 2004; leon et al., 2006a, b; ma et al., 2006). due to this property, apolp-iii is considered as a pathogen recognition receptor (prr). in their work, halwani et al. (2000) reported on interaction of g. mellonella apolp-iii with ltas of bacillus subtilis, enterococcus hirae, and streptococcus pyogenes. they also demonstrated that e. hirae lta promoted binding of apolp-iii to e. hirae cells. in addition, binding of ltas by apolp-iii prevented loss of plasmatocytes caused by b. subtilis ltas in g. mellonella larvae. our recent study has demonstrated binding of g. mellonella apolp-iii to different gram-positive and gramnegative bacteria. the results suggested that, in addition to ltas, apolp-iii interacted with other cell surface components of gram-positive bacteria, since it bound to some of the tested bacteria lacking ltas in their cell walls, e.g., b. circulans (zdybickabarabas and cytryńska, 2011). lipid a and the carbohydrate part of the lps molecule are involved in interaction with apolp-iii. the binding causes conformational changes in the apolp-iii molecule, leading to rearranging and opening of the bundle of α-helices, similar to binding to lipid surfaces upon interaction with lipoprotein complexes (leon et al., 2006a, b). recently, a model of interaction between g. mellonella apolp-iii and e. coli lps aggregates has been proposed. apolp-iii disaggregated lps by forming proteinlps complexes. according to this model, the initial step is binding of apolp-iii to the lps carbohydrate moiety through ionic interactions. the second step, leading to strong lipid a binding, is likely to be driven by hydrophobic interaction. it has been calculated that four apolp-iii molecules can interact with 24 molecules of e. coli lps. analysis of similar complexes formed between apolp-iii and k. pneumoniae lps revealed that the complexes 61 contained three apolp-iii and nine lps molecules (oztug et al., 2012). whitten et al. (2004) demonstrated binding of apolp-iii to β-1,3-glucan, a fungal cell wall component. moreover, they showed that the survival rate of g. mellonella larvae injected with conidia of entomopathogenic fungus metarhizium anisopliae coated by apolp-iii was higher in comparison with that one in larvae challenged by non-coated conidia, indicating a protective role of apolp-iii against fungal infection. binding of g. mellonella apolp-iii to the cell surface of different yeasts and conidia of filamentous fungi has also been demonstrated (zdybicka-barabas et al., 2012). an analysis of in vitro treatment of candida albicans, zygosaccharomyces marxianus, and fusarium oxysporum with apolp-iii revealed morphological and metabolic activity changes, suggesting a role of this protein not only in fungi recognition but also in antifungal activity of hemolymph. apolp-iii as a signaling molecule and its role in antimicrobial activity induction as reported by dettloff et al. (2001a), shortly after injection into g. mellonella hemocoel, biotinylated apolp-iii was detected in the immunecompetent hemocytes, suggesting functioning of apolp-iii as a signaling molecule in insect hemolymph. in accordance with this finding, g. mellonella bacterial challenge led to formation of apolp-iii-lipid complexes, assembled into ldlp which were taken up by granulocytes (dettloff et al., 2001b; niere et al., 2001). it was reported that apolp-iii potentiated antimicrobial activity in insect hemolymph. an injection of apolp-iii into the hemocoel of g. mellonella larvae led to an increase in hemolymph lysozyme and anti-e. coli activity, similar to bacterial challenge (wiesner et al., 1997; halwani and dunphy, 1999; niere et al., 1999). the constitutive presence of apolp-iii in hemolymph and the fact that intrahemocoelic injection of apolp-iii (recombinant or purified from insects) resulted in a negligible increase in the soluble apolp-iii fraction in hemolymph, ruled out hormoneor cytokine related activity of the protein. evidence provided by dettloff et al. (2001b) for ldlp formation after bacterial challenge and uptake of lipid-bound apolp-iii by the hemocytes, indicated that activation of the immune system by apolp-iii might be rather connected with conformational changes and an increase in the lipid-bound fraction of the protein in hemolymph. intensification of hemolymph antimicrobial activity after apolp-iii injection could result from activation of antimicrobial gene expression, which was demonstrated in apolp-iiichallenged hyphantria cunea. in this insect, apolpiii injection induced the expression of lysozyme and cecropin-like peptides (hyphancins). in addition, apolp-iii was detected in h. cunea granulocytes which underwent degranulation and degradation upon e. coli immune challenge. the authors postulated a relationship between a local discharge of apolp-iii from the granulocytes in response to bacterial challenge and activation of immune response (kim et al., 2004). the existence of immune signals upstream of cell-bound receptors has been postulated by rahman et al. (2006). they found that lipophorin particles mediated recognition and inactivation of lps and bacteria in immune-challenged flour moth ephestia kuehniella larvae. moreover, an association of pattern recognition receptors, lectins as well as regulatory proteins activating prophenoloxidase with sub-population of lipophorin particles has been demonstrated (rahman et al., 2006). detoxification of non-self components the ability of apolp-iii to bind microbial cell wall components, e.g., lps, implies participation of the protein in detoxification processes. dunphy and halwani (1997) demonstrated that g. mellonella apolp-iii bound to lps isolated from the outer membrane of insect pathogenic bacteria xenorhabdus nematophilus, which reduced the lps toxicity and prevented g. mellonella hemocyte damage. the role of lipophorins, in which apolp-iii is an exchangeable component, in lps detoxification was suggested by kato et al. (1994a, b) in their study on bombyx mori. the results indicated that formation of the lipophorin-lps complex in b. mori hemolymph, similar to the lipoprotein-lps complex in mammalian serum, caused a striking decrease in lps biological activity, reflected by significant reduction in cecropin gene inducibility. as demonstrated by ma et al. (2006), antibodies against lps-binding proteins, such as immulectin-2, cross-reacted with proteins associated with purified lipophorin particles formed in g. mellonella hemolymph in vitro upon lps addition. the results also indicated that lipophorin particles responded to lps by forming insoluble aggregates sequestering lps into non-toxic complexes. apolp-iii can also be considered as an ltaneutralizing protein, since binding of g. mellonella apolp-iii to b. subtilis ltas prevented loss of plasmatocytes caused by lta, indicating protection of the insect against the toxin (halwani et al., 2000). antimicrobial activity of apolp-iii and synergistic action with defense peptides our study has revealed that g. mellonella apolp-iii exhibits antibacterial activity against certain gram-positive and gram-negative bacteria. among the most susceptible bacteria were salmonella typhimurium, k. pneumoniae, b. circulans, and listeria monocytogenes (zdybickabarabas and cytryńska, 2011). interestingly, apolp-iii also inhibited significantly growth of legionella dumoffii cultured in a medium supplemented with choline (palusinska-szysz et al., 2012). afm imaging and analysis of apolp-iiitreated bacteria revealed considerable alterations of the structure and nanomechanical properties of the cell surface, e.g. roughness, elasticity, and adhesion (fig. 2) (zdybicka-barabas et al., 2011). the results underline the important role of apolpiii in insect antibacterial defense, similarly to the role demonstrated for mammalian apoe in immune 62 fig. 2 afm analysis of k. pneumoniae cell surface alterations after treatment with g. mellonella apolp-iii. three dimensional (a), amplitude (b), and topography (c) images are presented. section profiles corresponding to lines in (c) are demonstrated in (d). 63 response against k. pneumoniae and l. monocytogenes (roselaar and daugherty, 1998; bont et al., 1999). in g. mellonella, it was demonstrated that apolp-iii acted synergistically with other defense proteins and peptides against bacteria. synergistic action of apolp-iii and lysozyme against m. lysodeikticus was suggested by halwani and dunphy (1999) on the basis of experiments on apolp-iii and ewl. in addition, they demonstrated an increase in hydrophobicity and the negative charge of the bacterial cells treated with apolp-iii, which could, to some extent, explain why apolp-iii enhances the antibacterial activity of cationic defense proteins and peptides. recently, we have presented evidence for increasing g. mellonella lysozyme muramidase activity in the presence of apolp-iii, leading to an increase in lysozyme perforating activity of the e. coli cell membrane. our research indicated that three defense factors present constitutively in g. mellonella hemolymph, namely apolp-iii, lysozyme, and anionic peptide 2, act in synergy against bacteria. apolp-iii increases the enzymatic (muramidase) activity of lysozyme, whereas anionic peptide 2 seems to stimulate the non-enzymatic lysozyme activity (zdybicka-barabas et al., 2012, 2013). moreover, an increase in cecropin a anti-e. coli activity in the presence of apolp-iii has been demonstrated (park et al., 2005). in addition to enhancing antibacterial activity of other defense proteins and peptides, involvement of apolp-iii in regulation of phenoloxidase activity in g. mellonella has been reported; however a possible mechanism of this phenomenon has not been explained yet (halwani et al., 2000; park et al., 2005). recently, gupta et al. (2010) have reported on apolp-iii participation in midgut immune defense of anopheles gambiae against plasmodium berghei. in a. gambiae g3 females, invasion of p. berghei ookinetes triggered a strong transcriptional activation of apolp-iii in the midgut epithelial cells. expression of apolp-iii in these cells stimulated antiplasmodial response, while silencing of apolp-iii by systemic injection of dsrna greatly increased plasmodium infection. contribution of apolp-i and apolp-ii, in addition to apolp-iii, in insect defense mechanisms against pathogens has been recently suggested (hanada et al., 2011). it has been found that apolp, consisting of apolp-i and apolp-ii, of the b. mori silkworms’ hemolymph is involved in resistance against staphylococcus aureus infection by suppressing the expression of virulence genes encoding αand βhemolysin. moreover, apolp also decreased expression of saers and rnaiii, important for activation of these hemolysin genes. it is possible that apolp-i and apolp-ii, together with apolp-iii, function in coordinated antimicrobial defense in insects. role of apolp-iii in cellular immune response analysis of the properties of g. mellonella hemocytes after treatment with apolp-iii in vitro and after injection of apolp-iii into larval hemocoel revealed impaired adhesion of plasmatocytes and subpopulation of granulocytes to glass slides (zakarian et al., 2002; whitten et al., 2004). since the ability to adhere to and spread on non-self surfaces is essential for hemocytes involved in cellular immune response, the finding suggested a role of apolp-iii in this arm of insect immunity. in the same study, delayed removal of apolp-iii-coated b. subtilis cells from the hemolymph was reported. given the reduced hemocyte adhesion upon apolpiii treatment, the authors postulated that apolp-iii may down-regulate nodule formation and/or phagocytosis (zakarian et al., 2002). on the other hand, binding of apolp-iii to yeast cells (s. cerevisiae) enhanced the phagocytic activity of g. mellonella hemocytes in vitro, suggesting importance of apolp-iii opsonizing activity for effective clearance of the invaders (wiesner et al., 1997). similarly, the findings described by whitten et al. (2004) presenting more effective in vivo nodule formation in larvae injected with apolp-iii could point towards a stimulating role of apolp-iii in cellular response. in support of this idea are the results presented by son and kim (2011) on the role of apolp-iii in activation of cellular response in diamondback moth, p. xylostella. knockdown of apolp-iii expression by rna interference caused a significant decrease in the apolp-iii level and resulted in considerable suppression of hemocyte nodule formation in response to bacterial challenge. injection of recombinant apolp-iii to p. xylostella larvae parasitized by an endoparasitic wasp cotesia plutellae restored the hemocyte activity. in addition, apolp-iii reduced pathogenicity of entomopathogenic bacteria x. nematophila toward p. xylostella larvae (son and kim, 2011). although the exact role of apolp-iii in cellular immune response is difficult to define clearly on the basis of available data, it seems that the effect of the protein activity depends on the pathogen. involvement in clot formation clot formation can be considered as an integral part of insect immune response, because in addition to sealing wounds and limiting loss of body fluids, a clot entraps microbes at the wound site, thereby preventing invading the hemocoel. moreover, upon activation of the po system, the entrapped pathogens can be more easily killed and eliminated. lipophorins have been identified as a common clotting factor in several insect species, e.g. g. mellonella, tenebrio molitor, l. migratoria, periplaneta americana, and leucophaea maderae (duvic and brehélin, 1998; altincicek et al., 2008; dushay, 2009). the presence of apolp-i and apolp-i/ii in the anopheles gambiae and drosophila melanogaster clots, respectively, has also been reported (scherfer et al., 2004; agianian et al., 2007). proteomic analysis revealed that apolp-iii, together with other apolipoproteins, was a component of g. mellonella net-like coagulation structures containing endogenous extracellular nucleic acids. moreover, apolp-iii was detected among specific rna-binding proteins, suggesting its role in extracellular rna-mediated immune response (altincicek et al., 2008). 64 apolipophorin iii and entomopathogens one of the strategies developed by entomopathogenic organisms to cope with the host immune system is decreasing of the apolp-iii level. reduction of the protein level is achieved by suppression of apolp-iii expression, which was described in p. xylostella parasitized by the entomopathogenic wasp c. plutellae (son and kim, 2011). another way involves proteolytic degradation of apolp-iii by extracellular proteinases produced by entomopathogenic bacteria during infection, e.g. pseudomonas aeruginosa elastase b and protease iv in infected g. mellonella larvae (andrejko et al., 2005, 2008, 2013; andrejko and mizerska-dudka, 2012). a very interesting strategy developed by entomopathogenic nematodes steinernema feltiae infecting g. mellonella larvae was reported by brivio et al. (2005, 2010). hemolymph of the infected larvae was depleted of humoral immune factors which were attracted by and adsorbed to specific nematode surface molecules. among proteins removed in this way from the insect hemolymph, a lipopolysaccharide binding protein (lbp), a peptidoglycan recognition protein lb (pgrp-lb), gloverin-like peptide and apolp-iii were identified. removal of the immune factors, including apolp-iii, from the insect hemolymph by s. feltiae seems to protect nematode symbiotic bacteria x. nematophila, which kill the insect host and establish suitable conditions for reproduction of the nematodes (brivio et al., 2005, 2010). summary apolipophorin iii is a multifunctional insect protein involved in lipid transport and immune response. in addition, its role during programmed cell death has been described in m. sexta skeletal muscles and neurons (sun et al., 1995). in insect immune response apolp-iii serves as a pattern recognition molecule. it binds and detoxifies microbial cell wall components, i.e. lps, lta, and β-1,3-glucan. apolp-iii activates expression of antimicrobial peptides and proteins, stimulates their antimicrobial activity, and participates in regulation of phenoloxidase activity in insect hemolymph. in addition, the protein is involved in cellular immune response, influencing hemocyte adhesion, phagocytosis and nodule formation, and in gut immunity. reduction of the apolp-iii level by entomopathogens through e.g., suppression of apolp-iii expression and/or degradation of the protein by entomopathogen proteases seems to be a common strategy to avoid host immune response and indicates that apolp-iii is an important component of insect immunity. although apolp-iii is the best studied apolipophorin in insect immunity so far, a literature review suggests that all the three apolipoproteins, apolp-i, apolp-ii and apolp-iii, function together in a coordinated defense against pathogens. acknowledgements the authors would like to thank 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505-514, 2002. sun d, ziegler r, milligan ce, fahrbach s, schwartz lm. apolipophorin iii is dramatically up-regulated during the programmed death of insect skeletal muscle and neurons. j. neurobiol. 26: 119-129, 1995. weers pmm, narayanaswami v, kay cm, ryan ro. interaction of an exchangeable apolipoprotein with phospholipid vesicles and lipoprotein particles. j. biol. chem. 274: 21804-21810, 1999. minireview isj 10: 84-93, 2013 issn 1824-307x minireview transcriptomic response to stress in marine bivalves q li, x zhao, l kong, h yu college of fisheries, ocean university of china, qingdao 266003, china accepted september 25, 2013 abstract marine bivalves have a set of unique capabilities to adapt to the complicated conditions owing to their habitats, living habits and feeding ways. meanwhile, marine bivalves can be the biosensors to monitor the quality of the intertidal zones or other habitats. it is interesting for every biologist to find out the mechanisms by which organisms adapt to environmental challenges and the factors limiting their adaptive capacities. the development of biotechnology over the past few decades has provided biologists with a vast repertoire of biosensors that allow testing mrna expression in response to environmental factors. this minireview is focused on the transcriptomic responses to abiotic and biotic stressors in bivalves and the relative methods to provide new perspectives as well as improve applications for bivalve biomonitoring studies. key words: transcriptome; stress; bivalve introduction marine bivalves are an important component of the ecosystem and biodiversity (dame, 2011), which have abundant species distributed worldwide from the intertidal zones to hydrothermal vents and cold seeps (bettencourt et al., 2010; boutet et al., 2011; egas et al., 2012). bivalve cultivation is one of the most important aquaculture industries globally (forrest et al., 2009; pawiro 2010). furthermore, marine bivalves possess the unique adaptation to problematic surroundings. in light of the important status of marine bivalves in ecosystem and economy and their high adaptations, they are the valuable organisms to be investigated about their molecular mechanism responding to the variable environment. it is also meaningful to find relative gene expression index in marine bivalves to be the monitoring standard of the surroundings. owing to no model organism in marine bivalves and the repetitive organization of the non-coding fraction in their genome, as well as their size, the development of the genome and transcriptome of them make a slow progress. thanks to the technical advance, marine bivalves have acquired growing concerns and their genomic databases have enriched increasingly, such as mytibase (http://mussel.cribi.unipd.it) for mytilus galloprovincialis and deepseavent ___________________________________________________________________________ corresponding author: qi li college of fisheries ocean university of china qingdao 266003, china e-mail: qili66@ouc.edu.cn (http://transcriptomics.biocant.pt:8080/deepseavent) for bathymodiolus azoricus. marine bivalves commonly inhabit variable and unstable conditions and most are the crisscross between anthropic zone and the nature. complex ecological habitats bring about all kinds of stresses in the lives of bivalves and determine their distributions and abundance. these stresses are mostly from some reasons as follows. firstly, intertidal zone that is one of the most important habitats for bivalves experiences large and sometimes rapid fluctuations caused by tidal fluctuations, rain and freshwater run-off (shumway, 1977). owing to this, intertidal bivalves are mostly exposed to multiple stressors including periodic hypoxia, hyposaline, temperature fluctuations and pollution (ivanina et al., 2012). meanwhile, in the recent past, anthropogenic inputs of contaminants, and combustion of fossil fuels and deposition of metals make the condition further complicated (doney, 2010). furthermore, some important cultured bivalve species, such as pacific oyster and blue mussel, are subject to the “summer mortality” (tremblay et al., 1998a; tremblay et al., 1998b; xiao et al., 2005; samain et al., 2007; lynch et al., 2012). summer mortality has been reported to occur during the summer months in several countries (myrand and gaudreault, 1995; cotter et al., 2010; fleury and huvet, 2012). this phenomenon is multifactorial, resulting from a complex interaction between organisms, environment and pathogens (samain et al., 2008).   84 secondly, climate changes will affect temperature extremes and averages, and hyposaline conditions in coastal areas due to extreme precipitation events and oceanic ph (tomanek, 2012). meanwhile, the changes of the environment can affect the distribution and abundance of the native species (johnson et al., 2011), even lead to change the competitive interactions between invasive and native species (lockwood et al., 2010; lockwood et al., 2011). lastly, it puts down to their living habits and feeding ways of themselves. most bivalves are filterfeeding and their mobility is not great. they cannot escape the stresses by moving away quickly and have to adjust to the changing surroundings other than swimming organisms. it is more valuable to decipher the mechanisms of their unique capacities to adjust to the variable environment. there are kinds of reasons to induce the responses of the marine bivalves, and the factors involved in the stresses have multiple modes of interaction. responses to the environmental stresses on the transcriptomic level are complicated and hard to explain by single gene or pathway. actually, even against the single stress, there are a lot of genes participated and the complex networks between genes and pathways. here we provide an overview about the transcriptomic responses to abiotic and biotic stressors in marine bivalves and the development of the relative technological methods to provide promising perspectives for a better comprehension of the mechanisms. abiotic stress responses temperature temperature has been shown to be one of the most important determinants of survival, growth and reproduction (helmuth et al., 2006; menge et al., 2008). understanding the underlying mechanisms by temperature driving organismal responses and physiological performance is becoming increasingly imperative as climate change alters habitat temperature (somero, 2010). meanwhile, sessile inhabitants of marine intertidal environments commonly face heat stress which is an important incentive in “summer mortality” (tremblay et al., 1998a; soletchnik et al., 2005). the largest changes in gene expression in response to heat-stress were among genes that encoded the molecular chaperone heat shock protein 70 (hsp70) and his family (lang et al., 2009; lockwood et al., 2010; chapman et al., 2011). the increased expression of molecular chaperones is a primary component of the cellular stress response and a key indicator of environment stress (lockwood et al., 2010). hsps, especially hsp70 and hsp90, can protect cells and organisms from thermal damages (zhao and jones, 2012) and are well known as molecular chaperones that help in the refolding of misfolded proteins and assist in their elimination if they become irreversibly damaged (zhao and jones, 2012). peroxinectin is upregulated during exposure to elevated temperature, which is reported in oysters (lang et al., 2009; chapman et al., 2011). peroxinectin may perform adhesive and defensive functions (lang et al., 2009), owing to higher temperature negatively impacting resistance to bacteria (chapman et al., 2011). transcriptomic responses to heat stress are, in part, characterized by the up-regulation at 32 oc of genes involved in proteolysis, which was specific to ubiquitin-mediated proteolysis in mytilus trossulus (lockwood et al., 2010). proteolysis is the directed degradation of proteins and in the context of environmental stress serves to degrade and remove permanently denatured proteins, an indicator of severe cellular stress (dahlhoff, 2004; kültz, 2005). heat shock can also affect the genes related to growth and reproduction. the relative genes such as suppressor of cytokine signaling-2, collagen, were up-regulated during heat stress (lang et al., 2009). salinity in intertidal zones, the salinity of seawater can strongly vary from nearly fresh water to highly saline (meng et al., 2013). some bivalves, such as the pacific oysters, are able to survive in a range of salinity from 10‰ to 35‰ (pauley et al., 1988). for mussels, the salinity may determine the outcome of competition between native and invasive mussels (lockwood et al., 2011). during hypo-osmotic shock, all reported changes in gene expression mainly focus on osmoregulation and osmotic stress signaling (lockwood et al., 2011; zhao et al., 2012; meng et al., 2013). osmoregulation influences the upregulation of genes encoding ion channel (lockwood et al., 2011; zhao et al., 2012; meng et al., 2013) and free amino acid (faa) metabolic key enzyme (meng et al., 2013), as well as the downregulation of the ion and amino acid transporter genes (lockwood et al., 2011). salt stress signal transduction pathways including calcium signaling cascade and phosphorylation regulation were observed to be up-regulated (zhao et al., 2012; meng et al., 2013). furthermore, faa metabolic pathways (including those for glycine, alanine, betaalanine, arginine, proline, and taurine) were activated, altering the osmotic status in the oysters (meng et al., 2013). in the study of mussels, ornithine decarboxylase, serving as the key regulatory enzyme in polyamine synthesis was highlighted, owing to different expression between two mussels, which regulated cell growth and cell viability and whose disruption can cause cancer or apoptosis (lockwood et al., 2011). moreover, many other types of metabolism, including immune responses and apoptosis, were shown to be enriched in the kegg pathway analysis. metals and chemicals most species of marine bivalves have a long history as sentinel organisms for monitoring the status of marine ecosystems (dondero et al., 2006; hines et al., 2007; milan et al., 2011; varotto et al., 2013). heavy metals including cu2+, cd2+, hg2+, ni2+, participate in the biological cycles of different groups of organisms (zapata et al., 2009), affecting their distribution and abundance (kudo et al., 1996). in previous studies, copper gives rise to the differentially expressed genes involved in the respiratory chain and stress response, development   85 and differentiation, cytoskeleton in chilean scallop (zapata et al., 2009). metallothionein is the most well-known antioxidant that protects against metal toxicity, and was verified in marine bivalves (venier et al., 2006; dondero et al., 2011; varotto et al., 2013). moreover, nickle modulated proliferation, growth and apoptosis as the same as copper, and highly regulated the genes encoding lipid metabolism (dondero et al., 2011). organic contaminants elicited defensin c and the defender against cell death 1 gene (whose defective function causes apoptotic death) that were under-represented in mussels other than from heavy metals (venier et al., 2006). similarly, in mussels, chlorpyrifos decreased in acetylcholinesterase activity in the gills markedly, and up-regulated the chitinase activity, which play a role in digestion and participate in the innate immune response (dondero et al., 2011). in addition, studies focused on the responses of mytilus edulis to benzo[α]pyrene found that organic substance mostly disrupt the cellular redox status (brown et al., 2006). notably, gene expression levels primarily depend on the functional specificity of cells composing different organs and tissues (venier et al., 2006). so that, to different tissues, the transcriptional responses to the pollution are specific. hypoxia oxygen deficiency is a common stressor in estuarine and coastal environments. intertidal molluscs are among the animal champions of hypoxia tolerance owing to a suite of metabolic adaptions that allows them to survive prolonged periods without oxygen. they adapt to the fluctuation of oxygen by changing energy management and resource utilizations. in crassostrea gigas, which exposed to hypoxia, an overall transcriptome study indicated many genes coding for enzymes involved in antioxidant defense and reactive oxygen species detoxification for the cellular redox balance except for genes related to stress exhibited over-expressed (sussarellu et al., 2010). meanwhile, some literatures were to characterize the some genes in bivalves and to define their potential regulation in the hypoxic response. hypoxia-inducible factor-1 (hf-1), as the key regulator of oxygen homeostasis in aerobic organisms under hypoxia, had been verified to play a critical role in reactive oxygen species (ros) production of hemocytes in c. gigas (choi et al., 2013). and likewise, amp-activated protein kinase α (ampkα) was showed to participate in the metabolic response during hypoxia in the smooth muscle of c. gigas (guévélou et al., 2013). however, crassostrea virginica has both a better tissue aerobic capacity to compensate for reduced oxygen availability and a lower sensitivity to hypoxia than c. gigas, with a compensatory increase in activities of citrate synthase and cytochrome c oxidase after 2 weeks of hypoxia (ivanina et al., 2011). other abiotic stresses the different conditions of different vertical locations in intertidal zone depend on the tides. the tidal fluctuations changed many environmental factors such as temperature and food availability of the surroundings. researchers caught this natural phenomenon and showed that low intertidal mussels altered their physiology very little with respect to the tide cycle, and mid-intertidal and high intertidal mussels reduced the gene expressions involved in metabolic processes (place et al., 2012). especially, in high intertidal zones, pathways associated with protein rescue, cellular repair and protein degradation and oxidative stress were activated (place et al., 2012). biotic stresses biotic stress mainly refers to the stress that occurs as a result of damage to plants and animals by other living organisms such as bacteria, viruses, parasites and microalgae. marine bivalves harbor an abundant and diverse microflora on their surface or inside their tissues. with evolution, marine bivalves have developed effective systems for maintaining their homeostasis and for controlling potentially harmful and pathogenic microorganisms and microalgae. the current literature shows that bivalves eliminate or limit the development of the microorganisms through different innate immune response in combination with other cellular mechanism, such as the apoptotic pathway by transcriptomic analysis (wang et al., 2010; de lorgeril et al., 2011; morga et al., 2011; brulle et al., 2012; moreira et al., 2012a; moreira et al., 2012b). in flat oyster (ostrea edulis), fas-ligand that was involved in the immune response against the parasite bonamia ostreae was observed upregulated. meanwhile, according to the previous results, fas-ligand is also associated in the apoptosis pathway with the inhibitors of apoptosis proteins (morga et al., 2011). furthermore, apoptosis as autophagy can be triggered by reactive oxygen species (ros), and up-regulated genes are related to respiratory chain and particularly in ros production (wang et al., 2010; de lorgeril et al., 2011). above all, the apoptosis pathway is the most important response to the biotic stresses. in addition to the apoptosis pathway, other immune-related genes were reported, such as ferritin and lysozyme, several immune pathways and processes including the toll-like signaling pathway and the complement cascade (wang et al., 2010; de lorgeril et al., 2011; moreira et al., 2012). except for the immune response to the microorganisms, some bivalves exposed to vibrio spp. (gestal et al., 2007; brulle et al., 2012; moreira et al., 2012b), parasite including bonamia (martíngómez et al., 2012) and perkinsus marinus (tanguy et al., 2004) were elicited that such cytotoxic response led to the rearrangement of the cytoskeleton. cytoskeleton is important in phagocytosis, and all phagocytosis processes are driven by rearrangement of the actin cytoskeleton (may et al., 2001). besides the microorganisms, dinoflagellates and other microalgae can produce a wide spectrum of toxic molecules. during seasonal harmful algae blooms (habs), many filter-feeding bivalves can   86 accumulate phycotoxins at extremely high levels, thus representing a serious threat to human health. until now, a few researches about the transcriptome of marine bivalve induced by harmful algae have been reported. manfrin et al., 2010 studied the molecular mechanism that m. galloprovincialis exposed to okadaic acid (oa) which is a lipophilic toxin. its results indicated that the effects of oa mostly concentrated in genes related to stress response, apoptosis and cell structure function (manfrin et al., 2010). these variations were also observed in the study that evaluated c. gigas hemocytes responses to purified pbtx-2 in vitro (mello et al., 2012). in both researches, there were no genes associated to immune or antioxidant, which needs more experiments to be tested and verified. interactive stresses previous researches tended to control a single variable to discuss the transcriptomic responses and acquire the master genes, because the single variable is easier to control under the laboratory conditions. however, the natural circumstances consist of multifactor and are multivariable other than the single variable. in addition, the effect of two stresses do not coincide with the effect of the mix including the two stresses (dondero et al., 2011). several studies focused on the transcriptomic responses to the environmental stresses owing to “summer mortality” (chaney et al., 2011; fleury et al., 2012) and interactions with environmental variables that induced changes in gene expression profiles and affected the fitness of organisms (chapman et al., 2009; chapman et al., 2011; philipp et al., 2012). beyond that, the hydrothermal vent mussel (b. azoricus) itself thrives in a condition with the darkness, extreme cold, high pressure and rich in methane and sulfides (egas et al., 2012) which is full of multi-stresses. summer mortality causes a serious influence to the aquaculture of bivalves, particularly oysters. environmental stress and pathogens are known to interact and lead to summer mortality outbreaks. differentially expressed genes associated with “immune response” biological process were significant up-regulated (chaney et al., 2011; fleury et al., 2012). moreover, genes of oysters associated with cell death and autopahgy would suggest that at least some proportion of genes is symptomatic that underwent “summer mortality” (chaney et al., 2011). in addition to abiotic stressors, pathogen is an important factor in inducing “summer mortality”. vibrio splendidus is associated with summer mortality of juvenile oysters (c. gigas) and make juvenile oysters reduce stress-response capacities (lacoste et al., 2001). similarly, the effects of vibrio may impair adult oyster immune defenses and cellular and immune functions that characterize the oyster capability to survive v. splendidus infections (de lorgeril et al., 2011). ostreid herpesvirus 1 (oshv-1) infections have been reported around the world and are associated with high mortalities of c. gigas in summer (segarra et al., 2010; dégremont 2011). oshv-1, same as v. splendidus, induced the oysters with significant changes in the expression of immune related genes (renault et al., 2011). the interesting studies by chapman et al. (2009, 2011) examined the transcriptomic responses of oysters to environmental stresses and land-use impacts, providing an extension of an earlier assessment of the relative gene expression patterns. response to environmental stressors, genes encoding electron transport chain are important discriminators for the levels of metals, organic pollutants and nutrients. in addition, a suite of genes involved in the regulation of cell volume and growth, energy metabolism and stresses can also be the indicators of the environmental quality (chapman et al., 2009, 2011). through environmental cluster analysis, the environmental ph and the temperature were by far the leading environmental factors governing gene expression patterns with minor contributions of salinity and dissolved oxygen (chapman et al., 2011). it is noteworthy that there is a strong negative correlation, suggesting genes that are upregulated by higher temperatures are also upregulated by lower ph and vice versa (chapman et al., 2011). the hydrothermal vent mussel survival in such extreme conditions requires unique anatomical and physiological adaptions. it has been reported that they rely on unique capabilities to detect and respond to micro associated molecular patterns such as lipopolysaccharides (lps), lipoteichoic acids, lipoproteins, peptidoglycan (pgn) and (1→3) β-d-glucans (bettencourt et al., 2010). under controlled hyperbaric pressure, genes of b. azoricus relative to heavy metal contaminants and oxidative stress differentially expressed, and the occurrence of glycosylation was changing with the elevanted hyperbaric pressure (bettencourt et al., 2011). furthermore, b. azoricus survives in reducing environments rich in methane and sulfides, owing to symbiotic association with methylotrophic or methanotrophic and thiotrophic bacteria (egas et al., 2012). enzymes involved in sulfur and methane oxidation have been found, but the molecular pathways underlying sulfur and methane oxidation within the hydrothermal vent mussel had no sufficient evidence (egas et al., 2012). at “sea-level” condition, b. azoricus can be used a model organism to explore more information. the main studies focused on the transcriptomic response to stress in marine bivalves are summarized in table 1. the transcriptomic approach suppression subtractive hybridization (ssh) ssh is a pcr-based technique that allows the identification of genes that differentially expressed between two conditions (diatchenko et al., 1996). since this technology came to being in 1984 (lamar et al., 1984), it has experienced several improvements to be more accurate and easier. until 1996, ssh application has been maturation (diatchenko et al., 1996; diatchenko et al., 1999). this application is a powerful tool for the study of differential gene expression and the identification of   87 table 1 main studies related to transcriptomic response to stress in marine bivalve study stress bivalve species strategy tanguy et al., 2004 perkinsus marinus crassostrea virginica and crassostrea gigas ssh and real-time pcr on heamocytes brown et al., 2006 benzo[a]pyrene mytilus edulis ssh and macroarray on digestive glands of control and experimentally contaminated mussels dondero et al., 2006 copper pollution gradient m. edulis microarray and real-time pcr on digestive gland of experimental contaminated mussels venier et al., 2006 metals and chemicals mytilus galloprovincialis microarray and real-time pcr on gills, digestive gland, muscles and mantle of naturally and experimentally contaminated mussels gestal et al., 2007 mix of dead bacteria ruditapes decussatus ssh on heamocytes of oysters exposed to the mix of dead bacteria masson et al., 2007 atrazine m. edulis monitoring the gene of dnak-type molecular chaperone chapman et al., 2009 land-use influences c. virginica microarray on gills and hepatopancreas of oysters lived in 11 creeks along the atlantic coast of the southeastern usa green et al., 2009 hypoxia saccostrea glomerata ssh on haemocytes, monitoring the expression of genes encoding anti-oxidant enzymes lang et al., 2009 heat stress c. gigas microarray and real-time pcr on gills of oysters exposed to high temperature prado-alvarez et al., 2009 perkinsus olseni r. decussatus ssh on heamocytes exposed to perkinsus olseni in different time point wang et al., 2009 vibrio alginolyticus pinctada fucata monitoring the expression of hsp 70 in the heamocytes of oysters responding to bacterial challenge zapata et al., 2009 copper pollution gradient argopecten purpuratus ssh and real-time pcr on post-larvae of scallop bettencourt et al., 2010 conditions of the hydrothermal vent field bathymodiolus azoricus rna-seq on gills dondero et al., 2010 nickel and chlorpyrifos m. galloprovincialis microarry and real-time pcr on digestive gland of mussels exposed to nickel, chlorpyrifos and the mix (nickel and chlorpyrifos) lockwood et al., 2010 osmotic stress m. galloprovincialis and mytilus trossulus microarray on gills of two species mussels sussarellu et al., 2010 hypoxia c. gigas microarray and real-time pcr on the digestive gland wang et al., 2010 perkinsus marinus c. virginica microarray and real-time pcr on gills bettencourt et al., 2011 hydrostatic pressure b. azoricus real-time pcr on selected genes of gills and mantles boutet et al., 2011 environmental factors of the hydrothermal vent field b. azoricus compare differentially expressed genes in gills of two groups, one is rich in methanotrophic bacteria and the other is rich in thiotrophic bacteria using ssh and microarray chaney et al., 2011 summer mortality c. gigas microarray on heamocytes of survival and mortal oysters chapman et al., 2011 environmental factors c. virginica microarray on gills and hepatopancreas of oysters lived in 11 creeks along the atlantic coast of the southeastern usa de lorgeril et al., 2011 vibrio spp. c. gigas dge analysis on heamocytes of oysters infected by virulent and avirulent strains fu et al., 2011 osmotic stress and bacterial challenge crassostrea hongkongensis monitoring the expression of hsp 90 in the heamocytes of oysters under stresses ivanina et al., 2011 cadmium and hypoxia c. virginica real-time pcr on selected genes of hepatopancreas lockwood et al., 2011 heat stress m. galloprovincialis and m. trossulus microarray on gills of two species mussels milan et al., 2011 temperature and salinity ruditapes philippinarum rna-seq and microarry on individuals of clams exposed to quick changes of temperature of salinity   88 brulle et al., 2012 vibrio tapetis r. philippinarum ssh and real-time pcr on heamocytes exposure to v.tapetis and v.splendidus egas et al., 2012 endosymbionts and free-living deep-sea bacteria b. azoricus rna-seq on gills fleury and huvet, 2012 summer mortality c. gigas microarray on muscle, gills, gonad of natural mussels in different time points martín-gómez et al., 2012 bonamia spp ostrea edulis ssh and real-time pcr on heamocytes exposed to bonamia spp mello et al., 2012 brevetoxin c. gigas real-time pcr on selected genes following heamocytes exposure to brevetoxin moreira et al., 2012a v. alginolyticus r. philippinarum and r. decussatus real-time pcr on selected genes following heamocytes exposure to vibrio moreira et al., 2012b vibrio anguillarum r. philippinarum rna-seq on heamocytes of clams exposed to alive and heat-inactivated vibrio morga et al., 2012 bonamia spp o. edulis ssh and real-time pcr on heamocytes place et al., 2012 pacific tides mytilus californianus microarray on gills, muscle and mantle of mussels inhabiting different vertical locations wang et al., 2012 v. alginolyticus pinctada martensii ssh and real-time pcr on heamocytes zhao et al., 2012 osmotic stress c. gigas rna-seq on gills of oysters exposed to different salinity gradient seawater choi et al., 2013 hypoxia c. gigas monitoring the effects of hypoxia-inducible factor-alpha on respiratory burst activity guevelou et al., 2013 hypoxia c. gigas monitoring the expression of amp-activated protein kinase α meng et al., 2013 osmotic stress c. gigas rna-seq on gills of oysters exposed to different salinity gradient seawater varotto et al., 2013 combined metal salts m. galloprovincialis microarray and real-time pcr on gills genes involved in specific biological functions, especially in organisms where genomic data are not available (zhang et al., 2001). in molluscs, most studies aim to identify the molecular basis of the most common pathologies reviewed in romero et al., 2012. some studies focused on several genes by ssh-cdna libraries, such as hsp90, ubiquitin gene response to osmotic stress and bacterial challenge and two catalase homologs response to bacterial infection and oxidative stress in crassostrea hongkongensis (fu et al., 2011; zhang et al., 2011), a dnak-type molecular chaperone exposed to atrazine in blue mussels (masson et al., 2007), and hsp70 in the haemocytes of pearl oyster responding to bacterial challenge (wang et al., 2009). some studies investigated genes of the same categories, and mostly aimed at immune-related genes (xu et al., 2009; martín-gómez et al., 2012; wang et al., 2012; gestal et al., 2007) and anti-oxidant genes (green et al., 2009). with the increase of the est databases in marine bivalves, studies containing more coverage of transcripts were undertaken, involved transciptome in response to parasites (tanguy et al., 2004; prado-alvarez et al., 2009; morga et al., 2011), virus (brulle et al., 2012), and heavy metals (zapata et al., 2009). the ssh technology is a quick and effective method to distinguish the genes differentially expressed by high specificity and sensitivity. it can isolate dozens of, even hundreds of genes differently expressed and is easy to operation. however, this technology bases on the hybridization, so that the genes that have no or little restriction enzyme cutting site cannot be isolated by the ssh. microarrays microarrays are on the basis of abundant genes or gene segments with known sequences. the construction of the numerous libraries has led to a significant increase in the number of ests in databases, which contain genes that are modulated in response to environmental stresses and can be used to design probes in microarrays. microarrays have various applications, including the analysis of gene expression analysis and genotyping for point mutations, single nucleotide polymorphisms (snps), and short tandem repeats (strs) (heller, 2002). in molluscs, microarrays were mostly used on the gene expression profiling of responses to environmental stresses. lockwood et al. (2010, 2011) analyzed the effects of heat stress and hypoosmotic stress between invasive and native mussels. the relative studies about gene expression profiles of the oysters exposed to hypoxia (sussarellu et al., 2010), heat (lang et al., 2009), and parasites (wang et al., 2010) were performed. moreover, microarrays were used to analyze the interactions of multifactor in oysters (chapman et al., 2009; manfrin et al., 2010; chaney et al., 2011; chapman et al., 2011; dondero et al., 2011; fleury et al., 2012). microarrays were also   89 used to decipher the effects of the environmental factors on gene expression in the deep-sea mussels (boutet et al., 2011). owing to the limitation that microarrays have to base on the known sequences, the studies about the marine bivalves with limited gene databases have only concentrated on several species that are important under the commercial point of view. meanwhile, the relevant researches are so few that the costing of microarrays in marine bivalves is relatively high. the next-generation sequencing technologies the occurrence of the next-generation sequencing technologies is a giant leap in genomic and transcriptomic research. they make large-scale sequencing possible by high-throughput and costefficiency (marguerat et al., 2010). although these powerful and rapidly evolving technologies have only been available for a couple of years, they are already making substantial contributions to our understanding of genome expression and regulation. there are three main commercially technologies, including roche (454 life sciences), illumina (solexa sequencing technology), and applied biosystems (life technologies/apg). the approach to exploit dynamic transcriptomes by the next-generation sequencing technologies termed rna-seq. the application of these technologies is a fast and efficient approach for gene discovery and enrichment of transcriptomes in non-model organisms. currently, transcriptomes have been sequenced for various marine bivalves. in relation to the responses to environmental stresses in molluscs, the researches concentrated in oysters to discuss the relative genes and pathways against osmotic stresses and virus infections (de lorgeril et al., 2011; zhao et al., 2012; menge et al., 2013). furthermore, there were some literatures about the immune system of mussels (philipp et al., 2012), and clams (moreira et al., 2012). the next-generation technologies make the sequencing of the non-model organisms possible. with the development and popularization of highthoughput sequencing technologies, the genomes and transcriptomes of more and more marine bivalves would be sequenced. conclusion the ecological status of the marine bivalves is always an important advantage to monitor the environmental quality of the intertidal zones. along with the fast development of technologies and analysis methods, the information of the molluscs genomes increased drastically. the genomic information is the basis for understanding how the mollusks respond to environmental stresses and solving important problems in the bivalve production such as the “summer mortality”. in this minireview, we summarized the recent studies about the transcriptomic response to stress in marine bivalve. owing to the characteristic of bivalve’s genome and no model organism, the development of the molecular studies made a slow progress in a period. however, advanced technologies bring genome and transcriptome of bivalve new insights and progressive directions. increasing researchers devote themselves to the mechanisms of bivalve adapted to the complex conditions at molecular level. nevertheless, we face several problems. due to the decreasing cost of the next-generation technologies, we need to increase the biological repeats to increase the accuracy of the data. to date, most experiments are based on the laboratory conditions. however, environmental factors interact with each other in the nature. the researches in the future should consider the combination of imitation and the nature. eventually, transcript levels are only a proxy for protein expression, and cannot be identical completely with protein expression because of post-translational modifications or other reasons. comparing the results of the transcriptome and proteome, we can explain the experimental consequences more comprehensively. acknowledgements the work in our laboratory on bivalve genomics is supported by the grants from 973 program (2010cb126406), and 863 program (2012aa10a405-6). references bettencourt r, pinheiro m, egas c, gomes p, afonso m, shank t, et al. high-throughput sequencing and analysis of the gill tissue transcriptome from the deep-sea hydrothermal vent mussel bathymodiolus azoricus. bmc genomics 11:559, 2010. bettencourt r, costa v, laranjo m, rosa d, pires l, colaco a, et al. out of the deep sea into a landbased aquarium environment: investigating physiological adaptations in the hydrothermal vent mussel 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inflammatory response, tunic and pharynx homogenate supernatants were separated on high pressure liquid chromatography and fractions were assayed for the po activity before and after lps inoculation, as well as before and after trypsin treatment which activates propo. the lps inoculation per se did not significantly change the basal po activity of the tunic homogenate supernatant (ths) and pharynx homogenate supernatant (phs) restricted in two confluent peaks, whereas a significant enhancement was observable after the trypsin treatment. this trypsin effect suggests that propo is the main component of the hplc separated fractions, and indicates that lps inoculation mainly challenges the pro-enzyme production by tunic cells and hemocytes, as well as the activation of the serine-protease pathway. the protein size analysis and dopa-mbth assay, disclose two active proteins of 90.0 and 170.0 kda differently contained in the two main chromatographic peaks. due to the sds activating effect on the proenzyme analyzed by sds-page, the size of propo could not be shown, whereas modulation of an oligomerization process of the 90 kda component is suggested. key words: ascidian; phenoloxidase; pro-phenoloxidase; hplc; inflammation; lps; ciona intestinalis   introduction in invertebrates, phenoloxidases initiate melanin synthesis in almost all organisms, and pathway products are involved in different biological activities. the “prophenoloxidase activating system” plays a key role in humoral and cellular immune response exerting a defensive role in arthropods, molluscs, annelids and ascidians. this system comprises an enzyme cascade that activates prophenoloxidase (propo) to phenoloxidase (po). after cells were stimulated by components of pathogen associated molecular pattern, the zymogen is activated via serine proteases (söderhäll and cerenius, 1998; cammarata et al., 2008; cammarata and parrinello, 2009; cerenius and söderhäll, 2013), hemocytes degranulate and ___________________________________________________________________________ corresponding author: matteo cammarata marine immunobiology laboratory department of biological chemical pharmaceutical science and technology university of palermo via archirafi 18, palermo, italy e-mail: matteo.cammarata@unipa.it release components of the inflammatory reaction (reviewed in cerenius et al., 2010). in ascidians, pos were at first identified by histochemical reaction in the tunic hemocytes (barrington and thorpe, 1968), and a quinonetanning system involved in the production of tunic scleroproteins was suggested (chaga, 1980). these enzymes, are copper-dependent orthodiphenoloxidases (sugumaran et al., 1988) which catalyses both the ortho-hydroxylation of monophenol (i.e., tyrosine) forming o-diphenol, 3,4dihydroxy-l-phenylalanine (dopa) and, then, the dehydrogenation of diphenol into o-quinones, which can polymerize producing insoluble melanin (nappi and seymur, 1991). non-self agents activate serine proteases that, in turn, convert propo into pos involved in tunic graft rejection (raftos et al., 1987a, b, 1988), mixed hemocyte alloor xenogeneic reactions in vitro (kelly et al., 1992; fuke, 1980), non-fusion reaction between alloor xenogeneic colonial ascidian partners (ballarin et al., 1996, 2002), and inflammatory response to lps (cammarata and parrinello, 2009). in ciona intestinalis genome, two genes, referred to cinpo-1 75 mailto:matteo.cammarata@unipa.it and cinpo-2 (genbank/embl accession numbers aj547813 cinpo-1 and aj547814 cinpo-2), encode proteins with predicted molecular masses of 92.0 kda and 86.9 kda, respectively (immesberger and burmester, 2004). an antibody against anti-cinpo1 confirmed the predicted molecular weight and cells localization (cammarata et al. 2008). in this ascidian, an inflammatory response after inoculation of erythrocytes (parrinello et al., 1984a, b), foreign proteins (parrinello, 1981), or lipopolysaccharide (lps) (parrinello et al., 2007), has been shown in tunic and pharynx where hemocytes and humoral components are involved. the inoculated lps permeates both tunic and pharynx tissues stimulating the expression of several inflammatory factors (reviewed in parrinello, 2009). such a response involves hemocytes (compartment/morula cells, granular amebocytes, and unilocular refractile granulocytes urgs) recruited into the inflamed tunic (parrinello, 1981; parrinello and patricolo, 1984). in the pharynx, compartment cells (hemocytes with large vacuoles) express citypeixcollagen 1α-chain (vizzini et al., 2008), galectin-like lectins (vizzini et al., 2012), tumor necrosis factor-α-like cytokine (citnf-α; parrinello et al., 2008, 2010), a mannose-binding lectin (bonura et al., 2009) and a cysteine-rich secretory proteins, antigen 5, and pathogenesisrelated 1 proteins (bonura et al., 2010). in a previous paper (cammarata et al., 2008), the zymogram of the whole tunic homogenate supernatant from lps inoculated ascidians, indicated that po activity could be related to 90.0 kda and 120.0 kda protein bands, whereas the density of a 170.0 kda band was weak. the hypothesis that size differences between active components, as an effect of lps inoculation, could be due to an oligomerization process, has been proposed. in the present study, to study the modulation of propo/po involved in the inflammatory response and the serine protease activation process, tunic and pharynx homogenate supernatants were separated on high pressure liquid chromatography (hplc), and fractions were assayed for the enzyme activity before and after lps inoculation, as well as before and after trypsin treatment. the results of chromatographic analysis, sds-page analysis, and dopa-mbth reaction showed that the po activity was confined in two confluent peaks that match proteins of 90.0 and 170.0 kda, suggesting a dimerization process. since, in samples from lps-treated ascidians, a significant enhancement was manifest after a trypsin treatment, propo production appeared to be modulated and a serine-protease pathway could be involved. material and methods ascidians, lps inoculation and sample preparation ascidians were gathered from termini imerese marinas (italy), maintained in aerated sea water at 15 °c and fed every second day with a marine invertebrate diet (coraliquid, sera heinsberg, germany). lps (escherichia coli 055:b5, lps, sigma-aldrich, germany) was prepared in sterile marine solution (ms: 12 mm cacl2, 11 mm kcl, 26 mm mgcl2, 43 mm tris, 0.4 m nacl, ph 8.0). according to previous papers, each specimen received 100 µg of lps in 100 µl of ms, inoculated into the median region of the body wall just under the tunic. ascidians, either untreated (naïve) or injected with 100 µl ms (sham), were used as a control. the ascidian tunic surface was cleaned and sterilized with ethyl alcohol. for tunic homogenate supernatant (ths) and pharynx homogenate supernatant (phs) preparation, tunic and pharynx tissues were excised in order to avoid the presence of connective tissue and the underlying epidermis in the sample, whereas the cuticle was resected by using a razor blade. samples were homogenized with an ultra-turrax (ika) homogenizer, in distilled water for 3 min on ice. homogenates were centrifuged at 27,000g for 20 min at 4 °c, and the resulting supernatants were dialyzed against pbs at 4 °c, divided into aliquots, and stored at -80 °c. since serine proteases were used to activate the propo cascade, according to the method of jackson et al. (1993), protease inhibitors were not added to the sample preparations. protein content determination protein content was estimated by the method of bradford (bradford, 1976) with bovine serum albumin (bsa) as a standard. hplc size exclusion chromatography ths and phs were subjected to size exclusion chromatography using biosuite 250, 10 μm sec, 7.5×300 mm column (waters) on a hplc system (shimadzu scientific instruments, north america). the column was washed with tbs (150 mm nacl, 10 mm tris, ph 7.4). 200 μl of each sample were injected into the column which was eluted with tbs at a flow rate of 1 ml/min for 30 min. the chromatogram was recorded with a uv detector at 280 nm (mau). the collected fractions were concentrated by centrifugation at 500g with microconcentrators (3k omega centrifugal devices nanosep), and the final concentrated samples were stored at -80 °c until use. assay of po activity in ths and phs fractions after hplc po activity was measured spectrophotometrically according to winder and harris (1991), by using l-dopa (3,4dihydroxy-lphenylalanine) as the substrate and 6 mm 3-methyl2 benzothiazolinone hydrazone hydrochloride (mbth) as a specific reagent. briefly, 45 μl of sample (ths or phs fractions) with 45 μl of trypsin from bovine pancreas (1 mg/ml) or 45 μl of distilled water as control, were incubated for 20 min at 20 °c in 45 μl reaction mixture containing 20 mm l-dopa and mbth in distilled water (dopa-mbth). after the reaction, dopaquinone was detected within 60 min at 5 min intervals by spectrophotometric measurement at 505 nm. po activity was expressed in units (u) per minute, where 1 u was equal to 0.001 δa505 min-1 mg-1 protein. since the kinetics of the reaction with dopa-mbth reached their highest level after 20 min incubation, the u value observed at that time was taken into account in all experimental approaches. 76 results electrophoresis and po activity po activity was assessed by polyacrylamide gel as described by cardenas and dankert (2000) with some modification. the phs, ths and the relative fractions showing po activity, were subjected to polyacrylamide gel performed according to the method of laemmli (1970) using a mini protean ii cell (bio-rad). the gels were calibrated with high molecular weight range standard protein (sigmaaldrich, usa). for the native electrophoresis sds was omitted from all solutions. to identify the po activity of the protein bands, the gels were washed twice with pbs-t (0.1 m nacl; 0.02 m kcl; 0.01 m kh2po4; 0.06 m na2hpo4, ph 7.4, 2.5 % triton x100), and a final wash of 10 min in pbs (0.1 m nacl; 0.02 m kcl; 0.01 m kh2po4; 0.06 m na2hpo4, ph 7.4). the gel was incubated in a solution containing 20 mm l-dopa and 6 mm mbth in distilled water. after 1 h of incubation, the gel was washed several times in distilled water. the molecular weights of the bands were calculated using the program alfaeasefc. the po activity of ths and phs hplc fractions is enhanced by trypsin in figure 1 a typical chromatographic profile, obtained by separating ths samples on a size exclusion column biosuite 250, is shown. at 5.0 min and 5.5 min of retention time, two main confluent peaks (1, 2 respectively) were identified by spectrophotometric analysis at 280 nm. the chromatographic profiles of known proteins separated on the same column were registered and the protein sizes marked on the hplc profile (fig. 1). the peak 1 and 2 matched proteins of mass similar or superior than that of the phosphorylase b (97 kda), whereas the subsequent major peaks matched proteins ranging from 13.7 to 67 kda. although the elution profile that characterized the chromatographic separation of phs samples differed from that of ths, the elution volumes of two low peaks were similar to those of the ths peaks 1 and 2 (fig. 1c inset). fractions from each peak were collected, pooled, tenfold concentrated and assayed for the po activity. dopa-mbth reaction disclosed that, in the absence of a trypsin treatment, the ths fractions showed a low po activity restricted to the confluent peaks 1 and 2. values of 68 ± 1 u and 84 ± 16 u for the ths peak 1 and 2 respectively, were recorded. a lower activity was found by analyzing phs peak 1 (5 ± 1 u) and 2 (51 ± 4 u) (fig. 1 bars). statistical analysis student’s t-test was used to estimate statistical significance. multiple comparisons were performed with one-way analysis of variance (anova), and different groups were compared by using tukey’s ttest. standard deviations were calculated on four experiments. p < 0.01 was considered statistically significant. fig. 1 size exclusion hplc separation of the c. intestinalis tunic homogenate supernatant. the ths was analyzed by size exclusion chromatography using biosuite 250, 10 μm sec, 7.5 × 300 mm column on a liquid chromatography hplc. elution was performed with tbs over 30 min at a flow rate of 1 ml/min. the pooled fractions from each ths peak (1, 2) were further analyzed trough the same chromatographic column (insets a and b). on the top, the molecular weights from hplc separation profile of proteins used as molecular weight standards: phosporylase b (97 kda); bovine serum albumin (bsa, 67 kda), enolase (46.7 kda), myoglobin a (18.7 kda) and rnasea (13.7 kda). inset (c): the active material extracted from pharynx (phs) was analyzed trough the same hplc column. absorbance peaks were monitored at 280 nm and po activity of the collected peaks was evaluated in the presence (gray column) and absence of trypsin (hatched column). 77 the activities of the whole ths was 59 ± 7 u respect to phs (22 ± 2 u) samples. as shown by the higher bars (fig. 1 bars), the trypsin treatment of each ths sample enhanced the po activity of ths peak 1 and 2 (213 ± 21 u and 194 ± 20 u respectively). likewise, after the trypsin treatment of the phs peak 1 fractions, the value of po activity was 51 ± 8 u, whereas a lower activity of the peak 2 was recorded (13 ± 4 u) (fig.1 inset c bars). due to the low absorbance at 280 nm and the low po activity of the phs chromatographic fractions, only ths was used for further analyses. the pooled fractions from each ths peak (1, 2) were further analyzed trough the same chromatographic column. the elution profiles, shown in figure 1 (a and b), revealed that each of them presented two confluent peaks with 280 nm absorbance patterns enriched than that corresponding to the first separation step. the absorbance of the peak 1 (inset a) is mainly enriched of 5 min peak (elution time correspondent to that of the peak 1) conversely the graph of the peak 2 (inset b) is enriched of 5.5 peak (elution time correspondent to the peak 2). sds pattern and zymograms the fractions from the ths peaks 1 and 2 were analyzed by sds-page analysis, and the dopambth reaction on gel electrophoresis was carried out in the absence of mercaptoethanol and denaturing temperature. the protein patterns of the peak 1 and 2 disclosed the same active components (figs 2a, b). comparison of these patterns with standard proteins separated in the same chemical-physical condition, showed a smaller active component of 90 kda and a greater one of 170 kda (figs 2a, b). the gels obtained from a same electrophoresis procedure and stained with coomassie blue showed that each peak contained 2 additional inactive components of intermediate sizes, and showed that the 90 kda protein was mainly represented in the peak 2 and the enzymatic reaction was more intense for the 170 kda band (figs 2a, b). to avoid the possibility of propo activation by sds and check for the effect of trypsin, an electrophoresis was carried out in native conditions (in the absence of sds and denaturing factors) of whole ths sample. an unique band could be found only when the samples were treated with trypsin (fig. 2c) lps inoculation enhanced the po activity of trypsintreated ths and phs hplc fractions ths and phs samples from ascidians inoculated with lps, prepared at 4 and 24 h post injection, were separated on the chromatographic column. the fractions from peak 1 and 2, separately pooled, were assayed with dopa-mbth before and after a trypsin treatment. tunic and pharynx samples from ascidians inoculated with ms were the controls and significance values were calculated with anova. in all ths (t) and phs (p) fractions, prepared at both 4 h p.i. and 24 h p.i., the po activity (from 50 to 84 u for ths and 22 to 41 u for fig. 2 electrophoretic patterns of the hplc separated fractions. sds-page and page-dopambth in the absence of sds and denaturing temperature. a: electrophoretic pattern of ths peak 1 (300 µg/ml) stained with coomassie blue (lane 1) and after treatment with dopa-mbth (lane 2). b: electrophoretic pattern of ths peak 2 (300 µg/ml) stained with coomassie blue (lane 3) and after treatment with dopa-mbth (lane 4). c: electrophoretic pattern of ths (300 µg/ml) under native conditions (7.5 % no sds), in the absence of trypsin (lane 5) and after treatment with 1 µg/µl of trypsin (lane 6). the phs) was at control levels (fig. 3). by treating samples from the peaks 1 and 2 with trypsin, the activity of treated ths 1, ths 2 and phs 1 was significantly increased to different levels, whereas no changes were recorded in the activity of treated phs 2 (53 ± 8 u similar to controls). in treated ths 1 the highest activation level was found at 4 h p.i. (408 ± 102 u), and it lowered to control level at 24 h p.i. in treated ths 2, the trypsin treatment enhanced the activity to a level below that of treated ths 1 (fig. 3). in spite of an increase after the trypsin-treatment of phs 1 at 4 h p.i. (153 ± 11 u), the enzymatic activity of phs 2 was at control level (fig. 3). a lower and not significant increase of po activity, with respect to the treated ths 1 and ths 2 samples at 4 h p.i., was recorded for ths samples injected with ms and treated with trypsin (fig. 3). discussion in a previous paper (cammarata et al., 2008), it has been shown that lps inoculation challenges inflammatory hemocytes (unilocular refractile granulocytes and granulocytes containing few large granules) which show po activity. the enhanced po activity found in ths has been related to the numerous inflammatory po-positive hemocytes which populated the tissue. 78 fig. 3 propo/po activity of ciona intestinalis ths and phs after inoculation of artificial sea water (ms) and after 4 or 24 h lps injection (100 µg/ascidian). the obtained samples were assayed before and after treatment with 1 µg/µl of trypsin (treated). multiple comparisons were performed with one-way analysis of variance (anova), and different groups were compared by using tukey’s t-test. standard deviations were calculated on four experiments. same letters indicate statistical significant differences. p < 0.01 was considered statistically significant. since tunic and pharynx have been examined to study the immune-related responses, both tissues were analyzed. the chromatographic profiles of ths and phs from naïve ascidians, and the dopambth reaction disclosed that two pos of distinct sizes (about 90 kda and > 90 kda) could be separated. the po activity of each ths peak was significantly enhanced by trypsin indicating that they contained both activated po and propo very similar in size. these findings agree with the idea that the proenzyme is produced by the tunic cells that populated the inflamed tissue and it is present in a major and minor form. the basal activity found in untreated samples could be dependent on artificial activation of propo due to the experimental procedures. two lower phs peaks overlapped elution volumes similar to those of ths separation and disclosed a low po activity enhanced by trypsin. phs preparations mainly contained extracts from vessel epithelia and hemocyte populations, and it is reasonable that these cells, although at a lesser extent, were engaged in propo production. due to the low protein content and po activity of phs fractions, ths fractions were used for 79 further analyses. the two main chromatographic peaks of ths were confluent indicating that proteins of intermediate size could be contained. in an attempt to further separate the proteins, the isolated fractions were subjected to a second chromatographic analysis. however, only an enrichment of the component that mainly characterized each peak was obtained. the sds activation effect on propo and hemocyanins is well know (decker et al. 2001), therefore the precise propo size, checked by sdspage, remains unrevealed, whereas the sizes (90 and 170 kda) of the active forms could be revealed. in this respect, to assay the po activity of the protein bands, the dopa-mbth reaction on page gel was carried out in the absence of sds and denaturing condition, and the pattern was compared with standard proteins separated in the same conditions. the po activity of the whole tissue supernatant as well as the dopa-mbth on page gel before and after trypsin treatment, supported the view that propo was mainly contained in the tunic fractions. this analysis, in accordance with the hplc profile, revealed that the po activity was exerted by the 170 and 90 kda proteins. analysis of the deduced amino acid sequences of the cinpo1 and cinpo2, identified in the c. intestinalis genome (immesberger and burmester, 2004), provided predicted molecular masses of 92 kda and 89 kda, respectively which can be reasonably referred to the 90 kda pos separated by hplc, whereas, according to cammarata et al. (2008), the 170kda po may be referred to a dimerization process. likewise, results obtained by examining pos from circulating hemocytes, showed two active forms of different size: a greater one (175 188 kda) and a smaller one (74 kda) (parrinello et al., 2003). we do not know the functional differences between the two forms. however the possibility exists that the dimeric one is an artifact due to sample preparation and biochemical methods. the 120 kda active form, reported by cammarata et al. (2008), remains to be explained, whereas the inactive proteins of intermediate sizes found in the sds-page pattern of the fractions could be additional components separated by the chromatography. although phs samples were not analyzed by sds-page, a similar protein pattern can be assumed on the basis of the chromatographic profile and po activity of the fractions a similar protein pattern may be assumed. in ascidians, the inoculated lps modulates the propo expression and activates cell proteases that in turn activates the propo. in fact, in both in the tunic and pharynx preparations, the po activity of the peak 1 and 2 significantly increased within few hours after lps inoculation, and the effect of trypsin treatment indicates that the challenged tissues mainly expressed propo. the lps inoculation modulated the activity of the two peaks, it was higher for the peak 1 at 4 h p.i. and very low at 24 h. conversely, at the latter time the activity of the peak 2 was lower but significantly higher than the control. the analysis of phs fractions supported the idea that a low effect was exerted by lps inoculation on po activity of the pharynx tissue. a low activity was mainly bound to the peak 1 at 4 h p.i., whereas it was insignificant for the peak 2. differences between ths and phs activities at the same p.i. time instead of supports the main role of tunic cells in the po activity as a response to lps stimulation, whereas the lower phs activity may be imputable to a lower amount of propo released by a minor number of po expressing hemocytes (i.e., univacuolar refractile granulocytes and morula cells). in addition, the possibility exists that the lps modulating effect could be related to propo releasing and activating steps or to the modulation of a presumptive dimerization process. acknowledgements this work was supported by a research grant from the italian ministry of education (prin 2006 and 2010-2011 to parrinello n), co-funded by the university of palermo. we thank 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and biotechnology, holy cross college, nagercoil-629 004, tamil nadu, india accepted december 12, 2013 abstract the hemolymph of the mangrove crab, e. tetragonum contains lectins specific for neugcα 2, 3 gal β1-4 glunac β1 linkage and o-acetyl sialic acids. the role of sialic acid specific lectins on natural immunity of the crab is studied by using several kinds of mammalian erythrocytes as pathogen model. injection of erythrocytes showing differential agglutinability with the lectins, induced augmentation of hemagglutinating activity suggesting an increase in the circulating lectins. a significant correlation was observed between in vivo clearances of exogenous erythrocytes with the extent of erythrocyte agglutination by the lectins. another correlation was observed between the susceptibility of erythrocytes to lectin dependent hemocyte mediated hemolysis and the extent of lectin mediated erythrocyte agglutination. this study documents that opsonization of foreign pathogen by the native lectins is an important step in hemocyte recognition, hemolysis and clearance of the pathogen. key words: innate immunity; opsonin; hemolysis; hemocytes; induction; clearance introduction the great success of arthropods in diverse environments of earth must certainly rely on efficient immune defenses capable of protecting these animals against the microbial invasion. lectins from the hemolymph of invertebrates have been regarded as potential molecules involved in immune recognition and phagocytosis of the microbes through opsonization (marques and barracco, 2000). lectins are proteins or glycoproteins usually without catalytic activity that have the ability to bind to specific carbohydrates expressed on different cell surfaces. their specificity is always determined by the type of carbohydrate to which they bind. due to the fact that lectins have the ability to bind carbohydrate and promote the agglutination of different cells, such as bacteria and other invading pathogens, it is reasonable to assume that these molecules may be regarded as having a potential role in invertebrate nonself recognition reactions. many recent studies have emphasized the possible role of lectins as nonself recognition molecules in invertebrate immunity (zhu et al., 2006; vasta et al., 2007; yang et al., 2011; jin et al., 2013). as with vertebrate immunoglobulins, lectins can agglutinate microorganisms and enhance their phagocytosis by ___________________________________________________________________________ corresponding author: viswambari r devi department of advanced zoology and biotechnology holy cross college nagercoil-629 004, tamil nadu, india e-mail: viswambaridevi@gmail.com acting as an opsonin (arason, 1996; gasparini et al., 2008; franchi et al., 2011), and are apparently synthesized by invertebrate immune cells (hemocytes). however in contrast to immunoglobulins, the specificity of invertebrate lectins is restricted only to glycoprotein and sugar residues. in spite of their apparent ubiquity, and the remarkable number of recent publications on their occurrence, structure, and specificity, the natural functions of lectins are still not fully understood. interactions between lectins and carbohydrates have been shown to be involved in various activities. therefore, different biological roles have been proposed for these molecules, including the cellular and tissue transport of carbohydrates, glycoproteins and calcium (goldstein et al., 1980; ravindranath and coopper, 1984), correlation to insect development (armstrong et al., 1996), cell adhesion, migration and apoptosis (perillo et al., 1995; ni and tizard, 1996). compared to other arthropodan groups, such as insects and horseshoe crabs, the current knowledge on lectin involvement in crustacean nonself recognition is still much less well established. the hemolymph of the mud crab, scylla serrata contains sialic acid specific lectin, which increased markedly on challenging with erythrocyte species that it agglutinated best (mercy and ravindranath, 1993). similar augmentation of agglutinin activity after administration of erythrocytes was also observed in the freshwater crab paratelphusa jaquemontii (maghil, 2001), and 162 anomuran crab emerita emeritus (jayasuriya, 2002). the enhancement of lectin activity in response to foreign cells suggests a specific lectin foreign cell interaction. most crustacean lectins seem to share a common specificity to n-acetylated aminosugars, particularly sialic acids (marques and barracco, 2000). sialic acids are a family of nand o-acetyl or n-glycolyl derivatives of nine carbon monosaccharide neuraminic acid (schauer, 1982), among which, n-acetyl neuraminic acid is the most common in nature. sialic acid residues are commonly encountered in the cell membranes of almost all deuterostome animals and are virtually absent in protostomes including crustaceans (schauer, 1982). hence, the occurrence of sialic acid lectins in the body fluids of crustaceans can indicate that these molecules could have a role in defense against exogenous sialylated pathogens (cominetti et al., 2002). however, their precise role in the above function remains to be elucidated. therefore, it was felt important to know the biological role of sialic acid binding lectins in the biology of crabs. the lectins in the hemolymph of crabs are likely to form an innate immunological defense of organisms which can be verified by inducing immune response with model foreign antigens. to verify the above hypothesis, the mangrove crab e. tetragonum, was taken for the study and challenged with erythrocytes as an antigen model. materials and methods sample collection the mangrove crab, episesarma tetragonum was collected for the study from the mangrove and fresh water regions of manakudy, kanyakumari district, tamil nadu. the crabs were maintained in plastic tubs with fresh water and mud. the water and mud were changed on alternate days and was fed with paddy grains. purification of lectin as reported elsewhere (devi et al., 2013), affinity purification of episesarma tetragonum agglutinin-1 (eta-1) was performed using cyanogen bromide activated sepharose 4b in an econo column (bio-rad) previously equilibrated with tbs at 4 °c. the elution of lectin (etl-1) was done with elution buffer that contained 100 mm glunac and collected 1 ml fractions on ice in polypropylene tubes containing 10 μl of 100 mm calcium chloride at a rate of 0.3 ml/min. the fractions were vortexed immediately after collection and kept on ice. fractions containing lectin were pooled on the same day and dialyzed against 1 mm cacl2 , at 4 °c for 3 h and the dialysate was then aliquoted, lyophilized (speed-vac, sawant), and stored at -20 °c. ha assays to determine physicochemical parameters the physicochemical properties of the purified lectin was determined by hemagglutination assays with serum samples under conditions of varying ph, temperature, bivalent cation of diverse concentration, edta and some chemical agents. table 1 differences in the time taken for clearance of erythrocytes from circulation before and after coating the erythrocytes with sub agglutinating concentrations of the hemolymph lectins of e. tetragonum time taken for clearance (min) erythrocytes ha titer (n = 9) before after dog horse human o 64 32 4 45 ± 4.98 60 ± 3.31 240 ± 4.52 20 ± 2.29 30 ± 3.05 180 ± 2.81 n = number of crabs studied ha assays were performed as described by devi et al., 2013. hemagglutination inhibition (hai) assay hemagglutination inhibition assay was performed to test the ability of various glycoproteins (fetuin, transferrin, porcine thyroglobulin, alpha acid glycoprotein, porcine and bovine submaxillary mucin, apotransferrin, bovine thyroglobulin) and sugars (glunac, galactose, neugc, maltose, mannose, lactose, galnac, neuac) to inhibit agglutination. hemagglutination inhibition titer was reported as the reciprocal of the lowest dilution of inhibitors giving complete inhibition of agglutination after 1 hour. sialidase treatment of sialoglycoprotein asialo fetuin was prepared by incubating 2 mg of glycoprotein (fetuin) with 0.1 unit of clostridium perfringens sialidase (sigma type x) in 400 μl of 5 mm acetate buffer, ph 5.5 for 2 h at 37 °c. as a control, fetuin was treated similarly without sialidase. hai assay was performed with purified lectin for sialidase treated and untreated fetuin against 1.5 % dog erythrocyte suspension. erythrocyte collection the erythrocytes selected for the experiments included dog, horse and human o. human o erythrocytes were obtained with thanks from kanya blood bank (nagercoil). equine blood was collected from the jugular vein and canine blood from cephalic vein. the blood samples were directly collected in sterile modified alsevier’s medium (30 mm sodium citrate, 77 mm sodium chloride, 114 mm glucose, 100 mg neomycin and 330 mg chloramphenicol). before use, the erythrocyte types were suspended and washed thrice in tris buffered saline (tbs: 50 mm tris-hcl, ph 7.5, 100 mm sodium chloride, 10 mm calcium chloride) and resuspended in the same buffer to get required cell suspension. 163 a) b) c) 164 fig. 1 effect of injection of 1.5 % (a); 2.5 % (b); 5 % (c) dog erytrocytes on the ha titer of the hemolymph agglutinins of e. tetragonum. induction experiments a) injection of erythrocytes in each experiment 0.1 ml of 1.5 %, 2.5 % and 5 % of dog, horse and human o erythrocyte suspension in 0.9 % sterilized saline, was injected into the crabs separately. all injections were made slowly into the soft arthrodial membrane between the coxa of the fourth preopod and the dorsal surface of the carapace. the injection site was blotted with cotton both before and after the injection of the erythrocytes. those crabs that bled at the injection site, which occurred if they moved vigorously during injection, were discarded. care was taken to ensure complete injection of the erythrocytes. b) collection of hemolymph samples to study the effect of erythrocytes injected into the hemocoel of crabs, on humoral agglutinin activity, hemolymph was collected at regular intervals (1, 2, 4, 8, 16, 24, 48 and 72 h) after the injection and ha assay was done with dog, horse and human o erythrocytes. clearance experiments a) injection of erythrocytes the erythrocytes selected for the study on clearance also included dog, horse and human o erythrocytes. in each experiment 100 µl of 1.5 % erythrocyte suspension in saline was injected into the hemocoel of crabs. the method of injection was same as stated for the experiment on induction. b) pretreatment of erythrocytes with lectins to find out whether the clearance of erythrocytes was enhanced consequent to lectin binding, erythrocytes were coated with purified lectin diluted to sub agglutination concentration. the sub agglutination concentration differed for different erythrocytes, as shown in table 1. resuspended 200 µl of washed and packed erythrocytes in 20 volumes of lectin (diluted to sub agglutinating concentration) and incubated the mixture for 1 h at 30 °c. the lectin coated erythrocytes were washed and resuspended in sterilized saline and were injected as stated earlier. the erythrocyte suspensions were examined under the microscope to ensure the presence or absence of clumps of erythrocytes. clumps were disrupted by gentle vortexing. the rate of clearance before and after lectin coating was compared. c) collection of hemolymph samples to study the clearance of injected erythrocytes from the circulation, hemolymph (100 µl) was collected with a micropipette at regular time intervals (5 minutes) until the injected erythrocytes completely disappeared from the circulation. hemolymph was added to 700 µl of double distilled water, and after mixing, the total volume was adjusted to 1 ml with double distilled water. estimation of hemoglobin the amount of hemoglobin was estimated using the cyanmethhemoglobin method, a hemoglobin kit manufactured by sigma diagnostics (india) pvt. ltd. baroda. product no. 72431 of qualigens diagnostics designed for in vitro estimation of hemoglobin. the cyanmethhemoglobin technique is the method of choice selected by the international commission for standardization in hematology (icsh). the method measures all hemoglobin derivatives except sulfhemoglobin. hemolysis to study the interaction of crab hemocytes on lectin treated erythrocytes, the hemocytes were separated using the method of soderhall and smith (1983). the hemocytes were washed twice to remove the contaminating proteins and used for hemolysis experiments. the hemocyte suspension was added to the lectin coated or uncoated erythrocytes and incubated for 1 h at 30 °c, and then the erythrocyte hemocyte mixture was centrifuged at 200 x g for 5 min. the supernatant was collected for estimation of hemoglobin content, which was measured following the cyanmethhemoglobin method. the control included lectin coated or uncoated erythrocytes without hemocytes. results physicochemical properties of purified lectin (etl-1) specific to dog erythrocytes the ha activity of the purified lectin was sensitive to ph and temperature. the ha was stable between ph 6.5 9.5 and at temperature ranging from 10 40 °c. among the cations tested, divalent calcium ions did not have any effect on ha titer, however magnesium at 10 0.1 mm concentration reduced the ha titer to 32 while at 100 mm concentration gave the normal ha titer. the metal ion chelator edta at very low concentrations (1 0.01 mm) decreased the ha activity (ha = 32). at concentrations from 1 5 mm, the ha activity increased one fold from the normal ha (128) and at concentration from 10 20 mm there was sudden decrease in ha activity, after which the ha activity was completely lost. the agglutinating activity was completely inhibited by chloroform while the activity was greatly inhibited by incubation with denaturing agents such as hcl and naoh. hemagglutination inhibition (hai) assay the inhibitory potency of assayed compounds on ha by etl-1 was as follows: fetuin > porcine thyroglobulin = transferrin > α-acid glycoprotein > psm > apotransferrin = bovine thyroglobulin. the purified etl-1 showed a remarkable inhibitory potency with fetuin containing sialic acids with α, 2-3 linkages. on the other hand, bsm and lactoferrin failed to inhibit the ha activity of etl-1. to further define the possible role of sialic acids as potent inhibitor of lectin, the sialoglycoprotein, fetuin was enzymatically modified and its derivative was examined for hai. sialidase treatment of fetuin reduced its inhibitory properties at 20 h. in order to find out if etl-1 was neuac or neugc specific, free sialic acids neuac and neugc were tested as 165 inhibitors of hemagglutination. neuac did not inhibit hemagglutination whereas neugc inhibited. however neugc linked glycoproteins were more inhibitory than free neugc. galactose that showed a) b) c) 166 fig. 2 effect of injection of 1.5 % (a); 2.5 % (b); 5 % (c) horse erytrocytes on the ha titer of the hemolymph agglutinins of e. tetragonum. very low inhibitory potency with crude agglutinin was a potent inhibitor of purified etl-1. profile of ha after injection of dog erythrocytes injection of dog erythrocytes enhanced the agglutinin activity of the hemolymph. the ha titer of induced agglutinins varied with time and concentration of dog erythrocytes administered. injection of 1.5 % dog erythrocytes augmented the ha activity to two fold with dog and horse erythrocytes at 4 hour post injection. human o erythrocytes showed insignificant variation in ha titer (fig.1a). after injection of 100 µl of 2.5 % dog erythrocytes the ha activity increased four fold with dog erythrocytes at 4 h, which showed gradual decrease and then heightened activity at 24 hours post injection (fig.1b). injection of 5 % dog erythrocytes showed six fold increase in ha activity with dog erythrocytes within 4 8 h of post injection (fig.1c). profile of ha titer after injection of horse erythrocytes injection of 1.5 % horse erythrocytes showed an enhancement of agglutinin activity between 8 24 hours with horse erythrocytes and between 4 8 hours with dog erythrocytes (fig. 2a). injection of 2.5 % erythrocytes, slightly depressed the ha activity with horse erythrocytes at 2 hours and then it showed a consistent increase and the peak of activity was observed between 8 16 h post injection. however with dog erythrocytes ha activity showed an augmentation only at 8 hours post injection (fig. 2b). injection of 5 % horse erythrocytes enhanced the ha activity with two peaks, one at 4 8 h and another at 24 h post injection with horse erythrocytes. but only a single peak of ha activity occurred with dog erythrocytes at 4 h post injection. human o showed no augmentation in ha activity (fig. 2c). profile of ha titer after injection of human o erythrocytes injection of 1.5 % human o resulted in four fold and two fold increase in agglutinin activity with dog and horse erythrocytes respectively at 4 h of post injection. however ha activity of dog erythrocytes was depressed at 8 16 h post injection, and regained its preinjection level within two days (fig. 3a). injection of 2.5 % human o erythrocytes induced and augmented agglutinin activity as evidenced in four fold increase in ha titer with dog erythrocytes and two fold increase with horse erythrocytes. human o showed very low ha titer (fig. 3b). similarly on injection of 5 % human o erythrocytes, six fold increase in ha activity was observed with dog erythrocytes after 4 h of post injection. however the animals were unable to counter the challenge and crumbled after 48 h of post injection (fig. 3c). clearance of erythrocytes with a view to elucidate the biological role of lectins in the defense strategy of the crab, e. tetragonum, an attempt was made to find out whether there is any correlation between erythrocyte clearance and their respective ha titers. a positive correlation was observed between lectin agglutinability of different erythrocytes and the time taken for complete clearance of the respective erythrocytes. the hemoglobin level in the hemolymph indirectly measures clearance of erythrocytes at different time intervals after injection of different erythrocytes. dog and horse erythrocytes that were strongly agglutinated by the agglutinins were cleared faster than the human o erythrocytes (table 1). faster clearance of lectin coated erythrocytes was also observed. the lectin coated dog erythrocytes were cleared within 20 min whereas uncoated dog erythrocytes were cleared only by 45 min from the hemolymph. similarly, the lectin coated horse erythrocytes were cleared in less than 30 min whereas uncoated erythrocytes cleared only by 60. on the contrary, the crabs required 240 min to clear the low agglutinating human o erythrocytes, 180 min to clear the lectin coated human o erythrocytes (table 1). hemocyte mediated hemolysis hemolysis was measured after mixing hemocytes with lectin coated or uncoated erythrocytes. lectins or hemocytes alone had very weak effect to bring about hemolysis of any of erythrocytes, whereas the hemocytes with lectin markedly facilitated hemolysis of erythrocytes (table 2). the results clearly established a positive correlation between hemocyte mediated hemolysis of erythrocytes and their respective ha titers. lectin coated dog and horse erythrocytes showed marked lysis when compared to lectin coated human o erythrocytes. discussion the ability to distinguish self from nonself is the fundamental aspect of immunity. lectin carbohydrate recognition represents a ligand receptor interaction that is universal in organisms (cambi and figdor, 2003). arthropods display both cellular (hemocyte mediated) and non cellular (humoral) responses against a wide variety of antigens and recognize self from non self molecules (gallo et al., 2011). lectins, non enzymic proteins that bind mono and oligosaccharides reversibly and with high specificity, occur widely in nature (sharon and lis, 1995). lectins are receptor specific proteins (muramoto et al., 1995) whose biological role is meagerly understood. considering the report that crustaceans, like other protostomians, are not capable of synthesizing sialic acids (segler et al., 1978), any physiological role for the lectin should be implied with caution. in the absence of any interaction of the lectin with crab tissue components due to paucity of sialic acids in the tissues, the lectin may function as a defensive tool, protecting the host by binding with pathogens such as bacteria and virus that may contain sialoglycoproteins or sialopolysaccharides. 167 in this study, the biological role of hemolymph lectin of the crab e. tetragonum is elucidated. erythrocytes showing differential agglutinability with the lectin are used as pathogen model. the lectin demonstrates specificity for the erythrocytes of a) b) c) 168 fig. 3 effect of injection of 1.5 % (a); 2.5 % (b); 5 % (c) human o erytrocytes on the ha titer of the hemolymph agglutinins of e. tetragonum. table 2 effect of hemocytes on hemolysis (measured as equivalent of µg of hemoglobin released) from erythrocytes coated with or without lectins purified from e. tetragonum hemoglobin content in erythrocytes (µg) treatment (n = 12) dog horse human o control 28 32 30 uncoated erythrocytes + hemocyte suspension 1± 0.24 0.86± 0.036 0.92 ± 0.05 lectin coated erythrocytes + hemocyte suspension 20.5±2.31 18±1.34 4.5±4.2 lectin coated erythrocytes without hemocyte suspension 0.5 ± 0.04 0.5 ± 0.07 0 different species. the high ha titer with dog and horse cells indicates the preponderance of the lectin specific sugar moieties on dog and horse erythrocytes. the low ha titer for human o indicates that the sugar residues on these erythrocytes may not have the proper conformation to be recognized by the hemolymph lectin. injection of dog erythrocytes preferentially augmented the ha activity with dog erythrocyte and, of horse erythrocyte augmented the ha activity with horse erythrocyte. this clearly revealed the induction of two different lectins (as reported in devi et al., 2013) in the hemolymph of crab, e. tetragonum. following injection of 1.5 %, 2.5 % and 5 % dog erythrocytes, the hemolymph lectin exhibited a slight decrease in the ha from the initial titer of 64 with dog erythrocytes to 32 between 1 2 h and an increase to 2, 4 and 16 fold respectively between 4 24 h. the normal titer value was resumed by 48 72 h. similarly injection of horse erythrocytes resulted in normal and low ha followed by an enhanced hemagglutinin activity, but at 16 h of post injection the hemagglutinin activity declined and then enhanced to attain the second peak, suggesting that the adaptive survival of the crabs depended mainly on the inductive property of the lectin. subsequent increase in ha activity and the peak activity at 4 8 h suggest that some other tissue may be secreting the lectin when the hemolymph lectins are used up for elimination of pathogen or injected erythrocytes. similar patterns of augmentation in lectin activity after administration of erythrocytes is also reported in earthworms (stein et al., 1987), insects (jayalakshmi, 2005), oyster, crassostrea gigas (hardy et al., 1977) fresh water snail, planorbarius corneus (ottoviani et al., 1986), millipedes (basil rose, 1999) and crabs (mercy and ravindranath, 1994; jayasuriya, 2000; maghil, 2001). in crabs an enhancement in agglutinin activity occurred at a short duration after the injection of erythrocytes. this suggests the possibility of release of the lectin from other tissues. presence of hemagglutinating activity in the hepatopancreas of the sand crab emerita emeritus (jayasuriya, 2000), fresh water crab, p. jacquemontii (maghil, 2001) and the mud crab, s. serrata (mullainadhan, 1984) support such a possibility. the important finding that emerges from this study is that, there is a distinct positive correlation between clearance time and ha titers with different erythrocytes. this correlation suggests that the efficiency of clearance of injected erythrocytes is reflected in the ha titer against different erythrocytes. the rate and time taken for clearance of erythrocytes coated with lectin revealed that the clearance is faster with both high agglutinating dog and horse erythrocytes. hence lectins coated on the erythrocytes made them to be easily recognized by the hemocytes and, clear them by hemolysis. thus lectins have been known as playing a central role in nonself recognition and clearance of invaders in invertebrate immunity. faster clearance and more effective hemolysis of lectin-coated erythrocytes clearly indicate that the hemolymph lectins may function as a recognition mediator between the foreign cells conclusions 169 from these observations it can be summarized that the crab, e. tetragonum is physiologically adapted to challenge foreign cells by enhancing the production 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sek-1 also exhibits tissue-specific activities in host innate immunity. our findings suggest that overlapping immune effect may exist between the tol-1 and sek-1. key words: c. elegans; tol-1; sek-1; tissue-specific activity; innate immunity introduction caenorhabditis elegans is a bacterivore species so that microbes represent both nutrient sources and also potential infection sources (garsin et al., 2001). diverse infection modes by pathogens have been reported in c. elegans (pujol et al., 2008; irazoqui et al., 2010). when infection happens, c. elegans appears to initiate behavioral defenses such as avoidance and reducing digestion rates (hasshoff et al., 2007; schulenburg and ewbank, 2007). in addition, c. elegans are known to regulate physiological processes including innate immunity through activating conserved neuroendocrine signal and neuropeptides (styer et al., 2008). a complex innate immune response is involved in the ___________________________________________________________________________ corresponding author: hongyu li gansu key laboratory of biomonitoring and bioremediation for environmental pollution school of life sciences school of pharmacy lanzhou university tianshui south road no. 222 lanzhou 730000, pr china e-mail address: lihy@lzu.edu.cn infection, evolutionary conserved signaling pathways, such as toll-like receptor (tlr) signaling pathway and p38 mapk pathway are employed to defense the pathogens invasion (akira et al., 2006). tol-1, a component of toll signaling pathway, encodes a tlr, functions in pathogen recognition and thus enables c. elegans to avoid a potential pathogen (pujol et al., 2001). aversion behavior indicates the favor of worm to the bacteria; it may also influence the ingestion of nematode. as the only tlr gene in c. elegans, however, tol-1 appears to play no roles in innate immunity (kanzok et al., 2004). nevertheless, tol-1 is required for proper innate immunity in the presence of certain gram-negative bacteria but it does not have a universal immune protection against pathogens (tenor and aballay, 2007). c. elegans has no specialized immune cells like vertebrate for resisting pathogens, the antimicrobial genes are expressed in tissues where the site is exposed to the pathogen infection environment (alper et al., 2007), including the tissues that are in contact with the exterior or ingested microbes. digestive tract (pharynx and intestine) is the primary route of infection (alegado et al., 2003; tenor and aballay, 2007), and is considered as the typical infection model to investigate the immune response. 228 mailto:lihy@lzu.edu.cn a) b) c) fig. 1 tol-1 and sek-1 were not involved in aversion behavior. a) wild type n2 worms avoided jxoiii and e. faecalis markedly. b) sek-1 mutants exhibited similar tendency while e. faecalis induced stronger avoidance. c) tol-1 mutation had no influence on the avoidance to the bacteria we examined. chemosensory system with the sensory cilia enables the worm to detect the various cues of food and danger, and then elicit chemotaxis or avoidance behavior (bargmann, 2006). nervous system regulates the innate immune responses to maintain the delicate balance between infection and health (kawli et al., 2010). it is obvious that multiple immune pathways are activated upon encountering a single pathogen in c. elegans and the host can distinguish different pathogens and elicit specific responses (bogaerts et al., 2010). the nsy-1-sek-1-pmk-1 p38 mapk pathway mediates resistance to bacterial pathogens and sek-1 mutants have enhanced susceptibility to pathogens (kim et al., 2004). p38 mapk pathway is not involved in aversion behavior, but sek-1 229 contributes to this physiological process in response to certain functional classes of gene inactivation by rnai (melo and ruvkun, 2012). sek-1 activities in different tissues exhibit specific patterns in modulating immune responses to infection. in sensory nervous system, sek-1 is required for a protective avoidance behavior to pa14, however, sek-1 shows a cell-autonomous regulation in intestinal immunity (shivers et al., 2009). downstream effectors of p38 mapk pathway like c17h12.8, f08g5.6, f56d6.2 and t24b8.5 act as immune-related genes, they are classified into cub-like genes, c-type lectin genes and shk toxin genes, respectively (troemel et al., 2006). based on the higher transcriptional levels (troemel et al., 2006), we choose these genes to investigate the immune responses to pathogenic bacteria infection. xanthomonas oryzae pv. oryzae (xoo) is a plant pathogen, gram-negative, causes serious bacterial blight of rice (hopkins et al., 1992). in our previous research, it is shown that xoo can infect c. elegans and trigger innate immune responses to defense against the adverse effects on the host organism through up-regulating the downstream effectors. given that immune-related components express in multiple tissues, different transgenic worms (shivers et al., 2009) are used in our research. here, we further report that aversion behavior is benefit for nematode survival and sek-1 is important in tissue-specific innate immunity in response to xoo infection. materials and methods bacteria and c. elegans strains the nematode strains of wild type n2, ig10 (tol-1(nr2033) i), zd193 (sek-1(km4) x; qdex4)， zd202 (sek-1(km4) x; qdex8), and zd260 (sek-1(km4) x; qdex11) were provided by the caenorhabditis genetics center (cgc). ku4 (sek-1(km4) x) were kindly gifted from ausubel lab of department of molecular biology, massachusetts general hospital. xoo strains philippine race pxo99 and japonic race jxoiii, were kindly supplied by prof. js wang, key laboratory of monitoring and management of plant diseases and pests, ministry of agriculture, department of plant protection, nanjing agricultural university (li et al., 2007). e. coli op50 and enterococcus faecalis (atcc29212) were used as positive and negative control, respectively. xoo were maintained in nutrient agar (na) liquid medium, e. faecalis was in brain heart infusion (bhi) medium, and op50 was in lb broth. microbial avoidance assays avoidance assays were generally performed as previously described (melo and ruvkun, 2012). briefly, bacterial overnight cultures were concentrated 20×, and 50 ul aliquots were dropped in the centre of 3cm diameter ngm plates about 1 h before use. at least 50 synchronized l4 stage worms were dropped directly onto lawns and plates were scored for aversion (a = n off lawn/n total) at the time points of 1, 2, 4, 8, 16, 24 h. three replica plates were prepared for each treatment and at least two independent trails were repeated. nematode ingestion behavior xoo strains were cultured at 28 °c and e. faecalis and e. coli op50 were cultured at 37 °c until the logarithmic phase. l4 stage wild type n2 and sek-1 mutant worms were added to 96-wells plates contained the same known concentration bacterial cultures. each well contained 70 80 worms and each treatment was performed in triplicate. for 24 h and 96 h, supernatants were collected to measure the od600 and worms were obtained through centrifuging to test the total protein concentration by lowry method. bacterial consumption was determined by δod600/protein (mg). body length measurement sek-1 mutants were treated as ingestion assay described above. for 24 h and 96 h, worms were collected by centrifugation and washed twice in m9 buffer. the tube was heated to about 40 50 °c for several seconds to make the worms straight and easy to measure body length. then worms were pipetted on a glass slide and covered with a coverglass. at least 30 worms were measured each group under a motic microscope equipped with motic image software advanced 3.2. killing assays killing assays were performed as described (tan et al., 1999), modified with spotting a small lawn in the centre of the plates (shivers et al., 2009). then the plates were incubated overnight and equilibrated to the room temperature. l4 stage worms were picked to the assay plates and transferred to fresh bacterial plates every 48 h to eliminate newborn larvae. assays for survival of zd193, zd202 and zd260 were performed with big-lawn to exclude the interference of aversion. a total of 60-75 l4 larvae for each genotype were assayed in two independent trials. worms were scored as dead and live every 24 h along the time course. quantitative rt-pcr synchronized eggs were seeded on e. coli op50 plates until l4 larvae, rna were extracted from l4 worms exposed to pxo99, jxoiii, e. faecalis and op50 for 24 h using triol (takara). cdna was generated using the primescript rt reagent kit with gdna eraser (takara) as indicated by the manufacturer. the qrt-pcr was performed on a bio-rad s1000 thermal cycler using sybr premix ex taqtmⅱ(takara). cycle threshold (ct) values were normalized to act-3. samples treated by pathogenic bacteria were compared to parallel samples feeding on e. coli op50. primers used were listed in table 3. statistics spss ver. 17.0 was used for data processing; kaplan-meier method and log-rank test were adopted for statistical analysis. rt-pcr statistical tests were calculated from op50-normalized cycle threshold values prior to conversion to relative fold change. the normalized values for induction expression for the 3 replicates were compared using a 1-sample t-test. 230 results tol-1 and sek-1 are not involved in aversion behavior previous work has shown that tol-1 is required for the worm avoidance behavior of pathogenic serratia marcescens (pujol et al., 2001), sek-1 activity in the chemosensory neuron is necessary for protective avoidance response to pseudomonas aeruginosa strain pa14 (shivers et al., 2009). in our study, the regular food source op50 exerted an attractive effect on wild type n2, as well as the xoo strain pxo99, that was, nearly no aversion. however, e. faecalis and another xoo strain jxoiii had a strong tendency to repel n2 worms (fig. 1a). avoidance worms for 16 h was shown in figure 2, it also indicated that part of n2 worms were out of the jxoiii and e. faecalis bacterial lawns (figs 2b, c) while almost all the worms still maintained in the pxo99 and op50 lawns (figs 2a, d). sek-1 and tol-1 mutation had no effect on the avoidance tendency against jxoiii and pxo99 (figs 1b, c), suggesting that tol-1 and sek-1 were not involved in aversion behavior. ingestion behavior is related to the body length and associate with the survival of worms aversion behavior is a common response to products released by pathogen (pradel et al., 2007), these substances may have an effect on ingestive efficiency of c. elegans (schmeisser et al., 2013). pathogenic bacterium itself also serves as cues to be detected and ingested. in our previous work, xoo pxo99 and jxoiii can infect the c. elegans like e. faecalis (bai et al., 2014). to determine whether nematode ingestion is related to the favor of pathogenic bacteria, we tested bacterial consumption for different time periods. consistent with the tendency of avoidance, ingestion of jxoiii and e. faecalis by both n2 and sek-1 mutants were reduced remarkably for 24 h in contrast to their normal food source op50 (figs 3a, b). bacteria that smelt or tasted unpleasantly might be avoided and not be taken up effectively (kaletta and hengartner, 2006). it indicated that nearly no aversion was occurred after the exposure to pxo99 like op50 (figs 1a, b), however, the consumption of pxo99 was significantly reduced for 24 h compared fig. 2 avoidance behavior of wild type n2 at the time point 16 h. several worms were out of the bacterial lawns of jxoiii and e. faecalis while almost all the worms were within the pxo99 and op50 lawns. (a): pxo99 (b): jxoiii (c): e. faecalis (d): op50 to the control (figs 3a, b). thus, pxo99 could escape detection by c. elegans but pathogenic virulence of pxo99 induced the worm reducing uptaking. it is intriguing that pxo99 and jxoiii ingestion increased as time went on and jxoiii had no significant differences compared with control group in sek-1 mutants for 96 h (fig. 3b). habituation to the environment because of long-time exposure or repeated stimulation is defined as a reason for decreasing in responding (rose and rankin, 2001). it was hypothesized that adaption to the environment conferred the worm greater chances to survive and sek-1 mutation might induce recognition defect of the worms when the exposure time is longer than 24 h. factors affect ingestion were not only related to the characteristics of pathogen itself but also the interaction between the table 1 body length of ku4 (sek-1) mutants treated with xoo and e. faecalis 24 h 96 h treatment body length (mm) (means ± sd) n p-value body length(mm) (means ± sd) n p-value pxo99 0.91±0.09 45 < 0.01 1.17±0.07 33 < 0.01 jxoⅲ 0.85±0.05 30 < 0.001 1.26±0.05 50 > 0.05 e. faecalis 0.73±0.07 37 < 0.001 0.92±0.11 42 < 0.001 op50 1.04±0.12 40 1.32±0.06 37 231 a) δ o d 60 0 / p ro te in [m g] fig. 3 worm ingestion behaviors after exposed to pathogens and the standard food source e. coli op50 for different time period. a) wild type n2 ingestion changed for 24 h and 96 h. b) bacteria uptaking of sek-1 mutants for 24 h and 96 h. host and invaders. to our knowledge, e. faecalis is a classic animal pathogen, colonizes and proliferates in the gut of worm (garsin et al., 2001). we also reported that pxo99 and jxoiii infected c. elegans in a similar manner with e. faecalis. it is obvious that e. faecalis has stronger pathogenicity than xoo in the worm host and animal pathogen has essential differences with phytopathogen. this might be one explanation for the more consumption of pxo99 and jxoiii in contrast to e. faecalis. when compared the data in figure 3a (24 h) to the pictures in figure 2 (16 h), it seemed like there was a correlation between ingestion of bacteria and worm body size. therefore, it was interesting to know if the same size difference could be seen with sek-1 mutants. it indicated that after 96 h, all the worms we tested had longer body size than 24 h and the tendency of body length was identical to the differences of ingestion behavior (fig. 3b, table 1). we found that ingestion of jxoiii was fewer than pxo99 at 24 h while after 96 h the consumption of jxoiii was more than pxo99 and had no statistical significant difference with the control group. these phenomena were also verified in the body size (table 1). it could be explained with that the pxo99 is more virulent than jxoiii (bai et al., 2014). behavioral avoidance promotes nematode survival to xoo and e. faecalis exposure pathogenic bacteria represent an environmental challenge that can influence the worm survival, and c. elegans has the ability to detect and avoid pathogens (pradel et al., 2007). avoidance behavior in small-lawn of pa14 is a natural choice response, and confers survival benefit to the nematode (reddy et al., 2009). in our experiment, wild type n2 and sek-1 mutant worms were not susceptible to both e. faecalis and jxoiii in small-lawn killing assay (figs 4a, c). compared with this, survival in the big-lawn assay in which pathogenic bacteria jxoiii were covered the entire plate performed in our previous work and other research work on identification of traditional chinese medicine for promoting immunity of c. elegans against e. faecalis in our lab (personal communication) exhibited distinguished differences. the survival differences between small-lawn and big-lawn assays could be attributed to avoidance of the bacterial lawn (figs 1a, b). consistent with the δ o d 60 0 / p ro te in [m g] b) 232 a) b) to l-1 s ur vi vi ng fr ac tio n n 2 su rv iv in g fr ac tio n c) se k1 su rv iv in g fr ac tio n fig. 4 kaplan-meier survival plots of c. elegans mutant strains and wild type n2 treated with xanthomonas strain pxo99 and jxoiii and standard food source e. coli op50. assays were performed on small-lawn plates. pxo99 group (dark gray solid line), jxoiii group (black dotted line), e. faecalis group (dark gray dotted line), op50 (black solid line). results that tol-1 mutants are not more susceptible to e. faecalis than wild-type worms (tenor and aballay, 2007), the tol-1 mutants in our experimental condition also revealed similar survival patterns to n2 treated by e. faecalis (fig. 4b). however, although tol-1 mutants avoided jxoiii strongly, it seemed no benefit for survival compared to the control (p < 0.0001) (fig. 4b, table 2). this might be ascribed to the importance of tol-1 in the innate immune response to gram-negative bacteria (tenor and aballay, 2007). in contrast to e. faecalis and jxoiii, pxo99 decreased the survival of wild-type n2, tol-1 mutants, and sek-1 mutants remarkably (figs 4a c, table 2). it has been proved that pxo99 is more virulent than jxoiii in our previous data (bai et al., 2014), together with the no avoidance results (fig. 1), it indicated that pxo99 could successfully avoid the detection of immune system and shorten the lifespan of c. elegans. tissue-specific sek-1 activities regulate immune responses to xoo and e. faecalis sek-1 is a key component of p38 mapk signal pathway and plays an important role in immune responses to pathogen resistance (liberati et al., 2004). despite sek-1 was not involved in nematode 233 table 2 the effect of xoo and e. faecalis on lifespan of c. elegans lt50 (in days) first and last death strains and genotype p-value a treatment mean sd min max n2, wild type pxo99 12.0 0.4 7 20 p < 0.0001 jxoⅲ 14.5 0.2 10 18 p = 0.38 14.2 0.4 8 20 p = 0.72 e. faecalis op50 14.6 0.3 9 21 ig10, tol-1(nr2033) pxo99 7.1 0.3 2 14 p < 0.0001 jxoⅲ 9.4 0.4 5 14 p < 0.0001 10.6 0.4 5 16 p = 0.12 e. faecalis op50 11.5 0.5 4 17 ku4, sek-1(km4) pxo99 6.7 0.4 1 15 p < 0.001 jxoⅲ 10.6 0.4 2 17 p = 0.73 12.2 0.5 5 16 p = 0.11 e. faecalis op50 8.9 0.6 1 19 ku4, sek-1(km4) pxo99 6.7 0.4 1 15 p < 0.0001 zd193 (intestine) pxo99 11.4 0.4 3 20 p = 0.73 zd202 (neurons) pxo99 9.2 0.7 5 16 p < 0.0001 zd260 (ciliated sensory neurons) pxo99 8.0 0.6 5 14 p < 0.0001 n2, wild type pxo99 12.0 0.4 7 20 a p-value (log rank test) compared with control group of op50. avoidance behavior to xoo and e. faecalis, we proposed that tissue-specific sek-1 expressions participate in innate immunity of c. elegans. based on that no aversion was occurred when wild type n2 and sek-1 mutants exposed to pxo99, and both small-lawn and big-lawn could supply enough bacterial sources, we chose n2 and sek-1 mutants survival curves from experiments on ‘small lawn’ and zd260, zd202 and zd193 survival curves from ‘big-lawn’ assay for comparison. sek-1 mutation caused increased susceptibility to pxo99 (p < 0.0001) while transgenosis compromise the damage in different degrees (fig. 5). consistent with previous study that intestine is the main position where infection and immune response occur (garsin et al., 2001), intestinal activity of sek-1 (zd193 strain) was sufficient for nematode innate immune responses to pxo99 (fig. 5, table 2), survival curve had no significant difference from n2. zd260 and zd202 strains, sek-1 expressed in ciliated sensory neurons and all neurons respectively, both were susceptible to pxo99 like sek-1 mutants (fig. 5, table 2). however, compared with sek-1 mutants, zd202 (sek-1 in neuron cells) could alleviate the damage of pathogen pxo99 (p < 0.01, fig. 5), zd260 (sek-1 in ciliated neurons) had no difference on survival (p = 0.23, fig. 5). it suggested that sek-1 expressed in neuron cells conferred the host capability to resist pathogen infection. consistent with the survival assay, transcriptional levels of the cub-like genes c17h12.8 and f08g5.6 were higher in zd202 than in zd260 (figs 6a, b). 234 p x o 99 -t re at ed w or m s su rv iv in g fr ac tio n fig. 5 survival plots of worms treated with xanthomonas strain pxo99. small-lawn assay results of wild type n2 and sek-1 mutants treated by pxo99 were chosen for control groups, which owing to that n2 and sek-1 mutants showed no avoidance towards pxo99 and enough bacterial resources could be supplied on small-lawn plates like big-lawn. big-lawn method was employed for transgenic nematodes zd193, zd202 and zd260, which sek-1 expressed specifically in intestine, neurons system, and ciliated sensory neurons. pxo99-treated wild type n2 (black solid line), sek-1 mutant (dark gray solid line), zd260 (dark gray dotted line), zd202 (black dotted line), zd193 (light gray dotted line). downstream genes c17h12.8, f08g5.6, t24b8.5, and f56d6.2, act as immune effectors and expression levels are enhanced significantly to defend infection attack (troemel et al., 2006). cub-like genes c17h12.8 and f08g5.6 were significantly upregulated in wild type n2 (figs 6a, b), together with the upregulated c-type lectin gene f56d6.2 (fig. 6c), it showed that pxo99, jxoiii and e. faecalis could activate p38 mapk pathway to defend against infection. sek-1 mutation led to no response that all these effector genes nearly had no transcript (figs 6a d). however, tissue-specific expression of sek-1 could rescue the defense response. zd193, sek-1 specifically expressed in the intestine, effector genes c17h12.8, f08g5.6, f56d6.2 and t24b8.5 were remarkably upregulated after treated by pxo99 and jxoiii (figs 6a d). among these results, jxoiii could induce higher gene transcriptional levels (figs 6a, b and d), it further verified that jxoiii was less virulent than pxo99. zd202 (sek-1 in neuron cells) and zd260 (sek-1 in ciliated neurons) could trigger lower gene expressions compared to zd193 (sek-1 in intestine) (figs 6a d). tol-1 did not participate the avoidance response (figs 1a, c) but it may be involved in immune response to gram-negative bacteria like xoo (fig. 4b). during the infection process, c17h12.8 expression level in tol-1 mutants increased significantly after treated by pxo99 and jxoiii than control op50, but obviously reduced compared to the wild type n2 (fig. 6a). other effector genes we investigated did not have fold changes (figs 6b d). discussion as a bacterivore species, c. elegans encounters various bacteria including food source and dangerous pathogens in its habitat. avoidance behavior and conserved innate immune response are usually employed to defense against pathogenic bacteria (reddy et al., 2009). e. faecalis and jxoiii are animal and plant pathogens respectively, it indicated that worms could avoid the two strains effectively in the small-lawn assay (fig. 1). avoidance may related to the characteristics of the molecules produced by bacteria, such as the cyclic lipodepsipentapeptide serrawettin w2 produced by serratia (pradel et al., 2007), and molecules including toxic shock syndrome toxin 1 (tsst-1) and staphylococcal enterotoxin c (sec) secreted by staphylococcus aureus (osanai et al., 2012). it is necessary to elaborate the mechanism of aversion by c. elegans, however, based on the fact that the 235 a) b) d) c) fig. 6 transcriptional levels of effector molecules downstream of p38 mapk pathway after exposed to pathogenic bacteria. cub-like genes c17h12.8 a) and f08g5.6 b), c-type lectin gene f56d6.2 c), and shk toxin gene t24b8.5 d). we examined the fold changes of these genes to testify the roles of tol-1 and tissue-specific sek-1 in worm innate immunity to defend against the xoo and e. faecalis. results were obtained from 2 independent biological replicate and normalized to the control act-3. error bars represent sd. *t-test, p < 0.05 comparison to op50 groups. issue we want to discuss is the tol-1 and sek-1 roles in aversion and innate immunity of worms, molecules induced avoidance response are not included in our study. compared to the jxoiii, another xoo strain pxo99 did not induce worm avoidance response. we speculate that because pxo99 is more virulent than jxoiii, it may escape the detection and recognition system of the worm and make the worm maintain in the bacterial lawn to induce greater damage. sek-1 locates downstream of nsy-1, which are components of p38 mapk pathway in c. elegans innate immune system (kim et al., 2002). sek-1 mutation led to more sensitive to e. faecalis while there was no change in response to xoo (fig. 1b). consistent with the importance of nsy-1 mutation in enhanced avoidance (mori, 2008), sek-1 mutation may also cause more susceptibility to e. faecalis (fig. 1b). tol-1 is important for nematode in pathogen recognition, and that tol-1 mutants are defective in their aversion of pathogenic serratia marcescens (pujol et al., 2001). however, tol-1 mutation seemed have no forward or reversal effect on the aversion in our study (fig. 1c); it suggested that tol-1 did not play a universal role in recognizing pathogens and triggering avoidance response. in addition, sek-1 was not involved in aversion to pathogenic xoo. aversion is an indicator of unpleasant odorant or taste sensed by c. elegans. in order to study whether avoidance can influence the ingestion behavior, we examined bacterial consumption with wild type n2 and sek-1 mutants for 24 h and 96 h. as regular food source, op50 consumption was the most (fig. 3). no aversion was exhibited when exposed to pxo99 (fig. 1), thus uptaking of this bacterium seemed more than jxoiii and e. faecalis in n2 (fig. 3a). however, when prolonging the exposure time until 96 h, consumption of bacteria all increased (fig. 3b). we propose two explanations for this issue, (i) adaption to odorant or chemical repellents may attenuate responses to these stimuli (colbert and bargmann, 1995; hilliard et al., 2005), so that ingestion of bacteria increased. (ii) sek-1 was 236 table 3 sequence information for primers used in qrt-pcr gene sequence name primer sequence 5'tgtcatttcaatggaggatattgt3' c17h12.8 c17h12.8 5'tgatggagttggaggatattga3' 5'cacaatgatttcaatgcgaga3' f08g5.6 f08g5.6 5' tgctttcagaacacagtcagg3' 5' tgatggtgacagttcaaagc3' f56d6.2 f56d6.2 5' ttccaaaaatgcccgagtag3' 5' agaccatcatgcccttcact3' t24b8.5 t24b8.5 5' gtaacgcagacaccacaggt3' 5'ccatcatgaagtgcgacattg3' act-3 t04c12.4 5'catggttgatggggcaagag3' not involved in avoidance behavior to xoo (figs 1a, b), nevertheless, it plays important roles in c. elelgans innate immunity to resist bacterial pathogens (kim et al., 2004). mutation in sek-1 impairs the defense capability of the host, and pathogens can escape the detection system of worms and enter into the digestive tract. in addition, virulence of pathogens is also a key factor to influence the ingestion behavior. in our previous study, e. faecalis is more virulent to nematode than jxoiii (bai et al., 2014), it may enter the host digestive tract easier than jxoiii as we expected. however, although jxoiii and e. faecalis could be both avoided by sek-1 mutants effectively (fig. 1b), jxoiii could be ingested more than e. faecalis (fig. 3b). this may due to differences in virulent factors such as extracellular enzymes or extracellular polysaccharides used by these pathogens (shen and ronald, 2002). lifespan assay is always used to evaluate pathogenic bacterial effect on c. elelgans and defense responses of various mutant hosts to pathogens (irazoqui et al., 2010). in order to testify the tol-1 and sek-1 roles in host innate immunity, we performed small-lawn killing assay using tol-1 and sek-1 mutants. based on the aversion of jxoiii and e. faecalis (fig. 1b), survival of sek-1 mutants treated by the two strains had no differences compared to the control (fig. 4c, table 2). however, similar aversion tendency did not induce semblable survival results in tol-1 mutants (figs 1c, 4b), nematodes treated with jxoiii exhibited marked lifespan differences compared with control op50 (fig. 4b, table 2). it indicated that tol-1 may participate the innate immune response to gram-negative bacterium xoo (tenor and aballay, 2007). although tol-1 and sek-1 are both important in host immune response to xoo, tol-1 is considered to be independent of p38 mapk pathway (aballay et al., 2003). however, transcriptional level of cub-like gene c17h12.8 increased when tol-1 mutants treated by xoo pxo99 and jxoiii, and other genes we examined nearly had no changes (fig. 6). we hypothesize that tol-1 and sek-1 may have overlapping roles in defense against the xoo, and the host integrates multiple cues to respond the environmental stimulus. interaction between tol-1 and sek-1 needs further investigation. p38 mapk pathway acts as a pivotal immune signaling module in c. elegans, it can be activated in a range of tissues where specific immune responses are triggered (bolz et al., 2010). intestinal epithelium and nervous system are the common focus for investigating immune defense mechanism (pukkila-worley and ausubel, 2012; takeda and ichijo, 2002). therefore, transgenic worms expressed sek-1 in ciliated sensory neurons (zd260), neurons system (zd202) and intestine (zd193) were used to study the roles of tissue-specific sek-1 in innate immune response to xoo infection. it showed that intestine expression could rescue the survival of worms while neurons system and ciliated sensory neurons expression just attenuated the reduced lifespan compared to sek-1 mutants (fig. 5, table 2). consistent with this, effector molecules downstream of p38 mapk pathway such as c17h12.8, f08g5.6, f56d6.2, and t24b8.5 had enhanced transcriptional levels in zd193 (intestine expression) (fig. 6). fold changes of c17h12.8 and f08g5.6 were higher in zd202 (neurons) than zd260 (ciliated sensory 237 http://legacy.wormbase.org/db/seq/sequence?name=r13h8.1;class=gene_name http://legacy.wormbase.org/db/seq/sequence?name=t04c12.4;class=gene_name http://legacy.wormbase.org/db/seq/sequence?name=t04c12.4;class=gene_name garsin da, sifri cd, mylonakis e, qin x, singh kv, murray be, et al. a simple model host for identifying gram-positive virulence factors. proc. natl. acad. sci. 98: 10892-10897, 2001. neurons) (fig. 6), it suggested that sek-1 expressed in neurons could confer the host limited resistance to the xoo. in conclusion, our findings reveal that sek-1 and tol-1 are not involved in c. elegans avoidance behavior and ingestion of nematodes is related to the aversion and also the characteristics of bacteria. in addition, tol-1 and sek-1 participate the immune response to the infection by xoo, sek-1 also exhibits tissue-specific activities in host innate immunity. overlapping effect may exist between the tol-1 and sek-1. hasshoff m, böhnisch c, tonn d, hasert b, schulenburg h. the role of caenorhabditis elegans insulin-like signaling in the behavioral 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regulates expression of immune response genes and contributes to longevity in c. elegans. plos genet. 2: e183, 2006. 239 cellular aspects of allorecognition in the compound ascidian botrylloides simodensis isj 11: 219-223, 2014 issn 1824-307x short communication cellular aspects of allorecognition in the compound ascidian botrylloides simodensis n franchi1, e hirose2, l ballarin1 1department of biology, university of padua, padua, italy 2department of chemistry, biology and marine science, faculty of science, university of the ryukyus, nishihara, okinawa, japan accepted july 1, 2014 abstract when colonies of the compound ascidian botrylloides simodensis contact each other at their cut surfaces, either fusion or rejection occurs. contact between genetically compatible colonies leads to the complete fusion of their tunics and vasculature within 24 h. conversely, the rejection reaction between incompatible colonies is characterized by the appearance of a melanic, necrotic band along the contact border. in the case of fusion, limited crowding of cytotoxic morula cells (mcs) was observed in the ampullae near the contact border. in rejection, limited tunic fusion occurred in the contact region and mcs were selectively recruited inside the ampullae near the cut surface: most of them leaked into the tunic where they changed their morphology and contributed to the formation of the necrotic region. granular amebocytes, like mcs, have granules well stained by eosin and were also seen inside the ampullae involved in the rejection reaction and along the contact border between incompatible colonies. immunohistochemical analysis using antibodies raised against botryllus schlosseri phenoloxidase (po) and mammalian il-1-α and tnf-α indicate that mcs were the only cells recognized by the anti-po antibody; they resulted immunopositive also to the anti-cytokine antibodies in both fusion and rejection, whereas granular amoebocytes were recognized by the latter antibodies only during the rejection reaction. key words: botrylloides simodensis; ascidians; colony specificity; hemocytes; morula cells introduction botryllid compound ascidians share the ability of intraspecific colony recognition (colony specificity) which enables contacting colonies to fuse their tunic and circulation when genetically compatible (taneda et al., 1985; saito et al., 1994). in the case of incompatibility, contacting colonies do not fuse and, frequently, a rejection reaction occurs. the latter, typically, manifests itself as a series of necrotic, melanic spots along the contact border (rinkevich, 1992; saito et al., 1994). in this reaction, the key role of the enzyme phenoloxidase (po) in the induction of cytotoxicity was clearly established (ballarin et al., 1995, 1998; shirae and saito, 2000; shirae et al., 2002; cima et al., 2004). po is stored inside the vacuoles of morula cells (mcs), an ubiquitous haemocyte type in botryllid ascidians, together with its polyphenol substrata and quinones ___________________________________________________________________________ corresponding author: loriano ballarin department of biology university of padua via ugo bassi 58/b, 35100 padua e-mail: loriano.ballarin@unipd.it (ballarin et al., 1995; shirae and saito, 2000; ballarin, 2008). in the course of the rejection reaction, a typical inflammatory reaction takes place, involving the selective recruitment of mcs in the blind endings of the peripheral tunic vasculature, called ampullae, facing the alien colony, their subsequent crossing the epithelium of the ampullar tips and migration into the tunic. here, the induction of cytotoxicity occurs through mc degranulation and the release of mc vacuolar content (hirose et al., 1990; sabbadin et al., 1992; shirae et al., 2002; cima et al., 2004; ballarin, 2008). temporary crowding of mcs inside the facing ampullae was observed also in the course of the interaction between genetically compatible colonies of b. schlosseri and botrylloides leachi but, in this case, the amount of mcs is significantly lower than that observed in contacting incompatible pairs and no migration in the tunic occurs (cima et al., 2006; zaniolo et al., 2006; ballarin and zaniolo, 2007). in b. schlosseri, mc chemotaxis and cytotoxicity is modulated by cytokines (cima et al., 2004, 2006). these immunomodulatory molecules are recognized by antibodies raised against 219 mailto:loriano.ballarin@unipd.it fig. 1 colonies of b. simodensis after 24 h from the contact at their cut surfaces. a: genetically compatible colonies a, b) showing complete fusion of the tunic; b: incompatible colonies (c, d) with a necrotic, melanic line clearly visible along the contact border. scale bar: 5 mm. mammalian il-1-α and tnf-α and are synthesized and secreted by mcs upon the recognition of nonself molecules (ballarin et al., 2001) and, in addition to mcs themselves, they can influence also the behavior of phagocytes (menin et al., 2005; menin and ballarin, 2008). the immunopositivity of mcs to the above-reported antibodies has been observed also in the course of the intensive rejection reaction observed when genetically incompatible colonies of b. leachi are brought into contact at their cut surfaces. in this case, also granular cells, considered mc precursors, resulted positive (ballarin and zaniolo, 2007). botrylloides simodensis is a japanese ascidian where a clear allorecognition reaction, either fusion or rejection, is observed when colonies contact at their cut surfaces. even in this case, the pivotal role of mcs and po in the rejection reaction was clearly demonstrated (shirae et al., 2002). in the present study, we continued our previous investigations on the cellular events in colony specificity of b. simodensis, using a panel of antibodies to compare the behavior of mcs in both fusion and rejection reactions at the contacting cut surfaces, with particular reference to the expression of putative cytokines. results were compared with what already known in the reference species b. schlosseri. materials and methods animals colonies of b. simodensis were collected near shimoda (shizuoka prefecture, japan). they were attached to glass slides and reared in culture boxes immersed in nabeta bay. cut surface allorecognition assay colonies were cut with a razor blade and pieces of the same size from different colonies were brought into contact at their cut surfaces on a glass slide and left to adhere for 2 h in a moist chamber. juxtaposed fragments of the same colony were used as reference controls (compatible pairs). slides were maintained for 24 h in plastic petri dishes filled with filtered seawater (fsw) and the outcome of the reaction at the contact area was then observed under a binocular microscope. immunohistochemical analyses of contacting colonies colony pairs were fixed in 4 % formaldehyde in fsw, dehydrated through a butanol series and embedded in paraffin. deparaffined sections (6 µm) were treated for 30 min with 1 % h2o2 in 80 % methanol to block endogenous peroxidase, for additional 30 min in 1 % skimmed powdered milk in phosphate-buffered saline (pbs: 8 g/l nacl, 0.2 g/l kcl, 0.2 g/l kh2po4, 1.15 g/l na2hpo4, ph 7.2) for the reduction of unspecific staining and incubated overnight with 10 µg/ml primary antibody raised against b. schlosseri po (frizzo et al., 1999), human recombinant il-1-α and human recombinant tnf-α (santa cruz biotech., santa cruz, ca, usa), in pbs. slides were then washed in pbs, incubated for 30 min in 50 µg/ml biotinylated anti-rabbit igg antibody (santa cruz biotech.) in pbs, washed again and incubated for 30 min in the avidin-biotinperoxidase complex (abc, vector labs., burlingame, ca, usa). after thorough washing in pbs, sections were finally incubated for 5 min in a solution of 0.025 % 3,3’ diaminobenzidine (dab) containing 0.004 % h2o2 and mounted with eukitt. positive sites appeared brown. reference slides were stained with hematoxylin and eosin. results fusion and rejection reactions in the case of compatible pairs, no cytotoxic reaction was observed and complete tunic fusion was observed after 24 h from the contact of the cut 220 fig. 2 paraffin sections of contacting genetically compatible and incompatible colonies of b. simodensis. a: lumen of a peripheral ampulla facing a compatible colony. limited recruitment of eosinophilic mcs (arrowheads) is observable. b: contact region between incompatible colonies (right and left of the dashed lines); arrows indicate the regions of fusion of the contacting tunics. c: lumen of a peripheral ampulla facing an incompatible colony. selective recruitment of eosinophilic mcs (dark arrowheads) and some granular amoebocytes (open arrowheads) are observable. d-f: contact border between incompatible colonies. massive leakage of mcs in the tunic (d), where they show altered morphology (e), together with some granular amoebocytes (f) can be observed. scale bars: 20 μm in e, f; 50 μm in a, c, d; 250 mm in b. surfaces (fig. 1a). limited cell crowding occurred in the ampullae near the contact border: most of them were mcs which, as already reported (shirae et al., 2002; ballarin and zaniolo, 2007), had granules well stained by eosin. cell leakage was never observed (fig. 2a). in the case of incompatible pairs, a clear necrotic, melanic band (fig. 1b) characterized the border between the cut surfaces after 24 h from the contact (shirae et al., 2002). limited tunic fusion was observed in the contact region (fig. 2b), as well as selective recruitment of mcs inside the ampullae close to the contact border (fig. 2c). many of these cells leaked from the ampullar lumen into the tunic, crowding along the border, where they showed altered morphology and contributed to the formation of the necrotic region (fig. 2d). granular amoebocytes like mcs, have granules stained by eosin and also increase their frequency inside the ampullae involved in the rejection reaction, although to a lesser degree than mcs, and in the tunic, close to the contact border (figs 2c, f). immunohistochemical analyses during fusion and rejection reactions circulating mcs resulted immunopositive to anti-po antibodies in both fusing and non-fusing colonies (figs 3a, b). a similar result was observed with the anti-cytokine antibodies (figs 3c-f). conversely, granular amoebocytes inside the ampullar lumen never resulted immunopositive to the assayed antibodies in the case of fusion, whereas a clear positivity was observed during the rejection reaction (figs 3d, f). no recognition the cells of the ampullar epithelium or of the tunic by the antibodies was observed. discussion allorejection reaction between contacting, genetically incompatible colonies, has been extensively studied in the compound ascidian b. schlosseri, where a typical inflammatory reaction occurs in the colonial vasculature facing the alien colony. in this species, the pivotal role of cytotoxic mcs as effectors of the reaction has been demonstrated (ballarin et al., 1995). these cells, directly involved in immunosurveillance, are able to sense non-self molecules (ballarin, 2008; ballarin et al., 2001) and, as a consequence of the recognition, they release immunomodulatory molecules recognized by antibodies raised against mammalian pro-inflammatory il-1-α and tnf-α (ballarin et al., 2001, 2005). in the case of rejection reaction, these molecules, released as a consequence of the recognition of soluble allogeneic factors diffusing 221 fig. 3 immunohistochemical analysis on paraffin sections of facing ampullae in fusion (a, c, e) and non-fusion (b, d, f) reactions between contacting colonies of b. simodensis. a, b: anti-po antibody. positivity is limited to mcs (dark arrowheads) in both fusion (a) and rejection (b); granular amoebocytes (open arrowheads) are never stained. c, f: anti-il-1-α (c, d) and anti-tnf-α (e, f) antibodies. mcs (dark arrowheads) are stained in both fusion (c, e) and non-fusion (d, f), whereas granular amoebocytes (open arrowheads) are stained only in the case of rejection (d, f), scale bars: 10 μm in a; 50 μm in b-f. from the circulation of the alien colony through the partially fused tunics, are responsible of the selective recruitment of mcs inside the facing ampullae which characterizes the early phases of the rejection reaction (cima et al., 2006). these cells, then, cross the ampullar epithelium and enter the tunic where they degranulate releasing their vacuolar content, mainly the enzyme po and its substrata (ballarin et al., 1998, 1998; cima et al., 2004) which are responsible of the formation of the melanic, necrotic spots clearly visible along the contact border (sabbadin et al., 1992). in b. simodensis, humoral factors involved in the rejection reaction have been partially characterized from the blood plasma: they are heat labile, resistant to dialysis and divalent cationdependent (saito and watanabe, 1984). in the same species, the rejection reaction between contacting growing edges of genetically incompatible colonies occurs in the form of a socalled “subcuticular rejection”. in the course of this reaction, a few hemocytes leak from the ampullar lumen into the tunic and contribute to the formation of small necrotic regions along the contact border which are scanty visible under the binocular microscope (hirose et al., 1997). however, if colonies were brought into contact at their cut surfaces (cut surface assay), a more intense and rapid cytotoxic reaction can be observed (hirose et al., 1990). in the present work, we performed this latter assay in order to have a clear view of the events occurring during the rejection reaction at the contacting cut surfaces of incompatible colonies of b. simodensis. as reported elsewhere (shirae et al., 2002), in the course of the reaction, mcs selectively crowd inside the ampullae near the contacting cut surfaces and result positive to the cytoenzymatic assay for po (shirae et al., 2002, present work). the selective recruitment, although less marked, was observed also in the case of fusible pairs. however, in the case of incompatible pairs, mcs leak from the ampullae and enter the tunic where they change their morphology and discharge their vacuolar content (shirae et al., 2002). in both compatible and incompatible combinations, mcs resulted strongly labeled by the antibodies against mammalian cytokines. this supports the idea that, analogously to b. schlosseri, mcs of b. simodensis release immunomodulatory molecules upon the contact with another colony. in b. leachi, which has a behavior similar to b. simodensis with regard to allorecognition (i.e., it gives a limited subcuticular rejection at the growing edges and an intense reaction at the cut surfaces), immunopositivity to anti-il1-α and anti-tnf-α was observed only in incompatible combinations. in b. simodensis, contrarily to our expectations, immunopositivity was observed also in the case of contact between compatible pairs, the only difference being represented by the presence of immunopositive granular amoebocytes in allorejection which were not observable in fusible pairs. during the rejection reaction, these cells, considered related to mcs, are recruited inside facing ampullae, migrate into the tunic and secrete molecules recognized by antibodies raised against mammalian proinflammatory cytokines. in b. schlosseri and b. 222 leachi, granular amoebocytes are considered the precursors of mcs, on the basis of common staining properties (cima et al., 2001; ballarin and cima, 2005) and, in b. leachi, they can be found along the contact surfaces of incompatible pairs (ballarin and zaniolo, 2007). as stated before, in b. schlosseri, molecules recognized by antibodies raised against mammalian il-1-α and tnf-α recognize mcs and modulate the rejection reaction. we assume that a similar role is exerted by the same molecules also in our species: this hypothesis is supported by the observation that the above-reported antibodies recognize the mcs involved in the rejection reaction at cut surfaces of incompatible colonies of b. leachi (ballarin and zaniolo, 2007). unlike b. schlosseri and b. leachi, b. simodensis granular amoebocytes do not show any detectable po activity. therefore, it results that, in b. simodensis as well as in b. leachi, cytotoxic cells can exert their immunomodulatory role even before their full maturation to morula cells. this resemble the behavior of circulating mammalian monocytes that, although not fully differentiated to functional macrophages, can contribute to the modulation of immune responses (saha and geissman, 2011; patel and davidson, 2014). further studies will be directed to a better clarification of the role of granular amoebocytes in b. simodensis and their functional relationships with mcs. acknowledgements this research was supported by the italian miur. we thank dr. yasunori saito (university of tsukuba) for helping in the preparation of the 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fuscus. biol. bull. 192: 53-61, 1997. menin a, ballarin l. immunomodulatory molecules in the compound ascidian botryllus schlosseri: evidence from conditioned media. j. invertebr. pathol. 99: 275-280, 2008. menin a, del favero m, cima f, ballarin l. release of phagocytosis-stimulating factor(s) by morula cells in a colonial ascidian. mar. biol. 148: 225230, 2005. patel h, davidson d. control of pro-inflammatory cytokine release from human monocytes with the use of an interleukin-10 monoclonal antibody. methods mol. biol. 1172: 99-106, 2014. rinkevich b. aspects of the incompatibility nature in botryllid ascidians. anim. biol. 1: 17-28, 1992. sabbadin a, zaniolo g, ballarin l. genetic and cytological aspects of histocompatibility in ascidians. boll. zool. 59: 167-173, 1992. saha p, geissmann f. toward a functional characterization of blood monocytes. immunol. cell biol. 89: 2-4, 2011. saito y, watanabe h. partial biochemical characterization of humoral factors involved in the nonfusion reaction of a botryllid ascidian, botrylloides simodensis. zool. sci. 1: 229-235, 1984. saito y, hirose e, watanabe h. allorecognition in compound ascidians. int. j. dev. biol. 38: 237247, 1992. shirae m, saito y. a comparison of hemocytes and their phenoloxidase activity among botryllid ascidians. zool. sci. 17: 881-891, 2000. shirae m, ballarin l, frizzo a, saito y, hirose e. involvement of quinones and phenoloxidase in the allorejection reaction in a colonial ascidian, botrylloides simodensis: histochemical and immunohistochemical study. mar. biol. 141: 659-665, 2002. taneda y, saito y, watanabe h. self or non-self discrimination in ascidians. zool. sci. 2: 433442, 1985. zaniolo g, manni l, ballarin l. colony specificity in botrylloides leachi. i. morphological aspects. inv. surv. j. 3: 125-136. 223 abstract introduction forests isj 11: 337-344, 2014 issn 1824-307x research report foraging and oviposition of thyrinteina leucoceraea (lepidoptera: geometridae) on introduced and native hosts in brazil sprayed with the protease inhibitor benzamidine cl oliveira1, a pallini filho2, w de s tavares3, mg de a oliveira4, je serrão5, jc zanuncio2 1movimento de educação promocional do espírito santo, associação da escola família agrícola de castelo, 29360-000, castelo, espírito santo state, brazil 2departamento de entomologia, universidade federal de viçosa, 36570-900, viçosa, minas gerais state, brazil 3departamento de fitotecnia, universidade federal de viçosa, 36570-900, viçosa, minas gerais state, brazil 4departamento de bioquímica e biologia molecular, universidade federal de viçosa, 36570-900, viçosa, minas gerais state, brazil 5departamento de biologia general, universidade federal de viçosa, 36570-900, viçosa, minas gerais state, brazil accepted november 25, 2014 abstract the protease inhibitor (pi) benzamidine may be an option for protecting introduced myrtaceae plants from insect pests. the foraging behavior of the larvae (number of larvae per plant) and oviposition (number of egg masses per plant and eggs per mass) of thyrinteina leucoceraea (lepidoptera: geometridae) females were evaluated on an introduced myrtaceae (eucalyptus grandis) and a native one (psidium guajava), both sprayed by the pi benzamidine in aqueous concentrations of 0.000, 0.125, 0.250, 0.375, and 0.500 mol.l–1 and with the adhesive spreader triton x-100 at 0.01 % (mg.ml–1) in water as a control. the foraging preference by t. leucoceraea was similar between the different concentrations of the pi benzamidine on the treated host plants and the control. the numbers of egg masses per plant and eggs per mass of t. leucoceraea were similar between the treatments, but this insect showed slighter oviposition preference on non-sprayed e. grandis plants than on those of p. guajava sprayed with different concentrations of the inhibitor pi benzamidine. similar foraging of larvae among treated plants and the lower reproduction of t. leucoceraea on treated e. grandis plants, show possibilities of using the pi benzamidine in the management programs of this herbivore, in this culture. key words: benzamidine; eucalyptus grandis; geometrid moth; protease inhibitor; psidium guajava   introduction insect herbivores need free amino acids (organic molecules with an amino group, a carboxyl group, and a specific lateral chain) and nitrogen, which can be obtained from the degradation of peptide bonds between the amino acids forming proteins, by extracellular enzymes named as proteases, in process called proteolytic cleavage (bazok et al., 2005). plants can express specific protease inhibitors (pis) in response to adverse abiotic or biotic factors, such as herbivory by insects (moreira et al., 2011). proteolytic enzymes in the gut of the insect herbivores (digestive system) can reduce the availability of amino acids when inhibited ___________________________________________________________________________ corresponding author: josé cola zanuncio departamento de biologia animal universidade federal de viçosa 36570-900, viçosa, minas gerais state, brazil e-mail: zanuncio@ufv.br by pis from plants, which may affect the physiological processes, such as lengthening the life cycle and reducing the fecundity and the fertility of insects (oliveira et al., 2005). natural (isolated from plants) or synthetic (produced in the laboratory and commercially available) pis can be used for the protection of plants against insect herbivores (xavier et al., 2005) with specific genes introduced via genetic manipulation to encode the pis, which induce resistance (delledonne et al., 2001), or with synthetic pis in food (artificial diets in the laboratory). these pis can lengthen development, reduce the reproductive parameters, and cause insect mortality (population control) (pilon et al., 2006). benzamidine is a competitive, reversible inhibitor of the trypsin (enzyme that acts upon proteins of the chyme), trypsin-like, and of serine proteases (primary amino acid, non-essential, which 337 mailto:zanuncio@ufv.br comprises of a majority of glycolipids in animal cells) (macedo and freire, 2011; ranjbar et al., 2011). the insecticidal potential of the synthetic form of pi benzamidine was tested to evaluate the response of the eucalypt defoliator, thyrinteina arnobia arnobia (lepidoptera: geometridae) to this inhibitor when pulverized on plants or mixed in food (marinhoprado et al., 2011). the weight of larvae and reproductive parameters of sugarcane borer, diatraea saccharalis (lepidoptera: pyralidae) were lower with food (artificial diet) treated with pis from seeds of soybean, glycine max (fabaceae) (soybean proteinase inhibitor, spi) (pompermayer et al., 2001). the larvae survival of australian bollworm, helicoverpa punctigera (lepidoptera: noctuidae) was lower with food (artificial diet) treated with a common inhibitor of the solanaceae family (winged tobacco, nicotiana alata proteinase inhibitor, napi) (dunse et al., 2010). the t. arnobia arnobia and the thyrinteina leucoceraea (lepidoptera: geometridae) (native species to brazil) are the main defoliators of eucalyptus, eucalyptus spp. (myrtaceae) plants in some regions in brazil (grosman et al., 2005; oliveira et al., 2010). monocultures (large-scale of a single culture in one area) of the native plants of myrtaceae in brazil, as those of the genera campomanesia, eugenia, and psidium, can be damaged by native lepidoptera (wessels et al., 2007). its larvae can feed and develop on introduced or native hosts of this family, such as eucalyptus spp. and guava, psidium guajava, respectively (holtz et al., 2003). lepidoptera defoliators of eucalyptus spp. can be controlled with biological and chemical methods, but their deficiencies depends on monitoring programs such as the use of light or pheromone traps, to observe their population levels (zanuncio et al., 2003, 2012; freitas et al., 2005). damage on tasmanian blue gum, eucalyptus globulus (myrtaceae) plants caused by insect pests is higher in susceptible materials than in resistant ones, although those susceptible have a higher concentration of secondary metabolic compounds that could be toxic, such as, phenols, essential oils, and tannins (rapley et al., 2004). the aim of this study is to evaluate the foraging behavior of larvae and of oviposition site selection by t. leucoceraea females on the rose gum, eucalyptus grandis (myrtaceae) (introduced host) and p. guajava (native host) plants, sprayed with different concentrations of synthetic pi benzamidine compared with triton x-100 as control. material and methods obtaining insects the larvae of t. leucoceraea were obtained from the mass rearing of this insect, which originated from individuals that were collected from e. grandis and p. guajava plants in the field and preserved in the original hosts at 25 ± 1 ºc, 12-h photoperiod and 70 ± 10 % relative humidity in rearing chamber in the department of animal biology (dba) of the “universidade federal de viçosa (ufv)” in viçosa, minas gerais state, brazil. thyrinteina leucoceraea pupae were collected with fig. 1 chemical structure of benzamidine. international union of pure and applied chemistry (iupac) name: benzenecarboximidamide. molecular formula: c7h8n2. molar mass: 120.15 g.mol–1. a forceps when they were attached to the e. grandis and p. guajava leaves and placed in plastic petri dishes (9 cm diameter × 1.5 cm height) lined with cotton wool. the adults obtained from these pupae were reared in wooden cages (0.60 m width × 0.60 m length × 1.00 m height) closed with a thin organza-type fabric. egg masses obtained from these adults were removed from the cages with a brush and placed on cotton strips inside the plastic petri dishes with a cotton swab moistened with water, per dish. soon after hatching, the larvae were placed in cages similar to those described, and they received leaves of the e. grandis or p. guajava plants, about 90 days old, kept in vases (one plant per vase), with a capacity of 20 l, with a vermiculitetype substrate (hydrated basaltic minerals) and without synthetic chemicals. the substrate of the vessels was moistened daily in the morning with 2 l of water irrigated uniformly on it. fourth-instar larvae were used for feeding preference and oviposition tests because they had higher food consumption rates and mobility on the host plants (marinho-prado et al., 2011). obtaining solutions aqueous solutions were prepared with pi benzamidine (fig. 1) in different concentrations of 0.000, 0.125, 0.250, 0.375, and 0.500 m (or mol.l–1) and at 0.01 % (mg.ml–1) of the adhesive spreader triton x-100 (chemical product that increases the efficiency of pi benzamidine by reducing the surface tension of the inhibitor sprayed on leaves, due to the increased power of wetting and adhesion) (fig. 2). these solutions were sprayed on e. grandis or p. guajava leaves as treatments: e. grandis + 0.000 m of pi; e. grandis + 0.125 m of pi + 0.01 % (mg.ml–1) of triton x-100; e. grandis + 0.250 m of pi + 0.01 % (mg.ml–1) of triton x-100; e. grandis + 0.375 m of pi + 0.01 % (mg.ml–1) of triton x-100; e. grandis + 0.500 m of pi + 0.01 % (mg.ml–1) of triton x-100; p. guajava + 0.000 m of pi; p. guajava + 0.125 m of pi + 0.01 % (mg.ml–1) of triton x-100; p. guajava + 0.250 m of pi + 0.01 % (mg.ml–1) of triton x-100; p. guajava + 0.375 m of pi + 0.01 % (mg.ml–1) of triton x-100; p. guajava + 0.500 m of pi + 0.01 % (mg.ml–1) of triton x-100, and the control with 0.01 % triton x-100. each plant was sprayed with 20.0 338 ml of pi benzamidine solution and compared with the control (without pi benzamidine). benzamidine is economic to be sprayed on plants (silva et al., 2010) and has no known negative effect on nontarget organisms (oliveira et al., 2006). statistical analysis the experimental design was completely randomized (dcr). data obtained from the tests of feeding and oviposition preferences were subjected to variance analysis (one-way, anova). a regression was adjusted, to explore and infer the relationship between the concentrations of the products and the plant species used as a model in the tests, and the r2 value was calculated by the equation obtained. the means of the number of t. leucoceraea eggs by mass were analyzed by the tukey test (p ≤ 0.05) using the software ‘r’ version 2.2.1 (ritz and streibig, 2005) provided by the federal university of viçosa (ufv). food preference test each treatment had four replications, with one plant each. a total of 120 thirty-fourth-instar t. leucoceraea larvae, without food for 12 h, were used per treatment. the feeding preference test was conducted in wood cages closed with organza-type fabric representing arenas, with six plants arranged in a hexagon and alternated by treatment and replication. the t. leucoceraea larvae were released in the center of the hexagon (arena), free to choose their hosts, who had the same chance of being colonized. the presence of larvae on the plants was recorded according to the position (layout) of the plants in the arena, time of observation, and treatment. nine evaluations were performed every hour after the start of the test and the last after 24 h. the larvae were removed from the plants after each evaluation. results and discussion the foraging (number of larvae per plant) of t. leucoceraea was similar between the hosts (introduced and native) with different concentrations of pi benzamidine and the control (water and triton x-100) (table 1). the positions of the plants and reading times also did not show effect on the foraging by larvae of this species (table 1). the similar foraging (number of larvae per plant) by t. leucoceraea on either ‘sprayed’ or ‘not sprayed’ plants (control), with different concentrations of pi benzamidine, suggests lack of detection of this compound by the chemoreception system of this insect on the hosts. these chemoreceptors are normally in the ovipositor and antennas of adults and in the mouthparts of immature (kim and mullin, 2003). protease inhibitors can be used in pest control, in agriculture and forestry, but can be recognized by larvae and nymphs, which can avoid plants with these substances (pereira et al., 2005). t. leucoceraea was collected from native plants of the family myrtaceae, grown in the vegetation belts, in brazil, where their populations could increase, and colonize eucalyptus spp. plants that were grown near them. however, treated plants or expressing pis may reduce the development, reproduction, and survival of this pest (pilon et al., 2006). the choice of the host for egg laying is made by the female (stadler et al., 2002) and the success of their offspring depends on suitable oviposition locations (site). however, t. leucoceraea larvae from the fourth-instar can have higher mobility and avoid contact with plants expressing or sprayed with pis, as observed for those of the fourth-instar of glanville fritillary, melitaea cinxia (lepidoptera: nymphalidae) and the european corn worm, ostrinia nubilalis (lepidoptera: crambidae), avoiding plants expressing toxic compounds (reudler talsma et al., 2008; suverkropp et al., 2008). compounds from plants, especially those produced after herbivore damage can affect development and cause mortality, especially of firstinstar larvae, which are more susceptible (scott et al., 2010). this was shown by the synthesis in higher quantity of jasmonic acid (vegetable endogenous regulator that can be extracted from the volatile portion of essential oils from oleaceae plants after herbivory). this compound becomes more concentrated in the plant and activates the expression of genes, to codify for the production of oviposition preference test t. leucoceraea females, from mass rearing in cages, were used in the oviposition preference test. the solutions with pi benzamidine and the control (water and triton x-100) were prepared similarly to the food preference test. the cages with a wooden structure, as described, were closed with organzatype fabric, with equidistant plants in a hexagon and alternated to arena formation. eucalyptus grandis and p. guajava plants had about 40 cm height and the same number of developed leaves. each plant was sprayed with 30 ml of one of the concentrations of pi benzamidine solution, according to the treatment, with a plastic spray bottle with a capacity of 500 ml and the control with only water and triton x-100 solution in the same quantity. t. leucoceraea females were mated in the rearing cages and released in the center of the hexagon forming arenas. the egg masses of this lepidoptera were removed, recording their position on the plant, reading time, and treatment. readings were taken 12, 24, and 36 h after release of the insects. fig. 2 chemical structure of triton x-100. molecular formula: c14h22o(c2h4o)n, with n = 9 or 10. molar mass: 647 g.mol–1. 339 table 1 number of t. leucoceraea (lepidoptera: geometridae) larvae recaptured on p. guajava (native host) or e. grandis (introduced host) (myrtaceae) plants to brazil in five concentrations (m or mol.l-1) of the protease inhibitor (pi) benzamidine values (mean ± standard error) per column per species do not differ by tukey test (p ≤ 0.05). control without ip benzamidine (opi) (0.01 % mg.ml-1 of aqueous solution of the adhesive spreader triton x-100) and treatment with pi benzamidine (wpi). pi, which have great toxic potential, mainly for the early instar lepidoptera (fortunato et al., 2007). t. leucoceraea showed a slighter oviposition preference (number of egg masses per plant) on e. grandis than on p. guajava plants, but this parameter was similar among different concentrations of the ip benzamidine by host-model and control (water and triton x-100) (fig. 3). similar values of the number of egg masses per plant and eggs per mass, per t. leucoceraea female, on e. grandis and p. guajava plants, with different concentrations of the pi benzamidine, and in the control with water and triton x-100, indicates a higher production of toxic compounds on this herbivore. the selection of the host for oviposition by lepidoptera females is related to learning by previous contact with them (paixão et al., 2013). t. leucoceraea individuals were collected in the field from host plants and kept with those of origin, and therefore, with previous experience potential in the host plant (solarz and newman, 2001). the nutritional value of the plant could also affect the oviposition site choice for monarch butterfly, danaus plexippus (lepidoptera: nymphalidae) females as observed, to plants that allowed better development of their larvae with nontoxic proper balance of nutrients (yeargan and allard, 2005; pereira et al., 2010). the slighter oviposition preference by t. leucoceraea females on non-treated e. grandis than on treated p. guajava plants indicates that this herbivore can recognize compounds from the introduced species that negatively affects their descendants, after treatment with different concentrations of the pi benzamidine (oliveira et al., 2005), despite the short time of contact of this defoliator with plants of the introduced species. the production of toxic compounds by e. grandis plants treated with pi benzamidine can be similar or higher than those of myrtaceae, native to brazil. however, t. leucoceraea females could recognize these compounds, which would explain their similar reproduction on e. grandis and p. guajava plants with different concentrations of pis, and in the control. this should be considered in pest control, because the oviposition preference was slightly higher on e. grandis plants and the application of pis could reduce reproduction of this herbivore. the number of t. leucoceraea eggs per mass was similar between the treatments (different concentrations of pi benzamidine) and the control (water and triton x-100) (one way, anova, p ≤ 0.05) (table 2). this parameter also showed similar values between the position of the plants and reading times (one way, anova, p ≤ 0.05) (table 2). the variation between plants in plots with different concentrations of pi benzamidine and the slight number of eggs per mass of t. leucoceraea females on plants without this pi, showed a preference for more suitable hosts for the development and reproduction of their offspring. females of herbivorous-insects could choose hosts for phagostimulant or phagoinhibitor substances, nutrients, and volatile substances expressed by them (ikeura et al., 2010; larue and welty, 2010). t. leucoceraea females might have identified the pi benzamidine on the leaves of host plants and reduced oviposition, since this compound would affect the development of its offspring. this agreed with the lowest reproduction of d. saccharalis after their larvae fed on an artificial diet treated with pi, p. guajava ip benzamidina opi wpi 0.000 16.00 ± 0.82 16.75 ± 2.17 0.125 19.00 ± 1.87 18.25 ± 1.38 0.250 17.00 ± 2.27 18.50 ± 1.85 0.375 18.25 ± 0.63 17.25 ± 2.53 0.500 16.50 ± 2.18 17.25 ± 1.70 e. grandis ip benzamidina opi wpi 0.000 16.50 ± 2.39 17.00 ± 1.96 0.125 18.25 ± 2.36 17.50 ± 1.85 0.250 18.50 ± 2.33 17.25 ± 1.75 0.375 17.50 ± 0.87 18.50 ± 1.71 0.500 17.50 ± 1.19 16.50 ± 1.85 340 table 2 number of eggs per mass of t. leucoceraea (lepidoptera: geometridae) on p. guajava (native host) or e. grandis (introduced host) (myrtaceae) plants to brazil in five concentrations (m or mol.l-1) of the protease inhibitor (pi) benzamidine values (mean ± standard error) per column per species do not differ by tukey test (p ≤ 0.05). control without pi benzamidine (opi) (0.01 % mg.ml–1 of aqueous solution of the adhesive spreader triton x-100) and treatment with pi benzamidine (wpi). from g. max grains (pompermayer et al., 2001), with lower weight and larval length, and higher larvae mortality of corn earworm, helicoverpa zea (lepidoptera: noctuidae) with three different pis [benzamidine, phenylmethylsulfonyl fluoride (pmsf), and n-α-tosyl-l-lysine chloromethyl ketone (tlck)] in cotton, gossypium sp. (malvaceae) plants (zhu et al., 2007). the possible adaptation of t. leucoceraea to an exotic host (e. grandis) was reduced with different concentrations of pi benzamidine. this was shown by foraging (number of larvae per plant) and similar numbers of egg masses per plant and eggs per mass by female on e. grandis and p. guajava plants, disagreeing with the development and reproduction of t. arnobia with e. grandis and gympie messmate, eucalyptus cloeziana (myrtaceae) without pis (holtz et al., 2003). native hosts can have a more developed defense system, such as, chemical and physical barriers, which reduce the digestibility and affect the present and future insect population (population control) (castells and berenbaum, 2008). these barriers can induce herbivores to seek new hosts, including the eucalyptus species, mainly because of the quantity and availability of this host, which is widely cultivated in brazil, and because of its lower capacity of defense. the foraging and reproduction behaviors of t. leucoceraea on e. grandis (introduced host) and p. guajava (native host) shows lack of adaptation of this pest to the introduced host treated with pi benzamidine. this makes it necessary to evaluate the specialization of t. leucoceraea to this exotic plant, because it could form new races adapted to certain hosts. spraying with pi benzamidine can reduce defoliation and oviposition of t. leucoceraea on e. grandis plants. this shows the possibilities of using this pi in the integrated pest management (ipm) programs in eucalyptus spp. cultures. acknowledgments the authors acknowledge dr. v osmar becker at the uiraçu institute in camacan, bahia state (brazil) for confirming the scientific name of thyrinteina leucoceraea rindge, 1961 (lepidoptera: geometridae) and global edico services of india for revising and editing the english of this manuscript. this study was supported by grants at the “conselho nacional de desenvolvimento científico e tecnológico (cnpq)”, “coordenação de aperfeiçoamento de pessoal de nível superior (capes)”, and “fundação de amparo à pesquisa do estado de minas gerais (fapemig)”. p. guajava ip benzamidina opi wpi 0.000 35.76 ± 1.82 37.31 ± 2.70 0.125 30.94 ± 2.55 32.85 ± 2.59 0.250 28.20 ± 2.21 29.20 ± 5.76 0.375 31.71 ± 2.47 28.50 ± 5.48 0.500 32.79 ± 2.59 35.78 ± 2.82 e. grandis ip benzamidina opi wpi 0.000 28.15 ± 3.15 18.47 ± 2.28 0.125 28.56 ± 3.35 18.64 ± 2.70 0.250 27.44 ± 3.26 29.82 ± 4.49 0.375 20.94 ± 3.08 26.20 ± 4.61 0.500 19.00 ± 2.76 27.09 ± 4.45 341 fig 3 number of egg masses of t. leucoceraea (lepidoptera: geometridae) females per concentration of protease inhibitor (pi) benzamidine on p. guajava (native host) (a) and e. grandis (introduced host) (b) plants, myrtaceae to brazil. one way, anova of regression (p ≤ 0.05). mean of the control (without spraying pi benzamidine; with 0.01 % mg.ml–1 of aqueous solution of the adhesive spreader triton x-100). references bazok r, barcic ji, edwards cr. effects of 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helicoverpa zea. pest. biochem. physiol. 87: 39-46, 2007. 344 research report isj 9: 212-222, 2012 issn 1824-307x research report microarray validation of molecular and cellular signaling in homarus americanus and penaeus monodon kj mantione, c kim, fm casares, gb stefano neuroscience research institute, state university of new york college at old westbury, old westbury, ny 11568-0210, usa accepted december 11, 2012 abstract previous studies have demonstrated that invertebrate neural tissues contain mammalian-like neurotransmitters, which activate specific cellular functions. therefore, it was of interest to attempt to identify these molecules via agilent gene expression microarrays. the array was used to analyze the transcriptional profiles of lobster and shrimp rna samples. we show dopamine, serotonin, and acetylcholine genes and their corresponding receptors are significantly expressed in lobster and shrimp neural tissues with a signal to noise value greater than 2. these signal molecules are directly related to previously discovered molecules in invertebrates, suggesting that they first appeared earlier in evolution and are necessary for an animal’s survival. key words: lobster; shrimp; neurotransmitter; microarray; acetylcholine; biogenic amines introduction invertebrate neural tissues contain neurotransmitters, i.e., biogenic amines, found in mammals (see (stefano, 1982, 1990, 1992). in regard to catecholamines, the neural tissue of mytilus edulis contains dopamine (da) and norepinephrine (ne) as well as the indoleamine serotonin (stefano and aiello, 1975 stefano et al., 1976, 1977; hidaka et al., 1977; twarog et al., 1977; malanga and young, 1978; satchell and twarog, 1978; stefano, 1982, 1990; zhu et al., 2005). these studies imply that elements of the neurotransmitter functions, i.e., enzymes and receptors, are present in the neural tissues of this organism as well (malanga and aiello, 1971; malanga, 1974 stefano et al., 1976, 1978; catapane et al., 1977, 1978, 1979, 1980 twarog et al., 1977; collins et al., 1980; hidaka et al., 1977; malanga and young, 1978; satchell and twarog, 1978; malanga and poll, 1979; stefano and catapane, 1980). given this documentation we sought to use microarray technology to validate previous studies documenting the presence of these neurotransmitters in lobster and shrimp neural tissues. previously we demonstrated cholinergic ___________________________________________________________________________ corresponding author: kirk j mantione neuroscience research institute state university of new york college at old westbury old westbury, ny 11568-0210, usa e-mail: kjmantione@sunynri.org signaling elements, (acetylcholine and respective receptors in lobster (zhu et al., 2006) as well as chemical messengers associated with catecholamine metabolism (casares et al., 2005). we also performed a similar analysis in mytilus edulis (gerber et al., 2007; mantione et al., 2009). thus, given their presence and action in invertebrate physiological systems it was of great interest to determine if human microarray chips would also show that they are present along with other processes associated with their signaling. the results of the present study support the observation that these signaling molecules appear to have evolved earlier than previously realized, given the many support processes now demonstrated, validating years of research on this topic. materials and methods homarus americanus and penaeus monodon were purchased live commercially. animals were then transported to the laboratory in chilled seawater (4 10 °c). in the laboratory, they were maintained as previously described in detail (stefano et al., 1994). neural tissues, lobster and shrimp ventral nerve cords were dissected and kept on ice until needed. agilent microarray gene expression array agilent human genome survey arrays were used to analyze the transcriptional profiles of rna 212 samples. the agilent human genome survey array contains 31,700 60-mer oligonucleotide probes representing a set of 27, 868 individual human genes and more than 1,000 control probes. sequences used for microarray probe design are from curated transcripts from the celera genomics human genome database (www.celeradiscoverysystem.com), refseq transcripts that have been structurally curated from the locuslink public database (http://ncbi.nlm.nih.bov/locuslink/refseq.html), highquality cdna sequences from the mammalian gene collection (mgc) (http://mgc.nci.nih.gov) and transcripts that were experimentally validated at applied biosystems. total rna from 1 lobster and 3 shrimp ventral nerve cords were isolated separately with the rneasy mini kit (qiagen, valencia, ca, usa). the tissue was lysed in 600 µl buffer rlt and homogenized by passing the lysate 5 times through a 20-gauge needle fitted to a 3 ml syringe. the samples were then processed following the manufacturer's detailed instructions. in the final step, the rna was eluted with 50 µl of rnase-free water by centrifugation for 1 min at 10,000 rpm. quality of the rna was analyzed using agilent 2100 bioanalyzer (agilent, santa clara, ca, usa) using the total rna nanochip according to manufacturer’s protocol. rna was reverse transcribed and the cdna was transcribed and labeled with cyanine-3ctp following manufacturer's protocol. to each chip, 2 µg of labeled crna targets were hybridized at 55 °c for 18 h. agilent microarray scanner software was used to extract assay signal and assay signal to noise ratio values from the microarray images. to determine expressed genes, the gene list was filtered by removing genes with a signal to noise value below two. the gene list was further filtered into moderately highly expressed and very highly expressed genes. data sets were managed using spotfire for functional genomics (tibco software inc., palo alto, ca, usa). results the previously discovered invertebrate neurotransmitter molecules include dopamine, as well as other biogenic amines, acetylcholine and serotonin. tables 1-4 list the associated genes that were significantly expressed as analyzed by the gene survey microarray (agilent) with a signal to noise value greater than 2 in untreated lobster nervous tissue. table 1 homarus americanus dopamine and serotonin pathway genes present dopamine receptors present drd1 dopamine receptor d1 drd2 dopamine receptor d2 drd3 dopamine receptor d3 drd4 dopamine receptor d4 drd5 dopamine receptor d5 serotonin receptors present htr1a 5-hydroxytryptamine (serotonin) receptor 1a, g protein-coupled htr1b 5-hydroxytryptamine (serotonin) receptor 1b, g protein-coupled htr1d 5-hydroxytryptamine (serotonin) receptor 1d, g protein-coupled htr1e 5-hydroxytryptamine (serotonin) receptor 1e, g protein-coupled htr1f 5-hydroxytryptamine (serotonin) receptor 1f, g protein-coupled htr2a 5-hydroxytryptamine (serotonin) receptor 2a, g protein-coupled htr2b 5-hydroxytryptamine (serotonin) receptor 2b, g protein-coupled htr2c 5-hydroxytryptamine (serotonin) receptor 2c, g protein-coupled htr3a 5-hydroxytryptamine (serotonin) receptor 3a, ionotropic htr3b 5-hydroxytryptamine (serotonin) receptor 3b, ionotropic htr4 5-hydroxytryptamine (serotonin) receptor 4, g protein-coupled htr5a 5-hydroxytryptamine (serotonin) receptor 5a, g protein-coupled htr6 5-hydroxytryptamine (serotonin) receptor 6, g protein-coupled htr7 5-hydroxytryptamine (serotonin) receptor 7, adenylate cyclase-coupled dopamine and serotonin metabolism genes present comt catechol-o-methyltransferase dbh dopamine beta-hydroxylase (dopamine beta-monooxygenase) ddc dopa decarboxylase (aromatic l-amino acid decarboxylase) maoa monoamine oxidase a maob monoamine oxidase b tdo2 tryptophan 2,3-dioxygenase th tyrosine hydroxylase tph1 tryptophan hydroxylase 1 tph2 tryptophan hydroxylase 2 dopamine and serotonin transporters present slc6a3 solute carrier family 6 (neurotransmitter transporter, dopamine), member 3 slc6a4 solute carrier family 6 (neurotransmitter transporter, serotonin), member 4 213 other dopamine and serotonin related genes present aldh5a1 aldehyde dehydrogenase 5 family, member a1 bdnf brain-derived neurotrophic factor caly calcyon neuron-specific vesicular protein cyp2d6 cytochrome p450, family 2, subfamily d, polypeptide 6 ephb1 eph receptor b1 gdnf glial cell derived neurotrophic factor gfap glial fibrillary acidic protein moxd1 monooxygenase, dbh-like 1 nr4a1 nuclear receptor subfamily 4, group a, member 1 nr4a3 nuclear receptor subfamily 4, group a, member 3 pdyn prodynorphin ptgs2 prostaglandin-endoperoxide synthase 2 (prostaglandin g/h synthase and cyclooxygenase) slc18a1 solute carrier family 18 (vesicular monoamine), member 1 slc18a2 solute carrier family 18 (vesicular monoamine), member 2 syn2 synapsin ii table 2 homarus americanus signal transduction pathway genes present camp/pka pathway activity adcy1 adenylate cyclase 1 adcy2 adenylate cyclase 2 adcy3 adenylate cyclase 3 adcy5 adenylate cyclase 5 casp3 caspase 3, apoptosis-related cysteine peptidase cdk5 cyclin-dependent kinase 5 creb1 camp responsive element binding protein 1 dusp1 dual specificity phosphatase 1 fos fbj osteosarcoma oncogene mapk1 mitogen-activated protein kinase 1 ppp1r1b protein phosphatase 1, regulatory (inhibitor) subunit 1b prkaca protein kinase, camp-dependent, catalytic, alpha p13k/akt pathway activity akt1 thymoma viral proto-oncogene 1 akt2 thymoma viral proto-oncogene 2 akt3 thymoma viral proto-oncogene 3 gsk3a glycogen synthase kinase 3 alpha pik3ca phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha pik3cg phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit gamma pla2 pathway activity alox12 arachidonate 12-lipoxygenase pde10a phosphodiesterase 10a pde4a phosphodiesterase 4a pde4b phosphodiesterase 4b pde4c phosphodiesterase 4c pde4d phosphodiesterase 4d pla2g5 phospholipase a2, group v plc pathway activity itpr1 inositol 1,4,5-trisphosphate receptor 1 plcb1 phospholipase c, beta 1 plcb2 phospholipase c, beta 2 plcb3 phospholipase c, beta 3 g-protein coupled receptor regulation activity adrb1 adrenoceptor beta 1 adrb2 adrenoceptor beta 2 adrbk1 adrenergic, beta, receptor kinase 1 adrbk2 adrenergic, beta, receptor kinase 2 app amyloid beta (a4) precursor protein arrb1 arrestin, beta 1 arrb2 arrestin, beta 2 grk4 g protein-coupled receptor kinase 4 grk5 g protein-coupled receptor kinase 5 grk6 g protein-coupled receptor kinase 6 snca synuclein, alpha (non a4 component of amyloid precursor) sncaip synuclein, alpha interacting protein 214 table 3 homarus americanus cell signaling genes present acvr2b activin a receptor, type iib bmp1 bone morphogenetic protein 1 ccl1 chemokine (c-c motif) ligand 1 ccl13 chemokine (c-c motif) ligand 13 ccl15 chemokine (c-c motif) ligand 15 ccl19 chemokine (c-c motif) ligand 19 ccl23 chemokine (c-c motif) ligand 23 ccl24 chemokine (c-c motif) ligand 24 ccnc cyclin c ccnd3 cyclin d3 ccnl1 cyclin l1 ccr6 chemokine (c-c motif) receptor 6 ccr9 chemokine (c-c motif) receptor 9 ccrl2 chemokine (c-c motif) receptor-like 2 cdc14a cdc14 cell division cycle 14 homolog a cdc23 cell division cycle 23 homolog cdc25a cell division cycle 25 homolog a cdk6 cyclin-dependent kinase 6 csf2rb colony stimulating factor 2 receptor, beta, low-affinity (granulocyte-macrophage) cxcl12 chemokine (c-x-c motif) ligand 12 cxcr3 chemokine (c-x-c motif) receptor 3 dapk1 death-associated protein kinase 1 dock1 dedicator of cytokinesis 1 epor erythropoietin receptor gdf2 growth differentiation factor 2 gdf8 growth differentiation factor 8 ifnk interferon, kappa il11ra interleukin 11 receptor, alpha il15 interleukin 15 il16 interleukin 16 il18bp interleukin 18 binding protein il23r interleukin 23 receptor il31ra interleukin 31 receptor a il7 interleukin 7 lats1 lats, large tumor suppressor, homolog 1 lepr leptin receptor lif leukemia inhibitory factor mobk1b mob kinase activator 1a myh11 myosin, heavy polypeptide 11, smooth muscle nrp1 neuropilin 1 osm oncostatin m pard3 par-3 partitioning defective 3 homolog pard6a par-6 partitioning defective 6 homolog alpha nampt nicotinamide phosphoribosyltransferase pf4 platelet factor 4 pin1 peptidylprolyl cis/trans isomerase, nima-interacting 1 prc1 protein regulator of cytokinesis 1 stat1 signal transducer and activator of transcription 1 tlr2 toll-like receptor 2 tnfrsf11a tumor necrosis factor receptor superfamily, member 11a tnfrsf25 tumor necrosis factor receptor superfamily, member 25 215 table 4 homarus americanus other neurotransmitter related genes present ache acetylcholinesterase bche butyrylcholinesterase chrm1 cholinergic receptor, muscarinic 1 chrm2 cholinergic receptor, muscarinic 2 chrm3 cholinergic receptor, muscarinic 3 chrm4 cholinergic receptor, muscarinic 4 chrm5 cholinergic receptor, muscarinic 5 chrna1 cholinergic receptor, nicotinic, alpha 1 (muscle) chrna10 cholinergic receptor, nicotinic, alpha 10 (muscle) chrna2 cholinergic receptor, nicotinic, alpha 2 (muscle) chrna3 cholinergic receptor, nicotinic, alpha 3 (muscle) chrna4 cholinergic receptor, nicotinic, alpha 4 (muscle) chrna5 cholinergic receptor, nicotinic, alpha 5 (muscle) chrna6 cholinergic receptor, nicotinic, alpha 6 (muscle) chrna9 cholinergic receptor, nicotinic, alpha 9 (muscle) chrnb1 cholinergic receptor, nicotinic, beta 1 (muscle) chrnb2 cholinergic receptor, nicotinic, beta 2 (muscle) chrnb3 cholinergic receptor, nicotinic, beta 3 (muscle) chrnb4 cholinergic receptor, nicotinic, beta 4 (muscle) chrnd cholinergic receptor, nicotinic, delta (muscle) chrne cholinergic receptor, nicotinic, epsilon (muscle) chrng cholinergic receptor, nicotinic, gamma (muscle) colq collagen-like tail subunit (single strand of homotrimer) of asymmetric acetylcholinesterase slc18a3 solute carrier family 18 (vesicular monoamine), member 3 slc5a7 solute carrier family 5 (choline transporter), member 7 slc6a2 solute carrier family 6 (neurotransmitter transporter, noradrenalin), member 2 tables 5 8 list the associated genes that were significantly expressed as analyzed by the gene survey microarray (agilent) with a signal to noise value greater than 2 in untreated shrimp nervous tissue. gene sequences detected with signal to noise values greater than 2 were considered to be present. given the logarithmic analysis supplied by the spotfire for functional genomics program (spotfire, somerville, maine, usa), any positive signal to noise value indicates gene presence is in greater amounts than background noise. furthermore, the gene copy number of the human transcriptome on the microarray chip is not identical to the gene copy number of the respective animal. according to this criterion, most of the genes detected in the lobster were between the signal to noise ratio of 2 to 3. the ratios for the chrna5, htr3a, maoa and th genes were between 3 and 4. the most highly expressed genes, all with a signal to noise greater than 10, were the ccl24, chrne, comt, cxcr3, drd4, moxd1, pde4b, and plcb2. for the shrimp genes detected, most of the genes detected were between the signal to noise ratio of 2 to 3. the ratios for the ccnd3, chrm1, drd5, htr1b, htr1e, htr3a, pde4c, plcb1, prc1, prkaca, and snca genes were between 3 and 4. finally, the most highly expressed genes, all with a signal to noise greater than 10, were the acvr2b, akt2, ccl19, ccl24, chrm2, chrm5, chrnb2, chrnb3, chrne, cyp2d6, drd4, gfap, htr3a, il16, il18bp, plcb2, th, and tph2. discussion as noted earlier, invertebrate ganglia contain biogenic amines, serotonin and acetylcholine as validated by gene expression microarray (mantione et al., 2009). based on these findings, which validate the current results, one can surmise that these chemical messengers emerged early during the course of evolution and were maintained (ottaviani et al., 1991, 1988, 1995, 2007; ottaviani and franceschi, 1996; stefano et al., 2009). in another invertebrate, biogenic amines in mytilus tissues have been demonstrated not only in the tissues but to exhibit pharmacological specificity in regard to tissue excitation and inhibition. dopamine, serotonin and acetylcholine have been implicated in the regulation of cilia activity, smooth muscle regulation and foot control (twarog and cole, 1972; hidaka and twarog, 1977; hidaka et al., 1977; twarog et al., 1977; catapane et al., 1978, 1979; malanga and young, 1978; satchell and twarog, 1978; malanga and poll, 1979; aiello et al., 1981). these studies demonstrate that the respective receptor mediated systems, exhibiting high specificity to various related agonists and antagonists occur in specific tissues. 216 table 5 penaeus monodon dopamine and serotonin pathway genes present dopamine receptors present drd1 dopamine receptor d1 drd2 dopamine receptor d2 drd3 dopamine receptor d3 drd4 dopamine receptor d4 drd5 dopamine receptor d5 serotonin receptors present htr1a 5-hydroxytryptamine (serotonin) receptor 1a, g protein-coupled htr1b 5-hydroxytryptamine (serotonin) receptor 1b, g protein-coupled htr1d 5-hydroxytryptamine (serotonin) receptor 1d, g protein-coupled htr1e 5-hydroxytryptamine (serotonin) receptor 1e, g protein-coupled htr1f 5-hydroxytryptamine (serotonin) receptor 1f, g protein-coupled htr2a 5-hydroxytryptamine (serotonin) receptor 2a, g protein-coupled htr2b 5-hydroxytryptamine (serotonin) receptor 2b, g protein-coupled htr2c 5-hydroxytryptamine (serotonin) receptor 2c, g protein-coupled htr3a 5-hydroxytryptamine (serotonin) receptor 3a, ionotropic htr3b 5-hydroxytryptamine (serotonin) receptor 3b, ionotropic htr4 5-hydroxytryptamine (serotonin) receptor 4, g protein-coupled htr5a 5-hydroxytryptamine (serotonin) receptor 5a, g protein-coupled htr6 5-hydroxytryptamine (serotonin) receptor 6, g protein-coupled htr7 5-hydroxytryptamine (serotonin) receptor 7, adenylate cyclase-coupled dopamine and serotonin metabolism genes present comt catechol-o-methyltransferase dbh dopamine beta-hydroxylase (dopamine beta-monooxygenase) ddc dopa decarboxylase (aromatic l-amino acid decarboxylase) th tyrosine hydroxylase maoa monoamine oxidase a maob monoamine oxidase b tdo2 tryptophan 2,3-dioxygenase tph1 tryptophan hydroxylase 1 tph2 tryptophan hydroxylase 2 dopamine and serotonin transporters present slc6a3 solute carrier family 6 (neurotransmitter transporter, dopamine), member 3 slc6a4 solute carrier family 6 (neurotransmitter transporter, serotonin), member 4 other dopamine and serotonin related genes present aldh5a1 aldehyde dehydrogenase 5 family, member a1 bdnf brain-derived neurotrophic factor caly calcyon neuron-specific vesicular protein cyp2d6 cytochrome p450, family 2, subfamily d, polypeptide 6 ephb1 eph receptor b1 gdnf glial cell derived neurotrophic factor gfap glial fibrillary acidic protein moxd1 monooxygenase, dbh-like 1 nr4a1 uclear receptor subfamily 4, group a, member 1 nr4a3 nuclear receptor subfamily 4, group a, member 3 pdyn prodynorphin ptgs2 prostaglandin-endoperoxide synthase 2 (prostaglandin g/h synthase and cyclooxygenase) slc18a1 solute carrier family 18 (vesicular monoamine), member 1 slc18a2 solute carrier family 18 (vesicular monoamine), member 2 syn2 synapsin ii in previous and current research, measures are taken to confirm gene expression including taqman probes and molecular methods as well as western blotting (hauton et al., 2005). the ability of microarray to corroborate with and/or confirm an expanse of previous research is demonstrated in this study of neurotransmitter molecules found in these invertebrates. given the comprehensive nature of a single microarray chip and the accuracy and precision of the data expressed by these chips, this research indicates that the use of microarray could be independently sufficient for determining gene expression given the validating preexisting data (nachmansohn, 1964; nagabhushanam, 1966; 217 table 6 penaeus monodon signal transduction pathways genes present camp/pka pathway activity adcy1 adenylate cyclase 1 adcy2 adenylate cyclase 2 adcy3 adenylate cyclase 3 adcy5 adenylate cyclase 5 casp3 caspase 3, apoptosis-related cysteine peptidase cdk5 cyclin-dependent kinase 5 creb1 camp responsive element binding protein 1 dusp1 dual specificity phosphatase 1 fos fbj osteosarcoma oncogene mapk1 mitogen-activated protein kinase 1 ppp1r1b protein phosphatase 1, regulatory (inhibitor) subunit 1b prkaca protein kinase, camp-dependent, catalytic, alpha p13k/akt pathway activity akt1 thymoma viral proto-oncogene 1 akt2 thymoma viral proto-oncogene 2 akt3 thymoma viral proto-oncogene 3 gsk3a glycogen synthase kinase 3 alpha gsk3b glycogen synthase kinase 3 beta pik3ca phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha pik3cg phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit gamma pla2 pathway activity alox12 arachidonate 12-lipoxygenase pde10a phosphodiesterase 10a pde4a phosphodiesterase 4a pde4b phosphodiesterase 4b pde4c phosphodiesterase 4c pde4d phosphodiesterase 4d pla2g5 phospholipase a2, group v plc pathway activity itpr1 inositol 1,4,5-trisphosphate receptor 1 plcb1 phospholipase c, beta 1 plcb2 phospholipase c, beta 2 plcb3 phospholipase c, beta 3 g-protein coupled receptor regulation activity adrbk1 adrenergic, beta, receptor kinase 1 adrbk2 adrenergic, beta, receptor kinase 2 app amyloid beta (a4) precursor protein arrb1 arrestin, beta 1 arrb2 arrestin, beta 2 grk4 g protein-coupled receptor kinase 4 grk5 g protein-coupled receptor kinase 5 grk6 g protein-coupled receptor kinase 6 snca synuclein, alpha (non a4 component of amyloid precursor) sncaip synuclein, alpha interacting protein hildebrand et al., 1974; marder, 1974; sullivan et al., 1977; davis and ocorr and berlind, 1983; cournil et al., 1984, 1994; siwicki et al., 1987; juorio and sloley, 1988; chiba and tazaki, 1992; ma et al., 1992; ma and weiger, 1993; cournil et al., 1995; rodriguez et al., 1995; destoumieux et al., 1997, 1999; mancillas et al., 1998; scholz et al., 1998; heinrich et al., 2000; peeke et al., 2000; antonsen and paul, 2001; harzsch, 2003; pulver et al., 2003; casares et al., 2005; cheng et al., 2005; tiu et al., 2005; casares et al., 2006; zhu et al., 2006; chang et al., 2007; brown-peterson et al., 2008; leelatanawit et al., 2008; li and brouwer, 2009; tinikul et al., 2011). this preexisting data serves as a validation of the current microarray results. in summary, it appears neural communication, which occurs in invertebrate neural tissues, including those innervating peripheral tissues originated earlier in evolution and was maintained 218 table 7 penaeus monodon cell signaling genes present acvr2b activin a receptor, type iib bmp1 bone morphogenetic protein 1 ccl1 chemokine (c-c motif) ligand 1 ccl13 chemokine (c-c motif) ligand 13 ccl15 chemokine (c-c motif) ligand 15 ccl19 chemokine (c-c motif) ligand 19 ccl23 chemokine (c-c motif) ligand 23 ccl24 chemokine (c-c motif) ligand 24 ccnc cyclin c ccnd3 cyclin d3 ccnl1 cyclin l1 ccr6 chemokine (c-c motif) receptor 6 ccr9 chemokine (c-c motif) receptor 9 ccrl2 chemokine (c-c motif) receptor-like 2 cdc14a cdc14 cell division cycle 14 homolog a cdc23 cell division cycle 23 homolog cdc25a cell division cycle 25 homolog a cdk6 cyclin-dependent kinase 6 csf2rb colony stimulating factor 2 receptor, beta, low-affinity (granulocyte-macrophage) cxcl12 chemokine (c-x-c motif) ligand 12 cxcr3 chemokine (c-x-c motif) receptor 3 dapk1 death-associated protein kinase 1 dock1 dedicator of cytokinesis 1 epor erythropoietin receptor gdf2 growth differentiation factor 2 gdf8 growth differentiation factor 8 ifnk interferon, kappa il11ra interleukin 11 receptor, alpha il15 interleukin 15 il16 interleukin 16 il18bp interleukin 18 binding protein il23r interleukin 23 receptor il31ra interleukin 31 receptor a il7 interleukin 7 lats1 lats, large tumor suppressor, homolog 1 lepr leptin receptor lif leukemia inhibitory factor mobk1b mob kinase activator 1a myh11 myosin, heavy polypeptide 11, smooth muscle nrp1 neuropilin 1 osm oncostatin m pard3 par-3 partitioning defective 3 homolog pard6a par-6 partitioning defective 6 homolog alpha nampt nicotinamide phosphoribosyltransferase pf4 platelet factor 4 pin1 peptidylprolyl cis/trans isomerase, nima-interacting 1 prc1 protein regulator of cytokinesis 1 stat1 signal transducer and activator of transcription 1 tlr2 toll-like receptor 2 tnfrsf11a tumor necrosis factor receptor superfamily, member 11a tnfrsf25 tumor necrosis factor receptor superfamily, member 25 219 table 8 penaeus monodon other neurotransmitter related genes present ache acetylcholinesterase bche butyrylcholinesterase chrm1 cholinergic receptor, muscarinic 1 chrm2 cholinergic receptor, muscarinic 2 chrm3 cholinergic receptor, muscarinic 3 chrm4 cholinergic receptor, muscarinic 4 chrm5 cholinergic receptor, muscarinic 5 chrna1 cholinergic receptor, nicotinic, alpha 1 (muscle) chrna10 cholinergic receptor, nicotinic, alpha 10 (muscle) chrna2 cholinergic receptor, nicotinic, alpha 2 (muscle) chrna3 cholinergic receptor, nicotinic, alpha 3 (muscle) chrna4 cholinergic receptor, nicotinic, alpha 4 (muscle) chrna5 cholinergic receptor, nicotinic, alpha 5 (muscle) chrna6 cholinergic receptor, nicotinic, alpha 6 (muscle) chrna9 cholinergic receptor, nicotinic, alpha 9 (muscle) chrnb1 cholinergic receptor, nicotinic, beta 1 (muscle) chrnb2 cholinergic receptor, nicotinic, beta 2 (muscle) chrnb3 cholinergic receptor, nicotinic, beta 3 (muscle) chrnb4 cholinergic receptor, nicotinic, beta 4 (muscle) chrnd cholinergic receptor, nicotinic, delta (muscle) chrne cholinergic receptor, nicotinic, epsilon (muscle) chrng cholinergic receptor, nicotinic, gamma (muscle) colq collagen-like tail subunit (single strand of homotrimer) of asymmetric acetylcholinesterase ctrl chymotrypsin-like slc18a3 solute carrier family 18 (vesicular monoamine), member 3 slc5a7 solute carrier family 5 (choline transporter), member 7 slc6a2 solute carrier family 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119-131, 2014 issn 1824-307x research report the protection of cpg odns and yarrowia lipolytica harboring vp28 for shrimp litopenaeus vannamei against white spot syndrome virus infection q yi1,2, r liu1, r sun1, 2, l wang1, z zhou1, m wang1, y liu3, j sun3, c madzak4, l song1 1key laboratory of experimental marine biology, institute of oceanology, chinese academy of sciences, 7 nanhai rd., qingdao 266071, china 2university of chinese academy of sciences, beijing 100049, china 3college of life science, normal university of tianjin, 393 binshui rd., tianjin, 300387 ,china 4umr1238 microbiologie et génétique moléculaire, inra/cnrs/inapg, cbai, bp 01, f-78850 thiverval-grignon, france accepted april 22, 2014 abstract the white spot syndrome is one of the most serious disease which has caused high mortalities and huge economic losses to shrimp culture. in the present study, the oral administrations with cpg odns and yarrowia lipolytica harboring vp28 (rvp28-yl) as dietary supplement for shrimp litopenaeus vannamei were conducted to evaluate their protective effects against wssv. after feeding for 15 days, the cumulative mortality and the copy number of wssv in cpg and rvp28-yl feeding shrimps were significantly lower when they were challenged by wssv, compared with those in control shrimps (p < 0.05). the caspase-3 activity was suppressed in rvp28-yl feeding shrimps but ascended in cpg feeding shrimps after wssv challenge. besides, the po activity in cpg feeding shrimps was significantly increased after feeding trial, and kept increasing post wssv challenge (p < 0.05). while the increased no production was observed both in cpg and rvp28-yl feeding shrimps after feeding trial and wssv challenge. in addition, increased mrna expression levels of stat and dicer were observed in cpg group post wssv challenge. these results together indicated that oral feeding of cpg odns and rvp28-yl could enhance the innate non-specific immune responses especially antiviral immunity of shrimps in varying degrees, and increase their resistance against wssv infection. key words: cpg odns; yarrowia lipolytica surface-display vp28; white spot syndrome virus; litopenaeus vannamei; disease resistance; antiviral immunity   introduction white spot syndrome virus (wssv) is one of the most hampered pathogens in shrimp culture, which has caused severe disease, leading to significant economic losses (johnson et al., 2008; haq et al., 2012). owing to the potential deteriorative environmental effects, some traditional medication such as the antibiotic and prophylactic chemicals have been gradually abandoned in application, and the enhancement of immunity of shrimp has been becoming the most promising strategy for disease control (li and xiang, 2013a). the performances of immunostimulants and vaccines have gained momentum by virtue of their potential use in inducing ___________________________________________________________________________ corresponding author: linsheng song key laboratory of experimental marine biology institute of oceanology chinese academy of sciences 7 nanhai rd., qingdao 266071, china e-mail: lshsong@ms.qdio.ac.cn the immune response and reducing the disease impact on crustaceans (hauton, 2012). cpg oligodeoxynucleotides (cpg odns), also called bacterial dna or synthetic oligodeoxynucleotides, have been proven to trigger innate immune responses in many animal species, and they are always employed as the well-known vaccine adjuvant in mammals (krieg, 2002). in mammals, cpg can be recognized by toll-like receptor 9 (tlr9) to trigger the signaling pathways, and in turn activate several transcription factors including stress kinase and nf-κb (sparwasser et al., 1998; choudhury et al., 2002). meanwhile, the proliferation of b lymphocytes and immune responses are subsequently induced, and the immunological events occur after cpg activation of immune system include increased antiviral immunity (krieg, 2002). in shrimps, it has been reported that cpg induce various innate immune responses and can be used in the control of virus disease for its immunostimulating properties 119   table 1 sequences of primers used in the present study primer sequence(5’-3’) sequence information wssv-f(forward) wssv-r(reverse) wssv-rt-f (forward) cgcctaccctgttgaatctg tttagtgtgtggtctccgtctc ccagttcagaatcggacgtt wssvvirus detection wssvvirus detection real-time pcr for wssv copies wssv-rt-r(reverse) wssv-taqman (probe) oligo(dt)-adaptor aaagacgcctaccctgttga tccatagttcctggtttgtaatgtgccg ggccacgcgtcgactagtac(g)17 real-time pcr for wssv copies real-time pcr for wssv copies the first strand cdna synthesis vp28-1f (forward) vp28-1r (reverse) vp28-2f (forward) vp28-2r (reverse) stat-f (forward) stat-r (reverse) dicer-f (forward) dicer-r (reverse) accaccatggatctttctttc ttactcggtctcagtgcca accaccatggatctttctttc ttactcggtctcagtgcca agcccctgtctgagcgaaa ggtgttctcttgtaaccttcatca ccggagatagaacggttcagtg cgataattcctcccaacacctg surface display of vp28 surface display of vp28 prokaryotic recombinant of vp28 prokaryotic recombinant of vp28 real-time pcr for stat gene real-time pcr for stat gene real-time pcr for dicer gene real-time pcr for dicer gene lgbp-f(forward) lgbp-r (reverse) ggtaaccagtacggaggaacga tactcgacgtgggtcttctcga real-time pcr for lgbp gene real-time pcr for lgbp gene ef-α(forward) catcaaggagaaactgtgct real-time pcr of internal control ef-α(reverse) gatggagttgtaggtggtct real-time pcr of internal control ( ; )chang et al., 2003 zhang et al., 2010 . for instance, the ros production, apoptosis and phagocytosis level of shrimp hemocytes increased after they were incubated with cpg odns (sun et al., 2013a). the respiratory burst level and phenoloxidase activity of macrobrachium rosenbergii hemocytes were enhanced after cpg odns treatment (chuo et al., 2005; sung et al., 2009). and it was also reported that stimulation of cpg odns could induce the expression of antiviral associated genes in l. vannamei. the copy number of wssv in those shrimps pre-injected with cpg odns was lower than that in untreated group after wssv challenge, and the survival rate in cpg pre-injected shrimps was significantly higher than that of the control (zhang et al., 2010). the accumulating evidences suggested that cpg odn might be one eligible candidate immunostimulant to endow shrimps resistance via enhancing the non-specific immune response against virus infection. however, the underlying mechanism of dietary cpg odns on antiviral immune responses and disease resistance of shrimp is still not well understood. recently, there are accumulating reports that the application of inactivated pathogens or protein compounds derived from pathogen by means of “vaccination” was effective for disease control in crustaceans, which provided promising strategies (kurtz and armitage, 2006; cong et al., 2008). vp28 is a main envelope protein of wssv acting as an important viral attachment protein for the virus to enter into the cell of shrimp (yi et al., 2004). it can interact with some host cellular receptors, such as the small gtpase (rab7), heat-shock cognate protein 70 (hsc70) and signal transducers and activators of transcription protein (stat) to initiate the virus infection (sritunyalucksana et al., 2006; liu et al., 2007; xu et al., 2009). it has also been documented that vp28 is one major vaccine candidate to exert immune protective effects against wssv infection in shrimp (witteveldt et al., 2004; fu et al., 2008; syed and kwang, 2011). for instance, the shrimp and crayfish vaccinated with vp28 protein showed significantly lower mortality after wssv challenge (witteveldt et al., 2004). because of the notable vaccine effect of vp28 against wssv infection in shrimp, several routes and vehicles have been developed, such as the direct injection of vp28 protein, oral delivery of vp28 dna vaccine , and prokaryotes carrying with vp28 recombinant protein( witteveldt et al., 2004; syed and kwang, 2011; du et al., 2013). the yeast yarrowia lipolytica is one of the most attractive microorganisms for the expression of foreign genes (madzak et al., 2004), which has excellent properties compared with bacterial expression system, such as naturally secretion of high amount of proteins on the surface, lack of pathogenicity and immunological properties served as a probiotic candidate (yue et al., 2008). in the present study, a 15 days oral administration was implemented in shrimp l. vannamei, 120   table 2 ingredient formulation of feeding diets. (units: g contained in 1 kg diet) ingredients cpg supplemental diet rvp28-yl supplemental diet basal diet fish meal shrimp meal soybean meal peanut meal dextrin wheat flour oil mixture vitamin mixture lecithin chitosan sodium alginate cpg powder rvp28-yl powder 300 150 150 50 20 150 50 10 20 50 50 0.04 300 150 150 50 20 150 50 10 20 50 50 1011cfu 300 150 150 50 20 150 50 10 20 50 50 which fed with cpg odns and y. lipolytica surface-display vp28 supplemental diets. some immune parameters, the expression of antiviral associated genes were measured at the end of feeding trial in shrimps as well as post wssv challenge. wssv copy number and the mortality were determined after the shrimps challenged by wssv. they were contributed to evaluate the immunostimulatory effects of cpg odns and y. lipolytica-vp28 on the innate immune response and disease resistance of l. vannamei, as well as to provide valuable references for their further application in shrimp aquaculture industry. materials and methods shrimp rearing healthy shrimp l. vannamei, approximately 15 cm in length and 20 g in weight, were collected from a local farm in tianjin, china, and acclimated at 20 ± 2 °c for 7 days before process. during the culture period, every 20 individuals were kept in one container, and the seawater was changed 60 % daily. from the experimental animals, gills of five shrimps in each group were randomly sampled to examine the presence wssv in vivo by pcr with specific wssv primers wssv-f and wssv-r (table 1) (yoganandhan et al., 2003). only healthy individuals were used in the following experiment. wssv and in vivo titration wssv virus stocks were purified from gill tissues of wssv infected shrimps via the method of differential centrifugation described by xu et al (xu et al., 2007). the copy number of wssv stock was quantified by the real-time pcr, and the stock solutions were diluted with pbs (0.1 m, ph 7.4) to a final concentration of 108 copies ml-1 and stored at −80 °c. to obtain the desired challenge pressure of wssv (ld50), in vivo titration experiment with serial dilutions of wssv stock was conducted according to the procedure described by previous report (fu et al., 2008). shrimps were challenged by an injection with different wssv dilutions, and cultured for addition 10 days. the dead shrimps were recorded and tested by pcr reaction for the presence of wssv. for the determination of desired challenge pressure, the cumulative dead shrimp were recorded to calculate the relationship between wssv dose and shrimp mortality. the median lethal dose was used in the following challenge experiments. cpg odn large-scale preparation five odns which were proved to be effective in mammals and aquatic animals were constructed in series into puc57 vector in our laboratory (zhang et al., 2010). the puc57-cpg was transformed into escherichia coli for following fermentation in lb medium (tryptone 10 g l-1, yeast extract 5 g l-1, nacl 10 g l-1) under the ampicillin selective pressure. the large-scale plasmid extraction was performed following the previous report (holmes and quigley, 1981). the plasmids were dissolved in 0.1 m phosphate buffer saline buffer (pbs, ph = 7.4), heated at 100 °c for 10 min and immediately cooled in ice-water mixture. the linear cpg odns was quantitated and stored in pbs buffer until use in -20 °c. generation of recombinant vp28 vp28 was expressed in e. coli system following the method described by previous reports (witteveldt et al., 2004). the strain y. lipolytica po1h and the vector pina 1317 were kindly supplied by cbai, agroparistech, 78850 thiverval-grignon, france. vp28 was ligated into the vector pina 1317 and then transformed into y. lipolytica po1h by lithium acetate 121   table 3 the scheme sampling experiments group set cpg odns rvp28-yl basal diet (control) samplingtime points no. of shrimp 20(×6) 20(×6) 20(×6) 20(×6) 20(×6) 20(×6) 20(×6) 20(×6) 20(×6) before oral administration post oral administration on the first daypost-stimulation on the third daypost-stimulation no challenge wssv challenge pbs challenge wssv challenge pbs challenge wssv challenge pbs challenge 20(×3) 20(×3) 20(×3) 20(×3) 20(×3) 20(×3) 20(×3) 20(×3) 20(×3) 20(×3) 20(×3) 20(×3) 10 20(×3) 20(×3) 20(×3) 20(×3) 20(×3) 20(×3) method (xuan et al., 1988). vp28 displayed on y. lipolytica and expressed in e. coli were designated as rvp28-yl and rvp28-ec, respectively. the primers for amplification of the full length orf of vp28 were presented in table 1. the rvp28-yl was induced in the optimized culture medium (50 mm sucrose, 1.32 g l-1 yeast extract, 25 mm nh cl, 2 mm k hpo , 1 mm mgso , 1 μm vitamin b ) at 28 4 2 4 4 1 °c overnight for an enlarge cultivation. the  recombinant  plasmid  (pet-30a-vp28) was transformed into the strains e. coli rosseta-gami (de3), which was cultured in the lb medium (tryptone 10 g l-1, yeast extract 5 g l-1, nacl 10 g l-1) at 37 °c and then the rvp28 was induced with the addition of iptg at the concentration of 1 mm. the concentrations of these two strains were calculated as cfu ml .-1 preparation of feeding diet the experimental diets were prepared following the manual procedure. the adding amount of cpg odns and y. lipolytica was 40 mg kg-1 and 1011 cfu kg-1 diet respectively (syed and kwang, 2011; sun et al., 2013b). the diets were sufficiently mixed and pressed into strips to obtain pellets, and stored at 4 °c until use. there were three kinds of diets prepared, cpg supplemental diets, rvp28-yl supplemental diets, and basal diets respectively. the ingredients of basal diet were listed in table 2. oral administration and wssv challenge five hundred and fifty shrimps were divided into three sets, each with three groups. in each group, there were twenty shrimps in triplicate as subgroups. three sets were fed by cpg, rvp28-yl and basal diets for 15 days trial, which were designated as cpg group, rvp28-yl group and the control group, respectively. the daily feeding diets were about 5 % of body weight per shrimp and nursed with five times. according to the actual intake response, the adjustment was conducted at any time. based on the result of in vivo titration, the lethal dose 50 (ld50) of wssv was calculated as 1×106 copies. at the end of 15 days feeding experiment of different supplemental diet, one group in each set was randomly selected for injection of wssv stock (5×107 copies ml-1, 100 μl for each shrimp) to calculate the survival rate. the other two groups received an injection of wssv stock (5×106 copies ml-1, 100 μl) or pbs (ph = 7.4, 100 μl), and every 20 shrimps in each group were regarded as one subgroup. six shrimps in each subgroup with 15 days’ feeding trial were randomly sampled after the 1st and 3rd days post wssv challenge, and the untreated shrimps in the basal diet feeding set were employed as blank group. mortality was recorded every day for 10 days post-challenge. the scheme of feeding and challenge experiments is shown in table 3. polyclonal vp28 antibody preparation and west blotting of rvp28 the purified vp28 recombinant protein was obtained and quantified, according to the previous description (zhou et al., 2011). six weeks old healthy adult rats were immunized by four times injection of vp28 protein in one and a half month to acquire the polyclonal vp28 antibody (anti-vp28). the serum was separated and then stored at -80 °c until used. the concentrations of two strains y. lipolytica-vp28 and e. coli-vp28 were adjusted to 108 cfu ml-1 after centrifuge at 10000×g for 10 min, the supernatant y. lipolytica-vp28 and both two strain precipitates were heated at 100 °c for 10 min, and then used in western blotting. the quantitative assay of vp28 in these two recombinants was analyzed by the quantity-one software (bio-rad 122   fig. 1 immunofluorescence and quantitative western blotting analysis. (a) the surface-display vp28 in y. lipolytica was presented in the green color (b), the control group was no signal (green) detected (d), bar = 10 μm. (b) quantitative western blot analysis of vp28 expressed in y. lipolytica system compared with vp28 expressed in bacterial system hold the same strain concentration. the purified vp28 protein as a standard. lane 1 to lane 5 was the purified prvp28 with the concentration of 0.5μgml-1, 1μgml -1 , 5 μgml -1 , 10 μgml -1 , 50 μgml -1. the amounts of lane vp28-ec, vp28-yl and vp28-yl supernatant were analysed by quanlity one software. data (mean ± sd) in each column with different letters are significant (p < 0.05) from each other. laboratories). after sds-page, the gel was transferred to the 0.45 μm nitrocellulose membrane, and then the western blotting was carried out following the methods by zhou et al. (2013) with slight modifications. the anti-vp28 at dilution 1:1,000 in 5 % bsa and goat anti-rat igg-hrp conjugate (sangon biotech, china) diluted at 1:4,000 in 5 % bsa were used as the primary antibody and the secondary antibody in the assay, respectively. the enhanced chemiluminescence staining method (ecl) was performed using luminol and hydrogen peroxide as substrates to detect the vp28 expression. meanwhile, the vp28 protein which expressed in e. coli system was purified as the standard. hemocytes collection three hundred microlitres precooled anticoagulant (115 mm glucose, 336 mm nacl, 27 mm sodium citrate, 9 mm edta na2·2h2o, ph 7.4) was preloaded into 1 ml hypodermic gauge needle and syringe, and the hemolmyph was collected from the pericardial cavity of each shrimp. hemolmyph was immediately centrifuged at 800×g, 4 ℃ to harvest the hemocytes and the plasma supernatants. at each sample collection point, six shrimps were prepared from each group, and the hemolymphs collected from every two shrimps in the same treated group were pooled together as one single sample. triplicate parallels were set for the following experiment. the measurements of immune parameters the po activity was measured according to the methods reported by zhou et al. (2013) with slight modifications. l-3, 4-dihydroxyphenylalanine (l-dopa) (sigma aldrich, usa) was used as substrate, and the formation of dopachrome was recorded spectrophotometrically at 490 nm. briefly, 100 μl hemolymphs samples from different groups were added in the 96-well microplate (costar, usa), and incubated with trypsin (solarbio, china) (1 mg ml-1) at room temperature for 15 min. then, 50 μl l-dopa (4 mg ml-1 in potassium phosphate buffer) was added into each well, and the optical density at 490 nm was immediately measured every two minutes by using a microplate spectrophotometer (biotek, powerwave xs2) for a period of 30 min. one phenoloxidase activity unit was defined as an increase of 0.001 absorbance value at 490 nm per min, and the maximum increase between the two adjacent time points was selected and regarded as one po activity unit. the ratio of enzyme activity unit to the total protein concentration was defined as the relative phenoloxidase activity, which was expressed as u mg-1 protein. the concentration of total protein was determined via the bca protein determination kit (beyotime, china). the no production of plasma samples was measured by the kit (nanjing jiangcheng, china) according to the previous report by shi et al (shi et al., 2012). the caspase-3 activity was detected by a caspase 3 activity assay kit 123   (keygen, china) according to the manufacturer’s instructions. the relative caspase 3 activity was expressed by od experiment/od control. quantitative real-time pcr analysis of wssv viral numbers and stat, dicer genes the absolute taqman real-time assay was performed to determine the wssv viral numbers according to the previous report (zhang et al., 2010). total genomic dna extraction was followed by the manuscript of dna extraction kit (takara, dalian, china), and the dna concentrations were determined by using nanodrop 2000 (usa). the real-time pcr was carried out to quantify the mrna expression of stat, dicer. total rna of hemocytes collected from each experimental group was extracted using trizol reagent (takara, japan). the first-strand cdna was obtained according to m-mlv rt usage manual protocol (promega, usa). the qpcr and the equation of 2-δδct to calculate the relative expression level of genes were performed as described by wang et al (wang et al., 2013). shrimp ef-1α (elongation factor) was selected as the internal control gene described by roux et al (roux et al., 2002). the primers for the synthesis of cdna template and the real-time pcr assays were presented in table 1. statistical analysis the data were analyzed by spss17.0 software using one-way anova and duncan test. all experiments were implemented in triplicate, and the values were given as means ± sd. differences were considered as significant at p < 0.05. results generation of recombinant y. lipolytica and assessment the vp28 protein displayed on the surface of y. lipolytica was detected by immunofluorescence assay. the positive signal was observed in green, while there was no fluorescence signal detected in the control (fig.1a). the recombinant y. lipolytica and e. coli were designated as rvp28-yl and rvp28-ec respectively. western blotting was used to determine and compare the concentration of recombinant vp28 protein in two strains with anti-vp28 polyclonal antibody (fig. 1b) by quality one software. the vp28 presented on the surface of y. lipolytica was about average of 63.2 μg ml-1 (108 cfu), and only 3.4 μg ml-1 in the supernatant of yeast. the vp28 productin in e. coli was about 23.8 μg ml-1 (108 cfu) (fig. 1b). at the same cfu, the vp28 protein generated by y. lipolytica was 2.7 fold higher (p < 0.05) than that generated from e. coli. and the vp28 protein in the supernatant of yeast culture was significantly lower, which confirmed that the y. lipolytica strain was more suitable for the preparation of feeding diet. the cumulative mortality wssv challenge experiment was performed following oral administration, and the mortality of shrimps was recorded (fig. 2). shrimps began to die after 2nd day in all groups. on the 6th day, the cumulative mortalities in the cpg and rvp28-yl group was 39.4 ± 3.0 % and 36.4 ± 2.1 %, fig. 2 comparative protection effects of cpg odns, rvp28-yl and basal diet feeding shrimps after wssv challenge, cumulative mortality rates from cpg group(■), rvp28-yl group(▲) and the control group (♦)were presented. significant differences of the control group and supplemental diet feeding groups were indicated with asterisk at p < 0.05. respectively, which was significantly lower than that in the the control group (67.0 ± 3.0 %) (p < 0.05). from the 7th day to the 9th day post wssv challenge, the cumulative mortalities in the control group were significantly higher than that in cpg and rvp28-yl group (p < 0.05). on 10th day, the cumulative mortality nearly reached 100 % in the the control group, whereas it was only 77.4 ± 2.1 % and 71.2 ± 2.1 % in cpg and rvp28-yl group (p < 0.05). wssv quantification after the feeding trial, wssv copy numbers were measured in the hemocytes of all shrimps, which were 23.41, 12.53 and 19.23 copies ng-1 dna in cpg, rvp28-yl, and the control group, respectively. after wssv challenge, the virus copy numbers in cpg and rvp28-yl group were significantly lower than that of the control group. on the 1st day after wssv challenge, the copy number increased in all shrimpswith no significant difference observed among the groups. on the 3rd day post challenge, the mean copy number in the control group increased to 1.30×106 copies ng-1 dna, while 5.18×105 and 3.65×105 copies ng-1 dna were detected in cpg and rvp28-yl feeding shrimps, respectively. the virus number in the control group was 2.5 and 3.5 fold higher (p<0.05) than that in the cpg and rvp28-yl groups. the caspase-3 activity in shrimp hemocytes the caspase-3 activity was recorded as relative ratio (od experiment/od control). it ascended in cpg group after feeding trial, which was 2.3 and 1.9 fold (p < 0.05) higher than that of rvp28-yl and the control group, respectively. the caspase-3 activity in cpg feeding shrimps increased significantly on the 1st day post wssv challenge (p < 0.05) and dropped to normal level on the 3rd day. as time progressed during experimental trials, there was no significant 124   fig. 3 quantification of wssv viral copies number in cpg, rvp28-yl and control feeding shrimps pre-challenge and post wssv challenge on the 1st and 3rd day. each symbol and vertical bars represented the means of triplicate assays with standard deviation (sd). significant differences of wssv copies number between the two supplemental feeding shrimps and the control feeding ones were indicated with asterisk at p < 0.05. alteration of the caspase-3 activity in rvp28-yl group. in addition, the wssv challenge resulted in significantly higher caspase-3 activity in the control group on both the 1st day and 3rd day post challenge, and the activity was 3.3 and 3.7 fold higher than that in cpg and rvp28-yl group on the 3rd day (fig. 4). the po activity and no production in shrimp hemocytes cpg feeding shrimps resulted in a significant increase (p < 0.05) in po activity throughout the experimental trial. after feeding trial, po activity in cpg group was significantly increased to 2.1 and 2.3 fold of that in rvp28-yl and the control group (p < 0.05). after wssv challenge, it was significantly higher (p < 0.05) than that in all other groups on the the 1st and 3rd day, respectively. furthermore, after pbs stimulation, the po activity also exhibited significantly higher level (p < 0.05) in cpg group. however, the values of po activity in rvp28-yl group displayed no significant alteration post feeding trial and wssv challenge. the po activity in the control group was significantly decreased on the the 1st and 3rd day post wssv challenge (p < 0.05) (fig. 5a). the no productions in cpg and rvp28-yl group were higher than that of the control group (p < 0.05) at the end of feeding trial. after wssv challenge, the production of no in all three groups increased on the the 1st day. it was 23.3 μm in cpg group which was significantly higher than that in rvp28-yl (13.0 μm) and and the control group (13.6 μm), respectively (p < 0.05). and on the 3rd day, it decreased in cpg group (15.1 μm), while increased to 17.23 μm in rvp28-yl group, which were both significantly higher than that in the control group (11.2 μm) (p < 0.05) (fig. 5b). the mrna expression of immune-related genes the mrna expression of stat and dicer exhibited different variation tendency post feeding and wssv challenge. the expression level of stat mrna exhibited no significant variation in each group post feeding trial. but it increased significantly in hemocytes of cpg fed shrimps on the the 1st and 3rd day post wssv challenge (p < 0.05), which was 15.5 fold higher than that of blank on the 3rd day post challenge (fig. 6a). the expression level of stat in the control group decreased significantly on the 3rd day compared with other groups (p < 0.05). the mrna expression levels of dicer in cpg feeding shrimps were up-regulated both after feeding trial and wssv challenge, which were significantly increased than that in other group (p < 0.05) (fig. 6b). there was no significant difference of dicer mrna expression in rvp28-yl group between wssv and pbs injected subgroups throughout the experiment. discussion in shrimp culture industry, immunological approaches including the use of immunostimulants and vaccination have been validated with beneficial effects on the prevention and control of wssv 125   fig. 4 caspsase-3 activity in cpg odns, rvp28-yl and control feeding shrimps, including post feeding and post wssv and pbs challenge on the 1st and 3rd day. each symbol and vertical bars represented the means of triplicate assays with standard deviation(sd). bars with different letters are statistically significant from each other in the same sampling point (p < 0.05). diseases (xu et al., 2011; haq et al., 2012). it has been demonstrated that cpg odns can trigger various immune responses to enhance the immune capability, and it could be used as an immunostimulant candidate (carrington and secombes, 2006). vp28 is one of main structural proteins of wssv participating in the virus entry into cells, and it has been considered as a potential “vaccine” candidate for crustaceans against wssv infection (johnson et al., 2008). in the present study, cpg odns and vp28 displayed on y. lipolytica surface (rvp28-yl) were employed by oral routes to investigate their protective effects of shrimps against wssv. since oral feeding is the basic approach for the intake of nutriments in all stages of shrimp life, it is generally applicable in shrimp aquaculture (syed and kwang, 2011). the yeast y. lipolytica has always been employed as a probiotic candidate for its immunological properties. in the present study, vp28 was highly expressed on the surface of y. lipolytica, and the new constructed y. lipolytica strain was employed as the vehicle of vp28. meanwhile, cpg odns were large-scale prepared via plasmid extraction with alkali method. after fed with cpg and rvp28-yl for 15 days, the shrimps were challenged with wssv, and the mortality was recorded for ten days. from 6th day to 10th day post wssv challenge, the mortality rates of shrimps in cpg and rvp28-yl group were significantly lower than that in the the control group. on the 10th day, almost all the shrimps died in the control group (100 % mortality rate), while the survival rate in cpg and rvp28-yl group were 22.6 ± 2.1 % and 28.8 ± 2.1 %, respectively. meanwhile, the wssv copy numbers were also significantly lower in cpg and rvp28-yl group than that in the control group on the 3rd day post wssv challenge. it is generally accepted that there is a relationship between the high survival rate of culture shrimp and the low virus load (jang et al., 2009). these results indicated that the replication and proliferation of wssv could be partially inhibited by cpg and rvp28-yl, and the oral administration of cpg and rvp28-yl enhanced antiviral immunity of shrimp against wssv infection. in invertebrates, hemocytes are generally regarded as the main component of immune defense system, which participate in the immune responses against pathogen, such as apoptosis, encapsulation, melanization, oxidation and so on (bachere et al., 2004). in the present study, shrimps fed with cpg and rvp28-yl displayed the obvious enhanced capability to reduce the mortality rate caused by wssv. some relative immune parameters and mrna expression of antiviral genes in hemocytes were then analyzed to address the innate immune responses of l. vannamei after oral administration. apoptosis, as one of vital cellular defense mechanisms, can eliminate the pathogen infected cells to avoid their delivering into surrounding cells, and it has been reported to exert comparably obvious function against wssv infection in shrimp (leu et al., 2013; wang and zhang, 2008). in the intricate apoptotic course, 126   fig. 5 phenoloxidase (po) activity(a) and no production(b) in cpg odns, rvp28-yl and control feeding shrimps, including post feeding and post wssv and pbs challenge on the 1st and 3rd day. each symbol and vertical bars represented the means of triplicate assays with standard deviation (sd). bars with different letters are statistically significant from each other in the same sampling point (p < 0.05). caspase protein family members are the central effectors, among which caspase-3 has been confirmed to be the crucial one and also the indicator used to mirror the level of apoptosis (fu et al., 2010). in our previous study, cpg odns have be confirmed to boost apoptosis in shrimp hemocytes (sun et al., 2013a). in the present study, a significant increase of caspase-3 activity was observed after the shrimps were fed with cpg odns, and it ascended further at early stage of wssv infection, followed by a decrease on the 3rd day post wssv challenge. the lower mortality rate and wssv copies, and the 127   enhanced caspase-3 activity suggested that cpg odns could induce apoptosis which might contribute to the effective protection for shrimps to eliminate wssv at the early stage of infection. in the control group, caspase-3 activity kept rapid increase after wssv challenge, which indicated that the higher apoptosis level induced by wssv could generate damage effects for shrimps. however, there was no obvious alteration of caspase-3 activity in the rvp28-yl group before and post wssv challenge. it indicated that the apoptosis induced by wssv could be suppressed in rvp28-yl feeding shrimps. it has been reported that vp28 could bind with the host cell receptor to reduce the possibility of wssv entry into cells (sritunyalucksana et al., 2012). therefore, cpg and rvp28-yl might contribute to the protective effect against wssv via inducing adequate apoptosis activity to eliminate wssv and inhibiting the cell infection. po is one of the significant components of propo system in crustaceans, and it is also the key enzyme to control melanism cascade (li and xiang, 2013b). it was documented that cpg odn could activate the propo system and enhance the phenoloxidase activity effectively in giant freshwater prawn (chuo et al., 2005). in the present study, po activity increased significantly after feeding trial in cpg group, indicating that po system was provoked by cpg odns stimulation. the enzyme activity in cpg odns feeding shrimps kept increasing after wssv challenge, which confirmed that the effect of cpg odns on the activation of po system was long-lasting. meanwhile, there was no significant difference of po activity in rvp28-yl group post feeding and wssv challenge, which was in agreement with the result obtained in oral administration of rvp28 in f. chinensis (fu et al., 2010). however, the po activity was significantly decreased after wssv infection in the control group, suggesting that wssv could inhibit po activity and immune response induced by propo system, then continue to infect into host cells. it was interesting that the po activity in rvp28-yl group was significantly higher than that in the control group, but it did not increase post wssv challenge, suggesting that rvp28-yl was functional in the defense against wssv. these results together suggested that cpg odns and rvp28-yl could partially neutralize the inhibitory effect of wssv on propo system, and enhance the protective immunity of shrimps. no is considered as an important signal molecule playing versatile roles in many physiological processes including immune defense. the production of no in cells is one important immune response, mediating an oxidative progress with reactive oxygen species such as superoxide anions to enhance the non-specific immunity against pathogenic invasion (colasanti and venturini, 1998; bogdan, 2001). haiqi et al. (2003) reported that cpg odn significantly stimulated no production in avian. in the present study, the no production in cpg group was significantly higher than that in the control group after feeding trial, and also significantly increased on the 1st and 3rd day post wssv challenge, suggested that the cpg odns induced no production contributed to the resistance against wssv infection. it was also observed that the no production in rvp28-yl group increased after feeding trial and on the 3rd day post wssv challenge. similar results were also reported in f. chinensis that rvp28 could significantly heighten the inos activity (fu et al., 2010). because inos regulated the production of no and it was involved in innate response against wssv in shrimp (jiang et al., 2006). it has been demonstrated that some potent inducers contribute to the antiviral immune response, such as antiviral immunoregularory factors and double-stranded rna (tassanakajon et al., 2013). stat is one of transcription factors that regulate antiviral pathways (darnell, 1997; li and xiang, 2013b), and the previous reports have documented that several virus replication and proliferation could be inhibited by the regulation of jak/stat pathway (darnell, 1997; decker et al., 2002; tassanakajon et al., 2013). stat deficient mice were more susceptible than wild ones when they were undergone the rna virus infection (durbin et al., 1996). stat in invertebrates was also confirmed to play roles in antiviral process which was similar to that in mammals. for instance, stat from shrimps could be activated when they were infected with wssv (dostert et al., 2005; chen et al., 2008), and the mrna expression of stat was down-regulated as wssv infection progressed (syed and kwang, 2011). in the present study, the expression level of stat mrna continued decreasing in the control group post wssv challenge, indicating that wssv might inhibit immune response induced by stat in the early stage of infection. furthermore, there were significant difference of stat expression in cpg group and rvp28-yl group compared to that in the control group post wssv stimulation, suggesting that cpg odns and rvp28-yl could remove the inhibition generated by wssv to activate stat expression. it indicated that cpg odns and rvp28-yl could induce the stat mediated antiviral response in l. vannamei. and the significant higher stat mrna expression on the 3rd day in cpg group after wssv challenge suggested that cpg odns was a more effective inducer for the stat-mediated antiviral immunity in shrimp. rnai has been accepted to be one of the most promising strategies to combat both dna and rna virus in invertebrate (robalino et al., 2004). dicer is a key enzyme involved in rna interference, which recognizes a viral rna and splices it to small rna to exerts a protective role for host against rna and dna virus (lee et al., 2004 (lee et al., 2004; kemp and imler, 2009) . dicer could inhibit the replication of hiv-1 virus, and the knockdown of dicer gene yielded the increased virus production (haase et al., 2005). in the present study, the mrna expression of dicer in cpg group was significantly higher than that in rvp28-yl and the control group after feeding trial, and a delayed up-regulation was observed in cpg group on 3rd day post wssv challenge, suggesting that cpg odns might induce the dicer expression to render the corresponding inhibitory effect on wssv replication. interestingly, the feeding of rvp28-yl didn’t lead to significant alteration of dicer mrna expression compared to control. after wssv challenge, there was also no significant difference of dicer expression between rvp28-yl and the control 128   fig. 6 the mrna expreesion level of stat (a) and dicer (b) genes relative to ef-α gene in cpg odns, rvp28-yl and control feeding shrimps, including post feeding and post wssv and pbs challenge on the 1st and 3rd day. each symbol and vertical bars represented the means of triplicate assays with standard deviation (sd). bars with different letters are statistically significant from each other in the same sampling point (p < 0.05). group, indicating that dicer was not involved in the immune defense caused by rvp28-yl. in contrast, cpg odns might contribute to the enhanced antiviral immunity of shrimps for the induction of dicer gene to interfere the replication of wssv. in summary, the oral administration with cpg odns and rvp28-yl in shrimps significantly induced the immune responses including po activity, no concentration, caspase-3 activity, and expressions of stat and dicer, which might endow shrimps with 129   enhanced protective capability under wssv challenge. cpg and rvp28-yl exhibited almost similar efficacy in terms of protective effect for l. vannamei against wssv infection through different mechanisms. the present results provided insights into the immunological prevention management in shrimp culture industry. acknowledgements the authors thank dr. zhenming chi from ocean university of china for the technical assistance. they are grateful to mr. xuyun geng and junli wei from institute of tianjin fisheries research for the guidance in shrimp rearing, and all the laboratory members for their technical advices. this research was supported by national basic research program of china (973 program, no. 2012cb114405), and shandong provincial natural science foundation (no. jq201110). reference bachere e, gueguen y, gonzalez m, de lorgeril j, garnier j, romestand b. insights into the anti-microbial defense of marine invertebrates: the penaeid shrimps and the oyster crassostrea gigas. immunol. rev. 198: 149-168, 2004. bogdan c. nitric oxide and the immune response. nat. immunol. 2: 907-916, 2001. carrington ac, secombes, cj. a review of cpgs and their relevance to aquaculture. vet. immunol. immunopathol. 112: 87-101, 2006. chang c-f, su m-s, chen h-y, liao ic. dietary β-1,3-glucan effectively improves immunity and survival of penaeus monodon challenged with 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farreri. plos one. 6, e18596, 2011. 131   results isj 10: 29-37, 2013 issn 1824-307x research report a novel third complement component c3 gene of ciona intestinalis expressed in the endoderm at the early developmental stages t hibino1, m nonaka2 1faculty of education, saitama university, 255 shimo-okubo, sakura-ku, saitama city 338-8570, japan 2department of biological sciences, graduate school of science, the university of tokyo, 7-3-1 hongo, bunkyo-ku, tokyo 113-0033, japan accepted march 29, 2013 abstract the third complement component (c3) in ascidian was reported to function as an opsonin to enhance phagocytosis and as a chemotactic factor for phagocytes, indicating that ascidian c3 works in mesodermal cavity as a humoral factor like vertebrate c3s. in the basal eumetazoa, cnidaria lacking mesodermal tissues, c3 was reported to work in an endodermal cavity. evolution of structure and function of c3 is still to be clarified. here we report the identification of the third c3 gene, cic3-3, in the genome of an ascidian, ciona intestinalis. phylogenetic analysis using the entire amino acid sequences of eumetazoan c3s indicated that cic3-3 possess a closer relationship to vertebrate c3, c4 and c5 than other ascidian c3s. although cic3-3 retained the α-β processing site and 6 cysteine residues in the c3a region, it lacked the intra-molecular thioester bond and the catalytic histidine residue. instead, cic3-3 had a unique insertion of about 70 residues long lys/arg-rich sequence. cic3-3 was expressed highly in the embryonic stages, but little in the adult in contradistinction to cic3-1 and cic3-2. the expression of cic3-3 in early embryonic stages was restricted to endoderm similar to cnidarian c3s. thus, the ascidian complement system could represent a unique evolutionary stage sharing a primitive endodermal function with cnidaria, and newly developed humoral function with vertebrates. key words: thioester-containing protein (tep); complement c3; innate immunity; immunogenetics; tunicate; chordate introduction the vertebrate complement system comprises more than 30 proteins present in serum or on cell surface, and plays a pivotal role in innate immunity. this system is triggered by three different activation pathways, the classical, alternative and lectin pathways. these three pathways merge at the proteolytic activation step of the complement component 3 (c3) into c3a and c3b. upon proteolytic activation, c3 changes its conformation exposing the intra-chain thioester bond at the molecular surface. the exposed thioester bond of c3b reacts with surface molecules of invading microbes and makes a covalent bond, resulting in covalent tagging of microbes with c3b. covalently attached c3b works as opsonin to induce phagocytosis, and also induces assembly of the ___________________________________________________________________________ corresponding author: masaru nonaka department of biological sciences graduate school of science the university of tokyo 7-3-1 hongo, bunkyo-ku, tokyo 113-0033, japan e-mail: mnonaka@biol.s.u-tokyo.ac.jp terminal components of complement (tccs: c6~c9) into a membrane-attack complex that can damage the membrane of certain pathogens (murphy, 2011). the proteins possessing a similar domain structure as vertebrate tccs are present in ascidian and amphioxus. however, these proteins may not be activated through the complement system of ascidian and amphioxus because they lack an essential domain for interaction with c5b (nonaka and kimura 2006). the released smaller c3a fragment is an anaphylatoxin to induce inflammation. the c3 subfamily including c3, c4 and c5 is a member of thioester bond-containing protein (tep) family, together with the non-complement tep subfamilies such as the α2-macroglobulin (a2m) and cd109 subfamilies. the c3 subfamily members are distinguished from the a2m and cd109 subfamily members by the presence of the anaphylatoxin (ana) and c-terminal of c3, c4 ,c5 (c345c) domains unique to the c3 subfamily (sekiguchi et al., 2012). genes orthologous to vertebrate c3 have been identified not only from invertebrate deuterostome such as sea urchin (al-sharif et al., 29 fig. 1 phylogenetic tree of tep family members constructed by the neighbor-joining method using the entire amino acid sequences. bootstrap values higher than 50 % are indicated in the tree. accession numbers of each entry are; human c3, c4a, c5, a2m, and cd109 (np_000055, p0c0l4, aaa51925, p01023,and np_598000), carp c3h1, c4-1, and c5-1 (baa36619, bab03284, and bac23057), ascidian, halocynthia roretzi c3 (baa75069), c. intestinalis cic3-1, cic3-2, cia2m-like, and cicd109-like (np_001027684, cac85958, xp_002124325, and np_001027688), sea urchin c3 and c3-2 (np_999686 and spbase: spu_000997), horseshoecrab c3 (aaq08323), amphioxus c3 (bab47146), coral c3 (aan86548), cnidarian nvc3-1 and -2 (ab450038 and ab450040), and fly tep1 (np_523578). 1998; hibino et al., 2006; rast et al., 2006) and amphioxus (huang et al., 2008), but also from protostomes such as horseshoe crab (zhu et al., 2005; kawabata et al., 2009) and spider (sekiguchi et al. 2012) as well as cnidarian coral (miller et al., 2007) and sea anemone (kimura et al., 2009). the presence of c3 in cnidaria indicated that the c3 gene has been established prior to the divergence of cnidarian from bilaterian (nonaka, 2011). in contrast to its wide distribution, c4 and c5 has only been identified in jawed vertebrate, suggesting that c4 and c5 were derived from a c3-like common ancestor by gene duplication in the early stage of jawed vertebrate evolution (nonaka and takahashi, 1992). a tunicate, ciona intestinalis (urochordata) has been an attractive research model for developmental biology for more than a century (satoh et al., 2003). the recent accumulation of genome-wide sequence information showed that not cephalochordate but urochordate is a sister group of vertebrate, indicating that c. intestinalis is one of the most important species for understanding the origin and evolution of vertebrates (dehal et al., 2002, putnam et al., 2008). the c. intestinalis genome analysis revealed that this animal possesses several genes for complement components: two c3s, three bf/c2s, 10 of c6/c7/c8/c9/perforin and so on. an ancestor of the two c3-like genes seems to have diverged from a common ancestor of vertebrate c3/c4/c5 and has duplicated into two genes in the ciona lineage (azumi et al., 2003a). using c. intestinalis, in-depth expressed sequence tag and large-scale oligo-dna microarray analyses have been advanced, which identified gene expression profile during the life cycle (azumi et al., 2003b; satou et al., 2002, 2003). interestingly, c3s, masp, factor b (bf), mbp and two genes of complement c6-like were expressed only in the adult stages. on the other hand, c1q-like and two other genes of complement c6-like were expressed in the middle of the embryonic stages and maintained their expression level during the adult stages (azumi et al., 2007). the absence of c3 expression during the developmental stages could be explained by one of the following two hypotheses: (1) since c. intestinalis develops directly and metamorphosis in a day after fertilization, protection against infection which is considered to be the most 30 important physiological function of c3 is unnecessary in this short period, or (2) unidentified c3 is working under the developmental stage. in this study, we report a novel c. intestinalis c3 gene, cic3-3, that belonged to a different clade from known ascidian c3 genes in a phylogenetic tree, and that was specifically expressed in endoderm of the embryos. materials and methods adults and embryos adults of the ascidian c. intestinalis were provided from misaki marine biological station, the university of tokyo through national bio-resource project (nbrp) of mext, japan. the adults were surgically dissected to draw eggs and sperm. fertilized eggs were incubated at devitellination medium containing 0.065 % actinase e (kaken co. ltd.) and 1.3 % sodium thioglycolate in sea water at a ph of approximately 10, to devitellinate chemically (satou et al., 2001). devitellinated embryos were reared at 18 °c in agar-coated plastic dished filled with filtered sea water containing 50 µg/ml penicillin and 100 µg/ml streptomycin. gene identification in c. intestinalis genome database deduced amino acid sequences of all computationally predicted proteins were downloaded from the website, ensembl c. intestinalis database (http://www.ensembl.org/) (hubbard et al., 2007), and the ghost database: c. intestinalis genomic and cdna resources (http://ghost.zool.kyoto-u.ac.jp/indexr1.html) (satou et al., 2005). a typical c3 protein contains multiple domains; a2m_n, a2m_n2, a2m, anato, a2m_comp, a2m_recep, c345c, whose profile hmms were downloaded from the pfam website (http://pfam.sanger.ac.uk/) (bateman et al., 2004). hmmer (eddy et al., 1998) was used to identify pfam domain profile matches to c. intestinalis protein models. the deduced amino acid sequences of identified protein models were aligned with those of known c. intestinalis c3, cic3-1 and cic3-2 and ciα2-macroblobulins to find out a novel c3 gene model. cloning of a novel c3 gene of c. intestinalis c. intestinalis cdna was synthesized from the adult tissues containing gills and blood cells, and used as a template for pcr amplification. to confirm the nucleotide sequences, especially exon-intron boundary, of the novel identified c3 (cic3-3) gene model, rt-pcr was performed using primers that were designed at the ends of 5’ utr (forward 5’-ttggaaagccgtactatgcgacacg-3’) and 3’ utr (reverse 5’-tgctttggcaatatacacgtggcagt-3’) of the gene model. probably the nucleotide length of the predicted gene model of 5.7 kbp was too long for rt-pcr, the entire length of cic3-3 could not be amplified by using these primers. we then designed other primers at the middle of the gene models (forward 5’-tggaacaatcgctgctgctgtaa-3’, reverse 5’-atgccttctgggaccacattcaa-3’). extaq dna polymerase (takara) was used for this pcr. the cdna fragments were cloned into pcr2.1-topo vector (invitrogen) and were sequenced using vector specific primers or gene specific primers. domain prediction and phylogenetic analysis domain structure of the cic3-3 was predicted by the smart program (http://smart.embl-heidelberg.de/) (letunic et al., 2002). the e-value for the domain confidence was assessed by hmmer3 on the smart program. multiple alignment of the amino acid sequences among c. intestinalis and human c3s was done by clustalw on the mega5 program (tamura et al., 2011), as well as by eyes. based on this alignment, phylogenetic trees were constructed using full-length amino acid sequence information or a2m_comp domain region that was extracted using by the smart program. the neighbor-joining (nj) method (saitou and nei 1987) using mega5 excluding gaps by pairwise deletion was performed. the reliability for internal branches was assessed by the 1000 bootstrap replications. gene expression analysis using ciona database and whole mount in situ hybridization the c. intestinalis protein database (cipro 2.5) integrates not only protein database, but also transcriptome database including large-scale est analysis and dna microarray data (endo et al., 2011). we extracted the expression data of the three cic3 genes from the website and integrated the data in a graph. whole mount in situ hybridization was performed based on the previously described protocol (ogasawara et al., 2001; satou et al., 2001) with some modifications. for antisense or sense ribonucleotide probe for cic3-3, 544 bp of the 3’ end of coding region that covers the full length of the c345c domain and subsequent stop codon for cic3-3 was cloned into ptac-2 with dynaexpress ta pcr cloning kit, and the probes were subsequently synthesized using digoxigenin (dig) rna labeling mix and t7 or sp6 rna polymerase (roche). embryos were fixed with 4 % paraformaldehyde in 0.1 m mops (ph 7.5), 0.5 m nacl at 4 °c overnight. the developmental stages of fixed embryos were determined following hotta et al. (2007). the fixed embryos were washed three times with pbst (phosphate-buffered saline containing 0.1 % tween-20), then partially digested with 2 μg/ml proteinase k in pbst for 20 min at 37 °c. they were washed twice with pbst, subsequently post-fixed with 4 % paraformaldehyde in pbst for 1 h at room temperature (rt), and then washed three times with pbst. after prehybridization at 50 °c for 1 h, the embryos were hybridized at 50 °c for 24 h in the following buffer. the hybridization buffer contained 50 % formamide, 5x denhardt’s solution, 100 μg/ml yeast rna, 0.1 % tween-20, and 0.2 μg/ml dig-labeled rna probes. after hybridization, the embryos were washed three times with 2xssc, 50 % formamide, 0.1 % tween-20 at 50 °c for 15 min, then washed three times with 1xssc, 50 % formamide, 0.1 % tween-20 (a) at 50 °c for 15 min. next they were washed twice with 1:1, (a): pbst at rt for 10min, and then washed three times with pbst at rt for 3 min. after the series of washing, 31 http://www.ensembl.org/ http://ghost.zool.kyoto-u.ac.jp/indexr1.html http://pfam.sanger.ac.uk/ http://smart.embl-heidelberg.de/ the specimens were blocked with 0.5 % blocking reagent (roche) in pbst at rt for 30 min. they were immersed in 1/2000 anti-dig-ap fab fragments (roche) diluted with pbst at rt for 6 h. the embryos were washed 4 times with pbst for 10 min and then washed twice with alkaline phosphatase buffer (0.1 m tris-hcl (ph 9.5), 50 mm mgcl2, 0.1 m nacl) for 10 min. for signal detection, the embryos were incubated with nbt/bcip in the alkaline phosphatase buffer at rt overnight. the stained embryos were dehydrated in a graded series of ethanol, and then cleared in a 1: 2 mixture of benzyl alcohol/benzyl benzoate. results identification of the third complement c3 gene in c. intestinalis to find gene candidates encoding multiple domains of typical thioester containing protein (tep) superfamily from c. intestinalis, all of the deduced amino acid sequences of the fgenesh gene models and the genscan gene models that computationally predicted from the c. intestinalis genome were searched by local hmmer program using profile hmms containing the a2m_n, a2m_n2, anato, a2m, a2m_comp and c345c domains. out of five gene models extracted by this analysis, four gene models matched with the already reported tep genes. the other gene model, fgenesh76597 or genscan101558, predicted a 3,759 bp open reading frame corresponding to a 1,253 amino acid sequence containing the a2m_n2, anato, a2m and c345c domains. the same gene was contained in the recently uploaded kh gene models (ver. 2012) in ghost database: c. intestinalis genomic and cdna resources (http://ghost.zool.kyoto-u.ac.jp/indexr1.html) (satou et al., 2005). this gene model, kh.c12.243.v1.a.sl1-1, with a longer nucleotide sequence than that of the fgenesh/genscan model predicted a 1,873 amino acid sequence containing the additional a2m_n domain at its n-terminus. based on these gene models, several primers were constructed at the end or at the middle of the sequences, and then a novel c3 gene candidate was cloned and sequenced. the cloned nucleotide sequences had a size of 5,946 bp (1,873 amino acid residues) that matched 98.7 % (5666/5739) to that of the kh gene model. the novel and the third complement c3 gene in c. intestinalis was designated as cic3-3. phylogenetic analysis using the neighbor-joining method the deduced amino acid sequence of the cic3-3 gene was aligned with the known eumetazoan tep superfamily genes using clustalw program (data not shown). the phylogenetic tree was constructed based on the entire amino acid sequences using the nj method (fig. 1). the phylogenetic tree showed the presence of three subfamilies, the c3, a2m and cd109 subfamilies, supported with bootstrap percentages of 52, 98 and 99 %, respectively. in the c3 subfamily, cic3-3 was grouped with vertebrate c3/c4/c5 with a 85 % bootstrap percentage, but not with other ascidian c3 lineage including cic3-1, cic3-2 and h. roretzi c3. this result together with a long branch length of cic3-3 indicates that cic3-3 is a highly derivative ascidian c3, whose evolutionary origin is still to be clarified by analyzing other ascidian species. structural features of cic3-3 to reveal whether cic3-3 conserves the primary structure as well as domain structures of the vertebrate c3 subfamily, the deduced amino acid sequences of cic3-3 were aligned with cic3-1 and -2, and human c3, c4 and c5. the smart domain search was also performed to find the multiple domains (fig. 2). the alignment and domain search showed that cic3-3 possesses a signal peptide for secretion, the α/β processing site (rxxr) for dividing into two subunit chains, two cys residues involved in an inter-chain disulfide linkage between the α and β chains and a possible activation cleavage site (ttr) by the c3 convertase (fig. 1, table 1), suggesting that cic3-3 is processed into αand β-chains held together with the inter-chain disulfide bond similar to mammal c3s. the c3a anaphylatoxin (ana) region of cic3-3 contained the six cys residues conserved by most c3a analyzed thus far. since cic3-1 and -2 possess only four of them, cic3-3 showed a higher conservation in the c3a region. however, cic3-3 lacked the thioester site, gcgeq, and the catalytic his residue for cleavage of thioester. moreover cic3-3 also lacked the two pro residues on both sides of the thioester site which are conserved even in human c5 lacking the thioester site. these results suggest that the 3d structure around the thioester site is markedly modified in cic3-3 (highlighted in yellow and blue in fig. 2, summarized in table 1). table 1 32 http://ghost.zool.kyoto-u.ac.jp/indexr1.html fig. 2 sequence comparison of cic3-3, cic3-1, cic3-2 and the human c3, c4b, and c5. the multiple sequence alignment of cic3-3 with human and other c. intestinalis c3s was performed with clustalw. a2m_comp domain region is boxed. proteolytic cleavage sites are shown in bold letters. conserved cys residues in the c3a anaphylatoxin region are marked (*). the inter-chain disulfide bridges between the α/β chains are shaded. the thioester sites, catalytic his sites and kr-rich insertion are also annotated in each colored box. 33 phylogenetic analysis of a2m_comp domain region to reveal independent loss of thioester site and catalytic his. to reveal whether loss of the thioester site and catalytic his occurred independently in cic3-3 and vertebrate c5 or not, we reconstructed a phylogenetic tree with the deduced amino acid sequences based on the a2m_comp domain located at the c-terminal side of the thioester site. although the size of the usual a2m_comp domain is approximately 260 amino acid residues long, this domain of cic3-3 expands to 336 residues due to an insertion of approximately 70 amino acid residues highly enriched in lys and arg. a similar lys/arg rich insertion has already been reported from two cnidarian c3s, nv3-1, nv3-2, although the insertion of cnidarian c3 was observed at the different region, much more c-terminal side. therefore, the insertion of the lys/arg rich sequence into c3 occurred at least twice independently during the eumetazoa evolution. the nj tree constructed using the amino acid sequences of the a2m_comp domain showed the essentially the same topology as the tree based on the full length information described above, except that cic3-3 is separated far from c3 family (fig. 3). the long branch of cic3-3 indicates that the primary structure of a2m_comp region of cic3-3 is highly divergent. overall, these results indicate that cic3 possesses well conserved domain organization similar to vertebrate c3/c4/c5 except for the thioester site and the subsequent a2m_comp domain. spatial and temporal expression of the cic3-3 gene to understand the gene expression pattern of cic3-3, we first analyzed transcriptome data on the c. intestinalis protein database (cipro) (endo et al., fig. 3 phylogenetic tree of tep family members constructed by the neighbor-joining method using the amino acid sequences of a2m_comp domain. bootstrap values higher than 50 % are indicated in the tree. the genes in this tree are same as figure 1. 2011), and compared gene expressions among cic3-1, cic3-2 and cic3-3. both cic3-1 and cic3-2 were not expressed before the metamorphosis except for very slight expression in the tailbud stage, while both microarray and est a) b) fig. 4 comparison of expression intensities among cic3-1, cic3-2 and cic3-3. cic3-1, -2 and -3 are shown in light blue, light green and red, respectively. the bars represent the est data, while the lines represent the microarray data (labeled as cic3-1m, -2m -3m). y axes of graphs a and b indicate relative expression levels. the results of the est and microarray analyses are shown on the left and right sides, respectively. a: expression profiles during the life cycle of c. intestinalis. the left side of the graph indicates the expression intensity for est data, while the right side of the graph denotes the expression intensity for microarray data. b: expression profiles of adult tissues. the bars denote expression level estimated by the est analysis. 34 data showed that cic3-3 was significantly expressed from the gastrula to the tailbud stage (fig. 4a). the expression of cic3-3 disappeared by the larva stage. after metamorphosis, cic3-1 and cic3-2 began to be expressed, whose intensities were getting stronger during maturation. in contrast to cic3-1 and cic3-2, cic3-3 was not expressed by juvenile and was slightly expressed from the young adult to mature adult stages. the intensity of cic3-3 expression in mature adults is approximately 1/5 of cic3-1 and 1/7 of cic3-2 (fig. 4a). the weak expression of cic3-3 was detected only in the blood cells. cic3-1 was ubiquitously expressed except for ovary and endostyle, and cic3-2 was expressed in heart and blood cell (fig. 4b). these expression data indicate that cic3-3 is expressed in a contradistinctive manner from cic3-1 and cic3-2. to identify the spatial expression pattern of cic3-3 during the development of c. intestinalis, we next performed whole mount in situ hybridization using rna probes of cic3-3. cic3-3 began to be expressed in the invaginated cells of the early gastrulae (st. 11) (fig. 5a). at the late gastrula stage (st. 13), almost all of the invaginated cells expressed cic3-3. the cic3-3 expression was then restricted in the anterior end of the embryos (st. 14), especially the anterior ventral side of the invaginated cells strongly expressed cic3-3 (figs 5d, e). the strong expression was observed in the endoderm of the trunk region, and weak expression was observed in the endoderm strand of the ventral midline of the tail region (st. 16 and 19) (figs 5f, g, h). at the mid tailbud stage (st. 21) the cic3-3 expression was reduced and restricted only in the endoderm cells around the endodermal cavity (figs 5i, j). these expression data indicates that cic3-3 is specifically expressed in the endoderm of embryos, and ceases its expression before hatching into the larvae. discussion it had been reported that the number of complement c3 gene is one in h. roretzi, and two in c. intestinalis (nonaka et al., 1999; marino et al., 2002; azumi et al., 2003). h. roretzi and c. intestinalis belong to the orders, pleurogona and enterogona, respectively, and are evolutionary far apart to each other (turon et al., 2004). the phylogenic analysis of c3 genes have indicated that the gene duplication event between cic3-1 and cic3-2 occurred in the enterogona lineage after the divergence from pleurogona (marino et al., 2002). the newly found cic3-3 clustered with vertebrate c3, c4 and c5, rather than with cic3-1, cic3-2 and h. roretzi c3, although bootstrap percentage to support this clustering was not very high. this finding indicates the presence of two ancient c3 lineages in basal tunicates, the cic3-1, cic3-2 and h. roretzi c3 lineage and the cic3-3 lineage. two and three c3 genes were reported from the genomes of a sea urchin, strongylocentrotus purpuratus, and an amphioxus, branchiostoma floridae, respectively (hibino et al., 2006; huang et al., 2008). thus all the basal deuterotomes whose genomes have been elucidated so far contain more than two c3 genes. phylogenetic analysis showed that the multiple c3 genes of each species form species-specific cluster, indicating that gene fig. 5 spatial expression pattern of cic3-3 detected by whole mount in situ hybridization in the early gastrula through the mid tailbud stage embryos. the scale bar in a indicates 20 µm, and the magnification is the same for all pictures. a-e: anterior is toward to the top, f-j: anterior is toward to the left. f, h, i, j: ventral is toward to the bottom. a: vegetal view of early gastrula (4.9 hpf, st. 11), b-c: vegetal view of late gastrulae (5.9 hpf, st.13), c: no hybridization signal with sense probes, d: vegetal view of early neurulae (6.35 hpf, st. 14), e: lateral view of d, vegetal is toward to the right, f: lateral view of late neurulae (7.4 hpf, st. 16), g: ventral view of f, h: lateral view of early tailbud (9.3 hpf, st. 19), i, j: lateral view of mid tailbud (10 hpf, st. 21), arrow indicates endodermal cavity, j: diagram of mid-tailbud corresponding to plate i. yellow, light blue and pink denote endoderm, nervous system and notochord, respectively. 35 duplications occurred multiple times in each lineage. cic3-3 is exceptional in this aspect, suggesting a unique evolutionary history of this gene. cic3-1 and cic3-2 retain almost all domains and structural features of vertebrate c3, suggesting that they function as the central component of the ascidian complement system. actually, the c3a fragment of cic3-1 was demonstrated to induce chemotaxis of c. intestinalis hemocytes in the same way as vertebrate c3a (pinto et al., 2003). in contrast, cic3-3 showed an unprecedentedly unique structure. first of all, cic3-3 lacked the thioester site believed to be essential for covalent tagging of invading microorganisms by usual c3. unlike vertebrate c5 which also lacks the thioester site but retains the basic residues of the thioester domain, cic3-3 has a totally different sequence in this domain. especially, the insertion of the highly lys/arg-rich sequence could have drastic structural and functional consequence since it provides extremely positive charge to this region. it is unlikely, therefore, that cic3-3 play a similar function as vertebrate c3. however, the c3a region of cic3-3 showed a higher conservation of cys residues than those of cic3-1 and cic3-2, implicating in inflammatory process as anaphylatoxin. in mammal, the c3 gene is mainly expressed in hepatocytes and macrophages (lambris, 1988). in the ascidian h. roretzi, gastric caecum and blood cells have been identified as the sites of c3 gene expression (nonaka et al., 1999). the paraffin sections of the stomach in the adult of c. intestinalis have shown that both cic3-1 and cic3-2 are expressed only in the one type of blood cell, but not in the wall of the stomach (marino et al., 2002). gene expression profile during the life cycle of c. intestinalis using the large-scale oligo-dna microarray showed that not only cic3-1 and cic3-2, but also masp, factor b, mbp and two genes of complement c6-like were expressed only in the adult stages (azumi et al., 2007). two other genes of complement c6-like were expressed in the middle of the embryonic stages and maintained their expression level during the adult stages. in this study, cic3-3 showed a totally different temporal expression pattern during the life cycle from the other complement component genes of c. intestinalis. cic3-3 is prominently expressed during the embryonic stages when the other complement genes of c. intestinalis are hardly expressed. in adult stages, in contrast, cic3-3 is expressed at a very low level, whereas the other complement genes are expressed abundantly. since interactions among components are essential for complement activation, these results suggest that cic3-3 functions outside of the complement system. whole mount in situ hybridization revealed that cic3-3 was first expressed in the invaginating endoderm of the embryos. c. intestinalis develops in a direct developing manner, and the larvae do not undergo the differentiation of a functional gut. thus, endodermal expression of cic3-3 does not necessarily indicate digestive function. when the gastrulation began, the expression started in the invaginated region, and it was continuously seen invaginating cells from the head endoderm through the endoderm strand to the ventral blastopore. after closure of the blastopore, it was strongly expressed around the endodermal cavity in the trunk. this expression pattern indicates the possibility that cic3-3 is involved in development of certain embryonic region. complement c3 genes have been reported from basic metazoans, cnidarian coral, swiftia 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allolobophora sp., dendrobaena sp., dendrodrilus sp., and octolasion sp., and their numbers and riboflavin contents are affected in species-specific ways by soil quality, as observed by flow cytometry and spectrofluorimetry. the most striking results were obtained in the case of epigeic dendrodrilus rubidus; in unpolluted soil its riboflavin content was high, but when the earthworm was resident in metalliferous (pb/znor ni-polluted) soils, or transferred experimentally from unpolluted to the polluted field soils the riboflavin content was significantly reduced. such extreme alterations in a cohort of immune-competent cells were not observed in e. andrei, d. veneta, or al. chlorotica transferred into metalliferous soils. worms from these three species were also transferred to zn/pb/cd-polluted and unpolluted soils from southern poland. it was observed that species-specific changes in riboflavin content occurred not only due to metal pollution, but also other edaphic factors, possibly including organic matter content/quality. hypothetically, riboflavin status (storage/mobilization) may depend on parasite-immune system balance, which is disrupted by soilderived stressors, including metals. key words: earthworms; amebocytes/immunocytes; chloragocytes/eleocytes; riboflavin introduction the prevailing view is that a high degree of homology between invertebrate and vertebrate immune systems is found for innate but not for acquired immunity, implying that invertebrates lack both specificity of responses to pathogen invasion and memory (söderhäll, 2010). little et al. (2005) cautioned against such a rigid dichotomous view on the basis that it is plausible that invertebrates may possess capacities analogous to specificity and memory but achieve these via mechanisms that are distinct from those of vertebrates. nevertheless, the main focus in the burgeoning field of invertebrate immunology to date has been on innate responses to immunological challenges. a seminal review of innate immunity (mydlarz et al., 2006) concluded that there are a number of fundamental common pathways operating across invertebrate phyla; these ___________________________________________________________________________ corresponding author: barbara plytycz department of evolutionary immunobiology institute of zoology, jagiellonian university krakow, poland barbara.plytycz@uj.edu.pl pathways include the prophenoloxidase pathway, systemic phagocytic cells, cytotoxic effector responses, and antimicrobial molecules. there is, therefore, no question that innate immunity in all invertebrate taxa, irrespective of their preferred habitats and associated eco-physiology, involve processes that may be considered in the words of handy et al. (2003) “essential to normal function at cell tissue or organism level”. for this reason it is unsurprising that a variety of immune parameters have been used not only in vertebrates (duramad and holland, 2011) but also in a wide range of terrestrial and aquatic taxa (galloway and depledge, 2001; auffret et al., 2006; plytycz et al., 2009a; holmstrup et al., 2010) to assess the adverse effects of environmental stressors, including inorganic and organic contaminants, with the ultimate aim of achieving sound risk assessments (hagger et al., 2006) and establishing regulatory frameworks (handy et al., 2003). contaminated field soils can present formidable risk assessment and management challenges not only because ‘real-world’ pollution events often comprise of mixtures of potentially 199 fig. 1 celomocytes of eisenia andrei in confocal microscope bright light (a) and blue light (b); a amebocytes, e eleocytes (phot. by grzegorz tylko). (c) flow cytometric density plot of celomocyte sample from e. andrei; fl-1h autofluorescence intensity; ssc-h cell complexity/granularity; (d) spectrofluorimetric analysis of parallel samples; top: emission spectrum with maximum (proportional to riboflavin content) at 525 nm; bottom: twomaxima of riboflavin-specific excitation spectrum. interacting toxic chemicals, but also because only a fraction of the total concentration of a given contaminant is available to soil-dwelling organisms (semple et al., 2004). estimating available fractions and modeling their deleterious impacts on target biota is not straightforward. for this reason, ectotoxicological risk assessment has been steadily moving away from reliance on geochemical measurements toward the direct assay of perturbations in functional parameters in sentinel organisms (van straalen and roelofs, 2008; roelofs et al., 2008). according to römbke and egeler (2009) oligochaete worms are the “most important organisms in soil ecotoxicology”, a status attributable to a combination of factors including the ecological services that they render in a number of natural and disturbed temperate and tropical soils, as well as pragmatic considerations such as size, sensitivity, availability of standardized test methods. a number of earthworm biomarkers representing different levels of biological organization have been developed, optimized, and deployed (gastaldi et al., 2007; guo et al., 2009; brulle et al., 2010), including immunotoxicity biomarkers (plytycz et al., 2009a, 2011a; homa et al., 2010). the vast majority of earthworm biomarker studies have entailed laboratory exposures to experimentally spiked soils. 200 table 1 characteristics of several earthworm species from unpolluted sites in wales (cholewa et al., 2006) and krakow (*kwadrans et al., 2008) in respect of body weights, celomocyte numbers, among them percentages of eleocytes, and riboflavin content in celomocyte lysates, the latter being either undetectable (-), or present from low (x) to very high (xxxxx) amounts (plytycz et al., 2006). in bold species with high percentages of riboflavinloaded autofluorescent eleocytes species (alphabetically) body weight (g) celomocyte numbers (x 106) eleocytes (%) riboflavin content in celomyte lysates allolobophora chlorotica 0,2 1,3 30-43 xx aporrectodea caliginosa 0,9 0,4 1 aporrectodea longa 1,9 1,6 0,2 dendrobaena veneta* 1,5 9,0 22 x dendrodrilus rubidus 0,2 1,1 21 x eisenia fetida 0,6 3,0 22 xxxx lumbricus castaneus 0,2 0,3 0,5 lumbricus festivus 1,0 2 0,05 lumbricus rubellus 0,6 0,8 0,5 lumbricus terrestris 4,0 1,9 0,5 octolasion cyaneum 1,5 1,5 22 octolasion tyrtaeum lacteum 0,4 1,3 11 octolasion tyrtaeum tyrtaeum 0,7 3,4 35 xxx laboratory exposures of ‘naïve’ worms to contaminated field soils (i.e., ‘semi-field studies’) or, much rarer, measurements of biomarkers in individual earthworms sampled from field populations inhabiting contaminated soils (i.e., ‘field studies’) are less commonplace. this mini-review brings together some of our recent field and semifield studies on metalliferous soils. earthworm celomocytes evidently the earthworm immune system is functionally efficient (bilej et al. 2011). conveniently, the immunocompetent cells, celomocytes, of earthworms can readily be non-invasively retrieved for ex vivo examination, whilst the immune system subsequently has the capacity to fairly quickly recover its potency. this makes the earthworm immune system very attractive for studies on the adverse effects of various factors. the celomocytes of all earthworm species contain amebocytes, being according to the ottaviani (2011) nomenclature classical immunocytes. in addition, some species possess a second and morphologically distinct cells freely floating in celomic cavity, i.e., the eleocytes, which are considered to be mature, detached, chloragocytes. eleocytes, but not amebocytes, exhibit autofluorescence under fluorescence and laser confocal microscopy which is confined to the characteristic granular inclusions (figs 1a, b). autofluorescent ‘self-marking’ predisposes these cells for analysis by flow cytometry (fig. 1c). studies by spectrofluorimetry has revealed that riboflavin (koziol et al., 2006) stored in the chloragosome granules of chloragocytes and eleocytes (plytycz et al., 2007) is one of fluorophores responsible for their autofluorescence (fig. 1d) the percentages of autofluorescent eleocytes among celomocytes, and absolute amount of riboflavin stored within eleocytes are both species-specific parameters (table 1). for example, both the frequency of eleocytes and their riboflavin content are high in the endogeic species allolobophora chlorotica, whilst these granular cells are apparently absent or very uncommon in another endogeic species aporrectodea caliginosa (fig. 2) (plytycz et al., 2011a). whether these inter-species differences reflect underlying differences in trophicresource partitioning or in the biochemistry of these superficially similar, often sympatric, species is presently unknown. metal pollution affects earthworm celomocytes previous experiments indicated that celomocyte numbers and their composition can be significantly modified in response to soil metal contamination (wieczorek-olchawa et al., 2003; homa et al., 2003) or by experimental exposures to metal-spiked soil (e.g., kwadrans et al., 2008; dutkiewicz et al., 2009; podolak et al., 2011) or metal-spiked filter paper (e.g., olchawa et al., 2007; homa et al., 2005, 2007, 2010). recent observations have showed convincingly the superiority of eleocyte-rich al. chlorotica and dendrobaena veneta over a. caliginosa and lumbricus rubellus (devoid of eleocytes) for investigation of immunotoxic effects of heavy metals (plytycz et al., 2011a). in these experiments adult worms of these four species were dermally exposed for 2 days to filter paper soaked with 1mm ni, cu, zn, cd, or pb chlorides. the amebocytes of a. caliginosa and l. rubellus were subjected to flow cytometric measurements of in vitro neutral red uptake (nr) (loading method adapted from weeks and svendsen (1997) and reviewed by svendsen et al., 2004). however, the nr uptake assay was found to be technically demanding, requiring a strictly normalized incubation period for all samples 201 fig. 2 analysis of celomocytes of allolobophora chlorotica and aporrectodea caliginosa, (a) flow cytometry, and b) spectrofluorimetry (based on plytycz et al., 2011a). to yield useful comparative data. in contrast, the riboflavin content in celomocyte lysates of eleocyterich species appeared to be a robust and convenient immune-function biomarker of environmental stress (plytycz et al., 2011a). in al. chlorotica, the number of celomocytes, the percentage of eleocytes, and the amount of riboflavin were significantly decreased in cuexposed worms; these cytometric parameters were less adversely affected by ni, zn, cd, and were unaffected by pb at the wide range of concentrations (plytycz et al., 2011a). as seen in figure 3, a decrease of riboflavin content in samples from metal-exposed al. chlorotica corresponds strongly with a decreased number of riboflavin-storing eleocytes. any generalizations and extrapolations may, however, be misleading as further studies have revealed that this relationship does not seem to hold under different experimental conditions (see below, and plytycz et al., 2009b, 2011b). 202 fig. 3 analysis of coelomocytes of allolobophora chlorotica after 2-day exposure to filter papers soaked with 1mm metal chlorides; (a) flow cytometry; (b) spectrofluorimetry; (c) mean percentages of autofluorescent eleocytes (afe); (d) mean riboflavin (rf) contents in coelomocyte lysates (in arbitrary units, au) (based on homa et al., 2010). description in the text. riboflavin depletion in celomocytes of dendrodrilus rubidus is a biomarker of metal soil contamination one of the aims of the series of subsequent experiments was to apply flow cytometric assessment of autofluorecent elecytes and/or spectrofluorimetric quantification of riboflavin storage in eleocytes as putative biomarkers of natural soil quality. in wales, unpolluted parkland at cardiff is inhabited by several earthworm species, while only litter-inhabiting epigeic species (almost exclusively dendrodrilus rubidus and l. rubellus) live in the shallow, heavily polluted soils associated 203 fig. 4 photos (a) of the reference site r (pontcanna) and post-industrial site c (cwymstwyth) from wales inhabited by several earthworm species (r) and a few of them (c), sampled for experiments on dendrodrilus rubidus and lumbricus rubellus. (b) contents of zn and pb in soil (empty bars) and whole bodies of d. rubidus (black bars) and l. rubellus (grey bars) from the reference r site and from three metal polluted sub-sites: cc, cs and ce (based on plytycz et al., 2009b, 2010a). with and abandoned zn/pb mine at cwymstwyth (see sites r and c in fig. 4a). the earthworms resident at the metalliferous soils tolerate the metals not by exclusion but by accumulative immobilization within certain tissues (fig. 4b). d. rubidus contains a dense population of autofluorescent eleocytes both in worms from unpolluted reference and metal-polluted sites (plytycz et al., 2009b, 2010b). in contrast, riboflavin content was uniformly very low in conspecifics inhabiting zn/pb-polluted field sites (plytycz et al., 2009b) and in soils aerially polluted with ni from a long-established and still active ni smelter (plytycz et al. 2010b). as illustrated in fig. 5a, a cluster of complex granular cells exhibiting strong autofluorescence (i.e., eleocytes) are very abundant in the sample from earthworms inhabiting an unpolluted habitat and in counterparts from zn/pbcontaminated soil. in spite of this, fig. 5b shows that spectra characteristic for riboflavin are obtainable only from control (i.e., unpolluted) earthworm samples but not in samples derived from earthworms from the zn/pb metalliferous soil. these observations indicate that other fluorophores, probably lipofuscins, are responsible for eleocyte autofluorescence in worms from metalcontaminated sites. it is interesting to postulate that the so-called ‘ageing pigment’ lipofuscin (yin, 1996) accumulates in the presumptive metal-mediated stressed cells of earthworms from polluted soils as a consequence of enhanced cytological damage and membrane turnover (regoli, 1992). on the other hand, there is a body of evidence that the presence of lipofuscin can itself under certain circumstances mediate cell damage, including the loss of lysosomal membrane integrity (brunk and terman, 2002). growing evidence reveals that cryptic/sibling speciation (genetically distinct, but morphologically very similar or indistinguishable organisms) is widespread amongst the clitellate annelids (erséus and gustafsson, 2009), including some earthworm ‘species’ (king et al., 2008; pérez-losada et al., 2009; stürzenbaum et al., 2009). one of the important implications of these findings is the possibility that the genetically distinct ‘lineages’ (the 204 fig. 5 main results of experiments on celomocytes of dendrodrilus rubidus from wales, from the unpolluted reference site r and abandoned zn/pb site c (see fig. 4); (a) analysis by flow cytometry in respect of percentages of autofluorescent eleocytes; (b) analysis by spectrofluorimetry in respect of riboflavin (rf) content; (c) riboflavin content (in arbitrary units) from the reference site r (considered as 100 %; black bars), or zn/pbpolluted or ni-polluted (dark bars) or transferred for 4 or 6 weeks from the reference soil to the polluted soil: rzn/pb or r-ni (grey bars); (based on plytycz et al., 2009b, 2010b). term being used advisedly instead of ‘cryptic species’ to cover the possibility of a lack of reproductive isolation and, therefore, conformity with the biological definition of ‘species’) are differentially responsive to environmental contaminants (morgan et al., 2007). a striking example of this microevolutionary phenomenon is seen in the differences in pah tolerance and metabolism amongst sibling species within the species complex of the polychaete capitella capitata (bach et al., 2005). we were alert to the possibility that the endogeic and cosmopolitan earthworm species d. rubidus also forms a species complex, the members of which may be differentially susceptible to metalmediated stress. as a device designed to minimize the possible confounding effects of cryptic speciation we performed a laboratory-based reciprocal transfer experiment involving the chosen reference and metalliferous field soils as well as their resident earthworms. we observed that the riboflavin content in celomocyte lysates prepared from d. rubidus sampled initially from the unpolluted 205 fig. 6 main results of experiments on dendrobaena veneta (dv, empty bars), eisenia andrei (ea, grey bars) and allolobophora chlorotica (ach, black bars) maintained for 4 weeks in metal-free natural soil samples (r, zm, sf) or soil samples heavily polluted mainly with lead, zinc, or cadmium (bf, bm) from southern poland); fold of riboflavin content in coelomocyte lysates when content in the r sample was considered as 1 (based on plytycz et al., 2011b). soil dropped significantly after maintenance for several weeks in zn/pbor ni-polluted soils (fig. 5c) (plytycz et al., 2009b, 2010b). riboflavin amount in celomocytes is not a universal biomarker of soil quality eisenia andrei, d. veneta, and al. chlorotica have been exposed in another experiment to metalliferous field soils samples from wales uk (piotrowska et al., 2009) and southern poland (plytycz et al., 2011b). celomocytes and riboflavin content were affected by soil quality in speciesrelated ways. for example, it was evident according to these immuno-toxicity variables that al. chlorotica is much more sensitive to soil metal pollution intensity than the two composting species examined in the studies (piotrowska et al., 2010, podolak et al., 2011; plytycz et al., 2011b). moreover, it was apparent that riboflavin depletion in al. chlorotica celomocytes occurs not only in metal-polluted soil samples but also in metal-free sandy-clay or loamysand natural soil samples. in contrast, the riboflavin content increased in the eleocytes of e. andrei and/or d. veneta transferred from the lab soil to metal polluted soil samples and also to the unpolluted sandy clay or loamy-sand soils (fig. 6) (plytycz et al. 2011b). in conclusion, various edaphic factors other than elevated metal concentrations can effectively act as stressors, and this may lead to species-specific alterations in riboflavin metabolism. putative role of riboflavin storage in earthworm chloragocytes/eleocytes riboflavin plays an important role in immunity of both plants (dong and beer 2000; zhang et al., 2009) and animals (araki et al., 1995; osame et al., 1995; verdrengh and tarkowski 2005; bertello et al., 2006), and plays a role in bacterial quorum sensing (rajamani, 2008). in particular, riboflavin has antioxidative effects (seekamp et al., 1999; iwanaga et al., 2007), antinociceptive and antiinflammatory activity in mammals (bertollo et al., 2006; mazur et al., 2008), and at high doses riboflavin possesses pronounced anti-septic properties (toyosawa et al., 2004; kodama et al., 2005). therefore an immunomodulatory role of riboflavin in human immunity is highly appreciated 206 (iwanaga et al., 2007; yazdanpanah et al., 2009; damian et al., 2010). in vertebrates, flavin deficiencies lead to diseases such as glossitis, cheilosis, and organic acidurias (powers, 2003). the potential adaptive advantages of possessing riboflavin-storing immune-competent cells with high bactericidal potency are evident to soil-dwelling earthworms, with their highly permeable integument rendering them potentially vulnerable to invasion by pathogenic bacteria. we have shown that riboflavin storage is universal in earthworm species, as it was detected both in attached chloragocytes forming the chloragogen tissue and in chloragocyte-derived eleocytes (mazur et al., 2011). moreover, we have shown that riboflavin acts as a chemoattractant for celomocytes of several earthworm species, both in eleocyte-rich species (al. chlorotica, e. andrei, d. veneta) and in species that are devoid of freefloating eleocytes (l. terrestris, l. rubellus, a. caliginosa) (mazur et al., 2011). this property of riboflavin is probably of adaptive value to earthworm species through the targeted recruitment of immune-competent celomocytes to the loci of pathogen invasion, and subsequently to 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couples tnf receptor 1 to nadph oxidase. nature 460: 1159-63, 2009. yin d. biochemical basis of lipofuscin, ceroid, and age pigment-like fluorophores. free radic. biol. med. 21: 871-888, 1996. zhang s, yang x, sun m, sun f, deng s, dong h. riboflavin-induced priming for pathogen defense in arabidopsis thaliana. j. integr. plant biol. 51: 167-174, 2009. 209 http://www.ncbi.nlm.nih.gov/pubmed?term=%22yazdanpanah%20b%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22wiegmann%20k%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22tchikov%20v%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22krut%20o%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22pongratz%20c%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22schramm%20m%22%5bauthor%5d 1department of evolutionary immunobiology, institute of zoology, jagiellonian university, krakow, poland allolobophora chlorotica acknowledgements we thank all our collaborators and co-authors for their invaluable contributions to our research outputs and ideas. the research reported herein was supported by funds from k/zds/001955 (bp). king ra, tibble al, symondson woc. opening a can of worms: unprecedented sympatric cryptic diversity within british lumbricid earthworms. mol. ecol. 17: 4684-4698, 2008. yazdanpanah b, wiegmann k, tchikov v, krut o, pongratz c, schramm m, et al. riboflavin kinase couples tnf receptor 1 to nadph oxidase. nature 460: 1159-63, 2009. combined effects of temperature and salinity on functional responses of haemocytes and survival in air of the clam ruditapes philippinarum isj 11: 163-173, 2014 issn 1824-307x review effects of pharmaceuticals on immune parameters of aquatic invertebrates v matozzo department of biology, university of padua, padua, italy accepted may 21, 2014 abstract pharmaceuticals are a large group of chemicals used either by humans for personal health or by agribusiness to enhance the growth and health of livestock. pharmaceuticals are considered to be emerging environmental contaminants. indeed, several studies have shown that these compounds continuously enter aquatic ecosystems. both pharmaceutical consumption and erroneous discharge of unused or expired medications make notable contributions to the introduction of pharmaceuticals into the environment. additionally, pharmaceuticals consumed by humans and livestock are not entirely absorbed by organisms and are excreted and passed into wastewater and surface water. although most pharmaceuticals are designed for human consumption, they can affect non-target organisms that share certain homologous receptors with humans. this review intends to summarise the most recent information concerning the effects of some classes of pharmaceuticals on the immune parameters of aquatic invertebrates. key words: pharmaceuticals; hemocytes; immune parameters; aquatic invertebrates introduction among emerging environmental contaminants, pharmaceuticals are a large group of chemicals used either by humans or by agribusiness to enhance the growth or health of livestock (heberer, 2002). although information concerning the total annual production and use of pharmaceuticals is generally fragmentary, the us environmental protection agency (epa) reported that pharmaceuticals, including prescription drugs, veterinary drugs and diagnostic agents, are produced in large quantities thousands of tons per year worldwide (http://www.epa.gov/ppcp/basic2.html). in this context, cleuvers (2003) reported that several kilotons of non-steroidal anti-inflammatory drugs (nsaids), a group of substances widely used to treat pain and inflammation, are produced yearly. in italy, the annual consumption of prescribed drugs was estimated at 209.58 tonnes of amoxicillin, 22 tonnes of β-blockers, 7.6 tonnes of antilipidaemics, 1.9 tonnes of ibuprofen, 26.67 tonnes of antacids and 6.4 tonnes of diuretics (calamari et al., 2003). in the united kingdom, 2.56 tonnes of fluoxetine (an ___________________________________________________________________________ corresponding author: valerio matozzo department of biology university of padua via ugo bassi, 58/b 35131 padua, italy e-mail: matozzo@bio.unipd.it valerio.matozzo@unipd.it antidepressant) were consumed in 2000 (sebastine and wakeman, 2003), whereas 22,266 million prescriptions were issued in 2007 in the united states (modern medicine pharmacy, 2010). zheng et al. (2012) reported that the annual consumption of antibiotics in china was approximately 180,000 tons. regarding veterinary medicine, sarmah et al. (2006) provided an exhaustive review on the use and environmental occurrence of veterinary pharmaceuticals worldwide. in aquaculture in particular, the intensive farming that has been developed throughout the world (mainly in asia) requires the application of many pharmaceuticals, mostly antibiotics. sapkota et al. (2008) reported that the type and the total amount of antibiotics used per year vary markedly on a country-by-country basis for the top 15 aquaculture-producing countries (china, indonesia, taiwan, india, philippines, and norway, in particular). at the same time, the authors observed that the absence of data for some countries was not necessarily indicative of a lack in antibiotic usage but, rather, a lack of information available in these countries (sapkota et al., 2008). in any case, of the 26 antibiotics examined from the fao list, oxytetracycline, chloramphenicol and oxolinic acid were the most commonly used antibiotics (sapkota et al., 2008). additionally, it has been estimated that approximately 75% of most of the antibiotics incorporated in feed used in aquaculture systems enter aquatic environments 163 mailto:matozzo@bio.unipd.it fig. 1 a scheme summarising the origins and fate of human and veterinary pharmaceuticals in the environment. directly and accumulate in sediments (richardson and bowron, 1985; halling-sørensen et al., 1998). overall, the main sources of pharmaceuticals in the environment are human and veterinary drug use, residues from pharmaceutical manufacturing and hospitals, and illicit drug use. humans, in particular, contribute to the presence of these substances in the environment when pharmaceuticals are used and when unused or expired medications are erroneously disposed of. exhaustive schemes of possible sources and pathways of pharmaceuticals in aquatic environments were reported by heberer (2002) and on the epa website (http://www.epa.gov/ppcp/pdf/drawing.pdf) and are summarised in figure 1. some pharmaceuticals are metabolised and converted by organisms into more easily excreted metabolites, others are converted into more soluble forms by the formation of conjugates, and other substances are excreted in an unaltered form (daughton and ternes, 1999). the excreted metabolites or the conjugated and unaltered parent compounds can then be subjected to further transformations in sewage treatment plants (stps), where the elimination rates vary markedly according to the construction and treatment technology used, the hydraulic retention time, the time of year and the performance of the stp (fent et al., 2006). although many pharmaceuticals show low environmental persistence, the main concern is that low persistence can be compensated by continuous introduction of these substances into aquatic ecosystems, where many compounds occur in the ng/l µg/l range (daughton and ternes, 1999; fent et al., 2006; sarmah et al., 2006; kümmerer, 2009; zheng et al., 2012). however, the levels of pharmaceuticals can be higher in untreated water. according to fent et al. (2006), aquatic organisms are particularly vulnerable to pharmaceuticals. indeed, due to the continuous introduction of pharmaceuticals into aquatic ecosystems, the exposure of aquatic organisms may be chronic and multi-generational (fent et al., 2006). therefore, a major concern is not necessarily the acute effects of pharmaceuticals on organisms but, rather, the manifestation of imperceptible effects that can accumulate over time to yield truly profound changes in the biochemical and physiological processes of organisms. effects of pharmaceuticals on immune parameters of aquatic invertebrates antibiotics antibiotics are largely used worldwide to treat disease and protect the health of animals. regarding the effects of antibiotics on non-target species, gust et al. (2013) recently evaluated the short-term effects (3 days) of environmentally relevant concentrations of antibiotics, as a mixture (ciprofloxacin, 100 ng/l; erythromycin, 50 ng/l; 164 http://www.epa.gov/ppcp/pdf/drawing.pdf novobiocin, 100 ng/l; oxytetracycline, 200 ng/l; sulfamethoxazole, 50 ng/l; and trimethoprim, 50 ng/l), on the immune responses of the pond snail lymnaea stagnalis. no significant effects were observed on haemocyte viability and count after 3 days of exposure, whereas intracellular levels of thiols were significantly decreased in snail haemocytes. additionally, phagocytic activity was significantly decreased by 28 % in the haemocytes of snails exposed to the antibiotic mixture compared to the control. at the level of immune-related gene expression, the antibiotic mixture increased toll-like receptor 4 (tlr4) mrna expression and reduced glutathione reductase (gr) mrna expression (table 1). hemocytes from the freshwater bivalve dreissena polymorpha were exposed in vitro to different concentrations (0.2, 1 and 5 μm) of the antibiotic trimethoprim (tmp, 5-[3,4,5trimethoxybenzyl]pyrimidine-2,4-diamine), and the potential genotoxicity and cytotoxicity were evaluated by the scge (single-cell gel electrophoresis) assay, apoptosis frequency and the lysosomal membrane stability test (nrra, neutral red retention assay) (binelli et al., 2009a). the results demonstrated that tmp markedly affected mussel haemocytes, even if cytotoxic and genotoxic effects were mostly observed at the highest tmp concentrations tested (table 1). in the same freshwater mussel species, different hemocyte parameters were also measured after in vivo exposure to three concentrations (1, 3 and 10 nm) of tmp for 96 h. the scge assay, the micronucleus (mn) test, apoptotic frequency measurements and the nrra assay were performed in mussel hemocytes (binelli et al., 2009b). the study demonstrated a moderate cyto and genotoxicity of tmp on mussel hemocytes. indeed, only a slight increase in dna damage was recorded by apoptosis induction and mn frequency, while significant differences in lysosomal membrane stability from baseline levels were measured with 3 and 10 nm at the end of exposure only (binelli et al., 2009b) (table 1). in the freshwater mussel elliptio complanata, the separate and combined effects of the antibiotics ciprofloxacin, erythromycin, novobiocin, oxytetracycline, sulfamethazole and tmp (commonly found in urban wastewater effluents) on mussel immune parameters were evaluated in vitro (gust et al., 2012). most of the tested antibiotics, individually or as mixtures, caused marked alterations in hemocyte viability, phagocytosis, lysozyme and cyclooxygenase (cox) activities (table 1). overall, the authors observed that antibiotics, alone and as mixtures, modulate the immune parameters of e. complanata at environmentally relevant concentrations. of the antibiotics tested, erythromycin, tmp and sulfamethazole each caused effects similar to those of the mixture, and no additive effects of the antibiotics were observed. gagné et al. (2006) evaluated the immunotoxic effects of antibiotics in the freshwater mussel e. complanata. hemolymph was collected and treated in vitro for 24 h with increasing concentrations of tmp, novobiocin, oxytetracycline, and sulfamethazole. while novobiocin and sulfamethazole decreased phagocytic activity, tmp and oxytetracycline increased it (table 1). the authors observed that phagocytic activity was negatively correlated with the number of polar functional groups of the compounds, suggesting that the potential of drugs to decrease phagocytosis was related to their polarity. these studies suggest that immunomarker responses can vary markedly, depending on drug type, animal species and methodological approach (in vitro or in vivo exposure). in this context, it is important to highlight that in vivo exposures can cause variations in the hemolymph levels of some endogenous factors, such as oestrogens, neuroimmune modulators and cytokine-like proteins, which have been shown to influence immune responses (canesi et al., 2007a). non-steroidal anti-inflammatory drugs non-steroidal anti-inflammatory drugs (nsaids) are the sixth top-selling class of drug worldwide, and some of them are sold over the counter (langman, 1999). among nsaids, ibuprofen (ibu) is a propanoic acid derivative (2-[4-(2methylpropyl)phenyl]propanoic acid) widely used as an analgesic, antirheumatic and antipyretic (fent et al., 2006; praveen rao and knaus, 2008). ibu is a nonselective inhibitor of both cyclooxygenase (cox)-1 and -2 isozymes. the effects of ibu (0, 0 + ethanol, 100, 500, and 1,000 µg/l) on hemocyte parameters of the clam ruditapes philippinarum (=venerupis philippinarum) were investigated after a 7-day exposure (matozzo et al., 2012). the exposure of clams to the highest ibu concentration significantly reduced their total hemocyte count (thc), whereas no significant changes were observed in both the diameter and volume of hemocytes. significant increases in hemocyte proliferation were recorded in clams that were exposed to the two highest tested concentrations of ibu. exposure of clams to 1,000 µg ibu/l significantly reduced uptake of the vital dye neutral red (nr) and increased hemolymph lactate dehydrogenase (ldh) activity, which is indicative of cytotoxicity. conversely, ibu did not induce dna fragmentation in hemocytes (table 1). overall, the results obtained demonstrated that ibu caused marked alterations in the immune parameters of clams and indicated several mechanisms of action of ibu, mostly at the cell membrane level (matozzo et al., 2012). ibu-mediated lysosomal membrane destabilisation was also demonstrated in hemocytes from r. philippinarum exposed to various concentrations of ibu for 35 days (aguirre-martínez et al., 2013). the authors stated that the level of toxicity calculated for ibu suggested that environmental concentrations in the μg/l range can be extremely toxic for the lysosomal membrane stability of clams (table 1). a series of studies has been performed to investigate the effects of nsaids, namely diclofenac (dcf), paracetamol (pcm) and ibu, on the hemocytes of the zebra mussel d. polymorpha. the first study demonstrated that environmentally relevant concentrations of dcf (2-[(2,6165 dichlorophenyl)amino]phenylacetic acid) induced negligible cellular and genetic damage because a slight decrease in lysosomal membrane stability was observed at the end of exposure at the highest concentration tested (parolini et al. 2011a) (table 1). one of the most used analgesic and antipyretic agents in human medicine is pcm (n-(4hydroxyphenyl)acetamide). to evaluate the effects of pcm on d. polymorpha, mussels were exposed to pcm environmental concentrations (1, 5 and 10 nm) for 96 h, and cyto-genotoxicity was determined in hemocytes by the lysosomal membrane stability test (nrra), the scge assay, the mn test and apoptotic frequency assessment (dna diffusion assay) (parolini et al. 2010). the results revealed moderate cyto-genotoxicity in mussel hemocytes because no primary dna fragmentation was measured by the scge assay and only a slight increase in fixed dna damage was recorded by apoptotic and mn frequencies. a significant reduction in lysosomal membrane stability was observed at 5 and 10 nm at the end of the exposures (table 1). lastly, mussels were exposed to environmentally relevant ibu concentrations (0.2, 2 and 8 mg/l) for 96 h, and cyto-genotoxicity was evaluated as reported in the studies above (parolini et al. 2011b). additionally, in this case, a slight cytogenotoxicity was found at the ibu concentration of 0.2 mg/l, and higher ibu concentrations were able to significantly increase both genetic and cellular damage (table 1). considering that organisms are most likely exposed to a mixture of substances in the environment, parolini and binelli (2012) investigated the effects of a mixture of the three nsaids mentioned above (dcf, ibu and pcm) on hemocytes from d. polymorpha. the mussels were exposed to different environmental concentrations of the mixture, and the cyto-genotoxic effects were evaluated by means of the neutral red retention (nrr) assay, the scge assay, the dna diffusion assay and the micronucleus test. exposure to the mixture induced significant cellular stress in bivalves, most likely due to increased oxidative stress, and this significantly increased dna fragmentation and the frequency of apoptotic and micronucleated cells (table 1). likewise, luna-acosta et al. (2012) evaluated the toxic effects of a mixture of two herbicides (diuron and isoproturon, each at 5 µg/l) and one pharmaceutical (ibu, at 5 µg/l) on the immune parameters of the oyster crassostrea gigas. no cell mortality was recorded, and phagocytosis was significantly inhibited by almost 50% after 6 h of exposure (table 1). additionally, exposure to the mixture significantly decreased catecholase-type phenoloxidase activity (by 20%), highlighting once again that a mixture of contaminants can exert more pronounced effects than a single substance. in a recent study, the in vitro effects of ibu on the immune parameters of the colonial ascidian botryllus schlosseri were evaluated (matozzo et al., 2014). hemocytes were exposed for 1 h to 100 and 1000 µg ibu/l, and the effects on hemocyte viability and morphology (shape factor), lysosomal membrane stability (nrra), phagocytic activity, apoptosis (tunel reaction), and hydrolytic (acid phosphatase) and oxidative (phenoloxidase and peroxidase) enzyme activities were evaluated. the exposure of hemocytes to ibu did not significantly affect cell viability but did increase the percentage of round cells. ibu significantly reduced both phagocytic activity and lysosomal membrane stability but significantly increased the percentage of hemocytes positive for tunel reaction (indicative of dna fragmentation). a significant decrease in the percentage of hemocytes positive for acid phosphatase was recorded at 1,000 µg/l, while no significant variations were recorded in the percentage of hemocytes positive for phenoloxidase and peroxidase (table 1). the results obtained indicated that exposure of ascidian hemocytes to ibu induces marked alterations in cell function. anticancer agents in aquatic environments, the occurrence of chemotherapeutic and immunosuppressive agents used to treat cancers is of increasing concern. among such chemicals, cyclophosphamide (cp) acts as a neurotoxicant (rzeski et al., 2004; xiao et al., 2007). intracellular enzymes transform cp into active alkylating metabolites, which crosslink with dna strands (anderson et al., 1995). a nonnegligible percentage of cp (up to 10 – 20 %) can be excreted unchanged (anderson et al., 1995; johnson et al., 2008). in aquatic environments, active compounds and metabolites show poor degradability and high persistence. consequently, cp and metabolites have been detected in waste and surface waters (buerge et al., 2006; johnson et al., 2008). at present, only one study has investigated the negative effects of anticancer agents in aquatic animals. canty et al. (2009) evaluated the cytotoxicity and genotoxicity of cp on hemocytes from the mussel mytilus edulis and celomocytes from the sea star asterias rubens following 7 days of exposure (18 to 180 mg/l). in mussels, no significant effects on nrr were recorded, whereas a significant increase in the induction of micronuclei and dna strand breaks was observed, with a strong correlation between micronuclei induction and dna strand breaks. in sea stars, no significant differences in nrr were observed between cpexposed animals and seawater controls. conversely, significant increases in micronuclei induction and dna strand breaks were detected after 5 and 7 days of exposure to 32 and 56 mg cp/l (table 1). lipid regulators blood lipid regulators are a class of pharmaceuticals that can be detected in the ng/l μg/l range in wastewaters and surface waters (fent et al., 2006). gust et al. (2013) evaluated the effects of a hypolipaemic mixture containing atorvastatin (50 ng/l), gemfibrozil (100 ng/l) and bezafibrate (100 ng/l) on the immune responses of l. stagnalis. the mixture increased intracellular reactive oxygen species (ros) levels (2.9-fold) and decreased thiol levels but did not affect the phagocytic capability of hemocytes. additionally, the hypolipaemic mixture increased (2.9-fold) nitric oxide synthetase isoform 1 (nos-1) mrna expression and decreased (0.4-fold) 166 tlr4 mrna expression (table 1). reduced thiol levels in hemocytes, associated with increased ros levels and nos expression suggested that the oxidative burst can have detrimental effects (i.e., inflammation) on snail hemocytes. in a recent study, camp/pka regulation and abcb mrna expression were assessed in hemocytes from the mussel mytilus galloprovincialis exposed in vivo to 0.3 ng/l fluoxetine for 1 week (franzellitti and fabbri, 2013). there is evidence that mammalian transcriptional regulation of the abcb1 gene encoding p-glycoprotein (pgp) is mediated through the phosphorylation activity of the camp-dependent protein kinase (pka). although this regulatory pathway needs to be more fully investigated in molluscs, the aforementioned study demonstrated that fluoxetine significantly decreased camp levels and pka activity and induced abcb mrna down-regulation. the authors stated that their study provides the first evidence for the camp/pka-mediated regulation of abcb mrna expression in mussels (table 1). overall, these results demonstrated that the impairment of transduction pathways induced by fluoxetine may affect the ability of mussels to cope with stressful conditions in the environment (franzellitti and fabbri, 2013). the effects of bezafibrate and gemfibrozil on immunocytes of mytilus spp were investigated both in vitro and in vivo (canesi et al., 2007a). in vitro exposure to both compounds induced rapid lysosomal membrane destabilisation, extracellular lysozyme release, no production and decreased phagocytic activity. the effect of fibrates were partly mediated by the activation of erk and p38 mapks (mitogen activated protein kinases) (table 1). in the in vivo experiment, mussels were injected with 0.01, 0.1 and 1 nmol/animal (corresponding to 3.61, 36.18 and 361.8 ng/g dry weight for bezafibrate and to 2.50, 25.03 and 250.35 ng/g dry weight for gemfibrozil), and hemocytes were collected after 24 h. both compounds caused a concentrationdependent lysosomal destabilisation and extracellular lysozyme release, with a 50 % effect at 0.1 nmol. conversely, phagocytic activity increased (+24 %) at the highest concentration tested (table 1). the results obtained indicated that environmental concentrations of hypolipaemic drugs can affect mussel immune function (canesi et al., 2007a). we have evaluated the effects of fluoxetine on the immune parameters of the clam v. philippinarum. clams were exposed to various fluoxetine concentrations (0, 1, 5, 25, 125 and 625 µg/l) for 7 days, and the effects on the total hemocyte count (thc), the diameter and volume of hemocytes, hemocyte proliferation, neutral red uptake (nru), and lysozyme activity in cell-free hemolymph (cfh) were evaluated (munari et al., 2014). a significant increase in thc values was observed in clams exposed to 25 µg/l compared with controls, whereas no significant variations were recorded in either the diameter or the volume of hemocytes. hemocyte proliferation increased significantly in animals exposed to 25, 125 and 625 µg/l compared with controls, whereas nru decreased significantly in the hemocytes of clams exposed to 1 and 5 µg/l (table 1). in an in vitro study, gagne et al. (2006) observed an induction of phagocytosis after the exposure of hemocytes from e. complanata to both bezafibrate and gemfibrozil (table 1). antihypertensive drugs at present, only two studies have evaluated the effects of antihypertensive drugs on the immune parameters of aquatic invertebrates. in the first study, snails (l. stagnalis) were exposed to a mixture of antihypertensive drugs, including atenolol (500 ng/l), furosemide (300 ng/l), hydrochlorothiazide (300 ng/l) and lisinopril (50 ng/l), for 3 days (gust et al. 2013). the mixture caused a decrease in phagocytosis and upregulated tlr4, nos-1, nos-2 and superoxide dismutase (sod) expression compared to the controls (table 1). the authors suggested that the decrease in phagocytic activity was a consequence of increased no production. gust et al. (2013) observed that a mixture containing psychiatric drugs, including venlafaxine (200 ng/l), carbamazepine (200 ng/l) and diazepam (10 ng/l), did not affect immunocompetence (defined by hemocyte density, viability, phagocytosis, ros and thiol levels) in l. stagnalis. however, the mixture induced significant changes in the expression of immune-related genes. tlr4, heatshock protein 70 (hsp70) and selenium-dependent glutathione peroxidase (segpx) gene expression was upregulated, while allograft inflammatory factor1 (aif-1), catalase (cat) and gr gene expression was downregulated (table 1). the gene expression induction suggested that the psychoactive substances led to glutathione-dependent peroxidase activity (segpx) and the protection response against protein denaturation (hsp70). the reduced cat and gr gene expression in hemocytes suggested decreased ros handling and inflammation, whereas the increased tlr4 expression in snail hemocytes was most likely indicative of either a strong inflammation signal or a compensation mechanism against the loss of tolllike receptor signalling. in the second study, hemocytes from d. polymorpha were exposed in vitro to five increasing concentrations (from 0.001 to 10 mg/l) of atenolol for 96 h (parolini et al., 2011c). hemocyte viability was significantly reduced after 48 h of exposure to 0.01 mg/l of atenolol, and cell viability decreased markedly after 48 and 96 h of exposure to 10 mg/l of atenolol (table 1). antidepressant and anticonvulsant agents one of the most frequently detected substance in surface waters is fluoxetine, the active ingredient of prozac® (metcalfe et al., 2010; bringolf et al., 2010). fluoxetine is a selective serotonin reuptake inhibitor (ssri) that is prescribed as an antidepressant in large amounts worldwide to treat depression and other psychological disorders (brooks et al., 2003; nentwig, 2007). gagné et al. (2006) demonstrated that an high concentration of carbamazepine (14 mg/l) is 167 table 1 effects of pharmaceuticals on immune parameters of aquatic invertebrates pharmaceuticals species/exposure immune parameters references antibiotics mixture (ciprofloxacine, erythromycine, novobiocin, oxytetracycline, sulfamethoxazole, trimethoprim) lymnaea stagnalis in vivo exposure haemocyte viability = haemocyte count = phagocytic activity ↓ thiol levels ↓ gene expression ↓↑ gust et al., 2013 trimethoprim dreissena polymorpha in vitro exposure dna damage ↑ apoptosis ↑ lysosomal membrane stability ↓ binelli et al., 2009a trimethoprim d. polymorpha in vivo exposure dna damage ≈ apoptosis ↑ micronuclei ↑ lysosomal membrane stability ↓ binelli et al., 2009a ciprofloxacin, erythromycin, novobiocin, oxytetracycline, sulfamethazole, trimethoprim (alone and as mixture) elliptio complanata in vitro exposure haemocyte viability ↕ ros levels ↕ thiol levels ↕ phagocytosis ↕ lysozyme activity ↕ no production ↕ cox activity ↕ gust et al., 2012 trimethoprim, novobiocin, oxytetracycline, sulfamethazole e. complanata in vivo exposure phagocytic activity ∩ gagné et al., 2006 nsaids ibuprofen ruditapes philippinarum in vivo exposure thc ↓ haemocyte diameter = haemocyte volume = cell proliferation ↑ nr uptake ↓ ldh activity↑ dna fragmentation = matozzo et al., 2012 ibuprofen r. philippinarum in vivo exposure lysosomal membrane stability ↓ aguirremartínez et al., 2013 diclofenac d. polymorpha in vivo exposure dna damage = apoptosis = micronucleui = lysosomal membrane stability ≈ parolini et al., 2011a paracetamol d. polymorpha in vivo exposure dna damage ≈ apoptosis ≈ micronuclei ↑ lysosomal membrane stability ↓ parolini et al., 2010 ibuprofen d. polymorpha in vivo exposure dna damage = apoptosis ≈ micronuclei ≈ lysosomal membrane stability ↓ parolini et al., 2011b diclofenac + paracetamol + ibuprofen d. polymorpha in vivo exposure dna damage ↑ apoptosis ↑ micronuclei ↑ lysosomal membrane stability ↓ parolini and binelli, 2010 168 ibuprofen + diuron + isoturon crassostrea gigas in vivo exposure cell mortality = phagocytosis ↓ catecholase-type phenoloxidase activity ↓ luna-acosta et al., 2012 ibuprofen botryllus schlosseri in vitro exposure haemocyte viability = % of round cells ↑ phagocytosis ↓ apoptosis ↑ acid phosphatase ↓ phenoloxidase = peroxidase = lysosomal membrane stability ↓ matozzo et al., 2014 anticancer agents cyclophosphamide mytilus edulis asterias rubens in vivo exposure lysosomal membrane stability (m.e.) = micronuclei (m.e.) ↑ dna damage (m.e.) ↑ lysosomal membrane stability (a.r.) = micronuclei (a.r.) ↑ dna damage (a.r.) ↑ canty et al., 2009 lipid regulators atorvastatin + gemfibrozil + bezafibrate l. stagnalis in vivo exposure thiol levels ↓ ros levels ↑ phagocytosis = nos1 expression ↑ tlr4 expression ↓ gust et al., 2013 bezafibrate gemfibrozil mytilus spp. in vitro exposure lysozyme release ↑ no levels ↑ phagocytosis ↓ lysosomal membrane stability ↓ canesi et al., 2007a bezafibrate gemfibrozil mytilus spp. injection lysosomal membrane stability ↓ lysozyme release ↑ phagocytosis ↑ canesi et al., 2007a bezafibrate gemfibrozil e. complanata in vitro exposure phagocytosis ↑ gagné et al., 2006 antihypertensive drugs atenolol + furosemide + hydrochlorothiazide + lisinopril l. stagnalis in vivo exposure phagocytosis ↓ tlr4 ↑ nos-1 ↑ nos-2 ↑ sod ↑ gust et al., 2013 atenolol d. polymorpha in vitro exposure haemocyte viability ↓ parolini et al., 2011c antidepressant agents fluoxetine mytilus galloprovincialis in vivo exposure camp ↓ pka activity ↓ abcb mrna ↓ franzellitti and fabbri, 2013 fluoxetine v. philippinarum in vivo exposure thc ↑ haemocyte diameter = haemocyte volume = cell proliferation ↑ nr uptake ↓ munari et al., 2014 169 venlafaxine + carbamazepine + diazepam l. stagnalis in vivo exposure haemocyte density = haemocyte viability = phagocytosis = ros levels = thiol levels = tlr4 ↑ hsp70 ↑ segpx↑ aif-1 ↓ cat ↓ gr ↓ gust et al., 2013 carbamazepine e. complanata n vitro exposure phagocytosis ↑ cell adherence ↓ esterase activity ↑ lipid peroxidation = gagné et al., 2006 carbamazepine m. galloprovincialis in vivo exposure lysosomal membrane permeability ↓ martin-diaz et al., 2009 carbamazepine d. polymorpha in vitro exposure hemocyte viability ↓ parolini et al., 2011c estrogens 17β-estradiol m. galloprovincialis in vitro exposure ros production ↑ dna damage ↑ protein carbonylation ↑ lipid peroxidation ↑ cat mrna ↑ sod mrna ↑ gst mrna ↑ koutsogiannaki et al., 2014 17β-estradiol m. galloprovincialis in vitro exposure hemocyte adhesion ↑ koutsogiannaki and kaloyianni, 2011 17β-estradiol (i), 17α-ethinylestradiol (i), edc mixture (ii) m. galloprovincialis in vitro (i) and in vivo (ii) exposure (i) lysosomal membrane stability ↓ (i) phagocytosis ↕ (i) lysozyme release ↑ (ii) lysosomal membrane stability ↓ (ii) phagocytosis ↑ (ii) lysozyme release ↑ canesi et al., 2007b 17β-estradiol m. galloprovincialis in vitro (i) and in vivo (ii) exposure (i) phagocytosis ↕ (i) oxyradical production ↑ (ii) lysosomal membrane stability ↓ (ii) phagocytosis ↕ (ii) lysozyme release ↑ canesi et al., 2006 17β-estradiol mya arenaria injection hemocyte viability = phagocytosis ↓ gauthier-clerc et al., 2006 symbols: =: no significant variations ≈: moderate effects ↓: decrease ↑: increase ↕: effects depending on experimental plan, namely exposure to a single substance or to a mixture, or to various concentrations of pharmaceuticals (see text for details) ∩: effects depending on drug type (see text for details) 170 necessary to increase phagocytic activity and to reduce cell adherence in e. complanata hemocytes exposed in vitro for 24 h. in that study, esterase activity was significantly increased to a threshold concentration of 0.7 mg/l, whereas lipid peroxidation was not affected (gagné et al., 2006) (table 1). in d. polymorpha (in vitro study), hemocyte viability was significantly compromised after 48 h of exposure to 0.01 mg/l of carbamazepine (table 1); however, exposure to 0.1 mg/l was able to cause a significant increase in cell mortality already after 24 h (parolini et al., 2011c). in mussels, a significant decrease in haemocyte lysosomal membrane permeability was observed after exposure to 0.1 10 μg/l of carbamazepine for 7 days (martin-diaz et al., 2009) (table 1). estrogens in the last decades, increasing attention has been given to evaluating negative effects of estrogens in aquatic organisms. one of the most documented effects of estrogens is the induction of vitellogenins, precursors of the egg-yolk proteins, vitellins, which provide energy reserves for embryo development (matozzo et al., 2008). however, it has been demonstrated that estrogens can also affect hemocyte parameters in aquatic invertebrates. koutsogiannaki et al. (2014) recently evaluated the effects of 17β-estradiol (e2) on oxidative parameters of m. galloprovincialis hemocytes. results demonstrated that exposure of hemocytes to 25 nm of e2 for 30 min caused a significant increase in ros production and, consequently, a significant increase of dna damage, protein carbonylation and lipid peroxidation. increases in mrna levels of the antioxidant enzymes cat, sod and glutathione stransferase were also recorded (table 1). in the same mussel species, incubation of hemocytes with e2 (5, 25 and 50 nm) caused a significant increase in adhesion of cells to extracellular matrix proteins, mostly to laminin-1, collagen iv and oxidized collagen iv (koutsogiannaki and kaloyianni, 2011) (table 1). the immunomodulatory role of e2 in mytilus hemocytes was investigated both in vitro and in vivo (canesi et al., 2006). in vitro exposure of hemocytes to e2 (5-25 nm) rapidly stimulated phagocytosis and oxyradical production; however, higher concentrations of e2 (50 nm) inhibited phagocytosis. in vivo (= injection) exposure of mussels to 5, 25 and 100 pmol of e2 for 6 and 24 h significantly affected hemocyte lysosomal membrane stability, phagocytosis, and extracellular release of hydrolytic enzymes (table 1). in addition, canesi et al. (2007b) demonstrated that both natural (e2) and synthetic (17αethinylestradiol, ee) estrogens can influence markedly hemocyte parameters in m. galloprovincialis. in vitro exposure of hemocytes, affected lysosomal membrane stability (decrease), phagocytosis (it generally increased at lower concentrations and decreased at higher concentrations) and lysozyme release (after e2 exposure only). in vivo exposure (= injection) of mussels to a mixture of endocrine disrupting compounds (edcs), including e2 and ee, induced a clear dose-dependent lysosomal membrane destabilization, a significant stimulation of the phagocytic activity and a significant increase in lysozyme release (table 1). specimens of the soft-shell clam mya arenaria were injected with 10, 20 or 40 nmol of e2, and the effects on hemocyte parameters were evaluated (gauthier-clerc et al., 2006). cell viability did not change during the exposure, whereas significant decreases in phagocytic capacity of hemocytes were observed in clams treated with 10 and 20 nmol e2 (table 1). overall, results of the studies above indicate that hemocytes of aquatic invertebrates are potential targets of edcs. concluding remarks although the impact of pharmaceuticals on aquatic environments needs to be more fully investigated, the data reported in the present review (summarised in table 1) indicate that a variety of drugs can markedly influence immune parameters of non-target species. in this context, further studies are needed to better understand the relationship between pharmaceutical-mediated immunomodulation and the capability of animals to respond to pathogens. nevertheless, efforts should be directed at evaluating the effects of drug mixtures because animals are more realistically exposed to complex drug mixtures in their environments. acknowledgements the english text was revised by american journal experts. references aguirre-martínez gv, buratti s, fabbri e, delvalls at, martín-díaz ml. using lysosomal membrane stability of haemocytes in ruditapes philippinarum as a biomarker of cellular stress to assess contamination by caffeine, ibuprofen, carbamazepine and novobiocin. j. environ. sci. 25: 1408-1418, 2013. anderson d, bishop jb, garner rc, ostroskywegman p, selby pb. cyclophosphamidereview of its mutagenicity for an assessment of potential germ-cell risks. mutat. res. 330: 115181, 1995. binelli a, cogni d, parolini m, riva c, provini a. cytotoxic and genotoxic effects of in vitro exposure to triclosan and trimethoprim on zebra mussel (dreissena polymorpha) hemocytes. comp. biochem. physiol. 150c: 50-56, 2009a. binelli a, parolini m, cogni d, pedriali a, provini a. a multi-biomarker assessment of the impact of the antibacterial trimethoprim on the non-target organism zebra mussel (dreissena polymorpha). comp. biochem. physiol. 150c: 329-336, 2009b. bringolf rb, heltsley rb, newton tj, eads cb, fraley sj, shea d, et al. environmental occurrence and reproductive effects of the pharmaceutical fluoxetine in native freshwater mussels. environ. toxicol. chem. 29: 13111318, 2010. 171 brooks bw, foran cm, richards sm, weston j, turner pk, stanley jk, et al. aquatic ecotoxicology of fluoxetine. toxicol. lett. 142: 169-183, 2003. buerge ij, buser hr, poiger t, muller md. occurrence and fate of the cytostatic drugs in the rivers po and lambro in northern italy. environ. sci. technol. 40: 7242-7250, 2006. calamari d, zuccato e, castiglioni s, bagnati r, fanelli r. strategic survey of therapeutic drugs in the rivers po and lambro in northern italy. environ. sci. technol. 37: 1241-1248, 2003. canesi l, ciacci c, lorusso lc, betti m, guarnieri t, tavolari s, et al. immunomodulation by 17βestradiol in bivalve hemocytes. am. j. physiol. regul. integr. comp. physiol. 291: r664-r673, 2006. canesi l, lorusso lc, ciacci c, betti m, regoli f, poiana g, et al. effects of blood lipid lowering pharmaceuticals (bezafibrate and gemfibrozil) on immune and digestive gland functions of the bivalve mollusc, mytilus galloprovincialis. chemosphere 69: 994-1002, 2007a. canesi l, lorusso lc, ciacci c, betti m, rocchi m, pojana g, et al. immunomodulation of mytilus hemocytes by individual estrogenic chemicals and environmentally relevant mixtures of estrogens: in vitro and in vivo studies. aquat. toxicol. 81: 36-44, 2007b. canty mn, hutchinson th, brown rj, jones mb, jha an. linking genotoxic responses with cytotoxic and behavioural or physiological consequences: differential sensitivity of echinoderms (asterias rubens) and marine molluscs (mytilus edulis). aquat. toxicol. 94: 68-76, 2009. cleuvers m. aquatic ecotoxicity of selected pharmaceuticals including the assessment of combination effects. toxicol. lett. 142: 185194, 2003. daughton cg, ternes ta. pharmaceuticals and personal care products in the environment: agents of subtle change? 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velvetbean; food intake; fat body; midgut introduction botanical pesticides can be an alternative to the use of synthetic pesticides, because they are rapidly degraded in the environment and have low toxicity to natural enemies and mammals (copping and menn, 2000). the plant that has shown the highest potential insecticide activity in the world is azadirachta indica a. juss (meliacea), through the synthesis of azadirachtin, a tetranortriterpenoid produced as a secondary metabolite. azadirachtin inhibits the growth, affects survival, cause repellence and feeding deterrence, reduces the fertility of females and causes anatomical abnormalities in several species of insects (martinez and emden, 1999; mordue et al., 2000). in insects, azadirachtin has direct cytotoxic effects on glands (sayah et al., 2002), reproductive organs (sayah et al., 1996) and ___________________________________________________________________________ corresponding author: josé eduardo serrão department of general biology federal university of viçosa 36570-000, viçosa minas gerais state, brazil e-mail: jeserrao@ufv.br intestine (nogueira et al., 1997; correia et al., 2009). in addition, it affects protein metabolism (huang et al., 2004, 2007) and enzyme synthesis in insects (lowery and smirle, 2000). the velvetbean caterpillar anticarsia gemmatalis (lepidoptera: noctuidae) is an important insect defoliator of soybean in north and south america (nascimento et al., 2003). the frequent use of pesticides to control this insect may be harmful to natural enemies acting in biological control (vianna et al., 2009), and also to contaminate the environment (jergentz et al., 2005). furthermore, the effect of products derived from a. indica has been poorly studied in soybean defoliating caterpillars. thus, the study evaluated the effects of neem on biological and morphophysiological parameters of a. gemmatalis caterpillars. material and methods anticarsia gemmatalis the velvetbean caterpillar was reared with artificial diet (greene et al. 1976) at 25 ± 1 ºc , 70 ± 79 fig. 1 biological data of anticarsia gemmatalis (lepidoptera: noctuidae) after larval feeding for 4 d on artificial diet with neem seed extracts (0.061 g.ml-1 of azadirachtin in parent material). (a) mortality, (b) food consumption, (c) larval weight gain daily, (d) pupal weight, (e) eggs per female and (f) relationship between pupal abnormalities and moths emergence. 10 % relative humidity and photoperiod of 14:10 (l:d) in the insect’s biological control laboratory of universidade federal de viçosa, viçosa, minas gerais state, brazil. preparation of neem seeds extracts ripened fruits of a. indica were collected from espírito santo state, brazil (18º 39’ s and 40º 51' w), pulped in water, air dried and stored at -2 ºc. the obtained seeds (500 g) were macerated in 1.5 l of ethanol and filtered with wattman-1 filter paper. the solvent was removed from the filtrate by evaporation under vacuum with rotary evaporator at 50 ± 5 ºc for five days. this process was repeated thrice, obtaining 90 ml of a dark solution for preparation of stock solution. the 10 % stock solution was prepared with 10 ml of a. indica seed extract diluted in 90 ml of 30 % ethanol. 80 fig. 2 pupal morphological malformations of anticarsia gemmatalis (lepidoptera: noctuidae) after larval feeding for 4 d with an artificial diet containing a neem seed extracts. a) 0, b) 10, c) 50, d) 100, e) 250, f) 500 and g) 1000 ppm. the azadirachtin present in the seed extract was quantified by high performance liquid chromatograph (hplc), using ultra violet detector at 217 nm according to the method proposed by schaaf et al. (2000). a total of 20 µl of 20 % a. indica seed extract solution were injected into a reserve-phase column (c18), with a flow of 0.6 ml min-1, and column pressure of 97 kgf. the solvent used was methanol and water (1:1). the presence of azadirachtin in the crude neem seed extract was found at 14.13 min in hplc, following the same pattern of pure azadirachtin (sigma-aldrich germany). the azadirachtin was obtained at a concentration of 12.18 μg of azadirachtin per μl of 20 % crude seed extract, corresponding to a stock solution of 6.1 g.l-1 of azadirachtin. effects on larval and pupal development of anticarsia gemmatalis the evaluations of the effects of neem seed kernel extract (nske) on the larval and pupal stages of a. gemmatalis were adapted from senthilnathan et al. (2006a, b). the 6-day-old third instar larvae were starved for four hours, kept individually in petri dishes (9.5 cm diameter) for four days receiving 1.2 g of artificial diet containing 0 (control), 10, 50, 100, 250, 500 and 1000 ppm of nske (0.061 g of azadirachtin per ml) daily. after this period, the caterpillars were fed on diet without neem extracts. twenty larvae were used for the experiment and it was replicated five times. larval mortality was assessed daily until the beginning of the pupal stage. food consumption was determined by subtracting the mass of the diet provided by the mass leftover, and it was evaluated only during the period of exposure of larvae to artificial diets with nske. the weight gain daily (wgd) of a. gemmatalis larvae was obtained by using the formula wgd = (wf wi) / t, where wf = weight in the last larval stage, wi = larval weight at the beginning of the experiment, and t = time in days between the third and last larval stage. the pupal weight and abnormalities were measured 24 h after the molt to the third instar, and the ratio of abnormal pupae and emergence of a. gemmatalis moths was also evaluated. reproductive parameters a total of 20 pairs of newly-emerged larvae of a. gemmatalis were caged individually in plastic tubes (25 cm diameter) and fed for four days on an artificial diet with different concentrations of nske (0, 10, 50 and 100 ppm), followed by evaluation of the number of eggs per female. the eggs were counted between the 3th and 6th days after the emergence, which corresponded to the peak of egg production of a. gemmatalis (greene et al., 1973). the treatments with 500 and 1000 ppm of nske were not employed, because no moths were obtained from these treatments. histology the third instar larvae of a. gemmatalis were fed on artificial diet containing 0 and 500 ppm of nske. three larvae per concentration were collected after 2, 3 and 4 days of feeding and were transferred to zamboni fixative solution (stefanini et al., 1967) for 24 h at 4 ºc. the midgut and fat body were dissected into fixative solution, dehydrated in a graded ethanol series and embedded in historesin jb-4. subsequently, slices 5 µm thickness were stained with hematoxyline and eosin and analyzed under light microscope. sds-page the analyses of protein profile of haemolymph and fat body of a. gemmatalis larvae were adapted from the methods described byhuang et al. (2004, 2007). briefly, the third instar larvae of a. gemmatalis were fed on artificial diet with 0, 1 or 500 ppm of nske for four days. samples of hemolymph and fat body of 10 a. gemmatalis larvae per concentration were collected and placed in 0.1 % edta solution, following homogenization in 125 81 fig. 3 morphological changes in the midgut of anticarsia gemmatalis larvae (lepidoptera: noctuidae) after feeding with control diet (a), 2 d (b), 3 d (c) and 4 d (d) on diet containing neem seeds extracts. note epithelium detachment (arrowheads) and disruption (double arrows) in larvae fed on neem extract. arrows globet cells, l lumen, epepithelium, mmuscle. bars = 20 µm. mm saline solution. these samples were centrifuged at 12000xg for 15 min, and the supernatant was collected for total protein quantification according to braford (1976). the proteins (50 µg) were separated by 12 % sdspage (laemmli, 1971), and the gel was stained with coomassie blue solution. statistical analysis food consumption, weight gain, weight of pupae and number of eggs per female were analyzed by regression models. mortality, pupal abnormalities and adult emergence were subjected to regression analysis of logit. results the larval mortality of a. gemmatalis increased with nske concentration in the artificial diet, reaching 100 % at 500 ppm (fig. 1a). all the concentrations tested affected food consumption, weight gain of larvae and pupae, and fertility of adult females (figs 1b e). the quantity of artificial diet consumed by a. gemmatalis larvae over four days decreased linearly with increasing concentration of nske in the diet (f1,138 = 187.19, r 2 = 81.00, p < 0.0001). the larvae fed on control diet (without nske) ingested 1400 mg of artificial diet, whereas those fed on nske consumed 1193, 1023, 877, 852, 823 and 200 mg of diets containing 10, 50, 100, 250, 500 and 1000 ppm of nske, respectively (fig.1b). the daily larval weight gain of a. gemmatalis was linearly reduced with increasing concentrations of nske, reaching negative values with 1000 ppm (f1,138 = 136.74, r 2 = 75.23, p < 0.0001) (fig. 1c). the pupal weight was exponentially reduced with increasing concentration of nske in the diet (f2,97 = 67.75, r2 = 63.47, p < 0.0001), with pupae from larvae fed on 250 ppm showing 50 % of weight reduction (fig. 1d). there was an inverse relationship between the number of abnormal pupae and the emergence of moths of a. gemmatalis with increasing doses of nske (fig. 1f). furthermore, adults did not emerge from abnormal pupae. the a. gemmatalis pupal 82 abnormalities manifested as small intense malformations in the thorax area to no pupation (fig. 2). the occurrence of these symptoms was proportional to the increased dosage of nske in the diet. the larvae fed on low doses of nske (10, 50 and 100 ppm) reached the adult stage but showed exponential reduction in the oviposition rate (f1,79 = 349.63, r2 = 91.04, p < 0.0001). the average number of eggs per moth was 85, 30, 13 and 5 for females from larvae fed on 0, 10, 50 and 100 ppm of nske, respectively (fig. 1e). furthermore, a. gemmatalis fed on diets containing 500 ppm of nske showed morphological changes in the midgut when compared with the control larvae, which presented columnar digestive cells with evident striated border (fig. 3a). in the larvae treated with the neem extract, the midgut cells were swollen and detached from the basal membrane after two days (fig. 3b), while there was complete destruction of cells in some regions of the midgut epithelium after three days (fig. 3c) and midgut atrophy, characterized by a narrowed lumen, at four days (fig. 3d). the fat body cells of a. gemmatalis control larvae showed large granules of lipids and proteins (fig. 4a). however, the nske treated larvae showed lower amounts of lipids and absence of protein accumulation (fig. 4b). furthermore, the protein expression pattern in the hemolymph was similar in a. gemmatalis larvae fed on diets containing nske (fig. 5). on the other hand, the protein expression in the fat body was inhibited by diets containing 500 ppm of nske, whereas, the protein profile of larvae fed a diet with 100 ppm nske was similar to that of the control larvae (fig. 5). discussion the nske was efficient in controlling a. gemmatalis larvae, which exhibited increase in mortality, decrease in food consumption, larval and pupal weight, pupal abnormalities, and lower reproductive capacity after feeding on diet with different nske concentrations. similar effects have also been observed for other caterpillars such as spodoptera littoralis boisduval (martinez and emden, 2001) and spodoptera litura f. (huang et al., 2004), cnaphalocrocis medinalis guenée (senthil-nathan et al., 2006b), plodia interpunctella hübner (rharrabe et al., 2008), helicoverpa armigera hübner (kumar et al., 2008), and tuta absoluta (tome et al., 2013) after exposure to neem derivatives. azadirachtin and other limonoids present in ethanolic nske may be the cause of the antifeeding effects found in a. gemmatalis. it has been reported that the addition of 6ß-hidroxigeduim, gedunin, nimbinen, salanin or azadirachtin in artificial diet caused lower nutritional rates in h. armigera and s. litura, and azadirachtin showed the highest activity at a lower dose (koul et al., 2003). therefore, the effects of nske found in a. gemmatalis larvae may primarily be attributed to the azadirachtin, because fig. 4 fat body of anticarsia gemmatalis larvae (lepidoptera: noctuidae) 4 d after feeding on control diet (a) and neem seeds extracts (b). note protein granule (arrows) control that are lacking in larvae fed on neem extract. llipids, n nucleus. bars = 10 µm. the other limonoids have been observed to be effective only at doses higher than those of azadirachtin (senthil-nathan et al., 2006b), and the quantities of these substances in the neem seed extract have been found to be generally lower than that of azadirachtin (caboni et al., 2002). the cellular changes in the midgut epithelium of a. gemmatalis larvae are related to the antifeeding action of nske, promoting decrease in weight gain in the larvae and pupae of this insect. it has been reported that hypertrophy and displacement of epithelial cells from the basal lamina reduces the digestive capacity (barbeta et al., 2008; correia et al., 2009). this hypertrophy may be the result of cytoplasm vacuolation, endoplasmic reticulum fragmentation, and microvilli and plasma membrane disruption, as reported in the midgut cells of schistocerca gregaria and locusta migratoria (orthoptera: acrididae) (nasiruddin and mordue, 1993), rhodnius prolixus (hemiptera: reduvidae) (nogueira et al., 1997) and aedes aegypti (diptera: culicidae) (ndione et al., 2007) after azadirachtin exposure. these effects may also be related to the decrease in the midgut cells basal membrane folds and associated mitochondria (nogueira et al., 1997) 83 and atpase activity (senthil-nathan et al., 2005), disrupting the ion transport across membranes and promoting excessive water influx. several studies have shown that pure azadirachtin exerts cytotoxic effect, in vitro, on insects (salehzadeh et al., 2003). furthermore, other compounds synthesized by a. indica such as nimbolide and epoxyazadiradione have been noted to cause disruption in plasma membrane and swelling of cells (cohen et al., 1996). unlike the midgut epithelial cells, the fat body did not show cellular disorganization, but exhibited reduced lipid droplets and proteins granules. the cytotoxic effects of neem extracts on the midgut epithelium of a. gemmatalis larvae may be responsible for the reduction in the amount of lipids and proteins in the fat body, related to the inability of the larvae to metabolize and convert ingested food to reserves of fat body cells. furthermore, together with alterations in the midgut morphology, azadirachtin has also been observed reduce the activity of the digestive enzymes in lepidoptera (timmins and reynolds, 1992; senthil-nathan et al., 2005, rharrabe et al., 2008). the alterations in a. gemmatalis fat body protein production may have caused changes in the larval and pupal metamorphosis, as well as reduced oviposition rates in adult moths. stored proteins are synthesized in the larvae of lepidoptera, and are used as metamorphosis precursors, in egg production, and nutrient source during the adult life of a. gemmatalis (canavoso et al., 2001). thus, the larvae fed on neem extract that survived the lethal effects of this substance and emerged as adult frequently presented reduced reproductive capacity. in a previous study, topical application of azadirachtin to spodoptera exempta walker larvae (lepidoptera: noctuidae) affected oogenesis and reproductive maturation in adult females of this species owing to reduced protein synthesis in the fat body in the adult (tanzubi and mccaffery, 1990). the nske caused morphological malformations in a. gemmatalis pupae, which prevent adult emergence. these deformities were more extreme with higher doses of this preparation. similar effects have also been reported in s. littolarlis larvae fed on artificial diet containing azadirachtin (martinez and emden, 2001). the abnormalities and inhibition of metamorphosis can be attributed to disruptions in the molting hormone synthesis and release under the action of azadirachtin (mordue and nisbet, 2000). this effect may explain the larval and pupal mortality of a. gemmatalis, because the increase in mortality coincides with the metamorphosis period. the disruption of a. gemmatalis lifecycle at different developmental stages by nske can reduce the amount of active ingredient to be applied to control this insect pest. in the present study, a. gemmatalis larvae that were fed a diet containing 100 ppm of nske showed < 20 % mortality rates, 40 % decrease in food consumption decrease, high rate of larvae with deformities, low number of adult emergence, and low oviposition rates, which limited the population growth in the next generation of this pest. thus, the use of insecticides based on azadirachtin is important in a. gemmatalis fig. 5 protein expression in hemolymph (lanes b, c, d) and fat body (lanes f, g, h) of anticarsia gemmatalis larvae (lepidoptera: noctuidae) 4 d after feeding an artificial diet with neem seed extracts. a) molecular weight standard (kda), b) hemolymph control, c) 500 ppm, d) 100 ppm of neem seeds extract, e) fat body control, f) 500 ppm, g) 100 ppm of neem seeds extracts. management programs owing to the lethal and residual effects of this compound. furthermore, the seed extract of a. indica ws found to 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suspended animation known as anhydrobiosis. notably, anhydrobiosis also renders the organism tolerant to several other physical stresses such as extremes of temperature, pressure and radiation. anhydrobiosis-based technologies are promising strategies to preserve crop plants as well as organs for transplant. in order to understand the relation between anhydrobiosis and tolerance to physical stresses, we submitted the anhydrobiotic nematode panagrolaimus superbus to diverse abiotic stresses when alive (hydrated) and in anhydrobiosis (desiccated). remarkably, our data revealed that hydrated p. superbus naturally displays considerable tolerance to ultra-low temperature (-196 °c), x-radiation (500 gy) and ultracentrifugation (400,000xg) in the tested conditions. more importantly, anhydrobiosis enhances nematode tolerance to ultra-low and high temperatures (+100 °c), but not to x-radiation or ultracentrifugation. these findings may help explain the successful wide distribution of p. superbus on earth, since extremes of temperature are the most common stresses confronted by this species. finally, due to its intrinsic survival potential (hydrated or desiccated), our data evidence the potential of p. superbus as a model in astrobiology. key words: anhydrobiosis; desiccation tolerance; x-radiation; extreme temperatures; ultracentrifugation introduction the phenomenon of anhydrobiosis (from the greek: "life without water") was first described over 300 years ago by antonie van leeuwenhoek and can be defined as a highly stable state of true suspended animation that certain organisms (within diverse groups including bacteria, yeasts, plants and small invertebrates) enter when exposed to very low relative humidity conditions (tunnacliffe and lapinski, 2003). while most organisms die in this scenario, these organisms lose 95 99 % of the body water content, replacing their intracellular aqueous milieu by an amorphous bioglass, composed of trehalose (erkut et al., 2011), intrinsically disordered proteins (boothby et al., 2017) and other elements, which literally arrests all biomolecules in space and time. this ametabolic state is stable for long periods of time; life is resumed ___________________________________________________________________________ corresponding author: tiago campos pereira dpto de biologia, ffclrp universidade de são paulo usp av. bandeirantes, 3900. bairro monte alegre ribeirão preto sp, brasil. cep 14040-901. e-mail: tiagocampospereira@ffclrp.usp.br when the organism is rehydrated (crowe et al., 1992, clegg, 2001; tunnacliffe and lapinski, 2003; rebecchi et al., 2007). notably, several anhydrobiotic organisms (or anhydrobionts) are able to tolerate different types of stress when desiccated (e.g., ionizing radiation, vacuum, extreme temperatures, high hydrostatic pressures, hipogravity, etc) (tunnacliffe and lapinski, 2003; hengherr et al., 2009; horikawa et al., 2009; jönsson et al., 2008; beltrán-pardo et al., 2013). therefore, anhydrobiotic organisms are extremotolerant, i.e., they may live in conditions similar to those ‘suitable for human life’, but can tolerate conditions of extreme abiotic stress when necessary (rampelotto, 2013). the phenomenon of anhydrobiosis displays an immense biotechnological potential in agriculture and biomedicine: the preservation of crops during severe droughts as well as organ preservation at room temperature for transplant. recently, an anhydrobiosis-based strategy for vaccine storage at room temperature was developed (alcock, 2010), illustrating the great potential behind this natural phenomenon. 86 fig. 1 tolerance of dessicated p. superbus to heat. n = 600 per group, per technical replicate. different letters indicate statistically significant differences (p < 0.05). panagrolaimus superbus is a free-living anhydrobiotic nematode of approximately 1 mm in length, dioic, which was first described by fuchs (1930). members of the genus panagrolaimus occupy several different niches, from antarctic, volcanic islands, temperate and semi-arid soils to terrestrial mosses (shannon et al., 2005; mcgill et al., 2015). it is closely related to the well characterized nematode caenorhabditis elegans. here we investigated the tolerance profile of p. superbus to extremes of temperature (-196 ºc and +100 ºc), x-radiation (100 and 500 gy) and hypergravitational force (400,000xg) in order to uncover the survival potential of this species in the hydrated and desiccated (anhydrobiotic) states, as well as the role of anhydrobiosis on these abilities. material and methods nematode maintenance panagrolaimus superbus, kindly provided by prof. tunnacliffe a (university of cambridge, uk), was maintained in the dark, at 20 ºc, on ngm (nematode growth medium) agar plates and fed with a layer of escherichia coli (op50 strain). mixed populations, composed of all developmental stages, were used in all experiments. desiccation, rehydration and viability assay ngm agar plates were rinsed with m9 buffer in order to dislodge and collect worms, which were subsequently washed three times with m9 buffer to remove excess of bacteria. worms were then immobilized on 0.45 µm supor filter membranes by vacuum filtration using a sartorius funnel. these membranes were placed in a sealed chamber containing a saturated solution of cuso4, for 24 h [pre-conditioning in 98 % relative humidity (rh)]. then, they were transferred to another chamber containing regenerated silica gel, for 24 h (desiccation in 10 % rh). desiccated worms were then submitted to different stresses (temperature, xradiation or ultracentrifugation), as described below. thereafter, membranes were placed in a chamber with distilled water for 24 h (pre-rehydration in 100 % rh) and then the membranes are immersed in m9 buffer for 3 h for worm rehydration. subsequently, we performed a survival assay using a modified version of protocol which has been used for isolated cells (krause et al., 1984). briefly, the supernatant was removed and erythrosin b dye was added (0.4 % w/v in m9 buffer). after 1 h, samples were washed three times with m9 buffer to remove excess of dye. dead worms stained in pink, live worms remained unstained. liquid nitrogen (-196 ºc) the exposure procedure to liquid nitrogen (n2) was based on previous experiments performed in tardigrades with some modifications (horikawa et al., 2008). after 24 h in regenerated silica gel chamber, desiccated worms (immobilized on supor membranes and placed inside closed 1.5 ml microtubes without paraffin) were immersed for 15 min, 30 min, 1 h, 1 week or 1 month directly in liquid n2 (-196 ºc). at the end of exposure, worms were thawed at room temperature for 5 min and submitted to the subsequent steps (pre-rehydration, rehydration and viability assay). negative control group (nc) was composed of hydrated worms 87 immobilized on membranes, placed inside microtubes and directly immersed in liquid n2. positive control group (pc) was composed of desiccated worms immobilized on membranes, placed inside microtubes but not immersed in liquid n2. experimental group (eg) was composed of desiccated worms immobilized on membranes, placed inside microtubes and directly immersed in liquid n2. three biological replicates were performed (n = 600 per group, per technical replicate. each technical replicate comprised all three groups: nc, pc and eg. the only exceptions were pc for 15 min, 30 min and 1 h, which are the same, since they are functionally equivalent). statistical analyses (one way anova) were performed for each time point (comparing the corresponding pc, nc and eg). heat (50 100 ºc) desiccated worms (immobilized on supor membranes and placed in 0.6 ml microtubes) were subjected to heating at different temperatures (50 ºc, 62.5 ºc, 75 ºc, 78 ºc, 81 ºc, 87.5 ºc or 100 ºc) using a thermal cycler (mastercycle eppendorf). treatment of samples started at 25 ºc with an increase rate of 5 ºc every 2 min until reaching the desired temperature (previously indicated), in which they remained for 5 min. this period of time (5 min) was chosen since it may represent acute ‘peaks of stress’ that occur in nature. since survival percentage of desiccated worms at 50 ºc is high, we assume that temperatures below it may not represent stressing conditions. in a second experiment, worms were exposed to 50 ºc for 15 min, 30 min or 1 h. nc was composed of hydrated worms immobilized on membranes and exposed to heating. pc was composed of desiccated worms immobilized on membranes but not exposed to heating. eg was composed of desiccated worms immobilized on membranes and exposed to heating. three biological replicates were performed (n = 600 per group, per technical replicate. each technical replicate comprised all three groups: nc, pc and eg). statistical analyses (one way anova) were performed comparing all groups (fig. 1) or within each group separately (nc or eg) (fig. 2). x-radiation in order to measure the tolerance of p. superbus to x-radiation, desiccated worms were immobilized on membranes and placed in petri dishes which were irradiated (100 or 500 gy) using the rs 200 biological research irradiator (rad source) located in the radiology section of the university of são paulo hospital fmrp/usp. after rehydration worms were divided into 10 equal samples and population sizes were determined throughout 10 time points (the following 10 days after stress). population growth (in percentage) was determined by dividing the final number of living worms (output) by initial number of worms (input). nc was composed of hydrated worms immobilized on membranes and exposed to radiation. pc was composed of desiccated worms immobilized on fig. 2 tolerance of p. superbus to 50°c for different periods of time. negative control (nc): hydrated worms immobilized on membrane and exposed to stress; experimental group (eg): desiccated worms immobilized on membrane and exposed to stress. n = 600 per group, per technical replicate. different letters indicate statistically significant differences (p < 0.05). #: marked groups are not statistically different. 88 fig. 3 tolerance of p. superbus to x-radiation. pc (positive control): desiccated worms immobilized on membranes but not exposed to stress; nc* (negative control*): hydrated worms immobilized on membranes but not exposed to stress; nc 100 gy (or 500 gy) (negative controls): hydrated worms immobilized on membranes and exposed to 100 gy (or 500 gy); eg 100 gy (or 500 gy) (experimental groups): desiccated worms immobilized on membranes and exposed to 100 gy (or 500 gy). n = input of 100 per group, per day, per technical replicate. different letters indicate statistically significant differences (p < 0.05). #: marked groups are not statistically different. membranes but not exposed to radiation. eg was composed of desiccated worms immobilized on membranes and exposed to radiation. an additional negative control group (nc*) consisting of immobilized hydrated worms not exposed to radiation was used in order to reproduce ‘population dynamics’ close to which is observed under normal conditions. three biological replicates were performed (n = 1,000 per group, per technical replicate. each technical replicate comprised all three groups: nc, pc and eg). statistical analyses (one way anova) were performed comparing only the groups within the same time point (figs 3, 4). ultracentrifugation (hypergravitational force) p. superbus worms were centrifuged at 400,000×g at 4 ºc (the working temperature of the equipment) for 5, 15, 30 min or 1 h using max-xp ultracentrifuge. desiccated immobilized worms, submitted to centrifugation were considered as the eg. hydrated immobilized worms, submitted to centrifugation were considered as the nc. nonimmobilized worms centrifuged while immersed in m9 buffer were considered as ‘m9 negative control’ (m9-nc). hydrated, non-immobilized worms, not centrifuged but kept at 4 °c for 1 h were considered as the ‘pc4 °c’. two positive controls were used. positive control 1 (pc1): desiccated, immobilized worms, kept at 4 ºc for 1 h but not centrifuged and then rehydrated. positive control 2 (pc2): nonimmobilized worms kept in m9 at 4 ºc for 1 h but not centrifuged. three biological replicates were performed, each one consisting of three technical replicates (each one comprising all three groups: nc, pc and eg). n = 600 worms/technical replicate. statistical analyses (one way anova) were performed comparing only the groups within the same time point (fig. 5). statistical analyses all experiments were performed in biological triplicates (each one consisting of technical triplicates) and data are presented as mean values and standard deviations. statistical analyses were performed using "one way anova" (with student newman post-hoc or student-newman-keuls for ultracentrifugation). statistical differences were considered significant when p ≤ 0.05. identical letters indicate those groups are not statistically different. distinct letters indicate those groups are statistically different. in some cases, one group may be statistically not different from only one specific group, within a larger set of groups indicated with a different letter. in these specific cases, those groups indicated with hashtag (#) are not statistically different. results tolerance to liquid nitrogen remarkably, a considerable percentage of hydrated p. superbus is tolerant to liquid nitrogen (196 °c) in the absence of any cryoprotectants for up to one month (22.6 % on the average of the five time points) (fig. 6). survival is 30.2 % after 15 min, decreasing to 9.8 % after four weeks submitted to ultracold conditions. more importantly, desiccated worms exposed to liquid nitrogen always displayed much higher, statistically significant, survival percentages (80.9 % on the average of the five time points) than nc group similar or higher than the pc group in all treatments (75.0 % on the average). therefore, hydrated p. superbus presents a natural tolerance to ultra-low temperature (indicated as nc), which is enhanced by anhydrobiosis in the long term (indicated as eg). 89 fig. 4 population growth of p. superbus exposed to x-radiation. pc (positive control): desiccated worms immobilized on membranes but not exposed to stress; nc* (negative control*): hydrated worms immobilized on membranes but not exposed to stress; nc 100 gy (or 500 gy) (negative controls): hydrated worms immobilized on membranes and exposed to 100 gy (or 500 gy); eg 100 gy (or 500 gy) (experimental groups): desiccated worms immobilized on membranes and exposed to 100 gy (or 500 gy). n = input of 100 per group, per day, per technical replicate. different letters indicate statistically significant differences (p < 0.05). tolerance to heating (50 ºc 100 ºc) the survival curve (fig. 1) evidenced that desiccation rendered worms tolerant to heating. viability percentages obtained after exposure to temperature gradient revealed a nearly linear, inverse correlation, with a significant decrease observed from 75 ºc above. this data also evidences that a small fraction of the desiccated population seems to be thermostable from 80 ºc to 100 ºc (7.5 % on the average of the three time points). as expected, worms in almost all nc (hydrated exposed to heating) died (data not shown). therefore, by comparing both groups (hydrated versus desiccated), our data evidences that anhydrobiosis confers partial heat tolerance. although high temperatures are lethal to hydrated p. superbus, a few worms were still alive at 50 ºc for 15 min (6.5 %; fig. 2, nc), a situation abolished by 1 h. remarkably, a high percentage (74.3 %) of desiccated worms remained viable after one hour at 50 ºc, evidencing a protective effect of anhydrobiosis to heat. however, this tolerance gradually diminishes. x-radiation viability analysis of irradiated worms throughout ten days revealed small but statistically significant decreases on the first, second and sixth days (differences observed among groups within the same day), especially in desiccated worms exposed to 500 gy (fig. 3). therefore, according to these experiments, x-ray doses of 100 and 500 gy were not lethal to worms within the period of analysis. conversely, analysis of population growth indicated an apparent negative effect of x-rays from the seventh day on, which is statistically significant at day ten. during this period, both hydrated and desiccated worms exposed to 500 gy presented stagnation of population growth (fig. 4) (on average both groups nc 500 gy and eg 500 gy halted at 103.9 % at day ten, compared to the average of 1,162.6 % of the other groups). therefore, our data evidence that anhydrobiosis does not confer tolerance against x-radiation in any tested condition, since there were no differences in survival or population growth between experimental groups and their respective ncs. ultracentrifugation experiments with hypergravitational forces revealed unexpected findings (fig. 5). notably, a high percentage (41.3 %) of desiccated p. superbus is tolerant to extreme hyperacceleration (400,000xg) for 1h. however, more surprisingly is the fact that hydrated worms (immobilized on filter membranes) presented a similar result (39.1 %), thus uncovering a natural tolerance of this nematode to extreme gforces. since the filter membrane, used as immobilization substrate for both previous groups, often collapsed during ultracentrifugation (potentially damaging the worms), we decided to evaluate nonimmobilized p. superbus. remarkably, such hydrated and freely swimming worms in liquid medium are virtually fully tolerant to such g-force (5 min 96.7 %; 15 min 97.2 %; 30 min 98.3; 1 h 98.0 %) (fig. 5). discussion p. superbus is an anhydrobiotic nematode able to thrive at diverse enviromental conditions. our data reveals that anhydrobiosis confers considerable resilience to high temperatures for short periods, a situation that might take place in natural environments as semi-dry soils (shannon et al., 2005). we also observed that p. superbus tolerates up to 1 h in a relatively high temperature (50 ºc). taken together, these data indicate that anhydrobiosis guarantees the perpetuation of the desiccated population when exposed to high temperatures for varying periods of time in natural environments. notoriously, p. superbus can be stored at -80 ºc for 24 h without compromising viability (mcgill et 90 fig. 5 tolerance of p. superbus to ultracentrifugation. n = 600 per group, per technical replicate. different letters indicate statistically significant differences (p < 0.05). pc1: positive control group 1, desiccated worms immobilized on membranes but not centrifuged, kept at 4 °c for 1 h. pc2: positive control group 2, worms kept in m9 buffer but not centrifuged, kept at 4 °c for 1 h. nc: negative control group, hydrated, immobilized worms, centrifuged at 4 °c for 1 h. pc4 °c: hydrated, non-immobilized worms, not centrifuged but kept at 4 °c for 1 h. eg: experimental group, desiccated worms, immobilized on membranes, centrifuged at 4 °c for 1 h. m9-nc: hydrated worms immersed on m9 buffer, centrifuged at 4 °c for 1 h. al., 2015). when comparing survival of hydrated versus desiccated p. superbus, both submitted to ultralow or high temperatures, the second group displayed higher viability in both stressing scenarios. therefore, acute tolerance of desiccated p. superbus to extremes of temperature is due to anhydrobiosis rather than a natural adaptation of this species to extreme cold environments (a common habitat) or to heat (which in fact is lethal). the comprehension, at the genetic, biochemical and physiological levels, of how anhydrobiosis renders the organism tolerant to -196 ºc for such long periods or to high temperatures may not only help the advancement of cryobiology and anhydrobiotic engineering, but also to understand the limits of life confronting physical stresses. when comparing survival percentages of desiccated worms to ultralow versus high temperatures, it is clear that anhydrobiosis confers higher tolerance for long periods at ultralow temperatures rather than at high ones. this may seem counterintuitive since anhydrobiosis is a phenomenon directly related to high temperatures (when dehydration/desiccation naturally takes place). this lower tolerance to higher temperatures is probably due to the transition point of the bioglass, an amorphous matrix composed in some species of non-reducing disaccharides and other proteins which seems to stabilize the structure and cellular constituents during anhydrobiosis (buitink et al., 2004; sakurai et al., 2008; hengherr et al., 2011). if the external temperature raises up to values above the glass-transition point, its own integrity is compromised, thus decreasing viability (hengherr et al., 2009). curiously, different anhydrobiotic organisms seem to present distinct glass-transition points (or other heat-stabilizing elements), as judged by the fact that they tolerate higher temperatures. these are the cases of some rotifers, tardigrades and nematodes which survive to brief exposure to +150 ºc (reviewed in tunnacliffe and lapinski, 2003; eisenback et al., 2013). radiation exposure is possibly the most studied stress in suspended animation in scientific literature (horikawa et al., 2006; watanabe et al., 2006; gladyshev and meselson, 2008; nilsson et al., 2010; beltrán-pardo, 2013, 2015). these studies have focused on the understanding of eukaryote’s resilience to ionizing radiations and can be compared to other radiation analyses in nonanhydrobiotic animal models such drosophila melanogaster. as previously observed in c. elegans (onodera et al, 2010), doses of 100 and 500 gy caused no viability decrease in p. superbus ten days after exposure to x-rays, probably due to eutely (low degree of somatic cell divisions in the adults), thus less susceptible to the harmful effects of radiation. however, the stagnation in population growth at 500 gy dose possibly reveals a sterilizing (chang et al., 2015) or egg-lethality effect of x-rays. more importantly, our data evidence that anhydrobiosis does not confer tolerance against x-radiation in any tested condition. other anhydrobiotic organisms, when exposed to intense radiation regimes, suffer extensive dna damage. however, they survive by activating unique dna repair systems, able to reconstruct all the genomic landscape (zahradka et al., 2006). our data suggest that such mechanisms 91 fig. 6 tolerance of p. superbus to liquid nitrogen. positive control (pc): desiccated worms immobilized on membrane but not exposed to stress; negative control (nc): hydrated worms immobilized on membrane and exposed to stress; experimental group (eg): desiccated worms immobilized on membrane and exposed to stress. n = 600 per group, per technical replicate. statistical analyses were performed for each time point separately. different letters indicate statistically significant differences (p < 0.05). are not present in p. superbus, or if so, they are not effective within the tested conditions (period and dose). deguchi et al. (2011) revealed that single-celled microorganisms are able to withstand (and even discretely thrive) when subjected to 400,000xg. since this experimental condition seemed to be an interesting physical stress to test anhydrobiosis’ protective effect in a multicellular organism, we submitted desiccated p. superbus to ultracentrifugation. we initially hypothesized that such extreme forces would be lethal, to both hydrated and desiccated worms, leading to sedimentation of intracellular components, affecting internal structures and body morphology. however, unexpectedly, we observed that hydrated worms are completely tolerant to such extreme physical stress. after a deep and extensive search in the literature we noted that beams and king (1936) showed that eggs of the nematode ascaris suum were able to withstand identical hypergravitational forces, presenting dividing cells 48 h later. the only other report is of morey-holton (2003), who mentioned in a review that ‘nematodes tolerate 105xg for brief periods’ (without reference), leading us to believe that it refers to the study with nematode eggs (beams and king, 1936). therefore, to our knowledge, this is the first time that an adult animal is shown to tolerate 400,000xg, orders of magnitude above conventional studies (1-100xg) (kim et al. 2007; sasagawa et al., 2003; qiao et al. 2013). curiously, desiccated worms display a much lower tolerance than hydrated ones. this may be due the fact that the bioglass, as a solid matrix (hengherr et al., 2009), is susceptible to damages in its structure and/or integrity due to the tensional forces experienced during the hyperaccelerations, and/or the movement/collapse of the membrane on which the worms are immobilized. all these stressing physical forces probably cause ruptures within the bioglass, thus decreasing the viability of the desiccated worms. the fact that adult p. superbus withstands hyperaccelerations may have implications in diverse and fundamental aspects of biology. perhaps the most important involves the physical limits that constrain the existence of life (rothschild and mancinelli, 2001). the present evidence that a multicellular organism tolerates such condition extends the range of possible inhabitable planets, thus setting the foundation to consider the existence of biological activities also in much more massive celestial bodies, which display much higher gravitational forces. it is also important to highlight that the natural and continuous exchange of mass among planets (pizzarello and cronin, 1998) also involves equivalent hyperacceleration forces for short 92 periods, either during the ejection of a rock caused by intense eruption or the impact of a meteorite. therefore, organisms located on such rocks might survive such events, helping understand the origin and distribution of life in the universe. conclusions our data evidence that p. superbus presents remarkable survival potential to freezing, radiation and hyperacceleration even in the hydrated state. however, anhydrobiosis potentiates its survival in extremes of temperature, providing the additional capacity needed to withstand severe droughts or freezing observed in its natural environments. the surprising observation that hydrated p. superbus is fully tolerant to such hyperacceleration (400,000xg for 1 h) extends the known limits of tolerance to gforces for adult animals in orders of magnitude, raising new and fundamental questions about the limits of life. finally, due to its intrinsic survival potential (in the hydrated and desiccated states), p. superbus might also be exploited as a model in astrobiology. acknowledgments the authors would like to thank gouvêa de lima as and bárbara aparecida santana a from radiology section of the university of são paulo hospital (fmrp/usp) for help us during x-rays irradiation procedures. we are also thankful to prof. roy edward larson (fmrp/usp) for granting us access to the ultracentrifuge. we also thank prof. de souza 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ningbo university, ningbo, zhejiang, 315211, pr china 2college of biological & environmental sciences, zhejiang wanli university, ningbo, zhejiang, 315100, pr china 3zhejiang marine development research institute, zhoushan, 316021, pr china accepted april 18, 2017 abstract the hemoglobins produced by tegillarca granosa have antibacterial activity toward some gram-positive and gram-negative bacteria. in this study, the genes encoding the recombinant proteins tg-hbiia and tg-hbiib were cloned from t. granosa hemocytes by rt-pcr, and the proteins were expressed in escherichia coli transetta (de3). the proteins were purified using a histrap ff affinity chromatography column under denaturing conditions and refolded at 4 °c by urea gradient dialysis, and the antibacterial activity of the recombinant proteins was determined. the tg-hbiia protein had antibacterial activity toward vibrio harveyi and pseudomonas putida, with the minimum inhibition concentration (mic) values of 65.8 ug/ml and 4.11 ug/ml, respectively. the tg-hbiib protein had antibacterial activity toward v. harveyi, p. putida and acinetobacter baumanii, with mic values of 158 ug/ml, 39.5 ug/ml and 79 ug/ml, respectively. they had no antibacterial activity against staphylococcus aureus, e. coli, b. firmus, b. subtilis, s. epidermidis, or v. parahaemolyticus. this study provides a basis for further research on the antibacterial function and mechanism of hemoglobin. key words: tegillarca granosa; hemoglobin; recombinant protein; antibacterial activity introduction hemoglobin (hb) is a respiratory protein containing iron that has multiple biological functions and is one of the most researched proteins in the protein family (riggs, 1991; terwilliger, 1998; jiang et al., 2007; katsu et al., 2010). hb is rare in molluscs, with most harboring copper-containing hemocyanin instead (lieb et al., 2006). however, tegillarca granosa, which belongs to the family arcidae, contains abundant hb in circulating erythrocytes (suzuki et al., 1992). in our previous studies, full-length cdnas and genomic dna of tg-hbiia and tg-hbiib were cloned from t. granosa. sequence and structural analyses revealed that tg-hbiia and tg-hbiib form a2b2 heterotetramers (wang, 2012). the expression of tg-hbs mrna in t. granosa hemocytes was markedly upregulated after vibrio parahaemolyticus, lps or pgn challenge (bao et al., 2011). the purified heterotetramers of tg-hbii exhibited antibacterial activity against ___________________________________________________________________________ corresponding author: yongbo bao college of biological & environmental sciences zhejiang w anli university 8 south qianhu road, ningbo zhejiang 315100, p.r. china e-mail: bobbao2001@gmail.com pseudomonas putida but no antibacterial activity against staphylococcus aureus, based on an agarose diffusion test and the minimal inhibitory concentration (mic) (bao et al., 2013). and the polypeptides from tg-hbii also exhibited antibacterial activity against v. alginolyticus, v. harveyi and v. parahaemolyticus (wang et al., 2015). these results indicated that tg-hbs might be involved in the immune responses against bacterial infection. many animals are known to that their hb can limit the growth of or kill bacteria as a defense mechanism. parish et al. (2001) found that intact hb tetramers, alpha and beta subunits, from human, exhibited considerable activity against gram-positive and gram-negative bacteria, and fungi (parish et al., 2001). many previous studies demonstrated that bovine hb is a rich source of antimicrobial peptides (daoud et al., 2005; nedjararroume et al., 2008; przybylski, 2015). antibacterial activities of hb from non-mammalian vertebrates such as crocodylus siamensis and japanese eel (anguilla japonica) also have been confirmed. hb, α-chain, β-chain and fragmented hb of c. siamensis can inhibit growth of gram-positive bacteria b. subtilis, b. amyloliquefaciens, and b. pumilus (srihongthong et al., 2012). zhang et al. (2013) isolated an antibacterial 150 table 1 primers used in the present study gene genbank number primer sequence product size tg-hbiia hq729976 f: ggatccgttgatgcagcagttgcaaat r: aagcttccattgatggttggtccagat 492bp tg-hbiib hq149306.1 f: ggatccggtgtcaacgaagcaatcaaag r: aagcttaatagtcgttttttctcatgc 483bp peptide from hb alpha in the liver of japanese eel. and they found that its synthetic peptide also had strong antibacterial activities against gram-positive and gram-negative bacterias. there are several hb of invertebrate have been reported. it reported that scapharca kagoshimensis hb had antibacterial activity against s. aureus, b. subtilis, micrococcus tetragenus (xu et al., 2015). in a word, nature hb has broad-spectrum antibacterial activity and different sources of hb with different antimicrobial activity. however, few study reported that recombinant hb have antibacterial activity. in the present study, we constructed tg-hbiia and tg-hbiib expression vectors, expressed these vectors by iptg induction, and purified the proteins by affinity chromatography. in addition, through the analysis of the recombinant hb antibacterial activity by in vitro bacteriostatic experiments, this work provides the basis for the further study of the antibacterial immunity function of hb and its mechanism. materials and methods experimental clams, collection of hemocytes and experimental bacteria t. granosa (average shell length approximately 30 mm) were purchased from an aquaculture farm (ningbo, china). after the t. granosa were vivisected and the blood was quickly drawn, the hemolymphs were centrifuged at 1000 rpm for 10 min to harvest the hemocytes. the cells were flash-frozen in liquid nitrogen and then stored at -80 °c for rna extraction. e. coli (atcc 35218), p. putida (cgmcc 1.0593), s. aureus (atcc 29213), v. harveyi, v. parahaemolyticus, and v. alginolyticus, b. subtilis, s. epidermidis, b. firmus, and a. baumanii are provided by zhejiang wanli college microbiology and environmental engineering key laboratory. cloning and expression vector construction total rna was isolated from the t. granosa hemocytes using trizol (invitrogen, usa) and was reverse transcribed using a reverse transcription system kit (promega, usa). the primers were designed to amplify the orf of the full-length tg-hbiia and tg-hbiib cdnas. all primers were synthesized by sangon biotechnology (china) and are shown in table 1. the amplified products with a size of approximately 500 bp were cloned into the pmd-18t vector (takara, japan), and the recombinant plasmids were named pmd-18t/tg-hbiia and pmd-18t/tg-hbiib. the recombinant plasmids were digested with bamhi and hindiii (new england biolabs, uk), which were then inserted into the corresponding sites of the expression vector pet30a (takara) to generate pet30a/tg-hbiia and pet30a/tg-hbiib. three positive clones for each product were sequenced at sangon biotechnology (china). expression and purification of tg-hbiia and tg-hbiib the pet30a/tg-hbiia and pet30a/tg-hbiib plasmids were separately transformed into e. coli transetta (de3) (transgen, china). for the expression of tg-hbiia and tg-hbiib recombinant proteins, the positive transetta (de3) cells were grown at 37 °c in 1 l lb liquid medium with 50 mg/ml of kanamycin. when the od600 reached between 0.5 and 0.8, the culture was induced with 1 mm iptg (aladdin usa). to determine the optimal conditions for the expression, the expression of the target proteins at 37 °c, 30 °c, 28 °c, 25 °c, and 20 °c after 2 h, 3 h, 4 h, 5 h and 6 h of induction was examined. then, a large number of recombinant proteins were expressed in the best condition. finally, the cells were harvested by centrifugation at 6,000 rpm for 20 min at 4 °c and stored for next step of the experiment. all of the wet cells were resuspended in lysis buffer (50 mm nah2po4·2h2o, 2 mm edta, 100 mm nacl, 0.5 % triton x-100) and disrupted by sonication on ice. the cell lysate was centrifuged at 12,000 rpm for 30 min at 4 °c. because the tg-hbiia and tg-hbiib were found in inclusion body, the insoluble fraction (i.e., inclusion body) from the lysate was resuspended in solubilization buffer (50 mm nah2po4·2h2o, 10 mm tris-hcl, 8 m urea, ph 7.2) at 5 ml per gram of wet inclusion body and centrifuged at 12,000 rpm for 30 min to pelletize the cellular debris. to purify the proteins, a 5 ml histrap ff affinity chromatography column (ge healthcare) was used in the akta pure protein chromatography system (ge healthcare). as a first step, the histrap ff column was equilibrated with 2~3 column volumes of distilled water and equilibrating buffer (50 mm nah2po4·2h2o, 300 mm nacl, 8 m urea, 10 mm imidazole, ph 7.2) at a flow rate of 0.5 ml/min. then, protein samples were loaded into the column by using equilibrating buffer. subsequently, the desired protein was eluted using elution buffer (50 mm nah2po4·2h2o, 300 mm nacl, 8 m urea, 200 mm imidazole, ph 7.2), automatically 151 collected in a fraction collector and stored at 4 °c. the collected proteins were analyzed by sds-page to verify the purity of the proteins. the components of the proteins were identified by mass spectrometry, and the concentrations were estimated using a bradford protein assay. the purified protein was denatured, so we needed to make the proteins form complexes. we added 1 mg of the purified recombinant tg-hbiia or tg-hbiib into a dialysis bag and added a slight excess of heme, ~1 mg. the samples were then dialyzed for 12 h at 4 °c using renaturation buffer (50 mm nah2po4·2h2o, 50 mm nacl, 1 mm edta, 2 m reduced glutathione, 0.02 m oxidized glutathione, 10 % glycerol, 10 % glycine, ph 7.2), which contained 6 m, 4 m, 2 m or 0 m urea. antibacterial activity of the recombinant protein an agar diffusion method was used for the preliminary detection of the antibacterial activity of the recombinant proteins (tg-hbiia, tg-hbiib) against the gram-negative bacteria e. coli, a. baumanii, p. putida, v. harveyi, v. parahaemolyticus, and v. alginolyticus and the gram-positive bacteria s. aureus, b. subtilis, s. epidermidis, and b. firmus. s. aureus, e. coli, p. putida, a. baumanii, b. firmus, b. subtilis and s. epidermidis were spread on lb solid medium and grown at 37 °c for 12 h. then, colonies were picked into 50 ml of lb in a liquid culture flask and cultured at 37 °c for 5 h with shaking. v. alginolyticus, v. harveyi and v. parahaemolyticus were spread on seawater solid medium in a liquid culture flask and grown at 28 °c for 12 h. then, colonies were picked into 50 ml of seawater medium and cultured at 28 °c for 5 h with shaking. all of the bacteria were counted using a hemocytometer, diluted into 105 cfu/ml bacterial suspensions, and 200 µl of each test microorganism suspension was spread onto lb or seawater solid medium. bacterial plates were cultured for 10min, and three oxford cups were then put into the medium. the recombinant proteins (tg-hbiia, tg-hbiib) and the phosphate buffer negative control (50 mm nah2po4·2h2o, 50 mm nacl, 1 mm edta, 2 m reduced glutathione, 0.02 m oxidized glutathione, 10 % glycerol, 10 % glycine, ph 7.2) were added to the oxford cup. s. aureus, e. coli, p. putida, a. baumanii, b. firmus, b. subtilis, and s. epidermidis were cultured at 37 °c for 12 h, and v. alginolyticus, v. harveyi and v. parahaemolyticus were cultured at 28 °c for 12 h. then, the oxford cups were unplugged, and the zone of inhibition was observed. to ensure the accuracy of the bacteriostatic experiment, we performed three replicates for each kind of bacterium. the mic of recombinant proteins (tg-hbiia, tg-hbiib) was determined using the broth dilution method. the bacterial suspensions and serially diluted recombinant proteins were added to 96-well plates at a ratio of 4:1 in a final volume of 100 µl for 10 wells. the 11th well without added recombinant protein served as a growth control. the 12th well contained 0.1 mg/ml trypsin as a positive control. the microplates of s. aureus, e. coli, p. putida, a. baumanii, b. firmus, b. subtilis, and s. epidermidis were incubated at 37 °c with continuous shaking. the microplates of v. alginolyticus, v. harveyi and v. parahaemolyticus were incubated at 28 °c with continuous shaking. after 16 ~ 20 h, the od value of each well was measured with an automatic microplate reader (spectramax 190, molecular devices, usa) set at 600 nm. the experiment was repeated 3 times. finally the clear bacterial growth was observed both in the growth control and in the 96-well plates, and the concentrations of the wells with no bacterial growth were designated as the value of the mic. results and discussion cloning and expression vector construction we cloned the tg-hbiia and tg-hbiib genes from t. granosa hemocytes by rt-pcr and the pcr products were inserted into pmd-18t vectors. the coding region of the mature tg-hbiia was amplified from the plasmid pmd-18t/tg-hbiia and cloned into the expression vector pet30a (+) to generate the pet30a/tg-hbiia. the plasmid was confirmed by a double restriction enzyme digestion of pet30a/tg-hbiia with bamhi and hindiii, and using pcr, we showed that the coding region of the mature tg-hbiia gene was 492 bp, as expected. in the same way, the coding region of the mature tg-hbiib gene was confirmed and was shown to be 483 bp, as expected. the result was also confirmed by sequencing (data not shown). then, the pet30a/tg-hbiia and pet30a/tg-hbiib were introduced into e. coli transetta (de3). to confirm the positive transetta (de3), tg-hbiia and tg-hbiib were amplified using pcr with t7 primers and were then detected by 1% agarose gel electrophoresis. we observed a single band of approximately 750 bp (fig. 1). it was confirmed that the recombinant expression vector was successfully constructed. fig. 1 agarose gel electrophoresis analysis of recombinant expression vector. lane 1, pet-30a/tg-hbiia; lane 2, pet-30a/tg-hbiib; lane m, dna marker. 152 expression and purification of tg-hbiia and tg-hbiib the approach used to produce the recombinant tg-hbiia and tg-hbiib proteins at high levels was to determine the optimal expression conditions by evaluating the induction time and temperature. after induction with iptg, the tg-hbiia gene was successfully expressed, and the highest expression was induced by 1 mmol/l iptg at 37 °c for 5 h (suppl. fig. 1). in the same way, the highest expression of tg-hbiib was induced by 1 mmol/l iptg at 28 °c for 4 h (suppl. fig. 2). the largest amounts of tg-hbiia and tg-hbiib were found in the precipitate when analyzed by sds-page analysis, after the samples were ultrasonicated. this result showed that they were mostly in insoluble inclusion bodies. to isolate the tg-hbiia and tg-hbiib, bacterial cells were collected and disrupted using ultrasonication. the inclusion bodies were obtained by centrifugation and dissolved in the solubilization buffer. the cell debris was removed by centrifugation, and the solubilized protein was purified by the histrap ff column in the denaturing condition. after purification and sds-page, single bands of the tg-hbiia and tg-hbiib at approximately 24 kda and 25 kda, respectively, were observed (fig. 2). to further determine the components of the proteins, mass spectrometry was performed. the results see figure 3 and in supplementary figure 3. the concentrations of tg-hbiia and tg-hbiib were 3.26 mg/ml and 3.71 mg/ml, respectively, as determined by the bradford protein assay. exogenous gene expression in e. coli is affected by many factors, such as induction temperature, iptg concentration, inducing time and genetic structure (lilie et al., 1998). experiments found that the expression of the vectors pet30a/tg-hbiia and pet30a/tg-hbiib was mainly fig. 2 sds-page analysis of purified recombinationt hemoglobins. lane 1, purified recombinationt tg-hbiib; lane 2, purified recombinationt tg-hbiia; lane m, broad range protein molecular weight markers affected by inducing time. although the expression of exogenous genes in e. coli is one of the most economic and convenient expression systems, the greatest obstacle is the inability to correctly fold recombinant proteins and form active proteins. gradient dialysis, via the ultrafiltration centrifugal method, can induce recombinant protein folding in vitro but this method is unable to restore the original fig. 3 the mass spectrogram of recombinationt hbiia from tegillarca granosa. 153 table 2 antibacterial activity of recombinant hemoglobin from tegillarca granosa bacteria gram the minimum inhibition concentration (mic) (g/ml） tg-hbiia tg-hbiib v. harveyi 65.8 158 p. putida 4.11 39.5 a. baumanii --79 e. coli ---- v. parahaemolyticus ---- v. alginolyticus ---- s. aureus ---- b. subtilis + ---- s. epidermidis + ---- b. firmus + ---- level (mayer and buchner, 2004). if the recombinant proteins were solubilized as inclusion bodies and refolded in the right process, then they would have biological activity (sings and panda, 2005). the recombinant proteins (tg-hbiia, tg-hbiib) in dialysis renaturation required the addition of a slight excess of heme in proportion. heme is an important component of hb, but it is also the active center of hb. without adding the heme, the protein could not be folded into its normal active protein form (perutz, 1979). each hb subunit needs one auxiliary heme. the fe2+ of the heme is oxidized easily in the renaturation process, but it may reduce the activity of the recombinant protein. additionally, heme was dissolved in ethanol. therefore, the excess heme and the residual urea also likely affected the protein activity. it was found that when 1 mg of recombinant protein was added to 1 mg heme, the result of renaturation was good. protein renaturation is a complex process that is affected by many factors.the protein spatial structure needs to be formed slowly, so the urea should be diluted gradually (vallejo and rinas, 2004; yu and tao, 2007). to prevent protein denaturation, the entire process should be performed at 4 °c. antibacterial activity of the recombinant protein the results of the bacteriostatic experiment are shown in table 2. we found that recombinant tg-hb exhibits great differences in its effects on various bacteria. the recombinant proteins have no antibacterial activity against s. aureus, e. coli, b. firmus, b. subtilis, s. epidermidis, v. parahaemolyticus or v. alginolyticus. the antibacterial activity of the recombinant proteins to v. harveyi, p. putida and a. baumanii is shown in figure 4. in the previous study, the tg-hblla and tg-hbllb had different amino acids, but they had similar tertiary structure, like 8 alpha helix. we also found that the recombinant proteins without heme have no antimicrobial activities in the pre experiment. and the potential heme binding sites of tg-hblla, tg-hbllb and s. inaequivalvis hbii were highly conserved, indicating that the function of hb play mainly related to its tertiary structure and function domain(jr et al., 1994; jr et al., 1995; wang, 2012). compared with the previous study, we found that the effects of the recombinant proteins on antibacterial activity on p. putida, v. parahaemolyticus and s. aureus are similar to those of natural tg-hbii (wang et al., 2014). this result indicated that recombinant fig. 4 the inhibition zone of purified recombinationt hemoglobin from tegillarca granosa. a, v. harveyi; b, p. putida; c, a. baumanii. 154 proteins are inducible and have high antibacterial activity against gram-negative bacteria, similar to natural protein. there are also some differences. the nature tg-hbii heterotetramers obtained by gel exclusion chromatography was exhibited antibacterial activity against e. coli, b. subtilis, b. firmus, and had no antibacterial activity against v. harveyi, a. baumanii (bao et al., 2013; bao et al., 2016). the tg-hbii was an a2b2 heterotetramers formed by tg-hbiia and tg-hbiib. the interaction between tg-hbiia subunit and tg-hbiib subunit may lead to different antibacterial results. in the previous study, the expression of tg-hbiia and tg-hbiib mrna was markedly upregulated after v. parahaemolyticus, but the recombination hb had no antibacterial activity against v. parahaemolyticus. we speculate that polypeptides from tg-hblla and tg-hbllb by enzymatic hydrolysis can resist v. parahaemolyticus (wang et al., 2015). in addition, that the hemolytic effects of the hemolysins produced during v. parahaemolyticus infection are responsible for enhanced hematopoeisis (honda and iida, 1993). our results also confirmed that protein with antibacterial activity can be expressed and assembled in e. coli (de-la-re-vega et al., 2006; zhao et al., 2007; mai et al., 2009). so far, most have focused on the natural hb and its peptides antibacterial research, while the recombinant hb and its peptides is less. niu and chen (2016) reported that recombinant hb from urechis unicinctus has antibacterial activity against s. aureus, m. luteus, e. coli, and v. parahaemolyticus. and their results are different from ours, which indicated that the antibacterial mechanism of different sources of hb may be different. at present, the antibacterial mechanism of hb is unclear. though hb has been found capable of producing ros and of exerting pseudoperoxidase and deoxygenase activities, which are involved in host defense (adachi et al., 2003; cheng et al., 2011; goodarzi et al., 2014). jiang et al. (2007) showed that human hb is oxidized to ferric iron when stimulated by virulence factors, resulting in a superoxide anion that can produce toxic derivatives such as hydroxyl radicals and hypochlorous acid and has a bactericidal effect. wang et al. found that purified tg-hbs catalyzed the oxidation of several phenol compounds in the presence of h2o2, with high affinity to guaiacol. and the predicted structure at their heme pocket was highly similar to that of horseradish peroxidase (hrp) and myeloperoxidase (mpo). it indicated that tg-hbs may function as peroxidase in the clam’s hemocytes. the authors considered that the antibacterial effect may be generated by the peroxidase activity (wang et al., 2014; wang et al., 2017). the heme moiety of hb can either act as an iron chelator or an oxidant, leading to bacterial and fungal cell wall damage (katsu et al., 2010). the diversity of antimicrobial activity of tg-hbs might be due to their antibacterial mechanism. the research of antibacterial mechanism of nature and recombinant tg-hb is under way. acknowledgments this research was supported by the national science foundation of china (31672678), natural science foundation of ningbo (2015a610258), zhejiang province public technology applied research projects (2016c33090), zhejiang major program of science and technology (2016c02055-9), and zhejiang natural science foundation (lq15c190001). references adachi k, hirata t, nishioka t, sakaguchi m. hemocyte components in crustaceans convert hemocyanin into a phenoloxidase-like enzyme. comp. biochem. physiol. 134b: 135-141, 2003. bao y, wang j, li c, li p, wang s, lin z. a preliminary study on the antibacterial mechanism of tegillarca granosa hemoglobin by derived peptides and peroxidase activity. fish shellfish immun. 51: 9-16, 2016 bao y, wang q, lin z. hemoglobin of the bloody clam tegillarca granosa, (tg-hbi) is involved in the immune response against bacterial infection. fish shellfish immun. 31: 517-523, 2011. 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https://www.ncbi.nlm.nih.gov/pubmed/?term=dhiravisit%20a%5bauthor%5d&cauthor=true&cauthor_uid=22648692 https://www.ncbi.nlm.nih.gov/pubmed/?term=thammasirirak%20s%5bauthor%5d&cauthor=true&cauthor_uid=22648692 156 suppl. fig. 1 sds-page analysis of recombinant tg-hbiia. lane 1, non-induced; lane 2, after induction by 37 °c, 2h; lane 3, after induction by 37 °c, 3 h; lane 4, after induction by 37 °c, 4 h; lane 5, after induction by 37 °c, 5 h; lane m, broad range protein molecular weight markers. suppl. fig. 2 sds-page analysis of recombinant tg-hbiib. lane 1, non-induced; lane 2, after induction by 28 °c, 3 h; lane 3, after induction by 28 °c, 4 h; lane 4, after induction by 28 °c, 5 h; lane 5, after induction by 28 °c, 6 h; lane m, broad range protein molecular weight markers. suppl. fig. 3 the mass spectrogram of recombinationt hbiib from tegillarca granosa. (az4)stem cells are defined as the cells not only can self-renew and produce daughter cells devoting to differentiate for ages,but also repair injury and maintaining cellular homeostasis.(az10)moreover,they use two main strategies for division,including isj 11: 103-108, 2014 issn 1824-307x minireview mechanisms of asymmetric cell divisions in drosophila melanogaster neuroblasts x jiang, c tang, h gao, h cui state key laboratory of silkworm genome biology, southwest university, chongqing 400716, p.r. china accepted april 4, 2014 abstract stem cells possess the properties of self-renewal and differentiation, and mainly rely on two strategies for division, including symmetric and asymmetric cell divisions. in this review, we summarize the latest progress on asymmetric cell divisions in drosophila melanogaster neuroblasts (nbs), which focus on the establishment of cell polarity, mitotic spindle orientation, the asymmetric segregation of cell fate determinants as well as cell-cycle control. here we also introduce five major cell fate determinants, including numb, prospero, brat, miranda, and pon, which are thought to be unequally segregated to the ganglion mother cells (gmcs) and play an important role in the formation of stem cell-derived tumors. key words: asymmetric cell divisions; cell fate determinants; neuroblasts; drosophila melanogaster introduction stem cells are defined as the cells which not only undergo self-renewal but also produce daughter cells devoting to differentiation for all ages (sada and tumbar, 2013). moreover, they employ two main strategies for division including symmetric and asymmetric cell divisions. both of them can regulate the stem cell and the differentiated cell population (sousa-nunes and somers, 2013). symmetric cell divisions produce two uniform daughter cells. however, asymmetric cell divisions lead to one daughter cell destined to differentiate and one stem cell, the latter is crucial for the development of multicellular organisms (yamashita, 2009). recently, comprehensive research on the mechanisms of asymmetric cell divisions has been done. specifically, drosophila melanogaster sensory organ precursor (sop) cells and neuroblasts (nbs) provide excellent models for studying the cellular and molecular mechanisms of asymmetric cell divisions (rusan and rogers, 2009). in contrast to sop cells, most nbs are derived from the embryonic procephalic and ventral neuroectoderm (wu et al., 2008). during embryonic neurogenesis, nbs delaminate from the epithelium ___________________________________________________________________________ corresponding author: hongjuan cui state key laboratory of silkworm genome biology institute of sericulture and systems biology southwest university #216, tiansheng rd., beibei district chongqing 400716, china e-mail: hcui@swu.edu.cn and undergo repeated rounds of asymmetric division to self-renew and to produce one proliferating nb and one small ganglion mother cell (gmc) that terminally differentiates (sawa, 2010). this process is strictly controlled, and disrupting it can lead to both uncontrolled proliferation and aberrant differentiation. nbs restricted self-renewal capacity also limits their usefulness as a true stem cell model. for this reason, we primarily focus on nbs. during asymmetric cell divisions, it has been proposed that nbs depend on external environmental factors and internal regulations (sada and tumbar, 2013). both of them could work together or independently (yamashita, 2009). for extrinsic asymmetric divisions, stem cells reside in microenvironment, which is called the niche (sada and tumbar, 2013). the niche is used to maintain the stem cells identity and proliferation. during stem cell divisions, the cell fate appears to be determined solely by their localization (yamashita, 2009). in general, there is only one daughter cell maintaining the stem cell identity in the niche area. for intrinsic asymmetric divisions, cell fate determinants are asymmetrically segregated into two daughter cells in mitosis. thus, one cell undergoes differentiation and the other one keeps the stem cell identity (roegiers and jan, 2004; knoblich, 2008). in most cases, there are four steps in the intrinsic asymmetric divisions (fig. 1). namely, the mother cell sets up an axis of asymmetry during the interphase of cell division, following by the cell polarized. after that, the cell fate determinants, such as numb, prospero, brat, miranda, or pon, are segregated towards the regions of the polarized 103 mailto:hcui@swu.edu.cn fig. 1 intrinsic asymmetric division in d. melanogaster nbs. there are four steps in the intrinsic asymmetric division. firstly, the symmetry of nbs is broken during the interphase. secondly, nbs becomes polarized. thirdly, the major cell fate determinants (diamond), such as numb, prospero, brat, miranda and pon are segregated to the basal cortex, and the apical proteins (oval) are located in the apical cortex. fourthly, the mitotic spindle orientation along the cell polarity axis is establishment so that the cell fate determinants are divided into the newly forming gmc which can generate two neurons. mother cell. finally, the mitotic spindle orientation along the cell polarity axis is established, so that cell fate determinants can been separated into the daughter cells. due to these four steps, one mother cell can generate two daughter cells which own different cell fates (gonczy, 2008). here, we summarize the recent progress on asymmetric cell divisions in d. melanogaster nbs and describe the mechanism which regulates asymmetric nbs divisions during development. we also focus on the establishment of cell polarity, the regulation of mitotic spindle orientation, and the asymmetric segregation of cell fate determinants as well as cell-cycle control (wu et al., 2008). establishment of cell polarity the central nervous system of d. melanogaster originates from the basal delamination of the surface neurectoderm, which inherits its epithelial apical-basal (a-b) polarity (gonczy, 2008). while the cell polarity is established by the asymmetric accumulation of the par complex, including baz (d. melanogaster homologue of c. elegans par-3), par-6, as well as the atypical protein kinase c (apkc) to form a crescent at the apical cell cortex (haenfler et al., 2012; chen and zhang, 2013), which is evolutionarily conserved. the par complex is originally expressed in the neuroectoderm, while keeping in the nbs after delamination (wu et al., 2008). baz is a potential component of the apical organizer containing three pdz domains, which can also interact with apkc. following nbs delaminating, baz is co-localized with inscuteable (insc) to the crescent at the apical cell cortex (matsuzaki, 2000). however, not only baz but also insc is undetectable during late mitosis in every cell cycle. as a small protein, par-6 contains one pdz domain and a n-terminal pb1 domain, which binds to a similar domain on the apkc (knoblich, 2008). furthermore, baz recruits cdc42, which in turn binds to the semi-crib domain of par-6 (goldstein and macara, 2007). the cdc42 is a small gtpase which is vital for par-6 localization (atwood et al., 2007). the 104 three compotents of par complex also recruit insc, pin (the adapter protein partner of insc), and gαi (a subunit of heterotrimeric g protein) to the neuroectoderm, which are required for a-b polarity. their apical localization is maintained during nbs delamination, and the complex is preferred to divide into the daughter cell which localizes in the apical cortex. while baz, par-6, and apkc are closely related, any one mutanted can cause the other proteins delocalized. in addition, they also direct the cell fate determinants to the basal gmcs (knoblich, 2008). thus, the par complex is crucial for asymmetric cell divisions since it provides essential positional information for cell division. spindle orientation in general, nbs are arose from neuroectodermal cells and divided perpendicularly to the epithelial plane with a horizontal mitotic spindle axis (wu et al., 2008), so their mitotic spindle rotates 90 degree. thus nbs may undergo repeated division along the a-b axis (kaltschmidt and brand, 2002). spindle orientation needs to be done with the involvement of the apically localized molecules as well as cell polarity establishment, any one of the molecules is indispensable, especially the protein insc. insc is undetectable in the neuroepithelial cell layer during each nbs cell cycle, and becomes detectable during nbs delamination and associates with the par complex through baz, pin, and gαi protein (cai et al., 2003). moreover, insc plays a sufficient role in spindle orientation, deletion of insc or baz leads to the randomization of the mitotic spindle and cell fate determinants delocalized in the basal crescent. nevertheless, ectopic expression of insc in the neuroepithelial cell layer can cause spindle move 90 degree (siller and doe, 2009). as an adapter protein, insc links the par complex to a tripartite protein complex gαi-pins-mud, which regulates nbs spindle orientation. it associates with the dynein-dynactin complex, directs the orientation of the mitotic spindle and coordinates with the a-b polarity axis, which plays an important role in recruiting and keeping one centrosome at the apical pole (saini and reichert, 2012). moreover pins-dlg-khc73 complex also regulates the spindle orientation. losing a functional par complex can active dlg-pins-gαi complex formation in embryonic nbs, as a result of the interaction between astral microtubules and khc73. interestingly, down-regulation of dlg or khc73 induces partial spindle-orientation defects without affecting apical pins-gαi cortical polarity (siegrist and doe, 2005). however the regulation mechanism is not clear yet. more data show that the accurate spindle alignment is required for normal nbs or gmcs fate. while mud mutant induces either spindle orientation defects or additional nbs numbers with normal cortical polarity. the transverse spindle causes symmetric divisions but not asymmetric divisions and generates two nbs but not gmcs any more (homem and knoblich, 2012). cell fate determinants the cell fate determinants such as numb, prospero, brat as well as their adapter proteins miranda and pon assemble in the basal cortical domain before the cytoplasm division, and then segregate into the basal gmcs (fig. 2) (paridaen and huttner, 2014). next we discuss those cell fate determinants in d. melanogaster nbs below. numb numb is the first defined asymmetrically partitioned determinant, which also belongs to a clathrin-associated sorting protein (clasp). recently it has been shown that numb contains two interaction domain including phosphotyrosine binding (ptb) domain and c-terminus (spana and doe, 1996). numb can be recruited and phosphorylated by the par complex. the phosphorylation state of numb determines that it forms a crescent on the basal cell cortex of the nbs, and finally it is excluded from the cortex (egger et al., 2007). numb also works as a tissue-specific component of the notch pathway. therefore, numb transfers intracellular signal to regulate cell divisions are related to different levels of notch activity (couturier et al., 2013). in general, numb reduces or blocks the activity of notch pathway through binding the endocytic protein α-adaptin, which is a component of the adapter protein-2 (ap2) complex that functions in clathrin-mediated endocytosis of transmembrane proteins (tajbakhsh et al., 2009). some studies reveal that numb interacts with the ear domain of α-adaptin, and they locate identically. furthermore, α-adaptin acts as an upstream factor or in parallel with notch pathway. in α-adaptin mutant, it blocks the interaction with numb and no longer locates asymmetrically, which is similar to the numb mutant that nbs overproliferate and form a tumor-like phenotype (ntelios et al., 2012). prospero homeodomain transcription factor prospero is another pivotal regulator in asymmetrically divisions (choksi et al., 2006). prospero is detected in all embryonic and larval nbs, which congregates in the cytoplasm. it translocates to the basal cell cortex and forms a cresent pattern at one end of the cell during cell divisions, rather than distributes evenly (matsuzaki, 2000). it migrates from the cortex into the nucleus, where it establishes the neural fate of the cell. in the end, prospero asymmetrically divides into one of the two progeny called gmcs. therefore, the gmcs have a different fate from that of its sister called nbs. moreover, prospero is an essential regulator of the gmcs development, which binds more than 700 genes. prospero binds to a majority of the temporal cascade genes, including kruppel (kr), nubbin (nub/pdm1), or grainyhead (grh), which regulate the timing of cell fate specification in nbs progeny. interestingly, prospero regulates baz, miranda, insc, as well as apkc, which directly regulate asymmetric nbs divisions. prospero also suppresses those genes which are required for stem cell self-renewal, as well as cell cycle related genes including cyclins a or e or string (the d. melanogaster homologue of cdc25) (choksi et al., 2006). when prospero is mutated in nbs, the cell cycle related genes are up-regulated, nbs undergo multiple rounds of division, fail to differentiate and induce stem cell-derived tumors (knoblich, 2008). 105 fig. 2 asymmetric cell divisions in d. melanogaster nbs. during the nbs mitosis, the apical apkc-par3-par6 complex (dark gray) is linked to the gαi-pins-mud complex (pale gray) through inscuteable. all of the proteins are involved in the asymmetric partitioning of cell fate determinants, establishment of cell polarity and the spindle orientation. while, the determinants (black), including miranda (mira), prospero (pros), staufen (stau), brat, numb, and pon are concentrated at the basal cortex. finally, most of the basal determinants are segregated into the basal daughter cell called the gmc. brat brain tumor (brat) encodes a member of the conserved c-terminal nhl family of proteins, including ncl-1, ht2a, and lin-41 (saini and reichert, 2012). apart from the nhl domain, brat is also characterized by a coiled-coil region and an n-terminal zinc binding b-box, which mediates its recruitment to the 3'utr of posterior identity gene hunchback (hb) through pumilio (pum) and nanos (nos) during early embryogenesis to repress translation of the hb (slack et al., 1998). brat is also considered as a transcriptional activator of prospero, and pros/brat mutants show complete loss of all gmcs by reason of that they corporately regulate gmc fate (betschinger et al., 2006). brat mutants have reduced prospero expression and show uncontrolled proliferation in the larval central brain which may induce tumor development (bello et al., 2006). miranda and pon except the three crucial regulators of nbs self-renewal, there are two important adapter proteins including miranda and pon. miranda is identified as a double-headed, double-tailed homodimer with a long central coiled-coil region (residues 150 700) flanked by non-coiled-coil n and c termini. the double-heads is essential for increasing avidity for specific binding partners (yousef et al., 2008). some reports suggest that miranda may interact with not only brat but also the rna binding protein staufen which acts as the prospero-mrna carrier. during mitosis, miranda localizes asymmetrically and colocalizes with prospero to the basal cell cortical crescent. at the end of telophase, both of them divide into gmcs. unlike prospero, miranda fades away shortly after cytokinesis in gmcs. for that reason, it is possible that miranda is degraded in a cell-cycle-dependent manner and its degradation is a consequence of cargo proteins release (shen et al., 1997). in addition, the extreme c-terminal 103 amino acids (residues 727 830) of miranda, which also contains multiple consensus apkc phosphorylation sites, are found to be crucial for those processes (yousef et al., 2008). the asymmetric localization of miranda depends on polarized baz activity and has no related with pins-gαi function. moreover, baz regulates localization of apkc to make miranda 106 http://www.sdbonline.org/fly/segment/hunchbk1.htm http://www.sdbonline.org/fly/gene/pumilio.htm http://www.sdbonline.org/fly/torstoll/nanos1.htm migrate from the apical cell cortical crescent to the basal through lethal (2) phosphorylation (izumi et al., 2004). anaphase-promoting complex/cyclosome (apc/c) also can regulate the asymmetric localization of miranda, as well as its interrelated cargo proteins including prospero, brat, and staufen. several apc/c core subunits mutants displayed that miranda is transferred from cortex to cytoplasm (slack et al., 2007). pon is a coiled-coil protein, which is not essential for its cargo protein, but pon assists to establish the a-b polarity. as an important component of a multimolecular machinery, pon colocalizes with numb (lu et al., 1998), knockdown of pon postpones numb’s crescent formation in metaphase, which may lead to a defect in nbs self-renewal (wang et al., 2008). cell-cycle control recent published data provide an evidence that cell cycle regulators can affect the asymmetric nbs divisions, cdc2/cdk1, aurora a, polo, cyclin e, and apc/c core components take action in pro-metaphase and metaphase. those regulators mutants directly affect asymmetric cell fate determinants’ localization (reichert, 2011). cdc2/cdk1 is essential for driving cells enter into mitosis, and cells lacking of cdk1 activity cause cell cycle arrest in the g2 phase (reichert, 2011). aurora a is a centrosomal kinase which can cause par6 phosphorylated and apkc auto-phosphorylated, and which reaches a peak in the early mitosis (barr and gergely, 2007). as another centrosomal kinase, polo also regulates cell cycle which is similar to aurora a. both of them are required for a subset of mitotic events, including centrosome maturation, spindle formation and orientation, as well as cytokinesis (knoblich, 2008). moreover, they also can act as tumor suppressors to prevent excess self-renewal (reichert, 2011). cyclin e regulates the g1 to s-phase transition (chia et al., 2008). it is necessary for converting symmetric nbs divisions into asymmetric divisions through downstream of hox genes (berger et al., 2005). during asymmetric divisions, cyclin e may inhibit prospero and facilitate its cortical localization in the nbs, which is closely related with its self-renewal during asymmetric divisions (berger et al., 2010). apc/c, in transient association with the activating subunits cdc20 and cdh1, can promote cell cycle transitions through several key processes including regulation of dna replication, centrosome duplication and mitotic spindle assembly as well as the mitotic cyclins disfunction and inhibition of chromosome separation (leismann and lehner, 2003). apc/c also can control axon growth and regulate synaptic size and transmission (juo and kaplan, 2004). concluding remarks d. melanogaster nbs provide a well-studied model system for illustrating the cellular and molecular mechanisms of asymmetric cell divisions. in this review, some viewpoints have been made to describe the establishment of cell polarity, the orientation of mitotic spindle, the asymmetric segregation of cell fate determinants, as well as cell-cycle control. here we focused on five major cell fate determinants, including numb, prospero, brat, miranda, and pon, which are thought to be unequally segregated to one of the two daughter cells. all or most of them relate with the formation of stem cell-derived tumors, however, the regulatory mechanism of this process is not clear yet. understanding of asymmetric cell divisions in d. melanogaster nbs provides some clues for stem cell behaviors, stem cell therapy, as well as cancer 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division in drosophila. protein sci. 17: 908-917, 2008. 108 death for survival: what do we know about innate immunity and cell death in insects isj 8: 162-172, 2011 issn 1824-307x minireview death for survival: what do we know about innate immunity and cell death in insects? dm cooper1,2, k mitchell-foster3 1institute for heart and lung health, st. paul's hospital, vancouver, british columbia, canada 2department of pathology and laboratory medicine, university of british columbia, vancouver, british columbia, canada 3 global health research program, university of british columbia, canada accepted august 16, 2011 abstract insects are the most diverse and prolific animal group on earth, and as such, important lessons can be taken from the elements that contribute to their evolutionary success. this review examines insect immunity and how insects combat infection with the pathogens they encounter: bacteria, viruses, fungi and parasites. structural barriers, cellular and humoral responses and cell death all respond to specific immunological threats and contribute to the robust repertoire of immune strategies employed by insects. we discuss the strategies used by insects to combat pathogen infection and focus on what is currently known about cell death and its role in insect immunity. key words: insect immunity; innate immunity; immune signalling; cell death; apoptosis; autophagy introduction over their 400 million years of evolution, insects have become the most diverse and prolific animal group to inhabit land. there are more than one million species of insects identified to date, with the true number of species estimated at 5 million (grimaldi and engel, 2005). insects play important roles in nearly all areas of terrestrial and freshwater ecosystems; they are pollinators, aid in decomposition and redistribution of nutrients, are custodians of forests and soil, and are vectors of human, animal and plant disease. the incredible diversity and evolutionary success of insects is due, in no small part, to robust defences against infection. the niches insects occupy bring them into intimate contact with pathogens; bacteria, fungi, viruses and parasites. of particular importance, insects transmit some of the most devastating human and livestock diseases. in 2008, worldwide cases of malaria, transmitted by anopheles mosquitoes, were estimated at 190-311 million with over one million deaths. dengue fever transmitted by aedes mosquitoes, leishmaniasis transmitted by phlebotomine sandflies, onchocercaiasis, african trypanosomiasis, and chagas’ disease all take an incredible toll on human ___________________________________________________________________________ corresponding author: dawn cooper institute for heart and lung health, st. paul’s hospital university of british columbia 166-1081 burrard street, vancouver, british columbia, canada, v6z 1y6 e-mail: dawn.cooper@hli.ubc.ca health, economic growth and development on a global level each year (dias et al., 2002; gubler, 2002; kabayo, 2002; frick and foster, 2003; desjeux, 2004; sharma et al., 2006). how insects combat infections and more importantly, survive the pathogen loads required to transmit human disease, has been the focus of much research. here we review insect survival strategies from structural barriers to infection, to cellular and humoral responses, and focus on cell death and its role in insect immunity. insect immunity: a general overview insects owe much of their success to a potent immune response that effectively combats a broad range of pathogens and plays a key role in mediating the interactions between pathogen and insect host. in insects, the first line of defence consists of structural barriers: the exoskeleton or cuticle, the peritrophic matrix, midgut epithelium and the chitinous lining of the trachea (lowenberger, 2001; hoffmann, 2003). once past these barriers, pathogens face a diverse repertoire of immune defences; cellular responses such as hemocytemediated responses (i.e., phagocytosis, melanization, and encapsulation) and humoral responses that include the production of reactive oxygen species and antimicrobial peptides (amps) (naitza and ligoxygakis, 2004; tanji and ip, 2005; ferrandon et al., 2007). cellular and humoral immune responses are triggered once a pathogen has been identified as non-self. in insect vectors this 162 mailto:dawn.cooper@hli.ubc.ca fig. 1 the toll pathway in d. melanogaster. the toll pathway is activated by fungi and gram-positive bacteria. toll signaling is activated by the binding of a processed form of the cytokine spätzle to the toll receptor. during infection, soluble receptors (pgrp-sa, pgrp-sd, gnbp1, gnbp3) recognize pathogens and trigger proteolytic cascades involving the serine proteases persephone and spe (spätzle processing enzyme). spe ultimately targets and cleaves spätzle. once bound to the toll receptor, signalling through toll requires the formation of the tisc complex (toll induced signalling complex), composed of three death domain containing proteins, myd88, tube and pelle. by a mechanism still unclear, cactus [a homologue of the mammalian inhibitor of nf-κb (iκb)], is phosphorylated, poly-ubiquitinated and targeted for degradation. once released, the rel transcription factor dif (dorsal-related immunity factor) enters the nucleus and binds to nf-κb response elements and induces the transcription of genes encoding antimicrobial peptides. process is mediated by pattern recognition receptors (prrs) that bind to structures on the surface of pathogens referred to as pathogen associated molecular patterns (pamps). several families of proteins are believed to be involved in distinguishing different classes of pathogens and stimulating appropriate immune responses. prrs recognize a limited but conserved set of pamps that include bacterial lipopolysaccharides (lps), peptidoglycan (pgns), lipotechoic acids (lta), mannans, and fungal 1,3glucans (royet, 2004a, b; warr et al., 2008). once prrs recognize invaders as non-self, they trigger a variety of defence reactions that either mediate pathogen killing directly through phagocytosis and melanization, or indirectly through the activation of proteolytic cascades and signaling pathways that control the expression of immune effector genes. the most rapid immune responses include the direct phagocytosis of microbial pathogens by hemocytes and the activation of the prophenoloxidase activating cascade that leads to the melanization of invading microorganisms (lowenberger, 2001). shortly thereafter, effector molecules such as anti-microbial peptides (amps) can be detected in the hemolymph. amps are a powerful component of the insect immune response; they are rapidly produced and have activity specific to a given class of pathogens. amps are synthesized by the fatbody, epithelia in the midgut or trachea, and hemocytes, and are regulated principally by the toll and immune deficiency (imd) signalling pathways (khush et al., 2001; lowenberger, 2001; ferrandon et al., 2007). immune signaling pathways in general, innate immunity in insects is governed by signaling pathways that trigger amp production, phagocytosis, encapsulation and 163 melanization. drosophila melanogaster has traditionally been the main model organism for studies of invertebrate immunity and knowledge gained from these studies has been applied to other invertebrate systems. in d. melanogaster, the production of amps is regulated either by the toll or imd (immunodeficient mutant) pathways (silverman and maniatis, 2001; hoffmann, 2003; ferrandon et al., 2004, lemaitre and hoffmann, 2007). the toll pathway regulates the responses to both fungi and gram-positive bacteria, and initiates the melanization cascade and blood cell proliferation (fig. 1). activation of the toll pathway during septic challenge triggers two d. melanogaster nf-κb transcription factor homologs, dorsal and dif, and the production of two amps, drosomycin and metchnikowin (ferrandon et al., 2007, lemaitre and hoffmann, 2007). the imd pathway is activated primarily by gram-negative bacteria, as well as a few gram-positive bacteria and fungi, and results in the induced expression of the amps, cecropin, drosocin, defensin and diptericin (kaneko and silverman, 2005, tanji and ip, 2005) (fig. 2). some immune-induced genes, such as diptericin, are dependent on a single signaling pathway, but others can be induced by both cascades suggesting interactions between the toll and imd pathways during septic challenge. the signaling cascades that control toll and imd-mediated transcription of immune effector genes are important mediators of immune responses against extracellular pathogens, such as bacteria and plasmodium (meister et al., 2005; vlachou et al., 2005; dong et al., 2006) that exist transiently in insect hemolymph. the ability of these pathways or others to mediate infections caused by intracellular pathogens, such as viruses are less clear. there is mounting evidence that d. melanogaster and mosquitoes activate signaling pathways that lead to the expression of a distinct set of genes when faced with viral infection. genomewide screens of d. melanogaster infected with drosophila c virus (dcv) revealed approximately 150 genes are induced 24 48 h after infection and many of these genes are not induced by challenge with bacteria or fungi (sabatier et al., 2003). this suggests that flies, and likely mosquitoes, use distinct strategies to counteract intracellular pathogens. the toll, imd and janus kinase-stat (jak-stat) signaling pathways have all been implicated in response to virus infection (harrison et al., 1998; hu et al., 2000; agaisse and perrimon, 2004; gilbert et al., 2005; smartt et al., 2009; ramirez and dimopoulos, 2010; luplertlop et al., 2011). while the jak-stat pathway appears to be involved in antiviral immunity, jak-stat responsive genes are not induced in immune responsive tissues or virus-infected tissues, only in uninfected and non-immune tissues (dostert et al., 2005). this suggests that the jak-stat pathway may represent a cytokine-mediated signaling cascade involved in communicating with non-infected cells (beutler et al., 2007). the induction of toll and jak-stat signalling pathways have also been reported in mosquitoes following infection with japanese encephalitis virus and in shrimp infected with white spot syndrome virus (lin et al., 2004; thoetkiattikul et al., 2005; georgel et al., 2007). one of the more established antiviral mechanisms in insects is rna interference (rnai). rnai is an ancient form of defence activated by dsrna to trigger host-mediated degradation of viral rna. components of the rnai pathway are conserved in fruit flies and mosquitoes where they have been reported to play a role in antiviral immunity. in d. melanogaster, rnai deficient mutants are highly susceptible to several viruses including dcv, cricket paralysis virus, flock house virus, drosophila x virus (dxv) and sindbis virus (galiana-arnoux et al., 2006; van rij et al., 2006; wang et al., 2006). similar trends have been observed in mosquito c6/36 cell lines and adult aedes aegypti challenged with dengue virus specific dsrna prior to infection with dengue virus (gaines et al., 1996; adelman et al., 2002; caplen et al., 2002; olson et al., 2002; sanchez-vargas et al., 2004). the same pattern was demonstrated for alphavirus infections in anopheles gambiae, and keene et al. (2004) showed that o’nyong nyong virus (onnv) replication was significantly reduced when the virus was co-injected with dsrna derived from the viral genome (keene et al., 2004). cell death and the immune response cell death is a key process that tailors hostpathogen interactions. intracellular infections, particularly viral infections, provoke a dynamic interaction between the host immune system and pathogen immune avoidance strategies to create a suitable environment for pathogen replication. it is difficult for a pathogen to infect a cell without activating any number of immune pathways, including cell death pathways (clouston and kerr, 1985; everett and mcfadden, 1999). cell death plays an important role in all multicellular animals; it is involved in the formation and deletion of structures during development, maintenance of tissue homeostasis, and eliminating abnormal or damaged cells. three forms of cell death have been defined based on the morphology of dying cells. type i programmed cell death, more commonly known as apoptosis, is an intrinsic form of cell death defined by nuclear condensation and fragmentation, dna laddering (200 bp fragments), blebbing or rounding of the cell, and the externalization of phosphatidylserine (thornberry and lazebnik, 1998; aravind et al., 2001; kornbluth and white, 2005). cells dying by apoptosis often fragment into membrane-bound apoptotic bodies that are readily phagocytosed and digested by macrophages or by neighbouring cells without generating an inflammatory response. apoptotic cell death requires energy in the form of atp (edinger and thompson, 2004). type ii, or autophagic cell death, is a process that denotes self-cannibalization through a lysosomal degradation pathway (edinger and thompson, 2004; levine and deretic, 2007; levine and kroemer, 2008). cells undergoing autophagic cell death are characterized by the presence of autophagic vacuoles (autophagosomes) and autophagolysosomes. autophagic cell death occurs primarily 164 fig. 2 the imd pathway in d. melanogaster. the imd pathway is activated in response to gram-negative bacteria. peptidoglycan recognition receptors-lc and –le (pgrp-lc, pgrp-le) recognize dap-type peptidoglycans (dap-type pgn) associated with gram-negative bacteria and activate imd through the recruitment of protein, imd. imd interacts with the adaptor, dfadd, which itself interacts with the caspase dredd. dredd, by an unknown mechanism, is involved in the cleavage of relish after phosphorylation by the ikk complex, which itself is thought to be activated by dtak1 and the adaptor dtab2 in an imdand dfadddependent manner. the ring domain of diap2 may be involved in the activation of dtab2. following relish cleavage, the rel domain translocates to the nucleus to induce the transcription of several genes, including those encoding antimicrobial peptides. dashed lines represent mechanisms of unknown function. when the developmental program requires massive cell elimination but also may be used to degrade intracellular bacteria and viruses (levine and kroemer, 2008). this process occurs independent of phagocytes. lastly, type iii cell death, often referred to as necrosis, involves the swelling of organelles, lysosome-independent vacuolation of the cytoplasm, and breakdown of the plasma membranes (fiers et al., 1999; proskuryakov et al., 2003; edinger and thompson, 2004). apoptosis as an immune response apoptosis is a process inherent to all eukaryotic cells and has been highly conserved through evolution (raff, 1998; hengartner, 2000; kaufmann and hengartner, 2001). apoptosis is an active process that is initiated by the cell and governed by gene activities that either induce or inhibit cell death. pro-death stimuli are either generated by neighbouring cells or originate within doomed cells. in all cases, a death stimulus will activate a cellular sensor that transmits a signal to downstream proteins, which initiate the appropriate response. the end point of the cell-death signaling cascade is the activation of an evolutionarily conserved family of cysteine proteases called caspases (thornberry and lazebnik, 1998; aravind et al., 2001; kornbluth and white, 2005). in mammals, apoptosis is triggered by death signals that result in the activation of initiator caspases and the subsequent activation of downstream effector caspases. activity of proand anti-apoptotic members of the bcl-2 family of proteins govern caspase activation and the apoptotic process. once initiated, caspase activity is dampened by several mechanisms, principally by the iap family of proteins. although many components of the apoptotic pathways are conserved in insects, the pathways themselves differ slightly between species. much of our current understanding about apoptosis in insects comes from studies in fruit flies. in d. melanogaster there are three initiator caspases (dronc, dredd and strica) and four effector caspases (drice, dcp-1, damm and decay). among these, dronc and drice are the core capases involved in apoptosis with dcp-1 playing a minor role only evident in certain cell types in the absence of drice. in contrast to 165 mammals, where caspase activation serves as the main point of control, cells in d. melanogaster experience chronic activation of the apical caspase dronc (fig. 3). cells survive because they express the inhibitor of apoptosis protein1, diap1 (hay and guo, 2006; steller, 2008). diap1 suppresses dronc activity, and therefore the activity of downstream caspases activated by dronc. the expression of the pro-death rgh proteins (reaper, grim, hid, sickle, jafrac2) disrupt diap1-caspase interactions, resulting in the activation of the caspase cascade (orme and meier, 2009). less is known about cell death signalling in other insects, though a significant amount of research has recently been focused on apoptosis in mosquitoes. fruit flies and mosquitoes are separated by approximately 250 million years of evolution and have been exposed to very different evolutionary pressures. the genome projects of a. gambiae and a. aegypti have revealed that mosquitoes have expanded families of caspases and iaps which may be due in part to exposure to pathogens ingested during blood feeding. although mosquitoes are a relatively new model with respect to cell death, studies indicate that signalling pathways appear to be conserved when compared to d. melanogaster (bryant et al., 2008; cooper et al., 2007a, b, 2009; liu and clem, 2011; wang and clem, 2011). it should be noted that an expansion of caspases has also been noted in other drosophila sp., although roles for these additional caspases have not yet been identified (bryant et al., 2010). as insects lack an adaptive immune response, one would predict apoptosis to play a central role in antiviral and anti-parasite immunity. induction of cell death is a powerful immune response because it has the potential to severely limit pathogen production and to reduce or eliminate the spread of pathogens within the host. the importance of apoptosis as an immune defence mechanism is borne out by the large number of animal viruses that encode proteins specifically to interrupt cell death pathways. to date, more than a dozen viral genomes have been shown to encode proteins that modulate apoptosis (teodoro and branton, 1997; benedict et al., 2002). viruses use various strategies to impede apoptosis; in some cases targeting a single point of the process is sufficient to affect the onset of the cell death program. some viruses interfere with cellular sensors before they trigger apoptotic cell death, some encode gene products that inhibit the sensors of cell cycle manipulations and others target double stranded rna activated protein kinase (pkr) (katze, 1995; teodoro and branton, 1997; barry and mcfadden, 1998; roulston et al., 1999; barber, 2001; benedict et al., 2002; hay and kannourakis, 2002). viruses may also encode gene products that directly block the apoptotic signaling cascade or prevent the activation of caspases. several dna viruses, including adenoviruses and epstein-barr virus (ebv) encode gene products functionally analogous to the anti-apoptotic protein, bcl-2. the cytokine response modifier a (crma) encoded by the cowpox viruses, p35 and iap proteins encoded fig. 3 schematic of the cell death pathway in d. melanogaster. both organisms provide a different paradigm for the induction of apoptosis through caspase activation. in c. elegans, the bh3-only protein, egl-1, is upregulated in cells destined to die. egl-1 is a pro-apoptotic protein that binds to and inactivates the inhibitor protein, ced-9. the release of ced-9 results in the activation of ced-4, which in turn activates the sole death caspase, ced-3. in d. melanogaster, the primary apoptotic caspase is dronc, a caspase-9 homolog with an nterminal card domain for interaction with the apaf1 homolog dark. dronc activation is primarily regulated by the rhg proteins (reaper, hid, grim, sickle, and jafrac2). together, these proteins promote cell death by binding to the inhibitor of apoptosis protein diap1, disrupting anti-caspase activity of diap1. once activated, dronc will cleave and activate the effector caspases drice and dcp-1. no firm role in apoptosis has been established for the d. melanogaster bcl-2 proteins (buffy and debcl). by the baculoviruses, and viral homologues of the cellular apoptosis inhibitor known as cflips all regulate caspase activity (birnbaum et al., 1994; clem and miller, 1994; thome et al., 1997; chaudry et al., 1999). smaller viruses, such as the rna viruses, have developed alternative strategies like inducing the expression of endogenous antiapoptotic genes, including host bcl-2 and iap genes (liao et al., 1997; teodoro and branton, 1997; liao et al., 1998; hay and kannourakis, 2002). in some cases, bcl-2 expression is able to modulate the 166 effects of viral infection and over-expression of bcl-2 allowed for the establishment of persistent infection (liao et al., 1998; su et al., 2001). rapid replication and dissemination have also been reported as tactics used by rna viruses to avoid apoptotic cell death (koyama, 1995; kurokawa et al., 1999). while apoptosis is a commonly reported and extensively studied immune response in mammals, comparatively less is known about apoptosis and immunity in insects. studies in drosophila cell lines have shown that flockhouse virus will induce apoptosis by depletion of diap1 through virusinduced inhibition of host protein synthesis in d. melanogaster (yoo et al., 2002; settles and friesen, 2008). because diap1 has a short half-life (35-40 min), reductions in protein synthesis would create depletion in the intracellular pool of diap1 resulting in apoptosis (yoo et al., 2002). apart from a small number of studies in drosophila, much of the evidence collected to date has been observational, showing only associations between infection and apoptosis. perhaps the most compelling evidence linking apoptosis to immunity has been observed in mosquitoes. for many years, arthropod-borne viruses, such as dengue virus and yellow fever virus, were believed to cause lytic infections in mammalian cells but persistent infections with only moderate cytopathic effects in mosquito cells. more recently, in vivo studies have provided strong evidence that apoptosis may play a role in mediating virus infections in mosquitoes and therefore may impact vector competence. apoptosis-linked pathologies have been described in a. aegypti infected with semliki forest virus (mims et al., 1966), in a. albopictus infected with sindbis virus (bowers et al., 2003), and in the salivary glands of culex pipiens quinquefasciatus infected with west nile virus (wnv) (girard et al., 2005, 2007). infections with other intracellular pathogens, such as plasmodium sp., have also been shown to cause pathologies resembling apoptosis in mosquitoes (hopwood et al., 2001; alolayan et al., 2002; hurd and carter, 2004; hurd et al., 2006). p. berghei infection causes apoptosis in midgut cells of a.. stephensi (han et al., 2000) and a. gambiae (vlachou et al., 2004), while p. gallinaceum infection is associated with apoptosis in a. aegypti (zieler and dvorak, 2000). in addition, caspase activity is associated with plasmodium infections in mosquitoes (han et al., 2000; zieler and dvorak, 2000; abraham et al., 2004; vlachou et al., 2004). although these studies are largely observational, they suggest that apoptosis is associated with vector competence in mosquitoes. vaidyanathan and scott (2006) showed extensive cell death in the midgut cells of a lab-derived strain of c. pipiens pipiens refractory to infection with wnv upon exposure to the virus (vaidyanathan and scott, 2006). additionally, reduced virus transmission is associated with apoptosis in the salivary glands of c. pipiens quinquifasciatis during the later stages of wnv infection. these studies demonstrate that host cells detect virus infection and attempt to clear the infection through induction of cell death. the events that trigger virus-induced apoptosis and the mechanisms that viruses use to evade apoptosis remain largely unknown; however, this field of research holds promise in illuminating new determinants of vector competence in mosquitoes. the strongest evidence linking apoptosis to insect immunity comes from a family of viruses known as the baculoviruses. baculoviruses are large dna viruses, most often associated with lepidoptera, that can trigger rapid and wide spread apoptosis in insect cells following replication in host cell cytoplasm, (reviewed in clarke and clem, 2003; clem, 2005, 2007). consequently, baculoviruses encode their own apoptotic supressors including viral iaps and caspase inhibitors (p35 and p49) to block premature cell death and promote virus multiplication (reviewed in clem, 2005, 2007). iaps have multiple functions that include binding and inhibiting caspase activity, regulating cell cycle progression, and modulating receptor-mediated cell cycle signal transduction (deveraux and reed, 1999, salvesen and duckett, 2002). p35 and p49 are pancapase inhibitors that prevent the apoptotic response in cells by binding to the active site of caspases (lannan et al., 2007). given that both iaps and caspase inhibitors function downstream of the events that trigger apoptosis, it is likely that the host cell initiates apoptosis in response to viral infection while the baculovirus inhibits cell death to promote cell survival. recently, liu et al. (2011) used an in vivo system to demonstrate michelob_x (mx), the mosquito ortholog of pro-apoptotic gene drosophila reaper, is specifically induced in larval c. nigripalpus midgut cells following infection with a baculovirus (liu et al., 2011). in the permissive mosquito c. quinquefasciatus, a slow induction of mx failed to induce apoptosis; infected cells eventually underwent necrosis due to high virus loads. in contrast, a rapid induction of mx within 30 min post-infection followed by apoptosis within 2 6 h post-infection occurred in the refractory mosquito, a. aegypti (liu et al., 2011). these data suggest a possible role for apoptosis in limiting viral infection. further research aimed at understanding apoptosis in insects, particularly those that vector disease, will provide a promising platform for new research into host-pathogen interactions. autophagy as an immune response autophagy, an intrinsic program that degrades cytoplasmic components, has been implicated as a process that responds to many different forms of stress, including microbial infection. typically, autophagy transpires at basal levels in all cells and serves as the major mechanism in homeostatic functions such as the turnover of damaged proteins and organelles, and is essential for survival, differentiation, and development (codogno and meijer, 2005). the autophagy-related genes (atg), a family of conserved genes which encode proteins required for various phases of the autophagic pathway, are found in organisms ranging from the simple unicellular saccharomyces cerevisiae, to the multicellular organisms including insects and mammals (galluzzi et al., 2008). in multicellular animals, conserved signalling pathways also 167 regulate autophagy. the mammalian target of rapamycin (mtor) kinase pathway plays a pivotal role as a sensor of cellular energy, growth factor and nutrient levels and will inhibit autophagy when these factors are abundant. when nutrients are limited, mtor is inactivated and autophagy is induced (reviewed in mcphee and baehrecke, 2009). upstream of mtor, members of the insulin receptor/class i phospho-inositide-3-kinase (pi3k) pathway also repress autophagy in a nutrientdependent context. additional signaling pathways such as the tumor repressor pten promote autophagy through antagonizing pi3k signalling (mcphee and baehrecke, 2009). whereas basal levels of autophagy ensure the physiologic turnover of damaged organelles, the massive accumulation of autophagic vacuoles has been linked to an alternative cell death pathway, type ii or autophagic cell death. whether the autophagic vacuoles associated with type ii cell death are the results of failed attempts to adapt to cellular stress or whether they represent a unique non-apoptotic form of cell death is debatable and beyond the scope of this review. below we discuss what is currently known about autophagy and microbial infections in insects and highlight current limitations in the field. as with apoptosis, relatively little is known about the role autophagy plays in immunity in insects. autophagy can be triggered in infected cells where it may act as a host defence mechanism to eliminate a pathogen without eliminating the cell (kirkegaard et al., 2004; levine and deretic, 2007). in mammalian systems, autophagy has been to shown to clear bacteria from the cytoplasm and restrict the spread or replication of several viruses including herpes simplex virus 1 (hsv-1), human immunodeficiency virus -1 (hiv-1) and sindbis virus (liang et al., 1998; gutierrez et al., 2004; andrade et al., 2006; ling et al., 2006; singh et al., 2006; talloczy et al., 2006). numerous studies have shown that insects contain all the machinery to induce and regulate autophagy; however, autophagy and/or autophagic cell death is almost exclusively observed during developmental periods when massive cell or tissue elimination occurs (malagoli et al., 2010). few reports exist describing an immune role for autophagy or autophagic cell death in insects. two exceptions, a study by shelly et al. (2009) and a study by cherry et al. (2009) have demonstrated that autophagy is observed in drosophila infected with mammalian vesicular stomatitis virus (vsv). here, induction of autophagy was associated with decreased viral replication and repression of autophagy led to both increased viral replication and pathogenesis in both cell lines and adult flies. autophagy can be controlled by the wellconserved phosphatidylinositol 3-kinase (pi3k)-aktsignaling pathway, which normally mediates autophagy in response to nutrient availability. interestingly, the activation of autophagy in response to vsv infection did not require virus replication, suggesting that cells were sensing the virus itself, perhaps through a pamp. in contrast to vsv infection, flies depleted of atg18, a conserved component of the pi3k-akt-signaling pathway, were not more sensitive to infection with dcv, suggesting that the autophagy-mediated antiviral response is protective against vsv but not dcv infection. why autophagy is observed with vsv infection but not dcv infection is not known but remains an interesting question. it is important to note that the field of autophagy and autophagic cell death is only now gaining adequate attention. as the field develops and more tools become available, valuable knowledge regarding the role autophagy plays in antiviral or anti-parasite responses in insects should be uncovered. this is of particular interest for vectors of disease as they must maintain tissue integrity and promote cell survival while carrying large pathogen loads. concluding remarks insects are armed against invading pathogens with a diverse and powerful repertoire of defences. innate immunity, indeed all immune responses, requires identification of a pathogen as non-self. insect prrs and pamps engage cellular machinery to eliminate pathogens through phagocytosis, melanization, proteolytic cascades, amps, rnai and the toll, imd, jak-stat pathways. insects and the pathogens they transmit evolve in concert; an arms race to outwit the other’s defence and invasion strategies in turn. insects are reliant on innate immunity to overcome infection and in the absence of acquired immunity; cell death can be a powerful mechanism for the elimination of pathogens that evade the innate immune response. as the death of an infected cell is often concomitant with the death of the infecting agent, cell death can promote efficient pathogen clearance. the abundance of anti-apoptotic proteins encoded by animal viruses attests to the importance of apoptosis as a measure of combating infections. although cell death is a comparatively new field in non-drosophilid insects, numerous studies now link cell death to immunity in insects. as new information is gathered on cell death signalling in insects and more tools become available to study the various forms of cell death, we can begin to ask questions about the signalling events that trigger cell death and strategies used by pathogens to counteract this response. the mosquito model, in particular, provides the opportunity for a comparative study of cell death and the regulation of specific immune processes in insects in which intracellular pathogens infect host tissues and multiply before being transmitted to humans. new ways of combating arboviral transmission must be developed and understanding how insects interact with the pathogens they carry is an important step. acknowledgements we apologise to many researchers who were not referenced owing to space limitations. we thank bucchop j for constructive comments on this manuscript. dmc is the recipient of natural sciences engineering research council (nserc), canadian institutes for health research (cihr) impact, and heart & stroke foundation of canada postdoctoral fellowships. kmf is supported by an alexander graham bell phd scholarship from cihr. 168 references araham eg islam, srinivasan p, ghosh ak, valenzuela jg, ribeiro jmc, et al. analysis of the plasmodium and anopheles transcriptional repertoire during 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xiiith scientific meeting of the italian association of developmental and comparative immunobiology (iadci), 22 24 february 2012, department of environmental and natural sciences, university of camerino, camerino, italy organizers: a vallesi, c alimenti, p luporini department of environmental and natural sciences, university of camerino, camerino, italy plenary lecture genomics, immune studies and diseases in bivalve aquaculture a romero, a figueras, b novoa instituto investigaciones marinas, csic, eduardo cabello 6, 36208 vigo, spain bivalve diseases are one of the critical bottle necks causing important and recurrent losses in bivalve culture. in some cases, the presence of infectious pathogens has caused the complete destruction of bivalve culture as it happened to the flat oyster (ostreaedulis) in europe. research on bivalve diseases has relied mainly on histological techniques and has been mostly focused on pathogen morphology and ultrastructure, effect of external factors on pathogens or infectivity, and on the development of immune and molecular diagnostic techniques. although critical advances have been reported in the last years on bivalve pathology, increased efforts should be placed on the application of “omics and physiology” to explain key physiological/immunological processes. transcriptomic analysis in parallel to detailed functional studies on gene expression and cell biology in in vitro and in vivo experimental models are needed. another important aspect is the identification of “resistance traits” and a deeper understanding of the processes which contribute to bivalve welfare in culture. also the definition of "abnormal mortalities" is critical for managing and legislating bivalve aquaculture. this clearly “opens the door” toward directed manipulation to increase and improve all relevant processes in modern intensive aquaculture systems. a future exciting research is in front of us. now, more than ever, the need to develop multidisciplinary international research, involving research groups and growers organizations to work on shellfish pathology, is more than evident. session 1. chairman: e ottaviani, university of modena and reggio emilia, modena, italy new insights in immune system components multiple faces of ascidian host defense n parrinello department of environmental biology and biodiversity, section of animal biology and anthropology, university of palermo, palermo, italy cell migration, phagocytosis, encapsulation, activation of complement and propo systems, cytotoxicity, tissue injury and wound repair, matrix production, endothelial and epidermis activity are components of the ciona intestinalis inflammatory responses. hemocytes are primarily responsible of the inflammatory activity. in several paper we reported that cytophilic humoral molecules with functional similarity to vertebrate pro-inflammatory factors can be modulated by lps challenge suggesting their involvement in defense. cellular responses include enhanced expression of: collagen-like molecules, galectins with opsonic properties, mannose binding (mbl)-like collectin and c3-like factor, citnfα, a component of the cap superfamily, propo activation. in addition, for the first time we showed that citnfα and galectins are constitutively expressed in the adult and larvae, while cytotoxic hemocytes exert a phospholipasedependent activity. since hemocyte proliferation has been claimed in the inflamed tissues as indicated by a massive infiltration, recently we examined the proliferative capability of the ascidian circulating hemocytes. the proliferating cell nuclear antigen (pcna) gene expression in unseparated and separated hemocyte populations was examined. taking advantage of the c. intestinalis whole genome sequencing, an in silico search for a pcnalike sequence (ncbi) was performed. the pcna putative dna binding domain was cloned from hemocytes (cipcna), the rna probe was synthesized, in situ hybridization (ish) assay was 35 carried out, and gene expression in short-time primary hemocyte was evaluated by semiquantitative real-time pcr. results revealed that cipcna gene expression increased after 24h culture in native conditions, and in the presence of a mitogen such as vegf. brdu labeling disclosed incorporation in granular amebocytes with small granules and hyaline amebocytes, while the highest expression characterized lymphocytes-like cells. in conclusion the ascidian inflammatory response appears to be a well preserved process in chordate evolution. morphological and functional observations on the circulating cells of the freshwater snail pomacea canaliculata a accorsi, e ottaviani department of biology, university of modena and reggio emilia, modena, italy the gastropod mollusc pomacea canaliculata is a freshwater snail used as a model for different ecological studies. recently, the function of this snail as intermediate host for nematodeand trematode-driven human pathologies, made p. canaliculata the subject of intense parasitological investigations. at present, the morphology of p. canaliculata circulating cells has been described only by means of tem observations on clumping activity, which evidenced the presence of granular and agranular cells. in the present study we analyzed the morphology and phagocytic activity of the circulating cells in normal conditions. morphological and cytochemical stainings showed different morphological figures: a) small cells with high nuclear/cytoplasmic ratio, b) cells with circular nucleus and uniform, smooth cytoplasm, c) cells with irregular nucleus and vacuolated cytoplasm and d) cells with eccentric and polymorphic nucleus and granular cytoplasm. histochemical and histoenzymatic stainings revealed specific positivity for each cell morphology. the vacuolated cells were pas-positive in the perinuclear cytoplasm and neutral red-negative and the acid phosphatase activity assay showed the presence of positive vesicles. in the cells with granular cytoplasm pas-positivity was limited to the cytosol, while the granular structures were neutral red-positive. immunocytochemical experiments indicated the presence of acth-like material in all the circulating cells with the exception of those with circular nucleus and uniform cytoplasm. with regard to the functional assay, we have observed phagocytic activity expecially in the vacuolated cells towards both heat-inactivated escherichia coli and staphylococcus aureus. summarizing, the circulating cells of p. canaliculata present some features observed in molluscan immunocytes, such as phagocytic activity and presence of acth-like material. further studies will help in elucidating whether the different cell morphologies and stainability correspond to specific activities or different maturation stages. age related properties of the adriatic clam chamelea gallina (l. 1758) hemocytes f mosca, v narcisi, d cargini, a calzetta, pg tiscar department of comparative biomedical sciences, university of teramo, teramo, italy in marine bivalve molluscs, the phagocytosis represents the main immune mechanism and the circulating hemocytes are able to recognize, engulf and destroy the foreign particles. the hemocytes possess chemotactic ability to migrate towards foreign particles and include them inside phagosomes. this ability is strictly depending on morphological activation through the projections of membrane ruffles or pseudopodia. on the other hand, the respiratory burst represents aspecific mechanism involved in the destruction of foreign particles. the present research was aimed to analyse the phagocytic activity in the clam chamelea gallina hemocyte, monitoring both their morphological spreading by cell circularity parameter and the production of nitric oxide (no) by micromethod assay and flow cytometry using a specific fluorescent probe. the parameters were monitored in different groups in order to verify the possible influence of the clam size on phagocytic response. a preliminary analysis was conducted by light microscopy to study the hemocytes morphology and the differential hemocyte count on the basis of their morphotype. this approach revealed the existence of granular and agranular hemocytes with a prevalence of agranular type. however, a significant increase of granular cell type was observed in the larger clams (>30 mm length). in such a group, a better phagocytic response was also detected both in terms of morphological spreading and nitric oxide generation. then, the phagocytosis assay was also applied on each hemolymph type purified by percoll method, revealing a better response in granular hemocytes. thus, the more intense phagocytic response observed in the hemolymph of larger clams seems to be ascribed to the greater concentration of granular hemocytes. a such variation in hemolymph morpho-functional characteristics appeared not dependent on environmental factors or on the presence of pathogens monitored by histology, but rather on clam aging. finally, in the interpretation of our results we have also to consider the potential role of hematopoietic processes in the life-long hemolymph maturation and, in such a way, further studies have to be directed to investigate the origin, life cycle and life span of the different hemocyte types. molecular characterization of macrophage migration inhibitory factor (mif) from mussel, mytilus galloprovincialis mg parisi1, m toubiana , v mangano , n parrinello , m cammarata , p roch 2 1 1 1 2 1marine immunobiology laboratory, department of environmental biology and biodiversity, university of palermo, palermo, italy 36 2ecologie des systèmes marins et côtiers (ecosym), cnrs ird, université montpellier 2, cc093, place e. bataillon, 34095 montpellier cedex 05, france macrophage migration inhibitory factor (mif) is a highly conserved cytokine which exerts wideranged activities. it is a central mediator of innate immunity and has been shown to correlate with regulation of macrophage functions, lymphocyte immunity and a number of inflammatory diseases. its neuroendocrine role as mediator to increase host responses to microbial endotoxins suggested that mif is at the crossroads of the endocrine and immune systems. mif homologues have been detected in a wide range of animal species suggesting such molecule has been conserved. in this study, three mif-related nucleotide sequences were identified from a m. galloprovincialis est library. structure analysis of consensus sequence, closely related to gastropod mifs, revealed presence of an orf coding for 115 aa, with a 5’ utr of 32 nt and the 3’ utr of 349 nt. as for other mifs, m. galloprovincialis orf does not include signal or c-terminus extension. among various dissected tissues, mussel mif was constitutively expressed principally in hemocytes and in mantle. polymorphism analysis showed several mif mrna variants in mussels from palavas (france) and from palermo (italy), some of them being translated in different aa sequences. 11 peptide variants in the palavas mussel and 14 peptide variants in the palermo specimens have been found. only 7 of the 18 different aa sequences were common to both mussel populations. nevertheless, residues involved in thiol-protein oxidoreductase activity and in a tautomerase isomerase activity were conserved in all the 18 m. galloproviancialis mif (mg-mif) variants. mg-mif gene possesses the same exon-intron organization as in human and mouse structure, except for one α-helix between aa 68 76, which is 7 aa shorten in mg-mif than in human mif. among the mollusks, mg-mif appeared to be closely related to p. fucata and haliotis, but not to c. farreri and to gastropod b. glabrata. mif gene expression was quantified by realtime pcr system following in vivo challenges of the mussels. results indicated a different mechanism of elimination of yeast, bacteria and fungi. expression of mif was significantly up regulated in hemocytes after 1 h of stimulation and returned to background 9 48 h following the challenge. identification and modulation of an allograft inflammatory factor-1 (aif-1) homolog in the medicinal leech, hirudo medicinalis f drago*1, t schorn*2, a accorsi3, pe sautière1, f croq1, c lefebvre1, c van camp1, a grimaldi2, j vizioli1 1lsmbfa ea4550-equipe activation microglial, bât sn3, université lille 1, 59655 villeneuve d’ascq, france 2department of biotechnology and life science, university of insubria, varese, italy 3department of biology, university of modena and reggio emilia, modena, italy * equal contribution allograft inflammatory factor (aif-1) is a 17 kda cytokine-inducible calcium-binding protein, produced by activated macrophages during chronic transplant rejection and in inflammatory reactions. in vertebrates, aif-1 plays a significant role not only in immune defense reactions, but also in responses to inflammatory stimuli. in central nervous system (cns), aif-1 is a sensitive marker associated to activated microglia and is upregulated following neuronal death or brain lesions. aif-1-like factors have been described in several metazoan groups and share a well conserved amino acid primary structure throughout evolution suggesting a common, functional role. the analysis of an est library from medicinal leech cns, revealed the presence of a gene, named hmaif-1, showing a high homology with vertebrate aif-1. immunohistochemistry and expression pattern studies were performed to determine the localization and the modulation of this gene in leech. the presence of hmaif-1 in naïve and experimentally challenged tissues has been established using antihuman aif-1 polyclonal antibodies. results showed that hmaif-1 is constitutively present in spread, cd68+ macrophage-like cells; a few days after experimental wounding of the body wall, the amount of these immunopositive cells increases at the lesion site. in cns, hmaif-1 is constitutively present in some microglial cells and, during nerve repair process, it augments at the cut end of injured nerve fibers. quantitative rt-pcr analyses on cultured nerve chains showed a weak modulation of the gene in the days following experimental injury. interestingly, the hmaif-1 gene is strongly upregulated a few hours after bacterial challenge. also if the functional role of hmaif-1 has to be elucidated, these data suggest that in leech this factor is involved in immune response and in inflammation events like its vertebrate counterparts. session 2. chairman: r valvassori, university of insubria, varese, italy new insights in immune system components molecular studies on phenoloxidases of compound ascidians l ballarin1, n franchi1, f schiavon1, sc tosatto1, i mičetić2, k kawamura2 1department of biology, university of padua, padua, italy 2laboratory of cellular and molecular biotechnology, faculty of science, kochi university, japan phenoloxidases (pos) constitute a family of copper-containing enzymes with orthodiphenoloxidase (catecholase) activity widely distributed among invertebrates. they exert a pivotal role in immune defences as they can induce cytotoxicity through the conversion of phenols to 37 quinones and the production of reactive oxygen species. in ascidians, po activity has been described and studied in both solitary and colonial species and the enzyme is involved in inflammatory and cytotoxic reactions against foreign cells or molecules as well as in the formation of the cytotoxic foci along the contacting edges of genetically incompatible colonies which characterises the nonfusion reaction of botryllids. expressed genes for two putative pos (cipo1 and cipo2) have been identified in c. intestinalis (immesberger and burmester, 2004). in the present study, we determined the cdna sequences of the pos from two colonial ascidians: botryllus schlosseri from mediterranean (adriatic) sea and polyandrocarpa misakiensis from japan. multiple sequence alignments clearly evidenced the similarity between ascidian po and crustacean propos whereas the analysis of the threedimensional structure of compound ascidian pos reveal high similarity with arthropod haemocyanins which share common precursors with propos. ascidian pos and arthropod propos grouped in the same cluster well separated from mollusc tyrosinases, and share the full conservation of the six histidines at the two copper-binding sites as well as of other motifs, also found in arthropod haemocyanins, involved in the regulation of enzyme activity. cytoenzymatic studies and in situ hybridisation (ish) indicated that the genes are transcribed inside morula cells (mcs), a characteristic haemocyte type in ascidians, at the beginning of their differentiation. sequence analysis allowed a better understanding of previous biochemical data and suggest some hypothesis for the regulation of enzyme activity. toll-like receptors: an evolutionary approach mr coscia, s giacomelli, u oreste institute of protein biochemistry, cnr, naples, italy toll-like receptors (tlr) are key molecules of the innate immune response, shared by vertebrate and invertebrate phyla. the basic toll/toll-like molecular structure consists of two well defined portions with different funtional roles, connected by a segment spanning the membrane. the ectodomain, consisting of a series of leucine-rich repeats (lrr), is able to recognize conserved pathogen-associated molecular patterns (pamps); the cytoplasmic region includes a toll interleukin-1 receptor (tir) domain. it is devoted to transduce the ligand-binding signal to the nucleus where transcription of immune effector molecules genes is triggered. several mechanisms such as genome duplication, gene expansion, retrotrascription, and alternative splicing, contributed to shape the tlr repertoire in the different phyla. a comparative analysis of the tlr structure could help identify the critical features of the molecular structure. porifera represents the most ancient metazoan phylum showing nucleotide sequences reminiscent of tlr. in cnidaria only one out of many tir containing receptors shows several lrrs in its ectodomain, arranged in a specific manner, the so called “protostome-type”. a larger number of lrrs has been found in a caenorhabditis elegans tlr sequence and one anellid tlr has been shown to be unique since does not share the “protostometype” features, and presents a structure similar to mammalian tlrs. a survey of the genome of the purple sea urchin strongylocentrotus purpuratus, a member of the phylum echinodermata, has revealed the presence of 222 toll-like receptor gene models. a great number of tlr sequences (36) has been found also in the cephalochordate branchiostoma floridae, while only two have been identified in ciona intestinalis genome. so far, at least 23 vertebrate tlrs have been identified, based on amino acid similarity, genomic structure, and ligand properties. they can be grouped into six major families. a unique feature of teleost tlrs is the presence, in addition to tlr5, of a soluble tlr5 molecule (tlr5s), which lacks the transmenbrane and tir domains in oncorhynchus mikiss and takifugu rubripes genomes. by reviewing the presence of tlrs in different species it appears clear that evolution used tlr molecules for different functions, ranging from immune response to developmental signaling and cell adhesion. whether the tlr cooptation in insect and vertebrate immunity represents a convergent evolution is still an unsolved question. preliminary studies on the complement system in the compound ascidian botryllus schlosseri n franchi, l ballarin department of biology, university of padua, padua, italy the complement system represents an important humoral component of the mammalian immune system. complement components can be subdivided in 5 gene families: c3/c4/c5, bf/c2, masp/c1r-s, c6/c7/c8a/c8b/c9 and factor i. until 1884, it was generally believed that the complement system was an unique feature of vertebrates since all attempts to identify complement components in invertebrates failed. in recent years, the genomic approach revealed the presence of complement orthologue genes in invertebrate deuterostomes, mainly in sea urchins and tunicates (ascidians). conversely, no complement genes were found in the genome of protostomes such as drosophila melanogaster and caenorhabditis elegans, suggesting that the complement system was established in the deuterostome lineage. genome analyses carried out in the solitary ascidian ciona intestinalis revealed that most complement gene families are present in urochordates. we recently carried out the assembling of est collections from the colonial ascidian b. schlosseri, obtained in our and other laboratories: we found multiple transcripts showing high similarity with vertebrate complement components such as c3, masp, mbl and c6. preliminary in silico studies revealed close relationships between botryllus c3, masp and mbl and orthologues from other chordates. in particular, c6 seems related with the 38 c6 proteins of the solitary ascidians c. intestinalis and halocynthia roretzi and share with them the absence of the fim domain which is responsible for the interaction with the other complement molecules in vertebrates. future studies will be devoted to the analysis of the expression of genes for complement components of b. schlosseri. a cd83-like molecule in sea bass (dicentrarchus labrax) related to immune responses against viral pathogens e randelli, f buonocore, p tranfa, g scapigliati department for innovation in biological, agro-food and forest systems, university of tuscia, viterbo, italy the cd83 cell surface marker is an important and intriguing component of immune system. it is considered the best marker for mature human dendritic cells, but it also plays an important role as a regulator of peripheral b-cell function, homeostasis and for thymic development of t cells. a cd83-like molecule was identified in sea bass (dicentrarchus labrax) by est sequencing of a thymus cdna library; the comparison of sea bass cd83 sequence with its homologs in other fish species and mammals shows some differences, with two cysteine residues conserved from fish to mammals and a high variability both in the total number of cysteines and in mature cd83 sequence peptide length. basal transcripts levels of cd83 mrna are highest in liver, followed by thymus. the in vitro treatment of head kidney leukocytes with lps resulted in a down-regulation on cd83 mrna leves both after 4h and 24h, whereas with poly i:c an up-regulation after 4h followed by a down-regulation at 24h was observed. an in vivo infection of sea bass juveniles with betanodavirus induced an increase of cd83 expression on head kidney leukocytes both after 6h and 24h and a decrease after 72h. on the other hand, an in vivo infection with p. damselae bacteria induced a decrease of cd83 transcipt levels after 6h and 24h and an increase after 72h. these findings suggest that sea bass cd83 expression could be modulated by viral and bacterial immune response. complement system of antarctic teleosts: sequence analysis and molecular structure of trematomus bernacchii c3* mr pinto1, s varriale2, d melillo1, u oreste2 1zoological research station “anton dohrn”, naples, italy 2institute of protein biochemistry, cnr, naples, italy the complement system (cs) is an ancient defense mechanism, which in higher vertebrates involves more than 30 secreted or membrane-bound proteins interacting each other when the system is activated. this system plays important roles in the immune response of vertebrate species, such as pathogen cell surface recognition, promotion of inflammation and coordination of the adaptive immune response. it includes three different activation pathways (classical, alternative, and lectin), converging towards the activation of c3, the third component of the cs. c3 activation results in the production of two proteolytic fragments: a small fragment, c3a, that mediates many immunological activities, and a large fragment, c3b, which undergoes a dramatic conformational change, culminating in the exposure of the buried thioester group, that, in turn, allows c3b interaction with the invading bacterial matrix. the cs of teleost fish has been investigated in many species. in contrast to mammals, it functions also at low temperature and seric titers of several components are drastically higher. moreover, teleost c3 is present in several isoforms that have been suggested to bind complement-activating surfaces with different competences. c3 genes have been sequenced, so far, in 14 teleost species, belonging to 8 different orders. our aim is to extend the c3 literature to an antarctic species, trematomus bernacchii (perciformes), which has been selected as model species to study the cold-adaptation of the immune system. t. bernacchii liver cdna has been amplified by reverse transcriptase-polymerase chain reaction using oligonucleotides corresponding to highly conserved nucleotide sequences of teleost c3 available in databases. this approach allowed the isolation of two c3-like clones, tbc3-1 and tbc3-2, whose sequencing completion has been performed by 3' and 5' race procedure. molecular models of tbc3-1 and -2 have been built using as a template the crystallographic structure 2a73 of human c3. in parallel, a model of the c3 molecule of the temperate species paralicthys olivaceus has been constructed using the same template. molecular dynamic simulations of tbc3-1 and p. olivaceus c3 models have been performed for 15 nsec and the flexibility of the calpha atoms of the backbone have been compared at the equilibrium. significant differences in the rmsf plots suggest higher flexibility of the antarctic molecule possibly due to the cold-adaptation evolution process. special session 1. chairman: l ballarin, university of padua, padua, italy iadci award gene expression profiles in mussels from the venice lagoon u rosani1, s domeneghetti1, a pallavicini2, p venier1 1department of biology, university of padua, padua, italy 2department of biology, university of trieste, trieste, italy global gene expression profiles in organisms selected to represent a given ecosystem currently support ecotoxicological investigations and create a conceptual bridge between the early organism responses and late population effects (steinberg et al., 2008). bivalve molluscs are constantly exposed 39 to a variety of environmental changes. as regards mytilus galloprovincialis, tens of thousands expressed sequence tags (ests) are currently available and a number of dna microarrays have been developed. in this study, we propose the analysis of hybridization data based on the whole mytibase transcript collection (venier et al., 2009) and referred to digestive gland and haemolymph of mussels sampled from selected venice lagoon sites in different years. essentially, we designed an agilent 15k oligoarray representing all the 7112 mytibase est clusters (3' utr 60-mer probes, 63 % the fraction of annotated transcripts at the moment). to assess the performance of the 15k oligo-platform we calculated precision, specificity and sensitivity on the whole set of one-color hybridization data, according to moreau et al. 2003. we used the principal component analysis and t-test to analyze the results and obtain lists of differentially expressed genes. in this way, we identified transcripts typical of digestive gland or haemolymph. annualand site-related expression trends were also investigated. spike-in controls allowed us a precise assessment of the expression values for relevant families of immune-related transcripts. effects of fluoxetine on immune parameters of the clam ruditapes philippinarum m munari, v matozzo, mg marin department of biology, university of padua, padua, italy among emerging environmental contaminants, pharmaceuticals and personal care products (ppcps) are a large group of substances used either by human for personal health and cosmetic reasons or by agribusiness to enhance growth or health of livestock. ppcps are produced in large quantities and comprise numerous chemicals, including prescriptible drugs, veterinary drugs, diagnostic agents, fragrances, lotions, and cosmetics. as a consequence, main sources of ppcps in the environment (aquatic in particular) are human activities, residues from both pharmaceutical manufacturing and hospitals, illicit drug use, veterinary drug use and agribusiness. among pharmaceuticals, fluoxetine (a selective serotonin reuptake inhibitor) is an antidepressant commonly used for treating depression and other psychological disorders. despite its wide use, information is lacking about the effects of fluoxetine on non-target species. to fill this gap, in the present study the effects of fluoxetine on some important immune parameters of the clam ruditapes philippinarum were evaluated for the first time. clams (25 per concentration) were exposed for 7 days to differing fluoxetine concentrations (0, 1, 5, 25, 125, 625 µg/l), and haemolymph was collected from the anterior adductor muscle. eight pools of haemolymph (from 3 bivalves each) were prepared for each experimental condition, and total haemocyte count (thc), neutral red uptake (nru), lysozyme activity in cell-free haemolymph (cfh) and haemocyte proliferation were measured. an increasing trend was observed in thc values, the difference being significant at 25 µg/l, with respect to controls. nru was shown to decrease significantly in haemocytes of clams exposed to 1 and 5 µg/l, compared with controls, whereas nru increased to control levels in clams exposed to the highest fluoxetine concentrations. haemocyte proliferation increased significantly in animals exposed to 25, 125 and 625 µg/l, with respect to controls. conversely, no significant alterations were observed in cfh lysozyme activity. although preliminary, the results obtained demonstrate that fluoxetine influence markedly immune parameters in clams, even at environmentally realistic concentrations. expression of genes involved in glutathione biosynthesis in the solitary tunicate ciona intestinalis exposed to heavy metals n franchi, d ferro, l ballarin, g santovito department of biology, university of padua, padua, italy exposure to metals is known to generate oxidative stress risk in living organisms, which are able to respond with the induction of antioxidant defenses, both enzymatic and non-enzymatic. glutathione (gsh) is considered to be important components involved in protecting cells, both as metal chelating agent and oxygen radical scavenger. in this work we used molecular techniques to characterise the nucleotide sequence of genes involved in glutathione biosynthesis (cigclcgclc, ci-gclm and ci-gs) in the solitary tunicate ciona intestinalis. we also studied the expression of the genes in question after in vivo exposure to cd, cu and zn, to expand knowledge on the relation of metal-induced oxidative stress and glutathione production, locating mrna expression by in situ hybridisation (ish). these genes exhibit a good level of sequence conservation with corresponding metazoan homologs, especially for residues important for the activity of the enzymes. phylogenetic analyses indicate that the three enzymes evolved in different ways, ci-gclc and ci-gs being mostly correlated with invertebrate proteins, ci-gclm resulting as sister group of vertebrate gclms. our in silico analyses of the cigs and ci-gclc promoter regions revealed putative consensus sequences similar to mammalian metalresponsive elements (mre) and antioxidant response elements (are), indicating that the expression of these genes may directly depend on metals and/or reactive oxygen species (ros). our data highlighted a statistically significant increase in gene expression, demonstrating that metal treatments have inducible effects on this gene. they can modulate gene expression not only through mres but also through ares, as a consequence of metal-dependent ros formation. the ish location of ci-gs and ci-gclc shows that the cells most involved in glutathione biosynthesis are circulating haemocytes. the data presented here emphasise the importance of complex metal regulation of cigclc, ci-gclm and ci-gs transcription, which can create an efficient detoxification pathway allowing c. 40 inestinalis to survive in the continued elevated presence of heavy metals in the environment. comparative sequence analysis of antimicrobial peptides in mytilus galloprovincialis and ruditapes philippinarum m gerdol1, r casagrande1, g de moro1, c manfrin1, p venier2, a pallavicini1 1department of life sciences, university of trieste, trieste, italy 2department of biology, university of padua, padua, italy starting from our previous genome wide analysis in mytilus galloprovincialis we have identified by sequence similarity search in our transcriptomics database of clam r. philippinarum a total of 43 new amps belonging to 5 different families of peptides with antimicrobial properties. in details 16 defensins, 3 mytilins, 3 myticins, 10 macins and 11 big defensins. the search for the mytimycin homologous did not produce any results. for each class of amp we have also identified from other molluscs all the homologous sequences stored in public databases. all this data was used to produce a phylogenetic representation of sequence similarity through a bayesian approach. these results allow us to have a better picture, although not definitive, of the distribution and the diversity of amps in these clam and in general allows us to enrich our knowledge regarding antimicrobial defenses in the phylum mollusca. moreover we have demonstrate that a wide transcriptomics approach aiming to identify families of amps, can be very useful for organisms whose genome has not yet been sequenced. this approach proves to be very flexible and adaptable to perform research on a wide range of non-model organisms. although very important aspects related to the specificity and mode of action, expression inducibility and genomic organization of genes coding for these amps require a series of devoted downstream studies, bioinformatics approach can provide fairly quick and relatively easy to identify the primary sequence of many amps, laying the groundwork for further research. special session 2. chairman: m cammarata, university of palermo, palermo, italy iadci award skin wound repair in adult xenopus laevis e bertolotti, a franchini department of biology, university of modena and reggio emilia, modena, italy restoration of tissue integrity and homeostasis after an injury is a fundamental property of all organisms and there is diversity in how this process occurs. the healing response can lead to complete regeneration of tissue structure or repair that involves collagen deposition and scar formation. the regenerative capacity of anuran amphibians depends on the developmental stage and declines as metamorphosis proceeds. few studies have addressed the ability of adult anurans to heal their wounds: skin repair occurred with scar synthesis in rana catesbeiana, while it was without scarring in young adult xenopus froglets. in this work, we investigated the repair of skin wounds in different aged (8 and 15 month old) x. laevis adults to determine the quality of the wound healing response. molecules (tnf-α, inos, mmp-9, α-sma) and selected genes (socs-3, tgf-β2), known to be involved in inflammatory responses and wound healing, were analysed by immunohistochemical reactions and quantitative rt-pcr. the histological results showed similar repair step sequences in different aged frogs: inflammation, new tissue formation and maturation with wound contraction and remodeling. a large infiltrate of neutrophils and macrophages was early seen in the injured area and then lymphocytes were also detected in the granulation tissue. this wound connective tissue was characterized by extensive angiogenesis and transformation of some fibroblasts into anti-α-sma immunoreactive myofibroblasts which contributed to scar formation. quantitative rt-pcr analysis demonstrated that the regulator of cytokine signaling, socs-3, was rapidly upregulated after the injury and maintained high levels during the process, while tgf-β2, an important tissue fibrosis promoter, increased when the new tissue was formed and high induced expression persisted later in the repair. the results demonstrated that xenopus skin regenerative capacity is lost with increasing age of the adult. the outcome of skin wound healing is similar to that of mammals with the formation of a scar-like tissue that may be related to an intense immune response, abundant granulation tissue and wound contraction. moreover, α-sma, socs-3 and tgfβ promote the tissue repair with expression patterns that seem to be different from those observed in scarless healing. the cd45 receptor in the teleost fish dicentrarchus labrax c marozzi1, f bertoni1, e randelli1, f buonocore1, am timperio2, g scapigliati1 1department of science for innovative biology, agroindustry, and forestry, university of tuscia, viterbo, italy 2department of ecological and biological sciences, university of tuscia, viterbo, italy the cd45 tyrosine phosphatase plays an important role in regulating t lymphocyte activation in vertebrate species, and in this work we describe some features of the cd45 receptor molecule from the european sea bass dicentrarchus labrax. by immunising mice with fixed thymocytes we obtained a monoclonal antibody (mab) able to stain in immunofluorescence both live leucoytes at high percentages in thymus and mucosal tissues, and in situ immunoreactive cells by immunohistochemistry in sections from fixed tissues. the obtained mab dlt22 (igg2 subclass) recognized in western blots of lysates from thymus, spleen, intestine and gills leucocytes mainly polypeptides at 180 kda and 130 kda, and 41 immunoprecipitated a 130 kda polypeptide from thymocytes lysate. the 130 kda polypeptide was analysed by nano-rp-hplc-esi-ms/ms, and gave peptide sequences homologous to fugu cd45, that were employed for the homology cloning of a partial sea bass cd45 cdna sequence. the obtained cdna sequence was employed to measure by quantitative pcr the transcription of the cd45 gene in basal conditions and in in vitro stimulated leucocytes, showing a modulation induced by lps, cona, pha, il-1, and poly i:c. when splenocytes were stimulated in vitro with cona and pha, a cell proliferation was measured together with a corresponding increase of leucocytes stained by dlt22. these data indicate that the dlt22 mab may recognise in sea bass cd45-associated functional stages of lymphocytes, like cd45-like developing lymphocytes in thymus, and peripheral cd45rolike lymphocytes in tissues, thus dating back to teleost fish the functional activities of these cell populations in vertebrates. analysis of antarctic skate immunoglobulin heavy chain genes mr coscia, e cocca, s giacomelli, mf califano, f cuccaro, u oreste institute of protein biochemistry, cnr, naples, italy cartilaginous fish are the oldest vertebrate class having an immune system consisting of immunoglobulin (ig), t cell receptors, and major histocompatibility complex. in contrast to sharks, ig have been poorly investigated in skates despite their high occurrence in all of the major oceans of the world. we have focused on igm heavy chain from the antarctic species bathyraja eatonii, bathyraja albomaculata, bathyraja brachyurops, belonging to the subfamily arhynchobatinae, and amblyraja georgiana, belonging to the subfamily rajinae. the rt-pcr analysis of the four species of antarctic skates yielded several secreted igm heavy chain sequences. the primers used were designed on igm sequences available in genbank from various chondrichthyan species allowing us to isolate several cdna clones partially encoding immunoglobulin heavy chains. nucleotide sequence identities calculated for the constant region domains ranged from 88.5% to 97.5% between species, and from 91.1% to 99.7% within species. a distance tree, including also sequences from the temperate species raja erinacea, showed two major branches, one containing arhynchobatinae sequences, the other one rajinae sequences. four distinct d gene segments were defined, two being present in all the species analyzed. southern blotting analysis revealed 5 15 genomic fragments of different length, carrying the gene locus encoding igm heavy chain. as revealed by the shannon analysis, the position variability for the aligned b. eatonii amino acid sequences, was distributed through all four constant domains. however, ch3 was the most variable domain, whereas ch4 exhibited the lowest sum entropy value, being the most conserved domain. b. eatonii cdr3 region length varied between 11 and 15 amino acid residues, resulting in overall higher length variability than that of leucoraja eglanteria cdr3 (mean length: 7.7 aa). the presence of more extra-cysteine residues, not involved in the intradomain disulfide bridges, observed exclusively in the antarctic skates, is the most distinct difference between the antarctic and non-antarctic skate species analyzed. notably, in the ch3 domain, the two extra-cysteines fall within the cxxc motif of thiol/disulfide oxidoreductases, which is essential for their catalysis of redox reactions. taken together, these results may contribute to fill the knowledge gap relative to antarctic rajidae in the field of fish immunology. session 3. chairman: m balsamo, university of urbino, urbino, italy environmental stress responses of the immune system the new contribution of eco-immunology to the deciphering of immune-neuroendocrine integration d malagoli department of biology, university of modena and reggio emilia, modena, italy the original aim of eco-immunology has been the description of the modalities by which biotic and abiotic factors may influence the immune functions in free-living organisms and the source of natural variation in the immune response of a selected model. further conceptualizations have followed, in order to investigate how the many components of the immune system interact and determine the outcome of an immune challenge. fundamental concepts introduced by ecoimmunology derive from observations on vertebrates. however, the basic principles introduced are of general validity and may be applied to the largest part of multi-cellular organisms. one of the basic assumptions of eco-immunology is that in absence of an immune stimulus, the energy expenditure for immune responses may be kept at a minimum level. the allocation of different amounts of energy in relation with the functional status may be achieved by mean of trade-offs, i.e., the re-distribution of the available energy among different systems and components. the intrinsic plasticity of the immune functions allows energy re-allocation among different organs and activities, resulting in a variety of trade-offs between different systems. several experiments have indicated that in vertebrate and invertebrate models the immune and the neuroendocrine systems present a deep functional interconnection, based on the sharing of a common pool of signal molecules. cells as different as vertebrate lymphocytes and molluscan immunocytes have both and independently been described as immune-neuroendocrine cells. the cross-talk between immune and neuroendocrine systems must have represented a fundamental advantage being maintained during the diversification of evolutionary distant organisms such as mammals and molluscs. accordingly to the view of eco-immunology, the advantage could derive by the intrinsic capability of 42 optimizing energetic costs by mean of trade-offs and a common pool of mediators. these features made the immune-neuroendocrine system an evolvable unit, allowing its great diversification among metazoans, while maintaining intact the functional cross-talk among its various components. the antioxidant system of tetrahymena thermophila d ferro, f boldrin, f cattalini, e piccinni, g santovito department of biology, university of padua, padua, italy the “free radical theory” correlates reactive oxygen species (ros) production with oxidative stress and cell damaging. the antioxidative potential of organisms is probably affected by stress conditions, but this is an open question due to the multiplicity of ros scavengers and their overlapping functions. with the aim to study this problem we projected some experiments using the ciliated protozoan t. thermophila, as model organism. the first step of the research was to characterize the genes codifying for some antioxidant enzymes: copper-zinc superoxide dismutase (cu/zn-sod), manganese superoxide dismutase (mn-sod) and catalase (cat). total rna has been purified from t. thermophila cells (sb210 strain) cultured in ppyg medium and the cells were harvested after three days during exponential growth. cdna sequences were obtained by rt-pcr, 3’and 5’race techniques. the primers for the amplification of cu,zn-sod, mn-sod and catalase cdnas were designed after cross analyses between ncbi and t. thermophila genome databases. the obtained data demonstrated that two cu,zn-sods, one mn-sod, and one cat are constitutively expressed. our results highlighted that a third cu,zn-sod gene, annotated in the tetrahymena genome database, seems not be transcribed. the nucleotide sequences of all genes have been compared with orthologous of other organisms and used for phylogenetic analyses. experiments on the in vivo quantification of ros production, performed with specific fluorescent probes, are now in progress to study the physiological responses during the different phases of t. thermophila life cycle and under various oxidative stress conditions (exposure to uv, peroxides and metals). the results will be correlated to the gene expression analyzed by rtqpcr, using the same primers employed for cloning and sequencing. other genes of antioxidant enzymes such as glutathione peroxidases will be characterized. role of mapk activation in mediating the response to thermal shock in trichoplax (placozoa) m betti, e cesarini, l guidi, m balsamo department of earth, life and environmental sciences, university of urbino "carlo bo", urbino, italy trichoplax adhaerens (placozoa) appears as a flat disc consisting of two cellular pseudoepithelia, which sandwich a loose, syncitial network of fiber cells. in culture, trichoplax reproduces by binary fission, whereby one half of the animal moves away from the other half until their connection is broken. sexual reproduction has never been observed, but in culture formation of putative oocytes in degenerating animals is routinely seen. trichoplax has a compact genome compared with that of vertebrates and of many other animals. all essential components of the bmp/tgf and jak/stat signalling pathways are present in the trichoplax genome, but these pathways appear to be incomplete in that they lack molecular components critical to signal transduction. in this work, the possible role of different mapks and stat1 in mediating the response of trichoplax adhaerens (clone panama) to thermal shock was investigated. western blot analyses with specific anti-phospho-mapk and anti-phosphostat1 were performed in animals exposed to cold or heat shock. our results indicate that in trichoplax signal pathways leading to differential mapk–like and stat1-like activation are involved in mediating survival to thermal shock. heavy metals induced morphometrical alterations in earthworms granulocytes a calisi, p pagliara, a leomanni, mg lionetto, t schettino department of biological and environmental sciences and technologies, university of salento, lecce, italy earthworms are very important organisms for soil formation and organic matter breakdown in most terrestrial environments and they are able to accumulate various organic and inorganic contaminants present in the soil. earthworm coelomic fluid is particularly interesting from a toxicological perspective for the development of novel cellular biomarkers of pollutant exposure. it can transport pollutants throughout the exposed organism and its cells (coelomocytes) are involved in the internal defence system. five cell types were observed in eisenia foetida and lumbricus terrestris coelomic fluid: leukocytes type i (basophilic) and ii (acidophilic), granulocytes, neutrophils, and eleocytes. the aim of the present work was to investigate possible heavy metals induced alterations in earthworms coelomocytes in view of future application as sensitive biomarker for soil monitoring and assessment applications. morphometric alterations were determined by image analysis on diff-quick® stained cells. a considerable enlargement of granulocytes was observed in heavy metals (cu, cd, hg) exposed earthworms with respect to control group. on the other hand, the other cell types did not show any changes in the cell size. the enlargement was quantified by measuring the area of 2d digitalised granulocyte images. heavy metals are known to interfere with a wide 43 range of metabolic functions and membrane transport mechanisms. this could result in an increase of intracellular osmolyte content, followed by osmotic influx of water and cellular swelling. moreover, in heavy metals exposed animals the increase in the granulocyte dimension was accompanied by cell rounding with loss of pseudopods. this effect could be ascribed to toxic chemical-induced reduction of the microfilament and microspine number. moreover, granulocyte enlargement was paralleled by an increased frequency of necrosis in earthworm coelomocytes. due to the important immunological role of granulocytes, the observed heavy metal induced adverse effects on these cells may increase the susceptibility of animals to diseases and reduce their survival ability. therefore, early subtle alterations in some of the components of the immune system can be used as early indicators of altered organism health. the authors demonstrated that heavy metals induced morphometric alterations in earthworms granulocytes with possible applications as sensitive, simple, and quick biomarker for monitoring and soil risk assessment. immunomodulation by tio2 nanoparticles in mytilus galloprovincialis c ciacci1, c barmo2, r fabbri2, b canonico1, g gallo2, a marcomini3, g pojana3, l canesi2 1disteva, university of urbino, urbino, italy 2dipteris, university of genoa, genoa, italy 3department of environmental sciences, university of venice,venice, italy the potential toxicity of engineered nanoparticles (nps) for humans and the environment represents an emerging issue, due to the continuous development and production of manufactured nanomaterials. since nps tend to end up in waterways, their possible impact on the immune function of aquatic organisms needs investigation. in mammals, interactions of nps with immune cells represent a major issue for both therapeutic use and possible detrimental health effects. conservation of the general mechanisms of innate immunity from invertebrates to mammals is a key feature that can be used as an useful basis for studying common biological responses to nps. we have previously shown that in the marine bivalve mytilus short term exposure to ntio2, one of the most widespread np in use, significantly affected immune parameters in vitro and digestive gland biomarkers in vivo. in this work, the effects of ntio2 on immune function were investigated in mussels exposed to ntio2 (1, 10 and 100 µg/l) for 4 days. detailed physico-chemical characterization of nps was performed. the results show that exposure to ntio2 induced significant changes in different functional and molecular immune parameters in mussel hemocytes. in particular, ntio2 induced lysosomal membrane destabilization, inhibition of phagocytosis and stimulation of ros and no production. flow cytometry analysis revealed effects also at mitochondrial level, as well as decreased total hemocyte count (thc) and changes in the percentage of hemocyte sub-populations. changes in expression of selected genes (antimicrobial peptides, stress proteins) evaluated by rt-q-pcr were also observed. both lysosomal and antioxidant biomarkers were affected in the digestive gland, indicating general stress conditions. these data indicate that exposure to ntio2, at concentrations in the low µg/l range, can affect the mussel immune function, with possible immunosuppressive/inflammatory effects. moreover, these data support the hypothesis that in bivalves transfer of nps from the digestive system to the hemolymph and circulating hemocytes may occur. biomarkers of immunotoxicity may represent sensitive indicators of how nps may cause alterations in the organism’s physiology, providing an indication of the sublethal impacts of np exposure, as well as an “early warning” of population level impacts. session 4. chairman: l abelli, university of ferrara, ferrara, italy environmental stress responses of the immune system amyloidogenesis and melanogenesis: correlated events induced by “stress condition” a grimaldi1, g tettamanti1, t schorn1, f pennacchio2, r valvassori1, e ottaviani3, m de eguileor1 1department of biotechnology and molecular sciences, university of insubria, varese, italy 2department of entomology “f. silvestri”, university of naples, naples, italy 3department of biology, university of modena and reggio emilia, modena, italy amyloidogenesis has historically been associated with pathology of the neurodegenerative diseases while recently it has been demonstrated also in non-pathological protein fold utilized by organisms from bacteria to humans. we showed that the parasitization promotes in insect host hemocytes several massive morphological and physiological modifications that mimic general stress conditions, in which phenomena such as amyloid fibril formation and melanin polymerization, occur. moreover the physiological amyloid fibrillogenesis is an evolutionary conserved biological pathway and the relationship between amyloidogenesis and stressors allows to surmise a new background of information on the effects of stress. zinc effect on the immunological competence of the sea urchin paracentrotus lividus gametes and embryos p pagliara1, l stabili1, 2 1department of biological and environmental sciences and technologies, university of salento, lecce, italy 2institute for coastal marine environment, cnr, taranto, italy 44 in recent years, pollution of marine environment by heavy metals has become an international problem. in the mediterranean sea the main heavy metals found are cadmium, mercury, lead, tin, copper and zinc. these metals show levels often easily measurable in the marine samples and can exert toxic effects on living organisms causing an alteration of biological activities. in particular in marine invertebrates heavy metals affect survival, growth, reproduction, metabolism and immunity. effects of these pollutants on gametes and various developmental stages of echinoids were investigated by several authors focusing on the induction of morphological anomalies. by contrast studies on the effect of heavy metals on the immunological competence are scant. furthermore, being gametes and early life stages usually more susceptible to toxicants than the adult, in the present work we analysed the effects of a high zinc ion concentration on several immunological parametrs of the sea urchin paracentrotus lividus gametes and embryos. comparing untreated (control) and zinc treated sea urchins, we evaluated the effects of this metal on jelly coat and seminal plasma haemolitic and lysozyme-like activites as well as antibacterial activity on vibrio alginolyticus. all the examined immunological parameters were significantly reduced by the addition of zinc after 24 hours of treatment. the subsequent sea urchin development was negatively influenced. when lower zinc concentrations were employed deviations from the normal developmental process were observed. our results confirm sea urchin gametes and early developmental stages as suitable sensors for acute toxicity tests and their immunological competence modifications offer the potential for the development of sensitive assays to investigate and monitor heavy metal marine pollution. stress and immunomodulation indicators in gilthead seabream (sparus aurata): long-term dominant-subordinates interplay affects phagocytosis by peritoneal cavity cells m cammarata, m vazzana, d accardi, n parrinello marine immunobiology laboratory, department of environmental biology and biodiversity, division of animal biology and anthropology, university of palermo, palermo, italy fish are highly sensitive to stressful conditions that effect their immune system and increase susceptibility to diseases. the social hierarchy (dominant/subordinate) of gilthead seabream (sparus aurata) specimens were identified by using the “feeding order” and “aggressiveness” parameters in specimen pairs. this hierarchic position appeared at 24 36 hours after pairing, and did not change during one year in experiment with two (dominant/subordinate) or three fish (dominant/subordinate β/subordinate γ). to characterise physiological stress, we measured blood plasma levels of cortisol, glucose, and lactate as well as osmolarity and observed that the levels of these stress markers were higher in subordinate individuals than in dominant ones. since the increased glucocorticoid levels exhibited during stress might also serve to restrain defence mechanisms we examined the relationship between the social rank and the effects on the innate immunity. in particular the activity in the peritoneal cavity cells (pcc) was estimated using a phagocytosis activity assay employing yeast (pa) and a cell chemiluminescent (cl) reaction subsequent to stimulus with zymosan. after a 24 h pairing period, the pcc activity, with respect to pa and cl, was significantly higher in the dominant and control fish as compared with the subordinate ones. however, after 15 days, these responses returned to control levels in dominant fish, and these levels were maintained for 6 months. in contrast, in β animals, the pcc activity was increased by 3-fold within one month and 2-fold after 6 months. the discriminant analysis revealed the influence of the social status, with a clear allostatic response of the subordinate fishes and the highly significant separation among groups at 15 days. wild dolphin transcriptomes: identification of the immune response to environmental contaminants through gene expression information a mancia1, 2, j baatz2, j kucklick3, t rowles4, r wells5, p rosel6, l wilcox6, j ryan7, a hohn8, l schwacke7 1department of biology and evolution, university of ferrara, ferrara, italy 2marine biomedicine and environmental science center, medical university of south carolina, hollings marine laboratory, charleston, sc, 29412, usa 3national institute of standards and technology, hollings marine laboratory, charleston, sc, 29412, usa 4noaa, national marine fisheries service, office of protected resources, silver spring, md, 20910, usa 5chicago zoological society, c/o mote marine laboratory, sarasota, fl, 34236, usa 6national marine fisheries service, southeast fisheries science center, lafayette, la, 70506 usa 7noaa, national ocean service, hollings marine laboratory, charleston, sc, 29412, usa 8noaa, national marine fisheries service, southeast fisheries science center, beaufort, nc, 28516, usa as top level predators, bottlenose dolphins (tursiops truncatus), are particularly sensitive to chemical and biological toxins that accumulate and biomagnify in the marine food chain. a dolphin’s exposure to such toxins can be assessed using standard analytical methods, but it is costly and requires the collection of multiple tissue samples. we are currently investigating the potential of screening for multiple contaminant and/or algal toxin exposure through their associated immunological and/or endocrine perturbations in bottlenose dolphins using microarray technology and gene expression profile analysis. if successful, the gene expression profile analysis could provide a cost 45 effective means to screen for indicators of chemical and biological toxin exposure as well as disease status in dolphins, and potentially other cetaceans. a newly developed dolphin oligo microarray representing 24,418 unigene sequences was used to analyze blood samples from 74 dolphins collected from 4 geographic locations in the south east usa (beaufort, nc, sarasota bay, fl, saint joseph bay, fl, sapelo island, ga and brunswick, ga). the georgia samples were selected due to the measured high concentrations of persistent organochlorine contaminants in their blubber. genes involved in xenobiotic metabolism, in development/differentiation and oncogenic pathways were found to be differentially expressed in ga dolphins compared to the other locations. hypothyroidism has been previously described in ga dolphins and, interestingly, a few of the genes that we identified are involved in the proper function of the thyroid. the analysis of ga animals alone, correlated with contaminant load measured, showed the activation of genes involved in stress response, dna repair and skin damages, uv and/or viral infection-induced. the transcriptomic data analysis will be a first step towards identification of markers/patterns indicative of exposure to chemical contaminants as well as marine toxins and will promote an understanding of toxic mechanisms and/or pathways that are currently not well understood in marine mammals. session 5. chairman: v arizza, university of palermo, palermo, italy immuno-active and antimicrobial molecules molecular basis of self/nonself recognition in ciliate mating type systems p luporini, c alimenti, a vallesi department of environmental and natural sciences, university of camerino, camerino, italy like numerous other organisms, ciliates alternate their life cycles between vegetative (mitotic) growth and sex. however, ciliate sex is unique for at least two major aspects: (i) it becomes manifest as temporary cytoplasmic fusion between cells that carry diploid sets of genes and may equally be identical or different in their genotypes, and (ii) is under the control of a genetic mechanism of mating-types which may either be only two as is the case in various species of paramecium and blepharisma, or multiple as is the case in various species of tetrahymena, euplotes, stylonychia and the hypotrichs in general. while the mating type binary systems recall the duality of the sex of the multi-cellular organisms, the multiple systems find much closer counterparts with the self/non-self recognition mechanisms that permit animals to react immunologically against invaders, and fungi and flowering plants to decide their evolution between self-sterility and self-fertility strategies. a persevering research interest on the multiple mating type systems of different species of euplotes lead us to obtain relevant information on the genes and the cell type-specific diffusible signal proteins (pheromones) that interplay in the control of these systems. functionally most relevant was the finding that, in full accord with their genetic determination provided by multiple sets of single-locus genes, these euplotes pheromones are represented by species-specific family of structurally homologous proteins which, as such, can compete with one another for binding to their cell receptors in either autocrine (self), or paracrine-like (non-self) fashion. cells grow in response to the pheromone binding that signals self, and temporarily shift to the sexual stage in response to the pheromone binding that signals non-self. functional activity of the ciliate euplotes raikovi pheromone in human cell lines s picchietti1, e catalani1, c belardinelli1, g casini1, am fausto1, a vallesi2, c alimenti2, d cervia1 1department for innovation in biological, agro-food and forest systems (dibaf), university of tuscia, viterbo, italy 2department of environmental and natural sciences, university of camerino, camerino, italy ciliates synthesize cell type-specific chemical signals, designated pheromones, that in association with their mating type systems control the switching between the reproductive (mitotic growth) and mating (non-reproductive, sexual) stages of their life cycles. in the protozoan ciliate euplotes, pheromones have been isolated and characterized for their genetic determination and molecular structures and interestingly, euplotes raikovi pheromones (designed as er-1, er-2, and so forth) have been functionally linked with growth factors of higher eukaryotes as for instance the epidermal growth factor and the cytokine interleukin-2 (il-2). however, the activity of pheromones in mammalian cells and their underlying cellular basis are currently unknown. in an attempt to elucidate the pharmacological features and the functional effects of er-1 in human cells, we have performed an in vitro study using jurkat cells (il-2 producing human t lymphocyte cell line, commonly used to study t cell signalling), that express functional il-2 receptors. in particular, the present study was designed to investigate the role of er-1 on relevant aspects of cell growth and cytokine gene expression and/or release. since the cross-talk among il-2 system, t-cells, and malignant cells plays an important role in brain tumors, we also sought to investigate the possible effects of er-1 on human glioma cell line (u-373 cells). our results provide the first insight into the impact of the euplotes raikovi pheromones on the physiology of human cells, showing that er-1 may increase the growth of tcells, but not glioma cells, cultured under restrictive culture conditions. in addition, er-1 was found to significantly increase gene expression levels and production of specific cytokines in jurkat cells. the work allowed us to parallel the events which occur in vitro with those occurring in the microorganism ecosystem and discover euplotes raikovi as a fruitful mine of compounds with bioactive properties in mammalian cells. 46 hepatopancreatic transcript expression in the crayfish astacus leptodactylus functional genomic analysis and the effect of two stereomers of the crustacean hyperglycemic hormone (chh) m tom1, a mosco2, a pallavicini2, c manfrin2, g de moro2, pg giulianini2 1israel oceanographic and limnological research, p.o.b. 8030, haifa 31080, israel 2department of life sciences, university of trieste, trieste, italy the crustacean hyperglycemic hormone (chh) is present in many decapods in different isoforms, whose specific biological functions are still poorly understood. this hormone plays a key role in crustacean stress responses (lorenzon, 2005). recently we obtained the first chemical synthesis of the chiral isoforms of the chh of astacus leptodactylus carried out by solid phase peptide synthesis coupled to native chemical ligation (mosco et al., 2012). the synthetic 72 amino acid long peptide amides, containing lor dphe3 and (glp1, d-phe3) were tested for their biological activity on hepatopancreatic transcript expression. sixteen a. leptodactylus females were used, divided into 4 female groups. two groups were injected by dor l-chh, respectively (0.5 µg/female in 100 µl phosphate buffered saline). a control group was injected by the hormone carrier and a fourth control group of females neither eyestalk ablated nor chh injected. rna extracted from hepatopancreas were sequenced using the illumina technology in order to: 1) establish the hepatopancreatic transcriptome from pooled rna and 2) to perform gene expression analysis from individual samples. preliminary results indicated that the expression profiles of three of the experimental groups, namely the native, the sham injected and the l-chh injected females are qualitatively quite similar and differ from the d-chh injected group. consequently, the statistical analysis was divided into two parts. first, the sham injected profiles were compared to those of the native females to demonstrate differences caused by the extirpation. second, the profiles of the four sham-injected females were compared to the two most different profiles of the d-chh and lchh injected individuals, respectively, to demonstrate putative effect of each of the two hormone isomers. the overall expression trend and level resulted from statistical testing is presented. our data clearly shown that the eyestalk-ablation did not make dramatic gene expression alterations in the hepatopancreas and that the effect of d-chh is much more pronounced that of the l-chh in both terms of number of altered genes and their abundance (higher rpkm). the effect of the d-chh in terms of transcript number is mostly attenuation of expression. 47 e randelli, f buonocore, p tranfa, g scapigliati isj 9: xx-xx, 2012 isj 9: 82-88, 2012 issn 1824-307x research report immunotoxic action of cyclosporin a on the humoral immune response of galleria mellonella pupae mj fiołka department of immunobiology, institute of biology and biochemistry, maria curie-skłodowska university, lublin, poland accepted may 17, 2012 abstract cyclosporin a (csa) was inoculated into the hemocel of pupae in the initial phase of the immune response (at the time of immunization) and in the effector phase of the immune response (within 18 h post immunization). the results obtained indicate suppression of the humoral immune response of pupae after treatment with the antibiotic csa in both the initial and the efector phase. the immunosuppressant decreased the lysozyme activity against micrococcus luteus and the activity of antibacterial peptides against escherichia coli in the hemolymph within 3 days after injection. the peptide activity decreased more rapidly in comparison to the activity of lysozyme. after 72 h incubation, the reduction in the lysozyme activity was about 55 % in comparison to the activity in immunized insects and only traces of activity against e. coli were observed. differences between the untreated and immunosuppressant-treated insects were statistically significant. the decrease in the lysozyme activity and the antibacterial peptide activity was correlated with loss of protective immunity against pseudomonas aeruginosa. key words: cyclosporin a; immunity; lysozyme activity; peptide activity; galleria mellonella introduction cyclosporin a (csa) is a cyclic undecapeptide isolated from the fungi tolypocladium inflatum and cylindrocarpon lucidium. it is a powerful immunosupressive agent used in transplantation immunology (britton and palcios, 1982; white and calne, 1982; thomson, 1983; thomson et al., 1984; weil, 1984; shevach, 1985). csa has clinical application in the treatment of autoimmune disorders (laupacis et al., 1982). in human monocytes and macrophages, csa induced apoptosis and inhibited neutrophil functions in vitro. csa appeared to be effective in lowering chemotaxis, superoxide anion production and lysozyme release induced by different agonists (spisani et al., 2001). cyclosporins exhibit potent immunosuppressive, antifungal, antiparasitic, antiviral and insecticidal activities. they are able either to kill the infected ___________________________________________________________________________ corresponding author: marta j fiolka department of immunobiology institute of biology and biochemistry maria curie-skłodowska university lublin, poland e-mail: mfiolka@poczta.onet.pl insect or to incapacitate its immune system (vilcinskas et al., 1999). cyclosporins showed insecticidal activity against mosquito larvae (weiser and matha, 1988). csa caused pathological changes in all tissues of culex pipiens larvae, but the targets of csa were mainly mitochondria, which inflated their cristae, disintegrated and changed into vacuoles (weiser et al., 1989). it was detected that in chironomus riparius larvae this immunosuppressant inhibited the p-glycoprotein related pump, which was able to remove xenobiotics out of the body fluid (podsiadlowski et al., 1998). the wax month galleria mellonella, a laboratory model species, is the subject of much current research on insect immunity (jiang et al., 2010). the studies in g. mellonella revealed that lipophorin, a major insect protein, is the csa-binding protein (vilcinskas et al., 1997). csa added to cultivation medium at sublethal concentrations inhibited phagocytosis of isolated g. mellonella plasmatocytes (vilcinskas et al., 1999). isolated plasmatocytes incubated with csa exhibited cytoskeleton alterations. csa enhanced nodule formation accompanied by melanization in g. mellonella larvae when injected at sublethal concentrations and coated on particles. 82 fig. 1 lysozyme activity in the hemolymph of g. mellonella pupae after immunization and injection with csa. samples of pupae hemolymph of: hn-non-immunized control pupae, hiafter immunization with lps, hi-c15 after immunization with lps and injection with csa at dose 15 µg/insect, hi-c22.5 after immunization with lps and injection with csa at dose 22.5 µg/insect. bars represent mean ± sd calculated from five independent experiments; b vs a p < 0.001, c vs b p < 0.05. csa suppressed the humoral immune response of g. mellonella larvae (fiołka, 2008). csa moderately decreased the lysozyme activity and significantly decreased the antibacterial activity of peptides against escherichia coli. immunosuppressive effects were expressed in larvae treated with cyclosporin a both in the initial and the effector phase of the immune response. insects with an immune response impaired by the csa action lost their protective immunity to the pathogen pseudomonas aeruginosa. since there are no data on the effect of csa on the humoral immune response in lepidopteran pupae, it was advisable to analyze the action of this immunossuppressant on the antibacterial activity and protective immunity in g. mellonella pupae. materials and methods target insects young pupae of the greater wax moth galleria mellonella l. (lepidoptera: pyralidae) were used as an insect model system to study the modulation of the antibacterial cell-free immune response in g. mellonella under the influence of csa. the target animals were incubated on dark honey drawn combs at 28 oc and 70 % relative humidity under total darkness. immunization and immunosuppressant the induction experiments were performed on 2to 3-day-old pupae removed from coccons directly before immunization. the humoral antibacterial response was generated by intrahemocelic inoculation of insects with lps of pseudomonas aeruginosa (sigma) (39 ng/pupae) using a hamilton micrometer syringe. immune hemolymph for antibacterial assays was collected 24 h after immunization. during the experiments, the insects were kept in an incubator at 28 oc and relative humidity of ~70 %. control hemolymph was collected from unvaccinated insects. each experimental group consisted of at least twelve animals. cyclosporin a (fluka) was dissolved in 80 % ethanol and 2 µl were injected into the hemocel of the pupae. hemolymph collection after incubation, in each case the hemolymph was collected and tested for antibacterial activity of lysozyme and antibacterial peptides, using the inhibition zone assays in agar plates. the pupae were bled by piercing the region of the thorax and gentle squeezing the insect body. hemolymph (5 µl volume) was taken up in capillaries and pipetted into ice-cold eppendorf tubes containing sterile water (dilution 1:5) with a trace of phenylthiourea to prevent melanization of the blood. however, the phenylthiourea was omitted in hemolymph samples used for determination of the lysozyme titres due to its inhibitory effect on the lysozyme activity (jarosz, 1994). samples of diluted hemolymph were used immediately for the antibacterial assays. antibacterial assay for lysozyme activity lysozyme activity was quantified by inhibition zone assays in agar plates as described by mohrig and messner (1968), using freeze-dried micrococcus luteus at the concentration of 0.7mg/ml of the assay medium. each 10 cm petri dish contained 10 ml of 0.066 m sörensen buffer (ph 6.4), 100 mg of agarose (sigma) and 0.7 mg of streptomycin sulphate (sigma) to inhibit the growth of bacterial contaminators. wells with diameters of 2.7 mm were punched in the agar layer and then 83 fig. 2 lysozyme activity in the hemolymph of g. mellonella pupae collected 24, 48 and 72 h after immunization and injection with csa in the initial phase of the immune response (at the time of immunization). hn-hemolymph of non-immunized, control pupae; hi-24, hi-48, hi-72 hemolymph of immunized pupae, collected 24, 48, 72 h after immunization; hi-c-24, hi-c-48, hi-c-72 hemolymph of immunized and injected with csa pupae, collected 24, 48 and 72 h after immunization. bars represent mean ± sd calculated from four independent experiments; b vs a p < 0.001, c1 vs b1, c2 vs b2 and c3 vs b3 p < 0.05. filled with the hemolymph samples to be assayed. different concentrations of hen egg-white lysozyme (sigma) were used as standard. diameters of the lytic zones were measured after incubation of the plates at 28 oc for 24 h. the antibacterial activity of hemolymph expressed in terms of lysozyme activity (ec.3.2.1.17) is given in equivalents to µg/ml eggwhite lysozyme. maximum activity after immunization was taken as 100 %. antibacterial assay for peptide activity the antibacterial activity of peptides was routinely recorded as the diameter of inhibition zones around the wells (diameter 2.7 mm) in a thin layer of soft (0.7 %) nutrient agar inoculated with the exponential phase cells of e. coli d31 (cgsc 5165), a bacterium sensitive to cecropin action (boman et al., 1974). pupal hemolymph to be assayed was added into the well (faye and wyatt, 1980). the assay plates were prepared by spreading 10 ml of soft agar medium on sterile 10 cm glass petri dish. nutrient broth contained streptomycin sulphate (100 µg/ml) and a few crystals of phenylthiourea to inhibit hemolymph melanization due to phenoloxidase activity. anti-e. coli activity in g. mellonella hemolymph samples is given in equivalents to µg/ml of the synthetic peptide of cecropin a hyalophora cecropia (sigma) used as the standard. inhibition zones were recorded around the wells after 24 h incubation at 28 oc. maximum activity after immunization was taken as 100 %. protective immunity the protective immunity (100 minus percent of mortality) against p. aeruginosa, a highly virulent bacterium for lepidoptera (strain h3), was calculated from the cumulative mortality of g. mellonella on day 2 due to p. aeruginosa septicaemia (jarosz, 1994). overnight broth cultures of the bacterial pathogen were microbiologically standardized by the agar colony count, and a cell suspension of the required density (about 0,3x102 in 2 µl for pupae) was prepared in saline w (weevers, 1966) a physiological salt solution for lepidoptera. during the 24 h postimmunization, pupae of g. mellonella treated with an immunosuppressant were challenged with twelve to fifteen lethal viable cells of the insect bacterial parasite. the insects that had been immunized with the p. aeruginosa lps but not given the immunosuppressant were also challenged with a multifold lethal dose of living cells of p. aeruginosa. the onset of the disease was observed then and mortality due to pseudomonas saepticaemia was recorded daily. statistical analysis the cochran-cox test was used to determine statistical significance. the differences between statistical parameters were considered significant at p < 0.05 and p < 0.001. results and discussion lysozyme activity the lysozyme activity in the hemolymph of g. mellonella pupae was determined after immunization with p. aeruginosa lps and injection of csa at different doses (fig. 1). afrer immunization, the lysozyme activity was significantly 84 fig. 3 lysozyme activity in the hemolymph of g. mellonella pupae after immunization and injection with csa in the initial phase of the immune response (at the time of immunization) and in the effector phase of the immune response (within 18 h post immunization); hn hemolymph of non-immunized, control pupae; hi hemolymph of immunized pupae, hi-ci hemolymph of immunized and injected with csa pupae in the initial phase of the immune response, hi-ce hemolymph of immunized and injected with csa pupae within 18 h post immunization in the effector phase of the immune response. bars represent mean ± sd calculated from four independent experiments; b vs a p < 0.001, c vs b p < 0.05. increased in comparison to the activity in the control non–immunized insects. previously, a correlation between lysozyme activity and induced immunity was revealed by stephens-chadwick (1970, 1975) and jarosz (1970, 1985, 1988). in the hemolymph of immunized pupae treated additionally with csa at a dose of 15 µg/insect, the lysozyme activity was slightly decreased. however, after a higher dose of csa (22.5 µg/insect), the activity was decreased significantly by about 40 % in comparison to the lysozyme activity in the hemolymph of the pupae that had been immunized but non-treated with immunosuppressant (fig. 1). the lysozyme activity in the hemolymph of immunized pupae and those immunized and additionally treated with csa at a dose of 15 µg/insect was analyzed 24, 48 and 72 h after immunization and inoculation with the antibiotic (fig. 2). the lysozyme activity was significantly decreased in the hemolymph of the immunized and additionally treated with csa pupae. effective suppression of the lysozyme-type cell-free immune response was detectable for 3 days. after 24 h, the reduction in the lysozyme activity was about 39 %, after 48 h 58 % and after 72 h 55 % in comparison to the activity in the immunized insects (fig. 2). the immunosuppressant was inoculated into the hemocel of the pupae in the initial phase of the immune response (at the time of immunization) and in the effector phase of the immune response (within 18 h post immunization). after the inoculation with the antibiotic in the initial phase, the lysozyme activity was decreased by about 33 % at time 0, but after injection of csa within 18 h post immunization, the activity was less inhibited by 20 % in comparison to the immunized pupae (fig. 3). however, after treatment with csa in both phases of the immune response, the differences were statistically significant. csa at a dose of 22,5 µg/ml effectively decreased the lysozyme activity in the hemolymph of g. mellonella pupae, while the larvae of this insect were more sensitive. csa at a dose of 15 µg/ml had already resulted in significant changes in the lysozyme activity (fiołka, 2008). the lysozyme activity after inoculation with csa did not decrease as rapidly in the pupal hemolymph as in the larval hemolymph. the research on larvae showed that the changes in the lysozyme activity were correlated with the changes in the protein level observed after immunoblotting with antibodies against g. mellonella lysozyme (fiołka, 2008). antibacterial peptide activity antibacterial peptide activity against e. coli in the hemolymph of g. mellonella pupae was analysed 24, 48 and 72 h after immunization of the pupae and injection of csa in the initial phase of the immune response. the peptide activity decreased more rapidly in comparison to the activity of lysozyme during 3 days. the activity against e. coli evaluated after 24 h was reduced by 50 %, and after 48 h by 53 %, in comparison to the activity in the hemolymph of pupae immunized with lps but not treated with the antibiotic (fig. 4). the differences between the untreated insects and those treated with the immunosuppressant were statistically significant. after 72 h incubation, only traces of activity against e. coli were observed. 85 fig. 4 antibacterial activity in the hemolymph of g. mellonella pupae collected 24, 48 and 72 h after immunization and injection with csa in the initial phase of the immune response (at the time of immunization). hn-hemolymph of non-immunized, control pupae; hi-24, hi-48, hi-72 hemolymph of immunized pupae, collected 24, 48, 72 h after immunization; hi-c-24, hi-c-48, hi-c-72 hemolymph of immunized and injected with csa pupae, collected 24, 48 and 72 h after immunization. bars represent mean ± sd calculated from four independent experiments; b vs a p < 0.001, c1 vs b1 and c2 vs b2 p < 0.05. injection of csa at time 0, immediately after immunization, resulted in a ca.74 % activity decrease in comparison to the activity in the immunologically stimulated pupae. the administration of the antibiotic within 18 h after the immunization with lps p. aeruginosa resulted in a 24 % activity reduction (fig. 5). in both cases, the differences were statistically significant. the results indicate that inhibition of the antibacterial activity against e. coli with csa in the effector phase was less effective than in the initial phase, as in the case of the lysozyme activity. previously, it was observed in larvae that the csa immunosuppressant administered in the initial phase of the immune response almost completely inhibited the activity against e. coli, while in pupae, the suppressive effect was less pronounced. it is known that, in g. mellonella larvae, the reduction in the titres of anti-e. coli peptide activity after injection of csa was associated with inhibition of peptide synthesis (fiołka, 2008). probably, in the pupae the effect is similar to that observed in the larvae. protective immunity the decrease in the lysozyme activity and antibacterial peptides in g. mellonella pupae allowed the enthomopathogenic bacterium to multiply in the insect celomic cavity. pupae with the immune response impaired by the csa action lost their protective immunity to the insect pathogen p. aeruginosa. in the csa-treated pupae, the protective immunity diminished to about 70 % of the maximal resistance detected in immunized insects. all the control non-immunized insects died due to p. aeruginosa bacteremia (fig. 6). however, the g. mellonella pupae were more susceptible to infection with a lethal dose of the insect pathogen p. aeruginosa. about 30 % fewer pupae than larvae survived infection with the enthomopathogenic bacterium after treatment of insects with csa. the correlation between the protective immunity against p. aeruginosa and the antibacterial activity of hemolyph in g. mellonella was observed by jarosz (1979, 1984b, 1985). these results indicate suppression of the humoral immune response of pupae after treatment with the antibiotic csa, both in the initial and the efector phase of immune response. the immunosuppressant decreased the activity of hemolymph against m. luteus and e.coli during 3 days after injection. the decrease in the lysozyme activity and antibacterial peptide activity was correlated with the protective immunity against p. aeruginosa. in pupae treated with another known immunosuppressant agent, hydrocortisone, reduced titres of the antibacterial peptide activity and a considerably decreased activity of hemolyph lysozyme were found (jarosz 1994a, 1994b). hydrocortisone substantially depressed the hemolymph bactericidal activities induced by enterobacter cloacae in two lepidopteran pupae, g. mellonella and pieris brasicae (jarosz, 1994b). the protective immunity dropped in the hydrocortisoneinjected pupae of g.mellonella, as in the csainjected pupae. this suggests that csa like hydrocortisone may affect the hemolymph in cells that are active in phagocytosis, and exhibits the immunotoxic action on the humoral immune response of g. mellonella pupae. 86 fig. 5 antibacterial activity in the hemolymph of g. mellonella pupae after immunization and injection with csa in the initial phase of the immune response (at the time of immunization) and in the effector phase of the immune response (within 18 h post immunization); hn hemolymph of non-immunized, control pupae; hi hemolymph of immunized pupae, hi-ci hemolymph of immunized and injected with csa pupae in the initial phase of the immune response, hi-ce hemolymph of immunized and injected with csa pupae within 18 h post immunization in the effector phase of the immune response. bars represent mean ± sd calculated from four independent experiments; b vs a and c vs b p < 0.001. fig. 6 protective immunity of g. mellonella pupae against p. aeruginosa after immunization (i), immunization and injection with csa (i-c) and of non-immunized pupae (n-i). bars represent mean ± sd calculated from free independent experiments; b vs a and c vs a p < 0.001. 87 references boman hg, nilsson-faye i, paul k, rasmuson t. insect immunity. i. characteristics of an inducible cell-free antibacterial reaction in hemolymph of samia cythia pupae. infect. immun. 10: 136-145, 1974. britton s, palacios r. cyclosporin a usefulness, risks and mechanisms of action. immunol. rev. 65: 5-22, 1982. faye i, wyatt gr. the synthesis of antibacterial proteins in isolated fat body from 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as the major cyclosporin-binding protein in the hemolymph of the greater wax moth galleria mellonella. comp. biochem. physiol. 117c: 41-45, 1997. weevers r de g. a lepidopteran saline: effects of inorganic cation concentrations on sensory, reflex and motor responses in herbivorous insect. j. exp. biol. 44: 163-175, 1966. weil c. cyclosporin a: review of results in organ and bone-marrow transplantation in man. med. res. rev. 4: 221-265, 1984. weiser j, matha v, zizka z, jegorov a. pathology of cyclosporin a in mosquito larvae. cytobios 59: 143-150, 1989. weiser j, matha v. the insecticidal activity of cyclosporins on mosquito larvae. j. invertebr. pathol. 51: 92-93, 1988. white djg, calne, ry. the use of cyclosporin a immunosuppression in organ grafting. immunol. rev. 65: 115-131, 1982. 88 abstract target insects immunization and immunosuppressant hemolymph collection antibacterial assay for lysozyme activity antibacterial assay for peptide activity protective immunity statistical analysis results and discussion italian society for developmental and comparative immunobiology (isdci) isj 8: 33-46, 2011 issn 1824-307x report of meeting xiith scientific meeting of the italian association of developmental and comparative immunobiology (iadci), 16 18 february 2011, hotel s. marco, monteortone, padua, italy organizers: l ballarin, f cima, v matozzo, p venier department of biology, university of padua, padua, italy plenary lecture live and let die: hemocytes in the crustacean war against infection vj smith scottish oceans institute, university of st andrews, scotland whilst it has long been known that the circulating hemocytes are major effectors in invertebrate immune systems much still remains to be learnt about these cells, as new work is starting to show. this presentation will give an overview of the hemocytes in decapod crustaceans and describe their various roles in defense against pathogens and opportunists. it will give consideration to their production, and likely development into mature functional units and discuss the range of bioactive proteins and factors that they produce. included here will be antimicrobial peptides, opsonins and recognition factors, all of which may act outside the cell. fluxes and changes in the size and composition of the hemocyte populations caused by certain extrinsic and intrinsic factors will be described and the question asked: is the hemocyte titer a reliable indicator of physiological or environmental stress? finally, the talk will highlight the importance of hemocyte death in host defense and, crucially, how the nature of this death may be of greater significance than previously thought. session 1. chairman: n. parrinello, university of palermo, palermo, italy non-self recognition and immunocyte activation expressed sequences denoting receptors and other proteins progressively reveal the molecular basis of the mussel immunity l varotto1, m gerdol2, s domeneghetti1, c manfrin2, mg procopio1, u rosani1, a pallavicini2, p venier1 1department of biology, university of padua, padua, italy 2department of life sciences, university of trieste, trieste, italy the current years testify the expansion of genomic/post-genomics studies, and the setting up of new software solutions for project/data management. in particular, massive dna sequencing accomplished with sanger dideoxy terminators or third-generation machines is rapidly increasing the sequence data publicly available for bivalve mollusks. as regards mytilus galloprovincialis, we further investigated mytibase (knowledge-base of 7,112 transcript sequences, assembled from 18,788 high-quality ests and partially devoid of functional annotation). mytibase includes almost all the nine domains denoting innate pathogen recognition receptors (ppr). in addition to the amp precursors, the abundance and molecular variety of mussel sequences with complement c1q or c-type lectin or fibrinogen-like motifs indicate a large repertoire of recognition molecules. evolutionary related to the tnf-like domain, the c1q domain characterizes proteins active in complement activation, modulatory immune functions, apoptotic cell clearance, coagulation, embryonic development and tissue homeostasis. the 168 predicted c1q proteins of m. galloprovincialis show remarkable sequence diversity, lack of central collagen-like repeats and possess the globular c1q motif known to provide great flexibility in the ligand binding; hence, they are expected to act as secreted prr. recent data referred to the most abundant c1q est cluster (mgc00284) indicated its wide expression and significant modulation in mussels infected with gram positive or gram negative bacteria (dci 34(9):92634, 2010). among the heterogeneous group of mussel transcripts with carbohydrate binding domains, the most abundant and highly diverse in sequence are those denoting (ca2+-dependent) ctype lectins and fibrinogen-related proteins (frep). they are regarded as candidate prr since many c-type lectin proteins support pathogen-specific responses in caenorhabditis elegans and speciesspecific frep expansion relevant to immunity occurred in snails and mosquitoes. 33 using a multiple search strategy, 1820 expressed mytibase sequences have finally been selected to design probes and build a dnamicroarray of immuno-related sequences. the performance of the new immunochip has been positively ascertained with hemocytes of vibroinjected mussels. antarctic teleost toll-like receptors s ferraresso1, s varriale2, mr coscia2, u oreste2, l bargelloni1 1department of public health, comparative pathology and veterinary hygiene university of padua, padua, italy 2institute of protein biochemistry, cnr napoli, italy the pattern recognition receptors (prr) of the innate immune system sense the invariant molecular patterns present in microorganisms. secreted prrs comprise ficolins, collectins, and pentraxins, whereas transmembrane prrs include the toll-like receptor (tlr) family and the c-type lectins. tlrs are present in both the invertebrate and vertebrate lineages; in teleosts, orthologs of mammalian tlr6 and tlr10 are absent, however the tlr11 family includes additional members. all tlrs share the same molecular architecture consisting of an n-terminal ectodomain including various numbers of leucine-rich repeat motifs, followed by a membrane-proximal cysteine-rich region, a transmembrane domain and a cytoplasmic tir domain. the innate immune response of antarctic teleosts seems to be more relevant than the adaptive response, and, in turn, tlr molecules could assume a more important role in triggering the immune system. in addition cold-adaptive modifications could have shaped the molecular structure of the tlrs in unpredictable manner. a fragment of cdna coding for tlr9 and tlr2 was pcr-amplified from trematomus bernacchii and chionodraco hamatus using primers designed on conserved regions across five publicly available teleost tlr9 and six tlr2 sequences. full-length sequences were subsequently obtained in a rapid amplification of cdna ends (race). t. bernacchii and c. hamatus tlr9 sequences are respectively 1056 aa and 1054 aa long and share similarities in structure with previously identified teleost tlrs. structural homology is conserved also for t. bernacchii (804 aa) and c. hamatus (802 aa) tlr2, both displaying 6 lrrs and an intracellular tir domain. all putative orthologs of tlr9 and tlr2 in the teleost fish were identified through blastp searches against either ncbi or ensembl databases. multiple alignments of teleost tlr9s and tlr2s were obtained using different approaches (muffit, muscle, codonalign) and sequence analyses at the amino acid as well as at the codon level were carried out to investigate the role of natural selection in the evolution of antarctic fish tlrs. molecular models of the ectodomains of both the sequenced tlr2s were constructed using the mammalian structure of mouse tlr2 (3a7c) as template. the obtained models, validated using the procheck programme were minimized with the gromax3.2 package. molecular dynamic simulations were performed in water for 10 ns. the resulting models were compared to the mammalian structure to identify the differing regions. phagocyte behavior during the colonial blastogenetic cycle in the compound ascidian botryllus schlosseri f cima department of biology, university of padua, padua, italy colonies of the ascidian botryllus schlosseri experience a cyclical generation change (takeover) during which adult zooids stop their filtering activity, their tissue undergo cell death by apoptosis and are progressively resorbed. in the meantime, a new blastogenic generation reaches its functional maturity, opens siphons and starts filtering. during the blastogenetic cycle, the phagocytic differentiation pathway of the circulating immunocytes plays a key role: the presence of both hydrolytic enzymes and mediators of local inflammation supports the hypothesis that the phagocytic cell line maintains characteristics of a primordial system with high functional versatility. phagocytes cyclically change their morphology and frequency. during the mid-cycle, hyaline amoebocytes are numerous in blood circulation, inside the tunic and free of moving on the surface of the tunic that internally covers the oral siphon, where they play an immunosurveillance role of the pharynx by recognizing and phagocytizing foreign particles, and, after exposure of colonies to bacterial spores, forming a transitory plug in the siphonal lumen by exocytosis of floccular and colloidal material rich of heparin, histamine and proteases. in the takeover, the frequency of hyaline amoebocytes falls abruptly since, by engulfing apoptotic cells, they change their shape becoming large and spherical macrophage-like cells. these scavenger phagocytes are massively recruited from the circulation to the dying tissues of old zooids, where they assure the clearance of senescent cells. this is fundamental for the progression of the takeover as during this phase, lasting 24-36 h, colonies do not feed and rely uniquely on the recycling of nutrients deriving from the digestion of senescent cells. the massive ingestion of effete cells causes, in turn, an increase in reactive oxygen metabolite production and nitric ion release leading to the death of phagocytes and subsequent clearance by other phagocytes, so that a “russian doll effect” can be observed. finally, large macrophage-like cells accumulate in the pharyngeal lacunae and are continuously eliminated through the peribranchial chamber and then the cloacal siphon with a discharging mechanism never previously described which continues in the first phases of the mid-cycle. the consistent disappearance of large phagocytes from the circulation is counterbalanced by a population of new, undifferentiated cells (hemoblasts) already beginning from the late takeover. 34 immune roles of a rhamnose-binding lectin in the compound ascidian botryllus schlosseri n franchi1, f schiavon1, m carletto1, f gasparini1, g bertoloni2, sce tosatto1, l ballarin1 1department of biology, university of padua, padua, italy 2department of histology, microbiology and medical biotechnology, university of padua, padua, italy the present communication describes the immune role played by a recently identified member of the rhamnose-binding lectin (rbl) family from the colonial ascidian botryllus schlosseri. b. schlosseri rbl (bsrbl) can activate phagocytes through: i) induction of their directional movement towards the source of the molecule; ii) modification of cytoskeleton, required for shape changes; iii) stimulation of the respiratory burst, and consequent production of reactive oxygen species (ros) with microbicidal activity, including superoxide anions and peroxides; and iv) increase in the ability to phagocytose foreign particles. rbl also induces the synthesis and release, by cytotoxic morula cells (mcs), of cytokines recognized by anti-il1α and anti-tnfα antibodies. at high concentrations, bsrbl induces degranulation of mcs and the consequent release of the cytotoxic enzyme phenoloxidase into the medium. results are consistent with the existence of cross-talk between b. schlosseri immunocytes (phagocytes and mcs). in addition, a three dimensional model for bsrbl is presented. evolution of the genetic mechanism of self/nonself recognition in the protozoan ciliate euplotes a vallesi1, c alimenti1, s federici2, g di giuseppe2, f dini2, p luporini1 1department of environmental and natural sciencesi, university of camerino, camerino, italy 2department of biology, university of pisa, pisa, italy in the phylogenetic trees (mostly based on comparisons of ssu-rrna gene sequences) of the nearly 100 morpho-species of the most cosmopolitan and ubiquitous ciliate, euplotes, the marine complex of species e. crassus-e. minuta-e. vannus systematically appears as the latest, deeply divergent branch. in relation to the mating type systems that represent the genetic mechanism by which euplotes, and ciliates in general, control the switching from the growth (asexual) stage to the mating (sexual) stage of the life cycle, this species complex has historically been credited by the evolution of multiple mating type systems characterized by: (i) a genetic control of the mating types provided by multiple alleles inherited at the single genetic locus mat and mutually regulated by relationships of hierarchical dominance (e. g. mat1>mat-2>mat-3 ad so forth), and (ii) the synthesis of mating type-factors (pheromones) represented by water-insoluble, membrane-bound proteins. working on e. crassus, we have first isolated and structurally characterized soluble, water-borne pheromones, thus demonstrating that (contrary to the historically held concept) this species and, most likely, the whole e. crassus-e. minuta-e. vannus species complex constitutively secrete their pheromones into the extracellular environment as it occurs in other, earlier branching (more ancient) euplotes species. then, based on the knowledge of pheromone amino acid sequences, it was possible to clone and structurally characterize a set of different pheromone coding genes. it was found that each cell type synthesizes one pheromone that is shared in common with other mutually mating compatible cell types, plus one (if mat-homozygous) or two (if mat-heterozygous) other pheromones which are cell type-specific. the implication that arises from these observations is that, in euplotes species, the mat-locus underwent evolutionary duplication. while early branching (more ancient) euplotes species (such as e. raikovi and e. nobilii) show this locus as single copy, e. crassus and its late branching related species would carry it replicated into two distinct copies. one copy would retain the ancestral state as multi-allelic locus deputed to control the synthesis of pheromones which are specific for each cell type, while the new copy would be manifested as mono-allelic locus deputed to control the synthesis of a pheromone which is shared in common by a group of different, but mutually interbreeding cell types. as such, this novel pheromone might function as a selfrecognition signal not for a single cell type, but for a population of different and interbreeding cell types, and ensure that mating pairs are formed only between different cell types of the same population. in vitro characterization of the cytokine drosophila helical factor d malagoli1, a accorsi1, d conklin2, m filaferro3, m mandrioli1, m pinti3, s sacchi4, e ottaviani1 1department of animal biology, university of modena and reggio emilia, modena, italy 2department of computer science and artificial intelligence, university of the basque country, san sebastián, spain and ikerbasque, basque foundation for science, bilbao, spain 3department of biomedical sciences, university of modena and reggio emilia, modena, italy 4department of biological sciences, george washington university, washington dc, usa drosophila helical factor (hf) is a protein discovered through the qt method, an algorithm specifically designed for finding helical cytokines. since in vivo experiments suggested the involvement of hf in drosophila melanogaster immunity, we have proceeded with the characterization of hf functions in the macrophagelike drosophila embryonic hemocytes, sl2 cell line. qpcr results demonstrated that hf gene is induced in the sl2 cell line, after either 6 or 24 h incubation with escherichia coli-purified peptidoglycan. the silencing of hf expression through rnai resulted in the reduced capability of synthesizing antimicrobial peptides (amp) after exposure to heat-inactivated e. coli. the effects of the recombinant peptide rhf 35 have also been tested in the sl2 cell line. rhf promotes the expression and triggers the release of hf from the hemocytes, and stimulates the synthesis of the antimicrobial peptides (amp) defensin and drosomycin, without any further immune stimulation. consistent with the output of the qt method, which predicts hf as a secreted protein, chromatin immune-precipitation experiments confirmed that hf does not bind dna, excluding that it acts as an immune-regulated transcription factor. finally, rhf neither exerts chemotactic action nor triggers bacterial phagocytosis in sl2 cells. altogether, our data supports the prediction that hf is an helical cytokine produced and secreted by the hemocytes and it is mainly involved in the regulation of the humoral component of the immune response of d. melanogaster. session 2. chairman: p. luporini, university of camerino, camerino, italy effector mechanisms of immune responses the mast cell: comparative aspects g chieffi baccari department of life sciences, 2nd university of napoli, caserta, italy mast cells (mcs) are paracrine cells, ubiquitous in connective tissue of the majority of vertebrates. they are best known for their involvement in hypersensitivity, allergic and anaphylactic reactions, but they are also implicated in angiogenesis, in inflammation as well as in natural and acquired immunity. the mast cells, together with the basophils, are the only cells producing histamine and expressing membrane receptors for ige (fceri). although fceri receptor represents a relatively recent acquisition, since ige appeared with the mammals, either histamine either fceri-like receptor are present in mcs of teleost fishes. further analogies among vertebrate mcs are: mediator contents (heparin, proteases, serotonin, phosphatases), expression of membrane receptors (c-kit, tlr, cr3) and response to substances inducing mc degranulation (compound 48/80, chlorpromazine, capsaicin, substance p, bacterial/parasitic toxins). the neuroendocrine activity (i.e. secretion of gnrh) in birds and the production of antimicrobial compound in fish (piscidins, pleurocidins) are some unique roles the mcs are known to play. among invertebrates mc-like cells has been described in ascidians. they contain histamine and heparin, tryptase and are induced to degranulate by compound 48/80. in arthropoda, some leucocytes show ultrastructural features of rodent mcs. a fascinating hypothesis suggests that the progenitor of mcs could have origin in an ancestral leukocyte showing phagocytic activity, in the context of a primitive local immunity. the mcs could have acquired new molecular strategies without losing many of the functions accumulated during million years of evolution. they should have preserved their defensive function against pathogens as predominant in non-mammalian vertebrates. the thymus in the tail regeneration of xenopus laevis tadpoles a franchini, e bertolotti department of biology, university of modena and reggio emilia, modena, italy the tadpoles of the frog xenopus laevis show significant regeneration capacity and are useful models to examine cellular and molecular mechanisms underlying the appendage regeneration. after amputation, most of the tail can be rebuilt during the larval life and this ability is gradually reduced as development proceeds towards metamorphosis. previous studies demonstrated different morpho-functional responses to tail amputation of different aged x. laevis tadpoles. unlike stage (st) 50, an high percentage of st 55/56 larvae showed limited regenerative efficiency and malformed and shorter new tails were observed. the immune cells were found to take part in the response to tissue injury, in determining the inflammation degree and success of repair process. in this work, the thymus from tail amputated xenopus larvae (st 50 and st 55/56) was investigated by histochemical and immunohistochemical reactions. the examination of st 50 revealed changes of thymic architecture, compared to unoperated controls, characterized by an increased number of multicellular epithelial cysts, mucous and myoid cells, and cells, mainly located in the medulla, immunoreactive to anti-tnf-α antibody. a significant higher number in cortical apoptotic figures was only detected 1 day after tail cut. the thymic structural modifications were more marked, and observed throughout tail regeneration process, in most of the older tadpoles. the cellular responses included significant increase of apoptotic pictures and reduction of the medullary area where large epithelial cysts containing secretory material and cell debris were seen. at the end of regeneration process the organ size was found to be reduced of about 40 %. compared to unoperated controls, a higher number of tnf-α immunoreactive cells was also observed. these findings show that tail cut provokes a stimulation of the thymic function and induction of molecules critical for organ constitutive processes indicating a possible role of the lymphocyte component in control of xenopus tail regenerative quality outcomes. the stage-dependent events occurring in regenerating tail microenvironment, i.e. degree of inflammatory response, may be related to thymic structural modifications. immune response in carabus lefebvrei dejean 1826 (coleoptera, carabidae): circulating hemocytes, phagocytosis and plasma phenoloxidase activities at different developmental stages pg giulianini1, a schiafone2, f. talarico2, a giglio2 1department of life sciences, university of trieste, trieste, italy 36 2department of ecology, university of calabria, arcavacata di rende, cosenza, italy carabus lefebvrei dejean 1826 (coleoptera, carabidae) is a helicophagous italian endemic ground beetle that lives in central and south apennines mountain forests. the pathogens and parasites can be the main causes of mortality for all life-cycle stages of carabid beetles. however, few morpho-functional data about immune system are available. in this study we have compared the cellular population in the hemolymph of c. lefebvrei adult, larvae and pupae by light and electron microscopy analysis and identified the hemocytes involved in phagocytosis after in vivo artificial nonself-challenges. moreover, the plasma phenoloxidase (po) activity were detected using a l-dopa substrate and enzyme activity was expressed as absorbance units at 492 nm/μl of hemolymph. total hemocyte counts revealed in pupae a higher number of circulating hemocytes compared to larvae and adults. four morphotypes of circulating hemocytes were found: prohemocytes, granulocytes, oenocytoids and plasmatocytes. the plasmatocytes were always the main circulating hemocyte type and the pupae percentage was lower than adults and larvae ones whilst the granulocytes were higher in pupae than in adults and larvae. after in vivo artificial non-self challenge treatments, all c. lefebvrei stages showed a non-specific immune response involving phagocytosis performed by plasmatocytes. the comparison of hemolymph po activity (measured as the increment of absorbance at 30 min with respect to 0 min readings) between larvae, pupae and adults revealed a significant difference among stages (kruskal-wallis rank sum test, p-value < 0.01). in particular po in adults showed a significant higher activity compared to larvae (pairwise comparisons using wilcoxon rank sum test with bonferroni correction, p < 0.05) and an highly significant higher activity compared to pupae (p<0.01). no difference was recorded among larvae and pupae po activities. the exarate pupal stage produced a mixture of terpenes, ketones, aldehydes, alcohols, esters and carboxylic acids as defensive secretion from abdominal glands. this glandular secretion has both a deterrent function against predators and a prophylaxis function against pathogens. from an ecological perspective, the chemical protection is an efficient barrier against pathogen, hence, the pupal stage invest more energetic resources in specific cellular defense responses rather than in po activity. gene expression profiles of the mussel myticin c, an antimicrobial peptide displaying high sequence variability l varotto, mg procopio, u rosani, p venier department of biology, university of padua, padua, italy. the mediterranean mussel mytilus galloprovincialis commonly appear more resilient than other bivalves to the environmental variation and, in particular, to infective agents and disease onset: for this reason mussels should have a potent immune system able to fight against microscopic aggressors. their hemolymph cells play a fundamental role in defining rapid and effective defense reactions. in response to bacterial antigens and viral-like polynucleotides, the haemocytes of m. galloprovincialis synthesize and release antimicrobial peptides (amp), effector molecules able to execute the killing of microbial cells. the amp gene expression may represent up to 25-40 % of whole transcriptome in the hemocytes of immuno-stimulated mussels and one of them, myticin c, showed remarkable transcript polymorphism and unique transcriptional profiles in individual mussels. myticin c is expressed at constitutive levels in the mussel haemocytes and substantially induced after immuno-stimulation. the peptide has neither been isolated nor has its antimicrobial activity been specifically studied. aiming to the functional characterization of such ‘natural antibiotic’ we have set up a real time pcr method allowing the accurate quantification of its expression levels, and an ish system allowing the localization of myticin c transcripts in situ with riboprobes. using these experimental approaches we investigated the expression of this effector molecule in the hemocytes of mussels injected with heat-killed bacteria (vibrio splendidus and micrococcus lysodeikticus) and poly i:c at different time points (from 1 to 48 h post-treatment) in individual mussels. overall, the antigenic cocktail resulted to be a more potent inducer of the myticin c expression, with the maximum levels observed at 24 h posttreatment. the injection of individual v. splendidus or m. lysodeikticus produced different myticin c expression patterns, with a certain degree of interindividual response variation. preliminary ish results referred to naïve mussels suggest that myticin c is expressed by specific haemocyte subpopulations. antibacterial activity and immunomodulatory effects on a sea bream cell line of myrtus communis and aloe arborescens extracts c bernini1, c belardinelli1, e ovidi1, f buonocore1, g scapigliati1, am fausto1, l abelli2, d volpatti3, a manfrin4, s picchietti1 1department of environmental sciences, university of tuscia, viterbo; italy 2department of biology and evolution, university of ferrara, ferrara, italy 3department of animal production sciences, university of udine, udine, italy 4department of fish pathology, istituto zooprofilattico sperimentale delle venezie, adria (ro), italy. the emergence of antibiotic-resistant bacterial strains is a challenging problem that has led to an urgent global call for new antimicrobial drugs. such compounds, potentially devoid of undesirable sideeffects, have been sought in particular from natural resources. we have been studying the pharmacological potential of leaf components of m. communis and a. 37 arborescens, deserving special interest to their antimicrobial activity for possible future applications in fish aquaculture. ethanolic extracts from the two plant species were essayed in vitro for their effects on listonella (vibrio) anguillarum and photobacterium damselae ssp. piscicida strains. these grambacteria are two major pathogens for the cultured gilthead seabream (sparus aurata), being the causative agents of vibriosis and pasteurellosis, respectively. furthermore, the immunomodulatory effects of the plant-derived extracts on a lps-stimulated s. aurata fibroblast cell line (saf-1) were evaluated. obtained results have provided a promising new perspective for the use of medicinal plants to prevent or oppose bacterial diseases in fish. chairman: e. ottaviani, university of modena and reggio emilia, modena, italy melanin production and innate immunity m de eguileor1, a grimaldi1, g tettamanti1, f pennacchio2, r valvassori1, e ottaviani3 1department of biotechnology and molecular science, university of insubria, varese, italy 2department of entomology “f. silvestri”, university of naples, naples, italy 3department of biology, university of modena and reggio emilia, modena, italy in vertebrates and invertebrates, melanin biosynthesis is of prime importance in immune responses. independently from their phylogenetic position, invertebrates can show either a humoral system, responsible for massive production of melanin coupled with a cellular response lesser important in pigment production, or only a cellular system, as in vertebrates, in which the entire melanin synthesis is concentrated in specific organelles, the melanosomes. we have employed a variety of techniques to confirm and to discuss the new evidences that in animal, from cnidaria up to men, the same nexus among melanin production/templation, redox status, cytoplasmic ph, acid phosphatase activity, presence of pro-inflammatory cytokines, adrenocorticotropin hormone (acth), and neutral endopeptidase-24.11 (nep)-like activity overexpression occurs. diversity and evolution of piscidin antimicrobial peptides m cammarata department of environmental biology and biodiversity university of palermo, palermo, italy endogenous antimicrobial peptides (amps) are widely distributed in nature and are considered ancestral elements in the evolution of innate immunity. a variety of amps has been described in all organisms, particularly in aquatic organisms such as teleost fish. the piscidins are a family of linear amphipathic peptides initially identified in the hybrid of american bass (morone saxatilis x morone chrysops). in order to increase knowledge about the role of leukocytes in the innate immune responses of dicentrarchus labrax, we cloned, sequenced and characterized an antimicrobial peptide belonging to the family of piscidins from the head kidney leukocytes. the complete dna sequence of piscidin 1 was obtained using degenerate primers designed from other piscidins; in situ hybridization experiments revealed that this antimicrobial peptide is expressed in the leukocytes from peripheral blood, peritoneal cavity and head kidney. finally, the study of the family diversity, biological role in host protection and immunomodulation of all the known piscidins already sequenced in fishes, assumes a crucial importance for the health of fish in aquaculture and for potential biotechnological uses of these molecules. isolation and cytotoxic activity of neurotoxin from the mucus of actinia equina (anthozoa, cnidaria) mg parisi1, m cammarata1, l stabili2-3, s piraino2, n parrinello1 1department of environmental biology and biodiversity, university of palermo, palermo, italy 2department of biological and environmental science, university of salento, lecce, italy 3institute for coastal marine environment, cnr; taranto, italy cnidarians evolved chemical and biological defenses against predators, parasites and pathogens by the production of bioactive substances. both anthozoans and medusozoans cnidocytes are known to produce cytolytic and neurotoxic molecules with antihistaminic activity or blocking cationic channels. some cnidarian toxic peptides, listed in the pharmacopoeias, are now used as therapeutic agents. a number of toxins with a molecular weight of near 20 kda (equinotoxin i, ii, iii) have been characterized from actinia equine (anthozoa), the common beadlet red anemone living in the intertidal waters of the mediterranean and european temperate atlantic coasts. this study aimed to isolate and characterize cytolytic molecules of low molecular weight for potential biotechnological applications. in extracts from mucus and isolated nematocysts, cytolytic activity was detected toward rabbit erythrocytes and human chronic myelogenous leukemia cells k562. using high performance liquid chromatography followed by mass spectrometry we isolated a new lytic peptide of about 6 kda from both the mucus and cnidocytes. the complete amino acid sequence of this peptide revealed that it is a 54 aa polypeptide with high sequence homology with sea anemone neurotoxin 1. these cytolytic molecules have also been studied in the animal body, fluid extracts and free cells of tentacles. identification of antimicrobial peptides of betadefensin type in lizard tissues l dalla valle1, f benato1, s quinzani1, l alibardi2 38 1department of biology, university of padua, padua, italy 2 department of biology, university of bologna, bologna, italy defensins are a widely studied family of cationic antimicrobial peptides characterized by the presence of a conserved cysteine-rich motif and a large number of basic amino acid residues. these peptides have been broadly isolated from insects, animals, plants and mammals and they are critical to innate immunity and protection against infection. the high resistance to infections in lizards has prompted us to search for the presence of antimicrobial peptides in these vertebrates. the availability of the anolis carolinensis genome and expressed sequence tag (est) sequences (broad institute, boston, usa) enabled us to identify 32 different defensin-like genes, using bioinformatic methods and 5’and 3’-race analyses. these anolis defensin genes, designated as acbd 1 to 32, are clustered within 5 different genomic scaffolds suggesting that multiple defensin loci may be present in reptiles. the deduced peptides vary from 60 to 111 amino acid residues and almost all share the common features for vertebrate defensins, including small size, net cationic charge and six conserved cysteines in the mature peptide. moreover, based on their cysteine organization, the identified anolis defensin like peptides resemble beta-defensin family members of birds and mammals. like in avian beta-defensins, one betadefensin gene of anolis contains two highly divergent, tandem copies of the six-cysteine motif at the c-terminus. the anolis beta-defensin genes present, like in birds, a variable gene organization, ranging from two to four exons. moreover, the expression of two of these genes in anolis appears to be regulated by alternative splicing processes. the constitutive expression of 10 different betadefensins in healthy anolis was detected by semiquantitative rt-pcr in brain, testis, stomach, intestine, kidney, muscle, skin, lung, liver and tongue but the levels and patterns varied for individual defensin genes. further studies on the antimicrobial activity of these peptides are under investigation. session 3. chairman: l. abelli, department of biology and evolution, university of ferrara, ferrara, italy immunity and environment immune defenses and interactions with the environment e randelli, f buonocore, d casani, c marozzi, l guerra, c bernini, am fausto, s picchietti, g scapigliati department of environmental sciences, university of tuscia, viterbo, italy. animal species live in a condition of homeostasis with the microbiome present in their body and in the environment, constituted by a great number of microorganisms, many of which can be potential pathogens. a dynamic equilibrium exists between aggression strategies developed by microorganisms and defense strategies invented by animal eukaryotes, with the immune system that must guarantee full immune defense in every environmental condition. employing as main investigated species the sea bass dicentrarchus labrax, we studied the basic asset of the immune system of a teleost species by defining the tissue distribution and the ontogenesis of lymphocytes, the involvement of lymphocytes during antigenic stimulation “in vivo” and “in vitro”, the cloning of genes coding for immunoregulatory molecules, and the expression of these genes during various immune stimulations. of great interest is the intestinal immune system of sea bass, very rich in t lymphocytes, and displaying a regional distribution of t cell subclasses. current research focus on functional organization of gut-associated lymphoid tissue, and our hypothesis is that the intestinal immune system could be related to food habits. work is in progress to study predatory and planctophagous fish species, searching for differences that could be present between fish species with different food habits. immune system and environmental induced epigenetic and genetic mutations g damiani1, e capelli2, e grego3, e tarantola2, v bertone2 1cnr, institute of molecular genetics, igm, pavia, italy 2department of animal biology, university of pavia, pavia, italy 3 department of animal productions, epidemiology and ecology, university of torino, torino, italy prokaryotes are able to distinguish between self and non-self dna by means of the restriction and modification systems and a recently discovered antivirus immunity. several features of these systems are functionally analogous to eukaryotic epigenetic systems involved both in the uptake of foreign dna and in the generation of new genetic information. recent analysis of de novo mutations in several complete genomes, demonstrated that the majority of new mutations are g:c→a:t transitions. in particular cpg sites mutate at a rate several times higher than other sites. this very biased spectrum of these mutations may be the result of two main environmental induced processes: deamination of methylated cytosines and ultraviolet light mutagenesis. these data suggest an important role of deaminases in the transformation of acquired epigenetic changes in stable dna mutations. mutagenic enzymes as aids, apobecs, adars, x family polymerases and reverse transcriptases are involved not only in the generation of new point mutations but also in environmental induced dna rearrangements, as mobilization of retroelements, virus immunity and microrecombination. dna and rna methyltransferases and deaminases are able to editing the dna and rna in the epigenetic processes and have additional roles in the uptake of foreign dna sequences, in the regulation of the rnai, in the stress induced mobilization of the formatting 39 retroelements, in the cell differentiation mechanisms, and in the acquired immunity. with the discovery of molecular machinery that promotes directional genomic changes in response to environmental stresses, a rethinking of evolutionary processes is needed. as proposed in literature, we can recognize that genetic information processing is the fundamental driving force that provides hereditary changes in biological systems. infection, immunity and the environment: connecting the dots using marine mammal transcriptomic signatures a mancia1,2, jc ryan3, gw warr1, l abelli2, f van dolah3, fmd gulland4, rw chapman5 1medical university of south carolina, charleston, sc, usa 2department of biology and evolution, university of ferrara, ferrara, italy 3noaa/national ocean service, charleston, sc, usa 4the marine mammal center, sausalito, ca, usa 5south carolina department of natural resources, charleston, sc, usa functional genomics analyses of an organism’s transcriptome can be informative of the interaction of genetic, disease and environmental factors. here we used a combination of microarrays and machinelearning analytical approaches to understand the impact of environmental infection and stress on marine organisms. in particular, we studied marine mammals because they are considered an ideal model for the assessment of immunological responses to pathogens and contaminants. in fact, as mammals that live their entire life (or most of it) in the sea, they act as integrators of the stressors present in the marine environment. marine mammals may have the potential to predict contaminant effects on health, and to be an indicator of infectious disease that may impact humans who have contact with the marine ecosystem through residence, work, or recreation near the coast. we tested the hypotheses that 1) individual wild dolphins could be assigned to their home regions and 2) individual sea lions could be assigned to a specific disease status category, using only their blood transcriptomic signatures as classifiers. the tools used were a dolphin peripheral blood leukocyte (pbl) cdna microarray specifically designed for studies of immune function and stress reactions and a custom oligonucleotide microarray generated from cross-hybridization probing of a canine microarray. microarray data of 151 wild dolphin pbl samples and 73 sea lion blood samples were analyzed using a machine-learning approach. artificial neural networks (anns) were able to correctly classify dolphins according to their site of sampling and sea lions according to their diseased status. these results suggest that a combination of microarrays and machine-learning analytical approaches would significantly improve the knowledge about the marine environment/organism interactions. insights into b cell heterogeneity in two antarctic teleost species l abelli1, f bertoni1, e. errica1, a mancia1, mg marchetti1, c zeni1, am fausto2, s picchietti2, mr coscia3, s giacomelli3, u oreste3 1department of biology and evolution, university of ferrara, italy. 2department of environmental sciences, university of tuscia, viterbo, italy 3cnr-ibp, napoli, italy. interest in fish immunity has risen recently due to the discovery of some peculiar features. in particular, many studies have focused on teleost immunoglobulins (ig) and their adaptation to the environmental conditions. ongoing research is studying the b lineage of two species living at subzero temperatures in antarctic seas, the red-blooded trematomus bernacchii (tb) and the hemoglobinless icefish chionodraco hamatus (ch). transcripts for the secreted and membrane form (1089 and 864 bp, respectively) of tb igµ were amplified by rt-pcr using specific primers. b cells were identified by immunohistochemistry (ihc) using mutually no cross-reactive polyclonal antisera against tb purified serum igm heavy (µ, 76 kda) and light (l, 25 kda) chains, and a polyclonal antiserum against ch purified serum igm. proteins extracted from tb tissues and separated by reducing sdspage were immunoblotted using the antiserum to homologous igµ. both secreted and membrane igµ transcripts as well as 76and 66-kda proteins were detected in tb head-kidney (hk), thymus, spleen, blood cells, gills and intestine, at apparently varied expression levels. quantitative ihc confirmed that ig+ cells were more numerous in lymphoid than mucosal organs, in all instances with a prevailing perivascular distribution, but also outlined some differences in size and relative density among cells expressing µ and l chains in the different tissues. quantitative ihc on available ch tissues also reported more ig+ cells in the hk and spleen than in the intestine, predominantly perivascular. furthermore, cross-reacting sera anti-tb µ and l chains suggested some degree of b cell heterogeneity. the occurrence along the intestine of tb and ch of reverse gradients (decreasing or increasing towards the anus, respectively) in the number of ig+ cells needs to be carefully interpreted in light of immune regulation and control of a huge parasitic load, qualitatively and quantitatively different at the gut levels. indeed, evidence is accumulating in both species that liver and exocrine pancreas would provide to ig transport toward the intestinal lumen as well as to local defence against parasite invasion (peritoneal side) and/or ascending infections. reported findings raise important questions about existence and function of other ig isotypes (and subtypes) in antarctic fish, that need to be addressed on a wider set of tissues using proper analytical tools. 40 immune challenge is reflected in the expression of a male sexual trait in the peacock blenny (salaria pavo) l locatello, m pizzolon, mb rasotto department of biology, university of padua, padua, italy the information conveyed by male secondary sexual traits and the potential benefits to choosy females are at the core of sexual selection theory. indicator models of sexual selection assume that sexual displays reflect the phenotypic⁄genetic quality of males and that females gain benefits from choosing high-quality males. in particular, when male traits signal the individual ability to resist parasite, females can gather both direct benefits in terms of avoidance of diseased mates and indirect benefits in terms of genes for the progeny. accordingly, if sexual traits represent honest signals of male health status, immune-compromised males are expected to exhibit a reduced signals’ expression. we addressed this prediction in the peacock blenny, salaria pavo, by injecting males with lipopolysaccarides (lps; dosage: 2 mg/kg), eliciting the immune system activation and leading to a remarkable oxidative stress in animals by an increased production of reactive oxygen species. we recorded changes in the expression of male secondary sexual traits (a pronounced head crest and a pair of anal glands), courtship behavior and ejaculate quality (sperm number, velocity and viability) between groups of males respectively exposed to the immune challenge or sham-injected. the immune treatment had a significant negative effect only on the yellow coloration of the head crest, both in terms of color extension and intensity. these results indicate that in the peacock blenny the head crest, a sexual signal whose coloring expression depends on antioxidants (i.e., carotenoids), may represent an honest advertisement of male quality assessable by females during mate choice. indeed, the immune system activation trough lps may result in depletion of bodily antioxidants, mobilized to contrast the oxidative stress at the expense of a carotenoid mediated sexual signals. moreover we found that the proportional change in head crest color area of lps treated males was positively related to the total area of head crest, i.e. males with less developed head crest are suffering the strongest lps-induced reduction in head crest color area. in agreement with the predictions of the condition-dependent indicator model of sexual selection, more ornamented males are better able to cope with the simulated infection, suffering lower detrimental effects of the activation of the immune system. chairman: g. scapigliati, university of tuscia, viterbo, italy immunoglobulins at the blood-brain barrier in trematomus bernacchii j vizioli1, s giacomelli2, c van camp1, mr coscia2, u oreste2 1 lsmbfa equipe activation microgliale ea4550, université lille 1villeneuve d'ascq, france 2 institute of protein biochemistry cnr naples homeostatic regulation of central nervous system microenvironment is crucial for normal neuronal function in vertebrates. a key component in this regulatory process is the blood-brain barrier (bbb), a selective barrier formed by the endothelial cells that line cerebral microvessels regulating the transport of compounds from the blood to the brain’s extracellular milieu. tight junctions between endothelial cells are the most important structural elements of the bbb. furthermore the choroid plexus epithelium and the arachnoid epithelium are barriers between the blood and the cerebrospinal fluid. recent data revealed that the brain is neither isolated nor passive in its interactions with the immune system and the so-called neonatal fc receptor (fcrn) seems to transport ig molecules in particular conditions. due to the unique features of their vascular system, the antarctic teleosts are very suitable model species to study the bbb and the ig occurrence in the brain compartments. the morphology of the brain of antarctic species resembles that of other teleosts but several important differences should be highlighted: regulatory areas of the brain, including the saccus vasculosus (probably equivalent to the mammalian choroid plexus) and the subependyma of the third ventricle are more developed. in some species the expanded ependymal lining forms ventricular sacs, not previously described in any other vertebrate. the data we collected about an unexpected abundance of transcripts of immunoglobulin heavy chain (igh) in the brain of trematomus bernacchii, prompted us to investigate the presence of the protein. an antiserum produced against ig light chains was used to perform immunohistochemical analyses on serial sections of a paraffin embedded t. bernacchii brain. a clear staining was observed in the epithelial cells surrounding the microvessels, indicating the presence of a specific fc receptor on their membranes. in addition a staining not assignable to the vascular system was observed at three different localizations: i) between the telencephalon and the tectum of the mesencephalon; ii) lining the nucleus diffusus of the inferior lobe, iii) and between the saccus vasculosus and the inferior lobe of the diencephalon. the data presented here, obtained by preliminary experiments, convinced us that the antarctic species are highly appealing as animal model for encephalic barriers studies. effect of seawater ph and temperature variation on the hemocytes of the crab carcinus aestuarii s lorenzon1, s d’antoni2, c de vittor1, ea ferrero2, a furlani2 1department of biological oceanography, national institute of oceanography and experimental geophysics, trieste, italy 2department of life sciences, university of trieste, trieste, italy 41 the impact of co2 increase on marine systems as well as the consequent ecosystem feedback is still largely unknown. the response of organisms to ph reduction represents one of the main steps in evaluating the impact of direct ocean storage of co2 to overall functioning of ecosystem. modified ph has additional impact on the immune system of invertebrates and hypercapnia impairs the ability of the crab callinectes sapidus to remove culturable bacteria from its hemolymph. in this study the effects of exposure were tested on the hemocytes of the crab c. aestuarii at two ph (7.0 and 6.5) and temperatures (10 °c and 18 °c) for three weeks and subsequent recovery at normal seawater ph (8.1) for one week. firstly the reduction of ph induced high mortality in the exposed animals in particular at the higher temperature when no animal survived until the end of the recovery period. looking at the total hemocytes counts (thc) a reduction of seawater ph induced a significant variation in the number of cells at both temperatures also during the recovery period. looking at cell size we recorded a significant increment in the hemocytes dimensions (area, mean diameter, length and width) already after 24h of exposure to reduced ph at both temperatures; this increase in dimension is also present during the recovery period. combined effects of temperature, salinity and ph on immune parameters of bivalve molluscs m munari, a chinellato, v matozzo, m bressan, mg marin department of biology, university of padua, padua, italy atmospheric levels of co2 are increasing owing to human activities and are responsible for progressive acidification of oceans, which may affect calcareous structures of organisms (bivalves in particular), and modify their physiological performance, survival and growth. in bivalve mollusks, hemocytes play an important role in immune responses against foreign materials. effects of ph, temperature, and salinity as foreseen in climate change scenarios on immune parameters of the clam chamelea gallina and the mussel mytilus galloprovincialis were investigated for the first time. an experimental flow-through system was setup to test simultaneously different temperature (22, 28 °c) and ph (8.1, 7.7, 7.4) values in three experiments performed at 28, 34, 40 psu salinity. total hemocyte count (thc), pinocytotic activity, and hemolymph lysozyme activity were measured in bivalves after 7 days exposure. at 28 psu, thc was significantly affected by temperature and ph in both species, whereas lysozyme activity was significantly influenced by ph only. at 34 psu, a significant effect of ph on pinocytotic activity of both clams and mussels was observed. at 40 psu, thc was significantly affected by temperature in both species, and by ph in clams only. in addition, a significant effect of ph was detected on pinocytotic activity of clams and mussels. results obtained demonstrated that immunomarkers allowed to highlight stress conditions in bivalves, and indicated differing immunomodulation patterns, on the basis of the experimental conditions tested. of the immune parameters measured, thc showed to be the most influenced by variations in environmental conditions. interestingly, the same pattern of statistical significance of temperature and ph effects was observed in both species at the three salinities, with lower impact at 34 psu, that represents optimal salinity value for mussels and clams. metal-induced antioxidant defense in the solitary ascidian ciona intestinalis n franchi, d ferro, e piccinni, l ballarin, g santovito department of biology, university of padua, padua, italy antioxidant enzymes play important roles in antioxidant responses caused by metabolic process or pathogen invasion. in many organisms, it has been reported that antioxidant enzymes participate in their innate immune defense against immunostimulant challenges such as β-glucan and sulfated polysaccharide, lps or viruses. cu,zn superoxide dismutase (sod, ec 1.15.1.1) is one of these key enzymes, highly conserved from invertebrates to vertebrates, involved in scavenging superoxide radicals into molecular oxygen and hydrogen peroxide. however, little is known about the responses of antioxidant enzymes like cu,zn sod in tunicates after exposure to environmental changes in heavy metal concentrations. the present research focuses on structural and functional studies of the cu,zn sod gene in the solitary ascidian ciona intestinalis. the gene sequence has been identified in genbank and the amino acid sequence of the codified protein has been compared with those deduced from orthologous genes in other metazoans, both vertebrates and invertebrates, to determine if the amino acids important for catalytic activity were conserved and whether the enzyme of c. intestinalis acquired special features, relatively to the primary sequence, during its evolution. the in silico analysis was extended to the promoter regions for the presence of regulatory sequences such as antioxidant response elements (are), metal response elements (mre) and xenobiotic response elements (xre). the available sequences were used for cladistic studies, providing some interesting inference on the molecular evolution of the c. intestinalis protein. using semi-quantitative rt-pcr technique, cu,zn sod gene expression have been evaluated as a function of exposure to three different metals (cd, zn and cu). for this purpose, specimens of c. intestinalis were divided into three experimental groups, which were exposed for 6, 24, 48, 72, 96 and 120 h at equimolar concentrations (10 μm) of single metal. untreated specimens were used as controls. cu,zn sod is mainly induced by cu and zn, metals that are components of the active site. besides, it was provided an initial estimation on the 42 localization of expression by in situ hybridization. the results indicate that the cells most involved in the expression of the considered genes are the hemocytes. characterization of selenium glutathione peroxidase in the antarctic teleost gymnodraco acuticeps f volpe, f boldrin, d ferro, e piccinni, g santovito department of biology, university of padua, padua, italy gpx activity consists in the detoxification of hydrogen peroxide, and is one of the most important ubiquitous antioxidant enzymes involved in antioxidant homeostatic control. moreover, gpxs are closely involved in the innate immune responses of animals, as evidenced by the rapid modulation of transcription during challenges with endotoxins, bacteria or viruses. during infection, the host often uses ros to destroy pathogenic invaders via phagocytosis; however, excess ros formed during the respiratory burst may also be harmful to the host. for this reason, the elimination of excess ros via antioxidant enzymes, is crucial for maintaining cellular homeostasis in the host. despite numerous previous studies on gpx from aquatic animals, the gene structure and expression of teleost gpxs have not been comprehensively studied. in particular, very little is known about the gpx of antarctic teleost. the antarctic species have an interesting evolutionary history because they have developed some adaptations that allow them to survive and to breed in waters where the temperature, oxygen and salt concentration deviate significantly from the average recorded in the temperate waters. they represent paradigmatic cases for adaptation to different temperatures and salinities in their environment. specimens of gymnodraco acuticeps, a teleost fish widely distributed in antarctic ocean, were sampled in the ross sea (terra nova bay, 74°42’s, 164°7’e) during the xvii italian antarctic expedition. cdna sequence of se-gpx has been obtained from hepatic tissue by a combination of rt-pcr, 3’ and 5’race techniques. the obtained nucleotide sequence and the deduced amino acid sequence have been compared with those of homologous genes already available in genbank and have been used for phylogenetic analyses. preliminary results were also obtained regarding the regulation of gene expression. chairman: p. venier, university of padua, padua, italy effect of zinc on immunological competence of the sea urchin paracentrotus lividus p pagliara1, l stabili1,2 1department of biological and environmental sciences and technologies, university of salento, lecce, italy 2institute for coastal marine environment, cnr, taranto, italy the levels of trace metals into the marine environment can be affected by human industrial and mining activity, as well as agricultural and domestic wastes, the combustion of fossil fuels and also by natural factors such as erosion, volcanic and tectonic activities. significantly higher concentrations have been detected in some coastal zones compared to those in oceanic regions. in the mediterranean sea the main trace metals found are cadmium, mercury, lead, tin, copper and zinc. since zinc has been known as a heavy metal less toxic to human health unless the concentration is abnormally high, its release is loosely regulated allowing considerable emissions of zinc from anthropogenic sources. a number of studies on the impact of heavy metals on the invertebrate immune system have been carried out in mollusks, crustaceans and oligochaetes whilst, little information is known about echinoderms. in this framework we analyzed the effects of a high zinc concentration on several immunological parameters of the sea urchin p. lividus taking into account that a variety of studies demonstrated the effect of environmental perturbations on invertebrate immunological competence. in particular, by comparing control (untreated) and zinc treated sea urchins, we evaluated the effects of this metal on coelomocytes hemolytic, protease and lysozymelike activities as well as antibacterial activity on vibrio alginolyticus. in addition, we analyzed changes in coelomocytes composition and morphology. all the immunological parameters considered were significantly reduced by the addition of zinc after 24 h of treatment, except for the protease activity. in addition the number of red spherule cells, usually activated by stress conditions, increased significantly. the modifications in sea urchins immunological competence may give an early indication of disease susceptibility thus suggesting to consider the examined defense mechanisms as potential biological indicators of metal pollution. immunomodulatory effects of low doses of hexavalent chromium [cr(vi)] in mytilus galloprovincialis c ciacci1, c barmo2, m betti1, r fabbri2, g gallo2, l canesi2 1disuan, university of urbino “carlo bo”, urbino, italy 2department of biology, university of genoa, genoa, italy chromium (cr) is a transition metal that exists in many different oxidation states in the environment, with cr(vi) and cr(iii) being the most stable forms. hexavalent chromium cr(vi) is an important contaminant considered a model oxidative toxicant that is released from both domestic and industrial effluents, and represents the predominant chemical form of the metal in aquatic ecosystems. on the other hand, cr(iii) is considered the biologically active form, representing an essential 43 microelement in mammals involved in regulation of metabolism. chromium has been also shown to affect the immune function, with immunostimulatory or immunosuppressive processes on lymphocytes, macrophages, cytokine production, and to modulate serotoninergic signaling. in this work, the immunomodulatory effects of cr(vi) were investigated in m. galloprovincialis in vitro and in vivo. short term in vitro exposure of mussel hemocytes to different concentrations of cr(vi) (0.1-100 μm) induced significant changes in hemocyte lysosomal membrane stability-lms, extracellular ros and no production, phagocytic activity and lysozyme release. dose-dependent decrease in lms and lysozyme release were observed, whereas increased ros and no production, and inhibition of phagocytosis were observed at lower concentrations. in in vivo experiments, mussels were exposed to sublethal cr(vi) concentrations (0.2-20 μm) for 4 days and both functional and molecular responses were evaluated. metal exposure induced dosedependent decreases in hemocyte lms and soluble lysozyme activity, and increased no production at lower concentrations. significant increases in transcription of the serotonin receptor 5-htr and of immune genes (myticin b, mgc1q) were observed at 2 μm, whereas downregulation of lysozyme and mytilin b were observed at the lowest concentration tested. on the other hand, expression of the antiapoptotic gene p53 was unaffected by metal exposure. overall, the results demonstrate that low concentrations of cr(vi) are not toxic to mytilus hemocytes but can modulate the immune response both at the functional and molecular level. stress effects of bacillus thuringiensis on rhynchophorus ferrugineus hemocytes m celi, m vazzana, b manachini, v arizza department of environmental biology and biodiversity, university of palermo, palermo, italy the heat shock proteins (hsps) are rapidly synthesized in the cells after an environmental stress exposition. various stress as such as cold, heat, heavy metal, water osmolarity, drying, organic and inorganic pollution, uv exposure, induce in the animal, the hsps expression. these are grouped in distinct molecular family, according to molecular size. most organisms have multiple genes that encode for members of this family. in particularly, the genes for the hsp70s, are traditionally divided into two groups: the majority of organisms have different genes coding for members of this family. the first group of genes can be induced rapidly under stress, but return to a normal level of expression in non-stressful conditions. previously we found that the entomopathogenic bacterium b. thuringiensis (bt), registered against another family of coleoptera, could be a potential pathogen of r. ferrugineus beetles. this curculionidae is a quarantine pest that attacks the palm trees. to study the pathogen-host relationship, we used the model of the bt-r. ferrugineus interaction. in particular, we focused on the bt stress-induced infections. we also studied for the first time the interaction between bt and r. ferrugineus hematocytes evaluating the expression of hsp70 in the supernatant of the hemocyte lysate (hls) obtained from larvae fed with bt. moreover the modulation of hsp70 in larvae of r. ferrugineus fed with artificial diet containing a sub-lethal dose of commercial bt was examined. this is the first time that the presence of hsp70 has been recorded in r. ferrugineus. the western blot analysis, using a mouse monoclonal antibody anti-hsp70, showed that the hsp70 expression was modulated in the time (3 h, 6 h, 12 h, 19 h, 24 h) in the response to bt treatment, highlighting that bt is a stress factor for the r. ferrugineus larvae. the protein expression was increased approximately seven fold after 3 h from treatment and after 6 h it returning to control value. previous data showed that bt interacts negatively with hemocytes of r. ferrugineus whose numbers decreased drastically in the hemolymph and that bt treatment causes growth inhibition. further investigation is needed to understand the possible correlation between the reduction hemocytes and modulation dell'hsp70. the mast cell: comparative aspects hemocyte proliferation influences metallothionein and phytochelatin levels in ciona intestinalis n franchi, e piccinni, l ballarin department of biology, university of padua, padua, italy we studied, through semiquantitative pcr and in-situ hybridization, the activities of the newly identified metallothionein and phytochelatin synthase genes from the solitary ascidian ciona intestinalis (ci-mt and ci-pcs, respectively) in response to 10 μm cdcl2. metallothioneins (mts) and phytochelatins (pcs) are involved in detoxification systems of many organisms. an appreciable number of data are available for metazoan, especially for vertebrate, but very few data are available for urochordates. as the latter occupy the peculiar phylogenetic position of invertebrate chordates, the research on mts and pcs in tunicates assumes a particular significance. cd strongly induced ci-mt, with a maximum at 4 days. ci-pcs showed maximum expression at the same time. this result is probably related to a cell proliferation event, rather than an effective ci-pcs gene activation. the hypothesis is supported by the strong induction of proliferating cell nuclear antigen (pcna) transcript after 4 days of treatment and the colocalization in the hemocytes of the different riboprobes used. in addition, in literature is reported an expression profile similar to that of cimt for c. intestinalis mannose binding lectins (cimbl). collectively, our data and data from the literature support the conclusion that hemocyte proliferation occurs in tunicate immune responses. session 4. chairman: u. oreste, institute of protein biochemistry, cnr, napoli, italy immunity: biotechnological and applicationoriented aspects 44 bivalve mollusk notifiable diseases: legislation, monitoring and diagnostic procedures m zambon, c magnabosco, t pretto, wg dundon, g arcangeli istituto zooprofilattico sperimentale delle venezie, laboratorio nazionale di riferimento per le malattie dei molluschi, legnaro (pd), italy in italy, the farming of bivalves has become an important sector of the aquaculture industry with nearly 200.000 tons produced per year compared to the 70.000 tons/year from fish farming. over the last ten years, the european community has published several regulations aiming at protecting bivalve production in each member state. these regulations foresee a variety of different actions including prevention and eradication of particularly aggressive diseases, constant surveillance to avoid the spread of new diseases, control of anomalous mortality, registration of farms and their categorization according their health status. listed diseases of mollusks, which are all caused by protozoans, are classified as either exotic (i.e. bonamia exitiosa e perkinsus marinus in pacific oysters) or non exotic (i.e marteilia refringens in mussels and flat oyster, bonamia ostreae exclusively in flat oysters) the o.i.e. (office international des epizooties, now referred to a the world organization for animal health), provides updated diagnostic procedures published in the manual of diagnostic tests for aquatic animals (revised in 2010) containing many different diagnostic procedures including cytology, histology, and molecular assays. due to the lack of circulating antibodies in bivalves, serological test are not available as diagnostic tools. likewise, no suitable cell lines are available to perform cytotoxicity studies. recently, in addition to the protozoan diseases mentioned above, an important virosis has been affecting pacific oysters, mainly in france and ireland, with heavy losses in production of up to 8090 % being reported in some cases. an oyster herpes virus-1 variant has been associated with the mortality observed although it is believed that it is not the only cause of the syndrome which is thought to be multifactorial (e.g. involvement of vibrio species, classic herpes virus, environmental conditions, farming typologies, etc.). the anomalous mortalities events, i.e. chamelea gallina and callista chione in the veneto, are often of uncertain origin. finally, the interaction between host and parasite is still the object of active study and has become an important challenge for research groups. construction of new antigen delivery systems for innovative vaccine formulations p de berardinis institute of protein biochemistry, cnr, napoli, italy two non-pathogenic scaffolds represented by the filamentous bacteriophage fd and the dihydrolipoyl acetyltransferase e2 protein of the bacillus stearothermophilus pyruvate dehydrogenase (pdh) complex able to deliver antigenic determinants, were designed. both of these systems offer the potential for a safe, inexpensive and efficacious delivery system to be used as vaccine vectors. based on a modification of the phage display technology a delivery vehicle in which antigenic determinants are inserted into the n-terminal region of the major pviii coat protein of filamentous bacteriophage fd, was developed. this system has proven to elicit full-spectrum of antigen-specific immune responses. in order to improve its efficacy, it was engineered a new bacteriophage vector to display, at the n-terminus of the piii protein, single chain antibody fragments (scfv) able to bind the dendritic cells-restricted surface molecule dec-205. it was demonstrated by in vivo tumor protection assay a potent inhibition of tumor growth by targeting fd particles displaying tumor antigens to dendritic cells. another antigen delivery system based on the e2 component from the pdh complex and capable of displaying large intact proteins on the surface of an icosahedral lattice which assembles into 24nm virus like particles (vlps), was also developed. in particular e2 self assembles into trimers which, in turn, aggregate to generate a 60-mer scaffold displaying up to 60 copies of a foreign antigen on the surface of the particle. thus e2 may be structurally advantageous for displaying gp160 hiv-1 trimeric antigenic proteins. in this context it was observed the induction of high titers of anti-hiv1 neutralizing antibodies by immunizations with hiv antigens displayed on e2. in addition e2 immunization did not induce ifn-γ production by cd4+ t cells and, for this low pro-inflammatory profile, epitopes from β-amyloid were displayed on e2 and the immune responses were studied. 3d modeling of pro-inflammatory molecules in selected teleost fish species f buonocore1, s costantini2,3, am facchiano2, e randelli1, g scapigliati1 1department of environmental sciences, university of tuscia, viterbo, italy 2laboratory of bioinformatics and computational biology, cnr, institute of food science, avellino, italy 3oncologic research center of mercogliano “fiorentino lo vuolo”, mercogliano (av), italy the inflammatory response is the reaction of all metazoan organisms to pathogen invasion that initiates when pathogen-derived molecules are recognized by specific pattern recognition receptors, expressed mainly on cells of the innate immune system. the successive expression of proinflammatory cytokines and chemokines limits pathogen spread, and attracts and activates immune cells to help in the elimination of the invaders. in this study we focused on the analyses of the 3d structures of three pro-inflammatory molecules (interleukin-1β, tumor necrosis factor-α, interleukin-8) from selected teleost fish species (oncorhynchus mykiss, dicentrarchus labrax, chionodraco hamatus) generated using as template 45 models those of experimental mammalian homologous proteins. these structures have been discussed with the aim to investigate the differences between them and mammalian counterparts and, moreover, to verify the presence of the actually known structural requirements for their biological activities. our data demonstrated that 3d modeling of pro-inflammatory cytokines is a potent strategy that helps to show evolutionary differences in the structure of these proteins. moreover, the differences should be taken into account when comparing the pleiotropic activities of these molecules during evolution especially for the consequences in the binding with their specific cell receptors and should advise against the use of recombinant human or murine proteins and human or murine specific antibodies when studying biological activities in teleost fish. finally, the 3d structures give good indication in the choice of the epitopes of these molecules useful for the production of fish monoclonal antibodies and for the designing of synthetic peptides for the binding to the specific cytokine cell receptors. computational prediction and experimental analysis of the secondary structure of the primary transcript encoding the immunoglobulin heavy chain of the antarctic teleost chionodraco hamatus s giacomelli1, s varriale1, mr coscia1, c. rivetti2, u oreste1 1institute of protein biochemistry cnr naples, naples, italy 2department of biochemistry and molecular biology, university of parma, parma, italy the primary mrna splicing for the membrane form was found to be atypical in the majority of the antarctic species, because it excluded two entire constant exons, but included additional 39nucleotide exons. genomic dna analysis, revealed that each 39-nucleotide exon fell within a long sequence that was the reverse complement of an upstream region. in the present work we analyze the structure of a synthetic rna corresponding to the pre-mrna encoding the membrane-bound immunoglobulin heavy chain of the antarctic notothenioid teleost species chionodraco hamatus. rna was synthesized from a recombinant 4567nt-dna template of the antarctic teleost chionodraco hamatus using the ribomax large scale rna production system (promega). the dna template was linearized by digestion with hind iii prior to in vitro transcription. the transcription reaction was carried out under the control of the t7 rna polymerase promoter. computational analyses of the synthesized rna from the c. hamatus species were performed at different temperatures by using mfold web server, and resulted in different pairing of the two antiparallel regions. the conformation obtained at 0 °c involved 3835 kcal/mol. to experimentally verify the reliability of the predicted structures we performed atomic force microscopy experiments. atomic force microscopy of the synthesized rna, unfolded and relapsed at 0 °c was carried out on dried samples with a nanoscope iiia instrument. topographic images showed double stranded regions characterized by a rod-like shape with a height of about 2 nm with globular features at the two ends of the rod corresponding to the more complex base pairing of the 5’ and 3’ rna termini and the rna loop. agreement between the predicted and the observed rna structure confirms the validity of the computational model. 46 chairman: p. venier, university of padua, padua, italy effect of zinc on immunological competence of the sea urchin paracentrotus lividus 3d modeling of pro-inflammatory molecules in selected teleost fish species f buonocore1, s costantini2,3, am facchiano2, e randelli1, g scapigliati1 microsoft word 267r   110   isj 9: 110-121, 2012 issn 1824-307x review genomics, immune studies and diseases in bivalve aquaculture a romero, b novoa, a figueras instituto de investigaciones marinas (iim)-consejo superior de investigaciones científicas (csic). eduardo cabello, 6. 36208-vigo, spain accepted june 5, 2012 abstract diseases are a critical bottleneck for the culture of bivalves causing important yield losses. the study of bivalve diseases has relied on histological techniques and has focused on pathogen morphology, the effect of external factors on the pathogens and infectivity, and on the development of immune and molecular diagnostic techniques. recently, significant advances in the study of bivalve pathology have been reported; however, increased efforts using “omics tools” are required to explain key physiological/immunological processes. transcriptomic analysis in parallel with detailed functional studies of gene expression and cell biology in in vitro and in vivo experimental models are needed. another important factor is the identification of “resistance traits” and a deeper understanding of the processes that contribute to the welfare of bivalves in culture. additionally, the definition of "abnormal mortalities" is critical for managing and legislating bivalve aquaculture. the new technologies clearly “opens the door” for the directed manipulation of bivalves to improve the modern intensive aquaculture systems. the future of bivalve research is exciting, and there is an obvious need to develop multidisciplinary international research studies involving research groups and growers organisations to work on shellfish pathology. key words: genomics; immunity; molluscs; diseases   introduction over the past few decades, the global production of aquaculture species has increased, and mollusc aquaculture has doubled its production in that time (fao, 2006). in 2006, the total production of molluscs was estimated at 14 million tonnes (fao fishery statistics) (lee et al., 2008), representing approximately one quarter of the total global aquaculture production. asian countries are the main producers of molluscs; china, japan and thailand produced more than 30,000 tons in 2006 (fao, 2006) and the production levels are increasing. mollusc production on a global scale is based on more than 42 different species; however, the top five cultivated mollusc species are the pacific oyster (crassostrea gigas), the japanese carpet shell (ruditapes philippinarum), the yesso scallop (patinopecten yessoensis), the blue and mediterranean mussels (mytilus edulis and m. galloprovincialis) and the blood cockle (anadara granosa). in 2008, europe produced 658,000 tonnes ___________________________________________________________________________ corresponding author: antonio figueras instituto de investigaciones marinas (iim)-consejo superior de investigaciones científicas (csic) eduardo cabello, 6, 36208-vigo, spain e-mail: antoniofigueras@iim.csic.es of molluscs, contributing 26 % of the total volume of aquaculture production in europe (16.6 % value) (fao, 2010). spain, france and italy are the main bivalve-producing countries, although other countries, such as greece and norway, are demonstrating an increase in production. spain is the major producer of mussels, representing 71 % of the total mollusc production. clam and cockle production represented 10.9 % of the total molluscs produced in 2008, mainly in italy. the culturing of clams is primarily based on the introduced japanese carpet shell (ruditapes phillipinarum) rather than local clam species (ruditapes decussates). oyster culture is based on the introduced pacific cupped oyster crassostrea gigas and the native oyster (ostrea edulis). oyster production is largely dominated by the french industry, which contributed 19.9 % of europe’s total mollusc aquaculture in 2008. the high mortality of bivalves in the larval stage and in juveniles and adults are the main problem in mollusc cultures. mortalities decrease the global bivalve population and cause dramatic economic losses. physical environmental factors, such as prolonged periods of high/low temperatures, extreme salinities and industrial and domestic pollution as well as the increase in bacterial and   111   viral pathogens in seawater leads to mortality at each developmental stage (pipe and coles, 1995; braby and somero, 2006; carstensen et al., 2010). the mortalities in bivalves have been associated with different viruses, bacteria and protozoa. bacterial and viral diseases in molluscs are associated with pathogens from the vibrio genus, including brown ring disease and summer disease (borrego et al., 1996; paillard, 2004; gómez-león et al., 2005; huchette et al., 2006), and with the herpesviridae, iridoviridae and picornaviridae families, among others (marteil, 1976; jones et al., 1996; hine and wesney, 1997; renault and novoa, 2004; batista et al., 2007). examples of diseasecausing protozoa are haplosporidium nelsoni, bonamia ostreae, marteilia refringens, mycrocitos mackini, m. roughley and perkinsus marinus (carnegie et al., 2003; berthe et al., 2004; burreson and ford, 2004; villalba et al., 2004). how can we fight these diseases? the effectiveness of any treatment against diseases in molluscs is limited by the physiology of the animals as well as by the characteristics of the culture system (berthe, 2008). molluscs lack an adaptive immune response; therefore, they cannot produce antibodies (bayne, 2003; ottaviani, 2011) and vaccination strategies are not possible for these animals. moreover, the culture system is in an open environment, which makes treatments in the field difficult, and the impact on the environment must be considered. therefore, therapeutic measures have not been applied to mollusc aquaculture in the open environment, although a number of treatments for diseases and parasites have been successful in the laboratory or on a reduced scale in the field (calvo and burreson, 1994; nel et al., 1996; faisal et al., 1999; friedman et al., 2007). rigorous procedures are applied to minimise the risk of introducing an infectious disease into a disease-free area. standards, guidelines and recommendations for the introduction of new stocks are provided at international, regional and national levels (ices, 2004; mapa, 2008; oie, 2011). moreover, important efforts have been made to improve diagnostic methods for diseases that affect molluscs (bondad-reantaso et al., 2001; oie, 2011). historically, most of the descriptions of mollusc pathogens are based on structural and ultra-structural studies. traditionally, the diagnosis of mollusc diseases has primarily been achieved using histological methods and transmission electron microscopy (tem) (boulo et al., 1989; azevedo et al., 1990; anderson et al., 1994; bushek et al., 1994; hervio et al., 1996; longshaw et al., 2001; howard et al., 2004). the limitations of these methods include the need for highly qualified personnel, the techniques are time-consuming, and they often demonstrate low specificity/sensitivity. to overcome the limitations of histology and tem for diagnosis, the european and national reference laboratories have developed, validated and standardised a series of rapid, specific and reliable molecular diagnostic methods. these entirely new dna-based assays allow the rapid detection of pathogens (stokes and burreson, 1995; berthe et al., 1999; le roux et al., 1999; carnegie et al., 2000; cochennec et al., 2000; walker and subasinghe, 2000; yarnall et al., 2000; kleeman et al., 2002; oie, 2011). health management policies in areas where disease outbreaks occur attempt to reduce the incidence or the severity of a disease (grizel et al., 1986) using the following strategies: changes in culture management procedures (korringa, 1950, lauckner, 1983; andrews and ray, 1988; montes et al., 2003), increasing the tolerance of the individuals to disease through stimulating the immune system (macey and coyne, 2005; xue et al., 2008; van hai et al., 2009), producing tolerant strains by genetic handling (ford and haskin, 1987; martin et al., 1993; gaffney and bushek, 1996; hand et al., 1998; nell et al., 2000; culloty et al., 2004; nell and perkins, 2006; samain et al., 2007; villalba et al., 2007) or replacing susceptible species with resistant ones (grizel and héral, 1991; mann et al., 1991). however, the introduction of foreign species could introduce pathogens, predators or competitors that may perturb the equilibrium of the ecosystem and affect adjacent areas or neighbouring countries (carriker, 1992; shatkin et al., 1997). genomics focuses on the study of genes and their function. the use of these tools can compensate for the near absence of clinical manifestations and symptoms in diseased molluscs by identifying key molecules (rnas or proteins) expressed in response to injures or disease. moreover, genomics can help determine the relationship between the genes and environmental factors in biological processes, such as diseases. these genetic markers will allow the identification of genes involved in the defence against pathogens, and the generation of specific bioassays to measure their production, activity or expression can be used as markers of host physiology, the activation state of the pathogen or the immune status of the host. the use of genomic tools will facilitate the study of molluscs and increase our understanding of the biological processes that are critical for the success of molluscan aquaculture (saavedra and bachère, 2006). several problems currently affecting bivalves would benefit from the genomics approach (saavedra et al., 2009). the presence of toxins of phytoplanktonic origin produces red tides and causes several diseases in humans after consumption (bricelj and shumway, 1998). genomic tools can be used to study the depuration processes for each toxin. additionally, the study of the larval stages at cellular and molecular levels, diet design, and growth as well as factors related to disease resistance or stress are aspects of bivalve physiology that can be studied using genomic techniques. the genomic approach: the beginning when the genomic approach was first initiated in bivalves, the focus was on the effects of environmental pollution. because of the small number of validated and biologically relevant markers, together with inadequate information on the cellular processes occurring in normal and   112   stressed mussels, the development of effective biomonitoring programmes was delayed (viarengo et al., 2000; moore, 2002). mussels (m. edulis and m. galloprovincialis) have been used as model organisms because of their ecological importance (gosling, 1992) and their economic value in shellfish farming (beadman et al., 2002). since the mid-1970s, because of the ubiquitous and sessile characteristics of the adult mussel, its efficient filtering activity, limited xenobiotic metabolism and plasticity to environmental changes, mussels have been used as bio-indicators of chemical pollution (goldberg and bertine, 2000; whitfield, 2001). different proteins have been identified in mussels after their exposure to toxic contaminants (lopez et al., 2001; aardema and macgregor, 2002). the relationship between changes in the pollution status of the water and changes in the protein profile, the generation of free radical scavengers, and the copper and zinc content have been studied in mussels from dirty and clean areas (gorinstein et al., 2006). the transcriptome of mussels was used to select markers that trace contaminants (venier et al., 2003b, 2006; dondero et al., 2006a). the first attempts to describe important immune-related genes in bivalves were performed using homology cloning; however, this approach was not successful because of the limited information on bivalve genes in databases. most of the information on bivalve innate immunity is based on biological activities, and only indirect evidence for the role of particular genes in the bivalve resistance against disease has been described. for example, the protistan perkinsus marinus is a lethal parasite in crassostrea virginica; however, the pathogen is not known to cause lethal infections in mytilus edulis or geukensia demissa. in 2001, anderson and beaven suggested that the anti-p. marinus properties of plasma proteins in mytilus edulis or geukensia demissa exhibit many hundredfold greater activity than the plasma proteins in c. virginica and may be responsible for the low susceptibility of this species. in another example, meyers et al. (1991) reported that crassostrea gigas is less susceptible to perkinsus marinus than c. virginica, suggesting that c. gigas is tolerant to p. marinus. it has been demonstrated that p. marinus proteases compromise the immune defence mechanisms of the eastern oyster (garreis et al., 1996) and favour the protozoan’s propagation (la peyre et al., 1996). faisal et al. (1998) and oliver et al. (1999) associated the presence of highly specific protease inhibitors in c. gigas with their lower susceptibility to p. marinus. the genomic approach: the next step the next step in the genomic approach was to describe the differentially expressed immune-related genes in bivalves using different methodologies, such as bac libraries, est collections from cdna or subtractive hybridisation libraries (ssh) or microarray technology. these methodologies have been used to select key genes linked to resistance that could be used as markers for selection, and to study in detail the interaction between the host immune system and different pathogens. bac libraries only two bac libraries have been reported in molluscs, one for the eastern and pacific oyster (cunningham et al., 2006; hedgecock et al., 2005) and the other for the zhikong scallops, chlamys farreri (zhang et al., 2008). the two libraries produced 73,728 and 7,680 clones, respectively, and the combined libraries contain 81,408 bac clones in total. the oyster bac libraries are available to the research community at www.genome.clemson.edu. because bac libraries facilitate the handling of long genomic fragments, their use should rapidly expand to include other intensively exploited commercial bivalves. cdna and ssh libraries several expressed sequence tag (est) collections from cdna or subtractive hybridisation libraries (ssh) have been generated in the last few decades. those libraries are powerful tools for the study of differential gene expression and the identification of genes involved in specific biological functions, especially in organisms where genomic data are not available (zhang et al., 2001; satou et al., 2002). the est approach was used to identify genetic markers in salmon (davey et al., 2001) and environmental stress indicators in the american oyster (jenny et al., 2002); it was also used to characterise immune genes in shrimp (gross et al., 2001). in molluscs, most studies have focused on analysing the immune system with the aim of identifying the molecular basis of the most common pathologies (saavedra et al., 2009). although these libraries have provided sequences that are homologous to previously described sequences, a significant number of the generated ests did not show similarity to known genes in a blast search using the available databases. in oyster, the first published library was constructed in c. virginica with the aim of identifying genes to be used as bioindicators of exposure to environmental pollutants, toxins and infectious agents (jenny et al., 2002). approximately 40 % of the 1,000 ests generated were novel sequences and demonstrated homology to c-type lectin and scavenger receptors, proteinases, metallothionein, precerebellin, collectins and antimicrobial peptides. gueguen et al. (2003) performed a similar study in c. gigas and described genes involved in defence mechanisms after exposure to four pathogenic strains of vibrio sp. this approach allowed the characterisation of 55 sequences out of 1,142 ests that have potential immune function. interestingly, the authors reported the expression of a low number of antimicrobial peptides and a high number of proteases, inhibitors of proteases and adhesive proteins. additionally, roberts et al. (2009) reported the construction of a library from crassostrea gigas hemocytes in which 2,198 ests were generated. suppression-subtractive hybridisation (ssh) libraries were also constructed in oyster. huvet et al. (2004) identified differentially regulated genes in c. gigas in the f2 progeny with different susceptibility to the summer mortality disease. in this study, several transcripts were detected that exhibited homology to heat shock proteins, and the antimicrobial peptide defensin b was identified. a   113   study of genes expressed in response to perkinsus marinus challenge was performed in the hemocytes and gills from c. virginica and c. gigas (tanguy et al., 2004). the genes involved in the immune system, cell communication, protein regulation and transcription, cell cycle, respiratory chain and cytoskeleton were detected and were overexpressed in challenged oysters. the ssh technology has also been applied to the identification of antimicrobial peptides belonging to the mussel defensin family in oysters (peatman et al., 2004; gueguen et al., 2006). the first clam cdna library was generated in ruditapes philippinarum infected with perkinsus olseni. kang et al. (2006) sequenced 1,850 clones in total, and 29 ests were shown to be related to immune genes, such as c-type lectins, lysozyme and cystatin b. moreover, lectins, which were the largest group of immune-function ests, appeared to be pathogen specific because they appeared after perkinsus infection but not after a vibrio infection. ssh libraries have also been constructed in carpet shell clams (ruditapes decussatus), one after bacterial stimulation of the clams (gestal et al., 2007) and a second one after perkinsus olseni infection (prado-alvarez et al., 2009). gestal et al. (2007) first identified clam myticin isoforms 1, 2 and 3, and clam mytilin. moreover, they reported a low number of ests related to amps. prado-alvarez et al. (2009) generated 305 differentially expressed sequences, 42 of which were associated with immunity and stress related functions; the adiponectin-c1q and dad-1 genes in r. decussates were also identified. finally, tanguy et al. (2008) reported 124 contigs and 1,814 singletons for r. decussatus, including immune genes with homologies to lectins and ferritins. in mussel, the first systematic production and annotation of ests was performed in m. galloprovincialis by venier et al. (2003a); however, due to the lack of available mussel sequences in the databases, most of the sequences did not exhibit homology. a small number of ests were homologous to immune-related genes, such as the β-glucan binding protein, the lps binding protein, and some proteasome components, lectins and antimicrobial peptides. these ests were combined with other cdna and ssh libraries (pallavicini et al., 2008) from bacteria-stimulated mussels. venier et al. (2009) constructed, sequenced and annotated 17 cdna libraries from different mediterranean mussel tissues (gills, digestive gland, foot, anterior and posterior adductor muscle, mantle and hemocytes) challenged with toxic pollutants, temperature variations and potentially pathogenic bacteria. in total, 24,939 clones were sequenced from these libraries generating 18,788 high-quality ests, which were assembled into 2,446 overlapping clusters and 4,666 singletons, resulting in 7,112 non-redundant sequences. this annotated catalogue represents a valuable platform for expression studies, marker validation and genetic linkage analysis, which was the basis for constructing the important mussel database, the mytibase (http://mussel.cribi.unipd.it) (venier et al., 2009). the information available in the mytibase led to the detection of a considerable number of antimicrobial peptides (amps). the importance of the amps in the bivalve immune response is reflected by the large number of recent publications on this subject, such as the high diversity of mytc (costa et al., 2009) and its antiviral activity and immunoregulatory properties (balseiro et al., 2011); the genomic organisation, molecular diversification, and evolution of myticin-c (vera et al., 2011); studies on the variability of the amps (rosani et al., 2011); the characterisation of big defensins and mytimacins (gerdol et al., 2012); mytilins (roch et al., 2008; parisi et al., 2009); and mytimycins (sonthi et al., 2012). microarrays the construction of the numerous libraries described has led to a significant increase in the number of ests in databases. these collections of ests contain genes that are modulated in response to environmental, chemical or biological stimuli and can be used to design probes in microarrays. microarrays have various applications, including the analysis of expression profiles, the detection of snps, genotyping analysis and checking for mutations. in oyster, jenny et al. (2007) constructed a cdna microarray in a consortium from norway, france and the us based on est libraries from several different tissues of c. gigas and c. virginica exposed to hypoxia, pesticides, bacterial and protozoan infections, hydrocarbons, hyperthermia, and summer mortality conditions. this cdna microarray is available to the research community and has already been used (lang et al., 2009). moreover, an in situ oligonucleotide microarray was developed by wang et al. (2010) using existing ests to analyse gene expression profiles during dermo disease caused by perkinsus marinus. in mussel, the first study using microarrays was performed by dondero et al. (2006b) in the mediterranean mussel, m. galloprovincialis. this microarray was enriched for genes involved in pollutant and xenobiotic responses. the same research group developed the mytarray 1.0, which includes 1,714 probes (venier et al., 2006) and also an oligo array using rnas from mussels injected with vibrio splendidus (venier et al., 2011). another oligonucleotide-microarray was developed using 4,488 different genes from m. galloprovincialis and m. trossulus to analyse the effects of heat stress on gene expression (lockwood et al., 2010) and to study the transcriptional responses of the two species to an acute decrease in salinity (lockwood and somero, 2011). the “big jump”: the next-generation sequencing technologies the “big jump” in genomic research will occur with the development of new-generation sequencing technologies (ngs). these new technologies have been developed because of the high demand for tools that deliver fast, inexpensive and accurate genome information. the primary advantage of ngs over conventional methods is the ability to produce large volumes of data inexpensively (in some cases in excess of one billion short reads can be achieved per run). multiple platforms for nsg   114   coexist in the marketplace, some having clear advantages for particular applications over others. commercially technologies are available from roche (454 life sciences), illumina (solexa sequencing technology), applied biosystems (life technologies/apg), helicos biosciences, the polonator, and the pacific biosciences (near-term technology). sequencing technologies combine various stages that can be broadly grouped as template preparation, sequencing and imaging, and data analysis. the unique combination of specific protocols distinguishes one technology from another and determines the type of data produced from each platform (harismendy et al., 2009; metzker, 2010). the two most common methods for template preparation are emulsion pcr (empcr) (dressman et al., 2003) and solid-phase amplification (fedurco et al., 2006). the empcr prepares sequencing templates in a cell-free system and generates a library of dna fragments. the dna is separated into single strands and is captured on beads (one dna molecule per bead). millions of beads are immobilised on a microscope slide, on an aminocoated glass surface or are deposited into individual picotiterplate wells, according to the platform used (polonator, life/apg and roche/454, respectively). solid-phase amplification is used to produce randomly distributed, clonally amplified clusters from the fragments onto a glass slide. this amplification can produce 100 200 million spatially separated template clusters (illumina/solexa). the four main sequencing and imaging strategies used include single-nucleotide addition or pyrosequencing (ronaghi et al., 1996, 1998), cyclic reversible termination (metzker, 2005), sequencingby-ligation (tomkinson et al., 2006) and real-time sequencing (eid et al., 2009) methods. a hard computational study is used after the generation of millions of reads to perform an alignment to a known reference sequence or an assembled de novo (pop and salzberg, 2008; chaisson et al., 2009; trapnell and salzberg, 2009). recently, the next-generation sequencing technologies have been applied to the study of different processes in molluscs. in non-bivalve molluscs, 454 sequences have been used in the fields of developmental biology, cell biology, larval ecology, ecotoxicology, parasitology, and chemical ecology for the investigation of different species, such as the marine snails littorina saxatilis (galindo et al., 2010) and ilyanassa obsolete (lambert et al., 2010), the pulmonated snails radix balthica (feldmeyer et al., 2011) and conus bullatus (hu et al., 2011), the mollusc crepidula fornicate (henry et al., 2010) and the photosynthetic slug elysia timida (wägele et al., 2011). in bivalves, this technology has been applied to investigate different physiological processes, such as shell deposition and repair in the antarctic clam (laternula elliptica) (clark et al., 2010) and biomineralisation in pinctada margaritifera (joubert et al., 2010). the technology has also been used to generate genomic tools for environmental monitoring in the manila clam, ruditapes philippinarum (milan et al., 2011). m. galloprovincialis and the yesso scallop (patinopecten yessoensis) have been used to study molecular aspects of their reproduction, the skewed sex ratios of their offspring and their adaptation to climatic and pollution factors (craft et al., 2010; hou et al., 2011). the developmental process has been analysed at different larval stages in the clam meretrix meretrix (huan et al., 2012). in an attempt to identify genes potentially involved in bivalve innate immunity, high-throughput sequencing was performed in the deep-sea hydrothermal vent mussel bathymodiolus azoricus (bettencourt et al., 2010) and in the manila clam ruditapes philippinarum using in vitro immune-stimulated hemocytes (moreira et al., 2012), producing more than 700,000 sequencing reads. in the next few years, the european project reproseed has planned to perform 454 sequencing projects in different bivalve species (p. maximus, m. edulis, m. galloprovincialis and r. decussates) using larvae, different adult tissues and hemocytes (stimulated with pamps in vivo and in vitro). the main objective is to enrich the database with specific immune-related sequences. moreover, the ontogeny of the immune system in bivalves and the immune response at the larval stages will be analysed. identification and characterization of relevant physiological and immune processes in bivalves genomic methods offer useful tools for studying the molecular basis of biological characteristics of great interest in aquaculture, such as disease resistance and stress. because information concerning the molecular mechanisms of the immune response in bivalves is scarce and fragmentary, the 454-pyrosequencing will lead to the identification of genes involved in the immune defence against infectious diseases. in this context, moreira et al. (2012) described a large number of immune related genes in ruditapes philippinarum, showing the presence of putative members of several immune pathways and processes, such as apoptosis, the toll-like signalling pathway and the complement cascade. this study also identified sequences from molecules that have not been described in bivalves, such as the complement pathway where almost all components were shown to be present. information obtained from the mytibase database was used by romero et al. (2011) to identify for the first time the most important molecules involved in the apoptotic process induced after exposure to uv light in mytilus galloprovincialis. apoptosis is a gene-directed mechanism that eliminates unnecessary or dangerous cells without the release of the intracellular components (kerr et al., 1972; tittel and steller, 2000). this mechanism is an essential biological process in all animals and is critical in many aspects of development and for the maintenance of immune system homeostasis (opferman and korsmeyer, 2003). moreover, apoptosis is a host defence mechanism against viral and bacterial pathogens (koyama et al., 2003; deleo, 2004). in mammals, two major pathways of apoptosis activation have been characterised in   115   detail. the intrinsic pathway, initiated in response to oxidative stress, genotoxic substances or specific signals, leads to the release of mitochondrial cytochrome c into the cytosol (armstrong, 2006). cytochrome c activates caspase-9 and the caspase signalling cascade through the formation of the apoptosome (li et al., 1997). the extrinsic pathway is activated by the death domain-containing transmembrane receptors through interaction with their extracellular ligands, which results in the direct activation of the caspase cascade and/or the induction of the mitochondrial membrane permeabilisation (bridgham et al., 2003). although genes encoding caspase proteins have been described in vertebrates, in almost all invertebrate phyla (lamkanfi et al., 2002) and a caspase 8-like gene has been reported in the gastropod haliotis diversicolor (huang et al., 2010), limited information regarding the genes that mediate this mechanism and their regulation under different stimuli is available for bivalves (sokolova, 2009; kiss, 2010). romero et al. (2011) characterised 6 different caspase genes in m. galloprovincialis and analysed their regulation under the experimental exposure to genotoxic substances and several pamps. the initiator caspase group was shown to be composed of 2 sequences with high homology to caspase-2 and caspase-8, whereas the executioner group was composed of 4 members with high homology to caspase-3/7. evaluation of the tissue expression patterns of the genes revealed extremely high expression levels within the gland and gills where the apoptotic process is highly active due to the clearance of damaged cells. the hemocytes also exhibited high levels of expression of these genes, likely due to the role of apoptosis in the defence against pathogens. to understand the mechanisms of caspase gene regulation, romero et al. (2011) treated hemocytes with uv light, environmental pollutants and pathogen-associated molecular patterns (pamps), and evaluated the apoptotic process using microscopy, flow cytometry and qpcr techniques. the results suggested that the apoptotic process is tightly regulated in bivalve molluscs by the overexpression/suppression of caspase genes; additionally, there was evidence of caspase-specific responses to pathogens and pollutants. the apoptotic process in molluscs has a similar complexity to that of vertebrates; however, apoptosis in molluscs exhibit unique features that may be related to their recurrent exposure to environmental changes, pollutants and pathogens imposed by their sedentary nature. it is important to highlight the considerable capability of molluscs to scope with different typologies of stress, since it represent a fundamental link between environmental changes, immunity and infection lethality. advancements in the understanding of the apoptotic pathway in mytilus galloporvincilais were achieved following the description of the mitochondrial pathway. romero et al. (unpublished data) described six novel genes related to the intrinsic pathway (bcl-2, bax, bi, p53, pdrg and dff) and studied the relative contribution of the intrinsic pathway to mitochondrial cell death through the inhibition of different stages of the intrinsic apoptotic pathway. concluding remarks the recent use of new sequencing technologies has drastically increased the number of mollusc genomic sequences in the databases. these sequences are the basis for understanding physiological processes and the immune response against diseases and for solving problems in the bivalve industry. genomic information will help find specific answers to a number of questions: can this information be used to discover specific genes linked to resistance? can the resistance be enhanced or manipulated by stimulating specific responses? can the resistant individuals be selected, and will this resistance be maintained and passed to descendants? will this information open alternatives to selection? 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identification of bac clones containing the genes involved in its innate immune system. mar. biotechnol. 10: 358-365, 2008. visions and perspectves isj 11: 1-3, 2014 issn 1824-307x visions and perspectves the importance of studying invertebrate immune-neuroendocrine functions e ottaviani department of life sciences, university of modena and reggio emilia, modena, italy accepted december 15, 2013 abstract the present paper illustrates various advantages of the use of invertebrates as an experimental model as these models provide important evidence in the field of evolutionary research and at the same time offer practical applications in the bio-medical and agronomy fields of research. key words: invertebrate; evolution; translational biology   introduction invertebrates approximately represent 96 % of all animal species, and thanks to their excellent capacity to adapt they can be found in almost every habitat of the earth, from the coldest areas, such as the poles to the warmest, such as the deserts. this unique combination of diversity and simplicity represent a great advantage for the study of defense mechanisms. however, caution must be taken when citing the "simplicity of the model", because nowadays it is well known that invertebrate systems, though simpler than those of vertebrates, are far from being simple. as far as the immuneneuroendocrine system is concerned, my group as well as others have revealed the involvement of numerous mediators in what that include mammalian-like molecules such as the neurohormone corticotrophin releasing hormone (crh); proopiomelanocortin products such as corticotropin (acth) and β-endorphin; small bioactive molecules such as norepinephrine, epinephrine and dopamine, steroid hormones such as glucocorticoids, cytokines such as il-1, il-6 and tnf-α and gaseous mediators such as nitric oxide (ottaviani et al., 1997). why study invertebrate immune-neuroendocrine functions? by analyzing the changes that different species have undergone in time, evolutionary studies highlight changes at the molecular level, as well as the mechanisms that allowed the accumulation of molecular variants and selection. but while changes ___________________________________________________________________________ corresponding author: enzo ottaviani department of life sciences university of modena and reggio emilia via campi 213/d, 41125 modena, italy e-mail: enzo.ottaviani@unimore.it have been found to be important we can now assert on the basis of our previous data (ottaviani and franceschi, 1996; ottaviani et al., 1997) as well as the molecular data now available in the literature on all the major families of proteins (antibodies, adhesion molecules, heat shock molecules, calcium-binding molecules, etc.) that conserved traits are highly significant and, in some cases, more relevant than changes. our research on the evolution of signaling molecules in immuneneuroendocrine functions (e.g., neuropeptides, hormones and cytokines) have revealed that these primary signaling molecules are conserved very well across species. despite the progressive modification of organs and systems, the mechanisms which govern the exchange of information between cells have been maintained. in this context, the molecular diversity of complex biological systems, such as the immune system seems to be derived from a single ancestral protein domain which by duplication, gene conversion, dna recombination, rearrangement and other molecular mechanisms have generated all the molecules of the immune system (marchalonis and schluter, 1994). immunocytochemistry, flow cytometry, ria tests and functional studies of some molecules in various invertebrate species show the presence of acth and β-endorphin-like molecules in cells with macrophage characteristics (ottaviani et al., 1997). such molecules have a physiological role in chemotaxis and phagocytosis, and the role of acth appears to be broader than that of β-endorphin. with respect to the neuroendocrine response, the results obtained in molluscs demonstrate that stress activates an axis that leads to the release of biogenic amines from immunocytes upon activation of both the crh and acth-dependent cascade (ottaviani and franceschi, 1996). overall, we are witnessing the presence of an axis crh-acth1    mailto:enzo.ottaviani@unimore.it biogenic amines as seen in vertebrates, which is even more notable, considering the observation that in invertebrates, in contrast to vertebrates, organs such as the hypothalamus, the pituitary and the adrenal gland are not present. moreover, recent data indicate that the transcriptional response of neurons to immunological stress in a mollusc, involves epigenetic modifications just like in vertebrates. here epigenetic changes occurred in the freshwater snail pomacea canaliculata after injection with lps in form of immunopositivity towards phospho (ser10)-acetyl (lys14)-histone h3 and enhanced cfos in the nuclei of small gangliar neurons. western blot analysis revealed a significant increase of phospho (ser10)-acetyl (lys14)-histone h3 in nuclear extracts 2 h after receiving an injection with lps. c-fos protein levels were significantly enhanced 6 h after lps injection (ottaviani et al., 2013). another example of conserved immuneneuroendocrine response is provided by cytotoxicity, present throughout metazoans. there is a remarkable conservation of a cytotoxicity type nklike cell from invertebrates to man. for instance, cells of mollusc were able to lyse target cells k562 pre-labeled with 51cr (franceschi et al., 1991). the components of signal transduction pathways in the modulation of the innate immune response have been highly conserved during evolution. kim and ausubel (2005) report that the tir-1 (toll/il-1receptor)/sarm (sterile alpha and heat/armadillo motif)-pmr (p38 mitogen-activated protein kinase family)-1/p38 mapk (map kinase) pathway is the ancestral immune signaling pathway of the common ancestor of nematodes, arthropods and vertebrates. on the whole, the findings reported above display a close parallelism between invertebrates and vertebrates in terms of molecules and of signaling pathways and this provides a valuable tool for studying the evolutionary mechanisms underpinning diversification of multicellular organisms. beside the interest for basic research this conservation presents potential application for translational research. for instance, in the biomedical research field, the isolation and purification of invertebrate immune-related molecules may provide new mediators in models for the development of new chemotherapeutic agents. furthermore, in agronomy, biological control praxes may find in invertebrate molecules potential factors for the prevention of the disasters caused by pests in agriculture. studies performed in marine invertebrates, i.e., sponges and ascidians, have demonstrated their capacity to produce secondary metabolites with unique structures and potent bioactivities providing an important source of new drugs for human and animal diseases and in particular against cancer (sandler, 2005; imperatore et al., 2012). peptides purified from sponges, ascidians, tunicates and molluscs have an antioxidant activity and cytotoxic effect on several human cancer cell lines such as hela, ags and dld-1 (suarez-jimenez et al., 2012). the biological activities of the secondary metabolites with anticancer and cytotoxic properties are exerted on the cytoskeleton, which plays a central role in cellular proliferation, motility and is deeply involved in the metastatic process associated with tumors (mollica et al., 2012). many anticancer substances isolated from sponges are already in clinical use or undergoing the final stages of clinical trials (laport et al., 2009). antimicrobial peptides such as cecropin a and b from the silk moth cecropia are able to inhibit the growth of and to kill yeast-phase candida albicans (andrä et al., 2001). moreover, a cecropin isolated from musca domestica is a bactericidal agent against clinical isolated multidrug-resistant escherichia coli (lu et al., 2012). in agriculture, several strategies have been developed for insect control, includes mating disruption (md), pheromone antagonists, pheromones and plant-based volatiles, attractantand-kill, and push-pull strategies (reddy and guerrero, 2010). many insect species communicate with each other by a variety of chemical signals called pheromones. in particular, sex pheromones attract one sex to the other so that mating can take place, a phenomenon common in the insect order of lepidoptera. sex pheromones are a complex mixture of chemicals and each species has it own specific blend. md technology uses synthetic products closely related to the pheromones produced by the female (known as a “calling” female) that are able to confuse males and limit their ability to locate calling females. for instance, the splat-orb, a pheromone formulation, provokes md in the oriental beetle (rodriguezsaona et al., 2010). another interesting study on md was conducted in guatemalan potato moth tecia solanivora using the following sex pheromone components: (e)-3-dodecenyl acetate, (z)-3dodecenyl acetate, and dodecyl acetate (mccormick et al., 2012). references andrä j, berninghausen o, leippe m. cecropins, antibacterial peptides from insects and mammals, are potently fungicidal against candida albicans. med. microbiol. immunol. 189: 169-173, 2001. franceschi c, cossarizza a, monti d, ottaviani e. cytotoxicity and immunocyte markers in cells from the freshwater snail planorbarius corneus (l) (gastropoda, pulmonata): implications for the evolution of natural killer cells. eur. j.immunol. 21: 489-493, 1991. imperatore  c, aiello  a, d'aniello f, luciano  p, vitalone r, meli r, et al. new bioactive alkylsulfates from mediterraneantunicates. molecules 17: 12642-12650, 2012. kim dh, ausubel fm. evolutionary perspectives on innate immunity from the study of 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the stress response from invertebrates to man. 3    http://www.ncbi.nlm.nih.gov/pubmed?term=mollica%20a%5bauthor%5d&cauthor=true&cauthor_uid=22614862 http://www.ncbi.nlm.nih.gov/pubmed?term=locatelli%20m%5bauthor%5d&cauthor=true&cauthor_uid=22614862 http://www.ncbi.nlm.nih.gov/pubmed?term=stefanucci%20a%5bauthor%5d&cauthor=true&cauthor_uid=22614862 http://www.ncbi.nlm.nih.gov/pubmed?term=pinnen%20f%5bauthor%5d&cauthor=true&cauthor_uid=22614862 http://www.ncbi.nlm.nih.gov/pubmed?term=reddy%20gv%5bauthor%5d&cauthor=true&cauthor_uid=20831959 http://www.ncbi.nlm.nih.gov/pubmed?term=guerrero%20a%5bauthor%5d&cauthor=true&cauthor_uid=20831959 http://www.ncbi.nlm.nih.gov/pubmed/20831959 the characteristic of immune parameters in zhikong scallop chlamys farreri and bay scallop argopecten irradians isj 11: 47-53, 2014 issn 1824-307x research report biochemical biomarkers of glyphodes pyloalis walker (lepidoptera: pyralidae) in exposure to tio2 nanoparticles n memarizadeh1, m ghadamyari1, m adeli2,3, k talebi4 1department of plant protection, faculty of agricultural sciences, university of guilan, rasht, iran 2department of chemistry, faculty of science, university of lorestan, khoramabad, iran 3department of chemistry, sharif university of technology, tehran, iran 4department of plant protection, faculty of agriculture, university of tehran, karaj, iran accepted january 28, 2014 abstract biochemical biomarkers and bioassays, due to their assumed immediate response after acute exposure of the organism to the stressor, are useful tools to gauging anthropogenic impacts. the toxicity of tio2-nanoparticles (tio2-nps) on the glyphodes pyloalis walker was assessed and the lc50 value obtained as 660.85 mg/l. the in vivo responses of g. pyloalis to sub-lethal concentrations of tio2-nps were surveyed by monitoring the activity of general esterases (est), peroxidase (pod) and glutathione s-transferase (gst), as biochemical biomarkers. activity of these biomarkers affected by exposure to tio2-nps and this could lead to the mortality or sub-lethal impacts. the effect of tio2-nps concentrations on the activity of these enzymes was correlated to the exposure time. the activity of est and gst was significantly decreased compared to the control, after 24 h of treatments. by increasing exposure time, the expression of est and gst was significantly increased. more pod expression was occurred at low concentrations (i.e. lc20 and lc30); however, at high concentrations, less pod activity obtained. it can be concluded that these enzymes are good early indicator of toxicity and in conjunction with acute toxicity studies allow adverse effects of tio2-nps to be predicted and managed. key words: tio2-nps; biomarker; glyphodes pyloalis; esterases; glutathione s-transferase; peroxidase introduction nanoparticles (nps) are particles with a diameter between 1 and 100 nm (buzea et al., 2007). they show unique physico-chemical properties which differ from those of their respective bulk materials; such as large surface area, charge and shape (handy et al., 2008). nps can be utilized in the agricultural practices and this can be result in both risks and benefits for the ecosystem. therefore, the evaluation of the safety of nps in the environment looks very important (kahru et al., 2008; cattaneo et al., 2009). due to unique characteristics of manufactured nps of titanium dioxide (tio2); such as chemically and biologically inert, stable toward corrosion and photocatalytic property, they are widely produced and consumed. hence, the potential uses of tio2-nps in various fields subjected them to the ___________________________________________________________________________ corresponding author mohammad ghadamyari department of plant protection faculty of agricultural sciences university of guilan, rasht, iran e-mail: ghadamyari@guilan.ac.ir ecotoxicological studies (linhua et al., 2009). cytotoxicity, phytotoxicity, lung inflammation and oxidative stress in mammals, plants and microorganisms have been reported as side effects of tio2-nps (ferin et al., 1992; warheit et al., 2007; wang et al. 2007, 2009; clemente et al., 2012). despite the fact that tio2 has been classified as innocuous to the organisms (who, 1996); recently the international agency for research on cancer (iarc) has classified this material as “possibly carcinogenic to humans” (iarc, 2010). till now, the vast majority of nanoecotoxicological studies with tio2-nps have been focused on their toxicity to aquatic organisms (clemente et al., 2012). lovern and klaper (2006) reported that daphnia magna exposed to filtered tio2-nps showed 100 % mortality at 10 mg/l; whereas, sonicated tio2-nps caused only 9 % mortality at 500 mg/l. federici et al. (2007) concluded that tio2-nps caused sub-lethal toxicity in rainbow trout (oncorhynchus mykiss) involved oxidative stress, organ pathologies, and the induction of antioxidant defense system such as reduced glutathione (gsh). zhu et al. (2008) 47 compared the toxicity of several metal oxide nps with their bulk counterparts to early developmental stages of zebrafish (danio rerio) and showed that neither tio2-nps nor bulk tio2 caused any toxicity to zebrafish embryos and larvae. klaper et al. (2009) demonstrated that gst and catalase (cat) are appropriate early biomarkers for prediction of physiological impacts and future toxicity of nps to daphnia. linhua et al. (2009) showed that superoxide dismutase (sod), cat and pod activities and lipid peroxidation (lpo) levels in various tissues of carps varied with the concentration and exposure time. jianhui et al. (2005) formulated dimethomorph with sodium dodecyl sulfate (sds)/tio2/ag nanomaterial as a photodegradable nanofungicide. guan et al. (2008) also used the same nanomaterials to formulation of imidacloprid. furthermore, they produced w/tio2/avermectin photodegradable microcapsules (guan et al., 2010). despite the investigation on nanoecotoxicological studies with aquatic organisms; so far, no published reports are available on acute toxicity and biochemical alteration (i.e., enzyme activities) caused by exposure of insect to tio2-nps. thus, due to potential for application of tio2-nps to formulation of photodegradable pesticides; we aimed to make the ecotoxicological assessment of tio2-nps exposure to an insect pest model species, glyphodes pyloalis. ests are detoxification enzymes which are involved in the insect physiology, metabolism, and xenobiotic detoxification (ishaaya, 1993). glutathione s-transferases (gsts) are multifunctional enzymes in the phase ii of pesticides metabolism (ezemonye and tongo, 2010). pod is an antioxidant enzyme which catalyzes the oxidation by hydrogen peroxide of a number of xenobiotics (linhua et al., 2009). because of the lack of knowledge about responses of insect est, gst and pod to tio2-nps, these enzyme activities were evaluated in g. pyloalis treated with tio2-nps at different exposure times to predict the impact of these nps at sub-lethal levels. materials and methods chemicals glycerol, ethanol, triton x-100, h2o2, bovine serum albumin, α-naphthyl acetate (α-na), β-naphthyl acetate (β-na), reduced glutathione (gsh), 1-choloro-2,4-dinitrobenzene (cdnb), 1,2-dichloro-4-nitrobenzene (dcnb), bromophenol blue, ethylene diamine tetraacetic acid (edta), tris and acetic acid were purchased from merck (germany). titanium isopropoxide (ti(i-pro)4) and guaiacol were purchased from sigma-aldrich (st louis, missouri, usa). fast blue rr salt was bought from fluka (buchs, switzerland). insects the glyphodes pyloalis was collected from infested mulberry orchards in the vicinity of rasht, iran. mass rearing of insects was carried out in the laboratory, under controlled conditions with 25 ± 2 °c, 70 ± 10 % rh, and 16:8 l:d. newly-ecdysed fifth instar larvae of g. pyloalis were used for bioassay experiments. synthesis of tio2-nps tio2-nps were prepared according to the method of trung et al. (2003) by hydrolyzing titanium isopropoxide which was added drop by drop into stock solution (i.e. ethanol and acetic acid in a ratio of 8:3 v/v with glycerol) at 10 °c, followed by rigorous stirring under an argon atmosphere for 3 h. then, the solutions were heated at 60 °c for 5 h or until the gelling reaction was completed. the dried precipitates were heated at 400 °c for 10 h, at the heating rate of 1 °c/min. bioassay with tio2-nps suspension the toxicity of tio2-nps suspension in water was assayed to newly-ecdysed fifth instar larvae of g. pyloalis using the leaf dip bioassay (memarizadeh et al., 2011). five serial dilutions of tio2-nps suspensions (250, 500, 800, 1000 and 1200 mg/l) were prepared and then sonicated for 30 min in a bath type sonicator (100 w, 40 khz) to disperse the nanoparticles. mulberry leaf discs (diameter 3.5 cm) were immersed in the dilutions for 45s. after drying of leaf discs, petioles of them were placed in a vial containing water to provide moisture. up to 5 synchronized fifth instar larvae of g. pyloalis were placed on each treated leaf disk. mortality was assessed after the treated larvae were maintained at 25 ± 2 °c, 70 ± 10 r.h. for 48 h. each experiment was replicated ten times. the criterion for death was that a larva did not move when prodded with a camel’s hair brush. treatment the newly-ecdysed fifth instar larvae of g. pyloalis were treated by different concentrations of tio2-nps suspensions (0, 290, 380, 475, 565 and 665 mg/l, equivalent to control, lc10, lc20, lc30, lc40 and lc50, respectively). treatments were conducted according to the bioassay method. over 3 days after treatments, representative samples were taken from survived larvae. the collected samples were placed in a deep freezer at -20 °c until biochemical assays were performed. preparation of samples for determining est activity, one larva was homogenized in 150 µl of 0.1 m phosphate buffer, ph 7.0 containing 0.05 % (v/v) triton x-100 using a glass hand-held homogenizer on ice. after homogenization, they were centrifuged at 12,000×g for 15 min at 4 °c and resulted supernatant was used in the assay. for gst assay, enzyme preparation was similar to that previously mentioned for est; however without triton x-100. for pod assay, each larva was homogenized in 150 μl of 50 mm phosphate buffer (ph 7.4) containing 0.1 mm edta. once homogenized, they were centrifuged at 12,000×g for 15 min at 4 °c and resulted supernatant was used in pod assay. determining pod activity the 1 ml reaction mixture was consisted of 450 µl of 50 mm phosphate buffer (ph 7.4) containing 45 mm guaiacol, and 100 µl of prepared supernatant. adding 450 µl of 50 mm phosphate buffer (ph 7.4) 48 table 1 log dose probit-mortality data for tio2-nps against g. pyloalis after 48 h n lc10(95% ci)a lc20 (95% ci)a lc30 (95% ci)a lc40 (95% ci)a lc50 (95% ci)a slope ±se χ 2 (df)b g. pyloalis 250 290.86 (151.02394.95) 385.51 (237.99492.06) 472.34 (326.8582.86) 561.88 (422.32683.51) 660.85 (525.57810.02) 3.59± 0.42 6.25 (3) athe lc values are expressed as part per million (ppm) and their 95 % confidence intervals (95 % ci) bthe value of p > χ2 larger than or equal to 0.05 indicates a significant fit between the observed and expected regression lines containing 225 mm h2o2, the reaction was initiated. then, the oxidation of guaiacol was followed at 470 nm with a spectrophotometer (cary 3) using an extinction coefficient of 26.6 mm-1cm-1 (bergmeyer, 1974). the pod activity was expressed as μmol.min-1mg protein-1. determining est activity esterase activity was determined based on the van asperen (1962) method. α-na and β-na were used as substrates. 12 µl of supernatant were added to per well of a microplate which containing 113 µl phosphate buffer (ph 7.0). after 3 min, adding 50 µl of 1.8 mm substrate solution, the reaction was initiated. following the addition of 50 µl of the fast blue rr salt, absorbance at 450 and 540 nm were measured in a microplate reader (awareness technology inc., florida, usa) for α-na and β-na, respectively. the formation of the α-naphtholand β-naphthol-fast blue rr dye complex was converted to a specific activity using standard curves, which were obtained from different concentrations of α-naphthol and β-naphthol mixed with fast blue rr salt (0.075 %), respectively (miller and karn, 1980). the est activity was expressed as nmol.min-1mg protein-1. determining gst activity gst assays were conducted according to the method of habig et al. (1974), using cdnb and dcnb as substrates. 15 µl supernatant, 100 µl of 1.2 mm substrate solution and 100 µl of 10 mm gsh were added to per well of a microplate. enzyme activity was determined by continuously monitoring the change in absorbance at 340 nm for 5 min at 25 °c with a microplate reader (awareness technology inc.). the gst activity was expressed as μmol.min-1mg protein-1. determining protein concentration protein concentrations were estimated by the bradford (1976) method, using bovine serum albumin as standard. statistical analysis three replicates were conducted for all the biochemical assays and data were subjected to analysis of variance (anova). statistical analyses were performed at p = 0.05 by tukey’s test using the sas software. results bioassay results table 1 shows the median lethal concentration (lc50), sub-lethal endpoints and the 95 % confidence limits which calculated from probit regression using the polo-pc computer program (leora, 1987) and based on finney (1971). pod activity the level of pod differed significantly among treatments (fig. 1). 24 h after g. pyloalis larvae treatment with lc20 and lc30 concentrations of tio2-nps, the highest pod levels were observed. results showed that by increasing the sub-lethal concentrations from lc10 to lc30 and by increasing exposure time, pod activity was significantly increased compared to the control. lc50 concentration of tio2-nps significantly decreased pod activities. however, they were not significantly changed at lc40 concentration in comparison to the control group (fig. 1). fig. 1 peroxidase (pod) activity in g. pyloalis exposed to sub-lethal concentrations of tio2-nps at different time intervals. different letters indicate that the specific activity of enzymes is significantly different from each other by tukey’s test (p < 0.05). est activity est activities of g. pyloalis treated with sublethal concentrations of tio2-nps are presented in 49 figure 2. analysis of variance showed that esterase activity, using both α-na and β-na, significantly affected by: 1) the tio2-nps concentrations, 2) time of exposure to tio2-nps and 3) interplay effect of concentration and exposure time. this means that the effect of tio2-nps concentrations on the esterase activity is correlated to the length of exposure time. assessment of est activity in treatments after 24 h showed that tio2-nps led to inhibition of these enzymes and thus decreased their activities. however, when α-na was used as substrate, by increasing the time of exposure to lc10, lc20 and lc30 concentrations, the est activities significantly increased (fig. 2). a b fig. 2 comparison of esterase activity in g. pyloalis exposure to different concentrations of tio2-nps over 3 days, using α-na (a) and β-na (b) as substrates. means followed by similar letters showed no significantly difference from each other by tukey’s test (p < 0.05). gst activity gst activities of g. pyloalis treated by sublethal concentrations of tio2-nps are presented in figure 3. when cdnb was used as substrate, increasing tio2-nps concentrations led to significant reduction in gst activities after 24 h; however, this reduction was steeper than when dcnb was used in the assay. furthermore, as depicted in figure 3, by increasing the exposure time of g. pyloalis to lc20, lc30 and lc40 of tio2-nps, gst activity significantly increased. results of analysis of variance showed that using both cdnb and dcnb, gst activity significantly affected by: 1) the tio2nps concentrations, 2) exposure time of larvae to tio2-nps and 3) interplay effect of concentrations and exposure time. this means that the increasing tio2-nps concentrations over exposure time could be enhance the conjugation of gsh to these nps and thus the enzyme activity was affected. a b fig. 3 comparison of gst activity in g. pyloalis exposure to different concentrations of tio2-nps over 3 days, using cdnb (a) and dcnb (b) as substrates. means followed by similar letters showed no significantly difference from each other by tukey’s test (p < 0.05). discussion since the growth in use of nps and nanotechnology in various fields mainly in agriculture is inevitable, ongoing study of nanotoxicity will be necessary in order to avoid their unpredictable complications (clemente et al., 2012). to minimize the adverse environmental effects of manufactured and engineered nanomaterials, it is critical to have a good understanding of their toxic potential (handy et al., 2008). the dose-mortality data for tio2-nps generated in the present study showed a lc50 value of 660.85 mg tio2-nps suspension liter −1 in g. pyloalis larvae. following the bioassay results, the g. pyloalis larvae were exposed to 290, 380, 475, 565 and 665 mg/l tio2-nps as lc10, lc20, lc30, lc40 and lc50, respectively. the experiments were designed to allow sub-lethal physiological effects of tio2-nps. the exposure time of 3 days was chosen to enable some biochemical responses of treated insects. lovern and kapler (2006) reported an lc50 of 5.5 ppm in d. magna which exposed to filtered tio2nps for 48 h. kim et al. (2010) reported a 70 % mortality rate in d. magna exposed to 5 mg/l of sigma aldrich tio2-nps for 21 days. wang et al. (2009) were estimated 80 mg/l of tio2-nps as lc50 50 and showed that tio2-nps are more toxic than their bulk counterparts to the caenorhabditis elegans. acute toxicity assays will not be able to provide sufficient information on the interaction of nanomaterials and organisms. whereas, sub-lethal effects can be indicated the impacts of nps on the physiology and survival of organisms. biochemical biomarkers as indicators of sub-lethal effects of a stressor can be used to early warning of population level impacts (de coen and janssen, 1997; klaper et al., 2009). biomarkers indicative of neurotoxicity (est), oxidative stress (pod) and phase ii biotransformation of xenobiotics (gst), as well as general mortality have been linked to populationlevel endpoints (jemec et al., 2007; paskerova et al., 2012). usage of these biomarkers in risk assessments is suitable diagnostic tool for the detection of specific contaminants well before real adverse effects can occur (nascimento et al., 2008). pollutants may increase the intracellular formation of reactive oxygen species (ros) which have been reported to affect the physiology, growth, and survival of organisms (filho, 1996; pandey et al., 2003). pod is the key enzyme in antioxidant defense systems to convert the resulting free radicals h2o2 to water and oxygen (linhua et al., 2009). over 3 time points of treatments, by increasing the concentration of tio2-nps from lc10 to lc30, the pod activities were increased and then at lc and lc concentrations were decreased. 40 50 therefore, results of the present study demonstrate that enhanced activities of pod at low concentrations could lead to the elimination of ros. this also could be an indication that exposure to low concentrations would yield less mortality in these treatments. because of the importance of general ests in the insect physiology, metabolism and detoxification of xenobiotics, in this study the possible role of these enzymes as a biomarker to determination of nps toxicity was investigated (ishaaya, 1993; memarizadeh et al., 2013). results of the present work showed that by increasing tio2-nps concentration ests activities were decreased due to their inhibition. also the lc50 concentration of tio2nps had the highest inhibition on the est activity, when α-na was used as substrate. since, an effective inhibition of α-esterases occurs at lc50 concentration; these enzymes may be as a good indicator of the tio2-nps toxicity. gsts are multifunctional enzymes of the phase ii biotransformation system which play a key role in metabolism of a broad variety of xenobiotics and endogenous compounds (ezemonye and tongo, 2010; ezeji et al., 2012; memarizadeh et al., 2013; zamani et al., 2014). gsts are able to conjugate the xenobiotics and their metabolites to the tripeptide glutathione (gsh) and making them soluble for easy excretion (oakley, 2011). in addition to detoxification role of these enzymes, gsh also has antioxidant properties. thus, gsh as a general stress indicator is a more useful diagnostic tool. our study revealed that the gst activity significantly affected by exposure to tio2-nps. so, increased tio2-nps concentrations led to significant reduction in gst activities. results also showed that the lc50 concentration of tio2-nps had the highest impact on the gst activity. data demonstrated that activities of detoxification and antioxidant enzymes altered by exposure g. pyloalis to tio2-nps and this can be lead to the mortality or sub-lethal impacts. furthermore, gst, est and pod activities mainly affected by lc50 concentration and thus, this concentration is a good early indicator of tio2-nps toxicity. the sub-acute toxicity of tio2-nps to carp (cyprinus carpio) was assessed by linhua et al. (2009). they showed that 100 and 200 mg/l tio2nps caused the significant decrease in sod, cat and pod activities suggesting that the fish exposed to tio2-nps suffered from the oxidative stress. as each biomarker may have different sensitivities depending on the mode of action of the chemical (jemec et al., 2007); the use of multiple biomarkers to evaluate toxicity of nps for insects will provide the most suitable tool to indicate the potential impacts of them. conclusion the results of this study suggest that sub-lethal effects of tio2-nps to g. pyloalis are related to concentration and length of exposure time. est, gst and pod as biochemical biomarkers are useful diagnostic tools to determination of nps toxicity. pod activity in different treatments indicates that these particles are causing oxidative stress; especially at a lower concentration than the lc50 concentration. therefore, the expression of pod in low sub-lethal concentrations of tio2-nps in addition to the inhibition of 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abstract the increasing levels of fluoride in the aquatic environments is a matter of concern for freshwater and marine ecosystems. although data on fluoride toxicity to aquatic organisms are increasing in the scientific literature, few data are available regarding the effects, at cellular level, on freshwater or marine invertebrates. in the present paper, we present data on the effects of in vitro exposure to sodium fluoride (naf) on the hemocytes of the manila clam venerupis philippinarum. results indicate that naf reduces cell spreading and phagocytosis in a dose-dependent way, through the alteration of the actin cytoskeleton. in addition, the compound decreases the stability of the lysosomal membranes, as revealed by the neutral red assay. in addition, the observed increase in hemocyte mortality and the parallel rise in dna fragmentation inside their nuclei, as revealed by the tunel assay, suggest a naf-dependent induction of apoptosis, in accordance with the capability of naf to induce oxidative stress, a known cause of apoptosis, as hemocytes represent the major defence weapon against foreign, potentially pathogenic microbes, the above results indicate a negative effect of naf on the immune status of v. philippinarum. key words: venerupis philippinarum; bivalve molluscs; sodium fluoride; hemocytes; immunotoxicity   introduction fluorine belongs to the viia group of the periodic table and is widely distributed in the environment, mainly as fluorite, a calcium salt of the ionic form fluoride. fluoride was considered essential for tooth and bone development in mammals, although, today, a lack of consensus is emerging on its possible role in human nutrition, development and growth (nielsen, 2009). high fluoride concentrations can induce detrimental effects on cells and organisms as they can act as general inhibitors of various enzymes (barbier et al., 2010). recent results indicate that, when present at high levels, the ion causes cell toxicity, mainly related to the induction of oxidative stress and the activation of the apoptotic pathway (barbier et al., 2010; gutowska et al., 2010; jacinto-alemán, 2010; wang et al., 2010; liu et al., 2011). fluoride, in fact, can increase the release of cytochrome c from mitochondria, inhibit antioxidant enzyme activity, rise the production of reactive oxygen species and activate map-kinase-dependent signaling pathways ___________________________________________________________________________ corresponding author: loriano ballarin department of biology university of padua via u. bassi 58/b, 35100 padua, italy e-mail: loriano.ballarin@unipd.it (barbier et al., 2010; inkielewicz-stepniak and czarnowski, 2010; basha et al., 2011; chen et al., 2011; izquierdo-vega et al., 2011). in addition, fluoride can interfere with fertilization (izquierdovega et al., 2008; izquierdo-vega et al., 2011), mammalian development (zhu et al., 2013) and amphibian metamorphosis (zhao et al., 2013). as regards the aquatic environment, fluoride is present in unpolluted waters at concentrations ranging from 0.01 to 0.3 mg/l, however its level can increase more than 100 times as a consequence of human activities (camargo, 2003). fluoride can accumulate in the exoskeleton of arthropods and in vertebrate bones, as well as in the soft tissues of some invertebrates such as mussels and barnacles (barbaro et al., 1981a). as for the toxic effects, fluoride can lead to reduced male and female fecundity, growth impairment and mortality in aquatic organisms (casellato et al., 2013). seawater or estuarine organisms are usually less affected by high fluoride levels than those living in freshwater (camargo, 2003): this probably explains the paucity of data available for marine animals. bivalve molluscs are filter-feeding organisms widely used in the assessment of the quality of the aquatic environment and fluoride toxicity has been reported in the freshwater mussels alasmidonta raveneliana (keller and augspurger, 2005) and 22   mailto:loriano.ballarin@unipd.it fig. 1 a-e: fixed hemocytes stained with giemsa’s solution. a: undifferentiated cell; b-c: granulocyte in the round (b) and spreading (c) morphology; d-e: hyalinocyte in the round (d) and spreading (e) morphology. f: tunel assay for dna fragmentation; arrowheads indicates positive nuclei with fragmented dna. g: giemsa-stained hemocytes with ingested yeast cells (arrows). h-i: neutral red assay on control (h) and naf-exposed (i) cell. bar = 5 μm. dreissena polymorpha (casellato et al., 2012; del piero et al., 2012). as invertebrates, bivalves rely only on innate immunity to prevent foreign, potentially pathogenic cells or microbes from entering the organism. circulating hemocytes represent their major defense weapon and a loss of hemocyte functionality can be deleterious for the whole organism. at least three hemocyte types have been described in the clam venerupis (previously ruditapes = tapes) philippinarum: phagocytes (including granulocytes and hyalinocytes), hemoblasts or undifferentiated cells and serous cells (cima et al., 2000). this organism is emerging as a reliable model species for studying the effects of xenobiotic at various levels and variations in various parameters, including those relate to immune responses, have been reported in this species as a consequence of the exposure to pollutants (matozzo et al., 2001, 2002, 2012; matozzo and marin, 2005; aguirre-martínez et al., 2013). in the present work we studied, in v. philippinarum, the effects of in vitro exposure to fluoride on hemocyte functionality, with particular attention to phagocytes in order to get information on their mechanisms of action. we used naf concentration ranging from 10 to 250 µg/ml, the lower values resembling the concentration values reported for polluted waters in the lagoon of venice (barbaro et al., 1981b) and the higher values being similar to those used for in vitro experiments with mammalian cultured cell lines (barbier et al., 2010). results indicate detrimental effects on phagocytosis, probably related to cytoskeletal alterations, lysosomal stability, and cell survival, due to the induction of apoptosis. materials and methods animals venerupis philippinarum from the lagoon of venice were kept in tanks containing filtered sea water (fsw) and a layer of fine calcareous sand to allow burrowing, in thermostatic rooms, at the temperature of 17 °c. the animals were fed with liquifry marine (liquifry co, dorking, uk) for at least 5 days before experiments were undertaken. fluoride solution a storage solution was prepared by dissolving sodium fluoride (naf) in filtered seawater (fsw) at the nominal concentration of 1 mg/ml. this solution was subsequently diluted in fsw to obtain the working solutions of 10, 50 and 250 μg/ml. fsw was used in controls. hemocyte collection hemolymph was collected from the anterior and posterior adductor muscle with a plastic syringe and diluted 1:1 with a solution of 0.38 % na citrate in fsw, ph 7.5, to avoid hemocyte agglutination. hemolymph was centrifuged at 780xg for 10 min and pellets were resuspended in fsw to give a final concentration of 106 cells/ml. sixty µl of hemocyte suspension were placed in the center of culture chambers, prepared as described elsewhere (cima, 2010) and left to adhere to coverslips for 30 min at room temperature. for each experiment, pools of hemocytes from various animals were used. hemocyte viability assay to estimate the effects of naf on hemocyte viability, cell monolayers were exposed to 10, 50 or 23   fig. 2 mortality index of hemocytes exposed to various naf concentrations. significant differences with respect to controls (fsw) are marked by asterisks. *: p < 0.05; **: p < 0.01; ***: p < 0.001 250 µg/ml of naf in fsw for 60 min, then incubated with 0.25 % trypan blue in fsw for 5 min at room temperature and observed in vivo under a leitz dialux 22 light microscope (lm) at 1250×. the frequency of blue (= dead) cells was finally evaluated and expressed as the mortality index. cell spreading assay after adhesion to coverslips, hemocytes were incubated for 60 min with naf at the concentrations reported above; fsw was used in controls. cells were then fixed in 1 % glutaraldehyde and 1 % sucrose in fsw, stained with 10 % giemsa solution in fsw and mounted as already described. the morphology of hemocytes was observed under the lm, at the magnification of 1250× (at least 300 hemocytes per slide).  computer-assisted image analysis (casting image nt) was performed on fixed hemocyte monolayers to evaluate the phagocyte shape factor, defined as in ottaviani et al. (1997). one hundred cells were considered in each experimental situation. lower shape factors indicate larger perimeters with respect to the areas and, therefore, an increased amoeboid shape. phagocytosis assay after adhesion of hemocytes to coverslips, cells were incubated for 60 min at room temperature with 60 μl of a suspension of yeast (saccharomyces cerevisiae) cells (yeast:hemocyte ratio = 10:1) in fsw containing naf at the concentrations reported above. hemocyte monolayers were then gently washed several times in fsw to eliminate uningested yeast, fixed as reported above, washed in phosphate buffered saline (pbs: 1.37 m nacl, 0.03 m kcl, 0.015 m kh2po4, 0.065 m na2hpo4, ph 7.5), stained with 10 % giemsa and mounted on glass slides as previously described. slides were observed under the lm at 1250× and the cells in 10 optic fields (about 200 cells) per slide were counted. the percentage of cells with ingested yeast was evaluated. neutral red retention assay lysosomal membrane stability was assessed using a modification of the neutral red (nr) retention assay (lowe et al., 1995), as previously described (matozzo et al., 2001). a stock solution of 0.4 % nr in fsw was prepared. working solution was obtained by diluting 10 μl of stock solution in 5 ml of fsw. after exposure of hemocytes to naf for 5, 15, 30 and 60 min, naf-containing fsw was discarded from culture chambers and 60 μl of nr working solution were added. ten minutes later, the slides were observed under the lm at 1250×. the percentage of hemocytes showing dye loss from lysosomes into the cytosol which, consequently, appears reddish-pink was expressed as the membrane stability index. assay for apoptosis to reveal dna fragmentation, cells were exposed for 60 min to naf, fixed and incubated in the terminal dutp nick-end labelling (tunel) reaction mixture (in situ cell death detection kit, roche) for 60 min at 37 ºc, according to the manufacturer’s instructions. subsequently, they were incubated with a peroxidase-conjugated antifluorescein isothiocyanate (fitc) antibody, stained with 0.63 mm 3-3’-diaminobenzidine (dab) in pbs containing 4 % hydrogen peroxide, washed in distilled water, mounted with acquovitrex (carlo 24   fig. 3 apoptotic index of naf-exposed hemocytes. asterisks: significant differences with respect to controls (fsw). ***: p < 0.001 erba) and observed under the lm. the presence of fragmented dna was revealed by dark brown staining and the percentage of positive cells was expressed as the apoptotic index. statistical analysis each experiment was replicated at least three times (n = 3) with three independent hemolymph pools; data are expressed as mean ± sd. at least 300 cells in 10 optical fields at 1250× were counted under the lm for each experiment. frequencies were compared with the non-parametric χ2 test. in the case of the shape factor, data were checked for normal distribution (shapiro-wilk's test) and homogeneity of variances (bartlett’s test). as anova assumptions were not fulfilled, the nonparametric mann-whitney u-test was used for comparison. results the hemocytes of v. philippinarum three hemocyte types have been described in v. philippinarum: undifferentiated cells (fig. 1a), phagocytes, and serous cells. phagocytes represent the majority of the circulating cells, amounting to about 80 % of the total cell number (cima et al., 2000), and include granulocytes (figs 1b c) and hyalinocytes (figs 1d e): both the cell types can assume a round (figs 1b, d) or a spreading (figs 1c, e) morphology. naf alters hemocyte viability and induces apoptotic cell death under control conditions, about 3 % of the hemocytes stained with trypan blue. this fraction significantly increased to values around 10 % upon the exposure to 10 and 50 μg/ml naf (p < 0.05 and p < 0.01, respectively). at 250 μg/ml naf, the cell mortality rose to about 23 % (p < 0.001 with respect to the control; fig. 2). a significant (p < 0.001) increase in the fraction of hemocytes positive to the tunel assay (fig. 1f) with respect to controls was reported for hemocytes treated with 50 and 250 µg/ml of fluoride (about 15 and 20 %, respectively; fig. 3). naf inhibits phagocytosis by clam hemocytes about 18 % of hemocytes could ingest yeast cells in controls (figs 1g, 4). when hemocytes were incubated with yeast cells in the presence of fluoride, a significant (p < 0.01) reduction in the fraction of phagocytosing cells was obtained for all the used concentrations (fig. 4). exposed hemocytes show changes in cell shape and internal membrane stability in control slides, more than 50 % of hemocytes had a spreading morphology, whereas most of the cells exposed to 250 μg/ml of naf, acquired a round morphology. the shape factor of control hemocytes (0.55 ± 0.15) rose significantly (0.61 ± 0.15; p < 0.001) when hemocytes were exposed at the highest naf concentration. in control hemocytes, the fraction of cells with nr-stained cytoplasm never exceeded 3 % and the dye accumulated inside cytoplasmic granules, probably lysosomes, of phagocytes (fig. 1h). under stress conditions, the dye leaked into the cytoplasm which assumed a pinkish color due to membrane alteration. in hemocytes exposed to 250 μg/ml naf, the percentage of cells with red cytoplasm (fig. 25   fig. 4 percentage of phagocytosing cells in the presence of various naf concentrations. significant differences with respect to controls (fsw) are marked by asterisks. **: p < 0.01; ***: p < 0.001. 1) was significantly (p < 0.01) increased after 5 min of exposure and reached the value of 7 % after 60 min of incubation (fig. 5). discussion fluoride has been recognized as a cause of toxicity for many aquatic organisms ranging from algae and aquatic plants to invertebrates and fishes (camargo, 2003). at the cellular level, it acts as an inhibitor of various enzyme activities, and is able to induce oxidative stress and, as consequence, stress responses through the activation of mapk-related transduction pathways and cell death by apoptosis (barbier et al., 2010). in the present paper, we analyzed the effects of in vitro naf exposure on the hemocytes of the clam v. philippinarum, a bivalve mollusc widely distributed along the west coast of the adriatic sea. as invertebrates, clams rely on innate immunity for their defense and hemocytes play a key role in the response towards potentially pathogenic microbe having entered the organism. moreover, hemocytes represent a selected cell population to investigate, at the cellular level, the acute effects of exposure to xenobiotics. the naf concentration used in our experiments ranged from 10 to 250 μg/ml: the lower values are close to the concentration values reported for polluted waters in the lagoon of venice (barbaro et al., 1981b) whereas the higher values, although below those reported from highly polluted waters close to aluminum smelters (ares et al., 1983; camargo, 2003), are similar to those used for in vitro experiments with cultured cell lines (barbier et al., 2010). as reported before, most of the v. philippinarum hemocytes are phagocytes and they can assume a spreading or a round morphology. according to previous hypothesis (cima et al., 2000), spreading and round cells probably represent different phagocyte differentiation stages, with spreading cells representing actively wandering cells which, once ingested foreign material, withdraw their cytoplasmic extensions and acquire a round morphology. in control hemocytes, the frequency of spreading cells amounts to about 50 % of the total circulating cells: this value is severely decreased in the presence of pollutants which can alter the cytoskeletal organization (cima et al., 1999). the alteration of the hemocyte cytoskeletal organization in the presence of pollutants has been reported also for other marine invertebrate species (fagotti et al., 1996; cima et al., 1998; olabarrieta et al., 2001; gómez-mendikute and cajaraville, 2003; menin et al., 2008; franchi et al., 2013). as spreading and phagocytosis involve the actin component of the cytoskeleton, the observation that fluoride hampers both the processes suggests that, naf can interfere with actin polymerization. similar conclusions were reached in studies on mammalian cells in culture (barbier et al., 2010). the negative effect on actin cytoskeleton can be the consequence of either a direct interaction with microfilaments or an alteration of ca2+ homeostasis: previous studies have reported that fluoride can act at both the levels within the cell (barbier et al., 2010), although further research is required to better define the relationships between fluoride and cytoskeleton. the observed reduction in phagocytic activity and spreading morphology may also be the consequence of cell death by apoptosis, which affects about 20 % 26   fig. 5 membrane stability index of hemocytes exposed to various naf concentrations for different periods of time. asterisks: significant differences with respect to the respective controls (fsw). **: p < 0.01; ***: p < 0.001 of the hemocytes at fluoride concentrations higher than 10 μg/ml. moreover, the similarity between the values of the mortality and the apoptotic index at the various concentrations, suggests that most of the observed mortality is due to apoptotic cell death. an increase of apoptosis consequent to the exposure to fluoride has been reported in various mammalian cells (gutowska et al., 2010; jacintoalemán et al., 2010; wang et al., 2010) where, the down regulation of bcl-2, the increase in cytochrome c release from mitochondria and the activation of both the intrinsic and the extrinsic pathway of cell death have been reported (barbier et al., 2010). the observed occurrence of apoptosis is likely related to the induction of oxidative stress: this hypothesis fits previous observations demonstrating that one of the main effects of fluoride on cells is the inhibition of antioxidant enzymes and the increase of nadph oxidase activity with the consequent production of reactive oxygen species (barbier et al., 2010; izquierdo-vega et al., 2011; liu et al., 2011). the trypan blue exclusion assay evidences the alteration of the cell plasma membrane permeability, a final step in the progression of apoptosis; conversely, the nr assay gives an indication of the stability of the membrane of acid cytoplasmic compartments, abundant in phagocytes in the form of lysosomes, where the dye usually concentrates. the alteration of the internal membranes leads to the leakage of lysosomal content in the cytosol, an additional factor negatively influencing phagocyte functionality, which was clearly observed after 5 min of incubation at 250 μg/ml naf. in conclusion, our results show that fluoride affects the functionality of v. philippinarum phagocytes and, since the reduction of immune functions can severely compromise the organismal survival, it can be deleterious for the whole biocoenosis. in addition, they 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development in bufo gargarizans tadpoles. ecotoxicology 22: 1123-1132, 2013. zhu jq, si yj, cheng ly, xu bz, wang qw, zhang x, et al. sodium fluoride disrupts dna methylation of h19 and peg3 imprinted genes during the early development of mouse embryo. arch. toxicol. 2013 [ in press]. isj 12: 29-37, 2015 isj 12: 29-37, 2015 issn 1824-307x research report silencing two main isoforms of crustacean hyperglycemic hormone (chh) induces compensatory expression of two chh-like transcripts in the red swamp crayfish procambarus clarkii c manfrin, l peruzza, lc bonzi, a pallavicini, pg giulianini department of life sciences, university of trieste, trieste, italy accepted january 14, 2015 abstract rna interference has frequently been applied to modulate gene function in organisms. with the aim of creating new autocidal methods based on neuro-endocrine disruptors for invasive populations of procambarus clarkii, we silenced the crustacean hyperglycemic hormone (chh) by injecting the corresponding dsrna. chh is a pleiotropic hormone that primarily regulates the mobilization of energy reserves and plays a pivotal role in stress responses. here, we describe two experiments aimed at testing whether chh silencing significantly alters important physiological aspects. the first experiment investigates the effects of chh silencing at the glycemic and transcriptomic level in the eyestalk. the second experiment explores the long-term effects of chh silencing and the effects on mortality and moulting rates. osmotic deficits and mortality were recorded in specimens injected with chh dsrna, whilst controls were injected with gfp dsrna. after 20 days, despite still silenced for chh, individuals that survived recovered a strong hyperglycemic response after serotonin injection due to the compensatory effect of two peptides belonging to the crustacean neurohormone chh protein family. key words: procambarus clarkii; crustacean hyperglycemic hormone (chh); rna interference; stress; survival   introduction the red swamp crayfish is an invasive species widely distributed worldwide (issg, 2012). its burrowing behaviour and high fertility make it very competitive and often winning against native species which inhabit the same habitat. the policies applied to contrast the red swamp crayfish invasion into new habitats so far have been unsuccessful. the use of autocidal methods appears to be a promising strategy. in fact, autocidal approaches are based on the target species' biology and therefore do not cause environmental contamination and do not impact non-target species (gherardi and angiolini, 2004). silencing key hormones via baits would allow selective disturbance of the target alien species and reduce adverse effects on native species, even those closely related to the alien species. in addition, this method is potentially easily applicable year round. the method is also relatively inexpensive, compared to the costs of trapping, the approach used so far to restrain the spreading of invasive species. ___________________________________________________________________________ corresponding author: piero giulio giulianini department of life sciences university of trieste via l. giorgieri, 5, 34127 trieste, italy e-mail: giuliani@units.it the crustacean hyperglycemic hormone (chh) controls many fundamental physiological functions such as glucose mobilization from glycogen depots during stress responses, moulting, reproduction and osmoregulation (brown, 1934; scharrer, 1952; huberman and aguilar, 1989; de kleijn, 1994; chung and webster, 2003; lorenzon, 2005; lorenzon et al., 2005; katayama et al., 2013; turner et al., 2013) and behavioural responses, such as aggression (aquiloni et al., 2012) and anxiety (lok et al., 1977). in the crayfish pontastacus leptodactylus, chh injection causes a short term increase in glucose levels and its reduction through eyestalk ablation resulted with a decrease to basal levels (mosco et al., 2012, lebaupain et al., 2012). recent studies highlighted that chh specifically modulates ionic and metabolic homeostasis in the blue crab discoplax celeste (turner et al., 2013) and a variety of other functions involving, for example, inhibition of ecdysteroid (chung and webster, 2003), methyl farnesoate (borst et al., 2001) and ovarian protein synthesis (khayat et al., 1998; avarre et al., 2001). furthermore, the involvement of two chhs in the production of primary urine and in its branchial reprocessing was recently demonstrated (turner et al., 2013). chha increases na uptake at the gills in the very dry + 29 mailto:giuliani@units.it table 1 experimental plans of experiment 1 and 2 experiment 1 groups gfpi g – dsgfp ser – g* ser – g* – x chhi g – dschh ser – g* ser – g* – x experiment 2 groups gfpi g – dsgfp ser – g* x chhi g – dschh ser – g* x 1st day 2nd day 4th day 1st day 20th day 26th day g: glycemia measurement, g*: glycemia measurements after 1, 2, 4, and 8 h from the serotonin injection, dsgfp or dschh: double strand gfp or chh injection (2.5 µg/g body weight), ser: serotonin injection: experiment 1: 1x10-8 mol/g body weight and experiment 2: 2x10-8 mol/g body weight, x: sacrifice of specimens for eyestalk rna extractions. (pre-wet) season and chhb significantly increases na uptake at the gills in the wet season. noteworthy, recombinant chh induces cellular and humoral responses in litopenaeus vannamei infected with vibrio harveyi, resulting in a higher survival rate in this group compared to controls ( + wanlem et al., 2011). recombinant chh also induces chh expression in p. clarkii hemocytes (kung et al., 2013). these data suggest an immunerelated role for chh. serotonin (5-ht) injection depletes endogenous chh peptide reserves and elevates glucose level both in p. leptodactylus and in squilla mantis (lorenzon et al., 2005). these results corroborate the held view that 5-ht has a strong hyperglycemic effect through chh release from the medulla terminalis x organ-sinus gland complex (mtxo-sg), mediated by modulation of xorgan cells electrical activity (sáenz et al., 1997). rna interference (rnai) is a very effective technique to inhibit expression of genes targeting specific sequences (manoharan, 2003). this procedure has been widely applied to develop new therapies (kim and rossi, 2007; yu et al., 2014), to formulate novel drugs and vaccines (lundstrom, 2014), to understand important and diversified physiological mechanisms (denlinger and armbruster, 2014; liu et al., 2014), to control pathogens (md ali et al., 2013; dinh et al., 2014) and invasive species (bandaranayake and yoder, 2013; deng et al., 2013; wynant et al., 2014). two chhs were found in the p. clarkii’s eyestalk transcriptome with different expression levels. chh1 resulted to be 6x more expressed than chh2 (manfrin et al., 2014). aiming at creating new pest management strategies based on the use of autocidal molecules administered via baits, we decided to explore the functional aspects of silencing this pivotal pleiotropic neurohormone. the combination of chh transcript-silencing and chh peptide depletion by serotonin was expected to cause a decrease in glucose levels. in this study, an unexpected hyperglycaemia was recorded. the new chh-like transcripts previously found in p. clarkii’s eyestalk transcriptome were up-regulated in eyestalks of experimental p. clarkii. it is difficult to explain the function and mode of action of these two chhs, but the chh depletion, along with the other chh-like transcripts, could affect the survival of p. clarkii in autocidal-based methods. materials and methods experimental designs experiments were designed to evaluate 1short-term effects of chh triggered by the dschh injection (experiment 1) and 2-long-term effects of chh and effect of dschh injection on the survival and moult rate of the individuals (experiment 2). experiment 1: two groups of procambarus clarkii males (10 individuals per group) were used in a four-day time course experiment as described in table 1. about 2.5 µg/g body weight of green fluorescence protein (gfp) or crustacean hyperglycemic hormone (chh) double strand rna were injected at the beginning of the experiment to crayfish of the control and treated group, respectively. serotonin (5-ht) was injected (1x10-8 mol/g body weight, sigma-aldrich), at day 2 and day 4. hemolymphatic glycemic levels were measured before the serotonin injections and 1, 2, 4 and 8 h after 5-ht injection. mortality and moulting events were recorded daily. rnas extracted from the eyestalks collected on the last day of experiment were subjected to qrt-pcr in order to prove the rnai-affected chh-silencing. experiment 2: 10 control p. clarkii males and 15 dschh rna-injected males were used in a twenty-six-day experiment aimed at examining the long-term effects of chhrnai injections on both glycemia and survival rate. as described above, 2.5 µg/g body weight of gfp or chh double strand rnas were injected at the beginning of the experiment. mortality was recorded on a daily basis for 20 days. on the 20th day 2x10-8 mol/g body weight of serotonin were injected to each individual of both groups. hemolymphatic glycemic levels were measured before and 1, 2, 4 30 and 8 h post serotonin injections as in experiment 1. the individuals' health was checked every day until the 26th day and on this day total rna was extracted from the eyestalks of both groups. the plan of experiment 2 is presented in table 1. animal husbandry all the p. clarkii individuals used in this study were collected from a drain inside the “bonifica del brancolo” (45°46' n, 13°30' e, go, italy). they were all adults, in intermoult and at non-reproductive stage. specimens were acclimatized for a week in 120 l tanks provided with closed circuit filtered, and thoroughly aerated tap water at ~18 °c, and fed fish pellets (sera granular, heisenberg, germany) three times a week. each male was maintained in an individual cage within the same tank to preserve the same environmental conditions for all experimental specimen. ethical note the following experimental procedures comply with the current italian law. no specific permits were required for this study, as it did not involve endangered or protected species. individuals were maintained in appropriate laboratory conditions to guarantee their welfare and responsiveness. after the experiments were completed, crayfish were euthanized by hypothermia. double-strand rna synthesis specific primers amplifying a green fluorescence protein contained in the cloning vector pegfp-n1 (genbank accession number u55762) and the crustacean hyperglycemic hormone (genbank accession number ab027291) were used. both dsgfp and dschh primers fused with t7 5’-tail sequence (underlined in table 2) were designed with primer3 (untergrasser et al., 2012) and checked for secondary structures and possible hairpin formation with oligocalc (kibbe, 2007). their sequences, along with the resulting amplicon sizes, are reported in table 2. standard pcrs were performed by go taq (r) g2 dna polymerase (promega), following these thermal conditions: 95 °c for 2’, 35 cycles at 95 °c for 30”, 57 °c for 30” and 72 °c for 45” with a final extension step at 72 °c for 5’. the resulting amplicons were agarose gel purified using e.z.n.a. gel extraction kit (omega bio tek) and used as templates for the double-strand rnas synthesized with the transcriptaid t7 high yield transcription kit (thermo scientific). the dschh rna probe was designed to silence the two p. clarkii chh genes chh1 and chh2 (manfrin et al., 2014), both involved in the hyperglycemic activity stimulation. the double-stranded rna codifying the green fluorescent protein (dsrna-gfp) has been widely used as non-specific control in a variety of rna interference studies (rnai) (westenberg et al., 2005; ponprateep et al., 2012). injection and hemolymph withdrawal double-stranded rnas were suspended in crustacean saline solution 0.5m (nacl 14.5 g, cacl2 0.72 g, mgso4 3.18 g, kcl 0.35 g, hepes 5mm, nahco3 0.5 g at ph 7.4 in a final volume of 1.2 l) with a final volume of 100 µl/animal. table 2 primer sequences and their relative amplicon sizes used to synthesize double strand-specific rnas and to evaluate chh isoforms gene expression id primer 5’-3’ sequence amplicon size set of primers used for dsrna synthesis dsgfp for taatacgactcactatagggcacatgaagcagcacgacttc dsgfp rev taatacgactcactataggggttcaccttgatgccgttcttc 304 bp dschh for taatacgactcactatagggtcagcttcctctcccaagac dschh rev taatacgactcactatagggtacttgccgacagtttggac 302 bp set of primers used in qrtpcr ef1-α for agatctgaaacgtggttttgtt ef1-α rev tcaatcttttccagaagttcgt 186 bp β-actin for agggcgtgatggttggtat β-actin rev ccgtgctcaatgggatattt 100 bp chh for gcttgaccgagtgtgtgaag chh rev tacttgccgacagtttggac 171 bp chhop for ccggctccttctacaaaatc chhop rev agtacgtcaactgccaaggc 65 bp chhip for gaaacggaatgcagaaaagg chhip rev gcaggaaaaggtcggataca 70 bp 31 injections and hemolymph withdrawal were performed through the abdominal hemolymph sinuses. to evaluate hemolymphatic glucose content, hemocytes were pelleted from the sampled hemolymph and the serum was kept on ice for later glucose measurement, which was performed using a glucose oxidase method (glucose liquid mono reagent, hospitex diagnostics, italy). the normal distribution of glycemic levels was verified with a shapiro-wilk test and homogeneity of variance across groups was checked with a bartlett test. the null hypotheses from both tests could not be rejected. hence, differences of glucose levels among the experimental groups were tested using non-parametric statistics, kruskal-wallis rank sum test with post-hoc wilcoxon rank sum test pairwise comparisons with bonferroni correction. box and whiskers plots were drawn with the boxplot command of r. gene expression level evaluation total rna from eyestalks was extracted by homogenization in trireagent rna isolation solution (sigma-aldrich) and purified with rneasy minelute cleanup kit (qiagen). reverse transcription and real time pcr reactions were accomplished by using the go taq r 2 step rtqpcr system (promega) and the pcr amplifications were performed in triplicates for each rna sample using the cfx96 real-time pcr detection system (bio rad) mounted on c1000 thermal cycler (bio-rad) with the following thermal profile: 95 °c for 2’, 38 cycles at 95 °c for 15”, 57 °c for 30” and 72 °c for 20”, and a final melting curve analysis from 65°c to 95°c with an increment of 0.5 °c every 5”. elongation factor 1alpha (ef1-α) and β-actin were selected as candidate reference housekeeping genes. amplification efficiencies were estimated by linreg v12.1 (ramakers et al., 2003). gene expression stability for the references ef1-α and β-actin was tested considering as output the comprehensive ranking values derived from the comparison of delta ct (silver et al., 2006), best keeper (pfaffl et al., 2004), normfinder (andersen et al., 2004) and genorm (vandesompele et al., 2002) software. data were computed using the bio rad cfx manager software (version 3.0.1224.1015) and the statistical analysis performed using rest-2009 (pfaffl et al., 2002). survival analysis the effects of silencing on survival were assessed using the treatment as variable. both exponential and weibull parametric models with censoring were chosen because some individuals outlived the experiment. statistical analyses were performed using r version 3.0.2 with library 'survival' (r core team, 2013; therneau, 2014). results and discussion glycemic effects the amount of 5-ht to be injected in the animals was assayed through a pilot trial. during the trial we injected 1x10-8 mol/g of 5-ht and measured the glycemic levels in hemolymph 0, 1, 2, 4 and 8 hours after the injection. after an h, glycemic levels were around 120 mg/dl, after 2 h around 160 mg/dl and at 4 h around 40 mg/dl (data not shown). this amount of 5-ht was then considered adequate to be injected in experimental groups, in order to deplete eyestalk endogenous chh reserves, according to lorenzon and colleagues (2005). in experiment 1, 24 h after the injection of dsrna both dschhand dsgfp-injected groups showed a hyperglicemic peak at 2 h post serotonin injection with a mean glycemia of 160.71 ± 9.01 mg/dl (dschh) and 135.04 ± 12.03 mg/dl (dsgfp). on the 4th day of the experiment the peak of glycemia of dschh-injected group was recorded at 2 h post serotonin injection with a mean glycemia of 79.14 ± 12.27 mg/dl, whereas the dsgfp-injected animals showed a glycemic peak of 90.41 ± 15.23 mg/dl 4 h post serotonin injection. no significant differences were recorded between the 2 experimental groups (fig. 1a). these results were possibly a consequence of the short duration of experiment 1. it was therefore assumed that the silencing duration was too short to reduce the chh transcript level. in fact, the effects of dschh-rna were evaluated after 4 days from the dsrna injection and the amount of 5-ht was not enough to deplete the major amount of endogenous eyestalk chh reserves. as a consequence, the duration of experiment 2 was expanded to 20 days and the amount of 5-ht injected in the 25 animals was doubled. the glycemic peak was recorded at 2 hours post serotonin-injection with a mean value of 89.54 ± 17.57 mg/dl for the dschh-injected group and 52.96 ± 16.66 mg/dl for dsgfp-injected animals and no significant differences were recorded (fig. 1b). considering the 2 experiments together, there is a bias in the data collected on day 2 because the mean glycemia at time 0 is rather high due to the stress of dsrna injection. in order to compare the peak at 2 hours (2 h) post serotonin injection in the 2 experimental groups at different days we decided to subtract the glycemia value at time 0 to the value of 2 h for each animal. this mean glycemia of dschh varies significantly among day 2, 4 and 20 (kruskal-wallis, p = 0.01) and on day 4 it is significantly lower than that on the day 2 (pairwise comparisons using wilcoxon rank sum test, p = 0.004) but no significant differences were recorded in mean glycemia between day 2 and 20 (figs 1a, b). the means of glycemia at peak time (2 h) of dsgfp do not differ among day 2, 4 and 20 (figs 1a, b). chh silencing three individuals from each of the chhi and gfpi experimental groups were used to evaluate chh expression level, as well as of the above mentioned chhop and chhip genes using the ef1a as reference gene normalized to the gfpi group (fig. 2). for both experiments, we were able to confirm the efficacy of the chh1-2-rna interference. however, the upregulation of chhop and chhip identified in the transcriptome of p. clarkii (manfrin et al., 2014) was observed only after 26 days of chh1-2 silencing. we observed an up-regulation of chhop (6 times higher than the gfpi group) and a highly 32 fig. 1 box plots of glycemia levels recorded in both the experiment 1 (a) and experiment 2 (b). in the time point axis -24 indicates withdrawal led the day before the 5-ht injection, 0 represents the withdrawal done just before the 5-ht injection and the others time-points are the hours when hemolymph was collected. 33 fig. 2 relative expression of chh and chh-like transcripts in eyestalks detected by qrt-pcr. chhi: chh interference group and gfpi: green fluorescent protein interference for both the two experiments. the δδct method was applied by using gfpi group and ef1-α as calibrator samples. chh1-2, chhop (chh homologous procambarus) and chhip (chh immune-related procambarus). significant over-expression of chhip (80 times higher than the gfpi group, p = 0.01) in the eyestalk of p. clarkii. a remarkable ability of decapods is their long-term survival rate even when bilaterally eyestalk-ablated. a possible explanation of this phenomenon is that the animals compensate the lack of eyestalk neuropeptides via their expression or the expression of peptides of the same family in ectopic tissues. similarly, the ability to perform a normal hyperglycemic response after 20 days of chhs silencing followed by serotonin application suggests that other chh family members could restore the hyperglycemia even when chh1-2 are selectively silenced. indeed, the induced expression of chhop and chhip after 20 days of chh1-2 silencing may be this compensating factor. due to the relatively low sequence similarity between chh1-2 and chhop-ip we hypothesized that their involvement in compensating glycemia is a secondary function. our results led us to suppose the possible involvement of chhop-ip in the immune and stress responses. in fact, their upregulation during experiment 2 was detected only in the specimens that survived the dschh-rna injection, since almost half of p. clarkii males died in the 10 days following the injection. another important aspect is the absence of moulting events in both experimental groups, in contrast with the shedding episodes recorded in specimens collected from the same area, during the same days and maintained at the same laboratory conditions, but not challenged with any treatment. this finding is likely to be related to the stress triggered by the injection of double-stranded rna. survival analysis during experiment 1, 5 animals belonging to the dschh-injected group died, as well as 3 from the dsgfp-injected group. the higher number of specimens per experimental group in experiment 2 allowed more accurate evaluation of survival rate, and this is presented in figure 3. following the kaplan-meier estimation, the fraction of subjects living during the time course of the experiment after the dsgfp or dschh injection was recorded, highlighting the higher mortality caused by the dschh injection. the highest mortality rate was reached between day 5 and 10 in the dschh rna injected group. after 20 days from the beginning of the experiment, about 53 % of the specimens belonging to the dschh group and 70 % of the specimens from the dsgfp group were still alive. no significant difference was detected between the two groups, even though osmotic deficits were recorded only in individuals challenged with dschh rna. fig. 3 survival analysis. dschh represents the group of p. clarkii specimens (15) injected with dschh-rna and dsgfp represents the animals injected with dsgfp-rna (10 individuals). + indicates the end of the observation and the presence of alive animals at this time. 34 fig. 4 osmotic deficit recorded in deceased p. clarkii specimens following dschhinjection. the detachment of the carapace from the first pleon tergite is visible in this picture. specimens found deceased following the dschh injection, as documented in figure 4, showed indeed a detachment of the carapace from the first pleon tergite, with the underlying membranous cuticle and epidermis becoming visible. this is an indication of osmotic deficit. in vivo experiments on shrimps (nagabhushanam and jyoti, 1977; mcnamara et al., 1990) and crabs (kamemoto et al., 1966; kamemoto and ono, 1969; kato and kamemoto, 1969; kamemoto and tullis, 1972; heit and fingerman, 1975; davis and hagadorn, 1982) demonstrated that the eyestalk ligation or ablation increases water influx. similarly, chh silencing produces an increase in hemolymph volume, that can lead to death. these findings highlight the fundamental role of chh in osmoregulatory processes suggesting that they might be controlled by a neuroendocrine mechanism, as already reported for other crustacean species (e.g., serrano et al., 2003, turner et al., 2013). the second scenario refers to the specimens that were able to face the chhs silencing, as shown by the glycemic levels presented in figure 1, where a compensatory effect was activated. glycemia increased along the time course experiment, to slowly decrease after 2 h from the initial 5-ht injection. what is responsible for the increased glycemic level if the two main chhs produced in the eyestalks are silenced? it is our opinion that there are many other molecules related to the chh family which were not silenced by our dsrna probes due to the difference in nucleotide sequences. they may have compensated the lack of the known chhs. to investigate this hypothesis further, we tested the expression level of two other chh transcripts, chhop and chhip. after 26 days, both of them resulted up-regulated in the eyestalk of p. clarkii survived to the dschh-rna treatment and significantly less expressed in specimens injected with dsgfp-rna. little is known about the possible involvement of the chh-superfamily in the immune response, but this study represents the first analysis from this point of view. silencing the main chhs in the red swamp crayfish outlined two different scenarios: 1 death in the specimens not able to face the lacking of the chh, 2survival of p. clarkii individuals able to activate secondary responses involving other chh-superfamily members. the modes of action and intermediates messengers associated to these pathways are still unknown, but chh depletion, along with other chh-like transcripts, could impact the survival of p. clarkii in autocidal-based methods. acknowledgements we thank the anonymous referees whose comments refined our thinking on 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crustacean hyperglycemic hormone levels in the edible crab cancer pagurus during emersion stress. j. exp. biol. 199: 1579-1585, 1996. westenberg m, heinhuis b, zuidema d, vlak jm. sirna injection induces sequence-independent protection in penaeus monodon against white spot syndrome virus. virus res. 114: 133-139, 2005. wynant n, santos d, vanden broeck j. biological mechanisms determining the success of rna interference in insects. int. rev. cell mol. biol. 312: 139-167, 2014. yu y, liao m, liu r, chen j, feng h, fu z. overexpression of lactate dehydrogenase-a in human intrahepatic cholangiocarcinoma: its implication for treatment. world j. surg. oncol. 12: 78, 2014. 37 short communication isj 11: 224-227, 2014 issn 1824-307x short communication predation of thaumastocoris peregrinus (hemiptera: thaumastocoridae) by atopozelus opsimus (hemiptera: reduviidae) in brazil tkr dias1, cf wilcken1, ep soliman1, lr barbosa2, je serrão3, jc zanuncio4 1departamento de proteção vegetal, universidade estadual paulista júlio de mesquita filho (unesp), faculdade de ciências agronômicas, 18610-307, botucatu, sp, brasil 2laboratório de entomologia florestal, embrapa florestas, colombo, 83411-000, pr, brasil 3departamento de biologia geral, universidade federal de viçosa, 36570-900 viçosa, mg, brasil 4departamento de entomologia, universidade federal de viçosa, 36570-900 viçosa, mg, brasil accepted august 19, 2014 abstract the bronze bug thaumastocoris peregrinus (hemiptera: thaumastocoridae) is an important pest of eucalyptus in several countries and the strategies for the integrated management of this insect in commercial plantations needs to be investigated. the predatory behavior of atopozelus opsimus (hemiptera: reduviidae) on t. peregrinus is described. adults of a. opsimus feed on nymphs and adults of the bronze bug and also present phytophagy. a. opsimus has potential as a natural enemy for the biological control of t. peregrinus. key words: atopozelus opsimus; bronze bug; predator   introduction thaumastocoris peregrinus (hemiptera: thaumastocoridae), the bronze bug, is an important pest in eucalyptus plantations (fig. 1a). this insect is one of the major pest of this culture in argentina, south africa, australia, chile, italy, malawi, kenya, new zealand, uruguay, zimbabwe and portugal (jacobs and neser, 2005; carpintero and dellape, 2006; martinez and bianchi, 2010; nadel et al., 2010; noack et al., 2011; laudonia and sasso, 2012; sopow et al., 2012; garcia et al., 2013). the bonze bug was detected in brazil, in 2008, in são francisco de assis, rio grande do sul state, and in jaguariuna, são paulo state, and it spread widely and rapidly to other regions with eucalyptus plantations. to date, t. peregrinus has been recorded in minas gerais, espírito santo, rio de janeiro, mato grosso do sul, paraná, santa catarina, and goiás states (wilcken et al., 2010; barbosa et al., 2010; savaris et al., 2011; pereira et al., 2013). in addition eucalyptus urophylla and e. grandis, the most susceptible species for the development and reproduction of t. peregrinus, represent the genetic basis of eucalyptus plantations in brazil (soliman et al., 2012). ___________________________________________________________________________ corresponding author: thaíse karla ribeiro dias departamento de proteção vegetal universidade estadual paulista júlio de mesquita filho (unesp) faculdade de ciências agronômicas 18610-307, botucatu, sp, brasil e-mail: thaiserdias@yahoo.com.br the bronze bug is 2 3 mm long, with a flattened body, a head with recurved mandibular plates, antennae with four segments, and red compound eyes. the adults of this insect have a light brown color and an asymmetric male genital capsule, oriented to the right or left side of the body (carpintero and dellape, 2006; noack et al., 2011). the eggs of t. peregrinus are black and laid in masses of 60 eggs, on leaves, branches, fruits, and stems of the plant (button, 2007; noack and rosa, 2007). the insect has five instars with 15 days of total nymph period (noack and rose, 2007; soliman et al., 2012). damage by t. peregrinus adults and nymphs is due to sap sucking, which induces leaf fall and decreases the photosynthetic area. infested trees initially show leaf silvering, followed by tanning, and because of these peculiarities the insect has been called the bronze bug (jacobs and neser, 2005; wilcken et al., 2010). high populations of this insect can cause plant death (wilcken et al., 2010). strategies for the management of t. peregrinus in commercial plantations are scarce, but the systemic insecticide imidacloprid, injected into the tree trunks has been effective in controlling this insect in the urban areas of australia (noack et al., 2009). however, chemical control in commercial plantations in brazil cannot be implemented, because there is no  registered insecticide for this insect. furthermore, the use of chemical insecticides is restricted on commercial plantations in brazil due to environmental concerns (zanuncio et al., 1994). 224   fig. 1 thaumastocoris peregrinus (hemiptera: thaumastocoridae) of eucalyptus leaf (a). atopozelus opsimus (hemiptera: reduviidae) preying on t. peregrinus (b). biological control with fungi, parasitoids, and predators is an alternative to the integrated management of t. peregrinus. the egg parasitoid cleruchoides noackae (hymenoptera: mymaridae), the predatory bug and the  lacewings hemerobius bolivari (neuroptera: hemerobiidae) and chysoperla externa (neuroptera: chrysopidae) have been reported as natural enemies of the bronze bug (barbosa et al., 2010; souza et al., 2012; garcia et al., 2013). atopozelus opsimus (hemiptera: reduviidae) is a bug predator zoophytophagous and consumes nymphs and adults of glycaspis brimblecombei (hemiptera: aphalaridae), an important pest on brazilian eucalyptus plantations (dias et al., 2012). it is native to the americas and recorded in brazil in são paulo, minas gerais, rio de janeiro and mato grosso states (elkins, 1954; dias et al., 2012). the importance of the forest sector to the brazilian economy and the introduction of t. peregrinus in the country make it necessary to use natural and native resources to reduce problems with this pest. this study reports the predatory behavior of a. opsimus on t. peregrinus. material and methods the predator a. opsimus and the prey t. peregrinus were obtained from the biological control of forest pests laboratory (lcbpf) of the plant protection department, faculty of agricultural sciences/unesp in botucatu, são paulo state, brazil. the experiment was performed at 25 ± 2 °c, 70 ± 10 % rh, and a 12-h photophase. the insects were placed in gearbox-type boxes (11x11x3.5 cm), covered with plastic, containing a eucalyptus leaf and a moistened cotton ball. the experiment was conducted with 10 replications, with one a. opsimus adult each and five prey (t. peregrinus adults). the insects were observed for one hour, being recorded the number of t. peregrinus preyed and the behavioral predation actions prior and after the predator attack. results and discussion it was verified that the stink bug a. opsimus feeds on t. peregrinus (fig. 1). the first actions before each predation were the antennae and prothoracic legs cleaning to all of observed predators. due to prey movement on the leaves, the predator walked during the chase and search. in capturing were used the antennae and prothoracic legs to surround, hold and manipulate the prey. the viscous and adhesive secretion present on the body and appendixes of a. opsimus facilitated the capture of t. peregrinus, hindering scape of the prey during predation. the predator sucks all the internal contents and moves the prey with tarsus help, in circular movement seeking more fluids. a. opsimus made phytophagy and some bugs inserted their stylet into the leaf nervures of eucalyptus to suck sap. this is a common behavior to this species, which also consume sugary products, coming from extrafloral nectaries and sugary shell that protects g. brimblecombei. the predators phytophagy are related to the production of viscous and adhesive essence (zhang and weirauch, 2013), that, in a. opsimus, helps on the prey capture (dias et al., 2012). the stylet of a. opsimus was preferentially inserted into the thorax of t. peregrinus, dorsally, 225   226   then ventrally. adult prey in mating were predated more easily, especially the male over the female. the attack in this condition was more frequent, always through the back and did not allow the prey any defense. the exoskeleton of t. peregrinus preyed was released next to the others consumed ones, after the intern content suction. a. opsimus showed agility and moved rapidly toward the prey. a. opsimus showed a consumption of 2.0 ± 0.81 t. peregrinus individuals per h. the nymphs of supputius cincticeps (heteroptera: pentatomidae) and of c. externa larvae also preyed on the bronze bug, with a consumption of 10.26 ± 1.58 (barbosa et al., 2010) and 10.43 ± 0.51 (souza et al., 2012) individuals in 24 h, respectively. these preliminary findings suggest that the efficiency of a. opsimus preying on t. peregrinus was higher than that of s. cincticeps and c. externa. a. opsimus was found in the field preying on nymphs and adults of t. peregrinus on the eucalyptus plants, where both species take refuge and shelter. the sequence of the predatory behavior of a. opsimus with cleaning actions of antennae and legs, walking chase and search, capture, prey immobilization, fluids suction and later disposal of prey exoskeleton together with others t. peregrinus before consumed. it was similar to that reported for this predator on g. brimblecombei, an exotic insect pest also introduced in brazilian eucalypt plantations (dias et al., 2012). both insects are small and are easily preyed on by a. opsimus. conclusion a. opsimus is a native predator of t. peregrinus, an important sucking prey on eucalyptus plantations in brazil. the bioecology and the effectiveness of a. opsimus need to be better studied to use this natural enemy for the biological control of the bronze bug t. peregrinus. acknowledgements to “conselho nacional de desenvolvimento científico e tecnológico (cnpq)”, “coordenação de aperfeiçoamento de pessoal de nível superior (capes)” and “fundação de amparo à pesquisa do estado de minas gerais (fapemig)” for financial support. global edico services edited and proofread this manuscript. references barbosa lr, santos f, barddal hpo, machado bo, wilcken cf, soliman ep. predação de thaumastocoris peregrinus por chrysoperla externa. embrapa florestas, comun. técn. 257, 2010. barbosa lr, santos f, wilcken cf, soliman, ep. registro de thaumastocoris peregrinus (hemiptera: thaumastocoridae) no estado do paraná. pesqui. florest. bras. 30: 75-77, 2010. button g. thaumastocoris peregrinus. in: forest facts. 2007. http://www.nc t forest. com/showpage.asp?id044 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ferreira-filho pj, et al. bronze bug thaumastocoris peregrines carpintero and dellapé (hemiptera: thaumastocoridae) on eucalyptus in brazil and its distribution. j. plant prot. 50: 210-205, 2010. zhang g, weirauch c. sticky predators: a comparative study of sticky glands in harpactorinae assassin bugs (insecta: hemiptera: reduviidae). acta zool. 94: 1-10, 2013. zanuncio jc, nascimento ec, garcia jf, zanuncio t v. major lepidopterous defoliators of eucalyptus, in the southeast brazil. forest. ecol. manag. 65: 53-63, 1994.   https://www.google.com.br/search?biw=1438&bih=677&q=zhang,+g.,+weirauch,+c.+sticky+predators:+a+comparative+study+of+sticky+glands+in+harpactorinae+assassin+bugs+(insecta:+hemiptera:+reduviidae).+acta+zoologica,+stockholm,+v.+94,+p.1%e2%80%9310+,+2013.&spell=1&sa=x&ei=z7veu56qhiplsascn4cwdg&ved=0cbkqbsga https://www.google.com.br/search?biw=1438&bih=677&q=zhang,+g.,+weirauch,+c.+sticky+predators:+a+comparative+study+of+sticky+glands+in+harpactorinae+assassin+bugs+(insecta:+hemiptera:+reduviidae).+acta+zoologica,+stockholm,+v.+94,+p.1%e2%80%9310+,+2013.&spell=1&sa=x&ei=z7veu56qhiplsascn4cwdg&ved=0cbkqbsga https://www.google.com.br/search?biw=1438&bih=677&q=zhang,+g.,+weirauch,+c.+sticky+predators:+a+comparative+study+of+sticky+glands+in+harpactorinae+assassin+bugs+(insecta:+hemiptera:+reduviidae).+acta+zoologica,+stockholm,+v.+94,+p.1%e2%80%9310+,+2013.&spell=1&sa=x&ei=z7veu56qhiplsascn4cwdg&ved=0cbkqbsga https://www.google.com.br/search?biw=1438&bih=677&q=zhang,+g.,+weirauch,+c.+sticky+predators:+a+comparative+study+of+sticky+glands+in+harpactorinae+assassin+bugs+(insecta:+hemiptera:+reduviidae).+acta+zoologica,+stockholm,+v.+94,+p.1%e2%80%9310+,+2013.&spell=1&sa=x&ei=z7veu56qhiplsascn4cwdg&ved=0cbkqbsga organisms/tissues/cells live successfully even if they are constantly in presence of different types of stressors isj 11: 286-297, 2014 issn 1824-307x research report functional amyloid formation in lps activated cells from invertebrates to vertebrates a grimaldi1, g tettamanti1, r girardello1, l pulze1, r valvassori1, d malagoli2, e ottaviani2, m de eguileor1 1department of biotechnology and life sciences, university of insubria, varese, italy, 2department of life sciences, university of modena and reggio emilia, via campi 213/d,41125 modena, italy accepted october 1, 2014 abstract lps stimulation provokes serious cellular stress with an increase of cytoplasmic reactive oxygen species (ros). we have investigated, among the different cellular defenses, amyloidogenesis as common physiological response to attenuate oxidative stress. optical and electron microscopic observations of the following lps activated cell lines [insect (larval hemocytes, iplb-ldfb and drosophila schneider’s s2 cells); mouse (nih3t3 embryonic fibroblasts); human (human umbilical vein endothelial cells (huvec), neutrophils, and mesenchymal stem cells] reveal that, all are characterized by irregular profiles, cytoplasmic empty vacuoles or by cisternae containing fibrillar material. the compartmentalized fibrillar material shows staining properties typical of amyloid fibrils. lps activation leads to ros generation, resulting in ph acidification. stimulated cells show pink cytoplasm in may-grünwald giemsa differential staining, giving a gross indication of a lower intracellular ph. moreover the activation of amyloidogenesis is also linked with an extensive production of acth and α-msh in all cultured cell types. we suggest that amyloidogenesis is a common, physiological cellular response to weak ros, starting when other anti-stress cellular systems failed to restore homeostasis. the morphological evidence and/or functional characterization of synthesized amyloid fibrils could be an early indicator of oxidative stress that may lead to a general inflammatory process. key words: lps; amyloid fibrils; ros; acth axis   introduction organisms/tissues/cells live successfully even if they are constantly exposed to different types of stressors. contacts with foreign organisms/molecules, such as bacteria, fungi, parasites, or chemicals, can represent serious stress and induce protective responses (nappi and ottaviani, 2000; iwasaki and medzhitov, 2010). the defense responses, that follow non-self recognition ___________________________________________________________________________ corresponding author: magda de eguileor department of biotechnology and life sciences university of insubria via jh dunant 3, 21100 varese, italy e-mail: magda.deeguileor@uninsubria.it list of abbreviations: acth = adrenocorticotropin hormone; α-msh = alpha melanocyte-stimulating hormone; lps = lipopolysaccharide; nep = neutral endopeptidase; rer = rough endoplasmic reticulum; ros = reactive oxygen species; tht = thioflavine t belong to immunity (nappi and ottaviani, 2000) nonetheless the basis of stress remains the imbalance between the rate of production and clearance of reactive oxygen species (ros) (droge, 2002; dowling and simmons, 2008; dickhout et al., 2012; pietraforte and malorni, 2014). as far as stress response is concerned, it consists of mixed and complex responses, involving the bidirectional communication between the immune and neuroendocrine systems (smith et al., 1991; weigent and blalock, 1995; ottaviani and franceschi, 1997; wright et al., 2000; scholzen, 2004; dores and lecaude, 2005; huising and flik, 2005; slominski et al., 2005; lovejoy and jahan, 2006; malagoli et al., 2007, 2011; rivest, 2010; caruso et al., 2012; falabella et al., 2012; grimaldi et al., 2012a; thyaga rajan and priyanka, 2012). a controlled intracellular level of ros is fundamental in promoting various physiological functions, but high ros concentration exerts damaging effects. the intensity of oxidative stress can stimulate proportioned intracellular responses ranging from synthesis of detoxifying molecules, housekeeping autophagy, acth/α-msh loop activation, and 286 http://www.sciencemag.org/search?author1=akiko+iwasaki&sortspec=date&submit=submit http://www.sciencemag.org/search?author1=ruslan+medzhitov&sortspec=date&submit=submit mailto:magda.deeguileor@uninsubria.it amyloid-driven synthesis of melanin up to extreme point of no return such as apoptotic cell death (cesaratto et al., 2004; kroemer et al., 2010; tillement et al., 2011; grimaldi et al., 2012a, b). as a consequence, a time-sustained and excessive ros production can be implicated in a lot of diseases such as chronic inflammation, cancer, neurodegenerative-diseases, and in senescence mechanisms (christen, 2000; simon et al., 2000; nogueira de sousa andrade et al., 2012). recently several authors demonstrated in different contexts that an important immune response as the melanin synthesis is strictly linked to physiological amyloidogenesis (berson et al., 2001; fowler et al., 2006; harper et al., 2008; maji et al., 2009; rochin et al., 2013; watt et al., 2013). amyloidogenesis has usually been associated with aetiology of the neurodegenerative diseases. even if the aggregation of proteins into amyloid fibrils is the hallmark feature of diseases (grenwald and riek, 2010) including alzheimer’s (ad), parkinson’s, and prion diseases it has been evidenced that amyloid is also a fundamental nonpathological protein folding retrieved from bacteria to humans (kelly and balch, 2003; cherny et al., 2005; chiti and dobson, 2006; fowler et al., 2006; maury, 2009; eisenberg and juker, 2012; falabella et al., 2012; grimaldi et al., 2012a). nowadays, it is well-demonstrated that the ability to form amyloid structures is not an unusual feature of the small number of proteins associated with diseases but it is a general property of polypeptide chains (kourie et al., 2002; huff et al., 2003; goldschmidt et al., 2010; schnabel, 2010, 2011). in mammalian tissues nonpathogenic amyloid structure that functions in melanosome biogenesis has been described by numerous authors (fowler et al., 2006; maji et al., 2009; rochin et al., 2013). melanosomes are subcellular organelles specialized in melanin synthesis in melanocytes and retinal epithelial cells. the melanocytes produce a glycoprotein called pmel17 able to polymerise into amyloid fibrils, on which melanin is assembled (berson et al., 2001; harper et al., 2008; watt et al., 2013). the amyloid fibrils appear to play a role in mitigating the toxicity associated with melanin formation by sequestering and minimizing diffusion of highly reactive, toxic melanin precursors out of the melanosome. as far as invertebrates are concerned, amyloidogenesis has been implicated in a variety of developmental functions (qiu et al., 1995; coulson et al., 2000). our recent data have shown the relationship between amyloid and melanin synthesis in phylogenetically distant metazoan (viz, cnidarians, molluscs, annelids, insects, ascidians and vertebrates) (grimaldi et al., 2012a, b), confirming the physiological role of amyloid fibrils also in invertebrates. protostomes and deuterostomes share the same nexus between melanin synthesis and amyloid fibril production, and the presence of melanin is indissolubly linked to amyloid scaffold. during invertebrate pigment synthesis the amyloidogenesis is sustained by the cross-talk between immune and endocrine systems, by the redox status/cytoplasmic ph modification, and by the cleavage of pro-protein precursors. more in detail the amyloid fibril formation is accompanied by the overexpression of adrenocorticotropin hormone (acth), melanocyte-stimulating hormone (α-msh), and neutral endopeptidase (nep) (falabella et al., 2012; grimaldi et al., 2012a, b). in all, several data suggest that the massive amyloid fibril formation is an ancient physiological cell response harmonically integrated with the stress response. in the present paper we show that different types of cells from diverse organisms can generate invariant responses against an oxidative stress agent by producing amyloid fibrils and in some cases melanin. the production of amyloid fibrils acts here as an efficient and prompt method to strive ros overproduction and to avoid severe damages that may end with apoptosis or necrosis. the highlighted relationship between amyloidogenesis and ros production is validated here with morphofunctional experiments and it allows to surmise a new background of information on the effects of oxidative stress. material and methods all experiments were performed in four independent replicates cell culture insect hemocytes were obtained from last instar larvae of heliothis virescens (lepidoptera). samples of hemolymph (40/60 μl per larva) were collected and transferred in eppendorf tubes containing an equal volume of buffer mead. hemocytes were pelletted by centrifugation of the hemolymph at 400g per 7 min at 4 °c. the hemocytes were resuspended in complete medium (grace’s medium, fbs 10 %, antibiotic-antimicotic solution 1 %, sigma) and were plated at concentration of 1x106 cells/ml into 24-well culture plates. the iplb-ldfb cell line derived from the fat body of the insect lymantria dispar (lepidoptera) and generously gifted by prof. ottaviani (university of modena and reggio emilia, modena), was used. the cells were cultured in ex-cell 400™ medium (jrh biosciences ltd., andover, uk) at 26 °c. drosophila schneider’s s2 cells were cultured in schneider’s drosophila medium (wvr), containing heat inactivated fbs 10 % (sigma-aldrich). cells were used at 70 80 % confluence, in 12-well (25 mm) plates (corning). nih3t3 mouse embryonic fibroblast cells are established from a nih swiss mouse embryo (sigma-aldrich). these cells were cultured in dmem medium and 10 % bovine calf serum. human umbilical vein endothelial cells (huvec) extracted from human neonatal umbilical cords were cultured in endogro media (warmed to 37 °c) chemicon (merck, germany). human polymorphonuclear neutrophils freshly isolated from whole venous blood collected from healthy donors, by ficollpaque plus (ge healthcare, milan, italy) centrifugation and then separated from erythrocytes by red blood cell lysis and centrifugation. mesenchymal stem cells derived from human adipose tissue, generously gifted by prof. bernardini (university of insubria, varese), were cultured in human mesenchymal-ls expansion. for each type of cells examined: 8x104 cells/ml were seeded on rounded glass coverlips (12 mm diameter, treated 287 with or without 0.1 % gelatinase) placed into 24-well (for light microscopy observation) or in 6-well (for electron microscopy observation) plates in suitable medium. cells were then either left undisturbed (control) or stimulated with 100ng/ml lps (from escherichia coli, serotype o55:b5, sigma-aldrich) for 30 min. after incubation with lps, cells on coverslips from 24-weel plates were fixed with 4 % paraformaldehyde in pbs, while cells from 6-well plates were fixed with 4 % glutaraldehyde in 0.1 m na-cacodylate buffer (ph 7.2) for tem. light microscopy and transmission electron microscopy (tem) after samples were fixed at 4 °c for 2 h in 4 % glutaraldehyde in 0.1 m na-cacodylate buffer (ph 7.2), the cells were pellet washed in 0.1 m nacacodylate buffer (ph 7.2) and post-fixed at 4 °c for 2 h with 1 % osmic acid in cacodylate buffer (ph 7.2). after standard dehydration in ethanol series, samples were embedded in an epon-araldite 812 mixture and sectioned with a reichert ultracut s ultratome (leica). cells were stained by maygrünwald giemsa differential staining. the differential staining depends on ph (alkaline ph increases blue and acid ph pink or reddish tinge in the stained specimens) thus giving a grossidentification of cytoplasmic ph showing a possible increase of ros production). all semithin sections were observed with a light microscope olympus bh2 (olympus, tokyo, japan) and visualized pictures were acquired with a ds-5m-l1 nikon digital camera system. thin sections were stained by uranyl acetate and lead citrate and observed with a jeol 1010 electron microscope (jeol, tokyo, japan). amyloid fibril characterization amyloid structures were identified according to le vine iii (le vine iii, 1999), by staining cells with thioflavine t and visualizing the amyloid-specific green/yellow fluorescence with an olympus bh2 microscope (excitation wavelength 465 nm). images were acquired with a ds-5m-l1 nikon digital camera system. indirect immunofluorescence staining cells were incubated with pbs containing 2 % bsa for 30 min before the primary antibody incubation (4 °c over night). the presence of acth, and its cleavage product alpha (α-msh), were assessed using: anti-human acth (1:50 dilution, sigma-aldrich) and anti-human α-msh (1:50 dilution, sigma) polyclonal antibodies, respectively. incubations with suitable secondary antibodies conjugated with tetramethylrhodamine (tritc) (1:200 dilution, jackson, immuno research laboratories, west grove, pa, usa) or alternatively conjugated to horseradish peroxidase (hrp) enzyme (sigma-aldrich) were performed for 1h at room temperature in a dark moist chamber. nuclei were eventually stained with 4’,6-diamidino-2phenylindole (dapi, sigma-aldrich). all washing and dilutions were performed with pbs and 2 % bsa. in negative control samples, primary antibodies were omitted. coverslips were mounted in vectashield mounting medium for fluorescence (vector laboratories, burlingame, ca. usa); slides were observed on olympus bh2 microscope (olympus). data were recorded with a ds-5m-l1 digital camera system (nikon). images were combined with adobe photoshop (adobe systems, inc.). results the common morpho-functional traits occurring in cells undergoing a stress were assessed in diverse animal species. selected types of cells, named in material and methods section, differ for cell lineages as well as for recognized function. freshly withdrawn insect hemocytes, cultured larval fat body cells (iplb-ldfb), drosophila schneider’s s2 cultured embryonic cells, nih3t3 mouse embryonic fibroblast cells, human umbilical vein endothelial cells (huvec), human neutrophils, and human mesenchymal stem cells have been examined after stimulation with lps at the shortest time generating observable morphological changes. our previous studies indicate that under stress conditions the insect hemocytes (falabella et al., 2012; grimaldi et al., 2012a, b) and human neutrophils (pulze et al., 2014) produce within endoplasmic reticulum cisternae a fibrillar material responsible for the assembling of an amyloidogenic scaffold. given the similarities between the fibrillar material released by hemocytes and neutrophils we investigated whether analogous responses could intervene also in lps stimulated iplb-ldfb cells, s2 cells, nih3t3, huvec, and mesenchymal stem cells. in particular, we hypothesize that the formation of fibrillar material reflects an amyloidogenic process following cytoplasmic acidification. both in invertebrates and vertebrates, the decrease of cytoplasmic ph promotes a bidirectional activation of stress-sensoring circuits (weigent and blalock, 1995; ottaviani and franceschi, 1997; wright et al., 2000; scholzen, 2004; dores and lecaude, 2005; huising and flik, 2005; slominski et al., 2005; lovejoy and jahan, 2006; carroll, 2008; rivest, 2010; caruso et al., 2012; grimaldi et al., 2012a, b; thyaga rajan and priyanka, 2012) that results in the release of stressrelated molecules such as acth, α-msh and nep. in particular acth provokes cell shape changes and chemotaxis (smith et al., 1991; caselgrandi et al., 2000; ottaviani et al., 2007; genedani et al., 2008; malagoli et al., 2011), until it is converted to αmsh by nep (cohen et al., 1996; caselgrandi et al., 2000; slominski et al., 2005). nep regulates the acth/α-msh balance and is also involved in the control of amyloidogenesis (iwata et al., 2004; hamaguchi et al., 2006; el-amouri et al., 2008; kubiak-wlekly and niemir, 2009; meilandt et al., 2009; greenwald and riek, 2010; hafez et al., 2011; falabella et al., 2012; grimaldi et al., 2012a, b; saido and leissring, 2012; grimm et al., 2013). morphology of control and lps-treated cells hemocytes tem analyses (figs 1a c) showed that, h. virescens larval granulocytes, defined as the main circulating cell type involved in the defence 288 http://cancerweb.ncl.ac.uk/cgi-bin/omd?specific http://cancerweb.ncl.ac.uk/cgi-bin/omd?yellow http://cancerweb.ncl.ac.uk/cgi-bin/omd?fluorescence fig. 1 effects of lps stimulation on cells: tem analysis. thin sections of unstimulated and lps activated cells (a z) showing that, at short time from lps incubation, significant morphological changes at the cytoplasmic level occur. activated cells (b, c, e g, i k, m p, r t, v z), lost the roundish shape typical of control (a, d, h, l, q, u), acquire irregular profiles. these cells exhibit the presence of dilated reticulum cisternae filled with fibrillar material (arrowheads), and of double/multiple autophagic vesicles containing organelles and cytoplasmic degraded components (o, j, p, t, x). (scale bar : 1cm = a, 1 μm; b, 3 μm; c, 1 μm; d, 4 μm; e, 2 μm; f, 1 μm; g, 0.5 μm; h, 2 μm; i, 1 μm; j, 0.5 μm ; k, 1 μm; l, 2 μm; m, 5 μm; n, 1.3 μm; o, 0.5 μm; p, 0.8 μm; q, 2 μm; r, 4 μm; s, 1 μm; t, 1.3 μm; u, 2 μm; v, 2 μm; w, 2 μm; x, 0.5 μm ;y, 3 μm; z, 1 μm). 289 processes of the tobacco budworm, were roundish cells characterized by cytoplasm filled with a central nucleus, rer, golgi apparatus, mitochondria, glycogen, and few small homogenous granules (fig. 1a) (grimaldi et al., 2012a, b). h. virescens granulocytes after lps stimulation presented enlarged reticulum cisternae, occupied by fibrillar material spatially organized in respect to an electron dense body. the compartmentalized fibrillar material was located around the nucleus and hid the cytoplasmic organules (figs 1b, c). iplb-ldfb cells electron microscopic observations of control cells revealed that iplb-ldfb cells, from the larval fat bodies of the gipsy moth lymantria dispar, were round in shape, with cytoplasm filled with nuclei, containing dispersed chromatin, small mitochondria, and abundant rough endoplasmic reticulum (fig. 1d). lps stimulated cells showed morphological changes only involving the rer cisternae that were dilated and filled with a massive amount of fibrillar material forming compact structured bodies (figs 1e, f). autophagosomes containing portions of the cytosol and membrane-like structures are evident (fig. 1g). drosophila s2 cells s2 cells display approximately a spherical morphology with a central nucleus and well distributed organules in the cytoplasm (fig. 1h). lps stimulation induced s2 cells to undergo a change in their phenotype: large reticulum cisternae were predominant in respect to other organules and occupied a significant portion of cytoplasm (figs 1i, k). stimulated cells show the presence of autophagic vacuoles enclosing cytoplasmic components (fig. 1j). nih3t3 mouse embryonic fibroblast cells electron microscopic observations of control nih3t3 cells showed flattened and spindle shaped cells with nucleus generally in central position. the cytoplasm contained organules and a moderately developed endoplasmic reticulum (fig. 1l). lps stimulated nih3t3 cells presented well-developed pseudopodia all around the perimeter (fig. 1m). empty vesicles and dilated cisternae of reticulum filled with fibrillar material were visible (figs 1n, o). often sequestered materials (cytosol and membrane-like structures) in membrane-bound vesicles were visible (fig. 1p). human umbilical vein endothelial cells (huvec) control huvec appeared as roundish cells with cytoplasm rich in organules (fig. 1q). lps stimulated cells displayed an irregular profile due to the presence of peripheral large dilated reticulum cisternae filled with fibrillar material (figs 1r, s). in the cytoplasm of activated cells, autophagic vacuoles, containing electron-dense elements and partially degraded material could be visible (fig. 1t). human neutrophils resting circulating neutrophils are spheroidal cells showing an irregular, multi-lobed nucleus and cytoplasmic granules (fig. 1u). upon lps administration, activated neutrophils were characterized by irregular profiles, and cytoplasmic empty vacuoles or by cisternae containing fibrillar material (figs 1v, w, y, z). autophagic vacuoles were visible (fig. 1x). amyloid fibrils and acth/α-msh synthesis in lpstreated cells the morpho-characterization of all cell types is showed in multi-panel figure 2. h. virescens hemocytes activation via lps of tobacco budworm hemocytes leads to ros generation, resulting in ph acidification evidenced by may-grünwald giemsa differential staining that marks a gross indication of a lower intracellular ph. amyloid fibril formation the fibrillar material observed in the hemocytes of h. virescens, showed staining properties typical of amyloid fibrils. h. virescens hemocytes were positive to thioflavine t staining, showing the typical yellow-green fluorescence, more evident in stimulated cells as compared to controls. acth/α-msh axis activation activation of h. virescens hemocytes resulted in extensive production of acth which, due to nep cleavage, turned to α-msh. iplb-ldfb cells lps stimulated iplb cells showed pink cytoplasm in may-grünwald giemsa differential staining. amyloid fibril formation the fibrillar material observed displayed thioflavine t staining properties typical of amyloid fibrils. acth/α-msh axis activation a significant increase in the production of acth and α-msh was observed. drosophila s2 cells lps-treated cells showed an increased ros production evidenced by reddish tinge in the stained specimens by differential may-grünwald giemsa staining. amyloid fibril formation the fibrillar material observed in the reticulum cisternae showed specific staining of amyloid fibrils being thioflavine t positive. acth/α-msh axis activation the activation of stress circuits resulted in the extensive production of acth and α-msh by lpsactivated s2 hemocytes. nih3t3 mouse embryonic fibroblast cells lps activated cells showed mostly pink cytoplasm as a marker of ph decrease. amyloid fibril formation the observed fibrillar material showed typical staining properties of amyloid fibrils showing a yellowgreen fluorescence after thioflavine t exposure. 290 fig 2 characterization of lps treated cells. light microscopy: a gross identification of cytoplasmic acidification is obtained with differential may-grünwald giemsa staining (panel a). the increased ros production responsible of cytoplasmic ph is evident by comparing unstimulated with stimulated cells (alkaline ph increases the blue tinge while acid ph the pink/reddish tinge in stained samples). immunocytochemical characterizations for acth and αmsh expressions (panel a, b) showing high levels of positivity in activated cells while in unstimulated cells the expression of melanocortin hormones is basal. immunocytochemical evidence of amyloid fibrils, detected with thioflavin t (yellow-green brightly fluorescence), is more evident in stimulated cells as compared to controls. nuclei are stained with dapi and marked in brilliant blue (panel b). 291 fig 3 schematic overview explaining the possible behaviour of cells due to lps activation. cells play a “tug of war” with a lot of events, to gain a dynamic equilibium. lps activation of cells results in an increase of ros levels. autophagic processes, amyloid fibril production and the concomitant cross-talk between immune and neuroendocrine systems with an activation of stress-sensoring circuit avoid to reach the point of no return toward the cell death. acth/α-msh axis activation an activation of stress circuits resulted in a massive presence of acth and α-msh in respect to the control. human umbilical vein endothelial cells (huvec) amyloid fibril formation the stimulated cells were thioflavine t positive. acth/α-msh axis activation activated cells, concomitant with the increase in acth and α-msh, have been showed. human neutrophils lps stimulated neutrophils showed pink cytoplasm in may-grünwald giemsa differential staining giving a gross indication of a lower intracellular ph. amyloid fibril formation the fibrillar material observed in the activated cells was positive with thioflavine t. acth/α-msh axis activation activation of stress circuits resulting in extensive production of acth and α-msh was observed in lps activated neutrophils. discussion basic biological principles in animals show conserved regulation of the involved processes. this is also true for cellular stress response, a complex and dynamic process that restores the homeostasis through highly conserved steps. cellular stress conditions promote in different animal models (invertebrates and vertebrates) the same massive morphological and physiological modifications (grimaldi et al., 2012a, b). insults, mimicking a stressful condition, provoke in all cell/tissue, detectable series of events that begin with the overexpression of ros. evidence presented here suggests that the imbalance of ros levels result in amyloidogenesis and acth/α-msh synthesis while other researchers have already demonstrated increased levels of inflammatory cytokines such as il-18 (fowler et al., 2006; malagoli et al., 2007; bossù et al., 2010; alboni et al., 2011; grimaldi et al., 2012a). in invertebrates amyloidogenesis is involved in immune response mediated by circulating hemocytes, whereas in vertebrates, it is combined with melanin production and packaging into melanocytes (fowler et al., 2006; maji et al., 2009; rochin et al., 2013). together with melanocortins the amyloid scaffold may template the deposition of pigment and this event gives a fundamental contribution to homeostasis restoration (falabella et al., 2012; grimaldi et al., 2012a, b). here, we strengthen the key role of amyloidogenesis in the cellular homeostasis suggesting that the amyloid production may buffer ros increase and attenuate oxidative stress. this may represent the primary function of amyloid fibril and is not linked to melanin synthesis. the role for amyloidogenesis has been hypothesized by several researchers in the contest of diseases such as alzheimer’s disease (goldsbury et al., 2008; dumont et al., 2009; salminen et al., 2009; millucci et al., 2012; jie et al., 2013). however our findings, by studying the behaviour of different cell types in culture after incubation with lps, indicate that the production of amyloid fibrils is an usual physiological cellular response occurring independently from melanin synthesis. accordingly is worth noting the common morphological pattern that follow lps treatment in tobacco budworm hemocytes, iplb-ldfb, s2, nih3t3, huvec, human neutrophils, and human mesenchymal stem cells. when stressed by lps these different cell lineages (mesoderm and ectoderm) all present enlarged reticulum cisternae filled with fibrillar material positive to thioflavine staining. the amyloidogenesis observed in cells from 292 fig 4 predictive value of amyloidogenesis in lps-stimulated cells. the detection of amyloidogenesis in any type of activated cells, here huvec (a h) and mesenchymal stem cells (i k) are suggested as example, gives a precocious evidence of an inflammatory process and consequence compensatory events. to validate the synthesis of amyloid fibrils in unstimulated (a)/stimulated (b) cells, and of molecules linked to stress response such as acth and α-msh (c, f unstimulated cells; d, e, g, h stimulated cells) an immunocytochemical picture can be proposed. alternatively the presence of fibrillar material in rer can be detected from an ultrastructural analysis. 293 various organisms occurs in parallel with the acth/ α-msh increased synthesis. the sum of evidences here presented is corroborated by the information available in literature about the spontaneous formation of amyloid fibrils as a consequence of the innate tendency of a lot of protein to form β-sheet aggregates (kelly and balch, 2003; fowler et al., 2006; maury, 2009; hye et al., 2014). in accordance with our observations, amyloidogenesis, has been demonstrated to be promoted by an imbalance of the redox state (christen, 2000; slominski et al., 2005; el-amouri et al., 2008; grimaldi et al., 2012a, b) and regulated by several pathways that include nep clearance activity (avoiding the fibrillar accumulation) (tanzi et al., 2004; russo et al., 2005; meilandt et al., 2009; salminen et al., 2009; nalivaeva et al., 2012). the amyloid fibril synthesis is sustained by the activation of stress circuits resulting in the extensive production of melanocortin acth and its cleavage to α-msh by nep activity (chiti and dobson, 2006; caruso et al., 2012; grimaldi et al., 2012a, b). in these respects, nep may function as a regulator of either amyloidogenesis or acth hormone levels as well (grimaldi et al., 2012a, b; pulze et al., 2014). ros are normal products of cellular metabolism, maintained at low levels in healthy cell. when ros production increases and persists at heavy load in the cytoplasm, protective mechanisms come into action and several antioxidant molecules and detoxifying enzyme are synthesized. we suggest that on the basis of the ros levels, anti-stress mechanisms and consequent cellular events can vary over a wide range, from protective autophagy and amyloidogenesis up to apoptotic or necrotic cell death (fig. 3). autophagy was often present in the cells here examined and probably induced by redox stress (cecconi and levine, 2008; he and klionsky, 2009; kroemer et al., 2010; perrotta et al., 2011; scherzshouval and elazar, 2011). in this context, autophagy has to be considered as a protective and, from an energetic point of view, “positive” mechanism because it allow to recycle the demised organelles and damaged cytoplasm. when autophagy is not sufficient to keep ros levels under control, i.e., ros levels are too high, transient amyloidogenesis may be promoted (esposito et al., 2006; dumond et al., 2009). in this case the result of buffering oxidative damages can be reached with higher energetic cost that include amyloid fibril synthesis and their subsequent cleavage due to nep action. apoptotic or necrotic cell demise only intervenes when the ros level exceeds restoration cell capacity. as a protective and detoxificant event, amyloidogenesis seems to be an ancient process, widely distributed in different cell types and evolutionary distant animals. not surprisingly, this ancient process has been integrated into additional physiological functions, that principally include packaging of melanin and/or the driving of pigment close to the non-self invader. in mammals neutrophils utilize amyloid fibrils during the formation of extracellular trap for harbouring and conveying various molecules against the invaders (pulze et al., 2014). we speculate suggesting that the additional capabilities of amyloid fibrils could be explained considering their intrinsic properties represented by affinity for oppositely charged protein and of nucleic acid (for example melanin and dna/rna, respectively), thus this scaffold could function like a reversible ion exchange polymer. as final remark, in consideration of their synthesis in the immediacy of an oxidative stress, the early detection of amyloid fibrils into the rough endoplasmic reticulum, easily evidenced by morphological observations and/or functional characterization (see for example the images referred to the lps stressed huvec and mesenchimal stem cells) (fig. 3), could be an early indicator of active networks that may lead to a general inflammatory process. conclusions amyloidogenesis is the common response of lps-treated cells from various animals and belonging to different germ layers. amyloid fibril formation is linked to cytoplasmic acidification and sustained to melanocortin overexpression. amyloidogenesis is proposed as a fundamental and ancient detoxifying event, that during evolution also acquired additional 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the epigenetic changes play the part of lymphocytes? e ottaviani department of life sciences, university of modena and reggio emilia, modena, italy accepted december 10, 2014 abstract different hypotheses have been suggested for the neurological memory storage in vertebrates, either based on the structural induction of synaptic plasticity or on chemical modifications, i.e., dna rearrangement. for invertebrates, dna rearrangements, and in particular the involvement of epigenetic mechanisms which in turn regulate gene expression, have been proposed. based on the deep link existing among immune and neuroendocrine functions, it is argued here that epigenetic changes could represent the basis for explaining the numerous observations reporting hints of immunological memory in absence of lymphocytes.   introduction memory is a wide term encompassing the storage of experiences into specific neurons and the trace of immune challenges into specific cells. memorized experiences and pathogens are accumulated during the life (rensing et al., 2009), but the fine mechanisms building up memory are still open to debate. different hypotheses have been suggested for neurological memory (see for review, peña de ortiz and arshavsky, 2001). one hypothesis proposes that the synaptic plasticity is the key event for memory consolidation, describing memory storage as the consequence of a structural modification. a second hypothesis considers a chemical modification, i.e., the memory storage is not at the synaptic, but at the genomic level. in this second view the storage of information is based on somatic dna recombination. such memory storage mechanism working in the brain neurons is similar to that observed in lymphocytes of the immune system. in this context, it is appropriate to stress the similarities between immune and neuroendocrine system workflow. studies by j. edwin blalock and colleagues have demonstrated in mammals a functional integration of the immune and the neuroendocrine systems. one of the most convincing findings has been that both systems share a series of endogenous and exogenous ___________________________________________________________________________ corresponding author: enzo ottaviani department of life sciences university of modena and reggio emilia via campi 213/d, 41125 modena, italy e-mail: enzo.ottaviani@unimore.it mediators that combat threats to the homeostasis of the organism (weigent and blalock, 1987). based on these similarities, habibi et al. (2009) surmized that dna rearrangements could be present in immune and nervous systems in order to accumulate permanent information and allowing the creation of long-term memory. a possible relationship between genome changes and memory formation could be also contained into epigenetic modifications, i.e., alterations in the chromatin structure, which in turn regulate gene expression (levenson and sweatt, 2006). the present contribution suggests to consider epigenetic modifications as one possible mechanism of immunological memory formation in invertebrates. epigenetics the term epigenetics was coined by conrad h. waddington in 1942. today, epigenetics has been defined as ‘‘the study of changes in gene function that are mitotically and/or meiotically heritable and that do not entail a change in dna sequence’’ (wu et al., 2001). the well-studied epigenetic mechanisms are the following: dna methylation, histone tail modification and microrna (mirna) or non-coding rna (tammen et al., 2013) (fig. 1). current status on the immune and neuroendocrine systems in invertebrates already 20 years ago, experiments of my and other laboratories have shown that in molluscs, as in vertebrates, there is a close functional correlation between the immune and the neuroendocrine 1    fig. 1 schematic summary of the epigenetic mechanisms acting on chromatin remodelling that are involved in gene regulation. systems. given the evolutive distance separating mollusca form vertebrates, we concluded that this relationship has a deep and ancient evolutionary root, predating the split between protostomian and deuterostomian lineages. it seems that nature has followed an economic strategy by reusing the same pool of signal molecules (neuropeptides, hormones and cytokines) preserved in an extraordinary way in the course of evolution (ottaviani and franceschi, 1997). with regards the immune system, invertebrates present only the innate immunity and consequently there are no lymphocytes challenging the comparative immunologists focused on immunological memory studies. despite the lack of a cellular substrate recalling the vertebrate lymphocytes, in literature several data in supporting the presence of immunological memory emerge (cooper, 1969, 1976; hostetter and cooper, 1973; karp and hildemann, 1976; hildemann et al., 1977, 1979a, b; karp and rheins, 1980). in our molluscan model, planorbarius corneus, experiments performed on the humoral and cellular components, as well as on the bacterial clearance are in favor of the existence of some form of immunological memory (ottaviani et al., 1986; ottaviani, 1992). repeated injections of bacteria staphylococcus aureus and escherichia coli into the foot of the mollusc induce specific and aspecific agglutinins. specific agglutinins observed in direct-agglutination tests showed an increased titer after the second injection. aspecific agglutinins evaluated in crossagglutination tests showed no changes in titer after the second injection (ottaviani, 1992). the in vitro bacterial phagocytosis experiments have shown a higher bacterial elimination across the entire timerange considered (30, 60, 90, 120, 150 min) in the snails that had already contacted the bacteria to be phagocytized (ottaviani, 1992). the bacterial clearance experiments have revealed that after the second (14 days) and third (73 days) bacterial injections, clearance rates are faster (ottaviani et al., 1986). also more recent investigations have provided further evidence of a potential immune memory in invertebrates, though the edge between real memory and the effect of immune priming is 2    blurred, especially in short living species (kurtz, 2004, 2005; brehélin and roch, 2008). on the whole, a careful revision of the existing literature provides indications that from protozoans (csaba et al., 1984) to vertebrates, some types of memory may be present. the absence of a lymphocyte-based system comparable to those of jawed vertebrates and lampreys (hirano et al., 2013), points towards alternative processes. the close developmental similarities recently observed between neurons and hemocytes in crustaceans (benton et al., 2014) allow to speculate that the mechanisms described for neurological memory could be present also in invertebrates. with the exception of the data reported in drosophila (lafave and sekelsky, 2009), mechanisms of somatic rearrangement of dna are unusual and not occurring at loci hosting invertebrate immune genes. as an alternative mechanism, epigenetic modifications of hemocyte chromatin could be surmized, as it has been also proposed by levenson and sweatt (2006) for neurological memory. epigenetic effects in invertebrates recently, we have analyzed epigenetic modifications in neurons of the mollusc pomacea canaliculata after injection of lps (ottaviani et al., 2013). following the treatment the phosphoacetylation of histone h3 correlates with the increase of c-fos protein levels in the nuclei of the small ganglionic neurons. these findings show the highly conserved interactions between immune and neuroendocrine systems at a molecular level. in contrast to the pattern of genome-wide dna methylation in vertebrates, dna methylation in invertebrates is relatively sparse (bird et al., 1979; suzuki and bird, 2008; field et al., 2004). epigenetics studies in molluscs suggest the presence of cpg methylation in mytilus edulis (bird and taggart, 1980), donax trunculus (petrovic et al., 2009) and crassostrea gigas, where dna methylation has important regulatory functions (gavery and roberts, 2010). moreover, in octopus vulgaris methylation is involved in gene regulation during the development (díaz-freije et al., 2014). beside dna methylation, mirnas present in metazoan are about 22 nucleotides in length and play a role in the control of animal development and physiology by altering the chromatin architecture (ambros, 2004). conclusions this article wants to trigger the attention of comparative immunologists towards those mechanisms widespread in metazoans that could explain numerous observations at present orphans of solid explanations. at present it is neither possible to state that immunological memory is present in invertebrates nor that epigenetic changes represent its basis. however, the presence of epigenetic mechanisms could represent a potential alternative to the lymphocyte-based memory in invertebrates, especially in consideration of the very few examples of somatic dna recombination. acknowledgements the author thanks prof. m mandrioli (university of modena and reggio emilia, italy) for the for the figure. references ambros v.the functions of animal micrornas. nature 431:350-355, 2004. benton jl, kery r, li j, noonin c, söderhäll i, beltz bs. cells from the immune system generate adult-born neurons in crayfish. dev. cell 30: 322-333, 2014. bird ap, taggart mh, smith ba. methylated and unmethylated dna compartments in the sea urchin genome. cell 17:889-901, 1979. bird ap, taggart mh.variable patterns of total dna and rdna methylation in animals.nucleic acids res. 8:1485-1497, 1980. brehélin m, roch p.specificity, learning and memory in the innate immune response. inv. surv. j. 5: 103-109, 2008. cooper el. chronic allograft rejection in lumbricusterrestris. j. exp. zool. 171: 69-73, 1969. cooper el. comparative immunology, prenticehall, inc, englewood cliffs, nj, 1976. csaba g, darvas z, lászló v, vargha p. influence of prolonged life span on receptor 'memory' in a unicellular organism, tetrahymena. exp. cell biol. 52: 211-6, 1984. díaz-freije e, gestal c, castellanos-martínez s, morán p.the role of dna methylation on octopus vulgaris development and their perspectives. front. physiol. 5:62, 2014. field lm, lyko f, mandrioli m, prantera g. dna methylation in insects. insect mol. biol. 13: 109115, 2004. gavery mr, roberts sb. dna methylation patterns provide insight into epigenetic regulation in the pacific oyster (crassostrea gigas). bmc genomics 11: 483, 2010. habibi l, ebtekar m, jameie sb. immune and nervous systems share molecular and functional similarities: memory storage mechanism.scand. j. immunol. 69: 291-301, 2009. hildemann wh, bigger ch, johnston, is. histoincompatibilityreactions and allogeneic polymorphism among invertebrates. transplant. proc. 11: 1136-1142, 1979a. hildemann wh, johnston, is, jokiel pl. immunocompetencein the lowest metazoan phylum: transplantation immunity in sponges. science 204: 420-422, 1979b. hildemann wh, raison rl, cheung g, hull cj, akaka l, okamoto j. immunological specificity and memory in a scleractinian coral. nature 270: 219-223, 1977. hirano m, guo p, mccurley n, schorpp m, das s, boehm t, et al. evolutionary implications of a third lymphocyte lineage in lampreys. nature 501: 435-438, 2013. hostetter rk, cooper el. cellular anamnesis in earthworms. cell. immunol. 9: 384-392, 1973. 3    karp rd, hildemann wh. specific allograft reactivity in the sea star demasteriasimbricata. transplantation 22: 434-439, 1976. karp rd, rheins la. a humoral response of the american cockroach to honeybee toxin demonstrating specificity and memory. in: manning mj (ed), phylogeny of immulogical memory, biochemical press, elsevier/northholland, pp 65-76, 1980. karp rd, rheins la. a humoral response of the americancockroach to honeybee toxin demonstrating specificity and memory. in: manning mj (ed), phylogeny of immunogical memory, biochemical press, elsevier/northholland, pp 65-76, 1980. kurtz j.memory in the innate and adaptive immune systems. microbes infect. 6: 1410-1417, 2004. kurtz j.specific memory within innate immune systems. trends immunol. 26: 186-192, 2005. lafave mc, sekelsky j.mitotic recombination: why? when? how? where? plos genet. 5(3):e1000411, 2009. levenson jm, sweatt jd. epigenetic mechanisms: a common theme in vertebrate and invertebrate memory formation. cell. mol. life sci. 63: 10091016, 2006. ottaviani e. presence of a memory-type response in the freshwater snail planorbarius corneus (l.) 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tuscia, viterbo, italy lecture evolution of immunity from invertebrates to vertebrates k buchmann laboratory of aquatic pathobiology, department of veterinary and animal sciences, faculty of health and medical sciences, university of copenhagen, denmark interspecific communication between various organisms in the environment is relying on a series of basic biological processes which can be found in both invertebrates and vertebrates. even the most primitive unicellular organisms, such as amoebae, must find a balance between tolerance to surrounding elements and highly protective responses against potentially pathogenic microorganisms. the balance is well illustrated by the process of phagocytosis: this is a way for a cell to import energy and nutrition to sustain life processes but the process can also engulf and inactivate invading pathogens when combined with a range of effector molecules. these events can be supported under different environmental conditions. we presume that primitive unicellular organisms, solitary or residing in colonies, during the relatively early history of the earth, more than 1000 mya, occupied anaerobic sediments. this environment challenged survival of the local inhabitants which possessed advanced cellular machinery to extract energy from the substrate and at the same time were able to recognize and respond to pathogen associated molecular patterns. this early development of pathogen and danger recognition receptors is still the basis for survival in more developed animals appearing after the cambrian explosion. although photosynthesis and thereby oxygen placed an additional evolutionary pressure on the early organisms, and thereby gave rise to new life forms, a crucial conservation of an anaerobic environment with its associated microorganisms in the intestine of higher animals, plays a pivotal role for host immunity. concordantly, the importance of the gut microbiota in advanced mammals, such as man, has been well documented for a number of immunologically determined diseases (e.g. diabetes and ibd). teleosts represent one of the earliest vertebrate groups with a developed adaptive immune system comprising mhc, tcell receptors and several classes of immunoglobulins. as a range of basic innate effector molecules are available as well, it is worthwhile to study basic immune mechanisms (both innate and adaptive) in fish. this is illustrated by the rainbow trout, oncorhynchus mykiss, which can be immunized against the enterobacterium yersinia ruckeri by several routes. vaccination studies using this host species show how timing and administration of antigen (injection, immersion, bath, oral) can 45   provide new insight into the delicate balance between development of vertebrate immunity and tolerance. in vitro and in vivo immunoreactivity of different betanodavirus species f pascoli1, v panzarin1, a guazzo2, g. scapigliati3, f. buonocore3, a. toffan1 1istituto zooprofilattico sperimentale delle venezie, legnaro (pd), italy 2university of padua, padua, italy 3dipartimento dibaf, university of tuscia, viterbo, italy betanodaviruses are the causative agents of the viral nervous necrosis (vnn), a devastating disease for the mediterranean mariculture. four different betanodavirus species are recognized, striped jack-, redspotted grouper-, tiger puffer-, and barfin flounder nervous necrosis virus (sjnnv, rgnnv, tpnnv and bfnnv), but there is little knowledge on their antigenic properties. recent studies indicate that the sjnnv and the rgnnv are antigenically distinct, constituting serotypes a and c respectively. the natural reassortant viruses rgnnv/sjnnv and sjnnv/rgnnv group within serotypes a and c respectively, indicating that the coat protein encoded by rna2 acts as major immunoreactivity determinant. the aim of this study was to investigate the possible in vivo cross-protection in sea bass of these two betanodavirus species using a rgnnv and a sjnnv in-house produced vaccine. ten ml of rgnnv (strain 283.2009) and sjnnv (strain 484.2.2009) were inactivated with 1% formalin for one week at room temperature. two groups of 60 juvenile european sea bass were ip immunized with 0.1 ml/fish of one inactivated virus solutions each. an additional group was mock vaccinated with pbs 0.01 m to act as positive control. after 30 days post vaccination, 10 fish from each tank were bled to death and sera collected for immunological analyses. the remaining fish were im challenged with 283.2009 rgnnv and kept at 25 °c for 28 days. considering that sea bass is poorly susceptible to sjnnv, challenge was performed with the rgnnv only. the mortality rate for each experimental condition was calculated and compared through statistical analysis. results showed a rps of 81.2 for inactivated rgnnv and 25.3 for sjnnv, confirming that the cross-protection of the two species is very limited. immunological analyses showed an appreciable seroconversion of the rgnnvand sjnnv-vaccinated fish against the homologues antigens, but very low cross-reaction against heterologous virus, confirming the in vivo results. recently, several betanodavirus outbreaks in sea bream occurred in the mediterranean basin and they were associated to a reassortant strain rgnnv/sjnnv. the data obtained in this study could be highly informative for the development of cross-protective vaccines against different betanodavirus genotypes. computational modeling as a tool to predict a more effective fish vaccination a madonia, c melchiorri, s bonamano, c bulfon1, f castiglione2, m galeotti1, m marcelli, f mosca3, pg tiscar3, d volpatti1, n romano department of ecology and biology, tuscia university, viterbo 1department of food sciences, section of veterinary pathology, university of udine, italy 2department of comparative biomedical sciences, university of teramo, italy 3institute of computing applications “m.picone” cnr ,rome, italy the validation of vaccine protocols and the determination of the optimal dose for the populations to be treated can be immunological simulated by innovative approach in bioinformatics sciences. recently we have administered an anti-vibriosis vaccine by an innovative route treatment for the farmed sea bass juveniles were vaccinated against listonella anguillarum (la) using a commercial formulation (sharing-plug) administered by double immersion or by double immersion and subsequent injection via i.p.; in both cases, fish have shown an appreciable protection against a subsequent infection. the in vivo findings were compared with the in silico experimentation through the use of the immunological model cimmsim, an agent based model which describes both the humoral and cellular response of immune system. the grid is threedimensional with periodic boundary conditions, representing a known volume of a lymphoid organ or peripheral blood. the vaccination steps were reproduced by injecting "un-harmful" antigen at those t-steps matching the in vivo experiment protocol. the challenge was simulated by injecting a known amount of “active” antigen in the grid. calibration tests were carried out to select the appropriate doses of vaccine and infectious bacteria concentrations to be used in the simulation experiments. the outputs of the model agree with the immunological analysis of ab production (antil/serum), bcr and tcr gene transcripts in the spleen, allowing to follow the temporal evolution of the immunological response all over the trials. consequently to appreciable results in vivo and in silico, a new vaccination protocol has been tested by using two pathogens: la and p.damselae sb. piscicida. data were compared with the silico model, suitably modified with the 46   new parameters of the experiment. thus, for the first time a c-immsim model has been applied for a multiple vaccination. the results have indicated a good performance of the model capacity to reproduce the in vivo experimentation. the use of an immunological simulator in fish vaccination trials confirms the suitability of abm models for the simulation of biological systems, providing an useful tool to be exploited as a virtual laboratory where experiments can be made prior to the design of a real in vivo or in vitro experimentation. catalase in antarctic fish f corrà1, e poloni1, e callegaro1, s bramuzzo1, m gerdol2, f buonocore3, g scapigliati3, a biscontin1, c de pittà1, r bakiu4, g santovito1 1department of biology, university of padua, padua, italy 2department of life sciences, university of trieste, trieste, italy 3department for innovation in biological, agrofood and forest systems, university of tuscia, viterbo, italy 4department of aquaculture and fisheries, agricultural university of tirana, tirana, albania when phagocytes are strongly activated, molecular oxygen can be converted into reactive oxygen species (ros) to damage the microbes. to scavenge the excess of ros, aerobic organisms have evolved enzymatic and nonenzymatic antioxidant systems. catalase (cat, ec.1.11.1.6) is an important member of the enzymatic antioxidant system and can effectively catalyze the decomposition of hydrogen peroxide (h2o2) to keep the balance between de novo h2o2 generation and efficient elimination, which is essential for innate immunity. with the aim to increase the knowledge on this proteins in antarctic teleosts, we characterized the genes codifying for cat in three species: trematomus bernacchii, trematomus hansoni and chionodraco hamatus. the gene sequences were determined by pcr amplification and sequencing, and by transcriptome analysis. multi-alignment analysis, performed with fish orthologous sequences, demonstrated high conservation of the amino acids involved in catalytic activity also in cat of antarctic species. however, some substitutions with polar amino acids, are characteristics of some residues close to the motifs that are important for the functionality of this protein. the cat gene transcription from various tissues (gills, heart, liver, and skeletal muscle) of t. bernacchii and c. hamatus was measured by qpcr. in both species, cat mrna is preferentially expressed in liver. cat activity was also measured in the same organs. again, the highest level is present in liver. the tissue-specific differences in the mrna and active protein accumulations are probably related to the physiological characteristic of these organs. more insights into evolution of igt genes from antarctic fish a ametrano, m vitale, u oreste, mr coscia institute of protein biochemistry, cnr naples, italy immunoglobulin t (igt) represent the most ancient ig isotype found only in teleosts, and specialized in mucosal immunity. in our previous studies, igt heavy chain constant region genes of the antarctic teleost trematomus bernacchii, belonging to the nototheniidae family, were characterized. these studies highlighted that t. bernacchii igt lack most of the heavy chain second constant domain, retaining only a few amino acid residues, such as proline, glycine and cysteine (coscia et al., marine genomics, 2015). we suggested that this region may act as a hinge region similarly to that found at the ch2-ch3 boundary of the antarctic igm heavy chain. analysis of cdna clones encoding igt heavy chain revealed the presence of three different sized igt transcript variants, named long (l), short (s) and shortest (sts). genomic analysis of t. bernacchii igh locus revealed the presence, within the intron between the exons coding for the entire first and second constant domains, respectively, of a reminiscence of the ancestral second exon. the current work is aimed at expanding our knowledge on evolution of igt genes to other antarctic notothenioid families. igt nucleotide sequences have been determined from chionodraco rastrospinosus (family channichthyidae), gymnodraco acuticeps (family bathydraconidae), pogonophryne scotti (family artedidraconidae), trematomus hansoni, and gobionotothen gibberifrons (family nototheniidae). the sequences were aligned with the three variants of t. bernacchii, and with the sequence from notothenia coriiceps, previously isolated from an online available n. coriiceps transcriptome data set. based on cdna sequence alignment, our analysis confirmed that the lack of almost the entire ch2 domain is a feature common to antarctic fish. in particular, g. gibberifrons and n. coriiceps, both belonging to the nototheniidae family, showed a shorter ch2 domain than t. bernacchii variant s. moreover, in c. rastrospinosus a duplicated region, 9-nt long, was found between ch2-ch3. this duplication is probably a trace of the event leading to the loss of most ch2 domain through transposable elements. analysis of deduced amino acid sequence of the duplicated region highlights the conservation of cysteine residues, suggesting that these residues could be required for the assembly of the two heavy 47   chains in light of the loss of most second constant domain. diversity evolution and immune function of fish lectins m cammarata, mg parisi marine immunobiology laboratory, dipartimento di scienze e tecnologie biologiche chimiche e farmaceutiche, university of palermo, conisma, palermo, italy fish are equipped with a complex lectin repertoire that, like mammals, are involved almost all the immune reactions. carbohydrate recognition and interactions mediated by lectins have been recognized involved in vertebrate innate immunity, not only for recognition of potential pathogens, but also acting in the agglutination, immobilization and other functional steps. in fish, c and f types lectins, galectins, rhamnose-binding lectin (rbl) and pentraxin have been identified in both in both cartilaginous and bony fish. in addition, selectins and other genes have been found in the currently available fish genomes. on the basis of our results on f-type (fbl) and rbl lectins we showed that: -lectin repertoires in fish are highly diversified and include not only representatives of the lectin families; described in mammals, but also members of lectin families described for the first time in fish species like the fbl and rbl; -these lectins have been identified in the eggs and embryos but also are present in the serum; -tissue-specific expression and localization of the diverse lectin repertoires and their molecular partners is consistent with their distinct biological roles in innate and adaptive immunity; -although some lectins may bind endogenous ligands, others bind sugars on the surface of potential pathogens; in addition to pathogen recognition and opsonization, some lectins display additional effector roles, such as regulation of immune functions. session 2. invertebrate immunity chairmen: magda de eguileor, università dell’insubria, varese, italy allorecognition in the ascidian botryllus schlosseri: the importance of amyloid n franchi1, m de eguileor2, a grimaldi2, r girardello2, l ballarin1 1department of biology, university of padua, italy 2department of biotechnology and life sciences, university of insubria, varese, italy   in the compound ascidian botryllus schlosseri, allorecognition manifests primarily as colony specificity, controlled by a highly polymorphic fu/hc locus, so that contacting colonies sharing at least one allele at the fu/hc locus can fuse into a chimeric colony; if no alleles are shared, a typical inflammation reaction occurs, with the recruitment of a specific hemocyte type, the cytotoxic morula cells (mcs), inside the tips of the ampullae (the blind termini of the tunic vasculature) extending towards the alien colony, their extravasation in the tunic and their final degranulation with the consequent release of factors inducing autocrine and paracrine cell and the formation of necrotic, melanic spots (points of rejection; pors) along the contact border. mcs are the first cells to sense nonself and are the source of cytokines that induce the recruitment of immunocytes at the inflammatory sites and the activation of phagocytes required for the clearance of foreign material. mcs store quinones, polyphenols and the enzyme phenoloxidase (po) inside their granules, representing their cytotoxic potential. although po, quinones and polyphenols are soluble factors, it is remarkable that the deposition of melanin and the cell death is confined to the immediate outside of the ampullar tips, suggesting that the diffusion of the enzyme and the products of its activity is, in some way, prevented in order to limit cytotoxicity to the immediate neighbourhood of degranulating mcs. with this idea in mind, we looked for factors released by mcs that could limit the spreading of cytotoxicity and melanisation. we found that mcs contain amyloid inside their granules and that amyloid fibrils are released in the surrounding medium upon mc degranulation forming a net-like scaffold entrapping po and melanin, thus limiting their diffusion. in addition, the search for genes and factor controlling both melanogenesis and amyloidogenesis, revealed an evolutionary conserved machinery involved in the processes and an unexpected cross talk between the two botryllus immunocyte types, i.e., phagocytes and mcs. this work represents the first demonstration of a physiological role of amyloid in protochordata immunity.  bio-molecular perspectives on anti-tumour properties of the ciliate toxin climacostol f proietti serafini1, e catalani1, l guerra1, mc belardinelli1, s picchietti1, am fausto1, e marcantoni2, f buonanno3, c ortenzi3, s zecchini4, c perrotta4, d cervia1,4 1department for innovation in biological, agrofood and forestsystems (dibaf), università of tuscia, viterbo, italy 2school of sciences and technologies, section of chemistry, university of camerino, camerino, italy 3laboratory of protistology and biology education, university of macerata, macerata, italy 4department of biomedical and clinical sciences “luigi sacco” (dibic), university of milano, milano, italy the incidence of melanoma skin cancer has risen sharply over past few decades, making 48   melanoma one of the most common tumours in caucasian worldwide. considering that metastatic melanoma is almost completely resistant to most current therapies and is linked with a poor patient prognosis, the need for additional drugs is urgent. several modern drugs, including those for cancer therapy, have been isolated from natural sources. herein we focused on the biological activities of climacostol, a natural toxin, now available by a new and straightforward synthesis, produced by the freshwater ciliated protozoan climacostomum virens. climacostol inhibits cell growth and reduces viability/proliferation of b16 mouse melanoma cells. to evaluate the climacostol therapeutic potential the pharmacological properties and the molecular signature associated with its actions have been investigated in vivo. we showed that climacostol treatment exerts an effective inhibitory action on the progression of b16 melanoma allografts, thus increasing animal survival and, at the same time, decreasing the expression of the proliferation markers pstat3, ki67, and survivin. . at molecular level, climacostol rapidly leads to dna damage and apoptosis. in particular, the signalling events responsible for the climacostolinduced pro-apoptotic effects rely on the upregulation of p53 network that, in turn, activates the intrinsic programed cell death pathway (mitochondrial dysfunction, ros, bax/cytochrome c, caspase-9/3). finally, our results indicated that climacostol inhibits the autophagic flux in melanoma cells, likely through the modulation of the akt/mtor pathway. an increasing number of evidence reveals that perturbation of the autophagic machinery contributes to tumour development. also revealing multiple connection points between pro-survival autophagy and detrimental apoptosis. overall these results indicate that climacostol is a powerful and efficacious anticancer agent that it may be considered for the design of cytotoxic new drugs for melanoma therapy. host-parasite interactions: what is the role of the antioxidant system in immune memory of the red flour beetle tribolium castaneum? k ferro 1, d ferro 1, f corrà 2, r bakiu 3, g santovito 2, j kurtz 1 1animal evolutionary ecology group, institute for evolution and biodiversity, university of münster, germany 2department of biology, university of padua, italy 3department of acquaculture and fisheries, agricultural university of tirana, tirana, albania the consequence of an aerobic metabolism is the production of oxygen derivatives named reactive oxygen species (ros). the production of ros, for their unstable electronic configuration, could lead to oxidative stress with harmful consequences on organisms, unbalancing redox status and promoting cell death. despite the damage potential of ros, their production has not been eliminated during evolution, since they can be involved in many physiological, immunological and molecular processes that contribute to fitness. the importance of ros homeostasis on cell survival is also demonstrated by effects of an abnormal level (lack or overproduction) that results in pathologies such as cancer, cardiovascular and neurodegenerative diseases in vertebrates. the role of ros in host-parasite interactions is defined by their contribution to innate immunity, promoting parasite death through active participation during infection processes. moreover, ros are also assumed to play a role in acquired immune responses, such as immune priming, i.e. a form of memory in invertebrates (roth et al., 2009). investigating the involvement of ros in host-parasite interactions, many studies focused on phenoloxidases and nadphdependent oxidases only, potentially neglecting the importance of other related proteins, such as antioxidant enzymes. superoxide dismutases (sod) and catalases (cat) have pivotal roles in the antioxidant system and contribute to ros homeostasis under many physiological conditions. in the present study, we characterize sod and cat genes in  red flour beetle tribolium castaneum. we used the most recent transcriptomic and genomic databases, in order to identify genomic regions that potentially code for these enzymes, and we studied the promoter regions of their genes for the identification of putative ros-related regulatory sequences. moreover, we performed an immune priming experiment with the gram-positive entomopathogen bacillus thuringiensis, in order to identify the potential role of sod and cat in immune memory of this species. the c1q domain-containing protein from the ascidian botryllus schlosseri manifests a cytokine-like behavio n franchi, l ballarin department of biology, university of padua, italy genes encoding complement component 3 (c3) have been extensively investigated in invertebrate genomes and traced back in evolutionary history to the early metazoan radiation. however, other components of the complement system, such as those related to the classical activation pathway, are still not much investigated. currently, the genes encoding for proteins with a c1q domain, probably the main components of the classical pathway, have been only partially investigated from an evolutionary perspective. these genes exist in many of the sequenced genomes, from both vertebrates and 49   invertebrates and functions have been described for some of the corresponding proteins. a c1q-like gene have been identified in the medicinal leech hirudo medicinalis where a c1qlike peptide elicits a chemotactic behavior that could be blocked using a human antibody against the gc1q receptor. c1q-like genes have also been found in the urochordate ciona intestinalis and the cephalocordate branchiostoma floridae where it has been demonstrated that the globular domain is able to recognize and bind mammalian antibodies initiating the classical pathway of complement activation. the globular head c1q domain is a lectin domain present in transcriptomes of amphioxus, lamprey, and several teleost fishes. few of these putative c1q-like proteins have been characterized; however, they can bind to a variety of carbohydrates. in botryllus schlosseri we have found, in our est colletion, a single transcript with c1q characteristics (bsc1q-like). the deduced protein contains two globular head c1q domains, a feature unknown in invertebrates. as regard vertebrates, we can find a similar architecture only in mammals, in the so called c1q/tnf-related protein 4 (ctrp4). this protein is very poorly studied and seems to be expressed in the hypothalamus and contribute to the modulation of food intake and body weight. our data, from the colonial ascidian, suggest a role for the bsc1q-like protein as mediator of the activation and degranulation of the cytotoxic hemocytes. both ish and icc demonstrate that both cytotoxic morula cells and phagocytes express the bsc1q-like mrna and protein; functional analyses demonstrate that the human antibody against globular head c1q is able to inhibit morula cell degranulation after bacterial challenge. it is not yet clear if it is possible to considered this molecule as member of the complement system in botryllus but future analyses will be directed to the study of the functional relationships between bsc3 and bsc1q-like as well as of the binding capabilities of the latter. the medicinal leech as a useful tool for studying haematopoiesis, angiogenesis and innate immune response a grimaldi, r girardello, n baranzini, g tettamanti, m de eguileor department of biotechnology and life science, university of insubria, varese, italy in the last decade, a large body of evidence has demonstrated that hematopoiesis, angiogenesis, immune response and cytokines regulating the occurrence of these processes have been highly conserved across vertebrates and various invertebrate phyla. here we provide a broad overview of medicinal leech hematopoiesis and we highlight the benefits of using the medicinal leech, having a simple anatomy and a repertoire of less varied cell types when compared to vertebrates, as a powerful model for studying basic steps of hematopoiesis and immune responses. in particular, leeches’ hematopoietic stem precursor cells (hspcs), deriving from the activated botryoidal tissue, seem to share with their vertebrates counterpart most morphofunctional and molecular mechanisms. hspcs are massively recruited from the bloodstream towards sites of inflammation in response to injury, bacterial injection, or experimental administration of cytokines and contribute to immune response, neovascularization and muscle regeneration. further, similar to vertebrates these cells are driven toward a leukocyte, endothelial or myogenic, differentiation pathway, as a function of the time course of vegf release to target cells. since investigations on hematopoiesis can greatly help scientists and clinicians to better understand the pathological processes behind blood disorders, cancers and muscle regeneration, hspcs of medicinal leech can be used as a powerful model system for understanding tissue stem cells and their role in angiogenesis, tissue regeneration and oncogenesis. cellular responses induced by multi-walled carbon nanotubes: in vitro study on the medicinal leech macrophages r girardello, n baranzini, m de eguileor, a grimaldi department of biotechnology and life science, university of insubria, varese, italy the intrinsic characteristics of engineered nanoparticles, such as multi-wall carbon nanotubes (mwcnts) are impressive and attractive for technology, however their environmental dispersion could be potentially hazardous to animal and human health. since the production and use of mwcnts is steadily increasing, intentional or unintentional environmental discharges may occur. for this reason, the development of new methods and the identification of reliable models to completely understand mwcnts effects are critical. here we propose a freshwater invertebrate, the medicinal leech, as alternative model to assess the effects of mwcnts on immune system. our previous studies have already demonstrated that in vivo mwcnt treatment induces the activation of leech’s macrophages. in this study, we focus on the direct effects of mwcnts on these cells. we used the consolidated experimental approach, based on the injection in the leech body wall of the biomatrice matrigel (mg) added with the cytokine allograft inflammatory factor-1 (hmaif1), to specifically chemoattract macrophages. 50   cells extracted from mg were cultured and characterized with the specific markers cd45 and cd68, confirming their belonging to the monocyte-macrophage lineage. primary macrophage cultures were then subjected to an in vitro treatment with mwcnts at different concentrations (2.5, 5, 10, 25, 50 and 100 µg/ml). our results indicate that leech macrophages, once in close contact with mwcnts, actively produce amyloid material to encapsulate the foreign bodies. we also demonstrated that mwcnts in vitro treatment cause the decrease of cell proliferation rate and the increase of the apoptotic rate. furthermore, since oxidative stress is linked with inflammation and amyloid production, reactive oxygen species has been evaluated, confirming that their production rate increases after mwcnt treatment. our combined experimental approaches, not only attest the ability of mwcnts in inducing a potent inflammatory response, but also confirm the medicinal leech as a good alternative model that can be improved and successfully used to study the possible harmful effects of any nanomaterial. moreover, since autophagic cell death pathway activation is emerging as a possible consequence of mwcnt treatment, in the future we will attempt to clarify this aspect in order to completely understand mwcntinduces toxicity. the human recombinant rnaset2 activates the initial phase of the inflammatory response in the medicinal leech n baranzini, r girardello, m de eguileor, f acquati, a grimaldi department of biotechnology and life science, university of insubria, varese, italy recent studies have demonstrated that rnaset2, the only member of the ribonuclease t2 family present in human genome, is involved in the control of tumorigenicity in ovarian cancer cells. furthermore its capacity to be a chemoattractant for numerous cells of monocytic-macrophage line and the possibility to act as an inducer of the innate immune response in vertebrates have been established. in fact, in tumor tissues the detectable cross-talk between cancer cells and the surrounding tumor microenvironment is based on these aspects. although several studies have been reported on the molecular features of rnaset2, the details on the pathways by which this evolutionarily conserved protein regulates the immune system are still poorly defined. in order to better elucidate these aspects, we report here the effect of the human recombinant rnaset2 injection and its role in regulating the innate immune response after bacterial challenge in an invertebrate model, the medicinal leech. this animal has been chosen for its very simple anatomy and for its rapid inflammatory response. indeed, after few hours from the injection, a large number of fibroblasts are visible in the connective tissue that appears completely remolded and infiltrated by numerous cells expressing the specifics macrophage markers cd68 and hmaif1. in order to confirm the ability of this ribonuclease to attract macrophages, we used a consolidated experimental approach based on the injection in the leech body wall of the matrigel biomatrice (mg), supplemented with the human recombinant rnaset2. after one week, the extracted mg sponges was infiltrated by numerous cells cd68+ and hmaif1+. moreover, in the leech body wall challenged with lipopolysaccharides (lps) or with the environmental bacteria pathogen micrococcus nishinomiyaensis, endogenous rnaset2 is highly expressed by the numerous macrophages migrating to the site of inoculation. taken together, these results clearly suggest that rnaset2 is likely involved in the initial phase of the inflammatory response in leeches. session 3. invertebrate immunity chairmen: davide malagoli, university of modena and reggio emilia, modena, italy genomic immune system of ciliates: dna elimination as a genome defense mechanism a vallesi, p luporini school of biosciences and veterinary medicine, university of camerino, camerino (mc) italy whole genome sequencing analyses are providing compelling evidence that proand eukaryote microbes, like multicellular organisms, have their life threatened by parasitic attacks. bacteria and archea face invading viral nucleic acids with an ‘inheritable dna-encoded immunity’ (known as the crispr-cas system) that recognizes foreign dna from self dna. in eukaryotic microbes that are exposed to invasions from both bacteria and viruses, bacteria are promptly made harmless either by digestion into food vacuoles, or by ‘domestication’ as symbionts. but the defence from viral attacks is much less effective. foreign viral sequences can randomly insert into the cell genome, and may disrupt or deactivate vital genes. to fight this threat, ciliates rely on a unique model of inheritable genomic immune mechanism based on the evolution of two genomes, a germ-line one lying in the cell micronucleus and a somatic one lying in the macronucleus. the germ-line genome characterized by an orthodox chromosomic organization exposed to invasive viral dna sequences is maintained transcriptionally silent. only the somatic genome characterized by a unique sub-chromosomic organization is expressed. it is generated de-novo in 51   coincidence with every sexual event from a copy of the micronuclear genome that is previously made free of any invasive dna sequence by the activity of a small rna-targeted dna-deletion mechanism, called ‘internal eliminated sequences (ies)-associated gene system’ by ciliatologists. the discovery, function and effects of this mechanism that eliminates invasive dnas from the developing somatic genome will be the object of this contribution. assessment of morphological and cellular responses after infection with living bacteria in the marine mussel mytilus galloprovincialis mg parisi1, m maisano2,t cappello2, s oliva2, a mauceri2, m toubiana3, m cammarata1 1marine immunobiology laboratory, dipartimento di scienze e tecnologie biologiche chimiche e farmaceutiche university of palermo, conisma, palermo, italy 2department of chemical, biological, pharmaceutical and environmental sciences, university of messina, messina, italy 3laboratoirehydrosciences montpellier, umr 5569 cnrs, ird, université montpellier, france bacterial strains of vibrio genus associated with temperate regions are linked to musselsborne infections. the sedentary nature of marine mussels, mytilus galloprovincialis, together with their filter feeding combine to ensure that they have the potential for considerable exposure to infective agents. the primary mechanism of bivalve internal defense involves hemocytes responsible for cellmediated immunity through a panel of activities such as phagocytosis, the release of cytotoxic molecules, reactive oxygen intermediates, lysosomal enzymes, po enzyme and lysozyme. in this work in vivo infection of m. galloprovincialis with living v. splendidus to collect hemolymph from the posterior adductor muscle 1, 3, 6, 9, 12, 24 and 48 h post-injection was carried out. previously we have found that when bacteria were injected into the circulation of the mussel, the number of living intra-hemocyte bacteria dramatically increased already after thirty minutes, suggesting intense phagocytosis, then decreasing until 24 h. the quantification by flow cytometry indicated a variation of proportions of the three cell categories. in our study, injection of living bacteria resulted in total hemocyte count (thc) higher than normal 24 48 h post-injection, suggesting proliferation and/or recruitment of hemocytes which are mainly concentrated in the site of infection. to compliment this, here histological and immunohistochemical assessment was performed using adductor musclein order to evaluate the morphological features and cellular response spost bacterial infection. the morphological analysis showed changes in tissue organization, with an altered cell volume and recruitment of hemocytes among fibers. the change of osmotic equilibrium across muscle cell membranes was observed by increased the staining of na-k atpase during the entire period of stimulation, and reduced immunopositivity of aquaporin (aqp) 1 h post infection but with an increasing trend in the all experimental steps. the investigation on cellular turnover showed a tendency to recover a regular tissue structure, as highlighted by an intense immunopositivity of proliferating cell nuclear antigen (pcna) from 24 h to 48 h post injection, as well as cell surface death receptor (fas) and the cysteine-aspartic acid protease (casp3) untilto 72 h post bacterial injection. thus, a detailed overview of the morphological and cellular responses in the mussel adductor muscle following infection with living bacteria was provided herein. involvement of exosomes in immune responses p pagliara, e carata, g m fimia, e panzarini, l dini dipartimento di scienze e tecnologie biologiche ed ambientali, università del salento, lecce, italy exosomes are small-secreted microvesicles interacting with surrounding cells and implicated in intercellular communication. besides their signaling function, these microvesicles also serve as a mechanism to dispose obsolete cellular material. exosomes transport proteins, lipids and nucleic acids in the form of mirna and mrna moreover, bearing cytokine or death receptors, they can bind to specific ligand of cells and induce signal transduction. every tissue fluid analyzed so far (milk, saliva, tears, urine, blood, etc.) has revealed the presence of exosomes and the range of organisms that release small vesicles, including exosomes, covers almost all known life forms. depending on the tissue of origin, they can regulate a large number of processes as brain development and function, tumor invasiveness and immune system function. about this last issue, it is already accepted that exosomes play a crucial role in host-pathogen interactions being produced during viral, parasitic, fungal and bacterial infections and could either promote or inhibit host immunity. just as exosomes can become agents of dissemination, they may otherwise act as agents of control within the body when unaffected. vesicles secreted by infected cells contain substantial amounts of pathogen molecules, which are sufficient to induce modifications in 52   non-infected neighboring cells or act as antigen presenters for the immune system. when antigen-presenting cells secrete exosomes bearing mhc bound to pathogen antigens, an activation of humoral and t helper responses occurs. studies on exosomes other than mammalian cells are scant, but still support the role of exosomes as a conserved mechanism of communication throughout evolution. the release of these microvesicles has been shown in some processes of invertebrate species (drosophila melanogaster, caenorhabditis elegans, hirudo medicinalis and crassostrea gigas) but their role in invertebrate immunity is poorly investigated. in this framework we searched for the presence of exosomes in the hemolymph of mytilus galloprovincialis, that could represent a good model in some biomedical research areas, such as inflammatory processes. by differential centrifugation, we isolated small microvesicles (10 100 nm, exosomes) and we started to study their release in naïve and lps-stimulated animals. their proteomic characterization and the investigation on their potential role in inflammatory response are in progress. sequence diversity and antimicrobial activity of myticalins m gerdol1, g leoni2, a de poli1, m mardirossian 1, pg giulianini1, p venier3, a tossi1, a pallavicini1 1department of life sciences, university of trieste, trieste, italy 2international school for advanced studies (sissa), trieste, italy 3department of biology, university of padua, padua, italy myticalins (mytilus cationic linear amps), first identified by an in silico transcriptome mining approach, represent the first family of antimicrobial peptides devoid of disulphide bridges ever reported in bivalve molluscs. while the protein precursors present conserved signal peptide and pro-peptide regions, they possess highly divergent mature peptide regions, which can be categorized within four subfamilies (a, b, c and d). in stark contrast with the other amps previously described in mussels, myticalins are not produced by circulating hemocytes, but they are mainly expressed in the gill tissue. following the demonstration of a significant antimicrobial activity by myticalin a4b, we selected and synthetized on solid phase six additional peptides, namely myticalin a8, b1, c6, c9, d2 and d5. minimum inhibitory concentration microbiological assays revealed that myticalins have a broad spectrum of activity, targeting both gram-positive and gram-negative bacteria, although the specificity and strength of action appears to be strictly dependent on the primary sequence of the peptide. in addition, we revealed that, in spite of the high number of sequence variants identified at the transcriptome level, just a limited number of myticalin loci are present in the genome of mytilus galloprovincialis. we investigated more in depth the sequence diversity of myticalin genes in a mussel population, highlighting that this amp family is characterized by presence/absence genetic variation. furthermore, we confirm that myticalins represent a taxonomically restricted gene family only found in mytilus spp. session 4. invertebrate immunity chairman: loriano ballarin, university of padova, italy telocytes in invertebrates n baranzini1, r girardello1, a grimaldi1, e ottaviani2, l pulze1, g tettamanti1, m de eguilleor1 1 department of biotechnology and life science, university of insubria, varese, italy 2 department of life science, university of modena and reggio emilia, modena, italy vertebrate telocytes (tcs) are stromal cells described in humans and rodens cavitary and non cavitary organs such as heart, gut, gallblader, uterus, lung, pancreas, mammary gland, skeletal muscles. telocytes, present in many types of tissues, are strategically located among specific resident cells, close to the capillaries and to the nerve endings. tcs are always characterized by a small cell body, containing the nucleus and very thin, long tubular processes named telopodes (tps) that are considered the ultrastructural peculiar trait of these cells. each telopode, long and convoluted is moniliform showing thin tubes (podomers) alternated with dilations (podoms). tcs may contact with each other and with virtually all type of cells forming a close network. the cross talk among tcs and the other cells can be direct via gap junctions, and/or indirect via the release of extracellular vesicles (multivescicular bodies, exosomes). tcs are hypothesized as an extensive intercellular information transmission system able to modulate homeostasis, stem cell activity, and other functions working as a primitive nervous system at the cellular level. our study shows the existence of these type of cells in invertebrate body wall and we firstly identified telocytes in medicinal leech body. an ultrastructural portrait and immunophenotypic characteristics as well as the possible role played by invertebrate telocytes has been proposed. first evidences of a complement system lytic pathway in invertebrates: data from the compound ascidian botryllus schlosseri a peronato, n franchi, l ballarin 53   department of biology, university of padua, padua, italy the complement system is well studied in mammals, where more than 30 proteins have been described, involved in the activation and regulation of this important humoral effector. however, the evolutionary history of the complement system is not yet fully elucidated and, in recent years, it has been widely demonstrated that complement system is more than just a defender against intruders. for instance, it is important for the clearance of apoptotic cells and corpses. botryllus schlosseri is a cosmopolitan ascidian, belonging to the phylum chordata, considered a model organism for the studies of the evolution of the immune system. studying the complexity of the complement system in botryllus, we identified a transcript, in our est collection, coding a protein containing the macpf (membrane attack complex/perforin) domain, shared by both most of the proteins involved in the lytic pathway and perforins. in vertebrates, we know that perforins are produced by t-lymphocytes and natural killer cells, whereas the activity of the proteins, with the macpf domain is regulated by the complement component c3/c5. comparing the domain’s topology of vertebrate c9 and our protein, called botryllus c9-like protein (bsc9), a high level of similarity results. to demonstrate that our c9-like protein can be considered a part of the complement system in b. schlosseri we evaluated the expression of bsc9 with respect to c3 activity. our previous data demonstrates that b. schlosseri c3 is activated by zymosan. we combined the microinjection of zymosan with and without a validated anti-c3 antibody (against human c3) to block the activity of c3. with this approach we studied in, time course, the expression of both bsc3 and bsc9 demonstrating that the anti-c3 antibody is able to inhibit the expression of bsc9. these results are confirmed in both ish and icc using the same antibody, and in vitro with the c3 inhibitor compstatin. in addition, a significant (p < 0.05) decrement of labeling with bsc9 antisense riboprobe and validated antibody anti hsc9 is observed when hemocytes are incubated with zymosan and compstatin. collectively, these results argue in favor of the presence of components of the lytic pathway in our model organisms. allograft inflammatory factor 1 (aif-1) is expressed in cns and hemocytes of the molluscan pest pomacea canaliculata and shows immune-related function a accorsi1,2, e ross1,2, j vizioli3, d malagoli4 1stowers institute for medical research, kansas city, mo, usa 2howard hughes medical institute, stowers institute for medical research, kansas city, mo, usa 3laboratoire prism (protéomique, réponse inflammatoire, spectrométrie de masse), u 1192 inserm, université lille 1, villeneuve d’ascq, france 4department of life sciences, university of modena and reggio emilia, modena, italy in mammals, allograft inflammatory factor 1 (aif-1), alias ionized calcium-binding adapter molecule 1 (iba1), is a 17 kda cytokine largely synthesized by macrophages during inflammatory responses. aif-1 is associated to activated microglia in vertebrates and its orthologues have been retrieved in microglial cells of the annelid hirudo medicinalis and in the hemocytes, digestive glands and gills of several molluscs. the analysis of the organ specific transcriptomes of the gastropod pest pomacea canaliculata revealed the presence of an aif-1like protein sharing the 49 56 % of identity with its vertebrate counterpart and a higher degree of identity with the aif-1 invertebrate orthologues. real-time pcr experiments indicated that pcaif-1 expression is detectable in all the tested organs, including hemocytes, digestive glands and gills, and the highest level of expression was retrieved in the pedal ganglia. twenty-four hours after the injection of 10 mg/ml of escherichia coli-derived lps, the pcaif-1 expression significantly increased in ganglia, digestive glands and gills. these observations suggest the involvement of pcaif-1 in the immune responses of p. canaliculata and point out a potential marker for microglial cells in one of the most invasive molluscan pests. first insights in the autophagic processes of mytilus hemocytes g camisassi1, t balbi1, k cortese2, r fabbri1, c ciacci3, c grande1, g. tassistro1, l vezzulli1, l canesi1 1dept. of earth, environment and life sciences (distav), university of genoa italy 2dept. of experimental medicine (dimes), university of genoa, genoa, italy 3dep. of earth, life and environment sciences (disteva), university of urbino,taly autophagy is a highly conserved and regulated catabolic process by which the eukaryotic cell degrades unnecessary, undesirable, or dysfunctional cellular components. the autophagic machinery is also used to remove invading intracellular pathogens and to direct them toward lysosomal degradation. therefore, autophagy represents an innate immune mechanism against microbial infection. in invertebrates, that lack acquired immunity, 54   autophagy will thus play a key role in the protection against potential pathogens. in aquatic molluscs, induction of autophagy by starvation and different environmental stressors has been demonstrated by different types of lysosomal reactions in the cells of the hepatopancreas. however, no information is available so far on the role of autophagy in the immune cells, the hemocytes. in this work, the possible role of autophagy in the response to bacterial challenge were investigated in the hemocytes of the marine bivalve mytilus. hemocytes were exposed to different strains of marine vibrios and functional responses were evaluated: lysosomal membrane stability (lms), lysozyme release, ros and no production, bactericidal activity. autophagy was investigated in the presence different specific inducers/ and inhibitors. the results show that in mussel hemocytes exposure to the autophagy inducer rapamycin induced a significant decrease in lms. a similar decrease was observed after challenge with strains of v. tapetis and v. corallyticus. in all experimental conditions, lysosomal destabilization was prevented by cell pretreatment with wortmannin, a potent inhibitor of class iii pi-3kinase, that plays a key role in autophagosome formation. although neither v. tapetis or v. corallyticus strains were able to induce activation of functional immune responses, no cytotoxic effects were observed. however, incubation with v. tapetis in particular resulted in rapid induction of autophagy, as clearly shown by transmission electron microscopy. the results show that in hemocytes determination lms may represent a simple and sensitive indicator of induction of autophagic processes. moreover, these data underline the possible role of autophagy in the protection of bivalve hemocytes towards the potential pathogenicity of certain vibrios. identification of nanoparticle-protein coronas in the hemolymph of marine bivalves: implications in particle recognition and immune response t balbi1, r fabbri1, m montagna1, a salis2, g damonte2, l canesi1 1department of earth, environment and life sciences (distav), university of genoa, genoa, italy 2centre of excellence for biomedical researchcebr, university of genoa, genoa, italy the development of nanotechnology will inevitably lead to the release of consistent amounts of nanoparticles (nps) in aquatic ecosystems. invertebrates are emerging as suitable models for evaluating the environmental impact of nps. however, np intrinsic properties, as well as those of the receiving medium, will affect particle behavior, bioavailability, uptake and toxicity. moreover, once within the organism, nps will interact with cells in a physiological environment, i.e. in biological fluids. in mammalian serum, nps associate with soluble components organized into a ‘‘protein corona’’, which affects particle interactions with target cells. the corona protein are particleand species-specific. the study on np-protein coronas in invertebrate systems is still at its infancy.  the protein composition of extracellular fluids of invertebrates is largely unknown, given the large diversity of phyla and species, and different proteins may be involved in the formation of np corona in different groups. in the hemolymph of the marine bivalve mytilus, the formation of a stable protein corona increases the immunotoxicity of amino-modified polystyrene nps  (ps-nh2). the putative c1q domain containing protein (mgc1q6) or extrapallial protein precursor, an immune-related acidic glycoprotein with high affinity for metal cations, was identified as the sole component of the ps-nh2 hard protein corona. these data represent the first demonstration of the identity of hard-corona proteins in the hemolymph of a marine invertebrate. since ps-nh2 retain their positive surface charge in both asw and hemolymph serum, studies were carried out with other types of nps (n-tio2 and n-ceo2) characterized by a net negative surface charge. the results show that both n-tio2 and n-ceo2 associated with extracellular sod (superoxide dismutase), the second most abundant protein in mussel hemolymph. in contrast, no stable association was found between hemolymph serum proteins and neutral/weakly charged nceo2. the results demonstrate that distinct hemolymph proteins are involved in the formation of a stable corona around different types of nps. these np protein interactions are particle specific, and strongly influenced by the net surface charge of the particle. the role of np-protein coronas formed in biological fluids of different invertebrate species, and their possible consequences in determining the impact of nps on innate immunity, is discussed. innate immune responses of european sea bass (dicentrarchus labrax) induced by dietary administration of vitamin d3 m dioguardi1, fa guardiola , m vazzana , a cuesta , m a esteban , m cammarata ,2 1 2 2 1 1marine immunobiology laboratory, department stebicef, conisma, university of palermo, palermo, italy 2fish innate immune system group. department of cell biology and histology faculty of biology, campus regional de excelencia internacional “campus mare nostrum”, university of murcia, murcia, spain 55   aquaculture the global production of fish, molluscs, crustaceans and other marine animals is an ever-growing industry. the major challenges on which its expansion depends comprise the diversification of farmed species, the improvement and optimization of nutrition and the control and prevention of diseases. one of the most important objective is the production of natural commercial diets with no adverse effects on the growth and health of the fish that guarantee high quality for consumers. with respect to the control and prevention of disease, the rapid development of aquaculture, in recent years, has led to the occurrence of diseases and problems associated with its intensive development, involving significant economic losses. a field of great interest is the application of stimulants of the immune system as additives in the diet to improve the efficiency of growth and to prevent and/or control of fish diseases. the effects of dietary administration of vitamin d3 on some innate immune parameters and on the expression of immune-related genes in head-kidney (hk) and gut were investigated in dicentrarchus labrax l. vitamin d3 (vd3) was orally administered to fish in a commercial pellet food supplemented with 0 (control), 3,750, 18,750 or 37,500 u kg-1of dry diet. furthermore, gut histology was also considered. this study showed a modulation in the activities examined in fish fed with the addition of vd3. just after 2 weeks of administration, diet supplementation with the vitamin resulted in an increase in phagocytic ability, while serum peroxidase content was increased in fish fed with all experimental diet after 4 weeks, no significant differences were observed in protease, anti-protease, natural haemolytic complement activities and total igm level. at gene level, fucose and rhamnose binding lectin transcripts were up-regulated in hk in fish fed with highest concentration of vd3supplemented diets after 4 weeks while in the gut was observed an up-regulation of hepcidin gene in fish fed with the different doses of vd . 3 these results suggest that vd3 may be considered of great interest for immunostimulatory purposes in fish farms.       review isj 8: 231-240, 2011 issn 1824-307x review immunomodulatory effects of tick saliva mi camargo mathias1, kc scopinho furquim2, ph nunes1 1department of biology, biosciences institute, unesp, av. 24 a, nº 1515, cx. postal 199, cep: 13506-900, rio claro, s.p., brazil 2department of veterinary pathology, fcav, unesp, via de acesso prof. paulo castellane, s/n, cep: 14884900, jaboticabal, s.p., brazil accepted december 6, 2011 abstract ticks are bloodsucking ectoparasites that cause great damage to host organisms, so these ectoparasites are of great importance in medicine and veterinary medicine. all the biological success achieved by ticks is due to the action of bioactive components present in their saliva, which are synthesized by the salivary glands. these components have great diversity of functions such as enabling feeding and the permanence of ectoparasites on hosts, since they modulate immune system acting as complement inhibitors, immunosuppressors, cytokine expression modulator and chemokine binders of hosts. in addition, these components are an important source of protective antigens. in this sense, salivary glands/saliva are considered a potential source of multifunctional molecules. in this context, many studies have been conducted aiming at searching to establish a better understanding on the biology and morphophysiology of some organs such as salivary glands, as well as elucidate the complex relationship of these ectoparasites with their hosts. such studies are conducted with the main objective of developing new immunobiological products aimed at the alternative control of ticks, as well as for the identification and isolation of bioactive molecules with pharmacological properties and with great therapeutic potential in the search for treatments for some diseases. key words: tick saliva; immunomodulation; immunosuppression; salivary glands introduction ticks are bloodsucking ectoparasites that can parasitize animals of different groups such as mammals, birds, reptiles, amphibians (keirans and durden, 2005; anderson and magnarelli, 2008) and even spiders, including ticks themselves (labruna et al., 2007). these ectoparasites have broad geographic distribution, and can be found in all regions of the planet (keirans and durden, 2005; anderson and magnarelli, 2008). ticks are animals belonging to the phylum arthropoda, subphylum chelicerata, class arachnida, subclass acari, order parasitiformes and suborder ixodes. these ectoparasites are divided into four families, namely: ixodidae, argasidae, nutalliellidae and laelaptidae, the first two being the most important, totaling 878 species (anderson and magnarelli, 2008). ___________________________________________________________________________ corresponding author: maria izabel camargo mathias department of biology biosciences institute unesp, av. 24 a, nº 1515 cx. postal 199, cep: 13506-900, rio claro, s.p., brazil e-mail: micm@rc.unesp.br ticks are arthropods of great medical and veterinary importance because they are vectors of bacteria, viruses, protozoa and helminthes to their hosts, affecting domestic animals, wildlife and humans (jongejan and uilemberg, 2004; dantastorres et al., 2010), besides causing considerable economic losses. considering that ticks remain attached to their hosts feeding for weeks, they must have a large, diverse and effective pharmacological arsenal to ensure their survival and feeding (wikel, 1999, sauer et al., 2000; francischetti et al., 2005; steen et al., 2006), which can be rapidly introduced into the host. thus, throughout evolution, salivary glands have specialized in the production of a secretion called saliva, which has such capability. the present review will focus only on immunomodulatory effects of tick saliva. salivary glands of ticks the salivary glands of ixodid (family ixodidae) are vital organs for the biological success of this group, since they have wide variety of functions such as the production of substances necessary for 231 the fixing and feeding of ectoparasites (binnington, 1978; walker et al., 1985; gill and walker, 1987). the presence of these organs and their role in hosts to give these ectoparasites the status of one of the most important groups within the group of arthropods. their salivary glands are also responsible for the transmission of infectious agents to other groups of animals (balashov, 1983). morphologically, salivary glands of ixodid ticks are formed by an excretory portion composed of a common excretory duct, intermediate ducts and acinar ducts responsible for collecting and secretion of saliva produced in the secretory portion. the latter consists of types i, ii, iii and iv acini, the latter present only in males (binnington, 1978; walker et al., 1985; fawcett et al., 1986; gill and walker, 1987; sonenshine, 1991; furquim et al., 2008a, b, 2010). type i acini are nongranular and act in the water balance of the ectoparasite, while type ii, iii and iv acini are granular and act in the feeding process (binnington, 1978; walker et al., 1985), in osmoregulation in the phase of large blood consumption (kaufman and sauer, 1982; sonenshine, 1991) and in reproduction (feldmanmuhsam et al., 1970). the salivary glands of ticks have no reservoir to store secretion, which is released as soon as it is produced (binnington, 1978; balashov, 1983; walker, 1985; nunes et al., 2008). type i acini consist of a large central cell, surrounded by several smaller peripheral cells (binnington, 1978; walker et al., 1985; fawcett et al., 1986; gill and walker, 1987). type ii acini are formed by types a, b, c1, c2, c3 and c4 secretory cells (binnington, 1978) and in females of r. sanguineus, and in addition to those, c5 and c6 cells have been recently described (furquim, 2007). it is known that a cells are involved in the secretion of the cement (binnington, 1978; walker et al., 1985; fawcett et al., 1986; gill and walker, 1987), and b and c are involved with the manipulation of the host response (binnington, 1978; walker et al., 1985). type iii acini, in turn, are composed of three cell types, d, e and f (binnington, 1978; walker et al., 1985; gill and walker, 1987; furquim, 2007), where d and e secrete components of the cement during attachment (binnington, 1978; walker et al., 1985; gill and walker, 1987), and f cells secrete substances related to the consumption of blood by the ectoparasite (binnington, 1978). the new classification of the salivary gland cells of rhipicephalus sanguineus established by furquim (2007) included a larger number of secretory types present in type ii acinus (females), unlike those previously described in literature (binnington, 1978, walker et al., 1985). moreover, the author has shown that these new types (c5 and c6), and f of type iii acini would be activated and inactivated at specific moments of the glandular cycle (when the tick had already fed for two and four days), while the other types would remain continuously active until the complete engorgement of the female, becoming inactive, beginning the death process (furquim et al., 2008a, b). this was evidence that the activity of c5, c6 and f cells would be essential for feeding and permanence of ticks in the host, therefore acting specifically in the manipulation of their immune response. type iv acinus, exclusive to males, consists of a single cell type, g (binnington, 1978; furquim et al., 2008a, 2010). in some ixodid, its product participates in the secretion of the cement (fawcett et al., 1986) and can also produce other secretions important in the transfer of the spermatophore to the female (feldman-muhsam et al., 1970). functions of the tick saliva the tick saliva is a mixture that plays a variety of roles during parasitism and non-parasitism periods, as: modulating the immune system of the host (fawcett et al., 1986; ribeiro et al., 1985; wikel, 1999; sauer et al., 2000); attaching the tick to the host skin through the secretion of cement to form the cone (fawcett et al., 1986); excreting excess water and ions from the food (blood) (sauer et al., 2000); secreting hygroscopic solution, which is deposited in the mouth region and absorbs atmospheric water, hydrating the ectoparasite during non-parasitism periods (sauer et al., 2000; bowman and sauer, 2004); producing secretions that lubricate the spermatophore during its transfer to the female during copulation (feldman-muhsam et al., 1970); releasing toxins that cause paralysis in the host (fawcett et al., 1986) and conveying pathogens to the host (fawcett et al., 1986; wikel, 1999; sauer et al., 2000; bowman and sauer, 2004). immunomodulation of host immune responses given the importance of components synthesized by type ii and iii acini for the feeding and fixing of ticks (binnington, 1978; walker et al., 1985), these components should be studied in more depth (jittapalapong et al., 2008). proteins, glycoproteins, lipoproteins and lipids are among products with pharmacological and immunological properties present in the saliva of ticks (binnington, 1978; wheeler et al., 1991; shipley et al., 1993), such as acid phosphatase, esterases, aminopeptidases, metalloproteases, calreticulin, esterase, prostaglandins, lipocalins (binnington, 1978; gill et al., 1986; jaworski et al., 1995; brossard and wikel, 2004; steen et al., 2006; harnoi et al., 2007; mulenga et al., 2007; kaewhom et al., 2008; konnai et al., 2010; oliveira et al., 2011), among many others. although the function of the cement of keeping the ectoparasite attached to the host has been widely reported, this substance has proteins that act both in the host modulation and in the formation and maintenance of the feeding lesion (mulenga et al., 2007). in this sense, the chemical composition of the cement consists of a mixture of antigenic and non antigenic proteins containing carbohydrates and lipids in the inner layers of the cone, the outer layers being formed by lipoproteins and glycoproteins (sonenshine, 1993). binnington (1978) studied the salivary glands of females of boophilus microplus and demonstrated the production of: a) glycoproteins by b, c1, c2, c3 cells of type ii acinus, and f cells of type iii acinus, b) acid phosphatase by the three types of acini, 232 mainly by type i acinus and by d cells of type iii acinus , c) protease by cell of type ii acinus, d) esterase by cells of all types of acini, especially b and c cells of type ii acini and e) lipoproteins by a cells of type ii acinus and d and e cells of type iii acinus. the tick saliva is a complex and sophisticated pharmacological arsenal effectively interacting with elements of the immune-inflammatory and hemostatic system of the host that can hold its defenses quickly since the first days of ectoparasite feeding (ribeiro et al., 1985; wikel, 1989; brossard and wikel, 1997, 2004; francischetti et al., 2005, steen et al., 2006). moreover, the tick saliva immunosuppresses the host, making these ectoparasites skilled transmitters of pathogens that are transmitted through their own saliva (wikel, 1999; singh and girschick, 2003; kovár, 2004). in this sense, the saliva of ticks plays vital functions in the hemostatic, inflammatory and immune processes of the host by: a) increasing blood flow (circulation) in the bite region through the secretion of vasoactive agents; b) inoculating anticoagulants that keep the host’s blood in the fluid form; c) inhibiting the inflammatory process in the host d) immunosuppressing the host and enabling the attachment of ticks, making their rejection by the host difficult (sauer et al., 2000). the following elements present in the saliva of ticks with pharmacological and immunological properties stand out: a) enzymes (apyrase, kininase), enzyme inhibitors, b) proteins homologous to host proteins, c) proteins that bind to immunoglobulins, d) lipocalin that bind to amines, e) agonists / antagonists of receptors, f) calciumcarrier elements (calreticulin), g) cytokine expression modulators, and h) other non-protein bioactive components such as prostaglandins (pge2) (steen et al., 2006; brossard and wikel, 2004). the action of the tick saliva is a complex process, since this mixture has quantitative and qualitative variations due to the different glandular cycle periods within the same species (binnington, 1978; mcswain et al., 1982; sauer et al., 1982; shipley et al., 1993; sanders et al., 1996; furquim, 2007; mulenga et al., 2007; xiang et al., 2009) or when considering different species (kazimírová, 2007). in addition, another important factor that contributes to the heterogeneity of the tick saliva and that should be considered is the fact that the immunereactive activity of tick-derived factors may vary depending on the host species parasitized (lawrie et al., 1999). harnnoi et al. (2007) demonstrated the occurrence of the expression of six types of metalloproteases along the glandular cycle of haemaphysalis longicornis. in addition, oliveira et al. (2011) detected the presence of molecules of non protein origin, prostaglandin (pge2) and purine nucleoside adenoside (ado), with strong immunomodulatory properties in the saliva of r. sanguineus and found that isolated molecules have different ways of action, whereas when combined, their action becomes enhanced. moreover, some elements produced by the salivary glands of ticks are likely to have more than one biological activity, for example, molecules that inhibit coagulation and enhance vasodilation contribute to the formation of fixation and feeding lesion, anti-inflammatory and immunosuppressive molecules those reduce host defenses impairing the feeding of ticks (wikel, 1996). the bioactive molecules present in the saliva of ticks of different species and genera are: apyrase, which inhibits platelet aggregation through the hydrolysis of atp and adp into amp and orthophosphate (titus and ribeiro, 1990; kazimírová, 2008), and prevents neutrophil aggregation and degranulation of mastocytes (ribeiro et al., 1985). prostaglandins (pgs), found in high concentrations in the saliva of some species of ticks (bowman et al., 1996) such as prostaglandin e (pge ), with anti-hemostatic, vasodilator and immunosuppressive activity (bowman et al., 1996), which inhibit platelet aggregation and cause vasodilation (champagne, 1994; ribeiro et al., 1985; kazimírová, 2008), suppress the production of interferon (ifn)-γ (hasler et al., 1983; betz and fox, 1991) and interleukin (il)-2 (hasler et al., 1983; betz and fox, 1991; krause and deutsch, 1991) and inhibit the bioactivity of il-2 in cells that dependent on this cytokine by reducing the expression of il2 receptors in these cells (krause and deutsch, 1991) thus, prostaglandins can inhibit the function of t lymphocytes (singh and grischick, 2003), and in ixodes dammini, inhibit il-2 production by these lymphocytes (ribeiro and spielman, 1986); prostacyclin (pgi ), found in the saliva of i. dammini, which blocks platelet aggregation, inhibits degranulation of mastocytes and induces vasodilation (ribeiro and spielman, 1986; kazimírová, 2008). elements that in dermacentor andersoni act in factors v and vii inhibiting intrinsic (activated tissue factor) and extrinsic pathways (activated collagen) of the blood coagulation cascade (gordon and allen, 1991). peptides found in the saliva of ornithodoros savigny also inhibit the extrinsic pathway of the coagulation cascade (ehebauer et al., 2002). variegin found in the saliva of amblyomma variegatum, which is a thrombin inhibitor (koh, 2007). anti-coagulant, which in r. appendiculatus inhibits the activity of factor xa or other components of the prothrombinase complex (limo et al., 1991). another factor xa inhibitor, tap protein, was found both in the saliva of o. moubata (waxman et al., 1990) and in o. savigny (joubert et al., 1998). kininase, found in i. dammini, has the capacity to act in the cleavage of bradykinin (enhanced by pge 2) (ribeiro et al., 1985) and reduce skin irritation (itching) caused by this substance, leading to reduced removal of ticks by self-cleaning mechanism through licking. molecules with antichemokine activity in adult ticks of the species r. sanguineus, which act against human chemokines cxcl8, ccl2, ccl3, ccl5, and ccl11 (vancová et al., 2010). according to these authors, males of i. ricinus have molecules that act against these chemokines, while in females of the same species, no activity against chemokines ccl5 and ccl11 was detected (vancová et al., 2010). elements that bind to growth factors transforming them. in a. variegatum, these elements react with growth 2 2 2 233 factor-β (tgf-β1), platelet-derived growth factor (pdgf), fibroblast growth factor (fgf-2) and hepatocyte growth factor (hgf), in d. reticulatus and r. appendiculatus with tgf-β1, fgf-2 and hgf and in i. ricinus and i. scapularis with pdgf (hajinická et al., 2010). peptides found in i. scapularis are potent inhibitors of the complement system (tyson et al., 2007). calcium-binding proteins (calreticulins), whose functions in the parasite-host relationship would facilitate feeding through their actions in the immune system, immunosuppressive and antihemostatic (jaworski et al., 1995; brossard and wikel, 2004; jittapalapong et al., 2008; steen et al., 2006). lipocalins, present both in ixodid and argasids, act in the modulation of the immune response by inhibiting platelet (keller et al., 1993) or complement aggregation (nunn et al., 2005; mans and ribeiro, 2008a), inflammation through association with histamine, serotonin, leukotrienes c4, d4, e4 (paesen et al., 1999; sangramnatdej et al., 2002; mans and ribeiro, 2008b; mans et al., 2008c) and induction of toxicoses (mans et al., 2001, 2002, 2003). metalloproteases detected in the saliva of i. scapularis with anti-fibrinogen activity (francischetti et al., 2003). peptides that form and maintain feeding lesion (mulenga et al., 2007). elements that are potent inhibitors of angiogenesis and proliferation of endothelial cells (francischetti et al., 2005). anti-inflammatory proteins that bind to lipocalin and histamines (mulenga et al., 2007). also with regard to the biochemical and functional complexity of the saliva, ribeiro (1987) reported that the pharmacology of tick saliva can adapt to specific homeostatic defenses of the host. in this sense, furquim et al. (2011) demonstrated that the secretory behavior of the salivary glands of females of r. sanguineus is modified according to the resistance of the host through immunization. these authors found that the glandular secretion of these females showed increased amounts of many components such as proteins, lipids, polysaccharides, acid phosphatase and calcium (probably calreticulin), which enhanced the pharmacological action and biochemical complexity of the saliva produced by them, thereby modifying its action on the parasite-host relationship. resistance acquisition of hosts in ticks, the salivary glands are important sites for producing antigens (wikel et al., 1978; gill et al., 1986; almeida et al., 1994; ferreira et al., 1996; szabó and bechara, 1997; jittapalapong et al., 2000a; nunes et al., 2011). therefore, after the first contact of these ectoparasites with some hosts, the latter develop resistance (wikel et al., 1978; gill et al., 1986; jittapalapong et al., 2000a, zhou et al., 2006). in this sense, many studies have been carried out to verify the acquisition of resistance by hosts when they are immunized by successive infestations (jittapalapong et al., 2000a, b; monteiro et al., 2008, 2011; caperucci et al., 2009, 2010; veronez et al., 2010; nunes et al., 2011) or by inoculation of extracts made of whole ticks (or parts of them) (wikel, 1981; ferreira et al., 1996; szabó and bechara, 1997; jittapalapong et al., 2000a, b, 2008). in relation to the modulating capacity of tick saliva, it is known that this mixture acts on hosts affecting the proliferation of lymphocytes (kopecky et al., 1999), decreasing the oxidative activity of macrophages (kuthejlova et al., 2001), inhibiting neutrophils, including their oxidative and phagocytic activity (ribeiro et al., 1990), inhibiting the activity of "natural killer-nk" cells (kubes et al., 1994), reducing the development of the primary igm response (fivaz, 1989; wikel, 1985), which remains evident for a few days after the infestation (wikel, 1985), and decreasing cytokine production by th1 lymphocytes (il-2 and ifn-γ) and cytokines of macrophages, while increasing the production of cytokines by th2 lymphocytes (il-4, il-5 and il-10) (wikel, 1999), in order to influence the class of immunoglobulin produced, causing the host to the preferential production of igg1 and ige immunoglobulins instead of igg2 (wikel, 1996). cytokines il-2 and ifn-γ are vital to the development of an effective immune response in the attachment and feeding lesion of the tick, including the recruitment, activation and proliferation of immunocompetent cells, thereby mediating an inflammatory response to the ectoparasite (singh and grischick, 2003). the acquisition of resistance by hosts is measured analyzing food and reproductive parameters of ticks infesting animals previously immunized (wikel, 1981; szabó and bechara, 1997; jittapalapong et al., 2000b), as well as analyzing the impact of this resistance on the salivary glands (sanders et al., 1996; jittapalapong et al., 2008; furquim et al., 2011; nunes et al., 2011) and intestine (caperucci et al., 2009, 2010; veronez et al., 2010) of different species. according to wikel (1981) and jittapalapong et al. (2000b, 2008), immunization obtained from antigens derived from salivary glands would stimulate the host immune response that would affect ticks of subsequent infestations due to the direct action of resistance acquired by the host in the secretory cycle of the salivary glands, reducing the efficiency of the feeding process and pathogen transmission by ticks (jittapalapong et al., 2008). considering that ticks are able to modulate local hemostatic reactions in the host, this ability can be affected by the immune status (resistance) of the host. in resistant hosts, large amounts of inflammatory cells are recruited and the expression of anti-coagulant molecules in the salivary glands of ticks is reduced, and this glandular alteration may hinder the consumption of blood by ticks (carvalho et al., 2010). recent studies have shown that the saliva of different tick species, among them r. sanguineus, modulates different steps of the biology of dendritic cells, such as maturation and migration (oliveira et al., 2008, 2010). anticoagulant proteins have been identified in the saliva of ticks such as boophilina (obtained from the saliva of b. microplus) (macedoribeiro et al., 2008) and ixolaris and penthalaris (obtained from the saliva of i. scapularis) (francischetti et al., 2002, 2004). according to turner et al. (2002), much information can be obtained on the resistance 234 table 1 examples of bioactive components in tick saliva tick species molecule name target/ function reference ticks in general salp15 (anti-alarmin) chemotactic properties of chemokines marchal et al., 2010 salp 25 antioxidant kovar, 2004 mif homologue (cytokine homologue) inhibits the migration of human macrophages similarly of humam mif kovar, 2004 ixodidae hbps (lipocalins with 1 or 2 binding sites) suppress inflammation by hystamin or serotonin binding kovar, 2004 hyalomma asiaticum bif inhibits lps-induced proliferation of b-cells yu et al., 2006 ixodes tslpi lectin pathway inhibitor schuijt et al., 2011 pgs inhibits lymphocytes l2 ribeiro and splielman, 1986 isac alternate complement pathway, interacts with c3 convertase valenzuela et al., 2000 salp20 c3 convertase tyson et al., 2007 salp15 impairs il-2 production and tcell proliferation; binds borrelia burgdorferi ospc, protects the spirochete from antibodymediated killing ramamoorthi et al., 2005 il-2 binding protein inhibits proliferation of human tcells and ctll2 cells gillespie et al., 2001 ixodes scapularis sialostatin l inhibits cathepsin l activity kotsyfakis et al., 2006 bip inhibitorof b-cell proliferation hannier et al., 2004 iris modulates t-lymphocytes and macrophage responsiveness, induce t-h2 type responses; inhibitor of homeostasis leboulle et al., 2002 ixodes ricinus irac i, ii, isac paralogues alternate complement pathway, interacts with c3 convertase daix et al., 2007 ornithodoros moubata omci c5, prevention of interaction c5 with c5 convertase nunn et al., 2005 evasin-1 binds chemokines ccl3, ccl4 and ccl18 frauenschuh et al., 2007 rhipicephalus sanguineus pge2 modulations of dcs oliveita et al., 2011 235 acquired by hosts sensitized to different tick species or even to the same species, because immunosuppressive molecules synthesized by the salivary glands are differentially expressed during tick feeding. in addition, the salivary glands of different species have different antigens, in quantities and concentrations also different (jaworski et al., 1990; inokuma et al., 1994). data are summarized in table 1. therapeutic properties and prospects of tick saliva the saliva of ticks has several types of molecules that modulate the immune-inflammatory and hemostatic system of their hosts (wikel and bergman, 1997; steen at al., 2006; hajnická et al., 2011). this complex mixture is considered to be a potential reservoir of multifunctional molecules, i.e., the saliva of these ectoparasites, as well as the saliva of hematophagous organisms in general has bioactive compounds of great interest (batista et al., 2010). thus, tick saliva has been the subject of different studies due to the great interest in the identification and isolation of bioactive molecules with vasodilating, anti-inflammatory, immunosuppressive and anticoagulant activity (oliveira et al., 2010). the tick saliva has presented mitigating action of angiogenesis (francischetti et al., 2005), as well as anti-tumor properties (simons et al., 2011). in relation to the bioactive molecules present in the saliva of ticks, the evasin-1, a chemokine binding protein is used in the idiopathic pulmonary fibrosis therapy (russo et al., 2011) a protein recently found in the saliva of a. cajennense that acts in the coagulation process, also showed cytotoxic activity in different tumor cells, among them pancreatic cells and melanomas (chudzinskitavassi et al., 2010). the effects of crude saliva are also promising, since it triggered cell death and morphological changes in human melanoma cells (sk-mel-28) and human pancreatic carcinoma (mia paca-2), without causing changes in normal human fibroblasts exposed to it (simons et al., 2011). previous results have shown that molecules present in extracts of the salivary glands of r. appendiculatus and a. variegatum were able to hold the growth of human hela cells (cervical cancer cells) through an apoptotic process (kazimírová et al., 2008). thus, ticks seem to be a source of bioactive molecules with great potential for the treatments for some diseases, including cancer, because as already mentioned, among the known species of ticks, as well as among genera, there are differences in bioactive compounds present in their salivary glands (kazimírová, 2008). moreover, the acquisition of resistance by the host can affect the biochemical composition of the salivary glands / saliva of ectoparasites that feed on them. this change becomes an additional factor that contributes to the quantitative and qualitative variation of bioactives found in the saliva of ticks (furquim et al., 2011). references almeida apg, bechara gh, varma rmg. crossreactivity between hard tick antigens. braz. j. med. biol. res. 27: 697-707, 1994. anderson jf, magnarelli la. biology of ticks. infect. dis. clin. north. am. 22: 195-215, 2008. balashov, yus. an atlas of ixodid tick ultrastructure. in: raikhel as, 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surface: in vitro experiment using hemocytes of the colonial ascidian botryllus schlosseri l ballarin1, n franchi1, f gasparini1, f caicci1, a miyauchi2, e hirose1, 3 1department of biology, university of padua, padua, italy 2hitachi research laboratory, hitachi ltd., omika, hitachi, ibaraki 319-1292, japan 3faculty of science, university of the ryukyus, nishihara, okinawa 903-0213, japan accepted february 23, 2015 abstract nano-scale nipple array on the body surface has been described from various invertebrates including endoparasitic and mesoparasitic copepods, but the functions of the nipple array is not well understood. using the hydrophilized nanopillar sheets made of polystyrene as a mimetic material of the nipple arrays on the parasites’ body surface, we assayed the cell spreading and phagocytosis of the hemocytes of the colonial ascidian botryllus schlosseri. on the pillared surface, the number of spreading amebocytes and the number of phagocytizing hemocytes per unit area were always smaller than those on the flat surface (mann-whitney test, p < 0.05 0.001), probably because the effective area for the cell attachment on the pillared surface is much smaller than the area on the flat sheet. the present results supports the idea that the nipple array on the parasites' body surface reduces the innate immune reaction from the host hemocytes. key words: hemocyte; innate immunity; nanopillar sheet; nipple array; endoparasite   introduction the surface structure of integumentary tissue influences its interaction with various factors in external environment. the moth-eye corneal nipple array is one of the most well-known examples, where the refractive index gradient reduces eye glare to decrease visibility to predators (e.g., bernhard, 1967; stavenga et al., 2006). similar surface structures have been described in aquatic or parasitic invertebrates such as annelids (hausen, 2005), entoprocts (iseto and hirose, 2010; nielsen and jespersen, 1997), echinoderms (holland, 1984), and tunicates (hirose et al., 1997, 1999). the size of nipples is almost uniform in each species but various among species: 150 20 nm in height in ascidians (hirose et al., 1997). although these nipple arrays may have various functions, it is often difficult to examine the functions using live materials. the development of nanoimprint technology has enabled the fabrication of polymer sheets with nanoscale structures such as nanopillars (kuwabara and miyauchi, 2008). using nanopillar sheets as a mimetic model for the nipple arrays, we can examine the properties of the nipple arrays in various experimental conditions, and we recently ___________________________________________________________________________ corresponding author: euichi hirose faculty of science, university of the ryukyus nishihara, okinawa 903-0213, japan e-mail: euichi@sci.u-ryukyu.ac.jp demonstrated the bubble repellency and anti-glare property of the nipple array in the water (hirose et al., 2013; hirose et al., in press). this biomimetic approach can contribute not only for better understanding the functions of biological structure but also for technological application of the functions. nipple array was also found on the cuticle surface of the body in some endoparasitic and mesoparasitic copepods infesting marine fish and invertebrates (østergaard and bresciani, 2000; hirose and uyeno, 2014). depending on the species, the size of each nipple is quite various: from1 µm to 20 nm in height. for these parasites, neither anti-glare nor bubble repellence would be important functions, since the parasite's body is enveloped in the host tissue. nomura et al. (2005) showed that the hela cells do not spread on the synthetic nanopillar sheet, and thus, the pillared surface may have suppressive effects on cell attachment of the host organisms. therefore, the nipple array on parasitic organisms may have protective function against the immunocytes of the host organisms. to test this hypothesis, hemocytes from a marine invertebrate was incubated on the nanopillar sheets as a mimetic model of the nipple array on the parasite integuments. ascidians are sessile, marine invertebrates and they have various endoparasites, including notodelphyid copepods (e.g., monniot, 1990). although integumentary ultrastructures were rarely 82 fig. 1. giemsa-stained hemocytes of b. schlosseri on hydrophilized polystyrene flat (a) and pillared (b) sheets. arrows indicate spreading hemocytes. scale bars = 10 µm. investigated, tiny nipple arrays were found in an endoparasitic copepod (uyeno, unpublished). as a primitive chordate, the colonial ascidian botryllus schlosseri is one of the model animals among marine invertebrates for studies on innate immunity and colonial allo-recognition (ballarin, 2008). a common vascular system is anastomosed within the colony, and several types of hemocytes circulate throughout the colony. to date, seven types of four categories of hemocytes were described from b. schlosseri and characterized morphologically and cytochemically for the functional characterization (ballarin and cima, 2005), and many of these hemocytes are involved in innate immunity and colonial allo-recognition (sabbadin et al., 1992; ballarin, 2008, 2012). moreover, immunocytotoxity to the botryllus hemocytes was utilized for the assessment of the toxic effects of marine pollutants (e.g., ballarin and cima, 2001, cima et al., 2008). in b. schlosseri, there are two types of amebocytes, i.e., hyaline amebocytes and granular amebocytes, that are respectively involved in phagocytosis and cytotoxity (ballarin and cima, 2005). the granular amebocytes are precursors of morula cells that are the major effector cells releasing phenoloxidase against foreign cells, such as bacteria and yeasts, and in the allogeneic rejection reactions (ballarin et al., 1995, 2005). in another colonial ascidian aplidium yamazii, phagocytic amebocytes are involved in the formation of cell aggregation to reject allogeneic colonies (ishii et al., 2008). to dates, it is uncertain whether the botryllid ascidians have endoparasites with nipple arrays or not, due to the lack of studies. nevertheless, b. schlosseri is convenient marine invertebrates to study the potential function of the nipple arrays reducing the immune responses of the host organisms, since the hemocytes involved in innate immunity has been well documented as mentioned above. in this study, we compare the cell spreading and phagocytic activity of the botryllus hemocytes on flat and nanopillared surfaces to elucidate the potential function of the nipple array on parasite integument to immune responses from the host animals. materials and methods animals colonies botryllus schlosseri from the venetian lagoon were attached on glass plates, reared in aerated aquaria in the laboratory and fed with liquifry table 1 the number of spreading amebocytes per optical field* trials cell density (× 106 cells/ml) flat sheet average ± sd pillared sheet average ± sd statistic difference# 1 1.5 1.77 ± 1.12 0.90 ± 0.83 p < 0.01 2 1.0 1.90 ± 1.51 1.10 ± 1.25 p < 0.05 3 2.1 2.40 ± 1.56 1.40 ± 1.17 p < 0.05 4 1.4 1.83 ± 1.13 1.00 ± 0.93 p < 0.01 5 1.5 2.13 ± 1.20 1.10 ± 0.94 p < 0.001 *averages are based on the cell counts in 30 optical fields for each. #mann-whitney test. 83 marine (liquifry co., dorking, england). the blastogenetic cycle of each colony was checked under a stereomicroscope, because the composition of hemocyte types changes depending on the phase of the cycle (ballarin et al., 2008). hemocyte collection for hemocyte collection, we used the colonies in which the combination of the developmental stages of zooid and buds was 9/8/2, i.e., phase b (see watanabe, 1953; sabbadin, 1955; manni et al., 2007). one to three systems containing 10 30 zooids were immersed in filtered seawater (fsw) containing 0.38 % na-citrate (ph 7.5) to prevent hemocyte clustering. the tunic vessels and zooids in the colonies were punctured with fine tungsten needles and hemocytes leaked from the colonies were collected in a micro tube on ice and incubated for 5 min to allow debris sedimentation. the supernatant was then transferred to another micro tube and centrifuged at 780 g for 10 min. pellets were re-suspended in fsw at 1 2.2×106 cells/ml. cell concentration was determined using a bürker hemocytometer. two hundred µl of hemocyte suspension were spread over on a polystyrene sheet (20×20 mm) that was hydrophilized by oxygen plasma etching (exam, shinko seiki) to imitate an organic surface. each sheet had a flat surface or arrays of 1-µm-high pillars of 0.5 µm diameter at 1 µm interval between the centers of the neighbor pillars (kuwabara and miyauchi, 2008). the hemocytes on the sheet were left to adhere to the polystyrene sheets for 30 min at room temperature. number of spreading amebocytes hemocytes on the sheets were fixed in fsw containing 1 % glutaraldehyde and 1 % sucrose for 20 min at room temperature, washed in distilled water and stained with 10 % giemsa’s solution for 10 min. the polystyrene sheets were mounted with distilled water on glass slides and the giemsa-stained hemocytes were observed under an olympus cx31 light microscope equipped with a ccd camera (infinity 2, lumenera corp.). the number of the spreading hemocytes was counted in 30 optical fields, at the magnification of 400x, for each sheet. one optical field was about 120×160 µm. the hemocytes having pseudopodia (e.g., filopodia and lamellipodia) were regarded as the spreading amebocytes. we made five replicates using different clones of colonies. these spreading amebocytes potentially include two cell types: hyaline amebocytes and granular amebocytes that are precursors of macrophage-like cells and morula cells, respectively (ballarin and cima, 2005). here, we did not distinguish the two types, because they were often difficult to be distinguished particularly on the pillared surface. the detachment of the amebocytes was also tested preliminarily. a sheet with hemocytes was gently dipped in fsw for six times before fixation, and the number of spreading hemocytes on the sheet was determined as reported above so to compare the cell number on the same type of sheet without dipping. the number of all types of hemocytes was also counted in 30 optical fields. phagocytosis activity ordinary baker's yeast (saccharomyces cerevisiae) was suspended in fsw at a concentration around 1.5×107 cells/ml and 200 µl of the yeast suspension were added on the flat and pillared sheet on which hemocytes were attached. after 1-h incubation at room temperature, the sheets were dipped in fsw to remove free yeast cells, and the hemocytes were fixed as described above. following giemsa’s staining, the number of hemocytes containing yeast cells was counted in 20 optical fields at the magnification of 400x for each sheet. we made four replicates: a pair of duplicate tests using the same colony and two independent tests using different clones of colonies. statistics the student t-test was used to compare the mean cell number in pillared and flat sheets or in the sheets with and without dipping, when the normality of the data distribution could be assumed, passing the kolmogorov-smirnov test. the mann-whitney test was used when the normal distribution was not supported. we performed the analyses using instat software (v. 3.1a, graphpad software, 2004). scanning electron microscopy the sheets attaching the hemocytes were fixed in 2.5 % glutaraldehyde in 0.1 m cacodylate buffer containing 1.7 % sodium chloride (ph 7.4) on ice. the specimens were dehydrated through an ethanol series and dried at critical point. the sheets were cut into small pieces and sputter coated with gold-palladium. the hemocytes on the sheet were examined with a jeol jsm-6060lv scanning electron microscope at 15 kv. results number of spreading amebocytes hemocytes of b. schlosseri adhered to the hydrophilized polystyrene sheet regardless of the presence of nanopillars. among the various types of the hemocytes on the sheets, amebocytes spread their cytoplasm and extended the pseudopodia on the flat sheet (fig. 1a) and the pillared sheet (fig. 1b). the number of these spreading amebocytes per optical field (ca. 120×160 µm) was always larger on the flat sheets than on the pillared sheets (table 1). significant differences were shown in the all five replicates using different clones of colonies (mann-whitney test, p < 0.05 0.001). dipping the sheets in fsw had no significant effect on the detachment of the spreading amebocytes on the pillared sheet (mann-whitney test, p > 0.05): cell numbers per optical field were 1.2 ± 1.34 (average ± sd) on the dipped sheet and 1.1 ± 0.96 on the control (no dipping). similarly, there was no significance on the flat sheet (mann-whitney test, p > 0.05): cell numbers per optical field were 1.57 ± 1.2 on the dipped sheet and 2.13 ± 1.22 on the control. however, the number of all cell types on the pillared surface was significantly decreased by dipping (mann-whitney test, p < 0.01): 7.63 ± 2.10 on the dipped sheet and 10.8 ± 2.83 on the control. significant difference was not found for the number of all cell types on the flat sheets (student t-test, p > 0.05): 8.9 ± 3.33 on the dipped sheet and 9.73 ± 2.61 on the control. 84 fig. 2. hemocytes of b. schlosseri on the flat (a) and the pillared (b, c) sheets, incubated with yeast cells. arrows indicate yeast cells contained in the hemocyte. scale bars = 10 µm. phagocytic activity phagocytosis of yeast cells was observed on both of the flat sheets (fig. 2a) and the pillared sheets (figs 2b, c). the number of the cell engulfing the yeast cell per optical field (ca. 120×160 µm) was always larger on the flat sheets than that on the pillared sheets (table 2). significant differences were shown in the all trials (mann-whitney test, p < 0.05 0.0001). cell morphology on the sheets both spherical and spreading hemocytes were found on the sheets, but many of the well-spreading, fattened cells were partially damaged in the specimens for sem observation (figs 3a, e). the hemocytes attached on the substrate with various forms of pseudopodia (e.g., lamellipodia and filopodia). on the flat surface, the hemocytes appeared to adhere to the substrate with the almost entire substratum-side of the pseudopodia (figs 3b d). on the pillared surface, the hemocyte adhered only to the tips of the pillars, and the pseudopodia did not adhere to the lateral surface of the pillars (figs 3f i). the pillars at the periphery of the lamellipodia were broken at their bases (arrowheads in fig. 3g) probably due to the shrinkage of the cells during the critical-point drying, indicating that the pseudopodia adhered to the pillars. the filopodia also adhere to the pillar tips, and they strode over the intervals between the pillars (fig. 3i). discussion when foreign materials are introduced in the ascidian body, hemocytes endocytize the small materials (phagocytosis) and adhered to the large materials (encapsulation) (e.g., anderson, 1971; wright and cooper, 1975; parrinello et al., 1984). these are primary innate immune reactions to protect the body from pathogens and parasites. the colonial ascidian b. schlosseri contains various types of hemocytes and some of the hemocyte types have phagocytic activity and some are involved in inflammatory responses (ballarin and cima, 2005; ballarin et al., 1993; ballarin, 2008). here, using the hydrophilized nanopillar sheets made of polystyrene as a mimetic material of the nipple arrays on the body surface of endoparasitic/mesoparasitic organisms, we assayed the cell spreading and phagocytosis of the hemocytes of the colonial ascidian b. schlosseri to reveal the influence of the nipple array on immunocyte functionality. the ascidian hemocytes well adhered to the hydrophilized polystyrene sheets regardless of the presence or absence of nanopillars. however, the number of spreading amebocytes was significantly larger on the flat surface than the pillared surface, indicating that the spreading of amebocytes was reduced on the pillared surface. moreover, when the hemocytes were incubated with yeast cells, the number of hemocytes phagocytizing yeast cells was table 2 the number of yeast-containing cells per optical field* trials cell density (× 106 cells/ml) flat sheet average ± sd pillared sheet average ± sd statistic difference# 1† 2.2 2.30 ± 1.53 0.40 ± 0.68 p < 0.0001 2† 2.2 1.75 ± 0.97 0.75 ± 0.85 p < 0.001 3 1.3 2.20 ± 0.95 0.95 ± 0.89 p < 0.001 4 1.5 1.45 ± 0.94 0.80 ± 0.68 p < 0.05 *averages are based on the cell counts in 20 optical fields for each. #mann-whitney test. †these two trials were performed as duplicate tests using the same colony. 85 fig. 3. sem micrograph of round and spreading hemocytes of b. schlosseri on the flat (a d) and on the pillared (e i) sheets. spreading hemocytes were partially damaged during the sample preparation (arrows in a and e). they extended pseudopodia on the flat substrate (b d) and lamellipodia on the pillared substrate (f). enlargement of the attachment between the cell and the pillar tips is shown in g. pillars at the cell periphery were broken at their bases (arrowhead in g). spreading hemocytes extended branching filopodia on the pillared substrate (h; enlarged in i). filopodia adhered to the pillar tips and strode over the intervals between the pillars (i). scale bars = 10 µm (a, e), 5 µm (b, d, h), 2 µm (c, f), 1 µm (g, i). also significantly larger on the flat surface than the pillared surface. the smaller number of spreading amebocytes on the pillared sheet possibly causes the smaller number of phagocytizing cells than that on the flat sheet. however, the number of spreading amebocytes does not simply indicates the number of phagocytic cells on the sheet, because some phagocytic hemocytes, e.g., hyaline amebocytes and granular amebocytes, would not spread on the pillared sheet. in any cases, the phagocytic activity in the same amount of blood is significantly reduced on the pillared surface. 86 the primary cause suppressing the cell spreading is supposed to be the reduction of the area of the cell adherence to the substratum. on the pillared sheets, the hemocytes exclusively adhered to the tip of the pillars (0.5 µm diameter-circle), whereas the hemocytes appeared to adhere to the flat sheets with almost the entire area of the cells. according to the specifications of the nanopillar sheet, the effective area for the cell attachment on the pillared surface is one fifth of the area on the flat sheet. less stable cell-attachment on the pillared surface may also explain the suppression of the phagocytic activity of the hemocytes. actually, cell adhesion to any materials involves several molecular steps, and opsonization by various molecules enhances phagocytosis of foreign materials. therefore, the present results suggest the potential function of nano-surface structures on endoparasites only from the viewpoint of cell spreading and phagocytosis but the real interactions between hosts and parasites should be more complex and remain uncertain. it should be also considered that microorganisms and/or debris might partly (or entirely) cover the surface and change the properties of the surface in natural condition. however, the nipple arrays of the body surfaces were often free from epibionts and debris in electron micrographs (e.g., østergaard and bresciani, 2000; hirose and uyeno, 2014). nomura et al. (2005) reported that the hela cells on the pillared sheet were easily detached by pipetting, probably because the area of the cell adherence to the substratum is small on the pillared surface. in the present study, dipping of the sheet significantly reduced the number of total hemocytes on the pillared sheets but not on the flat sheet. this is consistent with the results on hela cells. in contrast, dipping of the sheet did not reduce the number of the spreading amebocytes on the pillared and flat sheet, indicating that the spreading cells firmly adhered even on the pillared surface enough to remain on the sheet through dipping. in sem observation, the pillars at the periphery of the lamellipodia were broken at their bases; the cell shrinkage during the critical-point drying probably pull the pillars down. this may indicate that the pseudopodia attached to the pillar tips with considerable adhesive strength. it should be noted that the glutaraldehyde-fixation harden the cell and each pillar (0.5 µm diameter) was soften at the condition of critical-point drying (> 35ºc, > 7.4 mpa) in the preparation of sem specimens. nano-scale nipple array on the body surface has been described from various invertebrates including endoparasitic and mesoparasitic copepods (østergaard and bresciani, 2000; hirose and uyeno, 2014). in the host tissue, these parasites should evade or resist host immune responses, such as encapsulation by hemocytes. the present results showed the nanopillar structure significantly reduces cell spreading and phagocytic activity of ascidian hemocytes. the pillared surface would be difficult to adhere and easy to separate for the hemocytes, because of the small area for the cell attachment. accordingly, the nipple array on the parasite body surface may reduce the innate immune reaction from the host hemocytes. recently, synthetic micro-dimple arrays, 3 7 µm in depth, were shown to decrease friction of the surface (hirai et al., 2013), and nipple arrays may also make low friction surfaces. for instance, the parasites may be able to evade the encapsulation or have an extension of time until the completion of the encapsulation. the affinity of the pillared surface to the hemocytes would vary depending on the diameter of the pillars and the interval among the pillars. the nanopillar sheets used in this study is not exactly the same as those of the parasites' body surface, and, optimizing the parameters, the pillared surface may have lower affinity to the cells. acknowledgements we thank university of padua for inviting e hirose as a visiting scientist in 2014. references anderson rs. cellular responses to foreign bodies in the tunicate molgula manbattensis (dekay). biol. bull. 141: 91-98, 1971. ballarin l. immunobiology of compound ascidians, with particular reference to botryllus schlosseri: state of art. inv. surv. j. 5: 54-74, 2008. ballarin l. ascidian cytotoxic cells: state of the art and research perspectives. inv. surv. j. 9: 1-6, 2012. ballarin l, cima, f. immunotoxicity in ascidians: the case of organotin compounds. in: sawada h, yokosawa h, lambert cc (eds), the biology of ascidians, springer, tokyo, japan, pp 374-379, 2001. ballarin l, cima f. cytochemical properties of botryllus schlosseri haemocytes: indications for morpho-functional characterisation. eur. j. histochem. 49: 255-264, 2005 ballarin l, cima f, sabbadin a. histoenzymatic staining and characterization of the colonial ascidian botryllus schlosseri hemocytes. boll. zool. 60: 19-24, 1993. ballarin l, cima f, sabbadin a. morula cells and histocompatibility in the colonial ascidian botryllus schlosseri. zool. sci. 12: 757-764, 1995 ballarin l, menin a, franchi n, bertoloni g, cima f. morula cells and non-self recognition in the compound ascidian botryllus schlosseri. inv. surv. j. 2: 1-5, 2005. ballarin l, menin a, tallandini l, matozzo, v, burighel p, basso g, et al. haemocytes and blastogenetic cycle in the colonial ascidian botryllus schlosseri: a matter of life and death. cell tissue res. 331: 555-564, 2008. bernhard cg. structural and functional adaptation in a visual system. endeavour 26: 79-84, 1967. cima f, bragadin m, ballarin l. toxic effects of new antifouling compounds on tunicate haemocytes i. sea-nine 211 and chlorothalonil. aquat. toxicol. 86: 299-312, 2008. hausen h. comparative structure of the epidermis in polychaetes (annelida). hydrobiologia 535/536: 25-35, 2005. hirai y, yabu y, kaido m, suzuki a, shimomura m. ag micro-dimples prepared by self-organization and their friction properties. kobunshi ronbunshu 70: 193-198, 2013. 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(crustacea, copepoda). j. crust. biol. 20: 674-679, 2000. parrinello n, patricolo e, canicatti c. inflamatory-like reaction in the tunic of ciona intestinalis (tunicata) 1. encapsulation and tissue injury. biol. bull. 167: 229-237, 1984. sabbadin a. osservazioni sullo sviluppo, l’accrescimento e la riproduzione di botryllus schlosseri (pallas), in condizioni di laboratorio. boll. zool. 22: 243-265, 1995. sabbadin a, zaniolo g, ballarin l. genetic and cytological aspects of histocompatibility in ascidians. boll. zool. 59: 167-173, 1992. stavenga dg, foletti s, palasantzas g, arikawa k. light on the moth-eye corneal nipple array of butterflies. proc. royal soc. b 273: 661-667, 2006. watanabe h. studies on the regulation in fused colonies in botryllus primigenus (ascidiae compositae). sci. rep. tokyo bunrika daigaku b 10: 253-284, 1953. wright rk, cooper el. immunological maturation in the tunicate ciona intestinalis. amer. zool. 15: 21-27, 1975. 88 many plants and animals utilize the pigment melanin, capable of regulating a wide range of molecular interactions and metabolic processes, as a defense against any type of non-self(golkar et al isj 9: 153-162, 2012 issn 1824-307x research report amyloid/melanin distinctive mark in invertebrate immunity a grimaldi1, r girardello1, d malagoli2, p falabella3, g tettamanti1, r valvassori1, e ottaviani2, m de eguileor1 1department of biotechnology and life science, university of insubria, via j. h. dunant 3, 21100 varese, italy 2department of life sciences, university of modena and reggio emilia, via campi 213/d,41125 modena, italy 3dipartimento di biologia, difesa e biotecnologie agro-forestali, university of basilicata, via dell’ateneo lucano 10, 85100 potenza, italy accepted september 14, 2012 abstract protostomes and deuterostomes show the same nexus between melanin production, and amyloid fibril production, i.e., the presence of melanin is indissolubly linked to amyloid scaffold that, in turn, is conditioned by the redox status/cytoplasmic ph modification, pro-protein cleavage presence, adrenocorticotropin hormone (acth), melanocyte-stimulating hormone (α-msh), and neutral endopeptidase (nep) overexpressions. these events represent the crucial component of immune response in invertebrates, while in vertebrates these series of occurrences could be interpreted as a modest and very restricted innate immune response. on the whole, it emerges that the mechanisms involving amyloid fibrils/pigment synthesis in phylogenetically distant metazoan (viz, cnidaria, molluscs, annelids, insects, ascidians and vertebrates) are evolutionary conserved. furthermore, our data show the relationship between immune and neuroendocrine systems in amyloid/melanin synthesis. indeed the process is closely associated to acth-α-msh production, and their role in stress responses leading to pigment production reflects and confirms again their ancient phylogeny. key words: amyloid fibrils; melanin; acth, α-msh; neutral endopeptidase; invertebrate immunity introduction living organisms produce melanin as defense system against attack, harm, or injury coming from any type of non-self (golkar et al., 1993; nappi and ottaviani, 2000; petes et al., 2003; nappi, 2010). in vertebrates, it is well known that the biopigment shows its main quality neutralizing the potentially deleterious effects of sunlight (edelstein, 1977). melanin production is considered a widespread event, and becomes essential in those invertebrates where invaders such as parasites or fungi, are rapidly isolated and sequestered in a capsule made of pigment and hemocytes (carton et al., 2008). in general melanin, acting as scavenger of reactive oxygen species (ros), defends cells/tissues from the toxic effects of free radicals and it is manifested, from invertebrates up to man, in the areas of tissue repair, during regeneration process and in response to pathogens (de eguileor et al. 2000; gourdon et al., 2001; ballarin et al., 2002; nappi and christensen, 2005; lewis and pollard, 2006; nappi, ___________________________________________________________________________ corresponding author: annalisa grimaldi department of biotechnology and life science university of insubria via j. h. dunant 3, 21100 varese, italy e-mail: annalisa.grimaldi@uninsubria.it 2010; palmer et al., 2011). melanin biosynthesis is due to the activation of prophenol oxidase (pro-po) system present in cell and/or in body fluids. the pro-po activating system is best understood in crayfish pacifasticus leniusculus (söderhäll and smith, 1986), in silkworm bombyx mori (ashida and yoshida, 1990; yasuhara et al., 1995), in drosophila melanogaster (nappi and vass, 1993; fujimoto et al., 1995), in echinoderms such as holoturia tubulosa (roch et al., 1992), in ascidians (jhoanson and söderhäll, 1989; cammarata and parrinello, 2009; ballarin, 2012), and in cephalochordates (pang et al., 2004). specifically about biopigment synthesis, in cnidaria several papers show that pathogens or any kind of stressors, induces a localized melanization in sea fan corals. the pigment production is due to an augment of amebocyte melanosome production and to pro-po activity in the tissues (petes et al., 2003; mydlarz et al., 2008). in annelida (oligochaets and polychaets), several authors (porchet-henneret, 1987; valembois et al., 1988; porchet-henneret and vernet, 1992; beschin et al., 1998; fyffe et al., 1999; adamowicz, 2005; prochazkova et al., 2006) describe the efficient activation of both pro-po cascade in coelomic fluid and in a subpopulation of granulocytes, with the final 153 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t79-3v02wbx-9&_user=1413312&_coverdate=07%2f31%2f1997&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchstrid=1408595973&_rerunorigin=google&_acct=c000052656&_version=1&_urlversion=0&_userid=1413312&md5=a60a670737ce6f4b2271ab0d36c5b5b3#b1#b1 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t79-3v02wbx-9&_user=1413312&_coverdate=07%2f31%2f1997&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchstrid=1408595973&_rerunorigin=google&_acct=c000052656&_version=1&_urlversion=0&_userid=1413312&md5=a60a670737ce6f4b2271ab0d36c5b5b3#b4#b4 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t79-3v02wbx-9&_user=1413312&_coverdate=07%2f31%2f1997&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchstrid=1408595973&_rerunorigin=google&_acct=c000052656&_version=1&_urlversion=0&_userid=1413312&md5=a60a670737ce6f4b2271ab0d36c5b5b3#b4#b4 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t79-3v02wbx-9&_user=1413312&_coverdate=07%2f31%2f1997&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchstrid=1408595973&_rerunorigin=google&_acct=c000052656&_version=1&_urlversion=0&_userid=1413312&md5=a60a670737ce6f4b2271ab0d36c5b5b3#b5#b5 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t79-3v02wbx-9&_user=1413312&_coverdate=07%2f31%2f1997&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchstrid=1408595973&_rerunorigin=google&_acct=c000052656&_version=1&_urlversion=0&_userid=1413312&md5=a60a670737ce6f4b2271ab0d36c5b5b3#b5#b5 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t79-3v02wbx-9&_user=1413312&_coverdate=07%2f31%2f1997&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchstrid=1408595973&_rerunorigin=google&_acct=c000052656&_version=1&_urlversion=0&_userid=1413312&md5=a60a670737ce6f4b2271ab0d36c5b5b3#b6#b6 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t79-3v02wbx-9&_user=1413312&_coverdate=07%2f31%2f1997&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchstrid=1408595973&_rerunorigin=google&_acct=c000052656&_version=1&_urlversion=0&_userid=1413312&md5=a60a670737ce6f4b2271ab0d36c5b5b3#b11#b11 fig. 1 circulating hemocytes from insect and ascidian. (a-b) light microscopy: may grünwald-giemsa technique (the circulating cell cytoplasm is differently stained in relation to their cytoplasmic ph) (pink mark indicates acid ph); (c-d) light microscopy: masson-fontana technique: silver staining demonstrates melanin deposition (arrowheads); (e-h) fluorescence and light microscopy: thioflavine t (e-f) and congo red (g-h) staining recognize amyloid or amyloid-like structures; (j-k) fluorescence microscopy: immunocytochemical evidence of furin-like protein (in red), nuclei in blue are stained with dapi. 154 result of massive melanin production. an extensive literature is reported about humoral factors and cellular responses in molluscs, against any type of non-self (ottaviani and cossarizza, 1990; ottaviani, 2006; novoa et al., 2011). in particular, a classification of hemocytes has been proposed according to their different function in immune responses with particular regard to melanin synthesis (ottaviani, 1983; ottaviani and franchini, 1988; ottaviani et al., 1990, 1993; matricongondran and letocart, 1999; gorbushin and iakovleva, 2006; jiravanichpaisal et al., 2006; koropatnick et al., 2007; mahilini and rajendran, 2008; venier et al., 2011). moreover, several authors have described in mollusc bivalves and gastropod granulocytes, involved in wound repair or internal defense, the presence of cytoplasm membrane-limited granules containing “filamentous matrix” with acid phosphatase activity of unknown function (giamberini et al., 1996; matricon-gondran and letocart, 1999). induced melanization/encapsulation against non-self is well known also in arthropods (söderhäll and smith, 1986; ashida et al., 1990; hoffman and reichart, 2002; martin et al., 2007; gallo et al., 2011). with reference to insects, two cell types are responsible for the entrapment of the invaders and this process is generally accompanied by the phenoloxidase activity inducing the formation of the melanotic material. these events have been well-studied in insect host/parasitoid model (heliothis virescens/toxoneuron nigriceps) (ferrarese et al., 2005; falabella et al., 2012; grimaldi et al., 2012). the same implications are also evident in deuterostomes such as echinoderms (sea urchin, holoturians) (canicattì and seymour, 1991) and tunicates (sea squirts) (shirae et al. 2002; hirose, 2003; ballarin, 2008; cammarata and parrinello, 2009; ballarin, 2012). in tunicates, morula cells are able to recognize the presence of foreign elements and release phenoloxidase which induces melanin formation (hirose, 2003; ballarin et al., 2005; ballarin, 2012). in mammals, melanocytes are cells with the main function in synthesizing and packaging the brown pigments in melanosomes to protect the skin against ultraviolet radiation (uv). as previously mentioned, we have demonstrated that in the insect h. virescens larvae, during the earliest phase of the parasitization, melanin was packaged due to the production of large amount of amyloid fibrils, sharing these linked events with vertebrates, where, as suggested by fowler and coworkers (2006), amyloid fibrils template and accelerate the formation of pigment. the principal divergence in melanization process of insects and vertebrates is that it takes place in specific cell types (granulocytes and melanocytes, respectively), but in insect cells the phenomenon is faint in respect to that observed in the hemocel, where large amount of pigment are derived from sieric pro-po system reactions. on the contrary, in vertebrates, the massive melanin synthesis occurs intracellularly, i.e., in melanosomal organelles (grimaldi et al., 2012). on the basis of our previous data (falabella et al., 2012; grimaldi et al., 2012) and the evidence (previously mentioned), we surmize that protostomes and deuterostomes, show the same nexus between melanin production, and amyloid fibril production, i.e., the presence of melanin is indissolubly linked to amyloid scaffold that, in turn, is due to a combined redox status/cytoplasmic ph modification, pro-protein cleavage presence, adrenocorticotropin hormone (acth), melanocytestimulating hormone (α-msh), and neutral endopeptidase (nep) overexpressions. thus, in the present paper, using a variety of techniques we confirm our hypothesis. materials and methods hemocytes extraction and culture hemocytes from several species stimulated with lps injection (helix pomatia, heliothis virescens, ciona intestinalis) were collected by centrifuging the circulating fluid at 400 g per 7 min at 4 °c. the pellet washed with mead-pbs solution (1:1). the hemocytes were resuspended in complete medium (grace’s medium, fbs 10 %, antibiotic-antimicotic solution 1 %, sigma) and were plated at concentration of 1x106 cells/ml into 24-well culture plates. the b16-f10 murine melanoma cell line (derived from c57bl/6j mouse, d/d) was a generous gift from prof. douglas noonan (university of insubria, va, italy). b16-f10 murine melanoma cells were cultured for 24 h in dmem and 10 % fbs and then changed to dmem and 2 % fbs for additional 48 h. cells were plated on glass coverslips in 35-mm-diameter petri dishes containing the appropriate medium as described above. coverslips were washed with phosphatebuffered saline (pbs), ph 7.2, and the cells fixed with 2 % paraformaldehyde containing 0.3 % triton x-100 for 10 min at 37 °c, followed by washing three times with pbs. cells were blocked with 2 % bovine serum albumin (bsa) and 5 % goat serum in pbs for 1 h at room temperature or overnight at 4 °c and then incubated for 4 h at room temperature in primary antibody diluted in blocking solution. circulating cells from stimulated hirudo medicinalis ten leeches were stimulated, at the level of the 80th superficial metamere, with injection of matrigel (mg) (bd biosciences, mississauga, canada) (300 μl) added with lps. according to grimaldi and coworker (2008) after 1 week mg implants were harvested from the animals, minced in small pieces using sterilized razor blades and mechanically dissociated with a micropipette in 400 μl of tissue culture. cells were plated, cultured, maintained at 20 °c and examined histologically and immunocytochemically after 3 days from seeding. all cultures were performed in quadruplicate and processed as previously described. light microscopy, transmission electron microscopy (tem) (standard procedure) for routine tem, collected circulating cells were fixed with 2 % glutaraldehyde in 0.1 m nacacodylate buffer (ph 7.2) for 2 h. the pellet washed in 0.1 m na-cacodylate buffer (ph 7.2), was post-fixed at 4 °c for 2 h with 1 % osmic acid in cacodylate buffer (ph 7.2). after standard dehydration in ethanol series, samples were embedded in an epon-araldite 812 mixture and 155 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t79-3v02wbx-9&_user=1413312&_coverdate=07%2f31%2f1997&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchstrid=1408595973&_rerunorigin=google&_acct=c000052656&_version=1&_urlversion=0&_userid=1413312&md5=a60a670737ce6f4b2271ab0d36c5b5b3#b4#b4 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t79-3v02wbx-9&_user=1413312&_coverdate=07%2f31%2f1997&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchstrid=1408595973&_rerunorigin=google&_acct=c000052656&_version=1&_urlversion=0&_userid=1413312&md5=a60a670737ce6f4b2271ab0d36c5b5b3#b4#b4 fig. 2 immunocytochemical characterization of insect/ascidian circulating hemocytes. (a-f) fluorescence microscopy: expression of acth (a-b); nep (c-d); α-msh (e-f). sectioned with a reichert ultracut s ultratome (leica, nussloch, germany). semithin sections were stained by conventional methods (crystal violet and basic fuchsin), and with may grünwald-giemsa staining. differential may grünwald-giemsa staining depends on cytoplasmic ph (alkaline ph increases blue and acid ph pink or reddish tinge in the stained specimens), therefore is useful for a grossidentification of cells showing an increased reactive oxygen species production. pictures visualized on a microscope olympus bh2 (olympus, tokyo, japan) were acquired with a ds-5m-l1 nikon digital camera system. thin sections were stained by uranyl acetate and lead citrate and observed with a jeol 1010 electron microscope (jeol, tokyo, japan). amyloid fibrils detection amyloid or amyloid-like structures can be recognized by different techniques. amyloid fibrils exhibit strong affinity towards the dye congo red and thioflavine t (sipe and cohen, 2000). congo red and thioflavine t staining were performed according to grimaldi et al. (2012). specific fluorescence was visualized on a fluorescence microscope olympus bh2 through a filter set (excitation wavelength of 465 nm emission). images were acquired with a ds-5m-l1 nikon digital camera system. melanin detection following the manufacturer’s protocol (biooptica, milan, italy), the masson-fontana silver stain was employed to demonstrate melanin deposition. experiments were performed in triplicate. indirect immunofluorescence staining cryosections were treated for 30 min with pbs containing 2 % bsa before the primary antibody incubation (4 °c over night). the presence of acth, 156 http://cancerweb.ncl.ac.uk/cgi-bin/omd?specific http://cancerweb.ncl.ac.uk/cgi-bin/omd?fluorescence fig. 3 comparison between protostome and deuterostome. cytoplasmic ph condition (giemsa staining), melanin presence (masson-fontana technique), amyloid fibrils production (thioflavine-t and congo red staining), and furin expression are similar in h. medicinalis (annelid), h. pomatia (mollusc), h. virescens (insect), c.intestinalis (ascidian) circulating cells, and b16-f10 murine melanoma cell line (as positive control). and its cleavage product α-msh) (responsible for stimulation, production and release of melanin), due to nep activity, and furin were assessed using the following primary antibodies: anti-human acth polyclonal antibody (1:50 dilution, sigma, saint louis, mo, usa); anti-human α-msh polyclonal antibody (1:50 dilution, sigma); anti-cd10/calla (nep) monoclonal antibody (clone 56c6, diluted 1:50, thermo scientific, freemont, ca, usa), antifurin polyclonal antibody (1:50 dilution, santa cruz biothecnology, santa cruz, ca, usa). incubations with suitable secondary antibodies conjugated with tetramethylrhodamine (tritc) (1:200 dilution, jackson, immuno research laboratories, west grove, pennsylvania, usa) were performed for 1h in a dark moist chamber. nuclei were eventually stained with 4’,6-diamidino-2-phenylindole (dapi, sigma, italy). the pbs buffer used for washing steps and antibody dilutions contained 2 % bovine serum albumin (bsa). in control samples, primary antibodies were omitted, and samples were treated with bsa-containing pbs. nuclei were stained by incubating for 15 min with 4-6-diamidino-2phenylindole (dapi, 0.1 g/ml in pbs). coverslips were mounted in vectashield mounting medium for fluorescence (vector laboratories, burlingame, ca, usa); slides were observed on olympus bh2 microscope (olympus, tokyo, japan). data were recorded with a ds-5m-l1 digital camera system (nikon, tokyo, japan). images were combined with adobe photoshop® (adobe systems inc., usa). results starting from our previous data about the link between the melanin synthesis and the production 157 http://en.wikipedia.org/wiki/melanin of amyloid fibrils, that template the pigment in activated insect h. virescens hemocytes (falabella et al., 2012; grimaldi et al., 2012), here we described and characterized the morpho-functional events linked to the production of amyloid fibrillar material in relation to melanin and the concomitant events that take place in the immune cells under stress condition in different invertebrates (protostome and deuterostome). for instance in insects and ascidians, melanin production (figs 1 c, d) and amyloid fibril assemblage (figs 1 e-h) [in the reticulum cisternae (grimaldi et al., 2012)] are always sustained by several conditions such as an overproduction of ros responsible of ph variation (as previously validated by enzyme inhibition) (figs 1 a, b), the presence of a furin-like proprotein convertase cleavage (figs 1 j, k) that it is well known liberates a fibrillogenic fragment in melanosomal biogenesis (berson et al., 2003). in addition we have observed that during this amyloid/pigment productive phase, a cross-talk between immune and endocrine systems occurs. these intercommunications are mediated by neuromodulators with the activation of stresssensoring circuits to produce and release molecules such as acth and α-msh (figs 2 a-f). this scenario of cytoplasmic ph condition, amyloid fibrils production/melanin synthesis, and interaction between immune and neuroendocrine system (acth-α-msh presence) as reported by grimaldi and coworkers (2012) is validated also in different invertebrates (fig. 3). on the whole, our data, here presented, and regarding h. medicinalis (annelid), h. pomatia (mollusc), h. virescens (insect), c. intestinalis (ascidian), and b16-f10 murine melanoma cell line (as positive control) were added to available data present in literature, and summarized respectively in the figure 3 and table 1. discussion extensive and deep studies were carried out on the multiple biochemical and morphological aspects involved in the protective responses of the immune system in invertebrates. among the various potent weapons typical of a innate defense there are pro-po system and granulocyte activations (söderhäll and smith, 1986; jhoanson and söderhäll, 1989; ashida and yoshida, 1990; roch et al., 1992; nappi and vass, 1993; fujimoto et al., 1995; yasuhara et al., 1995; pang et al., 2004; cammarata and parrinello, 2009; ballarin, 2012). the humoral pro-po system that according to several authors corresponds in function to the activated complement (söderhäll, 1982; johansson and söderhäll, 1989) was recorded in several taxa but it is not the most important defense mechanism for all invertebrates (smith and söderhäll, 1991; nappi and ottaviani, 2000; cerenius and söderhäll, 2004; cerenius et al., 2008; cammarata and parrinello, 2009; ballarin, 2012). independently from their phylogenetic position, invertebrates can produce melanin especially by humoral system or specifically by cellular population. in the first case massive production of melanin is due to the activity of pro-po system that can be coupled with a cellular response lesser involved in pigment production. in the second one, the melanin synthesis is confined in cell where is concentrated in organules, the melanosomes. in any case it is interesting to underline that the production of melanin is always supported by the formation of amyloid fibrils, as well as the concurrent events, such as acth production, nep increment and αmsh formation. in several invertebrates, such as arthropods, melanin is massively produced in body cavity especially as pro-po system product, while amyloid fibrils production is due to exocytosis of circulating cells (named in different ways as granulocytes or amebocytes) that are able to produce a huge amount of amyloid fibrils that adhere to the non-self driving the pigment accumulation close to the invaders, avoiding the toxic melanin dispersion in hemocelic environment (ferrarese et al., 2005; falabella et al., 2012; grimaldi et al., 2012). in other invertebrates and vertebrates there is a coupled productive system (melanin/amyloid fibrils) concentrated in a specific cell type, the melanocytes where melanin on amyloid fibrils is stocked in melanosomes (fowler et al., 2006). after stimulation, invertebrate cells engaged in melanin production, degranulate and their products flow close to the non-self or, as in vertebrates, the melanocytes convey towards superficial surface the pigment that are utilized as protection against uv. summarizing it is interesting to highlight that melanin employment is always coupled, from invertebrates up to man, with a physiological production of amyloid fibrils. the trade-off in utilizing the coupled system amyloid/melanin may shift from the possibility to have two separated producers (humoral pro-po system for melanin, and granulocytes for amyloid fibrils) as recorded in insects, echinoderms and ascidians with the following assemblage of the two products, up to a single cellular producer of both products (pigment and amyloid fibrils) as in coelenterates, annelids, molluscs and vertebrates. an additional striking aspect (in the previously mentioned taxa) refers to the cells involved in the production of amyloid fibrils that after cytoplasmic accumulation, are exocytozed to sustain melanin production. these cells, belonging to freely circulating hemocytes, show the same phenotype with a nucleus localized in central position, surrounded by large reticulum cisternae filled with fibrillar material, spatially organized in respect to a central electrondense core (xing et al., 2008; grimaldi et al., 2012). all these features and related processes involved in amyloid fibrils/melanin synthesis in animal phylogenetically distant (viz., cnidaria, molluscs, annelids, insects, ascidians and vertebrates) could be interpreted as evolutionary conserved. these shared innate immune responses could be interpreted in invertebrates as a basic event, constituting an integral component of immunity, independently deriving from a mix of cellular and humoral or from exclusive cellular responses, while in vertebrate could be interpreted as a modest and very restricted event of innate immunity. indeed, in vertebrates the multiple and 158 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t79-3v02wbx-9&_user=1413312&_coverdate=07%2f31%2f1997&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchstrid=1408595973&_rerunorigin=google&_acct=c000052656&_version=1&_urlversion=0&_userid=1413312&md5=a60a670737ce6f4b2271ab0d36c5b5b3#b4#b4 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t79-3v02wbx-9&_user=1413312&_coverdate=07%2f31%2f1997&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchstrid=1408595973&_rerunorigin=google&_acct=c000052656&_version=1&_urlversion=0&_userid=1413312&md5=a60a670737ce6f4b2271ab0d36c5b5b3#b5#b5 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t79-3v02wbx-9&_user=1413312&_coverdate=07%2f31%2f1997&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchstrid=1408595973&_rerunorigin=google&_acct=c000052656&_version=1&_urlversion=0&_userid=1413312&md5=a60a670737ce6f4b2271ab0d36c5b5b3#b11#b11 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t79-3v02wbx-9&_user=1413312&_coverdate=07%2f31%2f1997&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchstrid=1408595973&_rerunorigin=google&_acct=c000052656&_version=1&_urlversion=0&_userid=1413312&md5=a60a670737ce6f4b2271ab0d36c5b5b3#b11#b11 http://www.sciencedirect.com/science?_ob=articleurl&_udi=b6t79-3v02wbx-9&_user=1413312&_coverdate=07%2f31%2f1997&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchstrid=1408595973&_rerunorigin=google&_acct=c000052656&_version=1&_urlversion=0&_userid=1413312&md5=a60a670737ce6f4b2271ab0d36c5b5b3#b6#b6 table 1 giemsa melanin amyloid furin acth α-msh references thioflavine t congo red cnidarians *** (1, 2) annelids polichets *** (3, 4) oligochets *** (5-8) hirudineans *** *** *** *** *** *** *** (here) (9,10) molluscs *** *** *** *** *** *** (here) (11-25) arthropods crustaceans *** *** *** *** (26-28) insects *** *** *** *** *** *** *** (here) (29-33) echinoderms *** *** *** (34) tunicates *** *** *** *** *** *** *** (here) (35-38) cephalochordates *** (39) vertebrates *** *** *** *** *** *** *** (here) (40) available data present in literature has been summarized. cnidarians: (1, 2) petes et al., 2003; mydlarz et al., 2008. annelids: (3) porchet-henneret and verner, 1992; (410) porchet-henneret et al., 1987; beschin et al., 1998; fyffe et al., 1999; de eguileor et al., 2000; adamowicz, 2005; prochazkova et al., 2006; grimaldi et al., 2008. molluscs: (11-25) ottaviani and cossarizza, 1990; ottaviani, 1983, 2006; ottaviani et al., 1990, 1993; gourdon et al., 1993; giamberini et al., 1996; ottaviani and franchini, 1998; matricon-gondra, 1999; gorbushin et al., 2007; koropatnick et al., 2007; martin et al., 2007; mahilini et al., 2008; novoa et al., 2011. arthropods: (26-32) söderhäll and smith, 1986; johansson and söderhäll, 1989; nappi and vass, 1993; hoffman and reichart, 2002; ferrarese et al., 2005; gallo et al., 2011; falabella et al., 2012; grimaldi et al., 2012. ehinoderms: (34) canicattì and seymur, 1991. tunicates: (35) ballarin, 2008; (36) ballarin, 2012; (37) cammarata and parrinello, 2009; (38) hirose, 2003. cephalochordates: (39) pang et al., 2004. vertebrates: (40) fowler et al., 2006 multifaceted responses belonging to acquired immunity can mask the basic innate responses due to the presence of numerous modulate answers against the non-self leading to a precise discrimination of individual pathogenic species. another aspect that must be considered is the evidence of bidirectional messages between immune and neuroendocrine system. thus amyloid/melanin production is close associated to acth/α-msh production, emerging here as molecule overexpression. their presence and function related to stress responses leading to pigment production reflect and confirm their ancient phylogeny (wilder, 1995; ottaviani and franceschi, 1996). 159 references adamowicz a. morphology and ultrastructure of the earthworm 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a review. dev. comp. immunol. 6: 601-611, 1962. valembois p, roch p, lassegues m. evidence of plasma clotting system in earthworms. j. invertebr. pathol. 51: 221-228, 1988. venier p, varotto l, rosani u, millino c, celegato b, bernante f, et al. insights into the innate immunity of the mediterranean mussel mytilus galloprovincialis. bmc genomics 12: 69-84, 2011. wilder rl. neuroendocrine-immune system interactions and autoimmunity. annu. rev. immunol. 13: 307-338, 1995. xing k, sheng yang, h, chen, my. morphological and ultrastructural characterization of the coelomocytes in apostichopus japonicus. aquat. biol. 2: 85-92, 2008. yasuhara y, koizumi y, katagiri c, ashida m. reexamination of properties of prophenol oxidase isolated from larval hemolymph of the silkworm bombyx mori. arch. biochem. biophys. 320: 23-24, 1995. 162 microsoft word isj-2014-365r uncorrected proofs isj 12: 19-21, 2015 issn 1824-307x letter to editor tumors in invertebrates: molluscs as an emerging animal model for human cancer g de vico, f carella department of biology, university of naples federico ii, naples, italy accepted december 31, 2014 to the editor we read with interest the recent paper by tascedda and ottaviani (2014) about the occurrence of tumors in invertebrates. in order to further reinforce your statements that “histological and molecular biology studies have proved the existence of tumors in invertebrates” we would like to add to some insights focusing on molluscan neoplasia, an emerging animal model for human cancer (walker, 2011; carella et al., 2013). it is known that neoplasia is a pathological process characterized by an overgrowth of a new tissue in the context of a pre-existing one, and consists of atypical cells, a term which incorporates the sum of the differences in morphological, biochemical and functional features of cancer cells relative to normal cells (carella et al., 2013). furthermore, neoplastic tissue is characterized by a self-growing, progressive, irreversible and nonfinalistic behavior (dianzani, 2005; de vico and carella, 2012). two predominant types of neoplasia have been reported in marine molluscs, viz. disseminated neoplasia, also called leukaemia or hemic neoplasia (hn), and gonadal neoplasia (carella et al., 2009). in hn neoplastic cells are represented by atypical hemocytes, which display high nucleus to cytoplasm ratios, diffuse chromatin patterns and pleomorphic nuclei, and usually infiltrate tissues and organs of affected individuals (auffret and poder, 1986; villalba et al., 2001). since the initial description of the disease (farley, 1969), its cause has not been clearly defined (barber, 2004). viral infection, genetic profile, environmental changes and anthropogenic pollution have been proposed as the causative factors. the prevalence of the disease ranges from 0.5 to 73.3 % , according to the species considered (e.g., crassostrea virginica, mytilus spp. and ostrea edulis) and geographical origins of molluscs. in the affected bivalves hn have been for a long time considered a phenotypically similar proliferative disease in the different shellfish species involved (walker et al., 2011). however, differences ___________________________________________________________________________ corresponding author: gionata de vico department of biology university of naples federico ii via mezzocanone, 8, 80134, naples, italy e-mail: gionata.devico@unina.it in neoplastic cell morphology, along with descriptions of neoplastic hemocyte subtypes, have frequently contradicted this assumption (lowe and moore, 1978; green and alderman, 1983). recently, we described at last two different types of leukemia in two different bivalve species (mytilus galloprovincialis and cerastodema edule), showing distinctive morphological and histo-pathogenetic behaviour of cells (carella et al., 2013). in particular, in mussels, two predominant types of neoplastic cells (a and b) have been described, differently to common cockle where one population of cells have been observed; atypical cells in the respective species also showed a distinct pattern of pcna (proliferating cell nucler antigen) expression, nuclear or cytoplasmic (carella et al., 2013). such difference could be indicator of a different mechanism of neoplastic cells initiation/progression in early and advanced phase of the disease, respectively, as also strongly supported by diaz et al. (2013). gonadal neoplasia is mainly represented by germinoma, which consists of a proliferation of atypical germ cells. germinoma has been described in several species of marine bivalve molluscs, and most consistently in some populations of mercenaria mercenaria, mya arenaria and razor clam, ensis arcuatus (barber, 2004; darriba et al., 2006). in the above species, the prevalence of the disease in a given population could remain underestimated particularly in early cases, where neoplastic cells may still go undetected if the tissue section examined does not happen to contain them (barber, 2004). in fact, the probability of correctly diagnosing the presence of gonadal neoplasia in molluscs increases with disease progression (carella et al., 2009). three evolutive stages of the disease could be observed in m. arenaria (barber, 1996; 2004), and four stages in mercenaria spp. (bert et al., 1993), according to the percentage of gonadal follicles involved and the extent of tissue invasion. although the aetiology or causes of neoplasia remains unclear, pollution by carcinogenic agents is implicated in the heavily exploited littoral zones of coastal waters. germinoma have been described also in the gastropod patella coerulea, accompanied by other gonadal developmental disorders (carella et al., 2009). many genes and pathways critically involved in neoplastic transformation and metastasis are 19 evolutionarily conserved in molluscs. some molecular evidence regarding stress biology and relationships to human biology is available in relation to the function of p53 superfamily members in bivalves. in particular, literature reports p53 (which is among the best known molecules involved in vertebrates carcinogenesis) is demonstrably involved in both bivalve hns and germinoma (olberding et al., 2004; st-jean et al., 2005; walker et al., 2011) both structurally and functionally, bivalve p53 family proteins are the most highly conserved members of this gene superfamily so far identified outside of higher vertebrates and invertebrate chordates (protein sequences are 67 69 % conserved with human p53). however, while in vertebrates p53, p63 and p73 originating from different genes, isoforms of p53 of bivalve molluscs are splice variants of a single gene (van beneden et al., 1997; kelley et al., 2001; muttray et al., 2005, 2007; goodson et al., 2006). the p53 protein was detected in tumor cells of molluscs mytilus edulis, mytilus trossulus, m. arenaria, spisula solidissima, crassostrea rhizophoae and crassostrea gigas. in particular, a homologue of the human hsp53 protein was cloned and characterized in m. arenaria affected by hn (kelley et al., 2001) and presents a domain ii-v dna-ligand, a transactivation domain and a domain mdm2 stored for 73 % compared to the human p53, suggesting that the molecular mechanisms that regulate the transcription of the p53 gene in mollusks are similar to those involved in the human gene (kelley et al., 2001). furthermore, in neoplastic hemocytes of m. arenaria, the mortalin (a member of the hsp70 family whose expression is strongly correlated with the levels of expression of p53 in sick bivalve) (wadhwa et al., 2002; siah et al., 2008) sequesters inactivated p53 in the cytoplasm. a similar phenotype, characterized by hsp70 cytoplasmic sequestration of p53 protein, has been observed in several human cancers (undifferentiated neuroblastoma, retinoblastoma, colorectal and hepatocellular carcinomas, and glioblastoma). moreover, clam hemocyte cancer is the only animal model thus far investigated where cytoplasmically sequestered wild-type p53 can be reactivated both in vitro and in vivo using both genotoxic and non-genotoxic therapies. results suggest that mortalin-based cytoplasmic sequestration of wild-type p53 in cancerous clam hemocytes can be reversed by treatment with antineoplastic drugs also employed against similar human diseases and will result either in transcription based apoptosis when the nucleus is accessible or non-transcription-based apoptosis when nuclear access is blocked (walker et al., 2012). based on these data, leukemic clam hemocytes is regarded as novel and easily accessible in vivo and in vitro models for human cancers displaying a mortalin-based phenotype, and marine bivalves as the most relevant and best understood model currently available for experimental studies by biomedical and marine environmental researchers. references auffret m, poder m. sarcomatous lesion in the cockle cerastoderma edule. ii. electron microscopical study. aquaculture 58: 9-15, 1986. barber b. effects of gonadal neoplasms on oogenesis in softshell clams, mya arenaria. j. invertebr. pathol. 67: 161-168, 1996. barber bj. neoplastic diseases of commercially important marine bivalves. aquat. living resour. 17: 449-466, 2004. bert tm, hesselman dm, arnold ws, moore ws, cruz-lopez h, marelli dc. high frequency of gonadal neoplasia in a hard clam (mercenaria spp.) hybrid zone. mar. biol. 117: 97-104, 1993. carella f, restucci b, maiolino p, de vico g. a case of germinoma in limpet (patella coerulea). j. invertebr. pathol. 101: 154-156, 2009. carella f, figueras a, novoas b, de vico g. cytomorphology and pcna expression pattern in bivalves mytilus galloprovincialis and cerastoderma edule with haemic neoplasia. dis. aquat. org. 105: 81-87, 2013. darriba s. razor clams and aquaculture: studies in galicia (nw spain). in: counago sd (ed.), world aquaculture society, aqua 2006, aquameeting abstract 797, 2006. de vico g, carella f. argomenti di patologia comparata dei molluschi: aspetti ecologici e sanitari, loffredo editore, naples, 2012. díaz s, villalba a, insua a, soudant p, fernándeztajes j, méndez j, et al. apoptosis frequency in cockles cerastoderma edule. j. invertebr. pathol. 113: 214-219, 2013. dianzani um, dianzani i, dianzani u. istituzioni di patologia generale, utet edizioni, torino, 2005. goodson ms, crookes-goodson wj, kimbell jr, mcfall-ngai mj. characterization and role of p53 family members in the symbiont-induced morphogenesis of the euprymna scolopes light organ. biol. bull. 211: 7-17, 2006. green m, alderman dj. neoplasia in mytilus edulis l. from united kingdom waters. aquaculture 30: 1-10, 1983. kelley ml, winge p, heaney jd, stephens re, farell jh, van beneden rj, et al. expression of homologues for p53 and p73 in the softshell clam (mya arenaria), a naturally-occurring model for human cancer. oncogene 20: 748758, 2001. lowe dm, moore mn. cytology and quantitative cytochemistry of a proliferative atypical haemocytic condition in mytilus edulis. j. natl. cancer inst. 60: 1455-1459, 1978. muttray af, cox rl, st-jean s, van poppelen p, reinisch cl, baldwin sa. identification and phylogenetic comparison of p53 in two distinct mussel species (mytilus). comp. biochem. physiol. 140c: 237-250, 2005. muttray af, cox rl, reinisch cl, baldwin sa. identification of delta n isoform and polyadenylation site choice variants in molluscan p53/73-like homologs. mar. biotech. 9: 217-230, 2007. olberding ke, kelley ml, butler ra, van beneden rj. a hect e3 ubiquitin-protein ligase with sequence similarity to e6ap does not target p53 for degradation in the softshell clam (mya arenaria). mutat. res. 552: 61-71, 2004. siah a, delaporte m, pariseau j, mckenna p, berthe fc. patterns of p53, p73 and mortalin gene expression associated with haemocyte 20 polyploidy in the soft-shell clam, mya arenaria. j. invertebr. pathol. 98: 148-152, 2008. walker cw, van beneden rj, muttray af, böttger sa, kelley ml, tucker ae, et al. p53 superfamily proteins in marine bivalve cancer and stress biology. adv. mar. biol. 59: 1-36, 2011. st-jean sd, stephens re, courtenay sc, and reinisch cl. detecting p53 family proteins in leukemia cells of mytilus edulis from pictou harbour, nova scotia. can. j. fish. aquat. sci. 62; 2055-2066, 2005. walker cw. mortalin in invertebrates and the induction of apoptosis by wild-type p53 following defeat of mortalin-based cytoplasmic sequestration in cancerous clam hemocytes. in: kaul sc, wadhwa r (eds), mortalin biology: life, stress and death, chapter 6, 97 doi 10.1007/978-94-007-3027-4_6, © springer science+business media, 2012. tascedda f, ottaviani e. tumors in invertebrates. inv. surv. j. 11: 197-203, 2014. van beneden rj,walker cw, laughner es. characterization of gene expression of a p53 homologue in the sift-shell clam (mya arenaria). mol. mar. biol. biotechnol. 6: 116-122, 1997. villalba a, carballal mj, lopez c. disseminated neoplasia and large foci indicating heavy haemocytic infiltration in cockles cerastoderma edule from galicia (nw spain). dis. aquat. org. 46: 213-216, 2001. wadhwa r, sugihara t, hasan mk, taira k, reddel rr, kaul sc. a major functional difference between the mouse and human arf tumor suppressor proteins. j. biol. chem. 277: 3666536670, 2002. 21 research report isj 11: 240-246, 2014 issn 1824-307x research report immune response of phyllophaga polyphylla larvae is not an effective barrier against metarhizium pingshaense jn enríquez-vara1,2, aw guzmán-franco1, r alatorre-rosas1, h gonzález-hernández1, a córdoba-aguilar2, j contreras-garduño3 1postgrado en fitosanidad, especialidad en entomología y acarología, colegio de postgraduados, km 36.5 carretera méxico-texcoco, montecillo, texcoco, edo. de méxico, 56230, méxico 2departamento de ecología evolutiva, instituto de ecología, universidad nacional autónoma de méxico, apdo. p. 70-275, circuito exterior, ciudad universitaria, 04510,coyoacán, distrito federal, méxico 3departamento de biología, división de ciencias naturales y exactas, universidad de guanajuato, campus guanajuato, noria alta s/n, noria alta, 36050, guanajuato, guanajuato, méxico accepted september 3, 2014 abstract previous research has uncovered that the cuticle of p. polyphylla larvae acts as a good nonimmunological barrier against m. pingshaense. in the present study we investigated whether p. polyphylla larvae also show a similarly robust immunological response against m. pingshaense. firstly, we estimated a median lethal dose (ld50) of blastospores to be injected into the hemocoel. secondly, we injected the estimated ld50 of blastospores into the hemocoel of larvae to quantify phenoloxidase (po), nitric oxide (no) and antimicrobial activity as a response against fungal invasion. in contrast to a previous report that showed that m. pingshaense is unable to kill p. polyphylla after topical applications, here we demonstrate that: (a) 100 % of p. polyphylla larvae died when blastospores were injected into the hemocoel and (b) when injecting the ld50 into the hemocoel of the larvae, immune response did not differ with control. our results imply that immunological responses do not protect p. polyphylla larvae against m. pingshaense infections. thus, the cuticle seems a better defense mechanism compared to po, no and antimicrobial activity. one proximate explanation for our results is that blastospores are not detected by the host’s immune machinery. an ultimate explanation is that there may be a resource-based tradeoff between non-immunological and immunological barriers, in which white grubs may be investing more in cuticle at the cost of po, no and antimicrobial activity. key words: non-immunological barriers; ecoimmunology; white grubs; metarhizium pingshaense   introduction understanding the basis of host resistance is an intriguing biological phenomenon given that pathogens are ubiquitous and impose a strong ___________________________________________________________________________ corresponding authors: jhony n. enríquez-vara postgrado en fitosanidad especialidad en entomología y acarología colegio de postgraduados km 36.5 carretera méxico-texcoco, montecillo texcoco, edo. de méxico, 56230, méxico e-mail: jhnavaten@gmail.com 240   jorge contreras-garduño departamento de biología división de ciencias naturales y exactas universidad de guanajuato, campus guanajuato noria alta s/n, noria alta, 36050 guanajuato, guanajuato, méxico e-mail: jcont@ecologia.unam.mx selective pressure on their host (schmid-hempel, 2011). in both vertebrates and invertebrates, resistance consists of both non-immunological and immunological barriers (hart, 2011; parker et al., 2011). the former could be a behavioral, mechanical and/or hostile environment against invaders (smilanich et al., 2009). on the other hand, the immunological defence prevents foreign agents to cause infection by humoral and cellular components. it is assumed that non-immunological and immunological barriers can complement each other’s defensive action against parasites and/or pathogens (e.g. dubovsky et al., 2013; fedorka et al., 2013; reviewed by moreno-garcía et al., 2013). however, just how complementary both barrier types are has not been studied in detail (parker et al., 2011). one pathogen type towards which a host can use both type of barriers is the mailto:jhnavaten@gmail.com mailto:jcont@ecologia.unam.mx 241   entomopathogenic fungi (lundgren and jurat fuentes, 2012; reviewed by arsavanitis et al., 2013). these fungi use mechanical pressure and enzymatic degradation to damage the insect cuticle and penetrate the host (hajek and st. leger, 1994). hence, the cuticle represents a first nonimmunological barrier against entomopathogens (wilson et al., 2001). however, after injury (i.e., by the fungus penetration), the insect host responds to fungal infection by producing antimicrobial peptides and a cellular response to protect the hemolymph from invasion (lemaitre and hoffmann, 2007). once the pathogen penetrates the cuticle, the insect immune response (humoral and/or cellular) in the hemolymph attacks the fungi by phagocytosis, lytic activity and the activation of phenoloxidase (po), the latter producing nodule formation, encapsulation and melanization (lavine and strand, 2005; bogus et al., 2007). in the present study we have investigated the immune response of the white grub, phyllophaga polyphylla, when infected with the entomopathogenic fungi metarhizium pingshaense. white grubs are soil dwelling herbivore insects that continuously interact with a large variety of pathogens (jackson and klein, 2006), including entomopathogenic fungi. the wide use of these microorganisms to regulate white grub species (shah and pell, 2003) has demonstrated a differential susceptibility of these insects to fungal infection (rodríguez del bosque et al., 2005; morales-rodríguez et al., 2010; nong et al., 2011; guzmán-franco et al., 2012). previous studies showed that p. polyphylla larvae are fairly resistant to infection by m. pingshaense when immersed in a conidial suspension of this fungus, with mortality never exceeding 20 % after 36 days of incubation (enríquez-vara et al., 2012; guzmán-franco et al., 2012). thus, these studies concluded that cuticle acts as a fairly good non-immunological barrier. however, whether the same defensive capacity applies when the insect’s immune system is challenged, is unclear. given that 20 % of infected animals is high enough, one would expect that, if the fungus penetrates the insect, immune response should complement the defensive action of the cuticle. one approach for testing this is via artificially by-passing the mechanical barrier imposed by the cuticle by injecting blastospores (the fungal form that multiplies inside the insect) into the host hemocoel. using this approach, we had two aims in the present study: a) finding a median lethal dose (ld50) of m. pingshaense blastospores; and, b) measuring immune response of p. polyphylla larvae after injection with different doses of m. pingshaense. for immune response, we assessed po, antimicrobial activity and nitric oxide (no), three key actors in the defense against parasites and pathogens in insects (reviewed in beckage, 2008). materials and methods insects third-instar phyllophaga polyphylla larvae were collected from corn fields in guanajuato, mexico (20° 02´30.12” n, 100 ° 28´36.4”). once collected, the larvae maintained individually in plastic cups (100 ml) at 20 °c with damp peat moss (growing mix®, canada) for 4 weeks before they were used in the experiment. production of blastospores the fungus metarhizium pingshaense isolate gc01 was used. enríquez-vara et al. (2012) and gúzman-franco et al. (2012) used this isolate against p. polyphylla. in both works the isolate gc01 was referred as m. anisopliae (morphospecie) but carrillo-benítez et al. (2013) used molecular methods to demonstrate that the isolate gc01 is indeed m. pingshaense. hence we will refer to the isolate gc01 as m. pinshaense. first, conidia were produced in petri dishes containing sabouraud dextrose agar medium (sda). after 20 days of incubation at 25 ºc in complete darkness, conidia from the medium were harvested with a sterile scalpel. conidia and mycelium were deposited into a sterile 50 ml volume centrifuge tube containing 30 ml of 0.03 % tween 80. the mixture of conidia and mycelium was stirred for 15 min. conidia were separated from mycelium by filtration trough sterile cloth and deposited into a new sterile 50 ml volume centrifuge tube. conidia concentration was estimated using a haemocytometer. conidial suspension was then inoculated and grown in 50 ml of sterile liquid medium containing yeast extract, sucrose and tween 80 (2:2:0.4 p/v). liquid medium contained in a 250 ml erlenmeyer flask and with a concentration of 1x106 con ml was incubated on a shaker at 120 rpm at 28 °c for three days. blastospores were harvested by filtration through sterile cloth and, to remove any remaining liquid medium, the suspension was centrifuged three times at 10,000 rpm for 10 min and suspended in phosphate buffered saline solution ph 7.4 (pbs) (sigma). the concentration of blastospores was determined using a haemocytometer. the percentage of viable blastospores was estimated prior to experiments using the plate count technique on sda (goettel and inglis 1997). in all cases more than 95 % were viable. survival of p. polyphylla larvae injected with m. pingshaense blastospores different groups of 30 third-instar p. polyphylla larvae were injected with different doses of blastospores of m. pingshaense (103, 104, 105 and 106 blastospores, in a total volume of 5 µl per larva) suspended in pbs. before injection, white grubs were anesthetized on ice and immobilized. the blastospore suspension was injected into the larvae hemocel through the dorsal surface at the junction between the second and third abdominal segments. injections were carried out using a 30-gauge needle fitted to a 1 ml syringe mounted on a calibrated micro-applicator. as control group, larvae were only injected with pbs. the larvae were transferred individually to 12-well cell culture plates (costar ®, corning inc. ny, usa) (1 larva per well), which contained a 2 cm diameter filter paper which had been moistened with 80 µl of sterile distilled water. the 12-well culture plates were incubated at 25 °c 242   in complete darkness and mortality was assessed every 24 h for 10 days. dead larvae were incubated at 25 ºc and 100 % rh for 7 10 days, to encourage sporulation thereby allowing fungal infection to be confirmed. data were analyzed using kaplan-meier survival curves, and the log-rank test was used to evaluate statistical differences between white grubs injected with pbs only or with different doses of blastospores of m. pingshaense. kaplanmeier survival curves were constructed for each treatment. the log-rank test was used to compare survival amongst curves constructed for each treatment. immune response of p. polyphylla against m. pingshaense infection the immune response of p. polyphylla against m. pingshaense infection was estimated by quantifying the production of po, no and antimicrobial activity in the insect´s hemolymph as a response to infection (see below). to achieve this, a lethal dose (ld50) concentration of blastospores was injected into the hemolymph. injecting a ld50 increased the survival time before death thereby allowing immune parameters to be quantified. the ld50 was estimated by dose-response assays. estimation of ld50 the estimation of ld50 was obtained using the same methodology described above with some modifications. twelve third-instar p. polyphylla larvae were exposed to four doses. based on the results of the previous experiment, a different set of doses was selected 5.103, 1.104, 5.104 and 1.105 blastospores of m. pingshaense in pbs. the complete experiment was repeated on two different occasions. larval mortality was recorded every 24 h for five days. mortality was corrected using abbott´s formula (abbott, 1925). data from the bioassays were analysed using a generalized linear model with binomial error and probit link in the statistical package genstat v. 8.0 (payne et al., 2005). the numbers of infected larvae were assumed to follow a binomial distribution with sample sizes equal to the number of larvae tested. before combining two replicates, a parallel model analysis was done for each replicate. first, a single line was fitted to data from replicates. second, intercepts were allowed to vary amongst the replicates and third, slopes were also allowed to vary amongst the replicates. if the single line model was the best for each replicate, then data from the two replicates could be combined. concentration causing 50 % infection (ld50) of larvae was estimated from best fit model and confidence interval for ld50 was calculated according to fieller’s theorem (fieller, 1944). quantification of immune parameters five groups of 12 larvae each were injected with the ld50 estimated previously (5.103 blastospores) to quantify po, no and antimicrobial production. these parameters were estimated in the hemolymph of larvae at five different times after injection (0, 2, 6, 12 and 24 h). the immune response of each of the five different times after injection was estimated in a different group of 12 larvae. a different set of five groups of 12 larvae were injected with only pbs and treated as described before. a total of 120 larvae were used for both treatments and times of quantification. all treated larvae were maintained as described before until hemolymph was collected. hemolymph collection for the hemolymph collection, the integument of each larva was surface sterilized with 70 % ethanol and then rinsed twice using sterile distilled water. hemolymph samples were obtained by cutting the third thoracic leg of each larva and four drops (approximate 30 µl) of hemolymph were collected into sterile and precooled eppendorf tubes (1.5 ml) containing 100 µl of pbs, and vortexed for 10 s. the mixture was centrifuged for 10 min at 10, 000 rpm and 4 ºc to remove hemocytes and cell debris. the supernatant was divided into three aliquots, two of 50 µl and one of 30 µl. the two 50 µl aliquots were mixed separately with 50 µl of pbs, while the third was placed in a 0.5 ml eppendorf tube and kept at -80 °c until required. the first 50 µl subsample was used to measure protein hemolymph content and po activity. the second subsample was used to estimate no production, and the 30 µl sample was used to estimate antimicrobial activity. all measurements were carried out immediately after hemolymph collection. protein content proteins were measured using the bca (pierce biotechnology, rockford, il) protein assay kit with bsa as the protein standard. two replicates of 10 µl of hemolymph/pbs mixture were used to measure the protein in each sample (see enríquez-vara et al., 2012). the absorbance was measured on a varioskan flash microplate reader (thermo fhiser scientific, waltham, ma) at 562 nm. po activity hemolymph po activity was measured using the method described by enríquez-vara et al. (2012). briefly, an aliquot that contained 40 µg of protein was placed in a 96 well microplate (corning inc, corning ny), and then dose-tittered to a volume of 50 µl of sample and pbs. to this mixture, 50 µl of l-dopa (4 mg/ml) was added to obtain a final volume of 100 μl. po activity was assayed spectrophotometrically with dopamine as a substrate. the slope of the curve was calculated by using the optical density at 490 nm. optical density readings were taken every minute for one hour at 30 °c (enríquez-vara et al., 2012). no production a colorimetric nitrate/nitrite assay kit (sigma) was used to prepare the standard curve and to estimate no in each sample following the manufacturer’s instructions. the basis of this technique is that nitric oxide is a highly unstable radical that rapidly reacts with other oxygen-reactive species to form stable products, such as nitrites, nitrates and toxic radicals (i.e., peroxynitrite). hence, the total nitrate and nitrite content is used to indirectly estimate the amount of nitric oxide in each sample. the amount of no (µm) in samples was fig. 1 survival of third-instar larvae of phyllophaga polyphylla injected with different concentrations of blastospores of metarhizium pingshaense. *p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, denote differences in survival between concentrations of blastospores and pbs lines by log-rank statistics. the n.s. indicates that the survival were not significantly different between lines. estimated by extrapolation with a standard curve of known concentrations. readings were performed at 540 nm. antimicrobial activity antimicrobial activity in the haemolymph samples was measured using the cup agar-diffusion assay technique (mohring and messner, 1968). the microbial activity was measured according to the methods described by bogus et al. (2007), with some modifications. briefly, the assays were performed in 90-mm petri dishes containing 66 mm sörensen buffer, ph 6.4 (10 ml), using micrococcus lysodeiticus (7 mg) as substrate, agar (100 mg) and streptomycin sulfate (0.7 mg). hemolymph samples (4 µl) free of insect cells were added to the petri dishes, samples formed a circle of three mm in diameter. the diameters of the lytic zones around the three mm diameter samples were measured after incubation of the petri dishes for 24 h at 37 °c. the antimicrobial activity of the insect hemolymph samples was expressed in equivalents of chicken egg white lysozyme. increasing concentrations of lysozyme (10 1000 ug/ml) were used as a standard for comparisons. the quantities of no and antimicrobial activity found were so small that we were unable to record them. data from po activity were analysed using analysis of variance (anova) with the statistical package sas v. 9.0 (sas, north carolina,usa). we compared po measurements between pbs (control) and injected with blastospores treatments, and their interaction with the time after inoculation. po data were lntransformed to meet assumption of normal distribution and equality of variances. results survival of p. polyphylla larvae injected with m. pingshaense blastospores significant differences were found in the survival rates of p. polyphylla larvae amongst all treatments compared (x2 = 145.86; p < 0.05; fig. 1). when the larvae were injected with 1.105 and 1.106 blastospores of m. pingshaense, some died within the first 48 hours post-injection and 100 % mortality was recorded at 120 hours. an intermediate effect in survival was obtained when the larvae were injected with 1.104 blastospores (fig. 1). the survival rate in larvae injected with 1.103 blastospores and larvae in the pbs control were similar and mortality was never greater than 10 % (fig. 1). immune response of p. polyphylla against m. pingshaense infection estimation of ld50 no evidence of non-parallelism (χi2 = 0.22, p > 0.05) or differences in intercepts (χi2= 2.93, p > 0.05) amongst replicates were found, justifying the pooling of data from separate replicates for further analyses. the ld50 value estimated for m. pingshaense was 5.2x103 (ci=3.3x103-7.5x103) blastospores. therefore, larvae were injected with 243   fig. 2 slope po activity expression according to pbs or blastospores and time. sample size was 12 larvae per time point and treatment. each bar indicates mean ± se. the n.s. indicates that the po activity were not significantly different between pbs and blastospores. the ld50 estimated to assess immune response parameters. quantification of immune parameters after injection of blastospores, only po production was activated. neither no nor antimicrobial activity was recorded. po production was similar in larvae injected with blastospores and pbs (f 1,150 = 0.16, p > 0.05, fig. 2), and this result was consistent throughout all measurement times (f4,150= 1.75, p > 0.05, fig. 2). discussion by injecting blastospores directly into the hemocel of white grub larvae, we found that nearly 100 % of individuals were infected after 120 h of incubation at the greatest doses. in perspective with previous studies using the same host-pathogen interaction (enríquez-vara et al., 2012; guzmánfranco et al., 2012), it seems that the p. polyphylla cuticle is a more effective barrier against m. pingshaense than po, no and antimicrobial activity. related to this, bogus et al. (2007) found that mortality associated with the topical application of conidia was explained by the cuticle thickness in three insect larvae. the cuticle may be important as a barrier in soil systems because it prevents the negative impact of abiotic factors (i.e., the damage to the cuticle due to friction with the soil), and therefore may favour resistance against a wide variety of pathogens and parasites (villani et al., 1999). as the epicuticle is more variable in its components than the procuticle, this could be implicated in the differential insect resistance to invaders (see golebiowski et al., 2008). it is unclear why the po quantities did not differ between the pbs and blastospore treated larvae. we propose that blastospores of m. pingshaense were not detected by the cellular or humoral innate immune response of p. polyphylla. related to this, it is known that the entomopathogenic fungi must be discreet to avoid being recognized as a foreign agent by the host’s immune response (wang and st. leger 2005; vilcinskas, 2010). poprawski and yule (1991) injected 3.106 spores of metarhizium anisopliae in phyllophaga anxia larvae, obtaining 42 % mortality. interestingly, we injected only 1.106 blastospores, which produced 100 % mortality. it is likely that spores are better detected by the insect immune response than blastospores (wang and st. leger, 2006), as well as by the fact that blastospores are the stage of replication of the fungus leading to a faster invasion of the host´s hemocel, which is not the case for spores (gillespie et al., 2000). notice, however, that the immune response caused by a fungal infection may vary according to the distinct host and fungal pathogen species. for example, when the fungus conidiobolus coronatus were inoculated in different insect species, the immune response based on the po levels determined varied: po levels decreased when g. mellonella was inoculated whereas no modification was found in diprion pini, but an increase was observed in calliphora vicina (bogus et al., 2007). in relation to metarhizium, even the 244   245   same host species can produce different immune responses. for example, g. mellonella larvae infected with m. anisopliae reduced po activity (slepneva et al., 2003), a result that could not be corroborated when the same host and fungus species were used (dubovskiy et al., 2013). again, one mechanism is that blastospores may become undetected. an explanation is needed as for why immune response was so reduced (po) or non-existent (no and antimicrobial activity) in p. polyphylla larvae in the face of a fungal infection. this may be related to the evolutionary ecology of immune response. it is known that non-immunological and immunological barriers are costly to produce and so their costs can limit the expression of other traits (schmid-hempel, 2005; mckean and lazzaro 2011; parker et al., 2011). for example, in acheta domesticus the investment in cuticle thickness (a nonimmunological barrier) decreased egg production and adult body size (bascuñan et al., 2010). as for immunological barriers, ardia et al. (2012) found that in insects, encapsulation response led to increased levels of po and co2 production but decreased levels of lysozyme. on the other hand, both barriers can conflict each other’s expression. in support of this, immunological barriers are inefficient in species whose non-immunological barriers are effective at combating parasites or pathogens (parker et al., 2011). for example, dubovskiy et al. (2013) found that po and lytic activity were lower in hemolymph that in cuticle in g. mellonella against m. anisopliae. thus, one explanation for our results is that the efficiency of the cuticle against m. pingshaense is traded-off with po, no and antimicrobial activity. in practical terms, the studies by enríquez-vara et al. (2012), guzmán-franco et al. (2012) and those we have shown here suggest that entomopathogenic fungi could be used as a strategy to control white grubs. however, since fungi need to break the cuticle to penetrate the insect, one way to facilitate fungal infection is to use nematodes. to this aim, we suggest the use of heterorhabditis nematodes (bedding and molyneux, 1982). future experiments should see whether this strategy is viable. acknowledgements jn enriquez-vara had a scholarship grant from the consejo nacional de ciencia y tecnología (conacyt). fundación produce guanajuato a.c. 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var. acridum. eukaryot. cell 4: 937-947, 2005. wang c, st. leger rj. a collagenous protective coat enable metarhizium anisopliae to evade insect immune responses. proc. natl. acad. sci. usa 103: 6647-6652, 2006. wilson k, cotter sc, reeson af, pell jk. melanism and disease resistance in insects. ecol. lett. 4: 637-649, 2001. time profiling molecular toxicity agnp isj 10: 69-76, 2013 issn 1824-307x review earthworm’s immunity in the nanomaterial world: new room, future challenges y hayashia,b ,p engelmannc a department of bioscience terrestrial ecology, aarhus university, vejlsøvej 25, 8600 silkeborg, denmark b inano interdisciplinary nanoscience center, aarhus university, gustav wieds vej 14, 8000 aarhus c, denmark c department of immunology and biotechnology, clinical center, university of pécs, szigeti u. 12, pécs h-7643, hungary accepted august 22, 2013 abstract since the advent of the nanotechnology era, the environmental sink has been continuously receiving engineered nanomaterials as well as their derivatives. our current understanding of the potential impact of nanomaterials on invertebrate immunity is limited to only a handful of initial studies including those on earthworms. recently, we reported selective accumulation of silver nanoparticles in the amoebocyte population of eisenia fetida coelomocytes in vitro. in this review, we give an overview of available literature on the life-history impacts on earthworms, and what we have learnt of the immune responses to nanoparticles with references to other invertebrate species and vertebrate counterparts. we discuss the significant contribution of amoebocytes as nanoparticle scavengers and suggest a possibility of studying inter-cellular communications in coelomocytes. implications from the leading researches in vertebrate models tell us that study of the nanoparticle recognition involved in cellular uptake as well as suband inter-cellular events may uncover further intriguing insights into earthworm’s immunity in the nanomaterial world. key words: nanomaterials, silver nanoparticles, immunogenicity, earthworms, coelomocytes, uptake   introduction it was not until the advent of the nanotechnology era that earthworms were on the verge of encountering the “unknown” delivered by the modern human society. the current definition of a nanomaterial adopted by european commission states “a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50 % or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm 100 nm”. naturally occurring ultrafine particles of the same size range have existed long before the emergence of nanotechnology. yet, already in 2005 the extensive review by oberdörster and colleagues (2005) pointed out possible scenarios of engineered nanomaterials posing threats to ecosystem health. the major concern stemming from rapid development and commercialisation of nanotechnology products is uncertainty of such novel formulations in the modes of action and routes of exposure to living organisms. ___________________________________________________________________________ corresponding author: yuya hayashi aarhus university, the inano house gustav wieds vej 14, 8000 aarhus c, denmark. e-mail: yuya@inano.au.dk the environmental sink has received, and is expected to continue receiving, commercially produced nanomaterials as well as their derivatives, environmental transformations and the fate of which have yet to be elucidated in particular in the soil milieus (reviewed in tourinho et al., 2012). immunity is a vital function to maintain organism’s well-being, and represents a sensitive physiological indicator that may be affected even at low concentrations of nanomaterials exposure. only a handful of studies exist so far to aid the current understanding of immune responses to nanomaterials in invertebrates, particularly earthworms. this includes our recent in vitro study on eisenia fetida exposed to silver nanoparticles (agnps) (hayashi et al., 2012) supporting molecular responses observed in vivo (hayashi et al., in press). studies on other earthworm species have been reported by van der ploeg and co-workers, where lumbricus rubellus was exposed to the carbon-based nanoparticle c60 fullerene in vivo (2013) and in vitro (2012) and likewise exposed to agnps (unpublished), as well as partially by the work of hooper et al. (2011) with e. veneta exposed to zinc oxide nps. in this review we seek to recapitulate what has been learnt from the initial studies on the effects of 69   mailto:yuya@inano.au.dk nanoparticles (nps) on earthworm immunity, with references to other invertebrate species and vertebrate counterparts. the upshot is that different types of immunocytes may respond to nps in a distinct manner intimately linked to the cell’s ability, and that previously uncharacterised aspects of earthworm immunity are emerging in the light of the nanotechnology era. sub-lethal impacts of nanomaterials on earthworms limited numbers of studies are currently available in the literature on the impact of nanomaterials on earthworms. carbon-based nanomaterials can affect the life-history traits of e. veneta (scott-fordsmand et al., 2008), e. fetida (li and alvarez, 2011) and l. rubellus (van der ploeg et al., 2011). common to all was reduced reproduction, which could result in a significant decrease in the population growth rate (van der ploeg et al., 2011). c60 fullerenes are also suggested to bioaccumulate in earthworms (e. fetida) following soil exposure (li et al., 2010), whereas carbon nanotubes did not accumulate in e. fetida (petersen et al., 2008a) or l. variegatus (petersen et al., 2008b) when depuration of the gut content was allowed. ecotoxicological screening of metal-based nanomaterials indicated significant reproductive failure in e. fetida exposed to silver or copper nps (heckmann et al., 2011). different types (size and coatings) of agnps disturb earthworm’s reproductive capacity at 500-1000 mg/kg soil in e. fetida (heckmann et al., 2011; shoults-wilson et al., 2011c; 2011b), and in l. rubellus significant reproductive toxicity was observed at concentrations as low as 154 mg/kg soil when the agnps were well dispersed in soil (van der ploeg et al., unpublished). bioaccumulation of agnps in earthworms seems relatively low (coutris et al., 2012; shoults-wilson et al., 2011c; 2011b; van der ploeg et al., unpublished), however, more sensitive endpoints indicate that earthworm’s physiological traits are affected already at sub-100 mg/kg soil, for example, avoidance (shoults-wilson et al., 2011a), enzyme activities (hu et al., 2012), oxidative stress responses (tsyusko et al., 2012) and tissue apoptosis (lapied et al., 2010). other types of nps (e.g. alumina, titania and zinc oxide) have also been tested on earthworms and reviewed elsewhere (tourinho et al., 2012), and two types of nps (copper and gold) were observed to biodistribute across the tissues, partly if not all, persisting their nanoparticulate forms (unrine et al., 2010a; 2010b). scarcely studied, however, is the potential immunological effect of nanomaterials; which leads us to limit our focus of the review on carbon-based nps and agnps, where insightful observations were revisited in this context. immunogenicity of nanomaterials recognition, uptake and inflammation: cutting-edge studies in vertebrates before remarking rather limited knowledge of the effects of nanomaterials on earthworm’s immunity, we begin by capturing potentially relevant prospects that arise from studies of mammalian cells. in general to all eukaryotic cellular machineries size/shape and surface chemistry of nanomaterials are the central parameters in the interaction with immune systems, for example, via biological ligand-receptor signalling (reviewed in dobrovolskaia and mcneil, 2007). within the past few years the concepts of dynamic assembly of nps and biomolecules were established (shemetov et al., 2012) and this has shed light on how the cells “see” and interact with nps in the context of receptor-mediated responses (reviewed in monopoli et al., 2012). supporting this notion, evidence of np uptake via conventional endocytic (e.g. lesniak et al., 2013; wang et al., in press) and phagocytic (e.g. lunov et al., 2011) pathways is emerging. this has a direct implication to innate cellular immunity, which relies mainly on non-self pattern recognition and macromolecule-marking (opsonisation) of particles for phagocytic clearance. on the contrary, relatively less explored is the inflammatory potential of nps as a result of direct receptor activation (e.g. bastús et al., 2009). this is beautifully vindicated by the activation of integrin receptor signalling in thp-1 cells (a human acute monocytic leukemia cell line) through binding of fibrinogen unfolded upon interactions with nps (deng et al., 2011). inflammatory responses can also be induced indirectly resulting from oxidative stress, a frequently described mode of action of nps (e.g. hayashi et al., 2012). agnps are a good example of nanomaterials that involve oxidative stress (foldbjerg et al., 2012; hayashi et al., 2012; jiang et al., 2013) and modulate pro-inflammatory cytokines including a catalogue of interleukins and tnf-α (reviewed in klippstein et al., 2010). earthworm’s immune responses to nanomaterials the relative simplicity of invertebrate immunity offers a potentially sensitive and accessible means of disentangling the complex interactions of nps and immune cells. to address this challenge, we have developed an in vitro model of e. fetida coelomocytes. in our recent report, we observed at the molecular level a cascade of stress responses initiating from oxidative stress genes to immune genes downstream following short-term exposure to agnps in coelomocytes and in thp-1 cells (hayashi et al., 2012). a similar set of genes was affected with a temporal shift when the worms were exposed in vivo to the agnps in a soil matrix (hayashi et al., in press). from evolutionary perspectives, a similarity between e. fetida coelomocytes and thp1 cells in the expression patterns of catalase (repressed over time), a well-known oxidative stress response gene, and myeloid differentiation factor 88 (myd88, induced over time), which encodes a central adaptor protein of toll-like receptors, was an intriguing observation that may comprise a part of immune responses to agnps in earthworms but also across the animal kingdom (hayashi et al., 2012). signal transduction, primarily through mitogen activated protein kinase (mapk) pathways, appears to coordinate the cross-talk between oxidative stress and immune responses to agnps, as implicated in the expression profiles of mek kinase 1 (mekk1) gene both in coelomocytes and thp-1 cells (hayashi et al., 2012), as well as in vivo 70   fig. 1 light (a) and scanning electron (b, c and d) micrographs of eisenia fetida coelomocytes. the light micrograph was imaged at x200 magnification with a phase-contrast microscopy. the scanning electron micrographs were imaged on the coelomocytes after paraformaldehyde fixation and gold-sputtering (nominal 30 nm in thickness). from morphological observations, panels b and c appear to be amoebocyte populations while panel d is most likely a chloragocyte (characterised by the granule-rich feature of chloragosomes). in earthworms (hayashi et al., in press). phosphorylation states of mapk families in earthworm coelomocytes were not examined, whereas in vitro studies of nps using haemocytes from mytilus species suggest rapid activation of mapk cascade members, namely p38 and jnks, with subsequent nitric oxide production (canesi et al., 2008; 2010a). the authors further reported effects on other related traits, such as oxidative burst and lysozyme activity. interestingly, these observations were made with the np concentrations at which lysosomal membrane stability, a sensitive indicator of cell viability, was not affected. when the molluscs were exposed in vivo to nps in an aquatic system, the digestive gland appeared as the likely target of nps with signatures of oxidative stress and haemocyte damages (canesi et al., 2010b). similarly, haemocytes from freshwater mussels (dreissena sp.) exposed in vivo to titania nps accumulated the nps intracellularly and showed reduced phagocytic activity as well as activation of erk1/2 and p38 mapk families (couleau et al., 2012). van der ploeg and colleagues (2013) exposed l. rubellus earthworms in vivo to c60 fullerenes for two different exposure durations (4-weeks and lifelong), and in both cases observed suppression of heat shock protein 70 (hsp70) gene while enzymes involved in antioxidant mechanisms were unaffected. of particular interest is significant suppression of coelomic cytolytic factor 1 (ccf1) gene in the lifelong experiment (van der ploeg et al., 2013); ccf1 is a known pattern recognition receptor in earthworm’s immunity (for review see bilej et al., 2010). tissue injuries without histological signatures of inflammation were observed both in 4 71   weeksand, albeit less severely, lifelong-exposed worms in parallel with decreased ccf1 expression (van der ploeg et al., 2013). on this basis, the authors suggested immunosuppressive effects of c60, rather than a result of coelomocyte mortality. this was supported by their in vitro work, in which coelomocytes from l. rubellus were viable at a wide range of c60 concentrations while ccf1 was downregulated in concurrence with reduced phagocytic activity (van der ploeg et al., 2012). although these initial studies are only indicative of the extent to which the nanomaterials may interfere with the function of earthworm’s immune systems, early warnings are already given; that nanomaterials are a new class of environmental contaminants posing potential threats to earthworms. dissecting earthworm’s innate immunity earthworm immune system consists of cellular (coelomocytes) and humoral components (antimicrobial, cytolytic and pattern recognition molecules) directed towards non-self materials in a natural non-specific manner (reviewed in cooper et al., 2002). cytochemical, immunological and functional approaches characterised three major subpopulations (morphological observations by light and electron micrographs are shown in fig. 1), among which hyaline and granular amoebocytes participate in the cellular effector mechanisms (e.g. phagocytosis and encapsulation) while chloragocytes (eleocytes) contribute more to homeostasis and humoral immunity (engelmann et al., 2004; 2005; opper et al., 2010). fig. 2 illustrates the potential room for future challenges in facing the nanotechnology era, supported with evidence in earthworms or other multicellular organisms. amoebocytes as the scavenger of nanomaterials coelomocytes of l. rubellus, largely lacking free-circulating chloragocytes (cholewa et al., 2006), proved capable of internalising polymeric nps (hydrodynamic diameter of 45 ± 5 nm) apparently involving energy-dependent transport mechanisms (clathrinand caveolin-mediated endocytosis pathways) (van der ploeg et al., 2012). our recent study suggests that the hyaline subgroup of amoebocytes and pma-differentiated macrophage-like thp-1 cells, but not the monocytic phenotype of thp-1 cells, can accumulate agnps of the primary particle sizes around 83 ± 22 nm (hayashi et al., 2012). amongst the coelomocyte populations, the hyaline amoebocytes are known to adhere and engulf bacteria (engelmann et al., 2005), and may thus be considered as the invertebrate counterpart of macrophages. although np uptake mechanisms are largely unknown in coelomocytes, the macrophage-like thp-1 cells appear to effect macropinocytosis for the uptake of negatively-charged nps (lunov et al., 2011). in mammals, macropinocytosis initiates with cell membrane ruffling via actin rearrangement, suggesting an intriguing possibility of passive uptake of nps that are membrane-adhered (fig. 2). amongst invertebrates, ascidian haemocytes are able to engulf particles via a rgd motif-dependent macropinocytosis (ballarin and burighel, 2006), however, such mechanisms are not yet known in earthworms. another potential phagocytic pathway is via scavenger receptor class a that is expressed by both human macrophages and macrophage-like thp-1 cells, but not by monocytic thp-1 cells (lunov et al., 2011). scavenger receptors are conserved pattern recognition receptors known to bind lipids (lipopolysaccharides and modified lowdensity lipoproteins) and polyanions for phagocytosis. in particular, a macrophage receptor with collagenous structure (marco) is known to recognise and associate with nps for phagocytic clearance in mammalian cells (kanno et al., 2007). in invertebrates, haemocytes from insects (franc et al., 1996) and molluscs (liu et al., 2011) are known to effect scavenger receptor-mediated uptake of pathogens and apoptotic cells. to date, scavenger receptors are yet to be identified in earthworms; however, their ubiquitous presence suggests an unequivocally conserved role in innate immune recognition that may be involved in np uptake as in vertebrate counterparts (fig. 2). toxicological implications arising from selective cellular uptake of nanomaterials are profound. of metal-based nanomaterials that readily dissolve and liberate bioactive metal ions, agnps represent the most well-studied type of nps (e.g. liu et al., 2010). free ag+ ions, the product of oxidative dissolution, itself is highly biologically active and reacts with biomolecules (e.g. proteins and dna) of the cellular components in a similar manner as reactive oxygen species (ros). agnps and ag+ ions co-exist extracellularly and/or intracellularly, indicating a multitude of stress pathways not limited to those for the nanoparticulate form but including contribution of liquid-phase silver (e.g. beer et al., 2012; yang et al., 2011). intracellular uptake of agnps is likely to involve subsequent fusion with lysosomes that may accelerate oxidative dissolution of agnps under the acidic milieu (jiang et al., 2013). this implies that agnps may have a targeted impact on amoebocytes as a result of preferential accumulation and subsequent in situ molecular damages by liberated ag+ ions (hayashi et al., 2012; see fig. 2). time-course profiling of representative gene expressions, in parallel with flow-cytometric analysis of the intracellular ros level, favour the view that the amoebocyte populations are under oxidative stress that can signal-transduce to immune cascades downstream (hayashi et al., 2012; and fig. 2). recently, the amoebocyte populations, but not chloragocytes, were found to recruit calcium for activation (e.g. homa et al., 2013) and that they may possess a similar biochemistry of calcium signalling as in higher organisms linking stress responses to activation of immune systems (opper et al., 2010). studies on how agnps affect calcium signalling in amoebocytes may further illuminate the cross-talk between stress and immune responses as known for another highly-conserved signal transduction cascade, mapk pathways (fig. 2). 72   fig. 2. schematic illustrating the room and future challenges to progress further in our understanding of the effects of nanomaterials on earthworm’s immunity. see the main text for references to each heading. headings with a question mark show subjects that are relatively less understood in coelomocytes and in vertebrates. drawings are not to scale. me+; metal ions, ros; reactive oxygen species. inter-cellular communications: an indirect effect? phagocytes secrete cytokines as a biological means of cellular communication to initiate e.g. inflammation but also other immunological functions, such as acute phase responses by liver cells. as described earlier in this review, cytokine secretion/modulation is a documented effect of nps while much less is known for secondary impacts related to altered cytokine profiles (fig. 2). direct evidence, however, has not been discovered for conserved and novel cytokines in earthworms. in contrast, other invertebrate organisms (especially insects) have already provided sufficient data for cytokine-mediated immune functions (e.g. haematopoiesis) (malagoli, 2010; malagoli et al., 2012; söderhäll et al., 2005). the conservation and existence of proinflammatory cytokines in earthworms are not a fairy-tale; earlier we observed positive reactions of coelomocytes to monoclonal antibodies targeted for mammalian tnf-α (engelmann et al., 2002), and recently the work of fuller-espie and colleagues (2008) supports enhanced phagocytic activity of hyaline amoebocytes treated with mammalian cytokines (notably il-1β, il-2 and tnf-α). the coelomic fluid of earthworms is sometimes assumed in the immunological context equivalent to blood plasma in mammals, both representing a protein-rich immune-competent circulatory system. distinct from the mammalian counterpart is the existence of (migratory and sessile) chloragocytes involved in the regulation of essential minerals, haemoglobins and metallothioneins in response to natural stressors (molnár et al., 2012). this is probably by functional analogy with the hepatic/renal systems of vertebrates, and chloragocytes may contribute to regulation of the total protein balance in the coelomic fluid. for example in echinoderm, immunostimulants and invertebrate cytokines were able to regulate secretion of acute phase proteins from coelomocytes (beck et al., 2002). its relation to immunostimulation remains unclear, but in eisenia earthworms the expression and secretion of the cytolytic/antimicrobial molecule lysenin was increased in migratory chloragocytes upon gram73   positive bacterial challenge of total coelomocytes (opper et al., 2013). given that these characteristics partly resemble those of vertebrate hepatic cells, acute phase response in coelomocytes may await further investigations using nanomaterials as a tool to selectively target amoebocytes and modulate cytokine profiles. this also suggests an interesting feature of using coelomocytes as a mixedpopulation model integrating the cell-to-cell signalling events (especially of immune effector cells to hepatic-like cells) that is otherwise difficult to study in vertebrate in vitro models of monocultured cell lines. we also note that secretion of humoral factors and deposition of chloragosomes (specialised lysosome-like structures of chloragocytes) may lead to dynamic interactions of nps and these biomolecules (fig. 2). understanding immune responses to nanomaterials: the challenges in the light of our current understanding in nanomaterials and their immunogenicity, we have in this review given an account for a summary of available literature on the life-history impacts of nanomaterials on earthworms, and what we have learnt of the immune responses to nanomaterials. the phagocytic population of the coelomocytes, namely hyaline amoebocytes, seems a susceptible target of nanomaterials, as a result of which indirect responses by chloragocytes are conceivable. environmental toxicants (such as pesticides and heavy metals) compromise the host’s immune system. these pollutants inhibit the cellular immune functions (e.g. phagocytosis) while humoral components (e.g. lysozyme production) are elevated. at the dawn of knowledge about the impact of nanomaterials on invertebrate immunity, it is still obscured whether they influence cellular and/or humoral arms of immunity in a fashion that has not been previously documented. we have discussed a significant contribution of amoebocytes as np scavengers and proposed a possibility of studying inter-cellular communications in coelomocytes. implications from the leading researches in vertebrate models tell us that study of the np recognition involved in cellular uptake as well as suband inter-cellular events may uncover further intriguing insights into earthworm’s immunity in the nanomaterial world. acknowledgements we are grateful to three anonymous reviewers for their valuable comments on the manuscript. we acknowledge the financial support of the danish research council (fuu 1-5971-10000377) to yh, and of the medical faculty research foundation, university of pécs (pte áok-ka 2013/09) as well as berde botond visiting fellowship to pe. references ballarin 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gondikas ap, marinakos sm, auffan m, liu j, hsu-kim h, et al. mechanism of silver nanoparticle toxicity is dependent on dissolved silver and surface coating in caenorhabditis elegans. environ. sci. technol. 46: 1119-1127, 2011. 76   abstract review isj 10: 94-101, 2013 issn 1824-307x review the role of histones in the immune responses of aquatic invertebrates c nikapitiya1*, t dorrington2*, m gómez-chiarri3 1department of aqualife medicine, chonnam national university, chonnam 550-749, republic of korea 2center for structural molecular biology (cebime), department,of biochemistry, federal university of santa catarina, 88040-900 florianópolis, sc, brazil 3department of fisheries, animal and veterinary science, university of rhode island, 169 cbls, kingston, ri 02881, usa *equal contribution accepted october 4, 2013 abstract histones are primary components of eukaryotic chromatin and highly abundant in all animal cells. in addition to their important role in chromatin structure and transcriptional regulation, histones contribute to innate immune responses. in several aquatic invertebrate species, as well as in many other invertebrate and vertebrate species, the transcripts for core histones are upregulated in response to immune challenge and exposure to environmental stressors. histones show antimicrobial activity against bacteria and parasites in vitro and in vivo and have the ability to bind bacterial lipopolysaccharide and other pathogen-associated molecules. several mechanisms regulating and facilitating the antimicrobial action of histones against pathogens have been described in vertebrate and some invertebrate species, including the production of extracellular traps (ets) and the accumulation of histones in lipid droplets that can be selectively released in response to immune stimuli. further studies are needed to determine the mechanisms of action of histones in immune responses in aquatic invertebrates and investigate the potential use of histones in the treatment of infectious diseases in aquaculture. key words: antimicrobial responses; bivalves; histones; immunity; invertebrates; mollusks; pathogenic challenge introduction histones are major components of chromatin in eukaryotic cells, playing key roles in dna replication, repair, and recombination, transcriptional regulation, and cell growth control (parseghian and luhrs, 2006). the core histones (h2a, h2b, h3, and h4) constitute the basic structure of the nucleosome by forming an octameric complex, whereas the linker histones (h1) are involved in the generation of the higher-order chromatin structure which seal loops of dna and maintain the nucleosome structures condensed in a compact conformation. due to their important structural role, histones are very abundant in animal cells and most are highly conserved evolutionarily throughout eukaryotes. interestingly, ___________________________________________________________________________ corresponding author: marta gómez-chiarri department of fisheries, animal and veterinary sciences university of rhode island 169 cbls, 120 flagg road, kingston, ri 02881, usa email: gomezchi@uri.edu several other roles for histones have recently been described, including roles in cellular signaling and innate immunity (parseghian and luhrs, 2006; brinkmann and zychlinsky, 2012). histones have long been known to have antimicrobial activity (miller et al., 1942; hirsch, 1958), probably due to their cationic charge, which they share with many antimicrobial peptides. we review here evidence supporting a role for histones in antimicrobial defenses, with a special focus on aquatic invertebrate species, many of them of commercial interest for the aquaculture industry. we also speculate on the potential mechanisms of action of histones in the immune responses of invertebrate species based on evidence from studies in vertebrates and a few model and non-model invertebrate species. the evidence provided in this review and others (e.g. smith et al., 2010; noga et al., 2011) suggests that histones may be a target for use in the treatment and prevention of infectious diseases in aquaculture. 94 immune challenge leads to up-regulation of histones recent advances in gene library construction, sequencing, and cdna microarray technology have enabled the characterization of gene expression patterns in non-model species like aquatic invertebrates in response to a variety of environmental stressors, including immune challenges. these studies have revealed a large number of immune-related sequences, including the homologues for many genes involved in pathogen recognition, signaling, antioxidant and antimicrobial responses, and apoptosis, showing that aquatic invertebrates express many of the same genes that have previously been described in other invertebrates and vertebrates in response to viral, bacterial, or parasitic challenge (messier-solek et al., 2010; gosh et al., 2011; li et al., 2011; romero et al., 2012). among the many immune-related genes that have been described in these studies, several histone genes have been shown to be highly upregulated in aquatic invertebrates in response to pathogenic challenge and/or to possess antimicrobial activity (table 1). an up-regulation of histone gene expression in response to immune challenge is not restricted to aquatic invertebrates, but is a feature shared with several other invertebrate and vertebrate species. to provide some examples, rhesus macaque monkey kidney epithelial cells exposed to monkey pox virus, showed a steep up-regulation of histone genes (alkhalil et al., 2010), while zebrafish skin exposed to bacterium citrobacter freundii showed significant up-regulation of linker histone-like protein h1m (lu et al., 2012). these patterns of differential expression of histones in response to immune challenge suggest a potential role for histones in innate immunity. immune challenges, however, are not the only conditions in which histone up-regulation has been observed in aquatic organisms. for example, histones are up-regulated in rapidly growing oyster larvae (meyer and manahan, 2010), in oyster adults and larvae in response to many different environmental stressors (chapman et al., 2011; zhang et al., 2012), and in mussels maintained along a copper pollution gradient in the field (dondero et al., 2006), suggesting that histone gene up-regulation may be a general response to environmental stress (robinette and noga, 1998). there is additional evidence, however, supporting a role for histones in innate immunity. we review this evidence in the sections below. a role for histones in immune responses: histones have antimicrobial and lps-binding activity. the first report of the effects of histones on bacteria was by miller et al., (1942), who showed that calf thymus histones and histone-like cationic proteins (protamines) inhibited respiration in grampositive and gram-negative bacteria. hirsch (1958) demonstrated the antimicrobial activity of argininerich histones (h3 and h4) against a variety of bacteria under different ph and salt concentrations. decades later, hiemstra et al., (1993) identified three molecules with antimicrobial activity isolated from the lysosomal fraction of murine macrophages as lysine–rich histones h1 and h2b, suggesting that histones may have a role in immune defenses. subsequently, molecules with antimicrobial activity isolated from cells and tissues from a variety of organisms were identified as histones or histonederived fragments. antimicrobial molecules identified as histones (h1, h2a, h2b) by peptide sequencing have been described, among others, from a diverse set of tissues from humans and other terrestrial vertebrates (reviewed in parseghian and luhrs, 2006; kawasaki and iwamuro, 2008) and the tissues of many aquatic vertebrates (finfish, reviewed in smith et al., 2010). many peptides with antimicrobial activity are derived from the proteolytic digestion of intact histones. these include buforins i and ii, originally isolated from the korean frog bufo bufo gargarizans (cho et al., 2002; park et al., 1996; cho et al., 2009), oncorhyncin ii, an antimicrobial peptide derived from histone h1 isolated from rainbow trout (fernandes et al., 2004), parasin i (residues 1-19 of histone h2a), isolated from the skin of catfish parasilurus asotus (park et al., 1998), and hipposin (residues 1-51 of histone h2a), isolated from the atlantic halibut hippoglossus hippoglossus (birkemo et al., 2003). histones, either purified from biological samples or produced using recombinant technology, show antimicrobial activity in vitro against a variety of pathogens, including gram-negative bacteria like aeromonas and vibrios, gram-positive bacteria, fungi, viruses, and protozoa (reviewed in kawasaki and iwamuro, 2008). for example, histone (h2b) purified from skin secretions of o. mykiss exhibits powerful anti-bacterial activity against gram-positive bacteria with a minimal inhibitory concentration (mic) in the submicromolar range (fernandes et al., 2002). purified olive flounder histone h1-like protein extract from testis shows antimicrobial activity against gram-negative bacteria with minimal effective concentrations (mecs) of 1.4-12.0 μg/ml, gram-positive bacteria with mecs of 2.8-30.0 µg/ml, and yeast candida albicans with a mec of 2.0 μg/ml (nam et al., 2012). furthermore, recombinant human histones h2a and h2b efficiently kill promastigotes of the parasites leishmania amazonensis, l. major, l. braziliensis, and l. mexicana (wang et al., 2011). histones also have antimicrobial activity against several protozoan fish pathogens, including water molds (saprolegnia sp.) and the dinoflagellate amyloodinium ocellatum (noga et al., 2011). there is relatively less evidence of antimicrobial activity for histones h3 and h4. purified histones h2a and h4 from human meconium have antimicrobial activity (kai-larsen et al., 2007). in addition, histone h4 contributes to the antimicrobial activity of extracts of seb-1 sebocytes against staphylococcus aureus, and recombinant histone h4 showed antimicrobial activity against s. aureus and propionibacterium acnes (lee et al., 2009). invertebrate histones and histone-derived peptides also show antimicrobial activity against a wide range of microorganisms. a mix of core histone proteins h2a, h2b, h3, and h4, isolated from the 95 table 1 evidence supporting a role for histones in immune responses in aquatic invertebrate species species histone type accession number upregulation to challenge with technology used evidence for antimicrobial activity reference biomphalaria glabrata h4 dq117979 echinostoma caproni mass spectrometry and cdna not done bouchut et al., 2007 chlamys farreri h2a (39 n-terminal aa) dq418455 not induced by bacteria cdna yes (in vitro) li et al., 2007b c. farreri h1oo acute viral necrobiotic virus ssh not done chen et al., 2013 crassostrea virginica h4 hm130521 perkinsus marinus ssh yes (in vitro and in vivo) dorrington et al., 2011 c. virginica h2b bg624428 p. marinus est (microarray) not done wang et al., 2010 c. virginica h3.3 bg624455 p. marinus est (microarray) not done wang et al., 2010 c. virginica h2b-1 not done hplc yes (in vitro) seo et al., 2010 c. virginica h2b-2, 3, 4 not done hplc not done seo et al., 2011 haliotis discus discus h2aderived abhisin ef103384 bacteria g+/cdna yes (in vitro) de zoysa et al., 2009 macrobrachium rosenbergii h2a hg001454 viral and bacterial htg yes (in vitro) arockiaraj et al., 2013 meretrix casta and 4 other spp. h2a molluskin hq720143, hq720145 hq720148 not done pcr no sathyan et al., 2012 ruditapes philippinarum h2a 06-r-h12 (clone number) perkinsus olseni est not done kang et al., 2006 abbrevations: aa: amino acids; cdna: complementary dna; est: expressed sequence tags; hplc: high pressure liquid chromatography; htg: high throughput genomics,; ssh: suppression subtractive hybridization. hemocytes of the pacific white shrimp, have antimicrobial activity against micrococcus luteus. complete inhibition of the growth of a gram-positive bacterium was observed at 4.5 µm of purified histone h2a. on the other hand, a mixture of histones h2b and h4 was active at 3 µm (patat et al., 2004). recently, a recombinant form of h2a from the freshwater prawn macrobrachium rosenbergii was shown to possess antimicrobial activity against several gram-negative and grampositive bacteria (arockiaraj et al., 2013). in mollusks, histone h2b from the american oyster c. virginica has strong activity against gram-negative vibrio parahaemolyticus and v. vulnificus, two 96 human pathogens present in seafood (seo et al., 2010). an ortholog for buforin-i (1-39 residues of h2a), characterized in the scallop chlamys farreri, has broad-spectrum growth inhibitory activity against both gram-negative and gram-positive bacteria (li et al., 2007). further, abhisin derived from n-terminus of the abalone histone h2a (80% amino acid identity with buforin i), inhibits the growth of gram-positive and gram-negative bacteria and yeast (de zoysa et al., 2009). we have shown that recombinant histones h4 and h2b from the african clawed frog, xenopus laevis, which in the case of histone h4 is 100% identical at the amino acid level with those of other species such as the american oyster crassostrea virginica, have antimicrobial activity against the gram-negative bacteria escherichia coli and vibrio anguillarum, and the gram-positive micrococcus luteus at micromolar concentrations (dorrington et al., 2011). histone h4 protein levels of oyster in hemocyte lysate and cell free hemolymph are significantly increased in oysters experimentally challenged with the protozoan parasite perkinsus marinus, but neither histone h2b nor histone h4 inhibit the growth of this pathogen of oysters at the highest concentration tested (20 µm). these results suggest that p. marinus may have evolved resistance to the antimicrobial effects of histones (dorrington et al., 2011). alternatively, histone h4 up-regulation may be a general response to the stress caused by the pathogenic challenge (chapman et al., 2011; zhang et al., 2012) or histone h4 may have an indirect role in immunity against this protozoan parasite. further research needs to be done to determine the role of histones on immune defenses in oysters. interestingly, delivery of yeast cells expressing the recombinant american oyster histone h4 into the gut of brine shrimp artemia salina artificially challenged with v. anguillarum showed a significant and dose-dependent decrease of the bacterial load in brine shrimp (dorrington et al., 2011). these results support the idea that histones are potentially useful molecules in the development of novel drugs for therapeutic use against pathogenic infections in aquaculture (smith et al., 2010; noga et al., 2011). further, these experiments validate the use of oyster histone h4 in a yeast feed-based delivery system for the treatment of bacterial infection in aquaculture applications. another fascinating property of histones is their lipopolysaccharide (lps) binding ability. in humans, histones h2a and h2b show dose-dependent inhibition of the endotoxin activity of lps through binding to and the blocking of both the core and lipid a moieties of lps (augusto et al., 2003). therefore, binding of histones to lps released from gramnegative bacteria may also block the production of cytokines such as tumor necrosis factor alpha (tnfα) that can often leads to fatalities from toxic shock in humans (kim et al., 2002). the lps binding site is located in the c-terminal region of histones and all histones (but h2a1 and h4 in particular) from calf thymus are able to bind lps, with a greater affinity than the lps-binding antibiotic polymixin b (augusto et al., 2003). histones also have the ability to bind viral proteins like the human immunodeficiency virus (hiv) envelope glycoprotein gp120 and its receptor cd4 (mamikonyan et al., 2008). evidence of the potential role of histones as pattern recognition receptors (prrs) is the identification and characterization in catfish of a histone-like protein (similar to linker histone h1) in the membranes of non-specific cytotoxic cells (ncamp-1) that recognizes bacterial dna, oligodeoxynucleotides, and polyguanosine motifs, and has antimicrobial activity (connor et al., 2009). further research is needed to determine if histones have similar properties in invertebrate species. mechanisms of action of histone and histone derived compounds in immunity organisms have evolved specific mechanisms tightly regulating the motility and presence of free histones in the cytoplasm, membranes, and extracellular fluids (parseghian and luhrs, 2006), as well as facilitating the antimicrobial activity of histones against pathogens. one of such mechanisms is the formation of extracellular traps (ets), which was first described in vertebrate neutrophils. ets are part of the innate immune response and function through the release by dying neutrophils of granular proteins (such as elastase and myeloperoxidase) and chromatin (dna and histones) that form extracellular fibers to trap and kill both gram-negative and gram-positive bacteria, yeast, and parasites (brinkmann et al., 2004; urban et al., 2006; brinkmann and zychlinsky, 2007; guimaraes-costa et al., 2009; urban et al., 2009; brinkmann and zychlinsky, 2012; saffarzadeh et al., 2012). these fibers are covered in histones, which bring them into contact with bacteria more effectively, facilitating their antimicrobial and lpsbinding activities. neutrophils releasing ets undergo a particular cell death process named netosis that appears distinct from apoptosis and necrosis, is induced by inflammatory stimuli and pathogen-associated molecular patterns (pamps) such as lps and phorbolmyristate acetate (pma), and depends on the formation of radical oxygen species. during neutrophil cell death bynetosis, intracellular organelle membranes disintegrate leading to the formation of a complex composed of nuclear and cytoplasmic components (fuchs et al., 2007; metzler et al., 2011; mesa and vasquez, 2013). this phenomenon of nets production has also been described in fish such as the fathead minnow (palic et al., 2007a), zebrafish (palic et al., 2007b), goldfish (katzenback and belosevic, 2009), and carp (pijanowski et al., 2013). production of ets has also been observed in immune cells other than neutrophils (goldman and medina, 2012). the formation of ets has been also reported in a few invertebrate species, including the greater wax moth galleria mellonella (altincicek et al., 2008), marine crabs (unpublished results reported in smith et al., 2010), and the pacific white shrimp (ng et al., 2013). in g. mellonella, a small percentage of hemocytes appear to be the source of nets containing extracellular nucleic acids in the hemolymph during coagulation, which then induce degranulation of granulocytes and bacterial entrapment (altincicek et al., 2008). these studies suggest that a system of defense homologous to the nets is present in 97 http://www.copewithcytokines.de/cope.cgi?key=cytokine%20concentrations%20in%20biological%20fluids http://www.copewithcytokines.de/cope.cgi?key=saccharomyces%20cerevisiae http://www.copewithcytokines.de/cope.cgi?key=cell%20types fig. 1 potential roles and mechanisms of action of histones on immune responses in invertebrates. see text for a detailed explanation. amp: antimicrobial peptide; et: extracellular trap; lps: bacterial lipopolysaccharide; pamp: pathogen-associated molecular pattern; ros: radical oxygen species. several invertebrates and may be an ancient defense mechanism (smith et al., 2010). an alternative mechanism of action for histones in immunity has been recently described in the fruit fly drosophila melanogaster. histones bound to lipid droplets (fat storage organelles) in the cytosol of cells are released upon immunological stimuli, killing bacteria. furthermore, genetically modified flies lacking histones in lipid droplets are more susceptible to bacterial infection, and droplet-bound histones enhance fly survival to bacterial challenge (anand et al., 2012). these authors also describe that exposure of mice to bacterial stimuli leads to accumulation of histone in lipid droplets in the liver, suggesting that this may be an ancient immune defense strategy conserved through evolution (anand et al., 2012). another potential role for a histone on immunity has been described in an invertebrate model species, the nematode caenorhabditis elegans (studencka et al., 2011). a variant of linker histone h1 (his-24) interacts with heterochromatin protein 1 to regulate the transcription of many immune-related genes. interestingly, bacterial challenge leads to localization of a proportion of his-24 to the cytoplasm of intestinal cells in infected worms, which would facilitate the interaction of the histone with pathogens. these changes in localization of his-24 were dependent on the methylation status of the protein (studencka et al., 2011). based on the existing evidence in vertebrate and invertebrate organisms presented in this review, we hypothesize that histones could have multiple mechanisms of action in invertebrates (fig. 1). exposure of hemocytes to immune stimuli leads to up-regulation of the expression of histone genes. histones may be released after hemocyte cell death caused by apoptosis or a process similar to etosis in invertebrate hemocytes. additionally, in hemocytes and possibly in other cells, histones stored in lipid droplets may also be released to extracellular spaces upon immune stimuli. histones and fragments of histones derived from proteolytic activity, in combination with other molecules released into the hemolymph, would then be able to exert their antimicrobial and lps-binding activities against extracellular pathogens. based on their ability to bind dna and lps, histones may also be present on the surface of hemocytes, serving as receptors for pathogen associated molecular patterns (pamps). finally, histones may also be localized to granules and other structures in the 98 cytoplasm upon pathogenic challenge to become involved in pathogen killing intracellularly within phagosomes, in combination with radical oxygen species (ros) and other antimicrobial molecules. concluding remarks evidence supporting a role for histones in immune responses in invertebrate and vertebrate species include: 1) up-regulation in response to immune challenge, 2) biochemical identification of molecules with antimicrobial activity as histones or histone-derived peptides and 3) the ability of recombinant histones to kill a variety of pathogens and bind bacterial lps and other pamps. the mode of action of histones in immunity appears to involve mechanisms that facilitate direct contact of concentrated histones with pathogens, such as the release of extracellular traps (ets) as shown in many vertebrates and a few invertebrates (altincicek et al., 2008; brinkman et al., 2004; brinkman and zychlinsky 2007; ng et al., 2013) or the formation of lipid droplets in drosophila (anand et al., 2012). it has been postulated that antimicrobial histones have evolved as an ancient innate defense system component against pathogenic microorganisms that may have been coopted from the structural role of these abundant proteins as components of the chromatin structure of eukaryotic organisms (kawasaki and iwamuro, 2008; smith et al., 2010). it will be important to study the mechanisms that specifically regulate the transcription of selected histone genes in response to pathogenic challenge. histones are encoded by multigene families that occur in clusters (marzluff et al., 2002; albig et al., 2003), allowing for the differential expression of selected genes for histones subtypes (alami et al., 2003). it will also be important to study the potential role of posttranscriptional modifications on histone localization to immune-relevant organelles (ouvry-patat and schey, 2007; studencka et al., 2011). furthermore, due to the high levels of amino acid conservation of several histones, and in particular histone h4, between many diverse species, these proteins and peptides can be considered universal antimicrobial agents. histones and histone-derived peptides could be potentially useful targets in the development of novel drugs for the prevention and treatment of infectious diseases in the aquaculture industry. further studies should be done to determine mechanisms of action of histones in the immune responses of aquatic invertebrates, which would aid in the improved design of treatments (smith et al., 2010; noga et al., 2011). acknowledgements we thank two anonymous reviewers for their useful suggestions. our research was made possible by usdanricgp and nifa awards 200001264 and 2009-38925-19971, seagrant oyster disease research program award 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(lep: pyralidae) and their response to thermal stress v ghasemi1, s moharramipour1, jj sendi2 1department of entomology, faculty of agriculture, tarbiat modares university, tehran, iran 2department of plant protection, faculty of agricultural sciences, university of guilan, rasht, iran accepted october 27, 2013 abstract the hemocytes of ephestia kuehniella zell. are arranged in five main classes: prohemocytes (prs), plasmatocytes (pls), granulocytes (grs), oenocytoids (oes) and spherulocytes (sps). two other morphotypes of hemocytes namely vermicytes (ves) and podocytes (pos) were also distinguished in hemolymph of this species. hemogram was studied in five developmental stages of the insect including early ivth, late ivth, early vth and late vth instar larvae as well as prepupa. results indicated that total hemocyte count (thc) increased gradually with increasing in developmental stages and reached their maximal level in prepupa. also, total cell numbers in the late ivth and vth instar larvae and prepupa were significantly higher than that in the early ivth and vth instar larvae. an increasing trend was found in hemolymph volume (hv) and number of hemocytes in circulation (hic) with increasing in developmental stages of tested insect. pls and grs were the two abundant morphotypes of hemocytes in most stages. mitotic index (mi) of hemocytes was found to be high in the early part of each stage than in the later larval stage and in prepupa. heat and chill stresses were also assessed on hemocytes of 2-days old vth instar larvae. the findings revealed that high temperature (40 °c) caused a significant increase in thc principally pls, oes and the mi along with a drastic reduction in number of grs and hv. in contrast, chilling (4 °c) led to a significant reduction in thc, proportion of pls and mi with increase in counts of oes. changes in the cytological features of the hemocytes of incubated larvae are also discussed. key words: ephestia kuehniella; hemocytes; hemogram; heat and chill stress; cytological features introduction innate immune system of insects is classified as cellular and humoral responses. cellular immunity is hemocyte-mediated reactions like phagocytosis, nodule formation and encapsulation (lackie, 1988; strand and pech 1995; gillespie et al., 1997; irving et al., 2005). insect hemogram and their peculiar hemocyte combination in each stage are important as a case for development of environmental adaptability (sharma et al., 2008) since some vital physiological functions are performed by hemocytes. an exact analysis of insect hemogram and their hemocyte population dynamics throughout developmental stages should involve the simultaneous approach to a series of parameters including total hemocyte count (thc), ___________________________________________________________________________ corresponding author: saeid moharramipour department of entomology, faculty of agriculture tarbiat modares university p. o. box 14115-336, tehran, iran e-mail: moharami@modares.ac.ir differential hemocyte count (dhc), hemolymph volume (hv), absolute number of hemocytes in circulation (hic), mitosis index (mi), maximum daily production (mdp) of hemocytes through mitosis occurred in circulation and so on (arnold and hinks 1976; salt, 1968; shapiro, 1979). it is proved that insect hematological parameters are affected by biotic factors i.e. pathogens, nematodes and parasitoids and abiotic ones i.e. age, eclosion, sex, pesticides and starvation (sanjayan et al., 1996; sharma et al., 2008). like other abiotic factors, changes in temperature may also affect the hemocytic immune responses of insects. t here are several articles demonstrating the effects of temperature on hematology in lepidopterous insects. for example, pandey et al. (2010) showed that heat stress caused a significant variation in hemocytic immune responses of tropical tasar silkworm, antheraea mylitta (drury). in another study on danaus chrysippus larvae, it was demonstrated that chilling caused a decline in the number of hemocytes, however, heating elicited an 128 mailto:moharami@modares.ac.ir a b c d e f g h i j k fig. 1 light microscopy pictures of e. kuehniella hemocytes stained with giemsa. prohemocyte (a); plasmatocyte (b); granulocyte (c); oenocytoid (d); spherulocyte (e); vermicyte (f); podocyte (g); agglomeration of hemocytes (h); mitotic features of hemocytes: anaphase (i); telophase (j); cytokinesis (k). scale bar = 5 µm increase in circulating cells (pandey et al., 2008). also, mowlds and kavanagh (2008) indicated that incubation of galleria mellonella l. larvae at 4 or 37 °c for 24 h prior to infection by the yeast candida albicans berkhout led to an increase in hemocyte count and to the expression of genes encoding for antimicrobial peptides. nevertheless, no information is available on hematology of the mediterranean flour moth, ephestia kuehniella zell. so, in this study we emphasize on hemocyte profile and effect of temperature on composition of hemocytes of this species. e. kuehniella is a major pest of stored grain products, particularly flour. more importantly, its table 1 mean and range of head capsule width of first to fifth instar larvae of e . kuehniella head capsule width (mm) instar n* min max mean dyar’s coefficient 1 50 0.15 0.24 0.19 1.684 2 50 0.28 0.39 0.32 1.687 3 50 0.47 0.60 0.51 1.627 4 50 0.76 0.87 0.83 1.397 5 50 1.00 1.33 1.16 *n, no. of individuals examined 129 a b c d e f g h i fig. 2 phase contrast micrographs of e. kuehniella hemocytes. prohemocyte (a); plasmatocyte (b); granulocyte (c); oenocytoid (d); spherulocyte (e); podocyte (f); mitotic features of hemocytes: anaphase (g); telophase (h); cytokinesis (i). scale bar = 5 µm. eggs and larvae are widely used as a substitute host for the laboratory rearing of parasitoids and predators aimed for biological control (de clarcq et al., 2005; kim and riedl 2005; hamasaki and matsui 2006; paust et al., 2008) and research into the behaviour, biochemistry and molecular biology (corbet, 1973; rahman et al., 2004; rahman et al., 2007; jamoussi et al., 2009). some biological features like easy rearing under laboratory conditions, no need to special diet and short life span make this insect of great potential to serve as valuable material for laboratory research and commercial usage. in mass rearing programs, low quality and high mortality of insects may often occur due to fecal and microbial contamination especially fungi and bacteria (singh, 1977; stone and sims 1992). each microbial isolate has a range of temperature in which it grows most aggressively. for example, bugeme et al. (2009) found that the best germination and radial growth of the various isolates of beauveria bassiana (bals.) vuill. and 23 isolates of metarhizium anisopliae (metschnik.) sorok occurred at 25 and 30 °c. interestingly, insects can exploit this weakness by elevating their body temperature from optimum temperature of microbes (25-30 °c) in response to an immune 130 fig. 3 scanning electron microscopy of e . kuehniella hemocytes. prohemocyte (a); plasmatocyte (b); granulocyte (c); oenocytoid (d); spherulocyte (e); vermicyte (f). scale bar = 5 µm. attack through thermoregulatory behaviour. in this case, mostafa et al. (2005) reported that temperature and relative humidity had significant effects on cellular defense response of e. kuehniella larvae fed bacillus thuringiensis berliner. some earlier studies in arthropods have also shown that they respond to microbial infection and parasitism by altering their thermoregulatory behaviour and developing a behavioral fever (kluger et al., 1996). increased body temperature above normal levels through selection of warmer habitats has been shown to favor host survival in a number of host– pathogen associations (bronstein and conner 1984; louis et al., 1986; boorstein and ewald 1987; watson et al., 1993; inglis et al., 1996; adamo, 1998; karban, 1998; elliot et al., 2002). the above statements indicate the importance of temperature on immune responses of insects to microbes. this work represents a starting point in the study of hemocyte production in e. kuehniella and the importance of fever behaviour during immune challenges. the first aim of presented research is morphological characterization of circulating hemocytes. as larval instars are used for laboratory rearing of parasitoids, the hemocyte composition was determined in various developmental stages of this species. moreover, the changes in hemogram and hemocyte population of two-days old fifth instar larvae were evaluated after heating and chilling stress. table 2 micrometric measurements of hemocytes of e .. kuehniella size (µm) cytoplasm nucleus cell morphotype* width (mean ± se) length (mean ± se) width (mean ± se) length (mean ± se) pr 4.8-7.7 (6.2 ± 0.18) 5.0-7.8 (6.3 ± 0.18) 3.1-5.3 (4.0 ± 0.15) 3.2-5.3 (4.0 ± 0.15) pl 4.6-9.5 (7.3 ± 0.26) 10.1-18.2 (14.4 ± 0.47) 2.5-6.9 (5.5 ± 0.27) 3.6-8.1 (6.4 ± 0.27) gr 7.5-9.4 (8.8 ± 0.19) 8.7-11.3 (9.5 ± 0.23) 3.6-5.0 (4.5 ± 0.12) 3.8-5.1 (4.7 ± 0.10) oe 6.7-17.7 (9.8 ± 1.00) 7.1-19.4 (11.0 ± 1.11) 2.7-6.3 (3.7 ± 0.32) 2.9-6.5 (3.8 ± 0.32) sp 8.5-16.5 (14.2 ± 0.98) 10.5-17.9 (15.8 ± 0.85) 3.7-7.2 (4.7 ± 0.30) 3.6-7.4 (4.9 ± 0.31) ve 4.0-8.0 (4.9 ± 0.38) 23.0-34.4 (27.0 ± 1.06) 2.9-5.1 (3.8 ± 0.25) 4.0-8.0 (5.4 ± 0.45) po 3.4-5.1 (4.3 ± 0.15) 17.3-23.3 (20.9 ± 0.68) 3.0-4.2 (3.5 ± 0.13) 3.2-4.5 (3.8 ± 0.14) *pr, prohemocyte; pl, plasmatocyte; gr, granulocyte; oe, oenocytoid; sp, spherulocyte; ve, vermicyte; po, podocyte (n=30) 131 e e e b** a** c** d* cns bc ns bcn s b ab ab a 0 5 10 15 20 25 30 35 40 45 20000 30000 40000 50000 60000 70000 80000 90000 0 10 20 30 40 50 60 te m pe ra tu re ( °c ) th c (c el ls /m m 3 ) time (h) thc treatment thc control temperature (°c) a fig. 4 effect of heating (a) and chilling (b) on total hemocyte count of e . kuehniella larvae at different time intervals. each data point is the mean ± se of at least 10 individual. different letters indicate significant differences between groups (anova, p < 0.05). significant differences versus the respective controls were marked by **p< 0.01, *p < 0.05 and ns non significant at p < 0.05, student's t-test. materials and methods experimental insects eggs of ephestia kuehniella were provided by the insectarium of scientific and industrial research organization of iran. each 300 eggs were reared in a plastic container (25 cm length, 15 cm width, and 10 cm height) half filled with mixture of equal amounts of wheat flour and bran. powdered yeast (5 g) was also added to each jar. the rearing conditions were 26±1 °c, 75 % relative humidity in darkness. different larval instars of this moth were determined in accordance with the dyar’s law through the measurement of the head capsule width (dyar, 1890). also, body weight of different developmental stages of e. kuehniella was measured. hematological assays were carried out on the following developmental stages of the insect: l1: two-days old ivth instar larvae (2 days after ecdysis into the forth instar), l2: five-days old ivth instar larvae (2 days before ecdysis into the fifth instar), l3: two-days old vth instar larvae (2 days after ecdysis into the fifth instar), l4: six-days old vth instar larvae (2 days before prepupa stage) and l5: one-day old prepupa (pp). hemolymph collection the hemolymph was collected from subjected insect by amputating the foreleg with a pair of fine scissors sterilized in 70 % ethanol. a a ab c b a a dns d ns cd** cd** bc** b ** a** 0 5 10 15 20 25 30 35 40 45 20000 25000 30000 35000 40000 45000 0 10 20 30 40 50 60 te m pe ra tu re ( °c ) th c (c el ls /m m 3 ) time (h) b 132 table 3 body weight, hemolymph volume (hv), total hemocyte count (thc), hemocytes in circulation (hic) and daily hemocyte production by mitosis (mdp) in each developmental stage studied of e . kuehniella phase body weight (mg) hv (μl) thc (no. of cells×104/mm3) hic (no. of cells×104) mdp (no. of cells×103 /day) l1 8.15 ± 0.19e 1.05 ± 0.06d 1.31 ± 3.00×102e 1.38 ± 0.08×104d 1.09 ± 0.65×102c l2 12.22 ± 0.37d 1.44 ± 0.07d 1.94 ± 8.24×102d 2.82 ± 0.20×104d 0.52 ± 0.39×102c l3 19.77 ± 0.44c 3.81 ± 0.23c 3.18 ± 9.44×102c 1.21 ± 0.94×104c 11.37 ± 8.83×102a l4 27.18 ± 0.29a 6.04 ± 0.24b 4.36 ± 7.46×102b 2.64 ± 1.23×104b 6.21 ± 2.90×102b l5 22.58 ± 0.37b 6.90 ± 0.22a 5.64 ± 12.0×102a 3.83 ± 1.20×104a 6.53 ± 2.01×102b means with the same letters in each column are not significantly different at p < 0.05, tukey's test. values are expressed as mean ± se (n = 50). hemocyte profile light microscopy studies the hemocyte monolayers were prepared on clean glass slides and placed at room temperature for 20 min till dry. then, the hemocytes were stained with giemsa (diluted 7 times with distilled water and filtered before use) for 25 min, differentiated in saturated solution of lithium carbonate for 30 seconds and then washed in distilled water. after drying, permanent microscopy slides were prepared using canada balsam. hemocytes were observed under a light microscope (olympus bx51) and photographed with a dp 72 camera system powered with dic nomarski. phase contrast microscopy studies five µl of fresh hemolymph was mixed with an equal amount of anticoagulant buffer (naoh, 98 mm; nacl, 186 mm; edta, 17 mm; citric acid, 41 mm; ph 4.5) on clean glass slides and covered with a cover slip. since hemocytes begin to deteriorate after bleeding, a fresh preparation was made every 8-10 min. the samples were examined under the phase contrast microscope (olympus bx51) and images were taken using a dino-eye am 7023 b camera. scanning electron microscopy (sem) for sem studies, 5 µl of fresh hemolymph was mixed with an equal amount of anticoagulant buffer on clean, grease-free 1 cm2 microscope slides and smears were made by drawing a second slide across the first one at a 45° angle. the smears were allowed to air-dry at room temperature and were directly taken in 2.5 % glutaraldehyde in phosphate buffer saline (pbs) (0.1 m, ph 7.2) for 1 h, washed three times in pbs, post-fixed for 1.5 h with osmium tetra oxide (oso4) (1% in pbs), re-washed in pbs for three times, dehydrated in graded ethanol solutions in distilled water and finally dried with hexamethyldisilazane (hmds) for 10 min. the slides were mounted on specimen stubs, coated with gold in a spin coater and finally were observed under fesem (hitachi-s4160). all steps were carried out at room temperature. microscopy classification of the hemocytes was done based on identification key for hemocyte types set by gupta (1979). hemogram studies total hemocyte count (thc) for thc, 2 μl aliquot of hemolymph was mixed immediately in an eppendorf tube with 98 μl of tyson solution (nacl2.72 mm; na2so48.96 mm; glycerol 43.68 mm; methyl violet 0.061 mm). thc was conducted with a standard neubauer hemocytometer. the cells were counted using a light microscope and number of total hemocytes per cubic millimeter (mm3) was calculated using the following formula of jones (1962): hemocytes in x 1mm2 x dilution x depth factor of chamber no. of squares counted where dilution = 50 times, depth factor of the chamber = 10 (constant) and no. of squares counted = 5. differential hemocyte count (dhc) for dhc, permanent microscopy slides were prepared according to the method used in light microscopy studies. differential counting of hemocyte morphotypes was performed by classifying 200 cells per smear (arnold and hinks 1976). the various stages of mitotic division were distinguished based on the method of amaral et al. (2010) and number of hemocytes undergoing mitotic division was recorded in each 200-cell of a smear. measurements of cell and nucleus lengths and widths were made with a digital objective lens of light microscope (olympus bx51). a minimum of 30 cells of each morphotype were measured. hemolymph volume (hv) hemolymph volume was directly measured through collecting whole hemolymph of larvae using hamilton syringe. 133 hemocytes in circulation (hic) the total number of free hemocytes in circulation for any developmental stages is hypothetically derived from the formula thc × hv. maximum daily production (mdp) of hemocytes through mitosis occurred in circulation this hematological parameter is hypothetically determined as (mi × hic × 24) / 1000. thermal regimes three thermal treatments were utilized in this work. two-days old vth instar larvae weighing 20 ± 1 mg were used during the experiment. all larvae were placed inside a programmable, refrigerated test chamber, model mk53 (binder, tuttlingen, germany). to assess the effect of heat shocks on thc, hv and dhc of subjected larvae, the test chamber was scheduled to heat gradually at a constant rate of 0.05 °c/min starting from 27 °c to 40 °c. after 12 h exposing to 40 °c, larvae were cooled to 27 °c at the same rate of heating and were maintained at 27 °c for 24 h. for chilling assay the same trend was used and larvae were exposed to 4 °c for 12 h. control larvae were stored at 27 °c for whole of the experiment course. thc and hv were determined in subsequent series of time. dhc and mi were determined just after 12 h exposure of larvae to the thermal regimes. also, the possible cytological abnormalities of hemocytes were investigated after 12 h exposure of larvae to heat and chill stress and appropriate images were acquired with a dp 72 camera system powered with dic nomarski. statistical analysis means were analyzed using the spss program version 16.0 for analysis of variance (anova) and the means were grouped using tukey’s test (p<0.05). also, statistical comparison between treatment and control at each time point was performed using student’s t-test. results the life cycle of e. kuehniella includes five stages: egg, larva, prepupa, pupa and adult. also according to dyar’s law, five distinct instar larvae were identified (table 1). hemocyte profile five main hemocyte morphotypes were recognized in the hemolymph of e. kuehniella, i.e. prohemocytes, plasmatocytes, granulocytes, oenocytoids and sphrulocytes. prohemocytes (prs) prs are usually the smallest hemocytes found in the hemolymph, round or oval cells with variable sizes (4.8-7.7 μm wide and 5-7.8 μm long) (figs 1a, 2a, 3a and table 2). the nucleus is large, round, usually centrally located and almost filling the most part of cytoplasm. nuclear size is variable (3.1-5.3 μm wide and 3.2-5.3 μm long) (table 2). a thin peripheral layer of homogeneous cytoplasm surrounds the nucleus. plasmatocytes (pls) pls are small to large, polymorphic cells with variable sizes (4.6-9.5 μm wide and 10.1-18.2 μm long) (figs 1b, 2b, 3b and table 2). the cytoplasm is abundant and generally agranular or slightly granular. the cytoplasmic membrane may have filopodia. the nucleus may be round or elongate and is generally centrally located. nuclear size is variable (2.5-6.9 μm wide and 3.6-8.1 μm long) (table 2). scattered chromatin masses may be present in nucleus of these cells. granulocytes (grs) these are small to large, spherical or oval cells of variable sizes (7.5-9.4 μm wide and 8.7-11.3 μm long) (figs 1c, 2c, 3c and table 2). the nucleus is rounded and is centrally located with variable sizes (3.6-5 μm wide and 3.8-5.1 μm long) (table 2). the cytoplasm is characteristically granular and the cell membrane is usually articulated. oenocytoids (oes) oes are small to large, oval or spherical cells with widely variable sizes (6.7-17.7 μm wide and 7.1-19.4 μm long) and shapes (figs. 1d, 2d, 3d and table 2). the plasma membrane is generally table 4 differential hemocyte count (dhc) in each developmental stage studied of e . kuehniella % hemocyte morphotype (mean ± se) phase pr* pl gr oe sp mi l1 9.16 ± 0.64 b 61.86 ± 3.11 bc 16.36 ± 1.27 ab 2.46 ± 0.33 b 10.16 ± 1.55 a 3.30 ± 0.60 a l2 3.18 ± 0.19 d 75.05 ± 1.14 a 11.32 ± 1.21 b 5.14 ± 0.53 b 5.31 ± 0.65 b 0.78 ± 0.34 b l3 15.74 ± 0.69 a 53.34 ± 1.75 c 22.44 ± 1.99 a 2.24 ± 0.40 b 6.24 ± 0.78 ab 3.90 ± 0.67 a l4 5.48 ± 0.84 c 62.64 ± 4.18 abc 17.63 ± 2.88 ab 10.15 ± 1.12 a 4.10 ± 0.95 b 0.98 ± 0.27 b l5 3.76 ± 0.48 cd 69.26 ± 2.48 ab 11.56 ± 2.27 b 5.06 ± 1.71 b 10.36 ± 1.04 a 0.70 ± 0.25 b * (pr, prohemocyte; pl, plasmatocyte; gr, granulocyte; oe, oenocytoid; sp, spherulocyte; mi, mitotic index). means with the same letters in each column are not significantly different at p < 0.05, tukey's test. values are expressed as mean ± se (n=10). 134 a b fig. 5 effect of heating (a) and chilling (b) on hemolymph volume of e . kuehniella larvae at different time intervals. each data point is the mean ± se of at least 10 individual. different letters indicate significant differences between groups (anova, p < 0.05). significant differences versus the respective controls were marked by **p < 0.01, *p < 0.05 and ns non significant at p < 0.05, student's t-test. without micropapillae, filopodia or other irregular processes. the cytoplasm is slightly granular. the nucleus is small, round or elongate and generally eccentrically located (2.7-6.3 μm wide and 2.9-6.5 μm long) (table 2). spherulocytes (sps) these are small to large, ovoid or round cells with variable sizes (8.5-16.5 μm wide and 10.5-17.9 μm long) and usually larger than other hemocytes identified in e. kuehniella. (figs. 1e, 2e, 3e and table 2). the nucleus is generally small, round, central (3.7-7.2 μm wide and 3.6-7.4 μm long) (table 2) and generally obscured by the intracytoplasmic spherules that are characteristic of these cells. the number of the spherules varies from 4-6. two other morphotypes were also distinguished: vermicytes (ves) ves are extremely elongated cells (4-8 μm wide and 23-34.4 μm long) with slightly granular or agranular cytoplasm (figs. 1f, 3f and table 2). the nucleus may be located centrally or eccentrically (2.9-5.1 μm wide and 4-8 μm long) (table 2). 135 table 5 changes in differential hemocyte count (dhc) of two-days old vth instar larvae of e . kuehniella 12 h after treatment under different temperature regimes % hemocyte morphotype (mean ± se) temperature (°c) pr* pl gr oe sp mi 27 °c (control) 15.68 ± 0.65 a 51.78 ± 1.42 b 23.28 ± 1.27 a 3.18 ± 0.43 b 6.08 ± 0.33 a 4.10 ± 0.73 b 40 °c (heating) 15.18 ± 1.55 a 61.28 ± 0.79 a 11.68 ± 1.20 b 4.68 ± 0.51 a 7.18 ± 0.33 a 9.00 ± 0.59 a 4 °c (chilling) 13.46 ± 0.95 a 43.16 ± 2.21 c 29.26 ± 2.83 a 6.06 ± 0.51 a 8.06 ± 0.94 a 0.40 ± 0.18 c * (pr, prohemocyte; pl, plasmatocyte; gr, granulocyte; oe, oenocytoid; sp, spherulocyte; mi, mitotic index). means with the same letters in each column are not significantly different at p < 0.05, tukey's test. podocytes (pos) pos are very large in size (3.4-5.1 μm wide and 17.3-23.3 μm long), extremely flattened pl-like cells with several cytoplasmic extensions (figs 1g, 2f and table 2). the nucleus is generally large and centrally located and may appear punctuate (3-4.2 μm wide and 3.2-4.5 μm long) (table 2). an agglomeration of hemocytes (fig. 1h) was also observed in some profiles. some stages of mitotic division including anaphase (figs. 1i and 2g), telophase (figs 1j and 2h) followed by cytokinesis (figs 1k and 2i) to produce two identical daughter cells were also characterized in hemocytes of e. kuehniella. hemogram there is a gradual increase in body weight during larval stages, with maximum in l5, followed by significant decline in prepupa (f = 491.03, df = 4, p = 0.000) (table 3). in general, total hemocyte population of e. kuehniella showed an increasing trend with increase in larval instars (f = 424.75, df = 4, p = 0.000). it was also found that thc in the late stages (l2 and l4) and prepupa was significantly higher than that in the early stages (l1 and l3) (table 3). an increasing trend was found in hv of tested insect with increase in developmental stages (f = 198.21, df = 4, p = 0.000) (table 3). along with increase in thc and hv, a statistically significant increase in hic was observed (f = 330.63, df = 4, p = 0.000) (table 3). also, among different developmental stages of e. kuehniella, the highest level of hemocyte production through mitosis was observed in l1 and l3 because of maximum mitotic activity in these two stages (table 3). the differential hemocyte profile during the different developmental stages of e. kuehniella is provided in table 4. pls and grs were the most abundant hemocytes followed by the sps, oes and prs in all studied stages. we also observed significant variations of dhc between early and later part of each stage. generally, it is found that proportion of prs, grs, oes, sps (except in prepupa) and also mi in the early of each instar was significantly higher than that in the late part and prepupa. in contrast, pls were found to be in their maximum proportion at the end of each instar and also prepupa (table 4). thermal regimes the results of the effects of thermal stress on thc and hv of 2-days old vth instar larvae of e. kuehniella are presented in figs. 4 and 5. as it can be seen, the larvae exposed to 40 °c for 12 h showed a drastic increase in thc from 3.18×104 to 7.03×104 cells/mm3 (fig. 4a). interestingly, the increase in total number of hemocytes continued even 6 hours after returning of larvae to normal temperature (27 °c) reaching 8.21×104 cell/mm 3 (fig. 4a). also, hv significantly decreased from 3.81 μl to the lowest value of 2.1 μl after exposing the larvae to heat stress for 12 h (fig. 5a). in contrast, chilling for 12 h caused a significant reduction in thc of subjected larvae from 3.12×104 to 2.25×104 cells/mm3 (fig. 4b). when cold exposed larvae were returned to normal temperature (27 °c), total number of hemocytes gradually increased and reached to 3.03×104 cells/mm3 by increasing the incubation time (fig. 4b). no significant differences were observed in hv of control and cold acclimated larvae at various time series (fig. 5b). according to the results of thermal stress on dhc, variations in the proportion of different hemocytes were significant among three temperature treatments (table 5). while pls and oes percentages increased in heat-exposed larvae, a significant reduction in the counts of grs was recorded. in chilled larvae, on the other hand, oes rose in their number with a significant decline in pls. also, a little increase in grs was observed but it was not statistically significant. no significant differences were observed in the proportion of prs and sps under three temperature regimes. low temperature resulted in a decrease in mitotically dividing cells of e. kuehniella larvae but in contrast, high temperature caused a significant increase in mi (table 5). effect of thermal stress on cell-structure of hemocytes heating for 12 h led to vacuolization in the cytoplasm of pls (fig. 6a) and increase in number 136 http://en.wikipedia.org/wiki/cytokinesis fig. 6 cell-structure abnormality in larvae exposed to heat stress. vacuolization in cytoplasm of plasmatocytes (a) and rounded plasmatocytes loosing filipods (b) after 12 h exposing of larvae to 40 °c. scale bar = 10 µm. of rounded pls without filipods (fig. 6b). no considerable changes have been found in the cytological features of hemocytes of incubated larvae at 4 °c. discussion the present study provides detailed information of hemocyte profile and hemogram of the mediterranean flour moth, e. kuehniella. strand and pech (1995) divided the lepidopteran hemocytes into five classes on the basis of morphology, i.e. prohemocytes, plasmatocytes, granu locytes, oenocytoids and spherulocytes. similar results were also reported for other lepidopterous larvae, such as plutella xylostella (huang et al., 2010), ectomoyelois ceratoniae (khosravi et al., 2012), hyphantria cunea (ajamhassani et al., 2013), bombyx mori (tan et al., 2013). we also found all of these five morphotypes of hemocytes in e. kuehniella larva in this study. also, our results indicated that plasmatocytes and granulocytes are the most abundant hemocytes in the hemolymph of e. kuehniella. prior studies have shown that plasmatocytes and granulocytes were responsible for cellular immune responses in many lepidopteran insect larvae, such as g. mellonella (tojo et al., 2000) and manduca sexta l. (ling and yu, 2006) and together usually comprise more than 50 % of the hemocytes in circulation (lackie, 1988; ratcliffe, 1993). according to the results of cell measurements, it is shown that each cell morphotype is of a varied size. we also found various forms of each particular hemocyte in our tested insect. as shown in the results, two other morphotypes of hemocytes termed vermicytes and podocytes were distinguished in the hemolymph of e. kuehniella. these two morphotypes have not been recognized as distinct types in electron microscopic studies so far, primarily because ultrastructurally they appear similar to plasmatocytes (gupta, 1997). the origin of vermicytes and podocytes is unknown, but it is conceivable that they are derived from plasmatocytes and are considered as variant forms of plasmatocytes (gupta, 1997). this variation in the form and size has brought many problems in the classification of hemocytes because the insect blood cells are highly pleomorphic, and the particular form they present at any one time seems to depend on the age, developmental stage, nutritional state and species of insect as well as on the methods of collection and examination used by the investigator (jones, 1962; lai-fook and neuwirth 1972). the maintenance of circulating hemocytes in larval lepidoptera has been attributed to both the release of hemocytes from hematopoietic organs and the mitosis of hemocytes in the circulatory system (gardiner and strand 2000). the levels of mitotic activity in circulating hemocytes rarely exceed 1 % in almost all cases (jones, 1967a; jones and lin 1968; jones, 1967b), but it is shown that in e. kuehniella this activity varies with developmental stage of insect. the mitotic index of larvae in l1 and l3 reached 3.3 % and 3.9 %, while the dividing cells observed in l2, l4 and prepupa were 0.78%, 0.98% and 0.7%, respectively. in a parallel study on oncopeltus fasciatus dallas, feir and mcclain (1968) found that the mi was very low immediately after ecdysis into the fifth instar. they noted that mitotic activity began to rise at 23 hour post-ecdysis, reached its peak (4.06 %) in the 30-hr group, remained high until 74 hour, and then declined during the remainder of the stadium. these findings agree with the reports of sanjayan et al. (1996), who observed different values of the mitotic index in final instar and adult insects of spilostethus hospes fab. mitotic activity has been most consistently reported in the prohemocytes and hemocyte 137 differentiation studies in some insect species revealed that prohemocytes are the stem cells from which the other cells arise (gupta, 1991). in case of the silkworm, b. mori, it is proved that approximately 43 % of prohemocytes differentiate into plasmatocytes, granulocytes and spherulocytes (yamashita and iwabuchi, 2001). the population of prohemocytes in l1 and l3 is significantly higher than that in l2 and l4 and prepupa. the rapid decline in their counts coinciding with the increase in plasmatocytes and partially oenocytoids in the later part of larval instar suggests that their differentiation is probably toward the formation of plasmatocytes and oenocytoids at that time. the results indicated that cell population increased during larval development of e. kuhniella, and reached a peak during the prepupal period. also, thc was in its high value at the late stage of each larval instar than the early stage. it has been frequently reported that thc varies with the developmental stage and physiological condition of the insect. patton and flint (1959) noted that counts from periplaneta decreased at the molt and later increased post molt. also, crossley (1975) proved that hemopoiesis in insects is under endocrine control. in this case, akai and sato (1971) showed that the increase of ecdysteroids in hemolymph of b. mori would be resulted in more hemocyte production and release from the hematopoietic organs. so, it could be concluded that changes in thc during the developmental stages of e. kuehniella would be probably attributed to the variations in ecdysteroids and juvenile hormones (jh) titers. in many insect species, fluctuations in the number of hemocytes are influenced by the release of hemocytes from the hemapoietic organ and attachment of the cells to internal tissues (tu et al., 2002; okazaki et al., 2006). the number of hemocytes in circulation can change rapidly in response to stress, wounding or infection (gillespie et al., 2000; mowlds and kavanagh 2008). as low and high temperatures are a source of stress for insects, it is possible that the number of hemocytes was directly altered by the change in temperature. in this aspect, results of present study clearly indicated that compared to control temperature, heating caused a significant increase in thc and in contrast, those larvae incubated at 4 °c showed a significant reduction in thc. so, it seems that exposing the insects to high temperatures can increase the environmental fitness of larvae through a similar mechanism to thermoregulatory behaviour by increasing total number of hemocytes especially pls. there are several reasons to justify these changes in thc. cell cycle can be divided in three periods: interphase consisting g1 (pre-synthetic interphase), s phase (dna synthetic interphase) and g2 (post-synthetic interphase), mitosis phase and the final phase, cytokinesis, where the new cell is completely divided. in many cases it was found that the time taken for cells to divide decreases in response to an increase in temperature (francis and barlow 1988). moore et al. (1997) stated that temperature reduction from 37 °c to 30 °c caused a rapid decrease in the percentage of cells in s phase and accumulation of cells in g1. in another study, bloemkolk et al. (1992) showed that maximal cell density was observed at 37 °c. lower temperatures, in contrast, caused cells to stay longer in the g1 phase of the cell cycle. in parallel, our results showed that compared to chilling, heating caused a 2.2 folds increase in mitotic rate of hemocytes. so, it is concluded that increase in thc of e. kuehniella larvae exposed to high temperature may be attributed to more mitotic rate of hemocytes and more importantly increased hemocyte proliferation of hemopoietic organs. it was found that an inverse relationship exists between the thc and the hv after incubation of larvae at 40 °c. thus, it seems that another possible reason for the increase in thc is due to loss of body fluid as a result of desiccation. also, release of hemocytes attached to the internal organs of heat exposed larvae into the hemolymph circulation can be another reason of increase in thc. on the other hand, the low hemocyte counts in chilled larvae found to be primarily attributable to adherence to respected larval tissues. some authors indicated that low counts from unfixed larvae in heat may have been caused by increased clumping and hence, unavailability of circulating hemocytes. we could not find any significant relationship between thc and hv in chilled larvae. our results indicated that heating led to cell structure changes in pls. there are several evidences regarding the activation of hemocytes which are characterized by expanded filipods during immune challenge (kwon and kim 2007). however, in our experiment rounded pls without filipods were increased in response to heating condition. this phenomenon was occurred by heating without pathogenic infection. therefore, it could be expected that pathogenic infection would be the main reason for expanding filipods in pls in response to fever. further investigations are needed to be done to elucidate this kind of cell response. conclusion in this study, we determined the morphological characteristics of hemocytes of 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involvement of both granular cells and plasmatocytes in phagocytic reactions in the greater wax moth, galleria mellonella. j. insect physiol. 46: 1129-1135, 2000. tu zl, kobayashi y, kiguchi k, watanabe h, yamamoto k. effects of heavy-ion radiosurgery on the hemopoietic function of the silkworm bombyx mori. j. radiat. res. 43: 269-275, 2002. watson dw, mullens ba, petersen jj. behavioral fever response of musca domestica (diptera: muscidae) to infection by entomophthora muscae (zygomycetes: entomophthorales). j. invertebr. pathol. 61: 10-16, 1993. yamashita m, iwabuchi k. bombyx mori prohemocyte division and differentiation in individual microcultures. j. insect physiol. 47: 325-331, 2001. 140 http://link.springer.com/search?facet-author=%22alison+moore%22 http://www.sciencedirect.com/science/journal/00222011 http://www.sciencedirect.com/science/journal/00222011 http://www.sciencedirect.com/science/journal/00222011/61/1 watson dw, mullens ba, petersen jj. behavioral fever response of musca domestica (diptera: muscidae) to infection by entomophthora muscae (zygomycetes: entomophthorales). j. invertebr. pathol. 61: 10-16, 1993. yamashita m, iwabuchi k. bombyx mori prohemocyte division and differentiation in individual microcultures. j. insect physiol. 47: 325-331, 2001. minireview isj 9: 223-229, 2012 issn 1824-307x minireview the mechanisms of primordial germ cell determination during embryogenesis in molluscan species m obata, a komaru faculty of bioresources, mie university, 1577 kurimamachiya, tsu, mie 514-8507, japan accepted december 20, 2012 abstract primordial germ cells (pgcs) are the first germ cells distinguishable from surrounding somatic cells during embryogenesis. in many animal species pgc specification is generally classified into two modes: preformation, in which pgcs are determined by maternally inherited components in early development, and epigenesis, in which pgcs arise from proximal somatic cells by inductive signal in late development. in this review we focused on the process of pgc formation in molluscan bivalves and gastropods and compared the pgc specification modes of these two classes. several reports indicated that bivalves tend to adopt preformation, as maternally inherited germline-specific genes are transcribed in specific blastomeres differentiating into pgcs. in gastropods, maternal germline-specific genes are transcribed in mesodermal stem cells. pgcs seem to be epigenetically determined from mesodermal stem cells by inductive signals after the veliger larval stage, which indicates that pgc precursor cells not only generate germline tissues but also generate mesodermal somatic tissues. the common origin of germline and mesodermal somatic tissues is observed in the annelids platyhelminthes and cnidaria, and is considered to be an ancient mode of germ cell determination. we suggest that gastropods retain the ancient pgc specification mode, while bivalves switch their pgc specification mode to preformation. key words: primordial germ cell; vasa; early development, preformation; epigenetic; mollusc introduction stem cells are defined by the abilities to selfrenew and to differentiate into other cell types by asymmetric division (lin, 1997). they are classified by their differentiating abilities as unipotent, multipotent, pluripotent and totipotent stem cells. germinal stem cells (gscs) are pluripotent stem cells that can differentiate into almost every cell type except extraembryonic tissues. indeed, gscs are able to generate gametes and transmit the individual’s dna to the next generation. primordial germ cells (pgcs) are germline cells that can be genetically and morphologically distinguished from somatic tissues during early embryogenesis. the origin of the pgcs and the timing of pgc segregation from somatic cells during development have been reported for many animal species (extavour and akam, 2003). several studies have also been conducted on the origins of germline cells in molluscs, especially in bivalves and gastropods ___________________________________________________________________________ corresponding author: mayu obata faculty of bioresources mie university 1588 kurimamachiya, tsu, mie 514-8507, japan e-mail: mayu_obata@bivalve.sakura.ne.jp (woods, 1931; fabioux et al., 2004; swartz et al., 2008; kranz et al., 2010). in this paper we review the existing literature on germ cells determination during development in various animal species, in particular in molluscan bivalves and gastropods. first, we introduce two general mechanisms of germline specification during embryogenesis: preformation and epigenesis. second, we list genes that are important in molluscan gscs. finally, we compare the pgc specification modes in bivalves and gastropods. mechanisms of germline segregation from somatic tissues during embryogenesis preformation data from many previous studies on pgc origins have suggested that there are two distinct modes of pgc specification: preformation and epigenesis (extavour and akam, 2003). in some species, pgcs can be identified during early development by morphological features such as a large round nucleus, a single large nucleolus, relatively clear cytoplasmic organelles and granular cytoplasmic material. granular cytoplasmic material in particular is a useful pgc marker for many animal 223 mailto:mayu_obata@bivalve.sakura.ne.jp species. it is known under various synonyms like germ plasm, nuage, mitochondrial cloud and chromatoid body. in this study, we use the term “germ plasm”, as this is the most commonly used term in many animal species (saffman and lasko, 1999). germ plasm can be observed as electrondense granules containing mitochondria, proteins and rna. transcripts of many genes have been identified as germ plasm components. among these, the germline markers vasa and nanos are conserved among a wide range of animal classes, and these genes can thus be used as molecular markers for pgc identification. in some species, germ plasm is stored in oocytes before fertilization, and the maternally inherited cytoplasm is transmitted to the pgc precursor cells. this means that in some animal species the origin of pgcs is determined by maternal factors. this pgc specification mode is called preformation. preformation is reported in drosophila melanogaster (sonnenblick, 1950), anuran species (bounoure, 1939), most teleosts (yoon et al., 1997) and caenohabditis elegans (deppe et al., 1978; strome and wood, 1982). in d. melanogaster, germ plasm is assembled at the posterior poles of the oocytes before fertilization (reviewed by mahowald, 2001). it is then transmitted to the five posterior pole cells by unequal cleavage before blastoderm formation (huettner, 1923). these pole cells exclusively become progenitors of pgcs. transplanted germ plasm can induce pgc formation at ectopic sites (illmensee and mahowald, 1974; illumensee et al., 1976). moreover, disassembly of germ plasm prevents pgc formation (rongo and lehman, 1996). these results indicate that germ plasm is essential for germline formation in drosophila. many genes, such as vasa, oskar, tudor and gurken, are required for posterior germ plasm formation in drosophila (rongo and lehman, 1996). in particular, oskar has a critical role in germ plasm assembly at the posterior of the oocyte, as it is required for the posterior localization of the germ plasm components vasa, nanos and tudor (rongo and lehman, 1996). vasa acts as a translational regulator of gurken and oskar (mahowald, 2001). epigenesis in some species germ cells cannot be identified until late development and pgcs arise by inductive signals from surrounding somatic tissues. this pgc specification mode is called epigenesis. epigenesis is observed in mammals (saffman and lasko, 1999), urodeles (ikenishi and nieuwkoop, 1978), sea urchins (juliano et al., 2006) and cnidaria (noda and kanai, 1977). in mouse embryos, no maternally derived germ plasm has been found (eddy, 1975). unlike vasa transcripts in preformation species, mouse vasa orthologs cannot be used to identify pgcs in early development, because its protein is not localized to a specific subcellular region (toyooka et al., 2000). germ cells can first be identified by alkaline phosphatase staining at 6.5 days post conception (lawson and hage, 1994). pgc determination at the proximal epiblast is induced by signals from the extraembryonic ectoderm and endoderm. it is reported that bmp4, bmp8b and bmp2, members of the bone morphogenetic proteins (bmp) of the transforming growth factor β (tgfβ) superfamily, function as the inductive signals (hogan, 1996; lawson et al., 1999; ying et al., 2000; ying and zhao, 2001). extavour and akam (2003) reviewed the pgc determination modes of 28 metazoan phyla. in 23 of the 28 phyla it was suggested that pgcs are determined by epigenesis. this indicates that epigenesis is an ancient mode of pgc determination in metazoa. transcripts that play an important role in molluscan gscs germline-specific transcripts have been observed in many animal species, and their functions in pgcs have been described. in molluscs some gene transcripts have been isolated from germline cells and are used to identify the localization of pgcs and elucidate the mechanism of gametogenesis. vasa is an atp-dependent rna helicase of the dead-box family of proteins. it unwinds double-strand rna to promote the translation of target genes (hay et al., 1988; lasko and ashburner, 1988; carrera et al., 2000; raz, 2000; johnstone and lasko, 2001). one of the target genes of vasa translational control is nanos, which in turn regulates the translation of other genes (johnstone and lasko, 2001). vasa is widely used as a molecular marker for germlines including pgcs in many animal species (extavour and akam, 2003). in molluscs, vasa orthologs have also been isolated and are used for pgc identification and germline observation (fabioux et al., 2004; swartz et al., 2008; kranz et al., 2010; obata et al., 2010). fabioux et al. (2009) performed rna interference on the oyster vasa ortholog oyvlg in crassostrea gigas gonads and suggested that oyvlg has an important role in germ cell proliferation and early meiosis. nanos is a translational regulator in germline cells. its own translation is controlled by vasa. in insects it is also required for somatic patterning (lehman and nusslein-vorhard, 1991; lall et al., 2003). in molluscs, nanos ortholog transcription has been observed in ilyanassa obsoleta and haliotis asinina embryos. in i. obsoleta, nanos orthologs were transmitted to the 4d blastomere. nanos ortholog transcriptions remained in 4d derivatives including germline and somatic tissues at later stages of development (rabinowitz et al., 2008). inhibition of nanos orthologs by morpholino oligo injection resulted in larvae lacking all 4d derivatives. these larvae had no hearts and abnormal retractor muscles and intestines. in h. asinina embryos, nanos orthologs were transcribed in second quartet micromeres (2a-2d blastomeres), which give rise to the foot and ectoderm anterior. these results suggest that nanos functions in both germline and somatic tissue development in gastropod embryos. the tgfβ family is structurally conserved and plays an important role in cell proliferation and differentiation during development (ten dijke et al., 2000). in drosophila, tgfβ signal transduction pathways affect germline stem cell numbers and the 224 size of the stem cell niche (schulz et al., 2004). in rainbow trout, tgfβ increased spermatogonium proliferation and oocyte maturation (sawatari et al., 2007). in the bivalve c. gigas, a tgfβ ortholog (ogtgfβ) is found in the somatic tissues surrounding the spermatogonia and oogonia (fleury et al., 2008). results from an in vivo rna interference study on in male and female gonad in c. gigas suggested that og-tgfβ is required for spermatogonial and oogonial proliferation and oocyte maturation (huvet et al., 2012). the tgfβ superfamily may play an important role in embryogenesis and germ cell maintenance in many molluscan species. cell lineage and pgc location in spiral cleavage embryos in molluscs, the cleavage mode of early development is spiral cleavage. this form of cleavage is observed in many phyla like annelids, entoprocts, nemertines and platyhelminthes (nielsen, 2010). the spiral cleavage pattern was first shown more than 100 years ago (conklin, 1897). in spiral cleavage, embryos are divided into ab and cd blastomeres at the two-cell stage, and into a, b, c and d blastomeres at the four-cell stage. in many annelids and molluscs, a polar lobe is produced during each of the first two cleavages (figs 1, a1, a3). the two polar lobes are absorbed into the cd blastomere at the two-cell stage and the d blastomere at the four-cell stage (figs 1, a2, a4). at the third cleavage (which corresponds with the first quartet cleavage), a, b, c and d blastomeres divide synchronously and produce four micromeres toward the animal pole (1a, 1b, 1c and 1d) and four macromeres at the vegetative pole (1a, 1b, 1c and 1d) (henry et al., 2006) (fig. 1, a5). the following quartets (second, third and fourth) are oblique with alternating directions of spindles, resulting in the spiral cleavage pattern (figs 1, a6-a8). there are two types of spiral cleavage: early equal cleavage and unequal cleavage (henry et al., 2006). in early equal cleavage, the first four quadrants have an equal cleavage pattern until the 32-cell stage (van den biggelaar and guerrier, 1979; gonzales et al., 2007) (fig. 1, b). early equal cleavage is considered to be an ancestral condition of spiral cleavage (freeman and lundelius, 1992; henry, 2002). in unequal cleavage, the first cleavage is asymmetrical, which is caused by biased mitotic spindle orientation and polar lobe absorption. unequal cleavage results in larger blastomeres at the vegetative pole side of the embryo, which is derived from d blastomeres at the four-cell stage, and smaller blastomeres at the animal pole. spiral cleavage patterns and cell lineages are conserved among various animal species (nielsen, 2004, 2005; lambert, 2007). 3d blastomeres arise from d blastomeres through three quartet cleavages, dividing into large 4d and small 4d blastomeres at the fourth quartet cleavage (figs 1, a8, b6). the 4d blastomere differentiates into most mesodermal tissues, including germline cells (conklin, 1897). the specification of mesodermal lineages from 4d blastomeres by asymmetric cleavage is a common phenomenon in animal species with spiral cleavage patterns (wilson, 1892; conklin, 1897; woods, 1931). pgc segregation during embryogenesis in bivalves pgc specification modes have been reported for some molluscan species. the process and mode of pgc specification is different in each class, although pgcs commonly arise from 4d blastomeres. woods (1931) studied sphaerium striatum oocytes under light microscope and noted that the 4d blastomere divided equally into m and m1 blastomeres and that pgcs were generated from these blastomeres. he also reported that the germ plasm was localized at the vegetative pole in unfertilized eggs. germ plasm was specifically transmitted to d blastomere derivatives and finally segregated in germ cells in the late gastrula. recently, transcripts of the germline-specific gene vasa have been used as a germline molecular marker in some bivalve species (fabioux et al., 2004; kakoi et al., 2008; obata et al., 2010). fabioux et al. (2004) observed c. gigas vasa ortholog (oyvlg) transcription at the vegetative pole of fertilized eggs. the transcripts were specifically transmitted to one blastomere during early development. at the morula stage, oyvlg was observed in the 4d blastomere only. the 4d blastomere equally divided into m and m1 blastomeres, which then differentiated into pgcs. the oyvlg transcription pattern is identical to the location of germ plasm in s. striatum, which suggests that oyvlg is a general component of mollusc germ plasm, like in drosophila. kakoi et al. (2008) reported on the localization of the vasa ortholog sk-vasa in sakkostrea kegaki embryos. skvasa was transcribed throughout the embryo until the eight-cell stage. at the 50-cell stage, the transcripts were limited to the 2d descendant cell and a pair of cells located posteriorly, which were likely to be 4d blastomeres. in the late gastrula, strong sk-vasa expression was observed in the posterior mesoderm. germline-specific localization of sk-vasa could not be observed until the veliger larval stage. pgc segregation during embryogenesis in gastropods in i. obsoleta, the vasa ortolog lovasa accumulates during early development (swartz et al., 2008). the transcripts were observed ubiquitously throughout the first seven cleavage cycles, while the rna became more abundant in the d quadrant as the cleavage cycle progresses. maternally inherited lovasa accumulated at 4d specifically between the 28-cell and the 35-cell stage. at the 70-cell stage, lovasa transcription was limited to 4dl121 and 4dr121. swartz et al. (2008) suggested that 4dl121 and 4dr121 are likely to be homologous to the pgc lineage founder cells identified in s. striatum (woods, 1931). however, lovasa could not be detected after the 108-cell stage. 225 fig. 1 diagram of unequal and equal spiral cleavage. a) unequal spiral cleavage of ilyanassa obsoleta. a1) polar lobe formation at first cleavage. a2) two cell stage. a3) polar lobe formation at second cleavage. a4) four cell stage. a5) first quartet blastomeres at the 8 cell stage. a6) second quartet blastomeres at the 12 cell stage. a7) third quartet blastomeres at the 24 cell stage. a8) 4d blastomere formation at fourth quartet cleavage. b) equal spiral cleavage of crepidula fornicata. b1) two cell stage. b2) four cell stage. b3) first quartet blastomeres at the 8 cell stage. b4) second quartet blastomeres at the 12 cell stage. b5) third quartet blastomeres at the 24 cell stage. b6) 4d blastomere formation at fourth quartet cleavage. after third quartet cleavage, 4d blastomeres arise from 3d blastomeres by unequal cleavage in both spiral cleavages. 4d blastomere is shown as dark gray area.polar lobe (pl). kranz et al. (2010) observed transcription of vasa, nanos and pl10 orthologs in haliotis asinina embryos. the vasa ortholog hasvasa was ubiquitously expressed throughout the embryo until the 16-cell stage. strong transcription of hasvasa was observed in 4d blastomere at the 60–64-cell stage (3.5 hours post-fertilization (hpf)). at the gastrula stage, hasvasa was apparently distributed at the mesodermal band until the trochophore stage. hasvasa and haspl10 were colocalized in early veliger larvae. it was hypothesized that pl10 is transcribed in mesodermal cells, which also produce pgcs (rebscher et al., 2007). from these results, kranz et al. (2010) suggested that hasvasa is transcribed in undifferentiated multipotent cells that are precursor cells for pgcs and mesodermal somatic tissues. germline-specific transcription of hasvasa could not be observed until the veliger larval stage. in viviparus viviparus, pgcs have also not been identified in early development and 226 germline cells arise from the mesodermal pericardial epithelium (griffond, 1977). thus, in gastropods, maternally inherited germ plasm or mrna is not reported to be transmitted to pgcs, which is in contrast with the findings in bivalves. comparison of pgc specification modes in bivalves and gastropods in the bivalves c. gigas and s. striatum, maternally inherited germ plasm is transmitted to specific blastomeres by unequal cleavage from the first cleavage to the 4d blastomere formation. at the gastrula stage, germ plasm is distributed at germline-specific blastomeres only. thus it can be concluded that the pgc specification mode in these species is preformation, since maternally inherited germ plasm is specifically transmitted only to germlines in early development, like in d. melanogaster and c. elegans. in the case of s. kegaki, however, maternally inherited sk-vasa was ubiquitously transcribed during early development. although the signal became stronger in the 4d blastomere, germline-specific localization of sk-vasa could not be observed until the veliger larval stage. this suggests that pgc specification mode of s. kegaki is epigenetic, meaning that not all bivalve species may use preformation as the mode of pgc specification. however, more studies are required to clarify the pgc specification mode of s. kegaki. like in s. kegaki, vasa transcripts in gastropods do not have a specific distribution pattern during early development. in i. obsoleta, lovasa is more strongly transcribed in 4d blastomeres and the signal was transmitted to 4dl121 and 4dr121, which presumably differentiate into pgcs. from these results, swartz et al. (2008) suggested that maternal lovasa rna may be involved in germ cell specification. however, the localization of pgc could not be identified until the veliger larval stage in i. obsoleta, because lovasa transcription disappeared after the 108-cell stage. in h. asinina, transcription of hasvasa was observed in 4d blastomeres at 3.5 hpf. however, the transcription was observed in multipotent mesodermal cells, which differentiate into pgcs and mesodermal somatic tissues like heart and retractor muscles in veliger larvae. this suggests that pgcs are not established until the veliger larval stage, although maternal vasa is specifically transmitted to 4d blastomeres in gastropods. judging from these results, the pgc specification mode of gastropods appears to be epigenesis. rebscher et al. (2007) reported that the transcription pattern of the vasa ortholog pdu-vasa of p. dumerilli that is similar to that of h. asinina; ubiquitous distribution in most blastomeres during early development and specific localization at the mesodermal posterior growth zone (mpgz; mesodermal stem cells) that arises from the 4d blastomeres at the gastrula stage. specification of germlines from the mpgz occurred at the juvenile ii stage in late development. rebscher et al. proposed that pgc specification in p. dumerilli is a two-step process. first, a population of undifferentiated cells that can differentiate into both somatic tissues and pgcs is established in the mpgz. this process apparently requires maternally inherited germ plasm, suggesting that the establishment of mpgz is by preformation. pgc are then determined by induction signals from the mpgz in late development, which is the epigenetic mode of pgc specification. we suggest that this pgc specification mechanism is similar to that of h. asinina. the studies on h. asinina and p. dumerilli indicate that the germline-specific gene is distributed in the mesodermal stem cells, and not only the germlines. moreover, pgcs and somatic tissues arise from a common precursor cell, the mesodermal stem cells. in fact, in some animal species such as annelids (vincent et al., 2011), platyhelminthes (shibata et al., 1999) and cnidaria (noda and kanai, 1977), germ plasm is not only observed in germlines but also in the somatic stem cells. germline-specific genes like vasa and piwi have an important role in the maintenance of somatic stem cells as well as in germlines in these species (thomson and lin, 2009). pgc specification by induction of multipotent somatic stem cells is considered to be an ancient mechanism of germline formation. thus, h. asinina adopts an epigenetic mode for pgc specification, which may be the most ancient mode among molluscan species (kranz et al., 2010). on the other hand, the pgc specification mode used in some bivalve species like c. gigas and s. striatum is preformation. extavour and akam (2003) suggested that the basal pgc specification mode in metazoa is epigenetic. thus, we suggest that many bivalve species change their pgc specification mode from epigenetic to preformation. with this shift in germline specification mode, it is likely that germ plasm components like vasa lose their function in somatic stem cells. however, it is possible that not all bivalve species shift their germline specification mode to preformation, as s. kegaki apparently adopts the epigenetic germline specification. in conclusion, pgc specification modes are species-specific in molluscs, although 4d blastomeres are common precursor cells in both bivalves and gastropods. it is suggested that bivalves tend to use preformation while gastropods use epigenesis as their mechanism of pgc specification. koop et al. (2007) suggested that equally cleaving gastropods tend to use inductive signals, while unequally cleaving gastropods tend to rely on inherited determinants. kranz et al. 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zebrafish vasa homologue rna is localized to the cleavage planes of 2and 4-cell-stage embryos and is expressed in the primordial germ cells. development 124: 3157-3165, 1997. 229 activación de las gtp asas de rho por integrinas 1 isj 8: 109-131, 2011 issn 1824-307x review phagocytosis, a cellular immune response in insects c rosales immunology department, instituto de investigaciones biomédicas, universidad nacional autónoma de méxico, mexico city, mexico accepted june 20, 2011 abstract insects like many other organisms are exposed to a wide range of infectious agents. defense against these agents is provided by innate immune systems, which include physical barriers, humoral responses, and cellular responses. the humoral responses are characterized by the production of antimicrobial peptides, while the cellular defense responses include nodulation, encapsulation, melanization and phagocytosis. the phagocytic process, whereby cells ingest large particles, is of fundamental importance for insects’ development and survival. phagocytic cells recognize foreign particles through a series of receptors on their cell membrane for pathogen-associated molecules. these receptors in turn initiate a series of signaling pathways that instruct the cell to ingest and eventually destroy the foreign particle. this review describes insect innate humoral and cellular immune functions with emphasis on phagocytosis. recent advances in our understanding of the phagocytic cell types in various insect species; the receptors involved and the signaling pathways activated during phagocytosis are discussed. key words: insect; phagocytosis; hemocyte; innate immunity; signal transduction introduction multicellular organisms possess a series of systemic, cellular and molecular mechanisms, which allow them to protect themselves from infection by viruses, bacteria, fungi and protozoa. these mechanisms are collectively known as immunity. among the defense mechanisms taking place at the onset of an infection there are early systems, such as constitutive expression of antimicrobial peptides, recognition of microorganisms by patternrecognition receptors (prrs), and activation of phagocytic cells, involved in detecting and eliminating pathogens, through a wide variety of cellular responses. these responses are the innate immune systems. in vertebrates, such as mammals, the recognition of antigens by specific receptors of tand b-lymphocytes is yet another, more precise layer of the defense system, known as adaptive immunity. this adaptive response is activated at later times during the course of an infection. normally, leukocytes circulate in a resting state, not showing antimicrobial properties. upon recognition ___________________________________________________________________________ corresponding author: carlos rosales department of immunology instituto de investigaciones biomédicas unam apdo. postal 70228, cd. universitaria méxico d.f. 04510, mexico e-mail: carosal@servidor.unam.mx of microorganisms via prrs or via specialized phagocytic receptors, leukocytes develop a fully active antimicrobial and proinflammatory phenotype. this change is known as activation of phagocytic cells. the signals delivered to the cell by the various receptors determine the final activation stage of the leukocyte. at later times, these activated leukocytes can process and present antigens to specific tand b-lymphocytes to develop an adaptive immune response, which provides a much better and faster response to the same pathogen during a second challenge. for insects, the view is that they, like other invertebrates, depend only on its innate immune response to fight invading microorganisms; by definition, innate immunity lacks adaptive characteristics. however, there are some reports showing that priming drosophila with a sublethal dose of streptococcus pneumoniae protects against an otherwise-lethal second challenge of s. pneumoniae (pham et al., 2007). this protective effect has loose specificity for s. pneumoniae and persists for the life of the fly. while not all microbial challenges induced this specific primed response, a similar specific protection could be elicited by the fungus beauveria bassiana, a natural fly pathogen (pham et al., 2007). these results point out that insect immune responses can indeed adapt and suggest that insect hemocytes may also present an activation response similar to the one known in mammalian leukocytes. 109 millions of insect species live in practically every known habitat and ecological niche, though marine environments are an important exception. this diversity exposes insects to all sorts of infectious agents, such as viruses, bacteria, fungi and protozoa. insects have evolved an effective innate immune system that permits rapid and efficient responses against infectious agents. the innate immune system of insects consists of physical barriers, humoral responses and cellular responses (lavine et al., 2002; kanost et al., 2004). in addition, recent evidence suggests that insects also have adaptive immune responses as mentioned before, although these responses are not similar as those traditionally defined in mammals with band t lymphocytes. physical barriers include the integument and the peritrophic membrane. integument, the outer surface of an insect, is formed by a single layer of cells covered by a multi-layered cuticle (ashida et al., 1995). the peritrophic membrane or peritrophic matrix is a chitin and glycoprotein layer that lines the insect midgut. it is functionally similar to the mucous secretions of the vertebrate digestive tract and hence it acts as a physical barrier, protecting the midgut epithelium from abrasive food particles, digestive enzymes and some digestive pathogens (lehane, 1997; hegedus et al., 2009). however, due to its semipermeable nature, this matrix is not an efficient barrier to infection, particularly to viruses. together these structures form the first line of protection for the hemocel (the insect body cavity) and the midgut epithelium from invading microorganisms. in the case these barriers are breached, the humoral and cellular immune responses are activated. humoral immune responses include biosynthesis of antimicrobial peptides, activation of enzymes, such as lysozyme and the prophenoloxidase (propo) system, to regulate coagulation of hemolymph and production of reactive oxygen species (jiang, 2008; tsakas et al., 2010). cellular immune responses include nodulation, encapsulation, and phagocytosis (strand, 2008; tsakas et al., 2010). hemolymph, the liquid that fills the hemocel of an insect, has an analogous function to both blood and lymph in mammals. it is involved in transport of nutrients and waste products, although not transport of respiratory gases. in addition, it contains several types of free-moving cells or hemocytes. hemocytes originate from mesodermally derived stem cells that differentiate into specific lineages. the most common types of hemocytes are granulocytes, plasmatocytes, spherulocytes, and oenocytoids (lavine et al., 2002; meister et al., 2003). however, it is important to emphasize that not all these hemocyte types exist in all insect species (meister, 2004; michela et al., 2005; manachini et al., 2010; wang et al., 2010). hemocytes are essential for insect immunity, as shown in drosophila melanogaster larvae where plasmatocytes, making up approximately 95 % of circulating hemocytes, decrease in numbers during an infection (williams, 2007). also, by genetic ablation (defaye et al., 2009) or mechanical ablation (charroux et al., 2009b; nehme et al., 2011) of phagocytic hemocytes in drosophila, it was observed that in adult flies there is an increase in infection susceptibility to various bacteria including escherichia coli, bacillus subtilis and staphylococcus aureus. cellular immune responses are immediate after an invasion of the hemocel, while humoral responses appear several hours after an infection. based on data from coleopterans, although not shown in other insects yet, it is thought that humoral responses have the function of finishing up the invading microorganisms that escaped the initial cellular immune responses (haine et al., 2008). together, physical barriers, humoral, and cellular immune responses provide an effective defense system for the insect. these elements, however, do not work in isolation and in an orderly fashion. there is a complex interplay among them. for example, hemocytes and other insect cells produce molecules that increase hemocyte-microorganism binding (mohrig et al., 1979; wiesner et al., 1996; brivio et al., 2010; kim et al., 2010). these molecules are similar to the opsonins that increase phagocytosis of leukocytes in mammals. in addition, drosophila plasmatocytes, which are essential for phagocytosis, are also required during larval stages to induce fatbody (insect equivalent of the liver) cells to produce antimicrobial peptides after a bacterial infection (charroux et al., 2009b). in addition, in adult flies, plasmatocytes contribute to reduce the infection susceptibility to various bacteria including e. coli, b. subtilis and importantly s. aureus (defaye et al., 2009; nehme et al., 2011). these findings clearly indicate that there is an effective cross-talk between humoral and cellular immunity in insects. here, i will describe insect cellular immune functions with emphasis in phagocytosis and review recent findings on phagocytic hemocyte types, the receptors involved and the signaling pathways activated during this cellular response. insect cellular immunity hemocytes are responsible for a variety of defense responses in insects. many variations in insect immune responses exist due to the presence of millions of insect species, and we are just beginning to understand these variations (schmidhempel, 2005). however, a number of frequent innate immune responses have been described in most insects studied. these responses include nodulation, encapsulation, melanization, and phagocytosis. these general processes share common elements in terms of pathogen recognition, biochemical signals, and final clearance of the invading microorganism from the hemolymph. next, i briefly describe our current understanding on these insect innate immune responses. nodulation nodulation is a predominant cellular response in insects to large bacterial infections. it consists of formation of multicellular hemocyte aggregates that entrap large numbers of bacteria. it begins when hemocytes, together with antimicrobial molecules in the hemolymph, surround bacteria. hemocytes with their entrapped bacteria form small aggregates that grow by joining with additional hemocytes to form large nodules. the nodule is completed by covering 110 fig. 1 phagocytosis. the process of ingesting and destroying a particle begins with the particle recognition by special receptors on the cell. this initial interaction triggers a series of cellular signals that induce rearrangement of the actin cytoskeleton and membrane remodeling. the particle sits on a membrane depression, the phagocytic cup, and then it is surrounded by pseudopods. the membrane fuses around the particle and separates into the cell in the form of a new vesicle, the phagosome. in order to internalize large or numerous particles, the phagocyte recruits addition membrane from intracellular vesicles such as endosomes and the endoplasmic reticulum. the phagosome “matures” to form a phagolysosome by fusing with other vesicles such as lysosomes and undergoing acidification. inside the phagolysosome, the ingested particle is finally destroyed. it with layers of flattened hemocytes. in some cases, but not always, nodules are also melanized. these melanin-covered nodules are an effective way to isolate the invading bacteria from the hemolymph, since they have an impermeable wall between the nodule and the rest of the insect organism. the process of nodule formation is not completely characterized, but clearly eicosanoids are likely to be important for nodule formation in many insect species (miller et al., 1999; shrestha et al., 2009; zhao et al., 2009; shrestha et al., 2010) and prophenoloxidase (propo) and dopa decarboxylase (ddc) are also involved in this process at least in mediterranean fruit fly (medfly) c. capitata hemocytes (sideri et al., 2008). in addition, screenings for novel immune genes from an indian saturniid silkmoth (antheraea mylitta) larvae, and from the silkworm (bombyx mori) larvae, identified two proteins, noduler (gandhe et al., 2007) and reeler1 (bao et al., 2011) respectively, with a characteristic reeler domain in the fat-bodies of these insects. these proteins were essential in mediating nodulation response against e. coli k12 and b. subtilis bacteria challenge. encapsulation encapsulation is the response of hemocytes to large targets such as parasites, protozoa, and nematodes. hemocytes bind to the target in multiple cell layers until they form a capsule around the invader. the capsule is normally melanized at the end (ling et al., 2006). inside the capsule the invading organism is killed by reactive cytotoxic products or by asphyxia (carton et al., 2005; nappi et al., 2009). melanization melanization is the process of melanin formation. it is activated during wound healing and also in nodule and capsule formation against large pathogens or parasites in some lepidopteran and dipteran insects, such as manduca, pseudolusia and drosophila (lavine et al., 2001; lavine et al., 2002; kanost et al., 2004; nappi et al., 2009). the enzyme phenoloxidase (po) is key in this process. activation of propo to po is mediated by a serine proteinase cascade (cerenius et al., 2008; eleftherianos et al., 2011) in adult drosophila, but also in lepidopterans and requires prrs such as peptidoglycan recognition protein (pgrp) ( yoshida et al., 1996; ochiai et al., 1999) or beta-1,3-glucan recognition protein (betagrp) (ochiai et al., 1988, 2000). interaction of these recognition proteins with microbial peptidoglycan or beta-1, 3-glucan molecules initiates the activation of the propo cascade. then po binds to foreign surfaces including hemocyte membranes (ling et al., 2005), where it initiates melanin formation. po acts on tyrosine and converts it to dopa (marmaras et al., 2009). dopa can then be decarboxylated by ddc to dopamine or further oxidized by po to dopaquinone. both products are then further metabolized to eumelanin and finally melanin (marmaras et al., 2009). phagocytosis phagocytosis is the process by which cells recognize, bind and ingest relatively large particles (usually larger than 0.5 μm in diameter). phagocytosis is an evolutionary conserved cell response that was first described 130 years ago by elie metchnikoff (1845-1916) in sea-star larva; 111 where he observed the accumulation of cells he named phagocytes, around a rose thorn that he inserted into the larva to provoke an inflammatory injury ( metchnikoff, 1884; kaufmann, 2008; tan et al., 2009). phagocytosis is probably the oldest defense mechanism against microorganisms. during phagocytosis the target particle is first recognized by phagocytic receptors that activate various signaling pathways in the cell interior (jones et al., 1999). these signals lead to dramatic changes in the dynamics of the plasma membrane and the cytoskeleton. the membrane extends pseudopods around the particle, forming a cup that moves into the cell. within few minutes the membrane closes at the distal end, leaving a new plasma membranederived phagosome (yeung et al., 2006) (fig. 1). phagocytosis requires a rapid replenishment of plasma membrane. in mammalian leukocytes, endoplasmic reticulum-derived endosomes have been shown to be the source of this newly added membrane (bajno et al., 2000). in the next 40 minutes, the lumen of the phagosome becomes an environment capable of destroying the ingested particle. this process is called “phagosome maturation” and is the result of changes in the phagosome membrane through fusion with other membranous organelles including lysosomes (yeung et al., 2006) (fig. 1). phagocytosis is a fundamental cellular process performed by unicellular organisms and by particular cell types in multicellular organisms. in simple organisms such as amoeba and the slime mold dictyostelium discoideum, phagocytosis is used both in feeding and in defense (chen et al., 2007; cosson et al., 2008). in vertebrates, phagocytosis plays an essential role in embryogenesis and also in host defense mechanisms through the uptake and destruction of pathogens (greenberg et al., 2002). phagocytosis also contributes to inflammation and the immune response (rosales, 2005). in mammals, a subset of specialized cells, named professional phagocytes, is responsible for rapidly and efficiently ingesting invading microorganisms at sites of inflammation. these phagocytes are neutrophils, monocytes and macrophages (rabinovitch, 1995). neutrophils and monocytes circulate in the blood, while macrophages reside in tissues. in insects, phagocytosis is performed by a subset of hemocytes in the hemolymph (strand, 2008). professional phagocytes in diptera and lepidoptera have also been described as plasmatocytes and granular hemocytes, respectively (lavine et al., 2002). in agreement with this, plasmatocytes or granulocytes are the main phagocytic cells in most insects ( meister, 2004; castillo et al., 2006; lamprou et al., 2007; lemaitre et al., 2007; garcia-garcia et al., 2009). however, there is clearly a great deal of variability among different insect species. our knowledge of insect phagocytosis comes mainly from studies on the fruit fly d. melanogaster (lemaitre et al., 2007; stuart et al., 2008), on mosquitoes anopheles (blandin et al., 2007), which are vectors of the human malaria parasite plasmodium falciparum, or on the medfly c. capitata (lamprou et al., 2005; sideri et al., 2008). this however will change in the future, as more and more reports are coming out describing the phagocytic process by hemocytes from other insect species ( kanost et al., 2004; costa et al., 2005; mylonakis et al., 2005; borges et al., 2008; castro et al., 2009; garcia-garcia et al., 2009; amaral et al., 2010; manachini et al., 2010; wang et al., 2010). phagocytosis eliminates mainly two types of targets: microorganisms and “altered self” particles, represented by apoptotic cells. ingestion of apoptotic cells is important during tissue remodeling and embryogenesis when excess cells undergo programmed cell death (apoptosis) and removal (hopkinson-woolley et al., 1994). in insects, phagocytosis of dying cells is fundamental during embryogenesis, especially in the development of holometabolous insects such as d. melanogaster. in this fly, hemocytes eliminate the many apoptotic cells that appear during the process of metamorphosis (neufeld et al., 2008). despite the importance of this process in insect development, the mechanisms of phagocytosis of apoptotic cells in insects are still poorly known (sass, 2008). ingestion of microorganisms is also fundamental for insect defense against infections. however, it is becoming clear that hemocyte phagocytosis can control some, but not all bacterial infections. for example, it has been reported that e. coli (a gram-negative bacteria) is more readily phagocytosed than s. aureus (a gram-positive bacteria) by hemocytes from the mosquito a. gambiae ( levashina et al., 2001; hillyer et al., 2003), the fruit fly d. melanogaster (rämet et al., 2002), and the medfly c. capitata (lamprou et al., 2007). similar results were found for the phagocytosis of e. coli by cricket acheta domesticus hemocytes (garcia-garcia et al., 2009), but opposite results for phagocytosis of streptomyces lividans (a gram-positive bacteria) by the beetle zophobas morio hemocytes (garciagarcia et al., 2009). also in a. aegypti the main response of hemocytes to e. coli was phagocytosis, while the response to m. luteus was melanization (hillyer et al., 2003). these results should be taken with caution because nonvirulent bacteria are compared with pathogenic bacteria, therefore we cannot generalize the phagocytic response observed will be similar for all gram-positive or gram-negative bacteria. also, bacteria have been previously killed to perform these phagocytosis assays. thus, the phagocytosis response in vivo might also be different. together these reports suggest that indeed in insects several distinct molecular mechanisms controlling phagocytosis must exist. our knowledge of phagocytosis comes mainly from studies with mammalian cells and from these studies it is clear that there is much redundancy in this cellular response. different phagocyte membrane receptors and various elements of the phagocytic machinery seem to have similar and many times overlapping functions. despite this, much has been learned about phagocytic receptors for opsonins, such as antibodies and complement. opsonins are substances that cover the particle to be ingested and promote its phagocytosis. the beststudied phagocytic receptors are the receptors for antibody, termed fc receptors (garcia-garcia et al., 2002; swanson et al., 2004), and the complement receptors (jones et al., 1999; rosales, 2007). 112 however, our knowledge on the function of individual receptors of phagocytosis in professional phagocytes remains incomplete because these cells are not suitable for many genetic and molecular biology techniques, such as cdna overexpression or knockdown expression of molecules by rna interference (rnai). thus, studies of phagocytosis by d. melanogaster hemocytes have become very attractive because this is a genetically tractable system. in addition, the power of rnai analysis has been used to develop a molecular screen system for phagocytosis in the drosophila embryonic s2 (schneider line 2) cell line. these fruit fly cells present characteristics similar to mammalian macrophages and efficiently ingest bacteria in an actin-dependent manner (pearson et al., 2003). this system has also been adapted for high-throughput screening with rnai leading to identification of many phagocytosis-related genes (rämet et al., 2002; cheng et al., 2005). moreover, the system has also identified molecules involved in hemocyte interaction with microorganisms such as e. coli (rämet et al., 2002), s. aureus (stuart et al., 2005a), mycobacterium fortuitum (philips et al., 2005), listeria monocytogenes (agaisse et al., 2005; cheng et al., 2005), and candida albicans (stroschein-stevenson et al., 2006; stroscheinstevenson et al., 2009). similarly, the power of rnai has also been used in studies of phagocytosis by mosquito a. gambiae hemocytes ( levashina et al., 2001; blandin et al., 2002). despite the fact that drosophila s2 cells present characteristics similar to mammalian macrophages (pearson et al., 2003) the similarity of cultured cells to hemocytes remains an open question. thus the findings from the studies mentioned above still require in vivo validation. the gold standard is an increased susceptibility to specific infections of mutant insects, coupled with in vitro and in vivo data demonstrating an impaired phagocytosis. such an in vivo system has recently been described. a genome-wide in vivo drosophila rna interference screen was used to uncover genes involved in susceptibility or resistance to intestinal infection with the bacterium serratia marcescens (cronin et al., 2009). this system, in turn requires in vitro validation of the genes potentially involved in phagocytosis. but, things look promising since some of the identified genes overlap with those identified in a systems biology proteomic analysis of phagosomes isolated from cells derived from d. melanogaster (stuart et al., 2007) (see below). at the other end of the phagocytosis process, it is the destruction of the ingested particle within the phagosome. in mammals, phagosomes are not only important in innate immunity but also in adaptive immunity since they are active participants of the process of antigen presentation (houde et al., 2003). phagosomes vary in nature depending on the cell-surface receptors involved to recognize the target particle, the type of membrane used in their formation, the mechanism of internalization and the nature of the particle. once formed, the new phagosome undergoes a process known as phagosome maturation (kinchen et al., 2008). in this process the phagosome fuses with internal vesicles such as endosomes and lysosomes to become a mature phagolysosome (fig. 1). this mature compartment has an acid environment with highly active hydrolytic enzymes, which stop replication of bacteria and can kill many microorganisms (kinchen et al., 2008). the importance of the phagosome is also made evident by the fact that some microorganisms can alter the maturation process escape from being killed (fratti et al., 2001). thus, there is a great interest in understanding the complex biology of phagosomes. insect hemocytes and new techniques such as proteomics and computational modeling have also been helpful in elucidating the complexity of the phagosome (stuart et al., 2007). proteomics analysis of latex beadscontaining phagosomes from d. melanogaster s2 cells has confirmed the complexity of this organelle. close to 600 d. melanogaster proteins were identified to be associated with phagosomes, and many of them had mammalian orthologs, validating this as a model for mammalian phagocytosis. computational analysis has predicted that hundreds of protein-protein interactions take place in the phagosome and has identified several signaling pathways that could be initiated from this organelle including activation of the nuclear factor κb (nf-κb) and activation of mitogen-activated protein kinases (mapk) (fratti et al., 2001). together rnai and proteomics approaches with insect hemocytes have identified new genes and molecules important for phagocytosis, specially the putative receptors that insect hemocytes use to bind different microorganisms and signaling pathways activated during phagosome maturation. the relevance of these molecules for phagocytosis and host defense can now be tested in vivo. insect phagocytic receptors commonly for mammalian leukocytes, phagocytosis is initiated after the interaction of opsonins, on the surface of the particle to be internalized, with specific receptors on the phagocyte membrane (swanson et al., 1995). phagocytosis can also be triggered, in the absence of opsonins, through the interaction of phagocyte membrane receptors with specific molecules, such as lipids or sugars, which form part of the cell wall of many microorganisms (stuart et al., 2005b; stuart et al., 2008). these receptors are known as patternrecognition receptors (prrs) because they recognize discrete conserved molecular patterns within microorganism molecules. in insects, several potential prrs have been identified, and they can be grouped in various types: complement-like molecules, scavenger receptors, epidermal growth factor (egf)-like repeat-containing receptors, peptidoglycan recognition proteins (pgrps), integrins, and a highly variant receptor, down syndrome cell-adhesion molecule (dscam) (table 1). complement-like molecules thioester-containing proteins (teps) constitute an important group of proteins that includes the α2macroglobulin family of protease inhibitors and the c3/c4 complement factors in vertebrates. the thioester active site typical of and the α2macroglobulins and complement factors is present 113 table 1 insect phagocytic peceptors insect receptors ligands mammalian counterpart opsonin insect refs complement-like molecules tep vi candida albicans complement yes d. melanogaster s2 cells stroschein-stevenson et al., 2006; stroschein-stevenson et al., 2009 tep ii e. coli yes d. melanogaster s2 cells tep iii s. aureus yes d. melanogaster s2 cells tep1, tep3 e. coli, s. aureus a. gambiae moita et al., 2005 scavenger receptors croquemort apoptotic cells, s. aureus cd36 d. melanogaster franc et al., 1999; stuart et al., 2005a peste m. fortuitum sr-bi, sr-bii d. melanogaster philips et al., 2005 sr-ci e. coli, s. aureus ? d. melanogaster rämet et al., 2001 egf-like repeatcontaining receptors eater e. coli, s. aureus s. marcescens d. melanogaster charroux et al., 2009b; defaye et al., 2009; kocks et al. 2005; nehme et al., 2011 nimrod e. coli, s. aureus d. melanogaster kurucz et al., 2007 draper apoptotic cells, axon pruning, severed axons, s. aureus cd91 (lrp) d. melanogaster awasaki et al., 2006; freeman et al., 2003; hashimoto et al., 2009; macdonald et al., 2006; manaka et al., 2004 six-microns-under (simu) apoptotic cells, by glia in the nervous system d. melanogaster kurant et al., 2008 peptidoglycan recognition proteins pgrp-sc1a s. aureus mammalian pgrp* d. melanogaster garver et al., 2006 pgrp-lc e. coli d. melanogaster kurata, 2010; rämet et al., 2002 pgrp-le l. monocitogenes d. melanogaster yano et al., 2008 integrins α integrins e. coli, grampositive bacteria mammalian integrins medfly (c. capitata) lamprou et al., 2007 β integrins e. coli medfly (c. capitata) a. gambiae mamali et al., 2009; moita et al., 2006 dscam e. coli, s. aureus immunoglobulin ? yes a. gambiae d. melanogaster d. melanogaster dong et al., 2006; stuart et al., 2007; watson et al., 2005 * soluble molecules with no involvement in phagocytosis. tep, thioester-containing proteins; sr-ci, scavenger receptor class c 1; lrp, low-density lipoprotein receptorrelated protein; pgrp. peptidoglycan recognition protein; dscam, down syndrome cell-adhesion molecule. in the tep proteins. in insects, several teps are known in d. melanogaster (lagueux et al., 2000) and in a. gambiae (christophides et al., 2002). there are six tep proteins (tepi to vi) in the genome of drosophila, including tepv which is a putative pseudogene, and tepvi (also named mcr: macroglobulin complement-related) in which the thioester site is mutated (bou aoun et al., 2008). some of the teps are upregulated after a bacterial infection (lagueux et al., 2000), with tepiii being an 114 exception (bou aoun et al., 2011). their function was elucidated from an rnai screen in drosophila s2 cells, in which tepvi was found to bind and increase phagocytosis of c. albicans (stroscheinstevenson et al., 2006; stroschein-stevenson et al., 2009). similarly, tepii binds e. coli and tepiii binds s. aureus, and both increase phagocytosis (stroschein-stevenson et al., 2006) (table 1). in addition, teps also increase phagocytosis in a. gambiae (moita et al., 2005). because these teps are soluble molecules that bind to microorganisms and induce phagocytosis by insect hemocytes, they behave like typical opsonins. strengthen this idea is their structural relationship with mammalian complement components. in mammals, specific complement receptors bind the c3 complement factor deposited on microorganisms and promote phagocytosis (jones et al., 1999; rosales, 2007). however, insect complement-like receptors specific for binding teps on microorganisms have not been described so far. thus the mechanism by which some teps increase phagocytosis remains unclear. in addition, because tepvi does not have the classical thioester motif, it is possible that some teps may have alternative functions, such as protease inhibitors of the α2-macroglobulin family. scavenger receptors the scavenger receptors are unrelated multiligand receptors that share the capacity of binding to polyanionic ligands. for this reason they can bind multiple microorganisms and are thus important prrs (table 1). croquemort and its mammalian homolog cd36 are class b scavenger receptors. cd36 associates with integrins and mediates ingestion of apoptotic cells. similarly, croquemort is involved in phagocytosis of apoptotic cells during larval morphogenesis in d. melanogaster (franc et al., 1996; 1999) (fig. 2). in addition, croquemort has been reported to bind and induce phagocytosis of s. aureus in adult flies (franc et al., 1999), and this led to the characterization of cd36 as a phagocytic receptor for s. aureus (stuart et al., 2005a). because cd36 functions together with phosphatidylserine receptors (fadok et al., 2000), it was proposed that croquemort also required cooperation of phosphatidylserine receptor (psr) for phagocytosis of apoptotic cells in d. melanogaster (fadok et al., 2001). however, psr has been shown to be a nuclear protein and its role in phagocytosis is supported only by few psr lossof-function experiments (wolf et al., 2007). in mammals, other phosphatidylserine receptors have been described to be important for phagocytosis of apoptotic cells (kobayashi et al., 2007). thus, whether croquemort needs cooperation of phosphatidylserine recognition to promote ingestion of apoptotic cells remains an open question (kinchen, 2010). peste is another class b scavenger receptor identified with an rnai screen in drosophila s2 cells. peste can bind m. fortuitum (philips et al., 2005). this result has suggested that mammalian class b scavenger receptors may also be involved in recognition of mycobacteria. this idea is supported by experiments in which the mammalian class b scavenger receptors sr-bi and sr-bii conferred to non-phagocytic cells the capacity of ingesting m. fortuitum (philips et al., 2005). sr-ci is another unrelated type of scavenger receptor. scavenger receptor class c 1 (sr-ci) is expressed in drosophila hemocytes during embryonic development, and it can bind e. coli (a gram-negative bacteria) and s. aureus (a grampositive bacteria) (rämet et al., 2001). its expression is also upregulated in larvae after a bacterial infection (irving et al., 2005). however, the role of sr-ci in phagocytosis is not clear since phagocytic defects due to mutations of this receptor are very weak, about 20 % of wildtype cells (rämet et al., 2001; lazzaro et al., 2006). epidermal growth factor (egf)-like repeatcontaining receptors a new family of prrs that contain egf-like repeats to recognize different ligands is being documented in many species from c. elegans, insects, to mammals (kocks et al., 2005; kurucz et al., 2007). eater, a d. melanogaster protein, is a transmembrane molecule with 32 egf-like repeats in its extracellular domain. eater was the first egflike repeat receptor shown to be involved in bacterial recognition. it recognizes bacteria through its four nterminal egf-like repeats (kocks et al., 2005) and it is involved in phagocytosis (chung et al., 2011) (table 1). knockdown expression of eater in drosophila s2 cells resulted in reduced e. coli and s. aureus bacterial binding and ingestion. also, hemocytes from eater-deficient flies are impaired in phagocytosis of bacteria (kocks et al., 2005), and flies lacking the eater gene display more susceptibility to infection by ingested serratia marcescens and to injected gram-positive bacteria, such as s. aureus or e. faecalis (kocks et al., 2005; charroux et al., 2009b; defaye et al., 2009; nehme et al., 2011). nimrod is another d. melanogaster molecule similar to eater. it is a transmembrane protein with ten egf-like repeats in its extracellular domain. silencing nimrod expression in drosophila s2 cells results in less phagocytosis of bacteria, while overexpression increases adhesion of these cells to tissue-culture plates (kurucz et al., 2007), suggesting that nimrod may be both a phagocytic receptor and an adhesion molecule (table 1). draper is yet another transmembrane protein with egf-like repeats in its extracellular domain. it is expressed in glial cells and d. melanogaster hemocytes (freeman et al., 2003). it is important for phagocytosis of apoptotic cells in the central nervous system (awasaki et al., 2006; macdonald et al., 2006), by embryonic hemocytes, and by drosophila s2 cells (manaka et al., 2004) (table 1). recently, a molecule expressed on apoptotic cells was identified as a ligand for draper. this molecule, named pretaporter, is an endoplasmic reticulum protein that relocates to the cell surface during apoptosis to serve as a ligand for draper in the phagocytosis of apoptotic cells (kuraishi et al., 2009). draper is also involved in phagocytosis of both e. coli (gram-negative) and s. aureus (grampositive) bacteria, and recently it was reported that 115 fig. 2 phagocytosis pathways in drosophila hemocytes. draper initiates phagocytosis of apoptotic cells or s. aureus bacteria by binding to pretaporter, an endoplasmic reticulum protein that relocates to the cell surface of apoptotic cells, or to lipoteichoic acid on the surface of s. aureus. draper binds in a src kinase-dependent manner to shark, a non-receptor tyrosine kinase, similar to the mammalian syk. then, draper is able to interact with ced6 to promote phagocytosis. also, undertaker, a drosophila junctophilin protein, is required for draper-mediated phagocytosis. junctophilins couple ca2+ channels at the plasma membrane to the ryanodine receptors of the endoplasmic reticulum (er). the ryanodine receptor rya-r44f, the er ca2+ sensor dstim, and the ca2+release-activated ca2+ channel dorai are all in the same pathway promoting calcium homeostasis and phagocytosis. croquemort and six-microns-under (simu) are other phagocytic receptors in drosophila hemocytes, but their signaling pathways are still unknown. lipoteichoic acid serves as a ligand for draper in the phagocytosis of s. aureus by drosophila hemocytes (hashimoto et al., 2009). these reports confirm the idea that draper is indeed a multi-ligand prr. draper is a homolog of the c. elegans apoptotic receptor ced-1 (gumienny et al., 2001a, b), and similarly to this receptor, draper also initiates phagocytosis by interacting with ced-6, an adapter protein containing a phosphotyrosine-binding domain, via an itam motif in its intracytoplasmic tail (su et al., 2002). this motif is also present in the apoptotic receptor cd91/low-density lipoprotein receptor-related protein (lrp) (su et al., 2002). interestingly, in the mosquito a. gambiae a reverse genetics complementation screen (moita et al., 2005) designed to assign putative regulators of e. coli or s. aureus phagocytosis into separated functional groups clearly showed that ced-6 is functionally connected to the lrp for efficient phagocytosis of bacteria (blandin et al., 2007). together these results suggest that draper, ced-1, and cd91/lrp all activate a similar phagocytic machinery (see fig. 2). clearly, draper binds apoptotic cells and s. aureus to initiate 116 phagocytosis. however, it is not clear that the signaling pathway shown in fig. 2 is in fact the same one for both ligands. the model is a composite of the information available today. six-microns-under (simu) is another drosophila phagocytosis receptor, which is expressed in highly phagocytic cell types during development and required for efficient apoptotic cell clearance by glia in the nervous system and by hemocytes (macrophages) elsewhere. simu belongs to a conserved family of proteins that includes ced-1 and draper, and strongly binds to apoptotic cells, but does not require membrane anchoring, suggesting that it can function as a bridging molecule (kurant et al. 2008). phenotypic analysis revealed that simu acts upstream of draper in the same pathway and affects the recognition and engulfment of apoptotic cells, while draper affects their subsequent degradation (kurant et al., 2008; kinchen, 2010) (see fig. 2). peptidoglycan recognition proteins (pgrps) peptidoglycan recognition proteins (pgrps) are innate immunity proteins that are conserved from insects to mammals, recognize bacterial peptidoglycan, and function in antibacterial immunity and inflammation. pgrps have at least one carboxy-terminal pgrp domain (approximately 165 amino acids long), which is homologous to bacteriophage and bacterial type 2 amidases. mammals have four pgrps (dziarski et al., 2006a, 2010). they are secreted proteins expressed in polymorphonuclear leukocytes (pgrp1), in liver (pgrp2), or on body surfaces and in secretions (pgrp3 and pgrp4). all pgrps recognize bacterial peptidoglycan and three of them (pgrp1, pgrp3, and pgrp4 are directly bactericidal for both gram-positive and gram-negative bacteria (dziarski et al., 2010). insects have up to 19 pgrps, classified into short (s) and long (l) forms. the short forms are present in the hemolymph, cuticle, and fat-body cells, whereas the long forms are mainly expressed in hemocytes (royet et al., 2007; charroux et al., 2009a). the expression of insect pgrps is often upregulated by exposure to bacteria. insect pgrps activate the toll or immune deficiency (imd) signal transduction pathways (described later) or induce proteolytic cascades that generate antimicrobial products (steiner, 2004; dziarski et al., 2006b). several drosophila pgrps have lost this enzymatic activity and serve as microorganism sensors through peptidoglycan recognition. other pgrp family members, such as pgrp-sc1 (bischoff et al., 2006) and pgrp-lb (zaidman-rémy et al., 2006) have conserved the amidase function and are able to cleave peptidoglycan in vitro. however, the contribution of these amidase pgrps to host defense in vivo remains unclear. in pgrp-sc1/2-depleted drosophila larvae, an over-activation of the imd signaling pathway was detected after bacterial challenge in the gut (bischoff et al., 2006). in drosophila at least three pgrps have been shown to be important for phagocytosis of bacteria (table 1). pgrp-sc1a is relevant for phagocytosis of s. aureus (garver et al., 2006), while the membrane-bound receptor pgrp-lc is involved in phagocytosis of e. coli (gram-negative) but not gram-positive bacteria (rämet et al., 2002). interestingly, pgrp-sa was proposed to function in phagocytosis of s. aureus (garver et al. 2006), but this effect was not found in another study (nehme et al., 2011). the drosophila pgrp-le functions synergistically with pgrp-lc in producing resistance to e. coli and b. megaterium infections (takehana et al., 2004). it is expressed in two forms, the full-length pgrp-le acts as an intracellular receptor for monomeric peptidoglycan, whereas a version of pgrp-le containing only the pgrp domain functions extracellularly to enhance pgrplc-mediated peptidoglycan recognition on the cell surface (kaneko et al., 2006). pgrp)-le is also an important prr against intracellular bacteria such as listeria monocytogenes (yano et al., 2008). the pgrp-le-mediated intracellular response against l. monocytogenes infection includes induction of autophagy (kurata 2010) and induction of the gene listericin in cooperation with the jak-stat (janus kinase-signal transducers and activators of transcription) pathway (goto et al., 2010). integrins integrins are adhesion molecules found on the surface of virtually all cell types in mammals. they are responsible for binding to extracellular matrix proteins and to adhesion ligands on other cells (shattil et al., 2004; arnaout et al., 2007). insect hemocytes aggregate in multiple layers during encapsulation and bind to microorganisms during phagocytosis. these functions can be mediated by integrins (rosales, 2007) and indeed various integrins have been found in insect hemocytes (table 1). β integrins are important during encapsulation of wasp eggs by drosophila hemocytes (irving et al., 2005). α integrins are also relevant for encapsulation by m. sexta hemocytes (levin et al., 2005; zhuang et al., 2008). various α and β integrins are required for microbial recognition by granulocytes and plasmatocytes of p. includens (lavine et al., 2003). integrins are clearly important for phagocytosis of both e. coli (gram-negative) and s. aureus (gram-positive) bacteria by medfly hemocytes (foukas et al., 1998; lamprou et al., 2007; mamali et al., 2009), and for phagocytosis of e. coli by a. gambiae hemocytes (moita et al., 2006). down syndrome cell-adhesion molecule (dscam) down syndrome cell-adhesion molecule (dscam) is an ig superfamily receptor that is important for neural development and bacterial recognition in d. melanogaster (table 1). dscam was first identified as a molecule involved in determining neuron connections during development (schmucker et al., 2000). to achieve this, dscam expresses many different isoforms derived from splice variants generated by combining variable and constant gene regions, similarly to the gene rearrangement that generates antigen receptor diversity in b and t lymphocytes in mammals. it is estimated that dscam could have more than 38,000 isoforms (schmucker et al., 2000). in immune tissues, hemocytes and fat-body cells, dscam could express more than 18,000 different 117 fig. 3 toll or immune deficiency (imd) signal transduction pathways. a) activation of the transmembrane receptor toll requires a proteolytically cleaved form of an extracellular cytokine-like polypeptide, spätzle. after a complex of 4-spätzle:2-toll is formed the signaling pathway myd88/pelle/tube leads to phosphorylation and degradation of cactus (an iκb inhibitor) and translocation to the nucleus of the nf-κb transcription factors dorsal and dif. these factors in turn activate transcription of antimicrobial peptides (amp). b) the imd pathway is activated by binding of peptidoglycan (pgn) to the pgrp-lc receptor. this initiates signaling to the transcription factor relish. imd is connected to the caspase dredd via the adaptor protein fas-associated dd protein (fadd). dredd is believed to have two functions: upstream, it activates the tak1/ikkβ complex, which in turn phosphorylates relish; downstream, dredd cleaves phosphorylated relish. the n-terminal nf-κb module of relish then translocates into the nucleus where it activates transcription of antimicrobial peptides (amp). extracellular domains and also a similar number of soluble isoforms released to the hemolymph (watson et al., 2005). therefore, dscam could produce sufficient numbers of microbial recognition receptors to provide a wide range of specificity (neves et al., 2004; meijers et al., 2007). although, presently it is unclear how many dscam isoforms are produced by a single cell. dscam has been suggested to be important for phagocytosis of e. coli and s. aureus in a. gambiae (dong et al., 2006), and has also been found associated with phagosomes from drosophila s2 cells (stuart et al. 2007). in addition, there is an increase of secreted dscam isoforms after a bacterial challenge (dong et al., 2006). these characteristics have suggested that dscam might function similarly to antibody molecules in that they bind to microorganism and promote phagocytosis (stuart et al., 2008). however, it should be pointed out that data from drosophila is only from cultured cells and there is not evidence for a role of dscam in phagocytosis in vivo. also, the study in mosquitoes shows that silencing of dscam increases susceptibility to e. coli and s. aureus infections, but the connection with phagocytosis is weak. thus, further studies are needed to confirm the role of dscam as an antibody-like molecule that promotes phagocytosis. in addition, the mechanism for dscam expression is different from the mechanisms of adaptive immunity, which are based on selection and clonal expansion of lymphocytes (a process not found in invertebrate hemocytes), producing a single type of immunoglobulin receptor on each cell (blandin et al., 2007). 118 pallbearer pallbearer is an f box protein found in an in vivo screen for genes required for efficient phagocytosis of apoptotic cells by drosophila s2 cells. f box proteins generally provide substrate specificity to skp cullin f box (scf) complexes, acting as e3 ligases that target phosphorylated proteins to ubiquitylation and degradation via the 26s proteasome. pallbearer functions in an scfdependent manner and its involvement in phagocytosis of apoptotic cells in vivo suggests a role for ubiquitylation and proteasomal degradation in this cellular process (silva et al., 2007). nonaspanins nonaspanins comprise an evolutionarily conserved family of proteins with an essentially unknown function. they are characterized by a large n-terminal extracellular domain and nine putative transmembrane domains. three members in dictyostelium discoideum (phg1a, phg1b and phg1c) and drosophila melanogaster, and four in mammals (tm9sf1-tm9sf4) have been found (chluba-de tapia et al., 1997; schimmoller et al., 1998). genetic studies in dictyostelium demonstrated that phg1a is required for cell adhesion and phagocytosis (cornillon et al., 2000; benghezal et al., 2003; benghezal et al., 2006). in drosophila, phg1a/tm9sf4-null mutant larval hemocytes were sensitive to pathogenic e. coli (gram-negative), but not to s. aureus (grampositive) bacteria and tm9sf4-defective s2 cells showed reduced bacterial internalization (bergeret et al., 2008). these defects in phagocytosis were coupled to morphological and adhesion defects in mutant larval hemocytes, which had an abnormal actin cytoskeleton (bergeret et al., 2008). psidin psidin is a lysosomal protein required in hemocytes for both degradation of engulfed bacteria and activation of fat-body cells to produce antimicrobial-peptides (amp). for a long time, the role of drosophila phagocytes in the activation of the humoral immune response has not been clear. previous studies to determine whether hemocytes play a role in activation of amp production by the fat-body have relied on domino mutant larvae, in which proliferative tissues are disrupted and hemocyte numbers greatly reduced (braun et al., 1998). in these mutants, injection of e. coli results in normal activation of most amps, including diptericin (braun et al., 1998), which suggested that activation of imdand toll-pathways is independent of hemocytes. however, domino mutants failed to induce diptericin during erwinia carotovora (a gramnegative) gut infection (basset et al., 2000), suggesting that hemocytes could deliver a signal from the gut to activate imd signaling in the fat-body. psidin was then identified in a screen for mutant larvae unable to induce diptericin in response to injected e. coli (wu et al., 2001). more recently, drosophila larvae psidin mutants were not able to produce the amp defensin after e. coli (gramnegative) or m. luteus and b. subtilis (gram-positive) infections. this defect was not rescued by psidin expression in the fat-body (brennan et al., 2007). in addition, mutant hemocytes presented impaired phagocytosis, since they could not degrade the ingested bacteria, and psidin was found localized to lysosomes (brennan et al., 2007). thus, the psidin gene acts in larval hemocytes, where it is required for the phagocytic degradation of internalized bacteria and for the induction of defensin in the fatbody. these findings, whereby phagosome maturation is required for activation of humoral responses are reminiscent of the relationship in mammalian leukocytes, between endosome/phagosome function and immune activation. however, whereas the mechanisms for antigen processing and presentation in vertebrate adaptive immunity have been studied extensively (houde et al., 2003; amigorena et al., 2010), the connection between phagosome maturation and initiation of innate immunity are just beginning to be revealed. future studies will help understanding how phagocytosis and destruction of microorganisms in phagolysosomes are coupled to activation of innate immune responses. the receptors described above are all involved in insect (host)-microorganism (pathogen) interactions. most of them have been shown to somehow participate in phagocytosis. however, some of these receptors can be classified as pure prrs, that is receptors that have been selected during evolution for their ability to recognize conserved microbial structures that microorganisms cannot easily modify, as they are essential to their lifestyles. among them, we have pgrps and gnbps. some other receptors can be considered “true” phagocytic receptors, that is receptors that are required for fighting off specific pathogens. for example, teps and draper. several population genetic studies have been conducted with fruit flies, taking advantage of the complete genome sequencing of 12 lines of drosophila melanogaster and one line of d. simulans (jiggins et al., 2003; sackton et al., 2007). these studies find that pgrp and gram-negative binding protein (gnbp) genes are highly conserved. in contrast, drosophila tep genes evolve rapidly under positive selection (jiggins et al., 2006). pattern recognition receptors that trigger humoral immunity are evolutionarily rather static, but receptors required for phagocytosis show considerable genomic rearrangement and adaptive sequence divergence (lazzaro, 2008; sackton et al., 2010). thus, we should be alert to make the distinction between regular prrs that trigger drosophila systemic immune responses and phagocytic receptors, which may not be adequately classified as regular prrs. signaling pathways once a microorganism is detected by an immune cell, a series of signaling molecules are activated inside the cell to instruct it for different responses. these molecules follow particular signaling pathways that determine the final cellular response. in mammals the signaling pathways for phagocytosis are relatively well known, particularly for antibody and complement-mediated phagocytosis (rosales, 2007). in insects, the signaling pathways 119 fig. 4 phagocytosis pathways in mosquitoes hemocytes. in the mosquito a. gambiae two major signaling pathways for phagocytosis of bacteria have been defined with a genetics screen. the intracellular molecules ced-2, ced-5, and ced-6 are the homologs of known components of the two pathways that mediate phagocytosis of apoptotic cells in c. elegans. silencing ced-5 of ced-6 at the same time with identified regulators of phagocytosis permitted assignment of these regulators to one or the other pathway. one pathway, referred as ced-5, involves secreted putative opsonin tep4, the integrin bint, and the intracellular components ced-2 and ced-5. the other pathway, referred to as ced-6, involves the secreted proteins tep1, tep3, lrim1, and the transmembrane receptor lpr (low-density lipoprotein receptor-related protein). involved in humoral immune responses are relatively well described; but for cellular immune responses, the signaling pathways are only partially known. although signaling pathways for humoral responses are not directly related to phagocytosis, i will briefly describe them first, for comparison reasons, followed by what is known about the signaling pathways for cellular (phagocytic) responses. the humoral immune responses mainly involve the release of antimicrobial peptides by the fat-body, via the toll (valanne et al., 2011) and the immune deficiency (imd) pathways (kaneko et al., 2005). gram-positive bacteria and fungi predominantly induce the toll signaling pathway, whereas gramnegative bacteria activate the imd pathway (fig. 3). the toll signaling pathway d. melanogaster toll pathway was initially identified as a developmental pathway. it involves nf-κb signaling and is essential for embryonic development and immunity (ashok, 2009; valanne et al., 2011). from there, the subsequent characterization of toll-like receptors (tlrs) has reshaped our understanding of the mammalian immune system (kawai et al., 2011). activation of the transmembrane receptor toll requires a proteolytically cleaved form of an extracellular cytokine-like polypeptide, spätzle (shia et al. 2009), suggesting that toll requires cooperation of other prrs. this idea is supported by the fact that a mutation in a peptidoglycan recognition protein 120 (pgrp-sa) blocks toll activation by gram-positive bacteria (m. luteus, streptococcus faecalis, b. thuringiensis) and significantly decreases resistance to this type of infection (michel et al., 2001). toll activation is not only mediated by pgrps, but it requires gnbp1 (gram-negative binding protein 1) for gram-positive (m. luteus or enterococcus faecalis) bacterial infections (wang et al. 2006), and gnbp3 for fungal (beauveria bassiana, m. anisopliae, c. albicans, c. glabrata, saccharomyces cerevisiae, and aspergillus fumigatus) infections (gottar et al., 2006; mishima et al., 2009). in addition, the drosophila persephone protease activates the toll pathway when proteolytically matured by secreted fungal virulence factors. thus, the detection of fungal infections in drosophila relies both on the recognition of invariant microbial patterns and on monitoring the effects of virulence factors on the host (gottar et al., 2006). toll activation initiates the signaling pathway myd88/pelle/tube that leads to degradation of cactus and translocation to the nucleus of the nfκb transcription factors dorsal and dif (imler et al., 2002; tauszig-delamasure et al., 2002; valanne et al., 2011) (fig. 3a). the imd signaling pathway the imd pathway is activated by the pgrp-lc receptor (gottar et al. 2002) and initiates signaling to the transcription factor relish (choe et al. 2002), via the pathway fadd/dredd and the pathway tak1/ikkb ( kaneko et al., 2005; aggarwal et al., 2008; silverman et al., 2009) (fig. 3b). phagocytosis signaling pathways arguable, the most relevant cellular immune response in insects is phagocytosis. several signaling pathways have been described for this function in various insect species. in d. melanogaster hemocytes, an important phagocytic receptor is draper (table 1). this receptor mediates phagocytosis of apoptotic cells by activating shark, a non-receptor tyrosine kinase, similar to the mammalian syk. shark binds to draper via an itam motif present in the cytoplasmic tail of draper. in addition, draper/shark interaction is dependent on phosphorylation of draper by the kinase src (ziegenfuss et al., 2008). draper also interacts with ced-6 (su et al., 2002), an adapter protein containing a phosphotyrosine-binding domain, to initiate phagocytosis. thus, draper-mediated phagocytosis activates the draper/src/syk/ced-6 pathway (fullard et al., 2009) (fig. 2). in addition, it was recently found that undertaker (uta), a drosophila junctophilin protein, is also required for draper-mediated phagocytosis. junctophilins couple ca2+ channels at the plasma membrane to those of the endoplasmic reticulum (er), the ryanodine receptors (cuttell et al., 2008). draper, ced-6, uta, the ryanodine receptor rya-r44f, the er ca2+ sensor dstim, and the ca2+-release-activated ca2+ channel dorai were placed in the same pathway promoting calcium homeostasis and phagocytosis (cuttell et al., 2008) (fig. 2). as mentioned before, in the mosquito a. gambiae two major signaling pathways for phagocytosis of bacteria have been defined with a genetics screen (moita et al. 2005). one pathway, referred as ced-5, involves secreted putative opsonin tep4, the integrin bint, and the intracellular components ced-2 and ced-5 (fig. 4). another pathway, referred to as ced-6, involves the secreted proteins tep1, tep3, lrim1, and the transmembrane receptor lpr (blandin et al., 2007) (fig. 4). in medfly hemocytes, phagocytosis of bacteria involves the pathway integrin/src/fak/map kinases, that ends in activation of an elk-1-like protein (mamali et al., 2008a). activated elk-1-like is localized exclusively in the cell nucleus and it is associated with fak (mamali et al., 2008b) (fig. 5). map kinases can also be activated by other ligands such as lps and latex beads (lamprou et al., 2005). the signaling pathways from these stimuli to map kinases are still not defined. the signaling pathways for phagocytosis of bacteria by insect hemocytes are still incomplete. however, the pathways known resemble the pathways described in mammalian phagocytes. this suggests that many other signaling molecules described as relevant for phagocytosis in mammalian phagocytes will also be found to participate in phagocytosis by insect hemocytes. in support of this view, there are reports indicating that other signaling molecules are clearly involved in phagocytosis. it remains to place these signaling molecules within the corresponding signaling pathway. some of these signaling molecules participating in phagocytosis by insect hemocytes are mentioned next. rho rho is a family of small gtpases, homologous to ras, that includes rho, rac, and cdc42. these gtpases are involved in regulating the actin cytoskeleton (burridge et al., 2004). in drosophila rac1 (avet-rochex et al., 2007) and rac2 (williams et al., 2006) are relevant for phagocytosis. in medfly hemocytes rho gtpases participate in lps and e. coli phagocytosis (soldatos et al., 2003). fak focal adhesion kinase (fak) was first described as a kinase associated to focal adhesions, which are the cell adhesion structures where the actin cytoskeleton connects with the extracellular matrix via integrins. upon integrin engagement, fak is activated and functions as a docking molecule for src family kinases and other signaling molecules. fak plays a central role in various cell responses such as cell spreading and migration, cell proliferation and apoptosis (schaller, 2010). an insect fak homolog, dfak56 has been found in the central nervous system, epidermis, nerve cord, and visceral mesoderm of d. melanogaster (palmer et al., 1999). fak has been shown to be relevant for phagocytosis of e. coli by insect hemocytes (metheniti et al., 2001). syk spleen tyrosine kinase (syk) is a non-receptor tyrosine kinase with a clear and essential role in mammalian fc receptor and integrin signaling in phagocytes (rosales, 2007). in these cells, syk 121 fig. 5 phagocytosis pathways in medfly hemocytes. in medfly hemocytes, phagocytosis of bacteria involves the pathway integrin/src/fak/map kinase, which ends in activation of an elk-1-like protein. upon integrin engagement, fak is activated and functions as a docking molecule for src family kinases and other signaling molecules such as map kinases. map kinases then phosphorylate elk-1-like. active, phosphorylated elk-1-like is localized exclusively in the cell nucleus in association with fak. map kinases can also be activated by other ligands such as lps and latex beads, but the signaling pathways from these stimuli to map kinases are still not defined. using pharmacological inhibitors for all three types of map kinases (the extracellular signal-regulated kinases (erks), the c-jun n-terminal kinases (jnks), and the p38 mitogen activated protein) in medfly hemocytes greatly reduced bacterial ingestion, confirming the role for all map kinases in phagocytosis. activates cytoskeleton rearrangements, gene expression, and phagocytosis. in insects, the homolog of syk, shark is important for draperdependent glial phagocytosis (ziegenfuss et al., 2008) (fig. 3). src src is the prototype of the src family of tyrosine kinases (ingley, 2008). these kinases are involved in many cellular functions including cell proliferation, differentiation, and phagocytosis. similarly, src has been reported to participate in various drosophila functions including maintenance of epithelial integrity (langton et al., 2007), fusome development and karyosome formation (djagaeva et al., 2005), and phagocytosis (ziegenfuss et al., 2008). src also participates in phagocytosis by medfly hemocytes, in particular associated with fak (metheniti et al., 2001; soldatos et al., 2003; lamprou et al., 2007). map kinases map kinases are a group of evolutionary conserved signaling kinases involved in many important cellular functions, such as cell proliferation, differentiation, development, apoptosis, inflammation, and phagocytosis. three map kinase families are known: the extracellular signal-regulated kinases (erks), the c-jun n-terminal kinases (jnks), and the p38 mitogen activated protein (p38). in drosophila, rolled, basket, and dp38 are the homologs of mammalian erk, jnk, and p38, respectively (han et al., 1998; lim et al., 1999). they all have been implicated in phagocytosis by insect hemocytes. inhibition of erk in m. sexta hemocytes reduced phagocytosis of e. coli bacteria (de winter et al. 2007). also the use of pharmacological inhibitors for all three types of map kinases in medfly hemocytes greatly reduced 122 fig. 6 propo and ddc pathways. after bacterial challenge, membrane-bound prophenoloxidase (propo) is converted to active phenoloxidase (po) through limited proteolysis by serine proteases. active po then catalyzes tyrosine into dopa. another enzyme, dopa decarboxyilase (ddc) is also expressed on the surface of these cells and it transforms dopa into dopamine. po can also use dopa and dopamine as substrates to form quinones that are reactive intermediates for melanization, nodulation, and phagocytosis. map kinases activated by lps or bacteria seem to be relevant for secretion of the serine proteases needed for initial propo activation. phagocytosis (soldatos et al., 2003; lamprou et al., 2005, 2007). similarly, inhibition of a jnk-like protein in the mosquito a. albopictus cell line c6/36 resulted in reduced phagocytosis (mizutani et al., 2003). pi-3k phosphoinositide 3-oh kinase (pi-3k) is a phospholipid kinase that provides cells with a survival signal that allows them to withstand apoptotic stimuli. pi-3k is also involved in many other important cellular functions including cell migration, lymphocyte activation and phagocytosis (downward, 2004). in insects, pi-3k has been reported to participate in phagocytosis of bacteria in m. sexta (de winter et al., 2007) and endocytosis of lps by medfly hemocytes (soldatos et al., 2003). class i pi-3k (rusten et al., 2004; berry et al., 2007) and class iii pi-3k (juhász et al., 2008) also seem to be involved in autophagy in drosophila. propo the prophenoloxidase (propo) system is an important component of the innate immune system in insects. it is activated by bacteria infections and participates is several humoral and cellular responses including melanization, wound healing, encapsulation, nodulation, and phagocytosis (cerenius et al., 2008). after bacterial challenge, cell-free and membrane-bound propo is converted to active po (fig. 6) through limited proteolysis by serine proteases (cerenius et al., 2004; mavrouli et al., 2005). active po then catalyzes the formation of quinones that are reactive intermediates for melanization and nodulation (ling et al., 2005; jiang, 2008). ddc activation of propo seems to be insufficient to induce ingestion of bacteria by medfly hemocytes (lamprou et al., 2007). however, another enzyme, dopa decarboxyilase (ddc) is also expressed on the surface of these cells and it is reported to be important for nodulation, melanization, and phagocytosis, since treatment with small interfering rna (sirna) for ddc markedly blocked e. coli phagocytosis (sideri et al., 2008) (fig. 6). also, a microarray analysis in drosophila showed that ddc expression levels increased after a bacterial infection (de gregorio et al., 2002). ddc seems to be involved in wound healing, parasite defense, and behavior of insects (hodgetts et al., 2006). eicosanoids eicosanoids are active compounds biosynthesized by enzymatic oxygenation of arachidonic acid (aa) or other c20 polyunsaturated fatty acids. phospholipase a2 (pla2) is the enzyme 123 responsible for releasing aa from cellular phospholipids. free aa can be metabolized by cyclooygenase (cox) to produce prostanglandins (pg), by lipooxygenase (lox) to produce leukotrienes, lipoxins and other products, and by cytochrome p450-epoxygenase to produce epoxyeicosatrienoic acids (stanley et al., 2009). pla2 is expressed in fat-body cells and hemocytes and is increased after a bacterial infection (tunaz et al., 2003). many reports show that eicosanoids are involved in several immune responses including phagocytosis (figueiredo et al., 2008; castro et al., 2009; shrestha et al., 2009), nodulation (miller et al., 1999; zhao et al., 2009), hemocyte spreading (downer et al., 1997), elongation (miller, 2005), release of propo (shrestha et al., 2008), and protection against nematode invasions (hyrsl et al., 2011). concluding remarks phagocytosis is a well-established innate immune defense mechanism in higher organisms. it is fundamental for the host defense and also for stimulating the adaptive arm of the immune response. in insects, phagocytosis is also an innate immune response performed by hemocytes circulating in the hemolymph. among the different types of hemocytes, plasmatocytes and granulocytes are consistently reported as phagocytic in many insect species. the cellular and molecular mechanisms for hemocyte phagocytosis are relatively well described for drosophila, mosquitoes, and medfly. however, important variations in phagocytosis exist among different species. this underlines the fact that much new research is needed in more insect species to fully understand this cell process in insects. new techniques such as rnai and flow cytometry, together with systembased approaches such as genomics and proteomics will certainly generate exciting advances in our understanding of phagocytosis. the signaling pathways for phagocytosis are only partially known. several signaling molecules have been reported as relevant for phagocytosis, but we do not have information yet to place them in a particular signaling pathway. these gaps will certainly be filled as we continue studying how insects cope with the many pathogenic microorganisms they are exposed to. acknowledgements this work was supported by grant 48573 from consejo nacional de ciencia y tecnología, mexico, and by grant in205311-2 from dirección general de asuntos del personal académico de la universidad nacional autónoma de méxico (unam). references 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315211, pr china accepted june 5, 2014 abstract di-(2-ethylhexyl) phthalate (dehp) has many adverse effects on immunity and metabolic states. however, scarce information is available on its connection with toxicologically relevant proteomics response in marine invertebrates. in this study, gs-ms was employed to determine the bio-accumulated levels of dehp in clam venerupis philippinarum. after exposure to 0.4 mg l-1 and 4mg l-1 dehp, the bio-accumulated dehp in the clam foot was significantly increased in the first 24 h, and then sharply decreased from 0.203 ± 0.022 μg g-1 to 0.104 ± 0.011 μg g-1 , and from 1.689 ± 0.018 μg g-1 to 1.172 ± 0.012 μg g-1, respectively. comparative proteomic was conducted to investigate the global protein expression changes towards this contaminant exposure. twenty-eight proteins with significant differences in abundance were identified and characterized, among them six enzymes related to the glycolysis pathway were suppressed, and two members of tca cycle were induced. the activity and mrna expression level of malate dehydrogenase (mdh) were further assessed using qpcr and an enzymatic assay. the mdh activity and mrna transcript levels were both elevated compared to those in the ethanol control group. our findings indicated that a dehp-treated clam modulated host toxicological effect through depressing glycogen synthesis and activating tca cycle. key words: venerupis philippinarum; di-(2-ethylhexyl)phthalate; 2-dimensional electrophoresis ; glycolysis; tca cycle introduction the industrial revolution unleashes a vast variety of new chemicals into the environment, and it has been roughly estimated that approximately 1,000 2,000 new chemicals are produced each year (wei et al., 2010). phthalates are among these industrial chemicals and have been used for a variety of purposes, such as plasticizers that impart flexibility and durability to polyvinyl chloride products. among them, high molecular weight di-(2-ethylhexyl) phthalate (dehp) is gaining focus in the marine environment, as it is increasingly being used in construction materials and in numerous pvc products (lu et al., 2013). it was estimated that the concentrations of dehp in river water and sediments typically range from 8 to 25 mg l−1 and 1000 to 2000 mg kg−1, respectively (park and kwak, 2008). zhuang et al (2011) reported that the content of phthalate esters (paes) ranged from 18.77 to ___________________________________________________________________________ corresponding author: chenghua li 818 fenghua road ningbo university ningbo, zhejiang province 315211, pr china email: lichenghua@nbu.edu.cn 191.51 ng l−1 and from 171.50 to 1435.61 μg kg−1 in seawater and sediments, respectively, in the quanzhou bay of china, where dehp is a major component. cumulative evidence has revealed that dehp, as an estrogenic chemical, poses multiple health risks due to its ability to bind and activate estrogen receptors (er) in vitro (oh and lim, 2009; uren-webster et al., 2010). in rodents, dehp has been shown to decrease fetal testis testosterone production and reduce the expression of steroidogenic genes (borch et al., 2006). dehp has also been widely reported to produce reproductive and developmental toxicities, in mammals and fish, depending on the dose and exposure time (park and choi, 2007; svechnikova et al., 2007; paola et al., 2012). in aquatic animals, dehp can be absorbed, metabolized and largely accumulated in the tissue of a penaied shrimp (hobson et al., 1984). it can also affect or disrupt endocrine hormones (oh and lim, 2009) and interfere with the delicate balance of the immune system (chalubinski and kowalski, 2006). in zebrafish, dehp reduces fertilization success in oocytes spawned by increasing the levels of two 174 mailto:lichenghua@nbu.edu.cn table 1 the concentration of clam foot dehp determined by gc-ms peroxisome proliferator activated receptor (ppar) responsive genes (uren-webster et al., 2010). in our previous study, dehp is found to mediate immune responses in clams by triggering the production of reactive oxygen species and nitric oxide (lu et al., 2013). during the experiment, a strong decrease in movement is observed in dehp-treated clam compared to that of the control group. however its toxicological effects are still largely unknown in clams, although dehp has been shown to influence aquatic animals' energy metabolism. recently, proteomic analysis has emerged as an approach for the analysis of differential gene expression at the protein level and seems to be an effective way for analyzing the delicate effects of environmental toxicants based on comparisons of the patterns of proteomes after exposure to compounds of toxicological relevance (bandara and kennedy, 2002; kennedy, 2002; wetmore and merrick, 2004; dowling et al., 2006; benninghoff, 2007). the purposes of the present study were: (1) to determine the accumulated levels of dehp in clam foot; (2) to identify differentially expressed proteins in a dehp-treated clam foot; and (3) to clarify the expression and activity of malate dehydrogenase at mrna and protein levels. materials and methods sample preparation clams (venerupis philippinarum; 8.65 ± 1.24 g in weight) were purchased from ningbo, zhejiang province, china. the clams were acclimated in glass tank for a week before commencing the experiment. the temperature was maintained at 20 22 °c throughout the experiment, and the salinity of the supplied seawater was maintained at 30 ppt. the clams were randomly divided into four groups (50 individuals for each group). firstly, 200 mg dehp (sigma, shanghai, china, analytical purity >99.0 %) was dissolved in 25 ml absolute ethanol, then subsequently diluted with 25 ml seawater to obtain a stock solution of 4 g l-1. the dehp stress experiment was performed by exposing the clams to dehp with two final concentrations of 0.4 mg l−1 (4 ml stock solution + 18 ml absolute ethanol) and 4.0 mg l−1 (40 ml stock solution) in 40 l seawater. the control group was exposed to the same volume of absolute ethanol (20 ml). after 96 h of exposure, the clam foot from 20 randomly selected individuals were ground in liquid nitrogen for dehp concentration determination and protein extraction. assay of dehp richness in foot by gs-ms ten grams of foot samples from treated and control groups were collected, and then homogenized, respectively. the pellets were obtained by centrifuging at 5,000 rpm for 10 min. subsequently, dehp was extracted with 20 ml hexane for three times, then mixed together and dried in nitrogen conditions. the extracted dehp was dissolved into 1 ml hexane for gs-ms analysis. there were three biological replications for each sample. a working standard solution was prepared by ten times dilution of the stock solution (212 mg l-1) with hexane. we performed gc-ms analysis in trace dsq α ms (thermo) with tr-1 column (30.0 mm×0.25 mm×0.25 μm). the inlet was set at 250 °c and automatic injections of 1μl of extracts were performed in a splitless mode. the helium carrier gas flow was set at 1 ml min−1. the oven temperature began at 80 °c, then increased to 280 °c at 10 °c min−1, and kept finally at this temperature for 5 min. the ms detection was in selective ion monitoring operating mode (sim) at an electron impact energy of 70 ev. two or three mass fragments were selected for each compound. the most intense ion was used for quantification and the other ions were used for confirmation the presence of the compounds. the concentrations were expressed as (means±sd) μg dehp per gram of wet weight of clam foot. sample preparation for electrophoresis analysis the foot tissue (approximately 600 mg) was first dissolved in 1 ml lysis buffer (7 m urea, 2 m thiourea, 65 mm dtt, 4 % w/v chaps, 20 mm tris, ph 8.5, and 0.2 % v/v bio-lyte) and then homogenized at 20 kw for 2 min with a electric homogenizer (xinzhi, ningbo). the resulting mix was incubated on ice for 2 h, with vortex for 20 30 s every 15 min, and it was sonicated for 3 min with 5 s of ultrasound and 5 s resting intervals. after centrifugation at 12,000 g for 60 min at 4 °c, the supernatant was collected for desalt treatment. the protein samples were purified using a 2-d clean-up kit (ge healthcare, usa) according to the manufacturer’s instructions. protein was re-suspended in rehydration solution containing 7 m urea, 2 m thiourea, 4 % w/v chaps, 65 mm dtt, and 0.2 % v/v bio-lyte. the concentration of protein was determined using a protein quantification kit with bsa as a standard (sangon) and was further confirmed by 12 % sds-page. two-dimensional gel electrophoresis (2-de) the first-dimension of isoelectric focusing (ief) was performed with a powerpac basic/mini-protean® tetra cell system (bio-rad, usa) according to the readystriptm ipg strip instruction. briefly, proteins (300 μg) were diluted in 150 μl of rehydration buffer and loaded onto a ph 3 10 immobilize dry strip (ipg strip) (7 cm, linear, exposure doses exposure time foot dehp concentration 24 h 0.203±0.022 μg g-1 0.4 mg l-1 96 h 0.104±0.011 μg g-1 24 h 1.689±0.018 μg g-1 4 mg l-1 96 h 1.172±0.012 μg g-1 175 bio-rad). next, the strips were covered with mineral oil and rehydrated for 13 h at 20 °c. the voltage settings for ief were 250 v for 1 h, 500 v for 1 h, 1,000 v for 1 h, 4,000 v for 3 h, 4,000 v for 5 h and 6,500 v, to a total of 80 kvh. following the ief, strips were equilibrated for 15 min at room temperature in 2.5 ml of sds equilibration buffer (375 mm tris-hcl, ph 8.8, 6 m urea, 20 % glycerol, and 2 % sds) containing 20 mg/ml dithiothreitol for the first equilibration and 25 mg/ml iodoacetamide for the second equilibration. the gel strips were then washed with electrophoresis buffer (25 mm tris, 192 mm glycine, 0.1 % sds) and placed on a 12 % polyacrylamide gel containing 0.1 % sds. the gels were sealed with low melting point agarose, and the second dimension of gel electrophoresis was conducted at 70 v for 30 min and then 130 v for approximately 90 min until the dye front reached the end of the gel. the protein spots on 2-de gels were visualized by coomassie brilliant blue staining. the images of the gels were scanned using an image scanner gs-800 (bio-rad, usa). gel evaluation and data analysis were performed using pdquest 8.0 (bio-rad, usa). three replicates were conducted per sample to ensure gel reproducibility. mass spectrometry and protein identification spots with significant and reproducible changes (average protein spot intensities that changed 1.5-fold between control and treated gel sets) were manually excised from stained gels and subjected to in-gel trypsin digestion. after digestion, the supernatants were transferred into another tube, and the gels were extracted once with 50 µl extraction buffer (67 % acn and 5 % tfa). the peptide extracts and the supernatant of the gel spot were combined and then completely dried. samples were re-suspended with 5 µl 0.1 % tfa followed by mixing in 1:1 ratio with a matrix consisting of a saturated solution of α-cyano-4-hydroxy-trans-cinnamic acid in 50 % acn, 0.1 % tfa. 1 ul mixture was spotted on a stainless steel sample target plate. peptides were analyzed by matrix-assisted laser desorption/ionization tandem time-of flight (maldi-tof/tof) mass spectrometry using the abi5800 proteomics analyzer. proteins were unambiguously identified by searching against the non-redundant sequence database (ncbinr) and est database of clams and molluscs via the mascot program (http://www.matrixscience.com). fig. 1 protein expression patterns of venerupis philippinarum foot under different concentrations of dehp exposure: ethanol group (a); 0.4 mg l-1 dehp (b); 4 mg l-1 dehp (c). proteins were visualized by cbb r250 staining. arrows and spot numbers refer to differentially expressed proteins. enzymatic assays of malate dehydrogenase malate dehydrogenase (mdh) activity was determined by oxaloacetate reduction via the decrease in absorbance for nadh at 340 nm according to the mdh assay kit (jian cheng, nanjing). the assays were performed in 50 mm potassium phosphate buffer, ph 7.5, 1 mm oxaloacetate, 0.15 mm nadh, and the appropriate volume of the fraction (šukalović et al., 2011). activity was expressed as the mean values ± s.d u/mg (n = 5). the data were subjected to a t-test analysis , and p < 0.05 was considered to be a significant difference. confirmation of differential expressed protein by qpcr the publically registered clam mdh was selected for expression analysis. rna extraction and complementary dna (cdna) synthesis were described previously (lu et al., 2013). two clam 18s 176 http://www.matrixscience.com/ table 2 list of spots/proteins identified by ms analysis from venerupis philippinarum foot by 2-de following dehp treatment. spot no. protein name accession no.a matching species threshold （p<0.05)b score mr pi regulationc 1 transgelin am872804 crassostrea gigas 62 120 24944 8.32 ↓ 2 ltap xp_002121953 ciona intestinalis 49 50 63623 8.45 ↓ 3 triosephosphate isomerase ado27908 ictalurus furcatus 49 203 26824 6.90 ↓ 4 glyceraldehyde-3 phosphate dehydrogenase np_001080567 xenopus laevis 46 185 36017 7.60 ↓ 5 arginine kinase acp43447 pholas orientalis 47 735 81948 6.66 ↓ 6 arginine kinase acp43447 pholas orientalis 47 417 81948 6.66 ↑ 7 phosphoglycerate kinase bad17914 amia calva 48 151 42091 5.76 ↓ 8 enolase o02654 doryteuthis pealeii 49 138 47738 5.78 ↓ 9 glyceraldehyde-3 phosphate dehydrogenase np_001080567 xenopus laevis 47 190 36017 7.60 ↑ 10 voltage-dependent anion channel protein adi56517 haliotis diversicolor 48 207 30670 6.84 ↓ 11 fructose bisphosphate aldolase p53448 carassius auratus 49 117 39849 6.41 ↑ 12 malate dehydrogenase am874213 ruditapes philippinarum 59 97 24356 7.69 ↑ 13 coiled-coil domain protein xp_002154202 hydra magnipapillata 48 59 61209 8.95 ↓ 14 actin 1101351b pecten sp 49 340 41827 5.30 ↓ 15 phosphoglycerate mutase aax29976 clonorchis sinensis 49 99 29250 7.66 ↑ 16 malate dehydrogenase ejw88983 wuchereria bancrofti 49 258 36191 8.92 ↑ 17 h+-transporting atp synthase alpha subunit isoform 1 aam93478 petromyzon marinus 49 242 39603 9.53 ↑ 18 unknown protein ↑ 19 phosphoglycerate kinase bad17914 amia calva 48 113 42091 5.76 ↓ 177 fructose a. database accession numbers according to ncbinr b. results of mascot searching with scores higher than the thresholds indicated p<0.05 c. "↑" denotes that up-regulated spots, "↓" denotes that down-regulated spots rdna primers, cgtctttcaaatgtctgccctatc and gccgtatctcatgctccctctcc, were used to amplify a 115-bp fragment as an internal control, which verified successful reverse transcription and calibrated the cdna template. two gene specific primers for mdh (atctatggctgttatttccgac and tctctctcttcttcaagctcct) were designed to amplify an 196 bp product. real-time pcr amplification was performed using a rotor-gene 6000 real-time pcr detection system. we utilized the reaction components and thermal profiles suggested by the manufacturer. the 2−δδct method was used to analyze the expression level of the candidate genes, and the value obtained denoted the n-fold difference relative to the calibrator (ethanol group). the data were presented as relative mrna expression levels (means ± sd, n = 5), and subjected to a t-test analysis. results and discussion dehp accumulation in clam foot di-(2-ethylhexyl) phthalate (dehp) is a plasticizer and a ubiquitous environmental contaminant that may have adverse effects on organisms. the accumulated dehp concentrations in clam’s foot were presented in table 1. the higher dehp concentration was found in the first 24 h with 0.203 ± 0.022 μg g-1 and 1.689 ± 0.018μg g-1 at 0.4 mg l-1 and 4 mg l -1 exposure doses, respectively. no dehp was detected in the control group, indicating that clam foot had the powerful capacity for dehp enrichment at the first stage. as time progressed, the concentrations of dehp in the two exposure doses were sharply decreased, and arrived to 0.104 ± 0.011 μg g-1 and 1.172 ± 0.012 μg g-1 at the 96 h, respectively. this decrease of dehp informed that clam might have the special abilities to degrade or squeeze dehp from host by certain molecular mechanisms. 2-de image patterns in order to identify the proteins involved into eliminating dehp in foot tissue, the proteomic expression map under different concentrations of dehp exposure was investigated (fig. 1). within the 9 analytical gels, approximately 600 spots in each group were detected by pdquest 8.0. the analysis of protein data sets enabled the selection of 28 protein spots that are commonly altered in their expression between the experimental and ethanol control groups (fig. 1, arrow indicated), among which 20 protein spots displayed increased abundance and 8 showed decreased expression profiles (table 2). characterization of differentially expressed proteins a series of processes for protein spot picking, in-gel digestion and application to the target plate for abi5800 were automatically performed to characterize these differentially expressed proteins. as a result, 28 proteins with average protein spot 20 bisphosphate aldolase c p53448 carassius auratus 47 113 39849 6.41 ↓ 21 phosphoglycerate kinase caa45574 macropus eugenii 49 132 45274 7.98 ↓ 22 unknown protein ↓ 23 phosphoglycerate mutase ekc26210 crassostrea gigas 49 79 28792 7.66 ↓ 24 atp-dependent rna helicase xp_792082 strongylocentrotus purpuratus 49 51 90593 8.91 ↓ 25 unnamed protein product cby21625 oikopleura dioica 49 53 51639 5.80 ↓ 26 tropomyosin baf46896 balanus rostratus 49 453 32706 4.61 ↓ 27 unknown protein ↓ 28 myosin 1803425c mercenaria mercenariav 47 210 18105 4.47 ↓ 178 a) b) fig. 2 enzymatic activity (a) and mrna expression level (b) of mdh before and after exposure to 0.4 and 4 mg l-1 dehp. intensities were altered in the dehp-treated group and were successfully identified . among these proteins, 25 were successfully annotated and 3 did not yield unambiguous protein identification. among the identified proteins, 11 were involved in glycolysis (spot 3, 4, 7, 8, 9, 11, 15, 19, 20, 21, 23), and 10 members had at least a 1.5-fold decrease in intensity. an exception was observed for spot 9, glyceraldehyde-3-phosphate dehydrogenase (gapd), which had a 3.8-fold increase compared to its expression in ethanol control group (table 2). however, another gapd (spot 4) showed opposite expression patterns with 6.4-fold decrease towards dehp challenge. the distinct expression profiles of the two gapds indicated that they might be participated in the different metabolic pathways for dehp detoxication. mammals were known to possess two tissue-specific gapd isoenzymes of gapd-1 and gapd-2, which served as classical metabolic proteins involved in glycolytic energy production and maintenance of sperm motility (kuravsky et al., 2011). the decrease expression patterns of another gapd (spot 4) indicated a possible redirection of carbon flux from glycolysis to the pentose phosphate pathway for nadph regeneration. this mechanism had been proposed to be beneficial during oxidative stress due to the use of nadph as reducing equivalents by the thioredoxin and glutaredoxin antioxidant systems (costa et al., 2002; shenton and grant, 2003; shanmuganathan et al., 2004). meantime, an increasing number of diverse non-glycolytic activities of gapdh have been reported, including macromolecular transport, microtubule bundling, nuclear trna transport and apoptosis (ishitani and chuang, 1996; nahlik et al., 2003). ellington also reported that gapd could be up-regulated in apoptotic cells to 3-fold higher than that in non-apoptotic cells (ellington, 2001). the up-regulation of spot 9 indicated that cell apoptosis were induced on dehp exposure through unknown gapd-based regulatory network. it is known that the atp generated in the glycolytic pathway is the major energy source for several metabolic reactions. in dehp-fed rats, there is a significant suppression of glucose metabolism 179 observed in liver and muscle tissues (martinelli et al., 2006). in our study, 6 of the 11 glycolysis enzymes were detected with significantly down-regulated expression in clams under dehp stress, in which phosphoglycerate kinases and phospheglycerate mutases were identified two or three times (table 2). it was speculated that impaired glycolysis in the clam was linked to an abnormal glucose intermediate flux in the foot (mushtaq et al., 1980). the content of glucose-6-phosphate (g-6-p), fructose-6-phosphate, pyruvate, lactate, glucose-1-phosphate and glycogen in the foot will be addressed in our future work. dehp exposure also impacted expression levels of other energy metabolism-related proteins in our study, such as arginine kinase and atp synthase. arginine kinase, the equivalent of mammalian creatine kinase, can phosphorylate or dephosphorylate a phosphagen molecule, thus allowing the formation or consumption of atp. hence, arginine kinase regulates atp and proton buffering and, consequently, metabolism and energetic transport (ellington, 2011). the distinct expression profiles of two arginine kinases perhaps indicated that there was functional diversity among these enzymes (uda et al., 2012). the h+-transporting atp synthase alpha subunit isoform (spot 17) had significantly increased levels, which would directly increase the atp level. these up-regulations could compensate the significant down-regulation of the atp synthesis in glycolysis in response to the dehp exposure. mdh expression at protein and mrna levels towards dehp exposure the tca cycle, a sub-pathway of glycolysis, is a series of chemical reactions used by all aerobic living organisms to generate energy, and it provides precursors for the biosynthesis of compounds (chen and russo, 2012). two isoforms of mdhs, reversibly catalyzing the oxidation of malate to oxaloacetate in the tca cycle (spots 12, 16), were also identified with 1.97or 1.54-fold increases in expression compared to those of the ethanol group, respectively (table 1). to further elucidate its expression change in response to dehp exposure, mdh activity and mrna expression level were characterized and the results were shown in figure 2. dehp treatment increased malate dehydrogenase activities from 96.05 to 125.62 u/mg at 96 h (fig. 2a). the qpcr results revealed that the mrna expression level of mdh was elevated, with a 1.67-fold increase compared to the ethanol control group (fig. 2b). these results were consistent with the protein expression patterns revealed by 2-de analysis (fig. 1). a significant difference was observed not only in the enzymatic assay but also in mrna expression, according to the t-test analysis of the control and challenged groups. overall, the reference map reported here provided a useful tool for identifying protein pattern changes in the clam and clearly demonstrated the cellular response to dehp exposure. dehp triggered adjustments of the glycolysis pathway and the tca cycle, which are crucial for energy production and the maintenance of sufficient atp levels. acknowledgments this work was financially supported by the project of international cooperation in the zhejiang province (2012c24022), zhejiang key laboratory of aquatic germplasm resources of zhejiang wanli university (kl2013-4), chinese key technology r&d program (2011bad13b0903), and k.c.wong magna fund at 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http://www.ncbi.nlm.nih.gov/pubmed?term=fischer%20b%5bauthor%5d&cauthor=true&cauthor_uid=22147016 http://www.ncbi.nlm.nih.gov/pubmed?term=fischer%20b%5bauthor%5d&cauthor=true&cauthor_uid=22147016 http://www.ncbi.nlm.nih.gov/pubmed?term=schmidt%20js%5bauthor%5d&cauthor=true&cauthor_uid=22147016 http://joe.endocrinology-journals.org/search?author1=o+s?der&sortspec=date&submit=submit isj 11: yyy-xxx, 2014 isj 11: 132-142, 2014 issn 1824-307x research report side effects of immune response of colorado potato beetle, leptinotarsa decemlineata against the entomopathogenic nematode, steinernema carpocapsae infection l ebrahimi1, g niknam1, gb dunphy2, m toorchi3 1nematology lab., department of plant protection, faculty of agriculture, university of tabriz, tabriz, iran 2department of natural resource sciences, mcgill university, macdonald campus, quebec, canada 3department of plant breeding and biotechnology, faculty of agriculture, university of tabriz, tabriz, iran accepted april 24 , 2014 abstract entomopathogenic nematodes (epns) are lethal pathogens of agricultural insect pests. little is known about their sublethal effects on the insect hosts. the lethal effects of steinernema carpocapsae on fourth instar larvae of colorado potato beetle (cpb), leptinotarsa decemlineata were detected using soil infection and direct injection of the nematode into the hemocel. lc20 and lc80 values of 7.8 (3.0 13.4) infective juveniles (ijs) and 126.7 (91-206.7) ijs were obtained for the soil application method and 10.2 (8.7 11.4) ijs and 22.7 (19.73 28.0) ijs for direct injection, respecitvely. sublethal effects of s. carpocapsae on last instar larvae and subsequent surviving adults and phenoloxidase (po) activity in hemolymph of nematode-injected last instar larvae were investigated. sublethal effects included adult cuticular discoloration, deformation of the wings, legs and antenna and decreased fertilized egg production in females. considering cuticular discoloration in most treated insects, it is hypothesized that production of po in the insect larvae infected with an entomopathogenic nematode, s. carpocapsae might have costs for surviving adult insects. po specific activity in cpb against s. carpocapsae generally increased up to 48 h post injection. here in, the sublethal effects are discussed as a potential tread-offs of po production in nematode-injected insects. key words: steinernema carpocapsae; leptinotarsa decemlineata; po activity; cuticular discoloration   introduction colorado potato beetle (cpb), leptinotarsa decemlineata say (coleoptera: crysomelidae) is one of the most destructive pests of potato foliage and prone to developing chemical insecticide resistance (cutler et al,. 2005; hitchner, 2007). the environmental hazardous effects of chemical insecticides have made the use of biocontrol agents necessary. entomopathogenic nematodes (epns) are biological control agents of agricultural insect pests (burnell and stock, 2000). there are few studies on the sublethal effects of epns either on insect development or their immune system. heterorhabditis bacteriophora and its symbiotic bacterium photorhabdus luminescens, decreases larval feeding rate, wet weight gain, and frass production of schizura concinna larvae (milstead, ___________________________________________________________________________ corresponding author: niknam gholamreza nematology laboratory department of plant protection faculty of agriculture university of tabriz tabriz, iran e-mail: g_niknam@tabrizu.ac.ir 1980). late instar spodoptera littoralis larvae infected with steinernema riobrave exhibit decreased rates of leaf consumption and eat fewer meals in comparison to control treatments (alchanatis et al., 2000). galleria mellonella larvae infected with steinernema carpocapsae showed reduced silk production (simões et al., 2000). sublethal concentrations of heterorhabditis downesi and s. carpocapsae caused no effect on feeding rate of adults of the large pine weevil, hylobius abietis (girling et al., 2010). wing and elytra deformation of adult cpbs were reported when infected with s. feltiae and h. bacteriophora (ebrahimi et al., 2011b). awareness on entomopathogenic nematode�s sublethal effects helps to increase their efficacy as biocontrol agents. in that, how sublethal effects of biological control agents like those of chemical insecticides may affect ecological fitness of the insects due to morphological, physiological or behavioral changes (bao et al., 2009; girling et al., 2010; vidau et al., 2011). the cuticular phenoloxidase (po) is a melanizing enzyme at the wound site which limits infection; po in hemolymph is responsible for melanotic immune responses (pham and schneider, 132    https://www.researchgate.net/institution/university_of_tabriz/department/department_of_plant_breeding_and_biotechnology fig. 1 mortality (probit-transformed) of l. decemlineata prepupae injected with different doses of s. carpocapsae (solid triangles) and infected with different doses of s. carpocapsae in soil (solid squares). 2008); additionally, one po type is common to both cuticle and hemolymph (hiruma and riddiford, 1988). the po immune responses occur immediately against invading microbes in insects (castillo et al., 2011). po involves the formation of short-lived toxic substances (e.g., chemically reactive quinones) and long-lived products such as melanin (a brown-black pigment) both are deposited at wound sites and participating in antimicrobial responses including encapsulation and killing of microbial pathogens (castillo et al., 2011). the melanization cascade overlaps the humoral and cellular insect immune defenses (castillo et al., 2011). po is responsible also in sclerotization and tanning of the cuticle (arakane et al., 2005). insect cuticle is immunologically active in expressing po as well as antimicrobial peptides supplementing its physiochemical passive antimicrobial properties (brey et al., 1993; golkar et al., 1993). moreover, a correlation between increasing cuticular darkening and resistance to entomopathogens has been demonstrated in some insect species (barnes and siva-jothy 2000; wilson et al., 2001; armitage and siva-jothy 2005; dubovskiy et al., 2013). cellular encapsulation and capsule melanization of the epns in cpb is documented (thurston et al., 1994; armer et al., 2004; ebrahimi et al., 2011a, b) therefore, in the current study, lethal and sublethal effects of s. carpocapsae on cpb and the nematode interaction with po activity of the hemolymph of the insect were investigated. table 1 lc20, 50 and 90 values for s. carpocapsae against l. decemlineata prepupae a confidence limits method of infection slope±se chi-square lc20 (95% cl) lc50 (95% cl a) lc90 (95% cl) r 2 n soil infection 1.39±0.21 1.78 7.8 (3.0-13.4) 31.5 (20.4-43.0) 262.3 (168.6-555.2) 0.96 270 direct injection 4.84±0.62 4.7 10.2 (8.7-11.4) 15.2 (13.7-17.1) 28.0 (23.5-36.6) 0.94 225 133    fig. 2 adult l. decemlineata infected with s. carpocapsae during the prepupal phase showing a continuum in the pronotum discoloration (white boxes): (a) control (injected with ringer’s solution), (b h) injected with s. carpocapsae. materials and methods nematodes steinernema carpocapsae was obtained from the collection of nematology laboratory, university of tabriz, tabriz, iran. nematodes were cultured using the last instar greater wax moth larvae, galleria mellonella (woodring and kaya, 1988). infective juveniles (ijs) were stored in distilled water at 5 °c and used in all experiments within 30 days of emerging from the host. before starting the experiments, nematodes were kept at 25 °c for 2030 min. insects leptinotarsa decemlineata over-wintered adult colorado potato beetles were collected from potato fields in sarab (east azarbaijan province) in june, 2012 and were stored in plastic boxes (19.3 cm length, 14.3 width, 3.6 cm height; 20 larvae per box) with ventilated lids (26 ± 2°c, 50 ± 5 % rh and 16:8 (l:d) photoperiod) and received fresh potato leaves daily. offspring of the beetles were used for establishing the greenhouse colony (ebrahimi et al., 2011b). fourth instar larvae develop into prepupae after three days and prepupae were used in all experiments. fig. 3 adult l. decemlineata infected with s. carpocapsae during the prepupal phase showing a continuum in the ventral abdominal discoloration (white boxes): a) control (injected with ringer’s solution), b g) injected with s. carpocapsae. 134    table 2 percentages of adults of l. decemlineata showing the sublethal effects of s. carpocapsae number of replicates total number of insects anatomical deformations (%) ±se coloring wings legs antenna direct injection treated insects 3 65 44.62±8.08a 24.62±8.08a 21.54±9.81a 9.38±5.53a control 3 65 0b 7.81±1.56b 0b 0b soil application treated insects 3 47 52.92±9.22 a 20.97±10.5a 19.31±11.3a 8.47±3.5a control 3 45 0b 0b 4.4±7.6b 0b no significant differences are present between the values marked with similar superscripts (either lower-case letters or capital letters) (p < 0.05) galleria mellonella g. mellonella was reared under the same environmental conditions and in similar boxes as l. decemlineata, but on an artificial diet (1200 g wheat flour, 120 g beeswax, 300 g dried yeast, 600 g honey and 500 ml glycerol 99 %). comparison of two procedures for nematode infection; ijs in soil and direct injection of the nematodes for study of lethal effects both soil infection of the cpb with nematodes and direct manual injection of nematodes into the insects were used to determine the lc20, 50 and 80 values of the two methods and to establish lc values for examining sublethal effects on surviving insect from both methods. the soil infection experiments were conducted in plastic cylindrical boxes (3 cm diameter and 5 cm height) filled with 30 g autoclaved sandy soil (85 % sand, 10 % silt and 5 % clay w/w). distilled water (1ml) was added to each of the boxes and s. carpocapsae ijs were added at selected concentrations in 1 ml of water to the surface of the soil. the nematodes were used at rates ranging between 15 and 200 ijs per host prepupa (i.e., 15, 30, 100, 150 and 200 ij per prepupa). one prepupa was placed on the soil and the boxes were covered with ventilated lids to reduce desiccation. control boxes received 2 ml distilled water without nematodes (water content of the soil was 10 % (w/w)). 15 boxes were used for each nematode concentration and the experiment was replicated three times. mortality was recorded when the adults began to eclose in the control groups. all dead insects were dissected to ensure the presence of nematodes. an insulin syringe needle gauge (1 ml, soha, iran) was used to inject different concentrations of nematodes (i.e., 7, 10, 15 and 25 ijs in 20 μl ringer’s solution per prepupa) into the hemocoel of 15 prepupae, through the dorsal cuticle, the segment behind the pronotum. the ijs were counted in 20 μl aliquots to ensure the numbers were accurately delivered. fig. 4 deformation in the adult beetles infected with s. carpocapsae: a-b) deformed antenna, c) deformed antenna and wings, d) deformed leg, e) control for antenna and wings and f) control for leg. 135    fig. 5 a, b) thinning of the cuticle in color defected l. decemlineata adults; white and black arrows were used to show trachea and fatty tissue under cuticle, respectively. c) control. control groups received 20 μl ringer’s solution. the experiment was replicated three times. after injection, the insects were transferred to soil contained in plastic boxes moistened with 2 ml distilled water as previously alluded. mortality was recorded when the adults began to eclose in the control. all dead insects were dissected to ensure the presence of nematodes. sublethal effects direct injection method was chosen in the experiment on sublethal effects because of insertion of certain numbers of the nematode into the insect hemocoel. in addition, preliminary experiments showed the same kind of sublethal effects for both soil application and direct injection methods (which was reported in the results). each of 25 prepupae were injected with 11 ijs (lc20) in 20 μl ringer’s solution, the control group received the same volume of ringer’s solution. the experiment was replicated three times. morphological deformations and changes in cuticle coloring of emerged adult insects were recorded and digital images were recorded using a leica mz 125 stereomicroscope provided with a camera 2 3 h after emerging from the soil. adult insects that emerged from control and treated boxes were transferred to new chambers and fed with fresh potato foliage for two weeks. one male and one female were kept in separate chambers and numbers of the eggs per female and numbers of the hatched eggs were recorded. po specific activity and total protein quantification infective juveniles (28 ijs as lc80 and 11 ijs as lc20 in 20 μl of ringer’s solution) were injected into the hemocoel of each of 50 prepupae. prior to bleeding, prepupae were disinfected for 2 min in 70 % ethanol, and then placed on ice for 2-5 minutes. control insects (n = 50) were injected with 20 μl of ringer’s solution. other control group consisted of 50 prepupae wounded with needle of injection syringe. five prepupae were bled once at designated time intervals (0 min 48 h pi) by puncturing dorsal posterior of the abdominal cuticle. the blood was collected directly into a chilled (4 °c) 1.5 ml microcentrifuge tube. samples were kept at -80 °c until use. for the po activity assay, hemolymph samples were centrifuged at 4 °c and 12000xg for 10 min to pellet cell debris. aliquots (5 μl) of hemolymph supernatant were added to reaction buffer (195 μl 10 mm l-dopa and 600 μl 10 mm tris-hcl) and dopachrome formation followed spectrophotometrically (490 nm) (biochrom wpa biowave s2100 diode array spectrophotometer) in samples incubated at 35 °c table 3 number of the produced and hatched eggs in nematode-injected surviving and control adults of l. decemlineata in the sublethal experiment number of insects number of eggs per female hatching(%)±se %eggs in cluster nematodeinjected insects 12 48.3±22.3 a 24.8±21.6b 21.8±5.5b control 12 63.6±30.8a 81.3±6.0a 89.6±0.5a no significant differences are present between the values marked with similar superscripts (p < 0.05) 136    for 5 min in a water bath. there were three replicates for each sample. the experiment was repeated twice. to calculate po specific activity, the concentration of the total protein was determined using the bradford protein assay (bradford, 1976) with bovine serum albumin as the standard. hemolymph (5 μl) from each sample mixed with 795 μl distilled water was incubated with 200 μl of bradford reagent and the absorbance measured spectrophotometerically (595 nm, 10 min after mixing). po activity unit was defined as the change in optical absorbance unit at 490 nm per min. the specific activity was calculated as the units of enzyme activity per mg total protein. statistics lc20, lc50 and lc80 values for both soil infection and direct injection of the nematodes for lethal effects were obtained by probit analysis using sas software (sas institute, 2004). data were analyzed by analysis of variance and, when appropriate, means were evaluated by duncan’s multiple-range test (sas institute, 2004). lethal and sublethal experimental data were transformed into square root of (x+1) where needed, before analysis. results comparison of soil infection and direct injection of the nematodes methods for lethal effects lc20, lc50 and lc80 values for the two assays revealed a significant relationship between log dose and insect mortality of injected and soil infected insects (p < 0.05, fig 1, table 1), lc50 and lc80 values for the injection method being significantly lower than for the soil infection assay (about 2.1 fold and 5.7 fold, respectively), whereas lc20 values overlapped for the two infection methods. sublethal effects the most frequent significant (p < 0.05) sublethal effect of s. carpocapsae injection on surviving injected adult beetles of l. decemlineata was cuticular discoloration; the black spots on the pronotum and abdominal sternites were not formed completely and the pronotum and abdomen exhibited a yellow appearance (figs 2, 3; table 2). also, deformation in wings, legs and antennae of surviving adults was observed (table 2, fig. 4). thinning of the cuticle or increased transparency occurred in color defective adults resulting in visible trachea and other internal tissues (fig. 5). all mentioned sublethal effects were observed also in surviving insects from all five concentrations of the nematodes used in soil experiments (table 2). numbers of deposited eggs were not found significantly different between control and nematode-injected insects, while the egg hatching percentages were significantly lower in nematodeinjected insects (p < 0.05, table 3). leptinotarsa decemlineata deposits most of the eggs in clusters, but nematode-injected surviving beetles deposited most of their eggs individually rather than in the clusters (table 3). po activity and total protein quantification significant (p < 0.01) differences occurred in po units, total protein concentration and po specific activity in the hemolymph among nematodeinjected, ringer’s solution-injected and wounded insects over time. in the overall, po unit mean was the highest for lc80 injected insects and the lowest for wounded insects (f = 75.42, df = 3, p < 0.01, table 4). po increased significantly to highest values from 24 48 h pi in nematode (lc20 and lc80) injected insects. po activity was significantly higher for lc80 nematode-injected insects at 24 and 48 h pi than all other treatments (fig. 6). total protein values were lower in the hemolymph of the nematode-injected insects and ringer’s solution-injected than wounded insects (f = 45.57, df = 3, p < 0.01; table 4). in overall, total protein values were the highest at 48h pi for all groups which showed slight decreasing compared with 0 min pi in nematode (lc20) injected and ringer’s solution-injected insects (fig. 7). fig. 6 phenoloxidase units of nematode-injected, wounded and ringer’s solution injected insects in different time intervals. the results are expressed as mean ± sd. the data were statistically analyzed using anova and duncan’s multiple range tests with a specified significance level of p < 0.05. 137    table 4 mean comparison of phenoloxidase units, total protein concentration (mg/ml) and phenoloxidase specific activity among nematode-injected, wounded and ringer’s solution injected insects no significant differences are present between the values marked with similar superscripts (p < 0.05); n = 100 insects for each group in the overall, po specific activity was the highest in the hemolymph of lc80 nematodeinjected insects and was the lowest in wounded insects (f = 232.61, df = 3, p < 0.01; table 4). po specific activity in the hemolymph of the nematode-injected insects was significantly higher than wounded control and ringer’s solution injected insects from 30 min 16h pi (fig. 8). po specific activity was the highest at 24 h pi and 48 h pi for lc20 and lc80 injected insects, respectively and at 24 h pi ringer’s solution injected insects (fig. 8). in nematode-injected insects released bacteria were observed 10 h pi and bacteria concentration was increased with increasing the time. tissue damage was observed after 16 h pi. fat body lysis was the first obvious sign of tissue damage due to bacteria releasing and nematode activity. discussion comparison of insect infection with nematodes by the soil infection and direct nematode injection methods on lethal effect showed that slope of direct injection method was about three fold more than soil infection method. injecting the nematodes into the hemocel allows straightforward pathological expression of nematodes in the site of action, whereas in soil application, nematodes spend time navigating different barriers such as distances in the soil environment, penetrating of the host while dealing with insect behaviors and/or structural defenses to reach the site of action in hemocel (koppenhöfer et al., 2000; toubarro et al., 2009). nevertheless, in the soil method some of the applied nematodes penetrate into the hosts, therefore injection method was chosen for sublethal and po activity experiments. in the overall, surviving cpb adults sublethal effects including adult discoloration and thinning of the cuticle in color defective adults, anatomical deformations in wings, antenna and legs and decreased fertilized eggs when infected with low concentration of the nematodes in prepupa stage. none of these sublethal effects have been mentioned and documented in nematode-infected insects. the sublethal defect on cpb adult coloring is common here in, however there is no similar finding in the published studies related to nematode-infected insects. cuticular sclerotization is the process in which the cuticle is stabilized by incorporating phenolic compounds resulting in the formation of brownish colors of varying intensities, but nearly colorless and completely black cuticles can also be formed (andersen, 2010). black cuticles contain melanin which in some species is present in granules and in others the pigment is diffusely distributed (andersen, 2010). it should be noted that a phenotypic correlation has been demonstrated between increasing cuticular colour and insect resistance to entomopathogenic viruses in spodoptera exempta (reeson et al., 1998) and entomopathogenic fungi the coleopteran tenebrio molitor (barnes and sivajothy 2000; armitage and siva-jothy 2005), and the two lepidopteran insects spodoptera exigua (wilson et al., 2001) and g. mellonella (dubovskiy et al., 2013). therefore, defect in adult coloring and formation of lighter cuticle compared with control insects may impose the physiological cost of increased susceptibility against entomopathogens. in the sublethal experiments infective juveniles of nematodes were injected into the hemocoel in prepupa stage and two or three days later metamorphosis of the insects began. perhaps, secreting proteases by the nematodes or their symbiotic bacteria (hwang et al., 2003; cho and kim 2004; hinchliffe et al., 2010) induces apoptosis or less pronounced tissue damage on metamorphosing tissues such as the wings, antenna and legs of the beetles leading to such deformations. on the other hand, photorhabdus luminescens tt01 genome sequence revealed two loci with high similarity to the juvenile hormone esterase of cpb; the esterase regulates metamorphosis by inactivating juvenile hormones which maintain the larval stage (hinchliffe et al., 2010). it is possible that analogs to cpb juvenile hormone esterase produced by the symbiotic bacteria disrupt adult organogenesis during metamorphosis. additional experiments are needed to interpret the deformations. treatment total protein po unit po specific activity lc20 injected 5.15±0.33 b 0.106±0.01b 4.35±0.28b lc80 injected 5.13±0.27 b 0.122±0.01a 4.74±0.44a wounded control 6.28±0.17a 0.062±0.00c 2.01±0.07d ringer’s solution injected 5.15±0.27b 0.102±0.01b 3.92±0.24c 138    fig. 7 total protein concentrations (mg/ml) of lc20 and lc80 nematode-injected, wounded and ringer’s solution injected insects in different time intervals. the results are expressed as mean ± sd. the data were statistically analyzed using anova and duncan’s multiple range tests with a specified significance level of p < 0.05. decreasing fertilized eggs in surviving adults was the other sublethal effect of s. carpocapsae on cpb. deposition the eggs individually rather than in cluster could be a behavioral disorder that occurred in nematode-injected surviving adults; however immune activity and reproduction areoften negatively correlated in insects (siva-jothy et al., 2005; lawniczak et al., 2007) and reduced reproduction have been noticed as a trade-off for immune function against pathogens in insects (calleri et al., 2006). po is a key component in the immune system of insects and the main role of po in melanogenesis is converting phenols to quinones, which subsequently polymerize to form melanin (gonzález-santoyo and córdoba-aguilar, 2012). some species of entomopathogenic nematodes including s. carpocapsae are encapsulated and melanized in cpb (thurston et al., 1994; armer et al., 2004; ebrahimi et al., 2011a, b) as a part of the cpb immune system that displays some degree of resistance against the entomopathogenic nematodes. in the present study, po activity of nematode-injected cpb increased in the hemocel in a nematode dose-dependent manner compared to injection of ringer’s solution. increasing nematode concentration led to increased po activity which was coincident with the appearance of symbiotic bacteria in the hemolymph of the insects. yang et al. (2012) identified an insecticidal protein from xenorhabdus budapestensis which extensively activated the po cascade in g. mellonella which potentially caused larval g. mellonella death (yang et al., 2012). despite suppression of po activity by symbiotic bacteria of entomopathogenic nematodes in plutella xylostella (song et al., 2011; seo et al., 2012) and manduca sexta (eleftherianos et al., 2007, 2010), po activation in nematode-injected cpb larvae is expected since cellular encapsulation and capsule melanization is a common response of cpb against epns (thurston et al., 1994; armer et al., 2004; ebrahimi et al., 2011a, b) which implies the ij and its symbiont may overcome the insect defense system. the cuticular pro-phenoloxidases (propo) are, at least for some species, derived from the pool of hemolymphal propo (andersen, 2010). the epidermal cells in bombyx mori larvae can transport propo from hemolymph to cuticle (asano and ashida, 2001a, b). the cost of production and maintenance of the po system (including the propo-activating system) is likely to be high because melanin production, the final product of propo-activating system, is nitrogenrich, requiring substantial nitrogen or protein investment for its synthesis (blois 1978; lee et al., 2008). producing the regulatory proteins of the propo-activating system uses energy. the suppressive effect of immune activation on reproduction in insects is usually thought to be due to competition for resources between these two energetically expensive functions (adamo, 2008). perhaps, decreasing fertilized eggs in surviving adults is an indicator for such energy and nitrogen investments; egg production being dependent to proteins stored in the insect (wheeler et al., 2000). in present study, po specific activity of cpb against s. carpocapsae was generally increased 139    fig. 8 phenoloxidase specific activity of lc20 and lc80 nematode-injected, wounded and ringer’s solution injected insects in different time intervals. the results are expressed as mean ± sd. the data were statistically analyzed using anova and duncan’s multiple range tests with a specified significance level of p < 0.05. up to 48 h pi. po specific activity against entomopathogenic nematodes was investigated in g. mellonella. brivio et al. (2002) reported steinernema feltiae inhibited enzyme activity until 40 min pi compared with the control larvae. the juveniles of s. feltiae induced speedy suppression of phenoloxidase avoiding host humoral encapsulation. regardless of the nematode species, cpb shows effective but dose dependent immune defenses against entomopathogenic nematodes (dunphy and thurston 1990; armer et al., 2004; ebrahimi et al., 2011b), whereas g. mellonella is a susceptible host for the nematodes, so these differences are acceptable. walter et al. (2008) showed that live s. carpocapsae axenic infective juveniles and their exudates did not activate po in larvae of the lepidopteran g. mellonella and malacosoma disstria. as mentioned above, probably, insecticidal proteins of symbiotic bacteria are responsible for activation of po cascade in present study, while walter et al. (2008) used axenic nematodes. neither brivio et al. (2002) nor walter et al. (2008) noticed sclerotization of the infected insect’s cuticles and melanin formation. briefly, the level of total protein in the nematode as well as ringer’s solution injected insects was found lower than wounded control. as entomopathogenic nematodes (simões and rosa 1996; toubarro et al., 2009) and their symbiotic bacteria (caldas et al., 2002) induce proteases production inside the infected host insects, accordingly, it can be concluded that decline of total protein in the nematode treated insects in our study is the cost of the insects response against infection, however due to dilution of the hemolymph by ringer’s solution used in the experiment, decrease in total protein in nematode or ringer’s solution injected insects compared with wounded insects is expected. in conclusion, sublethal effects of s. carpocapsae on surviving adult cpb include discoloration and thinning of the cuticle in color defective adults, uniquely deformation in wings, antenna and legs, and decreasing 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http://www.ncbi.nlm.nih.gov/pubmed?term=shrestha%20s%5bauthor%5d&cauthor=true&cauthor_uid=21464604 http://www.ncbi.nlm.nih.gov/pubmed?term=kim%20y%5bauthor%5d&cauthor=true&cauthor_uid=21464604 http://www.ncbi.nlm.nih.gov/pubmed/21464604 http://www.ncbi.nlm.nih.gov/pubmed/21464604 http://www.ncbi.nlm.nih.gov/pubmed?term=yang%20j%5bauthor%5d&cauthor=true&cauthor_uid=22387345 http://www.ncbi.nlm.nih.gov/pubmed?term=zeng%20hm%5bauthor%5d&cauthor=true&cauthor_uid=22387345 http://www.ncbi.nlm.nih.gov/pubmed?term=lin%20hf%5bauthor%5d&cauthor=true&cauthor_uid=22387345 http://www.ncbi.nlm.nih.gov/pubmed?term=yang%20xf%5bauthor%5d&cauthor=true&cauthor_uid=22387345 http://www.ncbi.nlm.nih.gov/pubmed?term=liu%20z%5bauthor%5d&cauthor=true&cauthor_uid=22387345 http://www.ncbi.nlm.nih.gov/pubmed?term=guo%20lh%5bauthor%5d&cauthor=true&cauthor_uid=22387345 http://www.ncbi.nlm.nih.gov/pubmed/22387345 isj 11: 329-330, 2014 isj 11: 329-330, 2014 issn 1824-307x in memoriam anthony j. nappi 1937 2014   anthony j. “toni” nappi, parasitologist, invertebrate immunologist and biochemist, passed away at his home in la grange, illinois on october 18, 2014 at the age of 76. dr. nappi received his b.s. and m.s. degrees from the biology department at central connecticut state university in 1959 and 1964. he was awarded his ph.d. from the university of connecticut in 1969 where his studies concentrated on the fields of parasitology and insect immunology. he was the first graduate student to study under the direction of fred streams and it was in this laboratory that he became immersed in the study of hymenopteran and nematode parasites and how they both stimulated and suppressed the innate immune response of dipterans. his dissertation research laid the foundation for the study of cellular immune responses of drosophila against parasitoid wasps. in addition, toni convinced dr. john g. stoffolano, jr., a fellow graduate student, colleague and best friend during graduate school, that they should study hemocyte reactions and changes in musca domestica larvae during infection with the nematode heterotylenchus autumnalis and together they published several seminal papers on the subject.   329 in 1968 toni became an assistant professor of biology at the state university of new york in oswego, rising to the rank of professor in 1976. he taught numerous undergraduate courses, but his favorites were parasitology and immunology. in 1981, he was recruited as chairperson of the department of biology at loyola university of chicago where he remained as professor until his retirement in 2002. following retirement, he became a visiting professor of animal health and biomedical sciences at the university of wisconsin in madison where he directed research programs funded by the national science foundation and the national institutes of health. but toni’s activities were not restrained by the confines of stateside academic institutions. he also served for 20 years as the director of the overseas academic program in tropical biology at discovery bay marine laboratory in jamaica, where he annually taught tropical marine biology to undergraduate students. he also directed overseas academic programs in comparative immunology at the centre national de la recherche scientifique (cnrs), gif-sur-yvette, france and in immunogenetics at the institut pasteur, paris, france. he was a fulbright scholar at the université de bordeaux in france and he maintained very strong and long-standing research collaborations on drosophila immune systems with dr. yves carton at cnrs in gif-sur-yvette and the université paris. he also maintained active research collaborations with dr. enzo ottaviani at the university of modena and reggio emilia, italy. toni had a brilliant mind and coupled with his passion for science made it possible for him to see things others often missed. toni's excitement with science can best be described as pouring warm cola over ice, uncontrollable effervescence. he was creative scientifically and had the courage to stand by his convictions. at the same time he was willing to give of his time and ideas to aspiring young scientists. this nurturing attitude created an extremely popular and effective professor, teacher, and chairperson. i witnessed this first hand during the time toni spent at wisconsin. he invested an incredible amount of time with my graduate students and postdocs, giving advice, listening, and encouraging them in their efforts to become better at their craft. they are to this day deeply indebted to this gift toni gave them, as am i. like many scientists in the age of rapidly expanding technologies, toni’s scientific explorations of insect innate immunity to parasites and parasitoids evolved through the years. beginning with observational studies of insect cellular immune responses and encapsulation/melanization reactions against metazoan parasites, his research began to take on a biochemical emphasis in work on melanogenesis and this progressed to more detailed examinations of catecholamine metabolism   330 and its relation to insect cellular immunity. what perhaps tweaked his interests most during his final decade of research were his studies of the roles reactive oxygen and nitrogen intermediates play in immunity. overall, his research career resulted in more than 120 peer-reviewed publications, invited speaking engagements throughout france, and in germany, italy, the united kingdom, and taiwan and at many dozens of universities and research centers throughout the united states. both the nih and the nsf supported his research and he served on the editorial board of five different journals, including the invertebrate survival journal. his interests had great biological breadth; consequently, he w s a member of nine scientific societies. a t   oni was also an accomplished photographer, capturing action shots during college basketballs games and creating artistic images that challenged a viewer’s imagination. he loved italian and french food, some cooking but mostly eating, and wines from either country. he shared his happy life as husband of joyce for 55 years, father of lynn, kurt and paul, grandfather of amanda, isabelle and sebastien, and brother of andrew and mark. he will be greatly missed by family and many, many students, friends and colleagues throughout the world. bruce m. christensen department of pathobiological sciences university of wisconsin madison madison, wi 53706 molecular and cellular analysis of immunity in the phytoplasma vector euscelidius variegatus: exploiting immunity to improve biological control strategies 63 isj 14: 63-72, 2017 issn 1824-307x research report molecular and cellular analysis of immunity in the phytoplasma vector euscelidius variegatus: exploiting immunity to improve biological control strategies r tedeschi1, m monti1, e gonella1, g melchiori2, a alma1, m mandrioli2 1 disafa, university of torino, grugliasco, italy 2 department of life sciences, university of modena and reggio emilia, modena, italy accepted march 8, 2017 abstract insects depend on innate immunity only to defend themselves against pathogens and to regulate interactions with many other microorganisms, such as different kinds of symbionts. recently, it has been suggested that immunocytes could play a role in the vectorial capacity of insects leading to an increased interest towards primary immunocyte cultures. we analysed at molecular and cellular level the immune response of the leafhopper euscelidius variegatus with the aim to provide an in vitro model for studying the insect-microbe interactions. we in vitro cultured and kept alive for more than 3 months e. variegatus immunocytes that showed a mitotic capacity as well as adhesion and phagocytic activities. in situ hybridization revealed that the defensin gene is actively transcribed in cultured immunocytes, while cecropins were not recorded in this species. these promising results obtained with e. variegaus, a leafhopper frequently used as a laboratory experimental model of insect vector of phytoplasmas, will help in developing in vitro tools for the study of the interactions between these pathogens and their vectors. key words: innate immunity; circulating immunocytes; defensin, insect vectors; leafhopper introduction insects, as well as all multicellular organisms, are surrounded by many prokaryotic and eukaryotic microorganisms, playing different roles from beneficial to pathogenic. contrarily to vertebrates, insects rely on innate immunity only to defend themselves against pathogens (lavine and strand, 2002). however, beside entomopathogens, many other microorganisms may inhabit the insects’ body, such as mutualists, commensals and saprophytes (douglas, 2014). these microorganisms, which are differentially related to insects, exhibit a range of interactions with their immune system. for instance, obligate intracellular endosymbionts, which are strictly coevolved with their hosts, undergo strong genome reduction due to selective pressure that leads to the loss of many genes encoding target molecules of insect immune receptors; therefore they are able to escape defence responses (login and heddi, 2013). on the other hand, many endosymbionts have been reported to induce an immune response (nakabachi et al., 2005; anselme ___________________________________________________________________________ corresponding author: mauro mandrioli department of life sciences university of modena and reggio emilia via campi 213/d, 41125 modena, italy e-mail: mauro.mandrioli@unimo.it et al., 2006; ratzka et al., 2011). facultative symbionts as well have developed different strategies to escape or modulate insect mechanisms of immune response (eleftherianos et al., 2013), even often sharing with pathogens many molecular pathways recognized by the insects’ immune receptors (login and heddi, 2013). insect vectored plant pathogens, instead, were shown to either elicitate or suppress the immune responses (eliautout et al., 2016; vyas et al., 2015). in addition, insects regulate their immune response to control microbial infections by upor downregulating immune related genes in response to symbiotic microorganisms to maintain the microbiota balance (wang et al., 2009; login et al., 2011; weiss and aksoy, 2011; eleftherianos et al., 2013), avoiding detrimental immune induction which could result in disease-related dysbiosis (buchon et al., 2013). microbe-microbe interaction within the host’s body may be mediated by the insect immune system as well and some microorganisms can promote or depress the growth of another ones by elicitating the host production of immune-active molecules (douglas, 2014). this immune stimulation can be beneficial for the insect by contributing to the protection from pathogens and it may lead to the elimination of other harboured non 64 fig. 1 observation of e. variegatus immunocytes at the inverted microscope with a 20x (a) and 40x (b) phase contrast objective showing small immunocytes resembling prohemocytes (indicated by arrows) and large cells with abundant cytoplasmic inclusions here referred as granulocytes (indicated by arrow heads). both the immunocyte types resulted able to adhere to glass slides (c), phagocytize fluorescent microspheres (d) and express defensins (e). cells in photographs c, d and e have been counterstained with propidium iodide. bars = 10 m. symbiotic microorganisms, including vectortransmitted disease agents (weiss and aksoy, 2011). in these cases, the immune response induction may be favourable for the insects because, even though the role of human or plant pathogens on their insect hosts is poorly known, they may have detrimental effects for vectors’ life span and fecundity (hu et al., 2008; cassone et al., 2014; nachappa et al., 2014; alma et al., 2015; olson and blair, 2015). the insect innate immunity system is subdivided into humoral and cellular defense responses, with the production of antimicrobial peptides (amps) as a key process for both. amps are synthesized, predominantly, in fat body and released into hemolymph (tsakas and marmaras, 2010). some amps, such as defensis and cecropins, are common and highly conserved in different insect orders, while some others are more sporadic. insect defensins are active mainly against gram-positive bacteria, even if some insect defensins are also active against the gram-negative escherichia coli and some fungi. on the contrary, cecropins have a broad spectrum of activity against both gram-negative and gram-positive bacteria, as well as fungi (yi et al., 2014). new studies on immunocyte-mediated immunity highlighted the importance of cellular immune responses (tsaka and marmaras, 2010; goto and kurata, 2012). different types of immunocytes (frequently referred as hemocytes) can be found circulating in the insect hemolymph (marmas and 65 lampropoulou, 2009). they are produced during embryogenesis and within juvenile stages, and react to attack by pathogenic agents with different responses, including phagocytosis, encapsulation, melanization and production of antimicrobial peptides, as well (mandrioli et al., 2003; strand, 2008; buchon et al., 2014). recently, immunocytes have been shown to interact with symbionts and play a role in the vectorial capacity of insects (mandrioli, 2009; mandrioli et al., 2015a). as a consequence, the establishment of primary cultures of immunocytes has been widely increased in the last decades as a useful tool for the characterization of the immune response in different insects (mandrioli et al., 2015b). in particular, primary immunocyte cultures of insect vectors of plant pathogens has been proposed as new tools for studying the interactions between the pathogen and the host as well as the interplay between symbionts and immunocytes, leading to a better comprehension of insect vector competence (monti et al., 2014). here we analyse, at molecular and cellular level, the immune response of the leafhopper euscelidius variegatus, with aim to provide an in vitro model for studying insect immune response within the order hemiptera, with special regard to insect-microbe interactions. e. variegatus can be considered an optimal leafhopper model for such studies because, besides harbouring facultative symbionts (degnan et al., 2011), it is a vector of plant pathogens in the ‘candidatus phytoplasma’ genus, namely chrysanthemum yellows phytoplasma (‘ca. phytoplasma asteris) and flavescence dorée phytoplasma, and it was widely used as model species to study insectphytopathogen interactions (bressan et al., 2005; bosco et al., 2007; rashidi et al., 2014). fig. 2 amplification of the e. variegatus gene from genomic dna (a) and cdna (b) samples evidenced a 54 bp long exon 1 and a 225bp long exon 2 separated by a 601 bp intron (d). the presence of the intron was confirmed by splice site prediction (c) (indicated by arrows). 66 materials and methods e. variegatus primary cell cultures adults of euscelidius variegatus were obtained from laboratory lines reared on oats (avena sativa l.) in climatic chambers with 2025 °c and a photoperiod of 16:8 (l:d) hours (h), at the dipartimento di scienze agrarie, forestali e alimentari (disafa). they were used to establish cell cultures, according to monti et al. (2014). in particular, two female adults were washed in 0.115 % sodium hypochlorite, 75 % ethanol and milliq sterile water for 10, 30 and 20 seconds (sec), respectively. after drying on a filter paper for a couple of seconds, they were put in a single well of a sterile 24-well cell culture plate (costar®, corning, ny, usa) containing 1 ml of hert-hunter 70 (hh70) medium (marutani-hert et al., 2009). the hh70 medium has been supplemented with 10 ml/l lglutamine 200 mm solution (invitrogen, ca, usa), gentamicin (at a final concentration of 50 µg/ml, sigma-aldrich, mo, usa) and penicillin/streptomycin (sigma-aldrich, mo, usa) at a final concentration of 50 u/ml at 50 µg/ml, respectively. the antimycotic agent nystatin (sigmaaldrich, mo, usa) was also added to each medium at a final concentration of 100 u/ml. plates were incubated at 24 – 26 °c and 0.2 ml of medium was added every 48 h if necessary, whereas the observation of cell cultures and the evaluation of the cell growth were carried out daily using an inverted leica dmi3000 light microscope. functional assays by adhesion test and phagocytosis assay an aliquot of 200 µl from each immunocyte cell culture was collected and placed on a glass slide in an aseptical lab-tek chamber slide system (nunc, naperville, il, usa). immunocytes were let settled for 30 minutes (min) in presence of hh70 medium. thereafter, the slide was removed from the chamber slide system, stained with a 200 ng/ml propidium iodide solution and observed with a zeiss axioplan epifluorescence microscope. photographs were taken using a ccd camera as previously reported. for each cell culture, a phagocytosis assay was performed. briefly, a 200 µl aliquot was sampled and added to 100 µl of hh70 medium in a 0.2 ml tube previously covered and the material was then incubated with 0.1 µl of a fluorescent beads suspension for 30 min in soft oscillation, according to manfredini et al. (2008). after incubation, cells were cytocentrifuged onto glass slides, stained with a 200 ng/ml propidium iodide solution and observed with a zeiss axioplan epifluorescence microscope. search for defensin and cecropin coding genes genomic dna extraction was performed using the “sv genomic dna purification system” (promega, wi, usa) according to the manufacturer’s instructions. pcr amplification of an internal portion of the defensin gene was carried out using the primers evdef f (5’ atgcattcttccattactgctg) and evdef r (5’ cagctgcctcc cttcttgc). primers were selected by aligning the defensin coding sequences of the hemipterans triatoma brasilensis (ach57151), nilaparvata lugens (agk40896) and rhodnius prolixus (aao74624) available in genbank. the amplification mix contained 100 ng of genomic dna, 1 mm of each primer, 200 mm dntps and 2u of dynazyme ii dna polymerase (finnzymes oy, finland). amplification was performed with a thermal cycler at an annealing temperature of 54 °c for 60 sec and extension at 72 °c for 60 sec. rna extraction was performed with the “sv total rna isolation system” (promega), accordingly to the supplier’s suggestions. in order to complete the defensin coding gene identification, a rapid amplification of cdna ends (race) was performed following the method of frohman (1990). the amplified fragments were cloned with the “pgem t-easy cloning kit” following the promega protocols. race has been performed on cdna samples obtained by the total rna, extracted using tri-reagent tm (sigma) following the method described by the supplier, and the successive reverse transcription using oligo-dt primers and the access rt-pcr system (promega), according to the supplier’s protocols. pcr search for cecropin coding genes was carried using the primers evcec f (5’attggacaatcggaagctgg) and evcec r (5’cagttgcggcgacattng), designed after the alignment of the cecropin coding genes of the insects drosophila melanogaster (aaf57025), ceratitis capitata (xp_004534334) and bombyx mori (np_001037392). the choice of using those insects in place of hemipteran species is due to the absence of cecropin genes in the currently studied hemipteran species. sanger sequencing was performed at the bmr genomics (padua, italy), whereas sequence analysis was carried out using the clc sequence viewer software (madison, wi, usa) and using the splice site prediction (freely available at the address: http://www.fruitfly.org/seq_tools/splice.html) (reese et al., 1997). the e. variegatus defensin gene sequence can be retrieved from genbank under the accession number kx198127. semi-quantitative analysis by reverse transcription pcr (rt-pcr) of antimicrobial peptide (amps) expression in vitro in order to study the induction of defensin after bacterial challenges, e. variegatus immunocytes were incubated with a 109 cells/ml staphylococcus aureus (gram positive), escherichia coli (gram negative) and asaia sp. solutions for 0, 3, 6, 9, 12 and 24 h. after treatments (repeated in triplicates for each challenge), cells were centrifuged at 800g for 5 min at room temperature and the supernatant was discarded. rna extraction was performed with the “sv total rna isolation system” (promega), accordingly to the supplier’s suggestions. after extractions, rna quality and concentration were assessed with a nd-1000 spectrophotometer (nanodrop, de, usa). rt-pcr has been performed with the access rt-pcr system (promega), according to the supplier’s protocols. cytoplasmic actin was amplified (primers actf 5’agcaggagatggccacc-3’ and actr 5’tccacatctgctggaagg-3’) as a loading control, according to capone et al. (2013). for the 67 cytoplasmic actin, pcr reactions (20 cycles), the following parameters were used: annealing temperature 58°c; annealing time 40 sec, elongation time 45 sec. rt-pcr amplification was evaluated by electrophoresis in 1.2 % agarose gel (run 100v for 45 min). gel documentation was collected using a “gel doc xr”, digitally evaluated with “quantity one” (bio-rad lab, milano, italy) and normalized to the correspondent signals for cytoplasmic actin. three replicates were carried out for each induction. in situ hybridization the presence of defensin mrna in the e. variegatus immunocytes was studied using a defensin probe labelled with digoxigenin (dig) by end-labelling procedure (roche, switzerland). the in situ hybridization assay was performed using a non-radioactive procedure. briefly, immunocytes were cytocentrifuged at 800 rpm for 3 min. split cells were then fixed in pbs buffer containing 4 % paraformaldeyde and then permeabilized with pbs buffer containing 0.3 % triton x-100. cells were incubated with labelled probes for 16 hours at 42°c and subsequently washed at 42 °c in ssc 2x and 1x, respectively. after 30 min incubation with normal serum, cells were incubated with a fluorescein-conjugated anti-dig antibody for 2 h in the dark. nuclei were counterstained using a 100 ng/ml propidium iodide solution for 5 min at rt. fig. 3 alignment of the defensin aminoacidic sequences from drosophila melanogaster (dromel), pyrrhocoris apterus (purapt), triatoma brasilensis (tribra), nilaparvata lugens (nillug), rhodnius prolixus (rhopro), ceratitis capitata (cercap) and bombyx mori (bommor) revealed that e. variegatus (eusvar) defensin is well conserved, including the presence of six highly conserved cysteine residues (indicated by asterisks). 68 results on the basis of previous successful results with hemipteran immunocytes (monti et al., 2014), we isolated and in vitro cultured e. variegatus immunocytes in the hh70 medium with positive results and we kept cells alive for more than three months (fig. 1). cell counts showed a slightly declining cell number in the first 15 days with mitoses observed in plates starting after 12 days of in vitro maintenance. most of the observed immunocytes were small in size with the nucleus occupying the central part of the cellular body and they resembled typical insect prohemocytes (figs 1a-b). a second type observed consisted of cells larger than the previous with abundant cytoplasm containing cytoplasmic inclusions varying in shape from round to irregular or elongated that have been generally referred as granular cells (figs 1a-b). adhesion tests showed that e. variegatus immunocytes were able to adhere to a glass slide after 30 min incubation (fig. 1c). moreover, 72 % of cells were able to phagocytize fluorescent microspheres assessing that they are functional despite their in vitro maintenance (fig. 1d). pcr amplification with the defensin primers evidenced a product of about 800 bp using genomic dna as a template (fig. 2a) and an approximately 200 bp amplicon using cdna samples (fig. 2b); these results, as a whole, clearly suggest the presence of an intron. race pcr allowed the amplification of both the defensin cdna termini, allowing the identification of the complete defensin coding sequence in e. variegatus. sequence analysis revealed that the defensin gene consists of a 54 bp long exon 1 and a 225 bp long exon 2 separated by a 601 bp intron (fig. 2d). the presence of the intron was also confirmed by bioinformatic analyses performed using the splice site prediction (fig. 2c). the e. variegatus defensin consists of 92 aminoacidic residues exhibiting a 36 % to 60 % similarity with other insects, with an increase to 50 60 % if the comparison is limited to hemipteran defensins (fig. 3). sequence alignment also showed a conserved localization of 6 cysteine residues (generally referred as cysteine 3 8 in the defensin peptide) suggesting their involvement in the formation of three disulfide bridges in the e. variegatus defensin (fig. 3). in situ hybridization revealed the defensin gene was transcribed in the in vitro cultured immunocytes evidencing a bright fitc fluorescence in the cytoplasm (fig. 1e). the identification of the defensin gene allowed us to study its induction after bacterial challenges on in vitro cultured e. variegatus immunocytes (fig. 4). in particular, rt-pcr experiments clearly showed that s. aureus only induced the defensin gene expression after 6 h, whereas no induction has been observed after e. coli and asaia challenge (fig. 4). pcr amplification with the cecropin primers evidenced a 160 bp product using cdna samples obtained from the dipterans d. melanogaster and fig. 4 a) rt-pcr amplification of the e. variegatus defensin from immunocyte samples challenged with e. coli, s. aureus and asaia for times ranging from 0 to 24 h evidenced that defensin is constitutively expressed in e. variegatus, but its expression increased after a 6 h long exposure to the grampositive s. aureus. cytoplasmic actin has been amplified for each sample as loading control. b) graphical representation of the results obtained from experiments clearly evidences the induction of the defensin expression after exposure to the grampositive s. aureus. anopheles stephensi, the coleopteran tribolium castaneum, the lepidopteran b. mori, and the hymenopteran apis mellifera as a templates, whereas no amplification has been obtained in the hemipterans acyrthosiphon pisum and e. variegatus (fig. 5). discussion immunocytes play multiple functions in insects, including defence mechanism like nodule formation, phagocytosis, encapsulation and synthesis of antimicrobial peptides and other molecules (pandey 69 fig. 5 rt-pcr amplification of cecropin cdna in acyrthosiphon pisum (1), d. melanogaster (2), tribolium castaneum (3), b. mori (4), e. variegatus (5), anopheles stephensi (6) and apis mellifera (7) assessed the absence of cecropin hortologues in both the analysed hemiptera. and tiwari, 2012). interestingly, in the last years a role of immunocytes in the interaction with symbionts and in the vectorial capacity of insects has been suggested making their study a big challenge for the scientific community (mandrioli, 2009; mandrioli et al., 2015a). indeed, the immunocyte study is not simply related to immunity, but also to future applications in applied fields related for instance to the biocontrol of insects involved in the spread of plant diseases. up to date, immunocytes have been studied in several insect species and in particular in lepidoptera, where from 2 to 7 different cell types have been accurately described using morphological, histochemical and functional characteristics (chauvin, 1968; brehélin et al., 1978; brehélin and zachary, 1983, 1986; ahmad, 1992; butt and shields, 1996; hernandez et al., 1999; manfredini et al., 2008). in view of the large number of diverse immunocyte types (including their different ultrastructures, size and distribution in the insect body), it has been suggested the “multiple-cell theory” about hematopoiesis and hemocyte differentiation, which suggests the existence of separate immutable cell lines, each differentiating from a single germinal stem, that give rise to the different hemocyte types (akai and sato, 1973; gupta, 1985). in our current analysis, we recognized in e. variegatus two types of circulating immunocytes due to their substantial uniformity in the cellular morphology, in agreement with previous observation in hemiptera (monti et al., 2014). in particular, it seems that in e. variegatus the unique feature that marks distinctively a precise hemocyte variety is the presence of cytoplasmic granules, which are a prerogative of granular immunocytes. the other cells (even if they may be slightly different in size) are generally agranular and with a large nucleus. in the case of e. variegatus no other feature seems to justify the identification of further immunocyte types. as a whole, in view of the small size, the thin cytoplasm, which is a proof for a cell that is beginning its growth, and the absence of phagolysosomes or granules, we considered the small, agranular cells as prohemocytes, whereas the second cell type observed in e. variegatus can be referred as granular cells or granulocytes. literature data on hemiptera immunocytes are not abundant, but a previous study identified by phase-contrast microscopy seven morphological hemocyte types (prohemocytes, plasmatocytes, granular cells, cytocytes, oenocytoids, adipohemocytes and giant cells) in the species rhodnius prolixus, rhodnius neglectus, triatoma infestans, panstrongylus megistus and dipetalogaster maximus (azambuja et al., 1991). actually, not all of them are present in the different studied species: for example, adipohemocytes and oenocytoids were not observed in p. megistus and p. infestans, while giant cells were rarely found in any of the species studied. our proposal to refer to two type only is not unusual in literature, since also in honeybees and wasps few immunocyte types have been described (chauvin, 1968; manfredini et al., 2008) supporting a more unitarian interpretation of the cellular elements of hemolymph. this approach supposes that the various hemocyte types are merely stages, with separate functions, of a single cell line derived from a unique germinal stem i.e., the prohemocyte (ottaviani, 2005; manfredini et al., 2008). the “single-cell theory” relies therefore on the presence of transitional stages of some immunocyte types and on the existence of only prohemocytes and granular cells in tissue cultures and hematopoietic organs (lavine and strand, 2002). this is in line 70 with the great functional versatility of these complex and highly specialized cells, which is required in order to achieve a wide physiological flexibility that is necessary to undergo ready transformation in response to environmental stimuli and/or bacterial challenges (ottaviani, 2005; manfredini et al., 2008). according to literature data, immunocytes can grow and multiply also in vitro for an indefinite period, without the involvement of hematopoietic organs (gupta, 1985). even if with a low mitotic index, e. variegatus circulating immunocytes showed a mitotic capacity, suggesting that these prohemocytes are able of dividing not only as stem cells, as suggested in other insects (brehélin and zachary, 1986; franchini et al., 1996), but also when already circulating in the hemolymph. this aspect could be very important taking into account that immunocytes could interact with different symbionts and/or plant pathogens that can move within the insect body, where the number of immunocytes could be relevant for a proper immune response. molecular analyses and fish clearly showed that e. variegatus immunocytes are able to synthesize antimicrobial peptides belonging to the defensin family that are active against grampositive bacteria. this result was not unexpected since defensins form a family of antibacterial peptides that is widely distributed in insects (bulet et al., 1999; lamberty et al., 1999), including the presence of defensins expressed in insect cell lines (fallon and sun, 2000). as a whole, our results suggest the presence in e. variegatus of a gene coding for defensins, but the absence of genes coding for cecropins, in accordance to data obtained from the genome and transcriptome analyses of the brown planthopper n. lugens (bao et al., 2013). the absence of genes encoding antimicrobial peptides that are common in other insects, including defensins and cecropins, is not unusual in hemiptera since cecropins and defensins are also missing in aphids, as reported in the pea aphid a. pisum (the international aphid genomics consortium, 2010). these results are very useful in order to verify the chance of developing in vitro tools for the study of the interaction between phytoplasmas and the host insect vectors using e. variegatus as an experimental model. in order to plan in vitro analyses, it is essential to know the presence/absence of the most common molecules involved in the immune response, such as defensins and cecropins to verify if phytoplasmas may induce or modulate the insect immune response. indeed, as revised by bosco and d’amelio (2010), once in the hemocoel of the insect vector, phytoplasmas may circulate and multiply in the body cavity, and pass through the salivary glands before being excreted together with hemipteran saliva during successive feeding events. the innate immune system of insect vectors could therefore play a major role in enabling phytoplasma multiplication in and colonization of the insect body. this would be useful for clarifying the detailed physiological and immunological mechanisms in e. variegatus and could provide potential targets for the management of leafhopper phytoplasma vectors in the future. acknowledgement this work was supported by the project “iphyto” founded by 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immunity inside the digestive tract of the medicinal leech isj 8: 173-178, 2011 issn 1824-307x review innate and procured immunity inside the digestive tract of the medicinal leech ac silver1, j graf2 1department of internal medicine, section of infectious diseases, yale university school of medicine, new haven, ct 06520, usa 2department of molecular and cell biology, university of connecticut, storrs, connecticut 06269, usa accepted august 29, 2011 abstract especially when combined with unique biological adaptations, invertebrate animals provide important insights into innate immunity because the immune response is not complicated by adaptive immunity that vertebrates evolved. one such example is the digestive tract of the medicinal leech, hirudo verbana, which is unusual in two aspects, it contains a simple microbial community and it stores large amounts of vertebrate blood for a several months. in this review we will discuss aspects of the innate immunity of the leech and from the ingested blood that we term procured immunity to differentiate it from the immunity encoded by the leech genome. key words: medicinal leech; aeromonas; innate immuni y t   introduction the ability of medicinal leech to consume large quantities of vertebrate blood is a fascinating biological adaptation of this slow moving parasite and the underlying reason for the resurgence of its use in today’s surgical wards. in a single blood meal hirudo verbana, the hungarian medicinal leech, can consume over 5-times its body weight in blood. this process is facilitated by the release of vasodilators and anticoagulants that stimulate the blood flow and activate pumping of the leech (rigbi et al., 1987, 1996). the anticoagulants are so powerful that the bite site will continue to bleed for about 15 minutes after an engorged leech falls off its prey. while there are many uses of medicinal leeches, one particular use led to the approval of the medicinal leech as a medical device by the fda (rados, 2004). during reconstructive microvascular surgery, a major complication is venous congestion (whitaker et al., 2005; whitaker et al., 2009). while blood can enter the reattached tissue through the reconnected arteries, the blood does not exit at a sufficient rate, leading to a lack of oxygen and tissue necrosis occurs. application of medicinal leeches removes blood from the tissue allowing oxygenated blood to enter the wound. this procedure greatly increases the success rate in cases with this complication but also carries the risk of wound infections by one of ___________________________________________________________________________ corresponding author: joerg graf department of molecular and cell biology university of connecticut, 91 n. eagleville rd unit-3125, storrs, ct 06269, usa   173 e-mail: joerg.graf@uconn.edu the digestive-tract symbionts and thus a preemptive antibiotic therapy is suggested (whitaker et al., 2011). consuming such large blood meals requires several adaptations. the storage occurs in the largest compartment of the digestive tract, the crop, which accounts for most of the animal’s body. water and salts are removed from the ingested blood meal concentrating it into a viscous paste, which allows the animal to regain mobility (sawyer, 1986; zebe et al., 1986). interestingly, it has been reported that at least some of the erythrocytes remain physically intact inside the crop for several months (jung, 1955), but a recent report indicated that a portion of the erythrocytes are lysed by the digestive tract symbionts (maltz and graf, 2011). over a period of many weeks, the blood meal moves into the intestinum where the digestion of the blood meal and absorption of nutrients is thought to occur. the animals can survive for over six months between feedings. historically, hirudo medicinalis, the medicinal leech, from central europe was used for application on humans in france and germany. a combination of heavy collecting and loss or pollution of habitat led to a decline in the natural populations. this led to h. medicinalis being listed as an endangered species and the emergence of leech farms where the animals are raised and also bred in captivity (reviewed in graf, 2000). medical supply companies typically sold medicinal leeches as h. medicinalis but it was recently discovered that these animals are usually h. verbana, the hungarian medicinal leech (siddall et al., 2007). it seems likely that most   174 leech farms supplemented their breeding stock with field caught animals from eastern europe and thus inadvertently replaced h. medicinalis with h. verbana. there are some differences in the pigmentation but dna bar coding is the most useful technique in differentiating these species. given the paucity of h. medicinalis among commercially available animals, it seems reasonable that most research is done on h. verbana or perhaps hirudo orientalis. as all animals, the leech lives in close association with microorganisms that are thought to provide essential function for the leech. in the leech, the digestive tract is colonized by a relatively simple microbial community (worthen et al., 2006), which may be critical for preventing a premature degradation of the ingested blood meal. early culture-based studies reported the presence of a single bacterial symbiont that we identified as aeromonas veronii in the crop (graf, 1999). subsequently, a culture-independent study revealed the presence of a second numerically dominant bacterial symbiont, a rikenella-like bacterium (kikuchi and graf, 2007; worthen et al., 2006). in the intestinum, the same two species are highly abundant, but a greater diversity of species exists. diagnostic pcr of embryos revealed that the bacteria are transmitted from parent to offspring (rio et al., 2009). it is very intriguing that a gut community is this simple, even insects that have a highly alkaline midgut harbor a more complex community of microorganisms (broderick et al., 2004). a driving question is to determine which factors contribute to this unusual simplicity. one approach to address this question is to experimentally determine how specific the association is and identify mechanisms that are responsible. procured immunity a colonization assay allows one to assess the ability of different species or mutants to colonize an animal (graf, 1999). for the medicinal leech, we can introduce the bacteria that carry antibiotic resistance markers into the gut by adding the bacteria to a blood meal. by plating dilutions of the gut content onto antibiotic containing plates we can readily tell them apart from the native bacteria. a simple control provides insight into the changes that occur inside the gut (indergand and graf, 2000). an aliquot of the inoculated blood is incubated inside a microcentrifuge tube at the same temperature as the leech. a comparison of the density of colony forming units (cfu), or viable bacteria, allows one to deduce how well a strain is able to grow inside the leech gut and if that growth reduced inside the leech as compared to the blood in vitro control or in comparison to a native leech symbiont. an escherichia coli strain exhibited an interesting phenotype (indergand and graf, 2000). for the first 42 h after being fed to the leech, the number of e. coli that could be cultured from inside the leech decreased ~100 to 1,000 fold. interestingly, a similar decrease occurred in the in vitro blood control thus it seemed possible that the same activity that killed off the bacteria in the control could remain active inside the leech. interfering with the activation of the complement system in the blood meal prior to inoculating it with the e. coli strain either with heat, edta or egta with mg2+ permitted the e. coli strain to proliferate in blood, but in blood supplemented with inulin, the e. coli strain did not grow. this suggested that interfering with the activation of the complement system, perhaps via the classical pathway allowed e. coli to proliferate in the blood. when this strain was fed to the leech in heat-inactivated blood, the e. coli strain was able to proliferate and establish itself in the gut of the leech. these data clearly show a heat-sensitive property of vertebrate blood remains active inside the leech and contributes to the unusual specificity of the leech digestive-tract symbiosis. even at the strain level, aeromonads have been shown to have varying degrees of resistance to complement (janda, 1994), which could contribute to certain species or strains having a competitive advantage over others in their ability to colonize the leech. by feeding on vertebrate blood, medicinal leeches temporarily gain access not just to the nutritional value of the ingested blood meal but also acquire an immunological protection. we term this “procured” immunity to differentiate from the factors encoded by the leech genome. innate immunity antimicrobial peptides antimicrobial peptides (amps) are an important part in the innate immune response of vertebrates, invertebrates, and plants. they are a diverse group of compounds that have the ability to kill a broad spectrum of bacteria. the majority of amps are cationic and amphilic and their most common mode of action is to disrupt the bacterial cell membrane (ganz, 2003). as one might suspect they are most commonly found at the interface between the organism and environment (e.g., skin and digestive tract) in order to prevent microbial invasion (ganz, 2003). amps have been identified in the gastrointestinal tract of vertebrates and invertebrates but to our knowledge, none have been discovered in the leech gut. however, an expressed sequence tag library of the salivary transcriptome from macrobdella decora, revealed a c-type lectin that could possess antimicrobial activity (min et al., 2010). the amps hm-lumbricin and neuromacin, have been isolated in the h. medicinalis central nervous system and theromacin, theromyzin, and peptide b from the leech hemolymph of the distantly related theromyzon tessulatum (schikorski et al., 2008; tasiemski et al., 2004; tasiemski et al., 2000). theromacin and theromyzin were also detected in the leech intestinal epithelium, which makes it plausible that amps are present in the gut (tasiemski et al., 2004). a mutation in the a. veronii lpp, braun’s major outer membrane lipoprotein, led to a decreased ability for this strain to colonize the leech gut (silver et al., 2007b). lpp is involved in membrane stability and subsequent resistance to toxic compounds such as antibiotics, cationic dyes, edta and the surfactants sds and triton x-100 (hirota et al., 1977; sha et al., 2004). interestingly, some of these compounds share the core membrane disrupting characteristics as amps (i.e., amphilic or cationic) (ganz, 2003). the a. veronii lpp mutant exhibited an increased sensitivity to sds but the weakened outer membrane was not affected by possible changes in osmolarity within the leech crop and was not sensitive to mammalian complement (silver et al., 2007b). these data suggested the observed colonization defect of the lpp mutant was possibly due to the presence of amps secreted into the leech gut (silver et al., 2007b). besides membrane disruption and subsequent lysis, amps have also been shown to kill bacteria by binding intracellular targets (e.g. inhibiting dna, rna, protein, and cellwall synthesis) (brogden, 2005; nicolas, 2009). amps gain intracellular access through mechanisms such as receptor-mediated endocytosis, membrane permeabilization, the formation of toroidal pores, and transient membrane leakage (nicolas, 2009). under this scenario a hypothetical amp inside the leech gut would be able to traverse the membrane easier in the lpp mutant compared to wild-type a. veronii. this would lead to cell death and account for the colonization defect exhibited by this mutant, which provides indirect evidence for the presence of an amp present inside the crop. fig. 1 fluorescence micrograph of a. veronii in the crop of the leech. crop section of a leech inoculated with aeromonas 42 h after feeding. aeromonas (red) and nuclei (blue) were visualized by staining with an aeromonas-specific probe and dapi, respectively. bacteria were either not associated with leech immune cells, or associated with immune cells via surface-association or intracellular association. erythrocytes have a dark center surrounded by a red autofluorescence. copyright 2007 national academy of sciences, usa phagocytic immune cells phagocytic immune cells represent another essential component of the innate immune response against invading pathogens. immune cells referred to as amebocytes were first identified in the celomic fluid and connective tissue of h. medicinalis at which time it was proposed that they could play a role in phagocytosis (sawyer, 1986). more recently, in the leech glossiphonia complanata, macrophagelike cells, nk-like cells, and granulocytes were discovered in blood vessels, lacunae, and connective tissue (de eguileor et al., 2000b; de eguileor et al., 1999). when these leeches were stimulated via saline or lps prick on their outer surface, these immune cells traveled from the area adjacent to the gut to the wound site (de eguileor et al., 2000b). de eguileor et al. demonstrated the macrophage-like cells possess phagocytic activity (de eguileor et al., 2000a). interfere with phagocytosis (barbieri and sun, 2004). after the t3ss mutant or wild-type strain were fed to the leech, fluorescence micrographs of the gut revealed the leech immune cells phagocytosed the mutant at a much higher frequency than wild-type a. veronii (silver et al., 2007a). this was evidenced by the mutant appearing tightly associated with the nucleus of the immune cell (i.e., intracellularly located), while the wild-type strain remained on the outside of the cell, thereby resisting engulfment by use of its functional t3ss (fig. 1). these data demonstrated that phagocytic immune cells infiltrate the leech digestive tract and remove susceptible bacteria and that a. veronii overcomes this barrier of the innate immune response through the use of a functional t3ss (silver et al., 2007a). utilizing fluorescence in situ hybridization (fish), nucleated cells were identified in the gut of h. verbana after an ingested blood meal, however, it was possible these were sheep immune cells (silver et al., 2007a). the blood meal was reconstituted after several centrifugations and it was determined that over 95 % of the sheep derived leukocytes were removed before the leech was fed, thereby confirming these nucleated cells were of leech origin (fig. 1) (silver et al., 2007a). in the same study an a. veronii type iii secretion system (t3ss) mutant was shown to have a dramatically reduced ability to colonize the leech gut (silver et al., 2007a). the t3ss is a molecular needle complex that injects effector proteins into eukaryotic cells, which then elicit a multitude of host cell changes (hueck, 1998). in other gram-negative bacteria, many of these effectors contain n-terminal rho gtpase-activating protein (rhogap) domains, which if expressed in eukaryotic cells disrupt the actin cytoskeleton and another important aspect of the interaction of a. veronii and the leech immune cells is that a. veronii does not interfere with the phagocytosis of other cells. when the wild-type strain and the t3ss mutant were introduced together, the t3ss mutant still decreased dramatically in abundance, while the other strain proliferated (silver et al., 2007a). the lack of providing general protection may be an important aspect contributing to the specificity of the microbial community inside the digestive tract of h. verbana. the ability of several a. veronii isolates to colonize the digestive tract of h. verbana was examined by competing each against a native a. veronii isolate from h. verbana in a standard competition assay, using heat-inactivated blood, in which the test and control strains carry different antibiotic resistance markers (silver et al., 2011). strains isolated from another leech species, h. orientalis an eel, and several clinical isolates were   175 fig. 2 cross section of the medicinal leech depicting procured (complement) and innate (immune cells and amps) immunity. the digestive-tract symbionts, aeromonas and the rikenella-like bacteria, are shown. antimicrobial peptides are written in italics instead of bold to distinguish speculation versus evidence, as with complement and immune cells. competed against the control strain. it was determined that the strain isolated from h. orientalis colonized to comparable levels inside the crop of h. verbana as the control strain (silver et al., 2011). however, all of the remaining isolates had a significantly reduced ability to colonize the leech, which ranged from a less than 10-fold defect to a greater than 4,000-fold defect (silver et al., 2011). control experiments revealed that the colonization defect exhibited by the isolates was not due to an inability to proliferate in blood (silver et al., 2011). the inability of other closely related a. veronii isolates to colonize the leech crop further demonstrates the specificity of this microbial association. microbial derived factors   176 it remains likely that other factors in addition to the complement of the ingested mammalian blood, amps, and immune cells can contribute to the innate immunity in the leech digestive tract. the symbionts within the leech gut, a. veronii and the rikenella-like bacterium, could play an important role in supplementing the innate immune response within the crop. as proposed in other systems, the symbionts could prevent the colonization of pathogenic bacteria by occupying a specific niche or out competing pathogenic bacteria for nutrients. in addition, secreting amps, bacitracin, or anti-quorum sensing compounds could also shape the microbial community. the presence of a more diverse microbial population, which includes both a. veronii and the rikenella-like bacterium, in the intestinum suggests they are not secreting amps or if they are, their expression is downregulated in the intestinum. if the gut symbionts are not directly secreting amps they could be responsible for stimulating the gut epithelium to express increased levels of amps. the digestive tract of h. verbana represents an interesting evolutionary adaptation where the immune system of an annelid is supplemented by components of the immune system from a vertebrate on which it feeds (fig. 2). it is possible that the complement system of the ingested blood also remains active in the gut of other blood feeding animals. however, since many of these animals have not been amendable to the introduction of genetically manipulated symbionts during the feeding process, the series of factors that contribute to their specificity have not been revealed. in other model systems, such as the hawaiian bobtailed squid, euprymna scolopes, it is likely a series of factors contribute to the establishment of the normal microbiome (nyholm and mcfall-ngai, 2004; ruby, 2008) and inside hirudo verbana multiple factors such as hemocytes and ingested complement contribute to this specificity (silver et al., 2007a; braschler et al., 2003). acknowledgements we would like to thank v kask for the artwork. the research was supported by nsf career award mcb 0448052 and nih r01 gm095390 to jg.   177 references barbieri jt, sun, j. pseudomonas aeruginosa exos and exot. rev. physiol. biochem. pharmacol. 152: 79-92, 2004. braschler tr, merino s, tomas jm, graf j. complement resistance is essential for colonization of the digestive tract of hirudo medicinalis by aeromonas strains. appl. environ. microbiol. 69:4268-4271, 2003. broderick na, raffa, kf, goodman, rm, handelsman, j. census of the bacterial community of the gypsy moth larval midgut by using culturing and 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azzopardi ea, graf j, kon m, lineaweaver wc. preventing infective complications following leech therapy: is practice keeping pace with current research? microsurgery 29: 619-625, 2009. worthen pl, gode cj, graf j. culture-independent characterization of the digestive-tract microbiota of the medicinal leech reveals a tripartite symbiosis. appl. environ. microbiol. 72: 47754781, 2006. zebe e, roters fj, kaiping b. metabolic changes in the medical leech hirudo medicinalis following feeding. comp. biochem. physiol. 84a: 49-55, 1986. bivalves amps isj 8: 85-97, 2011 issn 1824-307x review molluscan antimicrobial peptides, a review from activity-based evidences to computerassisted sequences h li1*, m-g parisi2*, n parrinello2, m cammarata2, p roch3 1the shenzhen key laboratory for marine bio-resource and eco-environment, college of life sciences, shenzhen university, shenzhen 518060, people republic of china 2marine immunobiology laboratory, university of palermo, via archirafi 18, 90123 palermo, italy 3ecologie des systèmes marins et côtiers, cnrs-ird-université montpellier 2, cc093, place eugène bataillon, f34095 montpellier cedex 05, france * equal contribution accepted may 16, 2011 abstract antimicrobial peptides (amps) represent the most universal immune effectors. molluscs constitute the second largest animal phylum, after arthropods, in term of number of species. only a negligible number has been investigated regarding amps. the choice of the species to be studied relied on their economical importance and availability. first studies on molluscan amps dated from 1996 and were based on biological activities of biochemical-purified fractions. such approach released all the original structures we know, with biological activity sometimes different from one isoform to another. then, molecular biology techniques were applied to molluscan amps starting in 1999. complete screening of genome expression in various situations became available, as well as some exotic molluscs, the ones collected from deep-sea hydrothermal vent, for instance. full sequences of active peptides and precursors, and gene organizations were established. a breakthrough consisted in the discovery of numerous amp variants, even within the same animal. in addition, computer homology revealed the existence of already known amps in new studied molluscan species. key words: antimicrobial peptide; amp; defensin; mussel; oyster; bivalves; gastropods introduction thousands of bioactive compounds identified in marine organisms reveal that sea creatures constitute a large reservoir for pharmacologically active drugs. recently reviewed by (mayer et al., 2011), most of these active molecules are enzymatically synthesized. exceptions are rare and ___________________________________________________________________________ corresponding author: philippe roch ecologie des systèmes marins et côtiers cnrs-ird-université montpellier 2, cc093 place eugène bataillon f-34095 montpellier cedex 05, france e-mail: philippe.roch@univ-montp2.fr list of abbreviations: aa, amino acids; amp(s), antimicrobial peptide(s); cds, coding sequence; est, expressed sequence tags; ish, in situ hybridization; pcr, polymerase chain reaction; ssh, suppression subtractive hybridization consist on venoms and toxins, as regarding the gastropod genus conus with an estimated 50,000 different conotoxins (12-24 aa, 4-6 cysteines) reported for their neurotoxic effects, for instance (han et al., 2006). the first report on antibacterial activity observed in molluscs was from mucus of the giant snail achatina fulica. named achacin, the protein of 150 kda is composed of 2 subunits (iguchi et al., 1982; kubota et al., 1985). due to its large mw, such molecule cannot be assimilated to a peptide. also from gastropod, the sea hare, aplysia kurodai, were the bacteriostatic glycoproteins, aplysianin p, a and e, of 60-320 kda (kamiya et al., 1984; kamiya et al., 1986; kisugi et al., 1987; yamazaki et al., 1990). dolabellanin a from albumen gland (kisugi et al., 1989), c from coelomic fluid (kisugi et al., 1989), e from egg mass (kamiya et al., 1984), p from purple fluid (yamazaki et al., 1989), and b2 from skin and mucus (iijima et al., 2003) have been reported in another sea hare, dolabella auricularia. it was only in 1996, that true antimicrobial peptides (i.e., cationic, 4-6 kda) have been isolated simultaneously from the mediterranean 85 fig. 1 summary of the pathways regarding the discovery of mollusc amps, linking species and molecules to the applied technologies. mussel, mytilus galloprovincialis (hubert et al., 1996a) and the blue mussel, m. edulis (charlet et al., 1996). previous mini-review related to amps from marine invertebrates dated from 2004 (tincu and taylor, 2004). at that time, only the biochemical approach was presented regarding the sole mussels with 4 defensins, 2 mytilins, 2 myticins and one partially characterized mytimycin. since that time, knowledge on amps increased dramatically due to molecular biology techniques applied to invertebrates. more recent reviews included new discoveries, but were restricted to mussels, mytilus edulis, m. galloprovincialis and oysters, crassostrea gigas, c. virginica (fleury et al., 2008) or adding the venerid clams, ruditapes (li et al., 2009a). since that time, 35 new amps isolated from 12 different molluscan species were released (fig. 1). the present review will consider the same major mussel amps, now reported also from other molluscan species, and extended to some exotic amps grouped in as miscellaneous, organized chronologically moving from traditional antimicrobial activities assayed in biochemically-purified fractions, to recently applied computer-assisted nucleotide sequence investigations and data mining (table 1). activity-based evidences defensin is probably the best known and appropriate term related to an immune effector molecule. it was introduced in 1985, regarding human neutrophils (ganz et al., 1985) following the discovery of antimicrobial activity and gene organization of the so-called arginine-rich cationic peptides reported in rabbit and guinea-pig leukocyte lysates in the 60’s. later, reviewers regarding antigram positive peptides isolated from the flesh fly, phormia terranovae, forced the term “insect defensin” (lambert et al., 1989; cornet et al., 1995). mammalian and insect defensins resemble as 3 4 kda peptides with 6 cysteines engaged in three intra molecular disulphide bonds. meanwhile, knowledge of their 3-dimensional structures revealed crucial differences: 3 domains for insect defensins -one n-terminal loop, one amphipathic α 86 table 1 chronology of amp discovery in molluscs species amp name nature technology used date reference mytilus galloprovincialis defensin (mgd-1) aa hplc 1996 hubert et al. mytilus edulis 2 defensins (peptide a, b) aa partial hplc 1996 charlet et al. mytilus edulis 2 mytilins (a, b) aa hplc 1996 charlet et al. mytilus edulis mytimycin aa partial hplc 1996 charlet et al. mytilus galloprovincialis defensin (mgd-2) nucleotides cdna 1999 mitta et al. mytilus galloprovincialis 2 myticins (a, b) aa partial hplc 1999 mitta et al. mytilus galloprovincialis 3 mytilins (c, d, g1) aa hplc 2000 mitta et al. crassostrea virginica big-defensin nucleotides cdna 2001 database ruditapes philippinarum big-defensin rpd-1 aa partial hplc 2003 wei et al. mytilus trossolus mytilin-c nucleotides mrna 2004 database crassostrea virginica defensin (aod) aa hplc 2005 seo et al. biomphalaria glabrata 2 amps nucleotides est 2005 mitta et al. argopecten irradians irradians 2 defensins nucleotides est 2006 song et al. crassostrea gigas defensin (cg-def) nucleotides est 2006 gueguen et al. crassostrea virginica 2 defensins nucleotides bac 2006 cunningham et al. argopecten irradians big-defensin aibd nucleotides cdna 2007 zhao et al. crassostrea gigas 2 defensins (cg-defh1, -2) nucleotides mrna 2007 gonzalez et al. bathymodiolus azoricus mytilin nucleotides cdna 2007 bettencourt et al. ruditapes decussatus mytilin nucleotides ssh 2007 gestal et al. ruditapes decussatus 3 myticins (1-3) nucleotides ssh 2007 gestal et al. chlamis farreri histone h2a nucleotides cdna 2007 li et al. mytilus galloprovincialis myticin-c nucleotides ssh 2008 pallavicini et al. haliotis discus hannai defensin (hd-def) nucleotides cdna 2008 hong et al. mercenaria mercenaria big-defensin, defensin nucleotides ssh & cdna 2009 parrigault et al. haliotis discus discus abhisin nucleotides cdna 2009 de zoysa et al. bathymodiolus thermophilus defensin nucleotides ssh 2009 boutet et al. littorina littorea littorein activity hplc 2009 defer et al. crassostrea gigas prolin-rich (cg-prp) nucleotides est 2009 gueguen et al. argopecten purpuratus proline-rich amp aa hplc 2009 arenas et al. hyriopsis cumingii 2 defensins nucleotides est 2009 bai et al. haliotis discus discus defensin nucleotides cdna 2010 de zoysa et al. venerupis philippinarum big-defensin vpbd nucleotides est 2010 zhao et al. mytilus coruscus 2 mytilins (a, b) nucleotides cdna 2010 database mytilus coruscus 6 mytilins (3-8) nucleotides cdna 2010 database mytilus coruscus 9 myticins (1-9) nucleotides cdna 2010 database crassostrea virginica histone cv-h2b-1 aa partial hplc 2010 seo et al. crassostrea virginica histone cv-h2b-2, -3, -4 aa partial hplc 2011 seo et al. haliotis laevigata 2 antiviral molecules activity none 2011 dang et al. mytilus galloprovincialis 2 mytimycins (p, v) nucleotides cdna 2011 sonthi et al. gastropod species are grey boxed; bivalve species are open boxed. abbreviations: aa: amino acids; bac: bacterial chromosome; cdna: complementary dna; est: expressed sequence tags; hplc: high pressure liquid chromatography; mrna: messenger rna; ssh: suppression subtractive hybridization; database: information deposited in genbank and never reported in publication. note the numerous discoveries and the diversity of investigated species in the last years. helix and 2 c-terminal anti-parallel β-sheets (bonmatin et al., 1992), in contrast to the absence of α-helix in mammalian defensin. the term “plant defensins” introduced in 1995 regarding both mono and dicotyledon species (terras et al., 1995) added confusion, although related to insect defensins due to a cystine-stabilized α-helix motif. defensin the first molluscan defensin has been reported in 1996 from the mediterranean mussels m. galloprovincialis following purification using several steps of hplc applied to acidified plasma supernatant (hubert et al., 1996a). the first stepwise elution on c18 cartridge with 40 % acetonitrile resulted in several fractions displaying inhibitory growth activity against both escherichia coli and micrococcus luteus (lysodeikticus). the subsequent separations of the pooled active fractions on c8 then on c18 columns resulted in a pure 4 kda peptide named mgd-1. mgd-1 showed antibacterial activity also towards several vibrio 87 species. rimary-structure analysis revealed 39 aa including 8 cysteines and a modified undetermined aa residue in position 28. the presence of 2 extra cysteines and of one modified aa suggested that mgd-1 represented a new member of the arthropod defensin family. the same year, several cysteine-rich amps were isolated from the blood of immune-challenged mussels, m. edulis (charlet et al., 1996). the general strategy included solid phase extraction performed on acidified plasma supernatant, followed by hplc on aquapore od 300 column, and gel permeation hplc on ultra-spherogel sec 3000 and sec 2000 columns. final hplc purification step was on aquapore od 300 column. each fraction was assayed for antibacterial (e. coli and m. lysodeikticus) and antifungal (neurospora crassa) activities resulting in the partial characterization of 2 arthropod-like defensins, peptide a and b, containing 6 cysteines. labeled by mouse antiserum directed against synthetic defensin mgd-1 observed with confocal and electron microscopy, defensins were located in nearly half the circulating hemocytes: 16 % contained defensin alone and 32 % contained both defensin and mytilin (mitta et al., 2000c). such original and complex repartition reinforced the hypothesis on the existence of specialized hemocyte sub populations, never solved. despite long efforts regarding oysters only suggesting the presence of amps (hubert et al., 1996b), it was in 2005 that the first defensin-like, named american oyster defensin (aod), has been purified from boiled acidified gill extract supernatant of c. virginica (seo et al., 2005). the supernatant was initially purified by preparative continuous acidurea-polyacrylamide gel electrophoresis (cau– page). anti-e. coli activity was monitored during the 12.5 h of elution. final step of purification was by reverse phase hplc through c4 column. aod had 38 aa, including 6 cysteines, possessed a strong activity against gram-positive (lactococcus lactis and staphylococcus aureus) and gramnegative (e. coli d31 and v. parahemolyticus) bacteria, and high sequence homology to arthropod and mussel defensins. three-dimensional solution structure of mussel and oyster defensins was established analyzing purified native defensins, completed by synthetic or recombinant defensins, with a combination of matrix-assisted laser desorption/ionization time-offlight (maldi-tof), electro-spray ionization mass spectroscopy and nuclear magnetic resonance in spectroscopy with respect to hydrogen-1 nuclei (1hnmr). mgd-1 showed an arthropod defensins common cystine-stabilized alpha-beta motif (csαβ), but present an additional disulfide bond. the structure is characterized by an nh2-terminal α-helix followed by 2 anti-parallel �-strands connected by distorted loops and constrained by 4 disulfide bonds (yang et al., 2000). both oyster c. gigas and mussel defensins appeared with similar global fold but differed by the size of their loops and by the presence of 2 aspartic residues in cg-def (gueguen et al., 2006). interesting, the nonapeptide corresponding to loop 3 connecting the 2 β-sheets of mgd-1 retained antibacterial and antifungal activities as demonstrated using synthetic peptides modified to maintain the 3-dimensional structure by creating a non-native disulfide bond (romestand et al., 2003). the same nonapeptide was also found to be anti protozoa (trypanosoma brucei and leishmania major) and preventing the in vitro infection by virus hiv-1, but non-cytotoxic for human magic-5b cells (roch et al., 2004). increasing the net positive charge of the nonapeptides by selected aa replacements resulted in higher activities, whatever the target. on the opposite, unfolded linear sequences were not active revealing that the 3-dimensional hairpin structure is essential for activity. mytilin during the hplc process applied to m. edulis plasma and leading to defensin purification (see above), 2 isoforms of a novel peptide of 34 aa including 8 cysteines, with potent bactericidal activity named mytilins (isoform a and b) have been isolated (charlet et al., 1996). using similar hplc purification applied to m. galloprovincialis hemocyte granules, mytilin-b was recovered along with new isoforms c, d and g1 (mitta et al., 2000b). although these isoforms share a high degree of homology in their primary structure, including the canonical cysteine array, they exhibited complementary antimicrobial properties against distinct pathogens, including gram-positive and gram-negative bacteria, fungi and protozoa. also the kinetics of bacteriolytic effect was different according to the isoform, ranged from less than 3 min to 6 h. existence of multiple mytilin isoforms, engaged in different biological activities, suggested that mussels possess an extended arsenal of molecules to counter microbial infestation. confocal microscopy analysis using rabbit antimytilin-b antiserum demonstrated that mytilins are stored in 69 % of m. galloprovincialis circulating hemocytes, whatever alone (37 %) or in addition to defensin in different granules (11 %) or in the same granules (21 %). mytilin expressing hemocytes were particularly abundant in sinuses of adductor muscle, epithelia of gills, digestive gland and intestine (mitta et al., 2000c). they exerted their microbicidal effect within the cells through the process of phagosomemytilin granule fusion leading to the co-location of ingested bacteria and mytilins (mitta et al., 2000d). white spot syndrome virus (wssv), incubated with mytilin-b prior to injection into the shrimp, palaemon serratus, was no longer able to kill the shrimps (dupuy et al., 2004). in the absence of 3dimensional structure, one 10 aa fragment derived from the hypothesized loop 3 of mytilin-b, has been synthesized. it included 2 cysteines and its folding was supposed to form a stable β-hairpin structure. as obtained with complete mytilin-b, such synthetic peptide was able to reduce the shrimp mortality due wssv when incubated in vitro with the virus prior injection. the 3-dimensional structure of m. galloprovincialis mytilin-b has been established. it resembles the cs-αβ motif of defensin mgd-1, involving the same pattern of cysteine bonding (roch et al., 2008). one linear and 3 cyclic synthetic fragments corresponding to loop 3 sequence of mytilin-b have been synthesized. only the fragment 88 of 10 aa constrained by 2 disulfide bonds in a stable β-hairpin structure was able to prevent the shrimp mortality due to wssv. it was confirmed that the interaction occurred at the virus membrane level, and this in less than a min. in addition, such fragment inhibited the growth of several vibrios, micrococcus lysodeikticus and e. coli. on the opposite of what it has been reported with defensin fragments, increasing the positive net charge did not reinforce the antibacterial activity. moreover, it completely suppressed the antiviral one (roch et al., 2008). in a study involving several amps, synthetic mytilin-a has been assayed for anti protozoan parasite activity. no parasiticidal effect was observed, as well as any inhibition of trypanosoma cruzi growth. in contrast, leishmania braziliensis growth was inhibited by mytilin-a, since the first cell cycle and even after 3 cell cycles (lofgren et al., 2008). native mytilin-a purified from m. edulis, presented some activity against several human virus when added simultaneously with the cell target (vero, hep-2 and ma104 cells). however, replication of the herpes simplex virus (hsv-1) was completely inhibited only when the virus was preincubated with mytilin-a (carriel-gomes et al., 2007) suggesting direct interaction of the peptide at the virus membrane, as reported for mytilin-b and synthetic fragment (roch et al., 2008). myticin in 1999, a novel amp has been isolated from unchallenged m. galloprovincialis. named myticin, such amp was purified using 3 consecutive steps of hplc on c18 c8 c18 columns using linear gradients of acetonitrile, followed by edman degradation by electro spray ionization on mass spectrometer. two isoforms were obtained: myticina from hemocyte granules and cell-free plasma, and myticin-b only from hemocyte granules (mitta et al., 1999a). a partial sequence of 36 aa of myticin-a revealed the presence of 7 cysteines suggesting relationships with previously reported mussel amps. only 7 nh2-terminal aa sequence of myticin-b has been obtained and complete sequences were deduced from molecular biology data (see below). myticins showed antimicrobial activity against several gram-positive bacteria (m. lysodeikticus, bacillus megaterium, enterococcus viridans), but not against marine (vibrios, salmonellas) or nonmarine (brucella suis) gram-negative bacteria or protozoa (perkinsus marinus). only myticin-b inhibited the growth of the fungus, fusarium oxysporum (mitta et al., 1999a). mytimycin a cysteine-rich peptide, named mytimycin, has been isolated from the plasma of immunechallenged and untreated mussels m. edulis using reversed phase hplc purification. of an estimated molecular mass of 6.2 kda, mytimycin was strictly antifungal, inhibiting the growth of neurospora crassa and fusarium culmorum (charlet et al., 1996). only 32 aa from nh2-terminal sequence, corresponding roughly to half of the sequence, was released. reduction and alkylation indicated the possible presence of 12 cysteines, a unique situation regarding amps. miscellaneous a potent antibacterial histone h2b protein of 13.9 kda (123 aa) has been isolated from boiled acidified gill extract supernatant from c. virginica, and named cvh2b-1 (seo et al., 2010). only partial internal aa sequences have been released, confirming the similarity with c. gigas, m. edulis and m. galloprovincialis histone h2b counterparts. recently, 3 isoforms named cvh2b-2, -3 and -4 have been reported (seo et al., 2011). although active against gram positive, l. lactis, cvh2b-2 is inactive against s aureus. both cvh2b-1 and -2 had potent activity against the gram-negative oyster pathogen v. parahemolyticus as well as against the human pathogen v. vulnificus. in addition, cvh2b-3 and -4 had similarly strong activity against v. vulnificus. estimated concentration of histone h2b isoforms found normally within the oyster, were exceeding the in vitro inhibitory concentrations for vibrios, revealing their potent protective function. the big-defensin rpd-1 has been purified by a combination of precipitation, gel-exclusion and cation-exchange chromatography from the plasma of the manila clam ruditapes philippinarum (wei et al., 2003). of 24.8 kda, 11 nh2-terminal aa sequence has been published with no known homology at that time. activity was on gram-positive and gram-negative bacteria. a proline-rich amp of 5.1 kda has been purified by several hplc runs on c18 columns performed on hemocyte organelles collected from the chilean scallop argopecten purpuratus. the sequence of the 42 nh2-terminal aa did not contain any cysteine, but presented some homology with drosophila melanogaster ceratotoxin and human defensin hdb1 (arenas et al., 2009). one synthetic peptide of 31 aa has been designed in order to enhance hydrophobicity and to increase cationicity. both natural and synthetic peptides were clearly antifungal (f. oxysporum, saprolegnia parasitica, n. crassa) with weak effect against bacteria and no cytotoxicity toward fish cell line. increasing the positive net charge resulted in strong increase of antifungal activity confirming the importance of electrostatic interactions to develop the activity. littorein, an amphipathic cationic peptide of about 3.5 kda, has been observed in the plasma of the marine gastropod, littorina littorea (defer et al., 2009a). the peptide was prepared by hplc on c18 column, but not isolated in pure form. bactericidal activity against m. lysodeikticus and anti viral activity against hsv-1, were reported. the same authors investigated the same activities in the hemolymph of commercially important bivalves and gastropods. the broadest antibacterial activity was found in the bivalve, cerastoderma edule, whereas the highest activity was found in the oyster, ostrea edulis (defer et al., 2009b). also antiviral activity, but not cytotoxicity, was detected in c. edule. acid extracts from the other examined species were cytotoxic and presented less antiviral capacity then c. edule. as no pure fractions have been obtained, the molecular supports of activities remain unknown. 89 antiviral activity of the abalone haliotis laevigata was assessed against hsv-1 using a plaque assay technique (dang et al., 2011). hemolymph and lipophilic extract of the digestive gland substantially decreased the number and size of plaques. antiviral activity of hemolymph was rapid, in contrast to antiviral activity of lipophilic extract, which was greater when added 1 h after infection, suggesting that abalone have at least two antiviral compounds with different modes of action. computer-based evidences in the last decade, biochemical approaches were rapidly submerged by molecular biology-driven investigations, i.e. addressing mrna and genomic dna sequences. not only were the origins of rna diverse, from whole animal to dissected tissues or hemocytes, but also the techniques used: est, sage, ssh, dgge, 454 pyrosequencing, illumina, high-density microarrays, etc. regarding small proteins, frequently present in small quantities, including multiple isoforms with different biochemical properties, a real breakthrough was introduced by the molecular biology techniques, revealing amps in all the invertebrates analyzed so far. defensin using back-translation of the aa sequence from m. galloprovincialis mgd-1, a new isoform, mgd-2, has been obtained from a cdna library (mitta et al., 1999b). the signal peptide of 21 aa is followed by 39 aa of the mature defensin and 21 aa of the cterminal extension. the presence of 8 cysteines engaged in 4 intra chain disulfide bonds did not match with the 6 cysteines reported for m. edulis defensins peptide-a and -b (charlet et al., 1996). mgd-1 and -2 precursors were predominantly expressed in hemocytes. mature defensins were located in vesicles of hemocytes containing small granules, in large clear granules of another subclass of hemocytes and in some enterocytes. challenge with heat-killed vibrio alginolyticus triggered the plasmatic increase of mgd-1 concentration. the gene coding mgd-2 has been cloned and sequenced: one intron interrupted the signal peptide and another intron is located within the c-terminal extension (mitta et al., 2000a). remarkable was the fact that the sequence coding for the mature defensin is not interrupted. analyzed in southern blot, the defensin gene appeared present as a single copy in the genome. recent studies performed in real-time pcr shown that mgd-1 gene expression, the less expressed gene in untreated m. galloprovincialis compared to mytilin and myticin, was highly dependent on the season with higher expression in summer (li et al., 2009b). in addition, its expression was up regulated only by injection of v. splendidus (1 to 24 h) (cellura et al., 2007). using est libraries, the first oyster defensin was isolated from the mantle of the pacific oyster c gigas and named cg-def (gueguen et al., 2006). the deduced aa sequence starts with a 22 aa signal peptide followed by the 43 aa of the mature peptide. on the opposite of mussel defensins, cg-def precursor does not contain c-terminal extension. meanwhile, its genomic organization is similar to that of the mussel defensin genes. the in vitro antimicrobial effect of recombinant cg-def was tested in conditions mimicking the seawater environments. activity was directed against grampositive bacteria but no or limited activity was observed against gram-negative bacteria and fungi. cg-def in solution shares the cystine-stabilized alpha-beta (cs-αβ) motif with arthropod defensins, with an exception of the presence of an additional disulfide bond. this structure was similar to that of mussel defensin mgd-1 (romestand et al., 2003). however, cg-def and mgd-1 structures could be distinguished by the size of their respective loops and by the presence of two aspartic residues in cgdef. expression of cg-def gene was mainly in mantle edge as revealed by mass spectrometry analyses. cg-def mrna concentration was unchanged after bacterial challenge, which could be explained by its high level of continuous expression (gueguen et al., 2006). one year later, two new defensin isoforms, cg-defh1 and -2, were identified by cdna cloning technology from circulating hemocytes of c. gigas. after in silico translations, the precursor was of 73 aa, 43 of which representing the mature peptide. cg-defh1 and cgdefh2 aa sequences shared 86 % of identity and they belong to the "arthropod-molluscs defensin family". similarly to the expression behavior of cgdef in mantle, cg-defh2 is continuously expressed in hemocytes. in addition, the level of cg-defh2 transcripts decreases dramatically in circulating hemocytes following bacterial challenge. correlation with an increase in gills and mantle suggested migration of hemocytes expressing cg-defh2 towards tissues considered as representing the first defense barrier (gonzalez et al., 2007). sequence alignment of cg-defh1 and -2 suggested they aroused from a multigenic family displaying variety of gene structures (schmitt et al., 2010). consequently, different antimicrobial peptides from molluscs appeared to have been subject to distinct patterns of diversification. another economically important oyster species, c. virginica, was used to construct large-insert genomic bacterial artificial chromosome (bac) library. the library of 55,296 clones was screened with probes derived from selected oyster genes, including defensins, using high-density filter arrays. seven defensin positive clones were identified in this bac library which can be grouped in 2 clusters, probably resulting from only one gene copy (cunningham et al., 2006). some information recently emerged regarding the hydrothermal-vent mussel, bathymodiolus thermophilus. suppression subtractive hybridization (ssh) libraries were used to reveal the presence of defensin which expression is regulated by temperature (boutet et al., 2009). triangle snail mussel, hyriopsis cumingii, is the most important mussel species producing freshwater pearls in china. two defensins analogous to the c. gigas ones were identified among 1,165 singletons generated by est technology (bai et al., 2009). among the genes newly identified from an est library constructed from the whole body except 90 digestive tract and intestine of the bay scallop, argopecten irradians irradians, 10 ests out of 2,853 sequences were related to defensin (song et al., 2006). they were organized into 2 groups: one of 3 ests with a size of 518 nucleotides, the other of 7 ests with a size of 824 nucleotides. no more details were released. several genes involved in the defense against the parasite qpx (quahog parasite unknown) were identified in the hard clam, mercenaria mercenaria, using ssh libraries constructed from hemocytes (perrigault et al., 2009). one big-defensin and one hemocyte defensin homologous to the one of c. gigas were discovered. quantitative pcr revealed that the gene from hemocyte defensin was upregulated 14 to 48 days in gills after experimental infestation. defensins were also identified in gastropods. regarding the abalone, haliotis discus hannai, the major cultured species in china, sequence homology analysis of the 42 defensin-related sequences identified from an est library constructed from liver and kidney, revealed high similarity with arthropod defensins, including the 6 cysteines. non-expressed in untreated abalone, the defensin gene is triggered only in hepatopancreas after injection of v. anguillarum or s. aureus (hong et al., 2008). complete study of defensin has been performed in the disk abalone, haliotis discus discus, starting from cdna library constructed from whole tissues to tissue specific expression and regulation after bacterial challenge (de zoysa et al., 2010). the pro-defensin comprised a putative signal peptide of 18 aa followed by the mature defensin of 48 aa. the presence of 6 cysteines, the putative three disulfide bonds and 3-dimensional structure, joined to phylogenic relationship, suggested disk abalone defensin is related to arthropod defensins. constitutively expressed mainly in mantle and hepatopancreas, expression was up regulated principally in hemocytes 3 6 h and 24 48 h postchallenge with a mixture of bacteria mytilin the nucleotide sequence of mytilin-b precursor from m. galloprovincialis, one of the 5 mytilins purified by hplc, was obtained from a hemocyte cdna library (mitta et al., 2000b). the precursor starts by a signal peptide of 22 aa followed by the mature mytilin-b of 34 aa including 8 cysteines, and ended with 48 aa from the c-terminal extension. the gene coding for mytilin-b has been cloned and sequenced. its structure is similar to the mgd-2 one: one intron interrupted the signal peptide and another intron is located within the c-terminal extension (mitta et al., 2000a). analysis in southern blot suggested the presence of one copy of mytilinb gene per genome, with possibly another gene displaying homology. such last observation is in agreement with the evidence of numerous variants of mytilin (see below). the extended variability of mytilin has been investigated from circulating hemocytes focusing on mytilin-b (parisi et al., 2009). one mussel expressed simultaneously 2 to 10 different mytilin-b mrnas as observed in denaturing gradient gel electrophoresis (dgge), defining 10 individual dgge patterns. nucleotide substitutions were observed at specific locations, mainly within cterminus and 3’utr, leading to 36 nucleotide sequence variants and 21 different coding sequences segregating in 2 different clusters. most of the nucleotide substitutions were silent, and the number of pro-peptide variants was restricted to 4. finally, as the 2 aa replacements occurred within the c-terminus, mytilin-b mature peptide appeared unique. multiple sequencing of partial mytilin-b gene from one mussel revealed that 1 to 4 randomly distributed nucleotide substitution points occurred within intron-3. only one sequence out of the 91 analyzed contained 16 nucleotide substitution points. in addition, this sequence was the only one containing 4 out of the 6 nucleotide substitution points occurring within exon-4, that codes for most of the c-terminal domain, including the unique aa replacement. in addition, mytilin-b locus did not appear to be under directed selection (boon et al., 2009), a situation contradictory to myticin-c. mytilin-b gene expression slightly varied in m. galloprovincialis according to the season (li et al., 2009b). however, bacterial challenges modified its expression in hemocytes. strongly up regulated by v. anguillarum (24 48 h), expression was down regulated by v. splendidus and m. lysodeikticus (cellura et al., 2007), a situation confirmed by li et al. (2010), except regarding v. anguillarum. in a study considering also myticin, a mytilin of 34 aa, similar to the mussel counterpart, has been characterized in the clam, ruditapes decussates, using ssh technique (gestal et al., 2007). the clam mytilin differs by only 2 aa from mussel mytilin-c. expression level of clam mytilin increased 24 h and 48 h post-injection with the mixture of dead bacteria (m. lysodeikticus, v. splendidus, v. anguillarum) and on those injected with live v. anguillarum, as measured by real-time pcr. expression patterns were similar and confirmed the higher expression level at 48 h post-injection, as suggested by ssh. one mytilin-c complete mrna sequence isolated from the bay mussel m. trossulus, hemocytes has been deposited in genbank in 2004 (accession number ay730626). similarly, mytilin-a and -b complete mrna sequences from m. coruscus have been deposited in genbank in 2010 (accession numbers fj973154 and 5), together with mytilin-3 to -8 complete mrna sequences (accession numbers gu324718 to 23). regarding the deep-sea hydrothermal vent mussel, bathymodiolus azoricus, degenerated primers have been used in rt-pcr to evidence mytilin transcript in gills (bettencourt et al., 2007). mytilin mrna has been located in hemocytes underlying gill epithelium and its expression was induced by exposure to v. parahemolyticus (bettencourt et al., 2009) myticin myticin precursor nucleotide sequences of m. galloprovincialis had been established by cloning based on partial aa sequences. they consist of 96 aa with a signal peptide of 20 aa, followed by the mature myticin of 40 aa including 8 cysteines arranged in an original array, and a 36 aa cterminal extension (mitta et al., 1999a). southern 91 blot analysis suggested myticin gene is present as a single copy in the mussel genome. hemocytes are the main site for precursor production of myticins. nevertheless, mantle, labial palps, gills and digestive glands showed a faint band with the same mobility as that of hemocytes in northern blotting. myticin-b transcript has been observed by using in situ hybridization (ish) on 16 % of circulating hemocytes from untreated mussels, identified as small granulocytes. such expression pattern was not modified by bacterial challenges (li et al., 2010). myticin-b gene is subject to variation of expression according to season with lower expression in winter as observed during 3 consecutive years (li et al., 2009b). expression was also down regulated by injection of v. splendidus (1 to 12 h), and m. lysodeikticus (1 to 72 h), and to a less extent of v. anguillarum (1 to 9 h) (cellura et al., 2007). in a comparative study involving m. galloprovincialis from different geographical areas, myticin-b gene expression was also found down regulated, but with different kinetics following bacterial challenges and origin (li et al., 2010). first in silico data on myticin was obtained by production and sequencing of 3’end-specific est from multiple tissues of unchallenged, m. galloprovincialis (venier et al., 2003). a variety of genes potentially involved in stress responses, including the cysteine-rich peptide precursor myticin-a, were identified. later, primary and ssh libraries were prepared from hemolymph of m. galloprovincialis after heat-inactivated bacteria or poly i:c challenge. the 232 ests matching with myticin-a and -b displayed considerable sequence variability and revealed a third cluster proposed as myticin-c. analysis of the translated myticin-c yielded 74 and 25 variants of the precursor and active peptide, respectively (pallavicini et al., 2008). the individual sequences of myticin-c are unique for each mussel, independently of the geographic origin, age, sex or gonad maturation. comparative analysis of genomic and cdna sequences from the same individual showed that all detected variants shared a very high homology with the most frequent genomic isoforms, suggesting that variations were generated from the most common sequences (costa et al., 2009). meanwhile, the genetic mechanism(s) that maintains such high diversity is poorly understood. it has been suggested that the high sequence variability of myticin transcripts in m. galloprovincialis hemocytes reflected ancient hostpathogen interactions (pallavicini et al., 2008). using phylogeny-based models of sequence evolution and several statistical tests for neutrality, the role of natural selection process in the evolution of multiple variants of myticin-c has been confirmed. statistical tests rejected the hypothesis that all polymorphism within myticin-c loci is neutral (padhi and verghese, 2008), a result on the opposite to the analysis of mytilin-b polymorphism (parisi et al., 2009). analysis of differentially expressed genes in response to bacterial challenge in hemocytes of the carpet-shell clam, ruditapes decussatus, using ssh technique resulted in the identification of clam myticin-1, -2 and -3 (gestal et al., 2007). clam myticins share the cysteine array defining the 4 disulfide bonds which are the signature of the mussel myticin family. however, they constitute a separated sister taxon. the challenge with dead bacteria mixture or with alive v. anguillarum resulted in maximum 2xfold up-regulation of the clam myticins 48 h post challenge. here also it was suggested that the variations in aa sequences might be a response to the recognition of different pathogens. nine myticin complete mrna sequences, named myticin-1 to -9, isolated from mytilus coruscus have been deposited in genbank in 2010 (accession numbers gu324724 to 32). mytimycin such strictly antifungal peptide from m. edulis was reported in 1996 as partial nh2-terminus 33 aa sequence (charlet et al., 1996). mytibase, the first species-specific catalogue of m. galloprovincialis ests publicly available at http://mussel.cribi.unipd.it, has been constructed from different m. galloprovincialis tissues, including gills, mantles and hemocytes (venier et al., 2009). using bioinformatics, several hits related to mytimycin were identified. primers designed from a consensus sequence have been used to obtain the complete cds of 456 nucleotides. mytimycin precursor is constituted by a signal peptide of 23 aa, followed by the mature mytimycin of 54 aa and a c-terminal extension of 75 aa (sonthi et al., 2011). only 2 major aa precursor sequences emerged according to the geographical origin of the mussels: one shared by m. galloprovincialis from venice-italy and vigo-spain, and by m. edulis, the other belonging to m. galloprovincialis from montpellier-france, with 9 aa difference between the two mature mytimycins. the suspected presence of 12 cysteines has been confirmed and predicted disulfide bonds suggested the presence of 2 constrained domains joined by a 4 aa tract. mature mytimycin appeared more closely related to mytilin than to any other antibacterialantifungal peptide, possibly due to the c-terminal domain. intriguing was the presence of conserved canonical ef-hand-motif located in the c-terminal extension. mytimycin gene of m. galloprovincialis is interrupted by 2 introns. intron 2 presented 2 forms, a long one and a short one resulting from the removal of the central part of the long one, both being simultaneously present in each mussel (sonthi et al., 2011). miscellaneous two clusters from est library constructed from hemocytes of the gastropod, biomphalaria glabrata, maintained in non-axenic conditions showed sequence similarity to theromacin, the amp of the annelid leech, theromyzon tessulatum, particularly the cysteine array (mitta et al., 2005). the first proline-rich amp from mollusc, cg-prp, was identified from the oyster c. gigas. the cdna sequence codes for a 61 aa precursor with homologies to proline-rich amp, and was composed of a hydrophobic signal peptide of 37 aa followed by mature cg-prp, composed of an acidic region and a cationic proline-rich region. expression of cg-prp is restricted to hemocytes and induced following 92 http://mussel.cribi.unipd.it/ bacterial challenge. as cg-prp was located by immune cytochemistry in some hemocytes together with defensins, antimicrobial activity was suspected to be through synergy with defensins (gueguen et al., 2009). histone h2a was reported to participate in host defense response through producing novel amp from its n-terminus in the scallop, chlamys farreri. homology cloning and genomic dna walking have been used to identify the open reading frame of 375 bp coding for a polypeptide of 125 aa (li et al., 2007). as expression in hemocytes was not modified by bacterial challenge, it was concluded that h2a did not participate in immune response directly. partial recombinant h2a, restricted to 39 nterminal aa, has been produced. its antibacterial activity was 2.5 times higher toward gram-positive bacteria (m. lysodeikticus) than toward gramnegative bacteria (v. splendidus, v. anguillarum, v. vulnificus). histone h2a full-length cdna was cloned from disk abalone, haliotis discus discus, and a 40 aa amp, named abhisin, was identified from the nterminus of h2a sequence (de zoysa et al., 2009). abhisin shares 80 % aa identity with the buforin i from asian toad histone h2a. antimicrobial activity was investigated on synthetic abhisin, revealing growth inhibition of gram-positive (listeria monocytogenes), gram-negative (vibrio ichthyoenteri) bacteria, and fungi (yeast pityrosporum ovale). additionally, abhisin has cytotoxic activity against cancer cells but not against normal cells. h2a gene expression was significantly induced at 3 h post-infection with bacteria in abalone gills and digestive tract. so-called big-defensins have been identified using various techniques in several bivalves. they are proteins composed of an n-terminal hydrophobic sequence followed by the c-terminal cationic defensin portion, similar to those of mammalian defensins, particularly the disulfide array, but different from the «arthropod-mollusc defensins» (see above). the first one was deposited in 2001 as cdna from the oyster, c. virginica (genbank accession number bg624524). the second, named aibd, was released from the bay scallop, a. irradians, also as a cdna coding for a signal peptide of 28 aa followed by the mature peptide of 94 aa sharing homologies with bigdefensins from arthropods and amphioxus (zhao et al., 2007). constitutive expression in hemocytes was up regulated in the first 8h after injection of v. anguillarum, followed by a drastic increase at 32 h post-injection. in addition, recombinant aibd inhibits the growth of gram-positive and gram-negative bacteria, and possesses strong fungicidal activity. existence of a third big-defensin has been reported in the hard clam, mercenaria mercenaria after experimental challenge with the protistan parasite qpx (quahog parasite unknown) (perrigault et al., 2009). its expression was significantly induced in gills at 48 days post-infection. the fourth bigdefensin, vpbd, belongs to hemocytes of the manila clam, venerupis philippinarum. the precursor is composed of a signal peptide of 20 aa followed by the active molecule of 74 aa with a positive net charge of +5 and 36 % of hydrophobic residues. as others defensins, vpbd possesses remarkable microbicidal activity against grampositive and gram-negative bacteria, and showed strong fungicidal activity towards yeast (zhao et al., 2010). its expression was up regulated during the first 24 h after v. anguillarum challenge and its activity was directed toward a broad spectrum of bacteria species. two other amps from the oyster, c. gigas, gigasin-2 and -3, have been deposited in genbank in june 2003 under the accession numbers aj582629 and aj582628 respectively. alignments of the deduced aa sequences revealed that gigasin-2 is identical to defensin cg-defh2 and gigasin-3 closely related to cg-defh1 (gonzalez et al., 2007). surprisingly, some authors did not report the presence of amp-related sequences from cdna libraries constructed from different bivalves, such the ones from c. gigas and c. virginica (jenny et al., 2007), c. gigas, m. edulis, r. decussatus and bathymodiolus azoricus (tanguy et al., 2008), laternula elliptica (park et al., 2008), c. gigas (roberts et al., 2009) and chlamys farreri (wang et al., 2009). such apparent absence of existing amps highlights the limits of est technology, which is obviously non-exhaustive. concluding remarks it is remarkable that, on the opposite to the variety of structures characterizing arthropods amps, mollusc amps reported so far belong to a unique molecular family, i.e., the cs-αβ, with the exception of proline-rich amp from the chilean scallop, a. purpuratus, which did not contain cysteine. the 70’s have been the era of biological activitydriven technology completed by biochemical identification of the active molecules. such approach lead to the discovery of new aa sequences displaying antimicrobial activities and revealed unknown peptide frameworks with antibiotic capabilities. multiple molecules were discovered revealing the importance and universality of amps and establishing the basis of unsuspected innate immune mechanisms. since the mid-90’s, the field of amp exploded due to molecular biology techniques, confirming their existence in all the living creatures, including plants and bacteria. regarding only molluscs, several databases were made available, in addition to number of est libraries constructed for various purposes. nowadays, bio-informatic data are so numerous that data mining is applied also to molluscs sequences (yu and li, 2008; wang et al., 2008). meanwhile, nucleotide sequences from genomes or ests in themselves cannot demonstrate biological pathways, and functional assays must be performed to confirm, species by species, challenge by challenge, the real involvement of amps in anti infectious defense. only few mollusc families were investigated for amps, mainly the bivalve mytilidae, ostreidae and pectinidae (table 2). some information came from gastropods (haliotidae) but no one from cephalopods or polyplacophors. in addition, routinely applied molecular biology techniques 93 table 2 number of reported amps within the major classes of the mollusc phylum note that only 2 classes have been investigated and the discrepancy between the number of species and the number of reported amps in gastropods versus bivalves. allowed reporting amps only if closely-related sequences were already deposited in databases and mined by computer. in other words, molecular biology techniques alone cannot reveal new amp structures, probably underestimating the diversity of such universal immune effectors. biochemical purification linked to biological activity of isolated fraction represents the main approach capable to reveal new structures. meanwhile, sequence mining might represent an alternative approach which, using rational prediction models (torrent et al., 2011) and various algorithms like random forests (rf), 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(tokyo) 37: 2179-2182, 1989. yang ys, mitta g, chavanieu a, calas b, sanchez jf, roch p, et al. solution structure and activity of the synthetic four-disulfide bond mediterranean mussel defensin (mgd-1). biochemistry 39: 14436-14447, 2000. yu h, li q. exploiting est databases for the development and characterization of est-ssrs in the pacific oyster (crassostrea gigas). j. hered. 99: 208-214, 2008. zhao j, li c, chen a, li l, su x, li t. molecular characterization of a novel big defensin from clam venerupis philippinarum. plos one 5, e13480, 2010. zhao j, song l, li c, ni d, wu l, zhu l, et al. molecular cloning, expression of a big defensin gene from bay scallop argopecten irradians and the antimicrobial activity of its recombinant protein. mol. immunol. 44: 360-368, 2007. 97 isj 9: xx-xx, 2012 isj 9: 48-57, 2012 issn 1824-307x research report purification and biochemical properties of a salivary α-amylase in andrallus spinidens fabricius (hemiptera: pentatomidae) 48   a zibaeea, h hodaa, m fazeli-dinanb, c adepartment of plant protection, college of agriculture, university of guilan, rasht, 41635-1314, iran bdepartment of biological control, national institute of plant protection, amol, iran cdepartment of plant protection, college of agriculture and natural resources, university of tehran, karaj 31584, iran accepted february 29, 2012 abstract α-amylase is one of the enzymes that has crucial role in extra-oral digestion (eod) of hemipteran insects. an α-amylase was purified and biochemically characterized from the salivary glands of andrallus spinidens showing its considerable role in eod process. it was found an enzyme by specific activity of 4.22 u/mg protein, recovery of 14.67 % and purification fold of 13.83-fold as well as molecular weight of 26 kda. by using two buffer solutions, optimal ph of the purified α-amylase was found to be 9 for both universal and tris-hcl buffers. our findings revealed that the purified α-amylase had the highest activity at the temperatures of 35 and 40 °c, and were stable for 96 h at these temperatures. kinetic parameters of the purified enzyme show that both starch and glycogen, are the suitable substrates for the enzymatic assay, but a lower km demonstrated glycogen as a more appropriate substrate. among the cations used to show their possible involvement in active site of the enzyme, ca2+ 2+, mg and one concentration of cu2+ increased the α-amylase activity but na+ decreased the enzyme activity. triton x-100 increased the enzyme activity but sds, edta, egta and ttha decreased it, indicating involvement of metal ions in the active site of the purified α-amylase. key words: α-amylase; salivary gland; andrallus spinidens   introduction extra-oral digestion (eod) is a tool used by the majority of terrestrial predaceous arthropods to feed on relatively large preys. these predators obtain prey extraction and nutrient by refluxing or nonrefluxing application during injection of hydrolytic enzymes (cohen, 1995). abbreviation of prey handling time and increase in nutrient density of food are two major advantages for eod allowing small predators to consume preys larger than their body size. the basis of eod is a highly coordinated combination of biochemical, morphological, and behavioral adaptations that vary with different taxa (cohen, 1995). both nymphs and adults feed on several caterpillars like chilo suppressalis walker (lepidoptera: crambidae), naranga aenescens moore (lepidoptera: noctuidae) and helicoverpa armigera hübner (lepidoptera: noctuidae) (mohaghegh and najafi, 2003). najafinavaee et al. (1998) reported that a. spinidens is a specific caterpillar predator of rice fields in northern iran that has five generations per year having a critical role in regulation of rice pests’ population. proteases, lipases and α-amylases are the three major enzymes involved in eod of hemipterans. our findings in a previous study demonstrated that salivary glands of a. spinidens have two anterior, two lateral and two posterior lobes (zibaee et al., 2012a). general proteolytic activity in the salivary glands demonstrated optimal ph of 8 and optimal temperature of 40 °c when azocasein was used as substrate. by using specific substrates, it was found that trypsin-like, chymotrypsin-like, aminopeptidase and carboxypeptidase are the active proteases in the salivary glands of a. spinidens by maximal activity of trypsin-like protease in addition to their optimal ph of 8-9 (zibaee et al., 2012a). also, we characterized a andrallus spinidens is a potential bio-control agent of caterpillars that has been widely distributed around the world (nageswara rao, 1965). the insect has especially reported as a potential predator of rice pests in india, malaysia and iran (nageswara rao, 1965; manley, 1982; mohaghegh and najafi, 2003). _____________________________________________________________________________________________________________________________ corresponding author: arash zibaee department of plant protection, college of agriculture university of guilan, rasht, 41635-1314, iran e-mail: arash.zibaee@gmx.com mailto:arash.zibaee@gmx.com 49   tag-lipase from salivary glands of a. spinidens that had optimal ph and temperature of 9 and 40 °c, respectively (zibaee et al., 2012b). α-amylases (α1,4-glucan-4-glucanohydrolases; ec 3.2.1.1) are the hydrolytic enzymes highly widespread in nature. they catalyze the hydrolysis of α-d-(1,4)-glucan linkages in glycogen, starch and other related carbohydrates (strobl et al., 1998; franco et al., 2000). regarding eod importance in hemipterans especially a. spinidens as biocontrol agent, we have initiated a comprehensive study along with two other researches (zibaee et al., 2012a, b). so, the aims of the current study were the complete purification and characterization of salivary α-amylase to find its role in eod process of preys. materials and methods andrallus spinidens rearing colony of andrallus spinidens was established by adults collected from harvested rice fields in amol (mazandaran, north of iran), late september 2011. insects was reared on late instars of c. suppressalis l. (lepidoptera: crambidae) as prey and provided with wet cotton plugs fitted into small plastic dishes (2.5 cm diameter) as moisture sources at 25±1 °c and 80 % of relative humidity as laboratory conditions. sample preparation adults were randomly selected and the salivary glands were removed by dissection under a stereo microscope in ice-cold saline buffer 10 mm. bodies were cut separately by a scalper and salivary glands appeared. salivary glands were separated from the insect body, rinsed in ice-cold distilled water, placed in a pre-cooled homogenizer and grounded before centrifugation. an equal portion of tissue and distilled water were used to have a desirable concentration of the enzyme (w/v). homogenate were separately transferred to 1.5 ml centrifuge tubes and centrifuged at 13,000 rpm for 15 min at 4 °c. the supernatant were pooled and stored at -20 °c for subsequent analyses (zibaee et al., 2012a). α-amylase assay α-amylase activity was assayed by dinitrosalicylic acid (dns) procedure (bernfeld, 1955), using 1 % soluble starch (merck, darmstadt, germany) as substrate. ten microliters of the enzyme were incubated for 30 min at 35 °c with 35 μl of phosphate buffer (0.02 m, ph 7.1) and 20 μl of soluble starch. the reaction was stopped by addition of 80 μl of dns and heated in boiling water for 10 min prior to read absorbance at 540 nm. one unit of α-amylase activity was defined as the amount of enzyme required to produce 1 mg of maltose in 30 min at 35 °c. used negative control contained all reaction mixture but pre-boiled enzyme (for 15 min) to prove the enzyme presence in the samples. purification process purification process of the salivary α-amylase in a. spinidens was carried out based on englard and seifter (1990) and dennison (1999) procedures. the crude extract (40 ml) from salivary homogenates of a. spinidens adults was treated with ammonium sulfate at 4 °c to give fractions precipitated at 40 % and 80 % saturations. the precipitates were collected by centrifugation at 6,000 rpm for 15 min, diluted in 2 ml of tris-hcl (20 mm, ph 8.8) and dialyzed overnight at 4 °c against the same buffer. the enzyme solution was applied to a sepharyl g100 column, equilibrated with the same buffer. the column was run at a flow rate of 0.5 ml/min and 1.5 ml. amylase activity was measured as described in the previous section. fractions containing enzymatic activity were pooled and applied to a diethylaminoethyl (deae)-cellulose column, equilibrated with tris-hcl (ph 8.8). the enzyme elution was done at a flow rate of 0.5 ml/min with a linear nacl gradient (0 0.6 mol). fractions (1.5 ml/tube) were collected and their protein concentration and α-amylase activity were determined as described. in the final step, fractions containing the highest enzymatic activity were pooled and used as enzyme source. sodium dodecylsulphate polyacrylamide gel electrophoresis (sds-page) sds-page was performed according to the procedure described by laemmli, (1970). acrylamide concentration was 10 % for the separating gel and 4 % for the stacking gel. after running the gel at 100 mv as constant voltage, proteins on the polyacrylamide gel were stained with 0.2 % coomassie brilliant blue r-250 (hames 1998). β-galactosidase (116 kda), bovine serum albumin (66.2 kda), ovalbumin (45 kda), lactate dehydrogenase (35.5 kda), restriction endonuclease bsp 981 (25 kda), β-lactoglobulin (18.4 kda) and lysozyme (14.4 kda) were used as molecular mass standards. effect of ph on enzyme activity the optimal ph for amylase activity was determined using universal and tris-hcl buffers. the tested ph values were 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13. the enzymatic assay was done as described in the earlier section. effect of temperature on enzyme activity the effect of temperature on amylolytic activity was determined by incubating the reaction mixture for 30 min at the following temperatures: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70 and 80 °c. also the thermo-stability of the enzyme at the optimal temperatures was determined over 192 h (8 days). samples were maintained at 35 and 45 °c for 8 days followed by determination of residual activity as described in the earlier section. kinetic studies kinetic parameters of the purified α-amylase were calculated by using different concentrations of starch and glycogen as 0.05, 0.1, 0.2, 0.4, 0.6, 0.8 and 1 %. the assay procedure was carried out describing earlier. obtained data were inserted in sigma plot software (version, 6) and line regression was drawn by used concentrations of substrate and observed enzymatic velocity. effect of monoand divalent cations on α-amylase activity the effects of various cations on purified αamylase activity were investigated using cacl2, mgcl2, a) 50   b) fig. 1 column chromatography of the salivary purified α-amylase from adults of a. spinidens. a) sepharyl g-100 gel-filtration of the enzyme after ammonium sulfate (40 and 80 %) treatment. fractions 41 47 contained the highest enzymatic activity on starch (1 %) and collected for next steps. b) deae ion-exchange chromatography of the gel-filtrated α-amylase from a. spinidens. fractions 25 32 contained the highest enzymatic activity on starch (1 %) and used for other steps of the experiments. nacl, kcl, cuso4 and znso4. in case, 10 µl of a solution containing each concentration of ions (0, 0.5, 1, 3, 5 and 10 mm) and 10 µl of enzyme were pre-incubated for 10 min at ph 9 of universal buffer and 35 °c as optimal temperature. thirty microliters of starch were added to the mixture and experiment was continued as described above. effect of specific inhibitors on α-amylase activity the effects of enzyme inhibitors on amylolytic activity were studied using different concentrations (0, 0.5, 1, 3, 5 and 10 mm) of ethylene glycol-bis (βaminoethylether) n, n, n′, n′-tetraacetic acid (egta), triethylenetetramine hexaacetic acid (ttha), ethylenediamine tetraacetic acid (edta), sds and triton x-100. the purified enzyme (10 µl) was pre-incubated for 10 min at ph 9 and 35 °c with 10 µl of inhibitors (each concentration). fifty microliter of starch was added to the mixture and experiment was continued as earlier. other steps were carried out as mentioned earlier. kinetic parameters by using ic50 concentration of egta and ttha to measure further involvement of egta and ttha, ic50 concentration of each inhibitor were added to different concentration of starch (0.1, 0.2, 0.4, 0.6, 0.8 and 1%). then, observed activities were inserted in sigma plot software to calculate vmax and km values. protein determination protein concentration was determined either by measuring absorbance by the method of bradford (1976) using bovine serum albumin as standard. statistical analysis all data were compared by one-way analysis of variance (anova) followed by tukey's studentisized test when significant differences were found at p ≤ 0.05 (sas, 1997). differences between samples were considered statistically significant at a probability less than 5 % and marked in figures and tables with letters and asterisks. results and discussion saliva is the main source of enzymes involving in eod process of hemipterans. miles (1968) divided insect saliva into three categories; i) saliva of hemipterans contains almost exclusively α-amylase like in lygus disponsi linnavuori (hemiptera: miridae), while other carbohydrases are just present in the alimentary canal, ii) phloem feeder insects like aphids in which carbohydrases are the only salivary enzymes, iii) seed feeder insects whose saliva contains proteases and esterases. after amonium sulphate precipitation, samples were subjected to sepharyl g-100 column. among 73 collected fractions, fractions 41 47 showed the highest amylolytic activity (fig. 1a). these fractions were pooled and loaded onto deae ion-exchange chromatography. fractions 25 32 out of 47 gathered fractions showed the highest amylolytic activity (fig. 1b) then, they were pooled and used for sds-page showing purity of the enzyme. sds-page analysis revealed purity of the enzyme by molecular weight of 26 kda (fig. 2). after confirming purity of the enzyme by sds-page, biochemical assays revealed an enzyme with 4.22 u/mg protein of specific activity, a recovery percentage of 14.67 and a 13.83 purification fold (table 1). terra and ferriera (2005) reported that α-amylases in insects have a molecular weight of 48 52 kda. in our study, it was found a lower molecular weight that coincides with the hypothesis that salivary α-amylase is involved in preliminary digestion of starch and glycogen and it definitely has a few isozymes in comparison with midgut α-amylase that is involved in complete digestion of carbohydrates by considering the point that proteases fig. 2 denaturing sds-page to find purity and molecular weight of the salivary purified α-amylase from the salivary gland of adult a. spinidens. are the main salivary enzymes in predaceous bugs (zibaee et al., 2012a). ph and temperature are the two major factors that affect enzymatic reactions in several ways like substrate and enzyme stability, their combination, tertiary structure of the enzyme etc. it was observed that 9 is the optimal ph value for activity of purified αamylase in a. spinidens by using universal and trishcl buffers (fig. 3). terra and ferriera (2005) believe that enzyme optimal ph should be determined using different buffers to discount the table 1 purification of α-amylase in the salivary glands of a. spinidens adults total activity (µmol/min) total protein (mg) specific activity purification fold purification steps recovery (%) (µmol/min/mg protein) crude extract 2.59 ± 0.86 8.47 0.30 ± 0.07 100 1.00 (nh4) 51   2so4 (0 40 %) 2.08 ± 0.49 5.12 0.41 ± 0.03 80.03 1.33 (nh4)2so4 (40 80 %) 1.53 ± 0.32 3.14 0.74 ± 0.10 59.07 2.43 sepharyl g-100 0.80 ± 0.05 0.53 1.51 ± 0.17 31.00 4.95 cm-sepharose 0.38 ± 0.03 0.09 4.22 ± 0.34 14.67 13.83 all experiments were carried out at 30 °c. salivary gland homogenates were used in this experiment. starch (1 %) was used as substrate to find amylolytic activity. fig. 3 optimal ph determination of the purified α-amylase from salivary glands of a. spinidens adults. two bufferic solutions were used and statistical differences have been shown by the different letters (tukey’s test, p ≤ 0.05). 52   effects of chemical constituents present in the buffers and their ionic strength on enzyme activity. bezdi et al. (2008) reported ph 4.5 as the optimal ph of salivary α-amylase in eurygaster integriceps puton (hemiptera: scutelleridae). zeng and cohen (2000) reported that optimal ph 6 for α-amylase from l. herperus and l. lineolaris. mehrabadi and bandani (2009) found that salivary α-amylase of e. maura had the highest activity at ph 6 7. a. spinidens feed on caterpillar larvae whose ph is alkaline to overcome secondary metabolites of plants. so, salivary secretion of the bug must be alkaline adapting it to feed on these preys, a phenomenon that we have already found on salivary proteases and lipases (zibaee et al., 2012a, b). the optimal temperature of the purified α-amylase was found to be 35 40 °c (fig. 4a). also, it was found that the purified enzyme was stable for 96 h (3 days) at the optimal temperatures (fig. 4b). this value is lower than that observed for the α-amylase activity in blatella germanica (blatodea: blatellidae) 50 °c (applebaum, 1985) and bombyx mori (lepidoptera: bombycidae) 60 °c (kanekatsu,1978). also, it is more or less equal to that of the α-amylase activity in l. disponsi 37 °c (hori,1970), dolycoris baccarum (hemiptera: pentatomidae) 40 °c (hori,1969), cerambyx cerdo l. (hemiptera: cerambycidae) 35 °c (applebaum, 1985), e. maura l. 30 35 °c (mehrabadi and bandani, 2009) and higher than tenebrio molitor l. (coleoptera: tenebrionidae) 25 °c (barbosa et al., 1999). optimal temperature of salivary enzymes surprisingly corresponds to field temperature during june to september when a. spinidens is active on rice fields of iran. higher activity of the enzymes in a specific temperature of in vitro assays generally reflects the temperature of the environment where the organism feeds on the hosts. extremes in temperatures can also disrupt the hydrogen bonds that hold the enzyme in its three-dimensional structure leading denaturation of the protein (zeng and cohen, 2000). meanwhile, biological reactions are catalyzed by proteinaceous enzymes, and each enzyme has a temperature above which its three dimensional structure is disrupted by heat. therefore, biological fig. 4 optimal temperature and stability of the salivary purified α-amylase from salivary glands of a. spinidens adults. statistical differences have been shown by different letters for optimal temperature and for stability (tukey’s test, p ≤ 0.05). reactions occur faster with increasing temperature up to the point of enzyme denaturation, above which temperature enzyme activity and the rate of the reaction decreases sharply (agblor et al., 1994). lineweaver-burk analysis to find kinetic parameters of the purified salivary α-amylase in presence of two substrates, starch and glycogen, revealed that glycogen is slightly more specific for the enzyme. by using starch, the maximal velocity (vmax) of the purified enzyme was observed 7.35 u/mg protein and km was 1.04 % in comparison with 7.14 u/mg protein and 0.57 % for glycogen (fig. 5). since km has an inverse relationship with the substrate concentration required saturating the active sites of the enzyme, this indicates decreased enzyme affinity for the substrate (wilson and goulding, 1986). in the other words, km is the measurement of the stability of the enzyme-substrate complex and a high km would indicate weak binding while a low km would indicate strong binding (stryer, 1995). fig. 5 double reciprocal plot to show the kinetic parameters of the salivary purified α-amylase from the salivary glands of a. spinidens adults by using starch and glycogen (1 %) (1/v many fertilizers were used in agricultural ecosystems that might affect ecological levels in case of herbivores and carnivores. one of the side effects of these compounds could be on enzymes max = intercept on the 1/v ordinate, -1/k0 m = intercept on the negative side of the 1/[s] abscissa). 53                         fig. 6 effects of different concentrations of cations on the salivary purified α-amylase from the salivary glands of a. spinidens adults. statistical differences have been shown by different letters (tukey’s test, p ≤ 0.05). 54   that recruit ions in their active site. in fact, ions, especially divalent ones, work as cofactors and increase or sometimes decrease the enzymatic activity. in our experiments, amylolytic activity in salivary secretion of a. spinidens was increased in the presence of the divalent ions like mg2+, ca2+ and last concentration of cu2+ (fig. 6). but presence of na+ decreased the enzymatic activity, k+ and zn2+ showed no effects (fig. 6). saadati et al. (2008) observed inhibitory effects of cu2+ on salivary αamylase of e. integriceps but na+ showed no effects. mehrabadi and bandani (2009) reported that na+, ca2+ had no effects on salivary amylase of e. maura but mg2+ decreased it. hori (1969) stated that the polygalacturonase activity in the salivary gland of l. rugulipennis was greatly affected by salts in the medium. however, it has been reported that αamylases are metalloproteins that require calcium for maximum activity. calcium also affords stability for the amylases from a variety of sources, including insects, to both ph and temperature extremes (zeng and cohen, 2000). sds, edta, egta and ttha significantly decreased the activity of the purified salivary α-amylase 55   table 2 effects of different concentrations of inhibitors on purified salivary α-amylase in a. spinidens adults inhibitor concentrations (mm) specific activity (u/mg protein) ic50 (mm) control 0 3.41a sds 0.5 3.12a 1 2.45b 4.33 3 1.59c 5 1.21c,d 10 0.36d control 0 3.24b triton 0.5 3.38b 1 3.97a * 3 4.25a 5 4.33a 10 4.38a control 0 3.22a edta 0.5 3.15a 1 3a 3 2.14b 4.88 5 1.26c 10 0.14d control 0 3.25a ttha 0.5 3.14a 1 3.2a 3 2.52a,b 8.80 5 2.04b 10 1.57c control 0 3.18a dtc 0.5 3.18a 1 3.26a ¡ 3 3.11a 5 3.05a 10 3.09a control 0 3.23a egta 0.5 3a,b 1 2.18b 3 1.17c 3.76 5 0.87d 10 0.16e *) triton is an activator so no ic50 has been calculated. ¡) no statistical differences were observed so no ic50 was calculated. statistical differences have been shown by different letters (tukey’s test, p ≤ 0.05) for each compound seperately. in a. spinidens (table 2). dtc showed no effects but triton increased the enzymatic activity (table 2). these results clearly indicate that salivary α-amylase of a. spinidens is a metallo-enzyme that requires metal ions in its active site. edta is a general chelating agent that removes all metal ions from active site of the enzyme. egta and ttha are specific chelating agents of ca2+ and mg2+, respectively. inhibitory effects of both egta and ttha demonstrated the possible substitution of ca2+ and mg2+ in active site of the enzyme (podoler and applebaum, 1971; baker, 1983; andersson et al., 1989; kazzazi et al., 2005; feng et al., 2008; zibaee et al., 2008). using ic50 of egta and ttha along with different concentrations of starch revealed lower vmax caused by ttha and higher km value of egta (fig. 7). since, vmax of the purified enzyme by using ic50 concentrations of both inhibitors decreased in comparison with control, it can be concluded that both of these inhibitors can intervene in enzymesubstrate interaction. meanwhile, calculated k m value for egta was higher than that of ttha so it can be inferred that egta bind to the active site of the enzyme to remove ca2+ ion decreasing enzymatic activity. results of fig. 7 seems to verify statement of andersson et al. (1989) and feng et al. (2008) in replacing of mg2+ 2+ and ca in active site of enzymes. triton has been shown to be an activator on the majority of insect digestive enzymes like amylases, glycosidases and exopeptidases (terra and ferriera, 2005). edo in predacious bugs like a. spinidens facilitates their ability to utilize larger insects, but require a relatively longer time with their prey until they are completely satiated. this is because of their high investment in enzyme production and injection into the prey. this process requires time for the enzymes to act, and the subsequent necessity of recovering these enzymes (oliveira et al. 2006). short handling time and facultative phytophagy are important traits in biocontrol agents and largely based on the eod of predator on heteroptera (cohen, 1998). amylases might have crucial role in digestion of prey tissues, especially on glycogen which is the major carbohydrate stored in their body. fig. 7 double reciprocal plot to show the kinetic parameters of purified α-amylase from the salivary glands of a. spinidens adults by using ic50 concentration of egta and ttha (1 %) (1/vmax = intercept on the 1/v ordinate, -1/k0 m = intercept on the negative side of the 1/[s] abscissa). acknowledgement the project has been supported by a grant from the university of guilan (iran). we appreciate the assistance of the staff in the laboratories of insect physiology and toxicology. we highly appreciate two anonymous reviewers for their valuable comments and for improving the quality of the manuscript. 56   references andersson k, sun sc, boman hg, steiner h. purification of the prophenoloxidase from hyalophora cecropia and four proteins involved in its activation. insect biochem. 19, 629–637, 1989. agblor a, henderson hm, madrid fj. characterisation of alpha-amylase and polygalacturonase from lygus spp. (heteroptera: miridae). food res. int. 27: 321-326, 1994. applebaum sw. biochemistry of digestion. in: kerkut ga, gilbert ll (eds), comprehensive insect physiology, biochemistry and pharmacology: regulation, digestion, excretion, vol. 4, pergamon press, pp 279-307, 1985. baker je. properties of amylase from midguts of larvae of sitophilus zeamais and sitophilus granaries. insect biochem. 13: 421-428, 1983. barbosa pereira pj, lozanov v, patthy a, huber r, bode w, pongor s, et al. specific inhibition of insect α-amylases: yellow meal worm α-amylase in complex with the amaranth α-amylase inhibitor at 2.0 å resolution. structure 7: 10791088, 1999. 57   bernfeld p. amylases, α and β. meth. enzymol. 1:149-158, 1955. bezdi ms, pourabad rf, sadeghi h, golmohammadi g. some properties of αamylase in the salivary glands of eurygaster integriceps puton (hemiptera: scutelleridae). munis. entomol. zool. 3: 733-744, 2008. bradford m. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. analyt. biochem. 72: 248-254, 1976. cohen ac. extra-oral digestion in predaceous terrestrial arthropoda. ann. rev. entomol. 40: 85-103, 1995. cohen ac. solid-to-liquid feeding: the inside(s) story of extra-oral digestion in predaceous arthropoda. am. entomol. 44, 103-117, 1998. englard s, seifter s. precipitation techniques. meth. enzymol. 182: 285-305, 1990. feng c, song q, lü w, lu j. purification and characterization of hemolymph prophenoloxidase from ostrinia furnacalis (lepidoptera: pyralidae) larvae. comp. biochem. physiol. 151b: 139-146, 2008. franco ol, riggen dj, melo fr, bloch c, silva c, grossi mf. activity of wheat α-amylase inhibitors towards bruchid α-amylases and structural explanation of observed specificities. eur. j. biochem. 267: 2166-2173, 2000. hames bd. gel electrophoresis of proteins: a practical approach, oxford university press, new york, 1998. hori k. some properties of salivary amylases of adelphocoris suturalis (miridae), dolycoris baccarum (pentatomidae), and several other heteropteran species. entomol. exp. appl. 12: 454-466, 1969. hori k. some properties of amylase in the salivary gland of lygus disponsi. j. insect physiol. 16: 373-386, 1970. kanekatsu u. studies on further properties for an alkaline amylase in the digestive juice of the silk worm, bombyx mori. j. fac. text. sci. technol. 76: 1-21, 1978. kazzazi m, bandani ar, hosseinkhani s. biochemical characteristics of a-amylase of the sunn pest, eurygaster integriceps. entomol. sci. 8: 371-377, 2005. laemmli uk. cleavage of structural proteins during the assembly of bacteriophage t4. nature 227: 680-685, 1970. manley gv. biology and life history of the rice field predator andrallus spinidens f. 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immunity; aging; immunosenescence; c. elegans; drosophila introduction aging and its many attendants have fascinated people since antiquity, at least since the cato maior de senectute (cato the elder on old age) of cicero (44 bce). most contemporary research into aging is driven by interest in the human aging process and in interventions that attenuate the normal and pathophysiological effects of aging and help achieve the controversial goal of ‘successful aging’ (rowe and kahn, 1987). aging is the process of growing old. some professionals in aging research prefer the term senescence (which first appeared in the english language in the late 1600s) ________________________________________________________________________ corresponding author: david stanley usda/agricultural research service biological control of insects research laboratory 1503 south providence road, columbia mo 65203, usa email: stanleyd@missouri.edu to aging. operationally, senescence is the progressive, inevitable breakdown of the organism. in humans, senescence is expressed in many ways, including loss of muscle and bone mass, reduced metabolic rates, impaired reaction times, diminished memory functions, reduced sexual activity and lessened hearing, olfaction, vision and organ functions. among the changes associated with senescence is the diminished capacity of the immune systems and reactions to challenge, known as immunosenescence. immunosenescence is associated with increased occurrences of cancers and sensitivities to infections. however, immunosenescence is complicated because some innate immune functions, particularly inflammation, increase with age. this is sometimes referred to as ‘inflamm-aging’ (cannizzo et al., 2011) and is thought to contribute to ageand inflammationrelated health problems, including atherosclerosis and arthritis. while senescence, per se, is not a human 102 mailto:stanleyd@missouri.edu disease, a large research enterprise is deployed to understand the mechanisms of senescence and to ameliorate some of the conditions associated with it. senescence and death may be a universal feature of life, encapsulated in “no death, no life, no exceptions” by fulgham (1986). lanner and conner (2001), however, raised the question of whether the long-lived bristlecone pine, pinus longaeva, undergoes senescence. they analyzed several parameters of age-related declines, including tracheid diameter, annual shoot growth increments, pollen viability, seed weight, seed germinability and seedling biomass accumulation in bristlecone pines of widely varying ages, from 23 to 4,713 years. they found no significant differences within these and other parameters and concluded that this species does not senesce. there may be other such longlived species in certain relatively unexplored habitats, such as deep ocean floors, however, in the common experience, the life histories of individuals end in senescence and death. senescence and age-related immunosenescence has been recorded in several invertebrates. two invertebrates, the worm, caenorhabditis elegans, and the fruit fly, drosophila melanogaster, are model organisms for research into mechanisms of senescence and of prolonged life spans. of course, the goal of research with these models is to discover new knowledge on fundamental aspects of senescence that will contribute to our understanding of human senescence processes. in this essay, i will treat some of the available information on immunosenescence in invertebrates. the purpose is to move away from trying to understand human senescence and toward generating new ideas around the application of research into invertebrate immunosenescence to contemporary and emerging problems in agriculture. i begin with a brief discussion of mechanisms of senescence, to which we now turn attention. mechanisms of senescence a basic mechanism of senescence is the accumulation of damage at several levels of biological organization, including damage to molecules, to cells, to tissues and to intact organisms. one form of damage is oxidative damage, which arises from formation of the superoxide radical in mitochondrial electron transport chains. this idea was put forth in the 1950’s (harman, 1956) and continues to carry a great deal of weight (cannizzo et al., 2011; cerullo et al., 2012). the story is more complex, however, as partridge (2009) discussed two reports that draw the theory of oxidative damage into question. working with the worm, c. elegans, doonan et al. (2008) and van raamsdonk and hekimi (2009) report on the influence of silencing two genes encoding mn superoxide dismutases on lifespan. the deletions led to increased sensitivity to a superoxide generator, paraquat, but either increased or did not reduce lifespan. partridge (2009) discussed alternative explanations and concluded that superoxide damage may not attenuate lifespan in c. elegans. gems and doonan (2009) asked whether the oxidative damage theory of senescence is in error. they reviewed the outcomes of experiments designed to test the theory and concluded that many experimental results are not consistent with key predictions and suggested alternatives. one alternative is the molecular damage theory of senescence, which suggests that oxidative events are just one of several ways to induce life-shortening damage to molecules. several authors point out that senescence is not an adaptation. many organisms undergo reproductive diapause, including some insect species and the worm c. elegans (tatar and yin, 2001). diapause is a geneticallyand neurophysiologically-mediated state of sharply reduced activity. tatar and yin (2001) found that mortality rates of post-diapause drosophila are similar to the rates seen in newly emerged adults. they inferred that senescence is very much slower during diapause and, more importantly, the rates of senescence within a species exhibit considerable plasticity. the authors note that variable senescence rates are endocrine regulated. the point would be that plasticity in senescence rates is one mechanism of influencing immunosenescence and life spans, as seen in monarch butterflies (herman and tatar, 2001). zhan et al. (2011) note that the long-distance migrations of monarchs depends on endocrine organization of several physiological processes, including the juvenilehormone mediated increases in life span just cited. the idea of plastic rates of senescence may help understand how some insects, such as 13and 17year periodical cicadas, express very lengthy juvenile life spans. mis-regulation, or errors, of gene expression may be another senescence mechanism. micrornas (mirnas) are gaining recognition as an additional tier of regulating gene expression, particularly during development. they effectively silence gene expression by complementing the untranslated regions of some mrnas, thereby blocking translation of the mrna into protein. liu et al. (2012) forged a mirna based linkage between age and neurodegenerative diseases. mir-34 is one of the many mirnas that regulate gene expression during development. using the drosophila model, they found that expression of mir-34 is upregulated with increasing age. mir-34 mutants looked and acted normally in early adulthood, however, they suffered severely shortened life spans, reduced climbing ability and marked loss in brain integrity (indicated by appearance of many vacuoles in brains of older flies). mir-34 suppresses expression of a ecdysteriod-reglated gene, e74a. e74a is an antagonistically pleiotrophic gene; that is, it is beneficial in juvenile stages of drosophila development, but quite harmful in adults. mir-34 expression functions to silence the expression of e74a, one of the genes that can exert negative effects on older flies and increase risks of ageassociated diseases. mis-regulation of mir-34 expression can contribute to the senescence process and to age-associated diseases in insects and other invertebrates. a key point in understanding senescence is appreciation that the process is not an evolved trait, 103 in the sense that no genes responsible for senescence have been identified. rather, senescence is more accurately understood as an unregulated side effect, or, to be a bit more precise, unregulated side effects of life processes taking place in different time courses and by different mechanisms in each of the many systems that make up animals (partridge, 2010). at its basis, senescence is the outcome of many processes of increasing damage and decreasing functions. from a biomedical perspective, research into mechanisms of senescence has the goal of creating interventions that improve the health of older people and one outcome of improved health may be increased human life spans. in this essay i depart from the biomedical model with the proposal that some of the recent advances in invertebrate senescence may be applicable to some agricultural issues including aquaculture and insect pest management technologies. before addressing these issues directly, let us consider a brief sketch of insect immunity as a model of invertebrates, and document cases of naturally-occurring immunosenescence in invertebrates. peptides and proteins appear in the hemolymph of infected insects 6 12 h post-infection. again, far more detailed descriptions of humoral immunity, including non-self surveillance and the signaling pathways that lead to anti-microbial peptide expression are available (lemaitre and hoffmann, 2007). the hemolymph of most insects has a standing population of approximately 4 6x106 circulating hemocytes per ml hemolymph. the main immune effecter cells of lepidoptera are plasmatocytes and granulocytes. these cells clear microbes from circulation by phagocytosis and a process called nodulation, a form of encapsulation. nodulation begins with microaggregation of hemocytes with adhering microbes (fig. 1), which grow into nodules. in the last phase of nodulation, plasmatocytes surround the nodules and activate a propo system to melanize them. melanized nodules are finally attached to an internal organ or inner body wall and they can be a permanent record of previous infection. the darkened nodules remain in the body for the life of the insect and they are easily visible at 40x under a dissecting microscope (fig. 1). nodulation is responsible for clearing the majority of infecting microbes from circulation (haines et al., 2010). invaders too large for phagocytosis, such as parasitoid eggs, are encapsulated in layers of hemocytes within resistant insect hosts. these also are melanized and attached to the inner body wall or an internal organ. the melanization process produces reactive oxygen forms that may chemically kill the invaders. a sketch of insect immunity some readers may not be fully aware of insect immunity and this simple sketch is designed to facilitate reading this essay without referring to external literature. detailed treatments are available. strand and pech (1995) and strand (2008) are excellent reviews of cellular immunity. gillespie et al. (1997) review signal systems in insect immunity. eicosanoid signaling has been treated by stanley (2005; 2006). humoral immunity is detailed in lemaitre and hoffmann (2007). kanost and gorman (2008) review prophenoloxidase activation, a central process in insect immunity. an et al. (2012) report on a novel aspect of innate immunity, neuropeptidemediated prophylactic immunity in newly molted insects. immunosenescence in invertebrates the worm caenorhabditis elegans (nematoda: rhabditida: rhabditidae) is a well-known model animal for studies of development, aging and immunity. kurtz and tan (2004) reviewed immunosenescence in c. elegans. while the molecular mechanisms are not clear, it is certain that younger worms are far more resistant to bacterial infection than older worms. c. elegans is generally maintained on a bacterial lawn of escherichia coli (strain op50) in laboratory cultures. worms reared on living bacteria have shorter life spans than worms fed on killed bacteria, from which it is thought that e. coli can become an opportunistic pathogen in older worms. by extension, it seems immunocompetence declines as a worm ages. work on c. elegans has produced new understandings of the aging process, some of which are conserved in the evolution of all metazoans. invertebrates, including insects, express solely innate immunity, that is, non-specific immunity that does not depend on previous immune experiences. in insects and certain other invertebrates, the integument serves as an exoskeleton and also as a physical barrier to potential infectious agents (davis and engström, 2012). many microbes enter insect bodies via the gastrointestinal tract, although it is not a completely open path for them. insects express epithelial immunity, recorded in the salivary glands (abdelsadik and roeder, 2010), the gut (cronin et al., 2009), malpighian tubules (davies and dow, 2009), and tracheal epithelia (wagner et al., 2008) of drosophila and likely most other insects. epithelial immunity is generally a humoral response, based on induced expression of genes encoding a wide range of anti-microbial peptides. once infectious agents get into the open hemocel, insects detect and react to infection by launching humoral and cellular immune reactions. humoral reactions, again, involve biosynthesis of antimicrobial peptides and the enzyme lysozyme, and release of prophenoloxidase (propo) from circulating oenocytoids, a type of hemocyte. these age-related declines in invertebrate immune functions have been documented in several species, some of them outside the usual model animals. the cricket, gryllus assimilis, is a simple example in which park et al. (2011) recorded evidence for cellular immunosenescence. they used nodulation as a quantitative assessment of immune function, counting numbers of nodules formed in response to lipopolysaccharide (lps; prepared from the bacterium serratia marcescens) injections. nodulation increased with time following treatment and with increasing lps dosages. males, 104 fig. 1 microaggregation and nodulation reactions to bacterial infection in fifth-instar tobacco hornworms, m. sexta. hornworms were challenged by injecting the bacterium, s. marcescens, into the hemocel. for microaggregation, at 1 h post infection, hemolymph was withdrawn, diluted with buffer and placed on a microscope slide for observation and photography at 400x. the cells in these photographs range from 10 12 microns. for nodulation, at 4 h post infection the hemocel was exposed for photography at 40x. the nodules are 0.1 mm in diameter. photo by jsm. ages 0 to 3 weeks after adult emergence, produced about 12 15 nodules/male in response to experimental infection. nodulation declined to about 9 nodules/male at 4 weeks and to about 3/male at 5 weeks. total circulating hemocytes declined in parallel, from about 5x103 at ages 1 and 2 weeks, to about 3x103 at 3 weeks and to 1 to 2x103 at weeks4 and 5. declining hemocyte populations were attended by increased numbers of damaged hemocytes, from about 10 % to 60 % by week 5. the authors inferred that g. assimilis males undergo cellular immunosenescence. cellular immunosenescence has also been reported for the model fly, d. melanogaster (mackenzie et al., 2011). the authors injected fluorescently labeled escherichia coli into adult male flies, and then recorded the proportion of hemocytes able to phagocytose the bacterial cells. about 25 % of the hemocytes in flies age 1 week phagocytosed the microbes, which was reduced to about 16 % in flies age 4 weeks. they also considered the circulating hemocyte populations as a function of age, showing that hemocyte numbers declined in females, but not in males, as the flies aged from 1 to 4-weeks old. this work documented cellular immunosenescence in fruit flies with the suggestion that the decline in cellular immune functions may be one mechanism of increased susceptibility to disease in older flies. kurtz (2002) took a different approach to a similar conclusion. his research goal was to understand sources of variation in hemocyte phagocytosis in scorpionflies, panorpa vulgaris. he collected flies in the fall and allowed their offspring to overwinter in outdoor cages. the adults were collected the following spring as they emerged and males were used for experiments. he collected very small hemolymph samples (1 μl) for his phagocytosis assays. the author considered a wide range of variation sources; to list a few, these included genetic variation, development, gender and environment. he also studied variation associated with age, from which he determined that the ability of p. vulgaris hemocytes to perform phagocytosis declined sharply with age. i conclude that at least some insects, maybe most of them, undergo substantial declines in cellular immune functions as they age. aside from cellular immunity, aspects of insect humoral immunity may also decline with age. this is a controversial idea because several studies report increased transcripts of immune response genes in d. melanogaster as the flies age (pletcher et al., 2002). zerofsky et al. (2005) noted that while such transcripts increase with age, it remains unknown whether the increases are due to increased (and possibly chronic) exposure to microbes or due to age-related changes in immune system elements. for their work, zerofsky et al. used the expression of a gene encoding the antimicrobial protein (amp) diptericin (dpt) as a 105 measure of immune function. they found older females had higher dpt expression than younger ones in untreated, control flies. when challenged with infection with living bacteria, dpt levels increased at about the same rates for younger and older females, however, younger females reached highest dpt expression at 12 h post-infection, then rapidly declined. results with older flies differed, however, because dpt induction continued throughout the 48 h test period. the authors inferred that persistent induction of genes encoding amps in older flies accounts for higher levels of their indicator gene, dpt. they then challenged flies with killed bacteria. the killed bacteria induced more dpt expression in younger flies than in older ones. they understood from this observation that the ability to induce expression of amp genes declines with age. if this holds up, it might be concluded that insects undergo agerelated senescence of cellular and humoral immune functions. a study of hemocyte populations and propo activity in newly emerged and sexually mature field-captured adults of the damselfly, lestes viridis, complicates the common view of immunosenescence. rolff (2001) reported lower circulating hemocyte populations in newly emerged males and females than in older, sexually mature adults. he also registered higher propo activity in sexually mature females compared to younger ones and compared to older males. the propo data are consistent with recent work on honeybees, as noted in the next section. the hemocyte data run contrary to other findings that hemocyte populations decline with advancing age. this may follow from experiments with damselflies captured in the wild, with no information on the immune history of the individuals. individual organs can undergo immunosenescence. the mammalian brain includes a cell type known as microglia that makes up the brain’s innate immune system (streit and xue, 2010). these cells comprise about 12 % of the cells in the human brain and they serve important positive roles, including recognizing and responding to infections via pattern recognition receptors (block et al., 2007). microglia can also become overactivated by stimulations such as infection, environmental toxins or neuronal damage. in their over-activated states, microglia become important players in destructive neurodegenerative diseases, such as alzheimer’s and parkinson’s diseases. beyond the influence of over-activation, streit and xue (2010) report that microglia undergo senescence, characterized by several features, especially fragmenting the cytoplasm of microglia, and thereby lose their ability to protect neurons. they believe that this is a process of immunosenescence that leads to age-related neurodegeneration. invertebrates may also undergo cns-level immunosenescence, perhaps similar to the effects of mis-regulation of mir-34 in drosophila, mentioned earlier. in some instances physiological trade-offs, the subject of the next section, rather than senescence, may account for what looks like immunosenescence. let us move on to a few examples. ecological immunity: trade-offs between immune functions and other physiological systems apparent age-related losses of immune functions have been attributed to trade-offs among physiological systems. adamo et al. (2001) reported on immunocompetence in adults of the cricket gryllus texensis. they found approximately similar resistance to bacterial infections in nymphs and adults at ages 3 days and 14 days. adults over 4 weeks old, however, were very susceptible to bacterial infection, with less than 5 % survival. circulating hemocyte populations did not decline with age and phenoloxidase activity progressively declined in aging males but not in females. the authors interpreted their results in terms of physiological a trade off: as males enter the reproductive phase of adulthood, they trade immunocompetence for reproductive potential. it might be thought that the findings with g. texensis are at odds with outcomes of the experiments reported just above on the related cricket, g. assimilis. the differences in the two reports are probably due to experimental designs. park et al. (2011) used a direct measure of an immune function, that is, the ability to form nodules in response to infection in their assessment of immunocompetence. adamo et al. (2001) used an broader measure, namely, resistance to infection. also, park et al. (2011) removed the cost of reproduction by not allowing g. assimilis males to court and mate with females. i conclude that crickets are able to trade off immunocompetence for reproduction. honeybees, apis mellifera, trade off immunocompetence for the energy-demanding foraging phase of adult life. in their work on biochemical signaling in insect cellular immunity, bedick et al. (2001) tested the hypothesis that prostaglandins and other eicosanoids mediate nodulation reactions to infection in adult honeybees. the authors challenged newly-emerged adults with freeze-dried bacteria, serratia marcescens, and later determined numbers of nodules formed in response to the challenges. the young adults were competent to produce over 100 nodules/ bee at 4 h post-challenge. similar experiments with foraging bees revealed that bacteria-challenged foragers did not form nodules. on closer study, the foragers had virtually no circulating hemolymph and very few, if any, circulating hemocytes. the authors concluded that immunosenescence may occur in honeybees, but they could not judge whether it was linked to the age of the bees or to the ontology of tasks. the influence of age and task can be separated using the single cohort colony procedure. schmid et al. (2008) created precocious foragers and overaged nurses, then assessed hemocyte numbers. they found that age-right nurse bees had far more circulating hemocytes than over-aged nurses and precocious foragers similarly had far more hemocytes than age-right foragers. these data indicate the changes seen in honey bee hemocyte populations are related to age and not related to task. this would be consistent with a physiological trade-off of cellular immunocompetence for the 106 energetic costs associated with foraging flights. however, age-related declines in honeybee hemocyte populations differ from the findings of amdam et al. (2005). they reported the outcome of another reversion protocol that led to production of new hemocytes and what they took to be a reversal of immunosenescence in worker honeybees. the story is still more interesting, however, because schmid et al. (2008) also assessed hemocyte numbers in queens and drones, finding that all three phenotypes, workers, queens and drones undergo steep declines in circulating hemocytes with advancing age. queens live most of their lives within their colonies, which is not congruent with a direct trade-off of hemocytic immunocompetence for the costs of foraging. schmid et al. (2008) suggested the possibility that a more subtle trade-off may take place at the colony level, where all energy-withdrawing activities, both within and outside the hives, should be taken into account. the loss of hemocytes with increasing honeybee age is offset by maintaining phenoloxidase (po)-based immune functions (schmid et al., 2008). in workers, po activities increased over the first week of adult life, then leveled off. queens were different because po activity increased steadily with age, attaining twice the activities found in workers. po activity levels declined slightly with age in drones. maintaining pobased immune functions demands much less investment than maintaining standing populations of millions of hemocytes. it appears that honeybees may reduce the risks associated with trading off hemocyte-based immunity by retaining po-based immunity, as also seen in social leaf cutting ants, acromyrmex octospinosus (armitage and boomsma, 2010). here, changes in honeybee immunity illustrate the complexity of assessing immunocompetence over the life span of insects. bumble bee workers also undergo immunosenescence. in their research with two bumble bee species, bombus terrestris and b. lucorum, doums et al. (2002) used the ability to encapsulate a nylon filament as a measure of immunocompetence, a fairly direct measure of cellular immunity. in the range of 30-day aging periods, they recorded reduced encapsulation and slightly increased fat body sizes as workers aged. senescence did not influence hemocyte population sizes in these experiments. later work with b. terrestris, however, revealed a more complex picture (moret and schmid-hempel, 2009). these authors recorded reduced immunocompetence, measured by zone of inhibition assays, reduced hemocyte populations and reduced po activities in immune challenged workers as they aged. they also recorded increases in hemocyte populations and po activities in workers taken from older colonies. they interpreted their findings with respect to colony fitness, suggesting that older colonies are more prone to infection due to increased numbers of individual bumble bees and other factors. older colonies also produce sexuals, which are essential to the reproductive fitness of the colonies. in this view, the expectation is the older colonies would produce workers with enhanced immunocompetence. such pliable adjustments in individual immunity may optimize colony-level fitness. this view raises important questions about the mechanisms involved and also suggests that immunosenescence at the individual level may be altered somehow. as with honeybees, it is difficult to make absolute judgments about immunosenescence in eusocial contexts. practical significance of immunosenescence in invertebrates i finish this essay with a few comments on potential role of immunosenescence in two areas of applied invertebrate biology. these are untested speculations meant to stimulate discussion rather than suggest specific actions. although invertebrate immunosenescence is documented, it remains an open field with a great deal of new knowledge awaiting discovery. aside from tremendous contributions to understanding of human senescence and immunosenescence, knowledge of invertebrate immunosenescence may be applied to existing problems in invertebrate aquaculture and in insect pest management. aquaculture is a global-scale agricultural industry that produces large harvests of a wide range of edible invertebrates, including abalone, crab, clam, crayfish, oyster, mussel, scallop and shrimp for human consumption. the industry produces millions of metric tons of products with first sale values in billions of dollars (stentiford, 2011). aquaculture systems are subject to a wide range of disease agents, including bacterial, protistan, fungal, and viral afflictions. some lead to epizootics with very high mortalities and huge financial losses. others can lead to serious opportunity costs. in the case of lobster, for example, diseases have inhibited development of aquaculture systems (shields, 2011). a question, then, is whether the expanding knowledge of invertebrate immunity and immunosenescence can be applied to the goal of sustaining the health of commercial aquaculture systems. immunosenescence has not received sufficient attention in cultured invertebrate species; however, if the situation in insects is similar to other invertebrates, it is reasonable to expect reduced immune capacity in older invertebrates. the corollary, of course, is older invertebrates are more susceptible to disease than younger ones. aside from this sort of generalization, research into invertebrate immunity and immunosenescence may contribute to understanding and possibly mitigating the influences of global climate change on farmed and wild invertebrate species. matozzo et al. (2012), for example, investigated the influence of acidification and temperature, two parameters of global climate that potentially affect the health of seafood species. they worked with two bivalve species, the clam chamelea gallina and the mussel mytilus galloprovincialis. the authors maintained the animals for 7 days at three ph values, two temperatures and three salinity values. salinity of seawater, as practical salinity units (psu, a dimensionless quantity) is approximately 35. exposing c. gallina, to artificial seawaters with lower (28 psu) and higher (40 psu) salinities led to increases in circulating hemocytes at 22 oc. the 107 authors considered several other immune parameters, as well, and concluded that global climate changes can exert powerful effects on bivalve immunity by influencing hemocytes. we turn to the significance of insect immunosenescence in developing biological control technologies to manage insect pest populations. tunaz and stanley (2009) carried out a field study of infections in pest insects captured in agrarian fields, and reported that virtually all of the hundreds of examined pest insects had internal, melanized nodules, sure signs the insects had been infected in their pasts and had recovered from the infections to continue consuming crop plants. one of our conclusions from this work is insect immunity may impose limits on the efficacy of some microbial biocontrol agents or even naturally occurring microbes. taken in the context of immunosenescence, there is considerable interest in disabling insect immunity or greatly increasing the rate of immunosenescence. shrestha et al. (2010) reported on using gene silencing to disable insect immunity. they identified five genes that act in signaling and coordinating insect cellular immunity in the red flour beetle, tribolium castaneum. the five genes encode slightly different phospholipases a2 (pla2s), enzymes responsible for releasing arachidonic acid from cellular phospholipids, the first step in biosynthesis of prostaglandins. prostaglandins are key signal moieties in cellular immune reactions. silencing these genes by injecting dsrna constructs designed to separately complement mrnas for each pla2 led to reduced immunocompetence in experimental beetles. immunocompetence was determined by counting nodules, the quantitatively predominant cellular immune reaction to bacterial infections. this work serves as a proof of the concept that insect immunity can be disabled by silencing target genes. it also opens possibilities to silence genes encoding other key proteins in insect immune systems, such as genes acting in 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db, et al. genome-wide transcript profiles in aging and calorically restricted drosophila melanogaster. curr. biol. 12: 712-723, 2002. zhan s, merlin c, boore jl, reppert sm. the monarch butterfly genome yields insights into long-distance migration. cell 147: 1171-1185, 2011. 109 autophagy and autophagic cell death in bombyx mori isj 12: 103-108, 2015 issn 1824-307x minireview autophagy studies in bombyx mori l tian1,2, s li2 1state key laboratory of silkworm genome biology; southwest university, chongqing, 400716, china 2key laboratory of developmental and evolutionary biology, institute of plant physiology and ecology, shanghai institutes for biological sciences, chinese academy of sciences, shanghai 200032, china accepted march 4, 2015 abstract autophagy, which is well conserved from yeast to mammals, plays essential roles in development and diseases. using the domesticated silkworm, bombyx mori, as a model insect, several reports on autophagy have been made recently. autophagic features are observed in the midgut and fat body during the larval-pupal transition as well as the silk gland and ovarian nurse cells during the pupal stage. there are 14 autophagy related (atg) genes, including at least two transcript variants of atg1, predicated in bombyx. expression of most atg genes is consistent with the autophagy process in the fat body during the larval-pupal transition, and reduction of atg1 expression by rnai blocks this process. the molting hormone, 20-hydroxyecdysone (20e), and starvation induce autophagy in the fat body by upregulating atg gene expression and blocking the pi3k-torc1 pathway. meanwhile, autophagy precedes apoptosis in the midgut and other larval tissues during the larval-pupal transition, while the detailed mechanism is not illustrated yet. we assume that there are at least four future directions about autophagy studies in bombyx during the next years: (1) physiological functions of autophagy; (2) identification of new components involved in the autophagy process; (3) detailed molecular mechanism of autophagosome formation; (4) functional relationship between autophagy and apoptosis. key words: autophagy; atg genes; 20-hydroxyecdysone; bombyx mori   introduction macroautophagy (hereafter referred to as autophagy) is well conserved from yeast to mammals. autophagy was first discovered in the mouse kidney with membrane-bound compartments termed “dense bodies”, which were subsequently shown to include lysosomal enzymes (clark, 1957; novikoff, 1959). autophagy is responsible for bulk degradation of intracellular long-lived proteins, superfluous organelles, or clear of invading microorganisms, playing key roles in many physiological and developmental processes, such as cell survival, cell death, metabolism and innate immunity (yang and klionsky, 2010). under certain circumstances, weak autophagy helps cell survival, whereas extensive autophagy leads to cell death (shintani and klionsky, 2004). autophagy is involved ___________________________________________________________________________ corresponding authors: ling tian, sheng li key laboratory of developmental and evolutionary biology institute of plant physiology and ecology shanghai institutes for biological sciences chinese academy of sciences shanghai 200032, china e-mails: tianling@sibs.ac.cn, lisheng01@sibs.ac.cn in the utilization of intracellular lipids to maintain cellular energy homeostasis: during fasting condition, autophagy is triggered to enclose droplet parts for lipid degradation, while fed with high-fat diet, blocking of autophagy causes lipid accumulation (singh et al., 2009). moreover, abnormal autophagy is related to many human diseases, such as neurodegenerative diseases (alzheimer's and huntington’s) and tumors (yang and klionsky, 2010). during the last decade, autophagy has been a hot topic in the biology field. the process of autophagy is marked by the formation of autophagosome and autolysosome. autophagy is governed by a series of autophagy-related (atg) protein complexes, and the core machinery of autophagosome formation involves at least four atg protein complexes. autophagosome initiation requires the ulk1/atg1-atg13 protein kinase complex. autophagosome nucleation involves the beclin-1/atg6-pik3c3/vps34-atg14l complex. autophagosome expansion and completion is governed by two ubiquitin-like conjugating systems: atg5-atg12-atg16l1 and atg8-pe conjugates (boya et al., 2013; jin and klionsky, 2013). the atg12-atg5-atg16l1 complex is formed via atg16 103 homooligomerization and acts like a dimer at the outer membrane of the phagophore disassociated from the phagophore near the time of autophagosome completion. unlike canonical ubiquitination, atg12-atg5 conjugation is irreversible (mizushima et al., 2003). atg8-pe conjugation localizes on both the outer and inner membrane of the phagophore, and atg8-pe is the only atg protein remained in mature autophagosome. therefore, atg8-pe is usually used as a marker of autophagosome formation (levine and klionsky, 2004). finally, the mature autophagosomes fuse with lysosomes to form autolysosomes, in which bulk degradation of dysfunctional proteins, unnecessary organelles, and invading microorganisms is completed (klionsky et al., 2012). autophagy is triggered in response to various unfavorable conditions, such as starvation. starvation triggers autophagy by inhibiting pi3k-akt-torc1 pathway combined with inducing expression of some atg genes. under favorable conditions, torc1 phosphorylates and inactivates the ulk1/atg1-atg13 protein kinase complex to inhibit autophagosome initiation (levine and klionsky, 2004). under glucose deprivation, the energy sensor ampk not only inhibits torc1 activity, but also directly phosphorylates and activates the ulk1/atg1-atg13 protein kinase complex to induce autophagosome formation (lippai et al., 2008; egan et al., 2011). moreover, in response to amino acid starvation, the phosphorylation of beclin-1/atg6 by ulk1/atg1 is required for full autophagic induction (kim et al., 2013; russell et al., 2013). in insects, the molting hormone (20-hydroxyecdysone, 20e) signaling, including 20e-ecr/usp complex and its downstream transcriptional factors, induces autophagy by blocking pi3k-akt-torc1 pathway and upregulating atg genes (baehrecke, 2003; tian et al., 2013; tracy and baehrecke, 2013; liu et al., 2013, 2014; yin and thummel, 2005). apoptosis is the major type of programmed cell death (pcd) in organisms, autophagy is considered as the second type of pcd, and the relationship between autophagy and apoptosis is complex. under certain circumstances, autophagy prevents cell death from apoptosis, whereas extensive autophagy will cause cell death (shintani and klionsky, 2004). dying cells often display accumulation of autophagosomes, and adopt a morphology called autophagic cell death. it is usually agreed that this case is cell death with autophagy rather than cell death by autophagy (kroemer and levine, 2008). in drosophila, both autophagy and caspases function in parallel, contributing to autophagic cell death in the dying salivary gland during metamorphosis, but autophagy plays a more important role than caspases (berry and baehrecke, 2007; scott et al., 2007). moreover, autophagy, but not caspases, governs cell death in the midgut during metamorphosis (denton et al., 2009). a balancing crosstalk occurs between autophagy and caspase activity in the remodeling fat body, as the inhibition of autophagy induces caspase activity and the inhibition of apoptosis induces autophagy (liu et al., 2013, 2014). the domesticated silkworm, bombyx mori, emerges as a model organism not only for lepidopterans but also for general biology (xia et al., 2014). in view of the importance of autophagy, we here summarize the associated reports in bombyx and wish to inspire bombyx studies on autophagy in the future. autophagy detection in bombyx in insects, autophagy was first described in the larva of the butterfly, calpodes ethlius by morphological observation (locke and collins, 1965). guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes were recently emphasized by the autophagy consortium. methods corresponding to monitor the numbers or volume of autophagic compartments (e.g., autophagosomes or autolysosomes) and to measure autophagic flux are summarized (klionsky et al., 2012). morphological observation by transmission electron microscope (tem) is still the most important method to detect autophagic compartments. by observation using tem, the autophagic features, such as vacuoles, autophagosome, high-density bodies, and autolysosomes could be distinguished clearly in bombyx organs during metamorphosis (sumithra et al., 2010; franzetti et al., 2012; tian et al., 2013). atg8 is the most widely monitored atg protein, and atg8-pe is an efficient indicator of autophagy in mammals (klionsky et al., 2012). an antibody against bombyx atg8 was generated, which is able to detect the protein level of both atg8 and atg8-pe by western blotting (franzetti et al., 2012; tian et al., 2013) and the aggregated puncta of atg8 by immunochemistry (franzetti et al., 2012). the fusion of autophagosomes with lysosomes can be visualized by lyso tracker red staining, which successfully represents the trend of autophagy in the remodeling fat body as detected by tem (tian et al., 2013). in addition, the activity of acid phosphatase can partially and indirectly reflect autophagy (franzetti et al., 2012; tian et al., 2013). when autophagy is referred in bombyx and other lepidopterans, we suggest that at least two detection methods should be used simultaneously. for example, both tem observation and lyso tracker red staining are necessary and sufficient to detect autophagy in the bombyx fat body. atg genes and atg proteins in bombyx fourteen yeast or mammalian homologous atg genes (atg1, atg2, atg3, atg4, atg5, atg6, atg7, atg8, atg9, atg11, atg12, atg13, atg16 and atg18) were predicated in bombyx, most of which contain the conserved atg protein domains (zhang et al., 2009; tian et al., 2013). either atg1 or atg6 harbors a protein kinase domain at its n-terminus. crystal structure of atg8 reveals an ubiquitin-fold domain at its c-terminus, which is similar to atg8 proteins identified from other organisms. in addition, there are two helices at the n-terminus of bmatg8 (hu et al., 2010). until now, two transcript variants of bmatg1 were reported, which are evolutionary conserved with the orthologs from drosophila (casati et al., 2012). we found another atg1 transcript variant, which is shorter but more abundant than the previously reported ones (li et al., unpublished data). 104 induction of autophagy in bombyx 20e and starvation are the two important stimuli of autophagy in insects. in the anterior silk gland in bombyx, autophagy emerges right after the appearance of ecr-b1 protein as well as the transcripts of ecr, e74a and br-c, suggesting that 20e signaling induces autophagy in this organ (goncu and parlak, 2009; li et al., 2010). as revealed by a series of cellular, biochemical, molecular, and genetic studies in the fat body, 20e induces autophagy by upregulating atg gene expression and blocking the pi3k-torc1 pathway (tian et al., 2013). starvation can trigger autophagy in the absence of 20e in drosophila (chang and neufeld, 2010; jin and klionsky, 2013). in the bombyx fat body, starvation not only blocks torc1 activity, which phosphorylates and inactivates the atg1-atg13 complex in feeding condition, but also upregulates some atg genes (tian et al., 2013). nevertheless, the induction of atg1 expression in the mdigut is much less significant than that in the fat body (casati et al., 2012). notably, 20e reduces food consumption and causes starvation-like condition during molting and pupation in bombyx, providing a correlation between 20e and starvation in the induction of autophagy (wang et al., 2010). the observations on autophagy during the bombyx metamorphosis suggest that autophagy may play a role in preventing the onset of cell death under certain nutrient-deprivation conditions (romanelli et al., 2014). in addition, infected by a tachnid parasitoid, exorista bombycis，cell death is induced in the bombyx larval integumental epithelial cells with existence of atg5 protein and upreguation of atg5 expression, which are associated with signs of autophagy (anitha et al., 2014). this report may inspire the studies on how autophagy is involved in immune responses after parasitoid or pathogen infection. induction of autophagy in bombyx is summarized in figure 1. autophagic cell death and apoptosis in bombyx the relationship between autophagy and apoptosis is context-specific in drosophila (liu et al., 2013). during bombyx metamorphosis, it appears that autophagy precedes apoptosis in general, and that autophagy might contribute to cell death in a variety of larval tissues, e.g., in the midgut and fat body during the larval-pupal transition as well as in the anterior silk gland and ovarian nurse cells during the pupal stage. in the remodeling midgut, autophagy precedes apoptosis and gradually synergizes together to mediate its demise (franzetti et al., 2012). autophagic features were observed in the disintegrated perivisceral fat body cells, and considered to attribute to autophagic cell death (sumithra et al., 2010). in the peripheral fat body tissues isolated from the 5th abdominal segment, there was co-existence of autophagy and caspase activity during the larval-pupal transition (tian et al., 2012, 2013). in the anterior silk gland, autophagy appears earlier than apoptosis, and they might interact with each other during its degeneration process (goncu and parlak, 2008; li et al., 2010). during the middle stage of vitellogenesis, paraptosis precedes both apoptosis and autophagy, which in later stage operate synergistically to result in a more efficient elimination of the degenerated nurse cells (mpakou et al., 2006, 2008). fig. 1 induction of autophagy in b. mori. the upstream stimuli of autophagy in bombyx include 20e, starvation and parasitism. 20e induces autophagy mainly by upregulating atg gene expression and the inhibition of torc1 activity. 20e reduces food consumption and causes starvation-like condition. starvation leads to autophagy mainly by the inhibition of torc1 activity and partially by the induction of atg gene expression. parasitism might cause autophagy by the induction of atg5 expression. 105 in bombyx spc bm36 cells, amino acid starvation rapidly induces autophagy, inhibition of autophagy at the early stage of starvation results in necrotic-like cell death. in addition, parasitism causes autophagy preceding apoptosis in bombyx integumental epithelium (anitha et al., 2014). these data indicate that autophagy prevents bombyx cells and other lepidoptera insect cells from death at an early stage of non-favorable conditions (wu et al., 2011; romanelli et al., 2014). the underlying molecular mechanism of the functional relationship between autophagy and apoptosis requires further investigation in detail. future directions despite several reports on autophagy have been made in bombyx during the last years, autophagy is still a new research area in this model insect. we assume that there are at least four future directions about autophagy studies in bombyx during the next years. 1. physiological functions of autophagy previous studies suggest that autophagy plays essential roles in cell survival and cell death in bombyx and other insects. most likely, during bombyx metamorphosis, weak autophagy helps cell survival at the onset of cell death, whereas extensive autophagy exaggerates and eventually causes cell death. autophagy is suspected to recycle of cell components derived from larval midgut degeneration (franzetti et al., 2012, 2015). moreover, it has been long thought that autophagy is involved in the mobilization of stored nutrients in the larval fat body, but little evidence has been provided and no detailed studies has been reported. we suppose that autophagy might play essential roles in regulating cell fate and metabolism during bombyx metamorphosis. with the rapid development of powerful genetic tools, including systematic rnai, baculovirus-mediated overexpression, piggybac-mediated gene function, and talenor cas9-mediated genome editing, we should be able to deeply investigate the physiological functions of autophagy in bombyx within the coming years. 2. identification of new components involved in the autophagy process in recent decades, autophagy is well documented in yeasts, but many questions remain in high organisms. although many atg genes are conserved from yeast to insects to humans (chang and neufeld, 2010; malagoli et al., 2010), the molecular mechanism how atg proteins collaborate to form autophagosome and to originate autophagosome membrane are largely unknown in higher eukaryotes, and these questions are worthy of further investigation in bombyx. so far, only 14 homologous atg genes have been predicted in bombyx, we suspect that many more atg genes in this organism are still unknown. using those known atg proteins as baits to prey their interacted proteins via immunoprecipitation and yeast-two-hybrid, we should be able to identify some new components involved in autophagy in bombyx. this study will be very meaningful from both functional and evolutionary views. 3. detailed molecular mechanism of autophagosome formation once the new components in the four atg protein complexes have been identified, we should have a better idea about the detailed molecular mechanism of autophagosome formation in bombyx, at least from a comparative prospective. it will be very interesting to examine how the atg protein complexes are modified at the post-transcriptional levels. a detailed bioinformatic analysis revealed that the phosphorylation sites identified from mammalian ulk1 are not conserved in the insect atg1 proteins (li et al., unpublished data). we are currently investigating phosphorylation modifications of the bombyx atg1-atg13 protein kinase complex by ampk and torc1, which might be different from the drosophila atg1-atg13 protein kinase complex as well. a comparative analysis of the phosphorylation sties in the atg1-atg13 complex will shed light on the evolution of autophagosome formation from yeast to insects to humans. 4. functional relationship between autophagy and apoptosis the functional relationship between autophagy and apoptosis is complex, and it is context-specific in drosophila. although it is known that autophagy precedes apoptosis in bombyx, the detailed molecular mechanism has not been understood yet. in mammalian cells, atg4, atg5, and beclin-1/atg6 act as the molecular switches between autophagy and apoptosis (betin and lane, 2009; yousefi et al., 2006; wirawan et al., 2012), 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contraction. recently, myosin research has made considerable progress. however, the function of bombyx mori myosin remains unclear. in this study, we cloned the bmmyosin ii essential light chain (bmmyosin ii elc) gene from a cdna library of silkworm, which had an open reading frame (orf) of 444 bp encoding 147 amino acids (about 16 kda). after analyzing their sequences, bmmyosin ii elc was similar to the elcs of 27 other myosin ii types, which contained efh domain that bound ca2+. in addition, 28 sequences had five motifs, motifs 1 and 3 were relatively conserved. we constructed two vectors with bmmyosin to transfect mgc803 or bmn, monolayer wound healing of cells indicated they can promote cell migration successfully. for three fifth instar silkworms, bm306, bmnb, bmbc8, we mainly analyzed the change of bmmyosin ii elc from transcription and translation after infecting with nucleopolyhedrovirus (bmnpv). we found that gene expression of resistant strains were higher than susceptible strains at 12 h, while the result of the translation level was opposite that of the transcription level. through in vitro protein interactions, we found bmmyosin ii elc can interact with bmnpv ubiquitin. key words: bombyx mori; cell migration; myosin; ubiquitin   introduction khne (1859) proposed that myosin could be extracted from frog tissue. myosin, as the unit of myofibril, was composed of many heavy chains and light chains and divided into three areas, spherical head, neck and tail (kwon et al., 2014), (sweeney et al., 2010) regarded myosin as a kind of molecular motors , which played an important role in muscle contraction, chemotaxis cytoplasmic division, cell migration and vesicular transport signal transduction and so on (andruchov et al., 2006). myosin is actually a superfamily of chemicals. to date, we have found 24 kinds of myosin, and have divided them into traditional and unconventional myosin based on the myosin source. specifically, myosin ii is considered to be a traditional form of myosin. unconventional myosin refers to myosin not found in muscle myosin tissue, such as myosin i, iii, iv, v, exists only in the muscle cells, while myosin viii, xi and xii only exist in plants. in recent years, myosin ii has been a frequent topic of research, the protein coiled into the shape much like the letter y. myosin ii ___________________________________________________________________________ corresponding author: keping chen insitute of life science jiangsu university 301 xuefu road, zhenjiang 212013, china e-mail: kpchen@ujs.edu.cn has six protein polymers, including two heavy chains of about 220 kda, two essential light chain of about 17 kda and two regulatory light chain of about 20 kda (santos et al., 2007). according to the study, most myosin light chains were ef-hand superfamily members (kolodney et al., 1999). recently, some studies found that myosin plays a crucial role in cell growth and motion (holmes et al., 2000), and light chain of myosin ii has specific functions. therefore, the light chain of myosin has become a hot research topic. in higher mammal cells, phosphorylation of the myosin light chain can promote cell migration (straight et al., 2003). the function of myosin in silkworm (bombyx mori), a representative lepidopteran insect, remains unclear (wang et al., 2007). our previous unpublished research found that essential light chain of myosin ii was related with nucleopolyhedrovirus (bmnpv). therefore, we chose the mysin ii essential light chain of the b. mori (bmmyosin ii elc) infected with bmnpv (t3 strain) to study change of protein expression. then through protein interactions in vitro, we predicted the protein of bmnpv which interacted with bmmyosin ii elc. simultaneously, we attempted to determine whether bmmyosin ii elc promotes migration in insect and mammalian cells. 38 mailto:kpchen@ujs.edu.cn fig. 1 myosin ii essential light chain amino acid sequence homology analysis and the related genbank for 28 species analyzed here. biston betularia (aep43792); antheraea pernyi (agl33708); riptortus pedestris (ban21409); riptortus pedestris (agm32403); zootermopsis nevadensis (kdr17497); apis cerana (aey59302); harpegnathos saltator (efn85742); crangon spp (acr43477); marsupenaeus japonicus (add70028); procambarus clarkii (afp95338); drosophila melanogaster (aaa28710); pseudodiaptomus annandalei (agt28473); lepeophtheirus salmonis (aco13186); setaria digitata (act15365); caenorhabditis brenneri (acd86909); schistosoma mansoni (aar99584); bombyx mori (np_001040547.1); pararge aegeria (jaa88715); anopheles sinensis (kfb36116); aedes aegypti (abf18115); psorophora albipes (jaa94216); anopheles darlingi (etn67861); culex quinquefasciatus (eds45595); penaeus monodon (aet87131); litopenaeus vannamei (acc76803); gryllotalpa orientalis (aaw22542); bombyx mandarina (aci05250); papilio polytes (bam18881). identical residues are shaded in black, while similar residues are shaded in red, each species contains two efh domains,* indicates conservative sites. materials and methods analysis of bioinformatics on the essential light chain of bmmyosin ii the essential light chain of bmmyosin ii was used to find 28 similar protein sequences related to other invertebrates using the ncbi blast network service (http://au.expasy.org/tools/blast/). a neighbor-joining (nj) phylogenetic tree (dias et al., 2003) and the schematic distribution of conserved motifs among the defined gene clusters were found using online versions of mega5.0 and meme software. cell culture and monolayer wound healing assay in preparation for transit-2020 mediated transfections, mgc803 (human stomach cancer cells) and bmn (insect ovary cells) were plated in a 39 http://au.expasy.org/tools/blast/ fig. 2 neighbor-joining (nj) phylogenetic tree and schematic distribution of conserved motifs among the defined gene clusters. the unrooted phylogenetic tree (left side of the figure) from the complete protein sequence was depicted by the mega 5.0 program using the nj method. the tree shows the 28 phylogenetic subgroups (group i-group iii) with high bootstrap values. motifs were identified by means of meme software using the complete amino acid sequence of the 28 invertebrates (right side of the figure). 24-well plate at a confluency of 50 70 % (3 4×104 cells per well) in 500µl plating medium (dmem or tc-100 medium supplemented with 10 % fbs). before plating onto this plate, one black line was drawn on the underside of the well with a sharpie marker. the line would serve as fiducial marks for the wound areas to be analyzed. in preparation for making the wound, the free serum medium was used to prevent cell growth. two parallel scratch wounds of approximately 300µm width were made perpendicular to the marker line with a yellow p200 pipet tip (valster et al., 2005). then vectors were constructed with pcdna3.0-c-flag-myosin ii elc and piz-c-flag-myosin ii elc to transfect corresponding the cells of mgc803 and bmn. the wounds were observed using a microscope. images were taken at regular intervals over a 0 48 h period of both areas flanking the intersections of the wound and the marker lines. images were analyzed by digitally drawing lines (using adobe photoshop) averaging the position of the migrating cells at the wound edges. the cell migration distance was determined by measuring the width of the wound divided by two and by subtracting this value from the initial half-width of the wound (eccles et al., 2005). the preparation of bmmyosin ii essential light chain polyclonal antibody the recombinant plasmid pgex-5x-3-myosin ii elc was transformed into e. coli bl21, which were incubated at 37 °c in liquid lb culture media containing 50 mg/ml ampicillin. expression of the gsttag fusion protein was induced by adding iptg to a final concentration of 1 mm before another 5-h incubation. purification of the fusion protein was accomplished using glutathione-sepharose beads. polyclonal antibody of bmmyosin ii elc was prepared by immunizing a laboratory rat (rattus rattus) using purified myosin ii elc as antigen (zhang et al., 2012). 40 fig. 3 the motif 1 and motif 3 are highly conserved among all 28 myosin ii elc proteins. the overall height of each stack indicates the conservation of the sequence at that position, whereas the height of letters within each stack represents the relative frequency of the corresponding amino acid. (1) and (2) show motif 1 and 3 amino acid conservative analysis, respectively. rna isolation and cdna synthesis of midguts strains of bmbc8, bm306 and bmnb infected bmnpv midguts of the 5th instar larvae of strains bc8, 306 and nb infected bmnpv (1×109 pfu/ml) at 0 h, 6 h, 12 h, 24 h, 48 h, 72 h were collected and washed with cold pbs buffer (137 mm nacl, 2.7 mm kcl, 4.3 mm na2hpo4 and 1.4 mm kh2po4). then all samples were frozen immediately in liquid nitrogen and stored at −80 °c until extraction of rna. total rna was extracted from each frozen sample with triol (generay biotech rnaex). dna was removed through digestion with rnase-free dnase i at 37 °c for 20 min. the rna was further purified with phenol-chloroform and precipitated with ethanol. the rnas dissolved in depc-treated ddh2o were used to synthesize cdnas with a thermo scientific revertaid first strand cdna synthesis kit (thermo-fisher scientific, new york, usa) by following the manufacturer's instructions (zhang et al., 2012). qpcr analysis qpcr primers were designed using the primer select program of the primerselect software. the primer pairs were as follows: (myosin ii essential light chain) forward primer, 5’-ggacaaaatcctacagag-3’, reverse primer, 5’-ccctgagagtcttcttgtc-3’. a sybr premix ex taq ii kit (takara biotechnology co., ltd. dalian, china) was used. qpcr was performed using an abi 7300 sequence detection system (applied bio systems, darmstadt, germany) under the following conditions: an initial cycle at 95 °c for 30 sec., followed by 40 cycles of 95 °c for 5 sec., 60 °c for 31 sec., one cycle of 95 °c for 15 sec., 60 °c for 1 min., 95 °c for 15 sec., and 60 °c for 15 sec.. each reaction was performed in triplicate in 96-well plates. the relative expression level was calculated using 2-δδct, where δδct = δct (target gene)δct (actin gene), δδct = δct(target gene)δct (maximum) (gareus et al., 2006). western blot analysis the protein of midguts were collected and ground to powder in liquid nitrogen followed by ripa lysis buffer (50 mm tris-hcl ph7.4, 150 mm nacl, 1 mm edta, 1 % np-40, 0.5 % sodium deoxycholate, 0.1 % sds, 1 mm pmsf). the protein concentrations were quantitated by the method of bca, in which bsa was used as the protein standard. protein samples were equalized and electrophoresed by 12 % sds-page and electrotransfered to polyvinylidene difluoride (pvdf) membranes. the membranes were blocked with 5 % milk in tbs. following incubation with purified anti-bmmyosin igg, the membranes were washed and incubated with hrp-labeled anti-mouse igg. 41 the membranes were washed three times with tbst, and scanned using a tmb membrane peroxidase substrate system (kirkegaard and perry laboratories inc., gaithersburg, md, usa) or was detected by electro-chemi-luminescence (ecl) and exposed to film. gst pull-down to directly obtain a function protein, the expression vector pgex-5x-3 was constructed and used to express the recombinant protein gst-bmmyosin ii elc. the protein was suspended on glutathione-sepharose beads in wash buffer (25 mm tirs-hcl, 150 mm nacl, 1 mm edta), bmn and bmn infected with recombinant bmnpv (his-ubiquitin) after two days were collected. then ripa lysis buffer (50 mm tris-hcl ph7.4, 150 mm nacl, 1 mm edta, 1 % np-40, 0.5 % sodium deoxycholate, 0.1 % sds, 1 mm pmsf) was used and the recombinant protein was combined. they were palced in 4°c refrigerator overnight. after washing three times by washing buffer, the beads were loaded on the 12 % sds polyacrylamide gels. western blotting was used to analyze the expression of ubiquitin using his tag mcab. results bmmyosin ii essential light chain cloning and sequence analysis based on the bmmyosin ii elc gene sequence (genbank: dq534197) from ncbi, the full length cdna was cloned. sequencing showed that its length was 1086bp, orf was 444bp, encoding protein was 16kda and pi was 4.49. the sequence fig. 4 the vectors of piz/v5-c-flag-myosin ii elc and pcdna3.0-c-flag-myosin ii elc transfected bmn and mgc803. analysis of myosin ii elc by western blotting using flag monoclonal antibodies. found here was the same as the database sequence. on the base of bmmyosin ii elc, we found 28 similar protein sequences of other invertebrates on the ncbi blast network service website (http://au.expasy.org/tools/blast/) that were used to analyze homology. according to the predicted myosin ii elc structure domain we found that each species contained two identical efh domains, which were ca2+ binding sites (fig. 1). an un-rooted neighbor-joining (nj) phylogenetic tree was generated using mega5 software based on the alignment of the corresponding myosin ii elc complete protein sequences. the results showed that it was similar to the sequences of pararg aegeria. for statistical reliability, we conducted bootstrap analysis with 1,000 replicates. the 28 members of the myosin ii elc were subdivided into fig. 5 (a) monolayer wound healing of cells. phase micrographs of mgc803 human stomach cancer cells at various times after monolayer wounding (left side of the figure). mgc803 showed that the migration of pcdna3.0-c-flag-myosin ii elc transfected mgc803 at 0h, 24 h, and 48 h. mock showed that the migration of pcdna3.0 transfected bmn at 0 h, 24 h, and 48 h, there was no obvious migration at any time. phase micrographs of bmn insect ovary cells at various times after monolayer wounding (right side of the figure). bmn showed that the migration of piz/v5-c-flag-myosin ii elc transfected bmn at 0 h, 24 h, and 48 h. mock showed that the migration of piz/v5 transfected bmn at 0 h, 24 h, and 48 h. (b) quantification of cell migration using the monolayer wound healing assay. the means values of three measurements are shown for each point in time and condition. 42 http://au.expasy.org/tools/blast/ fig. 6 (a) analysis of gene of myosin ii elc expression was performed by rt-pcr , optical density between myosin and actin on bm306 at 0 h 72 h after infecting bmnpv, indicating expression was higher at 12 h than other time. (b) analysis of gene of myosin ii elc expression was performed by qpcr at 12 h for three strains of bombyx mori after infecting with bmnpv. graphs depict the mean relative fold changes and are representative of three replicate plates. (c) western blotting analysis of the expression levels of of myosin ii elc in three strains of bombyx mori after infecting with bmnpv at 12 h using its polyclonal antibody. three subgroups, designated groups i, ii and iii. bmmyosin ii elc was contained in group ii based on the clade with at least 50 % bootstrap support. according to the meme software (zhao et al., 2014 http://meme.nbcr.net/meme/cgi-bin/meme.cgi), bombyx mori had five motifs, followed by motif 5 (yellow) corresponding to 3 31 amino acids, motif 2 (blue) corresponding to 32 61 amino acids, motif 4 (pink) corresponding to 65 85 amino acids, motif 1 (light green) corresponding to 90 121 amino acids, and motif 3 (red) corresponding to 126 146 amino acids (fig. 2). all 28 sequences had motifs 1 and 3, and meme prediction showed that the amino acid sequences of motif 1 (light green) and motif 3 (red) were relatively conserved (fig. 3). bmmyosin ii essential light chain promoted bmn and mgc803 migration after the protein expressing successfully we selected two different cell lines, mgc803 and bmn, and constructed two vectors (pcdna3.0 and piz/v5) to encode bmmyosin ii elc protein fused to the flag epitope tag at the cooh terminus (c-flag-bmmyosin ii elc) ( clarke et al., 2007). two days later after transfection, western blotting and flag specific antibodies can be to detect whether the target protein transfection succeeded (fig. 4). monolayer wound healing of cells showed that the target protein can promote cell migration in two cell lines (shimura et al., 2012 fig. 5a). through quantitative calculation, we drew the curve of cell migration and the effect of bmmyosin promotion of bmn became more apparent (fig. 5b). transcription and expression levels of the essential light chain of bmmyosin ii where bm306, bmbc8, and bmnb infected bmnpv we selected three different silkworm strains, strain bm306 which is susceptible to npv and a near isogenic line bmbc8 and bmnb is resistant to npv (kang et al., 2011). the 5th instar larvae of bmbc8, bm306 and bmnb were infected with bmnpv virus and midguts were collected at 0 h, 6 h, 12 h, 24 h, 48 h and 72 h. the rt-pcr results showed that there exits a higher level about bmmyosin ii elc about bm306 at 12 h (fig. 6a). to further analyze the bmmyosin ii elc expression after bm306, bmbc8, bmnb infecting bmnpv at 12h (zhang et al., 2012), we found the expressed levels in resistant strains bc8 and nb treated with bmnpv were also significantly higher than the susceptible strain 306 by qpcr (fig. 6b). to further confirm the result of qpcr, antibody against the bmmyosin ii elc protein was used to perform western blot analysis of bmnpv-infected midgut of b. mori. the results showed that a specific band was observed clearly in controls and samples of strains bm306, while the samples of strain bmnb and 43 bmbc8 were relatively weak (fig. 6c), which is opposite of the results from qpcr analysis. bmmyosin ii essential light chain interacted with ubiquitin of bmnpv to further analyze the relationship between bmnpv and bmmyosin ii elc, we expressed fusion protein with gst-bmmyosin ii elc, interacting with bmn and bmn infected recombinant bmnpv (his-ubiqutin) in vitro (elena et al., 2005), we found bmmyosin ii elc was relevant with the ubiquitin of bmnpv (fig. 7). discussion the study of myosin, as a kind of superfamily of proteins, has progressed in recent years. the myosin family contained a conserved structure domain of efh, and a ca2+ binding site was involved the domain. experimental studies have found that when ca2+ combined with calmodulin complexes, the serine on the 19th (ser19) ( komatsu et al., 2007) of the myosin light chain 20 will be phosphorylated, leading to activation of the myosin head mg2+-atpase, which hydrolysised atp to produce energy to make the conformation change. the conformation will cause myosin to bind to actin (spudich et al., 2001), which plays an important function in motion. bmmyosin ii elc had the same efh domain based on bioinformatics prediction. recently, the research has shown that cell migration is a process that is critical at many stages of embryonic development, and is essential for tissue repair and immune function. in this article, we proved that bmmyosin ii elc promoted cell migration in bmn and mgc803. in addition, we speculated the efh domain may be involved in the important functions. the experiment showed that the heavy chain of myosin was a substrate for murf 1 ubiquitin ligase activity, and that the protein of myosin heavy chain was ubiquitinated by murf 1 in vitro (clarke et al., 2007). previous research in our lab found bmnpv ubiquitin was relevant to bmmyosin ii elc by co-ip (unpublished data), in this article we chose bmmyosin ii elc as bait to prove an interaction between the target protein and bmnpv ubiquitin via pull down technique. of the three different silkworm strains tested here, showed the expression of bmmyosin ii elc had the opposite result in transcription and translation level at 12 h. the expression of bmmyosin ii elc in resistant strain was higher than it was in the susceptible strain at the transcription level, while the result was opposite at the translation level. we speculated that silkworm infected bmnpv at 12 h belonged to late stage, in which dna replicated and expressed viral structural protein (faulkner et al., 1997). insect cells have different abilities to detect the presence of a virus infection and initiate an apoptotic program (lacount et al., 1997). in addition, baculoviruses are able to interfere with apoptosis by the expression of apoptotic inhibitors (prikhod'ko et al., 1996), which induce encoding bmmyosin ii elc mrna of silkworm which was resistant to npv increase. research indicated that after acmnpv infects tn-368 cells, fig. 7 analysis of the interaction between bmmyosin ii elc and bmn infected with recombinant bmnpv (his-ubiqutin) by pull down in vitro. western blot analysis was used to check the expression of ubiquitin using his tag mcab. (a) bmn infected recombinant bmnpv (his-ubiqutin) interacted with gst-bmmyosin ii elc, there existed ubiquitin band (~10 kda), gst-bmmyosin ii elc was detected using gst tag mcab (~42 kda). (b) bmn infected recombinant bmnpv (his-ubiqutin) interacted with gst, no detectable ubiquitin band was observed. gst was detected using gsttag mcab (~26 kda). (c) bmn was infected with recombinant bmnpv (his-ubiqutin) in two days, verifying the expression of bmnpv ubiquitin (~10 kda). actin moves into nuclei and subsequently is polymerized from g-actin to f-actin, leading to reorganization of the cytoskeleton. when f-actin polymerized, some activators are called wiskott-aldrich syndrome proteins (wasps) and a homolog of wasp (pp78/83) (acmnpv orf9) is encoded by all lepidopteran npv genomes combined with arp2/3 complex (goley et al 2006) to be involved in nucleating the formation of f-actin filaments. studies of acmnpv with mutations of pp78/83 suggested nuclear actin polymerization was required for the coordination of nucleocapsid development. in addition, since pp78/83 is a virion structural protein that is localized to the basal region of the nucleocapsids, it has the ability to cause actin nucleation and subsequent polymerization to release atp to facilitate the movement of the nucleocapsids through the cytoplasm (o'reilly et al., 1998). as bmnpv and acmnpv are closely related to homologous orfs showing ~90 % nt and ~93 % aa sequence identity (gomi et al.,1999), this shows that the mechanism was approximately the same. there were actin and atp binding sites in myosin head. when the virus needs a large amount of g-actin, the host was likely to initiate prevention and control mechanisms to reduce the expression of bmmyosin ii protein which was resistant to npv, and uncombined with actin to defend itself against viral infection. acknowledgements grants from the national basic research program of china “973” (2012cb114604), the national natural science foundation of china (no. 31301919), and a project funded by the priority academic program development of jiangsu higher education institutions supported this work. 44 references andruchov o, andruchova o, wang y, galler s. dependence of cross-bridge kinetics on myosin light chain isoforms in rabbit and rat skeletal muscle fibres. j. physiol. 571: 231-242, 2006. clarke ba, 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communities in most ecosystems and comprise a large proportion of macro fauna biomass. their activities are beneficial because they can enhance soil nutrient cycling through the rapid incorporation of detritus into mineral soil. however, mucus production associated with water excretion in earthworm guts also enhances the activity of other beneficial soil microorganisms. earthworms alter soil structure, water movement, nutrient dynamics and plant growth. the medical value of earthworms has been known for centuries. the extracts prepared from earthworm tissues have been used for the treatment of numerous diseases since they are valuable source of proteins, peptides, enzymes and physiologically active substances. several studies have shown that the earthworm extracts contain different macromolecules which exhibited the variety of activities, such as antioxidative, antibacterial, antiinflammatory, anticancer etc. some of these activities are involved in wound healing process, using the earthworm preparation. in some countries the earthworms are used as a part of healthy food. they have very high nutritive value because their bodies contain the high percentage of various proteins. besides the human food, the earthworms are used in the feeding of animals (fish, chicken, etc.). key words: earthworms; food; medicine; soil   introduction from evolutionary point of view the earthworms (ews) are very old species. they survived over a million years due to their ability of adjusting to different environmental conditions. their living place is damp soil enriched with organic substances. they are breading through the skin, and they are very sensitive on changing the temperature, the light and on the touch. during the winter they are burying in deeper layer to protect from low temperature, and during the summer and dryness to protect from dehydratation (brusca and brusca, 2003). their muscle system is built with circular (segments) and longitudinal muscles and with their shrinkage and spread the ews are able to move. the body of ews is covered with small fluffs, which is important in environmental adjustment and for search of the food in the soil. waste products of ew ___________________________________________________________________________ corresponding author: mira d. grdisa division of molecular medicine rudjer boskovic institute bijenicka 54, 10 000 zagreb, croatia email: grdisa@irb.hr diet enrich the soil with nutritive substances, which stimulate the growth of plants. however, the ews are very important source of diet for numerous animals in the soil. they are hermaphrodites, meaning that the each individual has both female and male systems for reproduction. this characteristic also contributes to well environmental adjustment, because that the animals are reproduced very easily. the eggs are hutched in soil protected with capsule, which arises from the secrets from clitellum (front part). the capsule protects the young worm until complete maturation (pechenik, 2009). the ews are the major decomposers of dead and decomposing organic matter, and acquire their nutrition from the bacteria and fungi that grow upon these materials. they decompose the organic matter and make the major contributions to recycling the containing nutrients. the ews occur in the warmest soils and many tropical soils. they are divided into 23 families, more than 700 genera, and more than 7,000 species. their size ranges from an inch to two yards, and are found seasonally at all depths of the soil (pechenik, 2009). 38   mailto:grdisa@irb.hr this review partially comprises the published information about versatile use of the earthworms. earthworms in soil fertility the role of ews in soil fertility is known since 1881, when darwin published the book entitled “the formation of vegetable mould through the action of worms with observations on their habits”. thereafter, several studies have been published (wardle, 2007). the soil macro invertebrates play a key role in soil organic matter (som) transformations and nutrient dynamics through perturbation and the production of biogenic structures, resulting in amelioration of soil fertility and land productivity (mora et al., 2003; barious, 2007). som is an important active carbon reservoir and fundamental component for soil fertility. it contributes to a number of soil properties, such as soil structure, porosity, water retention, cationic exchange and ph buffering capacity (lal, 2004; weil and magdoff, 2004). for that purpose the soil aggregates have been proposed as the structural units within the soil that control the dynamics of som and nutrient cycling (tisdall and oades, 1982; lavelle and spain, 2001; chevallier et al., 2004; fonte et al., 2007; hong et al., 2011). the major component of soil fauna communities are the ews. in cultivated tropical soil organic matter they are often related to fertility and productivity. in such ecosystem the invertebrate communities, especially ews, may play an essential role in som dynamics by the regulation of mineralization and humification processes (bouche, 1977; lavelle and martin, 1992). the effects of ews on soil biological process and fertility level differ in ecological categories. anecic species are active in deep mineral layers of the soil, endogeic species live in the upper mineral layer of soil, and epigeic species live on the soil surface (jones et al., 1994). mostly, the combinations of these ecological categories are responsible for maintaining the fertility of the soil (sinha et al., 2003; bhadauria and saxena, 2009). ews have important role in supplying the nutrients (n, p, k and ca) through production of aggregates and pores (i.e., biostructures) in the soil and/or on the soil surface, by affecting its physical properties, nutrient cycling and plant growth (scheu, 2003; mora et al., 2005). the effect of ews on the dynamics of organic matter varies depending on the time and space scale considered (mora et al., 2005). in the humid tropical environment endogeic ews accelerate initial som turnover through indirect influence on soil c as entry of microbial activity (haynes and fraser, 1998; parmelee et al., 1998). also it has been reported that ews increase the incorporation of cover crop-derived c into macro aggregates, as well as into micro aggregates formed within macro aggregates (fonte et al., 2007). thus, the increased transfer of organic c and n into soil aggregates implies the potential for ews to provide som stabilization and accumulation in agricultural systems. in addition, ews also increase nitrogen mineralization through direct and indirect effects on the microbial community. the studies have shown that the amount of soil nitrogen available for plants was produced more by activity of ews than the total input through the addition of slashed vegetation, inorganic and organic manure, recycled crop residues, and weeds (bhadauria and ramakrishan, 1996). an important role of ews is the huge increase in soil ph. the influence of ews on n cycling seems to be defined by the type of cropping system and the fertilizer applied (mineral versus organic) (postma-blaauw, 1996). furthermore, the ews can also enhance the nutrient availability in system with reduced human influence, with respect to tillage, less using mineral fertilizer, and low organic matter content (brown et al., 1998; cortez and hameed, 2001). the role of ews in the enhancing soil fertility is ancient knowledge, but now is better explain by scientific results. more details about this subject have been reviewed by bhaduaria and saxena (2010). the most important family of ews in enhancing agricultural soil is lumbricidae, which includes the genus lumbricus, aporrectodea, and several others. lumbricides originate from europe and by human activities they have been transported to many parts of the world. earthworms in medicine the use of ews in a medicine was documented at very early date, in 1340 ad. (stevenson, 1930; reynolds et al., 1972). moreover, in the folk medicine (north american indians, doctors in east asia) the ews have been used for the treatment of various diseases (cooper et al., 2004). traditional chinese medicine has also widely used the ews for a long time. the research on the pharmaceutical effects of ews has been initiated along with the development of biochemical technologies. many bioactive molecules which can be consider as potential drug have been detected in the ews. these molecules exhibited different activities, such as fibrinolytic, anticoagulative, anticancer, antimicrobial and thus may be exploited for the treatment of variety diseases (cooper et al. 2012). immunological recognition the ew (phylum annelida, family lumbricidae) is one of the first organisms in the evolution that possess immunological recognition and memory. the ews like the other complex invertebrates produce several types of leukocytes and synthesize and secrete the variety of immunoprotective molecules. they possess innate immunity, as well as some functions associated with the adaptive immunity (allogenic tissue rejection) (cooper et al., 1995, 1999). the celomocytes involved in innate immunity, play a central role in the ew immune system (phagocytosis, releasing of lytic factors) (stein et al., 1977, 1981; cossarizza et al., 1996; beshin et al., 2002). the ew celomocyte cells also provide immune functions and possess several cd markers (cd11, cd24, cd45ra, cd45ro, cd49b, cd54 and cd90) associated with innate immunity (engelmann et al., 2011). immuno-protective molecules synthesized and secreted from celomocytes induce agglutination, opsonisation and lysis of foreign material. in addition, they are 39   employed in clotting reactions and phenoloxidase cascade (cooper et al., 2002; mohrig et al., 1996). the carbohydrates, so called lectins are the target recognition fragments for the ew agglutinins (kauschke et al., 2000). the proteases are also very important factors in the immune system with their contribution to the destruction of foreign materials (clot formation, complement activation) (söderhäll and cerenius 1998). the patterns of celomic fluid protease can be considered as species specific in ew (mohrig et al., 1989; kauschke et al., 1997, 2000). the level of protease pattern and activity in celomic fluid might be considered as promising biomarker in environmental monitoring studies (kauschke et al., 2007). more details about ews proteases can be find in review paper of pan et al. (2010). fibrinolytic activity the fibrinolytic system is responsible for the proteolytic degradation of fibrin and therefore plays a role in hemostasis and thrombosis (cesarmanmaus and hajjar, 2005). intravascular thrombosis, as a result of aggregation of fibrin in the arteries, is one of the main causes of cardiovascular disease. the main component of blood clots is fibrin. the clots arise from fibrinogen after thrombin action. formation of fibrin clot and fibrinolysis are normally well balanced in biological systems. however, the thrombosis can occur if fibrin is not hydrolyzed as a consequence of any disorder. the usual outcome of such thrombosis is myocardial infarction. the fibrin clots are dissolved by fibrinolytic enzymes. for that purpose plasminogen activator (t-pa), urokinase and streptokinase are mostly used. these enzymes exhibit low specificity for fibrin and have undesired side effect and are also relatively expensive. therefore, the search for other fibrinolytic agent from various sources continues. the ews are an attractive source of the fibrinolytic enzymes and various physiologically active compounds. fibrinolytic enzymes, which are potent and safe, have been purified and studied from several species of earthworm, including lumbricus rubellas and eisenia fetida. its therapeutic and preventive effects on thrombosis-related disease have been clinically confirmed. the presence of fibrinolytic activity in coelomic fluid or tissue homogenate from ews has been reported previously. first isolation of the ews fibrinolytic enzymes (efe) were published in 1980’s. in crude extract of ew l. rubellus mihara et al. (1983) found the lumbrokinase that exerted the fibrinolytic and thrombolytic activities. many authors have isolated and characterized similar enzymes from different species (e. foetida, lumbricus bimastus) (lu et al., 1988; hrženjak et al., 1991, 1998; cheng et al., 1996; lin et al., 2000; xu et al., 2002; wang et al., 2003; li et al., 2003; 2012), and since that time, the medical value of ews has been investigated in more details. most earthworm fibrinolytic enzymes showed distinctive high stability and strong tolerance to organic solvents and high temperature. it was found that ew extracts could significantly diminish the coagulation of platelets and promote the dissolving of thrombi in the blood. because of that, they should be used for the treatment of cardiac and cerebro-vascular diseases. after oral administration to the patients, ew fibrinolytic enzyme reduced coagulation of fibrin and blood platelets (ryu et al., 1994; lijnen et al., 1995; gao et al., 1999: zheng et al., 2000), and had no adverse effects on the functions of the nervous system, respiratory system, cardiovascular vessels, liver and kidneys (stein et al., 1982; valenbois et al., 1982; hirigoyenberry et al., 1990; cho et al., 1998). the fibrinolytic enzymes have very specific way of absorption. they can exert the biological function in circulation after the transport into blood through intestinal epithelium (fan et al., 2001). on the other hand the fibrinolytic enzymes like urokinase and tissue plasminogen activator could be administrated by intaraperitoneal injection rather than orally. the potential use of fibrinolytic enzymes in the prevention and treatment of serious cardiac and cerebro-vascular diseases has been very attractive in medicine and pharmacology. besides their use as therapeutics, fibrinolytic enzymes could be also used in degradation of organic waste products from the food and livestock industry (nakajima et al., 2000). in addition the ews are very cheap source of biologically active molecules. however, many details about fibrinolytic enzymes from ews have been reviewed by grdiša et al. (2009). hemostatic activity of tissue extract of e. foetida (g-90) was also proved in an in vivo system (mataušić-pišl et al., 2011). because of remarkable degree of fibrinolytic and anticoagulation activity this extract exhibited the significant effect on bleeding and coagulation times, after administration in wistar rats. the effect was very similar to that of heparin. therefore, g-90 could represent a new source of fibrinolytic and anticoagulation enzymes suitable for future application in human and veterinary medicine. a similar effect has already been seen with a crude extract of earthworms l. rubellus (nakajima et al., 1993; cho et al., 2004; popović et al., 2005). the authors have shown that mixing of blood with that extract prolonged the clotting time. the studies of different authors have also indicated that the celomic fluid of ews exhibits other biological functions, including bacteriostatic (cooper et al., 2004; popović et al., 2005), proteolytic (nakajima et al., 1993; wang et al., 2003), cytolytic (hemolytic) (popović et al., 2001; procházková et al., 2006; mataušić-pišl et al., 2011), and mitogen activity (hrženjak et al., 1993). antitumor activity antitumor effect of the macromolecules from ews has been determined in in vitro and in vivo studies. however, the interest in efe has also been increased. it has been shown that efe isolated from e. foetida exhibits antitumor activity against the human hepatoma cells in vitro and in vivo (chen et al., 2007). hepatocellular carcinoma (hcc) is the fifth most common cancer and the third leading cause of cancer related mortality worldwide (sherman and takayama, 2004). it seems that efe induced apoptosis in these cells. the results indicated that efe could be used in treatment of hepatoma. moreover macromolecular mixture (g90) from the tissue homogenate of e. foetida 40   inhibited the growth of melanoma cells in vitro and in vivo (hrženjak et al., 1993). such effects have also been seen with coelomic fluid of e. foetida. isolated coelomic cytolytic factor 1 (ccf-1) was capable of lysing different mammalian tumor cell lines (bilej et al., 1995). similar antitumor effect of the earthworm extracts has been detected by different authors (chen et al., 2001; hu et al., 2002; xie et al., 2003; yuan et al., 2004). antipyretic and antioxidative activities antipyretic activity has also been detected in the ews lumbricus spp. and perichaeta spp. (hori et al., 1974), as well as in the paste obtained from ew lampito mauritii (balamurugan et al., 2007). this activity was similar to that obtained with aspirin (ismail et al., 1992). the paste from l. mauritii has also shown remarkable antipyretic and antioxidative actions in the treatment of peptic ulcer in rats (prakash et al., 2007). after paracetamol-induced liver injury in wistar rats, the hepatoprotective potential of extract from l. mauritii has been observed (balamurugan et al., 2008). the protection of human body from free radicals is very important since it is connected with the retention of progress for many chronic diseases. non-enzymatic antioxidants such as glutathione, vitamins c and e, tocopherol and ceruloplasmin protect the cells from oxidative damage (aldrige, 1981). the enzymatic antioxidants, such as superoxide dismutase, catalase and cyclooxigenase protect the cells from lipid peroxidation and they are very important scavengers of superoxide ion and hydrogen peroxide (scott et al., 1991). discovery of antioxidative activity in different ew preparations has been promising (grdiša et al., 2001; balamurugan et al., 2007). antibacterial activity during the 700 million years of their existence, ews have evolved in the environment replete with microorganisms, some of which threaten their existence, therefore they have developed efficient defense mechanisms against invading microorganisms. there is a variety of relationships between ew and microbes: (1) microbe as food for earthworm, (2) microbes as nutritive material for growth and reproduction, (3) microbes-mostly gram positive, pathogenic are digested by ew and thereby facilitate multiplication of useful microbes in the gut and (5) microbes are distributed to new places in soil (ranganathan, 2006; parthasarathi et al., 2007). the molecules which defend the ews from microbes have been detected in the celomic fluid of lumbricus and eisenia (stein et al., 1982; valembois et al., 1982). this activity is attributed to some proteins, such as lysozyme and fetidins (hirigoyenberry et al., 1990; milochau et al., 1997). few reports are also available regarding antimicrobial agents from ews tissue (cho et al., 1998; popović et al., 2005). wound-healing activity many scientists and medical communities have been searching for way to improve wound care and promote wound-healing. skin wound healing is a complex process characterized by re-epithelization and restoration of the underlying connective tissue. a number of overlapping phases are involved. during this process, keratinocytes, endothelial cells, fibroblasts and inflammatory cells proliferate and/or migrate to the site of injury, interacting both with each other and with extracellular matrix (gailit and clark, 1994). cell migration and tissue remodeling which take place during the course of the woundhealing process require controlled degradation of extracellular matrix and activation or release of growth factors (vassilli and saurat, 1996). along the line of neo-vascularisation, matrix-generating cells move into the granulation tissue. these fibroblasts degrade the provisional matrix and respond to cytokine/growth factors by proliferating and synthesizing new extracellular matrix. the agents with biomedical potential, prepared or isolated from the ews, have been used in wound care. characteristics of ews homogenate/paste, which could contribute to better healing of wounds, have been reported (hrženjak et al., 1993, 1998; popović et al., 2001; grdiša et al., 2001, 2004; cooper et al., 2004; popović et al., 2005; balamurugan et al., 2007; prakash et al., 2007). mitogen, antibacterial, hemostatic and antioxidative activities have an important influence on the healing and epithelization of wound. macromolecules from ews also stimulate the synthesis of egf and fgf, the factors involved in epithelization process (grdiša et al., 2004). the earthworm preparations from l. rubellus and e. foetida promoted wound healing (li et al., 2000; mataušić-pišl et al., 2010). both preparations shortened the healing time by increasing epithelization, granulation and synthesis of collagen. pasta, obtained from ew l. mauritii, kinberg, exhibited variety of activities, such as antiinflammatory, antioxidative and hepatoprotective activities (balamurugan et al., 2007; prakash et al., 2007). due to the properties of animal extracts, they should be considered in the treatment of wounds as well as different human diseases. thus, the ews could be handy and low cost source of bioactive molecules that are involved in wound healing process. earthworms as a food despite unusual perception, the ews have been used as food for humans. due to high nutritive value and abundance of the proteins they are basic of healthy diet. according to the literature data the ews contain about 60-70 % of proteins (zhenjun et al., 1997; medina et al., 2003; zhenjun, 2005). these authors have also reported that the presence of essential amino acids, especially a tyrosine, is much higher in the body of ews than it is recommendation of fao. besides the human diet, the ews have been used in the feeding of fish (vassilli and saurat, 2010), as well as the chicken (taboga, 1980). the group of scientist of royal society (london, uk) have published (2003) the study about nutritive properties of the earthworms and their usage in the diet of yekuana people from venezuela (paoletti et al., 2003). in their diet they 41   are using two species: andiorrhinus (amazonindrilus) motto (righi et al., 1999), which live in drift of sludge, it is white and known under the name “motto”, and andiorrhinus (andiorrhinus) kuru (moreno and paoletti, 2004), which lives in forest soil, and it is known under the name “kuru”. the yekuna people eat them raw (uncooked) or after treatment in water at the temperature around 60 70 °c or after drying in the smoke. these smoked ews are considered as speciality and the price is very high. beside using the ews as food the folk doctors also advise their usage against malaria and anemia. investigation on these two species of ews, paoletti et al. (2003) have found 18 amino acids incorporated in the proteins, which represents 64.4 72.9 % of total mass of the ews. also they have found 20 minerals, such as calcium and iron (the content of iron in these species is 10 times higher than in soya bean), and some elements in trace, as well as omega-3 and omega-6 fatty acids. according to this data, the ews “motto” and “kuru” are assumed as the best food for fulfilling all requirements of the human body. some fatty acids (palmitate, oleic, octadecanoic acids) have been detected in e. foetida (roubathsadiqui and marcel, 1995) with the highest content before complete maturation. the same authors declared that eisenia foetida also contain the high percentage of proteins (58 %). many restaurants in mexico have on their menu variety of ews, and in japan the ews are very appreciate as a food in combination with soya been or supplementation in juices. on the other hand in some part of asia, africa and south america the ews have been introduce 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oyster; crassostrea corteziensis; immune response   introduction the oyster crassostrea corteziensis (hertlein, 1951), also known as the pleasure oyster or the cortez oyster, is a species distributed from the gulf of california (also known as the sea of cortez) to panama (cáceres-martínez et al., 2008). production of the oyster worldwide is ~4.5 million tons/year (fao, 2010), and specifically in mexico, around 50,000 tons/year are produced, of which 1,500 tons correspond to the species crassostrea corteziensis (conapesca, 2013). this species possesses great economic and ecological importance; however, due to its sessile nature and feeding through filtration (150 l/24 h), it can accumulate biological and chemical contaminants, substances that can alter the physiology of these molluscs (zuykov et al., 2013). polycyclic aromatic hydrocarbons (pah) are chemical contaminants that are frequently found in aquatic ecosystems; they are characterized by having high persistence in the environment, high toxicity, and the capacity of bioaccumulation (ramdine et al., 2012). naphthalene is one of the most important pah in this type of ecosystem due to ___________________________________________________________________________ corresponding author: manuel iván girón pérez laboratorio de inmunotoxicología universidad autónoma de nayarit secretaría de investigación y posgrado bd. tepic-xalisco s/n, cd. de la cultura amado nervo c.p. 63190 tepic, nayarit, méxico e-mail: ivan_giron@hotmail.com its presence in the soluble fractions of petrogenic oils (hansen et al., 2007), which constitutes one of the most toxic fractions of petroleum for marine life (vijayavel et al., 2004; vijayavel and balasubramanian, 2006; zambrano et al., 2012). the mechanisms of toxicity of the pah are through the bonding of these compounds with the hydrophobic sites of the cell’s macromolecules, which causes alterations in the physiology of these, especially in the immune system (vijayavel and balasubramanian, 2006; zambrano et al., 2012). the immune system of the oysters is composed of cellular and humoral mechanisms such as phagocytosis, a process that involves hemocyte ingestion and destruction, while humoral mechanisms include the following: 1) phenoloxidase (po), an enzymatic pathway activated by components of the cell wall of microorganisms, resulting in melanin, a molecule with fungistatic properties (nappi and vass, 1993; vargas-albores and barracco, 2001; luna-acosta et al., 2011); 2) lysozyme, an enzyme that hydrolyzes the bond between the β-1,4, n-acetylmuramic acid and the nacetyl glucosamine localized in the peptidoglycan layer of the bacterial cell wall (mcdade and tripp, 1967; itoh et al., 2010), and 3) nitric oxide (no), a molecule produced by hemocytes in the presence of antigens, with high cytotoxic and microbicidal capacity (rivero, 2006); in addition, no reacts with the superoxide anion (o2 –) during the respiratory burst and generates peroxynitrite (onoo–), 30    mailto:ivan_giron@hotmail.com fig. 1 activity of lysozyme in digestive gland of oyster (n = 10) exposed to sublethal concentrations a) 1 µg/l, b) 20 µg/l, and c) 50 µg/l of naphthalene (n) during 1, 3, 5, and 7 days, and control oysters: acetone (a), and seawater (c). the data are represented as the  ± standard error of the mean. different letters between the bars of the same group indicate a significant difference (p <0.05). compounds with high oxidant activity (glinski and jarosz, 1997; gopalakrishnan et al., 2011). notwithstanding this, the effectiveness of the defense mechanisms of oysters can be altered by contaminants, which causes greater susceptibility in organisms to infectious diseases (gagnaire et al., 2007). studies on the immunotoxicity of pah in molluscs have demonstrated drastic changes in the immunocompetence of this type of organism (sauve et al., 2002; gagnaire et al., 2004; thiagarajan et al., 2007). however, the majority of studies have mainly centered on the cell’s immune response (pichaud et al., 2008; hannam et al., 2010; croxton et al., 2012; giannapas et al., 2012) and, to date, there are no studies to our knowledge on the effects of naphthalene on the humoral immune response of the oyster c. corteziensis. thus, the objective of the present work was to evaluate the effect of subacute exposure to sublethal concentrations of naphthalene on the parameters of the humoral immune response (the activity of lysozyme, phenoloxidase, and no production) in the oyster c. corteziensis, a species native to the eastern tropical pacific. materials and methods animals crassostrea corteziensis oysters of commercial length and weight (8 ± 2 cm and 70 ± 30 g, respectively, and approximately 7 months old) were acquired at a local market in the state of nayarit, mexico and were immediately transported to the laboratory for their depuration during a 30 day period prior to experimentation (adamo et al., 1997). for this purpose, the oysters were maintained in a 50 l recirculation system with filtered seawater (salinity, 26 ± 2 %, ph 8.7 ± 0.2, and temperature, 26 ± 2 °c, 12 h:12 h dark-light cycles) and constant 31    fig. 2 activity of phenoloxidase in digestive gland of oyster (n = 10) exposed to sublethal concentrations a) 1 µg/l, b) 20 µg/l, and c) 50 µg/l of naphthalene (n) during 1, 3, 5, and 7 days, and control oysters: acetone (a), and seawater (c). the data are represented as the  ± standard error of the mean. different letters between the bars of the same group indicate a significant difference (p <0.05). aeration. they were fed daily with dehydrated alga spirulina dissolved in filtered seawater (castillorodríguez and garcía-cubas, 1984). experimental groups after the depuration period, the oysters (n = 10) were placed in glass fish tanks with 5 l of filtered seawater (without food, under the previously mentioned conditions) during a 24 h period for their acclimatization. after this time, the organisms were exposed to sublethal concentrations of naphthalene (1, 20, or 50 µg l–1). the naphthalene (sigmaaldrich, 99 %) was taken from a stock solution (0.5 g/l), utilizing acetone as solvent at a 1:2 proportion (hydrocarbon:acetone). three experimental groups were utilized (n = 30) per treatment: experimental group i: oysters in seawater; experimental group ii: oysters in seawater with acetone, and experimental group iii: oysters in seawater with naphthalene. the oysters were exposed to the hydrocarbon during 1, 3, 5, or 7 days, with daily exchanges of water for each fish tank. during the experiment, the oysters were fed daily with alga spirulina. extraction of hemolymph and digestive gland the hemolymph was obtained from the adductor muscle using a 1 ml syringe (0.7 mm×30 mm). the hemolymph of three oysters was grouped together (~600 µl) and mixed for the determination of no production. for extraction of the digestive gland, we utilized tweezers and dissection scissors, immediately determined their weight, and processed these for determination of lysozyme and po. lysozyme activity lysozyme activity was determined by means of the turbidimetric method described by demers and bayne (1997). the samples of the digestive gland were homogenized in kh2po4 buffer 100 mm at 20 % (p/v) ph 5.9 and were centrifuged (1,000×g for 10 min at 25 °c). the supernatant (25 µl) was placed in a 96-well plate together with 175 µl of lyophilized 32    fig. 3 nitric oxide (no) production in hemolymph of oyster (n = 10) exposed to sublethal concentration a) 1 µg/l, b) 20 µg/l, and c) 50 µg/l of naphthalene (n) during 1, 3, 5, and 7 days and control oysters: acetone (a), and seawater (c). the data are represented the  ± standard error of the mean. different letters between the bars of the same group indicate a significant difference (p <0.05). micrococcus lysodeikticus dissolved in kh2po4 (0.15 %). we immediately determined absorbance (450 nm) at time 0 and afterward at 15 min. the difference of absorbance (δabs) was interpolated on a standard curve (0.041 0.25 u g–1) of chicken egg lysozyme (cel). po activity detection of po activity in digestive gland was performed through the measurement of the transformation of l-dihydroxyphenylalanine (ldopa) into dopachrome, as described previously by luna-acosta et al. (2010). prior to the determination of po, the digestive-gland samples were homogenized in pbs solution 0.1 m, ph 7.3, and centrifuged (1,400×g for 15 min at 25 °c). an aliquot of the supernatant was placed in a 96-well plate with 100 μl of l-dopa (4 mg ml–1) as substrate. absorbance was determined immediately (time 0) and after 5 h. δabs was determined at 450 nm in a plate reader. the difference of absorbance was expressed as po activity and was corrected by the concentration of protein. as target, we utilized pbs solution mixed with 100 μl of l-dopa. nitric oxide production nitric oxide (no) production was evaluated as described previously by tafalla et al. (2002) by means of the griess reaction, which quantifies the nitrites (no2 –) present in the hemolymph. fifty-μl aliquots of hemolymph, together with 50 μl of sulfanilamide at 1 % and 50 μl of 0.1 % n-(1naphthyl) ethylenediamine, were incubated for 10 min at ambient temperature in a 96-well plate. the absorbance of the samples was measured at 545 nm in a plate reader. the molar concentration of the nitrites of each sample was determined by a standard nano2 reference curve (10 100 mm) 33    34    and, as target we utilized pbs solution together with sulfanilamide and n-(1-naphthyl) ethylenediamine. no production was corrected by the concentration of protein. determination of proteins total concentration of proteins was measured by means of the bradford method (1976) with modification (girón-pérez et al., 2013). the sample (50 µl) was added into a 96-well plate together with 250 µl of bradford reagent. the plate was incubated at ambient temperature for 10 min and absorbance was determined at 545 nm in a plate reader. a known concentration of the albumin (0.25 5.0 mg ml–1) was utilized to perform a standard reference curve (r2 = 0.983). statistical analysis for statistical analysis, we employed sigmastat® ver. 3.5. statistical software. we determined the normality and homogeneity of the data variances with the kolmogorov-smirnov test and the levene f test. for normal data distribution, we utilized analysis of variance (anova) followed by the bonferroni subtest. for non-parametric data, we used the kruskal-wallis test followed by a multiple tukey-type comparison. the statistical difference was determined with a level of p < 0.05. results effect of naphthalene on lysozyme activity the exposure of 1 µg l–1 of naphthalene during 3, 5, and 7 days significantly diminished lysozyme activity in oysters with respect to controls (fig. 1a), while the 20-μg l–1 concentration of the hydrocarbon induced an increase in the activity of the enzyme on day 5 (fig. 1b). on exposing the oysters to 50 μg l–1 of this hydrocarbon, we observed an increase in the activity of this parameter on days 5 and 7 (fig. 1c). effect of naphthalene on phenoloxidase (po) activity the activity of po in c. corteziensis with 1and 50 µg l–1 of naphthalene during days 1 5 showed a significant increase with respect to that of the controls (figs 2a, c). on the other hand, we observed a diminution in this parameter after exposure to 20 µg l–1 of naphthalene on days 1 and 3 (fig. 2b), as well as exposure to 1 µg l–1 on day 3 (fig. 2a). effect of naphthalene on the nitric oxide (no) production the results indicate that the no production in organisms exposed to 1 µg l–1 of naphthalene during 1 day diminishes significantly compared with that of the control; notwithstanding this, the concentration of this molecule did not change significantly with respect to the control in oysters exposed during 3, 5, and 7 days to this concentration (fig. 3a). with regard to the effect of exposure to 20 µg l–1 of naphthalene, we observed that the no production increased in the exposed oysters during 1 day at this concentration, while organisms exposed during 5 days presented a diminution of this molecule (fig. 3b). on the other hand, oysters exposed to 50 µg l–1 presented an increase and a decrease of no at days 1 and 3 post-exposure, respectively (fig. 3c). on the other hand, on comparing the response time of each parameter evaluated in the oysters exposed to naphthalene, the results indicate that no is the most sensitive biomarker with respect to time, followed by po and lysozyme (fig. 4), because it is clear that no production is affected in the first 24 h post-exposure to the hydrocarbon. discussion in bivalve molluscs, an effective immune response is essential for the maintenance of the health of the organism; any alteration in the mechanisms of immunity can be accompanied by consequences in the development of infections and can even have repercussions on growth, reproduction, and survival (blaise et al., 2002). in this regard, there are studies that show that pah cause effects on the immune system in molluscs, which could induce stimulation or immunosuppression, thus causing diminution of resistance to diseases (gagnaire et al., 2006; matozzo et al., 2009; hannam et al., 2010). the majority of studies on bivalve molluscs have focused on the cellular immune response, and data are scarce in which the effect is reported of this type of compound on the humoral immune response. in particular, there are, to our knowledge, no reports in which the immunotoxic effect of naphthalene is evaluated in humoral defense mechanisms (po, no, and lysozyme) in c. corteziensis. to our knowledge, this is the first study that evaluated the parameters of the humoral immune response in this species native to the coasts of the eastern tropical pacific that, in addition, has economic importance in the mexican pacific. in the immune system of bivalves, lysozyme is one of the most important bacteriolytic agents against various species of bacteria (gopalakrishnan et al., 2011). this enzyme is found in mucosal secretions and digestive gland, and additionally is released by hemocytes during phagocytosis and participates in the inactivation of pathogens. notwithstanding this, the activity of lysozyme can be affected by the presence of contaminants such as pah (ordás et al., 2007; gopalakrishnan et al., 2011). in the present study, lysozyme activity in the digestive gland of the c. corteziensis oyster was modified by exposure to naphthalene, because the enzyme’s activity diminished significantly after exposure to 1 µg l–1 (on days 3, 5, and 7 postexposure) and increased significantly at concentrations of 20 µg l–1 (on day 5) and of 50 µg l– 1 (on days 5 and 7) of the hydrocarbon. studies referring the effect of pah on this parameter indicate that benzo[a]pyrene inhibits lysozyme activity in the hemolymph of bivalves (gopalakrishnan et al., 2009; matozzo et al., 2009; gopalakrishnan et al., 2011). however, it has also been demonstrated that exposure to mixtures of pah, such as crude oil, increases the activity of this enzyme (oliver et al., 2003; ordás et al., 2007). fig. 4 time of global response of the immunological parameters evaluated in oysters exposed to naphthalene (n). each line represents one oyster exposed to the hydrocarbons (oyster 1 10: 1 µg/l; 11 20: 20 µg/l; 21 30: 50 µg/l) at different times. a) nitric oxide (no) production (um/mg of protein); b) phenoloxidase activity (δabs/min/mg of protein), and c) lysozyme activity (u/min/g of tissue). po is one of the enzymes that participate in the humoral immune response in bivalves. this enzyme is key in the process of melanization and participates in the recovery of antigens through a capsule of melanin, or in the direct elimination of microorganisms by toxic quinones produced during the production of melanin (soderhall and cerenius, 1998). there are studies that show that po activity is a sensitive immune parameter to exposure to pah, because this parameter is observed to be affected on increasing or decreasing its activity due to exposure to these contaminants. for example, in the mussel mytilus edulis, in the oyster crassostrea gigas, and in the abulone haliotis diversicolor exposed to fluoranthene, benzo[a]fluoranthene, and benzo[a]pyrene, respectively, po activity increases significantly (coles and pipe, 1994; bado-nilles et al., 2008; gopalakrishnan et al., 2011), while in c. gigas oysters exposed to the soluble fraction of heavy fuel oil (hfo) (bado-nilles et al., 2009) and to the soluble fraction of light crude oil (lco) (bado-nilles et al., 2010), the activity of this enzyme diminished. in this study, the po activity of this enzyme decreased. in addition, in this study, the po activity in the digestive gland of the oyster c. corteziensis was modified by exposure to the three concentrations of naphthalene evaluated and showed dysregulation in this parameter, because the activity of the enzyme diminished to 1 µg l–1 on day 3 and to 20 µg l–1 on days 1 and 3 postexposure and increased significantly with respect to the control after days 1 and 5 at 1and 50 µg l–1. no is an ubiquitous signalling molecule with immunoregulatory and antimicrobial effects (macmicking et al., 1997; rivero, 2006); due to its non-polar nature, it can pass easily through the membranes of pathogens, causing damage to the dna, proteins, and lipids; in addition, no also reacts with the superoxide anion (o2 –) generated during the respiratory burst (fang, 2004). the hemocytes of various marine molluscs, such as the mussel m. edulis, mytilus galloprovincialis, the oyster c. gigas, and the clam ruditapes decussatus, are capable of producing no in response to immunological stimuli; notwithstanding this, their production can be altered by exposure to pah (franchini et al., 1995; nakayama and maruyama, 1998; arumugan et al., 2000; torreilles and romestand, 2001; stefano et al., 2002; tafalla et al., 2002; novas et al., 2004). gopalakrishnan et al. (2011) reported, in abulone (haliotis diversicolor) exposed to 0.05 mg l–1 de benzo[a]pyrene during 14 21 days, an increase in the production of no; however, in exposure periods of 3 7 days, no effect was observed. our results indicate that the production of no in naphthalene-exposed c. corteziensis oysters diminished significantly after exposure to 20 and 50 µg l–1 during 1 day, while after exposure to 1-, 20-, and 50 µg l–1 during 1, 5, and 3 days, respectively, an increase was observed in this parameter. evaluation of the mechanisms of defense, on the one hand, can provide early warning signals due to exposure to contaminants (biomarkers) and, on the other hand, can indicate the degree of resistance to infections (hannam et al., 2009), because an increase in the activity of an enzyme (lysozyme and po) or of some molecule (no) implicated in immunity can be interpreted as a response of the organism to protecting itself against 35    36    antigens, a response that can be modulated by extrinsic factors such as xenobiotics. however, excessive activation of the mechanisms of defense can be harmful for the organism, while the inhibition of these mechanisms can be interpreted as immunosuppression (huggett et al., 1992; thiagarajan et al., 2006). in this manner, changes in the activity of enzymes or in the concentration of molecules can affect the survival of organisms when these are exposed to antigens. the results obtained evidence the immunotoxic effect of naphthalene on the oyster c. corteziensis. however, the data reported in the literature suggest that this hydrocarbon possesses an immunotoxic potential that is less than other pah, such as pyrene, benzo(a)pyrene, fluoranthene, among others. notwithstanding this, it is necessary to perform more studies in this field, in that this investigative work is, to our knowledge, the first approximation reported of the effect of naphthalene on this native species of the tropical pacific. acknowledgments the first author of 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molecular responses of bivalves to toxic dinoflagellates    c manfrin1, g de moro1, v torboli1, p venier2, a pallavicini1, m gerdol1 1department of life sciences, university of trieste, trieste, italy 2department of biology, university of padua, padua, italy accepted october 31, 2012  abstract dinoflagellates and other microalgae can produce a wide spectrum of toxic molecules, which are the main responsible of shellfish poisoning syndromes. during seasonal harmful algal blooms (habs), many filter-feeding marine invertebrates, including bivalve molluscs, can accumulate phycotoxins at extremely high levels, thus representing a serious threat to human health. furthermore, habs also have a severe impact on the aquaculture sector due to the forced prolonged closure of large harvesting areas. although the targets and mechanism of action of many phycotoxins have been extensively studied on vertebrate model organisms, so far just a little attention has been focused on their effects on marine invertebrates. here we provide an overview about the molecular response of marine bivalves to phycotoxins, with a particular focus on toxins produced by dinoflagellates. even though large-scale genomic and proteomic approaches on molluscs are still hindered by the limited molecular knowledge of these organisms, a few studies exploiting the most recent technological advances provide promising perspectives for a better comprehension of the mechanisms involved in shellfish toxicity and for the identification of molecular markers of contamination. key words: bivalve; phycotoxin; harmful algal bloom; dinoflagellates; microarray; gene expression; rna-seq    introduction  the largest component of the universe of algae, estimated to comprise between one and ten million different species, is represented by unicellular microalgae. some of them can produce, through complex and not completely understood biochemical processes, toxic compounds of various chemical composition and modes of action, collectively named ___________________________________________________________________________ corresponding author: alberto pallavicini department of life sciences university of trieste via giorgieri 5, 34127 trieste, italy e-mail: pallavic@units.it 184   list of abbreviations: asp: amnesic shellfish poisoning; aza/azt: azaspiracid/ azaspiracid shellfish poisoning; pbtx: brevetoxin; da: domoic acid; dsp/dst: diarrhetic shellfish poisoning/toxin; dtx: dinophysistoxin; gym: gymnodimine; hab: harmful algal bloom; nsp: neurotoxic shellfish poisoning; oa: okadaic acid; pltx: palytoxin; psp/pst: paralytic shellfish poisoning/toxin, ptx: pectenotoxin; spx: spirolides; stx: saxitoxin; ytx: yessotoxin. phycotoxins. in response to favorable environmental conditions, toxic microalgae can proliferate and/or aggregate to form dense concentrations, called “harmful algal blooms” (habs). phycotoxins are responsible of a number of human illnesses associated with the consumption of contaminated seafood and, in some cases, with respiratory exposure to aerosolized toxins. in fact, filter-feeding shellfish, zooplankton, and herbivorous fishes can ingest these algae and act as vectors to humans either directly (e.g. shellfish) or through further food web transfer to higher trophic levels. although seasonal micro-algal blooms are considered as a natural phenomenon, their frequency of occurrence appears to have increased in the recent years. certainly, the worsening of the hygienic characteristic of the aquaculture areas, the transportation of ship ballast water, water eutrophication and global climate changes are factors which altogether provide favorable conditions for the spreading and the growth of toxic algae, thus contributing to the increase of threats for human by the consumption of contaminated shellfish. unfortunately, algal toxins are not detectable by sight or smell and contaminated seafood appears normal and in most cases they are heat stable and thereby largely unaffected by cooking. mailto:pallavic@units.it fig. 1 geographical occurrence of shellfish poisoning syndromes. asp: amnesic shellfish poisoning; azp: azaspiracid shellfish poisoning; cfp: ciguatera shellfish poisoning; dsp: diarrhetic shellfish poisoning; nsp: neurotoxic shellfish poisoning; psp: paralytic shellfish poisoning the consumption of contaminated seafood often results in shellfish poisoning syndromes, which are classified according to symptoms and the chemical nature of the toxins involved. figure 1 summarizes the geographical occurrence of the six main poisoning syndromes; although some are endemic of specific areas, their altogether distribution clearly points out shellfish toxicity as a global problem for human health, which consequently have an important economic impact on aquaculture worldwide. international laws promoted by environmental monitoring agencies and food safety associations, impose the routinely control of the toxicity of seawater and market shellfish stocks. while these monitoring strategies prevent episodes of massive intoxication, the closure of aquaculture hatcheries, sometimes even for a very prolonged time, is responsible of severe economic losses in this sector. in the following section, we provide a brief overview of the main human syndromes associated with contaminated marine bivalves consumption. the causative algae, the toxin structure and the most characterizing symptoms are reported in table 1. main classes of shellfish poisoning syndromes amnesic shellfish poisoning (asp) is caused by the consumption of domoic acid (da) contaminated food. this phenomenon was first documented in 1987 in canada, with 105 cases of acute human poisoning, including 3 casualties, related to the consumption of contaminated mussels. da, produced by marine diatoms belonging to the genus pseudo-nitzschia, is a watersoluble tricarboxylic acid that acts as an analog of the neurotransmitter glutamate and is a potent glutamate receptor agonist. in mammals da intoxication cause both gastrointestinal and neurological disorders as headache, disorientation, short-term memory loss, brain damage, and in severe cases it can also be fatal. diarrhetic shellfish poisoning (dsp), a syndrome characterized by diarrhea, nausea, vomiting and abdominal cramps, is one of the most common pathologies associated to habs worldwide. diarrhetic toxins (dsts) are lipophilic molecules such as okadaic acid (oa) and the structurally related dinophysistoxins (dtxs), produced by dinophyis and prorocentrum spp. oa selectively inhibits protein phosphatases and modifies the phosphorylation state of many regulators of cellular processes involved in metabolism and various cell activities, causing diarrhea because of the impairment of the sodium secretion control by intestinal cells. the marine biotoxins called azaspiracids (aza) cause a syndrome similar to dsp (azp), although they chemically differ from any previously known toxin found in shellfish. aza accumulates in bivalve molluscs that feed on toxic microalgae of the genus protoperidinium, previously considered to be toxicologically harmless. another widespread syndrome caused by contaminated bivalve molluscs is the paralytic shellfish poisoning (psp), caused by saxitoxin (stx) and its analogues (gtxs), globally indicated as paralytic shellfish toxins (psts). dinoflagellates of the genus alexandrium, in particular a. minutum, a. catenella, a. tamarense and a. fundyense, are the most numerous pst producers and are responsible for psp blooms all around the world (fig. 1). in fact, almost 2.000 psp cases are reported per year in human, with occasional fatal consequences. psts inhibit the voltage-dependent 185   table 1 main shellfish poisoning syndromes: summary of the toxins involved, causative algal species and symptoms in human toxic syndrome toxins molecular structure toxic algae symptoms in human asp – amnesic shellfish poisoning domoic acid (da) and analogues pseudonitzschia spp. gastrointestinal disorders, headache, disorientation, short-term memory loss, brain damage, death in severe cases (todd et al., 1993) azp – azaspiracid shellfish poisoning azaspiracids (aza) and analogues protoperidinium crassipes, azadinium spinosum. diarrhea, nausea, vomiting, abdominal cramps (satake et al., 1998). dsp – diarrhetic shellfish poisoning okadaic acid (oa) and dinophysistoxins (dtxs) dinophysis spp., prorocentrum spp. diarrhea, nausea, vomiting, abdominal cramps (hallegraeff, 1995). nsp – neurotoxic shellfish poisoning brevetoxins (pbtx) karenia brevis (formerly gymnodinium breve and ptychodiscus brevis) nausea, loss of motor control, muscular ache, asthma (wang, 2008). psp – paralytic shellfish poisoning saxitoxin (stx) and analogues (gtxs) alexandrium spp., gymnodinium spp. nausea, diarrhea, abdominal ache, shortness of breath, dry mouth, confused speech, tingling lips, tongue, neck, face, arms, legs and toes (clark et al., 1999). 186   cfp – ciguatera fish poisoning only palytoxin (pltx) and analogues in bivalves, other toxins are responsible of cfp in fishes (ciguatoxin, maitotoxin, etc.) ostreopsis spp. (pltx, ovatoxin and analogues) lethargy, muscle spasms, tremor myalgia, cyanosis, respiratory distress, rhabdomyolysis, death in severe cases (ramos and vasconcelos, 2010) others yessotoxins (ytxs) lingolodinium polyedrum, gonyaulax spinifera, prorocentrum reticulatum. diarrhea (tubaro et al., 2010) others pectenotoxins (ptxs) dinophysis spp. diarrhea (draisci et al., 1996) others spirolides (spxs) and gymnodimine (gyms) karenia selliformis (gyms) alexandrium ostenfeldii (spxs) neurological symptoms (munday et al., 2004) sodium channel conductance causing the blockade of neuronal activity. symptoms include nausea, diarrhea, abdominal ache, shortness of breath, dry mouth, confused speech, tingling or burning sensations. neurotoxic shellfish poisoning (nsp) is caused by brevetoxins (pbtx); the main symptoms are gastrointestinal and neurological, including nausea, loss of motor control and muscular ache. the formation of toxic aerosol by wave action produces respiratory asthma-like symptoms, even though no fatalities have been reported so far. pbtx is produced by karenia brevis and binds with high affinity to the voltage-dependent sodium channel, altering its sensitivity and inhibiting its inactivation. the sixth and last group of shellfish poisoning 187   188   is the ciguatera fish poisoning (cfp), an intoxication caused by a heterogeneous group of toxins, including ciguatoxin, maitotoxin, palytoxin (pltx) and others. while the main concerns for human health derive from the consumption of contaminated fish, pltx and its analogues, produced by species of the ostreopsis genus, can also be accumulated in bivalves. likewise ciguatoxin, pltx also lowers the threshold for the opening of voltage-gated sodium channels in synapses of the nervous system, determining severe neurological disorders, including lethargy, muscle spasms, myalgia, cyanosis, respiratory distress, rhabdomyolysis and even death in severe cases. beside the toxins classifiable among the six large classes listed above, microalgae can produce an extremely broad spectrum of additional toxic compounds. some species of dinoflagellates, such as lingulodinium polyedrum, gonyaulax spinifera and protoceratium reticulatum produce yessotoxins (ytxs), structurally related to pbtx and ciguatoxins. ytxs cause diarrhea and therefore they were initially wrongly classified as dsts, only to be later assigned to a novel independent group after the discovery of their different mechanism of action. pectenotoxins (ptxs) belong to a group of polyether-lactone toxins that, like ytxs cause symptoms similar to dsp. they are exclusively produced by dinophysis spp., which have a large distribution worldwide. gymnodimines (gyms) and spirolides (spxs) produced by karenia selliformis and alexandrium ostefeldii, respectively are emerging lipophilic marine toxins that belong to a heterogeneous group of macrocyclic compounds called cyclic imines. since their discovery in the early 1990s, gyms and spxs are well known due their fast acting toxicity in mouse bioassay, by blocking the nicotinic receptors and causing neurological symptoms. moreover, dinoflagellates are certainly capable of producing several harmful compounds which have not been fully characterized yet. as a matter of fact, the biosynthetic potential of microalgae is underestimated and many algal natural products, including toxins, remain yet to be discovered. therefore it is not surprising that the number of phycotoxins isolated from marine microalgae continues to increase exponentially. nevertheless, dinoflagellates are not the only source of marine toxins, as certain diatoms (such as the da producing pseudo-nitzschia spp.) and several species of seawater cyanobacteria produce compounds hazardous to human health. while these toxins are extremely diverse both by chemical composition and by physiological effects, these aspects will be not discussed in the present review, which is mainly focused on the effects of toxic dinoflagellates. for a more comprehensive overview suggesting the reading of more specific literature on the topic. toxicological studies on human and other vertebrate model organisms    the study of marine toxins has been historically connected with the hazard they represent to human health. therefore, the large majority of toxicological studies performed so far have been focused on model vertebrates or human cell lines. moreover, the most used method for marine algal toxins detection is the mouse bioassay developed by the japanese ministry of health and welfare. although this outworn method has been used worldwide as the main tool for shellfish toxicity biomonitoring for several decades, it is currently being replaced by other tests, such as the enzyme-linked immunosorbent assay (elisa) and the highperformance liquid chromatography (hplc), more sensitive and reliable. in parallel to this test, routinely used for the detection of contamination, many research studies investigated more specific aspects of the toxic effects of marine drugs, unraveling their molecular targets, their mode of action and the kinetics of accumulation and detoxification in model organisms. mice and other vertebrates have been repeatedly used as model organisms for in vivo studies to better understand the effects of several classes of phycotoxins, including aza, oa, stx, and ytx, on human. also cell lines provide useful models: a number of in vitro studies clarified the mode of action of diverse phycotoxins, including ytx in different vertebrate and invertebrate cell lines, pltx in mcf7 cells, stx in cultured mice neurons, oa and aza in multiple human cell lines. moreover, genomics and proteomics methods applied to vertebrate models contributed to elucidate the molecular pathways acting in response to shellfish poisoning. phycotoxin effects on bivalves    a direct comparison between the widely documented molecular effects of phycotoxins on vertebrates and bivalves is definitely hindered by several obstacles. one of the key factors is the specificity of interaction of many toxins with their molecular targets, which are often membrane channels. the sequence divergence among species explains why bivalves are often completely insensible to many toxins having a lethal effect on human and other vertebrates and vice-versa. in fact, biotransformation processes which likely reduce toxicity in bivalves, can sometimes produce analogues which are even more dangerous to human than the original compound. the specificity of interaction between phycotoxins and their molecular targets is refined to the point that polymorphisms to single ion channels can even lead to dramatic differences in sensitivity within bivalve populations. nevertheless, over the past decades numerous studies have been carried out to explore the specific effects of phycotoxins on marine invertebrates, even though their focus has been mainly addressed on physiological adaptations and behavioral modifications. this bias on non-molecular studies is caused by the still limited genetic knowledge of these organisms. in fact, before the next generation sequencing era, the main pool of bivalve genetic knowledge came from large est collections, but the recent technological advances have quickly led to http://en.wikipedia.org/wiki/lingulodinium_polyedrum http://en.wikipedia.org/w/index.php?title=gonyaulax_spinifera&action=edit&redlink=1 http://en.wikipedia.org/w/index.php?title=protoceratium_reticulatum&action=edit&redlink=1 189   the availability of many transcriptomes and even to the complete sequencing of the oyster genome. studies of physiological responses    the rich literature documenting the behavioral, physiological and histo-pathological alterations in contaminated filter-feeding molluscs, despite not providing outright molecular data, represents an important base of knowledge both for a better planning of molecular biology experiments and for the interpretation of the results in their biological context. hemocyte parameters  both the humoral and the cellular defense have a crucial role in bivalves immunity. the hemocytes are the main players in the response to parasites and pathogens, through a complex, non-adaptive, non-specific, innate immune system; microbial killing results from the combined action of the phagocytic process with a broad spectrum of humoral recognition and defense factors. therefore, the study of the effects of phycotoxins on bivalve immunity necessarily takes into account the monitoring of hemocyte parameters. the feeding on psp dinoflagellate species determines an increase of hemocyte counts, accompanied by changes in the hemocyte subpopulations and alteration of the phagocytic activity in some species. on the contrary, some studies report non-significant effects on hemocyte number, morphology, or functions in crassostrea virginica, while others evidenced a strong individual variability in the response in ruditapes philippinarum. histo-pathological studies on mussels exposed to a. fundyense and p. minimum detected an inflammatory response consisting of degranulation and diapedesis of hemocytes into the alimentary canal and, as the exposure continued, hemocyte migration into the connective tissue surrounding the gonadal follicles. also other classes of toxins can trigger immune responses: experiments by mello and collaborators showed the variation of various hemocyte parameters in different bivalve species affected by natural dsp blooms. also in this case, a speciesspecific response was observed, demonstrating the presence of vulnerable (e.g. perna perna) and unaffected (e.g. crassostrea gigas) species. moreover, also gyms can induce the alteration of hemocyte parameters in r. philippinarum, while ytx, pltx and oa can increase the phagocytic activity of mytilus galloprovincialis hemocytes. valve activity, filtration rate and depuration from toxins    the closure of the shells and the reduction of the filtration rate are normally occurring mechanisms played daily by bivalves in response to the variation of the tide levels. furthermore, these mechanisms are also used by bivalves for isolating themselves from the external environment, in presence of negative conditions, such as pollutants and toxins, as well. this mechanism has been extensively used to study the effects of phycotoxins on mussel physiology, even though sometimes passive valve closure can occur in response to psts, which notoriously cause adverse effects on some species, such as burrowing incapacitation. different species display different valve activity modulation in response to paralytic dinoflagellates, so the reduction of the filtration rate is a key parameter used for the classification of bivalves as susceptible or resistant to psp. other negative effects on valve closure and filtration activity have been reported in various bivalve species in response to microalgae producing different toxin classes, such as gymnodinium mikimotoi, heterocapsa circularisquama, pseudonitzschia and azadinium spinosum. despite the reduction of the filtration rate, prolonged habs may determine a significant accumulation of toxins in bivalve tissues. different farmed species are often characterized by highly variable detoxification rates, thus requiring a continuous monitoring of toxicity even for a long time after the bloom event. even though speciesspecific differences in depuration have been evidenced for many toxin classes, including psts and dsps, the most outstanding example is given by da. in fact, most bivalve species (mytilids, in particular) can depurate da very quickly, but others, such as pecten maximus, show an extremely low depuration rate, retaining the toxin even for several months in its visceral tissues. this differential ability of depuration between species is often crucial for the long-term management of bivalve aquacultures. feeding and excretion    pseudofaeces production is a particularly important pre-ingestive mechanism preventing the animal’s ingestive capacity from being exceeded, but it also facilitates the process of particle selection, whereby less nutritious particles can be rejected and the quality of the ingested material can be improved proportionally. the most complete comparative study concerning the feeding behavior of bivalves on toxic dinoflagellates is provided by hégaret and colleagues. clearance rates of five species of bivalve molluscs were assessed, following the exposure for one hour to three harmful-algal strains: prorocentrum minimum (psp and dsp), a. fundyense (psp), and heterosigma akashiwo (nsp). the analysis of faeces and pseudofaeces revealed species-specifc responses to the different harmful algae, indicating in most cases a preferential retention of harmful cells. the production of faeces and pseudofaeces varied appreciably between the different bivalve/algae pairs. effects on juveniles    the understanding of the physiological effects played by toxic algae on bivalve juvaniles is very important, especially for farmed species, in order to better understand how aquaculture activity is threatened by the habs. li and colleagues exposed 190   juveniles of r. philippinarum and perna viridis to a. tamarense (psp), measuring the scope for growth (sfg), and the growth rate. sfg was significantly reduced in both clams and mussels while r. philippinarum resulted to be the most sensitive to psp while considering the growth rate only. leverone and collaborators reported the effects on the clearance rate of juvenile bivalves of 4 different species in relation with a karenia brevis exposure (pbtx). both in a short and long-term exposure argopecten irradians resulted to be the most sensitive species, c. virginica the least responsive and p. viridis and mercenaria mercenaria displayed intermediated responses. on the contrary, da did not have a significant effects on feeding rate and shell valve clatter on the juvenile king scallops pecten maximus that, but registered a negative impact on their growth rate and survival. other effects    considering the different nature, composition and targets of marine toxins, it is likely that many additional effects, not described above, could be detected in different bivalve species. as an example, beside paralysis, psts can also produce increased mantel melanization and abnormal vitellogenesis in nodipecten subnodosus and argopecten ventricosus, production of white mucus and inhibition of byssus production in m. edulis and g. demissa. moreover, landsberg hypothesized a possible connection between neoplasia in certain species of bivalves, such as mya arenaria, with the presence of micro-algal blooms, even though specific surveys on the topic are still lacking. some phycotoxins such as oa and da, certainly have a genotoxic effect on bivalves. furthermore several classes of toxins (ptx, dsts, pltx) are known to be potent dysregulators of cytoskeleton dynamics in vertebrates and are likely to exert a similar action also in bivalves studies at molecular level a summary of the main studies focused on the molecular responses of bivalves to phycotoxins is reported in table 2 and discussed in detail below. toxin metabolism and biotransformation    many species of bivalve molluscs are capable of biochemical transformation of the toxins accumulated by filtration, thus generating novel metabolites not found in the causative algal species, suggesting that extremely complex mechanisms of selective accumulation and chemical or enzymatic conversion might be involved in the development of shellfish toxicity. while, in some cases, this could be interpreted as a strategy specifically developed to decrease toxin potency, in other cases these toxin derivatives are likely just the by-products of normal metabolic pathways. the metabolism of many classes of phycotoxins has been documented in a large number of bivalve species, even though comparative studies demonstrated significant differences in the ability of biotransformation between species, pointing out that different organisms might have adopted specific biochemical mechanisms as a defensive strategy to recurrent hab events. among the many bivalves able to transform marine toxins, the scallop patinopecten yessoensis is definitely one of the most active, since it is able to metabolize ytx, dtx, ptx and oa. the mussels m. galloprovincialis and mytilus edulis can metabolize aza, oa and, to some extent, also psts. episodes of pbtx conversion to less toxic analogues have been documented in many different bivalves, including the oyster c. virginica. although toxin biotransformations in bivalves are well documented, their biological significance is often unclear, since the modifications do not always result in a decrease of toxin potency. one of the few exceptions is represented by the esterification of oa and dtx1, which has been studied in a number of different bivalves, which is thought to play a role in the sequestration in lipid rich tissues and the conjugation to lipoproteins. while some of the toxin biotransformations are likely the effect of passive processes or enzymatic activities provided by symbiotic bacteria, in many cases they have been shown to be active processes catalyzed by bivalves themselves. nevertheless, despite the overwhelming evidence of phycotoxin transformation, just a few enzymes specifically involved in these processes have been isolated and described so far. an enzyme capable of hydrolyzing ptx and oa has been recently discovered in the digestive gland of the mussel perna canaliculus : the protein identified, an acidic serine esterase, did not show any similarity with known sequences and was active on a rather broad range of ptxs and oa esters. furthermore, two enzymes involved in different pst modifications have been isolated in peronidia venulosa and mactra chinensis, even though only partial amino-acidic sequences have been characterized: carbamoylase i can hydrolyze the carbamoyl mojety in both carbamoyl and ncarbamoyl psts, whereas sulfocarbamoylase catalyzes the hydrolysis of the n-sulfocarbamoyl mojety of the weakly toxic c-psts. the digestive gland is the main site of accumulation of different toxin species, so this tissue is assumed to be the most active in the bioconversion processes. therefore, not surprisingly, all the enzymes involved in toxin transformation so far identified have been isolated from this tissue and novel, yet unknown, enzymes are expected to be highly expressed and active in this tissue in response to habs. toxin binding and accumulation    despite the important role of digestive gland, in some cases other tissues may play a role in the accumulation of phycotoxins. in fact, some bivalve species can retain toxins for an extremely prolonged time in specific non-visceral tissues, exploiting toxin table 2 main molecular studies related to shellfish poisoning in bivalve molluscs study toxin class bivalve species strategy llewellyn et al., 1997 psts (stx) mytilus edulis, saximodus giganteus, spisula solidissima, donax deltoides and vepricardium multispinosum screening for saxiphin-like activity dizer et al., 2001 da mytilus edulis monitoring of the cholinesterase activity following intramuscolar da injection enzyme purification from the digestive gland (sulfocarbamoylase i) lin et al., 2004 psts mactra chinensis monitoring of oxydative stress related enzymatic activities choi et al., 2006 psts ruditapes philippinarum estrada et al., 2007 psts nodipecten subnodosus monitoring of antioxidant and hydrolitic enzymes in different tissues takati et al., 2007 psts acanthocardia tuberculata protein purification from the foot (pspbp) nzoughet at al., 2007 aza mytilus edulis isoelectric focusing and sec fernandezreiriz et al., 2008 psts mytilus chilensis monitoring of the activity of amylase, laminarinase and cellulase talarmin et al., 2008 dsts (oa) crassostrea gigas western blot malagoli et al., 2008 plt mytilus galloprovincialis immunoblot enzyme purification from the digestive gland (sulfocarbamoylase i) cho et al., 2008 psts peronidia venulosa sds-page and de novo sequencing; comparative analysis between control and contaminated bivalves nzoughet et al., 2009 aza mytilus edulis monitoring of prophenoloxidase, amylase activity and ros production in contaminated oyster hemocytes haberkorn et al., 2010 psts crassostrea gigas monitoring of antioxidant and hydrolitic enzymes in different tissues, with a particular emphasis on hemocytes estrada et al., 2010 psts nodipecten subnodosus microarray and real-time pcr on digestive glands of experimentally and naturally contaminated mussels manfrin et al., 2010 dsp (oa) mytilus galloprovincialis rossignoli and blanco, 2010 dsts (oa) mytilus galloprovincialis fractionation and enzymatic digestion of cellular fractions mackenzie et al., 2012 ptx and oa perna canaliculus enzyme purification from the digestive gland multibiomarker approach, assessment of several physiological parameters, including enzymatic activities gorbi et al., 2012 plt and ovatoxin mytilus galloprovincialis real-time pcr on selected genes following heamocytes exposure to brevetoxin mello et al., 2012 brevetoxin crassostrea gigas gerdol et al., 2012 psp mytilus galloprovincialis rna-seq on digestive glands of experimentally contaminated mussels gonzales et al., in preparation* dsts (oa) mytilus galloprovincialis *personal communication monitoring of histones gene expression levels 191   192   accumulation as a chemical defense towards natural predators. the better known phenomenon of chemical defense by phycotoxin accumulation is certainly the butter clam saxidomus giganteus, which selectively stores psts in its siphon epithelium, thus discouraging predation by siphon-nipping fishes. as a matter of fact, the occurrence of habs significantly influences the feeding behavior of many vertebrate predators, such as sea otters and shorebirds although proteins involved in toxin-binding, transport and accumulation have been described in many organisms, so far the molecular mechanisms leading to this selective retention in bivalves are almost completely unknown. when tested for saxiphilin-like activity, both m. edulis and s. giganteus, capable of accumulating very high levels of psts in their tissues, resulted to be negative, likewise spisula solidissima, donax deltoides and vepricardium multispinosum. nevertheless, a case of a psp-binding protein has been reported in the moroccan cockle acanthocardia tuberculata; in this species, a 181 kda protein named pspbp contributes to the prolonged retention of psts in the foot. beside psts, other classes of toxins can be conjugated to specific proteins produced by bivalves. rossignoli and blanco provided the first lines of evidence of a soluble cytoplasmatic component binding oa in mussel, which was identified as a high density lipoprotein. nzoughet and colleagues were able to identify a 45 kda protein weakly binding aza in the hepatopancreas of the blue mussel m. edulis and another unknown 22 kda protein which was apparently highly expressed only in contaminated mussels. even though only a little is known about bivalves phycotoxin-binding proteins, these few studies suggest that they may be involved in selective retention or detoxification, depending on whether the toxins will be used in the frame of a chemical defense strategy or simply metabolized. metabolic activities  although relatively few bivalve proteins related to habs have been identified so far, the alteration of a number of enzymatic activities, possibly related to both xenobiotic metabolism and stress response has been reported. some classes of phycotoxins can affect the overall metabolic rates of bivalve organs in a timeand doses-dependent way. a multi biomarker approach by gorbi and colleagues clearly showed that the activity of the na+/k+-atpase, the target of pltx, was strongly inhibited by ostreopsis ovata blooms in m. galloprovincialis, which can therefore be considered as susceptible organisms. consequently to this reduction, a significant alteration of other enzymatic activities was also observed (acetylcholinesterase in particular), while on the contrary no ros (reactive oxygen species) detoxifying enzymes were significantly altered, indicating that oxidative pathways are not involved in o. ovata toxicity. the cholinesterase activity was also monitored in m. edulis after intramuscular injection of da, highlighting just a moderate reduction after 48 h. other studies have been focused on the analysis of the effects of different toxins on the oxidative pathways; the monitoring of glutathione stransferase (gst), glutathione peroxidase (gpx) and superoxide dismutase (sod) activities and of lipid peroxidation in the clam r. philippinarum contaminated with psts highlighted only a modest involvement of these enzymes. in a different species (the giant lions-paw scallop nodipecten subnodosus) the same toxin class produced a significant alteration of both gpx (up-regulated) and sod (down-regulated) in gills, whereas no changes were observed in other tissues. moreover, catalase (cat) activity and lipid peroxidation were not markedly altered in any tissue. on the other hand, other studies observed a stronger correlation between phycotoxins and oxidative damage: in fact, cyanobacteria were able to produce a significant alteration of the activity of the two antioxidant enzymes gst and gpx in m. galloprovincialis and of cat in m. edulis. moreover, haberkorn and colleagues monitored ros production in psp-contaminated oysters, observing a linear correlation between pst accumulation and ros production in haemocytes. as we have reported in the previous sections, toxic microalgae can significantly alter the feeding of bivalves, with consequent effects on enzymatic activities linked to digestion. fernandez-reiriz and colleagues (2008) carefully examined the effects of a diet based on the toxic dinoflagellate a. catenella on mytilus chilensis, showing that mussels are able to adapt mechanisms which allow the feeding with toxic algae. the authors were in fact able to observe a logarithmic relationship for the assimilatory balance and the carbohydrate metabolism of the digestive gland, by monitoring the enzymatic activity of amylase, laminarinase and cellulase. the amylase activity of oyster digestive gland is also affected by the exposure to a. minutum, even though the changes observed are also largely dependent on the physiological status of bivalves. finally, the monitoring of several hydrolytic enzymes revealed marked alterations in different tissues of n. subnodosus exposed to psp. these enzymatic activities could be potentially used as molecular markers of psp contamination in scallops; in particular lipase, α-galactosidase, βglucosidase and α-mannosidase were altered in the digestive gland and trypsin and α-chymotrypsin in the gill, but other modifications were also observed in other tissues, including mantle and the adductor muscle. biomarkers of contamination: proteomic approaches  although there is no doubt that large-scale proteomic approaches can provide a tool of the utmost importance for the identification of molecular markers of aquatic pollution, to date just a very few studies have exploited this potential to explore the effects of phycotoxins on bivalves. 193   so far proteomic approaches have been mainly used to study the expression of proteins regulated by known molecular targets of toxins, such as in the case of protein phosphatases, the upstream effectors of the p38 mitogen-activated protein kinase, selectively inhibited by oa. an increase in the phosphorylation/activation of p38 mapk has been in fact observed in the heart of oa treated oysters and contributes to the increased phagocytotic activity in the immunocytes of pltxtreated mussels. one of the very few large-scale approaches concerned the identification of biomarkers of aza contamination in mytilus edulis; four proteins highly expressed in the digestive gland of toxic mussels were identified, namely cathepsin d, superoxide dismutase, glutathione s-transferase pi and a flagellar protein of bacterial origin. this data seem to suggest the activation of xenobiotic defense response in bivalves following azp blooms. the negative effects of a toxic cyanobacteria, cylindrospermopsis raciborskii, have been investigated using a similar approach on m. galloprovincialis. the expression of several structural proteins was remarkably altered, indicating a situation of stress and cytoskeletal destabilization. at the same time, other important proteins such as the mitochondrial hsp60, the major extrapallial fluid protein and a triosephosphate isomerase were significantly down-regulated in the toxic strain-fed mussels, highlighting a complex response of mussel to cyanotoxins. although the efforts put so far in large-scale proteomic studies have been very scarce, the possibility to identify potential biomarkers of contamination demonstrates that this represents a valid approach for better understanding the molecular mechanisms involved in shellfish toxicity. biomarkers of contamination: target genes focused approaches    the number of proteomic studies exploring the effects of phycotoxins on marine bivalves is quite narrow, but even less efforts have been put in genomic researches. the few studies focused on gene expression have been mostly aimed at the monitoring of a limited number of target sequences. mello and colleagues selected a few genes and assessed their expression in c. gigas hemocytes subject to in vitro pbtx exposure. the early activation of hsp70, cyp365a1 and fabp, genes related to stress and detoxification, suggest that oyster hemocytes activate a defense response which protects them from cytotoxic damage, which does not involve immune and antioxidant processes, as the expression of bpi, il-17, ecsod, prx6, gpx and sod was not altered. other studies based on gene expression, exploiting the knowledge gathered from previous experiments, have only been planned but not performed yet: an interesting oa biomonitoring approach based on the evaluation of the expression of genes critically important in oa-induced genotoxic damage has been in fact proposed. in particular, the authors claim that the expression of several histone variants, such as the histone h2a.z, is strongly down-regulated in response to harmful levels of oa, basing their hypothesis on preliminary data which will be published in an upcoming manuscript. biomarkers of contamination: wholetranscriptome scale approaches    to the best of our knowledge, only two studies have so far tried to tackle the issue of marine toxin effect on bivalves from a genomic perspective. the two studies, both performed on the mediterranean mussel m. galloprovincialis, investigated the effects of toxins on the gene expression of the main tissue of accumulation, the digestive gland, by using two different techniques. in the first study, focused on dsp, manfrin et al. assessed the effects of oa accumulation over 35 days of exposure by cdna microarray, identifying several transcripts candidates as oa-stress markers. although most of the sequences could not be linked to known metabolic pathways correlated to biotransformation, the up-regulation of several stress-related proteins, mainly linked to apoptosis, proteolysis and cytoskeleton destabilization, denoted a possible sufferance of oa exposed mussels. the preliminary observations collected from this experiment were further validated on the digestive gland of mussels subject to naturally occurring habs (unpublished data). the expression of 14 selected up-regulated genes was monitored by realtime pcr in samples collected during 2 dsp events occurred in the gulf of trieste. the analysis permitted to confirm 11 out of 14 genes as oaresponsive, highlighting that the results of experimental contaminations can be applied also on naturally occurring dsp events (fig. 2). the second and more recent study exploited the recent advances offered by the application of next generation sequencing technologies to rnasequencing. the study was aimed at the investigation of the possible molecular mechanisms activated or repressed in the digestive gland in response to the accumulation of psts produced by the toxigenic dinoflagellate a. minutum strain al9t over a time course of 5 days. the contamination with these toxins apparently only led to negligible effects on gene expression in mussel, which is an organism insensible to the paralytic effects of stxlike toxins, due to the resistance of its nerve voltage-gated sodium channel. therefore, the rnaseq experiment results seem to disprove the sporadic reports of negative effects of paralytic habs in mussel and support their classification as organisms refractory to psp. although preliminary, the two above mentioned studies were certainly able to give a better overview about the possible molecular effects of marine phycotoxins on bivalve molluscs and represent the first steps in moving the focus of dinoflagellate toxicity from a human-centered to a bivalvecentered point of view. there is no doubt that, thanks to the technological advancements and the increasing availability of bivalve sequence data, including fully fig. 2 validation of potential dsp-biomarkers in mussels digestive gland by quantitative rt-pcr. the expression of fourteen genes identified as differentially expressed in manfrin et al., 2010 were monitored in two samples collected during naturally occurring dsp-hab in the gulf of trieste (ts_09+ and ts_10+). the normalized fold expression values are shown in a log2 scale, significant (p< 0.05) upregulation is indicated by *. 194   195   sequence genomes, the design of similar studies performed on large panels of toxins will be much easier in the near future. conclusion    habs represent a serious and emerging issue for human health, so considerable research efforts have been put in the study of their toxicological effects on vertebrate model organisms, useful for a better comprehension of the dynamics of shellfish poisoning in human. comparatively, only limited data are available about the effects on bivalves, and the large majority of them concerns physiological aspects. while the lack of molecular studies is mainly caused by the still limited genetic knowledge of these organisms, there is an urge for further research in this field, as highlighted by the promising findings provided by the handful of molecular studies documented in this review. certainly, there is the need for combining large scale approaches (both on a proteomic and on a genomic level) for the identification of trustworthy biomarkers of contamination for a more effective biomonitoring of 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detect phenoloxidase (po) activity. no po activity was found in the hemocytes of snails. a low level of po activity (by dopa oxidation) was registered in the hemolymph without cells. addition of a specific po inhibitor revealed the lack of effect on enzyme activity in the hemolymph, whereas hydrogen peroxide has increased it. studying the electron paramagnetic resonance (epr) spectrum of dopaand dopamine-semiquinone indicates the peroxide-dependent dopamine oxidation. our results suggest that peroxidase, rather than phenoloxidase, plays a key role in the oxidation of dopa and dopamine in the hemolymph of l. stagnalis. it is just peroxidase activity that may be important in the formation of cytotoxic molecules, such as o-semiquinones, during snail defense immune reactions. key words: phenoloxidase; peroxidase; lymnaea stagnalis; immunity   introduction the enzyme phenoloxidase (po) oxidizes phenols to form melanin which plays an important role in various physiological processes, such as egg production in gastropods (bai et al., 1997), sclerotization of a new postmolt exoskeleton (terwilliger, 1999) and immunity of invertebrates (söderhäll and cerenius, 1998). highly reactive quinoid intermediates are generated during melanization (johansson and söderhäll, 1995; slepneva et al., 1999). these can be involved in cytotoxic reactions in defense mechanism (nappi and ottaviani, 2000). phenoloxidases occur as inactive precursors, termed the prophenoloxidases (propo). propo are activated by a proteolytic cascade system. the cascade is activated by microbial cell wall components (johansson and söderhäll, 1995; söderhäll and cerenius, 1998) or some chemical compounds (fisher and brady, 1983). po activity has been described for many invertebrates, including crustaceans, insects, and ___________________________________________________________________________ corresponding author: yana l. vorontsova laboratory of insect pathology institute of systematics and ecology of animals siberian branch of russian academy of sciences frunze str., 11, 630091 novosibirsk, russia yavoronts@yandex.ru molluscs (ashida and söderhäll, 1984; ashida and yamazaki, 1990; aspán and söderhäll, 1991; nellaiappan and sugumaran, 1996). lymnaea stagnalis is a freshwater gastropod snail, which is used as a model organism to investigate immunological defense mechanisms (sminia et al., 1973; van der knaap et al., 1993; plows et al., 2006). surprisingly, despite this, no study has been carried out to strongly investigate the presence of po activity in the hemolymph of l. stagnalis. only seppälä and co-authors reported the phenoloxidase-like activity determined in the hemolymph of l. stagnalis (leicht et al., 2013; seppälä and leicht, 2013). they consider the polike activity as an important parameter of snail defense against some immune elicitors (seppälä and leicht, 2013). unfortunately, the authors didn’t study whether the enzyme activity is present in the hemolymph or hemocytes. the hemocytes of l. stagnalis are the immunocompetent cells and play a key role in internal defense (van der knaap et al., 1981; adema et al., 1992). these cells take part in wound healing, encapsulation, and phagocytosis (sminia et al., 1973; van der knaap et al., 1993); they contain several lysosomal enzymes, including peroxidase that may be involved in intracellular killing (dikkeboom et al., 1984; sminia and barendsen, 1980). however, the 5 mailto:yavoronts@yandex.ru fig. 1 light microscopy of hemocytes from lymnaea stagnalis stained for phenoloxidase activity. scale bar = 10 µm. data on po activity in the hemocytes of l. stagnalis are lacking. therefore, the presence of the po activity in the hemolymph of l. stagnalis is still a problem. the aim of our study was to determine the po activity in the hemolymph and in the hemocytes of l. stagnalis. materials and methods snails lymnaea stagnalis (gastropoda, pulmonata, basommatophora) were collected at the littoral zone of lake chany, russia (54°30′-55°09′ n, 76°48′78°12′ e) and maintained under laboratory conditions (14/10 light/dark daily cycles, 20 ± 1 °c) in 5 l aquaria containing dechlorinated tap water supplemented with mussel shell as calcium source. the snails were fed with pesticide-free lettuce daily ad libitum, and the water was replaced once a week. chemicals 4-aminoantipyrine, catalase, 3,3′diaminobenzidine tetrahydrochloride hydrate (dab), diethylenetriaminepentaacetic acid (dtpa), 3,4dihydroxy-l-phenethylamine (dopamine), 3,4dihydroxy-l-phenilalanine (dopa), ethylenediaminetetraacetic acid (edta), formaldehyde, glucose, glutaraldehyde, hydrogen peroxide, phenylthiourea (ptu), potassium phosphate, sodium azide, sodium chloride, trishydroxymethylaminomethane (tris), were purchased from sigma-aldrich (usa). all solutions were prepared with bidistilled deionized water. hemolymph collection three adult snails (shell height: 25 mm) were rinsed with distilled water. the hemolymph was collected by stimulation of the foot sole, as described by sminia (1972). when the snail retracts into its shell, a drop of hemolymph is extruded through the hemal pore and collected with a micropipette. the collected hemolymph was mixed with either antiaggregant buffer (for hemocytes analysis) or phosphate-buffered saline (pbs: 50 mm phosphate buffer, 150 mm nacl), ph 7.2 (for peroxidase and phenoloxidase assays in hemolymph) (2 parts hemolymph: 1 part buffer) and was kept on the ice to prevent hemocyte clumping. hemocyte monolayers preparation the hemolymph with antiaggregant buffer (ab: 62 mm nacl, 100 mm glucose, 10 mm edta, 30 mm sodium citrate, 26 mm citric acid, ph 4.6) (1 part hemolymph : 2 parts ab) was centrifuged at 500 g for 5 min at 4 °c. the supernatant was removed and the hemocytes were rinsed twice in ab. hemocytes were then resuspended in pbs and 10 µl of hemocyte suspension (104 cells) were placed on a microscope slide. the slides were kept in a moist chamber at 22 °c for 15 min to allow hemocytes to adhere and spread. then hemocytes were fixed with either 1g4f fixative (1 % glutaraldehyde : 4 % formaldehyde) for peroxidase assay or acetone for phenoloxidase assay. acetone was used not only as fixative, but also as the activator of propo (fisher and brady, 1983). peroxidase assay the assay was carried out based on the method described by dikkeboom et al. (1984). to demonstrate the activity of peroxidase in the hemocytes, the fixed hemocyte monolayers were first covered with pbs containing 2.3 mm dab and 0.15 % hydrogen peroxide and then incubated at 22 °c for 30 min in the moist chamber. slides with hemocyte monolayers were rinsed with distilled 6 fig. 2 kinetics of dopa (a) and dopamine (b) oxidation in hemolymph of l. stagnalis, effect of adding of h2o2 (c) and catalase (d) on the rate of dopa oxidation. water. hemocytes were observed for the presence of brown deposits using a light microscope (zeiss; axioscope 40). hemocytes with brown coloration were described a peroxidase-positive cells, i.e., the hemocytes with peroxidase activity. the percentage ratio of the peroxidase-positive hemocytes was calculated. cells on control slides were incubated in media lacking dab or hydrogen peroxide. the effect of peroxidase inhibitor was checked by adding 0.8 mm sodium azide to the hemocyte suspension. the peroxidase activity in the hemolymph was assayed spectrophotometrically using 4-aminoantipyrine as a substrate (nicell and wright, 1997). each sample of hemolymph with pbs was then centrifuged at 500g for 5 min at 4 °c to remove the hemocytes. the hemolymph without cells (40 µl) was mixed with 0.85 mm hydrogen peroxide, 1.17 mm 4aminoantipyrine with 80 mm phenol and 0.2 m potassium phosphate buffer; ph 7.0, up to a final volume of 250 µl. the mixture was placed in a cuvette with optical path length of 1 mm and the absorption at 510 nm for 5 min was recorded using a uv-2401 (pc) ce spectrophotometer (shimadzu, japan). the effect of peroxidase inhibitor was checked by adding 0.1 mm kcn to the reaction mixture. determination of po activity after fixation with acetone, hemocytes were rinsed three times in pbs. thereafter, hemocytes monolayers were incubated with 100 µl of dopa (4mg/ml in pbs) at 22 °c for 15, 40, 60, 120 and 150 min in the dark moist chamber. then, the hemocyte monolayers were rinsed with distilled water and checked for the presence of dark-grey deposits (indicative of melanin formation) using a light microscope (zeiss; axioscope 40). po activity in the hemolymph was assayed spectrophotometrically by mixing 150 µl of hemolymph without cells with either 125 µl of dopa or dopamine (both 10 mm in pbs). the mixture was placed in the cuvette with optical path length of 1 mm and the absorption at 490 nm was detected. to study the effect of h2o2 and catalase on po activity, either 3 mm h2o2 or 470 u/ml catalase were added to the reaction mixture. the effect of the specific po inhibitor was checked by adding 18 µm ptu to the reaction mixture. determination of dopaand dopaminesemiquinone production the o-semiquinone radicals such as dopasemiquinone are very short-lived. however, these species form a comparatively stable complex with diamagnetic divalent metal ions allowing its study under static conditions (eaton, 1964; kalyanaraman et al., 1984; kalyanaraman, 1990). in our study, the dopaand dopamine-semiquinone radicals were detected by electron paramagnetic resonance (epr) method as metal complexes with mg2+ in the hemolymph of l. stagnalis. samples were prepared in tris-hcl-d (tris-hcl, ph 7.5, containing 50 µm dtpa). dtpa was used to decrease the rate of decay of the o-semiquinone radicals catalyzed by traces of transition metal ions. dopa or dopamine were dissolved in oxygen-free (argon-bubbled) trishcl-d to prevent the autoxidation of dopa and dopamine. hemolymph (70 µl) was mixed with 20 mm 7 fig. 3 epr spectra of semiquinones of 10 mm dopamine (a) and 10 mm dopa (c) obtained in hemolymph of l. stagnalis. effect of 2.5 mm h2o2 added in hemolymph on intensity of dopamine-semiquinone spectrum (b). amplitude of modulation was 0.16 g for spectra a and b, and 1.6 g for spectrum c. dopa or dopamine (100 µl) and 3.5 m mgcl2 (30 µl). formation of the o-semiquinones in the mixtures was detected at room temperature by epr method using an er 200-d src x-band esr spectrometer (bruker). to study the effect of h2o2 on dopaminesemiquinone formation, 2.5 mm h2o2 was added to the reaction mixture. the epr conditions were the following: field center, 3480 g; fields weep, 20 g; time constant, 1 s; microwave power, 2 mw; magnetic field modulation, 100 khz; and modulation amplitude, 0.16 g. statistical analysis the data were analyzed using the software sigmaplot for windows, version 9.0 (systant software, inc.). when necessary, a statistical analysis was used and the data were expressed as means ± se (n ≥ 5). significant differences between treatments were analyzed by student's t-test (p < 0.05) using the origin 6.0 program. results the hemocytes of l. stagnalis were analyzed to detect the po activity. no po-positive hemocytes were revealed after incubation of cells with dopa during 2.5 h (fig. 1). this indicates the lack of the po activity in the hemocytes of l. stagnalis. we have spectrophotometrically registered the low level of dopa oxidation in the hemolymph without cells. at the same time, the rate of dopamine oxidation was 5 times as high as that of dopa (figs 2a, b). adding 18 µm ptu to the hemolymph had no effect on the rates of dopa and dopamine oxidation (data not shown). the addition of hydrogen peroxide to the hemolymph increased the rate of dopa oxidation (fig. 2c). in contrast, the dopa oxidation rate was observed to decrease upon addition of catalase to the hemolymph (fig. 2d). enzymatic catechol oxidation is known to occur upon formation of the highly reactive radical species, o-semiquinones (kalyanaraman et al., 1984; kalyanaraman, 1990). using the epr spin stabilization with mg2+ we show that the dopaand dopamine-semiquinone radicals are produced by adding dopa or dopamine to the hemolymph of l. stagnalis, respectively. the intensity of the epr spectra obtained for dopamine was significantly higher than that of dopa (figs 3a, c). no influence on 8 fig. 4 (a) light microscopy of hemocytes from l. stagnalis with peroxidase activity. (b) effect of sodium azide on peroxidase activity in hemocytes from l. stagnalis. abbreviations: g, granulocyte; h, hyalinocyte; “+” show peroxidase positive staining hemocytes. scale bars = 10 µm. the epr spectrum was registered in the presence of the specific inhibitor of phenoloxidase, ptu (data not shown). the dopamine-semiquinone spectrum intensity increased with adding h2o2 to the hemolymph (fig. 3b). we have analyzed the hemocytes of l. stagnalis to detect the peroxidase activity. it was registered in granulocytes and hyalinocytes (fig. 4a). the quantity of peroxidase-positive hemocytes was 45 ± 2 %. adding sodium azide to a suspension of hemocytes inhibited the peroxidase activity in the hemocytes of l. stagnalis (fig. 4b). the peroxidase activity in the hemolymph of l. stagnalis was determined from the oxidation of 4aminoantipyrine. we have registered the high rate of 4-aminoantipyrine oxidation (fig. 5a). adding 0.1 mm kcn to the hemolymph reduced the rate of this reaction (fig. 5b). discussion phenoloxidase plays a key role in the immunity of invertebrates. this enzyme is known to be involved in encapsulation and phagocytosis of insects (carton et al., 2008) and some species of snails (aladaileh et al., 2007; scheil et al., 2013). only one group of researches reports that po or po-like activity of hemolymph is involved in the immune defense of the l. stagnalis (seppälä and jokela, 2010, 2011; leicht et al., 2013; seppälä and leicht, 2013). moreover, the authors carried out unusually long time incubation of hemolymph with dopa (6 h) to detect the po activity, named po-like activity (leicht et al., 2013; seppälä and leicht, 2013). this fact allows us to have some doubts on the real presence of po activity in the hemolymph of the snails because it is known that dopa can be oxidized not only by po, but by peroxidase too (kalyanaraman et al., 1984; puiu et al., 2010) and the question of the presence of phenoloxidase in the l. stagnalis hemolymph and its role in the immune defense is still ambiguous. our histochemical experiments revealed no po activity in the hemocytes of l. stagnalis. the level of dopa oxidation in the snail hemolymph was very low as compared with that in the insect hemolymph (lee and anstee, 1995; kryukova et al., 2011). the specific po inhibitor, ptu, was employed to verify the participation of po in dopa oxidation. the concentration of ptu we used (18 µm) should inhibit the po-dependent reaction completely (ryazanova et al., 2012). in our experiments, ptu has failed to inhibit the dopa oxidation in the hemolymph of snail which indicates that the dopa oxidation occurs under the action of other enzyme. moreover, we have detected a significantly higher level of dopamine oxidation in the l. stagnalis hemolymph which was not inhibited by ptu. these data allow us to suggest that the observed dopa and dopamine oxidation in the hemolymph is provided by the activity of peroxidase rather than po. this assumption is in fair agreement with the previous data testifying that the peroxidase is able to oxidize dopamine more effectively than dopa (kalyanaraman et al., 1984). actually, in our experiments, the addition of hydrogen peroxide to the reaction mixture has increased the dopa and dopamine oxidation rate, while the addition of catalase to the reaction mixture decreased it. based on spectrophotometrical results, we can conclude that po is not involved in the oxidation of dopa and dopamine in the hemolymph of l. stagnalis. it is known that the dopa and dopamine oxidation occurs through o-semiquinone radical formation (eaton, 1964; kalyanaraman, 1990). previously we have registered the dopasemiquinone radical by epr method in the hemolymph of insects. these highly reactive quinoid intermediates can be involved in cytotoxic reactions in the defense mechanism of insects (slepneva et 9 fig. 5 detection of peroxidase activity by oxidation of 4-aminoantipyrin in hemolymph of l. stagnalis (a), inhibitory effect of kcn (0.1 mm) (b). al., 2003; komarov et al., 2005). based on the results from these assays, we used the epr method to detect the o-semiquinone radical in the l. stagnalis hemolymph. the specific spectrum of very low intensity with dopa was observed as compared with dopamine (figs 3a, c). this result demonstrates the lack of the po activity in the hemolymph of snails. in order to identify the enzyme involved in dopamine oxidation, we added hydrogen peroxide and ptu to the hemolymph. the increasing intensity of the dopamine-semiquinone epr spectrum upon addition of h2o2 and the lack of ptu effect on the spectrum indicate to the peroxidedependent dopamine oxidation. as demonstrated earlier, peroxidase is present in the hemolymph of l. stagnalis and is involved in snail’s immunity (dikkeboom et al., 1984; adema et al., 1992; mohandas et al., 1992). sminia and barendsen (1980) registered the peroxidase activity in the lysosomal system of the spreading hemocytes of l. stagnalis. peroxidase histochemical reaction products are present in the golgi apparatus and in the lysosomes. we have also registered the activity of peroxidase in the l. stagnalis snail hemocytes of two types (fig. 4). furthermore, we have observed the activity of peroxidase in the hemolymph of snails using a typical substrate for peroxidase assay. enzyme activity decreased due to the addition of peroxidase inhibitor, kcn, to the hemolymph (fig. 5) which confirms the presence of the peroxidase activity in the hemolymph of l. stagnalis. gornowicz and co-authors (2013) have also detected the peroxidase activity in these snails. moreover, they revealed a significant increase of enzyme activity in the hemolymph of l. stagnalis naturally infected with digenean trematodes (gornowicz et al., 2013). thus, the peroxidase activity in the hemolymph is likely to play a key role in the host defense function. taken together, the results of this study show that peroxidase rather than po plays a key role in the dopa and dopamine oxidation in the hemolymph of l. stagnalis. it is only peroxidase activity that may be important in the formation of cytotoxic molecules such as o-semiquinones during snail’s defense immune reactions. acknowledgements we thank anonymous referees, who gave fruitful comments on the manuscript. the work was supported by the federal fundamental scientific research programme for 2013-2020 (vi.51.1.5) and the russian foundation for basic research, grants 13-04-02075 and 12-04-01057. references adema cm, harris ra, van deutekom-mulder ec. a comparative study of hemocytes from six different snails: morphology and functional aspects. j. invertebr. pathol. 59: 24-32, 1992. aladaileh s, nair sv, raftos da. induction of phenoloxidase and other immunological activities in sydney rock oysters challenged with microbial pathogen-associate molecular patterns. fish shellfish immunol. 23: 11961208, 2007. ashida m, söderhäll k. the prophenoloxidase activating system in crayfish. comp. biochem. physiol. 77b: 21-26, 1984. ashida m, yamazaki hi. biochemistry of the phenoloxidase system in insects: with special reference to its activation. in: ohnishi e, ishizaki h (eds), molting and metamorphosis, springer-verlag, berlin, pp 239-265, 1990. aspán a, söderhäll k. purification of prophenoloxidase from crayfish blood cells, and its activation by an endogenous serine proteinase. insect biochem. 21: 363-373, 1991. 10 bai g, johnston la, watson co, yoshino tp. phenoloxidase activity in the reproductive system of biomphalaria glabrata: role in egg production and effect of schistosome infection. j. parasitol. 83: 852-858, 1997. carton y, poirié m, nappi aj. insect immune resistance to parasitoids. insect science 15: 6787, 2008. dikkeboom r, van der knaap wpw, mueleman ea, sminia t. differences between blood cells of juvenile and adult specimens of the pond snail lymnaea stagnalis. cell tissue res. 238: 4347, 1984. eaton dr. complexing of metal ions with semiquinones. an electron spin resonance study. inorganic chem. 3: 1268-1271, 1964. fisher cw, brady ue. activation, properties and collection of haemolymph phenoloxidase of the american cockroach, periplaneta americana. comp. biochem. physiol. 75c: 111-114, 1983. gornowicz d, dmochowska k, zbikowska e, zolotowska k. total antioxidative status and the activity of peroxidase and superoxide dismutase in the haemolymph of lymnaea stagnalis (l.) naturally infected with digenean trematodes. j. mollusc. stud. 79: 225-229, 2013. johansson mw, söderhäll k. the prophenoloxidase activating system and associated proteins in invertebrates. in: rinkevich b, muller weg (eds), invertebrate immunology, springer, berlin, pp, 46-66, 1995. kalyanaraman b. characterization of o-semiquinone radicals in biological systems. methods enzymol. 186: 333-343, 1990. kalyanaraman b, felix cc, sealy rc. peroxidatic oxidation of catecholamines. a kinetic electron spin resonance investigation using the spin stabilization approach. j. biol. chem. 259: 7584-7589, 1984. komarov da., slepneva ia, glupov vv, khramtsov vv. detection of free radical formation in haemolymph of insects by epr spectroscopy. appl. magn. reson. 28: 411-419, 2005. kryukova na, dubovskiy im, chertkova ea, vorontsova yl, slepneva ia, glupov vv. the effect of habrobracon hebetor venom on the activity of the prophenoloxidase system and the generation of reactive oxygen species and encapsulation in the haemolymph of galleria mellonella larvae. j. insect physiol. 57: 796800, 2011. lee mj, anstee jh. phenoloxidase and its zymogen from the haemolymph of larvae of the lepidopteran spodoptera littoralis (lepidoptera: noctuidae). comp. biochem. physiol. 11b: 379384, 1995. leicht k, jokela j, seppälä o. an experimental heat wave changes immune defense ad life history traits in a freshwater snail. ecol. evol. 3: 48614871, 2013. mohandas a, adema cm, van der knaap wpw, sminia t. the effect of haemolymph extraction on distribution of lysosomal enzymes in lymnaea stagnalis haemocytes: a cytochemical study. comp. hematol. int. 2: 61-67, 1992. nappi aj, ottaviani e. cytotoxity and cytotoxic molecules in invertebrates. bioessays 22: 469480, 2000. nellaiappan k, sugumaran m. on the presence of prophenoloxidase in the haemolymph of the horseshoe crab, limulus. comp. biochem. physiol. 113b: 163-168. 1996. nicell ja, wright a. a model of peroxidase activity with inhibition by hydrogen peroxide. enzym. microb. technol. 21: 302-310, 1997. plows ld, cook rt, davies aj, walker aj. phagocytosis by lymnaea stagnalis haemocytes: a potential role for phosphatidylinositol 3-kinase but not protein kinase a. j. invertebr. pathol. 91: 74-77, 2006. puiu m, babaligea m, oimazu c, raducan a, oancea d. peroxidase-mediated oxidation of ldopa: a kinetic approach. biochem. engineer. j. 52: 248-254, 2010. ryazanova ad, alekseev aa, slepneva ia. the phenylthiourea is a competitive inhibitor of the enzymatic oxidation of dopa by phenoloxidase. j. enzym. inhibit. med. chem. 27: 78-83, 2012. scheil ae, hilsmann s, triebskorn r, köhler h-r. shell colour polymorphism, injuries and immune defense in three helicid snail species, cepaea hortensis, theba pisana and cornu aspersum maximum. results immunol. 3: 73-78, 2013. seppälä o, jokela j. maintenance of genetic variation in immune defense of a freshwater snail: role of environmental heterogeneity. evolution 64: 2397-2407, 2010. seppälä o, jokela j. immune defence under extreme ambient temperature. biol. lett. 7: 119122, 2011. seppälä o, leicht k. activation of the immune defence of the freshwater snail lymnaea stagnalis by different immune elicitors. j. exp. biol. 216: 2902-2907, 2013. slepneva ia, glupov vv, sergeeva sv, khramtsov vv. epr detection of reactive oxygen species in haemolymph of galleria mellonella and dendrolimus superans sibiricus (lepidoptera) larvae. biochem. biophys. res. commun. 264: 212-215, 1999. slepneva ia, komarov da, glupov vv, serebrov vv, khramtsov vv. influence of fungal infection on the dopa-semiquinone and dopa-quinone production in haemolymph of galleria mellonella larvae. biochem. biophys. res. commun. 300: 188-191, 2003. sminia t. structure and function of blood and connective tissue cells of the fresh water pulmonate lymnaea stagnalis studied by electron microscopy and enzyme histochemistry. z. zellforsch. mikrosk. anat. 130: 497-526, 1972. sminia t, barendsen la. a comparative morphological and enzyme histochemical study on blood cells of the freshwater snails lymnaea stagnalis, biomphalaria glabrata, and bulinus truncates. j. morphol.165: 31-39, 1980. sminia t, pietersma k, scheerboom jem. histological and ultrastructural observations on wound healing in the freshwater pulmonate lymnaea stagnalis. cell tissue res. 141: 561-573, 1973. 11 http://www.ncbi.nlm.nih.gov/pubmed?term=scheil%20ae%5bauthor%5d&cauthor=true&cauthor_uid=24600561 http://www.ncbi.nlm.nih.gov/pubmed?term=hilsmann%20s%5bauthor%5d&cauthor=true&cauthor_uid=24600561 http://www.ncbi.nlm.nih.gov/pubmed?term=triebskorn%20r%5bauthor%5d&cauthor=true&cauthor_uid=24600561 söderhäll k, cerenius l. role of the prophenoloxidase-activating system in invertebrate immunity. curr. opin. immunol. 10: 23-28, 1998. terwillinger nb. hemolymph proteins and molting in crustaceans and insects. americ. zool. 39: 589599. 1999. van der knaap w, boerrigter-barendsen lh, van den hoeven dsp, sminia t. immunocytochemical demonstration of a humoral defence factor in blood cells (amoebocytes) of the pond snail, lymnaea stagnalis. cell tissue res. 219: 291-296, 1981. van der knaap w, adema c, sminia t. invertebrate blood cells: morphological and functional aspects of the haemocytes in the pond snail lymnaea stagnalis. comp. haematol. int. 3: 2026, 1993. 12 119 isj 14: 119-128, 2017 issn 1824-307x review gene expression and regulation of molecules involved in pharynx inflammatory response induced by lps in ciona intestinalis a vizzini marine immunobiology laboratory, department of biological chemical pharmaceutical science and technology, university of palermo, via archirafi 18, palermo, italy accepted april 4, 2017 abstract in the ascidian ciona intestinalis, the pharynx (hemopoietic organ) connects the external environment to the gastrointestinal system for two main activities, respiration and food collection, potentially exposing the ascidian to high concentrations of pathogenic microorganisms. recently, evidence in c. intestinalis has indicated that the pharynx is involved in the inflammatory reaction induced by lipopolysaccharide (lps) injection into the body wall. immune-related genes such as cytokines, galectins, pro-po, cap are expressed in pharynx hemocytes and are up-regulated by the inflammatory agent lps. studies of the expression pattern of the immune gene clearly show that in c. intestinalis, as in vertebrates, immune gene expression can be regulated through alternative polyadenylation (apa) mechanism and gait element al 3’utr. key words: ascidian; inflammation; pharynx; lps; ciona intestinalis introduction inflammation is a rapid protective response to microbial infection, tissue injury, and insults (nathan, 2002; medzhitov, 2008). infection by microbial invaders is often implicated as the major promoter of inflammatory responses, though injury or trauma (in the absence of parasitic infection) and exposure to foreign particles/irritants/pollutants are also potent activators of inflammation (medzhitov, 2008), suggesting that this response likely evolved as a general adaptation to solve tissue damage (matzinger, 2002). a common explanation for why infection and trauma might evoke similar inflammatory responses is that both pathogens and wounding cause damage to cells and tissue, which implies that similar responses to trauma and infection could have advantageous results (bianchi, 2007). on an evolutionary level, inflammation is a highly conserved phenomenon and appears to be an important first line of defense for both invertebrates and vertebrates. many of the processes associated with the inflammatory cascade, such as chemotaxis and phagocytosis, are readily employed by unicellular organisms and were ___________________________________________________________________________ corresponding author: aiti vizzini department of biological chemical pharmaceutical science and technology university of palermo via archirafi 18, palermo, italy e-mail: aiti.vizzini@unipa.it later coopted as defensive components to maintain the integrity of more complex multicellular organisms (rowley, 1996). when host cells capable of innate immune responses encounter a pathogenic microbes or another foreign or host irritant, the inflammatory response initiates within minutes. the host cells first recognize the stimulus through a wide variety of sensing mechanisms involving transmembrane receptors. these interactions transmit signals to the nucleus, resulting in the activation and regulation of numerous genes via both transcriptional and posttranscriptional mechanisms (akira et al., 2006; medzhitov, 2007; ishii et al., 2008; beutler, 2009). some inducible gene products, such as antimicrobial peptides and complement factors, directly target infectious microorganisms. other inducible gene products, including proinflammatory cytokines and chemokines, activate endothelial cells and recruit immune system cells to the site of infection. the innate immune system is the major contributor to acute inflammation (akira et al., 2006; beutler et al., 2006). innate immunity is a conserved, complex and multi-pronged response to overcoming infection that is present in all metazoans. the ascidian c. intestinalis is a powerful model for studying innate immunity because it occupies a key phylogenetic position in chordate evolution and is considered the sister group of vertebrates (zeng and swalla, 2005; 120 delsuc et al., 2006; tsagkogeorga et al., 2009). ascidians possess an exclusively innate immune system, including inflammatory, humoral and cellular responses. in vertebrates, the development of the adaptive immune system is linked to the acquisition of the enzyme machinery encoded by recombination activating genes (rag) that provide for the rearrangement of immunoglobulin (ig) and t cell receptor (tcr) genes. analysis of c. intestinalis genome sequences has not revealed any of the pivotal genes and molecules for adaptive immunity, such as major histocompatibility complex (mhc) genes, tcrs, or dimeric igs (dehal et al., 2002; azumi et al., 2003; shida et al., 2003;). in c. intestinalis, challenge with pathogenassociated molecular patterns (pamps), such as gram-negative lipopolysaccharide (lps), induces inflammatory-like reactions in the pharynx (immunocompetent organ). these responses can induce several immunological phenomena, including the expression of characteristic innate immune genes and a repertoire of innate effectors. this review provides an overview of the molecular and cellular events that are involved in pharynx inflammatory response induced by lps in c. intestinalis. inflammatory response in the pharynx effector molecules following an lps challenge or the inoculation of a foreign agent into the ascidian body wall, several inflammatory events are initiated, including hemocyte recruitment into the inflamed tissue (parrinello, 1981; parrinello et al., 1984a, b; parrinello and patricolo, 1984). the well-studied organ involved in the inflammatory reaction induced by lps injection into the body wall is the pharynx. in ascidians, which are filter-feeding animals, the pharynx occupies an extensive part of the body. it consists of two epithelial monolayers perforated by rows of anteroposteriorly elongated elliptical ciliated structures, the stigmata (martinucci et al., 1988), which are aligned dorsoventrally. each row of stigmata is enclosed in a mesh of vessels (forming the so called transversal and longitudinal bars) that originate from the two epithelial leaflets, where the hemolymph, containing abundant mature and immature hemocytes, flows (de leo et al., 1987). the ciliated cells of the stigmata generate water currents, which are crucial for the two main activities of this organ, respiration and food collection that expose ascidians to high concentrations of microorganisms. the inflammatory response induced by lps challenge in the pharynx has been extensively studied, including the expression of characteristic innate immune genes and of a repertoire of innate effectors in pharynx hemocytes. granular cells and univacuolar refractile granulocytes are active in galectin and phenoloxidase (po) production (vizzini et al., 2012, 2015). the genome-wide analysis of c. intestinalis has provided a comprehensive picture of immune-related genes and annotated galectin. the sequences of two c. intestinalis genes encoding galectins, characterized by two carbohydrate recognition domains (crd) homologous but not identical, connected by a short linker peptide (bicrd) (cilgals-a and cilgals-b), have been deduced from ests and the genome (houzelstein et al., 2004). cilgals-a exhibits f4-crd-liker-f3crd organization which is typical of all vertebrate bi-crd galectin genes, and cilgals-b shows an f4crd-linker-f4-crd structure which is unknown in vertebrate genes. in situ hybridization assays and real time pcr show that both galectins appear to be inducible and promptly expressed after lps inoculation in pharynx hemocytes (vizzini et al., 2012) (fig. 1). in c. intestinalis, in vivo challenge of lps resulted in the upregulation of a mannose-binding lectin-like collectin (cimbl) (bonura et al., 2009) (fig. 1). collectins are a family of calciumdependent lectins that are characterized by their collagen-like domains and exert several functions, including complement activation, microbe agglutination, opsonization, and the modulation of inflammatory response. in pharynx hemocytes cimbl transcription results strongly upregulated by lps inoculation (fig. 1) and expressed by granular amebocytes and hemocytes with large granules (bonura et al., 2009). c. intestinalis has been shown to have an almost complete set of complement gene families (marino et al., 2002; azumi et al., 2003; nonaka and satake, 2010), suggesting the existence of complement cascade pathways in this ascidian. indeed, a specific chemotactic activity exerted on hemocytes by c3a, the anaphylatoxin produced by c3 activation, has been demonstrated in c. intestinalis, providing compelling evidence of the presence of a complement system-related inflammatory pathway in deuterostome invertebrates (pinto et al., 2003). this finding has been extended through the identification of the c3aspecific receptor, cic3ar, on hemocytes (granular amebocytes) (melillo et al., 2006). c3, which is constitutively expressed in the pharynx, is upregulated by lps injection. using two specific anticic3 and anti-cic3a polyclonal antibodies, it was found that the gene product was localized to the hemocytes of the pharynx vessels (identified as granular amebocytes) and in stigmata ciliated cells (giacomelli et al., 2012). hemocytes inside the pharynx vessels are also engaged in the po expression genes cinpo-1 and cin-po2 (vizzini et al., 2015). a basal expression of both genes was found in naïve ascidians, indicating an active role of the enzymes as a constitutive defense activity as befits innate-type defense factors. in the pharynx, expression gene analysis showed an increase of transcripts of these genes following lps inoculation (fig. 1). immune gene expression analysis of galectins, propo, and cytokines with a time course of 48 h after lps challenge, showed a peculiar upregulation at 1 4 h post injection, followed by a second wave of activation at the stage of 12 48 h (fig. 1). interestingly, a similar pattern of activation has been observed for ci-type-ix-collagen (vizzini et al., 2008), presumably involved as an inducer of inflammation as well as playing a role in the formation of granulation tissue, matrix, and tissue remodeling. these data are indicative of an early phase of inflammatory response activation and a late phase which precedes the resolution of inflammation. 121 fig. 1 cilgal-a,-b, cinpo-1,-2, cimbl, ci-typeix-collagen gene expression in c. intestinalis pharynx after inoculation of lipopolysaccharide (lps) into the body wall. receptors and signal transduction the initial sensing of cell infection is mediated by innate pattern recognition receptors (prrs), which include toll-like receptors (tlrs), rig-i-like receptors (rlrs), nod-like receptors (nlrs) and c-type lectin receptors (beutler, 2009; foster and medzhitov, 2009; takeuchi and akira, 2010). the intracellular signaling cascades triggered by these prrs lead to the transcriptional expression of inflammatory mediators that coordinate the elimination of pathogens and infected cells. in c. intestinalis, screenings of the genome sequence have identified homologues to human tlrs (htlrs) (azumi et al., 2003; sasaki et al., 2009), designated ci-tlr1 and ci-trl2 and composed of a toll/interleukin-1 receptor (tir) domain, a transmembrane, and a leucine-rich repeat (lrr) domain. ci-tlr1 and ci-trl2 result most homologue to htlr 7 and htlr 8. the tir domains of ci-tlr1 and ci-trl2 were most similar to htlr 4 and htlr 6 respectively. they show hybrid functionality since both bind the same ligands of htlr 2, 3, 5. the ci-tlr1 and ci-trl2 genes are expressed intensively in the stomach, intestine, and hemocytes (sasaki et al., 2009). ci-tlrs, like vertebrate tlrs, directly recognize pamps and trigger the transactivation of nuclear factor kb (nfκb) by orthologs of tlr-signaling factors such as myeloid differentiation primary response protein 88 (myd88), interleukin 1 receptor-associated kinase (irak), tumor necrosis factor receptor-associated factors (traf), inhibitor of kappa b protein (iκb), and nf-κb, as well as induce the expression of tumor necrosis factor α (ci tnfα) (fig. 2) (azumi et al., 2003; sasaki et al., 2009). in mammals, lps is recognized by a complex of tlr4 and myeloid differentiation protein-2 (md2) (miyake et al., 2000; dunne and o’neill, 2005; takeda and akira, 2005). in humans, md2 is a component of the md-2-related lipid-recognition (ml) superfamily which contains a large set of genes encoding proteins such as md-1, md-2, niemann-pick 122 fig. 2 inflammatory cascade: pathogens induce inflammation. tlrs activate an myd88-dependent signal transduction pathway. nf-κb is released from iκb and translocates to the nucleus where transcription is upregulated through binding to target inflammatory genes. type c2 (npc2) protein. ncp2 proteins play important roles in lipid metabolism and innate immune response. in c. intestinalis, the use of a subtractive hybridization strategy to identify differentially expressed lps sequences allowed the identification of a ci-npc2 mrna (vizzini et al., 2015a) that becomes upregulated after lps challenge and is preferentially expressed in hemocytes (compartment cells and signet ring cells) inside the vessel lumen of the pharynx. sequence analysis showed a significant homology of ci-ncp2 with several components of the ml superfamily, supporting a conserved evolution of the ncp2 protein and suggesting an evolutionary model based on gene duplication and sequence diversification of ml family components (vizzini et al., 2015a). this finding supported the hypothesis that the recognition of lps by tlr4 through md2 binding may have been acquired during the evolution of vertebrates and that c. intestinalis may respond to lps through a complex with other alternative lps sensors (i.e., the ci-ncp2 protein) before the differentiation of the md-2 protein in vertebrates (vizzini et al., 2015a). inflammation mediators inflammation consists of a tightly regulated cascade of immunological processes that are orchestrated by soluble immune signaling molecules called cytokines. the net effect of an inflammatory response is determined by the balance between pro-inflammatory and anti-inflammatory cytokines. in c. intestinalis, the recognition of lps (pamps) activates a signaling pathway that culminates in the activation of nf-kb and the transcription and translation of genes that lead to the expression of inflammatory cytokines such as transforming growth factor β (tgf-β), interleukin 17 (il-17), and tnf-α (azumi et al., 2003; sasaki et al., 2009). 123 tgf-β belongs to a family of regulatory cytokines that have pleiotropic functions in a broad range of cell lineages involved in numerous physiological and pathological processes, such as embryogenesis, carcinogenesis, and immune response (blobe et al., 2000; li et al., 2006; wharton and derynck, 2009). the tgf-β signaling process appears to be widely conserved in the animal kingdom since components of the pathway are characteristic of protostomes and deuterostomes (huminiecki et al., 2009). in comparative genome analysis of the tgf-β pathway genes in 33 species and in bilateria, at least one type ii receptor and multiple type i receptors were detected, and the ancestral bilaterian repertoire can be inferred as consisting of two type ii receptors and three type i receptors (huminiecki et al., 2009). in ascidians, the ancestral bilaterian tgf receptor repertoire is expanded to three type ii receptors: this is the first example of bilaterian tgf-β receptor duplication, mapping to chordates which is propagated through vertebrates (huminiecki et al., 2009). the signaling output of tgf-β elicits diverse cellular responses that are primarily mediated through the actions of smad transcription factors (massague, 1998; shi and massague, 2003; massague and gomis, 2006). in ascidians, at least two r-smads (one tgf-β and one bone morphogenetic protein), one co-smad, and one ismad have been described (huminiecki et al., 2009). citgf-β is structurally related to members of the superfamily, is synthesized as a large pre-proprotein composed of a hydrophobic signal peptide, a n terminal pro-domain, and a c-terminal active peptide, and also exhibits a cleavage site at a consensus site (rrrk) for generating a c-terminal domain. as with all members of the superfamily, the pro-domain shows a low degree of conservation for correct processing and secretion of the mature dimeric complex, and c-terminal active peptide exhibits high cysteine residues at invariant positions (munger et al., 1999). these are engaged in intramolecular disulphide bonds, resulting in the adoption of a tridimensional structure with a cysteine knot motif and shared secondary structures with two α helix and seven β sheets. in addition, an rgd motif is present with a potential binding of integrins that can activate the tgf-β1 present in lap-b conformation. in mammals, upon binding, it induces adhesion-mediated cell forces that are translated into biochemical signals which can lead to the liberation activation of tgf β from its latent complex (munger et al., 1999). citgf-β become transcriptionally upregulated during the inflammatory process induced by lps inoculation, suggesting that it is involved in the first phase (1-4 hrs) and significant in the secondary phase (48 h) of the inflammatory response in which cell differentiation occurs (vizzini et al., 2016) (fig. 3). this double peak of citgf-β mrna production could be related to the potential function of tgf-β as a pro-inflammatory cytokine and antiinflammatory molecule that, at the end of the reaction, helps to restore homeostasis. in situ hybridization assays show that the citgf-β gene is expressed in tightly packed hemocyte clusters within the vessel lumen by inflammatory hemocytes (granulocytes and univacuolar refractile granulocytes) in the pharynx vessels (vizzini et al., 2016). in mammals, tgf-β has a role in t cell differentiation during immune response, in particular for t helper 17 cells (th17), and in il-17 production (lohr et al., 2006). in humans, il-17 is a proinflammatory cytokine that plays a key role in the clearance of extracellular bacteria promoting cell infiltration and the production of several cytokines and chemokines. il-17 displays the capacity to induce other inflammatory effectors (benderdour et al., 2002; gaffen, 2004) and synergizes with other cytokines at the center of the inflammatory network (gaffen, 2004) to activate the nf-kb (hata et al., 2002). in c. intestinalis, il-17 genes (ciil17-1,ciil172, ciil17-3) show their involvement in the inflammatory response toward lps via pharynx tissue (vizzini et al., 2015b) (fig. 3). c. intestinalis il-17 genes share a prompt expression induced by lps inoculation, suggesting that they are involved in the first phase of inflammatory response, and a significant expression was also found in hemocytes (granulocytes and univacuolar refractile granulocyte) inside the pharynx vessels at 24 h post-inoculation (vizzini et al., 2015b). sequence and structural analysis of ciil-17s revealed that these genes shared similar features with vertebrate orthologs (human il-17a/f) which are involved in inflammation and host defense, including the production of proinflammatory cytokines such as tnfα, chemokines and antimicrobial peptides (iwakura et al., 2011). in c. intestinalis, tumor necrosis factor α gene (citnfα) is expressed either during the inflammatory pharynx response to lps or during the swimming larval phase of development. granulocytes and compartment/morula cells are citnfα-producing cells in both the inflamed pharynx and larvae (parrinello et al., 2010). the pharynx vessel endothelium also takes part in the inflammatory response. the presence of hemocyte nodules in the vessel lumen or associated with the endothelium suggests the involvement of citnfα in recruiting lymphocyte-like cells and promoting the differentiation of inflammatory hemocytes. in larvae, citnfα is expressed by trunk mesenchyme, preoral lobe, and tunic cells, indicating citnfα-expressing cell immigration events and an ontogenetic role (parrinello et al., 2008, 2010). in c. intestinalis, as in vertebrates, the complex interplay between multiple cytokines, cells, and the extracellular matrix is central to the initiation, progression, and resolution of inflammation. transcriptional and post-transcriptional regulation of gene expression in inflammation inflammation is a multicomponent response, and a crucial point of its control is at the level of gene transcription (mrna splicing, mrna polyadenylation, mrna stability, protein translation) which plays an instrumental role in controlling both the magnitude and duration of the inflammatory response. transcriptional and post-transcriptional mechanisms that modify mrna expression, stability, and/or translation provide for the rapid and 124 fig. 3 citgfβ, ciil-17s, citnfα gene expression in c. intestinalis pharynx after inoculation of lipopolysaccharide (lps) into the body wall. flexible control of this process and are particularly important in coordinating the initiation and resolution of inflammation (anderson, 2010). the use of alternative polyadenylation (apa) sites is a regulatory level by which cells can generate different protein isoforms with different functions or with mrnas differing in the length of their 3’ untranslated regions (3’utr) (batra et al., 2015). apa mechanisms can be divided into two major types. the simplest and most frequent are named untranslated region-alternative polyadenylation (utr-apa) in which the alternative poly(a) sites are located in the 3’utr of the mrnas, with the majority of them residing closer to the stop codon (proximal) compared to canonical poly(a) sites (distal). this kind of alternative mechanism results in the shortening of the 3’utr without changing coding capacity. the second type, named coding region-alternative polyadenylation (cr-apa), is a mechanism by which the alternative poly(a) sites reside in the upstream regions of genes. in particular, the less frequent intronic apa involves the recognition of a cryptic intronic poly(a) signal that involves premature polyadenylation within the coding region affecting the sequence of the protein (elkon et al., 2013). through the alteration of the 3’utrs, apa potentially regulates the stability, cellular localization, and translation efficacy of target rnas as 3’utrs serve as binding regions for factors that control these regulatory layers (i.e., micrornas and rna binding proteins). furthermore, 3’utrs of the mrna of proinflammatory proteins can contain regulatory elements, such as interferon-γ-activated inhibitor of translation (gait), that direct their degradation and/or translational repression (fox, 2015). lps challenge in c. intestinalis induces a crapa mechanism that generates an alternative transcript in two genes, named ci8 (vizzini et al., 2013) and cap (vizzini et al., 2016) (fig. 4). in particular, lps was able to weakly modulate the expression of the ci8long transcript and to induce the activation of a lps-induced apa mechanism responsible for the generation of a shorter mrna (ci8short). in fact, in silico analysis identified a noncanonical poly(a) site within the first intron of the annotated gene. sequence analysis showed that the ci8long-deduced amino acid sequence displays a protein domain homologous to components of the receptor transporting protein (rtp) family (mainland and matsunami, 2012). the rtp family consists of four members (rtp1-4): rtp1 and rtp2 are expressed in olfactory neurons and vomeronasal neurons, rtp3 is expressed in the liver, lungs and testes, and rtp4 is expressed in a wide variety of tissues, including lymph nodes, peripheral blood 125 fig. 4 lps challenge in the ascidian c. intestinalis induces a coding region alternative polyadenylation (crapa) event in the annotated ci8 and cap gene. intron-exon organization of the ci8 (a) and cap (b) gene. the thin lines represent the introns, and the open boxes indicate the exons. an intronic polyadenylation signal within the first intron leads to the transcription of variants named, respectively, ci8short and cicap-2 mrna. leucocytes, spleen, and thymus (mainland and matsunami, 2012). the action mechanism of this family of proteins is poorly understood, and the existence of several closely related family members with disparate phenotypes suggests a wide role for these proteins. the short isoform does not contain the rtp domain and does not display any other homologs in the data banks different from the c. intestinalis annotated transcript (vizzini et al., 2013). furthermore, in silico prediction demonstrated that the ci8long derived protein contains two transmembrane regions which are not present in the ci8short protein, suggesting that the short isoform may represent an lps-induced secreted form of the constitutively expressed gene. the ci8short expression profile showed a peak of activation within 1 h post inoculation, followed by a second wave of activation at 12 h. the tissue localization of the ci8short and ci8long transcripts showed that lps inoculation also induced a differential tissue localization of the two mrnas, probably related to the cr-apa mechanism. the ci8long transcript was expressed in some hemocytes of pharynx vessels, whereas the ci8short mrna appears to be strongly upregulated in compartment/morula and signet ring cells as well as in vessel endothelial cells and epithelium (vizzini et al., 2013). in c. intestinalis, lps induces an cr-apa event in the cap gene that generates a shorter mrna cicap-2 gene through an cr-apa site contained within the first intronic sequence and derived from the sequence of the first exon plus a stretch of nucleotides contained within the first intron with coding capacity (vizzini et al., 2016). cap proteins are a wide group of proteins that belong to the cysteine-rich secretory protein, antigen 5 and pathogenesis-related 1 superfamily which, it has been proposed, play key roles in the infection process and the modulation of immune responses in host animals (gibbs et al., 2008). the cicap-2 mrna potentially codifies for a deduced 51 amino acid long protein containing a predicted signal peptide of 22 amino acids and a 29 amino acid mature peptide that does not possess cap elements identified in the cicap protein (bonura et al., 2010; vizzini et al., 2016). expression studies performed in pharynx tissue showed that the cicap-2 mrna is upregulated a few hours after lps injection (1 4 h), while the lps injection procedure slightly modulated the expression levels of cicap. this data is in agreement with the colocalisation of the two mrnas within the hemocytes flowing through the pharynx vessels and epithelium of stigmata, and the net increase of cells expressing the cicap-2 mrna with respect to the cells expressing cicap after the lps challenge (vizzini et al., 2016). furthermore, an in silico analysis identified the presence of a gait element in the 3’utr of cicap2 mrna which was not identified in the 3’utr of cicap (vizzini et al., 2016). this gait element is a cis-acting rna element first identified in the 3’utr of the ceruloplasmin (cp) mrna in humans. in a more general way, gait elements have been found in several immune-related mrnas, showing an important role in gene-specific translation control in innate immunity (vyas et al., 2009). these elements form a stem-loop structure that is involved in the selective translational silencing of mrna transcripts. the gait complex identified in human myeloid cells is hetero-tetrameric, consisting of glutamyl-prolyl-trna synthetase (eprs), ns1associated protein 1 (nsap1), ribosomal protein l13a, and glyceraldehyde-3-phosphate dehydrogenase (gapdh) (fox, 2015). a search of the c. intestinalis genome showed annotated ortholog genes for eprs, ribosomal protein l13a, and gapdh except for nsap1. in this respect, ascidians seem to have a gait structure similar to the murine heterotrimeric gait complex, lacking nsap1 (arif et al., 2012; vizzini et al., 2016). 126 genes encoding cytokines are regulated at transcriptional and post-trascriptional level, mrna stability and at translational level (sariban et al., 1988). tnf-α is a key cytokine regulator of the immune process and of tissue homeostasis. a computational analysis of c. intestinalis was performed to identify cis-regulatory elements in the 3’utr of citnfα, and a gait element, au rich elements (ares), musashi binding element (mbe), and a tnf element were identified (vizzini et al., 2017). in humans, the biosynthesis of tnfα is tightly regulated at the transcriptional (beisang and bohjanen, 2012) and post-transcriptional levels (jensen et al., 2001; karpova et al., 2001, hao and baltimore, 2013). ares are present in the 3’utrs of many cytokine and inflammatory genes (lu et al., 2006; sandberg et al., 2008; wang et al., 2008). the production of tnf-α is mainly controlled posttranscriptionally by cis-elements located in the 3'utr which regulate mrna stability and translation efficiency. in addition to a class ii au-rich element (are2), another rna binding motif has been characterized about 150 nt downstream. this element contains a consensus core of auauuua, which is highly conserved in evolution and has been suggested to synergize with the are2 element in the repression of tnf-α mrna translation and in translation regulation (carpenter et al., 2014). finally, in the 3’utr of citnf-α an mbe was identified that is involved in mrna translational control during cell cycle progression and cancers (arumugam et al., 2012; fox et al., 2015). concluding remarks in c. intestinalis, inflammation is a complex reaction of host defense mechanisms (innate immunity), initiated by the presence of trauma, pathogenic microbes, or foreign materials, aiming at the neutralization of the insult and restoring normal tissue structure and function. this reaction proceeds via an overlapping pattern of events including coagulation, inflammation, 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research report lethal and sub-lethal effects of cypermethrin and glyphosate on the freshwater’s copepod, acanthocyclops robustus am houssou1,2, ej daguégué2, e montchowui1,2 1laboratory of research in aquaculture and aquatics biology and ecology, school of aquaculture of vallée, national university of agriculture, porto-novo, bp 43 kétou, republic of bénin 2laboratory of hydrobiology and aquaculture/faculty of agricultural sciences / university of abomey-calavi, bp 526 cotonou, republic of bénin accepted april 18, 2017 abstract the study aims to evaluate the acute and chronic toxicity of cypermethrin and glyphosate to a freshwater’s copepod, arcanthocyclops robustus. the acute sensibility was assessed by estimating lethal concentrations. then the chronic exposure allowed to assess the effects of low concentrations (0.2489 ppb and 0.4978 ppb respectively 10 % and 20 % of lc50 at 48 h of cypermethrin and 1.3 ppm and 2.6 ppm respectively for glyphosate) on the species. the estimated lethal concentrations at 1%, 50 % and 99 % were 2.353 ppb, 4.755 ppb and 9.610 ppb in 24 h, respectively 0.567 ppb, 2.489 ppb and 10.929 ppb in 48 h for cypermethrin. regarding glyphosate, the lethal concentrations 1 %, 50 % and 99% were 5 ppm, 19 ppm and 73 ppm in 24 h, respectively 8 ppm, 13 ppm and 21 ppm in 48-h. hatching was affected by 20 % lc50 of cypermethrin; only 20 % of females have hatched their eggs against 60 % in the control treatment. females and nauplii survival was affected by both pesticides. a. robustus is then more sensitive to cypermethrin compared to glyphosate. low concentration like 0.4978 ppb of cypermethrin could affect it population and then all the ecosystem biodiversity. key words: acanthocyclops robustus; chronic sensibility; cypermethrin; glyphosate; lethal dose introduction the use of chemical pesticides has greatly expanded in africa, as everywhere in the world, to fight against both the endemic diseases vectors and crops pests (lévèque and paugy, 2006). it results chronic contamination of all ecosystem (aquatic, terrestrial or atmospheric) (druart, 2011). only 0.1 % of sprayed pesticides reach their target, the rest is distributed in ecosystems and contaminate the land, water and air. the atmospheric fraction also finally regains, like atmospheric fallout, soil and surface waters during rainfall (haraguchi et al., 1994). soil leaching and soil-water dispersion of pesticides therefore lead to contamination of aquatic environments (kao, 2002; lalancette, 2012). it results various effects, on ecosystem balance and lead to the extinction of some species (adégbidi, 2000; fadoegnon and midingoyi, 2006; pesce et al., 2008; houssou et al., 2015, 2016). ___________________________________________________________________________ corresponding author: arsène mathieu houssou laboratory of research in aquaculture and aquatics biology and ecology school of aquaculture of vallée national university of agriculture bp 43 kétou, republic of bénin e-mail: arsnehous@yahoo.fr in bénin republic, aquatic ecosystems are contaminated with agricultural pesticides especially in the cotton basin (agbohessi et al., 2011), in the ouémé river (yèhouénou et al., 2006a and b). even, cypermethrin and glyphosate are two of the most used pesticides in food crops production in bénin (preliminary personal investigation: unpublished). the increasing of food crops production in the ouémé river basin causes a consequent increase of the use of cypermethrin and glyphosate, then the increasing of contamination of the ecosystem. freshwater’s copepods are an important part of the planktonic biodiversity. they are part of the first organisms that may present a quickly observable responses facing chemical pollution. due to numerous factors affecting the responses of these organisms to pollutant, specie in two different environments can present wide range of responses to the same substance. then, to efficiently monitor the status of ecosystems, it is important to have biological models for different environment and know their responses facing different type of pollutants. acanthocyclops robustus is one of the most abundant copepod species in the ouémé river basin (houssou et al., unpublished). nowadays, there 141 is no data on the sensitivity of plankton species to the different pollutants in bénin aquatic environments. the aim of this study is to evaluate the response of a. robustus exposed to cypermethrin and glyphosate, used in food crops production in bénin. material and methods test organisms: culture and rearing a plankton sample was taken in ouémé river at bonou (6°54'30.7"n and 2°26'58.0"e) by using plankton net of 50 µm mesh size. this sample was rushed to the laboratory in ambient conditions in absence of fixative. it was then cultured for 2 weeks in the presence of organic fertilizer (chicken droppings) at 0.6 g/l (agadjihouèdé et al., 2011). the species were then identified under photonic microscope and ovigerous females of a. robustus were isolated. a total of 10 females have been placed in a culture of phytoplankton (scenedesmus spp.) and rotifers (brachionus plicatilis). after hatching, the nauplii were kept in the culture until adulthood. the culture solution was renewed every two weeks. at the time of this study, culture has gone through four generations. chemicals cypermethrin is a high active synthetic pyrethroid insecticide. it has light yellow appearance with acidity (as h2so4) of 0.3 % (w/w) maximum. it toxicity classification is ii (moderatly hazardous). cypermethrin is lowly soluble in water (4 to 10 µg/l). in this study, cypermethrin is obtained in it supplied formulation (cyperforce®) containing cypermethrin 10 % ec (emulsifiable concentrate). glyphosate is a non-selective systemic herbicide. it is a weak organic acid. its molecular weight is 169.07 and its solubility in water of 12 g/l at 25 °c. glyphosate is used in this study as supplied formulation (kumark® (480 g/l). different concentrations of both pesticides were dissolved in water to have the needed concentrations of active ingredient. the structural formula of cypermethrin and glyphosate are following (see below). acute toxicity test test design and handling the experimental design for each pesticide is composed of twenty-eight (28) glass cup (petri box) (six concentrations and one control with four replications each) and seven (07) pillboxes. a pillbox containing the test solution of each treatment was used for the measurement of physico-chemical parameters. seven adults of a. robustus were placed in each glass cup immediately after distribution of the test solutions (28 individuals per treatment and a total of 168 per test). an initial test was performed to determine the concentration range to be used in the definitive test (data not presented). the definitive nominal concentrations used are: 0 (control), 1, 2, 4, 6, 8 and 10 ppb for cypermethrin and 0 (control), 2, 4, 8, 12, 16 and 20 ppm for glyphosate. each test lasted 48 h. the test solutions were not renewed and the copepods were not fed (usepa, 2002). the loss of active ingredients concentration in the 48-h was considered insignificant, view cypermethrin is non-volatile (vapor pressure = 3.1x10-9 mmhg at 20 °c; henry's constant = 4.21x10-7 atm.m3/mol) (sage pesticide, 2015). also its half-life time in the water is 14 days. glyphosate also is non-volatile (vapor pressure = 1.84x10-7 mmhg at 45 °c; henry's constant = 4.08x10-19 atm.m3/mol). the photoperiod was 16/8-h (light/darkness). temperature (26.49 ± 0.25° c), ph (7.11 ± 0.11) and dissolved oxygen (3.91 ± 0.19 mg/l) were measured daily in the different treatments. the individual’s mobility and mortality were monitored at 1-h , 24-h and 48-h of exposure. copepod was declared dead with lack of movement after a mild stimulus. chronic tests test design and handling hatching, survival of females and nauplii of a. robustus were studied in this part of the study. a total of 30 glass cups (petri box) of 50 ml each (three treatments with 10 replications each) was used for each pesticide. a pillbox (120 ml) was provided for each treatment allowing the measurement of physico-chemical parameters. two sub-lethal concentrations (10 % and 20 % lc50 in 48 h) were tested in addition to the zeroconcentration (control). after distribution of the test solutions in the experimental design, one ovigerous female of a. robustus was immediately placed in each cup. the feeding was ad-libitum with concentrated freshwater’s rotifer (b. plicatilis) and phytoplanktons. the test solution was renewed in all tests every 96-h. cypermethrin and glyphosate being non-volatile with half-life time in water more than 96-h, the test solution renewal time made insignificant the loss of active mater. the test lasted 10 days and the hatching was controlled daily. female and nauplii mortality have also been monitored daily. the photoperiod was 16/8-h (light/darkness). the temperature (24.73 ± 0.06°c), ph (6.67 ± 0.06) and dissolved oxygen (3.57 ± 0.12 mg/l) were measured daily in the different treatments. 142 table 1 cumulative mortality of acanthocyclops robustus (n = 28 each concentration) exposed to cypermethrin and glyphosate data analysis the copepod survival values were used to estimate the lethal concentrations (lc1-10-20-30-40-50-6070-80-90-99) at 24-h and 48-h. lethal concentrations were calculated using probit method in the computing program poloplus v.1.0. the chi-square test was used to assess difference in response other exposure concentration. the hatching rate, the death rate of nauplii and females were calculated of each treatment. the one way analysis of variance (anova) was used to assess variability of data other treatment (statistica v.7 software was used). results acute toxicity cumulative numbers of dead individual of a. robustus to increase concentration of cypermethrin and glyphosate during the exposure time (24 and 48-h) are presented in table 1. there is significant increased number of dead individual with increasing concentration for both pesticides. respectively two deaths were recorded in the control treatment (zero concentration) at 24-h and 48-h for cypermethrin exposure and four in the two periods for glyphosate exposure. in cypermethrin exposure, there was 100 % mortality from 8 ppb to 10 ppb (24-h) and from 6 ppb to 10 ppb (48-h). respective to the glyphosate exposure, a maximum of 53.57% (24-h) and 100% (48-h) was respectively observed at 16 ppm and 20 ppm. the estimated lethal concentrations of cypermethrin and glyphosate to 1 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % and 99 % of a. robustus population are respectively presented in tables 2 and 3. in case of cypermethrin, the median lethal concentration (lc50) at 24-h (4.755 ppb) was high than that of 48-h (2.489 ppb). regarding glyphosate, the lc50 was 19 ppm (24-h) and 13 ppm (48-h). sub-lethal effects the percentage of females that hatched their eggs according to different treatments is presented in table 4 respectively for cypermethrin and glyphosate. hatching was very low in females exposed to 20 % lc50 of cypermethrin. only 20 % of females have hatched their eggs against 60 % in the control and 10 % lc50 cypermethrin treatment. in the case of glyphosate contamination, the maximum hatching rate was 70 % in the control treatment. the lowest hatching was observed in the 20 % lc50 treatment. table 2 lethal concentration (lc1 to 99) and 95 % confidence limits of cypermethrin to acanthocyclops robustus point concentration (ppb) 24h 48h doses 95%cl 95%cl lc1 2.353 0.667 3.314 0.567 0.156 1.001 lc10 3.228 1.853 3.964 1.102 0.466 1.632 lc20 3.687 1.9324.464 1.457 0.734 2.015 lc30 4.058 2.415 4.778 1.783 1.015 2.355 lc40 4.405 2.912 5.080 2.119 1.332 2.705 lc50 4.755 3.451 5.409 2.489 1.706 3.099 lc60 5.134 4.048 5.817 2.924 2.160 3.59 lc70 5.573 4.705 6.417 3.474 2.726 4.290 lc80 6.134 5.389 7.495 4.251 3.454 5.472 lc90 7.006 6.145 9.839 5.623 4.526 8.126 lc99 9.610 7.745 20.352 10.929 7.698 23.230 chi-square 10.96 degrees of freedom: 2 p = 0.004 glyphosate cypermethrin concentration (ppm) no of mortality concentration (ppb) no of mortality 24-h 48-h 24-h 48-h 0 4 4 0 2 2 2 3 5 1 5 9 4 5 6 2 10 13 8 7 8 4 12 17 12 11 14 6 20 28 16 15 23 8 28 28 20 14 28 10 28 28 chi-square 15.076 14.203 23.385 17.624 df 22 22 22 22 143 table 3 lethal concentrations (lc1 to 99) and corresponding 95 % confidence limits of glyphosate to acanthocyclops robustus regarding the females which hatched their eggs, 100 % had died within 10 days of exposure to 20 % lc50 of cypermethrin (table 4). only 50 % of females had died under 10 % lc50 while in the control 33,33 % mortality was observed. respective to the females which have not hatched their eggs, 100 % mortality was observed with 20 % lc50 of cypermethrin against 75 % and 50 % respectively for 10 % lc50 and the control. in case of glyphosate exposure (table 4) 57.14 % of females which hatched their eggs was died in control treatment. in 10 %lc50 and 20 %lc50 treatment, 66.67 % and 80 % of died was observed respectively. for those which didn’t hatch their eggs in glyphosate exposure, 100 % was died under 10 % lc50 while 66.67 % and 60 % was noted respectively in control and 20 % lc50 treatment. the cumulative mortality of hatched nauplii during the study showed increase sensitivity to the exposure dose of cypermethrin (fig. 1). the dose of 20 % lc50 in 48-h (0.4978 ppb) was more toxic to the nauplii of a. robustus during the 10 days of exposure. a high mortality rate was also observed in the absence of contaminants (control). respective to glyphosate (fig. 2), 10 % lc50 and 20 % lc50 induced more mortality in nauplii. the average number of live nauplii obtained per female after 10 days of exposure is presented on figures 3 and 4 respectively for cypermethrin and glyphosate. in cypermethrin exposure, the lowest average number of nauplii per female was obtained with 20 % lc50 (9.5 ± 3.5 nauplii/female). this mean number is significantly different from those obtained in the other two treatments (anova 1, tukey test, p < 0.05). the averages of 21.67 ± 5.8 nauplii/female and 24.17 ± 10.96 nauplii/female were observed in the control and 10 % lc50 respectively. for glyphosate, no significant difference was observed between treatments. 21.5 ± 7.6 nauplii/female was the highest survival nauplii obtained in glyphosate exposure after 10 days. table 4 hatching rate and mortality of female of acanthocyclops robustus under sub-lethal concentrations of cypermethrin and glyphosate. control 10%lc50 20%lc50 hatching (%) cyperméthrin 60 60 20 glyphosate 70 60 50 mortality of females after hatching (%) cyperméthrin 33,33 50 100 glyphosate 57,14 66,67 80 mortality of females without hatching (%) cyperméthrin 50 75 100 glyphosate 66,67 100 60 point concentration (ppm) 24h 48h doses 95%cl doses 95%cl lc1 5 1 8 8 5 10 lc10 9 2 12 10 7 12 lc20 12 6 15 11 8 12 lc30 14 9 18 12 9 13 lc40 16 12 22 12 10 14 lc50 19 14 28 13 11 14 lc60 22 17 39 14 12 15 lc70 26 20 55 14 13 16 lc80 31 23 86 15 14 17 lc90 40 27 162 17 15 20 lc99 73 40 751 21 18 28 chi-square 28.08 degrees of freedom: 2 p = 0.000 144 fig. 1 cumulative mortality of hatched nauplii other cypermethrin treatments discussion the immobilization follow by mortality of a. robustus is higher when the exposure time is long and the concentration of cypermethrin or glyphosate is high. the increasing doses have therefore lead dysfunction of the nervous system causing paralysis. cypermethrin’s action has focused on interference with the functioning of the sodium channel in the central nervous system, stimulating the nervous discharges repeatedly, causing paralysis (sage pesticide, 2015). christensen et al. (2005) reported that cypermethrin at concentrations greater than 0.1 ppb causes immobilization of crustacean zooplankton in general and in daphnia magna particularly after respectively 6-h, 24-h, and in 48-h exposure. lethal doses determined showed that a. robustus is very sensitive to cypermethrin (cyperforce®). kuldeep et al. (2014) reported that cypermethrin is highly toxic to aquatic invertebrates. this high toxicity is observed on a. robustus with median lethal concentrations (lc50) 24-h and 48-h respectively 4.755 ppb and 2.489 ppb. in case of glyphosate, 19 ppm and 13 ppm was lc50 to a. robustus at 24-h and 48-h exposure respectively. these results showed that cypermethrin is more toxic to a. robustus than glyphosate, as golombieski et al. (2008) reported that insecticides are more toxic to aquatic organisms than other pesticides. chris (2009) also reported that glyphosate is less toxic to fish than other pesticide as: methidathion, beta-cyfluthrin, endosulfan et carbendazine. summarizing these observations, the sensitivity of aquatic species to a pollutant, is affected by both interspecific differences and the type of pollutant (see tables 5 and 6). takahashi et al. (2006) have also showed a large difference in sensitivity between life stages of specie exposed to same pollutant. they demonstrated that adult copepods are higher resistant to diazinon and carbaryl than nauplii. in the present paper, this aspect was not evaluated for a. robustus facing cypermethrin and glyphosate. but the high acute resistance observed by takahashi et al. (2006) in adult copepods to diazinon and carbaryl, confirmed the interspecific and pollutant difference effect in sensitivity of aquatic invertebrates. fig. 2. cumulative mortality of hatched nauplii other glyphosate treatments. 145 fig. 3. mean number of nauplii obtained per female of acanthocyclops robustus after 10 days of exposure to different doses of cypermethrin. plots having different letter are significantly different (turkey, p < 0.05). in aquatic ecosystems where shores are strongly used for agricultural production (as oueme river), concentrations higher than lc50 estimated for a. robustus (especially for cypermethrin) can easily be exceed in water. it would result a strong change in its population and zooplankton community in general (relyea, 2005). according to sarkar et al. (2005), the 96-h lc50 of aquatic invertebrates to cypermethrin are generally between 0.01 and 5 ppb. the observations of this study at 24-h and 48-h of exposure fit into this range and confirm the high sensitivity of the species. regarding the glyphosate, the lethal dose observed is largely high than that of roundup® (glyphosate) to the callanoïda phyllodiaptomus annae (1.06 ppm) (ashoka-deepananda et al., 2011). the difference may be due to the difference in used formulation of glyphosate (kumark® in the present study) also in the genetic difference between the two species of copepod (table 6). however, the department of agriculture of uas has reported that lethal doses of glyphosate roundup® to aquatic invertebrates varied between 4 and 37 ppm (ashoka-deepananda et al., 2011). the chronic effects tests showed that chronic exposure of a. robustus at sub-lethal doses of cypermethrin and glyphosate affects reproduction and survivorship of nauplii. in fact, chronic exposure to both pesticides caused mortality in ovigerous females before and after hatching (reproduction). hatching is strongly affected and survivorship of nauplii is also affected. the results confirm those of hanazato (2001) which showed that pesticides can affect population dynamics of freshwater zooplankton by reducing their survival rates, by affecting the eggs hatching and reducing their richness and specific diversity. lina et al. (2003) also showed zooplankton population reduction in the presence of cypermethrin. ratushnyak et al. (2005) showed that cypermethrin at concentrations above 0.002 ppb to 0.2 ppb does not affect the survival of freshwater’s invertebrates after 21 days of exposure. lutnicka et al. (2014) also showed that a concentration of 0.02 ppb of cypermethrin has no observable toxic effect on reproduction and growth of freshwater organisms. this justified the fact that a concentration of 0.2489 ppb (10% lc50, 48-h), had no significant effect neither on reproduction neither on the survival of a. robustus after 10 days of exposure. significant effects were then obtained with a concentration of 0.4978 ppb (20 % lc50, 48h) after 10 days of exposure. contrary to ratushnyak et al. (2005), wendt-rasch et al. (2003) have previously reported that cypermethrin causes a reduction in zooplankton population at concentrations greater than 0.13 ppb during 11 days of exposure. also lina et al. (2003) showed that copepods facing concentrations of cypermethrin greater than 0.03 ppb, presented firstly ovigerous females mortality and secondly an increase egg hatching rate among survivors. this demonstrates the variation of species responses to a single pollutant whether the environments are different. after 21 days of exposure of daphnia magna to different concentration (0.26 and 0.38 ppm) of glyphosate, no effect was observed on neonates’ survivorship and growth, but the reproduction has affected with increasing concentration (maycock et al., 2010). on freshwater copepods, concentration fig. 4 mean number of nauplii obtained per female of acanthocyclops robustus after 10 days of exposure to different doses of glyphosate. plots having different letter are significantly different (turkey, p < 0.05). 146 table 5 comparative toxicity of cypermethrin in different formulation to different planktonic crustacean species species cypermethrin formulation acute toxicity chronic effects references exposure time toxicity lc50 (ppb) concentration (ppb) effects ceriodaphnia dubia (cladoceran) pestanal® 48-h 0.23 0,0978 affected growth of neonate shen et al. (2012) daphnia magna (cladoceran) pestanal® 48-h 0.72 ashauer et al. (2011) daphnia magna (cladoceran) cypermethrin (cas no.52315-07-8) ≥0.1 significant reduction of the swimming ability. christensen et al. (2005) daphnia schoedleri (cladoceran) pestanal® 48-h 0,6 0.0005, 0.0054 and 0.054 all population parameters were significantly reduced. reduction in reproduction is observed martínezjerónimo et al. (2013) diaptomus forbesi (copepod) pestanal® 48-h 0.03 saha and kaviraj (2008) acartia clause (copepod) pestanal® 48-h 2.67 willis and ling (2004) pseudocalanus elongates (copepod) 48-h >5 acanthocyclops robustus (copepod) cyperforce® 48-h 2.49 0.25 reduced egg hatching in ovigerous females reduced nauplii survivorship present study greater than 0.01 ppm shouted reduce eggs hatchability (maycock et al., 2010). in the present study, a. robustus exposed to chronic doses 1.3 ppm and 2.6 ppm (respectively 10% and 20% of lc50 in 48-h) of glyphosate has presented any significant effects on hatching rate. significant effect on female survival after eggs hatching at 10% lc50 exposure (may be due to physical treatments). it is then observed a high resistance of a. robustus to sub-lethal doses of glyphosate. conclusion a. robustus, a copepod species present in ouémé river ecosystem, appeared very sensitive to cypermethrin. the median lethal concentration (lc50) is 4.755 ppb in 24 h and 2.489 ppb respectively in 48-h. this specie was less sensitive to glyphosate with respective lc50 of 19 ppm and 13 ppm. low concentrations rang of 0.2489 ppb and 0.4978 ppb of cypermethrin affected it reproduction by reducing egg hatching. the survival of ovigerous females and nauplii in the early development stages was also affected. regarding glyphosate, exposure to sub-lethal dose as 2.6 ppm also reduced survival factors of the species. low levels of cypermethrin in the aquatic environment can therefore significantly affect aquatic biodiversity. glyphosate in fact is less toxic for aquatic invertebrates. table 6 comparative toxicity of glyphosate in different formulation to different planktonic crustacean species species glyphosate formulation acute toxicity chronic effects references exposure time toxicity lc50 (ppm) concentration (ppm) effects daphnia magna (cladoceran) glyphosate ipa 48-h 31 4.05 significant reduction in survival significant reduction of body size significant decrease of fecundity cuhra et al. (2013) simocephalus vetulus (cladoceran) eskobat® 48-h 21.5 ≥6.4 age at first reproduction was delayed 2 to 4 days number of neonates/females was significantly reduced reno et al. (2014) notodiaptomus conifer (copepod) eskobat® 48-h 95.2 80 significant delayed of the sexual maturity reno et al. (2014) ≥160 n. conifer could not reach the adult stage phyllodiaptomu s annae (copepod) roundup® 48-h 1.06 ashokadeepanand a et al. 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32: 594-599, 2006a. yèhouénou ape, boko m, cornelis am, gestel v, ahissou h, lalèyè p, et al. organochlorine and organophosphorous pesticide residues in the ouémé river catchment in the republic of bénin. environ. int. 32: 616-623, 2006b. a short or full review on " the immune role c-type lectin in molluscs" for "invertebrate survival journal (isj)" isj 8: 241-246, 2011 issn 1824-307x minireview the immune role of c-type lectins in molluscs l wang1, l wang1,2, m huang1,2, h zhang1, l song1 1key laboratory of experimental marine biology, institute of oceanology, chinese academy of sciences, qingdao 266071, china 2graduate university of chinese academy of sciences, beijing 100049, china accepted december 7, 2011 abstract the phylum mollusca is one of the largest and most important group in the animal kingdom. recently, interest in molluscan immunity has increased due to their importance in worldwide aquaculture, their role in aquatic environmental science and their phylogenetic position, and a great number of immune molecules have been identified and characterized from molluscs. c-type lectins are a superfamily of diverse proteins with one or more carbohydrate recognition domains (crds) of ~130 amino acid residues. they recognize and bind to terminal sugars on glycoproteins and glycolipids and function in non-self recognition and clearance of invaders. this chapter provides a short review of c-type lectins in molluscs, including their structure, function and possible use in science and technology. key words: molluscs; c-type lectin; carbohydrate-recognition domain; non-self recognition; agglutination; phagocytosis; encapsulation introduction lectins are carbohydrate-recognition proteins that bind to specific carbohydrate structures endogenous to the host or presented by microbial invaders (drickamer et al., 1993; barondes et al., 1994), which make them the mediators of non-self recognition in the innate immune response (epstein et al., 1996). although they were first discovered more than 100 years ago in plants, they are now known to be present throughout nature (including the microbial world, wherein they tend to be called by other names, such as hemagglutinins, adhesins, and toxins). the first of the animal lectins shown to be specific for a sugar (l-fucose) was from the eel (watkins and morgan, 1952), and lectins in animals were further known since the purification of an agglutinin in the hemolymph of horseshoe crab (finstad et al., 1974). based on the structure, animal lectins have been classified into at least 13 lectin families, including c-type lectins and galectins, which are classic major families (kilpatrick, 2002). the c-type lectins, structurally characterized by double-loop composed of two highly conserved disulfide bridges located at the bases of the loops, are believed to mediate pathogen recognition and ___________________________________________________________________________ corresponding author: linsheng song institute of oceanology, chinese academy of sciences 7 nanhai rd., qingdao 266071, china e-mail: lshsong@ms.qdio.ac.cn play important roles in the innate immunity of both vertebrates and invertebrates due to their ability to bind specific carbohydrate in a ca2+-dependent manner (devi et al., 2010). the phylum mollusca is one of the largest and most important group in the animal kingdom, and there are about 200,000 living species distributed in terrestrial, freshwater and marine environments (zuschin, 2009). as invertebrates, molluscs lack adaptive immune system, but have evolved sophisticated strategies and rely exclusively on their innate immunity to defend themselves against a variety of pathogens (loker et al., 2004). since a sialic acid-specific lectin was first found in the slug limax flavus (miller, 1982), a number of lectins have been purified and characterized from molluscs. searching in the database of ncbi has revealed that c-type lectins attract much more attention, and totally 246 nucleotide sequences of molluscan c-type lectin, such as 119 from mytilus galloprovincialis, 8 from haliotis discus discus, 7 from chlamys farreri, and 3 from crassostrea gigas are identified. accumulating evidences have favored that these molecules differ significantly in the amino acid sequences and geometrical arrangement of carbohydrate-recognition domain (crd), and participate in many aspects of fundamental biological events, such as recognition of self and non-self, cell to cell interaction, serum glycoprotein turnover and so forth. this chapter reviews the 241 mailto:lshsong@ms.qdio.ac.cn http://www.ncbi.nlm.nih.gov/nuccore/?term=(c-type%20lectin)%20and%20%22molluscs%22%5bporgn:__txid6447%5d http://www.ncbi.nlm.nih.gov/nuccore/?term=(c-type%20lectin)%20and%20%22molluscs%22%5bporgn:__txid6447%5d http://www.ncbi.nlm.nih.gov/nuccore/?term=(c-type%20lectin)%20and%20%22molluscs%22%5bporgn:__txid6447%5d http://www.ncbi.nlm.nih.gov/nuccore/?term=(c-type%20lectin)%20and%20%22molluscs%22%5bporgn:__txid6447%5d interest arose around c-type lectins in molluscan animals, mainly in scallops, clams, oysters, mussels and snails, and especially highlights the diversity of their structure and functions. the crd structure of molluscan c-type lectin the ability of c-type lectins to discriminate self and non-self is determined by their broad selectivity of carbohydrate-binding site and the geometrical arrangement of crd (weis et al., 1998). the crd is a typical structure of most c-type lectins, which consists of 115-130 amino acid residues with several conserved motifs (drickamer et al., 1993; weis and drickamer, 1996). the crd always contains a double-loop structure, and the second loop also called long loop region is involved in ca2+-dependent carbohydrate binding. there are four ca2+-binding sites in this structure, among which the site 2 is known to be involved in the carbohydrate binding, and it is a useful simplification to predict the binding specificity of c-type lectins (drickamer et al., 1993; zelensky and gready, 2005). in this site, there are two conserved motifs determining the crd binding ability, and the first one is always glu-pro-asn (epn) or gln-pro-asp (qpd) in vertebrates (zelensky and gready, 2005). this motif seems to be more various in molluscan c-type lectins, and up to now seven types of motifs have been identified in molluscs, including epn, glu-pro-asp (epd), qpd, gln-pro-gly (qpg), gln-pro-ser (qps), tyr-pro-gly (ypg) and tyr-pro-thr (ypt). epn motif has been considered to determine the crd binding ability to mannose or similar sugar with the 3and 4-oh (weis et al., 1998). this motif is widely spread in molluscan c-type lectins, such as codakine from the tropical clam codakia orbicularis (gourdine et al., 2008), cflec-3, cflec-4 and cflec-5 from zhikong scallop c. farreri (zhang et al., 2009a, b, 2010) and aictl-9 from bay scallop argopecten irradians (wang et al., in press). epd motif is a conserved motif in c-type lectins of invertebrates, which has also been identified in molluscs, such as cflec-1, cflec-2, cflec-3, cflec-4 from c. farreri (wang et al., 2007; zheng et al., 2008; zhang et al., 2009a, b), as well as aictl-2, aictl-6, aictl-7 from a. irradians (zhu et al., 2009; kong et al., 2011; zhang et al., 2011). although the hydrogen bond donor asn in epn motif is replaced by an acceptor asp in epd motif, this replacement has no effect on agglutination towards microbes in d-mannose manner and specificity of carbohydrate binding in scallops (zhang et al., 2011). the motif qpd is only found in mcl-3 from manila clams ruditapes philippinarum (kang et al., 2006) and aictl-1 from a. irradians (zhu et al., 2008) and endows the ability to bind galactose similarly to vertebrate qpd motif in c-type lectins. ypt motif, which is quite different from motifs in vertebrates, is found in cflec-3 from c. farreri as well as aictl-9 from a. irradians and has wider binding spectrum including lipopolysaccharides (lps), peptidoglycan (pgn), yeast glucan, and even cpg oligodeoxynucleotide (data not published). the motifs qpg, qps, and ypd are unusual motifs only found in clhd from abalone h. discus discus (wang et al., 2008), meml from mytilus edulis (espinosa et al., 2010), cvml from crassostrea virginica (jing et al., 2011). qpg motif in clhd offered galactose binding ability which is similar to that of qpd motif (wang et al., 2008). the carbohydrate specificity of crds with qps and ypd motifs is still not well understood and requires further investigations. the second motif in ca2+-binding site 2, always trp-asn-asp (wnd) in vertebrates, has also been reported in invertebrate c-type lectins. the diversity of this motif in molluscs is even greater than that of the first one, and more than 10 motifs have been reported, such as wnd, trp-ile-asp (wid), trp-ser-asp (wsd), trp-his-asp (whd), phe-ser-asp (fsd), and leu-serasp (lsd). the first motif is believed to be the key switch in the specificity of binding with carbohydrate, and the second one can increase the affinity and specificity of this binding (drickamer, 1992; iobst and drickamer, 1994). however, the function of the second motif in invertebrate has not been studied thoroughly, which is very important for us to understand the mechanism of c-type lectin functioning as a pattern recognition receptor (prr). studies in other invertebrates implied that the clustering of multiple crds in one molecule endowed c-type lectin with broader spectrum and higher affinity of binding pathogen-associated molecular patterns (pamps) (watanabe et al., 2006; zhang et al., 2009c). to our knowledge, most of known molluscan lectins have single crd, but there are also multi-crd ones, such as cflec-3 with three crds (zhang et al., 2009b), cflec-4 and aictl-9 with four crds (zhang et al., 2009a; wang et al., in press). clustering of multiple crds may also result in wider specificity of carbohydrate binding in molluscs. for instance, cflec-3 with three crds can bind more pamps than cflec-1 and cflec-2 with single crd (data not published). however, the influence of potential cooperation of multi-crds for binding affinity in molluscan c-type lectins still remains of interest. considerable information has become available of the chemical groups on the lectin and on the carbohydrates that interact with each other and of the types of bond formed, primarily hydrogen bonds and hydrophobic interactions (sharon, 1993). moreover, during the past few years, the number of lectin primary and 3d structures has increased dramatically. interestingly, remarkable similarities have been noticed between the tertiary structures of lectins from diverse sources, in spite of the lack of primary sequence similarities (sharon and halina, 2004). further knowledge of lectin structure will deduct these ubiquitous recognition molecules with myriad exciting functions and applications. immunological functions of c-type lectins accumulating evidences have demonstrated the diversity of sequence and function of c-type lectin in molluscs. their functions in defense processes such as non-self recognition, microbe agglutination, induction of phagocytosis and encapsulation, anti-bacterial properties, will be discussed in detail below. pamps binding and non-self recognition the ability to distinguish self from non-self is 242 http://glycob.oxfordjournals.org/content/14/11/53r.full#ref-60#ref-60 one of the fundamental functions of immune system. due to the lack of adaptive immunity, invertebrate lectins play a major role in non-self recognition (janeway and medzhitov, 2002). c-type lectins specifically bind pamps on the surfaces of many pathogens, which provides them with the ability to recognize a wide variety of pathogens (kilpatrick, 2002; devi et al., 2010). there are increasing evidences that the senescent (i.e., apoptotic) cells are also recognized by lectins for their subsequent clearance by phagocytes. in recent years, many c-type lectins have been identified in molluscs, and their transcription levels increase after stimulation with pathogens or pamps, implying that they are involved in innate immune response (wang et al., 2007; zheng et al., 2008; yang et al., 2011). moreover, molluscan c-type lectins displayed high affinity to various pamps on the surface of pathogens, such as lps from gram-negative bacteria, pgn from gram-positive bacteria, glucan and mannan from fungi, and so on. for instance, a multi-crd lectin from scallop a. irradians (aictl-9) can bind lps, pgn, glucan and mannan (wang et al., in press). manila clam lectin from r. philippinarum can bind n-acetyl-d-galactosamine and mannan (bulgakov et al., 2004). it is noteworthy that scallop c-type lectins with the same first motif of ca2+-binding site 2 had different pamps binding spectrums. cflec-1 containing the motif epd could bind lps, pgn and mannan in vitro, while cflec-2 with the same motif could also bind zymosan besides these three pamps (yang et al., 2010, 2011). these special binding patterns may represent a ligand-receptor interaction that is involved in the recognition of various pathogens through the limited germline-encoded prrs and play key role in immune defense process. agglutination (microbes and erythrocytes) besides non-self recognition, molluscan lectins participate in innate immune responses, including agglutination, hemocyte phagocytosis as well as encapsulation, and even bactericidal effect (wang et al., 2007; zheng et al., 2008; yang et al., 2010, 2011). like other invertebrate lectins, molluscan c-type lectins have the property of agglutinating various microbes as well as vertebrate erythrocytes. for instance, most of scallop c-type lectins exhibited agglutinating activity towards various bacteria and fungi (wang et al., 2007; zheng et al., 2008; zhang et al., 2009b, 2010, 2011; kong et al., 2011). manila clam lectin from r. philippinarum agglutinated erythrocytes from sheep and rabbit (takahashi et al., 2008). moreover, purified lectins from the giant african snail achatina fulica can agglutinate not only bacteria, but also rabbit red blood cells (ito et al., 2011). interestingly, scallop c-type lectins with similar carbohydrate-binding specificity may distinguish different invading microbes in humoral immune system. for example, cflec-1, cflec-2, cflec-3 and cflec-5 from c. farreri, agglutinated e. coli, staphylococcus haemolyticus, pseudomonas stutzeri and pichia pastoris, respectively, though they all possessed mannose-binding specificity (wang et al., 2007; zheng et al., 2008; zhang et al., 2009b, 2010). additionally, pamps, with the same ypt, epd and epn motifs, multi-crd lectin aictl-9 agglutinated not only gram-negative bacteria e. coli and vibrio anguillarum, but also gram-positive bacteria bacillus subtilis (wang et al., in press), while another multi-crd lectin cflec-3 aggregated only the gram-negative bacteria pseudomonas stutzeri, although remarkably (zhang et al., 2009b). the agglutination of foreign particles by c-type lectins have been considered to enable phagocytic cells to recognize invading cells as non-self and therefore initiate the clearing (phagocytosis, encapsulation) process (devi et al., 2010). induction of phagocytosis and encapsulation even there is a great difference between vertebrate and invertebrate immunity, invertebrates share some similar innate immune defense mechanisms with vertebrates, such as encapsulation and phagocytosis (medzhitov and janeway, 2000; plows et al., 2005). molluscan c-type lectins have been reported to play significant roles in hemocyte phagocytosis and encapsulation. for example, manila clam lectins can significantly enhance the hemocyte phagocytic ability toward the bacteria and fluorescent beads (kim et al., 2006; takahashi et al., 2008). cflec-1 and cflec-2 from c. farreri can bind to the surface of scallop hemocytes and recruit them to enhance their in vitro encapsulation (yang et al., 2010, 2011). meanwhile, cflec-1 could also enhance the phagocytic activity of scallop hemocytes against e. coli (yang et al., 2011). the c-type lectin-enhanced hemocytes activity towards microorganisms suggested that these molecules could function as receptors to transduce extracellular signals into the cell, which was similar as the lectins in vertebrates (zelensky and gready, 2005). anti-bacterial properties the mechanism of humoral immune defenses in invertebrate mainly refers to a class of significant effector molecules, such as inducible antimicrobial peptides (amps), to be involved in a direct attack on infectious agents (hoffmann et al., 1999; roch, 1999). some identified molluscan c-type lectins can function in directly suppressing and clearing the microbes, although the underlying mechanism is not exactly known. purified mcl-4 from the plasma of manila clam r. philippinarum could markedly suppress the growth of alteromonas haloplanktis (takahashi et al., 2008). in addition, the recombinant c-type lectins from scallop c. farreri also inhibited the growth of bacteria, such as rcflec-1 inhibiting the growth e. coli and micrococcus luteus (wang et al., 2007), rcflec-2 suppressing the growth of e. coli (zheng et al., 2008). all these studies indicated the molluscan c-type lectins could also contribute to the host defense mechanisms as an effector molecule. the possible use of molluscan lectins in science and technology the activities of lectin are of advantageous within the immune system, both for self/non-self discrimination and interactions between components of the immune system. considering the high 243 abundance of c-type lectins discovered in molluscs as well as their functions in immune system, it is likely that many molluscan c-type lectins are of great significance. use in immunological research and disease control since there is no antibody-mediated immunity in the relatively simple invertebrates, abundant lectins with diverse expression profiles and bioactivities might function as effectors in the immune system. some molluscan c-type lectins, such as cflec-1 (wang et al., 2007; yang et al., 2011) and cflec-2 (zheng et al., 2008; yang et al., 2010), not only function to suppress the growth of microbes and clear the pathogen, but also play significant role in hemocyte phagocytosis and encapsulation. it may be that carbohydrate binding has evolved as a useful additional property amongst unrelated proteins fulfilling a variety of principal functions. future progress will elucidate the contribution of those lectins in mounting protective immune responses for molluscs against infection, which may promote the cognition of invertebrate immune system as well as the development of comparative immunology. furthermore, the abilities of those molluscan lectins to confer resistance to certain bacterial species have opened a new scope in the field of application in disease control for aquaculture animals. use in studying molecule interactions and developing chemical tools lectins are multivalent carbohydrate-binding proteins with the ability to agglutinate erythrocytes, bacteria and other normal and malignant cells displaying more than one saccharide of sufficient complementarity (barondes, 1981). c-type lectins were implicated as the indispensable players in carbohydrate recognition, suggesting the possible application in discrimination of various correlative microbes, and developing biochemical tools. molluscan c-type lectins has the property of agglutinating various microbes as well as vertebrate erythrocytes like other invertebrate lectins. lectins from the giant african snail and manila clam could agglutinate rabbit red blood cells and erythrocytes from sheep and rabbit (takahashi et al., 2008; ito et al., 2011), respectively. c-type lectins from scallops exhibited agglutinating activity of various bacteria and fungi (wang et al., 2007; zheng et al., 2008; zhang et al., 2009b, 2010, 2011; kong et al., 2011). some of lectins in other invertebrates are found specific in their cognition reactions, such as with human blood groups and somewhat bacteria, for instance, crude limulus polyphemus lectin agglutinated 96 % of coagulase-negative strains of staphylococci and none of the human strains of staphylococcus aureus (boyd, 1963; davidson et al., 1982). the special agglutination of lectins may lead to the future development of methodology for the differentiation of certain bacteria and erythrocytes. c-type lectins from molluscs have attracted much attention for their great diversity in structure and activity, and they also provide a model system to understand the molecular basis of how proteins recognize carbohydrates. because of their wide variety of sugar specificities, the molluscan c-type lectins are becoming attractive candidates for the development of biochemical tools in chromatography, blotting, and electrophoresis to purify and characterize the relative molecules and cellular structures. regarding the great diversity, the specific carbohydrate binding and recognition for molluscan c-type lectins still need further investigation, and the search for lectins in such diverse molluscan animals including their identification and characterization may provide the potential to uncover unique lectins, which may enrich the library of future biomedical tools. conclusions a variety of molluscan lectins have enabled greater insight into the diversity and complexity of lectin repertoires in invertebrates. the recent knowledge on the structure and functions of molluscan c-type lectins is underlined in this review. these identified molluscan c-type lectins differ in crd number and motif characterization, which endow them with different property fulfilling a variety of vital functions, and thus they are expected to be applied in several biological and biomedical aspects. the nature of the protein-carbohydrate interaction 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mollusca; immune response; energy demand   introduction the phylum mollusca includes at least 100,000 living species, making it one of the most diverse groups of animals with members occurring in almost every ecosystem on earth. not only does this group of animals colonise both terrestrial and aquatic habitats, they occur in extreme environments such as hot thermal vents and cold seeps. many molluscan species are considered seafood delicacies, while others are an important source of bioactive compounds, used to manufacture jewellery and are an intermediate host of human pathogens. since aquaculture of commercially important molluscan species has grown worldwide, and is likely to extend to additional species as a source of pharmaceuticals, it is becoming increasingly vital that the molluscan immune system ___________________________________________________________________________ corresponding author: vernon coyne department of molecular and cell biology university of cape town rondebosch, 7700, south africa e-mail: vernon.coyne@uct.ac.za is characterised with regard to its structure, function and regulation. it is well documented that high density monoculture results in increased stress and enhanced susceptibility to infectious disease in farmed molluscs (malham et al., 2003; harding, 2004; reddy-lopata et al., 2006; hooper et al., 2007; gestal et al., 2008; costa et al., 2009). additionally, it is becoming increasingly evident that climate change and environmental pollution significantly affect the health status of molluscs (cherkasov et al., 2007; anestis et al., 2008; ivanina et al., 2008). the molluscan innate immune system, which includes both a cellular and humoral component, has been well described by sokolova (2009). circulating hemocytes are integral to the molluscan immune system, being responsible for infiltration, aggregation, encapsulation, cytotoxic reactions and phagocytosis of foreign particles (hooper et al., 2007; gestal et al., 2008). ingested pathogens are neutralised by a hemocyte-mediated oxidative burst which generates reactive oxygen species (ros) that extensively damage the macromolecular   48 mailto:vernon.coyne@uct.ac.za fig. 1 diagrammatic summary of atp involvement in phagocytosis of a foreign body by a generalised molluscan hemocyte. energy-requiring processes are depicted by red arrows, while energy-generating processes are represented by blue arrows. the boxed question marks indicate processes that occur in macrophages and which may hypothetically occur in molluscan hemocytes as a consequence of the similarity between the cell types and conservation of function. pla2, phospholipase a2; cpla2, ca2+-dependent phospholipase a2; ipla2, cytosolic, ca2+-independent phospholipase a2; erk, extracellular signal-regulated kinase; pkc, protein kinase c (erk and pkc regulate phagocytosis in macrophages by inducing arachidonic acid production through activation of ipla2 and cpla2 isoforms; garcía-garcía and rosales, 2002); p2x7, p2x7 receptor (expressed by monocytes, including macrophages); s1p, sphingosine-1-phosphate (a phosphorylated lipid produced by sphingosine kinase (sp kinase) which is proposed to open a channel or transporter, allowing adp to enter the phagosome lumen and be converted to atp by adenylate kinase ( ). the atp binds to the luminal domain of p2x7( ), causing the receptor to transmit a signal across the membrane to activate the actin-assembly machinery on the cytoplasmic side of the phagosome; model proposed by kuehnel et al., 2009a); etc, electron transport chain. components of the pathogenic organism (goedken and de guise, 2004; tiscar and mosca, 2004; sokolova, 2009; donaghy et al., 2010). the oxidative burst is catalysed by membrane bound nadph oxidase that converts molecular oxygen to superoxide anion which is dismutated to hydrogen peroxide (buggé et al., 2007). hydrogen peroxide is a source of other toxic ros such as hydroxyl radicals and singlet oxygen, and is a substrate for myeloperoxidase which it converts to hypochlorous acid (murphy and decoursey, 2006; terahara and takahashi, 2008; buggé et al., 2007). in addition to the toxic effect of ros, phagocytosed microorganisms are attacked by a variety of lysosomal enzymes, such as lysozyme, βglucuronidase, acid and alkaline phosphatase, lipase and aminopeptidase, following the formation of the phagolysosome (gestal et al., 2008; sokolova, 2009). it is not the aim of this review to describe the innate immune system of molluscs in detail, but rather to consider the importance of atp as an essential component of the molluscan immune system (fig. 1). energy production in animal cells animal cells derive energy from mitochondria through oxidative phosphorylation, a process in which electrons are passed along a series of carrier molecules called the electron transport chain (etc), generating the vast majority of the atp required to   49 support cellular functions (watabe and nakaki, 2007). the electrons are initially generated from nadh and fadh2 and ultimately transferred to molecular oxygen (miyazaki et al., 2003). the mitochondrial outer membrane is freely permeable to small molecules and ions, while the convoluted, invaginated inner membrane is rich in cardiolipin, is impermeable to most small molecules and ions, including h+, and includes the enzymes responsible for oxidative phosphorylation, the co-factor coenzyme q (ubiquinone q), the f0f1-atp synthase and some carrier proteins (sas et al., 2007). the matrix, bordered by the inner membrane, contains the mitochondrial dna, chaperones, peptidases and a variety of enzymes that function in different metabolic pathways, including the citric acid cycle, fatty acid oxidation and the urea cycle. the mitochondrial etc consists of several enzyme complexes and cofactors (designated complexes i-iv, the last enzyme is often referred to as complex v); i: nadh ubiquinone reductase, ii: succinate ubiquinone reductase, iii: ubiquinol cytochrome c reductase, iv: cytochrome c oxidase (cox), v: f1f0-atp synthase (sas et al., 2007). energy generated by the passage of electrons down the etc creates a proton gradient across the membrane that drives atp synthase to produce atp from adp. the synthesized atp is used for energy-requiring reactions in the matrix and exported to the cytoplasm by the adenine nucleotide translocator in exchange for cytosolic adp (miyazaki et al., 2003). atp and the molluscan immune system immune cells require a constant supply of energy for basic housekeeping and specific immune functions (buttgereit et al., 2000). the latter includes migration, phagocytosis and cytotoxicity (krauss et al., 2001). consequently, immune stimulation results in increased biochemical activity and thus a greater demand for atp, while processes crucial for specific immune functions are rapidly impaired when immune cells are deprived of energy. phagocytosis, the core of the molluscan innate immune system, is an energy-requiring process (wu et al., 2009). phagocytosis is initiated when a foreign target is recognised and bound by a hemocyte (hughes et al., 1991; wu et al., 2009), resulting in activation of actin-dependent cytoskeletal changes (kuiper et al., 2008). actin polymerisation is considered to be primarily responsible for the formation of pseudopod protrusions around the target, as well as events that occur during early phagocytosis, including ruffle formation, membrane delivery, closure of the phagocytic cup and movement of the newly formed vesicles through the cellular cortex (kuiper et al., 2008). stockinger et al. (2006), using a scintillation proximity assay to study lysosome-phagosome interactions in vitro, showed that lysosome phagosome fusion required atp, actin polymerisation, calmodulin, other cytosolic factors and an absence of ca2+. indeed, a continuous supply of atp is required for f-actin polymerisation from g-actin, and to maintain the activity of nonmuscle myosin atpases that assist in actin and membrane recruitment, and provide motor activity around the phagocytic cup (garcía-garcía and rosales, 2002; kuiper et al., 2008). in fact, kuiper et al. (2008) propose that phagocytosis poses a formidable challenge to cellular energy homeostasis as a consequence of the significant requirement for atp to power the actomyosin-based micromechanical events associated with this process. adenylate kinase the molluscan innate immune system relies on recognition of conserved structures associated with microorganisms, termed pathogen-associated molecular patterns (pamps). lipopolysaccharides (lps), an essential component of the outer membrane of gram-negative bacteria, is a pamp that elicits a strong immune response in molluscs (araya et al., 2010; costa et al., 2009; hannam et al., 2010). once hemocytes detect pamps, they move towards the attractant by a process called chemotaxis (hooper et al., 2007). interleukins and related cytokines are small proteins called chemokines that regulate the effector phase of the immune response (ottaviani et al., 1995; tiscar and mosca, 2004; hooper et al., 2007). il-1α-, il-1β-. il2-, il-6and tnf-α-like molecules have been isolated from the hemocytes of two fresh water snails planorbarius corneus and viviparus ater (ottaviani et al., 1995). the study showed that il1α, il-2 and tnf-α played a role in hemocyte motility, phagocytosis and the induction of nitric oxide synthase. similarly, mytilus galloprovincialis hemocytes were shown to contain il-8, and recombinant human (rh)il-8 induced conformational changes and chemotaxis in the mussel hemocytes (ottaviani et al., 2000). changes in cell shape induced by rhil-8 were generated via protein kinase a and c pathways, leading to reorganisation of actin microfilaments. mouse raw 264.7 and j774a.1 macrophage cell lines exposed to either serum or sphingosine-1-phosphate (s1p), the inducing component in serum, were found to rapidly accumulate extracellular atp that coincided with actin assembly (kuehnel et al., 2009b). the atp was synthesised by adenylate kinase from adp released from the cells within 3 sec of exposure to s1p. the atp pool increased within 5 sec to levels sufficient for activation of the atp receptor p2x7r which links atp-dependent signalling to the actin assembly machinery (kuehnel et al., 2009b). van horssen et al. (2009), using embryonic fibroblasts from knockout mice lacking major atp-adp exchange enzymes, showed that adenylate kinase1 plays a significant role in providing atp for cytoskeletal functions. thus, adenylate kinase is a coupling factor between local atp supply and regulation of actomyosin assembly and other molecular motor behaviour, which is central to cell shape changes and cell motility (dzeja and terzic, 2009). besides a study by moraga and tanguy (2000) which showed that adenylate kinase is a molecular biomarker of sensitivity or resistance to the pesticides atrazine and isoproturon in the pacific oyster crassostrea gigas, there have been no studies to date regarding adenylate kinase from   50 molluscan hemocytes. since molluscan granulocytes strongly resemble vertebrate monocytes and macrophages both in structure and function (sokolova, 2009), it is highly likely that adenylate kinase plays a similar role in the molluscan immune system as that elucidated in mouse macrophage cell lines and thus warrants further investigation. protein kinase c as mentioned earlier, conformational changes and chemotaxis were initiated by rhil-8 via protein kinase a and c pathways in m. galloprovincialis hemocytes (ottaviani et al., 2000). protein kinase c (pkc) has also been detected in hemocytes from the gastropod snail lymnaea stagnalis and found to be phosphorylated (activated) in response to challenge with bacterial lps (walker and plows, 2003). besides pkc activation, lps has been shown to activate both mapk/erk kinase (mek) and extracellular-signal regulated kinase (erk), resulting in phagocytosis of escherichia coli by l. stagnalis hemocytes (plows et al., 2004). thus, in accordance to the classical map kinase pathway, activated pkc phosphorylates ras which phosphorylates raf1, which in turn phosphorylates mek which subsequently activates erk (garcíagarcía and rosales, 2002; plows et al., 2004; lacchini et al., 2006). in addition to phagocytosing invading pathogenic microorganisms, parasites too large to engulf are encapsulated by hemocytes which adhere to and spread over the invading organism and adjacent cells (walker et al., 2010). a model proposed by walker et al. (2010) to explain the signalling pathway leading to encapsulation of a foreign body by l. stagnalis hemocytes, includes a requirement for phosphorylated pkc and focal adhesion kinase during hemocyte adhesion and spreading. phosphorylated pkc is also required for activation and engagement of integrin which is integral to hemocyte adhesion and spreading. because protein kinase cascades amplify signals by serially phosphorylating target kinases, it is likely that atp utilization in phosphorylation reactions is quite significant (krauss et al., 2001). phorbol myristate acetate (pma), a synthetic analogue of diacylglycerol which can activate pkc (costa et al., 2009), has been shown to induce a rapid increase in oxygen consumption (oxidative burst) in neutrophils and lymphocytes (krauss and brand, 2000). similarly, pma induced oxidative activity has been reported in hemocytes from the common periwinkle littorina littorea (gorbushin and iakovleva, 2007), the freshwater snail biomphalaria glabrata (yoshino and humphries, 2008), the disk abalone haliotis discus discus and the spiny top shell turbo cornutus (donaghy et al., 2010). although the exact cause of this phenomenon is unclear, krauss et al. (2001) suggest that activated kinases such as pkc can control energy flow in the mitochondria of lymphocytes. in support of this hypothesis, pma induction of oxidative activity in hemocytes from h. discus discus and t. cornutus is thought to originate mostly from the mitochondria since incubation with carbonyl cyanide-ptrifluoromethoxyphenylhydrazone, which uncouples mitochondrial oxidative phosphorylation, resulted in strong inhibition of the oxidative activity (donaghy et al., 2010). mitochondrial respiratory chain a cdna microarray investigation of gene expression in hemocytes from immune-stimulated haliotis midae identified two etc genes, namely cytochrome b and cytochrome c oxidase iii, which were differentially expressed in comparison to unstimulated abalone hemocytes (arendze-bailey, unpublished). the role of these genes, and thus the electron transport system, in the abalone immune response was investigated by specifically inhibiting cytochrome b with antimycin a and measuring hemocyte immune parameters in vivo (van rensburg and coyne, 2009). antimycin a inhibits succinate oxidase and nadh oxidase, as well as mitochondrial electron transport between cytochrome b and c1 (park et al., 2007) by blocking electron flow in the q-cycle of complex iii (raha et al., 2000). the level of atp in h. midae hemocytes treated with antimycin a decreased by more than 50% of the atp levels present in control cells (van rensburg and coyne, 2009). furthermore, the study showed that phagocytosis of fitc-labelled vibrio anguillarum was significantly reduced in h. midae hemocytes exposed to antimycin a for 2 h as opposed to pbs, indicating the importance of the mitochondrial respiratory chain in the immune response of the abalone due to increased metabolic needs of the cell during immune-stimulation. phosphoarginine and arginine kinase although glycolysis and oxidative phosphorylation are the main pathways for energy generation, cells and tissues have other mechanisms for energy production and storage. for example, phosphagen molecules, such as phosphocreatine (pcr), can act as a temporary buffer during high-demand periods (cloutier and wellstead, 2010). since phagocytosis is such an energy intensive process (kuiper et al., 2008), a pcr buffer is thought to be crucial for coping with a sharp increase in energy demand since the conversion of one molecule of pcr to creatine (cr) regenerates one molecule of atp from adp without contribution from other cellular subsystems (cloutier and wellstead, 2010). indeed, pcr was found to decrease by 40 % in phagocytosing macrophages, indicating that between 1.5 to 2 times more atp is consumed by macrophages engaged in phagocytosis (loike et al., 1979). clearly, atp generation via this reaction is limited by the amount of available pcr, which can be replenished if the energetic state (atp/adp) is favourable. the enzyme creatine kinase catalyses the conversion of pcr to cr (kuiper et al., 2008; cloutier and wellstead, 2010). the importance of creatine kinase in maintaining pcr levels, and thus a reservoir of phosphate for atp synthesis in macrophages, is supported by the observation that both actin and creatine kinase-b (ck-b) are simultaneously recruited to the sites of action during the early phases of particle ingestion (kuiper et al., 2008).   51 creatine kinase, pcr and cr occur predominantly in vertebrates, but have also been identified in a polychaete chaetopterus variopedatus, some annelids, echinoderms, hemichordates, urochordates and sea urchin sperm (wyss and kaddurah-daouk, 2000; pineda and ellington, 2001; suzuki et al., unpublished data). invertebrates have been found to possess a variety of other phosphorylated guanidino compounds and their corresponding kinase, including phosphoarginine (arginine kinase, ak), phosphoglycocyamine (glycocyamine kinase), phosphotaurocyamine (taurocyamine kinase), phospholombricine (lombricine kinase) and phosphohypotaurocyamine (hypotaurocyamine kinase), which play a physiological role that is similar to pcr in vertebrates (wyss and kaddurah-daouk, 2000; uda et al., 2006). ak is the most widely distributed phosphagen kinase among the invertebrates (suzuki et al., 2003; uda et al., 2006). ak genes have been sequenced from a variety of molluscs, including the gastropods conus novaehollandiae (safavi-hemami et al., 2010), littorina fabalis (kemppainen et al., 2010), turbo cornutus (suzuki et al., 1997), haliotis madaka (suzuki and furukohri, 1994) and biomphalaria glabrata (fukunaga et al., unpublished data), the bivalves ensis directus (compaan and ellington, 2003), pholas orientalis (wong and chew, 2009b), meretrix lyrata (wong and chew, 2009a), calyptogena kaikoi (uda et al., 2008) and c. gigas (suzuki et al., unpublished data), and the cephalopods octopus vulgaris (suzuki et al., unpublished data) and nautilus pompilius (suzuki et al., unpublished data). clearly, investigation of the role of ak and phosphoarginine in generating sufficient atp for driving phagocytosis in molluscan hemocytes would be important in furthering our understanding of this energetically expensive process. in support of this point, microarray analysis of gill tissue from crassostrea virginica challenged with the protozoan parasite perkinsus marinus showed increased expression of a putative ak gene in comparison to uninfected control oyster samples (wang et al., 2010). vacuolar-type atpases once the invading organism has been engulfed by hemocytes and a phagosome has formed, phagolysosome biogenesis is the next step in the phagocytic process where the phagosome fuses with a lysosome containing various hydrolases that degrade the molecular components of the phagocytosed organism (steinberg et al., 2007). fusion between the phagosome and lysosome requires actin polymerisation and thus atp (stockinger et al., 2006). the internal ph of the phagolysosome becomes acidic in order for the hydrolytic enzymes to function effectively. acidification of the phagolysosome to a ph of less than 5.5 is accomplished by the action of vacuolartype atpases (v-atpases) which use energy generated from atp hydrolysis to move protons across the phagolysosome membrane (garin et al. 2001; steinberg et al. 2007; clarke et al. 2010). vatpase is a multi-subunit enzyme that occurs in all eukaryotes. however, there is only one published study of a molluscan v-atpase to date. huvet et al. (2004) used suppression subtractive hybridisation to investigate differential gene expression in resistant (r) and susceptible (s) c. gigas progeny to determine the genetic basis for the s oysters’ increased summer mortality rate. one of the cdna clones characterised from the subtracted library that was significantly up-regulated in r oysters compared to the s progeny possessed sequence similarity to a v-atpase from caenorhabditis elegans. interestingly, v-atpase mrna levels in mantle-gonad tissue sampled from r progeny challenged with the bacterial pathogen vibrio splendidus were seven-fold lower than v-atpase mrna levels in samples from unchallenged r oysters, possibly reflecting hemocyte migration from the location of the sampled tissue to the site of bacterial infection (huvet et al., 2004). stress and atp the importance of atp with regard to the molluscan immune system is reflected by the many studies conducted to elucidate the effect of abiotic conditions on the defence response of various commercially important marine shellfish. gametogenesis in bivalves is induced by elevated water temperatures which coincide with increased summer mortalities of the pacific oyster c. gigas. this is thought to be due to depression of the immune system as a consequence of depleted energy reserves. delaporte et al. (2006) showed that the adenylate energy charge decreased significantly during gametogenesis in c. gigas, and that this coincided with inhibition of key immune parameters such as the phagocytic and adhesive capacity of the oyster hemocytes. similarly, phagocytosis and hemolymph antimicrobial activity decreased in post-spawning c. gigas due to depleted energy reserves as a consequence of spawning which is induced by elevated water temperatures (li et al., 2010). the polycyclic aromatic hydrocarbon pollutant phenanthrene was shown to have an immunosuppressive effect on the immune system of the great scallop pecten maximus by reducing hemocyte membrane stability, phagocytic and cytotoxic activity possibly due to disruption of cell metabolism and atp production (hannam et al., 2010). indeed, elevated water temperature has been shown to enhance the deleterious effect of the trace metal pollutant cadmium on mitochondrial enzymes of c. virginica, including the etc, resulting in an energy deficiency (ivanina et al., 2008) which would be expected to compromise the animal’s immune system. concluding remarks a great deal of progress has been made in characterising the innate immune system of molluscs over the past decade. much of our understanding of the molluscan immune system at the molecular level has been extrapolated from information obtained from model invertebrates such as the fruit fly drosophila melanogaster and evolutionary conservation of the innate immune   52 system in vertebrates. however, many gaps remain regarding our understanding of the role that atp plays in the molluscan immune system. our ability to address the importance of atp in the immune system of molluscs will rely on a systems biology approach which will involve genomic, transcriptomic, proteomic and metabolomic characterisation of the molluscan immune system, which in turn, will require a holistic understanding of the physiology of these animals. such an approach is essential given the 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biomphalaria glabrata. dev. comp. immunol. 32: 554-562, 2008.   55 isj 11: yyy-xxx, 2014 isj 11: 109-118, 2014 issn 1824-307x review the role of lysosomal cysteine proteases in crustacean immune response fl garcia-carreño1, ma navarrete del toro 1, a muhlia-almazan2 1centro de investigaciones biologicas del noroeste (cibnor), calle ipn #195, la paz, baja california sur 23096, mexico 2centro de investigacion en alimentacion y desarrollo (ciad), carretera a la victoria km. 0.6. hermosillo, sonora 83000, mexico accepted april 22, 2014 abstract over the long course of evolution and under the selective pressure exerted by pathogens and parasites, animals have selectively fixed a number of defense mechanisms against the constant attack of intruders. the immune response represents a key component to optimize the biological fitness of individuals. two decades ago, prevention and control of diseases in crustacean aquaculture systems were considered priorities in most shrimp-producing countries, but knowledge was scarce and various pathogens have severely affected aquaculture development around the world. scientific contributions have improved our understanding of the crustacean immune response. several studies confirm the central role played by proteases in the immune response of animals, and the cooperative interaction of these enzymes in a wide variety of organisms is well known. this review summarizes the current information regarding the role of cysteine proteases in the immune system of crustacea and points to aspects that are needed to provide a better integration of our knowledge. key words: cathepsin; cysteine proteases; immune response; lysosome; regulation   introduction metazoa comprise approximately 1.3 million living species and the phylum arthropoda accounts for almost 95 % of the total. insecta, the most diverse class among this phylum, has a large evolutionary history; these species have colonized all environments and their immune systems are partly responsible for their biological success (vilmos and kuruz, 1998). in the sea, crustacea is a large and diverse subphylum that comprises about 67,000 species and could be considered aquatic counterparts of insects, but with a lower number of reported species. these species survive in an environment filled with a vast and diverse number of pathogenic agents. both groups, insects and crustaceans, share a close phylogenetic relationship, and consequently, similarities in their innate immune systems (regier et al., 2010). the american academy of allergy, asthma and immunology defines immune system as: “the network of cell types working together to defend and protect the body from invaders, such as viruses, ___________________________________________________________________________ corresponding author: adriana muhlia almazan centro de investigacion en alimentacion y desarrollo, a.c. carretera a la victoria km. 0.6. hermosillo sonora 83000, mexico e-mail: amuhlia@ciad.mx infections and disease” (http://www.aaaai.org/conditionsand-treatments/conditionsdictionary/immune-system.aspx). this definition applies to all species of arthropods; however, knowledge concerning the immune system of insects and crustaceans is still scarce, in contrast to their large number of species. moreover, the structure and function of the immune system differs among taxa and relates to their environmental conditions; thus, comparative studies are necessary to understand the way individual systems work and how they were shaped by natural selection. in the last 20 years, hundreds of studies about the immune system response of dipteran insects, as the fruit fly drosophila melanogaster, and the malaria mosquito anopheles gambiae, have been published (see jules hoffman publications http://wwwibmc.u-strasbg.fr/ridi/profil.php?equipe_id=10&lang=en) (dimopoulos et al., 2000; watson et al., 2005; pham et al., 2007; li et al., 2013). studies about crustaceans immune system sharply increased in the last 10 years, hundreds of studies on the freshwater crayfish pacifastacus leniusculus and some shrimp species of the genera penaeus, litopenaeus, marsupenaeus, and fenneropenaeus spp., are aimed to describe new proteins, specific-receptors and molecules participating as part of response mechanisms that regulate activation of the propo system, melanization and hematopoiesis among others   109 mailto:amuhlia@ciad.mx http://www.aaaai.org/conditions-and-treatments/conditionsdictionary/immune-system.aspx http://www.aaaai.org/conditions-and-treatments/conditionsdictionary/immune-system.aspx http://www-ibmc.u-strasbg.fr/ridi/profil.php?equipe_id=10&lang=en http://www-ibmc.u-strasbg.fr/ridi/profil.php?equipe_id=10&lang=en (söderhäll and cerenius, 1998; supungul et al., 2002; lin and söderhäll, 2012; jin et al., 2013). early studies of the crustacean immune system found proteases as part of the innate response. serine proteases were mainly described as participating in a series of enzymatic cascade pathways, but information about cysteine proteases and their physiological roles is scarce. the discovery of the first lysosomal cysteine protease, cathepsin c, in animal’s tissues, occurred in 1948 by gutman and fruton. no other physiological roles for cathepsins were described until cathepsins b and h were identified in 1970 (barret et al., 1998; turk et al., 2001). these enzymes were considered non-selective, being responsible for protein degradation in cells and only participating in lysosomes. a series of new functions and new enzymes have been identified recently, and some cathepsins and other lysosomal proteases are now recognized as a part of the immune system in vertebrate and invertebrate species. this review provides a comprehensive overview on the current state of knowledge related to crustacean lysosomal cysteine proteases, including those that have recently been described and whose roles include immune response. the most recent findings are discussed and new perspectives about further study of these enzymes are suggested. characteristics of lysosomal cysteine proteases proteases are involved in a plethora of physiological functions and pathologies. regarding the specificity of the enzyme, they are involved in two main functions: those proteases catalyzing nonspecific hydrolysis of proteins, like digestive proteases, and those catalyzing highly selective, limited, and efficient cleavage of specific substrates that control cell physiology (fuchs et al., 2013). the most abundant classes of proteases in human are, in order of abundance, metallo-, serine, and cysteine proteases (186, 176, and 143 genes, hence proteases). some may be grouped according to the site they act on and their function (puente et al., 2003). the cysteine proteases are those enzymes that include a cysteine residue in the catalytic site to hydrolyze peptide bonds (dunn, 1989). these proteases are distributed in all taxa and the clan ca, based on papain, is by far the largest among cysteine proteases, which are single polypeptide chain proteins around 30 kda. all those cysteine proteases, which are involved in immune reactions, belong to the clan ca, commonly associated with the innate and adaptive immunity processes. many of them are found in the lysosome as part of a group that, in concert with the proteasome, plays an important role in the cellular protein turnover pathway. among the lysosomal proteolytic enzymes, the aspartic proteases, in combination with the cysteine and serine proteases, are usually detected in vesicles of the endocytic pathway. besides their role in protein degradation within lysosomes, these enzymes in vertebrates are tissue-specific. they participate in antigen processing and presentation within early endosomes, zymogen processing, degradation of matrix constituents in the extracellular space, cytokine regulation, and in the initiation of apoptotic processes within the cytosol (brix, 2005; colbert et al., 2009). lysosomal cysteine proteases (lcps) have optimum activity in the slightly acidic and reducing milieu of lysosomes. as other proteases, these enzymes are synthesized as zymogens in order to yield activity at the right spot and time, preventing thus unwanted tissue damage (turk et al., 2001). some of the most studied members of the lcps family include cathepsins. various lysosomal cathepsins from vertebrates, such as l, s, c, f, b, x, h, k, v and w are cysteine proteases. cathepsins d and e are aspartic proteases, and cathepsin g, granzymes, and thymus-specific serine proteases (tssp) are serine proteases (colbert et al., 2009). interestingly, as several of the above mentioned enzymes perform the same task of protein degradation, it appears that the proteolytic processing machinery may be functionally redundant, which is supported by the fact that no effect can be detected by the lack of any cathepsin. however, specific functions have been reported for cathepsins expressed in a tissuespecific manner. some examples are bone remodeling by cathepsin k, processing of the major histocompatibility complex class ii by cathepsin s, and a lesser role by cathepsin l. the lysosomal cathepsin c is a major processing device that activates serine granule proteases, granzymes a and b, cathepsin g, neutrophil elastase, and chymase (turk et al., 2001); thus, some of them are required to activate others, and the loss of homeostasis in enzyme activity leads to pathologies. as with other lcps, cathepsins are also synthesized as inactive precursors that become activated after a limited proteolysis that removes the n-terminal propeptide. this propeptide is located on the active site (turk et al., 2001), and activation happens under acidic ph, autocatalytically in trans (one molecule activating another) (rawlings and salvesen, 2012). cathepsin disturbances in mammals have been associated with several malignancies. these enzymes are also involved in apoptosis; pathologies may appear when cathepsin inhibitors, instead, are down-regulated (colbert et al., 2009). since cathepsins b, l and c are the only lysosomal cysteine proteases identified and studied in crustaceans to date, they will be briefly described below (table 1). although caspases are predominantly cytosolic, these cysteine proteases were included in this review since they are involved in the response against infectious agents in vertebrates. recent evidence has shown that they also play a key role in apoptosis in arthropods. cathepsin b cathepsin b (ec 3.4.22.1) was one of the first described members of the large c1 family of lcps. the protein is synthesized as a pre-proenzyme and targeted to lysosomes, and it may exhibit both,   110 table 1 lysosomal cysteine proteases from crustacea species enzyme species tissue/organ protein size (aas) suggested function reference cathepsin b pandalus borealis mg exclusive 328 hydrolysis of food protein aoki et al., 2003 cathepsin b litopenaeus vannamei hm, gills, amp, m, mg, gut, gj 331 hydrolysis of food protein/intracellular protein hydrolysis stephens et al., 2012 cathepsin b fenneropenaeus chinensis muscle, gills, mg 331 may participate in antiwssv immune reactions li et al., 2013 cathepsin c penaeus monodon o, h, mg, m, p, b 449 perhaps involved in the immune defense qiu et al., 2008 cathepsin c eriocheir sinensis hm 427 involved in the innate immune system li et al., 2010 cathepsin c f. chinensis hm, mg, gills, gut 451 may play a role in the antiviral immune response wang et al., 2012 cathepsin l homarus americanus mg, gj 323, 322, 321 no details laycock et al., 1991 cathepsin l nephrops norvegicus es, s 324 cathepsin l may not work as a digestive enzyme le boulay et al., 1995 cathepsin l l. vannamei mg 326, 322 digestive enzyme le boulay et al.,1996 cathepsin l artemia franciscana embryos and young larvae 217 non lysosomal butler et al. 2001 cathepsin l metapenaeus ensis s, mg, h, es, t, o, g 322 hydrolysis of food protein intra and extracellularly hu and leung, 2004, 2007 cathepsin l f. chinensis s, gills, hm, gut, mg -- may participate in antiwssv immune reactions ren et al., 2010 es (eye stalk), mg (midgut gland), g (gut), gj(gastric juice), gills (gills), m (muscle), hm (hemocytes), s (stomach), h (heart), t (testis), o (ovary), p (plasma), b (brain), amp (terminal ampule) endopeptidase or dipeptidyl-carboxipeptidase activities (koga et al., 1991; hasain et al., 1992). the human cathepsin b proenzyme is a glycoprotein with 339 amino acids. all cathepsin bs include an extra 20-residue sequence encoding the highly flexible “occluding loop”, which is characterized by two adjacent histidine residues (his352, his353) and includes amino acid residues pro344 to cys371. the occluding loop confers dipeptidyl-carboxipeptidase activity to the enzyme (musil et al., 1991). cathepsin b compartmentalization within the lysosomes suggests an extensive role for intracellular protein degradation; however, its presence in the nucleus, cytoplasm, and plasmatic membrane indicates alternative roles in physiological and pathological processes. involvement of this enzyme in lysosomal-mediated apoptosis is supported by studies where cathepsin b is knocked out in mice. this enzyme is also found in tumor cells, and a role in invasion and metastasis has been proposed (guicciardi et al., 2000; colbert et al., 2009). cathepsin c cathepsin c (ec 3.4.14.1), better known as dipeptidyl peptidase i (dpp-i), removes dipeptides from n-terminus substrates and activates zymogens. as cathepsin b, it also belongs to the c1 family, and the mature enzyme is a tetramer with a tetrahedral arrangement already confirmed by crystallography (molgaard et al., 2007). in mammals, it is involved in the immune response and inflammatory disorders as arthritis (adkinson et al., 2002). there is evidence that indicates that cathepsins l and s are part of the activation mechanism of human cathepsin c, whose propeptide is removed to activate the mature enzyme (dahl et al., 2001). once active, this enzyme activates neutrophil-derived serine proteases and regulates development of acute experimental arthritis (adkinson et al., 2002). cathepsin l cathepsin l (ec 3.4.22.15) is an endopeptidase with a preference for aromatic residues. it is synthesized as a single chain   111 zymogen that is activated by limited hydrolysis (rawlings and salvesen, 2012). cathepsin l possesses two potential glycosylation sites, however only asn221 is derivatized. the catalytic site is formed by cys138, his276, and asn300. besides been located within the lysosomes at mannose-6-phosphate receptors, it is also located in the nucleus, cytoplasm, and outside the cell. cathepsin l specifically hydrolyzes autophagosomal membrane markers in lysosomes (takahashi et al., 2009). lack or inhibition of the enzyme in mammals leads to several biochemical and cellular alterations, including up-regulating of signaling pathways or turnover of defective collagen. the enzyme is also involved in exogenous protein digestion occurring before the presentation of specific antigen peptides by the major histocompatibility complex (mhc) to cd4+ t cells (onishi et al., 2004). caspases as one of the two major families of cysteine endoproteases, caspases are cysteinyl aspartatespecific proteases in (x-x-x-asp) motifs. these enzymes recognize tetra-peptide sequences on their substrates and hydrolyze peptide bonds after aspartic acid residues. they belong to the clan cd family c14 and rely on the catalytic dyad of his and cys, which is characteristic of this enzyme family (rawlings and salvesen, 2012; mcilwain et al., 2013). although they are predominantly cytosolic, these cysteine proteases were included in this review because they are involved in the response against infectious agents in vertebrates, and recent evidence points out they play also a key role in apoptosis in arthropods. the study of caspases in the nematode caenorhabditis elegans led to the discovery of apoptosis. fourteen nematode and insect caspases are listed in the handbook of proteolytic enzymes (rawlings and salvesen, 2012). since caspases may differ in the length of activation peptides and mature enzymes, they have been classified as procaspases-1, -2, -4, -5, -8, -9, -10, and -14, possessing longer activation peptides, and procaspases-3, -6, -7 -11, and -13 possessing shorter activation peptides (mcilwain et al., 2013). activation of procaspases with a short activation peptide is a two-step process: at first, a cleavage takes place by caspases or granzyme b, followed by an autoproteolytic step (zhivotovsky et al., 1999). apoptotic caspases can be divided into two classes: initiator and executioner caspases. initiator caspases (caspase-2, -8, and -9) are the apical caspases in apoptosis signaling cascades, and their activation is normally required for executioner caspase (caspase-3, -6, and -7) activation (stephen et al., 2010). executioner caspases are paramount in apoptosis; however, this is not their only function, since they are involved in a plethora of cellular functions, including proinflamatory cytokine interleukin processing, counteracting necroptosis, and mediation of cell immunity (rodrigue-gervais and saleh, 2013). participation of proteases in the immune system differences between vertebrate and invertebrate immune systems understanding the immune response of crustaceans has become a major issue in recent years because several pathogens have become catastrophic to shrimp farmers around the world. these include the white spot syndrome virus (wssv; sanchez-paz, 2010) and the recently described early mortality syndrome provoked by a bacterium (tran et al., 2013). efforts by researchers, institutes, and government bureaus have attempted to solve this problem. immune response, as all physiological functions, evolved gradually from a relatively simple immune system to more intricate and complex forms (kaufman, 2010). immunity in vertebrates involves two mechanisms: innate and adaptive immunity. for a long time, studies have demonstrated significant differences between vertebrate and invertebrate immune systems (vilmos and kuruz, 1998; müller et al., 2008). one of the more important differences is the lack of an adaptive immune response in insects and crustaceans, which means that there is no ability in invertebrates to produce immune cells specially designed to attack a specific antigen in a long term response, also called “immunological memory”, whose main purpose is to protect the host from re-infections and protect the immunologically immature offspring (welsh et al., 2004). to date, only the innate immune response present in flies, beetles, shrimp, crayfish, and crabs has been studied (faye, 1990; rinkevich and weissman, 1990; iwanaga et al., 1998; witteveldt et al., 2004). however, recent evidence indicates that these species have a resemblance of an adaptive immunity that is based on molecules other than antibodies, t-cell receptors, and mhc molecules, which was first discovered in snails (adema et al., 1997; zhang et al., 2004). an “alternative” adaptive immunity in crustacea was suggested in 1998 by flegel and pasharawipas, that hypothesized an “acquired tolerance of shrimp to viral pathogens”. subsequently, venegas et al. (2000) suggested a “quasi-immune response” of the kuruma prawn (marsupenaeus japonicus) to wssv. arala-chaves and sequeira (2000), after comparing vertebrate and invertebrate responses, concluded that an alternative adaptive immune response occurs in invertebrates, which is quite different to the adaptive system in vertebrates; invertebrates have a small number and less diverse set of receptors for immunostimulants than vertebrates. flegel (2009) suggested a heritable, antiviral immunity in crustaceans and insects that explains, through a viral accommodation process, the mechanism of heritable resistance to pathogens in arthropods; this is also one on the main characteristics of the adaptive immune response of vertebrates. recent studies report the first dscam (down syndrome cell adhesion molecule), a member of the immunoglobulin superfamily (igsf) in litopenaeus vannamei and penaeus monodon shrimp (chou et al., 2009, 2011), and the e. sinensis crab (jin et al. 2013). the dscam immunoglobulins in invertebrates are quite different from their vertebrate counterparts,   112 since they lack the transmembrane domain and the cytoplasmic tail; however, the single gene encoding dscam generates thousands of variants by alternative splicing; these molecules are found on the cell surface of hemocytes, and gene expression is induced by the presence of lipopolysaccharides, peptidoglycans, and glucans (brites et al., 2008; jin et al., 2013). watthanasurorot (2013) emphasized that the role of dscam in the crayfish p. leniusculus is similar to the generation of antibody diversity in vertebrates and that this specificity in immune recognition is part of the innate immune system of crustaceans. originally, the innate immune system of arthropoda was considered less sophisticated; currently, there is scientific evidence that supports a more complex role because it includes the protection exerted by physical barriers (epithelium) and rapid, efficient, and systematic responses to distinguish and destroy foreign (non-self) materials. innate immunity includes humoral and cellular components, that are triggered by signal transduction pathways and activated following an infectious challenge, mainly in hemocytes and plasma, as originally studied in d. melanogaster (for comprehensive reviews, see borregaard et al., 2000; liu, 2008; vazquez et al., 2009). the innate response in the crayfish p. leniusculus has been studied (liu et al., 2009; watthanasurorot, 2013) and includes responses, such as melanization, blood coagulation, production of antimicrobial peptides, phagocytosis of small pathogens, and encapsulation of invading parasites that are too large to be phagocytized by individual hemocytes (liu, 2008). a complex array of factors is activated when molecules are released from microbial structures, such as bacterial cell wall or double-stranded rna in viruses. in general, these factors are pattern-recognition proteins, such as ßglucan-binding protein, lipopolysaccarideand glucan-binding protein, masquerade-like protein/serine proteinase homologues, and lectins (maftuch et al., 2013). among invertebrate strategies to expand immune responsiveness is to produce a wide variety of antimicrobial peptides (amps; ghosh et al., 2011). amps are small, cation amphipatic molecules synthesized in the fat body of insects (hoffman and reichhart, 1997). in crustaceans, the first described amps were found in crabs, carcinus maenas (schnapp et al., 1996) and callinectes sapidus (khoo et al., 1999), and later in shrimp l. vannamei (destoumieux et al., 1997), these molecules specifically recognize chemical entities from microbial structures as part of an initial humoral response of the host against invasions of gramnegative bacteria, fungi, yeast, viruses, and protozoa (liu, 2008). in crustacean hemocytes amps are released to the hemolymph as a response to an infection (destoumieux et al., 2000; bulet et al., 2004). two major groups of shrimp amps are known: penaeidins and crustins. four classes and several isoforms of penaeidins have been identified in crustaceans, all are small peptides (5 7 kda), with specific characteristics as the c-terminal cysteinerich domain. penaeidin diversity relies on the variation of gene sequences and gene expression and in their activity against different microbes (ghosh et al., 2011). crustins are larger than penaeidins (7 14 kda) that contain whey acidic protein (wap) domains, including a four-disulfide-core motif of around 50 amino acids and eight highly-conserved cysteine residues (smith et al., 2008). several crustins have been classified into various types, according to their amino acid sequences and their wap domains; new proteins are described in freshwater and marine crustaceans containing up to two wap domains that confer the ability of inhibiting proteinases and exhibit antiviral and antimicrobial activities (chen et al., 2008; li and xiang, 2013). proteases as part of the crustacean immune system some of the more important molecules involved in the innate response of the arthropoda, mainly crustaceans, have been already mentioned. now, this review will focus on the proteases used to regulate defense responses, including antimicrobial peptide synthesis, melanization, and coagulation, which are carried in the hemolymph and are rapidly mobilized to activate the immune system (kawabata et al., 1996; iwanaga et al., 1998; gorman and paskewitz, 2001). over recent decades, a large amount of fundamental scientific information on the role of proteases in the immune system of invertebrates has been reported (gorman and paskewitz, 2001). studies in insects, mainly those species whose complete genome has been sequenced, such as the malaria mosquito anopheles gambiae, have identified a series of proteases as part of the responsive immune system, including serine proteases that participate in activating the prophenoloxidase (propo) cascade, trypsin-like proteases, serpins and their specific inhibitors, as part of the coagulation cascade (dimopoulos et al., 2000). the serine protease sp22d from a. gambiae is one of the first enzymes linked to the immune response; its transcriptional up-regulation occurs within 1 h after infection in immune related cell types, and its multi-domain organization suggests that it interacts with pathogen surfaces (gorman et al., 2000). in crustaceans, the lack of a completely sequenced genome has limited the identification and characterization of immune-related genes; therefore, there is less information available for these species. recently, tassanakajon et al. (2013) and jeswin et al. (2013) reported a list of molecules that may be involved in shrimp immunity, some of which will be described. in crayfish, the process of binding-pattern recognition proteins to a microbial molecular target causes propo activation to phenoloxidase (po), which is the terminal enzyme of the system that leads to the synthesis of melanin (sritunyalucksana and söderhall, 2000; cerenius et al., 2010). production of melanin, melanization, as a response to invaders, is a ubiquitous innate immune response that starts in hemocytes with the conversion of inactive propo to active po by a serine protease, the prophenoloxidase activating enzyme (ppa) and depends on a proteolytic cascade similar to the one   113 activating the toll pathway (söderhäll, 2010). shrimp genes encoding a pro-phenoloxidase, prophenoloxidase-activating factor, and a masquerade-like serine proteinase-like protein are involved in detecting and clearing bacteria, mediating hemocytes adhesion, and binding to bacteria cell walls (tassanakajon et al., 2013). serine proteases function as important regulatory proteins in activating the propo and clotting systems of the fleshy prawn fenneropenaeus chinensis, giant tiger prawn p. monodon, and whiteleg shrimp l. vannamei (supungul et al., 2002; jimenez-vega et al., 2005; dong and xiang, 2007; xue et al., 2013). trypsins and chymotrypsins participate in the immune response of marine and freshwater shrimp and crayfish by rapidly activating the immune response when pathogens are detected (xue et al., 2013). newly discovered enzymes, such as a chymotrypsin-like enzyme, have been found in crustaceans; the enzyme was isolated from the midgut gland of the redclaw crayfish cherax quadricarinatus. it has been suggested that it participates in the innate immune reactions of adult and juvenile crayfish since its transcripts were detected in all tissues, but mainly in midgut gland, intestine, hemocytes and muscle where this enzyme is over-expressed after a wssv challenge (fang et al., 2013). apoptosis and toll pathways two physiological mechanisms evolved to lead embryos development and deal with microbial infections in larval and adult stages. they are apoptosis and the toll pathway, with participation of cysteine proteases. apoptosis is a complex mechanism of defense against viral infections by inhibiting viral replication. although not exclusively, apoptosis is a process of cell destruction requiring cysteine proteases called caspases (wang et al., 2008). apoptosis in crustaceans is a highly regulated mechanism of cellular suicide that includes a series of consecutive steps, from sensing invading agents to mitochondrial changes in membrane permeability (menze et al., 2010; leu et al., 2013). caspases act as initiators and executioners in the process of apoptosis (see enzyme description above). in the crustacea, different caspases have been identified, all of them include the typical domain structure of mammalian caspases (wang et al., 2013). in the tiger prawn p. monodon, experimentally inoculated with the wssv, there is an increase in gene expression and enzyme activity of hemocyte caspase-3, which is present until apoptotic cell death (wongprasert et al., 2007). in the whiteleg shrimp l. vannamei, four enzymes were recently identified as lv-caspases 2 5, expressed in a tissue-specific manner. these were up-regulated after a viral challenge, suggesting a protective role in the defense against viruses (wang et al., 2013). various conserved molecules, acting as apoptotic regulators, have also been identified in crustacean proteomes, but further research should reveal their functions because they have not been confirmed. the toll pathway uses toll-like receptors (tlrs) to recognize gram-positive bacteria, fungi, and viruses to initiate an innate defensive reaction. in a review by valanne et al. (2011), nine toll receptors were found in the fly genome. invading pathogens activate the cellular response and systemic synthesis of antimicrobial peptides. activation happens when the proteolytically cleaved ligand spätzle attaches to the toll receptor, which then leads to activation of the nf-k b (dorsal) factor. the vertebrate tlr signaling pathway and the invertebrate toll signaling pathway share similarities and it is suggested they share similar function. recent information demonstrates a functional toll signaling pathway in crustaceans. identification of molecules, such as toll-like receptors (tlrs), the cytokine-like molecule spätzle, and the pelle protein, among others in several shrimp species, are similar to proteases reported in drosophila melanogaster, which suggests that a signal transduction system is part of the innate immune response of crustaceans (li et al., 2013). crustacean lysosomal cysteine proteases studying transcriptomes in shrimp has contributed to the ests list of proteins whose genes may be involved in the immune functions when organisms are infected by a virus, bacteria, or both (dong and xiang, 2007; tassanakajon et al., 2013). besides caspases, cathepsins are the more frequently reported cysteine proteases in the crustacean list of immune-related genes. probably the first reported cysteine protease was found in the gastric fluid of the american lobster homarus americanus (laycock et al., 1989). later studies report cathepsins l, c, and b; these are the only lysosomal cysteine proteases that have been identified and studied in shrimp tissues and species (table 1). in the crustacea, cathepsin c was first suggested to be involved in maturation of oocytes of the kuruma shrimp marsupenaeus japonicus (qiu et al., 2005). qiu et al. (2008) characterized the cathepsin c cdna in the giant tiger prawn p. monodon and proposed, for the first time, that it participates in the immune response of shrimp. experimental evidence indicates that cathepsin c mrna is over-expressed in midgut gland when stimulated by lipopolysaccharides (lps). increased levels of mrna of this enzyme were detected in hemocytes of the crab e. sinensis after a vibrio anguillarum challenge, peaking at 6 h after injection (li et al., 2010). other studies that analyzed the structure and phylogenetic relationships of shrimp cathepsin b are conclusive about its conserved regions, characteristic domains, and similarity to insect homologues; however, its function seems to vary among species. aoki et al. (2003) reported the first cathepsin b exclusively expressed in the midgut gland of the ghost shrimp pandalus borealis and suggest that the enzyme is a food protein hydrolase. later, stephens et al. (2012) found that cathepsin b mrna is expressed and the enzyme activated in several shrimp tissues, and suggested that the enzyme participates in extracellular hydrolysis of food protein as they compared the enzyme with other digestive enzymes as trypsin and   114 chymotrypsin. li et al. (2013) showed that cathepsin b participates in the antiviral immune response of the fleshy prawn as the amount of mrna increased after a wssv challenge. although cathepsin l has been proposed as part of the immune response of the fleshy prawn (ren et al., 2010), there is no information about the relationship between the enzyme digesting exogenous proteins and the production of antigenic peptides or any similar role, as observed in mammals, to date. to better understand the role of crustacean enzymes in the innate immune system, biochemical studies are required. acquiring data about the protein structure and their kinetic parameters of cysteine proteases will be useful, including their abilities to hydrolyze different substrates and to be inhibited by specific compounds. inhibition studies are essential, but there are no reports for these enzymes in crustaceans. since specific inhibitors have been tested on cathepsins in human cell lines in an attempt to control their activity in cancer cells, inhibition assays may help to determine how cathepsins function in uninfected/infected shrimp. as mentioned above, proteases do not act individually; instead, protease cascades may be activated through a mechanism of pattern recognition that controls coagulation, melanization, and activation of toll and apoptosis pathways. since experimental evidence shows a series of unidentified active proteases in zymogram gels of shrimp extracts, it is likely that not all molecules that form part of these complex mechanisms have been identified or their functions in crustaceans understood. differences between terrestrial and aquatic environments may explain some adaptive characteristics related to the structure and function of these enzymes, as observed by rojo et al. (2013) who demonstrated that cathepsin d in the midgut gland of clawed lobsters is a cold-adapted enzyme having structural modifications. immunohistochemical studies should provide information about extracellular enzymes functions as food protein hydrolases in the midgut gland and/or their role as intracellular lysosomal proteases actively participating in the innate immune response. concluding remarks some of the roles of lcps, as part of the immune system, were reviewed for the most studied vertebrate models and compared with roles of crustacean cysteine proteases. the absence of an adaptive immune system in crustaceans as that of mammals, which includes a mhc, t cells, and other components, may limit the roles of cysteine proteases, as they have been described; thus, we suggest that the enzymes are: (1) devoted only to essential functions, such as hydrolyzing food protein extracellularly and hydrolyzing lysosomal proteins intracellularly; and (2) involved in somewhat-related functions in the immune system, acting in a less specific manner than mammalian enzymes. studies of the crustacean immune system, including recent findings of molecules, such as the dscam, toll pathway receptors, and cathepsins suggest the innate immune response is more complex than expected. as research continues, there is an important opportunity to find new cysteine proteases, probably including a large group of caspases, unreported cathepsins, and other lysosomal aspartic and serine proteases. all the team players, their identities, characteristics, and functions may be integrated into the physiological and pathological mechanisms that allow crustaceans to overcome pathogens and develop resistance-states to viruses and bacteria in the sea. acknowledgements we acknowledge support from consejo nacional de ciencia y tecnologia (conacyt national council for research and technology, mexico) for grant 177954 to fgc. we also thank dr. iral fogel for critical reading of this manuscript. references adema c, hertel l, miller r, loker e. a family of fibrinogen-related proteins that precipitates parasite-derived molecules is produced by an invertebrate after infection. proc. natl. acad. sci. 94: 8691-8696,1997. adkison a m, raptis s z, kelley dg, pham ct. dipeptidyl peptidase i activates neutrophilderived serine proteases and regulates the development of acute experimental arthritis. j. clin. invest. 109: 363-371, 2002. aoki h, ahsan mn, watabe s. molecular cloning and characterization of cathepsin b from the hepatopancreas of northern shrimp pandalus borealis. comp. biochem. physiol. 134b: 681694, 2003. arala-chaves m, sequeira t. is there any kind of adaptive immunity in invertebrates? aquaculture 191: 247-258, 2000. barret aj, rawlings nd, woessner jf. handbook of proteolytic enzymes, academic press, london, uk, 1998. brites d, mctaggart s, morris k, anderson j, thomas k, colson i, et al. the dscam homologue of the crustacean daphnia is diversified by alternative splicing like in insects. mol. biol. evol. 25: 1429-1439, 2008. brix k. lysosomal proteases: revival of the sleeping beauty. in: madame curie bioscience database.landes bioscience, texas, usa, 2005. 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2015 to the editor how would we best inquire the challenging query for the likely shared allorecognition properties between taxonomically distant marine invertebrates? are the customary comparisons made with the mammalian immune systems satisfactory? the below discussion challenges this routinely employed research approach. as organismal genetic-homogeneity is assumed to be evolutionary beneficial in preventing innerorganism conflicts (dawkins, 1990; pal and papp, 2000), numerous natural and experimental transplantation situations have spawned considerable interest in the evolution of allorecognition, the non-pathogenically directed property of immunity (stewart, 1992; de boer, 1995; rinkevich, 1999). therefore, it has been proposed that preserving ‘individuality’ from soma and the germline littering by conspecific alien cells might have been the primeval function of the immune system, a theory that sees allorecognition as the possible key common background on which the diverse immunity has been developed (magor et al., 1999; rinkevich, 1999, 2012). following the aforementioned rationale, and if preservation of the individual identity was the original and salient function of the primitive immune system before it was harnessed to defend from invasive pathogens, we may expect to find intimate linkages between the allorecognition machineries in various phyla of multicellular organisms (grosberg, 1988). indeed, allorecognition phenomena exhibit suites of effector mechanisms, are ubiquitously recorded in diverse taxa of marine sedentary invertebrates, including sponges, cnidarians, bryozoans and urochordates, and are frequently documented in the field following direct tissue contacts between conspecifics (e.g., oka and watanabe, 1960; hildemann, 1979; rinkevich, 1999, 2002; cima et al., 2004; hughes et al., 2004; cerrano et al., 2007; fernàndez-busquets, 2008). these tissue-to-tissue contacts beget surprisingly complex sets of allorecognition phenomena, typified ___________________________________________________________________________ corresponding author: buki rinkevich israel oceanography & limnological research national institute of oceanography tel shikmona, po box 8030, haifa 31080, israel e-mail: buki@ocean.org.il by extreme allotypic diversity, a wide range of effector arms (many of which are simultaneously used for other purposes, such as feeding and competition; williams, 1991; rinkevich, 2011), allogeneic maturation, highly tuned immunological specificity, quasi-immunological memory, alloincompatible necrotic zones and fusion events that lead to chimerism (reviewed in oka and watanabe, 1960; grosberg, 1988; leddy and green, 1991; rinkevich 1996a, b, 1999, 2002, 2004, 2011; gaino et al., 1999; cima et al., 2004; cerrano et al., 2007). the genetics of allorecognition in marine invertebrates were elucidated in two model systems, the hydrozoan hydractinia symbiolongicarpus (cadavid et al., 2004; nicotra et al., 2009) and the colonial urochordate botryllus schlosseri (sabbadin et al., 1992; rinkevich, 1993; voskoboynik et al., 2013a). while the commonalities of allorecognition phenomena are above dispute (loker et al., 2004), the evolutionary basis for the effector mechanisms is not easily understood or illustrated yet. many of the related evolutionary claims, which are based on the striking superficial similarities between some genes and processes in marine invertebrates, are based on the claimed erroneous notion, that vertebrate immunity and marine invertebrate allorecognition are homologous, ‘stemming from the rationale that the early appearance of host defense indicates that same immune constituents are shared by most multicellular organisms; a sort of anthropocentrism’ (rinkevich, 2011). also, synthetic comparisons of marine invertebrates’ genes with seemingly counterpart vertebrate immune genes (the common approach taken by scientists) result in very limited valid information regarding the nature of allorecognition in marine invertebrates, as does the employment of deduced genomic sequences or gene homology comparisons (loker, 2004; rinkevich, 2011, 2012). the same holds true when the comparisons are made on the cellular level (peddie and smith, 1995; ballarin et al., 2001; khalturin et al., 2003; dunn, 2009). the literature thus reveals that we still do not really know what allorecognition in marine invertebrates is. as complex as allorecognition phenomena in marine invertebrates are, the historecognition attributes that accept/reject alien tissues are probably not associated with host-parasitic and disease 170   mailto:buki@ocean.org.il responses; thus host-parasitic/disease events (that are customary compared with the mammalian immune systems) have emerged as a serious obstacle to elucidating the nature of allorecognition (rinkevich, 2012). based on the aforementioned, we may reexamine the following query: are the salient properties of allorecognition systems in marine invertebrates conserved and thus shared by unalike taxa? this would be best summoned by analyzing allorecognition in one of the most morphologically simplest extant metazoans that do not possess a circulatory system (e.g., cnidarians; dunn, 1999) and comparing the results with results from one of the most developed metazoan groups that has a circulatory system operating in self/non-self recognition (e.g., colonial urochordates; rinkevich, 2002). both groups present copious in situ documentations for allogeneic and xenogeneic encounters. as the cellular components of allorecognition in both phyla are clearly dissimilar (e.g., the nematocysts in cnidarians and the morula cells in the tunicates; rinkevich et al., 1998; dunn, 1999; ballarin et al., 2001; rinkevich, 2012), it is advisable to establish cross-phyla comparisons at the molecular constituents that actually manifest allorecognition responses (in lieu of molecular comparisons with lists of mammalian immune related genes). this approach is further eased by the recent development in genome sequences in marine cnidarians and urochordates (e.g., rast and messier-solek, 2008; steele et al., 2011; voskoboynik et al., 2013b). a recent study (oren et al., 2013) illuminates the scientific insight that surfaced when employing the cross-invertebrate phyla comparisons approach and the need for additional, similar studies. oren et al. (2013) compared two independently developed allogeneic rejection transcriptomes, one from incompatible challenged colonies of the stony coral stylophora pistillata and the other from allo-rejecting partners of the ascidian b. schlosseri. this revealed common expression patterns of specific immunerelated genes and shared functional attributes expressed during allogeneic rejection. oren et al. (2013) disclosed 74 similar blast matches in the immune-related categories of the coral and the ascidian expression libraries, accounting for 37.2 % of the total immunerelated matches. within these matches, 43/74 cases were exact matches. two highly noticeable genes within this shared list of expressed genes were the immunophilins, cyclophilin a (cypa) and the fk506-binding protein (fkbp). the mrna expressions of the coral and ascidian immunophilins in allorecognition challenged colonies were restricted to the specific effector cell populations (nematoblasts and nematocytes in the coral and morula cells in the ascidian). furthermore, gene expressions were limited to only some of the effector cells within a population, disclosing disparities in numbers and location between naïve and immune challenged colonies. administration of the immunosuppression drug cyclosporine-a during ascidian allogeneic interactions inhibited both the fusion and the rejection reactions, probably through the inhibition of the ascidian morula cells’ movement and activation. the results of oren et al. (2013) illuminated the evolutionary shared immunocyte activation mechanisms that take place during allogeneic responses in these remotely affiliated taxa. these remarkable similarities are present at all allogeneic levels and include a similar inhibitory effect of immunosuppressive agents. cumulatively, the results showcase the need for additional studies targeting cross-invertebrate phyla comparisons (and less studies that target vertebrates/invertebrates comparisons) on the molecular pathways characteristic to allorecognition responses. acknowledgements this study was supported by a grant from the israel science foundation (68/10). references ballarin l, franchini a, ottaviani e, sabbadin a. morula cells as the major immunomodulatory hemocytes in ascidians: evidences from the colonial species botryllus schlosseri. biol. bull. 201: 59-64, 2001. cadavid lf, powell ae, nicotra ml moreno m, buss lw. an invertebrate histocompatibility complex. genetics 167: 357-365, 2004. cerrano c, calcinai b, di camillo cg, valisano l, bavestrello g. how and why do sponges incorporate foreign material? strategies in porifera. porifera research: biodiversity, innovation and sustainability. série livros 28: 239-246, 2007. cima f, sabbadin a, ballarin l. cellular aspects of allorecognition in the compound ascidian botryllus schlosseri. dev. comp. immunol. 28: 881-889, 2004. dawkins r. parasites, desiderata lists and the paradox of the organism. parasitology 100: s63-s73, 1990. de boer rt. the evolution of polymorphic compatibility molecules. mol. biol. evol. 12: 494-502, 1995. dunn sr. immunorecognition and immunoreceptors in the cnidaria. inv. surv. j. 6: 7-14, 2009. fernàndez-busquets x. the sponge as a model of cellular recognition. in: sourcebook of models for biomedical research, humana press, pp 75-83, 2008. gaino e, bavestrello g, magnino g. self/non�self recognition in sponges. ital. j. zool. 66: 299315, 1999. grosberg rk. the evolution of allorecognition specificity in clonal invertebrates. q. rev. biol. 63: 277-412, 1988. hildemann wh. immunocompetence and allogeneic polymorphism among invertebrates. transplantation 27: 1-3, 1979. hughes rn, manriquez ph, morley s, craig sf, bishop jdd. kin or self-recognition? colonial fusibility of the bryozoan celleporella hyaline. evol. dev. 6: 431-437, 2004. khalturin k, becker m, rinkevich b, bosch tc. urochordates and the origin of natural killer cells: identification of a cd94/nkr-p1-related receptor in blood cells of botryllus. proc. natl. acad. sci. 100: 622-627, 2003. 171   leddy v, green dr. historecognition of the cnidaria. in: warr gw, cohen r (eds), phylogenesis of immune functions, crc press, boca raton, pp 103-116, 1991. loker es, adema cm, zhang sm, kepler tb. invertebrate immune systems–not homogeneous, not simple, not well understood. immunol. rev. 198: 10-24, 2004. magor bg, tomoso a, rinkevich b, weissman il. allorecognition in colonial tunicates: protection against predatory cell lineages? immunol. rev. 167: 69-79, 1999. nicotra ml, powell ae, rosengarten rd, moreno m, grimwood j, lakkis fg, et al. a hypervariable invertebrate allodeterminant. curr. biol. 19: 583-589, 2009. oka h, watanabe h. problems of colony-specificity in compound ascidians. bull. mar. biol. sta. asamushi, tohoku univ. 10: 153-155, 1960. oren m, paz g, douek j, rosner a, amar ko, rinkevich b. marine invertebrates cross phyla comparisons reveal highly conserved immune machinery. immunobiology 218: 484-495, 2013. pal c, papp b. selfish cells threaten multicellular life. trends ecol. evol. 15: 351-352, 2000. peddie cm, smith vj. ‘lymphocyte-like’ cells in ascidians: precursors for vertebrate lymphocytes? fish shellfish immunol. 5: 613-629, 1995. rast jp, messier-solek c. marine invertebrate genome sequences and our evolving understanding of animal immunity. biol. bull. 214: 274-283, 2008. rinkevich b. immunological resorption in botryllus schlosseri (tunicata) chimeras is characterized by multilevel organization of histocompatibility alleles. a speculative endeavor. biol. bull. 184: 342-345, 1993. rinkevich b. immune responsiveness in colonial marine invertebrates revisited: the concourse of puzzles. in: söderhäll k, vasta g, iwanaga s (eds), invertebrate immunology, sos publications, fair haven, pp 55-90, 1996a. rinkevich b. links between alloresponses and their genetic background in colonial urochordates and cnidarians: evidence for the recognition of “nonself” as opposed to “self”. in: stolen js, fletcher tc, bayne cj, et al. 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with heterorhabditis bacteriophora, its symbiotic bacteria and metabolites a abdolmaleki1, zt maafi2, hr dastjerdi1, b naseri1, a ghasemi2 1department of entomology, agricultural sciences faculty, university of mohaghegh ardabili, tehran, iran 2iranian research institute of plant protection, agricultural research education and extension organization (areeo), tehran, iran accepted march 23, 2017 abstract in this study, virulence of the culture broth of photorhabdus temperata subsp. temperata and its aqueous and organic extracts were investigated for 3rd and 4th instars of pieris brassicae (lepidoptera: pieridae) larvae. virulent efficacy was found in all the treatments. however, the culture broth of bacteria showed greater mortality effect. also, this study presents the response of p. brassicae immune system factors, phenoloxidase, lysozyme and hemocytes, to infection by heterorhabditis bacteriophora, its symbiotic bacteria and their aqueous and organic extracts. results showed that after injecting infective juveniles (ijs) with phenoloxidase, specific activity was suppressed after primary increase. however, the phenoloxidase specific activity suppression was observed earlier after injecting p. temperata subsp. temperata, aqueous and organic extracts. also, results indicate more phenoloxidase activity of 4th instars larvae in comparison with 3rd instars larvae, which can show higher activity of phenoloxidase in the 4th larval stage. after injecting ijs into hemocel, the total hemocyte count was increased until five h post-injection and then decreased at seven hours postinjection. differential hemocyte count (dhc) indicated granolucytes and plasmocytes as frequent hemocytes after treatment. lysozyme consideration showed an increase after injecting living and heat-killed bacteria into hemocel. key words: hemocyte; lysozyme; phenoloxidase; entomopathogenic nematode; photorhabdus temperata subsp. temperata introduction pieris brassicae (lepidoptera: pieridae) is one of the most important pests of plants from the family brassicaceae. this pest has five larval stages of which the last two inflict the most damage on plants. currently, the most reliable management of this pest is provided by chemical insecticides (cartea et al., 2009). however, residues along with potential resistance development encourage the development of alternative measures. biological control-as a way to control pests like p. brassicae-has been taken into consideration (abdolmaleki et al., 2015). entomopathogenic nematodes (epns) are an efficient alternative to chemical pesticides. epns are from two families, heterorhabditidae and steinernematidae, including two genera, heterorhabditis ___________________________________________________________________________ corresponding author: arman abdolmaleki department of entomology agricultural sciences faculty university of mohaghegh ardabili, tehran, iran e-mail: arman.abdolmaleki@uma.ac.ir and steinernema respectively. epns have a symbiotic relationship with bacteria from the enterobacteriaceae family. genera heterorhabditis and steinernema have a symbiotic relationship associated with bacteria from genera photorhabdus and xenorabdus, respectively (ciche and ensign, 2003; martens et al., 2003). the infective juveniles (ijs) actively seek out insect hosts and penetrate the insect’s body, usually via natural openings. the symbiotic bacteria are released from the ijs into the hemocel. they proliferate and produce some metabolites causing septicaemia, which kills the host (gaugler, 2002). symbiotic bacteria produce antimicrobial metabolites, which help the epns proliferate and grow under monogenic condition and suppress microbial growth of secondary infections (webster et al., 1998). these ijs invade the hemocel. the symbiotic bacteria are then released from the nematode’s gut and cause septicaemia, which kills the host (akhurst, 1983; lewis et al. 1993; forst and clarke, mailto:arman.abdolmaleki@uma.ac.ir 74 fig. 1 analysis of proteins in aqueous extracts of 48 h culture broths of ptt. 2002). no adverse effects of epns have been proven on non-target insects, viz., predators and parasitoids (mbata and shapiro, 2010). heterorhabditis bacteriophora is one of the most frequent and important epns in controlling insect pests. virulence effect of this epn was earlier approved in erstwhile studies on p. brassicae (abdolmaleki et al., 2015). insects have varying immune strategies against pathogens and invaders. these strategies, including integument, cellular immune system, e.g., hemocytes and phagocytes, and humeral immune system, viz., phenoloxidase and lysozyme, are mainly responsible for keeping insects safe. an important step for successful infection by a biological control agent and killing of the host is to overcome these insect immune strategies (grewal et al., 2005; beckage, 2008). thus, considering virulence of biological control factors without investigating the response of the immunity system of the host seems irrational. several studies have been undertaken on the response of pests to epns and their symbiont bacteria. several studies showed that infection of insects by epns cause hemocyte responses (thurston et al., 1994; wang et al., 1994, 1995; steiner, 1996; armer et al., 2004); some indicated that epns and their symbiont bacteria are replete with different factors which they can suppress phenoloxidase activity and hemocytes (dunphy and webster, 1987; yokoo et al., 1992; brivio et al., 2002; walter et al., 2008). till date, different toxins, metabolites, and factors have been identified, which can have a suppressive effect on the host’s immune systems and help epns kill the host (kim et al., 2005; balasubramanian et al., 2009). to our knowledge, no study has been performed on the effect of h. bacteriophora, its symbiont bacteria, photorhabdus temperata subsp. temperata (ptt), and aqueous and organic extracts of this bacterium on p. brassicae immune system factors. the major purpose of this study was to approach a comprehensive view on the role of p. brassicae immunity system challenged with the epn species, h. bacteriophora. we selected this epn due to its potential control of the cabbage butterfly. in this study, total hemocyte counts, differential hemocyte counts, phenoloxidase activity, and lysozyme concentration were measured in response to infection h. bacteriophora, living and heat-killed symbiont bacteria of h. bacteriophora, ptt, and its aqueous and organic extracts. material and methods preparation experimented insect pieris brassicae eggs were collected from the cabbage fields of urmia (west azerbaijan province, iran). the larvae were fed fresh cabbage leaves cultivated in a research plot located at the iranian research institute of plant protection, tehran, iran, until the 3rd and 4th instars larvae appeared. the cabbages received no doses of chemical or even biological insecticides. preparation of epn and bacteria heterorhabditis bacteriophora strain hbiri1, used in the experiments, was from the soils of kurdistan province, iran (abdolmaleki et al., 2016). the nematode was maintained in the laboratory by passing through larval galleria mellonella. symbiotic bacteria, ptt, strain ptiri6 were isolated from surface-sterilized ijs of h. bacteriophora strain hbiri1 (abdolmaleki et al., 2016). preparation of bacterial metabolites aqueous and organic extracts of ptt were prepared as per shrestha and kim (2010), with 75 slight modifications. the bacteria were cultured on lb (liquid broth) for 48 h. the culture broth was collected and for 20 min centrifuged at 7,000 rpm. the supernatant was collected and mixed with the same volume of hexane in a new tube and incubated at room temperature. after two h of incubation, the hexane fraction was collected and dried under n2 gas and then re-suspended with dimethylsulfoxide (dmso) to form an organic extract. the remaining culture broth was used as an aqueous extract. aqueous and organic extracts were sterilized through filtering by a 0.22 μm pore size membrane and stored in a refrigerator at 4 °c until use. insecticidal activity bioassays were performed on the 3rd and 4th instars larvae of p. brassicae by injecting freshly cultured ptt, and its aqueous and organic extracts into haemocoel. ten µl (104 cell ml-1) of ptt and its metabolites were injected through proleg by a sterilized 25 µl hamilton syringe (hamilton co., reno, nev.). before injecting, the surface of the larvae was disinfected by immersing them in 1 % naoh for 10 s and then rinsed with distilled water. experiments were carried out at three replications and included 15 larvae. median lethal times (lt50) were calculated by counting dead larvae every 24 h for four days post-injection. after each injection, the syringe was rinsed three times with ddh2o, three times with 70 % ethanol, and then three times again with sterile ddh2o. ten microliters of lb diluted in saline and lb were injected into hemocel through proleg using sterilized 25 µl hamilton syringe as control for living bacteria, aqueous extract, respectively. however, ten microliters dmso was used as organic extract. expression of bacterial protein extract aqueous extract was analysed by using 10 % sodium dodecyl sulfate (sds) polyacrylamide gel electrophoresis (page) (hames and rickwood, 1981). proteins were stained with coomassie brilliant blue as per the manufacturer’s instructions. phenoloxidase activity measurement phenoloxidase activity and phenoloxidase specific activity of 3rd and 4th instars larvae of p. brassicae were considered when they were challenged by h. bacteriophora, living and heatkilled ptt, aqueous and organic extracts of ptt after one, three, six, 12, and 24 h after injection. in toto, 10 ijs per larvae, 10 µl of alive and heat-killed (104 cell ml-1) ptt, and 10 µl of aqueous and organic extracts were injected into hemocel of 3rd and 4th larval instars of p. brassicae through proleg. after injection, the larvae were reared under the same conditions and their behaviours and mortality were recorded. epn injection was performed by insulin syringe; however, others were injected by hamilton syringe. after each injection, the hamilton syringe was rinsed three times with ddh2o, three times with 70 % ethanol, and then three times again with sterile ddh2o. however, insulin syringe was changed after each treatment and new one was used. as control, 10 µl of pbs was injected into hemolymph for epn. however, 10 µl of lb mixed in saline was as control for living and heat-killed ptt experiments. however, 10 µl of lb and dmso were respectively injected as control for aqueous and organic extracts. heat-killed ptt was obtained by heating at 90 ºc for 30 min in a shaking water bath. the kill treatment was confirmed by plating 200 µl aliquots of the ptt suppression on nbta and incubating the plates for 48 h. no bacterial colony was observed on these plates. experiments were replicated three times. fig. 2 pathogenicity of ptt and its aqueous and organic extracts on 3rd and 4th larval stages of p. brasiicae. 76 table 1 pathogenicity of ptt and its aqueous and organic extracts against late 3rd and 4th instars larvae of pieris brassicae by hemocelic injection. treatment larval stage lt50 slope±se χ2a df ptt 3 38.84 (9.08-61-68) 3.63±0.51 3.85 2 4 44.73 (38.08-51.24) 3.51±0.49 0.11 2 aqueous extract 3 43.32 (37.55-48.96) 4.07±0.53 1.83 2 4 53.86 (45.63-63.30) 2.94±0.48 1.65 2 organic extract 3 49.89 (43.22-56.88) 3.64±0.51 2.88 2 4 57.58 (49.61-67.08) 3.21±0.50 1.05 2 a: pearson χ2 of the slope. total protein determination protein concentrations were measured by using bovine serum albumin as a standard, in accordance with the method of bradford (1976). protein concentrations of 3rd and 4th larval instars of p. brassicae were measured after one, three, six, 12, and 24 h after injection by h. bacteriophora, living and heat-killed ptt, aqueous, and organic extracts of ptt. experiments were replicated three times. lysozyme activity measurement lysozyme assay was performed in accordance with hernández-martínez (2010), with a little modification. lysozyme concentrations of 4th instars larvae of p. brassicae were measured when they were challenged with living and heat-killed ptt. for this approach, 10 µl of living and heat-killed (104 cell ml-1) ptt were injected into the hemocel through proleg by hamilton syringe. after each injection, the syringe was rinsed three times with sterile ddh2o, three times with 70 % ethanol, and finally three times again with sterile ddh2o. for assessing lysozyme concentration in different treatments, 1 ml hemolymph was obtained by puncturing larval proleg. petri dishes (diameter 8 cm, depth 1 cm) were filled with 20 ml phosphate buffer saline (pbs) containing 1 mg ml‾¹ of micrococcus luteus cohn (schroeter) atcc no. 4698 (sigma co., usa), with a final concentration of 1.5 % agar. after solidification of agar, the hemolymph was individually placed on it and incubated at 37 °c for 24 h. in each petri dish, 10 samples were assessed. for the consistency of the experiments, different dilutions of standard lysozyme (0.3, 01, 0.03, and 0.01 mg ml-1) (roche diagnostics sl, san cugat del vallés) were used as a control. lytic activity in the hemolymph was estimated as the lytic zone diameter around the sample. based on the halo diameters around the samples, the larvae were categorized into three groups ranging from 0 to 5 mm, from 5 to 10 mm, and from 10 to 15 mm. experiments were replicated 20 times. hematology for considering total hemocyte counts (thc) and dhc, 10 ijs by insulin syringe were injected into hemolymph of 3rd and 4th of p. brassicae through their prolegs. after one, three, six, 12, and 24 post-injection as interval times, hemolymph was collected by cutting the proleg. hemolymph (1 µl) was diluted with anticoagulant buffer (1 g of ascorbic acid, 2 g of citric acid in 100 ml of ringer’s solution). the thc was determined by placing the diluted hemolymph on a neubauer hemocytometer under a light microscope and reported as the total number of hemocytes per ml. for dhc, hemocytes monolayer was prepared and stained with 10 % giemsa solution and examined by oil immersion light microscopy. in toto, 200 cells were counted randomly for each slide and hemocyte types were evaluated as a percentage of total cells (gupta, 1979). after each treatment insulin syringe was changed and new one was used. statistical analysis probit programme was used to calculate lt50 (raymond, 1985). also, all means and variances of the treatments were analysed by one-way anova using spss software version 19 (ibm spss, 2010). all means were compared by tukey’s test (hsd) and it was used to determine significant differences among the treatments at the 0.05 level. the number of each hemocyte type number was reported as mean ± standard error (se). results expression of bacterial protein extract figure 1 shows proteins detected in aqueous extract of ptt. insecticidal activity pathogenicity of ptt was investigated and reported in figure 2 and table 1. lt50 values show more efficacy of ptt than its aqueous and organic extracts against 3rd and 4th larval instars of p. 77 table 2 total protein, phenoloxidase activity and phenoloxidase specific activity in pieris brassicae when challenged with heterorhabditis bacteriophora treatment larval stage time (h) total protein (mg)±se total activity (u)±se specific activity (u/mg)±se h. bacteriophora 3 1 16.30±0.29 13.09±0.43 0.99±0.00 3 0.76±0.02 1.33±0.03 3.65±0.26 6 0.77±0.08 1.90±0.07 1.93±0.09 24 2.62±0.05 3.06±0.07 1.35±0.01 control 1 0.46±0.04 1.42±0.04 3.09±0.20 3 0.14±0.01 1.12±0.01 8.11±0.72 6 0.37±0.11 1.33±0.09 4.40±1.39 24 1.37±0.06 2.25±0.06 1.64±0.03 h. bacteriophora 4 1 16.30±0.29 15.90±0.27 0.97±0.00 3 0.76±0.02 1.69±0.02 2.23±0.04 6 0.77±0.08 1.69±0.07 2.24±0.15 24 2.62±0.05 3.39±0.04 1.29±0.01 control 1 1.99±0.03 2.81±0.02 1.41±0.01 3 0.14±0.03 1.12±0.02 8.51±1.46 6 0.13±0.01 1.11±0.01 8.56±0.29 24 1.02±0.06 1.92±0.05 1.89±0.06 brassicae. the lowest lt50 was obtained from experiments carried out by organic extract. in comparison with two larval stages, more lt50 values were obtained in 4th instars. mortality percentages of 3rd f2, 6 = 127.73, p ˂ 0.05) and 4 th (f2, 6 = 89.89, p ˂ 0.05) larval instars treated by ptt and its aqueous and organic extract show more lethality of bacteria than two extracts. in all experiments, 4th instars larvae proved to be more resistant than 3rd instars (table 1, fig. 2). phenoloxidase activity, phenoloxidase specific activity, and total protein determination results show an increase in phenoloxidase until three h after injection. then, a decrease in specific activity was observed from six h postinjection of h. bacteriophora into the hemocel of larvae of p. brassicae. this increase and inhibition of phenoloxidase was observed in both treated larval instars of p. brassicae. comparison between specific activities of phenoloxidase of treated larvae by h. bacteriophora with control shows significant differences (f2, 32 = 56.61, p ˂ 0.001). also, significant differences were observed among specific activities of phenoloxidase in different times after injecting h. bacteriophora (f3, 32 = 54.33, p ˂ 0.001) (table 2). phenoloxidase specific activity in 3rd and 4th larval stages treated with ptt showed the same pattern as when treated with h. bacteriophora, albeit with some differences. in both treatments by living and heat-killed bacteria, inhibition was observed in specific activity of p. brassicae larvae. in comparison with h. bacteriophora treatments, preliminary increase of phenoloxidase specific activity was not demonstrated when larvae were treated by the bacteria. results showed significant differences among phenoloxidase specific activities in treated larvae by living and heat-dead bacteria and control (f4, 48 = 67.77, p ˂ 0.001). however, comparison of phenoloxidase specific activities showed significant differences in different interval times (f3, 48 = 45.96, p ˂ 0.001) (table 3). suppression effect pattern of aqueous and organic extracts on phenoloxidase specific activities were similar to the pattern when the larvae were injected by the bacteria. these results show significant differences among different treatments (f6, 64 = 47.61, p ˂ 0.05). also, results showed significant differences among phenoloxidase specific activities among different interval times (f3, 64 = 15.34, p ˂ 0.001) (table 4). lysozyme activity determination of the lysozyme activity of 4th instars larvae of p. brassicae, when challenged with living and heat-killed ptt, showed lyctic activity. results of lyctic activity were reported in two wayslyctic zone diameters and lysozyme concentrations. lyctic zone diameters showed that by passing postinjection time frequency of samples with high diameter zone increased (fig. 3). results of treated larvae with living and heat-killed bacteria showed significantly higher lysozyme activity than controls (f2, 171 = 84.94, p ˂ 0.001). also, results show nonsignificant differences between lysozyme activities of larvae treated by living and heat-killed bacteria. however, significant differences were demonstrated among results of different interval times (f2, 171 = 45.46, p ˂ 0.001) (table 5). 78 table 3 total protein, phenoloxidase activity and phenoloxidase specific activity in pieris brassicae when challenged with living and heat-killed ptt treatment larval stage time (h) total protein (mg)±se total activity (u)±se specific activity (u/mg)±se living ptt 3 1 1.89±0.06 2.72±0.05 1.44±0.02 3 6.20±0.27 6.67±0.25 1.07±0.01 6 8.75±0.17 8.99±0.15 1.03±0.00 24 12.43±0.03 12.36±0.02 0.99±0.00 heat-killed ptt 1 0.68±0.04 1.62±0.03 2.37±0.08 3 0.37±0.02 1.33±0.02 3.60±0.14 6 0.59±0.09 1.53±0.09 2.72±0.35 24 1.60±0.05 2.46±0.05 1.54±0.02 control 1 0.46±0.04 1.42±0.04 3.09±0.20 3 0.14±0.01 1.12±0.01 8.11±0.72 6 0.37±0.11 1.33±0.09 4.40±1.39 24 1.37±0.06 2.25±0.10 1.64±0.03 living ptt 4 1 2.13±0.05 2.94±0.05 1.38±0.01 3 6.44±0.13 6.88±0.12 1.07±0.00 6 8.98±0.17 9.20±0.15 1.02±0.00 24 12.66±0.02 12.57±0.02 0.99±0.00 heat-killed ptt 1 0.92±0.04 1.84±0.04 1.99±0.05 3 0.62±0.02 1.56±0.02 2.53±0.06 6 0.85±0.01 1.77±0.01 2.09±0.02 24 1.86±0.01 2.69±0.01 1.45±0.01 control 1 1.99±0.03 2.81±0.03 1.41±0.01 3 0.14±0.03 1.12±0.03 8.51±0.46 6 0.13±0.01 1.11±0.00 8.56±0.29 24 1.02±0.09 1.93±0.05 1.89±0.06 hematology thc values showed an increase early after injecting ijs into hemocel. p. brassicae larvae, challenged with ijs of h. bacteriophora, showed significant thc differences in different times postinjection (f). thc values showed an increase until five h post-injection, though seven h post-injection showed a decrease (fig. 4). investigation by dhc showed that more frequent hemocytes were granulocytes and plasmocytes. results of granulocyte counts show an increase through postinjection times and significantly count more than control (f7 = 113.05, p ˂ 0.001). plasmocytes showed a gradual increase in passing time and showed significant differences with results of control (f7 = 372.31, p ˂ 0.001) (fig. 5). discussion results of lc50 were in concurrence with lt50 values show high pathogenicity of ptt than its aqueous and organic extracts. also, aqueous 79 table 4 total protein, phenoloxidase activity and phenoloxidase specific activity in pieris brassicae when challenged with aqueous and organic extracts of ptt treatment larval stage time (h) total protein (mg)±se total activity (u)±se specific activity (u/mg)±se aqueous extract 3 1 0.94±0.04 1.85±0.04 1.99±0.09 3 1.95±0.06 2.78±0.06 1.42±0.02 6 5.97±0.19 6.46±0.18 1.08±0.01 24 12.38±0.37 12.31±0.34 0.99±0.00 organic extract 1 6.76±6.08 7.18±0.98 1.91±0.47 3 1.32±0.09 2.19±0.08 1.67±0.05 6 5.59±0.07 6.10±0.07 1.09±0.00 24 10.61±0.03 10.69±0.03 1.01±0.00 control (lb) 1 0.86±0.06 1.78±0.05 2.08±0.08 3 0.96±0.04 1.88±0.04 1.95±0.04 6 1.41±0.03 2.29±0.03 1.62±0.01 24 2.38±0.02 3.17±0.02 1.33±0.00 control (dmso) 1 0.14±0.03 1.12±0.03 9.02±1.98 3 0.09±0.03 1.08±0.02 14.51±4.39 6 0.08±0.01 1.07±0.01 14.16±1.91 24 1.28±0.06 2.17±0.05 1.69±0.03 aqueous extract 4 1 1.37±0.01 2.25±0.01 1.64±0.01 3 2.19±0.06 3.00±0.05 1.37±0.01 6 4.16±0.27 4.80±0.25 1.15±0.01 24 11.02±0.19 11.07±0.17 1.00±0.00 organic extract 1 0.75±0.04 1.68±0.04 4.89±0.76 3 1.89±0.02 2.73±0.02 1.49±0.01 6 3.02±0.25 3.76±0.39 1.22±0.02 24 9.89±0.05 10.04±0.08 1.03±0.00 control (lb) 1 0.75±0.04 1.96±0.03 1.85±0.03 3 1.89±0.02 2.08±0.03 1.75±0.02 6 3.02±0.25 2.60±0.02 1.48±0.01 24 9.89±0.05 3.86±0.03 1.23±0.00 control (dmso) 1 0.16±0.02 1.14±0.02 7.19±0.76 3 0.11±0.01 1.09±0.01 10.56±1.19 6 0.09±0.02 1.08±0.02 12.56±2.66 24 1.76±0.04 2.61±0.04 1.48±0.01 80 fig. 3 changes on the larvae distribution according to their lytic zone values observed for the different treatments and interval times post-injection. for each treatment and interval time, three groups of lysozyme concentration could be distinguished according to the diameter of the inhibition halo obtained in the lytic zone assays. extracts had more virulent effect than organic extract on 3rd and 4th instars larvae. these results are in agreement with the results of shrestha and kim (2010), who found higher pathogenicity of aqueous extract than organic extract of ptt on tribolium castaneum larvae. several reasons could affect higher pathogenicity of aqueous extract of ptt than organic extract. on the one hand, the effect of higher suppression of aqueous extract than organic extract found in the current study could be an important reason for this difference between aqueous and organic extracts. on the other hand, inhibiting eicosanoid biosynthesis by aqueous extracts, as found by shrestha and kim (2010), could be another reason for higher virulence of this extract than organic extract. also, higher virulence of ptt than its extract could be due to the simultaneous effect of both extracts in the infected host. however, more studies are required to find all the exact reasons. between the successful infection by epns and host immune response is a close relationship (li et al., 2007). therefore, investigation of their virulence without considering the host’s immune response seems irrational. insects usually respond to bacterial or parasite infections with humoral and cellular immune reactions (dunphy and thurston, 1990; feldhaar and gross, 2008). several types of hemocytes carry out the cellular immune responses, including phagocytosis, encapsulation, and nodule table 5 lysozyme concentration of (mg/ml) 4th larval instars of pieris brassicae when challenged with living and heat-killed ptt treatments time after injection (h) 1 3 6 living ptt 0.09±0.01de 0.18±0.02b 0.29±0.03a heat-killed ptt 0.11±0.02bd 0.16±0.02bd 0.31±0.02a control 0.03±0.01e 0.03±0.01e 0.03±0.01e different letters are significant treatments at p˂0.05, according to tukey's test. 81 fig. 4 changes in haemocyte populations of pieris brassicae larvae treated with heterorhabditis bacteriophora. means ± se are given. different letters in each time post-injection are significant at p ˂ 0.05, according to tukey's test. formation to pathogens and parasites (strand, 2008). also, humeral factors, such as phenoloxidase and lysozyme, play an undeniable role in the immunity of insects. the purpose of this study was to consider the cellular and humeral responses of 3rd and 4th instars larvae of p. brassicae when challenged with ijs of h. bacteriophora, its symbiont bacteria, and metabolites. results of phenoloxidase specific activity showed that all the treatments-h. bacteriophora, ptt and aqueous and organic extracts-caused suppression in both experimented instars of p. brassicae, though there were some differences in pattern. phenoloxidase specific activity values of 3rd and 4th instars larvae of p. brassicae treated by h. bacteriophora showed a decrease in six h postinjection after a preliminary increase. this increase could be due to the recognition of an invader; however, subsequent decrease might be related to release, proliferation, activity of symbiont bacteria and their metabolites production. in the study performed by rahatkhah et al. (2015), after injecting s. feltiae and h. bacteriophora into hemolymph of agriotes lineatus and g. mellonella, phenolooxidase inhibition was observed 16 h post-injection. also, results of yokoo et al. (1992) showed the inhibition effect on prophenoloxidase hemolymph of agrotis segetum larvae challenged with living and heatkilled steinernema carpocapsae. remarkable results were observed when p. brassicae larvae were challenged with living and heat-killed bacteria. both living and heat-killed ptt caused phenoloxidase specific activity inhibition in 3rd and 4th instars larvae of p. brassicae. however, in larvae treated by living and heat-killed ptt, suppression effect was observed early after living and heat-killed bacteria injection. in concurrence with this study, yokoo et al. (1992) found that living and dead xenorhabdus nematophilus inhibited phenoloxidase activity. it suggested that the suppression of phenoloxidase by both living and killed s. carpocapsae and its symbiotic bacteria was probably due to a factor in the living or dead responsible for the masking of the recognition protein that binds to laminarin. also, suppression by both living and heat-killed bacteria was possibly due to another factors present in living and heat-killed bacteria. for instance, dunphy and webster (1988a) considered x. nematophilus in g. mellonella hemolymph, and found that activation of prophenoloxidase was suppressed. the effect of aqueous extract, organic extract, and bacteria on phenoloxidase activity had the same pattern. in all these treatments, phenoloxidase activities were reduced early after treating. however, aqueous extract had more inhibition on phenoloxidase activity than organic extract. differences in inhibiting phenoloxidase activity may be due to the presence of different protein contents between two extracts. identifying protein contents of these extracts indisputably are necessary to determine the main reasons of difference between phenoloxidase activity inhibition of aqueous and organic extracts. a comparison of phenoloxidase activity revealed this truth: injecting h. bacteriophora ijs caused delayed inhibition on phenoloxidase activity. however, results showed phenoloxidase suppression early after injecting ptt or its extracts. in 82 fig. 5 changes in differential haemocyte populations of pieris brassicae larvae treated with heterorhabditis bacteriophora. means are given. different letters in each time post-injection are significant at p ˂ 0.05, according to tukey's test. pl: plasmatocyte, gr: granulocyte. the study by dunphy and webster (1988b), they found heterorhabditis heliothidis released its symbiont bacteria four to five h after injecting the epn. besides, wang et al. (1994) found that s. carpocapsae released its symbiont bacteria, x. nematophilus, and some four h after entering the host hemocel. the time required to release bacteria from ijs can reveal delayed phenoloxidase inhibition by epn in comparison with symbiont bacteria and their extracts. also, in all the treatments, 4th instars larvae showed higher phenoloxidase activity than 3rd instars larvae. this could be due to higher immune ability of 4th instars than 3rd instars larvae. the most important immunity factor in the case of bacterial infection is lysozyme (boman et al., 1991). hence, the lysozyme response of 4th instars larvae was considered against ptt infection. in the current study, an increase in lysozyme concentration was observed in response to living and heat-killed ptt and significant difference with control. however, non-significant differences were observed between treatment by living and heatkilled bacteria at the same interval. these results show no suppression activity of ptt on lysozyme activity. in concurrence with these results, dunphy and webster (1988b) found that h. bactetriophora and its symbiont bacteria, p. luminescens, do not inhibit lysozyme activity. hemocytes are one of the most important immune factors of insects against pathogens and invaders (li et al., 2007; strand, 2008). studies abound about the response of hemocytes against epns (alvandi et al., 2014; rahatkhah et al., 2015). their numbers rapidly increase during an infection, and they are responsible for several cellular defences. they can also take part in humoral reactions (strand, 2008). in the current study, thc and dhc values of p. brassicae larvae were investigated when they were challenged with h. bacteriophora. the results show an increase of thc early after injecting ijs into hemolymph. the thc increasing and significant differences treatments with control can show immunological activation of host due to the presence of foreign bodies (li et al., 2009). however, seven h post-injection of ijs thc were significantly decreased. this study is in concurrence with a study by abu-elmagd and el-kifl (1993) that considered cellular response of spodoptera exigua and a. segetum larvae challenged with h. heliothidis. also, these results agree with data obtained by rahatkhah et al. (2015) in a. lineatus against with h. bacteriophora and s. feltiae. symbiont bacteria of epns have a detrimental effect on the health of host haemocytes, causing a decrease in the number of hemocytes (bowen et al., 1998; cho and kim, 2004; brivio et al., 2005). this fact-along with the time required to release bacteria from ijs-can reveal the reason for the decrease in hemocyte numbers after a primary increase (dunphy and webster 1988b; wang et al., 1994). 83 in conclusion, our study indicates that several immune defenses in p. brassicae are engaged in the challenge against epns. a good understanding of insect host-pathogen relationships can help the progress of integrated pest management techniques. however, more study is necessary in this area for a deeper understanding of the immunity system of p. brassicae in its interaction with epns and its symbiont bacteria. acknowledgment we would like to thank prof. lewis e (department of nematology, university of california, davis, usa) and 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military service, he got his degree in natural sciences in naples in 1944, according to the instructions during the war period. he taught in secondary schools from 1945 to 1950. he was married with lidia martini (died in 2011, aged 90) who, together with her sisters, helped many wanted persons to took refuge in switzerland during the years of the nazi occupation of italy in 1943-45. prof. a. sabbadin was assistant professor of zoology at the university of padua, from january 1951 and of comparative anatomy from 1952. full professor of comparative anatomy in 1960; retired in 1991. teacher of experimental embryology from to 1964 and of general biology and zoology for the medical school from 1964 to 1974. director of the former institute of animal biology in the biennium 1964-1966 and of the marine station of the university of padua in chioggia (venice) from 1966 to 1980, where he began to restore the historical marine zoological collection now located in the museum of adriatic zoology "giuseppe olivi". visiting professor at the stazione zoologica in naples and the oceanographic institute in monaco in the early fifties of the last century; awarded a grant from the italian national research council (cnr) for research at the international embryology institute, hubrecht laboratory in utrecht in 1956. his research activity began with the study of gonadogenesis and sexual differentiation in amphibians, under the supervision of prof. e. vannini. he confirmed the role of interrenal blastema in gonadal development in rana dalmatina, where all the tadpoles develop female gonads and, only at the metamorphosis climax, in genetic males, the gonad turns to a testis. sabbadin demonstrated that antithyroidean compounds delay this inversion, in parallel with the retardation in metamorphosis. moreover, he showed that extirpation of the pituitary gland, in embryos, delays the inversion up to three months after metamorphosis. in 1952, fascinated by the colonies of botryllus schlosseri growing over the walls of the aquaria of the oceanographic institute of monaco, he decided to use ascidians to study sexual differentiation in chordates and established a laboratory for ascidian research in padua. the lab is still in activity, thanks to his disciples and his disciples’ pupils, and carries out researches in morphology, ecology, evolutionary biology, and comparative immunobiology. this laboratory absorbed all sabbadin’s successive research activity mainly focused on the colonial species botryllus schlosseri. his seminal papers on allorecognition, germ cell parasitism, genetics of pigmentations, induction of vascular budding, and inversion of polarity in zooids have stimulated the research of many scientists around the world. prof. sabbadin was member of the italian zoological society, italian association of developmental and comparative immunobiology, 66   67   italian embryology group, international society of developmental and comparative immunology. he was awarded the honorary membership by the italian zoological society and the italian association of developmental and comparative immunobiology, he was honoured in the tunicate name clavelina sabbadini brunetti, 1987. he continued to frequent the laboratory until he was 85. he died in padua on february, 19th 2016. l ballarin, p burighel, f cima, l manni, g zaniolo department of biology, university of padua, padua, italy http://www.marinespecies.org/aphia.php?p=taxdetails&id=103556 http://www.marinespecies.org/aphia.php?p=taxdetails&id=103556 1 isj 10: 77-83, 2013 issn 1824-307x review the molluscan hsp70s and their expression in hemocytes l wang, c yang, l song key laboratory of experimental marine biology, institute of oceanology, chinese academy of sciences, qingdao 266071, china accepted september 23, 2013 abstract the heat shock protein 70s (hsp70s) are a class of functionally related proteins involved in the folding and unfolding, translocation of other proteins, and stress responses in almost all organisms. as the most analyzed heat shock proteins, numerous hsp70s have been identified and characterized from bacteria, plants and animals. molluscan hsp70 is one of the largest and most important groups in the invertebrate hsp70 family. accumulating evidences have demonstrated the relevant physiological and ecological importance of hsp70 in response to pathogen infection and environmental stressors. this chapter reviews the interest arose around hsp70s in molluscan animals, mainly the recent research progress about the diversity of molluscan hsp70 family members, their sequence characters and expression profiles in hemocytes under various stressors. key words: mollusc; heat shock protein 70; expression profile; immune challenge; environmental stressor introduction heat shock protein 70 (hsp70) is one of the most abundant hsp families involved in the folding and unfolding, translocation of other proteins, and stress responses in almost all organisms. they consist of a class of functionally related proteins including hsp68, hsp70, hsc70, hsp75 and hsp78 (grp78), which are localized to distinct subcellular compartments including cytoplasm, mitochondria, and endoplasmic reticulum (er) (boorstein et al., 1994; mayer and bukau, 2005). the amino acid sequences of hsp70 family members are highly conservative from archaebacteria to humans, and there are two major functional domains, the n-terminal atpase domain and the c-terminal peptide binding domain (sung et al., 2001; mayer and bukau, 2005). the hsp70s are involved in a variety of physiological processes and perform complex functions, such as serving as molecular chaperones (gething & sambrook, 1992), involved in the regulation of apoptosis (böttger et al., 2008), and playing important roles in response to bacterial challenge (cellura et al., 2006), oxidative stress (golli-bennour and bacha, 2011) and various environmental stressors (cellura et al., 2006). recent studies in land snails sphincterochila species ___________________________________________________________________________ corresponding author: linsheng song institute of oceanology chinese academy of sciences 7 nanhai rd., qingdao 266071, china e-mail: lshsong@qdio.ac.cn suggested that hsp70 was also involved in the natural annual cycle of activity and aestivation and the survival strategy during desiccation and heat stress, and the adaptation of land snails to different habitats engenders the development of distinct strategies of hsp70 expression in response to stress (mizrahi et al., 2012). the mollusc phylum is one of the largest and most important groups in the animal kingdom, and around 130,000 extant species are described (haszprunar and wanninger, 2012). most of them live in freshwater or seawater, and they have to survive environmental perturbation from homeostasis, a situation generically described as stress. the production of acute phase proteins, such as the hsps, is regarded as a classical response against stressors. this chapter reviews the interest arose around molluscan hsp70s in the last 5 years, mainly in the diversity, sequence characters and their expression profiles in hemocytes under various stresses. the hsp70 family members in mollusc due to the important roles of hsp70s in the response against environmental stressors and the maintenance of homeostasis in molluscs, they have been studied extensively and the amount of their nucleotide sequences has increased noticeably during the past decades. there are totally 213 nucleotide sequences of molluscan hsp70 so far available in the database of ncbi, including 124 77 http://en.wikipedia.org/wiki/extant_taxon http://en.wikipedia.org/wiki/species http://www.sciencedirect.com/science/article/pii/s0960982212005921 http://www.sciencedirect.com/science/article/pii/s0960982212005921 table 1 the full length of cdna sequences encoding hsp70 in mollusc species species gene accession number reference snail biomphalaria glabrataembryonic hsp70.1 l44127 laursen et al., 1997 pomacea canaliculata hsc70 not released zheng et al., 2012 sea hare aplysia californica bip/grp78 nm_001204652 kuhl et al., 1992 oyster crassostrea gigas hsc70 aj305315 boutet et al., 2003b hsp70 aj318882 boutet et al., 2003b grp78 bad15288 yokoyama et al., 2006 grp94 ab262084 kawabe and yokoyama, 2009 other hsp70s see the reference zhang et al., 2012 ostrea edulis hsc70 aj305316 boutet et al., 2003a hsp70 af144646 boutet et al., 2003a oedcl5 af416608 piano et al., 2005 oedcld2 af416609 piano et al., 2005 crassostrea hongkongensis hsp70 fj157365 zhang and zhang, 2012 mussel mytilus galloprovincialis hsp70 dq178174, dq178175 franzellitti and fabbri, 2005 hsc70 dq178176, dq178177 franzellitti and fabbri, 2005 hsp70 ay861684 cellura et al., 2006 hsc70, hsc71 aj783714, aj783715 kourtidis et al., 2006 hsp70-2, hsp70-3, hsp70-4 aj783711, aj783712, aj783713 kourtidis et al., 2006 scallop argopecten irradians hsp70 ay485261 song et al., 2006 chlamys farreri hsp70 ay206871 song et al., 2006 mizuhopecten. yessoensis hsp70 ay485262 song et al., 2006 pinctada fucata hsp70 eu822509 wang et al., 2009 abalone haliotis discus hannai hsp70 dq324856 cheng et al., 2007 haliotis diversicolor hsp70 aco36048 unpublished hsc70 aco36047 unpublished clam laternula elliptica hsp70 ef198332. park et al., 2007 meretrix meretrix hsc71 hq256748 yue et al., 2011 tegillarca granosa hsp70 n936877 zhou et al., 2013 from bivalves, 77 from gastropods and 12 from cephalopods. the information about the full length cdna sequences encoding hsc70 and hsp70 in mollusc is summarized in table 1, and the species include snail (laursen et al., 1997; zheng et al., 2012), scallop (song et al., 2006; wang et al., 2009), oyster (boutet et al., 2003a and 2003b; piano et al., 2005; zhang and zhang, 2012; zhang et al., 2012), mussel (franzellitti and fabbri, 2005; cellura et al., 2006; kourtidis et al., 2006), abalone (cheng et al., 2007), and clam (park et al., 2007; yue et al., 2011). other cdna sequences encoding grp78 and grp94, the representatives of the grp members in the molluscan hsp70 family, have also reported in sea hare (kuhl et al., 1992) and oyster (yokoyama et al., 2006; kawabe and yokoyama, 2009; zhang et al., 2012). though only one hsp70 has been reported in some molluscan species, all eukaryotes are believed to have more than one gene encoding hsp70 proteins in their genomes. for example, there are at least 11 unique hsp70 genes in human (tavaria et al., 1996), 39 putative hsp70s in sea urchin (sodergren et al., 2006), and 10 putative hsp70s in fungus blastocladiella emersonii (georg and gomes, 2007). it is noteworthy that hsp70 gene family is remarkably expanded in c. gigas (zhang et al., 2012). a search of the genome sequence revealed that there were 88 members of hsp70 family in c. gigas, which were believed to play crucial roles in protecting cells against heat and other stressors (zhang et al., 2012). structural features of molluscan hsp70s the molluscan hsp70s share common structural and evolutionary features with homologues from other species (piano et al., 2005; kourtidis et al., 2006), including the highly conserved n-terminal domain and the diverse c-terminal 78 http://www.ncbi.nlm.nih.gov/nuccore http://www.ncbi.nlm.nih.gov/nuccore http://www.ncbi.nlm.nih.gov/nuccore domains (demand et al., 1998; fuertes et al., 2004; kourtidis et al., 2006). the highly conserved n-terminal domains of molluscan hsp70s usually shared three signature motifs (idlgttys, ifdlgggtfdvsil, and ivlvggstripkiqk) and one atp/gtp-binding motif (aeaylgkt) (wang et al., 2009; zhou et al., 2013). in spite of high conservation, there are still some small variations in the n-terminal domains of molluscan hsp70s. for example, there is an extra nqsq tetrapeptide in the atpase domain of hsc70s from o. edulis (boutet et al., 2003a) and c. gigas (boutet et al., 2003b), and there are two nonsynonymous mutations, y406i and g413e, in the atp/gtp-binding motif of hsp70 from different geographical populations of a. irradians (yang et al., unpublished data). congruous with the difference in their subcellular localizations and functions, the c-terminal domains of different hsp70s usually display low sequence homology with each other (demand et al., 1998; fuertes et al., 2004; piano et al., 2005), especially between hsp70 and hsc70 (fabbri et al., 2008). the tetrapeptide motif ggmp is an important element mediating cofactor binding to the hsp molecule by forming a structural entity together with the helical subdomain and the eevd motif (demand et al., 1998), and it has been once regarded as the peculiar sequence of hsc70s (fuertes et al., 2004; piano et al., 2005; fabbri et al., 2008). however, in some species, both of hsp70 and hsc70 contain ggmp tetrapeptide with variable numbers. for example, there are one, two, three and five ggmp tetrapeptides in the hsp70s from pearl oyster pinctada fucata, blood clam tegillarca granosa, pacific abalone haliotis discus hannai and argopecten irradians respectively (wang et al., 2009; zhou et al., 2013; cheng et al., 2007; song et al., 2006). therefore, it is necessary to investigate the effects of such structural variations on the expression profiles of hsp70 and hsc70 (fuertes et al., 2004), and these information could also provide insights into functional specificities of hsp70s (wang et al., 2009). moreover, there is a large amino acid deletion about 60 residues encompassing the end of the peptide-binding domain and a part of the c-terminal domain of hsc70 from o. edulis (kourtidis et al., 2006; fabbri et al., 2008). the molluscan hsp70s located in cytosolism, er, nuclear and mitochondrion always have the specific localization motifs gp(t/k)(v/i)ee(v/m)d, kdel, nucdisc and mitdisc, respectively. multiple alignments revealed that most of molluscan hsp70s localized in the cytosolism sharing the cytosolic localization motif gp(t/k)(v/i)ee(v/m)d (boorstein et al., 1994; demand et al., 1998; zhang and zhang, 2012; zhou et al., 2013). for example, 76 out of 88 hsp70s from c. gigas shared the motifs of gp(t/k)(v/i)ee(v/m)d, and they were predicted locating in the cytoplasm (yang et al., unpublished data). though eevd and eemd are both regarded as the cytosolic localization motifs, the effect of their sequence difference on structure and function still need further confirmation (zhang and zhang, 2012). besides, grp78 (yokoyama et al., 2006) and other seven hsp70s from c. gigas (yang et al., unpublished data) located in the er also contain the motif kdel. it is noteworthy that one hsp70 in oyster possessed a mitochondrial localization motif mitdisc, and this is the first mitochondrial hsp70 found in mollusc (yang et al., unpublished data). molluscan hsp70s are also classified into two groups of inducible hsp70s and cognate hsc70s at the present time, and they are closely matched to the corresponding hsp groups of other phylum in the phylogenetic analysis (fabbri et al., 2008). however, it is not always accurate to assign a hsp70 into a specific group according to the phylogeny relationship. for example, several hsp70s from oysters (boutet et al., 2003a and 2003b; kourtidis et al., 2006) and scallops (song et al., 2006) identified as inducible hsp70 proteins were clustered into hsc70 according to the phylogenetic analysis (fabbri et al., 2008). since there is limited information about the functions or activities of molluscan hsp70s, their classification is still not available currently. it has been reported that divergent evolution usually predominates when the members within one gene family acquire different functions (ohta and nei, 1994), and this is confirmed by inducible and cognate hsp70s, which belong to one family but display different expression patterns and functions. the phylogenetic reconstruction of molluscan hsp70s also indicates the occurrence of multiple duplication events in the evolution of hsp70 family, which is in agreement with the presence of multiple copies of the heat-inducible gene in molluscs. a phylogenetic analysis of 169 molluscan hsp70 proteins, including 88 from c. gigas, 12 from l. gigantean and 68 from other molluscs showed that 71 out of 88 c. gigas hsp70s were clustered together (zhang et al., 2012). it suggested that these genes were likely received significant positive selection and derived from oyster-specific expansions, and they might play major roles in oyster’s adaptation to heat and other stressors (zhang et al., 2012). expression of molluscan hsp70s in hemocytes under various stressors as the most abundant and well studied hsps, hsp70s are considered to play important roles in various physiological processes and protect organisms against various stressors. there are numerous studies to recognize the relevant physiological and ecological importance of molluscan hsp70s expression in response to the stresses resulted from changes of season and other environmental factors, such as temperature (cellura et al., 2006), heavy metal (boutet et al., 2003b; thompson et al., 2012; taylor et al., 2013), hypoxia (clark and peck, 2009; clark et al., 2013), ph (cummings et al., 2011), pollutants of pahs (song et al., 2006) and toxins (mello et al., 2012; mello et al., 2013), pharmaceuticals (gust et al., 2013) and bacteria challenge (cellura et al., 2006; song et al., 2006; cheng et al., 2007; xu and faisal, 2009). most of the information on hsp70 expression in molluscs was mainly obtained from five tissues including gill, digestive gland, muscle, mantle and hemocytes. in the gill of c. gigas, the expression pattern of hsp70s altered significantly at different temperatures. there were some hsp70 genes highly expressed at 79 normal temperature, and some genes were highly expressed at low temperature, while some other genes were highly expressed at high temperature (zhang et al., 2012). regardless of the expression in other tissues, the research progress about the expression of molluscan hsp70s in hemocytes under various stressors is summarized in this chapter based on the reports in the past 5 years. most molluscs have an open circulatory system composing of heart, blood vessels, sinusoids and hemolymph. as the major part of the hemolymph, hemocytes comprise the major component of the non-specific defense mechanisms, and they are involved in a series of cellular immune reactions (song et al., 2010; mello et al., 2012). the circulating hemocytes are able to migrate from the hemolymph to connective tissues, promote localized responses following injury or microorganism invasion (mello et al., 2012), and discriminate pathogenic and non-pathogenic bacteria. for example, the expression of hsp70 gene in mussel hemocytes increased significantly after v. anguillarum challenge, while v. splendidus and m. lysodeikticus could not induce the expression of hsp70 (cellura et al., 2006). recently, the expression of hsp70s in molluscan hemocytes have been investigated extensively against several environment stressors, such as high temperature (yang et al., unpublished data), heavy metal (taylor et al., 2013), pollutants of toxins (mello et al., 2012; mello et al., 2013), pharmaceuticals (gust et al., 2013), bacterial infections (wang et al., 2009) and seasonal changes (li et al., 2009), and their expression profiles are generally divided into three cases, up-regulated, invariable and down-regulated. when exposed to different stressors, up-regulated expression of hsp70s mrna was the general case observed in molluscan hemocytes. for example, the mrna expression of hsp70 in pond snail lymnaea stagnalis increased (2.6-fold) after they were exposure to the mixtures of four pharmaceuticals (gust et al., 2013). after incubation with the purified paralytic toxin of dinoflagellate alexandrium minutum, saxitoxin (stx), the mrna level of hsp70 in oyster hemocytes increased 2-fold (mello et al., 2013). moreover, the up-regulation of hsp70s expression in molluscan hemocytes usually displays a clearly time-dependent and dose-dependent pattern. the mrna level of hsp70 in hemocytes of c. gigas increased at 4 h after the hemocytes were incubated with 1000 μg/l of pbtx-2 (mello et al., 2012). at 6 h, 12 h and 24 h post heat stress treatment, the expression of hsp68 in c. gigas was up-regulated and relative mrna level was 3.78-, 16.11and 112.16fold of that in the control group, respectively (yang et al., unpublished data). after challenged by v. alginolyticus, the mrna expression of hsp70 in hemocytes of pearl oyster p. fucata increased to the maximum level at 4 h, and returned to control level at 32 h (wang et al., 2009). the mrna expression of hsp70 in hemocytes of zebra mussel dreissena polymorpha reached the highest level (2.8-fold) at 1 h post lps stimulation, and decreased at 2 h, and then increased again from 3 h to 6 h post-stimulation (xu and faisal, 2009). the mrna expression of hsp70 in hemocytes of blood clam t. granosa were all significantly up-regulated at 6 h after pb2+, cd2+ and cu2+ treatments, and peaked at 12 h after treatments (zhou et al., 2013). except for the frequently up-regulation of hsp70s, it is interesting that the expression of hsp70 could also be down-regulated under some stressors. for example, the expression of hsp70 was significantly down-regulated when the sydney rock oyster saccostrea glomerata was exposed to some heavy mental, such as zinc and copper (taylor et al., 2013), cadmium and lead (thompson et al., 2012). the excretory-secretory products (esps) from the larva of parasite schistosoma mansoni could reduce the hsp70 protein levels in hemocytes of its snail intermediate host biomphalaria glabrata, and the reduction in hemocytes of s. mansoni-resistant strain was less marked, while that in hemocytes of s. mansoni-susceptible snails was remarkable (approximately 70%) after infected by s. mansoni for 35 days (zahoor et al., 2010). regulation of molluscan hsp70 expression the up-regulation and down-regulation, as well as the dose-dependent and time-dependent expression pattern of hsp70 in the hemocytes of mollusks exposed to various stressors strongly suggested that the regulation mechanism of hsp70 expression was indeed complicated. generally, the expression of hsp70 genes is mainly regulated at the transcription level (park et al., 2007), and the regulation is mediated by direct interaction of heat shock transcription factors (hsfs) and their corresponding heat shock elements (hses) in the promoters of hsp70s (wu, 1995; buckley et al., 2001), and other indirect signaling pathways (buckley et al., 2001; park and liu, 2001; gourgou et al., 2010; zahoor et al., 2010). the interaction of hsfs and hses in the promoters of hsp70s is the prime strategy to regulate hsp70 expression. though molluscan hsf1s have been identified in the genome of m. trossulus, c. gigas and haliotis asinina, the relevant study on the regulation mechanism of molluscan hsp70 expression is at the very beginning. it has been reported that hsf1 of intertidal mussels (genus mytilus) releases from hsp70 and translocates into the nucleus in response to small increase of temperature, and remains inactive on the promoter until the mussels encounter a higher temperature (buckley et al., 2001). the regulation of hsp70 expression also involves other cell proteins and signaling pathways after hsf1 has been bound to the promoter, including the mitogen-activated protein kinases (mapk) signaling cascade (buckley et al., 2001; park and liu, 2001; gourgou et al., 2010) and the extracellular signal-regulated kinase (erk) signaling pathway (zahoor et al., 2010). in m. galloprovincialis, the increased phosphorylation of p38-mapk and c-jun n-terminal kinase (jnk) paralleled with the increased expression of hsp70, strongly supporting the involvement of mapk signaling cascade in the induction of hsp70 genes under various stressors (malagoli et al., 2004; kefaloyianni et al., 2005; anestis et al., 2007; gourgou et al., 2010). after m. galloprovincialis was exposed to 30 °c acute thermal 80 http://www.ncbi.nlm.nih.gov/protein/bak61501.1 http://www.ncbi.nlm.nih.gov/protein/abr15461.1 stress, the activation profile of p38-mapk phosphorylation was sustained and significant, while that of jnks was transient and relatively moderate (gourgou et al., 2010). this direct evidence demonstrated the principal roles of p38-mapk and jnks in transducing the stress signal via mobilization of specific transcription factors and the transcriptional up-regulation of hsp70 genes (gourgou et al., 2010). the erk signaling pathway has also been reported to regulate hsp70 expression in esp-challenged hemocytes of b. glabrata, in which the mitogen-activated protein-erk kinase 1/2 (mek1/2) inhibitor could significantly reduce hsp70 protein levels, and this might be a strategy employed by the parasite to manipulate the immune response of the intermediate snail host (zahoor et al., 2010). conclusion molluscan 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http://www.sciencemag.org/search?author1=xiaotian+zhu&sortspec=date&submit=submit http://www.sciencemag.org/search?author1=xun+zhao&sortspec=date&submit=submit http://www.sciencemag.org/search?author1=william+f.+burkholder&sortspec=date&submit=submit http://www.sciencemag.org/search?author1=alexander+gragerov&sortspec=date&submit=submit http://www.sciencemag.org/search?author1=craig+m.+ogata&sortspec=date&submit=submit http://www.sciencemag.org/search?author1=max+e.+gottesman&sortspec=date&submit=submit nematode antimicrobial peptides isj 9: 122-133, 2012 issn 1824-307x review nematode antimicrobial peptides dek tarr department of microbiology and immunology, arizona college of osteopathic medicine, midwestern university, 19555 n. 59th ave., glendale, az, 85308, usa accepted june 15, 2012 abstract there is currently insufficient data to determine the full spectrum of antimicrobial peptides (amps) that nematodes produce. defensins, nemapores, cecropins, and caenacins/neuropeptide-like proteins have been identified, but none of these is produced universally by all nematode species, and no single species produces all amp types. therefore, it seems unlikely that there is a core set of amps that can be considered “archetypal” for nematodes. additional information is also needed from underrepresented ecdysozoan and lophotrochozoan taxa to clarify the evolution of amps. to avoid generalizations that may later prove inaccurate, caution should be used when choosing “representative” sequences or taxa, and analyses should be interpreted conservatively when limited information is available. key words: antimicrobial peptide; caenacin; caenopore; defensin; nemapore; neuropeptide-like peptide   introduction let’s be honest-we often use the word “nematode” when we should say “c. elegans” (because no one actually says “caenorhabditis”). the overwhelming majority of nematode antimicrobial peptide (amp) research is performed using c. elegans, with ascaris suum a distant second, and along with drosophila melanogaster, c. elegans has become a popular model for the study of innate immunity. in general, this isn’t because there is significant interest in how nematodes defend themselves against potential pathogens. the interest is usually in human innate immunity, but humans (and other vertebrates) have that troublesome adaptive immune system that gets in the way of studying innate immunity, so an organism lacking adaptive immunity is an attractive alternative. findings in c. elegans are often generalized to all nematodes, even though no single species should be considered completely representative of the phylum. nematodes are an incredibly diverse group of organisms with species adapted to both free-living and parasitic lifestyles in a broad range of environments. it is possible (although i think unlikely) that there is a core set of defense molecules that ___________________________________________________________________________ corresponding author: d ellen k tarr department of microbiology and immunology arizona college of osteopathic medicine midwestern university 19555 n. 59th ave, glendale, az, 85308, usa e-mail: etarrx@midwestern.edu comprise an archetypal nematode defense system, but the wide variety of environments has almost certainly influenced the evolution of immune-related molecules in different lineages. there may also be similarities between species of divergent lineages that have experienced similar environmental pressures. the use of the model systems, c. elegans and d. melanogaster, naturally leads to comparisons between the two and to speculation regarding how innate immune molecules may have evolved. nematodes and arthropods represent two phyla within ecdysozoa (fig. 1), a superphylum that also includes nematomorpha (horsehair worms), loricifera, priapulida (penis worms), kinorhyncha (mud dragons), onychophora (velvet worms), and tardigrada (water bears) (telford et al., 2008). very little (if anything) is known about the amps produced by most of these phyla. further characterization of these groups will facilitate analyses of how nematode and arthropod amps have evolved. invertebrate amps can be broadly classified by structure: α-helical linear peptides, peptides that contain several cysteines that form one or more disulfide bonds, peptides with a large proportion of one or two amino acids, and peptides that are processed from larger precursors that do not have antimicrobial activity (bulet, 2004). this review will discuss four groups of amps identified in nematodes that represent three of these structural groups: defensins and nemapores (cysteine-stabilized   122 http://www.google.com/url?sa=t&rct=j&q=elegans&source=web&cd=1&ved=0cguqfjaa&url=http%3a%2f%2fen.wikipedia.org%2fwiki%2fcaenorhabditis_elegans&ei=lilct4unizhn4qt0nfm9cg&usg=afqjcnfdfpvy7bvrdidmypqvmteatezs2g mailto:etarrx@midwestern.edu fig. 1 representation of ecdysozoan phylogeny reproduced from telford et al. (2008). the tree is intended to show relationships only and is not drawn to scale. peptides), cecropins (α-helical peptides), and caenacins/neuropeptide-like proteins (glycine-rich peptides). these groups have not been studied equally-there is significantly more information on the cysteine-stabilized peptides compared to the other groups. reviews of c. elegans antimicrobial peptides are published regularly; i see no need to duplicate these efforts and will refer the reader to a recent review for information regarding wellestablished findings. in this review, i will focus on the general structure and function of nematode amps, the distribution of different amp groups across the phylum, and phylogenetic relationships. defensins defensins are by far the most studied of nematode antimicrobial peptides. the first nematode defensins identified and characterized were ascaris suum antibacterial factors (asabfs) and their homologs in c. elegans (kato, 1995; kato and komasku, 1996; zhang et al., 2000; kato et al., 2002; pillai et al., 2003). with the exception of asabf-6cys-α (minaba et al., 2009), these asabfs and ce-abfs have eight cysteines predicted to form four disulfide bonds. findings regarding expression of these peptides and the signaling pathways involved have been reviewed recently (bogaerts et al., 2010). they are often still referred to as abfs, but based on the conserved cysteine pattern, they are clearly similar to other invertebrate defensins (see below). for clarity, i will refer to the previously described abfs and their homologs in other nematode species collectively as nematode defensins. structure nematode defensins are part of the cysteinestabilized α-helix and β-sheet (cs-αβ) group of defensins that are evolutionarily divergent from mammalian defensins. the cs-αβ fold defines a superfamily that includes plant and invertebrate defensins, as well as arthropod toxins that target ion channels. this structural motif is characterized by six cysteines that form an α-helix and two β-sheets stabilized by three disulfide bonds (figs 2a, c). the proposed signature sequence for the cs-αβ superfamily is as follows: c-x(2,18)-c-x(3)-cx(2,10)-[gapsideryw]-x-c-x(4,17)-cxc (zhu et al., 2005). two smaller motifs are nested within the conserved cs-αβ fold: the cysteine-stabilized αhelix (csh) motif and the γ-core (fig. 2a). the csh motif was first described from arthropod toxins and venoms that act on ion channels (kobayashi et al., 1991). the motif connects the c-x(3)-c and cxc of the above superfamily signature, which accounts for two of the three disulfide bonds of the cs-αβ fold. the γ-core is proposed to be an archetypal structural motif found in all groups of cysteinestabilized host defense effector peptides (yeaman and yount, 2007). it is characterized by two antiparallel β-sheets separated by a short turn that can be generated by three isoforms of an enantiomeric signature sequence: x(1-3)-gxc-x(3-9)-c, c-x(39)-cxg-x(1-3), and c-x(3-9)-gxc-x(1-3) (yount and yeaman, 2004). within the proposed classification system based on this conserved motif, cs-αβ defensins are generally classified as having a γ-α structure (yeaman and yount, 2007).   123 the three disulfide bonds and conserved signatures described above are well conserved throughout the superfamily, while the presence and position of additional cysteines that form a fourth bond have been used to describe specific groups found in only some lineages. for example, the overwhelming majority of arthropod defensins described thus far have only three disulfide bonds, but drosomycin from d. melanogaster has a fourth disulfide bond formed from additional cysteines near the n and c termini (fig. 2a; landon et al., 1997). some mollusc defensins and the closely-related myticins also have eight cysteines (hubert et al., 1996; mitta et al., 1999), but the additional cysteines forming the fourth disulfide bond are located in the first β-sheet and at the c-terminus (fig. 2a; yang et al., 2000). this pattern is sometimes referred to as “mollusc-type” to differentiate it from the six-cysteine “arthropod-type” or “insect-type” pattern. the majority of nematode defensins have a cysteine array consistent with the “mollusc-type” pattern (zhang and kato, 2003; tarr, 2012); although only the structure for asabf-α has been experimentally determined (fig. 2c; aizawa et al., 2001). in general, these nematode defensins have a longer n-loop than mollusc eight-cysteine defensins (fig. 2a; tarr, 2012). asabf-6cys-α is a sixcysteine defensin that is missing the first and last cysteine of the typical eight-cysteine mollusc/nematode array (minaba et al., 2009). some rearrangement of bonding has occurred because these six cysteines do not correspond to those that form the three conserved disulfide bonds of the cs-αβ fold (fig. 2a), but the structure for this defensin has not been experimentally determined (minaba et al., 2009). it is unknown at this time whether the asabf-6cys-α cysteine array is restricted to nematodes. there have also been sequences identified that are consistent with the “mollusc-type” array, but are missing only the nterminal cysteine (tarr, 2012). it is not clear whether only six of the seven cysteines are participating in bond formation or if the extra cysteine facilitates dimer formation as has been suggested for some plant defensins (zhu et al., 2005). in addition to “mollusc-type” defensins, nematodes have sequences predicted to encode “arthropod-type” sixcysteine defensins (tarr, 2012; zhu et al., 2005), “arthropod-type” defensins with the addition of two n-terminal cysteines, and at least one potential drosomycin homolog (tarr, 2012). invertebrate defensins are generally produced as precursor molecules with an n-terminal secretory signal peptide. many arthropod defensins also have a pro-peptide located between the signal and mature peptides, while mollusc defensins tend to have the pro-peptide located c-terminal to the mature peptide (mitta et al., 1999; froy and gurevitz, 2003). the majority of nematode defensins have a signal peptide, and many of these also have a predicted c-terminal pro-peptide (tarr, 2012). however, some nematode defensins have a predicted pro-peptide located between the signal peptide and mature peptide (tarr, 2012), a domain organization previously thought to be found in arthropods, but not in molluscs or nematodes (zhang and kato, 2003; froy, 2005; rodriguez de la vega and possani, 2005). the cysteine array pattern and domain organization are not linked, i.e., nematode defensins that have an arthropod-like cysteine pattern are not necessarily the same as those with an arthropod-like domain organization (tarr, 2012). distribution in nematodes there are currently 86 nematode defensins identified from 25 species (tarr, 2012) representing clades i, iii, iv, and v of the phylogenetic framework based on small subunit (ssu) ribosomal dna sequences (blaxter et al., 1998). although clade ii (enoplia) has been studied for phylogenetic purposes (bik et al., 2010), there are too few sequences available to determine if defensins are present in this group or not. in general, defensins from clades i and iii have the “mollusc-type” cysteine pattern while the sequences from clades iv and v have more diverse cysteine patterns (tarr, 2012). very few nematode genomes have been sequenced, so it is premature to conclude that defensins are absent in many of the species for which a defensin sequence has not been identified yet. in spite of these limitations, there are some interesting observations when looking at the distribution of nematode defensins: (1) they are present in ascarids, but have not been identified in filarials; (2) they are present in xiphinema, but have not been identified in trichinella or trichuris; and (3) they are present in some species of meloidogyne, but not in others (tarr, 2012). molecular phylogeny based on ssu generally places ascarids (large intestinal roundworms) and filarial nematodes into clade iii, a grouping of zooparasitic nematodes that also includes pinworms and millipede-gut parasites (blaxter et al., 1998). nematode defensins are consistently present in ascarids: a. lumbricoides (3), a. suum (10), toxascaris leonina (2), and toxocara canis (5). in contrast, no defensins have been found in brugia malayi, dirofilaria immitis, litomosoides sigmodontis, loa loa, onchocerca volvulus, or wuchereria bancrofti (tarr, 2012). the lack of complete genome information can account for this in most of filarial species, but the genome for brugia malayi has been completed, and the absence of abfs was noted (ghedin et al., 2007). with defensins being widely distributed throughout nematodes, one interpretation of this observation is that defensins were lost in a filarial ancestor after the divergence of clade iii from clades iv and v. however, more recent molecular phylogeny using mitochondrial genome data from 36 species has concluded that clade iii is not monophyletic (park et al., 2011). furthermore, the analyses supported placement of ascaridida within rhabditida, and spirurida as a separate clade, i.e., ascarids may be more closely related to c. elegans than to filarial nematodes (park et al., 2011). within this context, it is not as surprising to see such a discrepancy between the groups, although it is still not clear why filarial worms do not have defensins. two aspects of filarial biology deserve further consideration in comparison to ascarids and free-living species such as c. elegans: filarial nematodes have arthropod intermediate hosts and some filarial species   124 (although not all) have a bacterial endosymbiont. it is possible that the production of defensins would be detrimental to the nematode endosymbiont or to an endosymbiont found in the arthropod vector. further studies should look specifically at vector-borne vs. non-vector-borne species as well as those that harbor bacterial endosymbionts vs. those that do not. molecular phylogeny has generally supported placement of xiphinema (a plant parasite, dorylaimida), and trichuris and trichinella species (vertebrate parasites, trichinellida) in clade i, which represents one of the earliest branches of nematodes (blaxter et al., 1998; bik et al., 2010). taxon sampling within this clade is minimal and conclusions should be drawn only tentatively, but within this framework, it is possible that defensins are present in dorylaimida and absent in trichinellida. defensins from dorylaimida have been found only in x. index, and there is no evidence yet from the other clade i lineages, mononchida and mermithida. therefore, it is also possible that defensins have been gained by x. index (or an ancestor) relatively recently. absence of defensins from trichinellida is supported by the completion of a draft genome for t. spiralis (mitreva et al., 2011), but again, there is not enough information to determine whether this is species specific or the general rule across trichinellida. as with molecular phylogenies of clade iii, it is not clear that clade i is monophyletic (bik et al., 2010; park et al., 2011), complicating interpretation of the findings. complete genomes from additional taxa are needed to determine where defensins have been lost or gained in clade i lineages. sequences encoding predicted defensins have been identified in meloidogyne hapla and m. javanica, but not yet from m. arenaria, m. artiellia, m. chitwoodi, m. incognita, or m. paranensis (tarr, 2012). genomes have been completed and published for both m. hapla and m. incognita, making it less likely the absence of defensins in m. incognita is due to lack of information. it is unclear why the presence of defensins is variable within this genus, but it does not seem to be consistent with molecular phylogeny based on ssu rdna sequences (holterman et al., 2009), or mitotic vs. meiotic parthenogenesis (m. incognita and m. javanica are both mitotic parthenogenetic species). a possible limitation on the identification of nematode defensins is the potential that the current consensus of conserved cysteines may not accurately reflect what is required for defensin function. while attempting to identify nematode defensins, there were cysteine-rich sequences that didn’t have the conserved invertebrate defensin pattern. structural characterization and antimicrobial testing of novel, cysteine-rich peptides will determine whether these are additional groups that are unrelated to defensins or if our current definition of “defensin” should be revised to include more variable cysteine patterns. activity previous work tested the activity of purified and recombinant asabf-α, and recombinant ceabf-2, and showed that these peptides have the greatest activity against gram-positive bacteria, with less activity against gram-negative bacteria and yeast (kato, 1995; kato and komasku, 1996; zhang et al., 2000; kato et al., 2002). the antimicrobial activity of defensins is probably due to their ability to form pores in microbial membranes. there are multiple models of pore formation, and the mechanism of action may differ between defensins or with membrane composition (ganz, 2003; pálffy et al., 2009). this is consistent with the proposed generalization that peptides with the γ-core motif interact with lipid membranes, resulting in pore formation or disruption of ion channels. differences in mechanisms of pore formation may be due to subtle differences within the γ-core as well as structural differences outside this motif (yeaman and yount, 2007). the three well-conserved disulfide bonds in the cs-αβ motif play a structural role in folding and stability. a fourth disulfide bond is present in some members of this superfamily, and is much more variable in its location. for mgd-1 from m. galloprovincialis, the fourth bond is suggested to contribute additional stability in a high-osmolarity environment such as seawater (yang et al., 2000). in contrast, drosomycin from d. melanogaster has a fourth disulfide bond that differs in its placement from that in mgd-1, and may contribute to its antifungal activity compared to the antibacterial activity observed in most insect defensins (dimarcq et al., 1998; zhu et al., 2005). the fourth disulfide bond in asabf-α is consistent with that in mgd-1, suggesting a role in increased stability, although this has not been experimentally verified. in addition to antibacterial and antifungal activities, some defensins have shown antiplasmodium activity that may correlate with a fiveresidue motif in the m-loop (fig. 2a; tian et al., 2008; gao et al., 2009). this motif was first identified as grsgg (the last g corresponds to the gxc of the γ-core motif) from the aeschna cyanea anti-plasmodium defensin acdef (shahabuddin et al., 1998). future studies should include testing activity against plasmodium in addition to antibacterial and antifungal activity to verify this motif. as noted above, many nematode defensins that have the “mollusc-type” cysteine pattern have a longer n-loop than seen in mollusc defensins/myticins (fig. 2a). this region is upstream of the cs-αβ fold and the functional significance is not known at this time. similarly, the defensins from x. index and some from caenorhabditis have an extended region between the conserved cxc and the last cysteine (tarr, 2012). the impact on the function of defensins with this c-terminal extension is also unknown. many nematodes produce several predicted defensins (tarr, 2012), but only the activities of asabf-α and ceabf-2 have been characterized (see above). the number of defensins per species may reflect differences in antimicrobial spectrum, differences in tissue localization, or both. there is some evidence of differential tissue expression for both asabfs (kato et al., 2002;  pillai et al., 2003; minaba et al., 2009) and ceabfs (kato et al., 2002), but neither activity nor expression have been investigated for most nematode defensins.   125 phylogenetic relationships the initial characterization of abfs showed that they are most similar to mollusc defensins and myticins. this led to a series of papers that suggested a common ancestor for mollusc and nematode defensins (zhang and kato, 2003), then convergent evolution of these groups (froy, 2005), and finally a lack of sufficient information to definitively establish the evolutionary relationship (rodriguez de la vega and possani, 2005). unfortunately, these studies were based on a limited number of nematode and mollusc sequences that underestimated defensin diversity in both groups. although the abfs represented two species traditionally considered to be relatively divergent that live in completely different environments (a. suum and c. elegans), these two species are still not completely representative of the phylum and as mentioned earlier, may not be as divergent as traditionally thought (park et al., 2011). several reports show that both molluscs and nematodes clearly have defensins with the “arthropod-type” cysteine pattern in addition to the “mollusc-type” pattern (charlet et al., 1996; zhu et al., 2005; de zoysa et al., 2010; xu and faisal, 2010; tarr, 2012). defensins with these different patterns are not distributed equally in nematodes. as stated previously, all ascarid and xiphinema defensins have a “mollusc-type” cysteine array (the spacing for asabf-6cys-α is still more similar to the mollusc spacing than the arthropod spacing). in contrast, clades iv and v have more diverse cysteine patterns, and none of the sequences identified from meloidogyne have the eight-cysteine “mollusc-type” array (tarr, 2012). a recent analysis included defensins from across nematode taxa, but was unable to definitively resolve phylogenetic relationships and did not support a clear “nematode” clade, suggesting a common origin for nematode and other invertebrate defensins (tarr, 2012). in contrast to previous studies that used either mgd-1 (froy and gurevitz, 2003; froy, 2005) or myticin (minaba et al., 2009) as the mollusc representative, this analysis used both, and did not place them in the same clade (tarr, 2012). the myticins are considered a different family of antimicrobial peptides based on lack of sequence similarity to mussel defensins (mitta et al., 1999), but the cysteine pattern is nearly identical and the myticins are clearly part of the cs-αβ superfamily (fig. 2a; zhu et al., 2005). in addition to mollusc defensins and myticins, there are mytilins, mytimycin, and mytimacins that seem to have conserved cysteine spacings that could indicate a cs-αβ fold (charlet et al., 1996; gerdol et al., 2012). these families should be investigated further to identify nematode homologs and determine their relationship to identified defensins. additional mollusc sequences as well as defensins from ecdysozoan taxa not currently represented in analyses should facilitate resolution of nematode phylogenetic relationships. studies of the cs-αβ and γ-core motifs have suggested that sequences with these motifs are descended from a common ancestor (zhu et al., 2005; yeaman and yount, 2007). three additional findings support this. first, an asabf-like sequence has been identified in the sponge suberites domuncula, making it the first defensin described from a poriferan. this peptide shows antimicrobial activity against gram-positive bacteria and hemolytic activity, as well as activity against a gastropod that sometimes grazes on the sponge (wiens et al., 2011). second, several groups of defensin-like peptides in fungi have been identified (zhu, 2008). this study used a framework that proposes three basic groups of invertebrate defensins: a group of defensins found only in neopteran insects referred to as classical insecttype defensins (citds), a group with broader taxonomic distribution called ancient invertebratetype defensins (aitds), and a group that includes drosomycin called antifungal plant/insect-type defensins (pitds) (dimarcq et al., 1998; froy and gurevitz, 2003). this study concluded that fungi and animals have all three groups of defensins, with the pitds being the only group that is also found in plants (zhu, 2008). this framework was based largely on arthropod defensins, and should be reevaluated to determine if it is still applicable with the addition of mollusc and nematode sequences. the recent analysis of nematodes included several arthropod sequences, but did not support these distinct clades (tarr, 2012). the last finding that provides evidence for a common ancestor of the cs-αβ defensins is the identification of two myxobacterial defensin-like peptides that might represent this ancestor (zhu, 2007). these defensin-like peptides from anaeromyxobacter dehalogenans (addlp) and stigmatella aurantiaca (sddlp) have the csh motif, and a domain organization with an n-terminal signal peptide and c-terminal pro-peptide (zhu, 2007). recombinant addlp has no antibacterial or antifungal activity, but has a predicted antiparasitic motif and activity against plasmodium falciparum (gao et al., 2009). in addition to the above findings that contribute to developing a coherent evolutionary history of the cs-αβ superfamily, an asabf-like peptide (hkabf) has been reported from the seahorse hippocampus kuda (wang et al., 2008). this represents the only cs-αβ defensin in vertebrates, and it is unclear whether it is the result of horizontal gene transfer (and if so, from what organism), or if there could have been a nematode parasite contaminating the source rna. nemapores caenopores are antimicrobial peptides from c. elegans with similarity to amoebapores from entamoeba histolytica, naegleriapores from naegleria fowleri, and mammalian nk-lysin and granulysin (leippe et al., 1991; leippe, 1995, 1999; banyai and patthy, 1998; herbst et al., 2002). the caenopores are encoded by 28 spp genes, several of which are involved in responses to various pathogens (see review by bogaerts et al., 2010; roeder et al., 2010; hoeckendorf et al., 2012). “nemapore” is a more general term used to describe similar peptides produced by any nematode species (tarr, 2012). as peptides from additional species are characterized, they may be given more specific names (ascaripores, brugiapores, etc.), but at this time, only caenopores have been studied experimentally.   126 structure nemapores are cysteine-rich antimicrobial peptides that belong to the saposin-like protein (saplip) superfamily. in addition to the antimicrobial peptides listed above, the saposin domain is also found in other membrane-interacting proteins, such as saposins and surfactant proteins, and can act either independently or as part of a multidomain protein (for a review of saplips, see bruhn, 2005). the saposin fold has a core of hydrophobic amino acids and six cysteines that form three disulfide bonds (figs 2b, d; bruhn, 2005). in contrast to the cs-αβ fold, the saposin fold does not have a clear γcore motif, so this may not be conserved among all cysteine-stabilized amps. a general consensus for the conserved cysteines differs substantially from that of the defensins: c-x(2)-c-x(21-31)-c-x(7-16)c-x(19-29)-c-x(3-7)-c (tarr, 2012). the bonding between c1-c6, c2-c5, and c3-c4 stabilizes a structure that is generally composed of five α-helices that form two bundles (bruhn, 2005). in nemapores, this structure has been confirmed experimentally only for caenopore-5 (fig. 2d; mysliwy et al., 2010). two sequences from prokaryotes, the bacteriocin as-48 from enterococcus faecalis and plasmid achromobacter secretion (pas) from vibrio vulnificus, do not have the conserved cysteines, but appear to maintain the saposin fold structure (gonzález et al., 2000; lee et al., 2006), suggesting the cysteines may have been a later addition to further stabilize the fold. another deviation from the canonical saposin domain is the “swaposin” domain found in some plant aspartic proteinases. the “swaposin” domain is a circular permutation of the saposin domain, composed of the c-terminal half of one saposin domain linked to the n-terminal half of a second saposin domain (ponting and russell, 1995). distribution nemapores have been identified from 46 species representing all clades (except clade ii due to lack of sequence information), and in every nematode species with a completed genome except m. incognita (tarr, 2012). the discrepancy between m. hapla and m. incognita was noted above, and the reason for the difference is no clearer for the nemapores than for the defensins. sequences encoding potential “swaposin” domains have not been investigated in nematodes. it is possible that these are present in m. incognita, although this would still not explain the discrepancy between the two closely-related species. the only other meloidogyne species with identified nemapores thus far is m. chitwoodi (tarr, 2012). caenopores have been proposed to be the best candidates for defense of c. elegans against microbes based on the large number of variants with varying antimicrobial spectra that are induced by exposure to different pathogens and active at an acidic ph (roeder et al., 2010). several species have a large number of predicted nemapores, including all caenorhabditis species with completed genomes and pristionchus pacificus. in contrast to defensins, filarials as well as ascarids have nemapores, although clade iii nematodes do not seem to have the high numbers found in some clade iv and v species (tarr, 2012). identified nemapores are predicted to have one to four saposin domains, with sequences predicted to have three or four domains found only in clade i and v species thus far (tarr, 2012). t. spiralis secretes a prosaposin homolog with four saposin domains and a single glycosylation site in the first domain (selkirk et al., 2004). activity the antimicrobial activity of caenopores has only been investigated for spp-1, spp-5, and spp12. all three are active against bacillus megaterium, but only spp-5 shows significant activity against escherichia coli (roeder et al., 2010; hoeckendorf et al., 2012). spp-12 is also active against b. thuringiensis and is involved in c. elegans resistance to this pathogen (hoeckendorf et al., 2012). the pore-forming activity of all three has been confirmed using liposomes, and in b. megaterium, saccharomyces cerevisiae, and dictyostelium discoideum for spp-1 and spp-12 (roeder et al., 2010; hoeckendorf et al., 2012). similar to amoebapores, spp-1, 5, and 12 are more active at acidic ph (leippe et al., 1991;  roeder et al., 2010; hoeckendorf et al., 2012). naegleriapores are produced as precursors with multiple saposin domains that are processed to produce multiple mature peptides, not all of which have pore-forming activity (herbst et al., 2004). some non-antimicrobial saplips are also produced as precursors, and in general, glycosylation seems to play a role in peptide stability (reviewed in bruhn, 2005). the only multi-domain nemapore that has been studied is the t. spiralis prosaposin, but there is currently no evidence regarding processing of the precursor (selkirk et al., 2004). in contrast to defensins, nemapores are hypothesized to have an additional role in nematode biology. bacteria aren’t just a potential source of infection for nematodes, but are a food source for many species. therefore, molecules that participate in bacterial killing, especially those expressed in the intestine, may be contributing to nutrition as well as to host defense. c. elegans spp-5 mutants do not store sufficient fat for normal egg production, providing evidence for spp-5 contributing to digestion (roeder et al., 2010). with the large number of nemapores expressed in some species, there is the potential that the sequences have diverged, with some playing a defensive role and others involved in nutrition. phylogenetic relationships previous analyses of caenopores (roeder et al., 2010) and nemapores (tarr, 2012) have been unable to resolve phylogenetic relationships of the saposin domains, due at least in part to the short length of the domain sequences. with the exception of mammalian sequences, phylogenetic analyses do not clearly separate nematode sequences from other eukaryotic sequences. they also do not provide evidence for defensive vs. digestive clades of nemapores (tarr, 2012). however, the analyses do suggest that the domains of multi-domain sequences are not the result of recent duplication of one of the domains (roeder et al., 2010; tarr, 2012). phylogenetic analyses have not been   127 performed that include the related bacterial sequences lacking the conserved cysteines. cecropins cecropins are an example of why it is necessary to look beyond c. elegans when characterizing nematode amps. a. suum cecropin p1 has been extensively characterized compared to other nematode amps, probably because it was originally isolated from pig intestine and thought to be a porcine cecropin (lee et al., 1989). this also highlights the potential difficulty of separating host dna from that of potential parasites, infections, and symbionts. cecropin p1 was later realized to be from the pig intestinal roundworm, a. suum (andersson et al., 2003), and a total of four cecropins are induced in response to pathogen injection into the pseudocoelom (pillai et al., 2005). structure in contrast to the defensins and nemapores, cecropins have an amphipathic α-helical structure that is not stabilized by disulfide bonds (sipos et al., 1992). therefore, a consensus sequence based on the number and spacing of cysteine residues is not possible for identification of new cecropins. a signature sequence proposed based only on insect cecropins (tamang and saier, 2006) was recently updated to include mosquito and nematode cecropins (tarr, 2012): [krden]-[kred]-[livmr]-[ed]-[rkghn]-x(0,1)[ivmalt]-[gvik]-[qrkha]-[nhqrk]-[ivta][rkfas]-[dnqke]-[gasv]-[livsatg][liveaqkg]-[rkqsgil]-[atgvsfiy]-[galivqn] this sequence appears to be cecropin-specific, but should not be used as the only criteria for identification of new cecropins. this proposed signature sequence is significantly different from the signature sequence currently defined in prosite (sigrist et al., 2010): w-x(0,2)-[kdn]-{q}-{l}-k-[kre]-[li]-e-[rkn] this signature (ps00268) is not currently cecropinspecific and begins upstream of the previous one, but may facilitate cecropin identification if the other signature is found to be too stringent. the prosite signature includes a tryptophan that is usually found as the first or second residue of the cecropin mature peptide, but is known to be absent from some cecropins (bulet et al., 2004). similar to many nematode defensins, nematode cecropins are predicted to be produced as a precursor with an n-terminal signal peptide and a cterminal pro-peptide (pillai et al., 2005; tarr, 2012). this domain structure is more similar to styelins from tunicates (zhao et al., 1997) than to insect cecropins, which are more likely to have a short propeptide immediately n-terminal to the mature peptide (boman and boman, 1989). for cecropin p4, the c-terminal pro-peptide has an inhibitory effect on the antibacterial activity of the mature peptide (ueno et al., 2008). distribution nematode cecropins have only been identified in ascaris and toxocara (pillai et al., 2005; tarr, 2012). these previous analyses used blast searches that did not rely on proposed signature sequences for identification, so it is unlikely that additional nematode cecropins have been missed because they deviate only slightly from these signatures. the presence of cecropins in such a limited group of nematodes suggests that a cecropin gene was acquired by an ancestor of ascaris and toxocara, but there is currently no evidence for the source of this transferred gene. activity cecropins p1-p4 from a. suum show activity against gram-positive and gram-negative bacteria, with weaker activity against yeast (pillai et al., 2005). studies of cecropin p1 suggest that it forms pores in bacterial membranes using a “carpet” mechanism in which the peptides are oriented parallel to the membrane, resulting in membrane destabilization once a threshold concentration is reached (pouny et al., 1992; gazit et al., 1996). phylogenetic relationships previous analyses have shown that nematode cecropins are monophyletic and may be more closely related to styelins from the tunicate styela clava than insect cecropins, although this relationship is not always conserved with changes in alignment parameters (tarr, 2012). in general, nematode cecropins seem to be more similar to dipteran than lepidopteran cecropins (tarr, 2012). within nematodes, clear orthologs are found between species, suggesting that more than one cecropin was already present in an ancestor of ascaris and toxocara (pillai et al., 2005). the cecropin family is part of the cecropin superfamily, which also includes the pleurocidin and dermaseptin families of toxic peptides (tamang and saier, 2006). sequences predicted to encode members of these families have not been found in nematodes (tarr, 2012). cecropins may have evolved from peptides found in bacterial ribosomal proteins. specifically, peptides from helicobacter pylori ribosomal protein l1 are similar to cecropins a and b from hyalophora cecropia (pütsep et al., 1999). whether the different families of the cecropin superfamily may have evolved from the same ribosomal peptides has not been investigated. caenacins and neuropeptide-like proteins the caenacins (cncs) and neuropeptide-like proteins (nlps) are related groups of glycine-rich peptides that have been most studied in c. elegans. two subgroups of these genes, the “cnc-2 cluster” (cnc-1 to cnc-5 and cnc-11) and the “nlp-29 cluster” (nlp-27 to nlp-31 and nlp-34), are induced by wounding and infection, but regulated by different signal transduction pathways, which have been characterized extensively (reviewed in bogaerts et al., 2010). i will refer to all nlps predicted to be antimicrobial as “antimicrobial nlps” to differentiate them from other nlp families that are not currently predicted have a role in innate immunity. structure the c. elegans “nlp-29 cluster” (nlp-27 to nlp-31 and nlp-34) overlaps with the previously identified   128 fig. 2 comparison of cysteine patterns and tertiary structures of nematode cysteine-stabilized antimicrobial peptides. a) the cysteine arrays for invertebrate defensins and a bacterial defensin-like peptide are aligned for comparison. cysteines that form disulfide bonds are color coded: the four cysteines that form the conserved disulfide bonds of the csh motif are shaded blue, the cysteines forming the remaining disulfide bond of the csαβ motif are shaded purple, the cysteines forming the fourth disulfide bond in nematode and mollusc sequences are shaded orange, the cysteines forming the fourth disulfide bond in drosomycin are shaded green, and the two non-canonical cysteines of asabf-6cys-α are shaded grey. the predicted structure is indicated above the alignment. the predicted γ-core and anti-plasmodium motifs are outlined. accession numbers: asabf-α (baa89497), ceabf-2 (np_491252), asabf-6cys-α (bac41496), mgd-1 (p80571), myticin (p82103), drosomycin (p41964), acdef (p91793), addlp sequence from gao et al. (2009). b) the cysteine array of caenopore-5 (spp-5) is shown with color-coded cysteines indicating the bonding pattern. the locations of αhelices as determined by mysliwy et al. (2010) are shown above the sequence. accession number for spp-5: np_509238. c) 3-d structure of asabf-α (pdb id: 2d56) with the location of the loops labeled. the α-helix is colored purple, β-sheets are colored blue, and the disulfide bonds are colored gold. d) 3-d structure of caenopore-5 (pdb id: 2jsa). the α-helices are colored purple and labeled to correspond with helices shown in 2b, and disulfide bonds are colored gold. yggwamide family, initially defined as nlp-24, nlp25, and nlp-27 through nlp-32 (nathoo et al., 2001), but later updated to include nlp-24 through nlp-33 (mcveigh et al., 2008). this family of antimicrobial peptides is characterized by an n-terminal signal peptide, arginine cleavage site, and a conserved yggyg motif (nathoo et al., 2001; mcveigh et al., 2008). the “cnc-2 cluster” is a subset of genes encoding caenacins that have been studied at the transcriptional level, but have not been the subject of investigations to clarify motifs defining specific families within this larger group. a cursory look at the amino acid sequences suggests the caenacins may also have the yggyg motif, but this has not been addressed directly in caenacin studies. in contrast to the other groups of peptides, studies of cncs and nlps have not focused on determining peptide tertiary structure. distribution a study of nlp diversity in nematodes was unable to identify sequelogs, a neutral term used by   129 the authors to indicate sequence similarity without implying common ancestry (varshavsky, 2004), of individual c. elegans yggwamide nlps (mcveigh et al., 2008). however, nlps with the conserved yggyg motif are present in anisakis simplex, bursaphalenchus xylophilus, b. macronatus, c. remanei, globodera rostochiensis, m. incognita, pristonchus pacificus, and strongyloides ratti (mcveigh et al., 2008). the study found only one or two sequelogs in each of these species, although some of these were predicted to encode several peptides (mcveigh et al., 2008). the initial study that characterized the yggwamide nlps identified a potential sequence in b. malayi (ai079056) that was not found in the more recent study (nathoo et al., 2001; mcveigh et al., 2008), making it unclear whether cnc/nlp antimicrobial peptides are found in filarials, or in the majority of clade iii nematode species (a. simplex was the only clade iii representative in the study). nlp antimicrobial peptides are thus far the only group to be found in m. incognita but not in m. hapla, although this study may have been completed prior to publication of the meloidogyne genomes (mcveigh et al., 2008). the t. spiralis genome had also not been published at the time this study was performed. only one nlp was identified in t. spiralis, but it was not an antimicrobial nlp (mcveigh et al., 2008). as pointed out in the study, antimicrobial nlps (and be extension, the related cncs) may be restricted to only a few species, but the data for most species are too incomplete to draw this conclusion at this time (mcveigh et al., 2008). activity to my knowledge, there have been no direct tests of the antimicrobial activity of purified or recombinant cncs or antimicrobial nlps. these groups have been studied in the c. elegans epidermal response to the nematophagous fungus drechmeria coniospora, but direct antifungal activity has not been demonstrated and no mechanism has been proposed (bogaerts et al., 2010; engelmann and pujol, 2010). phylogenetic relationships the phylogenetic relationships between all nematode cncs and antimicrobial nlps have not been investigated, but an analysis of c. elegans cncs and nlps has been published. this analysis supports a common ancestor of nlp-24 to nlp-34 (with the exception of nlp-26) and the 11 cncs compared to the other nlps (pujol et al., 2008). it is unclear at this time whether the cncs would be more appropriately named as additional antimicrobial nlps or vice versa, but the analysis does not support a clear division between these two groups (pujol et al., 2008). conclusion in spite of the lack of complete genome information for most nematode species, it is clear that none of the antimicrobial peptide groups studied thus far are expressed universally by nematodes. conversely, current results suggest that no single species produces all types of antimicrobial peptides. these observations suggest that there isn’t a single “archetypal” nematode innate immune system. understanding how different nematode species defend themselves against potential pathogens in their environment(s) will necessitate characterization of the defense molecules from the species of interest instead of reliance on a general description derived from c. elegans. the cell membrane is the common target of three groups of antimicrobial peptides (defensins, nemapores, and cecropins). this may become the general rule for nematode antimicrobial peptides, but structure and activity have not been investigated for the caenacins and neuropeptide-like 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hematopoiesis; bombyx mori   introduction in most insects, there are several types of circulating hemocytes (gillespie et al., 1997; lavine and strand, 2002; strand, 2008). in typical lepidopterans, like bombyx mori and manduca sexta, there are 5 types of hemocytes (akai and sato, 1973; beaulaton, 1979; han et al., 1998; ling et al., 2003b; tan et al., 2013). all circulating hemocytes are produced by either hematopoietic organs or through cell division while in circulation (gillespie et al., 1997; lavine and strand, 2002; ling et al., 2003c; strand, 2008; tan et al., 2013). most insect circulating hemocytes can be easily identified using light or fluorescent microscopy following different staining methods (gillespie et al., 1997; lavine and strand, 2002; ling et al., 2003b; ling and ___________________________________________________________________________ corresponding author: erjun ling key laboratory of insect developmental and evolutionary biology, institute of plant physiology and ecology shanghai institutes for biological sciences chinese academy of sciences shanghai, 200032, people’s republic of china e-mail: erjunling@sippe.ac.cn yu, 2006b). investigations on the development and differentiation of circulating hemocytes are a very popular research focus in the field of developmental biology (gillespie et al., 1997). insect hemocytes are a key component of immunity (gillespie et al., 1997; lavine and strand, 2002; strand, 2008). insects have no adaptive immune system, and they have to defend against foreign bodies via innate immune responses (gillespie et al., 1997; lavine and strand, 2002; kanost et al., 2004; strand, 2008; jiang et al., 2010; tanaka and yamakawa, 2011). insect innate immunity is composed of both humoral and cellular responses (gillespie et al., 1997; lavine and strand, 2002; kanost et al., 2004; strand, 2008; liu et al., 2009; jiang et al., 2010). humoral immune responses include the production of proteins like antibacterial peptides and prophenoloxidase (ppo) (gillespie et al., 1997; lavine and strand, 2002; kanost et al., 2004; strand, 2008; liu et al., 2009; jiang et al., 2010; tanaka and yamakawa, 2011). antibacterial peptides are produced primarily by hemocytes and fat bodies (gillespie et al., 1997; kanost et al., 2004; jiang et al., 2010), while insect     102   ppo is considered to be produced within hemocytes (ashida and brey, 1998). in lepidopteran insects, cellular immunity is primarily via granulocytes and plasmatocytes (lavine and strand, 2002; ling and yu, 2006b). however, in m. sexta, five types of circulating hemocytes all take part in the cellular response of encapsulation (ling and yu, 2006a). in drosophila melanogaster, plasmatocytes and lamellocytes are responsible for cellular immunity (lavine and strand, 2002). circulating hemocytes in d. melanogaster there are three terminally differentiated types of hemocytes, which are plasmatocytes, crystal cells and lamellocytes (lanot et al., 2001; evans and banerjee, 2003; wertheim et al., 2005; strand, 2008). all three types of hemocytes are differentiated from prohemocytes (strand, 2008), and they are involved in larval immune functions like phagocytosis, melanization and encapsulation (honti et al., 2013). approximately 90%-95% of circulating hemocytes are plasmatocytes that are responsible for phagocytosis of bacteria and dead host cells (lanot et al., 2001; wertheim et al., 2005; strand, 2008). crystal cells are not adhesive in vitro, occupy ~5% of the total hemocytes, and can produce ppo in drosophila (strand, 2008; tang, 2009). lamellocytes are a type of large, flat, adhesive hemocyte (strand, 2008). prohemocytes quickly differentiate into lamellocytes when drosophila is infected by parasites or during the process of metamorphosis. in drosophila, lamellocytes are responsible for encapsulating large parasites and other foreign bodies (lanot et al., 2001). in larvae of b. mori, the 5 types of circulating hemocytes are prohemocytes, granulocytes, plasmatocytes, spherulocytes and oenocytoids (akai and sato, 1973; han et al., 1998; ling et al., 2003b; tan et al., 2013). prohemocytes are progenitor stem cells which can differentiate into other types of hemocytes according to light and electron microscopy observations (beaulaton, 1979; yamashita and iwabuchi, 2001). in the silkworm, most circulating hemocytes are granulocytes that have many granules inside the cytoplasm (ling et al., 2003b). this type of hemocyte can recognize invading pathogens, which are then subjected to phagocytosis or encapsulation, and these cells are also important for wound healing (akai and sato, 1973; gillespie et al., 1997; strand, 2008; tanaka and yamakawa, 2011). in the silkworm, oenocytoids are the largest type of hemocyte in the silkworm (akai and sato, 1973; ling et al., 2003b). they are easily classified using light microscopy because of their large size and opaque appearance when compared to other hemocytes (wago, 1991). plasmatocytes are smaller than oenocytoids but larger than the other hemocytes (akai and sato, 1973; ling et al., 2003b). this type of hemocyte can adhere to the surfaces of foreign entities very quickly upon contact by asymmetrically extending pseudopods (ling et al., 2003b). plasmatocytes are the main type of hemocytes that are responsible for encapsulation of foreign bodies (strand, 2008). spherulocytes in the silkworm have no ability to adhere. thus, they must be observed immediately after spreading on a glass slide (ling et al., 2003b). just like granulocytes, spherulocytes have many obvious large granules in the cytoplasm (akai and sato, 1973; ling et al., 2003b). consequently, most of the 5 types of circulating hemocytes in silkworm larvae can be readily identified if a researcher is familiar with their morphological properties. although d. melanogaster and b. mori are two typical insect models for studying hemocyte differentiation and development, there are a number of differences in hemocyte nomenclature between the two species. based on the phagocytosis function, drosophila plasmatocytes, which are phagocytotic, appear to be more similar to silkworm granulocytes than to silkworm plasmatocytes (ribeiro and brehelin, 2006). in the silkworm, plasmatocytes are normally larger than granulocytes and plasmatocytes are the major hemocyte type that takes part in encapsulation (strand et al., 2008). thus, plasmatocytes in the silkworm are not exactly the same as plasmatocytes in d. melanogaster and these two types of hemocytes have different cellular functions even though they have the same name (ribeiro and brehelin, 2006). lamellocytes are probably the equivalent of the silkworm plasmatocytes due to their capacity to adhere to foreign surfaces (ribeiro and brehelin, 2006). oenocytoids and spherulocytes are both non-adhesive hemocyte types. it is thought that spherulocytes may function to transfer necessary materials to the cuticle via epidermal cells (sass et al., 1994). the silkworm spherulocytes have no equivalent in d. melanogaster (ribeiro and brehelin, 2006). oenocytoids are considered to be the main type of hemocyte that can produce ppo in lepidopteran insects (ashida and dohke, 1980; iwama and ashida, 1986; jiang et al., 1997; ling et al., 2005a). crystal cells in drosophila show strong similarities both in structure and function with silkworm oenocytoids (ribeiro and brehelin, 2006). however, recent studies indicate that other types of hemocytes and even insect hindgut cells have ppo. therefore, it is necessary to be cognizant of these differences when investigating insect hemocytes. the primary method to identify and classify insect hemocytes is according to hemocyte morphological differences determined via histochemical observations and specific physiological functions (gupta, 1985; brehelin and zachary, 1986). cell morphology is the basic method which is performed using a variety of microscopy formats, including light, electron, fluorescence, confocal and differential interference contrast (dic) microscopy. fluorescent staining with chemicals like acridine orange (ao) and propidium iodide (pi) has been used to stain silkworm hemocytes for classification (ling et al., 2003b). after staining, each type of hemocyte has a specific fluorescence, which makes it easier to identify them using fluorescent microscopy (ling et al., 2003b; ling et al., 2005a). granulocytes have many granules inside their cytoplasm which exhibit strong green fluorescence (ling et al., 2003b; ling et al., 2005a). spherulocytes also have many green granules after     103 http://www.ncbi.nlm.nih.gov/pubmed?term=breh%c3%a9lin%20m%5bauthor%5d&cauthor=true&cauthor_uid=16527302 http://www.ncbi.nlm.nih.gov/pubmed?term=breh%c3%a9lin%20m%5bauthor%5d&cauthor=true&cauthor_uid=16527302 http://www.ncbi.nlm.nih.gov/pubmed?term=breh%c3%a9lin%20m%5bauthor%5d&cauthor=true&cauthor_uid=16527302 http://www.ncbi.nlm.nih.gov/pubmed?term=breh%c3%a9lin%20m%5bauthor%5d&cauthor=true&cauthor_uid=16527302 http://www.ncbi.nlm.nih.gov/pubmed?term=breh%c3%a9lin%20m%5bauthor%5d&cauthor=true&cauthor_uid=16527302   staining with ao and pi. however, the green granules inside spherulocytes are much larger than those inside granulocytes (ling et al., 2003b; ling et al., 2005a). prohemocytes and plasmatocytes cannot be positively stained by ao, but their long and irregular shapes make them easily identifiable without such staining. oenocytoids can be stained by pi to show red nuclei (ling et al., 2003b). neutral red is also a good staining solution for the differentiation of hemocytes in m. sexta because these hemocytes exhibit different properties after staining (ling and yu, 2006b). monoclonal antibodies against specific types of hemocytes in m. sexta and pseudoplusia includens have been created and used for hemocyte identification, which is a novel way to study hemocyte development, differentiation and function (gillespie et al., 1997; gardiner and strand, 1999; levin et al., 2005). using monoclonal antibodies against manduca granulocytes and plasmatocytes separately, granulocytes were found to specifically phagocytose apoptotic cells while plasmatocytes phagocytosed injected fluorescent beads (ling and yu, 2006b). in drosophila, cell markers like antibody against p1 antigen (an uncharacterized surface marker) to differentiated plasmatocytes was also very useful (strand, 2008). thus, for future studies, it is important to find additional specific markers to label insect hemocytes and aid in identification. although there are diffferent methods that can be used to identify insect hemocytes as described above, most of them are not very convenient. for example, the use of monoclonal antibodies is inconvenient to identify large numbers of hemocytes within a short time frame, since this method requires fixation, washing and staining. there also are a number of hemocytes that are difficult to differentiate from each other using light microscopy (akai and sato, 1973; gillespie et al., 1997). for example, the younger granulocytes that contain very few granules are difficult to distinguish from prohemocytes, making it hard to classify them as a granulocyte or prohemocyte. also, the younger plasmatocytes that have no obvious pseudopods are similar to young granulocytes and/or prohemocytes (ling et al., personal observations). all in all, it is necessary to develop additional methods that provide convenience and precision in identifying hemocytes. just like other cells, hemocytes also undergo cell death, like necrosis and apoptosis, and insect hemocytes have been observed undergoing apoptosis (liu and ling, personal observation). however, hemocytes also can phagocytose apoptotic cells, making it difficult to distinguish them from apoptotic hemocytes. for example, in the silkworm and manduca, many hemocytes can phagocytose apoptotic bodies (ling et al., 2003b; ling and yu, 2006b). those hemocytes containing phagocytosed apoptotic bodies might be misleadingly considered as undergoing apoptosis if their nuclei are not counterstained by dapi (4',6-diamidino-2-phenylindole) when the tunel (terminal deoxynucleotidyl transferase dutp nick end labeling) method is used for identifying apoptosis. hematopoietic organs and hematopoiesis in insects, hematopoietic organs are the primary source of hemocytes (akai and sato, 1971; gillespie et al., 1997; strand, 2008). however, in different species of insects, there are likely many differences in hematopoietic organs and even hematopoiesis. in d. melanogaster, hemocytes are produced in two different ways with each occurring during different developmental stages (strand, 2008). when drosophila is still an embryo, some cells in the head or dorsal mesoderm differentiate into blood progenitors, and this process is under the control of transcription factors (grigorian et al., 2011). during the larval stage, hemocytes are produced by lymph glands, which are the drosophila hematopoietic organ. these lymph glands are located bilaterally along the anterior part of the dorsal vessel (jung et al., 2005). in lepidopteran larvae, hematopoietic organs and hematopoiesis are very different from drosophila. in the silkworm, there are four hematopoietic organs that are located in the mesoand meta-thorax (akai and sato, 1971; ling et al., 2003a). each hematopoietic organ is tightly attached to a wing disc, and the two anterior hematopoietic organs are larger than the two posterior ones (akai and sato, 1971; ling et al., 2003a; ling et al., 2006). these hematopoietic organs are circled by a layer of an acellular sheath, which is broken during the larval wandering stage to release hemocytes (akai and sato, 1971; han et al., 1998). within the organs many new hemocytes are produced inside the islets (akai and sato, 1971; ling et al., 2003a). hemocytes produced inside the hematopoietic organs are continuously released into the hemolymph during the larval stages. at the same time, circulating hemocytes, derived from the embryo, can still proliferate and differentiate into other types of hemocytes (akai and sato, 1971; gillespie et al., 1997; gardiner and strand, 2000; nardi, 2004; strand, 2008), which contributes to hematopoiesis. recently, an in vitro assay has demonstrated that the neighboring wing discs and fat body tissue are also important to hematopoiesis (wang et al., 2010), which has not been reported with drosophila. therefore, as the typical lepidopteran insect, the silkworm has a different type of hematopoiesis than does drosophila. as mentioned above, although insect hematopoietic organs are very important to hematopoiesis, hemocytes can also be produced through self cell division. in the silkworm, when the four larval hematopoietic organs were destroyed by pinpointed heavy ion beams or were physically extirpated via a surgical operation, the silkworm larvae did not die as predicted (ling et al., 2003c). very surprisingly, the number of circulating hemocytes in larvae with the four hematopoietic organs destroyed still increased during the wandering stage (ling et al., 2003c). when cell division of circulating hemocytes was studied, increased hemocyte division was observed in larvae with hematopoietic organs destroyed or extirpated as compared to naive larvae (ling et al., 2003c). these results indicate that cell division of circulating     104   hemocytes can contribute considerably to the total amount of hematopoiesis (ling et al., 2003c; tan et al., 2013). also, when the silkworm hematopoietic organs were targeted by heavy ion beams and were seriously damaged, the organs regenerated later via the assistance of circulating hemocytes (ling et al., 2003a). based upon the hypothesis that circulating hemocytes might enter the damaged hematopoietic organs to remove dead cells, silkworm larvae were injected with fluorescent beads, which served as a tracer to distinguish circulating hemocytes that phagocytosed the beads from hemocytes present in the hematopoietic organs. after an initial injection of fluorescent beads into larvae to label circulating hemocytes, the hematopoietic organs of these larvae were heavy ion beam irradiated. it then was demonstrated that circulating hemocytes containing phagocytosed fluorescent beads had entered the targeted hematopoietic organs in order to phagocytose and clear the dead cells (ling et al., 2006). subsequently, the invading hemocytes remained in the irradiated hematopoietic organs to induce the targeted organs to regenerate (ling et al., 2006). hormones may also affect insect hematopoiesis (ling et al., 2003c). silkworm hemocyte density increases abruptly during the wandering stage and then decreases during the spinning and prepupal stages (ling et al., 2003c). when silkworm larvae were injected with 20-ecdysone (20-e), hemocyte density significantly increased at approximately 12-18 h post injection (ling et al., 2003c). however, the application of a juvenile hormone analogue (methoprene) to the injected silkworm larvae kept the hemocyte level stable without an obvious change (ling et al., 2003c). in summary, hemocytes produced by hematopoietic organs are the main source of circulating hemocytes in the silkworm. however, hemocyte division in the circulation may also contribute considerably to hematopoiesis if the hematopoietic organs are destroyed, which is unheard of for mammalian hematopoiesis. during the hematopoietic process, hormones like 20-e and jh, also may take part in the regulation of hematopoiesis (ling et al., 2003c). hemocyte differentiation in the silkworm hematopoietic organs, a previous study demonstrated that there are prohemocytes, plasmatocytes, granulocytes and oenocytoids based upon electron microscopy observations (akai and sato, 1973). however, in the hematopoietic organs of spodoptera frugiperda and m. sexta for example, there are primarily prohemocytes and plasmatocytes (gardiner and strand, 2000; nardi et al., 2003; strand, 2008). in the silkworm, prohemocytes appear to differentiate into plasmatocytes in the hematopoietic organs before release (beaulaton, 1979). subsequently, in the circulation of the silkworm, these plasmatocytes then differentiate into granulocytes, spherulocytes and oenocytoids (beaulaton, 1979). obviously, there are still disagreements in regards to hemocyte differentiation even within one insect species of insect (like the silkworm) if the studies were performed in different labs. therefore, it is necessary to discover new methods and techniques to study hemocyte differentiation in order to minimize discrepancies in hemocyte descriptions. one such method to study hemocyte proliferation and differentiation is the use of tissue culture. when silkworm hematopoietic organs were cultured in grace’s medium supplemented with 10% heated silkworm plasma, large numbers of hemocytes were released during the process (nakahara et al., 2003; wang et al., 2010). after several days of tissue culture, most newly released hemocytes were plasmatocytes and prohemocytes (nakahara et al., 2003). but when using ao and pi to stain hemocytes immediately after the silkworm hematopoietic organs were physically split open to release hemocytes, we found there were mainly prohemocytes (60%-70%) and oenocytoids (30%-40%) (ling et al., 2005b). during the process of transient cell culture, prohemocytes newly released from silkworm hematopoietic organs could transform into granulocytes and plasmatocytes (ling et al., 2005b). yamashita and iwabuchi (2001) separated silkworm prohemocytes from other circulating hemocytes in a single cell culture and observed that prohemocytes can differentiate into plasmatocytes and granulocytes, and granulocytes can differentiate into spherulocytes. however, circulating prohemocytes did not differentiate into oenocytoids in vitro (yamashita and iwabuchi, 2001). in another study, circulating oenocytoids from p. includens and s. frugiperda were not labeled by 5-bromo-2-deoxyuridine (brdu), which indicated that circulating oenocytoids cannot proliferate via cell division (gardiner and strand, 2000). therefore, oenocytoids in lepidopeteran insects are likely derived only from hematopoietic organs. ppo is produced not only by hemocytes in insects, hemocytes have been considered as the only source of ppo (ashida and brey, 1998). however, recent studies have shown that there is also ppo in the hindgut and wing discs of the silkworm (diao et al., 2012; shao et al., 2012). therefore, it is necessary to address this topic and briefly summarize this research because it is important to know whether the ppo that is present in those tissues has a close relationship with circulating hemocytes, and whether ppo in the wing discs and/or hindgut can induce melanization as occurs in the hemocoel and that involves the participation of hemocytes (ashida and brey, 1998). ppo-positive hemocyte identification in most insects, oenocytoids can produce ppo (ashida and brey, 1998; hillyer and christensen, 2002; hillyer et al., 2003). since ppo has no signal peptide, it is thought to be released after cell lysis (ashida and brey, 1998). as described above, ppo is a good protein marker for identifying hemocytes that can produce ppo. the traditional methods to identity ppo-positive hemocytes are to use immuno-staining or in situ methods to detect ppo protein or transcription within hemocytes (jiang et al., 1997; ashida and brey, 1998). however, due to the absence of antibodies against insect ppo and other     105   limitations, ppo-positive hemocyte identification has not been extensively tried in most insects. yet there could be some simple methods to identify ppo-positive hemocytes, if hemocytes containing ppo can first be activated followed by staining using a substrate like l-dopa or dopamine. in insects, ppo is activated by the regulation of a cascade composed of several serine proteases and serpins (ashida and brey, 1998; kanost et al., 2004; jiang et al., 2010). however, many chemicals like the detergent sodium dodecyl sulfate (sds), cetylpyridinium chloride (cpc) and ethanol also can activate ppo by an unknown mechanism (ashida and brey, 1998). researchers have utilized this property to identify ppo-positive hemocytes in insects. since ethanol can activate insect ppo, a mixture containing 35% ethanol and l-dopa was used to activate ppo inside silkworm hemocytes for staining, by which many more types of hemocytes were found to contain ppo (ling et al., 2005a). using the same method, ling and yu (2005) found that most ppo-positive hemocytes were oenocytoids and a few spherulocytes and granulocytes also contained ppo in m. sexta. further studies indicated that 10% of hemocytes exhibited surface ppo (ling and yu, 2005), but oenocytoids showed no surface ppo (ling and yu, 2005). in m. sexta, oenocytoids were also found to encapsulate immulectin-2 coated agarose beads when a mixture containing ethanol and dopamine was used to identify ppo-positive hemocytes (ling and yu, 2006a). this was the first study to show that oenocytoids may take part in encapsulation. using the same methods that were performed to identify ppo-positive hemocytes in the silkworm and manduca, hemocytes that have ppo were also identified in mosquitoes. hillyer and colleagues (hillyer et al., 2003) used a formaldehyde solution (4%) to fix hemocytes followed by staining with a 2 mg/ml l-dopa solution, which resulted in the identification of ppo-positive hemocytes in the mosquitoes, aedes aegypti and armigeres subalbatus. in the mosquito culex pipiens quinquefasciatus, ppo-positive hemocytes were identified in larvae and pupae, and many more types of hemocytes were found to have ppo (wang et al., 2011). very interestingly, ppo-positive plasmatocytes were found only in larvae, and blood-feeding could specifically induce different types of ppo-positive hemocytes in adult female mosquitoes (wang et al., 2011). we are unsure as to why ppo-positive hemocytes in mosquitoes are so complicated and whether those ppo-positive hemocytes may be involved some specific innate immunity responses, like phagocytosis or encapsulation. these methods of activating ppo within cells should be tried with other insects in order to obtain a more complete picture of ppo-positive hemocytes in different insect species. ppo in the wing disc: a source of plasma prophenoloxidase in the silkworm in the silkworm, each hematopoietic organ is attached to a wing disc (akai and sato, 1971; han et al., 1998; ling et al., 2006). when wing discs were separated from hematopoietic organs for tissue staining and western blot assays, all results indicated that the silkworm wing discs contain ppo that can be released into the culture medium after a period of tissue culture (diao et al., 2012). this result was also verified by lc-ms/ms after the band containing ppo was excised for protein identification (diao et al., 2012). ppo protein was also found in the hindwing of tribolium castaneum (dittmer et al., 2011) and an in situ assay showed that a few free cells in the cavity of the wing disc contained ppo1 and ppo2 mrna (diao et al., 2012). silkworm wing discs are connected with the hematopoietic organs through many tube-like materials (ling et al., 2006). those cells in the cavity of the silkworm wing disc are likely hemocytes that were released from the hematopoietic organs and made their way to the disc via the tube-like connections. when ppo-positive hemocytes were broken within the wing disc, ppo was released into the wing discs. therefore, ppo released via the wing disc may contribute to the hemolymph ppo (diao et al., 2012). ppo is produced by epidermal cells in the hindgut a very familiar phenomenon is that silkworm larvae feed on green mulberry leaves but excrete black feces. various methods, including ppo activity staining, immuno-staining, western blot and in situ assays, were performed to show that the large epidermal cells and many small cells in the hindgut can produce ppo and release it into the hindgut lumen (shao et al., 2012). when ppo activity was inhibited by giving larvae phenylthiourea (ptu), the silkworm larvae excreted green feces. however, large amounts of bacteria then easily reproduced in the green feces. on the contrary, bacteria in the black feces were found to be dead (shao et al., 2012). this study demonstrated that, besides hemocytes, silkworm hindgut cells can also produce ppo in order to remove microorganisms in the feces through melanization, thereby avoiding contamination of the mulberry leaves and the surrounding living environment. utilization of drosophila macrophage-like cell line s2 cells to study ppo structure and activity to date, we know very little about the relationship of ppo structure and enzyme activitiy. drosophila has three ppo genes and when all three drosophila ppo genes were over-expressed in s2 cells, different biochemical properties of the three ppos were found (liu et al., 2012). ppo1 and ppo2 need additional cu2+ in order to subsequently become activated by ethanol and this was demonstrated by native gel separation of samples with or without cu2+ added during cell transfection. for ppo3, it has enzyme activity directly (i.e., auto-activation), without the need for activation by either ethanol or any other method, after over-expression in s2 cells. ppo3 also is able to induce auto-melanization if additional cu2+ is added (liu et al., 2012). when the predicted ppo3 structure was compared with those of ppo1 and ppo2, results showed that two key amino acids, around the active site pocket of ppo3, occupy less space than the comparable amino acids in ppo1 and ppo2. these two key amino acids in ppo3 make the entrance pocket larger such that there is     106   gap between the placeholder and the entrance, thereby permitting auto-activation since substrate can enter at any time (chen et al., 2012). when the two key amino acids in ppo3 were mutated to make the active site pocket smaller so that the placeholder occupies the entrance of the active site pocket without a gap, the mutant ppo3 acts like ppo1 and ppo2 and needs to be activated either by ethanol or serine protease (chen et al., 2012). conclusions in most insects, there are several types of circulating hemocytes (gillespie et al., 1997; lavine and strand, 2002; strand, 2008) and these hemocytes have been the focus of research on cell development and differentiation. insect hemocytes are important factors in innate immunity (gillespie et al., 1997; lavine and strand, 2002; strand, 2008). they can produce many immune proteins like antibacterial peptides and ppo (gillespie et al., 1997; ashida and brey, 1998; kanost et al., 2004; jiang et al., 2010). in addition, hemocytes are directly involved in cellular immunity like phagocytosis, encapsulation and nodule formation (gillespie et al., 1997). in the field of entomology, hemocytes are an attractive research arena for many scientists. using the silkworm as a model, scientists have been extensively studying hemocyte identification, development, differentiation and cellular immune responses. silkworm larvae have five types of circulating hemocytes that are produced by hematopoietic organs and released into the hemolymph. however, several types of circulating hemocytes also can proliferate through cell division, which contributed significantly to hematopoiesis after the larval hematopoietic organs were extirpated via a surgical procedure. novel types of hematopoiesis in the silkworm make this insect species a valuable model to study hemocyte-related activities in the future. now with the silkworm genome available (xia et al., 2004), we anticipate being able to explore insect hemocyte immune activities at the molecular level. acknowledgements this work was supported by the national basic research program of china (2012cb114605), national natural science foundation of china (31172151). references akai h, sato s. an ultrastructural study of the haemopoietic organs of the silkworm, bombyx mori. j. insect physiol. 17: 1665-1676, 1971. akai h, sato s. ultrastructure of the larval hemocytes of the silkworm, bombyx mori l. 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inhibition and micronucleus frequency in oysters (crassostrea corteziensis) exposed to chlorpyrifos ab benitez-trinidad1, yy bernal-hernández2, cl moreno-hernández2, im medina-díaz2, ml robledo-marenco2, bs barrón-vivanco2, d domínguez-ojeda3, ca romero-bañuelos2, mi girón-pérez2, ae rojas-garcía2 1posgrado en ciencias biológico agropecuarias y pesqueras, unidad académica de agricultura 2laboratorio de contaminación y toxicología ambiental, universidad autónoma de nayarit (uan), av. de la cultura s/n, col. los fresnos, cp 63190 tepic, nayarit, méxico 3escuela nacional de ingeniería pesquera, uan, bahía de matanchén km. 12, san blas, 63740 nayarit, méxico accepted september 15, 2014 abstract chlorpyrifos (cpf) is an organophosphorous pesticide (op) that has been widely used for both agricultural and domestic pest control. to date, there is little information regarding the effects of this pesticide on aquatic organisms, particularly oysters. the aim of this study was to evaluate acetylcholinesterase (ache) activity and micronucleus (mn) frequency in the oyster crassostrea corteziensis in laboratory exposure with cpf (20, 40, 60, 80, and 160 µg/l) and in a field study. the results showed that ache was reduced 60 82 % in oysters exposed to cpf, relative to the negative control. similar ache results were observed in oysters collected from the boca de camichín estuary in nayarit, mexico; with respect to genetic damage, evaluated through mn, treatment with cpf did not induce the mn frequency, nor did the oyster from the field study exhibit an increase in this biomarker. these results suggest that c. corteziensis is a sensitive model for evaluating the acute toxicity of op in laboratory studies as well in the field. in addition, it generates prospects on studying mechanisms through which the oyster could possess resistance to genotoxic agents, as well as its being a reliable model for evaluating the genotoxic effects of xenobiotics through the mn technique. key words: acetylcholinesterase; micronucleus; crassostrea corteziensis; chlorpyrifos   introduction marine pollutants have consequences at multiples biological levels (mix, 1986; malins et al., 1988; bolognesi, 1990; gopal and pathak, 1993) and have been traditionally documented in terms of chemical concentrations of contaminants; however, these measurements do not provide estimations of harmful effects on living organisms and are now complemented with the measurement of additional biomarkers that are indicative of such effects. biomarkers represent integral and measurable biochemical and physiological changes in organisms exposed to contaminants, thus indicating initial responses to environmental perturbations and contamination (mccarthy and shugart, 1990; bengtson and henshel, 1996; roy et al., 1996). for ___________________________________________________________________________ corresponding author: aurora elizabeth rojas-garcía laboratorio de contaminación y toxicología ambiental secretaría de investigación y posgrado universidad autónoma de nayarit cp 63155 tepic, nayarit, méxico e-mail: erojas@uan.edu.mx example, the measurement of acetylcholinesterase (ache) activity is widely used to evaluate the exposure and effects of anti-ache compounds (galgani and bocquené, 2000). ache is the enzyme responsible for the hydrolysis of the neurotransmitter acetylcholine into choline and acetic acid in the nerve synapse. ache inhibition is directly linked with the action mechanism of the organophosphorus (op) and carbamate pesticides that block the action of this enzyme (reigart and roberts, 1999). measurement of ache activity is widely employed in several species, including aquatic species (cajaraville et al., 2000; singh and sharma, 2005; bernal-hernández, et al., 2010). the rapid increase in the production and use of op and carbamate pesticides has raised concerns regarding their potential negative effects on humans and non-target wildlife populations. pesticides enter waterways from agricultural and urban run-off, groundwater discharge, and after direct application (schulz and leiss, 1999), and may be transported to estuaries and coastal waters (magni et al., 2006; ismail et al., 2014). 247 fig. 1 map of the boca de camichín estuary showing the three sampling stations. s1: station 1; s2: station 2, and s3: station 3. chlorpyrifos (cpf) is an op utilized for domestic and agricultural pest control. although cpf is a pesticide used worldwide, there is little information concerning its acute and chronic effects on aquatic organisms, particularly oysters. some reports have suggested that cpf has the potential to cause neuroand genotoxic effects in aquatic organisms, such as inhibition of ache and the formation of micronuclei (mn), respectively (shugart, 1995). in some cases, the formulated pesticides are found to be more toxic than the active ingredient, particularly to aquatic organisms (ali et al., 2009). mn frequency is one of the most widely used genotoxicity biomarkers in aquatic organisms. mn are small intracytoplasmic masses of chromatin resulting from chromosomal breakages during cell division. an increase in the frequency of micronucleated cells is thought to result from chromosomal and genomic damage caused by clastogens or spindle poisons (bolognesi and fenech, 2012). at present, the mn assay is applied in laboratory and field studies using hemocytes and gill cells from bivalves, or simultaneously in both cell types (barsiene et al., 2006; bolognesi and fenech, 2012). however, few studies have reported mn frequency in oysters. the oyster crassostrea corteziensis is a species native to the eastern tropical pacific where it is naturally distributed in mangrove zones from mexico to peru (stuardo and martínez, 1975). this species supports artisanal and commercial fisheries in northwestern mexico. the boca de camichín estuary situated in nayarit state is one of the largest oyster producers in mexico. this ecosystem is important not only for its oyster production, but also for its biologic diversity. high agricultural activity around the estuary is reported, with elevated use of pesticides. one of the most utilized insecticides in the ecosystem is cpf (gonzález-arias et al., 2010; rojas-garcía et al., 2011). therefore, the aim of this study was to determine ache activity and mn frequency in order to evaluate the degree of exposure to cpf and the integrity of genetic material, respectively, in vivo and in field study. materials and methods sampling collection for in vivo and in-field study, crassostrea corteziensis oysters approximately 4 8 cm in shell length were collected at the boca de camichín estuary in nayarit state, mexico. this area is characterized by its biological diversity and in part to the high-priority national wetlands (robledomarenco et al., 2006). three sampling sites at the estuary were chosen (fig. 1), taking into account the hydrographic and biological characteristics of the system. one site was localized toward the head of the estuary (s1: 21º44′06.8″n, 105º29′19.06″), another station was localized downstream and near 248 the mouth of the estuary (s2: 21º44′53.0″n, 105º29′39.0″w), and one was situated in the middle of the oyster-culture zone (s3: 21º45′41.1″n, 105º29′43.5″). the oysters were collected at each of the three sites at two levels: from the upper and the lower part of the oyster string. gills were dissected immediately and frozen until use to screen for total protein, ache activity, and mn frequency. exposure to cpf under in vivo conditions the oysters were carried to the laboratory, placed in 40-l tanks filled with filtered seawater, and acclimated for 15 days, during which time they were fed 2 l/day of microalgae species: chaetoceros spp. (6×105 cells/ml). for exposure to cpf, oysters were placed in aquariums with 10 l of aerated water and were acclimated to estuary conditions (26 ‰; 32 ± 1 ºc). ten organisms were utilized for each treatment. the organisms were divided into seven lots as follows: one for negative control (maintained under the same condition, but without treatment), one for positive control (treatment with mitomycin c as mn control) and five for cpf treatments (20, 40, 60, 80, and 160 µg/l). the organisms were incubated in 12-h:12-h dark-light cycles for > 96 h. each experiment was run in triplicate. at the end of exposure, the organisms were washed, opened, and the gills were excised to screen for total protein, ache activity, and mn frequency. for the present study, cpf (44.5 % emulsifiable concentrate [ec]) with the magnum l-480 brand was employed. total protein content and ache activity gills were homogenized (1:2 w/v) in an extraction buffer (20 mm tris-base, 1 mm edta, 1 mm dtt, 500 mm sucrose, 150 mm kcl, 0.1 mm pmsf, ph 7.6) (monserrat et al., 2002). the homogenate was centrifuged at 9,000 rpm for 30 min at 4 °c. the pellet was discarded and the supernatant (s9 fraction) was used to determine protein and ache activity. total protein was determined in fraction s9 according to the lowry method (lowry et al., 1951) using bovine serum albumin (bsa) as the standard. ache activity was performed according to ellman et al. (1961), modified by monserrat et al. (2002). enzyme activity was determined in triplicate using 300 µl of supernatant, 2700 µl of phosphate buffer (ph 8, 0.1 m), 100 µl of 0.5 mm ellman’s reagent (dtnb), and 20 µl of 0.5 mm acetylthiocholine iodide as substrate. the rate of absorbance change at 412 nm was recorded over 120 sec at 25 °c using a spectronic genesys 10 bio spectrophotometer (genesys, wi, usa). micronucleus (mn) determination cell preparation this technique was based on the methodology described by bolognesi et al. (1999) with some modifications. to prepare the cell suspension, gills were excised and cut into fragments with dissection scissors in 5 ml of saline solution. the cell suspension was centrifuged at 1,500 rpm at 25 °c for 5 min and then fixed in 5 ml of carnoy’s fixative solution (cfs) (3:1 methanol:acetic acid) for 20 min. afterward, the cell suspension was centrifuged and washed three times with cfs. the pellet was resuspended in 1 ml of cfs and treated with 1 ml of trypsin (0.00015 %). trypsin activity was blocked by the addition of 5 ml of cfs. the cell suspension was centrifuged and washed twice with cfs. finally the samples were resuspended in 1 ml of cfs and were maintained at 4 ºc until slide preparation. exposure to mitomycin c as positive control mitomycin c (mmc) is a dna cross-linking agent that has been extensively used as a reference mutagen in numerous mn studies (das and nanda, 1986; majone et al., 1987; scarpato et al., 1990, williams and metcalfe, 1992). oysters were placed in aquariums with 10 l of water (26 ‰). prior to the exposure, they were acclimatized for 24 h. subsequently, a group of 10 organisms with one replicate were exposed to mmc (2.5 mg/l) for 48 h. at the end of treatment, the oysters were sacrificed and their gills were separated to determine the presence of mn. the negative control group was run with one replicate. fig. 2 representative gill cells from crassostrea corteziensis (a) and a micronucleated gill cell (b). the micronucleus (mn) is indicated by an arrow beside the main nucleus. staining with the merck hemacolor kit (100x). 249 fig. 3 total protein content (a) and remaining acetylcholinesterase activity in oyster (crassostrea corteziensis) gills (b) exposed to different chlorpyrifos concentrations for 96 h. data are expressed as means ± standard deviations (sd) of three independent determinations. nc, negative control. the asterisk (*) indicates a significant difference from control animals (nc) at p < 0.05. criteria for scoring mn mn were identified according to following criteria described by fenech et al. (2003): (1) round and ovoid-shaped nonrefractory particles in the cytoplasm; (2) chromatin-like color and structure; (3) diameter of 1/3 1/20 of the main nucleus, and (4) particles completely separated from the main nucleus. figure 2 shows the cell morphology and micronucleated cell observed in the mn test. slides were analyzed by light microscope (carl zeiss, axiostar plus, göttingen, germany) at a magnification of 100x for nuclear abnormalities (mn) and 1,000 gill cells were scored. only intact cells were scored. all mn were checked by a second operator to ensure that unambiguous micronucleated cells were exclusively scored. statistical analysis the results were expressed as the average of three independent experiments. the results were analyzed by mann-whitney u test and dunn multiple comparison tests. p values < 0.05 were considered statistically significant. statistical analyses were conducted using the stata ver. 8.0 program (stata statistical software; stata corporation, college station, tx, usa) and graphpad prism 5.01 for windows (graphpad software, san diego, ca, usa). results and discussion the treatments with cfp did not cause mortality in the oysters, but we observed that the organisms closed their valves for 12 h. there are reports that suggest that these oysters are able to isolate themselves from a contaminated environment by closing their valves (kramer et al., 1989; mersch et al., 1993). in addition, in bivalve molluscs, contact with toxic compounds causes a reduction in filtration activity; thus, this might reduce exposure time (van der gaag et al., 1990; wrisberg et al., 1992). total protein content and ache activity the results showed that c. corteziensis oysters exposed to 80 and 160 µg/l had the highest protein concentrations (8.50 and 12.77 mg/ml, respectively) compared with those of the negative control; the opposite was observed at 20 and 40 µg/l (2.32 and 2.41 mg/ml) (p < 0.05) (fig. 3a). in addition, significant variations in remaining ache activity of gills exposed to different concentrations of cfp 250 fig. 4 total protein content (a) and remaining acetylcholinesterase activity (b) in crassostrea corteziensis oysters collected from the boca de camichín estuary. data are expressed as means ± standard deviations (sd) of three independent determinations. nc, negative control; s1, sampling site 1; s2, sampling site 2; s3, sampling site 3. u: upper part of string; l: lower part of string. the asterisk (*) indicates a significant difference from control animals (nc) at p < 0.05. during 96 h were recorded (fig. 3b). at the highest cfp treatments (80 and 160 µg/l), the remaining ache activity was 40 and 18 % with respect the control group, which confirms the inhibitory effects of cfp on oyster exposure. proteins and other oyster components vary considerably among species and among individuals of the same species, also depend on age, gender, size, environment, and season of the year, and are closely related with food organisms (maedamartínez et al., 2001). in this regard, average total protein content in the gills of oysters in this study was 5.93 mg/ml in cpf-treated organisms and 4.59 mg/ml in the negative control group. it has been reported that protein concentrations in the tissues of gills can be affected both positively and negatively by stress-associated physiological changes, as well as by the presence of chemicals (rank et al., 2007). on the other hand, in the field study no changes in protein content in c. corteziensis oysters was observed (fig. 4a), but all of the organisms collected at the estuary exhibited lower ache activities with respect the negative control, as illustrated in figure 4b; this indicates the presence of anticholinesterase compounds in the estuary and, possibly, op pesticides. oysters have been proposed as bioindicators of aquatic pollution, in addition to mussels (bebianno et al., 1993, 1994; viarengo et al., 1998; blasco and puppo, 1999; rodríguezortega et al., 2001), and data regarding the measurement of ache activity in oyster tissues are available in the literature (mora et al., 1999; dellali et al., 2001; bernal-hernández et al., 2010). previous studies suggest that ache activity is found in the gills, mantle, and adductor muscles of bivalves (bocquene et al. 1997; escartín and porte 1997; radenac et al., 1998; mora et al., 1999; monserrat et al., 2002; damiens et al., 2004). gills were chosen for measurement of esterase activities because gill tissue exhibited high activity levels and higher sensitivity to contaminants with respect to those of other tissues (mora et al., 1999). also, oyster gills are easy to dissect and have a relatively important and constant mass (apparently independent of field conditions) (mora et al., 1999). 251 fig. 5 micronucleus frequency (mn) in gill cells of the oyster crassostrea corteziensis exposed to chlorpyrifos for 96 h (a) and oysters collected in the boca de camichín estuary (b). values represent the means ± standard deviations (sd) of three independent experiments performed in triplicate. nc, negative control; pc, positive control: organisms exposed to 2.5 mg/ml of mitomycin c (mmc). s1, sampling site 1; s2, sampling site 2; s3, sampling site 3. u: upper part of string; l: lower part of string. the p value was determined by the mann-whitney u test and statistical significance was set at p < 0.05. micronuclei frequency mn frequencies in the different cpf treatments, as well as the control organisms, are depicted in figure 5a. in this study, we observed a low incidence of micronucleated gill cells in cpfexposed c. corteziensis, and no statistically significant differences in mn frequencies were observed between control organisms and those exposed to cpf (p > 0.05). on the other hand, in oyster gill tissues taken from the different sample sites at the boca de camichín estuary, none had significant differences in mn number with respect to that of the negative control (fig. 5b). our results agree with those reported in an oyster-farming area contaminated by cadmium and copper (burgeot and galgani, 1995). these authors found that mn frequency was not very sensitive to a pollution gradient and that it exhibited high interindividual variability. these results are similar to those reported in other bivalves, such as macoma balthica and mytilus edulus (barsiené et al., 1996). regarding mn frequency in oyster species, few studies are available in the literature. in crassostrea gigas, burgeot et al. (1995) found a baseline mn frequency of 2.8, while in crassostrea virginica, weis et al. (1995) found a baseline mn frequency of 4.8. few works are available on baseline mn frequency in bivalve molluscs. in this regard, table 1 shows that baseline mn levels reported in the literature in this organism range from 0.54 5.7 mn/1,000 cells. surprisingly, mmc did not cause an increase in mn number in oysters. to confirm the quality of the mmc reagent, we conducted human lymphocyte cultures with significant mn induction results (data not shown). mmc is a known mutagenic and clastogenic agent. sister chromatid exchanges, chromosome aberrations, and mn comprise genetic effects that can be observed during exposure in vivo to this compound (rudd et al., 1991; salvadori et al., 1994). our findings are important because the genotoxic effect of this compound is widely demonstrated in other species, such as mussels and fish. in these species, a lower concentration of mmc than that employed in this work causes an increase of 30 50 % in the baseline mmc number (majone et al., 1989; grisolia, 2002); this was also observed in mammal species such as rodents (hayashi et al., 1992; holtz et al., 2003). we observed that c. corteziensis oysters collected from the boca de camichín estuary had ache-level activity similar than that of oysters 252 table 1 micronucleus (mn) frequency in bivalve organisms nd: not determined; sd: standard deviation; pcb: polychlorinated biphenyl; ddt: dichlorodiphenyltrichloroethane; hcb: hexachlorobenzene; hch: hexachlorocyclohexane; cr: chromium; ni: nickel. exposed under in vivo conditions to cpf, which caused marked inhibition of ache activity but did not induce the presence of mn. these results confirm that ache activity in c. corteziensis oysters can serve as a sensitive biomarker to assess exposure to op, such as cpf (bernal-hernández et al., 2010). on the other hand, these results generate perspectives regarding the mechanisms through which oyster could possess resistance to genotoxic agents, as well as whether this is a reliable model for evaluating the genotoxic effects of xenobiotics. organism tissue compound mn/ 1,000 cells sd reference mytilus galloprovincialis gill zinc chloride 2.2 nd majone et al., 1987 crassostrea gigas heart cells benzo[a]-pyrene 2.8 ±2.3 burgeot et al., 1995 dreissena polymorpha hemolymph gill clastogens: mitomycin c (mmc) bleomycin di-methylar-sinic acid potassium chromate mitomycin c (mmc) bleomycin dimethyl-arsinic acid potassium chromate 1.2 4.4 6.9 3.2 6.1 6.7 5.6 5.4 ±1.0 ± 1.8 ± 2.4 ± 1.1 ± 1.4 ± 3.1 ± 1.9 ± 2.6 mersch et al., 1996 mytilus trossulus gill pcb, ddt, hcb, hch, and polybrominated diethyl ethers (pbde) 6 nd kopecka et al., 2006 mytilus edulis gill crude oil 2.4 nd baršienė and andreikė-naitė, 2007 macoma balthica gill contaminated zone 1.2-3.63 nd baršienė et al., 2008 mytilus edulis gill contaminated zone 1.7-3.3 nd baršienė et al., 2008 mytilus galloprovincialis hemolymph and gills urban and industrial pollution 5.5-5.1 nd taleb et al., 2009 crassostrea corteziensis gill chlorpyrifos 1.2 ±1.1 this study cerastoderma edule hemolymph chemical contaminant 0.4 ±0.6 ruiz et al., 2013 mya arenaria hemolymph organotin compounds, arsenic, polyaromatic hydro-carbons (pah) and pcb 15.4 nd debenest et al., 2013 mytilus galloprovincialis digestive glands hg 2+ 2.3 nd pytharopoulou et al., 2013 ruditapes philippinarum gill pah and trace metals such as cr and ni 4–16 nd sacchi et al., 2013 253 acknowledgments the authors thank ing. ml loza and ml llamas garcía for their technical assistance during oyster collection and experimental work. this research was made possible through grants nayarit-2006-c0166170 and promep/103.5/07/2746. references ali d, nagpure ns, kumar s, kumar r, kushwaha b, lakra ws. assesment of genotoxic and mutagenic effects of chlorpyrifos in freshwater fish channa punctatus (bloch) using micronucleus assay and alkaline single-cell gel electrophoresis. food chem. toxicol. 47: 650656, 2009. baršienė j, dedonytėa v, rybakovasa a, andreikėnaitėa, l, andersenc ok. investigation of micronuclei and other nuclear abnormalities in peripheral blood and kidney of marine fish treated with crude oil. aquat. toxicol. 78: s99s104, 2006. baršiené, j, tapia g, barsyte d. chromosome of molluscs inhabiting some mountain spring of eastern spain. j. mollus. stud. 62: 539-543, 1996. baršienė j. andreikėnaitė l, induction of micronuclei and other nuclear abnormalities in blue mussels exposed to crude oil from the north sea. ekologija 53: 9-15, 2007. baršienė j, andreikėnaitė l, garnaga g, rybakovas a. genotoxic and cytotoxic e�ects in the bivalve mollusks macoma balthica and mytilus edulis from the baltic sea. ekologija 54: 44-50, 2008. bebianno mj, nott ja, langston wj. cadmium metabolism in the clam ruditapes decusata: the role of metallothioneins. aquat. toxicol. 27: 315334, 1993. bebianno mj, serafim map, rita mf. involvement of metallothionein in cadmium accumulation and elimination in the clam ruditapes decussata. bull. environ. contam. toxicol. 53: 726-732, 1994. bengtson da, henshel ds. environmental toxicology and risk assessment: biomarkers and risk assessment. vol. v. astm, stp 1306, west conshohocken, pa, 1996. bernal-hernández yy, medina-díaz im, robledomarenco ml, velázquez-fernández jb, girónpérez mi, ortega-cervantes l, et al. acetylcholinesterase and metallothionein in oysters (crassostrea corteziensis) from a subtropical mexican pacific estuary. ecotoxicology 19: 819-25, 2010. blasco j, puppo j. effect of heavy metals (cu, cd and pb) on aspartate and alanine aminotransferase in ruditapes philippinarum (mollusca: bivalvia). comp. biochem. physiol. 122c: 253-63, 1999. bocquene g, roig a, fournier d. cholinesterases from common oyster (crassostrea gigas). evidence for the presence of a soluble acetylcholinesterase insensitive to organophosphate and carbamate inhibitors. febs lett. 407: 261-266, 1997. bolognesi c. carcinogenic and mutagenic effects of pollutants in marine organisms: a review. in: grandjean e. 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oceanology, chinese academy of sciences, qingdao 266071, china accepted november 12, 2013 abstract zhikong scallop (chlamys farreri) and bay scallop (argopecten irradians), two major aquaculture molluscan species in china, are different in some important traits such as life cycle, growth performance and temperature tolerance. in the present study, the malondialdehyde (mda) content and four immune parameters including phagocytic activity, respiratory burst and activities of superoxide dismutase (sod) and acid phosphatase (acp) of scallops under heavy metal exposure and bacteria challenge were measured by flow-cytometric method and immunochemistry assays to compare the immunity of these two scallop species. in the non-treated scallops, the phagocytosis of hemocytes, mda content, the activities of sod and acp in hepatopancreas of bay scallops were all significantly higher than those of zhikong scallops. after challenged with vibrio anguillarum, the ros level in hemocytes of bay scallops was significantly lower than that of zhikong scallops at 30 min, and the cumulative mortality of bay scallop was also significantly lower than that of zhikong scallop at 2nd-5th day. the exposure of pb2+ with different concentration induced significantly higher phagocytic activity, acp activities, sod activities, mda content in hepatopancreas and significantly stronger respiratory burst in hemocyte of bay scallops compared with those of zhikong scallops, while the hemocyte mortalities in bay scallops were significantly lower than that in zhikong scallops. the results collectively indicated that bay scallops had a higher level of immune potential than zhikong scallops, suggesting its greater capacity for stress response and immune resistance against pathogens as well. key words: chlamys farreri; argopecten irradians; immune defense; phagocytosis; respiratory burst; enzyme activities; lead exposure introduction the bay scallop argopecten irradians and zhikong scallop chlamys farreri are both dominant aquaculture species in china (guo et al., 1999; zhang and yang, 1999). the bay scallop, a hermaphroditic bivalve distributed along the atlantic coast of united states, was introduced to china as alternative species for aquaculture in 1982. the zhikong scallop is a dioecious bivalve native to the coast of china, korea, russian and japan. after having flourished for several years, massive summer mortality of both species has become a major constraint for the development of scallop aquaculture (zhang and yang, 1999; xiao et al., 2005). although the cause of mortalities has not ___________________________________________________________________________ corresponding author linsheng song institute of oceanology chinese academy of sciences 7 nanhai rd., qingdao 266071, china e-mail: lshsong@ms.qdio.ac.cn been accurately identified, it is believed that the mortalities are related to the combination of the deteriorating water quality, excessive stocking densities, pathogen infection and stock degeneration from inbreeding (linked to the original hatchery seed). anthropogenic contaminants, such as heavy metals, may partly be responsible for the increase in disease incidence by adversely affecting immunity, thus enhancing susceptibility to infection (pipe and coles, 1995; wootton et al., 2003). numerous studies have demonstrated that heavy metals could affect the hemocyte functions in molluscs, such as cell viability, cytoskeletal organization and phagocytic activity (fagotti et al., 1996; olabbarietta et al., 2001; sauvé et al., 2002; duchemin et al., 2008). among all of the heavy metals, lead is a toxic metal whose widespread use has caused extensive environmental contamination and health problems in many parts of the world. pb2+ can be accumulated in bivalves and cause immunosuppression and even lead to mortality. 141 mailto:lshsong@ms.qdio.ac.cn fig. 1 hemocyte mortality of bay scallops and zhikong scallops exposed to pb2+ at different concentrations. vertical bars represent the mean ± sd (n = 3), and bars with different letters are significantly different (p < 0.05). vibrio anguillarum has been reported one of the bacterial pathogens affecting scallops (song et al., 1997; zhang et al., 1999). increasing evidence indicates that the mortalities of scallops sometimes appear to be species specific, and that bay scallops not only endure higher temperatures in contrast with zhikong scallops but also grow faster (zhang et al., 2000; xiao et al., 2005). it can be inferred that there might be some immune mechanisms different from each other, which inspires our interests in the comparison of immune parameters in these two species after lead exposure and bacteria challenge. given the continuing prevalence of summer mortalities, there is a growing realization that knowledge of immunity and disease susceptibility in these aquaculture animals is still inadequate (harvell et al., 1999; falco et al., 2009; li et al., 2013). in the present study, the comparison of immune parameters (hemocyte mortality, phagocytic activity, respiratory burst, superoxide dismutase and acid phosphatase activities), malondialdehyde content, and the mortality between scallops c. farreri and a. irradians was conducted to better understand the immune potential of these two scallops, to improve health management practices as well as allow more efficient control in scallop farming. materials and methods source of specimens, lead treatment and mortality observation zhikong scallops (chlamys farreri) and bay scallops (argopecten irradians) were collected from a commercial farm (qingdao, china), and acclimated in a free-flowing aquarium system at salinity 30 ± 0.1 ‰, temperature 18 ± 1 °c, dissolved oxygen above 6.0 mg l-1 and ph from 7.7 to 8.2 for two weeks before assays. the scallops were fed on isochrysis galbana parke which was in logarithmic-growth phase at excess ration one time per day (at 10:00). the seawater was changed 100 % daily to ensure high water quality. zhikong scallops were one year old, averaging 56 mm in shell length, and bay scallops were 5 months old, averaging 51 mm in shell length. for the lead treatment experiment, 320 scallops (160 of zhikong scallop; 160 of bay scallop) were divided into eight groups. two groups of 40 animals maintained in normal seawater were employed as control groups. six groups of 40 scallops were exposed to pbcl2 for a period of up to 10 days at final concentration of 0.2 mg l-1, 0.3 mg l-1 and 0.5 mg l-1, respectively, according to the previous study (zhang et al., 2010). simultaneously, another 90 scallops from each species were employed to evaluate cumulative mortality under bacteria challenge. v. anguillarum m3, kindly provided by dr. zhaolan mo, was employed as pathogen in the challenge experiment. the bacteria were cultured in 2216e broth (tryptone 5 g l−1, yeast extract 1 g l−1, c6h5fe·5h2o 0.1 g l −1, ph 7.6) at 28 °c overnight, and centrifuged at 2000 g for 5 min. the pellet was suspended in pbs and adjusted to 1×108 cfu ml−1. for each species, 90 scallops were randomly divided into three groups (30 for each group), and two groups received an injection of 200 µl v. anguillarum suspension (challenged group) and 200 µl of pbs (control group) into the adductor 142 fig. 2 percentage of cells showing phagocytosis in bay scallops and zhikong scallops exposed to pb2+ at different concentrations. vertical bars represent the mean ± sd (n = 3), and bars with different letters are significantly different (p < 0.05). muscles, respectively. the treated scallops were immediately returned to seawater tanks. another 30 untreated scallops were used as the blank group. dead scallops were removed immediately and cumulative mortality was calculated for both species. hemolymph and hepatpancreas collection for the assays of phagocytosis, hemocyte mortality and respiratory burst assays, about 200 μl hemolymph was collected from the pericardium of each scallop by using a 23-gauge needle attached to a 2-ml syringe containing 1 ml tbs anticoagulant solution (0.05 mol l-1 tris-hcl, ph 7.4; 2 % glucose; 2 % nacl; 20 mmol l-1 edta). the hemolymph from ten scallops was pooled together immediately as one sample, and then divided into several aliquots for the following assays. three samples collected from 30 individuals were analyzed for each species.the hepatopancreas were collected from 30 scallops of each species in the lead treatment experiment, and ten of them were pooled together as one sample. the samples were homogenized in glass homogenizers containing pbs buffer on ice (ph 6.41, 15 mmol l-1). each homogenate was centrifuged at 13,000 rpm at 4 °c for 1 h. the supernatants were collected and stored at -80 °c for sod, acp activities and mda content analysis. quantitative analysis of phagocytosis two microliters of fluorescent beads (fluoresbrite yg microspheres, 2.00 μm; polysciences, usa) were added to 1.5 ml of ultrafiltered (0.2 μm) seawater (fsw). four hundred microlitre of each pooled hemolymph sample was immediately centrifuged at 3,000 rpm at 4 °c for 10 min. after removing the supernatant, the hemocyte pellet was resuspended in 400 μl of fsw. then 200 μl of each suspension was mixed with the fluorescent bead suspension and incubated at 18 °c for 1 h in the dark and this reaction was terminated by adding 250 μl of baker’s formol solution (4 % formaldehyde, 2 % nacl). the samples were analyzed by flow cytometer (facs vantage, bd, usa) and the flow cytometry data was analyzed by flowjo software (tree star, ashland, or, usa). the fitc staining cells were considered as the candidates of phagocytic hemocytes, and the microscopic examination was used to ensure that beads were internalized instead for adhered to the cells. the hemocytes with fitc fluorescence were recorded as phagocytic cells, while the hemocytes without fitc fluorescence were considered as non-phagocytic cells. the percentage of phagocytic cells was calculated as the following. percent of phagocytic cells = [number of hemocytes engulfing fluorescent beads (phagocytic hemocytes) / total number of hemocytes] × 100 %. observation of hemocyte mortality hemocyte mortality was recorded according to delaporte’s protocol (2003) with some modification. a total of 400 µl pooled hemolymph from control and pb2+ treated groups was labeled with propidium iodide (pi, at the final concentration of 20 µg ml-1) and incubated in dark for 10 min before flow cytometric analysis. pi fluorescence was measured at wavelengths above 630 nm. the hemocyte mortality was calculated as the percentage of hemocytes that had incorporated pi fluorescence relative to total hemocyte counts. 143 http://igitur-archive.library.uu.nl/dissertations/2002-1025-114139/c3.pdf table 1 sod, acp activities and mda content in hepatopancreas of zhikong scallops and bay scallops under lead exposure sod (u/mg protein) mda (nmol/mg protein) acp (u/g protein) pb2+ concentration (mg/l) zhikong scallop bay scallop zhikong scallop bay scallop zhikong scallop bay scallop control 40.6±6.9 a 114.0±24.3 bc 29.0±3.1 b 92.5±13.8 cd 146.6±13.3 a 432.4±56.9 bc 0.2 58.9±21.5 ab 218.7±38.0 d 52.5±9.1 b 155.6±18.2 ef 176.1±37.5 a 438.3±111.6 bcd 0.3 91.0±36.9 cd 212.1±58.8 bc 81.5±31.9 abcde 194.7±34.5 f 295.4±71.6 ab 544.3±131.7 d 0.5 153.1±43.1 cd 175.9±27.4 cd 92.3±14.5 c 135.5±17.3 def 295.1±65.1 b 536.9±76.2 cd the values were shown as means ± sd, n = 3. significant difference between two scallop species, and the various concentration of pb was indicated by different letters ( p < 0.05). flow cytometric respiratory burst assay the respiratory burst of both species was measured according to bass’s method using 2’, 7’-dichlorofluorescin diacetate (dcfh-da), a nonfluorescent fluorescein analogue, with some modifications (bass et al., 1983; hégaret et al., 2003). dcf production, quantitatively related to the ros production of hemocytes, was measured by evaluating the green fluorescence on the fl1 detector of the flow cytometer. for the detection of respiratory burst after pb2+ exposure, a 400 μl of pooled hemolymph from control and pb2+ treated scallops was mixed with 200 μl fsw. then 6 μl of dcfh-da (sigma) was added at a final concentration of 0.01 mm, and the mixture was incubated in the dark at 18 °c for 60 min. dcf fluorescence was measured at 500 530 nm by flow cytometer. respiratory burst of hemocytes was calculated as the geometric mean of the fluorescent peak on an fl1 histogram plot for each sample. for the detection of respiratory burst after bacteria challenge, a 400 μl aliquot from pooled hemolymph was mixed with 200 μl of v. anguillarum (od600 = 0.4). simultaneously, those hemolymph samples incubated with the fsw were used as control. then 6 μl of dcfh-da was added to each tube. the dcf fluorescence of each sample was measured with the flow cytometer at 30, 60, and 120 min after incubation with dcfh-da, and respiratory burst of hemocytes was calculated as above description. measurement of sod, acp activities and mda content in hepatopancreas the activities of sod and acp and the content of mda in hepatopancreas were measured by using the kits from jiancheng bioengineering institute (nanjing, china) according to manufacturer’s protocols. the mda content was expressed as nmol per mg of protein (nmol/mg protein). sod activity was defined as the ability of 1 mg protein to cause 50 % inhibition in 1 ml reaction solution, and expressed as unit activity per mg of protein in the sample (u/mg protein). acp activity was defined as the ability of 1 g protein to produce 1 mg phenol after incubation at 37 °c with the substrate for 30 min, and the activity was expressed as units per g of protein (u/g protein). the total protein content was measured with bca assay (beyotime biotechnology, china). statistical analysis data from the flow cytometer were processed by using the analysis software winmdi 2.8. for the immune parameters and mda content assays, the data were given in terms of means ± sd (n = 3). survival data were graphed by the method of kaplan-meier and compared using log-rank analysis with graphpad prism 5.0 software (lee and wang 2003; sohail et al., 2008). all of the data were subjected to one-way analysis of variance (one-way anova) followed by a multiple comparison using spss v15.0 (spss inc., chicago, illinois). differences were considered to be statistically significant at a p value of 0.05 or less. results the hemocyte mortality the flow cytometric analysis with pi fluorescence revealed that hemocytes mortality in the 144 http://www.google.com.hk/url?q=http://linkinghub.elsevier.com/retrieve/pii/s0022175904001152%23sec1&usg=afqjcnfc19oownnnnhs9hg2xvup4edjgcw&sa=x&ei=2579tnrzbiweciwrvpmf&ved=0cc4qygq fig. 3 ros levels of bay scallops and zhikong scallops exposed to pb2+ at different concentrations. vertical bars represent the mean ± sd (n = 3), and bars with different letters are significantly different (p < 0.05). control groups of bay scallops and zhikong scallops was 4.8 ± 1.1 % and 4.9 ± 0.7 % respectively. there was no significant difference between the controls of two species. after pb2+ exposure, the hemocyte mortality of zhikong scallops was significantly increased in a dose-dependent manner (p < 0.05). and the hemocyte mortalities of bay scallops at 0.2 or 0.5 mg l-1 pb2+ were significantly higher than those at 0.3 mg l-1 pb2+. under the same condition, the hemocyte mortality in bay scallops was lower than that of zhikong scallops except at 0.2 mg l-1 pb2+ (fig. 1). a significant difference between the two scallop species was observed at 0.3 and 0.5 mg l-1 pb2+ (p< 0.05). phagocytic activity of hemocytes in control groups, the percentage of cells showing phagocytosis in bay scallops (26.7 ± 2.4 %) was significantly higher than that of zhikong scallops (19.9 ± 0.8 %) (p < 0.01) (fig. 2). after pb2+ exposure, the phagocytosis of bay scallop hemocytes was significantly inhibited at the lowest pb2+ concentration (p < 0.05), while those at other pb2+ treatments were higher than that of the control group (fig. 2). and the phagocytosis in the 0.2 and 0.3 mg l-1 pb2+ treated zhikong scallop groups was significantly lower (p < 0.05) than that in the control (fig. 2). the percentage of cells showing phagocytosis in bay scallops was significantly higher than that of zhikong scallops (p < 0.05) in the 0.2, 0.3 and 0.5 mg l-1 pb2+ treatments. sod, acp activities and mda content in hepatopancreas of control and lead treated scallops sod activity in hepatopancreas of bay scallops in the control group was 114.0 u/mg protein, which was 2.8-fold of that in zhikong scallops (40.6 u/mg protein), and there was significant difference between them (p < 0.05) (table 1). after lead treatment, the significantly higher sod activity was observed in 0.2 mg l-1 pb2+ treated group of bay scallops compared with that of zhikong scallops (p < 0.05) (table 1). the acp activity in hepatopancreas of the control bay scallops (432.4 u/g protein) was significantly higher (2.9-fold) than that of zhikong scallops (146.6 u/g protein) (p < 0.05) (table 1). and bay scallops had a significantly higher acp activity in comparison with zhikong scallops when they were treated with pb2+ at corresponding concentration (p < 0.05), respectively. after pb2+ treatment, there was no significant change in the acp activity of bay scallops in comparison with controls. the acp activity in 0.5 mg l-1 pb2+ treated bay scallops were significantly higher than that of the untreated zhikong scallops (p < 0.05) (table 1). the mda content in hepatopancreas of bay scallops in the control group was 92.5 nmol/mg protein, and it was significant higher than that in zhikong scallops (29.0 nmol/mg protein) (3.19-fold, p < 0.05) (table 1). after pb2+ exposure, the mda content in different pb2+ treated bay scallop groups was 1.68-fold (p < 0.05), 2.10-fold (p < 0.05) and 1.46-fold (p > 0.05) of the controls, while that in zhikong scallop was 1.81-fold (p > 0.05), 2.81-fold (p > 0.05) and 3.18-fold (p < 0.05) of the controls, respectively. the mda contents in the pb2+ treated groups of bay scallops were significantly higher than those in pb2+ treated groups of zhikong scallops at the pb2+ concentration of 0.2 mg l-1, 0.3 mg l-1 and 0.5 mg l-1 (p < 0.05). 145 fig. 4 ros levels of zhikong scallop and bay scallop hemocytes challenged by v. anguillarum. vertical bars represent the mean ± sd (n = 3), and bars with different letters are significantly different (p < 0.05). variation of respiratory burst after lead exposure and v. anguillarum challenge there was no significant difference in the production of reactive oxygen species between bay scallops and zhikong scallops (1.56 vs 1.94) under control conditions (p > 0.05) (fig. 3). the respiratory burst in the two scallops both significantly increased (p < 0.05) after pb2+ exposure. in bay scallop, respiratory burst was about 3.05, 3.8 and 7.2 fold of the control after 0.2, 0.3 and 0.5 mg l-1 of pb2+ exposure, respectively. the respiratory burst in zhikong scallop was also significantly activated by 0.2, 0.3 and 0.5 mg l-1 of pb2+ exposure, and it increased by 1.91, 1.48 and 1.84 fold of the control, respectively (p < 0.05) (fig. 3). bay scallop had a significantly higher respiratory burst than zhikong scallop after the same level of lead exposure (p < 0.05) (fig. 3). the ros level in hemocytes of scallops challenged by v. anguillarum was significantly lower than that in the control (incubation with seawater) (p < 0.05). in the control groups, there was no significant difference in the ros production of two scallops (p > 0.05) (fig. 4). after bacteria challenge, the ros in the hemocytes of bay scallops was significantly lower than that of zhikong scallops at 30 min (p < 0.05), while it was comparable to that of zhikong scallops post 60 min and 120 min challenge (p > 0.05). compared to the control scallops, the respiratory burst in both scallops was significantly inhibited after v. anguillarum challenge (p < 0.05), which was different from the significant activation of respiratory burst after lead exposure. at 30 min after bacteria challenge, the respiratory burst was inhibited to be 0.43-fold of the control in bay scallops (p < 0.05) and 0.70-fold in zhikong scallops (p < 0.05), following 0.38 0.40 fold at 60 min (p < 0.01) and 0.20 0.25 fold at 120 min (p < 0.01) (fig. 4). there was a significant difference in the ros production of challenged zhikong and bay scallops at 30 min post challenge (p < 0.05). cumulative survival rate of scallops challenged by v. anguillarum the similar cumulative survival rates were detected in the blank (0 h) and control groups of the two scallop species. no scallop died in the first 5 days in the untreated zhikong and bay scallops (blank groups). in the control groups of both zhikong and bay scallops, the died scallop was firstly observed at 4th day, and mortality was of 3.3 % at the 4th and 5th day after injection (fig. 5). after bacteria challenge, no bay scallop died in the first two day, while the mortality of zhikong scallops occurred since the first day and quickly increased to 41.2 % on the second day. on the third day, the mortality of the bay scallops was 62.5 % and 94.1 % of the zhikong scallops died at 3rd day under the same condition. afterwards, all the zhikong scallops died in the 4th day post challenge, while there was still 25 % of bay scallops survival. the mortalities of bay scallops at 2nd 5th day post v. anguillarum challenge were significantly lower than those of zhikong scallops (p < 0.05). 146 fig. 5 cumulative survival rate in zhikong scallops and bay scallops under v. anguillarum challenge. bay: bay scallop; zk: zhikong scallop. cumulative survival rates of scallops (n = 30) treated by injecting 200 µl of v. anguillarum resuspended in pbs (od 600 = 0.4) (challenged group) (▲ in bay and ▼ in zk) and 200 µl of pbs (control group) (▽ in bay and △ in zk), and without treatment (blank group) (○ in bay and ◇ in zk) were plotted against the duration after challenge. discussion bivalves respond to environmental stress depending largely on the viability and functional capability of hemocytes (chu, 2000), and heavy metals as well as invading pathogens could affect the hemocyte functions in molluscs, such as cell viability, cytoskeletal organization and phagocytic activity (fagotti et al., 1996; song et al., 1997; zhang et al., 1999; olabbarietta et al., 2001; sauvé et al., 2002; duchemin et al., 2008). in the present study, the hemocyte mortalities were found to be less than 5 % in both healthy bay scallop and zhikong scallop under control condition, and this result was in agreement with previous reports from other bivalves. after pb2+ exposure, the mortality of scallop hemocytes increased in a dose-dependent manner, but it differed between the two scallop species. it has been reported that the percentage of dead hemocytes is a good indicator of physiological status especially before or during mortality events (paillard et al., 1996). in clams, a higher percentage of dead hemocytes was observed when they were experiencing massive mortalities (soudant et al., 2004). bay scallop exhibited lower hemocyte mortality than zhikong scallop under the same condition, indicating it bore stronger tolerance to environmental stress, and its physiological and immunological status were less influenced during heavy metal exposure compared with zhikong scallop. the phagocytosis assay has been developed as a common method to examine the ability of hemocytes to recognize and eliminate “non-self” material (chu, 1988). in the present study, bay scallops in the control and pb2+ treated groups all displayed higher phagocytosis ability than that of zhikong scallops. in eastern oyster c. virginica, the hemocytes with lower phagocytic ability exhibited a higher mortality (hegaret et al., 2004). pacific oysters with higher phagocytosis ability were less susceptible to the protozoan parasite perkinsus marinus infection (la peyre et al., 1995). in shrimp litopenaeus vannamei, the enhanced phagocytic activity could increase its resistance against vibrio alginolyticus (cheng et al., 2005). the percentage of cells showing phagocytosis in bay scallops increased after pb2+ exposure, while significantly decreased in zhikong scallops. the higher phagocytic potential of bay scallops in present study suggested that bay scallops seemed to have a possible advantage of resisting to environmental exposure compared with zhikong scallops. recent available evidence demonstrated that heavy metals enhanced the intracellular formation of ros and induced cellular oxidative stress (stohs and bagchi, 1995; gurer and ercal, 2000; pacheco et al., 2007). in the present experiment, pb2+ treatment significantly increased the ros generation of scallops, and the increased ros level lead to unspecific oxidation of proteins and membrane lipids, resulting in an increasing of malondialdehyde (mda). a higher mda content was observed in bay scallops compared with zhikong scallops before and after lead exposure. mda content along with ros results in present study possibly suggested the compromised immune function of scallops under metal exposure. the relatively gentle increase of mda content in bay scallops indicated they could offer a better buffer 147 capacity to alleviate metal induced damage than zhikong scallops. endogenous enzymes excreted and released by hemocytes during exocytosis and degranulation have a close connection with the functions of hemocytes (chu, 2000; cima et al., 2000). enzymatic activities in hemolymph have been studied as one of the immunity indicators in many bivalve species (hine and wesney, 1994; carballal et al., 1997). the inhibitory effects of lead on various enzymes would probably result in impaired antioxidant defenses by cells and render cells more vulnerable to oxidative attacks (gurer and ercal, 2000). significantly reduced superoxide dismutase (sod) activities have been observed in lead-exposed scallops (zhang et al., 2010). and higher sod activity was detected in japanese pearl oysters with low mortality from an infectious disease (uchimura et al., 2003). the higher sod activity after lead treatment indicated that bay scallop should have a somehow stronger ability than zhikong scallops to eliminate ros and to prevent the injury of oxidative stress resulting from metal exposure. acid phosphatase is a lysosomal marker enzyme playing important roles in destructing pathogens in lysosome, and its activity appears to be altered by stress conditions (cheng, 1989; suresh and mohandas, 1990). in the present study, acp activities in both hemolymph and hepatopancreas of bay scallop were higher than those of zhikong scallop before and after pb2+ treatment. the higher acp activity in bay scallop was consistent with its higher phagocytic ability (cheng, 1978). compared with zhikong scallops, bay scallops displayed higher acp activities, especially in hepatopancreas, suggesting their predominance in resisting to the environmental stress and invading pathogens. previous studies also revealed that some other immune factors in the hemolymph of a. irradians were significant higher than those in c. farreri (wang and sun, 2005). in the present study, ros in both scallop species was significantly lower than that in the control groups during the incubation of hemocytes with v. anguillarum, indicating the respiratory burst of both scallops was inhibited by bacteria challenge (lambert et al., 2003; labreuche et al., 2006). the reduced respiratory burst was possibly associated with impaired ability to kill bacteria, and increased susceptibility to infectious diseases (cai et al., 1994; johnston, 2001). furthermore, v. anguillarum was employed as the pathogen to challenge both scallops (cavallo and stabili, 2002), and the cumulative mortality of zhikong scallop at 2nd 5th day was significantly higher than that of bay scallop (p < 0.05). similarly, researches have previously reported that bay scallops not only grow fast in contrast with zhikong scallops but also endure higher temperatures (zhang et al., 2000; xiao et al., 2005). these results collectively favored that bay scallop had higher potential defense capabilities than zhikong scallop against pathogens and environmental stress factors. acknowledgements the authors would like to thank all the laboratory members for technical advice and helpful discussions. this research was supported by grants from national science foundation of china (no. 30925028 to l.s.), shandong provincial natural science foundation (no. jq201110 to l.w.), and the knowledge innovation program of the chinese academy of sciences (no. kzcx2-ew-qn201 to h.z.). references bass da, parce jw, dechatelet lr, szejda p, seeds mc, thomas m. flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane 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nematodes (epns) have been used against especially soil borne insect pests. epns are feasible and attractive for biological control, because of their virulence against various insect pests, host seeking ability, being usable with standard equipment, and long-term efficacy. in addition epns can be applied simultaneously with some pesticides. these properties make epns ideal biocontrol agent in integrated pest management. in the present study, effects of 4 widely used pesticides (glyphosate, chlorpyrifos-ethyl, captan, fosetyl-al) on virulence and mortality of three epn strains (heterorhabditis bacteriophora alman, h. bacteriophora hbh and steinernema carpocapsae dd-136) were examined at 24 and 48 h periods. all strains were able to infect galleria mellonella larvae averagely above 90 % rate, after 24 and 48 h treatments with all pesticides. however, some of the pesticides showed negative impact on the viability of the strains. especially, dd-136 and fosetylal seemed like incompatible, as the mortality rates were significantly higher than control for both 24 and 48 h. the results of the present study showed that it may be possible to use some epn strains with some pesticides. it is expected that the results of the study will provide useful information for future integrated pest management programs. key words: entomopathogenic nematodes; mortality; virulence; pesticides   introduction biological control provides safe and environmentally friendly control of insects, diseases and weeds, and it is raising its popularity every day with new developments and technologies. one of the most important biocontrol agents is entomopathogenic nematodes (epns), belonging to the families heterorhabditidae and steinernematidae (poinar, 1979). epns are soildwelling obligate endoparasitic organisms. they have control potential of many economic important insect pests (peters, 1996; susurluk et al., 2011; ulu et al., 2014), while safe for non-target organisms and environment (boemare et al., 1996; ehlers, 1996).they can be mass produced (ehlers, 2001) for widespread commercial use. epns seek for their hosts, penetrates through natural openings or intersegmental membrane, and release symbiont bacteria to the hemocoel of the host. the host is killed within 36 48 h and free-living third-stage ___________________________________________________________________________ corresponding author: i alper susurluk department of plant protection faculty of agriculture uludag university 16059, nilufer, bursa, turkey e-mail: susurluk@uludag.edu.tr infective juveniles (ijs) emerge from host cadaver (poinar, 1979; akhurst and boemare, 1990; brown and gaugler, 1997). although pesticides make pest control easier, they have negative impacts on the environment, human health and other various ecosystems because of the excessive use. integrated pest management (ipm) arose as a solution to problems associated with the excessive use of chemical pesticides to control pests, diseases and weeds, more than 50 years ago (hokkanen, 2015). with ipm, agricultural practices and control methods are used in a harmony and environmental risks are minimized. epns are environmentally safe organisms and they can be applied with standard pesticide sprayers or irrigation systems (georgis, 1990; wright et al., 2005). epns are known as resistant to various agricultural chemicals (rovesti and deseö, 1990; georgis and kaya, 1998). simultaneous applications with pesticides lead epns to become a possible option in ipm systems. pesticides are still the most convenient method for controlling pests in agriculture. however, the proportion of the other control methods such as biological, biotechnical, genetic, cultural, etc. are 111   mailto:susurluk@uludag.edu.tr 112   rising due to the negative effects of chemicals. although it doesn’t seem possible to take pesticides out of agriculture, it may be possible to lower their use or enhance their efficiency with some agricultural applications. epns are effective biocontrol agents, especially on soil-dwelling insects and they are known as resistant against several agricultural chemicals. the aim of the present study was to determine compatibility of different pesticides on commercial and a hybrid epn strain. thus, it may be possible to use epn simultaneously with common pesticides and enhance controlling while reducing labor cost. compatibility of the pesticides with epns has been investigated by researchers for more than 25 years (rovesti et al., 1988; rovesti and deseo, 1990; patel and wright, 1996; alumai and grewal, 2004; laznik and trdan, 2014; baimey et al., 2015). although there are many studies conducted, more investigations are still necessary as it has been showed that epn-pesticide compatibility can be also strain-specific (de nardo and grewal, 2003; garcíadel-pino and jové, 2005; gutiérrez et al., 2008; laznik et al., 2012; atwa et al., 2013). in the present study, effects of 4 different pesticides (fosetyl-al, glyphosate, captan and chlorpyrifos-ethyl) on virulence and mortality of heterorhabditis bacteriophora alman, h. bacteriophora hbh and steinernema carpocapsae dd-136 strains were examined. with the results of the study, reduction of application cost, effective control of pests with simultaneous use of chemical and biological control, and introducing new biological control agents for ipm are expected. materials and methods nematode strains three epn strains, h. bacteriophora alman, h. bacteriophora hbh and s. carpocapsae dd-136, were used in the experiments. hbh was obtained by hybridization of two native epn strains from turkey. the nematodes were produced in last instar larvae of greater wax moth, galleria mellonella (lepidoptera: pyralidae) according to white trap method as described by white (1927). the ijs were harvested from white trap and stored in flasks in ringer’s solution at 4 °c for a week before trials. pesticides especially, pesticides applied to soil were preferred as epns generally live under the soil and are effectively used against soil-borne pests. chlorpyrifos-ethyl usually is used against soil-borne pests as insecticide. fosetyl-al and captan are used for soil-borne pathogenic fungi, and glyphosate is for soil-borne pathogenic fungi, and glyphosate is applied against weed on fruit and vegetable fields in turkey. therefore, these active ingredients (table 1) were used in order to determine their toxicity on ijs of hbh, alman and dd-136 strains. results showed that, among all pesticides, fosetyl-al was the most toxic chemical for all strains at 24 and 48 h exposure times. fosetyl-al caused significant mortality rates on every strain ranging between 9.78 82.92 %, at both 24 and 48 h counts (p < 0.05) comparing with control. besides fosetylal, other pesticides also showed significant toxic effect on different strains at different exposure times. as seen from table 2, pesticides showed significant effects on mortality of the strains (p < 0.05). dd-136 strain showed significantly higher mortality than alman and hbh in all chemicals at both exposure times. mortality of alman and hbh strains also differed within pesticides (p < 0.05). statistical summary of the tests indicated in table 3. toxicity tests the 100 ij/10µl solutions of ijs were prepared in ringer’s solution and transferred in each well of 24-well plate for each nematode strain. one ml pesticides prepared at field doses (table 1) were added on nematodes in wells. the plates were incubated in shaker at 25 °c, 150 rpm and %70 rh, in order to prevent settling of pesticide+nematode mixture. the experiment was replicated four times. tap water used for control. dead and alive epns in each well were counted after 24 and 48 h, and their mortality rates were assessed (rovesti et al., 1988; patel and wright, 1996). virulence tests harvested nematodes from white trap were transferred into 100 ml erlen flasks with density of 2000 ijs/ml and field doses of the prepared pesticides were added on the epns. flasks were incubated in shaker at 25 °c. firstly, the nematodes were separated from the pesticide solutions with micro sieve and suspended in pure water for 2 h (hara and kaya, 1983). subsample was taken from pure water plus nematode solution for counting, afterwards a new suspension was prepared which has 50 ijs/larva, according to commercial dose. nematode virulence after 24 and 48 h of exposure to pesticides was tested against the last instar g. mellonella. one larva were placed for each well of the plates, the wells were filled with 10 % moist sterile silver sand (particle size 300 400 µm). the plates were sealed with parafilm and incubated at 25 °c. after four days, death larvae were collected to determine nematode infectivity. the death larvae were dissected under stereomicroscope in order to prove whether the larva killed by nematodes. tap water used for control. the virulence experiment was replicated 3 times with 10 larvae used per replicate. statistical analyses before analysis, all treatment data were corrected based on the control mortality (< 5 %) using abbott’s (1925) formula. toxicity and virulence test data were analyzed using one-way anova (analysis of variance) with jmpp®7.0 software. lsd (least significant differences) test (0.05 significance level) was used to determine the difference between applications. toxicity tests 113   table 1 information of the pesticides active ingredient classification commercial product manufacturer recommended field dose fosety-al fungicide placate platin chemistry 250 g/100 l water glyhosate herbicide roundup monsanto 300 g/100 l water chlorpyrifos-ethyl insecticide dursban 4 dow agro 200 ml/100 l water captan fungicide captan koruma 300 g/100 l water table 2 corrected mortality rates of epn strains exposed to different pesticides mortality (%) strains time control fosetyl-al glyphosate captan chlorpyrifos-ethyl alman 0ba 9.78ac 11.47ab 0.67bb 2.44bb dd-136 0da 52.97aa 27.30ba 25.72ba 13.40ca hbh 24h 0ba 18.78ab 1.21bc 2.76bb 0.93bb alman 0da 16.09ac 12.14bb 1.10dc 7.53cb dd-136 0da 82.92aa 32.14ba 26.40ba 16.63ca hbh 48h 0ca 65.30ab 3.88bcc 12.22bb 2.23cc different lowercase letters indicate statistical significance between treatments for each strain at 24 and 48 h separately (p < 0.05). different uppercase letters indicate statistical significance between strains for each treatment at 24 and 48 h separately (p < 0.05). results and discussion virulence tests the results of the virulence tests were not similar with toxicity, as the pesticides didn’t affect virulence of the strains as much as they did in mortality experiment. for 24 h, there were no significant difference on the virulence of alman and hbh strains exposed to the pesticides comparing with control (p > 0.05). dd-136 showed the lowest mortality and all pesticides significantly affected the virulence of the strain (p < 0.05). for 48 h, captan and chlorpyrifos-ethyl had a negative impact on the virulence of hbh strain, and fosetyl-al had a negative impact on dd-136. for both toxicity and virulence results, dd-136 strain was the most affected one among others (table 4). the results of the present study indicated that the most toxic pesticide was fosetyl-al for all strains. toxic effects of the other pesticides varied among strains and exposure time, but the least-toxic pesticide was chlorpyrifos-ethyl, which is one of the most widely used organophosphate insecticides all around the world, according to united states environmental protection agency. from another sight, the weakest strain was s. carpocapsae dd136 and h. bacteriophora strains were more tolerant than dd-136. according to baimey et al. (2015), all tested heterorhabditis species were more tolerant to glyphosate and fipronil than the steinernema species, which was a similar result with the present study. table 3 statistical summary of the toxicity tests toxic effects of the pesticides by strain 24h 48h fosetyl-al f=161.33, df=2;9, p<0.0001 f=56.08, df=2;9, p<0.0001 glyphosate f=45.38, df=2;9, p<0.0001 f=36.03, df=2;9, p<0.0001 captan f=149.40, df=2;9, p<0.0001 f=40.24, df=2;9, p<0.0001 chlorpyrifos-ethyl f=42.71, df=2;9, p<0.0001 f=25.12, df=2;9, p<0.0001 mortality rates of the strains by pesticide 24h 48h alman f=36.81, df=4;15, p<0.0001 f=49.63, df=4;15, p<0.0001 dd-136 f=113.67, df=4;15, p<0.0001 f=104.30, df=4;15, p<0.0001 hbh f=42.60, df=4; 15, p<0.0001 f=104.30, df=4;15, p<0.0001 114   table 4 virulence of pesticide-exposed epn strains on g. mellonella larva virulence (%) strain time control fosetyl-al glyphosate captan chlorpyrifos-ethyl alman 100a 96.6a 100a 96.6a 100a dd-136 100a 83.3bc 90b 86.6b 76.6c hbh 24h 100a 96.6a 100a 96.6a 96.6a alman 100a 93.3a 100a 96.6a 93.3a dd-136 100a 70b 93.3a 83.3ab 96.6a hbh 48h 100a 100a 93.3a 76.6b 73.3b different letters indicate statistical significance between treatments for each strain at 24 and 48 h separately (p < 0.05). study. likewise, results of the study conducted by negrisoli et al. (2010) indicated that, lorsbantm (chlorpyrifos-ethyl) caused the lowest mortality among other 18 insecticides. gutiérrez et al. (2008) conducted a study to determine effects of different pesticides, including glyphosate and chlorpyrifosethyl, on s. feltiae, and they stated that herbicides were more toxic then insecticides. unlikely, most toxic pesticide of the present study was fosetyl-al, which is a fungicide, and it is followed by glyphosate. their strain was s. feltiae and they did not use fungicides, this discordant result may be explained with strain and pesticide selection and interaction. according to another study performed by garcía-del-pino and jové (2005), h. bacteriophora and s. carpocapsae were both tolerant to fipronil while s. arenarium was sensitive. mortality rate were increased with the dose and exposure time, as expected. another aim of the study was to determine the effects of pesticides on virulence of the epn strains. steinernema carpocapsae dd-136 strain was the weakest strain, while alman didn’t show significant differences between pesticide treatments and control. virulence experiments were conducted with living ijs after exposing to the pesticides, and virulence results of the pesticides-exposed populations mostly didn’t show significant differences between control populations. it can be stated that pesticides didn’t affect the virulence of the strains. however, it must be taken into account that simultaneous applications of epns with these pesticides may lower effectiveness due to the toxic effect and mortality of strains. negrisoli et al. (2010) tried the effects of 18 insecticides on infectivity of three epn species. their results were significantly different from control; however, highest infectivity was recorded with lorsbantm (chlorpyrifos-ethyl) and matchtm (lufenuron), which can be stated as similar with the present study, as the highest virulence results were recorded for chlorpyrifosethyl and glyphosate. the study of gutiérrez et al. (2008) showed that virulence of s. felitae was not seriously affected by the chemicals, likewise in the present study. as it can be understood from former studies, there are lots of factors such as chemical type, strain, exposure time, different environmental conditions and etc., which have significant role on virulence, viability or other biological characters of epns. based on our findings, it may possible to apply epns with compatible pesticides within ipm programs in future, which will reduce application time and labor cost. however, there are incompatible pesticides that may need to be applied alone. further investigations may reveal new data for new pesticides and new species or strains. acknowledgements this study was supported by the scientific research projects unit of uludag university (bursa, turkey) [project number: kuap (z)-2015/48]. references abbott ws. a method of computing the effectiveness of an insecticide. j. econ. entomol. 18: 265-267, 1925. akhurst rj, boemare ne. biology and taxonomy of xenorhabdus. in: gaugler r, kaya hk (eds), entomopathogenic nematodes in biological control, crc press, boca raton, fl, pp 75-90, 1990. alumai a, grewal ps. tank-mix compatibility of the entomopathogenic nematodes, heterorhabditis bacteriophora and steinernema carpocapsae, with selected chemical pesticides used in turfgrass. biocontrol sci. techn. 14: 725-730, 2004. atwa aa, shamseldean mm, yonis fa. the effect of different pesticides on reproduction of entomopathogenic nematodes. turk. j. entomol. 37: 493-502, 2013. baimey h, zadji l, afouda l, moens m, decraemer w. influence of pesticides, soil temperature and moisture on entomopathogenic nematodes from southern benin and control of underground termite nest populations. nematology 17: 10571069, 2015. boemare ne, laumond c, mauleon h. the entomopathogenic nematodebacterium complex: biology, life cycle and vertebrate safety. biocontrol sci. techn. 6: 333-346, 1996. brown i, gaugler r. temperature and humidity influence emergence and survival of entomopathogenic nematodes. nematologica 43: 363-375, 1997. de nardo eab, grewal ps. compatibility of steinernema feltiae (nematoda: 115   steinernematidae) with pesticides and plant growth regulators used in glasshouse plant production. biocontrol sci. techn. 13: 441-448, 2003. ehlers r-u. current and future use of nematodes in biocontrol: practice and commercial aspects in regard to regulatory policies. biocontrol sci. techn. 6: 303-316, 1996. ehlers r-u. mass production of entomopathogenic nematodes for plant protection. appl. microbiol. biot. 56: 623-633, 2001. garcía-del-pino f, jové m. compatibility of entomopathogenic nematodes with fipronil. j. helminthol. 79: 333-337, 2005. georgis r. formulation and application technology. in:  gaugler r, kaya hk (eds), entomopathogenic nematodes in biological control, crc press, boca raton, fl, pp 173191, 1990. georgis r, kaya hk. formulation of entomopathogenic nematodes. in: burges hd (ed), formulation of microbial biopesticides: beneficial microorganisms, nematodes and seed treatments, kluwer academic publishers, drodrecht, the netherlands, pp 289-308, 1998. gutiérrez c, campos-herrera r, jiménez j. comparative study of the effect of selected agrochemical products on steinernema feltiae (rhabditida: steinernematidae). biocontrol sci. techn. 18: 101-108, 2008. hara ah, kaya hk. toxicity of selected organophosphate and carbamate pesticides to infective juveniles of the entomogenous nematode neoaplectana carpocapsae (rhabditida: steinernematidae). environ. entomol. 12: 496-501, 1983. hokkanen hmt. integrated pest management at the crossroads: science, politics, or business (as usual)? arthropod-plant inte. 9: 543-545, 2015. laznik ž, trdan s. the influence of insecticides on the viability of entomopathogenic nematodes (rhabditida: steinernematidae and heterorhabditidae) under laboratory conditions. pest manag. sci. 70: 784-789, 2014. laznik ž, vidrih m, trdan s. the effects of different fungicides on the viability of entomopathogenic nematodes steinernema feltiae (filipjev), s. carpocapsae weiser, and heterorhabditis downesi stock, griffin & burnell (nematoda: rhabditida) under laboratory conditions. chil. j. agr. res. 72: 62-67, 2012. negrisoli jr as, garcia ms, negrisoli crcb. compatibility of entomopathogenic nematodes (nematoda: rhabditida) with registered insecticides for spodoptera frugiperda (smith, 1797) (lepidoptera: noctuidae) under laboratory conditions. crop prot. 29: 545-549, 2010. patel mn, wright dj. the influence of neuroactive pesticides on the behaviour of entomopathogenic nematodes. j. helminthol. 70: 53–61, 1996. peters a. the natural host range of steinernema and heterorhabditis spp. and their impact on insect populations. biocontrol sci. techn. 6: 389-402, 1996. poinar go jr. nematodes for biological control of insects, crc press boca roton, fl, 1979. rovesti l, deseö kv. compatibility of chemical pesticides with the entomopathogenic nematodes, steinernema carpocapsae weiser and s. feltiae filipjev (nematoda: steinernematidae). nematologica 36: 237-245, 1990. rovesti l, heinzpeter ew, tagliente f, deseö kv. compatibility of pesticides with the entomopathogenic nematode heterorhabditis bacteriophora poinar (nematoda: heterorhabditidae). nematologica 34: 462-476, 1988. susurluk, ia, kumral na, bilgili u, acikgoz e. control of a new turf pest, dorcadion pseudopreissi (coleoptera: cerambycidae), by the entomopathogenic nematode heterorhabditis bacteriophora. j. pest sci. 84: 321-326, 2011. ulu tc, sadic b, susurluk ia, aksit t. virulence of four entomopathogenic nematode species for plum sawfly, hoplocampa flava l. (hymenoptera: tenthredinidae). inv. surv. j. 11: 4-10, 2014. white gf. a method for obtaining infective nematode larvae from culture. science 66: 302303, 1927. wright dj, peters a, schroer s, fife jp. application technology. in: grewal ps, ehlers r-u, shapiro-ilan di (eds), nematodes as biological control agents, cabi publishing, wallingford, uk, pp 91-106, 2005. research report isj 11: 143-148, 2014 issn 1824-307x research report oxidative stress in oysters (crassostrea corteziensis) exposed to naphthalene dg mendoza-lópez, mi girón-pérez, ca romero-bañuelos, ae rojas-garcía, bs barrónvivanco, im medina-díaz, ml robledo-marenco universidad autónoma de nayarit, secretaría de investigación y posgrado. boulevard tepic-xalisco s/n, cd. de la cultura amado nervo, c.p. 63190 tepic, nayarit, méxico accepted may 5, 2014 abstract naphthalene is a frequent pollutant in aquatic ecosystems that can affect the physiology of organisms such as molluscs. oyster crassostrea corteziensis is an endemic species from the tropical west pacific, with both ecological and economical importance. the objective of this study was to evaluate the oxidative damage in lipids and proteins in the tissue (gills and digestive gland), as well as the membrane stability in hemocytes of oysters c. corteziensis exposed to naphthalene (1 or 20 µg/l) sub-acutely (24 and 72 h). the results obtained indicate that under evaluated conditions, this hydrocarbon does not induce oxidation of lipids and proteins. however, the stability of the cell membrane of hemocytes diminished significantly in organisms exposed to 20 µg/l during 72 h. according to the obtained results, it can be suggested that stability of the hemocyte’s cell membrane is the most sensitive parameter to napthalene’s effect. it seems that compared to other hydrocarbons, naphthalene has low damage potential on oyster’s oxidative parameters. key words: naphthalene; oxidative stress; membrane stability; oyster; crassostrea corteziensis   introduction the physiology of molluscs can be altered by polluting substances in aquatic ecosystems (auffret, 2005; rodriguez et al., 2005; baqueiro-cárdenas et al., 2007; collin et al., 2010). bivalve molluscs accumulate a great quantity of water dissolved substances due to their sessile nature and filtration feeding habits (farrington et al., 1983; goldberg, 1986). oysters are one of the most commercially worldwide exploited groups of bivalves. it is calculated that the production of these organisms reaches 4.7 millions of tons per year (fao, 2012). pleasure oyster (crassostrea corteziensis), also known as cortez oyster, is a native species of the tropical west pacific, with ecological and economical importance. this mollusc species is distributed along the gulf of california and all through panama. only in mexico, approximately 1500 tons per year of this species are produced (conapesca, 2011). ___________________________________________________________________________ corresponding author: manuel iván girón pérez laboratorio de inmunotoxicología universidad autónoma de nayarit secretaría de investigación y posgrado bd. tepic-xalisco s/n, cd. de la cultura amado nervo c.p. 63190 tepic, nayarit, méxico e-mail: ivan_giron@hotmail.com a group of pollutants frequently detected in aquatic ecosystems are the polycyclic aromatic hydrocarbons (pahs). among these compounds is naphthalene, which has been found in water, suspended organic matter, and sediments, as well as in the tissue of molluscs, particularly, oysters (meador et al., 1995; noreña-barroso et al., 1999; botelloet al., 2002; guo et al., 2007; piazza et al., 2008; maskaoui and hu, 2009; ramdine et al., 2012; girón-pérez et al., 2013). this hydrocarbon possesses two rings of benzene and is included in the national priorities list (npl) of the united states (iarc, 2002). most of the naphthalene that goes into the environment is released into the air (which mainly results from combustion) from where it enters the soil and water by humid or dry deposition. by possessing only two aromatic rings and low molecular weight, naphthalene, compared to other pahs, has major water solubility. therefore, it has major bioavailability for organisms in this environment (iarc, 2002; iniesta and blanco 2005). several studies that have been made in different type of molluscs indicate that the pahs can induce the increase in concentration of reactive oxygen species (ros), which provokes oxidative stress to macromolecules such as lipids, proteins and dna (livingstone et al., 1992; manduzio, 2005; pichaud et al., 2008). however, up to this moment 143   table 1 lipid hydroperoxides and protein oxidation in oyster tissue (n = 3) exposed to h2o2 positive controls oxidation pbs h2o2 (3%) h2o2 (9%) lipid hydroperoxides (µmoles cumene hydroperoxide/gr tissue) gill 3.6 ± 1.0 4.0 ± 0.6 14.1 ± 0.04* digestive gland 3.1 ± 0.5 4.5 ± 0.6 14.3 ± 0.2* protein oxidation (µmoles/mg protein) gill 9.4 ± 2.2 6.8 ± 2.5 21.1 ± 5.8 digestive gland 23.0 ± 6.2 37.6 ± 12.3 33.0 ± 8.9 values represented as mean ± se, *p < 0.05 vs tissue exposed only to pbs there are no reports of the effect of naphthalene over oxidative stress in the c. corteziensis species. thus the objective of this study was to evaluate oxidative stress in lipids and proteins in tissue (gills and digestive gland), as well as the membrane stability in hemocytes of oysters c. corteziensis exposed sub-acutely to naphthalene. materials and methods animals and treatment crassostrea corteziensis oysters of commercial length and weight (8 ± 2 cm and 70 ± 30 g, respectively, and approximately 7 months old) were acquired at a local market in the state of nayarit, mexico and were immediately transported to the laboratory for their depuration during a 30 day period prior to experimentation (adamo et al., 1997). for this purpose, the oysters were maintained in a 50 l recirculation system with filtered seawater (salinity, 26 ± 2 %, ph 8.7 ± 0.2, and temperature, 26 ± 2 °c, 12 h:12 h dark-light cycles) and constant aeration. they were fed daily with dehydrated alga spirulina dissolved in filtered seawater (castillorodríguez and garcia-cubas, 1984). after the depuration period, the oysters were placed in glass fish tanks with 5 l of filtered seawater (without food, under previously mentioned conditions) during a 24 h period for their acclimatization. after this time, the organisms were exposed to sublethal concentrations of naphthalene (1 or 20 μg/l). the naphthalene (sigma-aldrich, 99 %) was taken from a stock solution (0.5 g/l), utilizing acetone as solvent at a 1:20 proportion (hydrocarbon:acetone). three experimental groups were utilized per treatment (n = 20/per group): 1) oysters in seawater; 2) oysters in seawater with acetone, and 3) oysters in seawater with naphthalene. the oysters were exposed to the hydrocarbon during 24 or 72 h, with daily exchanges of water for each fish tank. during the experiment, the oysters were fed daily with alga spirulina. concentration of lipid hydroperoxides for the determination of lipid hydroperoxides the ferrous oxidation-xilenol orange (fox) method was used, the tissue (gills and digestive gland) was homogenized with cold methanol (1:9 w/v) and was centrifuged to 1,000xg (3,600 rpm) during 15 min at 4 °c. a mix of reagents was prepared (400 µl feso4 0.25 mm, 400 µl h2so4 25 mm, 100 ml xilenol orange 0.1 mm) and it was incubated for 30 min. after that, 100 µl supernatant of the homogenized sample were added. the resulting mix was incubated in room temperature for 18 h in the dark. once incubating time passed, its absorbance was determined to 550 nm against a standard curve of cumenehydroperoxyde (monserrat, 2003; zanette et al., 2006). oxidation of proteins to determine the oxidation of proteins, the carbonyl group concentration was evaluated. from every analyzed tissue (gills and digestive gland), 0.4 g of each oyster were obtained and homogenized during 1 min in 1 ml of pbs. after that, it was centrifuged at 9,000 rpm during 20 min. from the centrifuged fraction 100 μl were taken and precipitated with 200 μl of trichloroacetic acid (tca) at 30 % with slow agitation. the precipitate was added with 500 μl of 2,4-dinitrophenylhydrazine (20 mm in ethanol). the mix was incubated in the dark for 1 h, agitating every 10 min. once incubation time was completed, it was precipitated with 200 μl of tca at 30 %. the precipitate was dissolved with 1000 μl of 6 mm urea. the concentration of oxidized proteins was determined at 366 nm and was calculated through molar extinction coefficient (22 mm/cm) (reznick and packer, 1994). membrane stability the hemolymph extraction of the posterior region of the adductor muscle was performed and the membrane stability of the hemocytes was determined through the measurement of neutral red retention (cantyet al., 2007; hannamet al., 2009). 50 μl of hemolymph in a microwell were incubated during 45 min at 4°c. a rinsing with saline solution was made in order to eliminate the non-added cells. after that, 200 μl of neutral red were added (0.004 %) and incubated during 3 h at room temperature. 144   once incubation time passed, it was rinsed with 200 μl of saline solution, and 200 μl of acidified ethanol were added. it was agitated during 10 min and its absorbance was determined at 545 nm. determination of protein concentration concentration of protein oxidation and membrane stability was adjusted according to the total concentration of proteins in the determined sample, which was measured through the bradford method (1976). positive controls as positive control of oxidative stress, samples of tissue (gill and digestive gland) (n = 3) were exposed to hydrogen peroxide (h2o2) (3 and 9%) during 24 h protected from light. after that, the concentration of proteins and lipid hydroperoxides in each sample was determined by following the techniques previously described. statistical analysis for statistical analysis, we employed sigmastat® (ver. 3.5. statistical software). we determined the normal distribution of the obtained data. for normal data distribution, we used analysis of variance (anova) followed by the bonferroni subtest. for non-parametric data, we used the kruskal-wallis test followed by a multiple tukeytype comparison. to compare statistical difference between two groups, either t-test or mann-whitney test were used. the statistical difference was determined with a level of p < 0.05. results previous to the determination of the oxidative damage in lipids and proteins of gills and digestive gland of oysters exposed to naphthalene, oxidation of these biomolecules was induced through the exposure of tissue at h2o2 (positive oxidation control). the obtained results showed an increase in the oxidation of lipids and proteins when the tissues were exposed at 9% h2o2. however, significant difference was shown only in the concentration of oxidized lipids (table 1). regarding the effect in vivo of naphthalene (1 µg/l) on the oxidation of lipids and proteins, no increase in the concentration of such molecules in the gills and digestive gland was detected, after 24 and 72 h exposition (table 2). on the other hand, the obtained results indicate that the naphthalene exposition (1 µg/l) during 24 and 72 h did not provoke alteration in the membrane stability of the hemocytes. regarding the oysters exposed to 20 µg/l of naphthalene, a decrease in this parameter was observed. however, this was only significant in the oysters exposed during 72 h (p < 0.05) (fig. 1). discussion in scientific literature, there are a number of studies that evaluate the effect of different pahs (individualized or mixed) about oxidative stress in different species of bivalve molluscs; however, up to this moment there are no studies published where table 2 lipid hydroperoxides and protein oxidation in oyster tissue exposed to naphthalene naphthalene (1 µg/l) naphthalene (20 µg/l) tissue treatment 24 h 72 h 24 h 72 h lipid hydroperoxides (µmoles cumene hydroperoxide/gr tissue) control 5.3 ± 1.6 4.0 ± 1.6 1.1 ± 0.1 1.6 ± 0.1 naphthalene 2.1 ± 0.5 10.4 ± 2.7 0.9 ± 0.2 1.0 ± 0.1 gill p>0.05 p>0.05 p>0.05 p>0.05 control 8.8 ± 1.8 13.0 ± 2.9 2.0 ± 0.3 1.7 ± 0.1 naphthalene 8.8 ± 1.9 11.1 ± 3.0 1.2 ± 0.2 1.9 ± 0.2 digestive gland p>0.05 p>0.05 p>0.05 p>0.05 protein oxidation (µmoles/mg protein) control 29.3 ± 3.8 36.6 ± 6.2 42.4± 9.7 3.6 ± 0.1 naphthalene 21.5 ± 3.5 20.3 ± 2.4 44.7 ±8.0 3.5 ± 0.1 gill p>0.05 p>0.05 p>0.05 p>0.05 control 16.2 ± 3.6 23.3 ± 4.0 55.0 ± 12.6 3.5 ± 0.07 naphthalene 28.1± 4.3 20.6± 3.9 43.7± 4.8 3.6 ± 0.1 digestive gland p>0.05 p>0.05 p>0.05 p>0.05 values represented as mean ± se, n = 20/per treatment 145   fig. 1 membrane stability a) oysters exposed to naphthalene during 24 h, b) oysters exposed to naphthalene during 72 h. c = control, filtered seawater, n = filtered seawater + naphthalene (℘ ± ee). n = 20/per treatment. the effect of naphthalene is evaluated on the oxidative stress of the oyster c. corteziensis. therefore, this study represents a first approximation on the effect of this hydrocarbon in such type of oyster with commercial significance in the tropical pacific. the obtained results in the bioassays with h2o2 (control) suggest major susceptibility of lipids to oxidation for the action of such agent, in comparison with proteins, which can be related with the structure of both types of molecules, since the double bound present in the lipid structures confer to these major susceptibility to oxidation, in contrast with the proteins that are more stable molecules. besides, there are some mechanisms of protein repair, such as disulfide bounds in cysteines and the formation of sulfoxides in methionines (hermes-lima, 2005). it has been reported that the exposition of mytilus galloprovincialis to benzo [a] pyrene for 7 days, provokes an increase in the lipidic peroxidation in gill (maria and bebianno, 2011). while the exposure of the molluscs pecten maximus to sublethal concentrations of phenanthrene (50, 100 and 200 µg/l) causes an increase in the levels of lipid peroxidation in hemolymph (ansaldo etal., 2005; hannam et al., 2010), after exposing the molluscs nacella concinnato diesel (0.05 and 0.1 %) during 24, 48 and 168 h, a significant increase was detected in the oxidized lipid concentration in both concentrations after 168 h of exposition. contrary to the previously stated, the obtained results in this study, by exposing the c. corteziensis to naphthalene, concur with the reported by lüchmann et al. (2011), who by exposing crassostrea brasiliana to diesel (2.5, 5, 10 and 20 %) during 96 h, found that there were no significant changes in the oxidation of lipids in the gill and digestive gland. pichaud et al. (2008) reported similar results by exposing bivalve mya arenaria to a mix of pahs (methylphenanthrene, naphthalene, 2methylnaphthalene, phenanthrene, anthracene, pyrene, benzo[a]pyrene, and fluoranthene) to a concentration of 47.6 ng/l applied through the consume of phytoplankton during 9 days. besides the pro-oxidative potential effect that pahs can have over molluscs, there are environmental factors that can modulate oxidative damage. in experiments made with c. gigas exposed to diesel (0.01, 0.1 and 1.0 ml/l) in different salinity conditions (35, 25, 15 and 9 ups), significant differences were observed in the lipid peroxidation over the different conditions, except for the salinity of 25 ups (zanette et al., 2011). in this study, the established salinity for the carrying out of the bioassays was of 28 ups (optimal salinity level for c. corteziensis), factor that might have had influenced in the oxidative potential of naphthalene, since external factors such as salinity, temperature and low food availability can influence the physiological state of the mollusc, which can provoke additional stress to the one provoked per se by the hydrocarbon. regarding the protein oxidation levels, in this study there were no significant differences observed in such parameter; contrary to what was reported by ansaldo et al. (2005) when exposing n. concinnato diesel 0.05 % during 168 h, who detected a significant increase regarding control. 146   membrane stability of hemocytes of c. corteziensis was significantly affected after the exposition of naphthalene (20 µg/l) during 72 h. these results agree with the ones obtained by hannamet al. (2010) by exposing scallops pecten maximus to sublethal concentrations of phenanthrene (50, 100 and 200 µg/l), which reported a reduction in the stability of the cell membrane. on the other hand, anton (2011) also reported destabilization in lysosomal membranes of hemocytes of pincta daimbricata, after the exposition (during 7 days) in soluble fractions of lubricants used in motor vehicles to a 20 % concentration. one of the possible mechanisms by means of which naphthalene can alter the structure of the lipid bilayer and membrane proteins,is through the induction of ros: molecules that can alter the biological functions of the membrane and induce cell damage (de la haba et al., 2013). the obtained results in this study indicate that under the evaluated conditions, naphthalene does not induce lipid and protein oxidation. however, naphthalene induces destabilization of cell membrane of c. corteziensis hemocytes, parameter that has major functional and physiological relevance. on the other hand, evaluated parameters in this study, could have been influenced by a number of factors, among others, the tested concentrations of naphthalene, the established times of bioassays and/or the intrinsic resistance of oyster species. acknowledgements the first author of the article received a grant from conacyt-méxico during postgraduate studies in the cienciasbiológico-agropecuarias (cbap) program of the autonomous university of nayarit. the work of the investigation was carried out within the framework of the fomix-nayarit 2009-c02131614 project. references adamo r, pelosi s, trotta p, sansone g. bioaccumulation and biomagnification of polycyclic aromatic hydrocarbons in aquatic organisms. mar. chem. 56: 45-49, 1997. ansaldo m, najle r, luquet cm. oxidative stress generated by diesel seawater contamination in the digestive gland of the antarctic limpet nacella concinna. mar. environ. res. 59: 381390, 2005. anton m.parámetros citológicos, inmunológicos y estabilidad lisosomal en hemocitos del bivalvo pinctada imbricata expuesto a fracciones solubles de lubricantes usados de vehículos, licenciatura (biología), universidad de oriente, cumaná, 2011. auffret, m. bivalves as models for marine immunotoxicology. investigative immunotoxicology, 2005. baqueiro-cárdenas er, borabe lj, goldaracenaislas cg, rodriguez-navarro j. los moluscos y la contaminación. una revisión. revista mexicana de biodiversidad. 78: 1s7s, 2007. botello av, garcía-ruelas c, ponce-vélez g. pah levels in bivalve mollusks from the mexican subtropical pacific. bull. environ. contam. toxicol. 69: 486-493, 2002. bradford mm. a rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dyebinding. analyt. biochem. 72: 248-254, 1976. canty mn, hagger ja, moore r, cooper l, galloway ts. sublethal impact of short term exposure to the organophosphate pesticide azamethiphos in the marine mollusc mytilus edulis. mar. pollut. bull. 54: 396-402, 2007. castillo-rodríguez z, garcía-cubas a. taxonomía y anatomía comparada de las ostras en las costas de méxico. ann. ins. cienc. mar. limnol. 13: 294-314, 1984. collin h, meistertzheim a, david e, moraga d, boutet i. response of the pacific oyster crassostrea gigas, thunberg 1793, to pesticide exposure under experimental conditions.j. exp. biol. 123: 4010-4017, 2010. conapesca. anuario estadístico de acuacultura y pesca, 2011. de la haba c, palacio jr, martínez p, morros a. effect of oxidative stress on plasma membrane fluidity of thp-1 induced macrophages. biochim. biophys. acta 1828: 357-364, 2013. fao. food and agriculture organization of the united nations. figis time-series query on: aquaculture.2012. http://www.fao.org/figis/servlet/sqservlet?file=/ work/figis/prod/webapps/figis/temp/hqp_1619 644301412387908.xml&outtype=html farrington jw, goldberg ed, risebrough rw, martin jh, bowen vt. u.s. 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responses of the coral c toledo-hernández1, cp ruiz-diaz1,2 1sociedad ambiente marino, po box 22158, san juan puerto rico 00931 2department of environmental sciences, university of puerto rico, río piedras campus, po box 70377 puerto rico accepted november 3, 2014 abstract corals are among the most ancient extant animals on earth. currently, coral viability is threatened, due in part to the increased number of diseases affecting them in recent decades. understanding how the innate immune systems of corals function is important if we want to predict the fate of corals and their response to the environmental and biological changes they face. in this review we discuss the latest findings regarding the innate immune systems of corals. the review is organized following the chronology of steps taken by corals from the initial encounter with a potential pathogen and recognition of threats to the orchestration of a response. we begin with the literature describing the repertory of immune-related receptors involved in the recognition of threats and the subsequent pathways leading to an immune response. we then review the effector responses that eliminate the threats described for corals. finally, we acknowledge the literature of coral microbiology to access the potential role of microbes as an essential constituent of the coral immune system. key words: immune response; coral diseases; coral-associated microbes; afferent and effector arms   introduction coral reefs are among the most ancient extant ecosystems. evidence of their existence can be traced back to the mesozoic era (veron, 1995). they are also among the most persistent ecosystems as they have survived several mass extinction events over geological time, and continued to thrive. ironically, during the last century reefs of the world have undergone unprecedented declines, primarily due to human derived stresses (mcclanahan et al., 2002). a combination of factors, which include water pollution (i.e., nutrient and sediment influxes), overfishing, rising water temperatures, ocean acidification and disease are causing declines in coral growth and coral cover. such degradation is readily observed in the indopacific region where reefs are disappearing at an average rate of 2 % per year (bruno and selig, 2007). in the caribbean, reefs are being lost even faster, i.e., at an average rate of 5.5 9.2 % per year (buddemeier and ware, 2003). given increasing human impacts, these pressures are projected to exacerbate damages to coral reefs worldwide and ___________________________________________________________________________ corresponding author: claudia patricia ruiz-diaz department of environmental sciences university of puerto rico, río piedras campus po box 70377 puerto rico e-mail:claudiapatriciaruiz@gmail.com these ecosystems are likely to suffer even more extreme stress than those already observed in the recent past. from the myriad of stressors threatening coral health, disease has perhaps, received the most attention by the scientific community during the last few decades. reports of emerging coral diseases have increased since they were first described in the early 1970s (antonius, 1973; richardson, 2012). currently, over 35 coral diseases have been reported (kline and vollmer, 2011), some of which have worldwide distribution, i.e., black band disease (bbd). others have narrower distributions, such as yellow band disease (ybd), which is reported exclusively in the caribbean (veron, 1995). diseases have affected over 80 coral species and their impacts have been so dramatic as to alter the seascape and the community structures of reefs (pandolfi and jackson, 2006). studies addressing the causations of coral diseases, as well as their prevalence, incidence, and their impacts on vital life history traits such as growth and reproduction, have dominated the scientific literature. for instance, several long-term field studies have shown that diseased corals exhibit slower growth rates and a reduced fecundity when compared to healthy ones (baird and marshall, 2002; petes et al., 2003; toledohernández et al., 2009; weil et al., 2009). far less   319 attention has been devoted to study the mechanisms by which corals recognize a threat and orchestrate an immune response to an insult. the fact that we do not fully understand how the immune system of corals works, undermines our capacity to comprehend the real causes and consequences of coral disease processes. the innate immune system of corals, as in all invertebrates, is responsible for maintaining the integrity and stability of the internal environment (homeostasis). adaptive immune-like systems have yet to be described in invertebrates. corals are thought to poses a rudimentary and rather simplistic immune system (alker et al., 2004; pollock et al., 2011; toledo-hernández et al., 2012). this notion has been supported in part by their primitive diploblastic body plan, typical of organisms from the basal metazoan phylogeny, concomitant with their similar macroscopic immune responses toward different immune insults. however, new molecular findings have shown that coral immunity is surprisingly complex, with some immune components such as immune-related genes and proteins homologous to those from vertebrates (miller et al., 2007; palmer and traylor-knowles, 2012). in this review we will present the latest findings regarding coral immune systems. for the purpose of this review, corals are identified as members of the scleractinia and gorgonacea orders. the review is organized following the chronology of steps taken by corals from the initial encounter with a potential pathogen and recognition of threats to the orchestration of a response. accordingly, we begin by summarizing the current literature concerning the repertory of immune-related receptors involved in the recognition of threats and the subsequent pathways to mount a response. we then review the effector responses that eliminate or mitigate the threats. finally, we acknowledge the literature of coral microbiology to access the potential role of microbes as integral constituents of the coral immune system. innate immune system of corals physical barriers are the first line of defense of any organism. for the most part, physical barriers are anatomical structures that inhibit or prevent a threat from affecting or entering the internal environment of an organism. in corals, such barriers are the mucus and sclerite layers that primarily settle on top of and within the coral ectoderm. coral mucus is a viscous fluid made of a complex mixture of compounds secreted by mucocytes. sclerites, on the other hand, are carbon calcium carbonate crystals of variable shapes, sizes and secreted by sclerocytes from gorgonian corals. coral mucus is involved in an array of functions ranging from feeding and sun protection to pathogen defense; the mucous layer hosts symbiotic microbes that prevent the settlement of noxious bacteria (brown and bythell, 2005). sclerites may function as a shield that prevents or retards the recruitment of microbes. for instance, we have documented that a layer of sclerites from the sea fan gorgonia ventalina, placed on top of culture media, delay the colonization and further penetration into the culture media of fungi isolated from sea fans. however, once threats overwhelm these physical barriers, the immune system activates. coral innate immune systems are no different from other invertebrates. it consists of two branches: 1) the afferent or sensing arm, and 2) the efferent or effector arm. the afferent arm is basically the component that recognizes the threat and activates an effector response, which will orchestrate a humoral and/or cellular response such as inflammatory and cytotoxic responses, plus phagocytosis, which will neutralize the threat. afferent component our knowledge regarding the recognition receptors of corals is very limited. most of what we currently know is based on a few microarray and transcriptional studies from a few coral species, i.e., acropora millepora and a. digitifera (miller et al., 2007), or from extrapolation from closely related organisms such as hydra or from more complex invertebrates such as the fruit fly, drosophila melanogaster. most of these studies report the presence of sequences that, due to their structural similarities with immune-related genes and proteins from other animals, are considered immune-related components of corals. moreover, investigations working on recognition receptors found in other invertebrates suggest that numerous coral immunerelated pathway components have yet to be identified. pattern recognition receptors (prrs) like all animals, corals rely on their capacity to detect non-self entities by means of pattern recognition receptors (prrs) to initiate an immune response. these receptors, which appear to be highly conserved across the animal kingdom, are complex proteins that bind to highly conserved cell wall structures from foreign entities collectively known as microbe-associated molecular patterns (mamp), such as flagellin, lipopolysaccharide (lps), lipoteichoic acid, and peptidoglycan. this are generally absent on hosts but are essential structural components in microbes (murphy et al., 2011). these bindings activate complex downstream signaling pathways that result in the expression, via transcription factors, of immune genes that initiate the proper effector responses, such as opsonization, phagocytosis, lysis, or the production of anti-microbial peptides. in corals, six distinct families of microbe detection receptors have been identified. three of these receptors are transmembrane sensors or receptors, i.e., toll-like receptors (tlrs), lectins and integrins. the remaining three belong to the families of intracellular receptors; the nucleotide-binding and oligomerization domain (nod)-like receptors (nlrs) and the complement-like system c3. however, it is important to highlight that, with few exceptions, the existing literature regarding prrs is purely descriptive. functional experiments are yet to be conducted, thus the roles of the majority of the prrs as immune constituent are unknown.   320 trans-membrane receptors toll-like receptors (tlrs) tlrs are perhaps the best-characterized of the prrs and one of the principal innate immune components of invertebrates. tlrs consist of an outer leucine-rich repeat domain (lrr) responsible for pattern recognition, and a cytoplasmic component toll/interleukin-1receptor (tir), which mediates signal transmission. complete tlrs (lrr and tir) have been identified in acropora digitifera (miller et al., 2007); while the cytoplasmic component tirs have also been identified in a. palmata, a. millepora and montastraea faveolata (miller et al., 2007; shwarz et al., 2008). several signaling pathways associated with tlrs have also been identified in these corals. these include the homologues of the myeloid differentiation primary response adaptor protein (myd88), which activates downstream signaling pathways, as well as homologues of the transcription factor nf-κb, which lead to cell death. mitogen-activated protein kinases (mapks), which are involved in the cellular responses to thermal stress among other roles, have been also identified in a. digitifera (miller et al., 2007; shwarz et al., 2008). lectins lectins are a family of trans-membrane receptors ubiquitous throughout the animal kingdom. lectins play an important role in cell recognition, including recognition of bacteria and viruses. these receptors contain one or more carbohydrate recognition domains, which allow binding to carbohydrate components from foreign entities in a calcium-dependent manner. lectin-like genes have been identified in several scleractinian corals. millectin for instance, has been identified and described in the scleractinian coral a. millepora (kvennefors et al., 2008). millectin contains a peptide recognition site and a carbohybrate-binding site, which under ca2+ dependent conditions has an affinity toward n-acetylglucosamine and other simple sugars such as mannose, glucose, and fructose. several studies have confirmed the role of millectin in recognizing non-self structures. for instance, functional experiments have revealed millectin binding to and agglutinating vibrio harveyi and v. corallilyticus as well as other potential pathogens (kvennefors et al., 2008). millectin has also been detected bound to zooxanthellae, further emphasizing its function in the recognition of nonself entities (kvennefors et al., 2008). immunohistochemistry studies have detected millectin encapsulated within nematocysts in the epithelial tissue lining the gastrodermis of the oral and aboral regions of a. millepora (kvennefors et al., 2010). as these regions are constantly in contact with potential pathogens, the authors hypothesized that millectin would be secreted upon pathogen recognition, preventing further penetration of the pathogen into the internal environment of the host. at the gene expression level, up-regulation of millectin has been detected after a. millepora fragments were exposed to bacterial lps (kvennefors et al., 2008). in this study, upregulation of millectin peaked 45 min. after lps exposure and gradually decreased within the next twelve hours, suggesting that overexpression of millectin and perhaps other c-type lectin genes primarily occurred during the first few minutes after pathogen recognition (kvennefors et al., 2010; brown et al., 2013). an additional lectin-like gene, namely techylectin has been described from the scleractinian coral oculina sp. (hayes et al., 2010). oculina tachylectin-2 is remarkably similar to the tachylectin-2 originally isolated from the japanese horseshoe crab tachypleus tridentatus, which has been shown to have broad-spectrum anti-microbial activity as well as a role in no-self recognition (kawabata and iwanaga, 1999). due to these similarities, oculina tachylectin-2 has been suggested to play a role in the coral immune system; however, functional experiments are yet to be conducted to confirm this argument. integrins integrins are surface receptors highly conserved across the animal kingdom, and employed in multiple functions, such as development and growth, wound repair and immune response. functional integrins always consist of at least two glycoprotein subunits, the α and β chains. these α β heterodimers are involved in cell-to-cell intra or extra cellular binding. in corals, specifically in a. millepora, several integrins have been identified (brower et al., 1997; knack et al., 2008). however, these have been associated to developmental roles, i.e. coral gastrulation (knack et al., 2008). the role of integrins as constituents of coral immunity has yet to be demonstrated. intracellular receptors nlrs microbes eluding trans-membrane surveillance encounter a second line of sensors with the capacity to recognize mamps at the intracellular level. these receptors are collectively known as nucleotide oligomerization domain (nod)-like receptors (nlrs). similar to extracellular receptors, nlr activation triggers multiple proinflammatory signaling pathways, which result in the eradication or inhibition of the invaders. the architecture of nlrs essentially consists of a c terminal lrr domain for microbe pattern recognition, an intermediary nod domain for nucleotide binding and modulation of nlr activity, and a n-terminal effector domain (kanneganti et al., 2007). genomic analyses have revealed that nlrs of a. digitifera are surprisingly diverse within the central domain containing a death-effector domain (ded), death domain (dd) and caspase recruitment domain (card), all of which play pivotal roles in apoptosis (hamada et al., 2012). complement system the complement system is a major effector mechanism of the innate immune system. its function varies from opsonizing pathogens for   321 further phagocytosis to lysis, tissue regeneration, or the clearance of immune complexes and apoptotic cells (trouw and daha, 2011). in higher vertebrates, complement activation proceeds via; 1) antibodyantigen binding; 2) mannose-binding lectin (mbl) and 3) spontaneous binding of a complement component to the pathogen surface (murphy et al., 2011). these pathways lead to the activation of complement factor c3, the main component of this system. as corals lack adaptive immunity, complement activation hypothetically should proceed either via mbl or spontaneously (cerenius et al., 2010). thus far, complement-like genes have been identified in three coral species, including the gorgonian corals swiftia exserta (dishaw et al., 2005) and gorgonia ventalina (burge et al., 2013), and the scleractinian coral a. millepora (miller et al., 2007). given the over-expression of complementlike genes from a. millepora and g. ventalina in response to challenge with bacterial pathogens and its localization in the epithelium, as in the case of c3 from a. millepora, these genes have been linked to the immune systems of corals (kvennefors et al., 2010; brown et al., 2013; burge et al., 2013). effector components humoral response humoral response in corals consists of the synthesis and release, upon activation, of a variety of chemical compounds, such as melanin, reactive oxygen species (ros), antimicrobial peptides (amp), and secondary metabolites whose main function is to kill microbes by: 1) opsonizing and agglutinating invaders, 2) permeabilizing the invader’s cell membrane, causing lysis, or 3) disrupting of their metabolism (ellis et al., 2011). these compounds may be constitutively present and stored in granules within epithelial cells or contained in free-living cells, in which case they can be recruited to the afflicted areas (palmer et al., 2008). alternatively, these compounds can be synthesized and released in situ upon local induction by mechanical stress, injuries or pathogen detection (destoumieux et al., 2000; ganz, 2003; geffen et al., 2009). antimicrobial agents have no functional specificity, meaning that these agents have a broad spectrum of activity against gram-positive and gram-negative bacteria, fungi, viruses and protists (otero-gonzález et al., 2010; ellis et al., 2011). that is not to say, however, that all antibacterial agents are equally effective against microbes (gochfeld and aeby, 2008). the size of the antimicrobial agent may impact structural components of the microbes differently. for instance, small peptides (< 23 amino acids long) mainly disrupt the integrity of the cell membrane of the invading entities, while larger peptides pose lytic properties, or may be proteins with specific domains that sequester essential nutrients from microbes (ganz, 2003). melanization melanin is notorious among invertebrates, including corals. melanization, the synthesis and deposition of melanin around foreign entities or wounded areas, is a relatively well-understood process in invertebrates, particularly in arthropods and crustacea (cerenius and soderhall, 2004). the enzyme phenoloxidase (po) is essential to the synthesis of melanin. po and its inactive form prophenoloxidase (propo), belong to the tyrosinase group of oxidases whose main role is to oxidize phenols (gonzalez-santoyo et al., 2012). melanin is derived from phenylalanine, which is hydroxylated to form tyrosine. upon activation, the proteolysis of propo activates po. once activated, po catalyzes the oxidation of mono-phenol (tyrosine) and diphenols (dopa) to form quinones. ultimately, nonenzymatic polymerization of quinones forms melanin (palmer et al., 2011). synthesis of melanin also leads to the formation of proteases, cytotoxic quinones and reactive oxygen and nitrogen intermediates that not only kill pathogens but also inflict self-damage. in summary, regulatory mechanisms of po such as the production of phenoloxidase inhibitor (poi), serine protease inhibitor, radical scavengers and anti-oxidant enzymes are produced to control the synthesis of melanin (cerenius and soderhall, 2004; cerenius et al., 2010). activation of po in response to dopa has been demonstrated in a. millepora and porites sp. (palmer et al., 2008). multiple melanin-synthesis pathways (tyrosinase-type and laccase-type pathways), and multiple pos (i.e., catecholase, cresolase and laccase), have also been identified in corals from several families, (i.e., pocilloporidae, acroporidae, merulinidae, mussidae, fungiidae, faviidae and poriferidae) (mydlarz and palmer, 2011; palmer et al., 2012). melanin synthesis in corals has also been shown during wound healing, pathogenic interactions and sustained elevated water temperatures (petes et al., 2003; mydlarz et al., 2008; palmer et al., 2008). furthermore, baseline po activity and the size and abundance of melanin-containing granular amoebocytes (the cells where melanin synthesis takes place), have been compared among several indo-pacific and caribbean corals (palmer et al., 2010). in fact, base-line levels of these immune constituents have been found to be good predictors of disease and bleaching susceptibility and thus have been related to coral immunecompetence. in other words, corals exhibiting lower base-line concentrations of the aforementioned melanization constituents are more susceptible to disease and bleaching than those with higher levels (palmer et al., 2011). reactive oxygen species (ros) ros are a group of highly reactive, relatively short-lived compounds i.e., 1o2, o2, h2o2, ho•, no•, which are by products of oxidation-reduction (redox) reactions of living organisms. in biological systems, ros have a diverse and dose-dependent impact. for instance, at relatively low doses, ros can function as signaling molecules, such as in immune defense and apoptosis (nappi and ottaviani, 2000). yet, at higher doses and since ros are not target-specific compounds, they produce oxidative stress (if the accumulation of ros exceeds the threshold of organisms to suppress them), which can inflicts damage to important cell components of both host and invader, such as dna, rna, proteins and lipids (lesser, 2006).   322 ros synthesis has been studied in corals as a way to measure environmental stress. for instance, mydlarz and jocobs (2006) studied the production of ros in the gorgonian corals pseudopterogorgia elisabethae, p. americana, euniceafusca spp., and lophogorgia chilensis after subjecting them to physical injury and thermal stress. they found that these stressors induced an oxidative stress that varied among species. in another study, saragosti et al. (2010) measured the production of ros in non-bleached and bleached stylophora sp and found that both produced a comparable ros concentration when maintained in the dark. however, under light regimes non-bleached corals increased ros production by two-fold when compared with bleached fragments, demonstrating that light increments induced the production of ros. on the other hand, tchervno et al. (2011) experimentally established that ros produced by zooxanthellae from the scleractinian corals seriatopora hystrix and stylophora pistillata and from the corals themselves maintained under thermal stress conditions, activated the caspase cascade, which promoted bleaching and apoptosis. they also demonstrated experimentally that exogenous ros activates the caspase pathway and apoptosis of montipora capitata fragments under conditions of low light and temperature, further showing the role of ros as a signaling pathway. antimicrobial peptides (amps) according to ganz (2003) amps are peptides with less than 100 amino acid residues, encoded by genes and synthesized by ribosomes from animal hosts. thus, this definition excludes amps synthetized by potential symbionts such as bacteria, fungi or protists. in addition to their antimicrobial properties, amps show immunomodulatory activities such as apoptosis and regulation of gene transcriptions (otero-gonzález et al., 2010). however, in general, little is known regarding amps of corals. in fact, damicornin from pocillopora damicronis is the only amp characterized for any coral thus far (vidal-dupoil et al., 2011). damicornis is a cationic, 40 amino acid amp, which inhibits gram-positive bacteria and fungi in vitro experiments. moreover, it is constitutively transcribed and stored in granular cells from the epithelium where it is released upon non-pathogenic immune challenge. transcript analyses have also revealed the presence of genes encoding potential anti-microbial peptides in corals. for instance, burge et al. (2013) working with the sea fan coral g. ventalina, identified gene homologues to the arenicin-2 gene from a polycheate worms and the royalisin gene from honeybee. furthermore, burge and co-workers have also observed an over expression of areicin-2 in response to the parasite aplanochytrium sp. parasite suggesting a role in host/pathogen interactions. the royalisin-homolog, on the other hand, was suggested to be active against grampositive bacteria and fungi. peroxidase enzymes the super family of peroxidase enzymes is another well-known enyzme family with multiple roles in the immune systems of organisms. for instance, peroxidase enzymes have been linked to the formation of ros (mydlarz and harvell, 2007), detoxification of active forms of oxygen (lesser, 2006), phagocytosis (rodríguez et al., 2003) and synthesis of melanin (mydlarz and harvell, 2007). peroxidases activity has also been linked to stress responses and has been identified as an immune constituent in corals. for instance, mydlarz and harvell (2007) were able to induce the production of peroxidases in g. ventalina colonies in response to fungal pathogen exposure. they also were able to show experimentally that commercial and sea fan extracted peroxidases can inhibit a. sydowii growth. however, they were unable to describe the mechanisms by which peroxidases acts against the fungal pathogen. secondary metabolites natural chemical compounds with antimicrobial properties, other than melanin, amp and peroxidase enzymes, have also been extensively documented in gorgonian and scleractinian corals. in fact, gorgonians are rich in secondary metabolites with anti-microbial properties. for instance, ospina and rodríguez (2006) isolated 9 polycyclized diterpenes from the gorgonian coral briareum polyanthes, all of which showed antimicrobial activities against human pathogens such as plasmodium falciparum and mycobacterium tuberculosis. similarly, shapo et al. (2007) reported that crude extracts from the gorgonian coral leptogorgia virgulata, likely containing homarine, showed inhibitory activity against echerichia coli and vibrio harveyi as well as other bacteria. uncharacterized antimicrobial agents have been also documented in over a dozen members of the plexauridae, gorgonidae and ellisellidae families (kim, 1994; kim et al., 2000a). some of these extracts, such as lipid metabolites extracted from g. ventalina colonies, have been shown to be active against a. sydowii (kim et al., 2000b; alker et al., 2001; dube et al., 2002). furthermore, the effectiveness of these extracts against pathogens appears to be dependent on the temperature and the location within the colony. for instance, crude extracts from the edges of g. ventalina colonies have shown higher inhibitory activity against a. sydowii than extracts from the center (kim et al., 2000b). on the other hand, crude extracts taken from colonies at temperatures over 30 °c inhibit the growth of a. sydowii less effectively than extracts at lower temperature colonies (alker et al., 2001). the effectiveness of extracts has also been linked to ontogenic factors. that is, extracts from sea fan recruits show higher activity against a. sydowii than extracts from mature sea fan colonies (dube et al., 2002). however, other studies contradict this argument (for detail see couch et al., 2013). scleractinian corals also possess secondary compounds with antimicrobial properties, though they have been less well studied than those derived from the gorgonians (gochfeld and aeby, 2008). gochfeld and aeby (2008) were able to show that crude extracts from three hawaiian corals i.e., montipora capitata, porites lobata and pocillopora meandrina, had some degree of antibacterial activity   323 against coral pathogens such as serratia marcescens, vibrio coralliityticus and v. shilo. however, the antibacterial activity of extracts varied among species and as a function of the state of health of the host. in another study, the scleractinian corals pocillopora damicorinis and stylophora pistillata were shown to continuously release chemical compounds that inhibit the growth of vibrio corallityticus upon sequential stress inductions (geffen and rosenberg, 2005; geffen et al., 2009). cellular response phagocytosis is a fundamental cellular response in innate immunity. phagocytosis is the process by which cells, some of which contain phagosomes, engulf foreign entities. the process is initiated upon recognition via actin-mediated mechanisms such as opsinization or in another instance, by cell-cell recognition (stuart and ezekowitz, 2008). phagosomes are organelles composed of over 140 proteins, including hydrolytic enzymes that neutralize the engulfed entity (garin et al., 2001). phagocytic cells target two main entities: pathogens and self material such as dead cells targeted for recycling. in addition, phagocytes also produce and release cytotoxic compounds such as ros, cytokines and melanin upon activation (rabinovitch, 1995). thus, phagocytosis is not only a cornerstone of host defense but also essential to tissue remodeling. in corals, cells capable of phagocytosis are collectively known as amoebocytes. these cells are the putative immune cells of cnidarians (mullen et al., 2004; ruiz-diaz et al., 2013). amebocytes are motile cells, primordially found spread across the mesoglea (mullen et al., 2004). a distinctive feature of amoebocytes is the presence or absence of granules. granules are amebocyte organelles that synthesize and store po and ros (olano and bigger, 2000; petes et al., 2003). the roles and tasks of amebocytes are variable and not yet fully understood, although they have been linked to wound healing and protection from infection, as shown in studies conducted mostly in gorgonian corals. for instance, during the wound healing process in the gorgonian coral swiftia exserta, amebocytes gathered as in an inflammatory response in the wounded area to clean up the cellular debris (olano and bigger, 2000). meszaros and bigger (1999) show that, amebocytes from the gorgonian plexaurella fusifera streamed and linedup at the edge of a wounded areas fusing and subsequently differentiating or dedifferentiating to form new epithelial cells. in the sea fan g. ventalina, a higher abundance of granular amebocytes has been observed in tissue areas infected by a. sydowii as compared to healthy tissues (petes et al., 2003; midlarz et al., 2008). similarly, diseased tissue fragments of g. ventalina placed on healthy corals induced an increase in the area occupied by amoebocytes when compared to healthy grafts (couch et al., 2013). direct evidence of the phagocytic capacities of amebocytes has been tested in functional experiments. for instance, olano and bigger (2000) documented phagocytosis in disassociated cells from s. exserta after being challenged with india ink and carmine. in this study, phagocytosis was also observed occurring in granular amebocytes and to a lesser extent in other cells such as scleroblats, epithelial cells and gastrodermal cells, after india ink and carmine were introduced to s. exserta via tissue trauma. likewise, phagocytosis has also been observed in disassociated cells from diseased and healthy tissue of diseased, as well as in healthy colonies g. ventalina (ruiz-diaz et al., 2013). in this study, disassociated living cells were incubated for several hours with fluorescent beads and the number of cells containing fluorescent beans was quantified. interestingly, the phagocytic index (defined as the number of cells containing fluoresce beads with respect to the total number of cells within 1 cm2 of sea fan tissue) was higher in diseased tissue when compared to healthy tissue from diseased colonies and from healthy colonies. these results suggested that diseased tissue was immunologically active previous to the experimental challenge. the ecological function of amebocytes in scleractinian corals has been also studied. however, as scleractian mesoglea is thinner than in gorgonians and amebocytes are smaller and fewer, the identification of amebocytes and their roles has been challenging (mullen et al., 2004; work and aeby, 2010). nonetheless, as in gorgonians, increased abundance of amebocytes in some scleractinians has been observed in immunologically challenged individuals. for instance, infiltrated granular amebocytes have been observed in pigmented tissues from porites species. in fact, the mean number of granular amebocytes per 100 μm2 of epidermal layer from pigmented porites was fourfold greater than from the epidermal layer of none pigmented tissue, suggesting a role as an immune constituent (palmer et al., 2008). moreover, amebocyte infiltration, plug formation due to the releasing of melanin from agranular amebocytes, and amebocyte differentiation to form epithelial tissue has been observed during the wound healing process in porites cylindrica (palmer et al., 2011). however, in other scleractinian corals the role of amebocytes as an immune constituent seems to be less prominent the in porites. for instance, infiltration of amebocytes has seldom been observed or not observed at all in tissues from monstastrea carvernosa under sedimentary-stress conditions (vargas-angel et al., 2007), in pigmented tissues from acropora millepora (palmer et al., 2008), or during wound repair in montipora capitata (work and aeby, 2010). role of coral-associated microbial community numerous studies have revealed that corals harbor a rich and diverse microbial assemblage that includes viruses, bacteria, archaea and several microbial eukaryotes. this microbial assemblage, referred to as a coral holobiont, varies widely as a function of coral species and the health conditions of an individual coral organism. furthermore, the microbial assemblage may vary within different parts of a single coral colony i.e., the microbial assemblage of the coral mucus layer is different from that of the internal coral tissue and the   324 skeleton (bourne and munn 2005; koren and rosenberg 2006; rosenberg et al., 2007; shnitorland and kushmaro, 2009). interestingly, microbe assemblages seem to be species-specific, suggesting that a beneficial relationship exists between corals and their holobiont. yet, in most cases, the roles of coral-associated microbes are unknown, due in part, to the difficulties in performing functional studies using culturing techniques. nonetheless, experimental evidence, much of which comes from cultured bacteria associated with coral mucus, suggests that certain microbes play a role as coral immune constituents. in this regard, several bacteria-associated mechanisms that play important roles in the protection of corals have been described, two of which we discussed bellow 1) compounds with anti-microbial properties, and 2) quorum sensing. microbes as producer of anti-microbial compounds coral mucus not only harbors a rich and diverse micro-flora assemblage, but also contains a diverse array of chemical compounds, some of which are produced by the coral itself, while others are produced by the microbes inhabiting the mucus. it has been rather difficult to track the source of bioactive compounds, i.e., host corals vs. symbionts, as most of the antimicrobial activity assays have used whole-colony extracts including other living organisms associated with corals. these data are essential to complement data information about the chemical structures of the antimicrobial compounds, their stereochemistry and estimates of their effective concentrations in the wild. these knowledge gaps, and the fact that the majority of the microbes associated with coral can not be cultured, precluded our ability to determine the roles of microbes as a constituent of the coral immune system. nonetheless, when researchers have been able to culture these microbes, a wealth of information has been obtained. for instance, in a functional experiment using cultured organisms, nearly 8 % of native bacteria from a. palmata mucus interfered with the ability of s. marcescens, the causal agent of white pox disease, to swarm and form biofilms in coral mucus thereby preventing the disease. the mechanism was reported to be inhibition of the enzyme activity involved in the breakdown of nutrients (krediet et al., 2013). however, further research must be conducted to elucidate the molecules involved in this inhibition. on the other hand, numerous studies have shown that bacteria associated with coral mucus produce anti-microbial compounds. for instance, ritchie (2006) found that 20 % of cultured bacteria isolated from the mucus of healthy a. palmata exhibited anti-microbial activity against grampositive and gram-negative bacteria, including s. marcescens. snit-orland and kushmaro (2009) and kvennefors et al. (2011) also showed, using agarplating experiments, that vibrios and pseudoalteromonas bacteria isolated from several scleractinian and gorgonians corals, produced antibacterial agents against gram-positive and gramnegative bacteria. quorum-sensing (qs) qs is a density dependent communication mechanism that coordinates the collective behavior of bacteria (fuqua and greenberg, 2002; chen et al., 2011; golbert et al., 2013). the qs communication mechanism in bacteria relies on hormone-like compounds that control the expression of genes involved with certain processes such as virulence, swarming, biofilm formation and others (skindersoe et al., 2008; tait et al., 2010; golberg et al., 2011, 2013; hunt et al., 2012; krediet et al., 2013). qs can be detrimental to hosts, if the behavior promoted by qs signaling activates the expression of virulence factors. however, the qs system may be beneficial to the host when the qs signals promote the production of compounds that regulate the growth of other noxious microbes. qs signals from bacteria associated with coral mucus have been demonstrated in numerous studies. for instance, golberg et al. (2011) reported that nearly 30 % of bacteria associated with coral mucus from several scleractinian and gorgonian corals, exhibited the production of qs signals. tait et al. (2010) also reported qs signaling in vibrios associated with diseased and healthy scleractinian corals. however, only one study has documented the ability of coral associated bacteria to inhibit quorum-sensing systems and thereby the progression of s. marcescens in sea anemone aiptasia pallida as a result of qs inhibition (alegely et al., 2011). the roles of other microbes found associated with corals such as viruses, archaeans, protozoans and fungi are unknown or undescribed, other than saying that members of some of these groups are known coral pathogens (cróquer et al., 2006; toledohernandez et al., 2012). fortunately, as culturing and molecular techniques continue to improve, so does our knowledge regarding microbes and their relevance to coral immune system function. conclusions corals are by no means simple creatures in terms of immune systems functionality. on the contrary, they possess a rather complex immune system that integrates a diverse array of recognition receptors and genes whose expression mediates the synthesis of countless cytotoxic compounds and cell responses that ultimately protect the coral from threats. it is also true that we are still ignorant of much of the detail of the coral’s immune system, although many advances have been made thanks to modern molecular techniques. clearly, future studies need to address, how climate changes will influence the coral immune system and its holobiont. in this regard, studies focusing on how environmental stressors such as elevated water temperature, and ultra violet radiation among others affect: 1) the capacity of corals to recognize threats, 2) the expression of immune-related genes and thus the synthesis of chemical compounds with microbial activities; 3) the base-line levels of immune constituents and 4) the microbial community associated. answering these questions will allow us to better understand the fate of corals under the current global changes scenario.   325 acknowledgements this study was 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the caribbean reef-building oral montastraea faveolata (anthozoa, scleractinia). dis. aquat. org. 87: 45-55, 2009. work tm, aeby gs. wound repair in montipora capitata. j. invertebr. pathol. 105: 116-119, 2010. 328 http://www.ncbi.nlm.nih.gov/pubmed?term=palmer%20cv%5bauthor%5d&cauthor=true&cauthor_uid=22896649 http://www.ncbi.nlm.nih.gov/pubmed?term=traylor-knowles%20n%5bauthor%5d&cauthor=true&cauthor_uid=22896649 http://www.ncbi.nlm.nih.gov/pubmed/22896649 http://link.springer.com/search?facet-author= gonzalez-santoyo i, córdoba-aguilar a. phenoloxidase: a key component of the insect immune system. entomol. exp. appl. 146: 1-16, 2012. microsoft word isj430   isj 13: 205-209, 2016 issn 1824-307x short communication application of maldi-msi for detection of antimicrobial peptides in tissues of the marine invertebrate arenicola marina al maltseva1, vv starunov1, pa zykin2 1department of invertebrate zoology, st petersburg state university, st petersburg, russia 2department of cytology and histology, st petersburg state university, st petersburg, russia accepted june 13, 2016 abstract maldi imaging mass-spectrometry (maldi-msi) is a highly informative approach combining morphology with molecular data. it is widely applied in neuroscience, plant science, cancer-biology, biomedicine, including clinical, and preclinical studies, but not for investigation of endogenous peptides/proteins or metabolites in marine invertebrates. we examined the informativeness of maldimsi for analysis of distribution of antimicrobial peptides (arenicins) in the polychete arenicola marina and concluded that it can be successfully used as a primary rough express screening method. key words: maldi-msi; antimicrobial peptides; polychete immunity; arenicola marina   introduction antimicrobial peptides (amps) are key players in innate immunity of diverse organisms, contributing to both effector and regulatory functions. they are among immune effectors most intensively studied during last 30 years (harder and schröder, 2016). amps are relatively small (not exceeding 100 amino acids) usually cationic polypeptidic molecules with a prominent inhibitory potential against various microbial pathogens. although amps were identified in a wide range of organisms including plants, vertebrate and invertebrate animals, protists and prokaryotes (boman, 2003; reddy et al., 2004; yount et al., 2006; otero-gonzález et al., 2010; pasupuleti et al., 2012; harder and schröder, 2016) different taxa are still very unequally studied in terms of amps diversity and functioning, and immunology in general. the wide distribution of amps makes them an attractive object for comparative immunology and description of amps functioning in less studied taxa (among which many marine invertebrates, e.g., polychetes) is in high demand. being very important in respect of both practical and theoretical points of view, amps attract attention in different aspects: structure, structurefunctional interrelation, mechanism of action, spatiotemporal pattern of expression and mechanism ___________________________________________________________________________ corresponding author: arina l maltseva department of invertebrate zoology st petersburg state university universitetskaya 7/9, st petersburg, 199034, russia e-mail: arina.maltseva@spbu.ru of its regulation. in this respect, the widening of methodological background for amps investigation in different taxa is an urgent task. maldi imaging mass-spectrometry (maldimsi) is a comparatively young and highly informative approach superposing morphological and molecular data, which was called "molecular histology" (stoeckli et al., 2001; walch et al., 2008). it allows characterization of spatial distribution of a wide spectrum of components (such as proteins, peptides, lipids, glycanes, hormones, secondary metabolites) in situ in a crude tissue material label free, without laborious procedures of processing histochemical slides, obtaining antibody and staining. the pioneering applications of maldi-msi for analysis of specific peptides in whole cells were performed on neuronal tissue of snails (jimenez et al., 1994; dreisewerd et al., 1997). nearly in that period, the effectiveness of maldi-msi usage in fresh tissue sections was proved (caprioli et al., 1997). since then maldi-msi became a routine method in neuroscience, plant science, cancerbiology, biomedicine, including clinical and preclinical studies (e.g., baluff et al., 2011; salzet et al., 2012). recent advances include in situ trypsinization for protein profiling, even in formalinfixed tissues (de sio et al., 2015), the use of specialized matrices for small molecules (shanta et al., 2012) and in situ derivation for direct detection of some neuromediators (shariatgorji et al., 2015). however, the applications of maldi-msi for investigation of endogenous peptides/proteins or metabolites in invertebrates are still not so numerous (e.g., esquenazi et al., 2008; bruand et 205    mailto:arina.maltseva@spbu.ru   fig. 1 maldi-msi visualization of arenicins (2,758.3 da ± 2da) in anterior (a, b) and main body (c, d, e) parts. a, c densitometry imaged slices; b, d arenicin-positive ms-signal in anterior or main body parts, respectively; e representation of principal body compartments by filtered ms-signals: celomic fluid (arenicins 2,758.3 da ± 2da, red channel), epithelium of body wall (m/z 1,212.9 ± 1, blue channel) and celothelium (1,006.3 ± 1, green channel). al., 2011); in respect to amps of invertebrates they are unique (kuhn-nentwig et al., 2014). this motivated us to examine the applicability and informative value of maldi-msi for characterization of distribution of amps (arenicins) in the marine invertebrate arenicola marina. material and methods animals adult lugworms (approx. 15 cm length) arenicola marina were collected from wild populations in the intertidal zone in the vicinity of the white sea marine biological station of saint petersburg university (russia). animals were maintained in permanently aerating static tanks with marine water for 5 7 days. tissue preparation for preparation of cryo-slides worms were frozen in liquid nitrogen after being anesthetized in 5 % mgcl2 in sterile marine water. immediately after freezing, cross-sections (15 µm thick) of anterior (first chaetigerous segments) and middle body (gillcarrying segments) parts were prepared with cryomicrotome (leica cm 3050s) at -18 °c. sections were thaw mounted onto indium tin oxide coated glass slides and drayed under vacuum in labconco speedvac for 30 min. fiducial points for microscopic and maldi-msi co-registration were drawn with edding 750 white marker and preparations were subsequently imaged with biorad gs-800 calibrated densitometer at 600 dpi. for calibration purposes, bruker peptide calibration standard ii was deposited near feducual points. sections were matrix-coated in automated matrix sprayer bruker imageprep (program version 2.0.1) using build-in “hcca_nsh04” protocol with matrix solution containing 7 g/l a-cyano-4-hydroxycinnamic acid (cas 28166-41-8), 50 % v/v acetonitrile and 0.2 % v/v trifluoroacetic acid. maldi-pictures obtaining and processing. spectra were obtained on maldi-tof bruker ultraflextreme mass-spectrometer with bruker flexcontrol v. 3.3 and fleximaging v. 3.0 software. laser intensity was set to 60 %, laser spot size was set to “small”, raster size 100 µm. actual laser spot size measured by matrix ablation was around 10 µm. spectra were collected in reflected mode with positive polarity, mass window 700 3,500 da, pulsed ion extraction delay 80 ns, matrix ions with masses less than 700 da were deflected. the preparation was scanned in bidirectional mode with 1000 shoots per pixel and random walk pattern inside pixel. maldi-msi and optical images were aligned in fleximaging by previously marked fiducial points. method was calibrated on spots previously 206      fig. 2 hematoxylin-eosin staining after maldi-imaging. a, b cross-sections through anterior (a) and middle (b) parts of the body; c fragment of the body wall; d longitudinal body musculature and celothelium; e, f celomocytes. abbreviations: c celomic cavity, cc celomocytes, ch chaetae, cm circular body musculature, coe celomic epithelium, cu cuticle, cue cuticular epithelium, d dissepiment, dbv dorsal blood vessel, g gut, lm longitudinal body musculature, vbv ventral blood vessel, vnc ventral nerve cord. deposited with bruker peptide calibration standard ii. spectra were preprocessed by baseline subtraction using tophat algorithm, no smoothing was done, spectra were normalized by total ion count (tic), peak-picking was done with snap algorithm. histology and light microscopy after maldi imaging slides were washed in ethanol to remove the matrix, stained with ehrlich’s hematoxylin-eosin following standard protocol and mounted in dpx mounting medium. the slides were examined with leica dm2500 light microscope equipped by leica dfc495 camera. results and discussion arenicins are short (21 residue peptides, 2.8 kda) peptides with strong antimicrobial activity, originally isolated from motile phagocytic cells of celomic fluid celomocytes (ovchinnikova et al., 2004). the spatiotemporal pattern of arenicins expression in lugworm body was later thoroughly characterized with immunohistochemistry and semiquantitative rt-pcr. although the expression of arenicins takes place in different compartments, the strongest signal was detected in cells of celomic fluid (maltseva et al., 2014). this allows to compare present results with earlier work to determine the applicability and scope of the approach chosen here. maldi-msi is used in either linear or reflected mode, the later with higher resolution. arenicins are short enough to be analyzed in reflected mode of tof/ms with resolution up to 20,000. identification of arenicins was done by previously known by lcesi-qtof mass of 2,758.3 da with mass window ± 2da (figs 1b, d, e red channel). for better structure representation automatic mass list filtering was done revealing two m/z of 1,212.9 ±1 and 1,006.3 ±1 colocalized with epithelium of body wall and celothelium respectively (fig.1e blue and green channels). correspondence of ms-signals to particular tissues/cells was established on the basis of histological staining of maldi-scanned slides (fig. 2). the staining indicates acceptable preservation of all compartments from which reliable signals were detected epithelium and musculature of the body wall (2c), celothelium (2d) and its derivates, gut (2a, b), celomocytes (2e, f). figure 1 shows that arenicin-positive signal in both anterior (1a, b) and main body (1c, d, e) parts was detected in celomic fluid, where strong arenicins expression was described. no specific signal was obtained from other body compartments, where arenicins were reported to be expressed (epithelium of the body wall and gut, extravasal 207      tissue, cuticle, ventral nerve cord (maltseva et al., 2014). the result obtained with maldi-msi only partially reproduced the previously reported data. there could be several possible not mutually exclusive explanations for this. (1) differential abundance of arenicins in different tissues. previous results demonstrated that the arenicins are more abundant in celomocytes than in other cells types (maltseva et al., 2014). the strength of the signal from body cavity was not maximal, but moderate (which could be the negative consequence of salt presence in tissues of marine animals). this allows to suspect that in the other compartments the abundance of arenicins was below the limit of detection. similarly, only amps ctenidins highly abundant within granules of spider hemocytes were detectable by maldi-msi, but not defensins, stored in small amounts (kuhn-nentwig et al., 2014). (2) differential accessibility of arenicins in different tissues. celomocytes of polychetes are rather fragile cells. thus, they are easily destroyed during contact with exogenous material as was reported, e.g., for celomocytes of nereis diversicolour during the process of implant encapsulation (porchet-hennere et al., 1987). this is also the case for a. marina celomocytes (our own observation). so, possibly arenicins are more easily extracted into the matrix from celomocytes than from other tissues this makes them detectable in these cells. this explanation does not exclude the first one. conclusion the first application of maldi-msi for study amps in marine invertebrates was performed and reported here. the obtained data additionally confirm that celomocytes are the major compartment of arenicins expression. although the present results only partially reproduced the output of alternative methods, the main compartment of arenicins expression was correctly detected. consequently, maldi-msi can be successfully used as primary rough express screening approach for characterization of distribution of amps in tissues of marine invertebrates. acknowledgments the opportunities for maldi-msi were provided by “center for molecular and cell technologies” research park, st. petersburg state university, russia. the work was supported by the russian foundation for basic research (rfbr, research grant № 16-34-60134). we thank dr m varfolomeeva for comments on the manuscript. references balluff b, schöne c, höfler h, walch a. maldi imaging mass spectrometry for direct tissue analysis: technological advancements and recent applications. histochem. cell biol. 136: 227-244, 2011. boman hg. antibacterial peptides: basic facts and emerging concepts. j. internal. med. 254: 197215, 2003. bruand j, sistla s, mériaux c, dorrestein pc, gaasterland t, ghassemian m, et al. automated querying and identification of novel peptides using maldi mass spectrometric imaging. j. proteome res. 10: 1915-1928, 2011. caprioli rm, farmer tb, gile j. molecular imaging of biological samples: localization of peptides and proteins using maldi-tof ms. analyt. chem. 69: 4751-4760, 1997. de sio g, smith aj, galli m, garancini m, chinello c, bono f, et al. maldi-mass spectrometry imaging method applicable to different formalinfixed paraffin-embedded human tissues. mol. biosyst. 11: 1507-14, 2015. dreisewerd k, kingston r, geraerts wp, li kw. direct mass spectrometric peptide profiling and sequencing of nervous tissues to identify peptides involved in male copulatory behavior in lymnaea stagnalis. int. j. mass spectrom. ion processes 169: 291-299, 1997. esquenazi e, coates c, simmons l, gonzalez d, gerwick wh, dorrestein pc. visualizing the spatial distribution of secondary metabolites produced by marine cyanobacteria and sponges via maldi-tof imaging. mol. biosyst. 4: 562-570, 2008. harder j, schröder jm (eds) antimicrobial peptides: role in human health and disease, springer international publishing, switzerland, 2016. jimenez cr, van veelen pa, li kw, wildering wc, geraerts wpm, tjaden ur, et al. neuropeptide expression and processing as revealed by direct matrix-assisted laser desorption ionization mass spectrometry of single neurons. j. neurochem. 62: 404-407, 1994. kuhn-nentwig l, kopp ls, nentwig w, haenni b, streitberger k, schürch s, et al. functional differentiation of spider hemocytes by light and transmission electron microscopy, and maldims-imaging. dev. comp. immunol. 43: 59-67, 2014. maltseva al, kotenko on, kokryakov vn, starunov vv, krasnodembskaya ad. expression pattern of arenicins the antimicrobial peptides of polychaete arenicola marina. front. physiol. 5: 497-503, 2014. otero-gonzález aj, magalhães bs, garcia-villarino m, lópez-abarrategui c, sousa da, dias sc, et al. antimicrobial peptides from marine invertebrates as a new frontier for microbial infection control. faseb j. 24: 1320-1334, 2010. ovchinnikova tv, aleshina gm, balandin sv, krasnosdembskaya ad, markelov ml, frolova ei, et al. purification and primary structure of two isoforms of arenicin, a novel antimicrobial peptide from marine polychaeta arenicola marina. febs lett. 577: 209-214, 2004. pasupuleti m, schmidtchen a, malmsten m. antimicrobial peptides: key components of the innate immune system. crit. rev. biotechnol. 32: 143-171, 2012. 208      porchet-henneré e, m'berri m, dhainaut a, porchet m. ultrastructural study of the encapsulation response of the polychaete annelid nereis diversicolor. cell tissue res. 248: 463-471, 1987. reddy kvr, yedery rd, aranha c. antimicrobial peptides: premises and promises. int. j. antimicrobial agents 24: 536-547, 2004. salzet m, mériaux c, franck j, wistorski m, fournier i. maldi imaging technology application in neurosciences: from history to perspectives. in: karamanos y. (ed.), expression profiling in neuroscience, neuromethods, springer science, pp 181-223, 2012. shanta sr, kim ty, hong jh, lee jh, shin cy, kim kh, et al. a new combination maldi matrix for small molecule analysis: application to imaging mass spectrometry for drugs and metabolites. analyst 137: 5757-5762, 2012. shariatgorji m, nilsson a, källback p, karlsson o, zhang x, svenningsson p, et al. pyrylium salts as reactive matrices for maldi-ms imaging of biologically active primary amines. j. am. soc. mass spectrom. 26: 934-939, 2015. stoeckli m, chaurand p, hallahan de, caprioli rm. imaging mass spectrometry: a new technology for the analysis of protein expression in mammalian tissues. nat. med. 7: 493-496, 2001. walch a, rauser s, deininger so, höfler h. maldi imaging mass spectrometry for direct tissue analysis: a new frontier for molecular histology. histochem. cell biol. 130: 421-434, 2008. yount ny, bayer as, xiong yq, yeaman mr. advances in antimicrobial peptide immunobiology. pept. sci. 84: 435-458, 2006. 209    minireview isj 10: 46-49, 2013 issn 1824-307x minireview the self/non-self dualism is still so marked as it was considered for a long time? m mandrioli, e ottaviani department of life sciences, university of modena and reggio emilia, modena, italy accepted may, 27, 2013 abstract for several decades the immune system has been described mainly as a molecular machinery aimed to recognize and eliminate all the non-self molecules or organisms. actually, recent evidences support the presence of a constant cross talk between the immune system and microorganisms that live within the host as symbionts resulting in the tolerance of non-self bacteria and yeasts. as a whole, the “defensive” role of immunity, described as highly prominent in several contexts of the modern biosciences, should be revised taking into account that the immune system defined during evolution which organisms have to be excluded and killed, and which have to be maintained. these new evidences support the idea that each animal is a dynamic and context-dependent entity with a mixed and tolerant self. key words: self/non-self; immunity; individual; symbiosis introduction the main property of the immune system is its ability to distinguish between not dangerous endogenous and exogenous structures (self) and harmful endogenous or exogenous entities (nonself). medzhitov and janeway (2002) reported three modalities of recognitions: “microbial non-self”, recognition of “missing self” and recognition of “induced or altered self” allowing to the innate immune system to discriminate between “infectious non-self” and “non-infectious self”. the recognition of "missing self", is based on the recognition of "markers of normal self” in which gene products and products of metabolic pathways are unique to the host and absent from microorganisms. the last strategy, i.e., the recognition of “induced or altered self”, allows the detection of markers of abnormal self that are induced by infection and cellular transformation. the discrimination between self and non-self occurs at the molecular level and it is mediated by specific cell structures, such as toll-like receptors (tlrs), receptors of t lymphocytes, mhc complexes and immunoglobulins (fig. 1). the microbial recognition is based on the presence of conserved molecular patterns produced by microorganisms referred as pathogen-associated ___________________________________________________________________________ corresponding author: enzo ottaviani department of life sciences university of modena and reggio emilia via campi 213/d, 41125 modena, italy e-mail: enzo.ottaviani@unimore.it molecular patterns (pamps), which include gram negative lipopolysaccharide and gram positive peptidoglycan. these and other pamps are recognized by receptors of the immune system and they are called pattern recognition receptors (prrs), also including tlrs. these receptors do not recognize and do not bind directly pamps, but act through other molecules resulting in a specific response for the different classes of pathogens. in mammals the recognition of lps by the macrophage receptor is realized by means of tlr4 and it involves the co-receptor cd14, localized on the membranes of macrophages, and the lpsbinding protein (lbp), present in the serum (perera et al., 2001). in drosophila melanogaster, the toll co-receptor is represented by spätzle. however, this molecule is in the form of pro-spätzle and the cleavage to spätzle and the binding to toll require the intervention of recognition molecules secreted by micetes or gram positive bacteria, that, in turn, activate a protease called spätzle-processing enzyme (spe) (lemaitre and hoffmann, 2007). a phylogenetic panorama in the social amoeba dictyostelium discoideum a specific cell type, called sentinel (s)-cells, has been suggested to play a role in both detoxification and immunity (chen et al., 2007). in particular, scells phagocytize bacteria and sequester toxins, through a toll/interleukin-1 receptor (tir) domain protein, tira. these findings suggest that an old cellular foraging mechanism is utilized for defensive functions and it supports the hypothesis about the     46 mailto:enzo.ottaviani@unimore.it fig. 1 the toll-like receptors and the nucleotide-binding oligomerization domain containing protein 2 (nod2) are involved in host defense and tissue repair responses so that their proper functioning promote the mucosal maintenance and the commensal homeostasis. in the presence of pathogens, the toll-like receptors and nod2 stimulates a pro-inflammatory response. modified from cario (2005). presence of an active system of pathogen recognition in eukaryotes before the appearance of multicellularity. sponges represent a very ancient form of multicellular animals, with filter-feeding behaviour. sponges are normally exposed to bacteria and tlrs are essential in mediating their innate immune responses among many different receptors that participate in the recognition of microbial pathogens. indeed, wiens et al. (2007) identified a tlr, a serine/threonine protein kinase (irak-41) and a novel effector caspase in the sponge suberites domuncula. alongside these studies, toll homologues have been found in arthropods, annelids and mammals (medzhitov and janeway, 2000; underhill and ozinsky, 2002; akira, 2003). the “danger” model “danger” theorists proposed the equivalence of "danger" with un-programmed tissue destruction, necrosis, or other signs associated to cellular distress (matzinger, 1998). on the basis of this "threat", the immune system would be able to recognize self from non-self avoiding the processes described by the “self-non-self” theories. the question posed by “danger” theory is indeed what happens when self changes. for example, the immune reactions against the changed tissues are not observed in organisms that undergo metamorphosis, puberty, pregnancy or aging. another example is provided by several tumors that are not rejected although many of them clearly express new or mutated proteins. even if intriguing, the “danger” theory has not been fully accepted and numerous criticisms arose by the scientific community. as reported by vance (2000) this perspective seems not only unnecessary, but potentially misleading. furthermore, the concept of “danger” is unable to support the three criteria used in rejecting the concept of self-non self because: 1) is not well defined, 2) there are many exceptions and 3) it does not consider fully the broad immunological phenomena that take part. a support in favour of the “danger” hypothesis has been obtained in experiments performed on the human eosinophilis that recognized and activated “danger” signal derived from damaged (necrotic) epithelial cells (stenfeldt and wennerås, 2004). furthermore, a relationship between degree of activation of eosinophils and the dose of necrotic epithelial cells has been observed (stenfeldt and wennerås, 2004). looking for an agreement: the interaction between the microbiome and the immune system according to a common view of immunity, the immune system should recognize and eliminate all non-self molecules or organisms. however, several evidences support the presence of a constant molecular dialogue between microorganisms and the host immune system in order to favour, for instance, both the establishment and maintenance of the intestinal gut homeostasis by promoting the tolerance of non-self bacteria and yeasts (medzhitov and janeway, 2002; cario, 2010; kaser et al., 2010). as a consequence of this interaction, the innate immune system in the gut, which includes many innate leucocyte populations and several types of intestinal epithelial cells, maintains a balanced immune response to the microbiota, despite its nonself nature (maloy and powrie, 2011). at the same time, the dysregulation of immune responses versus the commensal microbiota is related to intestinal inflammations occurring directly in consequence of disequilibria between the finely tuned proand antiinflammatory mechanisms within the gastrointestinal tract (dupaul-chicoine, 2010; maloy and powrie 2011). according to what observed in several mammals, the immune system activates tissueprotective innate defences that inhibit colonization and invasion by pathogens, but at the same time myeloid cells control circuits preventing harmful immune responses towards the intestinal microbiota (harrison, 2011). therefore, a selective modulation of the innate immune activation of downstream mediators presents an ongoing challenge to effectively target deleterious inflammatory responses whilst sparing host-protective immunity in the intestinal tissues (harrison, 2011).     47 recent studies greatly advanced our understanding of the mechanisms through which the gastrointestinal innate immune system can mediate differential host-microbial interactions in recognition and sorting of the broad luminal spectrum of diverse microbial products. in particular, toll-like receptors and nod2 are emerging as key mediators of innate host defense in the intestinal mucosa, crucially involved in maintaining mucosal as well as commensal homeostasis (cario, 2005, 2010). as reported in human gut, toll-like receptors may activate distinct signalling events via diverse cofactors and adaptor proteins mediating specific immune responses (cario, 2005). for instance, at least five different adaptor proteins have been identified in humans (myd88, mal/tirap, trif/ticam-1, tram/tirp/ticam-2, and sarm) (for review see o’neill et al., 2003) and they regulate different downstream signalling modules and interacting complexes resulting in activation of several transcription factors, such as nfκb, ap-1, elk-1, creb, stats, and the subsequent transcriptional activation of genes encoding proand anti-inflammatory cytokines and chemokines as well as induction of costimulatory molecules (cario, 2005). all of these diverse downstream effects are critically involved in the control of pathogen elimination, commensal homeostasis,and linkage to the adaptive immunity (cario, 2005). similarly, the expression of some genes related to the immune response is highly related to the microbiome composition since, for instance, microbial symbionts provide developmental signals that limit the proliferation of basophil progenitor cells and thereby prevent basophil-induced allergic responses in vertebrate, so that multiple populations of intestinal immune cells require the microbiota for their full development and function (gilbert et al., 2012; hill et al., 2012). as described by gilbert et al. (2012), the immune system may be formulated as having two “limbs”: an outward-looking limb that defines the organism that has to be protected from foreign pathogens, and an inward-looking arm that looks for potential dangers arising from within the organism itself (tauber, 1994, 2000, 2009; eberl, 2010; pradeu, 2011). this dualistic vision of immunity should be at present revised as a continuous negotiation of numerous interactions between the organism and its biotic environment (both “internal” and “external”). the “immune self” model of individuality, based on a clearly distinction of self and non-self reflecting the portray of the immune system as a defensive network against an hostile exterior world (that rejects anything that is non-self) should be revised considering animals as a sort of “mixed self”, using the metaphor suggested by pradeu (2011) or distinguishing three (in place of two) counterparts: self, non-self and nearby self, the last referring to non-self entities that are accepted as self by the immune system. interestingly, this last result is not due to an immune tolerance toward microbes nor to a strategy whereby the defensive factors are minimalized to prevent damage to the infected organism, but it relies on an active recruitment of symbiotic bacteria by the immune system (tauber, 2008a, b). in squids (mcfall-ngai et al., 2010) and mammals (hooper et al., 2012), elements of the host immune system have been co-opted to support the colonization, limitation, and persistence of symbiotic bacteria within the host. interestingly, if the immune system cannot be properly regulated to “accept” some symbionts, they need to be sequestered into specialized bacteriabearing host cells, such as the bacteriocytes, as reported in aphids (buchner, 1965). concluding remarks in accordance to the concepts reported above, the distinction between self and non-self may be today not so clear-cut as frequently thought. the “self-non-self” theory captures the favour of the majority of immunologists however, on the basis of the data reported by different laboratories, the existence of other modes of recognition should not be excluded. as lewis thomas (1974) commented discussing the concepts of self and symbiosis: “this is, when you think about it, really amazing. the whole dear notion of one’s own self marvellous, old free-willed, free-enterprising, autonomous, isolated island of a self is a myth”. as commented by gilbert et al. 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http://plato.stanford.edu/entries/biologyself/ isj 8: 5-14, 2011 isj 8: 5-14, 2011 issn 1824-307x review autophagy in lepidoptera: more than old wine in new bottle g tettamanti1, y cao2, q feng3, a grimaldi1, m de eguileor1 1department of biotechnology and molecular sciences, university of insubria, 21100 varese, italy 2laboratory of insect molecular biology and biotechnology, guangdong provincial key laboratory of agro-animal genomics and molecular breeding, college of animal science, south china agricultural university, guangzhou, 510642, china 3school of life sciences, south china normal university, guangzhou, 510631, china accepted december 17, 2010 abstract autophagy is a cellular pathway that leads to the degradation of proteins and organelles. this process is usually involved in the maintenance of cell homeostasis when the organism experiences nutrient starvation, but in holometabolous insects autophagy also intervenes in the demolition of larval tissues and organs during metamorphosis. this review summarizes the current knowledge about autophagy research in lepidoptera and discusses the use of moths and butterflies as models for tudying the roles and regulation of autophagy. it also gives insights into the cooperation between utophagy and apoptosis in cell death events that occur in lepidopteran in vivo and in vitro systems. s a   key words: autophagy; lepidoptera; bombyx mori; atg optosis genes; ap   introduction autophagy is a cellular self-eating process involved in protein and organelle degradation. although three types of autophagy (macroautophagy, microautophagy and chaperonemediated autophagy) are known, the term “autophagy” usually refers to macroautophagy, which will be the focus of this review. in autophagy, a membrane, called phagophore or isolation membrane, is formed in the cell and progressively expands and grows to engulf a portion of cytoplasm. this double-membrane structure finally closes to become an autophagosome. once the autophagosome membrane fuses with lysosomes, the content is degraded and the resulting macromolecules are recycled back into the cytosol (mizushima et al., 2008) (fig. 1). all these steps are regulated by autophagy-related (atg) genes, initially identified in yeast, but lately found in all eukaryotic organisms (see he and klionsky, 2009; inoue and klionsky, 2010 for a complete description about the regulatory mechanisms of autophagy). although autophagy can potentially degrade cytoplasmic proteins and any organelles, selective organelle degradation has been described in yeast and other cell models. pexophagy (manjithaya et ___________________________________________________________________________ corresponding author: gianluca tettamanti department of biotechnology and molecular sciences university of insubria via j.h. dunant 3, 21100 varese, italy   5 e-mail: gianluca.tettamanti@uninsubria.it al., 2010), mitophagy (narendra et al., 2008), nucleophagy (park et al., 2009) and reticulophagy (bernales et al., 2007) are responsible for specific dismantling of peroxisomes, mitochondria, nucleus and endoplasmic reticulum, respectively. a basal level of autophagy occurs in most tissues to allow an adequate turnover of the cell components. notwithstanding, autophagy represents an adaptation of the cell to starvation. if cells experience nutrient deprivation, they break down a part of their content to stay alive. but in some circumstances, stress-induced autophagy exceeds the safe threshold and/or cooperates with apoptosis pathways, leading to a form of cell death characterized by the accumulation of autophagosomes, known as type ii programmed cell death (type ii pcd), where type i pcd is more classical apoptosis (gozuacik and kimchi, 2007). type ii pcd allows the elimination of large number of cells in tissues/organs and is usually associated with developmental, differentiation and tissue remodelling processes, such as in insect metamorphosis. most evidence indicates that, at least in cells with intact apoptotic machinery, autophagy is primarily a pro-survival rather than a pro-death mechanism, but this issue is still under debate (kroemer and levine, 2008). autophagy is also involved in some human diseases since it can work as a cytoprotective mechanism to prevent the insurgence of specific pathologies, or it can be deleterious allowing, for example, the proliferation of microbes that subvert autophagy for replication (mizushima et al., 2008). all these features, together   6 isolation membrane autophagosome autophagosome-lysosome fusion autolysosome lysosome proteins ribosomes mitochondria breakdown products fig. 1 schematic model of macroautophagy. after induction, a portion of cytoplasm or specific organelles are surrounded by a growing membrane (isolation membrane or phagophore) that grows progressively, thus forming an autophagosome. the autophagosome fuses with a lysosome and in the resulting autolysosome, the hydrolytic enzymes break down the inner membrane of the autophagosome and its cargo. the macromolecules derived from the enzymatic digestion are released back to the cytoplasm through permeases and reused. with the identification of autophagy genes in higher eukaryotes, have led to the reappraisal of some experimental models suitable for studying autophagy, and in the last ten years the number of papers published on this topic increased substantially (klionsky, 2007). this review presents our current knowledge on the autophagic processes that occur during growth, development and metamorphosis in lepidoptera, highlighting why lepidoptera can be an excellent model for studying autophagy, while also pointing out some issues that still need to be clarified. why study autophagy in insects? in holometabolous insects, several organs are massively remodelled or even degenerate during metamorphosis and autophagic events occur extensively to eliminate those organs and tissues that are useful only during embryonic and larval stages (tettamanti et al., 2008b; malagoli et al., 2010) (fig. 2). because of the short life cycle and the wellcharacterized genetics, drosophila melanogaster has provided a useful model to dissect the molecular mechanisms and the physiological roles of autophagy. in drosophila several atg homologues and genes regulating autophagy have been identified and this has allowed the dissection of the different autophagic events occurring in this organism. scott et al. (2004) clearly demonstrated that starvation induces an autophagic response in the fat body, a nutrient storage and mobilization organ analogous to the vertebrate liver. moreover, development-associated autophagy, that is related to an increase in ecdysteroids, has been identified in the fly during oogenesis (nezis et al., 2009) and in specific tissues, such as midgut and salivary glands (rusten et al., 2004; berry and baehrecke, 2007; denton et al., 2009). the effects of ecdysone are mediated through a heterodimeric nuclear receptor and hormone binding to the receptor complex not only activates a transcriptional regulatory hierarchy including early and late genes, but also influences the autophagic process through the ptdins3k-akt/pkb-tsc1/tsc2-rheb-tor signalling pathway (malagoli et al., 2010). the atg genes identified up until now share evolutionary conservation and they have been proven to be essential for a correct initiation and completion of autophagy. flies with mutation or inactivation of atg genes including atg1, atg2, atg3, atg5, atg6, atg7, atg12 and atg18 are unable to carry out starvation-induced autophagy or fail to degrade larval organs at metamorphosis (scott et al., 2004; berry and baehrecke, 2007). one of the most intriguing findings is that cell death events occurring in larval midgut and salivary glands are characterized by features typical of both apoptosis and autophagy (jiang et al., 1997; martin and baehrecke, 2004). in addition, pcd of the larval midgut has been demonstrated to be strictly dependent on autophagy, while the canonical apoptotic pathway is dispensable although caspases are active in the dying midgut (denton et al., 2009). these observations make the whole story more complex and suggest an intricate relationship between autophagy and apoptosis. among insects, butterflies and moths have been widely used to study processes related to metamorphosis, because the larva is amenable to perform endocrinological, electrophysiological and developmental biology studies. several lepidopteran species have been used throughout the years to analyze demolition of body tissues/organs through autophagy, as will be reported below. if we also take into consideration the establishment of a wide repertoire of new molecular tools for several species that belong to this taxon, lepidoptera certainly represent an excellent model system for tackling a broad range of questions concerning autophagy. it is also worth noting that some of the most important fig. 2 autophagy and organ degeneration in bombyx mori larvae. during metamorphosis, the fat body is massively remodelled due to the intervention of autophagic processes: the pupal fat body (b) greatly differs from the larval one (a). (c) autolysosomes containing organelle debris are visible in the cytoplasm of cells undergoing autophagy. bars = 10 μm (a, b); 1 μm (c). arrowheads indicate the nuclei. species of high economic importance, such as those being bred for commercial purposes or phytophagous pests, are represented in this insect order. therefore a deep knowledge of the processes regulating metamorphosis, and cell death in particular, in these organisms could contribute to ameliorate crop production and the textile industry. the early age of autophagy in lepidoptera…   7 the early studies of autophagy in lepidoptera from the 1960s to the early 1980s were based on morphological analyses. a few years after the appearance of the term “autophagy” (de duve and wattiaux, 1966), locke and collins described in fat body of calpodes ethlius larvae the isolation of cell organelles within paired membraned derived from golgi (locke and collins, 1965, 1968). they not only described the complete sequence of autophagosome formation, but also suggested that the isolated compartment could be a region of massive lysis. a confirmation was obtained by using specific stainings for acid phosphatase, a lysosomal enzyme whose activity was detected in these “storage granules” (larsen, 1970, 1976). this massive cellular autolysis in the larva caused a reduction of mitochondria in the pupal fat body and a deep rearrangement of this tissue before the emergence of the adult (larsen, 1970, 1976). beyond the fat body, detection of autophagic compartments and increases in lysosome number had been reported in other larval/pupal tissues/organs, including midgut (misch, 1965), wing epithelium (nardi et al., 1991), silk gland (matsuura and tashiro, 1976; tashiro et al., 1976) and intersegmental muscles (beaulaton and lockshin, 1977; lockshin and beaulaton, 1979). all those early works established a close link between autophagy and its role in cell remodelling in insects undergoing complete metamorphosis. these old papers tackled two important features of the autophagic process. the first is the endless search for the source of the isolation membrane. although golgi and endoplasmic reticulum seem to be the best candidates, recently new information has emerged suggesting that mitochondria may provide a membrane source as well (hailey et al., 2010). this intriguing hypothesis could be the confirmation of what was previously seen in the bombyx mori silk gland, where cupshaped mitochondria function as a kind of autophagosome system, contributing to the autolytic process during the prepupal phase (matsuura and tashiro, 1976). the second issue concerns lysosomes. although detection of lysosomes per se cannot be considered a sufficient evidence to demonstrate the activation of the autophagic pathway, it is undoubted that activation of the lysosomal compartment marks the onset of tissue lysis (he and klionsky, 2009). some papers show an increase of acid phosphatase granules in degenerating silk gland of b. mori (matsuura et al., 1976; tashiro et al., 1976). this enzyme is synthesized at the end of the last larval instar, stored mainly in the golgi bodies and subsequently used (matsuura et al., 1976). the increase in enzyme activity is biphasic, with one peak at spinning/prepupal period and the other at early pupal phase (matsuura et al., 1976). this trend seems to explain the ultrastructural changes occurring in the silk gland and the role of the autophagic process: early “catabolic autophagy” provides energy and raw materials just after the   8 larva has ceased to feed, while subsequent autophagy is responsible for the demolition of the silk gland which is no longer necessary once the cocoon has been formed. the fat body differs slightly as elevated levels of acid phosphatase activity are visible later (6th day of pupation) (sumithra et al., 2010). more recently b. mori cathepsin b and d have been cloned and characterized. their activity is required for pcd of the fat body and midgut at metamorphosis (gui et al., 2006; lee et al., 2009). the expression patterns of these two lysosomal enzymes are similar to that of acid phosphatase and they are induced by 20hydroxyecdysone (20e). moreover, silencing through rnai precludes correct pupation in the larvae. all these features suggest that the two proteinases could also contribute to autophagy in these two organs undergoing tissue remodelling (gui et al., 2006). along with the identification of the two main actors of the autophagic process, the autophagosome and lysosomes, during the period of the ‘70s and ‘80s, several research groups analyzed the signals and the signal transduction pathway that regulate autophagy in lepidoptera. the onset of autophagy is triggered by 20e and the injection of this hormone in the body cavity of the larva induces an increase in the numbers of secondary lysosomes and mitophagy in midgut cells (radford and misch, 1971) and the fat body (de priester et al., 1979), while ligatures applied behind the brain-ring gland complex prevents the appearance of acid phosphatase-positive granules in degenerating cells (de priester et al., 1979). the sensitivity of the cells to ecdysone is agedependent, since injection of the hormone in larvae at the penultimate instar does not induce the emergence of autophagic compartments in the fat body (sass and kovacs, 1977). experiments performed ex vivo give additional information. administration of 20e to the fat body isolated from fifth instar larvae before the programmed occurrence of autophagy (critical period) sets in motion the self-digestion process (dean, 1978). moreover, fat body taken soon after the critical period continues with the autophagic sequence in hormone-free medium (dean, 1978). this not only confirms that autophagy is induced by ecdysone, but also demonstrates that, once the cells are commited to autophagy, the process does not require the continuing presence of the hormone for its completion. sass and colleagues (sass et al., 1983) identified camp as one of the possible mediators of the autophagic response induced by 20e in mamestra brassicae. the first peak of camp content in fat body cells observed during mid-late fifth larval instar overlaps the beginning of the formation of autophagic vacuoles and both events can be prematurely induced by injecting ecdysone in larvae at the beginning of the instar (sass et al., 1983). a slight variation in camp levels was reported also in labial glands, but this change, as well as that of other secondary messengers such as cgmp and ip3, occurs after the initial commitment to self-destruction (halaby et al., 1994), thus arguing against a true role of all these molecules on the signaling cascade induced by 20e. a second conundrum concerns protein synthesis. in fact, while an early drop in protein synthesis can be measured during pcd of this tissue (halaby et al., 1994; lockshin and zakeri, 1994; zakeri et al., 1996; jochova et al., 1997), komuves and colleagues demonstrated that 20e greatly enhances protein synthesis in the midgut and its inhibition by cycloheximide determines an impairment of the autophagic process (komuves et al., 1985). …and the modern age the beginning of twenty-first century witnessed the birth of a second age for the study of autophagy in lepidoptera. the beginning of expressed sequence tag (ests) projects in various lepidoptera species, the completion of genome sequencing in b. mori, the development of rnai, stable germline transformation and viral vectors for transient gene expression (daubnerova et al., 2009; lee et al., 2009), have made it possible to analyze in depth the autophagic processes occurring in these insects and to manipulate the expression of autophagic genes that have now been identified in the silkworm. the molecular base of autophagy bioinformatics analysis performed by zhang and colleagues (zhang et al., 2009) revealed that in the b. mori genome there are homologs of most of the atg genes originally identified in yeast and subsequently in higher eukaryotes. along with 11 atg genes, they found genes involved in the pi3k i and pi3k iii signal transduction pathway and in the formation of autophagosomes. in particular, most of these genes are involved in the two ubiquitin-like conjugation systems, atg8-pe and atg12-atg5atg16. the number of genes regulating autophagy has been recently widened by the identification of two paralogous target of rapamycin (tor) genes, bmtor1 and bmtor2 (zhou et al., 2010) (table 1). several of these genes are actively transcribed in different tissues during development and metamorphosis, or are up-regulated by starvation. the expression levels of bmatg8 and bmatg12 in silk gland show a progressive increase, reaching a plateau at pre-pupal stage, when autophagic features become evident (zhang et al., 2009). a similar pattern for these genes is observable in larval midgut cells that die at metamorphosis (cao y, unpublished data). it is worth noting that four atg genes (bmatg1, bmatg5, bmatg6, bmatg8) are expressed at high levels in b. mori peritracheal athrocytes (owa et al., 2008). these cells contribute to the regulation of the composition of hemolymph through the removal of certain molecules and therefore these genes could be involved in heterophagic lysosomal degradation, helping to maintain homeostasis during the development of the larva. both bmtor genes are up-regulated by two autophagy-promoting signals, starvation and 20e, although with different sensitivity (he and klionsky, 2009; zhou et al., 2010). since in higher eukaryotes the pathways through which hormones and nutrients regulate autophagy are different, but both converge   9 table 1 list of the principal autophagy-related genes identified in bombyx mori gene accession number protein function reference regulation of autophagy induction bmtor1 bmtor2 gu350772* gu350773* negative regulators of autophagy, rapamycin target zhou et al., 2010 bmatg1 bgibmga011986§ ser/thr protein kinase owa et al., 2008 autophagosome nucleation bmatg6 fj416328* component of class iii pi3-kinase complex owa et al., 2008 autophagosome expansion and completion bmatg3 fj416327* conjugates phosphatidylethanolamine to atg8 zhang et al., 2009 bmatg4 fj416326* cleaves atg8 at c terminus zhang et al., 2009 bmatg5 fj418152* conjugated to atg12 owa et al., 2008 bmatg7 bgibmga001467§ activates atg8 and atg12 zhang et al., 2009 bmatg8 fj416330* ubiquitin-like protein conjugated to phosphatidylethanolamine owa et al., 2008 bmatg12 fj416329* ubiquitin-like protein conjugated to atg5 zhang et al., 2009 bmatg16 bgibmga006504§ component of atg5-atg12 complex zhang et al., 2009 retrieval zhang et al., 2009 bmatg9 bgibmga012307§ interacts with atg2 zhang et al., 2009 bmatg18 bgibmga007298§ required for atg2 localization zhang et al., 2009 sequences from genebank are indicated with an asterisk, while those from silkdatabase (http://silkworm.genomics.org.cn/) are indicated with § on tor (he and klionsky, 2009), it is reasonable to hypothesize a key role for silkworm tor in the signalling pathway that regulates autophagy. growing interest in atg genes has led to the recent derivation of the crystal structure of bmatg8 (hu et al., 2010). atg8 is an ubiquitous protein among eukaryotes and, after its recruitment to the phagophore, is involved in the membraneexpansion step. bmatg8 has several residues and an ubiquitin-fold domain at c-terminus conserved in different species, thus implicating a central role in the autophagic pathway. although with small differences, such as the absence of an identifiable bmatg10, the identification, expression and structural characterization of the 25 autophagyrelated genes in the silkworm confirm the existence of a well-organized autophagy pathway in this insect (zhang et al., 2009), while the exact mechanisms of action of the autophagy pathway remain to be elucidated. the sequential gene activation triggered by 20e that leads to pcd in b. mori tissues has been characterized as well. the effects of 20e are mediated by a heterodimeric nuclear receptor formed by the ecdysone receptor (ecr) and ultraspiracle (usp). among the three isoforms of ecdysone receptor, ecr-b1 has been shown to actively take part in the onset of death processes in the silk gland. its protein levels reach a maximum just before the larval to pupal transformation when autophagosomes appear (goncu and parlak, 2009). although the main early and late genes involved in the cis-regulation downstream of ecr in b. mori are similar to that of drosophila (sekimoto et al., 2006), the recruitment of these genes is different in the two insects. this difference in behaviour is particularly evident when the regulation of autophagic and apoptotic process within the same tissue is dissected. accordingly, li et al. (2010) demonstrated that in the silk gland the expression of bmecr, bme74a, bme75c and bmbr-c peaks at the onset of the autophagic process, while bmbftz-f1, bmhr39 and bme75b are more likely involved in the initiation of apoptosis. a complex intertwining between autophagy and apoptosis a phenomenon that has been widely described in lepidopterans is that in many organs undergoing death during metamorphosis, autophagic and apoptotic features coexist: dna fragmentation, apoptotic nuclei and caspase activation have been detected in silk gland (goncu and parlak, 2008; li et al., 2010), fat body (muller et al., 2004; sumithra et al., 2010), midgut (tettamanti et al., 2007a, b; vilaplana et al., 2007) and other tissues (dai and gilbert, 1999; hoffman and weeks, 2001; kinch et al., 2003; mpakou et al., 2006, 2008) where autophagy has been shown to play a key role in their degradation. nuclear condensation and dna cleavage, the presence of unusual apoptotic bodies and the absence of identifiable phagocytes that cleanse the tissue by removing cell debris in certain organs, are three elements that are not typically seen in autophagy-mediated cell death. also the involvement of active caspases is under investigation, since it does not seem to be a feature exclusively linked to apoptosis. in fact, the larval midgut at the pupal stage exhibits positive immunostaining with an antibody specific for cleaved caspase-3 (tettamanti et al., 2007a; vilaplana et al., 2007), and ovarian nurse cells that degenerate during oogenesis are labelled by redvad-fmk, a specific in situ assay for activated http://silkworm.genomics.org.cn/   10 caspases (mpakou et al., 2006, 2008). expression of bmcaspase-c was also assessed by real-time pcr analysis in b. mori silk gland (li et al., 2010) and administration of a specific caspase inhibitor to dying motoneurons impairs the late phase of cell death which is autophagy-dependent (hoffman and weeks, 2001). the opposite situation has instead been described in manduca sexta fat body, where no evidence of activity of executioner caspases, such as caspase-3 and 7, was found (muller et al., 2004). an interesting alternative to the involvement of caspases in cell death processes mediated by autophagy has been provided by lockshin and colleagues (facey and lockshin, 2010). in their attempt to ascertain if type ii pcd really exists, they verified the possible occurrence of caspase action in m. sexta labial glands at metamorphosis. although no caspase activity was detected, they demonstrated an increase in lysosomal proteolytic activity when the gland disintegrates. they suggest that lysosomal proteases, cathepsin b in particular, may play the major proteolytic role similar to the apoptotic caspase cascade in mammals, since they cleave parp at late phases of the death process. summarizing these data, at least two settings that link autophagy and apoptosis in lepidoptera can be outlined: i) autophagy associated pcd, where autophagy is the driving force that promotes cell death and, although the autophagic machinery is functional, it does not involve precocious cytochrome c release, apoptosome formation and caspase recruitment (facey and lockshin, 2010); ii) caspase-dependent autophagic cell death that involves activation of effector caspases after loss of mitochondrial function (hoffman and weeks, 2001; kinch et al., 2003). there is also a series of situations in which the borders are not clear, since autophagy can be accompanied by dna fragmentation or nuclear condensation (dai and gilbert, 1999; muller et al., 2004), but no activation of executioner caspases (muller et al., 2004). the overlap between autophagy and apoptosis and the evidence that some morphological, biochemical and molecular features are not exclusive to either autophagy or apoptosis (berry and baehrecke, 2007; nezis et al., 2009) have led to a search for possible mediators common to these two processes. thus, besides a role for caspases in autophagic cell death, other factors such as inhibitor of apoptosis protein (iap) are now hypothesized to intervene in this self-digesting process. not only iap expression modifies during midgut remodelling in lepidoptera (parthasarathy and palli, 2007; vilaplana et al., 2007), but the iap protein bruce in drosophila is fundamental to autophagic processes (hou et al., 2008) and could provide a mechanistic link between autophagy and cell death (nezis et al., 2010). among the autophagic genes, atg5 has been suggested to be a molecular link between autophagy and apoptosis in mammals, since it plays a role in autophagosome formation, and is also involved in a pro-apoptotic signalling pathway through cytochrome c release and caspase activation (yousefi et al., 2006). in the b. mori silk gland, the expression pattern of bmatg5 suggests that it may function as a switch between autophagy and apoptosis during the prepupal stage as well (li q, personal communication). having demonstrated a concerted action between autophagy and apoptosis in the removal of larval tissues in lepidoptera, the obvious question is what could be a possible explanation for this synergy? an interesting paper by eisenberg-lerner et al. (2009) analyzes this issue and concludes that the co-occurrence of autophagy and apoptosis within the same tissue does not seem to represent mere redundancy. autophagy might cooperate with apoptosis to lead to cell death. in this way autophagy could work as a back-up system to ensure cell death if the apoptotic process failed, but it could also establish a partnership with apoptosis to maximize the death process. alternatively, autophagy and apoptosis could have different goals and autophagy would act as a pro-survival mechanism that helps cells to maintain homeostasis until a point of no return, after which apoptosis is activated and the cell dies. a third possibility is that autophagy may enable apoptosis by participating in the regulation of some molecular mechanisms of the apoptotic machinery. although all three of the hypotheses can be envisaged for lepidoptera, the current lack of sufficient information about the molecular mechanisms underpinning the interconnection between apoptosis and autophagy, and the complexity of the phenotypic features evidenced in the dying tissues in the larvae, do not yet allow us to disentangle this problem. on the other hand, the observation of both processes in almost all larval tissues of butterflies and moths provides experimental models ad hoc to address this issue. the recent identification of a large series of apoptotic factors in silkworm (zhang et al., 2010) could add new information about the connections between autophagy and apotosis in this animal model. two friendly models for studying autophagy the larval midgut is one of most used organs for studying cell death processes and autophagy in lepidoptera. the midgut represents the middle part of the alimentary canal of the insect and it is mainly involved in the digestion and absorption of nutrients. during metamorphosis, the epithelium formed during larval life is progressively displaced by a new epithelial layer that grows underneath and finally becomes the pupal midgut. the old tissue degenerates and forms the yellow body, a compact mass that is sloughed in the gut lumen, where the cells undergo cell death (tettamanti et al., 2007a; hakim et al., 2010). mitochondrial activity disappears in these cells (tettamanti et al., 2007a), dna fragmentation becomes visible (tettamanti et al., 2007a; vilaplana et al., 2007) and the expression levels for the anti-death factor iap are reduced along with an increase in caspase-1 mrna expression (parthasarathy and palli, 2007). positivity for cleaved caspase-3 antibody can be detected in these cells at an early stage of degeneration (tettamanti et al., 2007a; vilaplana et al., 2007) and the mrna of ice, the homologue of mammalian caspase-3, peaks during the prepupal/pupal stage, when apoptosis occurs in the yellow body (parthasarathy and palli, 2007). the   11 action of ice downstream of caspase-1 suggests that these caspases carry out various functions in the pcd machinery of larval midgut. autophagy contributes actively to this scenario and seems to be essential for the death of midgut cells. although dsrna-mediated interference (rnai) of atg genes such as has been carried out in drosophila (denton et al., 2009) has not yet been undertaken in lepidoptera, several results strengthen this hypothesis. autophagic compartments are visible in midgut cells during the whole pupal period (tettamanti et al., 2007a), in addition to observed increases in lysosomal enzymes in the cytoplasm (tettamanti et al., 2007a; vilaplana et al., 2007). moreover bmatg5, bmatg6 and bmatg8 are highly espressed at the prepupal stage, preceding the activation of autophagy in midgut cells (cao y, unpublished data). thus the midgut represents a valuable model for studying the relationships between autophagy and apoptosis, for analyzing development-related autophagy regulated by ecdysone, and for investigating starvation-induced autophagy since these cells are directly responsible for nutrient digestion in the larva. the midgut is also the site of entry for many pathogens in the larva and is the first barrier towards exogenous agents. since autophagy helps the cells to cope with stress that perturbs tissue homeostasis, this organ can also provide insights into the role of autophagy under pathological conditions. it must also be underlined that the midgut is more amenable to rnai approaches than other organs (huvenne and smagghe, 2010), therefore a functional screening of bmatg and other autophagy-related genes in this tissue is certainly conceivable. for b. mori and other lepidopterans, a large array of cell lines have been derived from the main tissues undergoing programmed cell death (sudeep et al., 2005; zhong et al., 2005). along with the undisputed advantages of experimentation in cell lines, the efficacy of in vitro systems to disentangle the intricate network between autophagy and apoptosis has already been demonstrated in the study of cell death processes of two different lepidopteran species (tettamanti et al., 2006; liu et al., 2007). in the iplb-ldfb cell line, established from lymantria dispar fat body, it is possible to selectively direct cells towards either apoptosis or autophagy (malagoli, 2008; tettamanti and malagoli, 2008). the same cell line has allowed the analysis of the effects of energy deprivation induced by glucose starvation (liu et al., 2007), or obtained by treating the cells with oligomycin a, an inhibitor of the mitochondrial atp synthase (tettamanti et al., 2006, 2008a). when spodoptera litura cells (sl) are deprived of glucose, the appearance of autophagic vacuoles is observed. autophagy can be reverted by readdition of glucose to the medium, but if starvation is prolonged for more that 48 hours, apoptosis initiates (liu et al., 2007). a quite different situation takes place in iplb-ldfb cells. although these cells have an apoptotic pathway that resembles that of mammals and apoptosis can be induced by suitable treatments (malagoli, 2008), administration of oligomycin a to the cells for 2 hours followed by different retrieval periods in control medium, determines the insurgence of apoptosis, necrosis, oncosis or autophagy. within 24 h, a small percentage of cells are positive for all four processes, but starting from 48 h autophagic cells become the predominant phenotype within the cell population (tettamanti et al., 2006). mitochondria are the primary target of autophagy and the concomitant increase in reactive oxygen species (ros) production generates a positive feedback loop that leads to cell death. despite the modified configuration of the actin cytoskeleton to try to preserve the remaining undamaged mitochondria, a massive decrease in cell number can be observed (tettamanti et al., 2008a). in this case, the dual role of autophagy can be easily assessed. the early pro-survival function of the autophagic process that enables the use of intracellular resources under starvation conditions and removes damaged mitochondria from the cytoplasm is rapidly overtaken, and autophagy becomes associated with cell death mechanisms. a proteomic analysis has been undertaken in these cells with the aim of identifying specific signals able to induce cell death (malagoli et al., 2009). in accordance with the autophagic mechanisms observed in dying cells, an up-regulation of proteins involved in cell metabolism, oxidative stress response and cytoplasm dynamics was found. but the most interesting result is a drastic reduction of imaginal disk growth factor (idgf)-like secretion after oligomycin treatment. thus, abolished release of this pro-survival factor could be associated with the activation of a signaling pathway leading to cell demise. unfortunately to date no lepidopteran cell lines in which ecdysone induces autophagy have been described. this could be mainly due to the fact that some lepidopteran cell lines are ecdysteroidresistant, while some others show differential responsiveness to these hormones (zhong et al., 2005; swevers et al., 2008). however, this is a gap that must be filled since this tool could be useful for studying developmental autophagy in vitro. what next? although much knowledge regarding autophagy in lepidoptera is available, many questions remain to be answered. 1) what is the role of programmed autophagy in larval tissues? autophagy could be necessary to achieve cell death in organs and tissues not readily accessible to phagocytes (dai and gilbert, 1999). alternatively, autophagy could help the apoptotic process by enhancing its efficacy or perhaps by offsetting its inefficiencies, but in any case is necessary to obtain large-scale histolysis (mpakou et al., 2006; tettamanti et al., 2007b). 2) can starvation have a role in developmental autophagy? programmed autophagy in larval tissues is switched on by ecdysteroids, but given that this self-digestion process is concomitant with the food starvation period that the larva experiences during metamorphosis, can nutrient deprivation contribute at least to maintain the autophagic process active, once started by the hormone signal? in other words, is it hormone signals or starvation   12 which plays the most important and direct role in the autophagic process in larval tissues/organs? 3) does autophagy support the development of certain new tissues that originate in the pupa and form the adult organs? the evidence obtained in the midgut suggests that the molecules derived from the massive autophagic digestion of the larval cells are absorbed and recycled by the new forming pupal midgut (tettamanti et al., 2007a, b), but conclusive proof of this hypothesis is still lacking. 4) is it possible to identify any factors that can switch autophagy from a pro-survival to a pro-death role as happens in iplb-ldfb cells? 5) in biological systems where autophagy coexists or cooperates with apoptosis, which are the molecular signals specific for initiating autophagy and apoptosis, rather than the mediators that regulate this cross-talk? 6) since in different tissues autophagy and apoptosis can intervene simultaneously or in an asynchronous manner, is it possible to define exactly the timing of their action in different tissues? 7) what about the evolution of the autophagic process in different organisms, i.e., yeast, drosophila, lepidoptera and mammals? a phylogenetic analysis of the autophagy-related genes that includes a wide collection of invertebrate species is not yet available. one of the most difficult issues is that in the published literature, the description of autophagic morphological characteristics is sometimes confused and autophagy in larval tissues/organs of lepidoptera seems to have its own peculiar features that are slightly different from those already found in yeast, drosophila and mammals. in order to have an accurate characterization and definition of the autophagic process in this model system, opportune biochemical and molecular markers are necessary. the present and future work in lepidoptera is and will be focused on the search for genes and proteins that initiate and regulate autophagy, as well as the identification of the complex interactions that relate autophagy and apoptosis. this will help us to understand what the roles are of this self-digesting process in different larval tissues both during the development of the animal and under physiological vs stress conditions. acknowledgements the authors are grateful to dr c mann for critically reviewing the manuscript and his helpful comments, and to e franzetti for image production. gt’s work was supported by the italian ministry of university and research (prin 2008, protocol 2008smmcjy) and by far 2009-2010 grants from the university of insubria. yc and qf were supported by grants from the “973” national basic research program of china (2005cb121002), the “863” national high technology and research program (no. 2006aa10a119; 2004aa2z1020). references beaulaton j, 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mori. bmc mol. biol. 11: 611, 2010. zhong y, imanishi s, kawasaki h. ecdysone responsiveness of several cell lines derived from bombyx mori. j. insect biotechnol. sericol. 74: 117-123, 2005. zhou s, zhou q, liu y, wang s, wen d, he q, et al. two tor genes in the silkworm bombyx mori. insect mol. biol. 19: 727-735, 2010. sessioni: isj 11: 54-65, 2014 issn 1824-307x report of meeting xvth scientific meeting of the italian association of developmental and comparative immunobiology (iadci), 12 14 february 2014, department of life sciences and biotechnology, university of ferrara, ferrara, italy organizers: l abelli, a mancia, mg marchetti, d lunardi, g zuccon department of life sciences and biotechnology, university of ferrara, ferrara, italy session 1. chairmen: l ballarin, university of padua, padua, italy; p luporini, university of camerino, camerino, italy evolution of the immune system lecture on the origin of lymphocytes: a review g scapigliati, e randelli, s picchietti, n nuñez, v stocchi, c bernini, f buonocore department for innovation in biological, agro-food and forest systems (dibaf), university of tuscia, viterbo, italy each individual of each species must defend against pathogenic microbes and thus is armed with molecules and cells recognizing and eliminating non-self. in this respect, immune defences are as old as first eukaryotes. main gene products recognizing non-self code for molecules having immunoglobulin domains, leucine-rich repeats, peptidoglycan-recognising proteins, cysteine-rich scavenger receptors, and lectins. the cells responsible for microbial recognition and clearance are the immunocytes, as they can be defined for all animal species, but in vertebrate species new classes of immunocytes appeared as a result of evolutionary changes: the lymphocytes. lymphocytes have an extraordinary capability of selecting antigens and maintain a memory and thus are indispensable for the homeostasis of vertebrate species. a main feature of lymphocytes is the presence of rag enzymes for somatic recombination of immunoglobulin-type and leucinerich repeats molecules, however, mechanisms of somatic recombination of non-self recognition molecules are present in invertebrates with paralogous sets of genes, molecules, and effector cells. on the basis of recent discoveries in comparative immunology, and with data obtained from our group, an hypothetical origin of somatic recombination mechanisms and of lymphocytic cells will be discussed. the earlier the smaller: evidence from the study of the pheromone and pheromone-gene structures in the protozoan ciliate euplotes a vallesi1, c alimenti1, g di giuseppe2, f dini2, p luporini1 1laboratory of animal biology and eukaryotic microbiology, university of camerino, camerino, italy 2department of biology, university of pisa, pisa, italy in protozoan ciliates, the mechanism of self/non-self recognition is evolutionarily associated with the genetic mechanism of mating types. within a species, these mating types may either be only two like sexes, or multiple, with the number limited to four, seven or eight, or virtually unlimited. the nearly 100 species of euplotes, the most ubiquitous and cosmopolitan ciliate, all appear to include virtually unlimited numbers of mating types that cells express by synthesizing water-borne signaling proteins, known as pheromones. the knowledge of the structures of these pheromones and of their coding genes has for long been based only on studies of euplotes species (i. e. e. raikovi, e. octocarinatus and e. nobilii) that branch into different central positions of the euplotes phylogenetic tree. more recently, we had the opportunity to analyze the pheromone and pheromone-gene structures in two euplotes species, i.e. e. petzi and e. focardii, that lie at the opposed extremities of the tree: e. petzi (together with e. sinicus) represents the earliest branch of the tree, while e. focardii (together with the e. crassus/e. minuta/e. vannus species complex) represents the latest branch. in e. petzi, we found that the pheromone sequences extend for 32 amino acids and that the pheromone genes have extremely reduced dimensions (715 nucleotides, telomeres included), because they practically lack the regulatory 5’ region (only 72 nucleotides). in e. focardii, at the opposite, pheromone sequences extend for 85 amino acids and pheromone genes include a long regulatory 5’ region (1357 nucleotides) for an overall gene extension of 1951 nucleotides. since the dimensions of the known pheromone and pheromone-gene sequences of e.   54 raikovi, e. octocarinatus and e. nobilii are intermediary between those of their homologs in e. petzi and e. focardii, it appears that an earmark of euplotes pheromone and pheromone gene evolution is a progressive increase of structural complexity, which presumes a parallel increase in complexity of the pheromone activities and of the regulatory mechanisms of pheromone gene expression. characterization of cellular and molecular responses of actinia equina (linnaeus, 1758) mg parisi, mr trapani, a damiano, g benenati, n parrinello, m cammarata department of biological, chemical and pharmaceutical science and technology, university of palermo, palermo, italy sea anemones (actininaria, cnidaria) are benthic sessile species that depend of their toxic venom for survival, providing to defense and predation. pore-forming cytolytic hemolysins, sodium channel toxins and potassium channel toxins have been isolated and characterized from a great number of sea anemones. however little is known about toxins compositions ant their cellular origin. aim of this research is to describe the cell types of internal cavity of actinia equina and their biological activity and characterize, after purification, cytolytic and antimicrobial molecules from different tissues. extracts from mucus and tissues showed cytolytic activity toward rabbit and sheep erythrocytes and human chronic myelogenous leukemia cells k562. a new lytic peptide of about 6 kda from percoled body fluid and tentacles extract has been isolated by sec-hplc followed by mass spectrometry and identified as a neurotoxin specific for sodium channels (aei) existing in the database. then, we showed clearly that the presence of cytolysins is not exclusive of nematocysts. a plaqueforming assay carried out with cell populations extracted from the percoled body fluid showed for the first time that anthozoan granulocytes are able to form plaque of lysis. we have separated the total population of free cells into three distinct discrete bands by discontinuous percoll gradient, and we have identified different cell types. cell lysate of each cellular band showed cytolytic activity towards different erythrocytes types and was inhibited by sphingomyelin. tentacles acid extracts were subjected to purification on sep pak c8 vac column followed by several hplc runs on c18. among the several peaks, two showed biological activity towards e. coli, s. aureus and c. albicans and were pooled and lyophilized. these first results on a. equina immunobiology lead us to explore the evolution of cnidarian immune response. the ampulla is a hemocyte reservoir but not an hematopoietic organ in the freshwater snail pomacea canaliculata d malagoli, a accorsi, e ottaviani department of life sciences, university of modena and reggio emilia, modena, italy scanty information is available on hematopoiesis in molluscs. in the land slug incilaria fruhstorferi, hematopoiesis has been described in the connective tissue associated with the vascular system, in absence of a hematopoietic organ. the only hematopoietic organs retrieved in gastropods are those located into the pericardial cavity of the freshwater snails, biomphalaria glabrata and planorbarius corneus. the south american freshwater snail pomacea canaliculata has been recently banned as a pest and invading species by the eu parliament, and novel information on its biology is welcome. we have morphologically analyzed the effects of repeated hemolymph collections on the components inside the pericardial cavity of p. canaliculata: the heart, the aortas, the ctenidial/pulmonary veins, the renal veins and the ampulla. the latter is a vesicle located just beside the heart, and it is connected with the anterior aorta. the principal function of the ampulla is to prevent an exceeding pressure into the heart chambers during mollusc retraction, but a role in hematopoiesis was also proposed. after 4 hemolymph collections, repeated on the same animal after 24-h intervals, significant changes intervened in the ampullar morphology, whereas the other components remained unchanged. into control ampullae numerous hemocytes were retrievable, especially inside the follicles formed by the inner epithelium. sporadic hemocytes were observed free in the ampullar lumen. light microscopy evidenced the absence of ampullar follicles after 4 hemolymph withdrawals and a very few hemocytes were still present into the ampulla. conversely, the number of circulating hemocytes remained constant after each hemolymph collection. immunohistochemical staining were performed with the anti-p-h3 (ser10) polyclonal antibody, which marks cells committed to mitosis. in control snails numerous positive nuclei were observed along the external side of the veins and into the pericardial lumen. no positivity was observed neither into nor around the ampulla or in circulating hemocytes. the number and the distribution of positive nuclei remained unaltered after repeated hemolymph collections. our data suggest that the p. canaliculata ampulla acts as a hemocyte reservoir but not as a hematopoietic organ. hemocyte renewal rate is not influenced by withdrawals and it takes place only within the pericardial lumen in correspondence of the principal veins. this situation closely resembles the hematopoietic regions of b. glabrata and p. corneus.   55 lecture evolution of mhc-g gene in primates r rizzo department of medical sciences, university of ferrara, ferrara, italy major histocompatibility complex (mhc)-g molecules are non-classical mhc class i antigens that differ from other classical class i mhc (-a, -b and -c) molecules for: (i) limited protein variability, (ii) presence of different membrane bound and soluble isoforms, generated by alternative splicing of the primary transcript, (iii) unique molecular structure characterized by three alpha domains (α13), presenting a reduced cytoplasmic tail, (iv) modulation of the immune response, and (v) restricted tissue expression to immune privileged sites (e.g. fetal tissues, thymus). mhc-g molecules play an important role in the suppression of the immune response. in particular, they interact with leukocyte inhibitory receptors inhibiting t lymphocyte and natural killer cell activation. considering the lineage that gave rise to old world and anthropoid monkeys, the new world monkey lineage separated about 38 million years ago (mya). the cotton-top tamarin (saguinus oedipus, saoe) that inhabits the central-south american continent is a typical example of this group. saoes presumably have functional mhc-glike genes instead of mhc-a and -b genes. thus, it has been proposed that mhc-g could be the ancestral mhc class i gene. old world primates (cercopithecinae subfamily) live in eurasia and africa and their mhc-g isoforms do not bear the α2 mhc-g domain due to the existence of stop codons in homozygosis mainly at position 164, that appeared in the cercopithecinae common ancestors after the separation from the human and pongidae lineages about 35 mya. gorillas and chimpanzees do not seem to have high polymorphism at mhc-g, while orangutans, that separated from the human lineage about 15 mya show more mhc-g alleles, exhibiting variability at the t cell receptor, nk and antigen binding sites. similar to gorilla and chimpanzee, humans also present low coding region polymorphisms, encoding only 14 common proteins. the selective forces acting at the regulatory regions of the mhc-g gene seem to be different from those acting at the coding region. the pattern of variation of the promoter region is characterized by two divergent lineages of haplotypes, which has been maintained by balancing selection in worldwide human populations and may modulate the fine balance between high and low expressing mhc-g haplotypes. since each primate species seems to have undergone a particular evolutionary pathway, further research studies are needed to understand the mhc-g 40 million year evolution. the study of mhc variation and evolution in the alpine chamois (rupicapra rupicapra): from targeted sanger sequencing to genomic enrichment? s fuselli1 , l bauzer2, r tonin1, a panziera1, a benazzo1, c mazzoni2, g bertorelle1 1department of life sciences and biotechnology, university of ferrara, ferrara, italy 2berlin center for genomics in biodiversity research, berlin, germany major histocompatibility complex (mhc) proteins are essential components of the immune response, and are coded by the most polymorphic of the vertebrate genes. being mediators between the organism and the environment, mhc-coding genes are excellent candidates for exploring adaptive processes and for estimating the genetic component of resistance to infections. accurate genotyping is essential to understand the role and evolution of mhc in natural populations, but it may be extremely challenging due to gene duplication and high sequence variation of these genomic regions. recently, mhc typing has been greatly improved by the advent of next-generation sequencing methods (ngs). in particular, the high coverage of ngs (i.e., the number of time each base is sequenced) allows an accurate representation of all variants that may be present in a multilocus system. mhc regions of interest are commonly enriched by pcr, and highcoverage ngs is obtained for tens or hundreds of individuals at the same time (amplicon-based approach). alternatively, mhc loci can be captured by hybridization with a probe and then sequenced by ngs (hybrid capture-based approach). compared to amplicons, hybridization is less stringent and potentially able to capture duplicated loci. in this study we set-up a hybrid-capture enrichment coupled with ngs (illumina miseq technology) to type the genetic variation at drb-like loci in the alpine chamois (rupicapra rupicapra). the approach is based on a probe obtained by the pcr amplification of the extremely variable exon 2 using known primers, followed by a low-stringency hybridization. in parallel, ngs sequencing of amplicons has been used to check the enrichmentby-hybridization results. these approaches were tested as a follow-up of a previous sanger sequencing study of a single exon that allowed us to infer an evolutionary process of stabilizing selection in different populations. we are currently analysing about 60 alpine chamois (rupicapra rupicapra) sampled in the dolomiti bellunesi national park (eastern alps). the hybridization approach has been performed in 4 individuals and 2 pooled-samples libraries, producing about 30 million short reads that map on the sheep genome. preliminary results show that the efficiency of the capture is limited and needs to be improved. since an epidemic of sarcoptic mange had his outbreak in 2009 the eastern alps, and produced a significant decline in many populations, we will also compare healthy and mange-affected individuals to test if a specific pattern of mhc genetic variation significantly predicts resistance to the parasite.   56 the production of amyloid requires cross-talk between immunocytes in the compound ascidian botryllus schlosseri l ballarin1, r girardello2, a grimaldi2, n franchi1, m de eguileor2 1department of biology, university of padua, padua, italy 2department of biotechnology and life sciences, university of insubria, varese, italy two main immunocyte types are present in the hemolymph of the compound ascidian botryllus schlosseri, i.e., phagocytes and cytotoxic morula cells (mcs). previous studies have demonstrated that mcs work as sentinel cells able to sense foreign molecules and, as a consequence, release cytokines which activate phagocytes leading to the synthesis and release of rhamnose-binding lectin. the latter, in turn, acts as a chemotactic factor for phagocytes and opsonizes foreign particles stimulating their clearance. in addition, upon the contact with foreign molecules, mcs can degranulate and release the content of their vacuoles, mainly phenoloxidase (po) and its polyphenol substrata. in a recent investigation on botryllus cytotoxic cells, we found abundance of amyloid inside mc vacuoles which likely, once released, act as a scaffold to prevent the diffusion of po and cytotoxicity to the whole organism. in addition, the study of the molecular cascade leading to the synthesis of amyloid, is revealing a non-classical pathway in which both the phagocytes and mc are involved. session 2. chairmen: e ottaviani, university of modena and reggio emilia, modena, italy; e adinolfi, university of ferrara, ferrara, italy the fight against pathogens lecture the p2x7 receptor for extracellular atp: from mediator of inflammation to regulator of tumoral immune infiltration m capece, e adinolfi department of morphology, surgery and experimental medicine, section of experimental pathology, oncology and biology, university of ferrara, ferrara, italy adenosine triphosphate (atp) is well known for its central role in intracellular metabolic processes. however, atp can also be released in the extracellular space activating two classes of receptors: p2x ion channels and g protein coupled p2y receptors. among those, the p2x7 receptor is well characterized for its ability to mediate the production and secretion of il1β, pge2 and other inflammatory mediators. p2x7 expression has been reported in a wide range of species including fish, amphibians, birds and mammals. in all these species, the receptor is mediating immune response against pathogens activating cytokine secretion and phagocytosis. in recent years, we have demonstrated that p2x7 receptor activity can increase intracellular levels of atp and mediate its secretion. the use of sensors enabling in vivo measurement of extracellular atp, allowed associating an increase in the peri-cellular levels of the nucleotide with almost all know inflammatory situations, including graft-versus-host disease, allergic and anti-cancer immune reactions. here we show for the first time that p2x7 receptor also regulates immune infiltrate in cancer. indeed, when developing experimental melanoma, c57/black 6 mice, lacking p2x7 expression, showed a virtually absent immune infiltrate if compared to wild type controls. the main cells involved seemed to be macrophage-dendritic cells and t lymphocytes, as demonstrated by f/480 and cd3 immunostaining. taken together all these data suggest a central role for p2x7 receptor in regulating a widespread range of immune responses. toxic secondary metabolites in predator-prey interactions among ciliated protists f buonanno1, a anesi2, g guella2, c ortenzi1 1laboratory of protistology and biology education, university of macerata, macerata, italy 2department of physics, university of trento, trento, italy predator-prey interactions involving numerous protist species have been extensively studied in the last years, and great attention has been paid to the repertoire of toxic secondary metabolites adopted in the immobilization and capture of prey, as well as in chemical defense against predators. in this context, particular attention was focused on the important role of specialized ejectable membrane-bound organelles, generally called extrusomes, and widely distributed in protists. these organelles are usually localized in the cell cortex and attached to the cell membrane, and they share a common characteristic in discharging their contents to the outside of the cell in response to mechanical or chemical stimuli. the chemical nature of protists’ extrusive compounds characterized to date is extremely variable, including proteins, carbohydrates, lipids, and dozens of additional classes of secondary metabolites. however few data are available to date for a particular group of protists, the ciliated protozoa. it is worthy of note that at least some of the secondary metabolites produced by ciliates have been demonstrated to show antibiotic, anticancer and pro-apoptotic properties in addition to their physiological functions. among these secondary metabolites, euplotin c produced by the ciliate euplotes crassus, and climacostol produced by climacostomum virens, have been shown to activate programmed cell death by impairing mitochondrial membrane potential and inducing ros generation. interestingly, it was also demonstrated for climacostol, antimicrobial activity against gram-positive bacteria and some fungal pathogens.   57 in conclusion, the toxic secondary metabolites from ciliated protozoa may set the basis for the development of a novel series of antitumor or antimicrobial agents. phagocytosis-induced apoptosis in the compound ascidian botryllus schlosseri f schiavon, n franchi, l ballarin department of biology, university of padua, padua, italy colonies of the ascidian botryllus schlosseri contain three blastogenetic generations: functional zooids, their buds and budlets on buds. generation change or take-over (to) occur cyclically and assure the recurrent renewal of the colony. during these events, lasting 24 36 h, diffuse apoptosis occurs in zooid tissues, as indicated by morphological, biochemical and molecular investigations. tissues are rapidly infiltrated by circulating phagocytes, selectively recruited by dying cells, which recognize and greedily ingest them. using hemocytes as selected cell population for investigation, we studied the transcription rate of three recently characterized genes involved in apoptosis, bsbax, bsaif1 and bsparp1, during the to as compared to colony developmental phases far from it. in addition, we observed that the massive ingestion leads phagocytes themselves to undergo apoptosis, probably as a consequence of the oxidative stress related to the sustained respiratory burst, as suggested by biochemical analysis. therefore, a large fraction of circulating phagocytes needs to be replaced by new, young hemocytes, entering the circulation at the end of the generation change. activity of salmonid antibacterial proteins and regulation of their expression in response to different bacterial pathogens m furlan1, u rosani2, s gambato1, m benincasa1, r gennaro1, a pallavicini1, p venier2, m scocchi1 1department of life sciences, university of trieste, trieste, italy 2department of biology, university of padua, padua, italy cathelicidins are a family of antimicrobial peptides characterized by conserved pro-peptide sequences. they represent an important component of innate immune response in vertebrates. two members of the cathelicidin group, cath-1 and cath-2, have been identified in salmonids and other fishes. both peptides have a variable glycine/serinerich c-terminal domain corresponding to the antimicrobial peptides. this study purposes to characterize their antimicrobial activity spectrum, their mode of action and tissue expression. different peptide fragments representing cath-1 and cath-2 of oncorhynchus mykiss, salvelinus fontinalis, salmo trutta fario and thymallus thymallus were chemically synthesized, and their antimicrobial activity tested against the most relevant fish pathogens such as yersinia ruckeri, aereomonas salmonicida, aeromonas hydrophila, vibrio anguillarum and lactococcus garvieae. the different peptides resulted to have mediumdependent antibacterial activity at micromolar concentrations. membrane permeabilization and hemolytic assays indicated that cath peptides rapidly kill target bacteria showing at the same time low toxicity against host erythrocytes. western blot analysis revealed that cath-1 is expressed in spleen and head kidney. cath-1 and cath-2 gene expression was then comparatively studied by in vivo experiments inoculating o. mykiss with one of the following formalin-inactivated pathogens: l. garvieae, y. ruckeri, a. salmonicida or flavobacterium psychrophilum. real-time pcr analysis performed on tissues collected at different time points demonstrated that both genes are expressed in most tissues and their expression is highly induced 24 h after the challenge. moreover, the expression pattern of cath-1 and cath-2 results different according to the different pathogen. these results suggest that cath peptides are endogenous antibiotics and an important element of salmonids innate immune response and open the possibility that their upregulation might be exploited in the protection of salmonids from infection and at the same time as a possible biomarker for diagnosis of bacterial diseases. igt from antarctic teleosts s giacomelli, f albanese, u oreste, mr coscia institute of protein biochemistry, ibp cnr, naples, italy igt, a novel teleost-specific ig isotype, also named igz, has been recently identified in the most studied species belonging to the main orders of teleost fish, such as salmo salar, oncorhynchus mykiss, takifugu rubripes, cyprinus carpio, ctenopharyngodon idella, dicentrarchus labrax, gasterosteus aculeatus, siniperca chuatsi and epinephelus coioides. this immunoglobulin isotype appears to be specialized in mucosal immunity since produced specifically by igt+ cells that represent an independent b lineage specialized in mucosal immunity. in the majority of the species investigated the igt heavy chain comprises four ig constant domains (ch), except in g. aculeatus and d. labrax where it presents only three ch domains. in this study, we isolated and analyzed the igt transcripts in the gut of the antarctic teleost trematomus bernacchii. we were also able to identify the igt nucleotide sequence in the liver transcriptome of another antarctic teleost, notothenia coriiceps. the phylogenetic analysis was carried out using the amino acid sequence alignment of the igt ch region of antarctic teleosts and other species using clustalx. based on the alignment, we constructed a nj tree of individual ch domains, supported by 1000 bootstrap replications, which revealed that both antarctic species had only three ch domains, ch2 being the missing domain. a limited diversity between the temperate and antarctic species was found, sharing a percentage of amino acid similarity of 24.7 80 %   58 for ch1, 16.7 88.5 % for ch3, 24.5 79.3 % for ch4. the cysteine residues, crucial for the ig domain folding, were found to be conserved and two extra-cysteines were identified in the second constant domain and in the secretory fragment. the analysis of n-glycosylation sites revealed the presence of one site in the second domain and two sites in the third domain. despite antarctic igt contained an n-glycosylation site and a cysteine residue in the secretory tail, this region showed low similarity to the canonical motif required for j-chain association. to clarify the polymeric assembly of igt, a biochemical analysis will be carried out. in addition, a preliminary analysis of amino acid sequences from a number of t. bernacchii specimens showed in some sequences the presence of a short insertion between ch1 and ch2. it will require further analysis to determine whether this insertion may act as a hinge, despite a lack of evidence for the hinge features typical of mammalian iga. lecture il-18 associates with microvesicles shed from human macrophages in response to p2x receptors stimulation d ferrari, e salaro, s gulinelli, d bozzato, c pizzirani, f di virgilio department of morphology, surgery and experimental medicine, section of pathology, oncology and experimental biology, university of ferrara, ferrara, italy extracellular atp, released upon cell stress, cell damage or as a consequence of necrotic cell death due to microbial infection or inflammation, acts as a danger signal alerting immune and nonimmune cells by activating their purinergic p2 plasma membrane receptors (p2r). p2r are classified into two subfamilies named p2yr and p2xr. in this report we show that pharmacological stimulation with extracellular atp induced in human macrophages the fast release of membrane microvesicles in which the inflammatory cytokine il18 was highly concentrated, while on the contrary it was almost absent from the culture medium. the pan p2r agonist atp and the p2x7 preferred agonist benzoyl-benzoic atp (bzatp), but not the p2yr active nucleotides utp or udp, evoked microvesicle shedding. the phenomenon was completely abrogated by pre-treatment of macrophages with the p2x inhibitor oxidized atp (oatp) and partially prevented by the non-covalent p2x7 inhibitors kn-62 a-740003 or a-438079. microvesicles release and il-18 content were greatly decreased in the absence of extracellular ca2+. disassembling of cytoskeletal microtubules or actin filaments did not impair formation and release of the microvesicles, while inhibition of ipla2 or cpla2 delayed it. interestingly, differently from il1β, accumulation of il-18 did not require pretreatment of cells with bacterial endotoxin (lps) as the pro-cytokine was already present in un-primed macrophages and did not decrease by incubating cells with the lps-binding antibiotic polymyxin b nor with the tlr-4 intracellular inhibitor cli-095. presented data point to a p2x-mediated release of il-18 loaded membrane vesicles from human macrophage. new evidences of conserved pathways in complement system dynamics from the colonial ascidian botryllus schlosseri n franchi, l ballarin department of biology, university of padua, padua, italy in recent years, it has been widely demonstrated that complement, although often depicted as a ‘first line of defense’, is more than just a defender against microbial intruders and acts as a tightly integrated surveillance system. it is not only important against microorganisms, but also for the clearance of apoptotic cells and corpses. botryllus schlosseri belongs, as vertebrates, to the phylum chordata and, for this phylogenetic trait, it is unanimously considered a reliable model organism for the studies of the evolution of the immune system. moreover it is also characterized by a peculiar life cycle with a cyclical, massive apoptosis. this two key features render b. schlosseri a good research tool for the study of the evolution of the complement system. here we report the first results on the expression of bsc3 and bsfactorb, both components of the alternative pathway (ap) of complement activation, which form the ap c3 convertase. since studies on mammalian models have shown that 80 % of the observed complement response is derived from ap convertase-mediated c3 amplification, even if initially induced by the classical pathway (cp), studies of complement activation in an organism that lack the adaptive immunity, as b. schlosseri, could lead to a better comprehension of the ap cascade and the behavior of c3 convertase not only in invertebrates, but also in vertebrates, mammals included. as in mammals, bsc3 is highly transcribed at basal level and over-expressed after incubation with non self (zymosan) while bsfactorb always shows limited expression. in the presence of compstatin, a 13-residue cyclic peptide able to inhibit the activation of c3 by c3 convertases, the percentage of phagocytosing hemocytes collapses. in the presence of both zymosan and compstatin, the transcription of bsc3 by hemocytes increases with respect to cells exposed only to zymosan: this suggests the presence of a conserved molecular machinery able to control and modulate b. schlosseri as well as the mammalian complement. early transcriptional immune responses in yersinia-injected rainbow trouts u rosani1, m furlan2, a manfrin3, a pallavicini2, m scocchi2, p venier1 1department of biology, university of padua, padua, italy 2department of life science, university of trieste, trieste, italy   59 3experimental zooprophylactic institute of venice, rovigo, italia the enterobacterium yersinia ruckeri is the causative agent of enteric red mouth disease (erm) leading to significant economic losses in salmonid aquaculture worldwide. infection may result in a septicemic condition with haemorrhages in the oral cavity, on the body surface and in the internal organs. knowledge about the immune response of rainbow trout against y. ruckeri is important in terms of control and prevention. adaptive immune responses related to y. ruckeri infection have been investigated by a number of authors. a few studies have measured in rainbow trout the expression of selected immune-related genes such as toll-like receptors (tlr), cxcd, il-1β, and some others. since the first defence line against bacterial pathogens mainly depends on the innate immune system, we undertook the global study of tissuespecific transcriptomes to depict the early response of juvenile oncorhynchus mykiss to 108 formalininactivated y. ruckeri cells. following i.p. injection, we collected the main tissues at different time points up to 96 h. after preliminary evaluation of the expression of cathelicidin genes in real time pcr, we selected 24h post-infection as informative time-point for rnaseq analysis. eight samples from gills, intestine, spleen and kidney were subjected to library preparation and illumina sequencing (paired 50 base reads). subsequently, a de-novo assembling was performed to obtain a comprehensive reference transcriptome and evaluate the differentially expressed genes for each pair of treated and control samples. about 1928 out of the 62 k assembled transcripts were differentially expressed in at least one tissue. kidney, spleen and intestine showed the greatest transcriptome modulation in terms of inflammatory and immune response to the bacterial antigens. pro-inflammatory cytokines like il1β, il6 and tnfα, complement proteins like c3, c5 and factorb and acute phase proteins like saa, hepcidin and also cathelicidins showed a significant induction and marked the early activation of the immune system in the treated trouts. effects of dietary vitamin d administration on innate immune response of sea bass (dicentrarchus labrax) 3 m dioguardi1, fa guardiola , m vazzana , a cuesta , ma esteban , m cammarata 1,2 1 2 2 1 1department of biological chemical pharmaceutical science and technology, university of palermo, palermo, italy 2department of cell biology and histology, campus regional de excelencia internacional “campus mare nostrum”, university of murcia, murcia, spain the aquaculture sector is a productive force rapidly growing in the world; therefore, over the past 30 years much has been done to improve the performance of fish growth by manipulating diets. currently, the optimization of diets and disease control are two main objectives to ensure the continued expansion of aquaculture. a new area of great interest to improve growth efficiency and to prevent and/or control fish diseases is the application of probiotics, prebiotics and stimulants of the immune system as additives in the diet, being a promising alternative to antibiotics. thus, the aim of the current study was to evaluate the potential in vivo effects of the dietary administration of vitamin d (vd ) on the humoral innate immune response (level of total igm antibodies, the activities of peroxidase, protease, and antiprotease) cellular (phagocytic activity) and biochemical parameters (cortisol, glucose) of the sea bass (dicentrarchus labrax). vitamin d 3 3 3 was orally administered to sea bass specimens in a commercial pellet food supplemented with 0 (control), 3750, 18750 or 37500 u kg-1 and fish were sampled after 2 and 4 weeks of treatment. serum and peritoneal cavity leucocytes were collected from specimens and innate immune biochemical parameters were evaluated. this study showed for the first time in sea bass a modulation in the activities examined in animals fed with the addition of vitamin d3 after 4 weeks. these results suggested that dietary vitamin d3 administration has an effect on the innate immune parameters of d. labrax, therefore this vitamin could be considered of great interest as immunostimulant when used as food additive in fish farming. session 3. chairmen: m cammarata, university of palermo, palermo, italy; mr coscia, ibp cnr, naples, italy immunity and biotechnology lecture molecular and cellular basis of mucosal immunity mr coscia, u oreste institute of protein biochemistry, ibp cnr, naples, italy mucosal surfaces represent a first line of defence facing many pathogens, and also monitoring harm-less antigens such as food, airborne particles or symbiotic microorganisms. the latter represent the majority of the encountered epitopes and require precise regulatory mechanisms from the host’s immune system to choose between ignorance and active suppression. on the other hand, a strong immune response is needed for the former. under these constraints, mucosae have evolved a complex immune system, anatomically and functionally distinct from the systemic counterpart. in mammals, mucosaassociated lymphoid tissues (malts) are the sites where sampling of antigens occurs and naïve t and b lymphocytes are stimulated. protection against pathogens is also due to malt antimicrobial components, in particular enzymes, lytic agents, lysozyme and various immune molecules, such as complement and immunoglobulins (ig). in teleost fish, malts are further distinguished in anatomically distinct compartments such as gut-associated lymphoid tissue (galt), skin-associated lymphoid   60 tissue (salt) and gill-associated lymphoid tissue (gialt). all malt types comprise different leukocytes, such as t and b cells, mast cells and macrophages. not long ago, only two isotypes of ig, igm and igd, have been described in teleosts, igm being considered the only player either in the systemic or mucosal immune responses. however, a third immunoglobulin isotype, named igt/igz, restricted to teleosts, has been discovered more recently and it has been reported to play a specialized role in mucosal immune responses. a number of studies has shown that in teleosts, like in mammals, the transport of mucosal ig occurs by a polymeric ig receptor which is expressed on the surface of epithelial cells. a review will be given to summarize the current knowledge of fish mucosal immune response with a brief reference to innovative mucosal vaccines. antimicrobial response in anemonia sulcata (cnidaria) mr trapani1,2, mg parisi1, d parrinello1, ma sanfratello1, g benenati1, l coquet3, t jouenne3, m toubiana2, m cammarata1 1department of biological, chemical and pharmaceutical science and technology, university of palermo, palermo, italy 2ecosym, university of montpellier 2-cnrs, montpellier, france 3plateforme de protéomique pissaro, university of rouen, mont-saint-aignan, france recently we analyzed the inflammatory response in anemonia sulcata, following injection of various substances, and observed specific reactions especially after injection of bacteria. moreover, we analyzed enzymatic activity of protease, phosphatase and esterase, showing how the injection of different bacterial strains alters the expression of these enzymes suggesting a correlation between the appearance of the inflammatory reaction and the modification of enzymatic activities. our study show for the first time in cnidarian, cellular and molecular responses following injection of bacteria. the sea anemones are a source of neurotoxins acting on potassium and sodium ion channels. a. sulcata is exposed to attacks from predators but is unable to retract the tentacles and actively capturing a broad spectrum of prey is subject to frequent breakage of the tentacles with subsequent bacterial infection. the chemical arsenal of a. sulcata is the optimal strategy for survival, mainly through the production of neurotoxins and by using antimicrobial molecules. by acid extraction, hplc purification and antibacterial assays, we isolated, purified and characterized an antimicrobial peptide towards micrococcus lysodeikticus. the mass spectrometry analysis and the sequencing, showed that antimicrobial peptide is present in the database as neurotoxin, named neurotoxin 2 (atx ii), na+ channel blocking toxin. atx ii can then be considered as a neurotoxin having antimicrobial peptide structural and functional characteristics. this multifunctionality could be an optimal survival strategy that allows these animals to be active predators through the production of neurotoxins and to resist to bacterial infections caused eventual rupture of the tentacles through the functionality by antimicrobial peptides. inflammatory effects of multi-walled carbon nanotubes in leeches r girardello, m de eguileor, g tettamanti, r valvassori, a grimaldi department of biotechnology and life sciences, university of insubria, varese, italy during last years nanotechnologies has developed considerably, occupying a pivotal role in scientific and technological research. among nanomaterials, multi-wall carbon nanotubes (mwnts) show high strength and flexibility and for these reasons they are very useful in different fields such as industry, electronics and medicine. despite these properties, mwnts seem to be toxic for organisms. our research is focused on the possible effects caused by carbon nanotubes exposure in a freshwater invertebrate model: the leech hirudo medicinalis. animals have been analysed after both a short time and a prolonged exposure to mwnts (1, 3, 6 , 12 h and from 1 to 5 weeks). our data obtained by morphological, immunocytochemical histoenzymatic and western blot analysis, show that mwnts treatment induce inflammatory responses: angiogenesis, fibroplasia and a massive macrophage migration take place. interactive effects of nanoparticles with other environmental pollutants on immune function and larval development of mytilus galloprovincialis t balbi1, a smerilli1, r fabbri1, c ciacci2, m montagna1, g gallo1, l canesi1 1distav, university of genoa, genoa, italy 2disuan, university of urbino, urbino, italy the increasing production of manufactured nanosized materials is leading to the release of nanoparticles (nps) and their products into the aquatic environment, thus posing a threat to aquatic biota. despite the growing concern over the potential biological impact of nps on marine organisms, little is known about their interactions with other pollutants. recent evidence indicates that the biological impact of nps may be due not only to the toxicity of nps themselves, but also to their capacity to modify the bioavailability, bioconcentration and toxicity of other common environmental pollutants, such as organic xenobiotics and heavy metals. the bivalve mytilus sp., largely utilized as a sentinel for marine contamination, has been shown to represent a significant target for different types of np, including ntio2, one of the most widespread np in use. in this work the effects of in vitro and in vivo exposures to n-tio2, alone and in combination with 2,3,7,8-tcdd and cd2+, chosen respectively as models of organic and inorganic contaminants, on   61 the immune function of mytilus galloprovincialis were investigated. the results demonstrate that combined exposure to n-tio2 and tcdd exerted interactive effects on phagocytic activity but not on lysosomal membrane stability-lms in vitro. these effects were confirmed in in vivo exposure to the mixture. antagonistic effects of n-tio2 and cd 2+ on no production and lysozyme activity were observed both in vitro and in vivo. on the other hand, no interactive effects were detected on expression of metallothioneins and immune-related genes lysozyme and toll-like receptor (tlr-i). cd2+ alone, but not in combination with n-tio2, also affected larval development. these data indicate complex and often unexpected interactions between nps and organic contaminants and heavy metals in marine bivalves. insights into the structure/activity relationships of m. galloprovincialis myticin c s domeneghetti1, r norante2, m franzoi1, a toffan3, m bellanda4, s mammi4, o marin2, p venier1 1department of biology, university of padua, padua, italy 2department of biomedical sciences, university of padua, padua, italy 3department of virology, experimental zooprophylactic institute of venice, rovigo, italia 4department of chemical sciences, university of padua, padua, italy one of the most intriguing amps of the marine mussel mytilus galloprovincialis is myticin c (mytc) because of its remarkable constitutive/induced expression levels, high transcript polymorphism and peculiar expression profiles in individual mussels. the conformation of both myticin c precursor and mature peptide (40 aa, 4.377 kda) is still unsolved but mainly depends on an evolutionary conserved array of eight cysteine residues. following stepwise and optimized protocols of chemical synthesis, we obtained the mature peptide and smaller analogs, useful to investigate the secondary structure in different experimental conditions by circular dichroism spectroscopy and nuclear magnetic resonance. aiming to determine the antimicrobial properties of the chemically synthesized mytc peptides and to identify the minimal active fragment we evaluated their bacterial growth inhibition (mic) and bactericidal (mbc) potency at two ph conditions on gram negative and gram positive bacteria able to cause human or fish/shellfish infections and diseases. opposite to recent findings (mar drugs, 2013, 11, 2328-2346), we could not verify any activity of our mytc peptides against the viral hemorrhagic septicemia (vhs) and infectious hematopoietic necrosis (ihn) viruses. next steps will focus on oxidative refolding, recombinant synthesis and purification of mytc. evidences for antimicrobial peptides in the colonial ascidian botryllus schlosseri f schiavon, n franchi, l ballarin department of biology, university of padua, padua, italy invertebrates have acquired many mechanisms of defence in order to overwhelm the risk of pathogen attack. in particular, the presence of various types of antimicrobial peptides (amp) guarantees an efficient response, making them able to kill both gram positive and negative bacteria, fungi and viruses. in ascidians, amps have been isolated and described in different species, such as in styela clava (clavanins and styelins) and in ciona intestinalis (the recent ci-mam). previous studies have revealed molecules, including phenoloxidase and rhamnose-binding lectins, with an antimicrobial effect also in botryllus schlosseri. here, we report that the growth of some bacteria strains is highly inhibited by extracts of haemocytes, showing alterations in their surface. moreover, molecular analyses allowed us to identify a botryllus sequence similar to styelins that is abundantly transcribed in phagocytes. session 4. chairmen p venier, university of padua, padua, italy; a mancia, university of ferrara, ferrara, italy immunity and environmental monitoring lecture the strange case of georgia dolphins: the effects of environmental changes on the transcriptome. a mancia1,2, l abelli1, j kucklick3, t rowles4, r wells5, p rosel6, a hohn7, f van dolah8, l schwacke8, j ryan8 1department of life sciences and biotechnology, university of ferrara, ferrara, italy 2medical university of south carolina, charleston, sc, usa 3national institute of standards and technology, charleston, sc, usa 4noaa/nmfs, office of protected resources, silver spring, md, usa 5chicago zoological society, c/o mote marine laboratory, sarasota, fl, usa 6nmfs/southeast fisheries science center, lafayette, la, usa 7nmfs/southeast fisheries science center, beaufort, nc, usa 8noaa/national ocean service, charleston, sc, usa land use and population densities influence contaminant concentrations in aquatic organisms. it is increasingly common to monitor the marine environment and establish geographic trends of environmental contamination by measuring contaminant levels in animals from higher trophic levels. in this work we chose the common bottlenose dolphin (tursiops truncatus) as a top level predator to monitor a site on the georgia coast   62 the us, known to be heavily contaminated by aroclor 1268, an uncommon pcb mixture primarily comprised of octathrough deca-chlorobiphenyl congeners. prior studies have suggested an association between high polychlorinated biphenyls (pcbs) concentrations and increased risk of infectious disease suggesting a causal link between persistent organic pollutants (pops) exposure, immune function and susceptibility to disease. we are currently investigating the potential for identifying pcbs exposure in bottlenose dolphins through screening for their immunological and/or endocrine perturbations associated with exposure using microarray technology and gene expression profile analysis. a newly developed dolphin oligo microarray representing 24,418 unigene sequences was used to analyze blood samples from 69 dolphins collected from 5 geographic locations (beaufort, nc, sarasota bay, fl, saint joseph bay, fl, sapelo island, ga and brunswick, ga). the georgia samples were selected due to the measured high concentrations of pcbs contaminants in their blubber. genes involved in xenobiotic metabolism, in development/differentiation and oncogenic pathways were found to be differentially expressed in ga dolphins compared to the other locations. hypothyroidism has been previously described in ga dolphins and, interestingly, a few of the genes that we identified are involved in the proper function of the thyroid. the analysis of ga animals alone, correlated with contaminant load measured, showed the activation of genes involved in stress response, dna repair and skin damage, uv and/or viral infection-induced. if successful, the gene expression profile analysis could provide a cost-effective means to screen for indicators of chemical toxin exposure as well as disease status in top level predators. the transcriptomic data analysis will be a first step towards identification of markers/patterns indicative of exposure to chemical contaminants and will promote an understanding of toxic mechanisms and/or pathways that are currently not well understood in marine mammals. characterization of superoxide dismutases in the antarctic scallop adamussium colbecki e mattiuzzo1, d ferro2, f cattalini1, l tallandini1, p irato1, l ballarin1, g santovito1 1department of biology, university of padua, padua, italy 2institute for evolution and biodiversity, westfälische wilhelms-universität, münster, germany superoxide dismutases (sods) are considered the most important and ubiquitous antioxidant enzymes, involved in cellular antioxidant defenses, maintaining the redox homeostasis during the aerobic cell metabolism. moreover, sods are also closely involved in the innate immune response of animals, as evidenced by the rapid modulation of transcription during challenges with endotoxins, bacteria or viruses. during infection, the host often uses reactive oxygen species (ros) to react to pathogenic invaders via phagocytosis. however, excess ros generated during the respiratory burst may also be harmful to the host, that use the antioxidant machinery to maintain low ros concentration in the cytoplasm. antarctic species are characterized by a large number of special physiological features that allow the life in the extreme environment. first of all, low temperature and salt concentration are physicochemical conditions that increase oxygen solubility and, consequently, the rate of ros formation. for this reason, a finely modulated antioxidant system is essential to prevent macromolecules oxidation that could result in dna damage, loss of membrane integrity and changes of protein activities. despite numerous previous studies on sods from aquatic animals, only few data are available for the sods of antarctic mollusks. in the present work, we studied the antarctic scallop adamussium colbecki, a bivalve mollusk widely distributed in the antarctic ocean; for the first time we characterized the gene structure and expression of adamussium sod. specimens were sampled in the ross sea (terra nova bay, 74°42’s, 164°7’e) during the xxi italian antarctic expedition. partial cdna sequences of cu,zn sod and mn sod were obtained from gonadic tissue by rt-pcr and ta-cloning. the obtained nucleotide and deduced amino acid sequences were compared with those of orthologous genes already available in genbank and the protein phylogenies were reconstructed. we also studied the gene transcription and enzyme activity in various organs and tissues, to investigate the biological fraction of sods in molluscs living in extreme environmental conditions. characterization of immune-related transcripts within the atlas gene database of procambarus clarkii (girard, 1852) c manfrin, g de moro, pg giulianini, a pallavicini department of life sciences, university of trieste, trieste, italy being procambarus clarkii an invasive species, showing high adaptability and resistance to diversified environmental challenges, such as high pollution levels, high organic and bacterial contents, it results of great interest analyzing its immunome. the knowledge of its immunity could help in characterizing possible aspects of its vulnerability in order to find new strategies for contrasting the continue spread of this alien invasive species. starting from an atlas gene database, obtained from a collection of 12 tissues, we depicted and described immune-related transcripts. in particular, eyestalk, brain, hemocytes, hepatopancreas, testes, ovaries, gills, green gland, ventral ganglia, cardiac muscle, y-organ-epidermis and tail muscle, have been sequenced with the illumina 2x100bp technology from oriented-cdna libraries generating 1.9 gb sequence data and resulting in a nonredundant, high quality subset of 97,976 different transcripts. we focused our attention on the several classes of immune-related transcripts, such as prophenoloxidases, anti-lipopolysaccharide factors, lectins, clottable proteins and proteins having   63 antimicrobial activity. for each immune-related transcript found, we detected its relative percentage expression in all the tissues sequenced creating a collection of immune-related transcripts characterized in p. clarkii and reporting the tissuespecificity for each of them. hereafter, we present some transcripts that have attracted our attention. two prophenoloxidases where detected, one had higher expression in the hemocytes and one in the gills. anti-lipopolisaccharide factors were most expressed in the hemocytes and in the gills. crustins were also detected and resulted to be mainly expressed in hemocytes, but also in the ovary. lysozymes where found highly expressed in the hemocytes, but also in the brain, in the hepatopancreas, in the gills and in the y-organ-epidermis. numerous lectins and ctype lectins resulted to be mainly expressed in the hepatopancreas. moreover, we detected clottable proteins, such class of transcripts was never reported in the red swamp crayfish since now and surprisingly the majority of them resulted to be electively expressed in the brain. this preliminary study helps in outline the possible tissue-specificity of some isoforms of immune-related transcripts and to characterize classes never reported before, such as clottable proteins. further functional assays could be led considering these data obtained in this preliminary screening, in order to outline the main defense mechanisms of this species. functional characterisation of the antioxidant system in drosophila melanogaster, after metals exposure c capriolo1, d ferro2, p cisotto1, c de pittà1, l ballarin1, g santovito1 1department of biology, university of padua, padua, italy 2institute for evolution and biodiversity, westfälische wilhelms-universität, münster, germany reactive oxygen species (ros) are a normal byproduct of aerobic metabolism playing an important role in cell signaling and immunity when their production is regulated or controlled by antioxidant enzymes. reduced expression and/or activity of these proteins lead to an excess of ros production and oxidative stress, accelerating aging and neurodegeneration. for example, amyotrophic lateral sclerosis (als) is a neurodegenerative disease caused by motoneuron loss and some familial cases (fals) are linked to mutations of superoxide dismutase type-1 (sod1). many animal models, such as drosophila melanogaster, are used as useful tool to study this disease, focusing the attention on the different mutant sod1s but not considering the complex relationships of functional complementarity between sod1 and the other components of the enzymatic antioxidant system. in the present work we study, for the first time, the gene expression, by qrt-pcr, of sod1 together with that of superoxide dismutase type-2, catalase, glutathione peroxidases and peroxiredoxins in wild type d. melanogaster, exposed to various concentration of copper or cadmium, used as prooxidants. the aim was to determine the adequate experimental condition to employ with d. melanogaster sod1 mutants. on the basis of our results, copper is the major inducer of all considered enzymes, and the dose of 1.0 mm seems to be the more suitable. catalase is temporally co-expressed with sod1, at least in males and the gene expression of other enzymes seems to be temporally uncorrelated to sod1. other important indication will be obtained by biochemical quantification of activity for all considered enzymes and the evaluation of ros production, that are now in progress. analysis of some hematological parameters of apis mellifera ligustica (spinola, 1806) populations in a polluted and non-polluted site s battistella, a giglio, a pallavicini, m zrnic, pg giulianini department of life sciences, university of trieste, trieste, italy main immune defences of the honey bees are the cellular responses represented by phagocytosis and melanisation. there are a number of factors that could impact on the honeybees immune system and, therefore, increase their susceptibility to disease and lower their survivorship such as: exposure to pesticides, in air, in pollen, in nectar and in water; fungicides from both field and in-hive treatments; varroacides; the pest varroa destructor; antibiotics used in in-hive treatments; fungal pathogens such nosema apis and the emerging nosema ceranae; bacterial and viral infections. more important, it must be highlighted the interactions among different chemicals and their synergic effect with diseases in the immune suppression of individuals and on the colony. the present study use simple and reliable methods that can clearly describe the state of health of the bees, such as total hemocyte counts (thcs) and the activities of the plasmatic phenoloxidase (po) and its inactive form (propo) to assess the immune competence of individuals. specimens of apis mellifera ligustica were collected from beehives located in s. giovanni (trieste, “control site”) and from hives placed in domio (trieste, “polluted site”) in summer and early autumn. both in summer and in autumn the statistical comparison showed a greater number of circulating hemocytes in bees from the site of domio (july: 1159333.3±123073.60 mean ± se, n = 15; october: 813333.3±50583.89 mean ± se, n = 15) compared to the numbers recorded in bees from s. giovanni (july: 834166.7 ± 55493.08 mean ± se, n = 12; october: 273333.3±33046.38 mean ± se, n = 12). the statistical analysis showed a trend towards significant difference in july (wilcoxon rank sum test, p = 0.063) and an highly significant difference in october (wilcoxon rank sum test, p < 0.00001). the honeybees from domio presented an highly significant lower po activity than those from s. giovanni (ancova: f3,206 = 38.73, p < 0.01, ns.giovanni = 17, ndomio = 18). also with regard to the enzymatic activity of the propo we recorded a significantly higher one in the   64 honeybees from s. giovanni in comparison to those from domio (ancova: f3,206 = 23.66, p < 0.01, ns.giovanni = 17, ndomio = 18). it should be noted that 52 % of the bees collected from hives of domio had 1 or 2 individuals of varroa destructor on the tergites of the thorax. the higher activities of po and propo in the honeybees from s. giovanni site is probably to be ascribed to the different quality of the environment in the 2 sites and thus it indicates a depression of non-specific immune competence and an increased susceptibility to varroa destructor parasites in the honeybees from domio. characterization of proxiredoxins’ coding genes in ciona intestinalis r benevenia1, d ferro2, l ballarin1, g santovito1 1department of biology, university of padua, padua, italy 2institute for evolution and biodiversity, westfälische wilhelms-universität, münster, germany ascidians represent an interesting model from an evolutionary and ecotoxicology point of view, because of their large distribution in temperate sea and their phylogenetic position of invertebrate chordates. immune responses imply an increase in oxygen consumption with a consequent risk of oxidative stress. with the aim to study the components of the antioxidant defense system in the solitary ascidian ciona intestinalis, we characterized gene sequences encoding peroxiredoxins (prxs), non-selenium peroxidases that are able to reduce hydrogen peroxide, organic hydroperoxides and peroxynitrite. thus they represent a class of important antioxidant enzymes, that protect cells against oxidative stress. in the genebank database five prx sequences are present, prx2, 3, 4, 6a and 6b. the multi-alignment analysis, conducted with orthologous sequences of vertebrates and invertebrates, showed that in ciona’s prxs the amino acids essential for their enzymatic activity are highly conserved, namely the catalytic tetrad consisting of proline, threonine, cysteine and arginine. preliminary phylogenetic reconstruction indicates that prx3 and 4 emerge as sister group of prxs of the respective vertebrates groups (or isoforms), while prx2, 6a and 6b have an uncertain position. a partial confirmation of phylogenetic results was obtained with the analysis of homology modeling, according to which prx3 and 4 present a structure similar to that of two vertebrate proteins, bos taurus prx3 and larimichthys crocea prx4, respectively, while prx2 and 6 six have a three dimensional structure similar to that of two invertebrate proteins, ancylostoma ceylanicum prx1 and arenicola marina prx6, respectively. the transcription of all these genes, measured by qrtpcr, resulted variable in different organs (intestine, ovary, pharynx, stomach). in particular, the ovary is the organ that expresses the highest level of messenger for all prxs. preliminary data were also collected about the possible circadian expression of prx. histological and molecular studies on igm expressing cells of the b-lineage of two antarctic teleost species d lunardi1, a mancia1, mg marchetti1, mr coscia2, u oreste2, l abelli1 1department of. life sciences and biotechnology, university of ferrara, ferrara, italy 2institute of protein biochemistry, ibp cnr, naples, italy life at sub-zero temperatures, such in antarctic seas, has selected especial adaptations of cell molecules, among them the immunoglobulins (ig). differently from species living in temperate water, many antarctic teleost bony fish showed peculiar protein structure and molecular dynamics of igm, acquired during radiation of the suborder notothenoidei (~24 mya). to gather further information about igm producing cells, we sampled two common antarctic species, the red-blooded trematomus bernacchii (tb) and the haemoglobinless icefish chionodraco hamatus (ch). our studies were focused on two main lymphoid organs: the head-kidney (hk), thought a primary lymphoid organ for cells of the b-lineage, and the spleen (spl), a secondary lymphoid tissue involved in effector b cells differentiation. the occurrence of secreted and membrane igµ transcripts, igm proteins and numerous cells containing igm has been demonstrated in both tissues of tb and ch. the combined use of rabbit polyclonal antisera against homologous/heterologous igm (in some instances specific for heavy or light chains) and b-specific transcription factors (with dna binding domains highly conserved throughout evolution) proved that, at least in ch, the hk houses both b-cell progenitors, b lymphocytes (especially abundant in the peripheral blood) and plasma cells, mainly accumulated around the arteries. these findings strongly suggest that the hk also behaves as secondary lymphoid tissue, in keeping with previous data from rainbow trout and other few teleost species. the peculiar characteristics of the igm molecule of many antarctic teleosts (e.g., three constant igµ domains in tb, only two constant igµ domains in ch, regarding the membrane antigen receptor) prompted us to start investigating in detail the organization of the b cell receptor (bcr) complex. preliminary data will be presented about the two accessory molecules cd79a (ig-α) and cd79b (ig-β).   65 antimicrobial peptides isolation from anemonia sulcata (cnidaria) isj 11: 182-191, 2014 issn 1824-307x research report first evidence of antimicrobial activity of neurotoxin 2 from anemonia sulcata (cnidaria) mr trapani1,2, mg parisi1, m toubiana2, l coquet3, t jouenne3, p roch2, m cammarata1 1marine immunobiology laboratory, department of biological, chemical, pharmaceutical science and technology, university of palermo, palermo, italy 2ecologie des systèmes marins côtiers, cnrs-université montpellier 2, place e. bataillon, montpellier, france 3plateforme de protéomique pissaro, cnrs-université de rouen, place e. blondel, mont-saint-aignan, france accepted june 6, 2014 abstract we investigated the antibacterial activity of anemonia sulcata (cnidaria, anthozoa) tentacle and body acidic extracts. biochemical purification consisted of first step on solid phase sep-pak c8 column followed by several hplc runs on c18 column using different conditions. anti-micrococcus lysodeikticus activity has been detected in 40 % acetonitrile fractions. the resulting purified molecule from tentacles had a molecular mass determined by maldi-tof mass spectrum of 4946,299 da and has been completely sequenced. its aa sequence revealed identity with the neurotoxin 2 (atx-ii), a na+ channel blocking toxins. consequently, atx-ii appeared to display a dual role as toxin and as antibacterial. key words: antimicrobial peptide; anemonia sulcata; atx ii; neurotoxin; micrococcus lysodeikticus introduction the sea anemone anemonia sulcata is a widespread mediterranean species. sea anemones are generally poisonous animals that spend most of their lives in a sessile form. capturing activities and defense mechanisms are strongly associated with toxin production (ruppert and barnes, 1994). the neurotoxin atx ii is a type 1 sodium channel toxin and consists of 47 amino acid residues, linked by three disulfide bridges. atx ii specifically binds to the sodium channel (site 3), thus delaying its inactivation during signal transduction; has a strong effect on crustaceans and insects and a weaker effect on mammals. these toxins specific for sodium channels have been thoroughly investigated also because they constitute a major fraction of the venom (moran et al., 2009). antimicrobial peptides are important defense molecules in marine invertebrates covering a broadspectrum of bacteria and fungi (boman, 2003; bulet et al., 2004). they are defined as molecules of less than 10 kda characterized by immediate and rapid response to invading microorganisms (bartlett et al., ___________________________________________________________________________ corresponding author: matteo cammarata marine immunobiology laboratory department of biological, chemical pharmaceutical science and technology university of palermo, palermo, italy e-mail: matteo.cammarata@unipa.it 2002). there is evidence that antimicrobial peptides are widespread in invertebrates (chisholm and smith, 1992), especially in tissues such as the gut and respiratory organs in marine invertebrates, subjected to a first exposure to pathogens. in spite of variations in structure and size, most of antimicrobial peptides are amphiphilic, displaying both hydrophilic and hydrophobic surfaces (tincu et al., 2004). amp are exciting candidates as new antibacterial agents due to their broad antimicrobial spectra, highly selective toxicities, and the difficulty for bacteria to develop resistance to these peptides (boman, 1998; lehrer and ganz, 1999; hancock et al., 2000). therefore, amps from marine organisms represent a largely unexploited resource that can afford design of new antibiotics with broad-spectrum antimicrobial activity (ovchinnikova et al., 2006). it is plausible that certain functional molecular motifs, such as amphipathic α-helices and β-sheets, would have featured predominantly in the arsenal employed by our distant eukaryotic ancestors. over time, these would have diversified through mutation and selection pressure to produce the great variety of amps and other molecules (smith et al., 2010). several amps have been isolated from phylum of cnidaria. have been reported the purification of a 40-residue amp from the mesoglea of a scyphozoid jellyfish, aurelia aurita (ovchinnikova et al., 2006). the peptide was named aurelin and exhibited activity against gram-positive and gram-negative 182 mailto:matteo.cammarata@unipa.it fig. 1 tentacles and body acid extracts of anemonia sulcata were loaded onto solid phase column (sep-pak c8). the resulting 10, 40 and 80 % acetonitrile fractions (10 μl) were tested for the antibacterial activity toward micrococcus lysodeikticus by filling wells performed in petri dishes containing trypticase soy agar (a). the 40 % active fraction have been also assayed at different dilution (b). bacteria. furthermore, a detailed biochemical characterization of the antimicrobial activity in hydra revealed the presence of conserved amps such as hydramacin-1 and novel taxon-specific amps such as arminin 1a and periculin-1 which have no counterpart in the transcriptomes of any other organisms (bosch et al., 2009). it could be possible to obtain, from this molecule, synthetic peptides with significant antibacterial activity and relatively low cytotoxicity to human erythrocytes, as in the case of stycholysin i and ii (tejuca et al., 1996), 2 poreforming cytolysins from the sea anemone stychodactyla helianthus. in this paper we reported i) the presence of anti-micrococcus lysodeikticus activity in acidic extracts from tentacles and body of a. sulcata; ii) the biochemical purification of the active peptide, and iii) its complete aa sequence revealing identity with neurotoxin 2 (atx ii). material and methods animals and tissues sampling anemonia sulcata (anthozoa) adults have been collected from termini imerese (palermo, italy) and maintained in oxygenated sea water at 18 °c. acid extractions were performed from tentacles and body. briefly, tentacle were separated from the animal body with a forceps and both suspended in tris buffered solution (tbs, 150 mm nacl, 10 mm trishcl, ph 7.4). after homogenization in ice bath with ultra-turrax for 5 min, 10 % acetic acid was carefully added. the 2 samples have been sonicated (branson model b15, danbury, ct) 3 times for 30 sec each, then centrifuged at 21,000xg, for 20 min at 4 °c. protein content of both supernatants was determined using the nanodrop nd-1000 spectrophotometer. antibacterial assays solid phase elution fractions have been freezedried and suspended in 50 μl sterile ultra pure water (upw). antibacterial activity has been tested against m. lysodeikticus by filling wells performed in petri dishes containing 10 ml of trypticase soy agar (tsa) and 3 ml of 0.5 mcfarland turbidity. each well received 10 μl of sample and petri dishes were incubated overnight at 37 °c. diameter of clear ring around the wells paralleled the antibacterial activity. liquid growth inhibition assay has been used to check for antibacterial activity in all hplc fractions. briefly, fractions were freeze-dried and suspended in 50 μl upw. twenty μl aliquots were added to 20 μl of a suspension of m. lysodeikticus (5x106 bacteria/ml) in poor-broth nutrient medium (1 % bactotrypton, 0.5 % nacl, w/v) in 96 wells microtiter plates. bacterial growth was measured at a600 with a microplate reader tecan (infinite 200 m) during 16 h at 37°c. minimal inhibitory concentration (mic) was obtained by testing serial doubling dilutions of selected purified peptide in liquid growth inhibition assay as above and corresponded to the lowest concentration that caused 100 % growth inhibition. minimal bactericidal concentration (mbc) was determined by plating the contents of the first 5 wells with no visible growth of bacteria onto tsa petri dishes. the lowest concentration that prevented any colony formation unit (cfu) as observed after 18 h incubation at 37 °c corresponded to the mbc. solid phase extraction and reversed phase hplc purification tentacles and body acid extracts were loaded onto sep-pak c8 vac cartridges (waters associates) equilibrated with acidified water (0.05 % tfa in upw). after washing with acidified water, three successive elutions were performed 183 fig. 2 tentacles and body acid extracts of anemonia sulcata were loaded onto solid phase column (sep-pak c8). minimum inhibition concentration of the 40 % active acetonitrile fraction was assayed toward micrococcus lysodeikticus obtained after incubation in the microplate reader tecan for 16 h at 37 °c. the data obtained show an antibacterial activity at concentrations up to 0.244 mg/ml. successively with 50 ml of 10, 40 and 80 % acetonitrile in acidified water. fractions were freezedried and reconstituted with upw. as the bulk of the antimicrobial activity was detected in the 40 % fraction, only this fraction was submitted to reversed phase hplc on a silica column c18 interchrom up5odb-25qs (250x4.6 mm). elution of the 50 μl sample was performed with a linear gradient of 0 60 % acetonitrile in acidified water over 60 min at a flow rate of 0.5 ml/min. fractions corresponding to absorbance peaks detected at both 280 and 225 nm were collected in polypropylene tubes, freeze dried, reconstituted in upw and tested for anti-m. lysodeikticus activity by the liquid growth inhibition assays described above. active fractions were then purified on the same reversed phase column, with a linear gradient from 20 60 % acetonitrile in acidified water at a flow rate of 0.5 ml/min. each collected peak was freeze-dried, reconstituted in upw and tested for anti-m. lysodeikticus activity. mass spectrometry the molecular mass of the active peak content was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with an ultraflex tof/tof (bruker daltonics). the 2,5-dihydroxybenzoic acid (dhb) matrix was prepared in acetonitrile/ultrapure water/tfa (20/80/0.1 %) for a final concentration of 5 mg/ml. one µl of sample dissolved in 0.1 % tfa was mixed with 1 μl of the matrix solution on the steel target and dry in ambient air. the calibration was performed with five peptide standards: angiotensin ii (mh+ = 1046.54), angiotensin i (mh+ = 1296.68), neurotensin (mh+ = 1672.91), acth clip 1-17 (mh+ = 2093.08) and acth clip 18 39 (mh+ = 2465.19). the data are acquiring with the flexcontrol software (version 3.3.108.0) in reflector mode and with an accelerating voltage in the ion source of 25 kv. spectrum was analyzed with the flex analysis software (version 3.3.80.0). 184 fig. 3 reversed phase hplc separation of the tentacles of anemonia sulcata. after pre-purification by solid phase extraction, the active material present in the fraction eluted with 40 % acetonitrile was analyzed on an silica column c18 interchrom up5odb-25qs 250x4.6mm; with a hydrophobic chain of 18 carbon atoms column. elution was performed with a linear gradient (dotted line) from 0 to 60 % acetonitrile in acidified water (0.05 % trifluoroacetic acid) over 60 min at a flow rate of 0.5 ml/min. absorbance peaks were monitored at 225 and 280 nm (blue and black full line respectively). histograms show the antibacterial activity of the peaks toward micrococcus lysodeikticus. amino acid sequencing the peptide is deposited on a glass fiber disc pre-coated with biobren plus (applied biosystems). the dried disc is introduce into a procise p494 automated protein sequencer (applied biosystems). the amino acids are determinate by multiple cycles of automated edman degradation coupled on-line to a microgradient system (model 140c) and to a hplc pth-amino acid analyzer (model 785a). the amino acid sequence of the peptide, deduced from the chromatograms generated by the acquisition data, was compared to sequences in uniprotkb/swiss-prot public protein sequence databases using blast tool from ncbi protein blast website. in silico analysis the in silico analysis allowed to determine which of the two neurotoxins purified had higher probability of being a factor antimicrobial. the database of prediction used were: apd; the antimicrobial peptide database, camp; collection of antimicrobial peptides and ampa; (wang and wang, 2004; shaini et al., 2009; torrent et al., 2009, 2012). apd: the antimicrobial peptide database is a site prediction based on two characteristic indices for the determination of antimicrobial power: wimley-white hydrophobicities and boman index (wang and wang, 2004). wimley-white hydrophobicities is a scale of hydrophobicity (kcal/mol) which defines the relative hydrophobicity of amino acid residues, the more positive is the value of this index, the more hydrophobic amino acids are located in that region of the protein. this scale is commonly used to predict the transmembrane alpha-helices of membrane proteins (white and wimley, 1999). the boman index (potential protein binding) is defined as the sum of the free energies of the side chains of amino acid residues divided by the total number of amino acid residues. a low value (≤ 1) indicates that the peptide has a higher antibacterial activity without many side effects, while a higher value (2,50 3,00) indicates that the peptide is multifunctional with activity similar to hormones (boman, 2003). the possibility of an alignment (clustalw2) for the structure homology was also verified using swissmodel. results purification of amp from the tentacles of a. sulcata antibacterial activity of the sep pak products the 10, 40 and 80 % acetonitrile (acn) fractions of the a. sulcata tentacle obtained through sep pak were tested to evaluate the antibacterial activity. preliminarily was determined the protein concentration of the same fractions, which showed a higher amount of protein in the fraction of 40 % acn (7.83 ± 2 mg/ml) compared to the 10 % acn fraction (0.98 ± 0.12 mg/ml) and a minimum amount in the protein fraction to 80 % (0.20 ± 0.09 mg/ml). the fractions were tested against different bacterial strains, highlighting specific antibacterial activity towards m. lysodeikticus. the activity was found only in the fraction at 40 % acetonitrile and was highlighted significant activity up to a dilution of 1/10 (fig. 1). following assays of samples on 96-well plate towards m. lysodeikticus and subsequent incubation in the microplate reader tecan for 16 h at a temperature of 37 °c, the data obtained show an antibacterial activity at concentrations up to 0.244 185 http://www.ncbi.nlm.nih.gov/pubmed?term=white%20sh%5bauthor%5d&cauthor=true&cauthor_uid=10410805 fig. 4 profiles of separation of the fraction 4 and fraction 5 of the tentacles of anemonia sulcata obtained by reverse phase chromatography with a gradient of acetonitrile from 20 to 60 % in acidified water (0.05 % trifluoroacetic acid) over 60 min at a flow rate of 0.5 ml/min. absorbance peaks were monitored at 225 and 280 nm (blue and black full line respectively). histograms show peaks active toward micrococcus lysodeikticus. mg/ml for the tentacles and antibacterial activity at concentrations up 0.436 mg/ml for the body, showing a higher potency of the extract sprawling respect to the body (fig. 2). at lower concentrations it has an exponential bacterial growth also confirmed by subsequent testing of the same samples on petri dishes. reverse phase chromatography the active molecules of the 40 % eluate were submitted to reversed phase hplc and eluted with a gradient of 0 60 % acetonitrile, yielding the chromatogram shown in figure 3. the graphs show a series of peaks from 30 min of entering the sample. all peaks were collected and assayed to m. lysodeikticus to try to identify potential antimicrobial factors. the assays show a high antibacterial activity in peaks collected between 34 and 46 min named fraction 1, 2, 3, 4, 5 and 6 (fig. 3). all the fractions which show high antibacterial activity were further purified by reverse phase chromatography with a gradient of acetonitrile from 20 to 60 %. the fractions 2 and 3 purified further show no significant peaks (data not shown), on the contrary fractions 4 and 5 show several peaks and in both fractions there is a single peak showing antimicrobial activity towards m. lysodeikticus (fig. 4). mass spectrometric analysis and sequencing the peak of fraction 5, which is found to be the most pure, was analyzed by mass spectrometry and sequencing. the mass spectrometric analysis showed the presence of two peptides, the first from the molecular weight of 4946,299 da present preponderantly and second, present in traces, from the molecular weight of 4808.174 (fig. 5). the sequencing by the method of edman detecting the presence of neurotoxin 2 (atx-ii) (wunderer et al., 1976) specific for the sodium channel with theoretical molecular weight of 4945.30 da in addition a small amount of blood depressing substance (bds ii) (doppelfeld et al., 1985; diochot et al., 1998) specific for potassium channels by the pm of 4773.12 da was also present (table 1). these results are in agreement with the results of mass spectrometry weight. 186 fig. 5 maldi-tof mass spectrum of fraction 5 using the matrix 2,5-dhb in reflectron mode. the peak obtained microsequence of atx ii is showed in the box. in silico analysis the in silico analysis showed in table 1 allowed to determine the antimicrobial potential of atx ii. the predicting database used were the antimicrobial peptide database (apd), collection of antimicrobial peptides (camp) and antimicrobial sequence scanning system (ampa) (wang and wang, 2004; shaini et al., 2009; torrent et al., 2009, 2012). apd is based on two characteristic indices for the determination of antimicrobial power: wimley white hydrophobicities and boman index (wang and wang, 2004). the hydrophobicity index is positive for atx ii and negative for bds ii, indicating that for the first molecule of the amino acids located in that region of the protein are more hydrophobic and therefore there is a greater potential that can play an antimicrobial function. even the boman index attaches a greater potential antimicrobial atx ii, in fact a lower value of this index (≤ 1) indicates that the peptide has a higher antibacterial activity. furthermore, aligning our sequences with those present in this database, atx ii showed a similarity of 35 % with active peptides towards gram + , gram and fungi , while bds ii showed a similarity of 35 % towards gram + and gram -. camp uses a algorithm prediction, support vector machine (svm), incorporated into the database, which also gives a probability score (0 1) for the prediction. the higher the probability, the greater the possibility that the prediction may be correct (shaini et al., 2009). according to this database both molecules, atx ii and bds ii, have the potential to be antimicrobial peptides, with a higher probability for atx ii. ampa uses of graphs showing the antimicrobial profile for the sequence of entry (s) and computes the probability of finding stretches of sequence in non-antimicrobial proteins (torrent et al., 2009, 2012). for bds ii is provided a probability value of 1 % to find the section provided in a non-antimicrobial protein. following the analysis in silico we can conclude that both molecules have a high probability of being factors and antimicrobials, this probability, it appears to be greater for atx ii. subsequently, all the toxins of a. sulcata were analyzed by prediction database, showing that neurotoxins specific for sodium channels have a higher chance of being antimicrobial factors compared to neurotoxins specific for potassium channels and, confirming that atx-ii presents all the characteristics suitable to be a factor antimicrobial together with atx-v, another neurotoxin specific for the sodium channels (table 1). using swiss-model was verified the possibility of an alignment (clustalw2) for the homology of structure between atx ii (target) and the chain a of the human beta-defensin 2 (1fd3a) (template). the quality of the model was verified by two programs: anolea and qmean. the alignment of the two sequences was also analyzed with the program dssp which defines the secondary structure, the geometric characteristics and 187 table 1 summary of the antimicrobial properties of all the known anemonia sulcata neurotoxins 188 the parameters indicated in red are congruent with neurotoxin antimicrobial properties. toxin uniprot/genbank accession number toxin family target wimley-white hydrophobicities (kcal/mol) apd total hydrophobic ratio(%) apd net charge apd boman index (kcal/mol) apd amp probability camp no-amp stretch (%) ampa atx-i p01533/ type i nav1 2.54 39 +2 1.15 0.988 0 atx-ii p01528/ type i binds to site 3. dmnav, scn2a and scn5a 1.39 38 +2 0.72 0.983 0 atx-iii p01535/ sea anemone short toxin family nav1 -0.02 33 +1 0.94 0.984 18 atx-v p01529/ type i nav 1.54 39 +3 0.76 0.979 0 sa5 ii p10280/ cnidaria kunitztype proteinase inhibitor/type ii 14.87 32 +4 2.28 0.889 10 kalicludin-1 q9twg0/ cnidaria kunitztype proteinase inhibitor/type ii kv1.2 13.97 32 +4 2.66 0.579 14 kalicludin-2 q9twf9/ cnidaria kunitztype proteinase inhibitor/type ii kv1.2 14.51 32 +8 3.01 0.827 5 kalicludin-3 q9twf8/ cnidaria kunitztype proteinase inhibitor/type ii kv1.2 13.47 33 +4 2.49 0.860 3 bds-i p11494/ cnidaria kunitztype proteinase inhibitor/type ii kv3.1, 3.2, 3.4 -2.73 34 +3 0.81 0.969 1 kaliseptin q9twg1/ type i kv1.2 10.76 38 +5 2.08 0.918 16 bds-ii p59084/ type iii kv3.1, 3.2, 3.4 -1.19 34 +2 0.95 0.928 1 orientation of the protein, showing the presence of alpha helices (h) and folds (s) common to the two molecules. although the structural model resulting show of the regions of incompatibility between the two molecules there are large regions compatible with a common structure in terms of energy (anolea and qmean) both as a possible presence of secondary structures common. discussion cnidarian are a source of neurotoxins acting on sodium and potassium ion channels (yamaguchi et al., 2010) or cytotoxic pore forming molecules (maisano et al., 2013; parisi et al., 2014). the sea anemone a. sulcata, is exposed to attack from predators with the tentacle always exposed. moreover, a. sulcata captures food actively wide spectrum of prey (chintiroglou and koukouras, 1992), that causes frequently the rupture of the soft tentacle with consequent bacterial infection. thus, the chemical arsenal of a. sulcata represents the preferred strategy to survive in such a habitat mainly with the produced neurotoxin together with antimicrobial molecules (moran et al., 2008). the present study describes isolation and characterization of a neurotoxins that possess also antibacterial activity against m. lysodeikticus. this neurotoxin was characterized and purified from the tentacle of an anthozoa a. sulcata by acid extraction, hplc purifications mass spectroscopy and antibacterial assays. more than fifty toxins specific for sodium channels classified on the basis of amino acid sequence (homma and shiomi, 2006). the types 1 and 2 are composed of a number of amino acid residues between 46 and 49, the type 3 is instead characterized by polypeptide chains ranging from a minimum of 27 to a maximum of 32 amino acid residues (homma and shiomi, 2005). a common origin of animal amps and toxins seems to be biologically expedient. despite the difference of their affected targets-the membranes of micro or macroorganisms, functioning conditions of both peptide groups impose similar requirements to molecule structure and its physicochemical characteristics, such as cationicity, often coupled with amphipathicity, resistance to enzymatic hydrolysis, and overall compactness. the best-known examples of these are the pardaxins of fish that were originally purified on the basis of their toxic, anti-predatory activity (lazarovici et al., 1986) yet have similar antibacterial potencies to the amphibian magainins and insect cecropins (oren and shai, 1996). the way these molecules are engaged in defense and aggression mechanisms includes temporary accumulation in reservoirs of specialized cells followed by eventual release to extracellular environment. it would be logical to assume that similar mechanisms of processing and secretion may be involved in both cases requiring the presence of the same signal sequences within peptide precursors. overlapping of biological properties, along with the sequence homology, might be a consequence of divergent evolution from a common ancestor (ovchinnikova et al., 2006). the neurotoxin atx ii is a type 1 sodium channel toxin and consists of 47 amino acid residues, linked by three disulfide bridges. atx ii specifically binds to the sodium channel, thus delaying its inactivation during signal transduction. this molecule has a strong effect on crustaceans and insects and a weaker effect on mammals. the research about these type of toxins specific for sodium channels have been thoroughly investigated also because they constitute a major fraction of the venom (moran et al., 2009). nature thus seems to ‘mix and match’ useful domains to create amps (smith et al., 2010) that could have mechanisms to destroy their targets compatible with other biological function. cnidarians have impressive strategies for locomotion, feeding and reproduction. its detailed study may allow better understand the diversification of the molecular novelties of these unique metazoan species (frazao et al., 2012). in the 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sea anemone toxin bds: significance for cns and biophysical studies. j. neurosci. 25: 87358745, 2005. 191 http://www.ncbi.nlm.nih.gov/pubmed?term=white%20sh%5bauthor%5d&cauthor=true&cauthor_uid=10410805 http://www.ncbi.nlm.nih.gov/pubmed?term=wimley%20wc%5bauthor%5d&cauthor=true&cauthor_uid=10410805 http://www.ncbi.nlm.nih.gov/pubmed/10410805?dopt=abstractplus## research report isj 8: 132-142, 2011 issn 1824-307x research report proteolytic activity in the midgut of ectomyelois ceratoniae zeller (lepidoptera: pyralidae), pomegranate carob moth m ranjbar, jj sendi, a zibaee department of plant protection, college of agriculture, university of guilan, 41635-1314, rasht,iran accepted july 11, 2011 abstract in this study, the proteolytic activity in the midgut of ectomyelois ceratoniae as the major pest of pomegranate was investigated to find nature of specific proteases and their properties for adopting possible pest management procedure. it was found that fourth and fifth instar larvae had the highest proteolytic activity as well as specific proteinases including, elastase, trypsin-like, chymotrypsin-like and two exopeptidases. the optimal ph of general protease was 10 and 9 for azocasein, casein and hemoglobin as substrate. the optimal temperature of the total proteolytic activity in the midgut of e. ceratoniae was found 30 and 35 °c by using azocasein and casein as substrates, respectively. in case of hemoglobin, the enzyme showed the highest enzymatic activity at temperatures from 15 to 35 °c. there was no enhancement in the proteolytic activity by using different cations but sds, citric acid and mercaptoetahnol significantly decreased the proteolytic activity in the midgut of e. ceratoniae. using specific proteolytic inhibitors including pmsf, tlck, tpck, e-64, dtt and phenanthroline revealed presence of serine proteases as the major proteases in the midgut of e. ceratoniae. key words: ectomyelois ceratoniae; midgut; protease; characterization   introduction insects obtain food to provide their necessary energy for biological processes. to obtain nutrients from ingested food, the macromolecules must be digested in the alimentary canal to smaller ones for absorption via midgut epithelium cells. to do so, gut is the important part of the insect to break down ingested carbohydrates, fats and proteins to their monomers. digestion process in the midgut is catalyzed by different enzymes known by their substrates namely carbohydrases, lipases and proteases (terra and ferreira, 2005). in fact, enzyme pattern in the gut of each insect represent its ability to utilization of substrate according to kind of diet. among mentioned digestive enzymes, proteases are the hydrolyzing peptidases that bind to protein a molecule from different positions for breaking-down them to more simple unites. these enzymes are divided into two groups; exopeptidases, that are separated an amino acid sequence at the end of peptide molecules and endopeptidases (proteinases) that break peptide bonds in the middle of molecule. based on location ___________________________________________________________________________ corresponding author: arash zibaee department of plant protection college of agriculture university of guilan, 41635-1314, rasht, iran e-mail: arash.zibaee@gmx.com characteristics and acidity endopeptidases are divided into the following types as: serine, cysteine, aspartic and metalloproteinases. endopeptidases are responsible for primary digestion of proteins converting them to oligopeptides. the oligopeptides resulting from proteinase action are digested from the n-terminal end by aminopeptidases and from the c-terminal end by carboxypeptidases, both enzymes liberating one amino acid residue at each catalytic step (terra and ferreira, 2005). ectomyelois ceratoniae zeller (lepidoptera: pyralidae) (larval-instars) is a major destructive pest of pomegranate and pistachio in iran so that its annual damage is between 15 to 90 % (behdad, 2002). the adults are gray butterflies that emerge on may and lay their eggs on june into the crown, on the rod, anther or internal surface of sepal of pomegranate. the eggs hatch after 10 days and the larvae penetrate into the fruit and fed on their internal parts (behdad, 2002). due to variation in the characteristics of insect digestive enzymes, it is necessary the better understanding of enzyme biochemistry to get knowledge on plant-herbivores associations (wilhite et al., 2000). selection of the target molecules like digestive enzymes is one of the most important aspects in the alternative pest control by providing plant protease or synthetic inhibitors. so, characterization of the digestive enzymes especially   132 proteases is mandatory to reach this avenue. based on this conception, the objectives of this study were: (i) determination of general and specific proteolytic activity by using general and specific substrates, (ii) effect of ph and temperature, (ii) effects of various cations, (iii) effect of general and specific inhibitors on proteolytic activity and demonstrating enzymatic inhibition by electrophoresis. materials and methods ectomyelois ceratoniae rearing intended to provide the population of insect, first the appropriate number of developmental stages is collected from the damage gardens. to obtain same age larvae, the collected individuals were grown on pomegranate fruit exactly the same as damaged garden under 16l:8d, 30 °c and 60 % relative humidity. e. ceratoniae dissection and sample preparation midguts of 150 larvae (10 larvae for each larval instar and 100 ones for enzyme characterization) were removed by dissection under a dissecting microscope in ice-cold distilled water. samples were rinsed in ice-cold distilled water and grounded with a handling hemogenizer. homogenates were transferred to 1.5 ml centrifuge tubes and centrifuged in 13000 rpm for 15 min at 4 °c. the supernatants for each tissue were pooled then stored at -20 °c for subsequent analyses. azocasein general proteolytic activity by using azocasein 2 % was measured based on a method described by elpidina et al. (2001). the reaction mixture consisted 100 µl of universal buffer solutions, 50 µl azocasein and 20 µl enzyme. after incubation at 37 °c for 60 min, proteolysis was stopped by addition of 150 µl of 10 % trichloroacetic acid (tca). precipitation was achieved by cooling at 4 °c for 120 min and it was centrifuged at 13,000 rpm for 10 min. an equal volume of 2 m naoh was added to the supernatant then the absorbance was recorded at 440 nm. blank solution consisted all mentioned portions except for enzyme solution. hemoglobin cohen‘s method (cohen, 1993) was used to assay general proteolytic activity in midgut by using hemoglobin as substrate. hemoglobin solution (50 µl) was added to 100 µl of appropriate buffer solution and incubation at 30 °c was initiated after addition of 20 µl of enzyme solution for 120 min. for termination of proteolysis, 150 µl of 10 % tca was added to the reaction mixture. precipitation was achieved by cooling at 4 °c for 45 min then the reaction mixture was centrifuged at 13000 rpm for 10 min. blanks solution contained all mentioned portions except for enzyme. the peptides liberated from hemoglobin were estimated using folin-phenol reagent at 650 nm (folin and ciocalteu, 1927). casein to measure general proteolytic activity by using casein, 50 µl of substrate solution was added to 100 µl of appropriate buffer. current solution was mixed by swirling and incubated at 37 °c for exactly 10 minutes. then, 150 µl of 10 % tca was added and the reaction solution was mixed by swirling and incubated at 37 °c for about 30 minutes. precipitation was achieved by cooling at 4 °c for 45 min then the reaction mixture was centrifuged at 13,000 rpm for 10 min. blanks solution contained all mentioned portions except for enzyme. the peptides liberated from casein were estimated using folin-phenol reagent at 650 nm (folin and ciocalteu, 1927). determination of optimal temperature (°c) on general proteolytic activity and stability the temperature range from 15 80 °c were used to find optimal temperature for general proteolytic activity in the midgut of e. ceratoniae by using three common substrates. the reaction mixtures were similar to described earlier but buffer solution was universal buffer at ph 9. specific proteolytic activity serine proteolytic activity trypsin-, chymotrypsinand elastase-like activities (as three subclasses of serine proteases) were assayed using a concentration of 1mm bapna (nabenzoyll-arginine-p-nitroanilide), 1 mm saapppna (n-succinyl-alanine-alanine-prolinephenylalanine-p-nitroanilide) and 1 mm saaapna (n-succinyl-alaninealaninealanine-p-nitroanilide) as substrates, respectively. the reaction mixture consisted 80 µl of universal buffer (ph 9), 10 µl of each mentioned substrate and 5 µl of enzyme solution. the reaction mixture was incubated at 37 °c for a period from 5 60 min before adding 30 % acetic acid to terminate the reaction. the absorbance of the resulting mixture was then measured spectrophotometrically at 410 nm by pnitroaniline release. cysteine protease for cysteine proteinase assay, benzyloxycarbonyl-arg-argp-nitroaniline was used as substrate. hydrolysis of the 1 mm final concentration of the substrates was determined by measuring the absorbance by p-nitroaniline after 30 min of incubation. the absorbance of different concentrations of p-nitroaniline was read at 410 nm to find extinction coefficient for specific activity calculation. to prove the specific proteolytic activity, a negative control were provided for each substrate separately containing all mentioned components except for enzyme pre-boiled at 100 °c for 30 min. exopetidase activity leucine p-nitroaniline (lpna) (1 mm) was used to find aminopeptidase activity in the midgut of e. ceratoniae. the reaction mixture consisted 80 µl of universal buffer (ph 8), 10 µl of each mentioned substrate and 5 µl of enzyme solution. the reaction mixture was incubated at 37 °c for a period from 5 60 min before adding 30 % acetic acid to terminate the reaction. the absorbance of the resulting mixture was then measured spectrophotometrically at 340 nm by p-nitroaniline release.   133 n-(3-(2-furyl) acryloyl)-l-phenylalanyl-lphenylalanine was used to find aminopeptidase activity in the midgut of e. ceratoniae. the reaction mixture consisted 80 µl of universal buffer (ph 8), 10 µl of each mentioned substrate and 5 µl of enzyme solution. the reaction mixture was incubated at 37 °c for a period from 5-60 min before adding 30 % acetic acid to terminate the reaction. the absorbance was read at 340 nm. optimal ph determination of specific proteases universal buffer (2 mm, ph range 3 14) was used to obtain the optimal ph of each specific protease and find possible ph dependency of each substrate. the reaction mixtures were similar to above but the ph of used buffer was varied from 3 14. also, negative controls as described above were considered in the experiment. effect of monoand di-valent cations on general proteolytic activity different concnetrations (1, 3 and 5 mm) of cations including k+, na+, ca2+, mn2+, mg2+, zn2+ and fe2+ were used to obtain potential alteration in general proteolytic activity in the midgut of e. ceratoniae. initially, 50 µl of each cation (different concentration) was added to universal buffer solution (2 mm, ph 9) and gently stirred for 10 min then, 50 µl of casein (1 %) added and additionally stirred for 10 min. incubation was initiated after adding of 20 µl enzyme for 60 min. proteolysis was stopped by addition of 150 µl of 10 % trichloroacetic acid (tca). precipitation was achieved by cooling at 4 °c for 120 min and it was centrifuged at 13000 rpm for 10 min. an equal volume of 2 m naoh was added to the supernatant and the absorbance was recorded at 440 nm. blank solution consisted all mentioned portions except for enzyme solution. effect of general and specific inhibitors general inhibitors selected general inhibitors in this experiments consisted sodium dodecylsulphate, urea, ethylendiamidetetraacetic acid and βmercaptoethanol in concentrations of 1, 5 and 10 mm. initially, 50 µl of each compound (different concentration) was added to universal buffer solution (2 mm, ph 9) and gently stirred for 10 min then, 50 µl of casein (1 %) added and additionally stirred for 10 min. incubation was initiated after adding of 20 µl enzyme for 60 min. proteolysis was stopped by addition of 150 µl of 10 % trichloroacetic acid (tca). precipitation was achieved by cooling at 4 °c for 120 min and it was centrifuged at 13000 rpm for 10 min. an equal volume of 2 m naoh was added to the supernatant and the absorbance was recorded at 440 nm. blank solution consisted all mentioned portions except for enzyme solution. specific inhibitors following compounds were used to find any alteration in the proteolytic activity of the midgut of e. ceratoniae regarding to specific used substrates; pmsf (phenylmethylsulfonyl fluoride, 1, 3, 5 mm); trypsin inhibitor, tlck (na-p-tosyl-l-lysine chloromethyl ketone, 1, 3, 5 mm); chymotrypsin inhibitor, tpck (n-tosyl-l-phenylalanine chloromethyl ketone, 1, 3, 5 mm); cysteine protease inhibitor e-64 (l-trans-epoxysuccinyl-leucylamido(4-guanidino)-butane, 1, 3, 5 mm), cystatin (1, 3, 5 mm) and metalloprotease inhibitors, phenanthroline, also, dtt (dithiothreitol, 1, 3, 5 mm) were used as cysteine activator. for e-64, cystatin and dtt no effects were observed (data not shown). electrophoresis zymogram electrophoretic detection (laemmli, 1970) of proteolytic enzyme was performed using resolving and stacking polyacrylamide gels of 10 % and 4 %, respectively, according to the method described by garcia-carreno et al. (1993) with slight modifications. non-reducing page was carried out at 4 °c in a constant voltage of 110 mv so that gelatin (0.5 %) were added in resolving gel. when dye reached at the bottom of glace, gel carefully separated and put in universal buffer for 15 min. then, gels were washed in water and immediately fixed and stained with 0.1 % coomassie brilliant blue r-250 in methanol-acetic acid-water (50:10:40) overnight. destaining was done in methanol-acetic acid-water (50:10:40) for at least 2 h. characterization of protease classes in sds-page zymograms using specific inhibitors was done according to garcia-carreno et al. (1993) with some modifications. a total of 50 µl of the enzyme extract was mixed with 30 µl of inhibitors at 5mm concentration including pmsf, tlck and tpck. electrophoresis and zymogram were carried out as described before. protein determination protein concentration was measured according to the method of bradford (1976), using bovine serum albumin (bio-rad, usa) as standard. statistical analysis all data obtained from a complete randomized design were compared by one-way analysis of variance (anova) followed by tukey's studentisized test when significant differences were found at p ≤ 0.05 (sas, 1997). differences between samplings (n = 3) were considered statistically significant at a probability less than 5 % and marked in figures and tables. results table 1 shows general and specific proteolytic activity in the various instar larvae of e. ceratoniae. although the enzyme activity was higher in 5th instars than 4th one but no significant differences was observed between them (table 1). similar results were obtained when specific proteolytic activity was assessed. on the other hands, 4th and 5th instar larvae of e. ceratoniae had the highest proteolytic activity (table 1). three general substrates were used to determine the optimal ph of general proteolytic activity in the midgut of e. ceratoniae. it was found ph 9 and 10 (azocasein) as the optimal ph (fig. 1). the enzymatic activity was from ph 3 7 then increased and reached to its maximum at ph 9 and 10 (fig. 1). all these mentioned substrates were used to find the optimal temperature for total   134 table 1 total and specific proteolytic activity (µmol/min/mg protein) in the different larval instars of e. ceratoniae larval instars general protease elastase trypsin chymotrypsin aminopeptidase carboxypeptidase 1st instar 23±2.41d 0.96±0.033d 1.28±0.17d 0.67±0.07e 4.11±0.27d 0.27±0.022d 2nd instars 104±11.28c 2.00±0.27c 4.56±0.31c 1.52±0.11d 9.56±0.81c 1.04±0.078cd 3rd instars 163±29.45b 2.71±0.47b 10.14±0.47b 2.11±0.41c 12.21±0.67b 3.14±0.67c 4th 231±81.22a 4.59±0.77ab 15.23±1.11ab 3.02±0.72b 15.77±0.75a 7.22±0.96b 5th 250±67.24a 5.02±0.88a 17.44±2.14a 4.31±0.87a 16.00±0.80a 9.71±0.88a 1 different letters show statistical differences among larval instars (tukey’s test, p ≤ 0.05). a) b) c) fig. 1 optimal ph determination for general proteolytic activity in the 5th instar larvae of e. ceratoniae. azocasein (2 %), hemoglobin (20 mg/ml) and casein (1 %) were used as substrates. statistical analysis was calculated by tukey’s test (sas software) and showed by different letters (p ≤ 0.05; n = 3).   135 a)   b) c) fig. 2 optimal temperature (°c) determination for general proteolytic activity in the 5th instar larvae of e. ceratoniae. azocasein (2 %), hemoglobin (20 mg/ml) and casein (1 %) were used as substrates. statistical analysis was calculated by tukey’s test (sas software) and showed by different letters (p ≤ 0.05; n = 3).   136 a) b) fig. 3 time course determination of the specific proteolytic activity in the midgut of e. ceratoniae. first, 80 µl appropriate buffer (in all range) incubated with 20 µl substrate at 35 °c as standard temperature in enzymatic assessment. after 10 min, 7 µl of enzyme added and allowed the reaction to continue for 60 min. at each time interval, absorbance was read at 405 nm for serine proteases and 340 nm for exopeptidases. proteolytic activity. in the case of azocasein, the enzyme activity sharply increased at ph 9 and 10 then decreased (fig. 1). when hemoglobin and casein were used as substrates, the enzyme activity increased at ph 8 and 9 with the highest activity at ph 9 then decreased so that the lowest enzymatic activity was obtained at ph 14 (fig. 1). the optimal temperature of the general proteolytic activity in the midgut of e. ceratoniae was found 30 and 35 °c by using azocasein and casein as substrates, respectively (fig. 2). in the case of hemoglobin, the enzyme showed the highest activity at temperatures 15 to 35 °c then decreased (fig. 2). maximal specific proteinases activity of the midgut extract was obtained in case of trypsin-like protease with the initial velocity of 40 at 405 nm/min (fig. 3). the initial velocities of activity in midgut extract on elastase and chymotrypsin-like were found 30 and 40 at 405 nm/min, respectively (fig. 3). in case of aminoand carboxypeptidases, the initial velocities were observed 30 at 340 nm/min for both enzymes (fig. 3). by using specific substrates and universal buffer, it was found alkali condition to optimal activity of specific proteases in the midgut of e. ceratoniae (fig. 4). the optimal ph for elastase, trypsin-like and chymotrypsin-like activity were obtained 9, 10 and 8, respectively while it was found 10 for amino and carboxypeptidases (fig. 4). tables 2 and 3 show the effects of some cations and general compounds on the general proteolytic activity in the midgut of e. ceratoniae. in case of cations, not only no enhancement of proteolytic was observed but also some cations decreased the enzymatic activity (table 2). in case of general compounds, urea and edta had no significant effects on the proteolytic activity but sds, citric acid and mercaptoethanol significantly decreased the enzymatic activity so that the most inhibition was observed in case of mercaptoethanol (table 3). different specific inhibitors were used to show the nature of proteases in the midgut of e. ceratoniae (table 4). e-64 (cysteine inhibitor), dtt (cysteine activator) and phenanthroline (metalloproteinase inhibitor) had no effect on the proteolytic activity so being cysteine and matalloproteinases was denied in the midgut of e. ceratoniae (table 4). instead, pmsf (serine protease inhibitor), tlck (trypsin-like protease inhibitor) and tpck (chymotrypsin-like protease inhibitor) significantly decreased the proteolytic activity so it was confirmed that serine proteases were the major proteases in the midgut of e. ceratoniae (table 4).   137          a) b)         c) d)   e) fig. 4 optimal ph determination of the specific proteolytic activity in the midgut of e. ceratoniae by using specific substrates. one way analysis (anova, tukey’s test) was used to determine statistical differences by various letters (p ≤ 0.05; n = 3). figure 5 demonstrates zymogram analysis of proteinase inhibitors effects on gelatin (0.5 %) hydrolytic activity in the midgut of e. ceratoniae, by using 8 % non-reducing sds-page. in control, there were three protease bands namely p1, p2 and p3 that p1 band was sharper than other ones (fig. 5). when pmsf was used as serine protease inhibitor, p1 change to a narrow band and p2 almost disappear and p3 completely removed. by using tlck as trypsin-like inhibitor, p3 completely disappeared and p1 and p2 were less sharp in comparison with control (fig. 5). tpck as chymotrypsin-like protease inhibitor decreased the sharpness p1 but made no significant changes in p2 and p3 (fig. 5). discussion this study depicted different proteases in the midgut of a serious pomegranate pest, e. ceratoniae. detailed experiments to clarify the nature of specific proteases revealed being serine proteases including trypsin, chymotrypsin and elastase types as well as two types of exopeptidases namely amino and carboxypeptidases. the higher activity of serine proteases in the midgut of e. ceratoniae showed a typical fact for lepidopteran larvae (johnston, 1993, 1995; broadway, 1995; gatehouse et al., 1999; hegedus et al., 2003; terra and ferriera, 2005; chougule et al., 2008).   138 table 2 effect of monoand di-valent cations on the proteolytic activity in the midgut of e. ceratoniae cations concentration (mm) relative activity (%) control 100 k+ 1 4.5* 3 6.5* 5 3.7* na+ 1 93.4 3 41.6* 5 13.3* ca2+ 1 103.7 3 108.8 5 100.7 mn2+ 1 17.1* 3 3.3* 5 0* mg2+ 1 49.5* 3 9.7* 5 0* zn2+ 1 105.8 3 98.8 5 104.8 fe2+ 1 53.1* 3 43.1* 5 21.4* 1. casein 1 % was used as substrate. 2. asterisks show statistical differences among used concentrations of cations (tukey’s test, p ≤ 0.05). table 3 effect of some general inhibitors on the proteolytic activity in the midgut of e. ceratoniae concentration (mm) urea sds edta citric acid mercaptoethanol control 100a 100a 100a 100a 100a 1 92a 28b 85a 99a 25b 5 82a 14c 92a 26b 38b 10 83a 14c 101a 19b 10c 1. all experiments were made at ph 9 and temperature of 35 °c. 2. different letters show statistical differences among used concentrations (tukey’ test, p ≤ 0.05). ph and temperature are the two main factors for characterization of enzymes in the biochemistry. both of these parameters provide suitable conditions to better affinity between enzyme and substrate complex as well as higher rate of breaking down of the complex. also, these parameters prepare a better situation of substrate accessibility for enzyme site of action (zibaee et al., 2011). in this study we found the optimal ph of proteolytic activity at 9 and 10 for both general and specific substrates except for elastase that it was obtained the highest activity at ph 8. the results of optimal ph in the current study confirm terra and ferreira (1994) on attribution the high ph of the lepidopteran gut to an adaptation of leaf-eating lepidopteran ancestors for extracting hemicellulose from plant cell walls. the alkaline ph is typical for the highest activity of proteases in the midgut of lepidopteran larvae. different earlier reports confirmed this finding including: spodoptera littoralis fabricius (lepidoptera: noctuidae) ph 11 (ishaaya et al., 1971); s. litura fabricius (lepidoptera: noctuidae),   139 fig. 5 zymogram analysis of the effect of proteinase inhibitors on gelatin hydrolytic activity in the midgut of e. ceratoniae, by using 8 % non-reducing sds-page. a: control (no inhibitor), b: pmsf 5mm, c: tlck, 5mm, d: tpck 5mm and e: molecular weight marker. ph 9, 10.5, and 11.0 (ahmad et al., 1976, 1980); heloithis zea stral (lepidoptera: noctuidae), ph 11 (klocke and chan, 1982); g. mellonella walker (lepidoptera: pyralidae), ph 10.5 and 11.2 (hamed and attias, 1987); helicoverpa armigera fabricius (lepidoptera: noctuidae), ph 9.5 and 10 (johnston et al., 1991); phtorimaea opercula zeller (lepidoptera: gelechidae), ph > 9.0 (christeller et al., 1992); manduca sexta, ph 8.5 (samuels et al., 1993); helothis virescens, ph 10 11 (johnston et al., 1995); lacanobia oleracea l (lepidoptera: noctuidae) ph < 11 (gatehouse et al., 1999) and mamestra brassicae l. (lepidoptera: noctuidae), ph 11 (chougule et al., 2008). temperatures of 30 and 35 °c were found as the optimal temperatures for activity of proteases in the midgut of e. ceratoniae by using three general substrates. to obtain the optimal temperature of proteases, it is mandatory to use different substrates because of the specificity of each substrate for temperature. for example, azocasein and hemoglobin have been kept at 4 °c but casein at 25 or 30 °c. so, using all these substrates could be attributed to real enzymatic activity not denaturation of the available substrates. biological reactions occur faster with increasing temperature up to the point of enzyme denaturation, above which temperature, enzyme activity and the rate of the reaction decreases sharply (zibaee et al., 2011). extremes in temperatures can also disrupt the hydrogen bonds that hold the enzyme in its threedimensional structure, denaturing the enzyme (zibaee et al., 2011). in this study, different concentrations of ions, general and specific inhibitors were used to verify the type of specific proteases and the nature of their activity. it was found no enhancement of the proteolytic activity by using cations and surprisingly the enzyme activity decreased. these findings have been proved by molecular analysis in the midgut proteolytic activity of lepidoptera. on the other hands, although insect serine proteases having structural features resembling vertebrate trypsins, they differ from these because they are not activated or stabilized by ions especially calcium (terra and ferriera, 1994). also, our results by using edta as the general chelating agents of ions in the active sites of enzymes showed no inhibition on proteolytc activity in the midgut of e. ceratoniae. another experiment that proved this finding was to use phenenthroline as the metalloproteinase inhibitor that showed no significant difference on the   140 proteolytic activity. the important point in using inhibitors was citric acid inhibitory effects. citric acid is a specific binding molecule to amino acids in the active site of the enzyme (especially histidine) and it makes inaccessible for enzymatic activity. as the important point, it was shown that pmsf, as the serine-protease inhibitor, and tlck, as the trypsin-like protease inhibitor as well as tpck as the chymotrypsin-like protease inhibitor, significantly decreased the proteolytic activity in the midgut of e. ceratoniae. because tlck is known to act as a specific inhibitor of trypsin-like proteases by binding to the histidine residue within the active site of the enzyme (anwar and saleemudin, 2002), the inhibition of the e. ceratoniae protease by tlck indicates that a histidine residue might be at the enzyme active site. these results correspond with the finding of anwar and saleemudin (2002), spilosoma oblique, josephrajkumar et al. (2006) in conogethes punctiferalis and chouglue et al. (2008) in mamestra brassicae. digestive enzymes of insects play dual functions in digestion and defense (zibaee and bandani, 2010) so that in response to inhibitor challenge, many insects undergo a series of adaptive changes to regain their normal feeding and growth, including over-expression of inhibitorinsensitive proteases. also, they could be a reliable target to develop pest management programs by considering host plant resistance having digestive enzyme inhibitor genes. using biotechnological approaches to target these enzymes, such as transgenic plants, may be a winning strategy for the development of effective bio-pesticides. this could be achieved through traditional breeding programs to select plants possessing higher degrees of resistance factor that are not easily degraded. acknowledgement this study was supported by a university of guilan grant. the authors highly appreciate h tabatabaee and m allahyari for their assistance. references ahmad z, saleemuddin m, siddiqui m. alkaline protease in the larvae of the army worm spodoptera litura. insect biochem. 6: 501-505, 1976. ahmad z, saleemuddin m, siddiqui m. purification and characterisation of three alkaline proteases from the larva of army worm spodoptera litura. insect biochem. 10: 667-673, 1980. anwar a, saleemudin m. purification and characterization of a digestive alkaline protease from the larvae of spilosoma oblique. arch. insect biochem. physiol. 51: 1-12, 2002. behdad e. major plant pests in iran. yadbood publishing, 2002. bradford m. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. analyt. biochem. 72: 248-254, 1976. broadway rm. are insects resistant to plant proteinase inhibitors? j. insect physiol. 41: 107-116, 1995. chougule np, doyle e, fitches e, gatehouse ja. biochemical characterization of midgut digestive proteases from mamestra brassicae (cabbage moth; lepidoptera: noctuidae) and effect of soybean kunitz inhibitor (skti) in feeding assays. j. insect physiol. 54: 563-572, 2008. christeller jt, liang wa, markwick np, burgess epj. midgut protease activities in 12 phytophagous lepidopteran larvae: dietary and proteases inhibitory interactions. insect biochem. mol. biol. 22: 248-254, 1992. cohen ac. organization of digestion and preliminary characterization of salivary trypsin like enzymes in a predaceous heteropteran, zelus renadii. j. insect physiol. 39: 823-829, 1993. doijode sd. seed storage of horticultural crops. new york: food products press, 2001. elpidina en, vinokurov ks, gromenko va, rudenskaya ya, dunaevsky ye, zhuzhikov dp. compartmentalization of proteinases and amylases in nauphoeta cinerea midgut. arch. insect biochem. physiol. 48: 206-216, 2001. folin o, ciocalteu v. on tyrosine and tryptophane determinations in proteins. j. biol. chem. 73: 627-650, 1927. garcia-carreno fl, dimes le, haard nf. substrate-gel electrophoresis for composition and molecular weight of proteinases or proteinaceous protease inhibitors. analyt. biochem. 214: 61-69, 1993. gatehouse amr, norton e, davison gm, babbe sm, newell ca, gatehouse ja. digestive proteolytic activity in larvae of tomato moth, lacanobia oleracea; effects of plant proteinase inhibitors in vitro and in vivo. j. insect physiol. 45: 545-558, 1999. hamed mmb, attias j. isolation and partial characterization of two alkaline proteases of greater wax moth galleria mellonella l. insect biochem. 17: 653–658, 1987. hegedus dd, baldwin m, o’grady l, braun s, gleddie a, sharpe d, et al. lydiate m. midgut proteases from mamestra configurata (lepidoptera: noctuidae) larvae: characterization, cdna cloning and expressed sequence tag analysis. arch. insect biochem. physiol. 53: 30-47, 2003. ishaaya i, moore i, joseph d. protease and amylase activity in the larvae of egyptian cotton worm spodoptera littoralis. j. insect physiol. 17: 945-953, 1971. johnston ka, lee mj, gatehouse ja, anstee jh. the partial purification and characterization of serine protease activity in the midgut of larval helicoverpa armigera. insect biochem. 21: 389397, 1991. johnston kaja, gatehouse jh, anstee, jh. effects of soybean protease inhibitors on the growth and development of larval helicoverpa armigera. j. insect physiol. 39: 657-664, 1993. johnston kam, le c, brough va, hilder amr, gatehouse ja. protease activities in the larval midgut of heliothis virescens: evidence of trypsin and chymotrypsin-like enzymes. insect. biochem. mol. biol. 25: 375-383, 1995. josephrajkumar a, chakrabarty r, thomas g. midgut proteases of the cardamom shoot and   141 capsule borer conogethes punctiferalis (lepidoptera: pyralidae) and their interaction with aprotinin. bull. entomol. res. 96: 91-98, 2006. klocke ja, chan bg. effects of cotton condensed tannin on feeding and digestion in the cotton pest heliothis zea. j. insect physiol. 28: 911915, 1982. laemmli uk. cleavage of structural proteins during the assembly of the head of bacteriophage t4. nature 227: 680-685, 1970. leora software, polo-pc. a user guide to probit or logit analysis, leora software, berkeley, ca, 1987. samuels ri, charnley ak, reynolds se. a cuticle degrading proteinase from the moulting fluid of the tobacco hornworm, manduca sexta. insect biochem. mol. biol. 23: 607-614, 1993. sas institute. sas/stat user's guide for personal computers. sas institute, cary, nc, 1997. terra wr, ferreira c. insect digestive enzymes: properties, compartmentalization and function. comp. biochem. physiol. 109b: 1-62, 1994. terra wr, ferreira c. biochemistry of digestion. in: gilbert li, iatrou k, gill ss (eds), comprehensive molecular insect science (vol. 3), san diego, elsevier, pp 171-224, 2005. wilhite se, elden tc, brzin j, smigocki ac. inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors. insect biochem. mol. biol. 30: 1181-1188, 2000. zibaee a, bandani ar, malagoli d. purification and characterization of phenoloxidase from the hemocytes of eurygaster integriceps (hemiptera: scutelleridae). comp. biochem. physiol. 158b: 117-123, 2011.   142 isj 12: 214-224, 2015 isj 12: 214-224, 2015 issn 1824-307x research report paralogous gene conversion, allelic divergence of attacin genes and its expression profile in response to bmnpv infection in silkworm bombyx mori g lekha, t gupta, k trivedy, k ponnuvel genomics division, seri biotech research laboratory, carmelaram post, kodathi, bangalore 560 035, india accepted july 27, 2015 abstract the genomic organization, structure and polymorphism of attacin gene within the mulberry silkworm bombyx mori strains have been analyzed. genomic contig (aadk01007556) of b. mori attacin gene contains locus with two transcribed basic attacin genes, which were designated as attacin i and attacin ii. survey of the naturally occurring genetic variation in different strains of silkworm b. mori at the promoter and coding regions of two attacin genes revealed high levels of silent nucleotide variations (14 % per nucleotide heterozygosity) without polymorphism at the amino acid level (nonsynonymous substitution). we also investigated variations in gene expression of attacin i and attacin ii in silkworm b. mori infected with nucleopolyhedrovirus (bmnpv). two b. mori strains, sarupat, csr-2 which were resistant and susceptible to bmnpv infection respectively were used in this study. expression profiles of b. mori genes were analyzed using microarray technique and results revealed that the immune response genes including attacin were selectively up regulated in virus invaded midguts of both races. microarray data and real-time qpcr results revealed that attacin i gene was significantly up-regulated in the midgut of sarupat following bmnpv infection, indicating its specific role in the anti-viral response. our results imply that these up-regulated attacin genes were not only involved in anti-bacterial mechanism, but are also involved in b. mori immune response against bmnpv infection. key words: bombyx mori; attacin; microarray; genomic organization; differential expression   introduction insects fight bacterial infection, in part, through the extra cellular circulation of a variety of short, general antibacterial peptides (iwanaga and lee, 2005). although over 400 different innate immune peptides have been identified in eukaryotes (hoffmann et al., 1999), most insects produce relatively small number, fewer than 10 peptide classes and these have to effectively combat a wide range of potential pathogens. among the different antibacterial proteins produced in insects, attacin, a high molecular weight protein has a major role in insect innate immunity. the amino acid sequence deduced from cloned attacin cdna of b. mori revealed that the cdna encodes an attacin precursor protein (sugiyama et al., 1995). the putative mature protein of bombyx mori attacin ___________________________________________________________________________ corresponding author: kangayam m ponnuvel genomics division seri biotech research laboratory carmelaram post, kodathi bangalore 560 035, india e-mail: kmpvel@yahoo.com revealed varying levels of identity in amino acid sequences with those of hyalophora cecropia acidic (70.4 %) and basic (68.3 %) attacins and sarcophaga peregrina sarcotoxin iia (18.8 %). injection of escherichia coli cells into b. mori larvae resulted in rapid induction of the expression of b. mori attacin gene that continued at least for 48 h mainly in fat bodies and hemocytes (sugiyama et al., 1995). taniai et al. (1996) isolated a genomic clone encoding attacin from genomic library of b. mori, and determined the nucleotide sequence of the 5'-upstream region. mature attacin peptides are typically 190 214 amino acids in length (sarcophaga peptides are longer) and adopt a “random coil” structure in solution (gunne et al., 1990). this loose, flexible structure is devoid of disulfide bonds and does not take a rigid conformational shape which may allow relatively free amino acid substitution, explaining the low level of amino acid identity between attacin homologs in distant taxa. dushay et al. (2000) reported cloning of two closely linked attacin genes from d. melanogaster. a comparison of their protein coding sequences 214   mailto:kmpvel@yahoo.com revealed that the amino acid sequences were more highly conserved than the nucleotide sequences, suggesting expression of both the genes (wheelan et al., 2001). in this paper we present data on the quantity of polymorphism in the b. mori attacin genes and their expression profile in resistant and susceptible race. further, the genomic structure of the attacin gene was analyzed and compared with attacin sequences of selected indian silkworm strains. the structure of exon and intron as well as the phylogenetic relation of b. mori attacin gene to that in other insects were also compared and analyzed. it is also reported that attacin gene has two paralogous genes i.e., attacin i as well as attacin ii, both the genes are found to be expressed after bacterial infection (tanaka et al., 2008). the organization of both attacin genes and its position are explained in this report which are found to be located on the 6th chromosome. there are few antibacterial proteins such as gloverin, lebocin, serpin and these genes have been found to be involved in the immune response against the viral infection, especially against bmnpv infection (cheng et al., 2014). there is not much information about the role of attacin gene against bmnpv infection. a microarray analysis was carried out to identify the genes associated with bmnpv resistance. there are many antibacterial proteins found to be upregulated after bmnpv infection. among those antibacterial genes, the expression of attacin gene was significantly upregulated after bmnpv infection in the microarray analysis, indicating its prominent role in antiviral immunity. in the present study the differential expression of both attacin i and attacin ii genes has also been analyzed after bmnpv infection to know the role of these genes in the antiviral immune response in silkworm b. mori. materials and methods selection of silkworm races the silkworm bombyx mori races viz., sarupat and csr-2 were selected for the study, as these are known to be most resistant and most susceptible to bmnpv. these two silkworm races were used for the microarray as well as for quantification and gene expression analysis using qpcr. virus and inoculations b. mori multiple nucleopolyhedrovirus stock was maintained at this laboratory and used as viral inoculum. the viral inoculum was prepared by counting the number of viral polyhedra in a neubauer chamber. the oral inoculation of bmnpv occlusion bodies was carried out in healthy newly moulted ‘0 day’ fifth instar larvae (first day after 4th moult) of sarupat and csr-2 races with viral dosage of 40,000 polyhedral inclusion bodies (pib) per larva. three replications containing twenty-five silkworms were maintained for each silkworm race. similarly, the uninoculated control batches were reared separately under disease free environment. silkworms feeding on bmnpv-free mulberry leaves were placed in labelled boxes until feeding was complete and then transferred to a controlled room where they remained until the end of the experiment. collection of tissue bmnpv-infected fifth instar larvae (n = 6) were dissected and the midgut tissues was removed at different (6, 12, 18, 24, 30) h after post infection (hpi). they were quickly washed in diethylpyrocarbonate (depc)-treated solution and immediately frozen at -80 °c for further analysis.   rna isolation and cdna synthesis the rna was extracted from different tissues like hemocytes, midgut, fat body and cuticle with trizol reagent (invitrogen, usa), and then denatured in formaldehyde, formamide and electrophoresed in 2.0 % agarose gels. the first strand cdna was synthesized using dnase treated rna sample (2 μg) along with 1μl oligo (dt) (0.01mm) (eurofin india pvt ltd, bangalore) was added followed by incubation at 70 ºc for 3 min. finally, 1x reverse transcriptase buffer (4μl), 10 mm dntp (2 μl), 5 mm dtt (2μl) and m-mlv superscript iii reverse transcriptase (invitrogen, usa) (0.5 μl) was added to obtain a final volume of 20 μl. the reaction mixture was incubated at 42 ºc for 60 min and terminated by heating at 75 ºc for 10 min according to the manufacturer’s protocol. identification of attacin gene and genomic contig the cdna of attacin gene was already identified and deposited. the attacin cdna sequence was blast (blast) searched with b. mori genomic dna database (xia et al., 2004), for identification of corresponding contig homologous sequence for attacin gene. the genomic dna sequence showing homologous sequence to b.mori attacin gene was identified and subsequently translated to determine putative amino acid sequence. the amino acid sequence was further analyzed through conserved domain search for the presence of the two functional domains in attacin (http://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb/cg i). est expression in different tissues the specific expression of attacin i and attacin ii in different tissues were identified by performing blastn followed by retrieval of the est from the library. the different tissues selected are hemocytes, midgut, fat body and cuticle. microarray experiment and data analysis a genome wide oligonucleotide microarray containing 24,924 probes were used to investigate the gene expression profiles of bmnpv infected as well as control midguts of sarupat and csr-2 silkworm b. mori at 12 h after post infection. the complete sets of raw and normalized data from this study have been deposited in the ncbi gene expression omnibus (geo) repository. amplification of attacin gene in different silkworm races the genomic dna isolated from silk moths using standard protocols (nagaraja and nagaraju, 215   http://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb/cgi http://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb/cgi 1995) was used as template in the pcr reaction. the up and down gene specific primers for attacin gene in the b. mori genomic contig were designed using the software program primer3 (http://frodo.wi.mit.edu/cgibin/ primer3). the forward primer used was 5’-ggctggaaagctggaactaa3’ and the reverse was 5’ agtccatagcctgggaacct-3’. the reaction was done in an eppendorf thermal cycler, ptc200, using 20 μl reaction mixture containing 50-100 ng of genomic dna as template, 2.0 μl of 10x pcr buffer, 0.2 mm dntps, 1.5 mm mgcl2, 1 µl each of forward and reverse primers and 0.3 u of taq dna polymerase (mbi fermentas). the pcr schedule was 94 °c for 3 min followed by 30 cycles of 94 °c for 30 s, 50 °c for 30 s, 72 °c for 2 min and a final extension of 7 min at 72 °c. the pcr amplified products were purified through gel-spin column (bangalore genei) and m13 primer was used for the sequencing reaction. the amplified pcr product 699 bp length (fig. 3) was cloned in ta cloning vector with m13 sequences flanking the 5’ and 3’ region and sequenced with gene specific primer as sequencing primer. expression in sarupat and csr-2 midgut the bmnpv infected and control midgut samples were collected at different intervals of post infection from 0 to 30 h. the rna was isolated from the midgut tissues and cdna was synthesized. the cdna was used as template to quantify attacin i and attacin ii gene expression by qpcr. results tissue specific expression profile tissues were collected from haemocytes, fat body, midgut and cuticle of fifth instar third day larvae for tissue specific expression analysis. the attacin i and attacin ii expression was analyzed using forward primer 5’gcaggcaaggtcaatttgtt-3’ and reverse 5’ cggttgatgacgtcagagtg-3’ for attacin i. forward primer-5’tcgaggtcgtattgcagaca-3’ and 5’ggctcccacgaagatctgta-3’ of reverse primer for attacin ii. the reactions were conducted on a stratagene mxpro-mx3005p real-time pcr system (agilent technologies) using the sybr premix ex taq kit (takara), according to the manufacturer's protocol. each amplification reaction was performed using a 20 μl reaction mixture, under the following conditions: denaturation at 95 °c for 30 s, followed by 40 cycles of 95 °c for 10 s and at 55 °c for 30 s. the experiment was performed in triplicate and results were standardized to the expression level of the constitutive β actin gene. a non-template control (ntc) sample was also run to detect contamination, if any. the microarray analysis was carried out to investigate the gene expression profiles in silkworm b. mori against bmnpv infection. the results indicated that some of the antibacterial proteins including attacin gene were upregulated after bmnpv infection and thus indicating their potential role in the antiviral immune response (sagisaka et al., 2010). therefore, an attempt has been made to study the differential expression of attacin gene in bmnpv resistance. in addition to that the organization of paralogous attacin genes, their tissue specific expression and variation in the promoter and coding regions were analyzed. the attacin cdna sequence (accession no. s78369) was blast (blast) searched with b. mori genomic dna database and a single contig (accession no aadk01007556) possessing the attacin gene sequence was identified. two paralogous sequences similar to attacin gene sequence were present in the locus. further, there were three exon with two intron regions of 91 bp and 79 bp length respectively. these two gene sequences were arranged in a direction opposite to the single contig with a gap of 3.4 kb length and the paralogous genes were designated as attacin i and attacin ii (fig.1). the conserved domain analysis showed the presence of two sub domains the in g domain of attacin, similar to that of other insect attacins. the data indicates that both attacin i and ii genes are conserved across the taxa. attacin i revealed full length cdna of 831 bp length similar to the original cdna sequence (accession no. s78369), while attacin ii sequence matched partially with a length of 683 bp. analysis of 5’ regulatory sequences of both the attacin genes indicated that attacin i and ii possess fig. 1 the b. mori attacin genomic organization. the position of the genes and their transcriptional directions ( ) are shown underneath. the overall structure of attacin i and ii and the distance from the start codon (+1) to the functional parts shown in base pairs. 216   fig. 2 comparison of promoter regions of attacin i and ii of silkworm b. mori. the g-box, tata box and cap site are boxed. g-box, tata box and cap site followed by exon/intron region. the g-box and tata box were found to be located at two different positions on the 5’ region in both the attacin genes. the g-box was located in 105 bp region of attacin i gene, whereas, it was in 138 bp region in attacin ii gene. similarly, the tata box location was at 79 bp region in attacin i and at 84 bp region in attacin ii of the upstream region from the starting codon. interestingly, no difference was found in the cap site location (position at 52 bp) of both attacin i and attacin ii upstream region (fig. 2). the attacin gene sequences of pure mysore, daizo, nistari, nb4d2, csr19 and cdna sequence (acc. no. s78369) were compared using multiple alignment program (clustal w). the results indicate single nucleotide variations at 22 places (fig. 3). the sequences were clustered through phylogenetic analysis and the dendogram obtained indicated that all the multivoltine races formed a separate cluster, while, the bivoltine races formed another (fig. 4). further, nucleotide variation was analyzed for possible changes in the amino acid level. these genes exhibit high levels of silent nucleotide variations (synonymous substitution), but within the silkworm races they are not excessively polymorphic at the amino acid level, as most of the nucleotide variation did not yield changes in the amino acid sequence. among the 14-nucleotide variations observed within the amplified region, most of the changes caused no change in the amino acid sequence. the amino acid serine was coded by various degenerative codons due to which there were no changes at amino acid level. similar findings were observed in the other amino acids such as valine, isoleucine, leucine and alanine. est expression in different tissues the complete sequences of attacin i and ii were blast searched in the silk base database and the est sequences with maximum homology expressed in different tissues were retrieved. a total of 37 transcripts were retrieved from the est library, of which 34 transcripts belonging to attacin i and 3 transcripts were that of attacin ii. out of 34 transcripts, attacin i expressed maximum (44 %) in fat body tissues (fig. 5) followed by corpora allata (17 %). in case of attacin ii, among the three transcripts, two were expressed in the fat body while one was from corpora allata. 217   fig. 3 multiple alignment of attacin gene sequences from different silkworm races of b. mori. the intron regions are boxed. fig. 4 the evolutional tree was obtained by the neighbor-joining method based on the multiple alignments of attacin dna sequences of different silkworm strains. the numbers on each branch indicate the percentage of the most parsimonious trees, which were found in 1000 bootstrap replications performed with mega5 programme. 218   fig. 5 the pie chart indicating the number of attacin i transcripts in different tissues. microarray experiment and data analysis the genes associated with bmnpv infection were identified in sarupat (resistant) and csr2(susceptible) b. mori silkworm races since these races reveal divergent responses with respect to bmnpv infection. oligonucleotide microarray containing 24,924 probes were used to investigate the gene expression profiles in the midgut tissue of bmnpv infected and uninfected silkworms after 12 hours post infection (hpi). results revealed that, 735 and 589 genes were up-regulated and downregulated, respectively, at 12 hpi, in sarupat, whereas, 2183 genes were up-regulated and 2115 down-regulated in csr-2 (data not shown). it was observed that immune related proteins showed higher expression in bmnpv infected tissues, of which attacin i and attacin ii had a significantly up and down regulation in resistant and susceptible silkworm races, respectively (fig. 6). based on this data, it was concluded that attacin i was upregulated in the bmnpv infected sarupat midgut tissue, however, higher expression of attacin ii was found in bmnpv infected csr-2 being a susceptible race. to validate the expression of these genes, the primers were designed for attacin i and attacin ii for further qpcr analysis.  tissue specific expression profile the qpcr analysis revealed that the attacin i expression was higher in the fat body followed by midgut, cuticle and hemocytes (fig. 7). the decrease in the expression in the hemocytes possibly occurred because the viral infection damaged physical functions, resulting in the reduction of the gene expression (cheng et al., 2014). none of the earlier report indicate expression of attacin in the mid gut tissues. in the present study the significant amount of transcripts were found to be expressed in the midgut tissues. it has already been reported that the attacin gene is expressed in the fat body and similar findings has been observed in the present study. fig. 6 heat map of hierarchical clustering of differentially expressed genes in bmnpv infected and uninfected midguts at 12 h of post infection in sarupat and csr-2 (clustering type: hierarchical clustering, distance metric: pearson correlation). the colors in the heat map display the relative values of all tiles; green indicates the lowest expression, yellow indicates the intermediate expression, and red indicates the highest expression. the numerical values give the actual values on a log 2 scale, which were associated with each color. 219   http://www.ncbi.nlm.nih.gov/pubmed/?term=cheng%20y%5bauth%5d fig. 7 relative gene expression patterns of attacin i upregulated during bmnpv infection in sarupat. rna was isolated at 12 hours post infection. the relative expression levels of each gene was normalized using the ct values that were obtained for the housekeeping gene β actin amplifications run on the same plate. in each assay, the expression level is shown relative to the lowest expression level, which is arbitrarily set at one. all samples were tested in triplicate. the mean value ±sd was used for analysis of relative transcript levels for each time point using the δδct method. a non-template control (ntc) sample was also run to detect contamination if any. ■ bmnpv infected □-uninfected fig. 8 relative gene expression patterns of gene expression in bmnpv infected and uninfected samples of sarupat with attacin i. rna was isolated at 6 hourly intervals from 0 to 30h. the relative expression levels of each gene at different time points were normalized using the ct values that were obtained for the housekeeping gene β actin amplifications run on the same plate. in each assay, the expression level is shown relative to the lowest expression level, which is arbitrarily set at one. all samples were tested in triplicate. the mean value ±sd was used for analysis of relative transcript levels for each time point using the δδct method. a non-template control (ntc) sample was also run to detect contamination if any. ■bmnpv infected □-uninfected quantification of attacin i and ii gene expression in bmnpv infected larvae in addition to microarray data, the expression of attacin i and attacin ii in the midgut tissues was confirmed through qpcr analysis in the control as well as bmnpv infected silkworm races i.e., sarupat and csr-2 at different time intervals. in the bmnpv resistant race (sarupat), the expression of attacin i has gradually increased from 6 hpi to 18 hpi and then the expression was maintained steadily up to 30 hpi (fig. 8). the expression of attacin ii was lesser in sarupat, when compared with attacin i expression, which also showed a gradual decrease in the expression up to 18 hpi (fig. 9). in csr-2, which is a bmnpv susceptible race, the expression of attacin i in bmnpv infected samples were found to be lesser when compared to that of the control samples (fig. 10). on the contrary to sarupat, the expression of attacin ii increased from 0 h to 30 h in bmnpv infected larvae of csr-2. the highest level of expression of attacin ii gene was observed in csr-2 up to 18 hpi, thereafter the expression gradually decreased and steadily maintained up to 30 h of post infection (fig. 11). 220   fig. 9 relative gene expression patterns of gene expression in bmnpv infected and uninfected samples of sarupat with attacin ii. rna was isolated at 6 hourly intervals from 0 to 30h. the relative expression levels of each gene at different time points were normalized using the ct values that were obtained for the housekeeping gene β actin amplifications run on the same plate. in each assay, the expression level is shown relative to the lowest expression level, which is arbitrarily set at one. all samples were tested in triplicate. the mean value ±sd was used for analysis of relative transcript levels for each time point using the δδct method. a non-template control (ntc) sample was also run to detect contamination if any. ■bmnpv infected □-uninfected. discussion it is already known that b. mori genome consists of two attacin genes viz. attacin i and attacin ii which play an anti-bacterial role. however, the genome-wide microarray analysis followed by real time pcr revealed that attacin gene not only functions as an anti-bacterial gene but also has an anti-viral role in silkworm b. mori. in order to identify the specific role against bmnpv infection and to analyze differential expression of the attacin genes, an attempt has been made to analyze the expression profiles of these genes in bmnpv infected sarupat (bmnpv resistant) and csr-2 (bmnpv susceptible) silkworm races. the up regulation of attacin i gene in the bmnpv infected midgut samples of sarupat indicated that attacin i was specifically being expressed in response to bmnpv infection in the resistant race whereas the attacin ii gene expression was comparatively lesser in sarupat. in case of csr-2, the expression of attacin i in bmnpv infected samples were found to be lesser than that in the control samples. however, the expression of attacin ii was comparatively higher in the bmnpv infected samples of silkworm race csr-2. this observation indicates that among the two attacin genes, attacin i has association with bmnpv resistance. families of attacin-like peptides (usually two to four functional genes per haploid genome) have been identified in the lepidopteran species b. mori (sugiyama et al., 1995), h. cecropia (hultmark et al., 1983), hyphantria cunea (shin et al., 1998), trichoplusiani (kang et al., 1996), and heliothis virescens (ourth et al., 1994), as well as in the dipteran species s. peregrina (ando et al., 1987) and d. melanogaster (asling et al., 1995; dushay et al., 2000; hedengren et al., 2000). the antibacterial peptides are often conserved across evolutionarily distance taxa but, little is known about the level and structure of the polymorphism within different species (choe et al., 2002). sugiyama et al. (1995) cloned the attacin gene and its 5’ upstream regulatory region was characterized. in the present study it was observed that the two attacin genes of b.mori were arranged in opposite directions in a single contig with a gap of 4.2 kb length and these two genes were designated as attacin i and attacin ii. these genes are transcribed in opposite directions and interrupted at homologous position by two introns. the major difference between these two attacin genes is the size of exon iii. in attacin i it is 351 nt while in attacin ii it is only 203 nt. similar findings have also been observed in giant silk moth h. cecropia (sun and faye, 1995) indicating that the duplication of attacin gene as well as gene synteny is conserved within the insect taxa. kadalayil et al. (1999) showed that the promoters of several inducible insect immune genes possess gata sites 0 12 bp away from nfkappab binding site (nf-kb site) in functionally important promoter regions. clusters of gata and nf-kb sites are also observed in the promoters of two important mammalian immune genes, namely il-6 and il-3. in b. mori also the nucleotide sequence of both the attacin gene 5’-upstream region 221   fig. 10 relative gene expression patterns of gene expression in bmnpv infected and uninfected samples of csr-2 with attacin i. rna was isolated at 6 hourly intervals from 0 to 30 h. the relative expression levels of each gene at different time points were normalized using the ct values that were obtained for the housekeeping gene β actin amplifications run on the same plate. in each assay, the expression level is shown relative to the lowest expression level, which is arbitrarily set at one. all samples were tested in triplicate. the mean value ±sd was used for analysis of relative transcript levels for each time point using the δδct method. a non-template control (ntc) sample was also run to detect contamination if any. ■bmnpv infected □-uninfected. contains a lipopolysaccharide (lps) response element (nf-kb site), caat box and tata box. the cap site of both attacin i and attacin ii genes is located at a position of 52 bp in the upstream region. tanaka et al. (2008) reported that the cap site is very important for the active transcription of attacin genes thereby indicating that both b. mori attacin i and ii genes are actively transcribed after microbial infection. however, there is a variation in the position of different promoter elements of attacin genes which may affect transcription efficiency. lazzaro and clark (2001) analyzed natural genetic variation in alleles of d. melanogaster attacins a and b and observed that, the overall level of nucleotide diversity is high in each of these, but there is no excess of amino acid polymorphism. they also observed that, attacins a and b have experienced multiple paralogous gene conversion events. our results also revealed attacin gene polymorphisms at nucleotide level, but not at amino acid level, in the different silkworm strains studied. gloverin and lebocin seems to be lepidopteranspecific antibacterial peptides (axen et al., 1997) and have expression levels that were strongly induced in b. mori larval fat body by escherichia coli immune challenge. bmnpv infection also caused a strong induction of b. mori gloverin and lebocin gene expressions in larval midguts and this induction occurred in both b. mori strains for gloverin-3 and lebocin genes. the antiviral mechanism that occurs in the resistant b. mori strain is not due to resistance against the bmnpv invasion but rather due to the inhibition of bmnpv proliferation in the larval midgut (bao et al., 2009). the defense processes against bmnpv infection that occur in the resistant larvae might be regulated via interactions involving multiple genes (liu et al., 2000). huang et al. (2013) observed that antimicrobial peptides have an antiviral role in response to alphavirus replication in arthropods. they focused their study on the antiviral response of d. melanogaster innate immune system induced by rna replication of sindbis virus (sinv). further, they carried out microarray analysis in search for sinv replication sensitive genes. out of the 95 sinv replication genes identified, two of the genes were found to be antimicrobial peptides viz. attc and dptb. knocking out these genes either led to an increase in viral rna synthesis or defects in development in the presence of sinv replication complex. these findings clearly demonstrate the antiviral role of attacin in drosophila. choi et al. (2012) also demonstrated that, genes like attacin were significantly up regulated during viral infection. in the present study, similar attempts have been made to identify immune response genes in bmnpv infected silkworms using microarray techniques. attacin i and ii genes of b.mori were found to be differentially expressed after bmnpv infection thereby proving these genes to be bmnpv responsive. further, the work carried out by huang et al. (2013) also demonstrates the phenomenon of allelic 222   fig. 11 relative gene expression patterns of gene expression in bmnpv infected and uninfected samples of csr-2 with attacin ii. rna was isolated at 6 hourly intervals from 0 to 30 h. the relative expression levels of each gene at different time points were normalized using the ct values that were obtained for the housekeeping gene β actin amplifications run on the same plate. in each assay, the expression level is shown relative to the lowest expression level, which is arbitrarily set at one. all samples were tested in triplicate. the mean value ± sd was used for analysis of relative transcript levels for each time point using the δδct method. a non-template control (ntc) sample was also run to detect contamination if any. ■bmnpv infected □-uninfected. divergence and the functional diversity of attacin genes. drosophila consists of four attacin genes. the attaa, attb and attc genes are located on chromosome 2 while attd is located on chromosome 3. the atta and attb proteins share 98% identity with each other while attc is 73% identical to atta and attb. in spite of such close similarity the expressions of atta, attb and attd were not found to be sinv replication sensitive. among the four paralogous attacin genes only attc was found to be sinv replication responsive which suggests that only a single gene (attc) acquired the antiviral role while the other genes remained devoid of such function. in our study it was found that attacin i gene specifically was upregulated during bmnpv infection, in the resistant race. on the contrary attacin ii was found to be devoid of such expression. a list of immune protein genes has been identified from the microarray analysis that is bmnpv responsive and regulated by the innate immune pathways of b. mori. among these genes attacin i was found to demonstrate an anti-viral role which otherwise has always been reported for antibacterial activity. among the two reported attacin paralogous genes, attacin i acquired antiviral role which is unique when compared to its ancestral gene. however, more work is needed to determine the mode of action of attacin i as well as its molecular mechanism of effector molecules including upstream signaling cascades which will provide insight into the role of innate immunity response to viral infection in silkworm b. mori. acknowledgement this study was supported by grants from the central silk board, ministry of textiles, government of india, bangalore, india. references ando k, okada m, natori s. purification of sarcotoxin ii, antibacterial proteins of sarcophaga peregrina (flesh fly) larvae. biochem. 26: 226-230, 1987. asling b, dushay ms, hultmark d. identification of early genes in the drosophila immune response by pcr-based differential display: the attacin a gene and the evolution of attacin-like proteins. insect. biochem. mol. biol. 25: 511-518, 1995. axen a, carlsson a, engstrom a, bennich h. gloverin, an 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in vertebrate and invertebrate models, adrenocorticotropic hormone (acth) belongs to the melanocortin group of related peptides, which share a common precursor, pro-opiomelanocortin (pomc). functional experiments indicate that in invertebrates, acth plays a major role in several biological functions. acth, whose effects have been conserved during evolution more than its amino acidic sequence, is, directly or indirectly, able to contrast agents that perturb a body’s homeostasis. here we review evidence highlighting the involvement of acth and acth-like molecules in the response of invertebrate models versus immune, environmental and parasitic challenges. key words: acth; invertebrates; immunomodulation; stress response; parasitization   introduction adrenocorticotropic hormone (acth) is a small, bioactive peptide derived from the proopiomelanocortin (pomc) gene that was first found in the pituitary gland and later also found in other organs in both vertebrates and invertebrates (ottaviani et al., 1997). pomc is the common precursor for both the melanocortin-related peptides (acth/α-msh, β-msh and γ-msh) and the opioid β-endorphin, whereas the other members of the opioid/orphanin gene family, i.e., proenkephalin, prodynorphin and proorphanin, are mainly precursors of opioid peptides (dores and baron, 2011). it has been herein summarized and updated the information about investigations on the presence and role of acth and acth-like molecules in invertebrates. the presence of pomc-products in invertebrates in invertebrates, information on pomc peptides and their sequences have been provided for the flatworm schistosoma mansoni (duvaux-miret et al., 1990), the leech theromyzon tessulatum (salzet et al., 1997) and in two species of mussels, mytilus galloprovincialis and mytilus edulis (franchini et al., 1994; ottaviani et al., 1995; stefano et al., 1999). the analysis of pomc peptides in m. edulis indicated that there was poor conservation of the overall sequence but a higher level of similarity in ___________________________________________________________________________ address correspondence: enzo ottaviani department of life sciences university of modena and reggio emilia via campi, 213/d, 41125 modena, italy e-mail: enzo.ottaviani@unimore.it the peptides contained within pomc (malagoli et al., 2011). among the fragments of pomc, acth has been found using several techniques, including immunocytochemistry, flow cytometry and radioimmunoassays in different tissue of various species examined (table 1). moreover, in situ hybridization experiments using a bovine acth receptor cdna probe evidenced that m. galloprovincialis immunocytes express an mrna encoding for a molecule similar to acth receptor (ottaviani et al., 1998). several studies, based on mammalian-derived acth or anti-acth polyclonal antibodies, were performed on the mollusc gastropod, planorbarius corneus, where acth-like molecules have been found in both the immune and nervous systems. the hemolymph is characterized by two cell types, and acth-like molecules were detected in the cells endowed with phagocytic activity, i.e., the so-called immunocytes (ottaviani, 1983, 2011; ottaviani and franchini, 1988). the central nervous system (cns) of p. corneus and other gastropods is characterized by three pairs of ganglia (cerebral, pleural and pedal) located around the esophagus. acth-like molecules were found in a relatively small number of neurons in all the ganglia (sonetti et al., 2005). moreover, in p. corneus, acth immunopositivity was also detected in a distinctive class of glial cells that are comparable to vertebrate microglia. it should be stressed that acth-like molecules were not found in the neuroglial cells in the bivalve m. edulis, indicating that there are species-specific differences in expression, or that the expression may be transient and related to factors presently unknown (sonetti et al., 1994). 28   table 1 presence of acth-like molecules in invertebrates __________________________________________________________________________________________ protostomia mollusca immunocytes neurons neuroglial cells intestinal cells cc/ca refs planorbarius corneus + + + 1-3 viviparus ater + 4 lymnaea stagnalis + + 5-7 achatina fulica + 8 helix aspersa + + 9,10 mytilus edulis + 11,12 mytilus galloprovincialis + 13 insecta periplaneta americana + 14 leucophaea maderae + + 15,16 calliphora vomitoria + 17 heliothis virescens + 18 annelida eisenia foetida + theromyzon tessulatum + 19 nematoda 20 goodeys ulmi + 21 trichinella spiralis + 20 trematoda schistosoma mansoni + 20 deuterostoma tunicata neurons neural gland gonads ciona intestinalis + + 22,23 stiela plicata + + 24,25 halocynthia roretzi + 26 __________________________________________________________________________________________ corpus cardiacum/corpus allatum = cc/ca refs 13 = ottaviani, 1983; sonetti et al., 1994, 2005 4 = ottaviani et al., 1995 5 7 = boer et al., 1979; leung et al., 1990; ottaviani et al., 1991 8 = van noorden et al., 1980 9, 10 = marchand and dubois, 1982; marchand and colard, 1991 11, 12 = smith et al., 1990; sonetti et al., 1994 13 = franchini et al., 1994 14 = schols et al., 1987 15, 16 = hansen et al., 1986; smith et al., 1991 17 = franchini et al., 1996 18 = grimaldi et al., 2012 19 = cooper et al., 1995 20 = pryor and elizee, 2000 21 = leach et al., 1987 22 = georges and dubois, 1979 23 = fritsch et al., 1985 24 = pestarino, 1985 25 = pestarino and facchinetti, 1995 26 = kawahara et al., 2002 the different distribution of acth-like molecules in the various regions of the cellular body of invertebrates implies the presence of several biological functions, such as immunomodulation, stress response and parasitization. the role of acth in immunomodulation acth affects cell shape changes (cell motility), chemotaxis (non-random locomotion) and phagocytosis. using a computer-assisted microscopy image analysis, it was observed that mammalian acth (1-24) induces the motility of molluscan immunocytes via an adenylate cyclase/camp/protein kinase a-dependent pathway, as well as through the activation of protein kinase c (sassi et al., 1998). with regard to chemotaxis, it has been detected that the various fragments of mammalian acth have different effects on the migration of immunocytes. indeed, stimulatory effects were 29   observed for fragments (1-4), (1-13), (1-17), (1-24), (4-9), and (11-24), while an inhibitory effect was detected for fragment (4-11) and for the whole molecule (1-39) (genedani et al., 1994). the incubation of immunocytes with acth (1-24) induces cytoskeletal changes the freshwater snail viviparus ater (franchini and ottaviani, 1994). the non-activated immunocytes typically have a nucleus in the central position and a cytoplasm characterized by thin pseudopods. furthermore, bundles of microfilaments are distributed radially from the nucleus to the cell periphery, where they enter into cytoplasmatic protrusions. the incubation with acth (1-24) provokes morphological changes that become more evident after 30 min. the cells then show a polarized morphology, most of the cytoplasmic protrusions are retracted, and the elongated cells extend lamellipodia. the microfilaments are reorganized, with thin bundles surrounding the nucleus, and are seen under the plasma membrane, while phalloidin-positive areas are evident at the cell periphery on the lamellipodia. with regard to the distribution of microtubules, immunofluorescent positivity is observed in control cells but not in cell protrusions. after incubation with acth (1-24), the fibronectin is more abundant in areas where the cell contacts the substrate, whereas in control cells, it is predominantly localized at the cell periphery. in treated cells, the adenylate cyclase activity increases, while no modification in cyclic 3’-5’-nucleotide phosphodiesterase activity has been detected. phagocytosis shows a different behavior with respect to chemotaxis (ottaviani et al., 1994), and in contrast to the conventional paradigm, there is no direct correlation between the two processes in molluscan immunocytes. for example, acth (1-24) increases phagocytic activity, while it has no effect on chemotaxis. moreover, for both chemotaxis and phagocytosis, it has been observed that the effect of a single peptide is species-and dose-dependent. acth in stress response in vertebrates, acth has traditionally been associated with stress, i.e., the complex series of responses that the body generates when its balance and internal composition are threatened. the stress response starts with the production of corticotropinreleasing hormone (crh) by the hypothalamus, which induces the production of acth by the anterior pituitary cells. in turn, acth stimulates the synthesis and release of glucocorticoid hormones by the cells of the adrenal cortex. these hormones regulate the activity of the enzyme that catalyzes the final step of epinephrine biosynthesis from the adrenal medulla, whose activity is basically under the control of the sympathetic nervous system. what meaning could the presence of acth-like molecules have in animals that do not possess any of these sophisticated organs, such as the hypothalamus, pituitary and adrenal glands. a first point is that acth is found, together with crh (ottaviani et al., 1990), in cells endowed with phagocytic activity in invertebrates and human as well. this could suggest that acth was already present in vertebrate progenitors and that it was retained also in lymphocytes, an immune-related cell type retrieved exclusively in vertebrates. a second point is that antigenic stimuli cause in p. corneus a release of acth-like material (ottaviani et al., 1992), suggesting the existence of an interconnection between stress and immune responses also in invertebrates. in this respect, the initial studies assessed organisms for the presence of enzymes such as tyrosine hydroxylase and dopamine β-hydroxylase, which are responsible for the synthesis of biogenic amines (epinephrine, norepinephrine and dopamine), well-known mediators of the stress response (ottaviani et al., 1993). in order to understand the mechanism underlying the stress response, the p. corneus hemolymph was incubated with stress-related peptides, such as human acth and crh, and the levels of biogenic amines produced in both serum and immunocytes within 45 min were determined by hplc. it was observed an increase in biogenic amines in the serum and a concomitant decrease of both acthand crh-like molecules into immunocytes (ottaviani et al., 1992). this rapid increase of free biogenic amines in the serum is likely due to the release of biogenic amines by immunocytes. time-lapse experiments based on hplc have demonstrated that the addition of human crh to the p. corneus hemolymph increases the concentration of acth-like material in immunocytes. these results allowed to assume that the release of biogenic amines might be the final step of the sequential activation of crh and acth receptors. however, the pre-incubation of p. corneus hemolymph with an anti-acth antibody did not abolish the release of biogenic amines, suggesting that human crh could directly mediate the release of biogenic amines, and that the contribution of acth was not indispensable (ottaviani et al., 1992). on the whole, it appears that in an invertebrate model, a stress response axis is concentrated in a single cell type, i.e., the immunocyte, while the key mediator molecules are the same as in vertebrates. acth in parasitization in insects, the host/parasitoid interaction results in several events including the production of amyloid fibrils (falabella et al., 2012). this process called amyloidogenesis, involves the conversion of a soluble protein into insoluble and fibrillar protein aggregates known as amyloid fibrils (bhak et al., 2009). in humans, amyloidogenesis may lead to pathological events such as neurodegenerative or cardiovascular diseases (catafau and bullich, 2015), but in invertebrates and humans as well, it may also play physiological roles in melanin synthesis (grimaldi et al., 2012, 2014), in detoxifying events, and in storing peptide/protein hormones within endocrine secretory granules (maji et al., 2009). the inoculation of parasitic eggs in the tobacco budworm heliothis virescens larvae by the endophagous parasitoid toxoneuron nigriceps (hymenoptera) induces the production of amyloid fibrils. these fibrils are involved in the immune response, serving as a necessary scaffold and providing a template for melanin deposition 30   (tettamanti et al., 2008; bhak et al., 2009). in insects, melanin is massively produced in the hemolymph by the pro-phenoloxidase pathway. amyloid fibrils develop in consequence of exocytotic activity of circulating cells, which release amyloid fibrils that adhere to the non-self, driving the pigment accumulation close to the invaders (tettamanti et al., 2008; falabella et al., 2012; grimaldi et al., 2012). the exocytosis of amyloid fibril precursors by circulating cells suggest that immunocytes activation may play a pivotal role in contrasting parasite development. accordingly, in addition to the formation of amyloid fibrils, the parasitization triggers host immunocytes to release acth-like and melanocyte-stimulating hormone-like (α-msh-like) molecules and neutral endopeptidase (nep). as stated above, acth-like mediators are involved in the migration and activation of circulating cells, and α-msh-like molecules promote melanin synthesis and do not affect cell motility. the acth/α-msh loop is modulated by the newly synthesized nep localized on the cell surface of activated immunocytes. this enzyme is able to abolish the excitatory effect of acth-like molecules by converting them into α-msh-like molecules. besides, nep can also hydrolyze the amyloid fibrils (grimaldi et al., 2012). several aspects of this complex relationship wait for further elucidation, but present evidence indicate in the acth/α-msh loop a possible controller of immunocyte activation. this loop has been exploited and subverted towards immunosuppression by the parasite schistosoma mansoni (duvaux-miret et al., 1992). the presence of pomc-derived peptides and their release from s. mansoni has been detected. coincubation of adult worms with human polymorphonuclear leukocytes (pmn) or immunocytes from the mollusc biomphalaria glabrata led to the appearance of α-msh in the medium that was derived from the conversion of the parasitic acth by the host nep. in this way, the worm can escape immune reactions in humans and molluscs because α-msh inhibits the adherence and locomotor activity of both pmn and invertebrate immunocytes (duvaux-miret et al., 1992). concluding remarks in invertebrates, acth-like molecules are present in different tissues. exogenous acth exerts various biological functions, being involved in immune-neuroendocrine responses and parasitism. there is a deep correlation between the lymphocytes of vertebrates and immunocytes of invertebrates (scapigliati, 2013) because both contain several molecules, such as pomc-derived peptides and cytokines. as suggested by blalock and smith (1985) for mammalian lymphocytes, the immunocytes may also be described as an "immune-mobile brain". indeed, lymphocytes are able to recognize a variety of stimuli and to set up a correspondingly complex response in which primitive but very efficient forms of immune and neuroendocrine responses are intermixed. in invertebrates, there is no lymphocytes and immune and stress phenomena are triggered concomitantly in the same cell (malagoli et al., 2015), suggesting that antigens and stress were indistinguishable from the outset. this is probably the reason why antigens and stress evoke an overlapping set of responses in mammals that are mediated by the same pool of conserved signaling molecules (ottaviani and franceschi, 1997). the consequences of this 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ai de a pereira4, je serrão5, jc zanuncio6 1departamento de entomologia, universidade federal de viçosa, 36570-900, viçosa, minas gerais state, brazil 2departamento de fitotecnia, universidade federal de viçosa, 36570-900, viçosa, minas gerais state, brazil 3 agroscope institute for sustainability sciences iss, reckenholzstrasse 191, 8046 zürich, switzerland 4instituto federal goiano, campus urutaí, 75790-000, urutaí, goiás state, brazil 5departamento de biologia geral, universidade federal de viçosa, 36570-900, viçosa, minas gerais state, brazil 6departamento de biologia animal, universidade federal de viçosa, 36570-900, viçosa, minas gerais state, brazil accepted july 6, 2015 abstract plant feeding can improve development and reproduction of the stink bug supputius cincticeps (heteroptera: pentatomidae), an important biological control agent in south american agro-forestry ecosystems. however, defensive compounds of plants may negatively impact this predator. the development, reproduction and survival of s. cincticeps fed on mealworm, tenebrio molitor (coleoptera: tenebrionidae) pupae with bean (fabaceae), cotton (malvaceae), eucalyptus (myrtaceae), soybean (fabaceae), or tomato (solanaceae) leaves were evaluated. females and males were heavier and the number of nymphs produced per female, the oviposition period and the longevity of females of this predator were higher when fed on eucalyptus, soybean, bean, and cotton than with tomato leaves. leaves of those plants improved biological parameters of s. cincticeps, while tomato leaves showed antibiosis with lower reproduction and survival of s. cincticeps, probably due to toxic compounds. key words: antagonistic association; defense; development; natural enemy; reproduction   introduction zoophytophagous stink bugs of the subfamily asopinae, such as supputius cincticeps (heteroptera: pentatomidae) are considered important biological control agents of numerous pests in south american agro-forestry ecosystems (zanuncio et al., 2005, 2012, 2014). however, populations of asopinae predators often only reach significant levels after pest population peaks (münster-swendsen and berryman, 2005; neves et al., 2009; de menezes et al., 2013), especially in short cycle crops under conditions of intensive management, such as cotton (gossypium hirsutum, malvaceae), soybean (glycine max, fabaceae) and tomato (solanum lycopersicum, solanaceae) (vivan et al., 2002; malaquias et al., 2010; zanuncio ___________________________________________________________________________ corresponding author: wagner de souza tavares departamento de fitotecnia universidade federal de viçosa 36570-900, viçosa, minas gerais state, brazil e-mail: wagnermaias@yahoo.com.br et al.,2012). the absence of suitable prey (particularly lepidoptera) at the early crop stages could partially explain the asynchrony between population dynamics of pests and asopinae predators (júnior et al., 2004; coelho et al., 2009). furthermore, leaf hairs and plant secondary metabolites can affect the establishment and development not only of pests but also of the asopinae predators (holtz et al., 2006). predatory stink bugs are considered zoophytophagous or phytozoophagous depending on the importance of prey or plant for their development and reproduction (coll and guershon, 2002; lemos et al., 2009a). plant and animal food sources provide apparently amino acids and other nutrients to predators which cannot be obtained from each of the two food sources alone (eubanks and denno, 1999; freitas et al., 2006). reproductive and biological characteristics of predators can be positively affected by plant feeding (eubanks and denno, 2000; robinson et al., 2008; paleari, 2013), but plant secondary compounds can 179   mailto:wagnermaias@yahoo.com.br     table 1 effect of feeding of different plants and tenebrio molitor pupae on the duration (mean ± standard error) (days) of different nymphal stages of supputius cincticeps treatments instar bean cotton eucalyptus soybean tomato i 4.00 ± 0.00 a 4.00 ± 0.00 a 4.00 ± 0.00 a 4.00 ± 0.00 a 4.00 ± 0.00 a ii 4.94 ± 0.22 b 5.34 ± 0.16 a 3.40 ± 0.12 c 4.79 ± 0.14 b 5.49 ± 0.17 a iii 4.27 ± 0.17 b 5.10 ± 0.17 a 3.93 ± 0.08 b 4.10 ± 0.10 b 4.87 ± 0.12 a iv 4.53 ± 0.09 a 4.72 ± 0.09 a 4.70 ± 0.13 a 4.19 ± 0.12 a 4.91 ± 0.58 a v 6.35 ± 0.09 a 6.82 ± 0.16 a 6.39 ± 0.11 a 6.19 ± 0.15 a 6.77 ± 0.74 a total 24.09 ± 0.30 b 25.98 ± 0.34 a 22.43 ± 0.22 b 23.26 ± 0.35 b 26.05 ± 1.28 a ns = not significant; means (± standard error) followed by the same letter in a row are not significantly different by the scott-knott’s test at 5 %. number of insects = 375. also have negative effects on natural enemies (de clercq et al., 2000; holtz et al., 2010). phytophagy is commonly beneficial to asopinae predators (holtz et al., 2009; lemos et al., 2009b; malaquias et al., 2010), mainly during prey shortages (perdikis et al., 2007; holtz et al., 2009, 2010) or during periods of low nutritional prey (vivan et al., 2003; lemos et al., 2009b), and it has been shown to reduce cannibalism (laycock et al., 2006; leon-beck and coll, 2007; frank et al., 2010). the zoophytophagous predator s. cincticeps occurs on bean (phaseolus vulgaris, fabaceae), cotton and soybean crops in brazil (zanuncio et al., 2003, 2012; de castro et al., 2013a) preying mainly on species of diptera, coleoptera, hemiptera, and lepidoptera (zanuncio et al., 2005; lemos et al., 2009b; da silva et al., 2012). presence of this stink bug can reduce insecticide sprays (zanuncio et al., 1998, 2003; tavares et al., 2009) and it seems to be compatible with certain products (zanuncio et al., 2013; de castro et al., 2013a; malaquias et al., 2014). development and reproduction parameters of s. cincticeps are highest when fed on a diet composed of both, plant and prey, although negative effects have been observed when feeding on certain plant species (zanuncio et al., 2004, 2005; lemos et al., 2009b). therefore, this study aimed to investigate the effects of different food plants on s. cincticeps by comparing biological aspects of the predator when fed on mealworm, tenebrio molitor (coleoptera: tenebrionidae) pupae combined with bean, cotton, eucalyptus (eucalyptus cloeziana, myrtaceae), soybean, or tomato leaves. material and methods seeds seeds of bean variety majestoso, eucalyptus, soybean variety ufv 16, and tomato variety santa clara were obtained from the federal university of viçosa (ufv) in viçosa, minas gerais state, brazil and those of cotton variety brs aroeira from the germplasm bank of the brazilian agricultural research corporation (embrapa algodão) in campina grande, paraíba state, brazil. plant species were chosen due to their economic importance for grain, fruit, wood production, and their numerous potential pest species, mainly caterpillars, which can be preyed by s. cincticeps (zanuncio et al., 2004; lemos et al., 2009b). plants were grown in 500 ml plastic pots with a mixture of soil and humus from earthworms, eisenia fetida (haplotaxida: lumbricidae) and eudrilus eugeniae (haplotaxida: eudrilidae) in 4:1 ratio. plants were cultivated in a greenhouse, watered as needed and pests controlled manually. no chemical fertilizers were applied. supputius cincticeps nymphs of s. cincticeps were obtained from the mass rearing of the laboratory of biological control of insects (lcbi) of the institute of biotechnology applied to agriculture (bioagro) at the ufv. experiments all experiments were conducted at 25 ± 2 °c, 60 ± 10 % relative humidity and a 12:12 light:dark photoperiod in a biochemical oxygen demand (bod). seventy-five early second instar nymphs of s. cincticeps were transferred to 15 plastic pots (500 ml) (five nymphs per pot) per treatment. two pupae of t. molitor were introduced per pot three to four times a week. two cylindrical tubes (2.5 ml), one with distilled water and one with leaves of different plants, were inserted in the pots through the cover. plants were replaced after loss of turgidity. nymphs were fed on one of the following diets: t1 (bean leaves + t. molitor + water), t2 (cotton leaves + t. molitor + water), t3 (eucalyptus 180       fig. 1 effect of feeding of different plants and tenebrio molitor pupae on the weight (mg) of fifth instar nymphs and adults of supputius cincticeps. bars with the same letters do not differ significantly by the scott-knott’s test at 5 %. comparison was made within the same developmental stage feeding on different plants. number of insects = 210. leaves + t. molitor + water), t4 (soybean leaves + t. molitor + water), and t5 (tomato leaves + t. molitor + water). adults of s. cincticeps were sexed by the appearance of the external genitalia (de castro et al., 2013b) and weighed after emergence (< 24 h). duration (days) and survival per instar (%) and of the total nymph stage (%), weight of fifth instar nymphs (mg) and of male and female adults (mg) were recorded. three days after emergence, adults of s. cincticeps were mated placing each one couple into one plastic container (twenty pairs per treatment) (zanuncio et al., 2004). couples of s. cincticeps received water daily and two t. molitor pupae three to four times a week. adults were fed on the same combinations of t. molitor as prey and plant species as nymphs. males of s. cincticeps, which died during the experiment, were replaced by others of similar age and reared with the same conditions and treatments. egg masses of s. cincticeps were collected daily and placed in petri dishes with a moistened cotton ball and the nymphs counted 48 h after hatching. the number of eggs and nymphs per female and egg mass, respectively, and of egg masses; period of pre-oviposition, oviposition and post-oviposition, and of incubation (days); female longevity (days), and egg viability (%) of s. cincticeps were measured. statistics the experimental design was completely randomized (crd). the data were submitted to univariate variance analysis (anova) and the means compared with the scott-knott post hoc test at 5 % probability using the software sas version 9.0 (statistical analysis system, 2002) (supplier: ufv). homogeneity of variance and normality of errors were checked using q-q plots (wilk and gnanadesikan, 1968) and data were transformed when necessary. results supputius cincticeps fed on a mealworm pupae diet supplemented with leaf material of different plant species showed marked difference in their life history parameters according to the plant species. the duration of the total s. cincticeps nymph stage was significantly influenced by the plant food source (table 1). it was shorter with eucalyptus, soybean or bean leaves than with cotton or tomato leaves (table 1). duration of the first, fourth and fifth nymph instars were unaffected by the diet, whereas the second instar was significantly shorter on eucalyptus leaves compared to the other plant food sources (table 1). the third instar was significantly 181       table 2 effect of feeding of different plants and tenebrio molitor pupae on the survival (mean ± standard error) (days) of different nymphal stages of supputius cincticeps treatments instar bean cotton eucalyptus soybean tomato ii 85.33 ± 3.63 a 86.67 ± 5.11 a 93.33 ± 3.19 a 92.00 ± 3.27 a 66.67 ± 5.40 b iii 80.00 ± 3.38 a 78.67 ± 4.56 a 88.00 ± 4.28 a 86.67 ± 5.04 a 62.67 ± 4.31 b iv 73.33 ± 4.22 a 68.00 ± 5.79 a 82.66 ± 5.11 a 82.67 ± 5.12 a 51.33 ± 4.87 b v 70.67 ± 3.84 a 65.33 ± 6.01 a 80.00 ± 5.16 a 76.00 ± 5.59 a 44.67 ± 4.24 b total 70.66 ± 3.83 a 65.33 ± 6.00 a 82.67 ± 4.31 a 76.00 ± 5.58 a 44.66 ± 4.23 b means (± standard error) followed by the same letter in a row are not significantly different by the scott-knott’s test at 5 %. prolonged with cotton or tomato leaves as food source (table 1). weight of s. cincticeps was also significantly affected by the plant species fed (fig. 1). fifth instar nymphs were significantly lighter when fed with tomato leaves, compared to all other plants. this effect was visible in both sexes (fig. 1). overall survival of s. cincticeps nymphs, as well as stage-specific survival of second, third, fourth, and fifth instar nymphs was influenced by the plant species fed (table 2). survival was significantly higher with eucalyptus, soybean, bean, or cotton than with tomato leaves (table 2). the longevity of s. cincticeps females was significantly longer with eucalyptus, soybean, bean, and cotton leaves than with tomato leaves (table 3). while the total number of eggs per female and egg mass and the number of egg masses were similar among treatments (fig. 2), the pre-oviposition period of s. cincticeps was significantly longer with tomato leaves and the oviposition period significantly shorter. the post-oviposition period was similar between treatments (table 3). the number of emerging nymphs per egg mass was significantly higher with eucalyptus or cotton leaves than with soybean, bean or tomato leaves (table 3). egg viability of s. cincticeps was significantly lower with tomato leaves and higher with eucalyptus leaves, while the incubation period was similar between treatments (table 3). the number of surviving nymphs per female was significantly higher with eucalyptus, soybean, bean, or cotton leaves than with tomato leaves (table 3). discussion in the present study we could show that almost all life history characteristics of s. cincticeps were negatively affected when they were fed with tomato leaves, and some parameters were also inferior when the nymphs fed on cotton leaves compared to the other plant food sources. these differences could result from differences in food accessibility, the content of secondary plant defense compounds and/or differences in nutritional quality of these plants (júnior et al., 2004; holtz et al., 2009; malaquias et al., 2010). morphological and chemical properties of plants may influence their attractiveness for herbivores as well as the biological characteristics of herbivores (agrawal, 2000; hagenbucher et al., 2013; eaton and karban, 2014) and asopinae predators (hilker and meiners, 2002; degenhardt et al., 2003). while asopinae predators often benefit from additional plant feeding by enhanced growth and reproduction (holtz et al., 2009; lemos et al., 2009b; malaquias et al., 2010), morphological characteristics, e.g., trichomes and chemical characteristics, such as secondary plant compounds, may have negative effects on the predator. thereby influences on the life cycle may vary among plant species, as well as among stink bug species feeding on the same plant (júnior et al., 2004; lemos et al., 2009b; malaquias et al., 2010). similar studies have reported shortened durations of the nymph stage and higher fecundity of p. nigrispinus fed on cotton or whiteweed, ageratum conyzoides (asteraceae) without prey (júnior et al., 2004); fall armyworm, spodoptera frugiperda (lepidoptera: noctuidae) on cotton cultivars (de jesus et al., 2014), and cotton leafworm, alabama argillacea (lepidoptera: noctuidae) with cotton or fennel, foeniculum vulgare (apiaceae) (malaquias et al., 2014). however, the longer duration of the second and third instar, and of the total nymph stage as well as reduced longevity of s. cincticeps with cotton or tomato leaves in our study suggest negative effects, as were found for the longer duration of the nymph stage of brontocoris tabidus (heteroptera: pentatomidade) with cotton (coelho et al., 2009; torres et al., 2010). like tomato plants, cotton 182       table 3 effect of feeding of different plants and tenebrio molitor pupae on the reproductive parameters and longevity of supputius cincticeps treatments reproductive parameters bean cotton eucalyptus soybean tomato number of eggs/female 157.11 ± 20.03 a 125.76 ± 20.77 a 163.28 ± 18.94 a 165.73 ± 25.25 a 121.67 ± 18.05 a number of nymphs/female 120.38 ± 16.02 a 101.28 ± 17.42 a 142.00 ± 17.36 a 125.80 ± 21.64 a 59.78 ± 8.23 b pre-oviposition period (days) 8.81 ± 0.38 b 8.72 ± 0.51 b 9.39 ± 0.16 b 8.07 ± 0.73 b 10.78 ± 0.79 a oviposition period (days) 31.81 ± 3.44 a 25.36 ± 2.76 a 29.83 ± 3.17 a 28.00 ± 3.92 a 17.11 ± 2.25 b post-oviposition (days) 4.12 ± 0.68 a 3.80 ± 0.73 a 2.22 ± 0.42 a 5.47 ± 1.23 a 3.78 ± 0.62 a longevity (days) 45.42 ± 3.49 a 37.40 ± 3.12 a 43.50 ± 2.98 a 41.53 ± 4.18 a 28.22 ± 2.01 b egg viability (%) 74.11 ± 3.25 b 77.64 ± 2.25 b 86.06 ± 1.45 a 72.48 ± 3.57 b 53.30 ± 3.74 c incubation period (days) 5.91 ± 0.04 a 5.84 ± 0.07 a 5.81 ± 0.03 a 5.85 ± 0.08 a 5.85 ± 0.07 a number of eggs/egg mass 10.55 ± 0.49 a 11.30 ± 0.72 a 11.49 ± 0.46 a 11.95 ± 0.79 a 11.81 ± 0.64 a number of nymphs/egg mass 7.24 ± 0.55 b 8.97 ± 0.71 a 9.89 ± 0.44 a 7.83 ± 0.78 b 6.67 ± 0.52 b number of egg masses 14.69 ± 1.74 a 11.04 ± 1.66 a 14.28 ± 1.57 a 14.13 ± 2.01 a 10.89 ± 1.70 a means (± standard error) followed by the same letter in a row are not significantly different by the scott-knott’s test at 5 %. twenty couples per treatment. contains secondary plant metabolites, e.g., the terpenoid gossypol that is active against leaf feeding insects (hagenbucher et al., 2013). supputius cincticeps is a generalist predator that may utilize prey and leaf material of different plants species (lemos et al., 2009b) and thus it might not be adapted to metabolize tomato and cotton plant allelochemicals very efficiently. in contrast to generalist species that often possess enzymes to metabolize a broad range of defense substances to certain extend (krieger et al., 1971; li et al., 2004), specialist herbivores are able to detoxify the specific defense substances from their few host plants highly efficiently (mao et al., 2006; lampert et al., 2011). effects of plant feeding on life-history characteristics of predators can be negative, as with tomato plants, for s. cincticeps or in the case of podisus maculiventris (heteroptera: pentatomidade) where feeding on a vegetable diet (bean) negatively affected the development, weight gain and growth of nymphs compared to individuals fed only on t. molitor pupae (crum et al., 1998; weiser and stamp, 1998). however plant feeding can also have positive effects on s. cincticeps, when nymph stages are shortened, lifetime prolonged and reproduction is enhanced (zanuncio et al., 2004, 2012). shortened nymph stages of s. cincticeps would allow this predator to reach adulthood and reproduce faster (zanuncio et al., 2004, 2005; holtz et al., 2006). adult weight, mainly of females may indicate the reproductive success of predatory stink bugs, with heavier ones presenting greater longevity and reproduction (legaspi and legaspi, 2005; oliveira et al., 2005; lemos et al., 2009b). likewise the lower weight of s. cincticeps females with tomato leaves resulted also in reduced longevity, oviposition period, number of nymphs produced per female and egg mass, and egg viability of this predator. in contrast, these parameters were enhanced with eucalyptus, soybean, bean, and cotton leaves, suggesting a positive effect of these plants, as found for the reproduction of p. nigrispinus with eucalyptus (holtz et al., 2009, 2011; torres et al., 2010). the favorable effect on development and reproduction of s. cincticeps was similar to other asopinae fed on plant diets (holtz et al., 2009, 2011; lemos et al., 2009a). as secondary plant defense compounds tomato plants contain chlorogenic acid (the ester of caffeic acid and (-) -quinic acid) and tomatine, a glycoalkaloid (lambert, 2007; inbar and gerling, 2008; tian et al., 2012) that can inhibit acetylcholinesterase and thus interrupt the transmission of nerve impulses (friedman, 2002). 183       fig. 2 effect of feeding of different plants and tenebrio molitor pupae on the number of eggs and nymphs produced per week and survival of supputius cincticeps. bars representing same letters do not differ significantly by the scott-knott’s test at 5 %. comparison was made within the same developmental stage feeding on different plants. nymphs of p. maculiventris and p. nigrispinus that were reared with prey (lepidoptera) fed on chlorogenic acid, gossypol and tomatine of tomato plants developed slower and had other negative effects on development and reproduction (traugott and stamp, 1997; kaplan and thaler, 2010; evangelista et al., 2011). plant secretions (enzymes) and trichomes may interfere with searching behavior of natural enemies and thus reduce their foraging efficiency (gruenhagen and perring, 2001; bjorkman and ahrne, 2005; rodriguez-lopes et al., 2011). cotton trichomes inhibited searching behavior of natural enemies (mainly hymenoptera) on insect-herbivores (hagenbucher et al., 2013). especially glandular trichomes that excrete enzymes such as gossypol can be a major problem for herbivores and beneficials alike (evangelista et al., 2011). the glandular trichomes of the tomato variety heinz, for example, excrete exudates and other substances that stuck to the legs of p. maculiventris nymphs, which hamper its movement and feeding (de clercq et al., 2000; lambert, 2007; kaplan and thaler, 2010). to assess directly whether the presence of trichomes has interfered with food accessibility for s. cincticeps behavioral bioassays would have been necessary. in summary bean, cotton, eucalyptus, and soybean improved biological features of s. cincticeps, an important source of biological control. however, tomato had a negative impact on reproduction and survival of s. cincticeps, which may be due to toxic compounds acting by antibiosis on this predator. acknowledgments this study was supported by grants at the “conselho nacional de desenvolvimento científico e tecnológico (cnpq)”, “coordenação de aperfeiçoamento de pessoal de nível superior (capes)”, “fundação de amparo à pesquisa do estado de minas gerais (fapemig)”, and “fundação de amparo à pesquisa do estado da bahia (fapesb)”. 184       references agrawal aa. specificity of induced resistance in wild radish: causes and consequences for two 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mcq. effect of female weight on reproductive potential of the predator brontocoris tabidus (signoret, 1852) (heteroptera: pentatomidae). braz. arch. biol. techn. 48: 295-301, 2005. paleari lm. developmental biology, polymorphism and ecological aspects of stiretrus decemguttatus (hemiptera, pentatomidae), an important predator of cassidine beetles. rev. bras. entomol. 57: 75-83, 2013. perdikis d, favas c, lykouressis d, fantinou a. ecological relationships between non-cultivated plants and insect predators in agroecosystems: the case of dittrichia viscosa (asteraceae) and macrolophus melanotoma (hemiptera: miridae). acta oecol. 31: 299-306, 2007. robinson ka, jonsson m, wratten sd, wade mr, buckley hl. implications of floral resources for predation by an omnivorous lacewing. basic appl. ecol. 9: 172-181, 2008. rodriguez-lopes mj, garzo e, bonani jp, fereres a, fernandez-munoz r, moriones e. whitefly resistance traits derived from the wild tomato solanum pimpinellifolium affect the preference and feeding behavior of bemisia tabaci and reduce the spread of tomato yellow leaf curl virus. phytopathology 101: 1191-1201, 2011. statistical analysis system. sas user's guide. version 9.0. cary, sas institute, 2002. da silva rb, corrêa as, della lucia tmc, pereira aia, cruz i, zanuncio jc. does the aggressiveness of the prey modify the attack behavior of the predator supputius cincticeps (stal) (hemiptera, pentatomidae)? rev. bras. entomol. 56: 244-248, 2012. tavares wd, cruz i, petacci f, de assis sl, freitas sd, zanuncio jc, et al. potential use of asteraceae extracts to control spodoptera frugiperda (lepidoptera: noctuidae) and selectivity to their parasitoids trichogramma pretiosum (hymenoptera: trichogrammatidae) and telenomus remus (hymenoptera: scelionidae). ind. crop prod. 30: 384-388, 2009. tian dl, tooker j, peiffer m, chung sh, felton gw. role of trichomes in defense against herbivores: comparison of herbivore response to woolly and hairless trichome mutants in tomato (solanum lycopersicum). planta 236: 1053-1066, 2012. torres jb, barros em, coelho rr, pimentel rmm. zoophytophagous pentatomids feeding on plants and implications for biological control. arthropod-plant int. 4: 219-227, 2010. traugott ms, stamp ne. effects of chlorogenic acid and tomatine-fed caterpillars on performance of an insect predator. oecologia 109: 265-272, 1997. vivan lm, torres jb, veiga afsl. development and reproduction of a predatory stinkbug, podisus nigrispinus in relation to two different prey types and environmental conditions. biocontrol 48: 155-168, 2003. 186       vivan lm, torres jb, barros l, veiga afsl. population growth rate of the predator bug podisus nigrispinus (heteroptera: pentatomidae) and of the prey tuta absoluta (lepidoptera: gelechiidae) under greenhouse conditions. rev. biol. trop. 50: 145-153, 2002. weiser la, stamp ne. combined effects of allelochemicals, prey availability and supplemental plant material on growth of a generalist insect predator. entomol. exp. appl. 87: 181-189, 1998. wilk mb, gnanadesikan r. probability plotting methods for the analysis of data. biometrika 55: 1-17, 1968. zanuncio jc, batalha vc, guedes rnc, picanço mc. insecticide selectivity to supputius cincticeps (stal) (het., pentatomidae) and its prey spodoptera frugiperda (j. e. smith) (lep., noctuidae). j. appl. entomol. 122: 457-460, 1998. zanuncio tv, serrão je, zanuncio jc, guedes rnc. permethrin-induced hormesis on the predator supputius cincticeps (stal, 1860) (heteroptera: pentatomidae). crop prot. 22: 941-947, 2003. zanuncio jc, tavares wd, fernandes bv, wilcken cf, zanuncio tv. production and use of heteroptera predators for the biological control of eucalyptus pests in brazil. ekoloji 23: 98104, 2014. zanuncio jc, beserra eb, molina-rugama aj, zanuncio tv, pinon tbm, maffia vp. reproduction and longevity of supputius cincticeps (het.: pentatomidae) fed with larvae of zophobas confusa, tenebrio molitor (col.: tenebrionidae) or musca domestica (dip.: muscidae). braz. arch. biol. techn. 48: 771777, 2005. zanuncio jc, de freitas fa, tavares wd, lourenção al, zanuncio tv, serrão je. no direct effects of resistant soybean cultivar iac24 on podisus nigrispinus (heteroptera: pentatomidae). chil. j. agr. res. 72: 528-534, 2012. zanuncio jc, jusselino-filho p, ribeiro rc, castro aa, serrão je. fertility and life expectancy of a predatory stinkbug to sublethal doses of a pyrethroid. bull. environ. contam. toxicol. 90: 39-45, 2013. zanuncio jc, lacerda mc, zanuncio jsz, zanuncio tv, da silva amc, espindula mc. fertility table and rate of population growth of the predator supputius cincticeps (heteroptera: pentatomidae) on one plant of eucalyptus cloeziana in the field. ann. appl. biol. 144: 357361, 2004. 187   http://apps.isiknowledge.com/daisyoneclicksearch.do?product=wos&search_mode=daisyoneclicksearch&db_id=&sid=4e5jmoe17ijghgg2bhj&name=zanuncio%20jc&ut=000222254100012&pos=1 http://apps.isiknowledge.com/daisyoneclicksearch.do?product=wos&search_mode=daisyoneclicksearch&db_id=&sid=4e5jmoe17ijghgg2bhj&name=zanuncio%20jc&ut=000222254100012&pos=1 http://apps.isiknowledge.com/daisyoneclicksearch.do?product=wos&search_mode=daisyoneclicksearch&db_id=&sid=4e5jmoe17ijghgg2bhj&name=lacerda%20mc&ut=000222254100012&pos=2 http://apps.isiknowledge.com/daisyoneclicksearch.do?product=wos&search_mode=daisyoneclicksearch&db_id=&sid=4e5jmoe17ijghgg2bhj&name=junior%20jsz&ut=000222254100012&pos=3 http://apps.isiknowledge.com/daisyoneclicksearch.do?product=wos&search_mode=daisyoneclicksearch&db_id=&sid=4e5jmoe17ijghgg2bhj&name=zanuncio%20tv&ut=000222254100012&pos=4 http://apps.isiknowledge.com/daisyoneclicksearch.do?product=wos&search_mode=daisyoneclicksearch&db_id=&sid=4e5jmoe17ijghgg2bhj&name=zanuncio%20tv&ut=000222254100012&pos=4 http://apps.isiknowledge.com/daisyoneclicksearch.do?product=wos&search_mode=daisyoneclicksearch&db_id=&sid=4e5jmoe17ijghgg2bhj&name=da%20silva%20amc&ut=000222254100012&pos=5 http://apps.isiknowledge.com/daisyoneclicksearch.do?product=wos&search_mode=daisyoneclicksearch&db_id=&sid=4e5jmoe17ijghgg2bhj&name=espindula%20mc&ut=000222254100012&pos=6 the experimental design was completely randomized (crd). the data were submitted to univariate variance analysis (anova) and the means compared with the scott-knott post hoc test at 5 % probability using the software sas version 9.0 (statistical analysis system, 2002) (supplier: ufv). homogeneity of variance and normality of errors were checked using q-q plots (wilk and gnanadesikan, 1968) and data were transformed when necessary. references isj 12: yyy-xxx, 2015     89 isj 12: 89-102, 2015 issn 1824-307x report of meeting xvith scientific meeting of the italian association of developmental and comparative immunobiology (iadci), 18 20 february 2015, department of life sciences, university of trieste, and national institute of oceanography and experimental geophysics, trieste, italy organizers: s battistella1, e ferrero1, m gerdol1, p giulianini1, c manfrin1, s lorenzon2, a pallavicini1, m scocchi1 1department of life sciences, university of trieste, trieste, italy 2national institute of oceanography and experimental geophysics, trieste, italy session 1. chairman: a pallavicini, university of trieste, trieste, italy molecular immunology lecture solving the jigsaw puzzle of bivalve immune response m gerdol1, p venier2, a pallavicini1 1department of life sciences, university of trieste, trieste, italy 2department of biology, university of padua, padua, italy the recent developments of “omic” technologies has greatly simplified the identification of the molecules involved in the invertebrate immune response, permitting to outline complex pathways linked to pathogen recognition and clearance. in particular, thanks to the long-standing availability of a complete genome and due to its ideal use in laboratory experiments, drosophila can be now considered as a relatively well-known model organism for immunological studies in arthropods, the largest group of invertebrates. comparatively, the knowledge of the immunity of lophotrochozoa, and in particular of mollusca (the second largest group of invertebrates) is still very limited and fragmentary. the molecular study of filter-feeding marine bivalves has recently revealed only some key aspects of immune response in these organisms, which are constantly exposed to high loads of microorganisms, especially in coastal shallow waters and yet display a remarkable tolerance to these conditions. the strategies used by bivalves to cope with infection include, among the others, the massive expansion of pattern recognition receptors and the diversification of antimicrobial peptides. however, many open questions remain (e.g., which are the main players in viral recognition? which signalling pathways are activated in response to pamp recognition? how is the expression of antimicrobial effectors regulated?), and our knowledge of the interconnection between bivalve immune pathways is still largely deficient. using mytilus galloprovincialis as a model organism, we explored the available sequence resources and scientific literature to update our knowledge on the main gene-encoded elements of mussel immune response, from the recognition of pathogens to their elimination. we highlight the high complexity of this system, identifying and describing for the first time several molecular players whose existence had so far just been hypothesized in lophotrochonzoa. identification of host-pathogen interacting molecules of mytilus galloprovincialis using phage-display technology v torboli1, m gerdol1, f florian1, p venier2, a pallavicini1 1department of life sciences, university of trieste, trieste, italy 2department of biology, university of padua, padua, italy immunocompetent mollusc cells, in particular the circulating hemocytes, provide a rapid line of defence against potential pathogens. defensive reactions are triggered by the interaction between pamps (pathogen associated molecular patterns) and prrs (pattern recognition receptors). even though an increasing number of host-pathogen interacting molecules has been characterized in mytilus galloprovincialis, to date a strategy for a large-scale identification of these prrs has never been implemented. we used phage display technology, based on the ability to express exogenous peptides as fusions to capsid proteins on the surface of bacteriophages, to identify the prrs of mussel immunocompetent cells possibly     90 involved in the recognition of vibrio splendidus and v. aestuarianus. these gram-negative bacteria are among the most commonly found pathogens in coastal waters, and mussels, compared to other aquacultered bivalves, display a remarkable tolerance to their infection. our approach permitted us to express peptides representative of a mussel hemocyte cdna library on the surface of phage virions, which were subsequently selected by their interaction with pamps present on the surface of target bacteria. the complete complement of peptides that recognize pamps has be thoroughly analyzed by the use of next generation sequencing methods and bioinformatic tools. the comparison between selected and control samples revealed that a v. splendidus is likely recognized by a broader range of prrs compared to v. aestuarianus. overall, we identified 42 putative v. splendidusinteracting proteins, comprising both putative membrane-bound and extracellular prrs. while some of these results are consistent with literature available for mussels or other invertebrates (c-type lectins, freps, c1qdc proteins and apextrinrelated proteins), others are completely novel. in conclusion this innovative approach identified a number of previously unknown mussel prrs whose involvement in the innate immune response will be further characterized. regeneration of adult sensory tentacles and eyes in pomacea canaliculata a accorsi1, e ottaviani1, e ross2, k gotting2, a sánchez alvarado2,3 1department of life sciences, university of modena and reggio emilia, modena, italy 2stowers institute for medical research, kansas city, mo, usa 3howard hughes medical institute, kansas city, mo, usa regeneration is a wide-spread phenomenon in the animal kingdom, but the level of regenerative potential is significantly different among metazoan phyla. for instance, planarians are able to regenerate the entire body from a small fragment of tissue, while the amniote vertebrates display poor regenerative ability. regeneration is a process activated when body parts are severed, damaged or amputated. regeneration induces the re-activation of development pathways for the replacement of the missing structures and it promotes the overexpression of genes typically involved in the functioning of the immune system. the adult freshwater gastropod pomacea canaliculata (lophotrocozoa, mollusca) is able to regenerate de novo three sensory organs: the long cephalic tentacles, the short oral tentacles positioned near the mouth, and the complex camera eyes composed of cornea, lens and retina. after amputation of one of the three sensory organs, the transcriptome of the regenerating tissue was obtained after 2, 3, 6, 12 and 24 h and daily up to the end of the regeneration process, that lasted 14, 21 and 28 days, respectively. the obtained sequences were assembled using trinity software and the transcriptome of each time point was compared with that of the original tissue. this analysis uncovered 8.432 sequences involved in the regeneration process of the cephalic tentacle, 2.725 involved in the oral tentacle regeneration and 5.492 in eye regeneration. the temporal expression profiles of all the identified sequences during the whole regeneration process was analyzed in detail and the time points presenting comparable gene profiles were clustered together allowing the identification of 4 main phases in the regeneration process of the tentacles and the eye. for all the three sensory organs, the early phase is characterized by extensive gene downregulation, followed by two intermediate phases with the majority of genes markedly upor downregulated. the late and final phase displays only a minority of genes with the expression levels significantly different from the original tissue. these studies open the door to a detailed molecular and mechanistic dissection of regeneration in a member of the understudied lophotrochozoan super-clade. il-17 signaling components in bivalves: comparative sequence analysis and involvement in the immune responses u rosani1, l varotto1, m gerdol2, a pallavicini2, p venier1 1department of biology, university of padua, padua, italy 2department of life science, university of trieste, trieste, italy according to the cellular origin as well as their pleiotropic and synergic actions, the vertebrate cytokines are grouped in interleukins, chemokines and interferons. interleukin-17 (il17) was firstly recognized as cytolitic t-cell factor with proinflammatory activity and is the unique member of an interleukin class with no homology to any other known cytokine family. il-17s are produced by activated t lymphocytes and other cell types relevant to the host immunity such as the mucosal epithelial cells. basically, the il-17 family members are potent pro-inflammatory cytokines involved in host defense, autoimmunity and cancer development. the il-17 signaling pathway starts with the binding of il-17 homoor hetero-dimers to specific membrane-bound il-17r complexes. then, intracellular signal transduction proceeds through the tumor-necrosis factor receptor-associated factor 6 (traf6), which is a key adaptor also in the tlr and il-1r-signaling cascades, up to the transcriptional activation of several cytokines, chemokines and also antimicrobial peptides. the crucial element which makes possible the interaction between il-17rs and traf6 is the sefir domain (sef/il-17r domain), located on the cytoplasmic tail of il-17rs and display similarity with tir domain. in the context of cross-talking signal transduction cascades, the adaptor protein ciks, also known as act1 and containing trafbinding motifs, is expected to activate the canonical transcription factor nf-κb and induce the expression of selected gene sets.     91 until some years ago, il17 was reported as a vertebrate-exclusive molecule, likewise its downstream pathway. in 2006, three il-17 gene models were detected in the genome of the sea urchin strongylocentrotus purpuratus. since then, the presence of il-17, il17r and ciks molecules has been reported in several non-vertebrate organisms. in mollusks, il17 was firstly identified in c. gigas in 2008 and subsequently in p. fucata. more recently, five il17 genes have been identified in the c. gigas genome. following deep scanning of many bivalve sequence datasets, we have identified and phylogenetically compared 52 il17-like proteins and more than hundred il17 receptor and adaptor molecules. aiming to validate the m. galloprovincialis sequence findings, we also report expression data on five selected il17s, two il-17rs and the proximate ciksl adaptor, as measured in the hemocytes of mussels injected with a mixture of heat-killed bacteria. are crustacean hyperglycemic hormone (chh)like transcripts involved in immune response in decapods? c manfrin, l peruzza, lc bonzi, a pallavicini, pg giulianini department of life sciences, university of trieste, trieste, italy crustacean hyperglycemic hormone (chh) is a pleiotropic peptide originally identified in the eyestalk x-organ/sinus gland (xo-sg) complex, the major endocrine system of decapods. it belongs to the chh family, a superfamily of neuropeptides that controls many fundamental physiological functions such as molting, osmoregulation, modulation of glycemia, reproduction and behavioural responses, such as aggression and anxiety. with the aim of creating new autocidal methods based on neuroendocrine disruptors for invasive populations of procambarus clarkii, we silenced the crustacean hyperglycemic hormone (chh) by injecting the corresponding dsrna. the effects of chh silencing at the glycemic and transcriptomic level were investigated in the eyestalk, as well as the effects on mortality and moulting rates. after 20 days from the dsrna injection, an unexpected strong hyperglycemic response, achieved after serotonin injection, was recorded in surviving individuals. since a couple of new chh-like transcripts (named here chhip and chhop) previously found in p. clarkii’s eyestalk transcriptome were up-regulated in eyestalks of experimental surviving crayfish, we hypothesized their possible involvement in the immune and stress responses. to preliminarily test our hypothesis, we set up an additional experiment where we analysed the expression levels of chh and chh-like mrnas in eyestalk and hemocytes of p. clarkii injected with lipopolysaccharide (lps). the overexpression of chhip both in the eyestalk and hemocytes of treated individuals was recorded, along with the up-regulation of well-known immune markers in hemocytes, suggesting a role of chhip, and thus an involvement of chh superfamily, in the immune response. the homolog of allograft inflammatory factor-1 induce macrophages migration during innate immune response in medicinal leech t schorn1, f drago2, r girardello1, m de eguileor1, r valvassori1, j vizioli2, a grimaldi1 1department of biotechnology and life sciences, university of insubria, varese, italy 2université lille 1, laboratoire de spectrométrie de masse biologique fondamentale et appliquée, (ifr 147). bat. sn3 cité scientifique, 59655 villeneuve d’ascq, france (aif-1) is a 17 kda cytokine-inducible calciumbinding protein that in vertebrates plays an important role in allografts immune response and its expression is mostly limited to the monocyte/macrophage lineage. recently it was assumed that allograft inflammatory factor-1 (aif-1) was a novel molecule involved in inflammatory responses. to better clarify this aspect in the present study we investigated the expression of aif-1 after bacterial challenge and its potential role in regulating the innate immune response in an invertebrate model, the medicinal leech (hirudo medicinalis). the analysis of an est library from hirudo cns, revealed the presence of a gene, named hmaif-1/alias hmiba1, showing a high homology with vertebrate aif-1. immunohistochemistry using an anti-hmaif-1 polyclonal antibody showed that this protein is constitutively present in spread cd68+ macrophagelike cells. a few hours after pathogen bacterial injection in the body wall, the amount of these immunopositive cells increases at the injected site, co-expressing hmaif-1 and the common leukocyte marker cd45. moreover here we demonstrated that the recombinant protein hmaif-1 induces a massive angiogenesis and it is also a potent chemoattractant for macrophages. after rhmaif-1 stimulation, macrophage-like cells co-express the macrophage marker cd68 and the surface glycoprotein cd45, which in vertebrates is implicated in the integrin-mediated adhesion of macrophages and plays a key role in regulating the functional responsiveness of cells to chemoattractants. we therefore hypothesized that cd45 could play a role for leech macrophage-like cells activation and migration towards the inflammation site and we examined its potential effect on hmaif-1-induced signaling. new data on botryllus schlosseri amp: a transcripts analysis n franchi, l ballarin department of biology, university of padua, padua, italy the innate immune system provides an immediate response against infections in animals and plants. endogenous antimicrobial peptides (amps)     92 are essential effector molecules of this first line of defence allowing the direct killing of invading microorganisms. moreover amps have attracted increasing interest because of their potential as new antibiotics. to date various natural peptides from all kinds of organisms and synthetic derivatives have been characterized for their potential use as novel therapeutics. however, the number of candidate peptides undergoing preclinical or clinical evaluation is still very low. marine invertebrates are source of several amps and some of these have been isolated and characterized from different urochordate such as styela plicata (clavanins and styelins) and ciona intestinalis (ci-mam). in the colonial ascidian b. schlosseri, exploiting the transcriptome and the genome, we have been able to identify a styelin-like amp. we also carried out some preliminary experiments to investigate the chemical properties and the expression pattern of such gene in the presence of different pamps. characterization of peroxiredoxin in the antarctic teleost trematomus bernacchii am tolomeo1, r bakiu2, sp place3, g santovito1 1department of biology, university of padua, padua, italy 2department of aquaculture and fisheries, agricultural university of tirana, tirana, albania 3department of biology, sonoma state university, rohnert park, ca, usa immune responses imply an increase in oxygen consumption and formation of reactive oxygen species, with a consequent risk of oxidative stress. with the aim to study the components of the antioxidant defense system in the antarctic teleosts, we have characterized the genes codifying for peroxiredoxins (prdxs) in the emerald rockcod trematomus bernacchii. prdxs are a family of small (22 27 kda) non-selenium peroxidases that are able to reduce hydrogen peroxide, organic hydroperoxides and peroxynitrite, thus representing a class of important antioxidant enzymes that protect cells against oxidative stress. in the genome of t. bernacchii we have verified the presence of five of the six prdx’s isoforms so far known in vertebrates: prdx2, prdx3, prdx4, prdx5, prdx6. for isoforms 3 and 6, we also characterized two variants (a e b). multi-alignment analysis, performed with fish orthologous sequences, demonstrated high conservation of the amino acids involved in catalytic activity of the various prdx’s isoforms of t. bernacchii. however, some substitutions with polar amino acids, are characteristics of some residues close to the motifs important for the functionality of these proteins. the gene transcription of all t. bernacchii’s prdxs has been measured by rt-sqpcr, in various tissues (gills, heart, liver, spleen, and skeletal muscle). the gills are the organ in which the highest levels of mrna accumulation is present, with the exception of isoforms 3b and 6a which have a low tissuespecificity of expression. prdx 2-cys activity have been measured in the same organs, spleen and liver showing the highest levels. the tissue-specific differences in the mrna and active protein accumulations are probably in relation to the physiological function characteristic of these organs. humoral immunity in antarctic teleosts c bernini1, s mattiucci2, m santoro2, m gerdol3, a pallavicini3, d de pascale4, mr coscia4, f buonocore1, e randelli1, g scapigliati1 1department for innovation in biological, agro-food and forest systems, university of tuscia, viterbo, italy 2department of public health and infectious diseases, university of rome la sapienza, italy 3department of life sciences, university of trieste, trieste, italy 4institute of protein biochemistry, cnr, naples, italy the humoral immunity of antarctic teleosts is an aspect of particular interest due to their physiological adaptation to the extreme environment in which they live. the cold-blooded vertebrates evolved particular features to perform physiological functions at temperatures as low as -2°c so the immune system must defend fish from infections even in such conditions and humoral defences play a pivotal role. first of all we analysed the head kidney transcriptome of the antarctic fish trematomus bernacchii, an important model species for environmental and immunological studies. rna-seq data was generated using illumina paired-end sequencing, obtaining ~7 gbp of sequence data, which were assembled into 96,641 contigs. the sequence collection contains a relevant number of immunity-related transcripts among which, interestingly, the presence of genes relative to the toll-like receptor 4 signalling pathway not so common in fish species. we next examined the basal expression of different genes involved in immunity processes such as tcrrγ, tcrβr , igm, igt, and then we focused on the kinetics of expression of the same genes in spleen and head kidney tissues after tad-1 (psychrobacter sp.) in vivo stimulation (1,06x1010/ml in pbs) for 0 (control), 8, 24 and 72 hours. the results showed that a stimulatory effect was appreciable for immune related genes that are expected to be involved in different aspects of the immune responses. moreover we performed indirect elisa assays in order to evaluate the immunoglobulin secretion after in vivo stimulation of t. bernacchii with three different antigens tad-1 (1.58x108/ml), conalbumin (1.25 mg/ml) and dnp-klh (1 mg/ml)). after 60 days the analysis of the serum immunoglobulins suggested the presence of an immune humoral response against all these antigens ( tad-1 = 0,123 ± 0,035 ; conalbumin = 0.212 ± 0.19 ; dnp-klh = 0.309 ± 0.105 values of absorbance at 492 nm). finally, considering that there are evidences of massive infestation by parasites in icefish chionodraco hamatus (differently from what observed for t. bernacchii); we focused on investigating by indirect elisa assays the capability     93 of these fishes to recognize nematodes as self or non-self. the results taken together suggested relevant immunity-related results providing a new perspective for future immunological studies in these species. session 2. chairmen: l ballarin, university of padua, padua, italy; e ottaviani, university of modena and reggio emilia, modena, italy; m cammarata, university of palermo, palermo, italy evolution of the immune system lecture evolution of complement system p macor department of life sciences, university of trieste, trieste, italy the complement system is part of the immune system and in particular of the innate immunity. it is not adaptable and does not change over the course of an individual's lifetime. however, it can be recruited and brought into action by the adaptive immune system. the complement system is most famous for its role in immunity, orchestrating an exquisitely refined system for immune surveillance. at its core lies a cascade of proteolytic events that ultimately serve to recognize microbes, infected cells or debris and target them for elimination. the human complement system is composed of more than 30 serum and cell surface components, and most of these components show a characteristic domain structure, enabling to trace the evolution of the genes based on their structures. ongoing projects in both vertebrates and invertebrates revealed that most domains used by mammalian complement components are found in invertebrates. unexpectedly, the complement system of an invertebrate shows a similar level of complexity and efficacy as that of mammals. moreover complement components from different species demonstrated the capacity to cross-react. mounting evidence has shown that a number of proteins and proteolytic intermediaries in this cascade have, in themselves, other functions in the body, signalling through receptors to drive events that appear to be unrelated to immune surveillance. it seems, then, that the complement system not only functions as an immunological effector, but also has cell–cell signaling properties that are utilized by a number of non-immunological processes. the scope of the complement system’s function is indeed much greater than we might have imagined only a few years ago. the protein pheromone family of the ciliate euplotes petzi, the earliest branching species in the euplotes phylogentic tree c alimenti, a vallesi, p luporini laboratory of eukaryotic microbiology and animal biology, university of camerino, school of biosciences and veterinary medicine, university of camerino, camerino, italy. self/non-self recognition in ciliates relies on signaling proteins (pheromones) synthesized in association with a genetic mating-type mechanism, that regulates the cell switching between the growth (mitotic) and sexual (mating) stages of the life cycle. in euplotes species, these pheromones are freely released into the medium from where they can be purified in relative abundance. the knowledge of their molecular structures has so far been limited to four species, namely e. raikovi, e. octocarinatus, e. nobilii and e. crassus, that occupy varied positions in the euplotes phylogenetic tree. most research interest has now been focused on the pheromone family of e. petzi because of a major distinctive, phylogenetic trait of this species. together with e. sinicus, e. petzi forms the earliest branching clade in euplotes evolution. four structurally distinct e. petzi pheromones have so far been structurally characterized together with their coding genes. with respect to the other known euplotes pheromones, they show smaller dimensions (only 32 amino acids vs. up to 108 in e. octocarinatus), a higher density of disulfide bonds (four), and a folding in which molecular districts with no regular structures equal in extension districts with regular structures represented by one extended and two single-turn alpha-helices. considering that in the other euplotes species pheromones have structures dominated by a bundle of three regular alpha-helices, the minimal dimensions and the relatively simple architecture of e. petzi pheromones thus indicates that the structural evolution of euplotes pheromones involves a progressive increase of architectural complexity. and the finding that the e. petzi pheromone genes are practically half in dimensions the pheromone genes of the other euplotes species reinforced this indication. autophagic cell death in the insect cell line iplb-ldfb: evidence in absence of known mediators a accorsi, a bellelli, e ottaviani, d malagoli department of life sciences, university of modena and reggio emilia, modena, italy the larvae of holometabolous insects are privileged models for studying the programmed cell death (pcd). the iplb-ldfb cell line has been derived from the larval fat body of the gypsy moth lymantria dispar. iplb-ldfb cells can undergo apoptosis after oxidative stress, but they also present autophagic cell death after a 2 h treatment with the f1-f0 atp synthase inhibitor, oligomycin a. autophagic cell death seems activated in iplbldfb with an established program because the oligomycin a-treated cells can condition the culture medium making it lethal for untreated iplb-ldfb cells. our observation demonstrated that conditioning time is critical for the lethality of the conditioned medium (cm), and that 1 h is enough to attribute lethal effects to the cm. in order to characterize     94 which molecules could work as lethal signals into cm, we first tested the effects of the steroidal molting hormone, ecdysone, on the iplb-ldfb. unexpectedly, ecdysone did not induce neither autophagic nor other types of pcd in iplb-ldfb cells. proteomic analysis of cm revealed that several survival factors are significantly downregulated in oligomycin a-treated cells, but no ligands able to promote the autophagic pcd were identified. to explore the potential role of cm proteins as pro-death signals, proteases were added to cm. the removal of proteins from cm increased its lethal effects, supporting the hypothesis that ligands other than proteins may intervene in promoting the autophagic pcd. in order to shed light on the targets of the uncharacterized pro-death signals of cm, we assessed the expression and the activity of some factors in cells incubated in cm. on the basis of the proteomic analysis, we investigated the expression of the survival molecule imaginal disk growth factor (idgf)-like. qpcr experiments demonstrated that idgf-like expression remains at control levels in the iplb-ldfb cells incubated for 6, 12 or 24 h in cm. the activity of the cell-death related proteases, caspase 8, 9 and 3/7 was then analyzed through luminometric assays. the caspase activity in iplbldfb cells maintained for 12 or 24 h in cm is similar to that of control cells, allowing to exclude the involvement of caspase 8, 9 and 3/7 in the autophagic pcd of iplb-ldfb cells. in all we conclude that oligomycin a-treated iplb-ldfb cells quickly release in the medium one or more steroidal or glucidic factors able to induce autophagic pcd, that occur without the involvement of caspases. tracking the evolution of bivalve antimicrobial peptides m gerdol1, u rosani2, p venier2, a pallavicini1 1department of life sciences, university of trieste, trieste, italy 2department of biology, university of padua, padua, italy antimicrobial peptides (amps) are key components of the invertebrate immune system, acting as a first line of defense against invading pathogens. these highly diversified peptides are usually classified according to their amino acid sequence, structure and mode of action, but due to their rapid evolution driven by the host/pathogen interplay, the relationship between different families in distantly related phylogenetic classes are often still obscure. almost all the amps so far characterized in bivalve mollusks are cysteine-rich, and their disulfide pattern is generally used as the main criteria for their classification within one of the currently known families. nevertheless, to date, a clear and unambiguous scheme of classification for these amps is still missing, leading to potential problems in the correct identification of novel peptides, especially in the next generation sequencing era. furthermore, due to the limited genetic and genomic knowledge of these organisms, a comprehensive view of the evolution and the spread of different amp families across highly diversified bivalve species is still completely missing. here, taking advantage of a genomic and transcriptomic data-mining approach, we highlight that mytilins, myticins and mytimycins are amp families which are present in a very narrow taxonomical range of bivalves, which only include the order mytiloida. the analysis of novel sequences revealed by ngs approaches as well as conserved genomic features permit to investigate with an unprecedented depth the relationship among these amp families and their possible evolution from ancestral defensin-like genes. new data on phagocytosis-induced apoptosis in the colonial ascidian botryllus schlosseri n franchi, f schiavon, l ballarin department of biology, university of padua, padua, italy colonies of the ascidian botryllus schlosseri undergo cyclical generation change or take-over (to) during which diffuse apoptosis occurs in zooid tissues, as indicated by morphological, biochemical and molecular investigations. tissues are rapidly infiltrated by circulating phagocytes, selectively recruited by dying cells, which recognize and greedily ingest them. previous observations led us to suggest that phagocytes, once ingested apoptotic cells or corpses, undergo phagocytosis-induced apoptosis (pia). we already demonstrated, by western blot and immunocytochemical analyses, the release of cytochrome c by hemocytes and the expression of apoptosis-related molecules, such as bax and caspases, in phagocytes during the to. in order to corroborate the above assumption, we carried out new morphological analyses at the transmission electron microscope (tem) and started a molecular analysis of apoptosis in botryllus phagocytes looking for transcripts differentially expressed in phagocytes at to. we identified and characterized transcript sequences for bax, aif and parp (poly adp ribose polymerase), and studied their expression and the location of the corresponding mrna in hemocytes. the collected data clearly indicate the diffuse occurrence of pia among phagocytes which guarantee the disposal of apoptotic cells or corpses. in addition, they extend the classical view of pia, intended as a mechanism for the prevention of microbial diffusion within the organism, and reveal an undescribed role of the process in the control of asexual development and colonial homeostasis. expression study of molecular markers involved in staminality and differentiation in the colonial ascidians botryllus schlosseri f ballin, n franchi, l ballarin department of biology, university of padua, padua, italy     95 all multicellular organisms originate from a small set of totipotent embryonic cells that differentiate into a structured body plan during embryogenesis. the ability to generate an embryo from a single cell and the regenerative capabilities of metazoans suggest the presence of cell types with stem cell attributes. compound ascidians, like botryllus schlosseri, offer the opportunities to investigate the biology of both embryonic and adult stem cells thanks to the presence of sexual and asexual reproduction. in b. schlosseri new buds emerge as thickening of the peribranchial epithelium in a process call palleal budding. sometimes, a vascular budding occurs, with the development of new buds formed by circulating multipotent or pluripotent cells. these two kinds of budding processes render b. schlosseri a good research tool for the study of staminality. in b. schlosseri, during the cyclical generation change, an increase in the number of hemoblasts occurs which will replace, after their differentiation, the circulating cells undergoing apoptotic cell death. ascidian hematopoiesis occurs in close proximity to the pharyngeal vessels, in the so-called “hematopoietic nodules” and in the endostyle, the cells of which proliferate and migrate to regenerating organs in developing buds. despite the morphologic suggestions that hemoblasts are the precursors of all the circulating cell types, immunocytes included, there is a general lack of biochemical and molecular data supporting this assumption. here we report the first results on the characterisation of staminality and differentiation molecular markers such as abcg2, cd133 and gata2/3 considered hematopoiesis molecular marker in other deuterostomes. interleukin 17 genes as mediators of inflammatory responses in ciona intestinalis a vizzini, f di falco, d parrinello, ma sanfratello, c mazzarella, n parrinello, m cammarata marine immunobiology laboratory, department of biological chemical pharmaceutical science and technology, university of palermo, palermo, italy inflammation is a complex reaction of host defence mechanisms aiming at neutralization of an insult and restoring normal tissue structure and function. in human il-17 is t-cell derived cytokine plays a key role in the clearance of extracellular bacteria promoting cell infiltration and production of several cytokines and chemokines. here, we report on three ciona intestinalis il-17 homologues (ciil17-1, ciil17-2, ciil17-3). the gene organization, phylogenetic tree and modeling supported the close relationship with the mammalian il-17a and il-17f suggesting that the c. intestinalis il-17 genes share a common ancestor in the chordate lineages. real time pcr analysis showed a prompt expression induced by lps inoculation showing that they are involved in the first steps of inflammatory response. in situ hybridization assays disclosed that the genes transcription was upregulated in the pharynx, the main organ of the ascidian immune system, and expressed by hemocytes (granulocytes and univacuolar refractile granulocyte) inside the pharynx vessels. as in human, we can assume that ciil-17-like stimulates the release of citnfalpha, which synergizes with ciil-17 in its effects on cells and molecules of ciona intestinalis immunity system. in addition, a comparative evaluation with others molecules upregulated by lps challenge as citnf-alpha, phenoloxisases, peroxinectin, galectins and mannan binding lectin, have been evaluated in terms of temporal and quantitative gene expression. isolation and characterization of a lps induced md2-like protein in ciona intestinalis a vizzini1, a bonura2, v longo2, m. sanfratello1, d parrinello1, n parrinello1, p colombo2 1dipartimento di scienze e tecnologie biologiche, chimiche e farmaceutiche, università di palermo, palermo, italy 2istituto di biomedicina ed immunologia molecolare “alberto monroy” del consiglio nazionale delle ricerche, palermo, italy the md2 (myeloid differentiation factor-2) protein belongs to the ml superfamily. this group of proteins contain a specific lipid binding domain (ml domain) that plays an important role in lipid recognition and metabolism. in vertebrates, md-2 is involved in innate immune response as coreceptor in the lps/tlr4 signaling pathway; md2 recognizes and binds the bacterial lipid a and drives the tlr4 activation. two tlr isoforms, citlr-1 and citlr-2, were identified in ciona intestinalis with a tir domain most similar to human tlr4 and tlr 6 respectively. using a pcr-based subtractive hybridization strategy for isolation of differentially expressed genes between lps-challenged and naïve c. intestinalis, we identified a full-length cdna (855 bp) encoding for a 150 a.a. protein (cimd-2-like). in silico analysis showed that the deduced protein contains a signal peptide (1-19 a.a.) and an e1/der p2/der f2/ml domain-md2 related lipid recognition domain (21148 a.a) with similarities to ml(md-2 related lipidrecognition) domain identified in md-2 and npc2 (niemann-pick disease type c2). phylogenetic and structural analysis supported the close relationship with md-2 and npc2 suggesting that cimd-2-like originated from a common ancestor gene. furthermore, gene expression studies by realtime pcr demonstrated that this cdna is upregulated after lps injection in the body wall. in situ hybridization performed in controls and lpsinduced animals has shown that this gene is expressed in granular amebocytes, large granules hemocytes and urg (univacuolar refractile granulocyte) in pharynx, the main organ of the ascidian immune system. characterization of a novel lps-induced peptide from ciona intestinalis with immune modulatory activities     96 a bonura1, a vizzini2, a longo1, s vlah1, m. r melis1, f gervasi3, n parrinello2, p colombo1 1istituto di biomedicina ed immunologia molecolare “alberto monroy” del consiglio nazionale delle ricerche, palermo, italy 2department of biological, chemical and pharmaceutical science and technology, university of palermo, palermo, italy 3unità operativa di ematologia, ospedale civico, palermo, italy we previously published that lps injection in the body wall of the tunicate ciona intestinalis induced the activation of an alternative polyadenylation signal leading to the up regulation and modified tissue localization of a cdna (c8short) containing the first exon of a ci-rtp-like mrna. in silico analysis revealed that the synthetic polypeptide derived from the primary sequence of the c8short cdna displays a peculiar amino acids composition with a high percentage of hydrophobic residues (36 %), a negative net charge (-1) and a high content of proline residues (21 %) suggesting the possibility that this peptide can act as an host defense peptide. for these reasons, the immunological properties of the c8short peptide were studied by using human peripheral blood mononuclear cells in vitro. as a first result, we were able to demonstrate that c8short peptide did not show cytotoxic or/and haemophilic activities in vitro. furthermore, we observed that the c8 peptide displays some immune regulatory activities showing the ability to preferentially induce the proliferation of human cd4+ cells. following this line of evidence, we decided to perform a time course looking at the appearance of cd4+/cd25+ cells after c8short stimulation demonstrating that this peptide was able to select peptide-specific effector cells in healthy subjects. finally, by means of magnetic cell sorting, we were able to show that the c8short induces the secretion of the ifn-γ and il-17 inflammatory cytokines from human cd4+ cells. taken together, our data demonstrated that during the inflammatory response induced by lps injection in the body wall of ciona intestinalis, the organism reacts inducing an alternative poly-adenylation event leading to the over-expression of a truncated form of a longer protein that may have potential to be developed as a novel immune regulatory adjuvant. fish igt: the weird case of antarctic teleosts s giacomelli, f albanese, r esposito de lucia, u oreste, mr coscia institute of protein biochemistry, national research council (cnr), naples, italy teleost fish are known to possess three immunoglobulin heavy chain isotypes, igm, igd and the lastly discovered igt. in the present study, igt genes of the notothenioid antarctic teleost trematomus bernacchii and of a non antarctic notothenioid species, bovichtus diacanthus, were cloned and characterized. surprisingly, compared to igt from other teleost species, including the non antarctic notothenioid one, t. bernacchii igt lack the entire second constant domain with only a few amino acid residues left; the latter can be aligned to the c-terminus of b. diacanthus ch2 domain. furthermore, genomic analysis of both species revealed that, in the case of t. bernacchii, within the intron between the first and the second costant exons a reminiscence of the ancestral second exon is present. this remnant falls within a 29 bp duplicated region and shows different sizes of 51, 33, and 42 bp according to the genomic sequence analysed. in all cases this exon segments preserved the donor and acceptor splicing sites to be correctly spliced into the mature transcript, giving rise to different cdna variants. these findings are likely to have resulted from duplication events. phylogenetic analysis performed on each constant domain and on the entire constant region from all teleost igt available to date revealed that the loss of almost an entire exon together with the conservation of some amino acids in the remaining domain, such as proline, glycine and cysteine, could be interpreted as another specific and distinctive feature of the evolution of the antarctic fish genome. lecture evolution of β defensin and cathelicidin host defense peptides in primates a tossi department of life sciences, university of trieste, trieste, italy beta-defensins and cathelicidins are the principal families of host defence peptides in mammals and other vertebrate animals. the defensins are encoded by several paraloguous genes in each animal, and some play relevant and differentiated roles in immunity. four of these (bd1 bd4) show quite different patterns of evolutionary variations in primates, ranging from conservation to neutral evolution or positive selection for variation. cathelicidins are structurally quite different to defensins and only one is present in each primate species, including man. these peptides can have both antibiotic and host cell modulating activities, and were found to be under strong positive selection. by chemically synthesizing and comparing the structures and activities of several defensins and cathelicidins from different primates, as well as rationally designed variants, it has been possible to gather useful information on their roles, modes of action, and factors underlying their differing evolutionary patterns. identification of igd and igt immonuglobulins in sea bass (dicentrarchus labrax) gill transcriptome v stocchi1, n nuñez ortiz1, m gerdol2, a pallavicini2, a facchiano3, e randelli1, g scapigliati1, f buonocore1 department for innovation in biological, agro-food and forest systems, university of tuscia, viterbo, italy     97 1department of life sciences, university of trieste, trieste, italy 2laboratory of bioinformatics and computational biology, institute of food science, national research council (cnr), avellino, italy the gills of fish are, in terms of exposed surface, the biggest tissue of most teleost species.they are involved in the maintaining of fish homeostasis by the uptake of nutrients and substances, and by forming an active barrier against the entry of pathogens. the thin gill epithelium is a mucosal tissue at direct contact with the water environment, and contains a gill-associated lymphoid tissue (gialt) with macrophages/granulocytes, lymphocytes. to achieve a more comprehensive knowledge of gialt asset and function in fish we used the european sea bass as a model and we produced a whole gill rna transcriptome by deep sequencing using an illumina platform. immune-related sequences identified in the transcriptome contained all know components of fish innate and acquired immune system and, interestingly, we identified all t cell gene subsets including th1/th2/th17/treg cell subpopulations, thus suggesting their possible presence in the branchial epithelium. regarding b cells, different sequences of possible igt and igd transcripts have been evidenced and these sequences have been confirmed by cloning from gill cdna. we obtained a full-length sequence of a heavy chain secretory igt (accession number km410929), a partial membrane-bound igt and a full-lenght heavy chain igd. heavy chain secretory igt has been aligned with secretory form of o. mykiss (rainbow trout) that shows 46% of sequence identity with sea bass, and important amino acids involved in fundamental structural features of igt are conserved, like cysteines that forms interand intradisulphide bridges and tryptophans that are required for the folding of igsf domains. modelling of 3d structure of igt has been performed based on the mouse ig gamma-2a chain c region (pdb file: 1igt) and it showed a betasheet sandwich architecture of immunoglobulin-like topology. moreover, the basal expression of igt and igd was studied by real-time pcr in different organs and tissues of unstimulated juvenile sea bass. the highest igt and igd expression was found in peripheral blood leukocytes (pbl) followed by gills, liver and head kidney. finally, after an alignment of the different cloned igt isoforms, we identified a well conserved region in all sequences and we designed three peptides in this region that have been used to produce a polyclonal antibody against sea bass igt. a polyclonal antiserum for sea bass (dicentrarchus labrax) igt n nuñez ortiz, v stocchi, e randelli, f buonocore, g scapigliati department for innovation in biological, agro-food and forest systems, university of tuscia, viterbo, italy immunoglobulin t (igt) is one of the key effector molecules of jawed vertebrate’s adaptive immune system. igt is a monomeric immunoglobulin in serum and polymeric in the gut mucus, was first discovered in trout and has an important role in mucosal responses, being analogous to mammalian secretory iga. we are investigating immune responses in sea bass after mucosal (immersion) vaccination against inactivated nodavirus, and we believe that igt should play a role in anti-viral immunity. a necessary step is to have an antibody against sea bass igt in order to investigate by elisa the presence of igt in serum and mucus, and the presence of igt-bearing cells in tissues. in this respect, we immunized rabbits with synthetic peptides deduced from the full length cdna sequence and located in the surface-exposed sequence of sea bass igt peptide. of the two antisera we obtained, we selected one (raigt1) that resulted able to stain in western blot of splenocytes lysates a polypeptide at 74 kda in reducing conditions and at 150 kda in non-reducing conditions. by iif and flow cytometry of leukocytes, the raigt1 stained 40±24 % in head kidney, 34±24 % of iels, 28±17 % in spleen, 20±5 % of pbl and 5±3 % in gills. at the fluorescence microscope, live cells from these tissue showed a typical membraneassociated positivity. moreover, by using raigt1 we performed an indirect elisa assay platform to evaluate the presence of igt in sera and intestinal mucus of control and immunized fish. preliminary results showed a very poor content of igt in serum and a high content of igt in mucus, in line data obtained in trout. experiments are in progress to obtain igtenriched fractions of leukocytes by immunopurification procedures, and in a first attempt in spleen we obtained a 70% of positive cells in the raigt-purified fraction. the next step will be to analyze by rt-pcr the expression of band tlymphocytes marker genes in the obtained fractions. localization of igt expressing cells in sea bass dicentrarchus labrax (l.) s picchietti, l guerra, n nunez ortiz, f buonocore, d cervia, g scapigliati department for innovation in biological, agro-food and forest systems, tuscia university, viterbo, italy teleost fish have three heavy chain isotypes: μ and δ that correspond to the igm and igd classes found in all vertebrates with jaws, and τ that encodes the igt class, which is specific to fish. the expression of igm and igt are mutually exclusive,     98 leading to the existence of two b-cell subsets expressing either both igm and igd or only igt. to define the sea bass igt positive cells and to investigate their distribution in systemic and mucosal sites, a specific polyclonal antibody (raigt1) has been developed. by immunohistochemical analysis, igt+ cells were distributed more commonly within the epithelium and lp of the juveniles sea bass posterior intestine. in particular, the gut mucus was strongly positive to the raigt1, confirming, as previously reported in other teleost species, that the high concentration of secreted igt found in the mucus could reflect a specialized role in gut mucosal immunity. in addition, igt+ b cells appeared located in the epithelium of the gill lamellae, in the blood vassels and in the spleen and head kidney parenchyma. the presence of unique stained igt+ and igm+ b cells subsets were also analyzed. in situ hybridization of igt-mrna labelled cells further substantiates the presence of igt expressing cells in sea bass lymphoid tissues, revealing in the intestine a gradient from the anterior segment towards the anus. immunization of sea bass dicentrarchus labrax against nodavirus f buonocore1, n nuñez ortiz1, e randelli1, v stocchi1, a toffan2, f pascoli2, s picchietti1, targetfish consortium3, g scapigliati1 1department for innovation in biological, agro-food and forest systems, tuscia university, viterbo, italy 2zooprophylactic institute of venezie, legnaro (pd), italy 3www.targetfish.eu the sea bass is particularly sensitive to viral encefalophaty and retinopathy (ver) caused by betanodavirus, a rna retrovirus that induces high mortalities in wild and farmed fish species. we are currently investigating immune responses of sea bass against nodavirus by immunizing fish with formalin-inactivated verv, strain 283/i09. to evaluate humoral responses we used an indirect elisa for sea bass igm, and developed a capture elisa assay employing a rabbit antiserum against whole virus and a monoclonal antibody specific for the capsid protein. by using these assy we detected measurable amounts of specific igm in fish injected intraperitoneally with verv, whereas after mucosal (immersion) vaccination no or very few specific igm have been detected. immunohistochemistry (ihc) analysis of gills in immersion-immunized fish showed uptake of verv inside the epithelium and expression analysis of antiviral genes showed their upregulation. from these data we conclude that inactivation of virus by formalin damaged antigenicity of the virus. alternative strategies of immunization have been by oral administration of verv by adding to food recombinant capsid protein produced in pichia pastoris yeast or in tricoplusia ni insect model. ihc analysis of the intestinal tract showed uptake of the antigen in pichia-treated fish, and elisa assays revealed little but measurable igm titers in sera. experiments are in progress to evaluate results from additional immunization experiments and to evaluate alternative verv inactivation methods. alien species and their impact on aquatic animal health a manfrin, t pretto zooprophylactic institute of venezie centro specialistico ittico di adria (ro), italy invasive alien species (ias) are considered to be one of the major threats to native biodiversity, with the world conservation union (iucn) citing their impacts as “immense, insidious, and usually irreversible”. it is estimated that 11 % of the c. 12,000 alien species in europe are invasive, causing environmental, economic and social damage and it is reasonable to expect that the rate of biological invasions into europe will increase in the coming years. among them, marine and freshwater invasive species are really important not only from an evolutionary point of view, but even for their impact on aquatic animal health. 38 of 82 (46,3 %) fresh water species in italy are aliens and some of them can be carrier of old or new pathogens, which could be highly pathogenic for autoctonous animals. as many of them are endangered, an exotic disease or a more virulent pathogen could increase the prevalence of mortality and destroy all the population in a limited area but also all along the water catchment. epizootic ulcerative syndrome (aphanomyces invadans) in finfish and crayfish plague (aphanomyces astaci) in crustacean are typical examples of pathogens with the potential to cause an adverse effect on aquatic animal health. the first, which is exotic for europe, can also be transmitted by many ornamental fish species, imported from many extra eu countries every day, while the second one is an “old pathogen” who found “new carriers” like red swamp crayfish (procambarus clarkii) making its spread easier. new pathogens (e.g. red sea bream iridovirus, shrimp taura syndrome virus, etc.) or new genotype of old ones can spread not only in fish farms but also in the aquatic environment, leading to the destruction of entire populations. to avoid this catastrophic event is important to keep in mind that sanitary and import measures must be taken in order to prevent their introduction and establishment. invasive species and their parasites: the case of the atlantic blue crab callinectes sapidus rathbun, 1896 and its endoparasitic dynoflagellate hematodinium spp. in the mediterranean sea p pagliara, m zotti, l carrozzo, g mancinelli department of biological and environmental sciences and technologies, university of salento, lecce, italy coastal marine and transitional water ecosystems are threatened world-wide by invasions of non-indigenous invertebrate species, altering     99 community structure and ecosystem functioning. noticeably, the mediterranean sea is to date experiencing a massive rate of invasion of marine species. a number of alien mollusks, crustacean and fish have established in the mediterranean basin becoming, in some cases, invasive species, altering native benthic communities. sometimes the success of these introduced species is due to their ability to escape their natural enemies. moreover, alien species may transmit disease to native hosts (pathogen spillover), an event that has been given little consideration in the context of biological invasions. the blue crab callinectes sapidus is an alien species originating in western atlantic ocean and that was introduced, accidentally or deliberately, into asia, europe, hawaii and japan. especially common in estuaries, this species eats a large range of foods (eats clams, oysters, and mussels as well as almost any vegetable or animal matte). in its native habitats, the blue crab is subject to infection by the parasitic dinoflagellate hematodinium perezi representing a significant cause of mortality in many crab populations. in a survey performed along the apulian coast, we sampled individuals of c. sapidus and of native crab species, such as eriphia verrucosa. their hemolymph was screened for detection of parasite infections. our results indicated only in very few cases the presence of hematodinium spp. in c. sapidus and never in native crabs species. furthermore, the abundance of parasite cells was very low. a morphological characterization on the blue crab hemocytes was also carried out evidencing the presence of 2 main hemocyte types: the granulocytes and the hyalinocytes. is the immunecompetent gender-related in carabus lefebvrei (coleoptera, carabidae)? pg giulianini1, p brandmayr2, f cavaliere2, a giglio2 1department of life sciences, university of trieste, trieste, italy 2department of biology, ecology and earth science, university of calabria, arcavacata di rende, cosenza, italy trade-offs exist between immunity and other traits in order to maximize fitness. moreover, an insect’s ability to mount an immune response is related to their gender and age. this study tests this assumption by examining two current immunity markers in insect ecological immunology, phenoloxidase and lysozyme-like enzyme activities in carabus lefebvrei adults. this species is a helicophagous italian endemic ground beetle that lives in central and south apennines mountain forests. the plasma phenoloxidase (po) activity was detected using a l-dopa substrate and enzyme activity was expressed as absorbance units at 492 nm/μl of hemolymph. moreover, a turbidometric assay was used to measure lysozyme-like enzyme activity in the hemolymph at 450 nm. lysozyme-like activity was quantified using a standard curve and expressed as ue/ml. total and basal phenoloxidase (po) and baseline lysozyme-like enzyme activities were compared between females and males according to their age. results show no significant difference in po activity levels between females and males at the same age. higher levels in basal po activity were found in females (wilcoxon rank sum test: p = 2.57x10-8) and males (p= 0.001) in pre-reproductive phase than adults displeasing reproductive behaviour. total po activity increase with age in males (p= 0.009). the females displeasing reproductive behaviour show lower level of total po than males (p = 4.13x10-6). baseline lysozyme-like activity was significantly higher in adults displeasing reproductive behaviour than in young adults (p = 1.564x10-6) for both females (p = 1.4x10-5) and males (p = 0.002). no significant differences were recorded regarding gender in young adults while high levels of enzyme activity were found in females displeasing reproductive behaviour (p = 4.79x10-5) than males ones. results suggest that the basal po was significantly affected by age because in young adults resources were allocated to strengthen the cuticle by melanization after the metamorphosis forming a barrier against pathogens. the total po and lysozyme-like enzyme activities were affected by gender but only in adults displeasing reproductive behaviour. our hypothesis was that resources were invested to increase the activity of two enzymes involved in the humoral response in the reproductive phase preserving the fecundity and longevity of females and males. moreover, as longevity is of more importance for female than for male fitness, c. lefebvrei females invest more in immune defence. the size inequality of apis mellifera ligustica hypopharyngeal glands along a gradient of heavy metal pollution s battistella1, a giglio2, a ammendola1, a naccarato3, e simeon1, a tagarelli3, pg giulianini1 1department of life sciences, university of trieste, trieste, italy 2department of biology, ecology and earth science, university of calabria, arcavacata di rende, cosenza, italy 3department of chemistry and chemical technologies, university of calabria, cosenza, italy a number of anthropogenic activities, including farming and urbanization, have a significant impact on the environment and can produce an irreversible damage at the population level. to assess the effects of environmental stressors as metal pollution on apis mellifera ligustica populations, we analyzed the hypopharyngeal gland size and morphology of foraging bees along a urban-suburban gradient. foraging bees are collected from beehives located in s. giovanni (trieste, “urban site”) and in domio (trieste, “suburban site”) over two full activity periods in early summer (july). foraging bee heads from urban site (n = 4) and from suburban site (n = 2) were fixed and embedded in epon 812-araldite     100 mixture for light microscopy analyses. in order to perform morphometrical analyses of hypopharyngeal glands, the minimum and maximum diameters of the acini was measure used imagej program. diameters were expressed as mean±se and differences were assessed by nonparametric statistics. statistical analyses were performed using r version 3.0.1 software (r development core team 2013). the statistical comparison showed a smaller diameter of the acini of hypopharyngeal gland of bees from suburban site (0.080 mm ± 0.003), compared to those of urban site (0.114 mm ± 0.004). statistical analysis showed an highly significant difference in the size of the acini between the site of domio (wilcoxon rank sum test, p = 1.651x10-7) compared to the site of s. giovanni. metal contents were recorded for foraging bees (n = 20 from each site) and measured by inductively coupled plasma-mass spectrometry (icp-ms). recorded elements are as, bi, cd, co, cr, cu, ni, pb, sr, v, and zn. they are accumulated in two different rank order zn> cu> sr=bi> ni> cr> pb=co> v>cd >as in bees from urban site and zn> cu> sr >cr >ni>b =co=v>pb>as>cd in bees from suburban site. significantly differences in concentration of cd, cr and cu were recorded in bees from the two sites (wilcoxon rank sum test p<9.229x10-5 for cd, p < 0.00021 for cr and p < 0.00053 for cu). metal concentrations in the animal’s body reflect quantitatively pollution levels of sites. moreover, morphological data demonstrate that metal pollutions in the environment may have detrimental effects on the individual level and then on a. mellifera ligustica population. aif-1 impregnated matrigel: an important tool to study in vivo and in vitro behaviour of the leech hirudo medicinalis macrophages in response to mwcnts treatment r girardello1, m de eguileor1, r valvassori1, j vizioli2, f drago2, a grimaldi1 1department of biotechnology and life sciences, university of insubria, varese, italy 2université lille 1, laboratoire de spectrométrie de masse biologique fondamentale et appliquée, (ifr 147). bat. sn3 cité scientifique, 59655 villeneuve d’ascq, france the rapid development of nanotechnology and nanoscience during the last decade has led to the discovery of nanomaterials such as multi-wall carbon nanotubes (mwcnts), widely used in industry. aquatic ecosystems seem to be particularly susceptible to contamination by mwcnts with harmful consequences for the aquatic animals as well as for humans, who may come into contact with this nanomaterial through many pathways, such as inhalation, injection, penetration but also ingestion. we have recently demonstrated that in vivo treatment of h. medicinalis with water dispersed mwcnts causes a strong inflammatory process, inducing a massive angiogenesis and migration of cd45+ and cd68+ macrophages throughout the animal body wall. in order to better understand the mechanisms through which this cell population interacts with the nanomaterial, we used a consolidated experimental approach based on injection in the body wall of the leech of the biomatrix matrigel (mg), added with a specific macrophage chemoattractant, the cytokine allograft inflammatory factor-1 (aif-1) and/or with mwcnts. we observe that mwcnts alone are able to induce the migration of a reduced number of macrophages into the mg sponges while the presence of rhmaif-1 invokes a larger number of cells within the matrigel and mg pellets are rich in cells which are positive for both cd68 and hmaif1, specific monocyte-macrophage markers. ultrastructural analysis at tem suggests that short and curled mwcnts may be internalized by phagocytosis or during the process of matrix degradation, while straight and rigid mwncts seem to be able to pierce cell membranes during cells migration and are then found free in the cytosol. starting from these preliminary results, the next goal of our work will be to obtain in vitro expansion of macrophages primary leech cells that could be used as a sensitive method to evaluate the presence of the nanomaterial in contaminated water. for this purpose, nanotubes at different concentration will be then added at primary cell cultures to study some aspects of their effects on cell morphology, cell stress response, cell viability and death events. immunomodulation of cationic polystyrene nanoparticles in mytilus hemocytes c ciacci2, b canonico2, e bergami1, l canesi3, ka dawson4, i corsi1 1dipartimento di scienze fisiche, della terra e dell’ambiente, università di siena, italy 2dipartimento di scienze della terra, della vita e dell’ambiente, università degli studi di urbino “carlo bo”, italy 3dipartimento per lo studio del territorio e delle sue risorse, università di genova, italy 4centre for bionano interactions, school of chemistry and chemical biology, univ. college dublin, ireland plastic debris and their degradation products are ubiquitously present in the world's seas and oceans. engineered plastic nanoparticles derived from post-consumer waste as well as from mesomicroplastics via degradation pose a specific challenge to the ecosystem. the fate and impact of nanoplastics in the marine environment is almost unknown but it is very important due to their increasing abundance in the water column and food webs. the impact of microplastics on marine organisms will depend on a combination of parameters that determine the position of these particles in the water column. even though the existing data are too limited to determine a realistic natural concentration of microplastics in seawater, the potential for ingestion by commercially important species, however, remains a cause for concern. bivalves are of particular interest since their http://apps.webofknowledge.com/full_record.do?product=ua&search_mode=citationreport&qid=3&sid=z2le@o7de7mgc4ebii6&page=4&doc=31 http://apps.webofknowledge.com/full_record.do?product=ua&search_mode=citationreport&qid=3&sid=z2le@o7de7mgc4ebii6&page=4&doc=31     101 extensive filter-feeding activity exposes them directly to microplastics present in the water column. polystyrene (ps) is one of the most largely used plastics worldwide, used in food and industrial packaging, disposable cutlery, compact disc cases, building insulation, medical products and toys and can be considered as model for studying both fate and toxicity of nanoplastics in marine organisms. our recent findings on amine ps nps (ps-nh2) toxicity in sea urchin embryos underline that marine invertebrates can be biological targets of nanoplastics. the present study aims to investigate pathways of toxicity of 50 nm cationic ps-nh2 in hemocytes of mediterranean mussel mytilus galloprovincialis: a battery of functional immune assays was applied to investigate the short term in vitro effects of ps-nh2. several functional parameters have been evaluated: lysosomal membrane stability and lysosomal enzyme release, extracellular oxyradical production and nitric oxide (no) production, phagocytic activity, as well as proapoptotic processes at both plasma membrane and mitochondrial level. the results suggest that mussel hemocytes and their immune activity are affected by ps-nh2 nps. therefore, further research is necessary on specific mechanisms of nanoplastic toxicity and its cellular uptake in order to assess the impact on marine biota. immunomodulation and physiological responses of mytilus galloprovincialis as bioindicators of environmental change mg parisi1, m mauro1, g sarà2, m cammarata1 1department of biological, chemical and pharmaceutical science and technology, university of palermo, palermo, italy 2department of earth and marine science, university of palermo, palermo, italy oxygenation level temperature increases and changes in food availability are predicted to occur in the future. in such scenario, a global climate change (gcc), there is growing concern for the health status of wild and farmed organisms. bivalve molluscs, are important components of coastal marine ecosystems, and as sedentary and filter feeders, are good bioindicators of environmental conditions. the ability of organisms to maintain the immunosurveillance unaltered under adverse environmental conditions may enhance theirs survival capability. only a few studies have investigated the effects of changing environmental parameters on the mussels immunity. in the present study, the effects of different food concentration, temperature and oxygenation treatments were evaluated on immune parameters of mytilus galloprovincialis detected on digestive gland and hemocytes. bivalves were exposed to experimental conditions by increasing of six food treatment, to three different temperatures under conditions of normoxia and anoxia. the multifactorial analysis applied to the responses of the immune variables has showed a direct dependence of various enzymes production by temperature and food concentration. the stability of the lysosomal membrane was altered under conditions of thermal stress and food changing. the protein concentration of the lysate of hemocytes instead was most affected by the lack of adequate oxygenation. in addition, a correlation was carried out between mussels immunological effectors and physiological responses as clearance rate, measured by the removal of suspended particles from water flowing through experimental chambers, food absorption efficiency and rates of oxygen consumption by individual mussels. overall, information summarized in the present study indicated that climate changes can affect hemocyte and enzymatic functionality and the immune responses of this bivalve could be used as good environmental biomarkers. variation of environmental condition and diet act on immune parameters of mytilus galloprovincialis m mauro1, mg parisi1, g sarà2, m cammarata1 1department of biological, chemical and pharmaceutical science and technology, university of palermo, palermo, italy 2department of earth and marine science, university of palermo, palermo, italy the knowledge of the immunity mechanisms as environmental indicators and their alterations in the case of physical stress can be of fundamental importance in the environmental management programs. recently has been shown that environmental factors affect immune responses in some species of bivalves. in this study we assessed different enzymatic activities from digestive gland of m. galloprovincialis such esterase, phosphatase and phenoloxidase (po), involved in digestive inflammatory, detoxification and melanization processes. particularly, esterases catalyze the hydrolysis reaction of the ester bond. phosphatases modulates the removal of phosphate groups by producing phosphoric acid from esters and participate in the metabolism of sugars, nucleotides and phospholipids. the melanization cascade, in which phenoloxidase is the terminal enzyme, plays a key role in recognition of microbial infections in molluscs. it was also evaluated, as potential biomarker, the lysosomal membrane stability through neutral red assay on hemocytes taken from the posterior adductor muscle. specimens were maintained under conditions of normoxia and anoxia and they were subjected to various food amount and different temperatures.     102 the results showed that the enzymatic activities of esterases, phosphatases and po are higher during treatments with lowest temperatures and food amount. moreover, the production of po is higher in the conditions of anoxia. the lower values of enzymatic production have been detected under the levels of temperature, oxygen availability and food different than the optimum conditions for the mussels life cycle. during the normoxic treatment, the stability of the lysosomal membrane is highest at the average values of temperature and food concentration. the lowest values, instead, were measured at a temperature of 12 °c and 28 °c in anoxic conditions. investigation on the effects of three nanoparticles (zinc oxide, titanium dioxide, c60 fullerene) on haemocyte parameters of ruditapes philippinarum i marisa1, v matozzo1, d sheehan2, mg marin1 1department of biology, university of padua, padua, italy 2department of biochemistry and environmental research institute, university college cork, ireland bivalve hemocytes are involved in various biological processes, including wound and shell repair, shell production, transport and digestion of nutrients, excretion and immune defence. it is well known that variations in environmental parameters, such as temperature, salinity, ph, oxygen and nutrients can affect haemocyte functionality in bivalves. however, most of the studies (both in vitro and in vivo) have been focused on the evaluation of contaminant effects on hemocytes parameters. in this context, attention has recently been addressed to the evaluation of the effects of nanoparticles (nps) on bivalve hemocytes. nps are a class of emerging contaminants, and the immune system of aquatic organisms is considered to be a sensitive target for these contaminants. in order to provide information concerning np toxicity in clam species, in this study in vivo effects of nzno, ntio2, c60 fullerene on haemocyte parameters of the clam ruditapes philippinarum were investigated for the first time. in addition, protein damage caused by exposure to a mixture containing the three nps was assessed using 1-d redox proteomics. clams were exposed for 7 days to two concentrations of each nps and the effects on various cellular and biochemical parameters were evaluated in clam haemolymph at time intervals (after 1, 3 and 7 days). results showed that ntio2 is more active in promoting modulation of haemocyte parameters than nzno and c60. indeed, in nzno-exposed clams only a significant increase in haemocyte proliferation and low levels of dna damage were observed. c60 exposure caused alterations in haemocyte diameter, volume and proliferation at the beginning of the exposure only, and increased slightly dna damage and neutral red uptake (nru) at the end of the exposure. conversely, ntio2 exposure significantly increased total haemocyte count, diameter and volume of hemocytes, haemocyte proliferation and dna damage. variations in both nru and cell-free haemolymph lysozyme activity were also observed. clams exposed to the mixture showed a high level of protein damage, probably due to npinduced oxidative stress. although preliminary, results of the present study can contribute to understand better the mechanisms of action of nps in r. philippinarum, and in bivalve molluscs in general. dopa-containing proteins in the compound ascidian botryllus schlosseri isj 12: 109-117, 2015 issn 1824-307x research report insights on cytotoxic cells of the colonial ascidian botryllus schlosseri n franchi, l ballarin, f cima department of biology, university of padua, padua, italy accepted march 2, 2015 abstract morula cells (mcs) represent the most abundant circulating hemocyte of the compound ascidian botryllus schlosseri. they are cytotoxic cells involved in the rejection reaction between contacting, genetically incompatible colonies. upon the recognition of foreign substances, they degranulate and release their content, which contribute to the cell death along the contact borders. a major role in mcrelated cytotoxicity is exerted by the enzyme phenoloxidase (po) that converts polyphenol substrata to quinones which, then, polymerize to form melanins. during this reaction, reactive oxygen species are formed which are the cause of mc-related cytotoxicity. here, we carried out new analyses to investigate further the nature of mc content and its role in cytotoxicity. results confirm that po is located inside mc vacuoles together with arylsulfatase, iron and polyphenols/quinones, the latter probably representing ready-to-use cytotoxic molecules, deriving from the oxidation of dopacontaining proteins. in addition, small dopa-containing peptides, called tunichromes, are also present inside mcs. mc degranulation and po-mediated cytotoxicity are prevented by secretion inhibitors and by h89 and calphostin c. the observation that po activity is always detectable in mcs in the absence of protease treatment, and its inhibition by sulfites and sulfates, suggest a non-classical pathway of po modulation in botryllid ascidians. key words: botryllus; ascidians; morula cells; phenoloxidase; immune response; cytotoxicity   introduction phenoloxidase-containing cells represent an ubiquitous hemocyte-type in ascidians, constituting one of the most abundant circulating cell-type, featuring the presence of cytoplasmic vacuoles containing the enzyme phenoloxidase (po) in an inactive form. these cells are involved in various biological processes, such as wound healing, tunic synthesis and cytotoxicity (goodbody, 1974; wright, 1981; oltz et al., 1987; cammarata et al. 1997; ballarin and cima, 2005). in many ascidian species, phenoloxidase-containing cells are known as morula cells (mcs), for the berry-like morphology they assume after fixation (see ballarin, 2012 for references). according to various authors (smith and söderhäll, 1991; jackson et al., 1993; arizza et al., 1995; ballarin et al., 1998; shirae and saito, 2000; shirae et al., 2002; parrinello et al., 2003; cammarata et al., 2008), po is stored, analogously to arthropods (söderhäll and cerenius, 1998), as a pro-enzyme, although a clear demonstration is still lacking (ballarin, 2012). ___________________________________________________________________________ corresponding author: loriano ballarin department of biology, university of padua via ugo bassi 58/b 35121 padua, italy email: loriano.ballarin@unipd.it in the cosmopolitan compound ascidian botryllus schlosseri, colony specificity allows the discrimination between contacting genetically compatible and incompatible colonies. in the case of compatibility, colonies can fuse into a large chimeric colony with common tunic and circulation, whereas, when incompatible colonies are involved, they give rise to a non-fusion or rejection reaction characterized by the appearance of a series of necrotic foci along the contact area (rinkevich, 1992; sabbadin et al., 1992; saito et al., 1994). the non-fusion reaction is characterized by: i) partial fusion of the tunics along the contact borders, ii) crowding of a selected immunocyte population, represented by mcs, inside the lumen of the facing, sausage-like blind termini of the colonial vasculature, called ampullae, iii) fenestration of the apical ampullar epithelium and its crossing by mcs which degranulate and induce the observed cytotoxicity (sabbadin et al., 1992; ballarin et al., 1995, 1998). mcs are the effectors of the non-fusion reaction of botryllid ascidians and, once activated by the recognition of soluble unknown factors diffusing from the facing colony, they synthesize and release chemotactic molecules recognized by antimammalian-cytokine antibodies (cima et al., 2006). 109 mailto:loriano.ballarin@unipd.it fig. 1 mc under various experimental conditions. a: living cell; b: untreated, aldehyde-fixed cell; c: after eosin staining; d: after may-grünwald-giemsa staining; e: stained for arylsulfatase; f: stained for po; g: mbth-stained cells: h: redox-cycling stain for quinoproteins/dopa-containing proteins; i: immunopositivity to anti-tunichrome antibody; j: living cells degranulated in the presence of heterologous cfh; k: living cells in the presence of incompatible cfh and monensin. asterisks: unstained phagocytes. scale bar: 10 µm. as previously stated, they contain the enzyme po inside their vacuoles (frizzo et al., 2000) which is responsible of the cytotoxicity observed in the rejection reaction (ballarin et al., 1998, 2002; shirae and saito, 2000; shirae et al., 2002). in addition, simple cytochemical techniques suggest the presence, inside mc vacuoles, of po polyphenol substrata (ballarin et al., 1995; ballarin and cima, 2005), but the nature of these substances remains largely unknown. in the present paper, we report the results of new analyses on mcs to investigate further the nature of the vacuolar content. results obtained confirm that po is located inside mc vacuoles together with the enzyme arylsulfatase, iron, polyphenols/quinones and dopa-containing proteins/quinoproteins from which dopa-containing peptides, also present inside mc vacuoles and called tunichromes (oltz et al., 1987; taylor et al., 1997), probably derive. the enzyme activity is inhibited by sulfites and sulfates; po is released upon mc degranulation as inhibition of exocytosis prevents the po-related cytotoxicity. materials and methods animals colonies of botryllus schlosseri from the lagoon of venice were used. they were kept in aerated aquaria, attached to glass slides and fed with liquifry marine (liquifry co., dorking, england) and baker’s yeast (saccharomyces cerevisiae). 110 fig. 2 percentage of degranulated mcs at various incubation temperatures. asterisks mark significant differences with respect to the exposure in fsw or, when indicated, between different temperatures. **: p < 0.01; ***: p < 0.001. hemocyte collection hemolymph was collected with a glass micropipette after puncturing, with a fine tungsten needle, the tunic marginal vessel of colonies previously rinsed in 0.38 % na-citrate in filtered seawater (fsw), ph 7.5, to prevent clotting. it was then centrifuged at 780xg for 10 min and the pellet was re-suspended in fsw at the final concentration of 5x106 cells/ml. sixty µl of hemocyte suspension were placed in the centre of culture chambers, prepared as described elsewhere (ballarin et al., 1994), and left to adhere to coverslips for 30 min at room temperature. cytochemical and immunocytochemical assays on hemocytes staining with eosin and may grünwald-giemsa hemocyte monolayers were fixed for 30 min at 4 °c with a solution of 1 % glutaraldehyde, 1 % sucrose and 1 % caffeine, to prevent the release of the vacuolar content (ballarin and cima, 2005), in fsw. they were then stained with 2 % eosin for 5 min and rinsed in phosphate-buffered saline (pbs: 0.8 % nacl, 0.02 % kcl, 0.02 % kh2po4, 0.115 % na2hpo4, ph 7.2). alternatively, hemocytes were stained with commercial may grünwald solution (merck) for 2 min, followed by 5 min in 10 % giemsa solution in distilled water. coverslips were finally mounted on glass slides with aqueous medium (acquovitrex, carlo erba, italy) and observed under the light microscope (leitz, dialux 22) at the magnification of 1250x. arylsulfatase fixed hemocyte monolayers were immersed in 0.1 m sodium acetate buffer (ab) containing 1 % sucrose, ph 5.2, for 5 min. cells were then incubated for 60 min at 37 °c in a reaction mixture prepared by adding 8 mg/ml of p-nitrocatechol sulfate to a solution made by ab, water and a 8 % lead nitrate in volumetric ratio of 6:2:2 (goldfischer, 1965). after thorough washing in distilled water, hemocytes were treated with 1 % ammonium sulfide for 1 min and washed repeatedly in water. coverslips were finally mounted as described before and observed under the light microscope. positive sites appeared dark. phenoloxidase (po) fixed hemocyte were incubated for 60 min in a saturated solution of l-dopa (fluka) or 2 mm catechol in pbs. after a final washing in pbs, coverslips were mounted on glass slides with acquovitrex. positive sites appeared dark brown (hose et al., 1987). polyphenols/quinones living hemocytes were incubated in a 2-mm solution of 3-methyl-2-benzothiazolinone hydrazone chloride (mbth) in fsw, containing 0.4 % dimethylformamide for 5 min. the compound reacts with polyphenols and quinones giving a red product (gasparič et al., 1977; winder and harris, 1991; ballarin et al., 1995). cells were then washed in fsw and coverslips were placed, upside down, on a teflon ring (15 mm internal diameter, 1 mm thick), glued on a siliconized glass slide, smeared with vaseline and observed under the light microscope. dopa-containing proteins/quinoproteins after the adhesion to coverslips, hemocytes were fixed as described above, washed in pbs and incubated in a solution of 0.24 mm nitroblue tetrazolium (nbt; sigma) in 2-m potassium glycinate buffer (15 % glycine and koh 2 m to ph 10.0) and 20 mm sodium benzoate (flückiger et al., 1995). cells were then washed in pbs and coverslips mounted with acquovitrex. positive sites featured a dark blue color. 111 tunichrome hemocytes were fixed for 30 min in a solution of 4 % paraformaldehyde and 0.1 % glutaraldehyde in 0.4 m sodium cacodylate buffer, ph 7.4, containing 1 % caffeine. they were then washed in pbs, incubated 30 min in 3 % h2o2 in methanol to block endogenous peroxidase activity, washed again, and kept 30 min in a solution of 3 % powdered milk in pbs. cells were then incubated overnight in a rabbit polyclonal antibody raised against the tunichrome of the solitary ascidian phallusia mamillata (bayer et al., 1992; taylor et al., 1997), a generous gift of prof. s. scippa, university of naples, diluted 1/200 in pbs, washed and incubated in secondary, anti-rabbit igg antibody conjugated with biotin (calbiochem), diluted 1/100 according to the manufacturer’s instructions, for 30 min. hemocyte monolayers were washed again, and treated with avidin-biotin-peroxidase complex (abc, vector) for 30 min. positivity was revealed by incubation, for 5 min, in a solution of 0.025 % 3-3’ diaminobenzidine (dab, sigma) in pbs. the primary antibody was omitted in control slides. after a final washing, coverslips were mounted on glass slides with acquovitrex. evaluation of the cytotoxic effect of cell-free hemolymph (cfh) under various conditions hemolymph was collected as described above, from colonies which were previously blotted dry with filter paper, and centrifuged at 780xg for 10 min. the supernatant was then collected, referred to as cfh and used as incubation medium for both autologous (i.e., from the same colony) and heterologous (i.e., from incompatible colonies) hemocytes, prepared as described above, in the presence or in the absence of the secretion inhibitors 4-acetamido-4'-isothiocyanatostilbene2,2'-disulfonic acid (sits; korchak et al., 1980) and monensin (kuivaniemi et al. 1986), at the concentration of 1 mm and 1 µm, respectively, and of h-89 (10 µm) and calphostin c (1 µm), specific inhibitors of protein kinase a (pka; chijwa et al., 1990) and protein-kinase c (pkc; tamaoki, 1991), respectively. after 60 min of incubation, cells were washed with fsw, incubated in a solution of 0.25 % trypan blue in fsw for 5 min at room temperature and observed in vivo under a leitz dialux 22 light microscope (lm) at 1250x. dying hemocytes, with altered plasma membrane permeability, assumed a blue color. the percentage of blue cells was expressed as the cytotoxicity index. in another series of experiments, heterologous cfh was incubated for 30 min at 37, 45, 60, 80 °c and for 10 min at 100 °c before its use as incubation medium for hemocytes and the effects of the incubation at different temperatures on mc degranulation was then evaluated with the trypan blue assay. hemolymph collection and sds-page hemolymph from large colonies was collected and centrifuged as described above. the pellet was resuspended in lysis buffer (tris-hcl 50mm, ph 7.8, sucrose 0.25 m, sds 1 %, edta 5 mm, pepstatin 1 lg/ml, leupeptin 1 lg/ml, sodium orthovanadate 2 mm, naf 0.01 m, nonidet p-40 0.1 %, nethylmaleimide 5 mm, phenylmethylsulfonylfluoride 0.04 mg/ml). cell suspensions were then sonicated 2 min with a braun labsonic sonifier at 50 % duty cycles at 0 °c. cell lysates were boiled for 5 min, centrifuged at 12,000xg for 15 min and supernatants were diluted with an equal volume of sample buffer (0.5 m tris-hcl, 2 % sds, 10 % glycerol) and loaded in the wells of 12 % tris-glycine sds-page slab gels. each well of the gel received a volume of supernatant equivalent to 20 µg of protein. electrophoresis was run at the constant current of 10 ma/gel and gels were stained with the redoxcycling method for dopa-containing proteins according to paz et al. (1991). spectrophotometric assay for po activity hemolymph was collected as described above in the absence of anti-clotting agents. it was centrifuged as described above and the pellet was re-suspended in pbs. it was then subjected to sonication, as already described, for 1 min and centrifuged at 10,000xg for 10 min to obtain cell lysate. fifty μl of lysate were incubated with 950 µl of a saturated solution of l-dopa in pbs in the absence (control) or in the presence of na2so3, na2so4, reduced glutathione, 2-mercaptoethanol and dithiothreitol (dtt), at 1 mm concentration, and the reaction was followed for 5 min at 490 nm. one unit (u) of po activity determines an increase of 0.001 absorbance units/min at 25 °c (söderhäll and smith, 1983). results are expressed as percentage of inhibition of the po activity with respect to control. quantitative microanalysis for iron in epoxy sections colonies, fixed in 1.7 % glutaraldehyde buffered with 0.2 m sodium cacodylate plus 1.7% nacl at ph 7.4, were dehydrated and embedded in epoxy resin. thick sections (1 µm) of b. schlosseri blood lacunae were obtained with a lkb ultrotome. chemical analyses were done on sections with a cameca-camebax electron microprobe with a finefocused beam (1 μm diameter) operating in the wavelength-dispersive (wds) mode using ferrosilite as a synthetic end-member mineral standard for fe. operating conditions were 15 kv accelerating voltage and 25 na beam current; counting times were 20 s for peaks and 20 s for backgrounds. statistical analysis each experiment was carried out in triplicate. data are expressed as mean ± sd. they were compared with the χ2 test. results morphology of botryllus schlosseri mcs mcs are the most abundant circulating cells, their concentration amounting to 40 60 % of total hemocytes, depending on the colony, without significant variation in the course of the colonial blastogenetic cycle. they have a diameter of 10 15 μm and they assume, after aldehyde fixation, a typical berry-like morphology (figs 1a b) due to the presence of many vacuoles, about 2 μm in diameter, which occupy most of the cell volume. the vacuolar content assume a yellowish color after aldehyde fixation (fig. 1b). 112 fig. 3 cytotoxicity index for hemocytes exposed to fsw, autologous and heterologous cfh. in the last two cases, the effects of the addition of monensin, sits, h89 and calphostin were also evaluated. significant differences with respect to the exposure to fsw were marked by asterisks. ***: p < 0.001. cytochemical and immunocytochemical properties of mcs mcs assume a pinkish color when stained with eosin or may grünwald-giemsa(figs 1c, d). they resulted positive to the arylsulfatase assay and the reaction product was located inside the vacuoles (fig. 1e). in addition, the vacuolar content of mcs was able to oxidize polyphenol substrates such as l-dopa and catechol (fig. 1f). mc also reacted with mbth: in this case, vacuoles acquired a red color (fig. 1g). at alkaline ph and in the presence of potassium glycinate and nbt, vacuoles of most mcs assumed a dark blue color; no other cell-type showed positivity to these assays (fig. 1h). a similar result was obtained with anti-tunichrome antibody which recognized only mcs (fig. 1i). iron in hemocytes the electron microprobe, when focused on mc vacuoles, revealed the presence of iron at a concentration up to 90,000 times that of seawater (0.012 mg/l), with a mean value of 687 ± 28 mg/l. no detectable iron was evidenced when the beam was directed towards other cell types. effects of incubation of hemocytes in cfh from incompatible colonies when hemocytes were exposed to cfh from heterologous colonies, living mcs changed their morphology: they degranulated and vacuoles reduced to small vesicles with a brown pigmentation due to residual po activity (fig. 1j). the level of mc degranulation observed at 25 °c was significantly (p < 0.001) decreased by exposure of cfh at temperatures higher than 80 °c (fig. 2). in the presence of monensin, sits, h89 and calphostin c, no degranulation was observed (fig. 1k). a significant (p < 0.001) increase in the frequency of blue hemocytes, having assumed trypan blue as a consequence of altered membrane permeability, with respect to cells exposed to fsw or autologous cfh, was observed in hemocytes challenged with heterologous cfh. this increase was abolished when monensin, sits, h89 or calphostin c were added to heterologous cfh (fig. 3). electrophoretic analysis of hemocyte lysate when hemocyte lysate was subjected to sdspage analysis, and the gel was stained with nbt in potassium glycinate buffer, at alkaline ph, two bands of approximately 48 and 37 kda appeared (fig. 4). po activity of hemocyte lysate hemocyte lysate had a po activity of 0.044 ± 0.002 u. na2so3, na2so4, reduced glutathione, 2mercaptoethanol and dtt completely inhibited the po activity when added to the incubation mixture (data not shown). discussion invertebrate immune responses rely only on natural immunity and, in many cases, the recognition of non-self molecules triggers the 113 degranulation of circulating cells and the release of po. po is a copper enzyme able to convert polyphenols to quinones which, in turn, polymerise to melanin. it can induce cytotoxicity through either the production of reactive oxygen species and the induction of oxidative stress or the rapid conjugation of quinones with -sh groups on essential molecules (kato et al., 1986; passi et al., 1987; halliwell and gutteridge, 1999). in ascidians, po-containing cells (known as mcs in most cases) are involved in cytotoxic reactions against foreign cells or molecules, in both solitary and colonial species (akita and hoshi, 1995; cammarata et al., 1997; ballarin et al., 1998; hata et al., 1998; shirae and saito, 2000; shirae et al., 2002; parrinello et al., 2003; ballarin et al., 2005). mcs play a key role in the rejection reaction between contacting, incompatible colonies of botryllid ascidians (taneda and watanabe, 1982; ballarin et al., 1995; hirose et al., 1997; shirae and saito, 2000; shirae et al., 2002; cima et al., 2004). although it has been clearly demonstrated that po is directly involved in the induction of the cytotoxicity observed along the contact border (ballarin et al., 1995, 1998), uncertainties still persist on the sequence of molecular events controlling po activation in these organisms. cytoenzymatic assays demonstrate that the enzyme is located inside mc (frizzo et al., 2000, this report) and is released in the medium as mcs degranulate. degranulation is mediated by ion fluxes as demonstrated by its inhibition in the presence of the monovalent ionophore monensin and the anionchannel-blocking agent sits, in agreement with results obtained in human fibroblasts (uchida et al., 1979; kuivaniemi et al., 1986), human neutrophils (korchak et al., 1980), mouse pituitary cells (heisler and jeandel, 1989) and pollen tube (speranza and calzoni, 1992). in addition, degranulation appears to be mediated by transduction pathways triggered by activated g protein-coupled receptors, such as those involving pka and pkc (gomberts et al., 2002), as indicated by the inhibition of degranulation by h89 and calphostin c. the in vitro inhibition of cytotoxicity in the presence of the above-reported inhibitors is likely related to the inhibition of the release of po and its consequent absence in the surrounding medium. immunocytochemical analysis indicates the presence of tunichromes in mc vacuoles. tunichromes are polyphenol compounds of low molecular weight, with reducing activity, present inside mc vacuoles of various ascidians (oltz et al., 1987; bayer et al., 1997). similar molecules, from insect and ascidian hemolymph, were demonstrated to exert antimicrobial activity (meylears et al., 2002; cai et al., 2008). according to taylor et al. (1997), tunichromes represent fragments of larger dopacontaining proteins which likely are the natural source of po substrates. these molecules and their quinone derivatives have been effectively revealed inside mc vacuoles by redox-cycling in nbtglycinate buffer and are responsible of the red color observed inside vacuoles of mcs after the treatment with mbth. the presence of quinones inside circulating cells has been already reported in other invertebrate species, such as echinoderms fig. 4 sds-page of hemocyte lysates. lane 1: molecular markers stained with coomassie blue; lane 2: staining for quinoproteins. (kennedy, 1979). the role of these molecules is not clear: they probably act as ready-to-use cytotoxic molecules able to induce an oxidative stress after their release through their reaction with biological molecules or polymerization to melanin (nappi and vass, 1993). in some arthropods, a role of secreted quinones in defence against predators has been reported (machado et al., 2005). the rejection reaction of b. schlosseri shares many features with vertebrate inflammation, including the selective recruitment of a hemocyte type, its extravasation and its degranulation with the induction of cytotoxicity (sabbadin et al., 1992). we already demonstrated that the recognition of soluble factors diffusing from the contacting heterologous colony through the partially-fused tunics, activate the synthesis and the release, by mcs, of molecules able to induce their chemotaxis and crowding inside the ampullae facing the contacting colony, their migration through the ampullar epithelium into the tunic and their degranulation with the consequent release of po which is responsible of the observed cytotoxicity (cima et al., 2004, 2006). the podependent cytotoxicity is also observed in vitro, upon the exposure of hemocytes to the incompatible factors contained in heterologous cfh (ballarin et 114 al., 2002, 2005). our results indicate that these factors are relatively thermostable and are active in inducing degranulation when stored at a temperature range from 37 to 80 °c. a similar optimum storage temperature range was reported for the cfh of the japanese colonial ascidian botrylloides simodensis which is capable to induce non-fusion reaction when injected in recipient incompatible colonies (saito and watanabe, 1984). previous results indicate that po-driven cytotoxicity is due to the induction of oxidative stress, related to the production of superoxide anions in the course of the oxidation of polyphenols (ballarin et al., 2002). the production of reactive oxygen species is greatly increased in the presence of iron which acts as a catalyser of enzymatic reactions leading to the production of peroxides and hydroxyl radicals (halliwell and gutteridge, 1999). our analysis confirm the presence of iron inside mc vacuoles, already stressed by previous results (milanesi and burighel, 1978; ballarin et al., 1995), at a concentration much higher than that in the environment, indicating an active accumulation of the metal that is probably released during the degranulation event and facilitates the induction of the oxidative stress and the consequent cell death. it is generally assumed that ascidian pos, analogously to arthropod pos (söderhäll and cerenius, 1998), derive from the proteolysis of a zymogen (smith and söderhäll, 1991; jackson et al., 1993; arizza et al., 1995; ballarin et al., 1998; shirae and saito, 2000; shirae et al., 2002; parrinello et al., 2003; cammarata et al., 2008). however, in our cytoenzymatic assays, no previous treatments with proteases are required to detect po activity as the enzyme is already active, at least in part, inside mc vacuoles. a recent analysis of the aminoacidic sequence of botryllus po failed to reveal the presence of a prodomain and a cleavage site recognized by serine proteases, analogous to those found in arthropods, in agreement with the idea that some other mechanisms, different from the enzymatic cleavage, should be considered to explain the state of low activity of po inside mc vacuoles (ballarin et al., 2012). po activity is completely inhibited by reducing substances, such as reduced glutathione, 2mercaptotethanol and dithiothreithol, which probably prevent the oxidation of the polyphenol substrata: this raise the possibility that polyphenols themselves can act as reducing substances preventing or limiting po activity. in addition, they can be present in a “masked” form inside mc vacuoles, linked to sulfites or sulfates, that inhibit po activity (present results), already demonstrated inside mcs (ballarin et al., 1995). sulfates or sulfites can be removed by arylsulfatase, also found inside mc vacuoles, upon cell activation thus making the substrates available to po. alternatively, the acidic ph of mc vacuoles, revealed by their pinkish staining by eosin and may grünwald-giemsa, can inhibit po activity which becomes evident when, once degranulation has occurred, the enzyme is released in the medium characterized by a higher ph. these points need further investigations. therefore, future studies will be directed towards a better understanding of the relationship between po, arylsulfatase, and dopacontaining proteins/quinoproteins in botryllus cytotoxicity. acknowledgements we wish to remember prof. scippa s (died on 2012), from the university of naples, for her encouragement and the generous gift of the antitunichrome antibody. thanks are due to prof. molin g, department of geosciences, university of padua, for his help in microanalysis. this research was supported by the italian m.i.u.r. 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(pamps), in the transduction of immune signaling and in the elimination of potentially pathogenic microbes in gastropod and bivalve molluscs. a major focus will be put on the differences and peculiarities of the molecular immune system of the two major molluscan classes, which have developed specific adaptations to cope with diverse living environments, pathogenic and nonpathogenic microbes over the course of several hundred million years of independent evolution. intriguing but still poorly understood aspects, such as antiviral response and immune priming, will be also explored, highlighting the present challenges and opportunities connected to the application of modern genomics techniques to the study of the immune system in these fascinating metazoans. key words: mollusca; bivalves; gastropods; innate immunity; lectins; antimicrobial peptides; antiviral response introduction the phylum mollusca is one of the largest and most diverse group of metazoans, only second to arthropoda in number of described species. molluscs are highly heterogeneous in terms of size, morphology, adaptations to diverse habitats (terrestrial, freshwater and marine) and feeding and behavior, reflecting the massive radiation this phylum underwent during the cambrian period. the phylogeny of molluscs is still a matter of debate, as many different but equally plausible hypotheses have been proposed over the years, either based on morphological features, molecular data, or both. the reasons behind the apparently unsolvable topology of the molluscan tree of life are multiple and mainly need to be sought in narrow taxonomical sampling, focus on specific target clades and lack of appropriate outgroups (sigwart and lindberg, 2015). while molecular studies have long been expected to disentangle the complex evolutionary relationships among molluscs, this task still appears far to be accomplished and poses a great challenge for the interpretation of the results of comparative studies. ___________________________________________________________________________ corresponding author: marco gerdol university of trieste department of life sciences via giorgieri 5, 34127 trieste, italy e-mail: mgerdol@units.it despite these uncertainties, most of the over 100,000 estimated extant species are currently classified within two main classes, gastropoda and bivalvia. the former is thought to comprise more than 80 % living molluscs, including species adapted both to terrestrial and aquatic life, while the latter strictly includes aquatic organisms, altogether accounting for ~14 % of the described species (nicol, 1969). the remaining six classes (scaphopoda, cephalopoda, aplacophora, monoplacophora and polyplacophora), despite their unique peculiarities and key position for evolutionary studies, overall only account for less than 2 % of all extant mollusc species. the two major molluscan classes are likely to have diverged from each other during the cambrian explosion and are therefore separated by over 450 million years of independent evolution. although a broad range of species, from chitons, to tusk shells, land snails, squids, mussels and oysters are all classified under the same taxonomical umbrella as molluscs, such a large time of divergence permitted the development of highly specialized, novel genetic and morphological adaptations. all molluscs rely on a similar cellular and molecular framework of immune cells and molecules for surviving in challenging environments, often rich of potentially pathogenic microorganisms. in particular, free-living bacteria and viruses in seawater can reach considerable concentrations, in 104 the range of 10-6 and 10-7 ml-1, respectively (bezdek and carlucci, 1972; drake et al., 1998), posing a great challenge for filter-feeding animals, including bivalves. the powerful innate immune system of molluscs, which comprises a broad array of soluble and membrane-bound pattern recognition receptors (prrs) for the recognition of pathogens, a finely regulated cytosolic signaling machinery and a battery of effector molecules aimed at eliminating invading microbes and recruiting specialized immune cells at the site of infection, is partially shared by other large invertebrate phyla which, unlike vertebrates, are not equipped with an adaptive immune system. far from being simple and less developed than in vertebrates, the invertebrate immune system has followed slightly divergent evolutionary routes in different taxa over the past few million years. this complex evolutionary process, driven by environmental selective pressure, has led the development of remarkable and diverse immune capabilities, which are still poorly understood (loker et al., 2004). similarly, the ancient split between gastropods and bivalves, as well as their adaptation to different habitats and lifestyles, determined the evolution of unique features in their immune system. while extensive works have been carried out to document the genetic and molecular background of both gastropod (loker 2010, coustau et al., 2015) and bivalve (gerdol and venier, 2015; song et al., 2015; zhang et al., 2015) innate immune system, little attention has been paid so far at providing a comparative account of the differences between the two major molluscan classes. indeed, important observations provided more than a decade ago concerning differences in the agglutinating and opsonizing activity of bivalve and gastropod plasma have not been followed by extensive molecular comparative research. yakovleva and colleagues (2001) described these two alternative defense strategies as “lowpromiscuous” and “high-promiscuous” for gastropods and bivalves, respectively. it was hypothesized that, while the former comprised a set of plasma lectins with a broad spectrum of carbohydrate recognition, the latter mainly relied on plasma lectins with narrow binding specificity. it was also observed that the rate of phagocytosis in gastropods was highly dependent on the concentration of plasma lectins, as opposed to bivalves, where opsonization was likely triggered by membrane-bound lectins. as it will be pointed out below, these definitions still appear to be valid, and closely mirror the situation which is starting to emerge from the analysis of the large-scale molecular data collected over the past 15 years. this review will focus on this particular comparative aspect, exploring the massive amount of literature recently produced on this topic and presenting molecular data in a genomic context, whenever possible. unless differently stated, data concerning the size and the sequence diversity of immune-related gene families will be inferred from the available molluscan genomes as of march, 2017. namely, the reference genomes taken into account in this study are: crassostrea gigas v.9 and pinctada fucata v.2.0 for bivalve molluscs; lottia gigantea v.helro1, aplysia californica v.3.0 and biomphalaria glabrata v.asm45736v1 (unpublished) for gastropod molluscs. molluscan lectins: a molecular repertoire characterized by lineage-specific expansions and extreme diversification ever since the first molecular studies carried out in the early ‘80s it became evident that the immune response of molluscs largely relied on the production of a large number of soluble proteins with marked carbohydrate binding properties, acting as prrs (miller et al., 1982; renwrantz, 1983; pipe, 1990; fisher and dinuzzo, 1991). while most efforts have been put into their isolation of these molecules from hemocytes, the central players in molluscan cellular immunity, considerable results have been also attained from the mucus of land snails, which covers their entire bodily surface, acting as a barrier from a potentially pathogen-rich environment. like gastropod skin, bivalve gills and mantle represent main tissues of interface with the external environment, they are covered by mucus and constantly exposed to microbes present in the water column and conveyed to the intervalvar space by filter-feeding. while the majority of lectin-like proteins of bivalves have been described and isolated from hemolymph, the important role of bivalve pallial tissues in immune recognition is starting to emerge, as evidenced by the gillsor mantle-specificity of several prrs (gourdine and smith-ravin, 2007; jing et al., 2011). it is now clear from genome and transcriptome data mining that molluscan lectin-like molecules are extremely variable, both in terms of their carbohydrate recognition domain (crd), sugarbinding properties, and sequence diversity among members of the same lectin family (gerdol and venier, 2015; gorbushin and borisova, 2015; zhang et al., 2015). the high molecular diversity and the remarkable plasticity of crds allow, even in absence of the genetic re-arrangements typical of the vertebrate adaptive immune system, a very broad spectrum of recognition for invertebrate lectins (vasta et al., 2007). over the past few decades, scientific literature has evidenced some remarkable differences in the repertoires of these molecules in bivalve and gastropod molluscs. while some of these apparent discrepancies could be simply explained by the higher amount of efforts put into immunological research of economically important bivalves, wholegenome analyses fully confirm that all the main lectin-like families show a different size and degree of diversification between these two molluscan classes, as reported in the comparative summary in figure 1. this divergent evolution might be linked to environmental factors (terrestrial vs freshwater vs marine) and adaptation to the associated microbiomes and pathobiomes, as well as to the development of alternative lowand highpromiscuity defense strategies, as previously proposed by other authors (yakovleva et al., 2011). the most evident case is certainly that of c1q 105 fig. 1 domain organization and size of the main lectin gene families in two representative species for bivalvia (the pacific oyster crassostrea gigas) and gastropoda (the owl limpet lottia gigantea). domain containing (c1qdc) proteins, which are present in hundreds different variants in bivalves, compared to the few (usually less than 20) gene copies found in gastropods. the c1q domain takes its name from the homonymous protein complex of the vertebrate complement system, whose three main components are characterized by the presence of the globular head domain c1q. the astounding plasticity and binding properties of this domain led to an extraordinary evolutionary success in metazoans (carland and gerwick, 2010), as highlighted by the nearly 6,500 c1qdc protein sequences deposited so far in public databases. in protostomes, c1qdc proteins have been linked on multiple occasions to a lectin-like activity, such as in the snail cepaea hortensis (gerlach et al., 2004). however, since this first report in 2004, very scarce references have been made in literature to gastropod c1qdc proteins as prrs, with the only exception of some abalone proteins linked to lipopolysaccharide (lps) and peptidoglycan (pgn) response (bathige et al., 2016). comparatively, 106 c1qdc proteins have been the target of a much higher number studies in bivalves (zhang et al., 2008; gestal et al., 2010; kong et al., 2010; xu et al., 2012), where they have been often linked to hemocyte-specificity, upregulation in response to multiple pamps, bacterial agglutination and growth inhibition. this disparity concerning the reports in the two major mollusk classes is certainly correlated to very low number of c1qdc proteins found in gastropod genomes, as opposed to the a massive gene family expansion that occurred in bivalves (fig. 1). this event, which is estimated to have brought to the development of almost 1,000 different paralogs in mytilus spp., only involved pteriomorphia and imparidentia, but not freshwater unionoids and the basal branch of protobranch bivalves (gerdol et al., 2015). despite the low number of c1qdc proteins, gastropods produce several other lectin-like molecules, as evidenced by a large scale transcriptomic survey of the hemocyte transcriptome of the periwinkle littorina littorea (gorbushin and borisova, 2015), even though, on average, their number at the whole genome level appears to be lower than in bivalves (fig. 1). c-type lectins are the second largest lectin family in the pacific oyster. these molecules are characterized by a clect domain which can be often associated with other conserved modules, creating a broad range of domain combinations. ctype lectins can cover multiple biological functions, but one of the best documented is innate immune recognition, such as in the case mannose-binding lectin, a key component of the lectin pathway of the vertebrate complement system (zelensky and gready, 2005). the role of c-type lectins in the molluscan immune system has also been clearly established and thoroughly investigated (wang et al., 2011). in gastropods, the abalone hdhctl is involved in the agglutination of gram-negative bacteria (zhang et al 2014) and incilarins have been shown to be major components of the mucus of the land snail meghimatium fruhstorferi (yuasa et al., 1998). in bivalves, the immune role of c-type lectins has been best defined in scallops. different proteins, characterized by the presence of one to four clect crds, function both as prrs with a broad spectrum of recognition (lps, pgn, mannose and galactose) and as opsonins capable of enhancing the phagocytic activity of hemocytes (mu et al., 2012; huang et al., 2013). interestingly a ctype lectin of the eastern oyster crassostrea virginica has been implicated in mucosal immunity, due to its expression in the epithelial mucocytes of pallial organs (jing et al., 2011) and, similarly, codakines have been implicated in the recognition of pathogenic or symbiotic bacteria in the gills of codakia orbicularis (gourdine and smith-ravin, 2007). fibrinogen-related proteins (freps) constitute the third molluscan lectin family in terms of number of sequences per genome. like c-type lectins, freps also find their counterpart in key molecules of the lectin pathway of the vertebrate complement system, ficolins. the primary role of freps in invertebrates has been demonstrated to be linked to immune defense (hanington and zhang, 2011), as it had been already suggested by their isolation in the mucus gland of land slugs in the late ‘90s (kurachi et al., 1998). the most remarkable differences between bivalve and gastropod freps involve the somatic mutations that occur in the latter, an aspect which will be discussed in detail in the next section. many other lectins have been implicated in the immune recognition system of molluscs and, despite pertaining to smaller gene families compared to c1qdc, c-type lectins and freps, they almost invariably appear to be more diversified in bivalves than in gastropods. among these, the multifunctional f-type lectins are certainly worth of a mention as they have been linked to pamp recognition in bivalves (chen et al., 2011). despite its small size, the galectin gene family has been studied in detail (fig. 1). compelling evidence has been indeed produced linking the up-regulation of galectins to infection by perkinsus spp. (tasumi and vasta, 2007; kim et al., 2008) and various types of bacteria (zhang et al., 2011; maldonado-aguayo et al., 2014). in stark contrast with the carbohydrate-binding proteins reported so far, h-type lectins have been only studied in gastropods, whereas no information has been provided so far in bivalves. these constituents of the perivittelin fluid protect the fertilized eggs of snails from bacterial infections (gerlach et al., 2005; sanchez et al., 2006). although suel rhamnose/galactose-binding lectins cover a very similar role in sea urchin, they have been only marginally studied in molluscs so far, with the lone report of a gram-negative bacteria agglutinating protein in the mantle of the pearl oyster pteria penguin (naganuma et al., 2006). the field of lectinomics is in constant expansion and the popularity of high throughput sequencing technologies is bringing a new impulse to the discovery of novel molecules with carbohydratebinding potential in non-model species (gorbushin and borisova, 2015). for example, apextrin-like proteins, which have been recently described as novel pgn sensors in amphioxus (huang et al., 2014), have also been unveiled as prrs in mussels (estévez-calvar et al., 2011). in this case, the occasional association of the apextrin domain with a macpf domain would combine pamp-binding and pore-forming activities within the same protein product (gerdol and venier, 2015). interestingly, such a functional combination, reminiscent of the link between the lectin pathway and the terminal pathway of the complement system, has been reported in two other cases in molluscs. the first one involves a defense system from the eggs of the snail pomacea canaliculata, which uses a complex between a macpf poreforming and a limulus lectin l-6-like subunit for microbe recognition and killing (dreon et al., 2013). however, unlike mussel macpf/apextrin proteins, in this case the pore-forming and pamp-binding activities are provided by two distinct proteins encoded by two different genes. the second case is that of mytilectins, a multigenic family identified in mytilus galloprovincialis, which is characterized by a ricin-b 107 fig. 2 panel a: schematic domain organization of freps in mollusca. panel b: three-dimensional modeling of the fibrinogen-like domain of biomphalaria glabrata frep3. fbg: fibrinogen; ig: immunoglobulin. like fold. although the structure of the sugar-binding domain of these molecules vaguely resembles that of an agglutinin described in gastropods (arreguínespinosa et al., 2001), mytilectins: (i) are taxonomically restricted to mytiloids and a few other bivalve groups and (ii) sometimes display a cterminal aerolysin-like pore-forming domain (hasan et al., 2016). the very same structural pore-forming motif is also present in biomphalysins from land snails, although in this case there is no association with a lectin-like domain (galinier et al., 2013). clearly, additional studies will be needed to establish whether these hybrid proteins provide a functional equivalent to the terminal pathway of the complement system, which only appears to be present as a primitive version in protostomes (nonaka and kimura, 2006). at the same time, it is still largely unclear how the large arsenal of secreted molluscan prrs can transmit signals of infection within the cell and coordinate the cellular immune response. somatic diversification: towards a definition of immune memory in invertebrates? as briefly mentioned in the previous section, proteins containing a c-terminal fibrinogen-like domain (freps) are among the most important and best studied families of prrs in molluscs. the structural similarity between the c-terminal domain of mammalian fibrinogen and the horseshoe crab tachylectin first revealed that this three-dimensional fold (fig. 2) is shared by molecules involved in innate immunity and blood clotting, which therefore likely originated from a common ancestor. so far freps have been mostly studied in gastropods, and specifically in the snail b. glabrata, where they play a fundamental role in conferring resistance to infections by the digenean trematode schistosoma mansoni (gordy et al., 2015). this fascinating and complex host/pathogen relationship is characterized by patterns of resistance and susceptibility to infection which are governed by the somatic mutation of snail frep sequences (zhang et al 2004). since somatic mutations had long been long thought to be confined to vertebrate immune systems, this phenomenon has attracted a considerable attention, making freps the best candidate molecules for immune memory in invertebrates (milutinović and kurtz, 2016). indeed, it has been demonstrated that somatic mutation in freps could permit not just to establish, but also to maintain resistance to infection in snail populations (hanington et al., 2012). this is supported not just by the different susceptibility to infection of snails expressing different frep isoforms, but also by the acquisition of resistance towards secondary homologous infections through a mechanism of immune priming reminiscent of vertebrate adaptive immunity (portela et al., 2013). this idea was further reinforced by the observation of complexes between freps and highly variable mucin molecules produced by trematodes, in an interaction which could be seen as quite similar to that occurring between antigens and antibodies (moné et al., 2010). so far, somatic diversification has only been demonstrated for a small subclass of freps called igsf-freps which, in addition to the c-terminal fibrinogen-like domain, also contain one or two nterminal immunoglobulin-like domains (fig. 2) where the process of somatic mutation appears to take place (zhang et al., 2004). curiously, bivalves lack igsf-freps and, while similar proteins are present in aplysia, they are absent in lottia, revealing that they are likely to represent evolutionary innovations of heterobranch gastropods (gorbushin et al., 2010). although bivalve freps display a simpler domain organization (fig. 2) and lack significant homology to the sequences of b. glabrata, they are also extremely diversified. while events of allelic recombination or somatic mutation have been invoked to explain this remarkable sequence diversity (zhang et al., 2012), the high number of frep genes found in the oyster genome and in various transcriptomes of other species suggest that 108 the major driving force behind the hyervariability of bivalve freps is gene duplication, not somatic diversification (gerdol and venier, 2015; zhang et al., 2015). other authors have also revealed the presence of creps and greps, i.e., molecules whose structure is similar to that of igsf-freps, but whose n-terminal lectin-like region is either a c-type lectin or by a galectin domain (dheilly et al., 2015). while somatic diversification has not been observed yet for creps and greps, these molecules are absent in bivalves and therefore they probably also represent gastropod innovations. in bivalves, somatic mutation has been suggested, but not demonstrated yet in myticin c (vera et al., 2011), an antimicrobial peptide characterized by extreme levels of polymorphism and probably evolving under positive selection. however, in absence of definitive evidence, it is reasonable to assume that the factors underlying this sequence hypervariability might be linked to the extreme rate of heterozygosity of the mussel genome (murgarella et al., 2016). overall, while convincing evidence has been collected concerning the existence of immune somatic diversification in b. glabrata and possibly also in other gastropods where igsf-freps are present, the possibility that such mechanism of molecular diversification takes place also in bivalve mollusks still remains to be explored. however, as reported in the previous section, it is certainly noteworthy that lectin-like gene families underwent much more relevant expansion and diversification in bivalves, where somatic mutations have not been reported, compared to gastropods, where the lower sequence diversity at the whole genome level seems to be compensated by somatic diversification. immune memory in invertebrates is certainly a hot topic, which has been revolutionized by important discoveries in the past 20 years, which have introduced concepts such as immune priming and immune memory also in non-vertebrate metazoans. at the same time this aspect of invertebrate immunology is still poorly understood and applied research in this field is certainly complicated by the highly divergent strategies adopted by different phyla. as an example, the fascinating strategy adopted by arthropods to generate up to 10,000 different isoforms of the down syndrome cell adhesion molecule (dscam) (brites and du pasquier, 2015), is not used at all by other animals. as opposed to the presence of a hypervariable array of duplicated exons and complex alternative splicing patterns of insects, the genomic organization of dscam in molluscs is only consistent with the production of a single invariable protein product (gorbushin and iakovleva 2013). the broad repertoire of molluscan toll-like receptors converge in a highly conserved intracellular signaling pathway toll-like receptors (tlrs) have been recognized as central players in the innate immune response of vertebrates, where they have been implicated in the recognition of a broad range of bacterial, fungal and viral molecular patterns. in drosophila melanogaster, toll has a dual role in the determination of embryonic dorsal-ventral polarity and in the transduction of immune signaling in response to gram-positive bacteria upon the binding with the proinflammatory cytokine spätzle, which is in turn activated by an extracellular proteolytic cascade mediated by peptidoglycan recognition proteins (pgrps) and gram-negative binding proteins (gnbps). in many invertebrate organisms, tlrs have been linked with pathogen detection and the subsequent production of immune effectors through the activation of nf-b signaling, even though no spätzle-like cytokine has been identified yet in non-arthropod protostomes. genomic studies have further revealed that some invertebrates possess a very large repertoire of tlrs, as perfectly exemplified by the several hundred members identified in the sea urchin strongylocentrotus purpuratus (hibino et al., 2006). however, echinoderms are not an isolated case, as a similar number of these membrane-bound receptors have been found in other deuterostomes and protostomes, including the pacific oyster (zhang et al., 2015). compared to bivalves, gastropod genomes appear to possess a significantly lower number of tlr genes, ranging from 10 to 20 in the genomes which have been sequenced so far. despite this numerical difference, both bivalves and gastropods are characterized by the presence of two different sets of structurally divergent receptors which coexist (gerdol et al., 2017), i.e., single cysteine cluster (scc) and multiple cysteine cluster (mcc) tlrs. while the former type resembles vertebrate receptors due to the presence of a single set of nterminal and c-terminal-type leucine rich repeats (lrrs), the latter are more similar to drosophila toll, with two consecutive sets of nand c-terminal lrrs. so far no comprehensive functional study has been carried out to assess whether the high sequence diversity of molluscan tlrs mirrors a functional specialization in immune recognition, in embryonic development, or in both processes. however, an increasing number of reports suggest that at least some tlrs likely play a crucial role in mediating immune response upon infection. for example, mcctlrs have been linked to antibacterial and antiviral response in abalone (elvitigala et al., 2013) and a tlr of the snail b. glabrata was found to be strictly associated with specimens resistant to infections by the trematode s. mansoni (pila et al., 2016). convincing evidence concerning the immune function of tlrs and their role in regulating the production of amps in bivalves have been also produced by the observation of their up-regulation in response to experimental bacterial infections (toubiana et al., 2013; ren et al., 2014). surprisingly, in spite of the highly diversified repertoire of molluscan tlrs, a single molecule, named myd88, has been so far determined as they lone cytosolic partner and positive regulator of downstream intracellular immune signaling. although different myd88 isoforms have been characterized in bivalve and gastropod molluscs 109 (toubiana et al., 2013; ning et al., 2015), it seems quite unlikely that such a small number of intracellular proteins are able to mediate signals originated from hundreds different receptors, as such a massive diversification would allow the recognition of a potentially very broad range of ligands, requiring the subsequent production of specialized immune effectors. a recent genomic survey tried to fill this knowledge gap, pointing out that a very large amount of evolutionarily conserved cytosolic tirdomain containing (ectir-dc) proteins are present in bivalves, and enabling the possible characterization of novel tlr intracellular partners in the near future (gerdol et al., 2017). although the total number of ectir-dc proteins found in gastropods genomes is somewhat lower than that found in bivalves, some highly conserved gene families are present in both the two main mollusca classes. these also include sarm, a probable negative regulator of toll signaling. the other conserved tir-dc families identified in both gastropods and bivalves were: ectir-dc 2, 3, 5, 6, 8, 9, 11, 14 and 15. while the precise role of these conserved proteins remains unknown, their remarkable conservation across metazoans suggests an important function in the regulation of intracellular immune signaling, which might warrant future research in the near future. notwithstanding the elusive nature of the cytosolic interactors of tlrs, the downstream machinery responsible of cytosolic immune signal transduction appears to be well conserved in molluscs, mirroring almost perfectly that of vertebrates and showing a less relevant overlap with the simpler pathway of drosophila (fig. 3). in vertebrates, the first complex activated by myd88 and by other related adaptors is composed by the serine/threonine kinases irak1, irak2 and irak4, which associate with tnf receptor associated factor (traf) 6. this evolutionarily conserved protein, despite having been identified in multiple bivalve species, has never been characterized so far in gastropods (he et al., 2013; toubiana et al., 2014). nevertheless, genome data clearly reveal that traf6 is present as a single copy gene in l. gigantea, a. californica and b. glabrata. irak kinases on the other hand represent one of the few cases of significant divergence between vertebrates and mollusks: in particular, while proteins similar to irak4 have been described in abalone (ge et al., 2011) and mussels (toubiana et al., 2014), no sequence displaying convincing homology with irak1 and irak2 is encoded by molluscan genomes, leaving the molecular partners of irak4 and traf6 (if any) presently unknown. the next step of signaling involves the transforming growth factor-β activated kinase 1 (tak1) which, together with the two associated proteins tab1 and tab2, is well conserved also across all metazoans. the activated tak1 complex subsequently phosphorylates and activates the ib kinase (ikk) complex, which is composed by ikk and ikk, whose homologs have been identified in m. galloprovincialis and p. fucata (xiong et al., 2008; toubiana et al 2014). alternatively, tak1 can also activate the mapk pathway (not shown in fig. 3), which ultimately leads to the activation of the ap1 transcription factor complex in the nucleus, triggering the expression of various immunity and stress-related genes (gerdol and venier, 2015). the inhibitor of nf-b (ib) is the key molecule in the entire intracellular immune signaling cascade, as is it binds and sequesters nf-b in the cytosol. this inhibition can be reversed by phosphorylation by the ikk complex described above, which permits the dissociation between the transcription factor and its inhibitor. ib is also perhaps the molecular component of the pathway which has so far been the object of the most studies, both in bivalves (zhang et al., 2009; mu et al; 2010; valenzuelamuñoz and gallardo-escárate, 2014) and in gastropods (kasthuri et al., 2013; zhang et al., 2014). finally nf-b, whose homologs have been reported in different molluscan species (jiang and wu, 2007; huang et al., 2012; li et al., 2015), can migrate to the nucleus, where it turns on the expression of pro-inflammatory genes and immune effectors, such as amps (fig. 3). overall, while most of the key components of the canonical intracellular signaling pathway activated downstream of tlrs seem to be well conserved between bivalve and gastropod molluscs, it remains to be investigated whether the high number and diversity of tlrs can trigger parallel intracellular signaling routes which are not shared with vertebrates. as briefly mentioned above, the remarkable sequence diversification of tlrs and the high number of intracellular tir-dc proteins in these organisms could, at least in line of principle, allow a tailored immune response with the production of highly specific immune effectors. as this response is likely to be driven by the interaction between tlrs and tir-dc adaptors alternative to myd88, the diversity of such molecules between bivalve and gastropod molluscs is certainly a topic worth of attention. antiviral response: sting as a major cytosolic viral sensor while the molecular mechanisms adopted by molluscs and other invertebrates to manage infections by bacteria and eukaryotic parasites are starting to be unveiled, the genetic basis of their antiviral immunity remains nearly completely unknown. the reasons of this poor knowledge are multiple and mostly linked to the somewhat elusive nature of molluscan viral pathogens, their difficult isolation, identification and characterization. only in the very recent years a series of technological improvements have finally permitted to gain an in depth view of mollusc-associated virome through the application of next generation sequencing technologies, but this field can still be considered to be at its very early stages. taking into account the important economic losses inked to virus-induced mass mortalities in mollusc species of commercial interest (barbosa solomieu et al., 2015), this field of study will most likely encounter a great expansion in the years to come. o t her aut hor s hav e al r eady pr ovi d ed an 110 fig. 3 overview of the canonical intracellular immune signaling pathway activated downstream of tlrs upon pamp binding. only the main components are shown. proteins whose existence has not been demonstrated yet in molluscs are marked by question marks. excellent review of the complex molecular antiviral machinery present in oysters, whose components have been identified by homology with their vertebrate counterparts (green et al., 2015). this system comprises a broad range of membranebound and intracellular receptors and has a significant overlap with the intracellular signaling routes activated downstream of tlrs. the multifaceted antiviral response of molluscs possibly also involves autophagy, which could be mediated by the recognition of viral nucleic acids by tlrs located in endosomes, and the activity of the rnainduced silencing (risc) complex and rig-like receptors (rlrs). while all these components have been only marginally studied in mollusks, they appear to be present, for the most part, in the genomes of both bivalves and gastropods. far for providing a complete account of the entire complement of gene-encoded molecules involved in antiviral response, this section of the review will mainly focus on one of the key components of the cytosolic system of viral sensing, the stimulator of interferon genes (sting), which is missing in all the sequenced gastropod genomes. sting is a key regulator of intracellular sensing of viral nucleic acids which is usually bound to the endoplasmic reticulum membrane through nterminal transmembrane domains. upon viral 111 sensing, sting dimerizes and migrates to the perinuclear region, where it interacts with tbk1 (a key molecule also in tlr signaling), thereby activating the irf3 transcription factor and triggering the expression of interferon genes (fig. 4) (ishikawa et al., 2009). sting can either bind directly singleor double-strand nucleic acids or collect signals derived from a multitude of intracellular receptors with somewhat redundant functions (ma and damania, 2016), thereby acting as a major hub coordinating immune responses to viral infection. the different players of this cytosolic sensing pathway are sometimes difficult to be identified due to large sequence divergence between protostomes and vertebrates, and lineage-specific loss of the known vertebrate regulators upstream of sting (gerdol and venier, 2015). also, while irf-like sequences have been previously identified in mollusks (wang et al., 2013), no irf-3 and interferon-like sequences have ever been described in invertebrate organism, suggesting that the terminal branch of this pathway might be largely divergent between vertebrates and invertebrates. despite these differences, sting homologs can be readily identified in many protostomes, including bivalve molluscs, although these proteins present remarkable structural differences compared to their vertebrate counterparts: (i) they lack nterminal transmembrane domains for the anchoring to the er membrane; (ii) they are associated to an n-terminal tir-like domain, whose typical function is immune signal transduction (fig. 4). also, the tir/sting domain pair is repeated two times, suggesting that, unlike vertebrates, no dimerization is necessary for sting activation, as the homotypic interaction could possibly involve the two sting domains present within the same protein precursor. a large scale genomic comparative study has recently evidenced that similar tir/sting proteins are also present in brachiopods and polychaetes, even though only a single tir/sting domain pair is found in these organisms (gerdol et al., 2017). on the other hand, as mentioned above, no sting gene is present in the available genomes of gastropods and cephalopods. however, transcriptome data mining revealed that homologous sequences, with the brachiopod/polychaete domain configuration, are present in caenogastropoda, implying that the loss of sting might have occurred only in heterobranchia and patellogastropoda (fig. 4). another important difference between bivalves and gastropods within the sting pathway is linked to cgas, an important dna sensor containing a mab-21 domain, which can catalyze the production of the second messenger cgamp, activating sting (ablasser et al., 2013). although no cgas sequence has been ever formally described in mollusks, several mab-21 domain containing proteins can be identified in their genomes. however, while only a single-copy cgas-like gene is present in gastropods, the oyster genome encodes several dozen paralogous sequences, potentially offering a very broad array of sting activators with slightly different binding specificities. overall, this data seems to point towards a broad capability of recognition of foreign nucleic acids by bivalves through sting/cgas, opposed to gastropods, where sting is absent (with the exception of coenogastropoda) and its upstream cgas-like partners display low sequence diversity. obviously, as no functional evidence has been provided so far about the involvement of molluscan sting and cgas homologs in antiviral defense, the possible effects of the loss of this pathway in gastropods are a matter of speculation. by homology with vertebrates, where sting knockout determines an increased susceptibility to lethal viral infections (ishikawa et al., 2009), one might argue that gastropods are likely to be more susceptible to viral infections than bivalves, unless any compensatory, presently unknown strategy has been developed to cope with this loss. occurrence and diversity of antimicrobial peptides one of the most striking differences between gastropods and bivalves is the scarce amount of reports in the molecular immunology literature about antimicrobial peptides (amps) in the former, opposed to the large number of publications produced over the past two decades for the latter (li et al., 2011). this disparity is so remarkable that one might wonder whether this is linked a more developed arsenal of microbe-killing peptides or, simply, to the greater efforts put so far in antimicrobial research in bivalves. as a matter of fact, the available genomic and transcriptomic data is in agreement with literature reports, pointing out that gastropod molluscs lack several amp gene families which are present not only in bivalves, but often also in other protostomes (table 1). mussels (mytilus spp.) represent a remarkable example of the expansion and diversification of host defense peptides. classical biochemical approaches carried out in the early ‘90s permitted to isolate several secreted cysteine-rich amps from circulating hemocytes, where these molecules are usually stored as inactive precursors (charlet et al., 1996; mitta et al., 1999). thanks to the recent developments of ngs, we have learned that these peptides are produced by multi-genic families, named defensins, mytilins and myticins. despite remarkable differences in amino acid composition and organization of the peptide precursor, these mussel amps pertain to the same cysteinestabilized alpha-helix beta-sheet (cs-αβ) superfamily, which reunites diverse peptides characterized by a common structural motif, i.e., the presence of an alpha helix, followed by two antiparallel beta-sheets, whose position in the three-dimensional space is stabilized by three (or four) disulfide bridges. while the role of these cysteine-rich amps is certainly linked to pathogen killing, with a preferential activity against grampositive bacteria, it has also been suggested that they may act as immune modulators, with a cytokine-like function (balseiro et al., 2011). following the studies in mussels, defensin-like peptides, either characterized by the presence of six cysteine residues, like in arthropods or eight cysteine residues, were shown to be also present in 112 fig. 4 panel a: taxonomical distribution of tir/sting and cgas within mollusca. presence and absence are marked by green and red colors, respectively. brachiopoda were included as an outgroup phyla for lophotrochozoa. whenever present, the domain organization of sting is shown. panel b: schematic overview of viral sensing mediated by sting. sting can directly bind viral nucleic acids, forming a complex with tak1 and irf3, which is activated and translocated to the nucleus, where it triggers the expression of interferon genes. alternatively, sting can be activated by the interaction with cgamp molecules produced by cgas upon the recognition of foreign dna in the cytosol. molecules whose homologs have not been reported yet in molluscs are marked in red. oysters, clams and freshwater mussels, with either hemocyte or mantle tissue specificity (gueguen et al., 2006; peng et al., 2012; wang et al., 2015). it is noteworthy that cs-αβ peptides similar to bivalve and arthropod defensins have been reported as missing in some large invertebrate taxa, which also include gastropods (rodríguez de la vega and possani, 2005). this is confirmed by the absence of gene products sharing significant sequence similarity to any of the previously described invertebrate defensins in l. gigantea, b. glabrata and a. californica. however, this consideration cannot be extended to all gastropods, since a defensin peptide with six cysteines has been identified in the abalone haliotis discus, pertaining to the ancestral gastropod clade vetigastropoda (de 113 zoysa et al., 2010). this report adds further complexity to the evolutionary history of invertebrate defensins, whose common ancestry has been previously brought into question (tarr, 2016). big defensins display an similar taxonomic distribution, characterized by an apparent absence in gastropods and multiple reports in diverse bivalve species (zhao et al., 2010; rosa et al., 2011). despite their name, these amps are not related to the other invertebrate defensin-like peptides described so far, as they share the same fold of vertebrate -defensins. despite the lack of big defensins in the available gastropod genomes, a big defensin-coding sequence appears to be present in the transcriptome of the abalone haliotis tuberculata, mirroring the presence of classical defensins in abalones and extending the known taxonomical distribution of these amps, which had so far been found only in bivalve mollusks, horseshoe crabs and amphioxus (gerdol et al., 2012). as evidenced from sequence data mining, macins are the only class of cs-αβ peptides apparently present in all bivalves and gastropods species. this amp family comprises multifunctional peptides which are thought to be involved both in bacterial killing and wound healing and which display some variations in their cysteine array, which may present 4, 5 or 6 disulfide bridges (gerdol et al., 2012). consistently with reports in other protostomes (tasiemski et al., 2004), macins appear to be broadly expressed in various tissues and, in particular, they critically contribute to the antibacterial activity of the mucus of the giant snail achatina fucata (zhong et al., 2013). the repertoire of molluscan cysteine-rich peptides is growing at a fast rate as new amp families are identified either by conventional or by omic methods. once again, mussels have been a major target for amp discovery, as mytimycins (sonthi et al., 2011), myticusins (liao et al., 2013) and the enigmatic crp-i family, whose function still remains to be fully elucidated (gerdol et al., 2015), have been described in the past few years. comparatively, only a little attention has been put in the discovery of linear amps devoid of cysteines, and this finds a possible explanation in the difficulty of the discovery of these fast-evolving gene products by sequence similarity-based searches. the most relevant case of linear amps described in molluscs so far are probably molluscidins, low-complexity and highly cationic short peptides composed by several dibasic repeats, found in both oyster and abalones (seo et al., 2013, 2016). another small family of linear peptides, named cgprp, rich in pro and arg residues, has been identified in oyster hemocytes. although cgprp peptides do not show any detectable antimicrobial activity, they are significantly and synergistically able to enhance the effectiveness of defensins (gueguen et al., 2009). as far as gastropods are concerned, potent prolinerich amps with no similarity to any other known peptide have been isolated from the hemolymph of the marine snail rapana venosa (dolashka et al., 2011). while the research on linear cationic amps is still mostly based on classical biochemical isolation methods, the use of in silico approaches is currently proving to be a valid complementary tool in the discovery of novel amps in molluscs, as testified by the recent discovery of myticalins in m. galloprovincialis, a novel class of taxonomically restricted, hypervariable amps which display a broad spectrum of activity towards gram+ and grambacteria (gerdol et al., 2016). conclusions with over 100,000 extant species and their colonization of nearly all terrestrial, freshwater and marine environments, mollusks represent a unique case for the study of many aspects of evolutionary biology, including defense against potentially pathogenic microbes. over 450 million years of independent evolution, gastropod and bivalves have clearly developed peculiar molecular mechanism to tackle these challenges and the nature of these unique adaptations are starting to emerge, also thanks to the contribution of the increasing number of fully sequenced genomes available. we are quickly moving from the isolation and characterization of single molecules through classical biochemical methods to the possibility of mining entire genomes and transcriptomes, identifying dozens if not even hundreds of candidate immune receptors, signaling transducers, transcription factors and antimicrobial effectors. obviously, these two approaches are complementary to each other, and the combination between data mining and functional approaches is quickly providing new hints about the placement of several missing pieces in the highly complex puzzle of the invertebrate immune system. as far as molluscs are concerned, despite several recent discoveries of the utmost importance, such as the identification of a complete tlr signaling pathway and the discovery of molecular strategies providing immune memory, we are still missing large portions of the whole picture. our knowledge of the functioning of viral sensing systems for example is still extremely limited and almost exclusively limited to the components which are homologous to those of vertebrates. at the same time, the nature of invertebrate cytokines is still elusive, despite the high conservation of the signaling routes leading to their production. most importantly, no global approach has yet been effectively implemented for defining how the different molecules involved in pamp detection, in the activation of immune signaling and in the elimination of pathogens coordinate their activity. for example, while functional studies have permitted to elucidate the primary sequence and the binding specificity of several dozen molluscan lectins, it is presently unclear how such molecules transmit immune signals within the cell and, while some of these appear to work in a “seek and destroy” mode, thanks to the combination between prr and pore-forming modules, others are likely to trigger a highly specific and finely regulated cellular response, which probably involves membrane receptors, intracellular partners and cytokine-like 114 table 1 summary of the main antimicrobial peptide families reported so far in bivalve and gastropod molluscs. a brief description of the taxonomic distribution, expression pattern and spectrum of activity is also reported, whenever available amp type/family bivalvia gastropoda defensins present in multiple species; 6/8 cysteine residues; hemocyteor mantle-specific; activity against gram+ bacteria absent in most species; present in abalones; 6 cysteines; expressed in whole body; unknown spectrum of activity mytilins taxonomically restricted to mussels, 8 cysteines; hemocyte-specific; broad spectrum of activity absent myticins taxonomically restricted to mussels, 8 cysteines; hemocyte-specific; activity against gram+ bacteria; antiviral activity; might have immuno-modulating properties absent mytimycins taxonomically restricted to mussels; antifungal activity; hemocyte-specific; unknown spectrum of activity absent macins present in multiple species; 8/10/12 cysteine residues; expressed in whole body; unknown spectrum of activity present in multiple species; 8/10 cysteine residues; expressed in whole body; present in the mucus; broad spectrum of activity big defensins present in multiple species; 6 cysteines; expressed in whole body; broad spectrum of activity absent in most species; present in abalones; 6 cysteines; no expression data available other cys-rich peptides myticusins, crp-i; only reported in mussels so far; variable disulfide array; diverse pattern of expression and spectrum of activity not reported molluscidins only reported in oyster; linear peptide rich in dibasic repeats; gills-specific; broad spectrum of activity only reported in abalone; linear peptide rich in dibasic repeats; gills-specific; borad spectrum of activity pro-rich peptides cgprp, only reported in oyster; no antimicrobial activity but synergistically enhances the antimicrobial activity of defensins; hemocytespecific. myticalins, taxonomically restricted to mussels; gills-specific; broad spectrum of activity only reported in r. venosa; hemocytespecific; unrelated to bivalve pro-rich peptides; activity against gram+ bacteria molecules which still remain to be unveiled. overall, molluscs, with their divergent molecular strategies for pamp sensing and pathogen killing, certainly represent fascinating models for the study of 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c1q-domain-containing protein from zhikong scallop chlamys farreri with lipopolysaccharide binding activity. fish shellfish immunol. 25: 281-289, 2008. zhao j, li c, chen a, li l, su x, li t. molecular characterization of a novel big defensin from clam venerupis philippinarum. plos one 5: e13480, 2010. zhong j, wang w, yang x, yan x, liu r. a novel cysteine-rich antimicrobial peptide from the mucus of the snail of achatina fulica. peptides 39: 1-5, 2013. microsoft word isj436 isj 13: 291-297, 2016 issn 1824-307x research report virus-induced opposite effect on bombyx mori gene transcriptions y yin*, h xia*, f zhu, l chen, p lü, k chen institute of life sciences, jiangsu university, zhenjiang, 212013, jiangsu, p. r. china * these authors contributed equally to this work. accepted september 6, 2016 abstract bombyx mori bidensovirus (bmbdv) and bombyx mori nucleopolyhedrovirus (bmnpv) are serious pathogens of bombyx mori. in this study, we reported the changes of transcription level of several immune genes, including bmi, argo, dicer, cap1, cap3 and car, in bombyx mori midgut after exposure to bmbdv or  bmnpv. silkworm strains 798 (anti-bmbdv) and 306 (susceptible to bmbdv) were subjected to bmbdv infection, and nb (anti-bmnpv) and huaba (35) (susceptible to bmnpv) were subjected to bmnpv infection. the results showed that the transcription levels differ largely among different silkworm strains, and that the extent to which the gene transcriptions were affected by the viruses was different. however, both bmnpv and bmbdv viruses can reverse the transcription patterns of these genes when the silkworms were administered with the viruses compared with those control groups. the transcript levels of bmi and dicer were decreased in 798 and 306 strains that were inoculated with bmbdv compared with their respective controls, but were increased in nb and huaba (35) inoculated with bmnpv. the transcript levels of argo and cap3 were risen in 798, 306 and nb strains when inoculated with their respective viruses, but were decreased in huaba (35) strain. the transcript levels of cap1 were risen in all silkworm strains, while the levels of car were decreased in 798, 306 and huaba (35) strains, and increased in nb strain when inoculated with their respective viruses. these findings may contribute to more in-depth understanding on functions of these genes in virus infection and proliferation. key words: bmbdv; bmnpv; bombyx mori; qpcr; immune genes; expression level   introduction microorganisms, especially viruses, are harmful to the growth and propagation of bombyx mori. virus results in approximately 70 % of damage to the sericulture industry due to viral diseases. bombyx mori viruses can be divided into four categories: nuclear polyhedrosis (bmnpv), midgut polyhedrosis, virus flacherie and densovirus (bmbdv). bmbdv is a non-enveloped spherical virus that is composed of two single stranded dna segments, and it replicates mainly in the columnar cells of the midgut epithelium (hu et al., 2013). bmnpv is a circular, double stranded dna virus, and similar to bmbdv, bmnpv also enters through the columnar cells of the midgut epithelium and replicates in this type of cells (gomi et al., 1999). however, bmbdv infection shows up ___________________________________________________________________________ corresponding author: keping chen institute of life sciences jiangsu university zhenjiang, 212013, jiangsu, p. r. china 291   e-mail: kpchen@ujs.edu.cn as a chronic disease while bmnpv acts acutely, indicating that these two viruses differ pathogenically in their viral infection mechanisms. bombyx mori and other invertebrates use innate immunity as defense strategy against various pathogens. many molecules and biological processes are involved in the resistance to insect viruses, such as antimicrobial peptides, phenol oxidase-dependent melanization and encapsulation, apoptosis, phagocytosis and rnai (lipardi et al., 2003; galiana-arnoux et al., 2006; liu et al., 2006; yao et al., 2006; wang et al., 2006). nadph-oxidoreductase, lipase-1 and serine protease-2 genes have been reported to be anti-viral genes (uchida et al., 1984, ponnuvel et al., 2003, nakazawa et al., 2004). peptides genes that have been reported to have antimicrobial properties are gloverin-1, lebocin and attacin (bao  et al., 2009, 2010). several heat shock protein genes, including small heat shock protein, hsp70 cognate and hsc70/hsp90-organizing protein were reported to be related to viral resistance (bao et al., 2010). mailto:kpchen@ujs.edu.cn 292   molecules that are involved in the prophenoloxidase cascade system, such as trypsin-like serine protease, serine protease-1, serpin-5 and retinoid-inducible serine carboxypeptidase, also relate to viral resistance (bao et al., 2008, 2020; qin et al., 2012; zhou et al., 2013). cytochrome c oxidase subunit ii, ribosomal s3a and death associated protein (dap), which are involved in the apoptotic pathway, relate to the resistance to virus (chi et al., 2009; bao et al., 2010;  xu et al., 2010). molecules that involve in pattern recognition, such as the sialic acid binding ig-like lectins, relate to viral resistance. molecules involved in oxidative stress, such as thiol peroxiredoxin, glutathione s-trasferase omega and thioredoxin relate to the resistance to virus (zhao, 2007; bao et al., 2009;  zhou  et al., 2013). molecules involved in energy metabolism, such as arginine kinase, v-atpase c subunit, v-aptase b subunit and v-atpase h subunit relate to the resistance to virus (zhao, 2007; yang et al., 2009; bao et al., 2010). actin, myosin, or tubulin family proteins, such as suppressor of profiling 2, transgelin, actin-depolymerizing factor 1 and myosin heavy chain, relate to the resistance to virus (xu et al., 2005; zhao, 2007; bao et al., 2009). in the ubiquitin pathway, ubiqutin-conjugating enzyme e2 and 26s protease regulatory subunit 6b relate to the resistance to virus (bao et al., 2010). other molecules related to the resistance to virus include rab7, beta-n-acetylglucosaminidase 2 (glcnacase 2), amino acid transporter, potassium coupled amino acid transporter, insect intestinal mucin 3 and juvenile hormone epoxide hydrolase (bao et al., 2009; qin et al., 2012). it is reported that in bombyx mori, bmcaspase1 (cap1), bmcaspase3 (cap3), bmice (bmi), bmdcr-like (dicer), bmago2 (argo), carboxylesterase (car) may be related to the resistance to virus (tanaka et al., 2008). cap1, cap3 and bmi all belong to capases, a cysteine aspirate protease family. these proteins relates to apoptosis, necrosis and inflammation (alnemri et al., 1996). the family is important for maintaining a stable number of t cells, for tissue differentiation and regeneration, and also plays a significant role in neural development in mammals (shalini  et al., 2015). some members of the family also have an effect on tumor resistance, and have a potential role in maintaining the stability of the genome, metabolism, autophagy and aging (bernstein et al., 2001). dicer, belonging to rnase iii family, can recognize and incise foreign dsrna (jaskiewicz et al., 2008). it is also reported that the family has other functions like facilitating activation of rna-induced silencing complex (risc), repairing damaged dna and playing an important role in the reproduction and development of animals (ketting et al., 2001; wei et al., 2012). argo, belonging to argonaute protein family, is the necessary catalytic part of risc which plays important role in rnai (kai, 2013). besides, some reports suggest that argonaute protein can inhibit the expression levels of some genes, and stimulate the expression of others (chu et al., 2010). car belongs to caboxylesterase family which is a sublevel of the hydrolase family. current published studies mainly focus on its functions of hydrolyzing exogenous compounds and activating drugs. it is reported that carboxylesterase has the function of anti-virus (gao et al., 2007). however, it may also play an important role in virus multiplication (blais et al., 2010). this study aims at examining the transcription levels of these immune related gens, namely cap1, cap3, bmi, dicer, argo and car, in different strains of the silkworm after inoculation with bmbdv or bmnpv virus, in order to explore the effects of these viruses on the transcription levels of the virus-related genes. materials and methods the bombyx mori breeding and virus inoculation bombyx mori strains 798 (anti-bmbdv), 306 (susceptible to bmbdv), nb anti-bmnpv) and huaba (35) (susceptible to bmbdv) were all preserved in the institute of life sciences, jiangsu university.  the silkworm strains 798, 306, nb and huaba (35) were raised routinely with fresh mulberry leaves till the fifth instar before inoculation of viruses. the nb and huaba silkworm strains were each administered through the mouths with 5 µl of the bmnpv virus at a concentration of 2×108 pib/ml, and 306 and 798 strains were each administered with 5 µl of bmbdv virus at a concentration of 20 mg/ml. the silkworm midgut was isolated from the body at the time points of 24, 48, 72 and 96 h post inoculation of the viruses, washed quickly using pbs buffer and stored in an eppendorf tube with sample protector for rna/dna (takara, dalian, china). the tube was immersed in liquid nitrogen and then stored at -70 oc for further use. for comparison, a control group was set up for each strain of the silkworm without the virus treatment. rna extraction and reverse transcription into cdna trizol reagent (sangon biotech, shanghai, china) was used to extract total rna, and then transcribed into cdna using primescript™ ii 1st strand cdna synthesis kit (takara, dalian, china). specifically, the midgut was grinded into powder in liquid nitrogen and then transferred into an rnase-free eppendorf tube. after the addition of 1 ml trizol reagent, the solution was mixed and rested for 5 min before centrifugation at 12,000g for 5 min at 4 oc. the supernatant was transferred into a new rnase-free eppendorf tube, and 200 µl chloroform was added, mixed and incubated for 2 min. after centrifugation at 12,000g for 15 min at 4 oc, the supernatant was again transferred to a new rnase-free eppendorf tube. the solution was added with 500 µl isopropanol, mixed, and incubated for 10 min. the supernatant was discarded through centrifugation at 12,000g for 10 min at 4 oc. the precipitate was dried and added with 100 µl depc treated pure water. the solution was mixed gentlely and incubated for 10 min at 55 oc to obtain the rna solution. the cnda synthesis was carried out according to the manufacturer’s guide. briefly, an aliquot of 1 μl of oligo dt primer at 50 μm, 1 μl of dntp mixture (10 mm each), 5 μl of rna template, and 3 μl of rnase-free h2o were mixed in a microtube, and then incubated at 65 oc for 5 min. the tube was then immediately quenched on ice. to 293   table 1 the primer sequences used for qpcr gene name sense primer anti-sense primer bmi 5’ ctgccgaccaaccatacaag 3’ 5’ gaacataccaaccagccgtc 3’ argo 5’ ccatcgccagatcagagtaata 3’ 5’ gtcacggaacacgaacacct 3’ dicer 5’ gaagttctgaagccagtttcgttat 3’ 5’ tgaatgtcttgagttgtgggagc 3’ cap1 5’ tctcgcacgggcaccaat 3’ 5’ aacacagcaaccagcagacaat 3’ cap3 5’ gaaatacgctacgacatacga 3’ 5’ tctacgacttcaaagccaaac 3’ car 5’ tggaggaagtagtatcagc 3’ 5’ agtgtatcacgggcaatc 5’ tif-3 5’ agatgacggggagcttgatggt 3’ 5’ gagggcggaatgtacttgttgc 3’ the tube, 4 μl of 5×primescript ii buffer, 0.5 μl of rnase inhibitor, 1 μl of primescript ii rtase, and 4.5 μl of rnase-free h2o was added and then mixed gentlely. the reverse transcription reaction was carried out at 45 oc for 30 min, and then incubated at 95 oc for 5 min before cooled down on ice. qpcr cds (bgibmga006131-ta) of bmi, cds (bgibmga006946-ta) of cap1, cds (bgibmga004420-ta) of cap3, cds (bgibmga010406-ta) of argo, cds (bgibmga011542-ta) of dicer and cds (gibmga010988-ta) of car were obtained from the silkworm genome database. the primer sequences used for qpcr were listed in table 1. tif-3 was used as internal reference. the qpcr was performed with an applied biosystems 7300 according to aceq® qpcr (vazyme, naning, china ) specifications. the qpcr data was analyzed using 2-∆∆ct. results the transcriptional levels of the immune genes in silkworm strains 798 and 306 inoculated with bmbdv the transcription levels of bmi in the control groups increased steadily and significantly from 0 h to 72 h (fig. 1a). the highest level was reached at 72 h, and then the level began to decrease, which was probably because the virus replication rate had decreased after this time point. it is interesting that when the silkworms are inoculated with bmbdv, the expression level of bmi was significantly inhibited, maintaining at similar or lower level than on the first day (fig. 1a). on the contrary to bmi, the transcription levels of argo in the control groups decreased sharply during the first 24 h and then maintained essentially unchanged at very low levels (fig. 1b). however, for silkworms that were inoculated with the virus, the level began to increase notably after 24 h and remain at a relatively high level (fig. 1b). we notice a slight decrease of the expression in the virus-treated silkworms after 72 h, which may be contributed to the decreased virus replication rate similar to the case in bmi (fig. 1b). the transcription levels of cap1 was mostly unchanged in control 798 and decreased in control 306, while it increased notably in virus-treated 306 and increased briefly and then decreased in the virus treated 798 (fig. 1c). the data indicate that virus invasion can stimulate the expression of cap1, temporarily for 798 and relatively longer for 306. also, cap1 expression differs significantly between virus-resistant and -susceptible strains, both in untreated and virus-treated groups. the transcription levels of cap3 kept low in control 798 and even lower in 306. however, its level increased notably in the virus-susceptible 306 treated with bmbdv, and much more significantly in the resistant strain 798 treated with the virus (fig. 1d). this is quite different from the case for cap1 in the 798 strain, whose expression raised briefly and then remained below the starting level. these observations indicate that cap1 and cap3, though both belong to the caspase family, may function very differently during viral invasion. the transcription levels of dicer increased significantly in control 798 and even more significantly for 306 on the second day (24 h) of the fifth instar and remained at high levels thereafter (fig. 1e). like bmi, the up-regulation of dicer during the fifth instar indicates that these two genes may be required during metamorphosis. however, after virus treatment, dicer expression in 306 increased slightly on the second day (24 h) and then decreased to below the starting level after this time point. for virus-treated 798 strain, dicer expression remained always below the starting level (fig. 1e). bmbdv appears to inhibit the transcription levels of both genes in either virus-resistant or susceptible strains. the transcription levels of car increased significantly in control 306 and decreased in 798. after virus treatment, the expression in both 306 and 798 remained basically below the starting level (fig. 1f). fig. 1 the expression levels of different genes in midguts of 798 and 306 infected with bmbdv at different time points. a: bmi; b: argo; c: cap1; d: cap3; e: dicer; f: car. the transcription levels of immune genes in silkworm strains nb and huaba (35) inoculated with bmnpv the transcription levels of bmi decreased steadily in control silkworm strains nb (viral-resistant) and huaba (35) (viral susceptible), while increased notably for nb and significantly for huaba (35) after bmnpv treatment (fig. 2a). compared with bmbdv treatment, which inhibited bmi expression, bmnpv can stimulate the expression. although the two viruses have different effect on the gene expression, both have reversed the original expression patterns. the transcription levels of argo was enhanced dramatically in control huaba (35) strain, and decreased markedly in control nb (fig. 2b). after virus treatment, the overall expression increased for both strains (fig. 2b). the transcription levels of cap1 enhanced in control huaba (35) but decreased in control nb, while it increased dramatically in virus-treated huaba (35) and nb (fig. 2c). similar to bmbdv treatment, bmnpv can also stimulate the gene expression, but much more effectively. the transcription levels of cap3 decreased slightly in control nb and increased dramatically in control huaba (35) (fig. 2d). the overall expression after viral treatment generally increased and remained relatively stable, slightly above the starting level for both strains. the transcription levels of dicer decreased and remained below the starting level in untreated nb and huaba (35), while it increased notably in both strains after bmnpv treatment (fig. 2e). compared with bmbdv treatment, which inhibited dicer expression, bmnpv can stimulate the expression. although the two viruses have different effect on the gene expression, both have reversed the original expression patterns. the transcription levels of car enhanced markedly in control huaba (35) but decreased in control nb (fig. 2f). after bmnpv treatment, the expression decreased and remained below the starting level for huaba (35). for virus-treated nb, the expression increased slightly after 24 h and kept basically unchanged after that (fig. 2f). the results suggest that car function differently in nb and huaba (35), with nb down-regulated and huaba (35) up-regulated, whereas bmnpv virus can inhibit both the down-regulation of the former and up-regulation of the latter. 294   fig. 2 the expression levels of different genes in midguts of 798 and 306 infected with bmnpv at different time points. a: bmi; b: argo; c: cap1; d: cap3; e: dicer; f: car. discussion the transcriptional changes of six immune genes related to virus infections have been systematically investigated by qpcr. two different silkworm viruses bmbdv and bmnpv were used to introduce viral infections. both bmbdv and bmnpv can initiate viral infections to the silkworm midgut, thus the midgut was collected and analyzed, and a time course analysis was used to examine the transcriptional changes. the results clearly showed that the transcriptional levels of these genes were quite different in different silkworm strains, and transcriptional changes upon virus treatment were also quite different. when infected with viruses, the transcription levels of bmi and dicer decreased in 798 and 306, but enhanced in nb and huaba (35); the transcriptional levels of argo and cap3 enhanced in 798, 306 and nb, but decreased in huaba (35); the transcriptional levels of cap1 enhanced in 798, 306, nb and huaba (35); the transcription levels of car decreased in 798, 306 and huaba (35) and increased in nb. however, a conclusion could be drawn based on the results, that viral treatment can adversely affect the gene expression patterns. that is, no matter the gene was up-regulated or down-regulated in the silkworm, viral treatment can inhibit this up-regulation or down-regulation. it is interesting to note the transcription patterns of bmi and dicer are virus-specific. compared with bmbdv treatment, which inhibited bmi expression, bmnpv can stimulate the expression. the transcriptional levels of cap1 and cap3 were enhanced briefly and then decreased in bmnpv treated nb strain, which may be caused by the apoptosis-inhibitory genes encoded by baculovirus (beidler et al., 1995,  roy et al., 1995). the transcriptional levels of car enhanced in bmnpv treated nb, the resistant strain, and decreased in bmnpv treated huaba (35), the susceptible stain, suggesting that car has anti-bmnpv function. dicer belongs to rnase iii which can recognize and cut dsrna. the transcription level of dicer increased in control silkworm strains but decreased when bmbdv virus-treated strains (fig. 1e), indicating that the gene is related to virus infection. similar to dicer, the transcription of bmi, an 295   296   interleukin-1-beta converting enzyme that belongs to capase1, also decreased in the virus treated silkworms (fig. 1a). by contrast, the transcription levels of argo and cap3 enhanced in bmbdv treated silkworms (figs 1b, d). the anti-virus function of argonaute protein is mainly executed by rnai, and cap3 may involve in the resistance to diseases via translation to the corresponding protein. acknowledgement this work was supported by national natural science foundation of china (31372259 and 31572467), natural science foundation for universities of jiangsu province (10kjb180001), natural science foundation of jiangsu province (bk20160509), start-up research funding of jiangsu university for distinguished scholars (09jdg005), and priority 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profile in resistant and susceptible bombyx mori strains reveals resistance-related genes to nucleopolyhedrovirus. genomics 4: 256-262, 2013. isj 12: 173-175, 2015 isj 12: 173-175, 2015 issn 1824-307x letter to editor on the existence of possible pituitary adenylate cyclase-activating polypeptide adenylate type 1 receptor in earthworms l molnár1, e pollák1, i somogyi1, p engelmann2 1department of comparative anatomy and developmental biology, faculty of sciences, university of pécs, ifjúság u. 6, h-7624 pécs, hungary 2department of immunology and biotechnology, clinical center, university of pécs, szigeti u. 12, h-7643 pécs, hungary accepted may 29, 2015 to the editor neuropeptides, expressed by both invertebrate and vertebrate nervous systems (grimmelikhuijzen and hauser, 2012) play critical roles in both synaptic and non-synaptic signalling mechanisms regulating the functions of nervous, endocrine and immune systems (nässel and larhammar, 2013). in contrast to vertebrates the invertebrate nervous system contains high number of neurosecretory cells that produce and elaborate several neurohormones (most of them are peptides) that act on peripheral tissues e.g., body wall muscles, alimentary canal, excretory and genital organs (taghert and nitabach, 2012). however, some of the neuropeptides are neuromodulators in the central nervous system mediating the firing mechanism and synaptic transmission of neurons (van den pol, 2012). while broad range of neuropeptides is known from some invertebrates like planarians, roundworms, insects or molluscs (de haes et al., 2014), but less is known from the annelids, especially from earthworms. most of the neuropeptides, like proctolin, fmrfamide-like peptides, neuropeptide y (lengvari et al., 2001), substance p and acth-like peptide (aros et al., 1980), believed to be regulator molecules in earthworms had only been identified by immunocytochemical stainings. there are only a few neuropeptides, e.g., capa-peptides (herbert et al., 2009) and pituitary adenylate cyclase activating polypeptide (pacap)-like peptides (somogyvárivígh et al., 2000) which expression, pattern and function have investigated by various experimental protocols in details. pacap is a highly conserved member of the vip/secretin/glucagon peptide family found in neuronal elements of both the cns and several peripheral tissues of vertebrates. it acts as a pleiotropic neuropeptide via three heptahelical gprotein-linked receptors, one pacap-specific (pac1) ___________________________________________________________________________ corresponding author: peter engelmann department of immunology and biotechnology clinical center, university of pécs pécs, h-7643, szigeti u. 12, hungary e-mail: engelmann.peter@pte.hu receptor and two receptors that are shared with vip (vpac1 and vpac2). pacap has been implicated in a variety of central nervous functions, including hypophysiotropic function, learning and memory formation, psychomotor function, and the response to nerve injury. furthermore, it is a potent antiapoptotic and neurotrophic substance (vaudry et al., 2009). by means of immunohistochemistry pacaplike activity was identified in the central and peripheral nervous system of some earthworm species, namely eisenia fetida, lumbricus terrestris and l. polyphemus (reglödi et al., 2000). two distinct forms of pacap-like molecules (consist from 27 or 38 amino acids) were found in the cns of l. polyphemus of which the smaller molecule proved to be the predominant form (somogyvárivígh et al., 2000). applying whole mount preparations to immunohistochemical observations molnár and colleagues (2006) showed that each pacap-like molecules occurred in distinct neuron populations of both l. terrestris and e. fetida and concluded that these molecules might mediate distinct physiological processes in earthworms. the biological activity of the purified hplc fractions of both pacap27 and pacap38-like peptides isolated from the earthworm cns was investigated in in vitro experimental conditions. both pacap-like molecule fractions from earthworms have activated adenylate cyclase and possessed sequence similarities to mammalian pacap isoforms (somogyvári-vígh et al., 2000). occurrence, distribution and cellular transport of the specific pac1-receptor in neural structures of the ventral nerve cord ganglia in e. fetida was investigated by light and electron microscopic immunocytochemistry. the experimental results revealed that pac1-receptor-like immunoreactivity localized both on intracellular membranes (endoplasmic reticulum cisternae) and special domains of plasma membrane (synaptic membrane) of neurons as well. it suggests a compound transport and presentation process of the receptor molecule on the cell surface which supposes its role in synaptic transmission (molnár et al., 2008). 173   mailto:engelmann.peter@pte.hu a possible role of pacap-like peptides in mediation of both embryogenesis (boros et al., 2008) and posterior regeneration of e. fetida (várhalmi et al., 2008) was suggested based on the quantitative analysis of the expression of pacaplike peptides applying one of the most sensitive immunoserological methods, the radioimmunoassay. during both the embryonic development and segment regeneration the overexpression of pacap-like proteins was detected and a characteristic antero-posterior gradient found in regenerating ventral nerve cord ganglia suggesting that they had a strong influence on cell cycle and tissue differentiation in earthworms (boros et al., 2008, várhalmi et al., 2008). the expression of pac1-receptor was investigated by immunocytochemical and immunoserological methods (western blot and far western blot) during the embryonic development of e. fetida and showed its existence in germinal layers and developing ventral nerve cord ganglia (boros et al., 2010). according to the pacap and pac1/vpac receptor localization studies in vertebrates it was clear that their expression is not restricted only to neuroendocrine and separate vegetative organs (vaudry et al., 2009). we observed pac1-like immunoreactivity in coelomocytes by means of immunocytochemistry and western blot. among coelomocytes the amoebocyte subgroup expressed more abundantly this receptor, but whether this immunocyte is only a responder and/or a producer of the neuropeptide was not clear. to answer this, first immunoserological experiments (ria) were performed from coelomocytes of regenerating earthworms and proved the pacap-like immunoreactivity in this immune cell compartment as well (somogyi et al., 2009). these immunological cross-reaction based results should be further supported by molecular biological approaches to isolate and characterize the mrna transcripts for pacap and pac1 receptor from earthworms. recent molecular evidence from other phyla (pirger et al., 2010; lugo et al., 2013) and our findings strengthen the notion for evolutionary conservation of this neuropeptide family across animal kingdom. acknowledgements we acknowledge the financial support of medical faculty research foundation, university of pécs (pte-áok-ka 34039/10-06 and 2013/09), the jános bolyai research foundation of the hungarian academy of sciences, the european union and the state of hungary, co-financed by the european social fund in the framework of támop 4.2.4. a/211-1-2012-0001 ‘national excellence program’. the present scientific contribution is dedicated to the 650th anniversary of the foundation of the university of pécs, hungary. references aros b, wenger t, vigh b, vigh-teichmann i. immunohistochemical localization of substance p and acth-like activity in the central nervous system of the earthworm lumbricus terrestris l. acta histochem. 66: 262-268, 1980. boros a, reglödi d, herbert z, kiszler g, nemeth j, lubics a, et al. changes in the expression of pacap-like compounds during the embryonic development of the earthworm eisenia fetida. j. mol. neurosci. 36:157-65, 2008. boros a, somogyi i, engelmann p, lubics a, reglödi d, pollák e, et al. pituitary adenylate cyclase-activating polypeptide type 1 (pac1) receptor is expressed during embryonic development of the earthworm. cell tissue res. 339: 649-653, 2010. de haes w, van sinay e, detienne g, temmerman l, schoofs l, boonen k. functional neuropeptidomics in invertebrates. biochim. biophys. acta 1854: 812-826; 2015. grimmelikhuijzen cj, hauser f. mini-review: the evolution of neuropeptide signaling. regul. pept. 177: s6-s9, 2012. herbert z, zougman a, pollák e, boros a, kapan n, molnár l. identification of novel neuropeptides in the ventral nerve cord ganglia and their targets in an annelid worm, eisenia fetida. j. comp. neurol. 514: 415-432, 2009. krajniak kg. invertebrate fmrfamide related peptides. protein pept. lett. 20: 647-670, 2013. lengvari i, csoknya m, lubics a, szelier m, hámori, j. proctolin immunoreactive elements in the nervous system of earthworm (lumbricus terrestris). acta biol. hung. 45: 337-345, 1993. lugo jm, carpio y, morales r, rodríguez-ramos t, ramos l, estrada mp. first report of the pituitary adenylate cyclase activating polypeptide (pacap) in crustaceans: conservation of its functions as growth promoting factor and immunomodulator in the white shrimp litopenaeus vannamei. fish shellfish immunol. 35: 1788-1796, 2013. molnár l, pollák e, boros a, reglödi d, tamás a, lengvári i, et al. comparative anatomy of pacap�immunoreactive structures in the ventral nerve cord ganglia of lumbricid oligochaetes. ann. ny acad. sci. 1070: 427430, 2006. molnár l, pollák e, boros a, shioda s, nakajo s, tamás a, et al. pac1 receptor localization in a model nervous system: light and electron microscopic immunocytochemistry on the earthworm ventral nerve cord ganglia. regul. pept. 145: 96-104, 2008. nässel dr, larhammar d. neuropeptides and peptide hormones. in: galizia cg, liedo pm (eds), neurosciences from molecule to behavior: a university textbook, springerverlag, berlin heidelberg, pp 213-237, 2013. pirger z, lubics a, reglodi d, laszlo z, mark l, kiss t. mass spectrometric analysis of activitydependent changes of neuropeptide profile in the snail, helix pomatia. neuropeptides 44: 475-483, 2010. reglödi d, lengvari i, szelier m, vigh s, arimura a. distribution of pacap-like immunoreactivity in the nervous system of oligochaeta. peptides 21: 183-188, 2000. 174   somogyi i, boros a, engelmann p, varhalmi e, nemeth j, lubics a, et al. pituitary adenylate cyclase-activating polypeptide-like compounds could modulate the activity of coelomocytes in the earthworm. ann. ny acad. sci. 1163: 521523, 2009. varhalmi e, somogyi i, kiszler g, nemeth j, reglodi d, lubics a, et al. expression of pacap-like compounds during the caudal regeneration of the earthworm eisenia fetida. j. mol. neurosci. 36: 166-174, 2008. vaudry d, falluel-morel a, bourgault s, basille m, burel d, wurtz o, et al. pituitary adenylate cyclase-activating polypeptide and its receptors: 20 years after the discovery. pharmacol. rev. 61: 283-357, 2009. taghert ph, nitabach mn. peptide neuromodulation in invertebrate model systems. neuron 76: 8297, 2012. van den pol an. neuropeptide transmission in brain circuits. neuron 76: 98-115, 2012. 175   isj 12: 142-154, 2015 isj 12: 142-154, 2015 issn 1824-307x review role of hemocytes in invertebrate adult neurogenesis and brain repair pg chaves da silva1,2, i santos de abreu1,3, la cavalcante1,3, c monteiro de barros4, s allodi1,2,3 1laboratório de neurobiologia comparativa e do desenvolvimento, instituto de biofísica carlos chagas filho, universidade federal do rio de janeiro, ufrj, rio de janeiro, rj, brazil 2programa de pós-graduação em ciências biológicas biofísica, instituto de biofísica carlos chagas filho, universidade federal do rio de janeiro, ufrj, rio de janeiro, rj, brazil 3programa de pós-graduação em ciências biológicas fisiologia, instituto de biofísica carlos chagas filho, universidade federal do rio de janeiro, ufrj, rio de janeiro, rj, brazil 4laboratório integrado de morfologia, núcleo em ecologia e desenvolvimento sócio ambiental de macaé, nupem, universidade federal do rio de janeiro, ufrj, macaé, rj, brazil accepted april 20, 2015 abstract the repair of lesions of the central nervous system (cns) varies widely throughout the animal kingdom. at the level of neuronal replacement lie the major differences in cns regeneration. at one extreme are the amniote vertebrates (reptile, avian and mammalian groups), which have very limited capacity for neuronal replacement, and therefore for neural regeneration; at the other extreme, animals such as planarians (flatworms) and colonial tunicates can repair their entire cns after major injuries. these differences can be attributed to the abundance of multipotent and/or pluripotent stem cells and/or undifferentiated precursors among the general cell population. in this review we discuss recent advancements in knowledge of regeneration of the cns of invertebrates. we focus on ascidians, which are a sister group of vertebrates, but we also address other invertebrate groups. because neurogenesis is central to the events that allow regeneration of the adult cns, we address this issue focusing on crustaceans, which have provided a paradigm to study the mechanisms underlying this phenomenon. the attraction of hemocytes toward a neurogenic niche and respecification of these cells toward a neural fate has been strongly suggested. based on recent and emerging research, we suggest that cells of the blood lineage are not only associated with the roles that are generally attributed to them, but are the cells that either signal other cell types to differentiate into neural cells, or even eventually themselves transdifferentiate into neural cells. key words: neuroregeneration; neurogenesis; blood cells; stem cells; hematopoietic tissue; ascidians; crustaceans   introduction the ability of many animals to regenerate their whole body or substantial parts of the body is a remarkable biological phenomenon that is nonuniformly represented in different phyla. mammals, ___________________________________________________________________________ corresponding author: paula grazielle chaves da silva laboratório de neurobiologia comparativa e do desenvolvimento instituto de biofísica carlos chagas filho universidade federal do rio de janeiro av. carlos chagas filho 373 bloco g2-001 ilha do fundão, 21949-902 rio de janeiro, rj, brazil e-mail: p.chaves@biof.ufrj.br for example, have a limited capacity to regenerate and restore tissues and organs. mechanisms associated with natural regeneration include dedifferentiation, reprogramming and transdifferentiation (jopling et al., 2011). transdifferentiation, the switch of lineages to create another cell type, is often illustrated by the change of pancreas exocrine cells to endocrine beta cells following a lesion (bouwens, 1998). however, as will be emphasized in the following section and elsewhere, it is now believed that certain normal phenomena, such as adult neurogenesis, may involve transdifferentiation (see mezey and brownstein, 2015, for a review). 142   the role of mesenchymal stem cells and blood cells in the repair of lesions in the central nervous system of vertebrates mesenchymal stem cells (mscs) were originally identified as a population of fibroblastic cells that are found in the bone marrow of vertebrates, and that are distinct from the hematopoietic lineage (friedenstein et al., 1976). however, mscs, considered to be multipotent, can also reside in other adult tissues, including circulating blood cells (kuznetsov et al., 2001; anker et al., 2003; miura et al., 2003; rosada et al., 2003; salingcarnboriboon et al., 2003; vandenabeele et al., 2003; igura et al., 2004; seo et al., 2004; tsai et al., 2004; toma et al., 2009; delorme et al., 2010). a series of studies have proposed that mscs have a higher potential for differentiation than previously thought, including the ability to form both endodermal and ectodermal tissue (petersen et al., 1999; mezey et al., 2000; sanchez-ramos et al., 2000; woodbury et al., 2000; krause et al., 2001; woodbury et al., 2002; for a review see mezey, 2007). in recent years, some investigators have challenged the notion that multipotent stem cells are restricted in their potency to the formation of cell types that have originated from only one embryonic germ layer. several authors have reported that different stem cells can form cell types of other germ layers, a process termed transdifferentiation. dedifferentiation can also explain the regeneration of body structures. during tail regeneration in salamanders, for example, mature muscle fibers lose their myofibrillar structure, their nuclei become enlarged, and mononucleate cells proliferate to populate the specific mass of cells that are capable of growth and regeneration, the blastema. endogenous muscle fibers lying next to the site of experimental amputation dedifferentiate and form mononucleate cells, which constitute a proportion of the blastema (echeverri and tanaka, 2002). in addition to being regarded as possible candidates for the treatment of diseases affecting mesodermal tissues, due to the functional recovery observed in various animal models with neural lesions, mscs are being considered as potential candidates for neurological treatments. three main hypotheses have been proposed to explain mscmediated neurogenesis and neural repair: 1) transdifferentiation (sanchez-ramos et al., 2000; woodbury et al., 2000; mezey et al., 2000); 2) cell fusion (terada et al., 2002); and 3) paracrine activity through the release of soluble factors (urdzíková et al., 2006). while there is evidence for all three phenomena, the debate over the degree of contribution of each of these models continues. stem cells derived from the umbilical cord have also been the subject of research focusing on repairing lesions in the brain of mammals. human umbilical-cord blood cells proved to be able to differentiate into neurons in vitro (sanchez-ramos et al., 2001) and when transplanted into the developing rat brain (zigova et al., 2002). in adult rats, peripheral-blood progenitor cells  reduced behavioral and functional deficits associated with cerebral infarction (willing et al., 2003). more recently, the umbilical cord matrix has been confirmed as a suitable source of mscs for applications in neurodegeneration, due to their primordial nature, neural-like plasticity, and readily availability with no significant ethical concerns (leite et al., 2014; frausin et al., 2015). therefore, future therapies based on the use of peripheral blood cells for treatment of cns diseases are a real possibility. stem cells in invertebrates life-long growth without fixed limits is typical of some evolutionarily very successful groups of aquatic invertebrates, such as echinoderms, bivalve molluscs and decapod crustaceans. these animals continue to enlarge their organs as adults and can regenerate lost appendages and organs, which is in sharp contrast to mammals and most insects. interestingly, according to specialized literature, echinoderms (ebert, 2008), bivalve molluscs (schöne et al., 2005) and decapods (vogt, 2010, 2012a) only rarely develop neoplastic and agerelated diseases, although some species can live for more than 100 years. maximum ages range from 40 days to 72 years for decapods, 1 to 375 years for bivalves, and almost 200 years for echinoderms. there are indications that their stem-cell systems have co-evolved with their successful environmentadapted life histories, suggesting that study of their features may offer new insights into stem-cell biology. in fact, several types of adult stem cells, as well as some types of mature cells that are capable of dedifferentiating into multipotent progenitor cells have been identified (vogt, 2012b). hydrozoans (phylum cnidaria) have a great capacity for regeneration, and the presence of multipotent stem cells in these organisms plays an important role in their normal development and also in regeneration (gierer, 1977; chandebois, 1976). cnidarians are phylogenetically basal members of the animal kingdom and show unlimited regeneration capacity and immortality. immortality can be described as the asexual mode of reproduction that requires cells with an unlimited self-renewal capacity (watanabe et al., 2009). cnidarian stem cells can give rise to a number of differentiated cell types, including neuronal and germ cells. their phylogenetic position, at the base of the metazoan branch of the evolutionary tree, makes them an important link in clarifying the mechanisms of stem-cell biology that are common to both animals and plants (watanabe et al., 2009). among many stem-cell markers in cnidarians, the transcription factor forkhead box o (foxo) has been shown to modulate the proliferation capacity of stem cells in order to regulate the lifespan and delay aging (boehm et al., 2013). knowledge of the biology of stem cells of the blood-cell lineage has grown since the discovery that they can be isolated from various adult organs, cultured, made to differentiate into different cell types, and put back into host organisms. the special attention to stem cells has also resulted in lines of research aiming at determining the evolutionary origin and subsequent modifications of this cell type. in flatworms, undifferentiated stem cells called neoblasts are morphologically comparable to vertebrate hemocytoblasts (andrew, 143   1965), and are able to differentiate into all cell types during normal postembryonic development and regeneration (ehlers, 1985). stem-cell research has used drosophila (hartenstein, 2013) to genetically tag individual stem cells and to observe their ability to self-renew for long periods (morrison and spradling, 2008). this period may range from 7 (lópez-oneiva et al., 2008) to 25 days (nystul and spradling, 2007). the resident stem cells from different sources, including stromal (lópez-oneiva et al., 2008) and epithelial niche (nystul and spradling, 2007) are maintained in an undifferentiated state using a short-range intercellular signal. these mechanisms of stem-cell maintenance are important to understand both the regulation of homeostasis and the possible alterations that may occur during adulthood (morrison and spradling, 2008). the classification of blood cells of invertebrates, the hemocytes, is not yet uniform, and many different terminologies are used to identify these cells. in some cases, differences in terminology exist even for the same species. hartenstein (2006) attempted to reconcile the terminology for invertebrate blood cells with that used for vertebrates, based on highly conserved molecular pathways involved in the development and function of these cells. among the characteristics of both vertebrate and invertebrate blood cells, blood-cell lineages diverge from a common type of progenitor cell, the blood stem cell (hartenstein, 2006). in general, the classification of circulating hemocytes in invertebrates is based mainly on the amount of granules in the cytoplasm and the nucleus/cytoplasm ratio (johansson et al., 2000). in crustaceans, including the crab ucides cordatus (chaves da silva et al., 2010), three types of hemocytes can be identified: hyalinocytes (agranular cells), semigranular cells (cells with small granules), and granular cells (cells with large granules) (bauchau, 1981; söderhäll and smith, 1983; martin and graves, 1985; hose et al., 1990; johansson et al., 2000; van de braak et al., 2002;. battison et al., 2003). the hyalinocytes are considered the immature blood cell type (cochennec-laureau et al., 2003). in ascidians (phylum chordata, class ascidiacea), the largest and the most diverse class of the subphylum tunicata, which is considered a sister-group of vertebrates, the blood stem cell is termed the hemoblast and shares many characteristics, specification mechanisms and regulatory pathways with the blood stem cells of vertebrates (sawada et al., 1993; cima et al., 2001; ballarin and cima, 2005; de barros et al., 2007; ballarin and kawamura, 2009; de barros et al., 2009; de barros et al., 2014). hemoblasts have been extensively studied in the colonial ascidian botryllus primigenus, and a few studies have examined solitary species. in a colonial ascidian, two types of hemoblasts have been described, somatic and germ-line hemoblasts. they are morphologically similar and show multipotent capacity, but have different cell markers (kawamura and sunanaga, 2010). in the solitary ascidian styela plicata, hemoblasts were shown to be spherical cells with a high nucleus/cytoplasm ratio. they appear either circulating (figs 1a, b) or in the hematopoietic tissue (fig. 1c). their cytoplasm contains few or no granules and few visible organelles, and the nucleus contains one or more nucleoli (de barros et al., 2009, 2014). at most, they comprise 1-2 % of the celomic population (wright, 1981). hemoblasts can be found within the hemolymph or aggregated in compartments that maintain and regulate their fates in ascidians, the so-called hematopoietic niches or stem-cell nodules, such as the branchial vessels, intestine submucosa, and vascular ampullae of colonial ascidians (ermak, 1976; voskoboynik et al., 2008; rinkevich et al., 2013). although the clear morphological characteristics of ascidian blood progenitors have been difficult to identify reliably, due to a lack of maintenance and differentiation markers or of reports using molecular-biology techniques, nevertheless, important markers do exist, many of which are described in colonial ascidians. fig. 1 ascidian hemoblasts. (a) from phallusia nigra, observed by light microscopy after removal from circulating hemolymph (stained with toluidine blue). (b) from styela plicata, observed by transmission electron microscopy after removal from circulating hemolymph. (c) from s. plicata, obtained from hematopoietic tissue located in the intestine submucosa. 144   ascidian hemoblasts express a cd34-like antigen (medina et al., 2014). cd34 is a human hematopoietic molecular marker conserved in mammals, and is a glycoprotein involved in adhesion mechanisms (gallacher et al., 2000). although ballarin and cima (2005) reported the presence of the hematopoietic marker cd34 in botryllus schlosseri, recently, braden et al. (2014) did not find any homologue gene to cd34 in the same species. therefore, it remains an open question whether this gene is evolutionarily conserved in ascidians, since genetic sequencing has not yet been conducted in many ascidian species. based on investigations conducted on both solitary and colonial ascidians, germ-line cells strongly express a vasa homologue gene involved in the formation or development of germ cells (fujimura and takamura, 2000; brown and swalla, 2007). recently, the piwi ascidian stem-cell marker was identified in the colonial tunicate botrylloides leachi (rinkevich et al., 2010). piwi belongs to the evolutionarily conserved piwi/argonaute superfamily of rna interface effector proteins, which are essential for both self-renewal and maintenance of germ-line and somatic stem cells in various multicellular organisms (cox et al., 1998; carmell et al., 2007; brown et al., 2009). hemoblasts play a key role in tissue renewal during reproduction and regeneration. some of them differentiate into somatic-lineage cells, such as endodermal multipotent epithelial, cardiac, muscle and blood cells (burighel and cloney, 1997); and others into germ cells, known to regenerate the whole body of botryllid ascidians (sunanaga et al., 2006; rinkevich et al., 2013). this phenomenon can be explained when the proportion of hematopoietic stem cells found in humans (pike-overzet et al., 2009) is compared with that in ascidians: humans have 1 stem cell per 100,000 circulating blood cells, whereas botryllid ascidians have 1 per 5,000 (laird et al., 2005; brown et al., 2009). hematopoietic cells in invertebrates although the concept of a niche was initially proposed by schofield (1978) for mouse hematopoiesis, it is probably fair to say that the in vivo signaling properties of niche cells were first discovered in invertebrate germline stem cells, including drosophila and caenorhabditis elegans (xie and spradling, 2000; kiger et al., 2001; crittenden et al., 2002). studies on the regulatory functions of local cell types in hematopoietic niches of invertebrates have been reviewed by adams and scadden (2006). four main ideas derived from invertebrates were described in the review: 1) the number of stem cells in a niche is tightly regulated; 2) physical interaction among heterologous types of cells is important for the maintenance of the stem-cell state; 3) products of the niche provide the molecular basis for physical interactions and a balance of inhibitory and stimulatory signals governing stem-cell number and function; and 4) niche occupancy can impose 'stem cell-like' characteristics on some cells, even if they are not stem cells. therefore, the authors suggested that examination of the pathways and perhaps the structural components of invertebrate stem-cell niches may improve the understanding of how the specialized microenvironment can affect mammalian stem cells (adams and scadden, 2006). the hematopoietic tissue in invertebrates (hpt) is responsible for the production and release of circulating blood cells (hemocytes). in crustaceans the hpt is composed of a number of ovoid lobules, which form a thin sheet on the dorsal part of the stomach (foregut), and are surrounded by connective tissue (chaga et al., 1995; martin et al., 1993; johansson et al., 2000). this location makes crustaceans a suitable model for studying hematopoiesis, because the tissue can be readily isolated, and the proliferation of stem cells and their differentiation can be studied both in vivo and in vitro. in many crustaceans, the various types of hematopoietic cells are mainly characterized by their morphology as seen under either light or electron microscopy, and are described based on the amount of granules and/or through biochemical analyses (söderhäll and smith, 1983; johansson et al., 2000). however, the characterization of the hematopoietic cells depends on the species. according to van de braak et al. (2002), in the shrimp penaeus monodon, the type 1 cells are the presumed precursor cells that originate a largeand a smallgranular hemocyte, called type 2 and type 3 cells, respectively. the type 4 cells show typical features of interstitial cells. in contrast, five different cell types have been found in crayfish: the type 1 and 2 cells are the main proliferating cells in the hpt, whereas the other cell types are precursors of granular and semigranular cells (lin and soderhall, 2011). recently, noonin et al. (2012) demonstrated that the hematopoietic system in crustaceans is far more extensive than previously recognized. a specialized region of the hematopoietic system, called the anterior proliferation center (apc) and located near the brain, is proposed to constitute a multipotent stem-cell center. in fact, the apc is only one part of a much more extensive hematopoietic system. it contains actively proliferating cells in the anterior part of the tissue near the area linking the hpt to the brain (fig. 2). anatomical and morphological analyses showed that the apc lies within the cor frontale, or auxiliary heart, which pumps hemolymph to the brain and eyes through the cerebral and ophthalmic arteries, respectively. interestingly, in both areas there is neurogenesis. these data, together with the fact that brdu-positive cells were observed within the dorsal median artery, which comes from the posterior hpt forward to the brain, suggest that apc cells are multipotent stem cells (chaves da silva et al., 2013). the hypothesis is that apc cells probably establish a communication with the brain in order to populate the neurogenic niche located in the brain of crustaceans (sullivan et al., 2007), and possibly to interact with resident niche cells by releasing growth factors or even to transdifferentiate into neural progenitor cells (chaves da silva et al., 2013). although there is no evidence for transdifferentiation of hemocytes into neural cells, the study of benton et al. (2014) identified hemocytes as a source of adult-born neurons in crayfish, and demonstrated that the immune system is a key contributor to adult neurogenesis. 145   fig. 2 scheme of the crayfish procambarus clarkii, showing the hematopoietic system. (a) the posterior hematopoietic tissue (hpt-blue) extends bilaterally toward the head, raising the anterior proliferation center (apc-red). (b) scheme showing only the hematopoietic system and the brain. the dorsal median artery (dmared line) links the posterior hpt (blue) to apc (red), which is between the posterior-hpt and the brain. the apc surrounds the cor frontale muscles (cfm), within the cor frontale, an auxiliary heart that pumps hemolymph toward the brain through the cerebral artery (ca) (image modified from chaves da silva et al., 2013). (c) scheme of brain (illustrated in b) showing the cerebral artery (ca) entering the brain and bilaterally accessing the neurogenic niche (niche) on the surface of the accessory lobe (al). (ol) olfactory lobe. adult neurogenesis in invertebrates neurogenesis persists in the adult brains of various animals, and is a common phenomenon that is not correlated with their phylogeny or the overall complexity of their brains. based on the review by cayre et al. (2002), it is very likely that similar processes underlie neurogenesis in the adult brain of both invertebrates and vertebrates. in vertebrates, adult neurogenesis has been widely explored in specific regions of the brain, including the hippocampus of mammals (altman and das, 1965, gheusi and lledo, 2007; ninkovic et al., 2007). among invertebrates, adult neurogenesis has been analyzed mainly in arthropods, i.e. in several species of insects and decapod crustaceans, with no information available for numerous other taxa and countless species (schmidt, 2007). 146   fig. 3 scheme of the role of hematopoietic stem cells in the crustacean procambarus clarkii during adult neurogenesis and in the ascidian styela plicata during brain repair. in the crayfish, the anterior proliferation center (apc), a part of the hematopoietic tissue, produces multipotent stem cells that have access to the brain and populate the neurogenic niche, possibly differentiating into neurons. in the ascidian, after the injection of a neurotoxin into the brain, the hematopoietic stem cells migrate to the cerebral ganglion for repair. in insect brains, adult neurogenesis only occurs in the mushroom bodies, which are higher-order multimodal integration centers (cayre et al., 1994, 1996; gu et al., 1999; dufour and gadenne, 2006; cayre et al., 2007). however, neurogenesis does not occur in many insect groups, such as dictyoptera and acrididae (cayre et al., 1996) or in honeybees (fahrbach et al., 1995); and lasts only several days into adulthood in cockroaches (gu et al., 1999). in decapod crustaceans, new neurons are continuously produced in two brain areas: the optic lobes and the central olfactory system (harzsch and dawirs, 1996; schmidt, 1997; sandeman et al., 1998; sullivan et al., 2007), which makes them useful animal models for providing insights into adult neurogenesis. in the crayfish procambarus clarkii (phylum arthropoda; subphylum crustacea), adult neurogenesis involves at least three generations of precursor cells (sullivan et al., 2007). the firstgeneration precursor cells reside in a vascularized neurogenic niche. these bipolar niche cells also provide a stream along which their progeny migrate. the second-generation cells are migratory precursors that move toward the proliferation (medial and lateral) zones where the neuron cell bodies are grouped in clusters (clusters 9 and 10). the progeny differentiate into cluster 9 (local) and cluster 10 (projection) olfactory neurons, respectively (sullivan and beltz, 2005). interestingly, the first generation of neuronal precursors migrate away from the niche after each cellular division, suggesting that niche cells are not self-renewing and that the niche is supplied by another cellular source. several studies have shown, by different techniques, a close relationship between the neurogenic niche and the vascular system/blood cells: 1) dye-conjugated dextran injections within the dorsal artery showed that the neurogenic niche lies on a complex vasculature network (sullivan et al., 147   2007; sandeman et al., 2009); 2) the vasculature is connected to the niche beginning in the early stages of development (sintoni et al., 2012); and 3)  the mature brain is surrounded by blood vessels, as observed in semi-thin sections (zhang et al., 2009),  and blood cells are frequently found in the connective tissue below the neurogenic niche, showing similar morphology with one of the cell types in the niche (chaves da silva et al., 2012). taken together, these data reinforce the close relationship of the blood cells and vasculature with the neurogenic niche. blood cells are selectively attracted to the niche via the vasculature. in vivo experiments using blood cells (hemocytes) and cells from three different types of tissue were isolated and labeled with a fluorescent marker. they were then set on a crayfish brain culture in order to observe the affinity of these cells to the neurogenic niche. interestingly, only hemocytes, especially semigranular cells, were significantly attracted to the niche, particularly to the vascular cavity (benton et al., 2011). semigranular cells are derived from a precursor cell named type i, the hematopoietic stem cell (lin and söderhäll, 2011). recently, benton et al. (2014) have also shown that hemocytes are able to invade the neurogenic niche and that their descendants are able to differentiate into neurons. this suggests that the crustacean brain has an "open" niche which is populated by blood-born cells, and that they are able to differentiate into neural progenitors. these studies reinforce the classical view that stem cells possess greater differentiation potential than previously thought, as we stated above for vertebrates. in crustaceans as well, it seems that the ectodermal origin of embryonic neural tissues is not the only source of neurons in the adults. a morphological study, using transmission electron microscopy, also demonstrated a specific cell type in the niche, which is by far the most numerous cell type, and that shows characteristics which suggest a role in regulating transport from the blood into the niche cells. it lines a vascular cavity located in the center of the niche, which is confluent with the vascular system of the animal (chaves da silva et al., 2012). through different lines of reasoning, studies in the crayfish p. clarkii have led to the idea that cells emerging from the hematopoietic system, and circulating in the hemolymph may reach the neurogenic niche and transform into neuronal precursor cells (zhang et al., 2009; beltz et al., 2011; sintoni et al., 2012; chaves da silva et al., 2012). the reasoning is as follows: 1) ablation of the hematopoietic tissue results in a decrease of proliferating cells within the neurogenic niche (benton et al., 2014); 2) cells that surround the niche show similar morphology to cells of the apc (chaves da silva et al., 2013); 3) blood vessels, perivascular cells and hemocytes have the capacity to penetrate the tissue surrounding the neurogenic niche (zhang et al., 2009; benton, 2011; chaves da silva et al., 2012, 2013). these data suggest that blood stem cells may be a cellular source to supply the niche and also to ‘‘transdifferentiate’’ into a neural progenitor (for a review, see hartenstein, 2014). regeneration and repair in invertebrates many animals are able to regenerate at least to some degree, as a strategy to maintain form and function throughout life. many studies, closely linked with stem-cell research, have been conducted in different animal models, and have provided some of the most promising information to advance regenerative medicine. however, not all animal tissues have the same capacity to regenerate. the extreme example of loss of the regenerative function is the most complex and intriguing system of the body, the nervous system (tanaka and ferretti, 2009; bonfanti, 2011). some representative published data on the use of different invertebrate models for regeneration are shown in table 1. in metazoans in general, the capacity of the cns to regenerate decreases in parallel with an increase in complexity (brockes and kumar, 2008). however, tunicates, the group to which ascidians, the sister group of vertebrates, belong, have a high cns regenerative capacity. the first complete study of the regeneration of their brain used ciona intestinalis as the experimental model (mingazzini, 1891). the cns was ablated and the recovery of neural components was observed. this recovery is considered a unique phenomenon among chordates (jeffery, 2015). experiments using the same method of mechanical ablation of the cerebral ganglion of c. intestinalis showed that the entire cns can be regenerated within a few weeks (bollner et al., 1997; dahlberg et al., 2009). bollner et al. (1997) suggested that post-mitotic cells migrate to the redeveloping ganglion cells external to the cns, and dahlberg et al. (2009) showed that neurons supply cells for reformation of the central network. our group has recently shown that hematopoietic stem cells were present in the regenerating nervous tissue of the ascidian s. plicata, after neuronal damage was produced with 3-acetylpyridine (medina et al., 2014). in some groups such as annelids, the cns is efficiently and functionally regenerated following mechanical trauma (baylor and nicholls, 1971; jansen and nicholls, 1972), and the attraction of microglia/macrophages is a key step in the engagement of an adaptive response leading to axonal sprouting. this may suggest that an optimal regeneration requires microglia/macrophages for initiation of cns regeneration (baylor and nicholls, 1971). interestingly, regeneration in the leech hirudo medicinalis (phylum annelida) proved to remain possible when glial cells were destroyed by intracellular injection of protease (elliott and muller, 1983). in this context, we can ask what other cell types might be involved in the regeneration of the leech cns. in the leech, it has been suggested that blood cells are important to facilitate and accelerate the process of regeneration (boidin-wichlacz et al., 2012). furthermore, circulating blood cells also appeared capable of infiltrating the injured cns where, in conjunction with microglia, they limited the formation of a scar (boidin-wichlacz et al., 2012). in contrast, in mammals, cns injury leads to the generation of a glial scar that blocks the mechanism of regeneration by preventing axonal regrowth (fitch and silver, 2008). 148   table 1 some representative published data on the use of different invertebrate models for regeneration and neurogenesis as noted above, a hematopoietic site was found to be correlated with adult neurogenesis in crustaceans (noonin et al., 2012). however, although neurogenesis is associated with regeneration in adult animals, to better comprehend regeneration in crustaceans, the first step is to understand the cellular and molecular basis of nerve-fiber degeneration. we have addressed this issue by characterizing cellular and biochemical strategies peculiar to neurodegeneration in crustaceans (corrêa et al., 2005; chaves da silva et al., 2010, 2013). immune cellular features, such as: 1) the recruitment of granulocytes, and secondarily, of hyalinocytes to the lesion site; 2) the attraction of 149   a larger number of cells 48 h after the lesion (subacute phase) than 24 h after the lesion (acute phase); and 3) the presence of activated glial cells, revealed with microglia/macrophage markers, suggest that molecules released from granulocytes in the acute phase attract hyalinocytes, thus producing more undifferentiated cells that are able to differentiate into either mature blood cells or even other lineages (chaves da silva et al., 2010, 2013). according to hartenstein (2006), hemocyte progenitors are rarely released into circulation under normal conditions, but they can appear under 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(cibnor). laboratorio de referencia, análisis y diagnostico en sanidad acuicola, calle hermosa 101. col. los angeles, cp 83106, hermosillo, sonora. mexico 4departamento de ciencias químico biologicas y agropecuarias, universidad de sonora unidad regional sur, apartado postal 85390, navojoa, sonora, mexico accepted february 2, 2015 abstract betaine aldehyde dehydrogenase (badh) belongs to the aldehyde dehydrogenases (aldh) family, an ancestral group of enzymes responsible for aldehyde detoxification in several organisms. the badh enzyme catalyzes the irreversible oxidation of betaine aldehyde to glycine betaine (gb) an important osmoptrotector and osmoregulator accumulated in response to cellular osmotic stress. the badh enzymes have been extensively described in terrestrial organisms, but information in marine crustaceans remains scarce. research on crustacean stress-adaptive capacity to environmental stressors relates gb accumulation in response to salinity variations. although gb de novo synthesis is confirmed on crustaceans, its metabolic pathways and regulation mechanism are unexplored. in this work, the state of the knowledge of betaine aldehyde dehydrogenase enzymes in marine crustaceans is summarized, as a mechanism to overcome the deleterious effects of changes in temperature, salinity and dissolved oxygen concentration in seawater. the purpose of this review is to provide a more comprehensive overview to set the basis for exploring novel functions and properties of badhs on the response of crustaceans to environmental stress. key words: betaine aldehyde dehydrogenase; stress response; osmoregulation; glycine betaine   introduction besides being the largest and most dynamic reservoir of biomass on earth, oceans are the ultimate repository for a vast amount of discharged compounds via human activities (kennish, 1996). in addition, marine dynamics promote continuous changes in environmental factors such as temperature, salinity and dissolved oxygen concentration, which, in addition to the presence of marine pollutants, significantly affects marine wildlife. the deterioration of the environmental conditions in the oceans may eventually lead to warming, ___________________________________________________________________________ corresponding author: jesús a rosas-rodríguez departamento de ciencias químico biologicas y agropecuarias universidad de sonora unidad regional sur apartado postal 85390, navojoa, sonora, mexico e-mail: jrosas@navojoa.uson.mx eutrophication, acidification, oxygen depletion (hypoxia) and the accumulation of toxic compounds as ammonia and sulfides (fabry et al., 2008; hoegh-guldberg and bruno 2010; stiti et al., 2011). a long list of toxic contaminants has been found affecting the marine ecosystem. oil, gas and agricultural wastes are among the most studied pollutants affecting the marine environment. in addition, bioactive metabolites synthesized by microalgae in marine ecosystems, as various unsaturated aldehydes, are among the most toxic compounds(leflaive and ten ‐ hage, 2009 the interaction between the aldehyde functional group of such secondary microalgal metabolites with biological molecules increases its toxicity. nucleophilic varieties of primary amines and sulphydryl or hydroxyl containing molecules are the main class of compounds that can be targeted by aldehydes, and such interactions disrupt biological function and compromise cell integrity (taylor et al., 66   mailto:jrosas@navojoa.uson.mx 67   2005). thus, it is known that aldehyde accumulation causes cellular dysfunction through protein/dna damage, membrane destruction, and oxidative stress. detoxifying enzymes belong to the second line of cellular defense on aquatic organisms and contribute to their survival. aldehyde dehydrogenases (aldh) are an extensive group of enzymes involved in the oxidation of environmental aliphatic and aromatic aldehydes, endobiotic or xenobiotic metabolism and lipids peroxidation (jackson et al., 2011). oxidation of reactive aldehydes to their corresponding carboxylic acids, a major detoxifying pathway of aldehydes, occurs through the reaction catalyzed by aldhs (stiti et al., 2011). the resulting carboxylic acids, as glycine betaine (gb), are subsequently involved in cell growth and osmoregulation. furthermore, the aldh enzymes have a non-catalytic function as antioxidant, uv radiation absorption and direct binding to protect molecules from cellular stressors (vasiliou et al., 2013). betaine aldehyde dehydrogenase (badh) belongs to the aldh9 family and catalyzes the irreversible oxidation of betaine aldehyde to glycine betaine (gb, or n, n, n-trimethyl amine) which is an essential organic osmolyte accumulated in cells under hypertonic stress for cell volume and turgor control (burg et al., 2007; burg and ferraris, 2008; chen and murata, 2011). gb plays a key role in animals as an osmolyte and osmoprotectant to counteract cellular osmotic stress (perozich et al.,1999; julián-sánchez et al., 2007). studies on gb accumulation in marine organisms have demonstrated that it participates in several biological processes including arsenic accumulation (fujihara et al., 2003), osmotic adjustment (de vooys and geenevasen, 2002), modulation of feeding periods (treberg and driedzic, 2007), and prevention of accumulation of denaturing agents (trischitta et al., 2012). despite the role of badh in osmoregulation, most studies on badh enzymes are focused on terrestrial animals, bacteria and plants, while the information regarding its mode of action in aquatic organisms remains scarce (arakawa et al., 1987; pan, 1988; arakawa et al., 1990; valenzuela-soto and muñozclares, 1994; guzman-partida and valenzuelasoto, 1998; chern and pietruszko,1999; velascogarcía al., 1999; figueroa-soto and valenzuelasoto, 2000; muñoz-clares and valenzuela-soto, 2008). to date, several studies have been focused on understanding the biology of marine crustaceans, particularly of commercially important species such as shrimp, crabs and lobsters, whose physiological abilities to face environmental stress have been demonstrated through their permanence in the marine ecosystem. previous evidence confirms that crustaceans under conditions of osmotic stress may display response mechanisms such as accumulation of a variety of organic solutes (osmolytes) in an effort to counteract the resulting movement of water. these osmolytes may include free amino acids, polyhydric alcohols, quaternary ammonium or tertiary sulphonium compounds (jahn et al., 2006). few studies have focused on the accumulation of osmolytes in euryhaline marine invertebrates, including mollusks and crustaceans, under different osmotic stress conditions (pierce et al., 1995). however, the key enzymes participating in such regulatory mechanism, as badh, have not been studied, and their roles remain to be understood. the aim of the review is to summarize the current state-of-the-art on aldehyde dehydrogenases from marine invertebrates, especially those enzymes that allow crustaceans to overcome osmotic stress conditions, in order to explore new functions and properties of this fascinating group of enzymes. also, understanding the pathways to synthesize osmoprotectants may help to increase our knowledge about the adaptive mechanisms of these marine species and their differences with some other animal models. aldehyde dehydrogenases super family and betaine aldehyde dehydrogenase the large family of aldehyde dehydrogenases (aldh) may play two central roles: the enzymatic detoxification of diverse aldehyde compounds, and the synthesis of molecules such as retinoic acid, which participate in central cellular processes. the diverse functions of this family of enzymes includes production of biomolecules, modulation of cell survival, osmoregulation and osmoprotection, hormone interactions, among others (jackson et al., 2011). the number of aldh genes is constantly increasing, encoding proteins belonging to 24 distinct aldh families; however, the biological roles of some of these enzymes are still not elucidated (jackson et al., 2011; vasiliou et al., 2013). aldhs are a numerous and ancestral group of enzymes classified on various families: the mitochondrial and cytosolic aldh1 family is involved in retinoic acid signaling; mitochondrial enzymes from animals and plants (aldh2) are particularly important enzymes for human pathologies, they are associated with acetaldehydes from alcohol metabolism, and aldh2 dysfunction may contribute to a variety of human diseases. aldh3 family includes dioxin-inducible enzymes from animals, metabolizing aldehydes from lipid peroxidation; aldh4, also known as pyrroline-5-carboxylate dehydrogenases (p5c) are mitochondrial enzymes that catalyze the second step of the proline degradation pathway; the succinate-semialdehyde dehydrogenases aldh5, which is essential for the removal of the gaba metabolite succinic semialdehyde; the methyl malonate semialdehyde dehydrogenases (aldh6), and the animal and plant antiquitin group (aldh7) that participates in lysine catabolism; the cytosolic (aldh8) that participate on retinaldehyde metabolism; and betaine aldehyde dehydrogenases from animals (aldh9) and plants (aldh10) (vasiliou et al., 1999; julian-sánchez et al., 2007; swenby and picklo, 2009). the betaine aldehyde dehydrogenases (badhs, e.c. 1.2.1.8.), the central topic of this review, belong to the aldh9 family and play a key 68   table 1 aldh9 enzymes identified in various marine species organism/ enzyme tissue/ subcellular location protein size (kda) accesion no. kinetics parameters references horseshoe crab limulus polyphemus heart kmba: 133 m vmaxba:220 μmoles/min/ml kmnad: 22 μm kmnadp: 267 μm dragolovich, 1994 oyster/badh crassostrea virginica gills/ mitochondria 62.3 kmba: 360 μm vmax: 1.7 μmoles/min/ml kmnad: 350 μm perrino and pierce, 2000 fish haplochromis burtoni 54.8 xp_005919907 nd cod gadus morhua liver 54.3 p56533.1 kmba: 140 μm johansson et al., 1998 zebra fish danio rerio 55.2 np_958879 ling et al., 2012 plankton calanus helgolandicus 6.4 aep43737.1 lauritano et al., 2012 sea hare aplysia californica 56.1 xp_005091798.1 knudsen et al., 2006 elephant shark callorhinchus milii 56.9 xp_007893828.1 venkatesh et al., 2014 coelacanth latimeria chalumnae blood 53.4 xp_005994849.1 zardoya and meyer, 1997 green sea turtle chelonia mydas 51.7 emp34519.1 wang et al., 2013 role in the metabolic pathways of organisms. as previously mentioned, badhs participate in the betaine aldehyde oxidation to glycine betaine, an osmotically active solute synthesized in cells and released into the blood stream, acting as an osmoprotectant compound (muñoz-clares and valenzuela-soto, 2008). despite badhs is a source for gb accumulation for osmotic adaptation, animals badhs have been described as γaminobutyraldehyde and trymethylaminobutyraldehyde dehydrogenases (tmaba-dh) because their ability to accept other aminoaldehydes as a substrate (vaz et al., 2000). this implicates badh in the carnitine biosynthesis, an indispensable compound for the transport of activated fatty acids in mitochondria. moreover, high levels of l-carnitine have been reported in distinct invertebrates (özogul et al., 2013). to date, mammalian badhs are by far the most studied enzyme of this family (julián-sánchez et al., 2007). the human aldh9 was intensely studied during the 1980s and 90s. pietruszko (1997) postulated various roles for the enzyme, including the conversion of betaine aldehyde to betaine and its putative function as a defense against osmotic stress; however, the enzyme structure was not solved until 1998, when the cod liver betaine aldehyde dehydrogenase was described as similar to that of other aldhs, as aldh2 (mitochondrial) and aldh3 (induced by dioxins). each subunit of this tetrameric enzyme contains a coenzyme binding domain, a catalytic domain, and an oligomerization domain; and the conserved residues asn and cys comprising the substrate binding pocket were identified (johansson et al., 1998). yoshida et al. (1998) described the human aldehyde dehydrogenase gene family. a total of twelve genes encoding the enzyme were described, including group aldh9; this last group of genes is mainly expressed in liver, kidney, and muscle. soon thereafter, the first mitochondrial form of the rat liver badh was identified, and the existence of fig. 1 kinetic mechanism for choline oxidation and glycine betaine synthesis. cdh: choline dehydrogenase, badh: betaine aldehyde dehydrogenase, ba: betaine aldehyde, gb: glycine betaine. two isoenzyme forms (cytosolic and the mitochondrial), encoded by the same gene, was suggested (chern and pietruszko, 1999; pietruszko and chern, 2001). in recent years, developments in the field have led to an increase in the amount of information on aldh9 and badh genes, and on proteins from several species, with approximately 16,765 entries from pfam database (jackson et al., 2011). however, only a limited number of these enzymes have been biochemically and kinetically characterized. even though badhs play a key role in animal adaptive mechanisms, information about these enzymes in marine species is scarce (table 1). the crustacean badhs an increasing number of studies about aldhs from marine species has emerged from recent research developments, mainly as an effort to better understand their function, which is closely related to the ability of these animals to face stress conditions in their environment. the main function attributed to invertebrate badhs is their ability to synthesize glycine betaine (gb), an organic compound that can be produced in tissues and extracellular fluids of marine animals to balance their internal osmolarity with respect to the external environment (burg and ferraris, 2008). jahn et al. (2006) demonstrated for the first time that gb is accumulated in crustaceans as an organic solute involved in the osmotic adjustment during hyperosmotic stress and that metabolic responses occur mainly in the hepatopancreas of these species. the first isolated and characterized badh from crustaceans was reported by dragolovich (1994).the active enzyme was partially purified from mitochondria of the heart of the horseshoe crab limulus polyphemus, and showed high specificity for betaine aldehyde with a michaelis-menten constant (km) of 133 μm. the end product, gb, was demonstrated to be a competitive inhibitor of badh activity, and according to these findings, badh activity is modulated through the increase/decrease in concentrations of inorganic ions as k+ and na+. to the best of our knowledge, at present there are no reports of any amino acid sequences of partially purified, isolated or solved structure of crustacean badhs. thus, there is no evidence about the ability of crustacean’s badh to use efficiently aminoaldehydes as substrates, as is typically observed in mammalian enzymes (muñozclares and valenzuela-soto, 2008). in addition, there is a lack of information about the expression of badh in crustaceans as a response to environmental stress that correlates with gb accumulation. so far, badh activity of a small number marine invertebrate species has been detected using isolated mitochondria, such as that reported for l. polyphemus, whose enzyme is exclusively found in mitochondria (dragolovich, 1994). in the same way, badh activity has been found on the mitochondrial matrix of the oyster crassostrea virginica which controls the synthesis of gb. interestingly, the enzyme kinetics showed variations according to the extracellular ions concentration, being active even if the organism is not osmotically stressed (perrino, 2000). 69   fig. 2 proposed mechanism for osmotic response in crustaceans. intracellular choline intake is controlled by membrane choline transporters in response to ion concentration variations. short-term hyperosmotic stress is controlled through na+/k+ ions exchanges while long-term hyperosmotic stress requires osmolyte participation to maintain cellular homeostasis. choline is transported into the inner mitochondrial for gb synthesis and excreted to cytosol for osmoprotection and osmoregulatory process. betaine aldehyde dehydrogenase and the osmotic stress response osmotic stress is a recurrent condition for some marine species, particularly for those living in nearshore environments as estuaries where coastal rivers, streams, and the intense solar incidence and evaporation continuously change the salinity of sea water (true, 2012). most marine crustaceans are often considered as osmoconformers, with a high capacity of tissue water regulation that relies mainly on salt transport to prevent extracellular osmotic disturbances (foster et al., 2010). probably one of the more important adaptive traits of marine crustaceans is their high body fluid osmolality, similar to that of the seawater (near to 1,000 mosm). however, it has been demonstrated that when large variations in the water salinity are produced, the osmotic stress response of crustaceans involves gill transport mechanisms, changes in membrane permeability and a reduced production of concentrated urine (sherwood et al., 2004). also, a series of molecules including some enzymes participating in central metabolic pathways as oxidative stress proteins, chaperones, proteases, cytoskeletal proteins and detoxification of xenobiotics have been suggested as part of the response (tomanek, 2011). besides its well-known use of free amino acids as osmolytes, the synthesis of osmoprotectant quaternary bases in the mitochondrial matrix, such as gb, contributes in the adaptive response of marine organisms to high salinities. according to previous evidence, choline (n, n, n-trimethyl-βhydroxyethanolamine), which is supplied to shrimp 70   71   species as penaeus monodon via commercial feed formulations, is an essential dietary component and the precursor for the synthesis of gb (richard et al., 2011). previous studies have reported that in mammalian cells, choline is oxidized in kidney and liver mitochondria. a specific choline transporter in the inner mitochondrial membrane allows choline molecules to reach the inner side of the membrane, and the fad-linked membrane bound choline dehydrogenase (cdh) (ec 1.1.99.1) converts choline to betaine aldehyde; then the nad-linked betaine aldehyde dehydrogenase (badh) oxidizes betaine aldehyde to gb (porter et al., 1992; o'donoghue et al., 2009) (fig. 1). both enzymes, cdh and badh, are soluble proteins mainly found in the cytosol (grossman, 1989; figueroa-soto et al., 1999), and a small fraction of badh is also found in the mitochondria of vertebrates (chern and pietruzko, 1999). in crustaceans, mitochondria isolated from gills and hepatopancreas are able to uptake choline to convert it into gb, suggesting that the mitochondrial matrix may possess the required enzymes for choline metabolism (dragolovich, 1994). previous evidence showed, in addition, an accumulation of gb in hepatopancreas, gills and heart of the crab neohelice (chasmagnathus) granulata exposed to long-term hyperosmotic stress (72 h), demonstrating that gb regulation has an important role in the response to periods of prolonged stress (jahn et al., 2006). although the activity of choline dehydrogenase and betaine aldehyde dehydrogenase in crustaceans have not been characterized, the evidence of de novo synthesis of gb from choline suggests that crustaceans may possess mechanisms for the metabolism and transport of choline and gb (jahn et al., 2006). based on this information, a preliminary model for crustacean’s response to osmotic stress is proposed here (fig. 2). in this model, cellular choline, under hyperosmotic conditions, is taken up and subsequently transported to the inner mitochondrial membrane, where it is oxidized to gb and betaine aldehyde by cdh and badh activity, respectively. the synthesis and accumulation of gb in cells in response to long-term osmotic stress is important for the regulation of cell volume and protein stabilization. crustacean´s responses to hypoxia another environmental factor that continuously imposes a physiological challenge to crustaceans is hypoxia. according to previous studies, oxygen concentrations may vary from normoxic conditions (6 7 mg/l) to hypoxia (1 3 mg/l) in a diurnal cycle (puente, 2009); therefore, aquatic organisms display a set of biochemical strategies, as well as distinct metabolic pathways to ensure stress adaptation. these mechanisms involve a number of stress response proteins which are known to be highly regulated (palacios and racotta, 2007). according to the findings of jiang et al. (2009), the aldehyde dehydrogenase (aldh1 like) of the shrimp fenneropenaeus chinensis is differentially expressed in the hepatopancreas of shrimp exposed to hypoxia. although the badh of the shrimp penaeus vannamei has not been characterized, it may be involved in the response to the effects provoked by the white spot syndrome virus (wssv). this arises from the evidence of severe infection of shrimp after nad-dependent aldehyde dehydrogenase and hsp70 genes knockdown by double-stranded rna interference (dsrna). a conserved domain analysis identifies this enzyme as a member of aldh4 family, enzymes involved in pyrroline-5-carboxylate dehydrogenase activity. in addition, a beneficial interaction between these proteins, by promoting the inhibition of the virus replication process, was suggested. it was further proposed that aldh activity reduces the generation of toxic by-products, as aldehydes, during the oxidative stress caused by the observed hyperthermic protection of shrimp against wssv (lin et al., 2011). these results encourage the necessity to study other aldhs, such as aldh9 enzymes to elucidate their participation on stress response mechanisms in crustaceans. concluding remarks and future directions salinity oscillations, as other physical factors, including temperature and oxygen, affect the behavior, distribution, growth, reproduction, and survival of organisms inhabiting the oceans. such disturbances have shown to elicit adaptive responses that are crucial to protect the organism against subsequent exposure to similar environmental insult. when water salinity fluctuations occur, the osmotic pressure of the celomic fluid of marine invertebrates changes, which commonly leads to osmotic stress (torres et al., 2007; rhodes-ondi and turner, 2009). osmotic stress induces adverse effects over cellular ion regulation, which may lead to disruption of protein synthesis and protein damage (yancey et al., 1982; kültz, 2005; fiol and kültz, 2007). thus, osmoregulation is a central physiological mechanism intended to conserve the homeostasis of marine animals. the accumulation of gb is critical for crustaceans to achieve a full response against environmental stress, which requires a functional biosynthetic pathway for gb. enzymes catalyzing irreversible reactions are often considered as control sites of metabolic pathways, and the importance of badhs is evident from the fact that its principal substrate, betaine aldehyde, is exclusively synthesized from choline and seems to play a central role in controlling the abilities of marine crustaceans to deal with osmotic and environmental stress. the badhs investigated to date represent a heterogeneous group in accordance to their structure, localization and kinetic mechanism, relating these characteristics with enzyme participation at distinct metabolic levels. further investigations focused on badh biochemical and kinetic characterization is needed to increase the knowledge of stress regulation in 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(lamiaceae) essential oil (tp eo) and αpinene as a major component of this essential oil, were tested against ephestia kuehniella z. (lep.: pyralidae) for larvicidal activity, digestive enzymes and non-enzymatic (amount of macromolecules) responses. concentrations of 4.91 and 10.66 µl/ml were determined as lc50 values for tp eo and αpinene, respectively. the activities of α-amylase, triacylglycerol lipase, general protease, serine proteases (trypsin and chymotrypsin-like), carboxyand aminopeptidases significantly decreased when the larvae fed on the diet supplemented with tp eo and α-pinene but significant increases were observed in elastase-like proteinase. the amount of protein, glycogen and triglyceride decreased in the treated larvae by both treatments. the results showed that the digestive enzymes and non enzymatic activities of the treated larvae were decreased compared to the control. therefore, tp eo and α-pinene are suggested as botanicals for controlling flour moth larvae. key words: digestive enzymes; essential oil; flour moth; macromolecules; teucrium polium; α-pinene introduction in view of enzymology, digestive enzymes (such as amylases, lipases, and proteases) are commonly found in the midgut of insects. to provide metabolites and energy, these enzymes convert the food complexes into micro molecules (klowden, 2007). α-amylase catalyzes the endo-hydrolysis of long chains such as starch and glycogen (terra and ferriera, 2012). this activity converts them to maltose and other oligosaccharides, which are easily absorbed by the epithelial cells of insect gut (rahimi and bandani, 2014). the outer links of triacylglycerols are hydrolyzed by triacylglycerol lipases (tag-lipases). these enzymes preferentially release fatty acids (preferably unsaturated fatty acids) which are activated by calcium ions (terra and ferriera, 2012). insect proteases are the diverse groups of hydrolytic enzymes, which break peptide bonds in dietary proteins (terra and ferriera, 2012; subala and shivakumar, 2015). insects receive their required energy from ingested macromolecules such as carbohydrates, lipids and proteins. in insects, the most important lipid and ___________________________________________________________________________ corresponding author: najmeh sahebzadeh department of plant protection faculty of agriculture university of zabol, zabol, iran e mail: najmeh.sahebzadeh@gmail.com carbohydrate reserves are triglyceride and glycogen (or trehalose), respectively (klowden, 2007; nation, 2008). proteins are composed of amino acids, associated to molecular transformation, cuticular scleritization, and visual pigment synthesis, and even engaged as neural transmitters (klowden, 2007; ramzi et al., 2014). the flour moth, ephestia kuehniella (lep.: pyralidae) is a polyphagous and dangerous pest on the stored products that has been observed in cereals flour. the larvae cause high levels of damage by feeding and producing frass on the stored products (jallouli et al., 2013). currently, synthetic insecticides such as methyl bromide are most often used to control the stored products pests. however, concerns over the environmental pollution, residue levels, toxicity to non-target organisms and pesticide resistance are associated with conventional application of these insecticides, which compel us to attention to new compounds for pest control (rajendran and sriranjini, 2008; muthusamy and shivakumar, 2017). to manage the stored product pests, the application of botanical insecticides is considered as an appropriate alternative to synthetic pesticides (rajendran and sriranjini, 2008; karaborklu et al., 2011). plant secondary metabolites play insecticidal activities which could potentially act in anti-herbivore defenses against 183 agricultural pests (isman, 2006). lamiaceae plants are quite promising to develop as novel biopesticides for the control of pest (kim et al., 2013). teucrium polium is a member of this family already showing strong insecticidal activity on musca domestica (dip.: muscidae), callosobruchus maculatus (col.: chrysomelidae), and tribolium castaneum (col.: tenebrionidae) (bigham et al., 2010; hydarzade and moravvej, 2012; khani and hydarian, 2014). the major constituents of its essential oil are α-pinene (12.52 %), linalool (10.63 %) and caryophyllene oxide (9.69 %) (moghtader, 2009). a comprehensive understanding of plant essential oils (eos), with their active metabolites, and their interactions on physiology of the insect pests is necessary before using these compounds against the stored grain pests. accordingly, we examined the toxicity and biochemical properties of t. polium essential oil (tp eo) and α-pinene with a specific focus on digestive enzymes including αamylase, triacylglycerol lipase, general protease, serine proteases (trypsin and chymotrypsin-like), carboxyand aminopeptidases, as well as elastaselike proteinase of e. kuehniella. it is well documented that these enzymes alter during physiological process in insects. in addition, the effects of tp eo and α-pinene on amount of protein, glycogen, and triglyceride were also considered on the treated fourth instar larvae of e. kuehniella under laboratory conditions. materials and methods insect rearing the larvae of e. kuehniella were collected from the infected wheat flour in zabol, sistan and baluchestan province, iran. the insects were reared in rectangular plastic boxes (17×9×9 cm3). the insects were reared in the laboratory under controlled (25 ± 1 °c, 65 ± 5 % r.h., and 15l:9d) on an artificial diet (43 gr wheat flour, 6 gr yeast, and 20 ml of glycerin) (lima et al., 2001). essential oil preparation seeds of t. polium were ground and 40 g of powdered seeds was incubated at 500 ml of distilled water for 4 h in 100 ºc. water of obtained sample was taken by sodium sulfate and kept at 4 ºc prior to the experiment tests (negahban et al., 2007). α-pinene was purchased from sigmaaldrich (spain) and were applied without further purification. bioassay according to shahriari et al. (2017), forth instar larvae of e. kuehniella were randomly selected and were exposed to 500 mg of artificial diet containing 2, 3, 4.5, 6.7 and 10 µl/ml of tp eo, and 4, 5.6, 8, 11.3 and 16 µl/ml of α-pinene, respectively. control larvae were fed on the diet containing acetone as solvent for dilution of serial concentrations of tp eo and α-pinene. larval mortality was recorded after 24 h. the experiments were carried out using 180 larvae with 3 replicates in each concentration (total numbers were 360 larvae for both tp eo and αpinene concentrations). larval gut extraction the treated larvae of e. kuehniella were randomly and individually selected and their midguts were dissected under a stereomicroscope in icecold distilled saline solution (nacl, 10 mm). the midguts were rinsed in ice-cold distilled water and placed in a pre-cooled homogenizer. then the prepared midguts were grounded prior to be centrifuged at 13,000 rpm (20 min at 4 oc). the supernatants were stored at -20 oc for determination of digestive enzyme activities (ramzi et al., 2013). effects of tp eo and α-pinene on digestive enzymes α-amylase assay was carried out as described by bernfeld (1955). ten microliter of gut extraction and 20 μl of 1 % soluble starch as substrate were incorporated using phosphate buffer (50 μl, 20 mm, ph 7.1), mixed thoroughly and incubated for 30 min at 35 °c. eighty microliter dinitrosalicylic acid (to stop the reaction) was added and heated in boiling water for 10 min and the absorbance of this reaction was read at 545 nm. one unit of α-amylase activity was defined as the amount of enzyme required to produce 1 mg maltose in 30 min at 35 °c (ramzi et al., 2014). the method described by tsujita et al. (1989) was used to assay tag-lipase activity. twenty microliter of homogenated guts were incubated at 37°c with 100 μl of tris-hcl buffer (10 mm, ph 8) and 40 μl of p-nitrophenyl butyrate (27 mm) as substrate. the absorbance was read at 405 nm. one unit of enzyme released 1.0 nmol of pnitrophenol per min at ph 7.2 and 37 °c when pnitrophenyl butyrate was used as substrate. standard curve of p-nitrophenol was used to calculate the specific activity of this enzyme. general proteolytic activity was measured using azocasein (2 %) based on a method described by elpidina et al. (2001). the reaction mixture consisted of 20 μl of the enzyme solution, 100 μl of tris-hcl buffer (ph 8) and 40 μl of azocasein (2 %) as substrate. the reaction mixture was incubated at 37°c for 60 min before adding 150 μl of 10 % trichloroacetic acid (tca) to terminate the reaction. precipitation was achieved by cooling at 4 °c for 120 min and it was centrifuged at 13,000 rpm for 10 min. an equal volume of naoh (2 m) was added to the supernatant and the absorbance was recorded at 440 nm. the blank consisted of all mentioned portions except the enzyme solution of guts. trypsin, chymotrypsin and elastase-like activities were assayed using a concentration 1 mm of bapna (nabenzoyl-l-arginine-p-nitroanilide), 1 mm n-succinyl alanine alanine proline phenylalanine pnitroanilide (saapppna) and 1 mm saaapna (nsuccinyl-alanine-alanine-alanine-p-nitroanilide) as substrates, respectively. the reaction mixture included 35 μl of tris-hcl buffer (20 mm, ph 8 as literature recommended ph for serines in lepidopteran species), 5 μl of bapna, saapppna and saaapna substrates individually and 5 μl of enzyme solution. this mixture was incubated at 30 °c (10 min), and its absorbance was then read at 405 nm. to prove the specific proteolytic activity, a negative control was provided for each substrate separately containing all mentioned components 184 besides enzyme pre-boiled at 100 °c for 30 min (oppert et al., 1997). activities of the two exopeptidases in the midgut of e. kuehniella were obtained using hippuryl-l-arginine and hippuryl-lphenilalanine for carboxyand aminopeptidases, respectively. the reaction mixture consisted 35 μl of tris-hcl buffer (20 mm, ph 7), 5 μl of each mentioned substrate and 5 μl of enzyme solution. following by incubation (30 °c, 10 min), the absorbance was read at 340 nm. to prove the specific proteolytic activity, a negative control was provided as mentioned previously (oppert et al., 1997). effects of tp eo and α-pinene on macromolecules the amount of protein was measured using lowry et al. (1951) procedure. twenty microliter of homogenized sample was added to 100 µl of reagent, and incubation was made for 30 min at 25 °c. the absorbance was read at 545 nm (recommended by ziest chem. co., tehran-iran) (ramzi et al., 2014). to determine triacylglyceride content, a diagnostic kit (pars azmoon co., tehran, iran) was used to measure the amount of triacylglyceride in the fourth instar larvae. the reagent solution contained phosphate buffer (50 mm, ph 7.2), 4-chlorophenol (4 mm), adenosine triphosphate (2 mm), mg2+ (15 mm), glycerokinase (0.4 ku/l), peroxidase (2 ku/l), lipoprotein lipase (2 ku/l), 4-aminoantipyrine (0.5 mm), and glycerol-3phosphate-oxidase (0.5 ku/l). samples (10 μl) were incubated with 10 μl of distilled water and 70 μl of reagent for 20 min at 25 °c. the following equation was used to calculate the amount of triacylglyceride: mg/dl = ×0.01126. optical densities (ods) of samples and reagent as standard were read at 545 nm (fossati and prencipe, 1982). for estimation of glycogen, fat bodies of 20 larvae were cut and immersed to 1 ml of 30 % koh w/na2so4. tubes containing the samples were covered with aluminium foil (to avoid evaporation) and were boiled for 30 min. tubes were shaken and cooled in ice. then, 2 ml of 95 % etoh was added to precipitate glycogen from digested solution. the samples were again shaken and incubated in ice for 30 min. tubes were centrifuged 13,000 rpm for 30 min. supernatant was removed and pellets (glycogen) were re-dissolved in 1 ml of distilled water before being shaken. glycogen standard (0, 25, 50, 75 and 100 mg/ml) was prepared before adding phenol 5 %. the samples were incubated on ice bath for 30 min. finally, the absorbance of standards and samples were read at 490 nm (chun and yin, 1998). data analysis for estimation of lc50 and corresponding 95 % ci values, polo-plus software was used. biochemical data were estimated in a complete randomized design, and were compared by oneway analysis of variance (anova) followed by tukey’s test. differences between samples were statistically considered at a probability less than 5 % and marked in the tables and figures. results and discussion bioassay in this study, the lethal concentrations (lc50) values were observed in 4.91 µl/ml and 10.66 µl/ml for tp eo and α-pinene, respectively (table 1). by increasing of concentration, the mortality of larvae was increased. similarly, α-pinene was evaluated against t. castaneum larvae, at 24 and 48 h posttreatment, and lc50 of this compound was determined as 12.85 and 8.39 µl/ml, respectively (shahriari et al., 2016). bigham et al. (2010) found a significant oral toxic activity of t. polium on m. domestica (lc50 = 80ppm). lc50 values were showed contact toxicity of t. polium 1263.09 and 1469.72 μl/m2 on adult males and females of c. maculatus, respectively (hydarzade and moravvej, 2012). plant essential oils (eos) are aromatic compounds and complex mixture of monoterpenes. it is often observed that complex essential oil compounds are more efficacious than the pure combination (don-pedro, 1996; bakkali et al., 2008; kumrungsee et al., 2014). in our study, the oral toxicity of tp eo was higher than α-pinene on e. kuehniella. similarly, don-pedro (1996), ho et al. (1997), hori (1998), and kim et al. (2010, 2013) demonstrated that essential oil from different plant species (anise, rosemary, citrus, origanum and cumin) were more effective than the their pure secondary metabolites (trans-anethole, linalool, limonene, carvacrol and p-cymene) on different pests such as c. maculatus, myzus persicae s. (homoptera: aphididae), t. castaneum and sitophilus oryzae (col.: curculionidae). table 1 dose-response parameters of fourth instar larvae of ephestia kuehniella exposed to teucrium polium essential oil and α-pinene treatment lc50(µl/ml) x 2 (df) slope±se tp eo 4.91 (4.16-5.87) 2.05 (3) 3.16 ± 0.504 α-pinene 10.66 (8.61-14.91) 2.37 (3) 2.28 ± 0.526 tukey test, p < 0.05 185 table 2 activity of digestive enzymes in fourth instar larvae of ephestia kuehniella at 24, 48 and 72 h post treatment with lc50 value of teucrium polium essential oil and α-pinene digestive enzyme time (h) control tp eo α-pinene fvalue pr α-amylase (u/mg protein) 24 0.658±0.0049a 0.144±0.0024c 0.240±0.0053b 3794.32 0.0001 48 0.763±0.0085a 0.355±0.0034b 0.370±0.0090b 992.23 0.0001 72 0.762±0.0008a 0.290±0.0049c 0.417±0.0017b 6250.88 0.0001 tag lipase (u/mg protein) 24 0.562±0.0045a 0.352±0.0045b 0.346±0.0092b 340.02 0.0001 48 0.563±0.0046a 0.506±0.0058b 0.338±0.0052c 676.9 0.0001 72 0.589±0.0053a 0.456±0.0035b 0.328±0.0035c 658.24 0.0001 general protease (u/mg protein) 24 0.118±0.0046a 0.059±0.0043b 0.053±0.0050b 59.38 0.0001 48 0.120±0.0043a 0.088±0.0017b 0.074±0.0023b 50.89 0.0002 72 0.123±0.0033a 0.106±0.0038b 0.094±0.0026b 19.07 0.0023 trypsin-like (u/mg protein) 24 0.166±0.0032a 0.063±0.0031b 0.033±0.0038c 409.32 0.0001 48 0.165±0.0035a 0.067±0.0037b 0.058±0.0031b 371.03 0.0001 72 0.163±0.0040a 0.073±0.0030b 0.059±0.0030b 275.65 0.0001 chymotrypsin-like (u/mg protein) 24 0.092±0.0029a 0.049±0.0013c 0.080±0.0038b 58.59 0.0001 48 0.094±0.0034a 0.078±0.0003b 0.087±0.0025ab 7.33 0.0245 72 0.092±0.0035a 0.069±0.0008b 0.086±0.0012a 29.64 0.0008 elastase-like (u/mg protein) 24 0.103±0.0024b 0.117±0.0029a 0.119±0.0036a 7.73 0.0219 48 0.099±0.0029b 0.125±0.0032a 0.130±0.0040a 22.44 0.0016 72 0.096±0.0030c 0.164±0.0034a 0.132±0.0043b 60.91 0.0001 carboxypeptidase (u/mg protein) 24 0.297±0.0031a 0.202±0.0040b 0.189±0.0031b 17.22 0.0033 48 0.300±0.0041a 0.218±0.0018b 0.199±0.0058c 159.55 0.0001 72 0.300±0.0041a 0.226±0.0040c 0.260±0.0030b 94.55 0.0001 aminopeptidase (u/mg protein) 24 0.105±0.0031a 0.079±0.0029b 0.108±0.0031a 26.59 0.0010 48 0.106±0.0041a 0.073±0.0011b 0.068±0.0028b 57.86 0.0001 72 0.108±0.0041a 0.072±0.0080b 0.073±0.0027b 75.66 0.0001 statistical differences have been done within each time intervals and marked by different letters in each row (tukey test, p < 0.05). effects of tp eo and α-pinene on digestive enzymes the lower activity of α-amylases was found in the larvae fed on the diet containing lc50 concentrations of tp eo and α-pinene compared to the control (table 2). similarly, bigham et al. (2010) reported that tp eo decreased the activity of αamylase in the treated m. domestica larvae versus control. the reduced activity of α-amylase by plantbased compounds could imply their cytotoxic effect on the midgut epithelial cells those synthesize insect α-amylase (franco et al., 2002; senthil-nathan et al., 2006; zibaee and bandani, 2010). this result is consistent with previous studies which were demonstrated the reduction of α-amylase activities in pests such as e. kuehniella (shahriari and sahebzadeh, 2017), ectomyelois ceratoniae (lep.: pyralidae) (ramzi et al. 2013, 2014), glyphodes pyloalis (lep.: crambidae) (khosravi et al., 2011; yazdani et al., 2013), and pieris rapae (lep.: pieridae) (hashminia et al., 2011) after treatment with botanical toxins. midgut tag-lipase has a primary role to obtain dietary lipid as well as strong antiviral activity. we found a significant reduction in tag-lipase activity in the larvae of e. kuehniella fed on the artificial diet containing tp eo and α-pinene (table 2). yazdani et al. (2013) reported that lc50 concentration of some 186 fig. 1 effects of essential oil of teucrium polium essential oil and α-pinene on amounts of storage macromolecules (total protein, triacylglyceride, and glycogen) in the fourth instar larvae of e. kuehniella. statistical differences have been done within each time intervals and marked by different letters (tukey test, p < 0.05). essential oils such as increased the activity of taglipase in the treated g. pyloalis larvae versus control. khosravi et al. (2011) found similar resus while senthil-nathan et al. (2006) reported the lower activity of tag-lipase in cnaphalocrocis medinalis (lep.: pyralidae) midgut after treatment with azadirachta indica (melliaceae) extract. great utilization of exogenous lipids could increase the 187 activity of tag-lipase in insect midgut (yazdani et al., 2013). decreased activity of tag-lipase by botanical insecticide probably resulted to disturbance of digestion and absorption processes (senthilnathan et al., 2006; zibaee and bandani, 2010). treatment of e. kuehniella larvae with lc50 concentrations of tp eo and α-pinene showed the lower general proteolytic activity (table 2). senthil nathan et al. (2006) found a significant reduction in protease activity of c. medinalis fed on a diet containing bt toxins and botanical insecticides including neem seed and leaf extract of vitex negundo (verbenaceae). digestive proteolytic activity increased in the larvae of g. pyloalis treated by lavandula angustifolia (lamiaceae) (yazdani et al., 2013). the digestion of proteins in the first stage in insects mostly occurs under the effect of serine proteases (terra and ferreira, 2012). in our study, tp eo and α-pinene decreased the activities of trypsin and chymotrypsin-like protease but a significant addition was observed on elastase activity of the treated larvae (table 2). similar to our results, mojarab-mahboubkar and jalali-sendi (2016) demonstrated that activity of serine proteases decreased by artemisia annua methanolic extract except for elastase. zibaee et al. (2014) reported that activity of serine proteases decreased in the larvae of pieris brassicae (lep.: pieridae) treated with 0.5, 1 and 2 % concentrations of polygonum persicaria (polygonaceae) agglutinin. senthil-nathan et al. (2006), zibaee and bandani (2010), and ramzi et al. (2013) reported that plantbased compounds may affect the construction of some kinds of proteases and inhibition them to digestion ingestion proteins. it was shown that activities of two exopeptidases including amino and carboxypeptidases of treated e. kuehniella larvae with lc50 concentration of tp eo and α-pinene significantly decreased in comparison with the control larvae (table 2). ramzi et al. (2013, 2014) and zibaee et al. (2014) found no significant differences in the activities of aminopeptidase between control and treated larvae, while carboxypeptidas activity significantly decreased 48 h post-treatment by lectins extracted from plant source. exopeptidases hydrolyze single amino acids either from the nterminus or from the c-terminus (aminopeptidases and carboxypeptidases, respectively) at the end of a polypeptide chain (kanost and clem, 2012). the potential mechanisms of eo's and plant secondary metabolites on exopeptidase activities are still lacking. effects of tp eo and α-pinene on macromolecules in this study, amounts of protein, glycogen, and triglyceride in e. kuehniella larvae treated by tp eo and α-pinene decreased significantly versus control (fig. 1). several studies have been demonstrated that synthetic and botanical insecticides could alter the amount of carbohydrates, lipids and proteins in insects. the significant reductions in amount of protein in g. pyloalis treated with lethal and sublethal doses of l. angustifolia eo have been reported by yazdani et al. (2013). in a similar study, zibaee et al. (2011) showed that the amount of protein, glycogen (trehalose) and lipid as nonenzymatic components in hemolymph and fat bodies of eurygaster integriceps (hemiptera: scutelleridae) reduced after exposure to pyriproxifen. citrullus colocynthis (cucurbitaceae) agglutinin decreased the amount of glycogen and triglyceride levels in e. ceratoniae (ramzi et al., 2014). the reductions occurring by tp eo and αpinene in e. kuehniella might be due to accelerating in glycogenolysis at larval fat bodies, transport glycogen from fat body to hemolymph as a response to energy depletion when larvae were exposed to toxins (zibaee et al., 2011). lipid depletion after toxin treatments could be occurred because of alteration in its synthesis patterns (klowden, 2007), hormonal dysfunction for controlling lipid metabolism (steel, 1980) and the utilization of this metabolic reserve to generate insect energy demand (sak et al., 2006). results of this study indicate that tp eo and αpinene possesses larvicidal effects on e. kuehniella. moreover, the results demonstrated the lower digestive enzyme activities and macromolecules amount of treated larvae compared to the controls. in general, eos and their secondary metabolites show to be significant entomotoxic molecules that efficiently affect physiology and biology of pests without affecting non-target organisms. this study could play a significant role in integrated pest management in the stores. acknowledgments the authors wish to thank department of plant protection, university of zabol (zabol, iran) and university of guilan (rasht, iran) for providing the necessary facilities to carry out this work. references bakkali f, averbeck s, averbeck d, idaomar m. biological effects of essential oils a review. food chem. toxicol. 46: 446-475, 2008. bernfeld p. amylases, α and β. meth. enzymol. 1: 149-158, 1955. bigham m, hosseininaveh v, nabavi b, talebi k, esmaeilzadeh ns. effects of essential oil from teucrium polium on some digestive enzyme activities of musca domestica. entomol. res. 40: 37-45, 2010. chun y, yin zd. glycogen assay for diagnosis of female genital chlamydia trachomatis infection. j. clin. microbiol. 36: 1081-1082, 1993. don-pedro kn. investigation of single and joint fumigant insecticidal action of citrus peel oil components. pestic. sci. 46: 79-84, 1996. elpidina en, vinokurov ks, gromenko va, rudenskaya ya, dunaevsky ye, zhuzhikov dp. compartmentalization of proteinases and amylases in nauphoeta cinerea midgut. arch. insect biochem. physiol. 48: 206-216, 2001. fossati p, prencipe l. serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide. clin. chem. 28: 2077-2080, 1982. franco ol, rigden dj, melo fr, grossi-de-sa mf. plant α-amylase inhibitors and their interaction with insect α-amylase: structure, function and 188 potential for crop production. eur. j. biochem. 269: 397-412, 2002. hashminia sm, sendi jj, jahromi kt, moharramipour s. the effects of artemisia annua l. and achillea millefolium l. crude leaf extracts on the toxicity, development, feeding efficiency and chemical activities of small cabbage pieris rapae l. 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(lepidoptera: pieridae). j. plant protect. res. 54: 250-257, 2014. zibaee a, bandani ar. effects of artemisia annua l. (asteracea) on digestive enzymes profiles and cellular immune reactions of sun pest, eurygaster integriceps (heteroptera: scutellaridae), against beauvaria bassiana. bull. entomol. res. 100: 185-196, 2010. zibaee a, zibaee i, sendi jj. a juvenile hormone analog, pyriproxifen, affects some biochemical components in the hemolymph and fat bodies of eurygaster integriceps puton (hemiptera: scutelleridae). pestic. biochem. physiol. 100: 289-298, 201. isj 13: 134-146, 2016 isj 13: 140-152, 2016 issn 1824-307x research report effects of exposure to zinc oxide nanoparticles in freshwater mussels in the presence of municipal effluents c gagnon, m pilote, p turcotte, c andré, f gagné aquatic contaminants research division, water science and technology, environment canada, 105 mcgill, montreal, quebec, canada accepted may 12, 2016 abstract zinc oxide (nano-zno) nanoparticles are used in the production of transparent sunscreens and cosmetics, which are released into surface waters and municipal wastewater effluent. the purpose of this study was to examine the toxicity of nano-zno in the presence of municipal effluents to freshwater mussels elliptio complanata. mussels were exposed for 21 days at 15 oc to nano-zno and zncl2 in the presence of 10 % dilution of primary-treated municipal effluent. after the exposure period and 24-h depuration step, mussels were analyzed for total zn in gills and digestive gland, free zn, metallothioneins (mt), oxidative stress (glutathione s-transferase and lpo), endoplasmic reticulum stress (heat shock proteins and protein ubiquitination) and genotoxicity. the data revealed that although total zn loadings did not change with these treatments, zn levels in digestive gland were elevated in mussels exposed to nano-zno but not with zncl2 in the presence of municipal effluent. free zn levels in the gills were elevated in mussels exposed to the municipal effluent, but decreased in mussels exposed to nano-zno. mt in digestive gland showed a similar pattern and was negatively associated with free and total zn. gst activity was significantly reduced by both nano-zno and municipal effluent and was negatively correlated with mt levels, suggesting the involvement of mt in the sequestration of reactive oxygen species. discriminant function analysis showed that the municipal effluent related effects differed from the unexposed mussels and nano-zno exposed mussels in terms of the following responses: free zn in gills and digestive gland and gst activity. nano-zno related effects also involved gst activity, mt and protein ubiquitination, which suggests a combination of oxidative stress and reticulum endoplasmic stress. in respect with oxidative stress, the oxidative properties of nano-zno and zncl2 are dampened in the presence of the municipal effluent. key words: zinc oxide nanoparticles; municipal effluent; freshwater mussels; oxidative stress; reticulum endoplasmic stress   introduction nanotechnology is an exponentially growing industry with applications in many fields, such as electronics, optical instruments, drug delivery vectors and diagnostics (lee et al., 2008). nanomaterials are also used in consumer products, such as cosmetics, sunscreens and textiles (contado, 2015). the presence of nanomaterials from personal care products in complex matrices, ___________________________________________________________________________ corresponding authors: christian gagnon francois gagne aquatic contaminants research division water science and technology environment canada, 105 mcgill montreal, quebec, canada h2y 2e7 e-mails: christian.gagnon@ec.gc.ca; francois.gagne@ec.gc.ca such as urban runoff and municipal effluents, complicates the hazard assessment process. indeed, at the exposure characterization step, the identification and quantitation of low concentrations of nanoparticles in complex environmental samples (sediments, effluents) poses a major challenge at the analytical level. hence, the concerns raised by the public and regulatory community about their safety, fate and ecotoxicity are legitimate. zinc oxide nanoparticles (nano-zno) are used worldwide in sunscreens given their strong uv-light absorbing properties compared to other sunscreen formulations (mitchnick et al., 1999). sunscreen creams typically contain bulk particles of zno and titanium dioxide, which are in the form of white pastes. creams composed of nano-zno diminish light scattering on the skin and appear more transparent (mitchnick et al., 1999). moreover,   140 mailto:christian.gagnon@ec.gc.ca mailto:francois.gagne@ec.gc.ca   141 nano-zno possesses anti-microbial properties, which is perceived as an additional benefit for the consumer. as explained above, the release, fate and toxicity of these sunscreens are difficult to assess at present time and the resulting effects of nano-zno in complex environmental media, such as municipal effluent, are not well understood at the present time. with respect to nanomaterials, mussels and clams (bivalves) are considered to be at risk from contaminants associated with fine particles and colloids (canesi et al., 2012). these invertebrates are more susceptible to nanoparticles since they are sessile, live for long periods (years to decades) and especially feed on suspended particles, therefore accumulating large quantities of particles including nanoparticles. copper oxide nanoparticles were shown to accumulate in the digestive gland and initiate toxicity (lipid peroxidation or lpo) in the mussel mytilus galloprovincialis (gomes et al., 2012). in another study, mussels exposed to ceo2 nanoparticles and nano-zno accumulated as much as 21 and 63 mg/g of total ce and zn respectively, indicating that these nanoparticles are readily available to mussels (montes et al., 2012). the toxicity of metal-based nanoparticles cannot always be explained by the released component which shows different toxic properties. these toxic properties are associated with other properties of nanoparticles, such as size, form and surface properties (oberdörster et al., 2007). recent studies have shown that many nanoparticles of different sizes, forms and surface coatings are cytotoxic and genotoxic, leading to reticulum endoplasmic stress, oxidative stress and dna damage. reticulum endoplasmic stress involves protein chaperones (heat shock proteins 70 or hsp70) to restore the conformational integrity of proteins and tagging of denatured proteins by ubiquitin via the ubiquitinproteasome pathways involved in the elimination of irreversibly damaged proteins (mcdonagh and sheehan, 2006). ubiquitination also plays a role in the calcium carbonate biomineralization process of shells (fang et al., 2012). oxidative stress involves the mobilization of metals, such as iron (fe3+), copper (cu+) and zn (zn2+), leading to the production of reactive oxygen species. these processes are controlled by antioxidants and metallothioneins (mt) in the sequestration of divalent heavy metals and reactive oxygen species. the uncontrolled release of reactive oxygen species could lead to lipid peroxidation (lpo) and dna damage via the formation of 8-oxo-guanine (rocha et al., 2015; valko et al., 2006; formigari et al., 2007). recent evidence suggests that nano-zno and municipal effluents could induce oxidative stress in mussels, but less is known about the combined effects in freshwater mussels located near urban waste discharges. the purpose of this study was to examine the cumulative effects of nano-zno and municipal effluent exposure in freshwater mussels. mussels were exposed to dilutions of municipal effluent in freshwater and to instilled nano-zno given the poor solubility of this nanoparticle in freshwater (majedi et al., 2014). for comparison purposes, mussels were also exposed to zncl2. zn bioavailability was assessed in mussel tissues and biomarkers of stress were determined. biomarkers of stress were labile zn in tissues, mt, glutathione s-transferase (gst, a marker of oxidative stress and conjugation), reticulum endoplasmic stress (hsp70 and protein ubiquitylation), lpo and dna damage. an attempt was made to highlight cumulative effects or interactions of a typical chemically-treated municipal effluent and nano-zno for 21 days based on effluent concentration and forms of dietary zn in freshwater mussels. materials and methods mussel handling and exposure to municipal effluents mussels (elliptio complanata) were collected by hand during the first week of june 2012 in a pristine lake in the laurentians under a provincial permit. the mussels were transported dry in coolers at 4 oc and transferred to 300-l tanks filled with city of montreal tap water, which was charcoal-filtered and uv-treated. the mussels were held in the tanks at 15 °c under constant aeration for 4 to 10 weeks before initiating exposure. the mussels were fed three times a week with commercial coral reef feed enriched with 100 million/ml of pseudokirchneriella subcapitata algal suspensions. for the exposure experiments, mussels (n = 10 individuals) were placed in 60-l tanks receiving a continuous flow (0.1 0.2 l/h) of physico-chemically treated municipal effluent from a city of 1.5 million people. three aquarium per treatment was also included. the physical/chemical treatment consisted in reducing suspended matter down to the mg/l range using grid traps, flocculation (surfactants) and sieving. the municipal effluent exposure concentrations were 0, 3 and 10 % prepared in dechlorinated uv-treated tap water from the city of montreal (quebec, canada). mussels were exposed to 1 and 10 µg/l zn as nano-zno or zncl2 using an “instillation” technique. during exposure, 1 and 10 µg/l zinc solutions were added to 60 l of aquarium water. to ensure contact of the zinc with mussel tissues, 60 and 600 µg of either nano-zno or zncl2 were dissolved in 20 ml of aquarium water and placed directly over the mussel siphons (each mussel received 1 ml). the mussels were held under static conditions for one hour prior to continuous exposure to the municipal effluent. this process was repeated every 3 days for 21 days. at the end of the exposure period, mussels were allowed to depurate in clean aquarium water overnight (24 h). morphological characteristics were measured for mussel weight and shell length. soft tissue, gills, gonad and digestive glands were dissected on ice and weighed. sex was determined by examination of gonad smears on glass slides under a binocular microscope at 200x magnification. a portion of the digestive, gill and gonad tissues were processed for heavy metal analysis (including zn) using icp-mass spectrometry and nitric acid digestions as previously described (gagnon et al., 2006). the digestive gland and gills were homogenized on ice using a teflon pestle tissue   142 grinder in 100 mm nacl containing 20 mm hepesnaoh, ph 7.4, 10 µg/ml aprotinin (protease inhibitor) and 1 mm dithiothreitol. a part of the homogenate was centrifuged at 15000xg for 30 min at 4 oc and the supernatant (s15 fraction) was removed and stored at -85 oc until biomarker analysis. total proteins were determined using the protein-dye binding principle with standard solutions of bovine serum albumin for calibration (bradford, 1976). basic physico-chemical characteristics were determined using standard methods for total coliform counts, conductivity, ph, biochemical oxygen demand, dissolved organic carbon and turbidity. xenobiotic metabolism xenobiotic metabolism was characterized by monitoring changes in glutathione s-transferase (gst) and metallothioneins (mt) in the digestive gland. gst activity was determined using the absorbance microplate methodology as described elsewhere (boryslawskyj et al., 1988). the 15000xg supernatant or s15 fraction (50 µl) was mixed with 200 µl of 1 mm glutathione (gsh) and 1 mm of 1chloro-2,4-dinitrobenzene in 125 mm nacl containing 20 mm hepes-naoh, ph 6.5. the increase in absorbance at 340 nm was measured at 0, 10, 20 and 30 min at 30 °c (synergy 4, dynatek instrument, usa). enzyme activity was expressed as the change in absorbance/min/mg protein. metallothionein (mt) levels in the digestive gland were determined using a modified spectrophotometric assay (viarengo et al., 1997; gagné et al., 2010). briefly, total mt levels were determined by the addition of a strong reducing agent, phosphine, in the s15 fraction for 15 min prior to the addition of ethanol-chloroform solvent. the data were expressed as µmole of gsh/mg protein. the levels of labile zn were also determined in the digestive gland extracts using the fluoresecent probe methodology (gagné and blaise, 1996). briefly, 20 μl of the homogenate was mixed with 180 μl n-(6-methoxy-8 quinolyl)-ptoluenesulfonamide (tsq) probe in phosphatebuffered saline (140 mm nacl, 5 mm kh2po4, ph 7.4) containing 20 % dimethyl sulfoxide (dmso). fluorescence values were determined at 370 nm excitation and 490 nm emission. blanks consisting of 20 % dmso-phosphate-buffered saline and standard solutions of znso4 were used for calibration. data were expressed as nanograms zn per milligram protein. endoplasmic reticulum stress the levels of heat shock proteins of the 72-kda family (hsp72) and total ubiquitin levels were assessed using the enzyme immunoassay described previously (louis et al., 2010). briefly, the s15 fraction was diluted to 1 µg total proteins in 50 mm carbonate buffer (100 µl) ph 9.6, then added to high binding microplate wells (immulon-4) and allowed to stand 10 12 h at 4 oc in darkness. the wells were then washed twice with 200 µl pbs and blocked with pbs containing 1 % albumin for 30 min at room temperature. the wells were washed once with pbs, and 100 µl of either hsp72 or polyubiquitin polyclonal antibody (recombinant human hsp72 igg spa-812 and rabbit ubiquitin antibody; stressgen, usa) at 1/1000 and 1/5000 dilution in pbs containing 0.5 % albumin were added in each well. the wells were incubated at 37 °c for 60 min. the wells were washed three times in pbs, and 100 µl of secondary antibody (rabbit igc antibody linked to peroxidase) were added at 1/5000 dilution in pbs containing 0.5 % albumin. after 30 min at 37 °c, the wells were washed four times in pbs, and peroxidase activity was determined using 1 µm luminol and 10 µm h2o2. readings were taken after 1 min using a luminescence microplate reader, at 2 and 5 min (synergy-4, dynatek). the data were expressed as the rate of increase in luminescence/min. oxidative stress and dna damage lipid peroxidation (lpo) was determined in the liver homogenates using the thiobarbituric acid method (wills, 1987). a volume of 50 µl of the homogenate was mixed with 250 µl of 10 % trichloroacetic acid containing 1 mm feso4 and 75 µl of 0.7 % thiobarbituric acid (sigma chemical company) and heated at 70 oc for 10 min. the mixture was cooled at room temperature and centrifuged at 10 000xg for 5 min. a 100 200 µl aliquot of the supernatant was transferred to a 96well dark microplate and fluorescence readings were taken at 520 nm excitation and 600 nm emission. standard solutions of tetrametoxypropane (stabilized form of malonaldehyde) were prepared for calibration in the blank (homogenization buffer). results were expressed as µmole thiobarbituric acid reactants (tbars)/mg total protein in the homogenate. dna damage in liver was determined using the alkaline precipitation assay (olive, 1988), which is based on potassium detergent precipitation of dna-proteins. protein-free dna strand breaks that remain in the supernatant were determined using fluorescence spectroscopy (at 360 nm excitation and 450 nm emission) in the presence of diluent to prevent interference from the detergent (bester et al., 1994). the diluent consisted of 0.4 m nacl, 0.1 m tris-acetate, ph 8.2, 4 mm sodium cholate and 10 μm sybr® green dye. standard solutions of salmon sperm dna were prepared for calibration. the data were expressed as µg dna strand/mg total protein in homogenate. data analysis the exposure experiment consisted of 10 mussels per treatment aquarium and three replicate aquarium per conditions were used. tissue biomarkers were performed on 10 mussels using 2way factorial analysis of variance (municipal effluent concentration and form of zinc) after verifying for homogeneity of variance and normality using the levene’s and shapiro-wilks tests respectively. correlation analysis was also performed using the pearson product-moment method. the physiological changes induced by exposure to the municipal effluent, dissolved zn and zno nanoparticles were determined using discriminant function and factor analysis methods. all statistical tests were performed using statistica software (version 8). significance was set at α = 0.05. results mussels were exposed to a physico-chemically treated municipal effluent from a relatively large urban area (1.5 million residents). the undiluted effluent had a conductivity of 835 ± 50 µscm-1 and a ph of 7.2 (table 1). the suspended matter content and dissolved organic carbon content were 25 ± 5 and 82 ± 10 mg/l respectively. the biochemical oxygen demand was 35 ± 10 mg/l and the concentration of thermotolerant coliform bacteria was 2.9x106 ± 5x105 counts per 100 ml. total zn levels were determined in the soft tissues, gills and digestive gland by icp-ms spectrometry following complete acid-digestion of tissues (fig. 1). in mussels treated with nano-zno, factorial analysis of variance (anova) with effluent concentration and added nano-zno (or zncl2) as the main factors revealed that added nano-zno was significant with a marginal interaction (0.05 < p < 0.1) with the municipal effluent (fig. 1a). the only significant effect was a significant decrease in total zn in mussels exposed to 3 % effluent in the presence of 10 µg nano-zno/l compared to 3 % effluent exposure or control groups respectively. in mussels treated with zncl2, factorial anova revealed that neither the municipal effluent nor zncl2 exposures affected total zn levels in mussels (fig. 1b). zn levels in gills and digestive gland were expressed in percentage of total zn. in mussels coexposed to nano-zno, factorial anova revealed a significant effect for added zn concentrations (fig. 1c). the proportion of zn in gills decreased in mussels exposed to 3 % municipal effluent when nano-zno was present. in mussels exposed to 10 % municipal effluent, the decrease was only observed in mussels co-exposed to 10 ug zn/l as nano-zno, indicating an interaction between effluent concentration and nano-zno for gill zn levels. in mussels co-exposed to zncl2, factorial anova also revealed that zn concentration in gills was significant (fig. 1d). the proportion of zn was significantly reduced in mussels exposed to 5 % municipal effluent and 1 ug zn/l as zncl2. this effect was lost when the effluent concentration was 10 %, although lower in the presence of zncl2. correlation analysis revealed that gill zn levels were significantly correlated with total zn in mussels (r = 0.66; p <0.001) and the percentage of zn in gills (r = 0.86; p < 001), suggesting that the total levels of gill zn is strongly related to the proportion of zn in gills (table 2). the proportion of zn in the digestive gland was also examined in mussels exposed to the municipal effluent and to each form of zn. in mussels cotreated with nano-zno, factorial anova revealed a significant effect for both the municipal effluent and zn concentration, with a significant interaction between these two factors (p < 0.05). zn levels were somewhat increased in mussels exposed to 1 ug nano-zno/l only (fig. 1e). the levels of zn in the digestive gland was increased at 10 ug nano-zno/l in mussels exposed to 5 and 10 % municipal effluent, which suggests a particular-type of exposure. in mussels exposed to zncl2, the concentration of added zn as zncl2 was significant (p < 0.05) in the presence of municipal effluent (fig. 1 f). indeed, the proportion of zn in the digestive gland was somewhat higher in mussels exposed to 1 and 10 ug zn/l than in mussels exposed to the 5 and 10 % effluent concentrations. correlation analysis showed that the proportion of zn in digestive gland was correlated with zn levels in the digestive gland (r = 0.82; p < 0.001) and inversely correlated with either zn levels in gills (r = -0.66; p < 0.001) or proportion of zn in gills (r = -0.74; p < 0.001). ionic or labile zn levels were determined using a fluorescent probe method (fig. 2). in gills, factorial anova revealed that both effluent and nano-zno concentrations significantly influenced labile zn in gills (fig. 2a). labile zn in gills increased at the 3 % municipal effluent concentration and decreased afterwards at the 10 % effluent concentration. coexposure with nano-zno eliminated this increase at both concentrations (1 and 10 µg/l). in mussels exposed to zncl2, factorial anova showed that both effluent and zn concentrations had an effect on labile zn levels in gills (fig. 2b). gill zn levels were increased by exposure to the 3 % effluent concentration and returned to control levels at the 10 % effluent concentration. the addition of 1 or 10 ug/l of zn as zncl2 also mitigated the increase at 3 % effluent. digestive gland labile zn was also determined in mussels exposed to the municipal effluent, nano-zno and zncl2. in mussels exposed to nano-zno, factorial anova revealed a significant effect only for zn exposure concentrations (fig. 2c). in mussels exposed to nano-zno, the increase in labile zn in the digestive was not significant, while in table 1 physico-chemical characteristics of the municipal effluent temperature °c conductivity µs/cm ph redox potential mv nh4 + mg/l nh3 mg/l no3 mg/l dissolved oxygen mg/l day 0 24.7±1 302±3 8.06±0.05 148±5 0.19±0.05 0.011±0.005 0.8±0.1 9.1±0.2 day 5 24.7±0.5 312±5 8.05±0.05 145±5 0.24±0.05 0.0125±0.005 0.68±0.01 8.9±0.05 day 10 22.7±0.4 316±4 8.15±0,05 135±5 0.26±0.05 0.015±0.005 0.58±0.01 9±0.05 day 29 23.7±0.5 310±7 8.25±0.05 155±5 0.23±0.05 0.01±0.005 0.57±0.01 8.8±0.05   143 fig. 1 zn levels in soft tissues (a,b), gills (c, d) and digestive gland ( e, f) of mussels exposed to municipal effluent, nano-zno and dissolved zn. the letter a indicates significant difference compared to me controls. the letter b indicates significant difference from nano-zno and zncl2. mussels exposed to the municipal effluent only, labile zn levels were somewhat lower at 10 % effluent. in mussels exposed to both 10 % effluent and nano-zno (1 and 10 µg/l), labile zn levels were increased compared to mussels exposed to 10 % effluent alone. in mussels exposed to 5 % municipal effluent and 1 ug nano-zno/l, labile zn levels in the digestive gland were increased. in mussels exposed to municipal effluent and zncl2, factorial anova revealed significant effects for both zn concentrations and the interaction between effluent and zncl2 at p < 0.05 (fig. 2 d). in mussels exposed to 1 ug/l, labile zn levels were decreased at the 3 % effluent concentration and increased at the 10 % effluent concentration compared to mussels exposed to the effluent alone. correlation analysis revealed that gill labile zn was not significantly correlated with total zn or the levels free zn in the digestive gland and gills. digestive gland labile zn levels were significantly correlated with the gonado-moatic index or gsi (r = 0.26; p < 0.01). mt levels were determined in the digestive gland of mussels exposed to municipal effluent and either nano-zno or zncl2 (fig. 3). in mussels exposed to nano-zno and the effluent, factorial anova revealed a significant effect for both municipal effluent and zn concentrations (fig. 3a). the effluent alone was able to induce mt at the 3 % concentration, while nano-zno had no effect. in   144   145 table 2 correlation analysis of the biomarker data condition factor gsi mt dna damage lpo dg gst dg free zn gills free zn digestive gland hsp70 poly-ubiquitin zn gills zn digestive gland condition factor 1 -0.10 p>0.1 0.18 p<0.1 0.01 p>0.1 -0.06 p>0.1 -0.21 p<0.05 -0.01 p>0.1 -0.15 p>0.1 0.09 p>0.1 0.04 p>0.1 -0.26 p=0.01 -0.07 p>0.1 gsi 1 -0.01 p>0.1 -0.03 p>0.1 0.03 p>0.1 -0.04 p>0.1 -0.07 p>0.1 0.26 p<0.01 -0.16 p>0.1 -0.12 p>0.1 -0.02 p>0.1 0.002 p>0.1 mt 1 0.11 p>0.1 -0.09 p>0.1 0.32 p=0.001 0.28 p<0.01 -0.20 p=0.05 -0.02 p>0.1 0.33 p=0.001 0.01 p>0.1 -0.27 p<0.01 dna damage 1 0.80 p<0.001 -0.06 p>0.1 -0.01 p>0.1 0.13 p>0.1 -0.11 p>0.1 -0.07 p>0.1 0.05 p>0.1 -0.01 p>0.1 lpo digestive gland 1 0.04 p>0.1 0.04 p>0.1 0.02 p>0.1 -0.11 p>0.1 -0.16 p<0.1 -0.02 p>0.1 -0.05 p>0.1 gst digestive gland 1 0.11 p>0.1 -0.05 p>0.1 0.06 p>0.1 0.31 p=0.001 0.14 p>0.1 -0.02 p>0.1 free zn gills 1 -0.17 p>0.1 -0.16 p>0.1 -0.03 p>0.1 0.11 p>0.1 -0.05 p>0.1 free zn digestive gland 1 -0.13 p>0.1 -0.12 p>0.1 0.1 p>0.1 0.08 p>0.1 hsp70 1 0.20 p>0.1 -0.28 p=0.05 0.01 p>0.1 poly ubiquitin 1 0.002 p>0.1 -0.2 p<0.05 zn gills 1 -0.22 p<0.05 significant pairs are highlighted in bold and in shaded cells. marginal significance (0.5 < p < 0.1) is indicated in italics. mussels exposed to the 10 % effluent concentration, mt levels in the digestive gland were decreased at both zn concentrations. in mussels exposed to zncl2 and the municipal effluent, factorial anova revealed a significant effect for zn concentrations with a positive interaction between the effluent and zncl2 (fig. 3b). correlation analysis revealed that mt levels were correlated with labile zn in digestive gland (r = -02; p = 0.05) and labile zn levels in the same organ (r = -0.22; p < 0.05). the activity in gst was also determined in mussels exposed to effluent, nano-zno and zncl2 (figs 3c, 3d). in mussels exposed to nano-zno, factorial anova revealed a significant effect for both effluent and zn concentrations (fig. 3c). gst activity was not affected by either nano-zno or effluent but was significantly reduced by 10 ug/l nano-zno in the presence of 3 and 10 % effluent. in mussels exposed to zncl2 and municipal effluent, no significant effects were observed (factorial anova p>0.05 for effluent and zn concentrations). correlation analysis showed that gst activity was significantly correlated with condition factor (r = -0.21; p < 0.05) and mt in the digestive gland (r = 0.33; p = 0.001). oxidative stress was examined by determining lpo in the digestive gland (fig. 4). in mussels exposed to nano-zno, factorial anova revealed that the effluent concentration was the main factor, with no significant interaction between zn and effluent concentration (fig. 4a). lpo in the digestive gland was significantly reduced by the 3 % and 10 % effluent concentrations. lpo levels in mussels exposed to nano-zno only were marginally significant (0.05 < p < 0.1). lpo levels were not significantly correlated with the above parameters. in mussels treated with zncl2, factorial anova revealed significant effects for both effluent concentration and the interaction between effluent and zn concentration (figure 4b). in mussels exposed to zncl2 only (i.e., no effluent), lpo was increased at the highest concentration tested (10 ug zn/l). the presence of the municipal effluent eliminated this effect. dna strands in the digestive were also determined in mussels exposed to municipal effluent, nano-zno and zncl2. in mussels treated with nano-zno, factorial anova revealed a significant effect only for the interaction between effluent and zn concentration (fig. 4c). dna strand breaks were increased in mussels exposed to nanozno only at each concentration. the presence of municipal effluent decreased this response, suggesting loss of dna repair. in mussels treated fig. 2 labile zn levels in gills (a, b) and digestive gland (c, d) in mussels exposed to municipal effluent, nanozno and dissolved zncl2. labile zn levels were determined in mussels exposed to both municipal effluent and zn as either nano-zno or zncl2. the letter a indicates significance from me controls. the letter b indicates difference from zn concentrations. with zncl2, factorial anova revealed significant effects both for municipal effluent concentration and interaction between the municipal effluent and zn concentration (fig. 4d). dna strand breaks were significantly increased by zncl2 alone at 10 ug/l. the presence of municipal effluent significantly decreased this response at 3 and 10 % effluent. correlation analysis showed that dna strand breaks were significantly correlated with lpo (r = 0.80; p < 0.001). impacts on the endoplasmic reticulum were determined by following changes in heat shock proteins and protein ubiquitynation (fig. 5). factorial analysis of variance revealed that hsp70 was significantly induced by the municipal effluent at the highest concentration tested (10 %). nanozno alone had no effects on hsp70 levels. however, when nanozno was added in the presence of municipal effluent, the increase in hsp70 was lost. in mussels exposed to zncl2, co-exposure to the municipal effluent increased the levels of hsp70 as above but the influence of the municipal effluent was less important. protein ubiquination was also influenced by the presence of the municipal effluent and nanozno. protein ubiquination was significantly increased by the municipal effluent at the highest concentration (10 %) tested. this response was also enhanced in the presence of 1 ug/l zn as nanozno. this was not observed for zncl2 although an increase in protein ubiquination was observed with 1 ug/l (but not with 10 ug/l) zn as zncl2. correlation analysis revealed that protein polyubiquitin levels were significantly correlated with mt (r = 0.33; p = 0.001), gst in the digestive gland (r = 0.31; p = 0.001) and zn in the digestive gland (r = -0.22; p < 0.05). in the attempt to gain a global view of the effects of nanozno in the presence of municipal effluents, a discriminant function analysis was performed (fig. 6). the mean classification efficiency between controls, nanozno, nanozno+municipal effluent and municipal effluent alone was 60 % the results revealed that nanozno mediated effects differed from those when the municipal effluent was present in the following: gst, mt and protein ubiquination. the effects of the municipal effluent was best explained by changes in labile zn in gills, gst and labile zn in the digestive gland which suggest the mobilization   146 fig. 3 metallothionein levels (a,b) and gst activity (c, d) in mussels exposed to zncl2, nano-zno and municipal effluent. mt levels in the digestive gland were determined in mussels exposed to both municipal effluent and zn, as either nano-zno or zncl2. the letter a indicates significance from controls. the letter b indicates difference from zn concentrations. (release) of zn2+ in municipal effluents. indeed, me related effects were principally discriminated from nanozno and nanozno+municipal effluent by the axis which involved labile zn in gills and digestive gland. discussion in the present study, mussels were exposed to two forms of zn, nano-zno and zncl2, in the presence of a primary physico-chemically treated effluent. exposure to zncl2 did not change zn levels in the digestive gland or gill tissues. in mussels exposed to nano-zno, although whole body zn levels remained somewhat constant, a small increase in zn was found in the digestive gland. this suggests that mussels are exposed to nanozno by the digestive system. however, zn levels in the digestive gland were reduced when in presence of the municipal effluent. municipal effluent is rich in organic matter, which can compete for zn in the digestive gland. this interpretation is justified since mussels were allowed to depurate for 24 h without food before sacrifice. decreased metal concentrations in mussels exposed to municipal effluent have also been shown with this type of effluent (gagnon et al., 2014). indeed, the proportion of zn bound to the colloidal matter in the effluent was on the order of 70 %, which suggests a strong interaction with suspended matter. in another study, zn levels did not readily accumulate in mussels caged 8 km downstream from a primary treated effluent, although it was present in the effluent at concentrations on the order of 25 µg/l (gagnon et al., 2006). these studies suggest that exposure to the suspended material in effluents could influence the bioavailability of metals in exposed organisms. in mussels exposed to nanozno and the municipal effluent, the increase in zn levels in the digestive gland was observed only at the highest concentration (10 µg/l). in the presence of municipal effluent and zncl2, an increase was observed at 1 µg zn/l, but not 10 µg zn/l in the presence of the municipal effluent. labile zn (zn2+) levels in gill and digestive gland tissues were also examined using a fluorescent probe in mussels exposed to either form of zn in the presence (or not) of the municipal effluent. in mussels exposed to   147 fig. 4 oxidative stress (a, b) and dna damage (c, d) in mussels exposed to municipal effluent, nano-zno and zncl2. oxidative stress was determined by measuring changes in lipid peroxidation (lpo) in the digestive gland. the letter a indicates significance from controls. the letter b indicates difference from zn concentrations. nano-zno, labile zn2+ increased in the digestive gland, which suggests the mobilization of ionic zn in the digestive system. zn is considered an essential element which is highly regulated in mussels (lemus et al., 2013). in a previous study, the release of zn2+ from bulk zno and nano-zno in suspension accounted for most, but not all, of the toxicity in zebrafish (yu et al., 2011). mussels exposed to metal oxide nanoparticles revealed that zn was detected in the pseudofeces, indicating absorption through the digestive system (montes et al., 2012). moreover, scanning electron microscopic analysis of the pseudofeces revealed that intact nano-zno was not found, which suggests that nanozno was degraded and involved perhaps the release the zn2+. however, when mussels were exposed to the municipal effluent, labile zn decreased at the highest concentration (10 %), indicating the dampening effects of the municipal effluent with respect to zn mobility. interestingly, gill labile zn levels were increased at the 3 % effluent concentration only in control mussels, which suggests a transient increase in zn in tissues at low effluent concentrations. the explanation for this is unclear, but we could hypothesize that at low concentrations, the dampening effect of the suspended solid matrix is too low to impede the activity of zn2+ in these tissues. the levels of mt, a heavy metal-binding protein, were significantly increased in mussels exposed to 1 ug/l zncl2 alone, but mt were increased by 1 ug/l nano-zno in the presence of the municipal effluent, which suggests the release of zn ions to induce mt. in a previous study with e. complanata mussels coexposed to nano-zno and to a municipal effluent, the metallome was significantly impacted in the digestive gland (gagné et al., 2013). the study revealed that the metallome of mussels co-exposed to nano-zno and the municipal effluent was closer to that of zncl2-exposed mussels, which suggests the mobilization of zn2+. in another study, the freshwater mussel unio tumidus exposed to nanozno alone and in combination with other stressors, such as pesticides and temperature, increased mt and oxidative stress (falfushynska et al., 2015). however, the increase in mt in their study was not accompanied by increased zn bound to mt or by elevated zn in tissues. this is consistent with the marginal negative correlation between labile zn and mt in the digestive gland. this suggests that mt   148 fig. 5 endoplasmic reticulum stress in mussels exposed to municipal effluent, nano-zno and zncl2. endoplasmic reticulum stress was determined by measuring changes in heat shock protein 70 (hsp70) and ubiquitin staining in the digestive gland. changes in hsp70 are shown in a and b for nano-zno and zncl2 respectively. polyubiquitin protein staining is shown in c and d for nano-zno and zncl2 respectively. the letter a indicates significance from controls. the letter b indicates difference from zn concentrations. is involved in oxidative stress rather than in handling the release of zn in cells, which would explain the significant correlation with gst activity (r = 0.32; p < 0.001). gst activity was reduced by nano-zno, but not by zncl2, and this trend was more pronounced in the presence of the municipal effluent. the decreased gst activity was also found in daphnia magna exposed to cuo and zno nanoparticles (mwaanga et al., 2014). the decreased activity was associated with increased production of oxidized gsh, lpo and mt, but the responses were significantly attenuated in the presence of natural organic matter. in the present study, exposure to organic-matter-rich municipal effluent led to increased mt production and reduced gst activity, which suggests oxidative stress. however, lpo was only marginally higher in mussels exposed to nanozno alone, and this effect was lost in the presence of municipal effluent. exposure to other non-oxide nanoparticles, such as gold, produced similar effects in the marine bivalve ruditapes philippinarum (volland et al., 2015). gold nanoparticles were readily taken up by the digestive gland and produced no changes in oxidative stress, but affected phase ii antioxidant enzymes (gst) at the gene expression level. the lack of induction response from mt suggests that nano-zno could partition to other metal stores in cells, such as intracellular granules. this was confirmed in a study in which exposure of goldfish to nano-zno led to an increase in zn percentage in metal-rich granule, while zncl2 exposure led to increased zn in organelles (fan et al., 2013). the study revealed that mt levels were more influenced by zn2+ than by nano-zno, which mobilized first in metal-rich granule stores in cells. it is noteworthy that exposure to nano-zno and zncl2 increased dna strand breaks in the digestive gland, which was highly correlated with lpo in the digestive gland (r = 0.80; p < 0.001), suggesting oxidative stressmediated genotoxicity. analysis of covariance revealed that, in the case of nano-zno, lpo in the digestive gland was the main variable. this supports the hypothesis that genotoxicity was produced by oxidative stress. in the case of zncl2, lpo was still a significant factor, and zn concentration remained   149 fig. 6 discriminant function analysis of mussel responses to the municipal effluent and nano-zno. discriminant function analysis was performed with the biomarker data and zn tissue loadings. a mean classification performance of 67 % was obtained, which suggests that the effects of nano-zno differed from those of the municipal effluent. co-exposure to the effluent and nano-zno produced a response different from that of exposure to nano-zno that was mostly explained by the y axis. the parentheses on the axis indicate the first three biomarkers with the highest factorial weight. significant, which suggests that other mechanisms were at play. however, the presence of municipal effluent eliminated the increase in dna strand breaks. decreased strand breaks in mussels exposed to a primary-treated municipal effluent have previously been shown (gagné et al., 2011). indeed, dna strand breaks were initially decreased at low effluent concentrations and increased at higher effluent concentrations (> 10 %). evidence of endoplasmic reticulum stress was also observed in mussels exposed to both zncl2 and municipal effluent. nano-zno did not show any effects on heat shock proteins or protein ubiquitination. hsp70 levels were elevated in mussels exposed to 10 % municipal effluent and in mussels co-exposed to 1 µg/l zncl2 and 10 % municipal effluent. protein ubiquitination was affected in mussels co-exposed to 10 % effluent and 1 µg/l nano-zno, but the response was more significantly affected by zncl2 alone and in the presence of municipal effluent. in another study, nano-zno did not induce hsp70 protein levels in fish exposed to mg/l concentrations of nano-zno, which is consistent with the present findings (wong et al., 2010). the effects of nano-zno cannot be explained solely by the release of zn2+ in tissues. particle size and surface reactivity (cationic charges at the surface) could also disrupt metabolic processes at the macromolecular level. for example, a proteomic analysis of the digestive gland of m. galloprovincialis exposed to 10 µg/l cuo nanoparticles and cu2+ for 15 days revealed that oxidative stress (gst), proteolysis and apoptosis were produced for cuo nanoparticles while changes in cell adhesion proteins for cu2+ (gomes et al., 2014). protein identification analysis also revealed that the toxicity of cuo nanoparticles is not equal to that of cu2+ and results specifically in stress-induced changes in mitochondria and the nucleus (apoptosis). similar effects were also observed in oysters exposed to relatively high nano-zno concentrations (0.05 to 50 mg/l), leading to mitochondrial disruption and oxidative stress (trevisan et al., 2014). swollen mitochondria and loss of mitochondrial cristae were observed after 24 h in both gills and digestive gland, which suggests oxidative stress and apoptosis from loss of mitochondrial membrane permeability. the toxicity of municipal effluents to marine and freshwater mussels is much better understood than that of nanoparticles. based on discriminant function analysis, exposure of mussels to nano-zno and municipal effluent led to more significant changes on the x axis, which comprised the following endpoints: free zn in gills/digestive gland and gst, while some changes were found on the y-axis, which included the following endpoints with high factorial weight: gst, protein ubiquitination and mt. interestingly, co-exposure to nano-zno and municipal effluent produced an intermediate effect between that of nano-zno and municipal effluent alone. increased oxidative stress caused by municipal effluent could be the result of the   150   151 increased proportion of polyunsaturated lipids (rocchetta et al., 2014). freshwater clams (diplodon chilensis) collected at sites downstream of a sewage discharge point had increased proportions of omega-3 fatty acids (epa and dha), which are targets for lipid oxidation. levels of lpo and lipofuscin (age-related pigments) were also higher in clams collected downstream of the sewage discharge point. caged mussels placed downstream of a municipal discharge showed increased ampactivated protein kinase, indicating increased cellular energy consumption which, in turn, could increase oxidative stress (goodchild et al. 2015). oxidative stress was confirmed by increased superoxide dismutase and gst activity. the negative correlation between mt levels and both total zn and free zn in the digestive gland suggests that mt was involved in the sequestration of other metals or reactive oxygen species occurring in municipal wastewater. mt is also involved in the sequestration of reactive oxygen species, which confers antioxidant properties, and typically in the sequestration of heavy metals in cells (gagné et al., 2008; formigari et al., 2007). in conclusion, exposure of freshwater mussels to nano-zno released in municipal effluent could have an effect on the toxicity of municipal effluents. the oxidative properties of both nano-zno and zncl2 are reduced in the presence of municipal effluent. although no clear signs of oxidative stress were observed at the lpo level, lpo levels were strongly correlated with dna damage and gst activity was positively correlated with mt levels which were not associated with zn binding in the digestive gland. the municipal effluent will likely change the oxidative stress response to nanozno and zncl2 in freshwater mussels. acknowledgements the work was funded under the chemical management plan and the st. lawrence action plan of environment canada. the authors thank sophie trépanier for her technical assistance in mussel handling and exposure at the city of montreal ecotoxicology laboratory. references boryslawskyj m, garrood ac, pearson jt. elevation of glutathione-s transferase activity as a stress response to organochlorine compounds in the freshwater mussel sphaerium corneum. mar. environ. res. 24: 101-104, 1998. balshaw dm, philbert m, suk wa. research strategies for safety evaluation of nanomaterials. part iii: nanoscale technologies for assessing risk and improving public health. toxicol. sci. 88: 298-306, 2005. bradford mm. a rapid and sensitive method for the quantitation of microgram 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http://www.ncbi.nlm.nih.gov/pubmed/?term=mart%c3%adnez-rodr%c3%adguez%20g%5bauthor%5d&cauthor=true&cauthor_uid=25994271 http://www.ncbi.nlm.nih.gov/pubmed/?term=blasco%20j%5bauthor%5d&cauthor=true&cauthor_uid=25994271 http://www.ncbi.nlm.nih.gov/pubmed/?term=wong%20sw%5bauthor%5d&cauthor=true&cauthor_uid=19902187 http://www.ncbi.nlm.nih.gov/pubmed/?term=leung%20pt%5bauthor%5d&cauthor=true&cauthor_uid=19902187 http://www.ncbi.nlm.nih.gov/pubmed/?term=djurisi%c4%87%20ab%5bauthor%5d&cauthor=true&cauthor_uid=19902187 http://www.ncbi.nlm.nih.gov/pubmed/?term=leung%20km%5bauthor%5d&cauthor=true&cauthor_uid=19902187 http://www.ncbi.nlm.nih.gov/pubmed/?term=yu%20lp%5bauthor%5d&cauthor=true&cauthor_uid=21611643 http://www.ncbi.nlm.nih.gov/pubmed/?term=fang%20t%5bauthor%5d&cauthor=true&cauthor_uid=21611643 http://www.ncbi.nlm.nih.gov/pubmed/?term=xiong%20dw%5bauthor%5d&cauthor=true&cauthor_uid=21611643 http://www.ncbi.nlm.nih.gov/pubmed/?term=zhu%20wt%5bauthor%5d&cauthor=true&cauthor_uid=21611643 http://www.ncbi.nlm.nih.gov/pubmed/?term=sima%20xf%5bauthor%5d&cauthor=true&cauthor_uid=21611643 http://www.ncbi.nlm.nih.gov/pubmed/21611643 research report isj 11: 204-212, 2014 issn 1824-307x research report exposure to tributyltin chloride induces penis and vas deferens development and increases rxr expression in females of the purple snail (plicopurpura pansa) d domínguez-ojeda , ae rojas-garcía , ml robledo-marenco , bs barrón-vivanco , im medina-díaz 1 2 2 2 2 1escuela nacional de ingeniería pesquera, universidad autónoma de nayarit, bahía de matanchén km. 12, san blas, 63740 nayarit, méxico 2laboratorio de contaminación y toxicología ambiental, universidad autónoma de nayarit, av. de la cultura s/n, col. los fresnos, 63155 tepic, nayarit, méxico accepted june 26, 2014 abstract tributyltin (tbt) and its derivatives are widely used as antifouling paints for ships, resulting in their being released into the marine environment. aquatic invertebrates, particularly marine gastropods, are extremely sensitive to tbt and undergo changes in the imposition of male secondary sex characteristics in response to exposure. this study aimed to evaluate the development of imposex and the expression of the retinoid x receptor (rxr) in tissues of plicopurpura pansa (males and females) exposed to tributyltin chloride (tbtcl). the histological results showed a penis-like structure in imposexed female and an undeveloped vas deferens that lacked circular muscular layers. tbtcl treatment increased the messenger rna (mrna) of rxr in females with imposex. the highest level of mrna rxr was found in the digestive gland and penis-forming area in females under in vivo exposure compared with control females. these results indicate that tbtcl modulates mrna levels of rxr in females. mrna rxr in imposex females and females exposed to tbtcl only was similar to that of males, indicating that rxr might contribute to the development of imposex. to our knowledge, this study is the first to show that tbtcl induces imposex and biphallia in this snail species, and that this effect is accompanied by an increase in rxr expression. key words: tributyltin; imposex; retinoid x receptor (rxr); purple snail   introduction organotin compounds have been widely used as antifouling paints for ships and fishing nets since the 1960s, resulting in their continuous release into the marine environment (de mora, 1996; fent, 1996; champ, 2000). tributyltin (tbt) and its derivatives are endocrine disruptors that induce deregulation of vertebrate and invertebrate endocrine systems (golub and doherty, 2004). aquatic invertebrates, particularly marine gastropods, are extremely sensitive to organotin compounds such as tbt. consequently, they undergo changes in imposition of male secondary sex characteristics in response to exposure (maguire, 2000; oetken et al., 2004; sternberg et al., 2010), which is caused by a decrease in aromatase ___________________________________________________________________________ corresponding author: irma martha medina díaz laboratorio de contaminación y toxicología ambiental secretaría de investigación y posgrado universidad autónoma de nayarit 63155 tepic, nayarit, méxico e-mail: irmartha@hotmail.com activity (the enzyme that converts androgens to estrogens) (matthiessen and gibbs, 1998; mcallister and kime, 2003). this worldwide phenomenon represents one of the most detrimental consequences of pollution by anthropogenic chemicals and has led these compounds to being banned from antifouling paints in a number of countries; however, organotin compounds remain in the environment (nishikawa, 2006). to date, very low concentrations of tbt have been shown to induce imposex in marine gastropods (smith, 1980, 1981; bryan et al., 1993; horiguchi et al., 1997; matthiesen and gibbs, 1998; nishikawa, 2006; chacón et al., 2007; oehlmann et al., 2007). these abnormalities are the result of a masculinization process by which male sex organs develop a notable penis and vas deferens. in certain species, the growth of the vas deferens disrupts the structure and function of the oviducts, preventing normal breeding activity and causing population decline (bryan et al., 1986; gibbs and bryan, 1986; nakanishi, 2008). at present, the effects associated with tbt have been reported in 268 species of 204   mailto:irmartha@hotmail.com table 1 body size and weight of female and male purple snails used in flow-through exposure experiments (october to march, 2011) control females control males tbtcl-exposed females tbtcl-exposed males shell height (cm) 3.14±0.346 3.16±0.249 3.085±0.33 3.185±0.27 shell weight (g) 4.87±1.61 5.6±1.19 4.85±1.8 6.22±1.40 mean ± standard deviation (sd). there was no statistical differences between size and weight with exposure to tributyltin chloride (tbtci). intertidal gastropods worldwide, belonging to the orders vetigastropoda, mesogastropoda, and neogastropoda. neogastropoda is the most affected order, which contains 214 species, of which 101 are part of the muricidae family (titley-o'neal et al., 2011). the purple snail (plicopurpura pansa) is part of this affected group of species. this snail is an intertidal carnivorous gastropod that inhabits rocky intertidal beaches exposed to strong wave action. it is distributed in the pacific ocean, from the northwestern mexican coast to northern peru (keen, 1971). in addition, this gastropod possesses both economic and cultural importance. alternative mechanisms by which tbt induces imposex in gastropods include the following: 1) increase in androgen levels as a result of the tbtmediated inhibition of aromatase (spooner et al., 1991; bettin et al., 1996); 2) tbt-mediated inhibition of the excretion of androgen sulfate conjugates, with a consequent increase in androgen levels (ronis and mason, 1996); 3) an increase in the level of an alanine-proline-glycine-tryptophan amide neuropeptide in response to tbt, and 4) tbt interference in the release of penis morphogenetic/retrogressive factor from the pedal/cerebropleural ganglia (féral and le gall, 1983). another important mechanism proposed to induce imposex development is through the retinoid x receptor (rxr). this receptor is a member of the nuclear receptor superfamily of ligand-activated transcription factors that have been characterized in a wide variety of metazoan phyla and is highly conserved (bouton et al., 2005). rxr play a central role in a variety of nuclear signaling pathway, have multiple physiological functions, and play key roles in embryo patterning and organogenesis in mammals. rxr is known to act both as a ligand dependent transcription factor and as a common heteroor homodimer partner for many non-steroid fig. 1 hematoxylin and eosin semi-thin cross-section of the male penis of the purple snail 2 months after treatment with 10 μg sn/l of tributyltin chloride (tbtcl). abbreviations: it, interstitial tissue; ml, muscle layer; vd, vas deferens. 205   fig. 2 hematoxylin and eosin cross-section of the penis-like structure (2 mm in length) that developed behind the right tentacle of a female purple snail 2 months after treatment with 10 μg sn/l of tributyltin chloride (tbtcl). abbreviations: it, interstitial tissue; ml, muscle layer; vd, vas deferens. nuclear receptors. because 9-cis retinoic acid effectively induces imposex, rxr may play an important role in the development of imposex in gastropods (mangelsdorf and evans 1995; morriskay 1997; bouton et al., 2005; stenberg et al., 2008). recently, horiguchi et al. (2010) suggested that rxr might be involved in organotin-mediated induction of male-type genitalia (penis and vas deferens) in female rock shells (thais clavigera). although imposex studies have been completed in several gastropods, the development of imposex by tbt in the purple snail (plicopurpura pansa) has not been previously described. the aim of the present study was to evaluate the development of imposex and expression in the rxr gene (mrna) of female and male p. pansa exposed to tributyltin chloride (tbtcl). materials and methods collection of specimens experiments were performed with a gonochoristic prosobranch species, the purple snail, plicopurpura pansa neogastropoda: thaididae. in march 2010, live female and male purple snails were collected by hand at low tide at olas altas, mazatlán, sinaloa, mexico (23°12’ 29.87’’ n, 106°25’ 43.67’’ w). the specimens were immediately transported to the laboratory in plastic cage containing seawater from the collection site. once at the laboratory, the specimens were reared for 2 weeks in a laboratory aquarium to acclimate to artificial seawater (red sea salt®, israel). the purple snails were fed with pieces of squid (loligo spp.). fig. 3 temporal change in the incidence of imposex in female purple snails exposed to 10 μg sn/l of tributyltin (tbtcl) for 6 months in a flow-through system. 206   fig. 4 temporal change in the average penis length of female purple snails exposed to 10 μg sn/l of tributylin (tbtcl) for 6 months in a flow-through system. histological procedure the soft tissues (penis and penis-like structure) of males and imposexed females were extracted from the shell and were fixed with 10% formaldehyde for 12 h. after dehydration with ethanol (70, 80, 90, 96, and 100%), the tissues were immersed in xylene and then embedded in paraffin and sliced into 7 µm sections. the paraffinembedded sections were then stained with hematoxylin and eosin and examined under a light microscope. in vivo experiments with tbt before the experiments, the purple snails were manipulated to allow the selection of females, males, and imposex females. males were identified by the penis, which is located behind the right cephalic tentacle, a common feature among neogastropods. this organ has a characteristic inverted-cedilla form with a 2 mm width that becomes thicker at the base; females were identified by the absence of this characteristic and the presence of organs such albumen and capsule glands (domínguez-ojeda et al., 2009). shell length was measured with a vernier caliper with 0.05 mm precision and weight was determined on an adam digital scale (d = 0.01 g). the purple snails (50 total, including 20 females, 10 imposex-exhibiting females, and 20 males). first, the snails were evenly divided into two experimental groups by sex, with two groups containing 10 females and two groups containing 10 males. one group of each sex was treated with tributyltin chloride (tbtcl) (10 μg sn/l, obtained from lc50), and the remaining group was used as the control. the group of imposexexhibiting females was employed to compare what happens in the wild. stock solutions of tbtcl (sigma-aldrich co., st. louis, mo, usa, 96 %) for the exposure experiments were diluted with ethanol. females with imposex (n = 10) were treated under the same conditions as the control groups. each experimental group of purple snails was maintained for 6 months in an 8 l glass bottle (control females, exposed females, imposex-exhibiting females, control males, and exposed males) with a flowthrough system of artificial seawater (read sea salt®, israel) by means of an aeration pump. the temperature of the experimental seawater was maintained at 25 ± 1 °c and a salinity of 38 ± 2 salt per unit (spu). the purple snails in the two exposure groups were removed each month for imposex examination (presence or absence of penis) and for assays of rxr gene expression in several tissues. these snails were dissected to remove the ovary or testis, digestive gland, penisforming area or penis, and head ganglia according to sex (male or female). then, each sample, containing the respective tissue of 10 individuals according to sex, was prepared. the survival rate of the purple snails in all of the experimental groups was 100 %. table 1 shows the body sizes of the purple snails utilized in the experiment. isolation of total rna total rna was prepared from purple snail tissue using trizol reagent, according to manufacturer’s instructions (invitrogen life technologies, carlsbad, ca, usa). rna was quantified spectrophotometrically at od260, and purity was assessed by measuring the od260/od280 ratio. rna integrity was evaluated by electrophoresing rna samples in 1 % agarose gel. complementary dna (cdna) was prepared from 4 µg of total rna, using the superscript preamplification system for first strand synthesis and oligodt. assay for rxr gene expression to assay rxr gene expression in various tissues, 1 µl of cdna was subjected to polymerase chain reaction (pcr). the following program was used: denaturation at 94 °c for 10 min; 35 cycles of pcr with denaturation at 94 °c for 15 sec; annealing at 45.5 °c for 30 sec, and extension at 72 °c for 30 sec. forward and reverse primers were used: rxr, 5’-gattctggaggccgagattg-3’ and 5’-tggctctttctgggcatca-3’ (horiguchi et al., 2010) (product size, 150 pb), respectively. to 207   fig. 5. imposexed female purple snail with biphallia after 6 months exposure to 10 μg sn/l of tributyltin (tbtcl). abbreviations: db, double penis; m, mantle; rt, right tentacle; lt, left tentacle; ft, foot. scale bar: 2 mm. normalize rxr gene expression in the respective tissues, the expression of 18s ribosomal rna (18s rrna), a housekeeping gene, was used as a reference to observe the effects of upregulated and downregulated genes and was also assayed in these tissues. rxr gene expression in each tissue was normalized by dividing the value for rxr gene content by the value for 18 s rrna content. pcr products were electrophoresed on 2 % agarose gel and stained with ethidium bromide to visualize the pcr amplification products. relative intensity was determined using the sigma gel program (jandel scientific software). statistical analysis the statistical significance of any difference in imposex levels in tbtcl-exposed groups compared with the control groups was tested. the statistical significance of the incidence of imposex was determined by the fisher exact test, and one-factor analysis of variance (anova) was used for penis length. for rxr gene expression in each tissue, we evaluated the statistical significance of the differences between tbtcl-exposed groups and the control groups by mann-whitney u test. results histological features of normal male penis and imposexed female penis treated with tbtcl a semi-thin cross-section of the male penis shows that it possesses two vas deferens, smooth muscle cells surrounding the epithelium, and interstitial cells (fig. 1). in comparison, the imposexed female had an undeveloped vas deferens that lacked circular muscular layers in the interstitial tissues. in addition, the muscle cells and internal epithelium were not visible, possibly because of being at an early stage of differentiation (fig. 2). effects of tbtcl on imposex development in p. pansa the experiments confirm that tbtcl induces imposex in p. pansa females. the incidence of imposex was significantly higher in exposed females compared with control females (p < 0.001) (fig. 3). female penis length gradually, but significantly, increased during the first 2 months of exposure (p < 0.01) (fig. 4). furthermore, at month 6 of tbtcl exposure, one of the imposexed females presented a double penis, the length of each penis 2 mm (fig. 5). effect of tbtcl on rxr mrna expression tbtcl treatment increased the expression of rxr mrna in females with imposex (fig. 6a). the highest level of rxr mrna expression was found in the digestive gland (3.2-fold) and penis-forming area (2.5-fold) (p < 0.02) in tbtcl-treated females compared with control females. however, rxr expression was significantly lower in the gonad and head ganglia of imposexed females exposed to tbtcl compared with control females (p < 0.05) (fig. 208   fig. 6 expression of rxr mrna levels in several tissues of (a) plicopurpura pansa imposex females, and (b) females exposed to tributyltin chloride (tbtcl). female snails were exposed to tbtcl (10 μg sn/l) or 0.5 % ethanol (control) during 6 months. relative expression of rxr mrna was determined by pcr. changes in level of expression were determined by normalizing the data to 18s and were expressed relative to the control or to the female without imposex. data are given as the mean ± standard deviations (sd) from two experiments performed in triplicate. the asterisk (*) indicates significant differences vs. the control (p < 0.05). gon, gonad; dg, digestive gland; pa, penis area; hg, head ganglia 6a). less pronounced effects were observed in females without imposex that were treated with tbtcl (fig. 6b). these results indicate that tbtcl might modulate mrna levels of rxr in females. in addition, we investigated whether a similar increase in rxr mrna levels was recorded in males treated with tbtcl (fig. 7). no significant increase in rxr gene expression was observed in the digestive gland and penis-forming area of males exposed to tbtcl compared with control males. however, a significant decrease in rxr mrna expression was observed in the gonad, head ganglia, and spermatic gland (p < 0.05). discussion in this study, we demonstrated that 10 μg sn/l tbtcl induces imposex within 2 months in normal female p. pansa. this finding indicates that rxr might be important in the development of imposex in gastropods. low concentrations of tbt (<1 ng/l [such as sn]) have been shown to induce imposex in neogastropods (gibbs et al., 1988; gooding et al., 2003). tbt concentrations of this magnitude and greater have been measured in marine environments where imposex occurs (svavarsson et al., 2001). thus, there is much evidence implicating tbt as the cause of imposex among prosobranch gastropods. a previous study indicated that induction of imposex and penis growth required 1 and 2 3 months, respectively, in thais clavigera (horiguchi et al., 2010). this study also demonstrated that 10 μg sn/l tbtcl causes the penis-like structures observed in the females of treated purple snails to form, with these structures being histologically recognizable as penises with vas deferens. these structures resembled the organs of morphologically normal males and of imposexed females collected from the wild. it is noteworthy that a previous study by our group recorded similar or higher concentrations of tbt in wild p. pansa (domínguez-ojeda, 2012). several examples of intersex and sex reversal have been hypothesized as being induced by environmental chemicals in wildlife. furthermore, some studies have shown that organotins or ligands for nuclear hormone receptors (retinoid x receptors [rxr]) cause a decrease in the production of cytochrome p450dependent aromatase (cyp19) mrna in human ovarian granulose cells (mu et al., 2001). in addition, the rxr homolog in gastropod mollusks has been found to be responsive to 9-cis retinoic acid (ra), with tbt and 9-cis ra shown to induce imposex (nishikawa et al., 2004; castro et al., 2007). these findings indicate that the transcriptional mechanism for tbt action is conserved across phyla (iguchi and katsu, 2008). in this study, rxr expression in imposex females and females exposed to tbtcl was similar to that of males. this observation indicates that rxr might contribute to imposex development. mrna levels of rxr in tbtcl-exposed females might have been modulated in the population of p. pansa, as documented in other studies (bryan et al., 1986; gibbs et al., 1991; huet et al., 1996). also, in this study, we observed different values of rxr expression in different tissues, in different treatments, and in different sexual categories. these results are according to horiguchi et al. (2010); the authors observed different patterns of 209   fig. 7 effects of tributyltin chloride (tbtcl) on the rxr mrna levels in several tissues of plicopurpura pansa males. male snails were exposed to tbtcl (10 μg sn/l) or 0.5 % ethanol (control). after 6 months of treatment, relative expression of messenger rna (rxr mrna) was determined by pcr. changes in level of expression were determined by normalizing the data to 18s and were expressed relative to the control. data are given as the mean ± standard deviations (sd) from two experiments performed in triplicate. the asterisk (*) indicates significant differences vs. the control (p < 0.05). gon, gonad; dg, digestive gland; pa, penis area; hg, head ganglia; sg, spermatic gland. expression of rxr among tissues analyzed. rxr possesses ubiquitous, or quite widespread, expression patterns, similar to those of other nuclear receptors (rarβ and rarγ). rxr levels of expression are related with early developmental stages and are tissue-specific. in terms of differential expression, only in some instances does their distribution correlate with specific differentiating cell or tissue types throughout the organism. although rxr are expressed rather ubiquitously, their expression also depends on epigenetic regulation, e.g., modifications in histones and degree of methylation of cpg dinucleotides (dollé, 2009; laursen et al., 2012). the differences observed in the time required for the development of imposex in different species might be associated with differences in the rate of accumulation of organotins to a level that exceeds the threshold concentration for increased rxr expression in target organs/cells. this parameter might vary according to the characteristics of each species. in addition, some studies have found that tbt increases testosterone (t) titers in a doseand time-dependent manner in snails, with administration of t also inducing imposex (spooner et al., 1991; stroben et al., 1991). although the present study did not demonstrate the mechanisms by which tbtcl gives rise to imposex in p. pansa, it is, to our knowledge, the first to demonstrate that tbtcl induces imposex and biphallia in this species of snail, and that this effect is accompanied by an increase in rxr expression. acknowledgments the authors thank dr. o aracely patrónsoberano from the instituto potosino de investigación, científica y tecnológica, méxico (ipicyt) for technical advice on microscopy. this research was made possible through nayarit 2008-c01-93389 grants. references bettin c, oehlmann j, stroben e. tbt-induced imposex in marine neogastropods is mediated by an increasing androgen level. helgol meeresunters 50: 299-317, 1996. bouton d, escriva h, de mendonça rl, glineur c, bertin b, noël c, et al. a conserved retinoid x receptor (rxr) from the mollusk biomphalaria glabrata transactivates transcription in the presence of retinoids. j. mol. endocrinol. 34: 567-82, 2005. bryan gw, burt gr, gibbs pe pascoe 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venerupis philippinarum x chen1, r zhang1, c li1, y bao2 1school of marine sciences, ningbo university, ningbo, zhejiang province 315211, pr china 2zhejiang key laboratory of aquatic germplasm resources, zhejiang wanli university, ningbo 315100, pr china accepted october 13, 2014 abstract mercury (hg), one of the most hazardous and persistent contaminants, is widespread in the aquatic environment. to establish an effective hg monitoring strategy, the mrna expression profiles of four antioxidant enzymes, as well as sod enzymatic activities and mda content were investigated at two sublethal hgcl2 exposure doses of 5 and 50 μg l -1 in venerupis philippinarum gill tissue and hemocytes at 1, 2, 4 and 5 days. all parameters tested showed significant differences between the experimental and control groups at the various time points with tissue-specific manners. gst displayed a dose-dependent increase expression profiles in the two examined tissues. also decrease expression patterns were detected in trx and cyp414a1 in both gill and haemocytes with a significant positive relationship of 0.915 in the form tissue (p < 0.01). a positive relationship was found in those of sod expression and the sod enzymatic activities in hemocytes (0.683, p < 0.05). taken together, we found that gill tissue is more suitable for biomarker identification compared to that of hemocytes because of lower variation. this study provides new evidences that mrna expression of trx, cyp414a1 and gst in gill tissue has a strong potential as a biomarker for marine mercury monitoring. key words: venerupis philippinarum; mercury; gene expression; enzymatic activity   introduction the manila clam venerupis philippinarum is a major aquaculture species worldwide and is widely distributed in high densities in coastal intertidal areas. due to its wide distribution, long life cycle, high tolerance to salinity and temperature, ease of collection, and high bioaccumulation of heavy metals (laing and child, 1996; kim et al., 2001; baudrimont et al., 2005; liu et al., 2011a, b ,c; wu et al., 2011), the manila clam exhibits most of the criteria that define a bio-indicator for metal pollution monitoring and has therefore been considered a useful bio-indicator in marine biology and ecotoxicology (liang et al., 2004; ji et al., 2006; zhang et al., 2011a, b, c; liu et al., 2013a, b). recent toxicological studies have elucidated distinct biochemical and genetic responses of v. philippinarum to heavy metals ___________________________________________________________________________ corresponding authors: chenghua li ningbo university 818 fenghua road ningbo, zhejiang province 315211, pr china e-mail: lichenghua@nbu.edu.cn yongbo bao zhejiang wanli university 8 south qianhu road ningbo, zhejiang 315100, pr china e-mail: bobbao2001@gmail.com and other toxic contaminants, further supporting the proposition that v. philippinarum may be useful for monitoring marine and coastal pollution (blasco and puppo, 1999; ji et al., 2013; nasci et al., 2000; moraga et al., 2002; irato et al., 2003; wu et al., 2013a, b ,c; zhang et al., 2011d, 2012a). mercury (hg) is a widely distributed pollutant and has been listed as a high priority pollutant by many international agencies because of its persistence in environments and high toxicity to organisms (jiang et al., 2006). increasing evidences have demonstrated that the mechanism underlying mercury toxicity is closely related to the generation of reactive oxygen species (ros) and the perturbation of antioxidative defense systems (larose et al., 2008; liu et al., 2011a). organisms can hold in check and detoxify the production of ros with antioxidative defense systems comprising of a wide variety of enzymatic and non-enzymatic molecules to balance the harmful effect of ros, which include superoxide dismutase (sod), glutathione-s-transferase (gst), cytochrome p450s (cyps) as well as non-enzymatic molecule of thioredoxins ( trxs). sods are metalloenzymes that represent one of the important lines of defense against ros, particularly against the superoxide anion (wang et 298 al., 2013). sods can catalyze the dismutation of superoxide into molecular oxygen and hydrogen peroxide (fridovich, 1995). gsts are a superfamily of multifunctional phase ii enzymes primarily involved in the detoxification of both endogenous and exogenous electrophiles (zhang et al., 2012b). moreover, gsts have been found to play a critical role in mitigating oxidative stress in all life forms (lee et al., 2008), and gst activities have been widely used as potential biomarkers for monitoring environmental pollution (hoarau et al., 2002; shailaja and d'silva, 2003; cunha et al., 2007). trxs are members of an evolutionarily conserved family of redox-active proteins and function to scavenge reactive oxygen species (ros) (wang et al., 2011). cytochrome p450s (cyps) comprise one of the largest and most versatile heme-thiolate protein families. they can catalyze the oxidation of a wide variety of exogenous compounds or xenobiotics. cyp414a1 is classified as a member of a new subfamily of cytochrome p450 (zhang et al., 2012c). cyp414a1 has been shown to respond to various xenobiotic stresses and could potentially serve as a candidate biomarker of heavy metals. the lipid peroxidation product malonyldialdehyde (mda) has also been widely utilized as an indicator of oxidative status (roméo and gnassia-barelli, 1997). to establish a rapid and accurate detection system for hg in marine environments, specific biomarkers are the first priority. as filter-feeding organisms, molluscs expose to various environment pollutants including heavy metals in the aquatic environment. gills of the bivalves remain in direct interaction with the surrounding aquatic environment and their biochemical profiles reflect the adverse environmental impact on the animal (gregory et al., 2002) and the study of biochemical biomarkers in this organ is usually employed in environmental monitoring studies (medeiros et al., 2008; zanette et al., 2008). meanwhile, hemocytes of molluscs are the first line of defense against environmental exposure, through phagocytosis and stimulation of respiratory burst and widely used as models in environmental toxicology, in order to determine the target of heavy metals’ toxic effects (bouki et al., 2013). in the present study, the expression levels of four antioxidant enzymes and malondialdehyde (mda) contents were assayed in venerupis philippinarum gill and hemocytes following two doses of hg exposure. their correlations were further analyzed to select reliable multi-indicators for hg biomonitoring. materials and methods experimental clams, hg exposure, and tissue collection venerupis philippinarum (wet weight 8.65 ± 1.24 g) were purchased from ningbo, zhejiang province, china and acclimated for three days prior to the experiment. the temperature of the aerated artificial seawater (salinity 25 psu) was maintained at 18 20 °c throughout the experiment. in the challenge experiment, approximately 450 clams were randomly divided into three tanks (90 cm×60 cm×40 cm), each containing 150 individuals in 30 l seawater. the clams in the two treatment tanks were exposed to different concentrations of hgcl2, either 5 or 50 μg l-1. the low experimental concentration (5 μg l-1) of hg2+ can be found in heavily polluted seawaters along china coast (zhang, 2001). the untreated individuals were selected as a control group. the hemolymphs from the control and challenge groups were collected using a sterile 5 ml syringe containing 1 ml of alseve buffer (als buffer: 60 mm glucose, 27.2 mm sodium citrate tribasic, 9 mm edta, 385 mm nacl, ph 7 and 1000 mosm/l), then centrifuged at 800 rpm for 5 min to harvest the hemocytes. gill were were sheared using sterilized scissors and tweezers, mixed together, and then ground into a fine powder using liquid nitrogen at 0, 1, 2, 4 and 5 days following hgcl2 exposure. all the samples were then stored at −80 °c for rna or protein extraction. we performed five replicates for each experimental groups as well as for the control group. temporal expression profiles of candidate genes under hgcl2 exposure total rna was isolated from clam hemocytes or gills using trizol reagent (invitrogen). first-strand cdna synthesis was performed using the mmlv first strand cdna synthesis kit according to the manufacturer’s instructions (bio basic inc.). the expression levels of sod, gst, trx and cyp414a1 were measured by qpcr. the primers used for qpcr are shown in table 1. two clam β-actin primers were used to amplify a 121 bp fragment as an table 1 primers used in the present study primer sequence (5’—3’) product size gene id β-actin (forward) ctcccttgagaagagctacga β-actin (reverse) gataccagcagattccataccc 121bp ef520696.1 sod (forward) gtgcaggtcctcactataaccca sod (reverse) gacaactcgtgaccacctttacc 226 bp gq384412 gst (forward) cattgcccgtgcttactattgac gst (reverse) tgttcctttcttcctcgttatcc 207 bp gq384392 trx (forward) ggacgttgatgatgtttcggaggt trx (reverse) tttccagttcatcagcatcagccc 125bp jf499393 cyp414a1 (forward) aggaccgaggtcatgtttag cyp414a1 (reverse) ggatttagagttgtcgccag 147bp hq234335 299 fig. 1 time course expression of sod in gill tissue and hemocytes of v. philippinarum after exposure to different doses of hgcl2 as measured by qpcr at 0, 1, 2, 4 and 5 days. internal control to verify successful reverse transcription and to calibrate the cdna template. qpcr amplification was performed using a rotor-gene 6000 real-time pcr detection system. real-time pcr amplifications were performed in a total volume of 20 μl containing 10 μl of 2×sybr green mix (takara), 4 μl of the 1:20 diluted cdna, 1 μl of each primer (10 mm) and 4 μl of pcr-grade water. the qpcr parameters included a denaturing step at 95 °c for 10 min, followed by 40 cycles of 95 °c for 15 s, 60 °c for 20 s, and 72 °c for 20 s. melting analysis of the amplified products was performed at the end of each pcr to confirm that a single pcr product was generated. the 2−δδct method was employed to analyze the expression level of candidate genes (li et al., 2011). the values obtained denoted the n-fold difference relative to the calibrator. the data are presented as relative mrna expression levels (means ± sd, n = 5). sod activity assay sod activities were determined by the method of inhibition of tetrazolium salt wst-1 reduction to formazan with xantine/xantine oxidase used as a superoxide generator. briefly, 0.3 g of gill tissue was homogenized in liquid nitrogen and resuspended in 3 ml normal saline. hemocytes from 18 ml of hemolymphs were used for protein extraction. for the enzymatic assay, the supernatant was collected by centrifuging at 3,000 rpm for 10 min. protein concentration was quantified following bradford (1976). absorbance was monitored at 550 nm. the total sod activity was expressed in u/mg of protein, with one unit of sod being defined as the amount of sample that produced 45 to 50 % inhibition under the assay conditions. mda content assay mda content was measured by the formation of thiobarbituric acid (tba) reactive substances (tbars) using commercial kits (jiancheng bioengineering institute, china). briefly, the reaction mixture containing 0.2 ml tissue homogenate, 0.2 ml 8.1 % sds, 1.5 ml 20 % acetic acid buffer (ph 3.5), 1.5 ml 1 % tba and 1 ml distilled water was heated at 95 � for 60 min in a water-bath, then cooled and centrifuged at 3000 rpm for 15 min. the absorbent value was assayed at 532 nm to calculate the content of mda according to the following formula: ps odod odod mdacontent cs cm ÷× − − = in the formula, odm is the od value of the measured sample, ods indicates the od value of the standard sample in the kit, odc is the od value of the control, s is the concentration of the standard sample (10 nmol/ml) and p is the protein content (mg/g tissues). statistical analysis all data were subjected to one-way analysis of variance (anova) followed by the multiple duncan test to test for differences between the treatment and control groups at each sampling time. significant differences between the treatment group and the corresponding control group at each time point are indicated with one asterisk for p < 0.05 and two asterisks for p < 0.01. spearman correlation analysis was conducted with spss 19.0 for windows (spss). significant differences between the treatment group and the corresponding control group at each time point are indicated with one asterisk for p < 0.05 and two asterisks for p < 0.01. results and discussion expression profiles of sod in gill tissue and hemocytes at the mrna and protein levels the mrna relative expression of sod in gill tissue and hemocytes upon hgcl2 exposure are shown in figure 1 and sod activities are indicated in figure 2. divergent expression trends of sod mrnas 300 fig. 2 sod activities in gill tissue and hemocytes of v. philippinarum exposed to hgcl2 (5 μg l-1, 50 μg l-1) for 0, 1, 2, 4 and 5 days. were detected not only in different tissues but also under different exposure doses. these results are consistent with the the fact that higher variation expression profiles of metalloenzymes like sods (kim et al., 2007). at a concentration of 5 μg l−1 hg exposure, the expression level of sod in hemocytes increased during the first 2 d and then decreased gradually until 5 d. in contrast, the expression profiles of sod in gill tissue decreased at the first stage and then sharply increased to its peak expression level at 4 d with a 1.22-fold increase over that of the control group (p < 0.05). when hg concentration increased to 50 μg l−1, the induced sod expression occurred earlier compared to that in the lower doses group. the peak expression of sod was detected at 2 d with a 2.46-fold increase in gill tissue (p < 0.01) and a 4.78-fold increase at 1 d in hemocytes. regarding to sod activities, hg treatment resulted in a significant decrease sod protein activities in gill tissue and a slight increase in hemocytes (fig. 2). an unpaired, two-tailed t-test between the control and treatment groups showed there is a significant difference in sod activity at 4 d (p < 0.01) following the higher level of hg exposure. however, no significant differences were observed at other time points. it was well-documented that hg could induce oxidative stress in organisms by generating ros via the mitochondrial electron transport chain (lund et al., 1991; fang et al., 2013), and the change in superoxide radical content was correlated with the induction or depression of sod. sods represent the first line of defense in biological systems against oxidative stress caused by excessive reactive oxygen species (ros), in particular superoxide anion. many researchers had analyzed sod activity under hg stress in bivalves other than v. philippinarum. verlecar et al. (2008) reported inhibited sod activity at day 5 and increased sod activity at day 15 in the digestive gland of the mussel perna viridis after exposure to 45 μg l−1 hg. exposure of the mussel dreissena polymorphato to 40 μg l−1 hg for 5 days resulted in an increase in sod activity in the digestive gland (faria et al., 2009). however, company et al. (2004) reported that sod activity remained unchanged in the gill of the mussel bathymodiolus azoricus exposed to 20 μg l−1 hg for 24 h. in general, the response of sod activity to hg exposure differs depending on the tissue, concentration, and exposure time. in the present study, the fluctuation of sod mrna expression and the alteration of sod activity together indicate that sod was involved in countering the oxidative stress caused by hg exposure. a positive relationship between sod mrna expression and sod activity was detected only in hemocytes (p < 0.05) (table 2). this discrepant finding might be attributed to the existence of different isoforms of sod in clam. mrna expression was quantified as one of isoform types of sods, whereas sod activity was quantified in terms of total sod activity (fang et al., 2013). the increased sod mrna expression in hemocytes at low concentration illustrates the fast adaptive response of sod to hg stress (fang et al., 2012). the decline of sod mrna expression and sod activity might be ascribed to the stimulation of other antioxidants to defend against ros toxicity, as suggested by verlecar et al. (2008) and jo et al. (2008). the discrepancies in the responses of different antioxidants are common among other researches, and several observations also highlighted that these defenses do not vary in a synchronous way, and have different time-courses of activation both at the transcriptional and translational levels (frenzilli et al., 2004; regoli et al., 2011; giuliani et al., 2013). these results suggest that, in addition to being influenced by enzymatic activity, sod can be regulated by hg at the molecular level in v. philippinarum. chatziargyriou and dailianis (2010) reported that the inhibition of se-gpx activity by hg could promote a shift in the balance between oxidants and antioxidants in favor of oxidants, resulted in the enhancement of hg induced oxidative and genotoxic effects. on the other hand, increased enzymatic activity and a 301 table 2 determination of correlation coefficients among the different examined parameters gill hemocytes sod level sod activities gst level trx level cyp414 a1 level mda content sod level sod activities gst level trx level cyp414 a1 level mda content sod level 1 1 sod activities 0.085 1 0.683* 1 gst level 0.573 -0.061 1 0.695* 0.4 1 trx level 0.427 0.317 0.378 1 0.817** 0.354 0.609 1 cyp414 a1 level 0.573 0.427 0.561 0.915** 1 -0.329 -0.476 0.277 0.024 1 mda content 0.5 0.207 0.183 0.171 0.39 1 -0.622 -0.22 -0.006 -0.5 0.659* 1 *statistical significance was denoted by p < 0.05, ** statistical significance was denoted by p < 0.01 versus the respective control group significant attenuation of hg toxicity were measured in se-treated cells exposed to hg in all cases. it’s helpful to reveal sod's ability to attenuate hg-mediated ros production, as well as the role of se-gpx. gst expression patterns following hg2+ exposure gsts play a critical role in mitigating oxidative stress in all life forms (hoarau et al., 2002), and gst activity has been widely used as a potential biomarker for monitoring environmental pollution (umasuthan et al., 2012). in the present study, similar expression trends of gst were detected not only in different tissues but also at different exposure doses (fig. 3). the gill gst transcripts was induced with a dose-dependent and time-dependent manners and reached its maximum level at 50 μg l−1 exposure dose with a 2.45-fold increase. in the hemocytes, the peak expression level was detected at 1 d with a 2.12-fold increase in the higher exposure dose group. many studies had indicated that metals/metalloids such as hg were potent inductors of sulfur metabolism through an increase in gsh synthesis (sobrino-plata et al., 2009). in addition, the level of intracellular gsh was directly proportional to gst activity, which leaded to an increase in the expression level of gst (nugroho and frank, 2012). in the freshwater prawn acrobrachium malcolmsonhii, gsh content was found to be higher in the tissues of test prawns, suggesting the formation of glutathione conjugate to eliminate hg in test prawns (yamuna et al., 2012). oxidative stress from hg exposure was also considered to elicit antioxidant gene expression, such as expression in the gsh s-transferase system. in our previous studies, gsts have shown high elevations in mrna levels under heavy metal stress, thus serving as useful biomarkers (rhee et al., 2007). trx expression patterns following hg2+ exposure thioredoxin (trx) proteins perform important biological functions in cells by changing the redox state of proteins via dithiol disulfide exchange. the expression level of trx following hg2+ exposure is shown in figure 4. at 5 μg l−1 hg2+, trx in gill tissue was constantly down-regulated and reached its minimal level at 5 d with a 0.2-fold decrease compared to the untreated group (p < 0.01). however, a very different expression profile was detected in hemocytes at this lower exposure dose. trx mrna levels increased sharply to a peak at 2 d with a 12.0-fold increase, and then recovered to the original level at 5 d. at the higher concentration of 50 μg l−1, an increased expression pattern was detected in gill tissue with a 3.72-fold increase relative to the untreated group at 2 d. surprisingly, no significantly change was detected in hemocytes at this exposure dose. as a molecule involved into counteracting oxidative stress and scavenging ros, trx serves as an electron donor for some peroxidases such as peroxiredoxins and is reduced by thioredoxin reductase to the reduced dithiol state (gonzalez et al., 2010). stadtman (1993) indicated that heavy 302 http://www.ncbi.nlm.nih.gov/pubmed?term=yamuna%20a%5bauthor%5d&cauthor=true&cauthor_uid=23033656 fig. 3 time course expression of gst in gill tissue and hemocytes of v. philippinarum after exposure to different doses of hgcl2 as measured by qpcr at 0, 1, 2, 4 and 5 days. metals participated in a metal-catalyzed fenton type reaction with superoxide or peroxide molecules to generate highly toxic hydroxyl radicals. thus, the observed induction of trx in the first stage could be interpreted as a response to this oxidative stress. a similar experiment by wang et al. (2011) indicated that trx was also significantly induced by 40 μg l−1 cd exposure at 96 h. it has been demonstrated that trx oxidation was significantly correlated with the inhibition of total trxr activity during heavy metal treatment (myers, 2012); therefore, trxr activity should be investigated in our future work. cyp414a1 expression patterns following hg2+ exposure previous studies had provided evidence that hg2+ modulated the expression of cytochrome p450 1a1 (cyp1a1) by affecting its transcriptional and posttranslational levels (amara et al., 2010). in the present study, the expression level of cyp414a1 is shown in figure 5 and exhibits a dose-dependent expression profile. at the lower level of hgcl2 exposure, the decreased expression profiles were both identified in the two examined tissue with different amplitudes. the minimal expression levels were detected at 5 d, with a 0.22-fold decrease in gill tissue and a 0.46-fold decrease in hemocytes. when hg2+ concentration increased to 50 μg l−1, we found that the expression of cyp414a1 was both elevated sharply to its peak levels following 1 d of heavy metal exposure in the two tissue. afterwards, the expression of cyp414a1s was both down-regulated slightly, and minimal expression levels were detected at 4 d. fig. 4 time course expression of trx in gill tissue and hemocytes of v. philippinarum after exposure to different doses of hgcl2 as measured by qpcr at 0, 1, 2, 4 and 5 days. 303 fig. 5 time course expression of cyp414a1 in gill tissue and hemocytes of v. philippinarum after exposure to different doses of hgcl2 as measured by qpcr at 0, 1, 2, 4 and 5 days. activation of the nf-κb and ap-1 (activator protein-1) signaling pathways had been demonstrated to be directly involved in the induction of cyp1a by hg stimulation (korashy et al., 2008). although the expression level of ap-1 was not investigated in that study, the induction of ap-1 and cyp414a1 was detected by our lab in the same species following di(2-ethylhexyl) phthalate (dehp) exposure (lu et al., 2013). an analogous study revealed that the expression of cyp414a1 was significantly up-regulated by cd exposure but sharply down-regulated by cu exposure (zhang et al., 2012c). a possible explanation for the distinct expression profiles following differential hg exposure doses is that no acute toxic effects were reported at the administered low dose. however, sublethal effects are subtler and take more time to become evident. higher doses of hg could potentially increase the toxic burden, with sub-lethal effects seen more rapidly (amachree et al., 2014). mda content analysis lipid peroxidation was a main indicator of oxidative damage, and mda was considered to be an ideal biomarker for this process. mda levels following hg2+ exposure were investigated in our study, and the results are shown in figure 6. in hg-treated gill tissue, mda content did not change significantly at the two different exposure doses. however, related to the control group, mda content in the hemocytes decreased significantly at 4 d at the higher hg concentration and at 5 d at the lower hg concentration. mda content of hemocytes after hg treatment was 46.48 % and 45.56 % lower at the higher and lower hg concentrations compared to the control group, respectively. these results demonstrated that hg exposure modified the production of mda in clams. most previous studies in different species had revealed significant increases in mda content following exposure to environmental pollutants (prakash et al., 1995; giguere et al., 2003; charissou et al., 2004), implying that higher oxidative stress was produced during that period. tavazzi et al., (2000) reported that lipid peroxidation of human erythrocytes also is deeply affected by oxidative stress. however, a negative relationship between hg exposure and mda activity was found in our study. we speculated that this result might be due to the following reasons. hg had a high affinity for gsh, which was the primary intracellular antioxidant agent and could bind to, and cause the irreversible excretion of, gsh, leading to the depletion of gsh and an increase in mda (quig, 1998). however, in the hemocytes of clams exposed to hg, a decrease in mda levels was associated with an increase in sod levels. this decrease could result in an intensification of antioxidant systems, including sod, limiting mda formation. it was well known that mehg was the most toxic form of hg in the environment, which was converted by aquatic microorganisms (compeau and bartha, 1985). this process could not be achieved under our lab conditions, leading to a reduction in the strength of the toxic effect and minimal ros production. in contrast, the induction of antioxidant enzymes (sod, cyp414a1 and gst) and the non-enzyme member (trx) was capable of suppressing this lower ros level at an early stage, preventing oxidative stress-induced damage to lipids. furthermore, zhang et al. (2014) reported that the decrease in mda might result from an intensification in production by the antioxidant systems, an interpretation consistent with our results. the depletion of mda levels might also be related to the faster transformation of mda into other metabolites (giarratano et al., 2010). further work should be conducted to investigate the changes in ros levels before and after hg exposure to better understand these mechanisms. 304 fig. 6 mda content of gill tissue and hemocytes of v. philippinarum exposed to hgcl2 (5 μg l-1, 50 μg l-1) for 0, 1, 2, 4 and 5 days. relationships between examined parameters to establish multiple biomarker detection system and improve the accuracy of detection technique, correlations analysis of these candidate biomarkers both in gill and hemocytes are further assessed and the result is showed in table 2. there is a significant positive relationship between the expression levels of trx and cyp414a1 expression in gill (0.915, p < 0.01) and trx and sod expression in hemocyte (0.817, p < 0.01). multiple factors correlation analysis also indicate a higher consistent expression pattern among sod, gst, cyp414a1 and mda content (p < 0.05). no statistically difference are detected among other examine parameters. conclusions our work presented here addressed that mrna expression of trx, cyp414a1 and gst in gill tissue has a strong potential as biomarkers for marine mercury monitoring. this findings supported that gills was promising tissue for further biomarker identification. more importantly, multiple biomarker detection system sounded to be promising choice in order to improve the accuracy of detection technique. the changes of ros levels before and after hg exposure should be further investigated to fully understand the interaction between ros production and antioxidant enzymes. acknowledgments this work was financially supported by the project of international cooperation in the zhejiang province (2012c24022), an open grant 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abstract the hematopoietic process by which blood cells are formed has been intensely studied for over a century using several model systems. an increasing amount of evidence shows that hematopoiesis, angiogenesis, immune response and the regulating these processes (i.e., cytokines) are highly conserved across taxonomic groups. over the last decade, the leech hirudo medicinalis, given its simple anatomy and its repertoire of less varied cell types when compared to vertebrates, has been proposed as a powerful model for studying basic steps of hematopoiesis and immune responses. here, i provide a broad overview of h. medicinalis hematopoiesis and i highlight the benefits of using leech as a model. key words: leech; hematopoietic precursor; stem cells; vegf; hematopoiesis   introduction in vertebrates, hematopoiesis occurs throughout embryonal, fetal and/or adult life and has been evolutionarily conserved. primitive hematopoiesis in vertebrates first occurs in the extraembryonic yolk sac, where aggregates of cells, forming “blood islands”, give rise to both precursors of endothelial cells (localized at the periphery of the cellular aggregates) and hematopoietic stem precursor cells (hspcs) (localized at the central part of the cluster) (choi et al., 1998; saha et al., 2004). both hematopoietic and vascular progenitors derive from a common precursor of mesodermal origin, known as hemangioblast (baron, 2003; bailey and fleming, 2003). a second round of hematopoiesis, termed definitive hematopoiesis, occurs in the mesodermal aorta/gonad/ mesonephros (agm) region of the embryo and gives rise to cells that will seed the fetal liver and the bone marrow in mammals and from which all mature blood cell types of adult organism derive (dzierzak, 1999; matsuoka et al., 2001). in adult life, hspcs primarily reside in the bone marrow, but during homeostasis and/or under stress conditions they circulate in blood and lymph, reaching other hematopoietic and nonhematopoietic organs (massberg et al., 2007; si et al., 2010). thus, injury, infections and inflammation can all induce stem cell niches outside the bone ___________________________________________________________________________ corresponding author: annalisa grimaldi department of biotechnology and life sciences university of insubria via j. h. dunant 3, 21100 varese, italy e-mail: annalisa.grimaldi@uninsubria.it marrow (martinez-agosto et al., 2016), which, under specific conditions, can support hspcs proliferation and differentiation. in particular, in sites of tissue inflammation several cell types, including activated endothelial cells, fibroblasts, macrophages, and other innate immune cells, provide a wide range of hematopoietic growth factors required for hspcs homing, retention, and function (granick et al., 2012). since hspcs circulate in the body periphery, express pattern recognition receptors (prr) and respond to conserved microbial and viral molecular patterns, it has been hypothesized that they represent cells actively involved in innate immune and inflammatory responses (nagai et al., 2006). prr are signaling receptors that recognize conserved pathogenassociated molecular patterns (pamps) and endogenous danger-associated molecular patterns (damps) in order to trigger immune responses. indeed, upon detection of pamps, hspcs preferentially differentiate towards mature myeloid immune cells to fight infection (de luca et al., 2009). these evidences suggest that hspcs not only represent a key source of leukocytes in the bone marrow, but also play a crucial role in peripheral tissues, where they act as a source of both mature effectors for immediate local leukocyte needs and innate immune cells, which orchestrate inflammatory responses and mediate tissue repair (granick et al., 2012). in the last decade, a large body of evidence has demonstrated the occurrence of significant similarities in hematopoiesis, angiogenesis and immune response among vertebrates and various invertebrate phyla (hartenstein, 2006; hartenstein and mandal 2006). in particular, the host defense in hirudo medicinalis (annelida, hirudinea) is extremely 257 mailto:annalisa.grimaldi@uninsubria.it efficient and relies exclusively on innate immunity, which provides a strong nonspecific response characterized by the proliferation and migration of immune cells towards the sites of infection. in response to surgical wounds, grafts or injections of stimulating factors (i.e., lps, gram-positive bacteria, cytokines and growth factors), hspcs are massively recruited from the bloodstream towards sites of inflammation, where they differentiate into mature leukocyte cells with migratory and phagocytic capacity (de eguileor et al., 1999; 2000a, b, 2004; tettamanti et al., 2003a, b; 2006; grimaldi et al., 2006, 2010; schorn et al., 2015a, b). these myeloid lineage-derived cells, such as macrophages, granulocytes and natural killer cells (nk), perform different types of responses in relation to the antigen size. indeed, small dimensions antigens are phagocytized, whereas larger ones induce both cytotoxic responses and encapsulation. interesting, immune challenges and surgical lesions in leeches not only give rise to immune responses based on the same process of cell lineage commitment, proliferation, differentiation and migration (schikorski et al. 2008) involved in tissue regeneration, but are also regulated by the same key molecules, such cytokines and growth factors (schorn et al., 2015a, b; tettamanti et al., 2006). given its simple anatomy and the less varied repertoire of cell types with respect to vertebrates, h. medicinalis displays a clear and easily detectable pattern of responses evoked by different stimuli and for this reason, it represents a powerful model system for studying the basic steps of both hematopoiesis and immune response (grimaldi et al., 2006; schikorski et al., 2009). in this review, i will describe the hematopoietic process of h. medicinalis immune competent cells. specifically, i will depict the hematopoietic environments representing their site of origin, the various hspcs-derived cell types and the cellular responses and the regulating factors that are engaged in these processes. the leech body organization h. medicinalis shows a parenchymatous body characterized by a relatively simple anatomy. underneath the cuticle and the epithelium, muscles are organized in three layers of fibers circularly, obliquely and longitudinally disposed. in each layers, muscles are organized in fields which are in close contact to each other, surrounded by a scant extracellular matrix. within the entire thickness of the muscular sac, blood vessels are almost absent (de eguileor et al., 2003, 2004; tettamanti et al., 2004). the musculocutaneous sac is separated from the digestive tube by a thick layer of loose connective tissue, containing the botryoidal tissue located close to the digestive system (fig. 1a). the botryoidal tissue and the origin of h. medicinalis hspc in leeches, immune competent cells have a mesodermal origin (grimaldi et al., 2006) and to date the botryoidal tissue has been defined as the hematopoietic organ from which myeloid lineagesderived leucocytes arise. this tissue is surprisingly versatile, since it not only displays myelo/erythroid and storage functions (fischer et al., 1976;sawyer, 1986; de eguileor et al., 2001b), but is also involved in hematopoietic cells production (grimaldi et al., 2006) and angiogenesis (de eguileor et al., 2001a, b). the botryoidal tissue is composed of clusters of cells of mesodermal origin, broadly ranked in two cellular types: botryoidal and endothelial-like cells, linked by desmosome-like junctions and well separated from the connective tissue by a basal lamina. botryoidal cells are large, roundish or oval and their cytoplasm contained multiple randomly distributed granules of different sizes and staining properties. by contrast, endotheliallike cells display a cytoplasm which is poor in organules and granules, are thin and linked with their neighbors by junctions (figs 1b, c). fig. 1 botryoidal tissue. general view at optical microscope of the body of an unlesioned h. medicinalis (a). under the cuticle and epithelia, large layers of muscle tissue (m) are visible. the botryoidal tissue (arrowhead) is localized between muscles and gut (g), is immerse in a loose connective tissue (ecm) and is formed by two type of cells: the large and oval botryoidal cells (b) and smaller and flattened endothelial-like cells (arrowheads). detail at tem (c) of botryoidal cells (b) showing a granule-filled cytoplasm and endothelial cells (arrowheads) characterized by smaller size, flattened shape, and by an agranular, electrondense cytoplasm. bar in a: 100 μm; bar in b: 25 μm; bar in c: 5 μm. 258 fig. 2 drawing representing the proposed model of the hematopoetic and vasculogenesis process in h. medicinalis. left to right: from solid cords of botryoidal tissue cells (a) a vascular lumen (v) is shaped through a dehiscence process (b-d). during this process, in the new vessel cavity (v), lined by endothelial cells (arrowheads) cluster of hematopoietic stem cells (hspc) become evident (b, c). after crawling between endothelial adjacent cells, the hspc leave the bloodstream and differentiate into mature leukocyte (e). the beginning of vasculogenesis, involving the early formation of the vascular system, is characterized by a marked remodeling of the botryoidal tissue. immediately after the induction of particular stimuli, such as surgical lesions, bacterial infection or the injection of specific growth factors, cells within the botryoidal tissue change their shape from a solid cord of cells to a tubular, pre-vascular structure (figs 2a-d). therefore, the diverse functions of botryoidal tissue are related to the varying physiologic demands occurring during the life cycle of leeches. the process of angiogenesis in hirudo body wall, which is virtually avascular in normal conditions, represents a mechanism for the fast recruitment in the stimulated lesioned area of a large amount of cells involved in immune defense and wound healing. indeed, concurrently to neovessel formation, groups of closely associated hspcs become evident in the centre of the immature vessel lumen (figs 2b, d). as the vessel grows, these circulating precursor cells lose cell-tocell contacts to move freely within the lumen. thus, both botryoidal and endothelial cells contribute to 259 the creation of tissue niches promoting hematopoietic cells maintenance (survival and selfrenewal) and regulating precursors cells migration, quiescence and differentiation. the leech hematopoietic precursors cells show the typical morphology of blast-like cells and, as in vertebrates, are multipotent progenitors that give rise to all types of mature blood cells. as such, they express several surface markers commonly used to identify vertebrate endothelium and myeloid hematopoietic progenitors, in particular the cluster of differentiation cd34, cd117, cd33 and cd45 markers (wells et al., 1996; crocker and varki 2001; guo et al., 2003; hildbrand et. al, 2004; kiel et al., 2008; raaijmakers et al., 2008). these markers have been related to the functional need of maturing cells, and can be upor down-regulated by different cytokine and growth factors. in particular, cd117, cd33, cd34 and cd45 are tipically localized on clustered or isolated precursors within the neo-lumen and in cells adhering to vessel lumen. as in vertebrates, these populations of cells are also positive for the vegf (vascular endothelial growth factor) receptors flk1 and flt1, confirming their responsiveness to this cytokine (damert et al., 2002; tettamanti et al., 2003a). most of these hspcs, conveyed into the lesioned/stimulated area by vessels, adhere to the luminal wall and, after crawling between endothelial adjacent cells, leave the bloodstream, disperse in the surrounding connective tissue and differentiate into mature leukocytes committed to mediate the inflammatory response (grimaldi et al., 2006; fig. 2e). as previously reported for vertebrates, several factors play a key role in this interaction, such as lselectin, talin and vinculin. in particular, leech hspcs that extravasate are positive for l-selectin, which is a typical marker for hematopoietic cells adhering to the inner vessel wall and destined to leave the blood stream. subsequently, extravasating hspcs express talin and vinculin, whose role is to link collagen fibers during migration across the extracellular matrix (grimaldi et al., 2006). structural and functional characterization of leech mature leukocyte population the specific nomenclature of mature leech immunocytes, first established almost 20 years ago by de eguileor and collaborators (1999), includes macrophages, two types of granulocytes and natural killer cells (nk). these cells, which have been characterized by ultrastructural and immunohistochemistry analyses and are involved in the leech defense system, not only display features and behaviors typical of those found in vertebrates, but also deploy the same molecules used as immune cell markers for vertebrates. indeed, several reports (de eguileor et al., 2000a, b; grimaldi et al., 2004, 2006; macagno et al., 2010; schorn et al., 2015a, b) have provided extensive evidence for the occurrence, in leech, of a wide range of cds proteins which are commonly used as immune cell markers, as for mammalian cds. in vertebrates, the structure and function of many surface markers cd have been deeply investigated and are strictly linked to a specific cell type or to a specific function. for instance, cd45 is a cell surface glycoprotein that is implicated in integrinmediated adhesion of macrophages in vertebrates (roach et al. 1997; zhu et al., 2011; st-pierre and ostergaard, 2013) and is thought to play a role in regulating the functional responsiveness of cells to chemoattractants (roach et al. 1997; mitchell et al. 1999). the cd14 and cd68 markers are expressed on cells from the myelomonocytic lineage, including monocytes and macrophages. in particular, cd14 is a glycoprotein receptor for lps that promotes phagocytic uptake, whereas cd68 is an antigen primarily expressed as an intracytoplasmic molecule in human macrophages. cd11b (springer, 1990) and cd11c are macrophage and granulocytesassociated proteins (cabanas and sanchez-madrid, 1999) while cd56 and cd57 antigens are expressed by cells with natural killer activity (robertson et al., 1996). the use of antibodies raised against mammalian cd antigens to detect immune cells in the leech is also supported by several data from the literature concerning earthworm and sipunculids. by means of cytofluorimetric assays with mouse antihuman monoclonal antibodies, cossarizza and colleagues (1996) were able to distinguish two types of celomocytes in the annelid eisenia foetida, whereas blanco and collaborators (1997) identified celomocyte surface antigens cross reacting with anti-human cd14, cd11b and cd11c in sipunculids, a phylum closely related to annelids. the observed cross-reactivity of sipunculid and earthworm leukocytes with anti-human cd markers is a further confirmation that cell recognition and immunodefense system share similar characteristics and effector molecules in leeches and vertebrates. the morphological characteristics and functions of each of these immune cells are described in detail below, including molecular markers with which they have subsequently been associated. macrophage-like cells phagocytosis is a primordial aspect of innate immunity and is highly conserved in all metazoan. in h. medicinalis, macrophages-like cells resemble the mammalian monocyte/macrophage lineage cells and represent the cell population primarily involved in non-self recognition. characterization of these cells was achieved by ultrastructural analysis, acid phosphatase reaction and immunohistochemistry using polyclonal antibodies directed against the human macrophage and leukocytes markers cd68 and cd45 (schorn et al., 2014). these cells are characterized by pseudopodia and numerous phagolysosomes in their cytoplasm (fig. 3a). both ultrastructural morphological analyses and acid phosphatase reaction positivity confirmed that activated macrophages migrating towards non selfproducing tissues or towards injured or grafted areas display a strong phagocytic activity (de eguileor et al., 2000a, b; girardello et al., 2015a, b; schorn et al., 2015a, b). numerous cd68+ and cd45+ macrophage-like cells are also recruited in animals injected with lps or with the pathogen bacterium micrococcus nishinomiyaensis (fig. 3b). as in mammals, immune challenge in leech body wall induces the migration and accumulation at the 260 fig. 3 tem (a, c, e) and immunohistochemical analyses of h. medicinalis mature leukocytes (b, d, f). macrophage-like cells (a, b) are characterize by ruffled surfaces, phagolysosomes in the cytoplasm (arrowheads) and by the expression of the specific macrophages marker cd68 (b). nk-like cells show in their cytoplasm a welldeveloped endoplasmic reticulum (arrow), numerous dense granules (arrowheads) and express the specific superficial marker cd56 (d). granulocytes of type i (arrowhead in e) and of type ii (arrow in e) are characterized by a large number of electron dense granules in their cytoplasm and expressed the cd11b marker (f). bar in a, c: 6 μm; bar in b, d, f: 10 μm; bar in e: 2 μm. 261 injury site of macrophage-like cells with phagocytic (de eguileor et al., 2000a, b; schorn et al., 2015a, b) and antimicrobial activity and the ability to produce proteins and peptides, such as hmtheromacin and destabilase (schikorski et al., 2008; hildebrandt and lemke, 2011). phagocytosis of foreign living (yeast, bacteria) and non-living material (sulfate spheres, nanomaterial such as multiwall carbon nanotubes mwcnts) represent an effective and simple mechanism to engulf and eliminate non-self foreign bodies. when antigens penetrating into the body are of small dimension they are simply phagocytized, while when foreign bodies are too cumbersome to be phagocytozed (i.e., group of spheres, aggregates of nanomaterial such as multiwall carbon nanotubes and/or parasites), they are encapsulated and afterward melanized (de eguileor et al., 2000a; girardello et al., 2015b). an interesting outcome of recent studies has highlighted that, during the encapsulation process, leech macrophages produce a large amount of amyloid fibrils, which are used as barrier to restrain non-self-material. indeed, several reports describing and characterizing the morphofunctional events sustaining the amyloid fibril assembly in relation to melanin production have underlined the relationship between melanin synthesis and the production of amyloid fibrils to template the pigment. therefore, amyloidogenesis has been proposed as a fundamental detoxifying event (grimaldi et al., 2012a) that also acquired physiological additional functions during evolution by packaging melanin and driving the pigment towards a non self-invader, both in vertebrates (fowler et al., 2006) and in invertebrates (falabella et al., 2012; grimaldi et al., 2012b). the nk-like cells leech nk-like cells are involved in host defense against bacterial and viral infections, in resistance to parasites and in the regulation of hematopoietic cell growth and differentiation. they are similar to those described in vertebrates (vivier et al., 2008), in other invertebrates annelids such as oligochetes (cossarizza et al., 1996; quaglino et al., 1996), polychetes (porchet-henneré et al., 1992) and molluscs (franceschi et al., 1991). these data strongly support the hypothesis that a primitive natural killer cell-like activity appeared early in phylogenesis and the same function has been preserved throughout phylogenetic development. these cells are characterized by a low nuclear/cytoplasmic ratio and their cytoplasm displays a well-developed endoplasmic reticulum, numerous mitochondria, and dense granules (fig. 3c). activated nk cells express cd56 and cd57 (fig 3d) on their surface and have cytolytic activity. when these cells come in close contact with the membrane of target cells, they exocyte the granules present in their cytoplasm in order to carry out their citolytic role (de eguileor et al., 1999, 2000a, b). granulocytes cells belonging to this class contain dense cytoplasmic granules and are characterized by the expression of the two surface marker cd11b+ and cd11c+ (figs 3e, f). based on size and shape of their granules, two different granulocytic types have been identified: granulocytes type i and granulocytes type ii. these cells show a different behavior in relation to the dimension and shape of their granules. granulocytes type i, characterized by small and round granules, can respond to several stimuli by degranulation when a large dose of bacteria (escherichia coli) is injected in the leech body wall. granulocytes of type ii, with large irregularly shaped granules showing complementary profiles, are activated when large antigens crosses the epidermis (de eguileor et al., 1999, 2000b). control of hematopoiesis and vasculogenesis hematopoiesis, neovascularization and leukocytes differentiation are complex multistep processes, spatially and temporally harmonized and triggered by extracellular signals conveyed by extracellular matrix components and soluble cytokines. strikingly, several similarities have been demonstrated between the hematopoietic process in h. medicinalis and vertebrates by several investigations, thus highlighting the conserved function of the underlying signaling pathways and transcription factors regulating proliferation, differentiation and lineage commitment (tettamanti et al., 2006; de grimaldi et al., 2008, 2009, 2010). previous studies in leech have indeed demonstrated the importance of a number of cytokines, including vegf (vascular endothelial growth factor), egf (endothelial growth factor), bfgf (basic fibroblast growth factor), gm-csf (granulocyte macrophage colony-stimulating factor) and tgf-β (transforming growth factor), during angiogenesis and associated hematopoiesis (tettamanti et al., 2003a, b, 2006; grimaldi et al., 2006). an interesting outcome of these investigation showed that injection in leeches of human vegf, bfgf or gm-csf promoted vascular growth, immunocytes migration and differentiation. in this review, i have added to the quoted data about vegf, the new discoveries concerning vasculogenesis, hematopoiesis, and muscle tissue regeneration. vegf and ras have different role in vasculogenesis and hematopoietic processes in vertebrates, vegf mediates its biological effects by binding to a family of transmembrane tyrosine kinase receptors, mainly vegfr-1 and vegfr-2 (neufeld et al., 1999). vegf is a potent proangiogenic molecule and a mitogen/survival factor or a chemoattractant for endothelial cells (koch et al., 1994; gerber and ferrara, 2003; wang et al., 2008) under physiological and pathological conditions. moreover, vegf165 (the most abundant and biologically active isoform) is a potent chemoattractant for cd34 positive cells and it is critical for the migration of cd34+ hematopoietic stem cells from the bone marrow into a tumor mass (lee et al., 2006). in leeches, the vegf165 isoform is synthetized and released in the extracellular matrix by muscle tissue and its activity is mediated by binding to specific vegf-like receptors (flt1/vegfr-1 and flk-1/vegfr-2 or kdr) expressed on endothelial-like cells (perletti et al., 2003; de eguileor et al., 2004). 262 fig. 4 cells expressing only gfp (in green) maintained a cord shape (a) and a low signal for polymerized f-actin (in red) is visible in the small endothelial cells (arrowhead) interposed among botryoidal cells (b). gfp/ras61 transformed cells (b) show a transformed phenotype and lacunae vessel (v) lined by flatten endothelial (arrowheads) cells highly positive for polymerized f-actin are visible. after vegf administration a dehiscence process led the compact cell cords to gradually line new cavities (v) in which hspc cells proliferate and express the cd33 (c) and cd34 (d) markers (in green). hspc exposed to a sustained and continuous source of vegf maintain their undifferentiated phenotype as precursor cells (e). hspc exposed to a low concentration of vegf differentiate in muscle fibres (m) (f). n, nucleus. bars in a, b: 25 μm; bars in c, d: 10 μm; bar in e: 600 nm; bar in f: 1 μm. the growth of new vessels is characterized by a combination of two distinct events, i.e., an initial vasculogenic step followed by extensive angiogenesis. a vegf stimulus induces the transformation of the botryoidal tissue into new immature vessels (vasculogenic step) from which new branches and capillaries can further outgrow, following a classic angiogenic pattern (i.e., an extensive development of new vessels from preexisting ones) (de eguileor et al., 2001a). an interesting outcome of recent investigation in leeches highlighted another mechanism regulating vasculogenesis, based on the gtpase ras protein and map/erk kinase cascade. in vertebrates the ras gtpase controls signal transduction pathways (carpenter 2000; kranenburg and moolenaar, 2001) leading to gene expression changes and cell motility through the 263 map/erk kinase cascade (ehrhardt et al., 2002). moreover, it is known that ras signaling, even if its mode of action is not completely understood, is capable of driving an angiogenic switch in primary endothelial cells in the absence of other angiogenic factors (meadows et al., 2004). interestingly, the ras protein plays an important role in leech vasculogenesis as well. during this process, endothelial cells express ras and are characterized by marked cellular changes. they become thin, flat, and tapering of these cells allow the definition of a lumen and the increase in diameter and length of new capillary. moreover, constitutively activated mutant ras61 expression in stably transfected botryoidal cells causes vessels cavities formation also in total absence of vegf, whereas cells expressing only gfp show the typical solid cord structure of un-stimulated tissue (figs 4a-d). such relevant modification of botryoidal tissue phenotype is due to the cytoskeletal reorganization of endothelial cell. indeed, ras/map/erk signalling is directly involved in the morphological modification of botryoidal tissue cells by acting on the polymerization and spatial arrangements of endothelial cell’s actin filaments (grimaldi et al., 2013). therefore, despite the fact that both botryoidal and endothelial cells cooperate in the formation of new blood vessels, the first step of new vessel formation in leeches, identified as a vasculogenic phase, seems to be mainly regulated by the ras/map/erk cascade. vegf also play a crucial role in promoting the formation of an extensive vessel network through the entire avascular body wall and in the recruitment of hematopoietic precursors cells. the different pattern of expression of vegf receptors on endothelial and botryoidal cells could explain the different behavior of these cells to vegf stimulus (grimaldi et. al., 2006). when vegf is readily available, such as after injection in the leech body wall, it acts as chemoattractant for endothelial cells and induces the migration and assembly of numerous vessels in the muscular body wall. however, a highly localized vegf concentration, achieved for instance by implantation of a vegfhydron pellet within the musclecutaneus sac, induces a massive production of circulating cells, localized in the cavities of neo-vessel lumen and expressing specific markers such as cd33, cd34, cd31, ve-cadherin, c-kit and the vegf receptors flt1/vegfr1 and flk-1/vegfr-2 (tettamanti et al., 2003; grimaldi et al., 2006). strikingly, these cells strongly resemble the circulating hematopoietic and endothelial progenitor cells described in vertebrates (hatzopoulos et al., 1998) and recently a direct correlation has been established between hematopoietic/endothelial precursor cells and muscle regeneration processes (grimaldi et al., 2009, 2010). indeed, the hematopoietic precursors cells of hirudo, deriving from the botryoidal tissue and extravasating into the extracellular matrix, not only express the same molecular markers of vertebrates hematopoietic stem/progenitors, but also gives rise to the vessel-associated myoendothelial cells involved in vertebrate muscle regeneration and are able to produce terminally differentiated muscular cells (figs 4e, f). therefore, hematopoietic precursors cells of leech seem to possess the capacity to activate diverse genetic programs in response to peculiar environmental stimulation. when these cells are exposed to a sustained and continuous source of vegf slowly diffusing from physiological ecm ‘‘sinks’’, they proliferate and principally maintain their undifferentiated phenotype as precursor cells. as the vegf concentration decreases in the ecm surrounding the muscle fibres, the proliferation stimulus diminishes, permitting these hematopoietic/endothelial precursors cells to enter in a differentiating program instead (grimaldi et al., 2009, 2010). byopolimer matrigel as a tool to isolate leech hspc the isolation and culture of leech hematopietic precursors offer a unique and accessible tool to study leech hematopoietic and endothelial lineage development (ballarin et al., 2008; grimaldi et al., 2008, 2009) and the role of cytokines in the recruitment, commitment and hematopoietic cells differentiation. recently, a new method has been proposed, based on injection of the biopolymer matrigel (mg) supplemented with different kind of growth factors in the leech body wall. after injection in the leech body wall, the biopolymer form a pellet and the signaling molecules diffusing out from this artificial microenvironment induce the hematopoietic precursors to migrate towards it. when the biopolymer attains a high cell density due to the recruitment of specific cell populations, it can be retrieved and used as a vector to prepare cultures of the infiltrating cells. thus, unlike standard methods to localize hspcs in adult tissues, to extract and culture them, the present approach provides a method by which hematopoietic precursor cells move from the tissues in which they reside into the injected biopolymer supplemented with a specific cytokine. the use of biocompatible and bioreabsorbable materials forming a 3d polymer scaffolds thus represents not only a remarkably effective tool for studying and charactering cells involved in leech immune responses, but also represents an easy system to select specific cell populations for in vitro culture, expansion and differentiation analyses (grimaldi et al., 2008, 2009, 2011; ballarin et al., 2008; girardello et al., 2015b). moreover, the role of several cytokines in the recruitment, commitment and differentiation of hematopoietic cells has been also confirmed by injecting mg supplemented with different kind of growth factors in the leech body wall. a comparative analysis of biopolymer in vivo-sorted stem cells indicates that vegf recruited cells of a hematopoietic/endothelial phenotype expressing cd34, cd117 and the vegf receptors flt1/vegfr1 and flk-1/vegfr-2, whereas the cytokines monocyte chemoattractant protein-1 (mcp-1/ccl2) and the allograft inflammatory factor-1 (hmaif-1) recruit cells that are of an early myeloid lineage and expressing cd11c, cd14, cd 45, and cd68 (figs 5a-d). 264 fig. 5 after 1 week in vivo the mg supplemented with vegf or mcp-1 was removed and the cells infiltrating the matrigel sponge were plated out. cells recruited by vegf (a) show the blast like phenotype (b) and express the cd34 marker (c), while cells recruited by mcp-1 (d), differentiate in macrophages (e) and express cd68 antigen (f). bars in a, c, d, f: 10 μm; bar in b: 2 μm; bar in e: 1 μm. concluding remarks the main conclusion of the studies reported above is that hspcs precursor cells in the leech, deriving from the activated botryoidal tissue, seem to share with their vertebrates counterpart most morpho-functional and molecular mechanisms. in leeches, these cells are recruited and activated in response to injury, bacterial injection, or experimental administration of cytokines and contribute to immune response, neovascularization and muscle regeneration. further, similar to vertebrates these cells are driven toward a leukocyte, endothelial or myogenic, differentiation pathway, as a function of the time course of vegf release to target cells. strikingly, as in vertebrates, the key molecules playing pivotal roles for guiding and regulating the hematopoietic cells recruitment, proliferation and differentiation are different from those involved in endothelial cells differentiation and neo vessel formation. in particular, vegf is mainly involved in promoting stem cell recruitment and proliferation, while the gtpase ras protein and map/erk kinase cascade, remodeling the endothelial cells cytoskeleton, regulates the new vessel formation. for these reasons, leeches, involving similar cellular mechanisms, can be considered a simple step-by-step model for studying the regulation of vasculo-genesis and hematopoietic cell biology. moreover, since investigations on hematopoiesis can greatly help scientists and clinicians to better understand the pathological processes behind blood disorders, cancers, muscle regeneration, hspcs can be used as a powerful model system for understanding tissue stem cells and their role in angiogenesis, tissue regeneration and oncogenesis. finally, it is well known that numerous factors, i.e., ethical considerations, stricter controls, improvements in animal welfare and housing, have led to a growing number of restrictions in the number of animal species available for experimentation. significantly, the use of leech as an experimental model for studying hematopoiesis effectively avoids many of these inconveniences. therefore, although some differences between vertebrates and invertebrates models do exist, h. medicinalis can be considered as a new emergent model in hematopoiesis research. in this context, the recently developed biopolymer in vivo cell sorting method, which allows the isolation of specific cell populations in relation to the cytokines utilized and the possibility to subsequently culture these cell types, is invaluable for studying hspcs characteristics. acknowledgements the author wish to thank belluco s for drawing the botryoidal tissue involved in hematopoietic process, acquati f for critical reading and english revision of the manuscript. references ballarin l, cammarata m, cima f, grimaldi a, lorenzon s, malagoli d, ottaviani e. immuneneuroendocrine biology of invertebrates: a collection of methods. inv. surv. j. 5: 192-215, 2008. 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2018 abstract elie metchnikoff was a russian scientist known as the pioneer of innate immunity. in particular, he was awarded the nobel prize for discovering the process of phagocytosis and its significance in the development and disease. here, we endeavor to demonstrate the enduring fascination of his scientific research, in particular the experiment involving the first observation of a macrophage reaction in the sea star. this applies to both adult and larvae immunity studies. recent work on sea star larval cellular immunity and adult immune systems using modern expansions of molecular and cellular techniques shows that it is a continually exciting research field that cannot just be consigned to history. finally, aspects of human scientific roles from the zoologist to embryological experiences to the father of innate immunity can teach us much about the oft-neglected added value of multidisciplinary knowledge and integration in animal science research. key words: metchnikoff, phagocytosis, zoology, immunobiology, sea star larvae introduction in 1882, elie metchnikoff (ilia mechnikov, 15 may 1845 15 july 1916) pioneered the study of cellular immunology and initiated the scientific process that led him to the discovery of phagocytosis. although the phenomenon of endocytosis by leukocytes (more related to pinocytosis) had already been described 30 years before, metchnikoff clarified the defensive function of phagocytosis, highlighting its importance in immunity where it is a nodal point of the immunity network. he devoted much of his life to studying the different aspects of phagocytosis and related immunological phenomena. it took 25 years of hard work for the theory of phagocytosis to be recognized: the first experimentally based theory in immunology. this struggle finally paid off in 1908, when the nobel laureate was awarded to metchnikoff, together with paul ehrlich, who had developed the methods for standardization of antibody activity in immune sera (gordon, 2008). in line with his zoological interest and scientific education, he adopted an evolutionary and comparative ___________________________________________________________________________ corresponding author: matteo cammarata dipartimento di scienze della terra e del mare università degli studi di palermo viale delle scienze ed.16, palermo, italy e-mail: matteo.cammarata@unipa.it approach to understand the animal biology of different organisms, from protozoan to exotic primates. however, he normally preferred simple organisms, convinced that they “affording as they do infinitely simpler and more primitive conditions than those in man and vertebrata, really furnish the key to the comprehension of the complex pathological phenomena which are of special interest in medical science”. despite his weak eyesight, he used light microscopy and scientific illustration to considerable effect (metschnikoff, 1884) to investigate the early development of germ layers, and focused on the biological processes of development (racine, 2014). metchnikoff displayed a passionate interest in science and natural history from an early age, and was thus encouraged by his mother to pursue a scientific career in the life sciences. while at the university in kharkov (1863-1865), he published his first scientific paper on the histology of vorticella, a genus of protozoa, and dedicated himself to studying marine life on the small island of helgoland in the north sea (1864). he was greatly influenced by charles darwin's theory of evolution and his scientific works and darwinism inspired theories. interest in invertebrate marine organisms and their development accompanied elie metchnikoff on mailto:matteo.cammarata@unipa.it 235 his travels around europe, including the baltics, germany and the mediterranean area including naples and messina. during this period, he made his first scientific discovery of the sexual and asexual alternation of generations in nematodes and subsequently identified intracellular digestion in planarians; this study influenced his later work. in naples, he had been working on a doctoral thesis with kowalensky, who described the tailed larval form in ascidians containing a dorsal neural tube. only then did zoologists begin to realize that the tunicate should be placed within the chordate. elie metchnikoff returned to st. petersburg to complete doctoral studies on the embryonic development of the cuttlefish of genus sepiola and crustaceans such as nebalia (racine, 2014). metchnikoff’s objective was a comparative study of the development of germ layers in vertebrate and invertebrate embryos. specifically, he sought to establish a common link in the evolution of vertebrates and invertebrates, and to confirm darwin's theory of the animal common ancestor. he was appointed at imperial novorossiya university of odessa professor of zoology and comparative anatomy at only 25 years of age. therefore, with a view to his early studies, we consider metchnikoff as a comparative embryologist who, to explore his first hypothesis on the mesoderm origin of endodermal structures then extended his observations on mesodermal digestive processes to a new theory of immunity. in 1873, metchnikoff’s life was greatly disrupted by the death of his first wife and by a consequent attempt of suicide. he remained absent from scientific life for the next nine years. the messina experiments in 1882, metchnikoff left odessa and started up a private research laboratory in messina, where the turning point in his career took place. looking at motile cells in a transparent sea star larva, he was struck by a novel idea: to imagine that similar cells could serve as a defense of an organism against dangerous intruders. this idea was the basis for his theory of phagocytosis. to test his hypothesis, metchnikoff introduced a rose thorn into the body of a sea star larva, whereupon motile cells rapidly encircled the foreign body. metchnikoff considered this process to be similar to the inflammatory response found in animals with vascular systems, where white blood cells gather at the site of inflammation. he hypothesized that the mobile cells in sea star larvae, or phagocytes, were the evolutionary ancestors of the mesodermal cells of vertebrate. these cells have a dual role: a primitive digestive function, ingesting particulate nutrients, and a protective function, ingesting foreign materials/invaders and maintaining the integrity of the organism. these preliminary studies did not garner sufficient appreciation, so metchnikoff spent much of his time underlaying his phagocytic cell theory with fierce polemics against the seemingly alternative humoralist theory of that time. he devoted much of his subsequent scientific work to developing his theory of phagocytosis in natural immunity. to support his work and theory, in 1901 he published the treatise “l'immunité dans les maladies infectieuses” (immunity in infectious diseases). phagocytosis has also been extensively studied from microbiological aspects. elie metchnikoff used a series of newly described bacteria in his experiments, including commensal and pathogenic agents such as syphilis and anthrax (metchnikoff, 1984a,b). finally, he turned to gut flora and aging, coining the term 'gerontology' (martin and gillen, 2013). from 1895 to 1910, metchnikoff’s cellular phagocytosis theory (cellular versus humoral effectors) became integrated with the development of the humoralist position, with the humoral basis of bactericidal defense starting to define innate versus acquired immune processes. the novelty of the sea star macrophage after the myth since metchnikoff, sea star larva mesenchyme cells have been extensively used to investigate their morphogenetic functions (crawford and chia, 1978; dan-sohkawa et al., 1980; crawford and abed, 1983; kominami, 1985; crawford, 1990; crawford et al., 1997; kaneko et al., 2005). as in echinoids, mesenchyme sea stars cells appear at the tip of archenteron during gastrulation and are widely dispersed throughout the blastocoel (chia, 1977, dan-sohkawa et al., 1980). when the embryo reaches the early stage of a bipinnaria larva, the mesenchyme cells amount to 110 cells per individual (kominami, 1985). these cells change gradually in morphology from a rounded to attenuated shape as they develop cellular processes. mesenchyme cells sustain their embryonic shape by exerting mechanical tension against the fibrous component of the extracellular matrix (crawford, 1990; crawford et al., 1997; kaneko et al., 2005). this idea derived primarily from ecm scanning and transmission electron microscopy observations of sea star pisaster ochraceus embryos (crawford, 1990, crawford et al., 1997). however, since they had only a little experimental evidence available, crawford and colleagues could only hypothesize that fibrous ecm supports the shape of the body wall, while the mesenchymal cells modify it by changing its length. afterwards, kaneko and coworkers (2005) studied the embryogenesis of the sea star asterina pectinifera by using a monoclonal antibody (4h11 mab), specifically recognizing a fibrous component of embryonic ecm. through these experiments, they evidenced that 4h11 mab caused an abnormal distribution of the fibers, and, in turn, an anomalous distribution of mesenchyme cells, in addition to morphological abnormality and delay in the ectoderm and the endoderm. furukawa and coworkers (2009) characterized the sea star (a. pectinifera) larval mesenchyme cells by studying their structural and functional properties. confocal immunofluorescence, nomarski, and transmission electron microscopy (tem) revealed the mesenchyme cells to be responsible for the construction of a dynamic network structure under the body wall; indeed, most of them were typically located under the endodermal and ectodermal wall, while others were dispersed in the blastocoel. in 236 fig. 1 the echinoderms development and mesenchymal cells in the larval and adult stage. the blastocoelar and pigment precursor cells, differentiated during the blastula stage, are respectively placed in the body cavity and in the ectoderm at the gastrula stage. the pigment precursors become pigment cells and the blastocoelar cells differentiated into four cell types (amoeboid, filopodial, ovoid and globular) in the larvae. in adults, six cell types (red and colorless spherule, small, discoidal, polygonal and vibratile) of mesenchymal cells can be found particular, the mesenchyme cells tended to distribute unevenly along the ciliary band in the ectodermal wall. however, details of their role in defense activity have been lacking for a long time. referring to metchnikoff’s idea that during echinoderm gastrulation, mesenchyme cells are capable of phagocytosis almost immediately after reaching the coelomic cavity, silva (2000) investigated the starting point of phagocytic activity of mesenchymal cells. this study was performed in embryos of the sea urchin lytechinus variegatus by microinjecting the yeast saccharomyces cerevisiae into the blastocoele. here, secondary mesenchymal cells were first detected phagocytosing injected yeast, through pseudopodia emission and internalization, during the mid-gastrula stage (silva, 2000). this is the point where mesenchymal cells leave the tip of the gut rudiment. therefore, the mid-gastrula represents the development stage during which a biological capacity for distinguishing between self and non-self begins to be established in the l. variegatus embryo. in this regard, further data were later derived from injection experiments performed on a. pectinifera (furukawa et al., 2009) phagocytic behavior. the mesenchyme cells responded to almost all foreign materials (i.e. polystyrene beads), displaying an active phagocytic function and forming aggregates to eliminate them. morphological simplicity and optical transparency of embryonic and larval stages coupled with techniques for transgenesis and gene perturbation in the echinoderms model, as well as the sequenced genome of the purple sea urchin (strongylocentrotus purpuratus) lend additional depth to the field of echinoderm immunity. in particular, sea urchin genome sequencing, carried out a century after the metchnikoff’s discovery, highlighted the genomic complexity of the echinoderms immune system. the availability of this genomic sequence enables us to reconsider metchnikoff's cellular immunology model using a series of modern molecular tools. using them, we can investigate fundamental issues of animal immunity, including those shared with vertebrates. in this context, furukawa and coworkers (2012) studied the molecular mechanism that regulates 237 mesenchyme cell dynamics during the immune response to foreign bodies in a. pectinifera. in this sea star apsrcr1 protein, the orthologue of a vertebrate scavenger receptor cysteine-rich-domaincontaining protein, serves as an opsonin against bacteria in the larval defense system. apsrcr1 protein is released extra-cellularly by mesenchyme cells and promotes their phagocytosis ability and aggregate formation. more recently, ho and coworkers (2016) analyzed the larval immune response in the purple sea urchin. they characterized five distinct larval cell types and defined their role in immune response. the larval response to pathogenic bacteria resulted in changes in gut morphology, cell behavior and alterations in gene expression levels, showing a complexity of reactions that way exceed the morphological simplicity of the larva. larval filopodial cells are also responsible for the up-regulation of sptransformer (sptrf) genes following immune stimulation (hirano, 2016). in sea urchin larvae, sptrf gene expression is restricted to a subset of blastocoelar cells localized in the blastocoel and functioning as the primary larval phagocytes able to protect the host. in the adult, also, the expression of the sptrf protein seems to be limited to the phagocytes (smith and lun, 2017). immune cells in larvae and in adult stage larvae and adults have different lifestyles, since the echinoderm larval stage does not extend beyond two months, compared with decades spent as adults. the morphologically simple larva is planktotrophic, while the more complex adult feeds primarily on kelp and other benthic algae. both similarities and differences between the larval and adult immune systems exist, despite larvae and adults sharing the same genomic background (rast et al., 2006). currently, we do not know if the few hundred immune cells of larvae give rise to the millions of circulating coelomocytes in the adult. while the adult immune cell types have been identified (smith and davidson, 1992; smith et al., 2010), the larval immune cells are not well characterized. some larval and adult cells (fig. 1) show the same morphology and express the same set of genes. adult coelomocytes are generally classified into at least six types (smith et al., 2010) and express homologs of genes involved in the immune response in many animals, including vertebrates. indeed, complement factors (gross et al., 2000), a complex srcr repertoire (pancer, 2001) and multigene families of diverse toll-like receptors (buckley and rast, 2012), as well as aif-1 (barca et al., 2017) are differentially expressed in coelomocyte subpopulations. regarding the larval stage, the mesenchymal cells are known to possess immune functions (smith et al., 2010), being able to recognize and phagocytose bacteria or yeast injected into the body cavity, or blastocoel (silva, 2000). in particular, in the sea urchin larva, two cell types have been recognized as major immune effectors (hirano, 2016). these are the filopodial phagocytes, mainly placed in the body cavity, and the pigment cells that are found in the ectoderm. this last cell type is characterized by the red pigment echinochrome a, and is considered responsible for antioxidant, antimicrobial and anti-inflammatory activities (mcclendon, 1912; calestani et al., 2003). the red spherule cells of the adult are morphologically similar to the larval pigment cells. furthermore, they share the expression of key molecules for notch signaling (ransick and davidson, 2006) and for pigment production (calestani et al., 2003). in addition to phagocytic activity, similarities between larval filopodial cells and small adult phagocytes are also evident in the up-regulation of sptrf genes following immune stimulation (hirano, 2016). in larvae, blastocoelar cells are the only cell type able to express the sptrf genes (smith and lun, 2017). conversely, in the adult, these proteins are expressed also in polygonal phagocytes and their strong up-regulation occurs when challenged with pamps and heat-killed marine bacteria (nair et al. 2005; brockton et al. 2008; majeske et al. 2013). regarding the differences, the larval globular cells have not been found in adults, and no equivalents of the adult vibratile cells have been observed in the larva (ho et al., 2016). for gene expression, a differential pattern has been evidenced among the subfamilies of immune receptors expressed in the two developmental stages, as takes place with tolllike receptors (buckley and rast, 2012). furthermore, since larval and adult cells express different amounts of effector molecules, a stagespecific gene regulation is made available. conclusions by using invertebrate organisms to make predictions of immune function in other animals, metchnikoff introduced a new field of biology: comparative immunology. the power of comparative immunology derived from observing invertebrates is evident at both cellular and molecular levels. indeed, the macrophages are considered a clue to a common evolution of immune and neuroendocrine systems (ottaviani and franceschi, 1998). on the other hand, the characterization of several immune active molecules, e.g. lectins (prokop et al., 1968), antimicrobial peptides (boman and hultmark, 1987; li et al., 2011), toll-like receptors (lemaitre et al., 1996; medzhitov et al., 1997), and rna interference (fire et al., 1998) as well as the evidence for molecular variability (smith et al., 2010), have expanded our understanding of invertebrate immunity and revealed shared features among animals across phylogeny and evolution. the larval and adult immune cell repertoire similarity unveils new scenarios on the history of animal life, in which the information and adaptation are related in a fitness action that crosses over individual morphology. although the implications of metchnikoff’s studies are well known to have characterized the birth and evolution of vertebrate immunity, the reconstruction of metchnikoff scientific history after so many years, and the recent development of invertebrate immunity constitute some beautiful instructive chapters from a single cell to mammals, including echinoderm larvae (ballarin and cammarata, 2016). sectorial or confined science areas are often used for aspecialized destination in education, 238 namely the application of study and project and grant finalization. several times over, the story of the “man metchnikoff” and its scientific history has clearly shown that multidisciplinary approaches and knowledge add a value that should not be confined within rigid sectoral limits. acknowledgments this paper is dedicated to prof. enzo ottaviani, who encouraged mc to write this thematic review following the italian meeting of developmental and comparative immunobiology. this work was supported by mc ffr (university of palermo) and a research grant from the italian ministry of education (prin 2010-2011 n. 20109xzepr_007). references ballarin l and cammarata m. lessons in immunity. from single-cell organisms to mammals academic press elsevier, london, uk,, 2016. barca a, vacca f, vizioli j, drago f, vetrugno c, verri t, et al. molecular and expression analysis of the allograft inflammatory factor 1 (aif-1) in the coelomocytes of the common 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"ilya ilyich mechnikov (elie metchnikoff) (1845-1916)". embryo project encyclopedia, 2014. issn: 1940-5030 http://embryo.asu.edu/handle/10776/8018. ransick a, davidson eh. cis-regulatory processing of notch signaling input to the sea urchin glial cells missing gene during mesoderm specification. dev biol. 297: 587-602, 2006. rast jp, smith lc, loza-coll m, hibino t, litman gw. genomic insights into the immune system of the sea urchin. science 314: 952–956, 2006. silva jr. the onset of phagocytosis and identity in the embryo of lytechinus variegatus. dev comp immunol. 24: 733-739, 2000. smith lc, davidson eh. the echinoid immune system and the phylogenetic occurrence of immune mechanisms in deuterostomes. immunol today 13: 356–362, 1992. smith lc, ghosh j, buckley km, clow la, dheilly nm, haug t. echinoderm immunity. adv exp med biol 708: 260-301, 2010. smith lc, lun cm. the sptransformer gene family (formerly sp185/333) in the purple sea urchin and the functional diversity of the anti-pathogen rsptransformer-e1 protein. front. immunol.. 8: 725, 2017. tamboline cr, burke rd. secondary mesenchyme of the sea urchin embryo: ontogeny of blastocoelar cells. j. exp. zool. 262: 51–60, 1992. tillmann s, bernhagen j, noels h. arrest functions of the mif ligand/receptor axes in atherogenesis. front immunol. 4: 115, 2013. http://embryo.asu.edu/search?text=cambridge%20university http://embryo.asu.edu/search?text=cambridge%20university http://embryo.asu.edu/handle/10776/8018 8 isj 16: 8-14, 2019 issn 1824-307x short communication low intensity light effects on survivability, biomass, tissue protein and enzyme activities of the earthworm eudrilus eugeniae (kinberg) csk mishra, s nayak, s samal* department of zoology, orissa university of agriculture and technology, college of basic science and humanities, bhubaneswar-751003, india accepted january 28, 2019 abstract the biological activities of invertebrates are influenced by environmental factors. surface feeding soil animals such as epigeic earthworms are likely to be influenced by the type and intensity of light unlike deep dwelling species which live in dark. this study reports the effects of low intensity light exposures on the survivability, biomass, vermicomposting efficiency, tissue protein, lipid peroxidation and activities of three stress enzymes, acetylcholinesterase, lactate dehydrogenase and catalase of the vermicomposting earthworm, eudrilus eugeniae. consistent exposure of the animal to low intensity white, blue, green and red led lights for 42 days in semi-decomposed organic substrate indicated decreased protein level, increased lipid peroxidation and enzyme activities in the animal with respect to those kept in dark. the study further indicated that darkness favours survivability, biomass gain and vermicomposting efficiency in this earthworm. key words: earthworm; eudrilus eugeniae; light; biomass; protein; enzymes introduction the lives of many animals including the invertebrates are influenced by light. it has been reported (nuutinen et al., 2014) that the native dew earthworm lumbricus terrestris was most active under ambient light. earlier studies (von frisch and kupelwieser, 1913) on daphnia had indicated that the animal swims towards red light source and away from a blue light source. the onychophoran euperipatoides rowelli was not attracted by light but instead significantly avoided illumination with wave lengths ranging from uv to green light (beckmann et al., 2015). it has been further reported that sensitivity to blue light is wide spread among invertebrates with monochromatic vision which has been attributed to the maximum distribution of energy of solar radiation at 480 nm (menzel, 1979; bowmaker and hunt, 1999; wang et al., 2003; kelber and roth, 2006).the nematode caenorhabtidis elegans when exposed to high energy blue light indicated stress with a spurt in the production of reactive oxygen species (ros) (abdel-rahman et al., 2017). among the invertebrates, ___________________________________________________________________________ corresponding author: suryasikha samal department of zoology orissa university of agriculture and technology college of basic science and humanities bhubaneswar-751003, india e-mail: suryasikha.777@gmail.com behavioural and physiological responses of annelids to light intensity and colours have not been adequately investigated. earthworms are extremely sensitive to environmental changes and have been widely used as model bioindicators. they have also been proved useful to evaluate soil contamination due to elevated concentrations of xenobiotics (samal et al., 2017, nayak et al., 2018). the effects of environmental factors such as soil moisture, ph, pesticides, animal manure and temperature on the bioactivities of earthworms had been studied earlier by several workers (grant, 1955; roots, 1956; barker, 1959; lee, 1959; wheatley and hardman, 1968; madge, 1969; reynolds, 1973; edwards and lofty, 1977; viljoen and reinecke, 1992). mishra et al. (2018) have reported that eudrilus eugeniae, exposed to variable temperatures for different time durations exhibited significant alteration in tissue protein and activities of some stress enzymes. earthworms show unconditioned response to light which is a complex function of a number of variables including light intensity and number of prior presentations. eisenia fetida when exposed to uv radiation shows depressed growth rate with reduction in fecundity, also decreased cocoon fertility by as much as 70% (hamman et al., 2003). owa et al. (2008) evaluated the suitability of various light colours on an african species of earthworm hyperiodrilus africanus. results indicated that red 9 fig. 1 percent change in biochemical parameters of e. eugeniae with different light exposures. a) protein content b) lpx. the bars in graph indicate mean ± sd values. t1 (no light/dark), t2 (white light), t3 (blue light), t4 (green light), t5 (red light) light was the most suitable which enhanced the bioactivity of the animal relative to white, blue and green lights. e. eugeniae, a detritus feeder and common vermicomposting earthworm is native to africa but now widely distributed in certain regions of the tropics and subtropics including the agricultural fields in india and sri lanka (sivasankari et al., 2013). with the above back drop, this experiment was designed to study the effects of low intensity lights of various colours on the survivability, biomass, tissue protein, lipid peroxidation and activities of the enzymes, acetyl cholinesterase, (ache), lactate dehydrogenase (ldh), catalase (cat) of e. eugeniae. materials and methods experimental set up the earthworm eudrilus eugeniae was procured from the vermiculture unit of the government quality control laboratotry, odisha, india and then acclimatized in semi-decomposed cattle dung in a rectangular earthen pot (40 cm × 40 cm) in dark for 7 days. then earthen pots of size (30 cm × 30 cm) were labelled as t1 (no light/ dark), t2 (white light), t3 (blue light), t4( green light), t5 (red light) respectively as treatment pots. the treatment pots for each light source was taken in triplicate. each pot was filled with 1 kg of semi decomposed cattle manure. twenty clitellated pre weighed earthworms of identical size were transferred to each treatment pot. thermocol sheets (thick sheets made of pulp) were used to cover the pots. 0.5 watt led lights of white, blue, green and red colours were fixed at the center of the covered thermo cool sheet on the inner side for uniform illumination of the treatment pots except t1, taken as control with no light source. on the day 1, one earthworm was randomly sampled from each treatment pot for biochemical analysis. this procedure was repeated every seventh day up to 42 days. biochemical analysis for biochemical studies the earthworms were washed properly with distilled water after removal of the gut content and then cut into small pieces. the whole body tissue was homogenized with phosphate buffer (in 1:4 ratio, ph 7.4, 0.05 m) using a tissue homogenizer (remi) and then centrifuged at 10,000 rpm for 10mins using a table top refrigerated centrifuge (remi). the supernatant was a) b) 10 collected and stored at -20 °c in a deep freezer (celfrost) for further use. lowry’s method (lowry et al., 1951) was followed for estimation of protein at 700 nm by taking bovine serum albumin as standard. the level of lipid peroxidation (lpx) in the tissue sample was measured as malondialdehyde (mda) at 532 nm in an acidic medium according to the method of ohkawa et al. (1979). ldh was determined in a medium containing phosphate buffer (ph 7.4, 0.05 m), 7.5 mm nadh and 30 mm sodium pyruvate at 340 nm as per cabaud and wroblewski (1958). the activity of ache was determined through ellman et al. (1961) assay by taking 2 mm dtnb and 1 mm acetylcholine iodide substrate. cat assay was done as per cohen et al. (1980) at 340 nm using uv-vis spectrophotometer (systronics). protein was expressed in mg/g tissue, lpx in nmol/mg protein whereas all the enzymes were expressed as u/mg protein. enzyme kinetics was done for each enzyme by calculating km value from line-weaver plot. the reciprocal of activity of ldh was plotted against the reciprocal of 5 different concentrations of substrate (nadh) such as 0.04, 0.09, 0.13, 0.19 and 0.25 mm. kinetics activities were measured by taking reciprocal of 5 different concentrations of substrate (0.02, 0.04, 0.06, 0.08 and 0.1 mm) for ache and 4 substrate concentrations (0.06, 0.18, 0.22, 0.27 mm) for catalase. earthworm biomass, survivability and vermicompost yield on the day 42, the surviving earthworms from each treatment pot were collected, counted and weighed for recording the final biomass and survivability. percent survivability and change in biomass were assessed with respect to the number and biomass of the earthworms released at the beginning of the experiment. percent vermicompost production was calculated with respect to the quantity of organic substrate. statistical analysis of the data for anova was done with the help of spss 6.0 software. results and discussion the amount of tissue protein indicated a nonsignificant variation between treatments. fig. 1a presents the percent change of protein over the experimental period of 42 days. maximum variation was observed in t2 with 44% decrease in protein level followed by t5 with 11%, on day 42. the protein level increased by 33% with respect to t1. however t3 and t4 did not show much variation in protein levels. highest protein level (268.25 mg/g tissue) was observed in t4 on the 28th day and the minimum (92.49 mg/g tissue) in t5 on the 21st day. although variation was observed in the lpx levels of the earthworms between treatments it was not statistically significant.t2 showed a maximum percent increase of 37.42% in the lpx level. a decrease of 9% in lpx level was observed in t1. t5 showed 18% increase in the level of lpx (fig. 1b). minimum lpx of 0.0184 n mol/mg protein was recorded in t1 on the 28th day. lpx was found to be maximum (0.263 n mol/mg protein) in t2 on the 28th day of observation. fig. 2 line-weaver plot indicating enzyme kinetics of a) ldh activity b) ache activity and c) catalase activity in e. eugeniae a) b) c) 11 fig. 3 percent change in biochemical parameters of e. eugeniae with different light exposures. a) ldh b) ache and c) catalase . the bars in graph indicate mean ± sd values. the star mark “*” indicates significant variation, p < 0.05. t1 (no light/dark), t2 (white light), t3 (blue light), t4 (green light), t5 (red light) the results of enzyme kinetics for ldh, ache and catalase were demonstrated in fig 2. km value of ldh, ache and catalase were calculated from the lineweaver-burk plot. the values for ldh, ache and catalase in e. eugeniae were found to be 0.34 mm, 0.17mm and 3.92µm respectively. in t1 the percent change in ldh activity was found to be maximum with 7% increase. however, t2 showed 6% decrease in this enzyme activity (fig.3a). t3 did not show much variation in the activity over the experimental period. t1 showed the maximum ldh value of 0.247 u/mg protein on the day 1. substantially lower activity of 0.011 u/mg protein was observed in t2 on the 35th day. statistical analysis however did not indicate significant variation in ldh activity between treatments. ache activity (fig. 3b) showed a wide variation among different treatments. while 23% and 13% decreased activity were observed in t2 and t5, t1 showed 15% increase in its activity over a period of 42 days. the maximum ache activity (0.147 u/mg protein) was recorded in t4 on the 28th day and minimum activity (0.007 u/mg protein) in t5 on day 42. significant variation (p<0.05) in the ache activities was observed in the earthworm between treatments. the percent change in cat activity over the period of 42 days is presented in fig 3c. the percent change in the enzyme activity was the highest in t2 with 31% increase over the experimental period. t5 also indicated an increase of 5% during the same period. t1, t3 and t4 showed a decrease in activity by 5%, 6% and 4% respectively. the minimum cat activity (0.040 u/mg protein) was observed in t1 on day 1 and the maximum (0.233 u/mg protein) in t2 on the day 28. the variation in cat activity between treatments however was not statistically significant. the survivability rate was found to be maximum of 85% in t1 followed by t3 and t4 with 70%and 60% respectively. t2 showed a minimum percent survivability of 20%. results showed that darkness was the most suitable environment for survival of the earthworm (fig. 4a). a maximum of 57% increase in biomass was observed in t1 and maximum decrease in t2 over the experimental period. t3 and t4 did not show noticeable variations (fig. 4b). this indicates that the earthworms were most stressed under white light with increased loss in biomass and underwent the minimal stress at dark with increase in biomass. the maximum earthworm cast was observed in t1 (87.5%) and the minimum (12.5%) in t2. this signifies that earthworms in dark were the most efficient in converting the organic substrate into vermicompost whereas earthworms exposed to white and red lights were the least efficient (fig. 4c). earthworms in general are highly sensitive to environmental changes and indicate wide fluctuations b) c) a) 12 fig. 4 percent a) suvivability, b) biomass, c) vermicompost yield of e. eugeniae with different light treatments. the bars in graph indicate mean ± sd values. t1 (no light/dark), t2 (white light), t3 (blue light), t4 (green light), t5 (red light) in their number, biomass and metabolism in response to alterations in soil chemical quality. e. eugeniae, because of its epigeic nature is likely to be influenced by variations in ecological factors such as light and temperature (mishra et al., 2018). in the present study, a variation was observed in vital biochemical parameters such as protein, lpx, activities of ldh, ache and catalase in the earthworm exposed to low intensity led lights with darkness as control. owa et al. (2008) conducted an experiment to evaluate the suitability of various light colours on an african species of earthworm hyperiodrilus africanus and found the bioactivity and vermicompost production were maximal in earthworms exposed to red light relative to dark, blue, green and white lights. however, our results indicated that, differently from h. africanus, the most favoured condition for all the parameters evaluated in e. eugeniae is complete darkness. saint-denis et al. (1998) have worked on four antioxidant enzymes i.e., catalase (cat), glutathione peroxidase (gpx) , glutathione reductase (gr) and glutathione-s-transferase (gst) in earthworm e. fetida exposed to different concentration hydrogen peroxide (h2o2). they have reported a significant relation between stress inducing hydrogen peroxide and the enzyme activities. hamman et al. (2003) reported that e. fetida when exposed to uv radiation, showed depressed growth rate with reduction in fecundity, also decreased cocoon fertility by as much as 70%. our results corroborate these earlier findings which support the hypothesis that earthworms in general are photophobic and perform their maximal bioactivity in dark. light even at low intensity increase the physiological stress on this earthworm resulting in lesser survivability, biomass, vermicomposting efficiency. light stress could also cause depletion in protein, metabolic enzyme activities with enhanced lipid peroxidation. this finding is supported by the observations of ravikiran and aruna (2010) who have reported that e. eugeniae shows age-related variations in these parameters with higher lpx in aged earthworms relative to young ones. jayanti et al. (2016) have made a comparative study of various biochemical responses in three earthworm species exposed to pesticide and metal contamination in soil. they have observed that when the earthworms e. eugeniae, perionyx ceylanensis and perionyx excavates were exposed to rising concentrations of the pesticide carbaryl and the heavy metal lead for different periods of time, there was an initial increase in protein content in response to low pesticide and a) c) b) 13 metal concentrations. when the concentrations increased there was subsequent decrease in tissue protein level in all the three earthworms tested. also the present study indicated a depleted protein level in earthworms exposed to lights relative to dark. abdel-rahman et al. (2017) conducted an experiment on the nematode c. elegans to study the impact of exposure to light emitting diode (led) domestic lighting and found that high-energy blue light (blue and black emitting lights) induces stress in the animal, affecting egg hatching, development, population, progeny, locomotion and survival. the stress response was also associated with the production of reactive oxygen species (ros). our results are in line with these findings. mishra et al. (2018) have reported that e. eugeniae exposed to variable temperatures from 4 °c to 40 °c for different time durations exhibit significant alteration in tissue protein and activities of stress enzymes. light exposure like non optimal temperature, could cause stress on this earthworm thus influencing its survival and bioactivity. it is further established that different light colours could affect the earthworm differentially. on the whole, our data indicate that e. eugeniae is most active under dark condition with maximum tissue protein and minimal stress enzyme activities. darkness too favours maximal survivability, biomass gain and vermicompost production. white and red lights exert the maximal stress resulting in an increase in lpx level and cat activity. blue and green lights are relatively less stressful in comparison to red and white lights. behavioural and physiological responses of other epigeic species of earthworms to light colors and intensity need further investigation. references abdel-rahman f, okeremgbo b, alhamadah f, jamadar s, anthony k, saleh ma. caenorhabditis elegans as a model to study the impact of exposure to light emitting diode (led) domestic lighting. j. environ. sci. and health a. 52: 433-439, 2017. barker rj. notes on some ecological effects of ddt sprayed on elms. j. wildl. manag. 22: 269-274, 1958. beckmann h, hering l, henze mj, kelber a, stevenson pa, mayer g. spectral sensitivity in onychophora (velvet worms) revealed by electroretinograms, phototactic behaviour and opsin gene expression. j. exp. biol. 218: 915922, 2015. bowmaker jk, hunt dm. molecular biology of photoreceptor spectral sensitivity. in adaptive mechanisms in the ecology of vision (ed. archer sn, djamgoz mba, loew e r, partridge jc , vallerga s), netherlands: springer, pp. 439-462,1999. cabaud pc, wroblewski f. colon-metric measurement of lactic dehydrogenase activity of body fluids. am. j. clin. path. 30: 234, 1958. cohen g, dembeic d, marcus j. measurement of catalase activity in tissue extracts. anal. biochem. 34: 30-38, 1980. edward c, lofty jr. biology of earthworms, chapman and hall, london, 1977. ellman gl, courtney dk, andreas v, feather stone rm. a new and rapid colorimetric determination of acetylcholinesterase activity. biochem. pharmacol. 7:88-95, 1961. grant, wc. studies on moisture relationships in earthworms. ecology. 36: 400-407, 1955. hamman a, momo fr, duhour a, falco l, sagario mc, cuadrado me. effect of uv radiation on eisenia fetida populations: the 7th international symposium on earthworm ecology·cardiff·wales·2002. pedobiol. 47: 842845, 2003. jeyanthi v, paul jaj, selvi bk, karmegam n. comparative study of biochemical responses in three species of earthworms exposed to pesticide and metal contaminated soil. environ. proces. 3: 167-178, 2016. kelber a, roth ls. nocturnal colour vision–not as rare as we might think. journal of exp. biol. 209: 781-788, 2006. lee ke. earthworm fauna of new zealand. new zealand. dept. of scientific and industrial research. bull. biblio. 454-460, 1959. lowry oh, rosbrough nj, farr al, randall rj. hartree-lowry and modified lowry protein assays. journal. biol. chem. 193: 265, 1951. madge ds. field and laboratory studies on activities of 2 species of tropical earthworms. pedobiol. 9: 188, 1969. menzel r. spectral sensitivity and color vision in invertebrates. in comparative physiology and evolution of vision in invertebrates, vol. 7/6/6 a (ed. h. autrum), heidelberg; berlin: springer 503-580, 1979. mishra csk, parida ss, mohanta kp, samal s. thermal stress induced alterations in tissue protein, lipid peroxidation and activities of lactate dehydrogenase, acetylcholinesterase and catalase in the earthworm eudrilus eugeniae (kinberg). curr. j. appl. sci. techn. 27: 1-8, 2018. nayak s, mishra csk, guru bc, samal s. histological anomalies and alterations in enzyme activities of the earthworm glyphidrillus tuberosus exposed to high concentrations of phosphogypsum. environ. monit. assess. 190: 529, 2018. nuutinen v, butt kr, jauhiainen l, shipitalo mj, sirén t. dew-worms in white nights: highlatitude light constrains earthworm (lumbricus terrestris) behaviour at the soil surface. soil biol. biochem. 72: 66-74, 2014. ohkawa h, ohishi n, yagi k. assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. anal. biochem. 95: 351-358, 1979. owa so, peters s, dedeke ga, aladesida a. effects of light colour and oscillator frequency on earthworm bioactivity. afr. j. agric. res.3: 29-36, 2008. ravi kiran t, aruna hk. antioxidants enzyme activities and markers of oxidative stress in the life cycle of earthworm eudrlus eugeniae. ital. j. zool. 77: 144-148, 2010. 14 reynolds jw. earthworm (annelida: oligochaeta) ecology and systematics. in 1st soil microcommunities conference of national technical information services, springfield, usa (pp. 95-120), 1973. roots bi. the water relations of earthworms ii, resistance to desiccation and immersion, and behaviour when submerged and when allowed a choice of environment. j. exp. biol. 33: 2944, 1956. saint-denis m, labrot f, narbonne jf, ribera d. glutathione, glutathione-related enzymes, and catalase activities in the earthworm eisenia fetida andrei. arch. environ. contam. toxicol. 35: 602-614, 1998. samal s, sahoo s, mishra csk. morphohistological and enzymatic alterations in earthworms drawida willsi and lampito mauritii exposed to urea, phosphogypsum and paper mill sludge. chem. ecol. 33: 762-776, 2017. sivasankari b, indumathi s, anandharaj m. a study on life cycle of earthworm eudrilus eugeniae. int. j. res. pharm. life sci. 1: 64-67, 2013. viljoen sa, reinecke aj. the temperature requirements of the epigeic earthworm species eudrilus eugeniae (oligochaeta)—a laboratories study. soil biol. biochem. 24: 1345-1350, 1992. von frisch k, kupelwieser h. über den einfluß der lichtfarbe auf die phototaktischen reaktionen niederer krebse. verlag von georg thieme, 1913. wang f, dong s, huang g, wu l., tian x, ma s. the effect of light color on the growth of chinese shrimp fenneropenaeus chinensis. aquacul. 228: 351-360, 2003. wheatley ga, hardman ja. organochlorine insecticide residues in earthworms from arable soils. j. sci. fd. agric.19: 219-222, 1968. 66 isj 16: 66-71, 2019 issn 1824-307x short communication immune priming of galleria mellonella larvae with bacillus thuringiensis affects coagulation and phenoloxidase activity upon subsequent infection m sułek # , l vertyporokh # , p waleczko # , i wojda* # these authors equally contributed to this work department of immunobiology, institute of biology and biochemistry, maria curie-skłodowska university, lublin, poland accepted march 29, 2019 abstract immune priming is a phenomenon that allows invertebrates, which are devoid of acquired immunity, relying on memory t-cells and antibodies, to achieve better protection after subsequent infection. in this short report, we present new investigations of the immune response of primed galleria mellonella larvae after infection with bacillus thuringiensis. we compared two related aspects of immune response – hemolymph coagulation and the activity of phenoloxidase in the primed and non-primed larvae after the injection of the lethal dose of b. thuringiensis. the in vivo assay showed that coagulation of hemolymph in response to the bacterial injection occurred more efficiently in the primed animals in comparison to the non-primed ones. further, we showed that the activity of phenoloxidase was also higher in the primed, infected larvae. both parameters of insect immune response may contribute to the increased resistance of primed g. mellonella to further infection with b. thuringiensis. key words: greater wax moth; hemolymph coagulation; insect immune memory; insect immunity; melanisation introduction insects possess only innate immune mechanisms to fight infections (buchmann, 2014). the cellular branch of their defence system involves hemocytes and comprises phagocytosis or entrapping intruders in structures called nodules and capsules (falabella et al., 2012). the humoral branch is based on soluble factors and involves inter alia: synthesis of antimicrobial peptides and activity of enzymes such as phenoloxidase and transglutaminase. antimicrobial peptides directly destroy invading microorganisms mainly by perforation of their membranes (hillyer, 2016). phenoloxidase and transglutaminase are involved in the synthesis of melanin and clot formation, respectively. in principle both melanization and coagulation require coordinated activity of the humoral and cellular defence components. both ___________________________________________________________________________ corresponding author: iwona w ojda department of immunobiology institute of biology and biochemistry faculty of biology and biotechnology maria curie-sklodowska university akademicka 19, 20-033 lublin, poland e-mail: wojda@poczta.umcs.lublin.pl processes are triggered in response to wounding and infection and serve as the basis for subsequent interaction of host's molecules with intruding microbial cells (wojda and vertyporokh, 2017). during coagulation, soluble hemolymph components are converted into an insoluble clot. in this process, insect hemocytes degranulate, releasing microparticles of reversed membrane polarity, exposing negatively charged lipids from the inner leaflet of the membrane bilayer. this attracts other hemocytes and all components necessary for coagulation. in many insect species lipophorins including apolipophorin iii serve as a protocoagulant (theopold et al., 2002). a protocoagulant is a substrate for transglutaminase, i.e. an enzyme catalysing the formation of isopeptidic bonds converting coagulogen into insoluble coagulin. the exact mechanism of insect coagulation is unravelled. li et al. (2002) identified proteins involved in hemolymph coagulation in g. mellonella. besides the known members of coagulation system, like lipophorins, they found components of a prophenol-activating cascade, supporting the idea that both coagulation and melanization systems work together during clot formation. haine et al. (2007) showed that hemolymph clotting in insects is 67 enhanced when associated with non-self and is an important feature of immune response. it localises immune effectors near injury and creates compartmentalisation of the open hemocel, preventing intruder invasion. the phenoloxidase system is activated after injury or infection and is involved in synthesis and deposition of a brownblack pigment called melanin, occurring at the site of injury or infection (krautz et al., 2014). this process is catalysed by phenoloxidase (po), a copper-containing enzyme oxidising phenols to quinones, which further undergo polymerisation and form insoluble melanin. in physiological conditions, this enzyme exists as an inactive precursor called prophenoloxidase (propo), which is released by hemocytes (oenocytoids in g. mellonella) upon immune challenge and activated by limited proteolysis by a cascade of calciumdependent serine proteases (kanost et al., 2004). melanin and intermediate products such as quinones and free radicals are highly toxic to invading microorganisms. the process is tightly controlled by serine protease inhibitors – serpins, which are released simultaneously with other components of the propo system (cerenius et al., 2008). the need of control arises due to the toxicity of free radicals and other intermediate products to host tissues as well. prophenoloxidase from g. mellonella was purified and its mass was established at 66.2 kda (kopacek et al., 1995; demir et al., 2012). the model insect used in this study is the greater wax moth galleria mellonella. in nature it lives in beehives, or, more often, in slices of stored wax, feeding with wax and pollen (wojda, 2017). it can be infected via the oral route with its natural pathogen bacillus thuringiensis. the toxins of this bacteria (cry and cyt) cause gut perforation, thereby allowing bacteria to reach the hemocel and colonise the insect body. injured cuticle is another gate of infection. both infection routes lead to the presence of bacteria in the hemocel and to the development of septicaemia, which is a cause of g. mellonella death (wojda, 2017). fig. 1 the scheme presenting the design of experiments concerning coagulation (a) and po assay (b). in both types of experiments, one group of larvae was injected with pbs (non-primed larvae, np), while another one with the low dose of b. thuringiensis (60 cfu/larvae, primed larvae, p). after 72 hours, both groups were injected with the lethal dose of b. thuringiensis (6×10 3 cfu/larvae; infected larvae). in case of coagulation assay, the amaranth red was added to the injection mixture to the final concentration of 2%. consequently the naive larvae were injected with 2% amaranth red only (control, c). larvae were left at 28 ˚c for 1 and 3 hours. then, the hemolymph was collected for checking dilution factor and for po assay. the hemolymph from the naive larvae (n) was also collected for po assay 68 there is an increasing number of reports regarding insect immune priming (for the review see cooper and eleftherianos, 2017). this term concerns increased resistance upon subsequent infection with the same or another pathogen. the regulatory mechanism of this phenomenon is unknown but attracts considerable attention because it reveals that, despite possessing only the innate immune mechanism, the insect can somehow "remember" previous infection and respond more effectively upon re-encountering of a given pathogen (chambers and schneider, 2012). we have published before that g. mellonella larvae primed with a low dose of b. thuringiensis were more resistant for further infection with a high dose of the same bacteria in comparison to the nonprimed ones (taszłow et al., 2017). we have already shown that increased resistance of g. mellonella larvae pre-exposed to a low dose of b. thuringiensis correlated with enhanced hemolymph activity without stronger immune-related gene expression, suggesting differences in the protein turnover in the infected larvae (taszłow et al., 2017). in this short report we continue this research, presenting that priming affects also coagulation process and phenoloxidase activity in the hemolymph of g. mellonella, which may also contribute to increased resistance of the primed larvae to further infection. materials and methods insects and infection the larvae of galleria mellonella (lepidoptera: pyralidae) come from breeding conducted in the department of immunobiology, maria curiesklodowska university, lublin. the larvae were reared on honeybee nest debris in darkness at 28 °c. to infect them, the last instar larvae of 200 mg weight were injected in the last proleg with the indicated number of vegetative bacillus thuringiensis cells (b. thuringiensis kurstaki hd1, bacillus genetic stock centre, the ohio state university, department of biochemistry) in pbs buffer (137 mm nacl, 2.7 mm kcl, 10 mm na2hpo4, 2 mm kh2po4, ph 7.4). bacteria to be injected were grown in lb medium (1% bactotryptone, 1% nacl, 0.5% yeast extract) at 37 °c until od600 reached about 1.0; next, they were centrifuged at 8,500×g and suspended in pbs buffer to appropriate density. the number of bacteria was estimated based on od600 and cfu (colony forming units) counting. 6×10 1 and 6×10 3 bacterial cfu in pbs buffer, in the volume of 5 µl were injected into each larvae for priming and for infection, respectively. bleeding the larvae and preparation of cell free hemolymph to obtain hemolymph, the larvae were anaesthetised in ice-cold water, ethanol sterilised and injured with a sterile needle. hemolymph was collected to eppendorf tubes kept at 4 °c and containing a few crystals of phenylthiourea to prevent melanization. it was then centrifuged at 200×g for 5 min to pellet hemocytes, and then at 20,000×g for 10 min at 4 °c. the cell-free hemolymph was kept at -20 °c if necessary. experiment design and hemolymph in vivo coagulation assay for our needs, we developed an in vivo coagulation assay based on the assay published by haine et al. (2007) with modifications. the coagulation index was measured as the dilution factor of an amaranth red dye injected into the larval hemocel. in this method, the dye is incorporated into the coagulating hemolymph. the more dye is incorporated, the less it will diffuse through the open hemocel and the hemolymph obtained will have less intensive colour. the amount of the dye in the hemolymph obtained from individuals is compared to the in vitro dilution factor of amaranth red. to prepare a calibration curve, a serial dilution of 2% amaranth red was prepared and 5 l of each dilution was added to 195 l of the ringer solution (172 mm kcl, 68 mm nacl, 5 mm nahco3, ph 6.1) in a 96well microplate. the absorbance was measured at 520 nm. then, the calibration curve was made based on the obtained od versus the dilution factor of amaranth red. to check in vivo coagulation in the infected, non-primed and primed larvae, one group of the larvae was primed with 60 cfu of b. thuringiensis / larvae in pbs buffer while another one was injected with pbs only. the priming dose (6 × 10 1 cfu) was chosen on the basis on the preliminary experiments analysing the effect of different bacterial doses on the viability of g. mellonella larvae. because all injected doses appeared to produce some mortality, we have chosen very low number of cfu for the injection. approximately 70% of larvae injected with this dose survived, as we reported before (taszłow et al., 2017). both groups (primed and non-primed) were left at 28 ˚c for 72 hours, and then infected with 6×10 3 b. thuringiensis in 2% amaranth red. as a control, 5 l of 2% amaranth red alone was injected into naive larvae. the detailed scheme of the experiment is presented in the fig. 1a. one and 3 hours after the second injection, the hemolymph from each larva was collected individually after injury near the head. the hemocytes were sedimented as described above. five microliters of cell-free hemolymph from each larva were added to 195 l ringer solution to measure the absorbance. the measurement was performed in triplicate for each larva and the average value was taken. the obtained values were re-calculated into the coagulation index based on the prepared calibration curve. experiment design and phenoloxidase activity assay for phenoloxidase assay the priming was performed as described above for coagulation assay. after 72 hours both groups (primed and nonprimed) were challenged with 6×10 3 cfu of b. thuringiensis in pbs and the hemolymph was collected 1 and 3 hours later for phenoloxidase assay. the detailed scheme of workflow is presented in the fig. 1b. the hemolymph from 10 larvae of each group was collected in every independent experiment and the cell-free hemolymph was obtained as described above. the phenoloxidase activity in such prepared hemolymph was assayed. 69 fig. 2 coagulation index in the primed (p) and non-primed (np) larvae at the indicated time-points after injection with the lethal dose of b. thuringiensis; (c) control larvae (amaranth red injected to naive larvae). the experiment was performed as shown in fig.1a. two independent experiments were performed in which the coagulation was assayed individually for each larva on the total number of 14-16 larvae in each group (+/sd, standard deviation). values with different letters are significantly different (p < 0.05; kruskal-wallis one way anova on ranks, tukey’s post-hoc test) po catalyses melanin synthesis from the coreless substrate l-dopa (l-3,4dihydroxyphenylalanine). synthesised melanin causes an increase in the absorbance values. cellfree hemolymph containing 80 g of protein in the volume of 2 l was added to 16 l of buffer a (50 mm tris-hcl, 150 mm nacl, 5 mm cacl2) in a 96well plate and left for 20 min at room temperature. afterwards, 180 l of 2 mm l-dopa in buffer b (50 mm na2hpo4, 50 mm nah2po4) were added and the absorbance at 490 nm was measured immediately (time 0) in relation to a sample containing water instead of hemolymph and after specified time-points after substrate addition: 5, 10, 15, 20, 30, 45, 60, 75, 90, and 120 min. the protein concentration was estimated using the bradford method (bradford, 1976). statistical analysis for statistical analysis, sigma stat 4.0 and statistica 13 software were used. kruskal-wallis one way anova on ranks, tukey’s post-hoc test, and student t-test for depended samples were used. normality of data was checked with the shapiro-wilk test. the differences were regarded as significant at p < 0.05. results and discussion we have found differences in both coagulation and phenoloxidase activity in the primed and nonprimed g. mellonella larvae after the infection with b. thuringiensis, which may contribute to the differences in their susceptibility to these bacteria reported before (taszłow et al., 2017). the coagulation index in the primed larvae was five-fold higher than in the non-primed ones 1 hour after infection but not at 3 hours time point (fig. 2). this means that the coagulation process in the primed larvae is faster and more efficient than in the nonprimed ones. as mentioned in the introduction, hemolymph can coagulate on the surface of infecting microorganisms or groups of microorganisms. coagulation serves as one of the first immediate reactions to infection (theopold et al., 2002; haine et al., 2007). by the time the transduction pathways are activated and synthesis of antimicrobial peptides occurs, quick coagulation may prevent or rather limit proliferation of an intruder (hillyer, 2016). it is worth to mention here, that insect circulatory system is open and therefore insects are not in danger of thrombosis (trenczek et al., 1988). that is why the coagulation of hemolymph 70 fig. 3 po activity measured in the primed (p), and non-primed (np) g. mellonella larvae at the indicated timepoints after infection with the lethal dose of b. thuringiensis (+/sd, standard deviation); n – naive larvae. three independent experiments were performed. in each experiment, hemolymph from ten larvae was pooled. student t-test for depended data showed significant differences between values in the primed and non-primed larvae both 1 and 3 hours after infection (p<0.05; exact p values are given in the figure) in insects is more important for innate immune defence than blood coagulation in mammals, the latter one mainly preventing blood efflux. furthermore, micro cloths around the intruder, apart from separating it from the rest of the body, may serve as a danger signal participating in switching on the immune response (ming et al., 2014). the po activity was higher in the primed larvae in comparison to the non-primed both 1 and 3 hours after the infection (fig. 3). probably, like it was in case of coagulation, the activation of po is quicker in the primed larvae in comparison to the nonprimed. it is worth to mention here, that at the later time points after infection, i.e. 7, 9 and 11 hours, there were no differences in the activity of po between the primed and non-primed groups of larvae (taszłow and wojda, data not shown). we noticed that 3 hours after infection the activity of po was lower than at one hour, respectively in the nonprimed and primed larvae (p=0.00842 for the nonprimed and p=0.002 for the primed; not shown in the figure). it is known that b. thuringiensis secretes some products as virulence factors that may inhibit activation of this enzyme. higher phenoloxidase activity in primed animals is likely to have an influence on their condition by affecting both cellular and/or humoral aspects of insect immunity. this enzyme in non-challenged larvae is present in hemolymph at a very low level but after injury or in response to infection, it is released from oenocytoids with simultaneous activation of zymogen into an active enzyme (bidla et al., 2009). synthesized melanin deposited on the surface of intruder may allow its faster phagocytosis. also, nodulation could be affected. as mentioned in the introduction, melanin can be deposited around the pathogen(s) during nodule formation and intermediate products releasing during melanin formation are highly toxic to pathogens closed in such structures (falabella et al., 2012). additionally, the fact that the defence molecules in the primed animals stay active longer in the hemolymph (taszłow et al., 2017), enhanced po activity may somehow influence the turnover of defence molecules, i.e. a process that is totally unknown in insects. considering the results presented here, the question may arise whether higher bacterial dose used for priming would enhance the coagulation and melanisation processes upon recurring infection. however, it is difficult to check, due to the very high pathogenicity of b. thuringiensis. on the other hand, the high pathogenicity may explain the fact that priming effect can be achieved by relatively low number of bacteria. it seems that even 60 cfu inside the hemocel may pose a significant immune challenge necessary to "leave an imprint" on the insect immune system to give a better protection against recurring infection. summarising, we have found here that two aspects of g. mellonella immunity are affected by priming: hemolymph coagulation and po activity, which may contribute to the higher resistance of the primed larvae to re-infection. the question remains about the mechanism regulating this phenomenon. 71 in other words, how did the larvae "learn the lesson" from the first encountering of a pathogen during priming? further studies are needed to understand the way the innate immunity can be modulated to protect insects against repeated infections. the discovery of the improved immune response in invertebrates have permanently switched our classical thinking of the features of innate and acquired mechanisms of defence. a fascinating answer may be provided by further studies on insect "immune memory". acknowledgement this work was partially financed by grant 2015/17/n/nz6/03500 from the national science centre, poland. we are grateful to dr. joanna czarnecka for her help in data analysis. references bidla g, hauling t, dushay ms, theopold u. activation of insect phenoloxidase after injury: endogenous versus foreign elicitor. j innate immun. 1: 301-308, 2009. bradford m. a rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. analyt. biochem. 72: 248, 1976. buchmann k. evolution of innate immunity: clues from invertebrates via fish to mammals. front. immunol. 5, e459, 2014. cerenius l, lee bl, soderhall k. the propo system: pros and cons for its role in invertebrate immunity. trends immunol. 29: 263-271, 2008. chambers mc, schneider ds. pioneering immunology: insect style. curr. opin. immunol. 24: 10-14, 2012. cooper d, eleftherianos i. memory and specificity in the insect immune system: current perspectives and future challenges. front immunol. 8: e539, 2017. 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greater wax moth galleria mellonella. insect sci 24: 342-357, 2017. wojda i, vertyporokh l. insect immune system in defence of organism integrity. kosmos 4: 541551, 2017. 197 isj 15: 197-202, 2018 issn 1824-307x research report nosema ceranae changes semen characteristics and damages sperm dna in honeybee drones g borsuk1*, m kozłowska2, m anusiewicz2, j paleolog3 1institute of biological basis of animal production; faculty of biology, animal sciences and bioeconomy; university of life sciences, akademicka 13, 20-950 lublin, poland 2department of botany and mycology; institute of biology and biochemistry; faculty of biology and biotechnology; maria curie-skłodowska university, akademicka 19, 20-033 lublin, poland 3department of zoology, animal ecology & wildlife management; faculty of biology, animal sciences and bioeconomy; university of life sciences, akademicka 13, 20-950 lublin, poland accepted june 07, 2018 abstract in this study, we aimed to determine how infection with nosema ceranae spores affected semen volume, sperm concentration, and fragmentation of sperm dna in honeybees. a total of 120 one-dayold drones were marked with queen bee marking numbers, and equally divided into two groups. drones in the infected group were individually fed with honey syrup containing 200 000 n. ceranae spores per 1 µl, while those in the uninfected (control) group were fed honey syrup without spores. the groups were then placed in two separate colonies. fourteen days later, ejaculate was collected from the drones and was analyzed for semen volume, sperm concentration per 1 µl semen, and sperm dna fragmentation. compared to uninfected controls, the n. ceranae spore-infected drones showed significantly decreased semen volume and sperm concentration, as well as a higher percentage of sperm dna fragmentation. key words: nosema ceranae; apis mellifera; sperm; drone; dna fragmentation; chromatin dispersion introduction one cause of apis mellifera colony loss or depopulation is the intestinal parasite nosema ceranae, which infects honeybees with the same intensity year-round (martin-hernandez et al., 2007; giersch et al., 2009; tapaszti et al., 2009). the parasite causes immune system suppression in honeybees (antúnez et al., 2009). nosema spp. destroys intestinal epithelial cells, thereby limiting nutrient absorption and increasing energy requirements (fries et al., 1996; mayack and naug, 2009). microsporidia lack functional mitochondria, and are thus fully dependent on the energy produced by the host (agnew and koella, 1997). infection with nosema spp. spores leads to declining protein levels in infected workers, resulting in hypopharyngeal gland atrophy and changes in the fatty acid composition of the hemolymph (roberts, 1968; wang and moeller, 1970; malone et ___________________________________________________________________________ corresponding author: grzegorz borsuk institute of biological basis of animal production faculty of biology, animal sciences and bioeconomy university of life sciences akademicka 13, 20-950 lublin, poland e-mail: grzegorz.borsuk@up.lublin.pl al., 1995, 1998). this limits the development of young bees and leads to honeybee colony weakening, depopulation, and collapse (higes et al., 2008, 2011, 2013; botías et al., 2012). honeybee colonies in poland commonly exhibit infection with n. ceranae spores (michalczyk et al., 2013). n. ceranae spores infect bees and queens as well as juvenile and adult drones, which transmit the infection (traver and fell, 2011). nosema apis infection does not impact the acceleration of maturation or the undertaking of earlier mating flights by infected individuals (tofilski and kopel, 1996). older drones infected with n. apis exhibit spores in their reproductive tissues and ejaculate, which may cause vertical/sexual transmission of the infection and deterioration of reproduction performance and reflects sperm damage caused by nosema spp. (peng et al., 2015). n. apis and n. ceranae represent microsporidia from the kingdom of fungi (adl et al., 2005; huang et al., 2013). both nosema species are obligatory parasites of adult bees causing similar severe bee infections worldwide (klee et al., 2007). therefore, results obtained for n. apis can be extrapolated to those for n. cerana, but n. ceranae is more virulent towards apis mellifera honey bees (huang, 2011). 198 in the present study, we investigated the hypothesis that n. ceranae damages sperm dna in honeybees. drones were individually infected with n. ceranae spores. we then examined the effects of infection on semen volume, sperm concentration per 1 µl semen, and sperm dna fragmentation relative to uninfected drones. material and methods nosema ceranae spore collection we obtained 30 dead bees from each of the 50 winter-killed colonies that originated from the apiary of the university of life sciences in lublin. these bee samples were homogenized in 30 ml of distilled water, and the homogenates were applied to a bürker hemocytometer. we counted the spores within each square under a light microscope at 400× magnification. infection intensity was assessed as the number of spores per honeybee. winter deaths characterized by the marked presence of nosema spp. spores were subsequently subjected to polymerase chain reaction (pcr) analysis using specific primers for n. ceranae 218mitoc and n. apis 321apis. we mixed 300 μl honeybee homogenate with 180 μl lysis buffer and 20 μl proteinase k, and then isolated total dna using the dneasy blood and tissue kit (qiagen) in accordance with the manufacturer’s instructions. to identify the nosema species, total dna was analyzed using the primers 218mitoc and 321apis, as described by martin-hernandez et al. (martin-hernandez et al., 2007). pcr was performed using the taq pcr core kit (qiagen). in a total volume of 30 µl, the reaction mixture included 6 µl of the dna sample, 9.85 µl h2o, 3.3 µl 10× pcr buffer, 6.6 µl q solution, 2.95 µl mg+2, 0.66 µl dntp mix, 0.125 µl forward primer, 0.125 µl reverse primer, and 0.39 µl taq. the pcr conditions were as follows: 10 min at 95 °c; followed by 35 cycles of 30 s at 95 °c, 30 s at 55.8 °c, and 45 s at 72 °c; and then a final extension of 7 min at 72 °c. the pcr product was analyzed in 5% agarose gel under uv light. we selected the winter-killed colonies that were infected solely with n. ceranae spores. homogenates including only n. ceranae spores were used to prepare honey-water syrup (1:1 ratio) containing 2 × 106 spores/µl. this syrup was administered to 120 one-day-old honeybee workers for 5 days, and was then replaced with spore-free syrup. for the next 14 days, the workers were kept in wooden cages of 12.5 × 12.5 × 4 cm, maintained under laboratory conditions (25 °c; h= 65%). after this period, they were euthanized with co2 for 10 min. once again, the hemocytometric method was applied to determine the number of spores. these spores were used to prepare a honey-water syrup (1:1) containing 200 000 n. ceranae spores per 1 µl to be administered to one-day-old drones. this procedure ensured good viability of the n. ceranae spores, such that they would have high ability to infect immediately and effectively the honeybee drones in the further experiments. drone breeding and infection the investigations were conducted in june 2017, using drones originating from one apis mellifera carnica queen. to obtain drones of the same age, the queen was caged for two days on a single drone comb using a queen excluder. then the queen was released, and the comb remained caged to protect it from further oviposition. on postoviposition day 23, the comb was transferred to a 34 °c incubator to induce drone emergence. the emerging drones were individually marked using queen bee marking numbers. a total of 120 drones were evenly divided into two groups: uninfected and infected with n. ceranae. using a micropipette, the control/uninfected drones were individually fed 2 µl of honey:water (1:1) solution, whereas drones in the infected group were fed 2 µl of honey:water (1:1) solution containing 200 000 n. ceranae spores per 1 µl. then, the drones were placed in separate colonies, with 60 uninfected drones in three colonies and 60 n. ceranae spore-infected drones in another three colon (20 drones per colony were free living). all colonies were nosema spp. free, as confirmed by pcr tests. the exit were barred by queen excluders to prevent the drones from leaving the colonies. on day 14 after emergence, the drones were captured and their semen was collected using a 1 µl calibrated micropipette. the semen volume was measured using an electronic caliper following the method of czekońska et al. (2013b). then the semen collected from each drone was divided into two samples of equal volume. one sample was used to determine the sperm concentration, and the other sample was used for dna fragmentation analysis. sperm concentration determination with a muse flow cytometer sperm concentration was determined using a flow cytometer and a muse count and viability kit from merck. the sperm was diluted 20× in phosphate-buffered saline (pbs), and then mixed with 380 µl of muse count and viability reagent. the mixture was vortexed for approximately 30 s, incubated at room temperature for 5 min, and then vortexed again for 10 s. samples prepared in this manner were then measured three times using the muse flow cytometer. sperm dna fragmentation (sdf) to quantify the sperm dna fragmentation, we used the sperm dna fragmentation (sdf) test from halosperm® (halotech dna sl, iso 13485), in accordance with the manufacturer’s instructions. this assay is based on sperm chromatin dispersion. the sperm samples were diluted with a 400× pbs solution. agarose was dissolved at a temperature of 95 °c for 5 min, and then cooled to 37 °c. we then combined 50 µl diluted sperm with 100 µl of agarose. next, 8 µl of the mixture of sperm and agarose was mounted on horizontal microscopic slides provided in the kit, and covered with a cover glass. the preparations were transferred to a refrigerator and kept at 5 °c for 5 min 199 table 1 semen volume and sperm concentration of infected and uninfected drones (mann-whitney tests) trait number uninfected drones nosema ceranae infected drones statistics mean mean z p semen volume (μl) 60 1.18 0.37 9.48 0.00001 sperm concentration (× 106/μl) 60 10.06 4.93 9.49 0.00001 until the agarose set. then the cover glasses were removed, and the slides were submerged in “solution 1” (denaturant agent) and incubated at room temperature for 7 min. next, solution 1 was removed, and the slides were dried, submerged in “solution 2” (lysis solution), and incubated at room temperature for 20 min. the slides were then rinsed in distilled water, dried, placed in a horizontal position, and dehydrated by submersion in 70% ethanol for 2 min. after removal of the 70% ethanol, 100% ethanol was applied for 2 min. next, the slides were dried and stained with “solution 3” (eosin staining solution) at room temperature for 7 min. finally, the slides were dried, and then stained with “solution 4” (thiazine staining solution) at room temperature for 7 min. in this manner, we prepared 20 samples total from the infected drones and 20 samples total from the uninfected drones (40 samples total). we then counted the sperm cells in five fields of vision from each sample using a nikon eclipse ni bright-field microscope at 400× magnification. in each field of vision, we counted 10 sperm cells with fragmented and degraded dna and normal spermatozoa. we calculated the percentage of sperm with fragmented dna using the following formula: fragmentation + degradation sdf(%) = x 100 total cell count statistical analysis statistical analysis was performed using sas software version 9.5 (statistical analysis system institute, cary, nc). comparisons between the uninfected drones and n. ceranae-infected drones were performed using mann-whitney tests. percentage data were arcsine-transformed (sokal and rohlf, 1981). results a significantly higher volume of semen was collected from the uninfected drones than from the n. ceranae-infected drones (p ≤ 0.05; table 1). the concentration of sperm in semen was also significantly higher in the uninfected drones (10.06 × 106/μl) than in the n. ceranae-infected drones (4.93 × 106/μl) (table 1). in the present study, we analyzed 1000 spermatozoa from each uninfected and infected drone. sperm cells with undamaged dna were characterized by a round central core with a large circular aureole/halo along the perimeter of the sperm head (fig. 1a). in spermatozoa with damaged dna and nucleotides, we observed a central core with a small elongated aureole/halo that tightly adhered to the sperm head, which was formed by dispersed dna fragments (fig. 1b). degraded sperm exhibited no aureole/halo (fig. 1c). fig. 1 representative photographs of sperm after the sperm dna fragmentation (sdf) test. (a) healthy sperm with no dna fragmentation. (b) sperm with dispersion of dna fragments. (c) degraded sperm 200 fig. 2 percentage of sperm dna fragmentation. *p < 0.05 for the difference between sperm from uninfected drones and n. ceranae-infected drones (mann-whitney test; z= 13.38 p= 0.00001). error bars represent standard deviation of the data we observed a markedly higher percentage of dna fragmentation among sperm cells from n. ceranae-infected drones (48.82%) than from the uninfected drones (13.56%) (fig. 2). discussion the volume of semen collected from the uninfected drones in this study was similar to the collected volume of 0.9 to 1.1 μl reported by czekońska et al. (2013b, 2015). strikingly, only 0.37 μl of this volume was collected from the n. ceranae-infected drones in this study. in fact, the sperm concentration in the infected drones was the lowest ever reported from a. m. carnica drones. the concentration of sperm per 1 μl of semen generally ranges from 6.76 to 11.95 × 106/µl, depending on the size of the drones and study (woyke, 1960; moritz, 1981, 1984; rinderer et al., 1985, 1999; berg and koeniger, 1990; duay et al., 2002; czekońska et al., 2015). we assessed sperm chromatin dispersion using a sperm dna fragmentation (sdf) test, which is widely used for examinations of daphnia, bull, wild boar, and even human sperm (fernández et al., 2003, 2005; enciso et al., 2006; gómez et al., 2016). the sdf test is a simple, accurate, and reproducible method for analyzing sperm dna fragmentation (breznik et al., 2016), which is routinely used for sperm analysis in medical laboratories and is an important tool for diagnosing male infertility (jay, 1963). spermatozoa in drones are produced during the preimaginal period (bishop, 1920; fukuda and ohtani, 1977; czekońska et al., 2013a), beginning at the larval stage and ending at the pupal stage (snodgrass, 1956). during the first week of adult life, spermatozoa are transferred from the testes to seminal vesicles, where they are stored until copulation (snodgrass, 1956; woyke, 1958; de graaf and jacobs, 1991). the reduced sperm volume and concentration observed in the n. ceranae-infected drones (table 1) may have been caused by undernourishment of the infected drones during the transfer of sperm from the testes to seminal vesicles, which lasts approximately one week. prior studies demonstrate that n. ceranae spreads into many tissues and affects the sexual function of infected drones (roberts and hughes, 2015; ciereszko et al., 2017). n. ceranae-infected hosts also suffer intestinal epithelial cell damage, limited nutrient uptake, and increased energy requirements (fries et al., 1996; mayack and naug, 2009). nosema infection reduces protein levels in infected honeybees, leading to hypopharyngeal gland atrophy (wang and moeller, 1970; malone et al., 1995), and changing the fatty acid composition of hemolymph (roberts, 1968). this condition of energy deficiency, limited protein availability, and disturbed fatty acid metabolism leads to undernourishment, which likely resulted in the production of sperm with dna fragmentation in the n. ceranae-infected drones in our present study (fig. 2). ciereszko et al. (2017) demonstrated that honeybee spermatozoa are susceptible to the toxic effects of imidacloprid, which aggravates sperm parameters. moreover, peng et al. (2015) reported that nosema apis infection reduced drone fertility and lifespan, and detected spores in the ejaculates of infected drones. these authors questioned whether infected drones actually leave their colonies to undertake mating flights. if so, they could additionally pose a threat to the mating success of females, since drones can transmit the infection vertically. our findings suggest that if infected drones undertake the mating flight, their reproductive success will only be achieved by approximately 51% of spermatozoa, as approximately 49% will be unable to fertilize eggs due to the phenomenon of sperm chromatin dispersion and semen volume. our present results confirmed the hypothesis that n. ceranae infection causes damage to sperm dna and decreases both the semen volume and sperm concentration in honeybee drones. this is the first demonstration that these phenomena may cause the poorer reproduction performance of n. ceranae-infected drones. sperm with damaged dna are incapable of fertilizing eggs, which may decrease queen fecundity and contribute to honeybee colony depopulation. 201 references adl sm, simpson agb, lane ce, lukeš j, bass d, bowser ss, et al. the new higher level classification of eukaryotes with emphasis on the taxonomy of protists. j eukaryot microbiol. 52: 399-451, 2005. agnew p, koella jc. virulence, parasite mode of transmission, and host fluctuating asymmetry. proc r soc lond b biol sci. 264: 9-15, 1997. antúnez k, martin-hernandez r, prieto l, meana a, zunino p, higes m. immune-suppression in the honey bee (apis mellifera) following infection by nosema ceranae (microsporidia). environ microbiol. 11: 2284-2290, 2009. berg s, koeniger n. larger drones (apis mellifera) have more offspring. proc. german zoological society, 83rd meeting, frankfurt am main, gustav fischer verlag, 1990. bishop gh. fertilization in the honey-bee. i. the male sexual organs: their histological structure and physiological functioning. j exp zool. 31: 224-265, 1920. botías c, martin-hernandez r, días j, garcíapalencia p, matabuena m, juarranz a. the effect of induced queen replacement on nosema spp. infection in honey bee (apis mellifera iberiensis) colonies. environ microbiol. 14: 845-859, 2012. breznik pb, kovačič b, vlaisavljević v. are sperm dna fragmentation, hyperactivation, and hyaluronan-bindin gability predictive for fertilization and embryo development in in vitro fertilization and intracytoplasmic sperm injection? fertil steril. 99: 1233-1241, 2013. ciereszko a, wilde j, dietrich gj, siuda m, bąk b, judycka s, et al. sperm parameters of honeybee drones exposed to imidacloprid. apidologie. 48: 211-222, 2017. czekońska k, chuda-mickiewicz b, chorbiński p. the effect of brood incubation temperature on the reproductive value of honey bee (apis mellifera) drones. j. apic. res. 52(2): 96-105, 2013a. czekońska k, chuda-mickiewicz b, chorbiński p. the influence of honey bee (apis mellifera) drone age on volume of semen and viability of spermatozoa. j. apic. science. 57: 61-66, 2013b. czekońska k, chuda-mickiewicz b, samborski j. quality of honeybee drones reared in colonies with limited and unlimited access to pollen. apidologie. 46: 1-9, 2015. de graaf dc, jacobs fj. tissue specificity of nosema apis. j invertebr pathol. 58: 277-278, 1991. duay p, de jong d, engels w. decreased flight performance and sperm production in drones of the honey bee (apis mellifera l.) slightly infested by varroa destructor mites during pupal development. genet mol res. 1: 227232, 2002. enciso m, lopez-fernandez c, fernandez jl, garcia p, gosalbez a, gosalvez j. a new method to analyze boar sperm dna fragmentation under bright-field or fluorescence microscopy. theriogenology. 65: 308-316, 2006. fernández jl, muriel l, goyanes v, segrelles e, gosálvez j, enciso m. simple determination of human sperm dna fragmentation with an improved sperm chromatin dispersion (scd) test. fertil steril. 84: 833-842, 2005. fernández jl, muriel l, rivero mt, goyanes v, vazquez r, alvarez jg. the sperm chromatin dispersion test: a simple method for the determination of sperm dna fragmentation. j androl. 24: 59-66, 2003. fries i, feng f, da silva a, slemenda sb, pieniazek nj. nosema ceranae n. sp. 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glutamine md venkatesh, mvv subramanyam* laboratory of physiology, department of life science, bangalore university, bangalore 560 056, india accepted july 11, 2018 abstract organisms are subjected to a variety of stress and physiological impact in the event of drug administration. drug extrusion plays a critical role in the survival of organisms. the present study elucidate the role of glutamate transporter, eaat2 in survival of model organism silkworm injected with azaserine, a glutamine analogue and offer a possible explanation for a contrasting observation of greater survival at higher dosage and lesser survival in lower dosage. our results have shown a dosage dependent inhibition of gamma glutamyl transferase (ggt) activity, differential expression of eaat2 transporter along with concomitant changes in residual concentration of azaserine, glutamate and cysteine that undermine cysteine transport. the current results also suggest that survival is dependent on eaat2 transporter in cysteine-glutamate/azaserine transport and rejuvenation of atp formation as observed in higher but not in lower dosage received worms. key words: azaserine, glutamate, eaat2, ggt, gsh, atp, rnai, bombyx mori introduction amino acid transporters play a crucial role in the metabolic dynamics in living organisms and these transporters primarily facilitate in cell signaling, that leads to either pro or anti apoptotic pathways. amino acid l-cystine is relevant in biological systems in wide array of processes and is a part of the antioxidant tripeptide molecule glutathione (gsh), which is composed of amino acids, glutamate, cysteine and glycine. cystine is transported by several amino acid transporters like excitatory amino acid transporters eaat1 and eaat2, system asc in invertebrates and system xc in vertebrates (lewerenz et al., 2013). eaat1 and eaat2 co-transport cysteine with (3na+/1h+/aa), as symporter with glutamate that has higher affinity to glutamate and as an antiporter with 1k+ (chen and swanson, 2003). system asc is a na+-dependent antiporter that transports cysteine directly to exterior of the cell (christensen, 1990). once inside the cell, cystine is reduced to cysteine by the gsh or thioredoxin reductase (mandal et al., 2010) and incorporated into gsh that protects the cells against oxidative stress (os) under various stressors, while ___________________________________________________________________________ corresponding author: mvv subramanyam laboratory of physiology department of life science bangalore university bangalore 560 056, india e-mail: muthangi@bub.ernet.in the catabolism of gsh involves externalization of cysteine through gamma glutamyl transferases (ggt/γ-gt) (dringen, 2000; hanigan, 2014). therefore a critical role is played by cystine/cysteine in maintaining cellular homeostasis, redox status and gsh metabolism under os (bannai et al., 1989). during an os there may be elevated externalization of glutamate that alters the status and can competitively inhibit cystine transport into the cells (lewerenz et al., 2013). the enzyme ggt is a glycoprotein located on the outer surface of the cell membrane, critical to cellular detoxification and amino acid transport (keillor et al., 2005). ggt also uses glutathione as an acyl donor substrate and transfers its glutamyl moiety to the acceptor substrate such as amino acid with higher affinity to l-cysteine and l-glycine and release cysteinyl glycine across the cell membrane (allison, 1981). ggt has primary role in gsh catabolism and aids cystine/cysteine balance to maintain redox state. cellular atp is a biological source of energy and needed adequately during any form of stress, not only to counter stress and survive but also for normal cellular homeostasis and functioning. drug azaserine is a glutamine analogue and is a competitive inhibitor for glutamine (moore and le page, 1957) and it can interact with cellular biomolecules thereby causing free radical generation and impairment in atp generation (revathi et al., 2012). it also inhibits ggt irreversibly by binding covalently to the active site of mailto:muthangi@bub.ernet.in 266 table 1 sequence of the primers for mrna of eaat2 gene and tubulin for real time pcr genes primer sequence product size accession number swtub-fp: tcgtcgagccctacaactct swtub-rp: actcggtgaggtccacattc 222 bp nm_001043419.1 bm-glutran-qfp: agatcacagcattctggatagcttgctg bm-glutran-qrp: tgtgccagatctgtaagacacgactcgt 187 bp nm_001253896.1 the enzyme (gardell and tate, 1981) and also a non-competitive inhibitor in low km system for glutamine but has no effect on high km system for glutamine uptake (hsu, 1980). the present work is based on a contrasting results obtained on azaserine treated silk worm larvae with higher survival rate that received higher dosage as compared to lower dosage. this prompted us to investigate the possible externalization of azaserine at higher dosages for subsequent higher rate of survival. transport of azaserine/cysteine across membrane has been critically examined in erythrocytes (yeldiz et al., 2009). we presume that azaserine-cystine transporter may be crucial and may pave way for azaserine into the cells/tissue because of its structural similarity to glutamine. here we experimentally demonstrated the impact of varied dosages of azaserine on inhibition of cystineglutamate transporter eaat2. the aim of the present study includes the expression of the gene eaat 2 in silk worm bombyx mori through qpcr. rnai studies were attempted in silencing the expression of the gene eaat2 in order to authenticate the presence and functionality of the gene in b. mori. the other studies include the impact of azaserine residuality on ggt, gsh and atp levels that plays a crucial role in the survival of injected larvae. materials and methods experimental animals healthy larvae of iv instar silk worm bombyx mori were procured from licensed private rearing houses and were maintained until they attained v instar in laboratory conditions at 23 °c, humidity 75% and 12 l/d cycle, rearing of larvae was according to improved method of krishnaswami (1978). 45  5 larvae were injected with each concentration of azaserine. all the experiments were repeated for minimum six times (n=6). chemicals all chemicals viz azaserine, cystine, glutamate, glutathione, 5-5’dithio (bis) nitrobenzoic acid (dtnb), γglutamyl-p-nitroanilide (gpna), glycyl-glycine, creatine phosphate were obtained from sigma aldrich chemicals (st louis, usa). rna isolation kit, ds rna, dntp’s, cdna synthesis kit were obtained from chromous biotech (india). all other reagents were of hplc grade and were procured from himedia (india). fig. 1 percentage of survival in larvae of b. mori injected with dosages of azaserine. significance is represented as x, y, z. †significance was calculated by manova and post-hoc test was done using spss11.5 and significance was considered at p < 0.05 https://www.ncbi.nlm.nih.gov/nucleotide/nm_001043419?report=genbank&log$=nuclalign&blast_rank=15&rid=5t6gk9e0014 267 fig. 2 a) representative agarose gel photograph from control, 1 and 5 mm azaserine injected larval tissues viz, mid gut (mg), posterior silk gland (psg), fat body (fb) of b. mori. b) qrt-pcr analysis of the cystine – glutamate transporter gene eaat2 in control, 1 and 5 mm azaserine injected larval tissues. data are shown as mean ± sem (n = 6). †significance was calculated by manova and post-hoc test was done using spss11.5 and significance was considered at p < 0.05 estimation of residual azaserine, cysteine and glutamate estimation of residual azaserine, cysteine and glutamate in larval tissues of b. mori were performed with a slight modification of earlier method of vasilescu et al. (2013). briefly, the tissue were extracted with methanol and were loaded on to the c-18 column (20 μl) with mobile phase that had two components a: phosphate buffer (0.01 m, ph = 3.60) and methanol in a ratio of 2:1 (v/v), while b: acetonitrile, in a final mixture of a:b = 75:25. azaserine, cystine and glutamate were resolved on uv high pressure hplc (agilent technologies, usa, model 1120). glutamate-cystine transporter gene expression glutamate-cystine exchanger gene sequence was obtained through blast search of silk worm genome with data sourced from ncbi. primers were designed and synthesized for silkworm tubulin (housekeeping gene) and eaat2 gene (cystineglutamate transporter) under study. rna extraction and real time quantitative polymerase chain reaction (qrt-pcr) for eaat2 gene (glutamate transporter) total rna in the larval tissues viz mid gut, posterior silk gland and fat body were extracted using lysis buffer-β-me mixture containing guanidine hydrochloride (chromous rna isolation kit). samples were treated with rnase free dnase 1 to remove genomic dna residual from rna samples. 5 μg of total rna was reverse-transcribed using 1 μg of oligo-dt primer, 2.0 μl of dntp (2.5 mm), 10 u of ribonuclease inhibitor (rnasin) and 1.0 μl mmulv rt (20 u/μl). the mixture was incubated at 42 °c for 1 h and the reaction was terminated by incubating the mixture at 92 °c for 2 min. the realtime pcr primers for target transcripts were designed using the complete cdna sequences from ncbi (table 1). a real-time run was taken on abi step-one real time pcr (applied biosystems, usa). briefly, real-time pcr reactions were performed with 50.0 μl reaction solution containing 2.0 μl first strand cdna, 2.0 μl of each forward and reverse primers, 25.0 μl of 2× sybr green master mix and 20.0 μl of rnase-free h2o2. the thermal cycling conditions include 5 min at 94 °c, 5 s at 94 °c, 10 s at 55 °c, 10 s at 72 °c and 5 min at 72 °c for 40 cycles. the output from real time software was analyzed. the eaat2 gene and tubulin transcript levels were estimated by using the formula 2δδct where δct represents the difference in ct values between target gene and tubulin. rt-pcr data were normalized with tubulin mrna levels and relative mrna levels were expressed as 2δδct values. 268 fig 3 a) representative agarose gel photograph from control and ds rna injected larval tissues viz, mid gut (mg), posterior silk gland (psg), fat body (fb) of bombyx mori . b) qrt-pcr analysis of the cystine –glutamate transporter gene eaat2 in control and ds rna injected larval tissues data are shown as mean ± sem (n = 6). †significance was calculated by manova and post-hoc test was done using spss11.5 and significance was considered at p < 0.05 rna interference studies on eaat2 gene in b.mori the dsrna for bm eaat2 was synthesized using chromous rna kit. dsrna of 40 μg was injected twice into each individual larvae with first injection on the 4 day of final instar and the second injection was at 2 h prior to the collection of tissues. further the expression of the gene and role of rnai was assessed by qrt-pcr. estimation of atp the tissue atp was estimated with modification of earlier procedure by korenberg, (1950). the principle involved for the above method is, atp reacts with glucose in the presence of mg2+ and hexokinase, forming glucose 6-phosphate which is further oxidized by glucose 6-phosphate dehydrogenase with simultaneous reduction of tpn. the rate of reduction of tpn is measured by observing the rate of change of optical density at 340 nm. in these coupled reactions, the final rate of reduction of tpn is equal to the rate of formation of atp with or without creatine phosphate and expressed as μm of atp formed/unit time. estimation of reduced glutathione glutathione was determined in larval tissues according to boyne and ellman (1972) based on 55’dithio (bis) nitrobenzoic acid (dtnb) reaction with compounds containing sulphahydryl groups. the final yellow colored reaction mixture was read at 412 nm and expressed as μm/mg tissue. determination of γ-glutmyl transpeptidase (ggt) activity ggt activity was assayed according to grisk et al. (1993), using γglutamyl-p-nitroanilide (gpna) as donor substrate while glycyl-glycine as glutamate acceptor for the transpeptidation reaction. p-nitroaniline formed was read at 405 nm and expressed as mm substrate transformed/ml/min. statistics data are shown as the mean  sd of six observations. changes between groups were analysed by manova and further tested by the bonferroni posthoc test, kaplan-meier test for survival analysis were assessed using statistical package for social sciences (spss) software (huberty and olejnik, 2006) and p < 0.05 was considered significant, statistically significant data are presented in the text. results survival of silk worm b. mori injected with 1 and 5 mm azaserine v instar larvae were injected with 1 and 5 mm azaserine and the percentage of survival were noted after 24 h of injections. fig. 1 shows significant increase in survival percentage in larvae injected with 5 mm in comparison to 1 mm. 269 fig. 4 residual values (ng /mg tissue) of (a) cysteine (b) azaserine,(c) glutamate in control, 1 and 5 mm azaserine injected larval tissues viz, mid gut, posterior silk gland, fat body from larvae of b. mori through hplc. †significance was considered at p < 0.05. significance was represented as α, β and γ for mid gut, p ,q and r for posterior silk gland and *, $ and # for fat body tissue specific expression of glutamate-cystine transporter gene eaat2 in b. mori larval tissues showed significant changes in expression of eaat2 gene, midgut tissue showed a significant decrease in expression of eaat2 with increase in concentration of azaserine (fig. 2a and b) and in larval posterior silk gland there was a significant up regulation in 5 mm azaserine injected in contrast to 1 mm. larval fat body showed an increase in expression of eaat2 with azaserine injections. silencing of bm eaat2 gene in b. mori through rnai studies expression profiles of the gene eaat2 in normal and dsbmeaat2 rna injected larvae in three tissues viz mid gut, posterior silk gland, fat bodies were compared. eaat2 gene was found to be silenced in all the three tissues in dsbmeaat2 rna injected larvae (fig 3a and b). the present results provided an evidence for the functionality of transporter gene eaat2 in b. mori. 270 fig. 5 effect of azaserine on atp synthesis in (a) mid gut (b) posterior silk gland and (c) fat body of larvae of b, mori. values are expressed in terms of μm atp formed/ mg protein/ min and represented as mean ± sem (n = 6). † significance was calculated by manova and posthoc test was done using spss11.5 and significance was considered at p < 0.05. significance was represented as α, β and γ for mid gut tissues, p, q and r for posterior silk gland tissue and *, $ and # for fat body tissues assayed in control, 1 and 5 mm azaserine injected larval groups + and represent the atp synthesized in presence and absence of creatine phosphate respectively residual concentration of cystine, azaserine and glutamate in larval tissues of b. mori residual concentration of amino acid cystine in larval tissues injected with 1 and 5 mm azaserine were assessed using hplc agilent zorbax column (c-18). significant decrease in cystine was observed in mid gut of larvae injected with 5 mm azaserine whereas traces of cystine were observed at 1 mm azaserine injected. cystine was undetectable in posterior silk gland of treated larvae. larval fat body showed significantly decreased concentrations of cystine with increased concentrations of azaserine (fig. 4a). the residual concentrations of azaserine were significantly evident only in mid gut of larvae injected with 1 mm azaserine whereas undetectable in mid gut of larvae injected with higher dosage. residual concentration of azaserine in posterior silk gland increased with azaserine dosages (fig. 4b). however fat body tissues were found to have no azaserine irrespective of concentrations. in the larvae injected with 1 mm azaserine showed a significant increase in the values of glutamate in mid gut and posterior silk gland, however fat body showed an insignificant change in glutamate levels. in the 5 mm azaserine injected larval tissues, mid gut and fat bodies showed a decrease in glutamate levels while elevated levels were observed in posterior silk gland (fig. 4c). interestingly ds rna injected larvae showed insignificant values of l-glutamate. 271 fig. 6 effect of azaserine on glutathione (gsh) in mid gut, posterior silk gland and fatbody of larvae of b. mori. values are expressed in terms of μm gsh/mg tissue and represented as mean ± sem (n = 6). † significance was calculated by manova and posthoc test was done using spss11.5 and significance was considered at p < 0.05. significance was represented as α, and γ for midgut tissues p, q and r for posterior silk gland tissue and * and # for fat body tissues assayed in control, 1 and 5 mm azaserine injected larval groups effect of azaserine on atp formation in b. mori tissues were assayed for atp formation in the presence (+) and absence (-) of creatine phosphate. the larval mid gut and posterior silk gland showed a significant increase in atp formed in 5 mm azaserine injected while 1 mm azaserine injected larval tissues did not show atp formation in the presence of creatine phosphate. in the larval fat body tissue atp formation showed a significant decrease in 1 mm injected and no significant changes in higher dosage (fig. 5). effect of azaserine on glutathione (gsh) levels in tissues of b. mori glutathione is an antioxidant molecule. the larval mid gut tissue reveals significant increase in gsh activity at higher dosage while the posterior silk gland showed a significant increase in gsh activity in lower dosage. a significant increase in gsh activity was observed in fat body at both the dosages of azaserine injected (fig. 6). effect of azaserine on gamma glutamyl transpeptidase (ggt) levels in tissues of b. mori ggt being membrane bound and externalizes gsh, significant inhibition of ggt activity by 7 folds was noted in mid gut tissues at both the dosages. the larval posterior silk gland showed inhibition only in 1 mm injected while the 5 mm injected showed no change, contrastingly the fat body showed significant elevation in ggt activity at both the dosages of azaserine injected (fig. 7). fig. 7 effect of azaserine on gamma glutamyl transpeptidase (ggt) in (a) mid gut (b) posterior silk gland and (c) fatbody of larvae of b. mori. values are expressed in terms of μm ggt/mg tissue and represented as mean ± sem (n = 6). †significance was calculated by manova and posthoc test was done using spss11.5 and significance was considered at p < 0.05. significance was represented as α and β for mid gut tissues, p and q for posterior silk gland tissue and * and # for fat body tissues assayed in control, 1 and 5 mm azaserine injected larval groups. † (significance between control and among dosages is considered and not between types of tissues for all the assays) 272 survival analysis survival analysis among the test groups (dosages) were assessed using kaplan-meier test. the outcomes of the analyses show significant difference in survival of larvae injected with 1 mm and 5 mm dosages of azaserine. the results show better survival at higher dosages of injections than lower dosages. the average survival post 24 h was about 17% with 1 mm injections and 30% with 5 mm injections (fig. 8). discussion azaserine is a known analogue of glutamine and often used as a drug. as the drug dosages become important in different stages of treatment, it is imperative that higher dosages have severe impact compared to lower dosages and the ld50 is a norm in deciding admissible drug dosages, however the present study has the contrasting result of lesser survival of larvae at lower dosages and greater survival at higher dosages, as such the concept of ld50 is not relevant in the present context. azaserine inhibits ggt irreversibly (hsu, 1980), our results have shown that ggt is inhibited by azaserine at both dosages in mid gut. in the posterior silk gland inhibition was evident at lower dosage and higher dosage has no effect on ggt activity contrary to these the fat body had significant elevation in ggt activity at both dosages. these observed changes could be due to induced changes in redox status (garama et al., 2015) and the gsh catabolism along with intracellular and extracellular cystine pool as reported (paolicchi, 2002). in sporodoptera littoralis, a lepidopteran, the increased ggt activity is due to the os induced by the spores of baveria bassiana (mirhaghparast et al., 2013). in our studies azaserine has impacted ggt differently with different dosages in the tissues studied. however the mechanistic of activation or inhibition of ggt was not attempted in the present study. as gsh is a non-enzymatic antioxidant, it becomes imperative in understanding gsh metabolism in stressed state. our results showed an increase in gsh activity in mid gut at higher dosage, at lower dosage in posterior silk gland, both dosages in fat body and these changes are tissue specific. these outcomes could be due to azaserine induced redox changes. in phytophageous insect such as lymantria dispar have higher gsh activity in the mid gut in order to defend against pro oxidant effect of plant allelochemicals and xenobiotics (maturga et al., 1997). our results with mid gut are concurrent with the above as gsh changes are imminent for survival of insect species (buyukguzel fig. 8 kaplan-meier statistical analyses depicting significant differences in survival between dosages of azaserine in b. mori 273 and kelender, 2007; poupardina et al., 2008; vijayvel and balasubramanian, 2009; ahemad, 2011; fahmy, 2012) and also gsh can nonenzymatically react with different ros and can be a free radical scavenger (winterbourn, 1994). cystine (cyss-) is an oxidized, whereas cysteine (2xcys) is reduced form. in gsh homeostasis, intracellular cysteine is a part of tripeptide gsh and externalized by ggt. during the process cysteine is converted to cystine and can make an entry either in acidic or alkaline forms, whereas cystine in neutral form could arise due to extracellular protons, thus makes cystine impermeable (bannai and kitamura, 1981). interestingly our results for the first time show no traces of cystine in the tissues that showed residual azaserine, clearly emphasizing azaserine cysteine transport in silk worm b. mori and further emphasized by expression of eaat2. excitatory amino acid transporters co transports glutamine with cysteine or aspartate (shankar et al., 2001) and uptake of cysteine and glutamate are mutually competitive, eaat2 inhibitors also inhibit cysteine uptake (chen and swanson, 2003), however the increased residual glutamate concentration in certain tissues can be attributed due to co transport mechanism involving na2+ even though eaat2 was inhibited. our results of eaat2 inhibition by azaserine are consistent with the above particularly in mid gut and posterior silk gland. interestingly the cysteine uptake has been totally inhibited in tissues showing residual azaserine however eaat2 expressions was up regulated in fat body. insect eaat’s have been cloned and compared to mammalian eaat’s and they share high degree of amino acid identity, comparable kinetics and pharmacological properties (donly and caveney, 2002). silk worm b. mori eaat2 amino acid sequence shares 83% homology with lepidopteran insect chilo suppressalis especially in fat body and mid gut (xu et al., 2015). larval neutral amino acid transporters show different dependency on ph and an increased uptake was seen at alkaline ph (hannigan, 1993; sacchi et al., 1994). the injection of azaserine can induce changes in the ph in insect system as the drug is itself acidic in nature and may impair glutamate transport as indicated by down regulation of eaat2 (castanga, 1997). in the present study down regulation of eaat2 transporter in posterior silk glands injected with both dosages can be attributed to the presence of residual azaserine in glandular epithelium. the expression of eaat2 gene was silenced using a double stranded rna (dsrna) that degrades the target mrna (fire et al., 1998). our studies confirm the presence of eaat2 gene in b. mori and further established with silencing of the same gene by introducing ds rna. several rnai studies have been performed in bombyx mori embryos and larvae to understand the role of various genes in developmental process and immunity (quan et al., 2002; rajagopal et al., 2002). the mode of dsrna introduction, the quantity or dosages of dsrna have shown variations with different tissues of bombyx mori (terenius et al., 2011). in the present study 80 μg of dsrna was injected to bring about effective silencing of the gene. biological form of energy is the atp molecules that define the status of cells ability to survive or to succumb to the critical conditions of any form of stress or disease. 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gx ,fang q, ye gy. de novo assembly and characterization of central nervous system transcriptome reveals neurotransmitter signaling systems in the rice striped stem borer, chilo suppressalis. bmc genomics. 16: 525, 2015. yildiz d, atli m, yildiz h, oztas h. the effect of azaserine on cysteine transport in erythrocytes. trakia j. sci. 8: 1-10, 2009. combined effects of temperature and salinity on functional responses of haemocytes and survival in air of the clam ruditapes philippinarum isj 13: 116-121, 2016 issn 1824-307x review aspects of eco-immunology in molluscs v matozzo department of biology, university of padua, padua, italy accepted april 5, 2016 abstract there is increasing interest in the international scientific community in eco-immunology, a relatively new discipline combining the knowledge from immunology, biology, ecology, physiology and biochemistry. the integrative approach of different scientific disciplines provides a useful perspective, mostly from an evolutionary point of view, into the understanding of the basic mechanisms of the immune responses, as well as the complex host-parasite interactions. however, while knowledge concerning factors affecting the immune response is increasing, more efforts should be made to determine the physiological mechanisms that regulate these responses. this need is particularly pressing in investigations in invertebrates because they are model organisms widely used to study the basic mechanisms of innate immunity and to assess environmental health (the immunomarker approach in biomonitoring studies). in this context, this review focuses on some of the ecoimmunology aspects of molluscs. key words: eco-immunology; hemocytes; immune responses; molluscs   introduction what is the cost of the immune response? this is probably one of the most important questions that stimulates debate among experts in the field. in invertebrates in particular, very few data about the metabolic cost of the immune response are available despite the fact that these organisms have a comparatively simpler immune system compared with vertebrates. according to schmid-hempel (2003), these costs may be “evolutionary costs” (a variation of a component of the immune system may affect growth and reproduction of an organism and vice versa) and “use costs” (the cost to maintain the immune function and to deploy a response). these fundamentals suggest that the immune defence can be costly and cannot always be maintained at maximum levels (ardia et al., 2012). consequently, determining the cost of the immune response assumes a considerable significance when considering the influence of both extrinsic and intrinsic factors on the immunosurveillance status of organisms. in this regard, it is well known that a relationship exists between stress and the immune responses (ottaviani and franceschi, 1996; lacoste ___________________________________________________________________________ corresponding author: valerio matozzo department of biology university of padua via ugo bassi, 58/b, 35131 padua, italy e-mail: valerio.matozzo@unipd.it et al., 2001a, b, c), as well as between the neuroendocrine and immune systems, in both vertebrates and invertebrates (ottaviani et al., 2008; demas et al., 2011). these perspectives have promoted (at least in part) the development of eco-immunology, or ecological immunology (sheldon and verhulst, 1996; rolff and siva-jothy, 2003; ottaviani et al., 2008; demas and nelson, 2012). demas et al. (2011) highlighted that in the last few years, the two research areas that have attracted remarkable attention among experts of eco-immunology of both vertebrates and invertebrates are (i) the energetics of immunity and (ii) the relationship between stress and immunity. in terms of the cost, eco-immunology assumes the immune defence must be minimised (ottaviani et al., 2008). to achieve this goal, organisms can activate pathways, which involve immune and neuroendocrine components (ottaviani et al., 2008). regarding the relationship between stress and immunity, this research area is expanding within the field of eco-immunology (biondi, 2001; ottaviani and malagoli, 2009). however, the debate on this topic is heated because a clear relationship between stress and immunity is not often obvious. in particular, several studies on invertebrates have demonstrated that stressors sometimes increase, while other times decrease, the immune response, depending on several factors, such as the animal 116 species tested, the type and duration of stress, and the immune parameters measured. despite such controversial data, invertebrates are considered an attractive model to study ecoimmunology because of the relatively simple mechanisms that support their innate immune system and their propensity to undergo chemical, biological and physical manipulations, which allows researchers to study the effects of both biotic and abiotic factors on the immune response (ellis et al., 2011; cuvillier-hot et al., 2014). although some invertebrate species use body structures or barriers (e.g., shells and external mucous substances) to protect against the surrounding environment, including pathogens, they rely on their innate immune reaction to protect against internal non-self materials. invertebrates lack antibodies, and the innate immune response is not specific to a particular pathogen. however, some authors stated the limit between “innate” and “adaptive” immunity may be fuzzy and artificial (vinkler and albrech, 2011). these authors suggest invertebrates with a long lifespan should have some kind of acquired immunity. indeed, some invertebrates, including molluscs, can live several decades or more than a hundred years (bodnar, 2009). for example, the arctica islandica species is a 374-year-old bivalve species (schöne et al., 2005; wanamaker et al., 2008). this is an interesting topic that will be a subject for discussion among eco-immunologists in the coming years. the eco-immunology of molluscs among invertebrates, molluscs are one of the most studied groups in terms of their immune functions. like other invertebrates, molluscs rely on an innate, non-lymphoid immune system, the main protagonist of which is the hemocyte. the hemocytes circulate freely in the hemolymph, where they play an important role in wound and tissue repair, shell production and repair, and the immune response (immunocytes) (cheng, 1981; hine, 1999). in molluscs, the immune reaction towards non-self particles involves several mechanisms, including recognition, phagocytosis, encapsulation, intracellular digestion, and the production of cytotoxic substances and antimicrobial peptides. this review does not intend to summarise the large amount of data concerning the involvement of mollusc hemocytes in the immune response. rather, attention is given to some of the ecoimmunology aspects of molluscs. as stated above, eco-immunology focuses on the effects of both biotic and abiotic factors on the immune system among taxa. because the immune defence is energetically costly, it is assumed that organisms under stressful conditions decrease resource allocation to immune system defence (moret and schmid-hempel, 2000). in molluscs, several studies have demonstrated variations in the immune response after exposure of the organisms/cells to environmental contaminants (cajaraville et al., 1996; oliver and fisher, 1999; donaghy et al., 2009; matozzo, 2014). however, there is increasing evidence that the immune response of molluscs can be modulated by both biotic and abiotic factors other than environmental contaminants. for example, variations in the number of circulating hemocytes were recorded in two economically important clam species (ruditapes philippinarum and r. decussatus) after challenge with the pathogenic bacteria vibrio p1 (experiments with both the bivalve species) and after starvation (experiment with r. philippinarum only) (oubella et al., 1993). the number of circulating hemocytes increased significantly in both species after short-term (0 to 72 h) and long-term (0 to 7 days) challenge experiments, whereas one week of fasting caused a marked reduction (by 65 %) in the number of hemocytes in r. philippinarum. to explain these variations, the authors suggested that a reversible migration of hemocytes from the tissues to the hemolymph, or vice versa, occurred in clams (oubella et al., 1993). these cellular mechanisms can be important in the cases of infections, as the capability for hemocyte redistribution in the organism may be interpreted as an increase in the immunosurveillance by providing immunocytes at sites of pathogenic aggression (oubella et al., 1993). experimental infection of molluscs with parasites offers a useful tool for the study of specificity and host immune resistance to parasitic infection. in a recent study, gorbushin and borisova (2014) implanted echinostomatid rediae, himasthla elongata, to the specific iteroparous long-living host, coenogastropod littorina littorea (common periwinkle). neither young nor mature rediae survived in the recipient periwinkles in the course of 30 days post-implantation, suggesting a strong immune response of the host. the strong immune response (production of toxic humoral immune factors, encapsulation of the implants and increased hemocyte counts) was already evident during the first week after implantation. conversely, rediae from the same microhemipopulation showed perfect survival rates in primary in vitro axenic cultures. based on the results obtained, the authors stated that low investment in l. littorea annual reproduction would result in increased investment in self maintenance and immune mechanisms, causing the general resistance to the trematode infestation. this resistance should be relatively higher in long-lived iteroparous gastropods compare to semelparous short-lived molluscs, such as pulmonates (gorbushin and borisova, 2014). regarding starvation, oubella et al. (1993) observed a return to initial hemocyte densities once feeding had resumed in r. philippinarum. since hemocytes play an important role in nutrient transport, an accumulation of hemocytes in storage tissues could allow the utilisation of these metabolic reserves by starved molluscs (oubella et al., 1993). differential food quality was shown to influence markedly phagocytic activity, reactive oxygen species (ros) production, and abundance of freefloating hemocytes in crassostrea gigas and r. philippinarum (delaporte et al., 2003, 2006). in the antarctic bivalve, laternula elliptica, reductions in the number of hemocytes were observed in animals subjected to starvation, compared to constant feeding (husmann et al., 2011). conversely, in the same bivalve species, analyses of immune response genes revealed that most transcripts were 117 more affected by injury (both valves of the animals were cracked and the siphon was cut at two places) rather than starvation (husmann et al., 2014). overall, the genes were upregulated in the hemocytes of young, fed individuals after acute injury, while only minor variations in expression were found in young animals under starvation conditions and in older individuals. the authors suggested that the stress response of l. elliptica depended on the nature of the environmental cue and on age (husmann et al., 2014). two exhaustive reviews on the effects of changing environmental parameters on the mollusc immune response are provided by matozzo and marin (2011) and anisimova (2013). at the cellular level, for example, phagocytic activity is affected by variations in temperature (hégaret et al., 2003; cheng et al., 2004a; monari et al., 2007), salinity (cheng et al., 2004b; matozzo et al., 2007), and air exposure/anoxia (pampanin et al., 2002; matozzo et al., 2005). recently, attention has also been given to the evaluation of the effects of reduced seawater ph (as predicted in a global climate change scenario) on the immune parameters of bivalves (bibby et al., 2008; matozzo et al., 2012). considering that environmental parameters vary seasonally and the reproductive cycle of animals is regulated by these parameters, seasonal and gender-related differences in the function of the immune system have been investigated in molluscs. for example, a seasonal variation pattern in the parameters of hemocyte function was recorded in clams (r. philippinarum) collected from different sites of the lagoon of venice (matozzo et al., 2003). indeed, variations in the functional response of hemocytes appear closely dependent on seasonal variations in both environmental parameters and the physiological status of clams (matozzo et al., 2003). in the same clam species, different hemocyte parameters, such as the total hemocyte count (thc), the hemocyte size frequency distribution, the endocytotic activity, and the activities of lysozyme, acid phosphatase, superoxide dismutase (sod) and catalase (cat), were measured during the prespawning period to assess whether the two sexes reach the spawning period with a different immunosurveillance status (matozzo and marin, 2010). that study demonstrated that gender-related differences in immune parameters can occur in clams during the pre-spawning period and indicated that the hemocytes from females were more active compared with those from males (matozzo and marin, 2010). a relationship between thc and the temperature-dependent reproductive cycle has been found in mytilus galloprovincialis from the lagoon of venice; the hemocyte number was lower in the summer during the spawning period compared with the spring and winter (pipe et al., 1995). in mya arenaria, the percentage of hemocytes with ingested fluorescent particles (indicative of phagocytic activity) and cell viability varied significantly among the sampling sites (the st. lawrence estuary and saguenay fjord, québec, canada), whereas no influence of gender was observed (gagné et al., 2008). however, in the same study, a slight gender-related difference in the number of hemocytes was recorded, with females having somewhat fewer hemocytes compared with males. spawning can be a stressful condition for the immune response of molluscs. in mytilus edulis, a relationship between lowered phagocytic activity and spawning has been observed (fraser et al., 2013). in some cases, spawning can reduce the phagocytic activity of mussel hemocytes by 60% when compared with hemocytes from mussels collected after the spawning phase (fraser et al., 2014). in the same mussel species, the phagocytic activity of the hemocytes from females was significantly reduced after exposing cells to 10-5 m of mercuric chloride. conversely, a significant reduction in the phagocytic activity of the hemocytes from males was recorded at 10-4 m of mercuric chloride, a 10-fold higher concentration, suggesting that the hemocytes from females were more sensitive to metal exposure compared with those from males (brousseau-fournier et al., 2013). other environmental characteristics can strongly affect hemocyte parameters in molluscs. in a recent study, clams (r. philippinarum) were collected for one year in two sites of the lagoon of venice characterised by different environmental conditions: a seaward site close to a lagoon inlet where high hydrodynamism and frequent shipping activity occur, and a landward site with low hydrodynamism and riverine inputs (matozzo et al., 2012). the measured hemocyte parameters highlighted an overall better condition for clams collected from the seaward site; however, no clear differences in contamination levels of sediments were observed between the two sites. this study suggests the unique environmental conditions (differences in salinity, total chlorophyll, sediment grain size and organic matter) of the two sampling sites affected the hemocyte parameters in bivalves. in addition, these results indicate animals experiencing different environmental conditions can respond differently to the experimental exposure to contaminants. to test this hypothesis, clams (r. philippinarum) were collected from two sites of the lagoon of venice with different environmental conditions: marghera, which is characterised by a relatively high contamination level and is a location where clam fishing is restricted, and chioggia, a site inside a licensed area for clam culture with lower contamination levels (matozzo et al., 2013). a number of hemocyte parameters, such as thc, the diameter and volume of hemocytes, and the lysozyme activity in both the hemocyte lysate and the cell-free hemolymph, were measured soon after clam sampling, after 7 days of acclimation in the laboratory and after 1, 3 and 7 days of exposure to copper. the results revealed a persistent difference in the hemocyte response of clams from the two sampling sites before and after exposure to copper, indicating bivalves with a different ecological history respond differently to experimental exposure to contaminants. tide or distance from the shore can also influence mollusc hemocyte parameters. it has been demonstrated that the hemocytes of clams (m. arenaria) exposed for a longer period of time to the air (low tide) are more sensitive to the toxic effects of metals (alix et al., 2013). the spatial distribution of bivalves on the shore (upper, middle and lower) 118 can also affect their immunocompetence. in m. arenaria, animals from upper and middle ranges showed markedly lower phagocytic activity compared with those from the lower range (beaudry et al., 2013). similarly, bivalves (m. arenaria) that are collected closer to the shore can have a higher number of circulating cells compared with clams from beds further offshore (gagné et al., 2009). a relationship between population characteristics and the response of immunomarkers has been demonstrated in clams (m. arenaria). in particular, clam density was significantly correlated with the viability of hemocytes, while residual clam density (clam density not related to distance from the estuary) was correlated with the viability of hemocytes and the phagocytic activity (gagné et al., 2008). in addition, the study demonstrated that when clam density was corrected for salinity differences (estuary), there was a significant correlation with hemocyte activity, phagocytosis and cellular energy expenses. considering that immunocompetence biomarkers are strongly associated with clam population metrics, the authors suggest that the immunomarkers could serve as predictive biomarkers, not only for clam health but also for population health as well. in conclusion, to date, eco-immunology studies on molluscs have largely dealt with both the constitutive immune defences and the effects of pollutants on immunomarkers. according to ellis et al. (2011), the understanding of the effects (also cumulative) of environmental stressors and biotic factors on mollusc immunocompetence is an important topic, especially when considering that interspecific differences in response to the same environmental stressor can often occur among mollusc species and among different invertebrate species in general. consequently, in the field of ecoimmunology, there is a need to increase knowledge on the effects of a wider range of stressors, alone or in combination. this is particularly true in a climate change scenario, as variations in various environmental factors could occur concurrently. a deviation from the optimum for some environmental factors (e.g., temperature, salinity, oxygen, and ph) may have deleterious effects on the physiological performance of animals, including immunosurveillance. a reduced immunosurveillance of animals can in turn have negative consequences for the entire population. in this context, in order to increase the 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of evolutionary ecology. proc. r. soc. lond. 270b: 357-366, 2003. schöne br, fiebig j, pfeiffer m, gleb r, hickson j, johnson ala, et al. climate records from a bivalve methuselah (arctica islandica, mollusca; iceland). paleogeogr. paleoclimatol. paleoecol. 228: 130-148, 2005. sheldon bc, verhulst s. ecological immunology: costly parasite defences and trade-offs in evolutionary ecology. trends ecol evol 11: 317321, 1996. vinkler m albrecht t. phylogeny, longevity and evolution of adaptive immunity. folia zool. 60: 277-282, 2011. wanamaker ad, heinemeier j, scourse jd, richardson ca, butler pg, eiriksson j, et al. very long-lived mollusks confirm 17th century ad tephra-based radiocarbon reservoir ages for north icelandic shelf waters. radiocarbon 50: 399-412, 2008. 121 abstract introduction 158 isj 15: 158-168, 2018 issn 1824-307x research report effect of essential and non-essential elements on cellular immune system of cotton bollworm, helicoverpa armigera hübner (lepidoptera: noctuidae) a baghban, jj sendi*, a zibaee department of plant protection, faculty of agricultural sciences, university of guilan, 41635-1314 rasht, iran accepted april 11, 2018 abstract three concentrations of 12.5, 25 and 50 mg/kg of cadmium and 25, 50 and 100 mg/kg of copper were added to artificial diet and fed to larvae for 7 days. total hemocytes count (thc) at 12.5 mg/kg concentration of cadmium significantly declined, but at 50 mg/ kg of cadmium and all treatments of copper, the thc was significantly enhanced. as for differential hemocytes count (dhc) prohemocytes were reduced at 12.5 mg/kg of cadmium treatment and all concentrations of copper, while granulocytes in all treatments of cadmium were increased. the phenoloxidase activity and nodulation were increased versus entomopathogenic fungi, beauveria bassiana (balsamo) vuillemin and latex beads. the adverse influence of stresses on immune responses of pest can be an effective solution for insect pest control. key words: beauveria bassiana; cadmium; copper; helicoverpa armigera; nodulation; thc; dhc introduction the rapid development of industrialization and urbanization has generated heavy metals contamination globally (zhou et al. 2012). some heavy metals such as cadmium (cd), nickel (ni), arsenic (as), chromium (cr) and lead (pb) have been increasing to dangerous levels for human, plant and animals (sharma and agrawal, 2005) but essential micronutrients; copper (cu), iron (fe), manganese (mn), nickel (ni), and zinc (zn) are required by organisms in low concentrations (epstein, 1972; marschner, 2011). although organisms are able to regulate small amounts of essential metals generally, in excess these metals may become toxic (kabata-pendias, 2010; chaffai and koyama, 2011). among heavy metals, cadmium risks and toxicity are well documented. cadmium accumulation is higher in leaves than other parts of plants (marschner, 1983). accumulation of cadmium in insects that feed on plants have been demonstrated (lindqvist, 1992). cadmium is involved in oxidative stress at the cellular level leading to the production of reactive oxygen (mirčić et al., 2010) which causes many structural and functional disturbances such as decreased stability ___________________________________________________________________________ corresponding author: jalal jalali sendi department of plant protection faculty of agricultural sciences university of guilan 41635-1314 rasht, iran e-mail: jjalali@guilan.ac.ir of the lysosomal membrane. they also cause increased lipid peroxidation (korsloot et al. 2004), and decreased activity of anti-oxidative and detoxification enzymes (lijun et al. 2005, augustyniak et al., 2009). at the organismal level, cadmium affects feeding indices, food consumption and digestibility (fountain and hopkin, 2001, van ooik et al., 2007), secretion of neurohormones (ilijin et al., 2009) and synthesis of hormone receptors (cervera et al., 2006; planelló et al., 2010). copper is a biostatic metal that in organisms has an important role in cytochrome oxidase c (bertini et al., 1994) and the structure of proteins such as prophenoloxidase, hemocyanin and super oxide dismutase (sods); nevertheless high concentration of copper may cause denaturation and dysfunction in proteins (huang et al., 2012). insect immunity is divided into two major defense systems against infectious agents, cellular immune and humoral immune. in cellular immune, different types of hemocytes are active but in humoral immunity, anti-microbial peptides or phenoloxidase (po) have been produced (cerenius and söderhäll, 2004; stanley and miller, 2006). there are indications that the humoral and cellular responses are well coordinated. there is an overlap between them, since several humoral factors affect the hemocytes function and these, in turn, are an important source of many humoral molecules such as phenoloxidase (lavine and strand, 2002; jiravanichpaisal et al., 2006). cellular immunity are phagocytosis, nodulation and encapsulation (lavine and strand, 2002). hemocytes have been 159 fig. 1 the effect of different concentrations of cadmium on thc of h. armigera. treatment columns sharing the same letter are not significantly different under the tukey tests (p≤ 0.05) well studied in lepidopterous larvae such as pseudoplusia includens, manduca sexta and bombyx mori, because they possess an adequate volume of hemolymph and a huge number of hemocytes which makes them a convenient system in which to study these cells. the hemocyte types described in lepidoptera include prohemocytes, plasmatocytes, granulocytes, oenocytoids and spherule cells (strand, 2008). pos are vital enzymes that cause melanization. pos are presented as inactive precursor (ppo) in insect hemolymph and are activated in response to wounding or infection as a part of the innate immune response (cerenius et al., 2008). granulocytes and plasmatocytes are adhesive in nature. after the entrance of invaders to the hemocel, micro-aggregations of granulocytes and plasmatocytes are initiated on microorganisms. this process is started by changing of circulating hemocytes from non-adhesive to adhesive cells that are able to bind to microorganisms (lavine and strand, 2002). these micro-aggregations will eventually lead to the formation of nodules. in the later stages of nodule formation, melanization takes place within the nodules (khosravi et al., 2014). it has been well documented that insect genotype (rantala and roff, 2006), sex and different stage (rantala et al., 2007; jalali and salehi, 2008), quality and quantity of food and feeding indices (rantala et al., 2003; yang et al., 2007; baghban et al., 2014), crowding (wilson et al., 2003) and physical activity (ahtiainen et al., 2006) affect the immune functions of insect. however, the role of immune system versus pollution has received less attention. if pollution affects the immune response (e.g., nodulation) in insects, the enhanced or declined immune response may act as a different behavior resistance against entomopathogenic fungi, bacteria fig. 2 the effect of different concentrations of copper on thc of h. armigera. treatment columns sharing the same letter are not significantly different under the tukey tests (p≤ 0.05) 160 fig. 3 hemocytes from american cotton bollworm h. armigera larvae stained by giemsa for light microscopy observation. plasmatocyte (pl), granulocytes (gr), oenocytoids (oe) and spherolucyte (sph) or parasites. this could affect the parasitism rate in herbivorous insects in metal polluted areas. hence, increasing or decreasing the number of hemocytes and type of them are indicator of response versus heavy metals. in this study, the effect of cadmium and copper were evaluated on total hemocytes count (thc); differential hemocytes count (dhc); po activity and finally immune response versus entomopathogenic fungi, beauveria bassiana, a common biological control agent and latex beads, the latex particles that cause cellular immune response on helicoverpa armigera as an insect model due to its high fertility rate, non-diapausing larvae and ease of rearing on artificial diet. materials and methods experimental insect and treatments the larvae of helicoverpa armigera were collected from tomato farms in astaneh-ye ashrafiyeh city (37°15′35″n 49°56′40″e) in the north of iran. the larvae were reared on artificial diet (powdered cowpea, wheat germ powder, yeast, sorbic acid, ascorbic acid, sunflower oil, formaldehyde and water) (shorey and hale, 1965) in transparent plastic containers (10×5×5 cm) at 26 ± 2 °c, 65 ± 10% relative humidity and a photoperiod of 16l:8d and after rearing them for three generations in the laboratory then the third instar larvae were used for experiments. the chloride salt of cadmium and copper (sigma-aldrich co., usa) were used and dissolved in distilled water to make stock solutions of 2000 ppm. the stock solutions then were diluted to make a series of different concentrations of heavy metals. the selected concentrations of heavy metals were considered as the amounts of these metals in different plants. hence 12.5, 25, and 50 mg/kg diet of cadmium and 25, 50 and 100 mg/kg of copper diet were considered (deng et al., 2004; liu et al., 2007; parizanganeh et al., 2010; nazir et al., 2011). newly hatched larvae reared in a plastic container on artificial diet until first and second instar. the third instar larvae were reared separately due to high cannibalism. heavy metal treatment continued for 7 days until third instar larvae were 24 h old in all assays. total hemocytes count (thc) and differential hemocytes count (dhc) for thc and dhc, the larvae were heat-fixed at 60 °c for 5 min following the method of rosenberger and jones (1960). this will prevent the adhesion of hemocytes to tissues and cause the release them to hemolymph. for thc, 10 µl hemolymph was mixed immediately in an eppendorf with 290 µl of physiological saline (0.098 m naoh, 0.186 m nacl, 0.017 m edta, 0.041 m citric acid, ph 4.5) (amaral et al., 2010). then, the total hemocytes numbers were counted on an improved neubauer hemocytometer. for dhc 5 µl of hemolymph were collected directly on a clean slide and a smear by another slide was made and dried at room temperature. the air-dried smears were stained by 1 to 10 diluted stock giemsa (merck, germany) and after 14 min were washed by distilled water. in order to distinguish cytoplasm from nucleus of hemocytes, 161 fig. 4 the effect of different concentrations of cadmium on dhc of h. armigera. treatment columns sharing the same letter are not significantly different under the tukey tests (p≤ 0.05) smears were dipped in saturated lithium carbonate (lico3) for 5 sec and then dried at room temperature. finally, smears were fixed in canada balsam (merck, germany) and dhc was performed by classifying 200 cells per smear (gujar and kalia, 2005; jalali and salehi, 2008, wu et al., 2016) based on the identification key provided by gupta (gupta, 1979). po activity assay for measuring po specific activity, 10 µl of hemolymph was diluted with 90 µl of ice-cold sterile phosphate buffered saline and then vortexed. samples were frozen at -20 °c for 48h. we used ldopa (sigma-aldrich co., usa) as substrate. samples were centrifuged at 5,000 g at 4 °c for 5 min. then 50 µl of hemolymph-buffer supernatant was mixed with 150 µl of l-dopa (10 mm). po activity was measured at 490 nm during the linear phase of the reaction. specific activity was calculated by dividing absorbance with protein content in hemolymph using a microplate reader (awareness technology inc., florida, usa) (catalán et al., 2012, khosravi et al., 2014). protein determination the method of bradford (bradford, 1976) was used for determining total protein, using bovine serum albumin (bio-rad, munchen, germany) as the standard. beauveria bassiana culture b. bassiana isolate iran403c was grown in sterile petri dishes containing pda (potato dextrose agar) at 25 ± 1 °c. after 14 days, spores were harvested from pda plates with sterile scalpel and distilled water containing 0.01% of tween-80 and finally, 1×104 spores/ ml concentration was adjusted by hemocytometer. immune response assay after 7 days of treatment, larvae were immobilized on ice for 5 min and surface sterilized with 70% ethanol. after injection with 1×104 spores/ml and 1 to 10 distillated latex beads with distilled water by a 10 µl hamilton syringe, the larvae were transferred to rearing jars and were provided with fresh diet. the control larvae were injected with distilled water containing 0.01% of tween-80 (1 µl) alone. effect of fungal spore on nodulation 24 h post injection were determined. hemolymph was collected from each larva, then samples in 3 replicates were poured into a hemocytometer, and the number of nodules was counted (franssens et al., 2006, seyedtalebi, 2017). statistical analysis all data are presented as means ± se and data were subjected to analysis of variance (anova) using sas 9.1 software. differences among treatments were compared using tukey's multiple range tests. differences among means were considered significant at p≤ 0.05. all experiments have three replicates and each replicate with ten larvae. results effect of cadmium and copper on thc the results showed that while the number of hemocytes at 12.5 mg/kg concentration of cadmium was significantly decreased but at 25 and 50 mg/kg concentration of cadmium it was significantly increased compared to control (f= 18.56; df= 3; p≤ 0.05) (fig. 1). on the contrary the thc after copper treatment showed an increasing trend and in all concentrations it was significantly higher than control (f= 16.46; df= 3; p≤ 0.05) (fig. 2). 162 fig. 5 the effect of different concentrations of copper on dhc of h. armigera. treatment columns sharing the same letter are not significantly different under the tukey tests (p≤ 0.05) effect of cadmium and copper on dhc five forms of hemocyte were observed (fig. 3). the dhc result demonstrated significant decline in prohemocytes at 12.5 mg/kg concentration of cadmium compared with the control (f= 7.07; df= 3; p≤ 0.05). while the number of plasmatocytes were significantly decreased (f= 26.7; df= 3; p≤ 0.05) and that of granulocytes were increased in all concentrations of cadmium compared with the controls (f= 12.5; df= 3; p≤ 0.05). on the other hand no changes in the number of oenocytoids and spherule cells were observed between treatments and their controls (fig. 4). the prohemocyte dhc results in copper treatments declined significantly compared with the control (f= 11.94; df= 3; p≤ 0.05) but in all other hemocyte types no significant differences were seen between treatments and the control (fig. 5). effect of cadmium and copper on po activity on the measurements of po activity, a key enzyme in immunology and immune responses of all insects showed that there was significant enhancement in its activity at 50 mg/kg concentration of cadmium compared with the control but no significant difference was observed between 12.5, 25 mg/kg concentrations of cadmium and the control (f= 24.37; df= 3; p≤ 0.05) (fig. 6). in case of copper treatments the activity of po did not show significant changes and remained same in all treatments compared with control (f= 1.6; df= 3; p≤ 0.05) (fig. 7). fig. 6 the effect of different concentrations of cadmium on po enzymes activity of h. armigera. treatment columns sharing the same letter are not significantly different under the tukey tests (p≤ 0.05) 163 fig. 7 the effect of different concentration of copper on po enzymes activity of h. armigera. treatment columns sharing the same letter are not significantly different under the tukey tests (p≤ 0.05) effect of cadmium and copper on nodule formation the formation of nodules as an indicator of cellular response was also studied (fig. 8). there were significant differences in the numbers of nodules after injection of b. bassiana and latex beads (f= 28.66; df= 3; p≤ 0.05 and f= 27.95; df= 3; p≤ 0.05, respectively) at maximal concentration of cadmium compared to control (fig. 9). copper treatment versus b. bassiana injection in all concentrations of copper showed no significant differences compared to control (f= 4.3; df= 3; p≤ 0.05). however, after injection of latex bead in all concentrations of copper, the nodule formation was sharply increased compared to control. treatments with 25 and 100 mg/kg concentration of copper showed significant differences compared with the control (f= 7.77; df= 3; p≤ 0.5) (fig. 10). discussion increase or decrease in the total number of blood cells are considered a common response to stressors present in the environment (perez and fontanetti, 2011). pipe and coles (1995) demonstrated that decrease in the total number of hemocytes under stress condition could be a consequence of cellular lysis, reduced replacement or migration of the cells from the circulation to the tissues. our result showed that at 12.5 mg/kg concentration of cadmium the thc was significantly decreased, possibly because hemocytes in low concentrations of cadmium move from circulation to the tissues. it seems at low concentration of cadmium, midgut epithelium was damaged thus causing the migration of hemocytes to this tissue fig. 8 nodule formation against b. bassiana spore in american cotton bollworm h. armigera larvae 164 fig. 9 the effect of different concentrations of cadmium on immune response (nodulation) of h. armigera versus entomopathogenic fungi, b. bassiana and latex bead. treatment columns sharing the same letter are not significantly different under the tukey tests (p≤ 0.05). columns with the same color are compared together henceforth leading to decline in the number of circulating hemocytes (perez and fontanetti, 2011). some studies have shown an increase in the number of hemocytes in midgut tissue of exposed animals that indicated a tissue injury with inflammatory process (de godoy and fontanetti, 2010; nogarol and fontanetti, 2010; perez and fontanetti, 2011). the thc increase under heavy metal stress can be explained by their potential to produce metallothioneins under exposure to heavy metals (roesijadi et al. 1997). metallothioneins are cysteine rich proteins that bind to heavy metals and scavenger them (kägi and kojima, 1987; demoor and koropatnick, 2000). in addition, some other studies have shown that hemocytes could transport heavy metals intracellularly (in lysosomal vesicles or within the cytoplasm) (robinson and ryan, 1988). our previous report (baghban et al., 2014) showed that the relative growth rate and the amount of lipids was reduced due to cadmium but reverse was true in case of as for copper treatment. cadmium produced free radicals and it is possible at high concentrations of cadmium, hemocytes were activated due to high oxidative stress. hence, migrated to injured epithelial cells and produced metallothioneins. it seems in all concentration of copper h. armigera have not a big challenge to heavy metal stress and increasing in thc was a response to enhancing growth (body size) (ghasemi et al., 2013) and/or copper transport to fat body. our previous report has shown significant increase of lipid storage and relative growth rate (rgr) in copper treatments (baghban et al., 2014). in the dhc assay, our results showed that at 12.5 mg/kg concentration of cadmium, the mean counts of prohemocytes were declined as the hemocytes developed into other hemocytes types. however, when the concentration of cadmium reached to toxic levels (50 mg/kg concentration of cadmium), hematopoietic organs produced stem cells (prohemocytes) for transformation to other hemocytes especially granulocytes. probably granulocytes through their granules transported cadmium intracellularly and/or migrated to the injured tissue. in a similar study victor (victor, 1993) reported in paratelphusa hydrodromous herbst that cadmium induced change in the ratio of hemocytes thus increasing granulocytes and prohemocytes number. our results also showed that in all concentrations of copper the percentage of granulocytes show no significant differences but prohemocytes were significantly reduced compared to the control. based on thc and dhc results, we understand that the stress of copper may not have reached to the point causing significant changes in percentage of involved hemocytes and granulocytes or other hemocytes that normally react to increasing amounts of copper in hemolymph and carrier copper and/or produce metallothioneins. present results showed that po enzyme activity significantly increased at 50 mg/kg concentration of cadmium but at 12.5 and 25 mg/kg of cadmium and all concentrations of copper no differences were observed. our data support previous findings that have shown increasing po activity in epirrita autumnata borkhausen larvae after exposure to heavy metals (van ooik et al., 2007; van ooik and rantala, 2010). dubovskiy et al. (2011) mentioned that probably enhanced po activity may be the result of oxidative damage of the midgut epithelial cells and discharge of some immune mediators into the hemocel. this hypothesis is supported by the reports that the rate of hemocytes apoptosis in spodoptera litura fabricius larvae increases with an 165 fig. 10 the effect of different concentrations of copper on immune response (nodulation) of h. armigera versus entomopathogenic fungi, b. bassiana and latex bead. treatment columns sharing the same letter are not significantly different under the tukey tests (p≤ 0.05). columns with the same color are compared together increasing nickel concentration in the diet (xia et al., 2005). a radical-related and antioxidant-dependent process such as apoptosis or oxidative stress can induce the level of melanization (dubovskiy et al., 2011). on the other hand, our results showed treatment with cadmium could change thc and dhc. the fact that humoral and cellular immunity are closely dependent, has been demonstrated in several studies (lavine and strand, 2002; jiravanichpaisal et al., 2006). however, some processes such as apoptosis that are radical-related and antioxidant-dependent ultimately cause changes in hemocyte counts. these changes have direct effect on immune response, as in dubovskiy et al. (2011). there exists discrepancy between reports by various researchers on the subject which could be related to the nature of heavy metals, their concentrations incorporated, exposure time and the species chosen for the study (lorenzon et al., 2001). there is a positive correlation between immune response and the number of circulating hemocytes (rantala et al., 2000). this reason probably is the strongest reason for the present result that indicates the high dose of cadmium cause enhanced nodulation reaction in the larvae of the cotton bollworm. several studies have also reported that pollution by heavy metals can increase immune response (encapsulation) of insects (van ooik et al., 2007; van ooik and rantala, 2010; dubovskiy et al., 2011). here we considered both po activity and nodulation showing higher activity at maximal dosage of cadmium. some studies have shown low-level and short-term metal exposure could increase immune responses, whereas higher concentrations or a longer exposure may inhibit the very same response (van ooik et al., 2008). however, in the present study it was found that at high levels of cadmium and 25 and 100 mg/kg concentrations of copper, immune response were enhanced. mirhaghparast et al. (2013) mentioned that the maximum number of nodules occurred 12 h after b. bassiana injection and 24 h after latex bead injection. it seems that due to enhanced oxidative stress in the higher concentration of cadmium immune functions may delay the responses to b. bassiana compared to treatments with copper. the duration of treatment was 7 days and the response of h. armigera to heavy metal pollution showed itself in the changes in number of hemocytes. this response could affect the infection of entomopathogenic fungi or latex bead and enhanced nodulation rate. however, if the duration of exposure or if the concentration of heavy metals extends, the immune response might be differently affected. conclusion this study clearly showed that the immune responses of h. armigera are affected by cadmium and copper. immune response of insect is affected by various factors which could alter their performance. the present results and other studies showed immune responses (like nodulation, encapsulation, and po activity) are affected by environmental stress (in this case heavy metals pollution) and this effect was imposed variously. the nature of the contamination, the duration of exposure to pollution, contamination concentrations and animal species variously affect immune responses. whether these responses favor organism or disfavor it has to be pointed and incorporated in our pest manipulation strategies. acknowledgments the deputy of research, university of guilan, which is greatly appreciated, supported this research. we thank dr. roya khosravi and miss malahat mojarab for their technical assistance and mr. ali assaran for reading the text. 166 references ahtiainen jj, alatalo rv, kortet r, rantala mj. immune function, dominance and mating success in drumming male wolf spiders hygrolycosa rubrofasciata. behav. ecol. sociobiol. 60: 826-832, 2006. amaral imr, neto jfm, pereira gb, franco mb, beletti me, kerr we, et al. circulating hemocytes from larvae of melipona scutellaris (hymenoptera, apidae, meliponini): cell types and their role in phagocytosis. micron. 41: 123129, 2010. augustyniak m, babczynska a, augustyniak m. does the grasshopper chorthippus brunneus adapt to metal polluted habitats? a study of glutathione‐dependent enzymes in grasshopper nymphs. insect sci. 16: 33-42, 2009. baghban a, sendi jj, zibaee a, khosravi r. effect of heavy metals (cd, cu, and zn) on feeding indices and energy reserves of the cotton boll worm helicoverpa armigera hübner (lepidoptera: noctuidae). j. plant. prot. res. 54: 367-373, 2014. 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epirrita autumnata: a possible causal trigger for the crash phase of population cycle, pp. 89-96. in, annales zoologici fennici,. helsinki: suomen biologian seura vanamo, 2007. zhou j, shu y, zhang g, zhou q. lead exposure improves the tolerance of spodoptera litura (lepidoptera: noctuidae) to cypermethrin. chemosphere 88: 507-513, 2012. 338 isj 15: 338-345, 2018 issn 1824-307x short communication distinct immuneand defense-related molecular fingerprints in sepatated coelomocyte subsets of eisenia andrei earthworms k bodó1, d ernszt2, p németh1, p engelmann1,* 1department of immunology and biotechnology, clinical center, medical school, university of pécs 2department of physiology, medical school, university of pécs accepted september 4, 2018 abstract during phylogenesis different types of immunocytes such as amoebocytes and eleocytes have developed in earthworms to defend the host against microbial pathogens. previously we applied a cell sorting-based approach to untangle the morphological and functional properties of these aforementioned coelomocyte subsets. in order to compare their constitutive gene expression patterns, cell-sorting was performed and followed by semiquantitative rt-pcr in the distinct, separated coelomocyte subpopulations of unmanipulated eisenia andrei earthworms. we targeted a variety of genes with diverse functions ranging from pattern recognition through intracellular signaling to oxidative stress. several immune-related genes (ccf, tlr, lumbricin, lurp, myd88) were only manifested in the amoebocytes. in contrast, other immune response genes (lysozyme, lysenin), lysosomal hydrolases (cathepsin l and cathepsin c) and cystatin b were expressed in both subpopulations. in addition, cell signaling molecules (myd88, pkc1) and oxidative stress-related genes (cu/znsod, mnsod) were mainly observed in amoebocytes, while other stress-related genes (cd-metallothionein, catalase) were apparent in both subsets. we conclude that these characteristic differences of the molecular signatures manifest in the functional heterogeneity of distinct coelomocyte subtypes. key words: eisenia andrei, coelomocytes, cell sorting, gene expression, immune response, oxidative stress introduction evolutionary conserved immune mechanisms are reported from diverse invertebrate organisms (loker et al., 2004). a surprising complexity and close cooperation between cellular and humoral immune components can be observed in several invertebrate models including earthworms (cooper et al., 2002; cooper and roch 2003; bilej et al., 2010; engelmann et al., 2016b). earthworm coelomocytes are divided into amoebocyte and eleocyte subpopulations. similarly to other invertebrate immunocytes, these cells are derived from the mesoderm (eleocytes are considered to be originated from the gut surfacelocated, liver equivalent chloragogenous tissue), and possess various functions during the immune response (engelmann et al., 2005). in this regard, ___________________________________________________________________________ corresponding author: péter engelmann department of immunology and biotechnology clinical center, medical school, university of pécs pécs, h-7643, szigeti u. 12, hungary e-mail: engelmann.peter@pte.hu amoebocytes are mainly involved in the phagocytosis and encapsulation (fuller-espie, 2010), while eleocytes have no phagocytic properties, but they produce a handful of bioactive molecules (stein et al., 1977; valembois et al., 1985). recently we applied a cell-sorting-based approach to separate these distinct coelomocyte subsets upon their light-scatter properties (from the perspective of physical parameters; size and granularity). after separation we characterized their differences in morphological, cytochemical, functional and lectin-binding properties (engelmann et al., 2016a). in the last two decades several immune proteins have been identified in earthworms; however, little is known about their differential gene expression in the coelomocyte subgroups (bilej et al., 2010; engelmann et al., 2016b). aware of the phenotypic and functional differences in the coelomocyte subsets, we aimed to analyze the distinct expression patterns of several immune and stress-related target genes in the separated amoebocytes and eleocytes. mailto:engelmann.peter@pte.hu 339 table 1 list of primers were applied for semi-quantitative rt-pcr experiments target gene gene bank accession # sequence (5'-3')a amplicon size (bp) tlr jx898685 att gtg tca aac gcc ttc gc 123 gtc ggc gat ctc ttc caa ca ccf af030028 cat taa gcc gac gtt gct gg 145 cgt cct gta gca tcc gtt gt lbp/bpi jq407018 ggt tcg acc tcc gac gat ac 107 ggt caa cag ggc gtc cat ta lysozyme dq339138 gtc gca tgg atg tcg gat ct 120 gcg agc agt cca tct gag tt lumbricin kx816866 act cgg aac gca aga acc aa 139 ggt tct gcg tga cct cct tc lurp kx816867 ggt cga gag aat caa ccc aac ta 133 ctt gtg agc gat gtc ggc ta lysenin d85846 tga tcc aca ctg gtg ctt cc 117 cag gtg cca agg aga aga ag myd88 eh670202 tgc gag tac agg ctc gtt aac 100 cgt gca gat gtg gtt tag ga mekk 1 eh672240 caa gga acg atc cca ttc at 147 gta tca tgg tgc aac caa cg pkc1 dq286716 ttt tat gcg gcc gaa gtc a 120 gtc ggc gat ttt gca gtg a mt aj236886 ctt gtt gct gca caa act gc 129 ttt cca cat ttg ccc ttc tc catalase dq286713 tac aaa ctg gtg aac gcc ga 139 aaa ggt cac ggg tcg cat ag cu/znsod kr106132 tgc caa gtt tga agt gac gg 103 ttt gcc aag atc gtc cac ca mnsod ku057379 ccg aag aaa agc tgg ctg aa 91 tgt cct ccg ccg ttg aat cystatin b bp524680 tgg agg gga tgc ttt gca tt 123 acg cag aca agg tac gaa ga cathepsin b ho001247 tcc tgc ctt tcc aga ttc att t 90 gaa cca cag gag ccc tga tc cathepsin c gr228740 cgg cta ctt ccg cat cgt t 120 agc gcc tgc tca gaa gga cathepsin l ey892565 caa cgg ctg ttt cct atc caa 110 gaa aac aca cga tgc aat gca coi hq534065 gga ttt gga aac tga ctt c 312 tcg ttc tag tcg aag ccc ac 18s ay365460 att aag cca tgc atg tct aag cac 135 ctt tgt ggc atg tat tag ctc cag rpl17 bb998250 gca gaa ttc aag gga ctg ga 159 ctc ctt ctc gga cag gat ga β-actin jq038870 atg tgg atc agc aag cag gag ta 90 atc gcc gag atc gga atc tt aupper and lower sequences represent forward and reverse primers materials and methods earthworm husbandry adult eisenia andrei earthworms were maintained in breeding stocks at standard conditions (molnár et al., 2012). prior to coelomocyte harvesting, earthworms were placed onto moist tissue paper allowing defecation to avoid contamination during coelomocyte collections. coelomocyte harvesting coelomocytes were isolated as we described earlier (engelmann et al., 2004) and enumerated by 0.14% trypan-blue dye-based exclusion. cell sorting and flow cytometry collected coelomocytes were resuspended in lumbricus balanced salt solution (lbss) (engelmann et al., 2005) supplemented with 1% fetal bovine serum (fbs, biowest, nuaillé, france) and 5 mm edta (sigma-aldrich, hungary) to prevent cell aggregation. coelomocytes were sorted according to their basic forward and side scatter (fsc/ssc) characteristics reflecting their cell size and granularity, respectively. sorting procedure was performed by a facsaria iii (bd biosciences) cell sorter as we described earlier (engelmann et al., 2016a). the efficacy of sorting and the coelomocyte viability was controlled by 7-amino-actinomycin d (7-aad) using a facscalibur flow cytometer. 340 hematoxylin-eosin staining sorted coelomocyte subsets (80 µl of 5 x 105/ml) were spread onto glass sides using cytospin 3 (shandon, thermoscientific, waltham, ma, usa) apparatus. hematoxylin-eosin staining was employed following standard protocols. rna isolation, cdna synthesis and semiquantitative rt-pcr total rna was isolated from unseparated coelomocyte, sorted amoebocyte and eleocyte samples using nucleospin® rna isolation kit (macherey-nagel, düren, germany) according to the manufacturer’s protocol. the quality and quantity of rna samples were measured by nanodrop 1000 spectrophotometer (thermoscientific). following dnase i digestion (sigma-aldrich) the reverse transcription reaction was performed by hi-capacity reverse transcription kit applying random hexamers (thermoscientific). dnase i-treated total rna was reverse transcribed and subsequently used in the pcr reactions. gene specific primers were designed based on the available sequences from ncbi genbank database and their major characteristics are detailed in table 1. the following pcr conditions were applied: an initial denaturation step at 95 oc for 10 min, followed by 35 cycles of denaturation at 95 oc for 30 s, annealing at 54 oc for 30 s, and elongation at 72 oc for 30 s. amplification cycles were terminated by a final extension at 72 oc at 10 min. finally, pcr mixtures were analyzed on 1% (w/v) agarose gel, and pcr products were visualized by gelred (biotium, inc., fremont, ca, usa) dye. gel pictures were photographed by geldoc xr system (biorad, hercules, ca, usa). results and discussion pattern recognition receptors (prrs) are attributed to separated amoebocytes to analyze the distinct gene expression patterns we separated the coelomocyte subpopulations based on their physical manifestation (size and granularity) (fig. 1a). postsort cell viability measurements (7-aad staining) indicated a high survival rate (82-85%) for sorted amoebocytes, while we were not able to evaluate the ratio of alive/dead eleocyte due to their high autofluorescence. hematoxylin and eosin staining was performed to check the efficacy of the sorting process. majority of the sorted population was composed by hyaline amoebocytes (fig. 1b), whereas a small percentage of granular amoebocytes appeared as well. separated eleocytes (fig. 1c) were easily perceptible by their small nucleus and the cytoplasm filled with chloragosomes. fig. 1 preand post-sorting analyses of coelomocyte subsets and cytochemical properties of sorted populations. total coelomocyte population (a) was separated to amoebocyte (b) and eleocyte (c) subpopulations upon their physical manifestations. post-sort analyses demonstrated that amoebocytes were mainly undamaged; however, eleocytes showed a certain fragility evidenced by the increased amount of debris. hematoxilin-eosin staining revealed mixed total coelomocytes prior to sorting while homogenous amoebocytes (b) and eleocyte subpopulations (c) can be observed following the separation. scale bars: 100 µm. representative dot-plots are presented from three independent experiments. 341 innate immunity operates with a panel of prrs to discriminate between non-self and self structures. recently, some unique and evolutionary conserved prr molecules have been identified in e. andrei earthworms (bilej et al., 2010; engelmann et al., 2016b). first, we investigated the expression of pattern recognition receptor (prr) genes including coelomic cytolytic factor (ccf), toll-like receptor (tlr), and lps-binding protein/bacterial permeability-increasing protein (lbp/bpi). ccf is an unique lps, peptidoglycan and β-1,3glucan/n,n’-diacetylchitobiose-binding protein that is expressed at higher level in the chloragogenous tissue and lower level in large coelomocytes (beschin et al., 1998). in the case of coelomocyte subsets, we found that ccf was only expressed in separated amoebocyte subpopulation, but it was not present in sorted eleocytes (fig. 2a). first evidence of annelid tlrs was emerged from the analyses of polychaete and hirudean species (davidson et al., 2008; cuviller-hot et al., 2011). shortly, the coding sequence of tlr was identified in e. andrei (eatlr). eatlr expression level was relatively low in coelomocytes (škanta et al., 2013) and -according to our observationonly occurred in amoebocytes (fig. 2a), however its presence is not restricted exclusively to immunocompetent tissues (škanta et al., 2013; engelmann et al., 2016b). further genomic investigations have shown the high diversity of tlrs in annelid earthworms (fjøsne et al., 2015). recently, a homologue of evolutionarily conserved lbp/bpi molecule was isolated from e. andrei. the highest expression level of ealbp/bpi was observed in coelomocytes, seminal vesicles, while the lowest level appeared in the intestine (škanta et al., 2016). we observed that lbp/bpi expression only occurred in amoebocytes, but not in eleocytes (fig. 2a). these findings confirmed the notion that molecular recognition of pathogens is dedicated mainly to the amoebocyte subpopulation. in comparison to the earlier functional studies (valembois et al., 1985; valembois and lasségues, 1995), our results support the concept that amoebocyte subpopulation is priorly involved in the pathogen-triggered phagocytic response. distinct antimicrobial molecular fingerprints of coelomocyte subsets nowadays, antimicrobial proteins (amps) are recognized as the first line of defense against microbial pathogens (boman, 1991; zasloff, 2002). lysozyme is a highly conserved amp present in many different organisms ranging from plants to human. previous investigations have revealed that e. andrei lysozyme represents strong sequence similarity with other invertebrate lysozymes (josková et al., 2009). furthermore its expression is increased in coelomocytes upon gram-positive and gram-negative bacteria exposure. we found that both separated coelomocyte subpopulation expressed this amp, however we observed a more augmented lysozyme expression in separated amoebocytes compared to eleocytes (fig. 2b). lumbricin is a distinctive earthworm amp that was initially isolated from lumbricus rubellus. so far several additional lumbricin homologues have been described from other annelid species (cho et al., 1998; wang et al., 2003; schikorski et al., 2008; li et al., 2011). its expression was observed in several tissues, but not in coelomocytes (li et al., 2011). recently, we have identified the coding sequence of lumbricin and its novel-related peptide (lurp) in e. andrei. lumbricin and lurp show close relationship with other lumbricin homologues (bodó et al., 2019). additionally, we observed that lumbricin and lurp exert ubiquitous expression in several earthworm tissues (including coelomocytes), but their highest expression was evidenced in the foregut. among other tissues lumbricin and lurp expression was the lowest in coelomocytes. as for their distribution in the subpopulations we observed that only amoebocyte subpopulation turned out to be weakly positive, but eleocytes were negative for these genes (fig. 2b). a species-specific bioactive molecule lysenin has been described from eisenia earthworms. lysenin protein family consists of sphingomyelin(and phosphocholine)-binding molecules, and possess diverse biological activities (cytotoxicity, antimicrobial activity, and opsonization) (engelmann et al., 2016a). our immunohistochemical analysis showed that mostly eleocytes were the lysenin expressing cells (opper et al., 2013), in contrast to previous observations where chloragocytes were suggested as the major players in lysenin production (ohta et al., 2000). lysenin expression was manifested in both subpopulations (fig. 2b) that is concordant with our previous flow cytometrybased observations (opper et al., 2013). conserved signaling molecules in coelomocyte subsets our knowledge is very limited concerning on the intracellular signaling in earthworm immunity (engelmann et al., 2011). we gained recent knowledge of different signaling pathways and we tested their expression pattern in the separated coelomocyte subsets. certain signaling pathways such as mapk cascade are fundamental and evolutionarily conserved; since several publications are available from various organisms (sakaguchi et al., 2004; ragab et al., 2011). hayashi et al., 2012 observed down-regulation of mekk1 level in silver nanoparticle (agnp)-exposed eisenia coelomocytes. we measured that mekk1 expression is present in the isolated amoebocytes, besides we found a very weak signal in the eleocyte population (fig. 2c). innate immunity is largely dependent on the engagement of tlrs. following pamp recognition intracellular molecular events are initiated by the cytosolic components of tlr signaling pathway, one such is the myd88. hayashi et al., 2012 have observed a delayed induction of myd88 gene in coelomocytes upon agnp exposure. myd88 was only expressed in the amoebocyte subpopulation that is relevant with the amoebocyte-restricted tlr expression (fig. 2c). protein kinase c (pkc) has fundamental functions in cellular homeostasis, and its role is well implicated in the immune response (larsen et al., 2002). earthworm pkc1 and pkc2 were recently 342 fig. 2 expression patterns of (a) pattern recognition receptors; prrs, (b) antimicrobial peptides amp, (c) signaling pathway genes, (d) metal-, oxidative stress-induced molecules, (e) hydrolytic proteases, and (f) housekeeping genes in coelomocytes of eisenia andrei. (a) tlr, ccf, and lbp/bpi; (b) lysozyme, lumbricin, lurp and lysenin; (c) mekk 1, myd88, and pkc1; (d) mt, cat, cu/znsod and mnsod; (e) cyst b, cath b, cath c, and cath l; (f) coi, 18s, 7 rpl17 and β-actin. total coelomocytes (t), and separated eleocytes (e) and amoebocytes (a), with h2o as pcr negative control. representative images are presented from three independent experiments. partially cloned (brulle et al., 2006). homa et al., (2013) found that a phorbol ester (pma), a potent activator of pkc caused the proliferation of earthworm coelomocytes. interestingly, in the course of in vitro pma administration we observed that pkc is not involved in the ca2+-dependent activation of coelomocytes (opper et al., 2010; engelmann et al., 2011). among coelomocyte subgroups only the separated amoebocytes evidenced the expression of pkc (fig. 2c). in fact, the amoebocyte subpopulation has a crucial role in the pathogen recognition, and in the downstream inflammatory response evidenced by the selective expression of signal transduction molecules and antimicrobial factors. expression of oxidative, metal stress genes in separated coelomocyte subsets in addition to immune response-related genes, we examined the expression patterns of other defense-related genes involved in metaland oxidative stress. metallothioneins (mt) are intensively studied metal-sequestrating proteins and ubiquitously expressed in a wide variety of organisms including earthworms (calisi et al., 2014; kowald et al., 2016). in recent years, earthworms are frequently applied as a sentinel organism to evaluate metal contaminations in soil (calisi et al., 2014). homa et al., (2005) previously described that e. fetida coelomocytes are able to accumulate various metal ions. earlier results demonstrated that 343 mt expression is mostly attributed to chloragocytes (morgan et al., 2004); however we found that mt manifestation occurred in both separated amoebocyte and eleocyte (free-floating chloragocyte) subpopulations. in contrast to the previous data we observed weak signal in eleocytes, while amoebocytes had a stronger mt expression (fig. 2d). all living organisms possess a diversity of antioxidant defense mechanisms (wang et al., 2015). catalase (cat) is one conserved key enzyme of oxidative stress, and it exists in many different cell types including earthworm coelomocytes (brulle et al., 2006). cat expression was observed in both sorted coelomocyte subpopulations (fig. 2d). amoebocytes are involved in the early immune response against pathogens, but probably both subpopulations participate in maintaining the normal cellular homeostasis. another oxidative stress-related enzyme, cu/zn-sod has been recently cloned and characterized in e. fetida (xiong et al., 2012). the predicted amino acid sequence was excavated that genetic distance of cu/zn-sod in e. fetida was far from other invertebrate sod molecules. indeed, it showed strong sequence similarity with homologue sequences from tubifex tubifex and l. rubellus (xiong et al., 2012). in addition, the sequence of mnsod has been recently assessed in e. andrei (roubalová et al., 2018). interestingly, its role in innate immune responses now has been elucidated (wang et al., 2015). in contrast to mt and cat expression cu/zn-sod and mnsod were only manifested in separated amoebocyte subpopulation (fig. 2d). indeed, following the phagocytosis the intracellular “killing” in amoebocytes is mediated by the free reactive oxygen (and nitrogen) species that needs to be terminated by certain antioxidative enzymes (e.g. cat, sod). hydrolytic endopeptidases and their inhibitor are present in coelomocyte subsets cathepsins (cath) are ubiquitous lysosomal proteases involved in many aspects of the cell life cycle. cath b, cath l and cath c have been identified in several invertebrate organisms. undoubtedly, cath b is one of the most typical member of this molecular family that takes essential part in the immune response against bacterial infections (balaji et al., 2002). cath b is involved in the regulation of apoptosis, and this lysosomal protease is implicated to be involved in the immune mechanisms of the echinoderm, apostichopus japonicus (chen et al., 2016). cath l is also identified in several invertebrates including the leech theromyzon tessulatum. cath l was involved in the phagocytic responses of leeches (lefebvre et al., 2008). cystatin b (cyst b) is an endogenous cathepsin inhibitor, which localization was observed in the cytosol, mitochondria and nucleus (kopitarjerala, 2015). previously, cystatin b gene was described in the leech t. tessulatum, and upregulated after bacterial challenge (lefebvre et al., 2004). interestingly, leech coelomocytes possess a differential expression of cathepsin l (in chloragocytes and amoebocytes) and cystatin b (only in chloragocytes) (lefebvre et al., 2008). according to our results all of the observed cathepsins (cath b, c and l) were present in the sorted amoebocyte subpopulation, while only cath c is absent from the sorted eleocytes (fig. 2e). interestingly, cath c expression evidenced a low expression in total coelomocytes, while it appeared relatively higher in isolated amoebocytes. the inhibitor of these lysosomal proteases, cyst b was expressed in both coelomocyte subpopulations (fig. 2e). their immune function in e. andrei is still unknown; however we cannot rule out the option that these proteases might be the possible regulators of lysenin-mediated cell lysis and also involved in the phagocytic machinery (engelmann et al., 2016a). we have chosen four „housekeeping” genes to prove the intact rna quality of the sorted coelomocyte subsets. all of tested genes including coi, 18s, rpl17 and β-actin expressed in both populations, however rpl17 and β-actin genes showed a lower level in eleocytes (fig. 2f). conclusions taken together, hereby we report initially the differential expression patterns of immune and defense-related genes in sorted coelomocyte subsets of e. andrei. our results verify the previously observed cytochemical, immunological and functional differences of coelomocyte subsets at the molecular level. accordingly, amoebocytes are the main effector cells participating in pathogen recognition, and elimination. eleocyte subpopulation is mainly involved in the stress response and production of bioactive molecules. these results provide fine details about the substantial molecular functions of separated coelomocyte subsets. acknowledgement we acknowledge the financial support of medical school research foundation (pte-áokka 2017/04), university of pécs, the ginop-23215-2016-00050 and efop-361-16-2016-00004 grants. p.e. was supported by the jános bolyai research scholarship of the hungarian academy of sciences. the present scientific contribution is dedicated to the 650th anniversary of the foundation of the university of pécs, hungary. references balaji kn, schaschke n, machleidt w, catalfamo m, henkart 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cloning, characterization of copper/zinc superoxide dismutase and expression analysis of stress-responsive genes from eisenia fetida against dietary zinc oxide. comp. biochem. physiol. 155c: 416-422, 2012. zasloff m. antimicrobial peptides of multicellular organisms. nature. 415: 389-395, 2002. microsoft word isj385 isj 12: 188-194, 2015 issn 1824-307x review presence and conservation of the immunoglobulin superfamily in insects: current perspective and future challenges m mandrioli1, m monti2, r tedeschi2 1department of life sciences, university of modena and reggio emilia, modena, italy 2department of agricultural, forest and food sciences, university of turin, turin, italy accepted july 6, 2015 abstract numerous proteins that contain a bona fide immunoglobulin domain have been identified in the last decade showing that immunoglobulin-like proteins are quite common in metazoans. in particular, recent surveys identified more than 140 immunoglobulin-like proteins in drosophila melanogaster, anopheles gambiae and bombyx mori. a well-studied example of immunoglobulin-like protein is the drosophila down syndrome cell adhesion molecule (dscam) that, accordingly to comparative molecular analyses, showed a high conservation in diptera, hymenoptera and coleoptera, together with a conserved presence of alternative splicing that permitted insects to possess an unsuspected molecular complexity of their innate immune system. at a functional level, immunoglobulin-like proteins seem to be capable of reacting to pathogen challenges and may contribute to the defense against infection so that they are candidates as immune effector molecules in insects. preliminary findings on insect-borne plant and animal diseases suggest a possible role of the immunoglobulin-like proteins in the vectorial capacity. key words: immunoglobulin-like proteins; insects; innate immunity; non-self recognition   introduction the evolution of immunity has been frequently regarded as a sort of tinkering resulting in jawed vertebrates (gnathostomes) in the presence of conserved “building blocks”, including immunoglobulins, t-cell receptors (tcrs) and polymorphic mhc class i and class ii molecules, as well as a thymus and compartmentalized secondary lymphoid tissues (zapata and amemiya, 2000). a relevant role has been also played by the evolution of the recombination-activating gene (rag)mediated v(d)j recombination and the somatic hypermutation of immunoglobulin genes that brought to the synthesis of hypervariable immunoglobulins (igs) that can bind to their nominal antigens with exquisite specificity and neutralize the harmful effects of non-self antigens (zouali, 2001; flajnik, 2002). urochordates and cephalochordates do not have an adaptive immune system involving the somatic rearrangement of the antigen receptor genes so that the status of their immune system ___________________________________________________________________________ corresponding author: mauro mandrioli department of life sciences university of modena and reggio emilia via campi 213/d, 41125 modena, italy e-mail: mauro.mandrioli@unimore.it seems to reflect a “primitive” pre-rag stage. in particular, an immunological “big bang” of the adaptive immunity occurred in an ancestor of jawed vertebrates, where the insertion of a transposon into an immunoglobulin superfamily gene member initiated the antigen receptor gene rearrangement via the rag recombinase (bernstein et al., 1996; agrawal et al., 1998; hiom et al., 1998; schatz, 1999; flajnik, 2002, 2014). interestingly, numerous proteins that contain bona fide immunoglobulin domains have been identified in invertebrates confirming that gnathostome igs evolved from previously present molecules that gained a primary role in immunity (pancer et al., 1998; blumbach et al., 1999; azumi et al., 2003). indeed, proteins containing immunoglobulin-like domains are frequently involved in activities other than defense in invertebrates, and just some of them participate to immune responses, as already reported in cephalochordates (cannon et al., 2002; haire et al., 2004) and for the “molluscan defense molecules” identified in the pond snail lymnaea stagnalis (sun et al., 1990; hoek et al., 1996). even though none of the invertebrate molecules that bear an immunoglobulin-like domain resembles gnathostome antibodies, the fibrinogen-related proteins (frep) from the freshwater snail biomphalaria glabrata can recognize through its ig188   like domain a wide range of pathogens (from prokaryotes to eukaryotes) and different categories of freps seem to exhibit a functional specialization with respect to the encountered pathogens (zhang et al., 2008). in the last years, the publication of the wholly sequenced genomes of different insect species allowed a deep analysis of the evolution of their immune systems. in particular, proteins containing immunoglobulin-like domains seem to be quite common and, at least part of them, play relevant roles in the immune response making insects valuable models to understand the extent of roles played by the immunoglobulin superfamily (igsf) in the insect immunity. from one to thousand: current state of the art of the insect immunoglobulin superfamily immunoglobulins have been considered as typical molecules of gnathostomes since the identification of hemolin (previously called p4), an immune protein isolated from the lepidopterans hyalophora cecropia and manduca sexta (sun et al., 1990; ladendorff and kanost, 1991; lindströmdinnetz et al., 1995). indeed hemolin has been the first ig-like molecule isolated in invertebrates and it has been assessed that it was also involved in the immune response (sun et al., 1990; ladendorff and kanost, 1991; lindström-dinnetz et al., 1995). hemolin is present in low, but significant, amounts in the hemolymph of untreated larvae and pupae, and its level increases 18 30-fold after injection of live bacteria into the insect body, as a consequence of an increased synthesis at the fat bodies (rasmuson and boman, 1979; faye and wyatt, 1980; ladendorff and kanost, 1991). functional analyses clearly assessed that h. cecropia hemolin was able to bind to bacterial cell wall components in vitro, forming a complex with two other hemolymph proteins (sun et al., 1990), whereas m. sexta hemolin was able to bind not only to bacteria (triggering a protective response involving humoral and cellular reactions), but also to hemocytes inhibiting their aggregation in vitro (ladendorff and kanost, 1991; eleftherianos et al., 2006, 2007). as a whole, hemolin was not only belonging to the immunoglobulin superfamily, but also played a relevant role in immunity, probably as a pattern recognition molecule, which discriminates between self and infectious non-self by the recognition of molecules unique to microorganisms, such as lps (schmidt et al., 1993). since this first identification, the immunoglobulin superfamily has been greatly enriched in insect genes encoding proteins with at least one immunoglobulin domain. the ig domain is highly conserved and it has a representative sandwich structure of two opposing antiparallel βpleated sheets, stabilized by a disulphide bridge, that make it easily identified by bioinformatics analyses (bork et al., 1994; halaby et al., 1999). indeed, searches of the interpro and pfam databases showed several types of ig domains such as i-subtype, i-subtype 2, c2-set_2, v-set, v-type, ig, i-set and ig-like (huang et al., 2009). these domains are found in cell adhesion molecules, cell receptors, antigens, cell surface glycoproteins and other proteins (wang and springer, 1998; ossiboff and parker, 2007; soroka et al., 2010) and they are at the basis of specific adhesion and recognition capability, including cell-cell adhesion, cell-surface recognition and pathogen recognition (hutter et al., 2000; hynes and zhao, 2000). recent surveys identified more than 140 igsf proteins in drosophila melanogaster (vogel et al., 2003), 138 igsf proteins in anopheles gambiae, with 85 of them increasing their amount after induction with plasmodium, gram-negative or grampositive bacteria (garver et al., 2008), and more than 150 igsf genes containing at least one immunoglobulin domain have been predicted in bombyx mori (he et al., 2014). at a functional level, recent reports showed that insect igsf proteins are essential for the immune response (i.e., garver et al., 2008). for instance, in a. gambiae, the igsf protein down syndrome cell adhesion molecule (dscam) is involved in the defense against bacteria and plasmodium (dong et al., 2006) and hemolin-like proteins have been also isolated in the silk glands of galleria mellonella in response to bacterial challenge and might mark apoptotic cells for elimination by hemocytes (shaik and sehnal, 2009). multiple sequence alignments and phylogenetic analyses indicated that, despite igsfs evolved rapidly, 58 % of anopheles igsf genes has orthologues in d. melanogaster and aedes aegypti even if they tended to share more sequence similarity with aedes than with drosophila (garver et al., 2008). for instance, 10 genes are homologous between a. gambiae and d. melanogaster, but not with a. aegypti, 28 genes are orthologues between the two mosquito species only, 23 igsf genes are uniquely present in anopheline and two of these share similarity with apis mellifera only (garver et al., 2008). gene ontology annotation indicated that insect igsf members are involved as cellular components and in different molecular functions and biological processes (garver et al., 2008; he et al., 2014). indeed igsf proteins were implicated in signal transduction and cell communication and they can be expressed in a wide range of tissue, such as testis, ovary, fat bodies, midgut, integument, hemocytes, malpighian tubules and head, as clearly assessed in b. mori (he et al., 2014). a well-studied example of a member of the ig superfamily is the drosophila dscam, whose gene comprises clusters of variable exons flanked by constant exons (schmucker et al., 2000) (fig. 1). interestingly, fly cells can combine dscam constant and variable exons by mutually exclusive alternative splicing potentially generating more than 19,000 different extracellular domains (watson et al., 2005; brites et al., 2008, 2013). despite the absence of a somatic rearrangement similar to that of gnathostome immunoglobulins, dscam is at the basis of a large protein isoform repertoire with the potential for recognizing diverse ligands and epitopes (schmucker et al., 2000). secreted dscam isoforms have been detected in the hemolymph and hemocyte-specific loss of dscam impaired the efficiency of phagocytic uptake of bacteria, possibly due to reduced bacterial 189   http://www.sciencedirect.com/science/article/pii/s0378111914008130#bb0005 http://www.sciencedirect.com/science/article/pii/s0378111914008130#bb0015 http://www.sciencedirect.com/science/article/pii/s0378111914008130#bb0020 fig. 1 the dscam gene contains four variable exon blocks (indicated in red, blue, green and yellow) (a) and multiple isoforms are assembled using single variants from each block through an elaborate process of alternative splicing (b). each generated protein contain 10 immunoglobulin domain, 6 fibronectin type iii repeats, a transmembrane domain (tm) and a cytoplasmic tail. the dscam proteins show a isoform-specific homophilic binding (c) where the ig2, ig3, and ig7 variable domains match in two opposing isoforms (as represented by the same shape) at all three variable domains is required for protein binding. modified from schmucker et al. (2000). binding. furthermore, dscam is upregulated after infection in numerous arthropods (armitage et al., 2014). importantly, comparative analyses of dscamlike sequences show high conservation of orthologous dscam genes in diptera, hymenoptera and coleoptera (graveley et al., 2004; watson et al., 2005), together with a conserved expression of diverse dscam isoforms in immune-competent fat body cells and immunocytes among highly diverged insect species (watson et al., 2005). lastly, the molecular diversity of dscam transcripts generated through alternative splicing is highly conserved across the major insect orders suggesting a common (and unsuspected) molecular complexity of the innate immune system of insects. interestingly, the availability of numerous genomes and transcriptomes belonging to different insects could allow to deeply explore dscam presence, expression and alternative splicing in insects (palmer and jiggins, 2015). as a whole, insect igsf members seem to be capable of reacting to pathogen challenges and to control events that contribute to the defence against infection so that they are intriguing candidates as immune effector molecules in insects. immunoglobulin-like proteins and the insect vectorial capacity: from theory to the field several insects are involved in the transmission of pathogens to both animal and plant species. some of these insect-borne pathogens may circulate within the vector body after acquisition and eventually be inoculated into new hosts during insect salivation event (circulative pathogens), while others may bind to various sections of the foregut of vectors without host internalization (killiny et al., 2012). biological and molecular interactions in both of these models are very complex leading to a high specificity between a pathogen and insect vectors and determining transmission success and different efficiency. for instance, it has been demonstrated that the gut microbiota is able to decrease viral and parasitic infections in mosquito and tsetse fly vectors by activating their immune responses or directly inhibiting pathogen development (cirimotich et al., 2011a). in particular, cirimotich et al. (2011b) identified an enterobacterium that confers total refractoriness to p. falciparum through the production of reactive oxygen molecules. however, the mechanisms responsible for bacteria-mediated reduction in vectorial capacity can also be indirect. in this case, the bacteria seem to impede the development of malaria by priming the mosquito immune system (lefèvre et al., 2013). intrinsic factors of the vectors such as age, reproductive status, and body size can have important consequences on vectorial capacity, as well. for example, the host immune system may weaken with age, resulting in an increased susceptibility to pathogens (lefèvre et al., 2013). 190   moreover, genetic variability of both vectors and pathogen strains can differently act on these interactions, as reported for phytoplasmas (seemüller and schneider, 2007; suzuki et al., 2006). this could be translated in the insect capacity to only acquire the pathogen or transmit it with low or high efficiency (galetto et al., 2009). recent gene expression analyses of infected and non-infected insect vectors of plant and animal pathogens offer novel opportunities to exploit new knowledge about host-parasite interactions in the different pathosystems. in particular, this approach pointed out the activation of particular genes encoding antimicrobial peptides, such as defensin and cecropin. however, genes involved in pathogen recognition, such as those encoding lectins, and those encoding receptors that activate the innate immune response, such as toll-3 have been studied, together with genes encoding for members of the signal transduction pathways activated by toll-like receptors, such as jnk kinase (medeiros et al., 2004; fisher et al., 2014; vyas et al., 2014; padrón et al., 2014; colpitts et al., 2011; shigihara et al., 2008). concerning immunoglobulin-like proteins, so far little is known about their direct correlation with the vectorial capacity. it is reported for examples in anopheles gambiae an interplay between agdscam and the resistance to plasmodium infection. in the specific, this study demonstrated that a rnaimediated agdscam depletion in mosquitoes, which fed on mice infected with gfp-expressing p. bergei parasites, resulted in a statistically significant increase of oocystis numbers on the midgut as well as at 13d of feeding (dong et al., 2006). as well, in a. gambiae it has been reported that the immunoglobulin domain 6 gene (irid 6) seems to be involved in limiting plasmodium infection, and so in the resistance to the pathogen invasion, since it was strongly down-regulated in the gut during p. berghei ookinete invasion (garver et al., 2008). on the other hand, the plant virus tomato spotted wilt tospovirus (tswv) activates the immune system of its main insect vector, frankliniella occidentalis and homologues of gene members of the immunoglobulin superfamily of proteins, such as hemolin are among the genes activated after tswv infection (medeiros et al., 2004). in the light of these findings, it could be interesting to verify if the large protein isoform repertoire related to the ig superfamily is involved in the establishment of the different insect vectorial capacity (fig. 2). this role could be particularly intriguing since it could allow us to disrupt some pathogen-vector interactions leading to the blockage of transmission. in particular, it would be the starting point to guide strategies in order to block the transmission of vector-borne pathogens to plants or animals altering gene expression. however, deep analyses of insect vector transcripts under infected and non-infected conditions prove to be a pivotal tool for identification of genes involved in the host immunity response to pathogen infection and transmission. fig. 2 despite the conservation of the dscam genes, the repertoire of proteins that may be assembled is different among diverse species so that two phylogenetically related species may have very similar (but not identical) dscam repertoire. as a consequence, some pathogen genotypes could be detected in one species, but 191   undetected in the other (indicated by the arrow in each species) explaining the different vectorial capacity of insect for similar pathogenic species. conclusions most animals, including insects, have not acquired adaptive immunity, but present a large receptor diversity that increases the effectiveness of immune responses of individual animals. in view of the absence of the adaptive immunity, the insect immune system has been described as simple, term that has been often misinterpreted as evolutionarily inferior to the complex vertebrate immune system. this is a viewpoint that falsely equates the phylogenetic position with a sort of functional inferiority. insects are the most successful class of organism on the planet (e.g., gullan and cranston, 2000) and their simple immune system plays an important role in that success. at this regard, the insect open hemocel provides some advantages 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of the genes involved in autophagy are conserved from yeast to invertebrates and mammals, and genetic manipulations can easily be performed in drosophila in vivo, drosophila is an ideal model system for studying the function of autophagy in whole animals. studies of autophagyrelated gene mutants revealed the importance of autophagic functions in larval midgut cell death during morphogenesis (denton et al., 2009), and hematopoiesis in larvae (shravage et al., 2013). in addition to its essential functions in development, emerging evidence indicates that autophagy functions against invasion of host cells by pathogens such as intracellular bacteria, viruses, and protozoa (yano and kurata, 2011). here, we summarize and provide insight into the functions of autophagy in innate immunity in invertebrates. the first evidence that autophagy is crucial for protection against intracellular bacterial growth came from a study of group a streptococcus, which invades mammalian culture cells (nakagawa et al., 2004). many subsequent studies demonstrated that intracellular bacteria, such as salmonella typhimurium, listeria monocytogenes, and mycobacterium tuberculosis are eliminated by autophagosomes, indicating the critical role of autophagy in innate immune responses against such bacteria (huang and brumell, 2014). because studies were performed in cultured cells and autophagy-knockout animals are in most cases lethal, the critical role of autophagy in vivo was first demonstrated in drosophila by tissue-specific autophagy knockdown (yano et al., 2008). listeria utilizes the host endocytotic pathway to enter drosophila hemocytes, the hematopoietic cells that function in innate immunity. when listeria infects the body cavity, they invade the hemocytes because of their high phagocytic ability, but are quickly engulfed by autophagosomes for elimination. flies ___________________________________________________________________________ corresponding author: shoichiro kurata aoba 6-3, aramaki aoba-ku, sendai 980-8578, japan e-mail: kurata@m.tohoku.ac.jp in which the autophagy gene atg5 is specifically knocked down in hemocytes are susceptible to listeria infection, suggesting that autophagy is important for their survival against infection by listeria (yano et al., 2008). the importance of autophagy in protecting against invasive bacteria has also been demonstrated in another insect, tenebrio molitor, in which knockdown of the autophagy genes atg3 or atg5 leads to susceptibility to listeria in vivo (tindwa et al., 2015). although basal levels of autophagy function in the basically non-selective turnover of cytosolic molecules, autophagy is selectively induced for the degradation of aggregate proteins, damaged mitochondria, or invading bacteria (mizushima and komatsu, 2011). autophagosomes that surround the bacteria are spatially regulated, and autophagy is selectively induced upon the recognition of bacteria by the pattern recognition receptors (prrs) of the host. in drosophila cells, intracellular prrs, such as peptidoglycan-recognition protein (pgrp)le, recognize and bind to peptidoglycans of the bacterial cell walls, and this recognition is necessary for autophagy induction (yano et al., 2008). compared to listeria infection of drosophila cells, which is largely dependent on pgrp-le for induction, mammalian cells have similar, but more complicated systems: several triggers are involved in the induction of autophagy, including recognition by prrs such as nod2, ubiquitination of proteins around the bacteria, and destruction of the host endosomes used by the bacteria to enter the host cells (gomes and dikic, 2014). mammalian cells likely adapt to the strategies bacteria use to escape from autophagic elimination. ubiquitin may be one sign of autophagic engulfment (boyle and randow, 2013). ubiquitin is often observed around bacteria invading the cytosol by breaking the endosomal membrane. in mammalian cells, the adaptor proteins p62, ndp52, and optineurin recognize the ubiquitinated bacteria, and recruit autophagosomes to the bacteria by binding to lc3/atg8 protein located on the autophagosomes (huang and brumell, 2014). although ubiquitinated proteins clearly colocalize with the invading bacteria, it is unknown how the ubiquitin-modified proteins are identified. the e3 ligase lrsam1 is required for ubiquitination in mammalian cells upon salmonella infection, and the enzyme is essential for colocalization of the adaptor 155 mailto:kurata@m.tohoku.ac.jp proteins and bacteria (huett et al., 2012). the e3 ligase parkin is also involved in the induction of ubiquitin-dependent autophagy upon invasion by m. tuberculosis (manzanillo et al., 2013). parkin (park2 in mammals) is an essential e3 ligase for autophagic clearance of damaged mitochondria (mitophagy), but it also functions to induce autophagy upon mycobacterium infection (xenophagy), which limits bacterial replication. parkin is essential for mitochondrial maintenance in drosophila as well as in mammals, and its xenophagic function is also conserved. flies with a mutant parkin gene are susceptible to l. monocytogenes, s. typhimurium, and m. marinum infection (manzanillo et al., 2013), indicating striking similarities between the mitophagy and xenophagy mechanisms. in addition to its anti-bacterial functions, autophagy is important as an innate immune response against viruses in both mammalian cells and drosophila. vesicular stomatitis virus (vsv), a rhabdovirus that infects drosophila s2 cells, rapidly replicates when autophagy is inhibited (shelly et al., 2009). moreover, atg18 or atg7 knockdown flies exhibit increased susceptibility to vsv, suggesting that autophagy is essential for fly survival against vsv infection. the induction of autophagy upon vsv infection is dependent on the viral glycoprotein vsv-g, and toll-7, a drosophila toll-like receptor that interacts with vsv-g on the plasma membrane to trigger autophagy via a nutrient signaling pathway for autophagy induction, the pi3k-akt-tor pathway (nakamoto et al., 2012). extracellular signal-related kinase signaling has a role in the anti-rna virus activity in drosophila cells and fly intestine by coupling nutrient status to antiviral defense (xu et al., 2013). the precise mechanism underlying how autophagy inhibits viral replication, however, remains to be elucidated. the drosophila p62 homologue ref(2)p is an another candidate molecule that functions in the anti-viral response via autophagy. the ref(2)p gene is polymorphic, and some wild populations carrying restrictive alleles exhibit reduced multiplication rates of the sigma virus, a vertically transmitted rhabdovirus (carré-mlouka et al., 2007). in this case, it is not known whether the ref(2)p-dependent restriction of viral replication occurs via autophagy. autophagy has a critical role in anti-bacterial immunity not only in phagocytic immune cells, such as macrophages in mammals or hemocytes in flies, but also in epithelial cells. autophagy in mouse intestinal epithelial cells (iecs) inhibits the growth of s. typhimurium, an intestinal pathogen that invades iecs to cause inflammation (benjamin et al., 2013). mice with iec-specific knockout of atg5 are unable to eliminate s. typhimurium, resulting in dissemination of the bacteria to other organs. infection by s. typhimurium induces autophagy, especially at the apical side of iecs. this raises the possibility that the host quickly recognizes invading s. typhimurium to induce autophagy. the induction of autophagy against s. typhimurium is independent of nod2, an intracellular pathogen recognition protein that induces xenophagy (travassos et al., 2010), but rather depends on myd88, a signaling molecule of the toll pathway that leads to nuclear factor κb activation. how myd88 contributes to autophagy induction in iecs, however, remains to be elucidated. although in drosophila less is known about the autophagic role against invasive bacteria in the intestine, the powerful genetic techniques available for drosophila could provide new insight. in addition to its role in immunity, autophagy is related to inflammation. recent genome-wide association studies identified the autophagy-related genes atg16l1, irgm, and nod2 as risk factors for crohn’s disease, a chronic inflammatory bowel disease (hampe et al., 2007; rioux et al., 2007). the pathogenesis of this disease is also affected by environmental factors, such as commensal bacteria. the mechanism of inflammatory diseases caused by defective autophagy was studied in atg16l1 gene knockout mice: overproduction of interleukin1β, an inflammatory cytokine, in atg16l1-knockout macrophages is stimulated by commensal bacteria such as escherichia coli (saitoh et al., 2008), whereas dysfunction of paneth cells, immune cells that reside in intestinal crypts and secrete antimicrobial peptides and lysozymes, occurs in atg16l1-hypomorphic mice and iec-specific atg16l1 knockout mice (cadwell et al., 2008; cadwell et al., 2010; conway et al., 2013). interestingly, this paneth cell secretion defect is also dependent on enteric bacteria. although these studies elucidated the importance of autophagy (genetic risk) and enteric bacteria (environmental risk) in intestinal homeostasis, the molecular mechanisms remain unknown. a drosophila model with powerful genetic approaches might provide clues to the mechanisms. as summarized here, autophagy has a critical role in anti-bacterial and anti-viral immunity. although its role in animal survival is clear, autophagy is not a perfectly efficient mechanism for eliminating microbes, and thus some microbes evade autophagy to replicate in the host cells. the ability to avoid autophagy is not just dependent on the microbe species, but it is often observed that a portion of one kind of bacterium, such as listeria, is trapped by autophagosomes, while others are able to evade autophagy. this might be because autophagy is primarily a host catabolic system that functions in the innate immune response using prrs or other triggers for selectivity. because of the highly conserved systems of innate immunity between species, the genetic tools available for drosophila will facilitate significant progress in understanding the role of autophagy against microbe infection. further studies on autophagic function in innate immunity will provide new insights for clinical application. acknowledgments this work was supported by grants-in-aid for scientific research from the ministry of education, culture, sports, science, and technology of japan (mext, 24390014, 21117005, 21025002, 21113502, 21200070, 21249004, 21022005, 21590058); the japan society for the promotion of science; japan science and technology agency (jst); the program for the promotion of basic research activities for innovative biosciences (probrain); the strategic international 156 manzanillo ps, ayres js, watson ro, collins ac, souza g, rae cs, et al. the ubiquitin 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also an invaluable tool for basic research. this review focuses on how transplantation techniques have been used to understand the biology of three large lophotrochozoan phyla: annelida, nemertea and platyhelmintha. i describe how transplantation paradigms have uncovered fundamental principles regarding the embryology, immunology, endocrinology and regeneration biology of representative species within these three groups. in particular, embryologists have used blastomere transplantations to show that both mosaic and regulative development occurs in animals within the phyla. immunologists have used transplantation techniques to demonstrate that these invertebrates mount a variety of innate immune responses, some of which include surprising features that classically characterize adaptive immunity. endocrinologists have used transplantation experiments to uncover hormonal requirements for sexual development and maturation. meanwhile, regeneration biologists continue to address fundamental questions regarding tissue polarity, post-embryonic patterning, stem cell physiology, and the role of the nervous system in regeneration. along with recent technical and conceptual advances, transplantation remains a powerful tool for invertebrate research. key words: transplantation; annelida; nemertea; platyhelmintha; immunity; regeneration   introduction tissue transplantation can be defined as the removal of a piece of tissue from a donor organism, followed by grafting into a recipient or host. based upon the relationship between the tissue donor and host, transplants are classified into three basic categories: autografts, allografts, and xenografts (fig. 1). autografts are transplants between a donor and host who are the same individual (fig. 1a). allografts are transplants between different individuals of the same species (fig. 1b). xenografts are transplants between separate species (fig. 1c). additionally, the anatomical origin of the graft and its placement in the host further delineates transplantations into two subtypes: homotopic and heterotopic (fig. 1). a homotopic transplant involves tissue grafted into the identical anatomical location in the host compared to where it ___________________________________________________________________________ corresponding author: eduardo e zattara national museum of natural history smithsonian institution department of biology indiana university 915 e. third street, myers hall 150 bloomington, in 47405-7107, usa e-mail: ezattara@gmail.com was excised from the donor. heterotopic transplants involve tissue grafted into a new anatomical location in the host. the amount of tissue transferred may range from a single cell transplant to a graft of two complete organisms which freely exchange internal body fluids (also known as a parabiotic graft, fig. 1d). within these transplantation categories lies a continuum of variants, each determined by the nature of the question addressed by a given transplantation experiment. however, for simplicity’s sake, this review will not consider transplantations to include ectopic contacts made between tissues after selective ablation of cells or anatomy, such as in the generation of out of register blast cells by teloblastic bandlet slippage in leeches (shankland, 1984). tissue transplantation has been successfully used to interrogate many types of developmental and physiological interactions between tissues and cells. the first reported grafts of different animal pieces were made by trembley (1744) in hydra (cnidaria: hydrozoa). the first report of successful grafts on worms is that of morren (1829) in earthworms. since then, transplantation has been used with great success to address questions in the fields of embryology, immunology, endocrinology, and regeneration biology. while many such questions 247 fig. 1 generalized examples of transplantation types. color fill represents species, outline represents individuals, dark-to-light gradient represents anterior-to-posterior axis, and dashed lines represent planes of transection. a) autografts, in which tissue is grafted back to its donor individuals, and is transplanted into the same location (homotopic, left) or a different location (heterotopic, right). b) allografts, in which tissue excised from a donor is transplanted into the same location (homotopic, upper) or a different location (heterotopic, lower) of another individual from the same species. c) xenografts, in which tissue excised from a donor is transplanted into the same location (homotopic, upper) or a different location (heterotopic, lower) of another individual from a different species. d) parabiotic transplants, in which two individuals of the same species (allograft, left) or different species (xenograft, right) are conjoined and can freely exchange body fluids and diffusible molecules. 248 are biologically closely related and benefit from integrative approaches, they will be treated here separately by discipline and topic to reflect their historical segregation. lophotrochozoan worms: annelids, nemerteans and platyhelminths this article reviews the use of transplantation experiments to study embryology, immunology, endocrinology and regenerative biology within three phyla of invertebrate “worms.” this includes annelida, the segmented worms; nemertea, the ribbon worms; and platyhelmintha, the flatworms. these phyla belong to the lophotrochozoa, a main branch of the bilaterian tree, which has historically been understudied relative to the ecdysozoa (arthropods, nematodes and related groups) and the deuterostomia (vertebrates and other chordates, echinoderms and related groups) (simakov et al., 2013; henry, 2014). the mollusca, the fourth main lophotrochozoan phylum, are not included in this review because of their significantly derived body plan and antero-posterior axis, which makes comparisons difficult with members of worm-like taxa. annelida, the segmented worms, is a large phylum of over 17,000 species of marine, freshwater and terrestrial worms (zhang, 2013; bely et al., 2014). it comprises two main clades, the errantia and the sedentaria (fig. 2) (struck et al., 2011; weigert et al., 2014). most errantia are active benthic and pelagic marine worms. the sedentaria constitute several families of benthic burrowing worms, in addition to a large clade of freshwater and terrestrial annelids, the clitellata, which includes naidids (water nymph and sludge worms), crassiclitellates (earthworms), enchytraeids (potworms), lumbriculids (blackworms) and hirudines (leeches). annelids have a body plan composed of segmentally iterated celomic compartments and organs intercalated between two non-segmental terminal regions. they possess a body wall that, in larger groups, includes thick muscle layers (stephenson, 1930; brusca and brusca, 1990). given this body plan, transplantation procedures usually imply grafting pieces of the body wall, ventral nerve cord or brain in larger animals like earthworms, leeches and ragworms (errantia:nereididae). in smaller animals like sludge worms, water nymph worms, blackworms and many meiofaunal groups, grafting is technically challenging, due to small size, fragility, and a tendency to autotomize (self-amputate) injured segments. nemertea, the ribbon worms, is a smaller phylum of around 1,300 species. they are mostly marine, although a few freshwater and terrestrial representatives are known (andrade et al., 2012; zhang, 2013). it comprises three main clades: palaeonemertea, pilidiophora and hoplonemertea (fig. 2) (andrade et al., 2014). all members have an unsegmented body plan characterized by a unique proboscis apparatus used for prey capture and kept within the only body cavity. most organs are contained within a muscular body wall (gibson, 1972; brusca and brusca, 1990). ribbon worms are, by far, the least studied of the three phyla. however, they have unique developmental and physiological properties, including very high tolerance to injury and robust wound healing. this has made ribbon worms particularly amenable for complex grafting experiments (bierne, 1990). platyhelminthes, the flatworms, is a large phylum comprising nearly 30,000 free-living and parasitic species that inhabit oceans, freshwater and land (zhang, 2013). they comprise two main clades, catenulida and rhabditophora (fig. 2). most species are found in the latter clade, including better known taxa like polycladida, tricladida and the obligately parasitic neodermata (tapeworms and flukes) (laumer et al., 2015). flatworms have a relatively simple acoelomate body plan lacking a circulatory system. most possess a blind gut with only one opening that serves as both the mouth and the anus of the animal. some species do not even possess a gut (laumer et al., 2015). interestingly, the flatworm body plan is hypothesized to be similar to that of the urbilateria (starred in fig. 2), the last common ancestor of bilaterian animals (de robertis and sasai, 1996). free-living flatworms, and likely some of the parasitic lineages as well, have a remarkable system of cellular homeostasis in which mitotic divisions are restricted to a population of mesenchymal stem cells which gives rise to all differentiated cells. within this phylum, the tricladid freshwater planarians have received particular attention, mostly due to the amazing regenerative capabilities of some species in this group (bely et al., 2014). transplantation as a tool to understand embryonic development since the earliest days of experimental embryology, transplantation of embryonic tissues has been a crucial tool to test models of signaling and interaction between the components of developing embryos (see examples in hörstadius, 1928, 1937, 1975; gilbert, 2006; sweet et al., 2004). arguably, the most famous example is a series of xenografts between urodele embryos that led to the discovery of the spemann-mangold organizer (spemann and mangold, 1924). furthermore, many insights into the processes that control early development were gleaned from tissue grafting studies and transplantation of blastomeres, the latter being pioneered by hörstadius between the 1920s and the 1960s, and later continued by several research groups (reviewed by sweet et al., 2004). however, while these approaches were widely used to study deuterostomes including echinoderms, hemichordates, cephalochordates, tunicates and vertebrates, reports of transplantations experiments in annelid, nemertean and flatworm embryos are extremely rare (hörstadius, 1937; novikoff, 1938; nakamoto et al., 2011; shimizu and nakamoto, 2014). likely reasons for such dearth are the small embryonic size in many species and the common presence of extraembryonic layers that difficult accessing the embryos and keeping them alive after extraction long enough to perform experimental manipulations. among annelids, egg diameters range from 40 350 µm in marine species to 50 1000 µm in terrestrial direct developing species; nemertean eggs are ~90 120 µm in diameter; while flatworm 249 fig. 2 lophotrochozoan worms compose three of four major phyla: annelida, nemertea and platyhelminthes (weigert et al., 2014; andrade et al., 2014, 2015; laumer et al., 2015a; laumer et al., 2015b). phylogenetic tree showing relationships among themselves, and relationships to the fourth major phylum, the mollusca, and other metazoan phyla. families mentioned in this review are shown along with some major groups for each of annelida (blue shaded boxes), nemertea (red shaded boxes) and platyhelminthes (green shaded boxes), and other major metazoan phyla (small font, all caps). the three main branches of bilateria are indicated by a grey box. the star indicates the location of the urbilateria, the hypothetical last common ancestor of all bilaterian animals. eggs go from 130 150 µm in marine species to the large 550 1000 µm eggs of terrestrial tricladids (novikoff, 1938; anderson, 1973; fernández and stent, 1982; henry and martindale, 1994, 1998; dohle, 1999; alvarado, 2003; maslakova et al, 2004; pernet and jaeckle, 2004; arenas-mena, 2007; martín-durán and egger, 2012). in contrast, xenopus laevis frogs lay 1000 µm eggs, while the triturus newts used to discover the spemannmangold organizer are 2000 µm in diameter. even in recent years, most experimental manipulations of annelid, nemertean and flatworm embryos have used cell ablation, lineage tracing and molecular interference of gene function rather than grafts (see for example shankland, 1984; martindale and shankland, 1988; henry and martindale, 1998; shimizu and nakamoto, 2014), attesting to the technical challenges of transplantation in smaller embryos. another reason for the scarcity of transplant studies amongst these three animal groups is the general assumption that most spiralians display mosaic development. this means that their blastomeres operate on fixed developmental programs and lack interaction with other cells during ontogeny. in contrast, numerous deuterostomes 250 studied during the late 19th and early 20th centuries display robust cellular interactions during regulative development, a type of embryogenesis in which cells alter their fates to compensate for environmental perturbations. in fact, up until world war ii, much of the field of experimental embryology focused on questions pertaining to regulative development, predominantly in sea urchins (driesch, 1892; hörstadius, 1928, 1950) and amphibians (lewis, 1904; mcclendon, 1910; harrison, 1918; huxley and beer, 2015). for these reasons, the use of transplantation studies in annelids, nemerteans, and flatworms has historically attracted far less attention than in their deuterostome counterparts. ironically, the few transplant studies that were performed in these phyla helped uncover the existence of mosaic development amongst many spiralians, and subsequently reduced researchers’ interest in these animals. hörstadius (1937) grafted together pieces of embryos older than the 4-cell stage from the nemertean ribbon worm cerebratulus lacteus and reported that they differentiated as they would have before the transplant. novikoff (1938) developed a technique to isolate blastomeres and polar lobes (cellular extrusions used to asymmetrically segregate embryonic determinants) from the 60 µm early embryo of the honeycomb worm sabellaria vulgaris (annelida: sabellaridae), and fused them together in novel combinations. as in nemerteans, he found that in all cases both donor and host cells continued their original developmental programs with no evidence of the inductive interactions found in many deuterostome embryos. it was not until more than half a century later that several transplantation studies in the sludge worm tubifex tubifex (annelida: naididae) finally uncovered the existence of inductive interactions in spiralian embryos (nakamoto et al., 2011; shimizu and nakamoto, 2014). in the relatively large tubifex embryos (~500 µm), two d quadrant micromeres, 2d and 4d, are necessary for embryonic axis formation, and their ablation results in a cell mass surrounded by epithelium. when isolated 2d and 4d micromeres were heterotopically grafted onto an intact embryo, the embryo developed a secondary axis, including terminal anterior or posterior structures. cell lineage tracing showed that while ectodermal and mesodermal structures in the secondary axis were derived from the donor tissues, the endoderm originated from the host. thus, the graft induced host endoderm to form a new axis. this work, along with studies of teloblastic bandlet slippage in leech embryos (shankland, 1984; martindale and shankland, 1988; wedeen and shankland, 1997), shows that after gastrulation, annelid embryonic development presents significant inductive interactions between cells and tissue layers. transplantation studies to understand embryonic development in annelids, nemerteans and flatworms are challenging. but new technologies developed for stem cell biology and invitro fertilization to allow manipulation of 50 100 µm mammalian eggs are opening the spectrum of tools available to perform grafts, even in very small embryos (sweet et al., 2004). combined with an ever-increasing toolkit to label cells, modify gene expression levels, alter genetic information, and perform high-resolution live imaging, transplantation experiments have a very promising future. with technologies enabling isolation, labeling and even genome editing of blastomeres, their grafting back into the original embryo or a new host will undoubtedly provide profound insights into the mechanisms of embryonic development in these animal groups. transplantation as a tool to understand invertebrate immunity a central question of immunology is how animals respond to invasion by foreign elements. this includes asking whether and how organisms achieve self vs. non-self recognition, what mechanisms they use to fight off foreign elements, and whether there are mechanisms to mount stronger responses upon repeated exposure to specific elements. transplantation experiments have proven to be a powerful approach in beginning to address these questions. most, if not all, multicellular organisms have the ability to distinguish self from non-self, and present some degree of reaction against foreign cells or substances (parish, 1977; coombe et al., 1984; bayne, 1990; tsutsui, 2004). this ability is known as innate immunity, and is characterized by a generic response to most foreign elements. in contrast, vertebrates also possess adaptive immunity, characterized by the production of immunoglobulins, specialized cell-surface receptors and a system of clonal cell selection and expansion that together provide memory and specificity to the immune response. while such adaptive immunity has been classically considered to be unique to vertebrates, the innate immune response is shared by vertebrates and non-vertebrates. however, a number of studies have shown that non-vertebrates also have immune-related molecules that confer a certain levels of immunological memory and specificity (kvell et al., 2007). tissue grafting facilitates the measurement of an organism’s capacity to recognize self versus non-self, and to explore the mechanisms of immunity (cooper, 1970; tettamanti et al., 2003; söderhäll, 2011). immune responses, both innate and adaptive, are usually based on cellular and humoral reactions (coombe et al., 1984; cooper and roch, 2003; salzet et al., 2006). cellular responses include phagocytocis, encapsulation of foreign elements too large to be engulfed by host cells, nodulation and wound healing. humoral responses are most commonly mediated by soluble factors produced by the circulating cells. these soluble factors are usually able to identify non-self elements, activate a signaling response and neutralize those elements (e.g. antimicrobial peptides, reactive oxygen species, melanization cascade, coagulation cascade) (coombe et al., 1984; loker et al., 2004; tsutsui, 2004). both cellular and humoral innate immunity mechanisms have been reported for most metazoans studied. however, the presence of adaptive-like immunity in non-vertebrates, defined 251 as the ability of the organism to show an enhanced response after repeated exposure to the same nonself elements, is still hotly debated. adaptive immunity has two main hallmarks: memory (the ability to retain information about past contacts with foreign elements) and specificity (the ability to mount a differential response against different nonself elements). the presence of adaptive immunity is a hypothesis with straightforward predictions for transplantations experiments. most organisms have some level of innate immunity, and are expected to reject tissue grafts recognized as non-self. however, if adaptive immune memory is present, a second graft from the same donor will elicit a stronger rejection response. if adaptive immune specificity is present, a second graft from a different donor species not previously encountered will fail to elicit a stronger response. transplantation paradigms have been used to study the ability of annelids and nemerteans to detect and react to non-self tissues (fig. 3a). amongst annelids, studies on ragworms (errantia: nereididae) have revealed that these worms can indeed distinguish self from non-self and mount a cytotoxic response to allografts and xenografts, leading to graft rejection (porchet-henneré et al., 1987). in almost all cases, the likelihood of graft rejection was found to be lower for xenografts of closely related species than for more distant ones (clark and clark, 1959; boilly-marer, 1974, 1976; cuvillier-hot et al., 2014), suggesting that these organisms use an evolving and divergent set of molecules in order to distinguish self versus nonself. likewise, in the 1960s, duprat (1964) and cooper (1968) independently reported that earthworms (clitellata: crassiclitellata) are capable of rejecting body wall grafts. the speed of rejection correlated with the phylogenetic distance between donor and host. this led to a number of studies testing whether transplantation sensitized the host to repeated challenges from the same donor. work on lumbricus terrestris, eisenia fetida, aporrectodea trapezoides, dendrobaena veneta (lumbricidae) and eudrilus eugeniae (eudrilidae) revealed that most autografts surviving the procedure were accepted. in contrast, allografts were usually rejected, and the chance of rejection apparently correlated with the inferred genetic distance between the conspecific individuals. furthermore, xenografts were always rejected. rejection times showed a rough inverse correlation to phylogenetic distance (valembois, 1963; duprat, 1964, 1967; cooper, 1968, 1969, 1970; chateaureynaud-duprat, 1970; bailey et al., 1971; dales, 1978; parry, 1978). along with the ragworm data, these results support that self-recognition and histocompatibility are likely mediated by genetically encoded markers that diverge between lineages over time. tests for adaptive immunity in earthworms have given more ambiguous results (fig. 3b). some results showed that a first graft from a given donor species could alter the speed of rejection of subsequent grafts from the same species, but had no influence on rejection of grafts from a different donor species. this suggested the presence of memory and specificity (hostetter and cooper, 1973; cooper, 1975). this claim has been disputed, since results show both strengthening and weakening of subsequent graft rejection, and some results failed to be independently replicated (dales, 1978; parry, 1978). more convincing support for the existence of immune memory and specificity in annelids came from transplantation studies in leeches (tettamanti et al., 2003). body wall autografts in the medicinal leech hirudo medicinalis (clitellata: hirudinidae) caused only an inflammatory and angiogenetic response. in contrast, allografts and xenografts from the broad snail leech glossiphonia complanata (clitellata: glossiphoniidae) were rejected in the span of 7 days. rejection consisted in a characteristic sequence of events resulting in graft destruction. a second allograft or xenograft resulted in a similar series of events; however, if the second graft came from the same donor as the first graft, rejection was markedly accelerated, taking place within 3 4 days rather than 7 days. this stronger response was verified both in the short term (3 to 7 days between first and second graft) and long term (1 or 4 months between first and second graft). immunolabeling using antibodies against human cell surface markers revealed the presence of different cell types between second grafts from same versus different donor, hinting that this putative immune memory in annelids might be cell-mediated. surprisingly, there were no differences in rejection time between allografts and xenografts from a distantly related species, suggesting that selfrecognition does not depend on species-specific markers in leeches. there are numerous known cellular and molecular effectors in annelid immune response (cooper and roch, 2003; salzet et al., 2006; cuvillier-hot et al., 2014) but the key players in self versus non-self recognition are still mostly unknown, and are a prime target for study using transplantation experiments (coombe et al., 1984; loker et al., 2004). transplantation experiments in ribbon worms of the genus lineus (nemertea: lineidae) have also found support for immunological memory and specificity outside the vertebrates (langlet and bierne, 1973, 1982, 1984; bierne and langlet, 1974; bierne, 1985). thanks to a plastic body plan and robust wound healing, species in this genus are highly tolerant to grafting and can survive surgeries where partial or full pieces from the same or different worms are spliced together into multiparental chimeras (bierne, 1985, 1990). the resulting chimeric worms can be homoor heterospecific and combine grafts of the same or different sexes. despite being genetic mosaic individuals with up to 16 parents, many chimeras can survive for several years. this potential has opened the possibility of using transplantations to address several questions regarding phylogenetic and cellular aspects of graft rejection and immunity in nemerteans. bierne and langlet explored rejection of reciprocal grafts in six lineus species: l. ruber, l. viridis, l. longissimus, l. (=ramphogordius) lacteus, l. (=ramphogordius) pseudolacteus and l. (=ramphogordius) sanguineus (langlet and bierne, 252 1973; bierne and langlet, 1974). autografts and allografts were never rejected, and became integrated with the donor. xenografts produced various reactions ranging from integration to rejection. as in annelids, likelihood and speed of rejection were correlated with phylogenetic distance between donor and host (langlet and bierne, 1982; zattara et al., 2015). using a combination of three lineus species, langlet and bierne (1982) found that initial xenografts from l. sanguineus onto l. ruber where rejected after more than 15 days. a subsequent xenograft from the same donor species (same or different individual donors) was rejected in less than 9 days. in contrast, rejection of a subsequent xenograft from a third species still took over 15 days. by changing the time between the initial and subsequent grafts, they showed that this memorylike effect is present up to 80 days but has faded by 120 days. these observations support the hypothesis that the immune response to xenografts in lineus has species-specific mid-term memory, but that such memory does not last longer than 4 months. to identify the effector of the graft rejection response, langlet and bierne (1982, 1984) devised an ingenious experiment (fig. 3c): anterior (antecerebral) ends of l. sanguineus were homotopically grafted onto individuals of l. lacteus or l. ruber, or onto chimeras composed of a l. lacteus middle portion and a l. ruber posterior (intestinal) portion or vice versa. as expected, nonchimeric l. ruber hosts rejected the grafts in less than 20 days, while most non-chimeric l. lacteus had not rejected the grafts after 90 days. strikingly, when grafting onto chimeric hosts, the rejection response did not depend on the source species of tissues adjacent to the graft, but only upon the source species of the posterior (intestinal) portion. histological analyses showed cell-mediated lysis of donor tissue. since the strength of the rejection response depends on the donor species of the posterior (intestinal) portion, it is likely that the response effectors are cells located on the intestinal region that migrate anteriorly in response to signals coming from the anterior graft. in contrast to annelids and nemerteans, very little is known about immunity in free-living flatworms. reports of many grafting experiments done in tricladid planarians to address questions in regenerative biology (morgan, 1901, 1906; moretti, 1911; rand and browne, 1926; miller, 1938) hint at a phylogenetic signal in immunity. autografts and allografts are usually accepted by the host, while strength of rejection responses to xenografts somewhat reflects evolutionary distance between donor and host species. cellular immunity is the most likely effector of graft rejection, as phagocytic cells accumulate after injury near a wound site (morita and best, 1974; morita, 1991), and destroy or encapsulate the foreign elements. humoral immunity is likely present, since many orthologues to genes involved in the immunity in vertebrates have been found in the genome of schmidtea mediterranea (tricladida: dugesiidae) (peiris et al., 2014). on the surface, observations suggest that the immune system of free-living flatworms may have more “lenient” self-recognition compared to annelids and nemerteans, but this conclusion is still premature. none of the above studies aimed to characterize graft rejection or immunologic parameters. experiments designed to systematically test for variability in average graft survival times are still pending in flatworms, and will be essential for reconstruction of immunity in the urbilateria. in summary, transplantation experiments in annelids, nemerteans and flatworms show that their immune systems share several common features. all three phyla are capable of healing transplanted tissue, recognizing self from non-self, and mounting a rejection response against grafts recognized as non-self. similar capabilities have recently been reported in at least one representative of the fourth main lophotrochozoan phylum, the mollusca (yamaguchi et al., 1999), so it is likely that they were present in stem group lophotrochozoans, and perhaps even the urbilateria. the strength of the response tends to increase with the phylogenetic distance between donor and host, though the slope of this trend varies both between and within phyla. several lines of evidence point at cellular rather than humoral response as the main effector of graft rejection. in some annelids and nemerteans, there is evidence for memory and specificity of the immune response, although not with the characteristics of vertebrate adaptive immunity. while transplantation has given key insights into immunity in these lophotrochozoan groups, much more is still to be learned. what are the molecular players modulating self/non-self recognition and cellular responses to grafts? what roles do putative homologs of vertebrate immunity genes play in these groups? are the mechanisms behind memory and specificity common among these groups and thus inherited from a common ancestor, or do they represent independent evolutionary innovations? with the development of increasingly powerful model systems in each of these phyla, many of these experiments can now be revisited to molecularly dissect the mechanisms of lophotrochozoan immunity. transplantation as a tool to understand invertebrate endocrinology transplantation is a common tool in studies of invertebrate endocrinology, as it can be used to remove and re-implant suspected sources of hormones, and to test interactions between endocrine organs and developmental stages. differentiation and maturation of gonads and sexually dimorphic structures in species that cyclically develop and resorb their reproductive organs is an example of endocrine regulation that has been extensively studied in annelids, nemerteans and planarians using transplantation experiments. in ragworms (annelida: nereididae), removal of the brain induces precocious gonadal maturation and development of secondary sexually dimorphic traits found in reproductive worms. heterotopic autografts in which the brain is removed and reinserted in the coelom delay or inhibit this precocious sexual maturation (hauenschild, 1960; durchon, 1962). interestingly, the brain, either intact 253 fig. 3 transplantation paradigms to test for self versus non-self recognition and innate versus adaptive immunity. a) in the presence of a self-recognition system that is genetically encoded, increasing the genetic distance between donor and host is expected to increase the strength of the graft rejection response. experimental evidence suggests that annelids have stronger self versus non-self discrimination than nemerteans and flatworms. b) experimental paradigm to test for memory and specificity in immune responses. expected changes in mean rejection times depending on traits of the immune system (left) and data from tests in annelids and nemerteans (right). the “*” highlights disputed results. c) xenograft experiments to determine the effector mechanism of nemertean immune response. grafting the anterocerebral portion of l. sanguineus to the medium region of its sister species l. lacteus elicits a weak response (graft rejection time over 90 days); if the host is the more distantly related species l. ruber, the response is stronger (graft rejection time is less than 20 days). when l. sanguineus antecerebral regions are grafted to homo and heterospecific chimeras made from the medium and posterior (intestinal) portions, the response is determined by the species of the posterior (intestinal) portion and not by the species of the medial portion adjacent to the donor tissues. 254 or grafted into the coelom, is also necessary for regeneration of posterior segments (clark and bonney, 1960; hauenschild, 1960; clark and evans, 1961; golding, 1967b, 1974). brain homotopic transplants and heterosexual chimeras obtained by grafting two individuals of different sexes have been used to explore regulation of gonadal maturation and secondary sexual character re-development in ragworms. such experiments have shown that some sexually dimorphic traits (male and female swellings and male crenellations of the parapodial cirri) are determined by the genetic sex of the source tissue. in contrast, another dimorphic trait (male pygidial papillae) is not sex-specific, but is inhibited by female hormones (durchon, 1962; boilly-marer, 1974, 1976). a similar neuroendocrine control of sexual maturation has been described in nemerteans (bierne and rué, 1979; sivaradjam and bierne, 1981; vernet and bierne, 1988). in several species, removal and grafting of the brain has demonstrated that a gonad-inhibiting hormone is secreted from this organ (bierne, 1966; bierne and rué, 1979). however, while the ragworm brain hormone controls also growth and restorative regeneration (see above), the nemertean hormone affects exclusively sexual maturation (vernet and bierne, 1988). experiments with heterosexual chimeras made by allografting lateral halves of a male and female individuals of two species of lineus have shown that sexual maturation occurs in two phases: initial formation of new gonads, that develop according to the sex of each half, resulting in immature gynandromorphs; and unilateral sex reversal of one of the halves by a putative diffusible factor coming from the other half (bierne, 1975; sivaradjam and bierne, 1981). the dominant sex is species specific: l. sanguineus chimeras became all female, but l. ruber chimeras become all male. although studies of flatworm endocrine abilities are scarce (basch, 1986; pincus et al., 2013), similar transplantation experiments on the control of sexual maturation have been done in dugesia tigrina and d. gonocephala (kenk, 1941; okugawa, 1957). allografts of anterior or posterior pieces from sexually mature donors onto the complementary piece of an asexual host eventually induced gonad maturation and development of secondary sexual traits in the host. while these studies were not able to differentiate between hormonal induction and migration of stem cells from the sexual donor tissue into the asexual host, recent work supports the role of a diffusible factor in controlling sexual maturation (kobayashi et al., 2002; maezawa et al., 2014). transplantation as a tool to understand regenerative biology regenerative biology is probably the field that has been making use of transplantation experiments for the longest time (morgan, 1901). the regeneration of missing body parts is a fascinating process that implies a re-deployment of developmental trajectories into a post-embryonic context, and thus combines basic questions of embryology like cell and tissue differentiation, with unique problems of the adult context, like sources of stem cells, short and long range signaling pathways used to initiate regeneration after wound healing, restoration of correct body proportions and functional integration of the regenerated parts. transplantation experiments covered in this last section have been used to test hypotheses exploring three major regeneration topics: axial polarity and spatial information, roles of the nervous system and stem cell biology. the ability found within annelids, nemerteans and flatworms to re-develop missing body parts and regulate their morphology to adapt to changes in body condition has made them superb models to study developmental and physiological mechanisms of regeneration (bely et al., 2014). both regenerative ability and research efforts are unequally distributed across these three phyla. anterior (including complete head) and posterior regeneration ability is widespread in annelida (bely, 2006), but most transplantation experiments are restricted to larger species, like earthworms and errant polychaetes. within nemertea, only a few species show complete head regeneration; when present however, regeneration is fast and robust (bely et al., 2014; zattara et al., 2015), and can be studied using multiparental chimeras created by multiple grafting (bierne, 1990). distribution of regenerative ability is also patchy in plathyhelmintha, but it is particularly strong in a number of tricladid planarians (egger et al., 2006; bely et al., 2014). these planarians have been a long time favorite of regeneration research (elliott and sánchez alvarado, 2013) which have endured too large a number of transplantation experiments to be fully covered in this paper. thus, while brief mention of some key studies are made in the following paragraphs (morgan, 1901, 1906; moretti, 1911; rand and browne, 1926; santos, 1931; miller, 1938; chandebois, 1985; kato et al., 1999, 2001; kobayashi et al., 1999), the interested reviewer is referred to several excellent articles specifically reviewing past and present planarian regeneration research (salo and baguñà, 2002; reddien and alvarado, 2004; baguñà, 2012; elliott and sánchez alvarado, 2013). the parsing of positional information into axial polarity is a crucial step of early embryonic development, but also a fundamental requisite for adequate regeneration of lost body parts. is axial polarity an intrinsic property of tissues, or does it result from context-dependent interactions? most amputation experiments have shown that anteroposterior polarity is usually retained by the stump tissue, so that anterior wound surfaces regenerate anterior ends, and posterior wound surfaces regenerate posterior ends (fig. 4a, left). heteromorphic regeneration, where a stump regenerates a “wrong” body part, can very infrequently result in two-tailed or two-headed worms (fig. 4a, center). heteromorphic regeneration of annelids and flatworms is rare and usually seen only in very short fragments, or due to physical (morgan, 1901, 1902; gates, 1950; moment, 1951; kawamoto et al., 2005), pharmacological (fitzharris and lesh, 1969) or molecular interference (gurley et al., 2008; adell et al., 2009; petersen and reddien, 2009). heteromorphic regeneration is unreported in 255 nemerteans, even in very small fragments (coe, 1929, 1934). a second major topic in regeneration biology is the role of the nervous system in inducing or inhibiting the regeneration process (kumar and brockes, 2012). transplantation experiments have been widely used to explore this problem (fig. 4b). working with earthworms, avel (1930) made heterotopic autografts of ventral body wall from posterior segments onto the dorsal side of the anterior region. when such grafts included the ventral nerve cord, small ectopic heads regenerated at the point where the cord contacted the anterior suture of the graft; however, denervated grafts simply wound healed. a somewhat similar result was obtained by sayles (1939) after heterotopic autografts of nerve cord placed into the dorsal body wall of clymenella torquata (annelida: sedentaria). numerous experiments on several species of planarian have shown that grafting of anterior tissues, including brain ganglia, can inhibit regeneration of heads from adjacent anterior wound surfaces, and induce the formation of ectopic structures, depending on the anterior level at which the graft is placed (moretti, 1911; steinmann, 1925; gebhardt, 1926; goetsch, 1926, 1929; rand and browne, 1926; santos, 1929, 1931, 1931; miller, 1938). at the end of the 19th century, it was shown that heterotopic grafts in earthworms could result in the ectopic regeneration of body parts (morgan, 1897, 1901; joest, 1895, 1897; korschelt, 1897, 1898). is then tissue polarity an intrinsic quality or is it the result of induction by specific structures nearby? grafting together posterior pieces of earthworms by their anterior surfaces, followed by amputation on one of the pieces near the suture site still results in regeneration of a posterior end (fig. 4a, right), proving that antero-posterior polarity is not reversed by influence of the larger, uncut fragment (morgan, 1901). more recent observations made after coincidental grafting of amputated fragments of the polychaete parougia bermudensis (errantia: dorvilleidae) confirmed that grafted annelid fragments do not switch polarity; interestingly, this study also reported that fusion of contralateral regenerating connectives from the nerve cord results in development of a brain, irrespective of whether the connectives originate from the same or separate nerve cords (müller, 2004). nemerteans are even more resistant to polarity switching: transversely cut fragments split longitudinally and grafted together in opposite direction retain their individual polarity, and a head blastema forms at anterior end of each piece (coe, 1934). in contrast, posterior fragments of planarians grafted by their anterior surfaces and then amputated close to the suture line occasionally regenerated a head from a posterior surface (morgan, 1906), suggesting that tissue polarity in flatworms is more labile than in annelids or nemerteans. in contrast, it has been found that extirpation of the brain greatly inhibits posterior regeneration (fig. 4b, center and right) in earthworms (clitellata: lumbricidae), ragworms (errantia: nereididae) and catworms (errantia: nepthydae) (kropp, 1933; hubl, 1956; clark and clark, 1959; hauenschild, 1960; clark and evans, 1961; golding, 1967a, b). in earthworms, autografts of whole or minced brains onto the anterior end of decapitated worms fails to rescue posterior regeneration (kropp, 1933). on the other hand, transplantation of minced brain macerates does partially rescue regeneration on a fraction of decerebrated platynereis dumerilii and hediste diversicolor ragworms (hauenschild, 1960; clark and evans, 1961), suggesting that some secretion from the brain is necessary for posterior regeneration. generation of parabiotic chimeric individuals by grafting posterior fragments into intact or decerebrated hosts in different combinations indicate that such a secretion is a permissive factor, but that growth rates of the regenerate are autonomously regulated (golding, 1967b, c). no such inhibition of posterior regeneration by removal of the cerebral ganglia has been observed on nemerteans (vernet and bierne, 1988) or planarians, save for one anecdotal account (moretti, 1911). among the lophotrochozoan worms, planarians present the most amenable body plan for grafting, robust regenerative abilities and a dynamical patterning system (elliott and sánchez alvarado, 2013). these traits have made them a popular organism to study the dynamics of morphogenetic gradients, being used to test concepts like child’s gradients of physiological dominance (child, 1929), turing morphogenetic fields (turing, 1952; erneux et al., 1978), gierer-meinhardt systems (gierer and meinhardt, 1972) and positional information (wolpert, 1969). juxtaposition of tissues from different axial regions induces ectopic anatomy (possibly indicating the induction of a new body axis) in many grafting experiments. for instance, cutting out a circular plug of tissue and grafting it back but inverting its dorsoventral orientation induces formation of blastema-like structures at the graft boundaries that produce what appears to be an ectopic body axis (santos, 1929, 1931; kato et al., 1999). heterotopic autografts from anterior regions of the animal to posterior ones and vice versa also induces the formation of ectopic anatomy in host tissues (santos, 1931; kato et al., 1999; kobayashi et al., 1999). with an expanding functional toolkit, grafting experiments could be very useful in testing hypotheses about the molecular underpinnings of dynamic regulation of scale and proportion, and the mechanisms by which positional information is reset and re-interpreted after injury. a third topic of regeneration biology addressed by transplantation experiments is the neoblast hypothesis of regeneration (baguñà, 2012). neoblasts are proposed to be a population of reserve stem cells that migrate towards the wound site after amputation and serve as a source of new tissues in the regenerate. the term neoblast was originally coined after observations on annelid regeneration revealed the presence of certain spindle shaped cells with unusual staining properties and large nuclear-to-cytoplasm ratios, which seemed to migrate along the ventral nerve cord and accumulate at the wound site (randolph 256 fig. 4 transplantation paradigms to study polarity conservation, influence of nervous system and the role of neoblast in regeneration. a) transverse amputation (left) normally results in fragments that conserve their anteroposterior (ap) polarity, so that anterior wound surfaces form anterior blastemas (black) while posterior wound surfaces form posterior blastemas (white). in rare occasions in annelids and planarians, blastemas with reversed polarity appear, leading to heteromorphic regeneration. a middle portion autografted with a reversed ap axis and cut through (right) normally retains its polarity, except occasionally in planarians where its polarity is reversed. b) grafting or deviation of a nerve cord can induce the regeneration of ectopic structures (left). conversely, removal of the brain in some annelid groups (right) can inhibit posterior regeneration, which can be sometimes rescued by grafting a brain. c) the role of neoblasts (migratory stem cells) in regeneration can be tested by grafting together a fragment of a regeneration-competent worm with a fragment of an irradiated worm or an individual from a regeneration-deficient species (left), followed by amputation through the latter: neoblasts are expected to migrate to the wound site and rescue regeneration, a result usually seen in planarians. the ultimate test for this model is transplantation of a single neoblast into a regeneration-deficient host (right): a regeneration-competent worm is dissociated, its neoblasts are isolated, and a single neoblast is transplanted into the host. if neoblasts are present, they are expected to rescue regeneration. 257 1891, 1892). finding similar cells in tricladid planarians led to adoption of the term neoblast by flatworm researchers (elliott and sánchez alvarado, 2013). with the discovery that x-ray irradiation could abrogate regenerative powers in vertebrates, researchers of worm regeneration combined this technique with transplantation experiments to test whether grafting from an intact, regenerationcapable donor could rescue an irradiated, regeneration-disabled host (fig. 4c, left). within annelids, such use of transplantation was very limited, since the larger earthworms are rather poor regenerators compared to other groups of smaller size like water nymph worms (clitellata: naididae), pot worms (clitellata: enchytraeidae) and blackworms (clitellata: lumbriculidae). despite the difficulty and low survival of such transplants, zhinkin (1934) showed that irradiated posterior ends of the blackworm rhynchelmis limosella grafted onto a posteriorly amputated non-irradiated host were capable of regenerating, albeit with a delayed timing. in nemerteans, neoblasts have also been proposed as key to regenerative abilities in some species of lineus (coe, 1930, 1934), but irradiation has not been used to test this hypothesis. however, experiments using bi-specific chimeras made from xenografts from species with different regenerative abilities have shown that the results of amputation depends only on the origin of the injured tissue (bierne, 1967); in other words, grafting a fragment from a species with great regenerative ability cannot rescue regeneration in a poorly regenerating species host. these experiments indicate that migratory neoblasts are not crucial in nemertean regeneration. in contrast to the ambiguous and inconsistent support for the role of neoblasts in annelid and nemertean regeneration, their role in planarian regeneration, already well supported by classical and recent experiments in which grafts of healthy donors can rescue regeneration of irradiated hosts (wolff and dubois, 1947; guedelhoefer and alvarado, 2012) has been spectacularly demonstrated in the planarian schmidtea mediterranea (tricladida: dugesiidae) by a combination of classical techniques and recent molecular and cytological tools. by transplanting into a fully irradiated host neoblasts from a donor with a distinctive genotypic signature (fig. 4c, right), it was confirmed that just a single neoblast is sufficient to rescue regenerative ability (wagner et al., 2011). because donor and host cells were chosen to have different karyotypes, it was also possible to show that eventually all of the somatic tissues of the host eventually became replaced by the clonal progeny of that single neoblast, proving that in these flatworms, neoblasts are fundamental not only for regeneration, but for normal body-wide tissue turnover. neoblasts are necessary for planarian regeneration, but not sufficient. several species of dendrocoelid planarians (tricladida: dendrocoelidae) are incapable of regenerating a head if cut at a level posterior to the pharynx (morgan, 1904; egger et al., 2006). stéphan-dubois used a clever set of reciprocal transplantations between anterior and posterior regions, and between intact and irradiated worms, to find whether lack of neoblasts or improper activation was the cause for such regeneration-deficiency in dendrocoelum lacteum (stephan-dubois and kolmayer, 1959; stéphan-dubois and gilgenkrantz, 1961). her data showed that neoblasts are present, divide and migrate to an anterior wound to initiate a blastema at all levels of the body, but the posterior regions were not competent to induce the correct antero-posterior polarity in the blastema, causing an arrest of the process. a series of recent studies used next-generation sequencing of mrna to discover aberrant upregulation of the wnt pathway in the posterior regions of dendrocoelum lacteum, phagocata kawakatsui and procotyla fluviatilis (liu et al., 2013; sikes and newmark, 2013; umesono et al., 2013). notably, these researchers also demonstrated that regeneration deficiency can be rescued by downregulating canonical wnt signaling using rna interference against β-catenin. transplantation has been a crucial tool for research on regenerative processes in annelid, nemerteans and flatworms. it allowed elaborating mechanistic hypotheses that are now being investigated using an ever-increasing array of molecular techniques. with the advent of inexpensive massive parallel sequencing of dna and rna, and functional tools that can be used on adult animals of non-model species, the doors are open to take advantage of the wide range of regenerative abilities across the metazoa to help us crack the riddle of regeneration. concluding remarks since trembley’s experiments on hydra over 270 years ago, the use of transplantation as an experimental tool to study several aspects of invertebrate biology has resulted in important advances in the fields of embryology, immunology, endocrinology, and regeneration biology. however, the use of this tool is not limited to these four disciplines, and has also been used in other areas, like neurobiology (drewes et al., 1988) and even systematics (bierne et al., 1993). the main limitations to the use of transplants (beyond the imagination of the scientific mind) are the difficulty posed by low survival rates in animals that are highly intolerant of surgical procedures and the skill of the hands that make surgeries usually at a microscopic scale. with the advent of new technologies, these limitations might soon be lifted. it is worth noting that many of the newer tools, like high precision electronic micromanipulators, have been developed for use in areas more closely related to strongly funded biomedical research, placing them out of reach of invertebrate zoologists with more modest resources. however, this should not be seen as a constraint, but as an incentive to develop collaborations among research groups. furthermore, the increasing number of molecular tools available for organisms outside the select club of traditional model systems and the development of techniques allowing the tracking of fate of the grafted tissues (abdulreda et al., 2011; 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understand invertebrate endocrinology transplantation as a tool to understand regenerative biology concluding remarks acknowledgements references isj 13: 68-75, 2016 isj 13: 68-75, 2016 issn 1824-307x review genes of ancient microtubule-stabilizing proteins traveled through pre-cambrian echinoidea to advanced life forms of dry land and ended up in the human genome as the fusion oncogenes-oncoproteins eml1/eml1-abl/abl, and eml4/eml4-alk/alk jg sinkovics department of molecular medicine, the university of south florida morsani college of medicine, the cancer institute, st. joseph’s hospital, tampa fl, usa accepted march 4, 2016 abstract the genes eml1/4 of the echinodermata microtubule-stabilizing gene product-like 1/4 proteins eml1/4 of the sea cucumbers (holothuroidea) traveled through the evolutionary scale up to the human genome. human cells malignantly transformed by oncogenes abl or alk enlist the protein products eml1/4 for the activation and protection of the gene product oncoproteins abl or alk against destruction by ubiquitination, and for gaining virulence and chemotherapy resistance. neither the abl nor the alk genes act as oncogenes without fusion with another particular gene, such as eml1/4 in this case. a large number of ancient but conserved gene product proteins chaperon, protect and enhance oncoproteins. these mechanisms indicate that ancient cell survival pathways exist conserved in the genomes of advanced multicellular diploand triploblastic hosts (including homo). these genomic pathways are on special occasions constitutively reactivated in extant cells undergoing transformations for survival under adverse circumstances. extant cells under threat react by re-living scenarios that characterized life forms in the primordial physico-chemical universe. in the clinical practice these cells are recognized as chemoradiotherapy-resistant cancer cells undergoing a process of retrograde immortalization. key words: sea cucumbers; microtubule-associated proteins; oncogenes-oncoproteins; eml1/4 proteins   introduction paleologists presume that in the precambrian environment ribozyme-like nucleotides coexisted with precellular ribozyme-armed ribosomes. complex hypercycling rna nucleotides deriving from hot subterranean vents travelled freely between these units. the rna molecular protogenes were sought after, were competed for, and were readily accepted in the working machinery of the targeted ribosomes. the precellular ribosomes contained trna precursors that could align amino acids in disorderly chains without any major conflict between the gene product peptide conglomerates pre-existing in the host, and those newly synthesized by the guests. the phenomena of horizontal first rna, then dna gene transfers may be viewed retrospectively as the natural form of existence in the world of precellular ribozyme-armed ribosomes sharing and exchanging first rna, and ___________________________________________________________________________ corresponding author: joseph g sinkovics cancer institute st joseph’s hospital 3001 w dr martin luther king jr blvd tampa fl usa 33607 e-mail: sinkovi.joseph@baycare.org then dna genomes. with the unresolved episodes of dna’s appearance, spheroplasts appeared and fused into multicellular colonies mediated by fusogenic viruses, in the category first of rna, then of dna phages. such a candidate virus is an extant fusogenic dna phage of the protoplast mollicute acholeplasma laidlawii (sinkovics, 2016). acholeplasma cells still release their primordial exosomes and lytic, temperate or fusogenic viruses (phages). all cells do, but especially cells undergoing malignant transformation in their multicellular host, exude an abundance of exosomes, as a signature for their atavistic return to the primordial life style of their ancestors. the first eukaryotes might have been formed by virally fused prokaryota and crenarchaea spheroplasts. endosymbiosis with proteobacteria of the pre-fused spheroplasts formed the mitochondria in ancestral eukaryota in all categories of animalia. endosymbiosis of the pre-fused spheroplasts with cyanobacteria formed the chloroplasts in ancestral eukaryota of all categories of plantae. remnant members of a precellular virus world entered the primordial spheroplasts and their derivative fungal, plant and animal cells, thus creating the obligatorily 68   virus-carrier three domains of life in the present era of archaea, bacteria (prokaryota) and eukarya (eukaryota). the ancestors of the extant megavirales sealed their intimate relationship with ancestral amoebae. the liberal coalescence with alien genes has become strictly constricted, and the integrity of the individual dna genomes became heavily guarded. the formerly practiced liberal horizontal gene transfers exerted major influence on, but without actual up-rooting, of the natural vertical course of evolution (extensively referenced in sinkovics 2011, 2015, 2016). in the present article, the evolution of certain microtubules from archaea and prokaryota will be followed through echinodermata eukaryotes, where they acquired their stabilizing proteins, eml1/4 (echinoderm microtubule-associated protein). it will be shown that a vital physiological system installed over three billion years ago remains conserved throughout evolution, as it becomes part of a retrograde immortalization process in extant multicellular eukaryotic hosts, including homo. this process appears in the form of fusion oncogenes/oncoproteins in the human genome diagnosed at the clinics as the eml1-abl malignant lymphoma, and the eml4-alk lung non-small-cell adenocarcinoma. in a most remarkable initial study (bermudes et al., 1994), prokaryotic microtubules were observed by electron microscopy, but their tubulin protein composition was not immediately identified. these structures are already operational in the lower ranks of prokaryota: the gram-positive mycoplasma a. laidlawii contains them. cyanobacteria, the ancestors of chloroplasts in algae and plant cells, possess microtubules. if the term gamma subdivision of purple bacteria included proteobacteria, the ancestors of mitochondria in animal cells, the protofilaments and tubules reacting with antitubulin antibody were present in them. in spirocheta, heat-shock proteins cross-reacted with anti-tubulin antibodies, but hsp-independent microtubules also appeared (szathmáry, 1987). the contribution of microtubules, including flagellary undulipodia, from spirochetes to ancient eukaryota might have occurred from phagocytized spirochetes, or through routes of ectosymbiosis with spirochetes. spirochetes, especially spirochaeta bajacaliforniensis harbor longitudinally aligned tubulin microtubules of antitubulin antibody immunoreactivity (bermudes et al., 1987). if the first eukaryota emerged from virally induced fusion of a mycoplasma cell (a fusogen phag-carrier a. laidlawii) with crenarchaeal spheroplasts (sinkovics, 2016), the eukaryotic microtubules are of bacterial derivation. the similarity of protofilament interactions of bacterial and eukaryotic cells reveals that the bacterial microtubules represent the primordial structures that preceded eukaryotic microtubules evolutionarily (pilhofer et al., 2011). the ancient microtubular filamentous temperature-sensitive z ring proteins (ftsz) initiate and carry out the separation (by membrane abscission) of the daughter cells in the processes of cell divisions. energy derives from the hydrolysis of gtp to gdp. recruited proteins synthesize the new cell wall of the daughter cell. single-stranded protofilamental subunits form the arrangements for the circular z rings consisting of multistranded structures of microtubules. the z rings are capable of exerting scissor-like contractile force. the z rings form a scaffold for accessory proteins recruited for the separation of the daughter cell. the tubulin proteins of archaea and prokaryota become actinmyosin proteins in eukaryota. the ftsz system is shared by prokaryota and crenarchaea represented by sulfolobus acidocaldarius (with the exemption of thermoproteales). the three-genes cdv cell division operons and cdv proteins, and the endosomal sorting complexes are required for transport (escrt) of molecular cargo, thus contributing to membrane remodeling, cellular abscission (separating the membranes by cleavage of two connected cells), and to multivesicular body biogenesis. these two systems are shared in crenarchaea and eukaryota (lindås et al., 2008). some ftsz homologs (repx from the plasmid pxo1 of bacillus anthracis; tubz from the virulence plasmid pbtoxis of bacillus thuringiensis) are the replicators of the plasmids (makarova and koonin, 2010). the representative of a new crenarchaeaspecific ftsz-like 1 subfamily is the unique escrtiii-interacting tubulin protein of the crenarchaeon sulfolobus solfataricus (after the volcano solfatara di pozzuoli at naples, italy). pseudomonas fluorescens yielded another new ftsz-like 2 domain that conserved all motifs of the nucleotide-binding loops, but without a coiled coil domain (which is present in the ftsz-like 1 element) (makarova and koonin, 2010). the orderly assemblage of the ftsz formation in bacteria could be physiologically inhibited by the min system (discovered in e. coli minicells) and by nucleotid occlusion (no) (rowlett and margolin, 2015). to the call to google and wikipedia for a list of ftsz inbibitors in pathogenic bacteria and in eukaryota undergoing malignant transformation, a long lists of natural and synthetic molecular inhibitors appear including berberine, colchicine, chrysophaentin, curcumin, genistein, quercetin, plumbagin, resveratrol, sanguinarine, taxanes, totarol, and vincristine/vinblastine, etc. the extremely acidophilic (optimally growing at ph 2 on temperature 60 °c), autotroph, methanotroph (using methane as its sole source of energy), methylacidiphilum infernorum replaced its lost genes by horizontal receipt of numerous new genes, first from archaea, then from eukaryota, including ftszs, but without synthesizing any microtubules; it fixes formaldehyde; and exempted itself of prophages due to an active crisprassociated system (clustered regularly interspersed short palindromic repeats). its publication received extraordinary reviews (hou et al., 2008). in one particular case, a verrucomicrobia other than m. infernorum, the prosthecobacter dejongeii, has become the carrier of the btuba/b microtubuleencoding genes, whose gene product microtubular proteins are not of prokaryotic, but of eukaryotic structural and functional entities. thus, in this case (or may be in all cases), the primitive αβ-tubulin genes entered after their duplication in a p. ancestor from a primordial eukaryotic cell possessing proto-tubulins assembled into microfilaments (schlieper et al., 2008; martin69   galiano et al., 2011). thereafter the tubulin heteropolymers have undergone two separate but related evolutionary lines, one prokaryotic, one eukaryotic, both preserving their gtpase-activating domains. results the primordial echinodermata originated as crinoids in the precambrian sea 600 mya. extant echinoidea and echinozoa are represented by the sea cucumbers (stichopus; dendrochirotida; holothuroidea), sea urchins (strongylocentrotus), seastars (asterozoa) and sandfish, sand dollar (holothuriidae; clypeasteroida; dendraster). echinoidea larvae and embryos form from the unison of haploid sperm-fertilized haploid eggs (the sea urchin meiosis of oscar hertwig, 1875). the dividing sexualzellen, keimzellen, urkeimzellen, stammzellen carry the keimplasm of august weismann, 1883 5 (while gregor mendel’s articles rested on the shelfs of charles darwin, unopened and unread). the keimplasm is understood to consists of chromosomes packed with dna genes interspersed by rna introns. the first three divisions of the echinoidea zygote provide eight large cells of equal size. the fourth division yields macromeres and micromeres: lmics, large micromeres encoding the larval skeleton, that is the tubulovesical structures included; smics, small micromeres undergoing the fifth cell division yielding mesomeres for the ectoderm and endoderm of the entire embryogenesis, and micromeres (smics) for the germ line of the adult organism. the wnt8/notch/apc (wingless; integrated; adenomatous polyposis coli)/dishevelled/csk (casein kinase) and gsk3β (glycogen synthase kinase) pathway with intranuclear β-catenin activating the tcf/tcf (t cell factor), characterize the micromeres cells. gsk-phosphorylated cytoplasmic β-catenin is degraded by ubiquitylation; unphosphorylated β-catenin is transferred in micromere nuclei. at the 5th cleavage, histone 3 lysine 9 is trimethylated (h3k9me3), thus silenced. both lmics and smics are needed for the formation of the mesenchymal blastocoel. lmics direct the metamorphosis of the archenteron gastrula of the larva into the adult rudiment, from which the adult organism develops. calcium-transporting proteins maintain currents for communications between the cell organelles (endoplasmic reticulum, lysosomes, mitochondria). this article contains numerous illustrations in color, regretfully not reproduced here (wessel et al., 2014). note: in vertebrate mammalian (including human) cells undergoing the so-called malignant transformation, due to deficient apc/gsk, unphosphorylated β-catenin is transferred into the nucleus, where it activates oncogenes, tcf/tcf in particular (referenced in sinkovics, 2015, 2016). the hox (homeobox) gene cluster for bilateral to pentameral body patterning in the superphylum deuterostomia (cnidarian hydra, sea anemone, jellyfish) and echinodermata, from protohox to hox and parahox anterior and posterior genes evolved and duplicated way before the divergence of these basal animals. in some cases, parahox is more involved in neurogenesis than in pre-bilaterian axis determination (ferrier and holland, 2001; long et al., 2003; chorrout et al., 2006; quiquand et al., 2009; ikuta, 2011; byrne et al., 2016). the longest living animals are among invertebrate taxa, especially those practicing reverse ontogenesis (hydrozoa; meduzoa, referenced in sinkovics, 2016). individuals of the gold and black corals gerardia and leiopathes, the clam arctica of island, the tube worm lamellibrachia, the demosponge astrosclera live hundreds even thousands of years (referenced in giga community of scientists, 2014). it remains unresolved what genomic constitution is responsible for the lack of senescence and the consequential life span extending over a century for the red sea urchin strongylocentrotus franciscanus, in opposition to the four years life span for the green s. variegatus. of mammalian species, the naked mole rat (heterocephalus glaber) and the bat myotis brandtii live long in absence of an aging process or neoplastic transformations (referenced in sergiev et al., 2016 and sinkovics, 2016). the complement cascade and its interactions, or the lack thereof, with activator or inhibitor micrornas evolved in lipopolysaccharideresponding coelomocytes of strongylocentrotus (spu-mir) and in its relation, in the sea cucumber apostichopus japonicus (ajc3); thus, the technology is available for further studies (referenced in zhong et al., 2015). the strongylocentrotus’ major contribution is to the evolving recombination activating genes (rag1/2) of adaptive immunity. the ancestors of these transib transposonmediated v(d)j transposition-catalyzing proteincoding genes reside in the genomes of the sea urchins strongylocentrotus and lytechinus (fugmann, 2010; kapitonov and koonin, 2015). the target of the rag transposases, the v(d)j sequence, is not present in the sea urchins; it appears first in the gnathostomata sharks. this particular genomic sequence might have been acquired by an ancestral herpesvirus candidatus ebv (epstein-barr virus). the v(d)j-like sequence inserted into the ebv genome was discovered by niller and associates, in 2004. the paleoimmunology concerning the virally mediated insertion of the elements of the adaptive immune system into chondrichthyes ancestral cartilaginous sharks have been discussed by dreyfus in 2009 and 2011; and by sinkovics in 2011 and 2016. the sea urchins harbor numerous inserted active and inactivated retroviral elements acquired and propagated vertically and horizontally (gonzales and lessios, 1999). sea urchin and sea cucumber coelomocytes are primarily phagocytic amebocytes. the high diversity of their cdna responses indicates that versatile immune responses were operational in the immunoglobulin-free era of native immunity. at the level of the sea squirt (ciona) and the sea urchin (strongylocentrotus) operational chemokine ligands and their receptors were non-detectable (de faria and da silva, 2008; xue et al., 2015). the ciona operates two toll-like receptors (tlr), but the strongylocentrotus is well endowed with some 222 tlrs, which are paralogous with the tlrs of the 70   amphioxus (branchiostoma floridae) (satake and sekiguchi, 2012). in the sea cucumber (a. japonicus) two tlr genes were sequenced. ajtlr3 and ajtoll were 3484 bp and 4211 bp, expressed leucine-rich repeats, and extended transmembrane into the cytoplasm. the two tlr genes were widely expressed, but in different extent in various tissues, including dominantly the coelomocytes. these receptors responded to lps, peptidoglycans, polyinosinic and polycytidylic acids and zymosans implying broad responsibility to grampositive/negative bacteria and dsrna viruses (sun et al., 2013). the sp185/333 gene cluster encodes highly diversified gene product protein reactions in six element patterns to heat-killed marine bacteria; these reactions were restricted to distinguished cell lines. thus individual phagocytes produced mrnas for uniform protein (buckley et al., 2008; terwilliger et al., 2008; majeske et al., 2014). the presentation of the major histocompatibility complex class iirestricted antigens in vertebrate mammalians is regulated by the enzyme gamma interferoninducible lysosomal thiol reductase (gilt). the sea cucumber stichopus monotuberculatus expresses a 1529 bp gilt protein of molecular weight 23.8 kda. its gene contains four exons and three introns. the protein displays nfκband ifnγ-binding sites. endotoxin lps upregulates the expression of this gilt, the initiator of innate immune reactions (ren et al., 2015). the echinoderm microtubule-associated protein-like 1/4 activate and protect the human oncoproteins abl in acute t cell leukemia, and alk in non-smallcell lung adenocarcinoma the echinoderm microtubule-associated protein (emap) family is represented by one single member from echinodermata up to caenorhabditis and drosophila (emap-like protein elp-1). by the vertebrate mammalians, the family, emaps expanded to five members (eml 1-5). in echinodermata, they are present in centrosomes and regulate the eccentric location of the germinal vesicle in oocytes during meiosis (suprenant et al., 1993; hamill et al., 1994; miyazaki et al., 2006; houtman et al., 2007). the reader is encouraged to view in original the illustrations of the hamill and suprenant articles. microtubule-associated proteins (map) manufactured in the ribosomes, in general regulate microtubule formation and stabilization, thus the functioning of the entire cytoskeleton. the echinodermata eml4 gene analogs reappear in the caenorhabditis as its elp-1 gene (hueston et al., 2008), and in the drosophila as its doublecortindomain containing echinoderm-microtubuleassociated protein ortholog (bechstedt et al., 2008). the genomic locus of the human eml4 gene (gene id 27439; also known as gene ropp 120 restrictedlyoverexpressed proliferation-associated protein) is at the short arm of chromosome 2p21. the wd (tryptophan aspartic acid) repeat eml4 protein is of 981 aa with mass 108916 da up to 120 kda molecular weight (pollmann et al., 2006). in the mouse genome, the ortholog of the human gene is at chromosome 17 (gene id 78798). the human eml4 is activated by phosphorylation of its serine/threonine residues. its aa sequence 1-249 acts upon the microtubules in the mitotic spindle. hela cells with sirna-deactivated eml4 proteins could not take up [3h]-thymidine and failed to develop mitotic figures. these hela cells showed an inactive microtubule network (pollmann et al., 2006). of the newly evolved and recently discovered nuclear microtubule-binding proteins, eml3 is referred to as an echinoderm microtubuleassociated gene product. it guards that microtubules to correctly align the chromosomes in the metaphase (as tested in hela cells). there are newly discovered spindle-accumulating emap family proteins: eml3 “poorly characterized” as such. the emap-like protein 70 (eml2) destabilizes and reorganizes microtubules in the m phase of the cell cycle (tegha-dungu et al., 2008). some maps are described without claiming relationship to the echinodermata-related emap family (orbánnémeth et al., 2005). emap-like protein 5 (eml5) could be overexpressed in the human brain in both glial and neuronal cells. when overexpressed in the anterior temporal neocortex, it induces intractable epileptic seizures (sun et al., 2015). further discussions will be limited to the participation of eml1 and eml4 in the formations of human fusion oncogenes with proto-oncogenes abl and alk, thus encoding fusion oncoproteins eml1-abl and eml4-alk abelson mouse leukemia retrovirus proto-oncogene; anaplastic leukemia kinase). the cryptic translocation between genes eml1/eml1 at 14q32 and abl/abl at 9q34 forming t(9;14)(q34;q32) occurred in a young female patient diagnosed with t cell acute lymphoblastic leukemia (t-all). this interaction results in the deletion of tumor suppressor cyclin-dependent kinase 2a (p16), and the expression of tlx/tlx1 gene-product protein, which is a human homolog of the drosophila tailless gene, a cell cycle driver, encoded in the human genome as nuclear receptor nr2e1/nr2e1 gene-product protein. further upregulated pathways are erk1/2 (extracellular signal-related kinase); stat 5 (signal transducer and activatior of transcription); and the lyn kinase (abbreviation of united lck/yes kinases: lymphocyte kinase of the rous sarcoma retrovirus src family, and yamaguchi sarcoma retrovirus kinase). the coiled-coil domain of the eml1 protein is incorporated in the naturally formed 190 kda the fusion oncoprotein. removal of the coiled-coil domain disabled the oncoprotein. the technology for this molecular diagnosis involved fish (fluorescence in situ hybridization), race (5’rapid amplification of cdna ends polymerase chain reaction); constructs of open reading frame of exon 1 to 17 of eml1 amplified with primer eml1-f1 and -r. abl was amplified with primers abl1-f and –r. the eml1/del eml1 parts were ligated in the murine stem cell retroviral vector puromycin kit (clontech, palo alto, ca). imatinib sensitivity was tested for in cell cultures and expressed in growth curves. the abl proto-oncogene was known in other leukemias to fuse with genes bcr (breakpoint cluster region), nup (nucleopore-to-gene-promoter interaction), and mll (mixed lineage leukemia). in this case, the abl /abl oncogene/oncoprotein inhibitor imatinib mesylate induced durable 71   complete remission. figures depicting of the fusion oncogenes-eml1/alb are shown and referenced in the cited articles (de keersmaecker and huret, 2005; de keersmaecker et al., 2005; van etten, 2005). the reader is encouraged to view these figures in original. amplifications of egfr in adenocarcinomas, and fgfr1 in squamous cell carcinomas of the lung are common events treatable accordingly, with individually appropriate monoclonal antibodies, chemotherapeuticals, targeted therapy (gefitinib; erlotinib, imatinib; pd173074) and with small molecular inhibitors and monoclonal antibodies to neo-vasculogenesis. however, the recently emerged eml4-alk-driven lung adenocarcinomas require specific considerations (weiss , sos, seidel et al., 2011). the fusion oncogenes consisted of the inversion of the short arm of chromosome 2 juxtaposing the 5’ end of the eml4 gene with the end of the alk gene, so that intron 13 of the eml4 gene fused with intron 19 of the alk gene. the encoded oncoprotein assumes three isoforms (versions or variants 1, 2 and 3). variant 3 dominates in china (52 %); variant 1 inflicts caucasians (75 %) (zhao et al., 2015). all variants of the eml4/alk fusion genes replicate constitutively. figures of the fused oncogenes eml4/alk are depicted in cited article (choi et al., 2008). the reader is encouraged to view these figures in original (karachaliaou and rosell, 2014). all tumors with alk/alk involvement are referred to as alkomas (mano, 2012), these tumors only occasionally express v-kiras2 (kirsten rat sarcoma retroviral homolog), or egfr additional mutations, because of the belief that these co-mutations are mutually exclusive. in cases of second co-mutations, crizotinib therapy fails to control the disease (ulivi et al., 2015). in china, in addition to non-small-cell lung cancers of the well-recognized heterologous etiology of driver genes egf-r, v-ki-ras2, and v-raf (rat fibrosarcoma) oncogene homolog b (braf), tumors of eml4-alk etiology not responding to erlotinib or gefitinib, but responding to crizotinib emerged. most of these adenocarcinoma tumors were diagnosed in their advanced stages in young non-smoker women (zhao et al., 2015). occasionally elderly smokers can be involved (choi et al., 2008). in these patients, egfr. kras and eml4-alk rearrangements may co-exist (yang et al., 2016). even when diagnosed in early stages, these tumors are considered highly malignant, due to their undifferentiated histopathology (ren et al., 2015). otherwise, co-existence of the egfr and kras mutations, if any at all, are expected to be very rare (zhu et al., 2014). in india, the incidence of eml4alk lung cancers is 3 % (versus 33 % for egfr mutation-induced lung cancers). these eml4-alk tumors retained high sensitivity to crizotinib at 250 mg twice daily, as measured by 72 % progressionfree survival at 7 months and 77 % and 64 % overall survival at 1 and 2 years (doval et al., 2015). crizotinib is the specific inhibitor of alk/alk and is the therapy of choice for the elm4/alk tumors. erbb ligand (erythroblastic mouse leukemia retroviral growth factor human homolog) expressor eml4-alk tumors gain crizotinib resistance (kimura et al., 2015). eml4-alk tumors express the cd133 stem cell marker and the extracellular signal regulated erk pathway. these tumor cells undergo epithelial-to-mesenchymal emt transitions (guo et al., 2015). when stemness-associated molecules rise: aldh, aldehyde dehydrogenase, human sarcoma stem cell marker (lohberger et al., 2012); nanog (never aging celtic tribe); oct4 (octamerbinding transcription factor), the tumor cells acquire crizotinib resistance. in this case, rapamycin combined with crizotinib induces a synergistic action (oh et al., 2015). a second generation alk/alk tyrosine kinase inhibitor ap26113 (ariad pharmaceuticals) is in clinical trial at the university of milano-biocca, italy (ceccon et al., 2015). explanation: the human fusion oncogene/oncoprotein npm-alk/npm-alk (nucleolar phosphoprotein nucleophosmin) at t(2;5)(p23;q35) causes anaplastic large cell lymphoma. retroviral transfer of the fusion oncogene into mice induces the lymphoma (kuefer et al., 1997). discussion the so-called cellular malignant transformation (the clinical diagnosis cancer) is considered here to be the manifestation of an inherent faculty of the original rna/dna complex consisting of a process referred to as a retrograde immortalization of individual stem and/or somatic cells in a highly organized multicellular host organism, including homo. the process of cellular transformation clinically referred to as cancer is the expression of a backward genetic regression to the original level of evolution. that overheated over-irradiated and chemically imbalanced (mainly hyperacid due to an excess sulfuric acids) environment was populated by primordial multi-resistant cells. those were the circumstances at the edge, the unicellular microorganisms, and the diploand triploblastic macroorganisms have had organized themselves into. those cells were endowed with the faculties of reversed ontogenesis (as in cnidaria/medusozoa) and trans-speciation (sporulation of bacteria; encystation of giardia; the dauer phenomenon of caenorhabditis; existence in the state bordering autophagy in the life cycle of dictyostelia; transspeciation of the entire microtubular cytoskeleton in naegleria) with full recovery (referenced in sinkovics, 2016). a great deal of the ancient cell survival pathways remained conserved throughout evolution. the heat shock proteins hsp60s of crenarchaeota appear as predecessors of eukaryotic hsp, as their cell division pathway is eukaryota-like. the mammalian vertebrate descendants of the ancient heat shock proteins of the hyperthermic archaea archaeoglobus fulgidus or haloferax volcanii (rohlin et al., 1995; cox et al., 2008) now chaperon oncoproteins encoded in the human genome (calderwood and gong, 2012). the toxin expulsion pumps of the algae are used by malignantly transformed high level parenchymal cells of multicellular organisms (including homo) to expel chemotherapeutic molecules (referenced in sinkovics, 2015, 2016). oncogenesis induced by extrinsic factors (oncogenic viruses) elicits strong immune defense reactions. whereas, endogenously 72   induced by inserted retrotransposons, the so-called malignant transformation of individual selected cells receives full support consisting of vascularization; micrornas-directed chemokines, lymphokines, cytokines; and growth factors of 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patients with lung adenocarcinoma. thorac. cancer 5: 411-416, 2014. 75   131 isj 15: 131-140, 2018 issn 1824-307x research report effects of dietary botanical and synthetic astaxanthin on e/z and r/s isomer composition, growth performance, and antioxidant capacity of white shrimp, litopenaeus vannamei, in the nursery phase l xiaohui1,2,3, w baojie1,2, l yongfu1,2, w lei1,2, l jianguo1,2,* 1cas key laboratory of experimental marine biology, institute of oceanology, chinese academy of sciences, qingdao 266071, china 2laboratory for marine biology and biotechnology, qingdao national laboratory for marine science and technology, qingdao 266071, china 3university of chinese academy of sciences, beijing 100049, china accepted march 26, 2018 abstract astaxanthin (ast) is a beneficial dietary supplement in shrimp farming. however, the different effects of synthetic ast (s-ast) and natural ast (n-ast) on the postlarvae is unclear. these effects were compared by supplementation with a gradient of ast (0, 50, 70, 90, 140 ppm) for 35 days. growth parameters including weight and length gain, carotenoid content, ast content and isomer composition, antioxidant capacity (superoxide dismutase, catalase, glutathione peroxidase), survival rate and mrna expression levels for antioxidant enzymes in litopenaeus vannamei postlarvae were measured. the results indicated that postlarvae given n-ast had significantly higher growth performance and ast content. although there were different ratios of ast optical isomers in the diets, there was a similar optical isomer content in juvenile shrimp. the cis/trans ast ratio and antioxidant enzyme (sod and gpx) activities in n-ast90 were the highest among all groups. cumulative mortality in n-ast90, n-ast140 and s-ast70 were significantly lower than in the control and n-ast50. the mrna expression levels for the antioxidant enzymes (cmnsod and gpx) also increased for n-ast90-fed shrimp under stress conditions. these results suggest that an epimerase may exist in vivo and that an appropriate level of botanical ast in diets was approximately 90 ppm for l. vannamei postlarvae. key words: astaxanthin; l. vannamei postlarvae; vibrio introduction the pacific white shrimp, litopenaeus vannamei, constitutes a considerable part of the cultured shrimp around the world (liu et al., 2014). environmental stresses, i.e. intensive culture and environmental deterioration, induce the generation of excessive reactive oxygen species (ros) in crustaceans, and lead to disease (flegel, 1997; cesaratto et al., 2004; pelicano et al., 2004; ji et al., 2011). ast, the dominant carotenoid pigment found in shrimp, cannot be synthesized de novo by the animals themselves (yamada et al., 1990). it must ___________________________________________________________________________ corresponding author: jianguo liu key laboratory of experimental marine biology chinese academy of sciences laboratory for marine biology and biotechnology qingdao national laboratory for marine science and technology qingdao 266071, china e-mail: jgliu@qdio.ac.cn be derived from their feed. ast can reduce oxidative damage generated under stress conditions through its powerful antioxidant properties (supamattaya et al., 2005; wang et al., 2006; díaz et al., 2014). this ast property has generated considerable interest in the pigment and its potential functions. this interest has prompted us to analyze the impacts of ast on the physiological and biochemical characteristics of shrimp and on pathogen resistance in shrimp. ast is a red pigment widely applied in crustacean and salmonid aquaculture as a feed additive (chien et al., 2003; choubert et al., 2006; félixvalenzuela, 2006). among other things, it can regulate immune responses and improve disease resistance (zhang et al., 2013). synthetic ast is most commonly used for feed supplementation, and there are only a few reports related to the use of botanical ast in shrimp aquaculture. it has been shown that dietary supplementation with botanical ast (derived from haematococcus pluvialis) can significantly influence immune indicators in the mailto:jgliu@qdio.ac.cn 132 hemolymph of l. vannamei, increase the expressions of sod and gpx in the hepatopancreas, and increase resistance to white spot syndrome virus (wssv) infection (wang et al., 2015). it has also been reported that botanical ast has a positive impact on the color, weight and antioxidant ability in shrimp; however, the optimum concentration differs widely among species and developmental stages (parisenti et al., 2015). some studies have suggested that dietary supplementation with synthetic ast enhances growth in penaeus monodon and shortens the intermolt stage for postlarvae of the prawn penaeus japonicas (torrissen, 1995; petit, 1997). putman (1992) believed that supplementation with synthetic ast to increase pigmentation would increase consumer acceptance, as well as accelerate sexual maturity, improve fertilization and egg survival, and embryo development. little is known about any difference between botanical and synthetic ast when used as dietary supplementation for l. vannamei postlarvae. a previous study found that supplementation with h. pluvialis cell powder had no significant effects on survival and ovarian development, but did enhance shrimp coloration, antioxidant levels in tissues, and improved the proximate composition of female eriocheir sinensis (long et al., 2017). botanical and synthetic ast showed no significant differences in comparisons except for flesh ast content (chien and shiau, 2005). here, using h. pluvialis powder and synthetic ast as the feed additives, we investigated the growth performance, ast and carotenoid contents and antioxidant parameters in growing l. vannamei juveniles. the aim of this study was to provide practical suggestions for diet formulation and quality improvement of reared postlarvae. materials and methods experimental diets experimental groups were fed a basal diet supplemented with various amounts of haematococcus pluvialis cell powder (yunnan alphy biotech co., ltd, china), designated as n-ast50 (with 50 ppm carotenoids), n-ast90 (with 90 ppm carotenoids), n-ast140 (with 140 ppm carotenoids). in addition, there was one negative control group (no ast added) and one positive control group with synthetic ast (carophyll® pink 10%, dsm, s-ast70, with 70 ppm carotenoids). the proximate compositions, total carotenoids and ast contents of all diets are presented in table 1. all ingredients were completely mixed together and water was added to shape pellets. subsequently, the pellets were air-dried at room temperature and then kept at -18 °c until used. the ast content and composition of diets were analyzed using high-performance liquid chromatography (hplc, agilent 1200; agilent technologies, santa clara, ca, usa) according to the protocol of yuan (ronneberg et al., 1980; okada et al., 1994). the chromatograms of diets obtained using silica and chiral columns are presented in fig. 1e and fig. 1f. the data indicated that the cis/trans ast ratios were 0.13 and 0.2 in s-ast70 and n-ast diets, respectively. the ast optical isomer ratio in n-ast was 3s, 3’s : 3r,3’s : 3r,3’r = 30:3:1, and that in the s-ast70 diet was 6:2:1. experimental design litopenaeus vannamei postlarvae with initial weights of 0.01 ± 0.001 g (mean ± sd) were pre-cultured at the xindadi aquaculture co., ltd, weifang, china. subsequently, postlarvae were transferred into indoor plastic drums (length × width = 1 m × 1.2 m) with 800 l water, and divided into five groups, each in triplicate (each replicate had 2000 individuals). the postlarvae were fed five times daily at 5:00, 9:00, 13:00, 17:00, and 21:00 with an initial ration of approximately 1.6 g/10,000 individuals, which was increased 20% every day, for 35 days. diets were slightly adjusted, based on the feeding response, water temperature and residual feed. a water depth of 70 cm was maintained and 30% of the water was refreshed in the second week. the ph, salinity, water temperature, ammonia-nitrogen and dissolved oxygen were maintained at 7.8 8.2, 24 27‰, 28 31 °c, 0.06 0.08 mg/l, and 8.0 9.0 mg/l, respectively. after 35 days, juvenile shrimp were fasted for 24 h, and weighed. then half of the juvenile shrimp were stored in liquid nitrogen for future analysis. the remaining animals were challenged with vibrio parahaemolyticus as described below. table 1 proximate composition, total carotenoid and astaxanthin contents of experimental diets (% dry weight) items control s-ast70 n-ast50 n-ast90 n-ast140 proximate composition ash (%) 16.00 16.00 16.00 16.00 16.00 moisture (%) 8.00 8.00 8.00 8.00 8.00 crude protein (%) 37.79 37.59 37.62 37.62 37.62 crude lipid (%) 8.50 8.32 8.32 8.32 8.32 carotenoid (mg/kg) 18.35 74.38 47.59 95.19 142.78 astaxanthin (mg/kg) 0.40 26.27 22.94 45.87 68.81 note: n-ast50, 90 and 140 indicate that the final carotenoid contents are 50 ppm, 90 ppm and 140 ppm, respectively. s-ast70 means the final carotenoid content is 70 ppm. 133 fig. 1 the carotenoid and astaxanthin contents (a), cis/trans isomer ratios (b), astaxanthin geometrical isomer percentages (c), astaxanthin optical isomer percentages (d), hplc chromatograms of three diets using a silica column (e) and hplc chromatograms of diets using a chiral column (f), in litopenaeus vannamei postlarvae after a 35-day feeding trial. the significant differences are indicated by different superscripts (a, b, c, d, e and f) (p < 0.05, n = 3). determination of carotenoids and astaxanthin content and composition the surface water on thawed juvenile shrimp was removed using paper towels and then the animals were weighed and placed into 10 ml centrifuge tubes. subsequently, the juvenile shrimp were cut into fragments with operating scissors and homogenized with an s10 homogenizer (ningbo xinzhi biotechnology co., ltd). total carotenoids were extracted with acetone and measured with a uv-vis spectrophotometer (shimadzu, kyoto, japan) at 478 nm, according to the method of johnston (johnston et al., 2000). after enzymatic digestion with cholesterol esterase (ec 3.1.1.13, wako, japan), ast was measured by hplc at 478 nm following the protocol of yuan (ronneberg et al., 134 1980; okada et al., 1994). ast geometrical isomers were separated using a silica column (4.6 mm × 150 mm, particle size = 5 μm, phenomenex, inc. usa), with a mobile phase of hexane and acetone (83:17, v/v). ast optical isomers were analyzed with a chiral column (460 mm × 250 mm, particle size = 5 μm, daicel chiral technologies co., ltd, shanghai, china), with a mobile phase of acetonitrile and methyl t-butyl ether (65:35, v/v). the injection volume for both was 60 μl at a flow rate of 1.0 ml/min. assessment of immune indices each thawed hepatopancreas was placed into 2 ml pre-cooling physiological saline solution and homogenized in an ice bath with an s10 homogenizer, and then centrifuged at 1,700 g for 10 min at 4 °c to obtain supernatant for further analysis. the activities of sod, cat and gpx in the hepatopancreas were measured with a spectrophotometer at room temperature at 550 nm, 405 nm and 412 nm respectively, according to the manufacturer’s instructions. the enzyme detection kits were provided by nanjing jiancheng bioengineering institute (jiangsu, china). challenge with v. parahaemolyticus and expression of cmnsod and gpx mrnas on the 35th day, the hepatopancreases of three juvenile shrimp from each drum were sampled and placed in rnastore reagent (takara 9750; takara bio, tokyo, japan) and then stored in liquid nitrogen for later examination of the mrna expression levels of cmnsod and gpx. then, 60 randomly selected, juvenile shrimp from each group were placed into 25 l plastic drums with 15 l water. v. parahaemolyticus was added to the water every 24 h to maintain the bacterial density at 3×108 cfu/ml. the dead shrimp were counted every 24 h. after this, the hepatopancreases of three live juvenile shrimp from each drum were sampled for further analysis of mrna expression levels. hepatopancreases from animals not challenged with v. parahaemolyticus were taken as control. total rnas were extracted with rnaiso plus (takara 9750; takara bio, tokyo, japan) according to the manufacturer’s instructions. the rna concentration and purity were verified with a nanodrop 1000 spectrophotometer (thermo fisher scientific inc., ma, usa) (a260/a280) and its integrity was quantified by electrophoresis on a 1% agarose gel. the cdna synthesis was conducted using a primescript™ rt reagent kit with gdna eraser (perfect real time) (takara 9750; takara bio, tokyo, japan) based on the manufacture’s instructions. primers of each gene were designed based on the published l. vannamei cdna sequence using primer 3 software as presented in table 2 (wang et al., 2017). all primers were produced by ruiboxingke biological technology co., ltd. (qingdao, china). reaction conditions were also optimized. real-time quantitative rt-pcr was executed using sybr®premix ex taq™ⅱ (tlirnaseh plus) (takara rr820a; takara bio, tokyo, japan) according to the manufacture’s instructions. after amplification, a melting curve analysis verified that there was only one amplified product. expression levels of genes were calculated with the comparative ct method (2 -δδct), and the values mean n-fold difference was compared with the control. data analysis statistical analyses were performed with ibm spss statistics 19.0 (spss, chicago, il, usa). data were expressed as mean ± standard derivation (sd). one-way anova was used to examine whether significant differences existed among the groups. homogeneity of data was determined with the duncan’s multiple range test. a probability (p) value < 0.05 was regarded as significant. results total carotenoid content and astaxanthin composition the carotenoid content of all experimental groups (i.e. control, s-ast70, n-ast50, n-ast90 and n-ast140) showed no significant differences; however, all values were higher (p < 0.05) than that of the control (fig. 1a). the average ast level of postlarvae fed n-ast140 was higher than all other diets. the ast content of postlarvae fed n-ast90 and n-ast140 were significantly higher (p < 0.05) than that of postlarvae in other groups, particularly for n-ast140, in which the ast/total carotenoid ratio was the highest. table 2 primers for real-time quantitative rt-pcr gene name genbank number reference primer sequence (5'-3') annealing temperature (°c) product (bp) cytosolic manganese superoxide dismutase (cmnsod) dq005531 gomez-anduro et al. (2006) (f)atcactcacggactggttcc (r)gagagaaacgcccttgtgac 59 219 glutathione peroxidase (gpx) ay973252 fu et al. (2007) (f)cgtgcaaaaaggaccttggg (r)atacgcgatgcccctaacac 54.5 231 β-actin af300705y sun et al. (2007) (f)gtgcccatctacgagggata (r)taggacttctccagcgagga 56.5 233 135 fig. 2 the weight and length gains of l. vannamei postlarvae after a 35-day feeding trial. data with different letters (a, b, c, and d) are significantly different (p < 0.05, n = 30). quantification of astaxanthin geometrical isomers among the main ast geometrical isomers (i.e. all-trans, 9-cis and 13-cis), the all-trans isomer was the dominant form in postlarvae (fig. 1c). the proportion of the 9-cis isomer presented in postlarvae fed n-ast90 was significantly higher (p < 0.05) than that of others. the proportion of the trans isomer found in postlarvae fed n-ast140 was significantly higher than that for the other diets (p < 0.05). the 13-cis isomer of postlarvae showed no significant differences (p < 0.05) between control and experimental groups. the cis/trans ratio was highest in n-ast90 (fig. 1b). quantification of astaxanthin optical isomers the optical isomers of ast in white shrimp postlarvae include the 3s,3’s isomer, 3r,3’s meso form and 3r,3’r isomer, among which the 3s,3’s isomer and 3r,3’s meso form are the major forms. the 3s,3’s isomer of postlarvae fed n-ast140 was significantly higher (p < 0.05) than in other groups. the 3r,3’s meso content of postlarvae fed n-ast90 and s-ast70 were significantly higher (p < 0.05) than they were in others. the 3r,3’r isomer contents of postlarvae of all experimental groups were similar (fig. 1d). growth performance with different supplemented diets the weight and length gain of shrimp postlarvae fed botanical ast were both higher than those of the control and s-ast70 after 35 days (fig. 2). no significant differences for these two parameters was found between s-ast70 and the control. the weight gain and length gain had no significant differences among n-ast groups; however, the average weight and length gain both increased with increasing ast concentration but with only weight gain for animals fed n-ast140. antioxidant enzyme parameters although the results were not uniform, all the highest values for antioxidant enzyme activities were found in the n-ast groups. sod, cat and gpx activities were increased over the control (p < 0.05) the most in n-ast90, n-ast140 and n-ast90, respectively (table 3). table 3 hepatopancreas antioxidant enzyme activities of litopenaeus vannamei postlarvae after a 35-day feeding trial. values are mean ± standard deviation (sd). mean values in the same row for each type of experiment with different letters (a and b) are significantly different (p < 0.05, n = 3). dietary treatments hepatopancreas parameters control s-ast70 n-ast50 n-ast90 n-ast140 sod (unit mg-1 protein-1) 10.53±0.92a 12.89±0.73ab 14.07±2.33ab 17.32±1.71b 10.64±1.22a cat (unit mg-1 protein-1) 4.48±0.47a 4.72±0.89a 4.60±0.61a 5.10±1.31a 7.93±1.18b gpx (unit mg-1 protein-1) 123.63±5.95a 136.44±12.99a 109.08±4.79a 197.76±40.29b 108.24±14.15a note: n-ast50, 90 and 140 indicate that the final carotenoid contents are 50 ppm, 90 ppm and 140 ppm, respectively. s-ast70 indicates the final carotenoid content is 70 ppm. 136 fig. 3 the cumulative mortality (%) of l. vannamei postlarvae after being challenged with v. parahaemolyticus. postlarval challenge with v. parahaemolyticus and expression of cmnsod and gpx mrnas after being challenged with 3×108 cfu/ml v. parahaemolyticus for 72 hours, the cumulative moralities of postlarvae in all experimental groups were determined (fig. 3). the cumulative morality of postlarvae in n-ast90 and n-ast140 were significantly lower (p < 0.05) than that for postlarvae in other groups. the cmnsod mrna expression levels of unchallenged postlarvae in s-ast70 and all levels of n-ast were significantly increased (p < 0.05) compared with the control. its expression level was not significantly different (p > 0.05) among groups (fig. 4a). the gpx mrna expression levels of unchallenged postlarvae were significantly higher (p < 0.05) than the control, with the highest in n-ast90. its expression level was not significantly different (p > 0.05) in any of four groups receiving ast (s-ast70, n-ast50, n-ast90 and n-ast140) (fig. 4b). postlarvae in 15 l of water were challenged for 72 hours with 3×108 cfu/ml v. parahaemolyticus. the cmnsod mrna expression levels of challenged postlarvae in all experimental groups were significantly down-regulated (p < 0.05) compared to levels in the unchallenged animals. its expression level in postlarvae fed n-ast90 was significantly less downregulated (p > 0.05), but other groups had no significant differences (p > 0.05) from each other after being challenged with v. parahaemolyticus (fig. 4a). the gpx mrna expression levels of challenged postlarvae in all experimental groups also were significantly down-regulated (p < 0.05). its expression level in postlarvae fed s-ast70 was significantly more downregulated (p < 0.05) than the others, which showed no significant differences from each other after being challenged with v. parahaemolyticus (fig. 4b). discussion ast is a valuable dietary ingredient for crustacean juveniles and plays a prominent role in intermediary metabolism (niu et al., 2012; daly et al., 2013). the exact mechanism making botanical ast better than synthetic ast from a growth perspective is not completely understood. according to studies, 6-7 hours after oral intake of trans ast, the ast concentration in plasma is significantly elevated, with the proportion of the all-trans configuration diminished while the cis configuration increased, especially 13-cis (qsterlie et al., 2000). the cis configuration, especially 9-cis, has demonstrated a higher antioxidant capacity in vitro as compared with the all-trans configuration (liu and osawa, 2007). this indicates that the cis/trans ratio can be an indication of antioxidant ability. the cis/trans ratios in s-ast70 and the various n-ast diets were 0.13 and 0.2, respectively. in terms of thermodynamic structure, the trans configuration is more stable, whereas the cis, with higher antioxidant capacity, is more active (britton, 1995). the cis isomer level was 137 fig. 4 sod, cat and gpx mrna expression profiles of l. vannamei postlarvae fed various experimental diets. the animals were examined by rt-pcr before and after being challenged with v. parahaemolyticus. the actin gene was used for internal calibration. vertical bars represented the mean ± sd. significant differences are indicated with the different letters (a, b, a and b) (p < 0.05, n = 3). higher in the n-ast diets; however, no significant increase of the cis isomer was found in postlarvae fed n-ast. we suppose that the cis isomer is consumed directly to neutralize existing oxidant stress in the postlarvae. the results showed that the cis/trans isomer ratio was significantly higher in animals fed n-ast90, not n-ast140. it is presumed that the 9-cis ast might, as an antioxidant, participate directly in immune reactions without configuration transformation in vivo. accordingly, the 9-cis isomer content was lower in animals fed diet n-ast140 than in animals fed the other diets. in this study, the ratio of the optical isomers 3s,3’s : 3r,3’s : 3r,3’r in the n-ast diets was about 30:3:1, and that in the s-ast70 diet was 6:2:1. the ratio in the diets with n-ast agrees with previous studies (grung et al., 1992; turujman et al., 1997). however, the ratio in the diet with s-ast70 was not in accord with previous reports on synthetic ast, where the 3s,3’s : 3r,3’s : 3r,3’r ratio was ca. 1:2:1 (lorenz and cysewski, 2000). however, the distribution of these ast optical isomers in juvenile shrimp was 5:4:1 to 3:3:1, which was not so extremely different as that in the diets. the results above strongly indicate that juvenile shrimp might have isomerization ability in addition to selective absorption and deposition. carotenoids usually can hydroxylate into ast irreversibly. in this connection, existing ast might be involved in dehydroxylation first, and then hydroxylates again in vivo. it is likely that an epimerase exists in the ast metabolic pathway in juvenile shrimp. however, in the absence of additional evidence, this possibility needs further examination. several previous studies have found that dietary supplementation with h. pluvialis powder significantly enhances the length and weight gain of l. vannamei (chuchird et al., 2015; parisenti et al., 2015). p. monodon had a higher weight gain through supplementation with botanical ast and capsanthin (supamattaya et al., 2005). this study demonstrates that dietary supplementation with n-ast90 and n-ast140 significantly enhances both length and weight gain of l. vannamei juveniles. a previous study found that dietary supplementation with 50 ppm synthetic ast had no significant influence on the growth of p. monodon juveniles (boonyaratpalin et al., 2001). it has also been reported that neither 50 ppm of botanical ast nor 100 ppm of synthetic ast had a significant effect on the weight gain of kuruma prawn (marsupenaeus japonicus) postlarvae (chien and shiau, 2005). this situation is consistent with part of the present study in that no significant difference was found between the growth performance of control and shrimp fed s-ast70. however, the present study found that n-ast50 had a significant effect on shrimp growth performance. according to a previous report, ast was preferable to canthaxanthin because it produced nature-identical pigmentation and was more efficiently deposited (storebakken et al., 1987). it is highly likely that the different ast types, presented in diets, were selectively absorbed, which may also help to explain why the ast content of juvenile shrimp in n-ast50 was higher than that of s-ast70. alternatively, it can be assumed that n-ast in the supplementation can be better assimilated, converted and deposited in juvenile shrimp. it is unclear why weight gain of animals in the n-ast140 treatment appeared to be slightly less (but not significantly so) than in the n-ast50 treatment. excessive absorption of dietary carotenoids might lead to side effects such as reported for female e. sinensis (long et al., 2017). petri and lundebye (2007) proved that the organ distribution content of high doses of ast in rats after oral application was not increasing all the time. ast beyond some threshold may generate side effects, including energy-consumption during metabolism and excretion. the slightly lower weight gain may be 138 related to this. this interesting phenomenon needs to be investigated further. our results suggest that dietary supplementation with botanical ast was more effective than with s-ast in promoting postlarval growth and ast accumulation. sod, cat and gpx are major endogenous antioxidant enzymes. their expression levels reflect the level of reactive oxygen species, and protect tissue cells from oxidative damage (liu et al., 2007). higher sod values indicate that there are more superoxide radicals in cells that need to be removed. higher cat and gpx values reflect that there is more h2o2 in tissues that needs to be eliminated. overall, the antioxidant activity generated by n-ast was relatively higher than that by s-ast70. the level of cis geometrical isomers, known to have greater antioxidant activity than the all-trans isomers, was higher in botanical ast than in synthetic ast. sod and cat activities were both significantly decreased with increasing concentrations of dietary ast in hyphessobrycon callistus and l. vannamei (wang et al., 2006; zhang et al., 2013). some studies have indicated the ast has more powerful ros quenching activity than cat and sod, and that it could protect individuals from oxidative damage. however, the study also indicated that sod, cat and gpx activities of e. sinensis all exhibited a tendency of “low-high-low” with gradient concentrations of the h. pluvialis powder (long et al., 2017). in the present study, sod and gpx activities of juvenile shrimp both presented the “low-high-low” profile as well. cat activity of juvenile shrimp fed n-ast140 was significantly higher than in the other groups. these results suggest that appropriate levels of dietary botanical ast could enhance the expression of the enzymes. moreover, ast itself can eliminate ros, which would decrease the substrate level for the antioxidant enzymes (chuchird et al., 2015). the lower levels of substrates might lead to the decreased expression of antioxidant enzymes (gpx). ast participates in various metabolic processes, but it should be maintained at a proper level. higher ast concentration is not necessarily better in the cultivation of shrimp. in the present study, after the l. vannamei were challenged with v. parahaemolyticus, the cumulative mortality of juvenile shrimp fed n-ast90 and n-ast140 were significantly lower (p < 0.05) than that for other treatments. according to a previous study, provision of synthetic ast in the diet increased the survival of l. vannamei and significantly improved total hemocyte count, phagocytosis activity, phenoloxidase activity, and sod activity (chuchird et al., 2015). this is in the agreement with our results. the immune protective effects of ast might be attributable to its antioxidant property to counteract the stress from pathogens. our results strongly indicate that dietary supplementation with ast improves the tolerance of juvenile shrimp to the vibrio stress. growth performance and resistance to stress are important considerations for industrial aquaculture, but diet cost must also be considered carefully. thus, the n-ast90 diet would be preferable overall. the sod enzymes constitute a first-line defense that transforms superoxide anions (o2·-) into hydrogen peroxide (h2o2) and oxygen molecules. it plays a prominent role in the oxidative damage defense system (campacórdova et al., 2002; parrilla-taylor and zenteno-savín, 2013). in the present study, postlarvae fed n-ast or s-ast70 had higher expression levels of cmnsod mrna than the control under normal conditions. after being challenged with v. parahaemolyticus, cmnsod mrna expression levels were significantly reduced, usually below the control level. among these experimental groups, postlarvae fed n-ast90 had significantly higher (p < 0.05) mrna expression levels than other groups. gpx removes the h2o2 produced by the first-line defense system, thus helping to neutralize the effects of oxidative stress. gpx, even at a low concentration, can transform h2o2 (nordberg and arnér, 2001). the postlarvae fed n-ast90 had slightly higher gpx mrna expression levels than the control animals and animals fed other rations under normal conditions. after being challenged with v. parahaemolyticus, the expression levels of gpx significantly decreased as compared to the unchallenged animals. postlarvae fed n-ast had higher gpx mrna expression levels than s-ast70 fed animals, and the expression tendency was similar to that of cmnsod mrna. these results indicate that dietary botanical ast effectively relieves environmental oxidative damage through higher gene expression levels of antioxidant enzymes, which could counteract stress induced by reactive oxygen species in l. vannamei postlarvae. conclusions in summary, different ratios of ast optical isomers in diets had no significant effects on juvenile shrimp. this result suggests that an astaxanthin epimerase may exist in vivo. dietary botanical ast can improve growth performance, ast content and resistance to pathogenic bacteria. n-ast50 appeared to have a more significant effect than s-ast70 except cumulative mortality, although their ast concentrations are similar. our present data indicate that the appropriate concentration of botanical ast in diets is around 90 ppm for l. vannamei postlarvae. acknowledgments this work was supported by national natural science foundation of china (no. u1706209 and 31572639). we are also particularly indebted to dr. john van der meer, pan-american marine biotechnology association, for 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palermo, italy 2ecologie des systèmes marins et côtiers (ecosym), université montpellier 2-cnrs, cc 093, place e. bataillon, f-34095 montpellier cedex 05, france 3laboratory of experimental ecology and behaviour department of earth and marine sciences, university of palermo, italy accepted october 5, 2015 abstract immunological effectors of invasive species playing a role in addressing new colonization are still poorly studied. in the present study the cdna sequence of the defensin from a lessepsian invasive species, the red sea mussel brachidontes pharaonis, was cloned using race method. defensins are a class of widely known antimicrobial peptides (amps), oligopeptides with a broad spectrum of targeted organisms ranging from viruses to parasites. analysis of bpdef sequence (262 bp) revealed the presence of an orf coding for 81 amino acids. the full-length amino acid sequence showed the highest similarity to antimicrobial peptides mgd1 and mgd2 sequence from mytilus galloprovincialis. phylogenetic analysis suggested that bpdef belongs to the αβ defensin amps with a typical domain structurally characterized by a α helix and two β sheets. bpdef mrna is located in circulating hemocytes with small intra-cytoplasmic granules and with large granules. the transcription of defensin gene was modulated by the stress from temperatures and oxygenation condition. temperatures of 20 °c did not stimulate a bpdef transcription over a short time. at 30 °c the kinetics of bpdef gene transcription showed up regulation after one day, while it was down regulated after six days, both under normoxia and hypoxia conditions. key words: brachidontes pharaonis; antimicrobial peptides (amps); defensin; lessepsian mussel; environmental stress effect   introduction biological invasions are considered one of the main concerns worldwide they elicit ecological modifications (mostly detrimental) to ecosystem dynamics with tangible economic rebounds on local societies (ojaveer et al., 2015). invaders act by competing for space with native species, often intercepting trophic resources (carlton, 1992, 1999; garci et al., 2007). nevertheless, also invasive species, once arrived in a new recipient site, need to cope with new, often stressful, environmental conditions. exactly like all the other species, invaders need to deploy all possible arms to cope with new recipient conditions; their success within ___________________________________________________________________________ corresponding author: matteo cammarata laboratory of marine immunobiology department of biological chemical and pharmaceutical sciences and technologies university of palermo, italy e-mail: matteo.cammarata@unipa.it the naïve community will depend on their ability to adapt and keep up individual fitness as high as possible (lockwood et al., 2009, pascual et al., 2010; barbarro and abad, 2013). barbarro and comeau (2014) reported adaptation capacity of xenostrobus securis that ensure its dissemination within estuarine environments. most studies have examined the ability of invaders to occupy a new site by analyzing the role of functional and lifehistory traits (sarà et al., 2013) and several other ecological features, mainly through their effect on density-dependent processes affecting the persistence of invasive populations over time (lockwood et al., 2005). the shift in dominance of some specie (gestoso et al., 2013) can alter habitat structure and complexity with effects on trophic web relations. in contrast, few data have been gathered on sub-organismal (e.g., cellular) “arms” of invasive species that often represent the first line of adaptation which increases the probability of colonization of new geographic areas. 264 in particular, scantly data has been produced investigating the role of ecoimmunology in invasion biology; moreover, how immunological effectors of invasive species play a role in addressing new colonization is still poorly studied (lee and klasing, 2004). when an organism is subjected to stressful conditions as might be expected in a novel recipient site (thousands of miles far from the core of its biogeographic area), the first line of response aims to cope with new variable conditions by modifying immunological, biochemical, physiological and behavioral traits. here we chose the pharaonic mussel brachidontes pharaonis as a model species to study the ecoimmunology of invasive species. the pharaonic mussel is one of the 100 most aggressive alien 75 species in the mediterranean sea: it is a lessepsian (i.e., coming from the red sea through the suez canal) invasive species and it is currently spreading through the shallow intertidal zones of the western mediterranean basin (sarà et al., 2013). this species is a reliable model to infer on the role of immunological traits in coping with highly variable abiotic conditions of novel recipient habitats. in shallow waters, this species should be able to cope with extremely varying thermal conditions (from 5 °c to 32 °c) and water oxygen levels, often changing from normoxia to anoxia in only a few hours during nighttime and calm wind (sarà, 2009). here, we focused on the immunological response by antimicrobial peptides (amps). amps are very important components of natural defenses in most living organisms. these peptides are geneencoded, relatively small (< 10 kda), amphipathic (boman, 2003; sang and blecha, 2008). amps constitute the first line of innate immunity for mollusks exposed to various potential pathogens in aquatic environments. amps have been isolated from a wide variety of animals and plants (reddy et al., 2004) as well as bacteria and fungi, and they exhibit a broad-spectrum activity against grampositive and gram-negative bacteria, protozoa, yeasts, fungi, and viruses. in molluscs, amps have been characterized mainly in marine bivalves, such as the mussels mytilus edulis and mytilus galloprovincialis (charlet et al., 1996; mitta et al., 2000a). using biochemical approaches and molecular cloning, four families of cysteine-rich amps have been identified in mytilus spp.: defensins, myticins, mytilins, and mytimycin (mitta et al., 2000b; balseiro et al., 2011; li et al., 2011). defensins are relatively short antimicrobial peptides (12 100 amino acids in length) that are widely distributed in single-celled microorganisms, plants, invertebrate and vertebrate species (jenssen et al., 2006). defensins respond rapidly to invading microorganisms by forming pores or otherwise disrupting the microbial membrane (tincu and taylor, 2004). they can interact with the microbial dna or rna after penetrating the microbial membrane (boman et al., 1993; park et al., 1998). cationic defensins involved in host defense have been linked to acute inflammation (tincu and taylor, 2004). many defensins with a toxic action against prokaryotes appear relatively nontoxic in eukaryotes and some of them also have antitumor and antiviral activities (chen et al., 2001; zang et al., 2002). defensins are very attractive as potential therapeutic agents for pharmaceutical or agricultural applications because of their small size, which allows easy synthesis, their mode of action, broad spectrum activity, highly selective toxicity and their ability to generally circumvent bacterial resistancedevelopment mechanisms (jenssen et al., 2006). thus, the main objectives of the present study were to: (i) clone the full-length cdna of the defensin from b. pharaonis (bpdef), (ii) examine the structural characteristics of the deduced amino acid sequence and investigate the cellular localization of mrna for bpdef, and (iii) evaluate the quantitative gene expression profile of gill bpdef under thermal stress and hypoxia conditions. materials and methods sampling, animal maintenance, experimental treatments and cell preparation about 300 adult brachidontes pharaonis (2.3 ± 0.2 cm umbonal length) were collected in may 2014 from ponds in western sicily (salina ettore; lat.12°27’34” e; long. 37°51’42”n). once brought back to the laboratory, animals were acclimated for one month in a flow-through system of oxygenated (by aeration; dissolved oxygen [do] about 8 mg l-1) seawater at 20 °c, before starting experiments mirroring the seawater features of the sampling location (ezgeta-balic et al., 2013). water in the tanks was continuously aerated and changed daily, and specimens were fed regularly (3 times a day) with monoalgal culture of isochrysis galbana. laboratory procedures were designed to evaluate the immunological effects of sudden oxygen depletion at two different temperatures that b. pharaonis could experience in late spring and summer in shallow ponds at these latitudes. two different temperatures were applied in our experiments: 20 °c as a control temperature (mirroring that of pond seawater at the time of sampling and coincident with the water temperature in the acclimatization tanks) and 30 °c, which represents the mean of the maximal water temperatures recorded in the pond in late summer (sarà et al., 2000). thus, animals were divided into four groups: two groups were left at 20 °c, and two were moved to separated aquariums in which the water temperature was increased (through submergible heaters; tetratec ht 200w) by about 1 °c for 6 8 h to reach 30 °c in about three to four days. once 30 °c was reached, animals were acclimated at this temperature for at least two weeks. in the combination of hypoxia and 30 °c we have registered 4 % of mortality after 1 week. in the pond, and generally in shallow waters at this latitude, do can suddenly drop from normoxia (7 9 mg l-1) to anoxic and hypoxic conditions (0 2 mg l1; sarà g, pers. obs.), particularly during the night and calm wind conditions, but usually hypoxic events are never longer than 8 9 h. accordingly, two aquariums out of four (one at 20 °c and one at 30 °c) were exposed to a sudden decrease of do, by insufflating gaseous nitrogen (n2). thus, the do concentration was slowly moved from normoxia to hypoxia (to about 1.8 2.1 mg l-1) in not more than 2 h. the hypoxia event was maintained for at least 8 265 h. then the aquaria water was aerated again and the normoxia conditions quickly restored. the other two aquariums (one at 20 °c and one at 30 °c) were left with their initial unaltered normoxic conditions. at 30 °c the control values are referred to the sample acclimatized to 21 °c and moved for 8 h at 30 °c. temperature was measured with type-g thermo-loggers (ibutton; alphamac, inc canada) while do was controlled by the oxygen sensor four times a day. to carry out immunological analyses (see below for details), we sampled hemolymph and gill (see below) from animals (n = 5) from all tanks (treated and controls) into three successive temporal steps: (1) at the end of the hypoxia period about 8 h after the beginning of treatment, this point is considered as “0”; (2) at 24 h after the beginning of treatment and lastly (3) 168 h after the beginning of treatment. hemolymph (about 0.3 ml per mussel) was collected from the posterior adductor muscle with a 1 ml syringe containing 0.2 ml of the anti-coagulant modified alsever’s solution buffer (nacl 0.42 g, sodium-citrate·2h2o 0.8 g, citric acid·h2o 0.055 g, d-glucose 2.05 g, dw 100 ml). hemocytes were pelleted by 10 min centrifugation at 800g, 4 °c, suspended in ms (12 mm cacl2, 11mm kcl, 26 mm mgcl2, 43 mm trishcl, 0.4 m nacl, ph 8.0) and maintained in ice until in situ hybridization was performed. gills dissected from specimens were immediately soaked in rna later tissue collection (ambion, austin, tx), and stored at -80 °c until use. the amount of peptides recovered from bivalve gills suggest that most likely gills, and perhaps other epithelial tissues, naturally express endogenous amps (mercado et al., 2005). b. pharaonis body is too small to recover sufficient material to extract rna from hemocytes or digestive gland. instead mussel gills represent roughly 13 % of the smooth body weight portion. thus, this tissue is a reasonable source for new antimicrobial molecules. cloning and sequence analysis total rna from gills was isolated using an rnaqueoustm-midi kit purification system (ambion, austin, tx) and reverse transcribed by the cloned amv first-strand cdna synthesis kit (invitrogen). amplification was performed with primers (table1) designed from coding sequences from mytilus galloprovincialis of defensin mgd2b (def:af177539.1) (def-f1: tatgaaagcagtattcgtcttg; def-r1: ctccacatcgtcccttctca). the end-point pcr mix contained 1 μl of template, 0.4 μm of each primer, 0.8 mm of dntps and 0.625 u of gotaq polymerase (promega) in 25 μl final volume pcr program started with initial denaturation at 95 °c for 2 min, followed by 40 cycles including 30 sec at 95 °c, 30 sec annealing at 60 °c, 30 s elongation at 72 °c, and 5 min final elongation at 72 °c. a single band of 164 bp in size was detected. the amplicon was cloned into the pcr™ii vector (ta cloning kit, invitrogen) and its sequence revealed 94 % homology with the mgd2 of m. galloprovincialis. the full-length cdna sequence obtained with rapid amplification cdna ends (race) technology, using the generacer kit (invitrogen, usa). the kit allows only the amplification of full-length transcripts and the elimination of truncated mrna from the amplification process. the primers used to study the defensin amp of brachidontes pharaonic are showed in table 1. phylogenetic analysis similarity searches were performed using the fasta algorithm (http://www.ebi.ac.uk/ tools/fasta/). sequences were subjected to multiple alignments using clc workbench 6.4. a phylogenetic tree was made by the neighbor-joining method (nj) after table 1 primers used to study the defensin amp of brachidontes pharaonis name sequence (5’-3’) pcr objective gene racer 5’ cgactggagcacgaggacactga 5’ race def-bp 5’ race ccaccgcatcgtcctggaatggact 5’ race gene racer n 5’ ggacactgacatggactgaaggagta 5’ nested def-bp 5’nested tttggacagccaaacccagcagacg 5’ nested def-bp 3’ race tgtggcgtctgctgggtttgg 3’ race generacer 3’ gctgtcaacgatacgctacgtaacg 3’ race def-bp 3’ nested gcgtctgctgggtttggctgt 3’ nested generacer n 3’ cgctacgtaacggcatgacagtg 3’ nested 266 http://www.ebi.ac.uk/ 1000 bootstrap iterations by using clc workbench 6.4. distances were corrected for multiple substitutions and gap positions were excluded. the accession numbers are as follows: mytilus galloprovincialis mgd1, aad45117.1; mgd2, aad45118.1; mgd2b, aad52660.1; ruditapes philippinarum, aek78067.1; ornithodoros moubata, bac10304.1; ornithodoros moubata def-b, bab41027.1; crassostrea gigas, caj19280.1; drosophila melanogaster, aaf58855.1; bombus terrestris, aci14287.1; apis cerana, ach96412.1; anopheles gambiae, abb00987.1; branchiostoma belcheri,q86qn6.1; ornithodoros puertoricensis, acj04430.1; homo sapiens alpha1, aat68878.1; homo sapiens beta, eaw80482.1; oncorhynchusmykiss beta4, cbb12549.1; oncorhynchus mykiss beta3, cbb12548.1; oncorhynchus mykiss beta2, cbb12547.1; oncorhynchus mykiss beta 1, cbb12546.1; danio rerio beta 2, 001075023; danio rerio beta3, 001075024.1. in situ hybridization assay digoxigenin-11-utp-labeled riboprobes (digriboprobe) were produced according to the instructions of the manufacturer (roche diagnostic, mannheim, germany), and used at a final concentration of 1 µg/ml . the probe was generated by pcr amplifying a cdna fragment of 177 bp of cdna using the forward primer (ggatactgtggtggatggca) and the reverse primer (cgtctgttaatagagctgatcca). after sampling and centrifugation to remove anticoagulant, the cells were resuspended at 5.105/ml final concentration in marine solution (ms: 12 mm cacl2; 11 mm kcl; 26 mm mgcl2; 45 mm tris; 38 mm hcl; 0.45 m nacl; ph 7.4). then 100 µl of cell suspension was layered on a slide in replicate, and they were fixed in bouin’s fluid (saturated picric acid:formaldehyde:acetic acid 15:5:1) for 30 min in a humid chamber. after washing in pbst (pbs containing 0.1 % tween 20), the hybridization was carried out overnight with hybridization buffer (50 % formamide, 5x standard saline citrate (ssc), 50 µg/ml heparin, 500 µg/ml yeast trna, and 0.1 % tween 20). incubation temperature was at 42 °c. after washings with pbst followed by 0.3 % ssc/1 % tween 20 (twice for 10 min each), the anti-dig fab-ap (roche diagnostic) 1:500 was added, and, after 1 h incubation at room temperature, washed with pbst. finally, the cells were incubated in 5-bromo-4chloro-3-indolyl phosphate/nitro blue tetrazolium (sigma-aldrich) to reveal the presence of the riboprobe. real-time pcr analysis of bpdef expression animals were held in tanks at temperatures of 21 °c and 30 °c, and at each temperature two groups of animals were held under normoxia and hypoxia (> 2 pp of o2) conditions. the experimental design consisted of measurements over one week, due to a limited number of animals. gills were therefore taken at 0, 24 and 168 h after the heat and hypoxic treatment. gill tissue expression of the bpdef gene was examined by real-time pcr analysis with the sybr-green method (applied biosystems 7500 real-time pcr system). tissue expression was performed in a 25-μl pcr containing 2 μl cdna converted from 250 ng total rna, 300 nm bpdef forward (5’gtggcgtctgctgggttt-3’) and bpdef reverse primers (5’-gaatggacttacaatgtcgatgaca3’), 300 nm 28s forward (5’cacccgacccgtcttgtaa-3’) and 28s reverse (5’-ccatgacttgcgcacatgtt-3’) primers, and 12.5 μl power sybr-green pcr master mix (applied biosystems). the 50 cycles of the two-step pcr program consisted of initial polymerase activation for 3 min at 95 °c followed by a denaturing step at 95 °c for 15 s, and then annealing/extension was carried out at 60 °c for 45 s when the fluorescent signal was detected. each set of samples was run three times, and each plate contained quadruplicate cdna samples and negative controls. the specificity of amplification was tested by real-time pcr melting analysis. to obtain sample quantification, the 2-δδct method was used, and the relative changes in gene expression were analyzed as described in the applied biosystems use bulletin n.2 (p/n 4303859). the amount of bpdef transcript from the various tissues was normalized to partial sequence of 28s in order to compensate for variations in input rna amounts the 28s sequence was cloned from b. pharaonis using primers 28 sfc atccgtaaagacccgcctg and 28src (cagaatcgctacggacctcc). relative bpdef expression was determined by dividing the normalized value of the target gene in each tissue by the normalized value obtained from the untreated tissue. statistical analysis qrt-pcr data are presented as the means ± se. differences among tested groups and the control groups were analyzed by a one-way anova with post-hoc student-newman-keuls test. significance was accepted at the level of p < 0.05. results cdna cloning and sequencing of the bpdef in b. pharaonis a fragment of 164 bp was obtained after amplification and cloning using primers previously designed on the sequencemgd2 (af177539.1 205) of m. galloprovincialis. the full-length cdna of bpdef was obtained by the rapid amplification of cdna ends (race) method (fig. 1). analysis of the sequence of 262 nucleotides revealed the presence of an orf coding for 81 amino acids with a predicted molecular weight of 9.054 kda. analyses carried out with the program signal p.4 server nn show a potential signal peptide of 23 amino acids (a.a.) in the amino terminal portion followed by the mature peptide from a.a. 25 to a.a. 60 and a carboxy-terminal extension.this peptide has eight cysteine involved in 4 disulfide bridges, which hold the molecule in its active form. calculation of peptide parameters through the apd database prediction indicate a 44 % of total hydrophobic ratio 267 fig. 1 nucleotide and deduced amino acid sequence of the defensin from brachidontes pharaonis gills. 3’utr regions are described in lower case letters; the coding region is in upper case letters; the stop codon is indicated by asterisk. signal peptides from 1 to 23 a. a. positions are indicated in bold. mid-gray shading mature peptide is found at positions 25 60 and the eight cysteine conserved residue are in bold and underlined. arrows indicate 2 bactericidal stretches calculated by a server for prediction of protein antimicrobial regions: the first found in 29 to 41 and the second found in 43 to 63 a.a. positions. and + 1 of the total net charge. the grand average hydropathy value of the peptide (gravy) is equal to -0.106 and the wimley-white whole residue hydrophobicity of the peptide is 10.74 kcal/mol. the antimicrobial character of the molecule is suggested by the value of the protein-binding potential (boman index) of 1.74 kcal/mol. analysis carried out by an automated web server for prediction of protein antimicrobial regions (ampa) revealed a mean antimicrobial value of 0.244 and 2 bactericidal stretches, the first found in 29 to 41 and the second found in 43 to 63 a.a. positions (fig. 1). the identity of nucleotide and a.a. sequences was confirmed using the blast program (http://blast.ncbi.nlm.nih.gov/blast.cgi). the results of the comparison and a alignment with the sequences of m. galloprovincialis indicate an identity of 93 % and a similarity of 96 % with the antimicrobial peptide mgd1 and an identity of 78 % and a similarity of 84 % with the antimicrobial peptide of the same mgd2 mediterranean mussel (fig. 2). sequence identity and the common conserved sequence characteristics indicate that bpdef belongs to the αβ defensin family of amps. alignment of a.a. sequences shows the domain typical of αβ defensins structurally characterized by a helix and two sheets αβ (fig. 2). phylogenetic analysis the construction of the phylogenetic tree by the nj method and bootstrap analysis showed three different clusters (fig. 3). the first included αβ defensins of bivalve and gastropods (c. gigas, r. philippinarum, m. galloprovincialis, c. puortoricensis, o. moubata). the second included the α defensins of arthropods and some vertebrates (a. cerana, b. terrestris, d. melanogaster, a. gambiae, b. belcheri, p. japonicus, r. norvegicus m. musculus, h. sapiens) were organized into two related groups. the third included β defensins of vertebrates (d. rerio, o. mykiss, r. norvegicus m. musculus, c. hircus, h. sapiens). the bpdef sequence presented the closest relationships with the αβ defensins. 268 http://blast.ncbi.nlm.nih.gov/blast.cgi fig. 2 sequence alignment and structural organization representative of the bivalve molluscs defensin family amp. the conserved and identical residues in amp proteic domain are reported in black and conservative substitutions are reported in gray. arrows indicate the location of the hydro-phobic and positively charged residues in a domain typical of αβ defensins structurally characterized by a helix and two sheets. the eight conserved cysteine residues involved in disulfide bridges are represented by yellow shading. bpdef mrna localization to investigate the cellular localization of bpdef in b. pharaonis mrna, in situ hybridization assays were performed on circulating hemocytes obtained from the posterior abductor muscle of b. pharaonis specimens. two cell categories mainly based on size of intracellular granules were detected as follows: granulocytes with (i) small and (ii) large intracytoplasmic granules. both hemocytes with small intra-cytoplasmic granules (b, d) and with large granules (f) express bpdef mrna (fig. 4). controls with the sense strand probe were negative (a, c, e). bpdef expression under environmental stressors real-time rt-pcr enabled us to quantify defensin gene expression kinetics from b. pharaonis gills under thermal and hypoxia stressors. in all the condition under hypoxia the values are downregulated and reduced respect the normoxia condition from 5 to 20 times. timing of bpdef was shown as the following scheme (fig. 5): at 21 °c, under normoxic conditions, bpdef transcription declined to the lowest value after 1 day from t0, but reached at the control values (cnt) after 6 days. under hypoxia at 21 °c, the gene expression was down regulated 1 day after the treatment and this condition was maintained until day 6. at 30 °c the cnt values are referred to the sample acclimatized to 20 °c and moved for 8 hour at 30 °c. the kinetics of bpdef gene expression showed an increase to the control values after one day, while it was down regulated after six days, both under normoxia and hypoxia conditions. fig. 3 phylogenetic tree of defensins belonging to invertebrates (bivalve molluscs and insect) and vertebrates (fish and mammals). the tree was constructed by the nj method and bootstrap analysis. numbers represent the percentages of 1,000 bootstrap replicates in which the same internal branch was recovered. bars indicate the number of amino acid residue substitutions for site. the sequences are separated into three specific clusters: the molluscs αβ defensins, the vertebrate α defensins including humans, and αβ defensins of arthropods. 269 discussion in molluscs, hemocytes are the primary immune cells engaged in several innate immune reactions including phagocytosis, clotting, encapsulation, and the synthesis of several immune-related molecules. among these molecules, the antimicrobial peptides (amp) are characterized by their extremely heterogenic structures (bulet et al., 2004; li et al., 2011). based on primary structure and consensus cysteine array, defensin, mytilin, myticin and mytimycin amp families have been identified in the mussels m. edulis (charlet et al., 1996) and m. galloprovincialis (hubert et al., 1996; mitta et al., 1999a). in this work, the putative antimicrobial peptide of αβ defensins family, bpdef was cloned and characterized in the lessepsian b. pharaonis. outcompeting with the indigenous mussel mytilaster minimus, it had strong negative effects on survival and growth of the native species. like all the invasive species, it expanded its range, due to selection favoring strongly growth and reproduction, traits that may compete against immune function (carrington et al., 2015). the cdna of bpdef encodes a 81 a.a peptide. the putative propeptide contains a signal peptide of 23 a.a. and a mature peptide of 40 a.a.. the sequence contains features common to other amps: α-helices, overall positive charge, and strong hydrophobic regions. sequence alignment with other known big defensins showed the presence of 8 conserved cysteine residues forming the typical defensin consensus pattern. the boman index value, that estimates the potential or a protein to bind to a wide range of proteins, indicated the strong antibacterial feature of b. pharaonis defensin. two predicted bactericidal stretches were found in correspondence of the mature peptide. the arrangement of the cysteines, their neighboring a.a., the spacing between the cysteines and the high identity with the antimicrobial peptide mgd from m. galloprovicialis, further support the hypothesis that bpdef is a member of the defensin family. the structural homology study carried out relating to the crystallographic structure of mgd1 and the phylogenetic analysis indicates that the molecule belongs to the category of αβ defensins. using in situ ibridation (ish), cells expressing the defensin transcript have been identified: granulocytes with large and small cytoplasmic granules. amps as defensins, mytilin b, and myticins a and b, are produced as precursor molecules processed to active compounds within the hemocytes (mitta et al., 1999 a, b, 2000b). furthermore, previous studies have shown that defensins and mytilins are stored in granulocytes. in m. galloprovincialis immunocytochemistry at the ultrastructural level showed that defensins (i) are predominantly located in vesicles of a granulocyte subclass containing small granules, but (ii) are also found in large granules of the other granulocyte subclass containing large granules (mitta et al., 1999a, 1999b; cantet et al., 2012). in addition, no hyalinocyte resulted positive for amp mrna.. fig. 4 in situ hybridization assay: detection of transcripts encoding of bpdef in hemocytes withdrawn from abductor muscle of b. pharaonis. granulocytes with small cytoplasmic granules (a-b); granulocytes with large cytoplasmic granules (c, d, e, f) . hemocytes treated with bpdef antisense dig-riboprobe (a, c, e). control: hemocytes treated with bpdef sense dig-riboprobe (b, d, f and g). arrows indicate granules positive to riboprobe. (h) negative hyalinocyte. bar: 10 µm. in fig. 4g hyalinocytes cells (h) not showed transcript of bpdef contrary to granulocytes resulted positive to bpdef antisense dig-riboprobe. in mytilus spp. the mrnas encoding the amps are first translated into pro-peptides that share a common structure including a signal peptide in the amino terminal portion followed by the mature peptide and a carboxy-terminal extension position (parisi et al., 2009). the pro-peptides undergo intrahemocyte maturation and are stored in granules (li et al., 2011). environmental factors including temperature, oxygen, food availability, and contaminants can affect immune parameters of molluscs (fisher, 1988). 270 fig. 5 quantitative expression profile of bpdef in the gill at 21 °c and hypoxia treatment (a) and 30 °c and hypoxia treatment (b). the relative expression was calculated based on the standard curve and normalized to the 28s mrna level in gill at 0, 1, and 6 days. cnt values are referred to the sample acclimatized to 21 °c and moved for 8 h at 30 °c. data from qrt-pcr are presented as mean ± se. *p < 0.05; **p < 0.01; ***p < 0.001 significant level against control (0 and cnt). ox: normal oxygenation condition; hy: hypoxia treatment with o2 < 2 ppm. numerous studies have demonstrated that changes in environmental factors strongly influence mollusc immune parameters, even if temperature effects should differ among species. however, most studies carried out in the last decades have investigated the effects of single environmental factors, whereas a limited number of studies assess the multiple effects of abiotic factors are scant. at the immunological level, the ability of an organism to maintain the immuno-surveillance unaltered under changing environmental conditions may preserve its survival. the immune system is highly integrated with other systems and their effectors can respond to the stress resulting from widely fluctuating temperatures. here, the temperature of 21 °c did not stimulate a bpdef transcription over a short time. however, under normoxia, the persistence in the mesocosm at 21 °c maintain the production of bpdef mrna, which is greatly depressed under the unfavorable condition of hypoxia. the decrease of the expression of defensin gene could result from the mobilization of cells from tissues to hemolymph, in order to reserve a response to stress (matozzo and marin, 2011; cantet et al., 2012). at 30 °c, depleted oxygen seems also to contribute to the down-regulation of bpdef production, only after six days the values seem to be comparable to the beginning of the stimulation (ctn condition). effects of environmental variables on immune functions of mussels have been extensively studied due to the use as sentinel (shaw et al., 2011). roche (2001) demonstrated by northern blot that amp genes, defensin, mytilin b and myticin b were constitutively expressed in winter, while the expression of only mytilin and myticin b was detectable in summer. in addition, 271 heat-shock resulted in no change in mytilin b expression but in suppression of defensin in cold weather condition and its induction in hot weather condition. in conclusion, here we report for the first time the presence of mrna for an amp of a defensin family in a lessepsian b. pharaonis. the results enrich the basic research on the amp gene family in mussels, increasing our understanding of the immune system and laying a theoretical foundation for further practical application in studies of ecological immunology for understand immune function in the context of life-history traits. the assessment of gene transcription of antimicrobial peptides (amps) could provide new evidences for the ecological success of one specie in favor to others. acknowledgements this work was supported by mc ffr (university of palermo) and a research grant from the italian ministry of education (prin 2010-2011 n. 20109xzepr_007), co-funded by the university of palermo, italy. references balseiro 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anti-hiv-1 activity of cd8 antiviral factor. sci. 298: 995-1000, 2002. 273 http://www.ncbi.nlm.nih.gov/pubmed/?term=sang%20y%5bauthor%5d&cauthor=true&cauthor_uid=18983725 http://www.ncbi.nlm.nih.gov/pubmed/?term=readman%20jw%5bauthor%5d&cauthor=true&cauthor_uid=21683998 barbarro jm and abad mj. co-existence of two mytilid species in a heterogeneous environment: mortality, growth and strength of shell and byssus attachment. meps 476: 115-128, 2013. 326 isj 13: 326-335, 2016 issn 1824-307x review an overview of the main pathways of metabolic resistance in insects m panini1, gc manicardi2, gd moores3, e mazzoni1 1department of sustainable crop production, università cattolica del sacro cuore, piacenza, italy 2department of life sciences, university of modena and reggio emilia, reggio emilia, italy 3apreslabs ltd, harpenden, herts, uk accepted september 19, 2016 abstract insecticides have played and still fulfil a very important role in sustainable production of food, animal feed and also as protection against disease vectors. they act to suppress insect populations and, as a consequence of their use, insecticide resistance has evolved. an overview of insecticide resistance mechanisms in insects is given, focusing on the metabolic systems involved in xenobiotic metabolism in the class insecta. several enzyme families (e.g., esterases, mixed function oxidases, glutathione s-transferases) are involved in insecticide detoxification, sequestration and excretion and have differing relative importance within the various taxonomic groups. a brief discussion of their impact on control strategies is given. key words: insecticide resistance; esterases; p450; glutathione-s-transferases; synergists introduction insect pests represent a serious threat to agricultural production and vector disease control. the use of insecticides fulfils an important role in controlling populations of insect pests, but as a result of their continued application several resistance mechanisms allowing survival have evolved. during the last 50 years, increased use, overuse and even misuse of pesticides has led to the selection of resistance in more than 500 arthropod pest species. michigan state university developed an online database (aprd) (http://www.pesticideresistance.com) to list the resistant cases reported. following the first report of resistance at the beginning of the last century (melander, 1914), the number of cases continued to grow, with an exponential increase during the late 1970s and early 1980s (georghiou and lagunes-tejada, 1991). today, the order with the highest number of resistant species is diptera (27 %), followed by lepidoptera (25 %), hemiptera (17 %) and coleoptera (10 %) (whalon et al., 2012). diptera can have severe economic impact as many of them transmit diseases to humans and domestic animals, whilst others are pests of agricultural plants. in the other orders, many of the resistant species represent ___________________________________________________________________________ corresponding author: emanuele mazzoni department of sustainable crop production università cattolica del sacro cuore via emilia parmense, 84, i-29122 piacenza, italy e-mail: emanuele.mazzoni@unicatt.it a serious threat for agricultural production and are responsible for important agricultural yield losses causing problems for future food security. development of resistance depends upon a variety of genetic, biochemical and ecological factors such as generation time, fecundity rate, dispersal ability or fitness costs, together with the frequency, the dosage or the persistence of insecticide applications (brattsen et al., 1986; hemingway et al., 2002; kliot and ghanim, 2012; liu, 2015). the presence of different genotypes in a population can confer a selective advantage to some individuals and result in survival after insecticide exposure (feyereisen et al., 2015). as a result of continued insecticide application, the proportion of resistant insects increases compared to that of susceptible and the population becomes increasingly difficult to control (nauen, 2007) (fig. 1). insecticide resistance mechanisms it has been shown that insecticide resistance evolves predominantly by two mechanisms: the enhanced production of metabolic enzymes, which sequester or detoxify the insecticide, and/or mutations of target proteins, which make them less sensitive to the insecticide (fig. 2). a number of subsidiary physiological mechanisms which contribute to reduce insecticidal effects have also been described, e.g., a lower penetration of the chemicals or an increased excretion. a variety of different chemical classes, which act on different biological targets, have been 327 fig. 1 theoretical example illustrating the increase of insecticide resistance levels in a pest population. some individuals (red) with genetic traits allowing them to survive insecticide applications can reproduce; if the selection pressure is frequent, they easily become the preponderant part of the population. developed (http://www.iraconline.org/documents/moa-classification). however, many insect species have evolved mechanisms to overcome the toxicity of most classes of insecticide. thus, the possibility that they could also evolve resistance against potential new products, with different modes of action, has to be considered. resistance mechanisms do not always occur in isolation but often interact to enhance the level of resistance. the presence of combinations of different resistance mechanisms has been demonstrated in many insect populations and also in single individuals within a population. “cross-resistance” occurs when a single defence mechanism against one insecticide also confers resistance to other insecticides, even if the insect has not previously been exposed to the latter product. this phenomenon can result from physical factors that can affect chemically unrelated compounds, or nonspecific enzymes that attack functional groups of insecticides rather than specific molecules; indeed it is not only restricted to a specific chemical class but can involve insecticides with different modes of action. “multiple-resistance” occurs when different resistance mechanisms co-exist and confer resistance to different insecticides to which the organism has been exposed (oppenoorth and 328 welling, 1976; yu, 2008; panini et al., 2014). the occurrence of both cross-resistance and multipleresistance is of particular importance, because they can create increased difficulty for pest control. clearly, since pest insect populations are usually large in size and breed quickly, there is always a risk that insecticide resistance may evolve, especially when insecticides are misused or overused (soderlund and bloomquist, 1990; coleman et al., 2006). metabolic resistance metabolic resistance is a common defence mechanism, based on enzymatic systems that protect the insect by detoxifying/sequestering insecticide molecules. the enzymes involved are those insects have developed as protection against naturally occurring plant toxins (allelochemicals) such as alkaloids, terpenes and phenols, in order to overcome the potential toxicity of the plants they feed on (gatehouse, 2002; despres et al., 2007; war et al., 2012; heidel-fischer and vogel, 2015; rane et al., 2016). this could explain the rapid development of metabolic resistance against a very broad spectrum of insecticides that, in many cases, have direct or indirect botanical origin (isman, 2006). enzymes can detoxify xenobiotics into a nontoxic compound and/or into a form more suitable for rapid elimination from the body. resistant insects metabolise the insecticide faster because they possess forms of the enzyme with a higher catalytic rate, or higher quantities of the enzymes as a consequence of increased transcription or gene amplification. detoxification can be divided into phase i (primary) processes, consisting of hydrolysis or oxidation, and phase ii (secondary) processes, consisting of conjugation of phase i products with endogenous compounds, like glutathione, and their subsequent excretion from the body (li et al., 2007; hollingworth and dong, 2008; yu, 2008; berenbaum and johnson, 2015). in addition to these detoxification strategies that rely on enzymatic cleavage and excretion, sequestration is another important defence mechanism which several insects have evolved to tolerate xenobiotics. this is a common phenomenon among herbivorous insects, which involves the specific and selective uptake, transport and storage of plant secondary metabolites, preventing interference with the insects’ physiological processes (erb and robert, 2016; petschenka and agrawal, 2016). such behaviour was reported to be retained in mosquitoes whose haematophagy is probably a secondary adaptation to obtain high quality food needed for egg production (moore, 2015). the enzymes mainly involved in detoxification of xenobiotics in living organisms are transcribed by members of large multigene families of esterases, oxidases, and gsts. esterases esterases are a large group of phase 1 metabolic enzymes that are able to metabolise a variety of exogenous and endogenous substrates. their involvement in detoxifying insecticide molecules is well documented and it has been demonstrated that they can act against a broad range of chemical classes, including pyrethroids, organophosphates and carbamates (hollingworth and dong, 2008). potential involvement in neonicotinoid resistance (zhu and luttrell, 2015) and even against bt toxin (gunning et al., 2005) has also been reported. detoxification can occur through enzymatic cleavage or sequestration of the insecticide molecules. esterases catalyse the hydrolysis of ester insecticides into their corresponding acid and alcohol compounds; this increases the polarity of the insecticidal metabolites which can then be excreted more readily from the insect body. esterases can also sequester insecticides such that the toxic molecules are no longer available for interactions with target proteins (devonshire and moores, 1982; oakeshott et al., 2005; wheelock et al., 2005). esterases have been associated with insecticide resistance in many insect species as a consequence of quantitative and/or qualitative changes, resulting in the overproduction of the enzymes or in modifications of their structures (li et al., 2007). esterase overexpression can be due to either gene amplification or upregulation, or a combination of both. the most extensively studied example of insecticide detoxification by gene amplification is the overproduction of a specific carboxylesterase in the green peach aphid myzus persicae (hemiptera: aphididae) (field et al., 1988; bizzaro et al., 2005; rivi et al., 2013; bass et al., 2014). amplified esterases associated with insecticide resistance have also been found in mosquitoes of the culex genus (diptera: culicidae) (severini et al., 1994; raymond et al., 1998; hemingway et al., 2004) and other species, for example the brown planthopper nilaparvata lugens (stal) (hemiptera: delphacidae) (small and hemingway, 2000). in some species, e.g., aphis gossypii glover (hemiptera: aphididae) or b-biotype bemisia tabaci (gennadius) (hemiptera: aleyrodidae), the increased expression of esterases results from increased transcription levels, due to upregulation of the corresponding gene (alon et al., 2008; cao et al., 2008). esterase-based resistance can also occur through changes of the enzyme structure, which confers an enhanced ability to metabolise the insecticide. this mechanism was first described in the housefly musca domestica (diptera: muscidae) and became known as the “mutant ali-esterase theory” (oppenoorth and van asperen, 1960). the resistant insects showed an apparent decreased esterase activity compared to susceptibles, resulting from structural modifications of the enzyme that facilitated the hydrolysis of the insecticide but prevented or reduced the hydrolysis of the model substrates conventionally used to determine the esterase activity. two amino-acid substitutions (gly137asp and trp251leu) were considered as the basis of this resistance mechanism in houseflies as well as in other insect species belonging to the order of diptera (campbell et al., 1998; claudianos et al., 1999; carvalho et al., 2006). 329 fig. 2 schematic illustration of different possible resistance mechanisms known in insects: 1) penetration resistance; 2) enzymatic cleavage; 3) sequestration; 4) conjugation; 5) excretion; 6) target-site modification. additional mechanisms can influence esterase overproduction, including demethylation or chromosomal rearrangements. demethylation mechanisms can cause gene silencing and consequent reduction of esterase levels in e4 populations (field, 2000), but this is not true for the other esterase variant fe4, where no correlations were found between methylation levels and esterase activity (bizzaro et al., 2005). further differences were observed between e4 and fe4 m. persicae populations: amplification of isoform e4, leading to carbamate and ops resistance, is closely linked to chromosomal translocation a1,3 (field and devonshire, 1997). the same autosomal rearrangement was recently reported for the other esterase isoform, fe4, in italian populations of this aphid species, but only in populations with low esterase activity, meaning that translocation and esterase-based resistance are not always correlated. (rivi et al., 2012, 2013). monooxygenases mixed function oxidases (mfos), or microsomal oxidases, are a large family of phase 1 enzymes involved not only in the detoxification of xenobiotics, but also in the metabolism of endogenous substances such as hormones, pheromones or fatty acids. they are able to convert lipophilic compounds into polar metabolites that can be easily eliminated from the body; for that reason, they are mainly located in the digestive system (feyereisen 2005, 2015; liu et al., 2015). cytochrome p450 monooxygenases (p450s) are microsomal oxidases that belong to the group of heme thiolate proteins, so named because they show a characteristic absorbance peak at 450 nm (soret peak) in their reduced form when complexed with carbon monoxide. they catalyse the transfer of one atom of molecular oxygen to a substrate and the reduction of the second atom of oxygen to form water; the process requires the transfer of two electrons provided by nadph cytochrome p450 reductase (feyereisen, 2005; guengerich, 2008). the reaction is commonly described as: rh + o2 + nadph + h +  roh + h2o + nadp + due to the large number of enzymes and their substrate specificity, p450s are able to catalyse different reactions such as epoxidation, hydroxylation, n-dealkylation, o-dealkylation or desulphurisation; for that reason they play an important role in plant-insect interactions, as well as in the metabolism of many insecticide classes, including carbamates, organophosphates, pyrethroids and neonicotinoids (despres et al., 2007; yu, 2008; philippou et al., 2010; puinean, 2010; alptekin et al., 2016). the nomenclature used is that a single p450 is named as cyp followed by an arabic numeral to designate the family, a capital letter to designate the subfamily and an arabic numeral to designate the individual protein; each form is coded by its own gene. to date, more than 600 insect p450 genes have been characterised and genes belonging to the families cyp4, cyp6, cyp9 and cyp12 have been associated with insecticide resistance (feyereisen, 2005, 2015; li et al., 2007). the mixed-function oxidase system has been shown to occur in the fat body, malpighian tubes, and the midgut. by far the most intensively studied mixedfunction oxidase system is that of the house fly (markussen et al., 2010). because of the complexity of the p450 system and the difficulties in purifying these enzymes (due to their instability or problems in obtaining high yields), it is difficult to determine the mechanisms which induce resistance. however, it has been demonstrated that resistant insects can show increased levels of p450s and an enhanced monooxygenase activity. many cases of resistance correlated to overexpression of p450 activity have been reported in the literature and it is commonly considered to be caused by gene upregulation, probably through changes in the regulatory elements (feyereisen, 2005). although this is the usual mechanism described, cases of gene amplification or qualitative changes have also been reported (amichot et al., 2004; wondji et al., 2009; puinean et al., 2010). insect p450 enzymes may also activate certain types of insecticides, for instance the conversion of phosphorothioates (p=s) to phosphate (p=o). this can result in an increased 330 potency for inhibition of acetylcholinesterase by 3 or 4 orders of magnitude. p450s are also involved in the biosynthesis of ecdysone, juvenile hormone, and pheromone components (feyereisen, 1995). glutathione-s-transferases glutathione transferases (gsts) are a diverse family of enzymes found ubiquitously in aerobic organisms. they play a central role in the detoxification of both endogenous and xenobiotic compounds and are also involved in intracellular transport, biosynthesis of hormones and protection against oxidative stress (ketterman et al., 2011). although gst enzymes can be involved in substrate sequestration, they usually catalyse the conjugation of reduced glutathione (gsh) with electrophilic substrates, converting those reactive molecules into more water-soluble and non-toxic conjugates that can be more readily excreted from the body. specifically, they catalyse conjugations by facilitating nucleophilic attack of the sulphhydryl group of endogenous reduced glutathione (gsh) on electrophilic centres of a range of xenobiotic compounds, including insecticides or acaricides (konanz and nauen, 2004) and various plant toxins (despres et al., 2007). thus the xenobiotics have increased solubility and are excreted from the insect system by the formation of mercapturic acid derivatives (habig et al., 1974; enayati et al., 2005). gsts can also metabolise insecticides by facilitating their reductive dehydrochlorination or contribute to the removal of toxic oxygen free radical species produced through the action of pesticides (hayes et al., 2005). insect gsts are divided into two different groups (microsomal and cytosolic) according to their location within the cell, but only the latter has been implicated in the metabolism of insecticides. due to the broad range of substrates of the individual enzymes, they play an important role in resistance to different classes of insecticides, including organophosphates and pyrethroids. a ddtdehydrochlorinase gst has also been reported as being responsible for ddt resistance in houseflies and mosquitoes (enayati et al., 2005). annotation of the anopheles gambiae giles and drosophila melanogaster meigen genomes has revealed the full extent of this enzyme family in insects (enayati et al., 2005). gst-based resistance is generally due to an increased amount of enzyme, resulting either from gene amplification or overexpression (vontas et al., 2002; ranson and hemingway, 2005). gsts may also protect against pyrethroid toxicity in insects by sequestering the insecticide (kostaropoulos et al., 2001). additional metabolic resistance mechanisms: pgp pumps p-glycoprotein (pgp) transporters are integral membrane proteins that belong to the atp binding cassette (abc) superfamily, which utilise the energy derived from atp hydrolysis to translocate a variety of different metabolites and xenobiotics across cellular membranes (hollenstein et al., 2007). the action of pgp pumps in removing a broad range of toxic compounds from cells is well established as a mechanism of antibiotic resistance in bacteria and fungicide resistance in fungi (lage, 2003); in contrast very little is known about their physiological functions in insects. only recently abc transporters in insects have emerged as a putative mechanism which can contribute to resistance by facilitating efflux transport of insecticides and their metabolites derived from phase i and ii reactions (o’donnell, 2008). the involvement of pgp pumps in insecticide resistance has been documented in several insect species and it has been correlated to increased expression of genes encoding abc transporters (porretta et al., 2008; aurade et al., 2010; bariami et al., 2012). a survey of cases where the involvement of abc transporters in insecticide resistance is suggested has recently been reviewed by dermauw and van leeuwen (2014). abc transporters have been associated with resistance to insecticides with different modes of action, based on the quantification of transcript or protein levels and by synergism studies using abc inhibitors (buss and callaghan, 2008; dermauw and van leeuwen, 2014). furthermore, in different lepidopteran species a mutant allele has been discovered which confers resistance to the pore-forming cry1ac toxin from bacillus thuringiensis (bt) by a mechanism that is not related to toxin extrusion, but because it causes the loss of cry1ac binding to membrane vesicles (gahan et al., 2010; heckel, 2012). non-metabolic resistance mechanisms target-site resistance target site resistance is another important mechanism by which insecticide resistance is achieved and point mutations conferring insensitivity have been reported in many species. however, this is not a common mechanism by which insects become insensitive to plant toxins (despres, 2007; dobler et al., 2015). although mutations to the target protein of an insecticide can provide an insect with high levels of insensitivity, it would tend to be specific for a particular chemical class. conversely, detoxification mechanisms have the potential to confer cross-resistance between plant toxins and insecticides and because of their capability to act against a broad range of molecules. as a consequence, metabolic resistance becomes more advantageous at the evolutionary scale, as it allows insects to survive in non-homogeneous environments, where plant toxins might be less uniformly distributed and the probability to encounter them could be reduced. in this situation, metabolic resistance is often less costly than targetsite mutations, even if a real comparison of fitness costs among different populations can be very difficult or even impossible in field conditions (kliot and ghanim, 2012). in addition, there are several other mechanisms that may contribute at a more modest level. although individually they may be only moderate in their impact, they can act as important intensifiers of resistance when combined with the major mechanisms in the same insect. penetration resistance to reach its target, an insecticide must first penetrate the insect’s cuticle. penetration resistance occurs when insects have physico-chemical 331 alterations to the structure of their cuticle that results in a slower absorption of the chemicals or in a reduced amount of the insecticide passing through these physical barriers. this mechanism protects insects from a wide range of insecticides, but on its own confers low levels of resistance. indeed, it is usually found in combination with other forms of resistance, enhancing their effects. for example, a delayed and slower penetration can provide more time for the detoxification of the insecticide by phase 1 enzymes (oppenoorth and welling, 1976; scott, 1990; strycharz et al., 2013; kasai et al., 2014). recently, indirect evidence of the importance of this mechanism has emerged in the green peach aphid, where several cuticular proteins were found to be differentially expressed in neonicotinoid resistant populations (puinean et al., 2010). also, resistance to pyrethroid insecticides is associated with cuticle structure and composition in mosquitos (fang et al., 2015; wood et al., 2010). behavioural resistance behavioural resistance results from a change of insect behaviour in order to avoid the insecticide. this phenomenon is stimulus dependent and resistant insects can detect or recognise the danger and simply stop feeding or leave the treated area, walking or flying away (gatton et al., 2013). they can respond to lower concentrations of insecticide than normal insects, indicating the presence of receptors that allow development of the ability to detect the presence of insecticides (sparks et al., 1989; yu, 2008). there are very few documented examples of behavioural resistance, one being the avoidance of malathion baits (schmidt and labrecque, 1959). insecticide resistance management and metabolic resistance one aim of resistance management is to delay the evolution of resistance in pests. the best way to achieve this is to minimize insecticide use, but the intrinsic difficulties of controlling a phenomenon driven by evolutionary forces must be considered (hoy, 1998). one effective strategy involves the use of synergistic molecules in combination with insecticide products. synergists are non-toxic compounds that enhance the efficacy of insecticides. they are capable of inhibiting the enzyme systems of insects that metabolise or sequester insecticide molecules. as a result, insect sensitivity increases, with the possibility to overcome metabolic resistance conferred by increased levels of detoxifying enzymes (bernard and philogene, 1993; feyereisen, 2015). the best known synergist, piperonyl-butoxide (pbo), is widely used in household products against urban pests, but its application in agriculture remains limited to niche areas. recent works have demonstrated in vitro the ability of pbo and its analogues to specifically interact with resistanceassociated phase i metabolic enzymes of key insect pests, like m. persicae (panini et al., 2016) or b. tabaci (panini et al., submitted). in addition, chen and sun (1986) and kumar et al. (2002) demonstrated that the use of pbo in combination with a synthetic pyrethroid can delay the development of resistance in diamondback moth and mosquitoes, respectively. after several laboratory bioassays and field tests in these and other species, the concept of ‘temporal synergism’ was developed. if an insect is treated with a synergist a few hours prior to exposure to an insecticide, it allows time for the synergist to cross the insect cuticle and inhibit those enzymes involved in metabolic resistance, thus creating a sensitive or even hyper-sensitive insect and allowing a reduction of the insecticide dose to be subsequently applied (moores et al., 2005). temporal synergism can be achieved not only with a split application of synergist and insecticide but also by using formulation technologies which allow a differential time release of synergist and insecticide, even when applied simultaneously (bingham et al., 2007; mazzoni et al., 2010). resistance management is a component of integrated pest management, which combines chemical and non-chemical controls to seek safe, economical, and sustainable suppression of pest populations. alternatives to insecticides include biological control by predators, parasitoids, and pathogens (lacey et al., 2015). also, valuable approaches include cultural practices (crop rotation, manipulation of planting dates to limit exposure to pests, and use of cultivars that tolerate pest damage), mechanical controls (exclusion by barriers and trapping) and behaviour manipulation (use of artificial signalling like mating disruption, false trail following or mass trapping) (witzgall et al., 2010). since large-scale resistance experiments are expensive, time consuming, and might increase resistance problems, modelling has played a prominent role in devising tactics for resistance management. although models have identified various strategies with the potential to delay resistance, practical successes have focused primarily on reducing the number of insecticide treatments, diversifying their types and, above all, the mode of action (moa) of the insecticide employed. indeed, these strategies can be unsuccessful if the choice is made when the genetic background of the considered species and/or populations is unknown. also, moa rotating is effective only against target-site resistance mechanisms. conversely, it can fail when multiple or cross resistance mechanisms are present, a situation that is common in many agricultural and urban pests (bass et al., 2014; panini et al., 2014, 2015; abbas, 2015; riveron et al., 2015). only an in-depth knowledge of the genetic basis of insecticide resistance will allow a reduction of the environmental impact resulting from inadequate treatments. molecular diagnostic tools can be important from this point of view, although they are generally utilised to detect target site resistance mechanism s (cassanelli et al., 2005; bass et al., 2007; puinean et al., 2013; silva et al., 2014; chen et al., 2015; donnely et al., 2016; puggioni et al., 2016;). efforts should be made to improve the diagnosis of metabolic resistance allowing a reliable, fast and cost-effective tool. furthermore, the information obtained from these studies would provide details essential for 332 the informed synthesis of effective and environmentally friendly actives. references abbas n, shad sa. assessment of resistance risk to lambda-cyhalothrin and cross-resistance to four other insecticides in the house fly, musca domestica l. 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adipokinetic hormone; sulfakinins, insects; zophobas atratus   introduction the neuroendocrine systems of insects play a special role in the regulation of most of their metabolic ___________________________________________________________________________ list of abbreviations: adipokinetic hormone, akh; red pigment concentrating hormone, rpch; sulfakinin, sk; programed cell death, pcd; high performance liquid chromatography with a laser lightscattering detector, hplc-llsd; gas chromatography with flame ionization detector, gc-fid; gas chromatography mass spectrometry, gc-ms; trimethylchlorosilane, tmcs; n,o-bis (trimethylsilyl) trifluoroacetamide, bstfa; matrix assisted laser desorption ionization time of flight, maldi-tof; high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry, hplc/apci-ms; high-performance liquid chromatography/mass spectrometry, hplc/ms; electrospray ionization quadrupole ion trap mass spectrometry, esi qitms; thin-layer chromatography, tlc; phosphatidylethanolamine, pe; phosphatidylcholine, pc corresponding author: marek gołębiowski laboratory of analysis of natural compounds department of environmental analysis faculty of chemistry, university of gdańsk ul. wita stwosza 63, 80-308 gdańsk, poland e-mail: marek.golebiowski@ug.edu.pl processes as well as in their development. hormonal regulation plays a key role in many processes including molting and metamorphosis, for example the ecdysone and juvenile hormone (koeppe et al., 1985; riddiford, 1985; hutchins, 2003), hemolymph metabolite (proteins, carbohydrates and lipids) homeostasis (sulfakinins; audsley and weaver, 2009) as well as in energy metabolism during flight (akh; van der horst et al., 1997, van der horst et al., 2001; lorenz and gäde, 2009). in the 1920s, secretory cells were discovered in the brains of insects which control different processes in different parts of the insect's body. from 1917-1922, pioneering research into the mechanisms which regulate the metamorphosis of the gypsy moth caterpillar (lymantria dispar) was carried out by the polish entomologist stefan kopec (słocińska, 2009). there are two types of glands involved in the synthesis and release of peptides in insects: the exocrine and endocrine glands. the exocrine glands secrete compounds on the surface of the insect which serve to protect, either by acting as repellents or as attractants. an example of an attractant are pheromones which are a complex mixture of chemicals (martins and ramalho-ortigão, 2012; ottaviani, 2014). examples of endocrine glands are among others the prothorax, corpora allata which produce the juvenile hormone responsible for the process of transforming insects and the corpora cardiaca, which are adjacent to the heart and brain 225 and which secrete hormones that stimulate the mobilization of lipids in locusts (biej-bijenko, 1976). the fat body of insects is made up of trophic tissue and is rich in triacylglycerols, free fatty acids and cholesterol. the fat body is functionally equivalent to the liver and adipose tissue in mammals. a series of transformations of the intermediary metabolism of insects takes place in the fat body, which are under the strict control of hormones secreted by the neuroendocrine system. metabolites such as carbohydrates, proteins and fats are stored in the fat body. in insects, these compounds are the source of energy for activities such as metamorphosis, flight and egg formation. (fernando-warnakulasuriya et al., 1988; van der horst et al., 1997; ryan and van der horst, 2000; ziegler and ibrahim, 2001; guedes et al., 2006; arrese and soulages, 2010; snart et al., 2015). during the changes which occur in the body of the insect, the delivery of large amounts of energy are required and processes occur in the fat body which release trehalose, diacylglyceride and the insect hemolymph protein.. the basic lipid composition of certain selected species of insects is already quite well known. but by using modern chromatographic methods, e.g., gas chromatography or liquid chromatography, the contribution of individual groups of compounds in the lipids can be successfully determined. in many species of insect, a significant amount of free fatty acids and their esters and hydrocarbons or alcohols have been discovered. however, the exact impact of various environmental factors on the lipid profile in the fat body of insects is still not fully known. the role of the adipokinetic and sulfakinin hormones the adipokinetic hormone controls among other things the mobilization of reserves of energy to the muscles of wings by releasing trehalose, diacylglyceride and proline to the hemolymph. akh is a hormone pleiotropic, which not only affects the locomotor activity of the insect, but also regulates the synthesis of rna, proteins and free fatty acids in the fat body, the activity of the heart and the propagation of the insect (van der horst and ryan, 2012). the first peptide (pelnfspgwa) belonging to the family of akh the red pigment concentrating hormone (rpch) was isolated from a pink shrimp (pandalus borealis) in 1972 (fernlund and josefsson, 1972). currently, 40 different types of akh peptides are known (gäde and marco, 2009). their construction is made up of 8 10 amino acid residues. pyroglutamic acid residue is located at the end of the n-terminus of the amino acid chain and at the c-terminus there is carboxamide residue (van der horst and ryan, 2012). as a result of the binding of akh peptides with g protein receptors located in the fat body, there is a mobilization of carbohydrates. in table 1, the structure of the akh family of peptides is shown. another important group of peptide hormones in insects are sulfakinins (sk). in their structure, these myotropic neuropeptides have sulfated residues of tyrosine. by using high performance liquid chromatography (hplc), the peptide family of sulfakinins was isolated from an extract of cockroach l. maderace (peptide leum-sk-1) (nachman, 1986). sulfakinins were found not only in cockroaches (blattodea), including the american cockroach (periplaneta americana) and the madeira cockroach (leucophea maderace) and the housefly (diptera), including the bluebottle fly (calliphora vomitoria), the australian sheep blowfly (lucilia cuprina), the common fruit fly (drosophila melanogaster) and the grey flesh fly (neobellieria bullata). table 1 example structure of peptides of the akh family in various insect species insects peptide sequence references tenebrio molitor pqlnfspnwa (gäde and rosiński, 1990) zophobas rugipes pqlnfspnwa (gäde and rosiński, 1990) leptinotarsa decemlineata pqltftpnwa (gäde, 1989) onitis aygulus pqynfstgwa (gäde, 1997) pachnoda marginata pqlnyspdwa (gäde, 1989) coccinella septempunctata pqlnftpnwa (neupert, 2007) cheilomenes luncata pqlnftpnwa (neupert, 2007) locusta migratoria pqlnftpnwgta (stone et al., 1976; bogerd et al., 1995) schistocerca gregaria pqlnftpnwgta (hekimi et al., 1989; schulz-aellen et al., 1989) melanoplus sanguinipes pqlnftpnwgta (taub-montemayor et al., 1997) tribolium castaneum pqlnfstdwa (amare and sweedler, 2007) manduca sexta pqltftsswga (ziegler et al., 1985; bradfield and keeley, 1989) heliothis zea pqltftsswga (jaffe et al., 1986) bombyx mori pqltftsswga (ishibashi et al., 1992) apis mellifera pqltftsswga (lorenz et al., 1999) 226 fig. 1 overall diagram of the analysis of fat body lipids in zophobas atratus (gołębiowski et al., 2014). extracts from the fat bodies were prepared at the department of physiology and biology adam mickiewicz university in poznan. sulfakinins play a special role in insects’ process of eating by modulating the muscle contractions of the intestines and heart. sulfakinins also influence the inhibition of food intake in cockroaches and stimulate the secretion of digestive juices in the great scallop (pecten maximus) and the red palm weevil (rhynchophorus ferrugineus) (schoofs and nachman, 2006). sulfakinins control the amount of storage energy and also the composition and amount of free fatty acids and cholesterol, thus affecting the maintenance of homeostasis. the physiological properties of sulfakinins have a functional similarity to gastrin and cholecystokinin which occur in vertebrates (audsley, 2009). methods of analysis various methods of extractions are used in the isolation of compounds (analytes) from the matrix. the most popular method for the extraction of lipids is liquid extraction. chloroform and hexane are used to isolate medium polar and non-polar compounds (nelson et al., 1999; buckner et al., 2009). however, more often dichloromethane and petroleum ether are used (cerkowniak et al., 2013). one of the most important stages in the analysis of lipid composition is the group analysis. two common techniques that have been previously used for the separation of particular groups of compounds are thin layer chromatography (mardaus and buckner 1997) and high performance liquid chromatography (cerkowniak et al., 2013). increasingly popular is high performance liquid chromatography using a laser light-scattering detector (hplc-llsd) (gołębiowski 2012, cerkowniak et al. 2013; gołębiowski et al., 2013a). for the specific analysis of lipids extracted from the fat or glandular secretions of insects, either gas chromatography with a flame ionization detector (gc-fid) or gas chromatography combined with mass spectrometry (gc-ms) can be used (durak and kalender, 2007; gołębiowski et al., 2013b). mass spectra can be obtained using a mass spectrometer as a detector, from which test compounds can be identified. for the purposes of quantitative analysis, an internal standard method is normally used. in this method, an internal standard is added to a predetermined amount of sample, whose retention time will differ from all of the examined analytes. the relationship between the ratio of the concentrations and the ratio of detector response of the test compound and the internal standard is determined. figure 1 shows the schematic procedure for the determination of the lipid content in the fat body of the giant mealworm beetle (z. stratus) (gołębiowski et al., 2014). the extracts were separated into individual groups of compounds in the normal phase using high performance liquid chromatography with hplc-llsd. from the fractions, a sufficient quantity of lipids was collected, evaporated to dryness, added to the internal standard, and then silylated with n,o-bis (trimethylsilyl) trifluoroacetamide (bstfa) and trimethylchlorosilane (tmcs). free fatty acids can be analyzed as trimethylsilyl derivatives or as corresponding fatty acid methyl esters (gołębiowski et al., 2014; radzik-rant et al., 2014). derived lipids and native organic compounds can be analyzed fig. 2 techniques used in the analysis of the fat body. 227 table 2 examples of the use of analytical techniques in the analysis of fat body composition insects/ extraction solvent/ compounds/ reference techniques 11 bumblebee species, e.g.: bombus terrestris, b. lucorum chcl3/ch3oh (1:1, v/v) triacylglycerols. the tgs consisted predominantly of fas with an even number of carbons, mostly 18 or 16. (kofronova et al., 2009) hplc/apci-ms columns: two stainless steel nova-pak c18 columns (300mm×3.9mm, 150mm×3.9mm, a particle size of 4 µm) connected in a series. phase: acetonitrile (a) and 2-propanol (b) the gradient program was: 0 min: 100% of a, flow rate 1 ml/min; 108 min: 30% of a, 70% of b, 1ml/min; 150 min: 5% of a, 95% of b, 0.5 ml/min; 165 min: 5% of a, 95% of b, 0.5 ml/min, 177-100% of a, 0.5 ml/min; 180 min: 100% of a, 1 ml/min. maldi-ms an acceleration voltage of 20 kv and a 200 ns extraction pulse. desorption and ionization were achieved using a nitrogen uv laser (337.1 nm, with a 4 ns pulse of 300 µj, and the maximum frequency of 20 hz) with the laser power adjusted to 50-60%. bombus lucorum, b. terrestris, b. lapidarius, b. hypnorum, b. hortorum, b. confuses chcl3/ch3oh (1:1, v/v) triacylglycerols. the most abundant fatty acids in bumblebees tags contained 18 or 16 carbon atoms. (cvacka et al., 2006) gc column: db-wax (30m×0.25 mm, 0.25µm). conditions: 140 °c (0 min), then 5 °c/min to 230 °c (30 min). hplc/ms column: 250mm×4mm packed with biospher psi 100 c18, 5 µm. phase: acetonitrile (a) and 2-propanol/acetonitrile (3:1, v/v) (b). the linear gradient from 25% of b to 100% of b in 30 min, followed by 5 min at 100% of b. pyrrhocoris apterus folch procedure phospholipids. two phospholipid classes, phosphatidylethanolamine (pe) and phosphatidylcholine (pc), represent more than 80% of total phospholipids. (hodkova et al., 2002) esi qitms positive esi/ms and ms2 and/or ms3 spectra were recorded at 4.5 kv, with capillary voltage 8 v and capillary temperature 190°c. negative esi spectra were recorded at 4.5 kv, with capillary voltage -21.5 v and capillary temperature at 190°c. zophobas atratus chcl3/ch3oh (1:1, v/v) total and phospholipid fatty acid composition. the quantitatively major components were 16:0, 16:1, 18:0, 18:1, and 18:2n-6. (howard and stanley-samuelson, 1996) gc column: supelcowax 10 capillary column (30m×0.25 mm, 0.25µm). conditions: 2°c/min from 150 to 250°c with an initial 2 min hold. gc-ms column: supelcowax 10 capillary column (30m×0.25 mm, 0.25µm). conditions: 1°c per min from 170 to 220°c. rhodnius prolixus bligh and dyer procedure triacylglycerols. (pontes et al., 2008) tlc thin-layer chromatography developing solvent: hepane-etyl ether-acetic acid (60:40:1 v/v/v). visualization: 10% cupric sulfate (w/v) in 8% phosphoric acid (v/v) for 30 s. bombus pratorum, bombus terrestris chcl3/ch3oh (1:1, v/v) triacylglycerols. unusual fatty acids with 24, 26, and 28 carbon atoms were found in triacylglycerols. (cvacka et al., 2008; jiros et al., 2011) gc-ms column: hp-5 ms (30m×0.25 mm, 0.25µm). conditions: 40 °c (1 min), then 50 °c/min to 140 °c, then 3 °c/min to 320 °c (20 min). hplc-ms columns: two stainless steel nova-pak c18 columns (300mm×3.9mm, 150mm×3.9mm, a particle size of 4 µm) connected in a series. phase: acetonitrile (a) and 2-propanol (b). a linear gradient from 0 to 70% of b in 108 min (1.0 ml/min) was followed by a linear gradient to 100% b (150 min, 0.7 ml/min). zophobas atratus dichloromethane fatty acids, fatty acids methyl esters, fatty alcohols, sterols (gołębiowski et al., 2014; słocińska et al., 2013) gc-ms column: hp-5 (30m×0.25 mm, 0.25µm). conditions: from 80 (held for 10 min) to 320◦c at 4◦c/min, and then held isothermal for 20 min. 228 using gc-ms (gołębiowski et al., 2012; pannkuk et al., 2013a). triacylglycerol fractions can be analyzed using the maldi-tof technique (matrix assisted laser desorption ionization time of flight) (ayorinde et al., 1999; gidden et al., 2007; pannkuk et al., 2013b). figure 2 shows the different techniques used in the analysis of the fat body of insects and table 2 contains data on the most frequently applied analytical techniques used in the analysis of the fat body of insects. compounds in the fat body are mainly triacylglycerols, phospholipids, fatty acids, fatty acids methyl esters, fatty alcohols and sterols (table 2). free fatty acids, triacylglycerols and fatty acid esters (methyl-, ethyl-, decyl-, dodecyland tetradecyl-), alcohols, glycerol, and cholesterol (słocińska et al., 2012; gołębiowski et al., 2014) are examples of compounds which have been discovered using gc-ms in the analysis of the fat bodies of larvae and pupae z. atratus. large differences were noted in the case of the free fatty acid content of the larvae, where the acid content increased 24 h and 48 h after the tenmoakh injection. concentrations of free fatty acids detected in the fat body of larvae markedly increased under akh treatment. on the other hand, the contents of the free fatty acids found in the fat body of pupae decreased after an injection of tenmo-akh (gołębiowski et al., 2014). the total amount of lipids identified in the pupae after using akh was lower than the control (słocińska et al., 2012). in the case of lipids in larvae, an increase of cholesterol was observed 24 h after the introduction of the hormone, whereas after 48 h the amount of cholesterol decreased again (gołębiowski et al. 2014). conclusions the use of modern analytical techniques, specific and sensitive bioassays and molecular biology have rapidly accelerated the development of insect neuroendocrinology. the use of gc-ms allows both qualitative and quantitative determinations of the lipid composition of insects to be made, which means correlations can be observed between the state of development of insects and their energy demands as well as changes in the quantity and quality of lipids in the tested insect species. the fat body plays a particular role in many of the processes related to the metabolism and life processes of insects. when there is a high demand for energy, for example during the flight of an insect, a continuous release of energy is necessary. mobilization is governed primarily by carbohydrates and lipids, which are stored in the fat body (van der horst, 1997). the mobilization of metabolites occurs when relevant hormones are released by the neuroendocrine system. in certain insect species, usually more than one type of hormone exists. for example, in the fat body of l. migratoria, there are three different forms of akh (akh-i, akh akh-ii and iii) which among other things have different lengths of peptide chains. in his research, van der horst drew attention to the influence of akh on the amounts of energy, as well as the speed of its release. even after 1 min, a 200 % increase in the intracellular camp can be observed, compared to its initial level. upon further lapses of time, only decreases in the amount of the camp are recorded and after 30 min, camp levels are already at the same output level of a signaling molecule. this shows that the effective length of time of the akh action is very short (van der horst, 1997). however, a series of biochemical changes can take place which cause changes in the metabolism of lipids and proteins, but whose effects can be seen only after longer periods of time (for example after 24 or 48 h). the length of time depends largely on the nature of the hormone, the species, the physiological conditions and the stage of development of the test insect. in insects undergoing a complete transformation of the body, there is a total reorganization of the larvae, through the pupal stage right up to the final form of the insect (imago) (larsen, 1976). so-called apoptosis programmed cell death (pcd) occurs in the tissue of the fat body. this process takes place at different phases of the development stage. for example, in the tobacco hornworm (manduca sexta), pcd occurs during the 3 to 5 day larval stage (müller et al., 2004), and in the case of the silkworm (bombyx mori) pcd occurs 2 days before pupate (gui et al., 2006, lee et al., 2009). the process of transformation during metamorphosis can also vary, tissue can differentiate between pupal and imago (oberlander, 1985) and it may be subject to changes without moving the cells, or by moving the differentiation of larval cells again from primary cells (kaneko. 2011). during metamorphosis, in the majority of insects, cells experience a period of larval and pupal transition (larsen, 1976). in the course of intensive developmental changes, the lipid composition of the fat body also changes, especially free fatty acids and cholesterol (słocińska et al., 2012). insects do not synthesize steroids and therefore sterols (primarily cholesterol) are essential components of their diet (roller et al., 2010). the very different impacts of akh on the fat bodies of intensive feeding larvae and on 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report the mrna expression profiles demonstrating versatile roles of glutathione s-transferase genes in the mollusk chlamys farreri m wang1, l wang3,4,5, d ni1, q yi3,4,5, x wang6, z jia1, l song2,3,4,5* 1cas key laboratory of experimental marine biology, institute of oceanology, chinese academy of sciences, qingdao 266071, china 2laboratory for marine fisheries science and food production processes, national laboratory for marine science and technology, qingdao 266237, china 3liaoning key laboratory of marine animal immunology, dalian ocean university, dalian 116023, china 4liaoning key laboratory of marine animal immunology and disease control, dalian ocean university, dalian 116023, china 5dalian key laboratory of disease prevention and control for aquaculture animals, dalian ocean university, dalian 116023, china 6college of marine science and biological engineering, qingdao university of science & technology, qingdao 266042, china accepted august 27, 2018 abstract glutathione s-transferase (gst) is a superfamily of multifunction enzymes with varying catalytic roles in cellular detoxification to protect hosts against oxidative damage. in the present study, six gst genes were identified from chlamys farreri, including cfgstω, cfgstσ-1, cfgstσ-2, cfgstρ, cfgstζ and cfmgst. cfgsts shared high similarities with their counterparts from other species, and were clustered with their homologues into the corresponding clades in the phylogenetic tree, respectively. we investigated the distribution of their mrna transcripts in different tissues and their temporal expression profiles in hemocytes after microbe stimulations by quantitative real-time pcr. the six cfgst genes were detectable in all the tested tissues, including hemocytes, muscle, mantle, gill, hepatopancreas, and gonad. stimulations with various microbes drastically induced the mrna transcripts of all the cfgsts with different expression profiles. for examples, cfgstω could be induced by three kinds of microbes, including vibrio anguillarum, micrococcus luteus and pichia pastoris, whereas cfmgst could be only induced by v. anguillarum. these results indicated a powerful detoxification system of gsts in scallop. moreover, the distinct mrna expression profiles of cfgsts indicated their versatile and immune-challenge specific roles in the mollusk c. farreri. key words: chlamys farreri; glutathione s-transferase; innate immunity introduction the innate immunity acts as the first defense line for all multicellular animals and almost the only mechanism for invertebrates to protect themselves against microbial invaders (hoffmann et al., 1999). many innate immune responses, especially hemocytes-mediated phagocytosis, were accompanied with respiratory burst and followed by mass production of reactive oxygen species (ros) (liu et al., 2009; jia et al., 2018). the production of ros is an effective way to eliminate invading microbes; however, it has been already proved to be ___________________________________________________________________________ corresponding author: linsheng song dalian ocean university dalian 116023, china e-mail: lshsong@dlou.edu.cn; lshsong@qdio.ac.cn a double-edged sword (benedetti et al., 2015). low concentration of ros is beneficial for activating signaling pathways mediating various responses to kill or eliminate foreign invaders (he and klionsky, 2009). while extremely high levels of ros may be detrimental to biological macromolecules, and lead to cellular dysfunctions, increase cell damage and finally threaten hosts’ survival (martindale and holbrook, 2002). therefore, almost all the aerobic organisms have developed an antioxidant system to remove excessive ros and maintain the redox balance (halliwell, 2006). the antioxidant system is constituted by a series of antioxidant enzymes, such as superoxide dismutase (sod), catalase (cat), peroxiredoxin (prx), thioredoxin peroxidase (tpx), thioredoxin reductase (trx), glutathione peroxidase (gpx), glutathione reductase (grx), 303 glutathione-s-transferase (gst) and many other non-enzymatic antioxidant molecules (harris, 1992). among all these antioxidant enzymes, gst (ec: 2.5.1.18) is a superfamily of multifunction enzymes, which play varying catalytic roles in cellular detoxification and protect hosts from oxidative damage (strange et al., 2001). by now, at least 15 different classes of gsts have been identified and characterized in numerous organisms according to their structural, catalytic and immune features, including alpha (α), beta (β), delta (δ), epsilon (ε), kappa (κ), lambda (λ), mu (μ), omega (ω), phi (φ), pi (π), sigma (σ), tau (τ), theta (θ), zeta (ζ) and rho (ρ) (hayes et al., 2005). the microsomal gsts, members of the membrane associated protein in eicosanoid and glutathione metabolism (mapeg) protein family, also play pivotal roles in antioxidant reaction (morgenstern et al., 1982). although no criteria were developed to classify gsts in marine organisms, the expression profiles and enzyme activities of gsts have been investigated in some aquatic species, such as abalone haliotis diversicolor (ren et al., 2009), bay scallop argopecten irradians (wang et al., 2017a), disk abalone haliotis discus discus (wan et al., 2008; sandamalika et al., 2018), green-lipped mussels perna viridis (li et al., 2013), intertidal copepod tigriopus japonicas (lee et al., 2007), manila clam venerupis philippinarum (xu et al., 2010; li et al., 2012; zhang et al., 2012a,b; li et al., 2015), marine mussels mytilus galloprovincialis (wang et al., 2013; li et al., 2015), pearl oyster pinctada martensii (chen et al., 2011), razor clam solen grandis (yang et al., 2012), ridge-tail white prawn exopalaemon carinicauda (duan et al., 2013), and sea cucumber apostichopus japonicas (shao et al., 2017; zhang et al., 2017a,b). some of these gsts from aquatic species were involved in innate immunity and could respond to invading microbes, for examples, the sigma class gst from h. diversicolor was significantly induced post bacteria challenged (ren et al., 2009), while the mrna expression level of a gst gene in s. grandis was significantly up-regulated in hemocytes after being stimulated by β-1, 3-glucan (yang et al., 2012). the zhikong scallop chlamys farreri is one of the most important commercial species which is widely cultivated in the northern coastal provinces of china (li et al., 2015b; song et al., 2015). with the rapid expansion of intensive culture and environmental deterioration, scallops have frequently suffered from various diseases. the knowledge about the antioxidant system and its function in response to invading microbes may provide a better understanding of innate immune mechanisms in scallop and potential development of disease control strategies in scallop farming. in previous reports, several antioxidant enzyme genes have been identified and investigated in c. farreri, such as sod (ni et al., 2007; wang et al., 2018), cat (li et al., 2008), prx (cong et al., 2009), and gpx (mu et al., 2010). moreover, the cdna sequence of a pi (π) class gst and its expression profiles in response to benzo[α]pyrene exposure was also reported in c. farreri (miao et al., 2011). however, compared with other antioxidant enzymes in scallop, the information of gsts is rather rare and fragmentary and more investigation is needed to illustrate their exact roles in the innate immunity. in the present study, six novel gst genes were identified in c. farreri based on the analysis of expression sequence tag (est) sequences (wang et al., 2009) with the main objectives (1) to characterize the molecular features of cfgst genes (2) to detect the tissue distribution and temporal mrna expression profiles of their mrna transcripts, and (3) to compare these features to lead a better understanding of their versatile roles in c. farreri. materials and methods scallops, immune stimulation and sample collection adult scallops with an average 55 mm in shell length were collected from a local farm in qingdao, china, and maintained in aerated seawater at about 15 °c. approximately 120 scallops were employed for microbe stimulation assay. after acclimated for two weeks, 30 scallops were kept in tanks containing live vibrio anguillarum strain m3 (kindly provided by prof. zhaolan mo) at a final concentration of 1.0 × 108 colony forming units (cfu) ml-1, and defined as gram-negative bacteria stimulation group. another 30 scallops were transferred to the tanks containing live micrococcus luteus (28001, microbial culture collection center, china) at a final concentration of 1.0 × 108 cfu ml-1, and defined as gram-positive bacteria stimulation group. the third 30 scallops were transferred to the fungi-containing tanks with live pichia pastoris strain gs115 (pa17237, thermo fisher scientific, usa) at a final concentration of 1.0 × 108 cfu ml-1, and defined as fungi stimulation group. and the last 30 scallops were employed as the control group. five individuals from each group were randomly sampled at 0, 3, 6, 12, 24 and 48 hours post stimulation (hps), respectively. the hemolymphs were collected from the adductor muscle using syringes and centrifuged at 800 g, 4 °c for 10 min to harvest the hemocytes for rna preparation. hemocytes, muscle, mantle, gill, hepatopancreas and gonad from five untreated scallops were collected to determine the mrna transcripts distribution of cfgst genes. rna isolation and cdna synthesis raw rna was isolated from the hemocytes and other tissues of scallops using rnaiso plus reagent (9108, takara, japan). the first-strand synthesis was performed with m-mlv (m1705, promega, usa) using the dnase i (rq1, m6101, promega, usa) treated raw rna as template and adaptor primer-oligo(dt) as primer (table 1). the reaction were carried out at 42 °c for 1 h, terminated by heating at 95°c for 5 min. a homopolymeric tail was added to the 5` end of the cdna using terminal deoxynucleotidyl transferase (tdt, 2230, takara, japan) and dctp (u1221, promega, usa) and the obtained product were subsequently stored at -80 °c till use. cdna cloning of the full-length cfgst genes the full-length cdna sequences of cfgst genes were obtained by rapid-amplification of cdna ends (race) technique based on the analysis of est sequences (wang et al., 2009). all the primers 304 used in this assay were listed in table 1. all pcr amplification was performed in a tp-600 pcr thermal cycler (takara, japan). the pcr products were gel-purified and then cloned into the pmd19-t simple vector (3271, takara, japan), and then transformed into the competent cells escherichia coli strain top10 (cb104, tiangen, china). the positive recombinants were identified through anti-ampicillin selection and verified via pcr screening with sequencing primers m13-47 and rv-m (table 1). five positive clones were sequenced with a 3730xl automated sequencer (thermo fisher scientific, usa). bioinformatics analysis of sequences the search for protein sequence similarity was conducted with blast+ 2.2.18. the deduced amino acid sequences were analyzed with dnastar lasergene suite 7.1.0.44 using the editseq module. signalp 3.0 was employed to predict the presence and location of signal peptide. the protein domain and motif features were predicted by simple modular architecture research tool (smart) 5.1. a phylogenic nj tree was constructed with mega 5.05. to derive confidence value for the phylogeny analysis, bootstrap trials were replicated 1000 times. real-time pcr analysis of relative mrna expression levels the mrna expression profiles of cfgst genes were detected via quantitative real-time pcr (qrt-pcr). all qrt-pcr reactions were performed with the sybr premix ex taq (tli rnaseh plus, rr420, takara, japan) in a 7500 real-time detection system (thermo fisher scientific, usa). all the primers used in qrt-pcr assay were listed in table 1. the mrna expression leveld of cfgst genes were normalized to that of elongation factor 1 α (ef-1α) gene for each sample, according to our previous reports (wang et al., 2016b, 2017b). the comparative ct method (2-δδct method) was used to analyze the relative mrna expression level of gst genes (schmittgen and livak, 2008). all data were given as means ± s.d. (n = 5). the data were subjected to one-way analysis of variance (one-way anova) followed by a multiple comparison via ibm spss statistics 19.0.0.0, and the p values less than 0.05 were considered statistically significant. table 1 primers used in the present research primer sequence (5`-3`) brief information cfgstω-race-f1 ggtaatgaagtcgctgcctgctgt gene specific primer for 3` race cfgstω-race-f2 cttttataaaagttacgcagcagg gene specific primer for 3` race cfgstω-race-r1 aaaggacagaacctcatgctatacagc gene specific primer for 5` race cfgstω-race-r2 gaatctttagagtgtgatttgaga gene specific primer for 5` race cfgstσ-1-race-f1 gctgaccgagttctttaagta gene specific primer for 3` race cfgstσ-1-race-f2 taagaagaaaactttcgattcagt gene specific primer for 3` race cfgstσ-2-race-f1 acttcgaaagtgacgagactaagaagg gene specific primer for 3` race cfgstσ-2-race-f2 ctattcctaagtttgccaaaatcttcacaa gene specific primer for 3` race cfgstσ-2-race-r1 caagtaccggcagctgaccagtgggcatctttt gene specific primer for 5` race cfgstσ-2-race-r2 taatggtatcttcttcgaatgtttgcccgg gene specific primer for 5` race cfgstρ-race-f1 cagtttgcttatggggataagttcact gene specific primer for 3` race cfgstρ-race-f2 gccactgtggtacgatttggctgcgacata gene specific primer for 3` race cfgstζ-race-f1 ggctgatgcgtgtctggttcctcaggt gene specific primer for 3` race cfgstζ-race-f2 gaaacagttccctaccattgctcgtctaaa gene specific primer for 3` race cfgstζ-race-r1 acctgaggaaccagacacgcatcagccattgtc gene specific primer for 5` race cfgstζ-race-r2 ccattccattttacacctcgtccc gene specific primer for 5` race cfmgst-race-f1 ggaatgtaaaccaacgttatcggaccc gene specific primer for 3` race cfmgst-race-f2 ggatccggcaacagccctgatgtactt gene specific primer for 3` race cfmgst-race-r1 ggtccgataacgttggtttacattcct gene specific primer for 5` race cfmgst-race-r2 ggttagcgtacaccgactttcgaa gene specific primer for 5` race cfgstω-qrt-f tcgttagagtaaccaccagga gene specific primer for real-time pcr cfgstω-qrt-r atgctatacagccttagtttccc gene specific primer for real-time pcr cfgstσ-1-qrt-f agtttggtttggcgggag gene specific primer for real-time pcr cfgstσ-1-qrt-r tgcgtacttaaagaactcggtc gene specific primer for real-time pcr cfgstσ-2-qrt-f caccaccatctatctaaggacac gene specific primer for real-time pcr cfgstσ-2-qrt-r gtatcttcttcgaatgtttgccc gene specific primer for real-time pcr cfgstρ-qrt-f taccaagactccaagcctactacga gene specific primer for real-time pcr cfgstρ-qrt-r gtccttcaattctccttccagcca gene specific primer for real-time pcr cfgstζ-qrt-f gagataaggtgacaatggcgg gene specific primer for real-time pcr cfgstζ-qrt-r tttagacgagcaatggtaggga gene specific primer for real-time pcr cfmgst-qrt-f taacccggaggactgtgcca gene specific primer for real-time pcr cfmgst-qrt-r atgacaccttctgatgcgttccac gene specific primer for real-time pcr cfef-1α-qrt-f atccttcctccatctcgtcct internal control for real-time pcr cfef-1α-qrt-r ggcacagttccaatacctcca internal control for real-time pcr adaptor primer-oligo (dt) ggccacgcgtcgactagtact17vn olido (dt) primer for cdna synthetize adaptor primer ggccacgcgtcgactagtac anchor primer for 3` race adaptor primer-oligo (dg) ggccacgcgtcgactagtacg10hn anchor primer for 5` race m13-47 cgccagggttttcccagtcacgac vector primer for sequencing rv-m gagcggataacaatttcacacagg vector primer for sequencing 305 fig. 1 nucleotide and deduced amino acid sequences of six cfgsts (a: cfgstω, b: cfgstσ-1, c: cfgstσ-2, d: cfgstρ, e: cfgstζ, f: cfmgst). the nucleotides and amino acids are numbered along the left margin. capital letters indicated coding sequence, small letters indicated utrs. the gst_n/gst_c/mapeg domains are in shade. the single typical polyadenylation signal was underlined. the asterisk and bold font indicated the stop codon results identification and classification of cfgsts genes six different cfgst genes were identified from the est database and the full-length cdna sequences were obtained via race technique. based on the deduced protein sequences identities and phylogenetic analysis with other gsts, the cfgsts were classified into five classes, including two in sigma (cfgstσ-1 and cfgstσ-2) and one each in omega (cfgstω), rho (cfgstρ), zeta (cfgstζ) and the microsomal gst isoenzyme (cfmgst), respectively. the main sequence features of these gst genes were illustrated in figure 1 and table 2. the cdna sequences of these six cfgst genes were deposited to genbank 306 table 2 sequence features of the six gsts in scallop feature cfgstω cfgstσ-1 cfgstσ-2 cfgstρ cfgstζ cfmgst accession number gq240291 eu183306 gq240292 eu183305 gu361617 gq403696 est cl23ct28cn28 cl124ct131cn139 cl327ct342cn359 cl51ct57cn59 rscag0_004919 rscag0_001764 cdna length (bp) 945 1089 776 954 696 647 5` utr length (bp) 85 46 68 48 21 112 3` utr length (bp) 140 425 90 231 39 79 orf length (bp) 720 618 618 675 636 456 polyadenylation signal sites 1 1 0 1 0 1 deduced polypeptide length (aa) 239 205 205 224 211 151 domain information gst_n+ gst_c gst_n+ gst_c gst_n+ gst_c gst_n+ gst_c gst_n+ gst_c mapeg calculated molecular mass (kda) 27.65 23.22 23.02 25.76 24.20 16.86 theoretical isoelectric point 7.261 8.849 5.339 6.201 6.417 8.386 best hits by blastx (protein, taxa, e_value, score, identity) gstω-2, [haliotis discus discus], 1e-90, 279, 57% gstσ, [argopecten irradians], 2e-59, 199, 52% gstσ, [argopecten irradians], 6e-114, 334, 78% gstρ, [solea senegalensis], 4e-54, 185, 45% gstζ, [cyprinus carpio], 1e-84, 259, 59% mgst-1, [xenopus tropicalis], 4e-45, 156. 52% database under the following accession numbers: gq240291 (cfgstω), eu183306 (cfgstσ-1), gq240292 (cfgstσ-2), eu183305 (cfgstρ), gu361617 (cfgstζ) and gq403696 (cfmgst). cfmgst consisted of an open reading frame (orf) of 456 bp encoding a polypeptide of 151 amino acid residues with the calculated molecular mass of 16.86 kda, while cfgstω, cfgstσ-1, cfgstσ-2, cfgstρ and cfgstζ consisted of 239, 205, 205, 224 and 211 amino acid residues, respectively. among these five cytosolic cfgsts, cfgstω had the highest calculated molecular mass (27.65 kda) and cfgstsσ-2 had the lowest one (23.02 kda), which were consistent with most identified mammalian gsts with the calculated molecular mass ranging from 23 kda to 28 kda as heterodimers or homodimers. the theoretical isoelectric points of these six putative cfgsts proteins were calculated from 5.339 to 8.849. these six cfgsts were annotated using blastx algorithm and each of them showed high identities (from 45% to 78%) with those from other vertebrate or invertebrate species. the assignment of six cfgsts to the omega, sigma, rho, zeta and microsomal gst isoenzymes was clearly supported by the phylogenetic analysis of all these six cfgsts along with those previous identified ones from other vertebrate and invertebrate species. these six cfgsts were separated into five groups in the phylogenetic tree and each gst class formed their own clades (fig. 2). tissue distribution of cfgsts mrna the tissue-specific expression patterns of these six cfgsts mrna transcripts have been investigated in the present study. these six cfgst genes were detectable in all the examined tissues, including hemocytes, muscle, mantle, gill, hepatopancreas and gonad, although there were noticeable variations in the mrna expression levels among different tissues. the highest mrna expression levels of cfgstω, cfgstσ-1 and cfgstζ were found in hemocytes (fig. 3a,b,e), cfgstρ and cfmgst were found to be most abundantly expressed in hepatopancreas (fig. 3d,f), while the cfgstσ-2 mrna transcripts highest expressed in gill (fig. 3c). moreover, the mrna abundance of different cfgsts was also variable within one single tissue, cfgstσ-1 was the most abundant gst in hemocytes, while cfgstρ was the most scarce one (fig. 4). expression profiles of the cfgsts genes after v. anguillarum stimulation the mrna transcripts of cfgsts exhibited differential expression profiles post v. anguillarum stimulation (fig. 5). the relative mrna expression levels of cfgstω, cfgstσ-1, cfgstσ-2, cfgstζ and cfmgst were all significant up-regulated within 3 or 6 hps and reached to the peak at 12 hps, which was 26.18-fold, 13.19-fold, 23.08-fold, 18.28-fold and 15.81-fold of the origin levels (p < 0.05), respectively (figure 5a, b, c, e and f), while no significant change was observed in the mrna expression profiles of cfgstρ during v. anguillarum stimulation (fig. 5d). additionally, within the two sigma class cfgsts, the immune responses of cfgstσ-2 were more rapidly and intensely than those of cfgstσ-1 (fig. 5b,c). expression profiles of the cfgsts genes after m. luteus stimulation the m. luteus stimulation affected the mrna expression profiles of these six cfgsts differentially 307 fig. 2 consensus phylogenetic analysis based on the amino acid sequences of gsts from different organisms. the evolutionary history was inferred using the neighbor-joining method. the bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history of the taxa analyzed. all positions containing gaps and missing data were eliminated. the numbers at the forks indicated the bootstrap values. the dark circles stood for sequences from c. farreri. the sequences and their accession numbers are as follows, omega class: chlamys farreri (adf32018), crassostrea gigas (xp_011429380), danio rerio (np_001002621), haliotis discus discus (abo26600), haliotis madaka (alu63761), perna viridis (agn03944); sigma class: argopecten irradians (ang56313), c. farreri (acf25904), c. farreri (adf32019); hyriopsis cumingii (agu68336), pinctada fucata (jas04242), ruditapes philippinarum (aew46325); rho class: c. farreri (acf25903); cyprinus carpio (bas29983); ruditapes philippinarum (aew46331); sebastes schlegelii (anw83217); siniperca chuatsi (aci32418); solea senegalensis (bag12568); zeta class: chlamys farreri (add82544); cyprinus carpio (bas29981); oplegnathus fasciatus (ady80028); xenopus laevis (xp_018084636); microsomal: c. farreri (adf45336), gallus gallus (np_001129022), microtus ochrogaster (xp_005364596), osmerus mordax (aco10098), sinonovacula constricta (alc77324), xenopus tropicalis (np_001011245) 308 fig. 3 tissue distribution of six cfgsts mrna transcripts detected by qrt-pcr (a: cfgstω, b: cfgstσ-1, c: cfgstσ-2, d: cfgstρ, e: cfgstζ, f: cfmgst). the mrna expression level of cfgsts in hemocytes, mantle, gill, hepatopancreas and gonad were normalized to that of muscle. vertical bars represented mean ± s.d. (n = 5), and bars with different characters indicated significantly different (p < 0.05) (fig. 6). the relative mrna expression levels of cfgstω and cfgstρ were all significant up-regulated within 3 hps and reached to the peak at 6 hps, which was 27.03-fold and 28.73-fold of the origin levels (p < 0.05), respectively (fig. 6a,d), and those of cfgstζ were significant up-regulated at 6 hps and reached the peak at 12 hps (15.18-fold, p < 0.05, fig. 6e). while no significant difference in cfgstσ-1, cfgstσ-2 and cfmgst mrna expression was observed (fig. 6b,c,f). 309 fig. 4 quantification of abundance of different cfgst isoforms in hemocytes of untreated scallops. the abundance were calculated relative to ef-1α gene and shown as (ctgsts-ctef-1α) -1. vertical bars represented mean ± s.d. (n = 5), and bars with different characters indicated significant difference (p < 0.05) expression profiles of the cfgsts genes after p. pastoris stimulation only two cfgsts, cfgstρ and cfgstζ, were drastically induced during p. pastoris stimulation (fig. 7). the mrna expression level of cfgstρ was significantly up-regulated firstly at 3 hps (4.94-fold, p < 0.05) and then reached to the peak expression level at 6 hps, which was 18.36-fold of the origin levels (p < 0.05, fig. 7d). while the cfgstζ were significantly induced at 6 hps (6.53-fold, p < 0.05) and reached its highest expression level at 12 hps (18.78-fold, p < 0.05, fig. 7e). although these six cfgsts expressions in the normal group were slightly fluctuant throughout the experiment, no significant difference was observed (figs 5,6,7). discussion glutathione s-transferases are a well characterized protein family of multifunctional isoenzymes ubiquitously identified in many aerobic organisms from bacteria to animals, and play pivotal roles in the oxidative stress responses and detoxification pathways (hayes et al., 2005). in the present study, the full-length cdna sequences of six different gst genes, including cfgstω, cfgstσ-1, cfgstσ-2, cfgstρ, cfgstζ and cfmgst, were identified from c. farreri. their sequence features, high similarities with other previous identified gtss and the phylogenetic relationship collectively suggested that they are novel invertebrate gsts and may have similar function with gsts from other invertebrates. in the gst family, at least 15 different classes of gsts have been identified and characterized in numerous aerobic organisms according to their different primary structures, enzyme properties, physiological functions and immune activities (strange et al., 2001). according to their functional differences, gst isoforms would express differentially in various tissues. accumulating research achievements on tissue-specific expression profiles of gsts in aquatic organisms have revealed that gsts are generally abundantly expressed in the mantle, gills, hepatopancreas and gonad (li et al., 2008; ren et al., 2009; mu et al., 2010; xu et al., 2010; chen et al., 2011; li et al., 2012; yang et al., 2012; zhang et al., 2012a; duan et al., 2013; li et al., 2013; wang et al., 2013; shao et al., 2017), indicating that different tissue-specific expression pattern of gsts were associated with their differential susceptibility to antioxidant damage. in the present study, the mrna transcripts of the six cfgst genes could be detected in all tested tissues, including hemocytes, muscle, mantle, gill, hepatopancreas and gonad, suggesting that they would be involved in many crucial physiologic or immune processes of scallop. and, there were noticeable variations in the tissue-specific expression pattern of cfgsts. hemocytes have been demonstrated to play irreplaceable roles in the innate immune response of invertebrates mainly through phagocytosis, which was usually companied with oxidative stress, and tubules of gill filaments were confirmed to be the hematopoietic position in mollusks (li et al., 2017a). in the present study, almost all the six cfgsts were high expressed in hemocytes, and cfgstσ-1, cfgstσ-2, cfgstρ and 310 fig. 5 temporal mrna expression profiles of six cfgsts detected by qrt-pcr in scallop hemocytes post v. anguillarum stimulation (a: cfgstω, b: cfgstσ-1, c: cfgstσ-2, d: cfgstρ, e: cfgstζ, f: cfmgst). each values was shown as mean ± s.d. (n = 5), and bars with different characters indicated significant difference (p < 0.05) cfmgst were found to be most abundantly expressed in gills, indicating that these gsts would act as efficient immune effectors in scallop. while cfgstω, cfgstσ-1, cfgstσ-2, cfgstρ and cfmgst were highly expressed in hepatopancreas, which was consistent with the opinion that hepatopancreas was the major organ for detoxification of xenobiotics in marine invertebrates (doi et al., 2004). similar phenome has been observed in m. galloprovincialis, in which tissue distribution study revealed that mggsta, mggsts2, mggsts3 transcripts were highly expressed in hemocytes, while mggsts1 mrna was most abundantly expressed in hepatopancreas. additionally, previous reports have demonstrated that some low constitutively expressed gsts might 311 fig. 6 temporal mrna expression profiles of six cfgsts detected by qrt-pcr in scallop hemocytes post m. luteus stimulation (a: cfgstω, b: cfgstσ-1, c: cfgstσ-2, d: cfgstρ, e: cfgstζ, f: cfmgst). each values was shown as mean ± s.d. (n = 5), and bars with different characters indicated significant difference (p < 0.05) performed a crucial role in the detoxification process, while high constitutively expressed gsts might involve in protecting the cell against endogenous oxidative stress (zhang et al., 2012a). it could be speculated that cfgstσ-1 perhaps played a pivotal role in the detoxification process. so, we hypothesized based on these results that each of the gst classes with different tissues distributions might be involved in some specific physiological functions in the basal metabolism of scallop. mollusks highly rely on innate immunity, and hemocytes-mediated phagocytosis is considered as a main arm of innate immune defense strategies (song et al., 2015; wang et al., 2016a). infection of microbes could induce hemocytes-mediated phagocytosis accompanied with respiratory burst and 312 fig. 7 temporal mrna expression profiles of six cfgsts detected by qrt-pcr in scallop hemocytes post p. pastoris stimulation (a: cfgstω, b: cfgstσ-1, c: cfgstσ-2, d: cfgstρ, e: cfgstζ, f: cfmgst). each values was shown as mean ± s.d. (n = 5), and bars with different characters indicated significant difference (p < 0.05) followed by mass production of ros in various organisms ranging from invertebrate to vertebrate (halliwell, 2006; benedetti et al., 2015). compared with vertebrate gsts, rare information about the mrna expression profiles of different classes of gsts is available in mollusks, considering their indispensable roles in antioxidant system (song et al., 2015). in the present study, almost all the identified cfgsts were high expressed in hemocytes. so this tissue was selected as candidate for investigating the temporal mrna expression profiles of cfgsts post various microbe stimulations. among of the previous identified gsts from aquatic species, some could be induced by foreign stimulus or invading microbes and be involved in innate immunity (ren et al., 2009; mu et al., 2010; chen et al., 2011; li et al., 2012; yang et al., 2012; duan et al., 2013; wang et al., 2013; shao et al., 2017). in 313 the present study, it was observed that the mrna transcripts of these six cfgst genes all drastically increased after one or two kinds of microbe stimulation. for examples, cfgstσ-1, cfgstσ-2 and cfmgst could only respond to the stimulation of v. anguillarum, while both cfgstω and cfgstρ could be significantly induced by two kinds of microbe stimulation, which indicated that they could be involved in the innate immune response of scallop against different invading pathogens. interestingly, cfgstζ could respond to all the three kinds of microbe stimulation with similar expression profiles, indicating cfgstζ was involved in the innate immune responses to more microbes and its modulation to different invading microbes might share the similar mechanism. similar phenome has been observed in v. philippinarum, in which all the vpgsts showed differential response profiles depending on the concentrations of various toxicants and exposure times. additionally, cfgstσ-2 with low basal mrna expression level responded to invading v. anguillarum more rapidly and intensely than cfgstσ-1, similarly, the basal mrna expression level of escytmnsod in hemocytes was higher than that of esmtmnsod by approximately two times, which indicated that escytmnsod might play a more routine role in the physiological activity of crabs (wang et al., 2015). these differences in their mrna expression profile indicated that cfgstσ-1 might play a routine role in the detoxification process, while cfgstσ-2 would mainly be involved in the response to invading pathogens. in summary, the full-length cdna sequences of six gst genes, including cfgstω, cfgstσ-1, cfgstσ-2, cfgstρ, cfgstζ and cfmgst, were obtained from c. farreri. all the cfgsts were constitutively expressed in all the tested tissues and they were drastically but differentially induced post different microbe stimulation. based on these obtained results, it could be hypothesized that cfgsts were involved in the defense responses of c. farreri against bacterial infection. additionally, the difference in their temporal mrna expression patterns against various microbe stimulation indicated that cfgsts would play pivotal but different roles in the innate immune responses of scallop. acknowledgement this research was supported by the national natural science foundation of china (31530069). we are grateful to prof. zhaolan mo, yellow sea fisheries research institute, chinese academy of fishery sciences, for her kindly providing the v. anguillarum strain m3, and dr. alexia-ileana zaromytidou, springer nature, for her kindly help in english writing. we would like to thank the editor office and the expert reviewers for their constructive suggestions and enlightening comments during the revision. references benedetti s, nuvoli b, catalani s, galati r. reactive oxygen species a double-edged sword for mesothelioma. oncotarget 6: 16848, 2015. chen j, xiao s, deng y, du x, yu z. cloning of a novel glutathione s-transferase 3 (gst3) gene and 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may 16, 2016 abstract hemocytes are the main effector elements of gastropod anti-trematode defence reactions. elucidation of morphological and functional characteristics of hemocytes allows for better understanding of gastropod resistance mechanisms. hemocyte composition of planorbarius corneus revealed types of cells: granulocytes and hyalinocytes, which differ in granularity, nucleus-tocytoplasm ratio and spreading compatibility. flow-cytometric analysis suggested the presence of these cell types in the hemolymph of p. corneus and showed the differences in granulocyte/hyalinocytes ratio in non-infected snails and snails infected with different trematodes cotylurus sp., notocotylus sp., plagiorchis sp. and echinostoma sp. it was also shown that in snails with large shell diameter (34 37 mm), the ratio of cell types in the hemolymph is clearly biased in favour of granulocytes. key words: snails, trematodes, granulocytes, hyalinocytes, defense reactions, phagocytosis   introduction hemocytes are the principal effector elements of defence reactions in gastropods. they are involved in all stages of the defence reaction identification, isolation and elimination of foreign bodies and also in restoring the mollusc's internal environment after defence reaction (adema et al., 2000; connors, 2003). information on hemocyte morphological types and their functional activity remains fragmentary. inconsistencies in the current system of hemocyte classification make comparison difficult. in addition, most immunological researches in gastropods have been done only on few species of mollusc, mainly on genus biomphalaria. there is a need to expand the set of molluscs used in such work. planorbarius corneus is a widespread species and an intermediate host for many trematodes (faltynkova et al., 2004; brown et al., 2011). it attracts the attention as natural model object for studying hemolymph/ immunological assays (ottaviani and cossarizza, 1990; ottaviani et al., 1993), molecular genotyping (prokhorova et al., 2015), physiological and ecological research (otludil ___________________________________________________________________________ corresponding author: gennady l ataev department of zoology herzen state pedagogical university of russia 191186, moyka river 48 saint-petersburg, russian federation e-mail: ataev@hersen.spb.ru et al., 2004; stepan et al., 2012; zbikowska et al., 2013). however, the analysis of trematode infection on the hemocyte composition in p. corneus has not been done before. materials and methods snails planorbarius corneus (n = 275) molluscs were collected from pure water springs in leningradskaya oblast (russia). molluscs species were identified based on morphological criteria (gloer, 2002; alexeev and tsalolyhin, 2016). the molluscs were kept in 10 20 l plastic aquariums filled with a mix of tap water and filtered pond water (1:1) in climatic chamber at 21 ± 1 °c under 12-h light: 12 h dark photocycle. the water was aerated continuously and changed every 3 days. chalk was used for са 2+ source and ph stabilized at about 7.0 with sodium bicarbonate. all snails were fed on lettuce leaves. some of the snails were infected with cotylurus brevis (strigeidae) (n = 10), notocotylus ephemera (notocotylidae) (n = 12), plagiorchis multiglandularis (plagiorchiidae) (n = 14) and echinostoma spiniferum (echinostomatidae) (n = 10). the definitive hosts of all these digenea species are wild ducks. the trematode infection of molluscs was defined by cercarial shedding. mollusks were put to individually plastic cuvets in the early morning and at the end of afternoon for detection of cercarial 164 mailto:ataev@hersen.spb.ru shedding every other day during 4 weeks following collection. the extent of infection was defined by dissection of molluscs after hemolymph collection. for the experiment, molluscs with shell diameter of 22 37 mm were selected (most measured 26 29 mm). hemolymph collection, incubation and sample preparation for morphological typing of hemocytes of p. corneus (n = 107), haemolymph was collected using glass pipettes from the pericardial region of the snail (sminia and baredsen, 1980). from 3 to 6 hemolymph samples were done for every mollusc. the number of hemocytes in 1 µl of hemolymph was counted in cell-counting chamber (cell-line associates). a part of hemolymph specimens were used to prepare fixed smears. haemolymph was applied on polylysine-coated glasses and cells were allowed to settle in a humid chamber for 30 40 min. then smears were fixed with 4 % paraformaldehyde solution prepared on pbs, rinsed twice by pbs, and stained with erlich hematoxylin-eosin. observations of hemolymph smears were used in addition to live hemocyte analysis for hemocytes morphological typing. to study the hemocytes in vitro, hemolymph from individual snails was placed on plastic petri dishes and incubated in a humid chamber for 4 8 h at 22 24 °c. during the incubation the specimens were intermittently observed (every 10 20 min) using a phase-contrast microscope (leica dm 5000). for the majority of snails it the total number of hemocytes/µl were counted. cell counts and size measurements were preformed using imagescope software (cma, russia). every cell was measured twice in minimum and maximum diameters, cell areas were calculated. flow-cytometric assay hemolymph for analysis in the flow cytometer (coulter epics altra, beckman coulter) was collected in plastic eppendorf tubes with 20 mm edta. sample analysis was done immediately with the following cytometric parameters. forward lightscatter (fs) and side-scatter (pmt1) signals were collected for 30,000 cells from each sample and stored as list mode data files. fs gives a relative indication of cell size, while pmt1 is an indication of complexity, texture or granularity of cells. gating of the cells was performed to exclude dead cells and debris from subsequent analyses. ranges for distinguishing the cell groups characterized on different size and complexity have been chosen first for non-infected snails and also were applied for trematode-infected molluscs. in the flow-cytometric analysis of tematode-infection influence on the hemocyte composition number individuals with 26 29 mm shell diameter were used including 47 noninfected and 46 trematode-infected molluscs. phagocytosis assay fluorescein isothiocyanate (fitc)-conjugated escherichia coli and staphylococcus aureus bacteria were used to study phagocytic activity in the hemocytes. the bacteria were stained with fitc (sigma) according to the method of coteur et al. (2002). incubation was carried out in the dark at 4 °c for 24 h. then the material was washed in pbs (1.7mm kh2po4, 5.2mm na2hpo4, 150 mm nacl, ph = 7.4) and physiological saline solution (0.9 % nacl). bacterial cell concentration was 107 cells/ml. the bacterial suspension (80 100 µl) was injected into the foot of the mollusc (n = 33). a control group of molluscs was injected with buffered saline solution (n = 20). hemolymph was analysed using a fluorescent microscope (leica dm 5000) and flow cytometer at three, six and 12 h post injection. for flow-cytometric analysis haemolymph was quenched with 0.5 % trypan blue in pbs (serva). while fs and pmt1 characteristics were acquired in linear mode, fluorescence intensity at wavelength of 530 nm (pmt2, fitc) was acquired at log scale. this channel was used for the analysis of green fluorescence positive cells. the resulting files were analyzed using expo32 (coulter, hialeah, fl) software. to study phagocytic activity in the haemocytes in vitro, hemolymph from snails (n = 22) was incubated with a suspension of the fitclabeled st. aureus bacteria in a humid chamber at 22 24 °c. the final bacterial concentration was adjusted to 107 cells/ml. samples were analysed every 10 min during 6 h. statistical analyses data were analyzed using microsoft excel software (microsoft). differences between data groups were tested by student’s t-test for independent and dependent samples. differences were considered as significant at p < 0.05. results are shown as mean percentage and standard deviation. in order to test correlation between distinct criterions pearson’s correlation coefficient (r) was used. the significance was computed using t test in past software (http://folk.uio.no/ohammer/past). results morphological types of hemocytes based on cell morphology, two distinct types of p. corneus (n = 107) hemocytes were observed. majority of cells (70.5 ± 3.1 %) were granulocytes (figs 1а d), the spreading cells with dimensions of 9.5 ± 5.9 x 12.95 ± 7.9 µm (cell areas of 174.26 ± 34.18 µm2) and oval nuclei (diameter 4.1 ± 2.8 x 5.3 ± 2.2 µm). their average nucleus-to-cytoplasm ratio (n/c) was about 0.12. the internal part of the cell's cytoplasm contained numerous granules and vesicles. granulocytes form numerous filopodiae and seldom lobopodiae. the less numerous subpopulation was represented by (24.7 ± 2.3 %) hyalinocytes (fig. 1d), rounded cells with dimensions 6.1 ± 1.2 x 8.1 ± 1.5 µm (cell areas of 41,79 ± 6.54 µm2), containing spherical or oval nuclei (diameter 2.6 ± 1 x 3.3 ± 1.3 µm). the average nucleus-to-cytoplasm ratio of these cells was approximately 0.25, and they were capable of forming lobopodiae. the morphology of some cells (about 3 %) was similar to hyalinocytes (fig. 1f), but they were smaller (4.5 ± 0.4 µm diameter with average n/c of 0.48. 165 http://lingvo.yandex.ru/specimen/%d1%81%20%d0%b0%d0%bd%d0%b3%d0%bb%d0%b8%d0%b9%d1%81%d0%ba%d0%be%d0%b3%d0%be/lingvouniversal/ http://lingvo.yandex.ru/specimen/%d1%81%20%d0%b0%d0%bd%d0%b3%d0%bb%d0%b8%d0%b9%d1%81%d0%ba%d0%be%d0%b3%d0%be/lingvouniversal/ http://folk.uio.no/ohammer/past fig. 1 hemocytes from p. corneus. two main types of cells with different spreading compatibility and granularity were obtained. granulocytes (a d) contains many granules (g) and vesicles in abundant cytoplasm and form numerous filopodiae (f), rapidly spreading across the substrate. after long-term incubation in a humid chamber the majority of the granulocytes change to large cells with nuclei containing large amounts of scattered heterochromatin clumps (b). hyalinocytes (e, f) have thin cytoplasm and mainly oval or round shape and sometimes form one lobopodia (l). these cells slowly spread on the substrate. phase-contrast microscopy. bar = 5 µm. pools of hemocytes, detected by flow cytometry flow cytometry of the hemolymph of p. corneus molluscs detected two populations of hemocytes: small cells with a low number of granules, and larger, more granular cells. in terms of relative dimensions and granularity, these haemocyte populations correspond to the above described granulocytes and hyalinocytes, respectively (fig. 3). in non-infected molluscs (n = 47), the hyalinocytes account for 58.5 ± 6.5 % of all hemocytes, and the granulocytes 37.1 ± 7.3 %. however, the cell populations are not segregated evidently (figs 3e, f). the correlation between number of circulating hemocytes and snail size one µl of hemolymph from a non-infected p. corneus mollusc contains 439 ± 176 cells (from 215 to 1,089) (table 1). no significant difference in number of hemocytes in molluscs with different shell diameter was observed. however, correlation analysis of the hyalinocytes/granulocytes ratio and mollusc shell diameter established a reliable inverse correlation (r = -0.93, n = 31, p < 0.001). nevertheless, hyalinocytes predominate in all groups (table 1). the activity of hemocytes p. corneus hemocytes retain their ability to survive in the humid chamber up to 8 h. hemocytes from snails which had previously been injected with e. coli and s. aureus remained viable for 4 6 h. during the observation period granulocytes changed their shape. they gradually spread across the substrate, simultaneously moving through it. the hyalinocytes gradually became fixed on the substrate, while their shape remained almost unchanged (figs 2a, b).often, granulocytes formed aggregates containing up to 10 cells (fig. 1c). when kept in a humid chamber for more than 5 h, large cells with nuclei containing large amounts of scattered heterochromatin lumps formed the majority of the granulocytes (fig. 1b). flow cytometry showed high intensity fluorescence of p. corneus hemocytes 3 h post injection with fitc-labelled bacteria. s. aureus were phagocytosized in 78.5 ± 7.1 % (n = 25) of cases (figs 4a, b), e. coli in 49.3 ± 12.4 % (n = 8) of cases (fig. 4c). moreover, e. coli were preferably absorbed by hyalinocytes, whereas granulocytes exhibited preference for s. aureus. the fluorescent intensity of hemocytes in molluscs of the control group (n = 20) remained at the same level as the intact individuals. three h after fitc-labelled bacteria injection fluorescing structures with dimensions (2 4 um) significantly greater than the dimensions of the bacterial cells were observed in cytoplasm of the granulocytes (fig. 2c). fluorescent structures were found only in small proportion of granulocytes 4 6 h post injection. no such structures were observed in the hyalinocytes. 166 fig. 2 the functional activity of p. corneus granulocytes. a, b) demonstration of granulocytes motility. granulocyte (left) changes the shape and moves across the substrate. hyalinocyte (right) doesn’t move and remain almost unchanged. phase-contrast microscopy. bar =1 0 µm. c, d) phagocytosis of s. aureus by granulocytes. c) hemocyte of mollusc injected with fitc-marked s. aureus suspension 3 h post injection. fluorescing structures in cells (are indicated by arrows) are phagocytic vacuoles containing partially digested bacteria. d) hemocytes obtained by incubation in vitro with s. aureus during 30 min. phagocytic vacuoles containing bacteria appear in the hemocytes' cytoplasm (are indicated by arrow). ph = phase-contrast microphotography, fluo = fluorescence photomicrographs. bar = 10 µm. in the cytoplasm of hemocytes incubated with bacteria in vitro for 15 30 min, fluorescent structures with dimensions equivalent to the bacterial cells were observed (fig. 2d). following longer incubation periods, large fluorescent granules were observed in hemocytes similar to those described above for hemocytes in molluscs injected with bacteria. influence of trematode infection on the ratio of circulating hemocytes types the ratios of granulocytes to hyalynocytes in trematode-infected molluscs, and non-infected snails were clearly different (figs 3а d, represent the individual flow-cytometric profiles). in molluscs infected by c. brevis (n = 10), two distinct populations of cells were observed. hyalinocytes made up 32.9 ± 1.7 % and granulocytes 57.4 ± 8.4 % of all hemocytes (figs 3a, f). in molluscs infected by n. ephemera (n = 12), hyalinocytes and granulocytes comprise 34.1 ± 9.1 % and 56.1 ± 10.6 %, respectively, of all hemocytes (figs 3b, f). in molluscs infected by p. multiglandularis (n = 14), the respective populations of cells were similar to noninfected individuals: granulocytes 31.7 ± 5.1 % and hyalinocytes 56.3 ± 4.9 %. in this case, however, a boundary could be clearly discerned between the granulocytes and the hyalinocytes (figs 3c, f). in snails infected with e. spiniferum (n = 10), hyalinocytes comprised an average of 41.3 ± 8.1 % of hemocytes, and 38.1 ±11.5 % of granulocytes (figs 3d, f). table 1 number hemocytes and percentage of granulocytes and hyalinocytes in the hemolymph of p. corneus snails with different shell diameters according to flow-cytometric analysis. shell diameter 22-25 (n=10) 26-29 (n=18) 30-33 (n=10) 34-37 (n=9) number of hemocytes in 1 µl of hemolymph 356±212 459±195 462±154 480±172 percent of hyalinocytes 66.4±5.09 61.74±5.88 54.43±4.44 55.44±1.37 percent of granulocytes 29.56±4.23 33.6±5.57 40.28±4.98 40.28±6.13 167 fig. 3 a e. individual flow cytometric profiles of hemolymph from p. corneus trematodes infected (a d) and non-infected (e) snails. profiles showing a distribution of side scatter (ss, indicates relative granularity) and forward scatter (fs, ss, indicates relative size). 30,000 cells were analyzed from each sample. two distinct types of hemocytes were detected: hyalinocytes (region h) small cells with a small number of granules, and granulocytes (region g) larger, more granular cells. f. percentage of granulocytes and hyalinocytes in p. corneus none-infected and trematode-infected snails. the hyalinocytes/granulocytes ratio is different in non-infected snails and snails infected by distinct trematodes. discussion the snail-trematode host-parasite system is a commonly used model to study defence reactions of molluscs. cell reactions such as encapsulation, formation of a paleot around the parasite, and changes in the number of circulating hemocytes were firstly described for molluscs infected by trematodes (lie and heyneman, 1975; galaktionov and dobrovolsky, 2003). cell reactions to trematode infection are currently considered as a resistance mechanism in the host-parasite system (loker, 2010). as such, hemocyte composition and peculiarities of hemocyte activity are useful tools to characterize the immune system of snails and define their resistance mechanisms to the parasite. our results suggest the presence of two main cell types in p. corneus hemolymph: granulocytes and hyalinocytes. hyalinocyte and granulocyte populations exhibited distinct morphology, granularity rate, motility, and phagocytosis activity. flow cytometry analysis of the haemolymph confirmed the data obtained by microscopy. the populations of small, low granular hyalinocytes and large, more granular granulocytes were revealed. similar subpopulations of p. corneus hemocytes have been earlier described by ottaviani (1983). the above-referenced study described "round cells" and "spreading cells", were labelled by different groups of bioactive polypeptides, and exhibited differences in their organelle composition (ottaviani et al., 1991; ottaviani and franchini, 1988). large cells which remained viable for longer period of time than other hemocytes. morphologically similar cells have been described in the composition of the cellular capsules that form around degenerating sporocysts (cheng and galloway, 1970), allografts and xenografts of different snail tissues (cheng and 1984; sullivan et. al., 1993; orta and sullivan, 2000). it has been suggested that some granulocytes become hypertrophied cells in response to pathological changes in the recipient-mollusc's organism, while the rest merge to form megacytes (jourdane and cheng, 1987). it has also been suggested that granulocytes have greater resistance to disturbances of homeostasis (hahn et al., 2001). metabolites facilitating cell destruction accumulate during long-term incubation of mollusc hemocytes. large granulocytes (fig. 1b) appear to be more resistant to the toxic effects of the metabolites than other cell types. it is conceivable that the large granulocytes represent a specialised group of cells involved in encapsulation processes. the ability of granulocytes to spread across the substrate and to adhere to other cells confirms their principal roles in the process of encapsulating alien bodies (van der knaap and loker, 1990; loker, 2010). we observed significant variability not only in size, but also in morphology of granulocytes. after 168 fig. 4 phagocytosis of bacteria by hemocytes of p. corneus snails injected by bacteria suspension. flowcytometric analysis showed high intensity fluorescence of p. corneus hemocytes three hr post injection with fitclabelled bacteria. fluorescent positive (phagocyted) cells were detected at wavelength of 530 nm (pmt2, fitc). st. aureus were phagocytized in 78.5 ± 7.1 % (n = 25) of cases, primarily by granulocytes (b) and e. coli in 49.3 ± 12.4 % (n = 9) of cases, primarily by hyalinocytes (c). a) histogram of fluorescent intensity of hemocytes from snails injected with s. aureus. b) plots of phagocytosis of s. aureus, c) plots of phagocytosis of e. coli. percent of hemocytes which phagocytized bacteria is shown. several h of incubation, these cells can change their shape, dimensions, and number of pseudopodia. granulocytes of various forms (flattened and polygonal) have been described in the composition of cellular capsules around transplants and parasites (byrd and maples, 1969; lie and heyneman, 1976; loker et al., 1986; ataev and coustau, 1999). morphological changes in granulocytes during incubation graphically illustrate their polymorphic nature. previously described morphotypes of pulmonate granulocytes probably represented various phases in granulocyte differentiation. analysis of phagocytic activity of hemocytes also confirmed the existence of different functional hemocyte populations. flow-cytometric analysis of the uptake of fitc-conjugated bacteria by hemocytes in p. corneus demonstrated a reduction in fluorescence in hemocytes of phagocytised bacteria at 6 12 h post injection. haemocytes rapidly defended the mollusc's internal environment from pathogens. in the cytoplasm of granulocytes in molluscs injected with fitc-labelled st. aureus, large fluorescent granules were visualised after 1 3 h. these granules are likely to represent phagocytic vacuoles (phagolysosomes) containing partially digested bacteria. flow-cytometry analysis and microscopical analysis provided different granulocyte/hyalinocyte ratios. obviously such different results were due to the low ability of hyalinocytes to spread and adhere to substrates. also, some of the cells were lost during preparation of monolayers and were not counted. bacterial injection leads to a 12 19 % increase in the number of p. corneus circulating hemocytes. similar data have previously been obtained for bivalves. injections of bacteria into mussels and oysters also lead to an increase in the number of circulating hemocytes (hernroth, 2003; terahara et al., 2006). similar data have been obtained for bivalvia. introduction of gram-negative vibrio bacteria into mussels leads to a sharp reduction in the number of hemocytes. however, the relative number of hyalinocytes increases, which may prove that these cells are involved in antimicrobial response (allam et al., 2001). gram-positive s. aureus and gramnegative e. coli were phagocytised primarily by granulocytes and hyalinocytes respectively, there is functional differentiation between different types of hemocytes depending on type of antigen (parisi et al., 2008). the microbicidal activity of granulocytes has been confirmed by the antimicrobial peptides expression. defensins and myticins are accumulated in the granulocytes cytoplasm in bacteria-immunised bivalves and ensure the elimination of gram-positive bacteria and funguses (mitta et al., 2000а, b). hemocytes are involved in all steps of the snail anti-parasite response (loker, 2010). as such, changes in the concentration of circulating cells can be seen as one of important criterions in defense reactions (ataev and polevshchikov, 2004). immunization of snails by different antigens can induce the activation of hematopoiesis (ataev et al., 2000; azevedo et al., 2006; ataev and prokhorova, 2013; sullivan and belloir, 2014). experiments show that the local cell reaction results the immobilization of large number of cells in the tissue (avesedo et al., 2006; prokhorova et al., 2015). such processes are able to cause the changes in the amount and composition of circulating hemocytes. flow-cytometric analysis showed the predominance of hyalinocytes in non-infected snails. however, in molluscs with large shell diameter, the ratio of hemocyte types is clearly biased in favour of granulocytes (fig. 4). increases in hemocyte numbers and the predominance of granulocytes in molluscs with large shell diameter indicate that the mollusc's defence systems "acquire" greater resistance to the influence of various antigens during the individual's lifetime. 169 http://www.ncbi.nlm.nih.gov/pubmed?term=%22ataev%20gl%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22polevshchikov%20av%22%5bauthor%5d we have not observed significant differences in the number of hemocytes between non-infected and infected p. corneus molluscs. this might be due to the fact that we studied only naturally infected molluscs, for which the time of infection was unknown. therefore, we were unable to gather data as to the dynamics of the number of hemocytes during the course of trematode infection. such data, however, have been reported for biomphalaria glabrata (ataev and coustau, 1999). during infection of snails by echinostoma caproni, sharp changes in the number of circulating hemocytes were observed only during the first week post infection. subsequently, their levels declined to just above the number of hemocytes in non-infected molluscs. hemocyte populations were more clearly distinguishable in trematode infected p. corneus molluscs, suggesting that infection leads to differentiation of the hemocytes involved in cell response. moreover, the granulocyte/hyalynocyte ratios were different in molluscs infected by trematodes of different types (fig. 3f). apparently, differentiation of cell response in pulmonates depends on the species of parasite. similar results were described for biomphalaria tenagophila and b. glabrata snails infected by schistosoma mansoni (martins-souza, 2009). experimental infection resulted in early reduction of large and medium circulating hemocytes followed by an increase of small hemocytes. such a response was particularly intense in the parasite-resistant b. tenagophila. the authors assumed that hemocyte response is associated with the cellular response of resistant snails against the parasite. the peculiarity of cell response depends on the different development of the trematode inside the mollusc (bayne et al., 2001; galaktionov and dobrovolsky, 2003). one factor influencing the ratio of circulating hemocytes is the formation of a “paletot” around the parasite (galaktionov and dobrovolsky, 2003). as a result of the mutation of cell reactions and the adhesion of a significant number of hemocytes onto the tegument of the sporocyst, the ratio of circulating cells can also change. it also affects the ratio of circulating hemocytes, and, in particular, formation of multilayered hemocyte capsules around the parasites (ataev and coustau, 1999). this study confirms that trematodes closely interact with the internal environment of the mollusc, influencing the ratio of cell types in hemolymph. acknowledgments this 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der knaap wpw, loker es. immune mechanisms in trematode-snail interactions. parаsitol. today 6: 176-182, 1990. zbikowska e, wrotek s, cichy a, kozak w. thermal preferences of wintering snails planorbarius corneus (l.) exposed to lipopolysaccharide and zymosan. j invertebr. pathol. 112: 57-61, 2013. 171 271 isj 14: 271-281, 2017 issn 1824-307x research report evaluation of a novel, short polya signal from the bombyx mori bidensovirus m wang, q yu, y li, y zhang, d miao, z hu, q yao, k chen institute of life sciences, jiangsu university, 301 xuefu road, zhenjiang, jiangsu 212013, china accepted august 7, 2017 abstract bombyx mori bidensovirus (bmbdv) is the only virus which belongs to the new bidnaviridae family established by the international committee on taxonomy of viruses in 2012. the genes encoding the major capsid protein and dna polymerase (ppolb) overlap partially at their 3′ untranslated regions, forming an extremely short, dual-function polyadenylation signal/stop codon (bmbdv polya) to complete post-transcriptional modifications and terminate protein expression. nevertheless, the functionality and usefulness of this signal regarding foreign genes remain unknown. to determine the effect of the bmbdv polya on gene expression, the expression of the green fluorescent protein and firefly luciferase was evaluated with the bmbdv polya, compared with the much larger sv40 polya, under the control of the p5 or ie1 promoter. fluorescence microscopy and dual luciferase assay both revealed enhanced expression of these proteins in the presence of the bmbdv polya, meanwhile real-time qpcr also showed increased mrna levels. therefore, we conclude that the bmbdv polya is a promising, characteristic polya signal from an insect virus, and that it can promote gene expression at either the mrna or protein levels. additionally, this study also suggests that the bmbdv polya potentially alleviates restrictions associated with the size of inserted fragments during constructing recombinant viruses. key words: bombyx mori bidensovirus; polyadenylation; protein expression assay; real-time qpcr introduction polyadenylation is an intermediate process between transcription and translation, which usually comprises the subsequent cleavage of the pre-mrna and the addition of a polya tail (moore et al., 1985; wickens, 1990). this process depends on recruiting cellular rna processing factors, such as the cleavage and polyadenylation specificity factor (cpsf) and cleavage stimulation factor (cstf), to the carboxyl-terminal domain of rna polymerase ii (keller et al., 1991; venkataraman et al., 2005). then, cpsf and cstf recognize canonical aauaaa motifs and gu-rich sequences in the transcripts, respectively (murthy et al., 1995). cleavage occurs approximately 10 30 nucleotides (nt) downstream of the aauaaa motif. subsequently, polya polymerase adds a stretch of a residues to form the polya tail, which is involved in mrna stability and transportation from the nucleus to the cytoplasm ___________________________________________________________________________ corresponding authors: qin yao keping chen institute of life sciences jiangsu university 301 xuefu road, zhenjiang ,jiangsu 212013, china email: yaoqin@ujs.edu.cn email: kpchen@ujs.edu.cn (beilharz et al., 2007). to the best of our knowledge, a typical polya signal contains three elements of aauaaa for cpsf binding and a downstream gu-rich region for cstf binding, as well as a seven-t element for potential transcription termination ( westwood et al., 1993; jin et al., 2000). widely used polya signals, such as the sv40 polya and the hsvtk polya, are usually derived from viruses associated with mammals. in the present study, the sv40 polya was inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (bevss) (salem et al., 2015). additionally, the sv40 polya was inserted into a green fluorescent protein (gfp) gene expression cassette to test piggybac-mediated germline transformation of the silkworm b. mori (tamura et al., 2000). the potential of being used for expressing exogenous products of polya signal from insect viruses has not been studied very well. bmbdv is the type species in the new genus bidensovirus, which belongs to the bidnaviridae family that was established by the international committee on taxonomy of viruses in 2012 (adams et al., 2012). this virus can specifically infect the columnar cells of the midgut epithelium (hu et al., 2013) of silkworms, leading to chronic densonucleosis disease. bmbdv has evolved from a mailto:yaoqin@ujs.edu.cn mailto:kpchen@ujs.edu.cn 271 table 1 primers used for small insertions synthesized by annealing in this study primer one complementary strand sequence (5′-3′) ranges (nt) construction bdvpa (63) -acgcgtttgttggttttcaataaataaatgtattaaaatttat aaatgttttattcaattacaacatcagtcgac 2,896–2,958 pt-p5-gfp-bdvpa (63) pt-ie1-gfp-bdvpa (63) -tctagattgttggttttcaataaataaatgtattaaaatttat aaatgttttattcaattacaacatcagtcgac pgl3-p5-bdvpa (63) -tctagattgttggttttcaataaataaatgtattaaaatttat aaatgttttattcaattacaacatcaggatcc pgl3-ie1-bdvpa (63) bdvpa (92) -tctagaataaataaatgtattaaaatttataaatgttttattc aattacaacatcatccatataattagaatacaattcaccctta taaccaaatggtgtcgac 2,909–3,000 pgl3-p5-bdvpa (92) bdvpa (50) -tctagaataaataaatgtattaaaatttataaatgttttattc aattacaacatcagtcgac 2,909–2,958 pgl3-p5-bdvpa (50) bdvpa (38) -tctagaataaataaatgtattaaaatttataaatgttttattc agtcgac 2,909–2,946 pgl3-p5-bdvpa (38) bdvpa (26) -tctagaaaatgttttattcaattacaacatcagtcgac2,933–2,958 pgl3-p5-bdvpa (26) bdvpa (24) -tctagaataaataaatgtattaaaatttatgtcgac2,909–2,932 pgl3-p5-bdvpa (24) parvovirus ancestor from which it inherited a jelly-roll capsid protein and a superfamily 3 helicase (krupovic et al., 2014). just like other parvoviruses, bmbdv is a non-enveloped, linear dna virus that has a spherical, icosahedral structure (zhang et al., 2016). although the virus is only 22 nm in diameter, its genome consists of two non-homologous single-stranded dna molecules (vd1 and vd2; 6,543 and 6,024 nts, respectively), which are encapsidated into separate virions. the vd1 consists of four open reading frames (orfs), which encode three nonstructural proteins [ns1, ns2 and dna polymerase (ppolb)] (f. wang et al., 2011)and one structural protein (mcp, vp) (li et al., 2009). the mcp transcript and the ppolb transcript overlap for 10 nts at their 3′ untranslated regions (utrs). additionally, the distance between the stop codons of vp and ppolb is only 24 nts. as mentioned above, this means that there is a short polya signal (bmbdv polya) in the overlap regions of these two genes. this characteristic is similar as sv40 polya (contains two genes’ polya signal from complimentary strands), but the bmbdv polya is much shorter in length. to test whether this novel polya signal from an insect virus is potentially useful for expressing exogenous products in insect cell lines, an intact bmbdv polya containing an aauaaa motif and gu-rich regions was fused with a gfp-encoding gene (gfp) and a firefly luciferase-encoding gene under the control of the p5 or ie1 promoter, respectively. the p5 promoter (236 bp) is an early promoter of bmbdv, which is used to drive ns1 and ns2 gene expression (zhu et al., 2012). the ie1 promoter (632 bp) is a strong promoter that drives the expression of an immediate early gene in the baculovirus prototype autographa californica multiple nucleopolyhedrovirus (fu et al., 2015) , and its activity is stronger than that of the p5 promoter. then, the expression of gfp and firefly luciferase was assessed qualitatively by fluorescence microscopy and dual luciferase assay in different insect cell lines, respectively. to clarify whether the bmbdv polya affects the transcription level of foreign genes, real-time qpcr was used to test the relative content of gfp mrna obtained with the bmbdv polya, compared with the sv40 polya. because the more widely used polya signals have lengths of approximately 250 bp, the development of a much shorter, functional polya signal that has the potential to increase the vector carrying capacity would be beneficial. therefore, a dual luciferase reporter gene assay was performed to determine the core functional areas of the bmbdv polya that might play significant roles in gene expression. overall, determining the effect of the bmbdv polya on the expression of foreign genes driven by different promoters in insect cells may provide a theoretical reference for its potential application to other viral expression vector systems. materials and methods recombinant plasmid construction two different marker genes were used to test the roles of the bmbdv polya in gene expression levels under the control of the p5 or ie1 promoters. transfer vectors were constructed to generate many recombinants that express gfpand firefly luciferase-encoding genes. the primers used for constructing the plasmids are listed in table 1. to test the effect of the bmbdv polya on gfp 272 272 expression levels driven by the p5 or ie1 promoters, we designed two 63-bp long primers (nt 2,896 2,958) that are derived from the genes encoding mcp and ppolb. then, two restriction endonuclease sites (mlui and sali) were added at each end, and the small fragment of bdvpa(63) was synthesized by annealing. subsequently, the small dna was cleaved with mlui/sali to delete a redundant base to produce a sticky end. we had previously constructed two plasmids in our laboratory, pt-ie1-gfp-sv40 and pt-p5-gfp-sv40, each of which contains a gfp expression cassette. these two plasmids were digested with mlui/sali to delete the sv40 polya, and they were ligated to the 63-bp mlui/sali fragment of bdvpa(63) to generate two new plasmids: pt-ie1-gfp-bdvpa(63) and pt-p5-gfp-bdvpa(63). for the pt-ie1-gfp and pt-p5-gfp constructs, a 720-bp blni/sali fragment carrying the gfp gene was cloned between the blni and mlui sites of pt-ie1-gfp-sv40 (the template was pcr-amplified using gene-specific primers: sense, 5′-cctaggatggtgagcaaggg-3′; antisense, 5′-gtcgactacttgtacagctcgtcca-3′). the plasmids pt-ie1-gfp-sv40 and pt-p5-gfp-sv40 were digested with blni/sali to retrieve pt-ie1 and pt-p5, and then ligated with the 720-bp blni/sali fragment to produce the transfer vectors pt-ie1-gfp and pt-p5-gfp. to detect the impact of the bmbdv polya on firefly luciferase gene expression levels driven by the p5 or ie1 promoters, two small fragments of bdvpa(63) with different restriction endonuclease sites (xbai/sali and xbai/bamhi) were synthesized by annealing, cleaved with xbai/sali/bamhi to produce a sticky end, and inserted downstream of the firefly luciferase gene in the long fragments pgl3-p5-sv40 − and pgl3-ie1-sv40 − , which were retrieved from pgl3-p5-sv40 and pgl3-ie1-sv40 that are preserved in our laboratory, to produce two clones carrying the p5 or ie1 promoter upstream of the firefly luciferase gene with a downstream bdvpa(63) [pgl3-p5-bdvpa(63) and pgl3-ie1-bdvpa (63)]. to determine bmbdv polya core functional areas, a series of signal fragments containing characteristic sequences (nt 2,909 3,000, 2,909 2,958, 2,909 2,946, 2,909 2,932 and 2,933 2,958) were synthesized by annealing, and cleaved with xbai/sali to form sticky ends. then, these small fragments were inserted into the long fragment pgl3-p5-sv40 − , which was retrieved from pgl3-p5-sv40 by digestion with xbai/sali. thus, these novel plasmids were named as follows: pgl3-p5-bdvpa(92), pgl3-p5-bdvpa(50), pgl3-p5-bdvpa(38), pgl3-p5-bdvpa(24) and pgl3-p5-bdvpa(26). all transfer vectors were confirmed by restriction endonuclease and dna sequence analyses. transfection of fusion plasmids and detection of gfp expression a qualitative analysis of gfp production driven by the p5 and ie1 promoters in the presence of the bdvpa(63), compared to the sv40 polya, was conducted as follows: the bmn cells used throughout this investigation were grown at 27 °c in 25-cm 2 flask cultures containing tc-100 (life technologies, carlsbad, ca, usa) supplemented with 10 % fetal bovine serum (fbs; gibco-brl, grand island, ny, usa) and 1 % antibiotics (penicillin-streptomycin; hyclone gibco-brl). hi5 and sf21 cells were maintained at 27 °c in grace’s medium (life technologies) supplemented with 10% fbs and 1 % antibiotics. the cell confluency was approximately 80 % -95 % at the time of transfection. six groups were set up in the transfection experiments. they included p5 promoter-regulated plasmids (pt-p5-gfp-sv40 and pt-p5-gfp-bdvpa(63)), ie1 promoter-regulated plasmids (pt-ie1-gfp-sv40 and pt-ie1-gfp-bdvpa(63)) and control plasmids (pt-p5-gfp and pt-ie1-gfp). these fragments were prepared by diagnostic restriction endonuclease digestions prior to transfection (usually 3 μg of dna is needed to transfect one 25-cm 2 tissue culture flask). transfections were performed with cellfectin ii reagent liposomes (invitrogen, carlsbad, ca, usa) according to the manufacturer’s instructions (as follows). three micrograms of the experimental group plasmid and 200 μl of serum-free tc-100 medium were mixed. then, the mixture was incubated for 15 min at room temperature. then, 10 μl of cellfectin ii reagent liposomes reagent and 200 μl of serum-free tc-100 medium were added and incubated for 15 min at room temperature. finally, the dna/cellfectin ii reagent liposomes reagent mix was incubated for 30 min at room temperature. meanwhile, the sf9, hi5, and bmn cells were washed three times with serum-free tc-100 culture medium. then, the dna/cellfectin ii reagent liposomes mixture was added dropwise to the 25-cm 2 tissue culture flasks, which were placed in a 27 °c incubator. after 5 h, the medium containing the dna/cellfectin ii reagent liposomes reagent mixture was removed, and 10 ml of fresh grace’s complete medium was added to the hi5 and sf9 cells culture flasks, while tc-100 complete medium was added to the bmn cells culture flasks. then, these cells were again placed in a 27 °c incubator. green fluorescence emitted by gfp was observed by fluorescence microscopy 48 h after the transfections. dual luciferase reporter gene assay to qualitatively analyze the influence of the bmbdv polya on gene expression, a dual luciferase reporter gene assay was performed. bmn cells were grown at 27 °c in 24-well plates containing tc-100 supplemented with 10 % fbs and 1 % antibiotics. hi5 cells were maintained at 27 °c in the grace’s medium supplemented with 10 % fbs and 1 % antibiotics. the cell confluency was approximately 80 % 95 % at the time of transfection. six groups were co-transfected with renilla luciferase-expressing plasmids in the transfection experiments (the quantity of the firefly luciferase plasmids was 100 times that of the renilla luciferase-expressing plasmids). they included p5 promoter-regulated plasmids [pgl3-p5-sv40 and pgl3-p5-bdvpa (63)], ie1 promoter-regulated recombinant plasmids [pgl3-ie1-sv40 and pgl3-ie1-bdvpa (63)], as well as control plasmids without a polya signal (pgl3-p5 and pgl3-ie1). these plasmids were prepared by diagnostic restriction endonuclease digestions prior to 273 273 transfection (usually 1 μg of dna is needed to transfect one well of a 24-well plate). transfections were performed with cellfectin ii reagent liposomes (invitrogen, carlsbad, ca, usa) according to the manufacturer’s instructions (as mentioned above in the transfections with the gfp fusion plasmids). at 48 h after transfection, the complete medium in 24-well plates was removed. then, the hi5 and bmn cells were washed two times with 1×phosphate-buffered saline. afterwards, 200 μl of passive lysis buffer was added and incubated for 15 min at room temperature to ensure that the cells were fully lysed. the mixture was collected in 1.5-ml eppendorf tubes for the dual luciferase reporter gene assay. then, 100 μl of cell lysate was added into an rnase-free eppendorf tube and mixed with 50 μl of luciferase assay reagent ii (promega, madison, wi, usa). the value of the firefly luciferase activity, recorded as the f value, was measured by a berthold detection systems detector (berthold gmbh, pforzheim, germany). then, the eppendorf tube was removed, and 50 μl of stop & glo reagent (promega, madison, wi, usa) was added to terminate the firefly luciferase reaction. the value of the renilla luciferase activity, recorded as the r value, was also determined by the berthold detection systems detector. finally, the fold activity was assessed in triplicate by a two-tailed student’s t-test. transcription level analysis to test the differences in the numbers of gfp transcripts between recombinant plasmids with bdvpa(63) and the sv40 polya, real-time qpcr was used. total rna was extracted from the different groups of transfected cells that were used to measure gfp protein expression (described above) at 48 h. the cells were scraped, collected in 50-ml conical tubes, and pelleted by centrifugation for 90 min at approximately 3,000 rpm at 4 °c in a bench-top clinical centrifuge. the supernatant was removed, and the cell pellet was collected. trizol reagent (invitrogen) was used to extract the total rna, which was confirmed by 1 % agarose gel electrophoresis. the extracted rna was first quantified by spectrophotometry. total rna (500 ng) was treated with rnase-free dnase (vazyme biotech, nanjing, china) to degrade potential dna contaminants following the conditions recommended by the manufacture. then, the dna-free rnas were used as templates for cdna synthesis using two reverse primers(pfaffl, 2001) , using the cdna synthesis kit (vazyme biotech). the synthesized cdna was diluted 100-fold with rnase-free water for a qpcr analysis of gfp transcript levels. in the qpcr, the primer pairs used to generate a 146-bp amplicon of the gfp gene were gfp-474f and gfp-619r. an internal reference amplicon generated with the primer pair rrna-f and 28s rrna-r that targets the b. mori 28s rrna gene was used to normalize the reactions in hi5 cells(xue et al., 2010). another internal reference amplicon generated with the primer pair rpl3-f and rpl3-r that targets the b. mori rpl3 gene was used to normalize the reactions in bmn cells. gfp fusion plasmids without a polya signal served as a control group. sybr green super-mix kits (vazyme biotech) were used in the real-time qpcr. the amplification data were acquired by an abi prism 7300 system (applied biosystems, foster city, ca, usa). the effects of bdvpa(63) on gfp transcript levels were expressed relative to the 28s rrna (xue & cheng, 2010) or rpl3 rna levels. each of the amplifications was run in triplicate to calculate the experimental variance in a statistical analysis by a two-tailed student’s t-test. identification of bmbdv polya core functional areas to determine bmbdv polya core functional areas, a dual luciferase reporter gene assay was used. hi5 and bmn cells in 24-well plates were co-transfected with renilla luciferase-expressing plasmids and seven groups of firefly luciferase plasmids, including pgl3-p5-bdvpa(92), pgl3-p5-bdvpa(63), pgl3-p5-bdvpa(50), pgl3-p5-bdvpa(38), pgl3-p5-bdvpa(26), pgl3-p5-bdvpa(24) and the control plasmids without a polya signal (pgl3-p5). the co-transfections and dual luciferase reporter gene assay were performed as described above for the transfections with firefly luciferase fusion plasmids. the fold activity was evaluated in triplicate for statistical analysis. statistical analysis descriptive statistical analyses were performed using graphpad prism 5 software (graphpad software, inc.). the data are all from at least three independent biological replicates per experimental variable and presented as means ± standard deviations. the differences between control and experimental groups were assessed by a two-tailed student’s t-test and accepted as significant at p < 0.01or p < 0.001. results construction of transient expression plasmids the bmbdv genome contains a small number of genes whose distribution is very compact. especially, the genes encoding mcp and ppolb overlap partially. the mcp transcript starts at nt 1,423 and ends at nt 2,931, and the ppolb transcript starts at nt 6,287 and ends at nt 2,922 (wang et al., 2007). thus, the two transcripts have a 10 nts overlap region at their 3′ utrs. meanwhile, the stop codon of vd1-orf3 is located at nt 2,916, and the stop codon of vd1-orf4 is situated at nt 2,940, which are only 24 nts apart. thus, the 3′ utrs containing the overlapping regions with characteristic sequences (an aauaaa motif and gu-rich regions) were truncated and designated as bdvpa(63) (nt 2,896 2,958). to investigate the effect of the bmbdv polya on other genes, we constructed recombinant plasmids in which bdvpa(63) was inserted into expression vectors. bdvpa(63) was respectively fused to the 3′ terminal sequence of two reporters (genes encoding gfp and firefly luciferase) under the control of two different promoters, p5 or ie1 (fig. 1). the modified variants pt-ie1-gfp-sv40 and pt-p5-gfp-sv40 were used as starting model systems. then, the sv40 polya was replaced by bdvpa(63) or deleted directly in these two plasmids. thus, the new vectors were renamed pt-ie1-gfp-bdvpa(63), pt-ie1-gfp, pt-p5-gfp-bdvpa(63) and pt-p5-gfp (fig. 1a). 274 271 fig. 1 schematic diagrams of the constructs expressing two heterologous proteins. (a) recombinant plasmids that were used to express the gfp gene under the control of two different promoters, p5 or ie1. (b) firefly luciferase expression constructs that were used to test the activity of luciferase driven by the p5 or ie1 promoters. to complement gfp fluorescence measurements, a dual luciferase reporter gene assay was used to visualize the difference in heterologous expression between each pair of the recombinant plasmids. first, the sv40 polya was eliminated from the recombinant plasmids pgl3-ie1-sv40 and pgl3-p5-sv40, which were constructed previously in our laboratory, to form two novel plasmids named pgl3-ie1-sv40 and pgl3-p5-sv40 − , respectively. then, bdvpa(63) was fused to the 3′ terminal sequence of the firefly luciferase reporter gene. the expression plasmids pgl3-ie1-bdvpa(63) and pgl3-p5-bdvpa(63) were constructed successfully (fig. 1b). effect of the bmbdv polya on gfp expression to measure the level of foreign gene expression, various gfp fusion plasmids were respectively transfected into three insect cell lines (hi5, bmn and sf9). all the cells were observed at 48 h post-transfection by fluorescence microscopy. the gfp gene from recombinant plasmids with bdvpa(63) was expressed in the transfected cells. compared with the sv40 polya, more transfected cells produced green fluorescence, and greater fluorescence appeared in each cell (fig. 2). in addition, the fluorescence intensity of the cells transfected with plasmids controlled by the ie1 promoter was slightly greater than that of plasmids driven by the p5 promoter, which is consistent with the fact that the activity of ie1 is stronger than that of p5. moreover, almost equivalent fluorescence was displayed in both hi5 and bmn cells that were transfected with the p5 promoter-based gfp fusion plasmids containing the sv40 polya or bdvpa(63) (figs 2a, b, e, f), while no gfp fluorescence was 275 271 fig. 2 fluorescence microscopy images of cells displaying gfp expression at 48 h post-transfection. hi5 cells were transfected with pt-p5-gfp-sv40 (a), pt-p5gfp-bdvpa(63) (b), pt-ie1-gfp-sv40 (c), or pt-ie1-gfp-bdvpa(63) (d). bmn cells were transfected with pt-p5-gfp-sv40 (e), pt-p5-gfp-bdvpa(63) (f), pt-ie1-gfp-sv40 (g), or pt-ie1-gfp-bdvpa(63) (h). cells were photographed at 10× magnification. detected from the sf9 cells transfected with either the pt-p5-gfp-sv40 or pt-p5-gfp-bdvpa(63) plasmids, which is the same as the results obtained from transfections with the control plasmid (pt-p5-gfp) (data not shown). the plasmids containing the ie1 promoter with two polya signals both showed fluorescence in hi5, bmn (figs 2c, d, g, h) and sf9 cells, but the fluorescence intensity in the hi5 and bmn cells was much stronger than that in the sf9 cells (data not shown). one of the possibilities is that the activity of bdvpa(63) may be relatively low in the sf9 cells when the expression of gfp is driven by the p5 promoter. this finding laid the foundation for the selection of a cell line in the subsequent dual luciferase reporter gene assay. in brief, all these data suggested that bdvpa(63) in promoter-based vectors was more conducive to the expression of the gfp reporter gene, compared with the sv40 polya, in insect cells. effect of the bmbdv polya on firefly luciferase expression the differences of firefly luciferase expression from the recombinant plasmids with bdvpa(63) or the sv40 polya were detected by a dual luciferase reporter gene assay. luciferase activity was determined 48 h after co-transfection with renilla luciferase-expressing plasmids. the results revealed that the relative expression of firefly luciferase was higher when bdvpa(63) was used (fig. 3). however, the increased levels of protein expression differed depending on the inserted polya signal. compared with the sv40 polya, the greatest increase was detected from the plasmid pgl3-ie1-bdvpa(63) in hi5 cells (approximately 21.7-fold, fig. 3b), followed by pgl3-ie1-bdvpa(63) in bmn cells (up to 12.6-fold, fig. 3d), pgl3-p5-bdvpa(63) in hi5 cells (approximately 6.5-fold, fig. 3a) and pgl3-p5-bdvpa(63) in bmn cells (approximately 2.7-fold, fig. 3c). hence, bdvpa(63) was more beneficial for the expression of the firefly luciferase gene than the sv40 polya, which is consistent with the fluorescence microscopy results. all these data suggest that bdvpa(63) has the potential to promote the expression of different types of proteins in different insect cell lines. analysis of promoter-based gfp transcription because protein expression was remarkably enhanced in the presence of bdvpa(63), it was imperative to determine its role at the transcriptional level. gfp fusion plasmids were respectively transfected into hi5 and bmn cells, which were subsequently cultured for 48 h. total rna was extracted from the transfected cells. the differences between the number of gfp transcripts were confirmed by real-time qpcr using the b. mori cellular 28s rrna gene and the rpl3 gene as an internal reference to normalize the reactions (fig. 4). gfp fusion plasmids without a polya signal served as a control group. the real-time qpcr results showed that the relative gfp mrna content was higher in both types of insect cells when bdvpa(63) was used (fig. 4). however, the increased levels of gfp transcripts differed depending on the types of promoters and cells. the greatest increase was detected in hi5 cells when gfp expression was driven by the ie1 promoter (4.9-fold, fig. 4b), followed by the ie1 promoter in bmn cells (3.6-fold, fig. 4d), the plasmid pt-p5-gfp-bdvpa(63) in hi5 cells (2.8-fold, fig. 4a) and the plasmid pt-p5-gfp-bdvpa(63) in bmn cells (1.7-fold, fig. 4c). thus, the increased levels of gfp transcripts due to the insertion of bdvpa(63) was significantly higher than that of the sv40 polya. these results also indicated that the integration of bdvpa(63) downstream of the gfp gene enhanced the level of gene transcription in insect cells. 276 271 fig. 3 quantitative analysis of firefly luciferase expression to test the effects of the bdvpa(63), compared to the sv40 polya, on protein expression. hi5 (a, b) and bmn (c, d) cells were co-transfected separately with renilla luciferase-expressing plasmids and the firefly luciferase constructs containing bdvpa(63) or the sv40 polya, or the construct without a polya signal. all the cells were harvested at 48 h post co-transfection and subjected to dual luciferase reporter gene assay in three independent cell transfections. the values are expressed as the ratios of firefly luciferase expression relative to those of the control plasmids pgl3-p5 and pgl3-ie1. significance is indicated as ** p < 0.01 and ***p < 0.001. error bars denote standard deviations. identification of bmbdv polya core functional areas to determine bmbdv polya core functional areas that might play significant roles in gene expression, we performed a dual luciferase reporter gene assay. the characteristic sequences of the bmbdv polya were truncated, and a series of firefly luciferase plasmids with bmbdv polyas of different lengths were constructed, therefore, these novel plasmids were named as follows: pgl3-p5-bdvpa(92), pgl3-p5-bdvpa(50), pgl3-p5-bdvpa(38), pgl3-p5-bdvpa(26) and pgl3-p5-bdvpa(24) (fig. 5a). these five plasmids and the plasmid pgl3-p5-bdvpa(63) were co-transfected separately into hi5 and bmn cells with renilla luciferase-expressing plasmids. then, these cells were cultured for 48 h to confirm that the firefly luciferase gene was expressed. the discrepancies among the fold activity of the different forms of the bmbdv polya were measured. the relative firefly luciferase activities were high in hi5 (fig. 5b) and bmn (fig. 5c) cells that were transfected with plasmids containing bdvpa(92), bdvpa(63), bdvpa(50) and bdvpa(38), which all contained an aauaaa motif and gu-rich regions. interestingly, the highest relative firefly luciferase activity was observed in cells transfected with pgl3-p5-bdvpa(50) (figs 5b, c). nevertheless, the relative firefly luciferase activities of cells transfected 277 271 fig. 4 transcription analysis of gfp from different transfections. the constructs with bdvpa(63), the sv40 polya, or those without a polya signal were used to transfect hi5 (a, b) and bmn (c, d) cells, and the transfected cells were harvested at 48 h for total rna extraction. equal amounts of rna from different cell transfections were used as templates for amplification of a 146-bp amplicon of the gfp gene using the sybr green dye rt-pcr kit from vazyme biotech (nanjing, china) for the real-time quantification of the gfp transcripts. the b. mori cellular 28s rrna gene and rpl3 gene were used as housekeeping genes in the real-time qpcr to normalize the reaction. significant differences are indicated as **p < 0.01 and ***p < 0.001. error bars represent standard deviations from three independent cell transfections. with the plasmids pgl3-p5-bdvpa(24) and pgl3-p5-bdvpa(26), which contained incomplete bdvpa, in which the aauaaa motif and gu-rich regions that function independently, were reduced in both hi5 and bmn cells (figs 5b, c). hence, bdvpa(50) comprising the aauaaa motif, gu-rich regions, and 12 nts downstream was identified as the core functional region. it is noteworthy that a stop codon is contained within the aauaaa motif of bdvpa(50). thus, this core functional region acts as a combination stop codon and aauaaa motif (polya signal/stop codon) (fig. 5a). in summation, we hypothesize that the length of the bmbdv polya may affect its activity. meanwhile, both the aauaaa motif and the gu-rich regions are crucial for the formation of polyadenylation, the subsequent processing of downstream sequences, as well as the efficiency of protein synthesis. discussion since increasing the expression of heterologous proteins in insect cells has research and pharmaceutical applications (tani et al., 2008; kato et al., 2010), the sv40 polya has been widely used in many commercial baculovirus expression vectors. in this study, we generated a novel, short, dual-functional polya signal/stop codon from an insect virus. our most intriguing discovery is that the bmbdv polya promotes gene expression at both the 278 271 fig. 5 the identification of bmbdv polya core functional areas using dual luciferase reporter gene assay. (a) schematic diagram of the constructs containing a series of bdvpa with different lengths. (b) detection of firefly luciferase activity from transfected hi5 cells. hi5 cells were con-transfected with renilla luciferase-expressing plasmids and the firefly luciferase constructs with a series of polyadenylation signal fragments of different lengths. all the cells were harvested at 48 h post co-transfection and subjected to a dual luciferase reporter gene assay in three independent cell transfections. (c) measurement of the firefly luciferase activity in transfected bmn cells. bmn cells were co-transfected as described above. the values are expressed as the ratio of firefly luciferase activity relative to that of the control plasmid pgl3-p5.the aauaaa motif is denoted by purple and green boxes, and the gu-rich region is indicated by a yellow triangle. //indicates sequences omitted. error bars signify standard deviations from three independent cell transfections. mrna and protein levels in diverse cell lines, which distinguishes it from the sv40 polya, which increased mrna levels, but decreased protein production in the bevs (salem et al., 2015). these results also differ from an early report showing that the sv40 polya driven by the strong promoter p10 from autographa californica multiple nucleopolyhedrovirus reduces mrna levels and protein synthesis (van oers et al., 1999). therefore, the new, dual-functional bmbdv polya is a valuable for increasing mrna production and protein synthesis in insect cells. similar to the sv40 polya, the bmbdv polya is situated at the regions of genes in two complementary strands, and it contains at least two elements: an aauaaa sequence for cpsf binding and a downstream gu-rich region for cstf binding. one interesting observation in this study is that intact 279 272 bmbdv polyas of different lengths (bdvpa(92), bdvpa(63), bdvpa(50) and bdvpa(38)) resulted in different protein synthesis efficiencies. especially, the fold activity of bdvpa(50), comprising the aauaaa motif, gu-rich regions, and 12 downstream nt, was the highest among all the polya signals. a comparison of the four intact bmbdv polyas showed that bdvpa(63) has two partial overlapping aauaaa motifs. the second aauaaa motif is followed by a dinucleotide with a guanosine (g), whereas no g nucleotide is present in the dinucleotide right after the first aauaaa motif. the other three bmbdv polya signals (bdvpa(92), bdvpa(50) and bdvpa(38)) only have the second aauaaa motif. these data also indicate that additional sequences other than the canonical aauaaa motif are needed for the efficient processing of the 3′ end of promoter-controlled transcripts during insect cell transfections. additionally, the evidence obtained from this study confirms that the lack of a g in the dinucleotide after the aauaaa motif of 3′ utrs might be responsible for the weak 3′ end processing (salem et al., 2015). because of diverse truncated fragments right after gu-rich region, bdvpa(50), bdvpa(92) and bdvpa(38) resulted in different firefly luciferase activities. nevertheless, the exact reason for this remains unclear. one plausible explanation could be that sequences downstream of the gu-rich region have different secondary structures, thereby influencing the binding of cellular rna processing factors to the polya signal of transcripts and interfering with translation initiation, which is required for efficient protein synthesis. when the bmbdv polya was inserted downstream of gfpand firefly luciferase-encoding genes under the control of two different promoters, increased transcript levels and protein synthesis were both detected, compared with the sv40 polya. hence, the incorporation of the bmbdv polya into expression plasmids is very useful. it is unknown whether it has the same effect in insect transgenic vectors and bevss. as sf9 cells are a high transfection efficiency line, these cells are commonly used for the production of recombinant proteins. in the measurement of gfp expression, the fluorescence intensity in the hi5 and bmn cells was much stronger than that in the sf9 cells, the reason of which may be related to the specificity of bmbdv polya for cell lines. it is also unknown whether the increased mrna and protein levels can be achieved in mammalian systems. therefore, it is interesting to consider the detailed aspects that could affect the use of this new polya signal in different cell lines in future research. additionally, this short polya signal may overcome the obstacle of improving the carrying capacity of plasmid or viral vectors, thereby making it an alternative to the use of small promoters and intramolecular recombination, which requires two separate virions to infect the same cell (flotte, 2000; shevtsova et al., 2005). in summation, the bmbdv polya has the potential to be used for the effective and stable expression of heterologous rnas and proteins in insect cells. furthermore, our study provides a reference for constructing plasmids with a small insertion, as well as for researching the function of other polya signals. acknowledgements this work was supported by the national natural science foundation of china (grant nos. 31570150 and 31272507) and the jiangsu provincial natural science foundation (youth project, grant no. bk20160507). references adams mj, carstens eb. ratification vote on taxonomic proposals to the international committee on taxonomy of viruses (2012). arch virol. 157: 1411-1422, 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overlap. it is thought that dioecy was ancestral because it occurs in most classes of molluscs. hermaphroditism evolved independently several times, and sequential and simultaneous hermaphroditism are more closely related to each other than to dioecy. this publication presents a general review of sexual systems and strategies in terrestrial gastropods with special emphasis on mating, fertilization, presence of love darts, reproductive strategies (semelparity vs. iteroparity) and modes (oviparity, ovoviviparity, viviparity), production of eggs and egg cannibalism. key words: copulation; mating; oviposition; reproductive systems; sequential hermaphrodite; simultaneous hermaphrodite; terrestrial gastropods introduction most animal species belong to invertebrates. molluscs constitute the second most numerous type and the majority of them are gastropods (o’connor and crowe, 2005). it is estimated that the number of molluscs species detected so far varies from 80,000 to 135,000 worldwide (abbott, 1989). molluscs are a diverse group of animals due to their morphological forms, occurrence in different environments water (marine and freshwater) and land, ways of feeding, but also because of various types of reproduction (policansky, 1982). the main functions of their reproductive systems are: 1) production of male and female gametes: sperm and ova, 2) nutrition and storage of mature gametes, 3) transport of sperm produced by one specimen (autosperm) to reproductive ducts of another specimen, 4) reception of sperm produced by the same individual (allosperm), 5) providing appropriate environmental conditions for the ovum fertilized by sperm, 6) covering of zygote by protective and nutrient layers, 7) laying eggs (oviposition) and 8) resorption of remains and excess of products of the reproductive process (gómez, 2001). the aforementioned animals can be dioecious (gonochorism or dioecism) and can have separate ___________________________________________________________________________ corresponding author: kamila zając institute of environmental sciences jagiellonian university gronostajowa 7 30-387 kraków, poland e-mail: kamila.zajac12@gmail.com sexes (female and male gonads occur in different, separate individuals) or hermaphroditic (an individual contains both male and female reproductive organs). hermaphrodites can be simultaneous, which means that an organism has both male and female organs at the same time, or sequential during their life their sexes change (wilson and harder, 2003). among these, there is protandry (male-to-female change) and protogyny (female-to-male change), but the first one is more frequent in molluscs (larsen et al., 2013). sequential and simultaneous hermaphroditism are more closely related to each other than to dioecy (collin, 2013). phylogenetic evidence indicates that dioecy is ancestral in the mollusca phylum (kocot et al., 2011; smith et al., 2011; collin, 2013). monoplacophora, caudofoveata, polyplacophora, scaphopoda and cephalopoda are exclusively dioecious, while solenogastres are simultaneous hermaphrodites. among molluscs, gastropods and bivalves are the most diverse in terms of their reproductive systems they are dioecious, simultaneous, or sequential hermaphrodites. there are some exceptions, for example the cases of a sequential hermaphrodite in cephalopods, one simultaneous hermaphrodite in monoplacophora micropilina arntzi, and one genus (lepidochitona) with a simultaneous hermaphrodite within polyplacophora (eernisse, 1988; haszprunar and schaefer, 1996; lamprell and scheltema, 2001). a very interesting case of reproductive strategy is sex reversal, which means that an animal can change its sex during its life more than once (park et al., mailto:kamila.zajac12@gmail.com 200 2012). a specific case of reproduction has been observed in a few patellogastropods, which are protandric with some overlap. it differs from common protandry due to the fact that there is a short period of time during the development when both male and female gonads occur in an animal. after that, it is possible to distinguish only one type of gonad (collin, 2013). most studies devoted to reproductive systems and strategies concern marine and freshwater molluscs (guo et al., 1998; siddiqui and ahmad, 2002; chen et al., 2004; calvo and templado, 2005; collin et al., 2005; brante et al., 2011) and only some of them focus on terrestrial snails. a relatively small number of terrestrial gastropods are dioecious and most of them are sequential or simultaneous hermaphrodites (heller, 1993). dioecy occurs in some families of land neritomorpha (helicinidae, hydrocenidae) and in all land caenogastropoda. usually the sex ratio is 1:1, but there are some exceptions to this principle. the main advantages of the presence of this type of reproduction are: 1) increased fitness of the offspring and 2) decreased inbred depression in conjunction with preventing self-fertilization (leonard, 2010). the problems connected with reproduction can result from the impossibility to find a partner to mate. dioecious animals incur the same costs associated with reproduction (energy or mortality), both parents take care of the offspring (biparental care) if this behavior is present in species, and there are no differences in mortality and growth between the sexes (warner, 1988). simultaneous hermaphrodites can produce male and female gametes in the same gonad (ovotestis) or separately in the ovary and the testis (hodgson, 2009). this type of reproduction is common in organisms living in groups. detecting simultaneous hermaphrodites is relatively simple because the anatomical section is usually sufficient (policansky, 1982; collin, 2013). discovering sequential hermaphrodites is more complicated because sex change depends on many environmental factors and it is difficult to capture the moment of this phenomenon. depending on whether we deal with protandry or protogyny, there are dissimilarities in mortality and growth. in protandry, females have a higher mortality rate, males grow faster and female functions are more costly than male functions, while in the case of protogyny, the situation is reverse (warner, 1988). the main advantages of hermaphroditism are: 1) possibility of reproduction by self-fertilization because some species have difficulties in finding a partner to copulate, 2) higher probability of meeting a potential partner as every encountered individual can be a potential partner, 3) increased productivity due to the discharge of their functions and division of resources, 4) egg production takes place in stages, not at one time (crowley et al., 1998) and 5) increased variability in fitness (wilson and harder, 2003). the main disadvantage is that individuals have to put a lot of energy into growth and subsistence of both types of reproductive organs (hodgson, 2009; leonard, 2010). in literature one can find several hypotheses which explain the presence of hermaphroditism among animals. the most cited ones in papers are “low density model” and “size advantage model” which are connected with sequential or simultaneous hermaphroditism (munday et al., 2006). the low density model proposed by tomlinson (1966) assumes that individuals from populations with low densities are more likely to meet the suitable partner than in the case of dioecious organisms (borgia and blick, 1981). this model focuses on simultaneous hermaphrodites. the “size advantage model” is connected with sequential hermaphrodites and it was proposed by ghiselin in 1969 (ghiselin, 1969). this model is linked with the sex allocation theory and it was designed to explain the occurrence of protandry and its evolution. it states that if a small male and large female have a much higher reproductive efficiency, the male to female change will be favored along with an increase in the size of the animal. in protandry, small individuals (males) have higher reproductive fitness than larger males. it is the same in the case of females large females have higher reproductive fitness than smaller ones. this means that reproductive functions are better realized when the animal is of the appropriate size. moreover, males can increase their reproductive success by copulating with many females (warner, 1988; wright, 1988; erisman et al., 2009). sex change is present in plants and animals, not only in molluscs, but also in echinoderms, annelids, crustaceans, and fishes (policansky, 1982). there are many good reviews of sexual systems and strategies in molluscs (for example baur, 1994b; leonard, 1999, 2006, 2013; heller, 2001; davison and mordan, 2007; jordaens et al., 2007; yusa, 2007; collin, 2013; nakadera and koene, 2013), but in this paper we put emphasis on these issues in terrestrial gastropods and summarize the important data on this group of animals in the context of mating, fertilization, presence of love darts, reproductive strategies (semelparity vs. iteroparity) and modes (oviparity, ovoviviparity, viviparity), production of eggs and egg cannibalism in terrestrial gastropods. description of genitalia terrestrial gastropods can be dioecious and have separate sexes (e.g., cyclophoridae) or hermaphroditic as stylommatophorans. the reproductive system of dioecious gastropods consists of gonad and duct called coelomic gonoduct, both structures have a mesodermal origin. gonoduct is divided into two parts, bigger is called renal oviduct or renal gonoduct, and smaller, distal portion pallial gonoduct (fretter and graham, 1962). renal gonoduct is narrow, tubular structure in both sexes, whereas pallial gonoduct is different in males and females. in case of males, pallial gonoduct is wide with thin walls forming prostatic gland, which secrete semen. in females, walls of pallial gonoduct are divided into several parts which are able to secrete nutritious and protective substances, these are called albumen and capsule glands located side by side. on the pallial gonoduct there are situated two types of sperm pounches: bursa copulatrix and receptaculum seminis. bursa copulatrix obtains allospermatozoa and prostatic 201 liquid from the proccess of mating, while receptaculum seminis is used for storing these substances for future oocyte fertilization (gómez, 2001). another structure of reproductive system is muscular penis, usually located on the right side of the head, it was created from the anterior body wall (gómez, 2001). reproductive system of stylommatophoran hermaphroditic gastropods contains gonad with gonoduct, the carrefour, albumen gland, spermoviduct, free oviduct, vagina, vas deferens, epiphallus, spermatophore, penis, diverticulum, bursa copulatrix, penial flagellum, genital atrium and other auxiliary copulatory organs (gómez, 2001). in stylommatophorans occur single gonad called ovotestis, which produces oocytes and spermatozoa. ovotestis is located among patches of the digestive glands, it is composed of few or many rounded or pear-shaped sac acini and contain both male and female germ cells. ovotestis opens to gonoduct, also called hermaphodite duct, along which germ cells are transported. another structure of reproductive system is the carrefour, which consists of spermatheca or female sperm-storage organ for allosperm storage, protein coating of zygotes or oocyte fertilization (gómez, 2001). there is a huge variability in structures and morphology of the carrefour. for example, it may be divided into spermatheca and fertilization chamber, as in the case of trigonephrus gypsinus (dorcasiidae), or single spermathecal tubule beside the fertilization chamber, what is observed in oxychilus draparnaudi (zonitidae) or bradybaena fruticum (bradybaenidae) (flasar, 1967; brinders and sirgel, 1992; bojat et al., 2001). in some species there is variation in number of spermathecal tubules: 2 9 in a. arbustorum or 4 19 in c. aspersum (haase and baur, 1995; baminger and haase, 1999; koemtzopoulos and staikou, 2007; chase and darbyson, 2008). in the early phase of the sexual activity of the gastropod albumen glad has small size. it develops fully when the gonad is starting to leave the eggs. gastropods differ in the shape of the gland, for example rounded and shield-shape in agriolimacidae, while limacidae has this structure worn and tongue-shape. the size of the gland changes is negatively correlated with size of the gonad, e. g. when the gonad is large, the albumen gland is small and vice versa (wiktor, 1989). albumen gland produces albumen or perivitelline substances, whereas secretory cells secrete galactogen which is a polymere of galactose (duncan, 1975). the vas deferens is narrow, partly folded duct whose main function is transport of autosperm whereas highly muscular epiphallus participates in spermatophore formation. spermatophore is built of epiphallus and flagellum during copulation and has species-specific shape and taxonomic significance (mann, 1984; baur, 1998). it is composed of substances containing mucoproteins and glycosaminoglycans (mann, 1984). spermatophore is not present in all stylommatophorans, it is produced singly during mating and exchanged between partners inversely (mann, 1984). penis is a copulatory organ with huge diversity within stylommatophorans, but at the same time, it is not present in all species (reise, 2007). its shape is typical for a particular species. penis is used also for identifying a partner during mating and it affects success of copulation (gómez, 2001). external sperm exchange occurs in some stylommatophoran species from succineidae, polygyridae, helicodiscidae, limacidae and agriolimacidae families (emberton, 1994; reise, 2007). diverticulum is a bursa tract from which the spermatophore is taken during mating (barker, 2001). the length of this structure may be variable, van osselaer and tursch (2000) studied a population of 79 individuals of h. pomatia. they discovered, that 34 % of them were deprived of diverticulum, whereas at the rest of them, its length varied from 1 mm to 9 mm (van osselaer and tursch, 2000). in these species, where diverticulum is absent, the spermatophore is deposited in bursa copulatrix (barker, 2001), which is also called gametolytic gland (tompa, 1984). the main function of this organ is extracellular digestion and further resorption of excess gametes, secretions and residues of the spermatophore (beese et al., 2006). penial flagellum is responsible for production of spermatophore tail (gómez, 2001). genital atrium is the final section of the reproductive system, where male and female organs are reunited (wiktor, 1989). mating and fertilization it is estimated that the number of gastropod species inhabiting the earth ranges from 60,000 to 105,000 (bouchet et al., 2005), whereas there are about 35,000 species of terrestrial gastropods (van bruggen, 1995). terrestrial snails and slugs have worldwide distribution and can be found in diverse land environments, such as different types of forests, gardens, rock surfaces, steppes and dry habitats, desserts, humid biotopes, they are present in hydrocenoidea, helicinoidea, cyclophoroidea, rissoidea, littorinoidea superfamilies, and in many families within heterobranchia clade (bouchet et al., 2005). most terrestrial gastropods belong to stylommatophora superorder in heterobranchia, and the number of species is about 20,000 (solem, 1978) grouped in 71 to 92 families (emberton et al., 1990). stylommatophorans are air-breathing terrestrial gastropods characterized by two pairs of invaginable head tentacles (dayrat and tillier, 2002), most of which are simultaneous hermaphrodites. within gastropods, one can distinguish internal or external fertilization, but the first type is more frequent among terrestrial gastropods (nakadera and koene, 2013). external fertilization occurs in some primitive species of archaeogastropods (jarne and auld, 2006). fertilization is affected by many factors, such as the quality and size of sperm (werner and simmons, 2008; birkhead et al., 2009; pizzari and parker, 2009), which is selected by sperm competition and fertilization success (pitnick et al., 2009). schmera et al. (2016) compared the data on sperm length in 57 stylommatophoran species from 23 families (succineidae 1 species, chondrinidae 3, lauriidae 1, orculidae 1, pyramidulidae 1, vertiginidae 2, enidae 1, 202 clausiliidae 8, bothriembryontidae 2, odontostomidae 2, strophocheilidae 1, discidae 1, oxychilidae 3, zonitidae 1, limacidae 3, agriolimacidae 1, vitrinidae 2, arionidae 3, helicidae 10, bradybaenidae 1, cochlicellidae 1, helicodontidae 1, hygromiidae 7) from europe and south america, taking into account the breeding systems in order to test the hypothesis that sperm competition can favor the evolution of longer sperm (parker, 1993; parker and begon, 1993). their results indicate that in stylommatophorans, simultaneous hermaphrodites’ sperm length can be influenced by the breeding system, the age of sexual maturity, and shell size. moreover, they showed that sperm length increases with shell size (schmera et al., 2016). courtship and copulation can be done unilaterally, which means that one partner plays a specified role (male or female), while the other individual plays a reverse role during copulation, and usually, after one round of copulation the roles are changed, or reciprocally (both individuals play the male or female role at one time during copulation) (tompa, 1984; heller, 2001). moreover, mating can be done in different positions, such as shell-mounting or face-to-face. in their review, davison and mordan (2007) pay attention to the fact that the mating position within terrestrial stylommatophorans is constant across the evolution of most lineages. they focused on the mating behavior within land stylommatophorans (snails and slugs) and classified the genera into four categories: 1) face-to-face, simultaneous reciprocal, 2) face-toface, unilateral, 3) shell-mounting, simultaneous reciprocal and 4) shell-mounting, unilateral. they summed up the data on the mating behavior and presence of love darts in 93 genera from 35 families of terrestrial gastropods. the comparison of the data suggests that the face-to-face, simultaneous reciprocal behavior is the most common mating behavior within stylommatophorans (davison and mordan, 2007). terrestrial gastropods are able to reproduce by cross-fertilization, which means that gametes from different individuals are necessary for insemination, or by self-fertilization insemination can be done by a fusion of gametes produced by one specimen, but the first type is more frequent among terrestrial snails and slugs, whereas self-fertilization is more widespread in freshwater species and bivalves (duncan, 1975; peake, 1978; heller, 1993, 2001; jarne et al., 1993). usually, cross-fertilization is the preferred type of insemination, but self-fertilization gives them the opportunity to reproduce when mating is not possible, allowing them to occupy and colonize new areas due to the ability of reproduction even at low population density, and it also reduces the costs of male allocation (heller, 2001). selffertilization has also some disadvantages, such as low genetic diversity caused by limited recombination and possible inbreeding depression (chen, 1993, 1994; heller, 2001). self-fertilization evolved several times in few independent phylogenetically lines, it occurs in different species of terrestrial gastropods. heller (1993) summarized data about presence of this type of fertilization in 19 genera from 12 families, in veronicellidae (filicaulis, vaginulus), vertiginidae (vertigo, truncatellina), vallonidae (vallonia), partulidae (partula), achatinidae (achatina, archachatina), subulinidae (rumina), chondrinidae (chondrina), arionidae (arion), philomycidae (philomycus), succineidae (catinella, omalonyx, oxyloma, succinea), limacidae (deroceras, agriolimax) and polygyridae (triodopsis) (heller, 1993). in some cross-fertilizer species (e.g. arianta arbustorum, bradybaena fruticum) also selffertilization is possible, but fitness reduction is observed as a consequence (chen 1993, 1994; kuźnik-kowalska et al., 2013). one of the most interesting families in the context of fertilization is arionidae, belonging to stylommatophora. a typical genus for this family is arion which has the most species. in europe, 35 arion species (welter-schultes, 2012) occur and in these species both cross-fertilization and selffertilization are present, depending on the species. a. lusitanicus; a. hortensis; a. distinctus and a. owenii reproduce by cross-fertilization, whereas a. circumscriptus; a. silvaticus and a. intermedius reproduce frequently by self-fertilization. a. ater and a. subfuscus are able to crossand self-fertilize (foltz et al., 1982). love darts some terrestrial gastropods are able to produce love darts called gybsobelum or shooting darts (koene and schulenburg, 2005), which are hard, pointed structures composed of calcium carbonate, chitin or cartilage (hasse et al., 2002). the size of the darts varies from 1 to 30 mm, but usually is less than 5 mm and always correlated with the size of the animal. apart from radula and jaws, love darts and their shape can be used for the identification and classification of gastropods (chung, 1986). some species have one or more love darts which is an effect of repeatable evolutionary events (koene and schulenburg, 2005). presence of these structures is connected with face-to-face mating behavior and low-spired shape of the shell (davison et al., 2005; jordaens et al., 2009). davison and mordan (2007) summarize data about presence of love darts in particular families in helicoidea and limacoidea superfamilies, they occur in bradybaenidae, helicidae, helminthoglyptidae, hygromiidae, ariophantidae, urocyclidae, vitrinidae, zonitidae, philomycidae and dyakiidae families (davison and mordan, 2007; koene et al., 2013). among terrestrial slugs and snails can be observed diversity in dart structures. some species produce single dart which stays in body of dart receiver, then dart is rebuilt by dart shooter, as in the case of c. aspersum or h. pomatia (chung, 1987; chase, 2007). in other species, e.g., polymita muscarum, p. picta, euhadra subnimbosa, dart is retracted and reused, it does not remain in the partner’s body (koene and chiba, 2006; reyes-tur and koene, 2007; koene et al., 2013). love darts contain gland products which cause changes in female reproductive system after entering to the haemolymph (kimura et al., 2014). sticking darts into the body of the gastropod causes contraction, retraction of the shell, and inhibition of sexual behavior, or escape. it appears that the firing of 203 darts stimulates the dart shooter and it is a discharge of aggressive behavior (chung, 1987). dart donor is disadvantaged because of exposed on possible infections resulting from skin damages and process of sperm storage is modified (rogers and chase, 2002). according to the functions of love darts, there were many hypotheses which tried to explain their significance. adamo and chase (1988) postulated that the main function of love darts were encouragement the partner to mate, whereas diver (1940) claimed that these structures were involved in recognizing of individuals which belong to the same species. another explanation of their presence was the gift of calcium for the partner. all these hypotheses have been rejected. the function of love darts is not fully understood, but it is believed that they play a stimulating role during mating and they increase fertilization success of the dart receiver by enhancing the possibility of fertilizing eggs. lodi and koene (2016) in their review combined physiological, morphological and behavioural data about love darts in 23 species belonging to the helicoidea superfamily (helicidae, bradybaenidae, helminthoglyptidae). they reported that the common characteristic of dart shooting is increasing of male reproductive success by moving the mucus to dart receiver (lodi and koene, 2016). reproductive strategies in gastropods: semelparity and iteroparity in the case of each reproductive strategy, the most important aspect is the trade-off between fecundity, growth, and survivorship of individuals (perron, 1983). among molluscs, not only in terrestrial gastropods, there are two different reproductive strategies, namely semelparity and iteroparity, which differ from each other, but these are not alternative strategies (heller, 2001). an essential factor is the survival of adults in relation to the survival of juveniles (heller, 2001). semelparity means that an animal accedes to the reproduction only once during its life and after that it dies and the death is considered to be part of this strategy. an animal puts all its resources to maximize its reproductive success at the expense of its lifespan. in semelparous species, the cost of offspring production increases, while the cost of offspring decline decreases. in case of iteroparity, an animal reproduces many times during its life, which means in practice that it is able to reproduce every year (rantes et al. 2002). in iteroparous species, the cost of offspring production decreases and each additional offspring is less expensive. an animal allocates some resources to reproduction and spends the rest of them on growth and survivorship, which allows it to reproduce many times during its lifetime (roff, 1992). for molluscs iteroparous species are the most common; this strategy is identified with k-selection, while semelparity is linked to r-selection (macarthur and wilson, 1967). the r-selected species are characterized by production of numerous small offspring, high mortality among juveniles, and a short lifespan of an animal. juveniles mature quickly and are able to reproduce, but only a small percentage of them survive. k-selection is based on a small number of offspring as well as the comprehensive care of them. most juveniles survive until the reproductive age, and produce the next generation of animals (macarthur and wilson, 1967; begon et al., 2006). semelparity is usually connected with the annual lifespan and occurs in many families within heterobranchia clade, in succineidae (succinea putris; oxyloma retusum), euconulidae (habroconus semenlini), milacidae (milax gagates), agriolimacidae (deroceras reticulatum), vitrinidae (semilimax semilimax, s. kotulai, vitrina pellucida), arionidae (arion ater, a. subfuscus, a. fuscus, a. lusitanicus, a. intermedius), limacidae (bielzia coerulans), helicidae (theba pisana), hygromiidae (xeropicta vestalis, monacha cartusiana, m. haifaensis, cernuella virgata, helicella itala, trochulus hispidus), and many others (runham and laryea, 1968; heller, 1982, 2001; barker, 1988, 1991; south, 1989; lazaridou-dimitriadou and sgardelis, 1995; silva et al., 2009; örstan, 2010; welter-schultes, 2012; kuźnik-kowalska et al., 2013; proćków et al., 2013). an example of semelparous terrestrial gastropods is arion vulgaris accepted as one of the 100 most invasive species in europe (rabitsch, 2006). it is a serious threat to agricultural and horticultural crops, causing their damage (gren et al., 2009). this species lives about one year, but some slugs can live longer, and most of them die after laying eggs (davies, 1987; kozłowski, 2008). each individual in its lifetime lays between 240 and 540 eggs, from 12 to 124 white, round eggs per clutch (kozłowski, 2000; kozłowski and kozłowski, 2000), and after a month juveniles hatch. hatching can be spread over time and depends on the temperature; it takes place from september until the air temperature falls to 5 oc (kozłowski, 2000). many slug species are considered to be invasive species (kozłowski et al., 2010). among them, many have a life cycle and are semelparous (heller, 2001). kozłowski et al. (2010) showed the alien invasive slug species which are threat to crop plants. the biggest pests are a. distinctus, a. vulgaris, a. rufus, deroceras panormitanum, limax maximus and tandonia budapestensis (kozłowski et al., 2010). these species have economic importance causing crop damages, mainly fruits and vegetables. for example, a. vulgaris reduce strawberry yields in sweden by half (gren et al., 2009). these pest species lay a large number of eggs, for example a. distinctus lays 200 eggs, and a. rufus 415 eggs, from 8 to 229 per clutch. a. fasciatus poses a relatively small threat to crops, perhaps because of its life expectancy (15 25 months) and the number of produced eggs (104 123 eggs, from 10 to 30 per clutch) (weltherschultes, 2012). reproductive modes in gastropods: oviparity, ovoviviparity and viviparity among terrestrial gastropods, three different reproductive modes occur, that is oviparity, ovoviviparity and viviparity. the first one is the most common mode, which means that an animal is capable of laying eggs (heller, 2001). lodé (2012) proposed to replace the term ‘oviparity’ with ‘ovuliparity’ in the case of molluscs and arachnids 204 (lodé, 2012). the term ‘ovoviviparity’ can be defined as a special case or modification of viviparity. this way of reproduction is based on laying eggs by the mother, but the embryonic development proceeds inside the mother’s body in the egg shell at the expense of egg yolks. juvenile offspring destroys egg shells before hatching or just after egg laying (meier et al., 1999; markow et al., 2009). within this concept, one can distinguish ovoviviparity sensu lato, which means that incubation of eggs takes place in the reproductive tract at any time of the embryonic development of an animal. moreover, a part of the embryonic development takes place after laying eggs in the environment. ovoviviparity sensu lato is identified with egg retention. ovoviviparity sensu stricto lies in the fact that the embryonic development takes place in the parent’s body and juveniles are born or hatch immediately after laying eggs. this mode of reproduction has many advantages, and the main ones are: 1) decrease in the offspring mortality due to drought or predation, 2) increase in the offspring chances in food competition with juveniles hatched from eggs, 3) this mode of reproduction gives a chance to reproduce in unstable conditions, where it is difficult to predict the beginning of a rainy season during the year (tompa, 1979a; sulikowska-drozd, 2009). in some papers one can find the term ‘brooding’, which means that the embryonic development occurs wholly or partially inside the parent’s body, not necessary inside the genital tract (heller, 1993). among ovoviparous terrestrial gastropods, one can mention ferussacia folliculum (ferussaciidae), pupilla muscorum, p. sterrii (pupillidae), leptinaria unilamellata (subulinidae), pyramidula pusilla, p. umbilicata (pyramidulidae), balea biplicata, b. perversa (clausiilidae) and all species of lauria (lauriidae) (pokryszko, 2001; carvalho et al., 2009; welter-schultes, 2012), and many others. sulikowska-drozd and maltz (2012) studied the reproduction of ovoviviparous clausiliid balea fallax under laboratory conditions. layed eggs of this clausiliid hatch earlier than in other clausiliid species, and adult specimens preserve eggs for short periods. the size of the eggs of this species suggests that parents invest more in offspring, and some part of parental care is used to increase offspring fitness (sulikowska-drozd and maltz, 2012). sulikowska-drozd et al. (2012) showed that b. fallax is capable of egg-retention and there is no correlation between the body size of individuals and the number of preserved eggs (sulikowska-drozd et al., 2012). other ovoviviparous clausiliids are: vestia gulo, v. elata, v. turgida (sulikowska-drozd, 2009) and ruthenica filograna (szybiak, 2010; szybiak et al., 2015). sulikowska-drozd (2009) studied egg retention and ovoviviparity in three clausiliids, vestia gulo, v. turgida and v. elata. differences in number of retained eggs in v. gulo and v. turgida were observed. sulikowska-drozd (2009) claimed, that the number of produced eggs depends on the season of the year, whereas number of juveniles is probably connected with the habitat preferences. cannibalism has been observed in v. gulo and v. elata, but not in v. turgida (sulikowska-drozd, 2009). detailed knowledge about ovoviviparous gastropods is crucial in the understanding of the reproductive success of each species, especially in the context of endangered species. an example of ovoviviparity among terrestrial gastropods is the study carried out by heller et al. (1997), in which scientists worked on the reproductive biology and population dynamics of a minute snail lauria cylindracea. this species is characterized by low fecundity, but this type of reproduction is advantageous because of offspring survival. after hatching, juveniles are able to feed, grow and cope with flooding, drowning and drought. ovoviviparous gastropods can survive more easily in unfavorable environmental conditions, such as excess of water in some habitats (heller et al., 1997). viviparity is a mode of reproduction which consists in producing living offspring. in other words, viviparity means that ova are fertilized in the reproductive tract and stored inside the reproductive system of an animal, and juveniles are born fully formed. tompa (1979b) suggested that viviparity occurs very rarely among terrestrial gastropods, but it is possible in some cases. there is evidence that viviparity may be present in vaginulid pseudoveronicella zootoca, achatinellid tekoulina pricei and in acavid stylodon studeriana (solem, 1972; tompa, 1979a, b, 1984; heller, 2001). eggs and egg cannibalism among terrestrial gastropods, a common reproductive mode is oviparity, as mentioned in the previous chapter. gastropods can lay eggs in different places, such as in the forest litter, inside excavated holes or in arboreal places, and their size varies depending on the species (heller, 2001). the smallest eggs are laid by opisthostoma retrovertens, carychium tridentatum, vertigo pusilla, vallonia costata, v. pulchella, cecilioides acicula, c. genezarethensis, punctum pygmaeum, succinea oblonga, whereas the largest ones are produced by acavus haemastoma, a. waltoni, megalobulimus bronni, m. capillaceus, m. oblongus, m. popelairianus, m. rosaceus, m. terrestris, stylodon studeriana, powelliphanta superba, limax flavus (frömming, 1954; berry, 1964; baur, 1989; pokryszko, 1990; heller et al., 1991; heller, 2001). there is a correlation between the body size expressed as shell height and the size of the egg produced by an animal. for example, a small gastropod c. tridentatum (2 mm) produces small eggs of 0.4x0.3 mm, and similarly v. pusilla (2 mm) produces eggs of 0.6x0.3 mm (baur, 1989; pokryszko, 1990), whereas large m. popelairianus (230 mm) produces eggs of 51x28 mm (heller, 2001). bigger juveniles hatch from larger eggs, which means that egg size determines the size of juveniles and also their growth, survivorship and possible future reproductive success. bigger juveniles, which come from bigger eggs, are more resistant to starvation (tompa, 1984) and have longer development (baur, 1994a). gastropods with larger sizes can lay more eggs, a. circumscriptus (25 32 mm long) deposits 104 123 eggs, a. hortensis (30 40 mm long) up to 200 eggs, whereas a. vulgaris (70 140 mm long) lays up to 205 400 eggs and a. rufus (150 mm long) up to 500 eggs (welter-schultes, 2012). production of larger eggs has many advantages: hatched juveniles are bigger and have a higher growth rate and survivorship, acquiring food is easier, and bigger juveniles have higher chances of successful reproduction. on the other hand, bigger eggs require longer incubation and the risk of mortality before hatching increases (clutton-brock, 1991). heller (2001) presented a summary on animal and egg size on the basis of literature data, which shows that there is a correlation between these two properties (heller, 2001). laying eggs is part of life reproductive strategy, and interest in this subject has increased recently, especially in relation to life cycles. in the case of terrestrial gastropods, egg cannibalism can sometimes be observed, especially in the case of many stylommatophonan species. egg cannibalism delivers calcium to juveniles, and this component has great importance for snails during all their life, especially affecting their growth during the early stages of development (oosterhoff, 1976). baur (1994a) discovered that this phenomenon occurs in helicid a. arbustorum. newly hatched juveniles cannibalize deposited eggs, which are derived from the same parent. cannibalistic behavior occurred with varying frequency depending on the type of population. baur (1994a) showed that cannibalistic behavior was observed in 50 % of hatched offspring in subalpine forest, and in 87.8 % in lowland forest. moreover, it has been shown that the occurrence of cannibalism among juveniles is not connected with the egg size. the amount of energy and nutrients obtained by a snail during embryonic development does not cause cannibalistic behavior in juveniles of a. arbustorum (baur, 1994a). ożgo and bogucki (2006) showed that juvenile specimens of cepaea nemoralis in natural habitats radulated shells of live snails. the authors linked this behavior with ph and calcium content in the soil. in c. nemoralis cannibalism has also been observed. shell predation and cannibalism were present on acid soils or with low content of calcium. the occurrence of these effects can be explained as an adaptation to life in adverse conditions of the environment (ożgo and bogucki, 2006). conclusions studies on reproduction systems of terrestrial gastropods provide new, original data, which fill the gap in the current state of the knowledge. the main significance of this type of research is possibility of population management, what is very important in the context of the control of pests which can cause serious damages in agriculture, conservation of endangered and rare species or in the case of gastropods farming for culinary and other purposes. many papers concern about morphology of reproductive systems of species belonging to arionidae, agriolimacidae, limacidae, helicidae families, but other species should be explored additionally. studying reproductive systems with combination of phylogenetic data among many species will allow to obtain new information about reproductive strategies in terrestrial gastropods. acknowledgements we are grateful to jankowska a for proofreading. this work was supported by grants from the polish-norwegian scientific cooperation (pol-nor/201888/77) and jagiellonian university (ds/mnd/wbinoz/inoś/25/2016; 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competitiveness of simultaneous hermaphroditism versus dioecy. amer. nat. 162: 220-241, 2003. wright wg. sex change in the mollusca. trends ecol. evol. 3: 137-140, 1988. yusa y. causes of variation in sex ratio and modes of sex determination in the mollusca an overview. am. malac. bull. 23: 89-98, 2007. microsoft word isj429 isj 13: 210-220, 2016 issn 1824-307x review the use of insecticides to control insect pests m wojciechowska1, p stepnowski2, m gołębiowski1 1laboratory of analysis of natural compounds, department of environmental analysis, faculty of chemistry, university of gdańsk, ul. wita stwosza 63, 80-308 gdańsk, poland 2laboratory of chemical environmental risks, department of environmental analysis, faculty of chemistry, university of gdańsk, ul. wita stwosza 63, 80-308 gdańsk, poland accepted june 10, 2016 abstract pesticides are used as plants protection products. among those, insecticides serve as agents to control insects. when incorrectly applied, however these substances may negatively affect people's health and natural environment. administration routes of insecticides depend on many factors and vary from spraying to fertilizers. these different methods influence how insects prey and how pests develop. additionally, too frequent use of the same chemicals can lead to development of resistance of insects to these insecticides. in order to prevent occurrence of negative effects of insecticides on surroundings, the effects of these compounds should be studied. key words: pesticides; insecticides; pest insects   introduction insects are now the largest group of animals (tsakas, 2010). moreover, owing in large part to their staggering diversity, insects are in more different places in the world than virtually any other organism. there are insects in habitats ranging from the high arctic to tropical rainforests to petroleum pools to glaciers to mines a mile below the surface to caves to sea (resh and cardé, 2003). currently, there are more than a million described species and it is assumed that is only a small percentage of this large group of animals. only few species of insects live in seawater, whereas ca. 100,000 species (especially larvae) in habit freshwater after their secondary aquatic adaptation. the insects can to adapt the life in the water or on the water surface (chapman, 2013). on land, insects are present in all biotopes: in the mountains, caves, hot springs, polar zones. the greatest biodiversity of insects are found in warm climates. the warm climatic conditions favor the development of biodiversity, such as environmental conditions and food availability (resh and cardé, 2003; chapman, 2013). biological adaptation of insects allows them to adjust to specific environment and survive in almost ___________________________________________________________________________ corresponding author: marek gołębiowski laboratory of analysis of natural compounds department of environmental analysis faculty of chemistry, university of gdańsk ul. wita stwosza 63, 80-308 gdańsk, poland e-mail: marek.golebiowski@ug.edu.pl all climate zones. some species can even fall into a state of diapause as a way to survive adverse conditions. such insects exhibit high fertility, large populations reaching a million or a billion individuals, and ability to feed on everything that contains organic matter (from wood to blood). for some species as little as one ingredient is enough to survive, e.g., wine barrel sediment rich in acidic potassium bitrate (cream of tartar). insects’ life span varies between species: mayflies live for one day, queen of bees for 10 years, and termites can live up to 50 years. insects play a very important role in biocenosis, interaction of insects with other species in the environment is beneficial for participating parties. moreover, insects also take part inhuman life, health, and the economy. unfortunately, that role instead of being beneficial is harmful. insects can be parasites, human pathogens and carry diseases. they’re involved in transmission of malaria, yellow fever, typhus, plague, dengue, various forms of encephalitis, relapsing fever, river blindness, filariasis, sleeping sickness, and innumerable other debilitating or even fatal diseases, not just in warmer climes (resh and cardé, 2009). characteristics of selected harmful insects species harmful insects, in various developmental stages such as larvae or adults, attack different parts of plants: roots, stems, leaves. if plant is attack by harmful insects usually this plant die (chapman, 2013). 210   ceutorhynchus napi are 3.2 4 mm grey in color beetles. females of these beetles puncture plant stems and lay eggs inside. larvae hatch after approximately 10 20 days. during growth and development of larvae, abnormal bulbous growth appears on the stem, which then ruptures, leading to deformation of the stem (chapman, 2013). larvae also tunnel through stems leading to damage that can facilitate pathogen’s access to plants. this species produces one generation per year (taiz and zeiger, 2010). ceutorhynchus quadridens are brown beetles with a length of 2.5 3 mm, and possess characteristic whitish dots on the wing covers. in the spring the females lay eggs and their development takes up to 3 months. white larvae up to 6 mm has a brown head. its chrysalis is yellowish and has a long snout. the larvae are feeding inside and tunnel through stems without distorting them. this strong damage inhibits the plant growth, where as the tunnels create sites for infection (chapman, 2013). baris spp. are 3 4 mm beetles with a blue or dark green metallic sheen. females deposit eggs in the roots of plants. the larvae tunnel through the roots and poison the roots at the same time. the pupae are whitish, they don't like move, size similar to adults (chapman, 2013). this leads to inhibition of plant growth and allows for easier access of pathogens. wireworms the larvae elateridae are beetles with length of 7 15 mm and a brown-gray color. the larvae are covered with cooper color chitin. these insects feed on the underground parts of plants what damages the roots system leading to plants withering. click beetle larvae (wireworms, elateridae) are generally plant or detritus feeders, but some are predatory in soil or rotting logs (resh and cardé, 2003). bibionidae are flies with a stocky and hairy body (8 10 mm in length). females have gold-red coloration, males are black. the larvae bibionidae live in the soil, damage the seeds, and chew on the roots. this leads to disruption of water management in plants and excessive lowering of the plant level. the stout, dark-colored adults feed on flowers. the worm-like larvae are general detritivores and can be found in organic soils and compost heaps in abundance (resh and cardé, 2003). zabrus tenebrioides is beetle with length of 12 15 mm. this beetle is black in color, has a characteristic light brown abdomen, and red-brown antennae, and legs. in the fall, larvae damage germinating seeds. they feed on the leaf bases and blades, leading to their withering and death. this species, in contrast to other beetles, which are mostly carnivorous species are herbivorous (chapman, 2013). thripidae is a slender, black-brown insect with length about 1 2 mm and narrow wings. lives mainly on flowers, under the tree bark, or in plant litter. the legs of adults lack typical insect tarsal claws, but each tarsus has an eversible bladder-like arolium (resh and cardé, 2003). the larvae and individual adults damage the seeds. pathogens can easily use the damage to penetrate to the interior of the plant and infect it. often the infected plant dies (chapman, 2013). grubs the larvae of beetles (melononthidae) have a specific white, curved body with a darker head and 3 pairs of strong legs. grubs live in soil and can damage the root system of plants causing their withering and death (chapman, 2013). cabbage aphid (brevicoryne brassicae) reach a length of 2 2.5 mm, are gray-green in color and their bodies are covered with waxy coating. winged or wingless forms of adults can be distinguished. they are herbivores suck the juice from plants, partially digesting it, and the residues excrete. the larvae of these insects are similar to the adult form. they attack the inflorescence stems, petioles and leaf blades leading to withering and death of plants (chapman, 2013). phorbia brassicae belong to flies with a body length of 5 6 mm and have characteristic red spots on silver-white foreheads. females are bigger than males. this species exhibits a sexual dimorphism: the male is black-gray and the female is brown-gray. the larvae attack the roots causing the death of plants (chapman, 2013). pieris brassicae belong to the butterflies with white wings (brzeziński, 1922) with a span of 5 6 cm. female large whites have a pair of prominent black spots in the median area of the forewings, but these are absent in the male. the spots are visible on the underside forewings of both sexes. caterpillars of this butterfly are yellowish with black spots and have a body length of about 4.5 cm. they feed on leaf blades and siliques. caterpillars scrape the parenchyma off the bottom side of the leaf blades which often leads to the death of plants (www.learnaboutbutterflies.com). methods of insects pests control different procedures and methods can be used to control harmful organisms. 1) physical methods: according to their mode of action, physical control methods can be active or passive. the level of efficacy of active methods is proportional to both intensity of the energy and duration of its application to the target. active methods include thermal shock (heat, cold), electromagnetic radiations (microwaves, radio frequencies, infrared, ionizing radiations, uv and visible light), mechanical shock and pneumatic (blowing or vacuum). passive methods do not require further energy to achieve desired effect. examples are traps, airtight or hermetic storage, barriers of various kinds and trenches (vincent et al., 2009). 2) biological methods: their goal is to reduce the population of the pest below the economic threshold. examples would be: introduction of nonnative, natural enemy of pests into the environment: use of pathogens including bacteria, viruses and fungi that would infect pests. other insect can also be used against insects pests (commission directive 2008/113/ec of 8 december 2008 amending council directive 91/414/eec to include several micro-organisms as active substances; can ulu et al., 2016). 3) chemical methods: associated with the use of chemicals that affect particular insects. these compounds may be of natural origin, e.g., attractants, repellents, antifeedants, pheromones, 211   and hormones: synthetic origin, and/or combination of natural and synthetic compounds. chemical compounds and mixed compounds of synthetic and natural origin are called pesticides. chemical measures should be considered only as a complementary. in selecting a pesticide and the appropriate formulation, consideration should be given to its biological effectiveness (including residual activity where appropriate) against the pest concerned, the susceptibility of the target organism, the methods of application, its safety to humans, its toxicity to non-target organisms (who, 2006; who, 2012). pesticides pesticides are synthetic or naturally-occurring chemicals used to target and destroy harmful organisms. the goal of their use is to reduce or completely eliminate pests, weeds and diseases (sánchez-bayo and ortega, 2012; satyavani et al., 2012). pesticides can also affect plant’s growth regulation. the name comes from the latin words pestis pest and cedeo destroy. the number of pesticides used in the world is enormous: more than 1000 different pesticide formulations containing substances with high biological activity are available. the classification of insecticides by chemical composition and examples of commercial products is presented in the table 1. pesticides play a significant role in the quality and efficiency of agricultural production (rodrigues macedo and freire, 2011), they also impact the environment and unfortunately, not always in a positive way (bazok et al., 2012; cardoso and alves, 2012). the new pesticides are systemic, meaning they can be taken up by the plants and animals and distributed through their tissues without accumulating in any particular organ or structure (e.g., fatty tissues) (sánchez-bayo and ortega, 2012). plant protection products exhibit high toxicity toward targeted pests. additionally, these substances should show short persistence in the environment, a high susceptibility to degradation in such a way that after reaching its destination these chemicals would quickly disappear from the soil, water and air (grosicka-maciąg, 2011; lofty et al., 2013). apart from targeted pests, pesticides affect surroundings and organisms that live in the area where agents are used. that is why it is so important to study the effects of specific pesticides on environment prior to use of these substances. they should be applied in accordance with the principles of integrated vector management, an evidence-based decision-making process adapted to local settings, which rationalizes the use of vector control methods and resources and emphasizes the involvement of communities (who, 2006; who, 2012). the use of insecticides insecticides are commonly used to protect against insects in households, restaurants, hospitals, farms, forest plantations, etc. these substances protects from the harmful insect-borne diseases, insects pests in warehouses, and agricultural and forest pests (cardoso and alves, 2012). a list of insecticides is presented in the table 2. effective control measures must be based on a clear understanding of the bionomics and behavior of the target species. effective vector and pest control also requires careful training, supervision of control operations and periodic evaluation of the impact of the control measures on the targeted vectors or pests and on disease incidence or prevalence. chemical measures should be considered only as a complementary addition to basic sanitation, as far as possible. in selecting a pesticide and the appropriate formulation, consideration should be given to its biological effectiveness (including residual activity where appropriate) against the pest concerned, the susceptibility of the target organism, the methods of application, its safety to humans, its toxicity to nontarget organisms. in addition, the extensive use of chemical pesticides, which allowed farmers a better pest control and more effective producing food (meissle et al., 2010). one of the most known diseases carried by insects in tropical countries is malaria. its epidemic is associated with the mass occurrence of mosquito (anopheles maculipennis). even though in moderate climates mosquitoes aren't responsible for carrying malaria, these insects cause inconvenience, especially female mosquitoes that bite causing skin itching (gliniewicz et al., 2003). the spread of yellow fever and dengue as the vector mosquito aedes aegypti dispersed throughout the tropics, and of malaria to brazil with the introduction of its vector anopheles gambiae, are notable examples of human diseases vectored by introduced insects (simberloff, 2009). another harmful and troublesome insect particularly in the warmer seasons is a fly, especially housefly. the house fly, musca domestica, is a well-known cosmopolitan pest of both farm and home. this species is always found in association with humans or the activities of humans. this insect sits on food, faeces, and carrion, which makes it great candidate for spreading diseases and parasites. housefly carries pathogenic bacteria, dysentery, paratyphoid fever, anthrax, and different forms of invasive parasites (e.g., eggs of threadworms). the house fly has a complete metamorphosis with distinct egg, larval or maggot, pupal and adult stages. the house fly overwinters in either the larval or pupal stage under manure piles or in other protected locations. warm summer conditions are generally optimum for the development of the house fly, and it can complete its life cycle in as little as seven to ten days. fly also spreads agents that cause conjunctivitis. it is also an intermediate host for the larvae of some tapeworms (sanchez-arroyo and capinera, 2014). blue bottle fly (calliphora womitoria) with a characteristic blue color and a green fly (lucilla sericata) (who, 2008) are typical insects on farms and meat processing plants. flies such as drosophila melanogaster, drosophila funebris occur mainly where fruits, juices, and jams ferment. flies carry mold, bacteria, and are particularly dangerous when their development is carried in the faeces (who, 2008). from the sanitary viewpoint it is important to control cockroaches. their representatives are blattella 212   table 1 the classification of insecticides by chemical composition and examples of commercial products insecticide chemical compound commercial product organophosphorus insecticides polychlorinated insecticides carbamate insecticides neonicotinoids pyrethroids others teep tetraethyl pyrophosphate dianizion parathion paraoxon trichlorfon malathion bischlorophenyl: ddt metoxychlorine derivatives cyklodiene: aldrin dieldrin ceptachlorine endosulfan chlordon derivatives cycloparaffinic: hexachlorocyklohexane lindane chlorinated terpenes: camphenes pinenes carbanil carbofuran primor izolan aldikarb bindokarb thiamethoxan imidaclopirid firponil clothianidin natural: pyrethrins jasmolin cynarin synthetic: allethrin alphamethrin bioresmethrin cyfluthrin cypermethrin deltamethrin permethrin rosmethrin trousfluthrin fenvalerate calcium oxide boric acid trioxane nicotine zyklon b fyfanon 440ew anthon bovinox proxol danex azotox ditox triox anotex cesarex chlorophenothane dedelo dinocide didimac digmar genitox guesapon ixodex neocid r50 primor 500w marshal 250ds priflor ae primix al primix ae actara 25wg mospilan 20sp cyflok 50ew k-othrine bros insect spray abc ac insektum a 01 al spruzit 04ec decis 2.5ec germanica and blatta orientalis. they demonstrate a high mobility and exhibit an unpleasant odor coming from their glands. b. germanica is tolerant of a wider range of conditions and may even be found in dry places, provided it has access to water. b. orientalis generally prefers cooler, moist conditions. cockroaches spread many diseases; can carry about 40 species of bacteria, protozoa, viruses, and fungi which are dangerous to people and pets. german cockroaches usually prefer a moist environment with a relatively high degree of warmth. the insects are mostly scavengers and will feed on a wide variety of foods (who, 2006; jacobs, 2013). 213   all the mentioned insects are very unpleasant and dangerous, not only in our homes and catering points, but seem to be constitute a health hazard in hospitals where hygiene is the priority (who, 2006, 2008). as total coverage can rarely be achieved, the focus should be on areas where people congregate, e.g., high-density housing, schools, hospitals and areas where cases of disease or high vector densities have been recorded. the most common insect pests in hospitals are: ants (monomorium pharaonis, fleas (siphonoptera), mosquitoes (culicidae), flies (diptera), bedbugs (cimex rectularius) and others. these insects can be found in virtually all areas including kitchens, laundry rooms, patient's rooms and operating rooms (who, 2006; resh and cardé, 2009). in order to control insects hospital rooms are sprayed with insecticides from the group of pyrethroids, e.g., deltamethrin, permethrin, cypermethrin, alphacypermethrin, and the group of carbamates, eg. bendiocarb. elimination of harmful table 2 a list of insecticides and the insect species to which these substances apply to insecticide insects species insecticide insects species cyflok 50 ew cockroaches: blatella germanica l., getox cockroaches: blatella germanica l., blatta orientalis l. blatta orientalis l. black beetles litter, eg. spondylis buprestoides bugs (cimex rectularius) monomorium pharaonis l. monomorium pharaonis l. lepisma saccharina lepisma saccharina plodia interpunctella karate zeon 050 cs caterpillars pieris brassicae, pieris rapae vigonez ants (formicidae) aphids beetles thrips tabaci, kakothrips robustus delia antiqua hoplocampa brevis getox ultra cockroaches: blatella germanica l., bembecia hylaeiformis blatta orientalis l. monomorium pharaonis l. magus 200 sc tetranychidae at different developmental stages bugs (cimex rectularius) fleas mospilan 20 sp leptinotarsa decemlineata larvae and beetle lepisma saccharina aphids leucoptera xitella k-othrine 25 sc cockroaches: blatella germanica l., hoplocampa testudinea blatta orientalis l. dasyneura piri fly (musca domestica, calliphora vomitoria) caterpillars mosquites (anopheles maculipennis, culex pipiens) bugs (cimex rectularius) actara 25 wg leptinotarsa decemlineata monomorium pharaonis l. hoplocampa testudinea anthonomus pomorum actellic 500 ec sitophilus granarius aphids tribolium confuscum eriosoma lanigerum oryzaephilus surinamensis l. psylla piri 214   insects from hospital rooms is quite a challenge due to the variety of buildings and different features in each room. presence of sick people who might have a reduced immunity is an additional complication. insecticides should combines strong selective activity against key pests with low vertebrate toxicity (owens, 2002; larson et al., 2012). products used by people in their homes or in restaurants mainly contain compounds from the group of pyrethroids, for example deltamethrin, alphamethrin, tetramethrin. these are effective for controlling mosquitoes, flies, and other insects. additionally, repellents or substances acting dissuasive on mosquitoes can be used to protest against their bites. the formulations used for insect are available in different forms, e.g., sprays, concentrates, powders, sticks. all pesticides are toxic to humans to some degree. however, the doses that are acutely toxic to humans are usually far higher than those required for killing vectors and pests. the key to safe use of pesticides is to reduce to a minimum the possibilities of unsafe exposure during handling of hazardous chemicals. therefore, care in handling pesticides, particularly by spraying staff and persons living in sprayed houses, should be a routine practice and form an integral part of any program involving the application of pesticides (who, 2006; sánchez-bayo and ortega, 2012). change in crops technology brought economic importance to pest control. intensive fertilization of crops as well as protection efforts against weeds and diseases are factors contributing to the development of different insects. the latter can significantly reduce crop yields (kaniuczak, 2013). in recent years, many pollinators have declined in abundance and diversity worldwide, presenting a potential threat to agricultural productivity, biodiversity and the functioning of natural ecosystems (piiroinen et al., 2016). unfortunately, all agrotechnical simplifications, new plants species, changes in agroclimate favor development of species of harmful insects that haven’t been observed in the area ever before. changing climatesoil conditions, simplification of crop rotation (bazok et al., 2012), and use of heavy equipment for field work affects oil. all of these mentioned factors can cause development of extra species which are harmful to crops. insecticides are chemicals used for the protection of plants against harmful insects (satyavani et al., 2012). of course, only approved and registered insecticides should be applied. additionally, for effective treatments, these substances should be properly selected, applied at the optimum for the insecticide temperature while maintaining grace period and prevention. the crops should include effective management of target pests, decreased use of conventional insecticides, and reduced harm to non-target organisms (prischmann et al., 2007; gassmann et al., 2014). plant protection products should be used according to the label and with necessary precautions. the use of insecticides acting selectively on specific species allows for protecting the populations of beneficial insects such as bees, ladybugs, lacewings.  bumble bees are important pollinators whose populations have declined over recent years (kaniuczak, 2012; laycock et al., 2012). it is also possible to apply seed dressing, which eliminates the need for spraying. females are the main target of used insecticides as reducing the number of females in the population decreases the number of laid eggs. development of safer and more effective technologies insecticide significantly affected the longevity or reproductive capacity of emerged females, or the sex ratio of their progeny (el-wakeil et al., 2013). ecological farming is another area of growing plants. plant protection is seen in a different way than in conventional farming. the prophylactics in reducing weeds, pathogens or pests are preferred. only exceptional situations allow direct applying permitted plant protection means (szymona, 2009). number of available chemicals used to protect plants from harmful insects is very limited. insecticides used in agriculture are mainly pyrethriod synthetic compounds with molecular structure similar to natural compounds pyrethrins. pyrethroids are more resistant to oxidation and photo degradation. currently, organic farming uses two active ingredients: deltamethrin and lambdacyhalothrin (lofty et al., 2013; thany et al., 2015). deltamethrin was synthesized in 1973, it is photostable, soluble in organic solvents but poorly soluble in water. farmers, mainly at the beginning of the organic farm activity, demand biological plant protection products that could fully replace chemical pesticides. unfortunately, it is impossible. it results from a very short list of permitted products as compared to a wide spectrum of synthetic pesticides and from complete lack of some groups. it exhibits contact-stomach action (szymona, 2009) on insects such as: orthoptera (caelifera), bugs (aphididae, cimicidae), butterflies (p. brassicae), beetles (chrysomelidae, melolontha melolontha, tenebrionidae), diptera (flies, midge). deltamethrin is toxic to fish and bees by contact. lambdacyhalothrin belongs to a group of chemicals called pyrethroids. pyrethroids are manmade chemicals that are similar to the natural insecticides pyrethrins. it exists in two isomeric forms, dissolves well in organic solvents, but is insoluble in water. works mainly on contact and is used for bugs (aphididae, leafhoppers, cimicidae), butterflies (p. brassicae), beetles (leaf beetles, melolontha melolontha) (who, 2006). in recent years inhibitors of chitin synthesis and pyrethroids were introduced to protect forests against harmful insects. restrictive measures established by european union significantly narrowed the list of substances authorized for use in forests. number of allowed chemicals has been cut, but number of harmful insects did not decrease. forest protection and health program aim to assist, advise and support countries to protect the health and vitality of forests, forest ecosystems and trees outside forests, with special reference to insects, diseases and other harmful biotic and abiotic agents (moore, 2009). harmful forest insect and insecticides used to control them is presented in the table 3. 215   table 3 harmful forest insect and insecticides used to control them insect insecticide insecticide class fastac las 15 sc pyrethroids hylobiusabietis and other weevils sherpa 100 ec foray 76 b dimilin 480 sc * chitin synthesis inhibitor mospilan 20 sp neonicotinoids lymantria monacha, dendrolimus pini sherpa 100 ec * pyretroids panolis flammea, bupalus pinaria sherpa 100 ec pyrethroids dimilin 480 sc sherpa 100 ec pyrethroids diprion pini mospilan 20 sp neonicotinoids sherpa 100 ec pyrethroids acantholyda posticalis mospilan neonicotinoids larvae hymenoptera sherpa 100 ec pyrethroids foray 76 b torix vinidiana, operopthera brumata sherpa 100 ec pyrethroids cacoecia piceana l., exoteleia dodecella, butterfly caterpillars sherpa 100 ec pyrethroids imagines of cockchafer, coleophora laricella, springtails mospilan 20 sp neonicotinoids aphids (aphidoidea) mospilan 20 sp neonicotinoids agro pirymikarb 500 wg primor 500 wg pinoil 012 al. tetranychus urticae ortus 05 sc parthenolecanium pomeranicum pinoil 012 al. promanal 60 ec * not recommended by the fscforest stewardship council 216   decades ago, a new group of insecticides with a contact and stomach action (hardy, 2014) has been developed. these insecticides are called neonicotinoids because of their resemble to nicotine. these substances act mainly by contact and stomach on insects belonging to different rows, e.g., heteroptera, flies, and butterflies. neonicotinoids exhibit action against forest leafeating insect pests, thus have high toxicity toward targeted insects, but low toward warm-blooded animals and other insects that aren't at aim. in forestry neonicotinoids are used to control: lymantria monacha, beetles, dendrolimus pini, and other species of folivorous insects. mospilan 20sp with an active ingredient acetamiprid is an example of neonicotinoid used to protect crops and forests. it acts as neuropeptide acetylcholine agoniston the central nervous system of insects. this formulation is used to control number of sucking and biting insect sand acts both on the plant surface and inside by deep-penetrating into the plant. insects feeding on the sprayed plant will die. however, it is important that the insecticide does not adversely affect non-target insects, e.g., bumblebees, bees (laycock et al., 2012). through biting the plant tissue insects suck toxic nourishment which ruins their digestive system. this measure may be applied in cultivation of: potatoes (larvae and beetles, leptinotarsa decemlineata), rapeseed (ceutorhynchus assimilis, meligethes aeneus), fruit trees such as cherries (aphids), apples (cydia pomonella), pears, and plums (aphids) (sánchez-bayo and ortega, 2012). some of the chemicals used are forest stewardship council (fsc) certified. awarding the certificate was established in 1993, it promotes a worldwide system of responsible forest management. the use of the fsc logo indicates that the wooden product has a known origin and has no negative influence on the environment, which is socially beneficial and economically viable. the fsc label is dedicated for wooden products and means that the harvesting of wood for this product did not disturb the environment (https://us.fsc.org/en-us/certification). laboratory conditions can be used to evaluate the effect of insecticides on individual groups and species of insects (korrat et al., 2012). analysis of chemical compounds produced by insects that were treated with insecticides is a useful technique (georghiou and metcalf, 1961). such analysis is performed using a gas chromatography-mass spectrometry gc/ms (lockey, 1988; buckner, 1993; cerkowiak et al., 2013). different chemical compounds are present on the external body surface of insects such as lipids or body fat (gołębiowski et al., 2012; cerkowiak et al., 2015). larvae exhibit much higher accumulation of those compounds than adults (ahammao-sahib et al., 1994) due to the fact that the body fat supports the juvenile hormone during the transformation of the larvae. juvenile hormone (jh) is a sesquiterpenoid produced by the corpora allata and is present throughout nymphal and larval life in all insects (riddiford, 2009). the problem of insects’ resistance to insecticides the widespread use of insecticides leads to development of resistance of insects to the chemicals (roush, 1995) used to protect plants (feyereisen, 1999; hardy, 2014). insecticides eliminate susceptible individuals from the population while resistant individuals remain. subsequent generations will inherit traits that determine resistance and as a result, a population of resistant individuals is favored (manchao et al., 1995). pyrethroid with two active ingredients 1) deltamethrin and 2) alphamethrin (laycock et al., 2012) is used for folivorous pest control (lymantria monacha, dendrolimus pini and attacking conifers (hylobius abietis). the problem of resistance to chemicals is not limited to the insects, but also applies to plants and animals including nematodes, bacteria, mites, crustaceans, fish, amphibians, fungi, rodents and some species of weeds (malinowski, 1997). insecticide resistance is one of the best examples of microevolution, or evolution occurring on an ecological time scale. the study of insecticide resistance is important, both because it leads to a better understanding of evolutionary mechanisms operating in real time, and because of its economic relevance. the development of insecticide resistance in pest insects has been an increasing problem for agriculture, forestry and public health. agricultural practices usually include the systematic application of a wide array of active compounds at variable dosages and frequencies, which represent a wide range of selective regimes. therefore, identifying the molecular and genetic adaptations responsible for insecticide resistance will offer new opportunities for developing pest management strategies. the study of insecticide resistance makes it possible to classify adaptations into three main mechanisms: reduction of insecticide uptake, by reducing the permeability of insect cuticle, detoxification, through alteration in the levels or enzyme activities that degrade or sequester insecticides and, insensitivity due to point mutations in genes encoding for proteins that are the target site of insecticides (silva et al., 2012). in 1956, the world health organization (who) developed an international program to collect information about resistant populations of pests, methods of detection and evaluation of the resistance, and the use of alternative plant protection products. manufacturers of plant protection products designated international group of national associations of manufacturers of agrochemical products (gifap) to look at insects’ resistance to insecticides. within this group, a special committee was establishedinsecticide resistance action committee (irac)to provide expertise on insects’ resistance to specific pesticides. this action aims to help producers prolong usability of produced insecticides (malinowski, 1997; irving, 1995). immunity is associated with natural selection observed in the environment. darwin observed that only fraction of offspring survives and reproduces by passing their genetic traits to future generations (malinowski, 1997). 217   fig. 1 frequency of occurrence: sensitive homozygotes (aa), sensitive heterozygotes (ab) and resistant homozygotes (bb) in the population of insects subjected to an insecticide (based on malinowski, 1997). according to crow the resistance causes genetic changes in response to selection. changes can apply to individuals or to entire population. development of resistance and its subsequent inheritance depends on the selection factor which acts on the organisms in the environment such as for instance insecticide. of course, the frequency and time of use of the insecticide play an important role (malinowski, 1997, silva et al., 2012). populations resistant to insecticide can develop when: 1) few heterozygotes resistant to insecticides are present in the population, 2) resistant homozygotes are practically nonexistent in the studied population, 3) frequency of occurrence and gene dominance cause the maximum level of resistance and these genes will not be recessive, 4) genes responsible for resistance to the insecticide will be present in the final population (winter et al., 2005). initially, the population consists mainly of individuals that are sensitive homozygotes and few heterozygotes. no resistant homozygotes are observed. when the application of the insecticide extends in time, individuals cross breed and in the subsequent generations there are fewer sensitive homozygotes while heterozygotes and homozygotes become more resistant (tang et al., 1995; winter et al., 2005). as time passes, resistant homozygotes begin to prevail over the sensitive homozygotes and heterozygotes. this leads to the population consisting of resistant homozygotes (fig. 1) (winter et al., 2005). building resistance to insecticide depends on: 1. internal factors: • genetic the frequency of occurrence of resistance genes, their number, and dominance. expression of these genes as well as impact of insecticides applied to insects. the number of resistance genes in the initial population correlates well with the number of resistant individuals. dominant homozygotes are more resistant than heterozygotes that exhibit incomplete dominance. the lowest resistance will be observed in sensitive homozygotes. whether one single gene or combination of genes is present, this determines rate of selection of individuals and development of resistance. • biological include biotic and behavioral factors. biotic factors include the size of one generation in the population and how long this generation lasts. the development of resistance is higher when the duration of one generation is short. behavioral factors, i.e., isolation, population mobility, migration, have a large impact on the development of resistance. when the population is more isolated the process of building resistance will occur faster due to lower opportunity to exchange genetic pool. 2. external factors • operating factors associated with the use of the chemical compound such as: chemical structure, similarity of this structure to the previously used formulation, accumulation in the environment, formulation, and longevity of threshold selection, dose, the stage of development of insects, the application route (winter et al., 2005). cross resistance has to be considered while using insecticides (winter et al., 2005). it can occur when chemicals are very similar in structure. insects would then be resistant to the whole group of insecticides due to their structural similarities as the same genes regulate the resistance. it is assumed that the threshold selection influences the development of resistance and use the insecticide should remove 90 % of the population. it is clear that the inheritance of resistance is faster when genes are dominant. the applied dose of insecticide is an important factor. if the dose is so high that it causes the killing of sensitive homozygotes and resistant heterozygotes, the dominant genes would be functionally recessive, and only use of a lower dose will lead to their dominance (winter et al., 2005). 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who, 2008. http://www.urbanpestsbook.com/downloads/wh o_report_summary.pdf available 14.06.2016. who, pesticides and their application, for the control of vectors and pests of public health importance. six edition who, 2006. http://apps.who.int/iris/bitstream/10665/69795/1 /who_cds_ntd_whopes_gcdpp_2006.1 _eng.pdf available 13.06.2016 winter pc, hickey gi, fletcher hl. instant notes genetics, 3rd ed., 2010. 220   120 isj 16: 120-129, 2019 issn 1824-307x research report effects of steinernema carpocapsae (weiser) on immunity and antioxidant responses of glyphodes pyloalis walker m mallahi, a zibaee*, jj sendi, s jamali department of plant protection, faculty of agricultural sciences, university of guilan, iran accepted june 21, 2019 abstract the effect of steinernema carpocapsae (ira18) infection was studied on the mortality, immunity and antioxidant responses of glyphodes pyloalis walker larvae. the lc50 value of 582.9 infective juvenile per ml with confidence limit of 359.1-811.5 was obtained via bioassay against the larvae. injection of s. carpocapsae increased the number of total hemocytes after 1-6 h compared to intact and ringer-injected larvae while the highest numbers of plasmatocyte and granulocytes were recorded after 1 and 3 h. although intact larvae had a steady activity of phenoloxidase at different time intervals but those injected by s. carpocapsae showed the elevated enzymatic activity at 3-12 h. nematode injection significantly increased the activities of superoxide dismutase and catalase compared to intact and ringer-injected larvae, while no significant difference was observed in peroxidase activity. the injection with s. carpocapsae caused the highest activity of glutathione s-transferase using cdnb as reagent, but the enzymatic assay with dcnb showed no statistical differences among treatments. also, activities of ascorbate peroxidase and glucose-6-phosphate dehydrogenase significantly increased in the nematode-injected larvae. intact and ringer-injected larvae showed no statistical differences in the concentration of malondialdehyde but the highest amount was recorded in nematode-injected larvae. results of our study indicate that native isolate of s. carpocapsae cause mortality on the larvae of g. pyloalis and it interferes in the immune and antioxidant responses. key words: steinernema carpocapsae, glyphodes pyloalis, immunity, antioxidant system introduction different varieties of mulberry (morus spp.) are the only source of silkworm feeding to produce high quality cocoons (khosravi and jalali sendi, 2010). these varieties are attacked by different pests around the world but lesser mulberry snout moth, glyphodes pyloalis walker (lepidoptera: pyralidae), is a monophagous pest solely feed on mulberry leaves which has been widely distributed in usa, mexico, india, japan, iran, central asia and azerbaijan (kanchaveli et al., 2009). in 2002, a high population of g. pyloalis was reported thoughout mulberry orchards of northern iran which caused severe damages not only on shortage of available leaves for silkworm but also to transmission of densoviruses and picornaviruses to silkworm as an alternative host (watanabe et al., 1988; khosravi ___________________________________________________________________________ corresponding author: arash zibaee department of plant protection university of guilan rasht-qazvin highway, iran e-mail: arash.zibaee@gmx.com; arash.zibaee@guilan.ac.ir and jalali sendi, 2010). g. pyloalis has five generations per year and the fourth and fifth instar larvae cause the highest damages during cropping season. extensive feed on mulberry and feces remnants are the main damages which significantly reduces quality of leaves (khosravi and jalali sendi, 2010). because of environmental concerns on wide spraying, control of g. pyloalis is based on mechanical and cultural tactics by removing infested leaves and ploughing of soil around mulberry trees in winter although spraying with insect growth regulators (igrs) are inevitable in some cases (khosravi et al., 2014). nematodes are the multicellular organisms which have different life styles in environment i.e. free-living, predator, pathogens of plants, animals and even human (gaugler, 2002). several nematodes may be found on insect orders but a few are able to kill insects. nematode-insect associations are categorized as phoretic, commensalism, facultative or obligatory parasitism (grewal et al., 2006). among nematode taxa showing entomopathogeic characteristics, steinernematide and heterohabditidae have been demonstrated as mailto:arash.zibaee@gmx.com mailto:arash.zibaee@guilan.ac.ir 121 fig. 1 dose-response curve of g. pyloalis exposure to s. carpocapsae. graph and data given were calculated by polo-plus software. the most successful nematodes in biological control (grewal et al., 2006). mortality on host is imposed due to presence of bacteria, xenorhabdus spp. and photorhabdus spp. which have mutualistic symbiosis with steinernematids and heterorhabditids, respectively (poinar, 1990). infection is initiated by entrance of infective juveniles (ijs) though natural openings of body including mouth, anus, and spiracles. then, symbiotic bacteria are released into hemocoel that leads to septicemia and kills host within 24–48 h. almost thee generations are completed within host body, afterward ijs leave host cadaver and seeks for new hosts (dowds and peters, 2002). the immune responses of insects include all processes that protect them against bacteria, viruses, fungi, and parasitoids (lavine and strand, 2002; schmidhempel, 2005). these responses are divided into cellular and humoral immunities in which phagocytosis, nodule formation and encapsulation are devoted to cellular responses while production of reactive oxygen or nitrogen species, antimicrobial compounds and deposition of melanin though prophenoloxidase system are the features of humoral responses (söderhäll and cerenius, 1998; sivajothy et al., 2005). these reactions in addition to evolution of pathogenic routes of entomopathogens contribute in success of microbial control or evasion of target pest from biocontrol agent. agricultural products are subjected to many pests requiring pesticide use to prevent severe damages although these chemicals impose production costs, human or wildlife health and environment pollution. in order to reduce risks of chemical pesticides, the use of biocontrol agents is of interest within organic agriculture. entomopathogenic nematodes have been successful in recent decades due to their wide host range, growth capability on artificial media, stable pathogenicity and incorporation with some chemicals and fertilizers (shapiro-ilan et al., 2017). since control of g. pyloalis though safe procedures is of importance due to environmental concerns, the current study was done to determine pathogenicity of a native entomopathogenic nematode of s. carpocapsae in addition to immune and antioxidant responses of g. pyloalis larvae following immune challenge. understanding the physiological response of g. pyloalis to the pathogenic nematode and the level of infection on the larvae can provide an overview on the effectiveness of microbial control. material and methods insect rearing larvae of glyphodes pyloalis were collected from infested mulberry trees in the campus of university of guilan. specimens were kept at 24 ± 2 °c, 75 ± 5 % of relative humidity and 16l:8d of photoperiod, fed on mulberry leaves within containers (18×15×7 cm) which have been sealed by cheesecloth. leaves were daily replaced with new ones and remnants were cleaned to avoid potential infection. after pupation, males and females were discriminated based on morphology of distal abdomen and transferred to cages for mating. some leaves which their petioles were covered with wet cotton were put in cages as the place of oviposition. once eggs hatched, 1st instar larvae were transferred to rearing containers and provided with fresh leaves till 4th larval instars at the above-mentioned rearing condition (khosravi and jalali sendi, 2010). 122 fig. 2 effect of s. carpocapsae injection on the numbers of total hemocytes (a), plasmatocytes (b) and granulocytes (c) of g. pyloalis. statistical differences have been marked with different letters within each time interval (tukey test, p ≤ 0.05). a a a a a a b b c b a b b c c 0 200 400 600 800 1000 1200 1400 1600 1 3 6 12 24 n u m b e r o f to ta l h e m o c y te s × 1 0 4 time (hours) intact ringer s. carpocapsae a a a a a a b b c b a a b c c 0 10 20 30 40 50 60 70 80 90 1 3 6 12 24 n u m b e r o f p la sm a to c y te s × 1 0 4 time (hours) intact ringer s. carpocapsae a a a a a a a a b b a a b bc c 0 50 100 150 200 250 300 350 400 450 1 3 6 12 24 n u m b e r o f g ra n u lo c y te s × 1 0 4 time (hours) a) b) c) 123 fig. 3 effect of s. carpocapsae injection on phenoloxidase activity of g. pyloalis larvae. statistical differences have been marked with different letters within each time interval (tukey test, p ≤ 0.05) nematode rearing the iranian isolate of steinernema carpocapsae (ira118) was generously provided from insect pathology laboratory of shahid madani university and reared following inoculation on galleria mellonella fabricius larvae at 25 °c for emergence of ijs. emerged ijs were kept at 15 °c for two weeks before main experiments. bioassay of nematode the 4th instar larvae of g. pyloalis were used to determine virulence of s. carpocapsae. this is an iranian native nematode collected from meshkin shah, east azerbaijan with the collection code of ira18. after preliminary assay, the concentrations of 250, 500, 1000 and 2000 ijs per ml were prepared in ringer solution (121.5 mm nacl; 10 mm kcl; 2.1 mm nah2po4; 10 mm nahco3; 0.7 mm mgcl2; 2.2 mm cacl2; ph 6.8). then, 500 µl of the solution containing nematodes was separately pipetted onto filter paper (watman no.1) fitted into a glass petri dish (10 cm). the experiment was done by 150 larvae which 30 larvae were devoted to each concentration in thee replicates including control larvae which received only ringer solution. all petri dishes were kept at 25 °c and the filter papers were received 500 µl of sterile distilled water to keep moisture. mortality of larvae was recorded from 24 h following experiment initiation and prolonged for a week. immune challenge initially, the third thoracic segment of g. pyloalis larvae was sanitized with ethanol solution (70 %), and injected with 1 µl of a solution containing lc30 concentration of nematode prepared in ringer solution which has been calculated based on bioassay experiment. control larvae were injected by ringer only and a group of larvae leaved without any challenged named as intact group. thirty larvae were separately devoted to each treatments including intact, ringerand s. carpocapsae injected in thee replicates. after time intervals of 1, 3, 6, 12 and 24 h post-treatment, hemolymph was collected and immediately diluted in an anticoagulant solution prepared based on azambuja et al. (1991) containing 0.01 m, ethylenediamine tetraacetic acid; 0.1 m, glucose; 0.062 m, nacl; 0.026 m, citric acid (ph 4.6). then, 100 µl of the sample was pipetted onto a hemocytometer and the numbers of total and differentiated hemocytesplasmatocytes and granulocyteswere counted by direct observation under light microroscopy. the experiment was done at laboratory conditions of 25 ± 2 °c, 70 % of relative humidity and 16:8 (l:d) h. phenoloxidase assay phenoloxidase activity was assayed in all treatments according to a method described by wilson et al. (2002). briefly, 10 μl of hemolymph was transferred to a plastic tube (1.5 ml), then 100 μl of ice-cold phosphate buffered saline (20 mm, ph 7) was added and the samples were frozen to disrupt hemocytes. to determine phenoloxidase (po) activity in the defrosted solution, samples were poured into each well of a plate containing 20 ml of 10-mm l-dopa (3,4dihydroxyphenylalanine) as a substrate. after 5 minutes of incubation at room temperature, the absorbance was measured at 492 nm. antioxidant assays sample preparation three groups including 20 larvae were considered as intact, ringer and s. carpocapsae injected (lc30). after 24 hours, the larvae separately selected and homogenized in distilled water using a a a a a a b ab a ab ab d ab a b c 0 2 4 6 8 10 12 14 16 18 20 1 3 6 12 24 s p e c if ic a c ti v it y ( u /m g p ro te in ) time (hours) intact ringer s. carpocapsae 124 glass pestle. the homogenated samples were centrifuged at 20000 g for 20 min at 4 °c. the supernatants were stored at -20 °c for subsequent analyses. catalase assay the reaction mixture contained 50 μl of sample and 500 μl of hydrogen peroxide (1 %) which were incubated for 10 min at 28 °c before recording absorbance at 240 nm (wang et al., 2001). superoxide dismutase assay briefly, 50 μl of sample and 500 μl of reaction solution containing 70 μm of nbt (nitro blue tetrazolium), 125 μm of xanthine, both dissolved in phosphate buffer (20 mm, ph 7.1) were mixed thoroughly and gently shaken. then, 100 μl of xanthine oxidase (5.87 units/ml) dissolved in 2 ml of phosphate buffer were added to the initial mixture and incubation was initiated at darkness for 20 min at 28 °c. afterward, absorbance was recorded at 560 nm (mccord and fridovich, 1969). peroxidase assay thee component of reaction mixture including 50 μl of sample, 250 μl of buffered pyrogallol (0.05 m pyrogallol in 0.1 m phosphate buffer) and 250 μl of h2o2 (1 %) were mixed thoroughly prior to read absorbance at 430 nm every 30 s for 2 min. an extinction coefficient of oxidized pyrogallol (4.5 liters/mol) was used to activity calculation (addy and goodman, 1972). glutathione s-transferase assay the activity of glutathione s-transferase (gst) was assayed based on a method described by habig et al. (1974). briefly, 20 µl of cdnb (1-chloro-2,4dinitrobenzene, 20 mm) and dcnb (1,2-dichloro-4nitro-benzene, 20 mm) were separately added into 50 µl of reduced glutathione solution (20 mm), then 10 µl of enzyme solution was added and the absorbance was read at 340 nm after 5 min of incubation. ascorbate peroxidase assay a reaction mixture containing 50 μl of sample, 150 μl potassium phosphate buffer (67 mm, ph 7.0), 70 μl ascorbic acid (2.5 mm) and 200 μl h2o2 (30 mm) was prepared based on asada (1992). absorbance was recorded at 290 nm for 5 min in continuous manner. glucose-6-phosphate dehydrogenase assay the assay was done using 100 μl of tris-hcl (100 mm, ph 8.2), 50 μl of nadp (nicotinamide adenine dinucleotide phosphate 0.2 mm) and 30 μl of mgcl2 (0.1 m). afterward, 50 μl of water, 50 μl of the sample and 100 μl of gpdh (6 mm) was added to the initial mixture before recording absorbance at 340 nm (balinsky and bernstein, 1963). malondialdehyde assay the concentration of malondialdehyde (mda) was determined based on bar-or et al. (2001) in which 100 μl of 20 % trichloroacetic acid and 50 μl of the sample were initially mixed and centrifuged at 15000 g for 10 min at 4 °c. then, supernatant was mixed with 100 μl of 0.8 % thiobarbitoric acid (tba) reagent and re-incubated at 100 °c for 60 min prior to reading absorbance at 535 nm. mda concentration is defined as the amount of mda produced per mg protein with a molar extinction coefficient of 1.56 × 105 m−1 cm−1. protein assay the amount of total protein was assayed by the method of lowry et al. (1951) using a commercial kit manufactured by ziestchem company (tehan, iran). statistical analysis a complete randomized design was adopted for the experiments. data of bioassay were inserted in polo-plus software to calculated lc values. means were analyzed using tukey’s test and the statistical differences were considered at a probability less than 5 % and marked with different letters. results the used native isolate of s. carpocapsae imposed significant mortality against fourth instar larvae of g. pyloalis. figure 1; shows that the used concentrations led to 10-90 % mortality against the larvae with the lc50 value of 582.9 ijs per ml, confidence limit at 95 % of 359.1-811.5 and slope of 2.33 ± 0.54 2 (p ≤ 1.537, df=2). figure 2; shows the effect of s. carpocapsae at lc30 concentration on the numbers of total and differentiated hemocytes of g. pyloalis larvae. injection of the nematode augmented the number of total hemocytes at the time intervals of 1-6 h compared to intact and ringer-injected larvae, but the total hemocyte count sharply decreased at the two other time intervals (figure 2a). although injection of ringer solution only increased the number of plasmatocytes after 1 h, but nematode induced proliferation of plasmatocytes at all time intervals compared to other treatments with the highest number after 1 and 3 h (figure 2b). the highest number of granulocytes was obtained after 1 and 3 h post-injection while it decreased after these intervals (figure 2c). the larvae of g. pyloalis exposed to experimental treatments showed significant differences in phenoloxidase activity (figure 3). although intact larvae had a steady activity of phenoloxidase at different time intervals but those injected by s. carpocapsae showed an elevated enzymatic activity after 3-12 h post-injection (figure 3). although similar trend was found in the larvae injected by ringer solution, but phenoloxidase activity was significantly lower than nematodeinjected larvae (figure 3). figure 4 and 5; show the effect of s. carpocapsae injection on the antioxidant enzymes of g. pyloalis larvae compared to intact and ringerinjected ones. nematode injection significantly increased activities of superoxide dismutase and catalase compared to intact and ringer-injected larvae while both nematode and ringer injections 125 fig. 4 effect of s. carpocapsae injection on a) superoxidase dismutase, catalase, peroxidase and b) glutathione-stransferase activities in g. pyloalis. statistical differences have been marked with different letters within treatments (tukey test, p ≤ 0.05). led to an equal elevation of peroxidase activity compared to intact larvae (figure 4). although s. carpocapsae injection caused the highest activity of glutathione s-transferase activity using cdnb as reagent, but it showed no statistical differences among intact, ringerand nematode injected larvae using dcnb (figure 4). moreover, activities of ascorbate peroxidase and glucose-6-phosphate dehydrogenase significantly elevated in the larvae injected by s. carpocapsae but no statistical difference was found in ascorbate peroxidase between ringerand nematode-injected larvae (figure 5). finally, intact and ringer-injected larvae showed no statistical differences in the concentration of malondialdehyde but nematode injection significantly led to the highest level (figure 5). discussion entomopathogenic nematodes mainly the members of steinernematidae and heterohabditidae have gained significant attentions because of their efficient potential in biological control of insect pests (shapiro-ilan et al., 2017). this capability results from some features including rapid kill of hosts, searching ability, ease of application, potential long-term effects due to environmental persistence, safety to nontarget organisms and compatibility with several chemical insecticides (koppenhöfer and kaya, 2002; vashisth et al., 2013; shapiro-ilan et al., 2017). previous studies have shown significant variations of host mortality after treatment with entomopathogenic nematodes which has been attributed to host b b a b b a a a a 0 0,02 0,04 0,06 0,08 0,1 0,12 0,14 0,16 intact ringer s. carpocapsae a c ti v it y ( δ o d /m in /m g p ro te in ) treatments superoxide dismutase catalase peroxidase c b a a a a 0 0,02 0,04 0,06 0,08 0,1 0,12 intact ringer s. carpocapsae a c ti v it y ( o d /m in ) treatments glutathione s-transferase cdnb dcnb a) b) 126 fig. 5 effect of s. carpocapsae injection on a) ascorbate peroxidase, glucose-6-phosphate dehydrogenase and b) malondialdehyde activities in g. pyloalis. statistical differences have been marked with different letters within treatments (tukey test, p ≤ 0.05). preference of species/isolates, environmental adaptability and pathogenic mechanisms (shapiroilan et al., 2017). nevertheless, it has been suggested to use indigenous isolates of entomopathogenic nematodes which have led to more efficiency against target insects because of their compatibility to native habitats (griffin et al., 2005; lacey and georgis, 2012). in the current study, a native isolate of s. carpocapsae caused significant mortality on the larvae of g. pyloalis within five days after treatment. our findings showed larval mortality appeared a day post-injection or post-treatment for all concentrations. finally, the lc50 concentration of 582.9 ijs/ml were calculated based on recorded data. although these results should be compared with bioassays using other nematode species/isolates, but the mortality after 24 h and median lethal concentration found here may confirm efficiency of s. carpocapsae (ira18) against g. pyloalis. results of the current study demonstrated induction of immune system of g. pyloalis larvae though proliferation of hemocytes and higher activity of phenoloxidase. although the highest total hemocyte count was recorded one hour after nematode injection then it slightly decreased, plasmatocytes remained the highest number after 1 and 3 h while granulocytes kept the highest numbers for all time intervals. it has been found that releasing symbiont bacteria, xenorhabdus nematophila, of s. carpocapsae is initiated 2 h after incubation in host hemolymph (snyder et al., 2007). so it may be b a a c b a 0 0,1 0,2 0,3 0,4 0,5 0,6 intact ringer s. carpocapsae s p e c if ic a c ti v it y ( u /m g p ro te in ) treatments ascorbate peroxidase glucose-6-phosphate dehydrogenase b b a 0 0,02 0,04 0,06 0,08 0,1 0,12 0,14 0,16 intact ringer s. carpocapsae c o n c e n tr a ti o n ( n m /m g p ro te in ) treatments malondialdehyde a) b) 127 concluded that the initial increase of total hemocyte, plasmatocyte and granulocyte counts in g. pyloalis larvae may be due to recognition of s. carpocapsae within hemocoel and response of hemocytes to possibly encapsulate nematode. in contrast, reduction in numbers of hemocytes may be attributed to secretion of toxic effects of symbiont bacterial secretion against s. carpocapsae. the toxicity may be imposed by lipopolysaccharide, cytolysins, toxins and the pore-forming fimbrial subunit (herbert and goodrich-biair, 2007). these components lead to actin polymerization, destabilizing of cytoskeleton architecture, inhibition of phospholipase a2 and hemocyte apoptosis (kim et al., 2005; li et al., 2009; eleftherianos et al., 2010). li et al. (2009) observed a significant decrease of total hemocyte count in helicoverpa armigera hubner (lepidoptera: noctuidae) following infection with ovomermis sinensis after 4 and 8 h. similarly, lalitha et al. (2018) showed reduction of total hemocyte counts after 3 h of spodoptera litura fabricius (lepidoptera: noctuidae) injection with h. indica poinar et al. due to releasing toxins by photorhabdus spp. phenoloxidases are the significant components of insect immunity to complete nodulation/encapsulation responses, clotting of hemolymph, wound healing and production some toxic elements against invading microorganisms. upon infection, a cascade of serine proteases is activated to initiate activation of prophenoloxidase (zymogen) via proteolytic cleavage at enzyme polypeptide chain (nappi and chistensen 2005). afterward, activated phenoloxidase finalizes nodulation/encapsulation of invading microorganisms by deposition of melanin in peripheral hemocytes (cerenius et al., 2008). the highest activity of phenoloxidase in g. pyloalis was obtained 6 h following s. carpocapsae injection then it sharply decreased to 24 h. similar to hemocyte counts, initial activation of phenoloxidase is related to signal transaction following recognition of nematodes within larval hemocyte which led to increase of hemocyte counts and secretion of phenoloxidase. in contrast, suppression of phenoloxidase at other time intervals may be due to inhibiting signal transaction for immune responses including phenoloxidase by symbiotic bacteria. this phenomenon has already shown by brivio et al. (2002) who reported lipoxygenase-mediated propo activation pathway in galleria mellonella l. (lepidoptera: pyralidae) larvae following s. feltiae infection. oxidative stress refers to production of reactive oxygen species (ros) in response to environmental extremes like temperature, chemicals and microorganisms which should be overcome by host to prevent cell damage, protein denaturation, lipid peroxidation, inhibition of dna replication and mutation (felton and summers, 1995; zhang et al., 2015). in details, organisms, e.g. insects, utilize a set of components including catalases, peroxidases, superoxide dismutases, ascorbate peroxidases, glucose-6-phosphate dehydrogenase and malondialdehyde known as antioxidant components to prevent biological damages. the thee antioxidant enzymes, superoxidase dismutase, catalase and peroxidase, have a sequential activity in which the first one transforms superoxide anions to hydrogen peroxide and oxygen while the two other enzymes break down hydrogen peroxide in stressed tissue (gaetani et al. 1996; halliwell, 1999; zelko et al. 2002). ascorbate peroxidase destroys hydrogen peroxide though concurrent oxidation of ascorbate although glucose-6-phosphate dehydrogenase deals with oxidation-reduction and decontamination of oxidant agents though production of nadph to neutralize ascorbate peroxidase productions (asada 1984). glutathione s-transferase is one of the critical enzymes in detoxifying mechanisms against xenobiotics that removes products of lipid peroxidation or hydroperoxides once oxidative stress is induced within host tissues (rahimi et al., 2018). malondialdehyde is a peroxidation product of unsaturated fatty acids from phospholipids so it is an index of cell membrane damages. in the current study, injection of s. carpocapsae caused induction of antioxidant enzymes in g. pyloalis except for peroxidase and glutathione s-transferase once dcnb was used as substrate. in details, no statistical difference in activity of peroxidase may be attributed to sufficient activity of catalase to remove hydrogen peroxide. moreover, findings on glutathione stransferase indicate involvement one of the isozymes in antioxidant function of the enzyme. finally, induction of malondialdehyde in nematode injected g. pyloalis may refer to cell damaging function of secondary metabolites from symbiotic bacteria of s. carpocapsae. lalitha et al. (2018) believe that production of ros in infected insects with entomopathogenic nematodes is due to synthesis of secondary metabolites from symbiotic bacteria and immune function of host insects to limit microbial growth within hemocoel. these authors reported the highest activities of superoxide dismutase and catalase after 6 h, peroxidase after 24 h and glutathione s-transferase after 9 h following injection with h. indica. krystyna et al. 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responses of propylaea japonica (coleoptera: coccinellidae) exposed to high temperature stress. j. insect physiol. 73: 47-52, 2015. 157 isj 14: 157-164, 2017 issn 1824-307x research rereport molecular cloning and characterization of high mobility group box（hmgb）gene from beauveria bassianainfected silkworm, bombyx mori h chengxiang1,2, w han1,2, l dingding1,2, l ruilin1,2, g xijie1,2 asericultural research institute, jiangsu university of science and technology, zhenjiang 212018, china bchinese academy of agricultural sciences, zhenjiang 212018, china accepted april 18, 2017 abstract the cdna sequence of high mobility group box (hmgb) from bombyx mori was cloned by rapid amplification of cdna ends and submitted to genbank (the accession number was jf969272). the full-length cdna of bmhmgb included 3015 bp, with five exons and four introns. it contained a 313 bp 5′ utr, a 2114 bp 3′ utr with a polyadenylation signal sequence aataaa and a poly(a) tail. it encodes a 195amino acid polypeptide with a predicted molecular weight about 22991.9da and a theoretical isoelectric point 10.00. phylogenetic analysis revealed that bmhmgb is grouped in insect hmgb proteins. bmhmgb was mainly expressed in hemolymph, cuticles, midgut and fat body by rt-pcr. in these tissues, the relative expression of bmhmgb with beauveria bassiana infected silkworm was higher than the normal ones by fluorescent quantitative real-time pcr. therefore, bmhmgb may play an important function in the response to b. bassiana infection of silkworms. key words: bombyx mori; beauveria bassiana; high mobility group box protein; rapid amplification of cdna ends; qrt-pcr introduction high mobility group box (hmgb) proteins are one of the cytokines with diverse functions, exist at the intersection of foreign and endogenous molecules in all eukaryotes (andersson and tracey, 2011; malarkey and churchill, 2012; choi et al., 2016). these proteins have one or multiple hmg-box, which is an unique and versatile dna-binding domain. they can bind many substrates, such as different immunogenic nucleic acids, bended/loopped and unwinded dna, damage associated molecular pattern (damp), toll-like receptor (tlr) (stros, 2010; malarkey and churchill, 2012). as dna chaperones, they also influence many biological processes in chromatin from maintenance of genome integrity to transcription, such as dna replication and repair, recombination, transcription, genomic stability, so they throw a critical vote in decisions of life and death (gerlitz, 2009). hmgbs have another crucial role that they act as an endogenous immune signal to inform other cells that damage or invasion has occurred (yang and tracey, 2010). in the nucleic-acid-mediated ___________________________________________________________________________ corresponding author: hou chengxiang sericultural research institute jiangsu university of science and technology zhenjiang 212018, jiangsu province, china e-mail: cxhou587@163.com innate immune responses, hmgbs act as an universal sentinels, that is all exogenous nucleic acids must bind firstly hmgbs, then be sent to specific pattern recognition receptors and activate the innate immune responses (yanai et al., 2011). they also function as damps, which can activate inflammatory and induce immune responses to protect against infection and promote healing after tissue damage in organisms (manigrassoet al., 2014; choi et al., 2016). the silkworm, bombyx mori, an important economical insects and model organisms of lepidoptera, only has the natural immune system. beauveria bassiana is one of the major fungal pathogens for silkworms and can cause economic damages to the sericultural industry. during the silkworm defense pathogen-infected, immune recognition is the first step of the responses. in our previous studies, we found a different expressed gene bmhmgb with recognized function (hou et al., 2014). to further study it, we first cloned the full-length cdna of bmhmgb by rapid amplification of cdna ends. then the expression characteristics of bmhmgb was analyzed in different tissues and at different time in the same tissue after b. bassiana infected. these results provide some useful information for further study of the immune recognition mechanism of silkworm to fungi infection. https://www.ncbi.nlm.nih.gov/pubmed/?term=choi%20hw%5bauthor%5d&cauthor=true&cauthor_uid=27007252 https://www.ncbi.nlm.nih.gov/pubmed/?term=choi%20hw%5bauthor%5d&cauthor=true&cauthor_uid=27007252 158 materials and methods silkworm and b. bassiana strain in our study, the silkworm strain p50 and b. bassiana hn6 were provided by the sericultural research institute of the chinese academy of agricultural sciences. the larvae of silkworm were reared with mulberry leaves at standard temperature and humidity until the newly exuviated fifth instar larvae for the experiments. hn6 were originally inoculated from the cadaver of the infected silkworm to potato dextrose agar (pda), then isolated and cultured on pda for about 10 days at 25 °c. inoculation of b. bassiana conidia of b. bassiana were scraped from pda and diluted to 3×108 spores/ml using sterile distilled water (containing 0.01 % tween-80). the newly exuviated larvae were immersed in the conidia solution for 15 s. the control larvae were immersed in sterile distilled water (containing 0.01 % tween-80) for the same period. then regulated temperature and humidity to 28 °c and 95 % rh, the larvae were reared in these conditions. collection tissues the different tissues were collected from both the hn6-infected and control larvae at 8 h post-inoculation (hpi). first, hemolymph was collected, it was directly mixed with pre-joined trizol reagent (invitrogen) in an eppendorf tube. then the larvae were rapidly dissected to collect gonad, martensite, mid-gut, mid-silk gland, fat body and cuticles. these tissues from 10 15 larvae of fifth instar larvae were pooled as one sample. the tissues were quickly washed in diethylpyrocarbonate -treated pbs solution (137 mm nacl, 2.68 mm kcl, 8.1 mm na2hpo4, 1.47 mm kh2po4, ph 7.4) and immediately frozen in liquid nitrogen before stored at -80 ℃ refrigeratory. the collected time was 8, 10, 12, 15, 18, 24 and 36 hpi, respectively. the larvae hemolymph of 36 hpi was used to cdna cloning. cloning full-length cdna and sequencing of bmhmgb total rna was extracted respectively from different tissues by using a rnipuretotal rna rapid extraction kit (beijing boling kewei biotechnology) according to the manufacturer’s protocol. then the degraded condition of trna and its concentration was determined separately. subsequently the eligible trna from hemolymph of 36 hpi was used to synthesize the first-strand cdna according to the protocol of primescript™ rt reagent kit with gdna eraser (takara). full-length cdna of bmhmgb was synthesized using the first-strand cdna as templates and smarttm race cdna amplification kit (clontech). the specific primers for 5' and 3' race were designed according to the est sequence revealed in our previous study. 3'-race-ready cdna was prepared according to the clontech kit protocol. the specific primer p1 (5'-cgttgactgacaatgaagaagcgaacagttg3', 5’-tacttatgtcaacggacactgaaggtcgga-3’, 5’-cgttgactgacaatgaagaagcgaacagttg-3) and p2 (5'-caactgttcgcttcttcattgtcagtcaacg-3') were separately used for amplification of 5'and 3' -end in the race reactions. the pcr amplification progress was: 95 °c for 1m; 28 cycles of 95 °c for 15 s, 68 °c for3 min; then 72 °c for 10 min. the pcr products were gel-purified and cloned into pmd19-tsimple vector (takara). the positive recombinants were selected by anti-ampicillin after being transformed into escherichia coli top10, then pcr screening was done with m13 primers (f: 5'-tgtaaaacgacggccagt-3', r: 5'-caggaaacagctatgacc-3') and then sequenced. the sequences of bmhmgb were assembled with the obtained fragments from race and analyzed at the national center for biotechnology information (http: // www. ncbi. nlm. gov/blast). the amino acid sequence of bmhmgb was analyzed with the expert protein analysis system (http://www.expasy.org/) and smart program (http://smart.embl-heidelberg.de/). the work of multiple protein sequences and phylogenetic tree was employed by the clustal w and neighbor-joining method in mage 6.06 software. bootstrap trials were replicated 1000 times to derive the confidence value for the phylogeny analysis. rt-pcr rt-pcr method was used to detect the expression condition of bmhmgb in different tissues of silkworm. 1μl (500 ng) trna, extracted from different tissues, was used as template to synthesize cdna using the prime script tm rt reagent kit (takara). the pcr reaction was performed to describe the expression in these tissues or not. quantitative real-time pcr (qrt-pcr) of bmhmgb the expression level of bmhmgb in different tissues of silkworm was detected by qrt-pcr method. qrt-pcr was performed using 2 μl of diluted cdna in each 20 μl reaction mixture according to the manufacturer’s instructions of the sybr premix ex taqtm (takara). cdna templates were synthesized from the hemocyte, cuticles, fat body and midgut of b. bassiana -infected and control larvae at 8, 10, 12, 15, 18, 24 and 36 hpi. specific primers for bmhmgb (f: 5'-acagtctgaactgcttgatcca-3', r: 5'-aatcaaccagtgctccccaa-3') and β-actin (an endogenous control gene, f: 5'-aatggctccggtatgtgc-3', r: 5'-ttgctctgtgcctcgtct-3') were designed by primer premier 5.0 software. reactions were run in triplicate on a 7300 sequence detection system (applied biosystem). the thermal cycling parameters of qrt-pcr was: 95 °c for 30 s; 40 cycles of 95 °c for 5 s, 60 °c for 31 s; 95 °c for 15 s, 60 °c for 60 s, 95 °c for 15 s. each tissue contained the control and affected sample at different times and its β-actin was run in the same plate. the relative expression levels of bmhmgb gene were normalized using the ct values obtained from the β-actin amplification run in the same plate. the mean value ± sd and 2−δδct method were used to analyze the relative transcript levels of each time point. http://www.ncbi.nlm.gov/blast http://www.ncbi.nlm.gov/blast 159 1 a c a ata a g c g c t g a a at c g t g t g c t c g t t t g tat t g a g a at t t ta g t t g t c g c a g a at c a 6 1 g t c aa at c a a g a a ag c g at c a a a a c a at c a ac a a g c g a ac g g tat t c a ac a g ag t c at c a 1 2 1 g t c g at c c aac a ac a ac ag c ag c ag g ag c a a a at c a a ac tat t c a ac ag c a ac a a ag t c a 1 8 1 at tac a a c a g c ag t t g c ag c a ac a ac ag a at c t c c ag c a ag c t t t g c a ac ag c ag a ac c a 2 4 1 at c tt t g c ag c ag t c g t tac aac a ac a ac a a c a ac ag c ag c ag c a ag a ag ag c ag c a ac c 3 0 1 g ac c c t g c ag c ag at g c tac ag c ag c at c a ac ag c a g c ag c ac c a ag g c t tac a ac aa ac 1 m l q q h q q q q h q g l q q t 3 6 1 t tt g c ag c ag ac at t g c a ag t t ag t c ag g c t c a ag c c c a ag c a at ag c g c a ag c c c a ag c l q q t l q v s q a q a q a i a q a q a 4 2 1 ag c c t t ac ag c ac c a ag t t g c t c aat c a at ac ag c ag c a a c a ac a a ac t c t ac a ag a ac a a l q h q v a q s i q q q q q t l q e h 4 8 1 c at t c a ag c t g t t c ag c a ac a ac ag at ac a g g c ag c at t a c ag a g g c a at c t g c c ac t c t i q a v q q q q i q a a l q r q s a t l 5 4 1 ac ag g ag c tg c ag c aac ag g c ac ag c ag c aag c t tt ac ag c aag c aac ag t aa at a ag g c q e l q q q a q q q a l q q a t v n k a 6 0 1 c ag g at g c c c c g t t c a ag g c c at ac a a c a a g c c c c g c g g t c g c at g ac ag c t t at g c a t t r m p r s r p y n k p r g r m t a y a f 6 6 1 c t t t gt g c ag ac g t g c c g ag a ag a a c ac a a g a ag a a at a c c c t g at g t c ag t g t t at at t f v q t c r e e h k k k y p d v s v i f 7 2 1 t gc ag c att ct c g aaaa ag t g c gc ag ag ag g t gg aat ac a at g t c g g aaaa ag a aa a ac a a a f s k k c a e r w n t m s e k e k q 7 8 1 gcgg tt cc at g ag atg g ctg a ac ag g ac aagc at cg att c g actt g g ag atg c ag aac t a r f h e m a e q d k h r f d l e m q n y 8 4 1 tgtacc acc aaagg ac atg a aggtc ag aggg cgaaag agg c agc agt atg aaag accc t aa v p p k d m k v r g r k r q q y e r p * 9 0 2 t g c ac c a a a g c g c t c g c t a t c ag c at t c t t t t t g t t t t g c a a c g at g a a c g t t c g a a g g t 9 6 2 g aaag c t g g c aac c c ag ag t ac ac c at g g g c g at at t g c g aag g a ac t c g g c ag ac g t t g 1 0 2 2 g g cg g c cg c tg at c c g g ag ac t aag g c t a a at at g ac g c g c t at c t g aa aa ag ac a ag g c 1 0 8 2 g c g at at g at ag g g a a at g ac ag c g t ac a a g aa ag g t c c g t t ag c t c ag c c g cc g c c ac a 1 1 4 2 g c c g cc t g tc g t c cc c g c c at c g ag g ac g a g g g c g g ag ac t t c g at g c ag ac g aa g a at a 1 2 0 2 c aat t g a a at a at g t g at c g c t g t c g at ac ag c c t a a a at c ag g ac ag ag ag at t t a ac t 1 2 6 2 c g at t t t g a a g a t g a a a g t g c g c g a c ag t c t t t c c c c c t t t t t c c a g c c a a a t g t at a a c 1 3 2 2 g c a a t t g c t g t a t a c a t t a c t g a t t t g t t t t g t t c a t t a t t c a g g g g a t g a t t t a t t t t t 1 3 8 2 g t g c t g c g g g t t t g g g g a g c a c t g g t t g a t t g a g t t t a a t a g a a a t t g t a a t c t t g t t t t 1 4 4 2 t g t ta g ag g g g g g g t c g t c g t c g t c at t c a a g g a a g g g t c g ta a at t ta a g g a a g c g t t t 1 5 0 2 t g ac g tac g at c a g g t g t c ag a g ag g c t g g g t c t t t g a ac g a a a g at t g ag g a g t g a at c 1 5 6 2 t t c t g t t c t g ta g c g t g t tata g t t t t t ta c a a at t ta at t c t g at t c a c a g at t t ta c t 1 6 2 2 tata c t t t c a a ata c t c a a a atat t t t t g ta c g at t t t t t g t t t g t c at c t g a g a a at g a 1 6 8 2 g g t g t c a a ata g t t t tat t ta c a g c t ta a a a a a a c g g c c a a a c a c c tat g t c g t t t ta a a 1 7 4 2 a a a a a a a c c t c ta at t t tat g tat t c t t c a a c a a ata a at g at tat c t t ta g t t c t c a g a 1 8 0 2 t g a c g a at g t t g a a c a g at t t t g a at g a c a a a a at t t ta a c at g a a a at g a c t t t ta at t 1 8 6 2 g t t g c a a a c t t t t g ta a a c g a a a c t g a c g t tat t t tat g at c t ta c g t t g a c t g a c a at g 1 9 2 2 a a g a a g c g a a c a g t t g at t t t t t t tat t t t t c t t g ata c g a at t t c t t c g a g ata c a a g g 1 9 8 2 a a a a a a g ta g a c a atat g a c a at t g g c a g t ta a a a a a a a at t g t c at t c t c c c g c c a a a a 2 0 4 2 tat c g ta c c at g a a a g ta a a at c a a a a a g t g t c at g g c g c c c a a a a c at g t c t t g c g t c t 160 2 1 0 2 a c t tat g t c a a c g g a c a c t g a a g g t c g g a a at g g t g t g g n c c c t g t g g t t ta ata a a at c 2 1 6 2 g ta at ta c a a g c a at at tat c a g g a a c a a at c g at a c a g a c g t t t t c a g tat c a c g tata 2 2 2 2 at g ta a g t c a a a a a g tat t t c t tat ta a g t ta g c ata g a c g ta a g g a g g t c g g g a g c g a a 2 2 8 2 t g tat t g t t g t t g t t t t g t g t c g at t at g t at a g t t a a at t at at t c c t t t at t a a at at 2 3 4 2 g ta c t t t t g t t t g at t t c g atata at t tat a a at c g c g t t ta g t ta a a at at ta a g t ta c 2 4 0 2 g a a at t t tatat t t t g t g a at t g t t t g t t ta g t g a g t c t g t c t c g c g c c at t t c t g t t t g 2 4 6 2 at g c at g c ata c c a g c g at g t g t t t t t g ta c ata a a a c g a c t g c a a c c c g t g ata g ta c t 2 5 2 2 ta ata g ta g c t g c g c ta g g ta c ta c a atata g g t t at g tatat g at t t c t t c t g a a a a at 2 5 8 2 t c a at t c c a a at g a a at ta at t c g c t c at t g t t t t t c g t t t a c t g t c t g at t g t t c g t t t 2 6 4 2 g a a at c c g t t ta c g t t t t c a a a g a atata at t t c a a g tat t t t g a a a g a ata a a c g a ata 2 7 0 2 g t c c t c t t c t g ata a a a c ata c g a at g at g g t g t t c a a a c c at t t g atat g c ta a g t t c t 2 7 6 2 t t t ta a g t g c ata at a at g c ata a c t t g a a a c a c a c g t g t c g g c g t c t t ta c t c g g c a g a 2 8 2 2 a g a c t g t t ta a atat c t t t t t c g a a c g g a c at t a c c g ta a atata atat g tat g ta c c a g 2 8 8 2 t t t t t t t ta g at tat ta a a a a a a a c a c g a g at t t g a c c c c g c t t g g a g a g g c a a a g c g at 2 9 4 2 g a a g t g at t t ta a g t t g tat c g a a a a g g g c a at t g t t t ta a at t t g ta a a a c g c t c a c t g 3002 taccaaaaaaaaaa fig. 1 the full-length nucleotide sequence information of bmhmgb. the initiation and stop codons are boxed, the trailing signal (aataaa) is underlined. black area is the hmgb domains. green area is the coiled coil region. red word is n-myristoylation site (no. 12-17). results analyse of the bmhmgb full length and structure the full length cdna of bmhmgb protein was obtained by race, then sequenced, spliced and uploaded to genbank (accession no. jf969272). it contained a 313 bp 5'untranslated region (utr), a 2114 bp 3' utr and a 588 bp open reading frame encoding a 195 amino acids protein with a predicted molecular weight about 22991.9 da and a theoretical isoelectric point 10.00 by protparam software (https: //www.expasy) (fig. 1). a putative polyadenylation signal aataaa was detected in the 3'utr 310 bp upstream from the poly(a) tail. at the no.106-178 amino acid sequences of the polypeptide chain, there was a hmgb domain which is a dna binding site domain and this protein belongs to the hmgb family. there was a coiled coil domain at the no. 41 96 amino acid sequences (fig. 2). the sequence contained a n-glycosylation site (no. 12-17), three protein kinase c phosphorylation sites (no. 120-122, 140-142, 151-153) and five tyrosine kinase phosphorylation sites (no. 52-55, 75-78, 120-123, 149-152, 151-154) (https://blast. ncbi. nlm. nih. gov, http://smart.embl-heidelberg.de) (fig. 1). analysis of the amino acid sequence by tmpred revealed that, this protein had no remarkable transmembrane region. aligning the full cdna of bmhmgb to the genome sequence of b. mori, it was contained on the bm_scaf1_contig476, this contig has 44217 bp and the accession number is babh01000476.1. it showed that bmhmgb cotained five exons and four introns and each exon-intron boundary conforms with “gt-ag” rule (http://www.expasy.org/). fig. 2 domain architecture analysis of bmhmgb（green area is coiled coil region） javascript:domwin(6) 161 fig. 3 phylogenetic tree of bmhmgb and other homologous protein. the analysis of homologous sequences the phylogenetic tree was constructed according to the amino acid sequences of hmgb homologous genes by mage 6.06 (fig. 3). the results showed two separate clusters of vertebrate and invertebrate hmgbs, bmhmgb was grouped in insect hmgbs of the invertebrate group. in the invertebrate group, some insects which should live in water for some period, including anopheles gambiae, culex quinquefasciatus, aedes aegypti which showed high identity (75 %, 69 %, 66 %) with bmhmgb. confirmation of infection at approximately 48 hpi after b. bassiana spores infected the silkworm larvae, oily spots appeared on their body, which is one of the typical symptoms of the white muscardine disease. all of the infected larvae died in 60 hpi. the disease was further confirmed by the observation of hypha in hemolymph under a microscope and the appearance of the symptom of muscardine. expression of bmhmgb in different tissues the test result of rt-pcr revealed that bmhmgb was expressed in hemolymph, cuticles, malpighian tubule, mid-gut, fat body, mid-silk gland, post-silk gland, and gonad. in contrast, it had lower transcript level in gonad and malpighian tubule. it mainly expressed in hemocyte, cuticles, fat body and midgut (fig. 4). fig. 4 the expression of bmhmgb in different tissues. 1. hemocyte 2. cuticles 3. fat body 4. malpighian tubule 5. gonad 6. midgut 7. mid-silk gland 1 2 3 4 5 6 7 ←β-actin ←bmhmgb 162 fig. 5 the relative expression level of bmhmgb in normal tissues and b. bassiana-infected tissues (a, b ,c, d are hemocyte, cuticles, fat body and midgut, separately. ◊ is infected tissues, × is normal tissues. 1-7 is different time points post-inoculation, namely 8, 10, 12, 15, 18, 24 and 36 h). the relative expression levels of bmhmgb gene at each time points were normalized using the ct values obtained from the β-actin amplifications run in the same plate. all samples were tested in triplicate. the mean value±sd was used for the analysis of relative transcript levels for each time point using the δδct method. the infected and control individuals are blue and red, respectively. expression difference of bmhmgb in control and b. bassiana-infected tissues of silkworm according to the result of rt-pcr, the tissue-specific expression of bmhmgb transcripts at different times was examined by qrt-pcr (fig. 5). between the normal and infected tissues, the expression variety of bmhmgb was significantly different. in the control tissues, there was almost no variety in different times. while in the infected ones, there was significant variation. the relative expression of bmhmgb in the infected tissues was higher than the control ones, especially with the extension of the infected time. for example, at 24 hpi, the relative expression levels in the fat body was 6.11-fold, in the cuticles was 8.34-fold, in the midgut was 10.31-fold and in the hemolymph was 21.71-fold. discussion beauveria is an important entomogenous fungi with a very broad host range, high pathogenicity and adaptability. some beauveria species have a wide use in the biological control of agricultural and forestry pests, but some threaten severely to silkworm and other economic insects. so far, the mechanism of the silkworm how to recognize fungi and initiate immune responses against b. bassiana infection is still poor. in our previous study, we screened and identified bmhmgb, an up-regulating gene which encoding high mobility group box protein (hou e al., 2011, 2014). hmgbs are one kind of multifunctional proteins, architectural chromatin nuclear proteins which have been implicated as a mediator or alarmin of inflammatory and autoimmune diseases in multicellular organisms (voll et al., 2008). they act as an innate alarm and trigger in the initiation of host defenses or tissue repair, and they may be an integrators of signals that maintain the peace and stress, life and death (bianchi and celona, 2011). they participate the activation of tlrs and other cytosolic receptors in immune response (tian et al., 2007; kazama et al., 2008). in these activation progress, hmgbs act as an universal sentinels, that is all immunogenic nucleic acids of the exogenous microorganisms first be promiscuous sensed and binded by hmgbs, then be discriminative sensed by specific pattern recognition receptors and activate immune responses of host. hmgbs shuttle continuously between nucleus and cytoplasm to hunt the invading nucleic acid of virus (bonaldi et al., 2003). they bind to nucleic acid without specific sequences, so it is very difficult to evade hmgbs surveillance for virus (bianchi and celona, 2010). if 163 hmgbs knockout, the activation of tlr3, tlr7 and tlr9 by their cognate nucleic acids will be severely impaired so the cells can’t sense the invasion of pathogens (wang, 2009). now they have attracted considerable attention due to they can be recognized by various cell surface receptors and their potent pro-inflammatory activities associated with diverse and major human diseases (andersson and tracey, 2011; venereau et al., 2012; yang et al., 2015; eunjin et al., 2016). hmgb proteins include single or multiple hmg boxes. b box is a domain of induced inflammatory response and can efficiently activate macrophages to release tumor necrosis factor and other cytokines, while box a antagonizes it (li et al., 2003; andersson and tracey, 2011). when the host cells are exposed to pathogen-derived molecules, hmgbs can be actively secreted by immunological cells, or passively released by injured, apoptotic or necrotic cells (andersson et al., 2000; andrassy et al., 2008; tsung et al., 2005). the passively released hmgb can induce an inflammatory response, promote tissue repair and angiogenesis (yun et al., 2013). the innate immunity of insect emerges as the defensive frontline that coordinates the interaction between host and pathogen. insect can use apoptosis as another defense strategy, while pathogen synthesize anti-apoptotic proteins like p35 to prevent host cell death against its defense (narayanan, 2004). so, it is important to understand the interaction between host and pathogen, the defensive mechanisms about host response infection and the anti-infective mechanisms of pathogen. these are important for better controlling pathogen spread, researching efficient utilization of entomopathogenic fungi and developing new drugs. in our study, the relative expression of bmhmgb in the beauveria -infected tissues were higher than that of the control ones, especially in the hemocyte and midgut. among of these tissues, there was a remarkable difference in the hemocyte. at 24 phi, the relative expression of the infected-hemocyte was approximately 21.71-fold of that in the normal one. we suppose that after the invasion of b. bassiana, the response of natural immunity of host was activated causing the immune cells to become active, infected and apoptotic cells passively released hmgbs which led the transcript level of bmhmgb in infected tissues was increased. this may be one of the reasons that led the dysfunction of host and eventually caused its death. in summary, during beauveria infect the silkworm, a series of physiological and pathological changes and immune responsive reactions take place in the host. as a corresponding result, the transcript levels of some related genes also change. according to our study, we suppose that bmhmgb may play an important role in the interaction of silkworm and beauveria. further functional experiments of bmhmgb should be taken. acknowledgments this work was supported by the national natural science foundation of china 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https://www.ncbi.nlm.nih.gov/pubmed/?term=herrmann%20m%5bauthor%5d&cauthor=true&cauthor_uid=18300566 https://www.ncbi.nlm.nih.gov/pubmed/?term=kalden%20jr%5bauthor%5d&cauthor=true&cauthor_uid=18300566 https://www.ncbi.nlm.nih.gov/pubmed/?term=zhang+s%2c+zhong+j+and+2009 34 isj 13: 34-43, 2016 issn 1824-307x research report the immunosuppressive effects of continuous cpg odns stimulation in chinese mitten crab, eriocheir sinensis d zhao 1 , l song 2 , r liu 3 , z liang 3 , l wang 3 , m sun 3 , b zhu 1 1 dalian polytechnic university, dalian 116034, china 2 key laboratory of mariculture & stock enhancement in north china’s sea, ministry of agriculture, dalian ocean university, dalian 116023, china 3 key laboratory of experimental marine biology, institute of oceanology, chinese academy of sciences, qingdao 266071, china accepted february 4, 2016 abstract cpg oligodeoxynucleotides (cpg odns) have been widely used as a novel vaccine adjuvant in mammals due to its immune protection, long effectiveness and safety. in the present study, the long-term immune effects as well as immunosuppression of cpg odns was evaluated by comparing the immune parameters of chinese mitten crab, eriocheir sinensis after continuous or interval feeding with cpg odns-supplement diet for 21 days (designated as cc and ci group, respectively). in the ci group, the mrna transcripts of estolls (estoll1 and estoll2) and esmyd88 (adaptor molecule) in hepatopancreas maintained at a significantly higher level (p < 0.05) compared with the cc group after 7 days and 14 days feeding, while there was no significant difference between them at the 21st day. moreover, after a significant increase at 7th day, the expression level of eslitaf mrna [lipopolysaccharide-induced tumor necrosis factor (tnf)-α] in cc group decreased at 14th-21st day, while that in ci group kept increasing at 14th day, followed by a decrease at 21st day. the tnf-α in plasma of cc group was abnormally increased at the 21st day (p < 0.05) in cc group compared with the significant raise at 7th-14th in ci group. moreover, the phagocytic activity and reactive oxygens (ros) level of hemocytes in continuous cpg odns feeding crabs were significantly lower than those in interval feeding crabs. these results indicated that long-term and continuous cpg odns stimulation could reduce the activation of pro-inflammatory cytokine production, hemocyte phagocytosis and ros generation, displaying immunosuppressive effects on the immune system of crabs. key words: eriocheir sinensis; cpg odns feeding; immunosuppressive effects; toll-like receptor pathway; tumor necrosis factor-α; phagocytosis introduction dna from bacteria has been demonstrated to induce the immune response of hosts by a specific structure termed cpg motifs (yamamoto et al., 1992; tassakka and sakai, 2002). the cpg motifs possess typical palindromic sequences with unmethylated cytosine and guanine triphosphate deoxynucleotides (cpg odns), and they are important immunomodulators to induce or enhance various immune responses (klinman et al., 1996). in ___________________________________________________________________________ corresponding authors: beiwei zhu dalian polytechnic university 1qinggongyuan, dalian 116034, china e-mail: zhubw@dlpu.edu.cn linsheng song dalian ocean university 52 heishijiao street, dalian 116023, china e-mail: lshsong@dlou.edu.cn, lshsong@qdio.ac.cn vertebrate, cpg odns have been used as a kind of therapeutic agents to prevent host from various pathogens. after internalized by target cells, cpg odns could be recognized by toll-like receptors, and trigger various immune responses in mammals, such as phagocytic activities, the generation of reactive oxygen species (ros), and the production of pro-inflammatory cytokines (stacey et al., 1996; klinman, 2004; aguilar and rodriguez, 2007). for instance, after interacting with human tlr9, cpg odns activate the myd88 dependent tlr pathways, resulting in the production of immune factors, such as ifn-α and tnf-α. cpg odns is not only widely applied as effective vaccine adjuvants for mammalian models, its immunoenhancement effect have also been observed in some aquatic animals (stacey et al., 1996; zhang et al., 2010). accumulating evidences have demonstrated that cpg odns can effectively mailto:lshsong@dlou.edu.cn 35 activate the innate immune response in aquatic animals. for instance, cpg odns could induce phagocytic activity and respiratory burst activities of kidney cells and the lysozyme activity in serum of cyprinus carpio (tassakka and sakai, 2002). cpg odns have also been reported to activate macrophages and increase levels of superoxide anion, hydrogen peroxide, acid phosphatase (acp) and the bactericidal activity in ctenopharyngodon idellus, which was consistent with the results from paralichthys olivaceus (lee et al., 2003; meng et al., 2003). in addition, cpg odns could activate the prophenoloxidase (propo) system in macrobrachium rosenbergii (chuo et al., 2005). in our previous studies, cpg odns were found to promote regeneration of circulating hemocytes, and enhance the phagocytosis and ros production in litopenaeus vannamei (sun et al., 2013a). although immunostimulants can improve the resistance of hosts against pathogens, the immunosuppression has also been observed in some aquatic animal because of the abnormal administration of immunostimulants. for instance, long-term oral administration of peptidoglycan could decrease immune response of rainbow trout as well as catfish against the infection of vibrio anguillarum (matsuo and miyazono, 1993). β-glucan or glycyrrhizin could cause the immunity fatigue in shrimp after continuous applying into the diet (bai et al., 2010). cpg odns has been considered as one of prospective immunostimulants in aquatic animals. to our knowledge, recently studies are mainly focused on its immune stimulation mechanism as well as its short-term effects, while the dysimmunity of its long-term stimulation is still not well known. chinese mitten crab, eriocheir sinensis, is an important economic crustacean in china (chen et al., 2013). in recent years, massive mortalities of crabs have caused decline of productions and huge economic losses by infections of various pathogens (wang and gu, 2002; bricknell and dalmo, 2005; wang, 2011). immunostimulants can directly initiate the innate immune system of hosts, which would be benefit for invertebrates lacking the adaptive immunity (bricknell, 2005; ringø, 2011). the use of biological immunostimulants such as cpg odns is an innovative approach to substitute antibiotics for disease control in aquaculture (zhang et al., 2010). in our previous research, the enhancements of immuno-protection induced by cpg odns were observed in crab (sun et al., 2013b). in the present study, cpg odns was added into the dietary and fed crabs with two feeding strategies (interval and continuous) for 3 weeks. the mrna expression levels of tlr pathway related genes (estolls, esmyd88, tnf- transcription factor eslitaf), and the release of tnf-α in plasma as well as the ability of phagocytosis and generation of reactive oxygen species (ros) were examined to explore the effects of long-term cpg odns administration in crabs, hopefully providing theoretical guidance for the application of cpg odns in aquaculture as an immunostimulant. materials and methods crabs and cpg odns chinese mitten crabs, eriocheir sinensis, weighing approximately 20 g, were collected from a commercial farm in lianyungang, china, and cultured in flat-bottomed circular tanks (80×90 cm) at 18-23 ℃, ph 7.3-7.5, for a week before processing. cpg odns used in the present study were synthesized by sangon biotech co. (china) containing five cpg rich fragments (fig. 1), which were previously found effective to mammalian and aquatic animals (sun et al., 2013b, 2014). the cpg odns were sub-cloned into the plasmid puc57, and transformed into escherichia coli. the recombinant plasmids were extracted and purified by using alkaline lysis, then heated at 100 ℃ for 15 min and cooled down rapidly for fragmentation (sun et al., 2015). the linear and fragmented dna was then freeze-dried and stored at -80 ℃ for the further experiment. fig. 1 description of tandem odns sequence. five cpg odns in this figure previously used effectively in mammalian and aquatic animals were designed to connect one another in series to form a cpg-rich fragment in the present study. 36 table 1 the composition of the basic diets for crab diet preparation the ingredients for the basic diet of crabs were shown in table 1, and they were mixed evenly following the reported proportion (sun et al., 2013b). the linear and fragmented cpg odns were dissolved in the double distilled water and added into the basic diet at a final proportion of 40 mg kg -1 . the ingredients of the diet were well mixed and extruded by a pellet extruder. the pellets were dried by the microwave oven and stored at 4 ℃. cpg odns-supplement diet feeding experiments, hemolymph and tissue collection a total of 135 crabs were randomly selected from the flat-bottomed circular tanks and equally divided into 3 groups with same conditions as described above. in the first group, crabs were continuously fed with the basic diet during the whole experimental period (21 days) as a control (named b group). the crabs in the second group were alternately fed with the cpg odns-supplement and basic diet according to a modified interval strategy (sun et al., 2013b), 4 days feeding of cpg odns containing diet following 3 days feeding of basic diet (named ci group). in the third group, crabs were continuously fed with the cpg odns-supplement diet every day (named cc group) (fig. 2). all the crabs were fed twice a day to ensure the repletion at 8:30 a.m. and 6:30 p.m., and the water was changed every two days. six crabs were randomly sampled from three groups at the 0th, 7th, 14th and 21st day after feeding. the hemolymph (about 2 ml each crab) was collected from the last walking legs using a syringe (5 ml) with 2 ml pre-cooled anticoaglulant solution (1l: 29.835 g nacl, 18 g glucose, 38.426 g sodium citrate, 8.823 g trisodium citrate, 3.7224 g edta·2na, ph 7.3) and immediately centrifuged at 800g, 4 ℃ for 10 min to harvest hemocytes for determination of phagocytosis and ros. the supernatants (plasma) were stored at -80 ℃ for subsequent enzyme activity examination. hemolymph from two crabs was mixed as a single sample and there were three biological replicates for each group. the hepatopancreas was collected from crabs and added into 1.5 ml microcentrifuge tube with 600 μl trizol reagent (takara, japan), stored at -80 ℃ for rna extraction. quantification of estolls, esmyd88 and eslitaf expression by quantitative real-time pcr total rna was extracted from hepatopancreas samples using trizol reagent (takara, japan) according to the manufacturer’s protocol. the fist-strand cdna synthesis was carried out based on m-mlv rt usage information using the rq1 dnase (promega, usa) treated total rna as a template and oligo (dt)-adaptor as a primer (table 2) (invitrogen, usa). the synthesis reaction was performed at 42 ℃ for 1 h, terminated by heating at 95 ℃ for 5 min. the cdna mix was diluted to 1:30 and stored at -80 ℃ and quantitative real-time pcr fig. 2 the feeding strategy diagram. the crabs of basic group were continuously fed basic diet for 21 days (b group). the one of experiment groups was fed using the interval feeding method, which was 4 days feeding with cpg odns-supplement diet following 3 days feeding with basic diet for 3 weeks (ci group). another experiment group was continuously fed cpg odns-supplement diet until end of the experiment (cc group). ingredients composition (g/kg) ingredients composition (g/kg) soybean meal 250 phosphatidylcholine 5 peanut meal 50 cholesterol 5 shrimp meal 100 choline chloride 5 wheat flour 80 vegetable oil 20 corn flour 140 nah2po4·2h2o 2 fish meal 300 na2hpo4·12h2o 3 multi-vitamin 10 ca(h2po4)2·2h2o 3 vitamin c 2 sodium alginate 10 glycine 15 37 (qrt-pcr). the mrna expression levels of estoll1 (jx295852), estoll2 (agt21374.1), esmyd88 (aim45535.1) and eslitaf (lipopolysaccharide-induced tnf-α factor, kf892539) in hepatopancreas were detected by qrt-pcr in an abi prism 7500 sequence detection system (applied biosystems, usa). the specific primers of estoll1 (p1 and p2), estoll2 (p3 and p4), esmyd88 (p5 and p6), and eslitaf (p7 and p8) were used to amplify the corresponding products. the β-actin gene fragment from e. sinensis, amplified with primers p8 and p9, was chosen as a reference gene for internal standardization (table 2). the assay was carried out in a total volume of 10 μl, containing 5.2 μl of sybr green mix (takara, japan), 0.2 μl of each primer (10 μmol l -1 ), 1 μl of the 30 times diluted cdna, and 3.4 μl of depc-water. the thermal procedure for the qrt-pcr program was 50 ℃ for 2 min, 95 ℃ for 10 min, followed by 40 cycles of 95 ℃ for 15 s and 60 ℃ for 60 s. dissociation curve analysis of amplification products was performed to confirm that only one pcr product was amplified and detected. after the pcr program, the data were analyzed using abi 7500 sds software v2.0 (applied biosystems, usa). the relative expression of esmyd88, estoll1, estoll2 and eslitaf gene was analyzed by the 2 -δδct method (livak and schmittgen, 2001). all the data were given in terms of relative mrna expressed as mean ± s.e. (n = 3). measurement of tnf-α level in plasma tnf-α activity in plasma was determined by using the commercial fish tnf-α elisa kit (baolai, china) following the manufacturer’s instructions. briefly, the 96 wells plates coated by the purified fish tnf-α antibody, were incubated with 10 μl plasma (diluted 5-fold) from different groups at 37 ℃ for 30 min. after 5 times of washing, the plate was incubated with 50 μl horseradish peroxidase (hrp)-fish tnf-α antibody at 37 ℃ for 30 min. after the final washing of 5 times, chromogenic agent and stop buffer were added, and the absorbance was determined at 450 nm by a precision microplate reader (biotek, usa). analysis of phagocytic rate of crab hemocytes the phagocytic activity of hemocytes was measured according to the method modified from wu’s description (wu et al., 2007). briefly, hemocytes (10 6 cells ml -1 ) from different groups were washed and resuspended with 1×leibovitz l-15 medium. then they were incubated with the latexs beads (sigma, usa) marked fluorescent yellow-green in advance for 60 min with dark. after the incubation, hemocytes were centrifuged at 900g, 4 ℃ for 10 min and resuspended with 400 μl 1×leibovitz l-15 medium. the relative intensity of fluorescence was analyzed by using becton dickinson facc scan (usa). measurement of intracellular ros generation the levels of intracellular ros were detected by using the peroxide-sensitive fluorescent probe dcfh-da (beyotime, china) as a substrate according to the method from sun’s description (sun et al., 2015). briefly, hemocytes from different groups were collected and incubated with 10 μmol l −1 dcfh-da in leibovitz l-15 medium (gibco, usa) table 2 names and sequences of the primers used in this study primer sequence oligo(dt) -adaptor 5'-ggccacgcgtcgactagtac(t)17-3' estoll1 p1 ctccttcacctgccctaactgct p2 ctccagtttgtattgctgtgcgaaa estoll2 p3 cattgattgctcttacctgaaccta p4 ctgcaagctatctaggctcgttaag esmyd88 p5 gcaacaggtggactttgaggagtg p6 cacggacaaaccacgactaaacc eslitaf p7 gattccctgctgtatggatgact p8 ctttctggatgcgttgtttaacc β-esactin p8 gcatccacgagaccacttaca p9 ctcctgcttgctgatccacatc 38 fig. 3 real-time pcr analysis of estoll1 (a), estoll2 (b) and esmyd88 (c) mrna expression in hepatopancreas of crabs after feeding cpg odns-supplement diet. a comparison of the level of mrna (relative to actin mrna) among different time points was performed using student’s t-test. each bar represents the mean with the standard error (sd), n = 3. different letters indicate significant difference from each other. the significant differences among the control and treated groups were subjected to one-way analysis of variance (one-way anova) (p < 0.05). (containing 0.27 g l −1 kcl, 10.1 g l −1 nacl, 0.3 g l −1 cacl2, 2.0 g l −1 mgcl2, 0.5 g l −1 mgso4) at room temperature for 20 min. the supernatant was discarded after washing for three times and the pellet was suspended with modified leibovitz l-15 medium. the relative fluorescence intensity was analyzed using becton dickinson facs scan (usa) and lysis ii software. statistical analysis statistical analysis was performed using spss 16.0. data from three samples of each time-point were subjected to a one-way anova. when overall differences were significant at less than 5 % level (p < 0.05), duncan’s test was used to compare the mean values between individual treatments. results the mrna expression of estolls (estoll1 and estoll2) and esmyd88 in hepatopancreas after interval or continuous feeding with cpg odns estolls and esmyd88 were selected to detect the interval or continuous activation of cpg odns to the immune system of crabs. for estoll1, the mrna expression in ci group was significantly upregulated (p < 0.05, 0.27-fold) at 7th day, and no difference was observed at 14th and 21st day than that in control group. in cc group, the expression level of estoll1 mrna upregulated significantly at 7th day (p < 0.05) and deceased at 21st day (p < 0.05), which was 1.47-fold and 0.88-fold of that in control group, respectively (fig. 3a). 39 the mrna expression level of estoll2 in ci group was significantly higher than that in control group except the samples at the 21st day, which was 3.26-fold (p < 0.05) and 7.04-fold (p < 0.05) of that in control group at the 7 and 14th day, respectively. in cc group, the expression level of estoll2 mrna increased significantly at the 7th day (1.19-fold, p < 0.05) and 14th day (2.05-fold, p < 0.05) in comparison to that in control group, while it was 0.57-fold (p < 0.05) lower than that in ci group at 14th day. there was no significant difference in the mrna expression level of estoll2 among the three groups at 21st day (fig. 3b). esmyd88 is considered as an important adapter protein in the tlr signaling pathway. after interval feeding of cpg odns, its mrna expression level increased significantly at the 7th and 14th day, which was 2.36-fold and 4.58-fold higher (p < 0.05) than that in control group. after continuous feeding of cpg odns, the expression of esmyd88 mrna increased significantly (p < 0.05, 1.07-fold) at the 7th day and no significant difference was observed at 14th and 21st day comparing to the control group. compared with the cc group, the expression level of mrna was significantly higher at 7th day (0.63-fold, p < 0.05) and 14th day (1.73-fold, p < 0.05) in ci group (fig. 3c). alteration of eslitaf mrna after feeding with cpg odns diets the expression level of eslitaf mrna in hepatopancreas of crabs after the interval and continuous feeding of cpg odns was investigated. the expression of eslitaf mrna was significantly up-regulated from 7th to 21st day after intermittently fig. 4 real-time pcr analysis of lps-induced tnf- factor (eslitaf) mrna expression of hepatopancreas after feeding the cpg odns at the 7th, 14th and 21st day in the three groups. data presented as mean ± s.d. (n = 4). different letters indicate significant differences from each other. the significant differences among the control and treated groups were subjected to one-way analysis of variance (one-way anova) (p < 0.05). feeding cpg odns diet, which was 2.57-fold (p < 0.05), 1.42-fold (p < 0.05) and 0.79-fold (p < 0.05) higher than that in control group, respectively. in cc group, its expression reached the maximum level (2.53-fold, p < 0.05) at the 7th day, while no significant difference was observed at the 14th and 21st day compared with the control group (fig. 4). the comparison of tnf-α protein level in plasma after feeding of cpg odns diets the tnf-α protein level in plasma was determined using the fish tnf-α elisa kit after the feeding with cpg odns-supplement diet for three weeks. compared to the control group, tnf-α concentration in ci group was significantly up-regulated (0.60-fold, p < 0.05) at the 7th day, and then reached a peak (0.99-fold, p < 0.05) at the 14th day, while no significant difference was determined at 21st day. after continuous feeding, the concentration of tnf-α in cc group was up-regulated (0.22-fold, p < 0.05) at the 7th day, followed by a decrease at 14th day, and then abruptly increased (1.52-fold, p < 0.05) at the 21st day, which was 2.52-fold (p < 0.05) and 1.90-fold (p < 0.05) of that in control and ci group, respectively (fig. 5). the phagocytic rate of crab hemocytes after the feeding of cpg odns diets the phagocytic rate of hemocytes was determined after the feeding of cpg odns diets. the phagocytosis rate of crab hemocytes kept in high level after interval feeding with of cpg odns diets, which was 1.77-fold (p < 0.05), 1.69-fold (p < 0.05) and 1.14-fold (p < 0.05) of that in control group fig. 5 determination of plasma tnf-α in three groups after feeding cpg odns for 21 day using the commercially fish tnf-α elisa kit. each bar represents the mean with the standard error (sd), n = 4. different letters indicate significant differences from each other. the significant differences among the control and treated groups were subjected to one-way analysis of variance (one-way anova) (p < 0.05). 40 at 7th, 14th and 21st day, respectively. while in cc group, the phagocytosis rate was significantly upregulated at 7th (p < 0.05, 0.47-fold) and 14th (p < 0.05, 0.27-fold) day, and no difference was observed at 21st day compared with the control group. meanwhile, the phagocytosis rate in cc group was 0.17-fold (p < 0.05) and 0.25-fold (p < 0.05) lower than that in ci group at 7th and 14th day, and there was no significant difference determined at 21st day between ci and cc groups (fig. 6). the production of reactive oxygen species (ros) after continuous or interval cpg odns stimulation the ros level in hemocytes after continuous or interval cpg odns stimulation was recorded by the relative fluorescence intensity. ros level in ci group was significantly up-regulated at the 14th day (p < 0.05, 1.69-fold), and decreased at the 21st day (p < 0.05, 0.52-fold) compared with that in the control group. and after continuous cpg odns stimulation, the ros kept high level at the 7th and 14th day, and then significantly decreased at 21st day, which was 1.91-fold (p < 0.05), 2.30-fold (p < 0.05) and 0.41-fold (p < 0.05) of that in control group, respectively (fig. 7). discussion immunostimulants are well known to improve the resistance of hosts to pathogens, but they are not absolutely safe and even seriously damage physiological processes of organisms after the long-term and continuous administration. it was found that cpg odns could lead to the disruption of normal lymphoid tissue and multifocal liver necrosis after administered daily to mice for three weeks (heikenwalder et al., 2004). cpg odns could cause severe mortality of murine when they were co-administered with sub-pathological does of lps and d-galactosamine (sparwasser, 1997). nowadays, the short-term immunostimulatory effects of cpg odns have been investigated in aquaculture animals (ian bricknell, 2005; ringø et al., 2011). however, little was known about its long-term immune effects in aquaculture animals. in the present study, the immune parameters were investigated after continuous or interval feeding with cpg odns-supplement diet to evaluate its long-term immune effects in chinese mitten crabs. as a classical type of pamps, cpg odns are recognized by conserved prrs, such as tlr9 in mammals and tlr21 in chicken which were identified in the previous studies (bauer et al., 2001; keestra et al., 2010). in our previous studies, lvtoll1 and lvtoll3 were confirmed as the recognition receptors of cpg odns in shrimp l. vannamei (sun et al., 2014). recently, estoll1 and estoll2 have also been cloned from e. sinensis, and estoll2 was found to induce a strong immune response than estoll1 after interval challenge with lipopolysaccharide, peptidoglycan and zymosan (yu et al., 2013). in the present study, the mrna expression level of estoll2 and estoll1 was significantly up-regulated after the feeding of cpg odns-supplement diet, suggesting that estoll2 and estoll1 might be potential receptor of cpg odns to activate the downstream signaling pathways. fig. 6 hemocytes phagocytotic activity were determination after feeding cpg odns for three weeks. the phagocytotic activity was represented by the percentage of latexs beads (marked with fluorescent yellow-green) phagocytosed by hemocytes measured by flow cytometer. each bar represents the mean with the standard error (sd), n = 4. different letters indicate significant differences from each other. the significant differences among the control and treated groups were subjected to one-way analysis of variance (one-way anova) (p < 0.05). similarly, estoll2 showed a relative higher expression level than estoll1, indicating that estoll2 might induce a strong response against cpg odns stimulation than estoll1. moreover, the relative lower expression level of estoll2 mrna in cc group was observed for the first two weeks than those in ci group. this was consistent with the previous observation, in which the immune tolerance of bacterial lipoprotein could be induced through downregulation of tlr2 expression (wang et al., 2002). the results suggested that successive cpg odns stimulation could reduce the recognition sensitivity of the immune cells by decreasing the expression levels of cpg odns receptors, which would further affect the activation of downstream immunostimulatory cascade induced by cpg odn. after interacting with human tlr9, cpg odns could activate the myd88-dependent tlr signaling pathway (kurlander and pisetsky, 1996; häcker et al., 2000; mogensen, 2009). it has also been reported that the recognition of cpg dna requires myd88 in drosophila (schnare et al., 2000). accumulating evidences have proved the existence and the activation of myd88-dependent tlr pathway in crustacean. for instance, the myd88-dependent signaling pathway in fenneropenaeus chinensis and penaeus monodon was reported to involve in defense against wssv infection (wen et al., 2013; deepika et al., 2014). esmyd88 was found to have a key function in innate 41 immune defense of e. sinensis (li et al., 2013; huang et al., 2014). in the present study, the mrna transcripts of esmyd88 were significantly increased after the feeding of cpg odns. it was also found in l. vannamei that the injection of cpg odns could enhance the expression of lvmyd88, suggesting that the tlr pathways in crustacean could be activated by cpg odns (zhang et al., 2012). moreover, the expression level of esmyd88 mrna in cc group was significantly lower than that in ci group after two weeks’ feeding, which was consistent with the tendency of estoll2 mrna. considering the existence of conserved tlr pathways in crustacean, it could be inferred that the continuous cpg odns-supplement diet feeding could weaken the activation of the downstream signal transduction by adaptor molecule myd88 in crabs. it is reported that after the binding of cpg odns, tlr pathways are activated to produce various pro-inflammatory cytokines and other immune factors (stacey et al., 1996; klinman, 2004; aguilar and rodriguez, 2007). litaf is an important transcription factor in downstream of tlr signaling pathway, which is closely associated with transcriptional regulation of tnf- and other cytokines dependent on myd88 (tang et al., 2006; li et al., 2014). in the present study, the mrna expression of eslitaf in ci group kept a higher level compared with that in control group during the whole experiment, while it was dramatically decreased in cc group from 14th to 21st day. moreover, the level of pro-inflammatory factor tnf-α in plasma was consistent with the change tendency of eslitaf mrna in both ci and cc feeding groups at 7th -14th day. these results indicated that continuous feeding of cpg odns might induce a suppression regulation to the downstream of tlr signaling pathway. interestingly, the concentration of tnf-α in cc group was found significantly increased at the 21st day after cpg odns feeding. the serious tissue injury (multifocal liver necrosis and hemorrhagic ascites) was observed in mice after 20 days’ daily injection of 60 μg cpg odns, which is closely related to the abundant production of pro-inflammatory cytokines tnf-α (heikenwalder et al., 2004). the dramatic increasing of tnf-α at the 21st day in the present study might be related to the serious collapse and vacuolization observed in hepatopancreas of crabs after continuous cpg odns feeding (data not shown). these results further indicated that the repeated cpg odns stimulation could not only reduce the activation of tlr signaling pathway, but also suppress downstream transcription factors expression and pro-inflammatory cytokine production, and even lead to disorder of cytokine production after administration of three weeks. lots of evidences have proved that cpg odns could induce phagocytic activity and respiratory burst activities in various aquatic animals (tassakka and sakai, 2002; meng et al., 2003; sun et al., 2013a). phagocytosis is considered to be one of the powerful cellular responses and plays significant roles for the invertebrate lacking adaptive immune system (allen and aderem, 1996). in the present study, the phagocytic activity of crab hemocytes was fig. 7 changes of hemocytes reactive oxygen (ros) after three cpg odns feeding methods. the ros values were represented by the mean fluorescence intensity of hemoncytes measured by flow cytometer. each bar represents the mean with the standard error (sd), n = 4. different letters indicate significant differences from each other. the significant differences among the control and treated groups were subjected to one-way analysis of variance (one-way anova) (p < 0.05). dramatically enhanced after 7 14 days feeding of cpg odns, similar to the observation in shrimps (sun et al., 2013). moreover, the phagocytic ability of hemocytes in cc group was significantly lower than that in ci group. the neutrophils of cpg odn-treated mice showed the elevated phagocytic activities against bacteria and the increased production of reactive oxygen species (ros) through the up-regulation of phagocytic receptors (weighardt et al., 2000). moreover, various tlrs differentially promote phagocytosis through induction of a phagocytic gene program and tlr9 being the strongest mediator of this process by myd88 in mice (doyle et al., 2004). these results indicated that the declined phagocytic activity of hemocyte in continuous cpg odn-fed crabs in the present study may be caused by the suppression of estolls as well as phagocytic receptors. ros is widely considered as an indicator of phagocytic activity, and it contributes to the increased capability to kill bacteria (meng et al., 2003; waris et al., 2005). in the present study, the ros level of both ci and cc group reached the lowest level at 21st day after significant increase at 7th and 14th day, and the ros level in interval feeding crabs was significantly higher than that in continuous feeding crabs at 14th day. these results suggested that continuous cpg odns stimulation could suppress the efficacy of the hemocyte phagocytosis as well as the ros generation in comparison with interval cpg odns stimulation. 42 in conclusion, continuous cpg odns-supplement diet feeding could reduce the activation of estolls and adaptor protein esmyd88 as well as its downstream production of pro-inflammatory cytokine and hemocyte phagocytosis and ros generation. compared with the continuous feeding strategy, the interval cpg odns feeding could effectively activate immune response at the first two weeks although the suppression was also observed at the last week, indicating that the interval feeding strategies needs further optimization. we hope that this information can provide theoretical guidance for the application of cpg odns in aquaculture as 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developmental biology, pecs university, hungary 3cardiff school of biosciences, main building, cardiff university, cardiff cf10 3us, wales, uk accepted january 6, 2016 abstract mild electrostimulation of earthworms is commonly used for harvesting celomocyte-containing celomic fluid. we have noticed that frequent electrostimulations of worms can lead to the autotomy of posterior segments followed by their regeneration. our present study aim was to develop an autotomy model in the eco-physiologically contrasting species, eisenia andrei and aporrectodea caliginosa, using direct current (dc), pulsating direct current (pdc), or immersion in the noxious anaesthetic ms222. a. caliginosa was clearly more susceptible to autotomy than e. andrei, with both electrostimulation regimes and ms-222 exposure inducing autotomy in the former species but only repeated pdc stimulations inducing segment loss in the latter. the observations lend credence to the hypothesis that autotomy in earthworms is caused by factors impinging upon the nervous system; they also indicate that repeated pdc stimulations could be an effective and reproducible means of facilitating segmental autotomy so that the fundamental cytological and molecular-genetic mechanisms underpinning the tissue loss event and subsequent regeneration can be studied in depth. key words: autotomy; electrostimulation; lumbricid earthworms; regeneration; eisenia andrei; aporrectodea caliginosa   introduction autotomy, literally meaning ‘self-severing’ or ‘self-amputation’, is a phenomenon involving the loss and often the subsequent regeneration of body parts such as appendages. it is manifest in a number of invertebrate (e.g., arthropods and echinoderms) and vertebrate (e.g., salamanders and lizards) taxa where it usually serves as a selfdefense mechanism enabling a potential prey organism to escape or confuse an attacking predator (maginnis, 2006; fleming et al., 2007). invertebrates lacking appendages, such as annelid worms, are also known to adaptively autotomize body segments, followed by their regeneration, as an anti-predatory strategy (lesiuk and drewers, 1999), as well as for the disposal of the products of antimicrobial and anti-eukaryotic parasite encapsulation (field et al., 2004), and as a response ___________________________________________________________________________ corresponding author: barbara plytycz department of evolutionary immunobiology institute of zoology, jagiellonian university gronostajowa 9, pl 30-387 krakow, poland e-mail: barbara.plytycz@uj.edu.pl and mode of elimination of highly toxic levels of organic (paris-palacios et al., 2010) and inorganic (mendez-fernandez et al., 2013) residues. previously, we and others have used mild electrostimulation of lumbricid worms for the induction of convulsive body movements and concomitant extrusion of celomocyte-containing celomic fluid through body wall dorsal pores. this procedure, originally developed by roch (1979), served as an effective non-invasive method for harvesting immunocompetent cells and soluble factors for ex vivo studies (e.g. stankiewicz and plytycz, 1998; podolak-machowska et al., 2013; rorat et al., 2014). using this procedure, we have observed that celomocyte depletion from eisenia spp. during a regime of daily 1 min-duration electro-stimulations with current from a 4.5 v battery for 19 consecutive days was followed by a period of steady replenishment of the celomocyte counts without gross adverse effects on the worms (unpublished data). in contrast, we have found recently that multiply stimulations with the pulsating direct electric current from a 4.5 v cell phone electric charger leads to posterior segment autotomy in the eco-physiologically contrasting lumbricids eisenia andrei (a litterinhabiting ‘epigeic’ species) and aporrectodea caliginosa (an ‘edogeic’ inhabitor of mineralized soil). 11   mailto:barbara.plytycz@uj.edu.pl fig. 1 representative examples of fates of earthworms subjected to multiple electrostimulations with pulsating direct current (pdc) twice per day. (a) eisenia andrei (ea) soon after autotomy of several posterior segments on day 4 after 7 electrostimulations; (b) the same specimen of e. andrei 4 weeks later with regeneration blastema (bl); (d) aporrectodea caliginosa (ac) soon after autotomy of several posterior segments on day 2 after 4 electrostimulations, and (e) the same worm 12 weeks later with regeneration blastema (bl). the primary aim of the present investigations was to develop and evaluate a reproducible model of autotomy in e. andrei and a. caliginosa, and to establish whether the susceptibility of the two species to autotomy was comparable. apart from distinct ecological differences, these two earthworm species have other trait differences that are pertinent for our investigative purposes: e. andrei has a high proportion of free-floating, riboflavincontaining, eleocytes in addition to amebocytes in its celomic fluid, a.caliginosa does not (cholewa et al., 2006; rorat et al., 2014); a. caliginosa, but not e. andrei, is known to undergo facultative diapause under certain adverse environmental conditions (bayley et al., 2010), a physiological resting state that may be functionally linked to tissue regeneration in at least some earthworm species (fragoso and lozano, 1992); differential species sensitivity to toxicants (kula and larink, 1998), as exemplified by studies deploying cytological and dna-damage biomarkers, respectively, indicating that a. caliginosa is more sensitive to sub-lethal concentrations of zn (spurgeon et al., 2000) but less sensitive to sub-lethal concentrations of cd (fourie et al., 2007) than e. fetida. our study was motivated as a foundation for future studies to establish common lumbricid species as model organisms to probe the involvement of neurosecretions and the immune system in autotomy and regeneration. analogous studies on e. fetida using a combination of low frequency electromagnetic and continuous light stimulation were recently initiated by banovački and matavulj (2013). table 1 comparison of autotomized posterior segments of eisenia andrei (ea) and aporrectodea caliginosa (ac) body segments (range) mean±sd autotomized/discarded species (worm numbers) total numbers numbers % numbers % body mass ea (7) (93 105) 99 ± 4.1 (14 50) 30 ± 14.3 (13 35) 27 ± 10.3 (7 29) 21 ± 11.6 ac (8) (117 140) 129 ± 8.3 (5 51) 25 ± 16.8 (5 13) 18 ± 12.8 (3 17) 12 ± 5.3 12   13   materials and methods earthworms experiments were performed on adult (i.e., fully clitellate) eisenia andrei and aporrectodea caliginosa. e. andrei, originally bought from the commercial supplier ekargo (kepa slupska, poland), reared for several generations in the laboratory at the institute of zoology, jagiellonian university, kraków, poland. adult a. caliginosa were collected from the field (kleczany, southern poland) and then cultured at the jagiellonian university. both species were routinely maintained at 17 23 oc, and fed with a mixed diet comprised of dried/boiled nettle (urtica dioica) and dandelion leaves (taraxacum officinalis), boiled/dried tea leaves, and powdered commercial mouse pellets. within each species, individuals of similar body weights were used for each experiment. experimental scheme in each experimental series weighed earthworms were individually immersed once or twice per day in 5 ml physiological saline and subjected to 60 sec-duration electrostimulations either: (i) with direct current (dc) from a battery (sony 3r12; 4.5 v); or, (ii) with pulsating direct current (pdc) from the adapted cell phone chargers, i.e., alternating current/direct current (ac/dc) adaptors either (input: 230 v, ~50hz, 20 ma; output: 4.5 v, 400 ma) or (input: 230 v, ~50 hz, 0.05 a; output: 4.8 v, ~350 ma). both dc and pdc treatments induced extensive body movements connected with the expulsion of celomocytecontaining celomic fluid (podolak-machowska et al., 2013). in some experiments, electrostimulation was preceded by 3 min anaesthesia in carbonated drinking water by soda siphon. in another set of experiments worms were immersed for 1 min in 5 ml 0.5 % ms-222 solution (tricaine methanesulfonate; ethyl 3-aminobenzoate methanosulfonate salt; sigma-aldrich; fluca), that is a known strong irritant of worms and induces celomocyte extrusion (podolak-machowska et al., 2013). in all experiments, worms were individually kept in boxes with moistened filter paper or soil and inspected daily for general condition and/or incidence of autotomy. in the latter case anterior and posterior parts of worms were weighed and segments were counted. numbers of worms of both species used in particular experiments are given in respective figures and tables of the section results and discussion. some autotomized worms were transferred to clean moist soil, fed ad libitum as per the laboratory cultures (see above), and observed for several weeks for regenerative blastema formation. immersion fluid from various sets of experiments was inspected for the presence/absence of celomocytes in hemocytometer. in the case of e. andrei, several 2 ml samples of celomocyte-containing fluids were lysed in 2 % triton (sigma-aldrich) and used for measurements of riboflavin content using an ls50b perkin-elmer spectrofluorimeter as described previously (rorat et al., 2014); this procedure was not undertaken with a. caliginosa because the celomic fluid of this species does not contain appreciable numbers of riboflavin-laden, freefloating, eleocytes. results and discussion figure 1 shows representative examples of e. andrei and a. caliginosa soon after autotomy of posterior segments and, several weeks later, during blastema formation that evidently occurred earlier in the former species. the formation of a blastema indicates that autotomy potentially may be followed by regeneration of severed segments. it is pertinent that a detailed description of an analogous phenomenon in echinoderms was entitled “autotomy as a prelude to regeneration in echinoderms” (wilkie, 2001). the total numbers of segments were relatively constant within intact e. andrei and a. caliginosa (99 ± 4.1 and 129 ± 8.3, respectively). during autotomy induced by pdc from a cell phone charger, e. andrei discarded a higher proportion of its segments than did a. caliginosa (27 ± 10.3 % versus 18 ± 12.8 %, respectively), which corresponded to body mass losses of 21 ± 11.6 % versus 12 ± 5.3 %, respectively (table 1). these observations imply the existence of species-specific morpho-regional differences in the susceptibility to autotomy in these earthworm species. figure 2a and table 2 show that among worms stimulated once per day with pdc from the cell phone charger, all 12 e. andrei individuals exhibited autotomy between the 4th and 22nd days after the start of stimulation regime, while all 9 a. caliginosa in the treatment group autotomized significantly earlier at between the 2nd and 5th days. median autotomy times post-initiation of once-daily pdc stimulations were 8 days (e. andrei) and 4 days (a. caliginosa). thus, the median number of pdc stimuli required to induce autotomy was 8 (e. andrei) and 4 (a. caliginosa). figure 2b and table 2 show that a twice-daily regime of pdc stimulations induced autotomy more efficiently than once-daily stimulation. twice-daily pdc resulted in all 18 e. andrei individuals in the treatment group autotomizing their posterior segments between the 2nd and 11th days postinitiation, whilst we observed autotomy in one a. caliginosa individual as early as after the second electrostimulation on day 1, with all 14 a. caliginosa individuals in the treatment group exhibiting autotomy by day 5. thus, median autotomy time was reduced from 8 days to 4.5 days (e. andrei), and from 4 days to 3 days (a. caliginosa), with twice-daily compared with once-daily pdc stimulations. nevertheless, the median number of electro-stimuli required to induce autotomy was similar whether the stimuli were given onceor twice-daily: 8 stimuli (once-daily) versus 7-8 stimuli (twice-daily) in e. andrei, and 4 stimuli versus 5 6 in a. caliginosa, the period being consistently longer in e. andrei (table 2). [note that in the twice-daily treatments, it was not possible to resolve the number of stimuli required to induce autotomy to a fig. 2 incidences of autotomy after multiple electrostimulations (v) with pulsating direct current (pdc) applied: (a) once per day or (b) twice per day in eisenia andrei (ea) or aporrectodea caliginosa (ac). days of autotomy by the median worm of each species are marked by open and gray arrows (ea and ac, respectively). table 2 autotomy of posterior segments of eisenia andrei (ea) and aporrectodea caliginosa (ac) induced by multiply electrostimulation with pdc from the cell phone charger, performed once (1x) or twice per day (2x). start of experiments is considered as day 1 days of autotomy numbers of electrostimulations before autotomy worms times per day pdc treatments worm numbers autotomising / total range median range median 1x 12/12 4-22 8 4-22 8 ea 2x 18/18 2-11 4.5 3-22 7-8 1x 9/9 2-5 4 2-4 4 ac 2x 14/14 1-5 3 2-10 5-6 14   fig. 3 riboflavin (in arbitrary units au) derived from celomocytes extruded to immersion fluid by eisenia andrei worms stimulated for the first time (t1) and then 8 h later (t2) by pulsating direct current (pdc) from the phone charger or by direct current (dc) from the battery or pdc after anesthesia in co2-water (co2+pdc) or by immersion in 0.5 % ms-222. x±se, n = 4. single number]. the differential responses of the two eco-physiologically contrasting earthworm species to electrostimulation was not surprising in light of the fact that species-specific sensitivity to electric current has been observed in fish. for example, pdc treatment caused mortality of fingerlings of chinook salmon oncorhynchus tschawytscha (collins et al., 1954) while rainbow trout (salmo gairdneri) were resistant to similar treatment (maxfield et al., 1971). the aim of our subsequent experiments was to address the question of whether the autotomy of posterior segments in e. andrei and a. caliginosa is caused primarily by electric stimulation or by a massive loss of coelomic fluid containing coelomocytes and soluble factors. consequently, a comparison was made of the results of 5-day experiments involving two stimulations per day, performed on 4 groups of worms: 1) electrostimulated with pdc; 2) electrostimulated with dc; 3) electrostimulated with pdc after anesthesia in co2-water (lubics et al., 2002); 4) immersed in ms-222 without electrostimulation. after worm removal from immersion fluids, microscopic inspection revealed in all samples the presence of expelled celomocytes, among them amebocytes and eleocytes in the case of e. andrei, but amebocytes only in a. caliginosa. the observed species-related differences in cellular constitution of the immersion fluids was consistent with the fact that a. caliginosa is a lumbricid exclusively containing free-floating amebocytes in its celomic fluid, while e. andrei is a lumbricid known to contain both major celomocyte types (i.e., amebocytes and autofluorescent eleocytes) (cholewa et al., 2006; rorat et al., 2014). autofluorescence of e. andrei eleocytes derives mainly from yellow riboflavin (koziol et al., 2006; plytycz and morgan, 2011; rorat et al., 2014); this is macroscopically evident because the extrusion fluids acquire a subtle yellowish hue, and was subjected to quantitative spectrofluorimetric analysis (fig. 3). the amount of riboflavin tended to be higher in samples after pdc compared with dc electrostimulation, albeit the difference was statistically insignificant. attempts to suppress the inadvertent extrusion of celomocytes into immersion fluid by mild anesthesia in carbonated water (e.g., lubics et al., 2002) were only partly successful because small measureable amounts of riboflavin were still detectable in immersion fluid. table 3 results of 5-day attempts to induce autotomy of posterior segments of eisenia andrei (ea) and aporrectodea caliginosa (ac) by electrostimulation with pdc from cell phone charger, or with dc from battery, or pdc after co2 anesthesia (co2+pdc), or by immersion in ms-222, all treatments performed twice per day worms treatments worm numbers autotomising/total pdc 14/18 dc 0/4 co2+pdc 1 /4 ea ms-222 0/4 pdc 13/13 dc 1/3 co2+pdc 3/3 ac ms-222 2/7 15   16   table 4 celomocytes/riboflavin loss and posterior segments autotomy in eisenia andrei and aporrectodea caliginosa earthworms induced by repeated stimulations with pdc from cell phone charger or dc from battery without or with co2 anesthesia (co2+pdc) or by immersion in ms-222 eisenia andrei aporrectodea caliginosa repeated treatments loss of celomocytes and riboflavin autotomy loss of celomocytes autotomy pdc + + + + dc + + +/ co2+pdc +/+/+ + ms-222 + + +/ ms-222 is an effective anesthetic for ectothermic vertebrates such as fish (neiffer and stamper, 2011) and amphibians (e.g., jozkowicz and plytycz, 1998). however, ms-22 is noxious to earthworms (podolak-machowska et al., 2014), and in the present experiments was found to induce the ‘leakage’ riboflavin from e. fetida at levels comparable to those observed after pdc electrostimulation. second treatments of the same worms performed 8 h later resulted in the detection of negligible amounts of riboflavin in all but the carbonated-water/pdc treatment group (fig. 3). these findings indicate that worms lose most of their celomocytes during the first pdc, dc, and ms222 stimulations, and that carbonated-water anesthesia did not fully inhibit the effect. the 5-day-experiments confirmed that e. andrei is less prone to autotomy than a. caliginosa (table 3). all e. andrei and a. caliginosa autotomized after repeated electro-stimulation with pdc. however, none of 4 e. andrei autotomized tail segments during 10 successive (i.e., twice daily) dc treatments, while 1 of 3 a. caliginosa did; none of 4 e. andrei immersed in ms-222 autotomized tail segments while 2 of 7 a. caliginosa did; each of the 3 a. caliginosa immersed in carbonated-water before pdc electrostimulation discarded tail segments, while only 1of 4 e. andrei did (table 3). as summarized in table 4, e. andrei discarded posterior segments only after repeated electrostimulation with pdc. in contrast, a. caliginosa did after a variety of repeated stressors, both those involving electrostimulation alone, ms222 alone, and electrostimulation in combination with co2 anesthesia. thus, in this endogeic lumbricid species, autotomy can be induced by physical and chemical factors capable of extruding celomocytes, although the loss of immunecompetent cells may be a spurious concomitant effect of the treatments rather than in any direct way causative. it is conceivable that electrostimulation and ms-222 treatment both cause autotomy by impinging on the nervous system. the mechanism(s) connecting the stimuli inducing autotomy in lumbricid earthworms and the morphological manifestations of the phenomenon are as yet unclear. however, lesiuk and drewers (1999), who developed an apparatus for focal body compression of the freshwater oligochaete lumbriculus veriegatus to induce autotomy, observed that body fragmentation in this species could be blocked by nicotine pre-treatment. this suggests that nicotinic cholinergic receptors are involved in the autotomy reflex, and offers sustenance to the notion that the nervous system is fundamentally implicated in earthworm autotomy. extrapolating from l. variegatus to terrestrial oligochaetes should perhaps be done with caution because spontaneous self-fragmentation is a prominent asexual reproduction strategy characteristic of lumbriculus. nevertheless, the simple pdc method that we have described appears to reliably induce autotomy of posterior segments in e. andrei and a. caliginosa. this may serve as a convenient model for studies on cytological and molecular-genetic mechanisms underpinning autotomy and subsequent tissue/organ regeneration in these soil-dwelling metazoans. moreover, it will be interesting to compare the details of segment restoration in surgically-amputated and autotomized earthworms. acknowledgments we thank to gałuszka a for sampling and delivery of aporrectodea caliginosa worms. these investigations were financially supported by the grant b/nz4/01640 (k/pbo/000178) from the polish national centre of science, and by k/zds/004831. references banovački z, matavulj m. exposure to extremely low frequency (50 hz) electromagnetic field changes the survival rate and morphometric characteristics of neurosecretory neurons or the earthworm eisenia foetida (oligochaeta) under illumination stress. arch. biol. sci. 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(krakow), 46: 183188, 1998. taylor gn, cole ls, sigler wf. galvanotaxic reasponse of fish to pulsating direct current. j. wildlife manage. 21: 201-213, 1957. wilkie ic. autotomy as a prelude to regeneration in echinoderms. microscopy res. tech. 55: 369396, 2001. microsoft word isj439 isj 13: 298-308, 2016 issn 1824-307x research report transcriptome-wide analysis reveals candidate genes responsible for the asymmetric pigment pattern in scallop patinopecten yessoensis xj sun, lq zhou, zh liu, b wu, ag yang yellow sea fisheries research institute, chinese academy of fishery sciences, qingdao 266071, china accepted september 14, 2016 abstract yesso scallop patinopecten yessoensis is an economically important marine bivalve species in aquaculture and fishery in asian countries. the colors of the left and right shells are obviously distinct, typically having reddish-brown for the left and white for the right. this left-right asymmetric pigment pattern is a very unique phenomenon among invertebrates, whereas the molecular mechanisms that control regional differences in pigmentation are not clear. to better understand the left-right asymmetric pigment pattern, we apply illumina digital gene expression (dge) to characterize the gene expression profiles in left and right mantle tissues, and identify five differentially expressed genes, including cytochrome p450 and other four unknown genes. among the five genes, one gene shows significantly higher expression in the right mantle, while other four exhibit significantly higher expression in the left mantle. we further validate the dge results by using quantitative real-time pcr for p450, resulting in approximately 32-fold higher expression in the left mantle than that in the right mantle. these findings will not only help assist our understanding of the sophisticated processes of shell pigmentation in scallops, but also provide new insights into the adaptive evolution of phenotypes to maximize survival that underlie the left-right asymmetric pigment pattern in molluscs. key words: mollusc; patinopecten yessoensis; digital gene expression; shell color; mantle; cytochrome p450   introduction color variation is an interesting and a nearly universal mechanism for recognition, adaption, and camouflage in nature (jiggins et al., 2001; barbato et al., 2007; mckinnon and pierotti, 2010; maan and sefc, 2013). the different pigment patterns can occur in different individuals within and among populations, at different stages of development, and also probably in different regions of the body within a species, which are an excellent system to investigate how morphological differences arise (candille et al., 2004). for decades, the color traits continue to be of great interest to evolutionary biologists because of their general tractability, importance in studies of selection, and potential role in speciation (tanaka, 2006; mckinnon and pierotti, 2010). moreover, color traits are also be attracted by geneticists and breeding scientists because of their commercial value in livestock and aquatic breeding industry _______________________________________________________________________ corresponding author: aiguo yang yellow sea fisheries research institute chinese academy of fishery sciences qingdao 266071, china e-mail: yangag@ysfri.ac.cn (johansson et al., 1992; colihueque, 2010; fan et al., 2013). for the aquatic animals, the appearance of color pattern in fishes and molluscs can significantly influence consumer’s acceptance and affect the value of goods in markets (guan and he, 2009; colihueque, 2010). as a valuable trait in both scientific and commercial communities, studies aimed to clarify the genetic basis of traits related to color aquatic animals, are considered to be of great importance for the knowledge on micro-evolutionary forces, adaptive evolution and survival mechanisms (kittilsen et al., 2009; gunter et al., 2011; maan and sefc, 2013; takahashi et al., 2013). however, most of attentions have been focused on color variation or polymorphism among different individuals, whereas little is known about how regions of body in vertebrates and invertebrates acquire differences in their appearance, especially for regional mechanisms that control regional differences in pigmentation. one of the most obvious aspects of regional color variation in vertebrates, including teleosts, is the dorsal-ventral pigment patterns, in which a dark dorsal surface juxtaposes to a light ventral surface in the color of skin, scales, feathers, or hair 298 mailto:yangag@ysfri.ac.cn (fukuzawa et al., 1995; jiggins et al., 2001; candille et al., 2004; yamada et al., 2010). as reported, several mechanisms may contribute to regional differences in vertebrate pigmentation, including alterations in the determination or migration of melanoblasts (reedy et al., 1998), paracrine signal controlling (furumura et al., 1996; barsh et al., 2000), movement of pigment granules (marks and seabra, 2001), the asymmetric appearance of adult-type pigment cells (yamada et al., 2010), and t-box gene action (candille et al., 2004). in comparison with vertebrates, the mechanism underlying regional differences in pigmentation of invertebrates remain largely unknown. for molluscs, shell color variations are not only known to exist in various kinds of species, but also may occur in different regions of the body within a species (comfort, 1951; kondo and miura, 2010). the shells are specifically secreted by the mantle epithelium, where the anterior edge of the mantle tissue directs the formation of different structural layers of the shell, and controls the patterning of architectural and color features (jackson et al., 2006). molluscan shells contain a wide range of pigments, including melanins, indigoids, pyrroles, pterins, quinones and flavones, which are probably formed in the secretory cells and subsequently transported to the shell edges (comfort, 1951; nagai et al., 2007; zhang et al., 2012; bai et al., 2013; sun et al., 2015). recently, the molecular mechanism of shell color variations has been investigated in several bivalve species (bai et al., 2013; yue et al., 2015; ding et al., 2015), but regional differences in shell pigmentation within a species are still poorly understood. the yesso scallop patinopecten yessoensis is a cold water bivalve and naturally distributes along the coastline of the northern islands of japan, northern korean peninsula, and russian primorye, sakhalin and kurile islands (ito, 1991). due to its large and edible adductor muscle, p. yessoensis is becoming one of the most important maricultural species in northern china (xu et al., 2008; cai et al., 2014). the colors of the left and right shells are obviously distinct, typically having reddish-brown for the left and white for the right, showing typically left-right asymmetric pigment pattern (fig. 1). the pigmentation for left mantle tissue is slightly darker than the right one. this regional difference in pigmentation is a very unique phenomenon among invertebrates, which can serve as a striking example for studying asymmetric pigment pattern in invertebrates. to better understand the mechanisms underlying the region-specific differences in body morphology that give rise to different shell colors, detection of differentially expressed genes from the mantle is essential. in the present study, we applied illumina rna-seq and digital gene expression (dge) analysis to characterize the gene expression profiles, identified the differentially expressed genes between the left and right mantle, and help understand the molecular mechanism underlying the left-right asymmetric pigment pattern in this economically important species.         fig. 1 the shell picture for yesso scallop patinopecten yessoensis. the predominant color of left valve is reddish-brown, while the color of right valve is white. 299 table 1 the primer information used in quantitative real-time pcr (rt-pcr) gene_id primers size (bp) tm ( ) actin f: ccaaagccaacagggaaaag 163 55.0 r: tagatggggacggtgtgagtg comp51117_c0 f: ggctgtgcacatgtgtagtc 235 58.7 r: ccaacttccggctcaaaact comp96758_c0 f: taccgtagctgccctgaaaa 246 59.0 r: ctttctttcttggcggctgt comp97294_c0 f: cctacaacagcggattcacg 256 59.0 r: caactatacggtccggtcga comp99344_c0 f: atcggtgaaagggttgaggt 250 58.9 r: aagtgccacagttcggtaga comp99344_c1 f: acggtgtttgcttaggagga 216 59.0 r: cgacaggacagatgtgaggt materials and methods animal and tissue collection six scallops p. yessoensis (two-year old; 9.55 ± 0.42 cm) were obtained from a commercial hatchery in yantai, china. the scallops were cultured in sand-filtered sea water at 14 ± 2 °c for two weeks before processing. the rearing methods were accordance with the previous study (sun et al., 2015). the left and right mantle tissues of each scallop was dissected and stored in rnalater (ambion). total rna was isolated with trizol reagent (invitrogen) and checked using the nanophotometer™ spectrophotometer (implen, ca, usa) for rna purity and quality. rna integrity was assessed using the rna nano 6000 assay kit. ngs library construction and sequencing a total amount of 3 μg rna per individual was used as input, and rna samples of left mantle from three individuals were pooled in equal amounts to generate the mixed sample for the left mantle library. similarly, rna samples of right mantle from the same three individuals were used to generate the mixed sample for the right mantle library. four libraries, including two biological replicates, were used for the illumina sequencing in this study. sequencing libraries were generated using nebnext ultra™ rna library prep kit for illumina (neb, usa) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. the mrna was purified using poly-t oligo-attached magnetic beads. the first strand cdna was synthesized using random hexamer primer, and second strand cdna synthesis was subsequently performed using dna polymerase i and rnase h. after adenylation of 3’ ends of dna fragments, adaptors were ligated to prepare for hybridization. the fragments were purified with ampure xp system (beckman coulter, beverly, usa) to select cdna fragments of 150~200 bp. the adaptor-ligated cdna was kept at 37 °c for 15 min followed by 5 min at 95 °c. finally, pcr products were purified and library quality was assessed on the agilent bioanalyzer 2100 system. the clustering of the index-coded samples was performed using truseq sr cluster kit v3-cbot-hs (illumina) according to the manufacturer’s instructions. after cluster generation, the sequencing was carried out on an illumina hiseq 2000 platform that generated about 100 bp paired-end raw reads. quality control raw data (raw reads) of fastq format were first processed through in-house perl scripts, in order to remove reads containing adapters, reads containing ambiguous ‘n’ nucleotides (‘n’ ratio of more than 10 %) and reads with low quality (quality score of less than 5). all the downstream analyses were based on clean data with high quality. meanwhile, q20, q30 and gc-content of the clean data were calculated for estimating the quality of clean data. aligning raw reads and differentially expressed genes analysis all clean tags were mapped back onto the assembled reference transcriptome using rsem (li and dewey, 2011; sun et al., 2015). read 300 counts obtained from the mapping results for each gene were used to estimate gene expression levels for each sample. we performed differential expression analysis for the left and right mantle samples with biological replicates using the deseq r package, which provides statistical routines for determining differential expression in digital gene expression data using a model based table 2 summary statistics for sequencing and data quality of rna-seq sample raw reads clean reads clean bases error (%) q20 q30 gc content (%) left_1 10,061,486 9,952,872 1.00 g 0.03 98.10 93.56 42.17 left_2 19,330,562 19,176,083 1.92 g 0.03 98.22 93.88 41.16 right_1 16,885,161 16,737,085 1.67 g 0.03 98.19 93.76 41.35 right_2 18,024,935 17,853,507 1.79 g 0.03 98.12 93.59 41.56 note: left_1, the left-end sequencing of the left mantle tissue; left_2 the right-end sequencing of the left mantle tissue; right_1, the left-end sequencing of the right mantle tissue; left_2 the right-end sequencing of the right mantle tissue. on the negative binomial distribution. the resulting p values were adjusted using the benjamini and hochberg’s approach for controlling the false discovery rate (benjamini and hochberg, 1995). the differentially expressed genes were assigned with an adjusted p-value lower than 0.05. quantitative real-time pcr the seven tissues, including left mantle, right mantle, adductor muscle, gill, gonad, hepatopancreas and foot, were immediately dissected from the live scallops. these tissues were cleaned and then stored in rnalater at −80 °c. total rnas were extracted using trizol reagent (invitrogen) according to the manufacturer’s protocols. the qualities of rna samples were assessed as described above. three biological replicates were used for the analysis of quantitative real-time pcr on applied biosystems 7500 system. the primer information for quantitative real-time pcr was listed in table 1. the transcripts level of each gene was normalized to the expression of β-actin and the comparative ct method (2ct method) was used to calculate the relative gene expression of the samples (livak and schmittgen, 2001). the expression data were subsequently subjected to one-way anova followed by an lsd test for multiple comparisons, using spss 17.0 to determine whether there was any significant difference (p < 0.05). results overall statistics and reads for the four samples (left_1, left_2, right_1 and right_2), there are a total of 64,302,144 raw reads generated by illumina sequencing, which yields a total of 6.37 g clean bases after quality filtering that remove reads containing adapters or ambiguous nucleotides and low quality reads (table 2). the left mantle samples of left_1 and left_2 produces 1.00 g and 1.92 g cleaned reads, and the right mantle samples of right_1 and right_2 produces 1.67 g and 1.79 g cleaned reads. the cleaned reads produced in this study have been deposited in the ncbi sra database (accession number: srp059521). the similar values of q20 percentage and q30 percentage are found around 98 % and 94 %, and the same error rates of 0.03 % are observed in the four samples. the percentage of gc content for the clean reads was consistent among samples, varying from 41.1 % to 42.2 %. the obtained clean reads are then used for reads alignment with the reference transcriptome of p. yessoensis (sun et al., 2015), resulting in the high percentages of mapped reads of 85.00 %, 94.01 %, 93.13 % and 90.73 %, respectively. quality control of gene expression analysis to obtain more reliable results and avoid incorrect interpretation of gene expression data, four library samples including two replicates are used to estimate the robustness of abundance as a function of expression level and mapping reads (fig. 2). the colored lines representing transcripts with different fpkm values show the gene expression obtained at different sequencing depth. for example, the purple line tracks the performance for transcripts with fpkm > 150; the green line tracks the performance for transcripts with fpkm of 3-15. although the fraction of transcript generally increases with additional sequencing data, high expressed transcripts (> 3 fpkm) are more likely to be accurately quantified even at low mapping rates, while > 90 % mapping rates are required for the low expressed transcripts (< 3 fpkm) being accurately quantified. at 40% mapping rates (31,396 unigenes), 301 transcripts determined to be moderately expressed (> 1 fpkm) are estimated at within 15 % of their final fpkm values. furthermore, a significant linear correlation between gene expression results obtained by the two replicates for each sample, as showed in figure 3 (pearson correlation coefficient = 0.724 between left_1 and left_2; pearson correlation coefficient = 0.755 between right_1 and right_2). these results suggest that the present sequencing is necessary for accurate determination of the expression level of genes. fig. 2 the saturation analysis of sequencing data. x-axis represents the percentage of mapped reads to reference transcriptome (%); y-axis represents the fraction of genes within 15% of quantitative deviation. each color line represents the saturation curve of different gene expression levels in terms of fpkm intervals. a, left_1 sample; b, left_2 sample; c, right_1 sample; d, right_2 sample. gene expression analysis about 80,000 genes identified at different levels of expression for each library sample are selected for further analysis using deseq (anders and huber, 2010). the applied deseq package has pointed five significantly differentially expressed genes (padj < 0.01), including comp51117, comp96758, comp97294, comp99344_c0, and comp99344_c1 (table 3). among the five screened genes, one up-regulated gene shows significantly higher expression in the right mantle, whereas four down-regulated genes exhibit significantly higher expression in the left mantle (fig. 4). the highest obtained log2 ratio (fold change) in the up-regulated gene is 8.25, while the lowest log2 ratio among the 302 down-regulated genes is -4.68. functional annotation of the screened genes and qpcr validation among the five differentially expressed genes, only one (comp96758) is annotated by nr database which encodes cytochrome p450 3a31, while other four are not annotated by nr (table 3). among the four unannotated genes, three (comp97294, comp99344_c0, comp99344_c1) are enriched by gene ontology biological pathway, relating to colicin e1 (microcin) immunity protein, herpesvirus latent membrane protein 1 (lmp1), and glutamate-cysteine ligase, respectively. however, comp51117 is an unknown gene which is not annotated by any database so far. the validation of rna-seq results was performed using quantitative real-time pcr (rt-pcr) technology by the estimation of relative expression level (2ct) for the left and right mantle samples in additional animals. statistically, the significantly higher level of p450 expression is detected in the left mantle, approximately 32-fold higher than that in the right mantle (p < 0.01; fig. 5). the significantly different expression of p450 between the left and right mantle revealed by rt-pcr is in accordance with the rna-seq results. to examine the spatial mrna expression pattern of p450 in the adult p. yessoensis, quantitative rt-pcr was also used to detect the expression of p450 in five other tissues, including adductor, gill, gonad, hepatopancreas, and foot (fig. 5). combined with the results of p450 expression in the left and mantle tissues, the rt-pcr results indicate that the expression of p450 is significantly different among the scallop tissues (df = 6, f = 11.19, p < 0.01). the highest expression level was detected in the gill tissue, which is significantly higher than that in other tissues (lsd post hoc comparisons, p < 0.05). moderate expression of p450 was observed in the three tissues of left mantle, gonad, and hepatopancreas, showing no significant difference among them (p > 0.05). the above four tissues exhibited significantly higher expression levels than those of right mantle, adductor, and foot (lsd post hoc comparisons, p < 0.05), and the lowest expression of p450 was observed in the tissue of adductor muscle. discussion bivalve mantle is the key organ that secretes biomineralization proteins inducing shell deposition and pigmentation, which direct the formation of different structural layers of the shell, and controls the patterning of architectural and color features (comfort, 1951; wilbur and saleuddin, 1983; addadi et al., 2006; jackson et al., 2006). in the present study, two mantle tissues collected from the same scallops responsible for reddish-brown and white shell colors, were used to investigate the transcriptome differences by using illumina digital gene expression (dge) tag profiling. due to lack of genome resources, dge tags were mapped to the previously assembled transcriptome (sun et al., 2015), and identified five differentially expressed transcripts related to the left-right asymmetric pigment pattern. comparing with other genome-wide expression profiling platforms, such as microarray, illumina dge is an efficient and economic choice to study mrna expression level in non-model organisms without a reference genome, which provides a major advance in robustness, comparability, richness, technical reproducibility of expression profiling data (marioni et al., 2008; bai fig. 3 the correlation for two biological replicates of the left and right mantle assessed by pearson's correlation coefficient. et al., 2013). however, the development and selection of efficient algorithm and software for computing such a large number of short reads 303 generated by next generation sequencing systems is critically important for gene expression studies (kvam, 2012). several programs used for differential expression analysis have been developed for next generation sequencing technology, such as deseq (anders and huber, 2010), degseq (wang et al., 2010), edger (robinson et al., 2010), tspm (auer and doerge, 2011), and nbpseq (di et al., 2011). in the present study, deseq is chosen because it has relatively low rate of false positives and its algorithm performs more conservatively than edger and nbpseq (robles et al., 2012; guo et al., 2013). moreover, the complicated rna-seq experiments during the process of rna isolation and library preparation may cause some errors and table 3 identification of differentially expressed genes between the left and right mantle of patinopecten yessoensis gene_id right read counts left read counts log2 fold change pval padj nr description gene ontology description comp51117 107.09 0.35 8.2493 4.8641e-14 1.3877e-09 comp96758 16.56 372.56 -4.4913 1.0545e-14 4.5125e-10 cytochrome p450 3a31 cytochrome p450 comp97294 84.26 1574.07 -4.2235 5.318e-10 9.1033e-06 colicin e1 (microcin) immunity protein comp99344_c0 34.82 673.62 -4.274 3.4147e-15 2.9226e-10 herpesvirus latent membrane protein 1 comp99344_c1 9.10 233.46 -4.6806 7.555e-14 1.6166e-09 glutamate-cysteine ligase fig. 4 the volcano plots for rna-seq showing differentially expressed genes (right mantle vs. left mantle). x-axis represents log2 (fold change), and y-axis represents adjusted p-values in terms of -log10 (padj) for the rna-seq data. points of the plots represent transcripts that are significant and differentially expressed. green points represent transcripts with significantly lower expression level, while red circles represent transcripts with significantly higher expression level (p < 0.05). 304 biases for the estimation of gene expression (wang et al., 2009; bullard et al., 2010). additionally, the detection power of differential expression analysis for the count data is limited by biological replicates, rather than technical replicates (anders and huber, 2010; robles et al., 2012). therefore, to obtain the most reliable gene expression measurements in the present study, we performed the illumina dge analysis by using the left and right tissues collected from the same scallops, designing biological replicates, and further validated by rt-pcr. all these conditions have enabled us to narrow down the considerable number of differentially expressed genes to a small number of candidates, which warrant further investigation. fig. 5 validation of rna-seq results by quantitative real-time pcr and relative gene expression in different tissues of p. yessoensis. error bars represent the standard error of three replicates (n = 3). values marked with different letters differed significantly from each other (p < 0.05). five candidate genes were identified by illumina dge analysis, including cytochrome p450 and other four unannotated genes, to express significantly different between the left and right mantle, which are suggested to be responsible for the left-right asymmetric pigment pattern. although three of the four are enriched by gene ontology biological pathway, their functions are largely unknown due to the limited genomic resource. the p450 genes are found in the genomes of virtually all organisms, which have a wide spectrum of functions, contributing to carbon source assimilation, biosynthesis of hormones, structural components and degradation of xenobiotics in living organisms (werck-reichhart and feyereisen, 2000; nelson, 2013). in particular, the cytochrome p450 genes play critical roles in flavonoid biosynthetic pathways especially in plants, which are responsible for the variation of flower colors (tanaka, 2006; hatlestad et al., 2012). for animals, however, melanins are the essential compound in shell pigments of molluscs, and the visible pigmentation of the skin, hair and eyes in mammals and other vertebrates (comfort, 1951; d'ischia et al., 2015; williams, 2016). as indicated, the melanin biosynthetic pathway in molluscs is mainly regulated by tyrosinase, which are secreted from the mantle and transported to the shell layer (slominski et al., 2004; nagai et al., 2007; zhang et al., 2012; sun et al., 2015, 2016). the two classes of pigments, flavonoids and melanins have a high similarity in their properties, which are able to bind the same substrate of l-phenylalanine, sharing the same role for uv protection strategy (carletti et al., 2014). this suggests their possible common origin after endosymbiosis in the two different kingdoms (martin et al., 2002; carletti et al., 2014). moreover, several flavonoids may competitively 305 inhibit tyrosinase activity and enhance melanogenesis due to their ability to chelate the copper in the active site, interfering with mammalian pigmentation (kim and uyama, 2005; carletti et al., 2014; takekoshi et al., 2014). for marine molluscs, flavonoids are largely synthesized in microalgae and ingested by filter-feeding (goiris et al., 2014). we therefore speculate that the higher expression level of cytochrome p450 in the left mantle than the right mantle of p. yessoensis may be related to flavonoid biosynthesis due to the ingested microalgae, which could probably inhibit tyrosinase activity and promote melanin biosynthesis, and eventually result in the reddish-brown pigmentation in the left shell. the evidences for tissue-specific gene expression of p450 have been uncovered in this study. as reported, the gills of bivalve molluscs are composed of curtains of filaments held in alignment by apposed patches of adherent cilia, which is the first tissue in contact with xenobiotics (reed-miller and greenberg, 1982). the present results revealing the highest level of p450 expression in gill of p. yessoensis may suggest the potential role of p450 involved in degradation of xenobiotics (carletti et al., 2014). however, further study is still needed to investigate the functional role of p450 in this species. in the natural living condition of p. yessoensis, the left shell with brown-color is always at the upper side position, while the right shell with white-color is located at the bottom contacting with the seafloor. the nature living state of the scallops is potentially associated with their left-right asymmetric pigment pattern, implying the adaptive evolution of external phenotypes in this species. in the present study, the differently expressed genes between the two shells are speculated to be responsible for the appearance of dark color in the scallop, which may have the potential shielding effects relating to molluscan defensive adaptations against predators in order to maximize survival when they live in the natural environment. the present findings will provide insights into the molecular basis for the left-right asymmetric pigment pattern in scallops, as well as other molluscs. conclusion in conclusion, we detect the significantly different expression of cytochrome p450 and four unannotated genes between pigmented (left) and non-pigmented shells (right) in the scallop p. yessoensis by transcriptome sequencing of the mantle tissue collected from the same scallops. the identified genes are suggested to be associated with the left-right asymmetric pigment pattern in the scallops. the findings will provide insights into the molecular basis for the left-right asymmetric pigment pattern in molluscs, and supply valuable information for their adaptive evolution of external phenotypes, which may maximize survival of scallops in natural condition. acknowledgements this work is supported by the grants from the basic scientific research fund of ysfri (no. 2060302201516054), zhejiang provincial top 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reproductive status. the phenoloxidase activity was activated by trypsin and inhibited by phenoloxidase activity specific inhibitor phenylthiourea. our results demonstrated that both in males and females, there were no significant differences in the basal phenoloxidase activity between reproductive and virgin beetles, while the total phenoloxidase activity increased significantly in virgin specimens. thus, resources seem to be invested to increase the humoral response in pre-reproductive phase forming a barrier against pathogens and preserving the fecundity and longevity of both sexes. the hemolymph dopambth assay on polyacrilamide gel electrophoresis showed a high activity of monomeric form with an apparent molecular weight of 90 kda and a dimer of about 170 kda, also multimeric bands were present in both sexes. in the sds-page general protein pattern, specific bands were evident for reproductive and virgin males and females as biochemical markers of sexual difference in immunocompetence. reproducible differences in peaks were recorded in hplc analysis performed on virgin and reproductive males and females key words: ecological immunology; life history; hplc; phenoloxidase; sexual dimorphism; sds-page introduction the gender-related differences in immunological competency have become of great interest at both physiological and evolutionary level. previous studies on dimorphism of immune response have investigated optimal allocation of resources between immunity, survival and reproduction in males and females (sequeira et al., 1995; kurtz et al., 2000; cheng and chen, 2001; stoehr and kokko, 2006; calleri et al., 2007; mckean and nunney, 2005, 2008; nunn et al., 2009; matozzo and marin, 2010; schwenke et al., 2016). the outline of defence strategy may vary generally under environmental selective pressure as a result of a wide range of factors (ecological, genetic and evolutionary) closely related to different life histories for males and females (rolff, 2002; viney et al., 2005; stoehr and kokko, 2006; zuk, ___________________________________________________________________________ corresponding author: matteo cammarata laboratory of marine immunobiology university of palermo via archirafi 18, palermo, italy e-mail: matteo.cammarata@unipa.it 2009; restif and amos, 2010). reproduction, growth, development and immune responses contribute to an animal’s fitness and the trade-off between them is likely due to alternative allocation of limiting energetic resources (zuk and stoehr, 2002; rolff and siva-jothy, 2003; schmid-hempel, 2003; sadd and schmid-hempel, 2009; vincent and gwynne, 2014). it is important to appreciate that plastic physiological trade-offs between reproduction and immune competence are mainly a matter of optimization rather than maximization to preserve the individual survival. studies on insect immunity have measured a number of immune effectors related to both cellular and humoral reactions, focusing on defence variation in both evolutionary and ecological context (gillespie et al., 1997; nappi and ottaviani, 2000; schmid-hempel and ebert, 2003; schmid-hempel, 2003, 2005; sivajothy et al., 2005; ottaviani, 2005). the background levels of these traits in both sexes can be measured in the absence of infection (constitutive immunity) (rantala and kortet, 2003) or after presenting an immune elicitor (induced immunity) (adamo, 2004; mailto:matteo.cammarata@unipa.it 166 galicia et al., 2014). immune traits that are commonly measured include survivorship of pathogenic infection, count or activity of circulating haemocytes, antimicrobial activity, encapsulation and phenoloxidase activity (ratcliffe et al., 1985; adamo et al., 2001, 2004; mckean and nunney, 2001; rolff, 2001; ahmed et al., 2002; schwarzenbach et al., 2005; meylaers et al., 2007; stoehr, 2007; córdoba-aguilar et al., 2009; lindsey and altizer, 2009; winterhalter and fedorka, 2009; shi and sun, 2010; piñera et al., 2013; galicia et al., 2014; vincent and sharp, 2014; cappa et al., 2015). the prophenoloxidase-activating (propo) system comprises a complex cascade of serine proteases allowing the conversion of prophenoloxidase to phenoloxidase (po) (marmaras et al., 1996; gillespie et al., 1997; rolff and siva-jothy, 2003; schmid-hempel, 2005; sivajothy et al., 2005). this enzymatic complex has been involved in physiological processes such as the cuticular melanization and sclerotization and the defence reactions (wounding, clotting, melanotic incapsulation, production of cytotoxic molecules) (marmaras et al., 1996; moreno-garcia et al., 2012). the main role of po in melanogenesis is to convert phenols to quinones that subsequently polymerize to form melanin. in immune defence, natural activators of the propo system are pathogen cell surface molecules such as β-1,3 glucans from fungal cell walls and lipopolysaccharides (lps) and peptidoglycans from microbial cells. the melanin deposited onto the foreign target prevents the pathogen growth and reproduction and thus melanization is an important cell-mediated immune response in tissue repair and in pathogen sequestration (söderhäll et al., 1994; nappi and vass, 2001; cerenius and söderhäll, 2004; nappi and christensen, 2005; gonzález-santoyo and córdoba-aguilar, 2012). we recently demonstrated that carabus lefebvrei is a good new model system to test on the one hand immune strategies to enhance the fitness of each life stage (giglio et al., 2008; giglio and giulianini, 2013; giglio et al., 2016) and on the other hand gender-specific immune responses for po and lysozyme-like enzyme activities (giglio et al., 2016). c. lefebvrei is an italian endemic carabid beetle that lives in beech, oak, chestnut and pine forests of the central and southern apennines, from lower altitudes to about 1500 m a.s.l. it reproduces in spring, is active from april until september and hibernates as adults (thiele, 1977; turin et al., 2003; giglio et al., 2009). the habit of adults and larvae is typically that of a snail-eating predator and males are smaller than females (turin et al., 2003; giglio et al., 2012). in this study, to add new information, we characterized the specificity of propo enzymatic activity and hlpc and sds-page profiles of hemolymph fractions as immunity marker to evaluate the difference between males and females in c. lefebvrei immunocompetence. laboratory tests were designed to compare virgin adults in their prereproductive phase with reproductive females and males after mating. material and methods insect rearing and hemolymph collection c. lefebvrei females and males were handcollected in the catena costiera mountains (39°19’ n, 16°7’ e, 900 1000 m a.s.l.; southern italy, calabria) in early spring 2014. these adults are emerged in the early summer of previous year and hibernating under rotten pine barks in winter. in the laboratory, beetles were sexed looking at the copulatory apparatus and divided in groups (males and females) in 10 l plastic boxes, filled with 6.0 cm with moistened humus. the specimens were reared with a light regime of l/d = 15/9 h, 70 % relative humidity and a day/night room temperature of 23/18 °c. they were fed with snails (helix sp.) and daily observed until specimens show reproductive behaviour (mating events). after copulation, males were removed from the boxes to reduce the disturbance to females, which readily laid eggs. the eggs were transferred singly into 150 ml glass jars filled to a depth of 4.0 cm with moistened humus. egg production and larval developmental time were recorded every two days until pupal instar and imago appearance. the hemolymph was collected from newly emerged adults 15-days-old (virgin females, n = 17 and males, n = 16) in their pre reproductive phase and from reproductive females and males two days after mating events (see above) attesting their reproductive status (n = 15 reproductive adults for both groups). the animals were co2 anesthetized before hemolymph collection. the hemolymph was collected by puncturing adults at the ventral level of the pro-mesothorax articulation with a 29-gauge needle. the first droplet of 10 µl of hemolymph was collected. each hemolymph sample was immediately transferred into 190 μl ice-cold pbs (10mm sterile phosphate-buffered saline, sigmaaldrich) in a 1.5 ml eppendorf tube and centrifuged at 1,700g for 5 min at 4 °c. the cell-free hemolymph obtained as supernatant was collected and stored at -20 °c until enzymatic assays. phenoloxidase specificity, zymogen activation and specificity of reaction for determining enzyme specificity and zymogen activation, the po activity was measured spectrophotometrically according to winder and harris (1991), using 3,4-dihydroxy-l-phenylalanine (l-dopa, sigma-aldrich) as substrate and 3methyl-2-benzothiazolinone hydrazone hydrochloride (mbth, 6 mm) as a specific reagent. briefly, 40 µl of hemolymph-buffer solution were incubated for 30 min at 20 °c with 40 µl of pbs (na2hpo4 1m, kh2po4 0.01 m; nacl 1.5 m, ph 7.4) and 40 µl of dopa-mbth reaction mixture (20 mm l-dopa and mbth in distilled water). after the reaction, dopaquinone was detected within 60 min at 5 min intervals by spectrophotometric measurement at 505 nm. the po activity was expressed as units (us) for min, where one u was defined as the amount of enzyme required to produce an increases in the absorbance at 505 nm of 0.001 unit min-1 for mg of protein. phenoloxidase 167 table 1 phenoloxidase activity of cell-free hemolymph in reproductive and virgin males and females of c. lefebvrei the enzymatic activity of control (basal po), trypsin treated-hemolymph (total po) and hemolymph incubated for 20 min with po inhibitor was expressed as u/min/mg protein. values are the mean of six experiments performed in triplicate ± sd. means (±sd) followed by the same letter are significantly different a: respect to the control; b respect to the opposite reproductive stage; c: respect to the opposite reproductive sex (tukey t test p < 0.05). activity in hemolymph was detected without (control) and with the addiction of trypsin, which enzymatically actives po from its inactive zymogen, pro-po. we thus measured basal (control) and total po activity in both female and male samples at different reproductive status to check for specificity of the enzyme reaction, before l-dopa and mbth were added, the hemolymph-buffer solution was incubated (20 min at 20 °c) with trypsin or with the 1-phenyl-2-thiourea (ptu) or diethilthiocarbamate (detc) in pbs at 1 mm final concentration. this inhibitor acts by chelating the copper at the active site, and it is known to be one of the most effective po inhibitor (kahn, 1985; aspàn et al., 1995; klabunde et al., 1998). electrophoresis and po activity of haemolymph po activity was assessed by polyacrylamide gel as described by cardenas and dankert (2000) with some modification. the haemolymphs from reproductive and virgin females and males showing po activity, were subjected to electrophoresis in polyacrylamide gel performed according to the method of laemmli (1970) using a mini protean ii cell (bio-rad). the gels were calibrated with high molecular weight range standard protein (sigmaaldrich (usa). to identify the po activity of the protein bands, the gels were washed twice with pbs-t (nacl 0.1 m; kcl 0.02 m; kh2po4 0.01 m; na2hpo4 0.06 m, ph 7.4, 2.5 % di triton x-100), and a final wash of 10 min. in pbs 1x without triton x-100. the gel was incubated in a solution containing l-dopa 20 mm and mbth 6 mm in distilled water. after 1 h of incubation, the gel was washed several times in distilled water. in relation to the regression curve obtained by means of the software alpha ease fc molecular weights of the obtained bands were calculated. sds-page (15 %, unless otherwise indicated) was performed also according to the method of laemmli (1970) and proteins were stained with coomassie blue. gels were calibrated with molecular mass markers (sigmamarker low range 6,5 66 kda, sigmaaldrich), and the calculated kda was the average ± sd of 6 distinct analyses. prior to electrophoresis, samples were boiled for 5 min in a sample buffer containing 5 % β-2-mecaptoethanol as reducing agent, unless otherwise indicated. software alpha ease fc stand-alone v.4.0 (spot density tools) was used to calculate the integrated density value (idv: sum of all the pixel values after background correction/area) of the stained protein bands. hplc size exclusion chromatography the hemolymph from both reproductive and virgin females and males was subjected to hplc separation silica column c18 interchrom upsodb25qs 250x4.6mm on a liquid chromatography hplc system (shimadzu scientific instruments, ssi, north america). column was washed with tbs (nacl 150 mm, tris hcl 10 mm, ph 7.4). an injection volume of 200 μl was used at a flow rate of 1 ml/min for 30 min. the chromatogram was recorded with a uv detector at 280 nm (mau) in tbs. the eluate corresponding to each peak was collected in 1 ml/min fractions. the collected fractions were concentrated by centrifugation at 500g with micro-concentrators (3k omega centrifugal devices nanosep), and the final concentrated samples were stored at -80 °c until use. hemolymph protein content protein content was estimated by the method of bradford (bradford, 1976) with bovine serum albumin (bsa) as a standard using 100 µl of hemolymph-buffer solution for each sample and 900 µl of bradford reagent (sigma-aldrich). statistical analyses po enzyme activities were measured and compared among reproductive female and male treatments phenoloxidase activity reproductive virgin male female male female controls 8.98 ± 0.8 7.46 ± 0.65 4.51 ± 2.8 b 5.49 ± 1.2 b trypsin (0.5mg/ml) 11.41 ± 0.9 abc 9.03 ± 0.91 abc 18.6 ± 6.6 ab 20.6 ± 2.2 ab po inhibitors ptu (1 mm) 3.54 ± 0.66 a 1.25 ± 0.34 a 1.2 ± 5.3 a 1.2 ± 2.2 a detc (10 mm) 2.53 ± 0.84 a 3.63 ± 0.54 a 1.1 ± 3.1 a 1.2 ± 1.3 a 168 table 2 statistical significant summarising the comparison results of tukey t test of phenoloxidase activity of cellfree hemolymph in reproductive (rm) and virgin males (vm) and females (rf and vf) of c. lefebvrei control (basal po) trypsin (total po) rm rf vm vf rmt rft vmt vft control rm nss p < 0.05 nss p < 0.05 nss p < 0.05 p < 0.05 (basal po) rf p < 0.05 nss p < 0.05 p < 0.05 p < 0.05 vm p < 0.05 p < 0.05 p < 0.05 p < 0.05 vf p < 0.05 p < 0.05 p < 0.05 p < 0.05 trypsin rmt nss p < 0.05 p < 0.05 (total po) rft nss p < 0.05 vmt nss the enzymatic activity of control (basal po), trypsin treated-hemolymph (total po). samples as well as virgin ones. student’s t-test was used to estimate statistical significance. multiple comparisons were performed with one-way analysis of variance (anova), and different groups were compared by using tukey’s t-test. the software package statistica 5.5 (statsoft, tulsa, ok, usa) was used for statistical analyses. standard deviations were calculated on four experiments. pvalue ≤ 0.05 was considered statistically significant. results characterization of phenoloxidase activity and zymogen activation a lower po activity was found when the samples were assayed in the absence of activating enzyme treatment (control, table 1). whereas the enzymatic activity increased significantly when samples were incubated with trypsin indicating the existence of inducible proenzyme. the specificity of the po reaction was demonstrated in both females and males at different reproductive status by the specific inhibitor ptu and detc added to hemolymph-buffer solution before the activation with trypsin. po activity, after inhibition with ptu and detc, compared to untreated (control) or trypsin activated samples, was lowered (tables 1, 2). the residual enzymatic activity could be attributable to the other class of enzymes like laccases or peroxidases. a significant increase of the total po activity in trypsin activated samples were detected for females and males of c. lefebvrei at the different reproductive status (tables 1, 2). moreover, there were no significant differences in the basal po activity between reproductive and virgin adults, while the total po activity increased significantly in virgin specimens (p ≤ 0.05). sds page and po activity the sds page are conducted with the same total protein concentration to evaluate differences and similarity among reproductive and virgin females and males. specific bands of an apparent molecular weight of 90 and 105 kda clear mark the difference between virgin and reproductive males and females (fig. 1, lanes 1 and 4 arrows). another specific band of an apparent molecular weight of 45 kda seems to be specific for the adult females (figs 1a, c). the protein size analysis and dopa-mbth assay, disclosed two active proteins with an apparent molecular weight of 90.0 and 170.0 kda at different concentrations, whereas, due to the sds activating effect on the proenzyme, the propo activation process could not be shown, whereas modulation of an oligomerization process can be suggested (fig. 1b). high pressure liquid chromatography analysis the chromatographic profiles of reproductive males and females displayed several confluent peaks in term of elution time and concentrations except for peaks indicated by arrows (fig. 2a). peaks marked by arrows at 3.5, 3.75 and 5.75 min/ml showed higher concentration in reproductive females than males. whereas, at 11.5 and 18 min/ml elution time peaks showed that the concentration is lower in reproductive females than males. the chromatographic profiles of hemolymph for virgin male and female were overlapped (fig. 2b). during the 40 min of profile separation running, several different peaks are revealed but only one peak at 7.25 min/ml of elution time showed a very high different concentration in virgin female compared with males. in females, an overlapped of comparative chromatographic profiles in reproductive and virgin females was recorded (fig. 3a). several common peaks with similar amplitude were identified between reproductive and virgin but with one very clear difference in concentration for the peak at 7.25 min/ml of elution time in virgin females. in males, three additional peaks respectively at 11.75, 18 and 21.5 min/ml seem to be characteristic of virgin males compared with reproductive ones (fig. 3b). 169 fig. 1 electrophoretic patterns of the hemolymph show male and female samples at different reproductive status performed with sds-page/coomassie blueunder reducing conditions (a), sds-page-dopa-mbth in nonreducing conditions (b) and sds-page/coomassie blue in non-reducing conditions (c). the arrows indicate specific bands that clearly show the difference between virgin and reproductive males and females. st: standard; lane 1: reproductive males; lane 2: reproductive females; lane 3: virgin males; lane 4: virgin females. discussion the gender-related immunomodulation could be strategic for physiology, adaptation and evolution of animals. previous study on c. lefebvrei showed that gender differences occur only for po activity that was significantly higher in reproductive males than females but not for lysozyme-like enzyme activity (giglio et al., 2016). to exclude that other enzymes with similar activity (peroxidases, laccases and catalases) are involved in the reaction of the enzymatic activity, we characterized and displayed calcium-independent po activity enhanced by trypsin. phenylthiourea and detc, a specific po inhibitor, supported the reaction specificity and trypsin treatment significantly enhanced. our data on po activity, protein contents and hplc analysis confirmed that the investment strategy in immunocompetence of c. lefebvrei adults varies with reproductive status to balance for resource allocation between physiological and ecological costs in both sexes. after pro-po activation with trypsin, virgin females and males of c. lefebvrei showed higher values of total po enzyme activities compared with reproductive adults, while basal po enzymatic activities was significantly higher in reproductive ones. the higher level of pro-po enzyme in virgin adults confirms that the inducible po activity is likely to preserve the adult survivorship against infections until the reproductive phase to maintain an effective protection increasing the organism’s fitness. furthermore, the reduction of immune function in reproductive adults is a result of increased reproductive activity in absence of infection (rolff and siva-jothy, 2002; fedorka et al., 2004; otti, 2015). the protein content in hemolymph of the reproductive males and females were found to be about five time more concentrated than virgin status. the protein content from the hemolymph subjected to sds-page and stained with coomassie blue shows two specific bands with apparent molecular weight of 90 and 105 kda that clear mark the difference between virgin and reproductive adults. another specific band with an apparent molecular weight of 45 kda seem to be specific for reproductive females. the same band stained with dopa-mbth assay on polyacrilamide gel electrophoresis shows a canonical high concentration of monomeric/dimeric form showing an high activity, also multimeric form are present both in reproductive and virgin males and females. the overlapping of the chromatographic profiles of males and females sample at different reproductive status highlights that the haemolymph of reproductive and virgin females present the highest peaks in term of concentration, with the exception of the peak at 11.5 min/ml in both status and that 18.0 min/ml for reproductive status. the overlapping of the chromatographic profiles of reproductive and virgin females males shows in both cases the highest peaks for virgin status. this first finding strengthened the evidence that on the one hand the direction and magnitude of sex differences in immune-competence could be different for each component of immune defence (zuk and stoehr, 2002; stoehr and kokko, 2006; stoehr, 2007). one the other hand, males and females may emphasize different immune components in relation to age, mating, sexual antagonism and attractiveness or food availability (adamo, 2001; lawniczak et al., 2006; stoehr, 2007; córdoba-aguilar et al., 2009; winterhalter and fedorka, 2009; kivleniece et al., 2010; galicia et al., 2014; vincent and gwynne, 2014; vincent and sharp, 2014). c. lefebvrei males gain fitness by investing heavily in immunity to protect themselves from an exposure to parasites due to their larger number of mating events. the high level of activable 170 fig. 2 hplc separation of the hemolymph from both female and male at different reproductive status using silica column c18 interchrom upsodb-25qs 250x4.6mm on a liquid chromatography hplc system. elution was performed with tbs over 40 min at a flow rate of 1 ml/min. absorbance peaks were monitored at 280 nm. a) overlapping of the chromatographic profiles of reproductive males and females. b) overlapping of the chromatographic profiles of virgin males and females. the arrows indicate the different concentration of the confluent peaks of males and females. po proenzyme present in the virgin males and females support the hypothesis that in reproductive females and males the immune response decrease because they shift resources from propo hemolymphatic activity to other physiological systems involved in reproduction including production of eggs, reception and storage of sperm, fertilization and oviposition. in conclusion, here we found that the po activity and protein composition, show variation over the lifetime of c. lefebvrei males and females. the sexual dimorphism was recorded for po activity and marked by specific band in page and hplc analyses. this result confirms that immune function is not a simple, static process, but rather a dynamic system of interrelated mechanisms that are differentially effective and may not be generalizable among species. immunity and the ability to reproduce are closely related in order to maximize the fitness of each species (schmid-hempel, 2003; schulenburg et al., 2009) and sexual differences in immune investment are difficult to predict and the investment of high energies amount to immunocompetence is not always the best choice in term of fitness. 171 fig. 3 hplc separation of the hemolymph from both female and male at different reproductive status using silica column c18 interchrom upsodb-25qs 250x4.6mm on a liquid chromatography hplc system. elution was performed with tbs over 40 min at a flow rate of 1 ml/min. absorbance peaks were monitored at 280 nm. a) overlapping of the chromatographic profiles of reproductive and virgin females. b) overlapping of the chromatographic profiles of reproductive and virgin males. the arrows indicate the different concentration of the confluent peaks of males and females. acknowledgements this work was supported by the grant (n° a.001.2014.ex60) 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(lepidoptera: pyralidae) and its endoparasitoid pimpla turionellae (l.) (hymenoptera: ichneumonidae) d özyılmaz1, r özbek 1,3, h altuntaş2*, f uçkan1 1department of biology, faculty of science and literature, kocaeli university, kocaeli, 41380, turkey 2department of biology, faculty of science, eskisehir technical university, eskişehir, 26470, turkey 3current institution: fraunhofer institute for molecular biology and applied ecology, department of bioresources, giessen, 35394, germany accepted october 21, 2019 abstract investigation of the antioxidant and oxidative effects of dietary indole-3-acetic acid (iaa), a plant growth regulator, on pest galleria mellonella l. (lepidoptera: pyralidae) and its endoparasitoid pimpla turionellae (l.) (hymenoptera: ichneumonidae) was aimed in this study. different doses of dietary iaa (50-10,000 ppm) caused an increase in lipid peroxidation in the hemolymph of the host, g. mellonella (l.) and its endoparasitoid p. turionellae (l.). when compared to the control, higher doses of dietary iaa decreased cat, sod and gst enzymes’ activities in g. mellonella. at higher iaa doses, the activity of sod enzyme in the hemolymph of p. turionellae significantly decreased while cat enzyme activity showed no significant change when compared to the control. additionally, gst activity in the endoparasitoid larval hemolymph significantly increased at 500 and 1000 ppm iaa doses. these findings indicate that incorporating iaa in the diet of model host g. mellonella larvae leads to oxidative stress and, also negatively affects the survivability of both the host and its endoparasitoid. key words: pimpla turionellae; galleria mellonella; indole-3-acetic acid; host-parasitoid interaction; oxidative stress introduction auxins are plant growth regulators (pgrs) that are involved in many developmental processes, including cell division and enlargement, root initiation, vascular tissue differentiation and flowering (davies, 2010). indole-3acetic acid (iaa) is also one of the important natural auxins in most plants (davies, 2010). synthetic iaa products are used widely in agricultural processes like plant growth and development in order to increase productivity (kumar et al., 2001). because of the wide usage of these indolic compounds as phytohormones or pgrs in the environment, nontarget organisms such as biological control agents could be affected negatively. several previous studies reported that iaa caused adverse effects on survival, longevity, developmental time, hemocytes responses and hemolymph metabolites of various lepidopteran pest species (rup et al., 2002; kaur ___________________________________________________________________________ corresponding author: hülya altuntaş department of biology eskisehir technical university eskişehir, 26470, turkey e-mail: hyalcitas@eskisehir.edu.tr and rup, 2003; uçkan et al., 2011a; uçkan et al., 2014; uçkan et al., 2015; çelik et al., 2017). various authors also suggested that pgrs such as gibberellic acid (ga3), ethephon (etf) and iaa could be used instead of insecticides to control lepidopteran pests in integrated pest management programs (uçkan et al. 2011b; 2014; altuntaş et al., 2012; altuntaş, 2015a). on the other hand, uçkan et al. (2011a) reported that iaa adversely affects the life history traits of the endoparasitoid apanteles galleriae (hymenoptera: braconidae), an important natural enemy of g. mellonella. it is possible that these observed biological effects of iaa on various pests and parasitoid species could be associated with their immune responses. antioxidant systems and immune mechanisms in insects play an important role in the detoxification of several organic and inorganic environmental pollutants (felton, 1995). previously, researchers also showed that dietary pgrs, ga3, and etf, at sublethal doses caused adverse impacts on various antioxidant enzymes and led to oxidative stress in model insect g. mellonella (altuntaş, 2015b; altuntas et al., 2016). for these reasons, the adverse biological effects of iaa on insects mailto:hyalcitas@eskisehir.edu.tr 185 might be related to their physiological antioxidant capacities. in order to provide information about toxic modes of action of iaa on target and nontarget insects, we used the model host (greater wax moth) g. mellonella l. (lepidoptera: pyralidae) and idiobiont, solitary, pupal endoparasitoid p. turionellae l. (hymenoptera: ichneumonidae). it is well known that g. mellonella larvae are used as a model insect in ecotoxicological and ecophysiological investigations or model host for several parasitoid species in biological control programs because their culture rearing conditions are economic, easy and faster in the laboratory (altuntaş et al., 2016; kwadha et al., 2017). therefore, the effects of various doses (50-10,000 ppm) of dietary iaa on activities of superoxide dismutase (sod), catalase (cat), glutathione stransferases (gsts) and also lipid peroxidation level in host galleria mellonella and its pupal endoparasitoid pimpla turionellae larvae were investigated for the first time in this study. materials and methods insect rearing the laboratory colonies of the model host galleria mellonella and pupal endoparasitoid pimpla turionellae were reared at 25 ± 5 °c, 60 ± 5 % rh, and with a photoperiod of 12: 12 (l: d) in kocaeli university, turkey as described before (uçkan et al., 2015). first instar larvae of the host were maintained by feeding on artificial diet as described by altuntaş et al. (2016). the last instar larvae of g. mellonella were collected and put in jars including folder paper to facilitate pupation. then, host pupae used for parasitization by female adult p. turionellae. adult parasitoids were also reared on 50% (wt: vol) honey solution in cages (25 x 25 x 25 cm). bioassays selected doses (0, 50, 500, 1,000, 5,000, and 10,000 ppm) of iaa (merck 10 g, darmstadt, germany) were used in all experimental analyses. all doses of iaa were prepared in distilled water and homogenized with an artificial diet (altuntaş et al., 2016). to determine the effects of iaa on the antioxidant enzyme activity in g. mellonella and its parasitoid p. turionellae, newly hatched g. mellonella larvae were exposed to 5 g of host artificial diet including the selected doses of iaa. in parallel experiments, larvae were fed with an artificial diet containing distilled water; these were treated as the control group. thus, for each experimental and control assay, eight last instars g. mellonella larvae (0.25 – 0.30 mg) were used in three replicates (n = 24). in addition, selected last stage g. mellonella larvae treated or untreated with iaa doses were used pupated and provided as host to p. turionellae for parasitoid experiments. therefore, in each experimental analysis, eight 6d old p. turionellae larvae (almost 8 d after parasitization) were used in three replicates (n = 24 larvae). hemolymph collection and storage hemolymph samples were collected from the last stage g. mellonella and p. turionellae larvae for experimental analyses. each larva was pierced on the second foreleg with a sterile microneedle and the was hemolymph collected via a 10 µl glass microcapillary tube (sigma, st. louis, mo). ten microliters of hemolymph were collected from each of the eight larvae and immediately transferred into the same two ml eppendorf tube containing 0.001 mg 1-phenyl-2-thiourea in order to avoid hemocyte aggregation and melanization. during hemolymph collection, collection tubes were kept on ice and then stored at -80 °c until enzyme activity assays. on the same day of assays, samples were centrifuged at 7000 rpm for 10 min at 4 °c and the supernatant transferred to a new collection tube and keep on ice. antioxidant enzyme activities and malondialdehyde levels protein concentrations of hemolymph samples were determined by using bradford reagent (sigma) according to 96 well plate method, and bovine serum albumin was used to create a standard curve. sod and gst activities were evaluated by using commercial kits from cayman (cayman chemical, ann arbor, mi). the sod activity was measured at 450 nm in a microtiter plate (bmg labtech) using xanthine and xanthine oxidase systems and defined as u/mg protein. gst activity was read continuously at 340 nm for 5 min in a microtiter plate (bmg labtech) using 1-chloro-2,4dinitrobenzene (cdnb) and reduced glutathione (gsh) as substrates and the activity was defined as µmol/ min/mg per protein. cat activity analysis was performed according to chance and maehly (1995). the decrease in absorbance over a 10 min period at 240 nm due to h2o2 decomposition was measured in this assay. the absorbance of cat activity was read in uv/vis spectrophotometer and thus, the activity was defined as mmol/min/mg per protein. malondialdehyde (mda, a product of lipid peroxidation) levels in hemolymph samples were also determined using a commercially available kit protocol (cayman chemical, ann arbor, mi). according to the protocol, mda in hemolymph samples was incubated with thiobarbituric acid (tba) at 95 °c and thus absorbance was read at 530 nm in a microtiter plate (bmg labtech). the content of mda was determined as the µm/mg per protein. statistical analysis all data were represented as mean ± standard error (se). the spss software program (version 18.0 for windows, chicago, il) was used for statistical analysis. dose-dependent changes in the antioxidant enzymes and mda level were verified to be normally distributed. to compare means, anova (one-way analysis of variance) and to determine the significant differences lsd-post hoc tests (least significant difference) were conducted. the results obtained in the experiments were evaluated as being statistically significant at a 95 % confidence interval with p ≤ 0.05. 186 table 1 effects of various doses of iaa on cat, sod, gst activities and mda level in larval hemolymph of g. mellonella mean ± se* iaa doses (ppm) cat sod gst mda 0 0.62 ± 0.04a 8.51 ± 0.23a 1.15 ± 0.10a 69.22 ± 17.81a 50 0.56 ± 0.03a 8,51 ± 0,00a 0.82 ± 0.02ab 156.28 ± 24.13b 500 0.34 ± 0.02b 5.06 ± 0.23b 0.65 ± 0.12b 197.03 ± 20.05b 1,000 0.38 ± 0.02b 6.44 ± 0.23c 0.43 ± 0.07b 194.02 ± 27.52b 5,000 0.42 ± 0.02b 5.98 ± 0.23bc 0.48 ± 0.12b 157.65 ± 27.02b 10,000 0.43 ± 0.02b 5.75 ± 0.23bc 0.56 ± 0.09b 175.81 ± 37.01b *means ± standard errors within each column followed by the different letter (a-c) indicate significant differences (p ≤ 0.05, lsd test). cat: catalase (mmol/min/mg protein), sod: superoxide dismutase (u/mg protein), gst: glutathione s transferase (µmol/min/mg protein), mda: malondialdehyde (µm/mg protein) results and discussion our data showed that treatment of g. mellonella larvae with diet containing iaa caused a decrease in the activities of antioxidant enzymes; cat (f = 16.351; df = 5, 18; p < 0.05), sod (f = 56.692; df = 5, 18; p < 0.05) and gst (f = 8.448; df = 5, 18; p < 0.05) at high doses 500, 1000, 5000 and 10000 ppm as compared to control. in particular, significant reductions in cat and sod enzyme activities in the hemolymph of g. mellonella larvae were observed at 500 ppm (> 40 %), also, gst enzyme activity increased by more than 60 % at 1000 ppm. similar to present findings, altuntaş (2015) revealed that activities of antioxidant enzymes in larval hemolymph of g. mellonella did not change at higher doses of dietary ga3 but increased at lower doses of this pgr. on the other hand, shayegan et al. (2019) also showed dosedependent inducing effects of ga3 on sod and cat activity of helicoverpa armigera larvae. further, an important finding presented in this study was the sharp decrease observed in cat and sod activities in host hemolymph at 500 ppm dose of iaa similar with ga3 doses reported previously by altuntaş (2012). iaa treatment also caused an increase in g. mellonella mda levels at all doses. as compared to control, the most effective iaa dose 500 ppm increased mda levels in hemolymph of larvae by 185% (f = 2.918; df = 5, 18; p < 0.05) (table 1.). similar to previous studies conducted with mammals showed that exposure to different iaa concentrations increased the lipid peroxidation, inhibited antioxidant response in various rat tissues (tuluce and celik, 2006), and also decreased cat activity in the kidney of the f2 generation of mice (yılmaz et al., 2004). altuntaş (2015) also reported that activities of antioxidant enzymes in larval hemolymph of g. mellonella did not change at higher doses of dietary ga3 but increased at lower doses of this pgr. on the other hand, shayegan et al. (2019) showed dose-dependent inducing effects of ga3 on sod and cat activity of helicoverpa armigera larvae. therefore, our findings demonstrated that exposing g. mellonella to iaa via larval diet leads to oxidative stress by elevating mda levels; it is known that an increase in mda levels is an important marker for oxidative stress and occurs naturally during lipid peroxidation. keeping in mind the similarity between the response of model insect g. mellonella and mammals to iaa may assist in the improvement of novel insectbased screening systems to measure the toxicity of pgrs or other environmental chemicals instead of using of mammals in biomonitoring tests. in addition, induced oxidative stress by xenobiotics causes cell death either by necrosis or apoptosis mechanisms (kannan and jain, 2000). increases in apoptotic activities in different tissue cells of mice treated with iaa were observed in early studies by furukawa et al. (2004). in another study, it was also reported that a plant growth regulator, ga3 induced apoptotic and necrotic cell death and reduced cell viability in ga3 treated g. mellonella larvae when compared to untreated larvae (altuntaş et al., 2012). furthermore, çelik et al. (2017) demonstrated that lower doses of dietary iaa caused an increase in apoptotic indices in achoria grisella (lepidoptera: pyralidae) larvae. for these reasons, these findings imply that dietary iaa treatment may cause excessive apoptosis by suppressing the antioxidant defense system in the host g. mellonella larvae. the endoparasitoid, idiobiont and solitary wasp p. turionellae is the most effective biological control agent against several lepidopteran pest species including model insect and storage pest g. mellonella. therefore, it is conceivable that p. truionellae could be exposed to iaa broadly used in agriculture during the adult stage that feeds on honey, fruit, and nectar or during the larval stage 187 table 2 effects of various doses of iaa on cat, sod, gst activities and mda level in larval hemolymph of p. turionellae mean ± se* iaa doses (ppm) cat sod gst mda 0 0.011 ± 0.003a 8.48 ± 0.36a 0.03 ± 0.01a 31.75 ± 7.65a 50 0.026 ± 0.008b 8.10 ± 0.39a 0.04 ± 0.01a 77.54 ± 11.03a 500 0.010 ± 0.003a 6.49 ± 1.06b 0.09 ± 0.02b 116.60 ± 39.49b 1,000 0.009 ± 0.005a 6.79 ± 1.19b 0.07 ± 0.02b 257.26 ± 53.10bc 5,000 0.006 ± 0.001a 4.14 ± 0.49b 0.05 ± 0.01a 241.28 ± 24.74bc 10,000 0.008 ± 0.0016a 5.75 ± 0.94b 0.04 ± 0.01a 305.15 ± 30.86c *means ± standard errors within each column followed by the different letter (a-c) are significantly different (p ≤ 0.05, lsd test). cat: catalase (mmol/min /mg protein), sod: superoxide dismutase (u/mg protein), gst: glutathione s transferase (µmol/min/mg protein), mda: malondialdehyde (µm/mg protein) that feeds on host’s pupae. researchers have already shown the effects of iaa on different physiological properties such as developmental times, biochemical parameters, total, and differential hemocyte counts and apoptosis of different insects in the host-parasitoid relationship (uçkan et al., 2011a; 2014; 2015; çelik et al., 2017). zhao et al. (2017) also reported that treatment of aphids with dietary pgrs including iaa, naphthalene acetic acid (naa) and ga3, has negative effects on the parasitoids by reducing parasitism rates/abilities, emergence rate, and proportion of females. these negative influences of iaa3 on life-history parameters of parasitoids may be related to the suppression of antioxidant defense as well as immunological response. data obtained from this study also support the explanation stated above. therefore, in accordance with these previous studies (uçkan et al., 2011a; 2014; 2015; çelik et al., 2017; zhao et al., 2017), exposure of the endoparasitoid to iaa by host pupae increased mda levels increased significantly in a dosedependent manner (f = 12.045; df = 5, 18; p < 0.05), and altered sod, cat and gst activities in the larval hemolymph of p. turionellae with respect to control (table 2, p < 0.05). we found that endoparasitoid’s sod activity decreased in all iaa doses except at 50 ppm in comparison to untreated larvae (f = 3.829; df = 5, 18; p < 0.05). in contrast to the sod activity results, cat activity in the larval hemolymph of p. turionellae increased only at 50 ppm iaa dose compared to the control group, but no changes were observed in other doses of iaa. (f = 3.478; df = 5, 18; p < 0.05, table 2). however, gst activity in larval hemolymph of endoparasitoid increased at 500 and 1000 ppm doses of iaa as compared with control (f = 3.482; df = 5, 18; p < 0.05). mda level in larval hemolymph of p. turionellae also. this increase in mda level reached nearly more than 10 times that of the control group at the highest dose of iaa (table 2). interestingly, these results indicated that increase in lipid peroxidation was not inhibited despite the increase in gst activity, an important detoxification enzyme in the hemolymph of the larval endoparasitoid, at 500 and 1000 ppm iaa doses treatment. thus, this study, for the first time, showed that oxidative stress increased depending on the iaa3 doses the larvae of p. turionellae were exposed to through the host pupae. as a consequence, the iaa3-mediated toxic effects occurred in not only host antioxidant defense system, but also in the endoparasitoid p. turionellae. in addition, our study results are also important for the observation of toxic effects of iaa on trophic interactions between host and parasitoid species. in conclusion, iaa induced oxidative stress in the host and parasitoid insects could be a potential threat causing the negative influences on the survival of parasitoid species for biological control programs. thus, adult emergence time may happen in unfavorable environmental conditions. therefore, our study provides important information for the conscious use of iaa in agriculture so as to conserve the host-parasitoid interactions in the ecosystem. acknowledgments this research was supported by kocaeli university, scientific research projects commission, of turkey (project no: 2014068). authors would like to thank native speaker anthony david plancherel who is a lecturer at anadolu university for providing proof reading on this paper. references altuntaş h, kiliç a, uckan f, ergin e. effects of gibberellic acid on hemocytes of galleria mellonella l. 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hh, pan mz, sun yx, liu tx. the role of plant growth regulators in a plant– aphid–parasitoid tritrophic system. j. plant growth regul. 36: 868-876, 2017. isj 12: 118-128, 2015 isj 12: 118-128, 2015 issn 1824-307x review densovirus infection in silkworm bombyx mori and genes associated with disease resistance t gupta1, k kadono-okuda2, k ito3, k trivedy1, km ponnuvel1 1genomics division, seribiotech research laboratory, carmelaram post, kodathi, bangalore 560 035, india 2laboratory of sericultural science, department of science of biological production, graduate school of agriculture, tokyo university of agriculture and technology, 3-5-8 saiwai-cho, fuchu, tokyo, 183-8509, japan 3division of lnsect sciences, national institute of agrobiological sciences, 1-2 owashi, tsukuba, ibaraki 3058634, japan accepted april 13, 2015 abstract the silkmoth bombyx mori has been bred in captivity for around 5,000 years and it is now a completely domesticated species of the silkmoth. the larva of b. mori feeds only on mulberry leaves so it is a monophagous insect. silk cocoons obtained from this species are the primary source of commercial silk and this makes b. mori an economically important insect. however, the silk industry suffers significant losses due to various viral infections during the larval stages. one of the frequently affecting silkworm viruses is the b. mori densovirus (bmdv). the bmdv is further classified into two types: b. mori densovirus-1 (bmdv-1) and b. mori densovirus-2 (bmdv-2). however, bmdv-2 is excluded from the family of parvoviridae and is referred to a new family bidnaviridae. to date, three isolates of bmdvs have been reported. this virus has been found to be a causative agent of the commonly occurring fatal silkworm disease, ‘flacherie’. bmdvs have been found to be a highly diverse group of viruses. while most of the strains of b. mori are susceptible to bmdv, few races have been found to be completely resistant to the virus. studies have shown both dominant and recessive alleles to be responsible for the resistance. so far four genes have been reported conferring resistance against bmdv-1 and bmdv-2. these are the nid-1 and nsd-1 genes against bmdv-1 and nsd-2 and nsd-z genes against bmdv-2. details about densovirus with special reference to b. mori and the resistant genes present against it and the studies undertaken towards screening of bmdv resistant silkworm races have been discussed in this review. key words: bombyx mori; densovirus resistance; nsd-2; bidnaviridae   introduction the pathological condition of densoviruses (dvs) was studied in greater wax moth, galleria mellonella and these studies further led to the discovery of dvs (meynadier et al., 1979). ultrathin sections of tissues of heavily infected larvae were examined which revealed enlarged feulgen-positive nuclei. further studies of these histological sections showed the presence of electron-dense viral factories leading to production of thousands of small isometric particles (amargier et al., 1979). virus particles of sizes 20 22 nm with icosahedral non enveloped symmetry were isolated and then purified ___________________________________________________________________________ corresponding author: kangayam m ponnuvel genomics division seribiotech research laboratory carmelaram post, kodathi bangalore 560 035, india e-mail: kmpvel@yahoo.com from the dead larvae. chemical analysis revealed the presence of viral dna and other proteinaceous moieties. dvs were found to have biochemical and biophysical features resembling that of vertebrate parvoviruses. the analysis of virus particles showed that the capsid was made up of four proteins and contained a single stranded linear dna of 6 kb in size. in addition to this the dvs also shared the nuclear site of replication and the structural features with that of vertebrate parvoviruses and accordingly a new subfamily named densovirinae was established within the family of parvoviridae. dvs have been isolated from different insect orders including lepidoptera, hemiptera, diptera, dictyoptera and crustacea (bergoin and tijssen, 2010). the densovirinae subfamily densoviruses (dvs) belong to the family parvoviridae. viruses belonging to the parvoviridae 118   table 1 the present classification of the family parvoviridae (cotmore et al., 2014) family are known for having a wide host range. hence this family has been divided into two subfamilies namely, densovirinae and parvovirinae. viruses falling under the category of densovirinae infect invertebrates while those under parvovirinae infect vertebrates. the extension of taxonomy of the parvoviridae family is under review by the international committee on taxonomy of viruses (ictv). a set of proposals have been extended to introduce new species and genera into both the subfamilies, thereby resolving misclassified species and enhancing taxonomic clarity. consequently, the present classification of the family parvoviridae differs significantly from that of the old one (cotmore et al., 2014). the present classification includes affixes in the names of the genera which clarifies subfamily affiliation and reduces ambiguity that’s results from the vernacular use of parvovirus and densovirus to denote multiple taxon level. the present classification of parvoviridae family has been shown in table 1. similarly, the subfamily densovirinae has also been reclassified and comprises of five different genera. the classification of densovirinae subfamily has been discussed in detail as under. classification of densovirinae subfamily invertebrate densoviruses (dvs) form a distinguished subfamily (densovirinae) within the parvoviridae family and have recently been reclassified. all members belonging to the family of densovirinae have arthropod hosts with high sequence identities of 85 95 % (dumas et al., 1992). the present classification of densovirinae family includes five distinguished genera namely, ambidensovirus, brevidensovirus, hepandensovirus, penstyldensovirus and iteradensovirus as shown in detail in table 2. these five different genera have distinguished genomic organization with varying genomic sizes. the genetic make-up of dvs can be classified mainly into two types: the ambisense dvs that encode open reading frames (orfs) on both complimentary strands, while the monosense dvs that has only a single strand containing the orfs. overall dvs can be described as viruses having small isometric, non-enveloped capsids with a linear dna genome (table 3). however, viruses such as bmdv-2 do not match the description because unlike parvoviruses, its linear genomic dna is segmented and is twice as long. also, it codes for the enzyme dna polymerase (hayakawa et al., 2000). therefore, it has been reassigned to a new family bidnaviridae (tissen et al., 2011). the ambisense dvs package their single strands of dna in separate capsids so upon dna extraction double stranded genomes are obtained under high salt conditions. the brief description of the different genera of densovirinae subfamily is given below. ambidensovirus under the previous classification this genus was known as ‘densovirus’. ‘ambi’ is the new affix added to this name. this genus comprises of dvs following an ambisense mode of transcription. five different species have been grouped under this genus so far (table 2). the previously existing genus pefudensovirus has been incorporated into this genus. pfdv, bgdv1, cpdv, pcdv, dsdv, gmdv, hadv1, jcdv, mldv, pidv and addv are the viruses grouped under this genus. brevidensovirus this genus has the shortest (4 kb) genome among all the other genera of dvs (shike et al., 2000; zhai et al., 2008). they have been found to genus species viruses or variants subfamily parvovirinae-vertebrate hosts amdoparvovirus 2 2 aveparvovirus 1 2 bocaparvovirus 12 22 copiparvovirus 2 2 dependoparvovirus 7 23 erythroparvovirus 6 12 protoparvovirus 5 25 tetraparvovirus 6 10 subfamily densovirinae-arthropod hosts ambidensovirus 6 11 brevidensovirus 2 8 hepandensovirus 1 7 iteradensovirus 5 6 penstyldensovirus 1 4 119   infect several mosquitoes (including mosquito cell lines) and shrimp species. in addition to their small size, their genome varies from the other species of dvs by the absence of itrs. the itrs in this case are replaced with short (130 150) hairpin structures at the 3’ and 5’ ends. the coding sequence of their genome is organized into three overlapping orfs. the ns1 is encoded by a large left orf, the ns2 is encoded by the orf nested in the 5’ half of the orf coding ns1 and the vp capsid proteins are encoded by a short right orf. hepandensovirus the new genus hepandensovirus was mainly introduced to accommodate shrimp viruses under the family of densovirinae. these are called hepandensovirus to indicate the original name of these viruses, “hepatopancreatic parvovirus”. the virus group comprising pmohdv1, pchdv, pmohdv2, pmohdv3, pmedv, pmohdv4 and fchdv are included in this genus (table 2). penstyldensovirus this particular genus is another newly introduced genus under the family of densovirinae. the name stands for a siglum for penaeus stylirostris, the host and also the founding member of this species. pstdv1, pmopdv1, pmopdv2 and pstdv2 are the viruses included this genus. iteradensovirus this genus was previously known as iteravirus. the present classification has added the new affix ‘denso’ to the name. to date, these viruses have been described in five species of lepidoptera. bmdv, cedv, sfdv, dpdv, ppdv and hadv2 are the viruses included in this genus. members of the iteradensovirus genus possess a monosense genome of only 5.1 kb in size. this genus is characterized by the presence of two overlapping orfs in the left half encoding ns1 and ns2 polypeptides and a right half orf encoding the four or five vp proteins. the coding sequence of this genus has 230 nt itrs at both 3’ and 5’ end (bergoin and tijssen, 2010). however, the viruses of bmdnv2 and bmdv-z are excluded from this classification and is not included in the parvoviridae family as the viruses exhibit a bipartite genome and also codes for the enzyme dna polymerase. these have been put into a separate family of bidnaviridae. dvs can be described as small (25 nm), nonenveloped viruses with icosahedral symmetry having a single stranded un-segmented linear dna genome size of 4 6 kb in length. the genome of dvs contains two set of genes encoding nonstructural proteins (ns) and capsid proteins (vp). further, some dvs have a monosense genome organization wherein the gene products are encoded in tandem from a single dna strand while others have an ambisense genome organization wherein ns and vp coding sequences are located in the 5’ half of the complimentary strand. most dvs cause fatal diseases in their hosts. the members of this genus are characterized with a genome of about 6 kb in length possessing long (> 500 nt) inverted terminal repeats (itrs) (dumas et al., 1992; fediere et al., 2002). their genome has an ambisense mode of organization, with sequences encoding ns and vp proteins being located in the 5' half on the complementary strands. the two strands are encapsidated separately in equimolecular ratio during the course of virus morphogenesis. the ns polypeptides are encoded by sequences organized in three orfs on the ns strand designated by convention as the positive strand. a stop codon (taa) is the only codon that separates the left most orf encoding ns3 from the main orf encoding ns1. further, there exists a third orf that overlaps with the n-terminal region of ns1 and encodes ns2 in a different frame that of ns1. the p7 promoter controls the ns genes whose tata box and upstream promotor elements are located in the 3’ terminal sequence of the left itr. in case of the ns genes two transcripts sharing the same transcription start have been identified. the transcripts lead to the generation of two polypeptides of about 30 and 60 kda through the translation of the ns1/ns2 cassette by a leaky scanning mechanism. the ns1 aug codon through leaky scanning mechanism facilitates the second (ns2) aug codon (bergoin and tijssen, 2010). a single large orf occupying the 5’ half of the complementary strand consists of the vp gene. the p93 promoter controls the vp gene and this promoter located in the left most sequence of the right itr. the p93 promoter transcribes a single 2.5 kb vp mrna and four structural polypeptides vp 1 4 through leaky scanning mechanism. the itr sequences are highly conserved among members of the densovirus genus (bergoin and tijssen, 2010). tissue tropism dvs exhibit a wide range of tissue tropism. for example in g. mellonella, the virus replicates in almost all tissues except midgut epithelium. fat body, hypodermis, muscles, tracheal cells, malpighian tubules and hemocytes are among the most severely infected tissues (amargier et al., 1979). infection of ovaries and the central nervous system have also been reported (garzon and kurstak, 1968). tissue polytropism like this was observed in other dv-infected lepidoptera such as spodoptera littoralis, agraulis vanilla, diatarea saccharalis, pieris rapae and pseudoplusia includens. dvs infecting mosquitoes and crickets also show similar wide tissue tropism. contrary to this dvs belonging to the genus iteradensovirus replicate either exclusively (bmdv-1) or predominantly sibine fusca dv (sfdv) and casphalia extranea dv (cedv) in the midgut epithelium of the hosts (maeda and watanabe et al., 1978; watanabe et al., 1976; amargier et al., 1979; fediere, 2000). similarly periplaneta fuliginosa (pfdv) replicates exclusively in the hindgut epithelial cells of the host (suto, 1979). shrimp dvs target the tissues of ectodermal and mesodermal origin (lightner and redman, 1985). 120   table 2 taxonomy for the subfamily densovirinae (cotmore et al., 2014) *bmdv-2 has been excluded from the family densovirus because it comprises a bipartite genome, including a gene which encodes for dna polymerase. it is now referred to a new family bidnaviridae (tijssen and bergoin, 1995). host range studies have shown that the host range of dvs vary considerably from one isolate to another. dvs such as gmdv, cedv and addv have a host range apparently restricted to their original hosts (jousset et al., 1986; fediere, 2000), while other dvs such as jcdv and mythimna loreyi dv (midv) have a broad host range. the jcdv can replicate in aglais urticae, b. mori, chrysodexis chalcites, l. dispar, mamestra brassicae, m. oleracea, scotia ipsilon, genus species virus /virus variants abbreviation blattodean ambidensovirus 1 periplaneta fuliginosa densovirus pfdv blattodean ambidensovirus 2 blattella germanica densovirus 1 bgdv1 dipteran ambidensovirus 1 culex pipens densovirus cpdv hemipteran ambidensovirus 1 planococcus citri densovirus pcdv diatraea saccharalis densovirus dsdv galleria mellonella densovirus gmdv helicoverpa armigera densovirus hadv1 junonia coenia densovirus jcdv mythimna loreyi densovirus midv lepidopteran ambidensovirus 1 pseudoplusia includes densovirus pidv ambidensovirus orthopteran ambidensovirus 1 acheta domesticus densovirus addv aedes aegypti densovirus 1 aaedv1 aedes albopictus densovirus 1 aaldv1 culex pipiens pallens densovirus cppdv anopheles gambiae densovirus agdv dipteran brevidensovirus 1 aedes aegypti densovirus 2 aaedv2 aedes albopictus densovirus 2 aaldv2 aedes albopictus densovirus 3 aaldv3 brevidensovirus dipteran brevidensovirus 2 haemagogus equinus densovirus hedv penaeus monodon hepandensovirus 1 pmohdv1 penaeus chinensis hepandensovirus pchdv penaeus monodon hepandensovirus 2 pmohdv2 penaeus monodon hepandensovirus 3 pmohdv3 penaeus merguiensis hepandensovirus pmedv penaeus monodon hepandensovirus 4 pmohdv4 hepandensovirus decapod hepandensovirus 1 fenneropenaeus chinensis hepandensovirus fchdv lepidoteran iteradensovirus 1 bombyx mori densovirus* bmdv casphalia extranea densovirus cedv lepidoteran iteradensovirus 2 sibine fusca densovirus sfdv lepidoteran iteradensovirus 3 dendrolimus punctatus densovirus dpdv lepidoteran iteradensovirus 4 papilio polyxenes densovirus ppdv iteradensovirus lepidoteran iteradensovirus 5 helicoverpa armigera densovirus hadv2 penaeus stylirotris penstyldensovirus 1 pstdv1 penaeus monodon penstyldensovirus 1 pmopdv1 penaeus monodon penstyldensovirus 2 pmopdv2 penstyldensovirus decapod penstyldensovirus 1 penaeus stylirostris penstyldensovirus 2 pstdv2 121   table 3 densoviruses infecting silkworm bombyx mori and resistance genes character bmdv-1 bmdv-2/bmdv-z virion size 20nm 24nm genome topology linear linear genome molecule ssdna segmented ssdna genome size 5.0 kb 6.0 kb, 6.5 kb target tissues midgut columnar cell midgut columnar cell no. of genes 4 6 symptoms acute chronic resistance genes nsd-1(l-21 identified) nid-1(l-17) nsd-2(l-17 identified)/ nsd-z spodoptera littoralis, s. exigua but is incapable of replicating in g. mellonella. similarly, midv is infectious for chilo agamemnon, s. cretica, g. mellonella, ostrinia nubilalis, g. mellonella, pectinophora gossypiella, s. cretica and s. littoralis (fediere, 2000). pfdv has its host species extended to four other species of the genus periplaneta: p. americana, p. australasiae, p. brunnea and p. japonica (suto, 1979). mosquito dvs, aedes albopictus dv (aaldv) and a. aegypti dv (aedv) have a host range extending to different species. larvae of a. albopictus, a. cantans, a. caspius, a.geniculatus, a. vexans, culex pipiens, and culiseta annulata are all susceptible to per os infection with aedv (lebedeva et al., 1973). in the contrary, culex pipiens dv (cppdv) does not replicate in a. aegypti (jousset et al., 1986). glyphodes pyloalis, a pyralid infesting mulberry plantations of sericultural farms has been found to be the host for bmdv-1. further, the susceptibility of silkworm to bmdv-1 varied from one strain to another and the resistant strains could be selected for betterment of sericulture industry. the mode of inheritance of the resistance to bmdv-1 has been investigated and it was established that the nonsusceptibility is determined by two genes, nsd-1 (non-susceptibility to bmdv-1) and nid-1 (noninfection to bmdv-1), located at chromosome 21 and 17, respectively (eguchi et al., 2007). finally it is worth mentioning that despite their high virulence for their hosts, dvs do not appear to be able to replicate in vertebrates, including humans. molecular biology of dvs vertebrate parvoviruses consist of a small (4.5 5.5 kb), linear, single-stranded dna molecule composed of two major genes i.e., non-structural (ns) and structural (vp) genes located on the same strand. the replication proteins are encoded by the ns gene occupying the left half of the coding sequence. whereas the capsid proteins are encoded by the vp gene located on the right half of the coding sequence. short imperfect terminal palindromes (120 400 nt) flank the coding sequence. these terminal hairpins render the genome self-priming for complementary strand synthesis by host cell dna polymerase (cotmore and tattersall, 2007). alternative splicing mechanism is usually employed by vertebrate parvoviruses to regulate the expression of their overlapping genes. like their vertebrate counterparts, and in accordance with their small size (4 6 kb), dv genomes are limited in their coding capacities. however, the manner their two sets of ns and vp genes are organized and transcribed as well as the structure of their noncoding 3' and 5' extremities appear much diversified (bergoin and tijssen, 2010). the structural criteria retained for their classification by the international committee on taxonomy of viruses (ictv) take into account the number and size of orfs encoding ns and vp proteins, their location on the same strand (monosense) or both strands (ambisense) of the genome as well as the size, sequence and folding properties of the 5' and 3' extremities (tijssen and bergoin, 1995). gene expression strategies of dvs iteradensoviruses are 5-kb parvoviruses with typical j-shaped inverted terminal repeats of about 250 nucleotides and terminal hairpins of about 165 nucleotides. to date six iteradensoviruses have been reported, out of which gene expression strategies of five different iteradensoviruses viz., bmdv, cedv,  sfdv,  dpdv  and  ppdv have been identified (yu and tijssen, 2014). the small singlestranded dna genome contains several open reading frames and the transcription maps and expression of the viruses in this genus were explored. as for brevidensoviruses, the two nonstructural (ns) genes were expressed by overlapping promoters with alternate transcription starts at both sides of the ns1 start codon. similarly, it was found that all viral proteins (vp) had short 5’-untranslated regions and the transcripts started only 10 to 15 nucleotides upstream of the first atg. the presence of unfavorable kozak sequences promoted a leaky scanning mechanism 122   for vps leading to alternate transcription phenomenon. overall it could be concluded that both brevdensoviruses as well as iteradensoviruses have overlapping ns gene promoters that are responsible for different transcripts with varying length (yu and tijssen, 2014). near atomic structure determined by x-ray crystallography the structure of the recombinant bmdv-1 viruslike particle has been determined at 3.1-å resolution using x-ray crystallography (kauffmann et al., 2011). it is the first near-atomic-resolution structure of a virus-like particle within the genus iteradensovirus. the particles consist of 60 copies of the 55-kda vp3 coat protein. the capsid protein has a β-barrel "jelly roll" fold similar to that found in many diverse icosahedral viruses, including archaeal, bacterial, plant, and animal viruses, as well as other parvoviruses. most of the surface loops have little structural resemblance to other known parvovirus capsid proteins. in contrast to vertebrate parvoviruses, the n-terminal β-strand of bmdv-1 vp3 is positioned relative to the neighboring 2-fold related subunit in a "domainswapped" conformation, similar to findings for other invertebrate parvoviruses, suggesting domain swapping is an evolutionarily conserved structural feature of the densovirinae (kauffmann et al., 2011). bombyx mori densoviruses (bmdvs) bmdvs are mainly classified into two types, type i and type ii based on their genetic constituents. bmdvs are dna viruses. to date three isolates of bmdv have been reported. they are: (1) bmdv-1(ina isolate), (2) bmdv-2 (saku isolate and yamanashi isolate) and finally (3) bmdv-z (zhenjiang isolate). bmdv-1 contains a single-stranded dna that is about 5 kb in length. on the contrary, bmdv-2 possessing a split genome comprises of two types of single stranded linear dna molecules. bmdv-2 (yamanashi isolate) and bmdv-z (zhenjiang isolate) contain two ssdna molecules which are 6.0 and 6.5 kb in length. bmdv type i was found out to have a higher pathogenicity to silkworm than type ii. as per previous classification bmdv-1, -2 and -z were all classified into the genus named bidensovirus. however, recently bmdv-2 and bmdv-z have been excluded from the family of parvoviridae due to its unique bipartite genome and the presence of a dna polymerase motif of its own (wang et al., 2007; ito, 2008; kadono-okuda, 2010). symptoms associated with bmdv infection flacherie is one of the widespread and fatal diseases occurring in silkworms, resulting in massive loss in silk yield. it is caused when the silkworms feed on virus contaminated mulberry leaves. the midgut columnar cells of the larva are infected by bmdv. the infected cells carry hypertrophied nuclei wherein the virus multiplies anorexia and lethargy followed by flaccidity and inhibition of molting or metamorphosis are some of the common symptoms observed in most of the lepidoptera species. symptoms associated with bmdv infections start with larvae becoming whitish and progressively paralysis take place (meynadier et al., 1964; chao et al., 1985). death occurs in 2 20 days depending on the dv isolate, larval stage, virus concentration, and the way of infection: natural (epidemic) or experimental (per os or inoculation) transmission. body flaccidity is a major sign when silkworm larvae are infected per os with the bmdv and they die usually after seven days. all bmdvs infect only the columnar cells of midgut epithelium and they multiply only in the nuclei of columnar cells of the midgut epithelium. the infected larvae become flaccid and develop diarrhea due to loss of midgut tissue, become dark brown in color and finally die. the nucleoplasm of the infected cells can be densely stained with dna-chromophilic methyl green or feulgen reagent. studies have shown that flacherie can be caused solely by bmdv or it can also be caused by a combined infection of bmdv along with npv. the alimental canal of the diseased larvae appears pale yellow with very little internal content. however, the virulence and the symptoms of infection are different for each of the isotypes. bmdv-2 almost exclusively infects the columnar cells of midgut epithelium while bmdv-z can infect the columnar cells of midgut epithelium during the early stage of infection and is also able to infect the goblet cells of midgut epithelium (wang et al., 2007). the flacherie diseased silkworms were collected from different sericulture areas of southern india and screened for the bmdv-2 infection through qpcr analysis. results revealed high copy numbers of bmdv-2 in the midguts of the flacherie diseased silkworms ranging from an average of 2.9x105 to 6.4x109 (fig. 1). this finding confirmed the presence of viable bmdv-2 in quite high numbers in the field and its widespread association with flacherie disease in b.mori (kadono et al., 2014). densovirus resistance genes in b. mori as a matter of relief, few of the silkworm strains have been found to be completely resistant to the bmdv. this finding has made way for the development of bmdv resistant parental breeds. these specific strains remain unaffected irrespective of the quantity of virus inoculation. the crosses made between the resistant and the susceptible breeds showed that resistance was controlled by both dominant and recessive genes. nid-1 and nsd-1 are the genes discovered against bmdv-1 while nsd-2 and the nsd-z genes are the ones reported against bmdv-2, represented in table 2. hence, four resistant genes against bmdv have been reported so far (kadono-okuda, 2010). resistance genes against bmdv-1 nid-1(no infection with dv-1) is a single major gene that controls the susceptibility/ nonsusceptibility to bmdv-1. also it is the only gene in which the dominant allele is responsible for bmdv-1 resistance. nid-1 was detected quite recently. so far only five strains have been identified as nid-1carriers. on the contrary there are many nsd-1 and/or nsd-2-carrying strains. the lesser prevalence of the nid-1 gene may be attributed to the fact that this particular mutation occurred quite recently and 123   fig. 1 qpcr quantification of bmdv-2 in flacherie diseased silkworms collected from different sericultural areas of southern india thereby is not widespread like the nsd-1 and the nsd-2 genes. single pair back crosses carried out between resistant and susceptible strains has proved that the nid-1 is linked to the bm (black moth) and bts (brown tail and head spot) mutations of chromosome 17. its locus was determined at 31.1 cm (eguchi et al., 2007). in contrast to nid-1, nsd-1 (non-susceptibility to bmdv-1) provides resistance against bmdv-1 by a recessive mutation, expressed only during the homozygous state. its locus has been determined at 8.3 cm on chromosome 21 by eguchi et al. (1991) which was later further confirmed by mapping five linked random amplification of polymorphic dna (rapd) markers (abe et al., 1998). the mode of action of resistance still remains to be unclear but it is known that after infection of cultured embryos the resistant or the susceptible phenotype is expressed after the embryonic reversal stage, once the midgut is formed. another interesting finding was that both nsd-1 and nid-1 are independent genes involved separately in different aspect of the process of virus invasion and proliferation. when a cross was made between +nsd-1 and nid-1, the dominant susceptible allele of the nsd-1 gene versus the resistant allele of the nid-1 gene the nid-1 was found to be epistatic. even if a silkworm has +nsd-1/+nsd-1 on chromosome 21, it becomes resistant in the presence of a heterozygous nid-1 gene on the chromosome 17 (abe et al., 1987). resistance genes against bmdv-2 several studies carried out so far have shown that certain strains of b. mori are susceptible to bmdv-2, while, some are resistant (ponnuvel et al., 2010, 2011; watanabe and maeda, 1981). a supraoptimal temperature was observed to inhibit accumulation of bmdv-2 polypeptides in the midgut (kobayashi and choi, 1990). inhibition of dv-2 proliferation in the midgut of b. mori races resistant to the virus have been reported where 11 genes were upregulated (bao et al., 2008). in japan, a total of 154 b. mori races have also been reported as resistant to dv-2 (furuta, 1995). in india, 70 multivoltine and 28 productive bivoltine germplasm resources were collected and screened for bmdv2resistant/susceptible indian b. mori races (ponnuvel et al., 2010). four multivoltine races revealed the presence of bmdv-2 resistant gene. an unlinked mutation nsd-2 was discovered to control nonsusceptibility to bmdv-2 in b. mori. two rapd markers were identified to be linked to the nsd-2 gene, 4.7 cm away from the gene that controls non-susceptibility to bmdv-2. analysis of rflp inheritance patterns using probes specific to each of the 28 linkage groups of b. mori indicated that the non-susceptibility gene was linked to linkage group 17 with a linkage map of 30.6 cm with nsd-2 mapped at 24.5 cm and three closely linked cdna markers were identified (abe et al., 2000; ogoyi et al., 2003). the nsd-2, a putative transporter gene was isolated for the first time by positional cloning using bombyx genome information which revealed that a deletion of ~6 kb and an insertion of 34 bp in the region corresponding to 9 of 12 predicted transmembrane domains conferred resistance to bmdv-2. it was suggested that the complete membrane protein functions as a receptor for bmdv-2, and the site that the virus recognizes as a target is present in the deleted portion of the membrane protein, nsd-2. thus the full length amino acid transporter gene functions as the bmdv-2 susceptible gene and the truncated gene as the bmdv-2 resistant gene. the analysis of full length cdna as well as corresponding genomic dna sequences in the candidate gene revealed that, the structure of nsd-2 gene in the susceptible race had 14 exons, while, in the resistant race, there were only 5 exons. the ~6 kb deletion in the resistant race corresponded to the region from exons 5-13 in susceptible race (ito et al., 2008). 124   a) b) fig. 2 detection of bmdv-2 resistance through pcr amplification of genomic dna of various multivoltine races using primers of amino acid transporter region. a) appearance of specific band at 775 bp indicates susceptible genotype; b) appearance of specific band at 890 bp indicates resistant genotype. screening of indian b. mori germplasm races for nsd-2 genes in india, invaluable genetic silkworm b. mori stocks are maintained at the central sericultural germplasm resources centre (csgrc) at hosur of central silk board. seventy multivoltine and 28 productive bivoltine silkworm germplasm resources were collected from germplasm subjected to pcr analysis using two sets of primers viz. aa-trans1 to identify resistance to the bmdv-2 and aa-trans3 to identify susceptibility to the virus were utilized to analyze the genomic structure of nsd-2 gene (ito, 2008). the aa-trans1 forward primer (5’tctacgtgctttcatactacgtatc) was designed to have a binding site within exon 4 and the reverse primer (5’ttcctcacgtttctgaatttctcttg) within exon 14. the aa-trans3 forward primer (5’ ggtaagaggtccaacgctgttaagtt) on the other hand was designed to have a binding site at exon 13, 3’ flanking region and reverse primer (5’ttcctcacgtttctgaatttctcttg) within exon 14. the results of the study revealed that, most of the multivoltine as well as bivoltine silkworm germplasm races harboured either gene for susceptibility or genes for both resistance and susceptibility to dv-2 indicating a heterozygous susceptible condition. a total of nine multivoltine races screened for the presence of resistance/susceptible genes, of which six races possessed susceptible genes, while two races (mv76, mv-77) had resistant genes in a homozygous condition and one race (mv-73) had neither of the genes (fig. 2). with respect to bivoltine silkworm germplasm races, the races with genes for susceptibility/resistance as well as susceptibility included those most widely reared by sericulturists like csr2, csr4 etc. this justifies the widespread prevalence and severity of infection by bmdv-2 in the field. among bivoltine silkworm germplasm races, race ka revealed presence of bmdv-2 resistance gene, while, race m-iii recorded absence of genes for both bmdv-2 resistance as well as susceptibility. the above studies have thus provided evidence for availability of indian silkworm 125   germplasm resources with probable resistance to bmdv-2 (ponnuvel et al., 2010; murthy et al., 2014). the recent discovery of resistant genes in some of the b. mori races has led to the initiation of screening of resistant parental races for the development of bmdv resistant transgenic breeds. in this backdrop twenty indian bivoltine b.mori races were screened for dv-2 resistant gene (nsd-2) in the parental breeds selected for autumn specific breeding in north & north west india. among the these bitivoltine races, apsht-p5, bbe-198, bbe178, aps-4, aps-9 and bbe-266 were found to possess the bmdv-2 resistant gene nsd-2. the race apsht–p5 had a score of 100 % for the prevalence of nsd-2. the races bbe-198 and aps-4 with a score of 50 %, bbe-178 with a score of 37.50 % and aps-9, bbe-266 with a score of 25 % each were found out to be the potential candidates for the development of bmdv-2 resistant parental breeds. thus, this study can help in identifying dv-2 resistant parental breeds which will be used for breeding programmes for developing dv-2 resistant silkworm races. conclusion densoviruses can be classified as a diverse group of viruses having a wide host range. bmdvs among this group have proved to be the major agent for the flacherie disease in silkworms. the molecular aspect involved in the infection mechanism is yet to be unveiled. the two categories of bmdv i.e. bmdv-1 and bmdv-2 completely differ in their genetic make-up. bmdv-1 like their other counterparts has a single monosense genome while bmdv-2 unlike the group has a bipartite genome. the nsd-1, nid-1 and nsd-2 genes found in bmdv-1 and bmdv-2 respectively has initiated studies involving crossing a race harboring gene for resistance to bmdv-2 with an existing superior productive race widely used by sericulturists through conventional breeding followed by f1 hybrid production and backcrossing to introgress the gene. the introgression of the gene can be confirmed early through molecular biology techniques. studies have also been undertaken for screening out indian silkworm races bearing the resistant genes. resistance to bmdv-2 involves only a single gene so this feature can be further used for developing bmdv-2 resistant b. mori races in a shorter period of time using molecular biology techniques. however infection pertaining to bmdv-1 will require further studies for developing markers to screen bmdv-1 resistant silkworm breeds. in the case of bmdv-2 the resistant gene itself can be used as a marker for screening bmdv-2 resistant breeds. the ultimate target of bmdv studies would be to develop disease resistant silkworm breeds which can help the sericulture farmers to have higher silk yield and thereby enhance their economic conditions. however, the molecular aspect of virus infection is yet to be studied in detail in indian silkworm breeds. dv infection is host specific as well as tissue specific. studies related to the molecular mechanism of infection could further help in differentiating the various isolates of the bmdvs. the resistance developed in certain b. mori strains against bmdv-2 is also due to deletion of exons in the amino acid transporter. hence, further studies can be taken up in this direction to find out the specific receptors related with other viral pathogens. acknowledgements this research work has been supported through collaborative dst-jsps project jointly funded by department of science and technology (dst), ministry of science and technology, government of india as well as japan society for the promotion of science (jsps), government of japan (grant no: dst/int/jsps/p-187/2014,16th june 2014 to 15th june 2016). references abe h, sugasaki t, kanehara m, shimada t, gomi sj, ohbayashi f, et al. identification and genetic mapping of rapd markers linked to the densonucleosis refractoriness gene, nsd-2, in the silkworm, bombyx mori. genes genet. syst. 75: 93-96, 2000. abe h, harada t, kanehara m, shimada t, ohbayashi f, oshiki t. genetic mapping of rapd markers linked to the densonucleosis refractoriness gene, nsd-1, in the silkworm, bombyx mori. genes genet. syst. 73: 237-242, 1998. abe h, watanabe h. double infection of two densonucleosis viruses in the silkworm bombyx mori. jpn. j. appl. ent. zool. 31: 381-384, 1987. amargier a. vago c, 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mosquito culex pipiens pallens. j. gen. virol. 89: 195-199, 2008. 128   abstract 190 isj 14: 190-198, 2017 issn 1824-307x resarch report wnt1 promotes the proliferation of midgut epithelial cells in silkworm, bombyx mori l yang*, z wei*, m xu*, y zhao, h liang, p tan, h ma, t lu, z wang, h cui state key laboratory of silkworm genome biology (southwest university), 400715 china *these authors contributed equally to this work accepted may 4, 2017 abstract wnt genes are crucial members of at least three major signaling pathways that control diverse cellular behaviors, such as cell fate decisions, cell migration, and cell proliferation. wnt genes are involved in many important embryological events, including the development of the gastrula, heart, limb, and nervous system. in this paper, we identified and characterized bmwnt1 in midgut of silkworm larvae, a lepidopteran model insect. the gene expression analysis revealed that bmwnt1 is expressed in most organs although in different patterns. in vivo, the addition of dextran sulfate sodium (dss) promoted the proliferation of midgut epithelial cells with bmwnt1 up-regulation. in vitro, the down-regulation of bmwnt1 expression by rna interference significantly inhibited cell proliferation. taken together, we anticipate that bmwnt1 may contribute to cell proliferation and development in the silkworm midgut. key words: silkworm; bmwnt1; midgut; dextran sulfate sodium; cell proliferation introduction the homeostasis and regeneration of adult tissues require a balance between the production of new cells and the removal of old or damaged cells (cordero et al., 2012), and several signal transduction pathways, including egfr and jak/stat, wnt/wingless and notch, participate in these processes (ohlstein and spradling, 2007; liu et al., 2010) . the wnt/wingless and notch pathways maintain gut progenitors or iscs in a dividing non-differentiated state (lin et al., 2008). wnt/wingless proteins are secreted glycoproteins acting as signaling molecules that can trigger a cellular response and activate intracellular signal transduction (rao and kuhl, 2010). these proteins bind to receptors of the frizzled family and lrp5/6 co-receptors and initiate complex signaling cascades ( logan and nusse, 2004; kestler and kuhl, 2008). there are 19 members of the wnt family and 10 frizzled receptors in humans (rao and kuhl, 2010). the deregulation of the wnt signaling pathways has been reported to lead to the development of human diseases (moon et al., 2004; clevers, 2006). seven family members of the wnt family have been identified in drosophila, and they ___________________________________________________________________________ corresponding author: hongjuan cui state key laboratory of silkworm genome biology southwest university 400715 china e-mail: hcui@swu.edu.cn play a range of roles at multiple stages in the development of the organism (dhawan and gopinathan, 2003). wnt1/wingless is essential for the development of the wing disc and leg, and it is known to have a crucial role in the patterning of gonads (campbell et al., 1993; williams et al., 1993). in addition, wnt1/wingless is required for the maintenance and proliferation of intestinal stem cells (iscs), although it is not necessary for their survival in drosophila (lin et al., 2008). iscs that can maintain tissue homeostasis and replenish lost cells in response to tissue damage in drosophila were identified in the adult midgut (micchelli and perrimon, 2006; ren et al., 2010; micchelli et al., 2011) . however, tissue damage can also induce cell proliferation to replenish damaged cells (amcheslavsky et al., 2009). to address the function of wnt1 in the tissue of midgut, we chose the silkworm bombyx mori, which is not only an important economic insect for silk production but also a nice research model(izumi et al., 2016; matsumoto et al., 2016). bmwnt1 has been reported to be necessary for posterior segmentation in the embryos of b. mori, and the loss of the function of bmwnt1 results in severe defects in body segmentation and pigmentation (nakao, 2010; yamaguchi et al., 2011, 2013; zhang et al., 2015). however, few reports have focused on this gene in the silkworm midgut. in this paper, we identified and characterized bmwnt1 in midgut of silkworm larvae, a lepidopteran model insect we mailto:hcui@swu.edu.cn 191 fig. 1 multiple alignment of wnt1/wingless amino acid sequences from d. plexippus (ehj69660.1), h. armigera (ahn95659.1), b. mori (nm_001043850), a. gambiae (aat92559.1), d. melanogaster (np 523502.1), t. castaneum (efa04660.1), h. sapiens (np 005421.1) and m. musculus (np 067254.1). anticipate that bmwnt1 may contribute to cell proliferation and development in the silkworm midgut. materials and methods silkworm strain and cell culture silkworm strain dazao (p50) and silkworm bme cells were obtained from the state key laboratory of silkworm genome biology (southwest university, china). various larval tissues in different stages were dissected and stored at -80 °c. eggs were collected during different embryonic stages and stored as described above. the cells were cultured at 27 °c in grace medium (gibco) supplemented with 10 % (v/v) fbs (gibco) (xu et al., 2015). 192 rna extraction total rna was extracted with trizol reagent (takara) according to the manufacturer's protocol. after the digestion of the residual genomic dna using rnase-free dnase i (takara) for 30 min at 37 °c, 1 μg rna was reverse transcribed using m-mlv reverse transcriptase (promega) according to the protocol provided by the manufacturer. the cdna product was stored at -30 °c. bmwnt1 cdna fragment amplification by pcr primers based on the predicted est sequences in the silkdb were used to amplify the dna fragment of bmwnt1, and the sequences were as follows: bmwnt1-f 5’-tttgcacttgacctcgca-3’ and bmwnt1-r 5’ggcaagaacttgttcggaa-3’. the housekeeping gene bmactin3 was used as an internal control to standardize the variance among the different templates as described (jiang et al., 2012). the primer sequences were as follows: bmactin3-f 5’-ttcgtactggctcttc tcgt -3’ and bmactin3-r 5’-caaagttgatagcaattccct-3’. pcr was performed with hifi taq dna polymerase (transgen biotech) under the following conditions: 94 °c for 4 min; followed by 28 cycles of 94 °c for 40 s, 56 °c for 40 s, 72 °c for 1 min; and a final extension at 72 °c for 10 min. the pcr product was cloned into a peasy™-t5 zero cloning vector (transgen biotech) and sequenced at invitrogen (shanghai). bioinformatics analysis multiple sequence alignments were performed with clustalx version 1.81 with default gap penalties (thompson et al., 1997; tamura et al., 2007). quantitative real-time or semi-quantitative pcr analysis of bmwnt1 quantitative real-time pcr (qrt-pcr) was conducted with sybr® premix ex taq™ ii (takara) and the abi 7500 fast real-time pcr system (applied biosystems). the sequences of the primers: bmwnt1-qrt-f 5’-ggaactgctccacgagaaact-3’ and bmwnt1qrt-r 5’-ctcacggcaacctctatcca-3’. the housekeeping gene bmgapdh was used as an internal control, and the sequences of primers were bmgapdh-f 5’-cattccgcgtccctgttgctaat-3’ and bmgapdh-r 5’-gctgcctccttgaccttttgc-3’ (jin et al., 2014). the relative expression of genes was calculated with the 2−δδct method (livak and schmittgen, 2001). the online t-test software graphpad (http://www.graphpad.com/quickcalcs/ttest1.cfm) was used to evaluate the statistical significance of the results (p < 0.05). a semi-quantitative pcr analysis was performed as described previously bmactin3 was used as the internal reference. dss treatment silkworm larvae at the first day of the 4th instar were used for the feeding experiments. rounded mulberry leaves with a diameter of 3.5 cm were coated uniformly with 20 μl 3 % or 5 % (m/v) of dextran sulfate sodium (dss) (sigma), and 20 μl phosphate buffered saline (pbs) was used for the control group (tan et al., 2015). after the mulberry leaves were air dried, the silkworms were fed separately in 35 mm×10 mm cell culture dishes. paraffin embedding and h&e staining the midgut was dissected and gently rinsed in pbs, fixed in 4 % paraformaldehyde (pfa, sigma) for 2 h, rinsed in ethanol, embedded in paraffin, and sectioned at 5 μm. the paraffin sections were mounted on polylysine-coated glass slides. after 2 h at 60 °c, the sections were deparaffinized in xylene and subsequently rehydrated prior to staining. finally, the sections were stained with hematoxylin-eosin (h&e) and examined by microscopy (nikon 80i). the basal part of 8 silkworms in each group was measured, and the average thickness was obtained. knockdown of bmwnt1 in bme cells the dsrnas for bmwnt1 and egfp were generated by a ribomax large scale rna production system-t7 kit (promega) (payungporn et al., 2006). the primers were as follows: t7-bmwnt1-f 5’-taatacgactcactataggatgaagtgtctgt ggctgttagtgata-3’and t7-bmwnt1-r 5’-taatacgactcactatagg ctataaacacgtgtgcaccactttttc-3’. egfp was the control, and the following primers were used: t7-egfp-f 5’ taatacgactcactataggacgtaaacggccac aagttc-3’ and t7-egfp-r 5’ taatacgactcactataggtgctcaggtagtgg ttgtcg-3’. approximately 1×105 bme cells were seeded into 24-well plates for 24 h, and 500 ng dsrna was transfected into cells using x-treme gene transfection reagent (roche, switzerland) following the manufacturer's instructions. after 3 days of exposure to dsrna, the cells were harvested for qpcr and further experiments (xu et al., 2015). edu treatment and immunofluorescence staining the silkworms were fed edu (5-ethynyl-2’-deoxyuridine, invitrogen) at a concentration of 10 μg/g body weight for 8 h (tan et al., 2013). the midguts were dissected, fixed, paraffin embedded, sectioned, and stained with edu antibody for the immunofluorescence analysis according to the manufacturer’s protocol. according to the protocol provided by the manufacturer, the cells were harvested after 2 h incubation with 5 μg edu and stained with edu antibody for the immunofluorescence analysis. results bmwnt1 contains a wnt domain, and is highly conserved with other species. using the ncbi genbank and silkbd databases, primers from 1st base and 322nd base were designed and the wnt1 gene in silkworm was identified and named bmwnt1. the orf (open reading frame) is 1203 bp. the predicted protein contains 392 amino acids with a conserved wnt http://www.graphpad.com/quickcalcs/ttest1.cfm 193 fig. 2 temporal and spatial expression patterns of bmwnt1 in silkworm analyzed by semi-quantitative pcr and quantitative real-time pcr respectively. (a). expression of bmwnt1 in different tissues of l5d3 silkworm. si: silk gland; ma: malpighian tubule; ha: hemocyte; ov: ovary; te: testis; mi: midgut; fa: fat body; ep: epidermis; he: head; wh: whole body. (b) expression of bmwnt1 in different stages of silkworm midgut development. 3m: molting larvae of the 3rd instar; l4d1, l4d2, l4d3, l4d4: from day 1 to day 4 of 4th instar larvae; 4m: molting larvae of the 4th instar; l5d1, l5d2, l5d4, l5d5, l5d7: from day 1 to day 7 of 5th instar larvae; w2, w3: from day 2 to day 3 of wandering stage larvae; p1 p2, p3, p4, p5, p6: from day 1 to day 6 of pupation; bmactin3 was used as an internal control. (c) quantitative real-time pcr analysis of bmwnt1 in different tissues of l5d3 silkworm. (d) quantitative real-time pcr analysis of bmwnt1 in different stages of silkworm midgut development. 194 fig. 3 effect of dss on the silkworm midgut. (a) basal part was observed by h&e staining after dss treatment on different days. 8 silkworms in each group were used and the average thickness was obtained. the arrows point to the basal side of the midgut. scale bar = 25 μm. (b) thickness of the basal side of the midgut in panel (a) measured after 3 % or 5 % dss stimulation; the statistical analysis was performed with a two-tailed student’s t-test. pbs was used as the control. domain. the bmwnt1 protein has a calculated molecular mass of 44,739 da. the predicted isoelectric point (pi) of its mature peptide is 9.28. amino acids are highly conserved from invertebrates to vertebrates, especially in the wnt1 domain from the 57th to 392nd amino acids, which is highly conserved in insects such as lepidoptera (fig. 1). bmwnt1 is showed in differential expression profile the temporal and spatial expressions of bmwnt1 were analyzed in different tissues on day 3 of the 5th (l5d3) instar larva and different developmental stages by rt-pcr. agarose gel electrophoresis showed that bmwnt1 was highly expressed in the ovary, epidermis, head, testis, and malpighian tubule and was detectable in the silk gland, midgut, and fat body, although it was not detectable in the hemocytes. the silkworm expression was used as the control (fig. 2a). to further confirm this finding, qrt-pcr was employed, which showed similar results (fig. 2c). the mrna expression levels of bmwnt1 in the midgut at different developmental stages were detected by semi-quantitative pcr and real-time pcr. the developmental stages from the 3rd molting stage, 4th instar, 4th molting stage, 5th instar, wandering stage, and pupal stage were observed. bmwnt1 expression was gradually increased and showed three peaks at the 3rd molting stage, 4th molting stage and the first day of pupal stage (fig. 2b). qrt-pcr confirmed similar results (fig. 2d). dss treatment thickens the midgut basal part dss can damage the basement membrane organization (apidianakis et al., 2009; ren et al., 2010; tian et al., 2015). to verify the role of bmwnt1 during the development of the silkworm midgut, 3 % or 5 % dss were used and fed to silkworms. in dss treatment group, the midgut basal part was clearly thicker than the control (figs 3a, b). the body weight of dss treatment group had no significant change (data not shown). dss treatment promotes the midgut epithelial cell proliferation and bmwnt1 up-regulation to examine the proliferation of midgut epithelial cells, the silkworm was dissected after treatment with edu for 8 h following with 5 % dss treatment for 8 days. edu staining of the midgut was accomplished, and the result showed that the number of edu-positive cells in 5 % dss was dramatically higher than that in the control (figs 4b, c). the expression of bmwnt1 was also investigated, and the results showed that the bmwnt1 expression in 5 % dss was higher than that of control (fig. 4a). down-regulation of bmwnt1 by rna interference reduces cell proliferation in vitro bmwnt1 was down-regulated in bme cells under dsrna interference. the qrt-pcr showed that bmwnt1 expression was down-regulated successfully, and it was dramatically reduced by more than 70 % (fig. 5a). the edu retention analysis showed that the number of edu+ cells in the bmwnt1 rnai group was reduced distinctly relative to that in the control group, and it was reduced by more than 30 % (figs 5b, c). these findings demonstrate that the down-regulation of bmwnt1 inhibited cell proliferation in bme cells. 195 fig. 4 effect of dss on cell proliferation of silkworm midgut. (a) expression of bmwnt1 detected by qrt-pcr in the midgut after feeding with dss. (b) edu immunofluorescence staining of the midgut after edu treatment for 8 h following 5 % dss treatment for 8 days. scale bar = 25 μm. (c) edu-positive cells in panel (b) were counted, and the statistical analysis was performed with a two-tailed student’s t-test. pbs was used as the control. data are presented as the mean±sd (n≥3). *p ≤ 0.05 and **p ≤ 0.01 indicate statistically significant differences. discussion the wnt family has been extensively studied in mammals as well as in drosophila which comprising of several paralogous members. until now, 19 of wnt ligands in mammals and 7 in drosophila have been identified. the bombyx genome probably has a single copy of bmwnt1 gene and other paralogous members of the wnt gene family（dhawan et al., 2003). wnt1/wingless, which plays a crucial role in embryo development and tumorigenesis, is a key component in the wnt pathway, and its function in the silkworm midgut remains to be elucidated. here we mainly focus on the bmwnt1 gene. wnt1 is conserved in most insects including helicoverpa armigera and danaus plexippus, and bmwnt1 shares 88 % and 79 % identity with them respectively. as a holometabolic insect, the silkworm undergoes four stages including the egg, larva, pupa, and adult stages. at certain stages of development, internal tissues and organs undergo obvious changes (xu et al., 2012). since the midgut is responsible for digesting food and absorbing nutrients, the midgut epithelium is continually remodeled, with degenerated old cells replaced with new cells in the phase of molting (franzetti et al., 2012). wnt1 is mainly produced by the intestinal epithelial cells upon damage or stress, and this protein is apparently required for isc proliferation during tissue regeneration in drosophila (cordero et al., 2012). therefore, bmwnt1 might play a similar role in the silkworm midgut in maintaining tissue homeostasis. dss is a compound that can lead to excessive inflammation that resembles ulcerative colitis in humans (kawada et al., 2007), and it has been reported to promote the proliferation of drosophila midgut cells (amcheslavsky et al., 2009). in this paper, 3 % or 5 % dss were used to feed silkworms. after dss treatment the silkworm midgut epithelial cell proliferation was obviously promoted with 196 fig. 5 effect of bmwnt1 knockdown on cell proliferation in bme cells. (a) expression level of bmwnt1 detected by qrt-pcr after rna interference in bme cells. egfp dsrna was used as the control. (b) edu immunofluorescence analysis after 2 h incubation with 5 μg edu following rna interference for 3 days in bme cells. egfp dsrna was used as the control. scale bar = 50 μm. (c) edu-positive cells in panel (b) were counted, and the statistical analysis was performed with a two-tailed student’s t-test. data are given as the mean ± sd (n ≥ 3). *p ≤ 0.05 and **p ≤ 0.01 indicate statistically significant differences. bmwnt1 up-regulation. down-regulation of bmwnt1 by sirna can inhibit cell proliferation, which suggest bmwnt1 may be involved in regulation of silkworm midgut epithelial cell proliferation. dss has been reported to stimulate hedgehog (hh) signaling in drosophila, which promotes intestinal stem cell proliferation (apidianakis et al., 2009; tian et al., 2015). here, we found that the basal sides of midgut in the dss-treated group were visibly thicker than those of the control. during the growth process, the expression of bmwnt1 was also up-regulated in the dss-treated silkworm compared with that in the control and midgut epithelial cell proliferation was promoted, indicating that bmwnt1 may play an important role in maintenance of silkworm midgut homeostasis . moreover, wingless has been reported to be required for the proliferation and maintenance of iscs and hpz (the hindgut proliferation zone) in drosophila larvae (lin et al., 2008; takashima et al., 2008), which further supporting our findings. therefore, bmwnt1 may play important roles in the process of cell proliferation in silkworm, as it does in other species. in summary, our study with dss treatment of b. mori suggests a injury-stimulated activity for wnt1 in b. mori and a role for midgut epithelial cell proliferation. downregulation of bmwnt1 by rna interference inhibits bme cell proliferation. this injury-induced midgut basement membrane damage model should enable further analysis of the role of bmwnt1 signaling pathway in b.mori. acknowledgments this work was supported by the chinese national natural science foundation (31672496), the natural science foundation of chongqing (cstc2016jcyja0425), the research fund for the doctoral program of higher education of china 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1department of zoology, faculty of biology, herzen state pedagogical university of russia, russian federation 2department of immunology, institute of experimental medicine, saint-petersburg, russian federation accepted september 28, 2018 abstract we studied hemocytes of biomphalaria glabrata with the use of light and transmission microscopy and flow cytometry with lysotracker and syto62. these specific fluorescent dyes make it possible to distinguish cells differing in the amount of lysosomes and in transcriptional activity. we found that hemocytes of b. glabrata were represented by hyalinocytes and granulocytes (35.34 (27.38; 36.10) and 65.13 (64.11; 73.05) % respectively). granulocytes were generally strongly granulated and more metabolically active than hyalinocytes. this is the first report confirming the presence of these two main cell types in the hemolymph of b. glabrata with the use of specific fluorescent dyes. key words: biomphalaria glabrata, hyalinocytes, granulocytes, flow cytometry, lysotracker, syto62 introduction molluscs biomphalaria glabrata are broadly used in physiological, parasitological and comparative immunological studies. an interest in them is mainly due to their role as intermediate hosts in the life cycles of trematodes, some of which, such as schistosomes, are dangerous human pathogens. defence reactions of b. glabrata have been studied intensively for decades. as in other gastropods, the main component of their immune response are hemocytes, the cells of the hemolymph. morphology, functional activity and origin of hemocytes have been described in numerous studies (yoshino et al., 2013; pila et al., 2016; zhang et al., 2016). nevertheless, even the number of types of these cells remains a debated question. while most authors consider that b. glabrata has two types of hemocytes, hyalinocytes and granulocytes, some authors have reported four and more types (joky et al., 1983; delgado et al., 2001; cavalcanti et al., 2012). obviously, new methods and approaches ought to be employed in further studies of hemolymph cells. a promising technique of studying circulating cells of different animals is flow cytometry. differences in cell composition of uninfected molluscs b. glabrata and molluscs infected by schistosoma mansoni (trematoda) and angiostrongylus vasorum (nematoda) were found ___________________________________________________________________________ corresponding author: elena e. prokhorova department of zoology herzen state pedagogical university of russia 191186, moyka river 48, saint-petersburg, russian fed. e-mail: elenne@mail.ru with the use of this method (martins-souza et al., 2009; barcante et al., 2012). flow cytometry has also been used for the study of the hemolymph of planorbarius corneus and planorbis planorbis (ottaviani and cossarizza, 1990; ataev et al., 2016). flow cytometry with specific fluorochromes makes it possible to define functional characteristics of hemocytes and to verify the validity of their morphological hemocytes populations. however, specific cell dyes have never been used before for b. glabrata. in this study we carried out a complex analysis of hemocytes of b. glabrata with the use of light and transmission microscopy and flow cytometry with the use of specific fluorescent dyes. the first dye, lysotracker, accumulates in cell compartments with a lowered рн (macintyre nad culter, 1988). the second dye, syto62, is a cationic cyanine dye of nucleic acids (cosa et al., 2001; wlodkowic et al., 2009). a change of the fluorescence level in a stained cell may indicate the degree of chromatin condensation and a change in the rna content in it (grinchenko et al., 2014). if both dyes are used, the cells can be divided into populations differing as to the amount of lysosomes and transcriptional activity (kudryavtsev et al., 2012). material and methods we used snails b. glabrata of the laboratory brazilian strain (brazil) with a shell diameter of 1416 mm. the hemolymph was sampled from the blood sinus of the snail’s head with the help of a glass pasteur pipette. the hemolymph was mixed with chernin’s solution (1:1) (chernin, 1963) to dissolve it and to prevent agglutination of hemocytes. mailto:elenne@mail.ru 347 fig. 1 morphological types of hemocytes of molluscs b. glabrata. a–c phase contrast image of live cells, d–f – electron microscopic micrograph. a, d, e – granulocytes, c, f – hyalinocytes, b granulocyte (left) and hyalinocyte (right). abbreviations: ag –golgi apparatus, g – granules, mi – mitochondria n – nucleus, nu – nucleosis, p – pseudopodia, rer – rough endoplasmic reticulum. scale – 2 μm for in vitro morphological analysis of the hemolymph cells, freshly sampled hemolymph from individual snails (n=40) was smeared on teflonprinted glass microslides (well diameter, 5 mm) and analysed with the use of leica dm5000 light microscope equipped with the phase contrast. the cells of each snail were counted in cell-counting chamber according to a previously described technique (ataev et al., 2016). in brief, from 3 to 6 hemolymph samples were collected from every mollusc. hemocytes in 1 μl of hemolymph were counted in a cell-counting chamber (cell-line associates). for in vitro study, hemolymph from individual snails was placed into plastic petri dishes and incubated in a humid chamber for 4-8 h at 2224 °c. during incubation the samples were observed intermittently (every 10-20 min) under a phase-contrast microscope (leica dm 5000). cell counts and size measurements were made using imagescope software (cma, russia). total hemolymph from at least 10 snails was fixed at 4 °c using a solution of 2.5% glutaraldehyde in 0.05 m cacodylate buffer at ph 7.2. the samples were washed in 0.05 m cacodylate buffer, ph 7.2 and embedded in 1.5% low melting point agarose. the resulting agarose blocks were post-fixed in 1% osmium tetroxide in cacodylate buffer for 1 h. finally, the blocks were dehydrated through a graded ethanol series followed by acetone, and embedded in epon 812. ultrathin sections (30–50 nm thick) were double-stained with uranyl acetate and lead citrate, and observed using a jeol jem 1011 electron microscope. hemolymph from individual snails (n=15) for the flow cytometric assay was diluted with chernin’s solution (1:1) (chernin, 1963) and stained with syto62 (syto62® red fluorescent nucleic acid stain, emission maximum 680 nm) and lysotracker dyes (lysotracker® green dnd-26, emission maximum 511 nm) (prokhorova et al., 2018). flow cytometer bd accuri™ c6 (lasers with wavelengths of 488 and 640 nm) (bd biosciences) was used. for registration of fluorescence fl1 (533±30 nm) and fl4 (675±25nm) detectors were used. for each cell we assessed the forward scatter (proportional to the cell size), the side scatter (characterizing the cell structure) and the fluorescence intensity of the dye (mullaney et al., 1969; loken et al., 1976). from 5000 to 10000 events were analysed for each individual haemolymph sample. mathematical processing of the data was performed with the help of kaluza™ v.1.5 (beckman coulter, usa) and novoexpress (acea, usa) software. statistical analysis was performed with the 348 help of graphpad prizm and microsoft excel software. the results were represented as the median (ме) and the interquartile range (q25, q75). statistical significance was assessed with the help of non-parametric mann-whitney u-test. to obtain quantitative data about the populational composition of the hemolymph we constructed two variants of two-parameter bar charts: based on the values of the side scatter and the forward scatter and based on the values of the fluorescent unit of syto62 and lysotracker green dyes. our approach to the populational analysis of the hemocytes was based on the construction of hierarchic dendrograms. populations distinguished on the basis of the side scatter and the forward scatter were then separated based on the fluorescence intensity of both dyes (kudryavtsev et al., 2016). results most of the hemolymph cells of b. glabrata (n=40) were represented by granulocytes. these were the largest hemocytes (14.03±5.6 µm in diameter, n = 180), which could spread on the substrate. their nucleoplasmic ratio (ncr) varied from 0.08 to 0.13. these cells may form both filopodia and lobopodia. their nucleus is eccentric, irregular in shape (7.00±1.2 µm in diameter). it contains visible accumulations of condensed chromatin, mostly lying under the nuclear envelope. the nucleolus was not observed. the cytoplasm is rich in organelles. the centre of the cell is occupied by small dictyosomes, multivesicular bodies, mitochondria and a well-developed rer. free ribosomes and small glycogen accumulations could be seen in the cytoplasm (fig. 1). the second type of hemocytes was represented by hyalinocytes. they are smaller than granulocytes (7.21±1.01 µm in diameter, n = 57). the cytoplasm contains numerous free ribosomes, the golgi apparatus and a few mitochondria and other organelles. hyalinocytes never contained accumulations of granules, which were present in granulocytes. the nuclei of hyalinocytes (4.51±1.19 µm diameter) are eccentric, and condensed chromatin is diffused. a nucleolus is well visible in the centre of the nuclei. hyalinocytes may form infrequent lobopodia but cannot properly spread on the substrate (fig. 1). their ncr lies in the range of 0.19-0.27. according to the results of counts in the cellcounting chamber, one microliter of the b. glabrata hemolymph (n=55) contained 138 (107-169) cells. similar results were obtained with the use of flow cytometry: 141 (median 120.67 (68.29; 172.02) cells/µl. the cytometric assay showed that there were two hemocyte populations in b. glabrata (n=15) (fig. 2a): cells with lower values of forward scatter and side scatter (fslowsslow) and cells with higher values of forward scatter and side scatter (fshighsshigh). the former population corresponded to hyalinocytes (65.13 [64.11; 73.05]), while the latter corresponded to granulocytes (35.34 [27.38; 36.10]) (fig. 2в, table 1). hemocytes were further analysed based on their ability to absorb syto62 and lysotracker green dyes. cells with a high fluorescence level for both dyes were denoted as lthighs62high. cells with a low fluorescence level were denoted as ltlows62low. as a result, hyalinocytes and granulocytes separated into two groups of cells based on the intensity of accumulation of both dyes (fig. 2с). when the parameters of the forward scatter and the side scatter and the intensity of dye absorption were also taken into account, four groups of hemocytes could be distinguished. two of them were represented by hyalinocytes (fslowsslowltlows62low and fslowsslowlthighs62high), while the other two were represented by granulocytes (fshighsshighltlows62low and fshighsshighlthighs62high) (table 1). fig. 2 population of hemocytes of b. glabrata (n=15) distinguished based on the cytometric assay: а – populations of hemocytes distinguished by forward scatter and side scatter: fshighsshigh – granulocytes, fslow sslow – hyalinocytes; в – relative number of granulocytes and hyalinocytes; с – the main populations of hemocytes identified based on size, granularity and absorption of syto62 and lysotracker 349 table 1 populations of cells in the hemolymph of the snail b. glabrata populations of hemocytes identified based on morphological study granulocytes hyalinocytes percentage 73.3 ± 5.1 23.7 ± 3.2 populations of hemocytes identified based on fs/ss granulocytes hyalinocytes percentage 35.34 (27.38; 36.10) 65.13 (64.11; 73.05) characteristics fshighsshigh fslowsslow populations identified based on absorption of lysotracker and syto62 percentage 3.35 (2.06; 3.98) 31.97 (24.26; 37.27) 51.00 (42.92; 62.82) 7.02 (5.33; 11.86) characteristics ltlows62low lthighs62high ltlows62low lthighs62high discussion the number of hemolymph cells is an important characteristic of the state of the internal environment of the mollusc. its changes may be due to immunization resulting, e.g., from trematode infection (ataev and coustau, 1999; ataev et al., 2016). cell-counting chambers such as goryaev’s chamber or neubauer´s chamber or multiwell glass slides have been traditionally used for counting hemocytes. it has been found with the use of these methods that 1 µl of hemolymph of b. glabrata contains ca. 120-140 hemocytes (ataev and coustau, 1999; azevedo et al., 2006; pereira et al., 2008 and others). in this study we obtained similar results: both manual counts and a more objective method of flow cytometry yielded the result of about 140 cells in 1 µl of hemolymph of b. glabrata. the results of morphological studies of hemocytes agree with most reports about the main morphotypes of cells circulating in the hemolymph. hyalinocytes differ from other cells in having higher ncr values. they do not contain any accumulations of granules and cannot properly spread on the substrate. on the other hand, granulocytes spread on the substrate and contain accumulations of granules (abdul-salam and michelson, 1980; barcante et al., 2012; cavalcanti et al., 2012; yoshino et al., 2013; pila et al., 2016). granulocytes are more polymorphic as to size and granularity than hyalinocytes (cheng, 1975; joky et al., 1983). several researchers even distinguish two subpopulations of these cells. for instance, in a previous study we divided the granulocytes of planorbids p. corneus, b. glabrata and p. planorbis into two cell populations based in the results of a cytometric assay (prokhorova et al., 2018). cells of these populations differed in the degree of granularity, the size and the ability to accumulate specific lysosomal and nucleic dyes (prokhorova et al., 2018). it cannot be ruled out, however, that these two groups of granulocytes belong to the same cell type and that the differences noted between them are due to functional specialization. flow cytometry data indicate that hyalinocytes are the prevailing cell type in the hemolymph of b. glabrata. they made up, on the average, 65% of all hemocytes. this agrees with the data on biomphalarians as well as other planorbids (ataev et al., 2016; prokhorova et al., 2018). however, counts of hemocytes on fixed preparations showed that granulocytes were more numerous (table 1). granulocytes also predominate on fixed preparations of other studied snails such as p. corneus, p. planorbis and pomacea canaliculata (cheng, 1975; ottaviani, 1989; azevedo et al., 2006; cueto et al., 2015; ataev et al., 2016). this may be an artefact since hyalinocytes, which are less capable of adhesion, are lost in greater numbers than granulocytes during the preparation of fixed smears. at the same time, hemocyte concentration in 1 µl was similar regardless of whether it was determined by hemocytometer counts or by flow cytometry. this means that about 50% of hyalinocytes are lost during preparation of cells for microscopic observation owing to their lesser adhesive capacity. therefore, flow cytometry is preferable for a quantitative analysis of the cell composition of the hemolymph because it does not incur any loss of cells. flow cytometry is a more objective method in this respect. once the limits for the gating of cell population are set up properly, the proportion of circulating cells in numerous samples can be determined. however, this method needs to be adapted for each molluscan species. 350 functional state of the cells can be assessed with the use of lysotracker and syto62. based on the accumulation of these specific dyes, four populations of hemocytes were distinguished in b. glabrata. subpopulations of granulocytes and hyalinocytes were distinguished with the help of sequential gating based on the side scatter, the forward scatter and accumulation of the dyes. most hyalinocytes accumulate both dyes in small amounts. they contain lysosomes and are not very active metabolically. on the contrary, most granulocytes are 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(a) time course of encystment rate. closed circles, stock es-1, with slowly induced encystment; open circles, stock eq-1, with quickly induced encystment. in this measurement, rounded cells with an ectocyst layer were counted as encysting cells. (b) sds-page of total proteins contained in the cells of encysting colpoda, showing ef-1α expression level (arrows). the solubilized samples of stock es-1 and eq-1 were obtained at 0 to 6 h (labeled on the top of each lane) after the onset of encystment induction. cells were suspended in 1 mm tris-hcl (ph 7.2) at a low cell density (2,000 cells/ml) so that the resting cyst formation could be inhibited as much as possible. excystment induction was carried out by replacing the surrounding medium (encystment-inducing medium) of 10-day-old resting cysts by a fresh 0.05 % (w/v) wheat-leaf infusion. sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) sds-page was carried out according to laemmli’s method (laemmli, 1970) with a slight modification. the vegetative cells and encysting cells of c. cucullus nag-1 were solubilized in sds-page sample buffer containing 1% sds, 30 mm tris-hcl (ph 6.8), 5 % 2-mercaptoethanol and 10 % glycerol, followed by boiling for 3 min. a sample containing 5,000 cells was applied in each lane, and electrophoresed on a 10 % gel at 150 v. the gels were stained with a solution containing 0.2 % coomassie brilliant blue (cbb) r250, 45 % (v/v) methanol and 10 % glacial acetic acid, and then destained in a 27 % (v/v) methanol, 9 % glacial acetic acid solution. determination of partial nucleotide sequence of the ef-1α of c. cucullus nag-1 pcr amplification of c. cucullus nag-1 ef-1α (using takara prime star hs) was performed using genomic dna, and the nucleotide sequence was determined (genbank accession no. ab918921.1). amplification primers (sense: 5’-aagaacatgattaccggt; antisense: 5’-gaaccaggtaaggttggg) were designed based on the sequence of c. inflate (genbank, accession no. af056098.1). gene silencing by feeding rnai method the region (336 bp) of the open reading frame of c. cucullus nag-1 ef-1α (genbank accession no. ab918921.1) was amplified by pcr, followed by cloning into the litmus 28i vector (new england biolabs) between the two t7 promoters. for pcr, a set of primers connected with ecori-hf (new england biolabs) or hindiii-hf (new england biolabs) recognition sequences (underlined) and 4 extra nucleotides (5’-ccgc) (sense: 5’ ccgcgaattcaagaacatgattaccggt/antisen se: 5’ccgcaagcttgaaccaggtaaggttggg) 90 fig. 2 suppression of encystment and semi-quantitative rt-pcr in c. cucullus nag-1 cells whose ef-1α had been silenced by rnai. (a) time course of encystment rates of ef-1α-silenced cells (closed circles) and nonsilenced cells (open circles) of stock eq-1. in this measurement, rounded cells with an ectocyst layer were counted as encysting cells. (b) semi-quantitative rt-pcr of ef-1α, showing electrophoresis images of ef-1α and α-tubulin expression in nonsilenced cells (control), left lane, and ef-1α-silenced cells (3 days after induction of gene silencing), right lane. was used. escherichia coli strain ht115 was transformed by the introduction of the obtained constructs. in order to confirm that the e. coli strain ht115 might be transformed by the introduction of an ef-1α-gene-cloned litmus 28i vector, the vector was isolated from the e. coli ht115 strain, and the nucleotide sequence was determined. in this case, a set of primer sequences (m13 primer m3: 5’-gtaaaacgacggccagt/m13 primer rv: 5’-caggaaacagctatgac) was used. rnai gene silencing was carried out according to the method described previously (galvani and sperling, 2002; kutomi et al., 2012) with modifications. resting cysts of colpoda cucullus nag-1 were stimulated to encyst in 0.05 % wheat leaves infusion (culture medium), and cultured for about 12 h in this medium. thus, cultured cells were suspended using a thin pipette at a cell density of 10 cells/ml in culture medium containing 100 μg/ml ampicillin, 100 μg/ml tetracycline and 0.4 mm isopropyl β-d-thiogalactopyranoside (iptg). gene silencing was initiated by the addition of e. coli strain ht115 into the culture medium at the cell density of od600 of 0.25, which was transformed to produce double-stranded rna of ef-1α. for the control (nonsilenced colpoda), e. coli strain ht115 transformed by the introduction of a litmus 28i vector from which the ef-1α gene had been excluded was added into the culture medium at the cell density of od600 of 0.25. semi-quantitative rt-pcr total rna was extracted from c. cucullus nag-1 vegetative cells by an acid guanidinium thiocyanate-phenol-chloroform technique using isogen-ii (nippon gene co., ltd, tokyo, japan) according to the attached protocol. the total rna (4 μg) was reverse transcribed using goscript reverse transcription system (promega) according to the attached protocol. the 100 μl of pcr mixture for competitive pcr amplification (30 cycles using go taq green master mix [promega]) contained 5 μl cdna solution (containing 1 ng cdna) as a template. pcr amplification of ef-1α gene of c. cucullus nag-1 was performed using ef-1α primers 5’-taagtccacctccactgg (sense) and 5’-tggcggtttcgaacttcc (antisense), which had been designed based on the sequence of c. inflate (genbank, accession no. af056098.1). as an internal control for cdna quantity, the α-tubulin gene of c. cucullus nag-1 was amplified using the primers 5’-ctgaaactggtgctgg (sense) and 5’-cagtgtgttcaagaaggg (antisense), designed based on the sequence of colpoda sp. (genbank, accession no. x94348.1). amplified pcr products were electrophoresed in 2.0% agarose gels, followed by visualization with ethidium bromide staining. in order to confirm that each band was a pcr product of the ef-1α (194 bp) or α-tubulin (456 bp) gene, dna was extracted from each gel band using phenol, and their sequences 91 were determined (ef-1α: genbank accession no. ab976559.1; α-tubulin; genbank accession no. lc004697.1). the rate of encystment and excystment is expressed as a percentage of the total number of tested cells (142-161 cells). points (columns) and attached bars correspond to the means of six identical measurements (140 163 cells per measurement) and standard errors. results and discussion we compared the time course of ca2+/overpopulation-induced encystment initiation between two stock cultures of c. cucullus nag-1 cells, those in which encystment was slowly induced (stock es-1) and those in which encystment was quickly induced (stock eq-1) (fig. 1a). as shown in figure 1a, compared to stock eq-1 (open circles), encystment initiation of ‘stock es-1’ (closed circles) was significantly prolonged (p < 0.01 in 3, 4, 5, 6 h after the onset of encystment induction, mann-whitney test). sds-page of total proteins of the cells obtained from these two stocks showed that the expression of a protein around 48 49 kda which had been identified ef-1α (sogame et al., 2012b) was enhanced 1 hour after onset of encystment induction in stock eq-1 cells (fig. 1b-2), but several hours after onset of encystment induction in stock es-1 cells (fig. 1b-1). encystment occurred spontaneously in the culture medium. we silenced ef-1α expression in stock eq-1 cells by feeding e. coli containing knockdown plasmid, and examined the effect of ef-1α silencing on the spontaneously induced encystment during culturing (fig. 2a). compared to nonsilenced cells (fig. 2a, open circles), the encystment initiation of ef-1α-silenced cells was significantly suppressed (p < 0.01 at 4 days after initiation of culturing, mann-whitney test). we used competitive pcr to examine whether the ef-1α mrna expression in colpoda fed e. coli containing the knockdown plasmid was actually silenced. semi-quantitative rt-pcr showed that the amount of ef-1α mrna contained in the silenced colpoda was decreased (fig. 2b) while the amount of α-tubulin mrna used as an internal control was almost identical between the silenced and nonsilenced cells (fig. 2b). photomicrographs shown in figure 3a, b are motile cells surrounded by endocyst layers (en) just emerging from the mechanically ruptured ectocyst (ec) of a 10-day-old resting cyst, and they indicate that an ectocyst layer and endocyst layers are formed in ef-1α-silenced colpoda cysts (fig. 3b) identical to the case with the nonsilenced colpoda cyst (fig. 3a). in addition, there was no difference (p > 0.05, mann-whitney test) in the emergence rate (%) between nonsilenced (fig. 3c, ‘control’) and ef-1α-silenced cells (fig. 3c, ‘ef-1α silenced’) when excystment was induced by replacing the surrounding encystment-inducing medium with fresh 0.05 % (w/v) wheat-leaf infusion. these resuts indicate that ef-1α-silenced cells may ultimately become mature resting cysts despite the cyst formation was prolonged. fig. 3 excystment of c. cucullus nag-1 cells whose ef-1α had been silenced by rnai. (a), (b) photomicrographs of ef-1α-nonsilenced cells (a) and silenced cells (b), showing just-emerging cells surrounded by endocyst layers (en) through the rupture of the hard ectocyst layer (ec). (c) effect of rnai silencing of ef-1α on the emergence rate (%) in the excystment-induced cysts. left and right columns correspond to the ef-1α-nonsilenced (control) and -silenced cells. the photomicrographs (a, b) and the emergence rate (c) were obtained at 1 h after the onset of excystment induction in the resting cysts which had been spontaneously formed until the 10th day of culture of vegetative cells (stock eq-1) in normal culture medium or ef-1α-silencing medium. the results obtained in the present study suggest that colpoda ef-1α may play a role in early events in the encystment process. ef-1α is one of the subunits of translation elongation factor 1 composed of four different subunits (ejiri, 2002), and has multiple functions such as a translation in ribosomes (ejiri, 2002), bundling of actin filaments (kurasawa et al., 1996), severing of microtubules 92 (shiina et al., 1994) and regulation of the proteasome-dependent degradation of proteins (gonen et al., 1994). judging from the multiple functions of ef-1α, it is suggested that enhancement in the ef-1α expression level may be involved in the acceleration of morphogenetic events such as cyst wall formation by promoting protein translation in the regulation of protein disintegration, or in cytoskeletal dynamics such as microtubule disintegration. acknowledgements this work was financially supported by a sasagawa scientific research grant (#24-407) from the japan science society, by a basic scientific research grant (#140890) from sumitomo foundation and by a research fellowship from the japan society for the promotion of science for young scientists (#13j08784). references asami h, ohtani y, iino r, sogame y, matsuoka t. behavior and ca2+-induced cell signaling for encystment of colpoda cucullus. j. protozool. res. 20: 1-6, 2010. ejiri s. moonlighting functions of polypeptide elongation factor 1: from actin bundling to zinc finger protein r1-associated nuclear localization. biosci. biotechnol. biochem. 66: 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marsupenaeus japonicus x-w feng, l-j huo, x-z shi* shandong provincial key laboratory of animal cells and developmental biology, school of life sciences, shandong university, jinan, shandong, 250100, china accepted june 12, 2018 abstract myeloid leukemia factor (mlf) plays an important role in the development of drosophila. it is reported that mlf could inhibit white spot syndrome virus (wssv) replication in shrimp. however, the function and mechanism of mlf in shrimp antibacterial immunity are still elusive. in this study, we found mjmlf could be upregulated by vibro anguillarum challenge in hemocytes, gills, and intestine of kuruma shrimp. rnai assay showed that mjmlf could facilitate bacterial clearance in shrimp, and it was beneficial for shrimp survival post v. anguillarum infection. pull down assay showed that rmjmlf could bind to rmj14-3-3 in vitro. some antimicrobial peptide genes could be regulated by mjmlf. the results indicated that mjmlf might participate in the anti-bacterial immune reaction of shrimp. key words: shrimp; mjmlf; v. anguillarum; amps; mj14-3-3 introduction when organisms were challenged by invading pathogens, immune reaction could be initiated. immune response contains humoral reaction and cellular reaction (iwanaga et al., 2005; li et al., 2013). humoral reaction includes prophenoloxidase, clotting cascade, and the production of some antimicrobial peptides (amps), while cellular reaction consists of apoptosis, encapsulation, nodule formation, and phagocytosis (iwanaga et al., 2005; li et al., 2013). it is reported production of amps is one hallmark in host defense of drosophila, as well as some other holometabolous insects (lemaitre et al., 2007). when microorganism invades, the synthesis and secretion of potent amps are induced and accumulated in hemolymph. microarray studies indicate the gene expression program in drosophila go through intensive change due to the microbial infection. amp genes and some other immune responsive genes could be regulated by toll/imd cascades pathway in drosophila (de gregorio et al., 2002). in drosophila, the infection of some viruses (such as hiv-1 and cytomegalovirus) could also activate toll/imd pathway and induce the expression of some immune related genes. ___________________________________________________________________________ corresponding author: xiu-zhen shi school of life sciences shandong university jinan, shandong 250100, china e-mail: shixiuzhen@sdu.edu.cn myeloid leukemia factor (mlf) was discovered in acute myeloid leukemia (aml) caused by chromosomal rearrangement. mlf protein is a small nucleo-cytoplasmic shuttling protein, and contains a consensus 14-3-3 binding motif (gobert, et al., 2012). some mlf interaction proteins, such as mlf-interaction protein (mlfip) and mlf adaptor molecule (madm), were identified through yeast two hybrid screens (lim, et al., 2002; hanissian, et al., 2004). mlf is able to decrease the occurrence of trib1-driven acute myeloid leukemia, as tumor suppressor ccat/enhancer binding protein (c/ebp) could be stabilized by mlf (nakamae, et al., 2017; koschmieder et al., 2009). mlf plays an important role in regulating mechanosensitive signaling pathway, which is responsible for typical characteristics in neonatal rat cardiomyocytes (nrvcms). the overexpression of mlf in nrvcms could inhibit cell proliferation and increase apoptosis, while knockdown of mlf causes opposite results by protecting cell from apoptosis and increasing proliferation. besides, knockdown of mlf could upregulate d cyclins expression, which indicates mlf functions in regulating cell proliferation (yoneda-kato et al., 2005; rangrez et al., 2017). in addition, mlf is reported to function as proapoptotic regulator to modulate cell survival by associating with hop complex molecule (hax-1 (hs1 associated protein x-1)/htra2-omi/parl (presenilins-associated rhomboid-like)) (sun et al., 2017b). mlf is able to interact with hax1 and htra2 physically, and prevent htra2 from cleavage and activation. mailto:shixiuzhen@sdu.edu.cn 204 fig. 1 the temporal course of mjmlf in hemocytes, gills, and intestine after v. anguillarum injection at 0, 6, 12, 24, 48 h by qrt-pcr at transcriptional level. -actin was used as the control. the significance was detected by t-test between pbs group and infected group (p < 0.05, p < 0.001) it is reported that mlf1 could associate with 14-3-3 via a classic rsxsxp sequence in murine and human (lim et al., 2002). mlf1 could be redirected from cytoplasm into nucleus by mlf1-associated nuclear protein (manp), and also be regulated by 14-3-3 via binding to the 14-3-3 binding motif on mlf1 n terminus (winteringham et al., 2006). and in shrimp, 14-3-3 could inhibit the expression of some amps and negatively influence the akirin-relish function (liu et al., 2016). in shrimp, mlf functions importantly by inhibiting wssv replication and inducing hemocytes apoptosis (feng et al., 2017). in this study, the function of mlf in shrimp innate immunity against bacteria was studied. and results showed that mjmlf could enhance the exogenous vibrio anguillarum clearance, regulate the expression of some amps, interact with 14-3-3, and affect shrimp survival. mjmlf participated in the innate immune reaction of kuruma shrimp against v. anguillarum. materials and methods animal and tissue collection healthy kuruma shrimp marsupenaeus japonicus (6-8 g/individual) were purchased from an aquaculture market in jinan, shandong province, china and cultured in artificial aquarium with pumped air (sun et al., 2017a). three shrimp were randomly selected to extract total rnas from hemocytes, gills, and intestine with tripure reagent (bioteke corporation, beijing, china) following the manufactures instruction. expression profiles analysis after v. anguillarum challenge 30 l (1×106 cfu) v. anguillarum was injected into the last second segment of shrimp. meanwhile, the same volume of phosphate-buffered saline (pbs) (0.14 m nacl, 1.8 mm kh2po4, 2.7 mm kcl, and 10 mm na2hpo4) was injected in the other shrimp group, which were used as the control. tissues including hemocytes, gills, and intestine from v. anguillarum injected shrimp and pbs injected shrimp were collected at 6, 12, 24, and 48 h post injection for total rna extraction. total rnas were obtained from at least three shrimp, and reversely transcribed into the first stand cdnas with primer oligo-anchorr (feng et al., 2017). the obtained cdnas were diluted 20-fold as the templates for quantitative real time polymerase chain reaction (qrt-pcr). primer mjmlf-rtf1 and mjmlf-rtr1 were used as the previous report (feng et al., 2017). dsrna preparation a partial cdna fragment of mjmlf was amplified by pcr with primer mjmlf-fi and mjmlf-ri and used as template for dsrna synthesis (feng et al., 2017). the control dsgfp was produced with the same method (yang et al., 2015). the synthesized dsrnas were purified by hydroxybenzene and chloroform extraction. and the final dsrnas were dissolved in rnase-free water, and the concentration was adjusted to 1 g/l. rna interference (rnai) was performed as previously reported (shi et al., 2015). at 24 h post the second dsrna injection, shrimp hemocytes were collected for total rnas extraction. to test the rnai efficiency, the first strand cdnas were reversely transcribed and diluted 20-fold as the qrt-pcr templates with primer mjmlf-rtf2 and mjmlf-rtr2 (feng et al., 2017). bacterial clearance assay for the bacterial clearance, shrimp was divided into two groups, dsmjmlf group and dsgfp group. after rnai, 1×106 cfu v. anguillarum were injected into the shrimp abdominal segment. at 30 min after v. anguillarum injection, hemolymph was extracted from at least three shrimp. the hemolymph was gradiently diluted and spread onto the solid medium plates (1% tryptone, 0.5% yeast extraction, 0.1% fecl3, 3% nacl, 1.5% agar) (zhang et al., 2014). after the plates were cultured at 37 c overnight, the number of bacteria colonies was counted. results were analyzed by graphpad prism 5.0 software with students paired t test. survival rate assay for the survival assay, shrimp were divided into three groups, pbs group, dsgfp group, and 205 dsmjmlf group (each group containing 30 shrimp). rnai assay was performed as the above mentioned. and also, the same volume of pbs was injected into shrimp as the pbs group. after rnai, 1×106 cfu v. anguillarum were injected into shrimp of the three groups. the dead shrimp were numbered at 6, 12, 24, 48, 72, 96, 120, 144 h post v. anguillarum injection. the dsgfp-injected shrimp and pbs-injected shrimp were used as the controls. the p value was detected by graphpad prism 5.0 software with log–rank (mantel-cox) test. the expression detection of some amp genes v. anguillarum was incubated in liquid medium (1% tryptone, 0.5% yeast extraction, 0.1% fecl3, 3% nacl) overnight. the bacteria were fully washed by sterile pbs twice. after rnai, 1×106 cfu v. anguillarum were injected. the relative expression of some amp genes including mjalfe1, mjalfe2, mjcrustini-2, and mjcrustini-4 at 6 h post bacteria injection was detected by qrt-pcr in shrimp hemocytes. -actin was the internal control. the primers could be found in the previous report (bi et al., 2015; yang et al., 2017). p value was analyzed and accumulated by students t test. pull-down assay to detect the interaction of mjmlf with mj14-3-3, gst pull-down assay was performed. 200 g purified recombinant mj14-3-3 protein (rmj14-3-3, with gst tag) or gst tag protein (as the control) was incubated with the proteinlso gst resin (transgen biotech, beijing, china) respectively for 20 min at room temperature. the resins saturated with rmj14-3-3 or gst protein were sufficiently washed by pbs, till no protein could be detected in the wash solution. 200 g rmjmlf (with his-tag) were added into the resins separately, and incubated with gentle shaking for 2 h at room temperature. then the resins was adequately washed by pbs for at least six times to clean up the weak binding protein. the washing solution of the last time was collected as the wash fraction. and finally, the elution buffer (10 mm glutathione) was added to elute the protein on resin. the eluted fraction and the wash fraction were separated by 15% sds-page. sds-page gel was stained by coomassie brilliant blue g-250 (sangon biotech, shanghai, china). the purified rmj14-3-3, rmjmlf, and gst tag protein were also run on sds-page as the control. as the predicted molecular mass of rmj14-3-3 was 53.92 kda and that of rmjmlf was 52.62 kda, it was hard to separate the two bands on normal sds-page. so sds-page was run at 100 v for about 6 h to separate the two bands on the same lane. gst tag protein was used as the negative control. results mjmlf was upregulated by v. anguillarum to study the function of mjmlf in antibacterial immune response, qrt-pcr was carried out to analyze the temporal expression of mjmlf in hemocytes, gills, and intestine of shrimp after v. anguillarum challenge. the results showed that mjmlf could be upregulated in hemocytes, gills, and intestine by v. anguillarum challenge (fig. 1). the expression level of mjmlf in hemocytes and gills reached the peak at 24 h after v. anguillarum infection (fig. 1a and 1b), while in intestine it exhibited the highest expression level at 12 h post challenge (fig. 1c). these results indicated that mjmlf might be involved in the immune defense against v. anguillarum. fig. 2 bacterial clearance assay and survival assay in shrimp. the dsgfp was injected to use as control. (a) the interference efficiency of mjmlf in hemocytes was analyzed by qrt-pcr; (b) the bacterial clearance was analyzed by calculating the v. anguillarum number per milliliter hemolymph. the significant difference was calculated between dsmjmlf group and dsgfp group. the significances were calculated by t-test (p < 0.05,  p < 0.01). (c) the shrimp survival rate was analyzed by rnai and v. anguillarum challenge. the shrimp mortality was detected for calculation of the shrimp survival rate. the dsgfp group and pbs group were used as the control. the survival rate was analyzed with the kaplan-meier method. the significant differences were detected by log-rank test (p < 0.05) 206 fig. 3 the expression analysis of amps (mjalfe1, mjalfe2, mjcrustini-2, and mjcrustini-4) in shrimp hemocytes. (a) the knocking down efficiency of dsmjmlf in hemocytes was detected after the dsrna injection. the relative expression of mjalfe2 (b), mjcrustini-2 (c), mjcrustini-4 (d), and mjalfe1 (e) was analyzed by qrt-pcr at 6 h post the v. anguillarum challenge. the significant differences were analyzed by t-test (p < 0.05,  p < 0.01) v. anguillarum clearance rate decreased in mjmlf-silenced shrimp to determine the function of mjmlf in immune defense against v. anguillarum, rnai and bacterial clearance assay were performed. after knocking down the expression of mjmlf (fig. 2a), v. anguillarum were injected into shrimp. compared to the dsgfp-injected shrimp, the v. anguillarum number in dsmjmlf-injected shrimp was much higher, which indicated the clearance rate in the dsmjmlf-injected shrimp was much slower (fig. 2b). mjmlf could accelerate the bacterial clearance in shrimp. the result implied that mjmlf might have an important function in the immune defense against v. anguillarum in shrimp. the expression of some amps was downregulated in dsmjmlf-injected shrimp to test whether some effector molecules could be regulated by mjmlf, the expression of some amps (mjalfe1, mjalfe2, mjcrustini-2, and mjcrustini-4) was analyzed after rnai by qrt-pcr. after knocking down mjmlf (fig. 3a), the expression of mjalfe2, mjcrustini-2, and mjcrustini-4 decreased significantly in dsmjmlf-injected shrimp compared with the dsgfp-injected shrimp (fig. 3b, c, d), while there was no significant change for the expression of mjalfe1 (fig. 3e). the results indicated that mjmlf might protect shrimp from v. anguillarum infection by regulating some specific amps expression. 207 fig. 4 pull down assay was used to check the interaction of rmjmlf and rmj14-3-3. (a) purified recombinant rmj14-3-3 was bound to gst resin, and then rmjmlf was added. after sufficient washing, the eluted fraction was collected. rmj14-3-3, the wash fraction, the elute fraction, and rmjmlf were separated by 15% sds-page for 6 h at 100 v. (b) purified gst tag protein was used as the control and separated on 15% sds-page at 100 v for 2 h. the gels were stained by coomassie brilliant blue rmjmlf interacts with rmj14-3-3 in vitro it is reported that mlf could enhance apoptosis, and bind to 14-3-3. and mlf is negatively regulated by 14-3-3 (sun et al., 2015). in shrimp, mj14-3-3 is involved in regulating amps expression (liu et al., 2016). to understand the mechanism of mjmlf in antibacterial immunity of shrimp, gst-pulldown assay was performed to investigate the interaction of rmjmlf with rmj14-3-3. the results showed that rmjmlf could interact with rmj14-3-3 in vitro (fig. 4a), while no interaction was detected between rmjmlf and gst protein (fig. 4b). it demonstrated that rmjmlf had the binding activity to rmj14-3-3, but not the gst tag. and it has been reported his tag protein could not bind to gst tag protein (xu et al., 2017). the results suggested that rmjmlf could bind to rmj14-3-3 directly in vitro. discussion in this study, the role of mjmlf in antibacterial immune reaction was researched. as far as we know, it is the first report about the function of mlf in antibacterial immunity of kuruma shrimp m. japonicus. as reported previously, the expression of mjmlf was upregulated in hemocytes and hepatopancreas by wssv challenge. and mjmlf inhibits wssv replication by influencing hemocytes apoptosis. mjmlf could increase the survival rate and play an important function in antivirus immunology (feng et al., 2017). the expression pattern assay showed that expression of mjmlf was upregulated in hemocytes, gills, and intestine of shrimp when shrimp were challenged by v. anguillarum. the result suggested that the potent role of mjmlf in shrimp antibacterial immunity. to research the function of mjmlf in antibacterial immune reaction, rnai assay and exogenous bacterial clearance assay were performed. the v. anguillarum clearance rate was much slower in dsmjmlf-injected shrimp, which indicated the critical role of mjmlf in antibacterial immune reaction. besides, the shrimp survival rate in dsmjmlf-injected shrimp was lower than the control groups. mjmlf could be involved in shrimp antibacterial immune reaction. in drosophila, mlf could promote the expression of jnk (c-jun n-terninal kinase) (yanai et al., 2014). further research indicates that jnk pathway is a critical signaling pathway that initiating some relevant effector molecular (boutros et al., 2002; park et al., 2004; kallio et al., 2005; delaney et al., 2006). it is reported that dmlf could associate with dref (dna replication-related element-binding factor) and initiate bsk promoter. the interaction could further activate the jnk signaling pathway (yanai et al., 2014). in drosophila s2 cells, the jnk signaling pathway plays an essential role in regulating normal amps release (kallio et al., 2005). the production of amps could regard as local immune response relating to external environment (lemaitre et al., 2007). and in drosophila and other holometabolous insects, the synthesis and secretion of the potent amps could be induced and accumulated in hemolymph when invading microorganisms challenge, and it was the critical hallmark of host defense (lemaitre et al., 2007). in drosophila, most of the immune responsive genes are believed to be regulated by immune signaling pathway, such as toll and imd pathway (de gregorio et al., 2002). 208 in our study, we found mjmlf could positively regulate the expression of mjalfe2, mjcrustini-2, and mjcrustini-4. additionally, it is reported that mlf is able to interact with 14-3-3 with phosphorylated motif (lim et al., 2002). in shrimp, we detected their direct interaction in vitro by pull down assay. mj14-3-3 was able to regulate the expression of some amps (liu et al., 2016). and mj14-3-3 could interact with akirin, which suppressed the expression of some amps. akirin participates in imd-relish pathway via associating with relish and activate relish-regulated amp genes. the interaction of rmjmlf and rmj14-3-3 indicated that mjmlf might suppress the regulation of mj14-3-3 on amps expression by binding to mj14-3-3. so mjmlf might participate in shrimp antibacterial innate immunity by regulating the expression of some amps. further research such the sublocation of mj14-3-3 in hemocytes after knocking down mjmlf or co-expression of mj14-3-3 and mjmlf in insect cell lines would help us fully understand the mechanism of mjmlf in shrimp antibacterial immunity. in conclusion, mjmlf could protect shrimp from bacterial infection by regulating some amps expression, enhancing bacterial clearance, and affecting the shrimp survival rate. mjmlf might play a critical function in antibacterial immunity of shrimp. acknowledgement this work was supported by grant from national natural science foundation of china (no.31301879). reference bi wj, li dx, xu yh, xu s, li j, zhao xf, et al. scavenger receptor b protects shrimp from bacteria by enhancing phagocytosis 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antibacterial immunity of marsupenaeus japonicus. dev. com. immunol. 46: 356-363, 2014. isj 12: 274-277, 2015 isj 12: 274-277, 2015 issn 1824-307x research report virulence of four entomopathogenic nematode species for plum sawfly, hoplocampa flava l. (hymenoptera: tenthredinidae) tc ulu1, b sadic1, ia susurluk1, t aksit2 1uludag university, faculty of agriculture, department of plant protection, turkey 2adnan menderes university, faculty of agriculture, department of plant protection, turkey accepted october 20, 2015 abstract the yellow sawfly hoplocampa flava is an important pest of plum all around the world. larvae feed on the seed with damaged fruit falling prematurely. there are many studies on the control of other pests with entomopathogenic nematode (epn), but few on the control of plum sawfly. the present study was conducted to determine virulence of four epn species: heterorhabditis bacteriophora, h. marelatus, steinernema carpocapsae tur-s4 and s. feltiae tur-s3 against plum sawfly hoplocampa flava l. (hymenoptera: tenthredinidae) under laboratory conditions. for each nematode species, six different doses (3, 5, 7, 10, 15, 20 infective juveniles (ijs) /larva) were applied against last instar larvae of h. flava. assays were done in 24 well tissue culture plates filled with 10 % (w/v) moist silver sand. the most virulent species was h. bacteriophora which had ld50 and ld90 values of 6.51 and 15.46 ijs, respectively. the least virulent was s. carpocapsae tur-s4 with ld50 and ld90 values of 16.617 and 33.779 ijs, respectively. key words: entomopathogenic nematode; hoplocampa spp.; sawfly; virulence   introduction the yellow plum sawfly hoplocampa flava is a member of the hymenopteran suborder symphyta. the name of the group is coming from saw-like shape of ovipositors of the female. generally, adults are 4 7 mm length with reddish-brown in color and black legs. eggs are white, slim-long-elliptic, 0.4 0.5 mm length and larvae is cream, caterpillar-like, when the larvae mature length is 10 15 mm. the species has one generation per year in turkey, and the wasps overwinter within cocoon as larvae stage, in soil of a few centimeters deep. in the spring, matures emerge from cocoon while plum trees about to bloom and emerging continues during blossoming period. h. flava is considered to a major pest of plum fruits and heavy infestations can cause considerable economic losses. this monovoltine species spends most of its life-cycle as diapausing larvae in the soil and adult emergent flight usually coincides with the start of flowering of early plum varieties. the females lay eggs on the calyx and larvae emerge when the fruit begin to develop. larva can destroy up to several fruitlets during the ___________________________________________________________________________ corresponding author: ismail alper susurluk uludag university faculty of agriculture department of plant protection, turkey e-mail: susurluk@uludag.edu.tr course of feeding and damaged fruitlets fall off. sawflies are one of the important pest groups on pear, apple and plum. larvae of the sawflies have direct damage and fruits and apple sawfly can cause up to 100 % product loss. although economic importance of the pest, there are very few permitted agricultural chemicals for the control of sawflies. in addition, pollinator bees were exposed to these chemicals due to the spraying period, which is done while blossoming. moreover, zero tolerance policies and strict rules of importer countries are big obstacles for chemical control. herewith, biological control becomes an alternative method and because of sawflies overwinters in soil, epns can be considered as a suitable biocontrol agent. entomopathogenic nematodes (epns) belonging to the families heterorhabditidae and steinernematidae have been using to control especially soil-dwelling insect pests (ehlers, 1996; lacey and georgis, 2012). epns have several beneficial features, which make them an impotant bio-control agents. epns can inhabit almost all around the world with different geographical and climatic conditions (griffin et al., 1990; poinar, 1990; hominick et al., 1996; hominick, 2002), they are effective on more than 250 insects pests (peters, 1996) and safe for non-target organisms and environment (boemare et al., 1996; ehlers 2003), they can seek for their hosts (lewis et al., 1992; 274   grewal et al., 1994) up to 50 cm soil depth (susurluk, 2008), and they can be mass produced in liquid culture (lunau et al., 1993; strauch and ehlers, 1998; ehlers, 2001) for widespread outdoor applications. several commercial formulations of epns have being used on agricultural pests such as western flower thrips, sciarid flies, vine weevils, codling moths, cut worms etc. common characteristic of these pests is that they spend at least one biological stage within soil. since sawflies are overwintering within cocoon as larvae in the soil, controlling sawflies with epns are highly possible. epns provide long time effectiveness on the pest in applied areas, because of their long term persistence (susurluk and ehlers, 2008). therefore, an alternative control method for sawflies with epns is expected to be successful especially within integrated pest management. the aim of this study was to identify virulent strain of epn for the control of sawfly larvae and to determine virulence of four epns and to calculate their lethal doses as ld50 and ld90 on plum sawfly larvae in order to detect possibility of using these epns in plum orchards in turkey. materials and methods nematode species four different entomopathogenic nematode species, heterorhabditis bacteriophora, h. marelatus, steinernema carpocapsae and s. feltiae were used in the present study. species were isolated from different regions of turkey (bursa, ankara, bursa and izmir, respectively) and cultured in vivo on the last instar of galleria mellonella (lepidoptera: pyralidae) larvae as described by kaya and stock (1997). the species were identified by molecular methods (pcr-rflp) (unpublished data). all cultures were stored at +4 °c and they were reproduced in vivo on g. mellonella larvae before inoculations. two weeks old epn populations were used for the experiments. hoplocampa flava larvae over 2000 last instar sawfly larvae were collected from decayed plum fruits brought from aydin, turkey. virulence experiments in order to determine virulence and calculate lethal doses of epn species on sawfly larva, six doses 3, 5, 7, 10, 15, 20 ijs/larva were applied. all trials were conducted in 24-well tissue culture plates and replicated three times with 20 larvae for each replicate. distilled water was applied on 10 larvae with three replicates for control. for each well of the plate, a larva was put within 10 % humid sterile silver sand (particle size 300 400 µm). all doses were applied on sand, plates were sealed with parafilm and incubated at 24 °c for 4 days. after incubation, the cadavers inside the sand were washed with distilled water. each cadaver was dissected under a stereomicroscope to ensure nematode presence. after dissection, dead and alive larvae were counted and mortality ratios calculated. for determining lethal doses more precisely, higher doses were applied on sawfly larva to reach 100 % mortality. statistical analysis data of virulence experiments were analyzed by analysis of variance (anova) and followed by a least significant difference (lsd) test as posthoc comparisons with p < 0.05 significance level using jmp® 7.0 software. probit analysis performed for lethal dose calculations by xlstat® software. results and discussion results showed that sawfly larvae were susceptible to epn, however, there were no significant differences for virulence at the low doses (3 and 5 ijs). at 7 ijs and above, h. bacteriophora had significantly higher lethal effect than both steinernema species. mostly, s. carpocapsae had the lowest effect among the other species (fig. 1). as it can be estimated from the virulence experiments, h. bacteriophora had the lowest ld50 (6.514) and ld90 (15.455) among the other species while. s. carpocapsae had the highest ld50 (16.617) and ld90 (33.779). heterorhabditis bacteriophora were more effective than steinernema species on h. flava larvae at higher doses (>7 ijs/larva) (table 1). besides effectiveness of epns, their safety also supports their usings in plant protection or ipm systems. this aspect is endorsed by the european and mediterranean plant protection organisation (eppo), which has produced the document pm 6/3 (2), containing a positive list of invertebrate biocontrol agents. the list contains five epns used in biological control (eppo, 2002). therefore, they are encourged to farmers who especially do organic farming in many countries. consequently, epns as successful biological control agents have potential for use in biological control of many economically important insect pests in glasshouses, lawns, turfgrasses, pasture, nurseries, trees, mushrooms, orchards, soft fruits and vegetables. in these crops, table 1 ld50 and ld90 values of the species on sawfly h. flava larvae ld50 confidence interval (ld50) ld90 confidence interval (ld90) h. bacteriophora 6.514 3.991-8.361 15.455 13.07518.933 h. marelatus 8.617 6.941-10.008 18.946 16.835-21.931 s. feltiae 12.958 10.626-14.685 25.120 22.521-29.485 s. carpocapsae 16.617 13.410-18.956 33.779 30.102-40.250 275 fig. 1 virulence of four different entomopathogenic species with six different doses on h. flava larvae. for each dose, letters not connected by same letter are significantly different (f = 21.757; df = 23, 48; p < 0.0001) epns can commercially used against many insect pests such as; otiorhynchus sulcatus, frankliniella occidentalis, agrotis spp., agriotes spp., dorcadion pesudopreissi, sciarids, cydia pomonella, tuta absoluta, bothynoderes punctiventris, diabrotica virgifera virgifera and cassida vittata (lemma et al., 2004; susurluk, 2008; susurluk and ehlers, 2008b; saleh et al., 2009; toepfer et al., 2010; carrera et al., 2010; susurluk et al., 2011). a study was conducted by bélair et al. (1998) on foliar application of s. carpocapsae against early season apple pests. in 1992 and 1993, damage caused by apple sawyfly h. testudinea larva was reduced by 98 % and 100 %, respectively. oppositely, treatments in 1994 were not effective as first two years. in addition, curto et al. (2006) performed a study to determine virulence of s. feltiae on pear sawfly h. brevis. results of the two years study showed that, in the first year, application of s. feltiae significantly reduced the adult population of h. brevis in the next spring. in the second year, epns managed to reproduce and control sawfly larvae, but they could not reduce the fruit damage. both studies showed that s. carpocapsae and s. feltiae can have high virulence of sawfly larvae, which is inconsistent with the present study. on the other hand, there are some studies that have similar result with the present study. tomalak (2006) studied on potential of 2 epn species (h. megidis and s. feltiae) for control of 13 different orchard and urban tree sawfly species including h. flava. semi-field trials of the study revealed that, for all sawfly species (excluding arge berberidis), h. megidis was found more effective than s. feltiae. additionaly, nježić and ehlers (2014) performed a new study to control two plum sawfly species (h. minuta and h. flava) with s. feltiae, s. carpocapsae and h. bacteriophora. the study had both laboratory and field trials, and methodology for laboratory trials were similar with the present study. however, doses were higher (50, 100 and 200 ijs/larva) and different ages of the larva (1, 10, 20, 40 days) were considered. results of the laboratory trials showed that mortality was 92 100 % for 1 day old larvae. whereas no mortality was observed for older larvae. moreover, field trials showed that s. feltiae, s. carpocapsae and h. bacteriophora reduced adult population by 62 %, 47 % and 85 %, respectively. the data obtained from the both studies were consistent with the present study. as it can be estimated from the studies, different results obtained from same epn species under similar conditions, except target host. s. carpocapsae and s. feltiae were found effective against h. testudinea and h. brevis, while same epn species were found less effective against h. flava. it is thought that the main reason for the differences between effectiveness of the species was the target host. generally, heterorhabditis species tend to be more effective then steinernema, which can be described by cruising behaviour of heterorhabditis. the present study was one of the first applications of epns on sawfly. besides having significant virulence, most effective species was found as h. bacteriophora, which is the most common epn species in turkey (susurluk et al., 2001). even though further virulence studies and field trials are necessary, the result of the study showed hopeful results for controlling sawflies with epns. there is very limited scientific data about epns and the pest relationships, yet. thus, the study can provide additional information on the pest controlling by epns for the future. 276   acknowledgements this study was supported by the research fund of the university of uludag (bursa turkey) (project number: kuap (z)-2013/17). references bélair g, vincent c, chouinard g. foliar sprays with steinernema carpocapsae against earlyseason apple pests. j. nematol. 30: 599-606, 1998. boemare ne, laumond c, mauleon h. the entomopathogenic nematode-bacterium complex: biology, life cycle and vertebrate safety. biocontrol sci. technol. 6: 333-346, 1996. curto g, vergnani s, reggiani a. effectiveness of entomopathogenic nematodes in the control of sawfly (hoplocampa brevis) on pear orchards. programmei abstracts & participats of summit workshop cost actions 850 “biocontrol symbiosis”, 1-june, 2006, schloss salzau, germany, p 14, 2006. ehlers r-u. current and future use of nematodes in biocontrol: practise and commercial aspects in regard to regulatory policies. biocontrol sci. techn. 6: 303-316, 1996. ehlers r-u. mass production of entomopathogenic nematodes for plant protection. appl. microbiol. biot. 56: 623-633, 2001. ehlers r-u. biocontrol nematodes. in: hokkanen hmt, hajek aj (eds), environmental impacts of microbial insecticides, kluwer academic publishers, dordrecht, pp 177-220, 2003. grewal ps, selvan s, gaugler r. thermal adaptation of entomopathogenic nematodes: niche breadth for infection, establishment, and reproduction. j. therm. biol. 19: 245-253, 1994. griffin ct, downes mj, block w. tests of antarctic soils for insect parasitic nematodes. antarc. sci. 2: 221-222, 1990. hominick wm, reid ap, bohan da, briscoe br. entomopathogenic nematodes: biodiversity, geographical distribution and the convention on biological diversity. biocontrol sci. techn. 6: 317-331, 1996. hominick wm. biogeography. in: gaugler r (ed.), entomopathogenic nematology, cabi publishing, wallingford, uk, pp 115-143, 2002. lacey la, georgis r. entomopathogenic nematodes for control of insect pests above and below ground with comments on commercial production. j. nematol. 44: 218, 2012. lewis ee, gaugler r, harrison r. entomopathogenic nematode host finding: response to host contact cues by cruise and ambush foragers. parasitology 105: 309-315, 1992. lunau s, stoessel s, schmidt-peisker aj, ehlers ru. establishment of monoxenic inocula for scaling up in vitro cultures of the entomopathogenic nematodes steinernema spp. and heterorhabditis spp. nematologica 39: 385-399, 1993. nježić b., ehlers r-u. control of plum sawflies (hoplocampa minuta and hoplocampa flava) by entomopathogenic nematodes. j. nematol. 46: 212, 2014. peters a. the natural host range of steinernema and heterorhabditis spp. and their impact on insect populations. biocontrol sci. techn. 6: 389-402, 1996. poinar go jr. taxonomy and biology of steinernema and heterorhabditis. in: gaugler r, kaya hk (eds), entomopathogenic nematodes for biological control, crc press, boca raton, fl, pp 23-61, 1990. strauch o, ehlers r-u. food signal production of photorhabdus luminescens inducing the recovery of entomopathogenic nematodes heterorhabditis spp. in liquid culture. appl. microbiol. biot. 50: 369-374, 1998. susurluk a, dix i, stackebrandt e, strauch o, wyss u, ehlers r-u. identification and ecological characterisation of three entomopathogenic nematode-bacterium complexes from turkey. nematology 3: 833-841, 2001. susurluk a, ehlers r-u. field persistence of the entomopathogenic nematode heterorhabditis bacteriophora in different crops. biocontrol. 53: 627-641, 2008. tomalak m. potential of entomopathogenic nematodes for control of orchard and urban tree sawflies. prog. plant. prot. 46: 249-255, 2006. 277 http://apps.webofknowledge.com/full_record.do?product=ua&search_mode=generalsearch&qid=1&sid=s2v32hzcgkawdv3ofcn&page=1&doc=1 http://apps.webofknowledge.com/full_record.do?product=ua&search_mode=generalsearch&qid=1&sid=s2v32hzcgkawdv3ofcn&page=1&doc=1 http://apps.webofknowledge.com/full_record.do?product=ua&search_mode=generalsearch&qid=1&sid=s2v32hzcgkawdv3ofcn&page=1&doc=1 http://apps.webofknowledge.com/full_record.do?product=ua&search_mode=generalsearch&qid=1&sid=s2v32hzcgkawdv3ofcn&page=1&doc=1 34 isj 16: 34-47, 2019 issn 1824-307x report of meeting xxth scientific meeting of the italian association of developmental and comparative immunobiology (iadci), 13 15 february 2019, department of biology, ecology and life sciences, university of calabria, rende, italy organizers: a giglio 1 , p brandmayr 1 , f talarico 1 , a mazzei 1 , s marsico 2 , a naccarato 3 , f cavaliere 1 , ml vommaro 1 , mc granieri 1 1 department of biology, ecology and life sciences, university of calabria, rende, italy 2 department of pharmacy, heath and nutritional sciences 3 cnr-institute of atmospheric pollution research, rende i met you when i was very young, at the beginning of my professional life, and i immediately understood that you were reserving many surprises; i followed you in silence when your secrets have been revealed; how do you always be the same and always different, the "god problem" revealed. i admired your anatomy, simple, elegant, based on harmonic couplings of a wonderful domain, classic like a doric temple; i admired the flexibility of your body: the movements of the hips and elbows, your fingers that shape the socket. i was entranced by the dynamics of your dance. then i had the pleasure to be the only one to own you in the frost of polar ice, where you appeared to me alone, only wonder, generated by an evolutionary miracle. it is true that i have been unfaithful to you; i was attracted to other molecules, from the aesthetics of the baroque forms of the toll-like receptors, from the molecular labyrinth of the third factor of the complement, that is leafed in luminous fragments up to the small, but multipotent, anaphylatoxin. now i will not see the many sure marvels that you reserve to those who will follow you in the future but i have the memory to have loved the most beautiful of the molecules: immunoglobulin. by umberto oreste, past researcher at institute of protein biochemistry, national council of research, naples (italy) 35 session 1. chairmen: magda de eguileor, university of insubria, varese, italy and adriana vallesi, university of camerino, camerino (mc), italy cell-cell interaction mytilus hemocytes as a model to investigate the specificity of innate immunity in bivalve molluscs l canesi 1 , t balbi 1 , c ciacci 2 , a borello 1 , l vezzulli 1 , m auguste 1 1 department of earth, environmental and life sciences, university of genoa, genoa, italy 2 deptartment of biomolecular sciences, university of urbino, urbino, italy invertebrates represent 97% of animal diversity and present in virtually any ecosystem. this implies that each species should be able to adapt and survive in its environment by only relying on innate immunity. the mechanisms of immune specificity (sophisticated recognition systems for a wide variety of non-self material) and immune training/memory (capacity to mount a faster and more effective response upon re-exposure to a stimulus) are therefore central to the invertebrate ability to survive in diverse environments. mytilus galloprovincialis hemocytes represent a powerful in vitro model to study the specificity of innate immunity in bivalve molluscs. data obtained in response to both natural stimuli (different bacterial species and strains), and different types of nanoparticles-nps, as environmental contaminants are summarized. moreover, a key role for soluble hemolymph proteins was identified. a case study is represented by the extrapallial protein precursor (ep), that specifically mediates the interactions between hemocytes and bacteria carrying mannose-sensitive ligands (i.e. vibrio aestuarianus 01/032), resulting in efficient recognition, binding and killing. moreover, this protein represents the sole component of the biocorona formed around ps-nh2 (amino modified polystyrene nps), with consequent immunomodulatory effects on the hemocytes. the interactions between hemocytes, hemolymph proteins, selected types of bacteria and nps observed in vitro, at the basis of immune specificity, suggest that these mechanisms may also participate in immune training in vivo. to investigate this possibility, mussels were subjected to pulse exposure to ps-nh2 (10 g/l; 24h, 48h depuration and further 24h). at each exposure time, several functional and molecular parameters were evaluated, including the bactericidal towards v. aestuarianus. after the 1 st exposure, hemocyte lysosomal stability and mitochondrial potential were decreased, ep was upregulated, lysozyme and mytb were downregulated, lysozyme activity was increased. however, killing of v. aestuarianus was unaffected. after the 2 nd challenge, all stress parameters were recovered, immune-related genes were upregulated (epp, mytb and frep), and the bactericidal towards was increased up to 50%. the results indicate that repeated exposure to ps-nh2 results in immune training, where expression of selected genes may play a role in improving the defence against pathogens through common recognition mechanisms. this project has received funding from the european union‟s horizon 2020 research and innovation programme under the marie skłodowskacurie grant agreement no 671881. the often overlooked coming out of ciliates: biological and experimental benefits from accepting genetically identical conspecifics as sexual partners a vallesi, p luporini school of biosciences and veterinary medicine, university of camerino, camerino (mc), italy ciliates usually manifest sex in the form of conjugation, a unique phenomenon in which cells temporarily unite two by two in mating pairs to perform a mutual exchange of gamete-nuclei derived from meiotic products of their germinal micronucleus. the native view of conjugation as a spontaneous manifestation associated with environmental famine conditions was eventually denied by the milestone sonneborn‟s finding (pnas, 1937) that the most popular ciliate, paramecium, actually controls conjugation through a genetic mechanism of mating types. being only two in the paramecium species studied by sonneborn, these mating types were functionally equated to „male‟ and „female‟ sexes. and, as a consequence of this equation, conjugation was since thought of as a phenomenon committed to involve, as a rule, genetically distinct cells representing two „complementary‟ mating types. however, this is a wrong tenet adverse the evidence that many ciliates conjugate with no discrimination between sex identity and diversity. and from this no discrimination both the ciliate biology and the students of ciliate biology draw benefit. the ciliate biology, because the homo-sexual pairs (yet ineffective to reshuffle the species gene pool) multiply the opportunity for cells to practice conjugation which, in every case, determines the initiation of a new life cycle and the replacement of the cell „old‟ transcriptionally active somatic (macronuclear) genome with a completely new one generated from the permanently „young‟ transcriptionally inert germinal (micronuclear) genome. the students of ciliate biology, because homo-sexual pairs form without requiring physical interactions between sexually/genetically different cells. they form as well in cultures of cells of the same identity previously suspended with filtrates from cultures of conspecific cells of different identity. which immediately identifies species that interact sexually via water-borne mating signals (pheromones), and greatly facilitates the isolation and function-structure characterization of these signals directly from cell-culture filtrates the pheromone genes of the self/non-self recognition mechanism of the ciliate euplotes crassus generate multiple transcripts by an alternative splicing of ‘matryoshka’ introns 36 a vallesi, p luporini school of biosciences and veterinary medicine, university of camerino, 62032 camerino (mc), italy in ciliates, cell-type distinctive protein pheromones control a self/non-self recognition mechanism responsible for the cell switching between the vegetative and sexual stages of the life cycle. these signaling molecules are encoded by genes (pheromone genes) that in the cell somatic nucleus (macronucleus) represent the transcriptionally active versions of transcriptionally silent genes allelic at the same genetic locus mat of the cell germinal nucleus (micronucleus). in the course of evolution, in euplotes, the native single multiallelic mat locus underwent duplication among species which, such as e. crassus, form the latest branching clade of the phylogenetic tree. because of this duplication, e. crassus expresses two distinct families of pheromone genes instead of a single family, as is the case in species of earlier branching clades. we analyzed the structure and expression of a number of e. crassus pheromone genes representative of the two families. like their orthologs of other euplotes species, these genes show 5‟-leader regions that are much more extended than the coding regions, lack canonical regulatory sequences for gene transcription, and synthesize multiple transcripts (in addition to the pheromone-specific one) through the activity of two distinct transcription start sites and a mechanism of alternative intron splicing. these e. crassus introns have been found to be unique with respect to introns of all the other euplotes pheromone genes. they can be distinguished between „matryoshka‟ introns, residing one inside the other like russian dolls, and „non-matryoshka‟ introns. while the former possess canonical gta/tag splicing sites, the latter possess cta/tac splicing sites complementary to the canonical gta/tag splicing sites. this strongly suggests that both the dna strands of the e. crassus pheromone genes can be used as template for transcription. we are currently attempting to verify this hypothesis and assign a function to the products of the multiple e. crassus pheromone gene transcripts. study of viral infections in bivalves as a tool to trace antiviral host pathways u rosani 1 , s domeneghetti 1 , cm bai 2 , m shapiro 3 , p venier 1 1 department of biology, university of padua 2 yellow sea fisheries research institute, chinese academy of fishery sciences 3 department of applied mathematics and statistics, stony brook university viruses are the most abundant biological entity on earth and the presence of an antiviral system in almost every living organism further supports their global distribution. virus-induced selective pressure drive host population dynamics, can interfere with biological invasions and mediate evolutionary transitions. actually, the gene flux from viruses to eukaryotic organisms is suggested to drive the longterm evolution of host genomes while the evolutionary pressure of host antiviral defenses shapes viral genomes in a never-ending arms race. although most of the molecular components of antiviral vertebrate pathways have been traced in bivalve genomes, evidence supporting their activation during viral infections is lacking. aiming to obtain functional data on antiviral defenses in bivalves, we studied the behavior of different malacoherpesviruses in different hosts by highthroughput dna and rna sequencing. accordingly, we describe the virus-induced activation of different antiviral responses in bivalve and gastropod species. among other findings, we showed that an interferon stimulated gene mediates a significant editing of malacoherpesviruses dsrnas in the oyster crassostrea gigas and abalone haliotid diversicolor supertexta, and we provide evidence that, in the evolutionary time, such activity have shaped the genomes of these viruses. our results update the phylogenetic distribution of different antiviral pathways among invertebrates and support discussion on differences and commonalities in comparison to vertebrates. this work was partially supported by the vivaldi european project. two distinct lectin families with different glycanbinding specificity share a β-trefoil fold in mussels m gerdol 1 , y fujii 2 , y ozeki 3 , a pallavicini 1 1 university of trieste, department of life sciences, via licio giorgieri 5, 34127 trieste, italy 2 nagasaki international university, department of pharmacy, faculty of pharmaceutical science, 2825-7 huis ten bosch, sasebo, nagasaki 8593298, japan 3 yokohama city university, department of life and environmental system science, graduate school of nanobio sciences, 22-2 seto, kanazawa-ku, yokohama 236-0027, japan metazoans possess a plethora of highly diversified lectins, which enable the specific recognition of sugar motifs exposed on the surface of target cells. these molecular interactions regulate diverse biological functions such as cell adhesion, fertilization, food particle selection and immune recognition. over the course of evolution, marine invertebrates have recruited a number of different structural units to enable the recognition of glycans associated with invading pathogens. one of such units is the ricin b lectin domain, which adopts a characteristic β-trefoil three-dimensional folding and characterizes a number of carbohydrate-recognition proteins known as r-type lectins. through the combination of glycobiology, molecular biology and genomics approaches, we identified and functionally characterized two distinct families of β-trefoild lectins in mussels (bivalvia, mytilidae), named mytilectins and sevil-like lectins. in spite of a shared three-dimensional structure, the two lectin types display no primary sequence homology and a markedly different glycan specificity, as shown by glycan-array assays. indeed, while mytilec-1 (isolated in mytilus 37 galloprovincialis) recognized globotriose (galα14galβ1-4glc), sevil (isolated in mytilisepta virgata) could bind specifically to asialo-gm1 (galβ13galnacβ1-4galβ1-4glc). we further show that the two type of lectins display a peculiar taxonomic distribution and report the presence of members of the mytilecin family with an additional aerolysin-like pore-forming domain, which might endow such proteins with combined pathogen recognition and killing properties. while the role of β-trefoild lectins in the immune system of bivalves remains to be fully elucidated, we discuss the potential biotechnological application of these molecules. indeed, the ability to specifically recognize glycans expressed on the surface of cancer cells, together with the ability to trigger cell death through the activation of the mapk pathway and caspase-3/9 in a doseand timedependent manner, offer a great opportunity for the development of novel drugs or diagnostic tools based on mussel β-trefoild lectins. a first step in this direction has already been made with the computational design of mitsuba, an artificial lectin based on mytilec-1, able to recognize burkitt‟s lymphoma cells. effects of a nematode-based molluscicide on survival and antimicrobial peptide expression in pomacea canaliculata a montanari 1 , a accorsi 2 , m nasi 3 , d malagoli 1 1 department of life sciences, university of modena and reggio emilia, modena, italy 2 howard hughes medical institute, stowers institute for medical research, kansas city, mo, usa 3 department of surgery, medicine, dentistry and morphological sciences, university of modena and reggio emilia, modena, italy the freshwater snail pomacea canaliculata is a highly invasive species with a robust innate immune system based on cellular and humoral components and only a few predators in nature. to date, no specific and lethal pathogens have been reported for the commonly known golden apple snails, although this information could be crucial for developing sustainable and eco-friendly approaches for controlling p. canaliculata diffusion. in this context, we tested the effects on adult p. canaliculata of a commercial molluscicide developed against terrestrial slugs and constituted by the living nematode, phasmarhabditis hermaphrodita. at the standard temperature of animal maintenance, i.e., 25 °c, the molluscicide proved to be effective against adult specimens of p. canaliculata in a dose-dependent fashion. during one-week experiments, lethal effects have been observed at the highest concentration tested (17 g/l). after incubation at a sub-lethal concentration (1.7 g/l) the snails reduced the food intake and stopped eating while no evident effects were observed for the lowest concentration, 0.17 g/l. the molluscicide efficacy is also temperature-dependent since, especially for the 17 g/l concentration, significantly stronger effects have been observed at 18 °c whereas no effects on survival or feeding rate have been recorded after incubation at 30 °c. real time pcr experiments performed on animals exposed for 24 h at the concentration of 1.7 g/l revealed organand temperature-specific changes in the mrna expression of the antimicrobial peptide (amp) bactericidal/permeability-increasing protein (bpi). more in details, bpi mrna expression relative to control snails significantly dropped in the anterior kidney at 18 °c and in the gills at 25 °c. the other amps tested, lipopolysaccharide binding protein (lbp) 1 and 2, did not change their expression in any of the tested organ at any temperature, as well as bpi after incubation at 30 °c. on the whole, we have observed that at sublethal concentrations the molluscicide induced a reduction of feeding rate and negatively influenced the immune defense in key sentinel organs, namely anterior kidney and gills. our results indicate that nematode-based molluscicides may represent an efficient solution for a sustainable control of p. canaliculata spread. in vitro exposure to 2,2',4,4'-tetrabromodiphenyl ether (pbde-47) impairs innate inflammatory response v longo 1* , a longo 1* , c di sano 1 , d cigna 1 , f cibella 1 , g di felice 2 , p colombo 1 1 istituto di biomedicina e di immunologia molecolare del consiglio nazionale delle ricerche, palermo, italy 2 istituto superiore di sanità, national center for drug research and evaluation, rome, italy *equal contribution polybrominated diphenyl ethers (pbdes) are persistent organic pollutants that are added to numerous products to prevent accidental fires. even though there is little information on the health effects of pbde exposure, it is still of concern to humans because some types of pbdes can build up in the fatty tissues of the several aquatic and terrestrial animals entering the food chain (efsa panel, 2011). a few surveys demonstrated that pbdes bioaccumulate in human tissues with particular attention to human milk. recently, their presence has been correlated to several pathologies but little is known about their effect on the human innate immune system activity. in this study we investigated the effect of the congener 2,2',4,4'tetrabromodiphenyl ether (pbde-47) on the functional activity of the thp-1 human macrophages cell line and on ex vivo freshly isolated human basophils. cytotoxicity and genotoxicity studies showed that pbde-47 was able to induce toxic effects on the thp-1 cell line viability at concentrations ≥25µm. immune function of thp1 was studied after stimulation with bacterial lipopolysaccharide (lps) and pbde-47 exposure at concentrations granting macrophage viability. two dimensional electrophoresis showed modification of the proteome in the 3 µm pbde-47 treated sample and real time pcr and elisa demonstrated a statistically significant reduction in the expression of il-1β, il-6 and tnf-α cytokines. furthermore, pbde-47 was able to perturbate genes involved in cell motility upregulating cdh-1 and downregulating 38 mmp-12 expressions. finally, basophil activation assay showed reduced cd63 activation in pbde-47 treated samples. in conclusion, our study demonstrated that pbde-47 may perturb the activities of cells involved in innate immunity dampening the expression of macrophage proinflammatory cytokines (il-1β, il-6 and tnf-α) and genes involved in cell motility (mmp-12 and ecadherin) and interfering with basophil activation suggesting that this compound can impair innate immune response. benthic foraminifera as a model to evaluate contaminant induced stress responses: a confocal microscopy approach c ciacci 1 , l canesi 2 , e cesarini 1 , b canonico 1 , f frontalini 3 1 department of biomolecular sciences (disb), university of urbino carlo bo, urbino, italy 2 department of earth, environment and life sciences, university of genoa, italy 3 department of pure and applied sciences, university of urbino carlo bo, urbino, italy foraminifera are unicellular organisms enclosed in shell with a great abundance, and biomass in benthic marine habitat. traditionally, the study of foraminifera has been the domain of palaeontologists however, their living counterparts has recently waked up the interests of foraminiferologists for biodiversity, ecological and biomonitoring studies. several investigations have demonstrated the value of benthic foraminifera in detecting environmental contamination and their possible application as bioindicators of pollution in marine and transitional marine settings. the response of benthic foraminifera to adverse environmental conditions may be investigated in terms of density and diversity, assemblage composition, reproductive capability, morphologic abnormalities, and cellular ultrastructure. since very little is known about the cytological alterations of these organisms under stressful conditions, the ultrastructural changes induced by pollutant exposure are not fully understood. microscopy techniques have been traditionally used to study the diversity of benthic foraminifera in morphological, genetic, cytological and mineralogical perspectives. most of these techniques(i.e., transmission electronic microscopy) produce high-resolution images but they require extensive sample preparation, and do not allow in vivo examination. other approaches, such as confocal laser scanning microscopy (clsm) using fluorescent and fluorogenic probes can be instead applied to living cells. clsm allows qualiand quantitative studies of the ultrastructural and biochemical organization of the cell both under natural conditions and in response to environmental stress. furthermore, multifluorescence labelling permits simultaneous targeting of different organelles and physiological processes. in this work, clsm is utilized to evaluate the effects of model contaminants (hg and different types of nanoparticles-nps) in the benthic foraminifera, ammonia parkinsoniana. data are presented on oxyradical production, lysosomal function and lipid accumulation utilizing selective fluorescent dyes. the results indicate that the clsm approach can be successfully utilized to evaluate contaminant-induced stress responses in benthic foraminifera. session 2. chairmen: loriano ballarin, university of padua, padua, italy and davide malagoli, university of modena and reggio emilia, modena, italy priming and immunity modulation the evolution of specificity in immune priming j kurtz institute for evolution and biodiversity, university of muenster, muenster, germany contrary to prior believe, recent studies indicate that all animals possess forms of acquired immunity, such as immune priming in insects, which can even be specific. however, we know little about the evolution of basic characteristics of these forms of immunity. we thus tested whether the specificity of immune priming can evolve rapidly and to what extent the evolved phenotypes are linked to transcriptomic changes. using controlled evolution experiments, we selected the model beetle host tribolium castaneum for either specific or unspecific immune priming towards pathogenic bacteria. after 14 host generations of evolution, specificity of priming was not universally higher in the lines selected for specificity, but rather depended on the bacterium used for priming and challenge. for instance, the insect pathogen bacillus thuringiensis induced the strongest priming effect. these differences in priming specificity between the evolved populations were mirrored in the transcriptomic response, revealing an involvement of metabolic and transcription-modifying genes in immune priming. in vivo isolation and characterization of telocytes using supplemented biomatrices l pulze, a grimaldi, f ferraro, n baranzini, g tettamanti, m de eguileor university of insubria, department of biotechnology and life sciences, varese, italy the leeches (hirudo verbana) have proven to be a good model for deciphering basic biological processes for two main reasons: first of all, they have a reduced dimension and, despite a relative anatomical simplicity, share with vertebrates the complexity of immunological mechanisms and wound-healing processes; secondly, in animal kingdom there is a remarkable evolutionary conservation of biological responses, cell types, cellular mechanisms, and molecules. one of the most phylogenetically conserved system, from lower invertebrates to man, is the innate immune system that, strictly interacting with neuroendocrine one, guarantee a powerful protection to organisms. in invertebrates, in addition to a 39 plethora of cytokines, a wide range of immunocytes such as macrophages, nk cells, granulocytes and a new type of cells, the telocytes, are involved. leech telocytes (tcs) are stromal cells engaged in surveillance and protection spread in various tissues and strategically localized among resident cells, nearby the capillaries and the nerve endings. these cells, contacting via gap junctions, are organized in an extensive three-dimensional network, and show cell-cell contacts with other cell types and interactions with collagen bundles of connective tissue. the interactions among these and the other cells is obtained in two ways: physically, by direct cell–cell contacts and, chemically, via the release of microvesicles and exosomes, which can transport a variety of soluble factors involved in the regulation of different physiological processes. leech-tcs originate from circulating precursor cells and, once activated in response to chemical or physical stimuli, are able to change their morphology and behaviour, moving towards the injured area to participate in repair/regenerative processes. to better characterize leech-tcs we have isolated and cultured these cells. the injection of an appropriate combination of matrigel biopolymer, supplemented with selected factors in the leech h. verbana, has allowed to recruit these cells. while few migrating cells are present in control matrigel specimens lacking factors, an increased number of cells in relation to the time elapsed from the injection of the supplemented biomatrix, colonize matrigel specimens containing: monocyte chemoattractant protein-1 (mcp-1/ ccl2) or il-8 or ribonuclease t2 (rnaset2). the recombinant hvrnaset2 protein induces a connective tissue remodeling in the invertebrate model medicinal leech hirudo verbana n baranzini, f acquati, m de eguileor, g tettamanti, a grimaldi university of insubria, department of biotechnology and life science, varese, italy recent studies demonstrated that the ribonuclease rnaset2 modulates inflammatory processes in both vertebrates and invertebrates. this protein chemoattracts macrophages in vivo and its expression significantly increases after bacterial infections. moreover recent data obtained in our laboratory, demonstrated that the injection of the human recombinant protein rnaset2 in the leech body wall, a consolidated invertebrate model for studying both immune response and tissue regeneration, induces not only macrophages recruitment, but also a massive connective tissue remodeling. based on these data, here we evaluated a possible direct or indirect role of the leech rhvrnaset2, recently cloned in our laboratory in the synthesis of new collagen. the connective tissue reorganization and the cell types involved in this process were characterized in rhvrnaset2 injected leeches by means of histochemical staining and morphological analyses at optical and electron microscopes. the expression of newly synthesized pro-collagen1α1, together with that of fibroblast receptors (fgfb), was localized at tissue level by immunofluorescence. moreover, to evaluate the expression profile of pro-collagen1α1, western blot experiments were performed on protein extracts from leech body wall retrieved after different time elapse from rhvrnaset2 injection. double immunofluorescence assays were performed to correlate fibroblasts activation, collagen fibrils production and the consequent connective tissue remodeling with the expression of hvrnaset2. the data reported in this work provide compelling evidence in support of a pleiotropic role for rnaset2 in orchestrating an evolutionarily conserved cross-talk between inflammatory response and regenerative process, based on macrophages recruitment and fibroblasts activation coupled to a massive extracellular remodeling. towards decrypting stress response in tardigrade -transcriptome survey of paramacrobiotus sideralis during anhydrobiotic state c manfrin 1 *, i giovannini 2 *, r guidetti 2 , pg giulianini 1 , l rebecchi 2 1 university of trieste, department of life sciences, trieste, italy 2 university of modena and reggio e., department of life sciences, modena, italy *equal contribution anhydrobiosis is a highly stable state of suspended animation in an organism due to its desiccation, which is followed by recovery after rehydration. tardigrades are water-dwelling, eight-legged, segmented animal ranging about 0.5 mm in length as adults and are among the most resilient known animals, because many species of this phylum are capable of anhydrobiosis and cryobiosis at certain environmental conditions. however, physiological and stress mechanisms at the bases of extreme desiccation remain elusive. using the anhydrobiotic tardigrade paramacrobiotus sideralis, as a model organism, we have investigated stress-related transcripts. the transcriptomes of hydrated active animals and desiccated ones were compared. anydrobiotic tardigrades were obtained after exposing them at 18 °c initially to 80% relative humidity (rh) for 4 h, then to 50 % rh for 4 h, and finally at room temperature to 0-3 % rh overnight. a de novo transcriptome has been assembled through trinity v2.4.0 and annotated with trinotate v3.1.1. to identify high-quality and non-redundant transcripts, redundancy has been removed following evidentialgene tr2aacds pipeline and evaluated with busco. a total of 78832 contigs have been assembled and 2323 differentially expressed genes (degs) resulted following dessication (log2 fold change >1 and a false rate discovery ≤ 0,01). about 74% of degs were up-regulated, and 26% down-regulated (1710 and 612 contigs, respectively). among the 40 highest 50 up-regulated degs with an enriched 10to 200-fold during desiccation relative to hydrated conditions, 1/6th resulted unknown and three of them are secreted. within the highest induced and annotated transcripts immunerelated are the most represented such as 5 cytosolic-abundant heat soluble proteins (cahs2s), 2 cytochrome p450 proteins, 2 apolipophorins, 2 lysosome-associated membrane glycoproteins, and 1 annexin. among the 50 highly suppressed transcripts, half of them are unknown and suppressed annotated categories are mainly related to translation activity, rna silencing processes, transketolase activity, and structural arrangement. secretory-abundant heat soluble protein 1, and chaperone proteins have been also found downregulated. findings from these preliminary analyses confirm a huge suppression of metabolism following anhydrobiosis and confirm activation of intrinsically disordered proteins, (as cahs) to survive desiccation. susceptibility to entomopathogens and modulation of basal immunity in two insect models at different temperatures. m mastore 1 , s quadroni 2 , a toscano 1 , n mottadelli 1 , mf brivio 1 1 department of theoretical and applied sciences, university of insubria, varese, italy 2 department. of science and high technology, university of insubria, varese, italy in this work, we analysed the efficacy of different commercial bio-insecticides (steinernema feltiae, steinernema carpocapsae, heterorhabditis bacteriophora and bacillus thuringiensis) by valuating the mortality induced on two insect models, galleria mellonella (lepidoptera) and sarcophaga africa (diptera) after exposure to different temperatures (10, 20 and 30 °c). moreover, we investigated the effects of temperature on the basal humoral immunity of the two target insects; particularly, phenoloxidase (po) and lysozyme activity. our results show that g. mellonella is susceptible to all bio-insecticides at all the examined temperatures, except when infected at 10 °c with s. carpocapsae and at 30 °c with s. feltiae and b. thuringiensis. s. africa is more susceptible at 30°c to all bioinsecticides; whereas, when infected at 10 and 20°c, h. bacteriophora is the most efficient. temperature modulates po activity of both g. mellonella and s. africa, otherwise variations in lysozyme activity is observed only in g. mellonella. except for a possible correlation between the increased lysozyme activity and the delayed bt efficacy recorded on g. mellonella at 30 °c, a different resistance to bio-insecticides at different temperatures does not seem to be associated to variations of the host basal immunity, probably due to immunoevasive and immunodepressive strategies of these entomopathogens. the immune response of hermetia illucens larvae: preliminary evidence d bruno 1 , m mastore 2 , m de eguileor 1 , a grimaldi 1 , mf brivio 2 , g tettamanti 1 1 university of insubria, department of biotechnology and life sciences, varese, italy 2 university of insubria, department of theoretical and applied sciences, varese, italy the dipteran hermetia illucens (diptera: stratiomyidae), also known as black soldier fly, is a promising insect species for waste management, thanks to the ability of the larvae to grow on a wide variety of organic substrates. moreover, the high nutritional value of the larvae makes this insect useful for the production of feedstuff. despite the great interest on this species, information about the biology of this insect remain scarce, especially about the immune system. in the present study we performed a preliminary investigation on the cellular and humoral response of h. illucens larvae. to this aim we conducted a morphological analysis of hemocytes that are involved in the cellular response. moreover, we evaluated the main components of the humoral response after immune challenge with grampositive and gram-negative bacteria. in particular, we analyzed phenoloxidase and lysozyme activity in the hemolymph of the larvae, and mrna expression of antimicrobial peptides (amps) in the fat body. our results show an increase of phenoloxidase and lysozyme activity in infected larvae compared to controls. moreover, we observed a modification of amp expression 6, 12, and 24h after the immune challenge compared to naive larvae. this study provides preliminary information on the immune system of h. illucens. this knowledge will hopefully open up the possibility to optimize the rearing procedures of h. illucens larvae and to obtain high quality larvae for feed production. characterization of the complement system in a colonial protochordate: c3 complement receptors and opsonic role of c3 a peronato, n franchi, l schiavon, l ballarin department of biology, university of padua, padua, italy the complement system is one of the most ancient immune modulator mechanism of bilaterian metazoans, able to influence ancient cells and factors of both innate and adaptive immunity. three complement-activation pathways are known in vertebrates: the classical, the alternative and the lectin pathways: all of them converge on the cleavage of c3. the compound ascidian botryllus schlosseri is a reliable model organism for the study of immunobiology. as an invertebrate, b. schlosseri relies only on innate immunity for its defense and immunocytes. recently, in the same species, we demonstrated the presence of homologues of mammalian c3, bf, mbl and masp1, referred to as bsc3, bsbf, bsmbl and bsmasp, respectively. all the complement components identified so far, are 41 expressed by morula cells, the most abundant circulating hemocytes. in mammals, once the complement system is activated, a cascade of reactions that involves proteolysis and polymerization occurs resulting in the cleavage of the third complement component (c3) to c3a and c3b, the former exerting a chemokine–like activity, the latter acting as opsonin and, ultimately, activating the lytic pathway. the best-known receptor for c3a in mammals is c3ar, whereas cr1 is the receptor able to recognize and bind c3b on the microbial surfaces. in the present work, we described, in b. schlosseri, two new genes showing homology with vertebrate c3ar and cr1, respectively, and studied their transcription in the course of the colonial blastogenetic cycle. results indicate that their mrnas are located in different immunocyte types suggesting the presence of an important cross-talk between phagocytes and morula cells. in addition, we continued our analysis of the role of c3 in botryllus immunity by studying the modulation of bsc3 transcription during the colonial blastogenetic cycle and the effect of bsc3 knockdown on immune responses. only morula cells, and no other immunocytes type, were labelled by the antisense probe for bsc3ar, whereas phagocytes and young, undifferentiated cells known as hemoblasts were the cells stained by the probe for bscr1. both the bsc3ar and bscr1 genes are constitutively transcribed as almost all morula cells and phagocytes, respectively, resulted labelled by the antisense probe in the ish assay, independently of their previous challenge with zymosan, a known activator of b. schlosseri hemocytes. however, a modulation in the extent of transcription occurs during the colonial blastogenetic cycle as the amount of bsc3ar mrna abruptly decreased at to, whereas no differences were observed when ec and mc were compared. this is probably related to the renewing of circulating cells at to, when 20-30% of hemocytes undergo cell death by apoptosis and are replaced by new, differentiating cells entering the circulation in the same period. tentacles regeneration in anemonia viridis (anthozoa, cnidaria). r pernice 1 , c la corte 1 , d parrinello 1 , s barnayverdier 2 , p furla 2 , m cammarata 1 , mg parisi 1 1 department of earth and marine sciences, university of palermo, palermo, italy. 2 era team, ircan umr 7284, nice, france. the mechanisms for discriminating the “self‟‟ from “non-self” have evolved into a long history of cellular and molecular strategies, from damage repair to the co-evolution of host-pathogen interactions. not all immune responses are due to the presence of genetically foreign entities, but to the emission of danger/alarm signals from injured cells, such as those exposed to pathogens, toxins and mechanical damage. in this sense, the cnidarian capacity for regeneration could be considered an additional arm of innate immune defense. in this study, the immune responses and tissue regeneration in the temperate symbiotic sea anemone, anemonia viridis, induced by cutting tentacles were investigated. the experimental plan was carried out on groups of animals to which 10, 20 and 30 tentacles were respectively cut. protein extracts of bodies and tentacles were prepared 7, 14 and 21 days after cutting. morphological observations on tentacles in state of regrowth, measurements of the expression of proliferating cell nuclear antigen (pcna) as a regeneration marker and protease activities detection were carried out. starting from previous knowledge on the natural seasonal variability in biometric traits and enzymatic biomarkers of a. viridis, the activity of enzymes involved in inflammatory response such as protease, peroxidase and esterase were analyzed in the tentacle and body extracts. finally, haemolytic activity of the tentacle extracts was assayed against sheep erythrocytes. the injury elicited a significant increase in the phosphatase and peroxidase activities of the tentacles, in contrast to esterase that seems not been affected. sds electrophoretic analysis revealed a variable gelatinolytic and fibrinolytic activity in the tentacle extracts, while none fibrinolytic activity was observed in the body extracts. the immunoblot in both tentacular and body extracts showed a cross reaction with antipcna antibody and a significant difference between treatment and control groups. the pcna is more present in tentacles extracts than in the body samples. particularly, high positivity was detected in tentacular extracts after 7 and 14 days from cut of 10 and 20 tentacles. in perspective, we want to study at histological and molecular level how homeostatic tissues start the regeneration program while triggering immune response and their mediators of inflammation. selenoprotein t: an example of novel endocrine modulator in non-mammalian species s leo 1 , r mazza 1 , s imbrogno 1 , m filice 1 , a gattuso 1 , c rocca 1 , t angelone 1 , y anouar 2 , mc cerra 1 1 department of biology, ecology and earth sciences, university of calabria, rende (cs), italy 2 neuronal and neuroendocrine differentiation and communication laboratory, rouen-normandie university unirouen, france. selenoprotein t (selenot) is a thioredoxinlike protein expressed in mammals and in nonmammalian vertebrates. it possesses a selenocysteine (sec, u) within a cxxu motif, and this confers oxireductase functions. studies in mammals proposed selenot as a humoral modulator with a role in endocrine homeostasis, neuroprotection, and in myocardial response to ischemia/reperfusion (i/r). despite many recent research efforts, its endocrine/paracrine/autocrine functions are still to be fully described, particularly in non-mammalian species. in the goldfish (carassius auratus), a hypoxia tolerance fish model, three 42 selenot transcripts (gfselt1a, gfselt1b and gfselt2) have been detected. this study aimed to evaluate the cardiac expression of selenot, and its putative role as an endocrine/paracrine/autocrine modulator of the goldfish heart function under both normoxic and hypoxic conditions. by western blot and immunofluorescence we found that selenot is expressed in the heart, and the expression is increased under hypoxia. on ex vivo isolated and perfused goldfish heart preparations, under normoxia, exogenous pselt, a selenot-derived peptide, dose-dependently enhanced myocardial contractility by involving a modulation of membrane and sarcoplasmic calcium, and a camp-dependent signaling. under hypoxia, pselt unaffected goldfish myocardial contractility but reduced myocardial nitrosative stress. these data propose selenot as an evolutionary conserved protein with a protective potential against cardiac hypoxiadependent redox unbalance. they also pave the way to explore the role of the protein as a cardiac endocrine modulator in fish and a humoral intermediary of the communication between the heart and distal tissues and cells. session 3. chairmen: luigi abelli, university of ferrara, ferrara, italy and giuseppe scapigliati, university of tuscia, viterbo, italy fish immunity immunity in the teleost digestive tract s picchietti department for innovation in biological, agrofood and forest systems, university of tuscia, viterbo, italy teleost fish constitute the most abundant vertebrate group, exhibiting a high assortment of morphological variations of their gastrointestinal (gi) tract related to phylogeny, ontogeny, environment and feeding habits of each species. this variability is closely linked with several specialized functions, since the gi tract is involved not only in nutrient absorption and digestion, but also in water and electrolyte balance, and immunity. teleosts moreover, possess a complex gut microbiota, that shapes every physiological system of the host and even effects the intestinal morphology. because the gi tract is a main route for pathogen entry, its mucosa plays an effective defence against potentially harmful determinants of the environment, while it tolerates a diversity of harmless microbes and dietary antigens. the mechanisms allowing antigen uptake over the epithelial barrier play a crucial role for maintaining the gut homeostasis and regulate appropriate immune responses, and involve the presence of antigen sampling cells equivalent to mammalian microfold and dendritic cells. teleosts possess an extensive system for immune activation, and responses to antigen uptake have usually been reported higher after anal than oral delivery. gene expression profiling revealed functional specialization along the gi tract, and demonstrated that the establishment of mucosal immune responses is especially relevant in the posterior intestine. generally speaking, teleost fish do not contain lymphoid aggregates in the mucosa, however their gut harbors high numbers of diffuse t (cd8 + and cd4 + ) and b cells. igt + b cells outnumber the igm + cells in teleost gut and the igst produced display a preponderant and specialized role in mucosal immunity, being the key players in the defence against pathogens. importantly, commensal bacteria shape b cells and igs responses and in a parallel way, mucosal igs and secretory component allow the host to sculpt its microbial communities. also t cells, mostly those bearing t receptors, play an essential role in intestinal cell-mediated immunity and it seems they are important in tolerance or attack against the microbiota. studies evidenced that fish gut microbiota responds to dietary manipulations, and although the interplay between nutrition and immune system is well recognised, understanding the link between diet, gut microbiota and health in fish is only at the beginning. evolution of immune responses: similarities between fish lymphocytes and mammalian innate-like lymphocytes g scapigliati, s picchietti department for innovation in biological, agrofood and forest systems, university of tuscia, viterbo, italy innate lymphoid cells (ilcs) of vertebrates are a cluster of innate immune cells that are classified in three groups for the expression of defined transcription factors, functional characteristics, and phenotype. the lymphocytes of vertebrates are classically described as the cells responsible of adaptive responses, but experimental evidence suggest that subpopulations of mammalian lymphocytes may behave as ilcs, engaging nonself rapidly and without antigen restriction. the innate-like lymphocyte subpopulations have been mainly identified as γδ-t cells, mucosal associated invariant t cells (mait), and b1-b cells, are principally located in mucosal tissues, may be involved in human pathologies and their functions and tissue(s) of origin are not fully understood. the similarities in the morphology and immunobiology of immune system between fish and mammals have been established, but the homologies between fish lymphocytes and mammalian innate-like lymphocytes is an issue poorly considered in comparative immunology. increasing experimental evidence suggests that main fish lymphocyte populations could have developmental, morphological, and functional features in common with innate-like lymphocytes of mammals. however, despite these similarities and with the hypothesis that mammalian innate-like lymphocytes could be evolutionarily related to fish lymphocytes, information on possible links between γδ-t lymphocytes and and b1-b cells of fish and mammals is missing, our research is currently aimed to investigate these possible similarities in lymphocyte evolution. effects on immunity of exposure to microplastics in adult zebrafish 43 a mancia 1 , l abelli 1 , a benkhalqui 1 , c bertolucci 1 , mc fossi 2 , g limonta 2 , c panti 2 1 department of life sciences and biotechnology, university of ferrara, ferrara, italy 2 department of physical sciences, earth and environment, university of siena, siena, italy it is now widely accepted that microplastics (mps) represent a serious concern for aquatic environments, therefore assesment of biological pathways affected is crucially relevant. this study focused on variations of liver transcriptome, histology of gastrointestinal tract and gills, and locomotor activity of exposed fish along various days after treatment. adult zebrafish (3 groups, n=12 each) were fed for 20 days with dry food alone (controls) or supplemented with a mix of pristine high-density polyethylene and polystyrene microplastics (0.1 or 1 mg/l), ranging in size from <25 to 90 µm. the exposure to mps resulted in differential transcription of 324 genes in total, already affected at the lower dose, mainly involved in cholesterol biosynthesis (fatty acid degradation) and immunity pathways. up-regulation of transcripts subserving response to extra-cellular antigens, and downregulation of others involved in innate antimicrobial response, antiviral defense and maintenance of epithelial integrity highligted defective control of pathogen entry at epithelial barriers, confirmed by occurrence of histopathological signs in both intestine and gills. furthermore, variations in energy utilization likely accounted also for alteration of circadian rhythm of locomotor activity. immunodetection of igm, igt and pigr in mucosal tissues of antarctic teleost a ametrano 1 , a mancia 2 , s ballarin 2 , a benkhalqui 2 , g calderoni 2 , g pavani 2 , mr coscia 1 , l abelli 2 1 institute of protein biochemistry, cnr, naples, italy 2 department of life sciences and biotechnology, university of ferrara, ferrara, italy we have previously investigated the immune response at hepato-biliary level in the antarctic teleost trematomus bermacchii, a species belonging to the perciform suborder notothenoidei, the most abundant component of the fish fauna living in the antarctic ocean. by that time only the igm isotype was known and well characterized at molecular and biochemical levels in antarctic fish. over the past few years we have cloned and sequenced genes encoding other two key molecules of the mucosal immune system, igt and polymeric ig receptor (pigr) of t. bernacchii. the present study aimed at investigating the localization in mucosal tissues of igm, igt and pigr in an attempt to clarify the protein occurrence and transepithelial transport. biochemical and immunohistochemical data provided convergent data about specific mechanisms operating apical release of igt in exocrine way, as well as depicting peculiar (maybe ancestral) features compared with well known mechanisms described for polymeric igs transport in mammalian tissues. f-type lectin from serum of trematomus bernacchii (boulenger, 1902): purification, characterization and bacterial agglutinating activity. m dara 1 , p giulianini 2 , c manfrin 2 , mg parisi 1 , m cammarata 1 1 marine immunobiology laboratory, department of earth and marine sciences, university of palermo, palermo, italy 2 department of life sciences university of trieste, building q room 306 trieste, italy lectins belong to a protein family, present in almost all living organisms and involved in several biological processes, including immune responses. peculiarity of these proteins is the ability to bind carbohydrates due to their carbohydrate-recognition domains (crds). in fish, c lectin, f binding lectin (fbl), galectin, rhamnose-binding lectin (rbl) and pentraxin have been identified in both cartilaginous and bony fish. in addition, selectins and other genes have been found in the currently available fish genomes. the fbl, known as fucolectins, constitute the most recent lectin family identified and structurally characterized in teleosts. the fbl family is constituted by a large number of proteins exhibiting multiples of the f-type motif, either tandemly arrayed or in mosaic combinations with other domains. in the present study, a fbl has been purified and characterized from serum of the antarctic fish trematomus bernacchii by affinity chromatography on fucose-agarose column. assay of inhibition from carbohydrates in fact showed affinity of this lectin for the fucose. a convincing hemoagglutinating activity (ha) was detected towards rabbits red blood cells (rbc) and at lesser extent towards sheep erythrocytes. the ha activity was analyzed at different temperatures. it was maintained at temperature values comprised between 4 °c and 37 °c and was completely depleted after exposure at 50 °c. in sds-page analysis, the fbl exhibited an apparent molecular weight of 30 kda in non-reducing conditions and an increase to 32 kda after reduction. this difference is recognized as a classical shrinkage of f-lectins, due to the present of internal disulfide bridges. the f lectin present on the t. bernacchii transcriptome show a very similar and congruent structure with a theoretical mw 32.16 kda and an isoelectropoint of 5.21. bacterial agglutinant activity (ba) of serum and purified fractions was tested towards e. coli. the serum showed high activity after incubation at room temperature (18 °c), as well as in the fractions. the sequence, structure, sugars specificity, fucose inhibition, molecular weight, protein shrinkage and activity against bacteria collocate this molecule on f-lectin family and thus suggesting its involvement in host pathogen interactions. 44 mucosal immunity response of european eel (anguilla anguilla l.) after bacterial infections e conforto 1 , l vílchez-gómez 2 , ma esteban 2 , mg parisi 1 , d parrinello 1 , m cammarata 1 , fa guardiola 2,3 1 department of earth and marine sciences, university of palermo, palermo, italy 2 department of cell biology and histology. faculty of biology, campus regional de excelencia internacional “campus mare nostrum”, university of murcia, murcia, spain 3 centro interdisciplinar de investigação marinha e ambiental (ciimar), university of porto, porto, portugal recently, mucosal surfaces of fish, in particular skin and its secreted mucus, have attracted significant interest among immunologists. the external mucus layer that covers fish skin contains numerous immune substances poorly studied that act as the first line of defence against a broad spectrum of pathogens and infections through the epidermis. for the first time, this study aimed to characterize and describe different humoral immune defence parameters in the skin mucus of the european eel (anguilla anguilla) challenged by intraperitoneal injection with vibrio anguillarum and tenacibaculum maritimum. in order to do this, several immune-related enzymes as well as the bactericidal activity against fish pathogenic bacteria were evaluated in skin mucus of european eel at 24, 48, 72 hours post-challenge. the results demonstrated that european eel skin mucus showed significant increments in peroxidase and lysozyme activity at 48 and 72 hours after v. anguillarum injection respect to the other experimental groups (unchallenged and challenged with t. maritimum). in the case of antiprotease activity, an increase was observed at 24 hours in fish challenged with v. anguillarum respect to control group whilst this activity was not detected at 48 and 72 hours in any experimental group. contrarily, protease activity decreased in challenged fish at 48 regarding control group whilst this activity only was diminished in skin mucus from fish challenge with v. anguillarum respect to unchallenged fish. esterase activity showed only an increase at 72 hours in fish challenge with t. maritimum compared with the results obtained in skin mucus from the other experimental groups. finally, european eel skin mucus revealed higher bactericidal activity against photobacterium damselae than those observed against v. anguillarum. more concretely, bactericidal activity against v. anguillarum did not show any significant variations. however, the bactericidal activity, measured when the skin mucus was incubated with p. damselae, increased in skin mucus from fish challenged with v. anguillarum at 24 hours of trial. interestingly, t. maritimum grew in presence of skin mucus from all experimental fish which could mean that this substrate serves as nutrient source for this bacterium. the present results could give new insights into the mucosal immune system of this primitive species with potential application to the aquaculture. fa guardiola thanks the fundación séneca de la región de murcia (spain) for his grant (saavedra fajardo program, grant no. 20407/sf/17). session 4. chairmen: anita giglio, university of calabria, rende (cs), italy and piero giulianini, university of trieste, trieste, italy microbiota and invertebrate immunity insect immunity as affected by stress factors and associated microorganisms f pennacchio department of agricultural sciences, university of naples “federico ii”, portici (na), italy insects represent the most species-rich taxon of multicellular organisms. this biodiversity is paralleled by an equally rich diversity of associations between insects and microorganisms. the resulting metaorganisms are governed by complex molecular networks underlying their physiology and the intricate interactions they establish with other biological entities. insect immune barriers are suppressed by invading parasites and pathogens, which have developed effective virulence strategies, as a result of a long co-evolutionary process. however, the immune response is not only modulated by these biotic stress factors, but is also conditioned by insect associated microorganisms and several abiotic stressors. among these, poor nutrition and pesticides play an important role. in particular, neurotoxic insecticides are able to interfere with the cross-talk between the nervous and immune systems, often separately considered. unveiling the regulatory mechanisms of these physiological networks can be profitably done only at metaorganism level. this paves the way towards the development of new bioinspired strategies for pest control and pollinator protection. exposure to tio2 nanoparticles results in shift in hemolymph microbiome composition and immunomodulation in mytilus galloprovincialis m auguste 1 , a lasa 1 , a pallavicini 2 , c pruzzo, 1 l vezzulli 1 , l canesi 1 1 department of environmental, earth and life sciences, university of genoa, genoa, italy 2 department of life sciences, university of trieste, trieste, italy increasing attention has been recently given to the microbiome and its complex dynamics maintained within the host (human, animal, plant), and on the different factors that could affect their natural equilibrium. although invertebrates represent 95% of animal species, a minority of studies focus on this group. the interest in invertebrate-microbe interactions is also due to conservation of the mechanisms of innate immunity, and understanding cross talk between microbes and the immune system can lead to insights of broader relevance. marine invertebrates host a high microbial abundance and diversity, and alteration of the 45 microbiota due to stressful conditions and/or environmental changes has been linked with a compromised health status and susceptibility to disease. in particular, the presence of microorganisms in the hemolymph of healthy bivalves indicates that this ecosystem could contribute to host homeostasis. the expansion of nanotechnology is raising concern on the potential biological effects of nanoparticles (nps), including nano-oxides such as titanium dioxide-ntio2. its current addition to a variety of food attracted interest on the potential impact on mammalian gut microbiota. moreover, ntio2 has been shown to alter the microbiota of fish and the composition of planktonic bacterial communities. therefore, exposure to ntio2 may also affect the microbiota of marine invertebrates. in this work, the effects of ntio2 exposure (100 μg/l, 96 h) on hemolymph microbiota of mytilus galloprovincialis were investigated. the microbiome was analysed by targeted high-throughput sequencing of the 16s rrna gene using ion torrent technology. the results show that although similar in composition, the hemolymph core microbiome was less diverse in ntio2-treated than in control mussels. the composition of the microbial population was unequally affected by ntio2, with decrease in abundance of some genera (kistimonas, shewanella, vibrio) and increase in others (e.g. stenotrophomonas). moreover, determination of immune parameters revealed decreased hemocyte lysosomal membrane stability, increased serum lysozyme activity, and increased bactericidal activity of whole hemolymph. the results may partly explain the observed shift in microbiome composition induced by ntio2exposure. these represent the first data on the effects of nps on the microbiome of a marine invertebrate, and suggest an interplay between hemolymph microbiota and activity of the immune system. this project has received funding from the european union‟s horizon 2020 research and innovation programme under the marie skłodowskacurie grant agreement no 671881. when immunity and extracellular matrix matter: repair and regenerative events after echinoderm arm injury c ferrario 1,2 , a czarkwiani 3 , f bonasoro 1 , md candia carnevali 1 , p oliveri 3 , m sugni 1,2 1 department of envrionmental sciences and politics, university of milan, milan, italy 2 center for complexity and biosystems, department of physiscs, university of milan, milan, italy 3 department of genetics, evolution and environment, university college london, london, united kingdom arm amputation in echinoderms is a traumatic event that removes differentiated body parts and damages all tissue types. immediately after injury the repair phase begins. if phenomena typical of this phase, such as emergency reaction, inflammatory/immune response, wound closure and extracellular matrix (ecm) remodeling and deposition, do not properly occur, the following regenerative process may be prevented or ineffective. in this study, the brittle star amphiura filiformis (afi) was used as model to investigate the main repair and regenerative events after arm injury, with a specific focus on the involvement of immune and ecm genes and proteins. in this perspective, both microscopy and molecular analyses were performed to highlight similarities and differences between regeneration-competent (i.e. echinoderms) and noncompetent (i.e. mammals) animals. our microscopy results showed that both emergency reaction and re-epithelialisation are faster in brittle stars than in mammals. fibrosis, i.e. over-deposition of ecm due to an exaggerated inflammatory reaction, is not detectable in echinoderms as, instead, described for mammals, suggesting that immunity modulation may facilitate subsequent regeneration. our molecular analyses showed that afi-ficolin (an important gene in the immune response) is expressed in the first phase after injury, whereas almost all the selected ecm genes are not expressed at early stage of regeneration, suggesting an activation delay that may be directly connected to their regeneration efficiency, as proposed for other echinoderms and in contrast to most vertebrates. moreover, at advanced regenerative stages these same genes are differentially expressed, suggesting that the molecular regulation of ecm deposition/remodelling is different throughout re-growth. overall, our brittle star model shows similarities in terms of repair and regenerative events and timing with other echinoderm species already studied. however, differences emerge between echinoderms and mammals: indeed, all phenomena should occur following specific signals and timing to ensure effective regeneration after severe wounds. further quantitative analyses will allow a better understanding of immune system and ecm contribution to brittle star arm regeneration and of the evolutionary implications on the regeneration competence widespread in the animal kingdom. preliminary data on functional coelomocytes changes during echinaster sepositus arm regeneration f stasi 1 , ap miglietta 1 , j vizioli 2 , p pagliara 1 1 di.ste.b.a university of salento, lecce, italy 2 protéomique réponse inflammatoire spectrométrie de masse prism, university of lille, lille, france echinoderms are known to have the greatest capacity for regeneration among deuterostomes. the mediterranean red sea star echinaster sepositus is a suitable model for studying this phenomenon because it is able to regenerate arms following self-induced amputation (autotomy) or traumatic loss/damage. the overall regenerative process could be subdivided in three main phases: a repair phase, characterized by wound healing and edematous area formation; an early regenerative phase, characterized by first differentiation phenomena; and an advanced regenerative phase, with proper arm regrowth. previous studies 46 evidenced that in sea star the regenerative process of arm tip is considered mainly morphallactic, involving either pluripotent progenitor cells such as coelomocytes or/and differentiated cells, which may undergo dedifferentiation or transdifferentiation. moreover, coelomocytes are important elements involved in the repair phase of arm by forming a clot and in first immune response following the injury. we focused our attention on the role of coelomocytes, cells freely wandering in the coelomic fluid, during sea star arm regeneration. in this work, we described the morphofunctional changes of coelomocytes occurring during the repair phase in the red sea star e. sepositus following traumatic amputation of the distal third of one arm. we evaluated ros production and the aif-1 protein expression in coelomocytes at different times after amputation. an increase in ros production was evidenced 24h post amputation (p.a.), while the highest number of aif-1 positive cells was detected 48 h p.a. these preliminary data underline the interest of investigating the high regenerative capacities in this attractive model for further/future biomedical studies in regeneration. cellular immune response in harpalus (pseudoophonus) rufipes (de geer, 1774) (coleoptera, carabidae) a giglio 1 , s battistella 2 , f cavaliere 1 , pg giulianini 2 , a mazzei 1 , f talarico 1 , ml vommaro 1 , p brandmayr 1 1 department of biology, ecology and earth science, university of calabria, rende, italy 2 department of life sciences university of trieste, trieste, italy carabid beetles are among the most important groups of beneficial arthropods in the agroecosystem food chain where they are predators of many pests (including aphids, lepidopterans, slugs and diptera). previous studies have been shown that they are good models to investigate the negative effects of agrochemical used in agricultural management practices on natural enemies of insect pests. in ecological immunology, variation on immune capacity of insects is an early warning, highly sensitive biomarker to monitor the sub-lethal effect of toxicants introduced into environment as a result of industrial or agricultural activity. however, morpho-functional data about immune system of carabid are scanty in spite of their ecological relevance. in this study, we have investigated the immune function of harpalus (pseudoophonus) rufipes for their use in eco-toxicological monitoring. this species is a omnivorous predator, very common in calabrian (south italy) agroecosystems and acts as a predator against insect pests. tests performed on adult involve a general screen of cellular responses and include: characterization of circulating hemocytes and phagocytosis in vivo. the cellular population has been characterized by light and electron microscopy analysis and 4 morphotypes of circulating hemocytes were found: prohemocytes (0.55±0.11%), plasmatocytes (67±2.52%), granulocytes (29.32±2.35%), oenocytoids (0.73±0.20%). the phagocytosis assays were performed in vivo by injection of 0.9 µm carboxylate-modified polystyrene latex beads in order to identify the hemocyte types involved in phagocytosis. after 2h non-self challenge treatment, specimens showed a decrease of plasmatocytes and oenocytoid percentages (12.38±2.21% and 0.16±0.05%, respectively) and a non-specific immune response involving phagocytosis performed by granulocytes (82.74±2.54%). moreover, hemocytes with mitotic figures and non-differentiate cells were found in the hemolymph (2.28±0.37), thus confirming a continuously turnover . melanotic nodules have been found 2h after the immune challenge formed to immobilize the latex beads. herbicide exposure effects on cellular and humoral immunity of soil insects f cavaliere 1 , p brandmayr 1 , pg giulianini 2 , ml vommaro 1 , a giglio 1 1 department of biology, ecology and earth science, university of calabria, rende, italy 2 department of life sciences university of trieste, trieste, italy herbicides used in agriculture have known negative effects in non-target organisms. pendimethalin is a dinitroanaline herbicide used for pre-emergent control of annual weeds. this class of herbicides interfere with the structure and function of microtubules inhibiting the steps in the plant cell division responsible for chromosome separation and cell wall formation. beneficial soil invertebrates, such as insects that inhabiting agricultural areas and provide ecosystem services, are potentially threatened by herbicides. this study has been designed to assess the effect of pendimethalin exposure on harpalus rufipes, one the most common omnivorous predators in agricultural ecosystems involved in the pest control. adults from an uncontaminated area (organic wheat field of about 6 ha in a biological farm, 39°17'10.28"n, 16°42'28.33"e, 1150 m a.s.l.; macchia di tuono farm, san giovanni in fiore, calabria, southern italy) were placed in plastic boxes contained soil sprayed with activus® (a.i. pendimethalin; recommended field rate 4l per ha of active ingredient, from 330gr/l of commercial formulation). tests lasted for 21 days and at 20 °c and 8:16 l:d cycle. to assess the sublethal effects of exposure over the time, the total hemocyte counts and the haemolymph phenyloxidase (po) activity were measured after 48h, 1 week and 21 days of exposure as markers of immune system strength. exposed animals showed a decrease of thcs compared to controls at all times. basal and total po activities were highly significantly lower in exposed animals compared to controls at all time. the negative impacts on these immune parameters demonstrate that the exposure to herbicides modify the susceptibility of this species to pathogens. as a result, we assume that the exposure of h. rufipes adults to commercial formulation of pendimethalin in field may altered other basic life history traits such as reproduction, dispersal activity and predation with effects on the adult fitness resulting in changes 47 of the population structure and in a reduction of the biocontrol activity for pest species in agroecosystem. 251 isj 14: 251-258, 2017 issn 1824-307x research report transcriptional effect of serotonin in the ganglia of lymnaea stagnalis c benatti1,2, c colliva1, jmc blom2,3, e ottaviani1, f tascedda1,2 1 department of life sciences, university of modena and reggio emilia, modena, italy 2 center for neuroscience and neurotechnology university of modena and reggio emilia, modena, italy 3 department of education and humanities university of modena and reggio emilia, modena, italy accepted july 31, 2017 abstract the serotonin system (5ht) is highly conserved in both vertebrates and invertebrates, and numerous evidence supports a biological link between 5ht and numerous animal function. in the present paper we evaluated the transcriptional effects of a serotonergic stimulation on selected targets involved in 5ht signalling and neurotransmission in the central nervous system of the great pond snail lymnaea stagnalis. adult snails were treated acutely (6 h) or chronically (48 h) with either 5hydroxytrypthophan (5-htp 1mm), the immediate precursor of serotonin, fluoxetine (flx 1µm), a selective serotonin reuptake inhibitor, or a combination of two. the central ring ganglia were dissected and used for q-pcr gene expression analysis. transcription was strongly induced following a chronic, but not an acute, exposure to 5-htp in the ganglia of lymnaea. in particular, lymcreb1 and lymp2x mrna levels were decreased following a 6 h exposure and increased in snails receiving 5hydroxytryptophan for 48 h. interestingly, this effect was reduced when snails were exposed chronically to both 5-htp and flx, suggesting a role for sert in mediating the effect of 5hydroxytryptophan. these data suggest that l. stagnalis is suited to unravel the complexity of the serotonin signaling pathway. key words: serotonin; creb; lymnaea stagnalis introduction the serotonin is an ancestral complex neurotransmitter system that plays an important role in the regulation of many biological functions. serotonin has a fundamental role in the modulation of stress-induced excitability (arousal), in the defensive behavior (il-han et al., 2010), in the modulation of aggressive behaviors and in the control of anxiety. normally, for these studies, have been used small mammals (i.e., rats and mice) but this approach may not be always effective and is accompanied by many ethical and economical drawbacks (tascedda et al., 2015). the high cost of these studies and the increasing difficulties in obtaining permits for experimentation prompted researchers to look for other strategies. many researchers have attempted to solve the problem by using in vitro cell systems (alboni et al., 2013b, 2014) that have many important advantages. unfortunately, ___________________________________________________________________________ corresponding author: fabio tascedda department of life sciences university of modena and reggio emilia via campi 287, 41125 modena, italy e-mail: fabio.tascedda@unimore.it the obtained results were often limited and inconclusive in elucidating the basis of diseases and identifying effective therapeutic strategies (alberts, 2010). invertebrates, thanks to their relatively simple nervous systems and to the latest technique in genome sequencing and manipulation, are becoming a useful tool for the study of neuronal physiology and for best disease process characterization (ottaviani et al., 2013; tascedda et al., 2015). invertebrates lack self-awareness “autonoetic consciousness” (curren and chalsani, 2012), emotional behaviour reduced to its individual components. in particular, the pond snail lymnaea stagnalis, an aquatic pulmonate gastropod with a central nervous system (cns) consisting of ≈20,000 neurons organized in a ring of interconnected ganglia, has proven to be an extremely useful and accessible model to study fundamental aspects of cns function such as synaptic plasticity and associative memory. the serotonin (5-ht) neurons present in the cns of lymnaea are analogous to vertebrate 5-ht neurons that originate in the raphe nuclei. serotonin, with specific innervation, control central pattern generators and other important circuits of the cns. furthermore, through integrated 252 feedback information coming from the innervated areas, support general behavioural arousal (andrianov et al., 2015; gillette, 2006). in this model recent finding demonstrate a fundamental role of serotonin in the control of reproduction and behaviour (il-han et al. 2010, ivashkin et al. 2015) dysfunction in serotonin system regulation or serotonin levels are involved in the control of many biological functions (gillette 2006, deneris et al. 2012). understanding the transcription mechanisms associated with the hyper stimulation of the serotoninergic system is a key step to clarifying the fundamental aspects of these mechanisms. in this context, we sought to mimic the generalized activation of the serotonergic system by administering the rate-limiting 5-ht precursor 5hydroxytryptophan (5-htp) to freely moving animals. we used 5-htp because studies in rodents and molluscs have proven that exposure to 5-ht or tryptophan are less effective in elevating serotonin and related molecules, with shorter amplitude and duration. it is also possible that 5htp disrupt homeostatically regulated serotonin levels (lynn-bullock et al., 2004; marinesco et al., 2004; fickbohm et al., 2005). in this study, we evaluated the effects of 5htp on the serotonin related genes and on major intracellular systems related to serotonergic stimulation. the molecular machinery governing serotonin signaling has been cloned and characterized in lymnaea. serotonin is synthesized by tryptophan hydroxylase (lymtph) (koert et al., 2001) in the cytoplasm of the presynaptic serotonergic neurons, vesicle monoamine transporters (lymvmat) then package serotonin into vesicles, and upon fusion with the cell membrane, the neurotransmitter is released into the synaptic cleft and binds to specific receptors (lymhtr1 and lymhtr2) (sugamori et al., 1993; gerhardt et al.,1996). the concentration of 5ht is then regulated by the action of a specific transporter (lymsert) (sadamoto et al., 2008). the activity of serotonin receptors produces important intracellular changes related to camp and ca++ signalling (poser et al. 2001). these pathways, in invertebrates and in mammals, are both linked to serotonergic control of related stress responses and adaptive mechanisms in response to pharmacological treatment (kaang et al. 1993, vinet et al., 2003, 2004; blom et al., 2006). one target for the serotonergic stimulation is a modifying effect on the regulation of postreceptor pathways and genes related to the camp cascade in particular the transcription factor camp response element binding protein (creb) (kaang et al., 1993; marinesco et al., 2004). creb is known to regulate the downstream expression of camp-inducible genes, and is proposed to be involved in the control of many biological functions, in the regulation of brain homeostasis and in the response to pharmacological treatment (blom et al., 2002; alboni et al., 2010, 2011, 2013a). in lymnaea, analogue of creb (lymcreb1) has been cloned and characterized (sadamoto et al., 2004). materials and methods animals and colony maintenance laboratory-reared freshwater pond snails, lymnaea stagnalis (original stocks donated by vrije universiteit, amsterdam) were maintained in aquaria at the university of modena and reggio emilia (italy) in standard laboratory conditions: 21 23 °c, 12:12 h light/dark cycle (on at 08:00). adult animals having shell lengths of 20 to 25 mm were used in this experiment and were kept in 12 l tanks (30 mature snails in each) supplied with wellaerated water. they were fed pesticide-free lettuce and goldfish pellets three times a week, and the aquaria were cleaned on alternate days. every effort was made to minimize the number of animals used and their suffering. pharmacological experiments the following compounds were used for pharmacological treatments: 5-hydroxy-l-tryptophan (5-htp) 1 mm (sigma-aldrich); fluoxetine hydrochloride (flx) 1 μm (polichimica). solutions of specified concentrations were freshly prepared in boiled filtered water (fw) with 50 μm ascorbic acid (sigma-aldrich) in order to avoid 5-htp oxidation. we incubated adult snails for 6 or 48 h without food and aeration in 2 l aquaria, 15 specimens per 400 ml of experimental solution. untreated controls (naive adults) were left undisturbed in an equal amount of fw, a group exposed only to ascorbic acid in the same conditions was also included. after incubation the animals were anesthetized on ice for 10 min and the central ring ganglia was dissected out (buccal ganglia were excluded) and stored at 80 °c prior analysis. total rna extraction, reverse transcription, and real time polymerase chain reaction four central ring ganglia were pooled for total rna extraction, 4 6 replicates were analyzed for each group. total rna extraction and dnase treatment were performed using genelute™ total rna miniprep kit and dnase70-on-column dnase i digestion set (sigma aldrich) as previously described (benatti et al., 2011). five hundred ng of total rna was reverse transcribed with high capacity cdna reverse transcription kit (life technologies corporation) in 20 µl of reaction mix. mrnas were quantified by real-time quantitative polymerase chain reaction in roche lightcycler® 480 (roche diagnostics gmbh) using power up sybr green mix (life technologies corporation). specific forward and reverse primers were used at the final concentration of 300 nm (table 1). single pcr products were subjected to a heat dissociation protocol as previously described (caraci et al., 2016). cycle threshold (ct) value was determined by the lightcycler® 480 software (roche diagnostics gmbh). statistical analysis for quantitative evaluation of changes the comparative δδct method was performed, using as calibrator the average levels of expression of control snails. the stability of mrna expression of two reference genes (elongation factor 1-alpha, lymef1α http://journal.frontiersin.org/article/10.3389/fnbeh.2015.00279/full#b31 253 table 1 nucleotide sequence of the forward and reverse primers used for real-time pcr gene bank accession target product length type sequence ct (25 ng) ab041522.1 lymnaea stagnalis camp responsive element binding protein, lymcreb1 180 bp [49-229] fw gtcagcagggaatggtcctg 25 rv aaccgcagcaaccctaacaa jx524180.1 lymnaea stagnalis p2x receptor, lymp2x 150bp [1005-1155] fw gggatcgtcttcgtggtga 24 rv agttcctggccttcaacagat aj238276.1 lymnaea stagnalis neuropeptide y, lymnpy 188 bp [432-620] fw actcttggtgtcactgctcg 17 rv cttgcgccgtttctctttcc l06803.1 lymnaea stagnalis serotonin receptor 1, lymhtr1 126 bp [893-1019] fw actatctcatcctgtccttg 23 rv gatatccacatgtcacacac u50080.1 lymnaea stagnalis serotonin receptor 2, lymhtr2 115 bp [884-999] fw acacctggagtattctcatc 23 rv gaagtagttggtcacgttct fx185022 lymnaea stagnalis serotonin transporter, lymsert 177 bp [726-903] fw ataccgtaccttgtcatgtt 20 rvtgttgtagtaccaggagaca af129815.1 lymnaea stagnalis tryptophan hydroxylase, lymtph 179 bp [238-417] fw aggatacagtctaccgacag 18 rv tgagttcacggaaaactatt af484094.1 lymnaea stagnalis vesicular monoamine transporter, lymvmat 172bp [529-701] fw aacgtgtacatgactgtgac 22 rv aagccagtaaacattggtat dq278441.1 lymnaea stagnalis elongation factor 1-alpha, lymef1α 150bp [7-157] fw gtgtaagcagccctcgaact 16 rv ttcgctcatcaataccacca x15542.1 snail, beta-tubulin, lymtub 127 bp [92-219] fw gaaatagcaccgccatcc 16 rv cgcctctgtgaactccatct the accession number, the size (bp) of the pcr product obtained by amplification of the cdna (mrna) are given for each target. as indication of the relative abundances of each target average ct values in adult snails (25 ng, n = 4). and beta-tubulin, lymtub) was assessed using normfinder®, lymtub was the most stable gene across groups and was used for gene normalization. statistical analyses were performed using an analysis of variance (one-way anova). significant changes were determined by tukey post-hoc test (with p < 0.05 significance level). results effect of an exposure to 5-htp for 6 or 48 h on the expression levels of analogues of creb1 and p2x in the cns of l. stagnalis lymnaea creb1 (lymcreb1) is a homolog of mammalian creb that is expressed in the cns of lymnaea and is involved in synaptic facilitation (sadamoto et al., 2004, 2010). one way anova revealed a main effect of both an acute and a prolonged exposure to 5-htp [f (4;21) = 5.282, p = 0.004 and f (4;30) = 13.896, p < 0.0001 respectively; fig. 1a]. in particular, post hoc analysis showed that the expression of creb1 was significantly induced in snails exposed for 48 h to 5htp with respect to all the other treatment regimens (p < 0.001). the effect of 5-htp was reduced in presence of flx: lymcreb1 mrna levels of the 5htp/flx group were significantly higher than the control group (p < 0.05), while being significantly lower than the group receiving 5-htp alone (p < 0.01). in contrast, following a 6 h exposure we observed a significant decrease of lymcreb1 expression in the group exposed either to 5-htp or flx and to the combination of the two compounds with respect to control (p < 0.05) (fig. 1a). a similar trend was observed for lymp2x (fig. 1b); this purinergic receptor was recently identified and cloned in the cns of lymnaea (bavan et al., 2012). prolonged exposure to 5-htp resulted in an overall significant increase in lymp2x mrna in ganglia [f (4;30) = 22.506, p < 0.0001]. again, this effect was still present when snails were exposed to both flx and 5-htp, but was significantly reduced with respect to the increase observed following 5htp alone (p < 0.0001) (fig. 1b). when considering the effects of a 6 h treatment, one way anova revealed a main effect [f (4;20) = 6.257, p = 0.002; fig. 1b], indeed, p2x expression was reduced by 5htp, flx, and their combination with respect to control (p < 0.05). effect of a 5-htp exposure on the expression levels of components of the serotonergic system in the cns of l. stagnalis to date, two 5-ht receptor genes have been cloned in lymnaea: lymhtr1 and lymhtr2 (sugamori et al., 1993; gerhardt et al., 1996). lymhtr1 expression levels were affected by a 6 h exposure to 5-htp [f (4;18) = 5.714, p = 0.004; fig. 2a], while no effect was observed when treatment was protracted up to 48 h [f (4;29) = 1.609, p = 0.199; fig. 3a]. in particular post hoc test revealed that flx 1 µm was able to decrease the expression http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&id=399822766 254 fig. 1 effect of an exposure to 5-htp for 6 or 48 hs on the expression levels of analogues of creb1 and p2x in the cns of lymnaea stagnalis. adult snails were incubated in 1 mm 5-htp, 1 μm flx, or a combination of the two (5-htp/flx) for 6 or 48 h. untreated adults (ctrl) and a group exposed only to ascorbic acid (ascorbate) in the same conditions were also included. lymcreb1 (a) and lymp2x (b) mrna expression in the ganglia, with lymtub as endogenous control, were measured by real-time pcr. n = 4 7 pools of 3 snails each. data are represented as means ± s.e.m. and were analyzed with anova followed by tukey. ***p < 0.0001, **p < 0.01,*p < 0.05 vs ctrl; p< 0.0001, p < 0.05 vs ascorbate; °°° p < 0.0001, °° p < 0.01 vs flx; ## p < 0.01, # p < 0.05 vs 5htp. levels of lymhtr1 with respect to both control groups (p < 0.05). this down-regulation was blunted in the group exposed to 5-htp, alone (p = 0.072) or in combination with flx (p = 0.015). on the other hand, the expression levels of the other serotonergic receptor, lymhtr2, were not altered on our experimental conditions [f (4;19) = 1.140, p = 0.368 for 6 h; fig. 2b, and f (4;29) = 2.380, p = 0.075 for 48 h exposure; fig. 3b]. sadamoto and co-workers (2008) identified and characterized the localization of the of the serotonin transporter in lymnaea: lymsert. in our experimental conditions, no effect on lymsert mrna levels was observed following a 6 h treatment regime [f (4;21) = 2.010, p = 0.130; fig. 2c], while exposure to 5-htp for 48 h significantly increased the expression levels of serotonin transporter with respect to untreated controls [f (4;29) = 3.043, p = 0.033; fig. 3c]. we also evaluated in ganglia the transcriptional effect of a serotonergic stimulation on the ratelimiting enzyme in the synthesis of serotonin: tryptophan hydroxylase (lymtph) (koert et al., 2001). a main effect was revealed in animals experiencing a 48 h exposure to 5-htp [f (4;30) = 8.662, p < 0.0001; fig. 3d], while no effect was observed following a 6 h treatment [f (4;18) = 1.887, p = 0.157; fig. 2d]. in particular, lymtph mrna was significantly higher in the ganglia of ascorbic acid-exposed snail with respect to the levels found in both the 5-htp exposed groups (alone (p = 0.007) or in combination with flx (p < 0.0001) and in untreated control snails (p = 0.017) (fig. 3d). vesicular monoamine transporter (lymvmat) mrna levels were not affected in snails exposed to 5-htp, flx or their combination for 6 h with respect to control groups [f (4;19) = 2.058, p = 0.127] (fig. 2e). on the other hand, one-way anova revealed a main effect of a chronic treatment with 5hydroxytryptophan on vmat expression [f (4;26) = 11.300, p < 0.0001] (fig. 3e). we observed a significant decrease of lymvmat mrna in animals exposed to 5-htp for 48 h, alone or in combination to flx, with respect to control groups (p < 0.01) or to flx-treated snails (p < 0.01). 255 fig. 2 effect of a 6-h 5-htp exposure on the expression levels of components of the serotonergic system in the cns of lymnaea stagnalis. adult snails were incubated in 1 mm 5-htp, 1 μm flx or a combination of the two (5htp/flx) for 6 h. untreated adults (ctrl) and a group exposed only to ascorbic acid (ascorbate) in the same conditions were also included. lymnaea stagnalis serotonin receptors [lymhtr1 (a), lymhtr2 (b)] and transporter [lymsert (c)], tryptophan hydroxylase [lymtph (d)], vesicular monoamine transporter [lymvmat (e)] mrna expression in the ganglia, with lymtub as endogenous control, were measured by real-time pcr. n = 4 7 pools of 3 snails each. data are represented as means ± s.e.m. and were analyzed with anova followed by tukey. **p < 0.01,*p < 0.05 vs ctrl; p < 0.05 vs ascorbate. discussion here we demonstrated that specific transcription was strongly induced following a prolonged, but not an acute, exposure to 5-htp in the ganglia of lymnaea. in particular, lymcreb1 and lymp2x mrna levels were increased in snails receiving 5-htp for 48 h, and decreased following a 6 h exposure. interestingly, this effect was reduced when snails were exposed chronically to both 5htp and flx, suggesting a role for sert in regulating the effects of 5-htp. previous studies in vertebrates and invertebrates have shown that a treatment with 5htp is able to increase serotonin content in the cns, in both serotonergic and non-serotonergic regions (gartside et al., 1992; lynn-bullock et al., 2004; fickbohm et al., 2005). moreover, studies on isolated neuron in lymnaea have demonstrated that the precursor acts only indirectly through its conversion to serotonin and its effects are mediated by enhanced serotonin release, activation of its receptors and modulation of electrical activity (dyakonova et al., 2009). 256 fig. 3 effect of a 48-h 5-htp exposure on the expression levels of components of the serotonergic system in the cns of lymnaea stagnalis. adult snails were incubated in 1 mm 5-htp, 1 μm flx or a combination of the two (5htp/flx) for 48 h. untreated adults (ctrl) and a group exposed only to ascorbic acid (asc) in the same conditions were also included. lymnaea stagnalis serotonin receptors [lymhtr1 (a), lymhtr2 (b)] and transporter [lymsert (c)], tryptophan hydroxylase [lymtph (d)], vesicular monoamine transporter [lymvmat (e)] mrna expression in the ganglia, with lymtub as endogenous control, were measured by real-time pcr. n = 4 7 pools of 3 snails each. data are represented as means ± s.e.m. and were analyzed with anova followed by tukey. **p < 0.01,*p < 0.05 vs ctrl; p < 0.0001, p < 0.05 vs ascorbate; °° p < 0.01 vs flx. in our experimental conditions we demonstrated that 48 h exposure to serotonin precursor is able to influence also gene expression of selected targets and that some of these effects where significantly diminished in the presence of flx, a selective inhibitor of serotonin transporter. in the group exposed to flx alone for 48 h no alterations of gene expression of the evaluated targets were observed. this effect is in agreement with the results of yu rl and collaborators (2008) that have shown that in aplysia, 24 h treatment of paired pleural-pedal ganglia, induced the mrna of creb gene and protein. in aplysia, treatment with 5-htp has been demonstrated to potentiate serotonergic activity (marinesco et al., 2004), which, in turn, may increase intracellular levels of camp and cause a rapid and transient induction of cre-responsive genes (kaang et al.,1993). this effect is mediated by the phosphorylation of creb at ser119, this transcription factor is a key component in regulating synaptic plasticity both in physiologic and pathologic 257 conditions (kaang et al., 1993; sadamoto et al. 2010, blom et al., 2002; alboni et al., 2011). we observe a strong increase in creb mrna levels following a 48 h serotonergic stimulation in the cns of lymnaea which could be the basis for major behavioral changes induced by serotonin in different invertebrate models (il-han et al., 2011; andrianov et al., 2015). the lymp2x gene acts similarly to creb. currently, in the literature, there is no direct evidence of a link between the transcription of this receptor and the activity of the serotoninergic system. we can hypothesize that an increase in the number of receptors can lead to an increase in intracellular ca++ and a strengthening of creb's transcriptional activity. the link between creb and ca++ is widely demonstrated both in mammalian and invertebrate models (poser and storm 2001; ghosh-roy et al., 2010). studies on knockout mice have established the importance of vmat2 in regulating catecholamine and serotonin levels, and their release from neurons following a depolarizing stimulus (eiden and weihe, 2011). similarly, in our model a long lasting serotonergic stimulation caused a significant downregulation in lymvmat expression, this effect was still present even when snails were exposed to the combination 5-htp/flx, suggesting that this effect does not depend on sert functionality. it is possible that the increased activity of serotonergic neurons evoked by exposure to 5-htp may induce this down-regulation. conclusions our experiments show that stimulation of the serotoninergic system can induce specific transcriptional changes in the ganglia of l. stagnalis. furthermore, these data suggest that lymnaea ideally suited to unravel the complexity of the serotonin signaling pathway and may represent a good model to provide new insights on how serotonin can modulate different biological functions and its role in brain homeostasis. aknowledgements the authors would like to thank mr frigeri c for his precious help in setting up the acquaria, dr malagoli d for his consult in dissecting procedure. this work was supported by the grant "far 2016" from university of modena and reggio emilia, italy. references alberts b. model organisms and human health. science 330: 1724, 2010. alboni s, benatti c, capone g, corsini, d, caggia f, tascedda f, et al. time-dependent effects of escitalopram on brain derived neurotrophic factor (bdnf) and neuroplasticity related targets in the central nervous system of rats. eur. j. 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aspects of allorecognition isj 12: 233-236, 2015 issn 1824-307x letter to editor evolutionary aspects of allorecognition l ballarin1, l du pasquier2, b rinkevich3, j kurtz4 1department of biology, university of padua, padua, italy 2zoology and evolutionary biology, university of basel, basel, switzerland 3national institute of oceanogtaphy, haifa, israel 4institute for evolution and biodiversity, university of münster, münster, germany accepted september 8, 2015 dear editor, although frequently neglected, allorecognition phenomena exhibit suites of effector mechanisms, altogether featuring one of the main biological characteristics of living organisms. a recent workshop organized by the münster graduate school of evolution at the university of münster, germany, focused on the evolutionary aspects of allorecognition and the potential links to immune systems. the term allorecognition, generally defined as the capability of non-self recognition between conspecifics, encompasses apparently unrelated phenomena on various biological organizations, such as: i) recognition between different strains within a single bacterial species (gibbs et al., 2008); ii) mating type recognition in protozoans (luporini et al., 2006); iii) recognition between cell lineages in the multicellular reproductive structures of slime molds (hirose et al., 2011); iv) intraspecific hyphae recognition in fungi (glass et al., 2000); v) selfincompatibility in plants (takayama and isogai, 2005); vi) graft rejection in metazoans (karp and meade, 1993; bilej et al., 2010; eckle et al., 2013); vii) self-sterility in many animals; viii) colony specificity in marine colonial organisms. evolutionary considerations can provide a unifying framework to identify communalities of these systems and to shed light on their relation to immune systems, and in particular the evolution of specific recognition and memory within innate and adaptive immune systems (du pasquier, 2005; kurtz, 2005; litman et al., 2005). allorecognition systems seem to be an important evolutionary force that helps in shaping the wide diversity of contemporary immune systems providing animals with some useful tools, such as the receptor variability and polymorphism that are required for the efficient distinction between non-self and self (de boer, 1995; dionne, 2013). ___________________________________________________________________________ corresponding author: loriano ballarin department of biology university of padua via ugo bassi 58/b, 35100 padua, italy e-mail: loriano.ballarin@unipd.it indeed, colony specificity has been widely described in colonial invertebrate taxa with sessile modes of life, such as sponges (which can be assimilated to colonial organisms), cnidarians, bryozoans, tunicates and algae. in these organisms the survivorship of recruiting propagules is highly dependent on the ability to compete for the available substrate with other sedentary organisms, including conspecifics, and 'natural transplantations' frequently occur when colonies physically contact each other. in the latter case, either a fusion of conspecific colonies into a larger chimeric entity or a non-fusion reaction, that prevents colony fusion, occurs, depending on the presence or absence of shared alleles at a limited number of highly polymorphic fusibility/histocompatibility loci (sabbadin et al., 1992; rinkevich, 1993; cadavid et al., 2004; nicotra et al., 2009; voskoboynik et al., 2013). larger colonies deriving from fusion between conspecifics have undoubtedly some ecological advantages with respect to smaller colonies, such as enhanced competitive capabilities, improved survivorship following attacks by predators, shortened onset of reproduction and augmented fecundity, as a great number of zooids contribute to gamete production, or benefit in terms of resource sharing (buss, 1982; grosberg, 1988). fusion between conspecifics leads to chimerism, where cells of different genotypes are commonly intermingled in the new developing biological entity. in spite of the low attention reserved to this phenomenon by the scientific community, chimerism is widespread in nature and documented as well in various non-colonial taxa, including mammals (rinkevich, 2011). during evolution, it could have played a role in shaping the immune systems, by selecting allorecognition mechanisms whose elements could have been recruited afterwards in other types of immune responses. in contrast to the aforementioned benefits that may incur to chimeras, a competition between somatic and/or germ cell lineages of different origin may develop within chimeras, leading to somatic or germ cell parasitism, in which one of the cell lineages dominates and parasitizes the whole 233   234   chimeric colony (magor et al., 1999; rinkevich, 2011). hence the need for histocompatibility systems, as an acquired response to prevent withinorganism conflicts, and the appearance of genetic systems limiting fusion to genetically related and kin colonies (dishaw and litman, 2009; czárán et al., 2014; gilbert, 2015). the high degree of polymorphism of genes involved in allorecognition ('antigens' and receptors) is encountered in all organisms so far studied with many cases of convergence (dionne, 2013). this stresses the selective value of allorecognition, that may have played a role in the generation of specific immune systems of many metazoa, and which reached its highest complexity in vertebrates with the appearance of adaptive immune systems. those listed above were some of the major issues discussed on last july 7th-8th, at the workshop organized by the münster graduate school of evolution (westfälische wilhelmsuniversität münster, germany), entitled: “evolutionary aspects of allorecognition: from intraspecific conflicts to links with adaptive immunity” and intended for phd students. the workshop included three lectures held by l ballarin, fellow at münster university (general aspects of allorecognition: cells, molecules and physiological responses), b rinkevich (chimerism: will two walk together except they have agreed? (amos, 3:3)) and l du pasquier (analogies and homologies in the somatic generation of immune repertoires of metazoa). the workshop also included a 'knowledge café' session. the 'knowledge café' was an invaluable scientific exercise that allowed the direct involvement of phd students with the puzzles and the open questions associated with allorecognition, the origin of the polymorphism in genes involved in allorecognition, and the relationships between allorecognition and specificity in immune responses of invertebrates and vertebrates alike. the scientific discussions were not terminated at the end of the workshop but they continued, following activities like exploiting the web, future planned round tables and the collection of the emerging ideas and opinions in evolutionary aspects of allorecognition. general aspects of allorecognition: cells, molecules and physiological responses l ballarin allorecognition is a widely diffuse phenomenon in nature. shared feature of allorecognition are: i) the variability of the recognition proteins, related to the high polymorphism of the respective genetic loci; ii) the induction of an inflammatory response, characterized by the recruitment of immunocytes upon the recognition of allogeneic molecules, the degranulation of cytotoxic cells and the release of cytokines; iii) the induction of cytotoxicity which, in invertebrate, frequently occurs as a consequence of the activation of the melanin-producing enzyme phenoloxidase (po) and the production of reactive oxygen species. in the compound ascidian botryllus schlosseri, colony specificity, a type of allorecognition present in sessile, colonial invertebrates, manifests itself as rejection of genetically incompatible colonies, with the appearance of a series of necrotic, melanic spots along the contact border. cytotoxic morula cells (mcs), constituting the majority of circulating hemocytes, are the first cells to sense non-self molecules diffusing from the alien colony. upon their recognition, mcs degranulate and release immunomodulatory molecules able to recruit other immunocytes in the contact region. they also release po, which is responsible of the observed cytotoxicity. mcs also synthesize c3 precursors and store amyloid fibrils inside their granules, which poses the question of the role of c3 and amyloid in ascidian inflammation. since the synthesis of melanin is controlled by α-msh, produced by phagocytes, another unresolved questions concern the role of other immunocytes, i.e., phagocytes, in colony specificity and the presence of a cross-talk among different immunocyte types in ascidian inflammation. chimerism: will two walk together except they have agreed? (amos, 3:3) b rinkevich while immunity in all multicellular organisms (animals and plants alike) is highly efficient in dealing with parasites, in many taxa it fails to combat chimerism between conspecifics. indeed, natural chimerism is widely documented in nature, appearing in about ten phyla of protists, invertebrates and plants, vertebrates and mammals, including humans. as a matter of fact, when appearing, chimerism serves as important ecological and evolutionary tools in metazoans’ life history portraits, dictated costs and benefits for the genotypes involved. including in the list of benefits are the increase of genetic variability, sizedependent ecological qualities that are improved following chimerism (affecting growth rates, reproductive outputs, survivorship, competitive exclusion benefits, increasing tolerance against environmental drivers), the development of synergistic complementation, the assurance of mate location, and more. major costs include the threat of somatic and germ cell competition and parasitism, sexual sterility, the development of diseases (including cancers and autoimmune diseases), and organ malformations. clearly, natural chimerism is an evolutionary driven phenomenon. the major questions that will be asked are, why chimerism first appeared? what are the evolutionary benefits that support its existence, and why do not all multicellular organisms present scenarios for chimerism? analogies and homologies in the somatic generation of immune repertoires of metazoa l du pasquier the immune systems of metazoa are under pressure to diversify repertoires of recognition structures that enable individuals to survive in a 235   diverse and rapidly changing pathogenic and competitive environment. during evolution various solutions that meet this demand have been selected within the different phyla. first, large families of germline genes encoding the receptors can be generated by multiple duplications. some of these are used in allorecognition, for example polymorphic immunoglobulin superfamily members encoded by the alr 1 and alr 2 histocompatibility loci of hydractinia that involve homologous interaction between cell surface molecules of the same family that end up, launching alloaggression reactions. second, several phyla use combinatorial associations to somatically generate, repertoires of large numbers of specific receptors (that exceed the number of genes encoding them!) that can provide adaptive individual responses during ontogeny. the associations can be between peptides (within the families of leucine rich repeats, immunoglobulin domains, lectin domains, etc.). the combinatorial associations can also take place during genesis of the effector cells populations at the level of nucleic acid segments. at the rna level they can involve splicing like in the mysterious receptor called dscam in arthropods, the role of which in immunity is far from being clear, but the diversity of which is amazing. at the dna level they can involve somatic rearrangement with combinatorial association of exons (vdj recombination), conversion, mutation and generate repertoires of receptors with different adaptive characteristics that can be analogous, but not necessarily homologous to each other. somatic modifications (i.e., at the dna level) can be encountered in echinoderms (183/333 molecules), molluscs (frep molecules) and vertebrates (molecules of the leucine-rich repeat or immunoglobulin superfamilies) but are not due to homologous mechanisms but to analogy and convergence. the most recent discovery of such an analogy is that of the adaptive immune system of agnathans that, in three lymphocyte lineages similar to αβ, γδ τ cells and b cells of gnathostomes, makes use of leucine-rich repeats receptor genes, to generate somatically the repertoire of their immunoreceptors. they do it with the help of a member of the cytidine deaminase family, an enzyme homologous to aid, also involved in somatic generation of immunoglobulin superfamily receptors repertoires in gnathostomes. two aspects that can profoundly differentiate responses among metazoa and that need to be elucidated are: 1) how selection of repertoires is achieved, whether mhc analogues will be discovered in agnathans. 2) whether specific cell proliferation is induced after encounter with a non-self epitope in any invertebrate. this will condition whether secondary responses can actually be attributed to classical immunological memory or to persistence of ongoing responses or to yet unknown mechanisms. because of the involvement of mhc, the answers to these two questions will perhaps help placing allorecognition in the context of the evolution of adaptive immune systems. acknowledgements authors wish to thank the students ferro d, hamley m, knoblich k, kutzer m, talarico m, wieczorek b, wohlleben a, who took part in the discussions, as well as dr kloke v for her invaluable support and santander universities for funding. references bilej m, procházková p, silerová m, josková r. earthworm immunity. adv. exp. med. biol. 708: 66-79, 2010. buss lw. somatic cell parasitism and the evolution of somatic tissue compatibility. proc. natl. acad. sci. usa 79: 5337-5341, 1982. cadavid lf, powell ae, nicotra ml moreno m, buss lw. an invertebrate histocompatibility complex. genetics 167: 357-365, 2004. czárán t, hoekstra rf, aanen dk. selection against somatic parasitism can maintain allorecognition in fungi. fungal gen. biol. 73: 128-137, 2014. de boer rt. the evolution of polymorphic compatibility molecules. mol. biol. evol. 12: 494-502, 1995. dionne ms. comparative immunology: allorecognition and variable surface receptors outside jawed vertebrates. curr. opinion. immunol. 25: 608-612, 2013. dishaw lj, litman gw. invertebrate allorecognition: the origin of histocompatibility. curr. biol. 19: r286, 2009. du pasquier l. meeting the demand for innate and adaptive immunities during evolution. scand. j. immunol. 62 (suppl. 1): 39-48, 2005. eckle sb, rossjohn j, mccluskey j. alloreactivity. meth. mol. biol. 1034: 3-39, 2013. gibbs ka, urbanowski ml, greenberg ep. genetic determinants of self-identity and social recognition in bacteria. science 321: 256-259, 2008. gilbert om. histocompatibility as adaptive response to discriminatory within-organism conflict: a historical model. am. nat. 185: 228-242, 2015. glass nl. jacobson dj, shiu pkt. the genetics of hyphal fusion and vegetative incompatibility in filamentous ascomycete fungi. ann. rev. genet. 34: 165-186, 2000. grosberg rk. the evolution of allorecognition specificity in clonal invertebrates. q. rev. biol. 63: 277-412, 1988. hirose s, benabentos r, ho h-i, kuspa a, shaulsky g. self-recognition in social amoebae is mediated by allelic pairs of tiger genes. science 333: 467-470, 2011. karp rd, meade cc. transplantation immunity in the american cockroach, periplaneta americana: the rejection of integumentary grafts from blatta orientalis. dev. 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sabbadin a, zaniolo g, ballarin l. genetic and cytological aspects of histocompatibility in ascidians. ital. j. zool. 59: 167-173, 1992. takayama s, isogai a. self-incompatibility in plants. ann. rev. plant biol. 56: 467-489, 2005. voskoboynik a, newman am, corey dm, sahoo d, pushkarev d, neff nf, et al. identification of a colonial chordate histocompatibility gene. science 341: 384-387, 2013. isj 13: 18-22, 2016 isj 13: 18-22, 2016 issn 1824-307x research report celomic cells of the marine fireworm hermodice carunculata (annelida, polychaeta) a franchini, r simonini, e ottaviani department of life sciences, university of modena and reggio emilia, modena, italy accepted january 11, 2016 abstract we have examined the morphological and functional characteristics of the celomic cells in the marine fireworm hermodice carunculata. the cells differ in morphology, in relation to the presence of cytoplasmic granules, adhere to the glass, phagocytize zymosan a particles and contain acth-like molecules. we suggest the presence of only one cell type in the worm celomic fluid, i.e., the immunocyte described in other invertebrates. key words: polychete; hermodice carunculata; celomic cells; immunocyte   introduction dhainaut and porchet-hennerè (1988) described the presence of five main types of amebocytes in several species of polychetes. these defense cells were called granulocytes by baskin (1974) and it is to be emphasized that not all the cell types were observed in every polychete species; for example three types of granulocytes, that can be separated by selective agglutination with lectins, were reported in nereis diversicolor (m’beri et al., 1988; porchet-henneré, 1990). regarding the functions, it has been demonstrated that the cells play a role in cellular responses, including phagocytosis, cytotoxic reactions and encapsulation (porchet-henneré et al., 1987, 1992; porchethenneré and vernet, 1992; maltseva et al., 2014). other populations of free cells, such as eleocytes and erythrocytes, were described in the celom. they have almost no role in immune functions while are primarily involved in regeneration and respiration (vetvicka and sima, 2009). in perinereis cultrifera the eleocytes can be easily separated by sedimentation or weak centrifugation (bulet et al., 1983). in this paper, we have studied the cell types present in the celomic fluid of the marine fireworm hermodice carunculata and their possible involvement in immunity was examined. ___________________________________________________________________________ corresponding author: enzo ottaviani department of life sciences university of modena and reggio emilia via campi, 213/d, 41122 modena, italy e-mail: enzo.ottaviani@unimore.it materials and methods animals the bearded fireworm hermodice carunculata is a large size amphinomid polychete (25 30 cm length) living in the coastal areas of the central atlantic ocean and central and eastern mediterranean sea (ahrens et al., 2013). the specimens used in this study were collected on the cost of porto cesario (lecce, italy) and maintained in a recirculating aquaria system at 25 °c. celomic cells the celomic fluid of h. carunculata (about 200 μl/animal) was collected by using a sterile 2 ml syringe and cytocentrifuged (400 rpm for 2 min) onto slides by cytospin ii (shandon instrument, uk) and air-dryed. unfixed and fixed (4% pformaldehyde in phosphate buffered solution) celomic cells were analyzed with morphological, cytochemical and immunocytochemical methods and for functional tests. a total of 20 animals were used. morphological stains the celomic cells were stained with diff-quik kit (biomap snc, italy) and with may-grünwald and giemsa and then observed under an olympus bh-2 light microscope (olympus corporation, japan) equipped with a ds-5m-l1 digital sight camera system (nikon, japan). cytochemical reactions fixed cells were stained with periodic acidschiff (pas) reaction and sudan black b method for fats and phospholipids (see brancroft and stevens, 18   mailto:enzo.ottaviani@unimore.it fig. 1 celomic cells of hermodice carunculata stained with diff-quik kit and with may-grünwald and giemsa. hyaline cells (a, c) and granular cells with variable numbers of cytoplasmic granules (b, c) are shown. scale bar = 10 μm. 1996). unfixed cells were also incubated in specific medium to localize acid phosphatase (barka and anderson, 1962) and β-glucuronidase (watt, 1987) activities. immunocytochemical procedure unfixed cells were incubated with anti-acth (124) polyclonal antibody (biogenesis, uk) (1:250) an overnight at 4 °c (for the detailed method see ottaviani et al., 1990). the reactivity was visualized by an immunoperoxidase technique using avidinbiotin-peroxidase complex (hsu et al., 1981) and diaminobenzidine as substrate. nuclei were counterstained with hematoxylin. control of the immunocytochemical reaction was performed by substituting primary antibody with non-immune sera. cell adhesion and spreading for the adhesion assay, a drop of celomic fluid was left to settle on a glass slide for 20 min in a humidified chamber. the celomic fluid was then carefully and gently removed with a micropipette and the slides underwent the morphological stains as previously described. in vitro phagocytosis for in vitro phagocytosis assay, the celomic fluid was mixed with zymosan a particles (sigma, usa) (ratio 100 μg/ml celomic fluid), incubated for 30 min in a humidified chamber and subsequently stained with giemsa’s solution prior to observing cell phagocytic activity. results the morphological observations revealed the presence of cells with two main different structures in the celomic fluid of h. carunculata. the first one appeared irregularly shaped with a round or oval eccentric nucleus that surrounded an abundant hyaline cytoplasm (figs 1a, c). the other one differed for the presence of a variable number of intensely stained cytoplasmic granules (figs 1b, c). the majority of these inclusions were positive to 19   fig. 2 celomic cells stained with pas (a, b) and sudan black b (c) reactions. scale bar = 10 μm. pas and sudan black b reactions (figs 2b, c). the functional studies showed that after 20 min the cells adhered strongly to glass microscope slides. moreover, the cells actively phagocytized zymosan a after 30 min of incubation and single or grouped particles were seen inside different sized cytoplasmic vacuoles (figs 3a, b). an higher phagocytic activity was observed in cells with hyaline cytoplasm. all cells were also positive to the cytoenzymatic reactions for the tested hydrolytic enzymes, i.e., acid phosphatase (fig. 3c) and βglucuronidase. regarding the immunocytochemical results, acth-like material was detected in the cytoplasm of hyaline and granular cells (figs 4a c). discussion on the basis of our observations, we suggest that only one cell type is present in the celomic fluid of h. carunculata, although cells with hyaline or granular cytoplasm were detected. the conclusion is supported by the fact that, regardless different morphologies, the cells show the same behavior. indeed, they are able to adhere to the substrate and to phagocytize foreign material. this cell is to be considered the immunocyte, the invertebrate defense cell previously demonstrated to have functional characteristics of vertebrate macrophages (ottaviani, 2011; malagoli et al., 2015). similarly, two main types of celomocytes, endowed of phagocytic capacity, were recognized in the celomic fluid of another polychete, arenicola marina (maltseva et al., 2015). moreover, sheep red blood cells (srbc) injected in the celomic cavity of neoamphitrite regulus and a. marina are phagocytized by celomocytes (immunocytes) and then transferred to the heart-body or extravasal tissue, thus indicating a role in the removal of foreign material (braunbeck and dales, 1984). we also found that the phagocytic activity of h. carunculata cells is associated with the presence of acth-like molecules. an analogous correlation has been demonstrated for the phagocytic cells of several invertebrate species such as the annelid eisenia foetida and the molluscs planorbarius corneus, viviparus ater, lymnaea stagnalis and mytilus galloprovincialis (ottaviani et al., 1990, 1991, 1995; franchini et al., 1994; cooper et al., 1995). the present data are also in agreement with those emerged from comparative and morphofunctional studies performed on different aged m. galloprovincialis (ottaviani et al., 1998). two 20   fig. 3 hyaline (a) and granular cells (b) phagocytize zymosan a particles (arrowheads) and are positive to the cytoenzymatic reaction for acid phosphatase (c). scale bar = 10 μm. fig. 4 hyaline (a) and granular (b) celomic cells are immunoreactive to anti-acth antibody. negative control of immunocytochemical reaction (c). scale bar = 10 μm. 21   types of immunocytes, with hyaline cytoplasm or with cell inclusions containing lipofuscins, have been identified in the hemolymph. on the basis of the shared functions and the expression of common signal molecules, the cells are suggested to belong to a single cell type. the structural differences seem to be ascribed to cellular aging: the immunocytes with hyaline cytoplasm are the young stage, while cells containing lipofuscin inclusions are the adult one (ottaviani et al., 1998). acknowledgements the research was supported by prin grant (miur, italy) to eo and far grant (unimore, italy) to rs. the authors are grateful to dr s fai for providing the marine fireworms. references ahrens jb, borda e, barroso r, paiva pc, campbell am, wolf a, et al. the curious case of hermodice carunculata (annelida: amphinomidae): evidence for genetic homogeneity throughout the atlantic ocean and adjacent basins. mol. ecol. 22: 2280-2291, 2013. bancroft jd, stevens a. theory and practice of histological techniques, 3rd ed., churchill livingstone, edinburgh, 1996. barka t, anderson pj. histochemical methods for acid phosphatase using hexazonium pararosanalin as coupler. j. histochem. cytochem. 10: 741-753, 1962. baskin dg. the coelomocytes of nereid polychaets. cont. topics immunol. 4: 55-64, 1974. braunbeck t, dales rp. the role of the heart-body and of the extravasal tissue in disposal of foreign cells in two polychaete annelids. tissue cell 16: 557-563, 1984. bulet p, hoflack b, verbert a, porchet m. simple procedure to isolate coelomocyte-free oocytes from coelomic fluid of perinereis cultrifera grube (annelida: polycheata). experientia 39: 436-437, 1983. cooper el, franchini a, ottaviani e. earthworm coelomocytes possess immunoreactive cytokines and pomc-derived peptides. anim. biol. 4: 25-29, 1995. dhainaut a, porchet-hennerè a. xiii. haemocytes and celomocytes. the ultrastructure of polychaeta, microfauna marina 4, westheude w, herman co (eds), gustav fischer, verlag, stuttgart, pp 215-236, 1988. franchini a, fontanili p, ottaviani e. expression of pro-opiomelanocortin (pomc)-mrna in phagocytic hemocytes of mytilus galloprovincialis. in: argano r, cirotto c, grassi milano e, mastrolia l (eds), contributions to animal biology, halocynthia association, palermo, pp 233-236, 1994. hsu sm, raine l, fanger h. use of avidin-biotinperoxidase complex (abc) in immunoperoxidase techniques: a comparison between abc and unlabeled antibody (pap) procedures. j. histochem. cytochem. 29: 577580, 1981. malagoli d, mandrioli m, tascedda f, ottaviani e. circulating phagocytes: the ancient and conserved interface between immune and neuroendocrine function. biol. rev. camb. philos. soc. doi: 10.1111/brv.12234, 2015. maltseva al, kotenko on, kokryakov vn, starunov vv, krasnodembskaya ad. expression pattern of arenicins-the antimicrobial peptides of polychaete arenicola marina. front. physiol. 5:497. doi: 10.3389/fphys.2014.00497. ecollection 2014. m'beri m, debray h, dhainaut a. separation of two different populations of granulocytes of nereis diversicolor (annelida) by selective agglutination with lectins. dev. comp. immunol. 12: 279-285, 1988. ottaviani e. immunocyte: the invertebrate counterpart of the vertebrate macrophage. inv. surv. j. 8: 1-4, 2011. ottaviani e, capriglione t, franceschi c. invertebrate and vertebrate immune cells express pro-opiomelanocortin (pomc) mrna. brain behav. immun. 9: 1-8, 1995. ottaviani e, cossarizza a, ortolani c, monti d, franceschi c. acth-like molecules in gastropod molluscs: a possible role in ancestral immune response and stress. proc. biol. sci. 245: 215-218, 1991. ottaviani e, franchini a, barbieri d, kletsas d. comparative and morphofunctional studies on mytilus galloprovincialis hemocytes: presence of two aging-related hemocyte stages. ital. j. zool. 65: 349-354, 1998. ottaviani e, petraglia f, montagnani g, cossarizza a, monti d, franceschi c. presence of acth and β-endorphin immunoreactive molecoles in the freshwater snail planorbarius corneus (l.) (gastropoda, pulmonata) and their possible role in phagocytosis. regul. pept. 27: 1-9, 1990. porchet-henneré e. cooperation between different coelomocyte populations during the encapsulation response of nereis divesicolor demonstrated by using monoclonal antibodies. j. invertebr. pathol. 56: 353-361, 1990. porchet-henneré e, dugimont t, fischer a. natural killer cells in a lower invertebrate, nereis diversicolor. eur. j. cell biol. 58: 99107, 1992. porchet-henneré e, m’beri m, dhainaut a, porchet m. ultrastructural study of the encapsulation. response of the polychaete annelid nereis diversivolor. cell tissue res. 248: 463-471, 1987. porchet-henneré e, vernet g. cellular immunity in an annelid (nereis diversicolor, polychaeta): production of melanin by a subpopulation of granulocytes. cell tissue res. 269: 167-174, 1992. vetvicka v, sima p. origins and functions of annelide immune cells: the concise survey. inv. surv. j. 6: 138-143, 2009. watt me. acid hydrolases in hela cells: comparison of methods for light microscopy. stain technol. 62: 383-399, 1987. 22   isj 12: 176-178, 2015     176 isj 12: 176-178, 2015 issn 1824-307x letter to editor interactions between earthworm neuroendocrine and immune systems b plytycz1, aj morgan2 1department of evolutionary immunology, institute of zoology, jagiellonian university, krakow, poland 2cardiff school of biosciences, main building, cardiff university, cardiff cf10 3us, wales, uk accepted june 27, 2015 to the editor we read with interest the recent paper about the importance of studying invertebrate immuneneuroendocrine functions published in the “vision and perspectives” section of inv. surv. j. (ottaviani, 2014). the paper reinforces the profoundly important view that the neuroendocrine and innate defense systems of invertebrates and vertebrates can interact in concerted harmony (cohen and kinney, 2007). to consolidate the insights propounded in these seminal articles we would like to advocate earthworms as tractable and powerful models for studies on immuneneuroendocrine interactions. earthworms are relatively easy to culture and manipulate in the laboratory, and deep knowledge of particular aspects of their physiology and molecular genetics is burgeoning rapidly (stürzenbaum et al., 2009; liebeke et al., 2014). earthworms, as representatives of oligochaete annelids, are metamerically-segmented celomates with a closed circulation and a well-developed nervous system. a segmented celomic cavity communicates with the external environment via dorsal pores, thus the celom ubiquitously contains bacteria, protozoans and fungi, as well as abundant free-floating immunocytes and humoral factors that inhibit microorganism outgrowth (bilej et al., 2011). in common with other invertebrates, earthworms are devoid of adaptive immunity based on t and b lymphocytes and antibodies; these components are present in jawed vertebrates only. however, the earthworm immune system detects the conserved pathogen-associated molecular patterns (pamps) of microbes by pathogen recognition receptors (prr), among them the extensively studied toll-like receptors (coscia et al., 2011; skanta et al., 2013). evidently, earthworms have evolved efficient innate immunity with both cellular and humoral components (bilej et al., 2011). the immunocytes, according to the term coined out by ottaviani (2011) for invertebrate cells endowed with attributes ___________________________________________________________________________ corresponding author: barbara plytycz department of evolutionary immunology institute of zoology jagiellonian university, krakow, poland e-mail: barbara.plytycz@uj.edu.pl of vertebrate macrophages, are represented in earthworms by free-floating amebocytes derived from the lining of the celomic cavity (parry, 1975). in some lumbricid species amebocytes are accompanied by eleocytes, the latter being detached chloragocytes invested with granules containing species-specific amounts of riboflavin (vitamin b2). riboflavin is now recognized as a potentiator of immunocompetence and tissue regeneration capacity in earthworms and other organisms (plytycz and morgan, 2011; johnson et al., 2012). earthworms stressed by predators or physical/chemical irritants expel celomocytecontaining celomic fluid through the dorsal pores during spasmic body movements. this ability is commonly exploited for non-invasive retrieval of celomocytes for ex vivo studies and/or for temporal depletion of earthworm celomocytes and celomocyte-derived humoral factors. depletion of celomocytes is followed by their restoration (eyambe et al., 1973); amebocyte restoration is faster than eleocyte restoration (klimek et al., 2011; santocki et al., 2015). the earthworm central nervous system (cns) is a highly differentiated neuroendocrine structure which produces hormones, neurohormones and neurotransmitters (e.g., takahama et al., 1998; hartenstein, 2006; wilhelm et al., 2006; herbert et al., 2009; molnar et al., 2015a). the cns of earthworms is comprised of a ventral nerve cord (vnc) consisting of segmentally repeated ganglia joined longitudinally by connectives and radially by comissures. the first vnc ganglia are fused to form the suboesophageal ganglion which is connected by paired circumpharyngeal connectives to a dorsal cerebral ganglion, often loosely referred to as the ‘brain’. its anatomical location makes the earthworm brain easy to remove surgically. earthworms possess the remarkable ability to regenerate the cerebral ganglion within a few weeks (lubics et al., 2002; csoknya et al., 2003; okrzesik et al., 2013; molnar et al., 2015b). our experiments on adult earthworms (dendrobaena veneta) have shown that amputation of the anterior (braincontaining) segments, or direct surgical brain removal, caused an immediate and pronounced inhibition of reproduction. reproductive output       177 fig. 1 reproductive success during 8 consecutive weeks in dendrobaena veneta either untreated (blue dotted lines), or after surgical brain removal (red solid lines), or surgical brain removal in celomocyte-depleted worms (green dashed lines) measured as: a) numbers of cocoons per worm; b) numbers of hatchlings per worm; c) numbers of hatchlings per cocoon. [this is a modified version of a figure and table published in molnar et al., 2015b] subsequently recovered over a matter of weeks and, significantly, in tandem with restoration of brain integrity, including active neurosecretory cells. thus, the restoration of reproductive activity is a sensitive and non-invasive biomarker for tracking the progression of brain regeneration (okrzesik et al., 2013). our studies also included monitoring reproductive activity of unmanipulated (control) d. veneta and of their experimental counterparts subjected either to surgical brain extirpation only or to the dual treatment (i.e., celomocyte extrusion and brain extirpation). as before, reproduction was temporarily inhibited in all brain-extirpated worms and was concomitantly restored alongside brain regeneration, both events proceeding faster in subjects with an undisturbed immune system compared with celomocyte-depleted ones (molnar et al., 2015b) (fig. 1). on the other hand, restoration of celomocytes was slower in worms engaged in regenerating their extirpated brains then compared with counterparts possessing intact brains. reproduction was only slightly inhibited by celomocyte depletion in otherwise intact worms (okrzesik et al., 2013; molnar et al., 2015b).       178 collectively, our studies provide empirical support for the notion that the neural and immune systems of earthworms can be demonstrated to be functionally intertwined. the earthworm experimental model that we have briefly outlined above is unquestionably amenable to detailed examination by a variety of molecular-genetic platforms and by various spatially defining immune-localization microscopic methodologies. aacknowledgments this work was financially supported by the ministry of science and higher education (k/zds/004831) and by national centre of science, grant number b/nz4/01640 (k/pbo/000178) from poland. references bilej m, prochazkova p, silverowa m, jaskova r. earthworm immunity. adv. exp. med. biol. 708: 66-79, 2011. cohen n, kinney ks. exploing the neural-immune system interactions. an update. in: ader r (ed.), psychoneuroimmunology, vol. 1, 4th edition, elsevier academic press, burlington ma 01803, usa, pp 1-38, 2007. coscia mr, giacomelli s, oreste u. toll-like receptors: an overview from invertebrates to vertebrates. inv. surv. j. 8: 210-226, 2011. csoknya m, barna j, hiripi l, hámori j, elekes k. reorganization of monoaminergic systems in the earthworm, eisenia fetida, following brain extirpation. j. exp. zool. part a: comp. exp. biol. 296a: 18-29, 2003. eyambe gs, goven aj, fitzpatrick lc, venables bj, cooper el. a non-invasive technique for sequential collection of earthworm (lumbricus terrestric) leukocytes during subchronic immunotoxicity studies. lab. anim. 25: 61-67, 1991. hartenstein v. the neuroendocrine system of invertebrates: a developmental and evolutionary perspective. j. endocrinol. 190: 555-570, 2006. herbert z, pollák e, zongman a, boros a, kaplan n, molnar l. identification of novel neuropeptides in the ventral nerve cord ganglia and their targets in an annelid worm, eisenia fetida. j. comp. neurol. 514: 415-432, 2009. johnson sjr, raja se, vedha yb, amutha aeajk, dinesh s, durairaj sj, et al. autofluorescence in brdu-positive cells and augmentation of regeneration kinetics by riboflavin. stem cells develop. 21: 2071-2083, 2012. klimek m, kruk j, plytycz b. restoration of coelomocytes in the earthworm dendrobaena veneta. acta. biol. cracov. ser. zool. 54: 11-17. 2012. liebeke m, bruford mw, donnelly rk, ebbels tmd, hao j, kille p, et al. identifying biochemical phenotypic differences between cryptic species. biol. lett. 2014, doi: 10.1098/rsbl.2014.0615. lubics a, reglodi d, szelier m, lengvari i. timecourse of the regeneration of the earthworm cerebral ganglion with special reference to serotonergic elements. eur. j. anat. 6: 147-152, 2002. molnar l, pollak e, somogyi i, engelmann p. on the existence of possible pituitary adenylate cyclase-activating polypeptide adenylate type 1 receptor in earthworms. inv. surv. j. 12: 173175, 2015a. molnar l, pollak e, skopek z, gutt e, kruk j, morgan aj, plytycz b. immune system participates in brain regeneration and restoration of reproduction in the earthworm dendrobaena veneta. dev. comp. immunol. 52: 269-279, 2015b. okrzesik j, kachamakova-trojanowska n, jozkowicz a, morgan aj, plytycz b. reversible inhibition of reproduction during regeneration of cerebral ganglia and celomocytes in the earthworm dendrobaena veneta. inv. surv. j. 10: 151-161, 2013. ottaviani e. immunocyte: the invertebrate counterpart of the vertebrate macrophage. inv. surv. j. 8: 1-4, 2011. ottaviani e. the importance of studying invertebrate immune-neuroendocrine functions. inv. surv. j. 11: 1-3, 2014. parry mj. evidence of mitotic division of coelomocytes in the normal, wounded and grafted earthworm eisenia foetida. experientia 117: 449-451, 1975. plytycz b, morgan aj. riboflavin storage in earthworm chloragocytes/eleocytes in an ecoimmunology perspective. inv. surv. j. 8: 199209, 2011. santocki m, falniowski a, plytycz b. restoration of experimentally depleted coelomocytes in juvenile and adult composting earthworms eisenia andrei, e. fetida and dendrobaena veneta. appl. soil ecol. 2015 [in press]. skanta f. roubalova r, dvorak j, prochazkova p, bilej m. molecular cloning and expression of tlr in the eisenia adrei earthworm. de. com. immunol. 41: 694-702, 2013. stürzenbaum sr, andre j, kille p, morgan aj. darwin and his earthworms: genomics, proteomics and metabolomics. proc. r. soc. b 276: 789-797, 2009. takahama h, haibara k, oumi t, ukena k, morishita f, furukawa y, et al. immunohistochemical localizationof annetocin, an earthworm oxytocin-related peptide, and identification and ultrastructural characteristics of the annetocin-secretory cells in the oligochaete earthworm eisenia foetida. zool. sci. 15: 381-388, 1998. wilhelm m, koza a, engelmann p, nemeth p, csoknya m. evidence for the presence of thyroid-stimulating hormone, thyroglobulin and their receptors in eisenia fetida: a multilevel hormonal interface between the nervous system and the peripheral tissues. cell tissue res. 324: 535-436, 2006.   http://www.ncbi.nlm.nih.gov/pubmed?term=beryl%20vedha%20y%5bauthor%5d&cauthor=true&cauthor_uid=22150027 http://www.ncbi.nlm.nih.gov/pubmed?term=amutha%20k%5bauthor%5d&cauthor=true&cauthor_uid=22150027 http://www.ncbi.nlm.nih.gov/pubmed?term=amutha%20k%5bauthor%5d&cauthor=true&cauthor_uid=22150027 http://www.ncbi.nlm.nih.gov/pubmed?term=dinesh%20sm%5bauthor%5d&cauthor=true&cauthor_uid=22150027 http://www.ncbi.nlm.nih.gov/pubmed?term=jackson%20durairaj%20sc%5bauthor%5d&cauthor=true&cauthor_uid=22150027 http://www.ncbi.nlm.nih.gov/pubmed?term=elaiya%20raja%20s%5bauthor%5d&cauthor=true&cauthor_uid=22150027 http://www.ncbi.nlm.nih.gov/pubmed?term=elaiya%20raja%20s%5bauthor%5d&cauthor=true&cauthor_uid=22150027 isj 12: 158-165, 2015 isj 12: 158-165, 2015 issn 1824-307x research report effect of silicon on the morphology of the midgut and mandible of tomato leafminer tuta absoluta (lepidoptera: gelechiidae) larvae mc dos santos1, am resende junqueira1, vg mendes de sá2, jc zanúncio3, je serrão4 1faculty of agronomy and veterinary sciences, university of brasilia, 70910-970, brasília, distrito federal, brazil 2faculty of engineering, university of the state of minas gerais, 35930-314, joão monlevade, minas gerais state, brazil 3department of entomology, federal university of viçosa, 36.570-000, viçosa, minas gerais state, brazil 4department of general biology, federal university of viçosa, 36.570-000, viçosa, minas gerais state, brazil accepted may 12, 2015 abstract tuta absoluta (meyrick) (lepidoptera: gelechiidae) is an important insect pest causing serious losses to tomato plantations in brazil. some populations of t. absolute are reported to present insecticide resistance resulting in its control failure and the use of alternative control based on silicon, which is clean and sustainable, can reduce pesticide use, increasing fruit quality and protecting the environment. this study evaluated changes in the morphology of the midgut and the mandibles of t. absoluta larvae caused by feeding with compounds containing silicon. larvae of t. absoluta were fed on tomato leaves with different compounds containing silicon and the histology of the midgut of fourth instar larvae was analyzed. the mandibles of all larval stages were dissected and analysed by scanning electron microscopy. there were no changes in the morphology of the mandibles of the larvae of t. absoluta fed on silicon compared to the control group. larvae of t. absoluta from the control group and the treatments where the calcium silicate was applied to the soil had not differences in the morphology of the midgut epithelial cells, which had four cell types: digestive, goblet, regenerative and mycetocyte cells. in larvae of t. absoluta obtained from silicon-based treatments applied to the leaves, the midgut epithelium showed detachment of the basal membrane, which can characterize the possible effect of this toxic element to larvae of t. absoluta. key words: lycopersicon esculetum; fertilization; morphology; pest control   introduction the tomato (lycopersicon esculentum) is widely cultivated in the world and it is one of the most produced vegetables in brazil, which is currently among the top ten world producers (vilela, 2009). the tomato crop in brazil is expanding and modernizing, seeking greater productivity and quality to meet market demands, thereby generating more concern about pest control (vivan et al., 2002) that can cause substantial reductions in fruit quality (miranda et al., 1998; kennedy, 2003; mahanil et al., 2008). the tomato pinworm, tuta absoluta (lepidoptera: gelechiidae), which attacks leaves and flowers causing mines and fruit drilling throughout the cycle of tomato, stands out as the most important insect ___________________________________________________________________________ corresponding author: josé eduardo serrão department of general biology federal university of viçosa 36.570-000, viçosa, minas gerais state, brazil e-mail: jeserrao@ufv.br pest of this crop and has usually been controlled by multiple applications of insecticides (medeiros et al., 2009). however, the frequent use of insecticides may be harmful to the natural biological control, generates the production of foods with high levels of toxic waste, environmental contamination, besides the risk of selecting resistant insects streams (vianna et al., 2009). many insecticides have the midgut cells of the insects as a first target (forkpah et al., 2014), which is the main organ where digestion and absorption take place (lehane and billingsley, 1996). the midgut wall has three layers: two muscular layers and an epithelial lining of the lumen, composed of a single layer of cells that, in most insects, has three cell types: columnar or digestive, regenerative, and endocrine, while a fourth cell type, goblet cells, occurs in lepidoptera (lehane and billingsley, 1996). the epithelium surface is isolated from the food content by a non-cellular membrane, the peritrophic membrane or matrix (lehane and billingsley, 1996; terra, 2001).   158 mailto:jeserrao@ufv.br table 1 treatments used to evaluate the effect of silicon on the morphology of the midgut and mandible of tuta absoluta (lepidoptera, gelechiidae) treatments product amount (ton ha-1) t1 0.45 (ton ha-1) t2 0.9 (ton ha-1) t3 1.35 (ton ha-1) t4 agrosilíciotm soil 1.8 (ton ha-1) t5 2.0 (ton ha-1) t6 4.0 (ton ha-1) t7 6.0 (ton ha-1) t8 agrosilíciotm leaves 8.0 (ton ha-1) t9 0.5 (l ha-1) t10 1.0 (l ha-1) t11 2.0 (l ha-1) t12 sili-ktm leaves 3.0 (l ha-1) t13 0.25 % t14 0.50 % t15 0.75 % t16 silicic acid leaves 1.0 % t17 0.25 % t18 0.50 % t19 0.75 % t20 silicic acid soil 1.0 % in lepidoptera larvae, the peritrophic membrane is secreted by the midgut digestive cells (de priester, 1971; terra, 1988). this membrane, although it prevents direct contact of food with the digestive cells, allows the passage of digestive enzymes in the direction of the midgut lumen and the absorption of the products of digestion which are then eliminated with the faeces (terra, 1988) avoiding mechanical damage, impeding or preventing the entry of pathogens and partition the digestion process (terra, 1988; 2001). the digestive cells are the most abundant and are responsible for producing enzymes and absorbing products of digestion (terra, 1988). goblet cells are involved in the homeostasis and absorption of metabolites, a function performed in conjunction with the digestive cells (lehane and billingsley, 1996; terra et al., 2006). the regenerative cells are seen alone, paired or in nidi at the base of the epithelium and they play a role in cell renewal (serrão and cruz-landim, 1996a; martins et al., 2006). the endocrine cells are located at the base of the epithelium and they are characterized by the presence of a large number of cytoplasmic granules, which produce peptide hormones (serrão and cruz-landim, 1996b; neves et al., 2002). synthetic insecticides results in many ecological problems such as selection of resistance strains of insects, ecological unbalance and mammals intoxications. thus, it is necessary the use of programs of integrated pest management, which aim the reduction of the use of pesticides compatible with natural enemies (zanuncio et al., 2003). although silicon is not essential for most plants (savant et al., 1999), it is beneficial to improve plant resistance against some biotic and abiotic stresses (do gramaci et al., 2013; haynes, 2014). the application of silicon has increased plant resistance to insect pests (almeida et al., 2008; cherry et al., 2012; sidhu et al., 2013; keeping et al., 2014; pinto et al., 2014), mainly for its ability to accumulate in the plant cell wall (costa and moraes 2006), thereby increasing the synthesis of lignin and phenolic compounds (chérif et al., 1992; ghanmi et al., 2004), in addition to activating the endogenous chemical defenses of plants (gomes et al., 2005). the use of silicon in controlling defoliator insects occurs due to the action of the mechanical barrier provided by the deposition of this mineral in the cell wall of leaves (goussain et al., 2002; massey et al., 2006; kvedaras et al., 2009). tuta absoluta reared on tomato plants accumulating silicon show decrease in larvae and pupae survival and male and female weight (santos et al., 2012). the deleterious effects on insect larvae that feed on plants treated with calcium silicate have been suggested to occur due damages in the incisor teeth of the mandibles, affecting the   159 insect nourishment and development (goussain et al., 2002; kvedaras et al., 2009) but data about its effect on midgut remains unknown. this study evaluated whether treatment of tomato plants with silicon affects the morphology of the midgut and the mandibles of the tomato pinworm t. absoluta. materials and methods planting, experimental design and treatments the planting of seedlings of tomato, variety tospodoro obtained at embrapa vegetables, brasília, df, was carried out a greenhouse at the federal university of viçosa, viçosa, mg, brazil. the experimental plots of tomato plants grown in 3 l polyethylene pots, containing one plant in each with planting nitrogen-containing fertilizer (600 kg ha-1 ammonium sulfate), phosphorus (3300 kg ha-1 superphosphate) and potassium (330 kg ha-1 potassium chloride), whose amounts were calculated on the basis of soil analysis. three compounds used as sources of silicon were: agrosilíciotm (10.5 % si), sili-ktm (12.2 % si) and silicic acid (100 % si). the experimental design was randomized blocks with five replications, twenty treatments (table 1) and control (without addition of any compound containing silicon). the agrosilíciotm was added to the soil together to fertilizer planting, aiming to increase alcaline saturation to 70 % (ribeiro and guimarães 1999) in the treatments t1 to t4, and applied weekly by foliar sprays in treatments t5 to t8. the sili-ktm product was applied weekly only to the leaves, in the treatments t9 to t12, due to the fact that compound is not available for soil application. the silicic acid solution was applied weekly, in foliar application, treatments t13 to t16 and in the soil around the stems of plants, treatments t17 to t20. the first foliar application of the products was made 30 days after planting of tomatoes, a total of three applications at weekly intervals in treatments where agrosilíciotm was applied to the soil, since this product has a soil corrective effect (sommer et al., 2006). in all treatments with agrosilíciotm, sili-ktm and silicic acid soil were corrected with dolomitic lime (0.8 t ha-1) in order to raise the alkaline saturation to 70 %. release of t. absoluta eggs tomato leaves with t. absoluta eggs of the same age, from the mass rearing of the laboratory of agricultural entomology, federal university of viçosa, were sectioned in order to contain about 30 eggs approximately. each of these sectioned areas was fixed with the aid of a pin in the branches of the tomato plant caged in organza bags 40x70 cm involving the entire plant, seven days after the last foliar application of products containing silicon. light microscopy to evaluate the silicon effect in the midgut of t. absoluta, fourth instar larvae obtained from the treatments were collected and transferred to flasks containing 2 ml of zamboni fixative solution (stefanini et al., 1967). afterwards, the midguts of t. absoluta were dissected in insect saline solution (0.1 m nacl + 0.1 m kh2po4 + o.1m na2hpo4), dehydrated in a graded ethanol series (70, 80, 90 and 95 %) and embedded in jb-4tm historesin for 24 h. the samples were sectioned at 5 μm slices, stained with hematoxyline and eosin, examined and photographed in light photomicroscope. scanning electron microscopy to evaluate the silicon effect in the wear of the mandibles in all four larval stages, t. absoluta larvae, were collected each 12 h, two larvae per plant, and placed in flasks containing 2 ml of zamboni fixative. larvae were collected immediately before ecdysis, based on the duration of each larval instar (giustolin et al., 2002) to ensure greater exposure to treatments. the mandibles were removed, dehydrated in a graded ethanol series (70, 80, 90 and 95 %), transferred to hmds (hexamethyldisilazane) and after five min air drying. the mandibles were gold covered (20 nm) and analyzed under a scanning electron microscope leo vp1430 at the center for microscopy and microanalysis at the federal university of viçosa. results the midgut of t. absoluta larvae showed a single layered epithelium with four cell types: digestive, goblet, regenerative and micetocyte cells (figs 1a e). the digestive cells were tall, which apex showing a well-developed brush border and cytoplasm containing numerous vacuoles (fig. 1c). in the middle portion of these cells was found an oval nucleus with a predominance of decondensed chromatin (fig. 1b). the goblet cells were characterized by a cavity formed by invagination of the apical surface of these cells that showed a basal nucleus (figs 1a c). the regenerative cells were small, never reaching the midgut lumen, containing a relatively large nucleus in relation to the cytoplasm and were scattered between the base of the goblet and digestive cells isolated or forming nests till three cells (fig 1a). throughout the midgut epithelium of t. absoluta were found some large and globular cells with spherical nucleus and cytoplasm almost filled with small basophilic bacteria-like particles (fig. 1e), which allowed the characterization of this cell type as mycetocytes. a well-developed peritrophic membrane line the midgut lumen (fig. 1c). the midgut epithelium was seated on a homogeneous basal membrane and a layer of circular muscles was found externally, followed by an outer layer of longitudinal muscles. larvae of t. absoluta from the control group and treatments where calcium silicate was applied to the soil showed no differences in the structural organization of the midgut that was described above. larvae of t. absoluta obtained from siliconbased treatments applied to leaves showed changes in the morphology of the midgut, a characterized by the detachment of the basal membrane of the digestive epithelium (figs 1a, 1b), presence of cytoplasmic protrusions with strongly   160 fig. 1 histological sections of the midgut of fourth instar larvae of tuta absoluta (lepidoptera, gellechidae) fed on tomato plants treated with compounds containing silicon. a) midgut epithelium of larvae fed on plants exposed to agrosilíciotm leaves (40 t ha-1) showing digestive cells (dc), globet cells (gc) and regenerative cell nidus (rc) note basal membrane detached (bm), well developed brush border (bb) and apoptotic bodies (arrows) released to lumen (l) bar = 20 μm; b) midgut epithelium of larvae fed on plants exposed to agrosilíciotm leaves (40 t ha-1) showing basophilic apoptotic bodies (arrow) bar = 10 μm; c) midgut epithelium of larvae fed on plants exposed to silicic acid leaves (1,0 %) showing digestive (dc) and globet cells (gc) with vacuoles in the cytoplasm (v) notice the peritrophic membrane (pm) bar = 10 μm; d) midgut epithelium of larvae fed on plants exposed to sili-ktm leaves (30 l ha-1) showing detached basal membrane (bm) bar = 10 μm; e) midgut epithelium of larvae from plants the control group without any addition of compounds containing silicon and fed plants exposed to sili-ktm leaves (05 l ha-1) showing mycetocyte (my) characterized by basophilic granules bacteria-like n, nucleus with decondensed chromatin bar = 10 μm. basophilic content, some them containing the cell nucleus, which are released into the midgut lumen, similar to apoptotic bodies (figs 1a, 1b). the larvae of t. absoluta from all treatments including the control group, had six teeth in their mandibles without variation in the number or shape of them, and show no signs of damage from first to fourth instar (figs 2a, 2b). discussion the efficiency of silicon-containing products to control t. absoluta has been suggested to occur due its toxic and anti-feeding effect to the larval stage, playing a role as activator of tomato plants resistance (rodrigues et al., 2004; côté-beaulieu et al., 2009). however, there were no studies proving   161 this mechanism of silicon action, which is accumulated in higher concentrations as silica in the leaf tissues of plants (basagli et al., 2003; correa et al., 2005; gomes et al., 2005). in this study, one of the effects caused by the application of silicon in the leaves was the detachment of the midgut epithelium from the basal membrane, which leads to the reduction of digestive capacity in insects (barbeta et al., 2008; almeida et al., 2009). however, there are no significant difference in the structural organization of the midgut epithelium of t. absoluta between the larvae in the control group and those of silicon treatments applied to the soil, as previously described for other lepidoptera larvae (levy et al., 2004; pinheiro et al., 2008; sousa et al., 2009, 2010). the mechanisms of silicon action in the gut cells of insects are not yet fully understood. cellular detachment from basal membrane in the midgut of larvae of t. absoluta may indicate that the antifeeding action of the compounds containing silicon may be related to physiological effects resulting from their ingestion. compounds derived from neem seeds applied in leaves for use in integrated pest management also showed similar effects by altering the epithelial cells of the midgut of insects and, consequently, any effect on their development (mordue and nisbest 2000; ndione et al., 2007; barbeta et al., 2008; almeida et al., 2009, 2014). the midgut of alabama argillacea (lepidoptera: noctuidae) caterpillar fed on bacillus thuringiensis cotton leaves has morphological changes of goblet and digestive cells with increase in cytoplasm vacuoles amount, degeneration of muscle layer and absence of peritrophic membrane (sousa et al., 2010). the presence of cell protrusions like apoptotic bodies being released in the midgut lumen of t. absoluta larvae treated with silicon applied to leaves, suggest a cytotoxic effect of silicon. apoptosis is a morphological pattern of programmed cell death characterized by cell shrinkage (ihara et al., 1998). the intake of silicon may have a toxic effect caused by damaging the epithelial cells of the midgut and, therefore, a response would be the elimination of these cells by programmed cell death. toxins such as cry1ac from bt have similar effect, providing a toxic effect and causing apoptosis in cells of the insects midgut (zhang et al., 2005). the occurrence of small basophilic granules found in globular cells of midgut of t. absoluta larvae in this study are similar to structures called mycetocytes found in some insect that depend on the obligatory mutualism with microorganisms, mainly bacteria (moran and baumann, 2000). however, the occurrence of such cells accumulating microorganisms has not been reported to occur in lepidoptera and the identification of the microorganisms found inside mycetocytes of t. absoluta must be conducted in the future, in order to understand their function in the physiology of the insect or disease transmission to plants. our results showed no effect of silicon on the mandibles of t. absoluta, which have not wear signals in all larval stages. silicon is beneficial to plants, acting as an inducing resistance agent against pest insects, and the silification of the fig. 2 mandibles of tuta absoluta (lepidoptera, gellechidae) larvae; a) fed on tomato plants exposed to silicic acid leaves (0.5 %) bar = 20 μm; b) control group without any addition of compounds containing silicon bar = 20 μm epidermis prevents the penetration and chewing by insects due to the hardening of plant cell wall (datnoff et al., 2001). in larvae of spodoptera frugiperda (lepidoptera: noctuidae) fed on corn plants treated with calcium silicate occurs a wearing in the mandibles teeth (goussain et al., 2002). however, in eldana saccharina (lepidoptera: pyralidae) caterpillars fed on sugar cane treated with silicon, there was no significant effect in wearing in the mandibles (kvedaras et al., 2009). absence of wear in the mandibles of t. absoluta larvae fed on tomato plants treated with silicon, may be due to physiological characteristics of the plant and the feeding habits of these larvae. the tomato is a non-accumulating silicon plant because it absorbs little silicon through the roots (ma and yamaji 2006) and the increasing levels of this compound in tomato leaves is not proportional to their availability in the soil (pereira et al., 2003). the effect of mechanical protection of silicon in plants is attributed to its storage as amorphous silica (sio2nh2o) in the cell wall (datnoff et al., 2001). the agrosiliciotm (insoluble in water) applied to leaves forms only one layer of silica on the epidermis of the leaves while the sili-ktm (siliconsoluble liquid) forms a silica layer evidenced by its   162 polymerization with cuticular compounds of the plant (unpublished data). the larvae of t. absoluta feed and develop in the mesophyll of tomato leaves (medeiros et al., 2009) and, probably, due to the diet, to the compounds containing silicon does not penetrate the leaves of tomato plants and this plant not accumulate si (ma et al., 2001), the mandibles of t. absoluta have been not stressed. conclusion the foliar application of silicon-containing compounds in tomato plants was effective against the attack of t. absoluta caterpillars causing detachment of midgut cells from the basal membrane, which may result in digestion difficulties and larval mortality. 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  164 http://pt.slideshare.net/nirlene/situao-da-produo-de-tomate-no-brasil-5166847 http://pt.slideshare.net/nirlene/situao-da-produo-de-tomate-no-brasil-5166847 vivan lm, torres jb, veiga fsl, zanuncio jc. comportamento de predação e conversão alimentar de podisus nigrispinus sobre a traçado-tomateiro. pesq. agropec. brasil 36: 581587, 2002. zanuncio tv, serrão je, zanuncio jc, guedes rnc. permethrin-induced hormesis on the predator supputius cincticeps (stal, 1860) (heteroptera: pentatomidae). crop protec. 22: 941-947, 2003. zhang x, candas m, griko nb, rose-young l, bulla la. cytotoxicity of bacillus thuringiensis cry1ab toxin depends on specific binding of the toxin to the cadherin receptor bt-r(1) expressed in insect cells. cell death differ. 12: 1407-1416, 2005.   165 141 isj 15: 141-148, 2018 issn 1824-307x research report construction of a bombyx mori cell line that stably express the susceptible gene +nsd-2 of bombyx mori bidensovirus y zhang, p lü, q yu, r li, d miao, z hu, y yang, l chen, k chen*, q yao* institute of life sciences, jiangsu university, zhenjiang, china accepted april 01 , 2018 abstract bombyx mori bidensovirus (bmbdv) is the only type species established in the new genus bidensovirus within the new family bidnaviridae. the bmbdv infected silkworm midgut shows symptoms such as the nucleus of midgut columnar cells expanded. it is confirmed the bmbdv-sensitive gene +nsd-2, which encoding an amino acid transporter protein might be the receptor of bmbdv. the deletion of this gene causes the silkworm resistance to bmbdv infection. there is no permissive cell line for bmbdv infection has been established yet. the cell lines (bmn, etc.) are used broadly for silkworm viruses study are mainly resistant to bmbdv infection. therefore, the establishment of bmbdv permissive cell line is particularly important for studying bmbdv in vitro. in this study, we successfully constructed a cell line bmn (+nsd-2), which expressed +nsd-2 gene stably. the nsd-2 expression and localization could be detected in bmn (+nsd-2) cells via rt-pcr, western blot and immunofluorescence. the interaction between nsd-2 and the major structural protein vp of bmbdv was determined by co-ip. however, we failed to detect the replication of bmbdv. this cell lines constructed has the potential for identifying the protein binding to the nsd-2 and provide a basis for study of bmbdv co-receptors and invasion mechanism. key words: silkworm; bidensovirus; sensitive gene; cell lines; nsd-2 introduction bombyx mori bidensovirus (bmbdv) is the only virus type species of the bidnaviridae family established by the international committee on taxonomy of viruses (ictv) (adams et al., 2012). bmbdv is a pathogen that can infect columnar cells of the silkworm midgut and cause the densonucleosis of b. mori larvae. bmbdv contains two single linear dna (vd1 and vd2), which are spherical virus particles with non-envelope and a diameter of about 22-24 nm (wang et al., 2007; zhang et al., 2016). vd1-orf3 encodes the major structural protein vp with β barrel structure similar to parvovirus and glycine rich sequence at the n-terminal region (krupovic et al., 2014). the proteins are produced through a leaky scanning mechanism (pan et al., 2014; lü et al., 2017). vd2-orf2 encodes a minor structural protein p133 with 1161 amino acids and a molecular weight of133 kda, which is homologous to the c-terminus of the capsid protein of reovirus. p133 homologs are usually ___________________________________________________________________________ corresponding authors: qin yao and keping chen institute of life sciences jiangsu university 301 xuefu road, zhenjiang 212013, pr china e-mail: yaoqin@ujs.edu.cn, kpchen@ujs.edu.cn found in the outer coat of the capsid, which are closely related to host recognition and viral entry (kong et al., 2011; lv et al., 2011). the resistance of b. mori against the like-parvovirus strain bmdnv-2 is controlled by a recessive gene nsd-2 (abe et al., 2000; murthy et al., 2014). the nsd-2 linkage map was obtained by restriction fragment length polymorphism linkage method, and finally the non-sensitive gene nsd-2 was determined to be an amino acid transporter (ogoyi et al., 2003; ito et al., 2008), which is expressed only in the midgut and encodes transmembrane proteins with 12-transmembrane domains, and lack of the 9-transmembrane domains of the c-terminal of the amino acid transporter, will cause the silkworm to be non-sensitive to bmbdv. our group analyzed the transcription of +nsd-2 gene in different developmental stages of silkworm and sensitivity in different tissues by rt-pcr, the results showed that the +nsd-2 gene is only expressed in midgut of 1th-5th instar larvae nsd-2’ and +nsd-2’ genes of bmbdv zhenjiang strain (china) were cloned and sequenced. the results showed that the nucleotide sequences of nsd-2 'and +nsd-2' genes were identical with nsd-2 and +nsd-2, suggesting that the silkworm had the same resistance mechanism to two bmbdv strains infection (li et al., 2010; qin et al., 1996; ponnuvel 142 et al., 2010; eguchi et al., 2010). ito k et al detected the expression level of +nsd-2 and transcription of bmbdv from the anterior, middle, and posterior parts of the midgut of the sensitive silkworm by qrt-pcr, the results showed bmbdv-derived transcripts were clearly detected in all parts of the midgut and +nsd-2 strongly expressed in the posterior part of the midgut. these results suggest that the infectivity of bmbdv depends mainly on the expression level of +nsd-2 in the midgut and the viral infection is supported even by very faint expression of +nsd-2 (ito et al., 2016). however, the mechanism of +nsd-2 mediates the entry of bmbdv into the cell is still unknown. as a model insect, b. mori cell lines plays an important role in the study of immunology, insect pathology and insect virology (taniai et al., 2006; pan et al., 2010). up to now, more than 30 silkworm cell lines have been established all over the world (iwanaga et al., 2009). however, there is no report about the sensitive cell lines to bmbdv (hu et al., 2010; van et al., 2011; zheng et al., 2014; feng et al., 2017; ming et al., 2017; qi et al., 2017). at present, the widely used silkworm cell lines, b. mori embryo cell lines (bme) and b. mori ovary cell lines (bmn) are non-sensitive to bmbdv. as we know, it is hard to promote the virus study without the proper sensitive cell lines, so we tried to clone and express the +nsd-2 gene in bmn cells, and established a bmn (+nsd-2) cell line that can stably express nsd-2 protein. we hope that this cell lines can be used for subsequent identification of receptor binding proteins and the mechanism of bmbdv invasion into the host cells. materials and methods plasmid construction according to the full lengh of gene sequence of national center for biotechnology information search database (ncbi), the specific primers of +nsd-2 gene (ncbi reference sequence: nm_001130871.1) were designed, nsd-f:5 ' cgggatccatggattcaaatgggataaat-3' (underline means bamh ⅰrestriction endonuclease sites); nsd-r:5'-ccctcgagccgcttcctgcgatacc-3' (underline means xhoⅰ restriction endonuclease sites). pcr was amplified by high fidelity enzyme pfu dna polymerase (takara, dalian, china) with cdna extracted from the sensitive silkworm strain 306 midgut. the pcr program consisted of 5 min pre-denaturation at 94 °c, 35 cycles (94 °c, 30 s; 56 °c, 30 s; 72 °c, 2’30s), and 10 min at 72 °c. pcr products are purified by omega gel extraction kit (omega bio-tek), and the digested fragment was subcloned into the vector piz-v5/his also digested by bamh i and xho i (invitrogen, shanghai, china). the recombinant plasmid piz+nsd-2 was confirmed by sequencing. cell transfection and cell culturing plasmid extraction kit (omega bio) was used to extract plasmid piz+nsd-2 and pib-gfp (laboratory constructed) was used for subsequent transfection. bmn cells were cultured with tc-100 medium supplemented with 10% fetal bovine serum (fbs, gibco, in grand island, ny, usa) and 1% antibiotics (penicillin-streptomycin; hyclone gibco-brl (life) technologies, carlsbad, ca, usa) in t-25 flask. according to the manufacturer's instructions, the transfection was carried out with cellfectin ii (invitrogen, carlsbad, ca, usa). 2 μg piz+nsd-2 plasmid was mixed with 100 μl serum free tc-100 medium for 30 minutes; 5 μl cellfectin ii resgent liposome reagent was added and mixed with 100 μl serum free tc-100 medium for 30 minutes. finally, the dna/cellfectin ii resgent liposome reagent was mixed and incubated for 15 minutes at room temperature. at the same time, bmn cells were washed three times with serum-free tc-100 medium. then, the dna/cellfectin ii resgent liposome reagent mixture was added to the culture flask in a 27 °c incubator. after 6 h, the dna/cellfectin ii resgent liposome reagent mixture was removed and 2 ml fresh tc-100 complete medium (containing 1% chloramphenicol and streptomycin +10% fetal bovine serum) was added. 48 h post-transfection, zeocin (invitrogen, carlsbad, ca, usa) antibiotics with final concentration of 200 μg/ ml were added to screen stable cell lines. a new culture medium containing 200 μg/ ml zeocin antibiotics was replaced every 3 days. the morphological characteristics of the cells were observed and recorded. the passage was cryopreserved after 30 passages. pcr assay rna of bmn (+nsd-2) cells, bmn cells and the midgut from the susceptible silkworm (jing song) were extracted by trizol reagent (invitrogen); total rna (500 ng/μl) by rnase-free dnase (vazyme biotech, nanjing, china) to remove dna pollution, cdna synthesis kit according to instructions (vazyme biotech) synthesis cdna. 100-fold dilution of cdna was used as template for pcr amplification. pcr amplification procedure and primers are as same as the section of plasmid construction. table 1 primers for qrt-pcr gene sense primer 5’-3’ anti-sense primer 5’-3’ product size (bp) genbank accession number bm-rpl3 caaagtgaaatgggccagag agcacgagctacagtgaacga 235 nm-001126254.1 bmbdv-ns1 gttggtggtgaagggtttg gggagatagtttacactttggag 198 dq017268.1 bmbdv-vp agaaatgctggagccgat cctccactgcctgtaacttc 209 dq017268.1 143 fig. 1 bmn (+nsd-2) cells morphological character of the zeocin selection. (a) bmn cells. (b) zeocin selected cells, the transfected bmn cells started agglomerate from 4th days and (c) formed a few cell colones within 7-13 days. (d) the size of the cell colones appeared to increase over the following 5-8 days. (e) the number of the cell clones increased on 24 days. (f) the cells had nearly grown to confluent cell bottle after approximately 41 days in culture. (g) the morphology of bmn cells. (h) the morphology of bmn (+nsd-2) cells after 30 passages western blot bmn (+nsd-2) cells were lysated by weak ripa lysis buffer (beyotime biotechnology) including 50mm tris (ph 7.4), 150 mm nacl, 1% np-40, 0.25% sodium deoxycholate and 1% pmsf (cell signaling technology, usa). the lysates were collected and incubated on ice for 10 min. lysates were cleared by centrifugation at 12,000 g for 5 min at 4 °c. the total protein was separated by 12% sds-page and transferred onto 0.2 μm pvdf membranes (ge healthcare life sciences, germany). membranes were blocked with 5% (w/v) non-fat milk (sangon biotech) for 4 h at 37 °c, and then incubated overnight at 4 °c with a specific polyclonal antibody anti-v5 (1:2000; genscript, nj). after three rinses with tbst, the membranes were incubated at room temperature for 2 h with the goat anti-rabbit (1:3000) igg-hrp (bbi life sciences) secondary antibody diluted in tbst. membranes were washed three times with tbst, the results were detected by ecl kit (wanleibio). immunofluorescence assay bmn (+nsd-2) and bmn cells (5×105 cells/ml) were seeded into 35 mm glass bottom dish. cells were washed 3 times with pbs, fixed with 4% paraformaldehyde for 15 min, washed with pbs again; 0.5% triton x-100/pbs permeabilization for 144 10 min, pbs washed 3 times; 3% pbs/bsa was used to blocked at 37 °c for 30 min; v5-tag (genscript, nj) polyclonal antibody (1: 1000) as the first antibody, secondary antibody with cytm5-labeled antibody to rabbit igg (h+l) (kpl, usa). after three washes with pbs, the cells were observed under in leica tcs sp5 confocal laser microscopy. co-immunoprecipitation (co-ip) bmn (+nsd-2) cells were grown to approximately 80-90 % confluence in t-25 flasks (2), and pib-vp recombinant plasmid (laboratory construction) was transfected into bmn (+nsd-2) cells. 48 h post-transfection, the cells were washed twice with pbs and harvested by scraping. cells were pelleted by centrifugation at 1000 g for 10 min at 4 °c and resuspended in ripa lysis buffer. the sample were pre-cleared by centrifugation and then the immunoprecipitation antibody was added and the samples rocked at 4 °c for a minimum of 1 h. protein a/g plus-agarose (bioworld technology) was added to the samples and rocked overnight at 4°c. immune complexes were washed three times with pbs; 5× sample buffer was added to the beads and the immunoprecipitated material was separated by 12% sds-page, transferred onto pvdf membranes and analyzed by western blotting using the anti-vp and anti-v5 antibodies. virus infection experiments 5 x 105 bmn (+nsd-2) cells were seeded into 6-well plates and each well was added with bmbdv virus suspension (20 mg/ml) 100 μl, respectively. cells were harvested at 0 h, 2 h, 4 h, 8 h, 12 h and 24 h post-infection. each total rna was extracted using trizol reagent (invitrogen). 20-fold dilution cdna was used as template for qrt-pcr (rpl3 as a reference gene). the transcription of bmbdv genes vp and ns1 were detected. the list of primers is as follows (table 1). results construction of nsd-2 stable expressing cell line nsd-2 protein encoded by sensitive gene +nsd-2 is one of the important factors of virus infect cells. we transfected the recombinant plasmid piz-+nsd-2 into bmn cells and selected with 200 μg/ ml zeocin antibiotics. finally, we obtained cell lines with stable expression of +nsd-2 gene and named the bmn (+nsd-2) cells after 30 times. in our culture conditions, the cells began to bunch up after 4 days (fig. 1b) with the normal morphology of bmn cells as control group (fig. 1a), and formed clonal cells in 7-13 days (fig. 1c); clonal cells began to grow in the following 5-20 days (fig. 1d, e). the cells overspreaded the t-25 flask (fig. 1f) at about 41 days after the following culture. when the cell growth gradually stabilized, the cells adapted quickly to the new medium with 200 μg/ ml zeocin antibiotics, exhibited a good growth condition and could be stably passaged. from the final cellular morphology after 30 passages, there is no significant difference between the bmn cells (fig. 1g) and the bmn (+nsd-2) cells morphology (fig. 1h). the transcription of +nsd-2 gene in bmn (+nsd-2) cells the total rna of bmn (+nsd-2) and bmn cells were extracted and reverse transcripted to cdna as the template of pcr amplification. theoretically, the target size of sensitive and non-sensitive genes is 1878 bp and 591 bp, respectively. in fact, nsd-f and nsd-r primers used in the process of plasmid constructing for pcr amplification and the size of the amplified products from bmn (+nsd-2) cells is near 2000 bp (fig. 2a), and the size of the amplified products from bmn cells and bmbdv susceptive strains of silkworm (jingsong) midgut is near 600 bp and 2000 bp, respectively (fig. 2b), consistent with the the theoretical size. these experimental results indicate that the +nsd-2 gene fragment could stably express in bmn (+nsd-2) cells after the 30 passages. fig. 2 pcr amplification of the + nsd-2 gene in bmn (+ nsd-2) cells and jingsong strain of silkworm midgut, bmn cells extracted total rna. (a) m, dna marker. bmn (+nsd-2): bmn (+nsd-2) cells well collected and extracted total rna, and amplification of +nsd-2 gene by rt-pcr; (b) bmn: the amplification of cdna reverse transcription from bmn cells rna; jingsong: the amplification of cdna reverse transcription from jingsong midgut rna 145 western blot analysis of nsd-2 protein expression in bmn (+nsd-2) cells after 30 passages of bmn (+nsd-2) cells, the expression of nsd-2 protein was detected by western blot. after lysis, bmn (+nsd-2) cells and bmn cells were analyzed by western blot with v5 polyclonal antibody as the first antibody, and the theoretical size of protein was about 68 kda. in this experiment, a specific protein of about 68 kda was detected in bmn (+nsd-2) cells while it wasn’t found in bmn cells (fig. 3). these results demonstrate that nsd-2 protein could stably express in the passages of bmn (+nsd-2) cells. immunofluorescence analysis of nsd-2 expression in bmn ( nsd-2) cells nsd-2 is a transmembrane protein. therefore, we want to ascertain wether it is specific located on bmn (+nsd-2) cell lines membranes. treated bmn (+nsd-2) cells with v5-tag polyclonal antibody (1: 1000) as the first antibody, the cytm5-labeled antibody to rabbit igg (h+l) was used as the secondary antibody, and the bmn cells were used as negative controls. red fluorescence (fig. 4a) was detected in bmn (+nsd-2) cells while no red fluorescence (fig. 4b) was observed in the cell membrane of bmn cells. the results show that nsd-2 protein was mainly expressed in the cell membrane of bmn (+nsd-2) cells. interaction between vp protein and nsd-2 protein in bmn (+nsd-2) cells vp protein is the main structural protein of bmbdv, and nsd-2 protein is a viral receptor fig. 3 expression analysis of nsd-2 by western blotting in bmn (+nsd-2) cells and bmn cells. m, dna marker. (1) bmn cells; (2) bmn (+nsd-2) cells well collected and lysised，and subjectedc to western blot analysis with anti-v5 antibody candidate. however, the interaction between vp protein and nsd-2 protein is still unknown. the bmn (+nsd-2) cells transfected with pib-vp recombinant plasmid (laboratory construction) were collected for fig. 4 detection of nsd-2 protein in bmn (+nsd-2) cells by immunofluorescence assay. (a) red fluorescence was observed in bmn (+nsd-2) cells. (b) red fluorescence wasn’t observed in bmn cells, which as a negative controls. dapi staining shows unclear dna (blue). 146 fig. 5 in vitro interaction between vp and nsd-2. (a) the presence of viral structural protein vp and host protein nsd-2 in lysate of infected bmn (+nsd-2) cells by ib assay. (b) co-immunoprecipitation of vp with nsd-2 by anti-v5. (c) co-immunoprecipitation of nsd-2 with vp by anti-vp the co-ip experiments. vp protein and nsd-2 protein were detected in bmn (+nsd-2) cells (fig. 5a), the use of anti-v5 polyclonal antibody immunoprecipitation pulled down vp (fig. 5 b) and using anti-vp monoclonal antibody (laboratory preparation) immunoprecipitation pull down nsd-2 (fig. 5 c); while the corresponding protein band were not detected in the control group. virus infection in bmn (+nsd-2) cells nsd-2 protein plays an important role in bmbdv infection, whether bmn (+nsd-2) cells could mediate bmbdv into the cell is still unclear. qrt-pcr could quantitatively detect bmbdv genes vp and ns1 in bmn(+nsd-2) cells infected by bmbdv. in addition, we make the bombyx mori gene rpl3 as the internal reference standard. cultured bmn(+nsd-2) cells with bmbdv for 0 h, 2 h, 4 h, 8 h, 12 h, 24 h, the rna were extracted and reverse transcribed into cdna (rpl3 as the control of qrt-pcr). the theory sizes of vp and ns1 genes were 209 bp and 198 bp, respectively. as a result, the corresponding segments (fig. 6 a, b) were not detected in bmn (+nsd-2) cells. the results showed that bmbdv could not infect bmn (+nsd-2) cells only in the presence of nsd-2, indicates that there may exist co-receptors to mediate virus infection. discussion as an important economic insect and a model insect of lepidoptera, the silkworm has made important contributions to the development of society and the progress of science (mita et al., 2008). bmbdv is one of the most dangerous viruses, which causes decrease of cocoon yield in summer and autumn in china. therefore, the research of virus invasion mechanism has a positive meaning for preventing of the silkworm diseases. host tissues and specific receptors on cell surface are the main factors that determine the pathway of virus invasion, the way of diffusion and the disease characteristics of the host. the presence of virus receptors on the cell surface directly affects cells susceptibility to viruses. ito transfected the +nsd-2 gene into the resistant strain silkworm through germ-line transport system to restore the bmbdv-sensitive silkworm strain, indicated that nsd-2 protein was the essential factor for bmbdv infection. we cloned the +nsd-2 gene and constructed the recombinant plasmid piz-+nsd-2 successfully. the recombinant plasmid was transfected into bmn cells and selected by zeocin antibiotics. bmn (+nsd-2) cell lines, which stably expressed bmbdv sensitive gene +nsd-2, were successfully constructed. rt-pcr technology was used to detect the transcription of +nsd-2 gene in bmn (+nsd-2) cells, and the expression of nsd-2 protein was tested by immunofluorescence analysis and western blot. the results showed that bmn (+nsd-2) cell lines were able to express the +nsd-2 gene stably. the immunofluorescence experiment revealed that the nsd-2 protein of the c-terminal with v5-tag was mainly located in the cell membrane area of bmn (+nsd-2) cells. co-ip results showed that the interaction between nsd-2 and vp protein really existed. however, the results of qrt-pcr, routine pcr and immunofluorescence experiments showed that the virus genes was not detected in bmn (+nsd-2) cells. therefore, we speculate that nsd-2 may mediate virus entry with other co-receptors and suitable ambient conditions, such as temperature and ph. it is well known that the hiv receptor has cd4 molecules as well as the co-receptor ccr5 or cxcr4 (lee et al., 2017; liu et al., 2017). hiv-1 forms a trimer complex to invade target cells, namely hiv-1 gp120 envelope protein, cd4 molecule and a co-receptor. the majority of its co-receptors are chemotactic cytokine receptor. hepatocytes are the main target cells for hcv infection, and in the initial stage of hcv infection, some liver specific factors / molecules are required to be involved except for hcv receptor in vitro (ea et al., 2011). moreover, virus enters the cell has two ways: ph is independent on the fusion of the plasma membrane directly, and the receptor mediated ph dependent endocytosis. for example, cell invasion of hcvpp is ph dependent: cytoplasmic acidification inhibitor bafilomycin a1 can reduce the infectivity of hcvpp to huh-7 cells, and has dose response relationship. similar results are also observed with the treatment of chloroquine, a weakly alkaline drug. in addition, the temperature and ions also have great influence on the virus invading cells (zhao et al., 2009). 147 fig. 6 the virus infection experiment results. (a) qrt-pcr amplification of the vp gene in bmn (+nsd-2) cells from six different times; (b) qrt-pcr amplification of the ns1 gene in bmn (+nsd-2) cells from six different times; data are performed in triplicate in this study, a bmn cell line is established which stably expresses the potential bmbdv receptor protein nsd-2 of the silkworm. although the results show that bmbdv cannot invade the cell, the stable recombinant cell lines could still be applied to the study of nsd-2, a protein closely related to viral invasion and the authentication of the receptor binding proteins. the subsequent task may focus on the analysis and identification of other components of the co-receptor by means of co-ip and mass spectrometry. and the identified co-receptors will be added into bmn (+nsd-2) cells to find whether the virus can invade cells. moreover, the virus dose, ph and so on factors will be considered to be related to virus infection. the study of the viral receptor could deeply reveal the 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2016. zheng gl, li mm, li cy. establishment and characterization of three new cell lines from the embryonic tissue of holotrichia oblita faldermann (coleoptera: scarabaeidae). in. vitro. cell dev. an. 50: 483-488, 2014. zhao q, dong li. viral receptors and its research status. prog. vet. med, 2009. 174 isj 14: 174-181, 2017 issn 1824-307x research report quantitative comparison for some immune responses among eurygaster integriceps, ephestia kuehniella and zophobas morio against the entomopathogenic fungus beuveria bassiana fs seyedtalebi1, sa safavi1, r talaei-hassanloui2, ar bandani2 1department of plant protection, faculty of agriculture, urmia university, urmia, iran 2department of plant protection, college of agriculture and natural resources, university of tehran, karaj, iran accepted april 26, 2017 abstract some defense reactions including cuticular lipids, phagocytic activity, nodulation and hemolymph phenoloxidasec activity were compared in eurygaster integriceps, ephestia kuehniella and zophobas morio exposed to five different b. bassiana isolates. the cuticular lipids were stimulating on conidia germination possibly with no fungicidal or fungistatic ability in epicuticular fatty acids. there was correlation between all studied immune system reactions and the virulence of isolates to e. kuehniella and e. integriceps. de as a less virulent fungal isolate stimulated immune reactions in high levels in most experiments, despite of tv as the most virulent isolate. e. integriceps was the most sensitive host with lowered immune reactions. z. morio showed resistant with the highest nodulation. e. kuehniella had moderate sensitivity with maximal phagocytic and the phenoloxidase activity. the phagocytic activity was the highest at 30 min after fungus injection. the nodulation and the phenoloxidase activity were demonstrated at 6, 12 and 24 h after injection. the most nodulation rate was observed at 24 h. the phenoloxidase activity for the most isolates reached a maximum value for z. morio and e. integriceps after 12 h and in e. kuehniella after 24 h. our research provides an interesting perspective in host susceptibility to fungal infections. key words: b. bassiana; e. integriceps; e. kuehniella; z. morio; immune system introduction there are different defense barriers in insect body which entomopathogenic fungi should have an ability to overcome them. they infect insects by penetrating the cuticle, thus insect cuticle plays a very important role as a barrier to infection in host specificity (murrin and nolan, 1987; altre and vandenberg, 2001). not only chemical and physical toughness, but also the cuticular compounds have been demonstrated to have toxicity to invading fungi (ortiz-urquiza and keyhani, 2013). furthermore, insects have a well-developed innate immune system that allows a general and rapid response to infectious agents. the innate immune system of insects relies on both humoral and cellular responses. humoral immune responses include several antimicrobial peptides, enzymic cascades that regulate coagulation and melanization of haemolymph, and the production of reactive oxygen ___________________________________________________________________________ corresponding author: seyed ali safavi department of plant protection faculty of agriculture urmia university, urmia, iran email: a.safavi@urmia.ac.ir species (ros) and reactive nitrogen species (rns). the term cellular immune response refers to hemocyte-mediated responses, including phagocytosis, nodulation and encapsulation (lavine and strand, 2002). phenoloxidase is an oxidoreductase that catalyses the oxidation process of phenols present in the hemolymph to cytotoxic quinones (ashida, 1990). these quinones polymerize non-enzymatically to melanin. both quinones and melanin are toxic to microorganisms (soderhall and cerenius, 1998). the cellular reaction is started with phagocytosis. during the phagocytic process, cells ingest and eventually destroy foreign particles (rosales, 2011). nodule formation is initiated with the micro-aggregation of hemocytes, which entrap large numbers of microorganisms. these micro-aggregates grow in size by recruiting additional hemocytes. finally, the process ends with melanization into darkened nodules, which attach to the body wall or to various internal organs (franssens et al., 2006). variations in the susceptibility of insect species to fungal invasion may result from their immune potencies. reaction of insect immune system to fungal infection has been studied earlier, for example 175 fig. 1 effect of different cuticular extract on mean (±se) percentages of tv and de isolates spore germination. means in columns marked with different letters are significantly different (f-lsd, p < 0.05) lee et al. (2005) investigated cellular immune responses in the larvae of mamestra brassicae injected with three entomopathogenic fungi, beauveria bassiana, nomuraea rileyi and paecilomyces tenuipes, suggesting that the different virulence of each fungal species is caused by specific immune responses in the larvae. zibaee et al. (2014) demonstrated different levels of cellular immune response of chilo suppressalis against several entomopathogenic fungi including isolates of b. bassiana, metarhizium anisopliae, isaria fumosoroseus and lecanicilium lecanii. most researches have considered lepidopteran hosts (meshrif et al., 2011; ajamhassani et al., 2013; khosravi et al., 2014), but comparative studies among different insect species are not many. bogus et al. (2010) studied immune responses of three insect species including dendrolimus pini, galleria mellonella and calliphora vicina to the entomopathogenic fungus conidiobolus coronatus. our data describes and compares some different defense criteria including cuticular lipids, phagocytic activity, nodulation and hemolymph phenoloxidase activity in three host species from different insect orders. sunn pest, eurygaster integriceps, the flour moth, ephestia kuehniella and giant mealworm or superworm zophobas morio were compared for their immune reactions against five different b. bassiana isolates. materials and methods insects rearing sunn pest adults were collected from wheat fields of varamin, tehran province, iran. they reared in plastic boxes (30×30×50 cm) on wet wheat seeds (triticum aestivum var. pishtaz), and a piece of cotton soaked with water was used as a water source. folded strips of paper were hung in containers for oviposition. after hatching of eggs, nymphs were transferred to plastic shelves with pots of wheat and wet wheat seeds. fifth instar nymphs were used in experiments. the flour moth was prepared from colony in biological control laboratory in college of agriculture and natural resources, university of tehran and bred in plastic containers containing flour and yeast (10 g yeast per kg of flour), the fourth instar larvae were used in experiments. initial colony of z. morio was obtained from a pet store and reared in plastic containers containing wheat bran and pieces of potato. the last instar larvae of new generation were used in experiments. the rearing condition was 25 ± 1 ºc, 70 ± 5 % r.h. and 16:8 (l:d) h photoperiod for all insects. beauveria bassiana isolates five b. bassiana isolates encoded tv, oz, un, dv and de (soil origin) were grown on sabouraud dexterose agar (sda) and maintained at 25 ± 1 °c, 70 ± 5 % rh, and a photoperiod of 16:8 (l:d) h. cultures were scrapped after sporulation and conidia were obtained (goettel and inglis, 1997). virulence of these isolates have been studied previously (our unpublished data) on these insects, as tv and de were most and less virulent isolates while the other has moderate virulence on e. kuehniella larvae and e. integriceps nymphs (22.70 ± 1.39 and 32.5 ± 1.71 % mortality for de, 36.87 ± 2.82 and 64.17 ± 1.54 % for dv, 45.74 ± 1.84 and 66.67 ± 1.61 % for un, 58.43 ± 1.90 and 71.67 ± 1.67 % for oz, 89.15 ± 2.39 and 86.00 ± 1.53 % for tv on e. kuehniella and e. integriceps, respectively). none of the isolates affected the survival of z. morio larvae. labeling of b. bassiana conidia b. bassiana conidia were obtained from the 10 14 days old culture on pda. these conidia were suspended in 10 ml of phosphate buffered saline (pbs) (0.13 m nacl, 2.68 mm kcl, 8.1 mm na2hpo4 and 1.47 mm kh2po4, ph 7.4, autoclaved). they were then washed and resuspended in a sterile co3-hco3 buffer at ph 9.4 (9.5 ml 0.2 m na2co3 was mixed with 41.5 ml 0.2 m nahco3). the solution was made up to 200 ml and labeled by mixing this solution with 1mg of fitc (fluorescein isothiocyanate, sigma) on a shaker for 30 min at room temperature in complete darkness (rohloff et al., 1994). 176 fig. 2 effect of b. bassiana isolates conidia on mean phagocytosis percentage (±se) in different insect on different times, means followed by different letters differ significantly (f-lsd, p < 0.05) extraction of cuticular surface lipids insects were killed by freezing and washed several times with distilled water. after air drying, they were immersed in three different solvents separately, methanol (polar solvent), hexane (nonpolar solvent) and dichloromethane (solvent with intermediate polarity) for 30 min. the insects were picked up and the extracts were collected in glass petri dishes, dried in a hood and resolved in ethanol 96 %. after alcohol evaporation, residuals was weighted and solved in ethanol 96 % again. the extracts were maintained in freezer at -20 °c (wang and st. leger, 2005). effect of cuticular extract on conidia germination the petri dishes (9 cm diameter) containing thin layers of water agar (0.5 %) were inoculated with 150 μl of fungus suspension 106 (conidia/ml) of isolates tv and de. one hundred microliters of each extract (4 mg/ml) were pipetted on glass coverslips. after solvent evaporation, they were placed up-side down on the surface of the media in the petri dishes. glass coverslips without extracts were used as control. percentages of germinated conidia were estimated after 24 h under a light microscope at 40x magnification. three replicates were considered for each treatment. phagocytosis assay insects were immobilized in freezer for some min and were surface sterilized with ethanol 70 %. quantities of 20, 1 and 1 μl of 1×108 conidia/ml of fungal concentration (labeled conidia), were injected to giant mealworm larvae (between second and third thoracic segment), sunn pest nymph (between second and third legs) and flour moth (lateral abdominal section), respectively by a hamilton syringe. the treated insects were transferred to rearing containers. control larvae were injected with distilled water. insect hemolymph was collected at different intervals (15, 30, and 60 min post injection). insects were anesthetized on ice, surface sterilized with ethanol (70 %) and bled by cutting one of the prolegs for sunn pest nymphs and giant mealworm larvae or scratched body surface for flour moth larvae. the hemolymph was then collected and mixed with ice-cold anticoagulant solution buffer (0.098 m naoh, 0.186 m nacl, 0.017 m edta, 0.041 m citric acid, ph 4.5) in 30:70 ratio (anggraeni and ratcliffe, 1991). phagocytic activity was determined by counting the cells containing conidia in a neubauer hemocytometer observations were done on a zeiss florescent microscope. hemolymph samples were checked in 3 replicates and whole experiment was replicated one more time. effect of fungal conidia on nodulation quantities of 10, 0.5 and 0.5 μl of 1×106 conidia/ml of fungal concentration (labeled conidia) were injected according to the above mentioned method. hemolymph was collected at 6, 12, and 24 h after injection from chilled, surface sterilized (ethanol 70 %) larvae. ten microliter samples of hemolymph from each insect were mixed with 10 μl cold anticoagulant buffer. then, the number of nodules was counted under a light microscope at 20x magnification. hemolymph samples were checked in 3 replicates and whole experiment was replicated again. assay for po activity injection was the same as nodulation assay. according to hung and boucias (1996), 20 μl of collected haemolymph was mixed with the same volume of phosphate buffer (ph 7) at 4 °c. the samples were centrifuged (12,000×g) at 4 °c for 5 min. the supernatant (plasma) was collected and used for po activity. the phenoloxidase assays were carried out in 96-well plates. each well contained 20 μl enzyme, 20 μl substrate (20 mm ldopa) and 80 μl phosphate buffer, ph 7.0. the absorbance was recorded by a micro plate reader (biotek, usa) over 30 min with a one min interval at 490 nm. this assay was carried out with three replications for each treatment and the whole assay was repeated. 177 fig. 3 effect of b. bassiana isolates spores on mean number of nodule formation (±se) in different insect on different times, means followed by different letters differ significantly (f-lsd, p < 0.05) protein determination the protein content of the samples was determined according to lowry et al. (1951) using bovine serum albumin (biorad) as the standard. statistical analysis all experimental data were subjected to analysis of variance (anova) (p < 0.05). pooled data of two-time repeats of the whole assay were analyzed to determine possible significant differences among the treatments via f-lsd test post-significant anova. possible correlation between immune reactions and virulence of isolates was analyzed through pearson correlation coefficient (sas institute, 2002). results effect of cuticular extract on conidia germination according to results (fig. 1) all extracts (4 mg/ml) had stimulating effect on conidia germination and hexane extracts had significantly less effect compared with two others (f3, 36 = 759.05, p < 0.0001). there was no statistical difference between two isolates (f1, 36 = 0.00, p = 0.97), but the cuticular extracts of z. morio had the highest stimulating effect on the conidia germination, while cuticular extracts of sunn pest had less effect (f3, 36 = 50.58, p < 0.0001). however, there was no correlation between conidia germination in cuticular extracts and virulence of isolates (p > 0.05). phagocytosis assay with increasing time, percentage of phagocytosis was decreased and the most phagocytic activity was observed 30 min after injection (fig. 2). there were significant differences among the isolates at 30 min (f4, 15 = 48.59, p < 0.0001), 60 min (f4, 15 = 27.80, p < 0.0001) and 90 min (f4, 15 = 3.02, p < 0.0001) and between insects at 30 min (f2, 15 = 436.89, p < 0.0001), 60 min (f2, 15 = 251.36, p < 0.0001) and 90 min (f2, 15 = 23.99, p < 0.0001). as, the highest and lowest percentages were for e. kuehniella larvae and e. integriceps nymphs, respectively. isolate de had the highest effect. there was a negative correlation between the percentage of phagocytosis and the virulence of isolates on e. kuehniella larvae at 30 min (r= 0.91, p = 0.0002), 60 min (r = 0.84, p = 0.0019) and 90 min (r = 0.69, p = 0.026) and on e. integriceps at 30 min (r = 0.82, p = 0.0030), 60 min (r = 0.90, p = 0.0003) and 90 min (r = 0.91, p = 0.0002). effect of fungal conidia on nodulation with increasing time, the number of nodules was increased and the most nodulation was observed 24 h after injection (fig. 3). there were significant differences between the isolates at 6 h (f5, 18 = 35.55, p < 0.0001) , 12 h (f5, 18 = 70.01, p < 0.0001) and 24 h (f5, 18 = 98.37, p < 0.0001) and between insects at 6 h (f2, 18 = 25.04, p < 0.0001), 12 h (f2, 18 = 45.13, p < 0.0001) and 24 h (f2, 18 = 97.84, p < 0.0001), the highest and lowest numbers of nodules were for z. morio larvae and the e. integriceps nymphs, respectively. isolate de had the highest effect. there was a negative correlation between the number of nodules and the virulence of isolates for two insects, e. kuehniella larvae at 6 h (r = 0.94, p < 0.0001), 12 h (r = 0.92, p = 0.001) and 24 h (r = 0.94, p < 0.001) and e. integriceps at 6 h (r = 0.73, p = 0.0144), 12 h (r = 0.710, p = 0.0197) and 24 h (r = 0.72, p = 0.0177). assay of phenoloxidase (po) activity phenoloxidase activities were statistically different among the isolates at 6 h (f5, 18 = 8.76, p = 0.0003), 12 h (f5, 18 = 3.01, p = 0.04) and 24 h (f5, 18 = 0.04, p = 0.6990). moreover, the phenoloxidase activities of the insects varied significantly at 6 h (f2, 18 = 8.04, p = 0.0035), 12 h (f2, 18 = 16.72, p < 0.0001) and 24 h (f2, 18 =45.13, p < 0.0001); as the enzyme activity reached a maximum value in 12 h for z. morio and the e. integriceps and in 24 h for e. kuehniella (fig. 4). there was correlation between the phenoloxidase (po) activity and the virulence of isolates for two insects, e. kuehniella larvae at 6 h (r 178 fig. 4 effect of b. bassiana isolates spores on mean phenoloxidase (po) activity (±se) in different insect on different times, means followed by different letters differ significantly (f-lsd, p < 0.05) = 0.80, p < 0.0051), 12 h (r = 0.81, p = 0.0044) and 24 h (r = 0.92, p < 0.001) and e. integriceps at 6 h (r = 0.87, p = 0.0010), at 12 h (r = 0.65, p = 0.0404) and at 24 h (r = 0.71, p = 0.0209). discussion to clarify the relationship between host susceptibility and virulence of b. bassiana isolates, some defense reactions including cuticular lipids, phagocytic activity, nodulation and hemolymph phenoloxidasec activity in 3 different insect were compared against five different b. bassiana isolates. insects were selected according to our pervious study, sunn pest e. integriceps with high level sensitivity, the flour moth e. kuehniella with moderate sensitivity and giant mealworm z. morio as resistancec host to fungal infection. b. bassiana isolates were tv (the most virulent), oz, un, dv (moderate virulent) and de (the less virulent). insect epicuticle represents the first barrier to entomopathogenic fungi infection, its components are extremely heterogeneous and therefore have the potential to lead to different pathogen responses in particular insects (thompson, 1973; wang and st. leger, 2005). inhibition effect of epicuticular fatty acids on conidia germination has been reported previously (smith and grula, 1982; saito and aoki, 1983; bogus et al., 2010; urbanek et al., 2012). in current research, three solvents with different polarity were used for lipid extraction and all of them were stimulating on conidia germination. tv and de isolates were used for this stage of experiment and no difference was shown between them. the fungi isolates were not specific pathogens on three insects from this point of view. while, z. morio was resistant to infection by fungal isolates, as a result, it seems that there is no fungicidal or fungistatic ability in epicuticular fatty acids of these insects. in contrast, wang and st. leger (2005) showed that germination and appressorial formation of the specific locust pathogen, m. anisopliae var. acridum was completely down with locust schistocerca gregaria cuticular extract compared to other insects such as leptinotarsa decimlineata and magicicada septendecim as non-specific hosts. as biochemistry of e. integriceps, e. kuehniella and z. morio epicuticules are not clear, possibly other components except the fatty acids have inhibitory effects on fungal infection. of course supplementary studies are needed. immune system reactions were significantly different among insects and fungal isolates. our data showed that the most phagocytic activity (fig. 5) 30 min after fungus injection in all treatments; the lowest percentage was observed in e. integriceps. it seems that fungi could overcome to the immune reaction of sunn pest, because the reaction level is low and without remarkable changes in the time intervals. also, ajamhassani et al. (2013) reported that phagocytic activity of conidia of b. bassiana and i. fumosorosea isolates in hyphantria cunea larvae was reduced during the time. but, khosravi et al. (2014) showed increase in phagocytic activity of b. bassiana conidia in glyphodes pyloalis larvae during the time. we studied nodule formation in three times 6, 12 and 24 h after injection of fungal conidia. aggregation of hemocytes and entrapping of labeled conidia was seen and melanization was occurred during the time (fig. 6). the formation of nodules was increased during the time and the highest number of nodules was recorded for z. morio. microscopic examination showed that its nodules melanized extremely in comparison with them in other insects. therefore, the beetle has better performance than others in nodule formation. zibaee et al. (2011) demonstrated the most number of nodules were observed 3 h after injection of b. bassiana conidia in e. integriceps adults, while our studies showed the maximum number of nodules after 24 h fungus injection in fifth instar nymphs. 179 fig. 5 phagocytic activity, labeled conidia attach to hemocytes (100x magnification) insect cellular immune system is connected with quality and quantity of hemocytes which is different between insects. fungal isolate, the amount of injected inocula and the experimental method may be the reasons of differences among our results with the others. the phenoloxidase activity was different in three insects and in exposition to various fungal isolates. as, the enzyme activity reached a maximum value in 12 h for z. morio and e. integriceps and in 24 h for e. kuehniella. in the most researches, the level of phenoloxidase activity was increased in first hours of fungal infection and decreased subsequently (ajamhassani et al., 2013; dubovskiy et al., 2013; khosravi et al., 2014). high phenoloxidase activity during infection process is because of its important roles in immune reactions such as wound healing, encapsulation and nodule formation (lokstan and li, 1988). zibaee et al. (2013) demonstrated the higher phenoloxidase activity in 6 h after injection of b. bassiana conidia in adult e. integriceps. ezzati-tabrizi et al. (2013) investigated the phenoloxidase activity of plodia interpunctella and g. mellonella after injection of b. bassiana conidia, it was higher in p. interpunctella and reached to maximal value in 12 h after injection in both insects. the decline may be due to the immunosuppressive effect of fungal proteins or toxic metabolites. comparative studies about insect immune system are very rare. bogus et al. (2007) demonstrated encapsulation, nodule formation and phenoloxidase activity of g. mellonella, c. vicina and d. pini to c. coronatus conidia, among them c. vicina was unharmed against fungal infection and had the lowest immune reactions. they expressed that other mechanisms such as antiproteolytic capacity of host hemolymph may play a role as additional safety device. while in our study z. morio was resistant to fungal isolates with high level of phenoloxidase activity and nodule formation. as the immune system is costly to insects, they try to show most efficient defense at the lowest possible cost. according to our data, there was correlation between all studied immune system reactions and the virulence of isolates for two insects, e. kuehniella larvae and e. integriceps nymphs. de, as the least virulent isolate, highly stimulated insect immune reactions in more experiments, vice versa tv as the most virulent isolate. it’s may be because of fungal metabolites, growth of entomopathogenic fungi in the hemolymph of the host is associated with the secretion of toxins (secondary metabolites) by the pathogen (mazet et al., 1994; clarkson and charnley, 1996; bandani et al., 2000); secondary metabolites disable several immune mechanisms allowing the fungus to overcome and then kill its host (bandani et al., 2008; zibaee et al., 2011). different entomopathogenic fungal species and isolates have varied secondary metabolites (sowjanya et al., 2008; mohammadi sharif et al., 2010; molnar et al., 2010) and different fungal isolates may induce immune reactions in different levels. which may affect their host immune system reactions. in conclusion, the immune reactions of insects showed considerable effect on susceptibility to fungal invasion. these reactions varied by host types and fungal isolates. according to our data, e. integriceps was the most sensitive insect with lowest immune responses against fungal infection .conversely, z. morio was resistant to all fungal isolates with high anti-fungal reactions. obviously, further research is needed to find the other reasons and dimensions of insect resistance or susceptibility to fungal diseases. 180 fig. 6 aggregation of hemocytes and entrap of labeled conidia (6 h after injection, 100x magnification) and melanization (24 h after injection, 40x magnification) acknowledgments this publication is part of the first author’s thesis funded by the urmia university. we thank the biological control laboratory, college of agriculture and natural resources, university of tehran, karaj for technical support. references altre ja, vandenberg jd. penetration of cuticle and proliferation in hemolymph by paecilomyces fumosoroseus isolates that differ in virulence against lepidopteran larvae. j invertebr. pathol. 78: 81-86, 2001. ajamhassani m, jalali sendi j, zibaee a, askary h, farsi mj. immunological responses of hypentria cunea (drury) (lepidoptera: arctidae) to entomopathogenic fungi, beauveria bassiana 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host and nonhost cuticles by the specific locust pathogen metarhizium anisopliae var. acridum. eukaryot cell 4: 937-947, 2005. zibaee a, bandani ar, talaei-hassanlouei r, malagoli d. cellular immune reactions of the sunn pest, eurygaster integriceps, to the entomopathogenic fungus, beauveria bassiana and its secondary metabolites. j. insect sci. 11: 1-16, 2011. zibaee a, malagoli d. immune response of chilo suppressalis walker (lepidoptera: crambidae) larvae to different entomopathogenic fungi. bull. entomol. res. 104: 155-163, 2014. http://www.medscape.com/viewpublication/6659 http://www.medscape.com/viewpublication/6659 the effect of entomopathogenic fungus metarhizium robertsii on the generation of reactive oxygen species in galleria mellonella l 276 isj 15: 276-284, 2018 issn 1824-307x research report the effect of entomopathogenic fungi metarhizium robertsii of different virulence on the generation of reactive oxygen species in galleria mellonella larvae yl vorontsova1,*, ia slepneva2, aa alekseev2, vy kryukov1, mv tyurin1, vv glupov1 1institute of systematics and ecology of animals, siberian branch of russian academy of sciences, frunze str., 11, novosibirsk, 630091, russia 2voevodsky institute of chemical kinetics and combustion, siberian branch of russian academy of sciences, institutskaya str., 3, novosibirsk, 630090, russia accepted august 13, 2018 abstract the toxicity of reactive oxygen species (ros) plays a significant role in the immune response of insects. little is known about the effect of the virulence of entomopathogenic fungi on the generation of ros in a host. the aim of the study was to reveal whether the different levels of virulence cause the different ros production in insects. the half-lethal dosages of two metarhizium robertsii strains, of high and low virulence, which have shown a similar survival to galleria mellonella larvae after treatment, were used in the study. the rates of ros generation were determined in the cuticle, hemocytes and cell-free hemolymph of g. mellonella larvae 1, 3, 5 days after fungi treatment. we have shown that the level of ros production in the cuticle and hemolymph of g. mellonella larvae depends on the virulence of the m. robertsii strains. the influence of both fungal strains on the rate of ros formation in hemocytes was the same throughout the observation period. the host’s defense mechanism was activated in the cuticle under the treatment of both low and high virulent fungi strains. in the hemolymph, the activation of the immune response occurred only after treatment with low virulent strain. key words: entomopathogenic fungus; galleria mellonella; reactive oxygen species; virulence introduction the immune system of insects comprises a variety of mechanisms and elements of defense against pathogenic microorganisms. an important role in insect immune response belongs to the prophenoloxidase (propo) cascade which participates in the process of melanization (ashida and brey 1997; gillespie et al., 2000; whitten and coates 2017). during this process, the quinoid active intermediates, including o-semiquinone radicals, are generated. these can be involved in cytotoxic reactions in the insect and/or can cause the production of the reactive oxygen species (ros) such as superoxide anion, hydrogen peroxide, and hydroxyl radical (nappi and vass 1993; carton et al., 2009). earlier, we have studied ros generation in the hemolymph of galleria mellonella larvae infected with microsporidia vairimorpha ephestiae (lozinskaya et al., 2004). it has been revealed that ___________________________________________________________________________ corresponding author: yana l. vorontsova institute of systematics and ecology of animals siberian branch of russian academy of sciences frunze str. 11, novosibirsk, 630091, russia e-mail: yavoronts@yandex.ru the generation of ros varies depending on the stage of microsporidiosis development in the insect organism. currently, there are works on the study of ros in gut and hemolymph immunity, which report the influence of bacteria and parasites on the activity of ros (whitten et al., 2001; bae et al., 2013). it is known that metarhizium fungi invade insects through a cuticle by producing the cuticledegrading enzymes and secondary metabolites (e.g. destruxins) in the infected insects (ríosmoreno et al., 2017; ríos-moreno et al., 2018). host specificity and the virulence level of the fungi are determined by the wide range of the enzymes and toxins produced (kershaw et al., 1999; amiribesheli et al., 2000; charnley 2003; wang et al., 2012; hu et al., 2014; sbariani et al., 2016). as a rule, the highly virulent metarhizium strains produce a large quantity of destruxins (kershaw et al., 1999; wang et al., 2012). the virulence level significantly influences the pathogeneses and the development of host cellular and humoral immune response, in particular, the encapsulation and phenoloxidase activity (tyurin et al., 2016; seyedtalebi et al., 2017). the main reaction in the antifungal defense mailto:yavoronts@yandex.ru 277 fig. 1 mortality dynamics of g. mellonella larvae after treatment with two m. robertsii strains in concentrations leading to 50% survival, and in equal concentrations mechanism against low virulent pathogens is the encapsulation of the fungus, which is rapidly melanized. in the case of highly virulent strains, the fungi overcome, perhaps, the encapsulation and continue to grow (götz 1986; hung et al., 1993; wang and st. leger 2006). it was shown that the melanization and the encapsulation have a negative correlation with the level of destruxins production by fungi (wang et al., 2012). it is assumed that the ros participate in the destruction of the encapsulated invader via its cytotoxicity (butt et al., 2016; dubovskiy et al., 2016). the production of ros in the insect organism under mycoses has been studied insufficiently. earlier, we have studied the influence of acute infection caused by metarhizium robertsii (formely m. anisopliae) on the ros production in the hemolymph of g. mellonella larvae. our results have demonstrated a decreased production of the dopa-derived quinones/semiquinones in the hemolymph of the insects infected (slepneva et al., 2003). until now, the effect of fungi strains with different virulence on the ros generation in insects remains poorly investigated. we hypothesized that the low and highly virulent fungi strains cause the difference in ros production in the insect organism. in the present work, we have shown that the level of ros generation mediated by melanization in the hemolymph and in the cuticle of g. mellonella larvae depends on the virulence of the m. robertsii strains. materials and methods insects the larvae of the siberian line of the greater wax moth g. mellonella (lepidoptera, pyralidae) were obtained from the long-established laboratory population. the insects were reared in glass containers (0.7 l) at 28 °c in the dark, and fed an artificial diet containing corn meal (22.5%), honey (12.5%), glycerol (12.5%), beeswax (12.5%), wheat flour (10%), milk powder (12.5%), yeast (5%) and water (12.5%). the larvae of the 5th instar were used in the experiments. fungi two fungi strains p-72 and mak-1 were used in the study. both cultures belong to one haplotype of species m. robertsii that were established using 5′ ef-1α gene sequence analysis (kryukov et al., 2017). culture р-72 was isolated in 1972 from the colorado potato beetle leptinotarsa decemlineata say (coleoptera: chrysomelidae) on the latvia territory (serebrov et al., 2007). culture mak-1 was isolated in 2001 from locust calliptamus italicus l. (orthoptera: acrididae) on the west siberia territory (kryukov et al., 2017). the strain mak-1 is characterized by low virulence towards different insects (orthoptera, coleoptera, lepidoptera, diptera) due to the slow mycelia growth, the prolonged germination on artificial media and on insect epicuticular extracts, and the low toxicity of cultural broth towards insects (no paralysis of g. mellonella larvae after injection of a 6 day old cultural broth into hemocoel) (kryukov et al., 2011; tyurin et al., 2016). on the contrary, the strain p-72 is characterized by the high virulence to orthoptera, coleoptera, lepidoptera and diptera, the fast growth on an artificial media and on the epicuticular extracts, and the high toxicity of cultural broth to insects (100% paralysis of g. mellonella) (kryukov et al., 2011; tyurin et al., 2016). the production of destruxin а in the chapeck-dox broth was 11.7 ± 1.3 μg/mg of mycelium dry weight for p-72 and 3.1 ± 0.6 μg/mg for mak-1 (esm fig. s1, table s1). 278 fig. 2 ros production in the cuticle homogenate of g. mellonella larvae during the development of fungal infection by two m. robertsii strains. * p ≤ 0.05 in comparison to control; ** p ≤ 0.05 in comparison to p-72 strain on the third day the conidia for infection were grown on sabouraud’s dextrose agar at 26 °c for 10 days, harvested by scraping from sporulating cultures, airdried at 25 °c for 1 week and stored at 4 °c. in our experiments conidia stored two weeks at that temperature prior to inoculate the insects. for infections, conidia were suspended in sterile 0.03% tween-20 and vortexed for 1 min. suspensions were diluted to final concentrations of 5×107 and 5×108 conidia/ml. conidia concentration was determined using a neubauer hemocytometer. bioassay the insects were inoculated by dipping in a spore suspension for 10 s, with control larvae being immersed in 0.03% aqueous tween-20 only. the control and infected insects were kept at 28 °c in 9 cm diameter petri dishes (10 larvae/dish) lined with moistened filter paper. mortality was registered every 24 h during 10 days. there were 30 larvae per treatment and the whole experiment was repeated 3 times. chemicals 3,4-dihydroxy-l-phenilalanine (dopa), diethylenetriaminepentaacetic acid (dtpa), ethylene-diaminetetraacetic acid (edta), glucose, potassium phosphate, and sodium chloride were purchased from sigma-aldrich (usa). cp-h (1hydroxy-3-carboxy-pyrrolidine) was synthesized and kindly provided by dr kirilyuk from the novosibirsk institute of organic chemistry. all solutions were prepared with bidistilled deionized water. hemolymph collection 10 µl of hemolymph of g. mellonella were collected in 40 µl of cooled (4 °c) pbs-d (50 mm k, na-phosphate buffer containing 50 µm dtpa and 150 mm nacl, ph 7.4) by cutting the third proleg with a needle and by drawing hemolymph into the tip of a pipette. the sample was centrifuged at 500 xg for 5 min at 4 °c to remove hemocytes. the supernatant (plasma) was used for determination of the rate of ros formation. hemocyte collection hemocyte collection from the hemolymph of g. mellonella larvae was carried out according to kryukova and co-authors (2011). briefly, the hemolymph (60 µl) from 3 larvae was collected in a cooled (4 °c) anticoagulant solution (62 mm nacl, 100 mm glucose, 10 mm edta, 30 mm sodium citrate, 26 mm citric acid, ph 4.6). the sample was centrifuged at 500 xg for 5 min at 4 °c to pellet hemocytes. precipitate was resuspended and washed three times in cool anticoagulant and once in pbs-d. the suspension of hemocytes was used for determination of the rate of ros formation. samples of cuticle fragment the whole cuticle fragment (central part of every larva) was dissected in pbs-d and cleaned from fat body tissues. subsequently washed 3 times for 1 min in pbs-d and then homogenized in 50 µl pbs-d for 2 min at 6.5 m/s with a fastprep®-24 homogenizer (mp biomedicals, usa). the homogenate was used to determine both the rate of ros formation and the protein concentration (accordingly bradford’ method) (bradford, 1976) in the cuticle. determination of the rate of ros formation the g. mellonella larvae were topically infected with fungus as described above, and 1, 3, 5 days after the treatment the rates of ros formation were determined in the freshly prepared plasma, hemocytes suspension and homogenized cuticle fragments. 279 fig. 3 ros production in the hemocytes suspension of g. mellonella larvae during the development of fungal infection by two m. robertsii strains. * p ≤ 0.05 in comparison to control the cp-h (1-hydroxy-3-carboxy-pyrrolidine) spin trap (dikalov et al., 1997; slepneva et al., 1999) was used to measure the rates of reactive intermediates formation mediated by melanization in all g. mellonella samples. cp-h is oxidized nonspecifically by the highly oxidizing metabolites which results in the formation of the cp stable nitroxyl radical. the time-dependent accumulation of the cp radical in the samples was studied by monitoring the amplitude of the lowfield component of the epr spectrum. cp-h was dissolved in oxygen-free (argonbubbled) pbs-d. dtpa was used to decrease the self-oxidation of cp-h, catalyzed by the traces of transition metal ions. the mixtures of tested samples with 1 mm cp-h and 0.2 mg/ml dopa (phenoloxidase substrate) were placed into glass capillaries for epr measurements. note that dopa alone does not contribute to the rate of cp-h oxidation when used at the experimental concentration. the esr measurements were performed at room temperature using an er 200-d src x-band esr spectrometer (bruker). the epr settings were the following: field center, 3484 g; field sweep, 50 g; time constant, 200 ms; microwave power, 20 mw; magnetic field modulation, 100 khz; modulation amplitude, 2 g. statistical analysis the data were analyzed using the software sigmaplot for windows, version 9.0 (systant software, inc.), sigmastat v. 3.1 and statistica 6.0. a log-rank test with the following holm-sidak adjustment was used for a survival analysis. the recorded ros rate data were checked for normal distribution using the shapiro-wilk w test. one-way anova followed by the tukey’s posttest was used to estimate the differences in response to infection. the total number of larvae was used to determine the ros production within each time interval and upon each treatment was n= 15 larvae per time point. all the data are presented as means ± se. results the influence of metarhizium strains on the production of ros in the experiment, we used the half-lethal dosages of two m. robertsii strains: 5×107 for p-72 and 5×108 for mak-1 which have showed the similar mortality upon treatment with cultures (fig. 1). the dynamics of wax moth mortality after the treatments were almost the same (log-rank test: χ2 = 0.3; p = 0.58) and led to the similar mortality rate (45-47%). the larvae mortality after the fungal treatment with both of the strains differed significantly from the control one (χ2 > 20.9; p < 0.00001). as follows from figure 2, the rate of ros formation in the cuticle increased significantly (p < 0.05) during 5 days of the experiment upon treatment by both pathogens in comparison with the control one. specifically, the treatment by the strain p-72 increased the rate of ros formation gradually during 5 days of the experiment (p < 0.05). the strain mak-1 showed the maximal effect on the rate of ros formation in the cuticle on the third day after the treatment (6-fold as compared to control (p < 0.05) and about 2-fold as compared to p-72 treatment (p < 0.05)), and on the fifth day, the rate decreased almost to the value obtained by p-72 treatment (fig. 2). the influence of both fungal strains on the rate of ros formation in hemocytes was the same throughout the observation period. particularly, on the first day of infection, we observed a decrease in the level of ros generation compared to the untreated control (p < 0.05). no differences were observed between the control and infected insects on the third day. five days after the treatment, the level of ros formation doubled in the hemocytes of the larvae infected with both the strain p-72 and the strain mak-1 (fig. 3). 280 fig. 4 ros production in the hemolymph of g. mellonella larvae during the development of fungal infection by two m. robertsii strains. * p ≤ 0.05 in comparison to control; ** p ≤ 0.05 in comparison to mak-1 strain on the first day in the plasma of g. mellonella, the strain of high virulence, p-72, has no effect on the rate of ros formation during the experiment. at the same time, the strain with a low level of virulence, mak-1, significantly (p < 0.05) increased the rate of ros formation in the plasma beginning with the first day after the treatment. the level of ros formation was significantly higher (p < 0.05) as compared with the control one during three days of the experiment. however, the tendency was observed to the lowering of the level of ros formation in the plasma of the insects infected by mak-1 over the 5 days of the experiment (fig. 4). thus, when the two fungi strains were used in dosages equal to lt50, causing the identical insect mortality, they made the different impact on the rate of ros formation in the cell-free hemolymph. the dosage-dependent effect of mak-1 strain on ros production in plasma to check whether the increase in ros is due to either the low virulence of mak-1 or its high concentration, we have conducted an additional experiment, involving both the equivalent dosages of mak-1 (5×107) and of p-72 (5×107) and the equivalent mortality (45-50% after treatment with 5×108 mak-1 and 5×107 p-72). the treatment with mak-1 at a concentration of 5×107 caused the lower mortality rate (22% for 10 days; fig. 1). in this experiment, we showed that the ros formation in the plasma was elevated upon infection with the dosages of strain mak-1 rather than of p-72 (fig. 5). the smaller concentration of mak-1 conidia (5x107) resulted in the strongly pronounced increase of ros formation 3 and 5 days after the treatment (2.6-fold as compared to control; p < 0.05). the higher concentration of mak-1 conidia (5x108) also increased the level of the ros formation rate 1 and 3 days after the infection but to a lesser extent (1.5fold as compared to control; p < 0.05). discussion in the present study, we provide the data on the ros production mediated by melanization during the mycosis of g. mellonella caused by the treatment of two m. robertsii strains of different virulence. the cuticle is the first and the most important barrier for entomopathogenic fungi. the interaction of fungi with the insect’s cuticle surface activates the melanization process which entails the ros production. the efficacy of the interaction depends on adhesion force, as well as on the quantity and nature of produced enzymes and toxins that could affect the production of ros and thus the pathogens’ ability to infect its host (st. leger et al., 1988; hajek and st. leger, 1994; butt et al., 2016; lovett and st. leger, 2017). in our experiments, both of the strains cause a significant activation of ros production in the cuticle of g. mellonella larvae. the result demonstrates a strong response of the host’s cuticle to the invasion of fungi strains of high and low virulence (high and low production of destruxins, respectively). one and three days after the treatment of insects with fungi we observed a more significant increase in ros production for the less virulent strain mak-1 in comparison to p-72. the cellular immune response of insects to fungi pathogens is mediated by host hemocytes. it is known that some fungal bioactive metabolites are the immune modulators and could suppress the host’s immune response (vey et al., 2002; pal et al., 2007). in our experiments, some fungi metabolites are likely to inactivate ros production in hemocytes during pathogen penetration and only on the fifth day is the cell defense activated. it was found that the less virulent mak-1 strain activates ros generation in the plasma of infected insects, while the more virulent strain p-72 does not affect it. it could be concluded that the process of 281 fig. 5 the dosage-dependent effect of m. robertsii strains on the ros production in the hemolymph of g. mellonella larvae. * p ≤ 0.05 in comparison to control melanization in the hemolymph is not activated when the highly virulent strain of the fungus is used. it is known that some fungi bioactive metabolites are involved in the suppression of propo activation (gillespie et al., 2000; pal et al., 2007). it was shown that the po activity was inhibited upon the treatment of insects with the fungi of high virulence and activated with the fungi of low virulence (cao et al., 2016). moreover, the destruxin deficit mutant of m. robertsii led to a stronger melanization of hemolymph as compared to the wild type (wang et al., 2012). we obtained similar results by comparing the influence of m. robertsii (p-72) and cordyceps militaris on ros production mediated by melanization in g. mellonella after injection of blastospores into hemocoel. the fungus m. robertsii did not change the rate of ros generation in the hemolymph relative to control. however, the less virulent c. militaris caused an increase in ros production (vorontsova, slepneva, kryukov, unpublished data). it is known that c. militaris does not produce destruxins, and has dramatically reduced the number of genes, encoding proteases, lipases and secondary metabolites, compared to m. robertsii (zheng et al., 2011). the data are in agreement with our results that indicate that ros production is activated after the treatment with low virulent fungi. however, an increase in the titer of conidia of low virulent fungi had almost no effect on the production of ros. perhaps, this is due to inhibition of the production of ros in the hemolymph by increased level of destruxins. this, however, can be due to the action of other metabolites of entomopathogenic fungi (enzymes, cell wall components, etc.). probably, these effects are mediated by epithelial cells, which are the first to "take a blow" of the pathogen. in response to the pathogen penetration, these cells synthesize the various mediators of intercellular interaction, determining the activity of the cells of the insect organism, specifically, the activity of enzyme systems responsible for the generation of ros. it is worth noting that there are other virulence factors of the fungus (besides destruxins) which could affect ros production. a particularly, important factor, which determines the encapsulation and melanization of fungi in the hemocoel, is the recognition by the host immune system caused by collagen-like proteins, which mask ß-glucans on hyphal bodies’ cells (wang and st. leger, 2006). it is possible that the different strains of metarhizium are recognized by the immune system and encapsulated to varying degrees. as demonstrated earlier, the strains of the fungi of low virulence are rapidly encapsulated in the insects, whereas the highly virulent strains of the fungi are not encapsulated, continuing their development in the host (götz, 1986; hung et al., 1993). the encapsulation is known to entail the melanization and ros generation (nappi and vass, 1993; nappi and ottaviani, 2000; carton et al., 2008). further studies could be aimed at the dependence of the ros production level on the pathogen recognition factors because ros are considered as one of the defensive antifungal reactions. thus, infection with a low virulent strain promotes the activation of the immune responses of insects, leading, probably, to the prolongation of the host's life, while the highly virulent strain suppresses the activation of its immunity. similar results were 282 obtained for the plant pathogen fungi botrytis. it was revealed that ros production increased after the treatment with the low virulent strains as compared with the highly virulent ones (urbanek et al., 1996; ungler et al., 2005). in addition, whitten and coauthors (whitten et al., 2001) have shown that ros response was inducible by trypanosoma rangeli hemolymph infection, and the magnitude varied with the parasite strain and stage of development. conclusions the obtained data indicate that the level of ros generation in the plasma and in the cuticle of the infected g. mellonella larvae depends on the virulence of the m. robertsii strains. the strongly pronounced difference between the strains was observed in the cell-free hemolymph. the strain of high virulence, p-72, has no effect on the ros production in the plasma of the infected larvae, whereas, that of low virulence, mak-1, causes the increase in ros generation, depending on the dosages of fungi. it means that the host defense mechanism is activated in the cuticle under the action of both the lowand the highly virulent strains. on the contrary, in the plasma, the immune response is activated depending on the virulence of fungal strain. acknowledgments this work was supported by the federal fundamental scientific research programme for 2013-2020 years (аааа-а16-116121410124-8), interdisciplinary project of siberian branch of russian academy of sciences (grant аааа-а17117091270110-0) and russian foundation for basic research (grant 17-34-50162). references amiri-besheli b, khambay b, cameron s, deadman ml, butt tm. interand intraspecific variation in destruxin production by insect pathogenic metarhizium spp., and its significance to pathogenesis. mycol. res. 104: 447-452, 2000. ashida m, brey pt. recent advances in research on the insect prophenoloxidase cascade. in: brey pt, hultmark d (eds), molecular mechanisms of immune responses in insects, chapman & hall, london, uk, pp. 135-172, 1997. bae ys, choi mk, lee w‑j. dual oxidase in mucosal immunity and host-microbe homeostasis. trends immunol. 31: 278-287, 2010. bradford m. a rapid and sensitive method for the 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zhang s, et al. genome sequence of the insect pathogenic fungus cordyceps militaris, a valued traditional chinese medicine. genome biol. 12: r116. 2011. https://www.ncbi.nlm.nih.gov/pubmed/?term=sturm%20s%5bauthor%5d&cauthor=true&cauthor_uid=15633742 https://www.ncbi.nlm.nih.gov/pubmed/?term=stuppner%20h%5bauthor%5d&cauthor=true&cauthor_uid=15633742 https://www.ncbi.nlm.nih.gov/pubmed/?term=butt%20tm%5bauthor%5d&cauthor=true&cauthor_uid=15633742 https://www.ncbi.nlm.nih.gov/pubmed/?term=strasser%20h%5bauthor%5d&cauthor=true&cauthor_uid=15633742 https://www.ncbi.nlm.nih.gov/pubmed/?term=strasser%20h%5bauthor%5d&cauthor=true&cauthor_uid=15633742 https://www.ncbi.nlm.nih.gov/pubmed/15633742 284 table s1 destruxin a content in cultural broths of p-72 and mak-1 strains of m. robertsii strain dtx a, mg/l cultural filtrate mean ± se, n= 3 dtx a, mg/g dry weight mean ± se, n= 3 p-72 34.1 ± 0.6 11.7 ± 1.3 mak-1 4.1 ± 1.1 3.1 ± 0.6 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 6 8 10 12 14 16 18 20 t i m e , m i n i n t e n s i t y , m a u t y , d t x e d t x a d t x b a 0.0 20.0 40.0 60.0 80.0 100.0 120.0 6 8 10 12 14 16 18 20 t i m e , m i n i n t e n s i t y , m a u a u u m d t x e d t x a d t x b b 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 6 8 10 12 14 16 18 20 t i m e , m i n i n t e n s i t y , m a u m a u d t x a c fig. s1. hplc-uv chromatograms of m. robertsii culture broth samples. a p72 strain; destruxin (dtx) a concentration determined from this sample was 33 mg/l. b mak-1 strain; dtx a concentration was 6 mg/l. c dtx a standard (10 mg/l). destruxin a quantification in cultural broth was performed according to method of seger et al. (2004) with some modifications. fungal biomasses were removed by centrifugation (20000 xg, 30 min), pellets were dried at 70 c to a constant weight. supernatants were filtered on a 0.22 µm membrane. aliquots of the resulting filtrates were diluted 1:1 with the acetonitrile for hplc-dad. agilent 1260 infinity system equipped with a c18 column (diaspher 110-c18, 2.1 × 150 mm, 5 µm particle size, 30 c column temperature, 0.4 ml/min flow) was used with uv absorbance at 210 nm. a mobile phase consisted of water and acetonitrile (acn). the gradient was: 0 min, 30% acn; 20 min, 50% acn; 21 min, 80% acn; 21-27 min, 80% acn; 28-40 min, equilibration at 30% acn. calibration curve for dtx a was obtained with six concentrations (0.125, 0.25, 0.5, 1, 10, 50 µg/ml) and was linear in the range (r2 = 0.999). despite the lack of the standards we identified dtx e and dtx b on the basis of uv-spectra and literature data on the sequence of elution of the peaks on c18 columns. 259 isj 14: 259-270, 2017 issn 1824-307x research report effect of cyclic serious/medium hypoxia stress on the survival, growth performance and resistance against vibrio parahemolyticus of white shrimp litopenaeus vannamei s-y han 1,2,3 , b-j wang 1,2 , m liu 1,2 , m-q wang 1,2 , k-y jiang 1,2 , c-c qi 1,2,3 , l wang 1,2 1 key laboratory of experimental marine biology, institute of oceanology, chinese academy of sciences, 7 nanhai road, qingdao 266071, china 2 laboratory for marine biology and biotechnology, qingdao national laboratory for marine science and technology, qingdao 266237, china 3 university of chinese academy of sciences, 19 yuquan road, beijing 100049, china accepted august 1, 2017 abstract the effect of cyclic serious/medium hypoxia stress in the presence of hypoxia environment on shrimp is not well explored. the survival, growth performance, osmoregulation gene expression, digestive enzyme activity, histology, and resistance against vibrio parahemolyticus of the white shrimp litopenaeus vannamei reared under conditions of cyclic serious/medium hypoxia (csmh, 0.8 3.5 mg/l) versus normoxia (n, 6.4 7.5 mg/l) were investigated during an experimental period of 28 days. consequently, the cumulative mortality rate of csmh shrimp increased continuously. the weight gain percentage and length gain percentage of csmh shrimp decreased continuously. the na + /k + -atpase, cytoplasmic carbonic anydrase (cac), and glycosyl-phosphatidylinositol-linked carbonic anhydrase (cag) transcripts in the gill of csmh shrimp increased first and then returned to normal or decreased. the amylase, lipase, and trypsin activities in the hepatopancreas of csmh shrimp decreased continuously. the hepatopancreas of csmh shrimp showed histopathological lesions in a time-dependent manner. in the v. parahaemolyticus immersion challenge test, the mortality rate of csmh shrimp increased continuously. therefore, cyclic serious/medium hypoxia could reduce survival and growth performance of l. vannamei during long-term exposure, which was resulted from broken osmoregulation mechanism of the gill, and suppressed digestive enzyme activities of the hepatopancreas caused by growing histopathological lesions . meanwhile, cyclic serious/medium hypoxia would probably lead to outbreak of infectious diseases in the shrimp farming. key words: litopenaeus vannamei; cyclic serious/medium hypoxia; mortality; growth performance; histology; vibrio parahaemolyticus introduction hypoxia has been rising in marine ecosystems since the 1960s (diaz and rosenberg, 2008). biological and non-biological factors, such as photosynthesis, respiration, eutrophication, tidal cycles, weather conditions, global warming, stratification of the water column, and so on, often cause the occurrence of chronic hypoxia and cyclic hypoxia/normoxia in coastal and estuarine environments (rabalais et al., 2002, 2007; chen et al., 2007; stramma et al., 2008; mcallen et al., 2009; ___________________________________________________________________________ corresponding author: lei wang key laboratory of experimental marine biology institute of oceanology chinese academy of sciences 7 nanhai road, qingdao 266071, china e-mail: wanglei@qdio.ac.cn tyler et al., 2009). meanwhile, the bottom layer of pond waters, where shrimp spend most of their time, may become hypoxic or even anoxic, due to respiration of the organisms and decomposition of accumulated organic matter such as unconsumed feed and feces, especially at night (cheng et al., 2003). this occurs particularly in rearing ponds that do not use aerators, in which dissolved oxygen (do) concentrations may reach critical values, and shrimp can be exposed to hypoxia as do levels drop from 3 mg/l to less than 1 mg/l (chantal et al., 2008). most aquatic species can maintain an adequate oxygen uptake at do above 5 mg/l (gray et al., 2002; vaquersunyer and duarte, 2008; diaz and breitburg, 2009). below their specific optimum do level, aquatic species display physiological and behavioral adaptations to maintain satisfactory oxygen uptake rates (morris and taylor, 1985; 259 fig. 1 do levels in the seawater of tanks during 0-7 d (a), 8-14 d (b), 15-21 d (c), and 22-28 d (d). wannamaker and rice, 2000; bernatis et al., 2007). specific adaptations vary depending on the severity and duration of hypoxia. previous studies have investigated the observable effects of: acute hypoxia on survival, carbohydrate metabolism, immune response, apoptotic processes, comparative proteomics, and resistance against viruses (cota-ruiz, et al., 2015; martínez-quintana et al., 2016; wei et al., 2016; felix-portillo et al., 2016; cheng et al., 2002; sun et al., 2016; lehmann et al., 2016); chronic hypoxia on growth, reproductive expression, aerobic and anaerobic metabolism, antioxidant response, gene expression profile, and mitochondrial expression (coiro et al., 2000; brouwer et al., 2007, 2008; li and brouwer, 2009; dupont-prinet et al., 2013; pillet et al., 2016); cyclic hypoxia/normoxia on gene expression profile, growth, and reproductive expression (coiro et al., 2000; brown-peterson et al., 2011; li and brouwer, 2013); and hypoxia and reoxygenation on dna damage and oxidative and antioxidant states of shrimp (parrilla-taylor and zenteno-savín, 2011; li et al., 2016; garcía-triana et al., 2016). however, we know little about the effects of cyclic serious/medium hypoxia that may occur as part of a subsistence process in shrimp in an aquaculture system. the white shrimp, litopenaeus vannamei can suffer fluctuations of oxygen levels and experience hypoxia (parrilla-taylor and zenteno-savín, 2011), as they can convert aerobic into anaerobic metabolism to produce energy under these circumstances (soñanez-organis et al., 2012). in the present study, we would evaluate the survival, growth performance, and disease outbreak's possibility of l. vannamei under cyclic serious/medium hypoxia and analyze related mechanism in an aquaculture system. the osmoregulatory capacity measurement was confirmed as a convenient tool to monitor the physiological condition and the effect of stress in crustaceans (charmantier and soyez, 1994), and digestive enzyme analysis is an excellent tool for the physiologists as the animal grows and matures (lee et al., 1984). therefore, we investigated: (1) mortality and growth performance; (2) na + /k + -atpase, cytoplasmic carbonic anhydrase (cac), and glycosyl-phosphatidylinositol-linked carbonic anhydrase (cag) gene expression of the gill; (3) trypsin, amylase, and lipase activity of the hepatopancreas; (4) histology of the hepatopancreas; (5) resistance against vibrio parahemolyticus in l. vannamei reared under conditions of normoxia and cyclic serious/medium hypoxia during a 28 d experiment. a) c) b) d) 260 259 table 1 primers for the genes encoding na+/k+-atpase, cac, cag, and β-actin in shrimp gene nucleotide sequence (5ʹ→3ʹ) amplicon na + /k + -atpase cac cag β-actin fgtatccatccacgagactgag raaggtaggcattgttgaaagc fcccgtgcgacagtaacctaa rggctcctcgaagacaatcca facgagcaatgtgga rgtggaactgagcgaagatgt fgcccatctacgagggata r-ggtggtcgtgaaggtgtaa 135 bp 147 bp 126 bp 121 bp materials and methods experimental shrimp nine hundred healthy juvenile litopenaeus vannamei that were of similar size (mean weight 1.20 ± 0.03 g) were obtained from the ruizi seafood development co. ltd. (qingdao, china), where the experiment was also conducted. only shrimp in the intermoult stage were used for this study. they were placed in six 640-l cylindrical tanks with net cover (n = 150 per tank), and each 640-l cylindrical tank contained 500 l of aerated seawater (do 6.4 7.5 mg/l). the initial seawater was unfiltered and had the following characteristics: ph 8.0 8.4, salinity 30 31 ‰, total ammonia 0.022 0.038 mg/l, nitrite 0.015 0.032 mg/l, and nitrate 0.120 0.205 mg/l at 28 32 c. the shrimp were acclimated for 3 weeks under a ‘photoperiod’ (12 h light:12 h dark). they were fed three times daily with a commercial diet (41.52 % crude protein, 7.42 % lipid, and 12.03 % crude ash, supplied by yantai dale feed co. ltd, shandong, china) at 07:00 am, 11:00 am, and 7:00 pm, with a daily feeding rate that was 10 % of the weight of the shrimp. unconsumed feed and feces were removed with a siphon tube and 50 % of the seawater was replaced once daily. unfiltered seawater was prepared in two 1000-l cylindrical tanks to use for daily exchange. preparation of vibrio parahaemolyticus the pathogenic strain v. parahaemolyticus atcc 17802 was donated by the first institute of oceanography, state oceanic administration, people’s republic of china. the strain was cultured in tryptic soy broth (supplemented with 2 % nacl, difco) for 24 h at 28 c, and then centrifuged at 7155g for 20 min at 4 c (yeh and chen, 2009). the supernatant fluid was removed and the bacterial pellet was re-suspended in saline solution at 1×10 8 colony forming units (cfu) ml −1 as the stock bacterial suspension for the resistance test. experimental design for the do challenge following acclimation, we randomly divided the six 640-l cylindrical tanks into two groups. each group of three 640-l cylindrical tanks constituted three repetitions of either (1) normoxia (n) or (2) cyclic serious/medium hypoxia (csmh). the experiments were conducted over 28 d, and the photoperiod, feeding conditions, water exchange, and waste disposal were handled in exactly the same way as during the acclimation period. during each day, the n group was aerated enough to generate a do level of 6.4 7.5 mg/l automatically; however, the csmh group was aerated sufficiently to generate a do level of only 0.8 3.5 mg/l automatically, and was also characterized by having the lowest do in the early morning and the highest do in the afternoon with exposure to a do of 0.8 2 mg/l for 16 h and 2 3.5 mg/l for 8 h during every 24-h cycle (figs 1a-d). the do levels were monitored four times a day using a ysi model 55 do meter (ysi incorporated, ohio, usa) during the experimental period. measurement of mortality and growth performance and sampling procedure the number of dead shrimp in each tank was recorded every 24 h during the experimental period. shrimp were considered dead when they failed to move even when gently stimulated with a glass pipette. dead shrimp were removed to prevent fouling. twenty shrimp from each tank were randomly selected before the experiment (day 0) and on days 7, 14, 28 during the experimental period and replaced after their weights and lengths were measured. mortality and growth performance was evaluated in terms of their cumulative mortality rate (cmr), weight gain percentage (wgp), and length gain percentage (lgp) based on the following standard formulae: cmr (%) = 100 × (cumulative dead shrimp number)/(initial shrimp number); wgp (%) = 100 × (final weight-initial weight)/initial weight; lgp (%) = 100 × (final length-initial length)/initial length. three shrimp were randomly selected and removed from each tank before the experiment (day 0) and on days 7, 14, 28 during the experimental 261 259 period, and the hepatopancreas and gill of each shrimp were collected using sterilized scissors and forceps. three hepatopancreases were immediately ground in liquid nitrogen and stored at -80 °c prior to the digestive enzyme activity assay; three gills were immediately placed into rnalater (applied biosystems, austin, tx, usa) and stored at -20 °c until rna isolation and osmoregulation gene expression analysis. another three shrimp were randomly selected and removed from each tank before the experiment (day 0) and on days 1, 3, 7, 14, 28 during the experimental period; the hepatopancreas of each shrimp were also collected using sterilized scissors and forceps, then immediately fixed in 10 % formaldehyde to be used for histological study. assays of osmoregulation gene expression in the gill rna was extracted from the gill using an rna fast extraction kit according to the manufacturer’s protocol (fastagen, shanghai, china). rna solution (6 μl) was mixed with homogenates of the three gills from each tank (2 μl rna solution per gill). this 6-μl rna solution was employed for cdna synthesis using a transscript® one-step gdna removal and cdna synthesis kit according to the manufacturer’s protocol (transgen biotech co., ltd, beijing, china). the relative expressions of osmoregulation genes encoding na + /k + -atpase, cac, and cag were evaluated by relative quantitative real-time pcr (qpcr) using transstar top green qpcr supermix according to the manufacturer’s protocol (transgen biotech co., ltd). each of the aforementioned osmoregulation genes were analyzed with three replicates of each sample. β-actin was selected as a reference gene and specific primers were used for each gene (table 1). qpcr was performed with the following two steps: denaturation at 94 °c for 30 s and then 40 cycles of 94 °c for 5 s and 60 °c for 30 s. the melting curve analysis was performed at the end of qpcr to confirm the specificity of the pcr products, and relative expressions were determined using the 2 −δδct method (livak and schmittgen, 2001). assays of digestive enzyme activity in the hepatopancreas approximately 100 mg of hepatopancreas tissue was dissected from a single hepatopancreas. the three hepatopancreas tissues from each tank were mixed at a 1:5 ratio (w/v) with chilled tris-hydrochloric acid buffer solution (ph 7.6, 10 mmol l −1 ) and homogenized under ice-chilled conditions. the homogenates were then centrifuged at 10,000g, 4 °c for 30 min and the supernatants were collected for the assays. digestive enzyme activities, including trypsin activity, amylase activity, and lipase activity, were evaluated using commercial kits (nanjing jiancheng bioengineering institute, nanjing, china) according to the manufacturer's instructions. the activities of each of the above digestive enzymes were analyzed with three replicates of each sample. activities were expressed as a relative unit per milligram of soluble protein (u/mg protein). fig. 2 cumulative mortality rates of shrimp during the experimental period. each bar represents the mean value from three repetitions with standard error (se). *indicates a significant (p <0.05) difference compared with the n group. histology assays on the hepatopancreas the fixed hepatopancreas tissues were dehydrated using ascending concentrations of alcohol, cleared in toluene, embedded in paraffin, and sectioned with a rotary microtome at 5 μm. sectioned tissues were stained with hematoxylin and eosin (h&e), and examined with a light microscope (casado et al., 2001). resistance of shrimp to vibrio parahaemolyticus shrimp from the two groups were evaluated before the experiment (day 0) and on days 7, 14, 28 during the experimental period. at each evaluating point, there were four treatment groups (two challenged and two unchallenged treatment groups). 20 shrimp and 20 l of seawater from each 640-l cylindrical tank were transferred to two 40-l cylindrical tanks, 10 shrimp and 10 l of seawater per 40-l cylindrical tank. challenge testing was conducted by adding 100 ml of a stock bacterial suspension to the 20 l of seawater, resulting in immersion of the shrimp at 5×10 5 cfu/ml, while unchallenged testing required no further processing. other culture conditions during these treatments were handled in exactly the same way as the 640-l cylindrical tanks, except that no seawater was exchanged. dead shrimp were recorded for each treatment after 2 days. the mortality rate (mr) was expressed as: mr (%) = 100 × (dead shrimp number)/(initial shrimp number). statistical analysis the data are all presented as mean ± standard error (se). statistical analysis was performed using spss (version 17.0) (ibm, armonk, ny, usa), and the t-test was used to analyze differences between the two experimental groups. the significance level was p < 0.05. all images were generated with origin 8.6 software (originlab, ma, usa). 262 259 fig. 3 weight gain percentages (a) and length gain percentages (b) of shrimp during the experimental period. see fig. 2 for statistical information. results mortality of shrimp under cyclic serious/medium hypoxia compared with n shrimp, the cmr of csmh was not significantly (p>0.05) different on days 1, 3, but significantly (p < 0.05) higher on days 7, 14, 28. specifically, the cmr of csmh shrimp showed an increasing trend with time until 36.5 % on days 28 (fig. 2). growth performance of shrimp under cyclic serious/medium hypoxia the wgr and lgr of csmh versus n shrimp significantly (p < 0.05) decreased during the periods 0-7d, 7-14d, 14-28d, and 0-28d (fig. 3). osmoregulation gene expression of shrimp under cyclic serious/medium hypoxia the na + /k + -atpase and cag transcripts of csmh versus n shrimp significantly (p < 0.05) increased on days 7, then significantly (p < 0.05) decreased on days 14, 28 (figs 4a, c); the cac transcripts of csmh versus n shrimp significantly (p < 0.05) increased on days 7, then returned to normal on days 14, 28 (fig. 4b). digestive enzyme activity of shrimp under cyclic serious/medium hypoxia the amylase and lipase activities of csmh shrimp were not significantly (p >0.05) different from n shrimp on days 7, 14; and 7, respectively (figs 5a, b); but the amylase, lipase, and trypsin activities of csmh versus a) b) 263 259 fig. 4 na + /k + -atpase transcription (a), cac transcription (b), and cag transcription (c) of shrimp during the experimental period. see fig. 2 for statistical information. n shrimp significantly (p < 0.05) decreased on days 28; 14, 28; and 7, 14, 28, respectively (figs 5a-c). histology of shrimp under cyclic serious/medium hypoxia in the hepatopancreases of csmh versus n shrimp, stellate tubule lumen appeared dilatation on day 1, some vacuoles appeared on days 3, full-scale vacuoles generated on days 7, these dispersive vacuoles gathered into big vacuoles on days 14, and ruptured to make epithelial cell layer thinner on days 28 (figs 6a, b). resistance against vibrio parahaemolyticus of shrimp under cyclic serious/medium hypoxia all unchallenged shrimp that had been reared at different do levels survived. in contrast, deaths occurred among challenged shrimp. the mr of csmh versus n shrimp significantly (p < 0.05) increased on days 7, 14, 28 (fig. 7). discussion the course of hypoxia is known to influence the mortality and behavior of crustaceans (llansó, 1991; alexandra et al., 2010). many researchers have studied the effects of hypoxia on the survival and growth of shrimp. avault (1986) reported that macrobrachium rosenbergii were stressed when do fell below to 2 mg/l, and 0.5 mg/l is normally lethal. moreover, the survival of penaeid shrimp including penaeus setiferus, farfantepenaeus californiensis, vannamei, and m. rosenbergii were not greatly affected by chronic hypoxia (1.5 mg/l< do < 3 mg/l) (rosas et al., 1998; ocampo et al., 2000; racotta et al., 2002; cheng et al., 2003). however, li and brouwer (2013) pointed out that the weights and lengths of palaemonetes pugio exposed to cyclic hypoxia/normoxia (1.05 8.87 mg/l do) were significantly lower than for shrimp (6.34 mg/l do) in their natural habitats. coiro et al. (2000) found that a) b) c) 264 259 fig. 5 amylase activities (a), lipase activities (b), and trypsin activities (c) of shrimp during the experimental period. see fig. 2 for statistical information. chronic hypoxia (2 mg/l do) exposure resulted in more serious growth impairment of larval palaemonetes vulgaris than cyclic hypoxia/normoxia (2 8 mg/l do) in their natural habitats. in the present study, the cmr of csmh versus n shrimp increased continuously, and the wgp and lgp of csmh versus n shrimp decreased continuously. these results were consistent with previous hypoxia reports on growth, but different from those on survival. it is well known that growth of an organism is often the sublethal endpoint used and has been shown to be a more sensitive indicator of low do than survival (morrison, 1971; das and stickle, 1993; thursby et al., 1997), all current studies also proved that any form of hypoxia could cause growth impairment no matter whether they affected survival or not. as the ecological effects of hypoxia on the biota depend partly on their severity, duration and frequency and partly on the tolerance of the affected organisms to hypoxia (diaz and rosenberg, 1995; modig and ólafsson, 1998; sagasti et al., 2001), the incessant impairment of both survival and growth performance in the present study indicated that cyclic serious/medium hypoxia might be more severe than previous reported hypoxia forms. the hemolymph osmotic balance of invertebrates can be altered by environment changes (cameron and mangum, 1983; truchot, 1983). regulation of hemolymph osmotic pressure in crustacean mainly depends on inorganic ion and the concentrations of free amino acid, and the concentrations of inorganic ion are the most important contributors (pan et al., 2007). ion-regulation in crustacean is mostly accomplished by na + /k + -atpase, ca and other ion-transport enzymes in gill epithelium membrane (morris, 2001). it has been reported that short-term (≤3d) ammonia exposure of penaeus chinensis, macrobrachium nipponense, portunus pelagicus, and macrobrachium amazonicum increased na + /k + -atpase activity (lin et al.,1993; wang et al., 2003; romano and zeng, 2010; pinto et al., 2016 ), thus maintaining cell function and body fluid ammonia levels within a tolerable range (weihrauch et al., 2004), while down-regulated of na + /k + -atpase transcription of metacarcinus magister exposed to ammonia after 14 d was not a protective measure taken by the gill epithelium to prevent unwanted ammonia influxes, but may rather occur due to a toxic a) b) c) 265 259 fig. 6 photomicrographs of the hepatopancreases of n shrimp (a) and csmh shrimp (b). h&e stain (×400), scale bar = 100 μm. thick arrow indicates dilatated stellate tubule lumen; thin arrow indicates vacuole. effect of ammonia (martin et al., 2011). pan et al. (2016) also found that cac and cag transcription increased under short-term (2 d) low and high ph conditions in portunus trituberculatus, to maintain the acid-base balance, while wang et al. (2002) pointed out that a decrease in na + /k + -atpase activity resulted from impairment of the active transport mechanism for sodium ions was the primary cause of the deaths of f. chinensis after 14 d in acid and alkaline water. henry et al. (2006) indicated that ca transcription in carcinus maenas under short-term (4d) low salinity conditions remained elevated as an adaptation mechanism, and began to decline by 7 d because of a breakdown in the mechanism of transport-related protein induction, resulting in more difficult low salinity adaptation. in the present study, the na + /k + -atpase, cac, and cag transcripts of csmh versus n shrimp increased in the short term (7 d) , then returned to normal or decreased afterwards, which were similar to previous reports related to ammonia, ph, salinity stress. therefore, cyclic serious/medium hypoxia might break osmoregulation mechanism in shrimp after long-term exposure, leading to incessant death. some investigations have been available on the effects of do changes on the digestive enzymes in crustaceans, as they showed acclimation to low oxygen environment. for instance, brown-peterson et al. (2008) and li and brouwer (2013) found the transcriptional expression of papain-like cysteine proteinase in the hepatopancreas of palaemonetes pugio were significantly up-regulated after exposure to cyclic hypoxia/normoxia, both in the natural habitat and 7 d in the laboratory. these conclusions suggested increased amino acids by protein degradation, which may maintain blood glucose levels as energy source through the gluconeogenic metabolic pathway from non-carbohydrate carbon substrates (li and brouwer, 2009, 2013; brown-peterson et al., 2011). zeis et al. (2009) also considered that high-expressed glycolytic and proteolytic enzymes in the daphnia pulex were remained as a process to improve carbohydrate provision for the maintenance of atp production under hypoxia in their natural habitats. paschke et al. (2010) found that total proteases, trypsin and chymotrypsin activity in the hepatopancreas of lithodes santolla were not affected after 10 d chronic hypoxia. in the present study, amylase, lipase, and trypsin activities of csmh versus n shrimp decreased continuously, especially the most sensitive trypsin. obviously, cyclic serious/medium hypoxia as more severe hypoxia form had worse effect on digestive enzyme than previous hypoxia reports. many studies have indicated that low do levels reduce food consumption by crustaceans due to weakened digestive process (rosas et al., 1998; mcgaw, 2005). moreover, crustaceans showed a preference for proteins as a metabolic substrate under hypoxia, due to an increase of free amino acids in the hemolymph, which have the double function of helping to maintain osmotic pressure and providing metabolic energy as a consequence of anaerobic mechanism (taylor and spicer, 1987; hagerman and szaniawska, 1994; rosas et al., 1999). all things considered, sustained low trypsin activity in the present study was likely to decrease metabolic energy supply and disrupt osmotic balance, resulting in reduced survival and growth performance of shrimp. histological analysis of the hepatopancreas has been used as a practical means for assessing environmental stress in shrimp culture (saravana and geraldine, 2000; li et al., 2007; kuhn et al., 2010; qiu et al., 2016). limited study has described the effects of do changes on hepatopancreas histology in shrimp. sun et al. (2015) pointed out that the hepatopancreas of m. nipponense exposed to chronic hypoxia were structurally altered after 7 d, which could affect the vital physiological functions of prawns. similarly, we found the histopathological lesions in the hepatopancreas of csmh shrimp in a time-dependent manner. thus, the growing lesions in the hepatopancreas of shrimp might induce the problems related to digestive enzyme in our mentioned above study (rosas et al., 1995; franceschini-vicentini et al., 2009). the resistance of shrimp against pathogens is also affected by environmental do changes. moullac et al. (1998) found that hypoxia reduced the 266 http://apps.webofknowledge.com/oneclicksearch.do?product=ua&search_mode=oneclicksearch&sid=y14nmk1rgdrhcvf8nov&field=au&value=henry,%20rp&ut=6472427&pos=%7b2%7d&excludeeventconfig=excludeiffromfullrecpage&cacheurlfromrightclick=no 259 fig. 7 mortality rates of shrimp after + v. parahaemolyticus challenge during the experimental period. see fig. 2 for statistical information. resistance to vibrio alginolyticus injection in penaeus stylirostris over 24 h in consistent with variations of immunological parameters. mikulski et al. (2000) pointed out that hypercapnic hypoxia reduced the resistance to v. parahaemolyticus injection in l. vannamei and p. pugio over 48 h due to decreased parameter of the immune response. cheng et al. (2002) indicated that hypoxia reduced the resistance to enterococcus injection in m. rosenbergii over 96 h by reducing immune ability. brouwer et al. (2007) suggested that pmav, a novel gene involved in virus resistance, and immunity-related genes were down-regulated in p. pugio after 14 d chronic hypoxia. similarly, csmh shrimp exhibited increased mr with v. parahaemolyticus immersion continuously. in contrast, li and brouwer (2013) considered that both pmav and immunity-related genes were up-regulated in p. pugio in response to 10 d cyclic hypoxia/normoxia. therefore, effect of hypoxia on resistance against pathogens of shrimp might mainly depend on their severity and frequency, and cyclic serious/medium hypoxia as severe hypoxia form might also decrease resistance against pathogens due to reduced immunity in the shrimp. in sum, cyclic serious/medium hypoxia as variation in the abiotic environment increased biological vulnerability to pathogens, which probably would lead to outbreaks of infectious diseases in the shrimp farming (bachère, 2000; lightner et al., 2005). conclusions cyclic serious/medium hypoxia induced incessant impairment of survival and growth performance of l. vannamei during long-term exposure, which might be resulted from broken osmoregulation mechanism of the gill, and suppressed digestive enzyme activities of the hepatopancreas caused by growing histopathological lesions. meanwhile, the decreased resistance against v. parahemolyticus probably would lead to outbreak of infectious diseases in the shrimp farming under cyclic serious/medium hypoxia. acknowledgments this research was supported by the major science and technology projects of shandong province (2015zdzx05002) and sts key deployment project of chinese academy of sciences (kfzd-sw-106). references alexandra h, michael s, martin z, bettina r. behaviour and mortality of benthic crustaceans in response to experimentally induced hypoxia and anoxia in situ. mar. ecol. prog. ser. 414: 195-208, 2010. avault jw. seven years of pond research with the prawn (macrobrachium rosenbergii) in louisiana. aquacult. mag. 12: 51-55, 1986. bachère 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borealis and cancer pagurus d parrinello1, ma sanfratello1#, m celi2#, m vazzana1 1 dipartimento di scienze e tecnologie biologiche chimiche e farmaceutiche, sezione di biologia animale e antropologia biologica, università degli studi di palermo, italy 2istituto per l’ambiente marino costiero u.o. di capo granitola consiglio nazionale delle ricerche, granitola, trapani, italy # equal contribution accepted july 23, 2015 abstract cancer pagurus and cancer borealis and are edible crabs produced by economically relevant aquaculture. in this study the hemocytes and some plasmatic parameters of cancer borealis and cancer pagurus were examined. the cell features of the hemocytes were observed using light and scanning electron microscopy (sem). granulocytes, semigranulocytes and hyalinocytes were mainly identified on the basis of size, presence/absence and quantity of the cytoplasmic granules and the nucleus-to-citoplasma (n/c) ratio. sem observations were useful for disclosing the surface features of these cells, and the same characteristics were found in both crab species. a smooth surface distinguishes elongated hyalinocytes and a rough texture the irregular surface of spherical/ovoid granular cells. total (thc) and differential hemocyte counts (dhc) were performed, and the differences between the two crab species were disclosed. also we were valuated ph and osmolarity values, agglutinating activity and different protein contents of the hemolymph. key words: crustacean; jonah crab; brown crab; hemocyte classification   introduction hemocytes circulating in hemolymph play a key role in the innate immune response of crustaceans, and are the first line of defense against internal pathogens such as viruses, bacteria and parasites (bauchau, 1981). hemocytes can lead to phagocytosis, encapsulation, and the lysis of foreign cells (smith and söderhäll, 1983; ratcliffe et al., 1985; söderhäll and smith, 1986; johansson and söderhäll, 1989; söderhäll and cerenius, 1992). they also play an essential role in melanization (johansson et al., 2000). in addition, they release the humoral factors (including agglutinins) that are involved in phagocytosis and cell communication, leading to the degranulation of hemocytes (hose et al., 1987; johansson, 1995; johansson et al., 2000). however, the classification and functional morphology of crustacean hemocytes is often ___________________________________________________________________________ corresponding author: mirella vazzana dipartimento di scienze e tecnologie biologiche chimiche e farmaceutiche-sez. di biologia animale e antropologia biologica università degli studi di palermo, via archirafi 18, 90123 palermo, italy e-mail: mirella.vazzana@unipa.it controversial due to the methods and criteria used. classification is generally based on the presence/absence of cytoplasmic granules, and three types of circulating hemocytes have usually been reported as hyalinocytes (cells without evident granules), semigranulocytes (containing small granules) and granulocytes (with abundant cytoplasmic granules) (bauchau, 1981). however, granulocytes have also been called granular eosinophils or granular amebocytes, while semigranulocytes have been regarded as monocytes or intermediate cells, and hyalinocytes as phagocytes or pro-hemocytes (toney, 1958; stang-voss, 1971; ravindranath 1974; bodammer 1978; cornick et al., 1978; smith and ratcliffe 1978). johnston et al. (1973) distinguished two hemocyte types (α-and β-cells) in carcinus maenas, and these can be recognized on the basis of the presence/absence of glycogen-containing granules (william and lutz, 1975). in callinectes sapidus, clare and lumb (1994) identified three hemocyte types, hyaline cells, and small and large granulocytes. in panulirus homarus, meanwhile, four types of hemocytes (pro-hyalocytes, hyalocytes, eosinophilic granulocytes and   195 chromophilic granulocytes) have been described (manjula et al., 1997). finally, 11 cell types that are morphologically distinguishable have been described in homarus americanus (battison et al., 2003). each cell type is active in defence reactions, with the hyaline cells chiefly involved in phagocytosis, the semigranular cells in encapsulation, and the granular cells in the storage and release of the prophenoloxidase (propo) system and cytotoxic factors (giulianini et al., 2007; celi et al., 2015). the proportion of hemocyte types in the hemolymph varies among species. total hemocyte counts (thc) and differential hemocytes counts (dhc) have been reported as stress indicators (jones, 1962; le moullac et al., 1998; lorenzon et al., 2008), and may be valuable tools for monitoring the health status of crustacean species (jussila et al., 1997; mix and sparks, 1980; battison et al., 2003). the crustacean decapods cancer pagurus and cancer borealis are paired as sister species (harrison and crespi 1999), and are edible crabs produced by economically relevant aquaculture (robichaud and frail, 2006; stentiford, 2008). the data reported in the literature on the ecology of the two species are limited and fragmentary. the two species live in intertidal and subtidal habitats and consume a wide variety of prey (lawton and elner, 1985; creswell and marsden, 1990). the jonah crab, cancer borealis occurs along the east coast of north america, while cancer pagurus is found in the northeast atlantic ocean, along the coast of europe (galan and eriksson 2009). jonah crabs are commercially harvested in the u.s. and canada. the crustacean c. pagurus, which is commonly known as either the european edible crab or the brown crab, is the most commercially important crab species in western europe. both species are fished offshore using baited pots or other traps. the aim of this study is to provide further information on the cells and plasmatic aspects of the species described above. in particular, the paper examines the morphological characterization of circulating hemocytes under light and scanning electron microscopy (sem), the basic profile of the thc and dhc, and some plasmatic properties of the hemolymph. materials and methods animals fifteen specimens of the jonah crab cancer borealis (catch area northwest atlantic) and the same number of cancer pagurus (catch area northeast atlantic), weighing 500 ± 100 g each, were supplied by l.p.a pesca srl (rimini). the specimens were held in running seawater tanks  at the premises of another local company, prontomar srl (palermo), until the time of sale. prior to collection of the hemolymph, the crabs were maintained in a circulating seawater aquarium at the university premises (10 °c cancer borealis and 15 °c cancer pagurus) for about 48 h according to the work of vogan and rowley (2002). hemolymph sampling according to the work of söderhäll and smith (1983), 15 crabs of both species were anesthetized on ice for 10 min and the hemolymph was then withdrawn from the unsclerotized membranes of the cheliped. since extremely rapid coagulation occurred, a syringe (23-gauge needle) with an equal volume of an anticoagulant (glucose 100 mm, nacl 450 mm, sodium citrate 30 mm, citric acid 26 mm, edta 10 mm, ph 4.6) was used to prevent clotting. consequently, plasma, separated by centrifuging (400g) at 4 °c for 10 min, was used to evaluate some of the plasmatic parameters. total and differential hemocyte counts and cytological staining the total hemocyte number per ml (thc) was determined using a neubauer hemocytometer chamber. differential counts were performed on slides prepared with 100 μl of a diluted cell suspension (3x106 cells). the hemocyte monolayer was fixed with 1 % glutaraldehyde in 3.2 % nacl for 30 min at 4 °c. the hemocytes were stained with a may-grünwald solution (3 min) followed by a giemsa solution (1:10 dilution for 10 min), and dehydrated with ethanol. after immersion in xylene (6 min), the slides were closed  with  a eukitt mounting medium (fluka) (celi et al., 2013). the cells were then counted in random areas, and the numbers and relative proportions of hemocyte types were calculated by counting at least 200 cells on each slide. the cells were observed under a leica dmre microscope, and the dhc was determined using the following equation: number of different hemocyte cell types dhc (%) = the cell sizes (length and width) and nuclear to cytoplasmic (n/c) ratios were calculated with the image j software, which is an image processing program. plasma ph, osmolarity and protein assessment the hemolymph’s ph was measured with a glass microelectrode and a ph meter (crison, italy).  osmolarity was estimated with a freezing-point depression osmometer (roebling, berlin, germany). the total protein concentration of the plasma was estimated using a qubit fluorometer (life technologies) for sensitive fluorescence-based quantization assays in accordance with the manufacturer’s instructions. scanning electron microscopy (sem) fresh hemolymph from c. borealis and c. pagurus was dropped directly on to coverslips pretreated with 0.1 % poly-l-lysine. after adhesion, the monolayer was fixed in a cacodylate buffer (0.1 m, ph 7.3) containing 2.5 % glutaraldehyde for 30 min at 4 °c. the cells, which were washed with a cacodylate buffer, were post-fixed with 1 % osmium total hemocyte cells counted x 100   196 table 1 morphometric measures and differential count of hemocyte types from cancer borealis and cancer pagurus hemolymph cell types cancer borealis cancer pagurus % (dhc) 47.6±2.2 30±1.4 cell length (μm) 19.3±1.66 19.4±2.2 cell width (μm) 4.8±0.9 3.3±0.6 hyaline n/c (%) 25±4 36±9 % (dhc) 45.2±3.3 60±6.2 cell length (μm) 9.7±0.46 11.4±1.1 cell width (μm) 8.7±0.6 5.5±1 semigranulocytes n/c (%) 23±4 26±5 % (dhc) 7.2±2.4 9.4±0.3 cell length (μm) 13.0±1.7 11.3±1.5 cell width (μm) 8.8±1.33 6.6±0.9 granulocytes n/c (%) 16±1 23±2 dhc: differential hemocyte count; n/c: nucleus/cytoplasm ratio. values are expressed as mean ± sd and range n = 15. tetroxide for 30 min at 4 °c, dehydrated in graded alcohol and dried at the critical point. the preparations were mounted on stubs, gold coated in a sputter coater and examined under a leo 420 sem. hemolymph from three distinct specimens was examined. hemagglutination assay the hemagglutinating activity (ha) of two folddiluted samples was assayed in a 96-well microtiter u-plate containing a suspension of 1 % rabbit red blood cells (rrbc) or sheep red blood cells (srbc) in phosphate buffered saline (pbs-e: 6 mmol/l kh2po4, 0.11 mmol/l na2hpo4, 30 mmol/l nacl, ph 7.4). erythrocytes were supplied by the “istituto zooprofilattico della sicilia” (palermo, italy) and maintained in a sterile alsever’s solution (27 mmol/l sodium citrate, 115 mmol/l d-glucose, 18 mmol/l edta and 336 mmol/l nacl in distilled water, ph 7.2). tris-buffered saline (tbs; see below) enriched with 1 % rrbc and srbc with 0.1 % (w/v) gelatin was used as the reaction medium. twenty-five microliters of plasma were serially diluted and mixed with an equal volume of erythrocyte suspension and incubated at 37 °c for 1 h. the titer of the hemagglutinating activity (ht) was expressed as the highest dilution showing a positive score for agglutination. plasma from several specimens was separately assayed, and tbs was used in place of the plasma as a negative control. each assay was performed in duplicate and the ha titer was expressed as the average of the recorded values. agglutination of yeast a suspension of 100 mg of yeast in 10 ml of physiological solution was washed twice with physiological saline and centrifuged at 400g for 5 min. the yeasts were inactivated by an autoclave, washed twice in saline and divided into aliquots of 1 ml. an aliquot was centrifuged at 400g for 5 min and 25 µl of pellet was resuspended in 1 ml of tbs gel. the plate was prepared following the procedure described above and incubated for 1 h at 37 °c. the titer of the ht was calculated as set out above.   197 statistical analysis all values were from five samples in triplicate. data were given as arithmetic means ± standard deviations. we used analysis of the t test for a comparison of the n/c ratio between species. results light microscopy observations the c. borealis and c. pagurus hemocyte monolayers showed flattened and well-spread cells, and three morphologically distinguishable cell types were found. may-grünwald-giemsa cytological staining allowed us to recognize: 1. hyalinocytes (c. borealis: 19.3 ± 1.66 μm in length; c. pagurus: 19.4 ± 2.2 μm in length) that had an elongated shape and missing cytoplasmic granules; 2. semigranulocytes (c. borealis: 9.7 ± 0.46 μm in length; c. pagurus: 11.4 ± 1.1 µm in length) with a few granules; and 3. granulocytes (c. borealis: 13.0 ± 1.7 μm in diameter; c. pagurus: 11.3 ± 1.5 µm in diameter) (table 1) with a great number of evident cytoplasmic granules. the hyalinocytes had a central nucleus, a high nucleus-cytoplasm ratio. in c. borealis n/c ratio was 25 ± 4 %, significantly (p< 0.05) lower than in c. pagurus with n/c ratio of 36 ± 9 % and basophilic cytoplasm (figs 1a, d, g, l). in both species, the semigranulocytes had a central or an eccentric nucleus (c. borealis 26 ± 5 %; c. pagurus 23 ± 4 % n/c) (figs 1b, e, h, j), whereas the granulocytes had an eccentric nucleus. in c. borealis n/c ratio was 23 ± 2 %, significantly (p< 0.01) lower than in c. pagurus 16 ± 1 %, (figs 1c, f, i, k). the granulocytes mainly contained eosinophilic granules (eosin staining), which were large and few in number in the c. borealis hemolymph, and small and numerous in the c. pagurus hemolymph (figs 1i, k). only a few basophilic granulations were observed in granulocytes from both crab species. the semigranulocytes of c. borealis contained small basophilic granules, whereas the semigranulocytes in the cytoplasm of c. pagurus contained both basophil and eosinophilic small granules. when examined by an sem, hemocytes from both crab species had the same morphological features and similar measures as observed with light microscopy (figs 1d, e, f, l, j, k). the elongated hyalinocytes had a smooth surface, whereas an irregular surface characterized ovoid or spherical semigranular cells, and a rough texture of the cell surface distinguished spherical granulocytes. thc and dhc the thc was 4.7 ± 0.4x106 cell/ml in c. borealis and 4.4 ± 0.6x107 cell/ml in c. pagurus. in the first species of crab, the hyalinocytes and semigranulocytes were numerous and similar in number (45.2 ± 3.3 % and 47.6 ± 2.2 % respectively). in contrast, in the second species, the hyalinocytes were present in a lower proportion (30 ± 1.4 %) and the semigranulocytes a higher proportion (60 ± 6.2 %). finally, the granulocyte proportions were similar in each species (7.2 ± 2.4 % and 9.4 ± 0.3 %, respectively). hemolymph parameters and hemagglutinating activity to check for plasmatic parameters, the ph, osmolarity and protein content of 15 specimens of each crab species were recorded by examining the plasma samples prepared in the presence of an anticoagulant (1:3 plasma/anticoagulant ratio). the ph values were 7.2 ± 0.08 in c. borealis and 7.4 ± 0.04 in c. pagurus. the osmolarity was 958 ± 28 mosm/kg (c. borealis) and 762 ± 19 mosm/kg (c. pagurus), while the protein content ranged from 27.3 ± 5.1 μg/µl (c. borealis) to 38.8 ± 6.2 μg/µl (c. pagurus). the plasma from the two species exerted in vitro agglutinating activity in the absence of calcium cations, as revealed with rabbit and sheep erythrocytes and yeast (s. cerevisiae). the c. borealis plasma agglutinated both erythrocyte types up to a serial dilution of 1:64, whereas lower titers were found by assaying the c. pagurus plasma with rabbit (titer: 1:4) and sheep (titer: 1:2) erythrocytes, respectively. the agglutinin titers of the plasma samples from both species, assayed with yeast, were 1:16 and 1:2, respectively. discussion it is known that live crabs are generally very delicate animals that should always be handled with great care. in fact, different research reports the negative effects of transport on the physiology of crustaceans (lorenzon et al., 2008; woll et al., 2010). in this study, before starting our observations, we determined that the animals showed no physiological changes after 48 h of acclimatization following a short journey (about 20 min) compared to samples taken directly at the commercial point. morphological characterization, cell type identification, differential numbering of hemolymph cell populations of reared crab hemocytes, some plasmatic parameters and agglutinating titers of the plasma hemolymph may contribute to examinations of the physiological role of reared crabs and the monitoring of their health (celi et al., 2013). the cell features of circulating hemocytes from c. borealis and c. pagurus were examined with may-grünwald-giemsa staining, and using light and sem observations. according to the morphological criterion proposed by bauchau and plaquet (1973), hose et al. (1990), and sung and sun (2002), granulocytes, semigranulocytes and hyalinocytes in the hemolymph from both crab species were identified on the basis of the presence/absence, quantity and cytochemical features of the granules, size and n/c ratios of the cells. a similar classification has been adopted for hemocytes from other crustacean species (jussila et al., 1997; gargioni and barracco, 1998; hammond and smith, 2002; yavuzcan and atar, 2002, matozzo and marin 2010). fusiform hyalinocytes with a basophilic cytoplasm and a central nucleus were smaller than granule-containing cells, which had a higher nucleus/cytoplasm ratio. the cytoplasm of the semigranulocytes contained a central or eccentric nucleus, whereas the granulocytes always had an   198 fig. 1 features of the hemocyte types from cancer borealis and cancer pagurus hemolymph. c. borealis: a c: maygrunwald-giemsa stain; d-f: sem observations. a, d: hyalinocytes, b, e: semigranulocytes, c, f: granulocytes. c. pagurus: g i: may-grünwald-giemsa stain; l k: sem observations. g, l: hyalinocytes, h, j: semigranulocytes, i, k: granulocytes. bars: 10 μm. the arrows indicate the eosinophilic granules (eg) and basophilic granules (bg). eccentric nucleus. the cytoplasm of the granulocytes was packed with large and round granules, whereas the semigranulocytes contained a lesser amount of smaller granules. in both cases, the may-grünwald-giemsa staining disclosed that a large part of the granules were eosinophilic and differed between the two crab species; they were few in number and large in the c. borealis granulocytes, and small and numerous in the c. pagurus granulocytes. finally, fine eosinophilic granules were also contained in the cytoplasm of c. pagurus semigranulocytes. sem observations were useful for disclosing the surface morphology of these cells from the hemolymph, which showed the same main features in both crab species. the elongated hyalinocytes had a smooth surface, the ovoid or spherical semigranular cells an irregular surface, and the spherical granular cells a cell surface with a rough texture. hemocytes play a central role in the immune defenses of crustaceans (söderhäll and cerenius, 1992; zhang et al., 2006); in fact, the total and differential hemocyte counts provide a useful way of assessing the physiological state of an animal (battison et al., 2003). unfortunately, although a wide range of values are available, differences in   199 the classification schemes used have prevented the comparison of hemocyte profiles among different crustaceans. (rodriguez and le moullac, 2000). the thc for c. pagurus reported in this study is in accordance with the findings of vogan and rowley (2002) and lorenzon et al. (2008), but no dates are reported in the research for c. borealis. the thc of c. borealis is lower than for c. pagurus, but is in accordance with the range presented for other crustaceans (celi et al., 2013; filiciotto et al., 2014). probably, this difference is due to the different ecology and geographic distribution of the two species. the dhc have provided varying results between crustacean species. hyalinocytes and semigranulocytes were more represented in the circulating hemolymph of c. borealis and c. pagurus. conversely, in m. rosenbergii, hyalinocytes comprised 70 % of the total hemocytes and no semigranulocytes were found (vázquez et al., 1997). in sicyonia ingentis, 50 60 % of the circulating hemocytes were hyalinocytes, whereas the semigranulocytes and granulocytes comprised 30 % and 10 % of the total, respectively (hose and martin, 1989). high percentages of hyalinocytes (five to eight times more abundant than granulocytes) have been observed in the crab eriocheir sinensis (bauchau and plaquet, 1973), the lobster panulirus interruptus (about 56 %) (hose et al., 1990) and the crayfish procambarus clarkii (more than 70 %) (lanz et al., 1993). conversely, in the lobster h. americanus and the crab loxorhynchus grandis, the semigranulocytes reached more than 60 % of the total cell numbers (hose et al., 1990). although the significance of this marked variability in the relative proportions of each hemocyte type among crustacean species remains unclear, it does appear to be useful when it comes to characterizing the hemocytes of every reared crab species. in addition, preliminary morphological observations of hemocytes under a light microscope and sem could be useful for further fine structural studies, and may contribute to determining the functional activity of the cell types that may be involved in immunity. furthermore, although the plasmatic parameters were checked by using 1:3 diluted plasma in the presence of an anticoagulant, they could be health indicators in reared crustaceans (chang et al., 2007). several authors have reported the ph values of c. pagurus, but these were not identical (lorenzon et al., 2008; woll et al., 2010; barrento et al., 2011). we found similar ph values for both species that are also in according to the same authors above mentioned. many studies on crustaceans describe the osmolarity values in stress conditions (charmantier et al., 1989; charmantier and soyez, 1994; lignot et al., 2000). in this study also, different values of osmolarity for the two species examined were reported, with c. borealis having higher values. conversely, the protein content was lower in the plasma of c. borealis than c. pagurus. although hemolymph proteins are another important physiological parameter in crustaceans, the literature reveals wide variations in hemolymph protein concentrations (depledge and bjerregaard, 1989; lorenzon et al., 2011). the agglutinins that play a role in protecting against bacterial infections are another important component of the hemolymph in terms of an immune defense role (sahoo et al., 2007). the agglutinins are synthesized and discharged in the hemolymph by hemocytes (rodríguez et al., 1995). the presence of agglutinins in the hemolymph of both examined crab species was revealed by using mammalian erythrocytes and yeast as targets. the results with respect to the two species were different, with more agglutinating activity being revealed for c. borealis. since plasma samples were prepared with a cation-chelating agent, the possibility exists that agglutinins may have properties of calcium-independent lectins (basilrose et al., 2014). in addition, due to the hemolymph sample dilution (1:3 with anticoagulant), the agglutinin titers may be higher than those reported here. finally, it is of interest to disclose the hematological and plasmatic parameters of reared crabs, because the dhc, plasmatic parameters and agglutinin titer could be influenced by the moult cycle, diet, harvesting, diseases and environmental contaminants (lorenzon et al., 2007; 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shellfish res. 29: 479-487, 2010. smith vj, ratcliffe na. host defense reactions of the shore crab, carcinus maenas, in vitro. j. mar. biol. assoc. uk 58: 367-379, 1978. yavuzcan yildiz h, atar hh. haemocyte classification and differential counts in the freshwater crab, potamon fluviatilis. turk j. vet. anim. sci. 26: 403-406, 2002. smith vj, söderhäll k. ß-1,3-glucan activation of crustacean hemocytes in vitro and in vivo. biol. bull. 164: 299-314, 1983.     202 http://www.ncbi.nlm.nih.gov/pubmed/?term=pillai%20br%5bauthor%5d&cauthor=true&cauthor_uid=17200030 http://www.ncbi.nlm.nih.gov/pubmed/?term=mohanty%20j%5bauthor%5d&cauthor=true&cauthor_uid=17200030 http://www.ncbi.nlm.nih.gov/pubmed/?term=kumari%20j%5bauthor%5d&cauthor=true&cauthor_uid=17200030 http://www.ncbi.nlm.nih.gov/pubmed/?term=mohanty%20s%5bauthor%5d&cauthor=true&cauthor_uid=17200030 http://www.ncbi.nlm.nih.gov/pubmed/?term=mohanty%20s%5bauthor%5d&cauthor=true&cauthor_uid=17200030 http://www.ncbi.nlm.nih.gov/pubmed/?term=mishra%20bk%5bauthor%5d&cauthor=true&cauthor_uid=17200030 matozzo v, gallo c, marin mg. can starvation influence cellular and biochemical parameters in the crab carcinus aestuarii? mar. environ. res. 71: 207-212, 2011. 152 isj 16: 152-163, 2019 issn 1824-307x research report combined effects of surface waters and ceo nanoparticle in zebra mussels j auclair1, c andré1, c peyrot2, k wilkinson2, p turcotte1, c gagnon1, f gagné1* 1aquatic contaminants research division, environment and climate change canada, montréal, qc, canada 2chemistry department, montréal university, montréal, qc, canada accepted august 27, 2019 abstract cerium oxide nanoparticles (nceo) are currently used in many sectors of our economy, for instance as fuel additives and in ceramics for catalytic converters. as a result, there are concerns about their release and resulting toxicity in the aquatic environment. the purpose of this study was to examine the bioavailability and toxicity of nceo and ce(iv) in zebra mussels (dreissena polymorpha) in various types of surface water differing in organic matter, conductivity and ph. mussels were exposed to 100 µg/l ce as either nceo or ce(iv) for 96 h in 4 types of water: 1) green water (high conductivity and low total organic carbon), 2) brown water (low conductivity and high natural total organic matter), 3) 10% municipal effluent (high conductivity and high anthropogenic organic matter) and 4) controls, which consisted of dechlorinated tap water. after the exposure period, the mussels were analyzed for morphological changes, resistance to survive in air, triglycerides (fat reserves), oxidative stress (arachidonate cyclooxygenase and lipid peroxidation) and dna damage. evidence of aggregation was observed with nceo in most types of water, with the exception of the diluted municipal effluent. the data revealed that some of the effects of nceo were influenced by surface water properties. the mussels were more sensitive to air emersion when exposed to nceo in green water but not in the other water types and ce(iv) to all types of water, although a marginal decrease was observed in mussels co-exposed to the diluted municipal effluent. a general decrease in oxidative stress and lipid levels was observed with both forms of ce and all water types. ce(iv) in brown water did not reduce the levels of dna strand breaks compared with the controls. in conclusion, the sublethal toxicity of nceo could be modulated by the surface water from which the nanoparticle is suspended. key words: cerium; genotoxicity; lipids; nanoparticles; oxidative stress; toxicity introduction inorganic nanoparticles have attracted commercial attention owing to their special properties. for example, cerium oxide nanoparticles (nceo) are widely used in the biomedical area as antioxidants in biological systems (brunner et al., 2006), in the textiles and color industry (dawson, 2008) and as an additive in diesel fuel and in catalytic converters (trovarelli et al., 1997; jemec et al., 2012). ce is the most abundant element in the rare earth family and is increasingly used by our economy owing to the high redox properties of ce(iii)/ce(iv). the many industrial applications of this element have given rise to some concerns about its release into the environment and the possible impacts on aquatic life. indeed, ce ___________________________________________________________________________ corresponding author: françois gagné aquatic contaminants research division environment and climate change canada 105 mcgill, montréal, qc, canada h2y 2e e-mail: francois.gagne@canada.ca pollution originates from solid waste (electronic device disposal) leachates, industrial (ceramic industry) waste, municipal sludge and wastewaters (limbach et al., 2008; keller and lazareva, 2014). the persistence of nceo and other nanoparticles in the aquatic environment, bioavailability and toxicity are an important part of the risk assessment of nanomaterials (gagné et al., 2007). ce nanoparticles have a positive charge at the surface with a zeta potential of 30 mv, making this particle susceptible to aggregation from surface charge neutralization in a low-salt environment usually found in freshwater. however, the occurrence of natural organic matter was shown to limit aggregation by capping the nanoparticle (grillo et al., 2015). humic acid, a major constituent of organic matter, was used to stabilize engineered nanoparticles from aggregation. according to a recent survey, 6% of the ce was released in municipal plant effluent and in the receiving waters (limbach et al., 2008), perhaps through stabilization 153 by the dissolved organic matter. the issue of the interaction of not only the organic matter content but other properties such as conductivity, ph and suspended matter could complicate the behavior of the nanoparticle and resulting toxicity in commonly found types of surface water. the aquatic toxicity of ce and nceo revealed that exposure to ce leads to detection in fish liver and that it is not very toxic given the antioxidant properties (van hoecke et al., 2009; gonzalez et al., 2014). the antioxidant properties arise from the ce(iii) which could form at the surface of nceo. ingestion of nceo could compromise the digestive system in bivalves, which readily feed on suspended matter, including nanoparticles (gagné et al., 2007; canesi et al., 2012). indeed, bivalves are considered species at risk of nanoparticle contamination because they are filter feeders, sessile and live for a relatively long time. there is some evidence that ce could influence dna integrity depending on the redox properties of nceo. on the one hand, ce(iv) could lead to dna scission in the presence of sodium triphosphate, which forms the basis of a dna assay (wu et al., 2005). on the other hand, the presence of ce(iii) could protect dna against oxidative stress as observed in caco-2 cells in vitro, which lead to a decreased amount of oxidized nucleotides (vila et al., 2018). given that the organic matter or other ligands could interact at the surface of nceo, the equilibrium between ce(iii)/ce(iv) at the surface of the nanoparticle could be changed and modulate its toxicity in freshwater mussels or other organisms. hence, the toxic outcome of nceo in representative surface waters with differing chemistries, and the nature of organic matter are difficult to predict at this time. the purpose of this study was therefore to examine influence of 4 types of surface waters on the behavior of dissolved ce and toxicity of nceo in the freshwater mussel dreissena polymorpha. the surface water types were green (high conductivity, high ph and low in total organic carbon), brown (low conductivity, low ph and high natural organic matter), a 10% dilution of municipal effluent in green water (high conductivity, low ph and high “urban” organic matter) and the laboratory control water. health effects were determined by monitoring mortality during exposure to nceo and ce(iv) because nceo mainly consists of ce(iv) from ceo2. resistance to air emersion and weight loss were also evaluated to determine the mussels’ resilience against an additional stress. sublethal effects comprise triglyceride levels (energy reserves), oxidative stress and dna damage. an attempt was made to compare the effects of nceo and ce(iv) in the 4 types of surface water on the freshwater mussels. materials and methods cerium oxide nanoparticle a stock solution of cerium oxide nanoparticle (ceo2) from sigma chemical company (ontario, canada) was used in this study. according to the manufacturer’s specifications, np ceo2 suspension has a size ≤ 25 nm and forms a homogeneous suspension at a concentration of 10% wt in water. for the exposure regime, zebra mussels (dreissena polymorpha) were exposed to a nominal concentration of 100 µg/l total ce as either nceo or ce(iv) in dechlorinated tap water (controls), and three other types of surface water as described below. the ce concentration was well below the reported solubility for ce(so4)2 at 9.84 g/l (20 oc). the hydrodynamic diameter and zeta potential of nceo nanoparticles were measured at least three times using a dynamic light scattering instrument (mobius instrument, wyatt technologies, santa barbara, ca, usa) operating with a laser at a wavelength of 532 nm. the instrument was previously calibrated with standard suspensions of silver nanoparticles (calibrators for electron microscope) from tedpella (usa). three types of water were sampled in the st. lawrence river: green, brown and a 10% dilution of physico-chemical–treated municipal effluent to simulate urban pollution. controls consisted of uv and charcoal-treated dechlorinated tap water. in each of the surface waters, total organic carbon (toc), ph and conductivity were determined according to standard methods (apha, 2010). green water is characterized by a relatively high conductivity (200-290 μs/cm), low toc (< 4 mg/l) and a slightly alkaline ph (ph > 7.2) (table 1). table 1 surface water basic properties water type conductivity (µs/cm) total suspended matter (> 0.7 µm; mg/l) ph total organic carbon (mg/l) % measured ce (µg/l)1 aquarium 310 ± 10 <1 7.97 ± 0.3 2.1 ± 0.1 75% brown 83 ± 5 5 ± 1 8.12 ± 0.4 6.5 ± 0.4 100% green 276 ± 25 13 ± 1 8.12 ± 0.3 2.9 ± 0.2 82% effluent 10% 375 ± 20 3 ± 0.5 8.07 ± 0.5 3.5 ± 0.3 78% measured concentration in the aquarium after 1 h equilibrium (aeration, 15 oc). the data are expressed as the percentage of added total ce as nceo brown water is characterized by low conductivity (100–160 μs/cm), higher toc levels (> 5 mg/l) and 154 a slightly acidic ph (ph < 7). the surface waters (20 l) were collected 2–3 weeks before the onset of exposure; they were stored in the dark at 4 oc until the exposure experiments at 15 oc. the total levels of ce in the 4 surface waters were determined after 1 h of dissolution of nceo using icp-ms spectrometry after acidification with 1% v/v hno3 seastar grade bc. zebra mussels (n = 30) were exposed to each type of water with and without 100 µg/l total ce as either nceo or ce(so4)2 (ceiv) for 96 h at 15 oc under constant aeration. the control mussels were exposed to each type of surface water only. the exposure experiment was repeated 3 times. the surface waters were not renewed and the mussels were not fed during the exposure period. a subgroup of 10 mussels was kept aside for air-stress survival while the remaining 20 mussels were processed as follows. for tissue ce assessments, a subgroup of 10 mussels were placed in clean aquarium water overnight as a depuration step and the soft tissues were removed for ce determination using icp-mass spectrometry as described above. the tissues were acid-digested in 10% hno3 at 70-80 oc for 12 h and diluted to 1% with milliq water. for the biomarkers, the remaining group of 10 mussels were processed on ice for the shell length, total weight and the soft tissues weight were determined. the soft tissues were then stored at -85 oc with the homogenization buffer (at 20% weight/volume). the homogenization buffer consisted of 100 mm nacl, 25 mm hepes-naoh, ph 7.4, 1 µg/ml apoprotin and 1 mm dithiothreitol. air survival test after the exposure period, 10 mussels were kept aside to determine the air-time survival as previously described (gagné et al., 2015). in brief, the mussels were weighed and the shell length determined, and they were placed individually in open plastic containers at 80% humidity at 20 oc. they were maintained as such and weighed each day until evidence of mussel mortality (opened shells, no decrease in mussel weight). the time of death in days was recorded and the data were expressed as the mean day of mortality for each individual over the 12 treatment groups: 4 types of surface water alone, 4 types of surface water with ce(iv) and 4 types of surface water with nceo. biomarker analyses the soft tissues were allowed to thaw on ice for 30 min and homogenized still in melting ice using a teflon pestle tissue grinder at 4 oc. a portion of the homogenate was set aside for lipid peroxidation (lpo), total triglycerides (tg) and total proteins. the remainder of the homogenate was centrifuged at 15000 x g for 20 min at 4 oc and the supernatant (s15) was removed for arachidonate cyclooxygenase (cox) evaluation. total proteins were determined in the homogenate and s15 fraction using the protein-dye binding principle using standard solutions of serum bovine albumin for calibration (bradford, 1976). lipid peroxidation (lpo) was determined in soft tissue homogenates using the thiobarbituric acid method (wills, 1987). a volume of 25 µl of the homogenate was mixed with 175 µl of 10% trichloroacetic acid containing 1 mm feso4 and 50 µl of 0.7% thiobarbituric acid. the mixture was heated at 75 °c for 10 min. the mixture was cooled to room temperature and centrifuged at 10000 x g for 5 min to remove any precipitates. a 200 µl volume was transferred to a 96-well dark microplate, and fluorescence readings were taken at 540 nm excitation and 600 nm emission. standard solutions of malonaldehyde (tetramethoxypropane, sigma chemical company, on, canada) were made for calibration in the homogenization buffer. results were expressed as µg thiobarbituric acid reactants (tbars)/mg total proteins in the homogenate. the activity of arachidonate-dependent cyclooxygenase activity was determined in the s15 fraction of soft tissues using a fluorescence microplate approach (gagné, 2014). briefly, 25 µl of the s15 fraction was added to 175 µl of the assay mixture composed of 50 µm arachidonate, 2 µm of dichlrofluorescein and 0.1 µg/ml of horseradish peroxidase in 50 mm tris-hcl, ph 8.0 and 0.05% tween-20. the reaction was allowed to proceed for 20 min at 20 oc and fluorescence readings were made at each 5-min interval at 485 nm excitation and 528 nm emission in dark microplates (synergy 4, biotek microplate reader, usa). the data were expressed as the increase in fluorescence/min/mg proteins in the s15 fraction. finally, total triglycerides in soft tissue homogenates were determined using an adipored fluorescent reagent (lonza; walkersville, md, usa). a volume of 5 µl of adipored reagent was added to 100 µl of homogenate in a black 96-well microplate. after 10 min incubation time, fluorescence was measured at 485 nm excitation and 535 nm emission (synergy 4, biotek microplate reader, usa). data were expressed as relative fluorescence units (rfu)/mg proteins in the homogenate. data analysis the study design examines the influence of surface waters on two chemical forms of ce (nceo and ce(iv)) in terms of bioavailability and toxicity in zebra mussels. in this study, there were 12 treatments: mussels exposed to each type of water: aquarium, brown, green, effluent 10% alone (treatments 1-4); mussels exposed to 100 µg/l ce as nceo in each type of water (treatments 5-8); and mussels exposed to 100 µg/l of ce as ce(so4)2 in each type of water (treatments 9-12). a bioavailability factor (l/kg) was calculated as follows: ce in tissues (ug/kg) / ce in water (ug/l). data normality and homogeneity of variance were verified using the shapiro-wilk and bartlett tests respectively. the influence of surface water types (aquarium, green, brown and 10% effluent) and ce forms (control, ce(iv) and nceo) were examined using 2-way factorial analysis of variance. critical differences between treatments were determined using the least square difference (lsd) test. the trends between the data were also analyzed using the pearson moment correlation test. the biomarker data were also analyzed by principal component analysis to determine which effects explained most of the observed variance. significance was set at p < 0.05. all statistical analyses were performed with the sysstat software package (version 13.2, usa). 155 results the surface water characteristics were determined (table 1). the aquarium water consisted of green water for drinking water from the city of montréal treated with charcoal filtration and uv radiation to remove chlorinated products. the diluted municipal effluent was prepared with aquarium water. the green water in the st. lawrence river originated from the great lakes (canada) and is characterized by low total organic carbon (< 4 mg/l) and high conductivity (> 200 µs/cm). the brown water originated from the forest mountains of the laurentian shield and is characterized by high organic carbon content (> 4 mg/l), low conductivity (< 200 µs/cm) and a mildly acidic ph (6.8-7). however, the green water contained relatively high amounts of total suspended matter compared with the brown water and the diluted municipal effluent. although the total organic carbon contents of the green water and the diluted municipal effluent were statistically similar (t test, p > 0.05), they differed in their nature: where the organic matter of the green water consisted mainly of humic and fulvic acids, the diluted municipal effluent was composed of proteinaceous matter. the addition of nceo to these waters revealed that the nanoparticles remained in suspension at > 75% after 1 hr exposure time. this proportion increased to 100% in the organic-rich brown water, as expected. the properties of nceo in the various types of water were examined by dynamic light scattering analysis (table 2). predilution of nceo suspension to 1000 µg/l in milliq water produced no significant changes in the mean diameter distribution and zeta potential of nceo. dilution at this concentration produced no changes when the green and brown waters and diluted municipal effluent were used. in the aquarium water, the size distribution was somewhat increased compared with the other types of surface water. based on these data, we selected milliq water to pre-dilute the nceo suspension before the final concentration of 100 µg/l in each of the 4 types of water. diluting the nceo suspension at the exposure concentration for mussels in surface waters revealed no important changes in size distribution and zeta potential in the presence of aquarium brown and green waters after 1 h. the mean size increased from 50 to 81 nm in the presence of diluted municipal effluent (10%). after staying for 48 h in the water samples, the mean size of the nanoparticles increased to 81 nm (brown water), 108 nm (green water), 103 nm (aquarium water) and 322 nm (10% municipal effluent). this suggests that nceo tends to form aggregates in time, whereas natural organic matter tends to favor small aggregates of nceo (81-108 nm) compared with urban municipal effluents (322 nm). the bioavailability of ce was assessed in mussels exposed to ce(iv) and nceo in the various water types (table 3). in mussels placed only in the various water samples, the levels of ce ranged between 0.4 to 4.5 ng/g and were somewhat higher in brown waters (3.5 ± 0.7 ng/g) and diluted municipal effluents (2.3 ± 0.8 ng/g) compared to aquarium water (0.8 ± 0.3 ng/g). in mussels exposed to ceiv, total levels of ce in tissues significantly increased from the corresponding water controls. mussels exposed to ceiv in brown waters and diluted municipal effluent accumulated more ce compared to mussels exposed to ceiv in aquarium and green waters reaching 155 ± 35 ng ce/g soft tissues. the bioavailability factor was 0.4, 0.36, 1.4 and 1.6 kg/l in aquarium, green, the diluted municipal effluent and brown waters respectively. in mussels exposed to nceo, total ce levels were modestly increased for all water types compared to water controls reaching 22 ± 3 ng cd/g in mussels placed in brown water and the diluted table 2 nceo characterization in surface waters water sample nceo diameter (nm) 1 h zeta potential (mvolt) 1 h diameter (nm) 48 h zeta potential (mvolt) 48 h brown water 1000 µg/l 51 ± 3 -15.2 ± 3.1 50.0 ± 1.5 -16.1 ± 3.8 green water 1000 µg/l 52 ± 1 -19.1 ± 1.8 72.1 ± 4.5* -19.1 ± 1.5 10% municipal effluent 1000 µg/l 54 ± 2 -13.7 ± 3.0 235 ± 111* -12.5 ± 2.4 aquarium 1000 µg/l 88 ± 7 -14.0 ± 1 258 ± 22* -14.5 ± 1.0 brown water 100 µg/l 50 ± 3 -13.6 ± 3.8 81 ± 10* -20.0 ± 2.9 green water 100 µg/l 53 ± 6 -17.1 ± 3.8 108 ± 31* -19.2 ± 1.8 10% municipal effluent 100 µg/l 81 ± 24 -14.0 ± 1.7 322 ± 200 -14.5 ± 2.1 aquarium 100 µg/l 60 ± 15 -10.2 ± 2.5 103 ± 6* -14.7 ± 4.4 *indicates significant difference between 1 and 48 h. table 3 tissue ce loadings in mussels 156 waters/treatment control (ug/g) ceiv (ng/g) ce(iv) bioavailabilty3 nceo (ng/g) nceo bioavailabilty aquarium 0.8 ± 0.3 40 ± 71 0.4 13 ± 31 0.13 green waters 1.7 ± 0.5 36 ± 61 0.36 9 ± 41 0.09 brown waters 2.3 ± 0.8 155 ± 351,2 1.55 22 ± 61,2 0.22 10 % municipal effluent 3.5 ± 0.7 138 ± 301,2 1.38 22 ± 31,2 0.22 1significantly different from water control 2significantly different from aquarium water within ceiv or nceo treatments 3bioavailability in kg/l: concentration in tissues/concentration in water. municipal effluent. mussels exposed to nceo in brown water and diluted municipal effluent contained more ce in tissues compared to mussels exposed to nceo in aquarium and green waters. the bioavailability factor for the nanoparticulate form was low with factors of 0.1, 0.09, 0.22 and 0.22 kg/l in mussels exposed to the nanoparticle in aquarium, green, brown waters and diluted municipal effluent respectively. the resistance to air emersion in post-exposed mussels was examined as a means of assessing the general health status of the mussels exposed to nceo and ce(iv) in the 4 types of surface water (figure 1a-c). factorial 2-way anova revealed a significant (p < 0.05) interaction between surface water types and cerium form [i.e., nceo and ce(iv)]. in the mussels exposed to surface waters only, there was a significant decrease between time of death in brown water (11 days) and green water (13.2 days). in the mussels exposed to ce(iv), no changes were observed in the time of death in the mussels placed in aquarium water compared with the aquarium water alone. a decrease in time of death was observed for the mussels exposed to ce(iv) and the municipal effluent (10 days) compared with the mussels exposed to ce(iv) in the controls (13.2 days, p < 0.01) and in the mussels exposed to aquarium water only (13 days, p < 0.05). in the mussels exposed to nceo in aquarium water, no significant change in the time of death was observed compared with aquarium water. there was a significant change in time of death in the mussels exposed to nceo in aquarium water (13.2 days) compared with those exposed to nceo in green water (9 days, p < 0.05) and nceo in brown water (12 days, p = 0.05). of the mussels exposed to the municipal effluent, the mussels exposed to ce(iv) survived marginally less to air stress (9 days) compared with the mussels exposed to nceo and the municipal effluent (12 days; 0.1 < p < 0.05). weight loss after 4 days in air was also determined in the mussels exposed to the ce forms and different surface waters (figure 1d-f). two-way factorial anova revealed that only surface water types produced a significant effect in mussels (p < 0.05). with respect to the mussels treated with aquarium water, the weight loss was higher in the mussels in brown water (p < 0.05) and diluted municipal effluent (p < 0.05). the mussels in brown water displayed more weight loss than the mussels exposed to green water (p < 0.05). correlation analysis revealed that air-time survival was significantly correlated with initial weight loss (r = 0.32; p < 0.01) i.e., the mussels that lost more weight initially (after 4 days) survived less time in the air. the condition factor (mussel weight g/shell length in cm) was also evaluated (figure 2). twoway factorial anova revealed a significant interaction between surface waters and ce form (p < 0.001). with respect to aquarium water only, the other types of water (i.e., municipal effluent, brown and green waters) did not significantly influence the condition factor. the condition factor of the mussels placed in the diluted municipal effluent was higher compared with the brown water (p < 0.01) and the controls (p = 0.05). in the mussels exposed to ce(iv) in green water, the condition factor was marginally lower relative to the mussels placed in aquarium water only (0.1 < p < 0.05). in the mussels exposed to nceo in aquarium water, the condition factor was marginally higher compared with that of the mussels in aquarium water only (0.1 < p < 0.05). the condition factor was significantly higher in mussels exposed to nceo in green water (p < 0.05) and lower in the mussels in the diluted municipal effluent and nceo (p < 0.05) compared with the mussels in aquarium water alone. the mussels exposed to ce(iv) in aquarium water had a significantly lower condition factor than the mussels exposed to nceo in aquarium water (p = 0.05). the condition factor was significantly higher in the mussels exposed to nceo in green water compared with those exposed to nceo in the diluted municipal effluent (p < 0.001), but lower when compared with the mussels exposed to ce(iv) in 157 a qu ar iu m b ro w n e ffl ue nt g re en control 7 8 9 10 11 12 13 14 15 t im e o f d e a th ( d a y s ) a qu ar iu m b ro w n e ffl ue nt g re en ce(iv) 7 8 9 10 11 12 13 14 15 t im e o f d e a th ( d a y s ) a qu ar iu m b ro w n e ffl ue nt g re en nceo 7 8 9 10 11 12 13 14 15 t im e o f d e a th ( d a y s ) * * a b c a qu ar iu m b ro w n e ffl ue nt g re en control 0 5 10 15 20 w e ig h t lo s s ( % ) a qu ar iu m b ro w n e ffl ue nt g re en ce(iv) 0 5 10 15 20 w e ig h t lo s s ( % ) a qu ar iu m b ro w n e ffl ue nt g re en nceo 0 5 10 15 20 w e ig h t lo s s ( % ) d e f * fig. 1 influence of cerium form and surface waters on air-time survival. the mussels were exposed to ce(iv) and nceo in the 4 types of water for 96 h at 15 oc. after the exposure period, a subgroup of mussels was processed for assessment of air-time survival (a, b, c) and initial weight loss after 4 days (d, e, f), as described in materials and methods. the star * indicates significance from aquarium water green water (p < 0.001) and those exposed to green water only (p < 0.001). correlation analysis revealed that the condition factor was marginaly correlated with ce tissue concentration (r=-0.21, 0.1< p <0.05). oxidative stress was determined in the mussels by following changes in cox activity and lpo levels (figure 3 a–f). for cox activity, two-way factorial anova revealed a significant interaction between surface water types and ce form (p < 0.001). in the mussels exposed to surface waters only (figure 4a), cox activity was significantly lower in the mussels placed in green water compared with the mussels placed in brown water (p < 0.01) and aquarium water (p < 0.05), and marginally lower in the diluted municipal effluent (0.1 < p < 0.05). in the mussels exposed to ce(iv), cox activity was significantly lower (p < 0.01) than the activity in the mussels exposed to aquarium water (figure 4b). in the mussels exposed to ce(iv) in brown water, cox activity was significantly higher than in the mussels exposed to ce(iv) in green water (p < 0.05), diluted municipal effluent (p < 0.01) and aquarium water (p < 0.01). cox activity was significantly lower in the mussels exposed to ce(iv) and the municipal effluent compared with the mussels exposed to the municipal effluent alone (p < 0.01). in the mussels exposed to nceo (figure 4c), cox activity was significantly increased in the mussels exposed to nceo in green water compared with the mussels in green water only (p < 0.001). cox activity was significantly decreased in the mussels exposed to nceo in brown water compared with those in brown water only (p < 0.01) and the mussels exposed to nceo in green water (p < 0.001) and in aquarium water (p = 0.05). cox activity in the mussels exposed to aquarium (control) water and nceo was significantly higher than cox activity in the mussels exposed to ce(iv) in aquarium water (p < 0.01). the same was observed for the mussels placed in green water and the diluted municipal effluent. however, the reverse situation was observed when the mussels were exposed to nceo in brown water. correlation analysis revealed that cox activity was marginally correlated (r = 0.2; 0.1 < p < 0.05) with weight loss and ce tissue loadings (r = 0.2; 0.1 < p < 0.05), which suggests that cox activity 158 a qu ar iu m b ro w n e ffl ue nt g re en control 40 50 60 70 80 90 100 c o n d it io n f a c to r (g /c m ) a qu ar iu m b ro w n e ffl ue nt g re en ce(iv) 40 50 60 70 80 90 100 c o n d it io n f a c to r (g /c m ) a qu ar iu m b ro w n e ffl ue nt g re en nceo 40 50 60 70 80 90 100 c o n d it io n f a c to r (g /c m ) * * * fig. 2 influence of surface water and cerium on the condition factor of mussels. the mussels were exposed to ce(iv) and nceo in the 4 types of water for 96 h at 15 oc. after the exposure period, the weight-to-shell-length ratio was determined in the mussels. *indicates significance from aquarium water was associated with weight loss and decreased tissue ce loadings. oxidative damage was also followed by lpo levels (figure 3 d–f). two-way factorial anova revealed that only the water type was significant (p = 0.001). lpo levels were significantly increased in green water compared with either aquarium or brown waters (p < 0.001 and p = 0.01 for aquarium and brown waters respectively). lpo levels were also significantly increased in the diluted municipal effluent compared with the aquarium water (p < 0.05) and green water (p < 0.05). interestingly, these responses were lost when ce(iv) and nceo were present, indicating that these compounds have antioxidant effects. the levels of dna strand breaks were also determined in the mussels exposed to the different ce forms and water types (figure 4). two-way factorial anova revealed a significant interaction between the ce forms and water samples. among the mussels exposed to the 4 types of surface water, dna strand breaks were significantly lower in those exposed to brown water compared with aquarium water (p < 0.001), diluted municipal effluent (p < 0.001) and, marginally, with green water (0.1 < p < 0.05). in the mussels exposed to ce(iv), dna strand breaks were increased in the mussels exposed to ce(iv) in brown water compared with those in the brown water only. the mussels exposed to ce(iv) and brown water also had elevated levels of dna strands breaks compared with the mussels exposed to the lanthanide in green water. dna strand breaks were lower in the mussels exposed to ce(iv) in green water compared with those immersed in green water only (p < 0.01). the same was also true with the diluted municipal effluent. among the mussels exposed to nceo, dna strand breaks were lower in the mussels placed in green water compared with those exposed to aquarium water only (p < 0.05) and in the mussels exposed to ce(iv) in aquarium water (p = 0.05). dna strand breaks were marginally correlated with cox activity (r = -0.20; 0.1 < p < 0.05), suggesting that decreased dna strand breaks are associated with susceptibility to oxidative stress as determined by cox activity. the influence of surface waters and ce forms on triglyceride levels was examined (figure 5). twoway factorial anova revealed a significant interaction between surface waters and ce forms (p < 0.001). in the mussels exposed to surface waters only, triglyceride levels were significantly decreased in brown water compared with those exposed to aquarium water (p < 0.01), diluted municipal effluent (p < 0.01) and green water (p < 0.001). triglyceride levels were significantly higher in the mussels placed in green water than those placed in aquarium water (p = 0.001). in the mussels exposed to ce(iv), triglyceride levels in the mussels in aquarium water were lower compared with those placed in aquarium water (p < 0.05) and green water (p < 0.001). the same was found with the mussels exposed to ce(iv) and green water, where the increase in triglyceride levels observed in the mussels in green water was lost when ce(iv) was added. the levels were marginally higher in the mussels exposed to ce(iv) and brown water compared with the mussels placed in brown water alone (0.1 < p < 0.05). a marginal decrease in triglycerides was observed in the mussels exposed to ce(iv) and the diluted municipal effluent compared with the mussels exposed to the diluted municipal effluent (0.1 < p < 0.05). in the mussels exposed to nceo, the increase in triglyceride levels observed in the mussels exposed to green water was lost compared with those exposed to green water alone (p < 0.001). the same was found with the mussels exposed to the diluted municipal effluent and nceo, but it was marginal (0.1 < p < 0.05). triglyceride levels also decreased in the mussels exposed to nceo in brown water (p < 0.05) and the diluted municipal effluent (p < 0.05) compared with those exposed to nceo in aquarium water. triglyceride levels were significantly correlated with weight loss during air-time survival (r = -0.35; p = 0.001) and time of death (r = 0.24; p < 0.05), which suggests that weight loss occurs when 159 a qu ar iu m b ro w n e ffl ue nt g re en control 100 200 300 400 c o x ( r f u /m in /m g p ro te in s ) a qu ar iu m b ro w n e ffl ue nt g re en ce(iv) 100 200 300 400 c o x ( r f u /m in /m g p ro te in s ) a qu ar iu m b ro w n e ffl ue nt g re en nceo 100 200 300 400 c o x ( r f u /m in /m g p ro te in s ) a b c * * a qu ar iu m b ro w n e ffl ue nt g re en controls 0 10 20 30 40 l p o ( u g t b a r s /m g p ro te in s ) a qu ar iu m b ro w n e ffl ue nt g re en ce(iv) 0 10 20 30 40 l p o ( u g t b a r s /m g p ro te in s ) a qu ar iu m b ro w n e ffl ue nt g re en nceo 0 10 20 30 40 l p o ( u g t b a r s /m g p ro te in s ) * * * * * d e f fig. 3 change in oxidative stress in mussels exposed to ceria forms and surface waters. the mussels were exposed to ce(iv) and nceo in the 4 types of water for 96 h at 15 oc. after the exposure period, oxidative stress was measured in the mussels by cox activity (a, b, c) and lpo (d, e, f). *indicates significance from aquarium water triglyceride levels are lowered. triglyceride levels were significantly correlated with dna strand breaks (r = 0.56; p < 0.001). in an attempt to gain a comprehensive view of the interrelationships between the biomarkers, a principal component analysis was performed (figure 6). the analysis revealed that most of the variance was explained (62%) by three factors. among the biomarkers within each of the 3 components, the following responses had the highest factorial weights (> 0.5): dna strand breaks, triglycerides, weight loss, time of death, condition factor, tissue ce loadings and lpo. this suggests that ce in different types of surface water will have an impact on lipid damage by oxidation, changes in triglyceride levels, dna damage and general health status as determined by time of death in air, weight loss and condition factor. discussion nceo was fairly stable in the surface waters based on the total ce measurement, the mean diameter and zeta potential after 1 h. although the zeta potential was not affected after 48 h in the 4 types of surface water, the mean diameter of nceo was significantly increased in the aquarium, brown and green waters, which suggests no degradation or major transformation within the (in)organic matrix of the water types. there was a great deal of variability in the mean diameter of nceo in the diluted municipal effluent, so aggregation could not be confirmed statistically (p < 0.05). this is consistent with the observation that nceo ingested by the marine mussel mytilus galloprovincialis was not degraded in the gut and remained as nanoparticles (montes et al., 2012). mussels exposed to 10 mg/l nceo accumulated 62 µg/g of ce and rejected 21 mg of ce/g, which gives a bioavailability factor of 6.2 and suggests that mussels exposed to a continuous input of nceo in the environment would tend to accumulate the nano-lanthanide. the bioavailability factor obtained for nceo in this study was low with values at 0.13 (l/kg) in aquarium water but rose to 0.22 in brown water and diluted municipal effluent. the increase in 160 a qu ar iu m b ro w n e ffl ue nt g re en control 0,2 0,3 0,4 0,5 0,6 0,7 0,8 d n a b re a k s ( u g /m g p ro te in s ) a qu ar iu m b ro w n e ffl ue nt g re en ce(iv) 0,2 0,3 0,4 0,5 0,6 0,7 0,8 d n a b re a k s ( u g /m g p ro te in s ) a qu ar iu m b ro w n e ffl ue nt g re en nceo 0,2 0,3 0,4 0,5 0,6 0,7 0,8 d n a b re a k s ( u g /m g p ro te in s ) * * fig 4 dna strand breaks in mussels treated with cerium and surface waters. the mussels were exposed to ce(iv) and nceo in the 4 types of water for 96 h at 15 oc. after the exposure period, oxidative stress was measured in the mussels by cox activity (a, b, c) and lpo (d, e, f). the star * indicates significance from aquarium water ce bioavailability with ce(iv) was also observed by these organic-carbon rich waters. however, given that bioavaialbity could be tissue-specific and ce analysis were done in the whole soft tissues, we cannot exclude the possibility that some specific target tissues might have accumulated the majority of ce. according to the principal component analysis, changes to the oxidative status had a major effect in freshwater mussels. exposure to ce(iv) readily decreased cox activity in the mussels, and nceo was less potent in decreasing cox activity in the control mussels placed in aquarium water. the antioxidant effect of nceo was more evident only in the presence of organic-rich brown water. moreover, the decrease in cox activity was also associated with lower levels of dna strand breaks in the mussels placed in aquarium and green waters and in the diluted municipal effluent in the mussels exposed to ce(iv), which suggests a decrease in oxidative-mediated dna damage and repair activity. nevertheless, ce(iv) could interact with dna in vitro, leading to its reduction to the fluorescent ce(iii) form in the presence of sodium triphosphate (wu et al., 2005). dna scission was associated with the formation of ce(iii) from the reduction of ce(iv) by dna and sodium triphosphate. dna strand break levels were significantly higher in the mussels exposed to nceo than in the mussels exposed to ce(iv), regardless of surface water type (t test, p = 0.01), which suggests that properties other than ce(iv) of nceo produced dna breaks. a mixed-valence ce (ce+3/ce+4) state at the surface of nceo was observed for this nanoparticle (bhargava et al., 2016), which could account for the increase in dna breaks. this was consistent with dna damage in the nceo2-exposed freshwater bivalve corbicula fluminea (koehlé-divo et al., 2018). dna damage was associated with apoptosis (caspase) with the expression of anti-oxidative defense mechanisms and increased anaerobic activity (lactate dehydrogenase activity), which suggests that the removal of damaged cells was not affected. a qu ar iu m b ro w n e ffl ue nt g re en control 0 100 200 300 400 t ri g ly c e ri d e s ( r f u /m g p ro te in s ) a qu ar iu m b ro w n e ffl ue nt g re en ce(iv) 0 100 200 300 400 t ri g ly c e ri d e s ( r f u /m g p ro te in s ) a qu ar iu m b ro w n e ffl ue nt g re en nceo 0 100 200 300 400 t ri g ly c e ri d e s ( r f u /m g p ro te in s ) * * fig. 5 influence of cerium form and surface waters on triglyceride levels in mussels. the mussels were exposed to ce(iv) and nceo in the 4 types of water for 96 h at 15 oc. after the exposure period, triglyceride levels were determined in soft tissues. the star * indicates significance from aquarium water 161 -1,0 -0,5 0,0 0,5 1,0 fac tor( 1) -1 ,0 -0 ,5 0 ,0 0 ,5 1 ,0 factor(2) -0,5 0,0 0,5 1,0 f a c to r( 3 ) time of death (days) weight loss (%) conditon factor (g/cm) cox (rfu/min/mg proteins) tbars (rfu/min/mg proteins) dna strands (ug dna/mg proteins) trigycerides (rfu/mg proteins) tissue ce total variance explained: 60% fig. 6 principal component analysis of biomarker responses in mussels exposed to cerium and surface waters the antioxidant properties of ce(iii) have also been reported in previous studies (gonzalez et al., 2014). for example, in vitro cultures of fish cone receptor cells exposed to mixed valence nceo (ce3+/ce+4) led to decreased h2o2 levels and increased cell survival (bhargava et al., 2016). this highlights the ability of nceo to scavenge h2o2 in cells. in another study with zebra fish embryos, exposure to albumin-coated nanoceria produced no oxidative stress and preserved the antioxidant system (bhushan et al., 2016). zebra mussels exposed to nceo showed decreased lysosome number, catalase activity and lipid peroxidation (garaud et al., 2015). it appears that the ce(iii) state of nceo is responsible for superoxide dismutase mimetic activity, which could act as a primary radical scavenger (hecker et al., 2008). hence, the antioxidant effect of ce(iv) and nceo involves the formation of intermediate ce(iii). it is reasonable to propose that the surface water properties (organic matter?) could favor the formation of ce(iii) given that the antioxidant effect of nceo was observed in brown water. ce could also protect against lead-induced hepatotoxicity in crucian carp (ling and hong, 2010). the fish were exposed via intraperitoneal injection (10–40 mg/kg) for 14 days, after which a subgroup of animals was injected with 1.5 mg/kg of ce(iii). this treatment prevented ros accumulation and improved the hepatosomatic index of the fish. the surface properties of nceo, such as negative charge (i.e., less cationic form of ce as ce+4), protein corona formation, shape and zeta potential but not aggregation state, were associated with a loss of viability of hemocytes of m. galloprovincialis (sendra et al., 2018). indeed, the negative charge and the rounded shape of nceo favored the formation of cu,zn-sod activity in the hemolymph serum and favored more changes in lysosome membrane stability and phagocytosis activity. a decrease in oxidized dna bases was observed in caco-2 cells exposed to nceo in vitro, which further supports its antioxidant activity although the comet assay did not observe changes in tail length (vila et al., 2018). this corroborates our finding here, where decreased dna strand breaks were obtained in the mussels exposed to the nanoparticle in aquarium water compared with the control mussels. based on air-time survival and condition factor assessments, the present study integrated impacts 162 at different levels of biological organization (biochemical changes to change in health status and resilience to air stress). thus, the observed changes at the biochemical/cellular levels could be compared with changes at higher levels of organization to determine whether more severe impacts (toxicity) are manifest at the organism level. based on the data on air-time survival, nceo and ce(iv) were more toxic (mussels survived less in air) in green water compared with the mussels in green water alone. this was not accompanied by weight loss, although triglycerides were lower in the mussels exposed to nceo in green water. this was similar to the results of a previous study where the filtering activity of zebra mussels exposed to either bare or citrate-coated nceo was not affected, although some changes were found at the molecular level i.e., glutathione s-transferase and catalase gene expression (garaud et al., 2016). this highlights the need to measure changes not only at the biochemical level, but also in organism health (morphological changes, resistance to applied stress such as air exposure). the toxicity of nceo could also be modulated by the surface water characteristics in fish (gagnon et al., 2018). exposure to 10 µg/l of nceo was lethal in trout juveniles in both the aquarium and green waters, but not in the brown water, suggesting that the natural organic matter in brown water might have prevented toxicity, perhaps through the formation of the antioxidant ce(iii). however, this could be due to increased bioavailability in aquarium and green waters, since ce contents in gills were higher in fish exposed to nceo in the aquarium and green waters compared with those of fish in the brown water. some evidence exists that nceo could alter ionic homeostasis in fish gills, which could be another pathway leading to toxicity (xia et al., 2013). brain acetylcholinesterase was also decreased, suggesting downregulation of neuromuscular activity in fish. it also appears that co-occurrence of copper oxide with nceo in automobile catalyst systems could lead to toxicity in daphnids (jemec et al., 2012). while nceo was not toxic to daphnids, the presence of copper-nceo mixed oxides (at 20% cuo) reduced survival at concentrations of 80 mg/l or more, while coppernceo at 10% and 15% did not produce any effects. this suggests that a mixture of metal oxides could have potentiated the toxicity of nceo. more research will be required to further study the toxicity of mixed metal oxide nanoparticle formulation in aquatic organisms. in conclusion, the influence of 4 different types of surface water on the biochemical effects of nceo and ce(iv) was examined in mussels. the data revealed that both forms of cerium decreased triglyceride levels in aquarium and green waters, but this was prevented by organic-matter rich surface waters (brown and diluted municipal effluent). the mussels exposed to nceo survived less in air when exposed to green water compared with aquarium water. the biochemical effects of nceo and ce(iv) could be modulated by the surface water properties involving natural and anthropogenic organic matter in addition to conductivity and the presence of suspended matter. acknowledgments this research was funded by the chemical management plan of environment and climate change canada. the helpful assistance of joanna kowalceik 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2009. vila l, garcía-rodríguez a, cortés c, velázquez a, xamena n, sampayo-reyes a, et al. effects of cerium oxide nanoparticles on differentiated/undifferentiated human intestinal caco-2 cells. chemico-biol interact. 283: 38-46, 2018. wills ed. evaluation of lipid peroxidation in lipids and biological membranes. in: snell k, mullock b. (eds.), biochemical toxicology: a practical approach. irl press, washington dc, usa, pp.127-150, 1987. wu x, yang j, sun c, sun s, guo c, chen y. a fluorimetric method for the determination of nucleic acids using ce(iv) and sodium triphosphate. luminescence 20: 41-45, 2005. xia j, zhao hz, lu gh. effects of selected metal oxide nanoparticles on multiple biomarkers in carassius auratus. biomed. environ. sci. 26: 742-749, 2013. https://www.ncbi.nlm.nih.gov/pubmed/29539589 https://www.ncbi.nlm.nih.gov/pubmed/?term=ling%20q%5bauthor%5d&cauthor=true&cauthor_uid=19130280 https://www.ncbi.nlm.nih.gov/pubmed/?term=hong%20f%5bauthor%5d&cauthor=true&cauthor_uid=19130280 https://www.ncbi.nlm.nih.gov/pubmed/19130280 https://www.ncbi.nlm.nih.gov/pubmed/22614026 https://www.ncbi.nlm.nih.gov/pubmed/29704629 https://www.ncbi.nlm.nih.gov/pubmed/15685662 https://www.ncbi.nlm.nih.gov/pubmed/24099608 microsoft word isj477 isj 14: 233-240, 2017 issn 1824-307x visions and perspectives telomeres and telomerase in basal metazoa i udroiu, v russo, t persichini, m colasanti, a sgura department of science, university roma tre, rome, italy accepted june 12, 2017 abstract telomeres are protein-bound tandemly repeated simple dna sequences placed at the chromosome ends, their role being essential for maintaining genome integrity. severe telomere damage can trigger potential carcinogenic events such as chromosome fusions, whilst telomere shortening results (at least in mammals) in a protective mechanism known as 'replicative senescence'. in most metazoa, telomere shortening is avoided by the ribonucleoprotein telomerase. in this brief overview, we focused on the evolutionary conservation of telomeres and telomerase in basal metazoa (ctenophora, porifera, placozoa, cnidaria). in all these taxa, telomerase seems to be active and telomeres show the canonical ttaggg sequence. presence of telomerase activity and absence of telomere shortening is in accordance with the lack of senescence seen in basal metazoa, although a clear correspondence between the “demographic senescence” and “replicative senescence” remains to be elucidated. nonetheless, basal metazoa can be useful as model organisms in studies on the evolution of telomere biology, evo-devo investigations on the control of telomerase activity, the emergence of senescence, as well as telomere-independent effects of telomerase. key words: basal metazoa; senescence; telomerase; telomeres   introduction telomeres are specialized structures at the ends of eukaryotic chromosomes, consisting of protein-bound tandemly repeated simple dna sequences (armstrong and tomita, 2017). with a few exceptions, including plant and insect species, telomeric dna sequences is usually rich in guanine and is a repeat of six bases. the sequence is ttaggg/ccctaa in all vertebrates underlining that is highly conserved to protect genome (armstrong and tomita, 2017). telomeres fulfill unique and essential functions in genome integrity. in general, non-telomeric double-strand dna breaks (dsbs) are not tolerated and are rapidly repaired. this scenario, however, does not apply to telomeres, thanks to the association of telomeric dna with specialized proteins whose role is to organize the linear chromosome end into a stable structure (t-loop) that is not recognized by the cell as a chromosome break. in mammals, the t-loop is held together by seven known proteins, the most notable ones being trf1, trf2, pot1, tin1, and tin2, collectively ___________________________________________________________________________ corresponding author: marco colasanti department of science university roma tre viale g. marconi 446, 00146, rome, italy e-mail: marco.colasanti@uniroma3.it referred to the shelterin complex (blackburn, 2000; armstrong and tomita, 2017) (see also fig. 1). this structure is required to maintain chromosome physiology, and loss of telomeric dna or mutations of telomere-binding proteins triggers a series of events including chromosomal fusions (fig. 2) and genomic instability that ultimately compromise cell proliferative capacity and/or viability (blackburn, 2000; depinho, 2000; berardinelli et al., 2010; armstrong and tomita, 2017). all chromosomes loss a small amount of telomeric dna every time a somatic cell divides because of the noted “end replication problem”. to avoid continuous sequence loss from the telomeres in dividing cells, special mechanisms have evolved. in most eukaryotic organisms, the solution to the problem involves a ribonuclear complex, called telomerase that consists of an enzymatic part (transcriptase, tert) and a rna component (tr). this last functions like a template for the de novo synthesis of telomeric dna sequences. however, telomerase activity varies across taxa and differs in mortal and immortal cells (armstrong and tomita, 2017). it is well established that an active telomerase is required for unlimited growth; in fact, it is repressed in the majority of human somatic cells while its activity is higher in immortal cell lines, germline cells, stem cells, activated lymphocytes, 233 fig. 1 representation of a closed (capped) telomere. in mammals, specialized proteins, collectively referred to the shelterin complex, organize the linear chromosome end into a stable structure (t-loop) [modified from (berardinelli et al., 2016)]. and most of the tumor cells analyzed (schmitt et al., 1994; bolzan et al., 2000). note that loss of telomerase enzymatic function leads to progressive telomere shortening over time, eventually resulting in the disappearance of detectable telomeric dna. loss of chromosomal end capping has consequences in a wide range of cellular processes, including senescence, apoptosis, and carcinogenesis (harley et al., 1992; morin, 1996) (fig. 3). telomeres and telomerase in senescence telomere shortening progresses with each cell cycle due to the “end replication problem” up to reach a critically telomere length that induce cell cycle arrest and activation of senescence profile that contribute to tumor suppressor mechanism. though, sometimes cells lose the ability to senesce because, for example, of mutation in p53 protein. telomere shortening may initiate chromosomal instability through end-to-end fusion of unprotected chromosomes. in vitro, cells with extensive chromosomal instability succumb to crisis or m2 stage of replicative senescence, which is characterized by wide-spread cell death. however, a rare human cell can escape m2 by reactivating or up-regulating telomerase activity, which will result in indefinite cell proliferation, typical of tumor cells (armstrong and tomita, 2017). despite many factors have been involved in cell transformation, up to today telomerase activity seems to be the principal responsible of unlimited proliferation of tumor cells confirmed by the knowledge that most tumor cells express telomerase. more rarely, a cell may engage an alternative to telomerase for maintaining telomeres (bryan et al., 1997) that appears to involve dna recombination between the telomere sequences. nevertheless, activation of a telomere-maintenance mechanism does not seem to be enough to confer a cancerous phenotype, even when combined with p53 inactivation, which represses the senescence response. therefore, it appears plausible that other changes must occur at the cellular level for initiation of carcinogenesis process. telomeres and telomerase in basal metazoa telomeric dna of eukaryotes is formed by a long array of short tandem repeats. the ttaggg sequence is conserved in almost all metazoa (fig. 4) and, since this motif is shared also by their unicellular sister-group choanoflagellata (fairclough et al., 2013), it can be inferred that it represents the ancestral telomeric repeat. other patterns present among metazoa are likely to have appeared later during evolution. this should be the case of the ttaggc motif in nematodes, as their sister-group chaetognatha displays the standard metazoan sequence. similarly, we believe that the ttagg sequence found in most arthropoda represents an apomorphic character, since their sister-group onychophora still shows the common ttaggg 234 motif. finally, it must be added that among arthropoda diptera shows telomeres composed of transposable elements, which are added to chromosome ends by transposition, an alternative mechanism of telomere maintenance present in this taxon that does not show telomerase (frydrychova et al., 2004). telomere length has been studied in some basal metazoan species. southern hybridization of rsai/hinfi-digested genomic dna showed that telomere lengths are between 0.5 and 20 kb in leucosolenia, sycon (porifera) and pleurobrachia pileus (ctenophora) (traut et al., 2007). among cnidaria, using the same technique, telomeres measure 0.5 10 kb in chrysaora hysoscella, 1 20 kb in cyanea lamarcki, 4 20 kb in hydra vulgaris, 2-25 kb in nematostella vectensis (traut et al., 2007) and 3.5 kb in acropora surculosa (sinclair et al., 2007). using telomere restriction fraction (trf), lengths are 8.9 kb in acropora digitifera (tsuta et al., 2014), 21.0 kb in acropora millepora and 19.2 kb in both agaricia fragilis and madracis auretenra (zielke and bodnar, 2010). performing the single telomere length assay (stela), telomere length in cassiopea andromeda is valued 1 2 kb (ojimi and hidaka, 2010). finally, in galaxea fascicularis telomere length measured with stela was 4 kb and 15.6 kb with trf (tsuta and hidaka, 2013). as we can see, there is a great variability in telomere length measures, most probably due to differences in protocols and technique employed. nonetheless, all this data fall in the same order of size below 25 kb, similar to other taxa like nematodes fig. 2 mechanism of telomere fusion. break or replicative erosion leave telomeres uncapped, leading to end-to-end fusion, resulting in a dicentrici chromosome. (wicky et al., 1996), platyhelminths (koroleva et al., 2013), non-dipteran insects (vitkova et al., 2005) and fish (ocalewicz, 2013). a much greater variability, instead, is present among tetrapods, reaching the maximum in endotherms, with telomeres lengths ranging from 5 to hundreds of kb. this is thought to be the result of different evolutionary pressures and linked to telomereshortening senescence, lifespan and body mass (gomes et al., 2011). fig. 3 telomeric dysfunction/shortening. extensive telomeric erosion or telomere dysfunction activate several biological processes including activation of dna damage response, block of cell cycle, apoptosis and senescence [modified from (berardinelli et al., 2016)]. 235 a few studies investigated the presence of telomerase in basal metazoa (table 1). applying the telomeric repeat amplification protocol (trap) assay, telomerase activity has been detected in cassiopea sp. (ojimi et al., 2009), galaxea fascicularis (nakamichi et al., 2012), madracis auretenra and m. decactis (zielke and bodnar, 2010). traut et al. (2007), performing the same assay, found positive results in pleurobrachia pileus and aurelia aurita, but did not find telomerase activity in chrysaora hysoscella, cyanea lamarcki, hydra vulgaris, nematostella vectensis, trichoplax adhaerens, leucosolenia sp., sycon sp. and suberites domuncula. however, it must be underlined that the same authors stated “this may be merely due to technical reasons”. in fact, another study employing the trap assay on suberites domuncula found positive results (koziol et al., 1998). moreover, the gene encoding telomerase reverse transcriptase (tert) is present (table 1) in trichoplax adhaerens (robertson, 2009b), hydra vulgaris and nematostella vectensis (steele et al., 2011). from all these evidences, and from the detection of the tert gene also in choanoflagellata (robertson, 2009a), we deduce that telomerase is present in all basal metazoa. some of the studies on cnidaria cited above investigated also possible differences in telomere length and telomerase activity in the different tissues and/or life stages of the studied species. in cassiopea andromeda, longer telomeres were found within cells of the bell region of the medusa compared to those of polyps, asexual propagules, or other regions of the medusa (ojimi and hidaka, 2010), but no differences in telomerase activity was found between polyps and the bell region of the medusa (ojimi et al., 2009). in galaxea fascicularis, no differences in telomerase activity were found between somatic and gonad-containing tissues (nakamichi et al., 2012); moreover, no differences in telomere length of its developmental stages (sperm, planula larvae and polyps) were detected (tsuta and hidaka, 2013). on the other hand, differences were observed in the mean telomere lengths during development (sperm > planulae > polyps) of acropora digitifera (tsuta et al., 2014). finally, another study indicated that sperm of ctenactis echinata has longer telomeres than that of somatic tissues and no relationship between telomere length and the weight/age of individuals (ojimi et al., 2012); however, in that work a great variability was present both among replicates and among individuals. however, in recent years it has become apparent that telomerase (e.g., its protein subunit tert) exerts functions that are relevant to cell proliferation but unrelated to telomere maintenance, as shown in mammal models. among the growing list of telomere-independent functions of tert/telomerase is the ability of tert to amplify signaling by the wnt pathway, by serving as a cofactor of the β-catenin/lef transcription factor complex (park et al., 2009). other ascribed telomere-independent effects include demonstrable enhancement of cell proliferation and/or resistance to apoptosis (kang et al., 2004), involvement in dna-damage repair (masutomi et al., 2005), and fig. 4 phylogenetic tree showing telomere motifs in metazoa. tree after dunn et al. (2014). length of branches is arbitrary. rt: retrotransposons. references: 1: (vitkova et al., 2005); 2: (wicky et al., 1996); 3: (niedermaier and moritz, 2000); 4: (traut et al., 2007); 5: (nomoto et al., 2001); 6: (bombarova et al., 2009); 7: (barthelemy et al., 2008); 8: (meyne et al., 1989); 9: (castro and holland, 2002); 10: (li et al., 2007); 11: (sinclair et al., 2007); 12: (sakai et al., 2007); 13: (fairclough et al., 2013). rna-dependent rna polymerase function (maida et al., 2009). consistent with these broader roles, tert can be found associated with chromatin at multiple sites along the chromosomes, not just at the telomeres (masutomi et al., 2005, park et al., 2009). hence, telomere maintenance is proving to be the most prominent of a diverse series of functions to which tert contributes. however, the contributions of these additional functions of telomerase in basal metazoa remain to be elucidated. are telomeres and telomerase involved in basal metazoa senescence? presence of telomerase activity and absence of telomere shortening is in accordance with the lack of senescence seen in basal metazoa (finch, 1990). however, the issue about aging in coral reefs which is seen at a colonial level (rinkevich and loya, 1986) is open, as it is the possibility that this is linked or not to telomere shortening. in mammals, telomerase is differentially regulated in different tissues. this regulation also varies throughout development (table 2). at early stages, telomerase is highly expressed. later on, it is downregulated in a tissue-specific manner (forsyth et al., 2002). this downregulation reaches 236 table 1 telomerase in basal metazoa phylum species trap assay tert gene suberites domuncula + (1) (2) ? leucosolenia sp. (2) ? porifera sycon sp. (2) ? placozoa trichoplax adhaerens (2) + (3) ctenophora pleurobrachia pileus + (2) ? aurelia aurita + (2) ? cassiopea sp. + (4) ? chrysaora hysoscella (2) ? cyanea lamarcki (2) ? galaxea fascicularis + (5) ? hydra vulgaris (2) + (6) madracis auretenra + (7) ? madracis decactis + (7) ? cnidaria nematostella vectensis (2) + (6) references: 1: (koziol et al., 1998); 2: (traut et al., 2007); 3: (robertson, 2009b); 4: (ojimi et al., 2009); 5: (nakamichi et al., 2012); 6: (steele et al., 2011); 7: (zielke and bodnar, 2010). complete silencing in many species (like humans), where almost all adult tissues are telomerasenegative (with the notable exceptions of germ and hematopoietic cells). we can infer that in basal metazoa, as well as in ectotherm chordates, different levels of telomerase activity in the various tissues and/or developmental stages are due to different proliferating rates, while in endotherms an additional level of control exists, in the form of active silencing of telomerase, probably through alternative splicing. a correlation between telomere shortening and cellular senescence was proposed many years ago and is currently widely accepted (reviewed in (collado et al., 2007). senescence is defined as irreversible growth arrest due to reduced number of cellular divisions. hayflick and moorhead (1961) were the first to describe this limitative replicative potential; later others hypothesized that this mechanism was genetically defined and proposed that telomere shortening was correlated to cellular senescence (hastie et al., 1990; rodier and campisi, 2011). when telomeres reach a critical length, the cells arrest proliferation and acquire enlarged morphology expressing senescenceassociated gene like the β-galactosidase and p16. this response has been called “replicative senescence” (harley et al., 1992) and attribute to telomere function the role of “molecular clock” (kirkwood, 2011) that measure cell proliferative history. many works report that some human telomerase negative cells can be immortalized by introduction of htert, while someone require also inactivation of p16, calling in question telomere role in replicative senescence (kiyono et al., 1998; dickson et al., 2000; calado and young, 2012). as seen above, studies on telomeres and telomere biology in basal metazoa are quite scarce. moreover, the issue about aging in some of these species remains opens: in fact, the studies that indicated the onset of “senescence” in hydra oligactis (yoshida et al., 2006) and coral reefs stylophora pistillata (rinkevich and loya, 1986) evidenced an age-related increase of the mortality rate, thus at a population level. differently, biological senescence is usually intended (in vertebrates, for example) as a phenomenon due to (or at least correlated with) cellular senescence. cellular (or replicative) senescence is the phenomenon by which normal cells cease to divide, and it is the result of several biological mechanisms among which a preponderant role has the telomere shortening that ultimately triggers a dna damage response. it would be useful and interesting to investigate if the “demographic senescence”, noticed by the above-mentioned authors, is characterized by the appearance of the classical endpoints of cellular senescence (such as saβgalactosidase) and, more specifically, if it involves telomerase biology and telomere shortening (as demonstrated in mammals). finally, with the aim to better understand the role of telomere both in senescence and in cell proliferation, further studies on the t-loop and/or shelterin complex in basal metazoa should be performed, given the few available data. concluding remark in conclusion, we can infer that all basal metazoa display telomerase activity, as evidenced by our survey of the literature. this seems to point 237 table 2 telomerase activity in different species telomerase activity high medium low absent basal metazoa (1) whole organism fish (2) amphibian (3) germ cells somatic cells human fetus (4) germ cells, stem cells forming tissues human adult (4) germ cells hematopoietic cells dividing cells non-dividing cells references: 1: (ojimi et al., 2009; nakamichi et al., 2012); 2: (lau et al., 2008); 3: (bousman et al., 2003); 4: (forsyth et al., 2002). to an absence of telomere shortening and, consequently, an absence of replicative senescence in these taxa. unfortunately, the lack of a detailed molecular characterization of cellular senescence in basal metazoa is one major limitation in the field. future experiments, using both known and possibly new markers, are one of the crucial steps to understand if replicative senescence is really absent in all these taxa and when it first emerged in evolution. however, basal metazoa can be useful as model organisms not only in studies on emergence of senescence but also in evo-devo investigations on the control of telomerase activity and telomere biology in other cellular processes, such as carcinogenesis. in 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channel neurotoxin (ae1) from sea anemone (actinia equina) mg parisi, mr trapani, l cardinale, m cammarata marine immunobiology laboratory, department of biological, chemical and pharmaceutical science and technology, university of palermo, palermo, italy accepted september 19, 2016 abstract the diversification of anthozoan toxins played an important role in the ability to colonize various ecological niches. in this study we evaluated the hemolytic activity of hplc separated fraction of tentacle extracts of sea anemone actinia equina. toxic components from acid tissue tentacle extracts were investigated by size exclusion and reverse phase hplc to characterize cytolytic molecules. a novel low molecular weight active fraction was sequenced by maldi tof analysis and a protein correspondent to 5.4 kda sodium channel neurotoxin (ae1) from a. equina was identified. synthetic ae1 was assayed and it showed an hemolytic activity against mammalian erythrocytes in a dose dependent manner. cytolytic activity in addition to neuro-inhibitory function could be a further property of ae1 toxin repertoire. key words: actinia equina; hemolysin; na+ channel neurotoxin; sea anemone; ae1 neurotoxin   introduction 309 sea anemones are a group of water-dwelling predators (cnidaria: anthozoa: actiniaria). like sessile benthic organisms, sea anemones produce venoms to immobilize prey and protect themselves. furthermore their adaptive radiation has led to a high specific richness over the course of million years. cnidarians are a rich source of bioactive molecules like antimicrobial peptides, cytolytic proteins and neurotoxins (parisi et al., 2014). the presence, diversification and multifunctionality of the toxins may have played an important role in the ability of colonization and adaptation to ecological niches that change over time. the major fraction of the venom is represented by toxins, which are able to extend the action potential kinetics during the depolarization to the extracellular site on sodium (nav) channels in a membrane potential-dependent manner (stevens et al., 2011). type 1 and 2 na+ channel toxins are composed of 46 49 amino acid residues, except for a. equina toxin ae1 of 54 residues (lin et al., 1996), and cross-linked by three disulfide bridges. they have an anti-parallel β-sheet with four β-strands ___________________________________________________________________________ corresponding author: matteo cammarata department of biological chemical and pharmaceutical science and technology university of palermo, palermo, italy e-mail: matteo.cammarata@unipa.it and a highly flexible loop (arg-14 loop) after their most conserved residue, lacking of any α-helix (frazão et al., 2012). the abundance of toxic proteins extracted from different cnidarian venoms shows its importance in defense and predation actions by sea anemones. the presence of type 1 and 2 toxins seems to be taxonomical related; infact, actiniidae family shows only type 1 toxin, while either type 1 or 2 toxins are present in stichodactylidae family. type 3 na+ channel toxin is only identified in a few species and it is composed of 2732 amino acid residues (moran et al., 2009). na+ and k+ channel peptide toxins are able to paralyze preys immediately. thus, important targets for neurotoxins are voltage gated ion channels in excitable cell membranes (messerli et al., 2006). sea anemone peptides active on nav1 channels are able to produce these effects by acting from the extracellular side of plasma membrane. instead, cytolytic actinoporins permeate cell membranes and form transmembrane pores leading to cell lysis. the actinidae a. equina is commonly found in temperate coastal waters and it’s well known to possess hemolysins such as equinatoxins (anderluh and macek, 2002) and sodium and potassium channel peptide toxins (minagawa et al., 1998). its peptide toxins resulted from the whole body, tentacles or secreted mucus. moreover toxins have been found in specific organs located in a ring   mailto:matteo.cammarata@unipa.it   around tentacles base (acrorhagi) and in cellular components of gastrodermal fluid (parisi et al., 2014). 310 in the paper of lin et al. (1996) the neurotoxic lethal activity to crab na+ channel toxin (ae1) was distinguished from cellular lytic activity suggesting that the studied fraction have cytotoxic activity against erythrocytes because was partial overlapped with the 20 kda equinotoxin. in this study a low molecular weight hemolysin from acid tissue tentacle extracts of sea anemone actinia equina was separated by hplc. sodium channel neurotoxin (ae1) was identified by direct sequencing and its cytolytic activity against mammalian erythrocytes was confirmed by using a synthetic ae1. the hemolytic activity of the isolated molecule against eukaryotic cells has been detected highlighting its multi-functional role, known until now only for its interaction with sodium channels. materials and methods samples preparation the sea anemone actinia equina was collected from a rocky shore of capo gallo coast (palermo, italy). specimens were maintained in a closedcircuit aquarium at 20 °c with aerated sea water (150 l aquaria) and fed every second day with a marine invertebrates filter feeding diet (kent marine inc. wi usa). tentacles were dissected and suspended in tbs (150 mm nacl, 10 mm tris-hcl) in the presence of 10 % acetic acid (acid extract) (payne and ames, 1982). after homogenization in ice with ultra-turrax for 5 min, sample was centrifuged at 21,000g for 20 min at 4 °c and finally supernatant was recovered and stored at −20 °c. fractionation of tentacles crude extract membrane filtration was used for the fractionation of proteins that differ significantly in size. 500 μl of tentacle sample was added to the 10 kda nanosep devices and after 20 min of centrifugation at 6000g the filtrate was transferred from the bottom receiver to a new tube for storage. the sample with low molecular mass protein was filtered through the membrane, collected and stored at −20 °c as down fraction tentacle extract (dfte) separation by hplc chromatography dfte was subjected to size exclusion chromatography using biosuite 250, 10 μm sec, 7.5×300 mm column (waters) on a hplc system (shimadzu scientific instruments, north america). the column was washed two times with 30 ml of tbs. 200 μl of dfte was injected into the column and elution was carried out with tbs at a flow rate of 1 ml/min for 30 min. the chromatogram was recorded with a uv detector at 280 nm (mau). the collected fractions were concentrated by a microconcentrators (3k omega centrifugal devices nanosep). the recovered sample was analyzed using reverse phase c18 column on hplc and showed a single peak named reverse phase tentacle extract (rpte). sds page for separating small peptides crude extract from tentacles, dfte and rpte were analyzed in sds-page (16 % acrylamide) following a procedure for separating small peptides on polyacrylamide gels according to biorad miniprotean ii protocol. after electrophoresis, gels were stained with coomassie brilliant blue r-250 (himedia laboratories, india) and destained in a solution of 50 % (v/v) methanol and 10 % (v/v) acetic acid. samples were subjected to electrophoresis using a running buffer (0.025 m tris-hcl, 0.2 m glycine and 0.1 % sds at ph 8.4) at 80 v at room temperature for 1.5 h. at 80 v approximately 10 ma current per gel was generated at the beginning of the run that dropped slowly to about 3 ma. using these conditions, it was possible to maintain the running buffer and gels at room temperature during the entire migration. sequence determination after coomassie blue dye staining, acrylamide gel bands were excised and send to bms mass spectrometry and proteomics facility of st. andrews university (uk) to perform sequencing. n-gel digestion the gel bands were excised and cut into 1 mm cubes. then these were subjected to in-gel digestion, using a progest investigator in-gel digestion robot (digilab) following standard protocols (shevchenko et al., 1996). briefly gel cubes were destained by washing with acetonitrile and subjected to reduction and alkylation before digestion with trypsin at 37 °c. the peptides were extracted with 10 % formic acid. maldi tof/tof analysis the digest solution (0.5 μl) was applied to maldi target along with alpha-cyano-4hydroxycinnamic acid matrix (0.5 μl, 10 mg/ml in 50:50 acetonitrile:0.1 % tfa) and allowed to dry. maldi ms was acquired using a 4800 maldi tof/tof analyser (absciex) equipped with and: yag 355 nm laser and calibrated using a mixture of peptides. the obtained sequence was analyzed in the swiss-prot or ncbi nr databases. peptide synthesis and hemolysis assay after sequence identification the peptide aei (accession: aab46904.1) was synthesized from genscriptservice and hemolytic activity was tested toward sheep erythrocytes (rbcs) obtained by istituto zooprofilattico della sicilia in alsever solution (0.42 % nacl; 0.08 % c6h5na3o7; 0.045 % c6h8o7; 2.05 % d-c6h12o6; ph 7.2). erythrocytes were washed with pbs (kh2po4 6mm; na2hpo4 0.11 m; nacl 30 mm; ph 7.4) and centrifuged at 400g for 10 min at 4 °c. supernatant was removed and the cells were re-suspended in tbs gel 0.1 % to make a 1 % suspension.     311 fig. 1 a) coomassie stained sds-page gel for separating small peptides of a. equina tentacles tissue acid extract (lane 1), dfte extract fractionated by 10 kda centrifugal nanosep (lane 2) and purified fraction rpte (lane 3); b) chromatogram of the dfte sample separated on the size exclusion column (biosuite 250). elution with tbs at a flow rate of 1 ml/min for 30 min. absorbance was monitored at 280 nm. bsa, chimotripsinogen, ribonuclease a and cytochrome have been used as standard; c)microplate lysis assay toward sheep erythrocytes. total tentacles extract (tentacles), dfte and rpte showed lysis at the concentration of 0.900 µg/ml, 0.510 µg/ml and 0.430 µg/ml respectively. ce: control experiment carried out with rbcs and buffer in tbs gel 0.1 % lysates are serially diluted twofold and incubated with an equal volume of 1 % sheep erythrocyte suspension for 1 h at 37 °c. after centrifugation the absorbance of the supernatants was determined at 545 nm. the values were at least the mean of three experiments, each performed in duplicate; d) hplc chromatograms of a.equina dfte using reverse phase c18 column on hplc that show a single peak (rpte); e) sequencing of of purified sample by reversed phase hplc rpte by n-gel digestion/ maldi tof analysis. blast searching showed a correspondence with the ae1 toxin (aab46904) and delta-actitoxin-aeq2a (q9njq2.1). aminoacid in boxes are referred to signal peptide and pro-peptide. mature peptide extending from position 29 to 82 is mature peptide. asterisks indicate position of cysteines involved in three disulphide bridges.     for the microplate assay, 25 μl of fraction purified by hplc or serial (two-fold) dilution was mixed with an equal volume of rbcs suspension in 96-well round-bottom microtiter plates (nunc, roskilde, denmark). after 1 h incubation at 37 °c the lytic activity was recorded as the reciprocal of the highest dilution showing complete rbcs lysis. for the quantitative hemoglobin release evaluation, 500 μl of synthetic peptide (0.5 mg/ml) in tbs was mixed with an equal volume of freshly rbcs suspension (8x106 cells) in pbs. the mixture was incubated with continuous and moderate shaking at 37 °c for 2 h and centrifuged at 1500g for 5 min at 4 °c. the supernatant was separated and the amount of released hemoglobin (hb) was estimated by reading the absorbance at 545 nm in spectrophotometer. the degree of hemolysis was determined according to the equation: measured release spontaneous release % hemolysis = ------------------------------------------------x 100 complete release spontaneous release complete hemolysis was estimated suspending erythrocytes in distilled water. for each experiment three samples were assayed. in addition, after carrying out cytotoxicity assay, erythrocytes were recovered by centrifugation at 112g for 10 min at 4 °c and lying on glass slides to be observed with phase contrast microscope. 312 results electrophoretic analysis crude extract components from a. equina tentacles and the dfte were analyzed in sds page and reported in fig 1a (lane 1 and 2). a procedure for separating small peptides on polyacrylamide gels has allowed the identification of several bands in lane 1 and a band with a low molecular mass (<6kda) in lane 2. separation by hplc of low weight protein the dfte was analyzed by size exclusion chromatograph yielding the chromatogram shown in figure 1b. the chromatogram showed a single peak identified at 11 min of the elution time. the recovered sample was analyzed using reverse phase c18 column on hplc and showed a single peak (rpte) reported in fig. 1d corresponding to a 6 kda band approximately (fig. 1 a, lane 3). hemolytic activity the results of microplate assays (fig. 1c) showed the presence of hemolytic activity toward rbcs up to 1:64 serial dilution for crude extract of tentacles and to 1:8 for the dfte (0.572 abs) and rpte (0.482 abs). determination of sequence and synthesis of the hemolytic polypeptide n-gel digestion and maldi tof analysis of rpte showed several partial peptide masses which, by post source decay analysis and database searching, were attributed to one protein of 5.5 kda molecular weight. protein database search confirmed that the complete sequence corresponded to ae1 toxin (aab46904.1) and delta-actitoxin-aeq2a (q9njq2) isolated from a. equina (fig. 1e). cytolytic activity of synthetized neurotoxin ae1 figure 2a showed cell membranes lysis and the presence of erythrocyte ghosts after treatment of rbcs with the synthetic ae1 peptide. the synthetic ae1, starting with final concentration of 125 µg, was assayed for its hemolytic activity by a spectrophotometric quantization. lysis was recorded and after 1 h of incubation in polyethylene tubes a value of 58 % hemolysis was evaluated and the level of cytotoxicity decreased in a dose dependent manner (figs 2b and c). as shown in figure 2b, a visible hemoglobin release is present in tube that contained the synthetic hemolysin. it was almost transparent in the control. the time course of the reaction indicated a lysis after 10 min, but the stable value was obtained after 1 h (data not shown). the statistical evaluation was performed with one-way analysis of variance (anova), and different groups were compared by using tukey’s t-test. standard deviations were calculated on three experiments. p < 0.05 was considered statistically significant. discussion in sessile anthozoa, cytolytic polypeptides have been classified at least in four groups. they were classified based on their molecular weight in a) 5 8 kda peptides with antihistamine activity, b) ~20 kda pore-forming proteins with activity of lysis versus cell membrane inhibited by sphingomyelin, c) ~3040 kda cytolysins with or without phospholipase a2activity, and d) a putative group of proteins represented at present only by a 80 kda (stabili et al., 2015). two most known molecules isolated from the sea anemone actinia equina are isoforms of equinatoxin ii (eqtii). toxins can act on common sites or on different subtypes of specific channels for na+ showing toxic and biologically significant differences (bosmans and tytgat, 2007). in this work, a neuropeptide from acid (10 % acetic acid) extract of a. equina tentacles has been isolated by hplc and the lytic capability against mammalian erythrocytes has been demonstrated. the tentacles crude extract exhibited many bands in sds page and resulted mainly enriched in the component of 20 kda correspondent to cytolysin equinatoxin ii (eqii). instead, a new hemolysin of about 6 kda was isolated by filtration and size exclusion and reverse phase hplc. following mass spectrometry, the low molecular weight hemolysin was characterized, sequenced and identified as a na+ channels neurotoxin (ae1) (lin et al., 1996). the neurotoxic lethal activity to crab na+ channel toxin (ae1) was distinguished from cellular lytic activity suggesting that “in gel filtration on sephadex g50 of crude extract toxin(s) with lethal activity to crab was distinguished from hemolysis reasonable assumed to be equinotoxins the former appears at around fraction 26 and the latter fraction 21” (lin et al., 1996).      fig. 2 a) sheep erythrocytes (rbcs) observed at the light microscope after treatment with synthetic peptide ae1 (125 µg/ml). arrows indicate the ghosts of erythrocytes. (bar: 10 µm); b) release of hemoglobin after 30 min of incubation with synthetic peptide. control (ce), total lysis in distilled water (et) and lysis after treatment with synthetic peptide ae1 (125 µg/ml); c) hemolytic activity evaluation of synthetic ae1 from a. equina toward rbcs in tbs at different concentrations. ce: control of erythrocytes suspended in tbs; the values were at least the mean of three experiments, each performed in duplicate. **p < 0.01 and ***p < 0.001 respect to the control. in this study a hemolysin with low molecular weight from acid tissue tentacle extracts of sea anemone actinia equina was isolated by hplc starting with a <10 kda fraction in which the equinotoxin (20 kda) was clearly separated from ae1 containing fraction. in order to verify the hemolytic activity of this identified neurotoxin by sequencing, a synthetic peptide was also produced. the results of quantification assay for hemoglobin release by red blood cells lysis indicated that, the synthetic peptide ae1 shows also hemolytic activity as well as the purified hemolysin. 313 since voltage-gated na+ channels are responsible for the conduction of electrical impulses in most excitable tissues in the majority of animals, they have become important targets for the toxins of venomous animals, from sea anemones to mollusks, scorpions, spiders and even fishes (wanke et al., 2009). the discovery of cytolytic activity in addition to inhibitory effect could support the hypothesis of evolutionary trend to combine two venomous functions in one compound: ion channel inhibitor and membranolytic activity. another example of neurotoxic/cytotoxic proteins resides in the components isolated from the sea anemones stichodactyla mertensii and s. haddoni (veeruraj et al., 2008) in which hemolytic activity was estimated toward several types of erythrocytes.     moreover, molecules with variable functions are found in animals including vertebrate could also take advantage of the multi-functionality of some of their bioactive molecules (ovchinnikova et al., 2006; peigneur et al., 2011; trapani et al., 2014). the unique feature of a multiple role within one molecule is intriguing. the production of toxins is an energy demanding task for an organism and the dual effects contained within one molecule can be seen as an efficient and economic manner to save energy with a maximal pharmacological output (trapani et al., 2016). neurotoxin-like protein with cytotoxic activity against different human cancer cell lines was purified from the venom of the snake najahaje (el hakim et al., 2008). its mechanistic role in the cell death was explored too. cstx-1, the main neurotoxic acting peptide in the venom of the spider cupiennius salei possess membranolytic activities caused by its c-terminal tail (nentwig et al., 2012). this peptide exhibits neurotoxin function because it inhibits l-type ca2+-channels and acts as a lytic peptide toward prokaryotic and eukaryotic cell membranes. 314 evolution has ensured sessile animals as sea anemones to develop a cysteine-rich peptides capable of affecting different extracellular sites of channel proteins. the venom in fact is abundant of low molecular mass neurotoxins with differences in structure, site of action and target preference. the mechanisms enable specimens to subdue a broader range of prey even if some of them do not express specific ion channels that are targeted by neurotoxins. at the same time this mechanism could prevent the development of resistance to a single venom compound. although the cytolytic mechanism of action is unknown, the hypothesis of combination of two venomous functions in one compound is certainly an evolutionary fascinating strategy. acknowledgments this work was supported by mc ffr (university of palermo); a research grant from the italian ministry of education (prin 2010-2011 n. 20109xzepr_007), co-funded by the university of palermo, italy and it@cha, “ricerca e competitività 2007 2013” pon01 00625 research grants. references anderluh g, macek p. cytolytic peptide and protein toxins from sea anemones (anthozoa: actiniaria). toxicon 40: 111-124, 2002. bosmans f, tytgat j. voltage-gated sodium channel modulation by scorpion α-toxins. toxicon 49: 142-158, 2007. el hakim a, walaa h, salama e, mahdi m, wahby a. assessment of the phospholipase a2 patterns of some egyptian snakes. venom. bull. nrc. 33: 181-191, 2008. frazão b, vasconcelos v, antunes a. sea anemone (cnidaria, anthozoa, actiniaria) toxins: an overview. mar. drugs 10: 18121851, 2012. lin y, ishida m, nagashima y, shiomi k. a polypeptide toxin in the sea anemone a. equina homologous with other sea anemone sodium channel toxins: isolation and amino acid sequence. toxicon 34: 57-65, 1996. messerli s, greenberg, r. cnidarian toxins acting on voltage-gated ion channels. mar. drugs 4: 70-81, 2006. minagawa s, ishida m, nagashima y, shiomi k. primary structure of a potassium channel toxin from the sea anemone actinia equina. febs 427: 149-151, 1998. moran y, gordon d, gurevitz m. sea anemone toxins affecting voltage-gated sodium channelsmolecular and evolutionary features. toxicon 54: 1089-1101, 2009. kuhn-nentwig l, fedorova im, lüscher bp, kopp ls, trachsel c, schaller j , et al. a venomderived neurotoxin, cstx-1, from the spider cupiennius salei exhibits cytolytic activities. j. biol. chem. 287: 25640-25649, 2012. parisi mg, trapani mr, cammarata m. granulocytes of sea anemone actinia equine (linnaeus, 1758) body fluid contain and release cytolysins forming plaques of lysis. inv. surv. j. 11: 39-46, 2014. ovchinnikova tv, baladin sv, aleshina gm, tagaev aa, leonova yf, krasnodembsky ed, et al. aurelin, a novel antimicrobial peptide from jellyfish aurelia aurita with structural features of defensins and channel-blocking toxins. biochem. biophys. res. comm. 348: 514-523, 2006. payne sh, ames bn. a procedure for rapid extraction and high-pressure liquid chromatographic separation of the nucleotides and other small molecules from bacterial cells. analyt. biochem. 123: 151-161, 1982. peigneur s, billen b, derua r, waelkens e, debaveye s, bèress l, et al. a bifunctional sea anemone peptide with kunitz type protease and potassium channel inhibiting properties. biochem. pharmacol. 82: 81-90, 2011. shevchenko a, wilm m, vorm o, mann m. mass spectrometric sequencing of proteins silverstained polyacrylamide gels. anal. chem. 68: 850-858, 1996. stevens m, peigneur s, tytgat j neurotoxins and their binding areas on voltage-gated sodium channels. front pharmacol. 2: 71-75, 2011. trapani mr, parisi mg, toubiana m, coquet l, jouenne t, roch p, et al. first evidence of antimicrobial activity of neurotoxin 2 from anemonia sulcata (cnidaria). inv. surv. j. 11: 182-191, 2014. trapani mr, parisi mg, maisano m, mauceri a, cammarata m. old weapons for new wars: bioactive molecules from cnidarian internal defense systems.  cent. nerv. syst. agents med. chem. 15, 2015 [in press]. veeruraj a, arumugam m, ajithkumar t, balasubramanian t. isolation and biological properties of neurotoxin from sea anemone (stichodactylamertensii, s. haddoni). int. j. toxicol. 5: 14, 2008. wanke e, zaharenko aj, redaelli e, schiavon e. actions of sea anemone type 1 neurotoxins on voltage-gated sodium channel isoforms. toxicon 54: 1102-1111, 2009.   http://www.ncbi.nlm.nih.gov/pubmed/?term=bosmans%20f%5bauth%5d http://www.ncbi.nlm.nih.gov/pubmed/?term=tytgat%20j%5bauth%5d http://benthamscience.com/journals/central-nervous-system-agents-in-medicinal-chemistry/volume/15/ isj 13: 122-133, 2016 isj 13: 122-133, 2016 issn 1824-307x research report chemical and organic fertilizers affect physiological performance and antioxidant activities in myzus persicae (hemiptera: aphididae) m mardani-talaee1, a zibaee2, g nouri-ganblani1, j razmjou1 1department of plant protection, faculty of agricultural sciences, university of mohaghegh ardabili, iran 2department of plant protection, faculty of agricultural sciences, university of guilan, rasht, iran accepted april 6, 2016 abstract myzus persicae is a widespread and polyphagous insect that causes severe damages to hundreds of host plants. in the current study, zinc sulfate and vermicompost as chemical and organic fertilizers, were added into cultural soil of capsicum annuum to determine their effects on physiology and antioxidant activities of m. persicae. the aphids reared on zinc sulfate-treated culture showed the highest activities of general protease, trypsin, cathepsins, carboxypeptidase and lipase but activities of chymotrypsin and aminopeptidase were the highest in vermicompost-treated culture. although activities of α-amylase in the fertilizer-treated cultures were higher than control but activities of αand β-glucosideases showed the highest values in zinc sulfate and vermicompost treatments, respectively. aspartate aminotransferase and γ-glutamyl transferase showed the highest activity in the aphids reared on the vermicompost-treated culture but alanine aminotransferase activity got the lowest value in fertilizer-treated cultures. activities of aldolase and lactate dehydrogenase in the fertilizer-treated aphids were higher than those of control and vermicompost-treated aphids, but alkaline phosphatase showed the lower activity although activity of acid phosphatase decreased in vermicomposttreated aphids compared to other treatments. activities of antioxidant enzymes were found to be the highest in the aphids fed on vermicompost-treated culture including glucose-6-phosphate dehydrogenase, superoxide dismutase, peroxidase and ascorbate oxidase but catalase in vermicompost treatment had lower activity than control and zinc-sulfate treatments. also, malondialdehyde and rssr/rsh ratio demonstrated higher values in the aphids fed on zinc sulfateand vermicompost-treated plants than control, respectively. finally, the amounts of glycogen and triglyceride revealed the highest values in zinc sulfate-treated plants compared to other treatments. these results indicated significant effects of fertilizers on physiology and antioxidant activity of m. persicae which are important to be considered in integrated pest management programs. key words: myzus persicae; fertilizer; physiological performance; antioxidant indices   introduction the green peach aphid, myzus persicae (hemiptera: aphididae), is one of the most important insect pests worldwide because of its severe damages to many crop plants, vegetables and fruit trees as well as transmission of plant pathogenic viruses such as potato leaf roll virus (plrv), potato virus y (pvy), pepper mottle virus (pep mov), tobacco etch virus (tev) and cucumber mosaic virus (cmv) (hill, 1983; robert et al., 2000). although population outbreaks of m. persicae are ___________________________________________________________________________ corresponding author: arash zibaee department of plant protection faculty of agricultural sciences university of guilan, rasht, iran e-mail: arash.zibaee@gmx.com, arash.zibaee@guilan.ac.ir somehow suppressed by synthetic insecticides but strong selection pressure of chemical spraying induces resistance to the most registered insecticides so other control methods need to be investigated for appropriate control (bolandandam et al., 2004). although fertilizers are recommended to increase crop yields but they can affect pest populations leading to to use control procedures (patriquin et al., 1995; arancon et al., 2006; edwards et al., 2009). in fact, fertilizers can fluctuate amounts of defensive chemical components in plants which finally change ecological fitness and physiological performance of herbivorous insects (edwards et al., 2009; mardanitalaee et al., 2016). numerous studies have reported the effects of macronutrient chemical 123   mailto:arash.zibaee@gmx.com mailto:arash.zibaee@guilan.ac.ir fertilizers (such as n, p and k) on population dynamics of insect pests (lu et al., 2007). since fertilizers may result in higher growth rate and population increase of herbivorous insects through improving nutritional quality of host plants (edwards et al., 2009). moreover, fertilizers residue have raised a great concern of consumers in recent years because of their chemical constituent. so, organic fertilizers may be more appropriate because of their least effects on environment or residual contaminations. in case, vermicompost is an organic fertilizer produced through the interactions between earthworms and microorganisms in a mesophilic process from organic wastes. it reduces ph and c:n ratio in soil, stabilizes the organic matter and makes nutrients readily available to plants (yardim et al., 2006). soil amendment with vermicompost have reduced population growth rates in some herbivores such as manduca quinquemaculata, acalymma vittatum, diabrotrica undecimpunctata (yardim et al., 2006), leptinotarsa decemlineata (mardani-talaee et al., 2015), pseudococcus sp., teranychus urticae, m. persicae (arancon et al., 2002, 2006; edwards et al., 2009; mardani-talaee et al., 2016), and aphis gossypii (razmjou et al., 2011). in a previous study, we found that vermicompost increased levels of phenolic compounds in the leaves of bell pepper and thereby decreased life table parameters of m. persicae (mardani-talaee et al., 2016). on the other hand, our results demonstrated an induced resistance in bell pepper cultured in vermisompost-treated culture (mardani-talaee et al., 2016). in case, physiological parameters of treated and non-treated aphids by fertilizers must be determined to better understanding of observed changes. so, the current study was conducted to compare potential changes in physiological processes of m. persicae induced by fertilizers. in details, a chemical fertilizer (zinc sulfate) and vermicompost (30 %) were separately added into cultural soil of c. annum to find their effects on digestion, intermediary metabolism and antioxidant activities of m. persicae under greenhouse conditions. these findings will increase our understanding on beneficial or detrimental effects of fertilizers to better management of m. persicae in greenhouses. materials and methods plant sources, insect colony and fertilizer treatments the sandy loam soil collected from a fallow potato field in ardabil plain (38°14´ n and longitude 48°19´ e) was used to grow c. annuum (cv. california wonder). seeds of bell pepper, c. annuum were grown in an aphid-free greenhouse set at 25 ± 5 oc, 60 ± 5 % of relative hunidity and a photoperiod of 14:10 (l:d) h. the aphids were collected from a tomato field from meshkin-shahr (ardabil province), iran and transferred to cultured c. annuum at six-leaf stage. to maintain a suitable aphid colony, some aphids were transferred from infested plants to new young plants every week. experimental treatments here were; i) bell pepper grown in field-collected soil as control treatment; ii) bell pepper grown in the field-collected soil sprayed on 4 6 expanded leaves stages of c. annuum with zinc sulfate (provided by iranian soil and water research institute in karaj) at the concentration of 0.001 % (50 ml per plant). iii) bell pepper grown in the same soil amended with 30% of vermicompost (obtained from cattle manure of pars koud company, gorgan, iran) which contained 1.8 % n, 3.9 mg/kg p, ph of 7.3 and ec of 2.2 ds/m before planting. sample preparation for biochemical assays adults of m. persicae from control and fertilized cultures were randomly selected and homogenized in ice cold nacl solution (0.15 m) in proportion of 20 aphids per ml of saline solution. the samples were then centrifuged at 20000 g for 5 min at 4 ºc (dubovskiy et al., 2008). supernatants were collected and stored at -20 ºc to onset of the biochemical experiments (< a week). determination of digestive enzyme activities general proteolytic activity general proteolytic activity in control and treated aphids by fertilizers were determined based on the method of elpidina et al. (2001) using table 1 effects of fertilizers on activities of digestive proteases in m. persicae adults statistic (mean±se u/mg protein) treatments general protease1 trypsin chymotrypsin elastease cathepsinl cathepsinb aminopeptidases carboxypeptidases control 0.062±0.016 b 4.568±0.336 b 2.818±0.759 c 38.51±1.950 c 2.819±0.459 b 3.885±0.374 c 0.134±0.017 c 1.123±0.125 a vermicompost (30%) 0.040±0.008 b 2.829±0.154 b 12.143±0.432 a 60.15±1.890 b 14.97±1.430 b 6.000±1.150 b 5.150±0.797 a 0.657±0.302 b zinc sulfate 0.224±0.027 a 14.842±0.996 a 5.25±0.231 b 128.82±0.682 a 23.25±1.280 a 13.280±1.080 a 0.550±0.057 b 1.092±0.0519 a 1activity unit of all enzymes were u per mg protein except for general protease as od/min. the means followed by different letters in a column are significantly different [p < 0.01, tukeys (hsd)]. 124   azocasein (2 %) as substrate. briefly, 40 µl of azocasein solution, 80 µl of universal buffer [20 mm, (frugoni, 1957; mardani-talaee and zibaee, 2015)] and 20 µl of sample were incubated for 60 min at 37 ºc. then, proteolysis was stopped by adding 80 µl of 30 % trichloroacetic acid (tca) and precipitation was achieved by cooling at 4 °c for 5 min prior to centrifugation at 20,000g for 10 min. finally, an equal volume of naoh (2 n) was added and absorbance was read at 450 nm (microplate reader, awareness statfax, 3200, usa). specific proteolytic activity based on rahbe et al. (2003), activities of trypsin, chymotrypsin, cathepsin b, cathepsin l, aminoand carboxypeptidase were determined in control and fertilized-treated aphids. trypsin and chymotrypsin activities were assayed using 1 mm of bapna (nabenzoyldl-arginine-p-nitroanilide) and saapppna (n-succinyl-alaninealanine-prolinephenylalanine-p-nitroanilide), as substrates, respectively. substrates (20 µl) were separately added into 50 µl of universal buffer (20 mm, ph 8) and incubation was initiated by adding 10 µl of enzyme solution for 10 min at 30 ºc. the reaction was terminated by adding 100 µl of tca (30 %) and absorbance was read at 405 nm. cathepsin b and l activities were determined using z-ala-argarg 4-metjoxy-bnaphtylamide acetate (1 mm) and n-benzoyl-phe-val-arg-p-nitroanilide hydrochloride (1 mm), respectively. substrates (20 µl) were separately added into 50 µl of universal buffer (20 mm, ph 5) and incubation was initiated by adding 10 µl of enzyme solution for 10 min at 30 ºc. the reaction was terminated by adding 100 µl of tca (30 %) and absorbance was read at 405 nm. activities of exopeptidases were determined using hippuryl-l-arginine and hippuryl-l-phenylalanine as the specific substrates (1 mm) of carboxyand aminopeptidases, respectively. substrates (20 µl) were separately added into 50 µl of universal buffer (20 mm, ph 7) and incubation was initiated by adding 10 µl of enzyme solution for 10 min at 30 ºc. the reaction was terminated by adding 100 µl of tca (30%) and absorbance was read at 340 nm. α-amylase assay activity of α-amylase was determined based on the method of bernfeld (1955) using starch (1 %) and dinitrosalicylic acid (dns). briefly, 40 µl of starch solution (1 %), 80 µl of universal buffer (ph 7) and 20 µl of enzyme sample were incubated for 30 min at 35 ºc. then, 100 µl of dns was added and the tubes containing reaction mixture were additionally incubated for 10 min at boiling water. finally, 100 µl of the reaction mixture was poured into wells of microplate and absorbance was read at 540 nm. αand β-glucosidase assay according the method of silva and terra (1995), αand β-glucosidase activities were assayed by adding 20 µl of p-nitrophenol αglucopyranoside for α-glucosidase (5 mm) and pnitrophenol β -glucopyranoside for β-glocusidase (5 mm) (separately) in 50 µl of universal buffer (ph 7). the incubation was initiated by adding 10 µl of the enzyme solution for 10 min prior to read absorbance at 405 nm. lipase assay activity of lipase was determined using the method of tsujita et al. (1989) in control and fertilized-treated aphids. ten microliter of sample and 20 μl of p-nitrophenyl butyrate (27 mm) as substrate were added into 50 µl of universal buffer (ph 7), mixed thoroughly and incubated at 37˚c. after 1 min, 100 μl of naoh (1 m) was added and absorbance was read at 405 nm. determination of intermediary metabolism in m. persicae assay of alanine (ec 2.6.1.1) and aspartate (ec 2.6.1.1) aminotranferases this is a chlorometric assay using 2,4dinitrophenyl hydrazine to synthesize pyruvate hydrazine by combination of pyruvate with 2,4dintitrophenyl puruvate (thomas, 1998). based on a kit provided by biochem company (tehran, iran), reagent a (for ast) and reagent b (for alt) were incubated separately with reagent d for 5 min. then, 10 µl of the enzyme solution was added and incubation continued for 60 min. at the end, reagent c was added and absorbance was read at 340 nm. specific activity was calculated by dividing absorbance with protein content in sample. assay of γ-glutamyl transferase the assay was done by the method of szasz (1976) using a kit provided by ziestchem diagnostic company (tehran-iran). the reaction mixture consisted 50 μl of buffer reagent, 20 μl of substrate reagent (l-γ-glutamyl-3-carboxy-4-nitrianilide) and 10 μl of the sample. incubation was prolonged for 3 min and absorbance was read at 405 nm (ziestchemdiagnostic co., tehran-iran). specific activity was calculated by dividing absorbance with protein content in sample. assay of aldolase as the instruction of manufacturer, ziestchem diagnostics company (tehran-iran), 50 µl of buffer reagent, 25 µl of substrate reagent (fructose-1,6 di-phosphate), 10 µl of cofactor reagent (nadh) and 20 µl of sample were incubated for 5 min prior to read absorbance at 340 nm (pinto et al., 1969). assay of lactate dehydrogenase the method of king (1965) was used to evaluate activity of lactate dehydrogenase (ldh). to standardize volumes, 0.2 ml of nad+ solution was added to the test tubes and 0.2 ml of water was added to control test tubes, each containing 1 ml of the buffered substrate and 0.01 ml of the sample was also added to the test tubes. test tube samples were incubated for exactly 15 min at 37 °c and then arrested by adding 1 ml of color reagent (2,4dinitrophenyl hydrazine) to each tube and the incubation continued for an additional 15 min. then, the contents were cooled at room temperature, 10 ml of 0.4 n naoh was added to each tube to make the solutions strongly alkaline. at exactly 60 s after adding of alkali to each tube, the intensity of color was measured at 340 nm. 125   table 2 effect of fertilizers on activities of αamylase, glucosidases and lipase in m. persicae adults statistic (mean±se u/mg protein) treatments α-amylase αglucosidases ß-glucosidases lipase control 5.870±0.142 b 43.49±2.560 b 6.500±1.480 c 79.790±1.640 b vermicompost (30%) 7.120±0.194 a 29.32±1.894 c 25.320±9.120 a 51.19±1.059 c zinc sulfate 7.346±0.759 a 65.88±3.730 a 15.723±0.512 b 169.94 ±2.271 a the means followed by different letters in a column are significantly different [p < 0.01, tukeys (hsd)]. assay of acid (ec 3.1.3.2) and alkaline (ec 3.1.3.1) phosphatases the assays were carried out as described by bessey et al. (1946). the buffered substrate (phosphate buffer, 0.02 m, ph 7.2) was incubated with samples for 30 min. alkali was added to stop the reaction and to adjust ph for determination of product concentration. the spectral absorbance of p-nitrophenolate was maximal at 340 nm. the molar absorbance of p-nitrophenolate at 400 nm is approximately double that of p-nitrophenyl phosphate at 310 nm. on converting the pnitrophenolate into p-nitrophenol by acidification, the absorption maximum shifted to approximately 320 nm with no detectable absorption at 405 nm. determination of antioxidant activities in m. persicae catalase (cat) activity was measured based on the method of (wang et al., 2001). briefly, 100 μl of sample was added into 500 μl of hydrogen peroxide (1 %), incubated at 28 °с for 10 min and the activity was determined as the δa at 240 nm/min/mg protein. superoxide dismutase (sod) activity was determined based on mccord and fridovich (1969) as the reduced rate of nbt (nitro blue tetrazolium) by the superoxide anion due to xanthine oxidation by xanthine oxidase (mccord and fridovich, 1969). sample (100 μl) was added into 500 μl of the reaction solution containing 70 μm of nbt; 125 μm of xanthine; both dissolved in pbs and the xanthine oxidase solution [(100 μl; 10 mg of bovine albumin; 100 μl of xanthine oxidase (5.87 units/ml); dissolved in 2 ml of pbs]. the reaction mixture was incubated in darkness for 20 min at 28 °с prior to calculate the activity as differences in absorbance between a sample containing the mixture and a clean reagent mixture at δa 560 nm/min/mg protein. peroxidase activity was assayed based on by the method of addy and goodman (1972). the reaction mixture consisted 500 µl of buffered pyrogallol [0.05 m pyrogallol in 0.1 m phosphate buffer (ph 7.0)] and 500 µl of h2o2 (1 %) prior to adding 100 µl of enzyme extract. changes in absorbance was measured at 430 nm for every 30 seconds in 2 minutes. the peroxidase activity was calculated using an extinction coefficient of oxidized pyrogallol (4.5 litres/mol). ascorbate peroxidase assay was carried out according to asada (1984). the reaction mixture consisted 100 µl of sample and 250 µl of 67 mm potassium phosphate buffer (ph 7) containing 2.5 mm ascorbic acid and 200 µl of 30 mm h2o2. absorbance was monitored at 290 nm for 5 min. mda (malondialdehyde) concentration was determined by considering the process of lipid peroxidation due to formation of mda. based on bar-or et al. (2001), briefly, 100 μl of 20 % trichloroacetic acid was mixed with 100 μl of the sample prior to be centrifuged at 15,000g for 10 min at 4 °c. the obtained supernatant was mixed with 100 μl of 0.8 % tba reagent, and the mixture was incubated at 100 °c for 60 min prior to read absorbance at 535 nm. the mda concentration is reported as amount of mda produced per mg protein using a molar extinction coefficient of 1.56×105 m-1 cm-1. activity of glucose-6-phosphate dehydrogenase was measured based on balinsky and bernstein (1963). the solution containing 100 μl tris-hcl buffer (100 mm, ph 8.2), 0.2 mm nadp and 0.1m of mgcl2 was taken in a cuvette along with 100 μl of water and suitable aliquots of enzyme extract. the reaction was initiated by adding 100 of 6 mm glucose-6-phosphate and od increase was measured at 340 nm. the activity of the enzyme is expressed as μmol/min/mg protein. the concentrations of oxidized (rssr) and reduced (rsh) thiols were determined based on the method of khramtsov et al. (1997). initially, rssrs were decomposed for 20 min by 1 m of hydrochloric acid to form rsh prior to neutralize the mixture ph by sodium hydroxide. then, fifty microliters of the homogenate was mixed with 500 μl of 0.1 % dntb solution in pbs, and the mixture was incubated for 10 min at 37 °с. the absorbances of rsh and rsh and rssr were measured at 405 nm. the concentration of rssr was calculated as the difference between the final concentration of reduced thiols after reduction by hydrochloric acid (rsh and rssr) and the initial concentration of one rsh in the sample. the results are presented as the ratio of rssr to rsh. 126   table 3 effects of fertilizers on activities of transaminases in m. persicae adults statistic (mean±se u/mg protein) treatments ast alt γ-gt control 0.121±0.020 b 0.182±0.022 a 0.256±0.040 b vermicompost (30%) 0.242±0.044 a 0.137±0.027 b 0.680±0.247 a zinc sulfate 0.147±0.018 b 0.167±0.023 ab 0.389±0.088 b ast, aspartate amino transferase; alt, alanine amino transferase; γ-gt, γ-glutamyl transferase. the means followed by different letters in a column are significantly different [p < 0.01, tukeys (hsd)]. determination of storage macromolecules in m. persicae triglyceride determination the amount of triglyceride was measured using a diagnostic kit provided by pars azmoon company (tehran, iran) in m. persicae adults. briefly, 10 µl of sample was incubated with 70 µl of reagent solution containing phosphate buffer (50 mm, ph 7.2), 4-chlorophenol (4 mm), adenosine triphosphate (2 mm), mg2+ (15 mm), glycerokinase (0.4 ku/l), peroxidase (2 ku/l), lipoprotein lipase (2 ku/l), 4-aminoantipyrine (0.5 mm) and glycerol-3phosphate-oxidase (0.5 ku/l) for 20 min at 25 °c (fossati and prencipe, 1982). then, absorbance of sample mixture and reagent was read at 546 nm. the following equation was used to calculate the amount of triacylglyceride: mg/dl = glycogen determination bodies of 10 aphids were immersed in 1 ml of 30 % koh with na2so4; covered with foil (to avoid evaporation) and put in boiling water for 30 min. tubes then were shaken and cooled in ice. two milliliters of 95 % etoh was added and the tubes were shaken again and incubated in ice for 30 min. followed by centrifugation at 22,000g for 30 min, supernatant was removed and the pellets (glycogen) were re-dissolved in 1 ml of distilled water before being re-shaken. incubation continued after adding 5 % phenol for 30 min prior to read absorbance at 492 nm. standard of glycogen was prepared as 0, 25, 50, 75 and 100 mg/ml to calculate amount of glycogen in sample (chun and yin, 1998). protein determination the method of lowry et al. (1951) was used to determine protein concentration in the control and treated aphids (ziestchem. co., tehran, iran). statistical analysis normality of data was tested by kolmogorovsmirnov method prior to analysis by one-way analysis of variance (anova) followed by comparison of the means with tukey post hoc honestly significant difference (hsd) test at α = 0.05. results effects of fertilizers on digestive enzymes statistical differences were found in activities of general protease, serine proteases, cysteine proteases and exopeptidases (aminoand carboxypeptidases) in m. persicae fed on control and fertilizer-treated c. annum (table 1). activities of general protease (f = 27.14; df = 2, 8; p < 0.01), trypsin (f = 111.95; df = 2, 8; p < 0.01), elastase (f = 149.79; df = 2, 8; p < 0.01), cathepsin l (f = 80.84; df = 2, 8; p < 0.01), cathepsin b (f = 27.88; df = 2, 8; p < 0.01) and carboxypeptidases (f = 1.86; df = 5, 17; p < 0.01) were the highest in the aphids fed on the zinc sulfate treated-plants while the highest activities of chymotrypsin (f = 30.69; df = 2, 8; p < 0.01) and aminopeptidase (f = 36.37; df = 2, 8; p < 0.01) were found in vermocompost treatment (table 1). for all proteases, the lowest activities were observed in control treatments except for carboxypeptidase (table 1). the highest activities of α-amylase (f = 9.75; df = 2, 8; p < 0.01), αglucosidase (f = 9.30; df = 2, 8; p < 0.01) and lipase (f = 416.68; df = 2, 8; p < 0.01) were found in the aphids fed on c. annum treated by zinc sulfate although no significant difference was observed in α-amylase of the aphids fed on both zinc sulfateand vermicompost-treated plants (table 2). aphids fed on control and vermicomposttreated c. annum showed the highest and the lowest ß-glucosidase (f = 3.10; df = 2, 8; p < 0.01) activity, respectively (table 2). the lowest activities of α-amylase and ß-glucosidase were obtained in control but the aphids fed on vermicompost-treated c. annum showed the lowest activities of αglucosidase and lipase (table 2). effects of fertilizers on intermediary metabolism of m. persicae significant differences were found in activities of aspartate amino transferase (ast), alanine amino transferase (alt) and γ-glutamyl transferase (γ-gt) of m. persicae fed on different fertilizer 127   table 4 effects of fertilizers on activities of aldolase, alkaline and acid phosphatase and lactate dehydrogenase in m. persicae adults statistic (mean±se u/mg protein) treatments aldolase alp acp ldh control 0.424±0.099 b 0.104±0.002 a 0.337±0.033 a 0.259±0.049 b vermicompost (30%) 0.773±0.030 a 0.039±0.026 b 0.150±0.012 b 0.796±0.097 a zinc sulfate 0.931±0.034 a 0.068±0.017 b 0.353±0.027 a 0.671±0.166 a alp, alanine phosphatase; acp, acid phosphatase; ldh, lactate dehydrogenase. the means followed by different letters in a column are significantly different [p < 0.01, tukeys (hsd)]. treatments (table 3). ast (f = 4.73; df = 2, 8; p < 0.01) and γ-gt (f = 2.13; df = 2, 8; p < 0.01) showed the highest activities in m. persicae fed on vermicompost-treated c. annum while control and zinc sulfate treatments caused the lowest enzymatic activities (table 3). in case of alt, the aphids fed on both fertilizer treatments showed lower activity than control (f = 0. 94; df = 2, 8; p < 0.01) (table 3). aldolase (f = 1.90; df = 2, 8; p < 0.01) and ldh (f = 6.06; df = 2, 8; p < 0.01) had the highest activities in the aphids fed on fertilizer-treated c. annum compared to control while activity of alp decreased in these treatments (f = 3.36; df = 2, 8; p < 0.01) (table 4). finally, activity of acp (f = 20.54; df = 2, 8; p < 0.01) was the highest in m. persicae fed on control and zinc sulfate-treated c. annum (table 3). effects of fertilizers on antioxidant enzymes both control and zinc-sulfate treatments caused the highest activity of catalase in m. persicae but the aphids fed on vermicompost-treated c. annum had the lowest enzymatic activity (table 5; f = 4.10; df = 2, 8; p < 0.01). activities of peroxidase (f = 4.90; df = 2, 8; p < 0.01), superoxide dismutase (f = 5.33; df = 2, 8; p < 0.01), ascorbate oxidase (f = 6.52; df = 2, 8; p < 0.01) and glucose-6-phosphate dehydrogenase (f = 8.90; df = 2, 8; p < 0.01) were also the highest in the aphids fed on vermicomposttreated c. annum (table 5). significant differences were found in the amounts of malondialdehyde (mda) and oxidized to reduced thiols (rssr/rsh ratio) in control and fertilizer treatments (table 6). adults of m. persicae fed on zinc sulfate-treated and control plants showed the highest and the lowest values of mda, respectively (table 6; f = 7.82; df = 2, 8; p < 0.01) while the highest rssr/rsh ratio was found in the aphids fed on vermicomposttreated c. annum (table 6; f= 1.84; df = 2, 8; p < 0.01). effects of fertilizers on storage macromolecules in m. persicae significant differences were found in triacylglyceride (tag) and glycogen contents of m. persicae reared on different fertilizer treatments (table 7). m. persicae fed on c. annum treated by zinc sulfate had the highest amount of triglyceride (f = 4.31; df = 2, 8; p < 0.01) and glycogen (f = 65.46; df = 2, 8; p < 0.01) compared to other treatments (table 7). discussion according to the results obtained here, zinc sulfate and vermicompost fertilizers significantly affected physiological performance and antioxidant activities of m. persicae. researches have demonstrated that fertilizer treatment affect fecundity, life table parameters and physiological performance of herbivorous insects (edwards et al., 2009; razmjou et al, 2011; mardani-talaee et al., 2015) because soil amendment with fertilizers increases level of organic matters and soil biological interactions led to fertility and relative host plant resistance to pest damages (luong and heong, 2005). also, organic fertilizers, e.g., vermicompost, may increase amounts of phenolic compounds in host plants which definitely affect biological performance of insects, the phenomenon has been observed in m. persicae (mardani-talaee et al., 2016). adults of m. persicae fed on zinc sulfate-treated c. annum showed the highest activities of digestive enzymes while the aphids on control and vermicompost treatments had the lowest enzymatic activities except for chymotrypsin, aminopaptidase and β-glucosidase. these results imply on suitability of c. annum reared on zinc sulfate cultural soil. the suitability may be created due to higher amounts of nutrients or lower levels of plant secondary metabolites which prevent growth and development of insect via repellency or inhibitory mechanisms on digestive enzymes (terra and ferreira, 2005; nation, 2008). our previous studies revealed the effect of vermicompost to increase amounts of secondary compounds in m. persicae and leptinotarsa decemlineata (coleoptera: chrysomelidae) (mardani-talaee et al., 2015, 2016). in details, mardani-talaee et al. (2015) reported the higher contents of flavonoids, anthocyanins and phenolic compounds in potatoes cultured in the soils containing 30 % of vermicompost. similar findings were obtained in c. annum cultured in vermicompost compared to control 128   table 5 effects of fertilizers on activities of antioxidant enzymes in m. persicae adults statistic (mean±se u/mg protein) treatments catalase ascorbate oxidase gpdh sod po control 0.328±0.081 a 0.778±0.549 c 0.136±0.026 b 0.080±0.002 b 0.003± 0.001 b vermicompost (30%) 0.148±0.049 b 7.176±0.463 a 0.447±0.0216 a 1.860±0.310 a 0.013± 0.005 a zinc sulfate 0.119 ± 0.021 a 3.704±0.106 b 0.286±0.085 ab 0.834±0.029 ab 0.002± 0.001 b gpdh, glucose-6-phosphate dehydrogenase; sod, superoxide dismutase; po, peroxidase. the means followed by different letters in a column are significantly different [p < 0.01, tukeys (hsd)]. and zinc sulfate treatments (mardani-talaee et al., 2016). moreover, stevenson et al. (1993) found that phenolic compounds were responsible for development retardation of spodoptera litura (lepidoptera: noctuidae) reared on wild ground nut. haukioja et al. (2002) reported changes of consumption rate in epirrita autumnata (lepidoptera: geometridae) due to presence of phenolic compounds. finally, edwards et al. (2009) highlighted the role of phenolic substances to alleviate feeding performance in sap sucking insects. insects are depend on several processes involved in intermediary metabolism to gain their required energy for biological activities such as flight, reproduction and etc. intermediary metabolism relies on activities of transaminases to process amino acids for energetic demands, tissue construction and lipid oxidation to provide energy and metabolic water, besides processing of glucose via glycolysis and krebs cycle (nation, 2008). alt and ast are the two important enzymes in transaminase mechanisms of insects that catalyze alanine cycle in proline metabolism and facilitate conversion of aspartate and α-ketoglutarate to oxaloacetate and glutamate, respectively (nation, 2008). these two enzymes are involved in proline metabolism and providing some components for krebs cycle. although γ-gt is a transaminase but it transfers γ-glutamyl moiety of glutathione to a receptor for glutamate formation so it is important in γ-glutamyl cycle to synthesize and degrade glutathione and xenobiotic compounds (tate and meister, 1985). in our study, activities of ast and γgt significantly increased in the aphids fed on vermicompost-treated c. annum compared to control and zinc sulfate treatments while activity of alt decreased in both fertilizer treatments compared to control. the lower activity of alt in fertilized-treated aphids may be attributed to nondependence of energy production via proline metabolism or protein shortage in hemolymph and fat bodies because of disordered protein digestion. the second reason seems to be more important because alt is crucial to convert amino acids to each other and keto-acid. meanwhile, increased activities of ast and γ-gt may be due to energy demand via krebs cycle by aspartate and αketoglutarate conversions to oxalate and glutamate as well as detoxification of secondary metabolites entered into hemolymph. aldolase is an isomerase which make sugars available in initial steps of glycolysis (pinto et al., 1969). ldh involves in pyruvate conversion to lactate in anaerobic conditions and utilizes electrons to provide nadh from nad+ (nation, 2008). also, the enzyme is considered as an index of tissue damage in clinical chemistry (kaplan and pesce, 1996). the higher activities of aldolase and ldh in the aphids fed on fertilized-treated plants refer to glycolysis cycle for energy demand from one hand and potential tissue damages due to increased levels of plants’ secondary metabolites. mardanitalaee et al. (2015) reported higher activity of ldh in l. decemlineata adults fed on potatoes cultured in vermicompost (30 %) with regard to increased levels of secondary metabolites in the treated plants. acid and alkaline phosphatases are the two hydrolytic enzymes that detach phosphate groups from different molecules such as nucleotids, proteins and alkaloinds in acid and alkali media. the higher activities of these enzymes indicate digestion efficacy and nutrient absorption in midgut and appropriate transfer of dietary nutrients to fat bodies (senthil-nathan et al., 2006). lower activities of these enzymes mainly alp in fertilized-treated aphids indicate disturbance of dietary utilization of ingested food because of disordered performance of digestive enzymes. on the other hand, it can be concluded that impaired digestive process led to lower availability of nutrients in aphids mainly storage in fat bodies which affect activities of given phosphatases. antioxidant system is recruited to protect animal cells against ros (reactive oxygen species) by decrease level of lipid peroxidation, dna and protein damage (felton and duffey, 1991; dubovskiy et al., 2008). several enzymes are involved in antioxidant defense system including 129   table 6 effects of fertilizers on rssr/rsh ratio and malondialdehyde (mda) in m. persicae asults statistic (mean±se) treatments mda (nm/mg protein) rssr/rsh ratio control 0.674±0.157 b 0.977±0.132 b vermicompost (30%) 3.699±1.22 ab 1.626±0.773 a zinc sulfate 4.783±0.467 a 0.5200±0.0560 b the means followed by different letters in a column are significantly different [p < 0.01, tukeys (hsd)]. ascorbate peroxidases (apx), superoxide dismutases (sod), catalases (cat), peroxidases (pox), glutathione-s-transferase (gst), glucose-6phosphate dehydrogenase and nonenzyme antioxidants such as ascorbic acid, thiols, and αtocopherol (fridovich, 1978; dubovskiy et al., 2008). functionally, sod converts superoxide radical о2 into h2o2; cat and pox convert h2o2 into h2o; gst removes the products of lipid peroxidation or hydroperoxides from cells; thiols are important to protect cells against hydroxyl radical (oh-); nitroxyl radical (no−) and superoxide radical о2 (udupi and rice-evans, 1992; dubovsky et al., 2008). besides, thiol ratio (rssr/rsh) is an important index to highlight the lower amounts of reduced sh-groups (rsh) and the higher amounts of oxidated shgroups (rssr) due to oxidative stress (dubovsky et al., 2008). meanwhile, increased amount of mda is an evidence of oxidative stress in an organism (dubovsky et al., 2008). peroxidase, sod, ascorbate oxidase and gpdh had the highest activity in the aphids fed on vermicompost treated plants but activities of ascorbat oxidase and catalase were the highest in control and zinc sulfate treatments. although the highest amount of mda was found in zinc sulfate treatment but the aphids fed on vermicompost-treated plants showed the highest value of rssr/rsh ratio. enhanced activity of sod in m. persicae reared on vermicompost indicated the higher amount of h2o2. meanwhile, cat is known to be inhibited by accumulation of superoxide anions in oxidative stressed organisms so the lower activity in vermicompost treatments may be attributed to possible destruction of aphid tissues mainly gut due to increased amounts of secondary metabolites (kono and fridovich, 1982; pardini et al., 1988). in glycolysis, glycerol-3-phosphate shuttle transfers the reducing equivalents from cytoplasmic pool of nadph to the mitochondrial membrane (nation, 2008). then, nadph is re-oxidized by transferring electrons across the mitochondrial membrane rather than nadh itself (nation, 2008). moreover, ascorbate peroxidase enzymatically removes h2o2 with the concurrent oxidation of ascorbate (asada, 1992). nadph is the final reducing equivalent in gsh-gssg system to equilibrate chemical constitutions between dehydroascorbate and ascorbate. so, the higher activity of ascorbate oxidase should be compensated with increase in glucose-6-phosphate dehydrogenase activity. our results on the highest activities of both enzymes in the aphids fed on vermicompost-treated plants indicate more reliance of aphids to provide energy from glycolysis and to maintain the balance between produced oxidative reagents. malondialdehyde (mda) is an organic compound which naturally exist in biological media indicating oxidative stress. mda is produced due to lipid peroxidation of polyunsaturated fatty acids. in fact, reactive oxygen species produced in cells degrade polyunsaturated lipids and produce mda which is a reactive electrophile species leading to toxic stress in cells (wang et al., 2001; dubovsky et al., 2008). in our study, adults of m. persicae fed on zinc sulfate and vermicompost showed higher amounts of mda compared to control. it can be concluded that oxidative stress in fertilizer-treated aphids caused a high level of lipid peroxidation which is accompanied with the lower amounts of storage triglyceride mainly in vermicompost. our results demonstrated the highest rssr/rsh ratio in the aphids fed on vermicompost-treated c. annum. alteration of balance between oxidized thiols in an organism indicates the higher activity of radical oxidative species. wang et al. (2001) believe higher ratio of oxidized to reduced thiols is accompanied with increase in lipid peroxidation processes led to higher amount of mda. their conclusion is corresponds with our findings on the aphids fed on vermicompost-treated plants with the highest rssr/rsh ratio and mda concentration. insects are highly dependent on their stored nutrients to survive in environment (nation, 2008). dietary food is transferred to fat bodies and stored as three macromolecules, protein, glycogen and triglyceride (nation, 2008). amounts of the two measured macromolecule, glycogen and triglyceride, were the highest in the aphids fed on zinc sulfate-treated plants compared to vermicompost and control. in fact, c. annum reared in soil containing zinc sulfate is an appropriate host 130   https://en.wikipedia.org/wiki/organic_compound https://en.wikipedia.org/wiki/organic_compound https://en.wikipedia.org/wiki/oxidative_stress table 7 effect of fertilizers on storage macromolecules in m. persicae adults statistic (mean±se mg/ml) treatments tag glycogen control 1.136±0.029 ab 0.034±0.025 b vermicompost (30%) 0.032±0.032 b 0.042±0.009 b zinc sulfate 4.783±0.015 a 0.510±0.001 a the means followed by different letters in a column are significantly different [p < 0.01, tukeys (hsd)]. plant for m. persicae because of nutrient quality and lack of feeding inhibitors mainly secondary metabolites. in other two treatments, mainly vermicompost, shortage of nutrients due to impaired digestive process led to utilize storage glycogen and triglyceride to support energetic mechanisms via glycolysis or β-oxidation of lipids. the conclusion can be supported by altered activities of intermediary enzymes highlighted earlier. in summary, significant differences were observed in digestion, intermediary metabolism and antioxidant activities of m. persicae fed on c. annum cultured in control and fertilizer-treated soils. the physiological findings here and life table parameters reported in our previous 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its host m asadi, h rafiee-dastjerdi*, g nouri-ganbalani, b naseri, m hassanpour department of plant protection, faculty of agriculture and natural resources, university of mohaghegh ardabili, ardabil, iran. accepted april 20, 2018 abstract habrobracon hebetor say is an important ectoparasitoid wasp that can control pyralidae and noctuidae pests in agricultural crops. in this research, the effects of allium sativum l., rosmarinus officinalis l., piper nigrum l., salvia officinalis l. and glycyrrhiza glabra l. essential oils were investigated on the functional response of h. hebetor to its host. the gc-ms analysis showed that tetracosamethyl cyclododeca siloxan, alpha-pinene, caryophyllene, beta-thujone and aristolene were major constituents of mentioned essential oils, respectively. in the experiments; the mated females of h. hebetor (under 24 h old) were exposed to sublethal concentrations (lc30) of isolated essential oils for 24 h with fumigant exposure method. in the control, the treatment was performed by using distilled water. then, six treated wasps were selected randomly to densities of 2, 4, 8, 16, 32 and 64 ephestia kuehniella zeller 5th instar larvae for 24 h under 25 ± 1 °c, 60 ± 5% rh and photoperiod of 16: 8 (l: d) h. eight replicates were conducted for each host density in all treatments. the regression analysis based on holling model (1959) indicated the functional response type ii in the control, p. nigrum, s. officinalis and g. glabra and type iii in a. sativum and r. officinalis essential oils. also, r. officinalis essential oil and the control showed the longest (0.542 h) and shortest (0.411 h) handling times, respectively. the highest (0.047 h-1) and lowest (0.033 h-1) attack rates were also recorded in the control and r. officinalis essential oil, respectively. in addition, r. officinalis and g. glabra essential oils showed the maximum and minimum negative effects on the functional response type and it’s parameters in h. hebetor, respectively. these results indicated that g. glabra essential oil can be recommended with h. hebetor in integrated pest management. key words: ephestia kuehniella; plant essential oils; functional response; glycyrrhiza glabra; habrobracon hebetor introduction habrobracon hebetor say is an important ectoparasitoid wasp with special behavioral characteristics (idiobiont and gregarious) that has been applied successfully in many biological control programs in different regions over the world including iran (heimpel et al., 1997; yu et al., 2002; darwish et al., 2003; salvador and consoli, 2008; abedi et al., 2012; mahdavi and saber, 2013). the mass rearing of h. hebetor are performed on the larval stage of flour moth (ephestia kuehniella zeller) as laboratory host in different commercial insectariums (mudd and corbet, ___________________________________________________________________________ corresponding author: hooshang rafiee-dastjerdi department of plant protection faculty of agriculture and natural resources university of mohaghegh ardabili, ardabil, iran email: hooshangrafiee@gmail.com 1982). this parasitoid wasp till now has been applied under inundative and inoculative release programs against helicoverpa armigera (hübner), sesamia cretica (lederer) and ostrinia nubilalis (hübner) (hentz et al., 1998; baker and fabrick, 2000; navaei et al., 2002). one of the important behavioral features of natural enemies including parasitoids and predators is their functional responses. holling (1959, 1961 and 1966) characterized three types of functional responses. the functional response type i has a linear shape (hassell, 1978). in functional response type ii, the numbers of hosts attacked by natural enemies reach to a fix rate. most of the natural enemies show this type of functional response (hassell, 1978; luck, 1985; yu et al., 2002; abedi et al., 2012; mahdavi and saber, 2013; jarrahi and safavi, 2015). the functional response type iii also show sigmoid shape (holling, 1959; hassell, 1978). mailto:hooshangrafiee@gmail.com 170 combination of different pest management methods such as biological and chemical control has been recommended in ipm designs all over the world and the negative effects of different compounds on the biocontrol agents must be considered (abedi et al., 2012). abramson et al., (2006) studied the effects of citronella and alfazema essential oils on the fennel aphids, hyadaphis foeniculi passerini (hemiptera: aphididae) and it’s predator, cycloneda sanguinea l. (coleoptera: coccinellidae) and concluded that citronella essential oil showed the most adverse effects on this predator. in addition, poderoso et al., (2016) studied the effects of some plant extracts on developmental of the predator podisus nigrispinus (hemiptera: pentatomidae) and concluded that the examined extracts caused relatively high mortality on the adults of this predator and must be used with care, because they can affect the life cycle of this important biocontrol agent. therefore, natural enemies can be affecting by different botanical compounds that use against the insect pests. therefore, estimation of the functional response types and their parameters under treatments of botanical compounds including essential oils and extracts are very important factors in ipm programs. to date there have been no research conducted the effects of plant essential oils on e. kuehniella and functional response of h. hebetor; but, the researches about the lethal and sublethal effects of essential oils on this important biocontrol agent are available (seyyedi et al., 2011; hashemi et al., 2014; ahmadpour, 2017). in addition, chemical pesticides can affect host-finding behavior and behavioral responses of h. hebetor. rafieedastjerdi et al., (2009b) showed that profenofos, thiodicarb, hexaflumuron and spinosad negatively changed the functional response of h. hebetor. faal-mohammad ali et al., (2010) also concluded that chlorpyrifos and fenpropathrin changed the functional response of this parasitoid wasp to it’s host that these negative effects of pesticides can lead to inefficiency of natural enemies and outbreak of plant pests. therefore, the effects of different compounds must be investigated in assessment of natural enemies for biological control programs. the effects of azadirachtin, cypermethrin, methoxyfenozide and pyridalil also were studied by abedi et al., (2012); who stated that cypermethrin had the highest negative effects on h. hebetor. moreover, mahdavi and saber (2013) stated that malathion was compatible insecticide on the functional response of h. hebetor compared with diazinon in ipm programs. in addition, jarrahi and safavi (2015) concluded that proteus as a new formulated insecticide showed the highest negative effects on this parasitoid wasp compared with entomopathogenic fungus metarhizium anisopliae sensu lato and the control under laboratory conditions. hence, the main objective of the present research was to investigate the effects of above mentioned essential oils isolated from some selective medicinal plants on the functional response of h. hebetor to evaluate the possibility of these botanical compounds to be integrated with this important ectoparasitoid wasp in ipm programs especially for the management of stored products pest. materials and methods the present research was carried out during 2016-2017, in the department of plant protection, faculty of agriculture and natural resources, university of mohaghegh ardabili, ardabil, iran. rearing of the parasitoid wasp parent population of habrobracon hebetor wasps was provided by a private commercial insectarium (kesht-gostar pishgam, kermanshah province, iran), during 2016. then, the parasitoid wasps were reared under laboratory conditions in growth chamber that was set at 25 ± 1 °c, 60 ± 5% rh and a photoperiod of 16: 8 (l: d) h, on the larvae of flour moth (e. kuehniella) as laboratory host for parasitism activities. moreover, the honey solution (10%) was applied as food source for feeding of the adult parasitoids (rafiee-dastjerdi et al., 2008, 2009b). the ratio of parasitoid to host in our experiments was one female wasp to ten e. kuehniella larvae and the exposure interval of host to the parasitoid wasp was two days. isolation of essential oils the selected medicinal plants including garlic, allium sativum; rosemary, rosmarinus officinalis; black pepper, piper nigrum; sage, salvia officinalis and liquorice, glycyrrhiza glabra that was available in the iranian flora and contained suitable amount of essential oil were collected from different regions of islam-abad gharb city (34.11° n, 46.53° e) in kermanshah province, iran, during may 2017. the collected plants were dried at room temperature (25 °c) under shade. then, the parts of noted plants that contained the most insecticidal components including leaves of r. officinalis, s. officinalis and g. glabra, berries of a. sativum and seeds of p. nigrum were milled by electric grinder and 50 g of milled parts were added to 500 ml of distilled water and their essential oils were isolated by clevenger apparatus at 100 °c in 4h time for each plant (shiva parsia and valizadegan, 2015). the water of essential oils was removed by sodium sulfate and pure essential oils in special glasses were covered with aluminum coverage and stored in a refrigerator at 4 °c for using in experiments. chemical analysis of isolated essential oils chemical components of each essential oil were identified by using gas chromatography-mass spectroscopy (gc-ms/ company: agilent, series: 7890 b, manufacturer: usa). the comparative and original analyses are two common types of gc-ms analysis. the original analyses that apply about the essential oils and the other volatile compounds measure the peaks in relation to one another. in this method, the tallest peak is assigned 100% of the value and the other peaks being assigned proportionate values. the total mass of the unknown compounds is normally indicated by the parent peak. the value of this parent peak can be 171 used to fit with a chemical formula containing the various elements which are believed to be in the compound (hites, 2016). bioassays experiments for investigation the fumigant toxicity of isolated essential oils on the young females of h. hebetor (under 24 h old); different concentrations of essential oils that lead to mortality between 20-80% put on filter paper (2×2 cm) in 60 ml glass petri dishes as fumigant chambers by using a microaplicator. distilled water was used in control treatments. then, 20 adult females h. hebetor were released in each petri dish without the presence of the host and the petri dishes immediately were sealed with parafilm to prevent the exit of essential oils. honey solution (10%) was used for the feeding of wasps on small pieces of paper. each concentration of the essential oils was bioassayed in four replications and after 24 h of exposure; the number of dead wasps was recorded (shiva parsia and valizadegan, 2015). functional response experiments in the functional response experiments, the lc30 of each essential oil was applied as the low lethal concentration. in first experimental setup, eighty mated females (under 24 h old) of h. hebetor that previously not in the presence of the host were exposed to lc30 of selected essential oils that were put on filter papers (2×2 cm) by using a microaplicator in 10 cm petri dishes (volume 60 ml) for 24 h. all procedures were performed for the control treatments with distilled water. after 24 h, six treated wasps were selected randomly and transferred separately to the petri dishes with the different densities (2, 4, 8, 16, 32, and 64) of e. kuehniella larvae (5th instar) and were placed in growth chamber that was set at 25± 1°c, 60± 5% rh and a photoperiod of 16: 8 (l: d) h for 24 h. ventilation in the petri dishes were provided with pores in the lids of petri dishes and honey solution (10%) was supplied as food source for the parasitoids. the functional response experiments were performed in eight replicates in all treatments and the numbers of parasitized host larvae by the parasitoid wasps were recorded after 24 h. used model the model of holling (1959) regarding the functional response of different natural enemies was used in this study as explained below: na = attn0(1+athn0) na = number of hosts attacked by h. hebetor n0 = different densities of host (2, 4, 8, 16, 32 and 64 5th instar larvae of e. kuehniella) tt = total time of experiment (was 24 hour) a = attack rate (area of host discoverage) by h. hebetor th = handling time (time of handling) of h. hebetor to it’s host the other form of this equation is: a= (d+bn0)/(1+cn0) here, “a” is host density and “b”, “c” and “d” are estimated constants (hassell et al., 1977; juliano and williams, 1987; juliano, 1993). statistical analysis the logistic and non-linear regression models were applied to determine the types of functional response and for the estimation of the parasitoid attack rate and handling time under different essential oils treatments and the control, respectively, using sas v 9.1 software (sas institute, 2002). results chemical analysis of isolated essential oils the gc-ms analyses results of isolated essential oils are shown in tables 1 to 5. eleven major compounds from a. sativum essential oil, fourty-three compounds from s. officinalis and p. nigrum essential oils and fourty-four compounds from s. officinalis and g. glabra essential oils were detected. tetracosamethyl cyclododeca siloxan (15.82%) from a. sativum, alpha-pinene (9.99%) from r. officinalis, caryophyllene (36.03%) from p. nigrum, beta-thujone (25.63%) from s. officinalis and aristolene (20.14%) from g. glabra were detected as major constituents of each mentioned essential oil. table 1 chemical constitutents of allium sativum l. essential oil peak material retention time (rt) % of total 1 52', 6', 6'-trimethyl-cyclohexene 36.49 2.16 2 octasiloxane 36.87 3.64 3 5, 6, 8, 9-tetramethoxy-2-methylpep 37.38 5.99 4 n-methyl-1-adamantaneacetamide 37.50 7.97 5 silicone grease, siliconfett 37.56 8.80 6 1, 3-xylyl-15-crown-4, 2, 3-pinan 37.59 8.63 7 4-methoxy-3-(3-methoxyphenyl) 37.80 10.20 8 1, 4-cyclohexadiene,1, 3, 6-tris 37.92 10.57 9 1-amino-1-ortho-chlorophenyl 38.02 12.87 10 anhydro 5-hydroxy-3-piperonyl 38.14 13.34 11 tetracosamethyl cyclododeca siloxan 38.27 15.82 172 table 2 chemical constitutents of rosmarinus officinalis l. essential oil peak material retention time (rt) % of total 1 tricyclene 5.21 0.27 2 alpha-pinene 5.45 9.99 3 camphene 5.70 4.25 4 bicyclo [3.1.0] hex-3-en-2-ol 5.79 0.42 5 bicyclo [3.1.1] heptane, 6, 6-dimet 6.21 1.19 6 3-octanone 6.36 1.31 7 beta-myrcene 6.44 1.73 8 (+)-4-carene 6.94 0.37 9 benzene,1-methyl 7.10 0.96 10 dl-limonene 7.18 2.88 11 1, 8-cineole 7.26 6.11 12 gamma-terpinene 7.76 0.51 13 alpha-terpinolene 8.39 0.52 14 linalool l 8.63 2.17 15 chrysanthenone 9.29 0.55 16 bicyclo [2.2.1] heptan-2-one 9.85 7.78 17 bicyclo [3.1.1] heptan-3-one 10.23 0.57 18 borneol l 10.41 5.02 19 bicyclo [3.1.1] heptan-3-one 10.64 1.07 20 3-cyclohexen-1-ol, 4-methyl 10.70 1.01 21 alpha terpineol 11.10 1.67 22 estragole 11.29 0.91 23 bicyclo [3.1.1] hept-3-en-2-one 11.77 7.24 24 bicyclo [2.2.1] heptan-2-ol 14.50 5.71 25 caryophyllene 19.37 3.81 26 alpha-humulene 20.41 0.66 27 heptasiloxane, hexadeca methyl 36.26 4.78 28 3-(4-chlorophenyl)-4, 6-dimethoxy 36.35 1.21 29 1, 3-xylyl-15-crown-4, 2, 3-pinan 36.38 2.25 30 cyclononasiloxane, octadecamethyl 36.55 1.70 31 acetamide, 2-(adamantan-1-yl) 36.68 3.01 32 bistri, methylsilyl n-acetyl eicos 36.83 5.14 33 1, 1, 1, 5, 7, 7, 7-heptamethyl 37.22 0.77 34 6-phenyl-3, 5-dithioxo 37.33 1.09 35 cyclodecasiloxane, eicosamethyl 37.46 1.50 36 octadeca methyl cyclononasiloxane 37.56 1.26 37 1-amino-1-ortho-chlorophenyl 37.60 0.64 38 cyclodecasiloxane, eicosa methyl 37.69 2.71 39 5, 6, 8, 9-tethramethoxy-2-methylpep 37.82 2.25 40 1, 1, 5, 7, 7, 7heptamethyl-3 37.89 0.69 41 cyclononasiloxane, octadecamethyl 38.91 0.41 42 benzene, 2, 3-dimethyl 38.12 1.10 43 iron, monocarbonyl 38.23 0.82 173 table 3 chemical constitutents of piper nigrum l. essential oil peak material retention time (rt) % of total 1 bicyclo [3.1.0] hexane, 4-methyl 5.28 0.67 2 r-alpha-pinene 5.42 3.05 3 camphene 5.69 0.18 4 sabinene 6.15 3.96 5 2-beta-pinene 6.22 4.34 6 beta-myrcene 6.43 0.73 7 1-phellandrene 6.71 1.34 8 3-carene 6.84 5.17 9 alpha terpinene 6.94 0.16 10 benzene,1-methyl 7.09 0.58 11 l-limonene 7.19 7.02 12 1, 8-cineole 7.24 0.11 13 1, 4-cyclohexadiene,1-methyl 7.75 0.27 14 bicyclo [3.1.0] hexan-2-ol 7.93 0.30 15 alpha-terpinolene 8.33 0.10 16 (+)-4-carene 8.39 0.35 17 1, 6-octadien-3-ol, 3, 7-dimethyl 8.61 1.07 18 3-cyclohexen-1-ol, 4-methyl 10.67 0.64 19 beta fenchyl alchol 11.05 0.21 20 estragole 11.29 2.72 21 alpha-terpinene 16.50 2.41 22 alpha-cubebene 16.93 0.29 23 alpha-copaene 17.90 3.84 24 beta elemene 18.46 1.69 25 caryophyllene 19.51 36.03 26 azulene 19.96 0.38 27 alpha-humulene 20.43 2.94 28 trans-beta-farnesene 20.52 0.29 29 1, 6-cyclodecadiene 21.25 0.45 30 beta-selinene 21.42 2.70 31 aalpha-selinene 21.67 2.40 32 naphthalene 21.82 0.57 33 cyclohexene, 1-methyl 22.08 5.13 34 1-naphthalenol 22.26 0.36 35 delta-cadinene 22.49 2.52 36 cadina-1, 4-diene 22.70 0.21 37 cyclohexane methanol, 4-ethenyl 23.17 0.59 38 1, 6, 10-dodecatrien 23.55 0.58 39 (-)-caryophyllene oxide 24.09 1.45 40 pentalene, octahydro 25.24 0.79 41 bicyclo [4.4.0] dec-1-ene 25.56 0.24 42 copaene 25.67 1.09 43 4h-1, 3, 5-thiadiazin-4-one 32.52 0.09 174 table 4 chemical constitutents of salvia officinalis l. essential oil peak material retention time (rt) % of total 1 cis-salvene 4.00 0.15 2 tricyclo [2.2.1.0 (2, 6)] heptane 5.20 0.13 3 1s-alpha-pinene 5.41 3.73 4 camphene 5.69 3.36 5 bicyclo [3.1.1] heptane 6.20 0.84 6 beta-myrcene 6.43 0.35 7 benzene,1-methyl 7.09 0.82 8 dl-limonene 7.17 0.80 9 1, 8-cineole 7.25 10.56 10 1, 6-octadien-3-ol, 3, 7-dimethyl 8.70 0.27 11 beta-thujone 8.90 25.63 12 thujone 9.11 6.54 13 bicyclo [2.2.1] heptan-2-one 9.87 16.46 14 borneol l 10.37 1.69 15 3-cyclohexen-1-ol, 4-methyl-1 10.68 0.52 16 p-menth-1-en-8-ol 11.06 0.23 17 2 (3h)-furanone 11.27 0.23 18 benzene, 1-methoxy-4-(1-propenyl) 14.42 0.52 19 phenol, 2-methyl-5-(1-methylethyl) 15.06 0.27 20 caryophyllene 19.34 1.68 21 1h-cycloprop[e] azulene 19.96 0.77 22 alpha-humulene 20.42 2.81 23 ledene 21.66 0.47 24 1h-cycloprop[e] azulene 23.94 0.52 25 (-)-caryophyllene oxide 24.08 0.86 26 veridiflorol 24.33 4.71 27 1, 2-dihydropyridine 24.50 0.35 28 12-oxabicyclo [9.1.0] dodeca 24.77 1.37 29 trans-z-alpha-bisabolene epoxide 25.34 0.71 30 cyclopentan,1-methylen-2-vinyl 25.97 0.27 31 bicyclo [6.1.0] nonane 30.89 0.21 32 cembrene 31.50 0.13 33 1-naphthalenepropanol 31.79 4.27 34 5, 7-dimethoxy-1-naphthol 31.96 0.16 35 4-methoxy-3-(3-methoxyphenyl) 36.83 0.18 36 1, 3-xylyl-15-crown-4, 2, 3-pinan 37.17 0.39 37 methoxy-3-(3-methoxyphenyl) 37.39 0.82 38 cyclononasiloxane, octadecamethyl 37.45 0.23 39 etracosamethyl cyclododeca siloxan 37.56 1.16 40 hexasiloxane, tetradecamethyl 37.65 0.63 41 iron, monocarbonyl 37.00 1.76 42 silicone grease, siliconfett 83.10 0.73 43 1-naphthalene ethanol 37.89 1.20 44 5, 6, 8, 9-tetramethoxy-2-methylpep 38.04 0.55 175 table 5 chemical constitutents of glycyrrhiza glabra l. essential oil peak material retention time (rt) % of total 1 1s-alpha-pinene 5.40 0.38 2 linalool 8.63 1.02 3 alpha terpineol 11.07 0.61 4 lavandulyl acetate 18.29 6.26 5 bicyclo [3.1.1] hept-2-ene 19.23 0.97 6 caryophyllene 19.36 0.51 7 endo-2, 6-dimethyl-6-(4-methyl) 19.89 1.81 8 1h-cycloprop azulene 20.93 1.33 9 geranyl propionate 21.14 3.31 10 benzene,1-(1,5-dimethyl-4-hexen) 21.35 0.76 11 trans-beta-farnesene 21.40 1.16 12 3-buten-2-ol, benzoate 21.53 0.97 13 cyclohexene, 1-methyl-4-(5-methyl) 22.06 0.42 14 propanoic acid, 2-methyl 22.24 2.19 15 beta-sesquiphellandrene 22.49 0.48 16 butanoic acid, 3, 7-dimethyl 23.59 7.34 17 nerolidol 23.74 4.11 18 3-hexen-1-ol, benzoate 23.89 4.37 19 linalool l 23.93 0.71 20 caryophyllene oxide 24.15 1.90 21 3-cyclohexene-1-ethanol 24.26 0.61 22 2, 6-octadien 24.70 8.54 23 (e)-2-formyl-6-methyl 24.81 2.68 24 1, 6, 10-dodecatrien 25.09 2.52 25 naphthalene 25.34 2.55 26 aristolene 25.58 20.14 27 hinesol 25.67 1.90 28 beta-eudesmol 25.90 1.73 29 2-naphthalene methanol 25.96 2.21 30 beta-bisabolol 26.33 0.48 31 alpha-bisabolol 26.61 0.20 32 geranyl tiglate 27.04 3.79 33 acetic acid, 1-methylcyclopentyl 27.22 0.71 34 neryl propionate 28.19 1.67 35 geranyl acetate 28.31 0.84 36 benzyl benzoate 28.48 2.72 37 neryl 2-methylpropanoate 29.47 0.76 38 2-hexadecen 29.82 0.34 39 2-pentadecanone 29.92 0.58 40 neophytadiene 30.35 0.37 41 neryl acetate 31.01 0.51 42 hexadecanoic acid 31.10 0.40 43 geranyl benzoate 31.15 2.88 44 cyclohexene,1-methyl-5-(1-methyl) 31.49 0.30 176 table 6 the acute toxicity of selected essential oils on the adult females of h. hebetor treatment n slope ± e lc30 µl/liter air (95% cl) lc50 µl/liter air (95% cl) lc90 µl/liter air (95% cl) χ2 a. sativum 480 1.41±0.20 2.22 5.22 42.05 9.85 (1.14 3.31) (3.57 6.76) (29.59 74.68) r. officinalis 480 2.35±0.30 2.48 4.15 14.51 15.72 (1.60 3.28) (3.11 5.04) (12.09 18.88) p. nigrum 480 1.39±0.20 5.41 12.88 107.33 7.82 (2.84 7.93) (9.06 16.42) (73.00 205.55) s. officinalis 480 1.12±0.15 6.30 18.36 250.47 6.19 (3.21 9.62) (12.69 24.30) (154.06 551.65) g. glabra 480 1.08±0.12 8.72 26.51 401.83 15.22 (4.81 13.07) (18.68 35.38) (274.39 837.72) cl: confident limit, χ2: chi-square value. table 7 the logistic regression analysis of e. kuehniella larvae parasitized by h. hebetor. treatments coefficient estimate se χ2 p-value control p0 (constant) 2.1769 0.6643 10.74 0.0011 p1 (linear) -0.0116 0.0964 0.01 0.9044 p2 (quadratic) -0.0011 0.0036 0.09 0.7603 p3 (cubic) 0.00001 0.00003 0.11 0.7350 a. sativum p0 (constant) 0.0261 0.4235 0.42 0.9508 p1 (linear) 0.0868 0.0653 1.76 0.1841 p2 (quadratic) -0.0033 0.0025 1.81 0.1782 p3 (cubic) 0.00003 0.00002 1.41 0.2348 r. officinalis p0 (constant) 0.1075 0.4190 0.07 0.7975 p1 (linear) 0.0351 0.0640 0.30 0.5837 p2 (quadratic) -0.0012 0.0024 0.26 0.6075 p3 (cubic) 0.00001 0.00002 0.12 0.7306 p. nigrum p0 (constant) 1.5420 0.5193 8.82 0.0030 p1 (linear) -0.0229 0.0762 0.09 0.7635 p2 (quadratic) -0.0007 0.0028 0.07 0.7927 p3 (cubic) 0.00001 0.00003 0.14 0.7096 s. officinalis p0 (constant) 1.6186 0.5090 10.11 0.0015 p1 (linear) -0.0694 0.0744 0.87 0.3509 p2 (quadratic) 0.0016 0.0027 0.32 0.5727 p3 (cubic) -0.00002 0.00003 0.31 0.5764 g. glabra p0 (constant) 2.1836 0.6279 12.09 0.0005 p1 (linear) -0.0469 0.0891 0.28 0.5986 p2 (quadratic) -0.0004 0.0032 0.02 0.8990 p3 (cubic) 0.00001 0.00003 0.08 0.7764 se: standard error, χ2: chi-square value 177 fig. 1 functional response curve of h hebetor previously exposed to lc30 of selected essential oils and the control to different densities of e. keuhniella larvae bioassay the lc30 and lc50 values for a. sativum, r. officinalis, p. nigrum, s. officinalis and g. glabra essential oils against the females of h. hebetor are shown in table 6. the adult bioassays indicated that acute toxicity of r. officinalis essential oil on the female wasps of h. hebetor was higher than the others. also, g. glabra essential oil showed the lowest acute toxicity in this research. functional response type logistic regression model with linear and nonlinear parameters indicated the functional response types in the control and essential oils treatments (table 7). according to the results, the functional response type ii (p1< 0) were determined in the control and p. nigrum, s. officinalis and g. glabra and type iii (p1≥ 0) in a. sativum and r. officinalis essential oils, respectively (figs 1 and 2). 178 fig. 2 the percentage curve of parasitized larvae by h. hebetor previously exposed to lc30 of tested essential oils and the control functional response parameters the estimation results of handling time, attack rate and theoretical maximum attack rate values from treated wasps of h. hebetor are shown in table 8. accordingly, the control and r. officinalis essential oil treatments showed the shortest (0.411± 0.028 h) and longest (0.542± 0.058 h) values of handling time, respectively. also, the highest and lowest attack rate values were recorded in the control (0.047± 0.003 h-1) and r. officinalis essential oil (0.033± 0.003 h-1) treatment, respectively. in addition, the highest value of the theoretical maximum attack rate base on t/th was obtained in the control (58.35) and the lowest being in r. officinalis essential oil (44.28) treatment; however, the difference between r. officinalis and s. officinalis wasn’t significant. 179 table 8 functional response parameters in h. hebetor previously exposed to lc30 of essential oils treatment type of functional response attack rate (h) a ± se (lower-upper) handling time (h-1) th ± se (lower-upper) theoretical maximum attack rate (t/th) r2 control ii 0.047 ± 0.003 (0.042 0.053) 0.411 ± 0.028 (0.355 0.469) 58.35 0.93 a. sativum iii 0.036 ± 0.003 (0.023 0.042) 0.515 ± 0.055 (0.405 0.626) 46.57 0.86 r. officinalis iii 0.033 ± 0.003 (0.027 0.038) 0.542 ± 0.058 (0.425 0.659) 44.28 0.85 p. nigrum ii 0.039 ± 0.002 (0.034 0.043) 0.462 ± 0.036 (0.389 0.534) 51.99 0.85 s. officinalis ii 0.041 ± 0.004 (0.034 0.048) 0.530 ± 0.052 (0.426 0.635) 45.27 0.86 g. glabra ii 0.042 ± 0.002 (0.037 0.047) 0.444 ± 0.031 (0.381 0.506) 54.09 0.86 a: attack rate, th: time of handling (handling time),r 2: coefficient of specification, se: standard error. discussion studies about the effects of different plant compounds such as essential oils on the functional response of h. hebetor can be useful tool for forecasting h. hebetor success in ipm programs, especially in the management of stored pests. the essential oils are safe compounds for human and environment programs and many of them showed high toxic effects due to aromatic and biologically active vapours (yildirim et al., 2011). there is no study about the effects of selected essential oils on the other natural enemies; but, the effects of these essential oils were investigated on different insect pests; such as a. sativum essential oils on tribolium castaneum (herbst), r. officinalis essential oil on larvae of pseudaletia unipuncta (haworth) and trichoplusia ni (hübner), p. nigrum essential oil on rice weevil, sitophilus oryzae l. and rice moth, corcyra cephalonica (st.), s. officinalis essential oils on drosophila melanogaster meigen and bactrocera oleae (rossi) and g. glabra essential oil on potato tuber moth phthorimaea operculella (zeller); and showed suitable effects on control of mentioned insects (yildirim et al., 2011; yazdgerdian et al., 2015). in addition, there are few investigations about the effects of selected plant essential oils on the important ectoparasitoid wasp, h. hebetor (seyyedi et al., 2011; hashemi et al., 2014; ahmadpour, 2017). in our study, the tested essential oils showed different acute toxicity on the adult females of h. hebetor that are in agreement with the findings reported by seyyedi et al., (2011), who studied the impacts of isolated essential oil from ferula gummosa l. on the female wasps of h. hebetor and concluded that mortality of h. hebetor was increased after 24 h of exposure (lc50= 9.16 µl/liter air). moreover, hashemi et al., (2014) concluded that ferula assafoetida l. essential oil had high toxicity on h. hebetor. tetracosamethyl cyclododeca siloxan, alphapinene, caryophyllene, beta-thujone and aristolene as major components in a. sativum, r. officinalis, p. nigrum, s. officinalis and g. glabra essential oil are volatile and aromatic compounds that showed high toxicity against h. hebetor in our research. these compounds contain active molecules that have fumigant, contact, antifeedants and repellent mode of actions and can be considered as efficient insecticides against different insect pests especially in enclosed environments (yazdgerdian et al., 2015). accordingly, the sublethal concentrations of different essential oils and the other botanical compounds can have negative effects on natural enemies especially on the functional response parameters (croft, 1990; mahdavi and saber, 2013; jarrahi and safavi, 2015). the results of this research showed that the attack rate values in treated wasps of h. hebetor with sublethal concentrations of studied essential oils was lower than control; but, the handling time values were higher than control; this shows that these essential oils have changed the searching behavior and the other parasitism activities of h. hebetor; because, when handling time increase therefore attack rate decrease and this is a negative effect of a compound on a biocontrol agent. there is no research about the effects of plant essential oils on the functional response of h. hebetor; but, the researches on the insecticides effects in this case are available. mahdavi (2011) studied the effects of abamectin, carbaryl, chlorpyrifos and spinosad and reported functional response type iii in the control and all insecticides treatments that his results are in agreement with our results about a. sativum and r. officinalis essential oil. mahdavi and saber (2013) also concluded that 180 malathion had lower negative effects on the functional response of h. hebetor compared with diazinon in ipm programs; but, our results indicated that g. glabra essential oil was compatible compound with h. hebetor. because, this essential oil showed the lowest adverse effects on the functional response type and it’s parameters in this parasitoid wasp. according to the result of rafieedastjerdi et al., (2013) and nazeefullah et al., (2014); g. glabra also showed low toxic effects on against potato tuber moth phthorimaea operculella (zeller) and tribolium castaneum (herbst), respectively. rafiee-dastjerdi et al., (2009b) also reported that the functional response of h. hebetor under hexaflumuron, profenofos, spinosad and thiodicarb and control treatments was type ii, and their results are in agreement with our results about the control and p. nigrum, s. officinalis and g. glabra essential oils treatments. faal-mohammad ali et al., (2010) stated that the functional response type in h. hebetor under larval and pupal treatments with chlorpyrifos and fenpropathrin and in the control was type iii that their results are in disagreement with our results about the control, p. nigrum, s. officinalis and g. glabra due to differences of treatments, growth stage of parasitoid wasps and it’s response type to different densities of host. moreover, abedi et al., (2012) studied the sublethal effects of azadirachtin, cypermethrin, methoxyfenozide and pyridalil on the functional response of h. hebetor and concluded that among them based on obtained handling time values, cypermethrin showed the highest adverse effect on the host-finding behavior of this parasitoid wasp; but, in our study r. officinalis essential oil showed the highest effects on this important characteristic of this parasitoid wasp. in addition, jarrahi and safavi (2015) concluded that proteus as a new formulated insecticide (based on combination of thiacloprid and deltamethrin) in pupal stage treatment of h. hebetor showed the highest handling time and the lowest attack rate compared with metarhizium anisopliae and the control; because the results are same, in our study r. officinalis showed the highest handling time and the lowest attack rate values on h. hebetor and therefore is an incompatible essential oil with this parasitoid wasp. the theoretical maximum attack rate also in all examined treatments was different and the highest value of this parameter was recorded for the control that this is in agreement with the results obtained by rafieedastjerdi et al., (2009b); abedi et al., (2012) and mahdavi and saber, (2013). functional response studies under laboratory conditions may have low similarity to the results that obtained in the field conditions (munyaneza and obrycki, 1997). houck and strauss (1985) and darwish et al., (2003) concluded that laboratory functional response has important role in understanding of the relations between different natural enemies and their hosts in biological control programs. such studies can provide valuable informations for developers of biological control programs and release of natural enemies in agricultural crops. in conclusion, this research showed that isolated essential oils affected the functional response and quality control of this parasitoid wasp. this study indicated that there isn’t significant difference between g. glabra compared with the control and g. glabra essential oil hadn’t negative effects on the functional response of h. hebetor and it’s parameters including attack rate, handling time and theoretical maximum attack rate. therefore, g. glabra essential oil can be recommended as a suitable botanical compound in integration with h. hebetor in ipm programs. in this research, we investigated the effects of selected essential oils against the ectoparasitoid wasp h. hebetor for the first time. this study shows the potential of essential oils as effective and natural compounds on different insects. the authors recommend more researches about the effects of essential oils on the other natural enemies and also application of these compounds for management of insect pests especially in 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(coleoptera: curculionidae). romani. biotech. let. 16(6): 6702-6709, 2011. yu sh, roy mi, na jh, choi wi. effect of host density on egg dispersion and the sex ratio of progeny of bracon hebetor (hymenoptera: braconidae). j. stored prod. res. 39(4): 385393, 2002. 312 isj 14: 312-323, 2017 issn 1824-307x research report evolution of the digestive enzymes and bacterial changes of the gastrointestinal tract of the artemia urmiana during growth period h ahmadniaye motlagh 1 , o safari 1 , m farhangi 2 , m lashkarizadeh-bami 3 1 department of fisheries, faculty of natural resources and environment, ferdowsi university of mashhad, khorasanrazavi, iran 2 department of fisheries, faculty of natural resources, university of tehran, karaj, iran 3 department of aquaculture, faculty of agriculture, universiti putra malaysia, 43400 upm serdang, selangor accepted august 19, 2017 abstract as the digestive enzymes and gastro-intestinal (gi) bacterial community contribute to the health and nutrition of the organism, this study aims to evaluate the ontogeny of bacterial population and digestive enzymes activities in the gi tract of artemia urmiana from nauplii to the adult stage. artemia cysts were hatched under standard conditions and stocked at a density of 20 nauplii ml -1 for 15 days. samplings for growth, bacterial and enzymatic analysis were collected on days 1, 5, 10 and 15 of the experiment. the results indicated that the gi tract was sterile at the time of hatching. artemia gi tract was active after hatching by protease (0.282 ± 0.001 u mg -1 protein min -1 ), lipase (0.182 ± 0.001 u mg 1 protein min -1 ) and amylase (0.295 ± 0.001 u mg -1 protein min -1 ) secretion and increased during the experiment. in addition, a significant relation (p < 0.05) was observed between artemia total length and the activity of digestive enzymes (lipase (r 2 = 0.98) amylase (r 2 = 0.97) and protease (r 2 = 0.98). a significant relation (p < 0.05) was observed between total aerobic bacteria and digestive enzyme secretion (lipase, r 2 = 0.83; amylase, r 2 = 0.66 and protease, r 2 = 0.84) too. such a relation was observed between total bacillus spp. count and digestive enzyme activities (lipase r 2 = 0.82; amylase r 2 = 0.79; and protease; r 2 = 0.74). these results suggest that in addition to the chemical composition of food, total length, gi bacteria enzyme secretion and the interaction of these factors contribute in digestive enzymes ontogeny during the growth period. key words: artemia; health; nutrition; digestion introduction some aquatic organisms, consume bacteria as food and gastrointestinal (gi) flora can be a reflection of bacterial community swallowed with food or as food. bacteria population can play an important role as food by providing micronutrients such as essential fatty acids, vitamins, or digestive enzymes (olafsen, 2001). the gi flora is affected by several factors such as season (irianto and austin, 2002), species (cahill, 1990), diet, environmental conditions and the growth stage (hansen and olafsen, 1999). the microbial ecology of the gi tract of a variety of freshwater and marine organisms has been investigated (denev et al., 2009; zarkasi et al., 2016; ___________________________________________________________________________ corresponding author: hamidreza ahmadniaye motlagh department of fisheries faculty of natural resources and environment ferdowsi university of mashhad khorasanrazavi, iran e-mail: ahmadnia@um.ac.ir zhu et al., 2016). in addition, some researchers have studied the probiotic properties of some indigenous and non-indigenous bacteria in aquaculture, but information on artemia larval development (ontogeny) is scarce and no work has been published concerning the bacterial changes of artemia gut during development. investigations of nutritional requirements and feeding ecology of marine invertebrates requires a deep consideration of basic digestive physiology. (patel et al., 2012). structure and the physiology of the digestive system of crustaceans and other marine invertebrate undergo important ontogenetic changes with adaptation to different food types (andrés et al., 2010) and developmental stages (veróonica and gimenez, 2013). fulfilment of nutritional requirements of marine invertebrates, including crustaceans, plays a critical role during larval development. since digestion is considered as a key factor in metabolism, it may be essential in determining the availability of nutrients required for biological functions (mcconaugha, 1985). thus, the mailto:ahmadnia@um.ac.ir 313 table 1 physico chemical parameters of artemia hatching and rearing water (mean ± sd) ph salinity(gl -1 ) do (mgl -1 ) temperature (ºc) agent 8.3±0.5 35±2 4±1 29±1 hatching 8.3±0.5 60±2 6±1 29±1 rearing study of various aspects of the digestion physiology during ontogeny is essential to have a promising larviculture (applebaum et al., 2001). most of the reports on digestive enzymes in crustaceans have been confined to adult specimens; interactions between variations in gut morphology, digestive enzyme activities, and diet throughout early stages of life cycle are thoroughly unknown. changes in digestive enzyme activity during development have been reported in relatively few crustacean species penaeus setiferus (lovett and felder, 1990) artemia spp, (gawlicka et al., 2000), macrobrachium rosenbergii (kamarudin et al., 2011), litopenaeus vannamei (wei et al., 2014). despite digestive enzymes secreted by indigenous bacteria of gi are of high importance (suzer et al., 2008; wang et al., 2017), we found no study concerning the role of bacteria in enzymatic development of crustaceans. ceccaldi (1989) stated that digestive enzymes of crustacean species are diversified into proteases, among which trypsin is the major one, lipases and esterases, amylases, maltases and chitinases are also well-represented. determination of digestive enzyme profiles in puerulus, postpuerulus, juvenile and adult stages of the spiny lobster jasusedwardsii was made to find out the ontogenetic alterations in digestive proficiencies. a variety of enzymes was observed in juvenile and adult lobsters, exhibiting their potential to exploit different diets. according to the results, lobster was found out to be carnivorous (johnston, 2003) as dietary protein played more essential role than carbohydrate sources. gawlicka and co-workers in 2000 determined activities of trypsin, amylase, lipase and alkaline phosphatase in metamorphic larvae hippoglossus hippoglossus and in their artemia prey to estimate the importance of exogenous enzymes for atlantic halibut larvae. the calculated contribution of enzyme activities derived from artemia prey to the relatively high levels of enzyme activity in the digestive system of metamorphic larvae was less than 10 % for all enzymes except amylase, for which the contribution was estimated to be more than 50 %. based on the available literature, there is no information on gi bacterial ontogeny and the interaction between gi bacteria and digestive enzymes, this survey can open new viewpoint of the nutritional biology of artemia and other crustaceans. recently, artemia urmiana (günther, 1890) population as an indigenous species in urmia lake (in the north-west of iran) has been exposed to main challenges including drought and salinity increment (motlagh et al., 2012). therefore, indoor mass-culture of artemia can alleviate the pressure of aquaculture industry on natural stocks to collect following products (cyst and biomass). regarding the critical role of gi microflora and digestive enzymes on the health and nutrition of aquatic larvae, the aim of present study was to evaluate the digestive enzymes (protease, lipase and amylase) and bacterial ontogeny of the gi tract of the a. urmiana from nauplius to the adult stage as a vital point of artemia mass culture. materials and methods hatching and rearing of artemia urmiana nauplii of a. urmiana were hatched in the laboratory in cylindrical containers (2 l) of saltwater and maintained on a 12:12 light:dark cycle, under standard condition (sorgeloos 1986). artemia nauplii were transferred into 60 l polyethylene tanks with the density of 20 nauplii ml -1 and reared for 15 days (shahnaz jabari et al., 2015). physicochemical properties of hatching and rearing water, including temperature, dissolved oxygen, salinity and ph (table 1) were monitored daily according to standard methods (agh, 2007) for hatching and rearing periods. artemia urmiana feeding during the first five experimental days, nauplii were fed with backers yeast (saccharomyces cerevisiae) (lavens and sorgeloos, 1996). from the second day, after hatching 1.25 mg of baker yeast per 1000 nauplii in 400 ml saline water (35 gl -1 ) at 28 ºc, the distribution of solutions in rearing water was facilitated by passing them through a 150 micron mesh. from the sixth day onwards, the artemia were fed a diet including white wheat flour (11.24 %) and equal amounts of soybean meal and chickpea flour (44.38 %) provided by behparvar co. (iran). feeding was performed three times a day with a four-hour interval. the dietary chemical composition was analyzed using the standard methods (peterson and martin-robichaud, 1983). the crude protein, crude fat, dry matter and ash values were (12 % ± 0.93), (55 % ± 1.2), (97.5 % ± 0.77) and (5 % ± 0.35), respectively. table 2 shows the feeding schedule of artemia. growth monitoring biometry of artemia was performed on the first, fifth, tenth and fifteenth experimental days for the evaluation of growth. sampling was conducted by removing five samples of water with 10 ml volumes from each tank and measuring the total length of artemia by a micrometer (0.01 mm). 313 table 2 artemia feeding schedule over the experiment 11-14 10 9 8 7 6 5 4 3 2 1 experimental days 0.07 0.07 0.062 0.06 0.05 0.05 0.03 0.02 feed amount (g l-1) 0.0155 0.03 0.037 0.05 0.03 0.02 yeast 0.07 0.07 0.0465 0.03 0.0125 dry food microbial analysis the total aerobic bacteria count and total bacillus count in the gl of artemia (cfu g -1 artemia) were estimated on first, fifth, tenth and fifteenth days. a 70 % alcohol was used for washing nauplii, and the sterile saline water (35 g nacl l-1) was used for rinsing them to eliminate the sticking bacteria from their body surface. five ml of sterile saline water was gradually added to the samples in order to homogenize them. then, ten times serial dilution were prepared, and mediums bacillus cereus agar and marine agar 2216 (himedia©) were used to count the total aerobic bacteria and bacillus, respectively (rengpipat et al., 1998). bacillus cereus agar was incubated at 30 °c for 24 h, and marine agar was incubated at 29 °c for 24 h. enzymatic assays samplings for determining the activities of protease, lipase and amylase were conducted on first, fifth, tenth and fifteenth experimental days. the samples were first washed with cold fresh water and rinsed. then, the samples were stored in 15-ml falcon tubes and instantly transferred to a freezer (80 ºc) (paiva-maia et al., 2013). the samples were defrosted in laboratory conditions to extract the enzymes. the homogenates of the whole animal body were used in all evaluations. homogenization of the extracts prepared in physiological saline solution (0.9 % nacl) was performed by adding saline solution which obtained a total volume of 1.6 ml per sample. the homogenized solutions were centrifuged at 5,000g for 5 min. the enzymatic evaluations were conducted using the supernatants. the hydrolysis of casein at ph 8 was used to estimate the protease activity (paiva-maia et al., 2013), starch was used as the substrate to measure the amylase activity (worthington, 1991). in order to determine the lipase activity, olive oil emulsion substrate-gum arabic was used and the thawed samples were titrated at room temperature (worthington, 1991). statistical analysis all percentage data were transformed using the arcsine method. the leaven test was used to confirm the homogeneity of variance, and the kolmogorov-smirnov test was used to determine the normality of data (zar, 1999). the data were analyzed using one-way anova. duncan multiple range test was applied in order to find out if there were any significant differences among the treatments (p < 0.05). regression relations were conducted using spsstm version 19 (spss inc., chicago, usa). results the changes of total length in artemia urmiana as shown in figure 1, there are significant differences between total length of a. urmiana during sampling days. the significantly highest total length (3.5 mm) was observed in the day 15. fig. 1 the mean (±sd) total length (mm) in a. urmiana during sampling times with three replicates. different letters indicate significant differences (p < 0.05). 314 313 fig. 2 the mean (±sd) total aerobic bacterial count (×10 6 cfu g -1 ) in a. urmiana gi during sampling times with three replicates. different letters indicate significant differences (p < 0.05). the ontogeny of the gi bacterial in artemia urmiana the changes of the total aerobic bacterial count total number of aerobic bacteria in the gi of newly hatched artemia nauplii showed that the gi was sterile and free of bacteria. as shown in figure 2, the total number of aerobic bacteria shows a significantly (p < 0.05) increasing trend, up to the fifteenth day of the experiment. the average number of total aerobic bacteria in the gut showed significant differences between the fifth, tenth and fifteenth day of the experiment (p < 0.05). the results revealed no significant difference between the tenth and the fifteenth day. the changes of the bacillus spp. count no bacillus was reported in artemia gi at the first day of sampling but count increased later throughout the experiment. the final comparison between average bacillus spp. count shows a significant increase (p < 0.05) from zero (in day one) to 3.8 ± 0.8×104 (in day 15) (fig. 3). the ontogeny of digestive enzymes activities in artemia urmiana the changes in protease, amylase and lipase activities during the experiment are shown in figure 4. on the first day, the specific activities (u min -1 ) of protease, amylase and lipase in the artemia gut were reported to be 0.282 ± 0.07, 0.295 ± 0.05 and 0.182 ± 0.02, respectively. the lipase activity was higher than the other enzyme activities (p < 0.05) on day five. however, on the tenth and fifteenth days, significant differences were observed among all enzymes, and lipase exhibited the highest activity (p < 0.05). at the beginning of the experiment, amylase showed the highest activity with no significant difference with the others, however over the time amylase activity decreased and lipase activity was increased. fig. 3 the mean (±sd) bacillus spp. count (×10 6 cfu g -1 ) in a. urmiana during sampling times with three replicates. different letters indicate significant differences (p < 0.05). 315 313 fig. 4 the mean (± sd) activities (u mg protein -1 min -1 ) of digestive enzymes (lipase, amylase and protease) in a. urmiana during sampling times with three replicates. different letters indicate significant differences (p < 0.05). small letters (a d) and capital letters (a c) show differences during day post hatch and among enzyme activities during a specific day, respectively. polynomial models in figure 5 indicated that, there were significant difference (p < 0.05) between polynomial models of length and digestive enzyme activities including lipase (r 2 = 0.98), amylase (r 2 = 0.97) and protease (r 2 = 0.98). as well, figure 6 significant differences were found (p < 0.05) between polynomial models of the age of artemia (day post hatch) and digestive enzymes including lipase activity (r 2 = 0.99), amylase activity (r 2 = 0.99) and protease activity (r 2 = 0.99). significant differences were found (p < 0.05) between polynomial models of total aerobic bacteria count and digestive enzymes including lipase activity (r 2 = 0.83), amylase activity (r 2 = 0.66) and protease activity (r 2 = 0.84) (fig. 7). in addition, as shown in figure 8, there were significant differences between (p < 0.05) polynomial models of total bacillus spp. count and digestive enzymes including activities of lipase, amylase and protease (r 2 = 0.82, 0.79 and 0.74, respectively). discussion the ontogeny of the gi bacteria in artemia urmiana ontogeny of the bacterial flora of the gi tract of freshwater and marine organisms have not been studied yet and this issue requires more research (safari et al., 2014; wang et al., 2017). in the current study, no bacteria were detected in gi on the first day post-hatch which confirms the other researchers finding (ringø and gatesoupe, 1998). an increasing trend of total aerobic bacteria was observed. in fact, the total number of aerobic bacteria increased significantly with artemia growth and development of the attachment sites in the gi. this trend was also observed in total bacillus count as representative beneficial bacteria. this could be due to dietary consumption. in the present study, the artemia consumed s. cerevisiae for the first 5 days of the experiment. several researchers had proved the dietary effects on the intestinal microflora. the effect of the food sequence on the population of intestinal microbiota in larvae and juveniles of sparus aurata has been observed by weaning the early juveniles of this fish (savas et al., 2005). vibrio and pseudomonas were found out as the dominant genera after feeding juveniles with a compound diet depending on culture methods (silva et al., 2011). unfortunately, few studies have been conducted on artemia ontogeny, which requires further research. moreover, quantifying the main bacteria family throughout each ontogeny stage and using dgge technique are recommended as the future research topics. the ontogeny of digestive enzyme activities in artemia urmiana one of the most important factors affecting commercially successful larval production is survival rate that is affected by various factors-mainly, larval rearing period and larval nutrition (shields, 2001; turkmen et al., 2017). digestion of food to provide nutrients for growth, maintenance, motion, and reproduction is a very important function in an organism (hernández and murueta, 2009). the intake of sufficient amount of nutrients and their efficient digestion by an organism depends on its ability to select food and the capacity of its digestive enzymes (bautista 1983; silva et al., 2010). it is thus important to understand the profile of these digestive enzymes, as this can be used as a tool to develop food specific to the metabolic needs of species (pavasovic et al., 2004). however, we need to standardize specific histochemistry methods for further studies on live feed production industry including artemia. extended published records have focused on invertebrate digestive enzyme synthesis, regulation, and evaluation, including crustaceans (le vay et al., 2001; muhlia-almazán et al., 2008; andrés et al., 2010; 316 313 fig. 5 polynomial model fitting (a) lipase activity (u/mg protein. min -1 ), (b) amylase activity (u/mg protein. min -1 ) and (c) protease activity (u/mg protein. min -1 ) to length (l; mm) in a. urmiana with three replicates. a) b) c) 317 313 fig. 6 polynomial model fitting (a) lipase activity (u/mg protein. min -1 ), (b) amylase activity (u/mg protein. min -1 ) and (c) protease activity (u/mg protein. min -1 ) to day post hatch (d) in a. urmiana with three replicates. c) b) a) 318 313 fig. 7 polynomial model fitting (a) lipase activity (u/mg protein. min -1 ), (b) amylase activity (u/mg protein. min -1 ) and (c) protease activity (u/mg protein. min -1 ) to total count (tc; x 10 6 cfu g -1 ) in a. urmiana with three replicates. a) b) c) 319 313 fig. 8 polynomial model fitting (a) lipase activity (u/mg protein. min -1 ), (b) amylase activity (u/mg protein. min -1 ) and (c) protease activity (u/mg protein. min -1 ) to bacilus spp. count (bc; x 10 4 cfu g -1 ) in a. urmiana with three replicates. c) b) a) 320 313 veróonica and gimenez, 2013; wei et al., 2014). assessment of the digestive enzymes activity can be used as an indicator of larval growth rate, food acceptance and the digestive capacity. several workers have reported the activity of proteases, amylases, and lipases in crustaceans (figueiredo et al., 2001). but no report was found about a. urmiana digestive enzymes (protease, lipase and amylase) ontogeny during development. it has shown that many crustacean species with economic importance possess the necessary enzymes for the hydrolysis of carbohydrates; however, amylase has been poorly studied in crustaceans (pavasovic et al., 2004). the results of the current experiment showed that a. urmiana nauplii digestive system is active starting from hatching in production and secretion of digestive enzymes. these findings were in agreement with andrés et al. (2010) which indicated that all assayed enzymes were active from hatching time in spider crab, while lovett and felder (1990) reported that in penaeus setiferus no activities were present for pepsin and lipase in some developmental stages. maximum activities of protease, lipase and amylase were recorded in the fifteenth day of the experiment for a. urmiana which agrees with peak activities for all enzymes occurred during late zoea or early mysis larval stages of p. setiferus. this discrepancy can be species-specific, due to sampling methods and the definition of critical ranges of digestive enzyme activities during sample preparation and final optical density (od) reading. the activity of lipase was higher than others during the test that is consistent with other studies (johnston, 2003). the high lipase activity in all artemia stages reflects the importance of lipid as an energy reserve in crustaceans (icely and nott 1992; johnston, 2003) and its key role as an energy substrate for j. edwardsii puerulus (jeffs et al., 1999, 2001, 2002), h. americanus (sasaki et al., 1986) and artemia. according to johnston (2003), a significant change in lipase-specific activity during different post hatch days confirms that lipid utilization is not consistent throughout development. although the lipase activity was higher than other assessed enzymes, such an increment will not necessarily demonstrate higher absorption of fat in the digestive tract. results indicated that by shifting diet to a rich protein diet on the fifth day, the secretion of protease increased significantly. these findings are confirmed by other researchers who treated a. urmiana with different diets and reported that chemical composition of diets can alter the activities of digestive enzyme secreted by artemia (bami et al., 2011). similar results were obtained in the investigation of the ontogenetic changes in digestive enzymatic capacities of the spider crab (maja brachydactyla) (andrés et al., 2010). on the other hand, some hypothesis on considering diet as a non-essential factor for ontogenic changes in the activities of digestive enzymes may arise. instead, some developmental changes in enzyme synthesis or a secondary effect of change in the function and approximate size of the midgut throughout its distinction may be responsible for the ontogenic alteration of digestive enzyme activities (lovett and felder, 1990). results reported there was a significant relationship between artemia total length and the protease activity which it may suggest that larger artemia tends to consume foods that contain more protein content, like certain protozoa and bacteria. a similar trend was detected in ontogenetic changes in digestive enzyme activity of the spiny lobster (jasus edwardsii) that revealed a strong relationship between total enzymes activity and carapace length juvenile and adult lobsters (johnston, 2003). in the present study, we observed significant polynomial models between total length and digestive enzymes activities. these findings are in agreement with ribeiro and jones (2000) which stated that the evolution of trypsin and lipase activity in fenneropenaeus indicus showed a dependence on the length of the post larvae, therefore the enzymatic response increases with size and evolutionary stages in f. indicus. measurement of amylase and trypsin activities during different evolution stages of san francisco lake artemia showed that the secretion of these enzymes depends on nutritional requirements of artemia, and also the amount, chemical composition and the ingestion of food (harris et al., 1986). these researchers also have reported lots of changes in the activity of these enzymes in different developmental stages. these enzymes activity can fluctuate depending on the age and type of food. since the beneficial bacteria of gi tract like bacillus spp. are capable of secreting digestive enzymes (moriarty, 1998), it is likely that a part of the enzyme activity in the gi was associated with the bacteria. these suggest that in addition to chemical composition of food (bami et al., 2011), total length, evolutionary stages, gi bacteria enzyme secretion (motlagh et al., 2012), the interaction among these factors (augusto e et al., 2012) can influence on digestive enzymes activity during growth period. however, these findings may open a new area in live feed production industry. the results of this study demonstrated the role of gi bacteria and other factors in the secretion of digestive enzymes. the accurate discovery of the bacterial role in producing digestive enzymes may result in the selection of probiotics more compatible with crustaceans and more accurate application of prebiotics. however, it requires a precise identification of bacterial communities and their role in the production and secretion of each digestive enzyme. nowadays, new methods such as ngs and dgge have been introduced to obtain such an intention. apparently, more authentic 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266237, china 3university of chinese academy of sciences, 19 yuquan road, beijing 100049, china 4fisheries college, tianjin agriculture college, 22 jinjing road, tianjin 300384, china accepted june 1, 2017 abstract ammonia and nitrite levels caused by shrimp excreta and metabolic waste and organic detritus are important limiting factors in intensive aquaculture system, the purpose of this study was to determine how ammonia and nitrite accumulation caused by accumulated these compounds affected survival and growth performance of white shrimp litopenaeus vannamei. unconsumed feed, feces and seawater in treatment group were not removed or replaced over a 33-day period, while unconsumed feed and feces in control group were removed with a siphon tube and 60 % seawater was replaced once-daily. significantly higher ammonia and nitrite concentrations were accumulated in the seawater of treatment group from day 6 to day 33 and from day 9 to day 33, compared with control group, respectively. significantly lower survival rate, weight gain percentage, length gain percentage and specific growth rate were recorded in treatment shrimp, compared with control shrimp. significantly higher lipase, superoxide dismutase, and catalase activities, malondialdehyde content, relative expression of 4e-binding protein 1, p70s6 kinase, glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase mrna were detected in the hepatopancreas of treatment versus control shrimp. significantly lower protease, glutathione reductase and glutathione peroxidase activities, and glutathione content were detected in the hepatopancreas of treatment versus control shrimp. meanwhile, hepatopancreas in treatment shrimp showed disorganized tubules, blurred boundaries, decreased or disappeared b, r and e cells, injured connective tissue between liver tubule, infiltrated hemocytes, narrowed lumen, and vacuolization compared with control shrimp. these findings might indicate that ammonia and nitrite accumulation caused by accumulated waste in aquaculture tanks could significantly reduce survival, growth performance of l. vannamei with hepatopancreas damage, which was resulted from accumulated reactive oxygen species. therefore, ammonia and nitrite accumulation may significantly impact shrimp production in intensive aquaculture system. key words: litopenaeus vannamei; ammonia and nitrite accumulation; survival; growth performance; hepatopancreas introduction decapod crustaceans release nitrogenous waste as ammonia in aquaculture system, the end product of protein catabolism (regnault, 1987); nitrite is an intermediate product either during bacterial denitrification of nitrate or bacterial nitrification of ___________________________________________________________________________ corresponding author: lei w ang key laboratory of experimental marine biology institute of oceanology chinese academy of sciences 7 nanhai road, qingdao 266071, china e-mail: wanglei@qdio.ac.cn ammonia (mevel and chamroux, 1981). both ammonia and nitrite are the most common pollutant in intensified aquaculture or in recirculated water, they increase with culture duration which can increase to 0.81 mg/l total ammonia-n, 0.12 mg/l nitrite-n in the hatcheries and 6.497 mg/l total ammonia-n, 4.611 mg/l nitrite-n in the grow-out ponds even with frequent water exchange (chen et al., 1986, 1989). pond water in massive cultivation may accumulate high concentrations of ammonia and nitrite as the result of excretion by the animals in the system and the mineralization of organic detritus, 222 such as unconsumed food and feces (tacon et al., 2002; chen et al., 2012; ren et al., 2015). previous studies demonstrated that ammonia stress could affect a variety of physiological functions of crustaceans, including respiration, metabolism, excretion (racotta and hernandez-herrera, 2000; cheng and chen, 2001; miranda-filho et al., 2009; barbieri 2010; martin et al., 2011; ren et al., 2015), the immune system (yue et al., 2010; peaydee et al., 2014; ren and pan, 2014; chang et al., 2015; duan et al., 2015; wongsasak et al., 2015; pinto et al., 2016), osmoregulation (romano and zeng, 2007; romano and zeng, 2010), and apoptosis and molting (mugnier et al., 2008; liang et al., 2016). like ammonia, nitrite stress could also affect crustacean growth, respiration, metabolism (chen and cheng, 1995, 2000; mallasen and valenti, 2006; hong et al., 2009; xian et al., 2011), the immune system (tseng and chen 2004; wang et al., 2004, 2006; liao et al., 2012), excretion (chen and cheng, 1995, 2001, 2002) and apoptosis (xian et al., 2012; guo et al., 2013). furthermore, ammonia and nitrite could act synergistically, resulting in toxic effects that were greater than either compound alone (schuler et al., 2010; cheng et al., 2013; zhang et al., 2015). the tropical white shrimp litopenaeus vannamei has become an attractive cultivar for inland aquaculture in many parts of the world, including the usa, thailand, and china. it is widely cultured in extensive, intensive, and semi-intensive systems (frías-espericueta et al., 1999). due to the limitations and availability of land to construct, the culture of l. vannamei has been intensified to boom the production and economic benefit. in this intensive culture system, shrimp excreta and metabolic waste produced and organic detritus caused concern with water pollution and shrimp diseases, ammonia and nitrite levels are important limiting factors during the rapid accumulation of these compounds. it has been reported that the concentration of nitrite increases directly with culture period, and might reach as high as 20 mg/l in grow-out ponds of l. vannamei (tacon et al., 2002). however, we know little about the toxic mechanism of ammonia and nitrite accumulation caused by accumulated those compounds in intensive aquaculture system on shrimp. the aim of the present study was to evaluate the effects of ammonia and nitrite accumulation caused by accumulated waste in aquaculture tanks on l. vannamei, which would lead to develop a better understanding of the toxic mechanism of ammonia and nitrite accumulation caused by shrimp excreta and metabolic waste and organic detritus in intensive aquaculture system on shrimp. given that the hepatopancreas is the most important digestive gland in crustacean, and is involved in digestion, nutrient absorption, storage, diseases, as well as synthesis and excretion of digestive enzymes (bautista et al., 1994; rosas et al., 1995; li et al., 2008, franceschini-vicentini et al., 2009), and this organ in shrimp comprises branched tubules lined by different types of epithelial cell (e cells, r cells, f cells, and b cells), we investigated the effects of ammonia and nitrite accumulation caused by accumulated waste in aquaculture tank on the survival, growth performance and digestive enzyme activities, antioxidant status, metabolic gene expression, and histology in the hepatopancreas of l. vannamei. materials and methods experimental shrimp fresh, healthy, juvenile litopenaeus vannamei (mean weight 0.90 ± 0.02 g) were obtained from the ruizi seafood development co. ltd. (qingdao, china), where the experiment was also conducted. a total of 1200 shrimp were distributed among six 640-l cylindrical tanks with net cover (n = 200 per tank), every 640-l cylindrical tank contained 500-l aerated seawater (dissolved oxygen 5.4 6.5 mg/l). the initial seawater is unfiltered with ph 7.5 8.2, salinity 30 31 ‰, total ammonia 0.022 0.038 mg/l, nitrite 0.015 0.032 mg/l, and nitrate 0.030 0.065 mg/l at 28 32 c. they were acclimated for 2 weeks under a natural photoperiod (12 h: 12 h light: dark). the shrimp were fed three times daily with a commercial diet (41.52 % crude protein, 7.42 % lipid, and 12.03 % crude ash, supplied by yantai dale feed co. ltd, shandong, china) at 07:00 h, 11:00 h, and 19:00 h, at a feeding rate of 35 %, 20 %, and 45 %, respectively, representing a daily feeding rate that was 10 % of the weight of shrimp. the unfed feed and feces were removed with a siphon tube and 60 % seawater was replaced once-daily. unfiltered seawater (28 – 32 c, ph 7.5 8.2, salinity 30 31 ‰, total ammonia 0.022 0.038 mg/l, nitrite 0.015 0.032 mg/l, and nitrate 0.030 0.065 mg/l) were prepared in other three 1000-l cylindrical tanks to use for exchange daily. experimental design for ammonia and nitrite accumulation following acclimation, the experimental setup was comprised of two experimental groups, each group had three repetitions, each 640-l cylindrical tank was regarded as a repetition: (1) control group (three 640-l cylindrical tanks); and (2) treatment group (three 640-l cylindrical tanks). shrimp were experimented for 33 days in 640-l cylindrical tanks with net cover containing 500-l aerated seawater (dissolved oxygen 5.4 6.5 mg/l), the photoperiod and feeding condition was handled in exactly the same way as during the acclimation period. for the control tanks, the unconsumed feed and feces were removed with a siphon tube and 60 % seawater was replaced once-daily, to maintain low ammonia and nitrite concentrations in the tanks. by contrast, for the treatment tanks, unconsumed feed, feces and seawater were not removed or replaced, to allow ammonia and nitrite accumulation in the tanks, and seawater was only added to raise the volume in the tanks to 500 l as necessary. unfiltered seawater (28 32 c, ph 7.5 8.2, salinity 30 31 ‰, total ammonia 0.022 0.038 mg/l, nitrite 0.015 0.032 mg/l, and nitrate 0.030 0.065 mg/l) was still prepared in other three 1000-l cylindrical tanks to use for exchange daily for control tanks, and for adding to 500 l for treatment tanks. at the end of the experiment, shrimp were deprived of feed for 24 h before any experimental treatment. 223 table 1 primers for the genes encoding p70s6k, 4ebp1, got, gpt, gst, and β-actin in shrimp. primer pairs of p70s6k, 4ebp1, got, gpt were designed by primer5 according to transcriptome sequences in our lab, and gst, β-actin were from zhou et al.(2009) target gene sequence (5ʹ→3ʹ) p70s6k f-gcaagaggaagacgccata r-ccgcccttgcccaaaacct 4ebp1 f-atgtctgcttcgcccgtcgctcgcc r-ggttcttgggtgggctctt got fctacgacccaaagacctgt raccttgctcatttcatccc gpt f-atgtcgtttgtgggttttcg r-ttcgccttggtcacgctgt gst faagataacgcagagcaagg rtcgtaggtgacggtaaaga β-actin fgcccatctacgagggata r-ggtggtcgtgaaggtgtaa measurement of ammonia and nitrite concentrations ammonia and nitrite concentrations in the seawater of each tank were measured using the hypobromite oxidation method and naphthylethylenediamine photometric method (gb 17378.4 2007), respectively, every 3 days. hypobromite oxidation method: 10 ml seawater was added with 1ml 168 g/l fresh sodium hypobromite solution and mixed, the mixture was mixed with 1 ml 2 g/l sulfanilamide solution and then mixed with 0.2 ml 1 g/l ethylenediamine dihydrochloride solution, absorbance (y) of this solution was measured at 543 nm wavelength, and ammonia concentration (x) was evaluated in term of drawn working curve, y = 3.28x + 0.0155. naphthylethylenediamine photometric method: 10 ml seawater was added with 0.2 ml 10 g/l sulfanilamide solution and mixed, the mixture was mixed with 0.2 ml 1 g/l ethylenediamine dihydrochloride solution, absorbance (y) of this solution was measured at 543 nm wavelength, and nitrite concentration (x) was evaluated in term of drawn working curve, y = 3.303x + 0.0126. measurement of survival and growth performance and sampling procedure the number of dead shrimp from each tank was recorded every 24 h during the experimental period. shrimp were considered dead when they failed to move even when gently stimulated with a glass pipette. dead shrimp were removed to prevent fouling. 20 shrimp from each tank were randomly selected at the beginning and end of the experiment, respectively, and replaced after measuring weight and length. survival and growth performance was evaluated in terms of their survival rate (sr), weight gain percentage (wgp), length gain percentage (lgp), and specific growth rate (sgr) based on the following standard formulae: sr (%) = 100 × (final shrimp number)/(initial shrimp number); wgp (%) = 100 × (final weight-initial weight)/initial weight; lgp (%) = 100 × (final length-initial length)/initial length; and sgr (%/day) =100 × (ln final weight-ln initial weight)/days. 20 shrimp were randomly selected and removed from each tank at the end of the experiment (n = 60 for each group); the hepatopancreas from each shrimp was collected using sterilized scissors and forceps: five for each tank were immediately placed into rnalater (applied biosystems, austin, tx, usa) and stored at -20 °c until rna isolation and mrna expression analysis (n = 15 for each group); five for each tank were immediately stored at -80 °c to be used for the digestion and antioxidant parameter assay (n = 15 for each group); and ten for each tank were immersion fixed in bouin's solution for 18-h and then transferred to 70 % (v/v) ethanol (romano et al., 2015), until processing for histological study (n = 30 for each group). hepatopancreatic digestive enzyme activity and antioxidant status approximately 200 mg of hepatopancreas tissue was dissected from a single hepatopancreas, five hepatopancreas tissues for each tank were mixed at a 1:5 ratio (w/v) with chilled tris-hydrochloric acid buffer solution (ph 7.6, 10 mmol l−1) and homogenized under ice-chilled conditions. the homogenates were then centrifuged at 10,000×g, 4 °c for 30 min and the supernatants were then collected for the assays. digestive enzyme activities, including protease activity, amylase activity, cellulose activity and lipase activity, were evaluated using commercial kits (nanjing jiancheng bioengineering institute, nanjing, china) according to the manufacturer's instructions. each of the above digestive enzyme activities was analyzed 224 fig. 1 ammonia concentrations in the seawater of tanks during the experimental period; *indicates a significant difference between the treatment (blue circles) and control (black circles) groups. ammonia concentration could significantly increase in the treatment group versus control group. fig. 2 nitrite concentrations in the seawater of tanks during the experimental period.*indicates a significant difference between the treatment (blue circles) and control (black circles) groups. nitrite concentration could significantly increase in treatment group versus control group. with three replicates of each sample. the antioxidant status, including superoxide dismutase (sod) activity, catalase (cat) activity, glutathione peroxidase (gpx) activity, glutathione reductase (gr) activity, glutathione s-transferase (gst) activity, glutathione (gsh) content, and malondialdehyde (mda) content, was also evaluated using commercial kits (nanjing jiancheng bioengineering institute) according to the manufacturer's instructions. each of the above antioxidant status was analyzed with three replicates of each sample. activities were expressed as a relative unit per milligram of soluble protein (u/mg protein), and gsh and mda contents were expressed as the relative concentration per milligram of soluble protein (nmol/mg protein). hepatopancreatic metabolic gene expression assays rna was extracted from a single hepatopancreas using a rna fast extraction kit according to the manufacturer's protocol (fastagen, shanghai, china). rna solution from five hepatopancreases for each tank were mixed into 5 μl, 1 μl rna solution per hepatopancreas; this 5 μl rna solution was used for cdna synthesis using a transscript® one-step gdna removal and cdna synthesis kit according to the manufacturer's protocol (transgen biotech co., ltd, beijing, china). the expression of the metabolic genes encoding 4e binding protein 1 (4ebp1), p70s6 kinase (p70s6k), glutamic-oxaloacetic transaminase (got), glutamic-pyruvic transaminase (gpt), and gst was investigated. β-actin was selected as a reference gene and specific primers were used for each gene (table 1). the relative expression of each of the above metabolic genes was analyzed by relative quantitative real-time pcr (qpcr) using transstar top green qpcr supermix according to the manufacturer's protocol (transgen biotech co., ltd) with three replicates of each sample. qpcr was performed with the following two steps: denaturation at 94 °c for 30 s and then 40 cycles of 94 °c for 5 s and 60 °c for 30 s. the dissociation curve analysis was performed at the end of qpcr to confirm the specificity of the pcr products, and relative expressions were determined using the 2−δδct method (livak and schmittgen 2001). hepatopancreatic histology assays the fixed ten hepatopancreases for each tank were dehydrated in ascending concentrations of alcohol, cleared in toluene, embedded in paraffin, and sectioned with a rotary microtome at 5 μm. sectioned tissues were stained with hematoxylin and eosin (h&e), and examined with a light microscope (casado et al., 2001). statistical analysis the data were all presented as the mean ± standard error (se). statistical analysis was performed with spss (version 17.0) (ibm, new york, america), and t-test was used to analyze differences between two experimental groups. the significance level was p < 0.05. all images were generated with origin 9.0 software (originlab, massachusetts, america). results ammonia and nitrite concentrations there were no significant changes of both ammonia and nitrite concentrations in the seawater of control group during the experimental period (figs 1, 2). by contrast, there were a gradual increase in both ammonia and nitrite concentrations in the seawater of treatment group, such that they were significantly higher (p < 0.05) than the control group from day 6 to day 33 and from day 9 to day 33, respectively (figs 1, 2). 225 table 2 growth performance of shrimp after the 33-d experiment group sr (%) wgp (%) lgp (%) sgr (%) control treatment 86.83±3.01 34.56±2.92* 547.05±6.47 341.10±4.85* 62.80±1.35 39.60±1.14* 5.66±0.11 4.50±0.06* sr, wgp, lgp, and sgr represent survival rate, weight gain percentage, length gain percentage, and specific growth rate respectively. * indicates a significant difference between the treatment and control groups. survival and growth performance sr, wgp, lgp, and sgr of treatment shrimp were significantly lower (p = 0.001, p = 0.024, p = 0.031, and p = 0.042, respectively) compared with control shrimp (table 2). hepatopancreatic digestive enzyme activities protease activity of treatment shrimp was significantly lower (p = 0.036) compared with control shrimp; however, lipase activity was significantly higher (p < 0.001) in treatment versus control shrimp (fig. 3). no significant difference was observed in amylase activity (p = 0.210) and cellulose activity (p = 0.376) between the two groups (fig. 3). hepatopancreatic antioxidant status mda content, sod activity, and cat activity in the hepatopancreas of treatment shrimp were significantly higher (p = 0.006, p < 0.001 and p = 0.035, respectively) compared with control shrimp; however, gsh content, gpx activity, and gr activity in treatment shrimp were significantly lower (p < 0.001, p = 0.015, and p = 0.001, respectively) than control shrimp (figs 4, 5). meanwhile, no significant difference was observed in gst activity (p = 0.087) between the two groups (fig. 5). hepatopancreatic metabolic gene expression relative expression of p70s6k, 4ebp1, got, and gpt mrna of treatment shrimp were significantly higher (p = 0.003, p < 0.001, p = 0.035, and p = 0.005, respectively) compared with control shrimp (fig. 6). however, no significant difference was observed in the relative expression of gst mrna (p = 0.104) between the two groups (fig. 6). hepatopancreatic histology structure histology structure of 30 hepatopancreases in control shrimp appeared consistent look, and a representative figure (fig. 7a) was chosen. meanwhile, histology structure of 30 hepatopancreases in treatment shrimp also appeared consistent look, and a representative figure (fig. 7b) was chosen. compared with the control shrimp (fig. 7a), histology structure of hepatopancreas in treatment shrimp showed remarkable change, including disorganized tubules, blurred boundaries, decreased or disappeared b, r and e cells, injured connective tissue between liver tubule, infiltrated hemocytes, narrowed lumen, and vacuolization (fig. 7b). discussion previous studies (schuler et al., 2010; cheng et al., 2013; zhang et al., 2015) revealed the physiological effects of isolated and combined ammonia and nitrite on crustacean, the experimental nitrite and ammonia concentrations in aquaculture water were all achieved by adding dissolved nano2 and nh4cl, resulting in animals being exposed to steady-state concentrations of ammonia and nitrite. however, the present study adopted a new approach, which showed that ammonia and nitrite were accumulated constantly in the treatment tanks, and that concentrations of both were significantly higher in the seawater of treatment group than control group after a certain period of time, presumably the result of the accumulation and breakdown of unconsumed food, faeces and excretion by the treatment shrimp (tacon et al., 2002; chen et al., 2012; ren et al., 2015). therefore, this approach was a more accurate representation of fig. 3 protease, amylase, cellulose, and lipase activities in the hepatopancreas of shrimp after the 33-d experiment. *indicates a significant difference between the treatment (blue circles) and control (black circles) groups. lipase activity increased significantly in the treatment group versus control group; protease activity decreased significantly in the treatment group versus control group. 226 fig. 4 mda and gsh contents of the hepatopancreas of shrimp after the 33-d experiment. *indicates a significant difference between the treatment (blue circles) and control (black circles) groups. mda content increased significantly in the treatment group versus control group; gsh content decreased significantly in the treatment group versus control group. fig. 5 sod, cat, gpx, gr, and gst activities in the hepatopancreas of shrimp after the 33-d experiment. *indicates a significant difference between the treatment (blue circles) and control (black circles) groups. sod and cat activities increased significantly in the treatment group versus control group; gpx and gr activities decreased significantly in the treatment group versus control group. the toxic mechanism of ammonia and nitrite accumulation caused by shrimp excreta and metabolic waste and organic detritus in intensive aquaculture system on shrimp. though changes in water quality within the outdoor zero-water exchange culture system included accumulated total nitrogen and total phosphorus, decreased ph, and increased microorganism over the course of the 56-day experimental test period (tacon et al., 2002), we would like to focus more on ammonia and nitrite accumulation. on the one hand, ammonia and nitrate are the main toxic product of protein catabolism in the aquatic system, which are generated mainly in the mineralization process of organic wastes such as unconsumed feed and feces (chen et al., 1989; cheng et al., 2013; ren et al., 2015); on the one hand, ammonia and nitrate can increase the concentration of hydrogen ions in aquatic ecosystem, resulting in acidification (camargo and alonso, 2006), and are essential, life-sustaining, nitrogen-containing compounds used by many aquatic microorganisms (pinto et al., 2016). thus, ammonia and nitrite accumulation might be the primary pollution in intensive aquaculture system. previous studies on the chronic physiological effects of isolated and combined ammonia and nitrite on crustaceans were limited (miranda-filho et al., 2009); it was shown that survival of juvenile pink-shrimp farfantepenaeus paulensis was ≥ 90 % under ammonia concentrations (0.016 0.287 mg/l) after 75 days, whereas growth (carapace length and wet body mass) was reduced at ammonia concentration as low as 0.033 mg/l. by contrast, the present study indicated that ammonia and nitrite accumulation had significantly higher lethality but similar inhibitory effects on physiological processes involved in body mass increase in treatment shrimp; this could be because exposure to high levels of ambient ammonia induces increased energy expenditure that regulates osmotic and ionic stress (young-lai et al., 1991; spaargaren 1982; chen and cheng 1993b, c), resulting in reduced growth; meanwhile, there might be a resulting synergistic effect of ammonia and nitrite accumulation in that energy expenditure for osmoregulation in treatment shrimp according to previous report (cheng et al., 2013), which led to higher lethality in the present study. the activities of digestive enzymes in crustacea have a central role in their nutritional physiology, which can supply nutrition requirements and metabolic energy for body, and directly or indirectly regulate growth (van wormhoudt 1973; lee et al., 1984; lovett and felder 1990). as a cheaper, primary, and immediate source of energy for organisms under stress conditions (tseng and hwang 2008; wang et al., 2012), carbohydrate nutrition might have an essential role in the osmoregulation of shrimp because carbohydrates are often included in their diet (cruz-suarez et al., 1994; cuzon et al., 2004). however, the results of the present study showed that ammonia and nitrite accumulation had no effects on amylase and cellulose activity in the hepatopancreas of treatment shrimp, which was similar to previous findings for the activity of digestive carbohydrate enzymes under salinity stress (li et al., 2008; wang et al., 2014). thus, we speculate that digestive carbohydrate enzymes were already saturated with ammonia and nitrite accumulation, and that more dietary carbohydrate content could contribute to providing 227 energy for increased osmoregulation. the observed changes in the present study were characterized by a marked reduction in protease activity paralleled by a significant increase in lipase activity in the hepatopancreas of treatment shrimp, which might suggest the increased use of lipid as an energy source to respond to the increased osmotic and ionic stress, but the reduced use of protein to limit the internal accumulation of nitrogenous waste products induced by ammonia and nitrite accumulation (miranda-filho et al., 2009; pinto et al., 2016). meanwhile, the fact that the change of protease activity in the hepatopancreas was consistent with weight gain in shrimp supported previous review that lower protease activity contributes to reduced growth (xu et al., 1987, 1995). therefore, ammonia and nitrite accumulation might disrupt the synthesis and excretion of digestive enzymes in the hepatopancreas, resulting in reduced metabolic energy for survival and growth performance. in terms of the antioxidant status, assaying antioxidant enzymes can indicate the antioxidant status of organisms and serve as a biomarker of oxidative stress (kohen and nyska, 2002). sod and cat provide a first line of defense against reactive oxygen species (ros) because sod catalyzes the conversion of o2 • − to h2o and h2o2, and the latter is further degraded into h2o and o2 by cat, gpx, gsh, gr, gst, and multiple different peroxidases (dandapat et al., 2003; cheng et al., 2006; yeh et al., 2009; sinha et al., 2015). generally, higher sod and cat activities indicate that there are more ros present that need to be metabolized (chien et al., 2003; ross et al., 2001). therefore, significantly higher sod and cat activities in the hepatopancreas of treatment shrimp might indicate that ammonia and nitrite accumulation resulted in the increased accumulation of ros. our results also found that the lower gpx activity was in contrast to the increase in cat activity in the hepatopancreas of treatment shrimp, which was consistent with previous reports, given that gpx and cat function differently under stress conditions (dandapat et al., 2003; zhang et al., 2015). gpx catalyzes the reduction of h2o2 and a variety of lipid peroxides by using gsh, which is further oxidized to gssh. gssh is re-reduced to gsh via gr in a reduced form of nicotinamide-adenine dinucleotide phosphate-dependent reaction. the parallel augmentation of gr with gpx activity in liver tissue requires the proficient renewal of gsh (sinha et al., 2015). therefore, significantly lower gr and gpx activities and gsh concentrations in the hepatopancreas of treatment shrimp might indicate that the hepatopancreas had lost its function to renew its gsh capacity as a result of toxicity effect of ammonia and nitrite accumulation. in addition, mda is the final product of lipid peroxidation, providing direct evidence of the toxic processes resulting from ros metabolism (doyotte et al., 1997; lushchak, 2011). thus, the significantly higher mda concentration in the hepatopancreas of treatment shrimp might further reveal that accumulated ros might be the substance, which caused abnormal hepatopancreas function. gst activity remained unaltered in the hepatopancreas of treatment shrimp, suggesting its limited role in ammonia and nitrite fig. 6 relative expressions of p70s6k, 4ebp1, got, gpt, and gst mrna in the hepatopancreas of shrimp after the 33-d experiment. * indicates a significant difference between the treatment (blue circles) and control (black circles) groups. relative expressions of p70s6k, 4ebp1, got and gpt mrna increased significantly in the treatment group versus control group. detoxification. therefore, higher sod and cat activities were not sufficient to scavenge the accumulated ros resulted from ammonia and nitrite accumulation. in contrast, previous studies reported that sod and cat activities significantly decreased in response to either ammonia or nitrite, although the mda concentration significantly increased (liu et al., 2004; wang et al., 2004; liang et al., 2016). we speculate the main factor that might have caused this discrepancy in results might be the exposure to incessant ammonia and nitrite accumulation process rather than steady-state concentrations of ammonia and nitrite immediately in the present study. got catalyzes an important reaction in the molecular rearrangement involving amino acids linked to the citric acid cycle at two points (oxaloacetic and ketoglutaric acids); this is the most important mechanism for introducing reduction equivalents into mitochondria (urich, 1994). gpt predominates in organs where intensive glycogenesis occurs, such as the liver (urich, 1994; de la torre et al., 2000). high levels of got and gpt in the liver can result in excess liberation of these enzymes into the blood, which suggests that liver cells are damaged (vaglio and landriscina, 1999; kim and kang, 2004; wu et al., 2008). the significantly higher relative expression of got and gpt mrna in the hepatopancreas of treatment shrimp was similar to results reported in a previous study (jiang et al., 2014). therefore, this result might indicate that the hepatopancreas in treatment shrimp was damaged, which might be caused by accumulated ros, resulting in abnormal hepatopancreas function. translation initiation is a limiting step in protein synthesis (holz et al., 2005), and major effector of cell growth and proliferation (hay and sonenberg, 2004), key steps in processes involved in the growth response (anthony et al., 2001). javascript:void(0); javascript:void(0); 228 fig. 7 photomicrographs of the hepatopancreas of shrimp from control group (a) and treatment group (b) after the 33-d experiment. epithelial cell: e cell; r cell; f cell; b cell. the pathologies observation: injured connective tissue between liver tubule (ict); infiltrated hemocytes (ih); narrowed lumen(nl); vacuolization(v) . h&e stain (×400), scale bar =100 μm. histology structure of hepatopancreas in treatment shrimp showed remarkable change versus control group. target of rapamycin (tor) promotes cap dependent translation initiation through inactivation of its downstream effector 4ebp1 or phosphorylation of p70s6k (wullschleger et al., 2006). thus, significantly higher relative expression of 4ebp1 mrna in the hepatopancreas of treatment shrimp induced by abnormal hepatopancreas function would inhibit translation initiation, which might result in reduced growth performance. though significantly higher relative expression of p70s6k mrna appeared in the hepatopancreas of treatment shrimp, p70s6k might not be phosphorylated correspondingly due to abnormal hepatopancreas function, and further investigation is needed in this area. evidence suggests that the expression level of gst mrna is a crucial factor in determining the sensitivity of cells to a broad spectrum of toxic chemicals (mearns et al., 2006; zhou 2009); however, the relative expression of gst mrna remained unaltered in the hepatopancreas of treatment shrimp, which is consistent with the above test result that gst activity was unaltered; thus, this is not a useful indicator of the response of shrimp to ammonia and nitrite accumulation. all the remarkable change in the hepatopancreas of treatment shrimp corroborated that this organ was damaged by accumulated ros, which would lead to detrimental effects on any related physiological functions. b and r cells are the main sites for the synthesis of digestive enzymes and nutrient reserves, respectively (al-mohanna and nott 1986, 1987, 1989; caceci et al., 1988). e cells, as undifferentiated embryogenic cell, can supplement other types of cell by dividing and differentiating into other liver cells (hong et al., 2007). therefore, reduced or disappeared b, r and e cells might be the essential reason, which disrupted the synthesis and excretion of digestive enzymes in the hepatopancreas of treatment shrimp, resulting in reduced metabolic energy for survival and growth performance. in conclusion, ammonia and nitrite accumulation caused by accumulated waste in aquaculture tank could damage hepatopancreas in the l. vannamei, 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of mohaghegh ardabili, ardabil, iran accepted june 28, 2016 abstract nutritional indices of three geographic populations (miandoab, moghan and kalposh) of the fifth instar of spodoptera exigua (lepidoptera: noctuidae) on four sugar beet cultivars (dorothea, rozier, persia and perimer) were evaluated at 25 ± 1 °c, 65 ± 5 % rh and a 16:8 l:d photoperiod. the estimates of all the nutritional indices were affected by the interaction effect of the geographic population and the sugar beet cultivars. miandoab population reared on dorothea, and moghan and kalposh populations reared on perimer had the highest approximate digestibility (ad). miandoab and kalposh populations reared on rozier showed the highest efficiency of conversion of ingested food (eci) and efficiency of conversion of digested food (ecd) values. moghan population reared on persia showed the lowest ad values and the highest eci and ecd values. the present study showed that the nutritional indices of three populations, in most cases, were significantly different from with other, due to different environmental conditions that the pest collected from there. it is concluded that both geographic origins of the pest and host cultivar affected the nutritional indices of s. exigua larvae. key words: beet armyworm; feeding performance; geographic population; host cultivar   introduction sugar beet (beta vulgaris) is a root crop commonly used for sugar production and is an important food source (biancardi et al., 2012). iran is one of 50 countries in the world that produces sugar beet in large scales (yazdani and rahimi, 2012). the beet armyworm, spodoptera exigua (lepidoptera: noctuidae), is a polyphagous species, which feeds on more than 90 plant species of over 18 plant families (mehrkhou et al., 2012). it is a migratory species, originating from south asia, and occurs at latitudes between 35° to 40°s and 40° to 57°n in tropical, subtropical and temperate regions (zhang and zhao, 1996). this pest causes economic damages to sugar beet, corn and alfalfa in iran (farahani et al., 2011; mehrkhou, 2013; goodarzi et al., 2015). high migratory capacity of this insect facilitates the geographic dispersal of populations beyond its normal distribution range, and causes outbreak of its populations (kimura 1991; burris et al., 1994; adamczyk et al., 2003). management of s. exigua is ___________________________________________________________________________ corresponding author:  bahram naseri  department of plant protection faculty of agriculture and natural resources university of mohaghegh ardabili, ardabil, iran e-mail: bnaseri@uma.ac.ir difficult due to high migratory dispersal and rapid resistance development to pesticides (adamczyk et al., 2003; shimada et al., 2005). host plant quality could affect population dynamics of herbivorous insects by influencing their spatial distribution and the migration rates (howard and dixon, 1992). there are evidences documented that plants nutritional status can influence their susceptibility to several herbivores (mattson, 1980). minerals are one of the main nutritional requirements of insects among amino acids, vitamins, carbohydrates, lipids and sterols (dadd, 1985). insects require elements such as potassium, phosphorus and magnesium considerably higher than calcium, sodium (na) and chlorides during their development (dale, 1988). effect of mineral concentration of host plants on herbivorous insects that feed on them could be positive, neutral or negative (dale, 1988). nitrogen (n) affects life history of insects such as growth (kainulainen et al., 1996), survival (ayres et al., 2000) and reproduction (bentz and townsend, 2001), because it plays an important role in the production of amino acid and synthesis of protein (sterner and elser, 2002). generally, concentration of nitrogen in plant leaf is an indicator of food quality affecting host plant selection by polyphagous insects. phosphorus (p) is another nutritional element in plant tissues that could affect 221   survivorship (ayres et al., 2000), fecundity (popp et al., 1989), body size (busch and phelan, 1999), oviposition (skinner and cohen, 1994), growth rate (perkins et al., 2004) and population density of insects (schade et al., 2003). the enzyme cofactor potassium (k) (mengel et al., 2001) is an important element in host plant quality (leigh and wyn jones, 1984) and in the process of amino acids conversion into proteins. there are several studies regarding the biological parameters of s. exigua on different host plants such as cotton, pepper, pigweed and sunflower (greenberg et al., 2001), shallot, long bean, lady’s finger and chilli (azidah and sofianazirun, 2006), wheat, cabbage and pea (saeed et al., 2010), corn (mardani-talaei et al., 2012), soybean varieties (farahani et al., 2011; mehrkhou et al., 2012; mehrkhou, 2013) and beet varieties (karimi-malati et al., 2014). however, despite the importance of sugar beet as a rotational crop, and high economic damages caused by different populations of s. exigua to this crop (saghfi and valizadegan, 2014), no published papers are available on the nutritional indices of different geographic populations of s. exigua on sugar beet cultivars. therefore, the aim of this research was to study the effect of host plant cultivar and geographic origin of the pest on the nutritional indices of s. exigua. to understanding this, four commercial sugar beet cultivars and three geographic populations of s. exigua were used. despite using the same laboratory conditions for rearing of all the populations, observation of variations in nutritional responses of s. exigua that were collected from different geographic regions was expected. moreover, the concentration of some important minerals (n, p, k and na) in the leaves of the four tested sugar beet cultivars was measured and their relationship with food consumption and utilization by s. exigua was studied. materials and methods plant sources seeds of four different sugar beet (b. vulgaris) cultivars, including dorothea, rozier, persia and perimer were acquired from the plant and seed modification research institute of sugar beet (ardabil, iran). they were grown in the research field of the university of mohaghegh ardabili (ardabil, iran) in may 2014. selection of these cultivars was based on their importance as the most cultivated sugar beets in different regions of iran. for this study, the young leaves (with equal size) of various sugar beet cultivars at four leaf stage were transferred to a growth chamber at 25 ± 1 °c, 65 ± 5 % rh, with a 16:8 l:d photoperiod and used for the experiment. insect rearing larval colonies (fifth instar) of s. exigua were collected from three regions of iran that have the highest production of sugar beet. these regions included semnan (kalposh), western azarbayjan (miandoab) and ardabil (moghan). the main sugar beet cultivars planted in miandoab, moghan and kalposh regions were dorothea, rozier and perimer, respectively. kalposh population was collected from cultivar perimer, and miandoab and moghan populations were collected from cultivars dorothea and rozier, respectively. to eliminate the effect of prior feeding experience on local host plant and provide the same cultural conditions for three populations, larval population from each region was reared separately, for two generations, on another sugar beet cultivar (torbat). nutritional indices experiments nutritional indices (including weight gain, food consumption and feces produced by the larvae) were calculated gravimetrically (waldbauer, 1968). in this study, nutritional indices of fifth instar larvae were calculated because they were more measurable than the younger instars. for this purpose, 30 fifth instar larvae of s. exigua from each population were individually transferred into plastic containers (diameter 16.5 cm, depth 7.5 cm) with outlets covered by a mesh net for larval aeration. the fresh leaves of the tested sugar beet cultivars for each population were placed in each container. the petioles of the detached leaves were inserted in a water-soaked cotton to keep freshness. for pupal stage, pre-pupae were transferred into small plastic tubes (diameter 2 cm, depth 5 cm). the weights of the larvae were recorded daily before and after feeding until they finished feeding and reached prepupal stage. the provided fresh leaves and the remaining leaves and feces at the end of each experiment were weighed daily. the amount of leaf consumption per larva was calculated by subtracting the weight of remaining leaf from the weight of fresh leaf supplied at the end of each experiment. all the above-mentioned weights were recorded as percentage of dry weights. to calculate the dry weight of leaf, larvae and feces, 20 specimens of newly molted larvae and 20 specimens of the remaining leaves and feces produced were collected, weighed and dried at 60 °c for 48 h, then reweighed. nutritional indices were calculated via formulae described by waldbauer (1968): approximate digestibility (ad) = e-f/e; efficiency of conversion of ingested food (eci) = p/e; efficiency of conversion of digested food (ecd) = p/e-f; relative consumption rate (rcr) = e/a*t; and relative growth rate (rgr) = p/a*t. where, a = mean dry weight of insect over unit time, e = dry weight of food consumed, f = dry weight of feces produced, p = dry weight gain of insect, and t = duration of feeding period (days). determination of minerals in sugar beet cultivars leaf samples of four tested sugar beet cultivars were oven dried (at 60 °c) for 48 h, and then ground with electric mills. for determination of phosphorus, potassium and sodium, approximately 0.2 g of dried powdered leaves were digested by sulfuric acid, salicylic acid and h2o2. after digestion, phosphorus concentration was measured spectrophotometrically and absorbance was read at 470 nm using colorimetry method (waling et al., 1989). sodium and potassium concentrations were estimated flame photometerically (waling et al., 1989). nitrogen concentration was determined by kjeltec auto 1030 analyzer (emami, 1996). 222   data analysis (f = 33.60; df = 2.87; p < 0.01), persia (f = 15.34; df = 2.85; p < 0.01) and perimer (f = 36.63; df = 2.80; p < 0.01) showed the highest ad values as compared to the miandoab and moghan populations. among different sugar beet cultivars, within populations, the highest ad value was observed in cultivar perimer in moghan (f = 27.69; df = 3.114; p < 0.01) and kalposh (f = 36.97; df = 3.111; p < 0.01) populations, and cultivar dorothea in miandoab (f = 16.39; df = 3.107; p < 0.01) population. the lowest ad value was observed in cultivar persia in kalposh and moghan populations and cultivar rozier in miandoab population. nutritional indices of s. exigua were conducted using factorial design with two main factors (population in three levels and cultivar in four levels). the data were analyzed with two-way anova using sas program (sas institue, 1989) followed by comparison of the means with lsd test at α = 0.05. data for mineral concentrations were analyzed with one-way anova using sas program (sas institue, 1989) and mean comparison was done with lsd test at α = 0.05. before analysis, all the data were tested for normality by kolmogorov-smirnov method which were normally distributed. effect of population and sugar beet cultivar on eci value of s. exigua results the effect of population and sugar beet cultivar on eci value of the fifth instar of s. exigua is shown in table 2. among the three populations reared on the four sugar beet cultivars, miandoab population reared on cultivars dorothea (f = 5.61; df = 2.85; p < 0.01) and rozier (f = 34.32; df = 2.92; p < 0.01), and kalposh population reared on cultivar perimer (f = 31.16; df = 2.78; p < 0.01) showed higher eci values. miandoab and moghan populations reared on cultivar persia (f = 4.20; df = 2.89; p < 0.01) showed higher eci values than kalposh population. within populations reared on different sugar beet cultivars, miandoab (f = 80.36; df = 3.113; p < 0.01) and kalposh (f = 28.18; df = 3.111, p < 0.01) populations reared on cultivar rozier and moghan population (f = 40.14; df = 3.120; p < 0.01) fed on persia showed the highest eci values compared to the other cultivars. nutritional indices of s. exigua were studied in three geographic populations of this pest in response to feeding on four sugar beet cultivars. in this study, by using factorial design, both effects of population, sugar beet cultivar and their interaction on nutritional indices of s. exigua (table 1) were studied. the statistics in table 1 show that the effect of population, cultivar and population×cultivar for all the nutritional indices of s. exigua had significant difference at α = 0.01. effect of population and sugar beet cultivar on ad value of s. exigua the effect of population and sugar beet cultivar on ad value of the fifth instar of s. exigua is given in table 2. kalposh population reared on cultivars dorothea (f = 5.22; df = 2.80; p = < 0.01), rozier table 1 summary results of anova for the effect of population, sugar beet cultivar and their interaction on nutritional indices of fifth instar of spodoptera exigua ad = approximate digestibility, eci = efficiency of conversion of ingested food, ecd = efficiency of conversion of digested food, rcr = relative consumption rate, rgr = relative growth rate. (anova, p < 0.01) f df source of variation index 37.92 2 population ad 45.97 3 cultivar (%) 10.08 6 population×cultivar 336 error 26.31 2 population eci 128.00 3 cultivar (%) 17.40 6 population×cultivar 336 error 22.26 2 population ecd 84.60 3 cultivar (%) 13.22 6 population×cultivar 336 error 14.46 2 population rcr 198.47 3 cultivar (mg/mg/day) 53.60 6 population×cultivar 336 error 14.10 2 population rgr 6.10 3 cultivar (mg/mg/day) 10.32 6 population×cultivar 336 error 223   table 2 effect of geographic origin of spodoptera exigua and sugar beet cultivar on nutritional indices (mean±se) of fifth instar of this pest the means followed by different small letters in the same column for each population and different capital letters in the same row for each cultivar are significantly different (lsd, p < 0.01) effect of population and sugar beet cultivar on ecd value of s. exigua table 2 shows the effect of population and sugar beet cultivar on ecd value of the fifth instar of s. exigua. among the three populations reared on the tested cultivars, the ecd value of larvae collected from kalposh on cultivar perimer (f = 26.30; df = 2.78; p < 0.01) was higher than that of the other populations. however, miandoab population reared on cultivars dorothea (f = 5.56; df = 2.85; p < 0.01), rozier (f = 29.14; df = 2.88; p < 0.01) and moghan population reared on cultivar persia (f = 7.99; df = 2.89; p < 0.01) showed the highest ecd value when compared with any other population. among the different sugar beet cultivars, the highest ecd values on miandoab (f = 54.93; df = 3.122; p < 0.01) and kalposh (f = 19.54; df = 3. 105; p < 0.01) populations were observed on cultivar rozier and the lowest values were seen in cultivar dorothea. moghan (f = 29.08; df = 3.113; p < 0.01) population reared on cultivars persia and perimer showed the highest and lowest ecd values, respectively for larval population collected from this region. effect of population and sugar beet cultivar on rcr value of s. exigua the effect of population and sugar beet cultivar on rcr of fifth instar of s. exigua is shown in table 2. among the three populations reared on cultivars kalposh population moghan population    miandoab population   ad (%)   cultivar 88.95±0.95 b, a 84.52±0.99 a, b 85.41±1.27 a, b dorothea  93.77±0.50 a, a 81.81±2.34 a, b 70.56±2.51 b, c rozier  81.51±1.28 c, a 56.46±4.91 b, c 71.47±2.32 b, b persia  93.83±0.92 a, a 89.63±0.60 a, b 83.79±0.92 a, c perimer   eci (%)    4.00±0.30 c, ab 3.29±0.22 b, b 4.56±0.25 b, a dorothea  12.58±0.79 a, b 10.14±0.80 a, b 21.70±1.39 a, a rozier  8.72±0.83 b, b 11.97±1.13 a, a 12.61±1.01 a, a persia  6.79±0.66 b, a 2.48±0.20 b, c 4.95±0.21 b, b perimer   ecd (%)    4.55±0.36 c, b 3.93±0.29 c, b 5.53±0.35 c, a dorothea  16.65±1.93 a, b 12.61±1.11 b, b 30.88±2.28 a, a rozier  10.51±1.16 b, b 22.95±3.47 a, a 19.90±1.91 b, a persia  7.35±0.78f bc, a 2.79±0.23 c, c 5.93±0.26 c, b perimer   rcr (mg/mg/day)    6.32±0.31 b, b 10.29±0.49 a, a 5.58±0.32 a, b dorothea  2.47±0.15 c, a 2.03±0.18f b, ab 1.63±0.15 c, b rozier  2.07±0.14 c, b 2.03±0.18f b, b 2.91±0.23 b, a persia  9.26±0.48 a, a 2.56±0.24 b, c 5.46±0.21 a, b perimer   rgr (mg/mg/day)    0.26±0.01 a, b 0.32±0.01 a, a 0.24±0.01 c, b dorothea  0.29±0.01 a, a 0.20±0.01 bc, b 0.29±0.02 b, a rozier  0.18±0.01 b, c 0.23±0.02 b, b 0.37±0.01 a, a persia  0.21±0.01 b, b 0.16±0.01 c, c 0.27±0.01 bc, a perimer  224   table 3 the mean (±se) nitrogen, phosphorus, potassium and sodium contents in the leaves of four tested sugar beet cultivars used for feeding of spodoptera exigua larvae the means followed by different letters in the same column are significantly different (lsd, p < 0.01, *p < 0.05) rozier (f = 6.47; df = 2.86; p < 0.01) and perimer (f = 6.70; df = 2.81; p < 0.01), kalposh showed higher rcr values as compared to the other populations, whereas, moghan population reared on cultivar dorothea (f = 42.62; df = 2.84; p < 0.01), and miandoab population reared on cultivar persia (f = 96.39; df = 2.76; p < 0.01), showed higher rcr value than the others. within populations, cultivar dorothea showed the highest rcr value and cultivar rozier showed the lowest rcr value for miandoab (f = 65.03; df = 3.111; p < 0.01) population. cultivar dorothea in moghan (f = 173.89; df = 3.108; p < 0.01) population showed higher rcr value than other cultivars. kalposh (f = 125.80; df = 3.108; p < 0.01) population reared on cultivar perimer showed the highest rcr value and cultivar persia showed the lowest rcr value. effect of population and sugar beet cultivar on rgr value of s. exigua the effect of population and sugar beet cultivar on rgr of fifth instar of s. exigua is shown in table 2. moghan population reared on cultivar dorothea (f = 6.42; df = 2.76; p < 0.01), miandoab and kalposh populations reared and cultivar rozier (f = 9.73; df = 2.89; p < 0.01) and miandoab population reared on cultivars persia (f = 29.65; df = 2.86; p < 0.01) and perimer (f = 13.16; df = 2.78; p < 0.01) showed the highest rgr values. within populations, mindoab (f = 10.71; df = 3.114; p < 0.01) reared on cultivar persia, kalposh (f = 12.31; df = 3,108; p < 0.01) reared on cultivar rozier and moghan (f=11.74; df = 3.107; p < 0.01) population reared on cultivar dorothea showed the highest rgr values. cultivar dorothea in miandoab population, cultivar perimer in moghan population and cultivar persia in kalposh population showed the lowest rgr values. determination of minerals in the different sugar beet cultivars table 3 shows the minerals content of the leaf of the four tested sugar beet cultivars. no significant difference was observed for nitrogen content (f = 1.25; df = 3.8; p = 0.356) in the different sugar beet cultivars. however, the highest and lowest phosphorus contents (f = 8.72; df = 3.8; p < 0.01) were observed in cultivars dorothea and persia, respectively. cultivar perimer showed the highest potassium content (f = 22.40; df = 3.8; p < 0.01). the highest and lowest sodium content (f = 7.68; df = 3.8; p < 0.05) was observed in cultivars dorothea and persia, respectively. discussion in this research, nutritional indices in three geographic populations of s. exigua were measured in response to feeding on four tested sugar beet cultivars. significant differences were found for the nutritional indices of the pest among the different populations and cultivars. approximate digestibility (ad) is one of the main factors among nutritional indices showing nutritional value of food for insect and ability for food uptake through the stomach wall. variations in ad values show the differences in factors such as poor nutrient value, lack of balance, higher ratio of crude fiber and lower ratio of water (chapman, 1998). efficiency of conversion of ingested (eci) and digested (ecd) food are the other important nutritional indices showing ability of insects to incorporate ingested and digested food into growth and body matter (nathan et al., 2005). variations in ecd value indicate the overall changes of metabolized nutrient for energy demand (koul et al., 2004). the highest ad values within each population (table 2) on cultivars dorothea and perimer, might be due to lower nutritional value or higher crude fiber of these cultivars for feeding of s. exigua. in addition, cultivars dorothea and perimer showed the highest concentration of potassium. digestibility of food for lepidopteran insects is influenced by imbalanced diet or high content of crude fiber in the food (waldbauer, 1968). perhaps, one of the possible reasons for this unsuitability might be the role of potassium in host plant resistance against herbivore insects. potassium can affect the attractiveness of the plant to insects by profound effect on the shape and dissemination of primary metabolites in plant tissues (amtmann et al., 2008). cultivar nitrogen (%) phosphorus (%) potassium (%) sodium (%) dorothea 3.665±0.083 a 0.538±0.020 a 5.145±0.188 a 6.190±0.352 a* rozier 3.426±0.067 a 0.377±0.018 bc 3.590±0.159 b 4.783±0.162 b persia 3.463±0.114 a 0.292±0.004 c 4.090±0.221 b 3.763±0.324 b perimer 3.523±0.143 a 0.412±0.063 b 5.310±0.115 a 4.293±0.557 b 225   negative effects of high levels of potassium on insect populations could be due to reduced carbohydrate accumulation and amino acids elimination (baskaran et al., 1985), higher silica content and increase in the sclerenchymous layer (dale, 1988). within each population (table 2), higher eci and ecd values on cultivars rozier and persia indicated that larvae reared on these cultivars had higher capacity in converting ingested and digested food to body matter. moreover, cultivars rozier and persia showed lower concentration of potassium, phosphorus and sodium than other cultivars. insects need minerals such as iron, copper, calcium, sodium, potassium ions, phosphorus and sulphur in very small amounts (nation, 2001). although, in this study, no significant difference was observed for concentration of nitrogen in the leaves of the four sugar beet cultivars; however, some researchers (mattson 1980; scriber and slansky, 1981), reported that the amount of ingested food by the insects is directly related to the concentration of nitrogen in the host plants. nitrogen is a fundamental component of proteins and, consequently, for larval growth in initial instar (scriber, 1983). these results suggested that nutritional status of the host plant can influence the amount of ingested and digested food by the insects. it is well known that chemical and nutritional quality of the ingested food, development stage, energy spent by the insect, and rate of intake into body biomass influenced the amount of food digestion by the insects (david and gardiner, 1962; schoonhoven and meerman, 1978; kaplan et al., 2014). higher rcr value within each population (table 2) on cultivars dorothea and perimer that had higher leaf minerals indicated that the concentration of minerals, especially phosphorus and potassium influenced food consumption by s. exigua. variation in host plant phosphorus and continuous changes in insect body phosphorus affected growth, survival, and development time of the insect (perkins et al., 2004; huberty and denno, 2006). the highest rgr value (table 2) of miandoab population on cultivar persia, kalposh population on cultivar rozier and moghan population on cultivar dorothea suggested that these larval populations reared on these cultivars had the highest weight gain and shortest larval period. however, lower rgr value on cultivar perimer in these populations might be due to lower nutritional value of this cultivar. as reported by hwang et al. (2008), lepidopteran larvae showed higher growth rate and faster development time when they were reared on nutrient-rich food as compared to nutrient-poor food. moreover, despite the importance of host plant quality on nutritional requirement of the insects, some characters of the insects such as age, sex, physiological stress, reproduction, diapauses or migration (chapman, 1998; nation, 2008) and body mass (phillipson, 1981; schroeder, 1981), could be affected by their optimal nutritional requirement. moreover, other reason for rgr reduction on cultivar perimer, perhaps is the highest concentration of potassium in this cultivar. the results of this study indicated that different populations reared on local host plant had higher nutritional performances. higher ad and rcr values in miandoab population on cultivar dorothea (as local host plant) and in kalposh population on cultivar perimer (as local host plant) might be due to prior feeding experience of these populations on their local host plant. conclusions according to the results of this study, nutritional indices of s. exigua do not only differ among cultivars, but also, they vary among geographic populations of s. exigua. although, host plant quality affects nutritional indices of s. exigua, when compared with geographic origins of different populations, it is not solely responsible for this variation. on the other hand, it is better to consider suitability or unsuitability of host plants for each population separately. these results indicate that there is a variation among populations in terms of nutritional indices and this might be due to the differences in environmental conditions that the pest was collected from there. this study is the first evidence supporting the effect of geographic origin and host plant cultivar on the nutritional responses of herbivorous insects. in future works, genetic basis of such differences should be studied. the information derived from this study is useful in developing a 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systematic zoology and ecology, eötvös loránd university, pázmány péter u. 1. 1/c, h-1117 budapest, hungary *recent address: 3dhistech ltd. konkoly-thege u. 29-33. h-1121 budapest, hungary accepted may 20, 2016 abstract the presence of γ-aminobutyric acid (gaba) immunoreactive elements was observed in the sensory system of eisenia fetida (annelida, clitellata). among the primary sensory cells a high number of labelled cells was found in the epithelium. using whole-mount preparations and multispectral recording, the number and the distribution pattern of the immunopositive cells were determined in different body segments. our morphological analysis revealed four typical types of stained primary sensory cells, which could be responsible for the different role of the gaba mediated sensory functions. the peripheral processes of the primary sensory cells ramifying at the border of epithelium and muscular layer produced a basiepidermal network where gaba-positive fibres and their connections were observed. the central processes of the labelled cells projected directly to the ventral nerve cord (vnc) through the segmental nerves to form the ventrolateral and ventromedial sensory longitudinal axon bundles (slabs). inhibitory gaba sensory inputs could influence indirectly the activity of the giant motoneurons through the ventrolateral giant axons, and thus the contraction of the body organizing withdrawal and escape reflexes. applying ultrastructural investigations the synaptic connections of gaba-immunoreactive structures were identified both in the basiepidermal plexus, in the segmental nerves, and in the slabs of vnc suggesting multistep regulatory effect of gaba in sensory processing of earthworms. key words: primary sensory cells; sensilla; basiepidermal plexus; sensory longitudinal axon bundles; multispectral imaging   introduction primary sensory cells are distributed in the body wall of the clitellata between the supporting and glandular cells (coonfield, 1932; mill, 1978). these cells send sensory information to the vnc and influence the dorsal giant fibres and motoneurons, controlling rapid reflexes like withdrawal and escape (drewes, 1984). their anatomical features were described by langdon (1895) in lumbricus agricola (annelida, clitellata), who noted that most of the sensory cells are grouped (forming a sensillum), solitary cells being rare. the location of the sensilla follows a well ___________________________________________________________________________ corresponding author: lászló molnár department of comparative anatomy and developmental biology university of pécs ifjúság u. 6, h-7624 pécs, hungary e-mail: molnar@gamma.ttk.pte.hu identifiable pattern. most of the sensilla are found in a middle line of segments so named chaetae-row; however, there is a seemingly random pattern on both the anterior and posterior parts of segments. langdon counted 1,900 sensilla on the first segment and prostomium, 1,200 on the 10th segment and 700 on the 56th segment. knapp and mill (1971) estimated that the larger sensilla contain 35 45 sensory cells. these data were confirmed by many later workers and they described that distribution pattern and the numbers of the sensilla are similar in the sister species (myhrberg, 1979; aros et al., 1978; moment and johnson, 1979; jamieson, 1981; spörhase-eichmann, 1998; molnár et al., 2006; kiszler et al., 2012). based on their morphological characteristics the primary sensory cells were classified (retzius, 1892). five distinct types were detected by ultrastructural investigations (for review see mill, 1978, 1982; jamieson, 1981): penetrative uniciliate 172 and penetrative multiciliate sensory cells (aros et al., 1971b; knapp and mill, 1971) nonpenetrative multiciliate cells (aros et al., 1971a), phaosomal photoreceptors (hess, 1925a; röhlich et al., 1970) and basal ciliated sensory cells (myhrberg, 1979). the functions of the different types are partly known. results of morphological and electrophysiological investigations suggest that nonpenetrative multiciliare cells are mechanoreceptors, penetrative uniand multiciliar cells are chemoreceptors (mill, 1978). electrophysiological investigations showed that the sensitivity of sensilla is different in each part of the body, although the sensory cells have similar morphology (laverack, 1963; mill, 1978, 1982). this suggests that the morphology of the sensory cells does not determine precisely their function; the location is at least as important. the synchronization of sensory inputs and motor outputs of the nervous system is mediated by transmitter-specific neural structures (dorsett, 1978; mill, 1982). distinct groups of the primary sensory cells were determined by their neurochemical features (schpörhase-eichmann, 1997). transmitter-specific sensory cells have been described by many workers in several species, thus peptidergic (gesser and larsson, 1986; renda et al., 1987; curry et al., 1989; reglődi et al., 1999, 2000), aminergic (myhrberg, 1967, 1971; ehinger et al., 1971; spörhase-eichmann, 1998; csoknya et al., 2005) and gabaergic (telkes et al., 1996; spörhase-eichmann et al., 1997; koza and csoknya, 2004; molnár et al., 2006) cells however, the possible functions of the transmitter-specific cells remained unknown. electrophysiological observations were used to find connections between the different types of sensory cells and their function. the receptive fields of the segmental nerves were determined by knapp and mill (1967), who described that these are not concentrated in one segment but overlap to the adjacent segments. the neural processes of the sensory cells form a neural plexus at the border of the epidermis and the muscle layer called basiepidermal plexus (rude, 1966; myhrberg, 1967). the plexus includes both efferent fibres and sensory axons (langdon, 1895; hess, 1925; ogawa, 1939; mill, 1978), and the most probable role of this network is controlling the contraction of the longitudinal muscle and acting as a reinforcing agent in the peristaltic waves (miller and ting, 1949). however, there are conflicting opinions that this system is segmental or continuous in its organization (retzius, 1892; langdon, 1895; smallwood, 1923, 1926; hess, 1925; coonfield, 1932; ogawa, 1939; rude, 1966; myhrberg, 1967; mill, 1978). the central processes of the sensory cells, without branching enter the vnc via three segmental nerves. after entering they divide in a tshaped manner and form five longitudinal sensory axon bundles. these are the intermediolateral, intermediomedian, ventrolateral, ventromedian and the dorsal bundle (günther, 1971; dorsett, 1978). from calculations of knapp and mill (1971) it was concluded that the relationship between the sensory cells and number of sensory axons in the segmental nerves are 1:1 (mill, 1975, 1978, 1982). also, their investigations stated that chemoreceptive and tactile inputs enter the central nervous system (cns) primarily via the first and the third segmental nerves and from proprioceptors via the second nerve (knapp and mill, 1967). because of the close locations of the second and the third segmental nerves, later studies suggested that all three kinds of sensory input enter the cns via all three segmental nerves (mill, 1982). based on the results of previous studies we can conclude that the identification of transmitter specific sensory structures and their exact anatomical characterization -localization, distribution density are necessary to reveal the possible mechanism of the sensory system. therefore, the aim of present work has been to identify and map gaba immunoreactive (gaba-ir) sensory elements in peripheral (pns) and cns of the earthworm eisenia fetida, including detailed morphological description of the labelled structures. materials and methods animals experiments were carried out on 15 sexually matured eisenia fetida (annelida, clitellata) specimens. the animals were kept in plastic boxes containing soil under standard laboratory conditions (20 22 ˚c, 50 60 % humidity). specimens were anaesthetized in carbonated water and cut into pieces of several body segments and their guts were removed. after the preparation segments were fixed in dark at 4 °c in a freshly prepared mixture of saturated picric acid (3 ml), 25 % glutaraldehyde (1 ml) concentrated acetic acid (40 µl). most of the specimens were used as whole-mounts (n = 50) but the cns and various body regions were dissected out from a few specimens (n = 18). light-microscopic immunocytochemistry the following protocol was used in the case of the whole-mount and the dissected specimens as well. after several rinses in 70 % ethanol, fixed samples were washed in phosphate-buffered saline (pbs), then treated with 2 % triton-x-100 diluted in pbs for two nights at 4 °c. samples were incubated for 2 h in 10 % normal goat serum in 2 % triton-x100 to decrease background staining, and were afterwards incubated in 1:500 dilution of anti-gaba primary antibody (sigma chemical company, budapest, hungary) in 2 % triton x-100 buffer for 4 days at 4 6 °c. the specificity of the antibody to gaba was confirmed by the preincubation of the antibody together with gaba-glutaraldehyde-bovine serum albumin complex (1 mm of gaba). after preincubation, the immunohystochemical protocol resulted in no staining. by means of stereomicroscopy labelled structures were visualized using diaminobensidine (dab), avidinbiotin-horseradish peroxidase method, extravidin kit, (sigma chemical company, budapest, hungary). control samples (n = 15) were processed utilizing the above immunohistochemical staining protocol except for the omission of the primary antibody. stained samples were prepared in two different ways: 1) whole-mounts were cleared with buffered 173 glycerol (50 % pbs and 50 % glycerol) for 2 3 days then mounted between cover-slips in pure glycerol for microscopy, or 2) dehydrated in graded ethanol series and embedded into epoxyresin (durcupan acm) and were after sectioned in 1.5 µm thin serial sections using a reichert supernova ultramicrotome, attached to gelatine-chrome-alum covered slides and stained with toluidine blue. electron microscopic immunocytochemistry for electron microscopy the samples (n = 12) were fixed in a mixture of 4 % freshly prepared paraformaldehyde and 2.5 % glutaraldehyde for 2 h at 4 °c. after thorough washing with several changes of 0,1 m pbs (ph 7) they were post-fixed in ice-cold 1 % oso4 for 60 min. samples were dehydrated and embedded into durcupan acm resin. ultrathin serial sections were cut by reichert supernova ultramicrotome and collected on nickel grids. sections were etched in 1 % hio4, deosmicated with 1 % naio4 and then were transferred to several drops of distilled water. the preincubation was carried out applying normal goat serum for 30 min and then samples were incubated overnight with a polyclonal rabbit anti-gaba serum (sigma chemical company, budapest, hungary) diluted 1:300 in tris-buffered saline (tbs, ph 7.4) at 4 6 °c. after incubation process the samples were washed in several changes of 0.01 m tbs and placed to drops of goat anti-rabbit igg secondary antibody conjugated with 10nm colloidal gold particles (sigma) diluted 1:50 in tbs, for 2 4 h. finally the samples were rinsed in distilled water and dried at 35 °c. imaging nikon eclipse 80i microscope equipped with nomarsky optic was used to visualize the samples. at the level of light microscopy the detection of the dab signal was difficult due to the relative high pigment content of the body wall, thus a multispectral imaging technique was applied. multispectral images were recorded using a zeiss axio imager z1 with a cri nuancetm multispectral imaging system. the spectral recording of the whole-mount samples were in the spectra interval from 400 nm to 750 nm. the digitalization of serial sections was performed using a pannoramictm scan digital slide scanner (3dhistech ltd, budapest, hungary) with zeiss plan-apochromat objective (magnification: 20x, numerical aperture: 0,8 ) and hitachi (hv-f22cl) 3ccd progressive scan color camera (resolution: 0,2325 µm/pixel). 3dview scientific software application (version: 1.5, 3dhistech ltd, budapest, hungary) was used to make three-dimensional reconstructions from the digitalized serial tissue sections. ultrastructural examinations were carried out with jeol 1200 ex electron microscope system. cell counts after binarization of the dab spectral images the number of gaba-ir cells for relevant body segments was counted using particle analysis application of image j software (imagej 1.32j, wayne rasband, nih, usa). used samples were: 4 prostomium, 5 first segments, 12 postclitellar segments, and 5 posterior segments. eight clitella were measured separately because of the special cell pattern. as the body areas used for counting differed between samples, cell counts were standardized to a unit area of 5,000 μm2. the distribution of the cells was additionally investigated within four different midbody segments of similar size (216,500 μm2), the number of cells being counted in the dorsal, ventro-lateral and ventral part of the segments. analysis of variance (anova) was used for data analysis using the spss 17.0 for windows (spss inc., chicago, usa). results the applied antibody and the immunocytochemical process revealed a specific and reproducible staining of sensory structures in the pns and the cns of e. fetida. immunoreactivity in the body wall based on the investigations of whole-mount samples the distribution pattern of gaba-ir primary sensory cells were described in relevant body segments. stained cells were represented consequently in all segments of the animal, the highest density being detected in the first five and the last three posterior segments. applying multispectral recording the spectra of the dab were identified on each the recorded image (figs 1a e). the maximum density of dab measured was 430 nm. the spectra line of dab was separated from the pigment spectra (figs 1a, b). further the distribution pattern of the stained sensory cells was described based on normal photomicrograph and spectrum separated images. the prostomium and oral cavity (segments 1 to 5) in the anterior part of the animal, the prostomium and the oral cavity was studied in detailed. the labelled primary sensory cells in the prostomium have small sized circular soma (approximately 8 10 µm in diameter) and stand typically alone in the sensilla (fig. 2a). three dimensional reconstruction of the prostomium allowed more precise morphological analysis and showed that the most of the stained cells were located near the surface of the epithelial layer with short processes to the cuticle (figs 3a, c). others reached through the epithelium or were located at the base of it on the basal layer (fig. 3b). equal distribution was typical, the distance between the stained cells was in average 20 µm (min = 12, max = 25) in the investigated samples (fig. 2a). in the epithelium of the buccal cavity clearly labelled cell bodies were detected. the cells had typically small soma and their processes created a dense plexus (fig. 2b). in the dorsal and the lateral side of the pharynx most of the fibres were linear and run in parallel however, in the ventral region the dense network with crossings were typical (fig. 2c). midbody and anterior segments in the midbody segments most of the gaba-ir cells were concentrated into a lane which was extending in the middle of the segments, along the line of the chaetae (fig. 2d). these cells were 174 fig. 1 multispectral recording of the body wall epidermis of eisenia fetida after gaba immunostain. a) solitary and grouped sensory cell were also represented in the epithelial layer. b) spectra of the immunostain (dab), pigment and gland cells based on a cri multispectral recording (c). stained cells were detected on the dab spectral image (d, e). in the spectral segmentation process the gland cells (d) and the pigment (e) spectra were also defined and these images were used as negative control. sharp arrow: solitary sensory cell, double arrow: sensory cells into sensilla, dab: diaminobenzidine. direction a p anterior-posterior. bar: 50 µm generally grouped in sensilla which contain 2 8 stained cells and additionally up to 20 other unlabeled sensory cells. in the large sensilla (average 80 µm diameters) we counted about 20 40 sensory cells and gaba-ir ones were more than a half of these. however there were sensilla with high number of stained cells close around the chaetae, in the marginal part of the chaetae sac (fig. 2e). the observation of the deeper regions of the chaetae row revealed stained transversal fibres between the sensilla (fig. 2f). in the anterior and the posterior part of the segments, solitary sensory cells were generally, following random distribution pattern. the localization of the sensory structures of the chaetae row coincided with the running of the second segmental nerve (fig. 4a).this distribution pattern was typical for all midbody segments, except for the clitellar segments. here low number of the gaba-ir cells was found in dispersed location. the solitary cell type occurred generally, sensilla with stained cells were rarely detectable. however, high numbers of stained cells were concentrated in two thin lanes which were parallel to the long axis of the animal and localized the ventrolateral side of the segments. these cells were round, stained intensively and had very small (2 3 µm) soma. the lane was narrow in its anterior and posterior end (fig. 4b). the pattern described in the midbody segments was found in most segments of the animal. the labelled cells in the posterior segments had similar distribution pattern as in the anterior segments. 175 fig. 2 gaba-ir sensory elements in different body regions of e. fetida. a) labelled primary sensory cells in the epithelial surface of prostomium. b, c) stained structures in the epithelium of the oral cavity. d) the localization and the distribution of labelled cells and sense organs in midbody segment. thumbnail: two sensilla. e, f) sensilla with gaba-ir sensory cells in the chaetae row. stars: chaetae sac, sharp arrow: solitary sensory cells, arrows with double arrowhead: grouped sensory cells, arrows: the processes of the sensory cells. direction a p: anterior-posterior, d v: dorsal-ventral. bar: a e: 100 µm; f: 10 µm. 176 fig. 3 three-dimensional micrographs of stained gaba-ir structures in the prostomium. a) the whole reconstruction of the prostomium with labelled primary sensory cells and their central processes which run into the prostomial nerve. b) virtual tissue segment at high magnification with the detected gaba-ir sensory cells which localized in different part of the epithelial layer. c) stained primary sensory cells with short apical processes to the cuticle. dotted line: sensilla, sharp arrow: solitary sensory cells, arrows: processes of the sensory cells, pn: prostomial nerve. direction a p: anterior-posterior, l m: lateral medial. bar: a: 10 µm; b, c: 50 µm. the average number of gaba-ir cells varies between the analyzed body segments as follows: 600 ± 52 cells in the prostomium, 530 ± 40 cells in the first segment, 800 ± 65 cells in a postclitellar segment and 300 ± 17 cells in the posterior segments. most of the cells was identified in the clitellum (i.e., 1,300 ± 72, n = 8), where they were mostly distributed in one line. anova revealed significant difference between the cell numbers of different body parts (f(2, 10) = 78.45, p < 0.001). the prostomium and the body end segments bore the highest number of gaba-ir cells (fig. 5). within an average midbody segment, ca. 40 % of the cells are located in the dorsal side, ca. 25 %, in the ventral side and 35 %, in the ventrolateral side. the investigations on serial sections confirmed the result of the whole-mount observations: only the subpopulation of the primary sensory cells show gaba immunoreactivity. 177 fig. 4 distribution pattern of gaba-ir primary sensory cells in various segments. a) the position of the stained cells coincided with the position of the segmental nerves which were also stained. b) specific distribution pattern were revealed in the clitellum, where the stained cells were localized in the area of the adolescent bump. stars: chaetae sacs, sharp arrows: solitary sensory cells, arrows with double arrowhead: grouped sensory cells, i., ii., iii.: position of the segmental nerves. direction a p: anterior-posterior. bar: 100 µm. there were labelled cells represented together with non-labelled ones inside one sensillum, whereas the phaosomal cells (i.e., photoreceptors) were not stained as shown in figure 6a. we could not distinguish areas inside the sensilla, where the gaba expression was preferred, the stained cells localized randomly in the investigated sensilla. from morphological aspect the labelled cells were heterogeneous even in one sensillum, but particularly significant differences could be observed in the anatomical attributes of the labelled cells in the different body regions. the morphology of gaba-ir primary sensory cells in the sensilla of the prostomium long, extended gaba-ir cells with well-developed central processes were found in high numbers. the structures of the labeled cells in the sensilla of the postclitellar segments were more heterogeneous, although a few basic types could be identified: i. the characteristic features of the first form were: relatively small sized, nearly globular soma localized in the apical part of the epidermis, some ramifications of their central processes entered in the basiepidermal plexus, whereas the thickest branch runs into the segmental nerve (figs 6b, g). ii. the second type resembled bipolar cells, had a short peripheral and a long central process. the latter was essentially thicker, than in the other sensory cells (figs 6c, d) and entered directly, without any ramifications, into the segmental nerve. iii. the third type was mainly detected in the centre of sensilla, reached across the whole thickness of the epithelium and its process coming out from the soma sprouted and broke up into several fibres ended in the plexus (figs 6e, h). the solitary sensory cells were represented in low number, had thick central branches which without ramification run directly to the basiepidermal plexus (fig. 6f). small dendritic processes were detected in the apical part of these cells, which run in the top of the cuticle layer. these cell types were typically in the anterior and the posterior parts of the midbody segments (figs 6a, f, i) gaba-ir branches in the basiepidermal plexus the varicose gaba-ir central processes entered the basiepidermal plexus (figs 6h; 7a, b). putatively in the motor axons, dense core and clear vesicles with large diameter were located, whereas in the sensory axons, 0.5 1μm in diameter, small clear vesicles could be located (fig. 7c). by means of immunocytochemical observations it could be well detected that the central process of gaba-ir sensory cells frequently gave inputs to processes of the non-gabaergic sensory cells in the same sensilla (figs 2f; 6b). the gaba-ir central processes and their lateral branches with varicosities entered to the basiepidermal plexus which could be clearly identified in serial sections (figs 6c, e, h). the branches connecting two individual sense organs were observed in many cases (fig. 2f). the stained processes of the gaba-ir sensory cells frequently gave inputs to the non immunoreactive sensory cells localized into one sensillum (fig. 7d). the central representation of the gaba-ir sensory branches the ascending labelled sensory fibres in the segmental nerves and in the cns were followed both in whole-mount preparations and in its semithin serial sections. it was established that only two pairs of the five longitudinal sensory axon bundles, namely the ventrolateral and the ventromedial axon bundles, contained gaba-ir fibres. the intensive labelling of the ventrolateral axon bundles surrounding the ventral giant axons was conspicuous (figs 8a, b). 178 fig. 5 the distribution of gaba-ir sensory cells in the investigated body segments. the ultrastructural features of the gabaergic axon profiles were investigated in the neuropil of the ventral ganglion. selective stained branches could be observed in the ventrolateral (figs 8c, d) and the ventromedial (figs 8e, f) longitudinal axon bundles. the gold particles were uniformly distributed over the gaba-ir varicosities; vesicles and the cytoplasm were also labelled. the gabaergic axon profiles mainly contained pleomorphic agranular synaptic vesicles with an average diameter of 25 nm. moreover dense-core vesicles of about 50 nm in diameter were additionally seen in the gaba-ir profiles. discussion in present study we applied a controlled gaba immunohistochemical protocol to identify distinct sets of inhibitory -gabaergicstructures in the sensory system of the earthworm e. fetida. we showed that a subpopulation of the primary sensory cells was gaba immunoreactive. based on the result of the preabsorption tests and the negative control where we never received staining, we could assume that indeed gaba-containing cells and fibres were stained. the specificity of the applied serum was tested in several laboratories in invertebrates (spörhase-eichmann, 1997) and vertebrates (meza, 1998) as well and specific staining was achieved with it. the presence of gaba, known as an inhibitory neurotransmitter, is not customary in receptor cells (sepherd, 1994), though according to many earlier studies there are gabaergic sensory cells in vertebrates as well, which might play a role in fine modulation of the sensation (meza, 1998). our result demonstrates that gabaergic primary sensory cells are represented in all segments. the distribution of the stained cells was not randomly in the body surface, but there were well defined, constant patterns in relevant segments of body regions. the distribution pattern followed consequently the pattern of sensory cells which was described earlier by langdon (1895). this showed that the majority of the primary sensory cells are gaba-ir, so gaba could play an important role in the sensory regulation. stained sensory cells were found in high number and high density in the prostomium, moreover, these patterns were typical the first and the second anterior segments and the last three posterior segments. we applied a pixel based tissue reconstruction of digitalized serial sections to reveal the three-dimensional organization of the gaba-ir elements in the prostomium. based on this method we described the exact localization and the morphological features of the stained primary sensory cells. measurements showed that the number of the sensory cells per area is the maximum in the prostomium and in the anal segment. because of these results it could be presumed that these body regions had an important role in the gaba-mediated sensory function. in the midbody segments we revealed a typical pattern. most of the gabaergic cells grouped into sensilla and were concentrated in the chaetae rows. in the anterior and the posterior part of segments solitary sensory cells were randomly distributed and less number of sensilla were also located. observing whole-mount preparations we found that the localization of the stained cells of the chaetae rows was coincided with the running of the 2nd and 179 fig. 6 types of gaba-ir primary sensory cells in the epidermis of eisenia fetida. a) cells with oval somata in the epithelium of the prostomium. the basal localized phaosomal photoreceptors were not stained. b) type i cells in small size, localized at the top of the apical surface occurred frequently in sensilla. c, d) stained type ii sensory cell with branched processes. e h) labelled type iii sensory cell with elongated soma. f i) apically localised sensory cells which stand alone. g) stained cells grouped into sensilla. h) gaba-ir processes in the level of the basiepidermal and muscular plexus. gaba immunoreactive processes at the level of the basiepidermal and muscular plexus. j) stained cells with heterogeneous morphology grouped in sensilla. sharp arrows: cell bodies, double arrowheads: dendritic processes of sensory cells, arrows: central processes of sensory cells, arrows with empty arrowhead: phaosomal photoreceptors, dotted line: border of sense organs, e: epidermis, m: muscle layer, gc: gland celldirection l m: lateral-medial. bar: 10 µm. 180 fig. 7 gaba-ir processes in the basiepidermal nerve plexus. a, b) stained processes in whole mount sample. c) the ultrastructure of the basiepidermal plexus. d) gaba-ir central processes of the primary sensory cell in the plexus. arrow: stained processes, gc: gland cells, dotted line: border of sensilla, stars: motor axons, cross: sensory axons, sc: sensory cell, c: collagen fibres, arrows with empty arrowhead: possible synaptic connections. directions l m: lateral-medial. bar: a, b: 10 µm; c, d: 500 nm. 3rd segmental nerves showing this is the most important region of the gabaergic sensory mechanism. we revealed a typical pattern in the clitellum, where the stained cells concentrated into two parallel lines in the area of the adolescent bump. presumably these gaba-ir cells, represented in high number with central processes which run directly to the vnc have a role in shaping of the mating behaviour to perceive the body surface of the partner. in the sensilla of the postclitellar segments the labelled cells were heterogeneous but we could define some characteristic cell types, which could be responsible for different gaba mediated sensory functions. based on the characteristic location of the type i cells (they were situated at the top of the epithelial layer close to the body surface) we can conclude that they play a role in the direct sensory perception and the fast transmission of the primary stimulus. the type ii which resembled bipolar cells, because of the reach arborisation of their processes possibly has a role to support an integrative function in the peripheral sensory system. the type iii was mainly located in the centre of sensilla have probably function in the synchronization of the intersensillar sensory function. moreover, the processes of these cells make dense arborisation in the level of the plexus and because of this possibly make role to the intersensillar communication. the function of solitary gaba-ir sensory cells could be relatively simply interpret. according to the results of anatomical and physiological experiments they were supposed to be stretch receptors, because they inhibit to overstrain the epithelium of the body wall (edwards and kuffler, 1959; craelius and fricke, 1981). our results partly corroborate this hypothesis, since the central processes of the majority of solitary sensory cells directly project to the ventral ganglia without making any synapses with the basiepidermal plexus. similar cells were 181 fig. 8 location of gabaergic in the vnc. a, b) stained axon bundles and sensory cells in a whole-mount preparation (a) and in its cross sections (b). ultrastructure of gaba-ir axon profiles in the ventrolateral (c, d) and the ventromedial (e, f) sensory longitudinal axon bundles after postembedding immunogold staining. arrowhead: stained central sensory cell, i., ii., iii.: the position of the segmental nerves, mga: medial giant axon, lga: lateral giant axon, vl: ventrolateral axon bundle, vm: ventromedial axon bundle, sharp arrow: agranular vesicle, arrow with double arrowhead: granular vesicle, m: mitochondria. direction a p: anterior-posterior; d v: dorsal-ventral. bar: a, b: 100 µm; c e: 100 nm. found in some sensilla. moreover, most of the stained cells had fibres running to the ganglia (fig. 7). in the ventral ganglia the gaba-ir fibres were only concentrated into two sensory bundles (ventrolateral and ventromedial), and it is obvious that they have synaptic connections with the determined branches of the cns. the ventrolateral sensory longitudinal axon bundles formed a thick sheath around the ventral giant axons, which take a prominent role in the movement coordination and give a synapsis to them (bullock, 1945; coggeshall, 1965; mill, 1982; büschges and el manira, 1998). the ventral giant axons make synaptic contacts with the so-called 182 giant motoneurons, and affect the activity of the longitudinal muscles in the body wall (mill, 1978; jamieson, 1981). presumably gaba inputs influence indirectly the activity of the giant motoneurons through inhibiting the ventral giant axons, and thus the contraction of the longitudinal muscles of the body wall. it is more difficult to interpret the function of the gabaergic endings of the basiepidermal plexus. based on our current knowledge, sensilla contain mainly uniand multiciliar cells, which mostly take part in chemoreception (laverack, 1963; mill, 1978, 1982). the gaba-ir cells of the sensilla presumably inhibit not only the function of the certain structures of the cns, but they could modify and thus influence the activity of a part of the sensory cells. consequently they could play a role in the delicate modulation of the processes of the perceptibility, or in the formation of the receptive fields of the body wall. laverack (1963) reported on the excitatory and inhibitory structures of the plexus. on the evidence of electrophysiological observations isolated receptive fields could be identified in the epithelia of the body wall (mill, 1982). these structures support the presumption of the integrative function of the basiepidermal plexus. gaba, like a modulatory transmitter could be in the background of this function. based on ultrastructural investigation it was revealed that the labelled presynaptic profiles in high numbers contained small, pleomorphic, agranular vesicles. this morphological statement correlates with the gaba profiles described already in the cns in the vertebrates (somogyi and soltész, 1986; ottersen et al., 1988; gábriel and straznicky, 1995), and also in the invertebrates (wattson, 1988; telkes et al., 1996, ganeshina and menzel, 2001). summing up these immunohistochemical investigations where the majority of the sensory structures were gaba positive, showing that gaba has an important role in the sensory mechanism. based on the anatomical organization of the gabaergic neurons, presumably these cells have a modulator role in the intercellular and intersensillar communication at the level of the basiepidermal plexus. according to our results we can assume the gaba-ir structures of the sensory system mediated in the motor reactions and segment synchronization by modulating the activity of giant interneurons (both ventral and dorsal giant axons) of cns. acknowledgement we 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184 sepherd gm. neurobiology, 3rd edition, oxofrd university press, new yorkoxford, 1994. telkes i, csoknya m, buzás p, gábriel r, hámori j, elekes k. gaba-immunoreactive neurons in the central and peripheral nervous system of the earthworm, lumbricus terrestris (oligochaeta, annelida). cell tissue res. 285: 463-475, 1996. watson ahd. antibodies against gaba and glutamate label neurons with morphologically distinct synaptic vesicles in the locust central nervous system. neuroscience 26: 33-44, 1988. 185 gene function and cellular pathways in higher vertebrates, including humans, have increasingly shown to be highly conserved thr 162 isj 15: 131-140, 2018 issn 1824-307x research report erratum to: effects of dietary botanical and synthetic astaxanthin on e/z and r/s isomer composition, growth performance, and antioxidant capacity of white shrimp, litopenaeus vannamei, in the nursery phase l xiaohui1,2,3, w baojie1,2, l yongfu1,2, w lei1,2, l jianguo1,2,* 1cas key laboratory of experimental marine biology, institute of oceanology, chinese academy of sciences, qingdao 266071, china 2laboratory for marine biology and biotechnology, qingdao national laboratory for marine science and technology, qingdao 266071, china 3university of chinese academy of sciences, beijing 100049, china in the above article the names and surnames of the authors were reproduced incorrectly, and they should have appeared as below: xh liu1,2,3, bj wang1,2, yf li1,2, l wang1,2, jg liu1,2,* isj 13: 23-27, 2016 isj 13: 23-27, 2016 issn 1824-307x short communication cutaneous neoplasm in phaeotabanus litigiosus (diptera, tabanidae) collected on the marambaia island, rio de janeiro, brazil rr guimarães1,2,5, rr guimarães-junior2,3, rs harlan-ronald2, r rodrigues-guimarães4,5, fl carvalho6, mlc araújo-junior6, rw carvalho1 1escola nacional de saúde pública sérgio arouca-fiocruz, rio de janeiro, rj, brasil 2centro de educação e pesquisas em medicina ambiental cema, nilópolis, rj, brasil 3associação brasileira de ensino universitário abeu, nova iguaçu, rj, brasil 4centro universitário de barra mansa ubm, barra mansa, rj, brasil 5universidade estácio de sá unesa, rio de janeiro, rj, brasil 6instituto nacional do câncer inca, rio de janeiro, brasil accepted january 28, 2016 abstract a female specimen of phaeotabanus litigiosus (diptera: tabanidae) collected on marambaia island was found with a tumor in the abdominal integument. histopathological examination revealed an epithelial dysplasia with anisokariosis and hyperchromasia. this is the first record of a neoplasm found in tabanid collected from natural environment. key words: atlantic island; displasia; horse fly; insect disease; insect vector; neotropical region   introduction the family tabanidae comprises approximately 4,300 species distributed throughout the world. the tabanids are mechanical and biological vectors of many pathogens to humans and domestic and wild animals (foil 1989; turcatel et al., 2007). several biotic and abiotic factors determine the behavior of tabanid populations by varying the number of individuals according to different landscapes and over years and seasons (guimarães, 2015). interactive biotic factors are viruses, bacteria, fungi, helminths, other arthropods, fish, reptiles, amphibians and birds, which result in predation or parasitism. some of these factors or other intrinsic factors cause pathologies that can lead to mutations, birth defects, metabolic disorders and malignancies that affect, in some degree, the survival of individuals or populations (philip, 1965; nayar, 1977; savini and furth, 2004; asiain and márquez, 2009; ferreira, 2008, 2014). neoplasm in insects has been described from various causes, such as genetic factors, viruses infections, exposure to insecticides and experimental nerve severance. melanotic tumors are reported in drosophila melanogaster (diptera: drosophilidae) linked to genetic factors and viral ___________________________________________________________________________ corresponding author: ronald rodrigues guimarães centro de educação e pesquisas em medicina ambiental av. dr. getúlio vargas, 1797 centro-olinda, nilópolis, cep 26525-023 rio de janeiro, brasil e-mail: ronaldrguimaraes@gmail.com infections (taylor, 1969). insecticides can lead to tumor formation in musca domestica (diptera: muscidae) (cantwell et al., 1966). recurrent nerve severance induce formation of tumors in digestive tract of cockroach leucophaea maderae (blattodea: blaberidae) (matz and bergoin, 1984). the literature records no pathology related to cancer or tumors in tabanids. this study is related to a cutaneous tumor formation found in phaeotabanus litigiosus collected on marambaia island, rio de janeiro state, brazil. materials and methods during october, november and december 2013 tabanids attracted by equine bait (equus caballus) were collected through insect net, on marambaia island, municipality of mangaratiba, rio de janeiro state, at location known as vacaria velha (23°03'47" s and 43°59'16" w). this site is close to secondary tropical forest and a swamp with a small pasture area where a tied equine spent the day; these collections were made from the morning twilight and all day until around 20:00 h, at least one day in each of the three months. for collection and transportation of biological samples the instituto brasileiro chico mendes de biodiversidade do ministério do meio ambiente issued permit nº 33382-1, sisbio-ibama. the collected tabanid specimens were examined and identified according to literature (barretto 1950; coscarón and papavero 2009a); 23  mailto:ronaldrguimaraes@gmail.com some specimens were mounted on entomological pins and other specimens were preserved in alcohol 70º; in one of these specimens a cutaneous injury was found on frontal side of abdomen. the specimen was examined under stereomicroscope leica mz 16 and the injury was photographed with leica dfc 420 camera, at the entomology laboratory of museu nacional de história natural do rio de janeiro. the injury was excised, fixed in buffered formalin and sent to the laboratório da seção integrada de tecnologia em citopatologia do instituto nacional do câncer sitec/inca. the fixed material was prepared in histological sections stained with hematoxylin and eosin. the tabanid specimen and the blade with the histological sections are deposited in the entomological collection of centro de educação e pesquisas em medicina ambiental cema. results p. litigiosus occurs in brazil, in minas gerais, rio de janeiro, sao paulo and parana states. during the three months of collection vacaria velha, marambaia island, 85 specimens were collected. material examined: p. litigiosus, brazil, rio de janeiro, mangaratiba, marambaia island, vacaria velha [23°03'47 "s and 43°59'16" o], 4-5.xii.2013, guimarães jr. leg., guimarães det., (1 ♂ cema). the observed injury was located between the first and the second left abdominal tergite (fig. 1) about 0.5 mm in diameter. the injury showed rough and hardened surface. histopathologic examination showed a cavity formed by a simple squamous epithelium, with an area of abnormal multiplication of integumentary cells; abnormal cells presented increased size nucleus and little cytoplasm (anisokariosis). increased size nucleus presented granular coarsely chromatin (hyperchromasia), and evident nucleolus. there was no evidence of invasion of underlying corium or mitotic figures (fig. 2). discussion neoplasic formations in insects are rarely reported in the literature: the insects are highly resistant to carcinogenesis, as most adult cells are post-mitotic, unable to multiply. however, during the larval stages, the stem cells present in the imaginal discs may change that result in neoplastic formations. kirby and spence (1826) refer to external wounds as precursors of tumors in insects, which does not seem to be the case of examined specimen, because any other anatomical changes on outer surface was observed. as to integument tissue formations for the account of balazuc (1948) the meeting of a specimen of phytodecta variabilis (coleoptera: chrysomelidae) presenting a large tumor formation in prothorax without histopathology. white (1929) found similar structure to fibroma, originated from connective tissue in the thorax of a honeybee (apis mellifera (hymenoptera: apidae). a brain tumor, presumably formed by glial cells, was found in worker of formica pratensis (hymenoptera: formicidae) without differential diagnosis of abscess (brun, 1925). palm (1948) described a tumor formation in ‘corpus allatum’ of a male nymph of a species of gryllotalpa (orthoptera: gryllotalpidae); the tumor showed giant cells formed from the cytoplasmic and nuclear fusion of smaller cells with kariorrhexis, indistinct nuclear membrane with numerous beads of chromatin and dispersed core remains in the cytoplasm. a tumor in pharyngeal gland was described in a species of the genus bombus (hymenoptera: apidae): the affected gland showed a compact structure, displaced, enlarged, with abundant connective tissue, with no connection to the common duct, with numerous secretory ducts ending blindly. the cells showed signs of hypersecretion, degeneration and kariorrhexis (palm, 1949). salivary gland tumor were also observed in periplaneta americana (blattaria: blattidae) when the ducts were tied or removed (sutherland, 1963, 1967). fig. 1 cutaneous injury in phaeotabanus litigiosus collected on marambaia island, mangaratiba, rio de janeiro, brazil. the black bar measures 1 mm. photo obtained at entomology laboratory of museu nacional de história natural do rio de janeiro. 24  fig. 2 histopathological aspects of tumor found in phaeotabanus litigiosus collected on marambaia island, mangaratiba, rio de janeiro, brazil; a) cavity (cav) formed by a simple squamous epithelium (see) and epithelial displasia area (eda) (400x); b and c) cavity (cav) and an epithelial dysplasia area (eda) with anisokariosis, hyperchromasia, coarsely granular chromatin, evident nucleolus, and little cytoplasm (ah) (1000x). 25  among the invertebrates d. melanogaster is considered to be the most susceptible species to cancerous tumors, subject to tumors determined by variations in temperature, exposure to x-rays or by chromosomal inheritance and most carcinogenesis records on insects is made in d. melanogaster (wautier and wautier, 1952; slade, 2012). melanotic hereditary tumors in larvae of d. melanogaster tumorw strain involve encapsulation of caudal lipid bodies by hemocytes and hematopoietic organs. these hematopoietic bodies put in service a large number of blood cells, which originate lamelocytes and plasmatocytes which synthesize endogenous tissues (perotti and bairati, 1968; rizki and rizki 1974; nappi et al., 1984; silvers and hanratty 1984; william and hanratty 1984). the melanotic encapsulation performed by melanocytes is a characteristic response against aberrant tissues in drosophila and other insects (tascedda and ottaviani, 2014). the tumorosa-lethal mutation (tum-l) is temperature-dependent and also leads to excessive proliferation of hemocytes and the formation of neoplasic melanotic tumors in the larval hematopoietic bodies (luo et al., 1993). rous sarcoma virus determines chromosomal abnormalities, and tumor formation in d. melanogaster that did not seem to be a really neoplasic formation, as it is known in mammals (burdette and yoon, 1967; kirk et al., 1970). the polyhedrosis virus occurrs naturally in gilpinia hercyniae (hymenoptera: diprionidae) and determines proliferation of the germ cells forming a tumor that surrounds the external surface of the digestive tract (bird, 1948). abnormal proliferation of epidermal cells larvae of hyphantria cunea moth (lepidoptera: erebidae) is also caused by the nuclear polyhedrosis virus and determines polyhedral formations in hypertrophied nuclei, which differs from the changes found in classical neoplasms (watanabe, 1968). tumors in the digestive tract caused by experimental severance of recurrent nerve in l. maderae are formed by proliferation of epithelial cells and hemocytes encapsulated by granulocytes; some authors point inflammatory processes determined by microorganisms in the digestive tract is the cause of the alteration (scharrer, 1945a, b, 1948, 1949a, b; matz, 1965; taylor, 1969; matz and bergoin, 1984) the fact that these tumors may be transplanted and proliferate in other individuals from filtered cell or insect dna with recurrent nerve damage, although not present tumors, indicating a likely viral etiology (matz and bergoin, 1984). the fluorenone derivatives induce the formation of tumor injuries in the hypodermis, intestine and lipid bodies in m. domestica (cantwell et al., 1966). in literature, only d. melanogaster is recorded as susceptible to true tumors. thus, the cutaneous neoplasm record in adult tabanidae, collected in nature, is unique true record of cancer in other species of diptera. the histopathologic diagnosis differs from any other recorded in similar injuries related to other insects. the used methodology did not allow the study of the cause of injury. acknowledgements the authors thank dr. m couri, museu nacional de história natural do rio de janeiro, for tabanid specimen photography. the authors also thank escola nacional de saúde pública sérgio arouca ensp-fiocruz for financial support and capes (fellowship process nº 1383383). references asiain j, márquez j. new teratological examples in neotropical staphylinidae (insecta: coleoptera), with a compilation of previous teratological records. revista mexicana de biodiversidad 80: 129-139, 2009. balazuc j. la teratologie des coleopteres et experiences de transplantation sur tenebria molitor l. mémoires du muséum national d'histoire naturelle 26: 1-293, 1948. barretto mp. estudo sobre tabanidas brasileiros. xiii. o gênero 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tumors in an insect. science 102: 102, 1945a. william ms, hanratty p. alterations in the production of hemocytes due to a neoplastic mutation of drosophila melanogaster. j. invertebr. pathol. 44: 324-328, 1984. scharrer b. experimental tumors after nerve section in an insect. proc. soc. exp. biol. med. 60: 18489, 1945b.   27  ferreira rn. two physical abnormalities in coleoptera (cerambycidae, lucanidae) from rhode island, usa. arquivos entomoloxicos 10: 173-174, 2014. foil ld. tabanids as vectors of disease agents. parasitol. today 5: 88-96, 1989. guimarães rr. tabanidae (insecta: diptera): caracterização, ecologia e interação com a população quilombola da ilha da marambaia, rio de janeiro, brasil [ph.d. thesis]. escola nacional de saúde pública-fiocruz. p 187, 2015. 32 isj 17: 32-35, 2020 issn 1824-307x report of meeting 3rd general meeting and working group meetings of the cost action 16203: stem cells of marine/aquatic invertebrates: from basic research to innovative applications (maristem), december 3, 2019, metu-culture and convention center (metu-ccc), metu-ankara, turkiye organizer: a karahan middle east technical university, institute of marine sciences, erdemli-mersin, turkey this is an open access article published under the cc by license cost action 16203 maristem: state of the art and future perspectives l ballarin department of biology, university of padua, italy in its first two years of life, the cost action 16203 maristem has organised two general meetings, two working group meetings, three workshops, two training courses, was present to one dissemination meeting and provided supports for 18 stsms. three additional workshops are forecasted within the 3rd gp. meantime, the number of full member countries increased from 20 (at the beginning) to 24, plus 1 cost cooperating member, 2 nnc and 1 ipc, with the involvement of 61 institutions. in the next two years, efforts should be directed towards the completion of the deliverables, the organization of new workshops and meetings aimed at deep discussions of themes of interest, and an increased exploitation of stsms to support early career investigators. a perspective of aging in botryllid ascidians o ben hamo, r ben-shlomo, b rinkevich israel oceanographic and limnological research, national institute of oceanography, p.o. box 8030, tel shikmona, haifa 31080, israel aging, the universal phenomenon ruled by the progressive degradation of organisms, has been the subject of high interest throughout the history of biological sciences. almost all our knowledge on aging stems from studying solitary model organisms, while very little is known on aging of colonial organisms. it appears that studying colonial organisms may yield new understandings on aging that otherwise could not be studied in individual organisms. botryllus schlosseri, a marine colonial invertebrate organism, is the subject of our study. in this colony three levels of structural organizations are assigned, each representing different forms of aging. the first level of organization are the zooids, the basic and the temporary units of the colony. the second level of organization is the whole colony, that is the sum of all zooids and the matrix they are placed in. the third level of organization is the entity that assembles several genetically different conspecifics (the chimera), due to morphological fusions between colonial vasculatures, a natural occurring phenomenon. on the zooid level, aging is developed synchronically once a week at all functional zooids, also characterized by an aging marker. at the second level of organization, the colony stage, 100 colonies were followed throughout their life span, revealing (still under analyses) that about one third of the colonies proceed through a natural fission process, and these genets were found to live significantly more than colonies that did not go through fission. this longer lifespan suggests an evolutionary advantage of the fission. at the level of the chimera, the study has been focused on chimeras between mothers and their offspring. most of the offspring settled very close to their mothers (56 percent settle up to two centimetres from their mothers), leading to high prevalence of chimeras, resulting in fast degradation and death of the mothers compared to non-chimeric mothers. in contrast, chimerism between offspring resulted in a higher rate of growth and earlier onset of reproduction compared to the controls. these contrasting chimerism outcomes suggest for 33 divergent evolutionary trajectories. thus, this study presents, for the first time, the uniqueness of aging in each level of biological organization in colonial organisms and opens a door for further studies in order to gain new understandings on aging. new aspects in electron microscopy and hydra stem cell lineages w salvenmoser, b hobmayer institute of zoology and centre of molecular biosciences innsbruck, university of innsbruck, austria hydra simple body is composed of three independent cell lineages, all of which contain large pools of either epithelial or small, set aside adult stem cells. they represent ancestral stem cell types present when animal multicellularity evolved. epitheliomuscular stem cells of the ectoderm, which show features of undifferentiated and differentiated cell types, possess a vesicular trafficking system similar to bilaterians. in addition, the presence of large, in some cases huge, vacuoles implicate a function in transport of water and osmoregulation necessary to deal with a fresh water environment. here, we have used scanning (sem) and transmission (tem) electron microscopy using high pressure-frozen tissue samples from the gastric region. after producing thick sections and sem visualization, we found that the larger vacuoles connect via an elaborated canal system. this canal system could be connected to smaller vesicle types and larger macropinosomes dynamically forming at the apical epithelial membrane. thereby, active intake and processing of particles of larger size is facilitated. using ultra-thin section tem, we are able to visualize the contractile actin structure, a dense network of actin monomeric filaments, building the macropinosome pores. serial block-face scanning electron microscopy will be established in our lab, which will allow the detailed 3d reconstruction of this canal system and cellular ultrastructure in general. crosstalk between stem cells and differentiated tissues in planarians g gambino, l rossi, a salvetti department of clinical and experimental medicine, university of pisa, pisa, italy stem cell fate depends on surrounding microenvironment, the so called niche. for this reason, understanding stem cell niche is one of the most challenging target in cell biology field and have to be unravelled by in vivo studies. planarians offer this unique opportunity, as their stem cells, the neoblasts, are abundant, highly characterized and genetically modifiable by rna interference (rnai) in alive animals. however, despite impressive advances have been done in the understanding of planarian stem cells and regeneration, only a few information is available in defining signals from differentiated tissues, which affect neoblast stemness and fate. we took advantage of the stem cell repopulation process that follows low-dose x-ray treatment in planarians to identify genes, preferentially enriched in differentiated cells, whose expression is activated during the repopulation process. silencing by rnai of some of them impaired the stem cell repopulation, suggesting a tight extrinsic control of stem cell activity. among these genes, we identified djmap that is expressed in the nervous system. djmap rnai animals failed to regenerate thus indicating that it is involved in neoblast maintenance not only after low-dose x-ray treatment but also in untreated animals, thus paving the way for future studies in crosstalk mechanisms between djmap-positive neurones and neoblasts. stem cell molecular markers in the demosponge halisarca dujardini i borisenko saint-petersburg state university, russia sponges are one of the most ancient multicellular animals that occupy key evolutionary position at the base of the tree of life. many of metazoan-character signaling mechanisms and transcription factors appear in sponges. the appearance of stem cell systems should be studied from the simplest metazoa. it was shown that genes participated in germ line program are expressed in different cell types of sponges like choanocytes and archaeocytes, but expression patterns differ from class to class of porifera. we studied expression of myc (cell cycle regulator), pou (ortholog of oct4) and piwi (germ line associated rnase from argonaute family) with whole mount in situ hybridization in demosponge halisarca dujardini. all of these genes expresses in choanocytes. in addition transcripts are present in little patches of oocyte cytoplasm, by the structure corresponded to mesochyl cells ingested during oogenesis. these cells keep undigested in oocyte cytoplasm until cleavage begins. perhaps presence of choanocyte-specific transcripts demonstrates that “nutrient” cells in oogenesis are dedifferentiated choanocytes. jellagen®: a next generation matrix derived from jellyfish for regenerative medicine and cell culture a mearns spragg jellagen limited jellagen® limited is a marine biotechnologies company based in cardiff, uk, whose strategic mission is to exploit sustainable marine species and natural resources, to develop technical and scientific high value research and medical device products, meeting state of the art specifications. jellagen ltd’s first range of products targeting cell culture include: • jellagen®: a next generation collagen biomaterial derived from jellyfish to support cell culture, tissue engineering and regenerative medicine applications. • jellagen®-2d: jellyfish collagen pre-coated plates – for improved results in promotion of adhesion, growth & differentiation. • jellagen®-3d scaffolds: suitable for in vitro cell culture and tissue engineering jellagen ltd’s research focus has led to the launch of a revolutionary new 3d cell culture 34 hydrogel technology; jellagel™. unlike mammalian collagen, jellagel™ is free of disease vectors, nonspecific mirna and other contaminants typical of mammal-derived materials (e.g., proteins, polysaccharides). unlike synthetic materials, many of which are based on β-structured fibrous materials and produced chemically, jellyfish collagen is consistently bioresorbable and non-toxic to cells, from stem to lineage cells. jellagel™ is able to provide and maintain a realistic, near-native microenvironments for cells. encapsulation of cells has been shown to be synchronised with cell adhesion observed in all three dimensions, with cells showing no polarity as otherwise would be the case for 2d cultures. biocompatibility has also been shown to be amply demonstrated by the appreciable expression of cellular filopodia within the hydrogel matrix. basic approach of inflammation, injury and regeneration in anemonia viridis. mg parisi, c la corte, d parrinello, m dara, m cammarata laboratory of marine immunobiology, department of earth and marine sciences (distem), university of palermo, italy the potential for tissue regeneration is a powerful adaptive strategy essential to the survival of individuals. it allows to face wounds or loss of body parts induced by predation, anthropic actions or environmental factors. in light of the high probability of increasing levels of disturbances caused by injuries and the increasing possibility of invasion of microbes and foreign agents in the tissues of anthozoans, it is crucial to determine how the species respond to wounds and physical damage and understand the capacity of recovering and tissues regeneration. from this point of view, the regeneration capacity of anthozoa it could be considered an additional arm of innate immune defence and viceversa. therefore, from our work team, the inflammatory response in mediterranean anthozoan anemonia viridis (forsskal, 1775) has been studied following the injection of substances of various type and size. we observed strong and specific reaction, especially after the bacterial injection of escherichia coli and vibrio alginolyticus. then, we focused on the regenerative aspects of this species of anthozoan carrying out an experimental plan based on different numbers of tentacle cuts and the evaluation of the regenerative potential after 7, 14 and 21 days. morphological observations and histological analysis on the tentacle regrowth, as well as measures of expression of proliferating cell nuclear antigen (pcna) were carried out. protease, phosphatase and esterase activities were measured as survival markers. in perspective, we want to study at histological and molecular level how homeostatic tissues start the regeneration program while triggering immune response and their mediators of inflammation. in this context, we will focus on those bioactive molecules that, with their vast abundance, can be potentially used for biotechnological applications. the enigmatic xenacoelomorphs: what they tell us about the evolution, development and regeneration of animals b gavilán1, sg sprecher2, p martinez1. 1departament de genètica, microbiologia i estadística, universitat de barcelona, av. diagonal 643, 08028 barcelona, spain 2department of biology, university of fribourg, 10, ch. du musée, 1700 fribourg, switzerland the emergence and diversification of bilateral animals are amongst the most important transitions in the history of life on our planet. however, although numerous labs are currently working on selected questions of genomic and morphological evolution, no interdisciplinary networks are currently in operation. as a result, many critically positioned animal groups have not been analysed and a comprehensive mapping of their genomes and tissue systems remains elusive. our consortium brings together leading experts from different fields with the goal of elucidating the evolutionary and developmental origins of organismal and genomic complexity. our aim is to provide answers to the questions: how did complex body plans arise in evolutionary time? how are complex body plans “encoded” in the genome? as the first step, we will focus on the earliest stages in bilaterian evolution, probing the most elusive organisation of the genomes and microscopic anatomy in basally branching taxa, which are currently assembled in a clade named xenacoelomorpha. our team’s major long-term goal is to employ multidisciplinary approaches to decipher the genomic bases of the organisation and physiological roles of these organ systems. moreover, we are now using these animal systems to study the regeneration of some key organs and tissues, a project facilitated by the enormous potential for regeneration of these organisms. i will describe in the meeting the progress we have made on the understanding of tissue architectures, the evolution of their genomes and the kinetics of regeneration in brain and gonadal tissues. shiok meats cell-based clean shrimp meat s sriram, k yi ling ceo and cto, shiok meats pte. ltd., singapore shiok meats is a cell-based clean meat company, the first of its kind in singapore and south-east asia. our mission is to bring delicious, clean and healthy seafood and meats by harvesting from cells instead of animals. shiok meats will bring cell-based crustacean meats (shrimp, crab, lobster) to your table. our meats are animal-, healthand environment-friendly with the same taste, texture, more nutrients and no cruelty. “shiok” in singapore and malay slang means fantastic and delicious. this presentation is our technology, mission, team and what we do at shiok meats. 35 biosafety aspects for clinical applications of adult human mesenchymal stem cells uldis berzins1,2 1cilmes sunu tehnologijas, ltd 2riga technical university somatic cell therapy is a growing field of biotechnology and a promising alternative for the treatment of such conditions as graft versus host disease, digestive tract and inflammatory joint diseases, as well as cardiovascular and neurological diseases. despite the potential, many problems are yet to be solved, including (1) the lack of sufficient body of clinical data and more thorough knowledge about in vivo posttransplantation processes; (2) donor-specific differences and heterogeneity of final cell product: (3) the variable functional activity of cells depending on post-transplantation microenvironment, (4) the inability of available animal models to accurately predict clinical outcomes in humans. altogether, it implies many risks which result in inconsistent clinical outcomes. the main goal of biosafety is to mitigate risks and facilitate therapeutic efficiency which is achieved by manufacturing cell products in accordance with good manufacturing practice. one of the strategies for enhancement of therapeutic efficiency is cell product preparation according to individualized protocols, an approach chosen by cilmes sunu tehnologjas, ltd. to deliver patient-specific autologous therapeutics under the hospital exemption scheme (implementation of art 28(2) of eu regulation 1394/2007). 92 isj 16: 92-104, 2019 issn 1824-307x research report harpalus (pseudoophonus) rufipes as a model to study cellular and humoral immune defence strategies in coleopteran species f cavaliere1, p brandmayr1, pg giulianini2, ml vommaro1, a giglio1* 1department of biology, ecology and earth sciences, university of calabria, rende, italy 2department of life sciences, university of trieste, trieste, italy accepted june 3, 2019 abstract carabids are of special interest as environmental quality assessment indicators of exposure to xenobiotic and for pest control. in agroecosystems, they can be exposed to a wide range of pathogens and environmental pollution exerting a stronger selection on their innate immune systems. therefore, information on species-specific immunocompetence is necessary to complete the ecological framework of ground beetles. in this study, cellular and humoral responses were characterized in adults of harpalus (pseudoophonus) rufipes (de geer, 1774) to define a baseline knowledge for future ecotoxicological studies. the circulating hemocytes were characterized by light and transmission electron microscopy and in vivo assay performed by injecting latex beads to identify phagocytizing hemocytes. ultrastructural analyses revealed four morphologically distinct types of circulating hemocytes: prohemocytes, plasmatocytes, granular cells and oenocytoids. differential hemocyte counts showed that plasmatocytes and granular cells were the most abundant circulating cell types and granular cells exhibited phagocytic activity following immune challenge with latex beads. mitotic figures and non-differentiated hemocytes observed under light microscopy indicate a continuous cell turnover in the hemolymph. melanotic nodules found 2h after the immune challenge were formed to immobilize the latex beads. phenoloxidase (po) assays revealed an increase of basal po activity in hemolymph after immune system activation with lipopolysaccharide (lps). however, the lpsstimulated adults showed no significant variation in the lysozyme-like enzyme activity in hemolymph. based on these results, h. rufipes displays a rapid, non-specific immune response involving cellular and humoral effectors that both sequester and clear pathogens. key words: hemocytes; ultrastructure; phagocytosis; nodulation; phenoloxidase; lysozyme; carabid beetles introduction evolutionary and ecological studies on insect immunocompetence have provided evidence that the ability to resist disease is a life history trait that depends upon biotic and abiotic ecological factors including habitat quality, resource availability, life cycle and gender (moreno-garcía et al., 2014). ecological inputs generate high species-specific diversity in both constitutive and induced immune responses (schmid-hempel, 2003, 2005; sadd and schmid‐hempel, 2009; schulenburg et al., 2009). thus, information on the strength, speed and specificity in recognizing and processing pathogens ___________________________________________________________________________ corresponding author: anita giglio department of biology ecology and earth science university of calabria via p. bucci, i-87036 rende e-mail: anita.giglio@unical.it is of great interest in an increasing numbers of species for controlling pests (bulmer et al., 2009; hillyer and strand, 2014) as well as for reducing mortality of beneficial insects (james and xu, 2012). the innate immune system of insects includes cellular and humoral immune defences, which provide an active barrier against pathogens (hillyer, 2016). hemocyte-mediated immune responses include phagocytosis, nodule formation and melanotic encapsulation (ribeiro and brehélin, 2006; strand, 2008). phagocytic activity of hemocytes is a fundamental innate immune mechanism used to recognize (ottaviani, 2005; lamprou et al., 2007; rosales, 2011), ingest and kill pathogens or to remove apoptotic cells (marmaras and lampropoulou, 2009; tsakas and marmaras, 2010). nodulation and encapsulation are cellular immune responses against bacteria and parasites too large for phagocytosis (gillespie and et al., 1997; satyavathi et al., 2014; hillyer, 2016). the humoral 93 fig. 1 circulating hemocytes in adults of h. rufipes, giemsa stained for light microscopy observations. (a) prohemocyte. (b) plasmatocyte. (c) granular cell. (d) oenocytoid. scale bar: 5 µm immune response involves synthesis of antimicrobial peptides (amps), phenoloxidase (po) enzymatic cascade and production of reactive oxygen and nitrogen species. amps are secreted proteins with lytic activity such as lysozyme (muramidase) that play an important role in immune defence by performing a hydrolytic action against peptidoglycan of gram positive cell walls (ratcliffe et al., 1985; hultmark, 1996; gillespie et al.,1997; nappi and ottaviani, 2000; callewaert and michiels, 2010; wagner et al., 2015). it is present constitutively at a very low level in the hemolymph and increases upon challenge. the propoactivating system comprises a complex cascade of serine proteases allowing the conversion of propo to po (marmaras and lampropoulou, 2009; gonzález‐santoyo and córdoba‐aguilar, 2012). the po enzymatic system can be triggered by pathogen cell surface molecules such as β-1,3 glucans from fungal cell walls and lipopolysaccharides (lps) and peptidoglycans from microbial cells. this po enzymatic complex converts phenols to quinones that subsequently polymerize to form melanin (gonzález‐santoyo and córdoba‐aguilar, 2012). melanin is involved in physiological processes such as melanisation and sclerotization of cuticle, which improves it ability to act as a physical barrier to invading parasites and pathogens (wilson et al., 2001; dubovskiy et al., 2013). moreover, melanogenesis exerts antimicrobial activity in tissue repair and pathogen sequestration (wounding, clotting, melanotic encapsulation, production of cytotoxic molecules) (söderhäll et al., 1994; marmaras et al., 1996; nappi and vass, 2001; nappi and christensen, 2005; cerenius et al., 2008; gonzález‐santoyo and córdoba‐aguilar, 2012; hillyer, 2016). harpalus (pseudoophonus) rufipes (de geer, 1774) is a generalist predator that acts as a biological control agent of pests, feeding on seeds (honek et al.. 2003, 2005, saska et al., 2008, 2010; talarico et al., 2016; reshetniak et al., 2017) and invertebrates (holland, 2002; monzó et al., 2011; brygadyrenko and reshetniak, 2014) in different crops (holland, 2002; miñarro et al., 2009; monzó et al., 2011). a previous study demonstrated that this beneficial species is sensitive to agricultural management practices, especially the use of herbicides (cavaliere et al., 2019). in spite of their ecological role and sensitivity as exposure indicators, no data are available concerning the cellular and humoral effectors involved in recognition and immobilization of pathogens in the hemolymph of h. rufipes. to evaluate the immune function of h. rufipes adults, we morphologically characterized circulating hemocytes by light and transmission electron microscopy and measured a set of the most common immune markers used in ecological and evolutionary studies to define the constitutive and inducible immune defences of insects. phagocytosis after in vivo artificial non-self challenge with latex beads was analysed as a general measurement of the cellular immune response. the basal and total po and lysozyme-like enzyme activities after in vivo lipopolysaccharide challenge were investigated as immunity markers of the humoral defences. material and methods insects harpalus rufipes adults were collected using in vivo pitfall traps (plastic jars 9 cm in diameter) containing fruit as an attractant. the sampling was performed in a wheat field of 5 ha located on the sila mountain at 1240 m a.s.l., (39°16'58.05"n, 16°38'43.26"e, società cooperativa orti dei monti, torre garga farm, san giovanni in fiore, calabria, southern italy) in spring 2018. in the laboratory, beetles were kept in groups in 10 l plastic boxes filled to a depth of 6 cm with soil from the capture site. the specimens were reared for one week before hemolymph collection with a light regime of l15:d9, 60% r.h. and at a day/night temperature of 24/20 °c. adults fed on homogenized meat and fruit ad libitum. the experimental design was conducted in accordance with all applicable government and institution laws and rules. immune challenges lipopolysaccharide to assess the immune response to lipopolysaccharide (lps) treatments, we used a 10 μl hamilton syringe to inject 4 μl of lps (0.5 mg/ml phosphate buffer) from escherichia coli 0127:b8 (sigma-aldrich, l3129) into the hemocoel of cold anesthetized adults at the dorsal level of the 7th abdominal tergite. twenty-four hours after injection, the hemolymph was collected by 94 puncturing cold anaesthetized adults at the ventral level of the pro-mesothorax articulation with a 29gauge needle. a pool of 10 μl of hemolymph was collected from three specimens, immediately transferred into 90 μl ice-cold sterile phosphatebuffered saline (pbs, 10mm; sigma-aldrich) and centrifuged at 1700g for 5 min at 4 °c. parallel controls were run with a group of non-injected insects. the cell-free hemolymph obtained as supernatant has been collected and stored at -20 °c prior to measure po and lysozyme-like enzyme activities. in vivo phagocytosis to assess the ability of hemocytes to phagocytize, we used a 10 μl hamilton syringe to inject 4 μl of carboxylate-modified polystyrene latex beads (0.9 µm in diameter, aqueous suspension, 10% solids content, sigma-aldrich) diluted 1:10 in 0.15 m sterile pbs into the hemocoel as described above. after injection of latex beads, adults were incubated for 2 hours before microscopy analyses were performed. parallel assays were run with noninjected animals as control. light and transmission electron microscopy to estimate the number of circulating cells per μl of hemolymph (total hemocyte counts, thcs) without distinction of morphology and function, 3 μl of hemolymph were collected from control animals by puncturing cold anaesthetized adults at the ventral level of the pro-mesothorax articulation with a 29-gauge needle. hemocytes were counted in a bürker’s hemocytometer (carlo erba, italy) without dilution and observed with light microscopy (lm) (zeiss primo star) at 400x magnification. thc was expressed as the number of cells (mean±se) per ml of hemolymph. the differential hemocyte count (dhc) was calculated as the relative percentage of different cellular morphotypes circulating in hemolymph for each sample. the hemolymph (3 μl for each beetles) was collected (as described above) from control (n= 14) and latex bead treated (n= 12) beetles and mixed with 5 μl of pbs, spread on a poly-l-lysine coated slide and dried at room temperature for approximately 30 s. during this time, the hemocytes adhered to the glass. cells were then fixed in methanol for 10 min. after natural air-drying of the fixative, hemocytes were stained with giemsa (1:20 in distilled water) for 5 min and slides were rapidly washed with distilled water. we analysed approximately 200 cells per slide with light microscopy (zeiss primo star) at 1000x magnification. for transmission electron microscopy, a pool of 20 μl of hemolymph was collected from five specimens of both latex bead treated and noninjected control groups. beetles were cold anaesthetised and the last two abdominal segments laterally torn; a 29-gauge needle was inserted in the neck membrane and sterile phosphate-buffered saline (pbs, sigma) slowly injected. when the first drop of hemolymph coming out from abdomen of the beetles, it was collected in a microcentrifuge tube containing fixative consisted of 2.5 % glutaraldehyde, 1 % paraformaldehyde and 7.5 % picric acid in 0.1 m phosphate buffer, ph 7.4, with 1.5% sucrose for 2 h at 4 °c. samples have then centrifuged at 1700g for 5 min at 4 °c and the supernatant removed. pellets were rinsed in phosphate buffer, post-fixed with 1% osmium tetroxide in 0.1 m phosphate buffer for 2 h at 4 °c and rinsed in the same buffer. dehydration in a graded acetone series was followed by embedding in epoxy resin (sigma aldrich). ultrathin sections, cut with a pt-pc powertome ultramicrotome (rmc boeckeler), were examined with a jeol jem 1400 plus electron microscope (microscopy and microanalysis centre, cm2, laboratory of transmission electron microscopy university of calabria, italy) at 80 kv. measurements of hemocytes were taken with image-pro plus version 4.5 software (media cybernetics) on digitized images and processed as means±standard error. enzymatic assays phenoloxidase enzyme activity phenoloxidase (po) enzyme activity was measured in cell-free hemolymph of both untreated and lps-injected (treated) animals. basal po activity was assayed spectrophotometrically as the formation of dopachrome from 3,4-dihydroxy-lphenylalanine (l-dopa, sigma-aldrich). to determine the basal po, 10 μl of hemolymph-buffer solution, collected as described above, was mixed with 90 μl of l-dopa (3 mg/ml in pbs) in a microtiter plate. to measure the total po enzyme activity, 10 μl of the hemolymph–pbs mixture was added to 5 μl of bovine pancreas alphachymotrypsin (5 mg/ml pbs, sigma), and incubated for 5 min at room temperature to activate po from its inactive zymogen. subsequently, 85 μl of ldopa was added to the solution. the change in absorbance was recorded at 492 nm and 25 °c for 30 min in 1 min intervals using a plate reader (sirio s, seac). all samples were assayed in duplicate. the enzyme activity was measured as the slope (absorbance vs time) of the reaction curve during the linear phase of the reaction (vmax value; between 0 and 30 min after the reaction began). the slope of the reaction curve at vmax was plotted as absorbance per μl of hemolymph per min for specimens from each group. lysozyme-like enzyme activity lysozyme-like activity was assayed by turbidometric changes in the cell-free hemolymph based on adamo (2004). the decrease in absorbance over time indicates that lysozyme degrades cell walls of the lysozyme-sensitive grampositive bacterium micrococcus lysodeikticus. ten μl of hemolymph-pbs mixture (collection described above) from untreated and lps-injected (treated) animals was loaded into the wells of a 96-well microplate and mixed with 190 μl of a m. lysodeikticus (strain atcc 7468, dsmz) cell wall suspension (approximately 1.6x108 cell/ml of cold pbs). the turbidity reduction in the wells were read on a plate reader (sirio s, seac) at 450 nm and 25 °c for 45 min in 5 min intervals. all samples were assayed in duplicate. the enzyme activity was reported as the change in absorbance (absorbance 95 fig. 2 transmission electron microscopy of h. rufipes hemocytes, control. (a) prohemocyte shows a prominent nucleolus (nu) and high nucleus/cell surface ratio. (b-d) longitudinal sections of plasmatocytes. numerous small electron dense granules (gr) and mitocondria (arrowheads) are present in the cytoplasm. the plasmatic membrane is irregular and prolonged in filopodia (f) or pseudopodia (p). the rough endoplasmic reticulum (rer) is arranged in large cisternae (d-e) surrounding a heterochromatic nucleus with an evident nucleolus (nu) (c). (e) higher magnification of the plasmatocyte showing vesicles of smooth endoplasmic reticulum (ser) in the cytoplasm. (f) cross section of a young plasmatocyte. the cytoplasm contains a low concentration of rough endoplasmic reticulum (rer), free ribosomes, mitochondria, and golgi complex (g). (g) detail (from plasmatocyte in fig. d) of mitochondria with evident cristae. (h) higher magnification showing saccotubular compartment of golgi complex. scale bar: 500 nm (e,g,h) 1 µm (c,d,f), 2 µm (a,b) 96 fig. 3 transmission electron microscopy of h. rufipes hemocytes, control. (a-b) granular cell, longitudinal section. within the cytoplasm are found characteristic large electron dense granules (gr), mitochondria (arrowhead) and rough endoplasmic reticulum (rer) are evident in the cytoplasm. at the level of plasmatic membrane, endo and exocytosis activities are evident (black arrow). the irregular shaped plasmatic membrane is also interrupted by filopodia (f). the nucleus is lobate with densely packed heterochromatin adjacent to the nuclear envelope. (c) granular cell, cross-section. golgi complex and a large electron-dense vesicle (asterisk) are evident. (d) granular cell, detail of the cytoplasmic compartment. the high magnification shows mitochondria (arrowhead) and a granule (asterisk) containing tubular elements. (e) detail of cytoplasmic region of granular cell showing a multivescicolar body (mvb) limiting intraluminal vesicles and many vesicles of smooth endoplasmic reticulum (ser). (f) oenocytoid, transversal section shows the peculiar regular round profile and the homogenous cytoplasm. scale bar: 500 nm (d), 1 µm (b,e), 2 µm (a,c,f) vs time) of the reaction curve during the linear phase of the reaction (vmax value; between 5 and 15 min after the reaction began). the slope of the reaction curve at vmax was plotted as absorbance per μl of hemolymph per min, for both lps-treated and control adults. standards of enzyme activity were made using lysozyme from chicken egg whites (sigma) and a suspension of m. lysodeikticus as substrate. the standards were incubated and recorded simultaneously with the hemolymph samples to confirm that the assay progressed as expected (i.e. absorbance values decreasing). 97 statistical analyses statistical analyses were performed using r version 3.0.1 software (r development core team 2013). all immune parameters were measured (mean ± se) and compared between challenged and control adults. the differences were assessed by non-parametric statistics, i.e,. welch two sample t-test sum test, followed by post-hoc wilcoxon rank sum test pairwise comparisons, with bonferroni correction since the null hypothesis of the bartlett test could not be rejected. results hemocyte types and morphology the total number of circulating hemocytes in hemolymph of h. rufipes was 4.4 ± 0.2 x106 cells/ml (n=15). four morphological types of circulating cells were identified for their size, morphology and dye-staining properties: prohemocytes (figs 1a and 2a), plasmatocytes (figs 1b and 2b-h), granular cells (figs 1c and 3ae) and oenocytoids (figs 1d and 3f). fig. 4 transmission electron microscopy of h. rufipes hemocytes 2 h after in vivo artificial non-self-challenge with latex beads. (a) granular cell phagocytized a large amount of beads and phagosomes containing clusters or single latex beads. (b) higher magnification of cytoplasm. granules (gr) fusing with the phagosome are evident demonstrating their role as primary lysosomes. (c) detail of cytoplasmic region shows phagosome containing latex beads (lb) and large electron dense granules (gr). (d) phagocytizing granular cells show cellular fragmentation and membrane blebs suggesting an apoptotic process. (e) detail of aggregate phagocytizing granular cells becoming to secrete melanised inclusions (in). (f) detail of early aggregation of plasmatocytes in nodule formation. a: autophagosome, arrowheads: mitochondria, white arrows: septate junction, g: golgi complex, n: nucleus, nu: nucleolus. scale bar: 1 µm (b,c,d), 2 µm (a,e,f) 98 fig. 5 transmission electron microscopy of nodules 2 h after in vivo artificial non-self-challenge with latex beads. (a-b) in the outer region of the nodule, plasmatocytes are aggregated around the surface of the nodule. golgi complexes and mitochondria (arrowheads) are evident in the cytoplasm. the euchromatic nucleus (n) indicates a high transcription activity. (c-d) mature nodules show a necrotic core (nc) consisting of latex beads (lb), an extensive melanised matrix and necrotic granular cells (ng) ensheathed by flattened plasmatocytes (pl). (e) detail of cytoplasm showing septate junction (white arrows) between adherent plasmatocytes. (f) detail of golgi complex. (g) higher magnification of nucleolus. dense fibril (df) and granular (gc) components are evident. ch: chromatin. scale bar: 500 nm (c,f), 1 µm (e,g), 2 µm (d), 5 µm (a,b) prohemocytes are the smallest cells found in the hemolymph and display a spherical profile (5 μm x 6 μm in diameter) and an intense blue colour (basophilic) after giemsa staining (fig. 1a). the nucleus almost fills the cell and the nucleus/cell surface ratio is 0.7 in section (fig. 2a). a welldeveloped rough endoplasmic reticulum and small mitochondria occur in the cytoplasm. plasmatocytes are irregularly shaped cells with a maximum diameter up to 13μm (figs. 2b and c). the cytoplasm looks basophilic after giemsa staining (fig. 1b) and ultrastructural analyses show numerous electron dense granules with a mean diameter of 0.34±0.01μm (n=43) (figs 2b-d). the nucleus is large 7 μm x 2 μm, lobed and euchromatic with a prominent large nucleolus (fig. 99 2c). the rough (figs 2d, f and h) and smooth (fig. 2e) endoplasmic reticulum and the golgi complexes (figs 2g,h) are well developed. numerous elongated mitochondria with tubular cristae are observed (fig. 2g). the plasma membrane exhibits irregular pseudopodia (fig. 2b) and filopodia (fig. 2c). granular cells have an elliptical profile with a maximum diameter up to 13 μm (figs. 3a-d). the nucleus is large approximately 6.35μmx2.6μm, lobed and euchromatic with a prominent large nucleolus (fig. 3a-c). after giemsa staining, they exhibit a homogeneous cytoplasm containing acidophilic granulations (fig.1c). these granules are variable in electron density with a round irregular to elliptical profile and have a mean diameter of 0.55±0.02μm (n=58) (figs. 3a-d and e). in some cases, structured granules (fig. 3d) contains tubular elements. the cytoplasm contains rough endoplasmic reticulum, golgi complexes and elongated mitochondria (figs 3a-c). multivesicular bodies (mvbs, about 1 μm in diameter) were clearly identifiable (from 2 to 4 mvbs per section) adjacent to the plasma membrane trapping numerous intraluminal vesicles (69±12 nm in diameter, n =22) (fig. 3e). oenocytoids are rare compared to the other cell types encountered in the hemolymph (fig. 3f). they are round cells, approximately 16 μm x 23 μm in diameter, characterized by an eccentric nucleus. the cytoplasm has few organelles, although small oval mitochondria, free ribosomes, numerous polysomes and a rough endoplasmic reticulum are sometimes present. after giemsa staining, the oenocytoids exhibit a mild basophilic cytoplasm and a strong basophilic nucleus (fig. 1d). in vivo phagocytosis assay after 2 h in vivo artificial non-self-challenge with latex beads, granular cells mounted a strong and rapid phagocytic response. they present up to 40 phagocytized beads within the cytoplasm and become enlarged (figs 4a, b and d). phagocytizing cells occurred as either isolated (figs 4a, b and c) or aggregate cells (fig. 4e). granules fusing with a phagosome are evident, demonstrating their role as primary lysosomes (fig. 4b). extensive plasma membrane blebbing followed by separation of cell fragments into apoptotic bodies were found in some granular cells having their cytoplasm filled with a high numbers of latex beads (fig. 4d). melanised nodules are found in the hemolymph entrapping a large number of beads 2h after the challenge (figs 5a-d). each nodule has a necrotic core (figs 5c and d) consisting of latex beads embedded in an extensive melanised matrix and necrotic granular cells (fig. 5d) covered by a layer of flattened plasmatocytes (figs 4f, 5a, b). septate junctions are evident between plasmatocytes that aggregate around the surface of the nodules (figs 4f; 5a, b, and e). moreover, numerous mitochondria and wellstructured golgi complexes are present in the cytoplasm (figs 5a, b and f) as well as a large euchromatic nucleus with an evident nucleolus (figs 5b and g). differential hemocyte counts (dhcs) the dhcs performed in control beetles showed that plasmatocytes (67.15 ± 2.52 %) and granular cells (29.32 ± 2.35 %) are the main circulating hemocyte types, while prohemocytes (0.52 ± 0.11 %) and oenocytoids (0.73 ± 0.02 %) are rare in hemolymph (table 1). after in vivo artificial non-selfchallenge with latex beads, the relative percentage of granular cells (82.74 ± 2.54 %; wilcoxon rank sum test, p= 1.74310-5) increases significantly in hemolymph and 54.31 ± 2.88 % of them are phagocytizing cells. moreover, a significant decrease in the relative percentage of plasmatocytes (12.38 ± 2.21 %; wilcoxon rank sum test, p= 2.071 x 10-7) and oenocytoids (0.16 ± 0.05 %; wilcoxon rank sum test, p= 0.00797) are recorded. no significant differences are remarked in prohemocytes between control and latex-treated beetles (wilcoxon rank sum test, p = 0.1725). in the dhcs, cells with intermediate features (figs 2c, 3a) and hemocytes showing features of mitotic process (figs. 6a-e) are also observed in both control and latex bead treated specimens (“not determined” group in table 1). however, the increase of their relative percentage from 2.28 ± 0.37 % (ctrl) to 4.44 ± 0.95 % (latex) of circulating hemocyte after latex bead challenge is not significant (wilcoxon rank sum test, p = 0.4105). table 1 relative percentages of circulating hemocyte types after latex bead challenge (latex; n = 12) compared with the non-injected (ctrl; n = 14) beetles note. values are expressed as mean percentage ± standard error; significance ascribed as **p-value < 0.01 and ***p-value < 0.001 versus control prohemocytes plasmatocytes granular cells phagocytizing oenocytoids not determined ctrl 0.52 ± 0.11 67 ± 2.52 29.32 ± 2.35 0.73 ± 0.15 2.28 ± 0.37 latex 0.28 ± 0.11 12.38 ± 2.21*** 82.74 ± 2.54*** 54.31 ± 2.88 0.16 ± 0.08** 4.44 ± 0.95 100 fig. 6 mitotic hemocytes in adults of h. rufipes giemsa stained for light microscopy observation. (a) metaphase hemocyte. (b) anaphase. (c and d) telophase. (e) cytokinesis. scale bar: 10 µm po activity plasmatic basal po activity was significantly higher (wilcoxon rank sum test, p = 0.01828) in the lps-treated adults (0.007 ± 0.002 abs/μl/min; n=11) than that of untreated ones (0.002 ± 0.0005 abs/μl/min; n=18; wilcoxon rank sum test) (fig. 7). no differences were observed in the plasmatic total po activity (wilcoxon rank sum test, p = 0.6109) between lps-treated (0.005 ± 0.002 abs/μl/min; n=11) and untreated (0.005 ± 0.0007 abs/μl/min; n=18) specimens. lysozyme-like enzyme activities the baseline lytic activity of hemolymph was not significantly higher (wilcoxon rank sum test, p = 0.1132) in the lps-treated adults (0.0012 ± 0.0003 abs/μl/min; n=13) compared with untreated adults (0.0008 ± 0.0003 abs/μl/min; n=18) (fig. 7). discussion in the current study, we provide the first description of the immunocompetence in the generalist predator h. rufipes. based on light microscopy observations and ultrastructural analyses, we characterized four different types of circulating hemocytes: prohemocytes, plasmatocytes, granular cells and oenocytoids. the dhcs showed that plasmatocytes and granular cells are the most abundant circulating hemocytes. according to previous study (ribeiro and brehélin, 2006; martins and ramalho-ortigao, 2012), circulating oenocytoids are large cells with a low nuclear to cytoplasmic ratio. the thc levels are in agreement with previous studies on circulating hemocytes of other coleoptera species such as carabus lefebvrei (12 x 106 cells/ml; giglio et al., 2008), rhynchophorus ferrugineus (3,85 5,2 x106 cells/ml; manachini et al., 2011), dicladispa armigera (5,9 x 106 cells/ml; phukan et al., 2008). morphology and function of hemocytes have been extensively investigated in insects. however, it lacks a unified terminology to indicate types and little is known about coleopteran species. distinct classes such as prohemocytes, plasmatocytes, granular cells, coagulaocytes, oenocytoides and spherulocytes are identified in hemolymph of tenebrionidae (zhao and wang, 1992), coccinellidae (firlej et al., 2012), curculionidae (manachini et al., 2011), melolonthidae (akai and sato, 1979; gupta, 1979), scarabeaidae (giulianini et al., 2003) and chrysomelidae (phukan et al., 2008). the low number of cellular subpopulations and hemocytes with intermediate feature and mitotic cells identified in h. rufipes, indicate proliferation and differentiation activities in circulating hemocytes of the adult stage. further investigation can clarify if new circulating hemocytes in h. rufipes derive from germinal cells named prohemocytes and perform separate functions (ottaviani, 2005; strand, 2008; hillyer, 2016) or arise from replication of mature granulocytes such as in mosquitoes (king and hillyer 2013). besides, morphotype has the ability to turn into another type as observed in vitro in plasmatocytes of periplaneta americana, galleria mellonella and tenebrio molitor (gupta and sutherland, 1966) and in prohemocytes of bombyx mori (yamashita and iwabuchi, 2001). from an evolutionary ecology perspective, the immunocompetence of a species is a plastic life trait with costs of both maintenance and activation. ecological factors influence the diversity and complexity of the cellular and humoral immune responses among different taxa exposed to a wider range of pathogens exerting stronger selection on the immune system. we performed immune challenges with latex beads and lps to test separately the specificity of the cellular and humoral immune response in h. rufipes. our results indicate that the dynamics of cellular and humoral responses in this species are highly adapted to maximize fitness. h. rufipes engages a rapid, non-specific constitutive immune response to fight off pathogens. phagocytosis is the primary, rapid, non-specific, constitutive response of hemocytes to small particles such as bacteria. granular cells and plasmatocytes have been reported to be the predominant phagocytic cell in insects (gillespie et al., 1997). plasmatocytes carry out phagocytosis in diptera (lemaitre and hoffmann, 2007; govind, 2008; hillyer and strand, 2014; honti et al., 2014) and lepidoptera (ribeiro and brehélin, 2006). granular cells phagocytose pathogens in the larval stage of the coleopteran allomyrina dichotoma (hwang et al., 2015). furthermore, granular cells work together with other cell types to perform phagocytosis, oenocytoids in cetonischema aeruginosa (drury) larvae (giulianini 101 fig. 7 the basal and total po activities and lysozyme-like enzyme activity in non-injected (control) adults and treated one with lps for 24 h. the enzyme activity (slope) was recorded as absorbance units for μl of hemolymph per min. the box represents the interquartile range (iqr = q3 q1) and bars represent first (q1, top) and third quartiles (q3, bottom) of enzyme activity values from untreated and lps-treated beetles. the central horizontal black line indicates the median. the ends of dashed lines (ends of the whiskers) represent the lowest (minimum) datum and the highest (maximum) datum et al., 2003) or plasmatocytes in the larvae of red palm weevil rhynchophorus ferrugineus (olivier) (manachini et al., 2011) and in adults of melolontha melolontha (brehelin and zachary, 1986) and harmonia axyridis (firlej et al., 2012). in the adults of carabids, previous studies showed that plasmatocytes are involved in phagocytosis (giglio et al., 2008, 2015; giglio and giulianini, 2013). however, after artificial non-self challenge with latex beads, granular cells in h. rufipes exhibit a high degree of latex bead sequestration. moreover, the challenge elicits a change in the relative percentage of hemocyte subpopulations. the number of granular cells increases and they form aggregates, while the reduction of circulating plasmatocytes may be the result of their recruitment in the nodule formation that is a more efficient clearance mechanism for high concentrations of invaders. these findings show that there is a variability among insect taxa in the coordinated response against pathogens involving different subpopulations of hemocytes in phagocytosis 102 (hillyer, 2016) because each species modulates the specificity of its immune responses under the selective pressure of its particular environment (schmid-hempel, 2005; siva-jothy et al., 2005; sadd and schmid‐hempel, 2009). plasmatic po and lysozyme-like enzyme activities are two immune markers assayed to evaluate the disease resistance in insects (adamo, 2004). they are components of the induced response that differ in specificity and are only deployed after an invasive pathogen has been recognized. lps is a pathogen-associated molecular pattern (pamp) and triggers propo activation to provide melanin synthesis that may be involved in several physiological processes, e.g., cuticle sclerotization, wound healing and killing of entrapped parasites or pathogens with a high activating cost for the organism. the challenge with lps from e. coli elicited a significant increase of the basal po enzyme activity in hemolymph of h. rufipes, but it had no significant effect on total po and lytic activity 24 h after inoculation. since lysozyme acts mainly on the peptidoglycan, we expected lps has a moderate effect on lysozymelike enzyme activity. because the low number of beetles collected, it lacks mock-injected controls to validate fully our preliminary results. however, previous studies have shown that po activity displays a dynamic and complex activity after challenges with immune elicitors (korner and schmid-hempel, 2004; charles and killian, 2015). thus, we assume that the low level of total po recorded in h. rufipes may depend on the time posttreatment that the measurements are obtained. further investigation will clarify if the induction of the systematic propo cascade may produce through the time several highly reactive (ros) and toxic compounds that contribute in resistance to pathogens. in conclusion, our preliminary findings will provide a baseline information for further investigation on pathogen resistance and plasticity in this carabid model. moreover, the modulation of constitutive and induced immune responses may be used as biomarkers of exposure in an ecotoxicological context. acknowledgement the authors thank dr pietro tarasi (società cooperativa orti dei monti, torre garga farm) for making fields available for sampling for this study, dr antonio mazzei for the taxonomic identification of species. this work was supported by the ministero dell’ambiente e della tutela del territorio e del mare – ente parco nazionale della sila (calabria, italy) (grant no. 2678, xiii/4). references adamo sa. estimating disease resistance in insects: phenoloxidase and lysozyme-like activity and disease resistance in the cricket gryllus texensis. j. insect physiol. 50: 209–216, 2004. akai h, sato s. surface and internal ultrastructure of hemocytes of some insects. in: gupta ap (ed) 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reaction against the larvae of macracanthorhynchus hirudinaceus in laboratory-infected beetles. j. parasitol. 70(6): 1098–1101, 1992. microsoft word isj407.doc 94 isj 13: 94-101, 2016 issn 1824-307x research report dynamics of hemocyte subsets from ascidian halocynthia aurantium in response to tissue damage: a comparative analysis of flow cytometry vs confocal microscopy data an sukhachev1, is dyachkov1, ne zyumchenko2, iv kudryavtsev1,2, av polevshchikov1,2 1institute of experimental medicine, saint petersburg, russia 2far eastern federal university, vladivostok, russia accepted march 21, 2016 abstract the current study was aimed at investigating the dynamics of circulatory ascidian hemocyte subsets from halocynthia aurantium in response to tunic damage. by using flow cytometry and confocal microscopy, it was demonstrated that the relative amount of hemoblasts and hyaline amebocytes was increased 24 h after cutting ascidian tunic and subjacent muscle layer. by applying a broad panel of fluorescently labeled monoclonal antibodies against human adhesion molecules expressed by lymphoid and stem cells both assays allowed to detect two cross-reactive epitopes (cd54 and cd90) on the surface of ascidian hemocytes. upon that, the expression of cd54-like epitope was found to be downregulated on ascidian cells after tissue damage, whereas binding to cd90-like epitope was upregulated in all examined cell subsets. key words: ascidian; hemocytes; flow cytometry; confocal microscopy; tunic damage   introduction dynamics and renewal of cell subsets in multicellular organisms are among the basic issues of current biology. most explicitly, the importance of such scientific area is related to the discovery, the investigation and the potential clinical use of human and animal circulatory stem cells. in the meantime, a question regarding tissue-specific stem cells and dynamics of cell subsets in multicellular organisms was most clearly raised in fundamental studies done by zavarzin (zavarzin, 1953). there is an important evolutionary aspect in examining dynamics of cell subsets and mechanisms underlying functioning of stem cell systems. whereas stem cells of various potency in invertebrates are mainly amassed as tissue-specific regeneration nests, nodes and other structures, chordates and particularly vertebrates possess tissue-specific oligopotent stem cells as well as new system of circulatory pluriand multipotent stem cells displaying high plasticity and regenerative potential. this led to changes in tissue reparation process resulting from overall increase in level of organization of all chordates, particularly vertebrates, with more pronounced tissue ___________________________________________________________________________ corresponding author: alexander polevshchikov institute of experimental medicine 12 acad. pavlov str., 197376 saint petersburg, russia e-mail: alexpol512@yandex.ru specialization, but, mainly, from the appearance of close circulatory system. directed trafficking of circulatory stem cells is accompanied by increasing role of body integrative systems, enhanced nervous regulation, establishing network of soluble cytokines and growth factors, which are specifically demonstrated in models of tissue reparation caused by various insults. according to this viewpoint, investigation of dynamic changes in ascidian circulatory hemocyte subsets may provide an important evidence on the initial stages of developing system of mobile stem cells. in the larva stage, ascidians have close circulatory system, however it seems possible that the transition to a sedentary lifestyle of these benthic filter-feeders, coupled with loss of close circulatory system, operational principles of stem cell system are not altered (polevshchikov et al., 2005). this is one of the reasons why, over the years, ascidians have been extensively used for examining specialization of circulatory cells (sukhachev et al., 2013) as well as their involvement in defense and reparative reactions (smith, 1970). our study was aimed at analyzing dynamic changes in circulatory hemocyte subsets during response to tunic damage of the solitary ascidian halocynthia aurantium from the sea of japan, which were assessed by flow cytometry and confocal microscopy. 95 table 1 changes in the percentage of subsets of circulatory hemocytes isolated from ascidian h. aurantium in response to tunic damage assessed by flow cytometry hemocyte subset control 24 h after hemoblasts and hyaline amebocytes 12.04±0.66 14.98±0.93* macrophage-like cells 13.60±0.59 10.90±2.35 morula cells and granulocytes 66.96±1.86 64.15±3.35 total identified hemocytes 92.60 90.03 *hereinafter, significant differences at 24 h after tunic damage vs. control group are marked by asterisks (*: p < 0.05). data were analyzed by using student’s t-test and are presented as mean ± s.e.m., n ≥ 24 at each time point. materials and methods animals the solitary ascidian halocynthia aurantium (stolidobranchia: pyuridae) was used in experiments. animals were harvested during julyaugust 2013 2015 at the biological station “vostok”, marine biology institute, far east branch of the russian academy of science. prior to the study, animals were kept for 4 days in aquarium with running seawater and forced aeration. totally, more than 120 animals were used during the study. model of ascidian tunic damage tissue injury consisted of a standardized cutting (length: 1 cm, depth: 0.5 cm) on tunic and subjacent tissues including the wall of the circulatory. after the 1st tunic damage 9 ml of flowing from the wound hemolymph was immediately collected from each specimen (0 h, control) into test tubes with 30 mm edta (sigmaaldrich, usa) prepared in filtered seawater for preventing hemocyte clotting. to examine dynamics in circulatory cell subsets, 24 h later each ascidian repeatedly received another cutting followed by hemolymph collection and the same volume of hemolymph was collected. cell suspension was centrifuged at 100g for 10 min 4 ºс followed by washing in dulbecco’s phosphate buffered saline (paneco, russia), without calcium chloride and magnesium chloride, ph 6.0, containing 34 g/l nacl that matches its concentration in water from sea of japan. final cell concentration after washing was 107 cell per 1 ml of modified dulbecco’s solution. flow cytometry for performing flow cytometry assay, 500 μl of cell suspension (5×106 cells) were transferred into eppendorf tube and added with equal volume of cooled (4 °с) 8 %-formalin (sigma-aldrich, usa) for storage at 4 °с until use. cell samples were analyzed by flow cytometry on facscalibur™ (becton dickinson, usa) equipped with cellquest pro software, according to the earlier described method (sukhachev et al., 2015). cross-reactivity of surface antigenic epitopes of hemocytes from h. aurantium and adhesion molecules expressed by human peripheral blood leukocytes was evaluated using to the above-described method by using a panel of monoclonal mouse, allophycocyaninconjugated, anti-human antibodies (mabs) against cd166 (alcam, activated leukocyte cell adhesion molecule), cd117 (с-kit), cd29 (integrin β1-chain), cd34 (adhesion molecule typical of mammalian hematopoietic stem cells), cd15 (3-fucosyl-nacetylglucosamine), cd54 (icam-1, intercellular adhesion molecule), cd62l (l-selectin), cd62p (рselectin), cd90 (evolutionarily conserved thy-1 antigen, typical of nervous system and differentiating t cells), cd94 (c-type lectin found within nkg2 heterodimers of mammalian nk cells). allophycocyanin-conjugated mopc-21 immunoglobulin (all mabs from bd biosciences, usa) was used as an isotype-match control antibody for determining the extent of non-specific binding. ascidian cells were incubated with antibodies at concentrations recommended by the manufacturer. for the assay, 100 μl of cell suspension from each animal, harvested at different time points, were incubated with the primary antibody, in microtubes, for 3 h in the dark at rt (20 25 °с) followed by adding 500 μl of bd facs fixative solution (bd biosciences, usa) according to the manufacturer’s instructions. in order to properly exclude all events, which did not match morphometric parameters of ascidian hemocytes (by size and granularity) from analysis, gating strategy was applied based on particle distribution according to forward and side scattering. fluorescence data were summarized per 10,000 cells gated according to their size and granularity. the expression of antigenic epitopes crossreactive with anti-human abs raised against cd15, cd29, cd34, cd54, cd62l, cd62p, cd90, cd94, cd117, and cd166 was presented as a percentage of cells stained with specific abs as revealed by fluorescence of higher intensity than the upper threshold of isotype-match control antibody staining. 96 fig. 1 binding of anti-human cd54 mabs with ascidian h. aurantium hemocytes in response to tissue damage assessed by flow cytometry. staining of the cells with: a antibody of isotype control, b 0 h, c 24 h after tunic damage. m1-gate staining with isotype-match control antibody, m2-gate cd54-positive cells. x-axis: fl4 channel (logarithmic scale); y-axis: number of events (cells). confocal microscopy confocal microscopy was used to detect surface adhesion molecules using the panel of abs mentioned above. briefly, after washing, cell suspension was placed on the culture slides bd falcontm (becton dickinson, usa) followed by sedimenting the cells for 2 h in the dark at rt. hemocytes were then incubated with 50 μl of diluted antibodies and incubated for 2 h in the dark. after that, specimens were washed with phosphatebuffered saline followed by fixing with 4 %-formalin solution. immediately before analysis, specimens were additionally stained with dapi (sigma, usa) and mounted with mowiol. specimens were analyzed by using confocal microscope zeiss lsm 510 meta (germany) and lsm 510 (release version 4.2) software. additionally, image processing was performed using image browser software. fluorochrome-conjugated abs were excited by two lasers with wavelength 364 and 633 nm, and signals were detected using 435 485 nm and 650 704 nm fluorescence filters for dapi and allophycocyanin, respectively. spectrum channel pmt1 and pmt2, for detecting dapi and allophycocyanin, respectively, were coded by blue and red color. overall, >150 images including phase-contrast imaging were taken at magnification 40x as well as 63x for oil-immersion microscopy. statistical analysis statistical analysis was performed according to previous recommendations. data were presented as mean ± sem. the statistical analysis was made using statistic 7.0 software (statsoft, usa). results are presented as the mean and sem. the significance of differences was assessed using student's t-test. significance level was set at p < 0,05. results and discussion previously, by applying standard morphology methods (sukhachev et al., 2013) and flow cytometry (sukhachev et al., 2015) 5 main subsets of circulatory hemocytes, in ascidian h. aurantium were described: hemoblasts, granulocytes, hyaline amoebocytes, macrophage-like and morula cells. during the current study, we assessed dynamics of the major hemocyte subsets in response to tissue injury 24 h after tunic damage in the same species (table 1). it was demonstrated, that more than 90 % of circulatory hemocytes were available for phenotyping and included into analysis, which is a good yield given technical limitations of fig. 2 analysis of сd54-like marker expression in total population of circulatory hemocytes assessed by flow cytometry. x-axis: time after tunic damage; y-axis: percentage of positive cells. bars: black the cells stained with antibody of isotype control, gray the cells stained with mabs against cell marker. data expressed as mean ± s.e.m., n ≥ 12. 97 fig. 3 binding of anti-human cd54 abs to circulating hemocytes from the ascidian h. aurantium, assessed by confocal microscopy and stained with apc-conjugated abs (650 704 nm, red color) and dapi (435 485nm, blue color). left: 0 h; right: 24 h after tunic damage. flow cytometry. moreover, it was found that percentage of hemoblasts and morphologically similar hyaline amebocytes was significantly increased in the circulation 24 h after tunic damage. conversely, the amount of circulating macrophagelike and morula cells as well as granulocytes was only slightly, not significantly, decreased, probably due to their role in thrombogenesis. the capacity of antibodies to cross-react with homologous or analogous epitopes on hemocytes was examined in both intact and operated ascidians. in the last case, tissue repair was triggered tissue reparation. no binding to any hemocyte subset from h. aurantium was detected by flow cytometry or confocal microscopy was observed with the five mabs (cd15, cd62l, cd62p, cd94, cd166) used during the experiments. in the case of two abs (cd54 and cd90), binding to ascidian hemocytes was observed in both assays. however, the dynamics of expression of both the surface epitopes detected by these abs was different: the expression of cd54-like epitope was decreased after tissue damage, whereas, for cd90-like epitope, labelling was upregulated on all examined hemocyte subsets after tissue damage. cd54 (icam-1) belongs to immunoglobulin superfamily and is involved in the interaction between antigen-presenting cells and naïve t cells. molecules from immunoglobulin superfamily were unequivocally confirmed to be expressed in ascidians (dehal et al., 2002; сima et al., 2004). in addition, it was firmly established that ascidians lack tcr and bcr (dehal et al., 2002). therefore, in case of using monoclonal antibody against human icam-1 apparently it might be correct to interpret such data as detection of cross-reactive epitope on some hemocyte surface molecule belonging to immunoglobulin superfamily or containing, along with lectin, one or more immunoglobulin domains. moreover, a search for cd54-like molecules in ascidians has never been done before with antihuman cd54 abs (figs 1 3). table 2 dynamics of cd54and cd90-positive hemocytes isolated from ascidian h. aurantium in response to tunic damage assessed by flow cytometry cd54 cd90 hemocyte subset control 24 h after control 24 h after hemoblasts and hyaline amebocytes 1.76±0.52 5.19±0.88* 19.10±4.85 42.56±3.77* macrophage-like cells 2.79±0.71 8.79±1.81* 29.18±5.23 64.00±3.66* morula cells and granulocytes 8.24±1.46 3.52±0.62* 34.03±3.21 51.96±3.30* all comments as in table 1. 98 fig. 4 binding of anti-human cd90 to hemocytes from the ascidian h. aurantium in response to tunic damage assessed by flow cytometry. figure captions as in figure 1 expression level for molecule bearing epitope cross-reactive with human icam-1 was found to be at low level in intact animals (m2-gate, fig. 1b), which was downregulated by ~2-fold after tunic damage (fig. 2): intact ascidians contained 7.14 ± 1.04 %, 24 h later after tissue damage its expression declined down to 4.06 % ± 0.59 % out of total hemocyte population. by using confocal microscopy it was shown that intact ascidians contained 9.67 % ± 2.19 % icam-1-like-positive cells, whereas 24 h later after tissue damage its percentage decreased to 7.33 ± 3.84 %. it should be noted that a huge amount of granulocytes and morula cells was lost after placing them on the slides, whereas this preparation technique did not affect amount of phagocytes and hemoblasts by allowing to detect them at full amount. therefore, the quantitative data obtained by flow cytometry and confocal microscopy due to the features of these assays must be compared with caution. in addition, flow cytometry allowed assessing dynamic changes in binding of anti-human cd54 abs to the earlier described phenotypic subsets of hemocytes from h. aurantium. in particular, it was found that subset-specific dynamics in expression of cd54-like marker differed from that one described in total hemocyte population (table 2). morula cells were found to represent the majority of cd54positive cells (8.24 ± 1.46 %), whereas hemoblasts and hyaline amebocytes expressed this marker at the lowest level (0.23 ± 0.06 %). importantly, 24 h after the onset of tissue damage, only 3.52 ± 0.62 % of morula cells were positive to anti-cd54 antibody (p < 0.05), whereas in undifferentiated hemoblasts the percentage of positive cells increased from 1.76 ± 0.52 % up to 5.19 ± 0.88 % (p < 0.01), and in macrophage-like cells from 2.79 ± 0,71 % up to 8.79 ± 1.81 % (p < 0.01). by taking into consideration, that morula cells is the most abundant cell subset among circulatory hemocytes in h.aurantium (~66 % out of total cells, according to previously published data (sukhachev et al., 2015), it may be concluded that the decreased binding of anti-human abs to cd54 in total hemocyte population may be of importance, as this was exactly the hemocyte subset containing the highest amount of positive cells, therefore, being responsible for overall drop in expression level, despite the fact that its expression was shown to be upregulated in other hemocyte subsets. it seems essential that such cd54-like molecule most likely represents an analogue of adhesion molecule involved in inter-cellular interactions. in human, anti-cd90 abs bind to thy-1 homologue, which is typical of thymocytes and determines t-cell lineage differentiation. therefore, a search for a molecule with cross-reactive epitope on ascidian hemocytes by anti-cd90 abs is of interest in terms of evolutionary histology and comparative immunology. anti-human cd90 ab recognized a great number of hemocytes (figs 4 6) fig. 5 analysis of сd90-like marker expression in total population of circulatory hemocytes assessed by flow cytometry. figure captions as in figure 2. 99 fig. 6 binding of anti-human cd90 abs to circulating hemocytes from the ascidian h. aurantium assessed by confocal microscopy. figure captions as in figure 3. which, in intact animals, amounted to 30.79 ± 2.78 %, it rose up to 46.33 ± 2.85 % (p < 0.001) of total circulatory hemocytes (figs 4, 5). similar tendency was observed on specimens for confocal microscopy, wherein percentage of cd90-likepositive cells increased from 46.66 ± 19.06 % up to 70.00 ± 2.45 % (fig. 6). antibody against human cd90 was shown to bind to the all examined ascidian cell subsets. nonetheless, the amount of morula cells bearing cd90 cross-reactive epitope was 34.03 ± 3.21 %, i.e., 1.78and 1.17-fold higher than of hemoblasts, hyaline amebocytes and macrophage-like cells compared to total hemocytes (table 2). moreover, 24 h after tunic damage the percentage of cd90+like cells was upregulated in all examined cell subsets. analyzing the percentage of cd90+-like cells in various hemocyte subsets, it was found that it increased by 2.1-, 2.2and 1.8-fold in hemoblasts, macrophage-like and morula cells, respectively. this suggests both the direct participation of cd90 homologue/analogue and the cells expressing it in tissue repair as well as the presence, in ascidians, of evolutionary t cell precursors, which are involved in reparative responses. finally, three other mabs (cd29, cd34, cd117) used in our study provided an incomplete match between the data obtained by both the assays. whereas flow cytometry detected a very low binding for such abs, confocal microscopy allowed to observe positive staining only in some of the cells. however, in this case positive signal was detected not only along the contour of the cells, as shown for anti-cd90 abs (fig. 6), but on the entire area of the cells as exemplified for anti-cd29 abs (fig. 7). probably, the fluorescent staining detected by confocal microscopy, reflects the higher sensitivity of this approach as well as previously described autofluorescence emitted by granules of morula cells (sukhachev et al., 2015). in addition, one should not exclude the possibility of interaction between antibodies and epitopes located inside the cell cytosol, which, however, should be further investigated in detail. at least five major cell subsets were detected in the circulation of the ascidian h. aurantium. among them, hemoblasts, hyaline amebocytes, macrophage-like and morula cells (sukhachev et al., 2013). in terms of morphology, hemoblasts from h. aurantium virtually did not differ from hemoblasts of other ascidian species. in all tunicates, hemoblasts are characterized by a high nucleuscytoplasm ratio, rarely exceeding 5 μm in diameter (hirose et al., 2003). in addition, it is worth mentioning that morphologically ascidian hemoblasts are similar to mammalian lymphocytes so that many investigators call them as lymphocytelike cells (fuke, 2001). in response to tunic damage, an increased amount of hemoblasts as well as hyaline amebocytes, which might be histogenetically related to them (ballarin and cima, 2005), was observed in circulation. recently, flow cytometry was widely applied in various scientific areas (kudryavtsev et al., 2012). annually, a rising number of studies dedicated to investigating circulatory cells of invertebrates and lower vertebrates is published. during our study, we applied flow cytometry to examine hemocytes from the ascidian h. aurantium. we found that flow cytometry data on examined cell subsets are in good agreement with the results obtained by light microscopy and data published elsewhere (hirose et al., 2003; sukhachev et al., 2013, 2015). however, whether hemoblasts are able to differentiate only into circulatory cells or they represent stem cells with a broader range of potential lineage-specific transitions still remains unclear. at present, an open question is whether 100 fig. 7 binding of anti-human cd29 abs to circulating hemocytes from the ascidian h. aurantium assessed by confocal microscopy. figure captions as in figure 3. ascidians contain circulatory pluripotent stem cells as well as oligopotent stem cells in various tissues. however, it is generally accepted that hemoblasts form different morphotypes of circulatory cells and that the latter (e.g., morula cells) may take part in formation of distinct tissue structures, e.g., tunic layers (smith, 1970; smith and peddie, 1992; ballarin and cima, 2005). amount of morula cells as the most abundant subset of circulatory hemocytes decreases after tissue injury, because they are responsible for “wound sealing” (smith and peddie, 1992). altogether, it clearly justified the reason for performing quite a broad examination of changes in adhesion molecule repertoire on hemocytes in response to tissue damage. it is evident that response to tunic damage coupled with extensive loss of circulatory cells was paralleled with significant downregulation of these markers on morula cells and granulocytes. on the conversely, the only exclusion from this rule was the upregulated expression of the marker bearing epitope that cross-reacted with anti-human cd90 abs. quite possibly, upregulated expression on all examined hemocyte subsets of a molecule crossreacting with anti-cd90 abs exactly reflects an enhanced intercellular cooperation in response to tissue damage. by taking into consideration that morula cells and granulocytes are the most differentiated subsets of hemocyte population (chaga, 1998) as well as the fact that they are involved in the recovery of ascidian tunic in response to tissue damage these cell types were consumed in vivo much faster compared to the other ones. upon that, flow cytometry data showed that percentage of these cell subsets was insignificantly decreased (table 1). thus, by using flow cytometry and confocal microscopy, it was suggested that the relative level of hemoblasts and hyaline amebocytes was increased in response to tunic damage in ascidian h. aurantium. this process was accompanied by altered expression repertoire of membrane receptors, wherein some of them bear antigenic epitopes cross-reactive with antibodies against adhesion molecules of human lymphoid (cd54, cd90) and stem cells (cd90). however, in order to unequivocally conclude that ascidians possess an established system of circulatory stem cells, it is necessary to assess proliferative activity of stem cells both in circulation and at site of tissue damage. acknowledgements this study was supported by program №1326 of the ministry of education and science, russian federation, and grant of the russian fund of basic researches (№15-04-05093). references ballarin l, cima f. cytochemical properties of botryllus schlosseri haemocytes: indications for morpho-functional characterization. eur. j. histochem. 49: 255-264, 2005. chaga oyu. blood cells from ascidian styela (goniocarpa) rustica. i. histology analysis. tsitologiya 40: 31-44, 1998 (paper in russian). cima f. sabbadin a, ballarin l. cellular aspects of allorecognition in the compound ascidian botryllus schlosseri. dev. comp. immunol. 28: 881-889, 2004. dehal p, satou y, campbell rk, chapman j, degnan b, de tomaso a et al. the draft genome of ciona intestinalis: insights into chordate and vertebrate origins. science 298: 2157-2167, 2002. 101 fuke m. cell types involved in allogeneic contact reactions of the solitary ascidian, halocynthia roretzi. zool. sci. 18: 195-205, 2001. hirose e, shirae m, saito y. ultrastructures and classification of circulating hemocytes in 9 botryllid ascidians (chordata: ascidiacea). zool. science 20: 647-656, 2003. kudryavtsev iv, khaydukov sv, zurochka av, chereshev va. flow cytometry in experimental biology. ekaterinburg: rio uro ran, p.192, 2012 (paper in russian). polevshchikov av, kudryavtsev iv, dyachkov is, kharazova ad. theory of tissue parallelism in comparative immunology. vestnik. sanktpeterburgskogo universiteta 3: 68-74, 2005 (paper in russian). smith mj. the blood cells and tunic of the ascidian halocynthia aurantium (pallas). i. hematology, tunic morphology, and partition of cells between blood and tunic. biol. bull. 138: 354-378, 1970. smith vj, peddie cm. cell cooperation during host defense in the solitary tunicate ciona intestinalis (l). biol. bull. 183: 211-219, 1992. sukhachev an, dyachkov is, romanyuk ds, kumeiko vv, sinitsina vf, korolkova ed, et al. morphology analysis of hemocytes from ascidian halocynthia aurantium. tsitologiya 55: 901-906, 2013 (paper in russian). sukhachev an, dyachkov is, kudryavtsev iv, kumeiko vv, tsibulsky av, polevshchikov av. experience of using flow cytometry for analysis of circulatory hemocyte subsets from ascidian halocynthia aurantium (pallas, 1787). j. evol. biochem. physiol. 51: 246-253, 2015. zavarzin aa. essays on evolutionary histology of blood and connective tissue. moscow leningrad: ussr academic publishing 4: 720, 1953 (paper in russian). isj 12: 166-169, 2015 isj 12: 166-169, 2015 issn 1824-307x letter to editor insect anti-viral immunity: roles of prostaglandins and other eicosanoids d stanley1, l zhang2, y kim3 1usda/agricultural research service, biological control of insects research laboratory 2state key laboratory for biology of plant disease and insect pest, institute of plant protection, chinese academy of agricultural sciences, beijing, china 3department of bioresource sciences, andong national university, andong, korea accepted may 19, 2015 to the editor insect/microbe relationships are very complex, with an array of signaling systems acting in surveillance, detection and responses to the presence of beneficial, neutral and harmful microbial species within insects. the emerging view indicates prostaglandins (pgs) and other eicosanoids are responsible for essential signaling in activating and coordinating insect innate immune reactions to infections, including viral infections. the structures, biosynthesis and history of eicosanoids are detailed elsewhere (stanley and kim, 2014). reports of pg actions in insects emerged in the 1970s. loher (1979) reported that pge2 releases egg-laying behavior in a cricket species. dalton (1977) found that pge1 modulates salivary gland fluid secretion in a blowfly. stanley and his colleagues discovered that eicosanoids signal insect cellular immune reactions to bacterial infections (stanley-samuelson et al., 1991). this finding stimulated research into the biochemistry of pg production and pg signaling mechanisms. in this letter, we outline pg actions in insect immunity and then draw attention to recent reports on pg actions in anti-viral immunity. insect hemocytes make up the immediate defense against microbial infections and parasite invasions. cellular actions include phagocytosis, encapsulation and nodulation. phagocytosis and formation of melanotic nodules is responsible for clearing the majority of infecting microbes from hemolymph circulation. remaining microbes are cleared by production of antimicrobial peptides (haine et al., 2008). pgs mediate microaggregation and nodulation reactions (miller et al., 1994). these are complex cellular processes. for example, 184 gene products act in phagocytosis in drosophila s2 cells (stroschein-stevenson et al., 2006). phagocytosis is mediated by pgs in hemocytes prepared from wax moths, galleria mellonella (mandato et al., 1997) and spodoptera exigua ___________________________________________________________________________ corresponding author: david stanley usda/agricultural research service biological control of insects research laboratory 1503 south providence road, columbia, mo 65203 usa e-mail: stanleyd@missouri.edu (shrestha and kim, 2007), as well as from the blood sucking bug, rhodnius prolixus (figueiredo et al., 2008). hemocytes migrate to the sites of wounds and infection, and hemocyte migration also is signaled by eicosanoids (merchant et al., 2008). the first step in eicosanoid biosynthesis is the hydrolysis of aa from membrane-associated phospholipids (pls) by action of a phospholipase a2 (pla2). shrestha et al. (2010) showed that bacterial challenge increased pla2 activity in plasma and, separately, in hemocyte/fat body preparations from flour beetle, tribolium castaneum, larvae. we identified and cloned five t. castaneum genes encoding secretory pla2s (spla2s), tcspla2a through e, and showed that all five recombinant tcspla2s have pla2 activity. dsrna constructs specific to each of the five genes silenced expression of four genes from 24 to 72 h after treatment, 48 to 72 h for tcspla2d. in separate experiments, silencing each of these genes, except tcspla2c, suppressed flour beetle immunity, determined as nodulation following a standard bacterial challenge. hence, these four genes are directly involved in eicosanoid signaling. the insect cytokine, plasmatocyte spreading peptide (psp) stimulates spreading in subpopulations of lepidopteran plasmatocytes (clark et al., 1997). kim et al. (2008) found that psp also influenced hemocyte spreading in s. exigua. work in kim’s laboratory later showed that psp mediates hemocyte spreading via eicosanoid signaling (srikanth et al., 2011). later work revealed the actions of psp and pge2 in hemocyte spreading are coordinated via the small g protein, rac1 (park et al., 2013). expression of serac1 increased by about 8-fold at 2 h post infection (pi) and a dsrna construct silenced serac1 expression for at least 96 h following infection. silencing serac1 negated psp-stimulated hemocyte spreading, which was rescued by pge2 treatments. this clarified some of the intracellular cross-talk between psp and pg signaling. phenoloxidase (po) acts in several aspects of insect immunity, including melanization of encapsulated parasitoid eggs and newly-formed nodules. the s. exigua po is biosynthesized in oenocytoids as the zymogen prophenoloxidase 166 (ppo). the ppo is released from oenocytoids into hemolymph circulation by cell lysis. shrestha and kim (2008) showed that eicosanoids mediate the cell lysis. they found that treating hemocytes with pharmaceutical inhibitors of eicosanoid biosynthesis blocked the cell lysis, which was reversed by aa treatments. they generated more insight into eicosanoid actions, showing eicosanoids mediate the release of ppo via protein kinase c (shrestha and kim, 2009). pgs exert their actions in mammalian cells via cell surface g protein coupled receptors (gpcrs; bos et al., 2004). oenocytoids express a gpcr that mediates pge2-stimulated oenocytoid cell lysis (shrestha et al., 2011). this is the first demonstration of the mechanism of a pg action in insect biology, from which we infer that some pgs act via cell surface gpcrs. drawing on the biomedical background, pgs are generated via a cyclooxygenase (cox), an enzyme with two separate active sites. the first site converts aa to the hydroperoxyendoperoxide, pgg2, which is reduced by the peroxidase site to pgh2. pgh2 is a substrate for several enzymes that convert it into various pgs in a cell-specific manner. on the idea that insects also express coxs, pg biosynthesis has been recorded in several insect tissues using enzyme preparations similar to protocols appropriate to mammalian coxs. for one example, a fat body preparation from the tobacco hornworm, manduca sexta, produced pgs from radioactive aa (stanley-samuelson and ogg, 1994). insect genomic data bases facilitated searches for insect genes encoding cox proteins, which uniformly indicate insects do not have such genes, although two crustaceans do (varvas et al., 2009). this situation prompts the question of how to explain the presence and actions of pgs in insects? tootle et al. (2011) showed that pg signaling is necessary for the correct temporal expression of genes encoding eggshell proteins. more to the point, they showed that a peroxinectin (pxt) type peroxidase (pox) is responsible for synthesizing the pgs. park et al. (2014) investigated the idea that a pxt type pox acts in s. exigua immunity. the authors identified ten genes encoding peroxidases from s. exigua transcriptomes (sepox-a sepoxj) and designed dsrna constructs to individually suppress expression of each of them. each of the 10 genes was partially silenced by injecting their cognate dsrnas into experimental larvae. silencing each of these genes showed that two of the ten pox genes, sepox-f and -h act in hemocyte defense reactions because hemocyte spreading and nodule formation were inhibited in insects injected with dspox-f and dspox-h, but not in insects injected with the other 8 dsrna constructs. the influence of dspox-f and -h was reversed by coinjections with pge2, from which it was concluded that sepox-f and -h are pxt-like genes responsible for pg biosynthesis in s. exigua. more broadly, pxt genes may be responsible for producing pgs in most, if not all, insect species. insects express several mechanisms to protect themselves from viral infections. one of the most important mechanisms is caspase-mediated apoptosis and sloughing of virus-infected midgut epithelial cells (o’neill et al., 2015). constitutive plasma po may act as an anti-viral defense in lepidopteran larvae (popham et al., 2004). washburn et al. (1996) recorded hemocytic encapsulation of virus-infected cells in h. zea larvae. we have less information on eicosanoid signaling in insect anti-viral immunity, reviewed just below. treating gypsy moth larvae, lymantria dispar, with various inhibitors of eicosanoid biosynthesis increased larval susceptibility to its nucleopolyhedrovirus, ldmnpv, from which it was inferred eicosanoids act in insect responses to baculovirus infection (stanley and shapiro, 2007). the work was expanded to other lepidopterans, including the beet armyworm, s. exigua, the corn earworm, heliothis zea and the fall armyworm, s. frugiperda (stanley and shapiro, 2009). again, larvae were treated with a selected inhibitor and then challenged with their respective npv, semnpv, hzsnpv and sfmnpv. at least one of the inhibitors led to increased larval susceptibility to their npv and the influence of one inhibitor, indomethacin, was expressed in a dose-related manner. the overall inference was that eicosanoids act in insect defenses against npvs, however, these papers did not provide insight about specific eicosanoid-mediated defense mechanisms. insects clear some viral infections from circulation via nodule formation. using larvae of the greater waxmoth, g. mellonella, büyükgüzel et al. (2007), tested the hypothesis that eicosanoids mediate nodulation reactions to a vertebrate virus, bovine herpes simplex virus-1 (bhsv-1). they reported that viral infection increased nodulation in a near linear manner, from about 15 nodules/larva in controls to nearly 200 nodules/larva in experimental larvae injected with the highest viral challenge. nodulation increased with time pi to a high at 4 h. injecting the cox inhibitor, indomethacin, into experimental larvae followed by a virus challenge led to sharply decreased nodulation reactions. this work also showed that orally administered indomethacin reduced nodulation reactions to viral infection. in a similar manner, durmuş et al. (2008) found that challenging larvae of the waxmoth parasitoid, pimpla turioinellae, with bhsv-1, also induced nodulation, increasing from about 15 nodules/larva in controls to >50 nodules/larva in infected larvae. treating parasitoid larvae with one of three pharmaceuticals, indomethacin, the lipoxygenase inhibitor, esculetin, or the glucocorticoid, dexamethasone, inhibited bhsv-1induced nodule formation. the inhibition was reversed by treating experimental larvae with aa and the authors drew the reasonable conclusion that eicosanoids mediate nodulation reactions to viral infection in the wasp larvae. eicosanoids influence expression of host defense genes in the silk-producing insect, antheraea pernyi. zhang et al. (2015a) showed that bacterial, fungal and npv infections led to increased expression of the a. pernyi small heat shock protein, ap-shsp21.4, in midgut, hemocytes and fat body of infected larvae. the expression increases occurred in separate temporal patterns, at 3 h pi in fat body, at 24 h pi in midgut and at 12 h pi in hemocytes. injected sirna constructs silenced ap-shsp21.4 167 expression following npv challenge and expression of some immunity genes, particularly genes encoding defensin, toll1 and lysozyme after npv challenge. the influence of npv infection on apshsp21.4 expression was strongly inhibited by treating experimental larvae with pharmaceutical inhibitors of eicosanoid biosynthesis; treating larvae with the eicosanoid biosynthesis precursor, aa, induced ap-shsp21.4 expression. the authors inferred that ap-shsp21.4 acts in host defense against microbial infections and that eicosanoids mediate infection-related ap-shsp21.4 expression. in a related paper, they also reported eicosanoids act in expression of another small heat shock protein, shsp20.8 (zhang et al., 2015b). this work demonstrates a specific molecular mechanism of eicosanoid actions in insect immune reactions to microbial, including viral, infections. pgs and other eicosanoids are central operators in insect immune signaling and in crosstalk with other signal systems, including an insect cytokine. the identification of several genes involved in eicosanoid signaling creates a sound basis for continued fundamental research into these systems, with the expectation of practical applied outcomes (stanley and kim, 2014). acknowledgments mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the u.s. department of agriculture. all programs and services of the u.s. department of agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap. lz is working in the biological control of insects research laboratory, supported by the china scholarship council. references bos cl, richel dj, ritsema t, peppelenbosch mp, versteeg hh. prostanoids and prostanoid receptors in signal transduction. int. j. biochem. cell biol. 36: 1187-1205, 2004. büyükgüzel e, tunaz h, stanley d, büyükgüzel k. eicosanoids mediate galleria mellonella cellular immune response to viral infection. j. insect physiol. 53: 99-105, 2007. clark kd, pech ll, strand mr. isolation and identification of a plasmatocyte spreading peptide from hemolymph of the lepidopteran insect pseudoplusia includens. j. biol. chem. 272: 23440-23447, 1997. dalton t. the effect of prostaglandin e1 on cyclic amp production in the salivary glands of calliphora erythrocephala. experientia 33: 1329-1330, 1977. durmuş y, büyükgüzel e, terzi b, tunaz h, stanley d, büyükgüzel k. eicosanoids mediate melantoic nodulation reactions to viral 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wei g, li j, et al. eicosanoids mediate shsp20.8 gene response to biotic stress in larvae of the chinese oak silkworm antheraea pernyi. gene 562: 32-39, 2015b. 169 282 isj 14: 282-294, 2017 issn 1824-307x research report characterization of protease activity from the digestive tract and tentacles of isostichopus fuscus sea cucumber ac hernández-sámano 1 , x guzmán-garcía 2 , r garcía-barrientos 3 , f ascencio-valle 4 , a sierrabeltrán 4 , i guerrero-legarreta 1 1 biotechnology department, universidad autónoma metropolitana, 09340 mexico city, mexico 2 hydrobiology department, universidad autónoma metropolitana, 09340 mexico city, mexico 3 universidad politécnica de tlaxcala, 90180 tlaxcala, mexico 4 centro de investigaciones biológicas del noroeste s.c., la paz, 23096 baja california sur, mexico accepted august 12, 2017 abstract sea cucumbers possess evisceration mechanisms and regeneration capacity. the function of tentacles is to collect food particles. from our results, we suggest these organs could also be part of the digestive system. therefore proteases in the digestive tract and tentacles of isostichopus fuscus were partially characterized by histological and biochemical methods. digestive cells and regions, and secretory granules were observed by histological methods in both organs. proteolytic extracts of the digestive tract and tentacles showed peak activity at ph 6 and 8. the digestive tract extract had peak activity at 40 and 70 °c, whereas the tentacle extract peak activity was at 60 °c. both extracts showed activity at 0 to 10 °c. the extracts retained 67 to 75 % residual activity when incubated at 60 °c for 1 h. the effect of different ions and specific inhibitors suggested the presence of cysteineand metalloproteases in both organs. sds-page showed 6 proteins of approximately 40, 43, 49, 76, 106, and 147 kda in the digestive tract extract, and 5 proteins of approximately 44, 60, 81, 108, and 150 kda in the tentacle extract. native-page and zymography assays confirmed the presence approximately 100 kda proteases in both extracts. the tentacle extract had the highest proteolytic activity, suggesting that this organ could contribute to the digestion process of i. fuscus. key words: proteolytic enzymes; proteolytic extract; brown sea cucumber; holothurians; digestive system introduction sea cucumbers are marine invertebrates of phylum echinodermata, class holothuroidea. these holothurians play an important ecological role in the structure and functioning of marine benthic communities by consuming organic material (uthicke, 2001). sea cucumbers have acquired commercial importance as food items due to its high nutritional value (56 % protein, 2 % fat dry weight). they are specially demanded by consumers in south east asia (bordbar et al., 2011; purcell et al., 2012). isostichopus fuscus is the most common sea cucumber commercial species found in the pacific coast of the americas, from baja california, mexico to ecuador (maluf, 1991). its morphology consists in an elongated body. the mouth, located in the anterior section, is surrounded by 15 to 20 peltated ___________________________________________________________________________ corresponding author: isabel guerrero legarreta biotechnology department universidad autónoma metropolitana 09340 mexico city, mexico e-mail: isabel_guerrero_legarreta@yahoo.com tentacles. the anus is located in the posterior end (solís-marín et al., 2009) (fig. 1). the digestive system is formed by a digestive tract (mouth, pharynx, esophagus, stomach, intestine and cloaca) and anus (yang et al., 2015). tentacles function is related to selection and capture of food particles, the adhesive forces and shape of tentacles select these feeding materials (jaeckle and strathmann, 2012; yang et al., 2015). sea cucumbers are able to eviscerate as a defense mechanism against predators, although evisceration can also be activated by environmental and mechanical factors, such as high temperatures, low oxygen levels, and handling. this process represents economic losses when these animals are produced by aquaculture methods. even though, several holothurians have the ability of viscera regeneration (garcía-arrarás and greenberg, 2001; wu et al., 2013). proteases, or proteolytic enzymes, (ec 3.4.) catalyze the hydrolysis of peptide bonds during protein digestion and other biological functions. over the years, marine animals adapted to different environmental conditions, developing endogenous proteases with mailto:isabel_guerrero_legarreta@yahoo.com 283 fig. 1 morphology of i. fuscus. two sea cucumbers show the dorsal surface (ds); it shows mouth (m) surrounded by 20 peltate tentacles (t) in the anterior section and anus (a) in the posterior section. scale bar = 10 cm. unique properties. some of their distinctive features include high catalytic efficiency at low reaction temperatures, stability to extreme temperatures, and substantial catalytic activity/stability at neutral to alkaline ph (haard and simpson, 2000). marine proteases are classified as serine-, cysteine-, aspartyl-, and metallo-proteases (whitaker, 1994). in the present work, proteases in the digestive tract and tentacles of i. fuscus sea cucumber were identified and partially characterized by histological and biochemical methods to contribute understanding the digestive system of these holothurian. materials and methods reagents the following reagents were purchased from sigma chemicals (st. louis, missouri, usa): hematoxylin, eosin, casein, hemoglobin, bovine serum albumin (bsa), β-mercaptoethanol, pepstatin a, phenylmethylsulfonyl fluoride (pmsf), n--tosyll-lysine chloromethyl ketone hydrochloride (tlck), trypsin inhibitor (ti), ethylenediaminetetraacetic acid (edta). molecular weight markers were purchased from biorad (richmond, california, usa). all reagents were analytical grade. capture of specimens twelve isostichopus fuscus adult male specimens (385 g average weight, 23.25 cm average length), apparently healthy, were captured at espíritu santo island shore (24° 24´ n, 110° 18´ w and 24° 36´ n, 110° 27´ w), gulf of california, mexico, at 3 to 15 m depth. organism collection was carried out according to official regulations (mexican official regulation, nom-059-semarnat-2010). the corresponding authorities issued the respective collection license (license number sgpa/dgvs/00616/11). living sea cucumber specimens were placed in seawater at 10 to 15 °c for 24 h. after killing the animals by anoxia, the digestive tract and tentacles were dissected and washed with distilled water to remove organic residues and extra tissue, and immediately processed for histological characterization and preparation of protease extracts. histological characterization samples of the studied organs were fixed in 70 % ethanol for 24 h, and imbibed in paraffin using an automatic tissue processor (leica tp1020, leica biosystems, nussloch, germany). the method consisted in sample dehydration for 6 h with increasing ethanol concentrations (50 to 100 %), subsequent clearing for 70 min with ethanol-xylene and xylene, and infiltration for 2 h 15 min with paraffin-xylene and paraffin (leica eg1140 modular tissue embedding). 7 to 12 μm-thick sections were obtained using a microm hm315 rotary microtome (thermo fisher scientific, walldorf, germany). paraffin was removed from tissue sections by 25 min sequence with 100 % xylene, 100 to 70 % ethanol and distilled water. samples were then stained with hematoxylin-eosin (he), hematoxylin for 10 min, 15 min sequence with acid ethanol solution, water, ammonium water, distilled water and 80 % ethanol, and eosin for 5 min, followed by a sequence of 96 to 100 % ethanol, ethanol-xylene and xylene for 17.5 min total time (grant and tyler, 1983). preparations were mounted in a synthetic resin, and observed with a light microscope. multiple slides and observations were registered but only the representative ones were reported. brown staining was considered as an indicator of the digestive enzyme activity, including proteases, due that waste accumulation is stained in brown (byrne, 2001). 284 preparation of proteolytic extracts digestive tract and tentacle proteolytic extracts were obtained by homogenizing 30 g with 60 ml-20 mm phosphate buffer at 4 °c, ph 7 (anti-proteases were not used during tissue homogenization), and centrifuged at 10,000g for 30 min, 4 °c. the supernatant was separated and stored at -20 °c until used. protein content was analyzed by the method reported by lowry et al. (1951), using bovine serum albumin as standard. determination of proteolytic activity it was analyzed according to a modification of the method reported by anson (1938) for acid proteases, and by kunitz (1946) for alkaline proteases. acid protease analysis was based on the hydrolysis of 1 % (w/v) hemoglobin; the hemoglobin solution contained 0.025 m boric acid, 0.025 m phosphoric acid, and 0.25 m acetic acid. alkaline proteases were analyzed by casein hydrolysis (1 %, w/v casein in 20 mm phosphate buffer, ph 7). 80 µl proteolytic extract and 250 µl substrate (casein or hemoglobin at given ph) was incubated for 15 min at a given temperature. the reaction was stopped by adding 175 µl 25 % (w/v) trichloroacetic acid. samples were then centrifuged at 10,000g for 10 min, 4 °c. the supernatant containing peptides resulting from substrate hydrolysis was read at 280 nm. results were reported as specific activity, expressed in u/mg protein, where one proteolytic activity unit (u) was defined as the amount of active enzyme necessary to change the absorbance 0.001 unit/min at 280 nm, at the given experimental conditions (yamaguchi et al., 1983). therefore: u = (abs280 nm) (dilution factor)/ (0.001) (reaction time, min). effect of ph the extract proteolytic activity was measured on hemoglobin or casein substrate at a ph range from 2 to 10, 37 °c. at ph 2 to 5, the activity was analyzed using 1 % (w/v) hemoglobin; at ph 6 to 10 the substrate was 1 % (w/v) casein. enzymatic stability at a given ph was assayed by incubating the extracts at 25 °c for 1 h in the following buffers: 0.1 m kcl-hcl (ph 2); 0.2 m gly-hcl (ph 3, 4); 0.1 m phosphate buffer (ph 5 8); and 0.1 m tris-hcl (ph 9 10) (fu et al., 2005). specific activity was analyzed after incubating the samples at the optimal ph and temperature, by the method described before and reported as residual activity (ra, %). therefore: ra = (final specific activity) (100)/(initial specific activity), where the initial specific activity was obtained at optimal ph and temperature before incubation. effect of temperature proteolytic activity was also analyzed in extracts incubated from 0 °c to 80 °c, at ph of peak activity. activity was measured by the method described above. once the temperature of peak activity was obtained, the extract activity of tentacles and digestive tracts were assayed at optimal ph and temperature conditions. results were reported as residual activity (ra). effect of protease inhibitors the following protease inhibitors were studied: 5 mm pmsf and 10 mm tlck for serine-proteases; 10 mm edta for metallo-proteases; 0.6 g/l ti for trypsin; 1 µm pepstatin a for aspartyl-proteases; and 1 mm β-mercaptoethanol (reducing agent). the inhibitory effect was analyzed by incubating the proteolytic extracts and a given inhibitor (1:1, v/v) for 1 h at 25 °c (garcía-carreño, 1992; fu et al., 2005). proteolytic activity was also analyzed at the optimal ph and temperature conditions by the method described above. results were reported as residual activity (ra). effect of ions the following ions were studied (10 mm): mn +2 and cu +2 (sulfates), and k + , ca +2 , na +2 , zn +2 , mg +2 , ba +2 , hg +2 (chlorine salts). proteolytic extracts were incubated together with a given ion (1:1, v/v) for 1 h at 25 °c (qi et al., 2007; zhu et al., 2008; sun et al., 2011; wu hl et al., 2013). the activity was analyzed at optimal ph and temperature by the method described above. results were reported as residual activity (ra). electrophoretic analysis extracts were analyzed by sds-page, according to the method reported by laemmli (1970), using a mini-protean ii electrophoresis cell (biorad, richmond, california, usa), with 8 to 10 % stacking gels and 4 to 5 % concentration gels. previous to analysis, extracts were boiled for 1 min in order to denature proteins. 5 mg/ml sample and 200 to 45 kda mw markers (myosin: 200 kda, βgalactosidase: 116.3 kda, phosphorylase b: 97.4 kda, bsa: 66.2 kda, and ovalbumin: 45 kda) were applied to the gels. analysis were carried out at 150 v constant voltage, 30 ma, and 4 °c. the gels were stained with 0.1 % (w/v) coomassie blue r-250 solution (40 % methanol and 10 % acetic acid). image analysis were performed with a gel-doc 2000 image analyzer (biorad), fitted with a quantity one software (version 4.0, biorad). native-page was also performed at 15 ma/gel constant amperage, 150 v, 4 °c. 66 to 14 kda molecular weight markers (bsa: 66.2 kda, ovalbumin: 45 kda, glyceraldehyde-3-phosphate dehydrogenase: 36 kda, carbonic anhydrase: 29 kda, trypsinogen: 24 kda, trypsin inhibitor: 20 kda, and α-lactalbumin: 14.2 kda) were used. zymography assays were performed on native-page by a modification of the method reported by garcía-carreño (1993). the gels were immersed in 2 % casein solution (ph 6, 4 °c) for 1 h, incubated for 2 h at 60 °c, and washed with distilled water. finally, the gels were stained with coomassie blue r-250 and analyzed using a gel-doc analyzer, as described above. statistical analysis the results were subjected to analysis of variance (anova), complying with anova assumptions (homogeneity of variances and normal distribution), and tukey's multiple comparison tests using spss statistical package for windows (version 15.0). all analyses were carried out in 285 fig. 2 histological sections of digestive tract and tentacles of i. fuscus. hematoxylin-eosin staining. digestive tract: (a) intestine section with digestive cells (dc). (b) intestinal tube (it) and digestive cells (dc). (c) possible parasite (pa) in basophile staining and secretory granules (sg). (d) secretory granules (sg) close to an inclusion (in). (e) digestive cell (dc) region. tentacles: (f) digestive region epithelium (dre). (g) possible food remains (fr). (h) inclusion (in) aggregates in connective tissue (ct) in basophile staining and secretory granules (sg). (i) inclusions (in) in connective tissue (ct) in basophile staining. (j) digestive cell (dc) region in connective tissue (ct). scale bars: a, g, h, j, 20 µm; b, c, d, e, i, 30 µm; f, 10 µm. 286 fig. 3 effect of ph on proteolytic extracts obtained from the digestive tract and tentacles of i. fuscus. (a) ph effect at 37 °c. (b) ph stability (optimal activity conditions). results reported the mean of three replicates ± sd. statistically significant at *p < 0.05 level. triplicate, in three independent experiments; mean values ± standard deviations were reported. differences were statistically significant at p < 0.05 level. results histological characterization several structures were observed in the digestive tract and tentacles of i. fuscus, such as a dense connective tissue layer, dermal epithelium, cell aggregates, digestive regions, and secretory granules. the digestive regions stained in brown with he were present around damaged cells, observed as deformed cells, foreign material, and possible pathogenic agents. the digestive tract showed the presence of cells with digestive activity, observed in brown staining (fig. 2a). intestinal tubules were observed (fig. 2b); a possible platyhelminth-type parasite showing digestive activity due to secretory granules close to it, observed in brown staining (fig. 2c); secretory granules close to a cell type in basophilic staining (fig. 2d) named “inclusion” in the present paper; and a digestive cell region was observed in brown (fig. 2e). tentacles showed digestive regions in the epithelium (fig. 2f), included cells that stained brown, similar to the secretory cells in the digestive epithelium; food residues, possibly algae (fig. 2g); secretory granules observed in brown staining close to different cell types in basophilic staining (“inclusions”) of ovoid or spherical shape forming aggregates, immersed in connective tissue (fig. 2h); “inclusions” isolated in connective tissue (fig. 2i); and cells with digestive activity observed in brown (fig. 2j). the brown staining can be interpreted as digestive activity in both tissues. effect of ph proteolytic activity showed a peak (p < 0.001) at ph 6 and 8, 37 °c, with respect to other studied ph in proteolytic extracts obtained from the digestive tract and tentacles of i. fuscus. comparing both organs, tentacle extracts had a higher proteolytic activity (p < 0.001) than digestive tract extracts at ph 2 to 10 (fig. 3a). the digestive tract extracts were highly stable (p < 0.001) at ph 8, 94.5 ± 1.0 % residual activity (ra) whereas tentacles extract showed higher stability at ph 3 to 4, 95.3 ± 1.9 % ra (fig. 3b). 287 fig. 4 effect of temperature on proteolytic extract activity obtained from the digestive tract and tentacles of i. fuscus. (a) temperature effect (ph 6). (b) thermal stability (optimal activity conditions). results are reported as the mean of three replicates ± sd. statistically significant at *p < 0.05 level. effect of temperature peak proteolytic activity (p < 0.001) of digestive tract extracts was observed at 40 and 70 °c (assayed from 0 to 80 °c), whereas peak activity of tentacle extracts was at 60 °c. however, extracts of both organs showed proteolytic activity even at temperatures as low as 0 to 10 °c (fig. 4a). digestive tract and tentacles extracts were partially stable when incubated at 60 °c for 1 h, retaining 72.3 ± 1.0 % and 67.5 ± 0.1 % initial proteolytic activity, respectively. as compared to other studied temperatures, significantly higher stability (p < 0.001) was observed at 30 to 50 °c (86.9 ± 0.6 to 88.6 ± 0.9 % ra) in the digestive tract extract. significantly higher stability of the tentacle extract (p < 0.001) was at 10 °c (84.7 ± 0.9 % ra) and 30 °c (85.5 ± 0.6 % ra) (fig. 4b). effect of protease inhibitors in order to preliminary characterize the protease type, several protease inhibitors were assayed (fig. 5). the digestive tract extract was significantly inhibited (p < 0.001) at ph 6 by pmsf (79.4 ± 1.1 % ra) and pepstatin a (80.5 ± 1.9 % ra), followed by tlck (86.2 ± 1.9 % ra). ti, edta, and β-mercaptoethanol (a reducing agent) did not inhibited proteases (p > 0.05) at ph 6 (92.0± 1.9 to 98.8 ± 2.3 % ra). a significant inhibition effect (p < 0.001) was observed at ph 8 with ti and edta (71.2 ± 1.9 and 74.9 ± 1.8 % ra, respectively). tentacle proteolytic enzymes were inhibited (p < 0.001) at ph 6 by tlck (79.4 ± 0.9 % ra) and pepstatin a (83.3 ± 1.2 % ra); ti, pmsf, edta and β-mercaptoethanol had no effect at ph 6 (p > 0.05) (93.1 ± 1.2 to 99.7 ± 0.12 % ra). ti and edta inhibited (p < 0.001) proteolytic enzymes (81.9 ± 1.6 and 81.1 ± 2.3 % ra, respectively) at ph 8. effect of ions various ions were also assayed to preliminary characterize the proteases (fig. 6). the digestive tract extract proteolytic effect was significantly (p < 0.001) inhibited by ba +2 (74.8 ± 0.4 % ra), whereas ca +2 increased the proteolytic activity by 1 %. other studied ions (cl , na + , zn + , mn +2 , cu +2 , mg +2 , and hg +2 ) had no effect (p > 0.05) (90.8 ± 0.4 to 95.3 ± 1.2 % ra). tentacle proteolytic extracts retained 70.8 ± 0.1 % ra (p < 0.001) initial proteolytic activity 288 fig. 5 effect of protease inhibitors (ph 6) on proteolytic extracts obtained from the digestive tract and tentacles of i. fuscus. results are reported as mean of three replicates ± sd. statistically significant at *p < 0.05 level. when was incubated with ba +2 . conversely, these extracts were partially inhibited by mg +2 , cu +2 , mn +2 , zn +2 , hg +2 , and ca +2 (81.3 ± 0.3 to 88.5 ± 0.2 % ra). cl and na + showed no significant effect (p > 0.05) (90.5 ± 0.2 % ra). electrophoretic analysis six proteins of 40, 43, 49, 76, 106, and 147 kda apparent molecular weight were observed in sdspage of the digestive tract extract. similarly, tentacle extract sds-page showed five proteins (44, 60, 81, 108, and 150 kda) (fig. 7). nativepage of the digestive extract showed three proteins of 39, 42, and 109 kda apparent molecular weight; whereas the tentacle extract showed three proteins of 39, 68, and 106 kda (fig. 8a). 2 % casein-zymograms (ph 6, 60 °c) confirmed the presence in the digestive tract and tentacle extracts of 100 kda-proteolytic enzymes (approximately 109 and 106 kda, respectively) (fig. 8b). discussion some species of sea cucumber, mainly stichopus japonicas, had been studied by histological and biochemical methods (yang et al., 2015). however, there are scatter reports regarding i. fuscus proteolytic enzymes (hernández-sámano et al., 2015). histological characterization sea cucumber insoluble collagen content is around 70 % total protein (saito et al., 2002). therefore, the animal’s connective tissue contains a considerable amount of collagen, responsible of cellular binding and support (san miguel-ruíz and garcía-arrarás, 2007). our histological observations were similar to those reported by mashanov et al. (2004) in eupentacta fraudatrix. brown staining of aggregates in tentacles and digestive tract indicated that these cells main function is secreting proteases as digestive enzymes in both tissues. byrne (2001) reported waste deposits in the connective tissue, also stained in brown. cell aggregates with digestive activity have been mainly described in the intestine of other holothurians (estrada-flores et al., 1982; smiley, 1994; vergara and rodríguez, 2015). this is a defense mechanism around damaged cells or foreign particles, and protection against microbial invasion (vergara and rodríguez, 2015). for instance, cladolabes schmeltzii sea cucumber showed abundant brown-stained cells in the luminal epithelium and conjunctive tissue of three regions of the digestive tract (vergara and rodríguez, 2015). these authors reported that it is likely that amebocytes are involved in the formation of brown cells. amebocytes are cells acting as a defense against external agents (rodriguez et al., 2000). clumps of material stained in brown are probably a result of defense functions by these amebocytes. secretory granules, also stained in brown, function as storage compartments for secreted products. these are the main organelles involved in regulated secretion. transport from the formation to export sites is a multiple step process (pavelka and roth, 2010). the observed secretory granules are sphere-shaped structures of heterogeneous content, as reported by mashanov et al. (2004). these granules stain positive for neutral mucopolysaccharides and proteins in paraffin preparations. 289 fig. 6 ion effect (10 mm) on proteolytic extracts obtained from the digestive tract and tentacles of i. fuscus (optimal activity conditions). results are reported as the mean of three replicates ± sd. statistically significant at *p < 0.05 level. figure 2 showed the possible presence of a parasite. these are frequent in sea cucumbers as the habitat preference of holothurians is rocky, sandy or muddy environments, also a suitable substrate for parasites. it was reported that one third of parasites found in echinoderms lives on, or inside sea cucumbers, mainly in the digestive system and celom (jangoux, 1990). therefore, the observed structure is a possible parasite in the digestive tract of i. fuscus. moreover, “inclusions” may be proteolytic cystic parasites. the cells are presumably amebocytes. sea cucumber feed mainly of algae or microalgae such as rhodomonas and dunaliella. histological sections also showed particles of these feeding material, in agreement to the reports on sea cucumber's diet (lovatelli et al., 2004). the function of tentacles is the selective uptake of particles (schmidt-rhaesa, 2007). however, proteolytic activity in i. fuscus tentacles can also be related to the autolytic character and regeneration mechanisms of sea cucumbers. the autolytic character is favored by the presence of highly active endogenous proteases (sun et al., 2011). likewise, it has been reported that the regeneration process in holothurians involves mechanisms including serine-, cysteine-, and metallo-proteases (quiñones et al., 2002; lamash and dolmatov, 2013). based on our reported results reported in the present work, we suggest that the proteolytic activity of i. fuscus tentacles can be an additional digestive mechanism when evisceration takes place as a response to stress or defense against predators (garcía-arrarás and greenberg, 2001). that is, during the time the internal organs takes to regenerate (3 to 4 weeks) (san miguel-ruíz and garcía-arrarás, 2007), sea cucumbers could utilize the digestive activity located in the tentacles as part of the digestion system. this conclusion is based in the fact that only the middle part of the digestive tube is ejected through the anal opening, characteristic of order aspidochirotida. nevertheless, the anterior (pharynx and esophagus) and posterior (cloaca) regions are retained. regeneration after evisceration includes only the transformation of intestinal mesentery and the retained broken ends of esophagus and cloaca (vergara and rodríguez, 2015). the information obtained by means of histological techniques was mainly qualitative, indirectly showing the presence of proteolytic activity. proteolytic characterization complemented this findings. effect of ph proteolytic activity has been demonstrated in a variety of echinoderms (jangoux and lawrence, 1982). extracts obtained from the digestive tract and tentacles of i. fuscus also showed proteolytic activity, in agreement to other authors (fu et al., 2006) who previously demonstrated that protease activity of s. japonicus varies according to the anatomical region. peak activity at the studied ph range was similar to that reported for digestive tract crude extracts of s. japonicus (ph 5 and 8, at 37 °c). proteolytic enzymes where reported as metallo and serineproteases (fu et al., 2005). the presence of serine proteases, as trypsin and chymotrypsin (alkaline proteases) in holothurians, was fully demonstrated (fish, 1967). it has also been reported that collagenases (metallo-proteases) in the digestive tract show both trypsinand chymotrypsin-like activities with peak activity at ph 6.5 to 8.0 (yoshinaka et al., 1987; zefirova et al., 1996; fu et al., 2005). our results were in agreement to data reported by wu et al. (2013) and wu et al. (2013). these 290 authors found peak activities of proteases of s. japonicus at ph 6, 46 °c for cysteine-proteases at ph 8 to 9, 37 °c for metallo-proteases. to the best of our knowledge, there are no reports on protease activity of sea cucumber tentacles. it is suggested that proteolytic enzymes in this organ are of the same type as in the digestive tract, probably in order to start food digestion from the oral cavity, or as part of an alternative digestive pathway to the digestive tract. previous studies reported digestive enzyme activity in various fish species, depending on food habits (hidalgo et al., 1999). carnivore species have higher concentrations of acid proteases as compared to omnivorous or herbivorous species (jonas, 1983). sea cucumber is a detritivorous organism, i.e. obtains nutrients by consuming detritus (organic material, it typically including fragments of plants and animals as well as feces). therefore, alkaline and acid proteases are very active in this species. in our study, proteolytic stability was similar to that reported for s. japonicus cysteine-proteases (qi et al., 2007). however, our results in the alkaline region differ from these authors. protease activity in s. japonicus notably decreased at high ph, whereas it remains stable for i. fuscus. conversely, i. fuscus protease stability at basic ph was similar for s. japonicus metallo-proteases (wu et al., 2013). i. fuscus proteases were stable at a wide ph range. the behavior of these digestive enzymes could be also affected by ph due to sea cucumber’s habitat (fu et al., 2006). i. fuscus protease stability at alkaline ph was related to a basic environment where this holothurian develops (ph 8.4 to 8.5) (mercier, 2004). this ph is higher than the optimal for s. japonicus cultivation (ph 7.8) (wang and cheng, 2004; liu et al., 2004) and to species adaptation to a continuous feeding system involving ingestion of large amounts of seawater at neutral ph (guillaume and choubert, 1999). consequently, i. fuscus proteases showing peak activity at ph 6 and 8 can be possibly cysteineand metallo-proteases, respectively, as those reported for s. japonicas. these results were confirmed by studies with protease inhibitors and ions, as well as electrophoretic analysis, discussed below. effect of temperature the optimal proteolytic temperature range was similar to the data reported by qi et al. (2007), zhu et al. (2008), and wu et al. (2013) who observed that peak activity for s. japonicus cysteineproteases was at 46 to 50 °c. fu et al. (2005) also reported that metallo-proteases peak activity occurred at 37 °c. the optimal temperature range depends on the habitat’s physicochemical conditions. the environmental temperature of s. japonicus is as low as 3 to 4 °c, up to 24 to 26 °c, whereas i. fuscus lives at 8 to 12 °c, up to 30 °c and ever higher (lovatelli et al., 2004; lluch-cota et al., 2007). the higher water temperature causes i. fuscus proteases having optimal temperatures higher than for s. japonicus proteases. i. fuscus proteases also showed proteolytic activity at 0 to 10 °c. to the best of our knowledge, other studies were carried out from 20 °c fig. 7 sds-page of proteolytic extracts from i. fuscus. digestive tract extract (dt), tentacles extract (t). upwards (fu et al., 2005; qi et al., 2007; zhu et al., 2008; wu et al., 2013), but not as low as < 10 °c as in the present work, although (haard and simpson, 2000) reported that marine proteases are highly active at low reaction temperature. the effect of water temperature on enzyme activity was also reported in s. japonicus by gao et al. (2009). our research team reported that purified enzymes of aquatic animals have considerable higher activity than similar enzymes in land animals (garcía barrientos et al., 2006). from this information, it was concluded that, among a number of factors making aquatic enzymes more active than their counterparts in land animals, is the fact that aquatic enzymes are active in vivo at lower temperatures than enzymes from warm-blooded land animals (guerrero-legarreta et al., 2009). our results on proteolytic stability agreed to those reported in previous studies for acid and alkaline proteases of s. japonicus digestive tract extract (fu et al., 2005) and for cysteine-proteases, stable at 20 to 50 °c of the same species (qi et al., 2007; zhu et al., 2008; sun et al., 2011). even though, these authors found a significant reduction in proteolytic activity at temperatures above 50 °c (fu et al., 2005; qi et al., 2007; zhu et al., 2008; sun et al., 2011), as opposite to our results where residual activity was as high 86 to 88 % ra at 50 °c. from our results, it was concluded that proteolytic enzymes resistant to heat denaturation were present in the extracts obtained from i. fuscus. 291 fig. 8 native-page and zymography of proteolytic extracts from i. fuscus. (a) native-page. digestive tract extract (dt), tentacles extract (t). (b) zymography. digestive tract extract (dt), tentacles extract (t). effect of protease inhibitors the effect of several inhibitors on digestive tract and tentacle protease extracts, obtained from i. fuscus, agreed to other reports (fu et al., 2005). these authors described cu +2 -dependent serine and metallo-proteases in s. japonicus digestive tract. they also suggested the presence of collagenases (metallo-proteases). wu et al. (2013) also reported that s. japonicus metallo-proteases are strongly inhibited by edta. therefore, it was conclude that metallo-proteases are predominant in digestive tract and tentacle extracts of i. fuscus. aspartyl-proteases can also be present due to inhibition by pepstatin a. however, studies with purified proteases are necessary to confirm these results. effect of ions inhibition of i. fuscus proteolytic activity by ba +2 was a possible result of the presence of a metalloprotease matrix (mmp). this is a family of peptidases that regulate cellular behavior by depleting components in the extracellular matrix (collagen, gelatin) (nagase and woessner, 1999). wu et al. (2013) reported gelatinolytic metalloproteases, also part of mmp, in s. japonicus body wall. this fact suggested its involvement in sea cucumber autolysis. ziouti et al. (2006) reported selective inhibition by barium chloride, removing ca +2 from metalloprotease-2 matrix (mmp-2) catalytic center. ca +2 activation effect was reported for metalloproteases in s. japonicus (fu et al., 2005) and for cysteine-proteases in the same species (sun et al., 2011). the high concentrations of ca +2 and mg +2 in seawater (10 and 50 mm, respectively) is responsible for cell metallo-proteases activation (mayne and robinson, 1996). the presence of endogenous ions in the studied specimens encouraged the proteolytic effect. therefore, zn +2 net effect was possibly masked. in addition, presence of zn +2 -dependent metallo-proteases in mmp-2 has been demonstrated (ziouti et al., 2006). the high proteolytic activity observed with hg +2 can also be related to mmp activation mechanism, as mmp can be activated by proteases or in vitro by chemical agents, such as hgcl2 (visse and nagase, 2003). a further study, previously inhibiting proteases with 5mm edta, is recommended. currently, there are no reports in the literature related to ion effects on sea cucumber tentacle extract, although our results were similar to those obtained for the digestive tract extracts of the same samples. electrophoretic analysis electrophoretic profiles of i. fuscus digestive tract and tentacle extracts showed protein bands of molecular weight that coincide to previous reports (fu et al., 2005; qi et al., 2007; zhu et al., 2008; wu et al., 2013; lamash and dolmatov, 2013). these authors identified proteins of 35.5, 39.1, 45, 47, 53, 58, 63, 114 and 132 kda in s. japonicus and e. fraudatrix. in our study, the highest apparent molecular weights (147 and 150 kda) were similar for proteins in s. japonicus, reported by saito et al. (2002), cui et al. (2007), and park et al. (2012). according to these authors, these molecular weights correspond to collagen chains of 100, 135, and 130 kda, forming a 1α trimer. siddiqui et al. (2013) also found in bohadshia spp. (jaeger, 1833) a type i 292 collagen, with α1 as major component of about 138 kda. protease molecular weights found by zymography (109 and 106 kda) were similar to those reported by fu et al. (2005) in the digestive tract of s. japonicus, a 114 kda metallo-proteases. lamash and dolmatov (2013) also found a 132 kda metallo-proteases in e. fraudatrix gut, concluding that high molecular weight proteases in the digestive tube are not typical of extracellular proteases, but probably indicate the presence of a protein complex. likewise, proteases above 100 kda in digestive tract and tentacle extracts of i. fuscus were possibly protein complexes, suggesting that their similarity is related to the role of the studied organs digestive processes. in summary, proteolytic extracts obtained from the digestive tract and tentacles of i. fuscus showed presence of thermo-stable, active at low temperature acid and alkaline proteases. the studied proteases showed higher activity at alkaline ph, similarly to s. japonicas, the most studied species. it was also concluded that cysteineand metallo-proteases are possibly in the digestive tract and tentacles of i. fuscus. however, studies on 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1 gene vg esvaran, t gupta, a mohanasundaram, km ponnuvel* genomics division, seribiotech research laboratory, carmelaram – post, kodathi, bangalore – 560035, india accepted september 26, 2018 abstract microsporidiosis of the silkworm bombyx mori is caused by the highly virulent parasite nosema bombycis (nageli). the infection can be deleterious due to horizontal and vertical transmission, causing heavy damage to the sericulture industry. in recent years, molecular diagnostics has revolutionized the possibility to detect diseases in terms of rapidity and simplicity, however, most of them are time consuming, require sophisticated instruments and skilled personnel. in this study, the polar tube protein 1 gene of n. bombycis (indian isolate) was cloned, characterized and utilized for the development of rapid and simple loop mediated isothermal amplification assay (lamp) for detection of microsporidiosis in silkworm b. mori. the lamp reaction conditions were optimized to 65 °c for 60 min. the developed method demonstrated a higher sensitivity and the detection limit was found to be 2-fold higher than conventional pcr. this is the first report on loop mediated isothermal amplification assay that could be used to diagnose microsporidiosis at various developmental stages of the silkworm. this method serves as a robust alternative technique to conventional pcr and aids in the rapid diagnosis of n. bombycis infecting silkworm b. mori. key words: microsporidiosis, nosema bombycis, loop mediated isothermal amplification, polar tube protein 1 introduction sericulture is a labour intensive, important agro based industry that employs the rural poor and tribals. silk production is the major milestone for the sericulture industry which is affected by various silkworm pathogens that has a devastating effect on silk production, leading to heavy crop loss. among these pathogens, nosema bombycis (nageli) is a devastating microsporidian which causes microsporidiosis and is commonly known as pebrine disease in silkworm bombyx mori. these microsporidians are obligate intracellular spore forming parasites that infect numerous vertebrates and invertebrates (didier et al., 1998). they infect the host through a unique invasion apparatus the polar tube, a coiled structure connected to the anterior end of the spore. on appropriate stimuli, the polar tube everts out of the spore and ejects the sporoplasm into the host cell. three polar tube proteins have been identified so far and among them polar tube protein 1 (ptp 1) was predicted to ___________________________________________________________________________ corresponding author: km ponnuvel genomics division seribiotech research laboratory carmelaram – post, kodathi bangalore – 560035, india. email: kmponnuvel.csb@gov.in be involved in host cell adherence during penetration (keohane et al., 1998). it can be transmitted to silkworm off-springs through eggs laid by the infected mother moth by transovarial transmission. the diseased and dead larvae are also the sources of secondary contamination which spreads at a faster rate (bhat et al., 2009). the gold standard followed for pebrine detection is mother moth examination test through a microscope, which is laborious and time consuming. a rapid and sensitive method for detection would help the rearers to escape from crop loss. molecular diagnostics involving the detection of nucleic acid sequence or the antigen specific to the pathogen are widely used for disease identification and control (kaya ghosh and weiss, 2009). polymerase chain reaction has been used for pebrine detection using the normal conventional pcr with multiple primers targeting the eggs, and the quantitative real-time pcr for the detection of pebrine in eggs as well as hatched larvae (hatakeyama and hayasaka, 2003). even though pcr has been found to be the novel diagnostic technique contributing to the success of molecular diagnostics there have been few disadvantages that need to be overcome which include time, expertise and specialized equipments. hence, to fill these gaps in the field of molecular diagnostics a novel and new method was developed 353 fig. 1 primer designing chart and the location of ptp1 primer in the partial dna sequence (accession no. ky636450) of polar tube protein 1 gene of n. bombycis was used to construct the inner and outer primers. inner primers fip and bip comprise complementary sequence to f1 and sense sequence of f2, and sense sequence of b1 and complementary sequence of b2, respectively. outer primers f3 and b3 include the sequence of f3 and b3 respectively termed loop mediated isothermal amplification (lamp) (fu et al., 2016). in this method, the target dna sequence is amplified by strand displacement dna polymerase without 5’-3’ exonuclease activity that leads to the production of single stranded dna (notomi et al., 2000). it specifically detects genomic dna by using a set of two or three oligonucleotide primers specific to different regions of a target gene. the lamp method is found to be sensitive and can amplify even from few to 10 copies of target dna in less than 30 min (mori et al., 2001; nagamine et al., 2002). the simplicity and ease of detection make this technique more innovative. the results can be detected visually by turbidity caused by the white precipitate of magnesium pyrophosphate or by the use of fluorescent dyes like calcein, hydroxy naphthol blue (hnb) and the strand displacement activity of bst polymerase makes the dna denaturation step negligible (tomito et al., 2008). lamp is being extensively used to amplify dna for detection and for diagnostic purposes like examination of pathogens such as viruses, fungi, bacteria, and parasites (huang, 2010; chen et al., 2016; lee et al., 2017). this method has been widely applied in fields for on-site detection because of its low cost, high specificity, efficiency, simplicity of operation, rapidity and its major impact has been on effective disease diagnosis and food safety testing. in this study, lamp was employed to detect the microsporidian infection in silkworm b. mori using six distinct primers targeting the ptp1 gene region of the pathogen n. bombycis (indian isolate). the specificity and detection limit of the assay was evaluated using real-time pcr. the developed diagnostic method was compared with the conventional pcr and the method proved to be rapid, sensitive and specific for screening pebrine disease in b. mori. table 1 primer sequence for lamp and pcr f3 5’-tgcacaagtcactcaagc-3’ b3 5’-gctccgacattcgatgaa-3’ fip 5’-aagggccaagtggtgtaacagttttgttccagtaacgagtgtcc-3’ bip 5’-acggatcttcaagccttccacttttctaagtgcgttctgaggag-3’ loop f 5’-gcactggtcattggaacagc-3’ loop r 5’-catcctactgctccttgtggt-3’ 354 fig. 2 detection of n. bombycis infection through loop mediated isothermal amplification assay. a) visual detection of lamp reaction using hnb dye. b) agarose electrophoresis based detection of lamp products materials and methods inoculation and multiplication of pebrine spores the nosema bombycis spores (nik-1) were obtained from central sericultural training and research institute (csr&ti), mysore. the purified spores were counted using hemocytometer and diluted to a concentration of 1x106 spores/ml (shimanuki and knox, 2000). the 4th instar first day silkworms were fed with mulberry leaves smeared with n. bombycis spores at a concentration of 1x106 spores/ml while the control larvae were administered with water and subsequently fed with clean mulberry leaves. the dna was extracted from midgut tissues collected at different hours post infection (hpi): 12, 24, 48, 72, 96, 120 hpi from the artificially infected larvae and total dna was extracted from the water treated control larvae. apart from these tissues, egg, little tissue from the abdomen of moth were also collected and percoll purified spore was also used for dna extraction. dna extraction a small amount of tissue was excised from the silkworm moth and around 20 eggs were used for fast dna extraction (modified at the laboratory). the excised tissue or egg was placed in a microfuge tube and homogenized by addition of lysis buffer containing 0.1 m tris-cl, 1m nacl, 0.01m edta of ( 500 µl) and crushed using micro pestle. the crushed tissue was briefly vortexed and centrifuged at 13000 rpm for 5 min and the aqueous layer was transferred to a new microfuge tube. an equal volume of freshly prepared phenol: chloroform: isoamyl alcohol (24:24:1) were added and the contents of the tube were mixed gently. the tube was centrifuged at 13000 rpm for 5 min and the supernatant was transferred to a microfuge tube. an equal volume of isopropanol was added and the tubes were incubated at room temperature for 20 min followed by centrifugation at 13000 rpm for 5 min to pelletize the dna. the pelleted dna was washed with 70% ethanol and air dried. the dried pellet was dissolved in 50 µl of sterile distilled water and stored at -20 °c for further use. the percoll purified spores were disrupted using glass beads and the dna was extracted using the dneasy blood and tissue kit (qiagen gmbh, hilden, germany). detection of pathogen using conventional pcr the pathogen multiplication was analyzed through conventional pcr using the gene specific primer designed for targeting the ptp1 gene region. the amplification was performed using the following conditions: initial denaturation at 94 °c for 3 min, 30 cycles of 94 °c for 1 min, 55 °c for 30 sec, 72 °c for 30 sec and a final extension of 72 °c for 10 min. the amplified product was analyzed using agarose gel electrophoresis. cloning and sequencing of ptp1 the ptp1 gene sequence (gene accession no. kb909850) was blast searched in n. bombycis genome database and a sequence coding for n. bombycis was retrieved from the database. the retrieved sequence was used to design forward and reverse primers using primer3 programme (primer3http://frodo.wi.mit.edu/cgi-bin/primer3/). two pairs of primers specific for ptp1 gene of n. bombycis (forward 5’gatgagaattagatccttcaaac-3’ and reverse 5’-gctcagtttagcacacatggattattgcc-3’) were used for the study. the presence of ptp1 gene in n. bombycis infected samples was confirmed by cloning the purified pcr fragments using pgem-t easy vector. the plasmid dna was isolated from pcr verified positive clones and purified through dna purification column (promega, madison, usa) for sequencing with m13 primers in eurofins genomics india pvt. ltd., bangalore. the sequence was submitted to ncbi database (accession no. ky636450). 355 primer designing the conventional as well as lamp primers were designed using the ptp1 region of n. bombycis (accession no. ky636450) using the primer 3.0 and lamp designer software, respectively. six primers for lamp consisting of two outer primers, two inner primers and two optional loop primers were designed. the primer design was performed by using the online tool lamp designer version 1.15. (http://premierbiosoft.com/crm/jsp/com/pbi/crm/client side/productlist.jsp) and were checked for cross homology. all the parameters were set by default. the primer design chart and sequence are presented in fig. 1 and table 1, respectively. optimization of lamp reaction lamp was carried out in a 25 µl reaction consisting of 2.5 µl of 10x isothermal buffer (20 mm tris-hcl, 10 mm (nh4)2so4, 150 mm kcl, 2 mm mgso4, 0.1% tween 20), 2 mm mgso4, 3.5 mm dntp’s, 0.1% triton-100, 1m betaine, 120 mm of hnb (), 1.6 µm of inner primers (fip and bip), 0.4 µm of loop primers 0.1 µm of outer primers, 8u of bst polymerase, 1µl of template dna and finally sterile water was added to make the final reaction volume to 25 µl. the mixture was incubated at 65 °c for different time periods (15, 30, 60, 90 min) and the reaction was terminated at 80 °c for 5 min. detection of lamp products in our study hnb is used for visual detection of lamp products. hnb is used as the metal-ion indicator which monitors the change in the mg2+ ion concentration during lamp reaction and allows the positive reaction to be detected in sky blue color and the negative in violet which can be detected easily by the naked eyes. the amplified lamp product can also be resolved on agarose gel, which produces a ladder like banding pattern. copy number calculation of ptp1 gene using qpcr quantitative pcr was performed to calculate the copy number using sybr green i technology on agilent stratagene mx3005p qpcr instrument. the concentration was determined using the spectrophotometer and 10-fold serially diluted ptp1 plasmid samples were used as template to obtain the standard curve. pcr cocktail was made with 1 µl of template dna followed by addition of 1x sybr green master mix and 10 pmol of each primer. the reaction was performed for 40 cycles under the following reaction conditions: denaturation at 95 °c for 5 min followed by annealing at 55 °c for 30 sec and elongation at 72 °c for 1 min. fluorescence was detected at the end of each elongation phase. standard curve was established by plotting the threshold cycle (ct) on the y-axis and the natural log of concentration on the x-axis and the total copy number (xt) of the ptp1 gene was calculated by relating the ct value (yt) to the standard curve and then the single cell copy number of the ptp1 gene in the nosema infected silkworm samples was calculated by the following formula: plasmid copy number = pcn 6.023 x 1023 copy/mol x concentration of dna (g) pcn = dna length (bp) x 660 g/mol/µl where 6.023 x 1023 is the avagadro number and 660 daltons is the average weight of dna . the concentration of dna was measured using the uv spectrophotometer. fig. 3 optimization of lamp reaction time for detection of n. bombycis at 65 °c for 15, 30, 60, 90, 120 min along with non template control (ntc). a) visual detection using hnb dye. b) agarose gel based detection of lamp products 356 fig. 4 validation of lamp reaction targeting ptp 1 gene in detecting nosema in different infected moth samples sensitivity of lamp to analyze the sensitivity of lamp reaction the spore dna was used as the template. the sensitivity of lamp was determined by 10-fold serial dilution of the spore dna. the conditions for conventional and qpcr was followed as mentioned above and lamp was performed at 65 °c for 60 min and terminated at 80 °c for 5 min. the normal lamp reaction was carried out with bst 3.0 polymerase was also compared with the warm start master mix (neb#e1700s) using the dna of infected egg, larvae and moth and visualized using hnb dye at a concentration of 120 mm. results the multiplication of nosema spore was found to be sequential starting from midgut followed by trachea, fat body, malpighian tubules and finally infecting the whole body. the larvae were found to be susceptible immediately after molt and hence the infection was given immediately after the third molt. the multiplication was detected after 24 hours of post infection in the midgut using conventional pcr. the dna extraction was performed by modifying the usual method and this took approximately 30 min. this method is neither laborious nor requires any further purification steps and the dna can be directly used as a template for the conventional as well as lamp assay. cloning and sequencing of n. bombycis ptp1 gene revealed the presence of an open reading frame of 1230 bp, coding a polypeptide of 409 amino acids. its overall gc content was found to be 46.50%. the molecular weight (40.4 kda) and the isoelectric point (5.82) of the putative orf was predicted using the prot param tool. the coiled structure of polar tube is a distinctive invasion apparatus of microsporidia, plays a vital role in ejection of sporoplasm into the host cell. the major fig. 5 specificity of developed lamp assay targeting ptp1 gene in detecting the pathogen n. bombycis. lane 1, 2 and 3: npv, dnv-2, nosema infected genomic dna samples 357 fig. 6 agarose electrophoresis profile of lamp assay targeting the polar tube protein 1 gene of nosema infected silkworms at different hours post infection under laboratory conditions aminoacid coded by ptp1 was found to be proline (17.1%) with 10 positively and 14 negatively charged residues. hence, the n. bombycis ptp-1 sequence (ky636450) was chosen as the target region for designing the lamp. the positions of the primers within the (position 774 to 1043) ptp1 sequence are shown in fig. 1. the predicted size of the pcr product (f3/b3) was found to be 270 bp. the sequence of the lamp and conventional pcr primers are listed in table 1. to confirm the specificity of the lamp products, the 270 bp product was cloned into pjet vector and sequenced. the results showed that the 270 bp targeted region of the sequence was 100% homologous with the reported ptp1 sequence. the conventional pcr and microscopic examination was initially used to validate the pathogen multiplication. the lamp assay was performed using the infected moth as a template and the best results were obtained in 25 µl reaction with 2.5 µl of primer mix (40 pmol fip and bip, 20 pmol of loop forward and reverse, 10 pmol of f3 and b3 primers), 2.5 µl of 10x isothermal buffer, 3.5 mm dntps, 2 mm mgso4, 0.1% triton x-100, 1m betaine, 120 mm of hnb and 1µl of bst polymerase 3.0 with strand displacement activity. the reaction was performed in 0.2 ml tubes and kept in water bath at 65 ˚c for 60 min followed by termination at 80 ˚c for 5 min. the positive sample was observed in sky-blue color while the negative sample displayed the violet color (fig. 2a). after assessing the color change, the result was reconfirmed using a 1.5% agarose gel electrophoresis and as expected, the typical ladderlike pattern was observed on gel for the positive sample (fig. 2b). optimization of lamp reaction conditions the optimization of lamp reaction was achieved by performing the reactions at different temperatures and time points with the optimized components described above. positive results were obtained at 65 ˚c within the time interval of 30 to 90 min and prominent bands were found on agarose gel, in case of the samples kept at 65 ˚c for 60 min. lamp reaction at 65 ˚c for 60 min was optimized for ptp1 primer (fig. 3a,b). when the reaction time reached 30 min, the color of lamp products slightly changed and the intensity of ladder-like pattern on gel electrophoresis was low, indicating that the amplification efficiency of lamp was low at 30 min. therefore, the appropriate reaction condition of the established lamp method was set at 65 °c for 60 min. validation of the lamp products to further confirm the lamp products, the product was cloned in pjet vector and the pcr verified positive clones were forwarded for sequence analysis. the sequencing results showed that the 270 bp target fragment was 100% homologous to the n. bombycis ptp1 gene that was used for designing of the primers. these results indicated that the lamp products were specifically amplified from the ptp1 gene of n.bombycis. specificity of lamp the specificity of the lamp primers for the detection of n. bombycis was analyzed by using dna samples from different infected moths. a skyblue color and a ladder-like pattern was observed on agarose gel, in case of the positive samples, whereas the negative sample remained violet in color. the sky-blue color and ladder-like pattern of bands were generated by the dna of n. bombycis (fig. 4). the ptp1 primers were validated and used to test the cross reactivity with the samples infected with bmbdv and bmnpv. the ladder like banding pattern was observed only in the nosema infected samples while the silkworm b. mori infected with bidenso virus and nucleopolyhedrovirus (bmbdv 358 fig. 7 sensitivity comparison of lamp reaction using serially diluted spore dna of n.bombycis. a) visual detection of lamp products using hnb dye. b) agarose based detection of lamp products. c) agarose electrophoresis profile of conventional pcr product and the white arrow indicates the amplified ptp 1 gene with a product size of 270 bp and bmnpv) showed negative results under similar conditions (fig. 5a,b). conventional pcr with ptp1 specific primers (ptp1 f3/b3) also showed good specificity. this result indicated that the lamp primers can be used to specifically detect n. bombycis. the sensitivity was also tested on artificially inoculated silkworms wherein the pathogen was detected at 24 hpi using the lamp assay under laboratory conditions (fig. 6). sensitivity of lamp and pcr absolute quantification was done using realtime pcr for calculating the exact copy number of the plasmid cloned with ptp1 gene. to determine the detection limit, the number of ptp1 gene copies in 100 ng/µl of spore dna was calculated from their respective ct (cycle threshold) using the linear equation of ptp1 standard curve. the conventional pcr and lamp reactions were performed with tenfold serially diluted spore dna. pcr products were resolved through agarose gel electrophoresis while the products of lamp assay were detected by hnb-visualization as well as through agarose gel electrophoresis. a 270 bp band was specifically amplified and successfully detected by pcr when the spore dna template was diluted to 1x105 copies/ml and the ladder-like patterns of the lamp products were observed from 1x108 copies/ml to 1x103 copies/ml indicating that the detection limit was 1x103 copies/ml (fig. 7a,b,c). thus, lamp was 2-fold more sensitive than pcr. according to hnb-visualization and gel electrophoresis all the samples tested for reproducibility were positive thereby indicating that the established lamp had good robustness and reproducibility. further, the optimized lamp reaction with bst 3.0 polymerase was compared with the commercial master mix (warm start master mix, new england biolabs, cat no. e1700) using the infected egg, larvae and moth which also revealed similar results (fig. 8a,b,c). discussion microsporidiosis is a deadly disease caused by the microsporidia n. bombycis in silkworm b. mori. the pathogen is unique and can spread from mother moth to egg through transovarian mode of transmission and by the consumption of infected leaf (horizontal transmission) or contaminated egg surface (transovum transmission). the early detection of pathogen prevents the spread of the disease and crop loss. the ptp1 gene of microsporidia was found to be one of the essential genes involved in host cell invasion. the ptp1 gene was cloned, characterized and was found to be rich in proline content, a common feature of polar tube proteins found in the other microsporidians. it was already reported that the amino acid proline accounts for the essential component of polar tube proteins. it forms a fixed kink with polypeptide resulting in chain rigidity which is important for the polar tube protein during the discharge and passage of sporoplasm (keohane et al., 1996). many molecular-based methods have been developed for 359 fig. 8 comparison of lamp assay using bst 3.0 polymerase with warm start master mix. a) visual detection of lamp products using hnb dye. b) agarose based detection of lamp products amplified using bst 3.0 polymerase (lane 1 3) and commercial warm start polymerase (lane 4 6). lane 1 and 4: nosema infected tissue from the abdomen of silkmoth. lane 2 and 5: nosema infected eggs. lane 3 and 6: nosema infected larvae the detection of n. bombycis, however, such techniques were found to be very expensive and time consuming. lamp is a new on-site diagnostic method with straightforward principles, and comprehensive protocol that allows a semi-skilled person to perform the assay without any sophisticated instrument (notomi et al., 2000; abdullahi et al., 2015). in lamp reaction, the specific region of the dna sequence was amplified using bst dna polymerase and the reaction takes place at a high speed, under constant temperature conditions (60-65 °c) with high accuracy for the specific region (nagamine et al., 2002). in this study, we have developed and analyzed the lamp assay for detection of microsporidiosis in b. mori. it is considered as an effective gene amplification method to be used for pen side diagnostic tests, that can be performed at the field level without any technical expertise or instrument (parida et al., 2008). this method allows naked eye detection by addition of dna intercalating dyes like sybr green, picogreen, propidium iodide or metal-ion indicators such as calcein or hnb (hill et al., 2008). in 2014, lamp was developed for n. bombycis detection only in b. mori eggs using filter paper based card known as fta (flinders technology associates) card as template by targeting the large and small subunit of rrna using 4 primers and the eb1 gene was used as the target for detection of pathogen in the infected eggs (yan et al., 2014; ping et al., 2015). in our study, for the first time, we have used the lamp for detecting microsporidian n. bombycis at all stages of the silkworm i.e., egg, larvae and moth with the ptp1 gene as the target sequence for the detection of microsporidian infection. a new dna extraction method was followed to extract dna from midgut of the infected larvae, egg and moth which is advantageous in two different ways. firstly, when compared with the conventional method, the new method requires only minimal amount of tissue without the requirement of further purification. secondly, the new method allows extraction within 30 min when compared to the conventional method which involves longer incubation time to act as a rate limiting step (singh et al., 2011). hence, this method was employed for dna extraction and further used for conventional pcr, lamp and qpcr. the metal ion indicator was added prior to the reaction to avoid non-template amplification which is one of the major constraints of lamp. the lamp reaction was optimized using six different primers targeting eight regions of the ptp1 360 sequence of n. bombycis. nagamine et al. (2002), reported that the reaction time involved for lamp could be shortened using loop primers, however, it is not mandatory for the lamp reaction to take place. even though the two loop primers were optional, they were included in our study to accelerate the reaction and the optimization of lamp reaction conditions was achieved by performing the reaction at different time intervals, temperatures and primer concentrations. in few experiments, a higher level of detection limit was reported for the lamp when compared to the pcr while in other experiments, the detection limit result of lamp was equivalent to the pcr (bakheit et al., 2008; zhao et al., 2010; tang et al., 2011). the difference in outcome of these experiments is probably related to the the characteristics of the primers and the sequence of the specific region in dna that can affect the sensitivity and specificity of the molecular technique (white et al., 2006). the final lamp conditions comprised of 40 pmol of fip and bip, 10 pmol of f3 and b3, 20 pmol of loop f and r and 8u of bst 3.0 polymerase (new england biolabs, ipswich, ma). in the 25 µl of lamp reaction, even though 1m betaine (sigma-aldrich, bangalore, india) and 0.1% triton x-100 were optional, they were added to the reaction to prevent secondary structure formations in the gc rich sequence regions. since conventional pcr is usually used in the detection of disease, due to its simplicity and cost when compared to real-time pcr, the sensitivity of lamp was compared with conventional pcr. in this study, the plasmid containing ptp1 gene was serially diluted and used to compare the sensitivity of the lamp. results revealed that, the lamp had 2-fold higher sensitivity than the conventional pcr. however, the higher specificity and sensitivity has a disadvantage as it may lead to cross reactivity, which can cause non-template amplification during the reaction (mori et al., 2001). hence, filtered tips were used to prevent aerosol contamination and closed reaction tubes and proper disinfection of the pipettes with ethanol are recommended (wang et al., 2009). yan et al., (2014) has reported the detection of n. bombycis in single silkworm egg, however, our study showed positive results when dna was extracted from ten eggs and this may be either due to the spore load in the eggs or the differential dna extraction procedure. the comparison of our optimized lamp assay using bst 3.0 polymerase cocktail with that of the commercial lamp kit (new england biolabs, ipswich, ma) revealed that the developed method would be economical, reliable and field based assay sensitive in detecting the pathogen in all the developmental stages of silkworm. the previously reported electrochemical and ph based detection of lamp targeting different region of polar tube protein gene requires technical expertise and detection was found to be tedious compared to the developed lamp assay (xie et al., 2014; 2015). in conclusion, the developed lamp method was found to identify the infection of n. bombycis in all the stages of silkworm b. mori. it has a better sensitivity than conventional pcr. furthermore, the presence of the pathogen can be detected with the naked eye immediately after the lamp reaction by using hnb. this technique can thus be a preferable choice over the conventional standard pcr for detection of n. bombycis in silkworm seed centers. acknowledgement we thank central silk board, ministry of textiles, govt. of india, bangalore for providing the necessary facilities to carry out the research work. the authors are grateful to department of biotechnology (dbt), government of india for the financial support, grant number: bt/pr/5405/tds/121/14/2015 dated 09th feb 2017. references abdullahi uf, naim r, taib wrw, saleh a, muazu a, aliyu s, et al. loop-mediated isothermal amplification (lamp), an innovation in gene amplification: bridging the gap in molecular diagnostics; a review. indian j. sci. technol. 8: 1-12, 2015. bakheit ma, torra d, palomino la, thekiso om, mbati pa, ongerth j, et al. sensitive and specific detection of cryptosporidium species in pcr-negative samples by loop-mediated isothermal dna amplification and confirmation of generated lamp products by sequencing. vet. parasitol. 158: 11-22, 2008. chen x, ma l, qiang s, ma d. development of a loop-mediated isothermal amplification method for the rapid diagnosis of ascochyta rabiei l. in chickpeas. sci. rep. 10: 25688, 2016. didier es, maddry ja, kwong cd, green lc, snowden kf, shadduck ja. screening of compounds for antimicrosporidial activity in vitro. folia parasitol. 45: 129-139, 1998. fu zw , he zk, cai s, liu h, he x, li m, et al. quantitative pcr for detection of n. bombycis in single silkworm eggs and newly hatched larvae. j. microbiol. methods 120: 72-78, 2016. hatakeyama y, hayasaka s. a new method of pebrine inspection of silkworm egg using multiprimer pcr. j. invertebr. pathol. 82: 148151, 2003. hill j, beriwal s, chandra i, paul vk, kapil a, singh t, et al. loop-mediated isothermal amplification assay for rapid detection of common strains of escherichia coli. j. clin. microbiol. 46: 28002804, 2008. huang ch, lin wh, lien yy, hsueh sc. development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of chicken anaemia virus. j. appl. microbiol. 108: 917-924, 2010. ghosh k, weiss lm. molecular diagnostic tests for microsporidia. interdiscip. perspect. infect. dis. 2009: 926521, 2009. lee ms, su tu, lien yy, sheu sc. the development of loop-mediated isothermal amplification (lamp) assays for the rapid authentication of five forbidden vegetables in strict vegetarian diet. sci. rep. 7: 44238, 2017. mori y, nagamine k, tomita n, notomi t. detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. biochem. biophys. res. commun. 289: 150-154, 2001. https://www.ncbi.nlm.nih.gov/pubmed/?term=lin%20wh%5bauthor%5d&cauthor=true&cauthor_uid=19737344 https://www.ncbi.nlm.nih.gov/pubmed/?term=lien%20yy%5bauthor%5d&cauthor=true&cauthor_uid=19737344 https://www.ncbi.nlm.nih.gov/pubmed/?term=hsueh%20sc%5bauthor%5d&cauthor=true&cauthor_uid=19737344 361 nagamine k, hase t, notomi t. accelerated reaction by loop-mediated isothermal amplification using loop primers. mol. cell. probes. 16: 223-229, 2002. notomi t, okayama h, masubuchi h, yonekawa t, watanabe k, amino n, et al. loop-mediated isothermal amplification of dna. nucleic acids res. 28: e63, 2000. parida m, sannarangaiah s, dash pk, rao pvl, morita k. loop mediated isothermal amplification (lamp): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases. rev. med. virol. 18: 407-421, 2008. ping lj, wei c, wei yy, ying wj, long yj. detection of pebrine disease in bombyx mori eggs with the loop-mediated isothermal amplification (lamp) method based on eb1 gene. acta entomol. sinica. 58: 846-855, 2015. shimanuki h, knox da. diagnosis of honey bee diseases. agriculture handbook, washington dc, usa, 2000. singh hr, unni bg, neog k, bhattacharya m, wann sb. isolating silkworm genomic dna without liquid nitrogen suitable for marker studies. afr. j. biotechnol. 10: 11365-11372, 2011. tang mj, zhou s, zhang xy, pu jh, ge ql tang xj, et al. rapid and sensitive detection of listeria monocytogenes by loop-mediated isothermal amplification. curr. microbiol. 63: 511-516, 2011. wang y, lan qk, zhao x, zhu z, cheng y. development and application of loop-mediated isothermal amplification for detection of genetically modified crops. j. integr. agr. 42: 1473-1477, 2009. white pl, linton cj, perry md, johnson em, barnes ra. the evolution and evaluation of a whole blood polymerase chain reaction assay for the detection of invasive aspergillosis in hematology patients in a routine clinical setting. clin. infect. dis. 42: 479-486, 2006. yan w, shen z, tang x, li x, xiao s, yue y, et al. detection of nosema bombycis by fta cards and loop-mediated isothermal amplification (lamp). curr. microbiol. 69: 532-540, 2014. zhao x, li y, wang l, you l, xu z, li l, et al. development and application of a loopmediated isothermal amplification method on rapid detection escherichia coli o157 strains from food samples. mol. biol. rep. 37: 21832188, 2010. xie s, yuan y, chai y, yuan r. tracing phosphate ions generated during loop-mediated isothermal amplification for electrochemical detection of nosema bombycis genomic dna ptp1. anal. chem. 87: 10268-10274, 2015. xie s, yuan y, song y, zhou y, li z, chai y, et al. using the ubiquitous ph meter combined with a loop mediated isothermal amplification method for facile and sensitive detection of nosema bombycis genomic dna ptp1. chem. commun. 50: 15932-15935, 2014. https://www.cabdirect.org/cabdirect/search/?q=au%3a%22liu+jiping%22 https://www.cabdirect.org/cabdirect/search/?q=au%3a%22cheng+wei%22 aif-1 isj 12: 129-141, 2015 issn 1824-307x research report the allograft inflammatory factor-1 (aif-1) homologous in hirudo medicinalis (medicinal leech) is involved in immune response during wound healing and graft rejection processes t schorn1, f drago2, m de eguileor1, r valvassori1, j vizioli2, g tettamanti1, a grimaldi1 1department of biotechnology and life sciences, university of insubria, varese, italy 2inserm u1192, laboratoire de protéomique, réponse inflammatoire, spectrométrie de masse (prism), université lille 1, cité scientifique, 59655 villeneuve d’ascq, france accepted april 9, 2015 abstract allograft inflammatory factor-1 (aif-1) is a 17 kda cytokine-inducible calcium-binding protein that in vertebrates plays an important role in allografts immune response. since its expression is mainly limited to the monocyte/macrophage lineage, it was recently suggested that it could play a key role during inflammatory responses, allograft rejection, as well as in the activation of macrophages. to clarify this point we have focused our research on the possible role of aif-1 during the inflammatory response after injury in the leech hirudo medicinalis (annelida, hirudinea). this invertebrate is an excellent animal model since the responses evoked during inflammation and tissue repair are clear and easily detectable and have a striking similarity with vertebrate responses. moreover the analysis of an est library from h. medicinalis cns, revealed the presence of a gene, named hmaif-1/alias hmiba1, showing a high homology with vertebrate aif-1. our data show that the related protein, named hmaif-1, is constitutively expressed in unlesioned leeches and that dramatically increases 48 h after wounds and tissue transplants. immunohistochemistry experiments, using a specific anti hmaif-1 polyclonal antibody, shows that this factor is present in spread, cd68+ /cd45+ macrophage-like cells. a few days after experimental wounding of the body wall, the amount of these immunopositive cells increases at the lesion site. in conclusion here we propose that in leech hmaif-1 factor is involved in inflammation events like its vertebrate counterparts. key words: leech; cd45; aif-1; wounds; grafts   introduction the allograft inflammatory factor-1 (aif-1), also called mrf-1, iba1, and daintain, is an interferon-γ inducible cytoplasmic cytokine of 17 kda, (alkassab et al., 2007). it contains a ca2+binding ef-hand domain and has been identified first in chronic rejection of rat cardiac allografts (utans et al., 1995). aif-1-like factors, have been described in other groups of metazoans and share a similar aminoacid structure and a very well preserved functional role. aif-1 expression increases significantly after transplantation, wounds or bacterial infections both in vertebrates (utans et al., 1995; watano et al., 2001; deininger et al., 2000, 2002; autieri and chen, 2005; alkassab et al., 2007) ___________________________________________________________________________ corresponding author: annalisa grimaldi department of biotechnology and life sciences university of insubria via j. h. dunant 3, 21100 varese, italy e-mail: annalisa.grimaldi@uninsubria.it and in invertebrates, such as sponges (kruse et al., 1999), molluscs (de zoysa et al., 2010; zhang et al., 2011, 2013; li et al., 2012) and echinoderms (ovando et al., 2012). since the release of aif-1 is a ca2+-dependent mechanism, it seems that this protein may play a role in cell-cell interactions under inflammatory conditions (tanaka and koike, 2002). in particular, the ability to bind calcium allows developing distinct pathways of signal transduction, protein expression and cell cycle regulation during the activation of macrophages and microglial cells. therefore aif-1 results to be a modulator of the immune response during macrophage activation and tissue regeneration (alkassab et al., 2007; pawlik et al., 2008). interestingly, aif-1 shows the same functions and colocalizes with a leukocyte-specific member of the transmembrane ptpase family namely cd45, ubiquitously expressed on the surface of all nucleated cells of hematopoietic origin (alkassab et al., 2007; sommerville et al., 2012; jeong et al., 2013;   129 mailto:annalisa.grimaldi@uninsubria.it fig. 1 acid phosphatase (acp) reaction on cryosections (a, c, e, g, i) and tem analyses on ultra-thin sections (b, d, f, h, j) from h. medicinalis body wall unlesioned (a, b) and surgically wounded analyzed after at 24 h (c, d), 48 h (e, f), 72 h (g, h) and 7 days (i, j) from injury. compared to control sections (a, b), after injury numerous macrophages acp positive (red in c, e, g, i) are visible migrating among muscles (m), under the epithelium (e) and close to pseudoblastema (p). detail at tem of uninjured body wall of leeches (b) and wounded leeches (d, f, h, j). after injury macrophage-like cells moved in the connective tissue (ecm) with ruffled surfaces (arrowheads in d) and projections of variable thickness (arrowheads in j). in the cytoplasm phagocytized material (arrowhead in f) and phagolysosomes (arrowhead in h) are visible. bars in a, c, e, g, i: 100 μm; bar in b, f, h, j: 2 μm; bars in d: 4 μm.   130 li et al., 2013; schorn et al. 2014). cd45 is a cell surface glycoprotein that, in vertebrates, is implicated in integrin-mediated adhesion of macrophages (roach et al., 1997; zhu et al. 2011; st-pierre and ostergaard, 2013). it plays a role in regulating the functional responsiveness of cells to chemoattractants (roach et al., 1997; mitchell et al., 1999), affecting the normal feedback mechanisms that are required to maintain adhesion and phagocytic activity. indeed it has been reported that monocytes highly express aif-1 and cd45, whereas resident microglia express aif-1 but weakly and barely express cd45, confirming that both cd45 and aif-1 might be involved in macrophage migration (jeong et al., 2013). in vertebrates, despite the extensive investigation focused on both molecular characteristics and expression level of aif-1 during the inflammatory response or wound healing, the direct relationship between aif-1 and cd45 expression and macrophage activation/migration during the inflammation phase after injury or graft remains unclear. it is probably because the study of the immune response in vertebrates appears to be a difficult challenge, primarily due to the complexity of these organisms. we recently characterized in the central nervous system (cns) of the leech a gene showing high similarity with vertebrate aif-1, named hmiba1/alias hmaif-1 (genbank accession number kf437461, drago et al., 2014). in peripheral tissues, the protein is mainly located in the macrophages and its production increases in body wall after bacterial injection (schorn et al., 2014). we presently focused our research on the possible role of aif-1 during the immune response after injury and grafts in the leech hirudo medicinalis (annelida, hirudinea). this invertebrate, offering simpler anatomy and lacking complex feed-back control systems typical of vertebrates, represents a great alternative for studying basic steps of immune responses (de eguileor et al., 2000b, 2001b, 2003, 2004; grimaldi et al., 2006, 2009, 2011; schikorski et al., 2009). h. medicinalis is characterized by the absence of a true vascular system within the muscular body wall and by the presence of a specific tissues, the botryoidal tissue, located close to the digestive system and involved in hematopoietic cells production and in the formation of new vessels (grimaldi et al., 2006). the effects of lesion or grafts in leech body wall are rapidly induced and after 24 h the inflammatory phase is characterized by an influx of macrophages that are responsible of phagocytosis and immune cytotoxicity, clean the stimulated area and release various growth factors (de eguileor et al., 1999, 2000a, b; grimaldi et al., 2006; tettamanti et al., 2006). in parallel, remodeling of the botryoidal tissue induces the formation of new vessels and inside the lumen of these growing vessel clusters of hematopoietic precursors develop. these cells, after transendothelial migration, diffuse in the wounded area and differentiate into mature leucocytes that mediate the inflammatory response (grimaldi et al., 2006). in order to better understand the role of hmaif1 after wound healing and graft stimulations, immunohistochemistry and western blot studies have been performed to determine the localization and the modulation of this gene in leech body wall. the presence of hmaif-1 in uninjured, experimentally injured and grafted tissues was established using the specific rabbit anti-h. medicinalis aif-1 polyclonal antibody. ultrastructural analysis at electron microscope, the acid phosphatase enzymatic histochemical reaction and immunohistochemical analysis using the polyclonal antibody anti-cd68 and anti-cd45 macrophage cell markers were performed to characterize the cells involved in the immune response and expressing hmaif-1. material and methods animals and treatments leeches (hirudo medicinalis, annelida, hirudinea, from ricarimpex, eysines, france) measuring 10 cm were kept in tap water at 20 °c in aerated tanks. animals were fed weekly with calf blood. animals were randomly divided into separate experimental groups according to different protocols and treatments. each treatment (wounds or tissue collection for grafting) was performed at the level of the 80th superficial metamere. before each experiment, leeches were anaesthetized with a 10 % ethanol solution and then dissected. the body tissues were removed at specific time points after treatments. group 1: uninjured control leeches to provide information on normal body organization. group 2: leeches for each time points (24 h, 48 h, 72 h, 7 days) were injured at about the 80th superficial metamere with a razor blade, in order to assess the modulation of hmaif-1 during the wound healing. group 3: leeches for each time points (24 h, 48 h, 72 h, 7 days) were used as hosts and donors for autografts and allografts. surgical grafting was performed at the distal dorsal portion of leeches, about 2/3 from the oral extremity (at about the 80th superficial metamere): grafts were sutured with dafilon® surgical synthetic monofilament (b. braun) to avoid transplant loss due to contraction of the muscular body wall. grafted leeches were kept in moist chambers for a post-surgical recovery period of 24 h, and subsequently placed in water tanks. the rate of successful transplantation experiments for all graft types was 90 %. all leeches survived surgery and were able to move and feed following recovery from anesthesia. autograft-bearing leeches: at about the 80th superficial metamere from the oral sucker, a block of 2mm×2mm×2mm was excised and afterwards replaced in the same hollow; allograft-bearing leeches: h. medicinalis host received a block of 2mm×2mm×2mm body wall excised from the 80th superficial metamere of a conspecific individual. grafts were sutured with dafilon® surgical synthetic monofilament as indicated above. electron microscopy leech tissues, dissected from the area of wound or the graft, were fixed for 2 h in 0.1 m cacodylate buffer at ph 7.4, containing 2 %   131 glutaraldehyde. specimens were then washed in the same buffer and post-fixed for 1 h with 1 % osmium tetroxide in cacodylate buffer, ph 7.4. after standard serial ethanol dehydration, specimens were embedded in an epon-araldite 812 mixture. sections were obtained with a reichert ultracut s ultratome (leica, wien, austria). ultrathin sections (80 nm in thickness) were placed on copper grids, stained by uranyl acetate and lead citrate and observed with a jeol 1010 ex electron microscope (jeol, tokyo, japan). data were recorded with a morada digital camera system (olympus, tokyo, japan). for immunogold cytochemistry, samples were fixed for 2 h with 4 % paraformaldehyde and 0.5 % glutaraldehyde in phosphate buffered saline (pbs), then washed in the same buffer. after a standard step of serial ethanol dehydration they were embedded in an epon-araldite 812 mixture (sigma, st. louis, mo) and sectioned with a reichert ultracut s ultratome (leica, wien, austria). ultrathin sections (80 nm in thickness), after etching with naoh 3 % in absolute ethanol (causton, 1984), were incubated for 30 min with pbs containing 2% bovine serum albumin (bsa) and then for 1 h with the primary rabbit polyclonal antihmaif-1 antibody (working dilution 1:50). primary antibodies were visualized by immunochemical staining with secondary goat anti-rabbit igg (h+l)gold conjugate antibodies (ge healthcare amersham, buckingamshire, uk) (particle size, 10 nm) diluted 1:40 (incubation 30 min at room temperature). in control sections, primary polyclonal anti-hmaif-1 antibody was substituted with rabbit pre-immune serum (1:20,000) or primary antibody was omitted and sections were treated with bsa containing pbs and incubated only with the secondary antibodies. samples were counterstained with uranyl acetate in water and observed with a jeol 1010 ex electron microscope (jeol, tokyo, japan). data were recorded with a morada digital camera system (olympus). acid phosphatase reaction (acp) leech tissues, dissected from unlesioned animals and from area of wound or graft, were embedded in polyfreeze tissue freezing medium (oct) (polysciences, eppelheim, germany) and immediately frozen in liquid nitrogen. cryosections (7 μm in thickness), obtained with a leica cm 1850 cryotome, were rehydrated with pbs for 5 min, incubated with sodium acetate-acetic acid 0.1 m buffer for 5 min and then in the reaction mixture (sodium acetate-acetic acid 0.1 m buffer, 0.01 % naphtol as-bi phosphate, 2 % nndimethylformamide, 0.06 % fast red violet lb and mncl2 0.5nm) for 90 min at 37 °c. after washings in pbs, the slides were mounted with pbs/glycerol 2:1 and observed with the light microscope nikon eclipse ni (nikon, tokyo, japan). images were taken with the digital camera nikon digital sight dssm (nikon, tokyo, japan). indirect immunofluorescence staining serial cryosections (7 μm in thickness) were stained by crystal violet and basic fuchsine for a morphological view or used for immunofluorescence staining. slides, rehydrated with pbs for 5 minutes, were pre-incubated for 30 min with pbs containing 2 % bovine serum albumin (bsa) before the primary antibody incubation (1 h at 37 °c). the primary antibodies used were: rabbit polyclonal anti-human cd45 (twin helix, milano, italy) which reacts with leech hematopoietic cells (de eguileor et al., 2003) diluted 1:100, rabbit polyclonal anti-human cd68 (santa cruz biotechnology) which reacts with leech macrophages (grimaldi et al., 2006) diluted 1:100 and rabbit anti-hmaif-1 (drago et al., 2014) diluted 1:1000. the use of antibodies generated against mammalian cd antigens to detect macrophages in leech is supported by several data from the literature on leeches (grimaldi et al., 2004, 2006; de eguileor et al., 2000a, b) and on animals phylogenetically related to annelids (cossarizza et al., 1996) and sipunculids (blanco et al., 1997). the washed specimens were incubated for 1 h at room temperature with the appropriate secondary antibodies diluted 1:200 (abcam®, cambridge, uk): goat anti-rabbit fitc-conjugated (excitation 493 nm, emission 518 nm), goat anti-rabbit cy3-conjugated (excitation 562 nm, emission 576 nm), goat-anti rabbit cy5-conjugated (excitation 650 nm, emission 672 nm). double labelling experiments were performed as already described (grimaldi et al., 2009): a) to detect hmaif-1, hmaif-1/cd45 or hmaif-1/cd68 the anti hmaif-1 was applied first, then sections were incubated with the secondary antibody goat anti-rabbit fitc conjugated. after washing the samples were incubated with the antibody anti cd45 or anti cd68. subsequently, the sections were treated with the secondary cy5 conjugated goat anti-rabbit antibody; b) to detect cd45/cd68, the anti cd45 was applied first, then sections were incubated with the secondary cy5 conjugated goat anti-rabbit antibody. after washing samples were incubated with the antibody anti cd68 and subsequently with the secondary antibody goat anti-rabbit fitc conjugated. according to würden and homberg (1993), to inhibit binding of the primary antiserum of the second staining cycle to the goat anti-rabbit iggs that were applied in the first sequence, the sections were incubated with rabbit igg (jackson immunoresearch) at 1:25 for 2 h. nuclei were stained by incubating for 15 min with 49,6diamidino-2-phenylindole (dapi, 0.1 mg/ml in pbs, excitation 340 nm, emission 488 nm). in control samples, primary antibodies were omitted and sections, treated with bsa-containing pbs or with the rabbit pre-immune serum (1:20,000), were incubated only with the secondary antibodies. according to schnell et al., 1999, after immunocytochemistry, the sections were treated with 1 mm cuso4 in 50 nm ammonium acetate buffer (ph 5.0) for 15 min and then washed in distilled water and pbs. application of cuso4 for 10 minutes after immunohistochemistry substantially reduced tissue autofluorescence while preserving the specific fluorochrome signal. the slides were mounted in citifluor (citifluor ltd, london, uk) with coverslips and examined with a nikon fluorescence microscope or with a confocal laser microscope (leica tcs sp5). images were combined with adobe photoshop (adobe systems, inc.).   132 fig. 2 morphological (optical microscopy) and immunohistochemical (fluorescence microscopy) analysis of cryosections from h. medicinalis body wall unlesioned (a-c) and surgically wounded and analyzed after 24 h (d f), 48 h (g i), 72 h (j l) and 7 days (m o) from injury. numerous migrating cells (arrowheads in d, g, j, m) among muscle fibers (m) and close to the pseudoblastema (p) were visible in injured leeches. immunohistochemistry using the rabbit polyclonal anti-hmaif-1 antibody (red) marks the cells in active migration towards the injured area (arrows). nuclei were counterstained with dapi (blue). no signal is detected in control experiment where the primary antibody was omitted (c, f, i, l, o). bars in a l: 100 μm.   133 biochemical procedures h. medicinalis tissues from the unstimulated body wall or from wounded areas were frozen in liquid nitrogen and then homogenized with a mortar. for sds-polyacrylamide gel electrophoresis (sdspage), leech homogenates were suspended in extraction buffer 2x laemmli's buffer in the presence of a protease inhibitor cocktail (sigma, milan, italy); the particulated material was removed by centrifugation at 13000 rpm for 10 min at 4 °c in a refrigerated eppendorf minispin microcentrifuge. supernatants containing total protein extracts were denatured at 100 °c for 10 min and loaded on 10 % acrylamide minigels for sds-page analyses.molecular weights were determined by concurrently running broad range standards from bio-rad (bio-rad, richmond, ma, usa). western blot proteins separated by sds-page were transferred onto bio-rad nitrocellulose filters. membranes were then saturated with 5 % non fat dried milk in tris buffered saline (tbs, 20 mm trishcl buffer, 500 mm nacl, ph 7.5) at room temperature for 2 h, and incubated for 90 min with a rabbit anti-hmaif-1 antibody (1:5000 dilution in 5 % tbs-milk) or rabbit polyclonal anti-human cd45 igg (twin helix) diluted 1:1000. the membrane was washed three times with tbs-tween 0.1 % and then incubated with the secondary anti-rabbit igg antibody hrp-conjugated (jackson immunoresearch laboratories, inc., west grove, usa), diluted 1:5000. after washing, the immunocomplexes were revealed with luminol liteablot® plus enhanced chemiluminescent substrate (euroclone s.p.a., pero, italy). bands were normalized, using the image j software package (http://rsbweb.nih.gov/ij/download.html), with the housekeeping protein gapdh, which were detected with a rabbit polyclonal anti-human gapdh igg (proteintech™, chicago, usa) diluted 1:2000. the expression level of hmaif-1 in treated leeches was reported relatively to control uninjured animals. experiments were performed in triplicate and data represent the mean values ± sem. statistical significance was assessed by an unpaired student’s t test. results morphological, immunohistochemical and biochemical characterization of cells recruited in the wounded area ultrastructural and enzyme histochemical analyses as already described in previous works (grimaldi et al., 2004, 2006; tettamanti et al., 2004), following tissue damage, wound healing initiates with an inflammatory phase characterized by a massive migration of immune cells, fibroblasts and myofibroblasts-like cells, towards the lesioned area. wound size and retraction was then obtained by the formation of a pseudoblastema region formed by the myofibroblasts-like cells (huguet and molinas 1994, 1996; grimaldi et al., 2009, 2011). enzyme histochemical and ultrastructural analyses showed that in unlesioned leeches (figs 1a, b) a few cells were located in the connective tissue, underlying the body wall epithelium and surrounding the fields of muscle fibers and were acp positive. by contrast, after injury numerous migrating cells were highly positive for acp reaction (figs 1c, e, g, i), confirming their phagocytic activity. these migrating cells were clearly visible among muscle layers and reached the healing area at which they increased numerically in relation to the time elapsed after the lesions were inflicted. in particular, a significant increase of acp positive cells was mainly observed in the area surrounding the pseudoblastema 48 h after the injury (fig. 1e). ultrastructural analysis of injured tissues at tem highlighted the presence of numerous activated macrophages-like cells in the connective tissue close to the lesioned region (figs 1d, f, h, j). these cells were characterized by a certain degree of surface ruffling, pseudopodia, and their phagocytic activity was mainly evident 48 h after the injury (fig. 1f). hmaif-1 immunolocalization as our previous data showed (schorn et al., 2014), hmaif-1 was constitutively expressed in unlesioned animals (figs 2a c). this factor was mainly expressed in cells located in the connective tissue surrounding the fields of muscle fibres. cryosections of injured leeches analyzed after 24 h, 48 h, 72 h and 7 days from lesion (figs 2d o), were immunostained with the antibody against hmaif-1. cells expressing hmaif-1 were found dispersed in the extracellular matrix (ecm) surrounding the groups of muscle cells and close to the wound healing region of injured leeches (figs 2d l). our data showed that hmaif-1 expression dramatically increased in 24/48 h injured leeches, when a massive migration of cells towards the lesioned area was detectable (figs 2e, k, n). no signal was visible in negative controls experiments, in which primary antibody was omitted (figs 2c, f, i, l, o). characterization of hmaif-1+ cells in order to characterize the hmaif-1+ migrating cells, double immunofluorescent stainings were then performed on cryosections of 24 h, 48 h, 72 h and 7 days injured leeches body wall using the following primary antibodies combination: hmaif-1/cd45, hmaif-1/cd68, cd45/cd68. our data showed, in all sections, that the hmaif-1+ cells dispersed in the ecm surrounding the groups of muscle fibers (figs 3a, b) and close to the wound healing region (figs 3d, e) expressed also the common leukocyte marker cd45 and the macrophage cell marker cd68. control experiments performed in the absence of the primary antibodies were negative for all the samples (figs 3c, f). immunogold golds experiments confirmed the expression of hmaif-1 in cd45+/cd68+ macrophage-like cells (figs 3g i). biochemical analysis western blot assays were performed to assess the expression profile of hmaif-1 (figs 4a, b) and cd45 (figs 4c, d) in wounded leeches. compared to the basal expression level detected in unlesioned leeches, the amount of hmaif-1 protein significantly change in extracts of lesioned leech body wall, showing a pick of expression after 48 h from injury (fig. 4 b). gapdh   134 fig. 3 after injury numerous migrating macrophages (in yellow) located among muscle fibers (a, b) and close to the pseudoblastema (p) region (d, e) are cd45+/aif-1+ and cd68+/aif-1+. (c, f) negative control experiments where the primary antibodies are omitted. (g-i) immunogold staining (arrowheads) confirms the expression of aif-1, cd45 and cd68 in macrophages cells. bars in a c: 20 μm; bars in d f: 50 μm; bar in g: 100 nm; bars in h, i: 500 nm was used as internal reference and bands intensity appeared homogeneously distributed in the loaded samples (fig. 4a). a similar result was obtained analyzing the expression profile of cd45 (figs 4c, d) compared to the basal expression level detected in unlesioned leeches, the amount of cd45 protein significantly increased in body wall extracts of 48 h and 72 h lesioned leech (fig. 4d). as described above, gapdh was used as internal reference (fig. 4c). morphological and immunohistochemical characterization of cells recruited in the grafted area our previous data demonstrated that selftransplantation caused no rejection but only an inflammatory response, whereas host h. medicinalis   135 fig. 4 western blot analysis. protein extracts of unlesioned (u) and injured leeches after 24 h, 48 h, 72 h and 7 days from injury were probed with the anti-hmaif-1 antibody (a) and cd45 (b). the housekeeping protein gapdh was used as a loading control. in all the samples, the anti-hmaif-1 detected specific immunoreactive bands of about 18 kda (a), according to the molecular weight ladder. (b) hmaif-1 protein was quantified by densitometry from three experiments. **p < 0.05 compared with uninjured leeches. the anti-cd45 detected in all the samples an immunoreactive bands of about 145 kda, according to the molecular weight ladder (c). the housekeeping protein gapdh was used as a loading control. (d) cd45 protein was quantified by densitometry from three experiments. ***p < 0.01 compared with uninjured leeches.   136 leeches rejected both alloand xenografts (tettamanti et al., 2003). in this work, we focused on a possible role of hmaif-1 in the rejection processes. since h. medicinalis respond to allo and xenografts in identical way, in terms of tissue reaction and cell populations involved, the results here presented were relative only to autografts and allografts experiments. leech responses to autograft the grafted area of leeches was characterized by an acute inflammatory reaction involving cell migration among fields of muscle fibers (fig. 5a). these migrating cells, morphologically and functionally already described as macrophage-like cells (tettamanti et al., 2003) positively stained for acp reaction (fig. 5b). the acp+ cells, forming a clot surrounding the autograft, showed a low level of hmaif/cd45 expression (figs 5c, d). leech responses to allograft starting from 24 h after allograft, an acute inflammatory phase started with migrating immunocompetent cells through the ecm. these cells were involved in clot formation and in graft isolation from neighboring tissues. in the timespan of 7 days, non-self grafted tissue was completely surrounded and coated by host cells. most of these cells were macrophages which played a pivotal role with their phagocytic activity directed to remove cell and matrix debris. they were positively stained for acp reaction and highly co-expressed cd45 and hmaif-1 (figs 5e g; i k; m o). no signal was detected in control negative experiments of immunolocalization, were primary antibodies were omitted (figs 5h, l, p). discussion both in invertebrates and vertebrates, inflammation is an acute reaction triggered by different types of lesions and aimed to fulfill two functions: a cytotoxic function to kill infecting microbes and a repair function to regenerate damaged tissues. this process is mediated by specific cells such as macrophages and neutrophils that infiltrate the damaged tissue removing debris and controlling invading microorganisms. these cells synthesize different molecules such as growth factors and cytokines, inducing mesenchymal cell recruitment in the injured or infected area (jeong et al., 2013). in leeches as well proliferation and migration of immune cells are associated to important effects like angiogenesis and fibroplasia and are regulated by different cytokines and growth factors (tettamanti et al., 2004; grimaldi et al., 2006). moreover, in our recent studies we demonstrated that in the leech h. medicinalis the inflammatory factor hmaif-1 is constitutively expressed in untreated animals but is dramatically enhanced after microbial infection. it particularly promotes macrophages and vessels migration towards the stimulated area (schorn et al., 2014). it has been demonstrated that in leech the immune response is based on the same molecules involved in wound healing and regenerative process (schikorski et al., 2008). here we were interested in understanding the possible involvement of hmaif-1 in the regulation of inflammatory response in both wounded and grafted leeches. first we investigated the tissue-specific and temporal expression profile of hmaif-1 factor after different time points. indeed, we found high level of hmaif-1 expression in the tissue of wounded and grafted leeches. in particular, we observed a significant increase of hmaif-1+ cells migrating towards the wounded area or forming a clot around the non self-tissue after 24 48 h from surgery. on the other hand the level of hmaif-1 expression is very low in those cells forming a clot surrounding the autografts tissue. taken together, these findings suggest that, besides cell-mediated defense reactions, the cytokine hmaif-1 is also elicited during wound healing and graft recognition and rejection in leeches. a steady increase of hmaif-1 was mainly detected in the initial stages of inflammation, 24 48 h after surgery, and decline by 7-10 days after surgery. these data confirm that, in leech as well, hmaif-1 is involved in early events that trigger inflammation more than in the late ones (autieri et al., 2000; schorn et al., 2014). therefore aif-1 not only has been highly evolutionarily conserved in amino acid sequence but also shows a similar function in both vertebrates and invertebrates (yamamoto et al., 2011). characterization of migrating cells was achieved by ultrastructural analysis, acid phosphatase reaction and immunohistochemistry using polyclonal antibodies directed against human macrophage and leukocytes markers cd68 and cd45 (schorn et al., 2014). the ultrastructural morphology and acid phosphatase reaction positivity confirmed that the cells migrating towards the injured or grafted areas have a strong phagocytic activity. this observation is in agreement with the fact that phagocytosis is an important process for the repair/regeneration of damaged tissue because it increases the clearance of tissue debris, limits further destruction and facilitates repair (takahashi et al., 2007). moreover these results are consistent with previous data obtained after wounding and grafts (de eguileor et al., 2003; tettamanti et al., 2003; grimaldi et al., 2004, 2006). furthermore, double immunostainig experiments based on cd68 tissue expression confirmed the accumulation of macrophages both in the wound healing and grafted region but also highlighted that these cells co-expressed both cd45 and hmaif-1. as we recently demonstrated (schorn et al., 2014), hmaif-1 in leeches is expressed by macrophages and it is involved in macrophage recruitment in the injured area. we speculated that macrophages may recruit and/or promote the proliferation of other macrophages suggesting a pivotal role in initial inflammatory response. our results are consistent with what previous observed in vertebrates, and highlight that aif-1 plays an important role in linking injury, inflammatory and immune response in allograft tissue transplantation. furthermore, these data suggest for the first time that the early activity of macrophages, involved in the initial phase of immune response during wound healing or allograft   137 fig. 5 morphological at optical microscopy (a, e, i, m), acid phosphatase reaction (b, f, j, n) and immunofluorescence (c, d, g, h, k, l, o, p) analyses of cryosections from grafted h. medicinalis. 24 h from autograft a clot of macrophages cells (arrow in a) surround the graft (g). these cells are acp+ (arrow in b) and weakly express aif1 and cd45 (arrows in c, d). 24 h after allograft the clot of macrophages cells (arrows in e, i, m) surrounding the graft (g) are acp+ (arrows in f, j, n) and highly co-expressed hmaif-1 and cd45. no signal is detected in control experiments where the primary antibodies are omitted (h, l, p). bars in a-c: 50 μm; bars in d, e, g, m p: 200 μm; bars in f, h l: 100 μm. rejection, is mediated by both hmaif-1 and cd45 expression. indeed, like in vertebrates (utans et al., 1995), hmaif-1 is highly expressed in allografts by 24 h and remained elevated through 72 h and 7 days. this early and sustained expression of hmaif-1 in allograft is consistent with an ongoing allogeneic inflammatory response. the high density of infiltrating hmaif-1/cd45+ macrophages in injured and allografted areas could be sustained by cytokine-rich environment chemoactraction (de eguileor et al., 1999; tettamanti et al., 2003). the pool of cytokine, in turn, may up-regulate hmaif-1 expression inducing an initial inflammatory response in transplanted host leeches. these macrophages,   138 co-expressing hmaif-1/cd45 and cd68, cooperated to isolate and infiltrate the graft producing themselves a large amount of cytokines responsible of mitogenic and chemotactic events. this macrophages recruitment is a detrimental component of allograft rejection. on the other hand, in autografts, macrophages show a low level of hmaif-1 and cd45 expression. we speculate that differences in hmaif-1 and cd45 expression level are linked to the different role played by macrophages in response to wound/allograft and to autograft. we suggest that like in vertebrates (mokarrama et al., 2012), in leeches as well macrophages can have characteristics of anti-inflammatory or proinflammatory features. in allograft and wound healing, macrophages are mainly involved in inflammatory response and highly express hmaif-1 and cd45. in contrast, in autograft are mainly involved in regeneration of the body wall microenvironment and support tissue repair by producing anti-inflammatory cytokines which mediate angiogenesis, cell replacement and matrix remodeling while suppressing destructive immunity and low expressing hmaif-1 and cd45. concluding remarks results here presented show that the expression of the hmaif-1 significantly increases during the early phases of the inflammatory response and it is mainly exerted by activated macrophages. 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278-286, 2015 isj 12: 278-286, 2015 issn 1824-307x visions and perspectives concepts and tools for exploiting sessile bio-filters as early warning elements: introductory applications for marine ecosystem preservation m scalici1, l traversetti1, v malafoglia1,2, m colamartino1, t persichini1, m colasanti1 1department of science, university roma tre, viale g. marconi 446, 00146, rome (italy) 2isal-foundation, institute for research on pain, torre pedrera (rn), italy accepted october 29, 2015 abstract current evidence suggests that integrating diverse warning systems at different biological levels may not only increase the probability of detecting threats but also mitigate their impact. here, we propose the use of both molecular and morphological descriptors at different biological levels in sessile bivalves (a suitable biological model in monitoring programs) to collect information on the ecosystem health of coastal marine habitats. in this context, studies may be implemented on biomarkers to exploit some population features, with the aim to propose an actual monitoring program that predictively would provide possible scenarios on the species fitness and ecosystem changes. thus, the use of quality biotic elements may provide an objective environmental monitoring method and facilitate the development of sanitary, economic, and social strategies related to sustainable exploitation. key words: cellular and morphological biomarkers; environmental status; longand short-term disturbances; mytilus galloprovincialis; monitoring   introduction a number of international meetings have recently taken place with the goals of reducing human impacts on ecosystems and living resources and ameliorating the effects of natural resource exploitation. such meetings are being held at an increasing frequency largely in response to the united nations conference on environment and development in rio de janeiro, brazil (unced, 1992). this conference provided the impetus to stimulate politicians, scientists, and socio-cultural and socio-economic managers toward developing horizon 2020, a recently created european program for research and innovation proposed by the european parliament (2011). several attempts have been made to prevent, ameliorate or remedy the effects caused by human impact. in this context, the use of living systems approach (across various levels of biological organization) may be as important as the development of environmental monitoring tools (fasulo et al., 2015). indeed, biological models can act as descriptors of environmental health and serve ___________________________________________________________________________ corresponding author: marco colasanti department of science university roma tre viale g. marconi 446, 00146, rome, italy e-mail: marco.colasanti@uniroma3.it as ‘early warning systems’ (ewss). as an example, in marine habitats, many benthic taxa have been used frequently as sentinel organisms in monitoring programs to evaluate the impact of anthropogenic disturbances. the latter is essentially due to their life-history characteristics and to their relatively rapid response to anthropogenic and natural stress descriptors (roméo and gnassia-barelli, 1997; cheggour et al., 2001; griscom et al., 2002; lecoeur et al., 2004; baudrimont et al., 2005; machreki-ajmi and hamza-chaffai, 2008; smaouidamak et al., 2009; serafim and bebiano, 2010; ramos-gomez et al., 2011; wang et al., 2012). two different types of ewss, biomarkers and bioindicators, can be used to detect effects at different biological levels. the redefinition of both were first proposed by van gestel and van brummelen (1996) and then modified (and internationally accepted) by peakall and walker (1996). specifically, a biomarker is described as a set of responses to chemical exposure at the individual/sub-individual (molecular, cellular, histological, physiological, anatomical, morphological, and behavioral) level. instead, bioindicators can be considered as responses to changes of environmental conditions at the levels of population, species, community, ecosystem, and landscape (the latter in adams et al., 2001). then, biomarkers respond rapidly to stressors but often 278   279   provide less information on their effects at ecosystem level. conversely, while bioindicators provide direct information on the habitat status in terms of environmental health, they often show a long response time to environmental changes. this is why mccarty et al. (2002) emphasized the usefulness of bioindicators to address environmental risk issues and, at the same time, they stated that the current practice tends to focus on the use of biomarkers. practically, biomarkers better reflect short-term effects (stes) to direct exposure, while bioindicators are more appropriate to highlight long-term effects (ltes). thus, stes and ltes may operate at different levels of biological organization. nevertheless, changes at the molecular and cellular levels may drive consequences at population, community or ecosystem levels, based on bottom-up effects (lagadic et al., 2000; hagger et al., 2008). however, some difficulties can emerge in developing investigations on stressor-specific, and quantitative dose-response functions to assess environmental risks (see bartell, 2006). therefore, an integrated approach to evaluating the ecosystem quality is becoming a must in biomonitoring, thereby helping to detect pollution-induced effects at several levels of the biological organization (allen and moore, 2004). thus, the use of biomarkers and bioindicators in combination is needed for effective ecosystem monitoring and the prevention of adverse biological effects. environmental pollutants produced by the settlements of approximately three billion people (with their associated agricultural and industrial activities and domestic effluents) are continuously being discharged into the marine ecosystem (moore et al., 2004); as such, it is easy to understand why aquatic habitats are considered amongst the most imperiled (olson et al., 2002). exacerbating this situation, coastal systems are preferred by many species, some with high economic value, for reproduction, nursery, and maturation. because the sediments of coastal ecosystems act as a sink for a variety of contaminants, benthic fauna in particular are regularly exposed to contamination stressors (langston et al., 2010). although the disposal of polluting substances and systems via wastewater management mitigate human impact, it has become necessary to better understand and thereby prevent harmful environmental exposures. the water framework directive (wfd) of the council of the european parliament (2000/60/ec) was a fundamental step in promoting bio-monitoring and aquatic habitat restoration. here, we evaluate the use of biomarkers at different biological levels and apply morphological and molecular markers to investigate the ultimate repercussions of using this approach to monitor population characteristics such as growth, structure and dynamics (the latter being exploited for describing population predictive models). specifically, we propose an integrated approach exploiting sessile bivalves as biological ewss to contribute in monitoring the coastal marine habitat health. a suitable biological model: the mussel mytilus galloprovincialis the use of bio-monitoring requires the following actions: 1) choice of bio-model, 2) assessment of type and time of response to a specific stressor, 3) selection of protocols and tools (such as biotic indices and/or molecular threshold), and 4) management of environmental preservation and mitigation. choosing an adequate biological model is important. no model is universal because different organisms show diverse degrees of habitat adaptation. moreover, the choice of a model depends on the habitat being investigated, as well as on the operator’s expertise in both an environment and the organisms living therein. the structural and trophic complexities of marine habitats constrain the type of biological models can be used as potential ewss. due to their life-history characteristics and relatively rapid responses to anthropogenic and natural stresses, members of the benthic community, especially bivalves (griscom et al., 2002; wang et al., 2012), have frequently been used as sentinel organisms in monitoring programs. the mussel m. galloprovincialis, a common sessile bivalve that is easy to identify and collect, is among the most exploitable biofilters around the world and has considerable economic importance in terms of both aquaculture (its value as a harvested wild resource), and an ews in coastal monitoring projects (e.g., hagger et al., 2008; stankovic et al., 2011). indeed, m. galloprovincialis is a widespread and long utilized biological model in scientific projects funded at national and international level, promoted by different organizations (see protocols of united nations environment programme mediterranean action plan), and actually used within the project ‘systems biology’ (funded by ministry of education, university and research, miur-prin, italy), of which we are a functional operative unit (fasulo et al., 2015). acting as a sentinel organism, this bivalve species can help detect marine pollution because of its filter-feeding activity, capacity to bioaccumulate chemicals (cheggour et al., 2001; metian et al., 2009), sedentary lifestyle, widespread distribution, and abundance. thereby, bivalves can provide a time-integrated indicator of environmental contamination (figueira et al., 2011). specimens collected in the field and sacrificed for molecular and morphological assays should therefore provide an immediate response to acute and intense stress (short-term effects, ste) and an indication of chronic exposure to pollutants (longterm effects, lte). short-term effects cellular markers evaluating molecular responses to pollutants is an emerging method of assessing the impact of stress on the environment (pampanin et al., 2005; regoli and giuliani, 2014; benedetti et al., 2015). exposure to xenobiotics is commonly associated with the production of radical oxygen and nitrogen species (ros/rns), including superoxide radicals, hydrogen peroxide, nitric oxide, and peroxynitrite, which can impair cell signaling, alter gene regulation and cause cellular damage via protein oxidation, enzyme inhibition, lipid peroxidation and dna damage. it is notable that antioxidant enzymes, including catalase and superoxide dismutase (sod), provide primary defenses against oxidative damage, thereby increasing the tolerance of organisms to polluted environments and their persistence therein. however, chronic oxidative stress occurs when cellular homeostasis is altered because of persistent accumulation of contaminants and subsequent excessive production of ros/rns and/or impairment of cellular antioxidant defenses (valavanidis et al., 2006). conversely, recent data indicate that transcriptional and catalytic responses to environmental stressors do not necessarily correspond to functional changes (giuliani et al., 2013). an increasing number of studies have focused on the assessment of post-translational modifications (ptms) of proteins as a signal of toxicity. in particular, ros/rns can cause specific reversible and irreversible oxidative modifications to sensitive proteins that may change their activities and functions. under conditions of oxidative/nitrosative stress, thiols in cysteine residues within proteins are among the most susceptible oxidant-sensitive targets. these thiols undergo various reversible and irreversible redox alterations in response to ros and/or rns production (fig. 1). reversible oxidation, such as that occurring during s-nitrosylation, sglutathionylation and the formation of interor intradisulfide bonds, can play regulatory roles both in sub-stress scenarios and during organismal responses to acute oxidative stress (ascenzi et al., 2000; musci et al., 2006; dalle-donne et al., 2007; casadei et al., 2008). alternatively, chronic oxidative stress caused by specific pollutants can result in irreversible oxidation (e.g., protein carbonylation), leading to protein aggregation, inactivation and degradation. bivalves can be exposed to relatively high levels of pro-oxidants as a consequence of their filter-feeding activities. common pro-oxidant environmental pollutants, such as polychlorinated biphenyls (pcbs), polycyclic aromatic hydrocarbons (pahs), heavy metals and organochlorines, are bioconcentrated within bivalves and lead to oxidative stress (mcdonagh et al., 2006). previously, a 2de proteomic approach was combined with immunoblotting to investigate carbonylation in individual tissues in response to a variety of stressors and pollutants known to induce oxidative stress in both mussels and clams (mcdonagh et al., 2005; mcdonagh and sheehan, 2006). furthermore, increased s-glutathionylation of proteins was observed in gill tissues of mytilus edulis in response to h2o2 and menadione treatment (mcdonagh et al., 2005). numerous protein species (e.g., actin, protein disulfide isomerase (pdi), and other chaperones) in mytilus form intramolecular disulfide bonds following exposure to menadione-induced ros (mcdonagh and sheehan, 2007, mcdonagh and sheehan, 2008). thus, in the current proposal, we aim to identify protein species (e.g., actin, hsp70, carbonic anhydrase, catalase, sod, pdi, glutathione-stransferase, and troponin) that can serve as molecular biomarkers due to their tendencies to undergo ptm induced by ros/rns. some of these proteins may represent specific sub-proteomes for diverse xenobiotics. fig. 1 (a) ribbon diagram of β-actin (pdb id: 2btf); (b) cys272, his275 and glu276 residues are shown in balland-stick representation. the thiol in cys272 is an oxidant-sensitive target (i.e., s-nitrosylation and sglutathionylation). the image was generated using the program chimera (pettersen et al., 2004). 280   fig. 2 a) three-dimensional surface scans of a representative mussel valve. b) the positions of 10 landmarks (larger, darker circles) and 50 semi-landmarks (smaller, lighter circles) are shown. long-term effects morphological markers shell shape is a morphological indicator of stress in bivalves. in addition to markers recorded by eye or by traditional measurements, we focused on novel morphometric biomarkers detectable in bilateral organisms. slight asymmetries in bilateria may reflect an organism’s ability to overcome the effects of stress. thus, organisms deviating from perfect symmetry by showing either fluctuating asymmetry (i.e., non-directional deviation from perfect symmetry) or directional asymmetry (i.e., more growth on one side than the other) (graham et al., 2010) may be associated with some type of developmental noise. morphological asymmetries may therefore be useful as a general biological stress indicator and represent a suitable marker for lte (arambourou et al., 2012). to investigate morphological markers, landmark-based geometric morphometric methods can be used to quantify shell shape deformations. in particular, the spatial coordinates of threedimensional homologous locations (i.e., landmarks) and points that depend on them (i.e., semilandmarks) can be easily exploited after removing non-shape effects [for more details, see (adams et al., 2004)]. 281   in this context, captured images are analyzed based on spatial relationships between cartesian coordinates of landmarks and semi-landmarks. once a set of points (landmarks + semi-landmarks) is fixed on an image using specific software, the x-yz cartesian coordinates of each point are registered to obtain the distribution of the points on the studied surface (i.e., their configuration). the landmarks and semi-landmarks used to quantify shell shape and surface texture in the present proposal were chosen because strict homology in the investigated structures could not be determined (fig. 2); these landmarks have been previously utilized (serb et al., 2011) to capture general shell surface shape. an important advantage of this approach is that shape information from landmarks, points along curves and points on anatomical surfaces can be included in the same analysis. finally, all of the configurations of each specimen may be aligned using a generalized procrustes superimposition, a procedure that removes information on location orientation and rotation, and standardizes each specimen to a unit centroid size (cs i.e., the square root of the summed squared euclidean distance from each landmark to the specimen centroid, which provides an estimate of the size of the studied structure). during this procedure, semi-landmarks were permitted to slide along their tangent directions to minimize the procrustes distance between specimens. from the aligned configurations, a set of procrustes shape coordinates was obtained and used as shape variables in subsequent multivariate statistical analyses (mitteroecker and bookstein, 2008). the euclidean (procrustes) distances amongst the individuals in the morpho-space may be calculated to compare findings within and among groups. warp grids representing shape deformations can be associated with diagrams resulting from multivariate analyses of ordination to visualize morphological variation patterns and facilitate biological interpretation. population structure and dynamics studying valve structure also provides an opportunity to better understand population structure and dynamics. because this biological feature is considered an essential aspect of fishery management, assessments of population structure and dynamics can be used as tools to monitor the effects of external stressors on valve structure. to study these structures, we first decompose a population into cohorts defined by age classes. age can be evaluated by using external tags during recapture or by assessment of body size (i.e., shell size). several methods of length frequency analysis using modal progression techniques can be used to perform aging analyses, and aging mussels is a relatively easy undertaking due to the frequent use of m. galloprovincialis in aquaculture. therefore, is straightforward to create growth rate simulations of reared specimens (see fig. 3). 282   fig. 3 simulated natural growth rate (gray points and black continuous lines) and simulated growth rate after the appearance of a stressor (black dotted line). in alphabetical order: c = amplitude of the curve (providing an estimate of the influence of seasonality on growth trends); k = curvature parameter (describing how quickly individuals approach asymptotic length); l∞ = asymptotic length (theoretical max length); t0 = initial condition parameter (time when the specimens have zero length); ts = summer point (referring to the onset of the first seasonal oscillation relative to t = 0). 283   panel s1 when age and size may be associated, we can express their relationship by the von bertalanffy equation: l(t) = l∞{1-exp[-k(t-t0)]} where l∞ is the asymptotic length (theoretical max length), k the curvature parameter (describing how fast individuals approach the asymptotic length), and t0 the initial condition parameter (time when specimens have zero length). this growth model has been showed extensively suitable for the assessment of a large number of marine animals both vertebrate and non. once we obtain l∞, and t0, we may evaluate further population properties as well: 1) the total mortality index (the sum of natural mortality and the mortality due to fishing) by the powell-wetherall plot equation, that computes the asymptotic length and the ratio between the mortality coefficient and the curvature parameter (z/k); 2) the natural mortality (m) by the formula: logm = 0.0066 0.279logl∞ + 0.6543logk + 0.463logt where t is the mean environmental temperature; 3) the fishing mortality (f) subtracting m from z; 4) the expected mean life-time and the longevity: t1/2 = σ{[n(t)*t]}/n; tmax = (3/k) + t0 respectively, where n(t) is the number of individuals at the time t and n is the total number of individuals. for more details on the stock assessment concepts and tools, see sparre and venema (1996). fishery biology traditionally uses the von bertalanffy growth model equation to determine population properties such as mortality rates and decay model parameters (see panel s1). lengthfrequency data entry and manipulation can be used to create an information flow that enables assessments of capture probability and mortality, as well as evaluations of stock damage via virtual population analysis. these finding can be used to create predictive models. the evaluation of population structure and dynamics is essential in the management of aquatic living resources because this information can be used to evaluate population status. such evaluations are useful for in situ monitoring of population status and could have roles in introduction programs and in monitoring reintroduced individuals during restocking. in fact, population dynamics are strongly associated with biological processes and thus the adaptation of a species to its habitat. hence, environmental changes (such as those mediated by humans) can stress a population and induce alterations in valve shape and integrity (see also bressan et al., 2014) and, consequently, dynamics (fig. 3). such changes can have ecosystem-level consequences when stressed species show a some kind of relevance for the energy flow in an ecosystem. the threat of pollution to mussels is more evident when we consider that valve malformations may also compromise the actions of the mantle as a gonad. this can cause fitness reduction and consequent population reduction. in turn, the reduced mussel biomass may affect other species because many organisms, including economically important species, feed on m. galloprovincialis in both the larval and adult phases. perspectives monitoring programs exploiting a part of a cenosis (e.g., benthic taxa) often provide useful information on contamination than that obtained using populations and species. the two latter difficulties supply information concerning the relationships between causes and effects at level of community or ecosystem. indeed, several investigations on the effects of environmental human-mediated constrains on benthic organisms are developed (parker et al., 2011, range et al., 2011; talmage and gobler, 2011; bressan et al., 2014), but direct effects at food-web and ecosystem levels are never provided. this is due to the strong difficulties in extrapolating general proxies from populations or species, since on one hand, many synergic (natural or anthropogenic) drivers may act at higher levels of the hierarchical biological organization; on the other hand, higher levels show more and more complex processes of resistance and resilience. therefore, in this proposal, we focused on methods of highlighting the effects of pollutants on biological descriptors that are easy to survey and may be useful as early warning systems for the whole ecosystem health. here, we proposed 284   integrated tools for using natural resources as monitors to survey and mitigate the impact of pollutants on the environment and explored the use of predictive models to forecast the effects of contaminants. to fill this gap, future risks may be evaluated exploiting multi-level approaches in long-term experiments and exposure to multiple stressors. further research in the lab and in the field should be initiated and funded to better understand (1) the mechanisms, advantages and drawbacks of the integrated approach and (2) the criteria by which to select an appropriate strategy of management and/or preservation. acknowledgements the current proposal for using integrated methods is part of the systems biology project, a medium-scale italian project (http://www.aiatsicilia.it/indexpr.htm) funded by the ministry of education, university and research (miur-prin). the 36-month project (coordinated by the university of messina) involved 8 italian partners; 9 “associated partners” from countries in europe, north and south america; and 2 small/medium italian industries. grant number: 2010arblt7_001/008. references adams dc, rohlf fj, slice de. geometric morphometrics: ten years of progress following the 'revolution'. ital. j. zool. 71: 516, 2004. adams sm, giesy jp, tremblay la, eason ct. 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alkaline protease in activation of the antimicrobial activity in galleria mellonella larvae m andrejko, a siemińska department of immunobiology, maria curie-sklodowska university, akademicka 19, 20-033 lublin, poland accepted august 12 , 2016 abstract the role of pseudomonas aeruginosa metalloprotease alkaline protease in activation of the antimicrobial activity in galleria mellonella larvae was investigated. the results of our in vivo study showed that injection of alkaline protease at a sublethal dose enhanced the antimicrobial activity in the hemolymph of g. mellonella larvae as a result of induction of defense peptides synthesis. we observed that the antibacterial activity against e. coli appeared in the hemolymph 4 h after the injection of both metalloprotease or heat-killed p. aeruginosa, reached the maximum level 24 h post injection, and next decreased slightly. antifungal activity against a. niger was detected in the hemolymph 15 h and 24 h after the challenge in the case of the alkaline protease and p. aeruginosa cell treatment, respectively. we also noted that the antimicrobial activity level induced by the presence of the metalloprotease in the hemolymph was higher than the activity measured after the injection of the insects with p. aeruginosa. the results of our in vitro studies indicated that inducible antimicrobial peptides present in the hemolymph of proteaseor p. aeruginosa-challenged larvae were digested by alkaline protease. key words: antimicrobial activity; galleria mellonella; alkaline protease; pseudomonas aeruginosa   introduction the gram-negative bacterium pseudomonas aeruginosa is an opportunistic pathogen which can cause severe and lethal infections in patients with a variety of diseases. the bacteria produce several extracellular proteolytic enzymes that have been implicated as virulence factors. among them, there are elastase (pseudolysin) (kessler et al., 1998), lasa protease (staphylolysin) (kessler et al., 1993), protease iv, serine protease (engel et al., 1998), and alkaline protease (morihara, 1963; maeda and morihara, 1995; caballero et al., 2001). the alkaline protease (ec 3.4.24.40) also named aeruginolysin (apr) is a member of the serralysin family (morihara et al., 1973) and, hence, belongs to the metzincin superfamily of metalloendopeptidases (rawlings and barrett, 1995). apr is homologous to the 50-kda metalloproteinases secreted by serratia marcescens and erwinia chrysanthemi (maeda and morihara, 1995; stöcker et al., 1995). this protease is implicated in hydrolysis of many biologically ___________________________________________________________________________ corresponding author: mariola andrejko department of immunobiology maria curie-sklodowska university akademicka 19, 20-033 lublin, poland e-mail: mariola.andrejko@poczta.umcs.lublin.pl important proteins, including the α1-proteinase inhibitor (morihara et al., 1979), cytokines (parmely et al., 1990), complement factors (hong and ghebrehiwet, 1992), laminin (heck et al., 1986), matrix metalloproteinases (mmps) (twining et al., 1993), human γ-interferon, and tumor necrosis factorα (horvat and parmely, 1988; parmely et al., 1990). insects have developed a very effective immune system, the functioning of which relies on humoral and cellular innate mechanisms. cellular reactions, i.e., phagocytosis, nodulation, and encapsulation, are mediated by hemocytes, whereas antimicrobial peptides and proteins (amps) are important components of humoral immune response. antimicrobial peptides are mainly produced in the fat body or hemocytes and are then released into the hemolymph. an impressive arsenal of defense peptides differing in biochemical and antimicrobial properties has been reported in hemolymph of immune-challenged g. mellonella larvae (brown et al., 2008, 2009; cytryńska et al., 2007). in addition, differences in the peptide sets and the kinetics of appearance in the hemolymph were detected after insect infection with various bacteria and fungi (mak et al., 2010). the recognition system for a microbial pattern in g. mellonella is able to sense both microbial cell 269 270 wall components and endogenous immune stimulatory peptides generated by microbial metalloproteinases. the infectious non-self model postulates that the immune system is set into alarm by recognition of microbial pattern molecules. the danger model explains the activation of immune response caused by alarm signals from injured cells, such as those exposed to pathogens, toxins, or mechanical damage (matzinger, 2002; altincicek et al., 2009). it has been shown that innate immune systems of mammals and insects share a high degree of structural and functional homology. many studies have demonstrated that the greater wax moth g. mellonella may be used as an alternative model host for investigating virulence factors of human pathogenic bacteria, including p. aeruginosa (madziara-borusiewicz and lysenko, 1971; dunphy et al., 1986; jarrell and kropinski, 1982). a positive correlation in the virulence of p. aeruginosa mutants in mice and g. mellonella caterpillars was demonstrated (jander et al., 2000). a study which was to find out the role of the type iii secretion system in p. aeruginosa pathogenesis has revealed a high level of correlation between the results obtained in the g. mellonella model and cytopathology assays conducted with a mammalian tissue culture system (miyata et al., 2003). the recent studies performed in our laboratory indicated that p. aeruginosa elastase b degraded inducible antimicrobial peptides in the hemolymph of g. mellonella (andrejko et al., 2009, andrejko and mizerska-dudka, 2012), while elastase b injected at a sublethal dose was responsible for eliciting the humoral immune response in g. mellonella, and this protease seems to be a more potent elicitor than thermolysin (andrejko and mizerska-dudka, 2011). the diverse effects of two p. aeruginosa clinical isolates on the parameters of g. mellonella immune response indicated that this model of insect could be useful for analysis of the virulence factors of different p. aeruginosa strains (andrejko et al., 2013b). in an in vivo study, proteolytic degradation of peptides and proteins in g. mellonella body after infection by an entomopathogenic p. aeruginosa strain was demonstrated, confirming the important role of bacterial proteinases in its pathogenicity (andrejko et al., 2014). larval infection with p. aeruginosa strains differing in the profile of proteolytic enzymes induces synthesis of immune peptides, likewise injection of the saprophytic bacteria e. coli (andrejko et al., 2009; andrejko et al., 2014). in this study, we tested the level of antimicrobial activity in the hemolymph of g. mellonella larvae after the treatment with alkaline protease of p. aeruginosa clinical isolate. as we had shown earlier, this strain produced elastase b and alkaline protease when it grew in the lb medium and the minimal m9 medium, respectively. pcr analysis confirmed the presence of the apra gene coding for alkaline protease in the genome of the p. aeruginosa clinical strain used in this study (andrejko et al., 2013a). furthermore, protease profile analyses of the larval homogenates revealed that this isolate produces alkaline protease during infection (andrejko et al., 2014). we examined the effects of injection with alkaline protease on antimicrobial activity in g. mellonella larvae hemolymph. the kinetics and level of antimicrobial activity is compared to that obtained after injection of heat-killed p. aeruginosa cells. we also studied whether alkaline protease was able to degrade inducible antimicrobial peptides of g. mellonella in vitro. materials and methods microorganisms pseudomonas aeruginosa atcc 27853, an isolate with moderate virulence to the 7th instar larvae of g. mellonella (ld50 = 17 cells), and clinical strain pa 02/18 (received from the department of microbiology and epidemiology, military institute of hygiene and epidemiology in warsaw, poland) were used in this study. the bacterial cells were grown overnight at 37 oc in luria bertani broth (lb broth, sigma) or m9 minimal medium supplemented with monosodium glutamate (0.13 m), glycerol (0.1 m), and cacl2 (0.01 m). escherichia coli k12, strain d31, which is lps-defective and streptomycinand ampicillin-resistant (cgsc 5165) (boman et al., 1974), was grown in luria-bertani broth (lb broth, sigma) for 24 h at 37 oc and pelleted by centrifugation at 8,000g for 10 min at 4 oc. the filamentous fungus aspergillus niger 71 was grown on pda slides (5 % potato extract, 0.5 % dextrose, 1.7 % agar) at 28 oc until conidial spores emerged; it was then stored at 4 oc. insect immune challenge, hemolymph collection, preparation of hemolymph methanolic extracts the larvae of g. mellonella were reared in darkness on honey bee nest debris at 28 oc and 70 80 % humidity. for immune challenge, the larvae were injected with 0.8 μg/larvae of alkaline protease (chromatographic fraction) or heat-killed (98 oc; 30 min) cells of p. aeruginosa atcc 27853. the larvae were pricked with a thin needle plunged in the bacterial pellet from the 24 h culture of bacteria (approx. 5x104 bacteria/larvae). after the treatment, the larvae were kept at 30 oc in the dark on sterile petri dishes and the hemolymph was collected after the time indicated in the text. hemolymph samples were obtained by puncturing larval abdomens with a sterile needle. the out-flowing hemolymph was immediately transferred into sterile and chilled eppendorf tubes containing a few crystals of phenylthiourea (ptu) to prevent melanization. the hemocyte-free hemolymph was obtained by centrifugation at 200g for 5 min and subsequently at 20,000g for 10 min at 4 oc. pooled supernatants were stored at -20 oc until used. the acidic/methanolic extracts of the hemolymph containing antimicrobial peptides and proteins below 30 kda were prepared from cell-free hemolymph as described elsewhere (andrejko et al., 2005; cytryńska et al., 2007). protease purification alkaline protease was purified from the culture supernatant of the p. aeruginosa clinical strain pa 02/18. the bacterial cells were cultivated under aerobic conditions at 37 oc for 20 h, in m9 minimal 271 medium. then, the bacterial culture was centrifuged at 8,000g for 15 min at 4 oc to pellet the cells. the resulting clear supernatant was used as the starting material. proteins secreted into the growth medium were precipitated from the supernatant with ammonium sulfate (90 % of saturation). the precipitate was collected by centrifugation (8,000g for 15 min, 4 oc), dissolved in 50 mm tris-hcl buffer (ph 8.0), and dialyzed overnight against the same buffer. the dialyzed solution was fractionated using ion-exchange chromatography on deae-cellulose column (de 52, whatman). the column was equilibrated with 50 mm tris-hcl buffer, ph 8. proteins bound to the column were eluted with a linear gradient of 0 0.7 m nacl in the above buffer. the fractions were assayed for proteolytic activity and analyzed by zymography and immunoblotting as described below. the lyophilized samples were stored at -20 oc. proteolytic activity assay the alkaline protease activity was measured using a modified method described by howe and iglewski (1984). 0.7 ml of the buffer (20 mm trishcl, 1 mm cacl2, ph 8) and 0.3 ml of the enzymecontaining fraction were added to 10 mg of the hide powder azure substrate (sigma). the reaction mixtures (1 ml) were incubated at 37 oc for 1 h with constant rotation. the undissolved substrate was removed by centrifugation at 4,000g for 5 min. the absorbance of the reaction mixtures were then determined at 595 nm. zymography analysis gelatin zymography was conducted following the procedures described by caballero and coworkers (2001). the samples containing alkaline protease (0.5 1.0 µg protein) were electrophoresed under non-reducing (30 mm tris-hcl ph 6.8, 1 % sds, 5 % glycerol and 0.04 % bromophenol blue) conditions using 10 % sds page with 0.1 % gelatin. the gels were then soaked twice in 2.5 % triton x-100 for 15 min and incubated at 37 oc for 24 h in gelatin gel substrate buffer (50 mm trishcl, ph 8.0, 10 mm cacl2, 1 µm zncl2, 150 mm nacl). the gels were stained for 60 min in 0.2 % amido black and then destained in 10 % acetic acid. immunoblotting after resolution by sds-page, the proteins were electrotransferred onto pvdf membranes (90 min at 350 ma). the membranes were blocked with 5 % non-fat milk in tbs (10 mm tris-hcl ph 7.5, 0.9 % nacl). for detection of bacterial alkaline protease, the membranes were probed with rabbit antibodies against p. aeruginosa alkaline protease (1:1000) (kindly provided by dr. r. voulhoux, laboratoire d'ingénierie des systèmes macromoléculaires, cnrs umr7255, institut de microbiologie de la méditerranée, france). goat anti-rabbit alkaline phosphatase-conjugated antibodies were used (1: 30,000) (sigma) as second antibodies and visualization of the immunoreactive bands was performed with 5-bromo-4-chloro-indolylphosphate (bcip) and p-nitro blue tetrazolium chloride (nbt) (blake et al., 1984). antimicrobial activity assays antibacterial and antifungal activity was tested by the well diffusion assay against viable e. coli d31 (lb agar plates) and a. niger 71 (pda plates), respectively, as described previously (cytryńska at al., 2001; mak et al., 2010). wells on petri plates were filled with 4 μl of the samples of the hemolymph or hemolymph extract. the diameters of the e. coli d31 and a. niger growth inhibition zones were measured after 24 h incubation at 37 oc and 28 oc, respectively. the level of antimicrobial activity was calculated as described by hultmark and coworkers (1982). for evaluation of antibacterial and antifungal activity, commercial cecropin b (sigma) and amphotericin b (sigma), respectively, were used as standards. mortality assay for in vivo experiments, the last instar (7th) non-feeding, wandering g. mellonella larvae were injected with alkaline protease at doses in a range of 0.8 2.0 µg protein/larvae. sterile water was used for the control injection. after the challenge, insects were kept on sterile petri dishes at room temperature in the darkness. the mortality of injected larvae was monitored 72 h after injection of the enzyme. larval survival was calculated as a percentage (%) of live larvae (i.e., moving and exhibiting no change in the body colour) relative to the total number of larvae in the group. the experiments were performed in groups of 12 larvae and repeated three times. in vitro assay to evaluate the effect of p. aeruginosa alkaline protease on antimicrobial activity, the immune hemolymph (hemolymph collected from p. aeruginosaor protease-challenged larvae) or the acidic/methanolic extracts were incubated at 37 oc for 10 min in the presence of alkaline protease (0.03 0.17 μg of total protein). after incubation, 4 μl of each sample were used to determine the antimicrobial activity by the well diffusion assay (see above). for sds-page electrophoresis, appropriate volumes of sample buffer were added to the samples and they were stored at -20 oc until needed. sds gel overlay method (bioautography) detection of antibacterial activity after sdspage and subsequent renaturation of polypeptides in the gels was performed as described elsewhere (cytryńska et al., 2001). after resolution of the proteins, the gels were washed for 30 min in 2.5 % triton x-100 (bio rad) for sds removal. the gels were then washed in 50 mm tris-hcl ph 7.5, and subsequently in lb broth, for 30 min in each step. to localize the peptide bands with antimicrobial activity, the gels were overlaid with nutrient agar containing viable e. coli d31 and ewl (2.5 mg/ml) and incubated for 6 12 h at 37 oc. zones of bacterial growth inhibition were subsequently observed. other methods protein concentration was calculated using the bradford method with bovine serum albumin (bsa) as a standard (bradford, 1976). polyacrylamide gel electrophoresis of protein samples was performed by 10 % or 13.8 % glycine sds-page under reducing or non-reducing conditions according to laemmli (laemmli, 1970) or by tris-tricine sdspage (16.5 % t, 3 % c) as described by schägger and von jagow (1987). results alkaline protease was purified from the culture supernatant of p. aeruginosa by ion-exchange chromatography on deae-cellulose. the activity assay showed that the chromatographic fraction was able to hydrolyze hide powder azure, a substrate for p. aeruginosa alkaline protease (data not shown). when the fraction obtained was analyzed by the zymography method using gelatin sds-page, a clear protease band with an apparent molecular mass 52 kda was observed (fig. 1a). it corresponded to the molecular mass of p. aeruginosa alkaline protease (caballero et al., 2001; andrejko et al., 2013a). the 52-kda single protein band was recognized by specific anti-p. aeruginosa alkaline protease antibodies, confirming the presence of this protease in the chromatographic fractions (fig. 1b). fig. 1 identification of alkaline protease in the fraction obtained by ion-exchange chromatography on deae-cellulose. (a) zymography analysis. the samples (0.5-1 μg of protein) were electrophoresed under non-reducing conditions in 10% polyacrylamide gel containing 0.1 % gelatin. the gels were washed in triton x-100, incubated at 37 oc for 24 h in a specific buffer, and stained in amido black as described in materials and methods. the zymogram shown is a typical representative of at least ten independent experiments. (b) immunoblotting. the samples (ca. 5μg of protein) were resolved by sdspage, transferred onto pvdf membranes, and probed with anti-p. aeruginosa alkaline protease antibodies. a fragment of the membrane containing alkaline protease recognized by the antibodies is presented. m molecular weight standards. ap – alkaline protease band is indicated by an arrow. in order to estimate the sublethal dose, we tested the toxicity of p. aeruginosa alkaline protease to the 7th instar larvae of g. mellonella. four doses of alkaline protease (0.8, 1.2, 1.6 and 2.0 μg/larvae) were used. the insects were injected through the last proleg and mortality rates were determined over a 72 h period. the larvae treated with alkaline protease at the doses of 1.2, 1.6, and 2 μg/larvae exhibited ca. 30 %, 50 %, and 80 % mortality respectively, while the treatment with 0.8 μg/larvae of alkaline protease resulted in a 100 % survival rate (fig. 2a). dead larvae exhibited high melanization as well as loss of turgor and body integrity (fig. 2b). on the basis of these results, the dose showing no toxic effects on the larvae was chosen for immune challenge of the insects, i.e., 0.8 μg/larvae. for comparison, heat-killed cells of p. aeruginosa were used. highest level 24 h after injection, and was equivalent to the activity of ca. 3.93 μm and 3.24 μm of cecropin b, respectively. next, the antibacterial activity slightly decreased. in the control samples containing the hemolymph or hemolymph extract of untreated insects, no anti-e. coli activity was observed. moreover, the level of antimicrobial activity in the protease-challenged larvae was slightly higher in comparison with that measured in the insect hemolymph treated with heat-killed p. aeruginosa. for example, the activity level after injection of the alkaline protease was approximately 1.2fold greater in comparison to that detected in p. aeruginosa injected larvae, both 15 h and 24 h post challenge. the results suggest that, like the microbial elicitor, the alkaline protease strongly induced antimicrobial activity in g. mellonella hemolymph. the kinetics of in vivo changes in the antimicrobial activity level in the hemolymph of g. mellonella larvae in response to the treatment with the metalloprotease and p. aeruginosa cells was investigated. the insects were injected with alkaline protease (0.8 μg of protein) or heat-killed p. aeruginosa cells. the hemolymph samples were collected 4, 8, 15, 24, and 48 h after injection and antibacterial and antifungal activity levels were measured (figs 3, 4). additionally, an acidic/methanolic extracts of hemolymph containing antimicrobial peptides and proteins below 30 kda were also used as a source of immune peptides. antifungal activity toward a. niger was detected in the hemolymph collected 15 h after the challenge of g. mellonella larvae with the metalloprotease (fig. 4a). the activity in this case persisted at a high level, corresponding to the activity of approx. 140 μm of amphotericin b, even 48 h after the treatment. in contrast, in the p. aeruginosa-challenged larvae, antifungal activity equivalent to the activity of 80 μm of amphotericin b appeared in the hemolymph 24 h after the treatment; 48 h after immunization, it decreased and corresponded to the activity of 48.5 μm of amphotericin b (fig. 4b). the results presented in figures 3a and b showed that after metalloprotease injection, the appearance of antibacterial activity had similar kinetics to that obtained after the treatment with heat-killed p. aeruginosa. the well diffusion assay against e. coli demonstrated that antibacterial activity appeared in the hemolymph 4 h after injection of both the metalloprotease and bacteria. then, the activity gradually increased, reaching the 272 fig. 2 (a) mortality assay in g. mellonella larvae injected with alkaline protease. twelve individuals were used per each group. all values represent the mean ±sd of three independent experiments. (b) larvae injected with alkaline protease (0.8 μg/larvae). (c) larvae after injection of alkaline protease (2 μg/larvae). additionally, the antibacterial activity of the hemolymph samples was tested by bioautography after resolution of polypeptides by sds-page and subsequent renaturation (fig. 5a). to localize the polypeptide bands with antibacterial activity, the gels were overlaid with e. coli d31 (cytryńska et al., 2001, 2007). the results obtained using the bioautography method revealed that the defense peptides with molecular mass corresponding to cecropin b were mainly responsible for the anti-e. coli activity in the studied hemolymph samples. a striking difference between the level of antibacterial activity in the hemolymph and the hemolymph extract was observed. in contrast, no difference was detected in the case of antifungal activity (fig. 4). most probably, in addition to the antibacterial peptides present in the hemolymph extract, other polypeptide components of hemolymph, which are not recovered by methanolic extraction, are responsible for the antibacterial activity in hemolymph. on the other hand, the small difference in antifungal activity level between hemolymph and the hemolymph extract may implicate that mainly peptides and proteins with molecular mass below 30 kda recovered during the extraction procedure to a high extent, are responsible for the antifungal activity. the tris-tricine sds-page analysis of g. mellonella larval proteins isolated from hemolymph of immune-challenged larvae showed that the same new peptide bands with molecular mass below 6.5 kda appeared in the hemolymph samples in response to the injected microbial elicitor (p. aeruginosa) or alkaline protease (fig. 5b). the presence of additional peptides in the hemolymph correlated also in time with the antimicrobial activity detected in the hemolymph of the immunized insects. 273 fig. 3 kinetics of the antibacterial activity in the hemolymph of alkaline proteaseor heat-killed p. aeruginosachallenged g. mellonella larvae. (a, b) the larvae were injected with the alkaline protease (0.8 μg protein) or heat-killed p. aeruginosa cells. the hemolymph samples were collected at specified time points after injection and methanolic extracts were prepared as described in section 2. the hemolymph/hemolymph extract samples were analyzed using the radial diffusion assay on solid agar plates containing live e. coli. the antibacterial activity was calculated as a cecropin b equivalent. the diagrams present the results ±sd of three independent experiments. nh = hemolymph from untreated larvae; ne = hemolymph extract from untreated larvae. in order to test if amps, which are mainly responsible for antibacterial activity in the hemolymph of g. mellonella, are susceptible to p. aeruginosa alkaline protease, the activity in the hemolymph and the hemolymph extract from immune-challenged g. mellonella larvae was measured after in vitro incubation in the absence or presence of alkaline protease. for immunization, the larvae were injected with a chromatographic fraction containing alkaline protease (0.8 μg protein) and heat-killed p. aeruginosa cells. the well diffusion assay revealed that the antimicrobial activity was sensitive to the fraction containing alkaline protease. the results presented in fig. 6a clearly showed that incubation of the hemolymph or the hemolymph extract in the presence of a fraction containing 0.17 μg protein for only 10 min completely abolished the anti-e. coli activity. in turn, the fraction added to the samples at a concentration of 0.03 μg protein significantly decreased antimicrobial activity, for example by ca. 69 % and 76 % in the hemolymph of p. aeruginosa-challenged and protease-challenged insects, respectively. 274 fig. 4 kinetics of the antifungal activity in the hemolymph of alkaline proteaseor heat-killed p. aeruginosachallenged g. mellonella larvae. (a, b) the larvae were injected with the alkaline protease (0.8 μg protein) or heat-killed p. aeruginosa cells. the hemolymph samples were collected at specified time points after injection and methanolic extracts were prepared as described in section 2. the hemolymph/hemolymph extract samples were analyzed using the radial diffusion assay on solid agar plates containing live a. niger. the antifungal activity as an amphotericin b equivalent. the diagrams present the results ±sd of three independent experiments. nh = hemolymph from untreated larvae; ne = hemolymph extract from untreated larvae. 275 the observation presented above was confirmed by testing the antibacterial activity by the bioautography method after electrophoretic separation of amps in polyacrylamide gels. the data presented in fig. 6b showed e. coli growth inhibition zones in the hemolymph of the proteaseand bacteria-challenged larvae (lane 2 and 5, respectively). incubation of the same hemolymph samples in the presence of the proteolytic fraction, led to a significant decrease (lane 4 and 7) and complete abolition of the antibacterial activity (lane 3 and 6). the presented results indicate that p. aeruginosa alkaline protease, i.e., the main proteinase of the clinical strain growing in minimal medium, may be responsible for degradation of antimicrobial peptides in g. mellonella hemolymph. it should be added that the other protein components of hemolymph involved in antibacterial activity (fig. 6a) are highly sensitive to the alkaline protease activity, and this issue requires further investigations. fig. 5 (a) bioautography of the hemolymph of g. mellonella larvae after injection of alkaline protease or heatkilled p. aeruginosa cells. the samples (120 μg of protein) collected at the indicated time points after injection were separated by glycine sds-page and after renaturation their antibacterial activity was detected by the gel overlay method as described in the materials and methods. (b) analysis of peptide profiles in the hemolymph of alkaline proteaseor p. aeruginosa-challenged g. mellonella larvae and control larvae. the methanolic extracts were prepared from the hemolymph collected at specified time points after injection. the proteins and peptides (20 μg of protein) were resolved by tris-tricine sds-page and stained with coomassie brilliant blue. the peptide bands appearing after the challenge are indicated by asterisks. ne = hemolymph extract from untreated larvae. m = molecular weight standards. 276 fig. 6 the effect of p. aeruginosa alkaline protease on the antibacterial activity in g. mellonella hemolymph in vitro. (a) immune hemolymph (120 μg of total protein) or hemolymph extracts (20 μg of total protein) were incubated without (control) or in the presence of the alkaline protease. then, antibacterial activity was tested by the well diffusion assay and calculated as a cecropin b equivalent. the diagrams present the results ±sd of three independent experiments. (b) bioautography. the samples of the hemolymph (120 μg of protein) of proteaseor p. aeruginosa-challenged larvae were incubated with alkaline protease (0.03 and 0.17 μg) at 37 oc for 10 min. then, the samples were separated by glycine sds-page and, after renaturation, their antibacterial activity was detected by the gel overlay method as described in the materials and methods. hemolymph from untreated larvae (lane 1); hemolymph of protease-challenged larvae collected 24 h after the challenge (lane 2); hemolymph of protease-challenged larvae treated with the enzyme fraction (0.17 μg of protein) (lane 3); hemolymph of proteasechallenged larvae treated with the enzyme fraction (0.03 μg of protein) (lane 4); hemolymph of p. aeruginosachallenged larvae collected 24 h after the challenge (lane 5); hemolymph of p. aeruginosa-challenged larvae treated with the enzyme fraction (0.17 μg of protein) (lane 6); hemolymph of p. aeruginosa-challenged larvae treated with the enzyme fraction (0.03 μg of protein) (lane 7). discussion 277 it is known that induction of antimicrobial peptides and proteins in response to an immune challenge is one of the most important mechanisms of humoral defense in invertebrates (hetru et al.,1998). in addition, it has been shown that hydrolysis of g. mellonella hemolymph proteins by collagenolytic enzymes, such as thermolysin, results in formation of small-sized protein fragments which elicit innate immune responses (altancicek and vilcinskas, 2006). it was demonstrated that the protein fragments and lps induced expression of a similar spectrum of immune-related genes encoding antimicrobial peptides, such as gallerimycin, gloverin, impi and lysozyme (altancicek et al., 2007). as demonstrated by us previously, p. aeruginosa elastase b was responsible for eliciting the humoral immune response in g. mellonella larvae (andrejko and mizerska-dudka, 2011). in the present study, we were interested to determine whether injection of other p. aeruginosa protease, namely alkaline protease mediates activation of g. mellonella innate immunity. the results of our in vivo studies showed that like the microbial elicitor (p. aeruginosa), the alkaline protease injected at a sublethal dose strongly induced antimicrobial activity in insect hemolymph. after metalloprotease injection, the appearance of antibacterial activity had similar kinetics to that obtained after the treatment with heat-killed p. aeruginosa. in contrast, the antifungal activity 278 appeared in hemolymph of the protease-challenged larvae much earlier and persisted longer at a high level than in the hemolymph of the bacteria-injected insects. this suggested that the metalloprotease treatment induced broader spectrum of antimicrobial peptides and proteins in comparison with the heatkilled bacteria. the electrophoretic analysis of hemolymph peptides after protease treatment confirmed that the activation of innate immune response was correlated with subsequently synthesized antibacterial peptides. two peptide bands with molecular mass below 6.5 kda were observed in the hemolymph samples in response to the injected microbial elicitor or alkaline protease, but not in the hemolymph of the non-treated larvae. it is known that the immune system of the lepidoptera moth g. mellonella is able to distinguish between different classes of microorganisms and responds to the invading pathogen accordingly (mak et al., 2010). it can be assumed that injection of microbial elicitors, i.e. gram-negative bacteria p. aeruginosa or e. coli, to the hemocel leads to the synthesis of a similar set of peptides. however, further detailed research is required in the case of antimicrobial peptides appearing in the hemolymph of alkaline protease-challenged larvae. it is commonly known that among the enzymes of human pathogenic bacteria and fungi, thermolysin-like metalloproteinases seem to play a predominant role during pathogenesis (miyoshi and shinoda, 2000; altancicek et al., 2007). in our previous papers, we demonstrated that extracellular proteinases present in a p. aeruginosa culture supernatant degraded immune-relevant proteins and peptides in g. mellonella hemolymph in vitro (andrejko et al., 2005; andrejko et al., 2008, 2009; andrejko and mizerska-dudka, 2012). from this study, we concluded that p. aeruginosa alkaline protease may also be responsible for degradation/inactivation of inducible antimicrobial peptides in g. mellonella hemolymph. the results of series of experiments obtained with the well diffusion assay and with bioautography method for assessment of antimicrobial activity revealed inhibition of anti-e. coli activity in insect hemolymph incubated with alkaline protease. as presented in this paper, the larvae treated with 0.8 μg of alkaline protease survived and antimicrobial activity was simultaneously detected in their hemolymph. it cannot be excluded that alkaline protease induces g. mellonella immune response in two ways: (i) it is recognized by the insect immune system as non-self and (ii) it can act as a danger signal and activate proteolytic cascades in the hemolymph. similarly, the decrease in the antimicrobial activity observed 48 h after the challenge could be a result of: (i) silencing of the immune response by the insect immune system, or (ii) proteolytic activity of alkaline protease. the results of our study suggest that the first possibility is more probable, as the larvae injected with 0.8 μg of alkaline protease survived; however, the effects observed could be a result of both of these processes. bacterial proteolytic enzymes seem to have a dual role in pathogenesis. in the initial phase of infection, they activate the insect immune system, thereby enhancing the antimicrobial activity in the hemolymph of infected insects. next, as bacteremia progresses, proteolytic enzymes may degrade certain proteins/peptides e.g. defense peptides. acknowledgement the authors would like to thank prof. m cytryńska 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useful for analyzing the impact of the heavy metal stress on the worm metabolism. the epigeic earthworm e. fetida were exposed to 100 %, 75 %, 50 %, 30 %, 25 %, 15 % and 5 % of dried automobile service station waste mud. all the earthworms exposed in the 100 %, 75 % and 50 % concentrations didn’t survived within 10days. further experiments were conducted with 25 %, 15 % and 5 % concentration of wastes. each concentration level was tested with three replicates using 10 animals and the metabolic response after exposure to the heavy metal containing service station waste mud was assessed by ftir. furthermore we also emphasized that dna damage was confirmed with the use of other biomarker like comet assay. the peaks at 1045, 1080, 1236 cm−1 and 1650 cm−1 represented the overall susceptibility of nucleotides, phospholipids, dna and rna. nucleic acids and proteins were modified due to heavy metal accumulation. in flow-through, single cell gel electrophoresis revealed the degradation nuclear dna. heavy metals accumulation in the worms was measured and it was found that lead, zinc and copper accumulation increased in the treatment group. without the use of biomarkers for identifying ecological risks of land contamination, traditional assessment would be difficult to interpret. this new ftir based biomolecules study revealed a clear molecule shift in the exposed worms, due to heavy metal accumulation. key words: earthworm; eisenia fetida; ftir; dna fragmentation; heavy metals and biomolecules   introduction heavy metal pollution of soil is widespread across the globe and has caused biological problems, leading to potential toxicity to living organisms. recent research found that the atmospheric input of heavy metals to agricultural systems also significantly contributed to metal loading in soil (vidovic et al., 2005). given the surge in passenger vehicle usage it has become difficult to avoid exposure to the metals existing in our surroundings. the determination of the toxicity of metals is difficult because of the complex nature of their interactions with biological systems. earthworms, among the many kinds of soil organisms are considered potential bioindicators proven their usefulness in the evaluation of metal contamination in soil. significant positive correlations have been observed between the metal concentrations in the earthworm and the cadmium (cd), copper (cu), lead (pb) and zinc (zn) ___________________________________________________________________________ corresponding author: gobi muthukaruppan department of biotechnology school of bio-engineering srm university kattankulathur 603203, tamilnadu, india e-mail: gobicc@gmail.com concentrations in the soil (morgan and morgan, 1988). earthworm celomocytes possess the celomic fluid harboring cells, which are similar to mammalian leucocytes, are relatively easy to obtain, and may be useful to perform both bioassays on the same biological samples (burch et al., 1999; weeks and svendsen, 1996). the metabolic profile of healthy organisms can be compared to that of treated organisms and the variations noted could be used to identify the response to the stress. in fact, as the interaction of toxic agents with living cells causes molecular modifications in the organisms (salman et al., 2002; argov et al., 2004; beleits et al., 2005), other approaches based on fourier transform infrared (ftir) spectroscopy could become helpful to study the molecular modifications observed in living cells after exposure to pollutants (saxena et al., 2005). since dna damage may result in severe consequences for individuals, species, and ecosystems, it is regarded as an important indicator to be used in the assessment of earthworm health (reinecke and reinecke, 2004; casabé et al., 2007). in the current study, we analyzed heavy metal induced bio-molecule changes in e. fetida reflected by ftir spectroscopic features with   238 attenuated total reflectance (atr) mode and which, to the best of our knowledge, have not previously been reported. furthermore, we aimed to identify and quantify the dna fragmentation of exposed worms. materials and methods earthworm species exposed and experimental setup in the present study, automobile waste mud was collected from madurai, tamilnadu, india. the experimental beds were prepared with cow dung and automobile waste at 100 %, 75 %, 50 %, 25 %, 30 %, 15 % and 5 % levels in rectangular plastic tubs (of 12′′×17′′×51′′ size) in triplicates and control (cow dung only) was maintained. all the earthworms exposed in the 100 %, 75 % and 50 % concentrations didn’t survived within 10 days. further experiments were conducted with 25 %, 15 % and 5 % concentration of wastes. tests were conducted using laboratory cultured adult specimens of eisenia fetida, each concentration level was tested with 10 worms and tests were run for a period of 35 days. after completion of 35 days, earthworms from each concentration were taken and washed with distilled water. then, (pokarzherskii et al., 2000) the earthworm’s gut was cleaned and then these earthworm samples were lyophilized and powdered, further these lyophilized samples were subjected for ftir analysis. for ftir in the absorbance mode, samples were mixed with kbr (merck) or, for diffuse reflectance infrared fourier transform (shimadzu ftir-8400s) measurements, used as dry finely ground powder in a micro sampling cup. ftir atr spectra were collected at 4 cm−1 of spectral resolution after 250 scans in the 400 4000 cm−1 range. then spectra were baseline corrected and normalized using the absorption band at 1650 cm−1 belonging to the c–o group of protein structures. second derivative spectra were also collected for enhancing peak resolution to identify some low resolved infrared bands. comet assay earthworm coelomocytes were obtained using the modified protocol of reinecke and reinecke (2004). the extrusion fluid containing cells was centrifuged and the supernatant removed. the cell pellet was suspended and washed three times in phosphate buffer solution (pbs), using microcentrifugation, for 3 min at 380g. the concentration of cells in the final suspension was determined using the trypan blue exclusion method and dilutions calculated to be used for the exposures (in vitro) and for the comet assay, described below. the comet assay was conducted under yellow light, to prevent uv-induced dna damage, and performed nogueira et al. (2006), with a few minor modifications: normal microscope slides, not fully frosted slides, were used; the slides, were covered with the first agarose layer and left to dry to enable the adherence of the gel layer to the slides; only two layers of agarose were used (the first dried layer and the layer with the cells). visual scoring of cellular dna on each slide was based on table 1 physico-chemical parameters of automobile service station waste mud was analyzed and given below parameters values colour texture ph electrical conductivity(ms/cm) moisture content (%) bulk density (m/m3) porosity (%) n (%) p (%) k (%) cu (ppm) pb(ppm) cd (ppm) zn(ppm) fe(ppm) brownish black sandy clay loam 7.0-7.6 0.4-0.8 40-50 1.211.32 37.8-39.2 8.48.9 1.45 -1.67 12.012.9 47.35-50.06 40.25-42.64 86.7-89.36 98.2-101.0 5796.7-5800.1 the categorization of 100 randomly-selected cells. four specimens per dose were used along with the negative control. for positive control, the cells were treated with ex vivo with 100 µm h2o2 for 7 min at 0 °c. two slides per specimen were prepared and 100 cells per slides were scored by using autocomet software. residual content analysis earthworms from each concentration were taken and washed with distilled water. using above said method pokarzherskii et al. (2000) earthworm’s gut was cleaned and then these earthworms were used for further extraction. then to assess the total metal content of samples, soil and earthworm samples from all the treatments were acid digested to determine the total amount of selected metals. substrates used for acid digestion were dried for 48 h at 70 °c and weighed. after samples were digested in nitric acid and perchloric acid, the metal dry weight concentration was determined by using atomic absorption spectroscopy. the heavy metals concentration in medium, earthworm over 35 days and dna damage scores at percentage of cells in each damage class in coelomocytes of e. fetida after heavy metals exposure over 35days were subjected to suitable statistical transformations and the transformed values were evaluated by one-way analysis of variance in the microsoft excel statistical package. results table 1 summarizes physico-chemical properties of automobile service station waste mud and it was found (47.35 50.06 ppm cu, 40.25 42.64 ppm pb, 86.7 89.36ppm cd, 98.2 101.0 ppm zn and 5796.7 5800.1 ppm fe) that highest range of heavy metals.   239 fig. 1 ftir-atr over lapped spectra of e. fetida both control and 25% concentration spectra with molecular modifications are reported by a red line while control spectra are reported by black line. metabolic action of e. fetida against heavy metals investigated by ftir spectroscopy ftir analysis offers excellent information on the nature of the bonds present on the earthworm surface and allows identification of different functional groups on the cell surface which are capable of interacting with metal ions. changes in band frequency can also be used to estimate the relative importance of the various surface functionalities in metal sorption. the ftir spectra of e. fetida are as shown in figs 1 3 and the list of ftir band assignment is reported in table 2. ftir spectroscopy shows many bands belonging to the different functional groups of worm’s biomolecules which were resulted by the structural modifications caused by the presence of pollutants. as clearly evident from figs 13, all the bands were modified by the exposure to the heavy metals containing waste. the absorbance bands at 2925.81 cm−1 correspond respectively to the ch2 asymmetric and symmetric stretching of methylene group, which mainly monitor lipids. in the present study, the asymmetric band shift from 2925.81 to 2927.74 in 25 % levels respectively, due to heavy metal exposure. the sharp band observed at 1652.88 cm−1 and 1544.32 cm−1 corresponding to amide i and amide ii vibration of structural proteins, respectively, (casado et al., 2007). identified amide i bands absorption was at 1652.88 cm−1 shifted to 1650.95 cm−1 in 25 %, 1654.81 cm−1 in 15 % and 1652.88 cm−1 in 5 % level and it was due to zinc exposure. according to peak assignment (the peaks at 1045, 1080, 1236 cm−1 and 1650 cm−1), these peaks represent the nucleotides and phospholipids. there were shifts in these regions, which mean that heavy metals strongly affect nucleic acids. furthermore we also emphasized that this approach should be confirmed (nucleic acid) with the use of other biomarker like comet assay. comet assay figures 4 and 5 show dna damage scores at percentage of cells in each damage class in coelomocytes of e. fetida, after heavy metals exposure over 35 days at 25 %, 15 % and 5 % concentration. after 35 days of exposure the dna damage, evaluated with the parameter tail dna percentage and tail moment in the coelomocytes of e. fetida exposed to the heavy metals containing automobile service station waste. these findings clearly indicate a connection between accessible or rather available metal content and highest dna damage in the celomocytes of e. fetida. a significant concentration-related increase in the percentage of damaged cells was observed (4.91 ± 5.3 in 5 %, 8.49 ± 10.2 in 15 %, 12.42 ± 12.72 in 25 % and 7.57 ± 10.7 in positive control). this percentage differed significantly when compared with controls, at all assayed heavy metal concentrations. heavy metal accumulation the earthworms collected from different treatments groups, showed considerable accumulation of metals in their tissues. statistically   240 fig. 2 ftir-atr over lapped spectra of e. fetida both control and 15 % concentration spectra with molecular modifications are reported by a red line while control spectra are reported by black line. (anova), the difference among treatments for contents of metals in earthworms was significant. table 3 depicts the heavy metal contents of the automobile service station mud and the mean copper concentration of 25 %, 15 % and 5 % were 22.13 ± 0.32, 15.13 ± 0.007 and 5.34 ± 0.14 ppm, respectively. the pb concentration was 19.4 ± 0.36, 12.48 ± 0.08, 7.06 ± 0.11, 30.96 ± 0.15, 24.6 ± 2.27 and cd concentration was 12.7 ± 0.2 ppm, respectively. zn and fe content in the automobile waste was 43.03 ± 0.29, 29.59 ± 0.04, 10.4 ± 0.01, 924.23 ± 11.92, 404.57 ± 7.3 and 320.78 ± 3.65 (ppm), respectively. differences in the mean heavy metal concentrations between the lower and higher concentrations were significant at 0.05 %. table 2 shows that the accumulated heavy metal concentration in earthworm on 35th day, the cu and pb content in 25 %, 15 % and 5 % earthworm was 1.369 ± 0.042, 1.023 ± 0.04, 0.09 ± 0.001, 6.60 ± 0.095, 4.306 ± 0.02 and 2.2 ± 0.017 ppm, respectively. similarly, the cd, zn and fe content were 0.082 ± 0.004, 0.063 ± 0.006, 0.021 ± 0.001, 3.613 ± 0.006, 3.101 ± 0.003, 1.997 ± 0.013, 2.907 ± 0.004, 1.242 ± 0.3 and 0.007 ± 0.002, respectively. when compared to all heavy metals, fe accumulated more in earthworm tissues. this could have been the result of both heavy metal accumulation with time as well as the increased service station waste mud concentration of the substrate with feed. the difference in waste concentrations in the body tissues of the earthworms between the three groups of exposure was significant (p < 0.005) at the end of the experiment. discussion metabolic action of e. fetida against heavy metals investigated by ftir spectroscopy the aim of this work was to evaluate the effect of heavy metals on the biomolecule changes in the earthworm e. fetida. as ftir spectroscopy may be more sensitive to certain functional group (e.g. including polar bonds) as compared to ft-raman spectroscopy (kamnev et al., 2006), we attempted ftir analyses of worms in control and in the presence of heavy metals containing medium. there are striking differences in the overall ftir profiles between the control and heavy metal exposed worms. a most prominent feature of the metal stressed worm is the appearance of a relatively strong and well resolved ch2 asymmetric and symmetric stretching of methylene group at about 2925.81cm−1. lipids play an important role in the membrane fluidity. by affecting the conformation of membrane proteins, they govern exposure and diffusion of membrane component (palaniappan et al., 2010). in the control, where the amide i at about 1655.10 cm−1 dominates, there was shift to 1650.95 cm−1 in 25 %, 1654.81 cm−1 in 15 %. the shift observed of the amide bands in the zinc exposed tissues indicates important structural alteration in the existing proteins as suggested by toyran et al. (2008). further, the area of amide ii band observed at 1544.32 cm−1 in the control tissues shifted to 1541.02 cm−1 in 25 %. these changes reflect the loss of protein level in the zinc exposed tissues. this loss of protein may be due to increased protein   241 fig. 3 ftir-atr over lapped spectra of e. fetida both control and 5 % concentration spectra with molecular modifications are reported by a red line while control spectra are reported by black line. oxidation in the tissues with zinc exposure (takahashi et al., 1991; cakmak et al., 2006; carpene et al., 2007). the amide i and amide ii bands of cellular proteins at 1656 and 1544 cm−1 are asymmetric and the test showing higher intensity and modified shapes with respect to the same band of the worms is the control. this is likely to reflect some partial changes in the secondary structure of the cellular proteins form dominating α-helix to other possible conformations (naumann et al., 1993; bonnina et al., 1999). this reduced amide bands in the heavy metal exposed tissues indicates important structural alteration in the existing proteins as suggested by toyran et al. (2008). further, the area of amide ii band observed at 1544.88 cm−1 in the control tissues decreases and these changes reflect the loss of protein level of the heavy metal exposed worms. this loss of protein may be due to increased protein oxidation in the exposure worms (takahashi et al., 1991; cakmak et al., 2006; akkas et al., 2007). nucleic acids and proteins intensity change when an external perturbation (i.e. the increased pollutant concentration) is applied to the biological system (melin et al., 2004). this is due to accumulation of pb, zn and cu in e. fetida. some metals, such as hexavalent chromium (crvi), manganese (mn), and pb, as well as cd and arsenic (as), also reportedly inhibited the 8-oxo-gua repair system (bolin et al., 2006; hodges and chipman, 2002; lee et al., 2005; sava et al., 2004; singh et al., 2009) which in turn increased the mutation frequency. ft-ir spectroscopic technique is used to determine the biomolecular profile of micro-samples of coelomic fluid. presence of arginine and lysine and absence of glutamic acid under toxicological condition could be considered as markers of pollutants in the environment (joe et al., 2014). comet assay the comet assay is a genotoxicity assay for the detection of dna single strand breaks (singh et al., 1988) in single cells. the exposure to the heavy metals containing soil seem to have caused significant dna damages after 35 days of exposure compared with the control. the increase in dna damages was probably caused by the production and intracellular accumulation of ros, induced by the exposure of earthworms to heavy metals. evaluation of metal sublethal toxicity should always include biomarkers of dna damage because such damage may result in inappropriate gene expression and, subsequently, in more concerning genotoxic and mutagenic effects. cadmium is of environmental interest due to its high toxicity to organisms and because it is usually mined and extracted from zinc ores (mirsal, 2004). zinc is an essential element, but can be highly toxic when present at high concentrations. it is possible that cd and zn share an uptake pathway in organisms since they have similar electron configuration, as well as chemical and physical properties (brzóska and moniuszko-jakoniuk, 2001). cadmium induces disruption of dna repair leading to mutations that   242 table 2 list of functional groups present in ftir spectra of e. fetida assigned according to the identified bands of absorption peaks control 25% 15% 5% identified bands 1045.35 1045.35 1047.27 1043.42 phospolipids, dna and rna 1080.06 1081.99 1080.06 1080.06 c–o carbohydrates 1153.35 1153.35 11.53.35 1153.35 co–o–c asymmetric stretching: mainly glycogen and nucleic acids 1236.29 1236.29 1236.29 1238.21 c–n peptide group of nucleic acids 1456.16 1452.3 1456.16 1456.16 ch2 bending: mainly lipids with little contribution from proteins 1544.88 1541.02 1544.88 1544.88 amide ii: n–h bending and c–n stretching of the polypeptide and protein backbone 1652.88 1650.95 1654.81 1652.88 amide i: c=o stretching of proteins 2925.81 2927.74 2925.81 2925.81 ch2 asymmetric stretching: mainly lipids, with little contribution from proteins, carbohydrates, nucleic acids 3342.41 3301.91 3296.12 3299.98 amide a: mainly n–h stretching of proteins with negligible contribution from o–h stretching of intermolecular hydrogen bonding together with increased cell proliferation and blocked apoptosis could result in tumor formation (waalkes 2003; waisberg et al., 2003; hei and filipic, 2004). zn has an optimal intracellular range above or below which internucleosomal dna cleavage, chromatin condensation, and nuclear fragmentation are induced (krug, 2002). hwang et al. (2004) and seve et al. (2002) reported that metals such as zn and cd may have apoptotic and/or necrotic effects over cells of different organs. heavy metal accumulation earthworms are known to accumulate metals from the soil efficiently as observed by various authors (ireland, 1975a, b; labort et al., 1998; morgan and morgan, 1988; wright and stringer, 1980). the toxicity of heavy metal for earthworms increases with increasing the soil metal concentration (marinussen et al., 1997). earthworms predominantly take up heavy metals from soluble metal fractions (saxe et al., 2001; vijver et al., 2003). as a result, the possibility for fe metal to be bound to ions and carbonates (i.e. more soluble fractions) increases in ingested material. as a result, the metal content reduces in digested organic material due to bioaccumulations of more soluble fractions of metals in an earthworm’s gut or cutaneous tissues. in general, earthworms consume a great amount of organic waste to achieve appropriate nutrition, and during this process metals are liberated in free forms due to the enzymatic actions in their gut (suthar, 2007). furthermore, such available forms of metals are then absorbed by the epithelial layer of gut during the transiting of wastes through it. bioaccumulation of high concentration of metals is well documented (hsu et al., 2006). heavy metal accumulation depends on the exposure duration whereas the accumulation of fe, zn, cu, cd and pb is dependent upon the metabolic turnover. thereafter metal concentrations remain constant throughout the entire life span. the present study clearly demonstrates that statistically fig. 4 apoptotic dna fragment induced in e. fetida with mixer of heavy metals for 35 days exposure. lane m: 1 kp marker; lane 1: 25% treatment, lane 2: 15 % treatment; lane 3: 5 % treatment and lane 4: control.   243 fig. 5 dna damage scores at percentage of cells in each damage class in celomocytes of e. fetida, after heavy metals exposure over 35 days at 25 %, 15 % and 5 % concentration. significant at p < 0.05 % level significant accumulation of fe, zn, cu, cd and pb in the earthworm does take place as reported earlier (honda et al., 1984). it is known that earthworm chloragosomes function as the cation exchange system capable of taking up and retaining heavy metals (ireland, 1978; morgan and morgan, 1998), which are subsequently excreted by fractionation of the chloragocytes (fischer, 1976). conclusion without the use of biomarkers for identifying ecological risks of land contamination, traditional assessment would be difficult to interpret. therefore, the earthworm based biomarkers used in this study to measure ecological exposure to hazardous substance have identified risks to scientifically relevant organisms and indicated bioavailability of pollutants and their effects. this new ftir based biomolecules study revealed a clear molecular shift in the exposed worms, due to heavy metal accumulation and this is new to earthworm toxicology. the bio-molecule modification responses of heavy metals are best assessed exploiting one of the many budding in vivo techniques. besides, the short-term biomolecule assay (ftir study) on earthworms is an early indicator of long-term toxicity, which may result in dna damage. further, the diverse structural changes in the dna, (dna fragmentation) was visualized and quantified, which provide a promising basis for the development of sensitive biomarkers for ecotoxicological study. acknowledgement this research was supported by the department of science and technology (dst), new delhi, fast track young scientist grant sr/ft/ls007/2007. the authors would like to thank ugcnrcbs and mku-usic for laboratory assistance. table 3 various concentrations of accumulated heavy metals in the medium and as well as earthworm over 35 days exposure in the three different treatments setup heavy metals heavy metals concentration in the medium at the initial stage heavy metals concentration in the medium after 35 days exposure heavy metals concentration in the earthworm after 35 days exposure 25% 15% 5% 25% 15% 5% 25% 15% 5% cu 22.13±0.32 15.03±0.07 5.34±0.14 16.24±1.39 11.93±0.11 4.723±0.52 1.369±0.04 1.023±0.04 0.09±0.00 pb 19.4±0.36 12.48±0.08 7.06±0.11 9.21±1.00 6.94±0.15 4.176±0.16 6.606±0.09 4.306±0.02 2.2±0.01 cd 30.96±0.15 24.6±2.27 12.7±0.2 27.14±1.69 18.68±1.03 10.31±1.11 0.82±0.00 0.063±0.0 0.021±0.00 zn 43.03±0.29 29.59±0.04 10.4±0.01 35.55±1.08 21.0±1.91 7.207±0.41 3.613±0.00 3.101±0.0 1.997±0.01 fe 924.23±11.92 404.57±7.3 320.78±3.65 546.05±20.1 224.7±12.6 134.4±8.78 2.907±0.00 1.242±0.3 0.007±0.0 f value 16880.8* 7567.2* 21693.7* 1993.9* 805.19* 622.09* 7072.9* 450.0* 38399* mean ± sd; * significant at p < 0.05   244 references akkas sb, severcan m, yilmaz o, severcan f. effect of lipoic acid supplementation on rat brain tissue: an ft-ir spectroscopic and neutral network study. food chem. 105: 12811288, 2007. argov s, sahu rk, bernshtain e, salman a, shohast g, zelig u, et al. 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svendsen c. neutral red retention by lysosomes from earthworm (lumbricus rubellus) coelomocytes: a simple biomarker of exposure to soil copper. environ. toxic. chem. 15: 1801-1805, 1996. wright ma, stringer a. lead, zinc, cadmium content of earthworms from pasture in the vicinity of an industrial smelting complex. environ. pollut. 23: 312-321, 1980. 336 isj 13: 336-349, 2016 issn 1824-307x review glutathione s-transferase, catalase, superoxide dismutase, glutathione peroxidase, and lipid peroxidation as biomarkers of oxidative stress in snails: a review j bhagat, bs ingole, n singh biological oceanographic division, csir-national institute of oceanography, dona paula, goa, india accepted october 17, 2016 abstract antioxidant defense plays a crucial role in the response of an organism to pollutants. several processes stimulate the production of free radicals or deplete the antioxidant defense, which if not regulated properly, may cause oxidative stress in the organisms, leading to damage in dna, proteins or lipids. free radicals are also beneficial as it plays an important role in defense against infectious agents, and signal transduction. hence a delicate balance between antioxidants and free radicals is required. oxidative stress biomarkers are very useful in disease etiology and environmental toxicological studies. the increase in anthropogenic activities and environmental awareness has resulted in an explosive increase of research in the field of oxidative stress. snails are excellent organisms for environmental biomonitoring and contribute a major proportion of the invertebrate biomass. in our article, we have summarized the research carried out using glutathione s-transferase (gst), catalase (cat), superoxide dismutase (sod), glutathione peroxidase (gpx), and lipid peroxidation (lpo) in snails exposed to various toxicants and their implication in the environmental monitoring programs. in the end, we have discussed different factors affecting the variations in oxidative biomarkers response for a better understanding of the phenomenon. key words: antioxidant defense system; gastropods; oxidative stress; reactive oxygen species introduction gastropods are ubiquitous invertebrates in the aquatic environment and are commonly studied as a suitable bioindicator for contaminants (itziou and dimitriadis, 2011; abdel-halim et al., 2013). they are widely studied for their ability to accumulate higher amount of heavy metal and other toxic pollutants (baurand et al., 2014; bo et al., 2015). contaminants entering into the aquatic bodies from several sources are taken up by these organisms, and may disturb the free radical process. reactive oxygen species (ros) are potentially dangerous because of their highly reactive nature and hence are neutralized by several antioxidant defense systems. ros are also involved in hormonal responses, signal transduction, and several others physiological processes including heart's pumping, aging, and disease (andersson et al., 2011). there is usually a balance between production of free radical and antioxidant defense. oxidative stress occurs when there are excess amount of free radical due to faulty or lower antioxidant defense ___________________________________________________________________________ corresponding author: jacky bhagat csir-national institute of oceanography dona paula, goa-403004, india e-mail: bhagatjack@gmail.com system in the body. antioxidant defense system helps protect the cell from damages caused by free radical by restoring their level. antioxidant defense system involves both enzymatic and nonenzymatic free radical inactivation and scavenging processes. changes in antioxidant systems of aquatic organisms can serve as indicators for a variety of pollutant exposures related to oxidative stress. thus, it provides sensitive biochemical markers for exposure and toxicity in environmental monitoring. among antioxidant defense, [superoxide dismutase (sod), catalase (cat), glutathione stransferases (gst), glutathione peroxidase (gpx) and lipid peroxidation (lpo) are extensively studied (radwan et al., 2010; el-shenawy et al., 2012; abdel-halim et al., 2013; zheng et al., 2013; wang et al., 2014). glutathione-s-transferases are phase ii multifunctional enzymes, which play a critical role in conjugation of electrophilic compounds (phase i metabolites) on one hand, and in the defense against oxidative damage and peroxidative products of dna or lipids (oost et al., 2003) on the other hand. superoxide dismutases are a class of naturally occurring enzymes that repairs and prevent the oxygen metabolizing cells from the harmful effects of free radicals mainly superoxide. 337 sod catalyze the dismutation of superoxide radicals to oxygen and hydrogen peroxide. catalase plays a key role in antioxidant mechanism by converting hydrogen peroxide (h2o2) to water. glutathione peroxidase plays major role in reductive detoxification process by catalyzing the reduction of hydrogen peroxide, organic hydroperoxide, and lipid peroxides using reduced glutathione. lpo can affect membrane fluidity, association of biomolecules (membrane bound proteins or cholesterol) with membrane, organelle function, and cell health (halliwell and gutteridge, 1999).oxidative stress biomarkers are commonly used by aquatic toxicologists, where multiple contaminants are involved. oxidative stress may cause damage to macromolecules in cell including dna, proteins, and lipids (di giulio et al., 2008). gastropods are widely studied as bioindicator species because of their sedentary lifestyle, easy availability, high sensitivity, and higher bioaccumulation. in this review, we have summarized the current research on oxidative stress biomarkers in gastropods and their usefulness in environmental monitoring program (table 1). in the end, we have discussed different factors affecting the variations in oxidative biomarkers response for a better understanding of the phenomenon. table 1 glutathione-s-transferase (gst), catalase (cat), superoxide dismutase (sod), glutathione peroxidase (gpx), and lipid peroxidation (lpo) in gastropod __________________________________________________________________________________________ glutathione s-transferase (gst) organism organ studied exposure biomarker effects references bellamya purificata gills and digestive glands landfill leachate effluent and bpa increase li et al., 2008 bellamya aeruginosa hepatopancreas ethylbenzene increase zheng et al., 2013 bellamya aeruginosa hepatopancreas toxic cyanobacterium (microcystis aeruginosa) and toxic cyanobacterial cells mixed with a non-toxic green alga (scendesmus quadricauda) increase zhu et al., 2011 cantareus apertus digestive gland carbamate pesticide carbaryl increase leomanni et al., 2015 chilina gibbosa azinphos-methyl no observed effect bianco et al., 2013 eobania vermiculata digestive glands sites contaminated with heavy metals increase el-shenawy et al., 2012 gibbula umbilicalis whole tissue mercury chloride increase cabecinhas et al., 2014 helix aspersa digestive gland, kidney and mantle cavity forming tissue (mcft) napthalene saturated atmosphere no observed effect ismert et al., 2002 helix aspersa digestive gland sites contaminated with heavy metals increase abdel-halim et al., 2013 hexaplex (murex) trunculus digestive gland, gill, muscle cadmium, copper, carbofuran and lindane increase/decrease romeo et al., 2006 helix aspersa digestive glands imidacloprid increase radwan and mohamed, 2013 lymnaea luteola l hepatopancreas single walled carbon nanotubes decreases ali et al. , 2015 lymnaea luteola digestive gland zinc oxide nanoparticles decrease ali et al., 2012 lymnea luteola hepatopancrease gland single walled carbon nanotubes decrease ali et al., 2014 lymnaea stagnalis digestive gland cytosolic as well as microsomal gst activity of the digestive gland of lymnaea siagnalis has been examined wilbrink et al., 1991 monodonta lineata gill cd2+, cu2+ no observed effect cunha et al., 2007 nucella lapillus gill cd2+, cu2+ increase/decrease cunha et al., 2007 physa acuta abamectin increase/decrease ma et al., 2014 338 physa acuta imidazolium ionic liquids (ils) decrease ma et al., 2014 planorbarius corneus soft tissue and gonads chlorpyrifos no effect rivadeneira et al., 2013 theba pisana digestive gland sites contaminated with heavy metals increase radwan et al., 2010 theba pisana digestive gland copper (cu), lead (pb), and zinc (zn) increase/decrease radwan et al., 2010b lanistes carinatus chlorpyrifos for 28 days increase till 21 day and then decrease khalil et al., 2015 theba pisana digestive gland copper-based pesticides; copper oxychloride, copper hydroxide and copper sulphate increase el-gendy et al., 2009 __________________________________________________________________________________________ catalase organism organ studied exposure biomarker effects references achatina fulica kidneys and digestive gland cdcl2 and znso4 decrease at both exposure chandran et al., 2005 biomphalaria glabrata whole body soft tissue azinphosmethyl decrease in pigmented snail whereas nonpigmented snails are unaffected kristoff et al., 2008 biomphalaria arabica whole tissue plant molluscicide solanum nigrum increase al-daihan et al., 2010 biomphalaria alexandrina soft tissue atrazine and roundup decrease barky et al., 2012 biomphalaria glabrata whole tissue paraquat no significant change cochon et al., 2007 bellamya aeruginosa hepatopancreas cu-spiked sediment increase ma et al., 2010 bellamya aeruginosa hepatopancreas ethylbenzene increase zheng et al., 2013 cantareus apertus digestive gland carbamate pesticide carbaryl increase leomanni et al., 2015 chilina gibbosa azinphos-methyl increase bianco et al., 2013 eubania vermiculata methomyl and thiodicarb increase el-w akil et al., 1991 eobania vermiculata digestive glands sites contaminated with heavy metals increase el-shenawy et al., 2012 gibbula umbilicalis mercury chloride no significant change cabecinhas et al., 2014 hexaplex (murex) trunculus digestive gland, gill, muscle cadmium, copper, carbofuran and lindane increase/decreas e romeo et al., 2006 helix aspersa digestive glands imidacloprid increase radwan and mohamed, 2013 helix aspersa digestive gland sites contaminated with heavy metals increase abdel-halim et al., 2013 lymnaea natalensis whole tissue sediment and water from metal polluted sites increase siwela et al., 2010 lymnea luteola hepatopancrease gland single walled carbon nanotubes increase ali et al., 2014 lymnaea luteola digestive gland zinc oxide nanoparticles znonps increase ali et al., 2012 onchidium struma hepatopancreas and muscle cu2+ increase/decreas e li et al., 2009 physa acuta soft tissues abamectin increase ma et al., 2014 physa acuta viscera imidazolium ionic liquids (ils) increase ma et al., 2014 theba pisana digestive gland sites contaminated with heavy metals increase radwan et al., 2010 theba pisana digestive gland [copper (cu), lead (pb), and zinc (zn) increases radwan et al., 2010b theba pisana digestive gland copper-based pesticides; copper oxychloride, copper hydroxide and copper sulphate increase el-gendy et al., 2009 lanistes carinatus chlorpyrifos for 28 days increase till 21 day and then decrease khalil et al., 2015 __________________________________________________________________________________________ 339 __________________________________________________________________________________________ superoxide dismutase (sod) organism organ studied exposure biomarker effects references achatina fulica kidneys and digestive gland cdcl2 and znso4 decrease at both exposure chandran et al., 2005 achatina fulica viscera (digestive gland) triclosan increases w ang et al., 2014 biomphalaria glabrata whole body soft tissue azinphosmethyl decrease in pigmented snails whereas nonpigmented snails showed biphasic effect kristoff et al., 2008 biomphalaria alexandrina whole tissue and hemolymph schistosoma mansoni increase/decrease mahmoud and rizk, 2004 biomphalaria alexandrina soft tissue atrazine and roundup (glyphosate) decrease barky et al., 2012 biomphalaria glabrata whole tissue paraquat decrease cochon et al., 2007 bellamya aeruginosa hepatopancreas ethylbenzene increase zheng et al., 2013 bellamya aeruginosa hepatopancreas cu-spiked sediment increase ma et al., 2010 cantareus apertus digestive gland carbamate pesticide carbaryl increase leomanni et al., 2015 chilina gibbosa azinphos-methyl no observed effect bianco et al., 2013 gibbula umbilicalis mercury no significant change cabecinhas et al., 2014 lymnaea stagnalis hemocyte particulate agents (latex, escherichia coli, staphylococcus saprophyticus, zymosan and with phorbol myristate acetate phagocytic stimulation of the hemocytes resulted in a superoxide dismutase induction dikkeboom et al., 1987 lymnaea natalensis whole tissue sediment and water from metal polluted sites decrease siwela et al., 2010 onchidium struma hepatopancreas and muscle cu2+ increase/decrease li et al., 2009 oncomlania hupensis liver tissue arisaema erubescens and nerium indicum extracts increase zhang et al., 2009 oncomelania hupensis liver sanguinarine (50 oncomelania hupensis per 500 ml solution) increase sun et al., 2011 oncomelania hupensis cephalopodium and liver extracts of arisaema erubescens tubers by acetic acetal (aae), benzinum (bze), n-butanol (nbe) and chloroform (cfe) were increase ke et al., 2008 physa acuta abamectin increases at earlier exposure and later declines ma et al., 2014 physa acuta imidazolium ionic liquids (ils) decrease ma et al., 2014 __________________________________________________________________________________________ glutathione peroxidase (gpx) organism organ studied exposure biomarker effects references austrocochlea porcata soft tissues crude oil increase reid et al., 2003 achatina fulica kidneys and digestive gland cdcl2 and znso4 decrease at both exposure chandran et al., 2005 bellamya purificata landfill leachate effluent and bisphenol a (bpa) no observed effect li et al., 2008 cantareus apertus digestive gland carbamate pesticide carbaryl increases leomanni et al., 2015 eobania vermiculata digestive glands sites contaminated with heavy metals increase el-shenawy et al., 2012 helix aspersa digestive gland sites contaminated with heavy metals increase abdel-halim et al., 2013 lanistes carinatus chlorpyrifos increases khalil et al., 2015 lymnaea luteola l hepatopancrease gland silver nanoparticles increases ali et al ., 2014 340 lymnaea natalensis polluted sediment and water increases siwela et al., 2010 lymnaea luteola l hepatopancreas single walled carbon nanotubes decreases ali et al., 2015 lymnaea luteola l digestive gland zinc oxide nanoparticles increases ali et al., 2012 theba pisana digestive gland metal polluted sites increases radwan et al., 2010 theba pisana digestive gland copper-based pesticides; copper oxychloride, copper hydroxide and copper sulphate increase el-gendy et al., 2009 __________________________________________________________________________________________ lipid peroxidation organism organ studied exposure biomarker effects references achatina fulica viscera (digestive gland) triclosan dose dependent increase w ang et al., 2014 achatina fulica kidneys and digestive gland cdcl2 and znso4 increase at both exposure chandran et al., 2005 biomphalaria glabrata whole tissue paraquat increase cochon et al., 2007 biomphalaria arabica whole tissue molluscicide solanum nigrum increase al-daihan et al., 2010 biomphalaria alexandrina soft tissue exposure to pesticides atrazine and roundup (glyphosate) increase barky et al., 2012 bellamya aeruginosa hepatopancreas ethylbenzene no observed effect zheng et al., 2013 eobania vermiculata digestive glands sites contaminated with heavy metals increase el-shenawy et al., 2012 gibbula umbilicalis mercury no significant change cabecinhas et al., 2014 helix aspersa extremely low frequency (elf) 50-hz magnetic fields increase regoli et al., 2005 helix aspersa digestive gland sites contaminated with heavy metals increase abdel-halim et al., 2013 lymnea luteola hepatopancrease gland single walled carbon nanotubes increase ali et al., 2014 lymnaea natalensis whole tissue sediment and water from metal polluted sites increase siwela et al., 2010 lymnaea luteola l hepatopancreas carbon nanotubes increase ali et al 2015 lymnaea luteola digestive gland zinc oxide nanoparticles increase ali et al., 2012 lanistes carinatus chlorpyrifos increase khalil et al., 2015 oncomelania hupensis liver sanguinarine (50 oncomelania hupensis per 500 ml solution) increased but not significant sun et al., 2011 physa acuta soft tissue abamectin increase ma et al., 2014 physa acuta viscera oxicities of imidazolium ionic liquids (ils) increase ma et al., 2014 theba pisana digestive gland sites contaminated with heavy metals increase radwan et al., 2010a theba pisana digestive gland copper (cu), lead (pb), and zinc (zn) increases for all exposures radwan et al., 2010b theba pisana digestive gland copper-based pesticides; copper oxychloride, copper hydroxide and copper sulphate increase el-gendy et al., 2009 __________________________________________________________________________________________ reactive oxygen species (ros) oxygen is essential to life and also toxic at the same time due to formation of free radicals. the production of ros is a natural phenomenon, which is triggered by various external factors. oxidative stress is caused due to imbalance between cell's oxidative defense and the production of excess ros during the impaired oxidant defense mechanism (fig. 1). ros can be radical (superoxide, ˙o₂⁻; hydroxyl, ˙oh; peroxyl, ˙ro2; alkoxyl, ˙ro; hydroperoxyl, ˙ho2) or non-radical (hydrogen peroxide, h2o2; hypochlorous acid, hocl; ozone, o3; singlet oxygen, 1o2; peroxynitrite, onoo⁻). radicals are very reactive 341 fig. 1 overview of reactive oxygen species and oxidative stress that carry one or more unpaired electron, and is important in lipid peroxidation and dna damage (halliwell and gutteridge, 1999). the non-radicals are mainly responsible for causing oxidative stress. some chlorinated compounds can generate hydrogen radical from h2o2 by direct metal ionindependent reactions (halliwell and gutteridge, 2007). hydroxyl radical can be produced as a result of conjugation of transition metal ions and h2o2 during fenton reaction and hemolytic fission of o-o bond in h2o induced by uv radiation (halliwell and gutteridge, 1999).hydroxyl radical is strongly reactive and can cause more biological damage than any other ros. highly reactive ˙oh can abstract h atom from sugars, purines, and pyrimidines in dna bases and formvariety of products (breen and murphy 1995). ˙oh can also react with protein or lipid molecule to form oxidative damage products. ˙oh can be enzymatically metabolized to oxygen and water or converted to extremely reactive hydrogen peroxide. organic compounds are well-known prooxidants and accelerate the production of ros and also increase lipid peroxidation. during the metabolic pathway of oxidation of organic compounds, oxygen undergoes stepwise reduction coupled with generation of atp during electron transport, leading to production of ros. transfer of one, two or three electrons to oxygen result in production of o2 (superoxide radical), h2o2 (hydrogen peroxide) and oh(hydroxyl radical) respectively. ros are product of normal metabolism and plays an important role in cell signaling and pathogen defense. the superoxide anion can also be produced due to action of xanthin and hypoxanthin oxidase enzyme. it doesn't readily cross cell membrane, although it can pass through anion exchange protein. superoxide anions are readily converted to oxygen and hydrogen peroxide by the action of superoxide dismutase. however, due to imbalance between the production of ros and biological system’s ability of detoxification, ros get accumulated into the cell and causes deleterious effects. the ros formed can react with macromolecules (e.g. lipids, proteins, nucleic acids) and provoke protein denaturation, lipid peroxidation, dna damage and others. hydrogen peroxide can penetrate biological membrane and act as an intermediate in the production of other highly reactive ros species (eg. hydroxyl radical). hydrogen peroxide can be removed from the cell by several enzymes like catalase, glutathione peroxidase and peroxiredoxins. consequently, cells have evolved with several defense mechanisms to neutralize the damage caused by ros and their reactive products. the non-enzymaticmechanism involves quenching of ros by molecules like gsh, carotenoids or ascorbate. antioxidantenzyme is the major mechanism in scavenging the free radical produced as a result of various metabolic activities. snails in environmental monitoring molluscs have been used extensively for environmental monitoring programs. gastropods 342 account for eighty percent of all living molluscs, and have recently attracted attention because of their potential as sentinel organisms, thanks to the discovery of imposex due to tbt pollution (lima et al., 2011). gastropods perform an important role in marine food chain and are used as a source of food for many fishes and birds. they are omnipresent in the aquatic environment and are identified as a suitable bioindicator for heavy metal pollutions (selgrade, 1999, 2005; gundacker, 2000; galloway and depledge, 2001; galloway and handy, 2003; auffret, 2005; woolhiser et al., 2005; itziou and dimitriadis, 2011; abdel-halim et al., 2013). they transfer toxicants from lower to higher tropic level organism through food chain. they have limited ability to metabolize xenobiotics and thus they are prone to accumulate high concentrations of hydrocarbons (zheng et al., 2012). the studies involving the genotoxic and cytotoxic damage in snails exposed to pollutants are well documented (sarkar et al., 2008, 2011; an et al., 2012; bhagat et al., 2012; ma et al., 2014). oxidative stress biomarkers oxygen is indispensable for all the aerobic species but the reactive forms of oxygen such as the superoxide (oxygen with an extra electron) can lead to certain disasters. to combat these phenomenon, the organism has several mechanisms which keep these reactive species in control as they are also important in several beneficial processes. however, there are several processes which are interdependent on each other because they share many metabolites and products. antioxidant enzyme can be induced under slight oxidative stress but severe oxidative stress can lead to suppression of these enzymes (xu et al., 2009). gst glutathione s-transferase is a phase ii multifunctional enzyme and plays a critical role in conjugation of electrophilic compounds (phase i metabolites) on one hand and in the defense against oxidative damage and peroxidative products of dna and lipids (oost et al., 2003) on the other hand. the gst activity in marine species is greatly induced by the presence of xenobiotic contaminants in the environment. thus an increase in the gst activity in the marine organisms clearly indicates their exposure to such toxic contaminants. an increasing concentration of ros causes oxidative stress which may lead to increase in the gst activity. therefore, an increase in gst activity shows the high concentration of xenobiotic compounds present in the environment. gst has been proposed as a promising biomarker in snails exposed to aquatic pollutants (li et al., 2008). induction of gst activity has been reported in several studies on gastropods exposed to variety of pollutants. the studies concerning seasonal and developmental differences in gst in snails are limited. gsts are involved in the metabolic activation and deactivation of pah metabolites. an increased gst activity was observed in experiments with freshwater snail bellamya aeruginosa exposed to ethylbenzene for 7 days (zheng et al., 2013). however, gst activity at higher concentrations (450 and 1,000 µg/l) showed progressive decrease after 14 or 21 day of exposure which may be due to the poisoning effect on gst by extra ros (cunha et al., 2007). ismert et al., (2002) reported no change in gst activity in digestive gland, kidney and mantle cavity forming tissue (mcft) in snail helix aspersa exposed to naphthalene. the author reported 1.5 fold decreases in gpx activity in digestive gland in exposed snails. exposure to pesticides has shown induction or inhibition of enzyme activities. copper-based pesticides have been shown to cause decline in gsh content in digestive gland of land snail, theba pisana (el-gendy et al., 2009). rivadeneira et al. (2013) showed no effects on gst activity in freshwater snail planorbarius corneus exposed to organophosphate insecticide chlorpyrifos. in another study azinphos-methyl also failed to induce any effect on gst in b. glabrata (kristoff et al., 2008). in contrast, azinphos-methyl showed no change in gst activity in freshwater gastropod chilina gibbosa (bianco et al., 2013). more research in this context is needed to understand the detoxification mechanism of pesticides involving gsts. li et al. (2008) has investigated the effects of landfill leachate effluent and bisphenol a in b. purificata and reported decrease in the gsh content and increase in the gst content in treated snails. ma et al. (2014) has studied the toxic effects of abamectin (abm), in physa acuta and reported promotion of gst activity at the earlier periods of treatment (12 48 h) in exposed snails, and inhibition at the end of test. accumulations of heavy metal in shells and whole tissues of lymnaea luteola exposed to contaminated sediments have been studied by siwela et al. (2010). the author reported significantly higher concentrations of pb, cd, zn and ni in soft tissues and zn, cu and cd in shells of exposed snails. an increase in gst activity in h. aspersa was observed in the metal contaminated sites then in reference sites (abdelhalim et al., 2013). h. trunculus has been shown to accumulate heavy metals like cadmium (bouquegneau et al., 1988; dallinger et al., 1989), copper (romeo et al., 2006), mercury (catsiki and arnoux, 1987). increased activity of gst with increasing hg concentrations was observed in sea snail gibbula umbilicalis (cabecinhas et al., 2014). some studies have shown conflicting results with unaltered or lower gst activities in snails exposed to heavy metals. in vivo exposure to cadmium chloride showed no significant change on gst activity snails (monodonta lineata and nucella lapillus) (cunha et al., 2007). the author reported decrease in gst activity when n. lapillus was exposed to copper sulphate pentahydrate. snail h. trunculus has shown a decrease in gst activity when exposed to cadmium. decrease in gst activity after exposure to heavy metals can be due to direct action of metals on the enzyme or inhibition of gst by extra ros (shumilla et al., 1998). gsh tend to chelate with heavy metal before the heavy metal reacts with metallothioneins, which can also indirectly cause decrease in the gst activity. metals can also cause depletion of the gsh substrate by binding or oxidizing it (canesi et al., 1999). lower 343 reduced glutathione (gsh) content was observed in snail t. pisana collected from sites polluted with heavy metals (radwan et al., 2010a). negative correlation of gsh and heavy metal concentrations in digestive gland has been reported by the author. various factors can also influence the downregulation of the gst gene (roling and baldwin, 2006). ali et al. (2012) have also reported decrease in gst activity in digestive glands of l. luteola exposed to zinc oxide nanoparticles. seasonal variation in gst activity has also been reported in snails. highest activity of gst was observed in summer close to polluted sites with heavy metal in h. aspersa (larba and soltani, 2014). oxidative stress responses were correlated with increasing metal concentrations in soil samples in h. aspersa (larba and soltani, 2014). romeo et al. (2006) has investigated the effect of toxic metals (cadmium and copper) along with two organics (carbofuran and lindane) to snail h. trunulus. the author found that digestive gland and gill cells have been shown to accumulate more metals than muscle. this observation indicates that gsts are a suitable biomarker in snails exposed to heavy metals. cat catalase plays an important role in enzymatic oxidant defense by protecting the cell from hydrogen peroxide by converting them into oxygen and water. niyogi et al. (2001) has also reported positive correlation of cat activity with pah content in oyster s. cucullata. such a relationship has also been reported for different bivalve species exposed to hydrocarbonsthat suggest that oxidative stress may be induced by hydrocarbons. b. aeruginosa (reeve), exposed to ethylbenzene (5 1,000 µg/l) for 21 days has reported an increase in cat activity (zheng et al., 2013). increase in cat activity were also reported in snails exposed to imidacloprid (radwan and mohamed, 2013), zinc oxide (ali et al., 2012), carbon nanotubes (ali et al., 2015), abamectin (ma et al., 2014), imidazolium ionic liquids (ma et al., 2014). exposure to metal increases the production of ros including h2o2 which in turn induces the cat activity in the body. increased activity of cat was shown in h. aspersa exposed to metal dust containing cu, zn, pb, cr, ni and fe (nedjoud et al., 2009). snail h. trunculus when exposed to cadmium, carbofuran, and lindane, has shown increase in cat activity (romeo et al., 2006). radwan et al. (2010b) has reported increased level of cat in theba pisana exposed to copper (cu), lead (pb), and zinc (zn). cdcl2 and znso4 have also shown to induce cat activity in achatina fulica (chandran et al., 2005). chronic exposure to cuspiked sediment has shown to induce cat activity in b. aeruginosa (ma et al., 2010). there was no significant increase in cat activity in gibbula umbilicalis when exposed to mercury for 96 h (cabecinhas et al., 2014). however, there are also reports that cat activity decreases in the digestive gland of the h. aspera exposed to ni (zawiszaraszka et al., 2010). evidences from laboratory exposure of pesticides and insecticides to snails have shown conflicting results. significant increase in cat activity was reported in a. fulica exposed to 40, 69, and 118 mg/kg of triclosan but high concentrations of 200 and 340 mg/kg showed inhibition in the enzyme activity (wang et al., 2014). in c. gibbosa, significant increase in cat activity was reported in snails exposed to azinphos-methyl (bianco et al., 2013). in another study el-wakil et al. (1991) also reported significant increase in cat activity in eubania vermiculata exposed to methomyl and thiodicarb whereas a decrease in cat activity was observed when the snails were exposed to metaldehyde. thebapisana exposed to sublethal doses (40 % and 80 % of ld(50) after 48 h) of copper-based pesticides; copper oxychloride, copper hydroxide and copper sulphate have shown increase in cat activity (el-gendy et al., 2009). in another study, khalil et al. (2015) reported increase in cat activity in lanistes carinatus exposed to chlorpyrifos till 21 days and then a decline in enzyme activity was observed. biomphalaria alexandrina exposed to atrazine and roundup (glyphosate) has shown decrease in cat activity (barky et al., 2012). in another study cochon et al. (2007) has reported no significant change in biomphalaria glabrata exposed for either 4 or 48 h to 0.5mg/l of paraquat. sod significant increase in sod activity in hepatopancreas of onchidium struma was observed after one week exposure to cu2+ (range 1.35 to 4.20 mg/l), however in the muscle the increase in sod activity was not consistent. cadmium has shown to decrease sod activity in snail a. fulica (chandran et al., 2005). exposure to heavy metal results in increased production of ros, which is controlled by enzymatic and non-enzymatic defense in the cell. gsh act as a quencher of o2*, decrease in gsh level can lead to production of high amount of o2*, which in turn can inhibit sod activity in the cell. inhibition of sod activity was reported in a. fulica exposed to cdcl2 and znso4 (chandran et al., 2005). in sail (l. natalensis), exposed to sediment and water from metal-polluted sites, decrease in sod was observed (siwela et al., 2010). exposure to triclosan resulted in significant increase in sod activity in a. fulica (wang et al., 2014). similar results were also reported in the digestive gland of cantareus apertus exposed to carbamate pesticide carbaryl (leomanni et al., 2015). barky et al. (2012) found exposure to pesticides atrazine and roundup (glyphosate) tends to decrease sod in b. alexandrina. cochon et al. (2007) also observed sod activity was significantly decreased (27 %) in b. glabrata snails after 48 h of treatment with paraquat. a decrease in sod activity in pigmented b. glabrata was observed, after exposure to 5 mg/l azinphosmethyl for 48 or 96 h, however in the non-pigmented exposed snails the enzyme activity was found to be biphasic. bianco et al., (2013) reported no change in sod activity in c. gibbosa exposed to exposure to azinphos-methyl (0.2 20 μg/l). ke et al. (2008) observed an increase in sod activity in cephalopodium and liver in oncomelania hupensis exposed to four extracts of arisaema erubescens tubers by acetic acetal (aae), 344 benzinum (bze), n-butanol (nbe) and chloroform (cfe). the snail physa acuta exposed to oxicities of imidazolium ionic liquids (ils) showed decrease in sod activity (ma et al., 2014). it has been found that sod is inactivated by dismutation product, h2o2 over time (rhodes, 2000). glutathione peroxidase (gpx) gpx catalyzes the reduction of hydroperoxides including h2o2, using reduced glutathione. the level of h2o2 is dependent on the sod, which produces it and also on cat which uses h2o2 as a substrate. gpx activity also depends on the intake of dietary selenium and the level of reduced glutathione (gsh), which is involved in the reaction catalyzed by gst. hence a battery of biomarker is involved in detoxification of ros and these biomarkers are inter-dependent on each other. the increase in gpx activity is often observed in snails collected from sites contaminated by heavy metals (radwan et al 2010; abdel-halim et al., 2013). snails exposed to water from metal-polluted area have also been reported to induce gpx activity (siwela et al., 2010). in another study, li et al. (2008) has observed no change in gpx activity in snail b. purificata exposed to landfill leachate effluent and bisphenol a (bpa). exposure to zinc oxide nanoparticles resulted in increase in gpx activity in snail l. luteola. pesticides cause generation of free radical which increases the oxidative stress in the body. several investigations have reported increase in gpx activity in snails exposed to pesticides and insecticides. increase in gpx has been reported in cantareus apertus (leomanni et al., 2015), lanistes carinatus (khalil et al., 2015), and t. pisana (elgendy et al., 2009) exposed to various pesticides. in contrast, few studies have also shown decrease or no effects on gsh in organism exposed to pesticides. cacciatore et al. (2015) has reported significantly decreased glutathione (gsh) in planorbarius corneus exposed to azinphos-methyl and chlorpyrifos. in a study carried out by liu et al. (2015), decrease in gsh content were reported in goldfish carassius auratus exposed to dichlorvos. as gpx uses gsh as the reducing factor for the detoxification of hydroperoxide, unavailability of gsh might lead to decrease value of gpx. lipid peroxidation (lpo) lipid peroxidation has been studied extensively in environmental monitoring programs. malondialdehyde (mda) is a low molecular weight end product of lipid peroxidation and is widely used as a biomarker of oxidative stress. increased level of oxidative damage in terms of lipid peroxidation has been reported in various snail species exposed to laboratory or environmental contaminants (aldaihan et al., 2010; barky et al., 2012; el-shenawy et al., 2012; ali et al., 2015; ma et al., 2014). a widely used pesticides paraquat has shown a significant increase in lpo in b. glabrata (cochon et al., 2007). in another study barky et al. (2012) reported induction of lpo in b. alexandrina exposed to atrazine and roundup pesticides. lanistes carinatus when exposed to chlorpyrifos for 28 day has also shown induction of lpo (khalil et al., 2015). carbon nanotubes have shown to induce lpo in l. luteola (ali et al., 2015). some studies have shown no increase in lpo with exposure to contaminants e.g. zheng et al. (2013) has shown no change in lpo in b. aeruginosa (reeve), exposed to ethylbenzene. dose dependent increase in lpo was reported in digestive gland of achatina fulica exposed to broad-spectrum antimicrobial agent, triclosan (wang et al., 2014). in the field, sites contaminated with heavy metal have shown to induce lpo in snails (radwan et al., 2010a; abdel-halim et al., 2013). cu, pb, zn have also shown to increase lpo in t. pisana (radwan et al., 2010b). increased lipid peroxidation occurred in l. natalensis exposed to sediment and water from metal-polluted sites (siwela et al., 2010).higher values of lpo were reported in digestive glands of h. aspersa collected from metal contaminated sites by abdel-halim et al. (2013). elevated level of lpo was reported in kidneys and digestive gland in a. fulica exposed to cdcl2 and znso4 (chandran et al., 2005). cadmium doesn't generate ros directly, but can alter the gsh and mt level in the cell, which can lead to lpo of cell membrane (mahboob et al., 2013). it has been known that lpo products are complex hydroperoxides that exerts cytotoxic and genotoxic damage. it has been demonstrated that cat and sod inhibit lipid peroxidation (packer and fuchs, 1992). exposure of contaminants results in increase production of hydrogen peroxide which is destroyed by cat. increase activity of cat under these condition can effectively counteract lpo. increase in lpo was reported in oysters exposed to cu but cu in combination with gsh inhibitor (buthionone sulfoximine) has shown lower lpo values, suggesting that gsh has protective role to cu toxicity (conners and ringwood, 2000).exposure to cu in oysters (resulted in increased lipid peroxidation (lpo) (conners and ringwood, 2000) but at the same time, when exposed to cu and a gsh inhibitor, a lower lpo was reported suggesting a protective role of gsh to cu toxicity. collectively these data suggests that lpo along with other oxidative stress biomarkers such as gst, cat, and sod can help in better understanding of the oxidative stress phenomenon. conclusion and perspective variations in oxidative stress biomarkers also depend on food availability, spawning period and exposure to pollutants and water temperature (sheehan and power, 1999; hagger et al., 2010). however, several other factors such as mode of life, reproductive cycle, and feeding habit can also influence the oxidative stress biomarkers (sheehan and power, 1999). abiotic factors such as temperature, salinity and dissolved oxygen content also influence the levels of antioxidant enzymes. temperature and salinity significantly affect the biomarker response in an organism (cailleaud et al., 2007). weak seasonal changes in environmental parameters can result in lack of seasonal variations in the biomarker. higher temperature induces the oxygen consumption in the cell leading to elevated production of ros, which are compensated antioxidant defense (ronisz et al., 1999). increase in lpo activity at higher temperature is due to 345 increase in the polyunsaturation in mitochondrial membrane due to higher mitochondrial respiration, with increase in the formation of ros. use of multibiomarkers has become an increasingly popular approach to study environmental parameters and organismal health. combination of biomarkers gives a comprehensive picture and provides better insights to study the effects of pollutants. hence the appropriate combination of biomarkers is recommended for the study of oxidative stress. acknowledgement we would like to thank the director, csir-nio for all the facilities and encouragement and csir new delhi for financial support under project no psc0206. department of biotechnology (govt. of india), new 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1824-307x report of meeting xviith scientific meeting of the italian association of developmental and comparative immunobiology (iadci), 11 13 february 2016, department of biological and environmental sciences and technologies, university of salento, lecce, italy organizers: p pagliara, l stabili department of biological and environmental sciences and technologies, university of salento, lecce, italy session 1. chairmen: g scapigliati, università della tuscia, viterbo, italy and a vallesi, university of camerino, camerino (mc), italy insights into fish immunity lecture evolution of the immune response to pathogens mr coscia institute of protein biochemistry, cnr, naples, italy to survive, organisms must continually adapt to continually evolving invading organisms. hosts and pathogens are the key players of a continous conflict in which natural selection aids pathogens to increase virulence to escape host surveillance, and hosts to acquire adequate defence strategies. in both cases, these achievements are limited by several factors such as the genetic fitness and the number of genes required, much larger than that available in the genome. moreover, since the pathogen usually has a very shorter life than the host, it has to fix new mutations favoring virulence much faster than the host can evolve effective defense mechanisms. another constrain concerns the host that must avoid adverse effects which may derive from the defence system itself. once resistance and counter resistance are genetically assessed, both the host and the pathogen evolve in response to mutually exerted pressures. this is generally referred to as the “red queen paradigm”, that highlights the significance of biotic versus abiotic factors that lead to constant evolutionary changes. evolution acts at different levels: biotic factors mainly shape species diversity over short time periods, whereas changes in the physical environment such as climate changes drive evolution at a large scale, during much longer time. much of our current knowledge of infection biology is based on studies of the immune system in humans and mice. in contrast, much less attention has been paid to immune response in lower vertebrates. since many features of immune defence mechanisms have been acquired throughout evolution, studying the evolution of successful pathogen virulence mechanisms highlights the potential weaknesses in host immune defenses. on the other hand, investigating the defence mechanisms which species other than tetrapods have evolved to counter infectious agents may allow to identify novel molecules and strategies useful to manage an infection in the host’ s favor. so far, few attempts have been made at considering host and pathogen as interacting partners into a common evolutionary framework. a short overview on how the host-pathogen interaction has been shaped by evolution will be given. analysis of antarctic teleosts transcriptomes as a tool to explore adaptive immune responses f buonocore1, m gerdol2, a pallavicini2, c bernini1, s mattiucci3, d lucente4, r cimmaruta4, g scapigliati1 1department for innovation in biological, agro-food and forest systems, tuscia university, largo dell'università s/n, viterbo, italy 2department of life sciences, university of trieste, via giorgieri 5, 34127 trieste, italy 3department of public health and infectious diseases, section of parasitology, "sapienza" university of rome, p.le aldo moro 5, rome, italy 4department of ecological and biological sciences, tuscia university, largo dell'università s/n, viterbo, italy during the last decade the use of nextgeneration sequencing technology has become highly widespread to generate massive amounts of sequence data mainly due to its reduced costs and to the possibility of collecting simultaneously information useful both for transcriptome characterization and quantification. in the present work we analyzed two transcriptomes from the head kidney of the emerald rockcod (trematomus bernacchii) and from gills of the icefish (chionodraco hamatus). the sequences were generated with an illumina platform and, successively, de novo assembling procedures were performed to obtain the final transcripts. a first aim was to identify new genes related to adaptive immune responses by using as a comparison the 44   known genome of the antarctic fish notothenia coriiceps, which contains about 30.900 expected transcripts. we were able to confirm that in the icefish transcriptome about 20400 transcripts were present (66,1 %), whereas in the emerald rockcod we found about 19800 transcripts (64,2 %). moreover, orthologous proteins showed about 80 % amino acid identity considering chionodraco and trematomus transcriptomes. from these transcriptomes, a relative high number of sequences related to adaptive immune responses genes have been identified and confirmed by cloning from cdna, e.g., mhc-i, mhc-ii, beta2microglobulin, cd4, cd8alpha, igt, igd, etc. the identification of mhc-ii molecules provided the opportunity of evaluating the levels of genetic variation at a mhc-ii β locus in the icefish population from the ross sea. preliminary data suggest a genetic variability comparable to that reported for other fish species at both interand intra-individual levels. this finding allows exploring possible relationships occurring between the levels of genetic variation of the mhc in the icefish with respect to the parasitic load recorded in this fish host. preliminary data on the effects of chestnut skin extracted polyphenols on oncorhynchus mykiss blood and galt l parrillo1, e coccia1, mg volpe2, s costantini3, e varricchio1, m paolucci1,2 1dipartimento di scienze e tecnologie, università degli studi del sannio, via port’arsa 11, 82100 benevento, italy 2istituto di scienze dell’alimentazione-cnr, via roma 64, 83100 avellino, italy 3crom: centro ricerche oncologiche, mercogliano, italy agricultural by-products are a rich source of bioactive molecules, including polyphenol compounds or “polyphenols”. the immunostimulant and antioxidant properties of polyphenols may have a potential role for animal welfare and for the production of “ healthy “ feed. polyphenol-enriched feed has been finding application in animal farming, thank to polyphenol capability to improve the productive performance, immune response and health of livestock. for this reason, polyphenols may also represent a valid alternative to antibiotics and medicines currently employed in animal farming. the introduction of natural extracts in animal feed represent a booming business after the ban of auxinic antibiotics and it proves that invest resources in the search for plant extracts can deliver significant benefits. the interest in use natural substances, known and used since ancient times in the care of man, can certainly be considered innovative for the animal diet. the potential immunostimulant activity of polyphenols extracted from chestnut skin has been studied on lymphocytes isolated from blood and lymphocytes extracted from galt of the rainbow trout oncorhynchus mykiss. the assays used to evaluate the parameters of both innate immunity and acquired immunity were: superoxide anion production; phagocytosis; expression of pro and anti-inflammatory cytokines. our results indicate that superoxide anion production and phagocytosis decreased in both the blood and galt leucocytes incubated in vitro with concentrations of chestnut skin polyphenols ranging from 10 to 100 µg/ml. higher concentrations (500 and 5000 µg/ml) were instead stimulating both anion superoxide production and phagocytosis. chestnut skin polyphenols used in our experiments were also able to modulate the gene expression of immune-related cytokines, such as tnf-α and il-10. specifically, it was observed an upregulation of the proinflammatory cytokine tnf-α and a downregulation of the anti-inflammatory cytokine il-10 in blood and galt leucocytes. similar results were obtained with gallic acid and ellagic acid, although the effects were less evident, suggesting that the effects of chestnut skin polyphenols are depending on the mixture synergism between the various phenolic compounds. in the light of these preliminary results, we suggest that the addition of polyphenols to standard diet may improve the immune response of farmed fish. moreover, this study suggests the possible reuse of agri-food industry wastes as feed additives for farmed animals. strategies for detection and vaccination of juveniles european sea bass (dicentrarchus labrax) against betanodavirus n nuñez-ortiz1, f pascoli2, a toffan2, f buonocore1, s picchetti1, g scapigliati1 1dipartimento per l’innovazione nei sistemi biologici agroalimentari e forestali, università della tuscia, italy 2istituto zooprofilattico sperimentale delle venezie, centro di referenza nazionale (nrl) per le patologie dei pesci, molluschi e crostacei, legnaro (pd), italy encephalopathy and retinopathy virus (verv) or betanodavirus causes massive mortalities of the most important farmed species in mediterranean area, the european sea bass. in order to develop strategies for the control of virus infection and virus detection, we have studied the possibilities of vaccinating sea bass at young age (2-10 grams) through mucosal and intraperitoneal immunization using verv inactivated by differents ways. after inactivation of verv by differents protocols: formalin, bpl and heat treatment we have performed two control experiments using the differents inactivated verv by immersion and intraperitoneal administration. serum antigenspecific igm titers was determined by indirect elisa being specially significant after intraperitoneal immunization with formalin-inactivated verv. vervfree juveniles immunised by immersion in formalininactivated virus showed the presence and the uptake of verv in the gills by immunohistochemistry (ihc). quantitative pcr expression on gut and head kidney after intraperitoneal administration with formalin-inactivated virus showed modulation in the expression of antiviral gene isg-12 after 48 h in both organs and mx gene in gut after 48 h too but 45   induced detectable modulation of mx and isg12 genes was detected in the gills 24 h postimmersion. finally, challenge experiment using live verv were performed after immunization with formalininactivated verv, and we observed a 80 % increase in a relative protection value in intraperitoneal immunizated fish with respect to unimmunized fish. in the other hand, bath immunization resulted in no protection in in vivo challenge. in addition, by employing a rabbit antiserum against verv (pab 283) and a mouse monoclonal against verv capsid protein (mab 4c3) we have developed an elisa system to detect and quantitate the presence of verv in solutions and biological fluids. fish cytokines: il-4/13 in sea bass (dicentrarchus labrax), molecular characterization and expression analysis after in vitro and in vivo stimulation v stocchi, e randelli, m gerdol1, a pallavicini1, cj secombes2, t wang2, g scapigliati, f buonocore dipartimento dibaf, università della tuscia, viterbo, italy 1dipartimento di scienze della vita, università di trieste. trieste, italy 2scottish fish immunology research centre, school of biological sciences, university of aberdeen, zoology building, tillydrone avenue, aberdeen ab24 2tz, uk interleukin-4 (il-4) and interleukin-13 (il-13) are key cytokines in th2 mediated immune responses. these cytokines have been extensively studied in mammals where they show different roles with some overlapping bioactivities exploited through shared receptors. usually il-4 and il-13 are activated at the same time when the immune system recognizes the presence of an external pathogen. in teleost fish the situation seems quite different from mammals, in fact only recently, in 2007, a gene with some relation to il-4 and il-13 was found in the pufferfish genome and, successively, a second il-4/13 like gene was identified in zebrafish at a different locus. these two genes were called il-4/13a and il-4/13b depending on their position on fish locus and they have been successively identified in other species, like rainbow trout, atlantic salmon and medaka. in our work we have found in a sea bass gills transcriptome three il4/13 transcripts that have been mapped on the available sea bass genome and therefore identified as il4/13a1, il4/13a2 and il4/13b. the identified sequences have been confirmed by cloning on sea bass gills cdna and their expression has been studied by real-time pcr. basal expression analysis revealed a different expression of the il-4/13 genes in the various tested organs and tissues: in particular the il4/13b expression is very high in spleen, while the expression of il-4/13a1 and il4/13a2 is high in head kidney. moreover, we investigated the expression of the il4/13 isoforms in sea bass head kidney and spleen leukocytes after in vitro stimulation with the t cell mitogen agents pha and pma and after in vivo infection with nodavirus and vibrio anguillarum. the results have showed that il-4/13b is high responsive to all stimulations, whereas, in contrast, il-4/13a1 and il-4/13a2 are less up-regulated. successively, we have produced the three isoforms as recombinant molecules and we tested their action in vitro on leukocytes from head kidney and spleen. this is the first in-depth analysis of the biological activity of il-4/13 cytokines in sea bass and it will highly contribute to a broader understanding of the evolution of type-2 immunity in this species. session 2. chairmen: n parrinello, university of palermo, palermo, italy and p luporini, university of camerino, camerino (mc), italy ascidian immunity new data on the expression of molecular markers involved in stemness and differentiation in the colonial ascidian botryllus schlosseri f ballin, n franchi, l ballarin department of biology, university of padua, padua, italy cell types are often identified by determining which genes they express specifically. the use of specific antibodies or complementary rna probes allows the identification of the translational/transcriptional products: these molecules, also called “molecular markers”, show an unique expression pattern, frequently used to identify and isolate stem cell populations. in b. schlosseri, a compound ascidian, there are three important processes that suggest the presence of stem cells during the life cycle: i) embryogenesis, in which an embryo develops from a zygote, palleal budding where new buds emerge as thickenings of the peribranchial epithelium and vascular budding, i.e., the development of new buds within the vasculature by circulating multipotent or pluripotent cells. during the cyclical generation changes, which characterize the colonial blastogenetic cycle, an increase in the number of hemoblasts, i.e., undifferentiated circulating cells, occurs which will replace, once differentiated, the hemocytes undergoing apoptotic cell death. ascidian hematopoiesis occurs in close proximity to the pharyngeal vessels, in the so-called “hematopoietic nodules” and in the endostyle, the cells of which proliferate and migrate to developing buds. despite the morphologic suggestions that hemoblasts are the precursors of all the circulating cell types, immunocytes included, there is a general lack of biochemical and molecular data supporting this assumption. here we report the identification of hematopoietic molecular markers in b. schlosseri, very similar to those of vertebrates, their localization and expression profile during the blastogenetic cycle. characterization and genetic variability of tumor necrosis factor alpha (citnfα) in ciona intestinalis a vizzini, mg parisi, l cardinale, m cammarata dipartimento di scienze e tecnologie biologiche, chimiche e farmaceutiche, palermo, italy 46   the tumor necrosis factor superfamily (tnfsf) is involved in a lot of cellular signaling pathways like inflammation, apoptosis, lymphocyte homeostasis and tissue development. tnfsf ligands are bound to membrane. about half of different ligands encode proteolytic cleavage sites that can generate soluble forms holding biological activity. the study of evolution of genes and genomes allows to understand the role of different evolutionary forces, including natural selection. the structure and composition of a gene are inherited from the organism’s ancestors, so various driving forces could cause the change of structural and functional aspects of a gene, for a best adaptation to environment. in ciona intestinalis, a tumor necrosis factor-alpha (tnfα)-like gene challenged with bacterial lipopolysaccharide (lps) was cloned and sequenced. it encodes a propeptide of 312 amino acids (35.4 kda), shows a transmembrane domain from positions 7 to 29, a tace cleavage site and a mature peptide domain of 185 amino acids (20.9 kda). the diversity of mrnas from citnfα has been investigated in 30 ascidians, collected from termini imerese (italy), by denaturing gradient gel electrophoresis (dgge). dgge migration revealed different molecular patterns. all patterns observed were sequenced and variants were identified in 30 citnf-α. several comparisons have been made: (i) for each dgge pattern, all the sequences were aligned and clustered according to nucleotide sequences, (ii) the different cds were translated into pro-peptides and (iii) the resulting aminoacid sequences were compared. the evolutionary relationships were inferred using the neighborjoining algorithm (mega-6). site-by-site frequency spectrum-based statistical assays were used to test the hypothesis of polymorphisms within loci being neutral. all three used tests were in agreement with the hypothesis of negative selection pressure linked to transmembrane domain, propeptide and mature peptide. fu and li’s d and f tests suggested possible positive selection pressure linked to cooh-terminus region only. all statistical tests indicated possible negative selection pressure when applied to full cds sequences. tnf mrnas from invertebrate and vertebrate animals were used to construct a phylogenetic tree to study the evolutionary dynamic of tnf family gene. these results show that the tnf family gene has encountered remarkable changes in invertebrates, but conserved in vertebrates during history of evolution. growing complexity of the invertebrate complement system: evidences from colonial tunicates n franchi, l ballarin department of biology, university of padua, padua, italy the evolutionary history of the complement system is not yet fully elucidated. evolutionary studies suggest that the origin of the complement system can be traced back to the common ancestor of the eumetazoa as the genes for the c3, factor b and masp have been identified in sea anemones. nonetheless, no complement genes are present in the genome of the sponge amphimedon queenslandica or the choanoflagellate monosiga brevicollis and, although their presence in invertebrate protostomes is generally accepted, complement genes are missing also in drosophila and caenorhabditis, probably due to a secondary loss. as regards deuterostome complement system, it is well studied in mammals, where more than 30 different proteins have been described, involved in the activation and regulation of this fundamental humoral system able to modulate immunocytes behaviour, belonging to both innate and adaptive immune response. however, the mere report of the presence of c3, factor b and lectin pathway in a species cannot help in elucidating the evolution of the complement system. since adaptive immunity evolved in the presence of a functioning complement system, the presence of considerable and important interaction between complement and adaptive responses is not surprising. in particular, referring to the invertebrate-vertebrate transition, the description of the complement-mediated immune modulation requires the identification and characterization of additional factors, such as complement control proteins (e.g., factor h, c4bp, cr1, cd46, cd 55) and receptors in invertebrates chordates. here we report on the identification and analysis of transcripts for cr1 (c3b receptor), c3ar and two factors h in the colonial ascidian botryllus schlosseri that, for the first time are described in tunicates, the sister group of vertebrates. the localization of cr1 and c3ar on phagocytes and morula cells, respectively, open the possibility to use such molecules as molecular markers for immunocytes. in addition, the presence of a complement regulator, such as factor h, in tunicates suggests a higher level of complexity than that expected in an invertebrate. session 3.  chairmen: l ballarin, university of padua, padua, italy and p pagliara, università del salento, lecce, italy a spotlight on echinoderms lecture the sp185/333 system in the sea urchin lc smith george washington university, department of biological sciences, washington, usa the arms race between a host immune system and its pathogens drives diversity on both sides of the conflict. pathogens change their approaches for infection and virulence, and the host changes the diversity of the immune detection and effector proteins. the purple sea urchin, strongylocentrotus purpuratus, survives in the marine habitat with an innate immune system of the is both complex and sophisticated. there are several large gene families in the sea urchin genome that encode pathogen detection and immune response proteins. one of these is the sp185/333 gene family, which is composed of ~50 small genes that are tightly linked 47   in clusters. the genes share blocks of sequence called elements that are present in mosaic patterns and have a variety of repeats within the second exon, and each gene is surrounded by one or two types of microsatellites. these characteristics suggest that the genomic region may be unstable, which may drive sequence diversification of the genes, a benefit for keeping up in the arms race with pathogens. gene sequence diversity and expression patterns suggest that the encoded proteins may have effector functions. to test this, we have evaluated one sp185/333 recombinant protein (rsp0032), which binds specifically to vibrio and yeast, but not to bacillus. it also binds lps, β1,3-glucan, and flagellin with specificity and high affinity, but does not bind peptidoglycan. rsp0032 also binds phosphatidic acid (pa) and can deform liposomes composed of 10 % pa. rsp0032 is intrinsically disordered, however, when bound to lps or pa, it transforms to α helical suggesting “shapeshifter” activities for binding lipids, sugars and proteins. given that single sea urchins are capable of expressing up to 260 sp185/333 protein variants, and if each one has a range of overlapping binding activities that target simultaneously multiple pamps, this may facilitate highly effective and flexible host protection against a broad array of potential pathogens encountered in the marine environment. antimicrobial and antioxidant activity in some echinoderm species l stabili1,2, mi acquaviva2, ra cavallo2, c gerardi3, m narracci2, p pagliara1 1dipartimento di scienze e tecnologie biologiche e ambientali, università del salento, lecce, italy 2cnr-iamc, u.o.s. taranto, italy 3cnr-ispa, uos lecce, via prov.le lecce monteroni, lecce, italy echinoderms represent one interesting marine renewable resource and produce bioactive compounds related to their innate immune system. these invertebrates indeed are able to differentiate self from non-self through the production of soluble molecules and coelomocytes response playing an important role in the resistance to disease. therefore, they appear as a promising alternative valuable source of new compounds for drug development. in particular, the application of new marine antioxidants in foods, food supplements, nutraceuticals and medicine is recently considered from the perspective of benefits to human health. in this study, the antimicrobial and antioxidant activity of several echinoderm species was investigated. we focused our attention on the two sea urchins sphaerechinus granularis and arbacia lixula and on the sea star echinaster sepositus. coelomic fluid and coelomocyte lysate of each species were utilized for antimicrobial activity assay using the kirby bauer method (1966). the antioxidant activity of the samples was measured by two in vitro assays: the teac (trolox equivalent antioxidant capacity) assay based on a single electron transfer (set) reaction, using abts [2-2’-azino-bis (3ethylbenz-thazoline-6-sulfonic acid)] as chromogen and the orac (oxygen radical absorbance capacity) based on a hydrogen transfer mechanism (hat). both the antioxidant assays showed a higher antioxidant activity in the coelomocyte lysate compared to coelomic fluid for all the echinoderm species studied. moreover a. lixula cell lysate had the highest antioxidand activity both with teac and orac assay. these antioxidant values are comparable with those reported in the literature for various high antioxidant fruit and spice extracts. among the investigated species, the coelomocyte lysate of a. lixula showed a bacteriostatic activity against two emerging pathogenic bacteria pseudomonas aerugionsa and staphylococcus aureus and against the yeast candida famata. these results are noteworthy considering the resistance against antibiotics developed by bacteria and the need to control human infections. the antioxidant activity was also of interest since it is the first record for the investigated species and represents a potential for applicative purposes. bacterial challenge induces variation of the allograft inflammatory factor i (aif-1) gene expression in paracentrotus lividus f vacca1, a barca1, j vizioli2, f drago2, t verri1, p pagliara1 1dipartimento di scienze e tecnologie biologiche e ambientali, università del salento, lecce, italy 2laboratoire prism-umr inserm u1192. université de lille, villeneuve d’ascq france the phylum echinodermata is a very heterogeneous group of marine invertebrate species. after chordata, it represents the second largest group of deuterostomes. the interest for echinoderm immune defence system is steadily increasing in the last years, due to their phylogenetic, ecological, biotechnological and economical relevance. noteworthy, their basal position within the deuterostome lineage makes the analysis of their defense mechanisms highly relevant to understand the evolution of the deuterostome immune system as it now appears in vertebrata. suggestions derive from the systematic analysis of the immune genes and of their products, such as cytokines, i.e., a specific group of effectors molecules that play a variety of roles in the immune response. the allograft inflammatory factor-1 (aif1), a calcium-binding cytokine, has been identified as i) a key regulator of the immune response; ii) a central player of the self-intensifying cycle of inflammation; and iii) an important pathogenic factor in multiple inflammatory diseases in vertebrata. recent literature evidences that proteins of the aif1 superfamily are present in several phylogenetically distant species, all showing high similarity at the primary protein sequence level. these data suggest a significant conservation of the functional properties of aif-1 in the immune response. in the present work, we report on the immune response of the sea urchin paracentrotus lividus after challenge with micrococcus lysodeikticus, and we estimate the variation of cellular and humoral responses after bacterial injection. in particular, aif-1-like protein constitutive expression has been evidenced in p. lividus coelomocytes by immunocytochemistry performed 48   using a human anti-aif-1 antibody. subsequent mining of expressed nucleotide sequences (ests) databases allowed us to identify an aif-1-like mrna fragment sequence and to study its expression. notably, mrna levels were found to be up-regulated in coelomocytes at 24 h post-bacterial injection. overall, our data suggest novel hints on the aif-1 involvement and responsiveness to immune activation by bacteria in cells and tissues of p. lividus. x-ray photoelectron spectroscopy as a nonconventional analytical technique for bioorganic materials characterization: the sea urchin coelomocytes and the purple photosynthetic bacteria examination mr guascito1, d chirizzi2, p pagliara1, l giotta1, s rella1, d mastrogiacomo1, f milano3 1dipartimento di scienze e tecnologie biologiche e ambientali, università del salento, lecce, italy 2dipartimento di beni culturali, università del salento, lecce, italy 3istituto per i processi chimico fisici, uos bari, bari, italy x-ray photoelectron spectroscopy is a surface technique (depth profiling analysis 5 10 nm) that allows to analyze, in terms of chemical speciation, all elements, with the exception of h and he, present in different typologies of solid samples (inorganic and organic), as long as their atomic percent concentrations (at.%) are not below 0.01 0.l %, depending on the specific element. this technique potentially can also allow to obtain chemical imaging of surfaces with lateral resolution of 3�m. however, at the state of the art, the employment of this analytical technique as a nonconventional tool for the investigation of bio-organic materials (i.e., microorganisms and their related systems such as biofilms, extracellular polymeric substances (eps) and bio-adhesions), has been reported only in a limited number of papers. in this study, we report some preliminary results on the xps characterization of sea urchins coelomocytes and photosynthetic bacteria. the sea urchin arbacia lixula coelomocyte population is characterized by the presence of red cells, which number may increase in response to different stress. as red spherula cells accumulate around injuries and sites of infection, this analysis may help to understand what is the role of cell surface interacting with bacteria in addition to the action of echinochrome a, present inside the cells. in particular, here we compare the red and withe coelomocytes surface. furthermore, the characterization of purple photosynthetic bacteria (rhodobacter sphaeroides) able to interact with detrimental heavy metal ions, such as nickel and chromium has been performed, successfully highlighting both the immobilization of metals and surface modifications induced by the environmental stress. session 4. chairmen: e ottaviani, university of modena and reggio emilia, modena, italy and l stabili università del salento, lecce, italy invertebrate models suitable for the study of mammalian diseases lecture bivalves as a translational model in inflammation and cancer research g de vico and f carella dipartimento di biologia, complesso universitario di monte s. angelo, edificio vii, università degli studi di napoli federico ii, naples, italy historically, animal models have been used in the biomedical research to characterize disease progressions, evaluate the action of drugs and also discover new biomarkers. invertebrate models of human disease first appeared in the scientific literature in the late 19th century. currently, model species range from terrestrial invertebrates such as nematodes and insects, to freshwater and marine life including planarians, crustaceans and molluscs. among new invertebrate translational models in biomedical research, mollusc are emerging as a promising one in many areas, such as those concerning immune-neuro-endocrine system, neurodegenerative disorders, inflammatory lesions and cancer. along with an overview of the main features concerning both inflammation and cancer pathogenesis in bivalve molluscs, we will also stress the rationale concerning the potential use of bivalves as a translational model for early validation of new therapeutic approaches in both human inflammation and cancer. at the same time, we highlight the need for standardization of scientific terminology in this new field of investigation. amyloidogenesis in animal models m de eguileor, a grimaldi, g tettamanti department of biotechnology and life sciences, university of insubria, varese, italy the biomedical research depends on the use of animal models that can be utilised to understand, at different levels, the pathogenesis of human diseases. the ultimate goal of this approach is to develop and test new therapies to eradicate the examined diseases. many researchers interested in embriologically and genetically tractable disease models have opted for zebrafish. this animal can provide a variety of human diseases due to sophisticated mutagenesis and supply economic screening strategies on a large scale. furthermore, despite the advantages and the pre-eminence of the mouse in modeling human diseases there is a lot of aspects limiting the use of this animal in large-scale genetic and therapeutic screening. even if invertebrates lack structures and organs that can be involved in human pathologies it is important to bear in mind that there is a surprising degree of functional conservation in basic cellular biological processes between mammals and invertebrates (such as worms and insects). thus when there is a slow down or a total block in one of complex and basic processes, pathological events take place. amyloidogenesis represents a primitive, simple response, widespread in invertebrates and 49   vertebrates where innate immune signalling pathways is linked to stress responses. the critical role played by amyloid synthesis and deposition in several pathologies, could explain the structural resistance of these scaffolds and could provide the basis for developing new diagnostic and therapeutic approaches in all diseases in which the innate branch of the immune system has a pivotal role. serum amyloid a in marine bivalves: an acute phase protein of innate immunity s domeneghetti1, u rosani1, m gerdol 2, m franzoi1, a pallavicini2 , p venier1 1department of biology, university of padua, padua, italy 2department of life sciences, university of trieste, trieste, italy serum amyloid a (saa) is an evolutionarily conserved acute-phase protein, involved in many vertebrate biological processes, such as lipid metabolism and immunity. the rapid increase of saa can activate the immune system and promote inflammatory responses after injuries, infections or stress. although saa homologs are widespread in vertebrates, to date they have only been identified in limited number of invertebrate species. we traced the presence of saa genes along metazoan evolution by screening available genomic and transcriptomic data, finally retrieving 51 saa-like proteins in several protostome taxa. in detail, we identified saa homologs in 21 marine bivalves and we investigated the gene structure and expression patterns of mussel and oyster saas. although phylogenetic and structural analyses support a certain degree of conservation between vertebrate and invertebrate saa sequences, vertebrate saas are mainly expressed in liver, whereas invertebrate saas appear to be expressed in various tissues. using both qpcr and rna-seq approaches, we observed that the two mussel saa genes are mainly expressed in gills (mgsaaa), mantle and posterior adductor muscle (mgsaab), whereas c. gigas saa is expressed in significant amounts in mantle and gonads. we also confirmed the inducible nature of bivalve saa transcripts, observing the overexpression of mussel saas after challenge with pathogenic bacteria, although timing and extent of the induction were different for the two mussel saa genes. the overall results provide new insights into the evolution of these ancient immune-related proteins in invertebrates. research of inflammatory markers in the medicinal leech, hirudo medicinalis r girardello1, f drago2, m de eguileor1, r valvassori1, j vizioli2, g tettamanti1, a grimaldi1 1department of biotechnology and life sciences, university of insubria, varese, italy 2laboratoire prism-umr inserm u1192. université de lille, villeneuve d’ascq france the leech hirudo medicinalis is an interesting model to study inflammatory processes both in nervous system and in peripheral tissues. here we considered two different molecules involved in peripheral tissues as well as neural immune response: the macrophage migration inhibitory factor (mif), a chemotactic cytokine which mediate lps-induced responses, and the glia maturation factor gamma (gmfg), a member of adf-gelsolin superfamily, which seems to be involved in actin cytoskeleton remodelling and tlr4 endocytic pathway in response to lps. we identified in h. medicinalis two genes coding for products showing high similarity with mif and gmfg of vertebrates, respectively. immunolocalization experiments show a weak expression of both these proteins in the leech cns whereas a stronger signal is detected in peripheral tissues macrophages. further studies are needed to assess the expression levels of these molecules in leech tissues. however, this work shows that these molecules are good selective markers of activated macrophages in h. medicinalis, confirming the close correlation between the leech and vertebrates. moreover, these results suggest the possible presence of more well-conserved molecules across evolution and represent an interesting starting point to analyze the complex crosstalk occurring during the innate immune response as well as the neuroimmunity processes. francisella-like endosymbionts, potentially harmful to human health, are transported by the universally distributed species of the ciliate euplotes a vallesi1, d petrelli1, g di giuseppe2, a sjödin3,4, j thelaus3, e nilsson3, c öhrman3, g gutierrez pozo5, e villalobo5 1school of biosciences and veterinary medicine, university of camerino, camerino (mc), italy 2department of biology, university of pisa, pisa, italy 3division of cbrn defence and security, swedish defence research agency, foi, umea, sweden 4department of chemistry, computational life science cluster (clic), umea university, umea, sweden 5departamento de microbiología, universidad de sevilla, av reina mercedes 6 41012 seville, spain genome analyses of wild-type strains of two ecologically separated euplotes species, e. raikovi living in temperate sea waters and e. petzi living in the polar seas, revealed that both host bacteria in their cytoplasm. these bacteria have been identified with facultative intracellular gamma-proteobacteria of the genus francisella, which includes a number of closely related species well known as extremely infectious to a great variety of organisms. francisella tularensis, with its four subspecies, is a specialized intracellular pathogen capable of infecting both invertebrate and vertebrate hosts, humans included; f. noatunensis is the etiological agent of the fish disease known as francisellosis, and its two subspecies well adapt to different temperatures of their hosts; the francisella-like endosymbionts wolbachia persica, together with the freely living generalists f. philomiragia and f. novicida cause diseases in humans with a compromised immune system. the francisella 50   endosymbionts of e. raikovi and e. petzi have been successfully isolated and their genomes completely sequenced. they are genetically distant from one another and form two different clades in the francisella phylogenetic tree, which are distinct from the all other well-established francisella clades. the finding that francisella has equally colonized polar and temperate-water species provides evidence that this bacterium is more common and widespread than previously hypothesized, and confirms that free-living euplotes species and ciliates in general, with their worldwide distribution, may represent a natural reservoir of francisella in every aquatic environment. lymnaea stagnalis ganglia transcriptional activity after lps induced immune activation f tascedda1, c benatti1, d malagoli1, jmc blom2, e ottaviani1 1department of life sciences, university of modena and reggio emilia, modena, italy 2department of education and human sciences and paediatric oncology unit, university of modena and reggio emilia, italy the mechanisms by which the neuroendocrine and immune systems communicate and influence each other from invertebrates to vertebrates are well known and are among the most exiting areas of research in biology. moreover, environmental influences, such as inflammation or stress, play a key role in determining susceptibility to disease and in particular to nervous system linked diseases. until now, studies regarding the genetic mechanisms underlying neuroendocrine and immune interactions have used rodent models, while invertebrate models have been used to a much lesser extent. among gastropods, the freshwater snail lymnaea stagnalis is emerging as an important model to study immuneneuroendocrine functions from an ecological, parasitological and immunological point of view. in the present research, l. stagnalis, was used as an invertebrate model to study neuronal responses to lps induced immune activation. more precisely, we tested the hypothesis that transcriptional changes occur in molluscan neural cells in response to lps. adult snails were exposed to lps after which l. stagnalis ganglia were dissected, rna extracted and analyzed for expression levels of genes related to neural and immune plasticity, such as, aif-1and hsp70. preliminary data suggest that lps induced immune and neural activity alters plasticity related gene expression. characterization and neurothrophic effects of leech microglia-released extracellular vesicles (evs) t arab1, f drago1, i. prada2, c van camp1, f le marrec-croq1, j franck1, m wisztorski1, j-p gimeno1, m salzet1, p-e sautière1, c lefebvre1, c verderio2, j vizioli1 1laboratoire prism-umr inserm u1192. université de lille, villeneuve d’ascq france 2institute of neuroscience (in), cnr, milano, italy the leech hirudo medicinalis is a well-studied model in neurobiology because its central nervous system (cns) spontaneously repairs after a mechanical lesion. this process, leading in a few weeks to the synapse regeneration and a complete functional recovery, is linked to the activity of microglial cells migrating to the injured area. in leech, a few hours after the injury the activated microglia release an impressive amount of extracellular vesicles (evs) that appear to constitute an important element in the cross-talk between microglia and lesioned neurons. by differential centrifugation we separately isolated small (10 100 nm, exosomes) and bigger (100 1000 nm, ectosomes) evs. we also investigated the amount of exosomes and ectosomes released by naïve vs. atp-stimulated microglia. in order to assess the function of these microglia-released evs we characterized their proteomic and rna content and we started investigating their potential in neurite outgrowth. proteomic analyses of leech vesicles revealed the presence of many proteins typically present in mammalian evs, including several surface molecules, and the presence of specific elements in differentially-stimulated samples. functional assays were performed to assess the neurothrophic role of microglia-released evs on a mammalian neuron-like cell line (pc12). neurite outgrowth was measured upon incubation with extracellular vesicles issued from leech microglial cells. results show a significant increase in neurites outgrowth indicating that both leech exosomes and ectosomes can exert a neurothrophic effect on mammalian cells. the association of a specific neurothrophic phenotype with its protein and rna signatures would help to understand the role of these microglial evs in promoting cns repair. session 5. chairmen: p venier, university of padua, padua, italy and l abelli university of ferrara, ferrara, italy immune response in molluscs and cnidarians structure and distribution of astakine in the organs of the freshwater snail pomacea canaliculata a accorsi1,2, s benatti3, k gotting1, e ross1,2, a sánchez alvarado1,2, d malagoli3 1stowers institute for medical research, kansas city, mo, usa 2howard hughes medical institute, stowers institute for medical research, kansas city, mo, usa 3department of life sciences, university of modena and reggio emilia, modena, italy the freshwater snail pomacea canaliculata is an emerging pest in eu, and its immune system is a potential target for developing strategies of pest control. circulating hemocytes represent the cellular component of the p. canaliculata immune system. p. canaliculata hemocytes originate in the pericardial fluid, and are maintained in the ampulla, which may act as a hemocyte reservoir. astakine-1 is a hematopoietic cytokine first described in the crayfish pacifastacus leniusculus, and recently described also in the insect lygus lineolaris and in 51   the bivalve mollusc crassostrea gigas. bioinformatic analyses of a comprehensive p. canaliculata transcriptome demonstrated the presence of an astakine 1-like molecule (pc-astakine) also in this organism. pc-astakine is 121 aa and contains a domain characterized by 8 cysteins with a conserved distribution pattern homologous to the vertebrate domain prokineticin. further bioinformatic predictions suggest that the structure of pc-astakine may be similar to that one of p. leniusculus astakine-1. the distribution of pc-astakine gene expression was evaluated by qpcr. we have observed that all organs analyzed express pcastakine at detectable levels. however, high expression levels were detected in the ampulla, pericardial fluid and circulating hemocytes. the data suggest that pc-astakine may have a wide range of functions, including the regulation of hematopoiesis and the modulation of inflammatory responses, as previously reported for the human prokineticin-1. effects of lps injection on pc-astakine expression in the gastropod pomacea canaliculata s benatti1, a accorsi2,3, m nasi4, e ottaviani1, d malagoli1 1department of life sciences, university of modena and reggio emilia, modena, italy 2stowers institute for medical research, kansas city, mo, usa 3howard hughes medical institute, stowers institute for medical research, kansas city, mo, usa 4department of surgery, medicine, dentistry and morphological sciences, university of modena and reggio emilia, modena, italy astakine-1 is a prokineticin-containing factor and the first hematopoietic cytokine described in invertebrates. astakine-1 was firstly retrieved in the freshwater crayfish pacifastacus leniusculus, and recent experiments have demonstrated the presence of astakine-like molecules also in insects and molluscs, including the freshwater snail, pomacea canaliculata. in control conditions pcastakine is expressed in several organs, especially in the ampulla (reservoir of hemocytes and potential district of hemocyte maturation) and in the pericardial fluid (i.e. the hematopoietic tissue). by mean of qpcr experiments, we have analyzed the effects of the injection of 50 µg lps on the expression of the gene pc-astakine. our observations indicate that 24 h after the injection, the major modification of the pc-astakine expression was evident in the anterior kidney, a potential hemocyte reservoir, in which the expression of the gene decreased to almost undetectable level. in the pericardial fluid, ampulla and circulating hemocytes, the expression of pc-astakine dropped to less than 50 % with respect to the sham-injected control snails. the drop in the amount of mrna detected by qpcr could reflect an increased rate of translation and consequent degradation of the available mrna, rather than a decrease of the transcription rate. similarly, in the bivalve crassostrea gigas, it has been suggested that accumulated cg-astakine transcripts are largely translated under some environment stress, including immune stimuli. on the whole, our results indicate that the expression of pc-astakine and the translation rate of its mrna may be influenced by immune stimuli, and support the hypothesis that pcastakine may be involved in pomacea hematopoiesis and/or may have immune-related functions, as well. myticalins: a novel family of linear cationic amps from mytilus galloprovincialis identified by de novo bioinformatics analysis m gerdol1, g leoni2, p venier3, a tossi1, a pallavicini1 1department of life sciences, university of trieste, trieste, italy 2international school for advanced studies (sissa), trieste, italy 3department of biology, university of padua, padua, italy the de novo discovery of bioactive peptides by bioinformatics screening methods is undoubtedly experiencing a renewed impulse due to the increasing availability of genomic and transcriptomic data from non-model organisms. marine invertebrates in particular appear to be a vast and still largely unexplored resource due to their adaptation to diverse challenging environments. all the antimicrobial peptides (amps) described so far in mussels (mytilus spp.) display a structure stabilized by intra-molecular disulphide bridges. no linear amps, relatively widespread in other invertebrates, have been identified so far, arguably due to the fact that they are encoded by taxonomically-restricted genes with no sequence homology to other known amps. here we describe myticalins (mytilus cationic linear amps), a novel family of amps produced as pre-propeptides and characterized by the absence of cysteine residues and by a positive net charge. myticalins have been fully identified through a bioinformatics approach with no prior knowledge concerning their biological activity, by scanning the m. galloprovicialis transcriptome for potential amp sequences, based on isoelectric point and amino acid composition. although these amps appear to be largely diversified, they can be categorized into 4 main subclasses (a, b, c and d). we selected myticalin a-5, whose mature peptide sequence presents several pro-arg-x repeats, and chemically synthetized the predited mature peptide sequence. the synthetic peptide displayed a significant antimicrobial activity both against gram+ and gram bacteria, validating the effectiveness of our de novo approach. differently from other mussel amps, myticalins are mainly expressed in the gills tissue. although many aspects concerning the biological activity of myticalins remain to be clarified, they represent a promising class of peptides with potential biotechnological applications. 52   antiviral immunity in oysters infected by ostreid herpesvirus-1 u rosani1, s domeneghetti1, m gerdol2, g arcangeli3, a pallavicini2, p venier1 1department of biology, university of padova, padua, italy 2department of life sciences, university of trieste, trieste, italy 3istituto zooprofilattico sperimentale delle venezie, adria, italy marine bivalves do not possess an acquired immune system that allows selective responses based on immunological memory. conversely, bivalves are evolutionary successful species that live in an environment rich of viruses and bacteria, thus meaning that they have effective defense systems against pathogens. ostreid herpesvirus type 1 (oshv-1) is characterized by a 207 kb dsdna genome and has been associated to sporadic mortalities of several bivalve species since mid ’60. in 2008, a new oshv-1 genotype called μvar was reported to be the causative agent of massive mortalities of young oysters in france and, in the following years, its presence was reported worldwide. today, oshv-1 has become a problematic infective agent for the pacific oyster crassostrea gigas. genetic susceptibility and critical chemico-physical conditions could facilitate the overwhelming of the oyster immune system and result in an uncontrolled viral replication (lytic phase). parallel sequencing of host and pathogen rnas (dualrna-seq) has the potential to reveal the host-pathogen interactions during infection. in detail, the availability of a c. gigas sample (goro lagoon, north adriatic sea, italy) highly infected by oshv-1 μvar (up to 8.4×104 copies/ng dna) allowed us to analyze both oyster and viral transcriptomes. owing to the high sequence coverage we were able to describe the complete genome of an italian virus genotype with strong pathogenicity. in the analyzed oyster sample, we found genes with similarity to elements of the vertebrate interferon pathway and several other defense genes. overall, these facts indicate that oyster possesses an antiviral-specific immunity, mainly based on an interferon-like pathway, rna interference and programmed cell death. voltage-gated sodium channels neurotoxin from tentacles of sea anemone actinia equina exhibits cytolityc activity mg parisi, mr trapani, m cammarata animal biology section, department of biological, chemical and pharmaceutical science and technology, university of palermo, palermo, italy sea anemones are sessile benthic organisms and their evolution has led to a high specific richness in the course of millions of years. the presence, diversification and multi-functionality of the toxins may have played an important role in the ability of colonization and adaptation to ecological niches that change over time. venoms include peptide and protein toxins as 3 to 5 kda neurotoxins acting on voltage-gated sodium channels, 3.5 to 6.5 kda neurotoxins acting on voltage-gated kv1 potassium channels, 20 kda pore-forming cytolysins inhibited by sphingomyelin and serine protease inhibitors belonging to the kunitz-type family. type 1 and 2 na+ channel toxins are composed of 46 to 49 amino acid residues, except for a. equina toxin aei of 54 residues and cross-linked by three disulfide bridges. in this study toxic components from acid tissue tentacles extracts of sea anemone a. equina were investigated by size exclusion separation to characterize cytolytic molecules of low molecular weight. tentacles extracts with low molecular mass were filtered through a membrane (10 kda nanosep device). sample was subjected to size exclusion chromatography using biosuite 250, 10 μm sec, 7.5×300 mm column on a hplc system and collected fractions were concentrated through micro-concentrators to be tested for lytic activity. after electrophoretical analysis of lytic fractions, bands were excised and sent to sequencing by ngel digestion and maldi tof analysis. database searching and blast analysis allowed to identify a protein of 5,5 kda molecular weight correspondent to the ae1 toxin from a. equina. then, the peptide aei was synthesized from genscript (chemical peptide synthesis service) and it was assayed for its hemolytic activity using the sheep erythrocytes (rbcs) by a spectrophotometric quantization assay of percentage of hemolysis. lysis was recorded after 1 h of incubation in polyethylene tubes and 58 % and hemolysis was found when the targets were put in contact with the neurotoxin aei. current studies concerning the mechanism of action of the molecule and its structure are carrying out. the discovery of cytolytic activity in addition to neuroinhibitory effect shows the evolutionary trend to combine two functions in one compound: ion channel inhibitor and membranolytic activity. from cnidarian immunobiology to cultural heritage applications mr trapani, mg parisi, d parrinello, ma sanfratello, g benenati, g barresi, f palla, m cammarata department of biological, chemical and pharmaceutical science and technology, university of palermo, palermo, italy the study of cnidarians immunity, as model systems of metazoans, lead additional information on the first steps of the immunity evolution. the functions of the genes and cellular pathways in higher vertebrates are conserved during the evolution of metazoans, as shown by the discovery of homologues in cnidarians. these basal metazoans in fact, are far from "simples" in the range of methods at their disposal to deal with potential prey but also invading microbes and pathogens. they can give information about the invertebrates innate immune repertoire. we investigated the immunobiology starting from the inflammatory response in anemonia sulcata (cnidaria: anthozoa) following injection of substances different in type and dimension, to understand the effector mechanisms involved in this 53   process. we observed clear, strong and specific reactions especially after injection of bacteria and the alteration of the expression of enzymes (protease, phosphatase and esterase), showing a correlation between the appearance of the inflammatory reaction and the modification of enzymatic activities. from cnidarian phylum a large number of toxic compounds have been isolated. tissues and mucus produced by cnidarians may have a role in immune defense and contain a variety of toxins as neurotoxins, cytolysins and antimicrobial peptides, which can have multifunctional role. the bioactive molecules were purified by acid extraction and hplc purifications and characterized through biological assays, mass spectroscopy and peptide synthesis. here, we show the cnidarian bioactive molecules as antimicrobial peptides and enzymes in order to draw applications in fields ranging from pharmacology to cultural heritage. particularly, in the control of the microbic growth and especially in the tuning of biocleaning protocols, bioactive molecules with proteasic and esterasic activity have been used. these novel enzymes are active at temperature lower than 30°c, they need a reduced time of application and are safety for both operators and environment. thus they could provide an important contribution to the development of sustainable innovative protocols. session 6. chairmen: d malagoli, university of modena and reggio emilia, modena, italy and m cammarata, university of palermo, palermo, italy hotspots in metazoan immunity lecture insights into the evolution of dendritic cells s bisin, l gagliardi, d lunardi, g pavani, l abelli department of life sciences and biotechnology, university of ferrara, ferrara, italy the development in gnathostome vertebrates of an adaptive immune system, that guarantees fine discrimination of antigenic determinants, also involved main specializations of antigen-presenting cells (apc), which govern presentation to lymphocytes of antigens associated to self and nonself mhc molecules. the dendritic cells (dc), discovered first in mammals and later classified as conventional, plasmacitoid, interdigitating (idc), follicular, intra-epithelial (e.g., langerhans cells), dermal or tissutal, fall into the apc category. however, information about their appearance and evolution in vertebrates is still limited and fragmentary. dc were indirectly (high levels of mhc ii molecules) hypothesized in chondrichthyes, whereas are much better documented in teleostei (actinopterygii osteichthyes), paradoxically more than in heteroterm tetrapods. studies in rainbow trout, zebrafish, atlantic salmon and european sea bass (esb) convincingly reported about dc, at cytological, molecular (specific surface markers, cytokines and cpg receptors, lectin-induced agglutination) and functional (phagocytosis, migration, mhc-restricted antigen presentation, cytokines release) levels. the best characterized populations recall conventional dc, but studies in esb additionally detailed idc occurring in developing and adult thymus, likely engaged in thymocyte selection processes. furthermore, recent studies in rainbow trout identified mhc ii+cd8-α+ dc in the skin. this finding prompted to advance the hypothesis for a common origin for all mammalian dc that may exert antigen crosspresentation. germ line dynamics in bivalve molluscs l milani, a pecci, f ghiselli, m passamonti, mg maurizii department of biological, geological and environmental sciences, university of bologna, bologna, italy studies about germ line specification in mollusca have been neglected for years. available data based on few species indicate that molluscs exhibit the same cleavage mechanism of other spiralians, in which much of the mesoderm comes form the 4d blastomere, including the germinal tissue, whose specification has been identified by localizing the products of vasa orthologs (vasa is highly conserved in the animal kingdom). despite the relevance of bivalve molluscs in marine ecosystems and aquaculture, their mechanism of sex determination is still unknown, as well as the details of their seasonal gonad reconstitution (i.e., the gonad is reabsorbed after spawning and reconstructed at the beginning of the subsequent reproductive season). in order to determine general features of bivalve germ line development, we employed two antibodies produced against the vasa ortholog of ruditapes philippinarum (subclass heterodonta, family veneridae) to investigate three additional species. we chose two species of the subclass pteriomorphia, scapharca inaequivalvis (family arcidae) and crassostrea gigas (family ostreidae), and another species of the subclass heterodonta, mya arenaria (family myidae). the immunoreactivity of anti-vasa was confirmed by western blot in which single specific bands were obtained in each species, although of different molecular weight. on the other hand, the presence of two different bands in males and females of the gonochoric r. philippinarum has been previously related to sex-biased isoforms, already found in other animal species outside bivalves. the detection of a single band in the two pteriomorphia may be tentatively related to their state of proterandric hermaphrodites. m. arenaria is reported to be gonochoric, as r. philippinarum, but the analyzed specimens were all females, so we cannot exclude the existence of different sex-related isoforms, although no sign of a second band was present. the immunohistological data obtained support a conserved mechanism of proliferation of primodial germ cells (showing the vasa labeling at one side of the cell cytoplasm) among the simple columnar epithelium of the gut. moreover, their seasonal migration to the reconstituting gonad appears to be a common feature of bivalves. the 54   study of bivalve reproductive biology can clarify important aspects of their development, but can also be useful for conservation management and breeding programs. managing of procambarus clarkii by x-ray sterilisation of males: immunological competence after the irradiation e simeon, mk bravin, c manfrin, f piazza, s battistella, pg giulianini department of life sciences, university of trieste, trieste, italy procambarus clarkii (girard, 1852) is an invasive alien species spread worldwide. the sterile male release technique has been chosen in friuli venezia giulia as part of a strategy to control the red swamp crayfish. this technique consists on the release into the environment of sterile males which are sexually active and able to compete with untreated males for mating partners. recently the gonad damage induced by ionising irradiation were described (piazza et al., 2015), but no data are available on the immunocompetence of males after irradiation. the present study describes the hemocytes of p. clarkii males 20 days after irradiation at a dose of 40 gy by means of light and transmission electron microscopy. total (thc) and differential number of hemocytes (dhc) and activity of basal (po) and total plasmatic phenoloxidase (ppo) are also evaluated. untreated animals (ctrl), unirradiated animals injected with sterile saline (pbs), and carboxylated polystyrene latex beads (ltx) were used as controls. three types of circulating hemocytes were characterized: granular hemocytes (gh), semigranular hemocytes (sh) and hyaline hemocytes (hh). irradiated animals present highly significantly lower thcs (35357 ± 9643 hemocytes/ml, n = 7) in comparison with ctrl (666818 ± 78546 hemocytes/ml, n = 11), pbs (1006071 ± 184413 hemocytes/ml, n = 7) and ltx (368437 ± 98895 hemocytes/ml, n = 8) challenged ones. irradiated animals show significantly higher gh percentages (48.57 ± 6.2) in comparison to all controls (ctrl: 17.97 ± 2.1; pbs: 29.91 ± 3.2; ltx: 21.89 ± 3.4), but significantly lower hh percentages (17.55 ± 5.2) in comparison to controls (ctrl: 59.73 ± 4.9; pbs: 65.83 ± 2.7; ltx: 67.57 ± 4.0). the comparison of po and ppo activities among different groups do not evidence significant differences. the literature documents a survival of males irradiated with a dose of 20 gy at laboratory conditions of at least one year (aquiloni et al., 2009). the present study reports a decrease of about 95 % of circulating hemocytes and an increase in percentage of gh in males irradiated with a dose of 40 gy after 20 days. interestingly, the activity of po and ppo are not affected by ionising radiation. the decrease in circulating hemocytes is consistent with the cytological damages to the gonads that are described as progressive from the day of irradiation up to 30 days (piazza et al., 2015). it remains to be seen how long and how animals are able to offset this hemocyte decline and if this would preclude the survival in the wild. 55   isj 16: 15-24, 2019 issn 1824-307x research report sabella spallanzanii mucus contain a galactose-binding lectin able to agglutinate bacteria. purification and characterization m cammarata 1 * ,# , g benenati 2,# , m dara 1 , mg parisi 1 , d piazzese 1 , f falco 3 , l stabili 4,5 # equal contribution 1 department of heart and marine science distem, marine immunobiology laboratory, university of palermo, , italy 2 department of biological, chemical and pharmaceutical sciences and technologies stebicef, university of palermo, italy 3 istituto per le risorse biologiche e le biotecnologie marine (irbm), units of capo granitola italy 4 istituto per l’ambiente marino costiero, u.o.s. di taranto, cnr, taranto, italy 5 department of biological and environmental sciences and technologies, university of salento, lecce, italy accepted march 04, 2019 abstract lectins are present in almost all living organisms and are involved in several biological processes, including immune responses. in the present study, a calcium dependent galactose-binding lectin exhibiting an apparent mw of 43 kda has been characterized and purified from the mucus of the polychaete sabella spallanzanii by using both affinity chromatography and high-pressure liquid chromatographic methods. its agglutinating activity towards rabbit erythrocytes was significantly modified by the addition of calcium or edta. the activity was optimal at temperature values comprised between 4 and 18 °c, maintain a 50% of activity between 20 and 37 °c, was significant deleted after exposure at 50 °c, and was depleted at 90 °c. the s. spallanzanii galactose-binding lectin (ssgbl) was able to agglutinate bacteria and to preferentially recognize gram-negative bacteria. the strongest agglutinating activity was observed towards vibrio alginolyticus and escherichia coli, by contrast mucus agglutinated in a lesser extent both aeromonas hydrophyla and the grampositive micrococcus lysodeikticus thus suggesting its involvement in host pathogen interactions. key words: mucus; hemagglutinin; bacteria; galactose-binding lectin; s. spallanzanii introduction lectins are multifamily proteins present in almost all living organisms and due to their carbohydrate binding ability are involved in several biological processes (kaltner and stieltorfer, 1998; kilpatrick, 2002), including development, cell adhesion, glycoproteins interactions (kaltner and stierstorfer, 1998; kilpatrick, 2002), and immune responses (liao et al., 1994; arason, 1996). lectins and sugars constitute an evolutionary conserved recognition system, involved in innate immunity, able to mediate several effector functions. these ___________________________________________________________________________ corresponding author: matteo cammarata department of heart and marine science distem marine immunobiology laboratory university of palermo viale delle scienze ed. 16, palermo, italy e-mail: matteo.cammarata@unipa.it activities include agglutination, immobilization and opsonization towards microbial pathogens and complement activation, by either recognition of glycans exposed on potential pathogens either immunoregulation binding to carbohydrates on immunocompetent cells surfaces (turner, 1996; kilpatrick, 2002; loris, 2002; fujita et al., 2004; sharon and lis, 2004; vasta et al., 2004). lectins have been classified into various structural families such as c-type lectin, galectin and r-type lectin. the lectins are distinguished on the basis of conserved amino acid sequence motifs in their carbohydrate recognition domain (crd), structural folds and calcium requirements (aranson, 1996; turner, 1996; fujita et al., 2004; sharon and lis, 2004; vasta et al., 2004). galactose-binding lectins have been documented both in vertebrates and in invertebrates, and their involvement in humoral 15 immunological processes is well described (arason et al., 1996; vasta et al., 2004). moreover, many of them are able to bind β-galactoside carbohydrates other than monosaccharide galactose (hirabayashi et al., 2002; vasta et al., 2004). much evidence exists about the presence of galactose-binding lectins in marine invertebrates. in phylum annelida, a 29 kda galactose-binding lectin was characterized in the earthworm lumbricus terrestris (class oligochaeta) (hirabayashi et al., 1998). the primary structure of the earthworm lectin belongs to r-type lectin family that is involving ricin b-chain. on the other hand, annelid lectins that recognize galactose and others sugars were isolated from various marine worms including polychaeta and oligochaeta. each lectin has different characteristics on carbohydrate-binding specificities, metal requirement and primary structure. isolation, physicochemical properties, and, in some cases, biological activity and primary structure of such lectins have been described. amphitritin, a ca 2+ independent n-acetyl d-galactosamine-binding lectin with molecular mass of 30 kda was the first hemagglutinin isolated from a sea worm amphitrite ornata (garte and rissel, 1976). a 30 kda βgalactose-specific lectin was isolated and characterized from the sea worm chaetopterus variopedatus (mikheyskaya et al., 1995). curiously, this lectin revealed cytopathic effect induced by human immunodeficiency virus (wang et al., 2006). d-galactose-binding lectins (33-35 kda) were isolated from body walls of echiuroid (urechis unicinctus; oligochaeta) and marine worms (neanthes japonica and marphysa sanguinea) (ozeki et al., 1997). another 32 kda d-galactosebinding lectin isolated from the marine worm perinereis nuntia was shown to have qxw sequence in the polypeptides (kawsar et al., 2009). this sequence motif was seen in r-type lectin family. marine duster worm, sabella spallanzanii (phylum annelida, family sabellidae) is a representative tube worm in the mediterranean bay. its glandular epithelium secreting mucus often appears conspicuous and forming the so called “ventral shield”. mucus production, as in many invertebrates, constitutes a key factor determining the ability of many polychaete species to survive in their environment (beckwith, 1999; smith, 2002; davies and ogawa, 2011). as reported by storch (1988) mucus intervenes in fertilization and egg protection, consolidates the tunnel wall of burrowing polychaetes and may also play a role in the absorption of metabolites (mouneyrac et al., 2003; mastrodonato et al., 2005, dales, 1961; stabili et al., 2009). their defensive functions, such as cytotoxicity and lysozyme-like activity, (canicattì et al., 1992; stabili et al., 2009; giangrande et al., 2014) inhibit in vitro the growth of vibrio anguillarum, vibrio harveyi, pseudomonas aeruginosa and candida albicans (stabili et al., 2011). in the present study, we further investigated the defensive role of s. spallanzanii mucus and we reported the identification, purification and characterization of a novel galactose-binding lectin with agglutinating activity against rabbit red blood cells and several bacteria. this lectin was isolated by both affinity chromatography and high-pressure liquid chromatographic methods. results are discussed in the light of elucidating the involvement of mucus in prevention of pathogenic microorganism proliferation. material and methods chemicals, molecular biology reagents unless otherwise specified, chemicals and reagents were from sigma-aldrich (usa). animals, mucus collection and preparation sampling was undertaken in the harbor of brindisi (southern adriatic sea, italy) using scuba equipment (depth range = 5-15 m). about 200 adult specimens of sabella spallanzanii were collected and transferred to the laboratory. in order to stimulate the secretion of the mucus, all the individuals have been removed from the tube where they lived and kept for 30 min in a petri dish. within the secreted mucus, we checked for trapped material by microscopical observations, whilst we excluded any contamination of other excretion products by ph measurements. secreted mucus was collected and centrifuged at 12000 xg for 30 min at 4 °c and stored at -80 °c until used. it was ten folds diluted in tris-buffered saline (tbs) and filtered through 0.2 µm pore size before performing affinity chromatography. hemagglutination assay rabbit and sheep red blood cells (rarbc and srbc, supplied by istituto zooprofilattico della sicilia) were washed three times in phosphate buffered saline (pbs), centrifuged at 500 xg for 10 min at 4 °c and suspended at 1% in pbs containing 0.1% (w/v) gelatin. a volume (25 μl) of s. spallanzanii mucus or 25 μl of the dialyzed purified s. spallanzanii galactose-binding lectin (ssgbl) were serially (2-fold) diluted in pbs-gelatin in 96well round-bottom microtiter plates (denmark), and an equal volume of erythrocytes suspension was added. the hemagglutinating titer (ht) was measured after 1 hour incubation at 37 °c and expressed as the reciprocal of the highest dilution showing clear agglutination (ballarin et al., 2008). physical and chemical characterization to examine divalent cation requirement for mucus hemagglutination activity (ha), cacl2 and mgcl2 were added to the assay medium to obtain 3 mm each one final concentration. edta (10 mm) or egta (10 mm) were used to examine the effect of ca 2+ versus mg 2+ depletion. to examine the thermolability, mucus samples were incubated at 4, 10, 18, 37, 50, 70, and 90 °c for 20 min and cooled down for 10 min on ice before testing the ha. carbohydrate specificity hemagglutinating activity was assayed against rarbc in the presence of serially diluted saccharides as potential inhibitors (ballarin et al., 2008). a volume (25 μl) of s. spallanzanii mucus or 25 μl of the purified s. spallanzanii galactosebinding lectin (ssgbl) and 25 μl of a serially 16 diluted sugar. finally, an equal volume of erythrocytes suspension was added and after 1 hour incubation at 37 °c the ht was evaluated. inhibition experiments were carried out using decreasing concentrations (starting from 130 mm in pbs ph 7.4, 3 mm cacl2, 1% gelatin) of monosaccharides (l-fucose, l-rhamnose, dgalactose, d-glucose, d-mannose, nacetylglucosamine) and disaccharides (lactose, and lactulose). the same procedure was performed in the inhibition experiments having bacteria as target. bacterial suspensions and agglutination in order to evaluate the hemagglutinating activity the following bacterial strains were employed: vibrio alginolyticus, escherichia coli, aeromonas hydrophyla, staphylococcus aureus and micrococcus lysodeikticus. bacteria were grown to log phase in tryptic soy broth (tsb) containing 3% nacl at 25 °c, with continuous shaking (120 rpm) in a gallenkamp incubator. log phase was estimated by absorbance at 600 nm. the correspondence between cell number and spectrophotometric absorbance have been determined by serial dilution plate count method. bacteria were killed with heat incubating them at 121 °c, for 20 min, at 1 atm. for the agglutination assay, they were washed three times in sterile pbs, suspended in pbs containing 0.1% (w/v) gelatin to obtain 1x10 7 bacteria/ml and dispensed in 96 wells plate. plates were incubated at 18 °c over night. lectin purification lectin was isolated by a two-steps chromatography procedure. the first consisted of a galactose-agarose affinity chromatography column with elution with 0.1 m galactose in tbs, 3 mm cacl2, as previously reported (salerno et al., 2009). the elution step was monitored by absorbance at 280 nm and protein concentration in collected fractions was evaluated through bradford method (1976). after dialysis in tbs, 3 mm cacl2, these were tested for hemagglutinating activity towards rabbit erythrocytes, and those that exhibited the highest activity were pooled and analysed by sdspage (laemmli, 1970). in the second step, the collected fractions from the chromatographic procedure exerting hemagglutinating activity were applied to a highpressure liquid chromatography size exclusion column biosuite 250–10 µm sec 7.5 x 300 mm waters, 350 psi pressure, 280/254 nm (mau) and analysed by hplc method. phosphorylase b (97kda), bovine serum albumin (bsa, 67kda), enolase (46.7kda), myoglobin a (18.7kda) and rnasea (13.7kda) were used as calibration standards. affinity column purified fractions were then applied to high pressure liquid chromatography size exclusion column biosuite 250–10 µm sec 7.5 x 300 mm waters, 350 psi pressure, 280/254 nm (mau). phosphorylase b (97kda), bovine serum albumin (bsa, 67kda), enolase (46.7kda), myoglobin a (18.7kda) and rnase a (13.7kda) were used as calibration standards (fig. 3b). table 1 range of hemagglutinating activity (titer 1 ) of s. spallanzani mucus and the purified lectin (25 µg/ml) towards various erythrocytes and bacteria erythrocytes mucus isolated fraction rabbit red blood cells 512-1024 32-128 sheep red blood cells 0-2 escherichia coli 128256 16-32 vibrio alginolyticus 128-512 16-32 aeromonas hydrophila 64-128 8-16 staphilococcus aureus na na micrococcus lysodeikticus 32-64 8-16 protein content estimation protein content was estimated according to the bradford method using bsa as a standard. undiluted mucus showed a protein content of about 0.6 mg/ml while the best eluted chromatographic fraction had a protein concentration of about 0.2 mg/ml. polyacrylamide gel electrophoresis sds-page (16%) was carried under reducing (5% mercaptoethanol) and non-reducing conditions. to evaluate the molecular size, gels were calibrated with low molecular weight (6.5-66 kda) standard proteins. proteins were stained with coomassie brillant blue r250. results mucus hemagglutinating activity mucus agglutinating activity was tested towards both erythrocytes and bacteria. it showed almost no activity when sheep erythrocytes were used as target cells (ha titer = 2). otherwise, this matrix had a strong agglutinating activity when rabbit red blood cells were used in the test showing an average agglutination titer of 512 (table 1). this activity was calcium dependent because it was strongly affected by calcium depletion when 10 mm edta or 10 mm egta and 3 mm magnesium were added to the hemagglutination assay medium. rarbc hemagglutinating activity thermolability was tested performing the assay after 20 min preincubation of mucus at different temperatures. the optimum of the activity was recorded when temperature ranged between 4 and 18 °c and decreased after 20 min pre-incubation temperature ranging from 37 to 90 °c (fig. 1). almost no loss of biological activity was detected after two months storage of samples at -80 °c. 17 fig. 1 s. spallanzanii mucus preincubated 20 min at different temperature was tested towards rarbc. it to have an optimum agglutination temperature ranging between 10 °c and 18 °c and a reduced but not completely deleted activity even after 20 min pre-incubation step from 37 °c up to 90 °c mucus was able to preferentially recognize gram-negative bacteria; indeed, the strongest agglutinating activity was observed towards v. alginolyticus and e. coli, by contrast mucus agglutinated in a lesser extent a. hydrophyla and the gram-positive m. lysodeikticus and did not agglutinate the gram-positive s. aureus (table 1). carbohydrates inhibition test was performed by adding several carbohydrates in decreasing concentrations (final concentration ranging from 130 to 4 mm) to the assay medium. galactose and at lesser extent fucose revealed to have inhibition activity even at the lowest concentration (8 mm galactose, 16 mm fucose) used in the hemagglutination assay (table 2). ssgbl purification and characterization the ssgbl has been purified starting from a 20 ml diluted collected mucus applied on galactoseagarose column. the profile of the affinity purification is shown in figure 2. in a typical isolation, the eluted fractions, having a protein concentration ranging from 0.08 to 0.18 mg/ml, represented approximately 10-30% of the total mucus protein content loaded onto the column (0.61 mg/ml). the recovery in terms of hemagglutinating activity was about 25% and the 3% after hplc elution (table 3). the galactose eluted fractions, having the highest protein concentration, showed similar average hemagglutinating activity towards rarbc that ranged from 32 to 64 (table 2). the action of the active fractions was calcium dependent because it appeared magnified when the medium contained 3 mm calcium and was heavily affected by the addition of 10 mm edta or 10 mm egta, 3 mm magnesium, as already observed for mucus extracts. electrophoresis analysis on sds-page revealed that the purified lectin consisted of a single component with an apparent molecular weight of 45 kda, under reducing and non-reducing conditions (figure 3a inset) suggesting a monomeric organization of the effector responsible of the hemagglutinating activity. the eluted fraction from affinity chromatography was applied to a hplc size exclusion column and the obtained profile is shown in fig. 3a. from the hplc size exclusion step the purified lectin seems to have an approximate molecular weight of 43 kda (fig. 3b). the hemagglutinating activity of the purified fractions was maintained after 2 months at 20 °c, mildly affected when preincubated for 30 min at 50, 60 or 70 °c but reduced at 90 °c. neither purified hemoagglutinin nor mucus showed agglutinating activity towards srbc (table 2). table 2 inhibition of hemagglutination activity activity of the s. spallanzanii mucus or isolated lectin with re by various sugars inhibitor minimum concentration (mm) of sugar required for 100% inhibition of ha reaction serum isolated lectin d-galactose 25.0 mm 8.1 mm l-fucose 100.0 mm 16.5 mm d-mannose 130 mm ni rahmnose 130 mm ni arabinose, cellobiose, d-glucose, lactose, lactulose, maltose, mannan, n-ac-galactosamine n-ac-glucosamine, d-raffinose; = no inhibition for 200 mm of sugar concentration 18 fig. 2 galactose-agarose affinity chromatography profile and eluted fractions from mucus. a: diluted mucus was applied on a galactose-agarose column and elution step (1-20) performed with 100 mm galactose. the eluted fractions (11-14) show a protein concentration ranging from 0.08 to 0.18 mg/ml. b: hemagglutinating activity (ha) against rabbit erythrocytes induced by: mucus (1), purified lectin fraction n. 26 (2), purified lectin fraction n. 27 (3), erythrocytes control (4) ssgbl bacterial agglutination the purified ssgbl agglutinated both grampositive and gram-negative bacteria (table 1, fig. 4) and was inhibited by galactose (table 2). therefore, the ssgbl binding specificity varied significantly depending on the bacterial target used. in fact, it strongly agglutinated e. coli and v. alginolyticus, in a lesser extent a. hydrophyla and m. lysodeikticus (table 1) whilst no agglutination was found by using s. aureus. discussion comparative immunology is important to understand a fundamental aspect of immunology particular for the phylogenetic perspective (ballarin and cammarata 2016) and the study of annelids immunology have been deeply contributed (engelmann et al., 2018). lectins are important immunomediators in vertebrates and invertebrates (kuhlman et al., 1989; cooper et al., 1994; matsushita et al., 1996; tino and wright, 1996; odom and vasta, 2006; vasta, 2009). many carbohydrate binding proteins have been already described in annelida, both in marine and terrestrial species. kawsar et al., (2010) purified a 32 kda galactose-binding lectin from p. nuntia homogenates that showed hemagglutinating activity against human and rabbit erythrocytes. this lectin from p. nuntia revealed a clear antibacterial activity inhibiting the gram-positive growth in vitro. hirabayashi et al., (1998) isolated a 29 kda lectin from l. terrestris body extracts. these carbohydrates-binding molecules are highly specific for sugar moieties. on account of their capability to bind carbohydrates involved in attachment of potential pathogens to host, lectins can protect the animal preventing its invasion from pathogens. lectins are also involved in cell agglutination, recognizing structures on pathogens surface, they can opsonize them and enhance host phagocytic activity or activate the complement pathway (matsushita et al., 1996; cammarata et al., 2014). due to these properties, lectins evidenced in the table 3 purification steps of ssgbl purification stage protein (mg) ha titer tha specific activity tha/pc purification (fold) yield % mucus 12.5 256 5120 410 1 100 galactose-agarose 0.33 64 1280 3879 9.46 25 hplc 0.015 8 160 10600 26 3 ha: hemagglutinating activity; tha: total hemagglutinating activity. 19 fig. 3 (a) affinity column purified fraction from galactose-agarose applied to a high-pressure liquid chromatography (hplc) size exclusion column biosuite 250-10 µm sec 7,5 x 300 mm waters, 350 psi pressure, 280 black line /254 red line nm (mau). (a inset) sds page (16%) of the purified lectins under reducing conditions; standard proteins hplc profile; native size estimation of ssgbl by hplc. the arrow points to the elution position of ssgbl. (b) calibration standards (rhombus): phosphorylate b (97 kda); bovine serum albumin (bsa, 67 kda), enolase (46.7 kda), myoglobin a (18.7 kda) and rnasea (13.7 kda) body surface mucus can be considered potential antimicrobial agents. among annellida s. spallanzanii is one of the best known and abundant mediterranean sabellid. in this animal, a large amount of mucus is secreted when specimens are subjected to different stress conditions leading to suppose its involvement as protective compartment against microorganisms and/or epibiosis (stabili et al., 2011, 2014; giangrande et al., 2014) as already observed in other invertebrates (denny et al., 1989; weis et al., 1998; smith et al., 2010; ogawa et al., 2011, stabili et al., 2014). a defensive function in s. spallanzanii mucus was firstly suggested by canicatti et al., (1992) who evidenced a haemolytic activity in this matrix. recently, stabili et al. isolated a lysozyme-like activity and an in vitro antimicrobial activity in s. spallanzanii mucus towards some gram-negative bacteria (stabili et al., 2009) clearly indicating the role of this compartment in defending the worms from bacterial attack serving as medium into which the antibacterial substances are exuded. here a divalent-cation dependent lectin was newly discovered from mucus of sabellid, and we purified the ssgbl, a 43 kda monomeric galactose-specific lectin from s. spallanzanii by using both affinity chromatography and highpressure liquid chromatographic methods. its agglutinating activity towards rabbit erythrocytes was significantly modified by the addition of calcium or edta. the activity shows good thermal stability, at temperature values comprised between 4°c and 37°c including the range of natural environment in which these annelid lives. of particular interest, this lectin is a significant fraction (10-30%) of the soluble proteins of the s. spallanzani mucus supporting an important functional role of this molecule. galactose-binding lectins (gbls) have been discovered and isolated also from others annelids. hirabayashi et al. (1998) first isolated from the earthworm, lumbricus terrestris, a galactosebinding lectin of 29 kda inhibited by a wide range of galactose-containing saccharides. the lectin is composed of two homologous domains of 14.5 kda showing 27% identity among other gbls and contained multiple short conserved motifs, "gly-x-xx-gln-x-trp". another gbl with a molecular weight of 32 kda was purified from the pacific annelid perinereis nuntia ver. vallata by affinity chromatography showing a typical r lectin qxw sequence (kwasar et al., 2010). in addition to the agglutinating activity against rabbit erythrocytes, the ssgbl bind both gramnegative and gram-positive bacteria; this ability to recognize and agglutinate exogenous target appears calcium dependent, according to the c-type lectins mode of action. 20 fig. 4 the purified ssgbl displays the ability to agglutinate e. coli (a), v. alginolyticus (c) and m. lysodeikticus (e). e. coli (b), v. alginolyticus (d) and m. lysodeikticus (f) as negative control. ba: bacterial agglutination in particular, the strongest agglutinating activity was observed towards the gram-negative v. alginolyticus and e. coli present in coastal areas and in the harbors in which s. spallanzani lives, by contrast mucus agglutinated in a lesser extent a. hydrophyla and the gram-positive m. lysodeikticus, but not against staphylococcus aureus. these data correspond to the findings already recorded for the total mucus, suggesting that the ssgbl could represent an important effector responsible for the mucus defense role. the ability to recognize, bind and agglutinate bacteria has been well described among vertebrates in fish lectins (bianchet et al., 2002; odom and vasta, 2006; vasta et al., 2011), mammals (cash et al., 2006; vaishnava et al., 2011) and many invertebrates (malagoli et al., 2006) suggesting an antibacterial activity (matsui et al., 1994; tateno et al., 2002). among these, galactose-binding lectins have been described both in invertebrates and fish skin mucus, with bacterial agglutination properties. the skin mucus galectin from japanese eel exhibited agglutination of streptococcus difficile and e. coli (suzuki et al., 2003). further confirming their protective role (shiomi et al., 1989; mistry et al., 2001; suzuki et al., 2003; ogawa et al., 2011). in 21 general galectins are parts of the fish defense system and mainly exist in organs and tissues that delineate the body from its surroundings, such as epidermal club cells of the skin, esophagus and gills (nakamura et al., 2012). in this paper, we propose that the ssgbl is involved in the mucus body defense role, probably it is able to prevent bacterial invasion through the ability to agglutinate some bacteria strains. our data suggest that the ssgbl could represent an important effector acting in contemporary presence with hemolytic factors and lysozyme as a first defense mechanism against potentially pathogens. such synergic strategy is common and well known in invertebrates that lack acquired immunity and therefore reliant on mechanisms of innate immunity. this role is important taking into account that s. spallanzanii lives in eutrophic environments such as harbors where bacteria, including pathogens for humans, marine organisms, are abundant (barg and phillips, 1998). although the worms may be aggressed by bacteria, they are able to survive bacterial attack. unfortunately, by using the classical edman degradation technique, n-terminal sequencing seem to be blocked. microsequence analyses carried out by maldi spectra, despite showing strong peaks didn’t give matches and the nlc-esi msms only gave hits for keratin, and for the weaker sample a bit of bsa as well. the missing of significant match is plausible because the s. spallanzanii sequences are not well represented in the ncbi database and probably is not close enough in the ncbi database to give at least a match based on homology (data not showed). even though our knowledge about composition, production and roles of mucus in various marine invertebrates remains incomplete, our results contribute to the understanding of the mucus protective properties in the investigated polychaete (stabili et al., 2009; 2011). acknowledgments this work was supported by mc and mgp ftofr (university of palermo) and a research grant from the italian ministry of education (prin 20102011 n. 20109xzepr_007) references aranson gj. lectins as defence molecules in vertebrates and invertebrates. fish shellfish immunol. 6: 277-289, 1996. ballarin l, cammarata m, cima f, grimaldi a, lorenzon s, malagoli m, et al. immuneneuroendocrine biology of invertebrates: a collection of methods. inv. surv. j. 5: 192-215, 2008. ballarin l, cammarata m. “lessons in immunity: from single-cell organisms to mammals”, elsevier, london, 2016. barg u, phillips mj. environment and sustainability. fao fisheries circular no. 886fao, rome, fao. 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hplc; gc-mass; toxicity; noctuidae; histology   introduction chemical pesticides provide important protection of crops, animals, and humans from pests and pathogens, improper use in developing countries has resulted in insecticide resistance development, and various environmental pollution disputes (matsumura, 1975). chemicals including synthetic pesticides can sometimes form byproducts which can be linked to unwanted side effects, in beneficial insects (abudulai et al., 2001). increasing development of resistance to synthetic insecticides, promotes investigations into the potential of biorational chemistries isolated from plants (abudulai et al., 2001). plants are potential producer of novel chemical compounds which cannot ___________________________________________________________________________ corresponding author: s. senthil-nathan division of biopesticides and environmental toxicology sri paramakalyani centre for excellence in environmental sciences manonmaniam sundaranar university alwarkurichi 627 412, tirunelveli, tamil nadu, india e-mail: senthil@msuniv.ac.in; senthilkalaidr@hotmail.com yet be synthesized. estimations suggest that over 2000 plant species have the potential to identify and develop new chemistries to reduce bacteria, fungal, and insect/arthropod pests (klocke, 1989). the complexity in chemical compounds in biorational products can also make development of resistance by insect pests is more difficult (regnault-roger et al., 2002). these environmental issues have driven agricultural researchers to search for better ecofriendly based pesticides (wood and granados, 1991). andrographolide is a labdane diterpene lactone from andrographis paniculata, an annual herbaceous plant in the family acanthaceae, is native to india and sri lanka, which has been used for its therapeutic activity. the therapeutic value is for its impact on enzyme induction (meenatchisundaram et al., 2009). unfortunately the limitation to wider direct use as a therapeutic agent is due to poor solubility in water (xiaoa et al., 2013). acid phosphatase (acp, e.c.3.1.3.2) and alkaline phosphatase (alp, e.c.3.1.3.1) are 153   mailto:senthilkalaidr@hotmail.com enzymes, which supports to hydrolyse phosphomonoesters in acid or alkaline environments. epithelim of intestine in animals is a common place for alp which helps to provide phosphate ions for metabolic processes mainly in transphosphorylation reaction (sakharov et al., 1989). during larval moulting stage the acp and alp activity decrease and increase after moulting (miao, 2002;  ajamhassani et al., 2012). adenosine triphosphatases (atpases) helps to transport glucose, amino acids, and other organic molecules. atpases are present in the guts and nerve cells of the lepidopteron insects (horie, 1958). thus if a reduction in atpase efficacy could be caused, there would be a defect in the gut physiology of the insect. spodoptera litura (lepidoptera: noctuidae) is an economically important polyphagous pest in india, china and japan (wan et al., 2014), causing major loss to many vegetables and field crops. the pest is active throughout the year on a wide variety of crops. the larvae are especially serious pests during the seedling stage (chandrasekaran et al., 2012). synthetic pesticides or chemical insecticides used for controlling s. litura have started to fail as populations of s. litura start to develop chemical resistance (sintim et al., 2009; senthil-nathan et al., 2013). a. paniculata extracts have been reported as an antifeedant and ovicidal activity against insects (hermawan et al., 1993), but activity of andrographolide on s. litura has yet to be evaluated. hence, the objectives of the present study includes, isolation and characterization of andrographolide from andrographis paniculata and evaluation of andrographolide for its larvicidal efficacy, phosphatases and cytotoxic activities against spodoptera litura. materials and methods spodoptera litura culture s. litura larvae were collected from ricinus communis plant in nagercoil, tamil nadu, india was cultured and maintained according to senthilnathan and kalaivani (2005) and senthil-nathan et al. (2008a). larvae were cultured in the laboratory on r. communis plant leaves. the r. communis plant leaves were collected from approximately 45 days old plant. for leaf assays and mass culturing, in-between young and mature leaves were used. pre-pupae were provided with vermiculture soil as pupation sites. adult moths emerged from the pupation site were transferred to the cages and fed on a 10 % sucrose solution. the fine cloths containing eggs were removed every 24hrs and eggs present were surface sterilized in situ by dipping in 10 % formaldehyde solution for approximately 3 mts, then washing with distilled water. the eggs in the fine cloths were moistened and kept in aerated containers for hatching. the culture and experiments were carried out at 27 (±3°c), 65 % relative humidity, with a 14:10 light: dark cycle. isolation of plant compound andrographolide the ethanolic extracted plant material was analysed using chromatograph column chromatography with chloroform and methanol. solutes were eluted with different gradients of 90:10, 80:20, 70:30, 60:40, 50:50 and 30:60 chloroform: methanol. active fraction analyses by gc-ms revealed andrographolide as a major compound along with a few trace elements. further purification by column chromatography and analyses by hplc was compared with a purchased standard and chromatogram of andrographolide for confirmation. insect bioassay bioassays were performed with second, third, fourth and fifth instar larvae of s. litura using different concentrations. methanol treated leaves was used as control. five replication was performend with twenty larvae per concentration (n = 100). the fresh r. communis leaves (75 125 cm2) were sprayed with different concentrations of (5, 10, 15, 25 ppm) on both surfaces and allowed for dry. all the treatment instars larvae were starved for 4 h, then fed with treated leaves. the uneaten leaves were removed every day and provide fresh leaves. replication was done five times (totally n = 50). mortality was recorded every 24 h for all treatments. the percentage mortality was calculated using the formula (1) and corrections for mortality when necessary were done using abbott’s (1925) formula (2): (1) percentage of mortality = number of dead larvae number of larvae introduced ×100 (2) percentage of mortality = in t after t t t ×100corrected (1 n in c after t t t n ) the mean lethal concentration (lc50 and lc90) were calculated by subjecting mortality data to probit analysis (finney, 1971). from these results the developmental studies were performed. developmental studies s. litura 3rd instar larvae were used for developmental studies. individual larvae were reared in containers on the r. communis leaves treated with the lethal concentrations of active fraction as 3, 6, 9 and 12 ppm. control leaves were treated with methanol. the containers of the treated and control groups were maintained at 27 (± 2 °c) in a 14l:10d photoperiod at 85 % relative humidity. records were made daily to document deformities in the pupae and abnormalities in emerged adults. enzymatic profile preparation of enzyme extract treated larvae of third, fourth and fifth instars were used for enzyme activities at different concentrations of 3, 6 and 9 ppm respectively. the 154   extraction procedure as in applebaum (1964) and applebaum et al. (1961). anaesthetized larvae were dissected out and entire digestive tract was treated within ice-cold insect ringer’s solution. the gut contents, malphigian tubules and adhering tissues were carefully removed. the separated gut regions was weighed (in mg) and homogenized in volume 300µl of ice-cold citrate-phosphate buffer (ph 6.8) with tissue grinder for 3 min at 4 c. the homogenate was suspended in ice-cold buffer bringing the volume up to 1ml with citrate buffer. samples were centrifuged at 500xg, (eppendorf 5415c table top centrifuge) for one min and the resultant supernatants were collected as the enzyme source. histology the effects of the active fraction ingestion was studied on larvae of s. litura. treated and control larval gut tissue was fixed in bouin’s solution overnight. the blocks were cooled about 27 °c for 3 h and cut into 1.5 µm slices with an ultra-cryomicrotome (cryocut 1800, leica, germany). the slices were stained with delafield’s haematoxylin and counter-stained with eosin, and mounted after drying. the sections were observed and photographed under optika, flow series hbo light microscope (model: b-600 tifl-italy). statistical analysis each experiment was replicated five times and the data’s obtained from mortality were expressed in mean of five replications and arcsine-square root transformation of percentages was used to normalise the data. the analysis of variance (anova) was used to identify the difference in percentage of mortality and were fitted with linear regression using minitab®17 for lethal concentration. differences between the treatments were determined using the tukey-kramer hsd test (p ≤ 0.05). the highest mean difference detected by statistical testing are marked with letter “a” the next text lower “b” etc. (snedecor and cochran, 1989; sas institute, 2001). for enzyme activity linear regression technique of microcal software (originpro 8) was used to graphs. the lethal concentrations (lc50 and lc90) were calculated using probit analysis (finney, 1971). acid phosphatase (e.c.3.1.3.2) and alkaline phosphatase (e.c.3.1.3.1) bessey et al. (1946) procedure was used to carried out the experiment the 100 ul substrates were incubated for about 30 min and 1 ml of alkali (1.0m ph 8.0) was added to stop the reaction. the p-nitrophenolate spectral absorbance was maximal at 310 nm. the molar absorbance of pnitrophenolate at 400 nm is about double that of pnitrophenyl phosphate at 310 nm. on converting the p-nitrophenolate into p-nitrophenol by acidification, the absorption maximum is shifted to about 320 nm with no detectable absorption at 400 nm. estimation of adenosine triphosphatase shiosaka et al. (1971) protocols was followed during the experiment. in the gut sodium and potassium dependent atpase activities were assayed. fiske and subbarow (1925) methodology was adopted for quantitative assay of inorganic phosphorous liberated during experiment. in detail first the protein is precipitated with trichloroacetic acid, then the protein-free filtrate is treated with acid molybdate solution and the phosphoric acid formed is reduced by the addition of 1-amino-2-naphthol-4sulfonic acid (ansa) reagent to produce blue color. the intensity of the color is proportional to the amount of phosphorous present. results analysis of purified plant compound andrographolide the isolated plant compound from column chromatography analysed by hplc revealed the purity at 88 % (fig.1) when compared with the standard of andrographolide (fig. 2). the analysed compound eluted at the rt 3.01, which was detected at 224 nm compared with the standard chromatogram. the andrographolide standard eluted fig. 1 hplc chromatogram of standard andrographolide 155   fig. 2 hplc chromatogram of isolated compound andrographolide at the rt of 2.92. the comparison analyses confirms the compound isolated was andrographolide. mortality of s. litura against andrographolide the active compound andrographolide had pronounced larvicidal activity against s. litura. mortality rates of larvae increased gradually with increasing concentrations (5, 10, 15 and 25 ppm). second instar larvae had the highest mortality. the mortality percentage of andrographolide against s. litura is shown in (fig. 3). the leaf disc treated with andrographolide was permitted to feed and the larvae were observed at 24 h. mortality rate showed significant difference between second instar (f4,20 = 54.61, p ≤ 0.001), third instar (f4,20 = 56.62, p ≤ 0.001), fourth instar (f4,20 = 67.35, p≤0.001) and (f4,20 = 29.02, p ≤ 0.001) fifth instar larvae respectively. the greatest mortality rate was observed in all instar larvae at 25 ppm concentration. the results shown that the andrographolide had significant larvicidal activity. the lowest (5 ppm) concentration of andrographolide had the lowest mortality rate in all instars, but it caused several abnormalities in larval growth. low concentrations were ample enough to give prominent lc50 and lc90 values for all the instars. lethal concentration (lc50) for second instar larvae was observed at 8.31 and 28.86 ppm for lc90. in third instar larvae lc50 was observed at 9.59 and 30.78 ppm for lc90. for fourth and fifth instar larvae the lc50 and lc90 was observed at 10.38 ppm, 34.53 ppm and 11.33 ppm, 36.88 ppm respectively. fig. 3 percentage mortality of second, third, fourth and fifth instar of s. litura after treatment with andrographolide. means (sem±) followed by the same letters above bars indicate no significant difference (p ≤ 0.05) by using probit analysis. 156   fig. 4 morphological effects of andrographolide on s. litura. (a,b,c) larval pupal intermediate (d,e) pupal deformities (f) control pupa (g) treated and unhealthy adult (died) (h) adult emerged with wrinkled wings, molting disorder (shrinking of larval body) (i) healthy adult. behavioural effects of s. litura against andrographolide effective dosage of andrographolide produced a typical colour change, with fluid ooze out from the body of the s. litura larvae. larval-pupal intermediates due to growth-regulatory effects of andrographolide and immature adults were noticed in all the treated concentrations but was more prominent at the highest concentrations (fig. 4). in some cases pre-pupae were affected often causing death, or formation of abnormal pupae were noticed at higher concentrations of 9 and 12 ppm. in lower dosage (3 and 6 ppm) durations of the development life cycle became extended, larval growth was retarded and colour change occurred. at the concentration of 12 ppm larvae appeared unhealthy, with severely damaged cuticles. the adult emergence showed lower longevity, deformed wings and egg produced by the adults were nonviable, immature. 157   fig. 5 enzyme activity (acp) in (a) third, (b) fourth and (c) fifth instar larvae of s. litura after treatment with andrographolide. acp activity concentration used below the lc50 value showed reduction in enzymatic activity. acid phosphatase activity was reduced in larvae treated even at lower concentrations 3, 6 and 9 ppm. the acp inhibition was significantly different for third (f3,16 = 22.65; p ≤ 0.001), fourth (f3,16 = 42.01; p ≤ 0.001) and fifth (f3,16 = 49.99; p ≤ 0.001) instar larvae (fig. 5). when compared with control the estimated highest reduction of 69.18 % was observed in third instar larvae treated with 9 ppm concentration. the reduction in acid phosphatase level reduces metabolism by minimal release of phosphorus. the reduction observed suggests that the cause is predominantly due to the physiological activity of andrographolide. alp activity activity of andrographolide against s. litura showed alp activity reduction across all concentrations. it showed significant reduction at 9 ppm. inhibition rate was protuberant in third instar larvae compared with fourth and fifth instars of s. litura (fig. 6). the greatest reduction of 75.3 % was observed in third instars treated with 9 ppm concentration of andrographolide. atpase adenosine triphosphate was reduced in treated larvae compared with control larvae. the greatest reduction of 74.9 % occurred with treatment of 9 ppm andrographolide (fig. 7). the reduction rate of third, fourth and fifth instar larvae are significantly 158   fig. 6 enzyme activity (alp) in (a) third, (b) fourth and (c) fifth instar larvae of s. litura after treatment with andrographolide. different with each other (p ≤ 0.001). this reduction affects the physiology of the gut by inhibiting the transphosphorylation activity. this activity clearly implies the compound can produce significant influence on enzymatic profile. histological study of s. litura differences in the midgut region was observed in 3, 6 and 9 ppm treated larvae of s. litura. the histology of treated larvae showed damaged cellular components to the epithelial and columnar cells. the untreated larvae had well distinguished epithelial and columnar cells, peritrophic membrane, brush border membrane and microvilli (fig. 8a). larvae treated at 3 ppm showed some disturbed cells, epithelial cells were enlarged, getting to rupture and partially disintegrated gut lumen was observed (fig. 8b). in 6 ppm treatment, larvae showed damaged gut lumen and cytoplasmic regions of oozing cell material into the alimentary tract that mixed within the food column. columnar cells enlarged, brush boarder membrane was damaged with no clear identification of epithelial layer (fig. 8c). larvae at 9 ppm concentration showed damage to cell organelles such as brush boarder membrane being reduced, columnar cells becoming thin, enlarged spacing of cells, mixing with other cell organelles and post disruption of membranes (fig. 8d). at higher concentration of 12 ppm, the epithelial cells were broken and fully collapsed. there was no distinct columnar cells, epithelial layer, cytoplasmic disappeared and other cellular components appeared to have disintegrated completely (fig. 8e). the space between the cytoplasm appeared larger and very distinct. thus at each increasing concentration the damage of cells became more significant and obvious. 159   fig. 7 percentage reductions of enzyme activity (atp) in (a) third, (b) fourth and (c) fifth instar larvae of s. litura after treatment with andrographolide discussion mortality rate was highest in second instar larvae compared with all other instars, possibly because of their earlier developmental stage. hexane and chloroform extract of a. paniculata showed 100 % mortality at 1000 ppm treated mosquitoes, anopheles subpictus (elango et al., 2011). young instars were more susceptible than older instars, as insects mature the mortality rate was reduced. this observation may be due to the increased physiological power of a maturing larvae which has greater capacity to excrete or breakdown ingested toxins. earlier instars were observed to be more susceptible to andrographolide exposure, when compared with later instars. morphological changes in the lifecycle of the insect was due to inhibitory effects of andrographolide. similar results were reported from ethanolic leaf extract of tribulus terrestris, family (zygophyllaceae), on s. litura (gunasekaran and chellaiah, 1985). the darkish black colour pupae was observed and unable to emerge into adults due to ingested andrographolide. the deformed adults showed, darker wings and unable to fly for a longer distance. similar results were reported by narendran et al. (1999), of deformities in head size, length of the body, remains of old cuticle, and darkened colouration on wings when treated with bark extracts of cassia fistula and leaf extract of murraya koenigii, l. family rutaceae, at 1000 ppm. martinez and van emden. (2001) also showed that azadiractin induced a wide range of abnormalities such as larval, pupal and adult abnormalities and small sized adults of the lepidopteran, spodoptera littoralis. the enzyme activity was reduced due to ingestion of treated diet, which reduced gut enzymes such as acp, alp and atpase. reports suggest that these enzymes are the most sensitive to pesticide exposure (wu and lam, 1997; diamantino et al., 2001;  de almeida et al., 2014; ottaviani, 2014). decreased acp levels from treatments suggests a decreased release of phosphorous needed metabolism (energy), which caused decline rate of transport of metabolites of that further reduced the overall rate of metabolism (senthil-nathan and kalaivani, 2005, 2006; senthilnathan et al. 2005a, b). 160   fig. 8 histological section of the midgut region of fourth instar larvae of s. litura (a) control, (b) treated concentrations of 3 ppm, (c) 6 ppm, (d) 9 ppm and (e) 12 ppm of andrographolide. cccolumnar cells el epithelial layer glgut lumen bbmbrush border membrane 161   adenosine triphosphate (atp) is considered the energy currency of cells, particularly in the epithelial cells of intestine which aid transport and metabolites reabsorption, nutrients and secondary transport of ions (lechleitner and phillips, 1988; fogg et al., 1991; zibaee et al., 2010). as ingested food cannot be digested or oxidised, the metabolism stops, thus atpase levels are reduced. the digestion and absorption of nutrients occurs in midgut (selin-rani et al., 2016). histological studies revealed that andrographolide treatment disrupted cell and organelle integrity in larval midguts of s. litura. destruction of epithelial cells would reduce nutrition absorption of ingested food. the midgut cytoplasm were severely damaged at the highest concentration ingested by larvae. corruption of the epithelial layer due to andrographolide ingestion led to oozing of cytoplasmic components which mixed with food in the digestive tract. cell destruction leads to the reduction rate of digestion and food intake. similar observations were reported by senthil-nathan et al. (2008a, b) on s. exigua which ingested styrylpyrone in an artificial diet, and for s. litura treated with 100 ppm of melia extract. melia is a genus of flowering trees in the mahogany family, meliaceae (schmidt et al., 1997). the apoptotic histopathological changes support reports that andrographolide has strong effect on cell physiology, and midgut structure of s. litura larvae. the results that the active compound bound to specific receptors on the midgut may have led to blockage of the digestive tract. furthermore, the subsequently destructions of the transmembrane potential and the osmotic lysis of cells lining the midgut would have contributed to the death of the larvae (aronson and shai, 2001). the present study suggest that andrographolide derived from a. paniculata had acute toxicity to s. litura and also produced morphological changes in their lifecycles. the enzyme activity was also reduced due to ingestion of andrographolide through their diet. similarly, andrographolide treatment strongly disrupted the midgut cells. overall, our results gain evidence to develop novel and safer natural pesticides against the polyphagous pest s. litura. if compared to commercial pesticides, andrographolide is usually more biodegradable, eco-friendly and also decreases the chemical burden on the society. acknowledgement the project was full financially supported by king saud university, through vice deanship of research chairs. references abbott w. a method of computing the effectiveness of an insecticide. j. econ. entomol. 18: 265267, 1925. abudulai m, shepard bm, mitchell pl. parasitism and predation on eggs of leptoglossus phyllopus (l.) 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microbiota; pathogen; spoilage mechanism; molecular analysis; preservation background oyster, a bivalve mollusk, is a nutritious marine food resource that high in protein, vitamin a, vitamin b12 and zinc, but low in calories. many researchers analyzed various nutritional components from oyster ___________________________________________________________________________ corresponding author: huihuang h. ding university of guelph 50 stone road e, guelph, on n1g 2w 1, canada email: dingh@uoguelph.ca list of abbreviations: automated ribosomal intergenic spacer analysis, arisa; denaturing gradient gel electrophoresis, dgge; fluorescent in situ hybridization, fish; lactic acid bacteria, lab; polymerase chain reaction, pcr; restriction fragment length polymorphism, relp; temperature gradient gel electrophoresis, tgge; terminal restriction fragment length polymorphism, t-rflp and verified that they have functional activities (achour et al., 1997; shiozaki et al., 2010; anderson and beaven, 2001). with the increase of consumption, oyster farming grows fast, and it is the most popular mollusk aquaculture around the world. the top 6 countries contributing to oyster production are china, japan, korea, usa, france and mexico (heinonen, 2014). since 1970, the aquaculture of shellfish doubled every decades worldwide, and the demand is still increasing (dégremont et al., 2015). there are approximately 4 million tons of oysters consumed annually and half of them are eaten raw (fang et al., 2015). china produces over 2 million tons of oyster per year, which is mainly used to make oyster sauce (heinonen, 2014). in view of the fast growth of the oyster aquaculture, the impacts of disease and mortality on the yield of oysters attracted prompt attentions by government, farmers, and researchers. however, the research on oyster pathogenic bacteria is challenging due to a wide variety of oysters and aquaculture location worldwide. in previous studies, 375 vibro aestuarianus and vibro splendidus were reported to cause the summer mortality of c. gigas oysters in france (le roux et al., 2002; gay et al., 2004; garnier et al., 2007). furthermore, the introduction of nonnative oyster may lead to disease outbreak (beck et al., 2011). the observation of oyster mortality is the main sign of diseases in aquaculture oyster. preventing contamination and keeping pathogen-free environment is of vital importance in oyster farming (dégremont et al., 2015). on the other hand, the bacteria from fresh oyster were attracted more attentions because some of the bacteria can bring about the outbreak of human diseases. for example, vibrio parahaemolyticus is a pathogenic bacterium for oyster, which is also well-documented foodborne bacteria responsible for the outbreaks of shellfish-associated gastroenteritis and diarrhea correlated to seafood consumption in the united states (dalsgaard, 1998; liu et al., 2009). perishable oyster could cause serious foodborne problems in processing and distribution. microbial activity is mainly responsible for the changes in flavor, texture, and odor (cao et al., 2009; prapaiwong et al., 2009a; montanhini and neto, 2015). compared to terrestrial foods, oyster has shorter shelf life due to relatively higher levels of free ammonia nitrogen and high diversity of microbiota (madigan et al., 2014). the shelf-life of oyster could be affected by many factors, such as extrinsic factor (temperature, atmosphere), intrinsic factors (species, size, age, health and composition) and microbial flora load ( linton et al., 2003; cao et al., 2010; chen et al., 2016). among those factors, microbiota in oyster plays critical roles on oyster diseases, food safety, and spoilage. this article summarized the oyster microbiota, including the analysis approaches, environmental impacts, pathogenic bacteria, and the microbiota in different oyster tissues. analysis approaches for oyster microbiota conventional cultivation method was widely used to analyze the bacterial population, and to isolate them through streak plate method. it plays an important role to obtain the bacterial strains. cultivation method was used to investigate bacterial microbiota and dominant species in oyster, among which pseudomonas were accounted for one third of 321 isolates and reported as dominant bacteria (kueh and chan, 1985). this method has been widely utilized for oyster microbiota analysis to reveal the bacterial population and community in details ( colburn et al., 1990; cao et al., 2009, 2010; liu et al., 2009; song et al., 2009; fang et al., 2015). however, cultivation and following isolation for microbiota analysis was time and resource consuming with poor reproducibility (cao et al., 2009; prapaiwong et al., 2009a). the phylogenetic analyses of rrna genes from laboratory culture and isolates were applied to evaluate the microbiota, of which the efficiency were highly improved in oyster bacterial analysis and many species were identified by sequencing (prapaiwong et al., 2009a; green and barnes, 2010; lee et al., 2010; thupila et al., 2011). conventional cultivation method could result in overestimation or underestimation of the microbiological community, because many bacteria are naturally uncultivable and unsuitable media may lead to biased results (randazzo et al., 2002; chen et al., 2013). molecular approach shows more abundant of bacterial microbiota than cultivation method in oysters (romero et al., 2002). in the past decades, culture-independent methods of finger print profile were introduced to oyster analysis for bacterial microbiota and diversity, such as tgge (fernández et al., 2014) and dgge (chen et al., 2013; wood and arias, 2015), terminal restriction fragment length polymorphism (t-rflp) (garnier et al., 2007; fernandez-piquer et al., 2012), which revealed that oyster had high diversity in the bacteria. dgge was widely utilized in the characterization of the bacterial communities from farmed, retailed, and storied oysters. the fingerprints of dgge gel intuitively reflect the microbiota variation by the band changes, of which the band corresponding to the bacteria (more than 1 %) can be clearly profiled (chen et al., 2013; wood and arias, 2015). however, dgge method may also subjected to the inaccuracy on bacterial diversity evaluations resulted from dna extraction, pcr amplification, and sequencing errors from environmental samples (wintzingerode et al., 1997). this bias was also observed in wood’s study (wood and arias, 2015) when they applied dgge to reveal the bacteria in oyster, few bands from dgge couldn’t be amplified and identified. compared to dgge, t-rflp technique is more reproducible and accurate, but more expensive. both of them provide overview of the bacterial communities and the variation of dominant bacteria in oyster. real-time pcr and multiplex real-time pcr were also introduced to identify and track the target bacteria with higher efficiency and accuracy for the bacteria with lower abundant, especially pathogen community (ward and bej, 2006; nordstrom et al., 2007; kim et al., 2008a). the fish on the basis of the designed probe were used in different organ microbiota in oyster and the high abundance of the bacteria were observed (hernández-zárate and olmos-soto, 2006). the arisa approach also showed the high diversity of oyster gill microbiota effectively (zurel et al., 2011). however, because of the high cost the new technologies, such as metagenome and transcriptome, were not commonly used in previous oyster microbiota studies. environmental impacts on oyster microbiota the diversity and community of bacteria in raw oysters were affected by many factors. oyster is normally eaten by whole body, thus all tissues with its original microbiota are eventually consumed by human. the impacts of aquaculture environmental are of vital importance to original microbiota, because all attached initial bacteria from environment were closely correlated to the microbiota in the growing stages of oyster, harvest, sale, storage, and consumption. these factors include the location of the sea (cao et al., 2009; king et al., 2012; madigan et al., 2014; wood and arias, 2015), harvest season (parveen et al., 2008), water temperature (gonzalez-acosta et al., 2006; shen et 376 table 1 food borne pathogen in oyster aquaculture, sale and storage species/ location pathogen/ food borne pathogen* analysis method reference c. virginica oyster/ mobile bay, us v. parahaemolyticus* alkaline phosphatase-labeled dna probe procedures (kaufman et al., 2003) pacific oysters (c. gigas)/ arcata bay, us listeria sp.*, l. monocytogenes* culture and isolates (colburn et al., 1990) ostrea rivularis salmonella spp. culture (fang et al., 2015) oyster/ washington, us v. parahaemolyticus* culture (liu et al., 2009) oyster (c. gigas)/ france vibrio splendidus, vibrio aestuarianus, vibrio harveyirelated, shewanella colwelliana isolates genotyping by the 16s rrna and gyvb genes (saulnier, decker et al., 2009) pacific oyster (c.gigas)/ france v. aestuarianus real-time pcr (saulnier, de decker et al., 2009) commercial oyster/ new jersey coast, us shewanella algae, s. putrefaciens, photobacterium damselae subsp. damselae, 16s rrna genes sequencing (richards et al., 2008) raw oyster v. parahaemolyticus* real-time pcr (kim et al., 2008b) oyster/ louisiana, us salmonella*, v. parahaemolyticus* micro-is and api-20e systems (abeyta et al., 1986) retailed oyster/ shanghai,china v. parahaemolyticus* polymerase chain reaction (pcr) (yu et al., 2016) salted oyster (jeotkal)/ korea l. monocytogenes*, staphylococcus aureus*, v. parahaemolyticus* culture (song et al., 2009) c. gigas oyster/ mediterranean, france escherichia coli* culture (derolez et al., 2013) commercial oyster/ us v. vulnificus*, v. parahaemolyticus*, v. alginolyticus*, a. hydrophila* culture isolates with 16s rdna identification (prapaiwong et al., 2009a) c. virginica oyster/ dauphin island, us v. parahaemolyticus*, v. mimicus, v. vulnificus total bacteria and vibrio-specific denaturing gradient gel electrophoresis (wood and arias, 2015) oyster tissues v. parahaemolyticus* pcr detection (wang et al., 2010) pacific oyster (c. giga)/ atlantic coast, france v. aestuarianus, members of the v. splendidus group, v. natriegens, v. parahaemolyticus*, pseudoalteromonas sp. the dominant colonies were identified by phenotypic and genotypic characters (rflp) (garnier et al., 2007) c. virginica oyster/ mobile bay, us v. parahaemolyticus* direct plating method involving an alkaline-phosphatase-labeled dna probe (gooch et al., 2002) oyster l. innocua isolation and biochemical tests (colburn et al., 1990) oyster (crassostrea belcheri)/ thailand salmonella*, v. parahaemolyticus*, v. vulnificus* 16s rrna gene sequencing (thupila et al., 2011) raw oyster/ korean v. parahaemolyticus* real-time pcr (kim et al., 2008a) pacific oysters (c. gigas) v. parahaemolyticus* culture (ma and su, 2011) http://onlinelibrary.wiley.com/doi/10.1111/jam.12040/full http://onlinelibrary.wiley.com/doi/10.1111/jam.12040/full 377 zhe oyster (crassostrea plicatula)/ zhejiang, china v. parahaemolyticus* culture (shen et al., 2009) alaskan oysters/ us v. parahaemolyticus* multiplex real-time pcr (nordstrom et al., 2007) oyster (c. virginica)/ chesapeake bay, us v. parahaemolyticus* quantitative direct-plating method followed by dna colony hybridization (parveen et al., 2008) oyster/ mandinga grande lagoon, us v. parahaemolyticus* culture (flores-primo et al., 2014) raw oyster/ alaska, us v. parahaemolyticus* isolates identified by pcr (mclaughlin et al., 2005) live oysters vibrio spp. multiplex pcr and dna microarrays (panicker et al., 2004) oyster/ washington, us v. parahaemolyticus* multiplexed real-time pcr (ward and bej, 2006) oyster/ dauphin island bay, alabama, us v. vulnificus, v. parahaemolyticus* quantitative pcr (givens et al., 2014) raw pacific oysters v. parahaemolyticus* culture (liu et al., 2009) * food borne pathogen. al., 2009), aquatic environment (la valley et al., 2009; shen et al., 2009; azandégbé et al., 2012; king et al., 2012), and environmental stress (paillard et al., 2004; green and barnes, 2010). the initial bacterial communities from different areas are different. cruz-romero (2008b) reported that the initial bacterial communities in raw oyster (c. gigas) from cork harbor were dominant by aeromonas, vibrio, and pseudomonas. the results were similar to the reported bacterial communities of the oysters from yellow sea in china (cao et al., 2009), in which pseudomonas, vibrio were presented as the dominant bacteria. except pseudomonas and flavobacterium, ortigosa et al. (1995) reported that alteromonas, shewanella, deleya, and oceanospirillum were detected in the oysters from mediterranean coast. despite the location, the microbiota were different under controlled and natural environments (colwell and liston, 1960). in our previous study (chen et al., 2013), the dominant microbiota in the raw oyster gills were lactococcus, lactobacillus, enterobacter, and aeromonas. harvest season was one of the main factors responsible for different varieties of the oyster microbiota (parveen et al., 2008; wang et al., 2014b; roterman et al., 2015), which has been well demonstrated by molecular methods. prapaiwong et al. (2009a) observed that more vibrio vulnificus could be isolated from raw oysters living in relatively higher water temperature. in addition, the bacterial communities were correlated to oyster species (roterman et al., 2015). the water temperature can affect the bacteria loads in oyster. the correlation between seawater and mictobiota in oyster were revealed through isolates and rdna hybridization with phylogenetic probes, and most isolates unidentified corresponded to α-proteobacteria (pujalte et al., 1999). pathogenic bacteria in oyster the pathogenic bacteria related to oyster diseases and mortality, as well as human pathogens associated with aquaculture oyster were summarized as shown in table 1. among main human pathogenic bacteria, vibrio, aeromonas, salmonella, e. coli, listeria, staphylococcus, photobacterium, and shewanella have been extensively investigated in oyster aquaculture and storage (table 1). vibrio and aeromonas were the main genus of bacterial pathogenic for oyster. the traditional cultivation and identification, 16s pcr sequencing, real-time pcr, dgge, rflp, multiplex real-time pcr, and quantitative pcr were used to investigate the pathogenic bacteria and microbiota in oyster. real-time pcr and quantitative pcr were regard as the effective way in vibrio inspection, which were designed to reveal the existence of the target pathogen in oyster (nordstrom et al., 2007; kim et al., 2008b; saulnier et al., 2009). in view of the difficulty of identification, polyphasic approaches have been developed to identify potential pathogens associated with oyster diseases (paillard et al., 2004). vibrio species were reported as the main pathogenic species in the oyster leading to 8,000 illnesses per year in the united states (kaufman et al., 2003), which has been extensive studied regarding oyster diseases and mortality, and food safety (kaufman et al., 2003; panicker et al., 2004; nordstrom et al., 2007; liu et al., 2009; saulnier et al., 2009; yu et al., 2016). the pathogenic bacteria associated with public health are v. vulnificus, v. parahaemolyticus, vibrio alginolyticus, and aeromonas hydrophila in raw oysters (lorca et al., 200; prapaiwong et al., 2009a). the proliferation of v. vulnificus during storage at temperature abuse conditions (e.g., 7, 13, and 21 °c) makes the oyster unsafe (lorca et al., 2001). 378 the risk of raw and uncooked oysters resulting in gastroenteritis in consumers has been well described (kueh and chan, 1985; green and barnes, 2010). using vibrio-specific dgge and rflp approaches, the profiles of vibrio were clearly demonstrated (garnier et al., 2007; wood and arias, 2015). more v. parahaemolyticus have been found in the gills and digestive glands than those in other portions of the oysters (wang et al., 2010). prapaiwong et al. (2009a) showed that shewanella, vibrio, psychrobacter and a. hydrophila were also identified in raw oysters, quick frozen oysters, and high pressure processed oysters, whereas v. vulnificus was only detected in raw oysters. the potential risk of v. parahaemolyticus infection might increase, and recently yu et al. (2016) demonstrated that 33 out of 96 isolates showed resistance to two or more antimicrobial agents in shanghai, china. salmonella are regarded as one of the most common human pathogenic bacteria in shellfish; however, they were not detected in oyster either under high pressure treatment or other controlled storage conditions ( jones et al., 1993; bej et al., 1994; lópez-caballero et al., 2000). e. coli found in raw oyster by culture-dependent dgge method illustrated that they may have potential hazard for the ingestion of fresh oyster (chen et al., 2013). listeria monocytogenes were reported to be associated with foodborne outbreaks (colburn et al., 1990). l. monocytogenes and staphylococcus aureus have been presented to be killed using electron beam irradiation in salted oyster (song et al., 2009). the pathogenic bacteria for oyster can also lead to the death of oysters, which cause big losses in oyster farming and related industry. v. aestuarianus and v. splendidus were reported to be related to the summer mortality of the c. gigas in the sea in france. while in north america, v. tubiashii were found to be associated with the mortalities of hatchery-reared crassostrea virginica oysters and c. gigas (saulnier et al., 2009). garnier et al. (2007) demonstrated similar results in their study as v. aestuarianus was detected in 56 % of isolates while 25% of isolates contains v. splendidus group. microbiota in different oyster tissues bacterial microbiota in aquaculture, processing and preservation were studied in the past decades. the predominant bacterial communities were diverse in raw oysters. as list in table 2, the microbiota in oyster mainly included pseudomonas, vibrio, aeromonas, moraxella, shewanella, flavobacterium, acinetobacter, enterobacteriaceae, table 2 microbiota and analysis methods for oyster storied at different condition oyster species/ location treatment methods initial dominant microbiota spoilage or survival microbiota treatment conditions & duration analyzing method reference pacific oyster (c. gigas) natural flora pseudomonas, vibrio, achromobacter, flavobacterium, corynebacterium, alcaligenes, micrococcus, bacillus sp., enterococci na na culture and isolates (colwell and liston, 1960) pacific oyster (c. gigas)/ yellow sea, china refrigeration pseudomonas*, vibrionaceae*, shewanella, alcaligenes, enterobacteriaceae, moraxella, acinetobacter, flavobacterium, corynebacterium, staphylococcus, micrococcus, lactic acid bacteria, bacillus sp. pseudomonas*, vibrionaceae*, moraxella, flavobacterium, micrococcus, bacillus sp. storage at 5 ±1 °c for 12d culture and isolates (cao, xue and liu, 2009) pacific oyster (c. gigas)/ yellow sea, china ozonated water treated pseudomonas, vibrionaceae, shewanella, alcaligenes, enterobacteriaceae, moraxella, acinetobacter, flavobacterium, corynebacterium, staphylococcus, micrococcus, lactic acid bacteria, bacillus sp. pseudomonas*, vibrionaceae*, enterobacteriaceae, moraxella, flavobacterium, micrococcus, bacillus sp. ozonated water (5.0×10-6 g/l for 2 min) culture and isolates (cao et al., 2010) 379 pacific oyster (c. gigas)/ yellow sea, china refrigeration pseudomonas, vibrionaceae, shewanella, alcaligenes, enterobacteriaceae, moraxella, acinetobacter, flavobacterium, corynebacterium staphylococcus, micrococcus, lactic acid bacteria, bacillus sp. pseudomonas*, vibrionaceae*, moraxella, flavobacterium, micrococcus, bacillus sp. storage at 0 °c culture and isolates (cao, xue, liu et al., 2009) pseudomonas, vibrionaceae, alcaligenes, enterobacteriaceae, moraxella, flavobacterium, micrococcus, lactic acid bacteria, bacillus sp. storage at 10 °c c. gigas oyster high hydrostatic pressure bacillus, moraxella, acinetobacter, pseudomonas, micrococcus, coryneforms, flavobacterium, cytophaga, alcaligenes, agrobacterium bacillus control: 300 mpa for 2 min at 20 °c , 0 d isolated from agar plates incubated at 7 °c (linton et al., 2003) moraxella, acinetobacter, flavobacterium, cytophaga storage at 2 °c 14 d bacillus*, moraxella, acinetobacter storage at 2 °c, 28 d c. gigas oyster high hydrostatic pressure bacillus, moraxella, acinetobacter, micrococcus, coryneforms, flavobacterium, cytophaga, enterobacteriaceae, staphylococcus bacillus, micrococcus, alcaligenes, agrobacterium, staphylococcus control: 500 mpa for 2 min at 20 °c , 0 d isolated from agar plates incubated at 30 °c (linton et al., 2003) bacillus, moraxella, acinetobacter, pseudomonas, micrococcus, flavobacterium, cytophaga, alcaligenes, agrobacterium , staphylococcus storage at 2 °c, 14 d moraxella*, acinetobacter* storage at 2 °c, 28 d pacific oyster/coffin bay, australia refrigeration prosthecomicrobium, mycoplasma, helicobacter, terasakiella vibrio, arcobacter, pseudoalteromonas storage at 4 °c, 7 d 16s rrna pyro sequencing (madigan et al., 2014) sydney rock oysters/ australia refrigeration mycoplasma, spirochaeta, haloplasma pseudoalteromonas, vibrio, colwellia storage at 4 °c, 7 d (madigan et al., 2014) pacific oysters (c. gigas) high pressure aeromonas, vibrio, pseudomonas, maraxella, acitenobacter, micrococcus, coryneforms, lactobacillus, leuconostoc, enterobacteriaceae, bacillus shewanella, putrifaciens, pseudomonas, fluorescens 260 mpa for 3 min, stored at 2 °c, 14 d api identification system (cruz-romer et al., 2008a) pseudomonas spp.* 500 or 800 mpa for 5 min stored at 2 °c, 14 d commercial oyster/ us high pressure gammaproteobacteria, alphaproteobacteria, shewanella, vibrio, psychrobacter high pressures of culture isolates with (prapaiwong et al., 2009a) 380 flavobacteria, bacilli, actinobacteria, sphingobacteria 250 to 400 mpa for 1 to 3 min 16s rdna identificaiton commercial oyster/ us quick frozen na shewanella* (in winter); shewanella*, vibrio*, and psychrobacter * (in summer); psychrobacter* and vibrio (dominant in fall) quick frozen oysters were kept at -20 °c culture isolates with 16s rdna identificaiton (prapaiwong et al., 2009a) pacific oyster (c. gigas)/ tasmania refrigeration proteobacteria* spirochaetes, planctomycetes, verrucomicrobia, fusobacteria, firmicutes, tenericutes, cyanobacteria, bacteroidetes psychrilyobacter spp.* (phylum fusobacteria), fusobacteria, spirochaetes 4 °c t-rflp (fernandez‐pi quer et al., 2012) bacteroidetes* 15 °c & 30 °c oyster (c. plicatula) gill/ fujian, china refrigeration l. raffinolactis, weissella cibaria, lactococcus sp., lactococcus lactis subsp. lactis, e. mundtii, e. coli, aeromonas, lactococcus garvieae, a. hydrophila subsp. hydrophila lactococcus*, lactobacillus*, weissella confusa, c. difficile 10 °c, 4 & 8 d dgge (chen et al., 2013) lactococcus, weissella, enterobacter, aeromonas 4 °c, 6 & 12 d (chen et al., 2013) eastern oyster (c. virginica)/ dauphin island, us refrigeration v. parahaemolyticus, v. shiloi, v. vulnificus v. diazotrophicus, listonella anguillarum, v. vulnificus refrigeration at 6 ± 2 °c total bacteria and vibrio specific dgge (w ood and arias, 2015) oysters (tiostrea chilensis)/ chile room temperature na pseudoalteromonas species room temperature (18 °c) at 4, 25, and 100 h after harvest pcr 16s-23s rdna (romero, gonzález et al., 2002) c. gigas oyster/ south korea only for raw oyster test lactobacillus spp., v. alginolyticus, v. proteolyticus na na 16s rrna gene sequencing (lee et al., 2010) pacific oysters(c. gigas), deep bay, hong kong raw oyster pseudomonas spp.*, vibrio, acinetobacter, coliforms, aeromonas spp., flavohacterium, cytophaga, coryneforms, alcaligenes, micrococcus na na culture isolation and identification (kueh and chan, 1985) oysters (c.corteziensis , c. gigas and c. sikamea) commercial production proteobacteria, bacteroidetes, actinobacteria, firmicutes na na pyro sequencing approach of the 16s rrna gene (trabal et al., 2012) commercial oysters (c. corteziensis) different growth phases (post-larvae, juvenile, and adult) ß-proteobacteria (post-larvae, juvenile, and adult), spirochaetes (juvenile), actinobacteria (juvenile) na different growth phases pcr, rflp, tgge (fernández et al., 2014) 381 commercial oysters (c.gigas) -proteobacteria (post-larvae, juvenile, and adult) β-proteobacteria (post-larvae, juvenile, and adult) -proteobacteria (adult), bacilli (post-larvae, juvenile, and adult ) na mangrove oysters/ gbolokiri creek, nigeria depuration of oysters bacteria: bacillus spp., pseudomonas aeruginosa, proteus spp., vibrio spp., e. coli, s. aureus, acinetobacter sp., micrococcus sp., corynebacterium sp., lactobacillus spp. fungi: aspergillus niger, a. flavus, a. nidulans, penicillium spp., fusarium sp., rhodotorula sp. bacteria: bacillus, pseudomonas aeruginosa, proteus spp., vibrio spp., streptococcus spp., s. aureus fungi: nd brackish water treatment culture isolated bacterial (amadi, 2015) raw oyster bacteria: bacillus*, pseudomonas*, vibrio, streptococcus, proteus, lactobacillus, micrococcus, corynebacterium fungi: aspergillus, penicillium, fusarium storage at (30 ± 2°c) ambient temperature for 24 h oyster (crassostrea plicatula)/ fujian, china gill lactococcus*, photobacterium, weissella, lactobacillus*, enterococcus, enterobacter, leclercia, escherichia, spirochaeta, aeromonas, citrobacter lactobacillus*, lactococcus* modified atmosphere package dgge (chen et al., 2016) * dominant bacteria; nd: not detected; na: not available. photobacterium, alcaligenes, micrococcus, staphylcoccus, lactococcus, lactobacillus, corynebacetrium, and bacillus mycoplasma. in addition, fungi of aspergillus, penicillium, fusarium and rhodotorula were obtained in oyster. the cultivation sites, life stages (e.g. post-larvae at the hatchery, juvenile, and adult) and the oyster species (crassostrea corteziensis, c. gigas, and crassostrea sikamea) have an impact on the microbiological communities in oyster ( trabal et al., 2012; fernández et al., 2014). in addition to aforementioned microbiota, shewanella and photobacterium were identified in spoilage oysters (richards et al., 2008). pseudomonas and vibrionaceae were frequently detected as dominant spoilage bacteria in oyster storage. cao et al. (2009) studied the c. gigas from yellow sea in china, and results showed that pseudomonas and vibrionaceae were dominant bacteria in raw oyster which accounted for 22% and 20 % of the total bacteria, respectively. whereas, madigan et al. (2014) pointed out that two genera causing the spoilage of saccostrea glomerata and c. gigas oysters were pseudoalteromonas and vibrio. seasonal difference affects the microbiota in fresh oysters, thus it also determines the dominant micobiotas in spoilage oyster. psychrobacter appears to be predominant only in fall. quick frozen oysters primarily contained shewanella in winter, shewanella, vibrio, and psychrobacter in summer, and psychrobacter and vibrio in fall, and most common dominant genera of high pressure treated oyster were shewanella (15.7 23.9 %) and vibrio (21.4 22.6 %) from all seasons (prapaiwong et al., 2009a). 382 the initial bacterial communities have decisive effect on dominant spoiled bacteria microbiotas in oyster, because spoiled bacteria were demonstrated to be main bacteria detected in fresh oyster in the previous studies. for instance, cao et al. (2009) found that pseudomonas and vibrionaceae in fresh oyster were growing to be dominant bacteria after treatment and chilling storage. in addition, the dominant spoilage bacterial microbiota (e.g., bacillus, moraxella and acinetobacter) after high hydrostatic pressure treatment and storage were also found in fresh oyster (linton et al., 2003). it is worth noting that not all dominant bacteria in fresh oyster are eventually growing competitive and became dominant spoiled bacteria after storage. wood and arias (2015) found that c. virginica oyster were dominated by v. parahaemolyticus (44 %), followed by v. shiloi (21 %) and v. vulnificus (13 %), whereas v. parahaemolyticus was replaced by other nonpathogenic vibrio species (e.g., vibrio species, v. diazotrophicus, listonella anguillarum, v. vulnificus, and unidentified uncultured bacteria) after two weeks storage at 6 ± 2 °c (amadi (2015) found that the dominant bacteria are bacillus (20.8 %) and pseudomonas (16.7 %), whereas those of fungal species are penicillium species (45.4 %) and aspergillus flavus (34.1 %). the role of fungi in oyster deterioration and spoilage should be assessed in the future investigation. in oyster, the initial microbiota in different tissues was studied in previous reports as summarized in table 3. the oyster tissues including gill, stomach, gut, digestive glands and gonads, body fluid, rectal area, crystalline, lower intestine, digestive diverticulum, pallial fluid were detected by culture or molecular approaches. from table 3, the micriobiotas in different tissues of oyster harvested from different locations were different. early in 1960, colwell and liston (colwell and liston, 1960) analyzed microbiota in gill, stomach, and body fluid in the pacific oysters using cultivation and subsequent biochemical identification. in the past two decades, with the development of molecular analysis techniques for microbiology, the studies in microbiotas from different oyster tissues were gradually increased (table 3) table 3 microbiota in different oyster tissues tissues oyster species/ location microbiota analysis methods reference glands sydney rock oysters (saccostrea glomerata)/ australia -proteobacteria, -proteobacteria, fusobacteria, firmicute, spirochaetes, chlorophyta, cyanobacteria, actinobacteria rflp (green and barnes, 2010) digestive glands and gonads c. gigas oyster/ todos santos bay, mexico -proteobacteria, gram-positive bacteria with a low g+c fish (hernández-zárate and olmos-soto, 2006) stomach and c. virginica oyster/ louisiana, us mycoplasma, planctomyctes roche 454 pyrosequencing platform (king et al., 2012) gut phylotypes closely related to shewanella and chloroflexi gill and c. gigas oyster/ japanese pseudomonas,vibrio, flavobacterium, culture and isolates (colwell and liston, 1960) stomach pseudomonas,vibrio, achromobacter, flavobacterium, micrococcus, bacillus body fluid pseudomonas,vibrio, achromobacter, flavobacterium, corynebacterium, micrococcus, bacillus, enterococci culture and isolates rectum pseudomonas/vibrio, achromobacter, alcaligenes, flavobacterium, micrococcus, bacillus culture and isolates gill c. plicatua oyster/ fujian, china lactococcus raffinolactis, weissella cibaria, lactococcus sp., lactococcus lactis subsp. lactis, enterococcus mundtii, e. coli, aeromonas aquariorum, aeromonas jandaei, lactococcus garvieae, a. hydrophila subsp. hydrophila dgge (chen et al., 2013) gill c. gigas oyster/ todos santos bay, mexico cytophaga, flavobacterium, -proteobacteria fish (hernández-zárate and olmos-soto, 2006) and -proteobacterias, pseudomonas spp. and bacillus spp. pcr (hernández-zárate and olmos-soto, 2006) 383 gill c. pacifica methanobrevibacter, corynebacterium, macrococcus, streptococcus, prosthecochloris, flavobacterium, sphingomonas, paracoccus, maritalea, nevskia, schlegelella, paramoritella, shewanella, vibrio, moraxella, acinetobacter, endozocomonas, spongiobacter automated ribosomal intergenic spacer analysis (arisa) (zurel et al., 2011) c. savignyi methanobrevibacter, thalassobacter, endozoicomonas, spongiobacter, acinetobacter, moraxella, limnobacter, schlegelella, neisseria, stenotrophomonas, nevskia, vibrio, prosthecochloris, staphylococcus, flavobacterium, eudoria, corynebacterium, actinomyces stomach c. gigas oyster/ deep bay, hong kong, china pseudomonas spp., vibrio, acinetobacter, coliforms, aeromonas spp., flavohacterium, cytophaga, coryneforms, alcaligenes culture and isolation (kueh and chan, 1985) crystalline vibrio, acinetobacter, coliforms, aeromonas spp., alcaligenes digestive diverticulum pseudomonas spp., vibrio, acinetobacter, coliforms, aeromonas spp., flavohacterium, cytophaga,, coryneforms, alcaligenes lower intestine pseudomonas spp., vibrio, acinetobacter, coliforms, bacillus aeromonas spp., coryneforms, alcali genes, micrococcus gut and eastern oyster (c. virginica) bacterial groups include bacteria (eub338 i, ii, & iii), bacteroidetes (cf319a), and pseudomonas group i (pseudo120) t-rflp (pierce et al., 2016) pallial fluid gills c. gigas oyster/ shanghai, china vibrio, aeromonas, photobacterium, pseudoalteromonas, dokdonella, microbacterium, micrococcus, flavobacterium, psychrilyobacter, bacillus, granulicella, firmicutes, verrucomicrobia culture-independent dgge (w ang et al., 2014a). digestive glands vibrio, aeromonas, photobacterium, pseudoalteromonas, pseudomonas, dokdonella, microbacterium, micrococcus, flectobacillus, flavobacterium, bacillus, granulicella, verrucomicrobia residual tissues vibrio, aeromonas, photobacterium, dokdonella, microbacterium, micrococcus, flavobacterium, fusobacterium, bacillus, granulicella, verrucomicrobia as filter-feeding shellfish, oysters ingest nutrients and microbiology by gills. thus, the gills of oysters accumulate different types of microorganisms, including pseudomonas, vibrio, flavobacterium, lactococcus, aeromonas, leuconostoc, lactobacillus, bacillus, weissella, enterobacter, pseudoalteromonas and enterococcus, photobacterium, dokdonella, microbacterium, micrococcus, psychrilyobacter, granulicella, firmicutes,verrucomicrobia ( colwell and liston, 1960; hernández-zárate and olmos-soto, 2006; zurel et al., 2011; chen et al., 2013; wang et al., 2014a). colwell and liston (1960) separated the different part of the pacific oyster to 384 study the original microbiotas, which showed that pseudomonas, vibrio and flavobacterium were dominant bacteria by traditional cultivation methods. spoiled micriobiotas of oyster gill under 4 °c, 10 °c , and 20 °c storage could be clearly characterized by dgge, through which lactobacillus and lactococcus were found to be the dominant bacteria at various investigating temperatures (chen et al., 2013). other methods, including fish, automated ribosomal intergenic spacer analysis (arisa), pcr, were also used to investigate the oyster gill microbiotas (hernández-zárate and olmos-soto, 2006; zurel et al., 2011). the microbiotas in oyster stomach included pseudomonas, vibrio, achromobacter, flavobacterium, micrococcus, bacillus, miscellaneous, acinetobacter, coliforms, aeromonas, flavohacterium, cytophaga, coryneforms, and alcaligenes (colwell and liston, 1960; kueh and chan, 1985; hernández-zárate and olmos-soto, 2006). more microbiota information were obtained through roche 454 pyrosequencing platform by king (king et al., 2012). kueh and chan (1985) indicated that the microbiota communities in stomach, crystalline, digestive diverticulum, and lower intestine were different when studying the inner parts of pacific oysters (c. gigas). among those microbiotas, vibrio, acinetobacter, coliforms, aeromonas were detected in all analyzing parts. however, pseudomonas was previous regarded as main spoilage bacteria found in stomach, digestive diverticulum, and lower intestine (kueh and chan, 1985; cao et al., 2009). the bacteria in the parts of digestive diverticulum and glands were also studied to demonstrate the relationship among the digestive system and original microbiota. rflp was used and results showed that those microbiota were belonged to -proteobacteria, -proteobacteria, fusobacteria, firmicute, spirochaetes, chlorophyta, cyanobacteria, actinobacteria (green and barnes, 2010). fish revealed that -proteobacteria and gram-positive bacteria with a low g+c were dominant (hernández-zárate and olmos-soto, 2006). kueh and chan reported that the isolates from glands mainly belong to pseudomonas, vibrio, coliforms, aeromonas (kueh and chan, 1985). through culture-independent dgge technology, the dominant communities were clearly profiled (wang et al., 2014a). these bacteria were considered as the most commonly reported microbiotas in shellfish (xuyama and qusi, 1987). the microbiota were complex in whole oyster, because the high diversity in oyster gill, gland, stomach, body fluid, rectal area, and gut were all included in above microbiota studies. microbiota in oyster preservation oyster spoilage resulting in quality losses during preservation was investigated by many researchers (cruz-romero et al., 2008b; cao et al., 2010; xi et al., 2012; bunruk et al., 2013; chen et al., 2014). the shelf life and quality changes of raw and treated oysters were well documented. preservative methods, such as high-pressure treatment (lópez-caballero et al., 2000; prapaiwong et al., 2009b), chitosan coating (cao et al., 2009), and ozone treatment (cao et al., 2010; chen et al., 2014), have been proven to effectively slow down the reproduction of spoilage bacteria. in these studies, the spoilage bacteria were investigated by a culture-dependent method followed by traditional oyster isolate identification. the microbiotas in oyster were mainly affected by the preservation technologies as below. refrigeration temperature is the major impact factor for the microbiota in oyster during storage. different bacterial communities of spoiled oyster under various storage temperatures were summarized in table 2, which showed that storage temperature affects the dominant bacteria in the oyster microbiota. after stored at 0 °c, 5 °c and 10 °c, pseudomonas became the major species and took up to 42 % 66 % of detected microbiotas, and vibrionaceae was around 20 % (cao et al., 2009). abundant pseudomonas was also found in sampled oysters (tiostrea chilensis) stored at room temperature (18 °c) (romero et al., 2002). except pseudomonas, bacillus became dominant bacteria in the oysters if the storage temperature is up to 30 ± 2 °c (amadi, 2015). at phylum level, bacteroidetes became the dominant bacteria under 15 °c and 30 °c storage (fernandez‐piquer et al., 2012). the spoilage bacteria in the different species of oysters could be different at the same storage temperature. after the storage at 4 °c for 7 days, the spoilage bacteria were vibrio, arcobacter for pacific oysters, and pseudoalteromonas, while the spoilage bacteria were pseudoalteromonas, vibrio and colwellia for sydney rock oysters (madigan et al., 2014). the spoilage bacterial microbiota of pacific oyster (c. gigas) after 4 °c storage were psychrilyobacter spp., fusobacteria, spirochaetes (fernandez‐piquer et al., 2012). chen et al. (2013) revealed that the main spoilage microbiotas in the gill of oyster were lactococcus, lactobacillus, weissella confusa and c. difficile under 10 °c storage, while the main spoilage microbiota were lactococcus, weissella, enterobacter and aeromonas under 4 °c storage. furthermore, the impact of modified atmosphere packaging (map) on gill microbiotas suggested that the investigation on the mechanism of oyster spoilage microbiotas during preservation requires to be focused on different tissues as well (chen et al., 2016). high pressure treatment cruz-romero et al. (2008a) demonstrated that the dominant spoilage microbiotas in oyster were shewanella putrifaciens and pseudomonas fluorescens after 260 mpa treatment for 3 min and stored at 2 °c for 14 days, while the dominant spoilage bacteria was pseudomonas spp. after 500 or 800 mpa treatment for 5 min and stored at 2 °c for 14 days. high pressure can inactivate vibrio effectively in oyster. the vibrio spp. accounted for 44 % of the microbiotas in untreated oysters, while they were not detected in all high pressure treated oysters after storage at 2 °c for 14 days (cruz-romero et al., 2008a). however, prapaiwong et al. (2009a) demonstrated that the predominant 385 bacteria were shewanella, vibrio and psychrobacter (only in the fall) after treated by high pressures of 250 to 400 mpa for 1 to 3 min, in which vibrio were survived and became dominant bacteria. high hydrostatic pressures were also utilized in oyster treatment. after 300 mpa treatment for 2 min, the dominant bacteria were moraxella, acinetobacter, flavobacterium, and cytophaga after 14d of storage at 2 °c. after 500 mpa treatment for 2 min, the dominant bacteria were bacillus (90%), moraxella, acinetobacter (10%) after 28 d storage at 2 °c (linton et al., 2003). other technologies other treatments, such as ozonated water treatment, quick frozen and supercritical fluid co2 pasteurization, were also evaluated. the results of quick frozen treatment of oysters at -20 °c showed that the predominant bacteria were shewanella in winter, and shewanella, vibrio, and psychrobacter in summer as well as psychrobacter and vibrio in fall through 16s rdna identification (prapaiwong et al., 2009a). cao et al. (2010) used ozonated water (5.0×10-6 g/l ozone) to treat oysters for 2 min, and the diversity of initial microbiotas were higher than those of treated oyster, which were dominated by pseudomonas and vibrionaceae. as process of cold pasteurization, supercritical fluid co2 was also proven to reduce oyster-associated bacteria (meujo et al., 2008; meujo et al., 2010). map was introduced into oyster preservation and was illustrated that appropriate atmosphere composition can inhibit the growth of microbiology and change the bacterial communities in oyster gill (chen et al., 2016). the mechanism on oyster bacterial spoilage should be further investigated focusing not only on the loads and population of total bacteria counts, but also on the characterization of bacterial microbiotas in whole oyster and different tissues. prospective microbiological analysis in oyster is of vital importance as microbiotas are associated with oyster mortalities, shelf life, spoilage, and human diseases. most studies on oyster preservation were focused on calculating bacterial counts instead of the spoilage bacterial communities during processing or storage. however, the mechanism of oyster bacterial spoilage should be further revealed by discovering the bacterial microbiotas and re-evaluated the spoilage in different oyster species and tissues, instead of focusing on the loads and population of total bacteria counts. innovative molecular technologies have been introduced to further characterize microbiotas in oyster. these technologies have been reported as effective way for microbiota investigation, which provide more advantages to study microorganism profile than traditional cultivation. moreover, those high throughput technologies can be used not only on diversity investigation but also on better understanding of dominant microbiota and illustration of spoilage mechanisms. the microbiota in oyster was well revealed on the basis of the present literatures, while applying state-of-the-art technologies such as metagenome and transcriptome will further clarify the functional roles of bacteria and their co-relationship. aquaculture location and environmental condition, which determine the initial bacteria and affect the proliferation of dominant bacteria and food borne pathogenic bacteria in oyster, should also be emphasized. furthermore, although the entire oyster microbiota has been well studied to illustrate the dominant spoilage bacteria at the end of shelf life, the spoilage mechanism needs to be characterized by different tissues. as the part of oyster, the gill and gut with complex microbiological diversity are easily resulted in spoilage before unacceptability of entire oyster, which should be paid more attention at the beginning of spoilage. the novel technologies for multi-target pathogens detection can provide potential application to prevent the outbreak of oyster diseases and human foodborne illness. acknowledgements this work was jointly supported by national 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antennary processes into the host tissues, while the trunk and egg sacs remain outside the host. the ultrastructure of the epicuticle surface differs among the antennary processes, cephalothorax, and trunk. in the antennary process, the epicuticle appears fuzzy and is less electron-dense than other parts of the body. this loose cuticle structure may be related to the absorption of nutrients in the host hemolymph. the cephalothorax and trunk have an electron-dense epicuticle: there is an array of minute protuberances on the epicuticle of the cephalothorax, whereas the trunk cuticle has no protuberances. this array of protuberances might be involved in suppression of the host immune response, because the cephalothorax has direct contact with the host connective tissues and similar structures are found on other parasitic copepods inhabiting host tissue. key words: cuticle; fine structure; innate immunity; nipple array; parasitic copepod   introduction the body surface has many important roles as an interface between the inside and outside of the body. therefore, the surface structure of the integument has functional significance. the features of the integument surface can differ among species depending on their habitat and behavior. moreover, the features can differ among regions within an individual depending on the functions of each organ. it is possible that similar surface structures have multiple functions and play different roles in different species or different body parts. for example, submicron nipple arrays reduce light reflection on the compound eyes of moths by forming a refractive index gradient (bernhard, 1967; wilson and hutley, 1982), and this anti-glare property probably decreases their visibility by predators. similar nipple arrays have been found in various taxa of aquatic metazoans, such as annelids (hausen, 2005), entoprocts (nielsen and jespersen, 1977; iseto and hirose, 2010), echinoderms (holland, 1984), and ___________________________________________________________________________ corresponding author: euichi hirose faculty of science university of the ryukyus nishihara, okinawa 903-0213, japan e-mail: euichi@sci.u-ryukyu.ac.jp tunicates (hirose et al., 1997, 1999), although their functions are not well-understood. using synthetic sheets bearing nanopillars that mimic a nipple array, we previously demonstrated the anti-glare property of nipple arrays under water (hirose et al., 2015) and bubble repulsion on hydrophilic nipple arrays (hirose et al., 2013). copepoda is one of the most diverse aquatic metazoan taxa and includes numerous parasitic species. according to ho (2001), 3,521 copepod species have been reported from fish species, and more species are routinely being described. nipple arrays have been found on the integumentary cuticles of some parasitic copepods that insert their body either partly or entirely into their hosts (østergaard and bresciani, 2000; hirose and uyeno, 2014). since anti-reflection and bubble repulsion are not likely very important functions within the host body, the nipple arrays in these parasitic copepods may have other functions. on a synthetic multi-pillar surface mimicking a nipple array, the numbers of spreading and phagocytizing hemocytes per unit area are always smaller than those on a flat surface made of the same material (ballarin et al., 2015), suggesting that the nipple array helps parasites suppress the anti-parasitic activities of host hemocytes such as encapsulation. 134   cardiodectes shini is a mesoparasitic copepod found on the heads of pygmy gobies: the cephalothorax of c. shini is embedded in the tissue between the host epidermis and the skull, while the trunk is exposed outside the host. here, we compare the cuticular ultrastructure of the parts embedded in the host tissues and those outside the host. materials and methods a pygmy goby (eviota sp.) was collected by scuba diving at a depth of about 30 m off manza (okinawa island, ryukyu archipelago, japan) on july 17th, 2013. a female cardiodectes shini was attached to the host’s head, with the anterior half of its body embedded in the connective tissue near the eyes. the host goby with the parasite was fixed in 2.5 % glutaraldehyde, 0.1 m cacodylate, and 0.45 m sucrose, and stored at 4 °c. the part of the host head including the copepod was excised with razor blades under a binocular stereomicroscope. the excised specimen was rinsed with 0.1 m cacodylate and 0.45 m sucrose and post-fixed for 1.5 h in 1 % osmium tetroxide and 0.1 m cacodylate. then, the specimen was dehydrated through an ethanol series, cleared with n-butyl glycidyl ether, and embedded in agar low-viscosity resin (agar scientific, england). thick sections were stained with toluidine blue for light microscopy. thin sections were stained with uranyl acetate and lead citrate and examined using transmission electron microscopy (jem-1011, jeol, japan). results gross morphology the body of c. shini consists of a cephalothorax with well-developed, branching antennary processes, a slender neck, and a bean-shaped trunk (figs 1a, b). there was a pair of spiral egg sacs near the posterior end of the trunk. the c. shini cephalothorax was completely embedded in the host tissue, while the trunk was exposed entirely outside the host tissue and the neck situated in the transition zone between the inside and outside of the host tissue (fig. 1b). the host tissue surrounding the antennary processes was reddish due to host erythrocytes, while the tissue around the cephalothorax was transparent (fig. 1a). in the resin-embedded specimen, the resin did not harden evenly in the internal tissues of the body of c. shini, causing large holes in the histological sections (fig. 1c). the resin hardened well in the integumentary tissues and antennary processes, as well as in the host tissues, enabling us to examine these tissues histologically and ultrastructurally (see below). fig. 1 cardiodectes shini infesting the pygmy goby eviota sp. (a) live specimen. (b) schematic drawing of c. shini. (c) histological section stained with toluidine blue. ap, antennary processes; co, connective tissue of the host fish including muscle; ct, cephalothorax; ep, epidermis of the host fish; es, egg sac; mu, muscle of the host; ne, neck; tr, trunk. scale bars: 1 mm (a, b), 50 µm (c). 135   fig. 2 antennary processes of cardiodectes shini. (a) antennary processes (ap) in the host tissue (histological section stained with toluidine blue). (b) enlargement of the processes and host erythrocytes (er) in the space among the processes (histological section). (c) cuticle (cu) and epicuticle (epc) of the antennary processes (tem). asterisks indicate fibrous material on the cuticular surface. co, connective tissue of the host fish; mu, muscle of the host fish. scale bars: 50 µm (a), 10 µm (b), 200 nm (c). antennary process the antennary process is a nodular organ that originates from part of the antenna, which is the second appendage on the cephalothorax. in the histological sections, the antennary process was observed as an aggregate of sections of the branching processes that were heavily stained with toluidine blue (fig. 2a). it was located in a sinus enclosed by the host connective tissues; there was a considerable gap between the host tissue wall and antennary processes. there were many host erythrocytes in the spaces among the branching processes, whereas no host hemocytes adhered to or encapsulated the processes. in the electron micrographs, the antennary process was comprised of electron-dense cells that were covered with a moderately electron-dense cuticle (fig. 2b). the cuticular layer was about 1 µm thick and included a 200-nm-thick epicuticle. the surface of the epicuticle appeared fuzzy, and much fibrous material was seen (fig. 2c). cephalothorax the cephalothorax was spherical and embedded entirely in the host tissue. since the resin hardened poorly in the inner cephalothorax tissues, we could only examine the integumentary tissues, i.e., the cuticular layer and epidermis, in the histological and ultrathin sections. the cuticular layer was at least 5 µm thick and the surface was often directly in contact with the host connective tissues (figs 3a, b). the cuticle consisted of a fibrous matrix, and an electron-dense 10 20-nm-thick epicuticle formed the outermost layer of the cuticle (figs 3c, d). some electron-dense material was found in the cuticular layer beneath the epicuticle. electron microscopy showed a gap between the cuticle and host tissue, and no host cells adhered to the cuticular surface. many minute protuberances were crowded on the epicuticle; each protuberance was about 50 nm in height and 40 nm in diameter at the base, and the protuberances appeared to form a nipple array 136   fig. 3 cuticular layer of the cephalothorax (a-d) and trunk (e, f) of cardiodectes shini. a, b, and e are histological sections stained with toluidine blue, and c, d, and f are transmission electron micrographs. arrowheads indicate electron-dense materials beneath the epicuticle. facing arrows indicate the epicuticle layer. co, connective tissue; cu, cuticle; ep, epidermis of c. shini; er, erythrocytes; mu, muscle. scale bars: 10 µm (a, b, e), 200 nm (c, d, f). (fig.3c). in some areas on the cephalothorax, the protuberances were markedly elongated, and an array of long protuberances formed a 300-nm-thick layer over the epicuticle (fig. 3d). trunk the trunk was exposed outside the host tissue and was connected with the posterior parts of the cephalothorax via the neck. the cuticular layer was at least 15 µm thick (fig. 3e), while the epicuticle was about 30 nm thick and had no minute protuberances (fig. 3f). discussion c. shini is a mesoparasitic copepod that inserts its cephalothorax into the connective tissue of the head of the host fish. our observations revealed that the cuticular layer of c. shini differs in thickness and surface structures among parts of the body. the cuticular layer was ~1 µm thick on the antennary processes, ~5 µm thick on the cephalothorax, and ~15 µm thick on the trunk. in parasitic copepods, the cuticle is generally very thin and the integument is often suggested to be a site of nutrient uptake 137   138   especially in the species lacking alimentary tract (reviewed in bresciani, 1986). some copepods have microvilli-like projections (i.e., microvillosities) on the epicuticle that are supposed to function in the nutrient absorption in the host tissue (bresciani, 1986). for instance, in a pennellid copepod phrixocephalus cincinnatus wilson, 1908 that infects the eyes of flatfishes, the cuticle surface of the holdfast is covered with numerous, fine microvillosities that are occasionally in contact with host cells and may be involved in molecular exchange between parasite and host (perkins, 1994). if the antennary process of c. shini is a nutrient-absorbing organ, branching of the nodular processes might have been an adaptation to increase the surface area, and the copepod is thought to absorb nutrients in the host hemolymph via the cuticle layer. therefore, it is reasonable to believe that the processes have a thin cuticle, and the electron density of the epicuticle is less than on other parts of the copepod body because the cuticular layer of this organ should be permeable to soluble nutrients in the hemolymph. in comparison, the cuticle must be robust to maintain and protect the body elsewhere, especially the exposed trunk. a congener, cardiodectes medusaeus (wilson, 1908), invades the heart of lantern fishes, and the cuticle of frontal processes constituting the attachment organ is about 1 µm in thickness and covered with microvillosities (figs 8 10 in perkins, 1985). this species is supposed to absorb small molecules via the thin cuticle of the attachment organ in addition to the oral ingestion, considering the structural similarity with other specialized endoparasites (perkins, 1985). the fuzzy, fibrous material on the cuticle surface of c. shini may correspond to the microvillosities (asterisks in fig. 2c). the presence of the mouth tube suggests that c. shini feeds on the host’s blood via its mouth as in the congener, c. medusaeus (see uyeno, 2013). although parachordeumium amphiurae (hérouard, 1906), an endoparasitic copepod on brittle stars, has a functional digestive system, it has been suggested that it supplements ingestion by absorption through the epidermis (østergaard, 1998). on the outermost surface of the cuticle on the cephalothorax, there is an array of cuticular protuberances (nipple array) where the host tissues are in close contact with the copepod body. by contrast, these structures were not found on the cuticular surface of the trunk, which is located outside the host tissues, or antennary processes, which are located in a connective tissue sinus in the host. in other words, the nipple array was found on the cuticle that would interact closely with the host connective tissues. therefore, the presence or absence of cuticular protuberances is a clue to understanding the function(s) of the nipple array in c. shini. nipple arrays on the cuticular surface have been found in some parasitic copepods, including ophioika sp. infesting a brittle star (østergaard and bresciani, 2000) and mihbaicola sakamakii infesting the branchiostegal membranes of groupers (hirose and uyeno, 2014). the body of m. sakamakii is located in a pouch composed of thickened host epidermis; consequently, the copepod body is located outside the host tissue topologically. the protuberances are 20 40 nm high in ophioika sp., about 60 nm high in m. sakamakii, and about 50 nm high in c. shini (fig. 3c), although the protuberances in c. shini are sometimes elongated and form a 300-nm-thick layer (fig. 3d). host organisms usually have an innate immune system to eliminate foreign organisms within their body. since endoand mesoparasitic organisms are usually exposed to host tissues, they need to have a mechanism to resist or reduce host attacks. a nipple array might reduce cellular responses such as phagocytosis and encapsulation, because the cell-spreading and phagocytic activities of hemocytes are suppressed on synthetic nanopillar-covered sheets mimicking a nipple array (nomura et al., 2005; ballarin et al., 2014). copepod parasitism leads to local inflammation (dezfuli et al., 2003), but the nano-scale nipple array on the body surface might reduce the adherence and spread of host immunocytes. a nipple array might also reduce surface friction because synthetic micro-dimple arrays are a low-friction surface (hirai et al., 2013). for parasites embedded in host tissue, a low-friction surface would help retain some motility and allow tissue/cells around the body to detach. in c. shini, it is not clear whether the 300-nm-thick layer formed by the elongated protuberances has the same functions as a nipple array. the layer does form considerable space between the host tissue and body surface, which may reduce host attacks. the trunk cuticle of c. shini has no cuticular protuberances, probably because the function(s) of the nipple array is not necessary on the trunk, which is located outside the host and has no contact with host tissue. although a nipple array was not found on the epicuticle of the antennary processes, a dense epicuticle with protuberances might be unsuitable for a nutrient-absorbing organ. it is not known why the host tissues are not in close contact with the processes and no host hemocytes adhere on the cuticular surface. another mechanism may prevent host fish immune responses. in parasitic copepods, the presence of a cuticular nipple array is thought to be an adaptation to enhance resistance to host defensive responses. accordingly, similar structures are expected to be found on the cuticles of many other parasitic copepods inhabiting host tissues. to test this idea, a further survey of the cuticular ultrastructure of endo and mesoparasitic copepods is necessary. acknowledgements this study was partly supported by a university of the ryukyus strategic research promotion grant. references ballarin l, franchi n, gasparini f, caicci f, miyauchi a, hirose e. suppression of cell-spreading and phagocytic activity on nano-pillared surface: in vitro experiment using hemocytes of the colonial ascidian botryllus schlosseri. inv. surv. j. 12: 82-88, 2015. bernhard cg. structural and functional adaptation in a visual system. endeavour 26: 79-84, 1967. bresciani j. the fine structure of the integument of free-living and parasitic copepods. a review. acta zool. 67: 125-45, 1986. 139   dezfuli bs, giari l, konecny r, jaeger p, manera m. immunohistochemistry, ultrastructure and pathology of gills of abramis brama from lake mondsee, austria, infected with ergasilus sieboldi (copepoda). dis. aquat. organ. 53: 257-262, 2003. hausen h. comparative structure of the epidermis in polychaetes (annelida). hydrobiologia 535/536: 25-35, 2005. hirai y, yabu y, kaido m, suzuki a, shimomura m. ag micro-dimples prepared by self-organization and their friction properties. kobunshi ronbunshu 70: 193-198, 2013. (in japanese with english abstract) hirose e, uyeno d. histopathology of a mesoparasitic hatschekiid copepod in hospite: does mihbaicola sakamakii (copepoda: siphonostomatoida: hatschekiidae) fast within the host fish tissue? zool. sci. 31: 546-552, 2014. hirose e, lambert g, kusakabe t, nishikawa t. tunic cuticular protrusions in ascidians (chordata, tunicata): a perspective of their character-state distribution. zool. sci. 14: 683-689, 1997. hirose e, kimura s, itoh t, nishikawa j. tunic morphology and cellulosic components of pyrosomas, doliolids, and salps (thaliacea, urochordata). biol. bull. 196: 113-120, 1999. hirose e, mayama h, miyauchi a. does the aquatic invertebrate nipple array prevent bubble adhesion? an experiment using nanopillar sheets. biol. lett. 9: 20130552, 2013. hirose e, sakai d, shibata t, nishii j, mayama h, miyauchi a, et al. does the tunic nipple array serve to camouflage diurnal salps? j. mar. biol. assoc. uk 95: 1025-1031, 2015. ho js. why do symbiotic copepods matter? hydrobiologia 453/454: 1–7, 2001 holland nd. echinodermata: epidermal cells. in: bereiter-hahn j, matoltsy ag, richards ks (eds), biology of the integument, 1 invertebrates, springer, berlin, germany, pp 756-774, 1984. iseto t, hirose e. comparative morphology of the foot structure of four genera of loxosomatidae (entoprocta): implications for foot functions and taxonomy. j. morphol. 271: 1185-1196, 2010. nielsen c, jespersen a. entoprocta. in: harrison fw, woollacott rm (eds), microscopic anatomy of invertebrates, vol. 13, wiley, new york, pp. 13-43, 1997. nomura s, kojima h, ohyabu y, kuwabara k, miyauchi a, uemura t. cell culture on nanopillar sheet: study of hela cells on nanopillar sheet. japanese j. appl. phys. 44: 1184-1186, 2005. østergaard p. anatomy and development of an endoparasitic copepod parachordeumium amphiurae (hérouard) (cyclopidae, chordeumiidae) living in the brittlestar amphipholis squamata (delle chiaje). zool. anz. 236: 189-202, 1998. østergaard p, bresciani j. sem and tem study of the integument of ophioika sp. (crustacea, copepoda). j. crust. biol. 20: 674-679, 2000. perkins ps. iron crystals in the attachment organ of the erythrophagous copepod cardiodectes medusaeus (pennellidae). j. crust. biol. 5: 591-605, 1985. perkins ps. ultrastructure of the holdfast of phrixocephalus cincinnatus (wilson), a blood-feeding parasitic copepod of flatfishes. j. parasitol. 80: 797-804, 1994. wilson sj, hutley mc. the optical properties of 'moth eye' antireflection surfaces. opt. acta 29: 993-1009, 1982. uyeno d. two new species of cardiodectes wilson, 1917 (copepoda: siphonostomatoida: pennellidae) from gobiid fishes (actinopterygii: perciformes) in the western pacific ocean. zootaxa 3664: 301-311, 2013. gross morphology the body of c. shini consists of a cephalothorax with well-developed, branching antennary processes, a slender neck, and a bean-shaped trunk (figs 1a, b). there was a pair of spiral egg sacs near the posterior end of the trunk. the c. shini cephalothorax was completely embedded in the host tissue, while the trunk was exposed entirely outside the host tissue and the neck situated in the transition zone between the inside and outside of the host tissue (fig. 1b). the host tissue surrounding the antennary processes was reddish due to host erythrocytes, while the tissue around the cephalothorax was transparent (fig. 1a). in the resin-embedded specimen, the resin did not harden evenly in the internal tissues of the body of c. shini, causing large holes in the histological sections (fig. 1c). the resin hardened well in the integumentary tissues and antennary processes, as well as in the host tissues, enabling us to examine these tissues histologically and ultrastructurally (see below). antennary process cephalothorax trunk 164 isj 16: 164-172, 2019 issn 1824-307x research report sequence feature and expression profile of a tumor suppressor qm protein gene from hard clam meretrix meretrix xy cui1, jw cheng1, y wang1,2, b zhou1,2* 1department of marine ecology, college of marine life science, ocean university of china, qingdao 266003, china 2laboratory for marine ecology and environmental science, pilot national laboratory for marine science and technology, qingdao 266237, china accepted september 17, 2019 abstract the present study describes the molecular characterization and transcriptional features of a tumor suppressor qm protein gene, mmqm, in meretrix meretrix. the full-length cdna (819 bp) of mmqm consists of a 657 bp opening reading frame (orf) encoding a 218 amino acid protein with a calculated molecular mass of 25.3 kda and theoretical isoelectric point of 10.2. a ribosomal protein l10 signature, an sh3-binding motif, an antibiotic binding site, an amidation site and two protein kinase c phosphorylation sites were revealed from the mmqm sequence. phylogenetic analysis showed that mmqm is clustered with previously identified mollusk qm proteins. mmqm mrna transcripts were detectable in all of the examined tissues in a constitutive manner, and were significantly different from each other. after bacterial stimulation, the mrna transcripts of mmqm in the hepatopancreas significantly increased. hence, we conclude that mmqm could respond to pathogenic infections and it might play an important role in the innate immunity against microorganisms in the clam m. meretrix. key words: innate immunity; meretrix meretrix; qm protein; vibrio splendidus introduction the clam meretrix meretrix is a crucial commercial marine bivalve in coastal and estuarine areas of south and southeast asia. the rapid development of m. meretrix farming depends on its high economic value (liu et al., 2006; tang et al., 2006; li et al., 2011). recently, the aquaculture of m. meretrix has suffered from diseases, and many of these diseases are provoked by gram-negative bacteria (yue et al., 2010). especially, vibrio has been reported to be the main pathogenic bacteria causing extensive mortality of m. meretrix (wang et al., 2011). therefore, it is necessary to understand the innate immune defense mechanisms against vibrio of m. meretrix to provide new insights into health management and disease control in molluscan aquaculture (yang et al., 2011). many previous studies have focused on this field and have discovered several genes engaged in the immune response of m. meretrix, including serum ___________________________________________________________________________ corresponding author: bin zhou department of marine ecology college of marine life science ocean university of china qingdao 266003, china e-mail: zhoubin@ouc.edu.cn amyloid a (zou and liu, 2015), γ-aminobutyrate type a receptor-associated protein (gabarap) (zhang et al., 2014), heat shock protein (yue et al., 2011), iκb protein (yang et al., 2011), cysteine-rich intestinal protein (chen et al., 2014), macrophage migration inhibitory factor (mif) (zou and liu, 2016) and tetraspanins (wang et al., 2019). accumulating evidence has demonstrated that the qm gene is associated with tumor suppression and is engaged in the innate immune responses. in humans (inada et al., 1997; han et al., 2015), mammals (inada et al., 1997), fish (han et al., 2015), insects (zhou et al., 2019), arthropods (zhou et al., 2011), yeast (koller et al., 1996), and even fungi (wen et al., 2005) and plants (bhardwaj et al., 2010) the qm gene has been reported to respond to viral and bacterial infection. the sequence of qm is highly conserved, which further implies that qm has stabilized and significant functions across species (koller et al., 1996; inada et al., 1997; wen et al., 2005; xu et al., 2008; bhardwaj et al., 2010; oh et al., 2010; zhou et al., 2011; chen et al., 2015; han et al., 2015). in particular, recent studies have suggested that qm could facilitate immune response in mollusks, and that the expression level of abqm from haliotis discus could be significantly upregulated in gills https://www.sciencedirect.com/topics/immunology-and-microbiology/introspection https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/aquaculture 165 upon bacterial (vibrio alginolyticus, vibrio parahemolyticus and listeria monocytogenes) and viral (hemorrhagic septicemia virus) challenge, suggesting it regulates the defensive effect against pathogenic infections (oh et al., 2010). subsequently, cfqm protein from chlamys farreri was associated with its host defense against bacterial (vibrio anguillarum) and viral (acute viral necrobiotic virus) infections (chen et al., 2015). moreover, several studies have revealed that the qm proteins are involved in cell growth, differentiation and apoptosis (marty et al., 1993; lillico et al., 2002; zhang et al., 2004), suggesting this gene is associated closely with cell viability. the current study reports a qm gene (mmqm) from m. meretrix. the full-length cdna of mmqm was obtained via a rapid amplification of cdna ends (race) technique. then, the tissue-specific expression of the mmqm gene was investigated. finally, the immune response of the gene to bacterial challenges was analyzed. materials and methods experimental clams, immune challenge and sample collection a total of 300 healthy clams (m. meretrix) with an average shell height of 2 cm were purchased from an aquatic market in qingdao, china. the clams were maintained at approximately 21 °c for one week before processing. approximately 210 clams were employed for the microbe stimulation assay. vibrio splendidus strain jz6 was employed as the pathogen (liu et al., 2013). one hundred and five clams were chosen randomly to be kept in tanks containing live v. splendidus (108 cfu ml-1) suspended in seawater based on the findings of a previous report (wang et al., 2018) while the other clams were cultivated in seawater as the control group. after stimulation, hepatopancreas samples were randomly selected from both the challenged and control groups at 0, 3h, 6h, 12h, 24h, 48h and 96h, with 5 replications at each time point, and each replication was a mixture of 3 individuals. six kinds of tissues from unstimulated clams, including hemocytes, hepatopancreas, mantle, foot, gill and adductor muscle, were dissected and stored at −80 °c to analyze the tissue transcripts distribution of mmqm. particularly, the hemocytes sample was prepared by collecting the hemocyte pellet after centrifugation of hemolymph from three clams for 10 min at 800 g at 4 °c. one analytical sample consisted of three individual clams. five replicates were performed for each type of tissue. isolation of total rna and synthesis of cdna the total rna was isolated using trizol reagent (thermofisher, usa) according to the manufacturer’s protocol. the purified rna was precipitated and dried for 5 min, and then dissolved in 10 μl of depc-treated water. before cdna synthesis, 4 μl of rna was used to identify its quality by electrophoresis; the rna concentration was determined by measuring the absorbance at 260 nm in a nanodrop 2000c. the 10 μl cdna synthesis mixture containing 6 μl of rna (1 μg), 1 μl of rq1 buffer, 2 μl of rq1 dnasei (promega, usa) and 1 μl of rnase inhibitor (promega, usa) was incubated at 37 °c for 30 min. inactivation was performed at 65 °c for 10 min with 1 μl of rq1 stop solution before 1 μl of 50 mm oligo-dt was added, then heat denaturation was performed at 70 °c for 5 min. the reaction was terminated by cooling on ice for 2 min, and then centrifuged briefly. then, a mixture containing 5 μl of fs buffer, 1 μl of dithiothreitol (dtt, 0.1 m), 1 μl of rnase inhibitor, 5 μl of 2.5 mm dntps and 1 μl of superscript iii (thermofisher, usa) was briefly centrifuged. the reverse transcription was conducted for 1h at 55 °c and inactivation was performed at 70 °c for 15 min. molecular cloning of the mmqm cdna based on a partial cdna sequence (gr902610) of a homologue to the qm gene in clam m. meretrix obtained in a previous study (li et al., 2011), a pair of sequence-specific primers f1 and f2 (table 1) were designed using primer premier 5.0 and used for pcr amplification to clone the full-length cdna of mmqm. the pcr products were gel-purified and cloned into the pmd-19t simple vector (takara, japan). after being transformed into competent cells of escherichia coli strain top 10 (tiangen, china), the potentially positive recombinant clones were identified through anti-ampicillin selection and verified by pcr screening. the sequencing was carried out using an abi 3730 sequencer (thermofisher, usa). table 1 information of primers used in this study primer length (bp) sequence tm(°c) f1 27 5’-gagccaagttcaagttcccaggcagac-3’ 71.75 f2 27 5’-atcccagatggtgtcagtgtacagtac-3’ 68.54 qm-f 20 5’-cgagccaagttcaagttccc-3’ 64.05 qm-r 24 5’-ctgggataagctgatgtttctgtc-3’ 64.02 β-actin-f 21 5’-ttgtctggtggttcaactatg-3’ 60.99 β-actin-r 20 5’-tccacatctgctggaaggtg-3’ 64.74 166 sequence characterization and phylogenetic analysis of mmqm the cdna sequence of mmqm was analyzed using blast (www.ncbi.nlm.nih.gov/blast) and orf finder (www.ncbi.nlm.nih.gov/projects/gorf). the isoelectric point and molecular weight of the deduced protein were predicted using the expasy molecular biology server (web.expasy.org/compute_pi). the nucleotide sequence was translated into the amino acid sequence using the danman 8.08 software package, and the functional domains were predicted by the smart program (smart.embl-heidelberg.de). signal peptide and nuclear localization signals (nls) were predicted using signalp prediction and nls prediction programs (http://cubic.bioc.columbia.edu/cgi/var/nair). the phylogenetic analysis was conducted using clustal x2 and mega 6 programs. a neighbor-joining (nj) phylogenetic tree was constructed using mega 6 and tested for reliability with 5000 bootstrap replications. tissue-specific expression and expression profile of mmqm the transcripts of mmqm in various tissues from healthy m. meretrix and its temporal expression in the hepatopancreas after bacterial challenges were detected via rt-qpcr. all of the rt-qpcr assays were performed with sybr premix extaq (tli rnaseh plus, takara, japan) on an abi 7500 real-time detection system (thermofisher, usa). the expression of mmqm was normalized to that of the β-actin gene for each sample. the primers used in this assay were designed using perlprimer 1.1.21, and the information of all primers is shown in table 1. the relative expression of target gene was calculated by the 2-△△ct method (livak and schmittgen, 2001). all data are given in terms of relative mrna expression level as the mean ± standard deviation and were subjected to one-way anova followed by duncan's multiple range tests by using spss 17.0. differences were considered statistically significant at p < 0.05. results cdna cloning and sequence analysis of mmqm the full-length mmqm cdna (genbank accession no. ku301769) was 819 bp including a 657 bp open reading frame (orf), a 32 bp 5’-utr and an 129 bp 3’-utr with a canonical polyadenylation signal (aataaa). the nucleotide and deduced amino acid sequences are shown in fig. 1. the orf encoded a polypeptide of 218 amino acids with a theoretical molecular mass of fig. 1 nucleotide sequence and deduced amino acid sequence of mmqm. the start codon (atg) and stop codon (taa) are in bold and underlined. the polyadenylation signal sequence (aataaa) is in bold. the ribosomal protein l10 signature (adrlqtgmrgawgkpqgtvarv) is in a shaded background, and the predicted sh3-binding motif (rparcyr) is shaded with a line. the amidation site (mgrr) is boxed, respectively. the protein kinase c phosphorylation sites (scr and skk) are double-underlined, and the antibiotic binding site (nk) is in bold and wave-underlined 167 fig. 2 multiple alignment of mmqm protein with other known vertebrate and invertebrate qm proteins. analysis was performed using representative qm proteins from azumapecten farreri (akm12718), pinctada fucata (aan85578), penaeus japonicus (abs45569), danio rerio (aav34163), mus musculus (caa53061), and homo sapiens (aab27665) by clustalw. identical amino acids in all sequences are highlighted by a shaded background. the characteristic domains of mmqm are bold-underlined and named on the domain 25.3 kda and an isoelectric point at 10.2. several characteristic motifs were revealed in the deduced amino acid sequence of mmqm, including an amidation site (33mgrr36), an sh3-binding motif (4rparcy9), an antibiotic binding site (73nk74), two protein kinase c phosphorylation sites (137svr139, 168skk170) and a ribosomal protein l10 signature (108adrlqtgmrgawgkpqgtvarv129). neither a signal peptide nor a nuclear localization signal (nls) was detected in mmqm. homologous and phylogenetic analysis of mmqm the mmqm amino acid identity and similarity percentages were conducted using the blastx program, and the deduced amino acid sequence shared high identity to representative qm sequences belonging to the mollusca. furthermore, the deduced amino acid sequence shared high identities to representative qm sequences belonging to the mollusca qm protein sequences from c. farreri (84.0%) and p. fucata (83.0%) and had an 168 fig. 3 phylogenetic relationship of mmqm with known qm proteins. the tree is based on an alignment corresponding to full-length amino acid sequences, using clustalx2 and mega6. the number at each node indicates the percentage of boot-strapping after 5000 replications. all of the protein sequences, excluding mmqm, were downloaded from genbank. o. aries (abv64839), b. taurus (np_777185), m. musculus (caa53061), h. sapiens (aab27665), d. rerio (aav34163), l. crocea (acs93602), e. sinensis (ato74510), p. japonicus (abs45569), p. vannamei (aga16579), p. chinensis (anh58179), m. meretrix (ang56314), a. farreri (akm12718), p. fucata (aan85578) overall similarity to the sequences from p. japonicus (78.0%), danio rerio (78.0%), mus musculus (76.0%), and homo sapiens (77.0%). to identify the evolutionary conservation of characteristic motifs and active sites of qm proteins, multiple alignment was performed using different qm homologues, including mollusks (c. farreri, p. fucata), arthropods (p. japonicus), fish (d. rerio), and mammals (m. musculus, h. sapiens). the results demonstrated that the n-terminal and middle regions of mmqm were more conserved than the c-terminal region (fig. 2). moreover, it was revealed that an sh3-binding motif, two protein kinase c phosphorylation sites, an antibiotic binding site, an amidation site and a ribosomal protein l10 signature were highly conserved during the evolution of qm proteins. a phylogenetic tree was constructed by the nj method to investigate the position of mmqm in the context of evolution of the qm family (fig. 3). all of the qm proteins can be clearly recovered in three separate clusters that correspond to mollusks, arthropods and vertebrates in the phylogenetic tree. the vertebrate group included separate clusters belonging to mammalians (oivs aries, bos taurus, m. musculus, h. sapiens) and fish (d. rerio, l. crocea). the invertebrate consisted of mollusks (m. meretrix, c. farreri, p. fucata) and arthropods (eriocheir sinensis, p. japonicus, penaeus vannamei, penaeus chinensis) that showed two independent clusters. within the mollusk cluster, mmqm formed a single lineage with a high bootstrap value of 96%. these results indicate that mmqm is a member of the qm protein subfamily but that it is remarkably different from other known qm proteins. tissue-specific expression of mmqm the relative expression level of mmqm mrna in various tissues including hemocytes, adductor muscle, foot, mantle, gill and hepatopancreas was determined by qrt-pcr with β-actin as an internal control (fig. 4). the mmqm mrna was found to be constitutively expressed in all of the examined tissues. the hepatopancreas and hemocytes displayed the highest and lowest mmqm expression levels, respectively. the fold expression was calculated using the hemocytes as the basis for comparison. the adductor muscle, foot, mantle, gill and hepatopancreas demonstrated 3.59, 4.88, 15.14, 19.38 and 28.38-fold higher mmqm expression levels than the hemocytes (p < 0.05), respectively. in addition, the expression levels in all tissues were significantly different from each other (p < 0.05). 169 fig. 4 tissue expression level of mmqm. the fold expression of each tissue was normalized with that of hemocytes to determine the tissue-specific expression of mmqm. data are presented as mean ± sd for five replicates of rt-qpcr reactions using pooled tissue from three individuals. different letters indicate significant differences (p < 0.05) determined by one-way anova and duncan's multiple comparisons temporal expression profile of mmqm in hepatopancreas after bacterial stimulation transcriptional changes of mmqm in the hepatopancreas after immune challenge at different times were normalized to 0 h, and the temporal expression patterns of mmqm are shown in fig. 5. after cultivation with seawater that contained v. splendidus, the expression level of mmqm exhibited a significant increase at 3 h postchallenge (p < 0.05) and reached its highest level, representing a 7.50-fold higher level at 3 h than that in the control group. the expression level of mmqm gradually decreased from 6 h to 24 h, demonstrating 3.55 to 1.17-fold higher level than in the control group and had a fluctuation at 48h where it increased to 3.41-fold higher than the control group and then normalized at 96 h. the expression levels of mmqm did not exhibit any significant changes in the control group over time. discussion since the first qm gene was identified from humans, the qm gene has been found in a wide range of living organisms, especially in several kinds of marine invertebrate. all of these identified qm proteins of known marine invertebrates contain conserved signature sequences, such as an sh3-binding motif, a ribosomal protein l10 signature, an antibiotic binding site, a protein kinase c phosphorylation site, an n-myristoylation site, an n-glycosylation site, an n-acylation site, and an acyl-amidation site (oh et al., 2010; liu et al., 2014; chen et al., 2015). in the present study, the cloned mmqm was subsequently found to contain these characteristic features of qms. thus, mmqm was regarded as a candidate gene that included several characteristic motifs from marine invertebrate qms and may have a similar function as qms from other invertebrates. phylogenetic analysis showed that mmqm was grouped together with the qms from other mollusks, while qms from fish and mammals were clustered into the same subgroup, and these results were in line with the traditional classification (fig. 3). these results agree with those deduced from the homologous sequence analysis of mmqm. different species display tissue differences of qm at the transcript level, such as qm in c. farreri and h. discus (oh et al., 2010; chen et al., 2015). in the present study, m. meretrix qm was expressed in all of the examined tissues, including hemocytes, gill, foot, mantle, muscle, and hepatopancreas. the highest rna expression levels of qm were observed in the hepatopancreas, followed by the gill. lacking independent immune organs, in shellfish the hepatopancreas is the main immunity and detoxification organ, and the gill is part of the mucosal system, functioning as the first line of defense against pathogens in lower animals (wang et al., 2019). therefore, the high mrna expression levels of mmqm in the hepatopancreas and gill indicate that mmqm could participate in the innate immune system of m. meretrix. increasing evidence has shown that qm-like genes have important immune functions in a wide range of organisms (marty et al., 1993; lillico et al., 170 fig. 5 temporal expression analysis of mmqm in hepatopancreas post bacterial stimulation. relative expression level of mmqm at each time point was normalized with the expression level at the beginning of the challenge test (0h). data are presented as mean ± sd for five replicates of rt-qpcr reactions using pooled tissue from three individuals at each time point. different letters indicate significant differences (p < 0.05) determined by one-way anova and duncan's multiple comparisons 2002; zhang et al., 2004; wen et al., 2005; xu et al., 2008; bhardwaj et al., 2010; oh et al., 2010; liu et al., 2014). to further investigate the role of mmqm in immune responses, the present research detected the temporal expression profiles of qm in response to a bacterial challenge. the expression of the qm gene in the hepatopancreas was upregulated post bacterial challenge in m. meretrix. similarly, the qm gene in the hemolymph is upregulated after infection with acute viral necrobiotic virus and v. anguillarum in c. farreri (chen et al., 2015). after infection with v. anguillarum, the qm gene of l. vannamei is upregulated in various tissues, such as the hemolymph, hepatopancreas, gill, heart, intestine, and muscle (liu et al., 2014). based on these results, we demonstrated that qm expression can be induced by pathogen attack, further verifying that qm may play a positive role in the response to various pathogens and act as an essential component in host defense system. specially, the upregulation of the expression level of mmqm at 48h, which is different from the other results, suggests that mmqm might be involved in multiple immune response processes or be regulated by multiple immune response pathways and confirms the opinion that the invertebrate immune systems are not homogeneous, not simple and are not well understood (loker et al., 2004). in summary, we identified a tumor suppressor qm-like gene from m. meretrix and successfully cloned the cdna sequence of mmqm, whose deduced amino acid sequence was structurally related to the qm protein family. tissue expression analysis indicated that mmqm was expressed in all of the examined tissues and its transcriptional regulation was different among the tissue types. 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43: 301-309, 2015. zou l, liu b. the polymorphisms of a mif gene and their association with vibrio resistance in the clam meretrix meretrix. dev. comp. immunol. 62: 116-126, 2016. 285 isj 15: 285-293, 2018 issn 1824-307x research report an i-type lysozyme (cflyzi) involved in innate immunity is essential for the survival of chlamys farreri during vibrio stimulation mq wang1,3, bj wang1, m liu1, ky jiang1, l wang1,2,4* 1cas key laboratory of experimental marine biology, institute of oceanology, chinese academy of sciences, qingdao 266071, china 2laboratory for marine biology and biotechnology, pilot national laboratory for marine science and technology, qingdao 266237, china 3research platform for marine molecular biotechnology, pilot national laboratory for marine science and technology, qingdao 266237, china 4center for ocean mega-science, chinese academy of sciences, qingdao 266400, china accepted august 22, 2018 abstract lysozymes act as key components of the innate immunity in invertebrates and play a pivotal role in early defense against invading microbe infection. in this study, an i-type lysozyme homology was identified and characterized in zhikong scallop chlamys farreri (designated as cflyzi). the full-length cdna sequence of cflyzi contained a 702 bp open reading frame (orf), which encoded a polypeptide of 233 amino acids and contained a typical destabilase function domain. the mrna transcripts of cflyzi were detectable in all the investigated tissues, including hemocytes, muscle, mantle, gill, hepatopancreas and gonad with the peak level in hemocytes. being stimulated by vibrio splendidus, the mrna transcripts of cflyzi significantly increased in hemocytes. the cflyzi-suppressed scallops turned to be more susceptible to vibrio. all these results indicated that cflyzi acted as an efficient effector in the innate immunity and was also essential for hosts’ survival during vibrio stimulation in zhikong scallop. key words: chlamys farreri; innate immunity; invertebrate type lysozyme introduction the innate immunity, also known as non-specific immunity or in-born immunity, acts as first line for all the multicellular organisms and almost the only defense mechanism for invertebrates that protects hosts from microbial invaders (medzhitov and janeway jr, 1997). antimicrobial proteins and peptides (amps), also called host defense peptides (hdps), are ancient effector molecules of innate immunity, and provide a principal first line of defense against the microbial pathogens in all living organisms (hoffmann et al., 1999). among all the identified amps, lysozyme (ec 3.2.1.17), also termed as n-acetylmuramide glycanhydrolase or muramidase is an antimicrobial enzyme forming part of the innate immunity and also regarded as an important digestive enzyme in animals, especially in ___________________________________________________________________________ corresponding author: lei w ang cas key laboratory of experimental marine biology institute of oceanology chinese academy of sciences qingdao 266071, china e-mail: wanglei@qdio.ac.cn ruminant artiodactyls and filter-feeding organisms (daffre et al., 1994; boman, 1995). lysozyme makes up a large amount of proteins and peptides and ubiquitously presents in diverse organisms ranging from human to virus (johnson, 1998). based on catalytic characteristics, molecular features and original sources, lysozymes could be classified into several types, including bacteria type, chalaropsis type (ch-type), chicken/conventional type (c-type), goose type (g-type), invertebrate type (i-type), phage type and plant type (jielian et al., 2017). among all the animal origin types, c-type and g-type lysozymes are present in all the vertebrates, while invertebrates mainly produce i-type lysozymes and partially produce c-type, ch-type or g-type lysozymes (callewaert and michiels, 2010). moreover, i-type lysozymes exhibit multiple activities, such as chitinase, isopeptidase, muramidase and non-enzymatic antibacterial activities (van herreweghe and michiels, 2012). zhikong scallop chlamys farreri (mollusca; bivalvia; pteriomorphia) is a dioecious bivalve native to the coast of china, japan and korea, and weightily contributes to the aquaculture industry of 286 northern china (li et al., 2017b). however, scallop aquaculture industry has been experiencing mass mortality during summer period and suffering from extensive economic losses in the past decades (matozzo, 2016; huang et al., 2018). the complex interactions among environment, hosts and pathogen are regarded to be the main causes for such mass mortality of cultured scallops (wang et al., 2012). lysozymes make a major contribution to the accomplishment of innate immune responses (saurabh and sahoo, 2008). in previous studies, a g-type lysozyme (designated as cflyzg) exhibited inhibitive effect on the growth of both gram negative and gram positive bacteria with more potential activities against gram positive bacteria, and its single nucleotide polymorphisms (snps) were associated with resistance or susceptibility to vibrio (listonella) anguillarum (zhao et al., 2007b; li et al., 2009; 2013). in the present study, an i-type lysozyme gene (designated as cflyzi) have been cloned and investigated in zhikong scallop, and the main objectives of the present study were (1) to characterize the molecular features of cflyzi (2) to validate the tissue and temporal expression patterns of its mrna transcripts, and (3) to confirm the function of cflyzi via double strand rna (dsrna) mediated rna interference (rnai). materials and methods scallops, vibrio stimulation, temperature stress and samples collection vibrio splendidus was cultured in liquid 2216e media (hb0132-1, hopebiol, china) at 28 °c and 140 rpm overnight. the bacteria were collected by centrifugation at 4000 g for 10 min and re-suspended in filtered seawater. approximately sixty scallops were employed for vibrio stimulation assay. scallops were purchased from a local farm in qingdao, china, and maintained in aerated seawater at 20 °c. after acclimated for two weeks, thirty scallops were immersed with live bacteria v. splendidus at a final concentration of 1.0 × 108 colony forming units (cfus) per 1 ml, defined as vibrio stimulation group. the rest thirty scallops were employed as the control group. five scallops from the two groups were randomly sampled at 0, 3, 6, 12, 24 and 48 h post stimulation, respectively. for the temperature stress assay, modified from the previous report (ding et al., 2018), the scallops were randomly divided into five groups, and treated at 10 °c, 15 °c, 20 °c (as control), 25 °c and 30 °c for 7 days, respectively. five scallops from each group were sampled. hemocytes, muscle, mantle, gill, hepatopancreas and gonad from five untreated scallops were collected to determine the mrna distribution of cflyzi. rna preparation and cdna synthesis total rna was isolated from the hemocytes and other tissues using rnaiso plus (9108, takara, japan). the first-strand cdna was synthesized with superscript iv reverse transcriptase (18090010, thermo fisher scientific, usa) using the rq1 dnase i (m6101, promega, usa) treated total rna as template and adaptor primer-oligo (dt) as primer (table 1). the reactions were performed at 55 °c for 1 h, terminated by heating at 80 °c for 5 min, then a homopolymeric tail was added using dctp (4028, takara, japan) and terminal deoxynucleotidyl transferase (tdt, 2230, takara, japan), and then stored at -80 °c till used. cloning the full-length cdna of cflyzi in our previous studies, a transcript sequence homologues to previous identified i-type lysozymes was identified in c. farreri via assembling and screening public available expression sequence tags and transcriptomic data (wang et al., 2018b). and this transcript sequence was selected for further cloning of cflyzi. four gene-specific primers, cflyzi-race-r1/2 and cflyzi-race-f1/2, were designed to clone the full-length cdna of cflyzi via 5` and 3` rapid amplification of cdna ends (race) technique, respectively (table 1). all the pcr reactions was performed in a mj mini personal thermal cycler (bio-rad, usa), and pcr products were purified with monarch dna gel extraction kit (t1020s, neb, usa), ligated into the pmd18-t simple vector (d103a, takara, japan), and then transformed into the competent cells escherichia coli dh5α (cb101, tiangen, china). the positive recombinants were identified using anti-ampicillin selection and confirmed by pcr screening using the universal primers m13-47 and rv-m (table 1). five of the positive clones were sequenced in a prism 3730xl automated sequencer (thermo fisher scientific, usa). bioinformatics analysis of cflyzi cdna and deduced protein sequences blast+ 2.7.1 was employed to conduct the search for sequence similarities. the deduced amino acid sequences of cflyzi were analyzed by lasergene suite 7.1.0.44 using the editseq module. the presence and location of signal peptide was predicted by signalp 4.1. smart 7.0 was employed to analysis the function domains. multiple sequence alignments were generated with clustal omega 1.2.4 and visualized by sequence manipulation suite 2.0 using the multiple alignment show module. quantitative real-time pcr analysis of cflyzi mrna expression patterns the tissue and temporal expression patterns of cflyzi mrna in hemocytes were investigated by quantitative real-time pcr (qrt-pcr). all the qrt-pcr reactions were performed in a linegene k fqd-48a fluorescence quantitative pcr detection system (bioer, china) using the sybr premix extaq (rr420, takara, japan). all the primers for qrt-pcr were listed in table 1. for each sample, the mrna expression of cflyzi was normalized to that of elongation factor 1 α (ef-1α). the relative abundance of cflyzi mrna was calculated using comparative ct method (2-δδct method) as mean ± sd (schmittgen and livak, 2008). the data were subjected to one-way analysis of variance (anova) followed by a multiple comparison using ibm spss statistics 23.0.0.0, and the p values less than 0.05 were considered statistically significant. 287 table 1 oligonucleotide primers used in the experiments knock-down of cflyzi in vivo via rnai t7 promoter tagged primers egfp-dsrna-t7-f/r and cflyzi-dsrna-t7-f/r (table 1) were used to amplify the cdna fragments of enhanced green fluorescent protein (egfp) and cflyzi, and the resultant pcr products were purified to synthesize dsrna. the dsrna were produced via in vitro transcription according to the methods previously described (wang et al., 2018a; wang et al., 2011), and its concentration was quantified using nanodrop lite (thermo fisher scientific, usa) and adjusted to a final concentration of 1 mg ml-1. one hundred micrograms of cflyzi dsrna was injected into adductor of each scallop, and the control groups received an injection of 100 mg egfp dsrna or pbs, while the untreated scallops were employed as blank group. post dsrna injection, hemocytes from five scallops of each group were collected every 12 h and used for total rna isolation and cdna synthesis. the efficiency of gene silence was confirmed via qrt-pcr, and an optimum time point (36 h post dsrna injection) was selected for the vibrio stimulation and mortality comparison assay. vibrio stimulation and mortality comparison approximately four hundred scallops were employed for vibrio stimulation and mortality comparison assay according to our previous descriptions (wang et al., 2011; 2018a). these scallops were equally and randomly divided into four groups (one experimental group, two control groups and one blank group) and then each group was subdivided into three subgroups. at 36 h post dsrna injection, the scallops were stimulated with live v. splendidus at the final concentration of 1×108 cfus ml-1. the cumulative mortalities were recorded every 12 h. the t-test was used to verify significant differences in mortality levels between different groups, and the p values less than 0.05 were considered as statistically significant. results the molecular feathers of cflyzi the complete cdna sequence of cflyzi was obtained via 5` and 3` race technique and submitted to genbank with the accession number ku361831. it comprised 880 bp, containing a 105 bp primer sequence (5`-3`) brief information adaptor primer ggccacgcgtcgactagtac anchor primer for 3’ race adaptor primer-oligo (dg) ggccacgcgtcgactagtacg10hn anchor primer for 5’ race adaptor primer-oligo (dt) ggccacgcgtcgactagtact17vn oligo (dt) for cdna synthesizing cfef-1α-qrt-f atccttcctccatctcgtcct internal control for real-time pcr cfef-1α-qrt-r ggcacagttccaatacctcca internal control for real-time pcr cflyzi-cds-f atgtgcatttatttgtatcctaactct gene specific primer for cds cflyzi-cds-r ctagctgtgtgccgagcaacccat gene specific primer for cds cflyzi-dsrna-basic-f gacagatagataaaatacaagcaagat gene specific primer cflyzi-dsrna-basic-r cccacagtcatgccagtaggc gene specific primer cflyzi-dsrna-t7-f ggatcctaatacgactcactatagggatccgacagatagataaaatacaagcaagat gene primer incorporated with t7 promoter cflyzi-dsrna-t7-r ggatcctaatacgactcactatagggatcccccacagtcatgccagtaggc gene primer incorporated with t7 promoter cflyzi-qrt-f ctttgccacaggtagcgt gene specific primer for real-time pcr cflyzi-qrt-r tttcccacagtcatgccag gene specific primer for real-time pcr cflyzi-race-f1 ccggtgaccttcattgttcatcca gene specific primer for race cflyzi-race-f2 caactgtgagagctacgcacggatcca gene specific primer for race cflyzi-race-r1 acatcttcagcgtgtccttgttctcga gene specific primer for race cflyzi-race-r2 atcggccttatcttgcttgtattttat gene specific primer for race egfp-dsrna-basic-f cgacgtaaacggccacaagt gfp specific primer egfp-dsrna-basic-r cttgtacagctcgtccatgc gfp specific primer egfp-dsrna-t7-f ggatcctaatacgactcactatagggatccgacgtaaacggccacaagt gfp primer incorporated with t7 promoter egfp-dsrna-t7-r ggatcctaatacgactcactatagggatccttgtacagctcgtccatgc gfp primer incorporated with t7 promoter m13-47 cgccagggttttcccagtcacgac vector primer for sequencing rv-m gagcggataacaatttcacacagg vector primer for sequencing 288 5` untranslated regions (utr), a 73 bp 3` utr with a poly a tail, a polyadenylation signal site (aataaa) and an 702 bp open reading frame (orf). this orf encoded a polypeptide of 233 amino acid residues with a predicted molecular mass at approximately 25.19 kda and a theoretical isoelectric point (pi) of 7.35. a signal peptide (from m1 to a31) and a typical destabilase domain (from s119 to m227) were revealed in the deduced amino acid sequence. additionally, two specific motifs (from c122 to c127 and from w130 to k153) were also revealed (figure 1a). multiple sequence alignment of cflyzi with cflyzg showed that the homology between these two lysozymes was rather low (figure 1b). while pairwise sequence alignment showed that cflyzi exhibited higher homology to its invertebrate counterparts, for examples, cflyzi exhibited similarities of 60 % to i-type lysozyme of chlamys islandica (cab63451), 53 % to that of crassostrea rivularis (ady38955) and 45 % to that of mytilus galloprovincialis (ajq21515). fig. 1 sequence features and multiple alignments of cflyzi. a: sequence features. the nucleotides and amino acids were numbered along the left margin. the putative signal peptide was underline. the typical function domain was in shade. the specific motifs were double underlined. the stop codon was indicated by the asterisk. the polyadenylation signal (aataaa) site was boxed. b: multiple alignments of cflyzi with cflyzg. the black shadow region stood for positions with the same amino acids. similar sites were in grey. gaps were indicated by dashes 289 the tissue and temporal mrna expression patterns of cflyzi the qrt-pcr was employed to detect the tissue and temporal mrna expression patterns of cflyzi. the cflyzi mrna could be detected in all the investigated tissues. the peak level of cflyzi mrna was found in hemocytes, followed by gill, which were 9.18-fold and 3.39-fold of that in gonad, respectively (p < 0.05, figure 2a). hemocytes were selected to test the temporal mrna expression patterns of cflyzi post vibrio stimulation. the mrna expression levels of cflyzi were significantly up-regulated at 3 h post vibrio stimulation (4.55-fold compared with the origin level, p < 0.05), reached to the peak level at 12 h (9.09-fold, p < 0.05), and then down-regulated to the origin level at 48 h (figure 2b). additionally, cflyzi mrna expression levels in hemocytes were temperature-dependent. compared with the control group, the cflyzi mrna expression levels were stable at 15 °c and 25 °c, but significantly decreased at 10 °c and 30 °c, which was 0.53-fold and 0.22-fold compared with the origin level (p < 0.05), respectively (figure 2c). cumulative mortality of cflyzi-suppressed scallops the effect of rnai for cflyzi was confirmed by qrt-pcr. generally, 70% inhibition of mrna expression post dsrna injection was considered as a threshold for an effective rnai experiment (krueger et al., 2007). in the present study, the mrna abundance of cflyzi gene started to decrease at 24 h post dsrna injection and maintained rather low (less than 0.3-fold of the origin expression level) from 36 h to 84 h (figure 3a). so, 36 h post-dsrna injection was selected as the optimum time point for the following vibrio stimulation and cumulative mortality assay. without vibrio stimulation, the final mortality rates were 9.47%, 8.92%, 8.83% and 9.29% for normal, pbs injected, egfp-dsrna injected and cflyzi-suppressed scallops, respectively (figure 3b). being stimulated with vibrio, the cumulative mortality of cflyzi-suppressed scallops was significantly higher than those of control groups from 12 h post stimulation. the cflyzi-suppressed scallops died out at 72 h, while the cumulative mortalities were 51.35%, 58.15% and 57.69% for normal, pbs injected and egfp-dsrna injected scallops at the same time, respectively. the semi-lethal time for cflyzi-suppressed scallops was less than 48 h, while those of other groups were about 72 h (figure 3c). discussion i-type lysozymes play pivotal roles in invertebrate innate immunity and are considered to be the first barrier against invading microbes (saurabh and sahoo, 2008). in invertebrates, especially in marine invertebrates, recent research achievements indicated that i-type lysozymes were extensively involved in innate immune responses and exhibit more extensive activities than those of terrestrial invertebrates, due to various invading microbes in the aquatic environment (jielian et al., 2017). for examples, an i-type lysozyme from the asiatic hard clam meretrix meretrix expressing along fig. 2 tissue and temporal expression patterns of cflyzi. a: tissue distribution of cflyzi. b: temporal mrna expression patterns of cflyzi post vibrio stimulation. c: temporal mrna expression patterns of cflyzi under temperatures stress. bars with different characters stood for significant difference (p < 0.05) with larval development showed typical lysozyme activity and strong antibacterial activity against gram negative and gram positive bacteria, and its snps were correlated with the resistance to vibrio 290 fig. 3 rnai of cflyzi and its effect. a: the relative abundance of cflyzi mrna in scallops hemocytes. bars with different characters stood for significant difference (p < 0.05). b: mortalities without vibrio stimulation. c: mortalities post vibrio stimulation 291 and growth of hosts (yue et al., 2011; 2012; 2013). the intensive expression profiles and strong antimicrobial activities against bacteria of recombinant i-type lysozyme indicated its potential antibacterial roles in the sea cucumber apostichopus japonicas (wang et al., 2008; 2009; yang et al., 2010). a recombinant i-type lysozyme from the white shrimp litopenaeus vannamei showed a broad spectrum of antimicrobial properties with high antibacterial activities against vibrio species (chen et al., 2016), while an i-type lysozyme was potentially involved in the ontogenesis and immune defense in kuruma shrimp marsupenaeus japonicus (liu et al., 2016). additionally, the research achievements on i-type lysozymes in the eastern oyster crassostrea virginica indicated a possible adaptive evolutionary pathway for i-type lysozymes from host defense to digestion in bivalves (xue et al., 2004; 2007; 2010). although many i-type lysozyme genes have been identified from marine bivalves, to our best knowledge, no i-type lysozymes have been studied in zhikong scallop yet (jielian et al., 2017). in the present study, a novel i-type lysozyme, cflyzi, was identified and characterized from c. farreri. its molecular feathers, tissue and temporal expression patterns and potential function were also investigated. bioinformatics analysis revealed that cflyzi contained a typical destabilase domain as the same as previously identified i-type lysozymes, and exhibited high similarity with its invertebrate counterparts. two specific motifs were revealed, which was as same as the observation in razor clam sinonovacula constricta and manila clams venerupis (ruditapes) philippinarum (zhao et al., 2010; chen et al., 2018). moreover, similar with its counterpart from s. constricta, the deduced protein sequence of cflyzi contained high amount of cysteine residues (8.59%, 20 among 233 residues), contributing to its stability in the high osmolality seawater (chen et al., 2018). its sequence characteristics, high similarities with other previously identified i-type lysozymes collectively suggested that cflyzi is a novel member of i-type lysozymes and may have similar functions with its homologues from other invertebrates. the i-type lysozyme functions as the essential defender against invading microbes, and its mrna transcripts have been reported to be ubiquitously found in various tissues in marine invertebrates (jielian et al., 2017). in the present study, the cflyzi mrna transcripts were detectable in all the investigated tissues and such ubiquity indicated that it would process many important physiological functions, especially as the first barrier against invading microbes in innate immunity. the peak level of cflyzi mrna expression was found in hemocytes, followed by gill, and the variable tissue distribution of cflyzi mrna transcripts would be related with tissue function. mollusk hemocytes have been reported to be responsible for bactericidal activity by mediating numerous toxic compounds, such as lysozyme, lysosomal enzymes, nitric oxide, phenol oxidase and superoxide (li et al., 2008). gill was reported to be the first line against invading microbes in fish or lower animals, and a recent report demonstrated that tubules of gill filaments might be one of the potential hematopoietic positions in mollusk (li et al., 2017a). the abundance of cflyzi mrna in hemocytes and gill implied its pivotal roles in the innate immunity of scallops. hemocytes are the major immune cells and respond to invading microbes mainly via phagocytosis in mollusks, and this tissue is usually selected to investigate the fluctuation of immune related genes (canesi et al., 2002). in the present study, hemocytes were also selected to test the temporal expression patterns of cflyzi post vibrio stimulation and during temperature stress. it has been reported that the mrna scripts of i-type lysozyme could be induced by various invading microbes (jielian et al., 2017). after the vibrio stimulation, the expression levels of cflyzi mrna in hemocytes sharply increased and reached the peak at 12 h, which was consist with previous observation on other mollusks species (chen et al., 2018). hence, cflyzi could participate in innate immunity via acting as an important innate immune effector to kill or eliminate invading microbes. additionally, the mrna expression of cflyzi were significantly depressed at 10 °c and 30 °c, and high temperature could inhibit the mrna expression of cflyzi more efficiently, compared with cold shock. such susceptible to temperature, especially to heat shock, might provide valuable insights and potential clue to a possible treatment for large scale mortalities of cultured scallops in summer. as a major pathogen, the gram negative bacteria vibrio was believed to cause mass mortality of cultured scallop (zhao et al., 2007a). a previous study revealed that snps of cflyzg was speculated to be associated with the resistance or susceptibility of c. farreri to vibrio (zhao et al., 2007b; li et al., 2009; 2013). in the present study, the role of cflyzi against vibrio infection in scallop has been evaluated via vibrio stimulation and rnai technique. after vibrio stimulation, the cumulative mortality of cflyzi-suppressed scallops significantly increased and the semi-lethal time for cflyzi-suppressed scallops significantly shortened. these results suggested that the cflyzi was involved in innate immunity and essential for hosts’ survival during vibrio stimulation in c. farreri. in conclusion, the complete cdna sequence of a novel i-type lysozyme was identified and characterized in c. farreri. its mrna transcripts could be significantly induced by vibrio stimulation. the cflyzi-suppressed scallops turn to be more susceptible to vibrio. all these results indicated that cflyzi was an efficient effector involved in the innate immunity and also essential for hosts’ survival during vibrio stimulation in c. farreri. acknowledgements this research was supported by the national natural science foundation of china (u1706209), aoshan innovation project in science and technology from polit national laboratory for marine science and technology (2016askj07), science and technology service network plan (sts) major deployment project (kfzd-sw-106) and sts regional centre project (fujian province) from chinese academy of sciences. we are grateful to dr. 292 zhao lv, institute of oceanology, chinese academy of sciences, for his kindly 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abstract gibberellic acid (ga3) is usually used as a plant growth regulator to enhance the quality and quantity of crop yield. moreover, ga3 may also affect insects and other herbivores that rely on a particular plant for their living. hence, in the present study the defensive mechanisms of helicoverpa armigera (hübner) fed on the diet contating ga3 have been considered. the larvae fed with ga3 significantly exhibited cellular and humoral inhibitory responses such as, reduced nodule formation and phenoloxidase activity. antioxidant system was also affected; the lowest activity of peroxidase, catalase, superoxide dismutase, ascorbate peroxidise, glucose-6-phosphate dehydrogenase, and glutathione s-transferases was observed in control larvae while the highest activity was found in those larvae that were provided with a diet containing 800 µg/g ga3. the metabolites like triglyceride, glycogen, and cholesterol were reduced compared to the control. it is concluded that the use of plant growth regulators like ga3 not only does benefit plants for their growth and yield, but also can somehow help plants to withstand the impact of herbivores. hence, studies covering direct field collected insects from crop plants treated with pgrs would be beneficial for further studies. key words: cellular immunity; detoxifying enzymes; oxidative stress; phenoloxidase introduction helicoverpa armigera (hübner) (lepidoptera: noctuidae), is a cosmopolitan insect pest of various crops such as tomato, soybean, pea, cowpea, and chickpea (sharma, 2005). gibberellic acid (ga3) as a plant growth regulator is being used in certain crops to improve the quality and quantity of the yield. for example, tomato crop (gelmesa et al., 2012; ning and subroto, 2018), soybean, cowpea (leite et al., 2003; emongor, 2007), chickpea, and pea (bora and sarma, 2006; mazid, 2014). in addition, the adverse impacts of ga3 use on insects have also been worked out in some studies. for example, a reduction in fecundity and fertility and shortening of the longevity has been reported in bactrocera cucurbitae (coquillett) (diptera: tephritidae) (kaur and rup, 2002; kaur and rup, 2003a). abdellaoui et al. (2009) demonstrated that the use of ga3 in the diet led to higher mortality in spodoptera littoralis f. (lepidoptera, noctuidae) and also in locusta migratoria l. (orthoptera, acrididae). ___________________________________________________________________________ corresponding author: jalal jalali sendi department of plant protection faculty of agricultural sciences university of guilan rasht, iran e-mail: jjalali@guilan.ac.ir the effects were particularly evident in discrepancy occurred in exuviations. the damages were also traced in target tissues of foregut and gastric caeca of l. migratoria (abdellaoui et al., 2009). a decrease in most of the stored energy by ga3 has been reported in the hemolymph of galleria mellonella l. (lepidoptera: pyralidae) (uckan et al., 2011). altuntaş et al. (2014) showed undulating titre in haemolymph free amino acids in g. mellonella larvae with different concentrations of ga3 used. gibberellic acid also significantly reduced proteins, carbohydrates, and lipids in the ovaries of l. migratoria, as well as the amounts of ecdysteroid in ovaries and freshly laid eggs. in addition, ga3 significantly reduced both fecundity and fertility in l. migratoria (abdellaoui et al., 2015). to the best of our knowledge, there is no report so far considering ga3 on h. armigera and specifically looking into the mode of action of this beneficial compound. therefore, various aspects of its effect on h. armigera were investigated including life table parameters (shayegan et al., in press) and digestion (shayegan et al., in press), and here we report its effect on immunity and antioxidant system which has remained unexplored in the studied insects. immune responses in insects under the effect of various stresses including xenobiotics are depicted 49 fig. 1 the total hemocyte numbers in the sixth instar h. armigera larvae fed on control and different concentrations of gibberellic acid (c= 0 µg/g diet c800 = 800 µg/g diet). statistical differences have been done within each treatments and marked by various letters (at p< 0.05, tukey’s test) in the of form cellular and humoral responses. cellular responses often involve hemocytes (particularly granulocytes and plasmatocytes) that are directly responsible for processes such as phagocytosis and nodule formation against foreign bodies entering hemolymph. the humoral responses include antimicrobial peptides and prophenoloxidase system that are responsible for eliminating the pathogens (beckage, 2011). antioxidants are compounds that inhibit oxidation and removes reactive oxygen species (ros) which can damage the cells of any organism (felton and summers, 1995; akbar et al., 2012). similarly, detoxifying enzymes like general esterases and glutathione s-transferase react against external stimulants and are also considered as a part of insect defense mechanism (grant and matsumura, 1989; hemingway and karunaratne, 1998; zibaee and bandani, 2010; valizadeh et al., 2013). there are some works which explore the effect of ga3 on insects, including l. migratoria and g. mellonella (uckan et al., 2011; altuntaş et al., 2014; abdellaoui et al., 2015). it is a common belief that plant growth regulators (pgrs) like ga3 benefit plants in terms of their growth and yield. this compound can also somehow help plants to withstand the pressures implemented through herbivores. our main aim in the present study was to explore the mechanisms indulged in reduction of insect immunity and make them susceptible to various environmental pressures. materials and methods insect rearing and experimental conditions the larvae of helicoverpa armigera (hübner) were collected from tomato farms at the university of guilan in the city of rasht in northern iran (37°16′51″n 49°34′59″e). they were individually reared on artificial diet containing cowpea (204 g), yeast (30 g), wheat germ (30 g), ascorbic acid (3.5 g), sorbic acid (1.3 g), formalin (2.7 ml), refined sunflower oil (4 ml), agar (14 g), and distilled water (600 ml) (shorey and hale, 1965). the insects were reared on this diet for two generations and the third generation was provided with the same diet including gibberellic acid (suvchem, mumbai, maharashtra, india). the concentrations included 100, 200, 400, and 800 µg/g diet (denoted as c100, c200, c400, and c800, respectively). the rearing jars included transparent plastic containers (10×5×5 cm) maintained at 26 ± 1 °c, 65 ± 5% rh, and a photoperiod of 16:8 hours (l:d). the adult moths of both sexes were released into cages made of transparent pvc (14×9×20 cm), enclosed at the top with a fine-mesh net. a cotton wool soaked in 10% honey solution was provided to the moths. the hatched larvae were directly collected from the rearing boxes using a soft brush. they were reared on artificial diet including various concentrations of ga3. the desired concentrations of ga3 were made in distilled water and then were mixed with the diet. the controls received only equal amounts of distilled water in the diet. thirty first instar larvae were individually reared on the diet containing different concentrations of ga3. they were maintained on their respective diet up to the sixth instar larval stage. similarly, the controls were provided with their own diet. totally, 150 larvae were used in these experiments. total and differential hemocyte count (thc and dhc) the hemolymph of 48 hours sixth instar larvae (in all concentrations and controls) were collected from the first abdominal proleg. for thc a neubauer 50 fig. 2 percentage of granulocytes and plasmatocytes in the sixth instar h. armigera larvae fed on control and different concentrations of gibberellic acid (c= 0 µg/g diet c800 = 800 µg/g diet). statistical differences have been done within each treatments and marked by various letters (at p< 0.05, tukey’s test) hemocytometer (hbg, germany) was used. for this purpose, 10 µl hemolymph was mixed with 290 µl of anticoagulant solution (0.017 m edta, 0.041 m citric acid, 0.098 m naoh, 0.186 m nacl, ph 4.5) (gupta,1979; amaral et al., 2010). the dhc was counted using heat fixed larvae (larvae were immersed in hot distilled water (60 °c) for five min), after drying with blotting paper, the first abdominal proleg was excised and a drop of hemolymph was released on a clean slide and a smear was made using another slide. the air-dried smears were stained with 1 to 10 diluted stock giemsa (merck, germany) and after 14 min the slides were washed by distilled water. the smears were dipped for 5 sec in saturated lithium carbonate (lico3) for differentiation of cytoplasm and nucleus and then washed again in distilled water for a few minutes, dried at room temperature and then permanent slide was prepared in canada balsam (merck, germany). the cells were identified based on the morphological characteristics observed under a microscope (leica light-microscope) (baghban et al., 2018). two hundred cells were randomly counted from four corners and central part of each slide (wu et al., 2016). totally, 800 cells from four individuals were counted and percentage of each cell type was calculated. the number of cells in controls was also simultaneously recorded. immune response assay the latex beads were used to stimulate the immune system (borges et al., 2008) of h. armigera in order to explore the interaction of gibberellic acid and its immunity response (i.e. nodulation process). therefore, the larvae fed on ga3-mixed diet and control were injected with latex beads (10 distillate latex beads with distilled water using 10 μl hamilton syringe). the thc and dhc were counted at 3, 6, 12, 24, and 48-hour time intervals post injection as the method described above. effect of latex beads on nodulation response was counted at 3, 6, 12, 24, and 48-hour time intervals post injection. for this purpose the hemolymph was collected from each larvae, then samples in four replicates were poured into the hemocytometer, and then the number of nodules were counted (franssens et al., 2006, seyedtalebi, 2017). phenoloxidase activity assay for measuring phenoloxidase activity, 10 μl of hemolymph and 90 μl of ice-cold sterile phosphate buffered saline (pbs) (0.13 m nacl, 2.68 mm kcl, 8.1 mm na2hpo4 and 1.47 mm kh2po4, ph 7.4, autoclaved) were used. the l-dopa (3,4 dihydroxyphenylalanine) (10 mm, sigma-aldrich co., usa) was used as the substrate for assaying this enzyme using the method designed by catalán et al. (2012), with some modifications. for this purpose, samples were centrifuged at 5,000 g at 4 °c for 5 min. then 50 μl of hemolymph-buffer supernatant was added to 150 μl of l-dopa. the activity measurement of phenoloxidase was read at 490 nm during the linear phase of the reaction. calculation of specific activity was based on dividing absorbance with protein content in hemolymph using a microplate reader (awareness technology inc., florida, usa) (khosravi et al., 2014; baghban et al., 2018). protein contents were quantified by the method of lowry (1951) (using the manufacturer’s procedure, biochem co., iran). to compute the specific activity of all enzymes, the absorbance values were divided by protein content. 51 fig. 3 the effects of different concentrations of gibberellic acid (c= 0 µg/g diet c800 = 800 µg/g diet) on total hemocyte numbers in the sixth instar h. armigera larvae after injection of latex beads. statistical differences have been done within each time intervals and marked by various letters (at p< 0.05, tukey’s test) the antioxidant defense assays sample preparation nine larvae for every treated diet were randomly selected and the whole larvae were homogenized by a hand homogenizer in pbs (10 mm). then, the homogenate was centrifuged at 28.600xg for 20 min at 4 °c. supernatants were used to determine enzymes activities of h. armigera larvae. protein contents were quantified by the method of lowry (1951) (using the manufacturer’s procedure, biochem co., iran). to obtain the specific activity of all enzymes, the absorbance values were divided by protein content. peroxidase assay the method described by addy and goodman (1972) was used for determination of the activity of this enzyme. hence, 3 ml of pyrogallol buffer (0.05 m pyrogallol was mixed with 0.1 m pbs (ph 7.0)) were mixed with 500 μl of 1% h2o2. then, 50 µl of sample was added. the absorbance was recorded every 30 sec for two min at 430 nm. finally, the activity was calculated by an extinction coefficient of oxidized pyrogallol (4.5 liters/mol). superoxide dismutase assay according to the method of mccord and fridovich (1969), 80 µl of samples were added to 500 µl of reaction solution (70 µm of nitroblue tetrazolium and 125 µm of xanthine dissolved in pbs, and were mixed with 20 µl of xanthine oxidase solution containing 10 mg of bovine serum albumin). then, 100 µl of xanthine oxidase (5.87 u/ml) (dissolved in 2 ml of pbs) was added to the reaction mixture and the reaction was initiated in darkness at 28 °c for 20 min. absorbance was read at 560 nm. catalase assay catalase assay was performed following the method of wang et al. (2001) with slight modification. the reaction mixture containing 50 µl of the sample and 500 µl of hydrogen peroxide (1%) was incubated at 28 °c for 10 min. the absorbance was read at 240 nm. ascorbate peroxidase assay for ascorbate peroxidase assay, 750 μl of sample was added to 250 µl pbs (67 mm, ph 7). then, 70 µl of ascorbic acid (2.5 mm) and 200 µl of hydrogen peroxide (30 mm) were added to the reaction mixture. the absorbance was read at 290 nm for 5 min (asada, 1984). glucose-6-phosphate dehydrogenase assay according to the method of balinsky and bernstein (1963), 100 μl of tris-hcl (2-amino-2(hydroxymethyl) propane-1,3-diol; hydrochloride) (100 mm, ph 8.2) was mixed with 30 μl of mgcl2 (0.1 m), 50 μl of nadp (0.2 mm), 50 μl of water, 50 μl of the sample, and 100 μl of glucose-6phosphate dehydrogenase (6 mm). the absorbance of the reaction mixture was read at 340 nm. the detoxifying enzymes assay sample preparation nine larvae for every treated diet were randomly selected and the whole larvae were homogenized by a hand homogenizer in pbs (10 mm) and the homogenate was centrifuged at 13000 rpm for 20 min at 4 °c. supernatants were used for determining enzymes activities of h. armigera larvae. protein contents were quantified by the method of lowry (1951) (using the manufacturer’s procedure, biochem co., iran). to compute the 52 fig. 4 the effects of different concentrations of gibberellic acid (c= 0 µg/g diet c800 = 800 µg/g diet) on percentage of granulocytes in the sixth instar h. armigera larvae after injection of latex beads. statistical differences have been done within each time intervals and marked by various letters (at p< 0.05, tukey’s test) specific activity of all enzymes, the absorbance values were divided by protein content. esterase activity assay esterase activity was measured by α-naphthyl acetate and β-naphthyl acetate (10 mm) as substrates, separately. twenty μl of each substrate, 50 μl fast blue rr salt (1 mm), and 20 μl of enzyme solution were mixed and incubated for 1 min and then the absorbance was recorded at 450 nm (han et al., 1998). glutathione s-transferase assay twenty μl of cdnb (1-chloro-2, 4 dinitrobenzene, 20 mm) and dcnb (1, 2-dichloro-4nitro-benzene, 40 mm) were separately added to 10 μl of the sample and 1 mm glutathione. the gst activity was read at 340 nm/min/mg protein after 5 min (oppenoorth et al., 1979). amount of protein, glycogen, triglyceride, cholesterol, and glucose protein protein contents of the treated-larvae and control were assayed according to the method of lowry et al. (1951) (recommended by biochem co., iran). glycogen for glycogen assay, the fat body of the control and treated-larvae were plunged in tubes contained 1ml of lysis buffer (30% koh with na2so4), at first. then the samples of tubes were boiled for 20-30 min. then the tubes were shaken and cooled in ice. for precipitation of glycogen from the samples, 2 ml of 95% etoh was added and then was shaken again and cooled in ice for 30 min. then the samples were centrifuged done at 22,000 g for 30 min. the pellets (containing glycogen) were transported to new tubes and 1 ml of distilled water was added to the tubes and was shaken. the samples were mixed with 5% phenol and incubated in ice bath for 30 min. the absorbance of samples was read at 492 nm. a glycogen standard curve was used for calculating the glycogen concentration (chun and yin, 1998). triglyceride a diagnostic kit (pars azmoon co., iran) was used for triglyceride assay. the method was based on the method of fossati and prencipe (1982), the reaction mixture included 10 µl of sample and 70 µl of reagent containing phosphate buffer saline (50 mm, ph 7.2), 4-chlorophenol (4 mm), adenosine triphosphate (2 mm), mg2+ (15 mm), glycerokinase (0.4 ku/l), peroxidase (2 ku/l), lipoprotein lipase (2 ku/l), 4-aminoantipyrine (0.5 mm), and glycerol-3phosphate-oxidase (0.5 ku/l). the reaction was done for 15 min at 25 °c. the samples and reagent absorbance were read at 546 nm. to obtain of the amount of triglyceride, the following equation was used: mg/dl= (od of sample)/ (od of standard) × 0.01126 cholesterol cholesterol was measured based on richmond’s method (richmond, 1973) and by utilizing a total cholesterol assay kit (pars azmoon co., iran). this method is based on hydrolyzing cholesterol esters with cholesterol oxidase, cholesterol esterase and peroxidase. 53 fig. 5 the effects of different concentrations of gibberellic acid (c= 0 µg/g diet c800 = 800 µg/g diet) on percentage of plasmatocytes in the sixth instar h. armigera larvae after injection of latex beads. the statistical differences have been done within each time intervals and marked by various letters (at p< 0.05, tukey’s test) glucose glucose assay was performed by a diagnostic kit (pars azmoon co., tehran, iran) based on the procedure of siegert (1987). whole body (100 µl) added to 500 µl of 0.3 n perchloric acid and centrifuged at 12000g for 10 min. for determination of glucose concentrations, the supernatants were used. statistical analysis the data obtained are presented as means ± standard error. all data were tested for normality (kolmogorov-smirnov test) and homogeneity of variance (bartlett's test). the data were compared by one-way analysis of variance (anova). differences between the treatments were determined by tukey’s family error rate by minitab® statistical software. the statistical differences were considered at a probability less than 5%. results the reaction of cellular immunity of h. armigera against ga3 effects of ga3 on total hemocyte count (thc) are shown in figure 1 (f= 14.98; df= 4, 19; p< 0.05). as depicted in figure 1 the lowest thc are observed in those larvae that were reared on c800 diet. differential hemocyte counts (dhc) in the highest concentration of ga3 resulted in significant reduction of granulocytes (fig. 2) (f= 11.47; df= 4, 19; p< 0.05). this reduction was also evident (56.4% and 57.2%) for c400 and c800 diets, respectively. however, amount of 67.2% was recorded for granulocytes in control. percentage of plasmatocytes in any of the treatments did not significantly alter (fig. 2). figure 3 demonstrates the effects of ga3-diet treated diet on thc following injection of latex beads. a significant decrease in thc was detected six hours (f= 13.62; df= 4, 19; p< 0.05) and 12 hours (f= 32.01; df, 19= 4; p< 0.05) after injection of latex beads to the larvae fed on c400 and c800 diets, while there were no significant changes in these treatments at 3, 24, and 48 h after injection. percentage of granulocytes in c200, c400 and, c800 treatments did not change significantly after three hours, but significantly decreased after six hours (fig. 4) (f= 57.69; df= 4, 19; p< 0.05). percentage of these cells was lower than control in all treatments after 12 hours (f= 49.79; df= 4, 19; p< 0.05), while after 24 hours the percentage of cells was significantly lower only in c200 and c800 treatments (f= 4.86; df= 4, 19; p< 0.05). after 48 hours, the percentage of granulocytes did not change significantly in different treatments (fig. 4). on the other hand, after the latex beads infusion into larvae reared on different ga3 treatments, significant changes were observed in the percentage of plasmatocytes (fig. 5). after three hours, all treatments except c800 showed no significant differences with the control (f= 3.99; df= 4, 19; p< 0.05), while after six hours, the control showed lower levels than treatments above c100 (f= 5.34; df= 4, 19; p< 0.05). twelve hours after the latex beads injection, the percentage of plasmatocytes in the control larvae was significantly higher than those in the larvae fed with ga3 diet (f= 6.28; df= 4, 19; p< 0.05). after 24 and 48 hours, these cells were lower only in c400 and c800 treatments (f= 9.23; df= 4, 19; p< 0.05 and f= 39.72; df= 4, 19; p< 0.05, respectively). 54 fig. 6 the effects of different concentrations of gibberellic acid (c= 0 µg/g diet c800 = 800 µg/g diet) on nodule formation in the sixth instar helicoverpa armigera larvae after injection of latex beads. statistical differences have been done within each time intervals and marked by various letters (at p< 0.05, tukey’s test) the larvae fed on the diet containing ga3 significantly inhibited the nodule formation after injection of latex beads (fig. 6). in all time intervals except for 48 hours, the number of nodules in the larvae fed by c400 and c800 diet was lower than control (fig. 6). in all treatments, 24 and 48 hours after injection the phenoloxidase activity was enhanced. the larvae feeding on the highest concentration of ga3 (c400 and c800) demonstrated the lowest phenoloxidase activity in all time intervals (fig. 7). antioxidant systems of h. armigera against ga3 feeding on the diet containing ga3 caused different effects on antioxidant enzymes of h. armigera (table 1). the lowest activity of peroxidase and catalase was observed in the control larvae (0.063 and 0.794 u/ mg protein, respectively), while the highest activity was found in those larvae feeding on c800 diet (0.120 and 1.892 u/ mg protein respectively) (f= 26.48; df= 4, 44; p< 0.05 and f= 14.08; df= 4, 44; p< 0.05, respectively). the activity of superoxide dismutase, ascorbate peroxidase and glucose-6-phosphate dehydrogenase was the lowest in the control larvae, but it increased in ga3-diet fed larvae in a dosedependent manner (f= 148.03; df= 4, 44; p< 0.05, f= 615.48; df= 4, 44; p< 0.05 and f= 36.23; df= 4, 44; p< 0.05, respectively) (table 1). the reaction of the detoxifying enzymes of h. armigera against ga3 the larvae bred on higher concentrations of ga3 depicted a statistically significant raise in esterase activities only when β naphtyl acetate was used as a substrate (f= 40.06; df= 4, 44; p< 0.05) (table 2). in the case of glutathione s-transferase, enzymatic activities of larvae fed on c800 diet were significantly higher than those of control. effect of ga3 on protein, glycogen, triglyceride, cholesterol, and glucose content of h. armigera effect of ga3 on macromolecules in the body of h. armigera larvae are shown in table 3. no significant differences were found in the amount of protein and glucose, while the lowest amount of triglyceride was observed in larvae fed on ga3 diet compared to the control (f= 32.40; df= 4, 44; p< 0.05). the larvae fed on c800 had the lowest amount of glycogen and cholesterol (f= 11.21; df= 4, 44; p< 0.05 and f= 43.48; df= 4, 44; p< 0.05, respectively). discussion exogenous compounds could affect the number of circulating hemocytes (perez and fontanetti, 2011). our results indicate that feeding on diet containing gibberellic acid affect the number of circulating hemocytes, especially granulocytes. it is inferred from the reducing number of circulating hemocytes, that the hemocytes might have migrated to damaged tissues in order to phagocytize the remains of the damaged tissues (pipe and coles, 1995). we have noticed damages to the midgut under the effect of ga3 feeding (unpublished data). reports by abdellaoui et al. (2009; 2013) also reported damages implicated on the digestive system, which support our own results. the thc and 55 fig. 7 the effects of different concentrations of gibberellic acid (c= 0 µg/g diet c800 = 800 µg/g diet) on the phenoloxidase activity in the sixth instar h. armigera larvae after injection of latex beads. statistical differences have been done within each time intervals and marked by various letters (at p< 0.05, tukey’s test) dhc (granulocytes and plasmatocytes) reduction have been shown in g. mellonella under the effect of abscisic acid, another pgr (er and keskin, 2016). paradoxically, there is a report showing an increased thc in g. mellonella larvae fed on diet treated with gibberellic acid. however, dhc in granulocytes and plasmatocytes did not change significantly (altuntaş et al., 2012). insects show cellular immune defense by phagocytosis, encapsulation, or nodulation against invaders (er and keskin, 2016). in order to confirm the hypothesis that ga3 weakens the immune system, we undertook the experiment of injecting latex beads. a trend of reducing hemocytes confirms that the immunity of our insect has been affected by feeding on ga3 treated-diet. the decrease in insect's ability to confront foreign bodies is similar to the reports by altuntas et al. (2012) when using ga3 in the diet of g. mellonella. the mode of action of ga3 is rather variable and one of the possible modes could be that this hormone might act the way juvenile hormone (jh) does to the insect (kaur and rup, 2002; abdellaoui et al., 2015). structural similarity of pgrs to insect indigenous jh might reflect similar mode of action. several studies have shown that juvenile hormone and their analogues reduce the ability of insect larvae to form nodules by inhibiting cell proliferation (rantala et al., 2003; franssens et al., 2006; zibaee et al., 2012). table 1 antioxidant enzyme activities of sixth instar h. armigera larvae provided with different concentrations of gibberellic acid in their diet enzymes specific activity of enzymes (u/ mg protein) c c100 c200 c400 c800 peroxidase 0.063±0.003 c 0.097±0.004 b 0.111±0.002 a,b 0.108±0.002 a,b 0.120±0.006 a superoxide dismutase 0.073±0.002 d 0.292±0.033 c 0.408±0.011 b 0.430±0.005 b 0.606±0.003 a catalase 0.794±0.140 c 0.965±0.115 b,c 1.643±0.119 a 1.366±0.089 a,b 1.892±0.136 a ascorbate peroxidase 0.232±0.014 d 0.732±0.038 c 1.207±0.018 b 1.234±0.032 b 1.944±0.013 a glucose-6-phosphate dehydrogenase 0.199±0.003 d 0.269±0.004 c 0.366±0.010 b 0.383±0.003 b 0.502±0.021 a means with the same letters in a row are not significantly different at p < 0.05 (tukey’s test) 56 table 2 detoxifying enzyme activities of sixth instar h. armigera larvae provided with different concentrations of gibberellic acid in their diet enzymes enzyme activity (od/ min) c c100 c200 c400 c800 general esterases (α-naphtyl acetate) 0.054±0.007 a 0.053±0.001 a 0.055±0.002 a 0.055±0.005 a 0.056±0.004 a general esterases (β-naphtyl acetate) 0.077±0.004 c 0.101±0.007 b 0.095±0.012 b,c 0.127±0.004 a 0.138±0.001 a glutathione s-transferases (cdnb) 0.163±0.008 c 0.173±0.013 c 0.193±0.020 c 0.303±0.012 b 0.450±0.017 a glutathione s-transferases (dcnb) 0.270±0.017 b 0.320±0.028 b 0.486±0.044 b 0.486±0.046 b 1.273±0.093 a the activity of glutathione s-transferase using cdnb (1-chloro-2, 4dinitrobenzene) and dcnb (1, 2-dichloro-4nitro-benzene) as reagents and the activity of general esterases using α-naphtyl acetate and β-naphtyl acetate as substrates. means with the same letters in a row are not significantly different at p < 0.05 (tukey’s test) humoral immunity in insects is considered as complementary to cellular immunity by production of antimicrobial peptides and phenoloxidases (lavine and strand, 2002; beckage, 2011). the phenoloxidases are also a complementary part of cellular immunity in the last stage of encapsulation and nodulation processes, forming melanisation of both. the precursors of phenoloxidases are the prophenoloxidases which are maintained in hemocytes (mostly oenocytoids) (lavine and strand, 2002; beckage, 2011). we assume that the reduced activity of phenoloxidases might be due to reduction in cell number. although while counting dhc we did not count the oenocytoids and instead focused on immunocytes (i.e. granulocytes and plasmatocytes). however, thc reduction gives a clear picture of what was expected under the impression of ga3. the similarity in structure of ga3 to insect jh brings it to the mind that the inhibition of phenoloxidase activity is the same as what jh does. the jh has been shown to have inhibitory effect on phenoloxidase activity (hiruma and riddiford, 1988; rolff and siva-jothy, 2002; rantala et al., 2003). the antioxidant system enables the insect to inhibit oxidation reactions that produce free radicals damaging the cells (dkhil et al., 2015). increasing activity of peroxidase, catalase, superoxide dismutase, ascorbate peroxidise, and glucose-6phosphate dehydrogenase can be explained since this insect has faced exogenous stress, here ga3 (lyakhovich et al., 2006, altuntas, 2015; sezer and ozalp, 2015). glutathione s-transferase is a detoxifying and antioxidant enzyme that eliminates lipid peroxidation products or hydroperoxides of cells (dubovskiy et al., 2008). this enzyme also plays an important role in detoxification by increasing the solubility of toxic substances (grant and matsumura, 1989). esterases are important detoxifying enzymes which hydrolyse esoteric bonds in synthetic chemicals (hemingway and karunaratne, 1998). increasing activity of glutathione s-transferases and esterases is an important indication of the unpleasant effects of ga3 on the insect. mirhaghparast et al. (2016) reported increased activity of these two enzymes in c. suppressalis larvae under the influence of pyriproxyfen. the detoxifying enzymes and oxidative defense system work in coordination to increase adaptation of insects by deactivating and eliminating harmful compounds (felton and summers, 1995; hemingway and karunaratne, 1998; büyükgüzel et al., 2010; aslanturk et al., 2011). in assessing the effect of gibberellic acid on protein and glucose in the insect body no significant changes were observed. contrary to our report uckan et al. (2011) showed higher protein levels in all concentrations (50 to 2000 ppm) of ga3 in the larvae of g. mellonella. however, hemolymph free amino acids in g. mellonella larvae after using ga3 undulated among dosages (altuntaş et al., 2014). also, carbohydrate levels of larval g. mellonella decreased significantly after ga3 use. moreover, the amount of glycogen in zaprionus paravittiger (godbole and vaidya) (diptera: drosophilidae) and b. cucurbitae was reduced after using gibberellic acid (rup et al., 1998; kaur and rup, 2003b). triglycerides and cholesterol reduction was similar to what has been reported in other insects (rup et al., 1998; uckan et al., 2008). the lower level of total lipid, as well as triglyceride lipid biosynthesis in the experimental larvae, emphasizes the need of this macromolecule for energy demands due to induced stress by ga3. furthermore, the reactive oxygen species also cause cell death, mutation, and even death through degradation on macromolecules such as lipids (ryter et al., 2007). conclusion the pgrs can be used to increase the crop yield quality and quantity. however, we should be cautious about the interaction between the ga3, 57 table 3 amount of protein, glycogen, triglyceride, cholesterol, and glucose in sixth instar h. armigera larvae provided with different concentrations of gibberellic acid in their diet macromolecules (mg/ml) treatments c c100 c200 c400 c800 protein 3.560±0.061 a 3.681±0.046 a 3.656±0.070 a 3.808±0.066 a 3.712±0.063 a glycogen 0.251±0.004 a,b 0.242±0.007 a,b 0.232±0.012 b,c 0.270±0.004 a 0.207±0.001 c triglyceride 0.171±0.001 a 0.166±0.001 b 0.166±0.005 b 0.158±0.009 c 0.154±0.001 c cholesterol 1.260±0.018 b 1.454±0.048 a 1.107±0.047 b,c 0.990±0.043 c 0.763±0.032 d glucose 0.172±0.003 a 0.165±0.002 a 0.171±0.002 a 0.165±0.002 a 0.163±0.001 a means with the same letters in a row are not significantly different at p < 0.05 (tukey’s test) insect, and the crop plant. in the present and previous reports we demonstrated this effect which aimed at digestion, reproduction, and now the immunity. further studies concerning insects collected directly from treated and untreated crops would help for planning on the use of various pgrs. acknowledgements the authors express their sincere gratitude to the vice chancellor in research affairs, university of guilan for providing financial in the form of grant (1987). references abdellaoui k, halima-kamel mb, fatma a, soltani n, aribi n, hamouda mhb. effects of gibberellic acid on ovarian biochemical composition and ecdysteroid amounts in the migratory locust locusta migratoria (orthoptera, acrididae). int. j. pest manag. 61: 68-72, 2015. abdellaoui k, halima-kamel mb, hamouda mhb. insecticidal activity of gibberellic acid against spodoptera littoralis (lepidoptera, noctuidae) and locusta migratoria (orthoptera, acrididae). pest technol. 3: 28-33, 2009. abdellaoui k, halima-kamel mb, soltani n, aribi n, hamouda mhb. biochemical and histological effects of gibberellic acid on locusta migratoria migratoria fifth instar larvae. pestic. biochem. physiol. 107: 32-37, 2013. addy sk, goodman rn. polyphenol oxidase and peroxidase activity in apple leaves inocultaed with a virulent or an avirulent starin of erwinia amylovora. indian phytopathol. 25: 575-579, 1972. akbar smd, sharma hc, jayalakshmi sk, sreeramulu k. effect of pyrethroids, permethrin and fenvalarate, on the oxidative stress of helicoverpa armigera. world j. sci. technol. 2: 1-5, 2012. altuntaş h, kiliç a, uckan f, ergin e. effects of gibberellic acid on hemocytes of galleria mellonella l.(lepidoptera: pyralidae). environ. entomol. 41: 688-696, 2012. altuntaş h, uckan f, kiliç ay, ergin e. effects of gibberellic acid on hemolymph-free amino acids of galleria mellonella (lepidoptera: pyralidae) and endoparasitoid pimpla turionellae (hymenoptera: ichneumonidae). ann. entomol. soc. am. 107: 1000-1009, 2014. altuntaş h. determination of gibberellic acid (ga 3)-induced oxidative stress in a model organism galleria mellonella l. 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(astracea) extracts the sunn pest, eurygaster integriceps puton (heteroptera: scutelleridae). j. plant prot. res. 50: 48-54, 2010. riboflavin content of coelomocytes in earthworm (dendrodrilus rubidus) field populations as a molecular biomarker of soil metal pollution isj 13: 315-325, 2016 issn 1824-307x minireview unexpected results and open questions from experiments on regeneration in lumbricid worms b plytycz1, j bigaj1, a falniowski2, aj morgan 3 1department of evolutionary immunology, institute of zoology, jagiellonian university, krakow, poland 2department of malacology, institute of zoology, jagiellonian university, krakow, poland 3cardiff school of biosciences, main building, cardiff university, cardiff cf10 3us, wales, uk accepted september 20, 2016 abstract lumbricid worms are commonly subjected to noxious stimuli leading to expulsion of celomic fluid or to loss of body segments; therefore regeneration of lost segments and restoration of the depleted cellular and soluble components of celomic fluid are of fundamental importance for these animals. series of experiments was performed on regeneration abilities in well-defined epigeic species eisenia andrei, e. fetida and dendrobaena veneta, and endogeic aporrectodea caliginosa. efficient regeneration of the lost anterior or posterior segments was consistently observed in eisenia sp. in a sharp contrast, d. veneta regenerated amputated anterior segments or extirpated suprapharyngeal ganglia (‘brains’) while regeneration of posterior segments was never recorded so far in this species. in a. caliginosa a loss of posterior segments was followed either by compensatory body growth or by formation of regeneration blastema. in all species regeneration was cold-inhibited while was resistant to cadmium soil pollution. the efficiency of regenerative processes in e. andrei and e. fetida might be connected with quality and quantity of some components of their celomic fluid; lysenin is unique for these species and riboflavin is much more abundant in eisenia sp. than in other lumbricids investigated so far. key words: earthworms; autotomy; regeneration; celomocytes; amebocytes; riboflavin   introduction lumbricid worms are commonly subjected to sub-lethal predator attacks leading to loss of body segments. body segments may be lost also by selfamputation called autotomy (zoran, 2010). earthworms may be subjected to mechanical/chemical stimuli inducing spasmic body movements connected with expulsion of celomic fluid containing the celomocytes and soluble factors crucial for immunity (bilej et al., 2011; ottaviani, 2011). therefore regeneration of lost segments or restorations of the extruded cellular and soluble components of celomic fluid are of fundamental importance for these animals. segmental body organization coupled with easy noninvasive celomic fluid retrieval make them attractive models for qualitative and quantitative interspecies comparisons of regeneration capabilities (bely et al., 2006; bely and sikes, 2010; zoran, 2010). ___________________________________________________________________________ corresponding author: barbara plytycz department of evolutionary immunology institute of zoology jagiellonian university gronostajowa 9, 30-387 krakow, poland 315 e-mail: barbara.plytycz@uj.edu.pl we have investigated the effects of some endogenous and exogenous factors on the restoration of certain depleted components of celomic fluid as well as the regeneration of the lost body segments in three epigeic lumbricid species, dendrobaena veneta (dv), eisenia andrei (ea), and e. fetida (ef) delimitated by barcoding of the mitochondrial cytochrome c oxidase subunit i (coi) gene and supplemented in some specimens by mug/mug-like fluorophore (4-methylumbelliferyl βd-glucuronide) characteristics (fig. 1), by the methods described previously (rorat et al., 2014). we have examined two distinct cohorts of e. fetida: one that expresses (efm+) and one that does not express (efm-) a mug-like fluorophore. these three composting species are easy to maintain and culture under laboratory conditions. in addition, we investigated a fourth species, the endogeic aporrectodea caliginosa (ac), which normally inhabits mineralized soil and is not so easy to maintain. depletion of celomic fluid was achieved by electrostimulation-induced expulsion (e.g., rorat et al., 2014); loss of posterior segments was achieved either by surgical amputation or experimentally mailto:barbara.plytycz@uj.edu.pl 316 fig. 1 phylogenetic tree based on sequences of coi gene with clades corresponding to specimens of eisenia andrei (ea), eisenia fetida (ef) (some of them identified as efm+ or efm-), dendrobaena veneta (dv), and aporrectodea caliginosa (ac) delivered from laboratory cultures from france (lille l); hungary (pecs p), and poland (kluczbork k; slupsk s; jasienczyk j; kleczany ky). maximum likelihood tree, computed under tamura 3-parameter with invariable sites, bootstrap supports (10,000 replicates) given if > 50 %. coi sequences 654 bp long were analysed; most of them are already published (santocki et al., 2016a: genbank accession numbers km823553-km823574; swiderska et al., 2016: genbank accession numbers kx671534-kx671544); new sequences marked by asterisks are deposited in genbank (accession numbers kx781363-kx781391). 317 table 1 celomocyte restoration characteristics and regeneration of anterior and posterior segments in four lumbricid species: eisenia andrei (ea); e. fetida (efm+, efm-); dendrobaena veneta (dv); a. caliginosa (ac) lumbricid species epigeic, composting endogeic ef investigated factors ea efm+ efm dv ac anterior regeneration (y/?) y y y y ? lo st bo dy se gm en ts posterior regeneration with blastema formation (y/n) y y y n n/y amebocytes +++ f +++ f +++ f +++ f +++ f celomocytes autofluorescent eleocytes +++ s +++ s +++ s +++ s -- riboflavin ++++ m ++++ m ++++ m + m -+ fluorophores mug ++++ f ++ f ------ r es to ra tio n of ce lo m ic flu id c om po ne nt s hemolytic factors lysenin family proteins +++ f/s +++ f/s +++ f/s ---- presence/absence (+/-) of investigated factors and their restoration/regeneration abilities/rates considered as present/lacking (y/n), and fast (f), moderate (m) or slow (s). note that restoration of cellular and soluble components of celomic fluid and segment regeneration in eisenia spp. are relatively resistant to soil pollution, and regenerative processes in all species are inhibited at low ambient temperatures (for detail see text). induced self-amputation (autotomy) (see galuszka et al., 2015; kocinski et al., 2016); anterior braincontaining segments were either amputated or only cerebral ganglia were surgically extirpated (okrzesik et al., 2013; molnar et al., 2015). here we review the main results from the published and unpublished experiments. a summary of our findings is presented in table 1. restoration of some components of celomic fluid after experimental expulsion the investigated composting species, ea, ef, and dv, belong to lumbricids possessing two main populations of cells floating in celomic fluid, i.e., the immunocompetent amebocytes and autofluorescent chloragocyte-derived granular eleocytes (fig. 2a), the latter containing various nutritive substances, e.g., riboflavin (see plytycz and morgan, 2011). in contrast, ac is an example of lumbricid species possessing in celomic fluid almost exclusively the amebocytes. riboflavin is stored in high or moderate quantities in eleocytes of eisenia sp. and dv, respectively (rorat et al., 2014; santocki et al., 2014, 2016a), but is also present in the attached chloragocytes residing on the celomic surface of the alimentary canal of all investigated lumbricid species (mazur et al., 2013). controlled expulsion of celomic fluid through dorsal pores of the earthworms may be induced by various irritants like 5 % ethanol (cooper et al., 1995), ultrasounds (hendawi et al., 2004) or mild electric current (roch, 1979), the latter procedure modified and used in present studies (see gałuszka et al., 2015; kociński et al., 2016). drastic depletion of morphotic elements is followed by several weeklasting recovery process, originally described in lumbricus terrestris devoid of eleocytes (eyambe et al., 1991). in ac, another lumbricid species devoid of chloragocyte-derived eleocytes, restoration of amebocytes was even faster in cd-polluted soil than in the clean soil; in this species restoration was temperature-dependent, being slower at 6 oc than at 25 oc (galuszka et al., 2015). in the composting species investigated here restoration of amebocytes was consistently faster than that of eleocytes (klimek et al., 2012; santocki et al., 2016a, b), as exemplified in ea on figures 2b, c. a few weeks after electrostimulation-induced depletion, the number of eleocytes was still very low, and they were qualitatively different than their counterparts from the untreated worms, as they were much less prone to degranulation than the old eleocytes from untreated worms (santocki et al., 2016a, b). restoration of riboflavin content was faster than the increase of eleocyte numbers (fig. 2c); newly formed eleocytes store more riboflavin in their granules than mature eleocytes (klimek et al., 2012, santocki et al., 2016a, b); moreover, riboflavin present in fluid may derive also from the attached chloragocytes of chloragogenous tissue. fig. 2 some components of celomic fluid of eisenia andrei. a) group of 5 celomocytes in fluorescent microscope (left: bright field; right: blue light), among them 2 amebocytes (a) and 3 autofluorescent eleocytes (e); b-d) restoration of the components of celomic fluid after experimental depletion induced at time 0 by electrostimulation (4.5v, 30 sec), expressed as percentages of initial values (c) considered as 100 % (modified according to santocki et al., 2016b): b) numbers of amebocytes (an); c) numbers of eleocytes (en) and riboflavin (rf) content; d) content of mug fluorophore. 318 fig. 3 effects of anterior or posterior segments amputation on reproduction of composting worms. cocoons production in eisenia andrei (ea) and e. fetida (ef) either intact control worms (c) or those subjected at time 0 to surgical amputation of either 6 anterior segments (6a) or 6 posterior segments (6p), 8 worms in each group. note that in groups (6a) of both species cocoons were absent during the first 3 weeks and their production started between 4th and 6th week after surgery, while in both species cocoon production was unaffected in worms with amputated posterior segments (6p). numbers of cocoons in boxes containing 8 worms from either (c), (6a), or (6p) groups of ea or ef, calculated per worm and per week. restoration of components of celomic fluid was more efficient in juvenile than in adult worms (santocki et al., 2016a). contrary to our working hypothesis, the kinetics of restoration of amebocytes, eleocytes and riboflavin content in juvenile ea was not affected in worms maintained in soil polluted with cadmium at concentrations inhibiting worm growths and maturation (takacs et al., 2016, submitted). also in adult ea worms, similar exposure to cadmium polluted soil inhibited cocoon production with negligible effects on restoration of the depleted components of celomic fluid (rorat et al., in preparation). putatively metallothioneins are responsible for protection of cadmium exposed celomocytes (homa et al., 2015; rorat et al., in preparation). the mug fluorophore is a molecular marker of e. andrei (albani et al., 2003), but mug-like fluorophore is also present in some specimens of e. fetida (efm+) (rorat et al., 2014; santocki et al., 2016a; swiderska et al., 2016). the initial contents of mug/mug-like fluorophores in celomic fluid of ea was less depleted after electrostimulation and restored relatively quickly (fig. 2d); much faster than restoration of riboflavin (compare figs 2c, d) (santocki et al., 2016b; swiderska et al., 2016). the biological function and cellular source/s of mug/mug-like molecules is presently unknown. are mug-positive e. fetida earthworms the interspecific hybrids between ea and efm-? experiments aimed in answering this are in progress. 319 fig. 4 various forms of regeneration of amputated or autotomized posterior segments in aporrectodea caliginosa (ac): a) delayed formation of blastema, 3 months after amputation; b) very unusual non-pigmented blastema several months after autotomy; c) worm amputated and kept in cold for 3 weeks without rudiments of blastema, and then formation of tiny blastema within 2 weeks after transfer to room temperature. lysenin, i.e., hemolytic sphingomyelin-binding pore-forming protein (roch, 1979; cooper et al., 2002 de colibus et al., 2012; bokori-brown et al., 2016) and lysenin-related proteins were detected in all investigated specimens of eisenia sp., both ea and ef (the latter both efm+ and efm-) but not in d. veneta (swiderska et al., 2016). after experimental expulsion of celomic fluid, content of lysenin was restored faster in cellular fraction containing lyseninstoring eleocytes than in soluble fraction of celomic fluid (swiderska et al., 2016). however, lysenin is still a puzzling component of defense system of eisenia sp., as the presence of its main target, sphingomyelin, seems to be restricted mainly to vertebrate cells membranes (kobayashi et al., 2000; shakor et al., 2003). in contrast, both the eisenia earthworms (mclaughlin 1971) and the main invaders of their celomic cavity, i.e., bacteria, fungi, and small invertebrates, are devoid of sphingomyelin in the cell membranes (hori and sugita, 1993), thus in the earthworms its antimicrobial function may be performed by sphingomyelin-independent mechanisms (cooper et al., 2002; bruhn et al., 2006), e.g., due to its opsonizing properties (hayashi et al., 2013). putatively another function of lysenin in eisenia sp. earthworms might be induction of avoidance behavior of the potential vertebrate predators (like birds, hedgehogs, or moles) after irritating their mucosa with lysenin-containing celomic fluid. hypothetically, we cannot exclude that lysenin might be involved in wound healing and/or regenerative processes. regeneration of extirpated brains or amputated anterior brain-containing segments surgical extirpation of suprapharyngeal ’cerebral’ ganglia (‘brains’) or amputation of brain containing anterior segments of dv caused an immediate inhibition of reproduction that was recovered in tandem with restoration of brain integrity, including active neurosecretory cells (okrzesik et al., 2013; molnar et al., 2015). restoration of reproduction, thus also regeneration of vital brain centers, was markedly more efficient in worms with intact immune system than in those subjected to electrostimulation-induced loss of amebocytes, eleocytes, and soluble components of immune system. and vice versa, restoration of components of celomic fluid was faster in intact worms than in their counterparts engaged in regenerating their extirpated brains (molnar et al., 2015). such experimental model is unquestionably amenable to further detailed examinations of interactions between earthworm neuroendocrine and immune systems (plytycz and morgan, 2015). since a reappearance of temporarily inhibited cocoon production is a convenient biomarker of regeneration of the brain morphology and functions, the present studies included monitoring reproductive activity of unmanipulated (control) adult specimens of ea and ef, the latter either efm+ and efm-, and their counterparts with surgically amputated of either six anterior (brain-containing) or six posterior segments. as in dv, cocoon production was hardly affected by amputation of posterior segments, while was completely inhibited for several weeks in all brainless worms, and then was gradually restored (fig. 3). thus, amputation of anterior segments or brain extirpation/regeneration is an attractive model for studies of neurohormonal-immune interactions in the composting species. however, to date we observe that such invasive treatment causes mortality in ac worms. species-specific reactions to loss of posterior segments in the present studies either surgical amputation of posterior segments was performed or their controlled self-amputation (autotomy) was induced. autotomy of body segments followed by their regeneration is an adaptive strategy of lumbricid worms as anti-predator strategy and the disposal of encapsulated parasites or toxic products (lesiuk and drewers, 1999). experimental controlled autotomy of posterior segments may be 320 fig. 5 effects of temperature on regeneration of amputated posterior segments in e.andrei from kluczbork (a) or pecs (b). top: photos of blastema formation (yellow arrows) 17 days after amputation of 10 posterior segments in worms a) kept at 17 oc; b) transferred after amputation to 25 oc; c) transferred after amputation to 6 oc; d) transferred after amputation for 10 days to 6 oc and then to 25 oc (photos by majos p). note the lack of blastema in worms kept in cold. bottom: numbers of regenerating segments (sn) in earthworms adapted to either 8 oc, 17 oc, or 25 oc, estimated at the start of experiments (s) on week 0 (0w) and then during 8 subsequent weeks after surgery; means ± se (n = 4 worms per group); 0 no blastema. 321 induced in worms subjected to multiply electrostimulation. the pulsating direct electric current from a 4.5 v cell phone electric charger was necessary to induce it in e. andrei, while a. caliginosa were more prone for autotomy that was evoked not only by pulsating direct current but also by direct current from battery or immersion in noxious ms-222 solution. autotomized segments in ea were quickly replaced by newly formed once, while segment regeneration, if any, was significantly delayed in ac (kocinski et al., 2016). such reproducible models of autotomy may serve for studies on molecular-genetic mechanisms underpinning autotomy and subsequent regeneration. in a. caliginosa, reaction to amputation of posterior segments was very variable, either absent or significantly delayed (fig. 4). amputation of low numbers (e.g., 6 10) of posterior segments in ac did not induce blastema formation but was followed by the compensatory body growths, while amputation of 40 segments induced significantly delayed regeneration blastema formation (fig. 4a), that was not affected by cadmium soil pollution fig. 6 effects of amputation of posterior segments in dendrobaena veneta (dv), eisenia fetida (ef, either efor ef+), and eisenia andrei (ea) kept at 23 oc. bottom part: changes of a) body weights (bw) and b) numbers of regenerating segments estimated at the start of experiments (s) on week 0 (0w) and then during 7 subsequent weeks after surgical amputation of 10 posterior segments; means ± se (n = 3 worms per group); 0 no blastema. top part: photos of dv, ea, and ef taken 3 months after amputation of posterior segments at 25th segment after clitellum. note the lack of regenerating segments in dv, and their presence in ea and ef (yellow arrows).  322 323 (galuszka et al., 2015). in one instance we observed a long regeneration non-pigmented ‘tail’ in ac (fig. 4b). in several small specimens of ac amputated and kept for 3 months at 6 oc blastemas were absent but very thin “fragile” blastemas appeared soon after the worm transfer to 20 oc (fig. 4c). in e. andrei, amputation of body segments induced blastema formation that was rapid at 17 oc and 25 oc but was completely inhibited at low temperature (8 oc) (fig. 5, bottom). as exemplified on top of figure 5, amputation of 10 posterior segments in ea kept at 17 oc was followed by regeneration blastema formation that was faster in worms transferred to higher temperature (25 oc), while was absent in worms transferred to cold (8 oc), but that was quickly rescued after worm transfer to 25 oc (fig. 5, top). at high temperature, blastema formation was unaffected in ea kept in cadmium polluted soil (takacs et al., submitted) putatively due to protective role of metallothioneins (rorat et al., in preparation). similar cold-inhibition and cadmium resistance of blastema formation was observed in a. caliginosa (gałuszka et al., 2015). unexpectedly, we have so far been unable to observe blastema formation and regenerating posterior segments in d. veneta. figure 6 (bottom) shows that regeneration of the amputated ten posterior segments was consistently present and similar in ea and ef, the latter both ef+ and ef-, while was consistently absent in d. veneta. in this experiment initial body weights of worms were similar, but body weight gain was much faster in dv than in the both eisenia sp., thus it might be supposed that loss of a few segments only was neglected due to the fast body growth. however, when specimens of eisenia sp. and d. veneta were amputated at the 25th segment after clitellum, 4 months after surgery regenerated segments were clearly visible in ea and ef but not in dv (fig. 6, top). in conclusion, both autotomy and surgical amputation of posterior segments in ea and ef are followed by formation of regeneration blastema that is cold-inhibited but efficient at high temperatures. formation of regeneration blastema is either absent or significantly delayed or modified in ac, and consistently absent in dv. in other words, blastemainvolved epimorphic type of regeneration is common in eisenia sp., while d. veneta perform morphallactic type of regeneration. therefore comparative studies on these closely related and ecophysiologically similar species may serve as convenient models to delineate differences and similarities in the regenerative capabilities at the genetic, molecular and cellular levels (alvarado and tsonis 2006). summary and concluding remarks a summary of our main findings is presented in table 1. anterior regeneration is present in eisenia sp. and d. veneta, but head amputation was lethal for ac investigated so far. posterior regeneration with blastema formation was consistently present in eisenia sp., consistently absent in d. veneta, and absent or attenuated in ac. among depleted celomic fluid components, restoration of amebocytes was efficient in all species. autofluorescent eleocytes were present and restored slowly in eisenia sp, and d. veneta; riboflavin content was very high in eisenia sp. and lower in dv, but in both species regenerated in a moderate rate. mug/mug-like fluorophore was present and restored fast in all ea and some specimens of ef (efm+). hemolytic proteins of lysenin family are restricted to eisenia sp. why d. veneta, with a very efficient brain regeneration capability has a weak posterior segment blastema formation is an open question. is this connected with lower riboflavin content in dv eleocytes relative to the same cell type in ea and ef species? what is a source of the abundant riboflavin stores in eisenia spp.? is this connected with horizontal gene transfer as suggested earlier (sulik et al., 2012)? evidently, restoration of brain-containing anterior segments is of highest priority for all epigeic earthworms often exposed to predator attacks, but studies on endogeic ac shall be continued. loss of posterior segments may be followed by compensatory body growth and/or regeneration blastema formation. blastema formation is most efficient in eisenia sp., possessing in celomic cavity huge amounts of riboflavin and lysenin. participation of riboflavin in regeneration is well documented (johnson et al., 2012). participation of factors derived from celomic cavity of eisenia sp. in wound healing is extensively investigated (cooper et al., 2004; grdisa et al., 2004; matausijc-pils et al., 2010). at the place of wounds secreted lysenin with opsonizing properties could cover the damaged endogenous cells and aid the phagocytes to engulf them (hayashi et al., 2013). since wound healing consists of parallel processes of regeneration and scar formation (white et al., eolls), some celomic factors of eisenia species (e.g., riboflavin and/or lysenin) may act in favor of regenerative blastema formation. in other lumbricid species some external and/or endogenous factors might be necessary to elicit the latent regeneration abilities (bely and sikes, 2010). acknowledgements this work was supported by the national centre of science (b/nz4/01640, k/pbo/000178) and k/zds/005405 from the jagiellonian university. we thanks to students, gałuszka a, tengolics a, and takacs v for participation in studies and taking photos. we gratefully acknowledge the valuable comments and suggestions of anonymous reviewers. references albani jr, demuynck s, grumiaux f, leprêtre a. fluorescence fingerprints of eisenia fetida and eisenia andrei. photochem. photobiol. 78: 599602, 2003. alvarado as, tsonis pa. bridging the regeneration gap: genetic insights from diverse animal models. nature rev. genetics 7: 873-884, 2006. 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matausijć-pisl m, cupić h, kasuba v, mikecin am, grdisa m. tissue extract from eisenia foetida as a wound-healing agent. eur. rev. med. pharmacol. sci. 14: 177-184, 2010. mazur ai, klimek m, morgan aj, plytycz b. riboflavin storage in earthworm chloragocytes and chloragocyte-derived eleocytes and its putative role as chemoattractant for immunocompetent cells. pedobiologia 54s: s37-s42, 2011. mclaughlin j. biochemical studies on eisenia foetida (savigny, 1926), the brandling worm: i. tissue lipids and sterols. comp. biochem. physiol. 38b: 147-163, 1971. molnar l, pollak e, skopek z, gutt e, kruk j, morgan aj, et al. immune system participates in brain regeneration and restoration of reproduction in the earthworm dendrobaena veneta. dev. comp. immunol. 52: 269-279, 2015. okrzesik j, kachamakowa-trojanowska n, jozkowicz a, morgan aj, plytycz b. reversible inhibition of reproduction during regeneration of cerebral ganglia and celomocytes in the earthworm dendrobaena veneta. inv. surv. j. 10: 151-161, 2013. ottaviani e. immunocyte: the invertebrate counterpart of the vertebrate macrophage. inv. surv. j. 8: 1-4, 2011. plytycz b, morgan aj. interactions between earthworm neuroendocrine and immune systems. inv. surv. j. 12: 176-178, 2015. roch, p. protein analysis of earthworm coelomic fluid: i. polymorphic system of natural hemolysin of eisenia fetida andrei. dev. comp. immunol. 3: 599-608, 1979. rorat a., kachamakowa-trojanowska n., jozkowicz a., kruk j., cocquerelle c., vandenbulcke f., et al. coelomocyte-derived fluorescence and dna markers of composting earthworm species.  j. exp. zool. a ecol. genet. physiol. 321: 28-40, 2014. 325 rorat a, vandenbulcke f, galuszka a, klimek b, molnar l, morgan aj, et al. protective role of metallothionein in eisenia andrei regenerating depleted coelomocytes or lost body segments during temperature-dependent cadmium exposure [in preparation]. santocki m, falniowski a, plytycz b. restoration of experimentally depleted coelomocytes in juvenile and adult composting earthworms eisenia andrei, e. fetida and dendrobaena veneta. appl. soil ecol. 104: 163-173, 2016a. santocki m, klimek m, kruk j, płytycz b. preliminary report on coelomocyte system during ontogeny of the earthworm dendrobaena veneta. acta biol. crac. zool. 55/56: 65-71, 2014. santocki m, morgan aj, plytycz b. differential time course of restoration of experimentally depleted coelomocytes and fluorophores in the earthworm eisenia andrei. folia biol. (krakow) 64: 121-130, 2016b. shakor aa, czuryło ea, sobota a. lysenin, a unique sphingomyelin-binding protein. febs lett. 542: 1-6, 2003. sulik p, klimek m, talik p, kruk j, morgan aj, plytycz b. searching for external sources of the riboflavin stored in earthworm eleocytes. inv. surv. j. 9: 169-177, 2012. swiderska b, kedracka-krok s, panz t, morgan aj, falniowski a, grzmil p, et al. lysenin family proteins in earthworm coelomocytes comparative approach. dev. comp. immunol., doi: 10.1016/j.dci.2016.08.011, 2016. takacs v, molnar l, klimek b, gałuszka a, morgan aj, plytycz b. exposure of eisenia andrei (oligochaeta; lumbricidea) to cadmium polluted soil inhibits earthworms maturation and reproduction but not restoration of experimentally depleted coelomocytes or regeneration of amputated segments. folia biol. (krakow), 2016 [submitted]. white lm, roy s, gordillo gm, kalliainen lk, melvin ws, ellison ec, et al. wound healing and regeneration. physiology and maintenance vol. i encyclopedia of life support systems (eolss) zoran mj. regeneration in annelids. encyclopedia of life sciences (els). john wiley & sons, ltd: chichester, 2010. lumbricid worms are commonly subjected to noxious stimuli leading to expulsion of celomic fluid or to loss of body segments; therefore regeneration of lost segments and restoration of the depleted cellular and soluble components of celomic fluid are of fundamental importance for these animals. series of experiments was performed on regeneration abilities in well-defined epigeic species eisenia andrei, e. fetida and dendrobaena veneta, and endogeic aporrectodea caliginosa. efficient regeneration of the lost anterior or posterior segments was consistently observed in eisenia sp. in a sharp contrast, d. veneta regenerated amputated anterior segments or extirpated suprapharyngeal ganglia (‘brains’) while regeneration of posterior segments was never recorded so far in this species. in a. caliginosa a loss of posterior segments was followed either by compensatory body growth or by formation of regeneration blastema. in all species regeneration was cold-inhibited while was resistant to cadmium soil pollution. the efficiency of regenerative processes in e. andrei and e. fetida might be connected with quality and quantity of some components of their celomic fluid; lysenin is unique for these species and riboflavin is much more abundant in eisenia sp. than in other lumbricids investigated so far. 210 isj 15: 210-222, 2018 issn 1824-307x research report the antifungal activity of a thaumatin-like protein from oyster crassostrea gigas x niu1,3, q xu1,3, w wang1,3, z yu1,3, z liu1,3, c qu1,3, y liu1,3, c gong1,3, l wang1,2,3,4, l song1,2,3,4* 1liaoning key laboratory of marine animal immunology, dalian ocean university, dalian 116023, china 2laboratory of marine fisheries science and food production processes, qingdao national laboratory for marine science and technology, qingdao 266235, china 3liaoning key laboratory of marine animal immunology & disease control, dalian ocean university, dalian 116023, china 4dalian key laboratory of disease prevention and control for aquaculture animals, dalian ocean university, dalian 116023, china accepted june 07, 2018 abstract in the present study, a thaumatin-like protein (cgtlp) was identified from the oyster crassostrea gigas. the full-length cdna of cgtlp was of 913 bp with a 5’ untranslated regions (utr) of 98 bp, a 3’ utr of 80 bp, and an open reading frame (orf) of 735 bp encoding a polypeptide of 244 residues. the cgtlp gene was expressed ubiquitously in mantle, gonad, hemocytes, hepatopancreas, gill, and adductor muscle with the higher expression levels in adductor muscle, hemocytes, and hepatopancreas. immunofluorescence assay indicated that cgtlp was mainly distributed in the cytoplasm of hemocytes. the mrna expression levels of cgtlp in hemocytes were significantly up-regulated after the stimulations with mannan (13.69-fold, p < 0.05), pichia pastoris (8.85-fold, p < 0.05) and polyinosinic-polycytidylic acid (3.62-fold, p < 0.05), but did not change significantly after stimulations with lipopolysaccharide, peptidoglycan, and vibrio splendidus. the recombinant cgtlp protein (rcgtlp) significantly inhibited the proliferation of p. pastoris (p < 0.05), while no inhibition towards staphylococcus aureus and v. splendidus. rcgtlp also displayed obvious β-1,3-glucanase activity, while no enzymatic activity towards chitin. these results collectively indicated that cgtlp was a homologue of tlp, which might play a vital role in defensing against fungal infection in c. gigas. key words: crassostrea gigas; thaumatin-like protein; antifungal activity; immune response introduction thaumatin was firstly isolated from the seeds of plant thaumatococcus danielli for its exceptionally potent sweet taste (noh et al., 2016). subsequently, the proteins with similar amino acid sequences were identified in most of the plant species, which were termed as thaumatin-like proteins (tlps) (breiteneder et al., 2000). tlps are low molecular-weight (20-26 kda) proteins containing one thaumatin (thn) domain with sixteen conserved cysteine (cys) residues. the thn domain is peculiar to the thaumatin-like protein family. the cys form eight intra-molecular disulfide bonds to stabilize the proteins under extreme ph and temperature (hegde et al., 2014). most plant tlps exhibit antifungal ___________________________________________________________________________ corresponding author: linsheng song liaoning key laboratory of marine animal immunology dalian ocean university dalian 116023, china e-mail: lshsong@dlou.edu.cn activity against a range of pathogenic and nonpathogenic fungi (cao et al., 2016; wang et al., 2017), and they are up-regulated in response to a variety of fungal infections and stress (hegde et al., 2014). their activities against pathogenic microorganisms are related to the activities of β-1,3-glucanase (menu-bouaouiche et al., 2003), glucose polymers depredating (liu et al., 2010), chitinase (yasmin et al., 2017), and xylanase (fierens et al., 2007), as well as α-amylase inhibiting properties (franco et al., 2002). meanwhile, tlps have also been reported in fungi, such as irpex lacteus (garcia-casado et al., 2000) and cryptococcus neoformans (sakamoto et al., 2006) with emphases on gene structure and activities (blouin et al., 2018). in addition, tlps have also been reported to exhibit membrane-permeabilizing activity (meng et al., 2017). however, the detailed mechanisms of their antifungal action are still not completely understood. with the advances in biological technology and 211 genome information, tlps have also been found in nematode caenorhabditis elegans (kitajima et al., 1999), desert locust schistocerca gregaria (brandazza et al., 2004), coleoptera diaprepes abbreviatus, hymenoptera lysiphlebus, and hemiptera toxoptera citricida (shatters et al., 2006). for marine animals, the information about tlps are still very limited, and only the predicted tlps from c. gigas (xp_011414138.1), crassostrea virginica (xp_022333236.1), and mizuhopecten yessoensis (owf35002.1) have been referred in ncbi (https://www.ncbi.nlm.nih.gov/) based on genomic sequencing. the pacific oyster c. gigas is one of the most important cultured mollusk species in the world, and it contributes greatly to the economic development of the global aquaculture (yang et al., 2017). because of its economic and ecological importance as well as biological characteristics, c. gigas is becoming a model to investigate molluscan biology, development, innate immunity, and stress adaptation (guo et al., 2015). as a sessile marine animal living in estuarine and intertidal regions, c. gigas has to face the infections of various pathogens including bacteria, protozoans, fungi, and viruses (paillard et al., 2004; guo et al., 2015). pathogenic fungi, such as monilia, ostracoblabe implexa, sirolpidium zoophthorum, dermocystidium marinus, and lagenidium, can cause more than 95% mortality of the cultured crassostrea virginica in summer (chen et al., 2007). the characterization of antifungal molecules would benefit the knowledge of the marine animals immune defense mechanism (franco et al., 2002; liu et al., 2010; yasmin et al., 2017). in the present study, a novel homologue of tlp was identified from oysters c. gigas (designated as cgtlp). the mrna expression levels of cgtlp in hemocytes post immune stimulation and its antimicrobial activity were investigated. moreover, the β-1,3-glucanase and chitinase activities of the recombinant cgtlp protein were detected in vitro, which would provide more information to understand the immune defense roles of cgtlp in c. gigas. materials and methods animals, microorganisms, and drugs oysters crassostrea gigas with an average shell length of 12.0 cm were sampled from a local farm in dalian, china, and maintained in aerated seawater at approximately 20 °c and salinity of 30‰ for an acclimation period of seven days. the oysters were fed daily with spirulina powder at a dose of 1-3 g/m3 water. six-week-old female mice were purchased from dalian medical university (dalian, china). the bacterium staphylococcus aureus and fungus pichia pastoris were purchased from microbial culture collection center (beijing, china) and invitrogen (lifetech, usa), respectively. vibrio splendidus was separated from the infected patinopecten yessoensis (liu et al., 2013). the bacteria v. splendidus and s. aureus were cultured in 2216e media at 28 °c for 20 h and lb media at 37 °c for 20 h, respectively. the fungus p. pastoris was cultured in yeast extract peptone dextrose medium (ypd) media at 28 °c for 24 h. the cell cultures were harvested by centrifugation and the pellets were washed with phosphate buffered saline (pbs, 0.14 mol/l nacl, 3 mmol/l kcl, 8 mmol/l na2hpo4, 1.5 mmol/l kh2po4, ph=7.4) for three times, and then re-suspended in pbs at a final concentration of about 1×108 cfu/ml. escherichia coli trans5α (transgen, china), pet-28a (takara, japan) and bl21 (de3) (transgen, china) were used for gene cloning and protein expression, respectively. lps (from e. coli, sigma-aldrich, usa), pgn (from s. aureus, sigma-aldrich, usa), poly (i:c) (sigma-aldrich, usa) and man (from saccharomyces cerevisiae, sigma-aldrich, usa) were dissolved in pbs at a final concentration of 0.5, 1.0, 1.0, and 1.0 mg/ml, respectively. immune stimulation and sample collection for the analysis of cgtlp mrna distribution, six tissues including gonad, gill, mantle, adductor muscle, hemocytes and hepatopancreas from nine oysters were collected as three parallels. the tissues were kept in trizol reagent at -80 °c for rna extraction. four hundred and twenty oysters were randomly selected and separated into seven groups for fungus, bacteria and pathogen associated molecular patterns (pamps) stimulations to reveal the mrna expression of cgtlp. fungus p. pastoris, bacterium v. splendidus and four different pamps were used for immune stimulations. the oysters in the treatment groups (80 oysters in each group) individually received an injection of 100 μl live v. splendidus (1×108 cfu/ml in pbs), p. pastoris (1×108 cfu/ml in pbs), lps, pgn, poly (i:c) and man, respectively. the lps, pgn, poly (i:c) and man were prepared at the concentration of 0.5, 1.0, 1.0, and 1.0 mg/ml in pbs as described above, respectively. the rest oysters in the control group received an injection of 100 μl pbs. nine oysters were randomly sampled from each group at 0, 3, 6, 12, 24, 48 and 72 h after injection and divided into three parallels. the hemolymphs collected from three oysters were pooled together as one sample. there were three parallels for each time point. the hemocytes were kept in trizol reagent at -80 °c for rna extraction. rna isolation and cdna synthesis the total rna was obtained from tissues using trizol reagent according to the manufacture's protocol (invitrogen, usa) and quantified by nanodrop 2000 (thermo scientific, usa). the first strand cdna was reverse-transcribed based on manufacture’s protocol using the primescripttm 1st strand cdna synthesis kit (takara, japan). the cdna mix was diluted 50-fold and stored at -80 °c for subsequent sybr green fluorescent quantitative real-time pcr (qrt-pcr). the gene cloning of cgtlp the dna fragment of cgtlp (genbank accession no. xm_011415836.2) (zhang et al., 2012) was amplified using extaq dna polymerase (takara, japan). the information of the primers used in this study was listed in table 1. the pcr products were cloned into the pmd19-t (takara, japan) and verified by nucleotide sequencing in both directions. 212 table 1 primers used in this study bioinformatics analysis of cgtlp the amino acid sequence of cgtlp was analyzed using the sequence manipulation suite (http://www.bio-soft.net/sms/), and domains were predicted by using the simple modular architecture research tool (smart) (http://smart.embl-heidelberg.de/). the three-dimensional models of tlps were built by homology modeling with the swiss-model prediction algorithm (http://swissmodel.expasy.org/). the amino acid sequences of tlps from other species were obtained from ncbi (https://www.ncbi.nlm.nih.gov/) database. the clustal x1.81 and the sequence manipulation suite (http://www.bio-soft.net/sms/) were used to perform the multiple sequence alignment for tlps. a phylogenetic tree was constructed by the neighbor joining (nj) method using the software of mega 6.06. the reliability of the branching was tested by using bootstrap re-sampling (1000 pseudo-replicates). quantitative real time pcr analysis (qrt-pcr) of the mrna expression the relative mrna expression levels of cgtlp were measured by sybr green fluorescent qrt-pcr, which was performed with an abi 7500 real-time thermal cycler (applied biosystems, usa) according to the manufacture’s instruction with sybr premix ex taq (tli rnaseh plus) (takara, rr420a). a pair of specific primers, cgtlp-qrt-f and cgtlp-qrt-r (table 1), was used to amplify a fragment of 202 bp. the c. gigas elongation factor (cgef, nm_001305313.2) fragment amplified with primers cgef-qrt-f and cgef-qrt-r (table 1) was employed as the internal control (zhang et al., 2012). the mantle and the time of 0 h were used as the reference group, respectively. the mrna expression levels of cgtlp were determined by 2-δδct method (livak et al., 2001). all the data were given in terms of relative mrna expression as mean ± s.d. (n=3). the expression and purification of recombinant cgtlp the orf fragment of cgtlp without signal peptide sequence was cloned with primers cgtlp-recombinant-f and cgtlp-recombinant-r (table 1) with an nde ⅰ and xho ⅰ site at their 5' end, respectively. after cloned into pmd19-t simple vector (takara, japan) and transformed into e. coli trans5α, the target fragment was obtained by completely digestion with restrictive enzymes nde ⅰ and xho ⅰ, and subsequently cloned into the nde ⅰ/xho ⅰ sites of expression vector pet-28a (novagen, usa). the recombinant plasmids pet-28a-cgtlp were transformed into e. coli bl21 (de3) (transgen, china) for prokaryotic expression of cgtlp. the recombinant proteins of cgtlp (rcgtlp) were purified by ni2+ chelating sepharose column (sangon biotech, canada) and dialysed with the dialysate. the purified rcgtlp was examined by 15% sds-page, and visualized with coomassie bright blue r250. the purified protein was quantified by bradford protein assay kit (beyotime, china) and stored at -80 °c. preparation of polyclonal antibodies, western blotting, and subcellular localization of cgtlp in hemocytes three six-week-old female mice were immunized with rcgtlp protein to generate polyclonal antibody as the method described in the previous report (sun et al., 2014). in short, 300 µl of recombinant protein (about 200 µg) was mixed with an equal volume of freund's complete adjuvant, and injected into the abdomen of mice. two weeks later, three immunizations were conducted weekly with an equal volume of freund's incomplete adjuvant. one week after the fourth immunization, the serum was collected. rcgtlp was electrophoretically transferred onto a piece of nitrocellulose transfer membrane after a 15% sds-page. after blocked in tbs-t (10 mm tris-hcl, ph 8.0, 100 mm nacl and 0.05% tween primer purpose primer name sequence (5’-3’) clone primers cgtlp-5’-f ttaacatttggagataaagaact cgtlp-3’-r tgaaagtatttcaacaaattgtaat cgtlp-orf-f atgaacaagatagccatgaagt cgtlp-orf-r ttatccacagaagacgacatc qrt primers cgtlp-qrt-f gaagggcaatcgggagcata cgtlp-qrt-r gggcgtatgagaagtttgga cgef-qrt-f ctccacccaacatcaccact cgef-qrt-r ggatttcctttacggacacg recombination primers cgtlp-recombinant-f ggaattccatatgcacagaatccattatagaaac cgtlp-recombinant-r ccgctcgagtccacagaagacgacatc 213 20) containing 5% skim milk powder at 37 °c for two hours, the membrane was incubated with antiserum solution (1:1000, v/v) at room temperature (rt) for one hour, washed three times with tbs-t, and then incubated with hrp-labeled anti-mouse igg 1:2000 (v/v) at rt for one hour. after three times washing with tbs-t, the membrane was incubated with ecl detection reagents (amersham biosciences, usa) and exposed to film after the final washing with tbs-t for three times. the hemolymph was collected from healthy oysters with the equal volume of anti-aggregation solution (zhang et al., 2014). after washed with sterile seawater for three times, the hemocytes were seeded into cell culture dishes (nest, usa), and incubated in a humidifying box at rt for one hour. the hemocytes were fixed by 4% paraformaldehyde for 20 minutes, and treated with 0.5% triton x-100 (prepared with pbs) for 20 minutes to permeabilize the cell membrane. after washed with pbs-t for three times, the samples were blocked with 20 μl of 3% bsa at rt for 30 minutes, and then incubated with polyclonal antibody of rcgtlp diluted with 3% bsa in the ratio of 1:500 at 37 °c for one hour. the hemocytes were incubated with alexa fluer® 488 (life technologies, usa) labeled rabbit anti-rat secondary antibody (1:1000) at 37 °c for 30 minutes. then 4',6-diamidino-2-phenylindole (dapi, beyotime biotechnology, china) was added at the ratio of 1:1000 and incubated for five minutes to dye the nucleus. after washed three times with tbs-t, hemocytes were observed with an lsm 710 laser confocal scanning microscope (carl zeiss jena, germany). the assay of microbe growth suppression activity of rcgtlp the antimicrobial activities of rcgtlp against gram-positive bacterium (s. aureus), gram-negative bacterium (v. splendidus) and fungus (p. pastoris) were determined according to the method described in previous report (li et al., 2015). s. aureus, v. splendidus and p. pastoris were grown in lb medium 37 °c for 20 hours, 2216e medium 28 °c for 20 hours, ypd medium 28 °c for 24 hours to mid-logarithmic phase, respectively. the cultures were centrifuged at 800 g, 4 °c for 10 minutes, and the microorganism pellets were re-suspended with accordingly medium at the concentration 105 cfu/ml. fifty microliter of gradient diluent rcgtlp (protein concentrations at 0.500 mg/ml, 0.250 mg/ml, 0.125 mg/ml, and 0.000 mg/ml in pbs, respectively), 50 µl microorganism resuspension, and 1000 µl of accordingly medium were added into a 96-well microliter plate, respectively, and the plates were placed in an elisa reader at 28 °c with a shake uninterruptedly. od600 was measured every one hour to detect the growth of bacteria and fungi. accordingly mediums were used as blank controls. there were three replicates for each group. the β-1,3-glucanase and chitinase activities assays of rcgtlp the β-1,3-glucanase activity was measured with a β-1,3-glucanase (β-1,3-ga) assay kit (bc0360, solarbio, china). briefly, 100 μl of reagent 1 was added to 100 μl rcgtlp (protein concentrations were 0.500 mg/ml) in a centrifuge tube. the same volume of distilled water was employed as negative control. after incubated at 37 °c for one hour, 600 μl of reagent 2 were appended to the tube and incubated at 100 °c for five minutes. two hundred microliter of reaction mixture was added to a 96-well microliter plate, which was measured absorbance at 540 nm in an elisa reader. each experiment was repeated in triplicate. the chitinase activity of rcgtlp was measured with a chitinase assay kit (bc0820, solarbio, china) according to the instruction. in brief, 200 μl extracting solution and 400 μl reagent 1 was added to 400 μl rcgtlp (protein concentrations were 0.500 mg/ml), and 600 μl extracting solution was added to 400 μl rcgtlp (protein concentrations were 0.500 mg/ml) in a centrifuge tube as negative control. the tube was incubated at 37 °c for one hour followed by centrifugation at 5000 rpm, 4 °c for ten minutes. two hundred microliter of reagent 2 was added to the reaction mixture followed by incubation at 100 °c for seven minutes. two hundred microliter of reagent 3 and 400 μl of reagent 4 were added to the tube followed by incubated at 37 °c for 15 minutes. two hundred microlitre reaction mixture was added to a 96-well microliter plate, and the absorbance at 585 nm was measured in an elisa reader. each experiment was repeated in triplicate. statistical analysis the data were analyzed by using spss 18.0 and expressed as means ± standard deviation (n=3). the statistically significant differences among groups were designated at p < 0.05 by one-way analysis of variance (anova) (labeled with *). results the molecular feature, domain and spatial structure of cgtlp the coding sequence of cgtlp was retrieved from ncbi (xm_011415836.2). the complete cdna of cgtlp was of 913 with a 98 bp 5’ untranslated regions (utr), an 80 bp 3’ utr and an open reading frame (orf) of 735 bp (fig. 1a). the orf encoded a putative polypeptide of 244 amino acid residues with a predicted molecular weight of 25.97 kda and theoretical isoelectric point of 7.84. there was a signal peptide (from m1 to g20) in the deduced amino acid sequence of cgtlp, and a classic smart thn domain of tlp protein family from i23 to g244 (fig. 1a and fig. 1b). in the spatial structure of cgtlp, there were three domains and a cleft structure. domain ⅰ was formed by a β-sandwich with 11 β-sheets, domain ⅱ was made up by one β-sheet, and domain ⅲ was composed of three α-helical structures. the cleft structure was localized between domains ⅰ and ⅱ (fig. 1c). the phylogeny of cgtlp multiple sequence alignment was performed on the basis of the deduced amino acid sequences of cgtlp and some other tlps downloaded from ncbi. the deduced amino acid sequence of cgtlp shared higher similarity with tlps from other organisms, such as 94% similarity with thaumatin-like 214 fig. 1 the molecular feature, domain and spatial structure of cgtlp. a: the cdna and amino acid sequences of gene encoding cgtlp from c. gigas. the nucleotides and amino acids were numbered along the left margin. the putative signal peptide was underlined. the initiation codon and termination codon were boxed in red. b: the structural domain of cgtlp predicted by smart. the red area represented the signal peptide. c: the spatial structure of cgtlp predicted by swiss-model program protein from c. virginica (xp_022333236.1), 51% similarity with pathogenesis-related protein 5 like protein from m. yessoensis (xp_021343380.1), 49% similarity with arabidopsis thaliana pathogenesis-related protein 5 (np_177641.1) and 37% similarity with rhizoctonia solani 123e pathogenesis-related protein pr5k (kep50242.1), respectively (fig. 2). in the phylogenic tree, 28 tlp proteins from c. elegans, s. gregaria, aleuroglyphus ovatus, m. yessoensis, a. thaliana and r. solani 123e et al. were gathered separately as three major distinct branches including fungi, plantae, and animalia. there were three distinct clades in the branch of animalia, nemathelminthes, molluscs and arthropods. cgtlp was firstly clustered with tlp from c. virginica and pr-5 from m. yessoensis to form molluscan branch, which was a sister branch to nemathelminthes and arthropods (fig. 3). the distribution of cgtlp in different tissues the mrna transcripts of cgtlp were detected in all the examined tissues, including mantle, gonad, hepatopancreas, hemocytes, adductor muscle, and gill (fig. 4). the highest mrna expression level of cgtlp was detected in adductor muscle, which was 64.88-fold (p < 0.05) of that in mantle. the mrna expression levels of cgtlp in hepatopancreas (45.64-fold, p < 0.05)and hemocytes were also significantly higher (34.58-fold, p < 0.05) than that in mantle, respectively. nevertheless, the mrna expression levels of cgtlp were relatively lower in gonad and gill, which were 2.46-fold (p < 0.05) and 1.13-fold (p > 0.05) of that in mantle, respectively (fig. 4). the mrna expression pattern of cgtlp in hemocytes after different immune stimulations the temporal mrna expression of cgtlp in oyster hemocytes was examined at 0, 3, 6, 12, 24, 48 and 72 h after lps, pgn, poly (i:c), or man stimulation, respectively. compared with pbs control group, the mrna expression level of cgtlp in hemocytes was significantly increased at 12 h (6.56-fold, p < 0.05), maintained higher levels from 24 h to 48 h, and then peaked at 72 h (13.69-fold, p < 0.05) after man stimulation. the mrna expression level of cgtlp in hemocytes was significantly up-regulated to the peak level at 6 h (3.62-fold of the control group, p < 0.05) and then recovered to the original level at 12-72 h (p > 0.05) after poly (i:c) stimulation (fig. 5a). however, there was no significant change of cgtlp mrna expression in hemocytes after lps or pgn stimulation (p > 0.05) (fig. 5a). the temporal change of cgtlp mrna expression levels in hemocytes were also monitored after live v. splendidus or p. pastoris stimulation. the mrna expression of cgtlp in hemocytes was significantly up-regulated to 5.45-fold (p < 0.05) at 12 h post p. pastoris challenge, and then continuously 215 fig. 2 multiple alignment of cgtlp with other tlp family proteins deposited in genbank. the same amino acids were shaded in dark and similar amino acids were shaded in grey. species and gene accession numbers are as follows: crassostrea gigas (xp_011414138.1), crassostrea virginica (xp_022333236.1), mizuhopecten yessoensis (owf35002.1), schistocerca gregaria (aar97603.1), acyrthosiphon pisum (np_001313585.1), caenorhabditis briggsae (cap30301.1), arabidopsis thaliana (caa61411.1), theobroma cacao (eoy24665.1), rhodotorula (kwu43254.1), and folsomia candida (xp_021955579.1) increased to the peak level at 72 h (8.85-fold, p < 0.05) (fig. 5b). there was no significant change of cgtlp expression (p > 0.05) after the injection of v. splendidus during the whole experimental process (fig. 5b). the polyclonal antibody of cgtlp and its subcellular localization in hemocytes the recombinant plasmid pet-28a-cgtlp was transformed and expressed in e. coli bl21 (de3). after isopropyl β-d-thiogalactoside (iptg) induction, the whole cell lysate was analyzed by 15% sds-page, and an obvious band about 25.0 kda was observed (fig. 6a). the band was highly consistent with the predicted molecular mass (25.97 kda) of cgtlp. the purified rcgtlp was injected into the mice to obtain the immune serums. a clear primary reaction band was revealed by western blotting assay, indicating the high recognition specificity of the polyclonal antibody against cgtlp (fig. 6b). the localization of cgtlp protein in the hemocytes was analyzed by immunofluorescence assay with the polyclonal antibody acquired from 216 fig. 3 the phylogenetic tree of cgtlp and tlp homologues of other species. the phylogenetic tree analysis of the amino acid sequences of tlps was constructed by the neighbor-joining method and was bootstrapped 1000 times using the mega 6.06 software. the cgtlp was marked by a red triangle fig. 4 the mrna expression levels of cgtlp in different tissues of adult oyster. the cgef (elongation factor) gene expression was used as an internal control and mantle sample was used as the reference sample. each value was shown as mean ± s.d. (n = 3), and bars with different characters were significantly different (p < 0.05) 217 fig. 5 the mrna expression patterns of cgtlp in hemocytes. a: temporal mrna expression of cgtlp in hemocytes after lps, pgn, poly (i:c) and man stimulation at 0, 3, 6, 12, 24, 48, and 72 h, respectively. b: temporal mrna expression of cgtlp in hemocytes after v. splendidus and p. pastoris stimulations at 0, 3, 6, 12, 24, 48, and 72 h, respectively. the relative expression values were shown as mean ± sd (n=3). asterisk indicated significant difference from control (p < 0.05) mice. the positive signals of fitc labeled antibody to cgtlp protein were of the green fluorescence signal, which was mainly observed in the cytoplasm of the oyster hemocytes, while the nucleus of hemocytes were stained in blue fluorescence (fig. 7). the activity of rcgtlp to inhibit the growth of microbes the microbe growth inhibition activity of rcgtlp was assessed by detecting the microbe growth curve. within the detection time (12 hours) and concentration range (0.000-0.500 mg/ml), there was no obvious growth suppression activity against s. aureus and v. splendidus (p > 0.05) (fig. 8a and 8b). however, the growth of p. pastoris in 0.250 mg/ml and 0.500 mg/ml rcgtlp treatment groups was significantly suppressed (p < 0.05), compared with that in the blank group from 7 h to 16 h post treatment. moreover, the growth level of p. pastoris in the 0.500 mg/ml rcgtlp treatment group was significantly lower (p < 0.05) than that in the 0.250 mg/ml rcgtlp treatment group during 7-16 h (fig. 8c). the antifungal effect of rcgtlp to p. pastoris was strengthened with the increase of protein concentration. fig. 6 sds-page and western blotting analysis of rcgtlp protein. a: sds-page of rcgtlp protein. m: protein molecular standard (kda); lane 1: negative control for rcgtlp; lane 2: iptg induced rcgtlp; lane 3: purified rcgtlp. b: western blotting of rcgtlp protein. lane 4: western blotting of rcgtlp. m: pre-dyed protein molecular standard (kda) 218 fig. 7 fluorescent microscopy analysis of cgtlp (green) distribution in hemocytes of c. gigas. a: the hemocytes were observed in light field; b: the cgtlp was labeled green by polyclonal antibody of cgtlp and alexa flour 488 labeled second antibody in single section; c: the nuclei of hemocytes were stained blue by dapi in single section; d: the merged chart. the scale bar was 20 μm the β-1,3-glucanase and chitinase activities of rcgtlp in the β-1,3-glucanase activity assay, the od450 of experimental group with rcgtlp (0.250 mg/ml) was significantly higher (4.10-fold, p < 0.05) than that in control group without rcgtlp (fig. 9a). however, the od585 of experimental group with rcgtlp showed no significant change compared with control group in the chitinase activity assay (p > 0.05) (fig. 9b). discussion thaumatin-like proteins (tlps) have been reported to be involved in host responses against a wide range of stresses, such as pathogen/pest invasion, wounding, drought, and cold hardiness (leone et al., 2006). in this study, a homologue of tlp with a theoretical molecular weight of 25.97 kda was identified in oyster c. gigas. it was consistent with the previous reports that tlps are about 200 amino acids (zhang et al., 2012) with the molecular weight of 21-26 kda (petre et al., 2011). there was a thn domain in cgtlp contained, which was an exclusive domain of tlp families (leone et al., 2006). the deduced amino acid sequence of cgtlp shared high similarity with other known tlps, such as the thaumatin-like protein from c. virginica (xp-022333236.1) and the pathogenesis-related protein 5 from m. yessoensis (owf35002.1). the putative mature polypeptide of cgtlp was of cysteine-rich, which was consistent with the previous report that most tlps contained 16 conserved cysteine residues (fierens et al., 2009). these cysteine residues could be paired to form disulfide bonds, which were expected to be a relatively stable 219 fig. 8 the growth suppression activity of rcgtlp was detected by growth curve of s. aureus (a), v. splendidus (b) and p. pastoris (c). each value was shown as mean ± sd (n=3). asterisk indicated significant difference from control (p < 0.05) chemical structure to resist heat, degradation, and acid stress (liu et al., 2010). there was an n-terminal signal peptide in the putative polypeptide of cgtlp contained, which could target the mature proteins into certain organelles (anžlovar et al., 2003). the three-dimensional structure of cgtlp was similar to the crystal structures of plant tlps (ghosh et al., 2008). three domains and a cleft structure between domains ⅰ and ⅱ were identified in cgtlp. the domain ⅰ was formed by a β-sandwich with 11 β-sheets, which was highly conservative in tlp family members (clustal et al., 1994). the tlps from different species possess different domain ⅱ or domain ⅲ, which is determined by the number of β-sheet and α-helical in domain ⅱ or domain ⅲ, respectively. the cleft between domain ⅰ and ⅱ had an acidic, neutral, or basic nature for binding different ligands/receptors (min et al., 2004). the difference of the domain ⅲ can contribute to different enzymatic activity of tlps (abdin et al., 2011). all these results suggested that cgtlp was highly homologous to the tlps from fungi, plants, and animals (wang et al., 2014). a growing number of studies indicated that tlps had antifungal function, and played an extremely important role for the organisms surviving from the environment stress (anžlovar et al., 2003). the mrna expression of tlps could be induced when plants were exposed to biotic and abiotic stresses (misra et al., 2018). in the present study, the mrna transcripts of cgtlp were much higher in adductor muscle, hemocytes and hepatopancreas than that in other organs of c. gigas. hemocytes and hepatopancreas were the major immune organs of c. gigas (wang et al., 2018). because tlps were regarded as immune-related genes, it was reasonable that the mrna expression level of cgtlp was high in these immune organs of c. gigas. some fungi could cause oyster diseases by sticking and infecting the adductor muscle (bower et al., 1994). meanwhile, in the present study, the antifungal activity of rcgtlp was verified in vitro, so we speculated that the high expression of cgtlp in adductor muscle might be related to its anti-fungi activity. of course, it did not exclude that cgtlp played other physiological functions in adductor muscle (liu et al., 2010). the higher mrna expression in these tissues indicated that cgtlp might play an important role in the immune response of c. gigas (wang et al., 2018). in addition, significant changes of cgtlp mrna transcripts could be detected in the hemocytes after p. pastoris or man stimulation. similarly, it was reported that tlps could be induced by the presence of fungal molds (such as man, chitin, and β-1,3-glucan) and fungi puccinia triticina (zhang et al., 2017). tlps could also be induced by some kind of virus or viral simulacrum (kinkema et al., 2000). in this study, the mrna expression of cgtlp in hemocytes was up-regulated after poly (i:c) stimulation from 3 h (1.51-fold change, p > 0.05) and peaked at 6 h (3.62-fold change, p < 0.05). these results demonstrated that the cgtlp might also play an important role in the immune response to virus infection in c. gigas. no significant changes were observed in the mrna expression levels of cgtlp among control group, v. splendidus group, lps group, and pgn group during the whole experimental process, indicating cgtlp could not be induced by the presence of bacteria or the bacterial simulacrum. it was consistent with the findings that bacteria could not induce the mrna expression of tlp in pinus thunbergii (solano et al., 2013). similar to plant tlps, cgtlp protein was mainly distributed in the cytoplasm of the oyster hemocytes, indicating that the mature cgtlp might perform as cytoplasmic protein to play important roles in the innate immune response of c. gigas against fungal and viral infection, and resistance to stresses (gómez-casado et al., 2014). tlp family members have been reported to inhibit several kinds of fungi including taphrinomycotina, saccharomycotina, and basidiomycota (anžlovar et al., 2003; jung et al., 2005; hayashi et al., 2014). in the present study, the antifungal and antibacterial activities of rcgtlp were investigated in vitro. rcgtlp could inhibit the growth of p. pastoris, which was in line with previous reports that most of plant tlps exhibited antifungal activities (liu et al., 2010). interestingly, rcgtlp could not inhibit the growth of gram-positive bacteria, s. aureus and gram-negative bacteria, v. splendidus. similarly, 220 fig. 9 the absorbance of enzyme activity detection of rcgtlp. a: the absorbance of β-1,3-glucanase activity detection of rcgtlp. b: the absorbance of chitinase activity detection of rcgtlp. each value was shown as mean ± sd (n=3). asterisk indicated significant difference from control (p < 0.05) some plants tlps, such as oryza sativa tlp, a. thaliana tlp, and pinus monticola tlp, also did not display inhibitory effect on bacteria (futamura et al., 2005; zhang et al., 2007; singh et al., 2017). these results suggested that cgtlp might exert selective inhibitory activity to fungi. it has been reported that the inhibition activities of tlps to microorganisms owe to their β-1,3-glucanase or chitinase activities (menu-bouaouiche et al., 2003). for instance, tlp from musa acuminata could hydrolyze β-1,3-glucan (menu-bouaouiche et al., 2003), and tlp from picea glauca possessed chitinase activity (beleneva et al., 2011). some tlps even did not possess enzymatic activity (hernández-blanco et al., 2007; borad et al., 2008; miura et al., 2013), which exert their antifungal activities by permeabilizing cell membranes (brandazza et al., 2004; meng et al., 2017). in order to explore the possible antifungal mechanism of cgtlp, the β-1,3-glucanase and chitinase activities of rcgtlp were evaluated in vitro. rcgtlp displayed obvious β-1,3-glucanase activity, but no chitinase activity, suggesting that the antifungal activity of rcgtlp might be ascribed to its β-1,3-glucanase activity rather than chitinase activity. the different activities of tlps were related to the difference of their domain ⅱ and ⅲ in difference species (rep et al., 2002). the domain ⅱ and ⅲ of cgtlp were similar to the domain ⅱ and ⅲ of the tlp from musa acuminate (menu-bouaouiche et al., 2003), which presented β-1,3-glucanase activity. the antifungal activity of cgtlp was suspected to be determined by its special domain ⅱ and ⅲ structure. in summary, a novel cgtlp with a classical thn domain was identified from oyster c. gigas for the first time. the mrna transcripts of cgtlp were highly expressed in adductor muscle, hepatopancreas and hemocytes, and could be induced by fungal stimulation rather than bacterial stimulation. rcgtlp could significantly suppress the growth of p. pastoris, but not s. aureus and v. splendidus. the antifungal activity of rcgtlp might be ascribed to its β-1,3-glucanase activity rather than chitinase activity. this study expanded the knowledge on the functions of tlp in the antifungal immune defense system in oyster, and threw light on the evolutionary of tlps. acknowledgments we are grateful to all the laboratory members for continuous technical advice and beneficial discussions. this study was supported by a grant (no. u17062049) from national science foundation of china, ao shan talents cultivation program supported by qingdao national laboratory for marine science and technology (no. 2017astcpos13), earmarked funds from modern agro-industry technology research system (cars-49), the fund for outstanding talents and innovative team of agricultural scientific research, the distinguished professor of liaoning, dalian high level talent innovation support orogram (2015r020) and the research 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z, liu r, et al. the specifically enhanced cellular immune responses in pacific oyster (crassostrea gigas) against secondary challenge with vibrio splendidus. dev. comp. immunol. 45: 141-150, 2014. zhang y, shih ds. isolation of an osmotin-like protein gene from strawberry and analysis of the response of this gene to abiotic stresses. j. plant physiol. 164: 68-77, 2007. zhang y, yan h, wei x, zhang j, wang h, liu d. expression analysis and functional characterization of a pathogen-induced thaumatin-like gene in wheat conferring enhanced resistance to puccinia triticina. j. plant interact. 12: 332-339, 2017. isj 13: 186-190, 2016 isj 13: 186-190, 2016 issn 1824-307x minireview biologically active peptides in molluscs f tascedda, e ottaviani department of life sciences, university of modena and reggio emilia, via campi 213/d, 41125 modena, italy accepted may 27, 2016 abstract the immune and neuroendocrine systems of invertebrates, as well as vertebrates, share a common pool of molecules that have been conserved throughout evolution. now we add a new interface to this bidirectional interaction, demonstrating the involvement of the gut system, showing that these three systems use the same biologically active peptides. key words: neuropeptides; hormones; molluscs; immunocytochemistry; evolution   introduction homeostasis, coined by claude bernard (1965), is a healthy state that is maintained by the constant adjustment of biochemical and physiological pathways. the immune and neuroendocrine systems play a fundamental role in this complex mechanism that thanks to the fluid compartment wetting/covering the cells of the body, transfers biologically active peptides (bap), such as neuropeptides and hormones, to all the cells of the body. more importantly, the immune and neuroendocrine system of vertebrates and invertebrates produce the same bap and data in the literature show a bidirectional correlation between them (weigent and blalock, 1987; ottaviani and franceschi, 1997; ottaviani et al., 1997). here we review the presence and the possible roles of bap in molluscs. presence of biologically active peptides in molluscs the synthesis of bap was detected in the neuroendocrine, immune and gut system. moreover, in gastropods the entire periphery of the nervous system, including nerves, connectives and commissure is used for hormone release. the central nervous system (cns) of the freshwater snails lymnaea stagnalis and planorbarius corneus have been the most studied models for the presence of bap using immunocytochemical approaches (grimm-jørgensen, 1978; boer et al., 1979; schot et al., 1981; sonetti et al., 1994, 2005). especially interesting are p. corneus immunocytes which exhibit immunoreactivity for several vertebrate bap, thus presenting a framework ___________________________________________________________________________ corresponding author: enzo ottaviani department of biology university of modena and reggio emilia via campi 213/d, 41125 modena, italy e-mail: enzo.ottaviani@unimore.it comparable to that observed in the cns (schot et al., 1981). these bap, include bombesin, calcitonin, cholecystokinin (cck)-8, cck-39, gastrin, glucagon, growth hormone, insulin, met-enkephalin, neurotensin, oxytocin, pancreatic polypeptide (pp), somatostatin, substance p, secretin, serotonin, substance p, thyroglobulin, vasopressin and vasointestinal p (vip) (ottaviani and cossarizza, 1990; ottaviani et al., 1992), adrenocorticotropin hormone (acth), β-endorphin (ottaviani et al., 1990), α-melanocyte-stimulating hormone (α-msh) (franchini et al., 1994) and corticotropin-releasing hormone (crh) (ottaviani et al., 1998). other proopiomelanocortin (pomc)-derived peptides were also confirmed in immunocytes by the expression of pomc-mrna (ottaviani et al., 1995). furthermore, thyrotropin-releasing factor-like peptides were found in the hemolymph of l. stagnalis (grimm-jørgensen, 1978). the cns of p. corneus and other gastropods is characterized by three pairs of ganglia (cerebral, pleural and pedal) located around the esophagus. acth-like molecules are present in a relatively small number of neurons in all ganglia (sonetti et al., 2005). moreover, in p. corneus, acth immunopositivity is detected in a distinctive class of neuroglial cells, comparable to vertebrate microglia. however, acth was not found in the neuroglial cells of the bivalve mytilus edulis, indicating that there are species-specific differences in expression (sonetti et al., 1994). furthermore, immunoreactive responses have been observed in the snail cns for some vertebrate neuropeptides such as substance p, neuropeptide y, calcitonin gene-peptide (cgrp) and cck (sonetti et al., 1990). previous studies demonstrate different neurosecretory cells in the cns of l. stagnalis and helicella virgata using the alcian blue/alcian yellow method (wendelaar bonga, 1970; franchini and ottaviani, 1982). the comparison 186   mailto:enzo.ottaviani@unimore.it between histochemical and immunohistochemical techniques showed a low overlap between the classically evidenced neurosecretory cells and the baps detected by immunocytochemical procedures. however, in p. corneus some overlap is present, for example, cgrp-like peptide is observed in one cluster of yellow cells and cck8like has been detected in three different neurosecretory cells (sonetti et al., 1990). other baps such as thyrotropin-releasing factor-like (sonetti et al., 1994), dopamine-like-, serotoninlikeand vasotocin-like peptides (boer et al., 1984), were observed in the cns of l. stagnalis, somatostatin-like peptides in cns of limax maximus (marchand et al., 1984) and in the hepatopancreas, mantle edge and hemolymph of helix aspersa (marchand et al., 1989). likewise, steroids have been identified in ganglionic tissue as well. the presence of 17betaestradiol, involved in the stimulation of the release of nitric oxide, has been detected by stefano et al. (2003a), while canesi et al. (2006) described this molecule in hemocytes that have a known immunomodulation effect. furthermore, studies in the pedal ganglia of mussel have allowed to isolate a 266 bp fragment with 100 % identity to the human 17beta-estradiol gene (2003b). furthermore, baps have been detected in the gut system. many mammalian gastroenteropancreatic (gep) hormones have their counterparts in invertebrates (molluscs, annelids and arthropods), which are mainly localized in the nervous system (van noorden and falkmer, 1980). these gep hormone-like substances include insulin, somatostatin, glucagon, gastrin, secretin, vip, pp, substance p and enkephalin. role of biologically active peptides bap are involved in fundamental mechanisms that allow the survival of every form of life, such as, reproduction, growth, defense and homeostasis. with regard to reproduction, functional androgen and estrogen receptors, have been detected in gastropods, but their genetic basis has yet to be clarified (köhler et al., 2007). a different behavior with respect to octopus vulgaris where endogenous estrogens are involved in the reproduction (di cosmo et al., 2001). on the whole, although the literature suggests the presence of steroid-like molecules in molluscan tissues, their functional role is far from clear. in this context, some authors hypothesize the possibility of the biosynthesis of steroids in molluscs (fernandes et al., 2011), while others are doubtful that this can be achieved (scott, 2012). furthermore, although in aplysia californica gonadotropin-releasing hormone (gnrh)-like molecules have been identified, these do not seem to be involved in reproduction (tsai et al., 2010). also, insulin-like peptide was found in cerebral light-green cells of the cns of l. stagnalis (smit et al., 1988). these neurosecretory cells contain and release immunoreactive insulin that regulate various processes related to the growth of the snail body, such as, soft body parts and the shell (geraerts, 1976; joosse and geraerts, 1983; ebberink and joosse, 1985; ebberink et al., 1987). as far as defense is concerned, the acth fragment (1-24) has been suggested to play an important role. indeed, it is involved in cell shape changes (cell motility), chemotaxis (non-random locomotion) and phagocytosis. this immunocyte motility is activated by an adenylate cyclase/camp/protein kinase a-dependent pathway, as well as through the activation of protein kinase c (sassi et al., 1980). chemotaxis and phagocytosis display different behaviors, indeed in contrast to the conventional paradigm, there is no direct correlation between the two processes (genedani et al., 1994; ottaviani et al., 1994). furthermore, acth is involved in the stress response and as in mammals, the key mediator molecules are the same with the cascade of events following the same order and pattern: crh-acth-biogenic amines. in contrast, in invertebrates, unlike in mammals, there are no organs involved in the stress response, such as the hypothalamus, pituitary and adrenal glands; in invertebrates all the stress response related molecules are concentrated in immunocytes (ottaviani and franceschi, 1996). unity of living beings and the “game” theory of evolution at all times, biologists have been fascinated by the extraordinary variety of living forms. from the time when lucretius asked: "how can there be this infinite variety of things and what is behind this incredible variety of shapes and sizes?", philosophers asked themselves this important question over and over again, searching for more general unifying principles. one of the solutions that has emerged since the time of epicurus and lucretius has been to assume that it is the arrangement of the constituent elements and the relationships among them that generates different and new forms of living. in the case of biology the unifying principle was initially given by the cell: all living organisms are composed of cells that work basically in the same way, at least with regard to the main functions and properties, such as the production of protein and energy, proliferation, etc. today, however, we have come to the realization that the unification of living organisms is based on precise molecular mechanisms, that is, fundamental information making up all living beings, is contained in the nucleic acids. how then can we reconcile the unity of the living and the variety of forms of the species? such a result is obtained from nature in accordance with laws and extremely economic strategies that are revealed to us by what is the fundamental approach to the study of the living, i.e., evolution. traditionally, the study of evolution is presented as the study of changes that have gone through the taxa and different species in their story on earth. this approach emphasizes what changes and, at the molecular level, what mechanisms allow the accumulation of different molecular variants fundamental for the selection of mechanisms that govern them. however, the enormous mass of molecular data available today, suggests we should adopt a rather opposite approach supporting conservation compared to changes (ottaviani et al., 2001). in order to understand the most strategically 187   fig. 1 the biologically active peptides (bap) mediate the interactions among the immune (is), the neuroendocrine (nes) and gut (gs) systems. important biological functions, it is thus more interesting to search for what has been preserved rather than what has changed, which is true, for example, for most of the different proteins. overall, the data available today, as reported in this manuscript, seem to indicate that the major signaling molecules (neuropeptides, hormones, cytokines) have been preserved in an extraordinary way in the course of evolution. this suggests that, despite the progressive complication of organs and systems, the mechanisms that govern the exchange of information between cells, remained essentially unchanged. in other words, the flow of information among the different cell types that make up higher organisms, such as vertebrates, is exchanged using the same pool of signaling molecules. more importantly, animals apparently much less complex, such as the molluscs, use the same type of signaling molecules to exchange information. finally, lucretius’ statement still holds true: "what happens with the words and verbs is also true for the elements of matter: it is the combinatorial change of their movement, order and position which explains the very essence of beings and species". concluding remarks from this brief survey of baps emerges that a common pool of baps have been employed since the beginning of evolution to set up an integrated type of response capable of maintaining body homeostasis. alternative use of the same molecules for different functions is a widespread, economical strategy used throughout evolution. normally this hypothesis is supported by research studying the immune and neuroendocrine systems, but as 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the sequence included an open reading frame of 4,497 bp, encoding a protein of 1,498 aa with a calculated mass of 173 kda. structural analysis revealed that lvduox contains several domains. homology analysis revealed that lvduox exhibits 96.1%, 67.3% and 67.3% sequence identity with marsupenaeus japonicas, drosophila melanogaster and acyrthosiphon pisum duox, respectively. the mrna transcripts of lvduox were detected in all tested tissues. the mrna expression of lvduox was significantly induced in the midgut after vibrio parahaemolyticus e1 (vpe1) stimulation. after the level of h2o2 in the midgut increased, expression of the superoxide dismutase and catalase genes in the midgut increased significantly. these results suggested that the lvduox gene was upregulated in the midgut after the challenge by vpe1, and antioxidant genes were involved in the regulation of ros in the shrimp midgut. lvduox may therefore be a new target for intestinal disease research in the pacific white shrimp. key words: litopenaeus vannamei; duox; antioxidant gene; innate immunity introduction in recent years, owing to water pollution and overuse of antibiotics, the intestines of aquatic animals have been exposed to substantial threats and challenges. in the pacific white shrimp litopenaeus vannamei, the gut is surrounded by various types of bacteria because of its open anatomical structure. some pathogens and viruses induce inflammation in the mucosa (qi et al., 2017), thus resulting in damage to the guts of shrimps. vibrio harveyi, vibrio alginolyticus and vibrio parahaemolyticus e1 (vpe1) are common pathogenic bacteria affecting the production of farmed prawns (martin et al., 2004; qi et al., 2017), and they pose a great threat to shrimp health. after the balance of intestinal flora is altered, the immune and digestive systems of shrimp may be affected ___________________________________________________________________________ corresponding author: lei w ang key laboratory of experimental marine biology institute of oceanology chinese academy of sciences qingdao 266071, china e-mail: wanglei@qdio.ac.cn (artis, 2008; miyake et al., 2014; you et al., 2014). therefore, it is crucial to maintain gut-microbiota homeostasis (van baarlen et al., 2013; meng et al., 2018) and determine the mechanism of defense against invading pathogens. the reactive oxygen species (ros), which are produced by dual oxidase (duox), have been suggested to be involved in inhibiting pathogenic bacterial infection in the gut (ha et al., 2005; yang et al., 2016). ros include oxygen radicals and some oxidizing agents formed by the partial reduction of oxygen, such as superoxide (o2 -), hydroxyl (oh-), ozone (o3) and superoxide-derived hydrogen peroxide (h2o2) (juven et al., 1996; skulachev, 1998). they can damage the structure of dna and the membrane system in eukaryotic cells, and induce lipid peroxidation (wang et al., 2009). therefore, ros are considered to be a major cause of damage in organisms. five homologs of nicotinamide adenine dinucleotide phosphate (nadph) oxidase (nox1, nox2, nox3, nox4 and nox5) and two homologs of dual oxidase (duox1 and duox2) can produce ros (morand et al., 2009). duox, a transmembrane protein in host cells, is the 317 main enzyme that generates ros in epithelial cells of many organs (flores et al., 2010; kim et al., 2014). the structural organization of duox is highly conserved in all studied organisms (kim et al., 2014). duox comprises a transmembrane domain, a calcium-modulated ef hand domain, a nadph oxidase domain producing h2o2 and an extracellular peroxidase homology domain that converts h2o2 into hocl (sumimoto, 2008). h2o2 and chloride are important components of host gut immunity. in a study of duox knockdown in flies, duox has been found to be responsible for host resistance to gut infection in the gut epithelia (biteau et al., 2008; buchon et al., 2009; kim et al., 2014). in drosophila, h2o2 participates in the regulation of intestinal epithelial cell renewal by activating intestinal stem cell proliferation (abid et al., 2000). in anopheles gambiae, duox-dependent h2o2 is involved in gut permeability by forming a dityrosine network at the peritrophic membrane (kumar et al., 2010). in zebrafish, duox-produced h2o2 facilitates wound healing and has an antimicrobial function (niethammer et al., 2009; flores et al., 2010). in addition, in pacific white shrimp, h2o2 is produced and has a role in anti-microbial activity after pathogens enter the hemolymph (munoz et al., 2000; gomez-anduro et al., 2006). because the pacific white shrimp is a crustacean that relies on its innate immune system, cloning the duox gene has important implications for studies investigating resistance to pathogen invasion. however, the existence of duox gene in the pacific white shrimp had not been verified, and the mechanism of duox gene expression in the pacific white shrimp was unclear. in the present study, we cloned the full-length cdna encoding the duox gene from l. vannamei, which we designated lvduox. additionally, we investigated the expression of the duox gene and two antioxidant enzymes (superoxide dismutase and catalase) genes in the gut of l. vannamei after infection by vpe1. moreover, we detected h2o2 levels at different times after vpe1 challenge to determine the role of lvduox in the natural immune defense mechanisms in the gut of l. vannamei. the results may provide a new therapeutic target for intestinal diseases of l. vannamei. materials and methods animals and vibrio parahaemolyticus e1 challenge adult litopenaeus vannamei (average weight 11±0.12 g) was obtained from ruizi seafood development co. ltd. (qingdao, china). before the experiment, the shrimp were randomly allocated to six tanks (each containing 50 l seawater), including three control tanks (c1, c2 and c3) for the gene cloning, and three treatment tanks (v1, v2 and v3) for vpe1 challenge, with forty prawns in each tank, and the shrimps were acclimatized at 28±1 °c in aerated and filtered seawater (salinity 30‰) for a week and fed commercial pellets (supplied by da le co. ltd, yantai, china). during challenge, vpe1 was added into each treatment tank at a final concentration of 5×106 cfu/ml. the vpe1 strain was donated by dr. zhaolan mo from yellow sea fisheries research institute chinese academy of fishery sciences. rna extraction and cdna preparation total rna was extracted using a minibest universal rna extraction kit (takara, dalian, china) according to the manufacturer’s instructions. rna degradation and contamination were monitored on 1% agarose gels. the rna purity and concentration of each sample were checked using a nanophotometer® spectrophotometer (implen, munich, germany). the cdna synthesis was carried out by transscript ii one-step gdna removal and cdna synthesis supermix (transgen biotech, china) in accordance with the manufacturer’s instructions, then stored at -20 °c and used as the temple for cloning and pcr analysis. cloning and sequencing the template of cloning was the cdna of gut from the healthy shrimp. the partial sequence of lvduox cdna was obtained from the transcriptome database of our laboratory. the primers were designed based on this partial sequence by primer premier5.0 (table 1). two pairs of gene-specific primers, d501/502r and d301/302f, were used to clone the 5’ end and 3’ end of cdna of lvduox by the rapid amplification of cdna ends (race) technique according to the standard procedures. the other primers for cloning were designed based on the results of 5’ end and 3’ end, and were used by cloning the full-length cdna of the lvduox by pcr with the primestar® gxl premix (takara bio, inc., japan). the pcr products were cloned into the peasy®-t1 simple cloning vector (transgen biotech, china) and transformed into the trans5α chemically competent cell (transgen biotech, china), the positive recombinants were identified via anti-ampicillin selection. a sequence analysis was performed using a ceq 8000 automated sequencer (beckman coulter, inc., usa). the sequence similarity of cdna was analyzed using fasta and the basic local alignment search tool (blast) at the national center for biotechnology information (ncbi). the theoretical isoelectric point and molecular mass were calculated using the expasy proteomics server (http://web.expasy.org/compute_pi). the structural domains of the white shrimp lvduox were predicted using the simple modular architecture research tool (smart; version 7.0) (http://smart.emblheidelberg.de/). using the mega 7.0 software package, a neighbor-joining (nj) phylogenic tree was constructed using the full-length amino acid sequences of some published duox proteins downloaded from ncbi, and multiple sequence alignments of some duox proteins from ncbi using the bioedit software package. detecting the level of h2o2 to detect the level of h2o2 in the midgut of shrimp at different hours after the vpe1 challenge, three midguts were obtained from shrimps at 0 h, 3 h, 6 h, 12 h, 24 h and 36 h during the vpe1 stimulation. then, the samples were measured with a hydrogen peroxide assay kit (nanjing jiancheng bioengineering institute, nanjing, china) according to the manufacturer’s instructions. http://web.expasy.org/compute_pi http://smart.emblheidelberg.de/ 318 table 1 primers used for the pacific white shrimp lvduox analysis in the paper primers sequence(5’-3’) primers for 5’race d501r gttgttgtaccagccatcgt d502r gccgagcacaatccatctg primers for 3’race d301f catcttcatcttcgcgcacc d302f atctggtcttcggaacgtcg universal primers for race nup aagcagtggtatcaacgcagagt upml ctaatacgactcactatagggcaagcagtggtatcaacgcagagt upms ctaatacgactcactatagggc 3’cds aagcagtggtatcaacgcagagtac(t)30v n 5-ap-dg aagcagtggtatcaacgcagagtgggggggggghn primers of cloning vector m13f tgtaaaacgacggccagt m13r caggaaacagctatgacc primers for cloning the rest of sequence d503f tcagatggattgtgctcggc d504f cagatggattgtgctcgg d505f ttatttccagggctctgaagtgacg d506f agaacttccgcaggaggcattt d303r cacttcagagccctggaaataatca d304r aagagccagtagcccacggt d305r agatttcctgcgtcagacacct primers for qrt-pcr analysis lvd1f atcagatggattgtgctcggc lvd1r gactcaacggagccccaaga sod1f tgacgagagctttggatcattcc sod1r tgatttgcaagggatcctggtt cat1f ggctatggttctcgtacttccaagc cat1r gcattgtataggtcccttgttgca β-actin1f gcccatctacgagggata β-actin1r ggtggtcgtgaaggtgtaa gene expression by qrt-pcr to analyze the expression of lvduox in various tissues of the white shrimp, including heart, hepatopancreas, intestine, eyestalk, gills and proventriculaus, these tissues were obtained from three healthy white shrimps. to analyze the expression of lvduox, sod and cat of the midgut after it affected the vpe1, the midguts of shrimp (six shrimp per group) were extracted at 0 h, 3 h, 6 h, 12 h, 24 h and 36 h after the vpe1 challenge. the genetic expression of all the temples from these tissues was determined by quantitative real-time pcr (qrt-pcr). all the temples were carried out in triplicate with a total volume of 20 μl containing 10 μl 2×transstart® top green qpcr supermix, 2 μl of cdna, 0.4 μl each of the forward and reverse primers (final concentration 0.2 μm) and 7.2 μl of ddh2o. the qrt-pcr using sybr green i dye (transgen biotech, china) was performed using the following cycling conditions: denaturation for 30 s at 94 °c, followed by 40 cycles of 5 s at 94 °c, and 30 s at 60 °c. the gene expression analysis primers of lvduox, cat and sod are listed in table 1. 319 fig. 1 the nucleotide and deduced amino acid sequences of the l.vannamei dual oxidase (lvduox) cdna. the sequence has been deposited in the genbank (accession number mg734366). the cdna (4735 bp) contains a complete orf encoding a protein of 1,498 amino acid residues (residue number indicated on the left). the start codon atg and the stop codon taa are indicated by the rectangle. the primer sequences for the qrt-pcr analysis are indicated by the solid lines 320 fig. 2 comparison of the predicted domain structures of dual oxidases from different organisms. the names of the different domains are marked. the full name, abbreviation and accession number of different duoxs are listed in the table 3 statistical analysis the expression of β-actin gene was used as the reference gene of all the samples, and the comparative ct method (2-δδct) was used to analyze the expression level of lvduox and the other genes. the results are expressed as means ± standard deviation (sd). to compare the differences between the data of different groups in different hours, the statistical analysis of these data was performed by one-way analysis of variance (one-way anova) using spss statistics 24.0 software. the p < 0.05 was considered statistically significant. results sequence and domain structures of lvduox a 4,735 bp nucleotide sequence of lvduox was assembled and included an open reading frame (orf) of 4,497 bp, encoding a protein of 1,498 aa with a calculated molecular mass of approximately 173 kda and a theoretical isoelectric point of 6.98 (fig. 1). the cdna sequence of lvduox has been deposited in genbank under accession number mg734366. to determine the similarity of the complete domain structure of lvduox to those of other duoxs, we predicted the structural domains of the duoxs from different animals by using the simple modular architecture research tool (fig. 2). the deduced amino acid sequence of lvduox contains a signal peptide (1–21 aa), a peroxidase-like domain (33-557 aa), two transmembrane regions (593–615 aa and 988–1,010 aa), a coiled coil (726–766 aa), three ef-hand motifs (calcium binding region: 818–846 aa, 854–882 aa and 899–927 aa), a ferric reduction region (1,031–1,178 aa), a fad binding domain (1,214–1,317 aa) and a nadph binding domain (1,323–1,479 aa). the structural domains of lvduox were nearly the same as those of mjduox, and the peroxidase-like domain, transmembrane segment, ferric-reductase domain, fad binding domain and nadph binding domain were conserved among lvduox and the other duox proteins. a coiled coil was found only in lvduox and mjduox. moreover, the signal peptide was not present in dmduox and drduox1. as for the calcium binding region, three arthropod duoxs (lvduox, mjduox and dmduox) were predicted to have three ef-hand motifs, and the others were predicted to have two ef-hand motifs. 321 fig. 3 comparison of the amino acid sequence of dual oxidases from the pacific white shrimp and the others organisms using the clustalw program of bioedit software. the full name, abbreviation and accession number are listed in the table 3 322 table 2 amino acid identity of the pacific white shrimp lvduox gene compared to the others known duoxes sequences sequence homology and phylogenetic analysis sequence alignment was performed to determine the sequence identity of lvduox compared with the other duox proteins (fig. 3). lvduox shares 96.1% sequence similarity with mjduox, 67.3% with the insect duox (dmduox and apduox-like), 35% with the chordate duox (ciduox-b) and 36.2–39.2% with the other duoxs (table 2). to elucidate the evolutionary relationships between lvduox and other duoxs, a neighbor-joining phylogenic tree was constructed by using sequence alignments in mega software (fig. 4). in this phylogenic tree, lvduox formed a cluster with arthropod duoxs, including mjduox, dmduox, tcduox and cfduox. the xtduox2, btduox2 and the other duoxs formed another cluster. fig. 4 the neighbor-joining (nj) phylogenic tree constructed using mega 7.0 software package based on the amino acid sequences of duoxs from different organisms. the numbers at the forks indicated the bootstrap value. the scale bar represents the proportion of amino acid differences between sequences based on nucleotide substitutions per site. the species and protein sequences id are listed in table 3 entire duox 1 2 3 4 5 6 7 8 9 10 11 12 1.lvduox 2.mjduox 96.1% 3.dmduox 67.3% 67.0% 4.apduox-like 67.3% 66.9% 68.8% 5.spduox1 37.2% 37.3% 36.6% 35.6% 6.ciduox-b 35.0% 35.1% 33.4% 34.5% 37.2% 7.xtduox1 37.0% 36.6% 36.9% 35.5% 38.3% 43.5% 8.xtduox2 36.2% 35.8% 35.1% 35.7% 35.6% 40.6% 56.6% 9.cjduox2 38.1% 37.9% 37.5% 36.3% 39.4% 43.6% 61.5% 57.9% 10.hsduox1 39.2% 39.3% 37.3% 36.0% 39.0% 42.2% 60.5% 56.2% 64.7% 11.hsduox2 37.9% 37.8% 36.1% 35.0% 39.2% 41.8% 59.7% 56.4% 65.8% 77.2% 12.pcduox2 37.6% 37.8% 36.4% 34.3% 38.7% 42.5% 60.0% 56.0% 65.9% 74.8% 87.4% 323 table 3 amino acid sequence numbers, symbols, genbank accession numbers and nomenclatures used in the paper symbol accession number nomenclature 1.lvduox mg734366 litopenaeus vannamei 2.mjduox ab744213 marsupenaeus japonicus 3.dmduox np_608715 drosophila melanogaster 4.spduox1 np_001118237 strongylocentrotus purpuratus 5.drduox1 baf33370 danio rerio 6.cjduox2 xp_015727798 coturnix japonica 7.hsduox1 aai14939 homo sapiens 8.hsduox2 eaw77288 homo sapiens 9.btduox2 daa25263 bos taurus 10.rnduox1 aan33120 rattus norvegicus 11.rnduox2 np_077055 rattus norvegicus 12.ggduox2 xp_425053 gallus gallus 13.xtduox1 xp_002937936 xenopus (silurana) tropicalis 14.xtduox2 xp_002937935 xenopus (silurana) tropicalis 15.cfduox efn70161 camponotus floridanus 16.tcduox xp_970848 tribolium castaneum 17.apduox-like xp_001951113 acyrthosiphon pisum 18.ciduox-b faa00329 ciona intestinalis 19.bmduox jq768349 bombyx mori 20.pcduox2 xp_007121449 physeter catodon fig. 5 pacific white shrimp lvduox expression in various tissues of healthy shrimps (n=3). tissue distribution of cdna of lvduox was detected using quantitative real-time pcr. β-actin gene was used as the reference gene, and vertical bars represented mean ± sd 324 fig. 6 the levels of the h2o2 in the midgut of the pacific white shrimp following affected the v. parahaemolyticus e1 (vpe1). the level of the h2o2 was detected at different hours (0-36 h) using a hydrogen peroxide assay kit according to the manufacturer’s instructions analysis of lvduox expression in various tissues the qrt-pcr was used to detect the tissue distribution of lvduox gene expression, by using the β-actin gene as a reference. the expression levels of the lvduox gene were observed in different tissues, such as the heart, hepatopancreas, intestine, eyestalk, gills and proventriculus. the results showed that the expression of lvduox was higher in the gills than in the other tissues (fig. 5). h2o2 levels in the midgut after vpe1 challenge before vpe1 challenge, h2o2 was present at a low level in the shrimp midgut (0.45 mmol/g prot). after vpe1 stimulation, it increased at 3 h and peaked at 6 h (0.88 mmol/g prot), then declined gradually afterward (fig. 6); the results were consistent with the expression of the lvduox gene (fig. 7). analysis of expression of lvduox and antioxidant genes after vpe1 challenge during vpe1 challenge, the midguts of pacific white shrimps were obtained at 0 h, 3 h, 6 h, 12 h, 24 h and 36 h. the expression levels of the lvduox gene, superoxide dismutase (sod) gene and catalase (cat) gene in the shrimp midgut were determined using quantitative real-time pcr (fig. 7). the relative gene expression level of lvduox in the midgut increased significantly at 3 h (5.03+0.41) and 6 h (5.33+0.4) (p<0.05), then decreased gradually at 12–36 h. the expression of the sod gene decreased 3–6 h after the vpe1 challenge, then began to increase at 12 h (2.16+0.21), peaked at 24 h (3.53+0.22) (p<0.05) and then decreased significantly at 36 h. the expression level of the cat gene at 6–12 h was lower than that at 0–3 h, but significantly increased at 24 h (2.32+0.28) (p<0.05), then decreased to normal levels. discussion duox has been studied extensively in many model species, but there have been few reports in commercial aquatic animals. the role of duox in the innate immunity of the pacific white shrimp remains unknown. in this study, the full-length sequence of the duox gene of the pacific white shrimp was cloned and named lvduox, and was deposited in genbank under accession number mg734366 (not released). the orf was 4,497 bp and encoded a 1,498 amino acid protein with a theoretical mass of 173 kda, results similar to those for marsupenaeus japonicus (~173 kda), bombyx mori (~172 kda), danio rerio (~172 kda) and drosophila melanogaster (~178 kda). the amino acid sequence of lvduox has a higher identity to duox from arthropods than from other species. both structural domain comparison and sequence alignment indicated that lvduox was more similar to crustacean mjduox than to other duoxs (inada et al., 2013). thus, the duox gene appears to be highly conserved in different kinds of shrimp. the analysis of structural domains of lvduox revealed that a peroxidase domain, the transmembrane segment, the calcium binding region, a ferric reduction region, a fad binding domain and a nadph binding domain were conserved, and the signal peptide also was present in many duoxs expected for bmduox and drduox1. there was a coiled coil in lvduox and mjduox. the coiled coil is a structural motif in proteins, in which 2-7 alpha-helices coil together like the strands of a rope, 325 fig. 7 the expression of the genes (lvduox,sod and cat) in the midgut of the pacific white shrimp following affected the v. parahaemolyticus e1 (vpe1). the expression levels of the genes were detected at different hours (0-36 h) using quantitative real-time pcr, and β-actin gene was used as the reference gene, and differences were considered significant at *p<0.05 and dimers are common. the coiled coil plays a major role in cell recognition and signal transduction. therefore, the coiled coil may be a special domain distinguishing the duoxs of shrimps from those of other organisms. the nadph oxidase domain can produce h2o2, whereas the peroxidase domain can convert h2o2 into hocl. h2o2 and hocl aid in resistance to the intrusion of pathogens and provide an important immune defense mechanism in organisms that is necessary for the adaptive immune response. the calcium binding region formed by three ef-hand motifs was predicted in three arthropod duoxs (lvduox, mjduox and bmduox), whereas the others contained two ef-hand motifs (fig. 2). intracellular concentrations of ca2+ modulate bmduox enzymatic activity via the ef-hand motifs (hu et al., 2013). thus, we believe that the ef-hand motifs of lvduox may be involved in the response to ca2+ in a manner similar to the mechanism in the fruit fly. the mrna transcripts of lvduox gene were observed in all the detected tissues. lvduox had high expression in the gills, a respiratory organ that, like the intestine, directly contacts water and bacteria. in the midgut of shrimp infected by vpe1, the expression of lvduox increased significantly at 3 h after infection (p<0.05), peaked at 6 h (p<0.05), then began to decline and returned to its original level at 36 h. the trends in h2o2 levels in the midgut were consistent with the expression level of the lvduox gene. vpe1 stimulated the expression of lvduox, and h2o2 from lvduox participated in resisting the invasion of vpe1. as the sod and cat gene expression increased significantly between 12 and 24 h (p<0.05), the level of h2o2 declined gradually. we concluded that the high concentration of ros in the midgut induced the response of the antioxidant system to protect the organism from oxidative damage. initial research has revealed that the production of o2 in the hemocytes of pacific white shrimps is dependent on the concentration of bacteria (escherichia coli) (munoz et al., 2000). in addition, the expression of kuruma shrimp mjduox increases after white spot syndrome virus injection (inada et al., 2013). thus, our results suggested that foreign pathogens stimulate the expression of lvduox to participate in innate immunity, and the antioxidant genes regulate h2o2 levels, thus protecting the shrimp against oxidative damage induced by ros. in conclusion, we cloned the full-length cdna encoding lvduox. on the basis of sequence alignment and phylogenetic analysis, the lvduox was found to be highly conserved and to be more similar to arthropod duoxs than to vertebrate and echinoderm duoxs. the lvduox gene was expressed in all the main organs of the white shrimp and responds to invading pathogenic bacteria in the midgut. two antioxidant genes were involved in the regulation of h2o2 levels generated by lvduox. therefore, lvduox may be a new target for intestinal disease research. more studies are needed to clarify the regulatory mechanism of lvduox in the innate immunity system and to determine how to accurately control the expression of duox in shrimp to protect cells from ros damage. 326 acknowledgement this work was supported by the science and technology development fund project of shinan district of qingdao city (2018-4-001-zh), and the chinese academy of sciences sts regional center project (fujian province). we are grateful to all the laboratory members for their technical advice and helpful discussion. competing financial interests the authors declare no competing financial interests. references abid mr, kachra z, spokes kc, aird wc. nadph oxidase activity is required for endothelial cell proliferation and migration. febs lett. 486: 252-256, 2000. artis d. epithelial-cell recognition of commensal bacteria and maintenance of immune homeostasis in the gut. nat. rev. immunol. 8: 411-420, 2008. biteau b, hochmuth ce, jasper h. jnk activity in somatic stem cells causes loss of tissue homeostasis in the aging drosophila gut. cell stem cell 3: 442-455, 2008. buchon n, broderick na, chakrabarti s, lemaitre b. invasive and indigenous microbiota impact intestinal stem cell activity through multiple pathways in drosophila. 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and the immune system in drosophila. curr. opin. immunol. 30: 48-53, 2014. 210 isj 14: 210-220, 2017 issn 1824-307x research report combined toxicity of cadmium and lead on early life stages of the pacific oyster, crassostrea gigas j xie1,2,3, d yang1,2,3, x sun2, r cao1,2,3, l chen1,2,3, q wang1,2, f li1, c ji1, h wu1, m cong1, j zhao1,2 1key laboratory of coastal environmental processes and ecological remediation, yantai institute of coastal zone research (yic), chinese academy of sciences (cas), shandong provincial key laboratory of coastal zone environmental processes, yiccas, yantai 264003, pr china 2muping coastal environment research station, yantai institute of coastal zone research (yic), chinese academy of sciences (cas), pr china 3university of chinese academy of sciences, beijing 100049, pr china accepted may 8, 2017 abstract trace metals cause toxic effect on the early development period of marine animals, however only few studies address the toxic interactions of trace metals on bivalves. in the present study, the individual and combined toxicities of dissolved cadmium (cd) and lead (pb) on early life stages of the pacific oyster crassostrea gigas have been investigated. embryotoxicity, larvae mortality and genotoxicity were measured under single and combined exposure of the two tested metals. for embryotoxicity, the median effective concentration (ec50) values for individual cd, pb and their mixture were of 272.2 µg/l, 660.3 µg/l and 373.1 µg/l, respectively. the median lethal concentrations (lc50) for 96 h larval mortality were determined to be 353.3 µg/l, 699.5 µg/l and 205.5 µg/l for individual cd, pb and their mixture, respectively. moreover, the marking-dawson additive toxicity indices were 0.10 for embryogenesis and 1.40 for larval mortality indicating, respectively, an additive effect and a trend to synergism for the cd and pb combination. furthermore, dna strand breaks were observed in oyster embryos after individual cd, pb and their mixture exposure, and a significant positive correlation was demonstrated between embryotoxicity and genotoxicty. the current study suggests the toxicity of cd is higher than that of pb, and the cd-pb mixture is slightly more toxic than individual cd or pb to the pacific oyster. these data will be helpful to predict the toxicity of metal mixtures, and provide biological criteria for the implementation of marine water quality standards to protect these marine organisms. key words: embryotoxicity; larval mortality; genotoxicity; trace metals; mixture; crassostrea gigas introduction aquatic organisms are constantly exposed to complex mixtures of chemical pollutants dissolved in the water and tolerate their potential toxic impacts. ___________________________________________________________________________ corresponding authors: qing w ang jianmin zhao key laboratory of coastal environmental processes and ecological remediation yantai institute of coastal zone research (yic) chinese academy of sciences (cas) shandong provincial key laboratory of coastal zone environmental processes yiccas, yantai 264003, pr china muping coastal environment research station yantai institute of coastal zone research (yic) chinese academy of sciences (cas), pr china e-mail: qingwang@yic.ac.cn e-mail: jmzhao@yic.ac.cn among the chemical pollutants, trace metals (e.g., cadmium and lead) represent one of the most widespread and serious forms of environmental contamination (devi et al., 1996; zaki et al., 2016). both cd and pb are abundant, non-essential elements that are continuously accumulated in the environment as a result of industrial activities. they are classified as human and animal carcinogens by the us epa and international agency for research on cancer (iarc) (tchounwou et al., 2012). bivalve species, particularly their early life stages, are widely used for aquatic environmental bioassays because of their high susceptibility and ecological relevance to chemical toxic pollutants (his et al., 1999a; beiras et al., 2003). therefore, the early life stages of the pacific oyster crassostrea gigas have been widely used to assess the toxicity of pollutants, such as trace metals, pesticides, mailto:jmzhao@yic.ac.cn 211 herbicides, pahs and synthetic estrogenic hormones (bellas, 2006; wessel et al., 2007; akcha et al., 2012; mai et al., 2012, 2014; gamain et al., 2016). although many studies have demonstrated the toxicities of cd and/or pb to invertebrate embryos and larvae such as sea urchins (fernandez and beiras, 2001), mussels (beiras and albentosa, 2004; nadella et al., 2009), clams ( wang et al., 2009; fathallah et al., 2013), ascidians (bellas et al., 2001) and crab (bakker et al., 2017), the toxic effects of cd and pb on the early life stages of the pacific oyster are not reported. in fact, the marine environments are normally affected by more than one trace metal, and information on their interactions provides a more realistic assessment of their toxicity to marine organisms. the toxicity of a chemical can be enhanced (positive interaction or synergism), reduced (negative interaction or antagonism), or unaffected (no interaction) by the presence of another toxicant (marking and dawson, 1975). if two chemicals in a mixture do not affect the toxicity of each other, simple joint action is present. therefore, the combined toxicities of two chemicals are additive, which means their toxicities may be predicted by the summation of toxic potencies measured in toxicity units [tu]. previous studies showed that the combination of metals such as cu-ag/zn, mn-mo, hg-cd/pb/cu, cd-pb and zn-cd had significantly toxic effect on aquatic organisms (macinnes et al., 1981; coglianese and martin, 1981; morgan et al., 1986; fernandez and beiras, 2001; prato and biandolino, 2007; fathallah et al., 2013; gamain et al., 2016). however, to our knowledge, there is no literature about combined effects of cd and pb on early life stages of pacific oyster. the bohai sea is a nearly enclosed interior sea located in northeast china and is a well-known area for bivalves farming such as clams, oysters and scallops. however, contaminations by metals (such as as, cd, cr, cu, ni, pb and zn) have been reported in coastal areas and estuaries of bohai sea (gao et al., 2014). the concentration of cd in seawater of bohai sea ranged from 0.007 to 5.0 µg/l, and the concentration for pb was 0.058~27.17 µg/l (table 1). moreover, the cd and pb concentrations were 0.02~1463 µg/g and 0.62~1828 µg/g respectively in sediments. in order to compare the toxic effect of individual and combined metals to early life stages of crassostrea gigas, the embryotoxicity (percentages of abnormal larvae), genotoxicity (dna damage) and larval mortality of the oyster were investigated. in addition, the correlation between genotoxicity and embryotoxicity data was also analyzed. materials and methods chemicals and seawater reference toxicants (cdcl2 and pb(no3)2), formalin and dmso (dimethyl sulfoxide) were purchased from sinopharm chemical reagent (analytical grade, china). dispase, triton x-100, low melting point (lmp) agarose, normal melting point (nmp) agarose, and mem-alpha (minimum essential medium) were purchased from solarbio (beijing solarbio science & technology co., ltd, table 1 the concentrations of tested metals in bohai sea and this study (µg/l) cadmium lead bohai sea 0.007~5.0 0.058~27.17 this study 0.35 1.32 concentrations (µg/l) of cadmium and lead in bohai sea reported by gao et al. (2014), and measured in yantai in may 2015 (present study). china). seawater was collected from the coastal area of yantai (china) that is populated by natural oysters. then the seawater was filtered using membrane filter of 0.2 µm to eliminate debris and microorganisms. chemicals exposure and analysis metal stock solutions (100 mg/l) were prepared by dissolving cdcl2 and pb(no3)2 into double distilled water. for the exposure experiments, these working solutions were then diluted to reach the final tested concentrations. in the test of single toxicity, five concentrations of each chemical in geometric progression were prepared. for the combined exposure experiment, five treatments were prepared by addition of equal fractions of the chemical concentrations tested in the individual series. a preliminary study was conducted to define the lowest exposure concentration. briefly, according to the average concentration of cd and pb in bohai sea, the embryos and d-shaped larvae were exposed to 0.02, 0.2, 2, 20 and 200 µg/l cd and pb, respectively. then the percentage of normal d-shaped larvae and mortality were calculated and the lowest exposure concentration was determined as 20 µg/l for cd and pb. actual concentrations of cd and pb in the experimental solutions were measured according to the method of “the specification for marine monitoring” (gb17378 4-2007) by anodic stripping voltammetry (asv). the asv measurements were carried out using a voltammeter (va 797 computrace, metrohm inc.). the recoveries were 97 %~102 %, 90 %~98 % for cd and pb, respectively. all analyses were carried out thrice. the concentrations of metals tested in the individual and mixed series were summarized in table 2. embryotoxicity assay the oysters were purchased from a local aquaculture farm in yantai (shandong, china). mature male and female oysters were stripped to get the gonad. spermatozoa and oocytes were collected from 10 individuals and sieved separately through a 50 µm and 100 µm meshes, respectively. the eggs were fertilized with sperms in a ratio of 1:10 in filtered seawater. fertilization success was verified under microscope and embryos were placed in contaminated seawater and transferred to beakers for embryotoxicity and genotoxicity assays. each experiment was replicated three times using three different batches of oysters. for embryotoxicity assay, 212 fig. 1 oyster d-shaped larvae at 24 h post of fertilization. normal (a); abnormal: protruding mantle (b), convex hinge (c), indented shell margin (d). approximately five hundreds of embryos were incubated for 24 h (at 24 °c in the dark) in beakers containing 200 ml of filtered seawater. there were three replicates for each contamination exposed group and control. this incubation time enables the embryos to develop to the d-shaped larvae stage. following the exposure, the larvae of each beaker were fixed using 8 % formalin (0.5 ml/beaker) and the percentage of abnormal oyster larvae was recorded. for each assay beaker, three hundreds of larvae were directly observed under an inverted microscope (olympus, magnification ×200) to determinate the percentage of developmental arrests (stages blastula, gastrula or trocophore), normal and abnormal d-shaped larvae, such as incomplete shell, indented shell margin, protruding mantle and convex hinge (fig. 1), according to the criteria described by his et al. (1999a) and wessel et al. (2007). the median effective concentration (ec50) defined as the metals concentration that resulted in a 50 % reduction in normal d-shaped larvae number for each individual pollutant and the mixture. the calculation of ec50 was normalized to the mean percentage of larval abnormality in the control group using abbot’s formula (emmens, 1948), p = (pe-pc)/(100-pc) × 100, where pc and pe are control and experimental percentage response, respectively. the ec50s values and the lowest observed effective concentration (loec) were calculated by the probit method (newman, 1995) with spss 16.0 statistical software. comet assay for genotoxicity assays, embryos were incubated in 250 ml beakers at 24 °c for 16 h in the dark. this incubation time enables the embryos to reach unshelled larvae that can be enzymatically digested for comet assay. three replicates were performed per treatment, and each replicate contained a total of 20 ×104 larvae. cell isolation was performed by the method described by wessel et al. (2007) with slight modifications. following 16 h of exposure, the embryos were recovered by sieving with a 50 µm mesh. one milliliter of embryo suspension (about 2000 embryos) was incubated with 1 ml of collagenase solution at concentration of 1 g/l for 20 min at 37 °c with gentle shaking (150 rpm). the reaction was stopped by centrifugation for 10 min at 1300 rpm (4 °c). the cell pellet was then suspended in 1 ml of minimum essential medium (mem) at a final cell density of about 10×104 cells/ml. cell viability was determined for each sample by trypan-blue exclusion test (wessel et al., 2007). comet analysis was only carried out with cell suspension with good cell viability (trypan blue exclusion test > 80 %). the comet assay was performed on isolated cells following the method proposed by akcha et al. (2003), with some modifications. briefly, 50 µl of cell suspension (about 5000 cells) was added to 100 µl of 1 % low melting point (lmp) agarose solution, and then 85 µl of this mixture was deposited on a frosted microscope slide 213 table 2 nominal and measured concentrations of metals in the embryotoxicity, larval mortality and genotoxicity assays data are presented as mean ± standard deviations (n = 3). pre-coated with 1 % normal melting point (nmp) agarose. the slides were kept at 4 °c for 5 min to allow the agarose to solidify. then 90 µl aliquot of 1 % lmp were pipetted on the pre-coated slide. after the solidification of agarose, the slides were placed in ice-cold lysing solution in the dark at 4 °c for 1 h. alkaline treatment was performed for 20 min to allow dna unwinding in a freshly prepared electrophoresis buffer. electrophoresis was carried out at 25 v, 300 ma, for 20 min. slides were analyzed at 400× magnification using an optical fluorescence microscope (olympus bx 51) and an image analysis system (komet 5.5, kinetic imaging ltd.). dna damage was expressed as the percentage of total dna that has migrated from the head (tail dna %). a hundred of randomly selected nucleoids was analyzed on two replicate gels. larval mortality after fertilization, the zygotes were filtered with a 50 µm mesh and gently washed three times with filtered seawater, and then re-suspended in filtered seawater (fsw) at 24 °c. after 24 h of incubation, the d-shaped larvae were obtained and re-suspended in 250 ml glass beakers (approximatively 2×103 larvae/beaker), each containing 200 ml of different concentrations of trace metal solution (table 2). there were three replicates for the control and contamination exposed groups. the d-shaped larvae were fed with isochrysis spp at a concentration of 1 10×104 cells/ml three times daily. after exposure for 96 h, the larvae were sampled and anaesthetized using magnesium chloride solution to make them static. then the percentage of larval mortality was then assessed under inverted microscope, and three replicates tests were performed for each sample. the lc50, defined as the metal concentration that resulted in half maximal larval mortality compared to the control group, and their 95 % confidence intervals (cis) were calculated by the probit method (newman, 1995). interaction analysis to identify the type of interaction in binary mixtures of cd and pb, the additive toxicity index (ati) of marking and dawson (1975) and its 95 % ci were calculated. the effective contributions of two chemical (a and b) in a mixture are represented by the formula: s= am/ai + bm/bi, where a and b are chemicals, m and i are the toxicities (ec50's/lc50's) of the individual chemicals and the mixtures, respectively. s is the sum of effective concentrations which is modified by one of two procedures: either the additive index = 1/s−1 for s≤ 1.0 (greater than additive toxicity) or s (-1) + 1 for s≥ 1.0 (less than additive toxicity), and additive effects are demonstrated when s = 1. additive, synergism and antagonism effects are indicated by zero, positive, and negative values of this index, respectively. the inclusion of zero within the 95 % ci suggests a lack of significant deviation from additivity, while 95 % ci lying above or below zero indicates significant synergism or antagonism, respectively. the toxicity unit (tu) for combined pollutants was calculated using the formula: tu = ci/ec50i or tu = ci/lc50i, ci is the concentration of each toxicant in the mixture and ec50i/lc50i is the half maximal efficient concentration for each individual toxicant (sprague and ramsay, 1965). cd (µg/l) nominal 20 100 500 2500 12500 measured 23.24 ± 0.59 106.01 ± 7.55 480.04 ± 31.46 2129.55 ± 328.77 11566.71 ± 957.34 pb (µg/l) nominal 20 100 500 2500 12500 measured 19.61 ± 0.98 96.68 ± 1.86 389.95 ± 37.30 1784.86 ± 49.17 8948.38 ± 588.19 cd + pb mixture nominal 10 + 10 50 + 50 250 + 250 1250 + 1250 6250 + 6250 measured 9.49 ± 1.17 (cd) 10.54 ± 0.95 (pb) 45.75 ± 2.78 (cd) 46.14 ± 0.45 (pb) 253.31 ± 29.73 (cd) 236.75 ± 12.30 (pb) 910.61 ± 68.73 (cd) 1087.56 ± 45.31 (pb) 6170.22 ± 279.77 (cd) 5318.94 ± 239.26 (pb) embryotoxicity (tu) 0.05 0.24 1.29 4.99 30.72 larval mortality (tu) 0.04 0.20 1.06 4.13 25.07 214 fig. 2 (a) percentages (mean ± s.d., n = 3) of abnormal d-shaped larvae and tail dna following oyster embryos exposed to different concentrations of cadmium. asterisks indicate significant differences between exposed and control treatment (*p < 0.05, **p < 0.01, ***p < 0.001). (b) relationship between tail dna and d-shaped larvae abnormalities in oyster (p = 0.0007). data analysis regression linear analysis (microsoft excel, 2010) was used to assess relationships between dna damage and the percentage of abnormal oyster after pollutant exposure. spss 16.0 statistical software (spss inc., chicago, il, usa) was used for data analysis. percentage data were transformed (arcsine of the square root) before one-way anova, and presented in figures as non-transformed percentages. homogeneity of variance was tested using levene's test. the data were analyzed by one-way anova and tukey’s test was used to compare the results between the control group and the treated groups, and the differences between the treated groups. statistical significance was accepted when p < 0.05. results chemical analysis the actual cd and pb concentrations were determined in the exposure solutions and summarized in table 2. measured concentrations ranged from 71.4% to 116.2 % of the nominal concentrations. significant differences (p < 0.05) were observed between the nominal and measured concentrations. therefore, measured concentrations were used for the presentation and calculation of toxicity parameters. embryotoxicity and genotoxicity effect of individual cd on embryotoxicity is illustrated in the concentration-response curves (fig. 2a). cd could significantly affect the embryogenesis of the oyster at a concentration of 106.0 µg/l (p < 0.01), at which the inhibition was up to a 35.9 % decrease in number of normal d-shaped larvae compared to the control. at the highest tested concentration (11566.7 µg/l), the larvae abnormalities reached 96.5 %. the level of dna damage was significantly (p < 0.05) increased from 6.6 % to 38.1 % with increasing cd concentration. cd inhibited the embryonic development of c. gigas with an ec50 value of 272.2 µg/l (table 3), and the loec value of cd for embryogenesis was 106.0 µg/l. in addition, a strong positive correlation (r2 = 0.956, p = 0.0007) was observed between the dna damage level and the percentage of abnormal d-shaped larvae after cd exposure (fig. 2b). table 3 median effective concentrations (ec50), median lethal concentration (lc50), and lowest observed effect concentration (loec) for cadmium, lead and cadmium-lead mixture in embryotoxicity and larval mortality assays embryotoxicity ec50 µg/l (95% ci) larval mortality 96 h lc50 µg/l (95% ci) loec µg/l cd 272.2 (170.5~444.6) 353.3 (213.2~570.4) 106.0 pb 660.3 (453.5~1062.4) 699.5 (508.4~983.9) 96.7 cd + pb 373.1 (270.2~539.3) 205.5 (138.3~293.1) 91.9 215 fig. 3 (a) percentages (mean ± s.d., n = 3) of abnormal d-shaped larvae and tail dna following oyster embryos exposed to different concentrations of lead. asterisks indicate significant differences between exposed and control treatment (*p < 0.05, **p < 0.01, ***p < 0.001). (b) relationship between tail dna and d-shaped larvae abnormalities in oyster (p = 0.0041). a significant increase of abnormal d-shaped larvae was observed at a concentration as low as 96.7 µg/l for pb (p < 0.01), and the percentage of abnormal larvae reached 88.7 % at the highest tested concentration (8948.4 µg/l) (fig. 3a). the level of tail dna significantly increased from 6.1 % to 30.8 % with the increase of pb concentration (p < 0.001). a significant positive correlation (r2 = 0.898, p = 0.0041) was also observed between the level of dna damage and the percentage of abnormal d-shell larvae for pb (fig. 3b). the ec50 and loec values for pb was shown in table 3. figure 4 presents the percentage of abnormal dshaped larvae and the level of dna damage after exposure to the mixture of cd and pb. significant increase of dna damage level was observed at 0.24 tu (p < 0.001), corresponding to 91.9 µg/l (45.8 µg/l cd + 46.1 µg/l pb) for the mixture. a dosedependent manner on the genotoxic effect was observed after exposure to the mixture of cd and pb, and the percentage of tail dna increased from 5.1 % to 42.6 % with the increase of mixture concentration. the percentage of abnormal dshaped larvae was also significantly (p < 0.001) increased from the dose of 0.24 tu mixture compared to that of the control. after exposed to the highest concentration of 30.72 tu mixture, the abnormalities of d-shaped larvae reached 91.1 % (fig. 4a). there was also a strong positive correlation (r2 = 0.975, p = 0.0002) between level of dna damage and the percentage of abnormal dshell larvae after the mixture exposure (fig. 4b). the ec50 and loec value for metal mixture (cd + pb) was shown in table 3. fig. 4 (a) percentages (mean ± s.d., n = 3) of abnormal d-shaped larvae and tail dna following oyster embryos exposed to different concentrations of the mixture of two metals (cd + pb). the x axis shows the toxicity units (tu, tu = concentration/ec50). asterisks indicate significant differences between exposed and control treatment ( *p < 0.05, **p < 0.01, ***p < 0.001). (b) relationship between tail dna and d-shaped larvae abnormalities in oyster (p = 0.0002). 216 fig. 5 the mortality (mean ± s.d., n = 3) of d-shaped larvae after 96 h exposure to different concentrations of cadmium (a), lead (b) and the mixture of two metals (cd + pb) (c). the x axis shows the toxicity units (tu, tu= concentration/ec50). asterisks indicate significant differences between exposed and control treatment ( *p < 0.05, **p < 0.01, ***p < 0.001). larval mortality for both metals, no significant difference in larval number was observed at the lowest concentration (23.2 µg/l of cd, 19.6 µg/l of pb) compared to that of the control. cd induced a significant increase (p < 0.001) in larval mortality at the concentration of 106.0 µg/l (fig. 5a). for pb exposure, the larval mortality reached 83.4% at the highest concentration of 8948.4 µg/l (fig. 5b). the lc50 values and loec values for cd and pb were shown in table 3. similarly, the mortality of the larvae ranged from 17.6 % to 95.2 % at the value of lowest (0.04 tu) to highest metal mixture (25.1 tu) (fig. 5c). the lc50 and loec values for the metal combination were shown in table 3. interactions table 4 shows the additive toxicity index (ati) and 95 % ci of combination (cd + pb) for embryotoxicity and larval mortality. the result of ati for embryotoxicity is 0.10 (1.13, 1.55), and the zero value is contained within the 95 % ci for the index. therefore, the results suggested a simple additive effect of the mixture. whereas, ati and 95 % ci for larval mortality was 1.40 (0.07, 4.54), indicating a significant positive interaction (synergism) between these two metals (cd + pb). discussion the contamination of coastal ecosystems by trace metals has gained increasing attention in recent decades (his et al., 1999a; shahidul islam and tanaka, 2004; pan and wang, 2012). chemical analyses of pollutants in seawater allow a determination of the degree and nature of pollution, but they are not able to provide evidence as to the biological consequences, and evaluate potential risks for living organisms (chapman et al., 1987). biological monitoring using marine organisms, particularly in their early life stage, has been shown to be a sensitive approach to predict the potential risk of persistent pollutants like trace metals. although the effects of single metals on marine species have been well documented in terms of their toxicity and bioaccumulation (novelli et al., 2003; reicheltbrushett and harrison, 2005; fitzpatrick et al., 2008; 217 gopalakrishnan et al., 2008), the literature about combined toxicity of cadmium and lead on early life stages of the pacific oyster is still scarce. the present study aimed to investigate the embryotoxicity, genotoxicity and larval mortality of the pacific oyster under individual and combined metal (cadmium and lead) exposures. embryotoxicity and genotoxicity the embryo development of oysters was strongly affected by cadmium and lead at the tested concentrations. the results of ec50s (table 2) demonstrated that cd was more toxic to embryolarval stages than pb. these results are in concordance with that shown in meretrix meretrix (wang et al., 2009). in the current work, ec50 value of cd for the pacific oyster was 272.2 µg/l, which was similar to the ec50 (212.3 µg/l) value for this oyster (mai et al., 2012) and another oyster crassostrea rhizophorae (211.8 316.2 µg/l) (da cruz et al., 2007). similarly, ec50 value of cd on embryotoxicity was 502 µg/l for the mussel mytilus trossolus (nadella et al., 2009), 1925 µg/l for mytilus gallporovincialis (beiras and albentosa, 2004), 1014 µg/l for clams meretrix meretrix (wang et al., 2009) and 570.9 µg/l for ruditapes decussatus (fathallah et al., 2013). for pb, the ec50 for inhibition of embryogenesis was 660.3 µg/l in this study. however, the ec50s for sea urchin paracentrotus lividus were determined to be 482 µg/l (his et al., 1999b) and 509 µg/l (fernandez and beiras, 2001). it was also reported an ec50 value of 297 µg/l for clams m. meretrix (wang et al., 2009), and 256.5 µg/l for r. decussatus (fathallah et al., 2013). the embryos of the pacific oyster seem to be more resistant to pb exposure than the other reported marine bivalves probably due to the sensitivity of different species and the use of different methods for testing (different trace metal salts, or pool of gametes). the toxicity of cd in combination with pb was also investigated in this study. the ec50 value for the mixture (cd + pb) was 373.1 µg/l, which was comparable with the ec50 value (355.4 µg/l) of (cd + pb) to r. decussatus (fathallah et al., 2013). in addition, trace metals also induced dna damage in pacific oyster embryos after 16 h exposure. for cd, the lowest concentration (23.2 µg/l) significantly increased dna damage (p < 0.01). mai et al. (2012) also reported that 10 µg/l cd increased dna damage level significantly (p < 0.05). cd-induced dna damage was probably due to the increase of lipid peroxidation in various tissues (stohs and bagchi, 1995). previous studies have also reported that cadmium could directly induce dna damage through breaking the single strand and inhibiting the reparation of dna (hassoun and stohs, 1996; beyersmann and hechtenberg, 1997). our study showed that 96.7 µg/l of pb exposure was able to induce 6.1 % of dna damage in oyster embryos. it was reported that pb could cause dna damage by the production of ros, which initiated dna oxidation and subsequent damage (hsu and guo, 2002). oyster embryos exposed to the metal mixture also showed a significant increase of dna damage level (9.1 %) at the concentration of 0.24 tu (corresponding to 45.8 µg/l cd + 46.1 µg/l pb). in present study, significant positive correlation table 4 the additive toxicity index (ati) and 95% confidence intervals (±ci 95%) for binary combinations of cadmium and lead ati ci 95% + ci 95% embryotoxicity 0.10 -1.13 1.55 larval mortality 1.40 0.07 4.54 between embryotoxicity and genotoxicity was demonstrated after individual cd and the mixture exposure, whereas the correlation was lower for pb exposure. mai et al. (2012) also found a strong positive correlation between embryotoxicity and genotoxicity after metal (cu and cd) and pesticide (metolachlor) exposure. similarly, relatively high correlation between embryotoxicity and genotoxicity was reported in oyster embryos after exposure to benzo(a)pyrene, 17α-ethinylestradiol and endosulfan (wessel et al., 2007). larval mortality in the present study, the 96 h lc50 values of larval mortality for cd, pb and their mixture were 353.3 µg/l, 699.5 µg/l and 205.5 µg/l, respectively. the lc50s for r. decussatus were 453.6 µg/l, 877.8 µg/l and 565.6 µg/l (for cd, pb and their mixture) (fathallah et al., 2013), and 68 µg/l and 353 µg/l (for cd and pb) for clams m. meretrix (wang et al., 2009). in individual cd and pb exposure treatments, it was evident that the susceptibility of embryos were slightly higher than larvae. the findings from this study were consistent with those in other bivalves such as crassostrea virginica (calabrese et al., 1977), c. gigas (beiras and albentosa, 2004), m. meretrix (wang et al., 2009) and r. decussatus (fathallah et al., 2013). interactions additive effects have been observed in bivalves under mixtures of cadmium and lead exposure. the additive indices of the metal combination were 0.10 and 1.40 for embryo toxicity and 96 h larval mortality respectively. the combined toxicity in embryogenesis was slightly higher (10 %) than predicted by the additive model. however, the 96 h larval mortality bioassay showed a significant higher toxicity index, which would be considered as synergistic following the marking-dawson method (marking and dawson, 1975). in present study, we had observed additive or synergistic effects for combinations of cd and pb. additive effects have also been observed in bivalves for mixtures of cuag (coglianese and martin, 1981), mn-mo (morgan et al., 1986), hg-cd, hg-cu and cd-cu (fernandez and beiras, 2001), hg-cu, hg-cd and cu-cd (prato and biandolino, 2007), cd-pb (fathallah et al., 2013) and cu-ag (boukadida et al., 2016). however, other authors propose alternative views. hayes (1991) proposed that combinations yielding additive indices between 0.5 and 2 (corresponding to the range from -1 to 1 for the marking-dawson index) 218 should not be considered as significantly different from additivity. following this criterion, the interactions studied in this study would all be considered to be additive. cadmium and lead pollution in coastal area of china and environmental risk to native bivalves the coastal and estuarine areas in china are facing increasing metal pollution pressures. in seawater, the concentrations of cd and pb are as high as 5.0 µg/l and 27.17 µg/l respectively in bohai sea (gao et al., 2014), and 0.89 µg/l and 7.7 µg/l respectively in south china sea (zhang et al., 2015). moreover, high levels of metal concentrations are also found in sediments along the coastal and estuary areas in china. it has been reported that cd and pb concentrations were as high as 488.2 µg/g and 1828 µg/g respectively in sediments of jinzhou bay (fan et al., 2006; zhang et al., 2008), and 22.69 µg/g and 145.5 µg/g respectively in sediments of dagu drainage canal (zhang et al., 2012). according to the concentrations in seawater, current cd and pb pollutions have no adverse effect on early life stages of oyster and other native bivalves. however, the release of metals from highly polluted sediments may increase the potential risk of metal pollution. in addition, other highly toxic metals such as copper, mercury may coexist in the polluted environment. thus, it could not be ruled out that combined metal pollution may have an adverse impact on recruitment of native bivalves. conclusion in conclusion, the results indicated that oyster embryo was highly susceptible to cd and pb exposure, and combined metal exposure induced a positive interaction synergism effect on larval mortality of c. gigas. this study also suggested that cd and pb pollution in most coastal areas of china will not affect the recruitment of wild or cultivated oysters. however, the complex mixtures of different classes of contaminants may cause potential toxic effect on oyster embryos and larvae, especially at high-concentration areas. therefore, to predict the impact of combined contamination on marine organisms, it would be necessary to test not only binary mixtures, but also combinations of three or more metals, or even combinations of metals with organic pollutants. acknowledgements this research was supported by grants from the key research program of the chinese academy of sciences (grant no. kzzd-ew-14), the strategic priority research program of the chinese academy of sciences (xda11020702), natural science foundation of shandong province (no. jq201310) and the youth innovation promotion association of cas (2016196). references akcha f, hubert, fv, pfhol-leszkowicz a. potential value of the comet assay and dna adduct measurement in dab (limanda limanda) for assessment of in situ exposure to genotoxic compounds. mut. res-gen. tox. en. 534: 21-32, 2003. akcha f, spagnol c, rouxel j. genotoxicity of diuron and glyphosate in oyster spermatozoa and embryos. aquat. toxicol. 106-107: 104-113, 2012. bakker ak, dutton j, sclafani m, santangelo n. accumulation of nonessential trace elements (ag, as, cd, cr, hg and pb) in atlantic horseshoe crab (limulus polyphemus) early life stages. sci. total. environ. 596-597: 69-78, 2017. beiras r, albentosa m. inhibition of embryo development of the commercial bivalves ruditapes decussatus and mytilus galloprovincialis by trace metals; 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mar. pollut. bull. 74: 453-463, 2013. zhang l, shi z, zhang jp, jiang z, wang f, huang x. spatial and seasonal characteristics of dissolved heavy metals in the east and west guangdong coastal waters, south china. mar. pollut. bull. 95: 419-426, 2015. zhang yf, wang lj, huo cl, guan dm. assessment on heavy metals pollution in surface sediments in jinzhou bay. mar. environ. sci. 27: 178-181, 2008 [in chinese with english abstract]. zhang qj, wang l, zhao lj, sun hw, lu y. analysis and assessment of heavy metal pollution in sediments of tianjin harbor and dagu drainage canal in bohai bay, china. fresenius environ. bull. 21: 1777-1785, 2012. zaki ms, zakaria a, eissa ia, eldeen ai. effect of cadmium toxicity on vertebrates. electron phys. 8: 1964-1965, 2016. 72 isj 16: 72-83, 2019 issn 1824-307x research report the transformation of energy metabolism and endoplasmic reticulum stress regulation in pacific oyster crassostrea gigas under air exposure c gong 1,3,4 , c liu 1,3 , h li 1,3 , m li 1,3 , z liu 1,3 , w wang 1,2,3 , l wang 1,2,3,4 , l song 1,2,3* 1 liaoning key laboratory of marine animal immunology, dalian ocean university, dalian 116023, china 2 laboratory of marine fisheries science and food production process, qingdao national laboratory for marine science and technology, qingdao 266235, china 3 liaoning key laboratory of marine animal immunology and disease control, dalian ocean university, dalian 116023, china 4 dalian key laboratory of aquatic animal disease prevention and control, dalian ocean university, dalian 116023, china accepted april 29, 2019 abstract the pacific oyster crassostrea gigas is an important species living in the intertidal zones. it is of great significance to study the mechanism of oysters to adapt air exposure. in the present study, weighted gene co-expression network analysis (wgcna) with the transcriptome data of gills and adductor muscle was conducted to investigate the metabolic transformation of c. gigas under air exposure. go enrichment of modules specifically expressed in adductor muscle of oysters exposed to air for five, seven and nine days revealed the phased expression of respiratory chain, protein turnover and lipid metabolism, indicating the conversion of energy metabolism. during air exposure, “respiratory chain” and “ribosome biogenesis” were enriched in the muscle on the fifth day, suggesting that glycogen metabolism was dominant in the early stages of air exposure. on the seventh day, many terms about the regulation of proteolysis were enriched, indicating that carbohydrates were not be able to meet the metabolic needs in the oyster adductor muscle, and proteins began to be degraded for energy supply. the processes related to lipid metabolism were enriched on the ninth day. the extremely high glycogen content of c. gigas allowed it to maintain a basic metabolic activity for a long time with a conservative compensation strategy. go and kegg enrichments of the modules sensitive to air exposure in gills were mainly involved in “response to endoplasmic reticulum stress”, “endoplasmic reticulum (er) to golgi transport vesicle membrane” and “protein processing in endoplasmic reticulum”. it revealed that the mechanism of oyster adapting to air exposure was a complex regulatory network depending on the er. hub gene network and ppi network analyses found that some transcription factors containing zinc finger domains regulated the biochemical reactions for stress adaptation, indicating that the er, as a regulatory element sensitive to external stress, could regulate apoptosis, autophagy and protein degradation in gills of c. gigas under air exposure. these results would provide new insights into the adaptation of c. gigas to air exposure in terms of energy metabolism and homeostasis. key words: crassostrea gigas; air exposure; wgcna; transcriptome; er stress; energy metabolism introduction the pacific oyster crassostrea gigas is a world-wide marine bivalve distributing in the intertidal zones. it can adapt to a wide range of environmental stresses such as air exposure, salinity, temperature, acidification, and anoxia ___________________________________________________________________________ corresponding author: linsheng song dalian ocean university 52 heishijiao street, dalian, 116023, china e-mail: lshsong@dlou.edu.cn (zhang et al., 2015), and has become an ideal model to study the molecular adapt mechanisms of marine molluscs to environmental stresses, especially the air exposure. air exposure is a common stress to the species living in the intertidal zones, which is also considered as a complex stress consisting of hypoxia, drought, heat, and solar radiation. as a complex environmental stress, air exposure can influence various aspects of c. gigas such as metabolism, immune system and dna repair. so far, there have been many studies on the response of 73 oysters to air exposure. for instance, calnexin (cnx) and calreticulin (crt) were proved to function as endoplasmic reticulum (er) chaperones in c. gigas to adapt to air exposure (kawabe and yokoyama, 2010). upregulated glucose concentration in serum induced the synthesis of interleukin-17 and inflammatory response during air exposure (xin et al., 2016). serotonin could modulate apoptosis and redox during air exposure in c. gigas (dong et al., 2017). most of the previous studies were focused on functional verification of a key gene or gene family in a process under air exposure. the rapid development of omics provides systematic analysis for the comprehensive understanding of the powerful adaptability of oysters to the complex environment stress. omics is a science of collective characterization and quantification of pools of biological molecules. so far, transcriptome and proteome have been applied to investigate the response of oysters against stresses such as temperature, salinity and pesticides (epelboin et al., 2015; zhao et al., 2016; yang et al., 2017). transcriptome data from c. gigas exposed to air for eleven days have been collected and analyzed, and iaps are found to be highly expressed in the gills (zhang et al., 2012). however, the responses and the underlying mechanisms of oysters against air exposure cannot be described simply by the changes of a single gene or gene family. weighted gene co-expression network analysis (wgcna) is a system biology network algorithm based on the concept of a scale-free network to analyze a few highly connected nodes participating in a very large number of metabolic reactions (zhao et al., 2010). wgcna is an ideal systematic biology method to describe the correlation patterns among genes across microarray or rna-seq samples (langfelder and horvath, 2008). this systematical biology method has been used to study the response upon complex conditions in different species (carlson et al., 2006; gargalovic et al., 2006; horvath et al., 2006; dong and horvath, 2007). in oysters, wgcna was used to study the adaptability of various environmental stresses. for instance, faas metabolism associated modules were identified in oyster c. gigas by wgcna analysis with gill transcriptome in response to different salinity (zhao et al., 2016). it was also employed to verify the activating function of tnf under different neurotransmitter stimulation (liu et al., 2016). the increasing reports demonstrated that wgcna was an advantaged tool to find the core regulatory networks and key genes in complex processes from multi-samples. in the present study, a gene co-expression network was constructed to comprehensively understand the response and regulation strategies of pacific oyster c. gigas against air exposure. the air exposure related modules were identified, and several modules of them were found to be involved in energy metabolism and homeostasis. hub gene network and protein-protein interaction network were constructed to reveal the possible mutual control relationships between these modules. these genes and modules found in the present study provided informative clues to understand the mechanisms of homeostasis and energy allocation in oysters to adapt a wide range of environmental stresses. materials and methods rna extraction and quantitative real-time pcr about 120 pacific oysters, collected from zhangzidao aquaculture farm in dalian, china in september 2017, were acclimatized in aerated seawater feeding with microalgae at 20 ± 1°c for one week. subsequently, they were placed into a dry box and kept at room temperature of 20 ± 1°c for air exposure. nine oysters were sampled to collect the gills and adductor muscle at 10:00 every day. to reduce individual variations and improve result reliability, the tissues from three individuals were pooled together as one sample, and there were three replicates for subsequent rna extraction in each group. on the 8th day, most of the remaining oysters died, and the oysters under air exposure for 0-7 days (72 in total) were collected for the follow-up experiments. the total rna was extracted by using trizol reagent (invitrogen) and reverse transcribed using a cdna synthesis kit (transgen). nadh dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13-like (loc105341417) and hormone-sensitive lipase (loc105345018) were selected as target genes for qrt-pcr analysis. primers of target genes and the internal reference gene (oyster elongation factor, genbank accession no. nm_001305313) were designed by ncbi primer-blast (table 1). qrt-pcr was performed with the abi7500 fast real-time detection system (applied biosystems, usa) using sybr green fluorescent. the relative expression was analyzed by the 2 -δδct method (livak and schmittgen, 2001). table 1 primers used in this paper primers sequence (5'-3') p1 (loc105341417-rt-f) tgtctggcctggtccaattt p2 (loc105341417-rt-r) tgctcgaagtaagctccctg p3 (loc105345018-rt-f) aactcccagctgtgaactcg p4 (loc105345018-rt-r) gggtctctccagtccatcct p5 (ef-rtf) agtcaccaaggctgcacagaaag p6 (ef-rtr) tccgacgtatttctttgcgatgt 74 transcriptome data acquisition the raw transcriptome data, the rna-seq data of gills and adductor muscle from adult oysters crassostrea gigas under air exposure for 0, 1, 3, 5, 7, 9, 10 and 11 days were obtained from the sequence read archive (sra) (leinonen et al., 2011) database with the accession number srx093464 and srx093475–srx093489 from the first paper concerning the oyster genome sequencing (zhang et al., 2012). these rna-seq data were derived from illumina hiseq 2000 single-ended 49bp sequencing. data preprocessing and mapping to the genome the sra files were transferred to fastq files by sra toolkit v2.8.2. fastqc v0.11.5 was used to control the quality of raw data. the genome data and gene annotation files of c. gigas were downloaded from ncbi refseq database (ftp://ftp.ncbi.nih.gov/genomes/crassostrea_gigas). the filtered raw data were aligned to the genome using hisat2 v2.0.5 (pertea et al., 2016). the rpkm (reads per kilobase per million mapped reads) was quantified by stringtie v1.3.3b with the parameter “-e -b” (pertea et al., 2015). the quantified expression matrix was filtered by an r package ballgown with a variance across samples less than one (frazee et al., 2015). weighted gene co-expression network analysis the weighted gene co-expression network was constructed with an r package wgcna (langfelder and horvath, 2008). sixteen samples were clustered to detect outliers according to the tutorials of the package. the soft thresholding power β was detected with the criterion of approximate scale-free topology by the function picksoftthreshold. all raw reads from gills and adductor muscle transcriptomes of c. gigas were mapped to oyster genome. the scale-free topology model fit and the mean connectivity of the network were evaluated over a range of the soft threshold power β. to minimize effects of noise and spurious associations, the adjacency was transformed into topological overlap matrix (tom) to calculate the corresponding dissimilarity. then a hierarchical clustering tree (dendrogram) of genes was produced by tom. the branch of the tree was cut to identify modules by function cutreedynamic with the minimum cluster size 30. identification of air exposure related modules module eigengene (me) of a module was considered as a representative of a module’s gene expression profile. the me value (e) was calculated and a heatmap was drawn to visualize the express pattern of each module. the genes in the modules were annotated by a comprehensive annotation software suite trinotate v3.0.1 to predicate their potential biological roles in the response to air exposure (haas et al., 2013). the mrna sequences of c. gigas were aligned to swissprotkb database by blast v2.6.0 (altschul et al., 1990). the hmmer v3.1 (finn et al., 2011) was used to identify protein domains from pfam-a database. the results of gene ontology (go) (ashburner et al., 2000) and kyoto encyclopedia of genes and genomes (kegg) table 2 statistics of reads and mapping rate of rna-seq samples total reads %mappable dried_gills_0d 15775296 72.65% dried_gills_1d 17218677 73.16% dried_gills_3d 18281681 74.85% dried_gills_5d 16430916 73.56% dried_gills_7d 16612191 74.39% dried_gills_9d 17289062 75.29% dried_gills_10d 18058657 75.09% dried_gills_11d 19406328 71.46% dried_muscle_0d 18829273 82.05% dried_muscle_1d 15603618 80.63% dried_muscle_3d 18384113 79.48% dried_muscle_5d 15359448 80.45% dried_muscle_7d 16114873 81.08% dried_muscle_9d 16739672 80.86% dried_muscle_10d 15281286 81.08% dried_muscle_11d 16702190 79.52% (kanehisa et al., 2012) annotation terms were extracted by in-house perl scripts and imported into an r package annotationforge (carlson and pages, 2017) to make an orgdb organism annotation object. the r package clusterprofiler (yu et al., 2012) was employed to analyze the go and kegg enrichments for all modules under hypergeometric distribution with p-value < 0.05 and q-value < 0.05 (benjamini-hochberg method). network construction and visualization the top 5% genes with the highest connectivity in each module were defined as hub genes. the hub gene network was visualized by cytoscape v3.6.1. the gene list was mapped to the strongylocentrotus purpuratus (purple sea urchin) proteome (source from string-db “7668.protein.sequences.v10.5”) by blast v2.6.0 with the parameter “-evalue 1e-5 -max_target_seqs 1 -outfmt 6” to obtain the best match of orthologous genes. the corresponding gene list was imported into string database (https://string-db.org) to generate ppi network (szklarczyk et al., 2015). the network was then imported into cytoscape for functional annotation, manual curation, and grouping. results identification of air exposure related modules all raw reads from gill and adductor muscle transcriptomes of c. gigas were mapped to oyster genome (table 2). of all the 33790 genes, 15139 genes with a variance less than one across samples 75 were filtered to reduce the range of genes that need to be analyzed (table s1). the sample clustering analysis revealed that there was no obvious outlier (fig. s1). the adjacencies of the network were calculated using the soft thresholding power β = 5 (fig. s2). the number of genes per module was listed in table 3 and a heatmap was drawn to visualize the mes of all twenty-one modules (fig. 1). these modules were roughly divided into several categories: (i) specifically expressed in a sample (green, yellow, lightcyan, orange, etc.); (ii) specifically expressed during a certain period of time (tan, magenta, darkred, royalblue, and lightgreen); (iii) gradually changed over time (brown); (iv) tissue-specific expression (blue). air exposure related modules were defined as the modules specifically expressed at a certain stage of air exposure or significantly correlated with air exposure time. after the gene expression was normalized, the gene clusters specifically expressed in tissues were effectively filtered by the co-expression network of the dual-tissue samples under air exposure to greatly reduce the possibility of background noise. eight modules (green, yellow, lightcyan, midnightblue, lightyellow, greenyellow, salmon, grey60) were expressed differently at different air exposure time in gills. other six modules (black, red, cyan, darkgrey, darkturquoise, orange) exhibited periodic expression patterns on the 0, 5, 7, 9, 10 and 11th day in adductor muscle. the me of brown module increased in a time dependent manner during the exposure period, indicating that the expressions of these genes in oyster gills were highly correlated with exposure time. the above 14 modules were initially identified as air exposure related modules for further analysis. table 3 list of module size module gene numbers black 330 blue 13088 brown 1593 cyan 111 darkgrey 38 darkred 68 darkturquoise 62 green 847 greenyellow 145 grey60 98 lightcyan 106 lightgreen 94 lightyellow 74 magenta 446 midnightblue 107 orange 34 red 355 royalblue 73 salmon 132 tan 138 yellow 712 fig. 1 module eigengenes heatmap of 21 modules. the x-axis represents different samples, the y-axis represents the name of each module, and the color shade of the patch represents module eigengene (me) values. each row represents a module's general expression trend 76 fig. 2 important go terms enriched in different duration of air exposure. a schematic of important go entries enriched in gills and adductor muscle of c. gigas under air exposure for 0-11 days. go terms mentioned in the text were marked red. see table s2 for more details. functional enrichment of air exposure related modules go and kegg pathways were enriched for all the modules to gain a comprehensive understanding of the biochemical reactions in oysters under air exposure. fourteen modules specifically expressed in different duration of air exposure were first analyzed (fig. 2), and go terms enriched on the fifth, seventh and ninth day displayed a clear trend of convert from glucose metabolism to lipid metabolism. on the fifth day (fig. 3a), “respiratory chain” and “ribosome biogenesis” were enriched in the adductor muscle, suggesting that glycogen metabolism was dominant in the early stages of air exposure (fig. 3b). on the seventh day, many terms were enriched in the regulation of proteolysis, indicating that the carbohydrates could not meet the metabolic needs in oyster adductor muscle, and proteins began to be degraded for energy supply (fig. 3c). the trail-mediated apoptotic pathway was also enriched on the seventh day, indicating that the apoptotic pathway was initiated in this period. the processes related to lipid metabolism were enriched on the ninth day. the brown module was a special module whose me value increased with the time of air exposure in gills. go enrichment reveled that the gene clusters sensitive to exposure time in the brown module (table s2) were mainly involved in “autophagy”, “small gtpase mediated signal transduction”, “response to endoplasmic reticulum stress”, and “er to golgi transport vesicle membrane”. “protein processing in endoplasmic reticulum”, “snare interactions in vesicular transport” and “mitophagy – animal” were enriched pathways in kegg database (fig. 5a). the mrna expression pattern of energy metabolism related genes the expression levels of nadh dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13-like (cgndufa13, ncbi gene id: loc105341417, fig. 4a) and hormone-sensitive lipase (cghsl, ncbi gene id: loc105345018, fig.4b) in oyster adductor muscle were examined under air exposure. the expression level of cgndufa13 gradually increased in the first three days of air exposure, reached a maximum on the third day, and then gradually decreased. the expression level of cghsl displayed a similar trend, but peaked on the fourth day. hub gene connectivity and protein-protein interaction network of brown module to further clarify the biological significance of the brown module, a hub gene co-expression network and a protein-protein interaction (ppi) network were established. the top 5% genes with the highest connectivity in the module were chosen as hub genes (fig. 6a, table s3). “serine/threonine-protein kinase 17a” (loc105324979) was the center of the network and several hub genes were found to involve in erad pathway. for the lack of oyster protein interaction data, the oyster genes were mapped to the genome of purple sea urchin s. purpuratus, a model species of marine invertebrates, to characterize protein interactions of these genes (fig. 6b). the enrichment p-value of ppi was 0.0329 (< 0.05), indicating that the proteins in the same network reacted more frequently with each other than with a random set of other proteins. this network was centered by some transcription factors containing 77 fig. 3 gene expression pattern and go enrichment of three energy metabolism related modules. go enrichment dotplot, expression heatmap and me barplot of red (a), cyan (b), and darkgrey (c) module. the x-axis of dotplots represents the proportion of genes enriched to the total number of genes in the entry. the size of the dots represents the number of genes enriched. the x-axis of heatmaps and barplots represents different samples, from left to right: the oyster gills and adductor muscle under air exposure for 0, 1, 3, 5, 7, 9, 10, 11 days respectively. the y-axis of heatmaps represents module genes. the color shade of the heatmap represents the fkpm of module genes increasing from green to red. the y-axis of barplots represents the mes c2h2 type zinc finger which was involved in the regulation of endocytosis, rna transport, and ubiquitin mediated proteolysis (table s3). kegg pathway enrichment showed that the ppi network was mainly composed of endoplasmic reticulum-mediated n-glycosylation, endocytosis, and erad pathways. the centered gene loc583350 was annotated as an e3 ubiquitin-protein ligase mib2. discussion transcriptomes have been mainly analyzed to find differentially expressed genes (degs), which are limited to be applied directly to time-series expression data due to the differences in sampling rates and variations in the timing of biological processes (bar-joseph et al., 2003). wgcna is an ideal tool for merging genes with approximate expression patterns into modules, so that all the gene expressions can be separated into different subsets, and the key pathways can be revealed for time-series data. in the present study, a weighted gene co-expression network was constructed with the rna-seq data to reveal the adaptation mechanism of c. gigas to air exposure. the genes expressed in the various periods of air exposure were grouped into different modules in the me heatmap. the biological functions of these modules and the biochemical reactions occurred during the various stages of air exposure could provide helpful clues to understand the possible mechanisms of adaptability of oysters to the environmental stresses. it has been demonstrated that all the organisms have different strategies to deal with various stresses by mediating the energy metabolism to meet the increasing maintenance costs. in an energy-based stress classification, the overall scope of the values of an environmental factor experienced by an organism is divided into several ecologically and physiologically relevant ranges: optimal range, pejus range, pessimum range, and lethal range (pörtner and farrell, 2008). the judgment of the range conversion is mainly based on the proportion of the basic maintenance costs to the total energy expenditure of the organism. base maintenance costs refer to the maintenance of key cellular processes (e.g., protein turnover, ion balance, acid-base regulation, and anabolism of substances) and the basic activity costs of living organisms (e.g., circulation and excretion) (sokolova et al., 2012). adductor muscle has been considered as one of the important energy storage organs in c. gigas which provides the protein and energy for gametogenesis 78 fig. 4 the mrna expression patterns of energy metabolism related genes. the mrna transcripts of nadh dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13-like (a) and hormone-sensitive lipase (b) in oyster adductor muscle were detected by real-time pcr at 0, 1, 2, 3, 4, 5, 6 and 7 day after air exposure. each value was shown as mean ± s.d. (n = 3) and reproduction (berthelin et al., 2000; li et al., 2006). in the present study, the modules specifically expressed at the fifth, seventh and ninth day in the adductor muscle indicated the changes of energy allocation strategies and the response range during the exposure process (fig. 3). on the fifth day (red module), “respiratory chain” and “ribosome biogenesis” were enriched in the adductor muscle. as a rule, the exposures to pejus and pessimum stresses cause metabolic and atp turnover acceleration to compensate additional energy expenses for increased physiological activity, cellular maintenance, and damage repair (sokolova et al., 2012). in the present study, the increasing expression of cgnudfa13 (fig. 4a) demonstrated an increase of cellular energy demands for basal maintenance. the increased maintenance costs exacerbated the consumption of nutrients (especially glycogen). in general, glycogen makes up 20–40% of the oyster’s dry weight. as the main flavor component, the glycogen content is critical to oyster quality (li et al., 2017). the rich glycogen content of c. gigas can provide sufficient energy to maintain pressure from pejus to pessimun, and endow oysters the ability to adapt to prolonged air exposure. on the seventh day (cyan module), many terms were enriched in the regulation of proteolysis and trail-mediated apoptotic pathway, suggesting the metabolic compensation in pejus stress. the activation of trail-mediated apoptotic pathway in the present study proved the similar transfer of energy metabolism strategy for maintenance and repair in the middle stage of response against air exposure. the increased protein turnover, negative regulation of proteolysis, and activation of the apoptotic pathway suggested that the metabolic strategy of c. gigas turned into conservation and compensation to reduce negative impact of stress. on the ninth day (darkgrey module), enrichment of lipid metabolism-related processes indicated that prolonged hunger consuming glycogen made energy insufficient to maintain basic physiological activity. the up-regulated expression of cghsl, functioning in hydrolyzing triglyceride, was also detected in the oyster muscles under air exposure by qrt-pcr (fig. 4b). the representative signals of cell death, such as “neuropeptide signaling pathway” and “microtubule depolymerization” (linden et al., 2005; oropesa-ávila et al., 2013) were enriched on the tenth day (darkturquoise module). the enrichment of neuropeptide signaling pathway suggested that the lethal pressure at this time exceeded the tolerance range of oysters, and death was irreversible. er is responsible for folding and processing of nearly all polypeptides destined for secretion and plays a major role in maintaining cell homeostasis (frakes and dillin, 2017). it has been reported that there is a significant response to endoplasmic reticulum stress and endoplasmic reticulum associated degradation (erad) in gills of c. gigas under lead (pb) stimulation (meng et al., 2018) and heat stress (yang et al., 2017). er stress could be induced by a wide range of cellular environments and events, including increased levels of protein synthesis, impaired ubiquitination and proteasomal degradation, deficient autophagy, energy deprivation, excess or limitation of nutrients, dysregulated calcium levels, redox homeostasis, inflammatory challenges, and hypoxia (wang and kaufman, 2016). gills, as the respiratory and filtration organ of c. gigas, is the first line in contact with seawater (fabioux et al., 2015), which could dynamically adjust production of secreted proteins in response to environmental insult and nutrient availability (zhang et al., 2014). in the present study, the me of brown module increased gradually with the exposure time. this module was consisted of two clusters in the expression heatmap (fig. 5c). the expression of one 79 fig. 5 gene expression pattern and function enrichment of brown module. kegg (a) and go (b) enrichment dotplot of endoplasmic reticulum related module. the color of the dots represents adjust p-value. (c): expression heatmap of brown module genes, these genes can be divided into two clusters: the expression level gradually increases and decreases in gills over time. (d): kegg pathway of “protein processing in endoplasmic reticulum” (crg:04141). genes in brown module were marked red cluster increased over time and the other one decreased over time. their correlations with air exposure time indicated that the modules were the most important modules for oysters to adapt to air exposure. kegg enrichment showed that protein processing in endoplasmic reticulum was enriched in brown module (fig. 5b). it has been reported that er-related pathways function in the adaption of oysters to various environmental stresses, such as high temperature, heavy metal ions, and diesel (zhu et al., 2016; meng et al., 2018; müller et al., 2018). er stress related pathways were suggested to be inseparable for oysters to adapt to long time air exposure. erad pathway related genes and part of er stress element (e.g., perk, atf4, traf2) were significantly enriched in brown module (fig. 5d). er stress has been reported to activate perk pathway and regulate autophagy gene transcriptional program in response to amino acid starvation under short stress. for the prolonged stress, the response of er stress changes from promoting cellular survival to committing the cell to a apoptosis pathway mediated by traf2 (rozpędek et al., 2016). a hub gene network and a ppi network were constructed to further clarify the possible expression regulation and protein interactions of the brown module (fig. 6). the highly connected hub genes in a module work together synergistically and play important roles in the biological processes (yuan et al., 2017). the centered gene in the hub gene network was serine/threonine-protein kinase 17a, which had been reported to be a member of the death-associated protein (dap) kinase-related apoptosis-inducing protein kinase family encoding an autophosphorylated nuclear protein with a protein kinase domain in humans (lawrie et al., 2009). meanwhile, many erad-related genes were identified from the hub genes, indicating that this module was an endoplasmic reticulum-associated protein-mediated degradation and apoptosis regulatory network. a ppi network was further constructed to clarify the specific control mechanism of this complex control module. in ppi network, the autophagy and apoptosis related pathways regulated by er were obviously clustered in brown module. the center of the network was a group of transcription factors rich in c2h2 zinc finger domains to regulate various processes, such as ubiquitination, purine metabolism, jak-stat signaling, and rna transport. on pejus press, the ubiquitination pathway is activated to degrade the misfolded protein resulted from oxidative damage and energy deficiency. on pessimun press, the apoptosis is induced to avoid more damage caused 80 fig. 6 hub genes connectivity network and protein-protein interaction network of brown module. (a): hub genes connectivity network. network visualization of the interactions between top 5% highest connectivity genes in brown module. each node represents a gene, which is labeled with ncbi id. green nodes represent iaps. red nodes represent genes in erad pathway. see table s2 for the genelist of hub genes. (b): protein-protein interaction network. all genes were aligned to the sea urchin by blast to find the best match. edges with the interaction confidence < 0.8 were filtered. nodes information and the string enrichment of colored nodes can be found in table s3 81 by the inflammatory reaction. the regulation strategy of the transfer from damage repair to apoptosis according to the press greatly reduced the basic maintenance cost of c. gigas, which was suspected to be the key mechanism of c. gigas to adapt to harsh environments. the results indicated that this module was an er associated protein mediated complex regulatory network. oyster cells relied on er as a receptor for environmental stress to dynamically regulate various homeostasis-maintaining pathways (such as autophagy and apoptosis) through er stress and erad pathways to maintain the homeostasis in oyster gills. in conclusion, gene co-expression network analysis of transcriptome data indicated that c. gigas had evolved a set of air exposure adaptation mechanisms based on energy metabolism and regulation endoplasmic reticulum-related pathways. rich glycogen content provided sufficient energy to maintain stress induced damage in a pejus range. apoptotic pathway was activated when the stress increased to pessimum, and the overall energy strategy was changed from compensation to conservation. as a sensitive receptor for the external environment, er in the gill cell could adjust the strategy of homeostasis according to the magnitude of the environmental pressure, and rationally allocate the energy to meet the requirement for damage repair, transcriptional regulation and apoptosis. acknowledgment the authors are grateful to all the laboratory members for continuous technical advice and helpful discussions. this research was supported by national key research and development program (2018yfd0900606), a grant from national science foundation of china (no. u1706204), key r&d program of liaoning province (201703165 to l.s.), the earmarked fund (cars-49) for modern agro-industry technology research system, outstanding talents and innovative teams of agricultural scientific research in the ministry of agriculture, aoshan talents cultivation program supported by qingdao national laboratory for marine science and technology (no. 2017astcp-os13), dalian high level talent innovation support program (2015r020), the research foundation for distinguished professor in liaoning and talented scholars in dalian ocean university. references altschul sf, gish w, miller w, myers ew, lipman dj. basic local alignment search tool. j. mol. biol. 215: 403-410, 1990. ashburner m, ball ca, blake ja, botstein d, butler h, cherry jm, et al. gene ontology: tool for the unification of biology. nat. genet. 25: 25-29, 2000. bar-joseph z, gerber g, simon i, gifford dk, jaakkola ts. comparing the continuous representation of time-series expression profiles to identify differentially expressed genes. pnas. 100: 10146-10151, 2003. 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connectivity under different soft powers. generally, the minimum softpower with a scale free topology fit larger than 0.8 was chosen. review isj 12: 203-213, 2015 issn 1824-307x review the role of earthworm defense mechanisms in ecotoxicity studies r roubalová, p procházková, j dvořák, f škanta, m bilej laboratory of cellular and molecular immunology, institute of microbiology of the academy of sciences of the czech republic, v. v. i., vídeňská 1083, 142 20, prague 4, czech republic accepted july 24, 2015 abstract earthworms are important soil organisms that affect the soil structure by influencing organic and inorganic matter breakdown. earthworms are in permanent contact with soil particles via their permeable skin and digestive tract and are thus strongly affected by pollutants present in the soil. earthworms often live in very hostile environments with an abundant microflora and therefore have developed very potent defense mechanisms. these mechanisms have been described to be influenced by various types of organic and inorganic pollutants and also by the nanoparticles that reach the soil system. reduced abilities of earthworms to protect themselves against pathogenic microorganisms result in lower reproduction rates and increased mortality. in this review, a summary of the up-to-date data describing the effects of contaminants on the natural defense barriers and immune system of earthworms is presented. key words: pollution; immune system; earthworms; biomarker   introduction earthworms (lumbricidae, annelida) are protostomian organisms with a true celom that is filled with celomic fluid containing free celomocytes. the celomic cavity is metameric, and the segments are separated by transversal septa. each segment of the cavity interfaces with the outer environment via a pair of metanephridia and a dorsal pore that enables microorganisms to enter the celomic cavity. therefore, the celomic fluid is not aseptic and contains bacteria, fungi and protozoa from the outer environment. the growth of these microorganisms is kept under control by various cellular and humoral innate defense mechanisms that will be described in detail in the following section. earthworms are the most abundant invertebrates in the soils of temperate regions and are extremely important for soil formation (edwards, 2004). earthworms participate in nutrient cycling in terrestrial ecosystems and in the formation of the soil profile from the physical, chemical and microbial perspectives (bartlett et al., 2010). they improve its structure by increasing the macroporosity, which ___________________________________________________________________________ corresponding author: radka roubalová laboratory of cellular and molecular immunology institute of microbiology of the academy of sciences of the czech republic, v. v. i. vídeňská 1083, 142 20, prague 4, czech republic e-mail: r.roubalova@biomed.cas.cz affects aeration, water dynamics and organic and inorganic matter breakdown (wen et al., 2006; ruiz et al., 2011). earthworms are permanently in close contact with soil particles and microorganisms present in the soil via both a highly permeable skin and an alimentary tract (jager et al., 2003; drake and horn, 2007). therefore, they are significantly affected by the pollutants that reach the soil system and are thus well suited for the monitoring of soil contamination. different earthworm species have different effects on soil formation because of their different behavioral patterns. epigeic earthworms live above the mineral soil, rarely form burrows and preferentially feed on plant litter. endogeic species live below the surface, where they build predominantly horizontal burrows. these species ingest large amounts of mineral soils and humified material. anecic earthworms build permanent vertical burrows deep into the mineral soil layer and come to the surface to feed on decomposed plant litter and other organic residues (lee, 1985). two epigeic species, i.e., eisenia fetida and eisenia andrei, have been used for many years to monitor ecotoxicity. there are two sets of guidelines, i.e., those from the organization for economic cooperation and development (oecd) and those from the international organization for standardization (iso), for the assessment of the ecological risk of contaminated soil, the determination of the acute toxicity of chemicals on earthworms (oecd, 1984;     203 mailto:r.roubalova@biomed.cas.cz fig. 1 earthworms are affected by the presence of pollutants in the soil. hydrophilic contaminants enter the earthworm body predominantly through the skin, whereas hydrophobic substances enter via the digestive tract. pollutants are accumulated in earthworm tissues, which can result in tissue and cell disruption, such as progressive reduction of intestinal villi (iv) and chloragogenous tissue (ch) in earthworms kept in dioxin-polluted soil (b; roubalova et al., 2014). additionally, both cellular and humoral defense mechanisms are impaired by the soil contaminants. iso, 1993), and the effect on their reproduction (iso, 1998; oecd, 2004). earthworms have been described to bioaccumulate contaminants, such as various organic pollutants (jager et al., 2005), heavy metals (nahmani et al., 2007) and nanoparticles (canesi and prochazkova, 2014). they are able to take up chemicals from pore water through their skin and via soil ingestion. according to the model developed by belfroid et al. (1995), the ingestion of sediment can be the dominant uptake route of hydrophobic compounds with logkow values > 5. the presence of contaminants in the soil disturbs major physiological functions of earthworms, such as survival, nutrition, immunity, growth, and reproduction, and these effects depend on the matrix, exposure time, and the types and doses of the pollutants in the environment. in recent years, there has been a growing interest in increasing our knowledge of the biological responses of earthworms to pollutants in order to standardize a suite of biomarkers of the responses to soil chemical pollution (beliaeff and burgeot, 2002). biomarkers detect the effects of contamination at an early stage before sublethal effects, such as inhibition of growth and reproduction, become apparent. the biomarker approach represents a very useful tool in monitoring stress response to pollutants in field populations (kammenga et al., 2000; hankard et al., 2004). the choice of appropriate biomarkers is crucial for monitoring the effects of pollution on organisms. reactions to pollution may be monitored on various levels, the whole body level (viability, weight loss, reduction of reproduction, and escape reaction), the organ and tissue level (histopathological changes), the cellular level (decrease in the physiological conditions of the cells) and the molecular level (the upand down-regulation of the expression levels of     204 fig. 2 the general scheme of the innate defense mechanisms in earthworms. the first protective barrier of earthworms is the skin in combination with the secreted mucus that contains various antimicrobial factors. invading microorganisms are recognized by both soluble and membrane-bound pattern recognition receptors (prrs) that sense pathogen-associated molecular patterns (pamps). on the basis of this recognition, microorganisms are phagocytized by coelomocytes or agglutinated and subsequently encapsulated. moreover, genes encoding various humoral factors involved in the elimination of invaders are expressed, such as antimicrobial peptides (amps), cytolytic molecules, agglutinins, lysozyme and various soluble prrs that trigger the activation of the prophenoloxidase cascade. genes that are sensitive to the environmental changes, transcriptome profiling) (owen et al., 2008; asensio et al., 2013; calisi et al., 2013; roubalova et al., 2014; sanchez-hernandez et al., 2014; sforzini et al., 2015) (fig. 1). although these responses may indicate the disturbances at the level of populations, only few data link biomarker level with effects on the functioning of earthworms in ecosystems (maboeta et al., 2003; spurgeon et al., 2005; plytycz et al., 2009). the effects of pollutants on the defense mechanisms of earthworms     205 similarly to other invertebrates, earthworms rely on natural nonspecific innate immunity for defense and lack anticipatory, specific and lymphocytebased immune mechanisms. additionally, the natural barriers of earthworms represent the first line of protection against the invasion of microorganisms. a brief summary of earthworm immune mechanisms is shown in figure 2. in the following sections, the effects of various soil pollutants on the nonspecific defense barriers and the cellular and humoral mechanisms of immunity are reviewed. natural defense mechanisms and pollution the first nonspecific protective barrier in earthworms is the skin, which consists of the epidermis and a thin cuticle that covers the entire body. the epidermis is formed by a single-layer epithelium of supporting cells, basal cells that have an important role in wound healing and graft rejection, and secretory cells that secrete mucus containing mucopolysaccharide-lipid-protein complex (alves et al., 1984; bernaldo de quiros and benito, 1986) that serves as a lubricant during locomotion and contains several antimicrobial factors (valembois et al., 1986). the cuticle contains mucopolysaccharides that act as an antimicrobial barrier (rahemtulla and lovtrup, 1974). both cuticle and mucus production can be affected by the inorganic and organic contaminants as well as nanoparticles present in the soil. the exposure of the earthworms lumbricus rubellus and lumbricus variegatus to c60 fullerene nanoparticles has been described to result in cuticle damage with underlying pathologies of the epidermis and muscles (pakarinen et al., 2011; van der ploeg et al., 2013). furthermore, the exposure of e. fetida to sub-lethal concentrations of 1,2,4-trichlorobenzene     206 table 1 summary of recent studies involving genotoxicity assessment of various organic and inorganic pollutants tested species organic pollutant source reference e. fetida naphtenic acids constituents of petroleum, used in commercial and industrial applications (wang et al., 2015a) e. fetida di-n-butyl phthalates increase the plasticity of many materials (du et al., 2015) e. fetida benzo[a]pyrene the result of incomplete combustion (duan et al., 2015) e. fetida triclosan antimicrobial additive used in personal care products (lin et al., 2014) e. fetida metalaxy-m fungicide (liu et al. 2014) e. fetida azoxystrobin fungicide (han et al., 2014) e. fetida chlortetracycline veterinary antibiotics (lin et al., 2012) e. andrei b[a]p, tcdd by-products from a number of human activities (sforzini et al., 2012) e. fetida toluene, ethylbenzene and xylene associated with crude petroleum and petroleum products (liu et al., 2010) tested species inorganic pollutant source reference e. andrei cd, zn metals provided in the form of cdso4, znso4 (otomo et al., 2014) e. fetida cr, cu, ni, pb, zn soils subjected to chemical characterization and total main heavy metal quantification (zheng et al., 2013) l. castaneous, d. rubidus as concentrations of arsenic elevated due to mining (button et al., 2012) a. caliginosa, e. fetida cu, cd sites near roads with heavy traffic (klobucar et al., 2011) e. andrei be, al, ba, mn, fe, ni, zn, u deposition of mine tailings and sludge, runoffs from the aquatic system (lourenco et al. 2011) e. fetida ni, cr(iii), cr(vi) pollutants used in numerous industrial processes (bigorgne et al., 2010) e. fetida cd, pb toxic elements widely distributed in the environment (li et al., 2009) results in ultrastructure alterations of the cuticle and skin, and the reduction of mucus production by secretory cells. at higher concentrations, mucus production disappears, and the cuticle is loosened and weakened (wu et al., 2012a). exposure of the earthworm e. fetida to soil containing tetraethyl lead (tel) and lead oxide (a gasoline additive) causes ruptures of the cuticle and skin, extrusion of the coelomic fluid and inflexible metameric segmentation (venkateswara rao et al., 2003). cellular innate immunity the celomic fluid of earthworms contains different types of cells that are generally termed celomocytes. the nomenclature of celomocytes is based on differential staining, ultrastructure, and granular composition. there are two basic categories of celomocytes. amebocytes function primarily in immune reactions, such as phagocytosis, encapsulation, nodulation as well as humoral immune responses, and mainly nutritive     207 eleocytes (sima, 1994). celomocytes have been described to respond to a wide range of pollutants and therefore are often used in soil ecotoxicology assessment. at the cellular level, two immune system-related parameters have been used as sensitive sub-lethal endpoints in assessment of the toxicity of pollutants in earthworms: phagocytosis and nk-like cell activity. phagocytosis represents an important defense mechanism that begins with the recognition of non-self, which is followed by the engulfment and destruction of phagocytosed particles. engulfed material can be eliminated by proteolytic and lysosomal enzymes or by an oxidative burst that involves the production of highly reactive oxygen radicals. the inhibition of phagocytosis in earthworms that are exposed to various metals and organic substances, such as polychlorinated biphenyls (pcbs) and polychlorinated dibenzo-pdioxins/dibenzofurans (pcdds/fs), has been described (ville et al., 1995; fugere et al., 1996; fournier et al., 2000; sauve et al., 2002; belmeskine et al., 2012). silver nanoparticles have been shown to be accumulated predominantly in the amebocyte population of celomocytes with subsequent selective cytotoxicity of these cells (hayashi et al. 2012). furthermore, some celomocytes have been shown to possess cytotoxic activity similar to that of natural killer (nk) cells. these cells exhibit rapid response to allogenic structures and have been described to be involved in the rejection of allografts (suzuki and cooper, 1995). the nk-like cell activity has been demonstrated to be suppressed by polyaromatic hydrophobic hydrocarbons (pahs) (patel et al., 2007), pcbs (suzuki et al., 1995), and pcdds/fs (belmeskine et al., 2012). furthermore, flow cytometry has revealed a lower frequency of immune cells (amebocytes) in contrast with metabolic eleocytes in earthworms that have been exposed to metaland radionuclides-contaminated soil (lourenco et al., 2011). at the subcellular level, the lysosomal membrane stability system has been identified as a specific target of the toxic effects of contaminants (moore, 1990). lysosomal membrane integrity can be measured with the neutral red retention assay (weeks and svendsen, 1996). the stability of the membranes has been shown to decrease with increasing stress due to the presence of pollutants in the environment (moore, 1985; booth and o'halloran, 2001; booth et al., 2003). because many soil contaminants exert genotoxic activities that result in dna damage in the celomocytes, it is used as an important tool in environmental biomonitoring. the most widely used genotoxocity biomarker is the comet assay; this method has been shown to be appropriate for measuring dna damage in the individual cells of both vertebrates and invertebrates (singh et al., 1988; fairbairn et al., 1995; cotelle and ferard, 1999; faust et al., 2004; sforzini et al., 2012). in table 1, examples of organic and inorganic pollutants described in recent studies that cause dna damage are listed. humoral defense mechanisms molecules involved in innate immunity the celomic fluid of annelids exhibits numerous biological activities that are involved in the defense mechanisms against invaders (fig. 2). the recognition of microbial pathogens is mediated by pattern recognition receptors (prrs) that sense so-called pathogen-associated molecular patterns (pamps). these structures are common among microorganisms and include, i.e., the lipopolysaccharides of gram-negative bacteria, constituents of the peptidoglycan of gram-positive bacteria, β-glucans of yeasts and viral doublestranded rna. this recognition results in the activation of both cellular and humoral defense mechanisms, including the production of antimicrobial proteins and peptides (joskova et al., 2009), and the activation of an important invertebrate defense mechanism termed the prophenoloxidase cascade (beschin et al., 1998; soderhall and cerenius, 1998). to date, only two prrs in earthworms have been described, i.e., celomic cytolytic factor (ccf) (beschin et al., 1998; bilej et al., 1998, 2001) and toll-like receptor (tlr) (skanta et al., 2013), and these prrs recognize various pamps. the expression of ccf has been described to be significantly down-regulated in l. rubellus following lifelong exposure to c60 nanoparticles, which suggests the induction of immunosuppression (van der ploeg et al., 2013). dioxins have also been shown to affect the expression of ccf (roubalova et al., 2014). a wide range of antimicrobial molecules that are involved in killing the microorganisms that enter the earthworms´ bodies have been described. celomic fluid has been documented to contain various antimicrobial factors, such as lysozyme (çotuk and dales, 1984; joskova et al., 2009) and antimicrobial peptides (wang et al., 2003; liu et al., 2004; li et al., 2011). among the factors that are involved in humoral immunity, particular interest has been devoted to the cytolytic components that are secreted by celomocytes. the cytolytic activity of the celomic fluid was originally demonstrated on vertebrate erythrocytes and the resulting effect was described as hemolysis. the majority of identified hemolysins exhibit broad spectra of antibacterial and/or bacteriostatic activities against pathogenic soil bacteria (roch et al., 1991; milochau et al., 1997; eue et al., 1998). various types of pollutants, such as metallic compounds (brulle et al., 2008; mo et al., 2012) and tio2 nanoparticles (bigorgne et al., 2012), have been described to influence the expression of these molecules and therefore cause inappropriate immune response to invading pathogens. earthworm calreticulin is a highly conserved calcium-binding protein that has also been shown to be affected by the presence of various pollutants in soils (chen et al., 2011; roubalova et al., 2014). it participates in the regulation of ca2+ homeostasis, acts as a chaperone and is involved in the regulation of cell signaling (wang et al., 2012). it also plays a role in the stress response and immune reactions (goo et     208 table 2 list of pollutants that affect the activity and gene transcription of antioxidant enzymes pollutants that affect activities of antioxidant enzymes tested species type of pollutant enzymes affected by pollutants reference e. fetida decabromodiphenyl ether sod, cat, pod (zhang et al., 2014) e. fetida phenanthrene sod, cat, pod (shi et al., 2013) e. fetida multi-metal-contaminated soil (cd, cr, cu, ni, pb, and zn) sod (zheng et al., 2013) e. fetida phenanthrene, pyrene sod, cat (wu et al., 2012b) e. fetida chlortetracycline sod, cat (lin et al., 2012) e. fetida zno nanoparticles sod (li et al., 2011) pollutants that affect gene expression of antioxidant enzymes tested species type of pollutant genes affected by pollutants reference e. fetida naphthenic acids sod, cat (wang et al., 2015b) e. fetida 2,2',4,4'-tetrabromodiphenyl ether sod, cat (xu et al., 2015) e. fetida copper sulphate (cuso4) sod, cat (xiong et al., 2014) e. fetida silver nanoparticles sod, cat (hayashi et al., 2013) e. fetida zinc oxide (zno) sod, cat (xiong et al., 2012) e. fetida galaxolide, tonalide sod, cat (chen et al., 2011) al., 2005; kuraishi et al., 2007; silerova et al., 2007; gold et al., 2010). enzymes involved in oxidative stress aerobic organisms developed efficient antioxidant defense system to protect themselves against reactive oxygen species (ros). the major source of intracellular ros is the mitochondrial respiratory chain (han et al., 2001; ott et al., 2007), and these radicals are also produced in smaller amounts in other cell compartments, such as the endoplasmic reticulum, the plasma and nuclear membranes, and by some oxidases (mittler et al., 2004; del rio et al., 2006; navrot et al., 2007). free radicals were described to have an important role in cell signaling (mates et al., 2002; scandalios, 2005; mates et al., 2008) and protection against invading pathogens (babior et al., 1975; rossi et al., 1985; nacarelli and fuller-espie, 2011). oxidative stress induces dna modifications (bohr, 2002), direct oxidation and inactivation of iron-sulfur (fe-s) proteins (fridovich, 1997), lipid peroxidation (arai, 2014), and apoptotic events by means of caspase dependent pathways (bearoff and fuller-espie, 2011). under stressful conditions (e.g., exposures to uv radiation, organic and inorganic contaminants, extreme temperatures and biotic stress), the concentrations of ros increase, resulting in the development of oxidative stress and subsequent damage to cellular structures (foyer and noctor, 2005; gill and tuteja, 2010; tumminello and fullerespie, 2013). antioxidant enzymes are considered to be a primary defense that protects biological macromolecules from oxidative damage. three groups of these enzymes play significant roles in protecting cells from oxidant stress, i.e., superoxide dismutases (sods), catalase (cat) and peroxidases (pods) (mates, 2000). sods are a ubiquitous family of metal-containing enzymes that depend on bound manganese (mitochondrial sod), copper or zinc (intraand extra-cellular sods) for their antioxidant activity. sods efficiently catalyze the dismutation of superoxide anions into hydrogen peroxide, which is substantially less toxic than superoxide, and oxygen. cat and pods degrade hydrogen peroxide to water. both the enzyme activities and gene expression levels of antioxidant enzymes are frequently used to determine the effects of pollution on earthworms (table 2). conclusions this review summarized the data that have been published so far regarding the effects of various soil pollutants on the defense mechanisms of earthworms. the toxicities of these chemicals, which often enter the food chain, have been described to affect the immune system of not only invertebrates but also vertebrates, including humans. this article illustrated the various mechanisms through which the effects of pollutants are mediated on both the cellular and humoral components of the immune system. such disruptions of the abilities of earthworms to protect     209 themselves against invading pathogens has been shown to be closely related to the reduced reproduction and growth rates of earthworms and increased mortality. moreover, these immunological parameters can be used as reliable biomarkers for the detection of the pollutant-induced responses of soil organisms. acknowledgements this research was supported by the ministry of education, youth and sports (cz.1.07/2.3.00/30.0003), the institutional research concept rvo 61388971 and by the project “biocev biotechnology and biomedicine centre of the academy of sciences and charles university in vestec” (cz.1.05/1.1.00/02.0109) from the european regional development 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environ. toxicol. pharmacol. 38: 297-304, 2014. zheng k, liu z, li y, cui y, li m. toxicological responses of earthworm (eisenia fetida) exposed to metal-contaminated soils. environ. sci. pollut. res. int. 20: 8382-8390, 2013. review the role of earthworm defense mechanisms in ecotoxicity studies 105 isj 16: 105-112, 2019 issn 1824-307x research report expression analysis of mir-2005 and its target genes in apostichopus japonicus by vibrio splendidus challenged xx zhou, yq chang*, yy zhan, xl wang*, k lin key laboratory of mariculture & stock enhancement in north china’s sea, ministry of agriculture, dalian ocean university, dalian, 116023, china accepted june 25, 2019 abstract micrornas (mirnas) are important effectors in mediating host-pathogen interaction. mir-2005 is observed to be involved in immune response processes in apostichopus japonicus. in the present study, the putative target genes of mir-2005 in a. japonicus coelomocytes were predicted by bioinformatics analysis of transcriptome database and pcr approaches. a total of 506 potential targets were screened, and 187 targets were annotated. several immune-related target genes were identified in this study, such as sli3, cfhr5, fgl, a2ml, and rab9al. the expression patterns of mir-2005 and its potential targets were validated by quantitative real-time pcr in vibrio splendidus challenged a. japonicus. for further characterization, an overexpression experiment of mir-2005 at cellular levels was applied. accordingly, significant negative correlation expression profiles were detected between mir-2005 and two candidates targets, suggesting that sli3 and chrp5 showed high possibility to be the targets of mir-2005 in a. japonicus. altogether, this study will enhance our understanding in the context of mir-2005 modulating the interaction of a. japonicus after being challenged by v. splendidus. key words: mir-2005; sea cucumber (apostichopus japonicus); vibrio splendidus; overexpression experiment; spatial expression introduction the sea cucumber, apostichopus japonicus with medicinal effects and rich nutritional value for human consumption, naturally distributed along the coasts of east asian countries such as china, japan, korea and russia (sloan et al., 1984; chang et al., 2009). additionally, it is widely cultured as a significant aquaculture species in the above countries of east asia. the echinoderm immune system initiates a response when challenged with pathogen-associated molecular patterns (pamps), which are recognized through molecules known as pattern recognition receptors (prrs) (kawai et al., 2010). the combination of pamps and prrs activates many immune factors, such as antimicrobial ___________________________________________________________________________ corresponding authors: xiuli w ang yaqing chang key laboratory of mariculture & stock enhancement in north china’s sea ministry of agriculture dalian ocean university 52, heishijiao street, dalian, 116023, china e-mails: xiuliwang417@sina.com; yaqingchang@hotmail.com peptides, lectin, and complement through a series of cascade reactions to kill and eliminate invading pathogenic microorganisms, parasites and apoptotic bodies (fukuzawa et al., 2008). the immune response of a. japonicus is shown to be based on coelomocyte activity (chemotaxis and phagocytosis) (he et al., 2016), some humoral immune factors (lectins, opsonins and some bactericidal substances) (mai et al., 2009; wei et al., 2015) could also involve in the immune response that resist the invasion of pathogens directly. within such an intricate immune defense system, there are multiple layers of process for molecular regulation. micrornas (mirnas) are important effectors in complicated gene expression profiles through sequence-specific regulation, they could prove to be of significant functional importance in intricate molecular regulation. in a previous study, lv et al. (2015) observed that mir-200 could modulate the lipopolysaccharides (lps) priming and antimicrobial activities via augmenting toll-interacting protein (ajtollip). the negative expression profiles between mir-31 and its target ajp105 were also observed (lu et al., 2015). it’s indicated that reactive oxygen species (ros) accumulation could be stimulated, whether by mir-31 overexpression or ajp105 silencing. mir-137 and mir-2008 were predicated that could targeted the 3’-utr of betaine-homocysteine mailto:xiuliwang417@sina.com mailto:yaqingchang@hotmail.com 106 s-methyltransferase (ajbhmt), which induces respiratory bursts and affects hcy accumulation in coelomocytes (zhang et al., 2015). in addition, lv et al. (2017b) also found another target of mir-137, and identified the depressed expression profiles of mir-137 and its target gene 14-3-3ζ (aj14-3-3ζ) in both lps-exposed primary coelomocytes and vibrio splendidus-challenged a. japonicus (lv et al., 2017a). moreover, several putative targets of mir-92a were also identified by zhang et al. (2014). studies of drosophila and euarchonta (lu et al., 2008; zhang et al., 2008) suggested that gain and loss of mirnas can be affected by transposition and retrotransposition. thus, mirnas have been revealed as major regulators in various hosts-pathogen interaction processes of a. japonicus by repressing the transcription and post-transcriptional expression of target genes. however, although mirna libraries were constructed for understanding the physiological process of a. japonicus (li et al., 2012; chen et al., 2013; chen and storey, 2014; wang et al., 2014, 2015; sun et al., 2016), the studies on mirnas and their targets associated with immune response related skin ulceration syndrome of a. japonicus are relatively few. although, mir-2005 is found present only in strongylocentrotus purpuratus, lytechinus variegatus and patiria miniata based on mirbase records, the expression level of mir-2005 in sea cucumber was also discovered by wang et al., (2014) and zhong et al., (2015). wang et al., (2014) indicated that mir-2005 was significantly up-regulated in the tube foot of a. japonicus, followed by coelomocytes and respiratory tree. it’s suggested that the expression level of mir-2005 was not only highly expressed in the external tissues, but also in the internal environment of a. japonicus. moreover, mir-2005 was also observed to be involved in the immune response process in a. japonicus after the lps injection (zhong et al., 2015). in addition to mir-2005, the expressing levels of mir-2004, mir-133, and mir-137 that was altered along with the expression of a. japonicus complement c3 changing at diff erent time points after lps injection was also observed (zhong et al., 2015). according to the evidence resulting from zhong et al., (2015), it provided further proof for the important regulatory role of mir-2005 in a. japonicus immune system. to sum up, mir-2005 may be involved in the development and immunity defense process of a. japonicus. however, the knowledge of the regulation mechanism of mir-2005 in immune response is still very vague. as previously documented, coelomocytes are recognized to be one of the main components in echinoderm animal immune responses. the coelomocytes of a. japonicus are proved as an appropriate target to explore immune-related genes and physiological mechanisms in response to pathogenic challenge (dong et al., 2014). v. splendidus, as the gram-negative bacteria, is identified as a major pathogen that can cause skin ulceration disease in a. japonicus (zhang et al., 2006). therefore, v. splendidus was chosen as the pathogen to use to understand the regulation mechanism of mir-2005 in immune response by stimulating a. japonicus coelomocytes. fig. 1 predicted target mrnas of mir-2005. in this study, we report the identification of target genes of mir-2005 from the transcriptome database, including our transcriptome (unpublished) and that of sun et al., (2013) (accession no. gse44995). then, the relative expression of mir-2005 and its predictive targets were further investigated in v. splendidus-challenged a. japonicus coelomocytes. subsequently, overexpression was conducted in vitro to obtained the connection of mir-2005 and putative target genes. the present study can strengthen the understanding of the regulatory role of mir-2005 in host–pathogen interactions. additionally, this study could enhance the knowledge base of a. japonicus immunity upon being challenged by pathogens. material and methods target prediction of mir-2005 the 5’-untranslated regions (5’-utrs) and 3’-untranslated regions (3’-utrs) were regarded as the potential binding region between target mrna and mirna. the extracted utrs were obtained by in-house perl script according to the predicated open reading frame (orf) as previous study (zhou et al., 2018). the mir-2005 sequences, which were obtained from several previous works (wang et al., 2014; zhong et al., 2015), were used to predicate the target mrna by using the rna-seq sequences containing the extracted utrs. three software packages, including rnahybrid (rehmsmeier et al., 2004), pita (kertesz et al., 2007) and miranda-3.3 (ellegren, 2008), were used to predict the target mrnas, and the potential target genes were considered by the intersection of all three programs as previously described (zhou et al., 2016). the parameters of pita software were “-l 7-8”, and “-sc 140”, “-en -17” for miranda-3.3, while the default parameters were used and filtered by δg <= 20 kcal/mol in the rnahybrid. 107 table 1 primers used in qrt-rcr. animals and challenge experiment healthy sea cucumbers (average weight of 100 g) provided by the key laboratory of mariculture & stock enhancement in north china’s sea, ministry of agriculture (dalian, liaoning) were used in this study. for the challenge experiment, the pathogen bacteria vibrio splendidus (d4501) were chosen to cultured and harvested in our laboratory as previously described by (cheng et al., 2017). the v. splendidus were cultured at 28 °c overnight with shaking at 200 rpm. then centrifuged at 4000 rpm for 1 min (4 °c) to harvest the bacteria as previously described by cheng et al., (2017). for bacterial challenge experiment, the overnight cultured v. splendidus (d4501) was diluted with the phosphate buffered saline (pbs, 0.1 mm, ph 7.4) to achieve the working solution of v. splendidus (d4501) at the concentration of 107 cfu/ml. the challenge experiments were divided into a control group and a bacterial challenge group. a total of 60 sea cucumbers were divided into bacterial-challenged group and control group. the bacterial-challenge group (30 individuals) was injected with 100 l v. splendidus (d4501), while control group (30 individuals) was injected only in a tank containing pbs. the celomic fluids from five a. japonicus was collected at 0, 4, 8, 12, 24, 48 and 72 h post-injection. the samples centrifuged directly at 1000 × rpm for 5 min at 4 °c. after centrifugation, the supernatant was discarded, and snap-frozen in liquid nitrogen, then transferred to -80 °c until rna extraction. the validation of interaction between mir-2005 and target genes to evaluate the regulatory relationships between mir-2005 and its predicted targets, the expression level of mir-2005 and five predicted target genes were assessed by qrt-pcr. total rnas were extracted from coelomic fluid using trizol reagent (invitrogen, carlsbad, ca, usa) and the corresponding cdnas were synthesized with transscript mirna first-strand cdna synthesis supermix (transgen biotech, co., ltd., china). the sybr® primescripttm mirna rt-pcr kit (takara) was used for mirna and mirna quantification in the same samples by using described method with an abi 7500 real-time pcr machine (applied biosystems, foster city, ca, usa). the specific primer sets for mir-2005 and potential targets are shown in table 1. in our study, cytb and rnu6b (table 1) were served as a reference gene to normalize targets and mirna, respectively. in brief, each reaction was performed in a final volume of 16 μl containing 2 μl of the cdna as in a previous study (zhou et al., 2016a). three technical replications were performed for each qrt-pcr validation. pcr was conducted as follows: 94 °c for 30 s, 45 cycles of 94 °c for 5 s, and annealing temperature for 32 s. primary coelomocytes culture and mir-2005 over-expression in vitro in this study, coelomocytes were cultured as described by lu et al. (2015) and shao et al. (2015). the a. japonicus were dissected and the coelomic gene id gene name primer sequence (5’-3’) application isotig15273 serum lectin isoform 3 f: attgacagacacccttccac r: gacttcctgacctaacatcg real-time pcr isotig01560 fibrinogen-like protein f: gccaggatgtttatgacgct r: ttgttaccgaagccgtctct real-time pcr isotig15571 alpha-2-macroglobulin-like f: cggagagtaggtctgatgat r: gagtgacaaagagggaggtt real-time pcr comp279590_c0 complement factor h-related protein 5 f: gccagtagtatccatcatcc r: cgtgctccaacagattagtc real-time pcr comp275604_c0 rab-9a-like f: agccagtccttccatacgat r: ctcttgaacctctcctgtcct real-time pcr mir-2005 f: agtccaatagggagggcattgcagt r: universal mirna qpcr primer real-time pcr mir-2005m aguccaauagggagggcauugcag gcaaugcccucccuauuggacuuu mir-2005 mimics ncm uucuccgaacgugucacgutt acgugacacguucggagaatt negative control of mirna mimics cytb f: tgagccgcaacagtaatc r: aagggaaaaggaagtgaaag reference gene rnu6b f: acgcaaattcgtgaagcgtt r: universal mirna qpcr primer reference gene 108 fluids were filtered through a 300 mesh cellcribble to filter out large tissue debris, and then the coelomic fluids were centrifuged at 900 g 16 °c for 10min with the same volume of anticoagulant solution (0.48 m nacl, 0.019 m kcl, 0.02 megta, and 0.068 m tris-hcl, ph 7.6). the isotonic buffer (0.53 m nacl, 0.001 m egta, and 0.01 m tris-hcl, ph 7.6) was used to wash the harvested cells, and re-suspended in leibovitz's l-15 cell culture medium (invitrogen, usa) supplemented with gentamycin sulfate (100 μg × ml-1), penicillin (100 u ml-1), streptomycin sulfate (100 μg × ml-1), and nacl (0.39 m) to adjust the osmotic pressure. the coelomic cells that were diluted to 106 cells ml-1 were the suspended and dispensed into 24-well microplates and incubated at 16 °c for 12 h before the mir-2005 overexpression experiment. the mirna mimics and negative control of mirna mimics (ncm) were synthesized at genepharma and are shown in table 1. the rnase-free water was used to dissolve mir-2005 mimics and ncm to obtain a working solution of 20 μm. then 2 μl of mir-2005 mimics and ncm were mixed with an equal volume of hiperfect transfection reagents (genepharma, shanghai), and transfected into primary cultured cells. after 24 h post-transfection, the primary cultured cells were harvested and used for mir-2005 overexpression analysis. results prediction and analysis putative target genes of mir-2005 by using the query of the obtained utr reads in our previous study (zhou et al., 2018), the potential target genes of mir-2005 were screened using the program pita, miranda-3.3a and rnahybrid in that order. the veen plot shows the number of potential target genes that predicted by pita, miranda-3.3a and rnahybrid. as shown in fig. 1, 506 potential target genes of mir-2005 were predicted. the sequences of predicted target genes and the predicted binding sites for the mir-2005 are listed in supplementary table 1 and supplementary table 2, respectively. a total of 187 potential target genes were annotated (supplementary table 3). from the fig. 2 time-course expression patterns of mir-2005 and five predict target genes in a. japonicus after v. splendidus challenged at 0, 4, 8, 12, 24 and 48 h. 109 187 potential targets, five potential target genes (serum lectin isoform 3, sli3; fibrinogen-like protein, fgl; alpha 2-macroglobulin like, a2ml; complement factor h-related protein 5, cfhr5; and rab-9a-like, rab9al) were involved in immune defense against pathogens. both of sli and cfhr can possess the functions as opsonization (boackle et al., 2003). fgla, a2m and rab9a are also play vital roles in many physiological and biochemical reactions, including blood clotting and regeneration (lu et al., 2002; wu et al., 2014), regulate the prophenoloxidase (propo) activating system (jiang et al., 2006), biogenesis of lysosome-related organelles and regulates the degradation of cytoplasmic contents in the lysosome (nottingham et al., 2011). expression profile of mir-2005 and its target genes in v. splendidus-challenged a. japonicus to further understand the putative role of mir-2005 in a. japonicus immune defense in vitro, we selected five immune-related targets (sli3, cfhr5, fgl, a2ml, and rab9al) of mir-2005 using qrt-pcr method combined with the result of the bioinformatics analysis result. as shown in fig. 2, the first drastic cut was found in the expression of mir-2005 at 4 h (0.12-fold), and the second downward trend happened at 48 h. the expression pattern of sli3, rab9a and chrp5 all had two peaks, reached at 4 h and 48 h. functional analysis of mir-2005 and its target genes in vitro the primary coelomocytes of a. japonicus were cultured for gain-of-function experiment of mir-2005 (fig. 3). as shown in fig.4, the significant increase was obtained in the overexpression of mir-2005. the qrt-pcr results of sli3, a2ml and cfhr5 are also shown in fig. 4. the overexpression of mir-2005 decreased the mrna expression levels of sli3, a2ml and cfhr5 obtained in this study, and the downward trend of sli3 and cfhr5 were more significant than a2ml. discussion the echinoderm, with no specific immune cells, are mainly rely on the non-specific immune system that composed of cellular immunity and humoral immunity to complete the body's defense response. many complex molecular regulatory mechanisms are involved in the cellular immunity and humoral immunity of sea cucumber, one of the process is the reprogramming of expression of immune-related genes. the universal transcripts have been demonstrated that are across 70-90 % of mammal genome. however, more than 98 % of total transcripts are represented by non-coding rnas (ncrnas) (tay et al., 2014). accumulative evidence indicated that mirnas play essential role in multiple regulatory mechanisms of gene-expression to adapt and survive under pathogen infection (li et al., 2012; wang et al., 2014, 2015; sun et al., 2016; lv et al. 2017a,b; chen et al., 2018). thus, mirnas have been discovered as major regulators in different hosts-pathogen interaction processes of a. japonicus by blocking the expression of target genes. fig. 3 primary coelomocytes culture. in this study, we recognized that mir-2005 played a regulatory role in the interactions between a. japonicus and v. splendidus. the putative target genes of mir-2005 in a. japonicus coelomocytes were predicted by bioinformatics analysis. among the 506 potential target genes, five immune-related target genes (sli3, cfhr5, fgl, a2ml, and rab9al) of mir-2005 were found and selected to verified their relation expression patterns in a. japonicus under v. splendidus infection. two highly confident target genes (sli3 and cfhr5) are spotted in vitro and in vivo. both lectin and complement can be activated by the combination of pamps and prrs. lectin that exists on the surface of host cells or bacteria are necessary for the specificity recognition between humoral factors and foreign substances. sli and ca2+ dependent lectin has been found in the sea urchin and sea cucumber, which is able to recognize and attack foreign substances, and similar homologous to vertebrate cell membrane or soluble lectin (giga et al., 1987; himeshima et al., 1994; matsui et al., 1994; liu et al., 2012). after a. japonicus infected by v. splendidus, the expression pattern of sli3 was reached at 4 h and 48 h. similar expression pattern have been also observed in liu et al. (2012). the expression level of sli significantly increased in a. japonicus after they were infected by vibrio sp. for 3 days (liu et al., 2012). it is suggesting that sli may play important roles in the immune defense mechanism of sea cucumber against bacterial infection. moreover, the complement system is also playing a necessary role in the innate defense against common pathogens. as a complement control protein, cfhr is a member of the complement activation family regulators. five plasma proteins (cfhr1, cfhr2, cfhr3, cfhr4 and cfhr5) that each member binds to the complement component 3b (c3b) can comprise factor h related proteins (zhou et al., 2011; skerka et al., 2013; józsi et al., 2015). in human, cfhr5, composed of nine scr domains, which is the longest cfhr protein in the cfhr family (skerka et al., 2013). although, cfhr5 shares high 110 fig. 4 relative expression of mir-2005, rab-9a, sli3, and chrp5 in mir-2005 mimics transfected coelomocytes. ncm: negative control of mirna mimics; mir-2005m: mir-2005 mimics. * p < 0.05. sequence similarity with factor h. unlike factor h (fh), cfhr5 can binding to activated c3 and potentially enhance c3 deposition (zhu et al., 2018). one complement factor h gene and four-factor h-like genes were identified in danio rerio (sun et al., 2010), which highlights the possibility that all of the five genes were involved in the acute phase due to the fact that they were up-regulated by an lps challenge (sun et al., 2010). in our study, the expression pattern of cfhrs was similar to sli3 that were reached at 4 h and 48 h after a. japonicus infected by v. splendidus. by using an overexpression experiment of mir-2005 at cellular levels, significant negative correlation expression profiles were detected between mir-2005 and sli3 and cfhr5. it’s suggested that the abnormal expression of mir-2005 may have great effect in v. splendidus-challenged a. japonicus by targeting sli3 and cfhr5. based on the transcriptome and bioinformatics 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coffeae) in response to pesticide exposure s roy1, ak prasad2, g handique2, a barua2, s roy2 1natural product chemistry group, csir-north east institute of science & technology, jorhat, assam 785006, india 2entomology department, tocklai tea research institute, tea research association jorhat, assam 785008, india accepted november 2, 2016 abstract red spider mites (rsm) is one of the major pest of tea and reported from all tea producing regions around the world. chemical acaricides are the primary control method against this pest which induced biochemical changes in the rsm. in this study early expression of heat shock proteins have been observed in rsm exposed to commonly used acaricides viz., ethion, dicofol and fenpropathrin. in case of pesticide exposed rsm, hsp90 showed more prominent bands than the hsp70 in immunoblotted membrane, indicating that the intensity of expression of hsp90 in rsm against pesticides is higher than hsp70. hsp70 expression is higher in ethion and fenpropathrin induced rsm than that of dicofol. ethion and fenpropathrin treated rsm were not showing any difference when compared based on exposure time, whereas dicofol treated once showed more expression in short time. the expression of hsp90 is significantly higher in the dicofol treated samples followed by fenpropathrin, however ethion treated sample showed marginally higher hsp90 expression than control. key words: red spider mites; oligonychus coffeae; heat shock protein (hsp); ethion; fenpropathrin; dicofol introduction tea is currently produced by 30 countries including china, india, sri lanka, kenya, turkey and indonesia which contributes major part of the production (anonymous, 2013). tea is perennial plantations crop, provides a stable microclimate to thousands of pests which led to 5 55 % annual crop loss (hazarika et al., 2009). red spider mite, oligonychus coffeae nietner (acari: tetranychidae) is one of the major pest of tea and reported from most of the tea-producing countries. it is widely distributed, starting from afro-tropical region to australian, nearctic, neotropical, oriental and palearctic regions in 48 countries and feed on around 133 crops of these regions (roy et al., 2014). in last few decades, it caused a huge damage to tea plantations of north east india (roy et al., 2014). nymphs and adults of o. coffeae lacerate cells, producing minute characteristic reddish brown ___________________________________________________________________________ corresponding author: somnath roy entomology department tocklai tea research institute tea research association jorhat, assam 785008, india e-mail: somnathento@gmail.com marks on the upper surface of mature leaves, which turn red in severe cases of infestation, resulting in crop loss. the pest is present on tea all the year round, although numbers vary depending on season and reported to cause 17 46 % of crop loss per year (roy et al., 2014). chemical control is the primary mode of management of o. coffeae and a wide range of acaricides belonging to different chemical groups is currently used worldwide to control this pest. the difference in relative toxicity of different commonly used acaricides to o. coffeae was observed in plantations of the tea-growing region of india (roobakkumar et al., 2012; roy et al., 2012) as well as of japan (gotoh et al., 2001). in north east india, resistance in o. coffeae populations has been reported to a range of organochlorine (dicofol) and organophosphate (ethion) acaricides (roy et al., 2012). insects/mites are known to evolve resistance in about a decade after the introduction of a new pesticide by virtue of their genetic plasticity. the introduced toxicant present in the environment exerts a selection pressure on an insect population and only those individuals that can survive the toxic effect are able to reproduce. endemic population of a pest usually comprises a variety of 351 biotypes with minute differences from one another. pesticide selection may favour the fittest in the toxic environment. selection operates at biochemical, physiological, and behavioural level as well. zhao and jones (2012) reported that in insects heat shock protein (hsp) expressed in response of various stress factors. abiotic stress (including temperature, ultraviolet radiation, drought and anhydrobiosis, chemicals and metals), biotic stress (including parasites, pathogens) and developmental regulators and mutants can induce the hsp gene expression. hsp 70 and hsp 90 are also involved in various essential activities such as protein folding, localization and dehydration (king and macrae, 2015). hsp90 genes are also involved in tolerance and resistance not only to temperature but pesticides too. sun et al. (2014) studied the effect of pesticides on apolygus lucorum (meyer-dür) and subsequent expression of hsp90 gene. sarker and mukhopadhyay (2006), roobakkumar et al. (2012) and mukhopadhyay et al. (2016) reported enhancement of general esterase and glutathione-s-transferase as two defense enzymes responsible for development of resistance (biochemical resistance) in o. coffeae against acaricides in india. beside the xenobiotic detoxifying enzymes, synthesis of heat shock proteins could be another phenomenon by which insects respond to any abiotic stress (zhao and jones, 2012; zhang et al., 2015). in this paper, we compared the expression of hsps in o. coffeae exposed to commonly used different pesticides (acaricides) to assess the o. coffeae response against the effect of pesticides. material and methods chemicals the primary antibody anti-heat shock protein 70 and anti-heat shock protein 90 (monoclonal antibody, raised in mouse), ammonium persulfate, coomassie brilliant blue, phenylmethylsulfonly fluoride (pmsf), and nonidet were procured from sigma chemical co. (st. louis, mo). the molecular weight marker for polyacrylamide gel electrophoresis was purchased from genetix biotech asia pvt ltd. rabbit anti-mouse igg linked to alkaline phosphatase was procured from santacruz biotechnology, inc. all other chemicals used were of analytical grade, purchased from sisco research laboratories (mumbai, india) and e. merck (mumbai, india). acaricides used for induction of hsp are ethion 50 emulsifiable concentrates (ec) (aliphatic organothiophosphate: tafethion, rallis india ltd., mumbai, india), dicofol 18.5 ec (diphenyl aliphatics organochlorine: klin xl, krishi rasayan india ltd., delhi, india) and fenpropathrin 30 ec (fourth generation pyrethroid ester: meothrin, sumitomo chemicals india pvt. ltd., mumbai, india). maintenance of red spider mite a culture of red spider mite was maintained in the laboratory at 25 ± 2 ◦c and 70 80 % rh on a susceptible tea clone, tv1 by following the detached leaf culture method of roy et al. (2010). estimation of heat shock protein of 70 and 90 kda preparation of sample groups of two hundred adult mites each were taken in mature tea leaves and sprayed with selected acaricides viz., ethion (2500 ppm), dicofol (2500ppm) and fenpropathrin (500ppm) at their recommended dose. the mortality of rsm after 1 h of exposure to dicofol, ethion and fenpropathrin was 0%, 7.5 %, 7.5 % and after 6 h, mortality was recorded as 42.5 %, 65 %, 67.5 % respectively for three acaricides. the mites treated with water were taken as control. after 1 h and 6 h interval of spraying the treated mites were collected in a centrifuge tube. a 20 % homogenate of the whole body of control and treated red spider mite was prepared in 50mm tris buffer (ph 7.6) containing 0.1mm pmsf and 1 % nonidet and centrifuged at 10,000g for 10 min. the cytosolic supernatant was collected very carefully and protein content of the sample was measured following the method of lowry et al. (1951). methods for western blotting a 10 µl aliquot of cytosol with a concentration of 5 g/µl protein, prepared from control and treated mite samples containing total 50 g protein, was run through a 10 % sds-page at constant voltage (100v) for 2 h following the method of laemmli (1970) and blotted on a polyvinylidene fluoride membrane following the method of sambrook et al. (1989) as modified by roy and bhattacharya (2006). western blots were performed according to the method of sambrook et al. (1989) and transfer was carried out for 60 min at 50 v constant. antihsp70 monoclonal antibody raised in mouse (diluted 1:1,000) and anti-hsp90 monoclonal antibody raised in mouse (diluted 1:1,000) was used as primary antibody and rabbit anti-mouse igg linked to alkaline phosphatase (diluted to 1:500) was used as secondary antibody. the membrane was washed thoroughly and then incubated in the presence of the substrate (sigmafasttm bcip/nbt) results profile of the heat shock protein 70 and 90 the gel obtained from the sds-page was examined by immunoblotting with anti-hsp 70 monoclonal antibody. clear bands developed at regions of 70 kda and 90 kda respectively against hsp70 and hsp90 antibody. no other additional bands or smear are present on the immunoblot. the immunoblotted membrane of the hsp90 showed more prominent bands than the hsp70, indicating that the intensity of expression of hsp90 in red spider mite against pesticide namely ethion, fenpropathrin and dicofol is higher than hsp70 which is distinctly represented in the graph showing integrated density values (idv; calculated with the help of alpha easer software) (fig. 1). it is also clear that ethion and fenpropathrin induce hsp70 expression remarkably higher than dicofol. the exposure period-wise profile did not show any 352 fig. 1 graph showing integrated density values of each band of hsp70 and hsp90 developed in pvdf membrane (c: control, e1 h: after 1 h exposure to ethion, e6 h: after 6 h exposure to ethion, f1h: after 1 h exposure to fenpropathrin, f6h: after 6 h exposure to fenpropathrin, d1h: after 1 h exposure to dicofol, d6h: after 6 h exposure to dicofol). difference in ethion and fenpropathrin treated samples where as dicofol samples showed more expression in shorter time period. the expression of hsp90 is significantly higher in the dicofol treated samples followed by fenpropathrin (fig. 2). the level of hsp90 is marginally higher than control in ethion treated samples. discussion hsps are expressed in numerous tissues from several animal species, and their presence is often associated with a response to a harmful stress situation or to adverse life conditions. the highly conserved nature of hsp 70 and hsp 90 allows the use of a monoclonal antibody raised in mouse to detect the induced hsps in red spider mite. this is the only report of findings from pesticide-induced red spider mite which resemble those described in many other animals, including invertebrate species (zhao and jones, 2012). it is surmised that induction of hsps is an early response against prescribed doses of selected pesticide viz., ethion, dicofol and fenpropathrin. the hsp induction profiles of pesticide treated red spider mite clearly indicate the early response which can be a significant survival strategy of the test species. information about the induction of hsp in response to repeated or continuous exposures to pesticide is limited. pesticides, including organophosphate, organochlorine, and pyrethroid compounds, are widely used in agricultural including tea crop. in general, organophosphates are known to inhibit acetylcholine esterase (key enzyme of the cholinergic system, regulating the level of acetylcholine and terminates nerve impulses by catalyzing the hydrolysis of acetylcholine) mostly leading to death of the insect (stenersen, 2004). in the other hand, organochlorines are gaba-gated chloride channel antagonists and pyrethoid acts as sodium channel modulators (stenersen, 2004). all these pesticide classes affect the insect physiology in different way, ultimately causing stress to insect body. further, pesticides induce up regulation of detoxifying enzymes which in turn involves in glycolysis, the tca cycle and oxidative phosphorylation and this will lead to increase energy metabolism to support detoxification and the stress response (despres et al., 2007) as well as production of ros (reactive oxygen species) (du rand et al., 2015). the ros will then induce oxidative and heat shock stress responses (du rand et al., 2015). therefore the response of red spider mite to organochlorine (dicofol), organophosphate (ethion) and pyrethroid (fenpropathrin) with respect to hsps is highly significant. it is reported that the induced response of noctuidae spodoptera litura hsp90 to zinc was more sensitive than that of hsp70 (zhao and jones, 2012). the observation in this study also demonstrate that the intensity of expression of hsp90 in red spider mite against pesticide namely ethion, fenpropathrin and dicofol is higher than hsp70. joshi and tiwari (2000) suggested that a common set of gene loci encoding heat shock proteins is responsive to diverse environm ental 353 fig. 4 expression of hsp bands developed in pvdf membrane stresses in blowfly lucilia cuprina. hsp genes are induced and modulated in insects in response to environmental factors including abiotic and biotic stresses. it may be likely that via hsp activity, many pest and beneficial species will be able to adapt to global warming more than previously thought (zhao and jones, 2012). the function of hsps and other genes has been recently studied using dsrna interference (rnai) knock down techniques (papaconstantinou et al., 2010). this is the first report of hsp from red spider mites and can be of great importance while contemplating alternate methods of their control where any compound which can suppress gene expression of these hsp proteins can be employed as a new pesticide. from the present investigation it may be opined that the expression of hsp has a protective role in red spider mite. in future the pesticide-induced stress response can appear as an evident biomarker as indicated in our present study which may provide a great boost to biological control and reducing pesticide use. acknowledgement first author is grateful to dst-serb (sr/ft/ls-298/2012) for financial assistant. other authors extend their thanks to the director, tocklai tea research institute, tea research association, jorhat assam for providing laboratory infra structure and dst-syst [sp/yo/014/2015(c)] for funding the project. we thank both reviewers for critically reviewing the manuscript and for the help in the analysis of the result. references anonymous. planting commodities a stock taking. planters chronicles. 109:7, 2013. després l, david jp, gallet c. the evolutionary ecology of insect resistance to plant chemicals. trends ecol. evol. 22: 298–307, 2007. du rand ee, smit s, beukes m, apostolides z, pirk cww, nicolson sw. detoxification mechanisms of honey bees (apis mellifera) resulting in tolerance of dietary nicotine. sci. rep. 5:11779, doi:10.1038/srep11779, 2015. gotoh t, kitashima y, goka k, nagata t. susceptibility of the red spider mite, oligonychus coffeae (acari: tetranychidae), to acaricides on tea plants in japan. int. j. acarol. 27: 303-307, 2001. hazarika lk, bhuyan m, hazarika bn. insect pests of tea and their management. annu. rev. entomol. 54: 267-84, 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725, doi:10.1007/s12192-0140500-0, 2014. zhang lj, wu zl, wang kf, liu q, zhuang hm, wu g. trade-off between thermal tolerance and insecticide resistance in plutella xylostella. ecol. evol. 5: 515-530, 2015. zhao l, jones wa. expression of heat shock protein genes in insect stress responses. inv. surv. j. 9: 93-101, 2012. 1 isj 16: 1-7, 2019 issn 1824-307x research report effects of fluoride on primary cultured haemocytes from the marine gastropod haliotis tuberculata r ladhar-chaabouni1*, t houel2,3, j-m lebel2,3, a hamza-chaffai1, a serpentini2,3 1marine ecotoxicology ur 09-03, ipeis bp 805, 3018 sfax, tunisia 2normandie université f-14032 caen, france 3umr borea (biologie des organismes et des ecosystèmes aquatiques), mnhn, upmc, ucbn, cnrs-7208, ird-207, ibfa, université de caen normandie, esplanade de la paix, f-14032 caen cedex 5, france accepted january 3, 2019 abstract as a consequence of human’s activities, fluoride concentration in many aquatic ecosystems is significantly increasing. nevertheless, little is known about fluoride toxicity to aquatic life. in this study the effect of exposure to different concentrations of sodium fluoride (2, 10, 50, 250 and 1,250 μg ml−1) during 24 h on primary cultured haemocytes of the gastropod haliotis tuberculata was realized. results indicate no significant effect of naf on cell viability, lysosomal membrane stability, phagocytosis and ros production at concentrations of 2, 10, 50 and 250 μg ml−1. nevertheless, lysosomal membrane alterations, a decrease of phagocytosis and morphological changes of h. tuberculata haemocytes were observed at concentration of 1,250 µg ml−1 naf suggesting a potential impact of naf at high concentration in the environment. key words: fluoride; haemocytes; haliotis tuberculata; immune parameters; in vitro; primary culture introduction high exposure to fluoride may occur through a combination of natural and anthropogenic process as well as misuse of fluoride-containing dental care products (borke and whitford, 1999). the most obvious early toxic effects of fluoride on humans are dental and skeletal fluorosis (barbier et al., 2010; ullah et al., 2017). in mammalian cells, agalakova and gusev (2012a) showed that fluoride is an important modulator of the expression of genes implicated in apoptosis, amino acid phosphorylation, oxidative stress, cell cycle progression, chemotaxis, glycolysis, inflammation and signal transduction. furthermore, fluoride acts as an inhibitor of the activity of a broad range of enzymes (reddy et al., 2009; barbier et al., 2010; zuo et al., 2018). in unpolluted seawater, fluoride concentrations generally range from 1.2 to 1.5 mg l-1. however and as a consequence of human activities, these levels can increase more than 100 times (camargo, 2003). aquatic animals such as fish and invertebrates can take up fluoride directly from the water or via food(hemens and warwick, 1972; nell and livanos, ___________________________________________________________________________ corresponding author: rim ladhar-chaabouni marine ecotoxicology ur 09-03 ipeis bp 805 3018 sfax, tunisia e-mail: rladhar@yahoo.fr 1988; mondal and nath, 2015) and the toxicity of this element was reported in the freshwater mussels like alasmidonta raveneliana (keller and augspurger, 2005) and dreissena polymorpha (del piero et al., 2012). in mollusks, the cellular immune system is represented by haemocytes due to their ability to interact with foreign materials and to develop immune responses (galloway and depledge, 2001; hooper et al., 2007). a loss of haemocyte functionality due to pollutants like fluoride can be deleterious for the animal survival. few data are available regarding the effects of fluoride on haemocyte parameters of mollusks and marine invertebrates in general. thus, the aim of this work was to determine the in vitro effects of sodium fluoride on primary cultured haemocyte of the european abalone haliotis tuberculata. materials and methods specimens haliotis tuberculata were collected by france haliotis (plouguerneau, france), maintained in aerated seawater at 17 °c and fed ad libitum with a mixed algal diet (laminaria sp. and palmaria sp.). in the centre de recherche en environnement côtier (c.r.e.c., luc -sur-mer, bassenormandie, france), abalones were acclimated at least 2 weeks before the experiments began. mailto:rladhar@yahoo.fr 2 fig. 1 variations of haemocyte viability after exposure to 0, 2, 10, 50, 250 and 1,250 µg ml-1 of naf for 24 h using the mtt reduction assay. data shown are from three separate sets of experiments. each experiment was realized in triplicate primary cell cultures haemolymph was extracted from abalone by inserting syringe needles into the pedal sinus in the middle of the foot. haemocytes were counted with a hemocytometer and plated at a density of 300,000 cells per well in 24well plates (neutral red assay) or 500,000 cells per well in 12-well plates (mtt, flow cytometry analysis). after addition of three volumes of sterile artificial seawater (assw) (436 mm nacl, 53 mm mgso4, 20 mm hepes, 10 mm cacl2, 10 mm kcl, final ph 7.4), the cultures were maintained at 17 °c for 90 min to allow cells to adhere onto the bottom of the culture well. then, the assw was replaced by hank’s sterile 199 medium modified by the addition of 250 mm nacl, 10 mm kcl, 25 mm mgso4, 2.5 mm cacl2 and 10 mm hepes (final ph of 7.4) and supplemented with 2 mm l-glutamine, 100 µg ml−1 streptomycin, 60 µg ml−1 penicillin g and 2 mm concanavalin a. cells were incubated at 17 °c overnight (lebel et al., 1996; serpentini et al., 2000; mottin et al., 2010; ladhar-chaabouni et al., 2017). sodium fluoride solution a storage solution was prepared at a concentration of 40 mg ml−1 in hank’s sterile medium. then, dilutions in hank’s sterile medium to obtain the working solutions (0, 2, 10, 50, 250 and 1,250 μg ml−1) were realized. after cell cultures, the medium was aspirated and replaced by the naf solutions for 24 h. haemocyte viability assay cellular viability was estimated using a 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (mtt) reduction assay adapted to molluscan cell cultures as previously described (domart-coulon et al., 2000; ladhar-chaabouni et al., 2017). briefly, after 24 h incubation of the cells (17 °c) with 10% (v/v) of mtt solution (5 mg ml-1 of pbs), resulting product (formazan) was dissolved using an equal volume of isopropanol containing 0.04 n hcl. optical density was read at 570 nm and at 630 nm (reference) neutral red retention assay lysosomal membrane stability was assessed using neutral red (nr) retention assay as previously described (ladhar-chaabouni et al., 2017). after 1 h incubation of the cells (17 °c) with 10% (v/v) of nr stock solution (0.5% in pbs 1x), wells were washed with molluscan physiological saline (mps) (0.4 m nacl, 0.1 m mgso4, 20 mm hepes, 10 mm cacl2 and 10 mm kcl) and nr was extracted from lysosomes using 1% acetic acid in 50% ethanol. absorbance was estimated photometrically at 540 nm with 650 nm reference. phagocytosis and ros assays phagocytosis and reactive oxygen species (ros) production were determined using a flow cytometer (gallios, beckman coulter®), as previously described by mottin et al. (2010), latire et al. (2012) and ladhar-chaabouni et al. (2017) on abalone haemocytes, and 10,000 events were counted for each sample. for phagocytosis assays, 7 µl of bead solution (carboxylate-modified fluospheres®, yellow-green fluorescence, 1 µm diameter, molecular probes) was added to each well. after incubation at 17 °c in the dark during 60 min, the wells were scraped gently and the samples were centrifuged at 500 xg for 10 min at 4 °c. then, 300 µl of 3% paraformaldehyde was added to the pellet. the percentage of phagocytic cells was evaluated as the percentage of haemocytes that had engulfed at least three beads (i.e. immunoefficiency). ros production was evaluated using the 2’7’-dichlorofluorescein diacetate (dcfh3 da, sigma) method. after haemocytes incubation for 60 min at 17 °c in the dark with dcfh-da (final concentration of 100 µm), the wells were scraped gently and the samples were centrifuged at 500× g for 10 min at 4 °c. the resulting pellet was mixed with 300 µl of 3% paraformaldehyde (pfa). samples were stored at 4 °c until analysis. the results were expressed as the percentage of cells exhibiting fluorescence. data analysis each experiment was repeated three times, and the means were calculated from triplicates for each experiment. the results were processed by spss software. the statistical differences were assessed using the one-way anova followed by a post hoc test (tukey test) (p < 0.05 was considered significant). after statistical analysis the mean optical density (mtt and nr assays) in controls was assigned a value of 100%. for the phagocytosis and ros production, the mean percentage in controls was assigned a value of 100%. results variations of haemocyte viability and morphology after naf exposure as shown in figure 1, no significant difference in cell viability for haemocytes cultured for 24 h in the presence of different concentrations of naf (2, 10, 50, 250 and 1,250 μg ml−1) when using mtt assay. in the absence of naf, cells displayed many thin pseudopodia (fig. 2a). at concentrations of 2, 10, 50 and 250 µg ml-1 naf, the same shapes were observed (fig. 2b-e). nevertheless, after exposure to 1,250 µg ml-1 naf, changes in cell morphology were observed (fig. 2f) with the abundance of shrunk cells with no extensions. variations of haemocyte lysosomal membrane stability after naf exposure figure 3 showed a significant decrease (p  0.05) of nr staining of lysosomes after exposure to 1,250 μg ml−1 naf compared to controls. this decrease was 79% compared to the 100% control. fig. 2 variations of h. tuberculata haemocytes morphology after exposure to 0 µg ml-1 naf (a), 2 µg ml-1 naf (b), 10 µg ml-1 naf (c), 50 µg ml-1 naf (d), 250 µg ml-1 naf (e) and 1,250 µg ml-1 naf (f) for 24 h using light microscopy. arrow showed spreading cell with pseudopod; arrow head showed shrunk cells with no extension 4 fig. 3 variations of haemocyte lysosomal membrane stability after exposure to 0, 2, 10, 50, 250 and 1,250 µg ml-1 of naf for 24 h using the neutral red assay. data shown are from three separate sets of experiments. each experiment was made in triplicate. significant differences compared to controls are marked by asterisk (p < 0.05) variations of immune parameters after naf exposure figure 4 shows no significant variations of phagocytosis of abalone haemocytes after the exposure to 2, 10, 50 and 250 μg ml−1 of naf. nevertheless, the phagocytic activity was significantly inhibited (p  0.05) when cells were exposed to 1,250 µg ml-1 naf. this decrease was 24.25% compared to the 100% control. concerning ros production, figure 5 shows no significant variations of ros production by abalone haemocytes after exposure to different concentrations of naf after a 24 h of exposure. discussion in the present paper, we analyzed the effects of in vitro naf exposure on the haemocytes of the european abalone h. tuberculata. the results showed no significant influence of exposure to 2, 10, 50 and 250 μg ml−1 of naf on the viability, lysosomal membrane stability, morphology, phagocytic activity and ros production of haemocytes after 24 h of exposure. however a significant decrease of lysosomal membrane stability and phagocytic activity was observed after an exposure to 1,250 µg ml-1 of naf as well as changes in cell morphology. concerning cell viability, our results were in disagreement with those observed by ballarin et al., (2014) who showed a significant increase of mortality index of venerupus philippinarum haemocytes exposed to 10, 50 and 250 μg ml−1 of naf during 60 min. the authors showed that 23% of cells exposed to 250 µg ml-1 of naf stained positively with trypan blue indicating that cell membrane barrier function had been compromised. in the present study, cell viability was assessed using the mtt assay based on the assumption that mtt tetrazolium salt reduction to formazan occurs in the mitochondria of living cells due to the activity of mitochondrial dehydrogenases (in particular, succinate dehydrogenase). therefore, it can be assumed that the increased mtt-tetrazolium salt reduction rate is an effect of elevated succinate dehydrogenase activity. according to barbier et al., (2010), who outlined disruption of enzymes activities (mostly inhibition) by fluoride by binding to functional amino acid groups that surround the active centre of an enzyme, we expected an inhibition of succinate dehydrogenase activity of abalone haemocytes exposed to naf. curiously, this inhibition was not detected with naf concentrations used in this study. thus, higher naf concentrations should be tested to confirm the results of mendoza-shulz et al., (2009) who indicated that fluoride at micromolar concentrations can act as an anabolic agent and promote cell proliferation, whereas at millimolar concentrations it acts as an enzyme inhibitor like phosphatases, which play an important role in the atp production cycle and cellular respiration. after 24 h exposure to 1,250 µg ml-1 naf, morphological changes of h. tuberculata haemocytes were observed. in v. philippinarum the cell morphology changes were detected after an exposure to 250 µg ml-1 of naf during only 60 min of exposure. cell morphology changes in the presence of pollutants were also reported for other marine invertebrate species (olabarrieta et al., 2001; 5 fig. 4 variations of phagocytic activity after exposure to 0, 2, 10, 50, 250 and 1,250 µg ml-1 of naf for 24 h compared to the 100% control. each data point represents the mean percentage ± standard deviation of triplicate cultures. significant differences compared to controls are marked by asterisk (p < 0.05) gómez-mendikute and cajaraville, 2003; menin et al., 2008) suggesting that some pollutants can alter the cytoskeletal organization (cima et al., 1999). further research is required to determine the effect of fluoride on glycolysis in molluscan cells (as it was shown for mammalian cells). since glycolysis is a major atp source and its inhibition can disrupt the membrane-cytoskeleton interactions. using nr assay, ballarin et al., (2014) showed a decrease of lysosomal membrane stability of v. philippinarum haemocytes after exposure to 250 µg ml-1 of naf during only 5 minutes. in the present study, such decrease was not observed when using the same concentration during 24 h. we had use a concentration 5 times greater to induce lysosomal membrane destabilization of h. tuberculata haemocytes. a loss of lysosomal integrity was also observed in h. tuberculata haemocytes exposed to cadmium chloride (latire et al., 2012) and antidepressants (minguez et al., 2014). it appears that lysosome alterations vary according to the species as well as to the nature of contaminant. in addition to lysosomal membrane destabilization, it was shown that fluoride induced plasma membrane alteration and cytoskeleton disorganization leading to cells morphological changes (agalakova and gusev, 2011). in mammalian cells, such phenomenon was explained by the direct inhibition of glycolysis and depletion of cellular atp caused by fluoride (otsuki et al., 2005). indeed, fluoride induced accumulation of na+ and ca2+ in the rat erythrocytes, accompanied by ca2+ dependent k+ loss and morphological changes of the cells, can be partly explained by inhibition of na+-k+ and ca2+pumps due to atp depletion (agalakova and gusev, 2012b). flow cytometric evaluation of phagocytosis of fluorescent beads showed a decrease in the phagocytic activity of h. tuberculata haemocytes only at the highest concentration tested (1,250 μg ml−1 naf). ballarin et al., (2014) indicated that naf reduces v. philippinarum cell phagocytosis in a dose-dependent way. such results demonstrated that from species to species, the haemocyte phagocytic activity varies. such suggestion was also determined by sauvé et al., (2002) who showed that haemocyte phagocytosis varied with species after in vitro exposure to different metals (ag, cd, hg and zn). mazur et al., (1977) showed that naf markedly inhibited the macrophage phagocytosis due to interaction of this agent with cellular constituents, possibly contractile (and/or associated) proteins, which are direct or indirect determinants of cell deformability. further studies are needed to elucidate the effect of pollutants like naf on the function of contractile proteins and microtubules in molluscan haemocytes. concerning ros production, no significant variations after exposure to different concentrations of naf was observed in the present study although oxidative stress is a recognized mode of action of fluoride exposure that has been observed in vitro in several types of cells (ghosh et al., 2002; zhang et al., 2007; garciamontalvo et al., 2009). fluoride is thought to inhibit the activity of antioxidant enzymes such as superoxide dismutase (sod), glutathione peroxidase, and catalase (nobes et al., 1995; garcia-montalvo et al., 2009; miranda et al., 2018). the excessive production of ros leads to macromolecule oxidation, mitochondrial membrane depolarization, and apoptosis (barbier et al., 2010; giri et al., 2016). the last phenomenon was observed in v. philippinarum haemocytes exposed to 50 and 250 µg ml-1 naf during 60 min (ballarin et al., 2014). the authors suggest that apoptosis could be a consequence of the oxidative stress caused by the exposure to naf. suchocki et al., (2010) showed that the presence of ros leads to 6 fig. 5 variations of ros production after exposure to 0, 2, 10, 50, 250 and 1,250 µg ml-1 of naf for 24 h compared to the 100% control. each data point represents the mean percentage ± standard deviation of triplicate cultures the disruption of mitochondrial enzyme activity. scatena et al., (2004) showed that under increased ros level conditions, the activity of mitochondrial succinate, an enzyme responsible for mtt salt reduction, is inhibited. hence, the absence of significant increase of ros production in the present study could explain the stability of mtttetrazolium salt reducing mitochondrial enzymes activities in abalone haemocytes exposed to different concentrations of naf. in summary, our study has shed some light on the effect of naf on the primary cultured haemocytes of h. tuberculate. the results showed that naf at concentrations of 2, 10, 50 and 250 μg ml−1 didn’t have immunotoxic effects. nevertheless, a reduction of immune functions was observed at concentration of 1,250 µg ml−1 naf. further studies concerning molecular mechanisms of naf toxicity such the inhibition of glycolysis thus causing the depletion of cellular atp in h. tuberculate haemocytes are required. conflict of interest on behalf of all authors, the corresponding author states that there is no conflict of interest. references agalakova ni, gusev gp. fluoride-induced death of rat erythrocytes in vitro. toxicol. in vitro. 25: 1609-1618, 2011. agalakova ni, gusev gp. molecular mechanisms of cytotoxicity and apoptosis induced by inorganic fluoride. isrn cell biology. 2012a. agalakova ni, gusev gp. fluoride induces oxidative 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ghayor c, hebert v, galéra p, pujol jp, boucaud-camou e, et al. de novo synthesis and identification of collagen transcripts in hemocytes from the gastropod mollusc, haliotis tuberculata. j. exp. zool. 287: 275-284, 2000. suchocki p, misiewicz-krzemińska i, skupińska k, niedźwiecka k, lubelska k, fijałek z, et al. selenitetriglicerydes affect cyp1a1 and qr activity by involvement of reactive oxygen species and nrf2 transcription factor. pharmacol. rep. 62: 352-61, 2010. ullah r, zafar m.s, shahani n. potential fluoride toxicity from oral medicaments: a review. iran. j. basic. med. sci. 20: 841-848, 2017. zhang m, wang a, he w, he p, xu b, xia t, et al. effects of fluoride on the expression of ncam, oxidative stress, and apoptosis in primary cultured hippocampal neurons. toxicology. 236: 208-216, 2007. zuo h, chen l, kong m, qiu l, lü p, wu p et al. toxic effects of fluoride on organisms. life sci. 198: 18-24, 2018. https://www.hindawi.com/68737604/ https://www.hindawi.com/82950920/ https://www.hindawi.com/98137821/ https://www.hindawi.com/92142367/ https://www.hindawi.com/92142367/ https://www.hindawi.com/23193914/ https://www.hindawi.com/93691205/ https://www.hindawi.com/93691205/ https://www.sciencedirect.com/science/article/abs/pii/s0024320518300456#! https://www.sciencedirect.com/science/article/abs/pii/s0024320518300456#! https://www.sciencedirect.com/science/article/abs/pii/s0024320518300456#! https://www.sciencedirect.com/science/article/abs/pii/s0024320518300456#! https://www.sciencedirect.com/science/article/abs/pii/s0024320518300456#! https://www.sciencedirect.com/science/article/abs/pii/s0024320518300456#! isj 12: yyy-xxx, 2015 isj 12: 287-295, 2015 issn 1824-307x research report detection and preliminary characterization of antibacterial protein(s) in the serum of mud crab, scylla serrata v meiyalagan, m arumugam laboratory of pathobiology, department of zoology, university of madras, guindy campus, chennai 600 025, india accepted november 2, 2015 abstract serum of mud crab, scylla serrata has been found to possess significant antibacterial activity against some of the resident specific bacteria including bacillus sp. n1, bacillus flexus n3, escherichia coli as well as crustacean pathogenic bacteria viz., vibrio harveyi, v. alginolyticus and v. vulnificus. the physico-chemical characterization reveals the molecule responsible for antibacterial activity in the serum over 14 kda, stable in the ph range of 6 to 8 and between the temperatures 20 to 40 ºc. precipitation of respective molecule(s) with 75 % ammonium sulphate or the supernatant obtained after precipitating the protein with 10 % tca indicated that the molecule(s) responsible for serum antibacterial activity appear to be proteinaceous in nature. further studies demonstrated that antibacterial molecule(s) against e. coli and v. harveyi appeared to be trypsin and pronase resistant and the molecule(s) or domain responsible for antibacterial activity against bacillus sp. n1 and b. flexus n3 appeared to be protease sensitive, thereby implicating possible involvement of multiple antibacterial factors in the serum of mud crab, s. serrata. key words: mud crab; scylla serrata; serum; antibacterial activity; protein   introduction invertebrates, which contributes about 95 % of the extant species in the animal kingdom (ratcliffe, 1985; smith, 1991). among invertebrates, crustaceans are the second largest group, next to insects, in the phylum arthropoda comprising approximately more than 30,000 known species and they have a predominant role in aquatic food chain, especially as primary and secondary consumers as well as food for human (bachère et al., 2000). though, the invertebrate including crustaceans lack lymphocytes and functional immunoglobulins (iggs), this should not rule out the potential for the existence of not only innate immune mechanisms known in mammalian system but also unique form of innate immune mechanisms that might have been discovered with evolution of the first vertebrates (rowley and powell, 2007). invertebrates have developed unique defense mechanisms/modalities to detect microbial surface ___________________________________________________________________________ corresponding author: velayutham meiyalagan department of diagnosis and surgery school of dentistry at araraquara sao paulo state university rua humaita, 1680 14801-903 araraquara, sao paulo, brazil e-mail: meiyazhagan1982@live.com ‘antigens like’ lipopolysaccharides (lps), lipoteichoic acids, lipoproteins, peptidoglycans (pgn), 1,3-β-d-glucans, toll like receptors mediated antibacterial peptides (lemaitre et al., 1996; imler and hoffmann, 2000; underhill and ozinsky, 2002) and respond through hemolymph coagulation (iwanaga et al., 1978), melanin formation (sugumaran, 2002), agglutinin/lectin mediated complement activation (fujita, 2002), generation of reactive oxygen intermediates (rois), nitric oxide (no) (raman et al., 2008) and phagocytic system, encapsulation and nodule formation which cooperate with humoral immune reactors to kill invading pathogens (söderhäll and cerenius, 1992; bogdan et al., 2000). these mechanisms, which together compose the innate immune system, defend invertebrates from invading pathogens like bacteria, fungi and viruses (iwanaga and lee, 2005; rowley and powell, 2007). antimicrobial peptides/proteins are a major component of the innate immune defense system in marine invertebrates. these molecules are the first line of host defense in various species and the knowledge acquired over the last two decades on the identification and characterization of antimicrobial peptides/proteins in crustaceans has revealed their essential role in the immune response and also in the capacity of these animals to survive 287 mailto:meiyazhagan1982@live.com fig. 1 serum antibacterial activity against bacterial colonies (bacillus sp. n1, bacillus flexus n3, escherichia coli and pseudomonas aeruginosa) isolated from the scraps of injured/wounded cuticle of the mud crab, scylla serrata. each bar represents mean ± sd from three determinations. the difference in antibacterial activity between control and whole serum treated bacteria were statistically significant at *p < 0.05 level. infection. antibacterial peptides or proteins have been most extensively studied (yeaton, 1981; vasta and marchalonis, 1985; olafsen, 1995; smith and chisholm, 2001) due to the following reasons: (1) they occur in the hemolymph/serum of almost every crustacean species examined and (2) interact directly with foreign materials, particularly the potential microbial pathogens and thereby appear to serve as humoral recognition function in second line of defense. scylla serrata, is an economically important marine invertebrate distributed throughout the west pacific and indian oceans. it is the most important edible crab for commercial culture in the indo-west pacific region and commands a high price in both the domestic and export markets (samonte and agbayani, 1992). these crabs are in intimate contact with an environment rich in pathogenic bacteria, and are prone to infection by microbes at various stages of growth, losses due to disease can be enormous (hudson and lester, 1994). hence, it is necessary to understand the existing defense mechanisms in such animals and find ways of enhancing their natural immunity against infectious pathogens. in addition, from the identified antimicrobial peptides/protein families, only a few investigators have tested these molecules against crustacean pathogens such as marine vibrios that may cause severe infections to these animals. this study thus describe the serum antibacterial activity against resident specific as well as crustacean pathogenic bacteria and physico-chemical characterization of the naturally occurring antibacterial molecule in the serum of mud crab, s. serrata. materials and methods isolation and identification of bacteria the mud crab, scylla serrata collected from the backwaters of pulicat lake, were brought to laboratory, maintained in sea water (35 ± 2 ‰) and acclimatized for at least 3 to 4 days. the samples were collected from injured cuticular region of the crab, from these samples four distinct bacterial colonies were isolated and identified using a series of morphological, biochemical as well as molecular (16s rdna) studies as bacillus sp. n1, bacillus flexus n3, escherichia coli and pseudomonas aeruginosa (data not shown) using bergey’s manual of determinative bacteriology (holt et al., 1994). serum antibacterial activity from the cut end of dactylus region of the walking leg of the crab, the hemolymph was collected in the eppendorf tubes held on ice and allowed to clot at room temperature (rt). the clot was vigorously disturbed by using a glass rod, centrifuged (450g, 10 min, 4 °c) and the resulting clear supernatant (=serum) was used immediately. the serum antibacterial activity was tested by colorimetric method using mtt [3-(4, 5dimethylthiazol-2-yl) 2, 5diphenyltetrazolium bromide] against bacterial species isolated from s. serrata viz., bacillus sp. n1, bacillus flexus n3, escherichia coli and pseudomonas aeruginosa as well as other known crustacean pathogenic bacteria procured from ciba, chennai, including vibrio harveyi, v. vulnificus, v. alginolyticus, v. parahemolyticusand v. anguillarum (freimoser et al., 1999; sheena et al., 2003). 288 fig. 2 serum antibacterial activity against crustacean pathogenic bacteria (vibrio harveyi, v. alginolyticus and v. vulnificus). each bar represents mean ± sd from three determinations. the difference in antibacterial activity between control and whole serum treated bacteria were statistically significant at *p < 0.05 level. fig. 3 antibacterial activity in the serum of serum dialysate (mwco 12 to 14 kda) of the mud crab, scylla serrata. each points represents mean ± sd from four determinations. effect of dialysis on the serum antibacterial activity serum samples (500 µl) were extensively dialysed using dialysis tubing with mw exclusion limit of 12,000 to 14,000 da against tbs (50 mm tris-hcl; 115 mm nacl; 10 mm cacl2; ph 7.5; 300 mosm). the resulting dialysates were centrifuged (400g, 5 min, 4 °c) and the antibacterial activity of the supernatant was determined against four bacterial species including bacillus sp. n1, b. flexus n3, e. coli and v. harveyi. 289 fig. 4 ph stability of serum antibacterial activity of mud crab, scylla serrata. results represents consistent performance from three determinations. a. bacillus sp. n1; b. b. flexus n3; c. e. coli; d. v. harveyi 290 fig. 5 thermal stability of serum antibacterial activity of mud crab, scylla serrata. results represent consistent performance from three determinations. a. bacillus sp. n1; b. b. flexus n3; c. e. coli; d. v. harveyi fig. 6 effect of protein precipitating agent (75 % ammonium sulphate) on serum antibacterial activity of the mud crab, scylla serrata. each points represents mean ± sd from four determinations. ph and thermal stability serum samples (250 µl) were dialyzed against the following buffers (200 mm) at ph ranging from 3 to 12: acetate buffer (ph 3 to 6), tris-hcl buffer (ph 7 to 9) and glycine-naoh buffer (ph 10 to 12) and re-equilibrated by dialysis against tbs. the dialysates were centrifuged (400g, 5 min, 4 °c) and the resulting supernatant were tested for antibacterial activity. thermal stability of serum antibacterial activity was examined by holding 150 µl serum samples for 30 min at temperatures ranging from 10 to 100 °c. all samples were centrifuged (400g, 5 min, and 4 °c) and the clear supernatant was used to determine the antibacterial activity against bacillus sp. n1, b. flexus n3, e. coli and v. harveyi. effect of deproteinising agents and proteases the ice cold serum samples were mixed with 75 % saturated (nh4)2so4 and incubated for 45 min at 10 °c, centrifuged (400g, 5 min, 4 °c), resulting precipitates were finally dissolved in tbs, dialyzed and used for antibacterial assays. similarly, 400 µl serum samples was mixed with an equal volume of ice-cold tca solution (10 %) and incubated for 45 min at 10 °c, supernatant was collected, dialyzed and used to antibacterial activity. the serum samples (250 µl) were mixed with an equal volume of trypsin or pronase (final concentration 6 mg.ml-1), incubated for 3 h at 37 °c, centrifuged (400g, 5 min, 4 °c) and the supernatants were tested for antibacterial activity. controls consisted of serum mixed with heat inactivated (10 min, 100 °c) enzymes. statistical analysis the differences observed between control and experimental values were analysed for statistical significance using paired sample student’s t-test (bailey, 1995). results serum antibacterial activity the serum (1.224 mg protein) of healthy mud crab, s. serrata significantly inhibit the growth of the resident specific bacteria (isolated from injured cuticle of crab) viz., bacillus sp. n1, b. flexus n3 and e. coli (p < 0.05) but did not affect the growth of pseudomonas aeruginosa. similarly, the serum also inhibit the crustacean pathogenic bacteria including vibrio harveyi, v. alginolyticus and v. vulnificus (p < 0.05) but serum did not influencing the growth of v. parahemolytics and v. anguillarum (figs 1, 2). these observations reveals the presence of antibacterial molecule in the serum of this crab not only against resident specific gram-positive (bacillus sp.) and gram-negative (e. coli) bacteria but also against certain other crustacean pathogenic bacteria. based on these results obtained from the serum antibacterial activity tested, three resident specific bacteria including bacillus sp. n1, b. flexus n3, e. coli and one of the crustacean pathogenic 291 fig. 7 effect of protein denaturing agent (10 % tca) on serum antibacterial activity of the mud crab, scylla serrata. each points represents mean ± sd from four determinations. bacteria (v. harveyi) were chosen for further preliminary characterization of serum antibacterial activity (factor) of mud crab, s. serrata. physico-chemical properties the dialyzate obtained, after an extensive dialysis of serum of s. serrata, using dialysis tubing with a molecular weight cut-off (mwco) of 12 to 14 kda, showed effective antibacterial activity all the four bacterial species tested including, bacillus sp. n1, b. flexus n3, e. coli and v. harveyi (fig. 3). ph and thermal stability the serum samples dialyzed against the buffers at different (ph 3 to 12), the antibacterial activity was stable only between the ph range 6 to 8 and no activity was found on either side of this ph range against all the four bacteria tested (fig. 4). when serum samples were held at various temperatures (10 °c to 100 °c) for 30 min, the serum antibacterial activity against all the four bacterial species tested were found to be stable only between 20 to 40 ºc and the activity was completely lost at both the extremes of temperature (fig. 5). effect of deproteinising agents and proteases on serum antibacterial activity 292 the buffer re-dissolved precipitate, obtained after precipitation of serum proteins using 75 % ammonium sulphate, inhibited the growth of all the four bacterial species tested (fig. 6). on the other hand, the dialyzed supernatant, obtained after precipitating serum proteins with 10 % tca, did not inhibit the growth of any of the four bacterial species tested (fig. 7). in addition, when serum samples were treated with proteases like trypsin and pronase e (6 mg. ml-1; 37 °c; 3 h), serum incubated with pronase e completely inhibited the growth of e. coli and v. harveyi while no antibacterial activity were found against bacillus sp. n1 and b. flexus n3. while the serum samples incubated with tripsin failed to inhibit the growth of all the four bacterial species tested (fig. 8). discussion the earlier investigations reported the presence of antibacterial proteins/peptides in a variety of tissues including seminal plasma, hemolymph or serum of crustaceans (schnapp et al., 1996; destoumieux et al., 1997; majumder et al., 1997; jayasankar and subramoniam, 1999; hoq et al., 2003; amparyup et al., 2008) and they were found to be effective against both gram-positive and gram-negative bacteria. in the present study, the undiluted serum of mud crab, s. serrata appear to contain antibacterial factor(s) especially against bacillus sp. n1, b. flexus n3 and e. coli but not p. aeruginosa. similarly, when the undiluted serum was tested against other vibrio colonies also showed antibacterial activity against vibrio harveyi, v. alginolyticus and v. vulnificus, while it failed to show antibacterial activity against v. parahemolyticus and v. anguillarum. these results clearly indicate that the serum of the mud crab appeared to possess effective antibacterial activity not only against resident specific gram-positive fig. 8 effect of proteases on serum antibacterial activity of the mud crab, scylla serrata. the serum treated with proteases (6mg.ml -1 ) showed antibacterial activity against escherichia coli and vibrio harveyi. each points represents mean ± sd from four determinations. (bacillus sp.) and gram-negative (e. coli) bacteria, but also against certain specific species of vibrio isolates from other crustaceans. in order to study the physical/chemical characteristics of the molecule responsible for serum antibacterial activity, a dialysis experiment was carried out using dialysis tubing with mwco 12 to 14 kda. these observations clearly showed that the molecule responsible for antibacterial activity against these bacterial species over 14 kda, indicating the presence of larger antibacterial molecules in serum of mud crab s. serrata. while most of the antibacterial activity reported in the serum of decapod crustaceans are in the range of 3.7 to 12 kda (schnapp et al., 1996; khoo et al., 1999; relf and chisholm, 1999; herbinière et al., 2005; battison et al., 2008), there are few reports including the serum of s. serrata with larger native antibacterial molecule(s) with a size of 247 kda (makesh, 2006) or inducible antibacterial protein with a size of 36 to 64 kda (hoq et al., 2003). further analyses on the physical properties of antibacterial factor(s) present in the serum revealed that this antibacterial factor(s) against all the four bacterial species tested was stable in the ph range of 6 to 8 and between the temperatures 20 to 40 ºc. the protein present in the granular and semigranular hemocytes able to kill both gram-positive and gram-negative bacteria including some vibrio strains known to pathogenic to crustaceans with activity optimal at ph 6 9 stable to 50 ⁰c (hikima et al., 2003; tyagi et al., 2007). in contrast, relf and chisholm (1999) reported that the antibacterial protein from the granular hemocytes of the shore crab, carcinus maenas is stable after heating to 100 ⁰c. presence of antibacterial activity against all the four bacterial species in the precipitation obtained with 75 % ammonium sulphate or the supernatant obtained after precipitating the protein with 10 % tca indicated that the molecule(s) responsible for serum antibacterial activity appear to be proteinaceous in nature. the pre-treatment of serum samples with exogenous proteases namely trypsin or pronase did not affect the serum antibacterial activity against e. coli and v. harveyi. on the other hand, the proteases treated serum failed to show antibacterial activity against bacillus sp. n1 and b. flexus n3. these results clearly indicated that while the molecule(s) responsible for antibacterial activity against e. coli and v. harveyi appeared to be trypsin and pronase resistant, the antibacterial molecule(s) or domain responsible for antibacterial activity against bacillus sp. n1 and b. flexus n3 appeared to be protease sensitive, thereby implicating possible involvement of multiple antibacterial factors. further, these inferences also indicated that the serum molecule(s) responsible for antibacterial activity against e. coli and v. harveyi or bacillus sp. n1 and b. flexus n3 possibly appear to be larger sized heterogenous protein comprising non-specific protease sensitive and resistant molecule either with 293 294 different domains of native molecule(s) or subunits of a native antibacterial molecule(s). thus, this study provides the evidence that serum of mud crab, s. serrata possess multiple antibacterial protein molecules against host specific/resident specific bacteria as well as crustacean pathogenic bacteria. the isolation and characterization of these respective antibacterial molecules are required to understand the humoral immune mechanism of the crustaceans. references amparyup p, kondo h, hirono i, aoki t, tassanakajon a. molecular cloning, genomic organization and recombinant expression of a crustin-like antimicrobial peptide from black tiger shrimp penaeus monodon. mol. immunol. 45: 1085-1093, 2008. bachère e, destoumieux d, bulet p. penaeidins, antimicrobial peptides of shrimp: a comparison with other effectors of innate immunity. aquaculture 191: 71-88, 2000. bailey tjn. statistical methods in biology, 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marchalonis jj. humoral and cell membrane-associated lectins from invertebrates and lower chordates: specificity, molecular characterization and their structural relationships with putative recognition molecules from vertebrates. dev. comp. immunol. 9: 531-539, 1985. yeaton rw. invertebrate lectins: i. occurrence. dev. comp. immunol. 5: 391-402, 1981. isj 13: 102-110, 2016 isj 13: 102-110, 2016 issn 1824-307x research report are immune responses gender-related in carabus lefebvrei (coleoptera: carabidae)? a giglio1, p brandmayr1, m cammarata2, f cavaliere1, mr trapani 2, pg giulianini3 1dipartimento di biologia, ecologia e scienze della terra, università della calabria, rende (cs), italy, 2dipartimento di scienze e tecnologie biologiche, chimiche e farmaceutiche, università degli studi di palermo, palermo, italy 3dipartimento di scienze della vita, università degli studi di trieste, trieste, italy accepted march 29, 2016 abstract the “live hard, die young” theory predicts the evolution of gender differences in immunocompetence, with males having a weaker immune system than females. to test this hypothesis in carabus lefebvrei, total and basal phenoloxidase (po) activities and lysozyme-like enzyme activity were compared among males and females of different reproductive status. the sexual dimorphism occurred only in reproductively active adults and for total and basal po levels, while no significant differences were recorded between sexes in virgin adults. differences were not recorded for lytic activity between sexes. basal po and lytic activities decreased in both males and females after mating, while the total po value increased in males and decreased in females. thus, resources seem to be invested to increase the humoral response in pre-reproductive phase forming a barrier against pathogens and preserving the fecundity and longevity of both sexes. males preserve their survivorship in reproductive phase by increasing enzymatic levels in hemolymph to avoid fitness reduction due to the increased exposure to pathogen as result of mating. females shift resources from po and lytic activity to other physiological systems involved in reproduction in order to maximize their fitness. key words: ecological immunology; life history; lytic activity; phenoloxidase; sexual dimorphism   introduction studies on ecological immunology suggest that the variation and complexity in insect immune defence are closely related to different factors, intrinsic and extrinsic (schmid-hempel, 2003, 2005; schmid-hempel and ebert, 2003; sadd and schmid-hempel, 2009; schulenburg et al., 2009). intrinsic factors such as genetic and physiological trade-offs and extrinsic ecological factors affect the expression of immune effectors and result in a specie-specific immune strategy to resist or tolerate pathogens (zuk and stoehr, 2002; schmid-hempel, 2003; schmid-hempel and ebert, 2003; schmidhempel, 2005) sadd and schmid-hempel, 2009). moreover, immune function is costly in term of evolving, maintaining and utilizing an immune effector system and trade-offs exist between immune defences and life history traits as reproduction, growth and development that share common resources and contribute to an animal’s ___________________________________________________________________________ corresponding author: anita giglio dipartimento di biologia, ecologia e scienze della terra università della calabria via p. bucci, i-87036 rende (cs), italy e-mail: anita.giglio@unical.it fitness (zuk and stoehr, 2002; rolff and siva-jothy, 2003). organisms should thus optimize immune defence through the life cycle often according to their age and gender. females and males have different evolutionary challenges and they differ in a number of life-history traits. therefore, a sexual dimorphism appear in their investment in immune defence such as have been investigated in a wide number of insect species including coleoptera (córdoba-aguilar et al., 2009; shi and sun, 2010), diptera (mckean and nunney, 2001; ahmed et al 2002; schwarzenbach et al., 2005; winterhalter and fedorka, 2009; vincent and sharp, 2015), hymenoptera (cappa et al., 2015), lepidoptera (meylaers et al., 2007; stoehr, 2007; lindsey and altizer, 2009), odonata (rolff, 2001) and orthoptera (adamo et al., 2001; adamo, 2004; pinera et al., 2013; galicia et al., 2014). actually, sexual selection is predicted to be an important evolutionary force affecting the evolution of the dimorphism in immunocompetence (zuk and stoehr, 2002, schmid-hempel, 2003, 2005; mckean and nunney, 2008; nunn et al., 2009; zuk, 2009). forstad and karter (1992) outlined potential costs of parasite-resistance in terms of the evolutionary 102   103   process of sexual selection (known as the immunocompetence handicap hypothesis). the sexual dimorphic set of disease-resistance mechanisms have been demonstrated to be depended on environmental variations in terms of fitness-limiting resource availability (mckean and nunney, 2005) and related to differences between males and females in resource allocation for selfmaintenance (stoehr and kokko; 2006). immune defences may be costly for both sexes and parasites may negatively affect male mating success and female fecundity or survival for both sexes (rolff, 2002; zuk and stoehr, 2002; stoehr and kokko, 2006; zuk, 2009). based on bateman’s principle, male fitness is limited by access to females whereas female fitness is limited by offspring production and variation among male fitness is higher than among females. males are thus generally expected to have a “live hard, die young” strategy and increase their fitness by increasing successful mating events, while females invest in immune response to favour longevity (zuk and stoehr, 2002; zuk, 2009). however, males can invest more in immune defence than females in weak or absent sexual selection if the costs of infection to male mating success are high enough (stoehr and kokko, 2006). insect immunity involves the expression of a large array of cellular and humoral effectors to recognize and immobilize pathogens (gillespie et al., 1997; ottaviani, 2005; siva-jothy et al., 2005). humoral defences include the production of antimicrobial peptides (amps), reactive intermediates of oxygen or nitrogen and the prophenoloxidase enzymatic cascade (propo) regulating melanization of haemolymph (nappi and ottaviani, 2000). the propo-activating system comprises a complex cascade of serine proteases allowing the conversion of prophenoloxidase to po (marmaras et al., 1996; gillespie et al., 1997; rolff and siva-jothy, 2003; schmid-hempel, 2005; sivajothy et al. 2005). this enzymatic complex has been involved in physiological processes such as cuticular melanization and sclerotization and the defence reactions (wounding, clotting, melanotic incapsulation, production of cytotoxic molecules) (marmaras et al., 1996; moreno-garcia et al., 2012). the main role of po in melanogenesis is to convert phenols to quinones that subsequently polymerize to form melanin. in immune defence, natural activators of the propo system are pathogen cell surface molecules such as β-1,3 glucans from fungal cell walls and lipopolysaccharides (lps) and peptidoglycans from microbial cells. the melanin deposited onto the foreign target prevents the pathogen growth and reproduction and thus melanization is an important cell-mediated immune response in tissue repair and in pathogen sequestration (söderhäll et al., 1994; nappi and vass, 2001; cerenius and söderhäll, 2004; nappi and christensen, 2005; gonzález-santoyo and córdoba-aguilar, 2012). amps such as lysozymes are present constitutively at a very low level in the hemolymph and their level increase upon challenge. the lysozymes belong to the c-type enzymes (callewaert and michiels, 2010) and perform a hydrolytic action against the peptidoglycan of gram positive cell walls (ratcliffe et al., 1985; gillespie et al., 1997; nappi and ottaviani, 2000). they have also a low activity against gram-negative bacteria (yu et al., 2002) and a fungistatic property (fiolka et al. 2005). in the present study, the gender-specific and trade-off between immune defence and reproductive effort were tested using carabus lefebvrei, as the model system. this is an italian endemic carabid beetle that lives in beech, oak, chestnut and pine forests of the central and southern apennines, from lower altitudes to about 1500 m a.s.l. it reproduces in spring, is active from april until september and hibernates as adults (thiele, 1977; turin et al., 2003; giglio et al., 2009). the habit of adults and larvae is typically that of a snail-eating predator and males display smaller than females (turin et al., 2003; giglio et al., 2012). previous studies have shown different strategies in terms of cellular and humoral immune response to enhance the fitness of each life stage. the analysis demonstrated that the variation in speed and specificity of immune function across the developmental stages is correlated with differences in infection risk, life expectancy and biological function of the life cycle (giglio et al., 2008; giglio and giulianini, 2013). in this study, we measured basal and total phenoloxidase (po) enzyme activities and lysozyme-like enzyme activity as immunity markers involved in the pathogen resistance to evaluate the sexual dimorphism in c. lefebvrei immunocompetence. laboratory tests were designed to compare virgin adults in their prereproductive phase with reproductive females and males after mating. material and methods insect rearing and hemolymph collection carabus lefebvrei females and males were hand-collected in the catena costiera mountains (39°19’ n, 16°7’ e, 900 1000 m a.s.l.; southern italy, calabria) in early spring 2014. these adults are emerged in the early summer of previous year and hibernating under rotten pine barks in winder. in the laboratory, collected beetles were sexed and kept in groups (males and females) in 10 l plastic boxes, filled to a depth of 6.0 cm with moistened humus. the specimens were reared with a light regime of l/d = 15/9 h, 70 % relative humidity and a day/night room temperature of 23/18 °c. they were fed with snailsand daily observed until specimens show reproductive behaviour (mating events). after copulation, males were removed from the boxes to reduce the disturbance to females, which readily laid eggs. the eggs were transferred singly into 150 ml glass jars filled to a depth of 4.0 cm with moistened humus. egg production and larval developmental time were recorded every two days until pupal instar and imago appearance. the haemolymph was collected from newly emerged adults 15-days-old (virgin females, n = 17 and males, n = 16) in their prereproductive phase and from reproductive females and males two days after mating events (see above) attesting their 104   reproductive status (n = 15 reproductive adults for both groups). the animals were co2 anesthetized before hemolymph collection. the hemolymph was collected by puncturing adults at the ventral level of the pro-mesothorax articulation with a 29-gauge needle. the first droplet of 10 µl of hemolymph was collected. each hemolymph sample was immediately transferred into 190 μl ice-cold pbs (10mm sterile phosphate-buffered saline, sigmaaldrich) in a 1.5 ml eppendorf tube and centrifuged at 1700 g for 5 min at 4 °c. the cell-free hemolymph obtained as supernatant was collected and stored at -20 °c until enzymatic assays. phenoloxidase specificity, zymogen activation and specificity of reaction for determining enzyme specificity and zymogen activation, the po activity was measured spectrophotometrically according to winder and harris (1991), using l-dopa as substrate and 3methyl-2-benzothiazolinone hydrazone hydrochloride (mbth, 6 mm) as a specific reagent. briefly, 40 µl of hemolymph-buffer solution were incubated for 30 min at 20 °c with 40 µl of pbs (na2hpo4 1m, kh2po4 0,01 m; nacl 1.5 m, ph 7.4) and 40 µl of dopa-mbth reaction mixture (20 mm l-dopa and mbth in distilled water). after the reaction, dopaquinone was detected within 60 min at 5 min intervals by spectrophotometric measurement at 505 nm. the po activity was expressed as units (us) for min, where one u was defined as the amount of enzyme required to produce an increase in the absorbance at 505 nm of 0.001 units min-1. to check for specificity of the enzyme reaction, before l-dopa and mbth were added, the homogenate was incubated (20 min at 20 °c) with trypsin or with 1-phenyl-2-thiourea (ptu) in pbs at 1 mm final concentration. this inhibitor acts by chelating the copper at the active site, and it is known to be one of the most effective po inhibitors (kahn, 1985; aspàn et al., 1995; klabunde et al., 1998). basal and total phenoloxidase enzyme activity phenoloxidase (po) activity was monitored spectrophotometrically as the formation of dopachrome from 3, 4-dihydroxy-l-phenylalanine (l-dopa, sigma-aldrich). for determination of basal po, 20 μl of haemolymph-buffer solution were taken and mixed with 180 μl of l-dopa (3 mg/ml in pbs) in a microtiter plate. for determination of total po enzyme activity, 30 μl of hemolymph -buffer solution were added to 30 μl of methanol that chemically activates po from its inactive zymogen, prophenoloxidase (propo). the hemolymph-methanol mixture was incubated for 5 min at room temperature and 20 μl were mixed with 180 μl of l-dopa (3 mg/ml in pbs) in a microtiter plate. the basal and total phenoloxidase enzyme activity at 20 °c was recorded at 492 nm for 30 min in 5 min intervals using a plate reader (sirio s, seac). all samples were assayed in duplicate. the enzyme activity was measured as the slope (absorbance vs time) of the reaction curve during the linear phase of the reaction (vmax value; between 15 and 20 min for reproductive females and 0 and 5 min for all the others after the reaction began). the slope of the reaction curve at vmax was plotted as absorbance per μl of hemolymph per min for female and male of different mating status. lysozyme-like enzyme activity a turbidometric assay was used to measure lysozyme-like activity in the hemocyte-free plasma. the assay is based upon the lyses of the lysozymesensitive gram-positive bacterium micrococcus lysodeikticus. 10 μl of hemolymph-pbs mixture (described above) was loaded into the well of a 96well microplate followed by 190 μl of a micrococcus lysodeikticus (strain atcc 7468, dsmz) cell wall suspension (1.6x108 cell/ml of cold pbs). the reductions in turbidity in the wells were read on a plate reader (sirio s, seac) at 25 °c for 45 min in 5 min intervals at 450 nm. all samples were assayed in duplicate. the enzyme activity was reported as the change in absorbance (absorbance vs time) of the reaction curve during the linear phase of the reaction (vmax value; between 5 and 15 min after the reaction began). the slope of the reaction curve at vmax was plotted as absorbance per μl of hemolymph per min, for female and male samples at different mating status. standards of enzyme activity were made using lysozyme from chicken egg whites (sigma) and a suspension of m. lysodeikticus as substrate. the standards were run simultaneous with the hemolymph samples to confirm that assay was progressing as expected (i.e., absorbance values decreasing). hemolymph protein content protein content was estimated by the method of bradford (bradford, 1976) with bovine serum albumin (bsa) as a standard using 100 µl of hemolymph-buffer solution for each sample and 900 µl of bradford reagent (sigma-aldrich). statistical analyses statistical analyses were performed using r version 3.0.1 software (r development core team 2013). total and basal po enzyme activities, lysozyme-like enzyme activity and hemolymph protein content were measured and compared among reproductive female and male samples as well as virgin ones. the differences among vmax were assessed by nonparametric statistics, i.e., kruskal-wallis rank sum test (kwt) followed by post-hoc wilcoxon rank sum test pairwise comparisons with bonferroni correction (pwt), since the null hypothesis of the bartlett test could not be rejected. the box and whisker plots were drawn with the boxplot command. all values are reported as mean ± se in the text. results phenoloxidase specificity, zymogen activation and specificity of reaction a lower po activity (males 8.98 ± 0.90 u, females 7.46 ± 0.65; n = 5) was found when the samples were assayed in the absence of activating enzyme treatment, whereas it was raised by trypsin (males 11.41 ± 0.94 u, females 9.03 ± 0.91 u; n = 5). the specificity of the po reaction was demonstrated by the specific inhibitor phenylthiourea added to homogenates before the activation with trypsin. po activity of samples, compared to untreated or activated controls, was lowered (males 3.54 ± 0.66 u; females 1.25 ± 0.34 u). basal and total phenoloxidase enzyme activity highly significant differences were detected in total (kwt, p < 0.0001) (fig. 1a) and basal (kwt, p < 0.0001) (fig. 1b) po activity of c. lefebvrei among females and males of different reproductive status. in females, there were no significant differences in the total po activity (pwt, p = 0.062), while the basal po activity decreased significantly in reproductive specimens (pwt, p = 0.002). in males, the total po activity was significantly lower in virgin than reproductive ones (pwt, p < 0.0001), whilst a contrary trend was recorded in basal po activity (pwt, p = 0.014). in reproductive adults, the total po activity was significantly higher in males than females (pwt, p < 0.0001) as well as the basal po activity (pwt, p < 0.0001). a) b) fig. 1 total a) and basal b) phenoloxidase activity in c. lefebvrei measured as the slope of the reaction curve at vmax. the enzymatic activity of reproductive and virgin females and males was recorded as absorbance units for µl of hemolymph per min (for statistics see the text). the boxplot represents the interquartile range (iqr = q3q1) and bars represent first (q1, top) and third quartiles (q3, bottom) of phenoloxidase activity values from reproductive females and males and in virgin ones. the central horizontal black line indicates the median. the ends of dashed lines (ends of the whiskers) represent the lowest datum and the highest datum. 105   fig. 2 lysozyme-like enzyme activity in c. lefebvrei measured as the slope of the reaction curve at vmax. the lytic activity in reproductive and virgin females and males was recorded as units for µl of hemolymph per min (for statistics see the text). the boxplot represents the interquartile range (iqr = q3-q1) and bars represent first (q1, top) and third quartiles (q3, bottom) of phenoloxidase activity values from reproductive females and males and in virgin ones. the central horizontal black line indicates the median. the ends of dashed lines (ends of the whiskers) represent the lowest datum and the highest datum. in virgin adults, there were no significant differences in the basal (pwt, p = 0.164) and total (pwt, p = 1.0) po activities between males and females. lysozyme-like enzyme activity in the hemolymph plasma the results showed a significant difference in baseline lytic activity (kwt, p < 0.0001) (fig. 2) among females and males at different reproductive status. the enzymatic activity decreases significantly in both females (pwt, p < 0.0001) and males (pwt, p < 0.0001) at reproductive status. there were no significant differences between sexes in both reproductive (pwt, p = 0.370) and virgin adults (pwt, p = 0.349). hemolymph protein content the total protein content was recorded in hemocyte-free plasma of both males and female at different reproductive status and significant differences were detected (kwt, p < 0.0001) (fig. 3). there was no significant differences in plasmatic protein content between reproductive males and females (pwt, p = 0.163) and virgin males and females (pwt, p = 1.0). however, there was a highly significant increase in protein content in reproductive adults for both females (pwt, p < 0.0001) and males (pwt, p = < 0.0001) compared to virgins. discussion previous studies on dimorphism of immune response have investigated optimal allocation of resources between immunity, survival and reproduction in males and females (stoehr and kokko, 2006). the outline of defence strategy may vary generally under environmental selective pressure as a result of a wide range of factors (ecological, genetic and evolutionary) closely related to different life histories for males and females (viney et al. 2005; stoehr and kokko, 2006; restif and amos, 2010). in our study, reproductive males of c. lefebvrei showed high values of basal and total po enzyme activities compared with females after mating, while enzymatic activities did not differ between sexes in virgin adults. in spite of that, there was no sexual dimorphism in baseline lysozyme-like enzyme activity among females and males at different mating status. some studies have formerly found that the direction and magnitude of sex differences in immune-competence could be different for each component of immune defence (zuk and stoehr, 2002; stoehr and kokko, 2006; stoehr, 2007) and limited mainly by resource availability (mckean and nunney, 2005; galicia et al., 2014). in some species there is strong evidence that males and females may emphasize different immune components in relation to age, mating, sexual antagonism and attractiveness or food 106   fig. 3 protein content in c. lefebvrei cell-free hemolymph for reproductive and virgin females and males (for statistics see the text). the boxplot represents the interquartile range (iqr = q3-q1) and bars represent first (q1, top) and third quartiles (q3, bottom) of phenoloxidase activity values from reproductive females and males and in virgin ones. the central horizontal black line indicates the median. the ends of dashed lines (ends of the whiskers) represent the lowest datum and the highest) datum. availability (adamo, 2001; lawniczak et al., 2006; stoehr, 2007; córdoba-aguilar et al., 2009; winterhalter and fedorka, 2009; kivleniece et al., 2010; galicia et al., 2014; vincent and gwynne, 2014; vincent and sharp, 2014). our results show that the investment strategy in immunocompetence varies with reproductive status in c. lefebvrei adults, but further investigation is needed to clarify if change in immune responses may be often age-dependent for both sexes and if male may have a limited amount of resources to invest optimally in both the immune system and sexual attractiveness. we assume that the sexual dimorphism of po enzymatic activity in c. lefebvrei adults are due to a different strategy to balance for resource allocation between physiological and ecological costs in both sexes. moreover, the significant increase of hemolymph protein content was positively correlated with the reproductive status of adults and confirmed the specificity of enzymatic activities recorded in absence of infection. to exclude that enzymes with similar activity such as peroxidases, laccases and catalases are involved in recorded enzymatic activity, the propo activating system were characterized and displayed the calciumindependent po activity enhanced by trypsin (cerenius and söderhall, 2004). phenylthiourea, a specific po inhibitor, supported the reaction specificity with an inhibition of 86 % of activity in females and 68 % in males. some studies have provided evidence for a reduction of immune function as a result of increased reproductive activity in absence of infection (rolff and siva-jothy, 2002; fedorka et al., 2004; otti, 2015). however, natural selection may favour immunity if the infection pressure is high (schwenke et al., 2016). c. lefebvrei males gain fitness by investing heavily in immunity to protect themselves from an exposure to parasites due to their larger number of mating events. females mating one time have a reduced exposure risks compared to males. as a result, further investigation will be addressed to verify that reproductive females decrease the immune response because they shift resources from propo system and lytic hemolymphatic activity to other physiological systems involved in reproduction including production of eggs, reception and storage of sperm, fertilization and oviposition. high levels of both basal po and lysozyme-like enzyme activities recorded in virgin adults are likely to preserve the adult survivorship against infections until the reproductive phase. actually, the available data strengthen the leading role phenoloxidase and lysozyme-like enzyme activities play in disease resistance (adamo, 2004). studies on cricket that have estimated disease resistance based on sex differences have shown that lysozyme-like activity is maintained at a low level and increases in response to bacterial challenge (adamo, 2004; piñera et al., 2013; galicia et al., 2014). in many species, the degree of cuticular melanization and wound repair is a strong indicator of resistance to pathogens and it is correlated with po activity in the cuticle, hemolymph and midgut (barnes and siva-jothy, 2000; wilson et al., 2001). in c. lefebvrei, as the cuticle does not offer full protection before its complete melanization, basal po expression may be up-regulated to melanize the cuticle mainly in 107   108   newly emerging adults which are more exposed to wounds and thus more susceptible to infections. at the same time, this species shows an increase in predation activity before mating to accumulate metabolic resources so, as a result, it is exposed to potential infectious microorganisms via oral consumption of prey (snails). thus, the high level of lytic hemolymphatic activity in pre-reproductive phase of adulthood might increase humoral defence to maintain an effective protection increasing the organism’s fitness. in conclusion, here we found that two different immune effector systems, po activity and lytic activity, show variation over the lifetime of c. lefebvrei males and females. the sexual dimorphism was recorded for po activity, but not for lytic activity. this shows that measuring a single component of the immune system in not going to provide an overall indicator of immunocompetence. furthermore, c. lefebvrei reproductive males have higher values of both po and lytic activity than reproductive females. this result confirms that immune function is not a simple, static process, but rather a dynamic system of interrelated mechanisms that are differentially effective and may not be generalizable among species. immunity and the ability to reproduce are closely related in order to maximize the fitness of each species (schmidhempel, 2003; schulenburg et al., 2009) and sexual differences in immune investment are difficult to predict. at the last, lower enzymatic activity in reproductive females demonstrates that the investment of high energies 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celomocytes; phagocytosis; antimicrobial peptides; arenicins; polychetes; lugworm; arenicola marina   introduction polychetes are segmentary celomate invertebrate animals inhabiting marine or estuarine habitats being either benthic or pelagial. polychaeta is an ancestral group within the phylum annelida (struck et al., 2011). the molecular data unambiguously place it within lophotrochozoa animals (together with lophophorates and molluscs (halanych et al., 1995). the long held morphological view closely relates annelids with arthropods within articulata as both being segmented (dohle and scholtz, 1995). in accordance to any of these views polychetes are considered among taxa closest to a putative common ancestor of other protostome bilaterians (balavoine and adoutte, 2003), and so, are of particular evolutionary importance. immunity is among crucial evolutionary factors for speciation, surviving or extinction (loker, 2012). identification of the immunological features typical for the high-rank taxa (e.g., phylum or class) is an essential problem for comparative immunology. this can help in the understanding of the immune system history in macroevolution. elucidation of immune mechanisms in a particular species is also very important it allows to estimate diversity of immune ___________________________________________________________________________ corresponding author: arina l maltseva department of invertebrate zoology st petersburg state university universitetskaya 7/9, st petersburg 199034, russia e-mail: arina.maltseva@spbu.ru patterns within a phylum. this also may clarify some aspects of the species history. several comprehensive reviews of immunity of annelids are available (stein and cooper, 1983; salzet et al., 2006; tasiemski, 2008; vetvicka and sima, 2009), but there are still few immunological syntheses, done on particular polychete species. here we discuss the available data concerning aspects of immune responses of polychete arenicola marina. a. marina is a common inhabitant of sand flats in northern europe. it lives within the burrow, in permanent contact with surrounding sediment. a. marina is sensitive to quality of sediment in term of biochemical properties and toxicity (moralescaselles et al., 2008, 2009) and has been suggested as a test-species for ecotoxicological studies (ramos-gómez et al., 2011). living in habitats rich with microbes and spending their lives engulfing and disgorging the sediments, these animals obviously need an efficient immunity. like other invertebrates it relies on innate immune system. traditionally immunity is considered as a concord of cellular and humoral branches, so we will discuss known facts about cells and molecules involved in the lugworm’s defensive reactions. receptors several classes of pathogen recognizing receptors (prr) were identified in annelids: tlr, nlr, and lectins (ozeki et al., 1997; hirabayashi et al., 1998; molchanova et al., 2007; davidson et al., 2008; cuvillier-hot et al., 2011; škanta et al., 2013). 247   among them, only one lectin was recognized in a. marina as a putative prr aml-1 (vitashenkova et al., 2012). aml-1 showed no homology with any known lectin or other protein families, and is a representative of a new family of lectins with specificity directed towards n-acetylated carbohydrates (e.g., chitin). it was able to agglutinate different mammalian erythrocytes in vitro with clear preference to rabbit cells. this data are in line with hemagglutinating activity in a. marina body fluid, most strong toward rabbit red blood cells, demonstrated in early studies (dales, 1982). purified originally from cell-free celomic fluid, aml-1 was immunohystochemically identified in different types of celomocytes (both floating in celomic fluid and migrating through the tissues), nephridia cells and eggs within cytosolic granules in all cases (vitashenkova et al., 2012). this indicates that the role of aml-1 in lugworm immunity could be more complex than just agglutination in celomic cavity, and remains to be elucidated. celomocytes body wall of polychetes consists of 4 elements: thin cuticle produced by one-layered epithelium, and layers of transversal musculature underlined by ribbons of longitudinal musculature. the later are separated from the celomic cavity by celothelium (peritoneum). in a. marina, celomic cavity is divided by segmentary dissepiments only in the most anterior and posterior parts of body, while within main body it represents continuous volume. celomic cavity is filled by celomic fluid, where floating cells (the celomocytes) are present. those cells might also settle onto peritoneum and migrate through tissues. there are several types of celomocytes in polychetes, such as granulocytes (syn. amebocytes), eleocytes, and hemocytes. however, the latter two are absent in arenicolidae (rev. in vetvicka and sima, 2009), as well as in some other polychetes [e.g., hermodice carunculata (franchini et al., 2016)]. granulocytes (amebocytes) can be further subdivided into several subtypes, although relationships between them are not well understood. in arenicola these subtypes vary in their extent of development of granular apparatus and actin fiber networks (persinina and chaga, 1994a, 1998; chaga et al., 1998). chaga et al. separated the terms amebocytes (few granules, well developed actin fibers) and granulocytes (numerous granules, thin membrane associated actin network), which corresponded to granulocytes of types i and ii respectively in dhainaut and porchet-henneré nomenclature (dhainaut and porchet-henneré, 1988). both cell types originate from the same cell-source “juvenile cells” (persinina and chaga, 1994b). in a. marina, bacteria inoculated into body cavity are quickly cleared by phagocytic cells in the celom and tissues (fitzgerald and ratcliffe, 1983). granulocytes of both types actively phagocytize, individually or within aggregates. individual cells loaded by engulfed bacteria migrate into the tissues; cell aggregates finally mature into “brown bodies” (fitzgerald and ratcliffe, 1983), which can be discarded by nephridia (kermack, 1955). the same authors showed that there is no opsonic activity toward different bacteria in celomic fluid plasma, and that phagocytic celomocytes are able to discriminate between gram-positive and gramnegative bacteria (fitzgerald and ratcliffe, 1982). besides the absence of unambiguous interpretation of an interrelation among celomocytes subpopulations, an evident demonstration of the common hematopoietic area is also lacking. different derivatives of celomic peritoneum, including extravasal tissue, are most often proposed as candidates (rev. in gardiner, 1992; vetvicka and sima, 2009). cells of extravasal tissue (specialized part of ventral blood vessel) possess granular cytoplasm. along with celomocytes these cells participate in clearance of celomic cavity via phagocytosis (braunbeck and dales, 1984, 1985). melanin and ros production “brown bodies” contain melanin in polychete nereis diversicolor (porchet-henneré and vernet, 1992) and olygochete eisenia fetida (valembois et al., 1994). melanogenesis in invertebrates is related to the activity of phenoloxidase (po), and is accompanied by generation of a spectrum of highly toxic free radicals, including reactive oxygen species (ros) (e.g., rev. in nappi and ottaviani, 2000; cerenius and söderhäll, 2004; nappi and christensen, 2005) and formation of amyloid fibrils (falabella et al., 2012; grimaldi et al., 2012, 2014). appearance of “brown bodies” during clearance reaction in a. marina allows to suspect production of ros and activation of po during this process. coincidence of encapsulation and po-activation or both melanin and ros production was described in n. diversicolor (porchet-henneré et al., 1987; porchet-henneré and vernet, 1992) and in e. fetida (valembois et al., 1994), respectively. unexpectedly, the once carried testing for po-activity in whole celomic fluid failed to reveal any in a. marina (smith and söderhäll, 1991). possibly, po and related enzymes are activated in this species only after induction. in accordance to such suggestion, both production of melanin and activation of po in granulocytes ii was demonstrated in n. diversicolor after infection (porchet-henneré and vernet, 1992). ros production in invertebrates accompanies different immunological phenomena phagocytosis, encapsulation, and epithelial defense (e.g., rev. in nappi and ottaviani, 2000). defense-related ros production by celomocytes both in vivo and in vitro was demonstrated in the olygochete e. fetida (valembois et al., 1994; valembois and lassègues, 1995). in a. marina, diverse antioxidant enzymes (superoxide dismutase, catalase, glutathione reductase, peroxiredoxin, etc.) were identified, and their activity was demonstrated in different compartments (e.g., body wall, chloragog tissue, gills) under various stress conditions (seasonal temperature variation, pollution, etc.) (buchner et al., 1996; storch et al., 2001; loumaye et al., 2008; ramos-gómez et al., 2011). nevertheless, neither their involvement into immunity, nor the defensive ros production (or ros producing enzymes) were ever characterized in this species. so, both mechanisms of melanin production and defensive ros functioning in a. marina remain open questions. 248   antimicrobial activity early studies searched for antimicrobial activity in a. marina against gram-positive and gramnegative bacteria. no bacterial growth inhibitory activity was detected in celomic fluid plasma of neither intact animals nor ones induced by injection of killed bacteria into body cavity (dales and dixon, 1980). the same was shown 35 years later again there was no clear antibacterial activity in plasma of a. marina celomic fluid, and injection of microbes into body cavity did not stimulate its appearance (maltseva et al., 2014). instead, strong antimicrobial activity was detected in acidic extracts from celomocytes, and the peptides responsible for this activity arenicins were identified (ovchinnikova et al., 2004). the expression of arenicin-1 was later revealed in different compartments, though the strongest signal was found in celomocytes. this was established by different method, including immunohistochemisry, pcr (maltseva et al., 2014) and maldi-imaging mass-spectrometry (maltseva et al., 2016). expression of arenicins was constitutive in all the tissues, where it was detected (maltseva et al., 2014). within celomocytes (both granulocytes i and ii), arenicins are stored within cytosolic granules, which undergo fusion with phagosome. arenicins functioning this way participate in inactivation of the phagocytized pathogens, instead of being secreted into celomic cavity. importantly, presence of arenicins was detected in granules of cells of extravasal tissue, also possessing phagocytic activity (maltseva et al., 2014). apart from celomocytes, expression of arenicins was detected in epithelia of the body wall and gut (maltseva et al., 2014). similarly, amps of another annelid worm theromicin and theromyzin from a leech theromyzon tessulatum are expressed both in intestinal and epidermal cells (tasiemski et al., 2004). this supports the importance of arenicins as key components of both epithelial and systemic branches of host defense. arenicins are also present in a major part of nervous system of a. marina, midventral neuronal cord (maltseva et al., 2014). the expression of two amps by glia and neurons in response to injury and microbial challenge was also demonstrated in the medicinal leech hirudo medicinalis. this indicates the involvement of amps in both defense and regeneration (schikorski et al., 2008). the functioning of arenicins in the nervous system still remains to be elucidated although their presence in this compartment indirectly supports the multifunctional nature of amps in vivo. therapeutically perspective components several compounds with some potential for usage in medicine were characterized in worms of the genus arenicola (a. marina or a. cristata): hemoglobin (rousselot et al., 2006), fibrinolytic serine protease (zhao and ju, 2015), cell growth inhibiting and apoptosis inducing serine protease (zhao and ju, 2014), and arenicolsterol a (bin et al., 2005; wang et al., 2007). except for evident oxygen-transporting function of hemoglobin, natural function of those compounds in lugworms remains to be elucidated. nevertheless, up to date, arenicins represent most intensively studied a. marina compounds with therapeutic potential, so we will discuss their structure and activities in more details. primary structure of arenicins there are three isoforms of arenicins. they all are 21 amino acid peptides with 6 positively charged arginine residues for arenicins-1 and -2 (ovchinnikova et al., 2004) or 4 for arenicin-3 (sandvang et al., 2008). arenicins-1 and -2 differ by a single amino acid residue (val or ile in 10th position), and contain two cysteine residues linking the only disulfide bond. arenicin-3 is a bit more different and contains four cysteines forming two disulfide bonds. primary structure of arenicins displays considerable homology with previously described horseshoe crab amps, tachyplesins from tachypleus tridentatus (nakamura et al., 1988) and polyphemusins from limulus polyphemus (miyata et al., 1989). this is why we propose to consider all these amps within ‘tachyplesin/arenicin family’ (taf). other taf members are gomesin from tarantula spider acanthoscurria gomesiana (silva et al., 2000), androctonin from sahara scorpion androctonus australis (mandard et al., 1999), alvinellacin from pompeii worm alvinella pompejana (tasiemski et al., 2014) and capitellacin from capitella teleta (the later one was predicted by analysis of genome database (tasiemski et al., 2014). thus, identified up to date taf-related amps belong to two animal taxa polychetes and chelicerates. structures of arenicins and some other selected members of taf are represented in figure 1. noteworthy, among taf members only arenicins1 and -2 are characterized by a single disulfide bond while all other peptides contain two disulfide bonds. hence, the latter variant could be accepted as putative ancestral pattern. several amps unrelated to taf were revealed in other annelids species: perinerin from asian clamworm perinereis aibuhitensis (pan et al., 2004), hedistin from sandworm nereis diversicolor (tasiemski et al., 2007), lumbricins from different olygochete and leech species, theromacin and neuromacin from leaches theromyzon tessulatum and hirudo medicinalis, and others (rev. in tasiemski, 2008). there is still no evidence of presence of similar peptides in a. marina. spatial structure of arenicins as it was established by nmr spectroscopy (ovchinnikova et al., 2007; andrä et al., 2008), arenicins-1 and -2 in aqueous solutions adopt a conformation of β-hairpin formed by two antiparallel β-strands (cys3-val/ile10 and val13-cys20), and stabilized by one disulfide and nine hydrogen bonds. β-strands are connected by a type i’ β-turn. reduction of disulfide bond disrupts the β-hairpin structure, at least in a case of arenicin-2. the twostranded β-sheet in structure of both peptides has significant right-handed twist and in the case of arenicin-1 the β-sheet is bent. right-handed twist in the β-sheet deprives the arenicin-2 molecule surface of amphipathicity (ovchinnikova et al., 2007), while alternative study reported that arenicin1 retains amphipathicity in aqueous solution (andrä 249   fig. 1 primary structures of arenicins and some other taf-related peptides. the most conserved regions are highlighted with colour. two disulfide bonds are shown in the top part of the image; the bond absent in arenicins-1 and -2 shown as a dashed line. ‘z’ indicates pyroglutamic acid residue derived from glutamine in the gomesin structure; ‘r*’ indicates amidated c-terminal arginine residues in the tachyplesin-1, polyphemusin-1 and gomesin molecules. sequences of amps are taken from uniprot database with following id: arenicin-1: q5sc60, arenicin-2: q5sc59, tachyplesin-1: p14213, polyphemusin-1: p14215, gomesin: p82358. arenicin-3 and capitellacin are annotated up to date. et al., 2008). other taf-related amps with known spatial structure are characterized by similar βhairpin conformation (fig. 2). in more details amps adopting β-hairpin are reviewed by panteleev et al. (2015a). antimicrobial activity of arenicins arenicins at micromolar or even lower concentrations display significant antimicrobial activity towards the wide range of gram-positive and gram-negative bacteria, and fungi (ovchinnikova et al., 2004, 2007; lee et al., 2007; andrä et al., 2008; sandvang et al., 2008). several marine bacteria (vibrio alginolyticus, listonella anguillarum, and planococcus citreus) were also susceptible to arenicins (andrä et al., 2009). among the sensitive microbes there are some antibioticresistant strains including clinical isolates (sandvang et al., 2008; cho and lee, 2011b). antimicrobial activity of many cationic amps significantly decreases with increasing ionic strength (rev. in bowdish et al., 2005). however, arenicins-1 and -2 display comparable antibacterial activity in both low salt and high salt conditions (ovchinnikova et al., 2004) or retain decreased but significant activity even at high nacl level up to 500 mm (andrä et al., 2008). the data about fungicidal activity to candida albicans are contradictory. either it was retained at 150 mm nacl for arenicin-1 (park and lee, 2009) or decreased 4 5 fold at 100 mm nacl for arenicins-1 and -2 (ovchinnikova et al., 2004). the peptide nz17074, which is an artificial derivative of arenicin-3, demonstrated fungicidal activity moderately impaired at high salt conditions (wang et al., 2014). resistance to enhanced salt concentrations is considered to be evolutionary selected feature of arenicins essential for amps of marine animals (andrä et al., 2008, 2009). mechanism of antimicrobial action of arenicins it is accepted that the most common mechanism of toxic action of cationic amps is performed via permeabilization of the cytoplasmic membrane of the target cell (yeaman and yount, 2003). accordingly, many studies aimed to describe the “behavior” of arenicins in lipophilic media such as natural membranes, liposomes, micelles, lipid monoand bilayers. detailed data, obtained for arenicin-2, allowed to outline a model of its action on bacterial membrane. binding of the peptide to membrane due to electrostatic interactions with anionic phospholipid heads leads to conformational changes of the peptide molecule. partial unwinding of twisted β-hairpin occurs making it more planar and resulting in disclosure of amphipathicity of the arenicin-2 molecule (ovchinnikova, et al., 2007; salnikov et al., 2011). the next step is the formation of peptide dimers via parallel association of nterminal β-strands (cn↑↑nc type of association) (ovchinnikova et al., 2008; shenkarev et al., 2011). finally, the peptide dimers turn into the transmembrane orientation establishing ionconducting pores within the membrane constituted of 2-4 arenicins’ dimers in a complex with anionic lipids head groups (ovchinnikova et al., 2008; shenkarev et al., 2011, 2014; sychev, 2015). this mechanism is consistent with the toroidal pore model implying the formation of pores by both peptides and lipids with overall torus-like geometry (matsuzaki, 1999). experimental data concerning arenicin-1 imply a similar mechanism, though it may differ in some details (andrä et al., 2008, 2009; shenkarev et al., 2011). сellular membrane permeabilization also occurs during fungicidal action of arenicin-1 (park and lee, 2009; cho and lee, 2011a) and of arenicin-3-related peptide nz17074 (wang et al., 2014). however, the downstream events are important for killing of fungal cell. the action of arenicins on fungi is energy-dependent, since it is blocked by metabolic inhibitor (sodium azide) as well as by low-temperature conditions (+4 °c) (park and lee, 2009; wang et al., 2014). the latter is true only for fungi, but not for bacteria, which are equally 250   fig. 2 spatial structures of arenicins-1 and -2, tachyplesin-1 and gomesin. images were generated with chimera 1.11 software https://www.cgl.ucsf.edu/chimera/. susceptible to arenicin-1 action at both +37 °c and +4 °c (andrä et al., 2008). within a fungal cell, arenicins attack mitochondria leading to increased generation of hydroxyl radicals and other ros and activation of apoptosis, presumably via rossensitive metacaspases (cho and lee, 2011a; wang et al., 2014). according to choi and lee, increased intracellular ros production accompanies killing of bacterial cells by arenicin-1 as well (choi and lee, 2012). structure-function interrelation significance of the particular amino acid residues in the structure of arenicins for their antimicrobial activity was intensively investigated. arenicin-1, devoid of disulfide bond due to substitution of both cysteines by serines, was 2-4fold less active than the natural peptide (lee at al., 2007; andrä et al., 2009, 2011). substitution of cationic arg11 by neutral ala11 or deletion of nterminal dipeptide arg-trp decreased the activity of arenicin-1 towards gram-positive and gramnegative bacteria, and fungi by 2 3 times (cho and lee, 2011b; park et al., 2011). similarly, arenicin-1 analog with trp2 replacement retained only a quarter of antimicrobial activity compared with prototype peptide (panteleev et al., 2015b). another tryptophan residue (trp21) is also critical for antimicrobial activity of arenicin-1, as its replacement causes approximately 2-fold increase of minimal inhibitory concentration (panteleev et al., 2015b). the same is observed after replacement of all arginine residues by lysines (andrä et al., 2009, 2011). among other residues, ala6, val4 and val10 are also essential for antimicrobial activity of arenicin-1 (panteleev et al., 2015b). interaction with mammalian cells and molecules the degree of toxicity towards mammalian cells is critical for potential therapeutic agents. arenicins1 and -2 poorly discriminate between microbial and animal cells. these peptides exhibit high hemolytic activity against human erythrocytes (lee at al., 2007); and nucleus-possessing mammalian cells can also be damaged by arenicins. arenicin-1 was toxic to both cancer (jurkat) and normal (embryonic fibroblasts, astrocytes) human cells in vitro (andrä et al., 2008; panteleev et al., 2016). in vivo experiments revealed that arenicin-2 should be referred to as a class iii toxicity (20 > ld50 > 700 mg/kg) for cd-1 mice (dyachenko et al., 2012). at the same time, arenicin-3 seems to be non-toxic for mammalian cells, and displays very low hemolytic activity (hoegenhaug et al., 2011). deletion of n-terminal dipeptide arg-trp from the arenicin-1 sequence resulted in dramatic decay in hemolytic activity (cho and lee, 2011b). in nonhemolytic arenicin-3 there is no arg-trp dipeptide. thus, hydrophobic trp2 residue is essential for the interaction with erythrocyte membrane, however, its replacement with any of three different residues only moderately affects hemolytic activity of arenicin-1 251   https://www.cgl.ucsf.edu/chimera/ (panteleev et al., 2015b). other hydrophobic residues are also important for toxic activity of arenicin-1 towards erythrocytes. one of five valine residues at positions 4, 8, 10, 13, and 15 or alanine at 6th position can be replaced with more hydrophilic amino acid with significant decrease in hemolytic activity of arenicin-1. an analog of arenicin-1 with arg substituted for val8 retains the high antimicrobial activity with dramatically decreased hemolytic properties (panteleev et al., 2015b). similar activity shift was observed for shortened analog of arenicin-1 with deletion of two dipeptide fragments: tyr7-val8 and val13-leu14 (panteleev et al., 2016). the toxicity of those analogs towards nuclear human cells was significantly lower compared to the natural peptide. these analogs can be considered as a perspective base for the development of therapeutic variants of arenicin. another possibility to produce a novel therapeutics is related with non-hemolytic arenicin-3. its analog nz17074 with asn and his substituted for tyr5 and tyr17 respectively is currently at the preclinical stage as an agent against multiresistant gramnegative bacteria (fox, 2013). other than antimicrobial activities of arenicin-1 in mammalian system in respect of possible medical application are also highly important. the ability of arenicin-1 to form a complex with human complement protein c1q resistant to high salt concentration was demonstrated recently (berlov et al., 2015). previously similar data were shown for homologous peptide tachyplesin, which is able to interact with human c1q and activate complement cascade on the surface of tsu tumor cells (chen et al., 2005). thus, possible therapeutic application of arenicins may concern not only their antimicrobial action but also some immunomodulatory activities. there are still no direct data about particular site of c1q molecule responsible for interaction with arenicin. however, available reports on other βstructural amps (defensins and tachyplesin) point at the collagenous domain of c1q as the site involved in the interaction with peptides (van den berg et al., 1998; chen et al., 2005). possibly, ability to interact with c1q reflects a more general feature of arenicin to interact with collagenous proteins that may be functionally important in the organism of a. marina. conclusion immunity of the lugworm a. marina is still comparatively poorly studied. basically it relies on both systemic and epithelial branches of immune responses. several types of celomocytes were described. these cells are active phagocytes, able to aggregate under stress conditions, and capable to discriminate between gram-positive and gramnegative bacteria. though, no prrs on celomocytes surface were identified up to date. among immune effectors, antimicrobial peptides arenicins are most well characterized. these are constitutively expressed in a wide range of tissues including celomocytes and various epithelia. in celomocytes, arenicins participate in phagocytosis during fusion of cytosolic granules with lysosome without being exocytized into celomic fluid plasma. in the epithelia of body wall and gut, arenicins most probably are secreted into cuticle and intestinal cavity, respectively. involvement of the same amps into both systemic and epithelial immunity is rather uncommon, as usually epithelia and phagocytes produce their own set of amps (e.g., in humans αdefensins 1-4 in neutrophils, ll-37 in monocytes, αdefensins 5 6 in intestine and different β-defensins in skin and other borders (rev. in de smet and contreras, 2005)). arenicins are very potent antimicrobials that retain their activity in presence of salt, which is critical for marine organisms. β-hairpin structure relates them to members of tachyplesin/polyphemusin family of amps and is essential for realization their toxic action. although native arenicin-1 and -2 are highly toxic to different mammalian cells (a feature limiting their application in medicine), several modified analogs with great therapeutic potential were designed based on their structure. yet, many aspects of a. 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genetics and breeding, ocean university of china, qingdao 266003, china 5center for marine molecular biotechnology, national laboratory for marine science and technology, qingdao 266237, china 6cas center for ocean mega-science, chinese academy of sciences, qingdao 266400, china this is an open access article published under the cc by license accepted may 13, 2020 abstract as pattern recognition receptors (prrs), c-type lectins (ctls) have important roles in the recognition and clearance of pathogens by the innate immune system. in the present study, the first cherax quadricarinatus ctl gene (designated cqctl) was cloned and characterized. the complete cdna sequence of cqctl contained an open reading frame (orf) of 543 bp, which encoded a protein of 180 amino acids. a carbohydrate recognition domain (crd) containing four conserved cysteines (cys48, cys59, cys76, cys177) and the epd (glu80-pro81-asn82) and qpd (gln146-pro147-asn148) motifs were identified in the deduced amino acid sequence of cqctl. the deduced tertiary structure of cqctl revealed two α helices，five β sheets and two disulfide bonds. cqctl exhibited high similarity with previously identified ctls from other species. the mrna transcripts of cqctl were ubiquitously detectable in all the tested tissues, with the highest expression level in hepatopancreas. these results provide useful information on the potential role of cqctl in the innate immune system of c. quadricarinatus, and lay the foundation for further studies on the ctls of crustacean. key words: cherax quadricarinatus; c-type lectin; innate immunity introduction in the long evolutionary process, under the double selection pressure of pathogens and the environment, organisms gradually evolved two sets of complex immune mechanisms to protect the body from infection, namely innate immune system and acquired immune system. however, invertebrates lack adaptive immune which produce specific ___________________________________________________________________________ corresponding authors: mengqiang wang moe key laboratory of marine genetics and breeding college of marine life science ocean university of china qingdao 266003, china e-mail: wangmengqiang@ouc.edu.cn lei wang cas key laboratory of experimental marine biology institute of oceanology chinese academy of sciences qingdao 266071, china e-mail: wanglei@qdio.ac.cn, leiwang@qdio.ac.cn antibodies, and only have nonspecific innate immune. the innate immune system is composed of cellular immunity and humoral immunity, which has the ability to bind to the surface conservative molecules of pathogenic bacteria (iwanaga and lee, 2005, zelensky and gready, 2005). these evolutionarily conserved pathogen-associated molecular patterns (pamps) are essential for the survival of microorganisms, but they are not present in the host animals, so they become the fatal weakness of microorganisms (gottar et al.,2002; janeway and medzhitov, 2002). invertebrates distinguish "self" and potentially harmful "non-self" mainly by pattern recognition receptors (prrs), which recognize the invading pathogen pamps and transmit signals to innate immune cells (hoffmann et al., 1999; janeway, 1989). this recognition of prrs to pamps enables the host to respond quickly and extensively to infection, which is a typical characteristic of the innate immune system (yu et al., 2002). https://creativecommons.org/licenses/by/4.0/legalcode 109 table 1 nucleotide sequences of primers used in this study primers sequence (5'-3') brief information cqctl-race-f1 caaggctgaggtggtgagagg gene specific primer for race cqctl-race-f2 tccgttctggtgtatggctc gene specific primer for race cqctl-race-r1 ttgagccatacaccagaacg gene specific primer for race cqctl-race-r2 gctgacagatgaacggtaggag gene specific primer for race upml ctaatacgactcactatagggcaagcagtggtatcaacgcagagt universal primers for race upms ctaatacgactcactatagggc universal primers for race nup aagcagtggtatcaacgcagagt universal primers for race 3'cds aagcagtggtatcaacgcagagtac(t)30v n oligo (dt) for cdna synthesizing 5-ap-dg aagcagtggtatcaacgcagagtacgcgggggggggg anchor primer for 5’ race m13f tgtaaaacgacggccagt vector primer for sequencing m13r caggaaacagctatgacc vector primer for sequencing cqctl-qrt-f atggtgaaggcatgtgtgacg gene specific primer for real-time pcr cqctl-qrt-r gcagaccaaggtctcttgctca gene specific primer for real-time pcr 18s rrna-qrt-f aatggttggacgagaaggaa internal control for real-time pcr 18s rrna-qrt-r ccaactaaacaccctgctgata internal control for real-time pcr lectin is a membrane or soluble prr, and it plays a prominent role in autoimmune recognition and the clearance of invasive microbes (yu and kanost, 2004). among the lectins, c-type lectins (ctls) are the most widely studied proteins, which are a group of ca2+-dependent protein superfamily involved in agglutination and widely distributed in almost all organisms (dodd and drickamer, 2001; lakhtin et al., 2011). the main feature of the ctls superfamily is that it contains at least one carbohydrate recognition domain (crd), which has specific structures to identify specific sugars (dodd and drickamer, 2001). each crd contains four conserved cysteines and is involved in forming two pairs of disulfide bonds to stabilize the crd conformation (zelensky and gready, 2005). ctls perform a variety of immune functions, such as microbial agglutination, antibacterial or antiviral responses, cell adhesion, opsonization, phenoloxidase activation, nodule formation, phagocytosis, and maintenance of gastrointestinal immune system homeostasis (ofek et al., 2000; wang and wang, 2013; li et al., 2019). in recent years, ctls in an increasing number of crustaceans have been reported to participate in innate immunity against pathogens. for example, lvctl3 in pacific white shrimp penaeus vannamei reduced the mortality of shrimp infected by vibrio parahaemolyticus and white spot syndrome virus (wssv) (li et al., 2014). ptclec1 in the swimming crab portunus trituberculatus revealed antimicrobial activity, and promoted the clearance of vibrio alginolyticus in vivo and hemocyte phagocytosis in vitro (su et al., 2020). both fclectin and fc-hsl in chinese shrimp penaeus chinensis and pmav in black tiger shrimp penaeus monodon showed antiviral activity (luo et al., 2003; liu et al., 2007; sun et al., 2008). the red claw crayfish, cherax quadricarinatus, is a species native to northern queensland, australia and southeast papua new guinea (karplus et al., 2003). it is a kind of freshwater breed, which has been raised for human consumption since 1985, and has gradually become an important economic species (garcia-ulloa et al., 2003). the worldwide shrimp/crayfish cultivation industry is seriously affected by viral pathogens, particularly wssv, and the cultivation industry has led to significant economic losses (liu et al., 2013). however, the study of its molecular immunology is insufficient. only a few genes especially prrs have been identified, such as serum lectin (cql) (sanchez-salgado et al., 2019), toll receptor (cqtoll) (li et al., 2017), and hypervariable immunoglobulin domain-containing receptor (cqdscam) (li et al., 2015). therefore, it has become the focus of attention to understand the immune ability and defense mechanism of c. quadricarinatus. in our present study, the first c. quadricarinatus ctl gene (designated cqctl) was reported. the full-length cdna of cqctl was obtained via a rapid amplification of cdna ends (race) technique. the expression characteristics of cqctl were analyzed in different tissues. the purpose of this study was to preliminarily investigate the sequence characteristics and tissue distribution, and to provide useful information for its further study. 110 fig. 1 nucleotide and deduced amino acid sequence of cqctl. the predicted signal peptide was in underline. the polyadenylation consensus signal was indicated in double underscore. the wavy line was the sugar binding site. the predicted carbohydrate recognition domain (crd) was in shade. the asterisk indicated the stop codon. the amino acid sequence of cqctl has been submitted to genbank with the accession number mn944107 materials and methods crayfish and samples collection the healthy adult red claw crayfish used in this study were obtained from a crayfish farm in boxing, shandong province, china. to determine the tissue distribution of cqctl, tissues of the stomach, gill, testis, nerve, intestine, eyestalk, epithelium, muscle and hepatopancreas were sampled from five adult red claw crayfish, and immediately frozen in liquid nitrogen and then stored at -80 °c until total rna extraction. hemocytes were collected from the ventral sinus of five adult red claw crayfish using a sterile syringe which preloaded with an equal volume of anticoagulant buffer (nacl 450 mmol l-1, kcl 10 mmol l-1, edta·2na 10 mmol l-1, and hepes 10 mmol l-1, ph 7.45), centrifugation at 800 g for 10 min at 4 °c, the supernatant was removed, and immediately frozen in liquid nitrogen and then deposited at -80 °c until use. total rna extraction and cdna synthesis minibest universal rna extraction kit (9767, takara, china) was used to extract total rna from ten tissues of cherax quadricarinatus in accordance with the manufacturer’s instructions. degradation and contamination of rna were determined by electrophoresis on a 1% agarose gels. the quality and quantity of rna were checked using a nanodrop lite (thermo scientific, usa). synthesis of the first-strand cdna for the quantitative real-time pcr (qpcr) and the 3’-end race was carried out by transscrip® one-step gdna removal and cdna synthesis supermix (at311, transgen biotech, china) using anchored oligo(dt) primer (3'cds) according to the manufacturer’s instructions. to amplify the 5’-end of the cdna sequence, a poly (c) tail was added to the first-strand cdna using the terminal deoxynucleotide transferase (tdt). the obtained cdna was stored at -20 °c for cloning and expression quantity analysis. 111 fig. 2 (a) the three-dimensional (3d) structure of cqctl modeled by swiss-model. (b) the disulfide bonds formed by conserved cysteine cloning and sequencing of cqctl the partial cdna sequence of cqctl was derived from the transcript that was obtained by the transcriptome of c. quadricarinatus. based on the 901 bp mrna linear transcript, primers for cqctl (cqctl-race-f1/2 and cqctl-race-r1/2) were designed to clone the full-length cdna of cqctl by the race technique. the universal primer a mix (upml: upms= 1:5) combined with cqctl-race-f1 was used to amplify 3’end, and the anchor oligo(dg) primer 5-ap-dg combined with cqctl-race-r1 was used to amplify 5’end. subsequently, pcr products were used as the template for the nested pcr, which was performed with nested universal primer a (nup) and cqctl-race-f2 or cqctl-race-r2. the pcr was performed in 50 µl reaction volume containing 19 µl sterile distilled h2o, 25 µl of 2 × easytaq dna polymerase (ap111, transgen biotech, china), 2 µl of each primer (10 µmol l-1), and 2 µl of dna template (~10 ng l-1). the pcr amplification program included a denaturation for 5 min at 94 °c, followed by 30 cycles of 30 s at 94 °c, 30 s at 57 °c, and 45 s at 72 °c, and finally 10 min at 72 °c. the pcr products were gel-purified and ligated into the peasy®-t1 simple cloning vector (ct111, transgen biotech, china), and then transformed into the competent cells of escherichia coli trans1-t1 (cd501, transgen biotech, china). m13 primers (m13f and m13r) were used for colony pcr to identify the potentially positive recombinants, and the positive recombinant clones were picked for sequencing. the primers are listed in table 1. bioinformatics analysis of cqctl the nucleotide and amino acid sequence homologs of cqctl were searched via the blast program (http://blast.ncbi.nlm.nih.gov/blast). multiple sequence alignment of cqctl with ctls from other animals was generated using the clustalw2 (http://www.ebi.ac.uk/tools/msa/clustalw2/). the domain features of cqctl were predicted by the simple modular architecture research tool (smart, http://smart.emblheidelberg.de/). the location of signal peptide was predicted by signalp 3.0 program (www.cbs.dtu.dk/services/signalp). the structure of cqctl was predicted by the swiss-model server and repository (http://swissmodel.expasy.org/). the phylogenetic tree was constructed based on the amino acid sequences alignment by the neighbor-joining (nj) method with mega 7.0 software, bootstrap trials were replicated 1000 times. the potential disulfide bonds and their position were predicted by the scanprosite program (http://www.expasy.ch/tools/scanprosite/). quantification analysis of cqctl mrna expression the mrna expression quantities of cqctl in different tissues were checked by the qpcr technique. all qpcr reactions were performed with transstart® top green qpcr supermix (aq131, transgen biotech, china) using about 100 ng cdna template and 0.2 μm of each primer (table 1), in an abi 7300 real-time pcr system (thermo fisher, usa). the amplification program included a denaturation for 10 s at 94 °c, followed by 40 cycles of 5 s at 94 °c, and 31 s at 60 °c. the mrna transcriptional level of cqctl was normalized to that of the 18s rrna gene for each sample. three biological replicates were set for each tissue, and each experiment was performed in three independent replicates. statistical analysis the results of qpcr analyses were based on the melting curve analysis of the pcr products and 112 the comparative ct (2 -δδct) method (stomach as the reference tissue) was used to analyze the relative expression level of cqctl mrna (livak and schmittgen, 2001). final results were expressed as means ± standard deviation (sd). the statistical analysis was performed by one-way analysis of variance (one-way anova) followed by duncan's multiple range tests by using spss statistic 19.0 software to detect the significant intergroup differences. the p values less than 0.05 were considered as statistically significant. result cloning and characterization of cqctl cdna the full-length cdna sequence of cqctl was obtained by race and submitted to genbank under the accession no. mn944107. the gene contained a 543 bp open reading frame (orf), a 30 bp 5’-untranslated region (utr) and 389 bp 3’-utr, including a polyadenylation consensus signal site (aataaa) and a poly a tail, a total length of 962 bp (fig. 1). the orf encoded 180 amino acids with a calculated molecular weight of 19.89 kda and a theoretical pi of 4.57. a signal peptide containing 18 amino acid residues and a single crd domain were predicted by the smart program. there were conserved epd (glu80-pro81-asn82) and qpd (gln146-pro147-asn148) motifs in the crd domain, which determine the specificity of ligand binding (fig. 1). the protein homologous modeling was performed to generate the three-dimensional (3d) structure of the cqctl based on its amino acid sequence. the template protein was human ctl domain family 4 member c (pdb code: 4zes) (jegouzo et al., 2015). the tertiary structure of cqctl revealed two α helices and five β sheets (fig. 2a). two β sheets at the n-terminal (β1 and β2) and one at cterminal (β5), which are close together to form an antiparallel β sheet. the β3 and β4 sheets form the second antiparallel β sheet slice. the disulfide bond formed by cys48 and cys59 connect β1 and β2. another disulfide bond formed by cys76 and cys177 connect α1 and β5 (fig. 2b). multiple alignments and phylogeny relationship of cqctl the deduced amino acid sequence of cqctl exhibited high similarity with previously identified ctls, 34.56 % with penaeus merguiensis pmctl (ags42196) and 32.75 % with scylla paramamosain spctl (agl46986). the alignment of the protein sequence of cqctl was performed to determine its identity, compared with those of previously identified ctls (fig. 3). a comparison of ctls revealed that they had four conserved cysteines (cys48, cys59, cys76, cys177) involved in the formation of disulfide bonds, the nterminal of cqctl also contains two additional cysteines (cys26, cys37), indicating that cqctl is a standard long ctl. fig. 3 multiple alignments of cqctl with previous known c-type lectins (ctls). the triangle indicated four conserved cysteines, and the square indicated two additional cysteines. the same amino acid residues were shaded in black and the similar amino acids were shaded in grey. gaps were indicated by dashes to improve the alignment. the sequences and their accession numbers are as follows: penaeus merguiensis (ags42196), scylla paramamosain (ahh02662), eriocheir sinensis (aff59978), macrobrachium rosenbergii (afn20600), and pantholops hodgsonii (xp_005959959) 113 fig. 4 neighbor-joining (nj) phylogenic tree of cqctl constructed based on the protein sequences of ctls from different organisms. to derive confidence value for the phylogeny analysis, bootstrap trials were 1000 replicates. the black triangle indicated the ctl protein of c. quadricarinatus. the numbers at the forks indicated the bootstrap value. species and their protein accession numbers are as follows: scylla paramamosain (ahh02662), eriocheir sinensis (aff59978), penaeus merguiensis (ags42196), crassostrea gigas (xp_011415449), crassostrea virginica (xp_022293075), argopecten irradians (acs72239), danio rerio (xp_021327322), sinocyclocheilus rhinocerous (xp_016423812), labeo rohita (rxn24634), pygoscelis adeliae (xp_009320153), fulmarus glacialis (xp_009583679), tyto alba (xp_009974840), merops nubicus (xp_008945916), pantholops hodgsonii (xp_005959959), bos mutus (xp_014337887), homo sapiens (np_001007034), mus musculus (np_001004159), pseudonaja textilis (xp_026579441), protobothrops mucrosquamatus (xp_015681994), hoplocephalus stephensii (abp94113), and cryptophis nigrescens (acc67946) an nj phylogenetic tree based on amino acid sequences of cqctl and other animal ctls was constructed to evaluate their molecular evolutionary relationships. cqctl was clustered most closely with ctls of s. paramamisain, eriocheir sinensis, and p. merguiensis, and then with mollusks, including crassostrea gigas, crassostrea virginica, and argopecten irradians. the vertebrate ctls formed other clusters, including the fish ctls (danio rerio, sinocyclocheilus rhinocerous, and labeo rohita), bird ctls (pygoscelis adeliae, fulmarus glacialis, tyto alba, and merops nubicus), mammal ctls (pantholops hodgsonii, bos mutus, homo sapiens, and mus musculus) and reptile ctls (pseudonaja textilis, protobothtops mucrosquamatus, hoplocephalus stephensii, and cryptophis nigrescens). (fig. 4). the tissue distribution of cqctl mrna cqctl mrna transcripts were detected by qpcr in ten different tissues using 16s rrna as an internal control. cqctl mrna transcripts were detectable in all the tested tissues; the highest mrna transcripts level was in hepatopancreas, which was 22412.88-fold (p < 0.05) of that in the stomach, then muscle, epithelium, hemocytes, eyestalk, intestine, and nerve, which were 160.00-fold, 50.82-fold, 23.98-fold, 19.19-fold, 12.81-fold, and 12.75-fold (p < 0.05) of that in the stomach, respectively (fig. 5) the mrna expression levels were low in the stomach, gill, and testis. discussion ctl, as an important prr, is ubiquitous in animals and plants, and participates in innate immunity by non-self recognition, binding and clearing the invading pathogenic microorganisms (dodd and drickamer, 2001; vasta et al., 2004). in recent years, the research on ctl of crustacean has been gradually carried out. however, ctl has not 114 fig. 5 relative expression of cqctl in different tissues. the 18s rrna gene was used as an internal control to calibrate the cdna template for each sample. each vertical bar represents the mean ± sd (n = 3), and bars with different characters were significantly different (p < 0.05), while bars with same characters were not significantly different been reported in c. quadricarinatus. in the present study, a novel c-type lectin (cqctl) was identified in the red claw crayfish c. quadricarinatus, which was 962 bp and encoded a protein of 180 amino acids. we analyzed the sequence characteristics and tissue distribution, laying a foundation for further study on its functional characteristics. bioinformatics analysis revealed that cqctl contained a crd domain, which has conserved epd and qpd motifs. it is known that epn or qpd motifs are important to the binding of mannose or galactose and their derivatives, respectively (zelensky and gready, 2003; zelensky and gready, 2005). in many lower invertebrates, epn may also be replaced by epd or eps (wang and wang, 2013). cqctl contains epd and qpd motifs, indicating that it may be able to combine with mannose, galactose, and their derivatives. besides, the wnd motif is important to carbohydrate-binding and is highly conserved in vertebrate lectins (iwanaga and lee, 2005), whereas invertebrates show a lack of wnd motif or a mutation to a similar motif during evolution, for example, the lvlectin-1 and lvlectin-2 of the p. vannamei lack the wnd motif but still respond to listonella anguillarum and wssv (wei et al., 2012). like lvlectin-1 and lvlectin-2, cqctl does not have wnd or its similar motifs. the crd domain contains two conserved disulfide bonds (cys48-cys59 and cys76-cys177), which maintain the structural stability of cqctl and form a fundamental and stable two-double-loop structure (zelensky and gready, 2005). the n-terminal of the crd domain contains two additional cysteine residues, indicating that the cqctl crd is the “long” type, like the ctls of other aquatic organisms (yu et al., 2013; li et al., 2019). the amino acid sequence of cqctl had more than 30 % similarities with other identified ctls. phylogenetic analysis of the different ctls sequences showed that cqctl clustered with homologs from the s. paramamisain, e. sinensis, and p. merguiensis. the results indicate that cqctl is a new member of the ctl family. to investigate the potential functions of cqctl in red claw crayfish, the tissue expression of the cqctl gene was analyzed. the cqctl mrna transcripts could be detectable in all the sampled tissues; expression was highest in hepatopancreas. it is well known that the hepatopancreas is the main organ of immune defense in crustaceans (lee et al., 2000; jiravanichpaisal et al., 2006). hepatopancreas is an important tissue producing immune response factors, including humoral immune and cellular immune response (lee et al., 2000; wang et al., 2013). in previous studies, the expression level of ctls was highest in hepatopancreas, while the expression level was relatively low in other tissues, such as p. monodon (ma et al., 2008), p. merguiensis (rattanaporn and utarabhand, 2011) 115 and p. chinensis (xu et al., 2010). the high mrna expression levels of cqctl in hepatopancreas indicated that it might be involved in the innate immunity of red claw crayfish. in conclusion, the full-length cdnas of cqctl were cloned and characterized from the red claw crayfish, the structure of cqctl was modeled, and its tissue distribution was detected. cqctl was found to be widely distributed in tissues, with the highest expression in hepatopancreas, and may participate in the immune response. the results obtained from this study would provide useful information on the potential role of cqctl in the innate immune system of c. quadricarinatus,and lay the foundation for further studies on the ctls of crustacean. acknowledgements this work was supported by the national key r&d program of china (2019yfd0900401) and 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466-477, 2003. zelensky an, gready je. the c-type lectin-like domain superfamily. febs j. 272: 6179-6217, 2005. 330 isj 14: 330-339, 2017 issn 1824-307x research report functional analysis of hemolin gene from silkworm, bombyx mori immune and development x liu, j li, q wang, h xia, k chen institute of life sciences, jiangsu university, zhenjiang 212013, p.r. china accepted august 31, 2017 abstract hemolin, an immunoglobulin-like protein, is involved in the immunity and development of lepidoptera, but its exact functions are still unclear. in this work, the quantitative pcr and western blot analysis were used to study the effects of exogenous substances on hemolin transcription and translation in the silkworm midgut. also, rna interference was used to examine the effects of hemolin on wing disc development of silkworm. we found that the transcription and expression of hemolin gene could be up-regulated effectively after injection of inactive escherichia coli and cell wall components (lps and pgn) into silkworm midgut. however, no any changes in transcription and expression of hemolin were observed by the injection of bmnpv (bombyx mori nuclear polyhydrosis virus). the protein-protein interaction between hemolin and yippee was verified by co-immunoprecipitation (co-ip) in silkworm wing discs. in addition, the silence of hemolin gene by rna interference could lead to adult wing hypoplasia in most hatched moths. our study provides some useful information regarding the functions of hemolin associated with the immune and developmental mechanisms in silkworm. key words: bombyx mori; immunity; development; hemolin; yippee introduction hemolin, an immunoglobulin-like protein initially found in lepidopteran insects, is a member of the immunoglobulin superfamily and has been suggested to participate in the immune response to various pathogens, metamorphosis, and the regulation of embryonic development (schurmann et al., 2001). since it was first discovered in hyalophora cecropia in 1975, the genome sequences for more than 10 species of lepidoptera, including silkworm (bombyx mori), have been identified based on their structural similarity (faye et al., 1975; sun et al.,1990). more recently, a hemolin-like protein which shares high similarity with insect hemolins, named lvhemolin, has been identified from litopenaeus vannamei and could also play an important role in shrimp innate immune defense against bacterial and viral infections (zuo et al., 2015). ___________________________________________________________________________ corresponding author: xiaoyong liu institute of life sciences jiangsu university 301 xuefu road, zhenjiang 212013, p.r. china e-mail: liuxiaoyong@ujs.edu.cn list of abbreviations: bmnpv: bombyx mori nuclear polyhydrosis virus; pcr: polymerase chain reaction; qpcr: quantitative pcr; rnai: rna interference. transcription and expression of the hemolin gene mainly occur in the insect fat body. some of them could be secreted into the hemolymph to participate in the immune response. under normal circumstances, hemolin is present at lower levels in the hemolymph although its expression levels change with the developmental stages of the insect. however, upon infection by bacteria, hemolin titer in the hemolymph increases rapidly, strongly suggesting a functional role for hemolin in humoral immune defense. hemolins in lepidoptera own the conservative horseshoe-shaped structure composing immuneglobulin domains, which are similar to drosophila neuroglian cell adhesion molecule (terenius, 2008). therefore, they are thought to be the most common type of cell adhesion molecules and regulate the cellular immune response through binding to the cell surface ligand specificity. hemolin interacts with lipopolysaccharide (lps) in gram-negative bacteria, lipoteichoic acid in gram-positive bacteria, and β-1,3-glucan on the fungal cell wall (daffre and faye, 1997). it exhibits two binding sites for lps, one for the interaction with lipid a phosphate groups and the other for the interaction with the o-specific antigen and outer-core carbohydrates of lps (yu and kanost, 2002). this is suggestive of its function as a pattern recognition molecule in the cellular immune response. hemolin has also been shown to participate in the immune response to viruses in some but not all mailto:liuxiaoyong@ujs.edu.cn 330 species (terenius et al., 2007). for example, it is involved in antiviral resistance in bombycoidea, but not in noctuoidea (terenius et al., 2009). lepidoptera are susceptible to viral infection only in the larval stage. with the growth and development, they display an increased resistance against viruses and the hemolin gene expression is strongly up-regulated accordingly (kirkpatrick et al., 1998). moreover, growing body evidences suggest a functional role for hemolin in the development of insects. upon injecting hemolin-specific sirna, the h. cecropia pupae could incubate normally, but do not produce fertile eggs after mating (bettencourt et al., 2002). in plodia interpunctella, hemolin gene expression is significantly affected by molting hormone regulation before pupation, where the main site of expression is epidermis, and fat body also show some expression as well (roxstrom-lindquist et al., 2005). an intracellular protein, yippee, was initially identified from the proteins encoded in a drosophila imaginal disc cdna library in a yeast interaction trap screen as physically interacting with h. cecropia hemolin (roxstrom-lindquist and faye, 2001). subsequently, yippee and ypel (yippee-like protein) family of proteins, showing extremely high degree of sequence homology, were found in all virtually eukaryotes, including fungi, plants, and animals (hosono et al., 2004, 2010). yippee from silkworm (bmyippee) has shown to be highly homologous with the drosophila yippee. it encodes 121 amino acid residues with no signal peptide. a tissue specific expression element c/ebp locates in the promoter at the nf-kb sequence. bmyippee is expressed in pupae and eggs and distributed around the mitotic apparatus (xin et al., 2009; hosono et al., 2010). several lines of evidence suggest that yippee protein plays an important role in eukaryotes that is involved in the cell cycle, cell proliferation and growth (hosono et al., 2004, 2010; liang et al., 2010). however, little is known about the exact function of yippee in development process. silkworm is an ideal representative insect for studying immune, development, and metamorphosis in lepidopterans. using silkworm as a model organism has numerous advantages, including rich genetic materials, well-studied genetic background, and easy maintenance. therefore, the silkworm, bombyx mori, was used in this work to investigate the effects of hemolin in immune challenges and on the development of insect. in silkworm, the wing disc, one of the imaginal discs, is a key organ for the development of adult wing. in addition, both yippee and hemolin are present in silkworm. it is important to study the yippee-hemolin interaction in silkworm wing discs that will contribute significantly to the understanding of novel mechanisms involved in development process. in this paper, xenobiotics and pathogens were used to study the hemolin gene expression in the silkworm midgut. also, the interactions between hemolin and yippee in the wing discs of silkworm were investigated. the inhibition of hemolin expression at the pupal stage and its role in the development of wing discs indicate that hemolin is involved in the development of the silkworm wing. materials and methods chemicals, cell line and animals all reagents and chemicals used in this study were purchased from sigma-aldrich, gibco-brl, and invitrogen except as otherwise indicated. the strains of bombyx mori named jingsong × haoyue were stored in our laboratory, and all larvae were reared with fresh mulberry leaves at 27 °c. kunming mice and new zealand white rabbits were provided by jiangsu university laboratory animal center. bmn cells derived from ovary of b. mori and e. coli strains (dh5α, tg1, bl21) were kept in our laboratory. preparation of antisera against bmhemolin (hemolin from bombyx mori) and bmyippee (yippee from b. mori) protein sequence of hemolin in b. mori was obtained from ncbi database (genbank: acq82817.2) using protein prediction software dnastar 6.0. the primers were designed using primer design software “primer premier 5.0” based on the sequence corresponding to the predicted hydrophilic region. the forward primer was 5′-agaattcatgcaaaccaaagcttcgga-3′ and the reverse primer was 5′-actcgagctatttatcgttggcatcgtc-3′. protein sequence of yippee in b. mori was also obtained from ncbi database (genbank: babh01000166). the primers corresponding to the full-length open reading frame of yippee gene were designed as follows: forward primer, 5′-cgcggatccatgggtaaaatatttcttgatc3′; reverse primer, 5′-ccgctcgagcccttccttgtatctttga-3′. hemolin and yippee target fragments were amplified using the pfu dna polymerase (takala, dalian, china) and synthesized primers (generay, shanghai, china). the target fragments were cloned into t vector (takala, dalian, china) for amplification and sequence identification (sangon biotech, shanghai, china). after sequence identification, target fragments for hemolin or yippee were inserted into the expression vector pet-30a (takala, dalian, china) for the generation of recombinant plasmids, pet-bmhemag and pet-bmyipag, respectively. the recombinant plasmids were transformed into e. coli bl21 (de3) with kanamycin for screening. after iptg induction, the expressed proteins were subjected to sds-page and visualized by coomassie brilliant blue staining. the target proteins were cut and dried using a vacuum freezing dryer; then dried gels were grinded to powder for the preparation of antisera. the animal immunization against bmhemolin was performed in kunming mice using complete freund's adjuvant (sigma-aldrich, shanghai, china). the effects of polyclonal antiserum were tested at the third day after the fourth boosting injection of antigen. similarly, new zealand white rabbits were immunized 4 times using bmyippee protein as immunogen. collected blood serum from mice or rabbits with high sensitivity to target proteins was stored at -80 °c in a cryogenic refrigerator. 331 330 treatment of xenobiotics in bmn cells and silkworm larvae treatment of xenobiotic in bmn cells the bmn cell line, maintained in our laboratory, is derived from the silkworm ovary and has been wildly used for the studies of insect physiology, developmental biology, pathology, molecular biology and so on. after the recovery training, the cells were grown as an adherent culture in tc-100 insect cell culture medium supplemented with 10 % fetal bovine serum (fbs) (gibco, usa) at 27 °c. the cell culture medium was changed with fresh medium every 3 4 days. when the monolayer reached about 80 % confluence, 5 μl of xenobiotic solution was added to the medium in the form of inactivate e. coli dh5α, peptidoglycan (pgn), or lipopolysaccharide (lps) (sigma-aldrich, shanghai, china), respectively. each treatment group has three replicates. at 24 h after the addition of xenobiotic solution, the adherent cells from 3 flasks of each group were scraped and collected in sterilized ep tubes. after centrifugation at 600 rpm for 1 min, the harvested cell pellets were stored at -80°c till further use. treatment of xenobiotics in silkworm larvae silkworms were fed with mulberry leaves in a sterile environment. the fifth instar larvae were injected with inactivate e. coli (dh5α, about 5×10 7 bacterial cells each silkworm), peptidoglycan (pgn, 5 μl of 1 mg/ml solution), lipopolysaccharide (lps, 5 μl of 1 mg/ml solution), bmnpv (5 μl of virus, with 1.6×10 4 virus particles/ml), and pbs (5 μl) in separate treatment served as the control group. each group contained 30 larvae. the wounds were sealed with vaseline immediately after injection to prevent the loss of injected material and any inadvertent infection. at 9, 24, and 48 h post injection, 10 larvae from each group were placed on a sterile operating table and their midgut tissues were carefully dissected out, rinsed clean with pbs in a 1.5-ml centrifuge tube, and then immediately stored at -80 °c until further analysis. rna extraction and quantitative pcr (qpcr) the stored bmn cell pellets or extracted midgut tissues were taken from -80 °c and grinded in liquid nitrogen. total rna was extracted by the trizol method (transgen, shanghai, china). the quality of extracted rna was checked by agarose gel electrophoresis and stored at -80 °c. the primers for amplification of silkworm hemolin gene (genbank: ab115084.1) and α-tubulin gene were designed using primer design software “primer premier 5.0”. for hemolin gene, the forward primer was 5′-gtcactcggaactccccta-3′ and the reverse primer was 5′-gacactctgcccaaacctc-3′. while for α-tubulin gene, the forward primer was 5′-gtgttcagccaaaccaaa-3′ and the reverse primer was 5′-tacagcggagttcaaagg-3′. the amplification procedure was as follows: pre-denaturation at 95 °c for 10 min, followed by 25 cycles at 95 °c for 30 sec, 60 °c for 30 sec, and 72 °c for 30 sec. each sample was tested in triplicate. the relative expression was evaluated following the quantification equation, 2 -△△ct (livak and schmittgen, 2001). western blot analysis the collected midgut tissues were completely ground into powder in liquid nitrogen. ripa lysis buffer (strong) supplemented with phenylmethylsulfonate (pmsf) at a final concentration of 10 mm was then added to the ground powder and mixed well by vortexing. the mixture was centrifuged at 13000 rpm for 15 min. the supernatant containing total proteins was collected and protein concentration was determined by the bca protein assay kit (pierce). the total proteins from tissues were separated by electrophoresis on sds-page gel and transferred onto a nitrocellulose membrane. the membrane was blocked with 5 % w/v nonfat dry milk in tbst buffer for one hour, and then incubated with our prepared polyclonal antiserum (1: 500) for one hour at room temperature. after three 15-min washes with tbst buffer, the membrane was incubated with hrp labeled goat anti-mouse or anti-rabbit igg (1: 5000) (beyotime, shanghai, china) for one hour according to the source of used primary antibody and washed with tbst buffer 3 times. the primary antibody used for reference protein was anti-α-tubulin monoclonal antibody (1:5000). dab reagent kit (zsgb-bio, beijing, china) was used for signal generation. immunoprecipitation and immunodetection wing disc tissues from the fifth instar larvae were collected and ground into a powder in liquid nitrogen. total tissue protein lysate samples were prepared as described above. immunoprecipitation was performed according to the methods previously described (golemis, 2002). briefly, 40 µl of mouse polyclonal antiserum anti-bmhemolin was added into 1 ml of each lysate sample in a sterile 1.5-ml ep tube and incubated on ice for 2 h. in the control test, a mouse monoclonal antibody anti-vta1 (novus biologicals) was used. then, 30 µl of protein a/g agarose beads (cwbiotech, shanghai, china) was added, followed by a slow rotation for overnight at 4 °c. after the incubation, the beads were spun down and washed with ripa buffer 4 times. the immunoprecipitates were collected and suspended in 2×loading buffer. after boiling at 100 °c for 10 min, the samples were subjected to western blot analysis using polyclonal antiserum anti-bmyippee. alternatively, a parallel immnuoprecipitation reaction was performed with anti-bmyippee polyclonal antiserum. the final immunoprecipitates were subjected to western blot analysis using anti-bmhemolin polyclonal antiserum to detect bmhemolin protein in co-precipitates. rna interference of hemolin gene in silkworm pupae based on the sequence of hemolin gene, a blast homology search (http://www.ncbi.nlm.nih.gov/blast) was performed to avoid off-target effects on other genes or sequences. the sirnas, synthesized by shanghai genepharma co., ltd. (shanghai, china) as shown in table 1, were dissolved in diethylpyrocarbonate-treated water at three concentrations (0.4, 0.8, and 1.6 μg/μl h2o). a 5 μl aliquot of sirna solution was injected into each pupa in the position of the wing disc using a microliter syringe. correspondingly, the amount of injected sirnas was 2, 4, 332 https://www.baidu.com/link?url=vyz2io1hftshe1qclypvdhvw-wbg5wmyvajnea3jqu__awnayjvaagfujkx5_l3me-ce4bwyklnr80fp920_sa&wd=&eqid=8061e7e40004c47400000005590bdfd0 http://www.ncbi.nlm.nih.gov/blast 330 table 1 the sirna fragments of hemolin gene for rna interference the pairs of fragment sequence (5’-3’) base number (bp) pair 1 (rnaibmhem-1) f: ccguuaauuccggagacaatt r: uugucuccggaauuaacggtt 21 21 pair 2 (rnaibmhem-2) f: ccagcacaagggcuacuaatt r: uuaguagcccuugugcuggtt 21 21 pair 3(rnaibmhem-3) f: gcgaucugacguaucuauatt r: uauagauacgucagaucgctt 21 21 negative control f: uuguccgaucgugucacgutt r: acgugacacgaucggacaatt 21 21 and 8 μg per pupa in each group. to avoid leakage of sirna from the body, the wounds were sealed with vaseline immediately after the injection. five pupae were injected with 8 μg sirna each, whereas 25 pupae were injected with 4 μg or 2 μg sirna each, separately. one set of sirnas with random sequences were used as a negative control and injected alongside the experimental treatments. at 72 h after sirna injection, 5 pupae were collected for qpcr and western blot analysis. and the others were allowed growing under sterile conditions, hatched into moths for the observation of the wing changes in adult. results the efficiency of the polyclonal antisera the efficiency of prepared polyclonal mouse antiserum against bmhemolin or polyclonal rabbit antiserum against bmyippee was determined by western blot analysis. as shown in figure 1a, only a single band around 45 kda was detected by antiserum against bmhemolin using total protein extraction from pupae as a loading sample (lane 1), which is consistent with the predicted size (44.79 kda) of silkworm hemolin. meanwhile, a truncated peptide corresponding to hydrophilic region of bmhemolin expressed in e. coli bl21 was also detected as a major band around 21 kda although there were some slight miscellaneous bands (lane 2). it establishes that our produced polyclonal antiserum against bmhemolin could specifically recognize not only endogenous bmhemolin but also recombinant fragment of bmhemolin. as for bmyippee rabbit polyclonal antiserum, a specific band of around 14 kda was recognized (black arrow) using total protein extraction from pupae as a loading sample (fig. 1b), which is consistent with the predicted size (13.72 kda) of the silkworm yippee protein. our results indicate that both proteins are present in silkworm pupae, and both prepared antisera are suitable for future studies. fig. 1 western blot analysis for the generation of polyclonal antisera against bmhemolin and bmyippee. a) detection of bmhemolin by mouse polyclonal antisera against bmhemolin. lane m, protein marker in kda. lane 1, total protein extraction from the silkworm pupae as a loading sample. lane 2, a truncated peptide corresponding to hydrophilic region of bmhemolin expressed in e. coli bl21 as a loading sample. b) detection of bmyippee by rabbit polyclonal antisera against bmyippee. lane m, protein marker in kda. lane 1, total protein extraction from the silkworm pupae as a loading sample. the positions of detected proteins are indicated by the arrows. 333 330 effects of xenobiotics on the expression of bmhemolin gene transcriptional changes in bmn cells the bmn cells at about 80 % confluence were treated with xenobiotics by adding appropriate amount of inactivated e. coli (dh5α), peptidoglycan (pgn), and lipopolysaccharide (lps) into the culture medium. after 24 h, the total rna was extracted from cells, and qpcr was performed to determine the expression levels of bmhemolin gene. as shown in figure 2, the transcription of bmhemolin gene in bmn cells did not appear significant changes, compared with the control group (pbs), which is completely different from the effects observed in the larval body. transcriptional changes in midgut tissues of larvae on the third day of the fifth instar, the larvae were injected with inactivated e. coli (dh5α), bv virions of bmnpv, peptidoglycan (pgn), lipopolysaccharide (lps), and pbs at indicated concentrations as described in materials and methods section. total rna was extracted from the midgut tissues of larvae at indicated time points and the results of qpcr are shown in figure 3. comparing with the control group (pbs), the transcriptional levels of bmhemolin in group dh5α were significantly increased by 21.97, 26.05, and 22.61 times at 9, 24, and 48 h post injection, respectively. for the group pgn and group lps, the transcription of the bmhemolin gene was also up-regulated by the injection of peptidoglycan or lipopolysaccharide. however, the expression of bmhemolin gene did not appear to have any significant changes in the group bmnpv, with only a slight increase by 1.02, 1.11, and 1.07 times, at 9, 24 and 48 h post injection of virions, respectively, indicating that in silkworm, the bmnpv cannot induce bmhemolin transcription. fig. 2 transcriptional changes of bmhemolin gene from stimulants challenged bmn cells. a qpcr was performed at 24 h after adding exogenous substances in triplicate for each treatment. expression values were normalized to those of α-tubulin using the livak (2 -△△ct) method and the data were provided as the mean fold changes (means ± sd, n = 3) relative to the control group (pbs). the vertical axis indicates a relative mrna level of bmhemolin and the horizontal axis shows the addition of pbs (pbs), inactivated e. coli (dh5α), peptidoglycan (pgn), and lipopolysaccharide (lps), respectively, into the cultured medium. effects of xenobiotics on the changes of protein level of bmhemolin in the silkworm midgut following the same procedure as described above, on the third day of the fifth instar, the larvae were injected with inactive e. coli, bmnpv, pgn, lps and pbs. at 9, 24, and 48 h post injection, total proteins were extracted from the midgut tissues. western blot analysis was performed using polyclonal antiserum anti-bmhemolin as the primary fig. 3 qpcr analysis for the transcriptional changes of bmhemolin from stimulants challenged midgut at larval stage. a qpcr was performed at 9, 24, and 48 h, respectively, from stimulants challenged midgut tissues of silkworm at the fifth instar larvae. expression values were normalized to those of α-tubulin using the livak (2 -△△ct) method the data were provided as the mean fold changes (means ± sd, n = 10) relative to the control group (pbs). the injected stimulants shown on the horizontal axis are pbs as control, bv virions (bmnpv), inactivated e.coli (dh5α), peptidoglycan (pgn), and lipopolysaccharide (lps), respectively. 334 330 fig. 4 western blot analysis for the expression of bmhemolin protein from stimulants challenged midgut at larval stage. parallelly, a western blot analysis was performed at 9, 24, and 48 h from stimulants challenged midgut tissues of silkworm at the fifth instar larvae, respectively, as described in “materials and methods” section. numbers on the left indicate a protein marker in kda. the injected stimulants at the bottom are inactive e. coli (dh5α), bombyx mori nuclear polyhedrosis virus (bmnpv), lipopolysaccharide (lps), and peptidoglycan (pgn), respectively antibody to detect bmhemolin protein changes in the silkworm midgut tissues. the results are shown in figure 4. during 48 h post injection of inactive e. coli, bmhemolin protein levels were significantly increased at different time points (9, 24, and 48 h) (fig. 4a). however, there was no any bmhemolin protein to be observed by the injection with b. mori npv (fig. 4b). injection of lps or pgn resulted in a moderate increase in bmhemolin expression (figs 4c, d). these results are consistent with the observations on the changes of mrna levels after xenobiotics injections in which the stimulating effect of inactivated e. coli was much higher than that by lps or pgn injection. interaction between bmhemolin and bmyippee in the wing disc tissue the interaction of bmhemolin and bmyippee in b. mori wing disc tissue was verified by co-ip assays. immunoprecipitation with anti-bmhemolin or anti-bmyippee antisera was performed using total tissue lysates from wing disc, respectively, as shown in figure 5. the carefully washed immunoprecipitates were then subjected to sds-page electrophoresis. as indicated by black arrows on coomassie blue-stained gel, a separated band from immunoprecipitates with anti-bmhemolin antisera corresponding to the position of bmyippee protein (fig. 5a, lane 1) or the one from immunoprecipitates with anti-bmyippee antisera corresponding to bmhemolin (fig. 5b, lane 1) was excised using a sterile scalpel blade and followed by maldi-tof mass-spectrometric analysis (shanghai applied protein technology co. ltd, shanghai, china). two bands were identified as bmhemolin and bmyippee (data not show), implying that there is a direct interaction between two proteins in the wing tissue. to further verify the above results, the western blot analysis was performed. the immunoprecipitates from total wing disc tissue lysates with anti-bmhemolin antisera or with anti-bmyippee antisera were run on a 12 % sds-page gel, separately. the separated proteins were then transferred onto a nitrocellulose membrane. the anti-bmyippee antiserum was used as a primary antibody to detect bmyippee protein from co-immunoprecipitates with anti-bmhemolin antisera. parallelly, an anti-bmhemolin antiserum was used to detect bmhemolin protein from co-immunoprecipitates with anti-bmyippee antisera. correspondingly, a single band of bmyippee or bmhemolin protein was detected as shown in figures 5c and d. therefore, there is an interaction between hemolin and yippee proteins in the wing disc of silkworm. rna interference of hemolin in the wing disc at pupal stage the expression of bmhemolin gene after rna interference the sirna solution was injected into each pupa at the position of wing disc. after 72 h, the total rna was extracted and the changes of bmhemolin transcription were determined by qpcr. in an initial round of experiments, the injection of a high dose (8 μg/pupa) led to death of almost all the pupae. therefore, an injection with a low dose (2 μg/pupa) or mid dose (4 μg/pupa) was applied in subsequent experiments. the qpcr results for bmhemolin gene transcription in the pupal stage are shown in figure 6a. comparing with the negative control group (lane n), strangely, there were no significant effects on bmhemolin gene transcription by the injection of 2 μg or 4 μg per pupa with three different pairs of specific bmhemolin-sirna (lanes 1 3). as shown in figure 6b, the protein levels of bmhemolin after injection of sirnas in the pupa wing disc were also determined by western blot analysis. comparing with the control group (lane 1), the protein levels were slightly decreased by a 2-μg/pupa injection with either pair 1 (rnaibmhem-1) or pair 2 (rnaibmhem-2) of specific bmhemolin-sirna (lane 2 and 3). interestingly, the protein levels were dramatically decreased to almost 335 http://dict.cn/parallelly 330 fig. 5 co-ip assays for the interaction of bmhemolin and bmyippee in the wing disc tissue. a and b: coomassie blue stained sds-page for co-ip assays. a) co-ip assays were performed using total tissue lysates from wing disc with mouse anti-bmhemolin polyclonal antisera (lane 1) in which a mouse monoclonal antibody anti-vta1 was used as the control (lane 2). b) a revised co-ip was performed using total tissue lysates from wing disc with rabbit anti-bmyippee polyclonal antisera (lane 1). c and d) western blot analysis for co-ip. the immunoprecipitates with mouse anti-bmhemolin antisera or with rabbit anti-bmyippee antisera were then followed by immunodetection with rabbit anti-bmyippee antisera (c) or with mouse anti-bmhemolin antisera (d). lane m indicates protein marker in kda. the positions of bmhemolin and bmyippee proteins are marked by arrows. undetectable levels by a 4-μg/pupa injection of either pair 1 or pair 2 sirna (lane 6 and 7). for pair 3 sirna, both 2-μg and 4-μg injection per pupa did not lead to significant decreases in protein levels (lane 4, 8). therefore, the injection of 4-μg sirnas of pair 1 or pair 2 to each pupa was applied to observe the effects of decreased bmhemolin on the development of adult wings. phenotypic changes in adult wings after rna interference apart from those sirna-injected pupae that were collected at 72 h post injection for qpcr and western blot analysis, the rest of them continuously grew and eventually hatched into moths, and the phenotypic changes in adult wing were observed. by a low dose of injection (2 μg/pupa) with three different pairs of sirnas, no significant changes of adult wing were observed. however, by an injection of 4 μg/pupa dose with pair 1 or pair 2 sirna, the developed wings were not normal while there were no any phenotypic changes in other body parts. most of them tended to be in a short, shrinked, and unopened state, in which the injection of pair 2 was found more effective than that by the injection of pair 1 (fig. 7). among 20 hatched moths by the injection (4 μg/pupa) of pair 2 sirna, 8 moths exhibited abnormal wing development. from a repeated experiment using 10 pupae by pair 2 sirna injection (4 μg/pupa), 5 hatched moths were presented as abnormal, strongly suggesting that bmhemolin plays an important role in adult wing development. as for 4-μg/pupa dose of injection with pair 3, the adult wings did not change significantly compared with the negative control group. 336 330 fig. 6 rna interference of the bmhemolin gene. a) the relative expression of bmhemolin mrna at 72 h post injection of sirna. a sirna (2 or 4 μg) specific to bmhemolin was injected into each pupa at the position of wing disc. the expression levels were assessed using sirna random sequences for normalization. lane n, 1, 2, and 3 indicate that the injected sirna was random sequences, pair 1, pair 2, and pair 3, respectively, as described in table 1. b) western blot analysis of the bmhemolin protein levels in response to rnai by an injection of specific bmhemolin-sirna at 72 h. m, protein marker in kda. lane 1 4, 2-μg injections with random sequences, pair 1, pair 2, and pair 3, respectively. lane 5 8: 4-μg injections with the same sirna as that of 2-μg injections. the α-tubulin protein was used as a loading control. discussion in insects, innate immunity and development often share the same molecules, for example, sapecin and lectin protein in sarcophaga peregrina. they are up-regulated upon pathogen stimulation, mainly expressed in the fat body which is functional equivalent to mammalian liver and/or kidney and in some hemocyte species. meanwhile, they are also found to be expressed at specific developmental stages (natori, 2010). these proteins seem to play two independent roles. hemolin is such a kind of dual-function molecule. however, the addition of inactivated e. coli, bacterial cell wall components, lps and pgn, into cultured medium did not stimulate the expression of hemolin gene in bmn cells in this work (fig. 2). therefore, the pathogen cannot directly stimulate hemolin expression in cultured bmn cells. normally, hemolin works as a pattern recognition protein for identifying the infection source via cell signal transduction, and is engaged in phagocytosis to eliminate xenobiotics. it is proposed that hemolin is stimulated by exogenous pathogen in fat body and then released into the hemolymph where hemolin protein is capable of identifying the bacterial cell wall components, lipopolysaccharide and peptidoglycan, and gathering pathogenic microorganisms to eliminate pathogens (yu and kanost, 2002). truly, our studies confirm that the expression of hemolin gene could be up-regulated by the injection of inactivated bacteria or bacterial cell wall components (lps and pgn) into the midgut of fifth-instar silkworm larvae (fig. 3). as for lps and pgn injection, the up-regulation of hemolin gene expression was higher in the pgn injection group than that in lps injection group. a possible explanation is the high molecular weight of pgn. alternatively, an amino acid moiety in side chain, a part of the short peptide in pgn, serves as an easily recognizable pattern for the insect immune system, and therefore, results in a strong induction of hemolin gene expression. also, hemolin plays an antiviral role in some species, but in some other species, it has no effect on viral invasion. for instance, it is involved in antiviral resistance in bombycoidea, but not in noctuoidea (terenius et al., 2009). our experiment indicates that the b. mori nuclear polyhedrosis virus did not stimulate the up-regulation of hemolin gene expression in the midgut of fifth-instar silkworm larvae (fig. 3). the first identification of drosophila yippee as an interaction protein of h. cecropia hemolin is from the proteins encoded in a drosophila imaginal disc cdna library (roxstrom-lindquist and faye, 2001). therefore, we applied co-ip assay, using our prepared two polyclonal antisera with a high sensitivity and specificity in detection of endogenous bmyippee and bmhemolin (fig. 1), to verify their interaction in silkworm wing discs. our study demonstrates that there is a direct interaction between two proteins in the wing disc of silkworm (fig. 5). 337 330 fig. 7 morphological changes in adult wings after rna interference. the pupae were injected by 4-μg pair 1 or pair 2 of sirna and eventually hatched into moths. panel 1, no sirna injection as the control (a normal silkworm wing development state). panel 2, 4 μg of sirna injection with pair 1. panel 3, 4 μg of sirna injection with pair 2. the effects of hemolin on the wing disc development were further investigated in silkworm pupae by preliminary sirna interference. as expected, hemolin protein levels were dramatically decreased by an injection of 4 μg/pupa dose with pair 1 or pair 2 sirna in pupal stage (fig. 6, lanes 6, 7), which leads to subsequent adult wings hypoplasia in most hatched moths (fig. 7, panels 2, 3). it strongly suggests that hemolin plays a crucial role in adult wing development of silkworm. the puzzling observation was that the fluorescence qpcr results showed no significant reduction in bmhemolin gene transcription by sirna interference in pupal stage although a slight decrease was observed by the injection with pair1 or pair 2 sirna (fig. 6a). one possible reason is that our samples were taken from the parts of pupae which are not separated from the wing tissue. therefore, further experiments are required by the use of a sample specially isolated from wing tissues to confirm it. both the protein and gene structure of hemolin are similar to cell adhesion proteins (kanost et al., 1994; bettencourt et al., 1997). in fact, hemolin can function as a cell adhesion molecule in the immune system. but further experiments are needed to elucidate the role of hemolin in the development of insects to see whether the hemolin gene is associated with tissue reconstruction and how to play a functional role in insect development. thus, our works will be significant in studies on the functional role of hemolin in silkworm immunological and developmental processes in the future, enriching our knowledge of the immune regulation and mechanisms of insect metamorphosis. conclusions hemolin can work as a pattern recognition protein for identifying the infection source via cell signal transduction, and is engaged in phagocytosis to eliminate xenobiotics; hemolin plays an important role in development of wing disc of silkworm. acknowledgements this work was supported by national natural science foundation of china (31101673, 31372259); natural science and youth support foundation for universities of jiangsu (1281330023, 5621330001); natural science foundation of jiangsu province (bk20140539) and funded by the priority academic program development of jiangsu higher education institutions (papd). we wish to thank dr. yajing z for reorganizing and revising the manuscript. references bettencourt r, lanz-mendoza h, lindquist kr, faye i. cell adhesion properties of hemolin, an insect immune protein in the ig superfamily. eur. j. biochem. 250: 630-637, 1997. bettencourt r, terenius o, faye i. hemolin gene silencing by ds-rna injected into cecropia pupae is lethal to next generation embryos. insect mol. biol. 11: 267-271, 2002. daffre s, faye i. lipopolysaccharide interaction with hemolin, an insect member of the ig-superfamily. febs lett. 408: 127-130, 1997. faye i, pye a, rasmuson t, boman hg, boman ia. insect immunity. 11. simultaneous induction of antibacterial activity and selection 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insects as a pattern-recognition receptor. eur. j. biochem. 269: 1827-1834, 2002. zuo h, li h, wei e, su z, zheng j, li c, et al. identification and functional analysis of a hemolin like protein from litopenaeus vannamei. fish shellfish immunol. 43: 51-59, 2015. 339 189 isj 16: 189-208, 2019 issn 1824-307x report of meeting third international symposium ammr 2019. advances in marine mussel research, august, 26–28, 2019, palazzo grassi, chioggia, italy organizers: l ballarin, u rosani and p venier (chair) international committee: n bierne, á pérez diz, a figueras, m gerdol, b novoa, a pallavicini, u rosani and p venier total 58 delegates, from 14 countries and 30 different cities of the world, gathered in chioggia (ve) in the occasion of ammr 2019. the event was organized in partnership by the university of padova, department of biology and school of biosciences, under the patronage of veneto region, the town of chioggia and the horizon 2020 project vivaldi (see other sponsors at https://www.biologia.unipd.it/ammr-2019). news and views have been exchanged on i) genetic mussel traits, including a significant molecular evidence of gene presence/absence variation, ii) population markers and evolution of the mytilus species complex, iii) functional mussel responses in relation to potential pathogens and other factors. innovative experimental strategies applied to marine mussels as well as mussel contributions in goods and services confirm these fascinating marine invertebrates as a model of study and strategic resource for the future. the next ammr symposium is expected in 2021 in poland! oral presentations session 1. news and views on genetic and functional traits of the mediterranean mussel chairpersons: beatriz novoa, angel e. perez diz advances in genomics and immunity of mytilus galloprovincialis a figueras1, m gerdol2, r moreira1, m sendra1, b novoa1 1institute of marine research (iim), national research council (csic), eduardo cabello, 6, 36208, vigo, spain 2department of life sciences, university of trieste, via giorgieri 5, 34127, trieste, italy the mediterranean mussel (mytilus galloprovincialis) is a marine invasive species cultured all over the world andd also an appreciated resource in local aquaculture enterprises because of their robust production and resilience that translates into a reliable economic value. the sequencing of the mussel genome has revealed a very complex organization with high heterozygosity, abundance of repetitive sequences and extreme intraspecific sequence diversity among individuals, mainly in immune related genes. among those genes, antimicrobial peptides are the most expressed gene families in mussels, highly polymorphic and with antimicrobial effect against molluscs pathogens, but also against pathogens of lower vertebrates and humans. the combination of a complex genome with the adaptation of mussel immune system to a changing environment could explain this high variability, not only in immune-related genes, but also in the functional response among individuals sampled in the same location and date. disentangling the impact of pav by high throughput sequencing. implications for the interpretation of gene expression data m gerdol1, p venier2, a figueras3, a pallavicini1 1department of life sciences, university of trieste, trieste, italy 2department of biology, padova, italy 3consejo superior de investigaciones científicas (csic), vigo, spain recent genomic investigations have revealed that the mussel genome is characterized by an extreme level of intraspecific genetic variation, far superior to other marine invertebrates. while the high frequency of single nucleotide polymorphisms can in part explain the high heterozygosity rates observed in mussels, the most significant contribution to inter-individual genetic variability appears to be due to hemizygosity, i.e. to the presence of large genomic regions found in just one out of the two homologous chromosomes. unexpectedly, such “haploid” regions are not limited to intergenic sequence, but they include a high number of protein-coding genes, which are be consequently present in a single copy in the diploid genome. this unusual genomic architecture determines the presence of a set of “core genes”, invariably 190 present in all individuals, and underpins gene presence/absence variation (pav), as the result of the crossing between individual bearing different haplotypes. genes subject to pav are “dispensable” (i.e. they can be completely absent in some individuals) and they cover accessory functions, which appear to be mostly related with immune recognition and survival. this finding has profound implications for the interpretation of gene expression data derived from large-scale (i.e. rna-sequencing and microarray), as well as from sequence-targeted (i.e. q-pcr) approaches. indeed, the extreme mussel genetic heterogeneity now emerges as a key factor that should be taken into account since the very early phases of experimental design of genetic and molecular investigations. unfortunately, this aspect has been nearly completely ignored over the past two decades, often hampering the correct interpretation of gene expression profiles. we discuss a few key examples of gene families that have been recurrently, but incorrectly, implicated in the response to a multitude of different biotic and abiotic stimuli, showing that such results are artefacts related to the intrinsic dispensable nature of the investigated sequences. we also propose some guidelines for a correct and balanced interpretation of mussel gene expression data in future investigations, suggesting the use of conservative approaches that should always take into account as a paramount concern the extreme levels of inter-individual genetic diversity. innate immune memory in mussel (mytilus galloprovincialis): tolerance of hemocytes after a repeated contact with vibrio splendidus b novoa1, m rey-campos1, r moreira1, m gerdol2, a pallavicini2,3, a figueras1 1institute of marine research (iim), csic. eduardo cabello 6, 36208, vigo, spain 2department of life sciences, university of trieste, via giorgieri 5, 34127, trieste, italy 3istituto nazionale di oceanografia e di geofisica sperimentale – ogs, via auguste piccard, 54, 34151 trieste, italy mediterranean mussels (mytilus galloprovincialis) are sessile filter feeders that live in close contact with numerous marine microorganisms. as all invertebrates, mussels lack an adaptive immune system but they respond to pathogens, injuries or environmental stress in a very efficient manner. however, it is not known if they are able to modify their immune response when they reencounter the same pathogen. in this work we studied the transcriptomic response of mussel hemocytes before and after two consecutive sublethal infections with vibrio splendidus. the first infection significantly regulated genes related to inflammation, migration and response to bacteria. however, after the second infection, the differentially expressed genes were related to the control and inhibition of the ros production and the resolution of the inflammatory response. our results also show that the second infection with v. splendidus led to changes at transcriptional (control of the expression of pro-inflammatory transcripts), cellular (shift in the hemocyte population distribution) and functional (inhibition of ros production) levels. these results suggest a modified immune response of mussels after the second challenge oriented to tolerate and not to fight the infection, minimizing tissue damage. what does macrophage migration inhibitory factor do in mytilus galloprovincialis? u rosani1,2, s domeneghetti1, m gerdol3, f vallese4, e bortoletto1, g zanotti4, a pallavicini3, r tavano4, p venier1 1department of biology sciences, university of padova, padova, italy 2alfred wegener institute, waddenseastation sylt, list auf sylt, germany 3department of life sciences, university of trieste, trieste, italy 4department of biomedical sciences, university of padova, padova, italy the presence of cytokines in invertebrates has been debated for a long time. nowadays, it is clear that ancestral cytokine genes were present before the radiation of deuterostome, although their functional roles remain mostly elusive. macrophage migration inhibitory factor (mif) is a pleiotropic cytokine initially described as a proinflammatory factor in early 1930s. depending on the functional context and cell type, human mif can promote the development of inflammatory and malignant diseases or play a protective role supporting resistance to various infections. the current data on mif receptors and downstream signaling pathways reveal how intricate and multifaceted is the mif-mediated modulation of cell processes. hence, human mif has been renamed ‘most interesting factor’. searching for mif-like sequences in genomic and transcriptomic data of bivalves, we retrieved 148 sequences, with most of the analyzed bivalve species encoding two mif paralogues, namely one mif and one d-dt gene. we traced the genomic expansion of d-dt genes in the mytilidae family, describing 10 mif-like genes in the mussel m. galloprovincialis. the analysis of rna-seq and rt-qpcr data indicated enhanced tissue-specific expression of mif-like genes, possibly underpinning neofunctionalization of certain isoform in highly specialized mussel tissues, like the byssus. conversely, expression data scarcely supported the involvement of the mussel mif in innate immune responses. we produced a recombinant m. galloprovincialis mif protein (mgmif) in pichia pastoris and we tested its effects in mussel hemocytes as well as in human macrophages for comparison. finally, we verified if mgmif conserved the enzymatic functions typical of vertebrate mif and d-dt proteins, namely oxidoreductase and dopachrome methyl ester decarboxylase into 5,6dihydroxyindole (dhi). 191 proteomics analysis of sperm samples from two different mytilus species including hybrids a perez diz1, mr romero1, wj swanson2, dof skibinski3 1department of biochemistry, genetics and immunology, university of vigo, vigo, spain 2department of genome sciences, school of medicine, university of washington, seattle, usa 3institute of life science, swansea university medical school, swansea university, swansea, uk the study of the mechanisms that lead to the formation of new species is of special interest in marine ecosystems due to the lack of obvious barriers to gene flow. mussels of the genus mytilus are marine organisms with external fertilization able to hybridize where the distribution of two species overlap, allowing the study of reproductive isolation mechanisms in a natural scenario. because the formation of hybrids is so frequent between mytilus spp., it is likely that different types of reproductive barriers might be playing a role to preserve the genome integrity of each species, though the relative contribution and underlying molecular mechanisms of each are poorly known at present. choosing gametes as a direct research target is key in order to investigate about specific adaptations that, for example, enhance gamete performance and fertilization success during intraspecific rather than interspecific crosses, hence also helpful to elucidate the molecular mechanisms underlying reproductive isolation. comparative results from ongoing shotgun proteomics analysis on sperm samples from pure m. galloprovincialis and m. edulis mussels, including a few wild viable f1hybrids, will be presented and discussed from an evolutionary ecology viewpoint. immunostimulating mytilus galloprovincialis hemocytes: description of transcriptomic, mirnomic and functional capacities r moreira, a romero, m rey-campos, b novoa, a figueras institute of marine research (iim), national research council (csic), eduardo cabello 6, 36208, vigo, spain mediterranean mussels (mytilus galloprovincialis), as all invertebrates, do not possess adaptive immune response. however, the high resilience of mussels to biotic and abiotic stress is one of the reasons why this species is so interesting to study processes such as immune response. in this work, we sequenced the hemocytes transcriptome and mirnome to study the molecular basis behind this innate immune response. we stimulated the hemocytes with poly i:c, β-glucans, and lps to study the hemocytes response to these pamps. an immune transcriptome comprising 219,765 contigs was obtained after the assembly of all the available samples and around 20% of these contigs were identified. after discarding mirnas with very low expression, a total of 1,550 mirnas were identified. the expression analyses showed interesting and opposite results in the transcriptome and mirnome: lps was the stimulus which triggered the transcriptomic response with 648 differentially expressed genes (degs), meanwhile the mirnomic response was led by poly i:c, which triggered the expression of 240 mirnas. to further study the sequencing results we performed functional approaches. viability assays showed that the treatment with these pamps did not cause changes in hemocyte count 24hours after the treatment. in the short term, lps and poly i:c increased phagocytosis and glucans were able to trigger the ros production. in the long term, the no production was increased after poly i:c stimulation. pamps also affected the hemocytes morphology and mobile behavior: cells treated with poly i:c showed rounded morphology with condensed cytoplasm and increased their velocity over 20% when compared to controls. the dark matter of the mussel’s genome: long non-coding rnas as key players in mytilus c gallardo-escárate1, a figueras2, b novoa2 1interdisciplinary center for aquaculture research (incar), universidad de concepción, concepción, chile 2instituto de investigaciones marinas (iim), csic, vigo, spain. the complexity of the genomes in marine organisms and their functional information have been defined for only a small proportion of them. here, close to ∼1% of the genome is transcribed into protein-coding (mrna) and non–protein-coding (ncrna), where dna elements controlling the gene expression involves ∼0.5%. these facts suggest that the genome in marine species is dominated by a “dark matter,” which is mostly nonfunctional. however, this portion of the dna is pivotal for the evolutionary process and greatly exploited in bivalves such as mussel to face with the continuously changing marine environment. this study aimed to explore the role of ncrnas in mytilus, reviewing the functional implications of long non-coding (lncrnas) in the immune response of mussels exposed to pathogens and habs, and how ncrnas are modulated in individuals exposed to contrasting environments and also to marine pollution such as microplastics. interestingly, the analysis of lncrnas revealed that these transcripts are involved in relevant biological processes such as immune system and local adaptation in mytilus. herein, a comparative transcriptome analysis was conducted between m. galloprovincialis and m. chilensis. our results provide the first identification of lncrnas in mytilus and evidence that lncrnas are key players in the biology of mytilus. distribution and features of dui in mytilidae a burzynski, b smietanka, m lubosny department of genetics and marine biotechnology, institute of oceanology pan, sopot, poland double uniparental inheritance (dui) of mitochondrial dna was first discovered in mytilus 192 mussels. ongoing debate on the origin and age of this phenomenon is hindered by the lack of firm knowledge about its taxonomic span. here we will present mitogenomic data covering the whole mytilidae family, revealing a complex pattern of dui – related features. session 2. the mytilus species complex in a changing environment: from the past to the future chairpersons: cynthia riginos, nicolas bierne mussel melting-pots: hybridization and admixture in the mytilus edulis complex of species n bierne institut des sciences de l’evolution de montpellier, university of montpellier-cnrs-ephe-ird, montpellier, france the mytilus edulis complex of species has become a flagship system for the study of hybridization and genetic admixture in the sea. we have studied natural zones of contact between species of the complex and have demonstrated extensive introgression through semi-permeable species barriers, reconstructed the history of divergence and secondary gene flow, and started to understand the isolation factors at play to maintain the integrity of species genomes. extensive sampling and surveys of new previously unstudied areas have recently allowed to evidence new zones of contact between native and non-native lineages introduced via human activities (mainly maritime traffic). i will present and compare the patterns of admixture observed in natural hybrid zones, anthropogenic hybrid zones and lab crosses experiments. this comparison of independent admixture events revealed parallel patterns of admixture that suggest a highly polygenic determinism of reproductive isolation in mytilus mussels. anthropogenic hybridization reshuffles genetic backgrounds which might open new evolutionary routes for invasive populations. twin introductions by divergent mytilus galloprovincialis lineages are associated with high levels of genetic admixture with a native congener in australia i popovic1, a matias1, n bierne2, c riginos1 1school of biological sciences, university of queensland, st lucia, queensland 4072, australia 2institut des sciences de l’evolution umr 5554, université de montpellier, cnrs-ird-epheum, france introduced species can impose profound impacts on the evolution of receiving communities with which they interact. if native and introduced taxa remain reproductively semi-isolated, humanmediated secondary contact may promote genetic exchange across hybrid zones, potentially impacting native genetic diversity and invasive species spread. here, we investigate the contributions of past and ongoing (post-introduction) gene flow between the invasive marine mussel, mytilus galloprovincialis and a morphologically indistinguishable and taxonomically contentious native australian taxon, mytilus planulatus. using transcriptome-wide markers, we demonstrate that two contemporary introductions of m. galloprovincialis into southeastern australia originate from genetically distinct lineages from its native range in the mediterranean sea and atlantic europe. contingent on resolving species relationships, we also provide strong evidence that both introductions are associated with high levels of recent admixture with the native genomic background. increased resolution of genomic species relationships and demographic modelling validated that m. planulatus sampled in tasmania are representative of the endemic australian taxon but share a strong genetic affinity to northern m. galloprovincialis. demographic inferences indicated latepleistocene divergence times and low levels of historical gene low between the tasmanian endemic lineage and northern m. galloprovincialis, suggesting historical isolation between native and introduced taxa of at least 100,000 years prior to present day contact. accurate species identification, however, will require the use of many genetic markers, presenting significant impediments for rapid and efficient identification of introduced and endemic populations. the results of this study build upon previous genetic studies investigating m. galloprovincialis introductions and its interactions with endemic southern hemisphere lineages. our findings also demonstrate the utility of genomic data for detangling contemporary invasive introgression from signatures left by historical gene flow and recent divergence histories in native and introduced marine taxa when species boundaries are not well-defined. variable patterns of hybridization and locusspecific introgression in hybrid zones of mytilus edulis and m. trossulus assessed by the traditional set of markers p strelkov1, m skazina1, m katolikova1,2, a gagarina1,3, a ivanova1, r vainola4 1st. petersburg state university, st. petersburg, russia 2murmansk marine biological institute, murmansk, russia 3zoological institiute russian academy od sciences, st. petersburg, russia 4finnish museum of natural history, university of helsinki, helsinki, finland different hybrid zones between the same lineages can vary in extent of hybridization and locus-specific introgression. in blue mussels it was long ago demonstrated on the example of zone between mytilus edulis and m. trossulus in the baltic sea, where both hybridization and introgression at some but not all the markers appeared to be extensive. here we (1) review literature and original data on the genotypic structure of samples from five different zones between m. edulis and m. trossulus 193 at historically most commonly used taxonomic molecular markers (allozymes and pcr-based me 15-16, efbis, its, mal), (2) assess variation among zones in the extent of hybridization, (3) calculate allele frequencies in regional sympatric populations of hybridizing species and estimate the rate of introgression into their gene pools. our direct comparisons demonstrate that not only the baltic zone, but all of them have individualistic features and that most popular pcrbased “diagnostic” taxonomic markers are not universally informative, in contrast to orthodox allozymes. this should affect the performance of traditional method of taxonomic identification of mussels when mussels are identified by their genotypes characterized by one or few markers from the same limited set. this approach implies that diagnostic loci are universally diagnostic while backcrossing is always limited, which is not true for most hybrid zones considered. the research was supported by rsf (project no. 19-74-20024). a farewell to allozymes? reanalysis of the classical dataset on blue mussels (mcdonald, seed, koehn 1991) m katolikova1,2, p strelkov1,3 1st. petersburg state university, universitetskaya emb. 7/9, st. petersburg 199034, russia 2murmansk marine biological institute, kola scientific center, russian academy of sciences, vladimirskaya str. 17, murmansk 183010, russia 3murmansk arctic state university, kapitana egorova str. 16, murmansk 183038, russia development of electrophoretic techniques for separating enzyme allelic variation triggered a dramatic recovery of the empirical population genetics in the 1970s. mussels mytilus were among the model objects for allozyme-based population genetic studies. these studies, firstly, played a remarkable role in the establishment of a global scientific trend to consider natural selection as a predominant cause of spatial population divergence (e.g. salinity-associated variation at lap locus). secondly, they were to a large part responsible for substituting this trend by historical divergence and secondary contact paradigm, postulating the existence of three historical lineages of mytilus: m. edulis, m. trossulus and m. galloprovincialis and describing their world-wide distribution and population structuring. since that time, numerous genetic methods exploiting different types of molecular markers have been developed for taxonomic and population genetic purposes. a wide range of effective statistical tools has also evolved, allowing a more thorough analysis of genetic data. this helped to reveal hitherto unknown spatial genetic patterns in marine organisms, mussels in particular. against the background of this technological and informational leap, there has been a rising skepticism concerning the reliability of allozymes as molecular markers. direct calls to disregard findings inferred by allozymes have even been heard. in our talk we stand up for allozymes and demonstrate their validity as neutral markers. we claim that allozyme loci can provide accurate information on taxonomy, population structuring and hybridization between ancient lineages in mussels. noteworthy, primary indication of genetic differentiation between divergent mussel lineages was recognized using a multi-locus analysis of allozyme genes. we consider the old allozyme data against the contemporary background by reanalyzing the initial data set from one of the basic papers addressing m. edulis, m. trossulus and m. galloprovincialis differentiation and distribution (mcdonald j., seed r., koehn r., 1991. allozymes and morphometric characters of three species of mytilus in the northern and southern hemispheres, marine biology, 111, 323-333, https://doi.org/10.1007/bf01319403). we use a model-based statistical technique implemented in structure (pritchard et al., 2000) to give a better resolution of this classical example of allozyme based population study and to compare the results with the findings obtained by other types of genetic markers in the last 25 years. we are deeply grateful to dr. john mcdonald, who kindly shared with us the precious historical data. the research was supported by projects аааа-а19-119011690138-0 by mes of russia and 19-74-20024 by rsf. adaptive potential in a changing ocean: unraveling the genomic architecture of a climate stress response in blue mussels (mytilus edulis and m. trossulus) in the gulf of maine se kingston, p martino, db carlon department of biology and schiller coastal studies center, bowdoin college, brunswick, u.s.a. as coastal marine environments change rapidly, sessile habitat-building benthic species populations will either adapt or face local extinctions. in an effort to assess the adaptive potential of sentinel marine calcifiers in the gulf of maine, we used next-generation sequencing techniques to map the response to multivariate climate stress in wild populations of blue mussels, mytilus edulis and m. trossulus. genotype-bysequencing methods were employed to assay tens of thousands of loci across the genomes of individual mussels (n=655) also phenotyped for calcification rate under predicted future climatestress (-0.3 ph units, +3 ºc, and limited food availability). rna-seq was used to estimate changes gene expression levels under the predicted future climate-stress (n=24). we leveraged genome-wide association models to infer the underlying genomic architecture of blue mussel calcification under future multivariate climate stress. the stress calcification trait is heritable (0.19 < h < 0.41, 95% ci) and likely associated with 2-10 genomic regions; 208 transcripts demonstrate differential expression in future climates stress conditions. upregulation of heat shock proteins, constituents of metabolic pathways, and membrane processes (including ion binding) appear to be 194 important components of the stress response at the expression level. genetic variation associated with resilience to environmental stressors associated with climate change predicted over the next 50-100 years does exist in extant blue mussel populations. further exploration of this response at the juvenile life stage and through multiple generations of climate stress exposure will further inform predictions of population persistence in the face of a rapidly changing environment. analyses of distribution and long-term dynamics of sympatric mussels mytilus edulis and m. trossulus in the white sea using the semi-diagnostic conchological marker v khaitov1,2, m katolikova3, a zaichikova4, p safonov1, t korotkova1, y marchenko1, p strelkov1 1biological faculty, saint-petersburg state university, saint-petersburg, russia 2kandalaksha state nature reserve, kandalaksha, russia 3murmansk marine biological institute, kola scientific center, russian academy of sciences, murmansk, russia 4biological faculty, moscow state university, moscow, russia blue mussels m. edulis (me) and m. trossulus (mt) form mixed populations and hybrid zones in the north atlantics. dynamic of these systems are poorly understood. mussels exhibit two discrete morphs: t-morphotype (tm, underdevelopment nacreous layer under the ligament nympha) and emorphotype (em, nacreous layer is well developed). we investigated the association between morphotype and species genotype in the white sea and revealed that mussels with tm are mostly mt whereas mussels with em me. we further analyzed current (2013-2018) distribution of morphotypes along the gulf of kandalaksha and inspected old collections from the area, in particular annual samples from four monitoring sites in kandalaksha harbor area obtained through 20022018. populations with high concentration of tm were from areas associated with active or abandoned sea ports. mussels with tm were rare in collections sampled before 2000. rapid rise in tm frequencies in populations of kandalaksha harbor area happened between 2002 and 2014. however, tm frequencies experienced some decreasing therein last years. high concentration of tm in areas associated with sea ports is in a good agreement with the hypothesis of introduction of mt into the white sea by oceanic transport. rapid expansion of tm around kandalaksha harbor in the very beginning of the century we relate to the massive freshwater discharge from local hydroelectric power plant in 2000, which was catastrophic for marine intertidal communities. after the extinction of intertidal mussel populations, the areas were colonized by more opportunistic mt. recent decline in tm frequencies we relate to competitive exclusion of mt by me in more stable hydrological situation in the area in last years. the research was supported by rsf (project no. 19-74-20024). hybridisation in a future ocean: a mechanism for adaptation of mussels to multiple environmental challenges rp ellis1, rd houston2, ma urbina3, ae todgham4 1department of biosciences, university of exeter, exeter, uk 2the roslin institute and royal school of veterinary studies, university of edinburgh, uk 3departamento de zoología, universidad de concepción, chile 4department of animal sciences, university of california, davis, usa climate change is one of the greatest challenges facing society in the 21st century, widely considered as the most pervasive threat to global marine biodiversity, as well as the predominant factor determining the value of goods and services that marine ecosystems will continue to provide. nonetheless, despite an exponential increase in research addressing this topic over the past decade, our understanding of ecosystem level responses remains limited by a significant predominance of single species, short term, single stressor experiments. adaptive capacity of longer lived, marine metazoan groups, across relevant timescales, however remains unresolved. the marine mussel complex has a unique global biogeography, offering a natural analogue for determining adaptive capacity to climate change stressors and the role hybridisation plays in genetically determining physiological tolerance to abiotic stress. during a 3.5-year fellowship high-throughput next-generation sequencing technology is being employed to sequence 24 mussel populations from across their global range (12x eu pops.; 6x californian pops. and 6x chilean pops.), with populations selected according to prevailing environmental conditions that encompass three key environmental stressor gradients – namely temperature, salinity and elevated co2. this preliminary ascertainment of snps is subsequently being used to develop a high-density genotyping tool (snp array), generating a 50k array that will enable segregation of samples based on mussel speciation, introgression and back-crossing, as well as broad scale global biogeographic population structure, and regional local adaptation. subsequently, by employing this novel technology alongside measures of whole organism physiology and performance the overarching question ‘does hybridisation confer an advantage to multi-stressor exposures in an ecologically and commercially important bivalve species?' will be addressed. this project will therefore develop a unique, industryrelevant, resource that will significantly advance understanding of ecological physiology and evolutionary biology. 195 session 3. mussels and mussel-associated microorganisms in the light of evolution chairpersons: paola venier, k. mathias wegner macrobial and microbial symbionts of mussels. direct and indirect effects of parasite invasions km wegner1, me feis1,2, f demann1, c buschbaum1 1department of coastal ecology, awi alfred wegener institute for polar and marine research, waddensea station sylt, list, germany 2station biologique roscoff, roscoff, france biological invasions often have negative impacts on native biota. this is particularly true if the invasive species is a parasite. blue mussels mytilus edulis in the north sea were invaded as a new host of the parasitic copepod mytilicola intestinalis in the 1930ies starting a new coevolutionary arms race. here, we explore the evolution of parasite and host traits along separate fronts of the invasion and how infection with the new parasite affects host physiology directly. however, next to direct effects, this host-parasite interaction can also have profound indirect effects that feed back on mussel fitness. these include changes of gut microbiota, resistance to secondary infections but also the interactions with epibionts as well as predators indicating that indirect effects can outweigh the direct effects. microbiome diversity in blue mussel (mytilus edulis l.) during larval development s mcmillan1, a desbois1, m crumlish 1, ad hughes2, av laudicella2, j taggart1, m bekaert1, s carboni1 1instutute of aquaculture, university of stirling, stirling, scotland, uk 2scottish association for marine science, oban, scotland, uk global demand for the common or blue mussel (mytilus edulis) continues to increase. one barrier to increased production is the reliable availability of mussel spat. to this end, the saichatch consortium was formed to develop a pilot-scale mussel hatchery, supporting the optimization of various production traits via targeted research. this effort includes investigations on feeding regimes and the monitoring for harmful bacteria. disease outbreaks due to bacterial pathogens are a risk in all intensified aquaculture systems (kesarcodiwatson et al., 2009) but especially within bivalve hatcheries. despite being an essential primary food, marine algae are also an important source of bacteria within a hatchery system (dubert et al., 2015). thus, the aims of this investigation were to evaluate the effects of selected microalgae species on growth and survival of the mussel larvae and describe the evolution of the total m. edulis microbial community abundance and diversity during larval ontogeny and between the different microalgal feeds. five different algal diets (chlorella vulgaris, cylindrotheca fusiformis, monodopsis subterranean, nannochloropsis oceanica, and a mix of chaetocerous calcitans f. pumilus and isochrysis galbana mix) were fed to m. edulis larvae, that otherwise were reared under common conditions. samples from each dietary group were aseptically collected at 2 days post fertilization, and then weekly for 4 weeks, until animals reached settlement. total dna was extracted before libraries of bacterial 16s ribosomal rna hypervariable regions v3-v4 were amplified, indexed and pooled. sequence data was analysed at the family level to generate euclidean similarities calculated from standardised and log-transformed otu abundances. correlations between family relative abundance over time and between treatments were calculated using kendall's tau method (kendall, 1938). sequence data revealed that bacterial populations were not significantly different among diets, except for larvae fed with m. subterranean (r2 = 0.5328, p < 0.001). however, diversity did increase significantly with time (r2 = 0.8965, p < 0.001), suggesting microbiome development is largely endogenous indicating that microbiome composition was affected more by development than diet. to our knowledge this is the first report detailing mussel larvae microbiome diversity during ontogeny and will further our understanding of microbe/host interactions during early development. methods developed here can be applied in future studies to understand the role of the microbiome and what affects it. experimental infection of mytilus edulis by two vibrio splendidus-related strains: evaluation of virulence, the influence of the origin of mussels and season on their sensitivity m charles1, 2, s trancart2, e oden2, m houssin1, 2 1normandy university, university of caen normandy, borea, cnrs-7208, ird-207, mnhn, upmc, ucn, esplanade de la paix, 14032 caen cedex 4, france 2labéo frank duncombe, 1 route de rosel, 14053 caen cedex 4, france in 2014, france was affected by high and unusual mass mortality of mussels affecting several important production areas along the french coast of the atlantic and english channel, where production losses have reached 90 to 100 %. the phenomenon again affected farms on the west coast of france in the first quarter of 2016. the pathogenic bacteria of the genus vibrio has been considered as one of the causes of these events. vibrios are among the major bacterial pathogens of marine organisms and have been identified as pathogens for several bivalve molluscs that may cause high economic losses. during mussel mortality events, different vibrio splendidus strains were isolated from french moribund mussels and were linked to mortality. in this study, two strains from a vibrio population assembly isolated during disease events in oysters (crassostrea gigas) and mussels (mytilus edulis) in 2014, and described as highly virulent for bivalvia, were used for infection assays. 196 pathogenicity in the mussel, m. edulis, of these two vibrio splendidus-related strains – vibrio crassostreae 7t4_12 and v. splendidus 3g1_6 – were tested every month, respectively for a period of 13 and 9 months; in order to determine whether physiological and morphological conditions, impacted by the season, played a role in susceptibility to bacterial infections. two concentrations were tested, one close to the natural concentration of vibrio spp. in seawater (~105 cfu/ml) and another much higher (~108 cfu/ml). in addition, those experiments were conducted on mussels of the same age but from five different areas, in order to verify whether the origin of the mussels' capture could affect their sensitivity to vibriosis. the observed mortalities are significantly different depending on the strain injected and the tested concentrations. vibrio splendidus is more virulent than v. crassostreae at the highest tested concentration and the tested environmental concentration has no pathogenic effect, whatever the used strain. besides, mussels from one specific origin are significantly less sensitive to infection than others. however, the parameter that seems to be the most decisive in the sensitivity of animals to bacterial infection is the season; a criterion known to have a particular impact on the physiological and morphological conditions of the animals. the highest mortalities due to infection were observed in autumn and in spring. reproductive status impacts host susceptibility toward infection; the highest concentration seems to cause a specific stress during the reproductive period leading to forced egg laying, promoting the fragility of mussels and their susceptibility to infections. nanoparticle exposure affects the microbiota composition of mussel hemolymph: possible interplay with the host immune system m auguste1, a lasa,1 t balbi1, a pallavicini2, l vezzulli1, l canesi1 1department of earth, environmental and life sciences, university of genoa, genoa, italy 2department of life sciences, university of trieste, trieste, italy bivalves host high microbial abundance and diversity, and alteration of their microbiota, in both tissues and hemolymph, in response to stressful conditions has been linked to a compromised health status and susceptibility to diseases. the hemolymph associated microbiota has been investigated in oysters in response to pathogens and to temperature stress and infection. in contrast, less information is available in other species of widely farmed bivalves such as mytilus spp., that are generally not affected by mortality events, being less sensitive to changes in environmental conditions and microbial infection. however, mussel microbiota may be affected by exposure to environmental contaminants. the recent expansion of nanotechnology has led to increasing concern on the potential consequences of exposure to nanoparticles-nps for human and environmental health, thus attracting interest on their potential effects on mammalian gut microbiota. in this work, data are presented on the effects of np exposure on hemolymph microbiome of m. galloprovincialis evaluated by targeted highthroughput sequencing of the 16s rrna gene (v4 hypervariable region) using ion torrent sequencing technology. two model nps were utilized: ntio2, one of the most widespread np in use, and amino modified nanopolystyrene (ps-nh2), as a model nanoplastics, that represents an emerging threat for marine ecosystems. exposure conditions were 100 and 10 µg/l, respectively, for 96 h. the results indicate that both ntio2 and psnh2 induced shifts in microbiota composition of mussel hemolymph, with specific effects on abundance of different genera, depending on the np type. the effects were associated with distinct changes in functional immune parameters, indicating an interplay between the host innate immune system and its microbiota. these represent the first data on the effects of np exposure on bivalve microbiome. these studies may represent the basis for better understanding how exposure to both contaminants and natural stressors may shape the interactions between the microbiota and the immune system, and the consequent impact of human activities on the health of aquatic ecosystems. work supported by the eu commission h2020 project pandora (ga 671881). session 4. the weird case of transmissible cancers in mussel species chairpersons: k. mathias wegner, nicolas bierne a single clonal lineage of transmissible cancer identified in two marine mussel species in south america and europe ma yonemitsu1, rm giersch1, m polo-prieto2, a simon3, f cremonte4, ft avilés5, n merinovéliz5, m hammel3, eav burioli6, af muttray7, j sherry8, c reinisch8, sa baldwin9, sp goff2,10, m houssin6,11, g arriagada5, n vázquez4, n bierne3, mj metzger1 1pacific northwest research institute, seattle, wa, 98122, usa 2howard hughes medical institute, chevy chase, md, 20815, usa 3isem, univ montpellier, cnrsephe-ird, montpellier, 34095, france 4laboratorio de parasitología (lapa), instituto de biología de organismos marinos (ibiomar) (cct conicet cenpat), puerto madryn, u9120acf, argentina 5instituto de ciencias biomedicas, facultad de medicina, universidad andres bello, santiago, 2531015, chile 6research and development, labéo frank duncombe, saint-contest, 14280, france 7environmental resources management, vancouver, british columbia, v6e 2j3, canada 8environment and climate change canada, water science & technology directorate, burlington, ontario, l7s 1a1, canada 197 9chemical and biological engineering, university of british columbia, vancouver, british columbia, v6t 1z3, canada 10department of microbiology and immunology, columbia university medical center, new york, ny, 10032, usa 11fre borea, mnhn, upmc, ucn, cnrs-7208, ird-207, université de caen normandie, caen, 14032, france transmissible cancers, in which cancer cells themselves act as an infectious agent, have been identified in tasmanian devils, dogs, and four bivalves. we investigated disseminated neoplasia affecting geographically distant populations of two species of mussels (m. chilensis in south america and mytilus edulis in europe). sequencing alleles from three loci (two nuclear and one mitochondrial) provided evidence of transmissible cancer in both species. phylogenetic analysis of cancer-associated alleles showed that cancers in both species likely arose in a third species of mussel (m. trossulus), but these cancer cells are independent from the previously identified transmissible cancer in m. trossulus from canada. unexpectedly, cancers from m. chilensis and m. edulis are nearly identical, showing that the same cancer lineage affects both. thus, a single transmissible cancer lineage has crossed into two new host species and has been transferred across the atlantic and pacific oceans and between the northern and southern hemispheres. genotype typing of transmissible cancers in the mytilus edulis complex of species m hammel1,2, a simon1, c arbiol1, j-f pépin3, jb lamy3, a benabdelmouna3, i bernard4, a villalba5, e burioli6, m houssin6, g charrière2, d destoumieux-garzon2, j welch7, m metzger8, n bierne1 1isem, univ montpellier, cnrs, ephe, ird, montpellier, france 2ihpe, univ montpellier, cnrs, ifremer, univ perpignan, via domitia, france 3eureka modélisation, brest, france 4santé, génétique, microbiologie des mollusques, ifremer, la tremblade, france 5centro de investigacións mariñas, consellería do medio rural e do mar, xunta de galicia, vilanova de arousa, spain 6labéo, caen, france 7department of genetics, university of cambridge, downing street, cambridge, uk 8pacific northwest research institute, seattle, usa cancer cells are egoist entities that derived from and escaped the immune system controls of a multicellular organism. cancerous cell lines usually become extinct with the death of their host; however some cell lines -called transmissible cancers acquired the potential to infect a new host from the same or even another species. we studied the population genetics of one of those newly discovered transmissible cancers which affect mussels of the mytilus edulis complex of species. a transmissible cancer originating from m. trossulus was discovered to affect populations of this same species in british columbia. moreover, european populations of two other species of the complex, m. edulis and m. galloprovincialis, have been suspected to be the host of a transmissible cancer that also originated from m. trossulus. here, we used snp genotyping to screen a sample of 2500 mussels for genetic chimerism, the hallmark of transmissible cancers. we confirmed the existence of m. trossulus cancer in european mussels but at low prevalence. we then analyzed the genotypes of infected individuals from each species. we observed polymorphism among neoplasias sampled in different individuals suggesting either multiple emergences or deviation from clonal propagation. the cancer genotypes proved to be intermediate between reference samples of the three species. these results suggest that cancer emerged in individuals from a hybrid or an ancestral population. session 5. mussels as biological models for in vitro and in vivo toxicological testing chairpersons: loriano ballarin, laura canesi internalization, uptake routes pathways and effects of polystyrene nanoplastics in hemocytes of mytilus galloprovincialis through an in vitro approach m sendra, a saco-beiroa, b novoa, a figueras institute of marine research (iim), national research council (csic), eduardo cabello 6, 36208, vigo, spain among marine litter, plastic represent 60-80% of litter on the ocean. plastics trigger a threat for aquatic ecosystems. degradation and fragmentation of plastics to small fragments led to the formation of microplastics (mps<5 mm) and nanoplastics (nps<1µm). the most representative plastic found in the ocean and widely used is polystyrene (ps). due to primary characteristics of nps (such as small size, high surface area and high reactivity), ps nps can stablish colloidal interactions with cells and therefore unwanted effects. among marine organisms, bivalves and particularly their immune system is recognized as a target to assess nanomaterials toxicity. since the hypothesis that internalization and different effects in hemocytes exposed to ps nps could be observed and that different nominal size of ps nps could affect from distinct way to internalization routes and immune responses, several goals were proposed: i) to analyze the internalization of ps nps with different sizes (50 nm, 100 nm and 1 µm) in granulocytes of mytilus galloprovincialis, ii) to study the location of ps nps in lysosomes as a target organelle as well as the effects in alkalinisation of the lysosomal lumen, iii) to assess how the ps nps exposure could affect to different cell processes and iv) to study the immune capacity of hemocytes infected with vibrio splendidus previously exposed to different size of ps nps. the most internalized particles were 1 µm ps nps when the same number of particles (1.8·107 198 particles·ml-1) was used for all nps, however when the same concentration was used (10 mg·l-1) 50 nm ps nps were the most internalized followed by 100 nm and 1 µm nps. when hemocytes were exposed to ps nps differences in the uptake of the particles were observed. the main route for 50 nm nps were caveolar and clathrin-mediated endocytosis while phagocytosis inhibition did not show significant differences. on the other hand, when hemocytes were exposed to 100 nm and 1 µm ps nps phagocytosis pathways played a significant role in particles internalization. after internalization all ps nps were located in lysosomes. autophagy induction, modulation of gene expression and decrease in phagocytic capacity were also observed after ps nps exposure. using marine mussels for testing the ecosafety and the efficacy of nanostructured cellulose sponges for remediation of zinc in seawater g liberatori1, g grassi1, a fiorati2, c faleri3, g protano1, c punta2, i corsi1 1department of physical, earth and environmental sciences (instm local unit), university of siena, siena, italy 2department of chemistry, materials, and chemical engineering “g. natta” (instm local unit), politecnico di milano, milano, italy 3department of life sciences, university of siena, siena, italy despite the growing interest in using nanotechnologies for environmental remediation, potential impacts on marine ecosystem has been underestimated so far. in order to select ecofriendly and sustainable materials and avoiding unexpected toxic side effects especially on marine biota, in the present study an eco-safety design concept has been developed using marine mussels as environmental indicators. a novel nanostructured cellulose-based sponge (cns) has been used for validating this eco-design concept, based on their effective sorbent ability of heavy metals removal from seawater. specimens of mytilus galloprovincialis widely used as bioindicator of coastal pollution were used as model in laboratory studies, able to show clear response at both cellular and morphological levels even after acute exposure (48 h in vivo). the effects of zn (< lc50), cns alone and cns-treated zn contaminated waters (cns t-zn), were evaluated in mussels’ haemocytes and mantle tissue. lysosomal membrane stability (lms) by neutral red retention time assay and micronucleus (mn) frequencies, were both significantly affected by zn exposure (10 mg/l) while, a significant recovery was observed in mussel’s haemocytes exposed to cns t-zn waters, which showed no differences with cns alone. from macroscopic observations, mussels’ mantle exposed to zn resulted clearly damaged with the edge almost lost and absent/wilted siphons, fused together to build deformed and no more functional structures. upon exposure to cns t-zn, they appeared distended and vigorous resembling the normal morphology as observed in controls and cns alone exposed mussels. from histological analysis, zn-treatment caused a significant rupture of the mantle’s columnar epithelium, the loss of epithelial cilia and a production of a copious amount of mucus associated with an increase of acidic mucosubstances respect to the neutral ones. both neutral and acidic mucopolysaccharides of the mantle edge were still present in cns-t zn group as well as in controls and cns alone. finally, total zn levels measured in exposure media at time 0 and after 24 hours, confirmed the absorption capacity of cns toward zn in seawater and the reduction of zn bioavailability to exposed mussels. ecotoxicological risk assessment for the herbicide glyphosate and its degradation product ampa: analysis of host and microbiota response in the mussel mytilus galloprovincialis m milan1, s iori1, g dalla rovere1, m smits1, s ferraresso1, m babbucci1, mg marin2, l masiero2, j fabrello2, l carraro1, b cardazzo1, t patarnello1, v matozzo2, l bargelloni1 1department of comparative biomedicine and food science. university of padova, viale dell’università 16, 35020 legnaro (pd), italy 2department of biology, university of padova, via bassi 58/b, 35131 padova, italy glyphosate has been the most widely used herbicide worldwide over the last three decades, raising increasing concerns for its potential impacts on environmental and human health. recent studies revealed that glyphosate occurs in soil, surface water, groundwater and drinking water, and residues can be found at all levels of the food chain, such as plants, animals, and even in humans. while research has demonstrated that glyphosate can induce a broad range of biological effects in exposed organisms, the global molecular mechanisms of toxicity remain unclear, in particular for marine species. in a recent study, gene expression analysis was used to evaluate the effects of glyphosate on the mussel mytilus galloprovincialis, revealing the disruption of several key biological processes including energy metabolism and ca2+ homeostasis, cell signalling, and endoplasmic reticulum stress response. here, the impact of glyphosate and its degradation product aminomethylphosphonic acid (ampa) is assessed for the first time in m. galloprovincialis following exposure for 7 and 21 days to environmentally realistic concentrations of ampa (100 μg/l), glyphosate (100 μg/l), and a mixture of ampa and glyphosate. considering that glyphosate targets the 5-enolpyruvylshikimate-3phosphate synthase (epsps) enzyme in the shikimate pathway found in plants and some microorganisms, gene expression analysis (rnaseq) was analysed in combination with the characterization of the digestive gland microbiota to assess whether glyphosate may affect bacterial symbionts of animals living in the marine environment. overall, this study provides a novel overview on host-microbiota interactions induced by exposure to realistic concentrations of glyphosate and ampa. 199 multi-level responses of mytilus galloprovincialis to environmental stressors mg parisi, a giacoletti, c la corte, g sarà, m cammarata department distem, university of palermo, palermo, italy maintaining homeostasis requires the proper functioning of all individual physiological processes, including the immune system network, both influenced by environmental conditions. multiple stressors are widely known to affect marine organisms over time and space, exerting negative effects already as individual factors, further generating hard to predict synergistic and interactive effects on ecosystem functioning. no study actually links functional traits (ft) and immune mechanisms to multiple stressors. here the effects of food concentration, temperature and hypoxia were studied on ft such as clearance rates (cr), assimilation efficiency of food (ae), respiration rates (rr) and on enzymatic scavengers of reactive oxidative species (catalase, cat; superoxide dismutase, sod; glutathione s-transferase, gst and peroxidase) in the digestive gland of the bivalve mytilus galloprovincialis. mussels were exposed to three temperatures (12, 20 and 28°c) in normoxic (8 mg o2 l-1) and hypoxic (0 1.5 mg o2 l-1) conditions and fed with food concentrations ranging from 0.9 to 3.5 mg of chlorophyll (chl-a) l-1. all three factors significantly influenced cr, which at the maximum temperature resulted negatively correlated to chl-a concentration. maximum ingestion rate decreased from 12°c to 28°c, with a significant negative effect highlighted by hypoxia. instead ae was influenced only by temperature. rrs were modulated by both temperature and food, while different oxygen concentrations did not exert a significant effect until coupled with temperature. in both anoxic and normoxic samples, cat increases at higher temperatures and at higher food concentrations. greater availability of food and an optimal temperature of 20°c positively modulated the process of dismutation carried out by sod. the analysis showed as temperature, oxygen concentration and food availability had a significant effect on the gst of mussels. peroxidase was highly significant between temperature and food and even a difference emerged from different oxygen conditions. also in this case it is evident as food can act as a buffer to adverse environmental conditions and specimens under normoxic conditions are better able to maintain homeostasis at high temperatures. overall, these results can be used to determine the efficacy thresholds and facilitate the interpretation of functional and immunological biomarker monitoring. this approach, applied during significant temperature increases, can be a step forward in determining an environmental evaluation criterion in the population of coastal marine mussels. contribution to the study of molecular effects of copper on mytilus galloprovincialis during reproductive period m piscopo1, g lettieri1, v mollo1, a ambrosino1, f caccavale1,2, j troisi3, f febbraio4 1department of biology, university of naples federico ii, naples, italy 2department of biology and evolution of marine organisms, stazione zoologica anton dohrn, napoli, italy 3theoreo srl–spin‐off, company of the university of salerno, salerno, italy 4institute of protein biochemistry, national research council of italy, naples, italy successful reproduction is a determining factor for the survival of species. in this century, major risks affecting reproductive health are for those species that fertilize externally, such as several marine invertebrates, since several marine ecosystems are polluted by different types of xenobiotics. among the xenobiotics present in seawater, a relevant role is played by heavy metals whose release into the marine environment has increased their levels to large extents within the past few decades. copper is one of the most interesting heavy metals, because in small quantities, it is essential, being involved in several physiological functions, such as redox reactions, oxygen transport, cellular respiration, free radical defense, neurotransmitter synthesis, and neuronal myelination, but can be toxic above certain threshold concentrations. in this study, we have evaluated the effects induced by 24 hr exposure to a subtoxic copper concentration on the reproductive system (gonads, spermatozoa, and protamine‐like proteins) of mytilus galloprovincialis. protamine‐like proteins represent the main basic protein component of sperm chromatin of this organism. after exposure, we found accumulation of this metal in gonads, spermatozoa, and protamine‐like proteins of exposed mussels, as indicated by inductively coupled plasma–mass spectrometry analyses. moreover, altered expression levels of mt10 and protamine‐like proteins genes in spermatozoa and gonads, respectively, of exposed mussels were registered by real‐time polymerase chain reaction analyses. we analyzed also the dna binding affinity of protamine‐like proteins extracted from spermatozoa of exposed mussels. the results showed a higher dna binding affinity and a different dna binding mode in exposed mussels. moreover, an increased amount of nacl was required for the release from sperm nuclei of pl‐iii, the main protamine‐like proteins component. interestingly, protamine‐like proteins extracted from spermatozoa of exposed mussels promoted dna oxidative damage in the presence of h2o2. these results demonstrate that also tolerable copper amount could affect the properties of protamine‐like proteins and determine negative effects on mytilus 200 galloprovincialis reproductive system. finally, these analyses could be useful to develop quick and efficient chromatin‐based genotoxicity tests for pollution biomonitoring programs. mytilus galloprovincialis haemocytes activity as biomarker for evaluation of methylmercury effect c la corte, mg parisi, d parrinello, m cammarata department distem, university of palermo, palermo, italy biomonitors offer a direct measure of metal pollution, as it affects the local ecosystem. bioindicator organisms filter feeders, like bivalves, accumulate trace metals in their tissues and, by virtue of their wide geographic distribution, they are been used in many countries to monitor contaminants in the ecosystem. in an attempt to identify cellular markers for revealing pollution, this study examined the effects of different concentrations of methylmercury (ch3hgcl) on mytilus galloprovincialis haemocytes activities and morphology. the ch3hgcl concentrations we used for examining the effect on immunocytes were not toxic, as indicated by trypan blue dead cell exclusion test. thus, we evaluated the effect of three organometal sub-lethal concentrations on cellular morphology and the efficacy of phagocytosis towards yeasts and bacteria, the maintenance of lysosomal membrane integrity and the ability to release cytotoxic molecules. the results provided evidence of the ch3hgcl immunotoxic effects on haemocyte viability and morphological changes induced by cytoskeleton alterations. thus, a morphometric cellular parameter, such as spreading ability, was used as a complementary method. in particular, the haemocytes exposed to the xenobiotic have significantly reduced the phagocytic percentage and phagocytic index of the haemocytes towards the yeast. ch3hgcl could also act on cytoskeletal network, as indicated by changes in haemocyte morphology and spreading capacity causing immunosuppression. with regard to this, it is known that cytoskeletal alterations lead to reduced phagocytic activity, due to the decreased ability of haemocytes to adhere to the substrate and interact with targets. the cytotoxic activity of m. galloprovincialis haemocytes towards erythrocytes and the activity revealed from lysis plaque assay has not been altered by adequate concentrations of methylmercury in the medium. the results confirm that the mediterranean mussel m. galloprovincialis, which lives in an anthropogenically influenced environment, is a suitable model organism for environmental assessment of the quality in natural ecosystems. session 6. advancing tools and strategies chairpersons: stefano carboni, paola venier effect of temperature during broodstock conditioning in blue mussel (mytilus edulis l.): a lipidomics perspective va laudicella1, s carboni2, mk doherty3, pd whitfield3, ad hughes1 1scottish association for marine sciences, oban, uk 2institute of aquaculture, faculty of natural sciences, university of stirling, stirling, uk 3division of biomedical sciences, university of the highlands and islands, inverness, uk blue mussels (mytilus edulis l.) account for over a quarter of total bivalve produced by european aquaculture. the reliance of the industry on natural spatfall is one of the principal bottlenecks for mussel production in europe, as these are highly unpredictable and fluctuate between years. a possibility to overcome such limitation is constituted by the development of industrial mussel hatchery technology, which should provide a continuous input of high-quality juveniles all year round. conditioning adults’ gonads is a fundamental practice in hatcheries, as it allows synchronous spawning and high-quality gametes production. temperature is between the main factors influencing the gonad development in mollusks; the rearing of adults at higher temperatures triggers gonad development and spawning in several bivalve species and enhances, at the same time, their metabolism and filtration rates. temperature has a major effect on bivalve lipid compartment; in fact, as poikilothermic organisms, mussels maintain constant the fluidity of membranes by modifying the sterol content and unsaturation degree of their cellular membranes. in this study, we tested two different conditioning programs on commercial size m. edulis adults: a single stage conditioning (ssc) and a cold and hold (c&h) treatment. the conditioning lasted for 80 days with two sampling points (40 and 80 days). the effects of the two conditioning programs were evaluated on the gonad ultrastructure, via histological examination of gonads, and on gonad lipidome, through untargeted lipidomics analysis of the gonads. lipidomics analyses were performed with a high-resolution lc-ms platform both in positive and negative ionization modes. the results suggest that ssc enhanced the nutritional requirements of mussels, due to higher metabolic cost, inducing gonad resorption and atresia. at the same time, the two treatments had a profound effect on the mussels’ gonad lipidome. mussels evidenced shifts in gonad membrane lipids after 40 days of conditioning. such changes were more severe by the end of the trial and included the alteration of triglycerides (tg) composition in female gonads. 201 the results of this study suggest the importance of a correct broodstock conditioning process. keeping mussels at higher temperatures resulted deleterious in term of fecundity. moreover, prolonged exposure to high temperature modified the tg composition in female gonad. tg contained in female gonads are energy source for the offspring during the early development, resulting in possible downstream issues during larval development. mytimap: a tool for making publication quality species distribution maps for mytilus mussels c riginos1, j thia1, a simon2, p borsa3, i popovic1 1school of biological sciences, the university of queensland, st. lucia, australia 2institut des sciences de l’évolution, université de montpellier, montpellier, france 3institut de recherche pour le developpement, montpellier, france creating maps of mussel species distributions can be tedious and time consuming. existing public resources of species distributions are often confounded by species misidentifications in the absence of genetic validation. moreover, judgements based on personal (= expert?) knowledge and artistic license make the generative process opaque and non-reproducible. in an attempt to circumvent these issues, we present mytimap, an r based scripting tool for automating the creation of mussel species distribution maps. underlying mytimap is a growing database of species records based on compilations of results from existing genetic studies. mytimap allows the user to specify criteria for determining species distributions such as types of molecular markers and number of genotyped individuals per location. known hybrids zones can be added to maps or excluded. stylistic options include geographic extent, map projections, and color assignment. mytimap is an open source project hosted on github with an r shiny interface for map creation. the intention is for continued development to be communal such that new studies can add their findings to the database. creation of a map will produce a list of references underlying the data such that end-users can cite data contributors (thus incentivizing data contribution). estrogens in bivalve revisited: lesson learnt from mytilus l canesi1, a miglioli1, c ciacci2, t balbi1 1department of earth, environment and life sciences-distav, university of genoa 2department of biomolecular sciences -disb, university of urbino “carlo bo”, italy natural vertebrate estrogens are continuously discharged into the aquatic environment, where they are ubiquitous at ng/l concentrations. in particular, estrone-e1; 17β-estradiol-e2; and 17-αethinylestradiol-ee, have been included in the eu watch list of contaminants of emerging concern (cecs) for monitoring of surface waters (decision 2015/495). bivalve molluscs have been widely utilized to investigate the effects of estrogenic compounds, one the most widespread class of endocrine disrupting chemicals-edcs. however, knowledge on endocrine regulation in bivalves has considerably increased in the last decade, with the identification of receptors/signaling pathways/effectors involved in steroid signaling, synthesis and metabolism: from these studies, a considerable debate emerged on the role of ‘natural’ steroids in bivalve physiology, in particular in reproduction. this is a key point to understand both the basic endocrine mechanisms and the possible impact of estrogenic chemicals in this relevant invertebrate group. in this work, we will present available information on the effects and mechanisms of action of estrogens in the mussel mytilus spp, that, widespread in coastal and estuarine ecosystems, are most likely affected by exposure to estrogenic edcs. recent advances in steroid uptake and metabolism, and estrogen receptors-ers in molluscs, as well as in estrogen signaling in vertebrates, will be considered. the results so far obtained with 17β-estradiol and different estrogenic compounds demonstrate specific effects on immune function, development and metabolism in mussels, from cellular to organism level. transcriptomic data obtained from the digestive gland adult mussels exposed to estrogenic compounds confirm alternative estrogen signaling pathways that are supported by new observations at the cellular level. in vitro and in vivo data show, through independent lines of evidence, that estrogens act through, non-genomic signaling pathways in mussels. in this light, regardless of whether bivalves synthesize estrogens de novo or not, and despite their ers are not directly activated by ligand binding, estrogens can interact with multiple signaling components, leading to modulation of different physiological functions. increasing knowledge in endocrine physiology of mussels will provide a framework for a better evaluation and interpretation of data on the impact of estrogenic edcs in bivalves. mytilus galloprovincialis protamine-like proteins are new bactericidal molecules active also against antibiotic resistant bacteria m piscopo1, r notariale1,2, e montana1, g tenore3 , m guida1, a basile1 1department of biology, university of naples federico ii, naples, italy 2department of biochemistry, biophysics and general pathology, university of campania "luigi vanvitelli", naples, italy 3department of pharmacy, university of naples federico ii, naples, italy the discovery of antibiotics with novel mechanisms of action is a critical issue to overcome the serious problem of growing numbers of bacteria resistant to conventional antibiotics. sperm nuclear basic proteins are the chromosomal proteins that are found associated 202 with dna in sperm nuclei at the end of spermiogenesis. protamine-like proteins are one of the three types of sperm nuclear basic proteins, and represent a structurally and functionally intermediate group of proteins between the histone and protamine type. protamine-like proteins represent the major acid-soluble protein components of the mussel mytilus galloprovincialis sperm chromatin and consist of the protamine-like proteins pl-ii, pl-iii and pl-iv. the aim of this study was to investigate the antibacterial activity of these proteins since, to date, there are reports on bactericidal activity of protamines and histones, but not on protamine like proteins. we tested the bactericidal activity of these proteins against gramnegative bacteria: proteus mirabilis, proteus vulgaris, pseudomonas aeruginosa, salmonella typhmurium, enterobacter aerogenes, enterobacter cloacae, and escherichia coli as well as on grampositive bacteria: enterococcus faecalis and two different strains of staphylococcus aureus. the results show that mytilus galloprovincialis protamine-like proteins exhibited bactericidal activity against all bacterial strains tested with different minimum bactericidal concentration values, ranging from 15.7 to 250 µg/ml and also on the clinical isolates of the same bacterial species. interestingly, these proteins were active against some bacterial strains tested that are resistant to conventional antibiotics. for their possible therapeutic use, we investigated the tossicity of these proteins. we found that these proteins showed very low toxicity as judged by red blood cell lysis and viability mtt assays and seem to act both at the membrane level and within the bacterial cell. antibacterial proteins have a potential as alternative treatments to standard antibiotic therapies but oral administration would most likely result in the proteins being degraded in the digestive system. in order to analyze this aspect we generated an in vitro model of gastrointestinal digestion of pl-proteins and tested the bactericidal activity of the product obtained on a gram-positive and a gram-negative strain. we obtained the same results with respect to undigested protamine-like proteins on the grampositive bacterium. in conclusion, this work presents the first evidence obtained for mytilus galloprovincialis of bactericidal activity of protaminelike-proteins. hemolymph extraction sites and 3d-visualization of the cardiovascular system and related structures of the blue mussel (mytilus edulis) m eggermont1, p cornillie2, n nevejan1, p bossier1, m dierick3, d adriaens4, p sorgeloos1, t defoirdt5, am declercq1§ 1laboratory of aquaculture and artemia reference center, faculty of bioscience engineering, ghent university, ghent, belgium 2department of morphology, faculty of veterinary medicine, ghent university, merelbeke, belgium 3department of physics and astronomy, ghent university, x-ray engineering bvba zwijnaarde, belgium 4research group evolutionary morphology of vertebrates, ghent university, ghent, belgium 5center for microbial ecology and technology, faculty of bioscience engineering, ghent university, ghent, belgium bivalve hemolymph is used in a broad range of research domains such as eco(toxico)logy and immunology. however, the lack of a detailed description of hemolymph withdrawal protocols and locations (adductor muscles and heart) raises questions regarding the exact origin of the aspirated hemolymph and doesn’t exclude the possibility of contamination with other body fluids which may have led to biased conclusions. a good description of the species-specific anatomy is lacking for many bivalves but is essential for a correct hemolymph withdrawal. in this study we visualized and discussed the cardiovascular anatomy of the blue mussel (mytilus edulis) and generated three-dimensional (3d) reconstructions based on micro-ct and histological images. other structures, such as the gastrointestinal system, the muscular system and body cavities, were included as well because of their close relationship to the cardiovascular system. hemolymph withdrawn from the posterior adductor muscle originates from small spaces and fissures between the muscle fibers that are connected to at least one hemolymph supplying artery, more specifically the left posterior gastrointestinal artery. hemolymph withdrawal from the heart is less straightforward. it is possible to puncture the pericard, anterior aorta and ventricle to collect a limited volume of hemolymph, however caution should be taken for contamination from the pallial cavity. drainage of the pallial fluid prior to hemolymph extraction is therefore essential. the different hemolymph extraction sites were clearly visualized in 3d. this study resulted simultaneously in a detailed description and visualization of the anatomy of mytilus edulis useful to many research areas. furthermore the described protocols and techniques to visualize the anatomy in 3d can easily be reproduced and adapted to other bivalve species. more with mussels ac smaal1,2 1aquaculture and fisheries group, wageningen university and research, wageningen, the netherlands 2hz university of applied sciences, aquaculture team, vlissingen, the netherlands marine bivalves like mussels have been cultivated for ages and are recognised as a sustainable low food chain resource that acquires feed from natural resources in their environment. they provide a rich source for human nutrition and an associated economic value for local communities. total mussel aquaculture production amounted 1.9 million tons in 2015 with a landing value of 3.2 billion us $. besides human nutrition, the mussels provide food for birds and benthos, a habitat for a large number of species, they regulate water quality and sequester carbon and nitrogen. mussels are used to 203 mitigate fish farming impacts. as eco-engineers, epibenthic bivalve beds are used for coastal defence and nature conservation. shellfish restoration is a worldwide issue that not only aims to bring back the bivalves but also restore the facilitation functions of bivalve beds. they produce significant amounts of shell material that has many applications. the mussels also provide cultural services through shellfish gardening as a community issue. these functions can be defined as ecological goods and services. this concept provides a framework for description and analysis of the role of bivalves in the ecosystem, and a basis for addressing a wide range of topics, benefits and controversies related to the use of bivalves for production, habitat restoration, water quality and coastal management. understanding goods and services may improve management decisions. a way to improve decision making is to valorise the goods and services. the economic revenues of aquaculture are based on the market value of harvestable products, while the economic values of ecosystem services are often not adequately quantified and not fully captured in commercial markets. both for market and political decisions, techniques are needed that can be used to make economic values of mollusc ecosystem services explicit, provided full knowledge of the goods and services is available. a comprehensive analysis of their ecosystem functions is needed to better make use of the good and services. in the presentation, the state of the art in multiple use of mussels will be reviewed on the basis of a number of case studies. _________________________________________ poster presentations mussel aquaculture in china c-m bai1,2, l-s xin1,2, c li1,2, c-m wang1,2 1yellow sea fisheries research institute, chinese academy of fishery sciences, qingdao, china 2laboratory for marine fisheries science and food production processes, qingdao national laboratory for marine science and technology, qingdao, china although mussel aquaculture existed since 1950s in china, large-scale commercial aquaculture initiated in the early 1970s and expanded rapidly in the early 1980s due to the development of breeding, seed collection and longline culture techniques. the mussel production has accounted for near 30% of the total mollusk production in late 1980s. in early 1990s, the development of the mussel aquaculture industry began to slow down, because its’ space was occupied by the relatively high value oyster and scallop aquaculture. and the proportion of mussel production in molluscs dropped to about 6% in late 1990s. during the last two decades, the annual production of both mussels and molluscs increased steadily in china, while the proportion of mussel production in molluscs was always around 6%, and mussels were always the fourth most cultivated marine bivalves following oysters, clams and scallops. in 2017, the total mussel production from aquaculture amounted about 0.93 million tons in china, accounting for 53% of global mussel production. more than 50 species belonging to 18 genus of the family mytilidae distributed naturally along the coastline of china. in northern and southern china, the primary species of mussels cultured are the blue mussel (mytilus edulis) and the green mussel (perna viridis) respectively. in the middle of china, the thick shell mussel (mytilus coruscus) is the primary species farmed. depending on the abundance of mussel spat in nature and the market value of specific mussel species, mussel seed are either collected from the wild or produced in hatcheries accordingly. for the thick shell mussel, all spats come from hatchery broodstock now. mussels are cultured by three basic methods in china: rope culture, lantern net culture, and bottom culture. rope culture is a more widely accepted method for mussel aquaculture due to its high productivity and relatively low cost. the grow-out period may take 6 -12 months after which mussels can attain the market size (8-10 cm). mussel is relatively resistant to disease compared to its’ counterparts, and the loss which mussels might encounter during the grow-out period is attributed mainly to bad weather (e.g. typhoon), predators and bloom of harmful plankton. it is also worth to mention that mussels are viewed as kind of fouling organisms in many aquaculture areas of china, because the large numbers of juvenile mussels produced by wild populations can foul the culture facilities of other species. how to control the fouling of mussels is still a challenge for many farmers and scientists in china. preliminary data on immune priming in the mediterranean mussel mytilus galloprovincialis l ballarin, v matozzo department of biology, university of padova, padova, italy molluscs, like all the other invertebrate, rely only on innate immunity for their defence. the latter has been traditionally associated with low specificity and lack of immune memory. however, in the last decade, the presence of short-term immune memory, referred to as “immune priming”, was revealed in representatives of various invertebrate phyla. in the present work, we studied the response of the mussel mytilus galloprovincialis to single or double exposure (second one after 7 days from the first) to the gram-positive bacterium bacillus clausii. one day after the 1st or 2nd exposure, the digestive gland and the gills were collected from exposed and unexposed animals (controls) frozen in liquid nitrogen and stored at -20°c. haemolymph was also collected from the adductor muscle and haemocytes were obtained by centrifugation and resuspended in filtered seawater (fsw). the following parameters were measured: superoxide dismutase (sod) and catalase (cat) activities on tissues; total haemocyte count (thc), haemocyte volume (hv), haemocyte diameter (hd) and the percentage of phagocytosing cells on haemocytes. 204 no variations in sod and cat activities were observed in exposed animals with respect to their control after a single exposure, whereas a significant decrease in cat activity was observed after the 2nd exposure. no significant differences in the thc and phagocytosing activity were observed whereas significant increases in hv and hd were observed after the exposures without any significant variations between the two exposures. collectively, these data represent a first attempt to study immune priming in m. galloprovincialis. further studies are required using a more appropriate stimulus (b. clausii is not a natural pathogen for m. galloprovincialis) and changing the interval between the 1st and the 2nd exposure. risk factors analysis for cadmium contamination in mytilus galloprovincialis harvesting areas of marche region (italy) for the purposes of chemical sanitary survey em epifanio1, f agnetti1, f barchiesi2, m latini1 1istituto zooprofilattico sperimentale dell'umbria e delle marche "togo rosati", terni, italy 2istituto zooprofilattico sperimentale dell'umbria e delle marche "togo rosati", ancona, italy cadmium (cd) is the heavy metal most presents in the shellfish (jernelöv, 2013). mussels are one of natural accumulators of cadmium from the environment (efsa, 2009). this happens because metals are accumulated by these filter feeding organisms through water, ingestion of suspended sediments or food (butler & timperley, 1996). sources of cd into the environment come from human activity but also from natural reservoir. commission regulation (ec) 1881/2006 sets maximum levels of cd in edible bivalve mollusc (wet weight) at < 1.0 mg/kg. according to regulation (ec) 854/2004, bivalve molluscs can be harvested and commercialized only from classified areas. the classification is based on escherichia coli contamination of bivalve molluscs. the classification can be correctly established only through a complete and preliminary sanitary survey. the sanitary survey is a procedure that studies the sources of fecal pollution and the environment characteristics of an area used for the bivalve mollusc production. to ensure public health shellfish harvesting areas have to be periodically sampled and monitored. frequency of sampling and geographical distribution of sampling point are chosen by competent authority but, unlike during fecal bacteria monitoring, the plan of a sanitary survey is not a legally requirement for chemical contaminants.therefore, a correct monitoring for chemical contaminants could be very difficult and expensive for public resources. this study applies the concept of sanitary survey on cadmium control on mytilus galloprovincialis harvesting areas. it was discovered the most representative areas to control the cd contamination in mussels and created a model that can help to build a better monitoring program following specific risk that is the base for any type of survey (shi et al, 2016). the study was applied in the marche region (italy) mussels harvesting areas. six main risk factors for cd contamination in the environment were studied: type of industrial activity, presence of ports, agriculture land, rainfall, presence of rivers, sea sediments. these factors were compared with the historical cd contamination in mussels in a ten years period (2009-2018). five out of six factors (type of industrial activity, presence of ports, agriculture land, rainfall, presence of rivers) were confirmed as risk factors for cd contamination in mussels. the greater the presence of risk factors the greater the quantity of cd found in mussels. the health surveillance also for cd pollution can therefore be used in order to optimize time and locations of sampling for monitoring. characterisation of the intracellular protozoan mpx in mussels, mytilus edulis linnaeus, 1758 g fichi, s carboni, je bron, j ireland, mj leaver, g paladini institute of aquaculture, university of stirling, stirling, uk ciliates have been reported as pathogens of many species of economically important bivalves. mussel protozoan x (mpx), is an uncharacterised intracellular ciliate of mussels and has been widely reported in mytilus spp. around the world. in order to characterise this ciliate, mytilus edulis samples were collected from a site on the west coast of scotland, and four different fixatives for histological examination were tested. fresh preparations of mussel digestive glands were also examined by laser scanning confocal microscopy. intracellular ciliates were prepared by laser capture microdissection and partial sequences of small subunit ribosomal rna gene and of large subunit ribosomal rna gene were generated, using phyllopharyngea primers. methacarn solution proved to be the best fixative for both histological and molecular characterization. the morphological and molecular investigations confirmed that this ciliate belongs to the class phyllopharyngea, order rhynchodida. however, this organism does not belong to any known family, genus or species, therefore, a new description is necessary, following further morphological analyses. most mussel samples containing mpx displayed mild to moderate infections, with no signs of necrosis or haemocytic response, although a single sample displayed a severe infection (∼103 ciliates per section). the localization of this ciliate in tissues other than the digestive gland, the presence of necrosis in infected tissue of the most severely infected mussel and the binary fission of this ciliate have been observed here for the first time. we also report the first observation of the live ciliate isolated from tissue. although mpx remains of unknown significance to the mussel industry, tools and protocols described will be useful in further characterizing these and other ciliates (subclass rhynchodia) known as pathogens for bivalves. 205 nmr-based metabolite profiles in mytilus galloprovincialis: experimental set-up and preliminary data r frizzo1, t riello1, e schievano1, s mammi1, p venier2 1department of chemical sciences, university of padova, padova, italy 2department of biology, university of padova, padova, italy metabolomics studies founded on mass spectrometry (ms) or nuclear magnetic resonance (nmr) have rapidly expanded in the last decade. this and other current ‘omics (genomics, transcriptomics, proteomics) complement each other and contribute to a holistic view of organism responses to stimuli. in fact, the profiles of all small molecules detectable in biological fluids or tissues capture a further phenotype level, that of conditiondependent and time-dependent metabolome changes. we have evaluated different protocols in order to process the whole hemolymph, its acellular fraction and other soft tissues of the mediterranean mussel mytilus galloprovincialis, relying on available metabolite databases and suitable software for the identification and representation of mussel metabolite profiles. mussels maintained in standard conditions or kept in air at 4 °c were used in the procedural setup. the nmr profiles resulting from the analyzed tissue matrices allowed the identification of amino acids, fatty acids, organic acids and typical osmolytes. these profiles represent the starting point for hypothesis-driven basic and applied research, such as the quality assessment of marketable stocks. spat settlement and growth of mytilus galloprovincialis on the ropes in gökçeada island i keskin, a ekici department of aquaculture, faculty of aquatic sciences, university of istanbul, istanbul, turkey rope collectors were used as a settlement material for black mussels in gökçeada-kaleköy, settlement time, quantity, growth rates of black mussels and the occurrence of other fouling organisms were evaluated monthly from june 2017 to november 2018. average values of chlorophyll-a 0.32±0.31 μgl-1, water temperature 19.9±4.93 °c, salinity 35 ± 2.07‰, ph 8.12 ± 0.04 and total particulate matter (tpm) 14.95 ± 10.48 mgl-1 were recorded in the sea water. mussel spat started to be observed in the rope collectors after the first six months. the spat length was 3.23±1.17 mm in march 2018 (n=100) and 31.79±6.20 mm in october 2018. the growth of black mussels on the rope collectors was mostly influenced by the temperature (p<0.01). significant differences in growth rates were detected in all months, except april and may when mussel spat were detected in abundance (p<0.05).polychaeta, barnacles, tunicates and musculus costulatus bivalves were also detected in the rope collectors. these results strongly indicate that black mussel farming could be carried out commercially in the region. the spat settlement time was march in the aquaculture operations while large amounts of spat settled on the rope collectors in the following april and may, reaching 3.1 cm length size in a period of eight months. also, rope collectors are suitable for monitoring the growth of both mussels and fouling species. application of monoclonal antibodies in mytilus galloprovincialis hemocytes subpopulation sorting m mendoza1,2, s magadán1,2,3, c canchaya1,2 1department of biochemistry, genetics and immunology. school of biology, university of vigo. vigo, spain 2biomedical research centre. university of vigo. vigo, spain 3center for evolutionary and theoretical immunology. university of new mexico, albuquerque, united states all metazoans have evolved in a constant “arms race” with their pathogens, developing specific cell-types to detect and neutralise them. the mussel mytilus galloprovincialis, as well as other molluscs, presents only innate immune system: humoral and cellular mediated response. carballal et al. described two major types of cellular-subtypes in m. galloprovincialis hemolymph: basic and acidophilic granulocytes, and hyalinocytes, both similar to other mussel species. afterwards, garcía-garcía et al. and andreyeva et al. characterised these populations’ functions and responses. these cell populations, which have specific roles in the immune cellular mediated response, have not yet been characterised at molecular level. to characterise them, we need first to separate each population individually. for this purpose, we are improving the isolation of each cell-type using fluorescence activated cell sorting. because there is not yet any single specific cell-surface antibody described for m. galloprovincialis hemocytes, we tested antihuman or mice monoclonal antibodies (moabs). these antibodies have been previously used to characterise bivalves hemocytes by flow cytometry (e.g. anti-cd25 and anti-cd34 and other moabs suitable to identify human myeloid or lymphoid cells). however, the staining pattern produced by most of them suggested a non-specific reactivity. this led us to make an in silico search in m. galloprovincialis transcriptomes of homologous sequences to surface markers or cds useful for the identification of leukocytes. finally, potential useful sequence candidates were identified to characterize mussel hemocytes. 206 halobacteriovorax isolated from marine water of the adriatic sea to challenge v. parahaemolyticus in mytilus galloprovincialis d ottaviani1, g angelico1, s pieralisi1, e rocchegiani1, m latini1, f leoni1, f mosca2, pg tiscar2 1istituto zooprofilattico sperimentale dell’umbria e delle marche “togo rosati”, ancona 2facoltà di medicina veterinaria, università degli studi di teramo, teramo the public impact of pathogenic vibrios in bivalves is relevant and current decontamination processes are not always effective. the aim of this work was to test predation efficiency “in vitro” of a halobacteriovorax strain isolated from marine water of the adriatic sea, named hbxco1, towards 17 vibrio and 7 non-vibrio strains. moreover, to test “in vivo”, at the laboratory scale, its applicability for decontamination of mytilus galloprovincialis from v.parahaemolyticus. 16s rrna analysis and double layer agar plating technique were used for identification and predation efficiency of hbxco1, respectively. mussels artificially contaminated by v.parahaemolyticus and hbxco1 were subjected to depuration and analysed after 24, 48, 72 h. mussels contaminated with target pathogen without hbxco1 were used as control. hbxco1 preyed all v. parahaemolyticus, vibrio cholerae non-o1/o139 and vibrio vulnificus strains, but not v. alginolyticus and non-vibrio strains. in the depuration experiment, over the test period, we obtain in the control a progressive increase of v.parahaemolyticus counts while the opposite trends in the hbxco1 treated group, with a 2 log reduction of pathogen counts.to the light of these results, we believe that hbxco1 represents a potential candidate for development of biocontrol strategies of pathogenic vibrios in bivalves from harvesting to trade. the study was supported by the health ministry financed project rc005/2016. genetic variability of mytilus galloprovincialis (mollusca: bivalvia): the genetic pattern of an invasive marine species pa oyarzún1, je toro2, jj nuñez2, jpa gardner3 1centro de investigación marina quintay (cimarq), universidad andrés bello, quintay, chile 2instituto de ciencias marinas y limnológicas (icml), facultad de ciencias, universidad austral de chile, independencia 631, valdivia, chile 3centre for marine environmental and economic research, victoria university of wellington, p o box 600, wellington 6140, new zealand the genetic characteristics of introduced species have a significant impact on their ability to establish and spread. the blue mussel (mytilus galloprovincialis), native to the mediterranean coast, is a leading invasive species from the intertidal coasts of the world (i.e. america or south africa). here, we use mitochondrial dna sequence data to investigate the genetic diversity and phylogeographic structure of invasive versus native populations. we also evaluated whether genetic diversity in invasive populations could be explained by the genetic characteristics of the native sources from which they derived. the phylogenetic analysis revealed two main lineages indicating a clear separation between the afro-european and south american populations. on the contrary, we found no evidence of genetic structure in the invasive range in the southern pacific. this was probably the result of a recent arrival of m. galloprovincialis to the coasts of america. we also detected the spatial mixture of both lineages in the sampling locations of the chilean coast, giving rise to high levels of genetic diversity in some areas compared to the population of native mussels. this could lead (potentially) to a better fitness form of these individuals and thus increase the viability of the population and the invasiveness of this species. these results point to the need to better study the populations in which lineages are mixed and, if necessary, to intensify control efforts in them. molecular effects on spermatozoa of mytilus galloprovincialis exposed to severe hyposaline conditions: a case of fertility preservation strategy due to gamete plasticity m piscopo, g lettieri, m maione, ma ranauda, e mele department of biology, university of naples federico ii, naples, italy climate changes are affecting species physiology, pushing environmental tolerance limits and shifting biogeographic distribution ranges of marine organisms. in addition to temperature and ocean acidification, global climate changes can also occur through changes in seawater salinity. salinity levels may change also by anthropogenic factors such as mining activity and agricultural and industrial processes. salinity is considered one of the most significant environmental stressors for marine bivalves. mussels, in close proximity to coasts and in estuaries, in fact, periodically experience hypo-saline stress, particularly during intense precipitations. many bivalve species, when experiencing water with high temperatures and low salinity, have the skill to acclimatize, or migrate to deeper water which is cooler and more saline. mytilus galloprovincialis, is distributed in the north atlantic ocean, mediterranean sea and black sea and in recent years has invaded new places, including south africa, japan and california. this mussel is a common member of the intertidal estuarine and coastal areas where it can meet different salinities. mytilus galloprovincialis can survive at elevated heat exposure, but it is susceptible to hyposalinity, while the native pacific coast m. trossulus is more tolerant to hypo-saline conditions but vulnerable to heat stress. since salinity represents a critical factor in reproduction of marine species, we analyzed the responses of mytilus galloprovincialis spermatozoa to hyposaline stress. we exposed mussels, in laboratory tanks, for 24 hours at 18°c to control (35.9 psu) and three 207 hyposaline (17.1; 22.6 and 26.2 psu) conditions, and evaluated the expression of sperm hsp70 and protamine-like proteins genes. further we analyzed the electrophoretic pattern, the dna binding and the release from sperm nuclei of protamine-like proteins. for all experimental approaches used, the results obtained at 17.1 psu condition were very similar to those obtained in control condition, while alterations were always recorded at 22.6 and 26.2 psu conditions. particularly, at 22.6 and 26.2 psu, was observed: 42.5 and 17.1-fold increase in hsp70 expression, respectively and hypo-expression of plii/pliv protamine-like proteins genes. further, electrophoretic mobility shift assays and saltinduced release of nuclear proteins from sperm nuclei, revealed alterations in the pl proteins/dna binding, in these two hyposaline conditions. the similarity between the results obtained in control and in the more severe hyposaline condition (17.1 psu) could indicate a phenomenon of fertility preservation strategy due to gamete plasticity. traceability of bivalves (mytilus galloprovincialis) throught the application of stable carbon and nitrogen isotopes f rampazzo1, o giovanardi1, c gion1, m formalewicz1, g romanelli1, a scarpato1, f tosi2, g arcangeli2, g franceschini1, d berto1 1istituto superiore per la protezione e la ricerca ambientale (ispra). national institute for environmental protection and research, chioggia (ve), roma (rm) 2istituto zooprofilattico sperimentale delle venezie, national reference center for fish, molluscs and crustacean diseases, legnaro (pd) the traceability of agricultural products is regulated by european parliament regulation n.178 / 2002, uni en iso 22005: 2008 and reg. (eu) no. 1379/2013 chapter iv article 35 of the european parliament and of the council of 11 december 2013 laying down mandatory information to be included on the label of fishery and aquaculture products. traceability is therefore of great importance in terms of guaranteeing the origin of the products offered to the consumer, but also the sustainability of the fish resource for the protection of the natural heritage. molluscs, including mussels (mytilus galloprovincialis), have a chemical-nutritional composition that undergoes changes during the year, related to site, season and reproductive period. the purpose of this paper was to explore the possibility to distinguish the origin of mussels coming from different geographic areas through the analysis of stable carbon and nitrogen isotopes (13c, 15n). mussel were sampled in different geographic areas during the period 2015-2017. subsamples of ten specimen from each area were homogenized and freeze dried after separation of muscle from the shell. isotopic analyses were performed using a delta v advantage, coupled with an elemental analizer chn flash 2000 (thermo fisher scientific), using glutammic acid and sucrose as standard reference material. analytical precision was 0.2%. 13c values of mussels ranged from -23.584 ‰ to 17.101 ‰ (mean value -20.951 ±1.470 ‰), while 15n from 5.337 ‰ to 8.345 ‰ (mean value 6.627±0.964 ‰). from the plot 13c vs 15n a good separation from mussel from diffent geographical areas was observed. the application of isosource mixing model to the mussel isotopic data showed as the phytoplancton was the major bivalves carbon source (mean 56 ± 12%), while the urban waste discharges (mean 18 ± 15%), treated and untreated, showed higher influence in the area of koper with a contribution of 54%. the contribution of the particulate organic matter of terrestrial origin resulted lower (9±6%), probably due to the distance from the river mouths. isotopic approach seems to be a promising tool for the traceability of bivalves. this evidence was confirmed also by the shift of the isotopic values of the mussel transplanted in a different geographical area. the isotope analysis of lightweight elements is still at the experimental stage in determining the origin of most matrices, including fish products, while for others agricultural products (wine, honey, vinegar, fruit juice; camin et al., 2016 and the cited authors) the experimental process has already been concluded and has come to regulate the use of this technique in controls protocols. invasions create competitors: how novel interactions among native and invasive parasites influence coevolution with blue mussel hosts km wegner1, me feis1,2, l gottschalck1 1department of coastal ecology, awi alfred wegener institute for polar and marine research, waddensea station sylt, list, germany 2station biologique roscoff, roscoff, france within parasite communities infecting the same host, ecological theory predicts that two species occupying the same niche should evolve distinct niche use to avoid direct competition. biological invasions can however create situations, where competition could not select for different niche occupancy and closely related parasites species find themselves competing for the same host resources for the first time since their lineages split. such novel interactions cannot only alter the evolutionary trajectories of both parasite species, but will also feed back onto established coevolutionary interaction of native parasites with the host. here, we show how the invasion of the parasitic copepod mytilicola orientalis creates potential competition with the established congeneric parasite mytilicola intestinalis, and how this novel menage a trois feeds back on the immune response of the blue mussel host mytilus edulis. from a series of controlled infection experiments that manipulate competition among the parasites we can show that, although both species occur in the same section of the mussel gut, competition between the parasites is weak and shows similar impact on host condition in either simultaneous or sequential infections. triplet transcriptomics of matching host (m. edulis) and parasite samples (m. 208 intestinalis and m. orientalis) however revealed that the novel interaction of the invader with the host changes the transcriptional activity of many more genes and processes than the interaction with the established and coevolved parasite. our results therefore not only show the utility of biological invasions of parasites to study coevolutionary processes, but also shows that responses to novel host-parasite interactions can lead to massive reactions on the molecular level that are not reflected in host or parasite phenotypes. water quality monitoring station in the bivalve purification centre of giulianova city (abruzzo region, italy): one-year results f di giacinto1, g mascilongo1, m scattolini2, d pichinelli2, mì berti1, n ferri1 1istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’, teramo, italy 2cimar s.r.l., lungomare spalato 17, giulianova, teramo, italy european “hygiene package” lays down hygiene requirements to be respected for the commercialization of live bivalve molluscs. one of the first requests for the purification centres is the availability of good quality water. apart from microbiological parameters, no specific technical references are listed in the legislation to monitor water quality in the purification system. the collaboration between “cimar s.r.l.”, the purification and dispatch centre (it 001 cdm ce) located at harbour of giulianova city (abruzzo region, italy), and the research institute “istituto zooprofilattico sperimentale dell’abruzzo e del molise (izsam)” has been instituted to enhance management system of water quality to be adopted by the centre. a control station for the continuous monitoring of water intended for purifying shellfish has been installed at the inlet of water circuit. chemical/physical and biological parameters, are registered continuously. for over a year, a multiparametric probe has been recording once per hour: ph, oxidation reduction potential (orp), temperature (t), dissolved oxygen (do), conductibility (cond.) and salinity (sal.). in july 2019, it has been added biomonitoring through the biological early warning system (bews) “mosselmonitor®”, based on the behavioural responses of mytilus galloprovincialis to disturbances. electromagnetic sensors, glued on eight mussels, monitor valve movements every one minute. abnormal behaviors are registered and sent as early warning signals linked to potential water contaminations or to any other mussel disturbances. three types of alarms are considered: closure of shell valves for a long period (alarm c); rapid openings (alarm a); decrease in the average distance between valves (alarm d). one-year results of chemical and physical monitoring showed the following average values from july 2018: t = 13 °c; ph = 7,82; orp = 106 mv; cond. = 53 ms/cm; sal. = 34,85 ppt; do = 95%. no sudden and unusual changes have been registered in the water, only seasonal fluctuations. during the biomonitoring period, no alarms related to water contamination have been registered. several signals for alarm c and few alarm d were recorded, due to the temperature increasing of almost three degrees. in the same yearly period, results of official controls and the company haccp self-controls, were negative for all parameters foreseen in the current legislation. innovation is essential to make more efficient and sustainable the management of purification centre, as well as to guarantee food safety. 60 isj 16: 60-65, 2019 issn 1824-307x report of meeting 2nd general meeting and working group meetings of the cost action 16203: stem cells of marine/aquatic invertebrates: from basic research to innovative applications (maristem), november 28-30, 2018, marine biology station – laboratoire arago, banyuls-sur-mer, france organizer: a-m genevière sorbonne université, cnrs, biologie intégrative des organismes marins (biom) f-66650 banyuls-sur-mer, france introduction to maristem stem cells of marine/aquatic invertebrates: from basic research to innovative applications l ballarin department of biology, university of padova marine/aquatic invertebrates constitute the largest biodiversity and the widest phylogenetic radiation on earth, from morphologically simple organisms (e.g., sponges, cnidarians), to the more complex mollusks, crustaceans, echinoderms, and protochordates. today, adult marine/aquatic invertebrate stem cell (misc) biology is of prime research and medical interest. however, studies on stem cells from organisms outside the classical vertebrate (e.g., human, mouse, and zebrafish) and invertebrate (e.g., drosophila, caenorhabditis) models have not been pursued vigorously. these organisms contain a variety of misc-types that allow the production of a large number of novel bioactive-molecules, many of which are of significant potential interest for human health. miscs further participate in aging and regeneration phenomena, including whole-body regeneration. for years, the european misc-community has been highly fragmented and has established scarce ties with biomedical industries in an attempt to harness miscs for human welfare. thus, it is important to (i) consolidate the european community of researchers working on miscs; (ii) promote and coordinate european research on misc biology; (iii) stimulate young researchers to embark on research in misc-biology; (iv) develop, validate, and share novel misc tools and methodologies; (v) establish the misc discipline as a forefront interest of biomedical disciplines, including nanobiomedicine; and (vi) establish collaborations with industries to exploit miscs as sources of bioactive molecules. in order to fill the recognized gaps, the ec-cost action 16203 “maristem” has recently been launched. at its initial stage, the consortium unites scientists from 24 ec countries, cooperating countries, and near neighbor countries. general meeting (speakers in alphabetical order) evo-devo of non-embryonic development in colonial ascidians a alié1, l hiebert2,3, p simion4, m scelzo1, f brown2,3, s tiozzo1. 1sorbonne université, cnrs, laboratoire de biologie du développement de villefranche-sur-mer (lbdv), 06230 paris, france 2departamento de zoologia instituto biociências, universidade de são paulo, são paulo, brazil 3centro de biologia marinha (cebimar), universidade de são paulo, são paulo, brazil 4isem, université de montpellier, cnrs, ird, ephe, montpellier, france ascidians of the styelidae family regroup more than 550 species and comprise both solitary and colonial forms. whereas solitary species can reproduce only sexually, colonial ones have the ability to also propagate asexually by different modes of non-embryonic development and often can regenerate the body completely after injury1. we recently generated a robust phylogeny of styelidae based on 20 new transcriptomes, showing that asexual reproduction has been acquired twice 61 in this family by convergence2. notably, the species polyandrocarpa zorritensis acquired asexual reproduction independently of all other species and displays a unique mode of bud formation that is a combination of epithelial morphogenesis and proliferation of putative blood stem cells3. in order to understand the molecular bases of convergent acquisition of budding across ascidian species, we are conducting a comparative transcriptomic study of budding tissues between distant colonial species (e.g. including polyandrocarpa zorritensis and botryllus schlosseri). in addition, we will generate transcriptomes of single blood cells in colonial and solitary species with an emphasis on candidate totipotent stem cells to provide molecular-level description of blood cells in ascidians, and pave the way to their functional characterization during budding. taken together, this ongoing work will help to elucidate the plastic evolution of non-embryonic development in ascidians, in order to better understand why colonial species are able to propagate asexually, while solitary species are not. 1tiozzo s. et al. (2008) regeneration and stem cells in ascidians. in: bosch tcg (ed) stem cells. springer, dordrecht, pp. 95-112. 2alié a. et al. (2018) convergent acquisition of nonembryonic development in styelid ascidians. mol biol evol. 35: 1728. 3scelzo et al. in prep. tissues regeneration and in hospite microalgae proliferation in the photosymbiotic marine flatworm symsagittifera roscoffensis (xenacoelomorpha, acoela) x bailly sorbonne université, cnrs, station biologique de roscoff, place georges teissier, 29680 roscoff, france some marine animals evolved long-term functional partnership with photosynthetic microalgae they reared inside the animal tissues. the biology and physiology of the green flatworm symsagittifera roscoffensis show how a population of around 100.000 photosynthetically active green unicellular algae (tetraselmis convolutae) is controlled beneath the epidermis of this animal. the non-photosymbiotic juvenile animal must ingest (not digest) micro-algae found in the surrounding environment or die (after 15 days in the lab) if no ingestion occurred. once ingested, algae divide, confer the green color to the animal and supply energy, releasing in the tissues various photosynthates, the unique source of food for the animal. controlling the life cycle in captivity of this flatworm – including the induction of photosymbiosis (i.e culture of the free living photosynthetic partner) allows having access to any developmental stages in order to explore the intimate trophic relationship and other features. in the animal tissues algae are fully dedicated to photosynthesis as suggested by the absence of typical structures expressed in the algae free-living state for which lot of energy is allocated, such as the synthesis of body-wall (complex polysaccharides) and the movement flagella. the in hospite algae take advantage of the animal nitrogen waste and recycle them. at the very beginning of the establishment of the symbiosis algae also recycle the uric acid crystals that accumulate in the non-photosymbiotic juveniles flatworm. intertidal natural s. roscoffensis colonies submitted to submarine groundwater discharge, enriched in nitrogen also show that the flatworms are nitrate interceptors. in the natural environment, animals are exposed, several hours each day, to sun rays and are adapted to overcome the excess of sun (including uvs) and high oxygen concentration (from photosynthesis) in their tissues. beside coping with putative detrimental physiological conditions (oxidative stress) s. roscoffensis also exhibits strong capacities of tissue regeneration including brain. after more than a decade of functional exploration related to s. roscoffensis, tuning the techniques for completing life cycle in captivity investing huge efforts in genomics/transcriptomics, morphological (cell types) characterization advances, many conditions are met for starting a formal exploration of the molecular and cellular mechanisms underlying brain and peripheral nervous system regeneration. corals as sources of bioactive molecules r benchaouir coraliotech, monaco the corals, organisms relatively under-studied compared to many other species over the world, must represent a very diverse source of active substances with potential applications in the fields of human well-being and health. coraliotech, young startup of marine biotechnology located in monaco, proposes an ecological technology of production of active products (proteins and peptides) originating from corals. based on genetic engineering and biotechnologies, coraliotech uses the coral genetic information provided by its scientific partners (such as the centre scientifique de monaco) to clone genes of interest before implementation of an artificial process of production and purification of the corresponding proteins. our technology does not require the use or exploitation of the coral itself. in addition to guaranteeing the preservation of natural ecosystems, our production activity also has the advantage of being clean (waste of essentially biological nature), safe (fully automated processes) and scalable (possibility for a rapid rise towards industrial productions). after r&d proof of concept, the process is performed at pilot scale before initiation of product evaluation assays. most of our innovative effects are valorized through patenting. our b2b activity targets more specifically the pharmaceutical, biotech and cosmetic companies. finally, coraliotech aims to progressively enrich its product pipeline and contribute, through its scientific partnerships, to a better knowledge of coral biology. 62 marine/aquatic invertebrate stem cells as promising models in environmental toxicology: future prospects and research needs d drobne research group for nanobiology and nanotoxicology, department of biology, biotechnical faculty, university of ljubljana, slovenia, http://www.bionanoteam.com most of ecotoxicity studies have been carried out in whole organisms at various developmental stages. selected cell lines have been used less frequently to elucidate the mechanisms of toxicity. there are many reasons for that, but the lack of proper research models and biomarkers to identify physiological modes of toxic action of environmental pollutants is among the most important ones as pointed by g. vogt (2011) in his paper entitled “hidden treasures in stem cells of indeterminately growing bilaterian invertebrates”. however, it is also true that advance of a scientific discipline could generate new research models to better address scientific questions. this holds true for aquatic invertebrate stem cells and their potential applicability in environmental toxicology as proper research models. for example, a number of environmental toxicology studies have already been done using digestive gland cells, hepatopancreas of isopods (crustaceans) as a test model. in addition to existing endpoints, one could use also stem cells of hepatopancreas located at the blind ends of the digestive gland tubules (hepatopancreas) which are resembling the apical meristem of plants (zimmer, 2002). also, hematopoietic organs provide an environment where undifferentiated stem cells could be used to measure of response (endpoint) to variable conditions and agents. another possible biomarker of cellular development and morphogenesis is the alteration of the cholinergic system, which is involved in embryonic development. paraoanua et al (2007) report that locally produced acetylcholine might function as an intercellular signal, modulating the proliferation of stem cells. in our previous study, we have demonstrated altered activity of ache to be related to altered sea urchin early development (mesarič et al, 2015). further, echinoderms represent a phylum with exceptional regenerative capabilities that can reconstruct both external appendages and internal organs (reinardy et al., 2015). understanding how regenerative processes respond to changing environmental conditions (contamination of ecosystem and variation in normal weather patterns) is paramount to predict the future vulnerability or success of these keystone marine animals. it is a privilege of maristem project to transform the tremendous potential of research outcomes on marine invertebrate stem cells into guidelines for animal (humans included) health and environmental protection. mesaric t, et al. sperm exposure to carbon-based nanomaterials causes abnormalitiesin early development of purple sea urchin (paracentrotus lividus). aquatic toxicology 163: 158-166, 2015. paraoanu le, et al. expression and possible functions of the cholinergic system in a murine embryonic stem cell line. life sciences 30: 80, 2375-2379, 2007. reinardy hc, et al. tissue regeneration and biomineralization in sea urchins: role of notch signaling and presence of stem cell markers. plos one 10: e0133860, 2015. zimmer m. nutrition in terrestrial isopods (isopoda: oniscidea): an evolutionary-ecological approach. biol rev. 77: 455-493, 2002. vogt g. hidden treasures in stem cells of indeterminately growing bilaterian invertebrates. stem cell rev. 8(2): 305-17, 2012. stem cells cell in sponges (porifera): an update a ereskovsky1,2 1institut méditerranéen de biodiversité et d'ecologie marine et continentale (imbe), aix marseille university, cnrs, ird, avignon university, marseille, france 2department of embryology, faculty of biology, saint-petersburg state university, saint-petersburg, russia sponges (porifera) are thought to be the sister group of all other animals and the earliest branching multicellular lineage of extant animals and as such a key group for understanding of the evolutionary history of animal stem cells and their regulation. sponges are known to possess remarkable reconstitutive and regenerative abilities and high cell dynamic. there is a widespread opinion that all sponges cells are capable of transdifferentiation and under certain conditions exhibit properties of pluripotency. however, the experiments on the regeneration and reaggregation of dissociated cells, have shown that not all cells exhibit the properties of stem cells. sponges do not have well-established stem cell lineages. furthermore, presumable stem cells differ between four sponge classes. the most consistent model of the stem cell system is elaborated for fresh-water demospongiae. according to this model demosponges have two stem cell lineages: archaeocytes and choanocytes. both express the ortholog of the stem cell marker piwi and show the proliferation activity in the intact sponges (funayama, 2018). during regeneration in demosponges these cell types play an important role: they give rise to the new exopinacoderm and participate in the restoration of the choanosome structures. additionally, the archaeocytes and choanocytes have ability for (trans)differentiation to various cell types during the restoration processes after sponge tissue dissociation (borisenko et al. 2015; lavrov, kosevich, 2016). despite the importance of archaeocytes as stem cells of demosponges, there is still no ultrastructural characterization of this type of cell; moreover, there are contradictory and unclear interpretations of the morphology of this cell type. however, both calcarea and many homoscleromorpha do not have mesohyl cells similar to demosponges archaeocytes. in these sponge clades choanocytes and pinacocytes exhibit properties of polypotentiality, as follow from gametogenesis, experiments on regeneration, and 63 cell dissociation. these cells can directly transdifferentiate into other cell types without archaeocytes-like stage (ereskovsky et al. 2015; lavrov et al. 2018). finally, it is necessary to emphasize the importance of models diversification: the comparison between different sponge taxa may help to shed light on the diversity of stem cells in porifera and their properties. financial support by the russian science foundation n° 17-14-01089 is gratefully acknowledged. coral skeletal proteins and their function in mineral formation t mass university of haifa, department of marine biology, the leon h. charney school of marine sciences, mt. carmel, haifa 3498838, israel coral biomineralization is important at the organismal, ecosystem, and global scales, yet the biological component has not been well understood. in particular, identities, roles, and environmental susceptibility of the proteins retained in coral skeleton were previously unknown. understanding the cellular and molecular responses of stony corals to ocean acidification is key to predicting their ability to calcify under projected high co2 conditions. of specific interest are the links between biomineralization proteins and the precipitation of new calcium carbonate (caco3), which potentially can provide a better understanding of the biomineralization process. to address this, we developed a novel coral tissue cultures to investigate the biophysical mechanism of calcification in corals. our goals were to establish an experimental system in which calcification is facilitated at the cellular level, while simultaneously allowing in vitro manipulations of the calcifying fluid, and to study the mineral initiation mechanism in corals. viable cell cultures of the hermatypic, zooxanthellate coral, stylophora pistillata, have been maintained for 6 to 8 weeks. using an enriched seawater medium with aragonite saturation state which mimiks open ocean surface waters ( arag ~4). we have shown that within 72 hr after isolation, cultures of separated coral cells aggregate into proto-polyps and form an extracellular organic matrix (ecm) and precipitate aragonite crystals at a rate comparable to the intact organism and with geochemical properties similar to parent skeleton. responses of molluscan cells to ultra-low temperature exposure n odintsova, y kipryushina, m maiorova, k yakovlev, a boroda national scientific center of marine biology, far eastern branch of the ras, vladivostok, russia the study is focused on the alterations that occur in mussel larval cells both in standard culture conditions and in response to ultra-low temperature exposure. development of this direction is important for understanding the mechanisms of cold susceptibility of marine organisms. the main pathways of molluscan cell death have been found to be associated with mechanical cellular disruption caused by the freezing-thawing processes themselves, as well as with apoptosis and necrosis. apoptosis was not the main death pathway after a freeze-thaw cycle, but it was induced in a significant part of mussel cells immediately after thawing and depended mostly on the cryoprotectant used. nothing is known about the causes of apoptosis in mollusks now, although this phenomenon is described in different classes of mollusks. we suggested that the use of the apoptotic inhibitors, known to mammalian cells, could provide a higher yield of viable cells after thawing. for comparison, we used primary mouse embryonic fibroblasts and human colon tumor cells. additionally, the analysis of nuclear aberrations (such as few multipolar mitoses or the absence of a division spindle in mitotic cells) has been shown to be a useful tool for assessing cell disruption of molluscan cells regardless of the used cryoprotectant. the best cryoprotectant for bivalve cells was 5% dimethyl sulfoxide without any additives. only staurosporine resulted in evident apoptosis in molluscan larval cells. unfortunately, we did not reveal apoptotic inhibitors that could significantly reduce apoptosis in molluscan cells after freezing-thawing. this study was partially supported by the cost. reverse development and stem cells in the cnidarian turritopsis dohrnii r pennati1, mp miglietta2, y matsumoto2, s mercurio1, f bonasoro1, g scarì3, c gissi4, s piraino5. 1department of environmental science and policy, university of milan, italy 2department of marine biology, texas a&m university at galveston, usa 3department of biosciences, university of milan, italy 4department of biosciences, biotechnology and biopharmacology, university of bari, italy 5disteba department, university of salento, italy the medusa of the mediterranean jellyfish turritopsis dohrnii can revert into the preceding polyp stage, completing a full morph rejuvenation, by a process known as reverse development (rd). rd is achieved through different stages ending with the formation of a ball-like cyst from which eventually a new stolon will form. stem cell proliferation and differentiation are supposed to play key roles during rd. hydrozoans possess a population of stem cells, called interstitial cells, characterized by a large nuclear to cytoplasmic ratios and prominent nucleoli. cells with these characteristics are mainly present in the manubrium and in the canal system of t. dohrnii medusa, as revealed by tem analysis. in cnidarians, orthologs of c-myc and sox2 have been found expressed in stem cell lines of hydra polyps and in clytia hemisphaerica medusae and planula larvae. we identified homologues of these genes in t. dohrnii and studied their expression pattern. we found that 64 they are mainly expressed where i-cells are localized in the medusa. their expression is turned off during the early stages of rd, whereas transcripts can be detected again during late rd stages. moreover, by edu incorporation assays, we demonstrated that proliferation is an active process in the medusa and during the late stage of rd. our results suggest that stem cells proliferation and differentiation may play a role primarily during the cyst stage and the formation of the stolon. stem cells in marine invertebrates an overview b rinkevich national institute of oceanography, haifa, israel stem cells are unspecialized cells in multicellular organisms that have the capabilities to differentiate into other types of cells and can also renew themselves to produce the same type of stem cells. most interesting are the adult stem cells (ascs; somatic stem cells) that are found throughout the body after development, and in the vertebrates are the cellular tools used particularly to replenish dying cells and to regenerate damaged tissues (as multipotent cell types). in these organisms they form all/many cell types of the organ from which they originate. in contrast to what is known from the vertebrates, many marine invertebrates reveal significant different characteristics for ascs. these include high abundancy of ascs in marine invertebrates (up to 30% of total cells), pluriand even totipotency, very limited dependency on niches (also the unique appearance or transitory niches), the frequent dedifferentiation and transdifferentiation associated with ascs, the expression of germ cells markers in somatic stem cells (e.g., specific markers for the germ line are equally expressed in somatic stem cells, such as piwi; further highlighting that there are no boundaries between somatic and germ cells lineage in many marine invertebrates) and the consideration of ascs as units of selections and units of regeneration. in a wide range of marine invertebrates, ascs may emerge de novo and are contributors to dramatic changes in biological features, such as whole body regeneration, re juvenilization, torpor phenomena (hibernation and aestivation) and more. ascs in marine invertebrates may also reveal unique phenomena such as germ cell transformation to somatic stem cells and vice versa. ascs of marine invertebrates differ structurally from the typical ascs in the vertebrates and even between different marine phyla (such as the interstitial cells in hydrozoans, the neoblasts in flat worms, the archeocytes in sponges and the lymphocyte-like cells in tunicates), and there are unknown tumors of ascs, rarely any neoplastic or age-related diseases. the above and other characteristics may point to new perspectives for the evolutionary forces that dictate the development of ascs. tissue crosstalk is required to induce a potential stem cell based regenerative response in the anthozoan cnidarian nematostella vectensis a amiel, s ferreira, k foucher, e röttinger cnrs inserm, university côte d’azur – institute for research on cancer and aging, nice (ircan), france whole body regeneration in anthozoan cnidarians is poorly understood but has recently been investigated using the sea anemone nematostella vectensis. in this organism, while cell proliferation is required for regeneration, stem cells have yet to be identified. in order to highlight the location of the potential stem cells involved in regeneration in n. vectensis, we focused our efforts on a very detailed characterization of the regenerative capacities of various body parts. in addition, to gain a better understanding of the cellular mechanisms involved in the regenerative response, we analyzed the cellular behavior during regeneration using pulse and chase as well as irradiations experiments. our analysis revealed that body parts depleted of a specific structure are unable to regenerate. further, we have shown that this particular structure is essential for inducing proliferation at the amputation site. the analysis of cell behavior during regeneration suggests that two populations of potential stem cells, located in different structures of the animal body, are reactivated in response to the injury. taken together, our results strongly suggest that a tissue crosstalk is required to induce a potential stem cell based regenerative response in the anthozoan cnidarian n. vectensis. insights in the evolution of mechanisms controlling commitment of neural stem cells j duruz, r bruggmann, mjg trujillo-sprecher, sg sprecher department of biology, university of fribourg, fribourg, switzerland. commitment of stem or progenitor cells to undergo terminal divisions and differentiation is a critical step during development, tissue maintenance or regeneration. while the molecular processes that are involved in cell cycle control and cell growth are conserved among eukaryotes the processes that mediate the progressive commitment of stem cells and progenitors remains largely elusive. we have recently identified the zinc finger transcription factor glass to play a critical role in neural progenitors to mediate commitment towards a defined photoreceptor cell fate in the fruit fly drosophila melanogaster. in order to explore if this function of glass is evolutionarily conserved, we have analyzed the expression of glass in the marine 65 annelid platynereis dumerillii. interestingly in platynereis we could not detect expression of glass in photoreceptors. similarly, analyzing published single cell rnaseq data of the flatworm schmidtea mediterranea indicates that opsin genes are not coexpressed with glass. these findings suggest that in distinct animal clades different developmental mechanisms act to specify a similar neural fate. to further explore the diversity of neural stem cells and progenitors we have initiated a single-cell transcriptomic in cnidarians and acoels. application of nano titanium dioxide treated surfaces for marine organisms growth inhibition v vrecko cinkarna, celje, slovenija as producer of nanomaterial titanium dioxide we have been asked to search for the solution of slippery access to the sea on the concrete surfaces at the beaches. the challenge is to find a solution that could be applied to existing surfaces and would sufficiently slow the growth of marine organisms on the treated objects. we have prepared several mixtures of the fast setting cement based material and applied them to the demonstration object. to be able to understand the mode of action of the photocatalytic surfaces on the growth of marine organisms, we approached biotechnical faculty of university of ljubljana to help us in characterization and understanding of the desired and eventual unintentional effects of our materials. we plan to observe the dynamics of the growth of organisms in one year period. 1 isj 17: 1-10, 2021 issn 1824-307x research report a putative insulin receptor involved in immune response of chinese mitten crab eriocheir sinensis l wang1,3, h chen1, l qiu1, l wang1,2,4, l song1,2,4* 1key laboratory of experimental marine biology, institute of oceanology, chinese academy of sciences, qingdao 266071, china 2laboratory of marine fisheries science and food production processes, qingdao national laboratory for marine science and technology, qingdao 266235, china 3qingdao key laboratory for marine fish breeding and biotechnology, yellow sea fisheries research institute, chinese academy of fishery sciences, qingdao 266071, china 4liaoning key laboratory of marine animal immunology and disease control, dalian ocean university, dalian 116023, china this is an open access article published under the cc by license accepted december 9, 2020 abstract insulin plays important roles in metabolic homeostasis during environmental challenges. the insulin receptor is a key molecule to receive and transduce insulin signals. in the present study, a novel insulin receptor was identified from the chinese mitten crab eriocheir sinensis (designated as esir). the coding region of esir gene was 3573 bp in length and encoded 1190 amino acids with all the functional domains of mammal insulin receptors, including furin-like domain, receptor l domain, transmembrane domain, and tyrosine kinase domain. phylogenetic analysis showed that the esir shared the closest evolutionary relationship with the insulin receptor from macrobrachium rosenbergii. cell transfection experiments confirmed that esir proteins were localized on the cytomembrane. the mrna transcripts of esir were widely distributed in various tissues with higher abundance in hepatopancreas and eyestalk of e. sinensis. after aeromonas hydrophila stimulation, the expression level of esir mrna decreased from 3 h to 6 h, and then increased at 12 h. the conserved structure and subcellular localization of esir together with its sensitivity to a. hydrophila stimulation implied that esir was probably involved in immune response of e. sinensis. the present study provided clues for the further investigation about the evolution and function of the insulin signaling pathway in invertebrates. key words: aeromonas hydrophila infection; chinese mitten crab; immune response; insulin receptor introduction insulin plays important roles in metabolism, fecundity, growth, immunity, and aging (de meyts, 2004). the modulation effects of insulin are mediated primarily via the insulin receptor. this receptor belongs to the superfamily of tyrosine kinase receptors, and it is always located on cytomembrane (white and kahn, 1994). the binding of insulin to its receptor initiates a cascade of intracellular signal transduction, including autophosphorylation of tyrosine kinase domain and the interaction of multiple molecules with insulin receptor. the key molecules in the downstream pathway are the insulin ___________________________________________________________________________ corresponding author: linsheng song key laboratory of experimental marine biology institute of oceanology, chinese academy of sciences qingdao 266071 e-mail: lshsong@dlou.edu.cn receptor substrates (irss), which are protein substrates of the intrinsic tyrosine kinase activity of insulin receptor, transmitting the signal to downstream cascades (taniguchi et al., 2006). vertebrate insulin signaling pathway possesses single insulin and several insulin receptor family members, including insulin receptor, insulin-like growth factor receptor (igfr) and insulin receptor-related receptor (irr). however, the increasing evidences demonstrate that the insulin signaling pathway in invertebrates has unique characteristics. there are multiple copies of genes in their genome encoding insulin-like peptides (ilps) but only one copy of receptor and irs gene (mao et al., 2018b). the relative simplicity of the insulin signaling components, together with the diversification of ilp, implies the functional diversification of the insulin signaling pathway in invertebrates (guirao-rico and aguade, 2011). 2 compared to higher animals, invertebrates face more severe environmental challenges, such as frequent food shortages and pathogen infection (karpac and jasper, 2009). the activation of immune system and maintenance of homeostasis are energetically costly. therefore, the metabolic regulation to environmental stress is crucial for the long-term survival of invertebrate (broughton and partridge, 2009). as a crucial synthetic metabolic signaling pathway, insulin action is always inhibited in order to enhancing the resistance to environmental stress. for instance, bacterial infection leads to the activation of toll signaling in drosophila melanogaster, which suppresses the insulin signaling, extending the survival against bacterial pathogens (mccormack et al., 2016). loss-of-function for the insulin receptor homolog in caenorhabditis elegans larval dramatically increases the oxidative stress tolerance and adult lifespans compared to the wild-type counterparts (tatar, 2001). these studies collectively indicate that the insulin signaling pathway is critical for invertebrate survival during environmental stress. the chinese mitten crab eriocheir sinensis is an important aquaculture crustacean in asian areas (sang et al., 2016). it was found that ilp in e. sinensis participated in the immune response against aeromonas hydrophila infection by providing more glucose (wang et al., 2020). investigation of the potential metabolism and immune related genes, such as insulin receptor in e. sinensis, is necessary to elucidate the homeostasis regulation during stress resistance, which might be helpful to develop strategy for economic and efficient aquaculture. the purposes of this study were to (1) identify the insulin receptor homologue from e. sinensis (designated as esir), (2) characterize the its expression at subcellular and tissue levels, and (3) investigate its response against a. hydrophila stimulation to better understand the homeostasis regulation role of esir during the immune response. materials and methods crab and bacteria stimulation adult chinses mitten crabs eriocheir sinensis (about 50  5 g) were obtained from a commercial farm in qingdao, china and maintained in aerated freshwater at 25 °c for one week before the experiments. a total of 30 crabs were randomly divided into two groups for aeromonas hydrophila challenge experiment. the crabs in the control group received an injection of 50 l pbs, while the crabs in bacteria stimulation group received an injection of 50 l a. hydrophila suspension (3 × 106 cfu /ml, diluted in pbs). three individuals from each group were randomly sampled at 0, 3, 6, 12, and 24 h post challenge. the hepatopancreas tissue was collected and stored in liquid nitrogen for total rna extraction. in addition, the hepatopancreas, eyestalks, gills, muscles, stomach, hemolymph and hematopoietic tissues were collected from three crabs in control group at 0 h for gene cloning and tissue expression analysis. rna isolation and cdna synthesis total rna was extracted from the tissues using trizol reagent (invitrogen) according to the manufacture’s protocol. the rnase-free dnase i (promega) was used to digest the genomic dna from the total rna. first-strand cdna synthesis was carried out based on m-mlv reverse transcriptase using the total rna as template and oligo (dt)-adaptor as the primer (table 1). the reactions were incubated at 42 °c for 1 h and terminated by heating at 95 °c for 5 min. the cdna mixtures were diluted to 1:30 and stored at -80 °c for subsequent gene cloning and qrtpcr (qu et al., 2018). gene cloning and sequence analysis blastp analysis of all crab protein sequences revealed that a sequence (vn_glean_10002430, esir) was homologous to the insulin receptor table 1 nucleotide sequences of primers used in this study primer sequence (5’-3’) brief information adaptor primer ggccacgcgtcgactagtact17 oligo (dt) for cdna synthesizing esir-f1 atgcagcgctacaaccagat gene specific primer for cds esir-r1 acacggttgtctcactgcgg gene specific primer for cds esir-f2 taccggactcagatctcgagatgcagcgctacaaccagatc primer for vector constructing esir-r2 taccgtcgactgcagaattcgcacggttgtctcactgcggg primer for vector constructing esir-f3 ggcagagtcgccacagaacc gene specific primer for qrt-pcr esir-r3 agtgggtcggagcagtagcg gene specific primer for qrt-pcr β-actin-f gcatccacgagaccacttac internal control for qrt-pcr β-actin-r ctcctgcttgctgatccacatc internal control for qrt-pcr 3 table 2 the insulin receptors used in multiple alignment and phylogenetic analysis species protein accession number homo sapiens insulin receptor aaa59452.1 xenopus laevis insulin receptor cab46565.1 danio rerio insulin receptor b acc77575.1 ciona intestinalis insulin receptor xp_002125750.3 aplysia californica insulin receptor 2207309a drosophila melanogaster insulin receptor aac47458.1 anopheles gambiae insulin receptor xp_320130.3 bombyx mori insulin receptor np_001037011.1 macrobrachium rosenbergii insulin-like receptor akf17681.1 sinonovacula constricta insulin-like peptide receptor ayv97262.1 lymnaea stagnalis insulin-like peptide receptor caa59353.1 apostichopus japonicus insulin-like peptide receptor pik45733.1 acanthaster planci insulin-like peptide receptor xp_022110929.1 identified from other species (the threshold of e-value was 1 x 10-5). a pair of specific primers (table 1) was designed to amplify the full length cdna of esir from cdna library. the searches for protein sequences similarity of esir were conducted with blast algorithm at the national center for biotechnology information (https://blast.ncbi.nlm.nih.gov/blast.cgi). the expert protein analysis system (https://www.expasy.org) was used to analyze the deduced amino acid sequence. the protein domain was predicted with smart (http://smart.embl-heidelberg.de). multiple sequence alignment of the esir with other insulin receptors was performed with the online multiple alignment program (http://espript.ibcp.fr/espript/cgi-bin/espript.cgi) and optimized manually. a phylogenetic tree was constructed by the maximum likelihood algorithm with the seaview software based on the insulin receptors in different species (table 2) (gouy et al., 2010). the reliability of the branching was tested by bootstrap resampling (100 pseudo-replicates). plasmid construction, hek293t cell culture and transfection to assess the subcellular location of esir protein, the target encoding region of esir was amplified by primers (table 1) and inserted into p-egfp-n1 expression vector (transgene). hek293t cells were cultured in dulbecco’s modified eagle’s medium (d-mem, gibco brl, gaithersburg, md) supplemented with 15 % fetal bovine serum (fbs, transgene) at 37 °c and 5 % co2. the recombinant plasmid pegfp-esir was transfected into hek293t cells with lipofectamine ltxtm and plustm reagent (invitrogen). the control group was transfected with the p-egfp-n1 plasmid. after cultured at 37 °c for 48 h, the cells in the experimental group and the control group were washed, fixed with 4 % paraformaldehyde for 10 min, stained with the dii staining solution, and photographed under a laser confocal microscope (mao et al., 2018a). real-time fluorescence quantitative pcr (qrt-pcr) the qrt-pcr was carried out in an abi prism 7500 sequence detection system with a total volume of 10 μl. the primers used in the present study were listed in table 1. the fragment of crab actin gene was employed as an internal control. all data were given in terms of relative mrna expression using the 2−δδct method (schmittgen and livak, 2008). statistical analysis all data were given as means ± sd and subjected to one-way analysis of variance (one-way anova) followed by a multiple comparison (lsd). differences were considered significant (labeled with * or letters) at p < 0.05 or extremely significant (labeled with **) at p < 0.01. results molecular characteristics and multiple sequence alignments of esir a potential insulin receptor in e. sinensis (esir) was revealed by bioinformatics analysis, which was deposited in genbank under accession no. mn232176. the coding region of the esir was of 3573 bp and it encoding a peptide of 1190 amino acids. the predicted molecular size was 132.2 kda and the theoretical isoelectric point was 6.43. smart conserved domain analysis revealed that there were a furin-like domain (2-67 aa), a receptor l domain (82-209 aa), five fu domains (229-505 aa), a transmembrane domain (534-556 aa) and a tyrosine kinase domain (602-858 aa) in the deduced amino acid sequence of esir (fig. 1a). multiple https://www.expasy.org/ 4 5 fig. 1 structure prediction and multi-sequence alignment of esir. (a) structure prediction of esir by smart analysis, which contained a furin-like domain, a receptor l domain, five fu domains, a transmembrane domain (tm), and a tyrosine kinase domain (tyrkc). (b) multiple sequence alignment of esir extracellular region, transmembrane region and intracellular region with insulin receptors in other species. the red shadow region indicates all sequences share the same amino acid residue, and the blue box indicates the amino acids with similarity more than 50 %. gaps are indicated by dots to improve the alignment 6 fig. 2 phylogenetic relationship of the insulin receptors in different species alignments showed that esir exhibited relatively low similarity in the extracellular region, while shared high identity in intracellular region with other insulin receptors (fig. 1b). the phylogenetic analysis of esir phylogenic tree was constructed by the maximum likelihood method. all insulin receptors were clustered together according to phylum. esir was firstly clustered with the insulin receptor from macrobrachium rosenbergii, constituting a sub-branch of crustacean insulin receptors. this branch was then clustered with other arthropods insulin receptors. in addition, insulin receptor from urochordata shared closer relationship with vertebrate insulin receptor (fig. 2). subcellular localization of esir protein a recombinant pegfp-esir plasmid was constructed and transfected into well-growing hek293t cells and observed under a laser confocal microscope. the recombinant vector was successfully transfected into hek293t cells, and the signal of green fluorescent protein (green) was present throughout the cell. the positive signal of esir fusion protein with egfp (in green) was co-localized with the dii-stained cell membrane (in red) (fig. 3). distribution of esir mrna in different tissues qrt-pcr was performed to detect the distribution of esir mrna in different tissues of e. sinensis. the mrna transcripts of esir were detected in all the tested tissues, including hematopoietic tissue, stomach, muscle, gills, eyestalks and hepatopancreas, and hemocytes with the highest expression level in hepatopancreas, which was 94.00-fold (p < 0.05) of that in hematopoietic tissue. higher expression levels of esir mrna were also observed in eyestalks and 7 fig. 3 subcellular localization of esir in hek293t cells. (a) esir protein (green signal) was expressed on cell membrane. (b) dii (red signal) stained cells. (c) esir protein was co-located with dii stained cell membrane. (d) the transfected cells showed normal morphology. (e) control group egfp (green signal) expression in the whole cell. (f) the control group cells showed normal morphology gills, which were 43.70 and 41.15-fold (p < 0.05) of that in hematopoietic tissue, respectively. the expression levels of esir mrna in muscle, stomach and hemocyte were 27.10, 20.16 and 1.43-fold (p < 0.05) of that in hematopoietic tissue, respectively (fig. 4). temporal expression of esir mrna in hepatopancreas after a. hydrophila infection the expression of esir mrna in hepatopancreas changed significantly after a. hydrophila infection. it decreased firstly from 3 h (0.09-fold of that in control group, p < 0.01) to 6 h (0.52-fold of that in the control group, p < 0.05), then increased to 1.62-fold (p < 0.05) that of the control group at 12 h, and finally returned to normal level at 24 h (fig. 5). discussion the insulin receptors have been well studied deeply since the protein fragments on the cell membrane was first discovered to specifically bind insulin in 1970 (de meyts, 2004; house and weidemann, 1970). these evidences confirm that the insulin receptors regulate metabolic homeostasis in a systemic manner and reallocate energy during stress response. however, only a few insulin receptors have been described in crustacean species, and their roles in maintenance of homeostasis are far from well understood. in the present study, a homologue of insulin receptor (esir) was identified from the chinese mitten crab e. sinensis. the extracellular portion of esir protein contained a cysteine rich region with a furin-like domain, a receptor l domain and five fu domains, which were cysteine rich repeats (fig. 1a). this domain architecture in the extracellular portion has also been reported in many other invertebrates, such as m. rosenbergii and daphnia pulex (boucher et al., 2010). in vertebrate, the extracellular portion of ir consists of two l-domains, a cysteine rich region， and three fibronectin type iii (fniii) domains (hernandez-sanchez et al., 2008). most invertebrates possess more ilps, but only one insulin receptor (mao et al., 2018b). the unique domain composition in the extracellular region suggests that the ligand-receptor contact can be diverse in invertebrate. the intracellular portion is responsible for ligand-induced signal transduction and phosphorylation of second-messenger proteins inside cells (shu and steiner, 2000). the architecture of functional domains in this region of esir is same as that in other vertebrates. alignment of the esir with the other insulin receptors from invertebrates and vertebrates revealed that the 8 fig. 4 the expression of esir mrna transcripts in different tissues of e. sinensis detected by quantitative rt-pcr. different letters (a, b, c, d) represent statically significant differences (p < 0.05) intracellular components were less variable than the extracellular parts, indicating that the insulin signal transduction was conserved (fig. 1b). further evolutionary analysis showed that insulin receptors from different species were clustered together according to the phylogenetic relationship of the species. there was an independent replication event between chordate and invertebrate insulin receptors. in invertebrates, esir shared the closest homology with the insulin receptor in m. rosenbergii, and constituted a sub-branch with that of other arthropods (fig. 2). these results indicated the highly conservation of insulin receptors throughout evolution. the insulin receptors distribute in nearly all cells surface, where they specifically bind to insulin to activate intracellular signaling cascades and cause a series of physiological reactions, and no insulin receptor has been found in the cytoplasm (hernandez-sanchez et al., 2008). in the present study, the recombinant pegfp-esir plasmids were transfected into hek293t cells, and the esir protein was found to be localized on the cytomembrane of hek293t cells, which supported our assumption that the esir protein was an insulin-like membrane-bound receptor (fig. 3). together with the prediction of esir domain, it was speculated that esir was anchored to cytomembrane by the transmembrane domain. as important molecules in metabolic process, the insulin receptors are widely distributed in various tissues. esir mrna transcripts were detected in all examined tissues, indicating its basic physiological function (fig. 4). in crustacean, hepatopancreas functions crucially in carbohydrates metabolism while eyestalk plays an important role in synthesizing and secreting the endocrine hormones (roszer, 2014; nguyen et al., 2016). the higher expression levels of esir mrna in hepatopancreas and eyestalk implied the potential roles of esir in metabolism and endocrine. previous studies showed that the activation of toll-like signaling triggered by infection interfered with insulin signaling pathway in rat liver. the survival rate of d. melanogaster carrying loss-of-function for the insulin receptor increased after bacterial infection (karpac and jasper, 2009). these results implied that the insulin signaling pathway played important roles in antibacterial immune responses. in the present study, the expression of esir mrna in hepatopancreas decreased significantly from 3 h to 6 h post a. hydrophila stimulation (fig. 5). it was speculated that the activated immune response inhibited esir expression during this time, thereby limiting glycogen synthesis in hepatopancreas. these results were consistent with previous report that the mrna expression level of esilp decreased significantly in hepatopancreas of e. sinensis after a. hydrophila stimulation (wang et al., 2020). meanwhile, the decreased esir expression might also be involved in immune modulation during bacterial infection. it has been reported that a. hydrophila stimulation could significantly elevate the 9 fig. 5 the expression of esir mrna transcripts in hepatopancreas after a. hydrophila stimulation. statistical significance is indicated by single (p < 0.05) or double (p < 0.01) asterisks activity of phenoloxidase in e. sinensis (jia et al., 2018). the loss-of-function of insulin receptor was also found to promote melanization and phenoloxidase activity in drosophila (mccormack et al., 2016). it has been reported that the metabolic statuses (glycolysis/tca cycle) varied greatly in crustacean during the early or late stage of infection (su et al., 2014). compared to glycolysis, tca cycle costs less glucose for atp production. therefore, the upregulated esir at 12 h indicated a metabolic shift to promote the glucose transport and glycogen synthesis in hepatopancreas of the challenged crabs. these results collectively suggested that the insulin receptor (esir) played important roles in both metabolic and immune modulation during immune response. acknowledgement the authors were grateful to all the laboratory members for their technical advice and helpful discussions. this research was supported by national key r&d program (2018yfd0900606), a grant (no. 31530069) from national science foundation of china, the fund for outstanding talents and innovative team of agricultural scientific research, the distinguished professor of liaoning (to l. w.), aoshan talents cultivation program supported by qingdao national laboratory for marine science and technology (no. 2017astcp-os13), dalian high level talent innovation support program (2015r020), and the research foundation for talented scholars in dalian ocean university (to l. w.). references boucher p, ditlecadet d, dubé c, dufresne f. unusual duplication of the insulin-like receptor in the crustacean daphnia pulex. bmc evol. biol. 10: 305, 2010. broughton s, partridge l. insulin/igf-like signalling, the central nervous system and aging. biochem. j. 418: 1-12, 2009. de meyts p. insulin and its receptor: structure, function and evolution. bioessays 26: 1351-1362, 2004. gouy m, guindon s, gascuel o. seaview version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. mol. 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7: 85-96, 2006. tatar m. a mutant drosophila insulin receptor homolog that extends life-span and impairs neuroendocrine function. science 292: 107-110, 2001. wang l, chen h, wang l, song l. an insulin-like peptide serves as a regulator of glucose metabolism in the immune response of chinese mitten crab eriocheir sinensis. dev. comp. immunol. 108: 103686, 2020. white mf, kahn cr. the insulin signaling system. j. biol. chem.. 269: 1-4, 2016. isj 13: 281-290, 2016 isj 13: 281-290, 2016 issn 1824-307x research report the impact of zinc oxide nanoparticles in freshwater mussels exposed to municipal effluents gagné f, auclair j, trépanier s, turcotte p, pilote m, gagnon c aquatic contaminants research division, environment and climate change canada, 105 mcgill, montreal, quebec, canada. accepted august 12, 2016 abstract zinc oxide nanoparticles (nano-zno) are used in the production of transparent sunscreens and cosmetics, which are released into the environment through municipal effluents. the purpose of this study was to examine the toxicity of nano-zno to freshwater mussels (elliptio complanata) in the presence of municipal effluents. mussels were exposed for 21 days at 15 oc to 1 and 10 µg/l nanozno, and zncl2 in the presence of a physico-chemically treated municipal effluent (3 and 10 % v/v). after the exposure period and a 24 h depuration step, mussels were analyzed for free zn in gills, metallothioneins (mt), oxidative stress (production of malondialdehyde (mda) during lipid peroxidation), gonad alkali-labile phosphate (alp) levels and genotoxicity. gill mt levels were increased at 10 µg/l nano-zno and zncl2 and in the presence of the municipal effluent. mt levels were positively correlated with free zn in gills and negatively correlated with mda levels, indicating its involvement in the prevention of oxidative stress. however, mda levels were significantly related to dna damage in gills, indicating that mt induction did not prevent oxidative-mediated damage in cells. gonad alp levels were increased by exposure to zncl2 and to the highest concentration of municipal effluent. dna strand breaks were increased in mussels treated to nano-zno indepentely of municipal effluent. multivariate discriminant function analysis revealed that control mussels differed from mussels exposed to the municipal effluent and from those exposed to nano-zno or zncl2 alone. when the municipal effluent was added, changes in mda, mt and labile zn were produced and formed another cluster, suggesting a change in the toxicity of the municipal effluent in the presence of nano-zno. key words: zinc oxide nanoparticles; municipal effluent; freshwater mussels; oxidative stress; dna damage; metallothioneins; alkali-labile phosphates   introduction nanotechnology is an area of intense commercial interest and development. products derived from nanotechnology are used in the production of many consumer products, such as cosmetics, textiles and sunscreens (contado et al., 2015). with the growing presence of nanoproducts in personal care products, municipal effluents and urban runoffs, there are concerns that these compounds with emerging properties at the nanoscale could alter the toxic properties of complex mixtures such as effluents. indeed, ___________________________________________________________________________ corresponding author: françois gagné aquatic contaminants research division environment and climate change canada 105 mcgill, montreal, quebec, canada h27 2e7 e-mail: francois.gagne@canada.ca legitimate concerns have also been raised by the public and regulatory agencies about their safety to human health and ecosystems. given their strong uv absorptive capacity, sunscreens composed of zinc oxide nanoparticles (nano-zno) are currently used worldwide as effective protection against sunlight (cole et al., 2016). sunscreen lotions usually contain bulk suspensions of zno and titanium dioxide, which form a white film on the skin. however, lotions composed of nano-zno are transparent, while retaining their uv light absorption properties. nano-zno also possesses antimicrobial properties, an additional benefit. notwithstanding this, little is known about the release, fate and toxicity of these sunscreens at the present time. the toxic behaviour of nano-zno in complex matrices such as municipal wastewaters is not well understood. in addition, the zn content of municipal effluents was shown to be in the order of 10 -50 281   mailto:francois.gagne@canada.ca ug/l (gagnon et al., 2006) and a recent study on the acute toxicity of zno nanoparticles revealed that nano-zno could pose a risk to aquatic organisms (adam et al., 2015). the 5 % hazard concentration for nano-zno was 60 µg/l and 30 µg/l for zncl2 which suggests that municipal effluents enriched in zn nanoparicles could represent a risk to aquatic life. bivalves (clams and mussels) are considered at risk of contamination by suspended solids and colloids (canesi et al., 2012). they are sessile organisms which feed on suspended matter, which leads to the bioaccumulation of large quantities of particles, including nanoparticles and their aggregates. for example, a study on mytilus galloprovincialis has shown that copper oxide nanoparticles accumulate in the digestive gland, leading to lipid peroxidation (gomes et al., 2012). in another study, mussels exposed to cerium oxide nanoparticles and nano-zno accumulated large quantities of ce and zn at mg/g levels (montes et al., 2012). the toxicity of nanoparticles is due to more than the release of their components (i.e., leaching of zn2+ from nano-zno) (gagné et al., 2008a). indeed, toxic interactions could arise from the size, reactive surface properties and coatings of nanoparticles, in addition to the leaching of their components. recent studies have shown that nanoparticles can lead to oxidative stress and genotoxicity, which cannot be explained solely by the release of their components. oxidative stress involves the mobilization of iron(iii), copper(ii) and zn(ii), which are sequestered by metallothioneins (mt) (formigari et al., 2007; gagné et al., 2008b). the mobilization of ionic metals could lead to the production of reactive oxygen radicals, which could result in lipid peroxidation and dna damage through the formation of 8-oxoguanine (valko et al., 2006; rocha et al., 2015). in snails, a 3-week exposure to 7 mg/l of nano-zno lead to increased malondialdehyde and nitric oxide with decreased glutathione s-transferase activity in both the hemolymph and tissues (fahmy et al., 2014). exposure to nano-zno also increased total lipids and cholesterol levels in snails which suggest decreased mobilization of energy reserves in snails. however, in clams exposed to a more realistic concentration (10 ug/l) for 7 days of either nanozno or zncl2, dna damage in hemocytes were higher in the former form of zinc (marisa et al., 2016). significant increases in superoxide dismutase and catalase activities suggested that oxidative stress was at play in clams exposed to nano-zno but not for zncl2. recent studies have also shown that both nano-zno and municipal effluents could induce oxidative stress in aquatic organisms (gillis et al., 2014; gagné et al., 2015), but less is known about their combined effects in freshwater mussels downstream from municipal effluent discharge sites. the purpose of this study was to examine the toxicity of nano-zno and municipal effluents in freshwater mussels. mussels were exposed to increasing concentrations of municipal effluent and to nano-zno. additional mussels were exposed to zncl2 for comparison purposes. the levels of free zn in gills were examined in conjunction with mt, oxidative stress (as determined by the malondialdehyde (mda) assay for lipid peroxidation) and dna damage endpoints. the interaction of nano-zno and municipal effluent exposures was examined based on both the effluent and zn concentrations in freshwater mussels. materials and methods mussel handling and exposure to municipal effluents and zinc forms mussels (elliptio sp.) were collected by hand in a pristine lake in the laurentians under a provincial permit in june 2012. the mussels were transported dry at 4 oc and transferred to 300-l tanks filled with uv-treated dechlorinated city of montreal tap water. the mussels were held in the tanks at 15 oc under constant aeration for at least 6 weeks before initiating exposure. the mussels were fed three times a week with commercial coral reef feed enriched with pseudokirchneriella subcapitata algal suspensions (100x106 algae/ml). for the exposure experiments, n = 20 mussels were placed in 60-l tanks receiving a continuous flow (0.15 l/h) of physico-chemically treated municipal effluent from a city of 1.5 million people. the physical/chemical treatment consisted in reducing suspended matter down to the mg/l range by means of grid traps, flocculation (surfactants) and sieving. the exposure concentrations were 0, 3 and 10 % diluted in dechlorinated uv-treated city of montreal tap water (qc, canada). mussels were exposed to 1 and 10 µg/l zn as nano-zno or zncl2 using an “instillation” technique. to ensure contact of the zn solutions with the mussels, 60 and 600 µg of either nano-zno or zncl2 were dissolved in 20 ml of aquarium water and placed directly over the mussel during active filtratrion (each mussel received 1 ml over the siphons). control mussels received only the aquarium water. the mussels were held under static conditions for one hour prior to continuous exposure to the municipal effluent. this process was repeated every 3 days for 21 days. at the end of the exposure period, mussels were allowed to depurate in clean aquarium water overnight (12 h). morphological characteristics were determined for mussel weight and shell length. soft tissues, gills and gonad were dissected on ice and weighed. sex was determined by examination of gonad smears on glass slides under a binocular microscope at 200x magnification. the gills and gonad were homogenized on ice using a teflon pestle tissue grinder in 145 mm nacl containing 10 mm hepes-naoh, ph 7.4, 10 µg/ml aprotinin and 1 mm dithiothreitol. part of the homogenate was centrifuged at 15,000xg for 30 min at 4 oc and the supernatant (s15 fraction) was removed and stored at -85 oc until biomarker analyses. total proteins were determined using the protein-dye binding principle with standard solutions of bovine serum albumin for calibration (bradford, 1976). metal metabolism metal metabolism was characterized by monitoring changes in labile zn and metallothioneins (mt) in gill tissues. labile zn levels in tissues were determined using a fluorescent 282   probe method (gagné et al., 2008b). fluorescent probe tsq (n-(6-methoxy-8-quinolyl)-ptoluenesulfonamide) was prepared in 20 % dimethyl sulfoxide (in 5 mm kh2po4, ph 7.4, containing 125 mm nacl) at 50 µm concentration. a volume of 150 µl of the probe was mixed with 25 µl of the gill s15 fraction for 10 min and fluorescence readings were taken at 400 nm excitation and 485 nm emission (biotek instruments, usa). standard solutions of zinc sulfate were prepared for calibration. the data were expressed as ng zinc/mg protein. metallothionein (mt) levels in gills were determined using a modified spectrophotometric assay (viarengo et al., 1995; gagné et al., 2010). briefly, total mt levels were determined by the addition of a strong reducing agent, phosphine, in the s15 fraction for 15 min prior to the addition of ethanolchloroform solvent. the data were expressed as µmole of glutathione (gsh) per mg protein. data analysis the exposure experiment consisted of 20 mussels per treatment aquarium. tissue biomarkers were performed on all mussels (n = 10 mussels) using 2-way factorial analysis of variance [municipal effluent concentration, zinc concentration (for dissolved and nanoparticulate) and their interaction] after verifying for homogeneity of variance and normality using levene’s test and the shapiro-wilks test, respectively. post-hoc analysis was performed using fisher’s least square difference test. correlation analysis was also performed using the pearson product-moment method. the physiological changes induced by exposure to municipal effluent, nano-zno and zncl2 were determined using discriminant function and factor analysis methods. all statistical tests were performed using statistica software (version 8). significance was set at α = 0.05. results oxidative stress and dna damage the municipal effluent resulted from physicochemical treatment with the following characteristics (ph 7.8; conductivity 350 µsxcm-1; total ammonia 0.8 mg/l and dissolved organic carbon content 16 24 mg/l). previous investigation of the zinc content in the municipal effluent indicated that total zn content was in the range of 10 25 ug/l, i.e., in the same range of each of the applied zn forms. nano-zno were diluted in bidistilled water and the particles exhibited an initial size range of 50 ± 10 nm determined using dynamic light scattering, but the mean size increased to 440 ± 70 nm when diluted in aquarium water. this suggests aggregation of nanozno in the presence of tap water. no change in the zeta potential was observed (-35 mvolt). the general condition of the mussels was assessed based on the mussel’s weight/shell length (condition factor or cf) and the gonado-somatic index (gsi). in mussels exposed to nano-zno, the condition factor was affected only by zn exposure concentrations (fig. 1a). cf decreased at 10 µg/l for both forms of zn, while municipal effluent concentrations were not significant. however, the presence of municipal effluent removed the significance at 10 ug/l nanozno. in mussels exposed to zncl2, a two-way factorial anova revealed that zn concentration only was significant (p < 0.05) (fig. 1b). however, only a small decrease in cf was found in mussels exposed to 10 µg/l zn and to the 3 % municipal effluent concentration. in mussels exposed to nano-zno, the gsi was significantly affected by both the municipal effluent and by both forms zn (fig. 1c). the gsi was increased by 1 µg/l zn in the presence of 3 % and 10 % municipal effluent concentrations. in mussels exposed to zncl2 and municipal effluent, no significant change in the gsi was observed. there was no significant correlation between cf and gsi. malondialdehyde (mda) levels were determined for lipid peroxidation in gill homogenates using the thiobarbituric acid method (wills, 1987). a volume of 50 µl of the homogenate was mixed with 200 µl of 10 % trichloroacetic acid containing 1 mm feso4 and 50 µl of 0.67 % thiobarbituric acid and heated at 70oc for 10 min. the mixture was cooled and centrifuged at 10,000xg for 5 min. a 200-µl sample of the supernatant was transferred to a 96well dark microplate, and fluorescence readings were taken at 520 nm excitation and 600 nm emission. standard solutions of tetramethoxypropane (stabilized form of malondialdehyde) were prepared for calibration in the blank (homogenization buffer). results were expressed as µmole thiobarbituric acid reactants (tbars) per mg total protein in the homogenate. dna damage was determined using the alkaline precipitation assay (olive, 1988), which is based on the potassium detergent precipitation of dna proteins. protein-free dna strand breaks which remain in the supernatant were determined using fluorescence spectroscopy (at 360 nm excitation and 450 nm emission) in the presence of a special buffer to control for the interference of detergent traces (bester et al., 1994). the diluent consisted of 0.4 m nacl, 0.1 m tris-acetate, ph 8.2, 4 mm sodium cholate and 10 μm sybr® green dye. standard solutions of salmon sperm dna were prepared for calibration. the data were expressed as µg dna strand/mg total protein in the homogenate. the levels of vitellogenin-like proteins were determined in the gonad of both male and female mussels as described elsewhere (gagné, 2014). briefly, the s15 fraction was treated with 35 % acetone at 4 oc and the high molecular weight proteins were precipitated at 10,000xg for 5 min at 4 oc. the protein pellet was washed in 50 % acetone and treated with 1 m naoh for 30 min at 40 oc. the released phosphates were determined using the phosphomolybdate methodology at 640 nm absorbance (stanton, 1968). the data were expressed as µg of inorganic phosphates/g gonad. metal metabolism was assessed by measuring changes in labile zn and mt expression in gills (figs 2a-d). in mussels exposed to nano-zno and the municipal effluent, a significant interaction between municipal effluent and zn was obtained (fig. 2a). labile zn was increased at the 3 % effluent concentration and returned to control levels 283   fig. 1 morphological characteristics of mussels co-exposed to municipal effluent and both forms of zn. the condition factor was determined by the mussel total weight / shell length ratio for nano-zno (a) and zncl2 (b) and municipal effluent. the gonado-somatic index is reported in mussels treated with nano-zno (c) and zncl2 (d) and municipal effluent. the letter “a” indicates significant difference from controls while the letter “b” indicates significance from municipal effluent concentrations. the data are expressed as the mean with standard deviation from n = 8 mussels. at the 10 % municipal effluent concentration owing to the high organic charge of the municipal effluent at this concentration. no significant change in labile zn was observed for either form of zn. in mussels exposed to zncl2 and the municipal effluent, a significant interaction between zn and municipal effluent exposure concentrations was observed, with the municipal effluent concentration being the dominant factor. labile zn was significantly increased in mussels exposed to the 3 % municipal effluent concentration and to 1 µg/l and returned to control levels at 10 µg/l zn and 10 % municipal effluent. both forms of zn reduced the increase observed with the 3 % municipal effluent concentration and produced no changes on their own. mt levels in gills were determined in mussels exposed to the municipal effluent and to the two forms of zn. in mussels exposed to nano-zno and the municipal effluent, a significant interaction between the municipal effluent and zn concentration was obtained, with the municipal effluent concentration being the dominant factor. mt levels were significantly increased with nano-zno at 1 and 10 µg/l zn in the absence of municipal effluent. when municipal effluent was present, the increase resulting from exposure to nano-zno was eliminated owing to the complex nature of the effluent matrix. the municipal effluent alone was able to significantly increase gill mt in mussels. in mussels exposed to zncl2 and the municipal effluent, a positive interaction between the exposure concentration of zn and the municipal effluent concentration was obtained. in the absence of municipal effluent, mt levels were significantly increased at 10 µg/l, but this effect was lost when the municipal effluent was present. correlation analysis (table 1) revealed that mt levels were significantly correlated with labile zn (r = 0.36; p = 0.001) and gsi (r = -0.31; p < 0.01). gonadal vitellogenin-like proteins (vtg-like proteins) levels were determined as an indirect assay for based on alp levels in high molecular 284   fig. 2 metal metabolisms in gills of mussels exposed to municipal effluent, nano-zno and zncl2. labile zn was determined in gills for nano-zno (a) and zncl2 (b) and municipal effluent. the gonado-somatic index is reported in mussels treated with nano-zno (c) and zncl2 (d) and municipal effluent. the letter “a” indicates significant difference from zn controls while the letter “b” indicates significance from municipal effluent concentrations. the data are expressed as the mean with standard deviation from n = 8 mussels. weight proteins (figs 3a-d). the data revealed that alp levels marginally differ between males and females (0.1<p<0.05), with alp levels somewhat higher in females than in males in control mussels. mussels were at the post-spawning/resting phase, which explains the absence of differences between males and females. in addition, the presence of vtg-like proteins was also investigated by high resolution gradient gel electrophoresis and silver staining (results not shown). it corroborated the changes in alp levels. alp levels were significantly induced at the 10 % municipal effluent concentration in both males and females, whereas nano-zno had no significant effect on alp levels in males. vtglike proteins where increased by nano-zno and zncl2 but only for the lowest concentration of 1 µg/l zn. however, a significant increase in vtg-like proteins levels was observed in male and female mussels exposed to 1 µg/l zncl2, but the levels returned to control values when the municipal effluent was present. correlation analysis revealed that vtg-like proteins were not significantly correlated with any of the parameters tested, which suggests that the estrogenic nature of the municipal effluent does not explain the changes observed with the other endpoints. the extent of tissue damage was assessed by mda and dna strand breaks in gills (figs 4a-d). in mussels exposed to nano-zno and the effluent, both factors were significant for mda levels in gills, with the zn concentration being the dominant factor. mda levels were decreased at the 10 % municipal effluent concentration, and this decrease was lost in the presence of nano-zno (fig. 1a). in mussels exposed to zncl2 and the municipal effluent, only the municipal effluent concentration was significant in respect of gill mda levels (fig. 1b). mda levels were significantly reduced at the 10 % effluent concentration, but this decrease was lost in the presence of 1 and 10 µg/l zncl2. correlation analysis revealed that gill mda was significantly correlated with mt in gills (r = -0.27; p = 0.01). dna strand breaks were also determined in mussel gill tissues. in mussels exposed to nanozno and municipal effluent, only the zn concentration was significant (fig. 4c). dna strand 285   table 1 correlation analysis of biomarker data condition factor gsi mt gills dna damage gills mda gills alp gonad free zn gills condition factor 1 -0.10 p>0.1 -0.02 p>0.1 0.02 p>0.1 0.02 p>0.1 0.16 p=0.1 -0.01 p>0.1 gsi 1 -0.32 p=0.001 -0.05 p>0.1 0.01 p>0.1 -0.12 p>0.1 -0.07 p>0.1 mt gills 1 -0.15 p>0.1 -0.27 p=0.01 -0.07 p>0.1 0.36 p<0.001 dna damage gills 1 0.54 p<0.001 0.14 p>0.1 -0.08 p>0.1 mda gills 1 -0.11 p>0.1 0.15 p>0.1 alp 1 -0.11 p>0.1 breaks were increased in mussels exposed to 10 µg/l zn only and to the 10 % municipal effluent concentration. in mussels exposed to zncl2 and the municipal effluent, exposure to zn was the significant factor (fig. 4d). the levels of dna strand breaks were increased in mussels exposed to 1 and 10 µg/l zn in the presence of the 10 % municipal effluent concentration. correlation analysis revealed that dna strand breaks were significantly correlated with mda in gills (r = 0.53; p < 0.001), indicating that oxidative stress contributed to dna damage. this was further supported by analysis of covariance of dna strand breaks against mda levels, which showed that only mda levels were significant and not the exposure concentrations of nano-zno, zncl2 and municipal effluent. in the attempt to gain an overall picture of the effects of municipal effluent and each form of zn, discriminant function and factor analysis was used (fig. 5). the total variance of the biomarker data was explained at 86 % with a mean classification efficiency at 53 %, which suggests some similarities between the responses for nano-zno, zncl2 and municipal effluent alone and in combination. the municipal effluent response patterns differ markedly from the controls and both forms of zn and involved the following effects: free zn, cf, dna damage, mt, gsi and alp (vitellogenin-like proteins). the response pattern for nano-zno and zncl2 was more closely related, which suggests a common mechanism, perhaps the involvement of dissolved zn2+ or low molecular size zn. this holds true when the municipal effluent was present where the effects are associated with the first component on the x axis: mt, alp and gsi. when each form of zn was present with the municipal effluent, a different response pattern was observed, which was explained by the x axis with the following major biomarkers: mt, mda and labile zn. it is noteworthy that the combined exposure effects were more closely related to either nano-zno or zncl2 than to the municipal effluent, indicating that zn concentration-mediated effects were generally more predominant in the presence of municipal effluent. discussion in the present study, mussels were exposed to two forms of zn-nano-zno and zncl2-both in the presence and absence of a primary physicochemically treated effluent. nanoparticles with low zeta potential (between -40 to 40 mvolts) are expected to form aggregates at low salt concentrations found in freshwater (gagné et al., 2015). the surface charges of the nanoparticles could be canceled by salts in the media which results in aggregation. in mussels exposed to nanozno, the following changes were observed: decreased cf and mda levels with increased gsi, mt and dna strand breaks. the induction of mt suggests the mobilization of zn ions or reactive oxygen species given the dual role of mt in sequestration of divalent ions and reactive oxygen species (gagné et al., 2008b). given that mt levels were negatively correlated with mda levels and positively correlated with free zn in gills, the data suggest that mt was involved in the prevention of oxidative stress and the mobilization of free zn in gills. this was also observed in another recent study with the freshwater mussel unio tumidus (falfushynska et al., 2015). the effects of nano-zno on mt levels and oxidative stress end points, such as protein carbonyl levels and superoxide dismutase, revealed that protein carbonyl levels were also decreased, suggesting the involvement of oxidative stress in addition to the release of zn from the nanoparticles. in another study, exposure of pacific oysters crassostrea gigas to nano-zno led to oxidative stress and mitochondrial dysfunction (trevisan et al., 2014). the study revealed that gills were the primary target for nano-zno owing to its function in trapping fine particles and directing them to the digestive system. increased mda levels and glutathione-dependent peroxidase with lower glutathione reductase activities in addition to ultrastructural alterations of mitochondria were also observed in gills. finally, in a previous study with elliptio complanata, exposure to nano-zno also led to increased mt and mda levels in the digestive 286   fig. 3 levels of vitellogenin-like proteins in male and female mussels exposed to municipal effluent and nanozno. the levels of alkali-labile phosphates were determined in the gonad of mussels exposed to each form of zn and to the municipal effluent. the letter “a” indicates significant difference from zn controls. the letter “b” indicates significance from municipal effluent controls. the data are expressed as the mean with standard deviation from n = 4 males and females mussels. gland (gagné et al., 2013). the study revealed that the metallome profile in the digestive gland of mussels exposed to nano-zno differed from that of mussels exposed to zncl2. these studies support the hypothesis that nano-zno toxicity is not associated solely with the release of zn2+ ions in tissues. nano-zno, but not zncl2, also caused genotoxic effects in mussel gills related to mda levels in gills. exposure to municipal effluent decreased gill dna breaks which suggest reduced dna repair activity. exposure to both forms of zn and to the municipal effluent led to an increase in dna strand breaks, which suggests that increasing effects on dna strand breaks in mussel exposed to either form of zn were maintained in the presence of the municipal effluent. there are few studies in the literature on the genotoxicity of nanozno in mussels. nano-zno was found to cause genotoxic effects in the fruit fly drosophila melanogaster (carmona et al., 2015). in larvae hemocytes, a significant increase in both dna damage and mda levels was observed for nanozno, which suggests genotoxicity and involves the production of reactive oxygen species. nano-zno genotoxicity was shown to be size dependent, with small nanoparticles (26 nm diameter) not being genotoxic while larger nanoparticles (78 and 147 nm diameter) induced micronuclei formation in human lymphoblastoid cells (yin et al., 2015). mussels exposed to municipal effluents had elevated levels of alp, an indirect assay for vitellogenin-like proteins in e. complanata mussels. alp levels were induced for mussels exposed to 1 µg/l of nano-zno and zncl2, but not to 10 µg/l. the increasing effect in alp was lost when the municipal effluent was present, which suggests a dampening effect of the zn-induced increase in alp by the municipal effluent. an explanation for this increase is lacking at the present time. one possibility is that production of vitellogenin-like protein was somehow enhanced at the low zn concentration given that vitellogenin synthesis involves the mobilization of zinc and binds zn in oocytes (thompson et al., 2001). increased vitellogenin gene expression was also observed in sexually-immature rainbow trout exposed to cdbased quantum dots (gagné et al., 2010). interestingly, gene expression of estradiol receptor 287   fig. 4 biomarker of tissue damage in mussels exposed to municipal effluent and zn. gill lipid peroxidation was determined in mussels exposed to nano-zno (a) and zncl2 (b) and to municipal effluent. the levels of dna strand breaks are also shown in mussels exposed to nano-zno (c) and zncl2 (d) and to municipal effluent. the letter “a” indicates a significant difference from zn controls. the letter “b” indicates significance from municipal effluent concentrations. the data are expressed as the mean with standard deviation from n = 8 mussels. was also more strongly expressed which confers some endocrine-disrupting activity of cd and znbased nanoparticles. further research will be necessary to understand the mode of action of increased vitellogenin signaling and to confirm whether nano-zno or zncl2 could induce vitellogenesis in mussels. it is noteworthy that both the municipal effluent and nano-zno led to increased mt levels, but the effects were not additive. the maximum induction of mt for nanozno alone was about twice that in the controls, compared to 1.6 to 2.1 fold in mussels exposed to the municipal effluent (3 and 10 %) and 10 µg/l nano-zno. induction of mt in the mussel meretrix meretrix exposed to treated municipal effluent was observed (wan et al., 2015). signs of oxidative stress were also observed through increased superoxide dismutase and catalase activity and mda levels. increased mt levels and oxidative stress were associated with increased concentrations of pb, cr and zn in lasmigona costata mussels collected downstream of municipal discharges, which shows an impact directly in the receiving environment (gillis et al., 2014). hence, disruption of metal metabolism and oxidative stress is likely to occur in mussels exposed to nano-zno through municipal effluent. in conclusion, exposure to nano-zno for 21 days leads to a series of changes in freshwater mussels: decreased cf, increased mt, increased alp and increased dna strand breaks. for mussels exposed to municipal effluent for 21 days, the following biomarkers were significantly influenced: gill labile zn (increase), mt (increase), alp (increase), and mda (decrease). the biomarkers mt and alp (vitellogenin-like proteins) were therefore affected by both the municipal effluent and zn, which could reach saturation in their response and overspills to other physiological targets such as labile zn and mda levels in gill tissues for mt. there is no evidence of overspill effects for alp levels since no correlation with the other biomarkers was observed. the toxicity of municipal effluent to freshwater mussels could be modified by the addition of nano-zno. in conclusion, the effects of nano-zno could change in the presence of municipal effluents for freshwater mussels. when alone, the effects of me differ from those of nano-zno, zncl2 and controls. the effects of 288   fig. 5 discriminant function of biomarker responses in mussels exposed to municipal effluents and zn. the total variance was explained at 85 % with both components. the biomarkers in parentheses are the three most correlated biomarkers with each of the two components of the x and y axis. nano-zno were more similar to those of zncl2 based on free zn levels, mt levels and dna damage. the levels of vtg-like proteins were influenced by both 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http://www.ncbi.nlm.nih.gov/pubmed/?term=mccall%20mj%5bauthor%5d&cauthor=true&cauthor_uid=26248212 http://www.ncbi.nlm.nih.gov/pubmed/?term=fenech%20m%5bauthor%5d&cauthor=true&cauthor_uid=26248212 http://www.ncbi.nlm.nih.gov/pubmed/26248212 http://www.ncbi.nlm.nih.gov/pubmed/?term=yu%20lp%5bauthor%5d&cauthor=true&cauthor_uid=21611643 http://www.ncbi.nlm.nih.gov/pubmed/?term=fang%20t%5bauthor%5d&cauthor=true&cauthor_uid=21611643 http://www.ncbi.nlm.nih.gov/pubmed/?term=xiong%20dw%5bauthor%5d&cauthor=true&cauthor_uid=21611643 http://www.ncbi.nlm.nih.gov/pubmed/?term=zhu%20wt%5bauthor%5d&cauthor=true&cauthor_uid=21611643 http://www.ncbi.nlm.nih.gov/pubmed/?term=sima%20xf%5bauthor%5d&cauthor=true&cauthor_uid=21611643 http://www.ncbi.nlm.nih.gov/pubmed/21611643 25 isj 16: 25-33, 2019 issn 1824-307x research report the first identification of a malectin gene (cfmal) in scallop chlamys farreri: sequence features and expression profiles mq wang 1,3 , bj wang 1 , m liu 1 , ky jiang 1 , l wang 1,2,4* 1 cas key laboratory of experimental marine biology, institute of oceanology, chinese academy of sciences, qingdao 266071, china 2 laboratory for marine biology and biotechnology, national laboratory for marine science and technology, qingdao 266237, china 3 research platform for marine molecular biotechnology, national laboratory for marine science and technology, qingdao 266237, china 4 cas center for ocean mega-science, chinese academy of sciences, qingdao 266400, china accepted march 05, 2019 abstract malectin is a newly discovered lectin of the endoplasmic reticulum (er) that might be involved in innate immunity. information about the roles of malectin in innate immunity is scarce. in the present study, a novel malectin gene (designated as cfmal) from the zhikong scallop chlamys farreri was identified and characterized. sequence features, tissue distribution, and temporal expression profiles were investigated to infer the potential functions of cfmal in innate immunity. the complete cdna sequence of cfmal comprised 1,111 bp and contained an open reading frame of 909 bp, which encoded 302 amino acid residues. a malectin domain and a transmembrane region were identified in the predicted protein sequence. cfmal mrna transcripts were detectable in hemocytes, muscle, mantle, gill, hepatopancreas, and gonads. cfmal expression was highest in hemocytes. stimulation with vibrio splendidus increased cfmal expression in hemocytes, gill, and hepatopancreas. the mrna transcripts of cfmal and three related genes, including binding immunoglobulin protein, heat shock protein 90 kda β member 1 protein and er degradation enhancing α-mannosidase like protein 1, increased in scallop hemocytes during an artificial er-stress. our results indicate that cfmal might not only be involved in er-stress, but may also play a role in innate immunity of scallops. key words: chlamys farreri; innate immunity; malectin introduction lectins are a large family of evolutionally conserved proteins that bind terminal sugars of glycoproteins or polysaccharides; they act as pattern recognition receptors (prrs) of the innate immune system (weis and drickamer, 1996). based on carbohydrate ligands, subcellular localization, and dependence on divalent cations, animal lectins could be classified into several groups including c-type, f-type, i-type, l-type, p-type, s-type (also known as galectin), and x-type lectins (also known as intelectins), discoidins, and pentraxins (also known as pentaxins) (dahms and hancock, 2002; kilpatrick, 2002; arasu et al., 2013; jia et al., 2016; shao et al., ___________________________________________________________________________ corresponding author: lei w ang cas key laboratory of experimental marine biology institute of oceanology chinese academy of sciences qingdao 266071, china e-mail: wanglei@qdio.ac.cn, leiwang@qdio.ac.cn 2018; tian et al., 2018; wang et al., 2016a; wei et al., 2018). lectins localized in the endoplasmic reticulum (er) are termed as er-resident lectins (aebi et al., 2010). most of these ubiquitous lectins participate in host-pathogen interactions and in immunomodulation (cambi et al., 2005). malectin is a highly conserved membrane-anchored er-resident lectin; it was first identified in xenopus laevis in 2008 and specifically recognized glc2man9glcnac2 (g2m9) in newly synthesized glycoproteins (schallus et al., 2008). accumulating research shows that malectin is induced by er-stress and is associated with folding defective glycoproteins to reduce their secretion (galli et al., 2011; yang et al., 2018). what’s more, de novo characterization of the spleen transcriptome of the large yellow croaker pseudosciaena crocea stimulated with polyinosinic:polycytidylic acid (poly ic) revealed that malectin might be involved in antiviral responses (mu et al., 2014). moreover, 26 three genes, including binding immunoglobulin protein (bip, also known as 78 kda glucose regulated protein), heat shock protein 90 kda β member 1 protein (also known as 94 kda glucose-regulated protein, grp94), and er degradation enhancing α-mannosidase like protein 1 (edem1), were previously reported that exhibited close relationship with mal during er stress (galli et al., 2011; qin et al., 2012; merulla et al., 2013). in scallops, grp94 might play an important role in the innate immune defense of the yesso scallop patinopecten yessoensis (also known as mizuhopecten yessoensis) (wang et al., 2018c). however, the potential roles of malectin in innate immunity are still unclear. scallops represent an important aquaculture species with commercial, ecological, and evolutionary importance (matozzo, 2016; tascedda and ottaviani, 2016; gerdol, 2017; jielian et al., 2017). as invertebrates, scallops lack clonally derived immunoglobulins and t-lymphocytes based adaptive immunity, and depend on their innate immune system to eliminate non-self-particles and to kill invading pathogens (song et al., 2015). in the past two decades, many prrs have been identified in marine scallops, especially in the bay scallop argopecten irradians and the zhikong scallop chlamys farreri. these prrs include c-type lectin (mu et al., 2012), galectin (song et al., 2011), lipopolysaccharide (lps) and β-1,3-glucan binding proteins (su et al., 2004), leucine-rich repeat-only proteins (wang et al., 2017), peptidoglycan recognition proteins (ni et al., 2007), scavenger receptors (liu et al., 2011), thioester containing proteins (zhang et al., 2007), and toll-like receptors (wang et al., 2011). these research achievements have enhanced the understanding of the potential functions of these prrs in invertebrate innate immunity (song et al., 2015). therefore, in the current study, we used zhikong scallops to explore the potential roles of malectin in invertebrate innate immunity. we identified a malectin gene from c. farreri (designated as cfmal) and we analyzed its expression induced by various stimuli, which indicated its potential role in innate immunity. the main purposes of our present work were: (1) to describe the sequence features of cfmal; (2) to investigate the expression profiles of cfmal; and (3) to predict the potential functions of cfmal in innate immunity. materials and methods scallops, in vivo vibrio stimulation, and sample collection adult scallops (average 5 cm in shell length) were collected in a local farm in qingdao, china, in summer; they were maintained in aerated seawater at approximately 20 °c. vibrio splendidus strain jz6, which has been proved to be a main kind of pathogens for scallop and widely used for the stimulation (wang et al., 2019a; wang et al., 2019b), was cultured in liquid 2216e media (hb0132, hopebiotech, china) at 28 °c with shaking at 180 rpm overnight. bacteria were collected by centrifugation at 4000 g for 20 min, and then re-suspended in filtered seawater. fifteen scallops were immersed for 12 h in filtered water containing live v. splendidus at a final concentration of 1.0 × 10 8 colony forming units per ml at 20 ºc, which constituted the vibrio stimulation group. hemocytes, muscle, mantle, gill, hepatopancreas and gonads from both infected and control scallops were collected for cfmal mrna expression analysis. primary cultured hemocytes, in vitro er-stress induction, and sample collection primary cultures of scallop hemocytes were prepared as previously described (each time point has 5 repetitions, and each repetition was a mixture of 3 individuals, wang et al., 2014). briefly, the hemolymph was withdrawn using a sterile syringe from the adductor muscle and diluted (1:3) in modified anticoagulant alsever's solution (3.36 g·l −1 edta, 20.8 g·l −1 glucose, 22.5 g·l −1 nacl and 8 g·l −1 sodium citrate, ph = 7.0, 1000 mosm). approximately 1.0 × 10 5 scallop hemocyte cells were suspended in 200 μl complete dulbecco’s modified eagles medium (high glucose, fi101, transgenbiotech, china) supplemented with 10% transserum eq fetal bovine serum (fs201, transgenbiotech, china), 10% scallop serum, 100 u·ml −1 penicillin and 100 μg·ml −1 streptomycin (fg101, transgenbiotech, china). cells were added to tc-treated multiple well plates (24 wells, cls3527, corning costar, usa) and incubated for 12 h at 21 °c in 5% co2. thapsigargin (tg, t9033, sigma-aldrich, usa), tunicamycin (tun, 654380, sigma-aldrich, usa) and lps (l2630, sigma-aldrich, usa) were added to corresponding wells at a final concentration of 300 ng·ml −1 , 10 μg·ml −1 , and 10 ng·ml −1 , respectively. these stimuli were considered er-stress induction groups, according to previous reports (urano et al., 2000; yoshida et al., 2001; wang et al., 2015). among them, tg specifically could inhibit the fusion of autophagosomes with lysosomes; the last step in the autophagic process. the inhibition of the autophagic process in turn induces stress on the er which ultimately leads to cellular death (ganley et al., 2011). tun is an inhibitor of glycosylation that disturbs protein folding machinery in eukaryotic cells. tun causes accumulation of unfolded proteins in cell er and induces er stress (namia et al., 2016). while the expression of ccaat/enhancer binding protein (c/ebp) homologous protein (chop), which is an er stress-induced transcription factor, induces apoptosis. and a previous study demonstrated that lps-induced chop expression does not induce apoptosis, but activates a pro-il-1β activation process (nakayama et al., 2009). untreated primary cultures of scallop hemocytes were used as a control. cells from each experimental group were sampled at 0, 3, 6, 12, 24 and 48 h after stimulation. rna isolation, cdna synthesis, and full-length cdna cloning total rna was isolated using transzol up (et111, transgenbiotech, china). first-strand cdna was synthesized using easyscript first-strand cdna synthesis supermix (at301, transgenbiotech, china) with dnase i (rnase-free, gd201, transgenbiotech, china). raw rna was used as template, and adaptor primer-oligo (dt) as 27 table 1 primers used in the present research primer sequences (5`-3`) brief information adaptor primer ggccacgcgtcgactagtac anchor primer for 3` race adaptor primer-oligo (dg) ggccacgcgtcgactagtacg10hn anchor primer for 5` race adaptor primer-oligo (dt) ggccacgcgtcgactagtact17vn olido (dt) for cdna synthesizing cfef-1α-qrt-f atccttcctccatctcgtcct internal control for qrt-pcr cfef-1α-qrt-r ggcacagttccaatacctcca internal control for qrt-pcr cfmal-race-f1 gcctccgatgacaccagcacc gene specific primer for race cfmal-race-f2 ctctgtaaactgtaaatatcagatcagggg gene specific primer for race cfmal-race-r1 cttgcatatacacggctacacgccgac gene specific primer for race cfmal-race-r2 gcgaatgtccaggaagtgcggctc gene specific primer for race cfmal-cds-f atggcgctgcgagccgca gene specific primer for cds cfmal-cds-r ttacagtttacagaggcagaagaggagagg gene specific primer for cds cfmal-qrt-f agattcgctcaaagtcggg gene specific primer for qrt-pcr cfmal-qrt-r cgctgagtgggatttcgt gene specific primer for qrt-pcr cfbip-qrt-f ggtcttcttcaggtatcagcag gene specific primer for qrt-pcr cfbip-qrt-r cttatcttcctcagcaaacatttcc gene specific primer for qrt-pcr cfgrp94-qrt-f tcccagacgacgaacttaatcca gene specific primer for qrt-pcr cfgrp94-qrt-r gttacccattattgccagagtgtcc gene specific primer for qrt-pcr cfedem1-qrt-f agcaccagttaaggattctaatgtt gene specific primer for qrt-pcr cfedem1-qrt-r ccacttcctccatacgacttg gene specific primer for qrt-pcr m13-47 cgccagggttttcccagtcacgac vector primer for sequencing rv-m gagcggataacaatttcacacagg vector primer for sequencing *the efficiency of cfef-1α-qrt-f/r, cfmal -qrt-f/r, cfbip-qrt-f/r, cfgrp94-qrt-f/r and cfedem1-qrt-f/r were 98%, 103%, 101%, 97% and 99%, respectively primer (table 1), according to the manufacturer instructions. subsequently, a homopolymeric tail was added using terminal deoxynucleotidyl transferase (ep0161, thermofisher, usa) and dctp (10217016, thermofisher, usa). we previously identified a malectin homologue sequence of c. farreri by using available public transcriptomic data (wang et al., 2018b). and it was selected to clone the full-length cdna sequence of cfmal. gene-specific primers, cfmal-race-r1/2 and cfmal-race-f1/2 (table 1), were designed using primer premier 5.00 to obtain the full-length cdna sequence of cfmal using the rapid-amplification of cdna ends (race) method. all pcr reactions were performed in an mj mini personal thermal cycler (bio-rad, usa). the pcr products were directly ligated into the peasy-t3 cloning vector (ct301, thermofisher, usa). after transformation into phage resistant chemically competent cell escherichia coli strain trans1-t1 (cd501, transgenbiotech, china), positive recombinants were selected using transcult lb agar plates (ampicillin, cp111, transgenbiotech, china) and verified by pcr screening with vector primers m13-47 and rv-m (table 1). three positive clones were sequenced in a 3730xl automated sequencer (thermofisher, usa) by genscript biotech (nanjing) inc. bioinformatics analysis of cdna and protein sequences the protein sequence of cfmal was deduced and analyzed by the editseq module of lasergene 7.1.0.44. cfmal and the three related genes were identified from the genome of c. farreri using blast+ 2.8.0, as described previously (galli et al., 2011; wang et al., 2007). the protein sequence similarity search was also conducted by blast+ 2.8.0. the presence and location of signal peptides and functional domains were predicted using signalp 4.1 and simple modular architecture research tool (smart) 8.0. a phylogenetic tree was generated with mega-x 10.0.1 using the neighbor-joining (nj) method. bootstrap trials were replicated 1,000 times to derive a confidence value for phylogenetic analysis. analysis of mrna expression patterns via quantitative real-time pcr (qrt-pcr) the expression of cfmal was analysed by qpcr in several tissue form control and vibrio infected animals as well as in hemocytes during induced er-stress. all qrt-pcr reactions were carried out using transstart green qpcr supermix udg (aq111, transgenbiotech, china) in a linegene k fqd-48a fluorescence quantitative pcr detection system (bioer, china). all the primers 28 fig. 1 sequence features and phylogenetic relationships of cfmal. a. nucleotide and predicted protein sequences of cfmal. the nucleotides and amino acids are numbered on the left margin. the function domain is shaded. the low complexity is boxed. the transmembrane region has a double underline. the stop codon is indicated by asterisks. the polyadenylation signal site (aataaa) is underlined. b. phylogenetic tree based on the protein sequences of different malectins. the nj model was used to infer the evolutionary history. the numbers at the branches indicate the bootstrap value (%). the accession numbers of these sequences are as follows: apis mellifera, xp_006563359; aplysia californica, xp_005104301; biomphalaria glabrata, xp_013067932; chlamys farreri, ayb71126; crassostrea gigas, xp_011422439; cynoglossus semilaevis, xp_016898532; homo sapiens, np_055545; maylandia zebra, xp_004558571; mizuhopecten yessoensis, xp_021354488; mus musculus, np_780612; oreochromis niloticus, xp_005473062; pelodiscus sinensis, xp_006114823; pomacea canaliculata, xp_025090097; rattus norvegicus, np_001014005 and salvelinus alpinus, xp_023857162 29 fig. 2 spatial mrna expression patterns of cfmal. mrna expression levels in hemocytes, mantle, gill, hepatopancreas, and gonads of five adult scallops were normalized to that of muscle. vertical bars represent mean ± sd (n = 5); different letters represent statically significant differences (p < 0.05) for qrt-pcr were designed with perlprimer 1.1.21 (table 1). the threshold cycle (ct) slope method, based on serial two-fold dilutions of cdna, was used to confirm that all pairs of these primers had similar efficiency (pfaffl et al., 2001; wang et al., 2018a). for each sample, the expression level of target genes was normalized to that of elongation factor 1 α (cfef-1α). the relative mrna abundance of target genes was determined using the comparative ct (2 −δδct ) method (schefe et al., 2006; schmittgen and livak, 2008). data are presented as mean ± sd (n = 5, each time point has 5 repetitions, and each repetition was a mixture of 3 individuals); data was subjected to one-way analysis of variance, followed by a multiple comparison using ibm spss statistics software 25.0.0.0. p < 0.05 was considered as statistically significant. results molecular features of cfmal and its phylogenetic relationship the full-length cdna sequence of cfmal obtained via race was submitted to genbank under the accession number mg546685. the complete cdna sequence of cfmal was 1,111 bp long and consisted of a 40 bp 5’ untranslated region (utr), a 3’ utr of 162 bp, and an open reading frame (orf) of 909 bp. a polyadenylation signal site (aataaa) was revealed upstream of the polya tail. the orf encoded 302 amino acid residues with a predicted molecular mass of 33.665 kda, and an isoelectric point of 5.210. a malectin domain (from v 61 to i 220 ) and a transmembrane region (from t 278 to c 300 ) were identified in the predicted protein sequence by smart analysis; no signal peptide was revealed (figure 1a). blast+ search revealed that cfmal shared high identity with its homologues from m. yessoensis (97% identity), crassostrea virginica (75% identity) and pomacea canaliculata (68% identity). phylogenetic analysis showed that cfmal clustered with its counterparts from m. yessoensis and formed a sister branch to their homologue from crassostrea gigas (figure 1b). tissue distribution of cfmal mrna transcripts the tissue distribution of cfmal mrna transcripts was detected by qrt-pcr using cfef-1α as an internal control. cfmal mrna transcripts were detectable in all the sampled tissues; the highest expression was that of hemocytes, which was 6.98-fold (p < 0.05, relative to muscle), followed by gill (4.23-fold, p < 0.05) and hepatopancreas (3.98-fold, p < 0.05). after stimulation with vibrio for 12 h, cfmal expression increased significantly in hemocytes, hepatopancreas, and gill (20.07-, 7.36-, and 7.16-fold, respectively, relative to muscle with no stimulation, p < 0.05). no significant differences were observed in muscle, mantle, or gonads, before and after stimulation with vibrio (figure 2). temporal expression of cfmal and related genes during er-stress induction cfmal and the three related genes (cfbip, cfgrp94 and cfedem1) were identified from the genome of c. farreri using blast+ 2.8.0, according to previous description (wang et al., 2007; galli et al., 2011). the expression patterns of these genes were 30 fig. 3 temporal mrna expression patterns of cfmal and cfmal-related genes during er-stress. vertical bars represent mean ± sd (n = 5); different letters represent statically significant differences (p < 0.05). a. cfbip b. cfgrp94 c. cfedem1 d. cfmal analyzed by qrt-pcr. the expression of these four genes all increased after hemocytes were stimulated with tg, tun, or lps. after 3 h of tg stimulation, the expression of cfbip increased significantly (3.82-fold, p < 0.05), reached a peak at 12 h (9.12-fold, p < 0.05), and returned to basal levels after 48 h. after 3 h of tun stimulation, the expression of cfbip increased significantly (4.24-fold, p < 0.05), reached a peak at a 6 and 12 h (5.96-fold and 6.19-fold, p < 0.05, respectively), and returned to basal levels after 48 h. after 3 h of lps stimulation, the expression of cfbip increased significantly (3.97-fold, p < 0.05), reached a peak at 12 h (8.87-fold, p < 0.05), and decreased at 24 and 48 h after stimulation (3.86and 4.24-fold, respectively, p < 0.05 for both; figure 3a). after 6 h of tg stimulation, the expression of cfgrp94 increased significantly (3.27-fold, p < 0.05), reached a peak at 12 h (6.12-fold, p < 0.05), and returned to basal levels after 24 h. after 6 h of tun stimulation, the expression of cfgrp94 increased significantly (2.96-fold, p < 0.05), reached a peak at 12 h (8.20-fold, p < 0.05), and returned to basal levels after 24 h. after 3 h of lps stimulation, the expression of cfgrp94 increased significantly (2.97-fold, p < 0.05), reached a peak at 12 h (6.29-fold, p < 0.05), and returned to basal levels after 48 h (figure 3b). expression of cfedem1 peaked after 6 h of tg stimulation (3.17-fold, p < 0.05), and returned to basal levels after 12 h. expression of cfedem1 peaked after 6 h of tun stimulation (3.31-fold, p < 0.05), and returned to basal levels after 12 h. expression of cfedem1 peaked after 6 h of lps stimulation (4.96-fold, p < 0.05), and gradually returned to basal levels after 48 h (figure 3c). expression of cfmal peaked after 12 h of tg stimulation (5.17-fold, p < 0.05), decreased significantly after 24 h (2.95-fold, p < 0.05), and returned to basal levels after 48 h. expression of cfmal increased only after 12 h of tun stimulation (3.28-fold, p < 0.05). expression of cfmal peaked after 12 h of lps stimulation (3.36-fold, p < 0.05) and returned to basal levels after 48 h (figure 3d). 31 discussion malectin is a newly discovered er-resident lectin, which specifically recognizes g2m9 in newly synthesized glycoproteins (schallus et al., 2008). recent research indicates that malectin might play potential roles in innate immunity (mu et al., 2014; wang et al., 2018c). however, information about the role of malectin in innate immunity is scarce. in the present study, we identified a novel malectin gene (cfmal) in zhikong scallop c. farreri. we analyzed cfmal sequence features, its tissue distribution, and temporal expression profiles, in order to predict its potential functions in innate immunity. bioinformatics analysis revealed that cfmal contained a typical malectin domain, and exhibited high identity with its invertebrate counterparts. additionally, in the nj phylogenetic tree cfmal clustered with its homologues from m. yessoensis and c. gigas. the conserved function domain has high similarity with that of other invertebrates. these phylogenetic relationships suggest that cfmal belongs to the invertebrate malectin family. to investigate the potential functions of cfmal in scallops, the tissue distribution of its mrna transcripts was analyzed. cfmal mrna transcripts could be detectable in all the sampled tissues; expression was highest in hemocytes, followed by gill and hepatopancreas. hemocytes play pivotal functions in invertebrate innate immunity (jia et al., 2017; jia et al., 2018). gill is a potential hematopoietic position in mollusks and is the first line of defense against invading microbes in lower animals (li et al., 2017). the hepatopancreas is considered as main immune organ in crustaceans and mollusks (wang et al., 2016b). the high abundance of cfmal mrna transcripts in these tissues indicates that it might be involved in the innate immunity of scallops. cfmal expression in these tissues was significantly up-regulated by vibrio stimulation, especially in hemocytes, which confirmed this hypothesis. to further investigate the potential roles of cfmal in scallops, the temporal expression profiles of cfmal and three related genes was investigated in hemocytes stimulated with tg, tun, or lps. in previous report, the expression of mammalian bip, grp94, edem1 and malectin is up-regulated during tg-induced er-stress (galli et al., 2011). in the present study, the expression of cfbip, cfgrp94, cfedem1 and cfmal increased in scallop hemocytes after stimulation with either tg, tun, or lps. tg and tun induced similar gene expression modification in hemocytes, which confirmed the hypothesis that cfmal might play a role in er-stress of scallops. while lps treated hemocytes showed slightly differences compared to the other stimuli, indicating cfmal might also be involved in innate immunity of scallops. in conclusion, a novel malectin gene (cfmal) was identified and characterized in c. farreri, including sequence features and expression profiles. the expression of cfbip, cfgrp94, cfedem1 and cfmal increased in scallop hemocytes after stimulation with either tg, tun, or lps. the present study provides useful information about the potential functions of cfmal in scallops. acknowledgements this research was supported by national natural science foundation of china (u1706209), aoshan innovation project in science and technology from national laboratory for marine science and technology (2016askj07), science and technology service network plan (sts) major deployment project (kfzd-sw-106) and sts regional centre project (fujian) from the chinese academy of sciences. we would like to thank the editor and the two expert reviewers for their constructive suggestions and enlightening comments during the revision. references aebi m, bernasconi r, clerc s, molinari m. n-glycan structures: recognition and processing in the er. trends. biochem. sci. 35: 74-82, 2010. arasu a, kumaresan v, sathyamoorthi a, palanisamy r, prabha n, bhatt p, et al. 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and characterization of a thioester-containing protein from zhikong scallop chlamys farreri. mol. immun. 44: 3492-3500, 2007. 138 isj 17: 138-146, 2020 issn 1824-307x research report expression profiles of immune-related genes in coelomocytes during regeneration after evisceration in apostichopus japonicus hf dang1, x han1, y guo1, q li2, sg ye1, j liu1, rj li1* 1ministry of agriculture and rural affairs key laboratory of mariculture & stock enhancement in north china's sea, dalian key laboratory of marine animal disease control and prevention, dalian ocean university, dalian 116023, china 2department of ocean technology, college of marine and biology engineering, yancheng institute of technology, yancheng, 224051, china this is an open access article published under the cc by license accepted june 15, 2020 abstract a representative echinoderm, the sea cucumber apostichopus japonicus has a special regeneration mechanism. the sea cucumber has no specific immune tissues or organs. coelomocytes of sea cucumbers are involved in their cellular and humoral immunity. in this study, expression profiles of the main immune-related factors in sea cucumber coelomocytes were detected during coelomocyte regeneration after evisceration in a. japonicus. immune-related factors cu/zn superoxide dismutase (cu/zn sod), catalase (cat), c-type lysozyme (c-lyz), cathepsin d (ctsd), melanotransferrin (mtf), toll-like receptor (tlr), c-type lectin (c-lct), complement 3 (c3), myeloid differentiation factor 88 (myd88), nuclear factor kappa-b (nf-κb), nf-κb inhibitor (ikb), tnf receptor-associated factor 6 (traf6), peroxiredoxins (prx), nitric oxide synthase(nos), caspase-2 (casp-2), phenoloxidase (po), and glutathione peroxidase(gpx), heat shock protein 70 (hsp70) were detected by real-time fluorescent quantitative pcr at different time points during regeneration. the main immune-related genes in sea cucumber coelomocytes were significantly differentially expressed after evisceration, and an upregulation was observed for the majority of the considered genes. in summary, the discharge of viscera had a significant effect on expression of immune-related genes of sea cucumber coelomocytes. the expression level of each gene had a certain correlation with the sea cucumber regeneration process. the results provide reference data for the immune response of coelomocytes during regeneration. key words: sea cucumber; regeneration; coelomocyte; immune-related factors; expression profile introduction the sea cucumber apostichopus japonicus is classified in the phylum echinodermata, class holothuroidea and is a representative echinoderm (zhang et al., 2017). sea cucumbers have a defense mechanism against environmental changes or pathogenic infection. in hostile or detrimental environments such as high salt stress or anaerobic conditions, the internal organs of sea cucumbers are excreted (sun et al., 2013). sea cucumbers in an appropriate environment regenerate new organs, including intestines, respiratory trees and gonads (yuan et al., 2019). the sea cucumbers regeneration mechanism is an important model for studying the cellular and molecular mechanisms of ___________________________________________________________________________ corresponding author: ruijun li dalian ocean university heishijiao street 52, dalian 116023, china e-mail: liruijun@dlou.edu.cn regeneration (garcia-arraras et al., 1998). sea cucumbers lack specific immune tissues and organs. coelomocytes and their immune factors are important in hostile or detrimental environmental conditions (xue et al., 2015). and there was an interested question about what the expression profiles of the main immune-related factors in sea cucumber coelomocytes would be after removing the internal organs. when sea cucumbers were infected by pathogens, coelomocytes defend against them by phagocytosis, identified coelomocyte immune factors attack foreign substances in coelomic fluid, remove invading microorganisms and repair damage (jiang et al., 2018). cu/zn superoxide dismutase (cu/zn-sod) (gao et al., 2014), catalase (cat) (gao et al., 2014), c-type lysozyme (c-lyz) (tian et al., 2014), cathepsin d (ctsd) (jiang et al., 2018), melanotransferrin (mtf) (qiu et al., 2014), toll-like receptor (tlr) (jiang et al., 2018), c-type lectin https://www.sciencedirect.com/science/article/pii/s105046481830130x https://www.sciencedirect.com/science/article/pii/s105046481830130x https://creativecommons.org/licenses/by/4.0/legalcode https://www.sciencedirect.com/topics/immunology-and-microbiology/immunoglobulin-enhancer-binding-protein 139 table 1 primers for qrt-pcr (c-lct) (wei, et al., 2015), complement 3 (c3) (zhou et al., 2011), myeloid differentiation factor 88 (myd88) (lu et al., 2013b), nf-κb (wang et al., 2013), nf-κb inhibitor (ikb) (lu, et al., 2013), tnf receptor-associated factor 6 (traf6) (lu et al., 2013a), peroxiredoxins (prx) (wang et al., 2011), nitric oxide synthase (nos) (shao et al., 2016), caspase-2 (casp-2) (ye et al., 2016), phenoloxidase (po) (jiang et al., 2014), glutathione peroxidase (gpx) (wang et al., 2011b ), heat shock protein 70 (li et al., 2018) and other immune-related factors are reported to be involved in response and removal of invading pathogens. based on our previous study on the regeneration of coelomocytes after evisceration by a. japonicas (li et al., 2018), we selected 18 immune-related factors reported to be involved in pattern recognition receptor pathways, immune enzymes, and other immune-related factors. expression profiles of immune-related genes in regeneration after evisceration were used to research immunological characteristics and immune defense mechanisms of the immune factors in coelomocytes after evisceration. this study provides reference data for studying the immune regulation mechanism of echinoderm regeneration. immune-related factors accession no. primer sequences (from 5′ to 3′) references pattern recognition receptors (prrs) toll-like receptor jq743247 f: acgaaagcgatttagcc r: gagcccgtggtgagatg jiang et al., 2018 myeloid differentiation factor 88 kf032818.1 f: ggaaacgagaggaggagagacg r: tccagacagtagcagacgaaagc lu et al., 2013 tnf receptor-associated factor 6 kf032819.1 f: aggagcgggaaaggaagcag r: tagccgtagagcgccgtgtag lu et al., 2013 nf-κb jf828765.1 f: tgaaggtggtatgcgtctgg r: ttgggctgctcggttatg li et al., 2018 ikb kf032816 f: acaggagtcgtttgatgattgg r: gtttcttcttgtgtttggcgttc lu et al.,2013 complement 3 hq214156 f: gcgttgtttcgttcaacaagggga r: gccattcactggaggtgtggca jiang et al., 2018 cytochrome b 7802877 f: tgagccgcaacagtaatc r: aagggaaaaggaagtgaaag wang et al.,2016 enzymes phenoloxidase kf040052 f: cagcagttacaagtgggatg r: ccagtcacgaagaccagaat jiang et al.,2014 cu/zn superoxide dismutase jx097096 f: tcgggcactattaccttc r: accattatcatcggcttc jiang et al., 2018 catalase jq776634 f: ctcccaactacttcccaaac r: gtccgacaagacctcacg jiang et al., 2018 c-type lysozyme kf773759 f: gtaccacggagcaggagt r: cacagacaagcggagacc jiang et al., 2018 cathepsin d jf430592 f: ctcccaactacttcccaaac r: gtccgacaagacctcacg jiang et al., 2018 glutathione peroxidase jf769857 f: ggatgtgtgtgtctagtggtgaa r: gaattactcccaggttcctgact wang et al., 2011 nitric oxide synthase kt366016.1 f: ttgggctgctcggttatg r: tgatgcaagagactgctgga li et al., 2018 peroxiredoxins jf769853 f: tattcactcctggctgctctaag r: tgtgaagtcacagcaggtatcag wang et al., 2011 other immune-related factors caspase 2 kc972624 f: caagttccacacgacacagg r: gcagtctttgttcgttccgt li et al., 2018 heat shock protein 70 eu930813.1 f: gccgcatcccttgtaagaag r: agttcaaattgaccgaggcg li et al., 2018 melanotransferrin hq260578 f: ggtgggtgatgcctgttg r: agctgaggtgggttcgtt jiang et al., 2018 c-type lectin hq728281 f: tcggatctaacttggacg r: ttaccctgcgaatgactt jiang et al., 2018 http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search&db=nucleotide&doptcmdl=genbank&term=kc972624 140 materials and methods experimental animals and sampling healthy apostichopus japonicus (55 ± 10.2 g, mean ± sd) were collected from a local aquatic farm (dalian, liaoning province, china) and acclimated in an indoor aquarium with well-aerated sea water at 17 19 °c for 1 week before experiments. to study expression of coelomocyte immune-related factors during regeneration, evisceration was induced by intracoelomic injection of 1.2 ml 0.35 m kcl. total coelomocytes from a. japonicus at pre-evisceration, 2 hours post evisceration (hpe), 6 hpe, 12 hpe, 36 hpe, 3 days post evisceration (dpe), 5 dpe, 7 dpe, 14 dpe, 21 dpe, and 35 dpe were sampled. the health sea cucumber before evisceration was as the control group (0 hpe), and each group had three biological replications. the coelomocyte sample was put into rnase-free centrifuge tubes. after centrifugation at 5000 rpm for 10 minutes at 4 °c, the supernatants were removed. and the coelomocyte precipitations were stored at -80 °c for further analysis. total rna extraction and cdna synthesis samples of coelomocytes were taken from -80 °c. total rna was extracted by triozl (life technologies, usa). rna integrity was detected by 1 % agarose gel electrophoresis. concentration and purity of rna were determined by a micro nucleic acid analyzer (nano drop 8000, thermo, usa). coelomocyte rna was reverse transcribed using the primescript™ rt reagent kit gdna eraser (takara, japan). reverse transcription occurred as follows: first-strand cdna was synthesized using primescript™ 1st strand cdna synthesis kits (takara, japan) as a 10.0 μl mixture (1.0 μl 50 μmol/l oligo dt primer, 1.0 μl 10 mmol/l dntp mixture, 0.8 μg total rna and the remaining rnase-free ddh2o complemented the total volume of 10 μl) incubated at 42 °c for 2 min. to the 10.0 μl mixture, 4.0 μl 5 × primescript™ buffer, 0.5 μl 40 u/μl rnase inhibitor, 1.0 μl 200 u/μl primescript™ rnase and 4.5 μl rnase-free dh2o were mixed for incubation at 42 °c for 15 min, 85 °c for 5 s and cooling on ice. products were used as cdna and stored at -20 °c. quantitative real-time pcr relative mrna levels of immune-related factors were determined by quantitative real-time pcr (qrt-pcr). primer pairs for immune-related factors were reported (table 1) and verified for amplicon sizes and specificity by melting curve analysis and agarose gel electrophoresis. all cdna sample for qrt-pcr, we used sybr premix ex taq™ ii kits (takara, japan) in 20 μl reactions of 10 μl 2 × sybr premix ex taq™ ii, 0.8 μl 10 μmol/l forward and reverse primer, 0.4 μl rox reference dye ii, 1 μl cdna template and 7 μl sterile distilled water. the program was 95 °c for 30 s, followed by 40 cycles of 95 °c for 10 s, 55 °c for 25 s and 72 °c for 25 s. each samples amplification melting curve was a single peak, and the ct value was collected, followed by data processing and analysis. statistical analysis the ct values of amplification curves were analyzed by the 2-δδct method. all experimental data were expressed as mean ± sd and all experimental data were analyzed with spss 19.0 version (spss inc., il, usa), including t-tests for comparison of two sets of data, one-way anova for multiple sets. the level of significance was defined as p < 0.05. results expression of major immune genes related to pattern recognition receptors in sea cucumbers expression of major immune factors in the pattern recognition receptor in sea cucumber coelomocytes after evisceration was shown in figure 1. after evisceration, expression of tlr in sea cucumbers was significantly up-regulated and was 3.26 times than the control group at 3 dpe (p < 0.05). expression of tlr in sea cucumbers was significantly down-regulated and was 0.0813 times the control group at 5 dpe, 0.1090 times at 7 dpe and 0.0274 times at 35 dpe. expression of ikb after evisceration was significantly higher than the control group at 3.65 times at 6 hpe, 2.42 times at 12 hpe, 2.91 times at 3 dpe, and 4.44 times at 21 dpe (p < 0.05). expression of nf-κb after evisceration was significantly higher than the control group at 12 hpe, 36 hpe and 3 dpe (p < 0.05). the highest value was at 3 dpe, at 11.15 times the control group. expression of c3 after evisceration was significantly higher than the control group at 6, 12, and 36 hpe and 3, 5, 7, 14, and 21 dpe (p < 0.05). the highest value was at 3 dpe, at 22.30 times the control group. expression of myd88 after evisceration was significantly higher than the control group at 6 and 36 hpe and 3 dpe (p < 0.05). the highest value was at 3 dpe, at 26.27 times the control group. expression of the traf6 gene after evisceration was significantly higher than the control at 12 and 36 hpe, and 3 dpe (p < 0.05). the highest value was at 3 dpe, at 43.04 times the control group. within 35 days of regeneration in sea cucumbers, the overall trends for ikb, nf-κb, c3, myd88, and traf6 were up-regulation. tlr was significantly up-regulated first, then significantly down-regulated. these six genes were all up-regulated at 3 dpe. c3 was continuously up-regulated throughout the regeneration process except for the initial 2 hpe and 35 dpe. expression of c3 was significantly up-regulated within 35 days of regeneration. expression of immune enzyme genes of sea cucumbers after evisceration expression of immune enzymes in coelomocytes after evisceration was shown in figure 2. expression of cat first rose, then decreased at 2 to 12 hpe, 36 hpe to 5 dpe, and 5 dpe to 35 dpe, with peaks at 6 hpe and at 3 dpe and 14 dpe. expression of cat was highest at 3 dpe, at 8.38 times the control group (p < 0.05). expression of ctsd rose first and then decreased at 2 to 12 hpe, 36 hpe to 5 dpe, and 5 dpe to 35 dpe, with peaks at 6 hpe, and 3 and 14 dpe. expression of ctsd was the highest at 6 hpe, at 4.85 times the control group (p < 0.05). expression of c-lyz rose first and then decreased at 2 to 12 hpe, 36 hpe to 5 dpe and 5 dpe to 35dpe, with peaks at 6 hpe, and 3 dpe and 14 dpe (p < 0.05). expression of c-lyz was highest at 3 dpe, 141 fig. 1 expression of major immune genes related to pattern recognition receptors in sea cucumber coelomocytes after evisceration. abbreviations: toll-like receptor (tlr), nuclear factor kappa-b (nf-κb), nf-κb inhibitor (ikb), complement 3 (c3), myeloid differentiation factor 88 (myd88), tnf receptor-associated factor 6 (traf6) figat 2.46 times the control group (p < 0.05). expression of gpx rose and then decreased at 2 to 12 hpe, 36 hpe to 7 dpe, and 7 dpe to 35 dpe, with peaks at 6 hpe, and 3 dpe and 14 dpe. expression of gpx was highest at 3 dpe, at 24.17 times the control group (p < 0.05).expression of po rose and then decreased at 2 to 12 hpe, 36 hpe to 7 dpe, and 7 to 21 dpe, with peaks at 6 hpe and 3 and 14 dpe. expression of po was highest at 3 dpe, at 9.69 times the control group (p < 0.05). in addition, expression of prx was significantly lower than the control group at 3 dpe (p < 0.05), rising first and then decreasing at 2 to 12 hpe, 12 hpe to 7 dpe, and 7 to 35 dpe, with peaks at 6 hpe, and 3 and 14 dpe (p < 0.05). expression of prx was highest at 3 dpe, at 2.21 times the control group (p < 0.05). expression of prx was lowest at 35 dpe, at 0.12 times the control group (p < 0.05). expression of cu/zn sod rose and then decreased at 2 to 12 hpe, 36 hpe to 5 dpe, and 7 to 35 dpe, with peaks at 6 hpe, and 3 and 14 dpe. the expression of cu/zn sod was the highest value at 14 dpe, at 5.12 times of the control group (p < 0.05). expression of nos rose and then decreased at 2 to 12 hpe, 36 h to 5 d, and 14 to 35 dpe, with peaks at 6 hpe, and 3 and 21 dpe. expression of cu/zn sod was highest at 21 dpe, at 57.18 times the control group (p < 0.05). within 35 days of regeneration, the overall trend for cat, ctsd, c-lyz, gpx, po, cu/zn sod, nos was up-regulation. these seven genes, except for cu/zn sod, were significantly up-regulated at 3 dpe. cu/zn sod was significantly up-regulated at 14 dpe. prx was significantly up-regulated at 3 dpe, while was significantly down-regulated at other time points. expression of other immune factors in sea cucumber coelomocytes after evisceration expression of other immune factors in coelomocytes after evisceration was shown in figure 3. in addition to casp-2, all other immune factors studied showed a trend of increasing first and then decreasing at 2 to 12 hpe and 36 hpe to 5 dpe, peaking at 6 hpe and 3 dpe (p < 0.05), with significant up-regulation at 3 dpe (p < 0.05). the casp-2 gene showed a downward trend within 35 days after evisceration, reaching the lowest value at 3 dpe, at 0.01 times the control group (p < 0.01). expression of mtf rose first and then decreased at 2 to 12 hpe, and 36 hpe to 5 dpe, with peaks at 6 hpe and 3 dpe (p < 0.05). expression of mtf was 142 fig. 2 expression of immune enzymes in sea cucumber coelomocytes after evisceration. abbreviations: catalase (cat), cathepsin d (ctsd), c-type lysozyme (c-lyz), glutathione peroxidase (gpx), phenoloxidase (po), peroxiredoxins(prx), cu/zn superoxide dismutase (cu/zn sod), nitric oxide synthase(nos). *p < 0.05 highest at 3 dpe, at 15.36 times the control group (p < 0.05). expression of hsp70 rose and then decreased at 2 to 12 hpe, and 36 hpe to 5 dpe, with peaks at 6 hpe and 3 dpe. expression of hsp70 was highest value at 3 dpe, at 49.78 times the control group (p < 0.05). expression of c-type lectin rose and then decreased at 2 to 12 hpe, and 36 hpe to 5 dpe, with peaks at 6 hpe and 3 dpe. expression level of hsp70 was highest at 3 dpe, at 114.49 times the control group (p < 0.01). within 35 days of regeneration, the overall trend for mtf, hsp70, and c-type lectin were up-regulation, with significant up-regulation at 3 dpe. casp-2 was significantly down-regulated at 3 dpe. discussion pattern recognition receptor-associated immune factor after evisceration, sea cucumber bodies were sensitive and fragile. the pattern recognition receptor (prrs) pathway of sea cucumbers was important for evisceration and regeneration because sea cucumbers were echinoderms that lack acquired immunity, and their defense mechanism relies on prrs to identify and eliminate pathogens. in this study, tlr was significantly up-regulated at 3 dpe after evisceration and then significantly decreased, which might be a protective mechanism that was conducive to sea cucumber regeneration. significant up-regulation of tlr might be because when microorganisms destroy physical barriers of the sea cucumber, tlr activated a signaling pathway of immune cells by significantly up-regulating and specifically recognizing the conserved molecular structure of the pathogen pamp (liu et al., 2017), which allowed sea cucumbers to eliminate more pathogens, thereby promoting regeneration. in our previous study, the total number of luminal cells in sea cucumbers returned to previous levels 6 hours after evisceration and the ability to divide cells 3 days after evisceration was relatively stronger than at other time points (li et al., 2018). tlr4 was down-regulated when it differentiates into human neural stem cells in vivo (grasselli, et al., 2018). in addition, expression of tlr4 was down-regulated during differentiation of cervical tumor cells into cervical tumors (ma et al., 2018). therefore, we hypothesized that on the third day after evisceration, sea cucumber coelomocytes began to differentiate and form coelomocytes with different immune functions. in this study, both traf6 and myd88 were significantly up-regulated in the prrs pathway compared with the control group, and the difference 143 fig. 3 expression of other immune factors in sea cucumber coelomocytes after evisceration. abbreviations: caspase-2 (casp-2), melanotransferrin (mtf), c-type lectin (c-lct), heat shock protein 70 (hsp70) fold was significantly higher than other genes; traf6 and myd88 belonged to adaptor proteins, from which we can speculate the significant up-regulation of both traf6 and myd88 in the prrs pathway compared to the control group was to increase the sensitivity of the recognition pathway. traf6 was an ubiquitin-ligase. when activated, it produced short protein chains on itself and other proteins. traf6 acted as a switch that determines which signals are turned on in cells (sun et al., 2014). studies showed that traf6 was involved in receptor-mediated activation of multiple signaling pathways in adult muscle fiber regeneration (hindi et al., 2012). when traf6 was knockout in adult mouse satellite cells, it caused severe muscle regeneration defects (hindiet al., 2016). when the expression level of traf6 was significantly decreased, the proliferation of zebrafish cardiomyocytes was significantly inhibited (seeger et al., 2013). these findings indicate that traf6 was important in the process of cell regeneration. myd88 was a key linker molecule in the tlr signaling pathway and important in the transmission of upstream information and disease development. myd88 was an adaptor protein that mediates toll-like receptor and interleukin-1 receptor signaling. up-regulation of myd88 promoted fusion of exogenous myoblasts with damaged muscle fibers during muscle regeneration (hindi et al., 2017). we hypothesized that the up-regulation of traf6 and myd88 in the regeneration process of sea cucumbers increased the sensitivity of the recognition pathway and enhanced the proliferation of coelomocytes, resulting in more immune responses that facilitated recovery of sea cucumbers. c3 was continuously up-regulated during the entire regeneration process except the first 2 hours after evisceration and the 35th day after regeneration. we hypothesized that the complement pathway was important in the process of regeneration of coelomocytes. pleurodeles waltl complement component c3 was also expressed early in development and its expression increased with the development of the immune system (gueguinou et al., 2014). the hepatocyte proliferation ability of mice was impaired when c3 is defective, and proliferation of hepatocytes is restored when c3 was normal. complement was activated in skeletal muscle injury in mice and was a key to skeletal muscle regeneration (markiewski et al., 2004). genetic ablation of complement c3 resulted in impaired muscle regeneration following cardiac 144 cardiomyopathy-induced injury in mice (zhang et al., 2012). these studies indicated that the complement pathway was important in the process of regeneration, indicating the good agreement with our current results. enzyme-related immune-related factors after evisceration, the sea cucumber immune response was important to ensure normal regeneration. the immune response of the sea cucumber was significantly enhanced in regenerative tissue (zhang et al., 2017). because of a lack of acquired immunity in invertebrates, sea cucumber coelomocytes immune enzyme factors were important in regeneration. in our study, all immunoenzyme genes were significantly expressed on the third day after evisceration, except for sod, which was significantly expressed on the 14th day. in addition, the difference in expression of the nos gene was significantly greater than other immunoenzyme-based genes and it was the most significantly up-regulated immune enzyme. nos is expressed during peripheral nerve regeneration and functions in repairing nerve-induced regeneration (gonzálezhernández et al., 2015). in addition, nos was significantly up-regulated in liver endothelial cells and regulates the proliferation of hepatic sinusoidal endothelial cells to regenerate the liver (yokoi et al., 2010). therefore, we hypothesized that nos participated in regeneration of sea cucumbers and was important in regeneration. prx was down-regulated throughout the regeneration process and might be a protective mechanism to promote regeneration. prx were a ubiquitous family of cysteine-dependent peroxidases with antioxidant and anti-apoptotic functions. down-regulation of oxidoreductase mediated an increase in oxidative stress and induces apoptosis in cancer cells (yoshida et al., 2011). when dprx5 was inhibited, drosophila were more sensitive to oxidative stress and had a higher rate of apoptosis (radyuk et al., 2009). in a study on the effect of peroxiredoxin i gene silencing on tgf-β1-induced fibroblast proliferation and collagen synthesis, when the prx-1 gene was silenced, jnk activation was promoted by increased ros levels, leading to cell proliferation (chang et al., 2006). these results indicated that inhibiting peroxiredoxins was beneficial to increasing body cells. in the antioxidant system of our study, cat, ctsd, c-lyz, gpx, po, sod, and nos were up-regulated. we hypothesized that during regeneration, prx induced apoptosis of damaged cells by down-regulation and enhanced the antioxidant system's defense response to adverse factors in regeneration, promoting regeneration. when genes for cat, ctsd, c-lyz, gpx, po, sod in the antioxidant system were up-regulated during 3 dpe of regeneration, prx was also significantly up-regulated. the antioxidant activity of prdx5 promotes the development of bovine scnt embryos in vitro (wang et al., 2017). therefore, it was speculated that the antioxidant system was over-expressed, resulting in damage to the sea cucumber body, thereby affecting the regeneration of sea cucumber. at this time, the up-regulation of prx made the expression of sea cucumber antioxidant system relatively stable. it maintained the sea cucumber body's regenerative function. sod in the antioxidant system was the first act in scavenging reactive oxygen species (zhang et al., 2007). in the antioxidant system in our study, cat, ctsd, c-lyz, gpx, po, sod and nos were all up-regulated on the third day after the evisceration. sod was significantly up-regulated on day 14 after evisceration. of course, the function of this particular mechanism in regeneration remained to be further studied. other immune-related factors after evisceration, sea cucumber bodies were rapidly enhanced for immunity against external pathogenic microorganisms, for a good environment for regeneration. this result indicated that c-lct, hsp70, mtf, and casp-2 were involved in regeneration of sea cucumbers. casp-2 was consistently down-regulated during 35 days of regeneration and significantly down-regulated on 3 dpe. as the core enzyme of apoptosis mechanisms, the caspase protease system directly caused cell disintegration (takle et al., 2007). as a member of the caspases, caspase-2 induced apoptosis in response to both extrinsic and intrinsic signals. it was important in cell proliferation and differentiation. in studies involving caspase-2 in cell cycle promotion and ar activation, the activity of caspase-2 was important for the proliferation of androgen dependent prostate cancer cell lines lncap (taghiyev et al., 2011). caspase-2 was necessary for skeletal muscle differentiation and muscle formation. the activity of caspase-2 was significantly increased during skeletal muscle cell differentiation (boonstra et al., 2018). these findings indicated that increased caspase-2 was conducive to cell proliferation. whereas caspase-2 was down-regulated within 35 days of evisceration in this study, this might be due to the fact that the visceral regeneration of sea cucumber was similar to embryonic development, involving a large number of natural cell deaths, and a large number of new cells are formed, thereby forming new internal organs. caspase-2 deficient mice accelerated cell death in motor neurons during development (bergeron et al., 1998). caspase-2 was down-regulated during the 35 days’ period of regeneration, causing apoptosis of naturally dead cells during regeneration to create new internal organs. and on the third day, caspase-2 was significantly down-regulated. this may indicate that the third day of the regeneration was the beginning of coelomocyte differentiation in sea cucumbers. all this results indicated down-regulation of caspase-2 was important in this process. in this study, the selected immune-related genes in the coelomocytes of sea cucumber were significantly expressed at 3 dpe, except for cu/zn sod, which was significantly up-regulated at 14 dpe. after evisceration, sea cucumber pattern recognition receptor-associated immune factors ikb, nf-κb, c3, myd88, traf6 showed an overall upward trend within 35 days. tlr was first significantly up-regulated then significantly down-regulated. the overall trend for sea cucumber enzyme-related immune factors cat, ctsd, c-lyz, gpx, po, cu/zn sod, nos was up-regulation. these seven genes were significantly upregulated at 3 dpe except 145 for cu/zn sod, which was significantly up-regulated at 14 dpe. the other immune-related factors of sea cucumbers, mtf, hsp70, and c-lct were up-regulated. the overall trend was up-regulation with significant up-regulation at 3 dpe. casp2 was significantly down-regulated throughout this process. acknowledgments this work was funded in part by the state key research project “marine environment 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molecular characterization and expression analysis of a complement component 3 in the sea cucumber apostichopus japonicus. fish shellfish immunol. 31: 540-547, 2011. http://eurheartj.oxfordjournals.org/content/34/suppl_1/p1447.short http://eurheartj.oxfordjournals.org/content/34/suppl_1/p1447.short https://www.medsci.cn/sci/submit.do?id=8ee93780 http://link.springer.com/10.1007/s00595-010-4484-9 http://link.springer.com/10.1007/s00595-010-4484-9 http://link.springer.com/10.1007/s00595-010-4484-9 http://link.springer.com/10.1007/s00595-010-4484-9 http://link.springer.com/10.1007/s00595-010-4484-9 estresse alimentar no quinto estdio do predador brontocoris tabidus (heteroptera: pentatomidae): efeito na reproduo, isj 13: 389-396, 2016 issn 1824-307x research report prey scarcity at the beginning of fifth instar: effect of eucalyptus urophylla (myrtaceae) plants on reproduction, longevity, and weight of the predator brontocoris tabidus (heteroptera: pentatomidae: asopinae) r pinto1, jc zanuncio2, w de s tavares3, fl fernandes4, lr junqueira5, je serrão6 1faculdade de engenharia, universidade do estado de minas gerais, 35930-314, joão monlevade, estado de minas gerais, brasil 2departamento de entomologia/bioagro, universidade federal de viçosa, 36570-900, viçosa, estado de minas gerais, brasil 3departamento de fitotecnia, universidade federal de viçosa, 36570-900, viçosa, estado de minas gerais, brasil. current address: riau andalan pulp and paper (rapp), asia pacific resources international holdings limited (april), plant health program, pangkalan kerinci, 28300, riau province, indonesia 4universidade federal de viçosa, instituto de ciências agrárias, 38810-000, rio paranaíba, estado de minas gerais, brasil 5programa cooperativo em proteção florestal (protef), instituto de pesquisas e estudos florestais (ipef), avenida comendador pedro morganti, no 3500 bairro monte alegre, 13400-970, piracicaba, estado de são paulo, brasil 6departamento de biologia geral, universidade federal de viçosa, 36570-900, viçosa, estado de minas gerais, brasil accepted november 15, 2016 abstract intervals without prey during the fifth instar and nutrient quality may affect reproduction, longevity, and weight of the zoophytophagous predator brontocoris tabidus (heteroptera: pentatomidae: asopinae). this asopine was reared on eucalyptus urophylla (myrtaceae) trees under field conditions at 23 ± 6 oc, 76 ± 9 % rh and 13:11 (dark:light) h photoperiod. the experiment was developed with 60 b. tabidus nymphs individualized in organza bags (31 cm long × 21 cm diameter). one group of nymphs received only tenebrio molitor (coleoptera: tenebrionidae) pupae and water without e. urophylla trees (t1). other nymphs were reared on e. urophylla trees and fed with t. molitor pupae and water during the second, third, and fourth instars and twenty of them per group after zero, five, and 10 days from the beginning of the fifth instar making up the treatments t2, t3, and t4, respectively. the period without prey increased duration of the fifth instar for males in the t3 and t4 while female weight was lower in the t4. the oviposition period was shorter and the number of egg masses of b. tabidus was lower in the t1 than in the t2, t3, and t4. the highest egg numbers were found in the t2 and t3 with about 4 times more eggs than in the t1. the number of nymphs was low and the percentage of nymph hatching higher in the t1. the interval of five and 10 days without prey from the beginning of the fifth instar did not affect the duration and survival of this instar and supplementation with e. urophylla increased the reproductive capacity of the predator b. tabidus. key words: insect predator; plant feeding; starvation; supplementation; survival; zoophytophagy   introduction asopine predators can be used in the biological control of defoliator caterpillars (lepidoptera) in eucalyptus (eucalyptus spp., myrtales: myrtaceae) ___________________________________________________________________________ corresponding author: wagner de souza tavares departamento de fitotecnia universidade federal de viçosa 36570-900, viçosa, estado de minas gerais, brasil e-mail: wagnermaias@yahoo.com.br plantations (tavares et al., 2013, 2014, 2015). the most common asopine predators are brontocoris tabidus, podisus maculiventris, podisus nigrispinus, and supputius cincticeps (heteroptera: pentatomidae) feeding on coleopteran and lepidopteran specimens (mohaghegh et al., 2001; pereira et al., 2008; zanuncio et al., 2014). asopine predators have diversified feeding behavior, including plant material in their diet 389 mailto:wagnermaias@yahoo.com.br table 1 duration (days) and survival (%) of fifth instar brontocoris tabidus nymphs (mean ± se, standard error, and variation interval) fed on tenebrio molitor pupae after zero (t2), five (t3), and 10 (t4) days from the beginning of the fifth instar on eucalyptus urophylla or without plants (t1) (tr. = treatments) at 23 ± 6 ºc, 76 ± 9 % rh, and 13:11 (dark:light) h photoperiod. duration (days) tr. females males survival (%) t1 9.3 ± 0.20 a (7.0-10.0) 9.3 ± 0.39 b (7.0-17.0) 88.3 ± 0.05 a t2 11.0 ± 0.52 a (8.0-15.0) 10.6 ± 0.36 ab (7.0-14.0) 90.0 ± 0.04 a t3 12.1 ± 0.65 a (8.0-18.0) 11.1 ± 0.47 a (8.0-18.0) 91.6 ± 0.04 a t4 11.7 ± 0.82 a (8.0-23.0) 11.5 ± 0.58 a (8.0-18.0) 93.3 ± 0.03 a means followed by the same letter, per column, do not differ between them by the tukey’s honest significant difference (hsd) test (p > 0.05). (zanuncio et al., 2000, 2004, 2011a). in the laboratory, b. tabidus females fed on mealworm pupae, tenebrio molitor, (coleoptera: tenebrionidae), eucalyptus urophylla seedlings, and water were heavier and had higher oviposition rates than those fed only on this prey and water (zanuncio et al., 2000). supputius cincticeps had higher egg production fed on mealworm pupae, e. urophylla trees, and water in the field (zanuncio et al., 2004) and mealworm larvae, common bean pods, phaseolus vulgaris (fabales: fabaceae), and water in the laboratory (mourão et al., 2003). the importance of plant material for asopine predators suggests that these insects might obtain water and nutrients from these plants (coundron et al., 2002; holtz et al., 2011; castro et al., 2015). food quality and quantity affect the life cycle, reproduction, and longevity of asopine predators (wittmeyer et al., 2001; holtz et al., 2009; zanuncio et al., 2011b). intervals without prey and protein levels in nutrients affect body weight, and total number and viability of eggs (mourão et al., 2003; ramalho et al., 2008; holtz et al., 2009). prey shortages and plant material may affect the life cycle of asopine predators and, consequently, their efficiency to control insect-pests (molina-rugama et al., 1998; lemos et al., 2001; zanuncio et al., 2012). the objective of this work was to evaluate duration and survival during the fifth instar and the reproduction and longevity of b. tabidus adults after zero, five, and 10 days without prey from the beginning of the fifth instar. material and methods experimental site this research was carried out in the laboratory of biological control of insects (lcbi) and in the insectarium of the department of entomology (den) at the “universidade federal de viçosa (ufv)” in viçosa, minas gerais state, brazil. brontocoris tabidus was mass reared in the vicinity of the insectarium (20° 45' s × 42° 52' w and 648 m altitude) in organza bags (31 cm length × 21 cm diamater) on e. urophylla branches (approx. threeday-old trees) at 23 ± 6 °c, 76 ± 9 % rh, and 13:11 (dark:light) h photoperiod. fifth instar brontocoris tabidus nymphs a total of 900 first instar b. tabidus nymphs reared in the insectarium was divided in three groups of 300 each, into organza bags on e. urophylla branches from the second to the beginning of fifth instar. fifth instar nymphs were reared in groups of 20 individuals per bag on e. urophylla branches in the field. nymphs were supplied ad libitum with mealworm pupae and water in 5 ml plastic containers. these nymphs were weighed at the beginning of the fifth instar before feeding to obtain a similar number of males and females per treatment. although it is not possible to determine sex during the nymph stage, heavier asopine nymphs usually become females (mohaghegh et al., 1998). first instar b. tabidus nymphs are not predators, feeding only on their eggshell and water (pires et al., 2011). description of treatments eighty fifth instar b. tabidus nymphs were separated from each group of 300 individuals and divided into four treatments with eight of them individualized into the bags on e. urophylla tree branches with mealworm pupae supplied daily and water ad libitum. a group of nymphs was maintained without e. urophylla plants, feeding only on mealworm pupae and receiving water (t1). at the beginning of fifth instar, the nymph groups had periods of zero, five, and 10 days without prey constituting the treatments t2, t3, and t4, respectively. assessment of nymphs the duration (days) and survival (%) of fifth instar nymphs were verified daily. 390 assessments of adults brontocoris tabidus adults obtained from nymphs of the treatments t1, t2, t3, and t4 were weighed just after their emergence and sexed, based on their genitalia appearance (lemos et al., 2005; guedes et al., 2007). these adults were mated on the fourth day of adult age (zanuncio et al., 2006) and those from t2, t3, and t4 placed into the bags on branches of e. urophylla plants (zanuncio et al., 2004). adults received a mealworm pupa and water daily. those of t1 received only pupae of this prey and water in similar bags without e. urophylla branches. the number of b. tabidus pairs, per treatment, varied according to males and females obtained, with 23, 25, 20, and 22 for treatments t1, t2, t3, and t4, respectively. assessment of eggs brontocoris tabidus egg masses were collected daily and the number of eggs was counted. these egg masses were maintained in plastic petri dishes (9.0 cm diameter × 1.2 cm height) with a moistened cotton ball at 24 ± 2 °c, 70 ± 6 % rh, and 12:12 (dark:light) h photoperiod in the laboratory. general assessments the male and female weight (mg) and the preoviposition, oviposition, and post-oviposition periods (days), besides the number of eggs, nymphs, egg masses, eggs per egg mass, nymphs per egg mass, percentage of nymph hatching, and longevity of males and females (day) were recorded. statistical analysis the results were analyzed using the cochran’s c test (cochran, 1941), bartlett’s test (bartlett, 1937), and lilliefors test (lilliefors, 1967) to determine the homogeneity of variance and normality, respectively. the duration of the fifth instar, adult weight, and reproductive data were transformed to √x, except for the percentage of nymph hatching which was transformed to arcsen x. the data was subsequently analyzed using a oneway anova and tukey’s honest significant difference (hsd) test at 5 % probability (tukey, 1949). results survival of b. tabidus fifth instar nymphs varied from 88.3 to 93.3 % without difference between treatments (table 1). the duration of this instar was longer for males in the t3 and t4 than in the t1 (table 1). the duration of the fifth instar for females was similar between treatments (table 1). the weight of b. tabidus females was lower only for those in the t4, while male weight was similar between treatments (table 2). the pre-ovipostion and post-oviposition periods of b. tabidus were similar between treatments (table 3). however, the oviposition period was about 4 times longer with mealworm pupae and e. urophylla trees than with only pupae (11.8 days), regardless of the period of prey shortage (table 3). brontocoris tabidus females fed only mealworm pupae presented a lower number of egg masses (3.6) than in the other treatments (table 3). the number of eggs per female was similar in the treatments where nymphs received pupae after zero, five, and 10 days from the beginning of fifth instar and about 4 times higher (410.5, 452.8, and 332.2, respectively) than for those fed only mealworm pupae (table 3). the number of eggs per egg mass was similar between treatments (table 3). the number of nymphs was higher for b. tabidus females on e. urophylla and fed on mealworm pupae after zero, five, and 10 days from the beginning of the fifth instar (259.9, 299.2, and 201.2 nymphs, respectively) than only with pupae (86.5 nymphs) (table 3). the number of nymphs per egg mass was similar between treatments, but the percentage of nymph hatching was higher with only mealworm pupae (table 3). the longevity of b. tabidus males and females was greater in the treatments with e. urophylla and mealworm pupae feeding, than with only the prey (table 3). beyond this, b. tabidus females with prey and eucalyptus lived longer than those fed only prey. females fed only prey, and those fed prey and plant material had a 60 % and 80 % survival rate after 25 days of the adult stage, respectively. all females of the treatment fed only prey died after 45 days, while 60 % of those fed on prey and plant material were still alive at the end of this period (fig. 1). table 2 weight (mg) of brontocoris tabidus males and females (mean ± se, standard error) fed on tenebrio molitor pupae after zero (t2) (n = 25), five (t3) (n = 20), and 10 (t4) (n = 22) days from the beginning of the fifth instar on eucalyptus urophylla or without plants (t1) (n = 23) (tr. = treatments) at 23 ± 6 °c, 76 ± 9 % rh, and 13:11 (dark:light) h photoperiod weight (mg) tr. males females t1 103.2 ± 1.56 a (90.4-118.9) 128.3 ± 3.32 a (79.8-152.1) t2 102.6 ± 3.29 a (81.0-132.3) 135.4 ± 5.03 a (104.7-205.3) t3 95.8 ± 3.94 a (64.4-133.4) 131.5 ± 3.69 a (100.8-188.3) t4 92.1 ± 4.65 a (47.2-134.1) 111.3 ± 4.74 b (69.7-157.4) means followed by the same letter, per column, do not differ between them by the tukey’s honest significant difference (hsd) test (p > 0.05). 391 table 3 characteristics (char.) of pre-oviposition (days) (pr), oviposition (days) (ov), post-oviposition (days) (po), number of egg masses (em), number of eggs (eg), number of eggs/egg mass (eg/em), number of nymphs (ny), number of nymphs/egg mass (ny/em), longevity of females (days) (lf), longevity of males (days) (lm), and nymphs hatching (%) (nh) of brontocoris tabidus (mean ± se, standard error, and variation interval) fed on tenebrio molitor pupae after zero (t2), five (t3), and 10 (t4) days from the beginning of the fifth instar on eucalyptus urophylla or without plants (t1) at 23 ± 6 °c, 76 ± 9 % rh, and 13:11 (dark:light) h photoperiod treatments char. t1 t2 t3 t4 pr 10.8 ± 0.97a (6.0-22.0) 12.6 ± 1.35a (6.0-29.0) 13.2 ± 1.42a (3.0-26.0) 13.5 ± 1.39a (6.0-30.0) ov 11.8 ± 1.94b (1.0-33.0) 48.0 ± 4.14a (2.0-86.0) 49.6 ± 5.38a (7.0-108.0) 45.0 ± 5.48a (1.0-84.0) po 5.8 ± 1.05a (1.0-17.0) 7.0 ± 1.02a (1.0-18.0) 5.3 ± 0.65a (1.0-16.0) 5.5 ± 1.38a (1.0-14.0) em 3.6 ± 0.45b (1.0-8.0) 9.4 ± 1.00a (2.0-18.0) 12.9 ± 1.65a (2.0-29.0) 9.5 ± 1.26a (2.0-20.0) eg 110.0 ± 14.59b (19.0-205.0) 410.4 ± 40.41a (57.0-815.0) 452.8 ± 49.87a (82.0-890.0) 332.5 ± 40.43a (40.0-633.0) eg/em 32.2 ± 3.47a (9.0-52.0) 40.4 ± 2.18a (24.3-70.6) 39.4 ± 3.78a (20.5-95.0) 38.5 ± 3.16a (10.0-88.5) ny 86.4 ± 14.32b (23.0-180.0) 259.8 ± 33.77a (42.0-609.0) 299.2 ± 33.07a (54.0-600.0) 201.5 ± 21.23a (54.0-366.0) ny/em 29.8 ± 3.36a (11.5-60.0) 26.0 ± 2.77a (5.2-67.6) 26.4 ± 2.76a (13.5-57.8) 26.0 ± 2.76a (8.2-62.0) lf 29.0 ± 2.37b (8.0-51.0) 62.0 ± 4.85a (23.0-111.0) 65.0 ± 6.06a (8.0-110.0) 63.0 ± 7.02a (17.0-114.0) lm 29.0 ± 2.97b (6.0-52.0) 66.0 ± 6.50a (11.0-135.0) 55.0 ± 6.65a (23.0-122.0) 62.0 ± 7.50a (5.0-127.0) nh 79.6 ± 2.90a (51.8-96.0) 62.4 ± 4.50b (16.5-95.5) 66.8 ± 2.65ab (40.9-85.8) 64.0 ± 4.04b (27.4-85.7) means followed by the same letter, per line, do not differ between them by the tukey’s honest significant difference (hsd) test (p > 0.05). discussion the survival of fifth instar b. tabidus nymphs was higher even after 10 days without mealworm pupae than that for those fed daily on pupae and river red gum seedlings, eucalyptus camaldulensis, flooded gum, eucalyptus grandis, and e. urophylla (zanuncio et al., 2000) or only mealworm pupae (jusselino-filho et al., 2001). this suggests that asopine nymphs can store reserves in the fifth instar to reach adult stage even with prey shortages during the final instar. beyond this, these results indicate that eucalyptus spp. trees can be a better food source for b. tabidus than its seedlings. supputius cincticeps and p. nigrispinus also presented higher survival rates with leaves of upland cotton, gossypium hirsutum (malvaceae) and e. urophylla in their diets when fed on cotton leafworm caterpillars, alabama argillacea (lepidoptera: noctuidae) (lemos et al., 2001). the increased duration of b. tabidus fifth instar nymphs that became males and the decrease of female weight without prey show the importance of food quality for predatory asopines. this was also shown for p. nigrispinus (as podisus connexivus) with longer nymphal period when fed on lighter (3.1 and 11.6 mg) than on heavier (72.4 and 171.0 mg) a. argillacea caterpillars (santos et al., 1995). the similar duration of the fifth instar b. tabidus nymphs between treatments was greater than that for this predator fed only mealworm pupae (jusselino-filho et al., 2001) or domesticated silk moth caterpillars, bombyx mori (lepidoptera: bombycidae) (5.8 days) (oliveira et al., 1999). the temperature in the field may have contributed to increasing the duration of the fifth instar b. tabidus nymphs. the duration of this instar was shorter while mean minimum and maximum temperatures were 16.67 ± 3.03 °c and 27.61 ± 2.99 °c with an average of 23.0 ± 6.56 ºc during the period when b. tabidus nymphs were 392 fig. 1 survival (%) and longevity (days) (mean) of brontocoris tabidus females on eucalyptus urophylla fed on tenebrio molitor pupae after zero (t2), five (t3), and 10 (t4) days from the beginning of fifth instar or without plants (t1) at 23 ± 6 ºc, 76 ± 9 % rh, and 13:11 (dark:light) h photoperiod. maintained on plants in the field. temperature affects b. tabidus metabolism, reproduction, longevity, and behavior (zanuncio et al., 2006, 2011a). the duration of the fifth instar of s. cincticeps nymphs varied from 26 to 28 days at 15 °c and from 6.3 to 6.8 days at 28 °c when fed on house fly larvae, musca domestica (diptera: muscidae) (zanuncio et al., 2005). the duration of fifth instar p. nigrispinus nymphs fed on a. argillacea caterpillars varied from 15.3 to 17.5 days at 17 °c and from 6.3 to 8.0 days at 28 °c (medeiros et al., 2003). this predator had a longer duration of the fifth instar at 10 and 20 °c, 13 and 23 °c and 15 and 25 °c than at a constant temperature of 27 °c (torres et al., 1998). the longer duration of the fifth instar found in this study for b. tabidus may be due to lower temperature and/or to temperature variations in the field. the lower weight of b. tabidus females in the t4 than in the t2 and the similar reproduction between treatments suggests that this predator can survive from prey shortages after receiving prey daily. hence, fifth instar nymphs of this predator can survive in the field even after 10 days without prey from the beginning of the fifth instar. brontocoris tabidus uses the energy from food for development, and resource shortages can increase the duration of nymphal stages and reduce adult weight (zanuncio et al., 2000; oliveira et al., 2005; lemos et al., 2009). beyond this, the energy obtained can be used for two or more competitive processes, such as reproduction and general survival (zanuncio et al., 1996; jusselino-filho et al., 2003) and asopine predators with continuous feeding on prey complete their development in a shorter time period with greater weight (torres et al., 2000). this is important, because heavier podisus rostralis (stål, 1860) females had higher egg production than lighter ones (zanuncio et al., 2002). conversely, food shortages reduced p. nigrispinus oviposition period, viability, and total number of eggs per day (vivan et al., 2003; ramalho et al., 2008; holtz et al., 2009). the longer pre-oviposition period of b. tabidus fed on mealworm pupae and e. urophylla trees in this study than for those fed only b. mori caterpillars was similar to that for this predator with seedlings of e. camaldulensis, e. grandis, and e. urophylla and mealworm pupae (8.3 days) (zanuncio et al., 2000) or only mealworm larvae (7.6 days) (jusselino-filho et al., 2001) suggesting that eucalyptus spp. plant substances may increase the pre-oviposition period of this predator. differences in the oviposition period (period between the first and the last egg mass is laid) of b. tabidus with and without e. urophylla plants show that this predator has better reproductive potential with plants in its diet. the oviposition period presented by this predator was longer compared to that with seedlings of three eucalyptus species (e. camaldulensis, e. grandis, and e. urophylla) in the laboratory (22.1, 26.0, 22.5 days, respectively) (zanuncio et al., 2000) indicating that eucalyptus (an exotic plant in the new word) may be appropriate for b. tabidus. this predator can reproduce across almost its entire lifespan on eucalyptus spp. plants with short post-oviposition period (zanuncio et al., 2000; coelho et al., 2009). high b. tabidus survival on e. urophylla branches results in a higher number of eggs, nymphs, and egg masses per female of this predator than those reported with only mealworm larvae (98.2 eggs and 2.5 egg masses per female) (jusselino-filho et al., 2001) or b. mori caterpillars (oliveira et al., 1999). similar results were found for p. nigrispinus fed mealworm pupae and plants (g. hirsutum cv. precocious cnpa1 or tomato, lycopersicum esculentum cv. ipa5, solanaceae) (oliveira et al., 2002) and s. cincticeps with e. cloeziana leaves (zanuncio et al., 2004). these findings reinforce the hypothesis that eucalyptus spp. plants increase the reproductive capacity of predatory stink bugs. 393 the longevity of b. tabidus males and females with e. urophylla branches and mealworm pupae was shorter than that found for this predator fed b. mori caterpillars (104.5 days for males and 93.8 days for females) (oliveira et al., 1999), indicating the better quality of the later for this predator. however, the longevity of b. tabidus fed on mealworm pupae and eucalyptus plants was 4 times greater than that of those fed only on this prey in the laboratory (jusselino-filho et al., 2001). the high b. tabidus female survival in the treatments with prey and plant material is significant, because it allows this predator to reproduce over longer periods of time with a high egg production in the field. brontocoris tabidus is a generalist predator of eucalypt pests used in classical biological control in brazil (menezes et al., 2013; guanabens et al., 2014). eucalypt plants could be a food source to rear this predator for release in plantations of these species (zanuncio et al., 2000; pereira et al., 2008; pires et al., 2011). the interval of up to 10 days without prey from the beginning of the fifth instar with eucalyptus plants did not affect the adult stage duration and survival for b. tabidus. this plant increased the longevity and reproductive capacity of b. tabidus, suggesting that this predator should be reared on 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(heteroptera: pentatomidae). chil. j. agric. res. 72: 528534, 2012. zanuncio jc, lacerda mc, zanuncio junior js, zanuncio tv, silva amc, espindula mc. fertility table and rate of population growth of the predator supputius cincticeps (heteroptera: pentatomidae) on one plant of eucalyptus cloeziana in the field. ann. appl. biol. 144: 357-361, 2004. zanuncio jc, lemos wp, lacerda mc, zanuncio tv, serrão je, bauce e. age-dependent fecundity and fertility life tables of the predator brontocoris tabidus (heteroptera: pentatomidae) under field conditions. j. econ. entomol. 99: 401-407, 2006. 396 294 isj 15: 294-301, 2018 issn 1824-307x research report molecular characterization of a defender against apoptotic cell death 1 gene (cfdad1) from the mollusk chlamys farreri mq wang1,3, bj wang1, m liu1, ky jiang1, l wang1,2,4* 1cas key laboratory of experimental marine biology, institute of oceanology, chinese academy of sciences, qingdao 266071, china 2laboratory for marine biology and biotechnology, qingdao national laboratory for marine science and technology, qingdao 266237, china 3research platform for marine molecular biotechnology, qingdao national laboratory for marine science and technology, qingdao 266237, china 4center for ocean mega-science, chinese academy of sciences, qingdao 266400, china accepted august 27, 2018 abstract the defender against apoptotic cell death 1 (dad1) was a negative regulator to inhibit the apoptosis process. in the present study, a dad1 gene (designated as cfdad1) was identified in zhikong scallop chlamys farreri. its full-length cdna sequence of cfdad1 contained a 342 bp open reading frame (orf), which encoded a mature protein of 113 amino acids. a dad domain was revealed from the deduced protein sequence of cfdad1. the mrna transcripts of cfdad1 could be detectable in all the investigated tissues, including hemocytes, muscle, mantle, gill, hepatopancreas and gonad, among which the maximum expression level were found in hemocytes. being infected with vibrio splendidus, staphylococcus aureus or yarrowia lipolytica, the mrna expression levels of cfdad1 in hemocytes were all significantly up-regulated. moreover, the apoptosis level of cfdad1-suppressed group was significant higher than those of control groups. all these results indicated that cfdad1 was efficient negative regulator of apoptosis and involved in the innate immune responses of scallop. key words: apoptosis; chlamys farreri; innate immunity introduction apoptosis, also termed as programmed cell death (pcd), was one of the most fundamental processes essential for normal growth and regular development in multicellular organisms (nagata, 2018). the main characteristic morphological features of apoptosis included cellular shrinkage, chromatin condensation, chromosomal dna fragmentation, global mrna decay, membrane blebbing and nuclear condensation (banfalvi, 2017). apoptosis was considered as a pivotal component of various biological processes, such as chemical induced cell death, embryonic development, hormone dependent atrophy, normal cell turnover, proper functioning of the immune defense system, and so on (lockshin, 2016). several factors required ___________________________________________________________________________ corresponding author: lei w ang cas key laboratory of experimental marine biology institute of oceanology chinese academy of sciences qingdao 266071, china e-mail: wanglei@qdio.ac.cn for execution or regulation of apoptosis were rather conserved in evolution, including caspases (galluzzi et al., 2016), inhibitor of apoptosis proteins (iaps) (kocab and duckett, 2016), and also defender against apoptotic cell death 1 gene (dad1) (zhang et al., 2016). however, the activation of apoptotic pathways and the interaction between the key genes differ between the main taxonomic groups of invertebrates, and it is important to investigate the diversity of the apoptotic pathways found in the animal kingdom (estévez-calvar et al., 2013). dad1, a regulatory protein to inhibit the apoptosis process, was first described and characterized as a negative regulator of mammalian cell death that may act downstream of the b-cell lymphoma 2 (bcl2) protein (nakashima et al., 1993). the function of dad1 has been well investigated in multicellular model organisms, for examples, mutants in mouse dad1 exhibited aberrant morphology, developmental delay and increased apoptosis during embryogenesis (nishii et al., 1999; hong et al., 2000), while over expression of dad1 inhibited developmental apoptosis during 295 embryogenesis in caenorhabditis elegans (zhang et al., 2016). these research achievements indicated that dad1 would play irreplaceable roles in regulating apoptosis and cell viability. compared with the extensive identification and investigation of dad1 in vertebrates, although dad1 has already been identified and primary characterized in the bay scallop argopecten irradians (zhu et al., 2008), black tiger shrimp penaeus monodon (molthathong et al., 2008) and kuruma shrimp marsupenaeus japonicas (zheng et al., 2016), the information on dad1 in marine invertebrate is still rare and fragmentary. zhikong scallop chlamys farreri (mollusca; bivalvia; pteriomorphia) is an important and representative species of mollusk, not only for its economic and ecological importance, but also for its increasing value in the invertebrate innate immunity investigation (matozzo, 2016; tascedda and ottaviani, 2016; gerdol, 2017). the research on dad1 in c. farreri would enhance the understanding of its potential functions in apoptosis process and invertebrate innate immunity. however, no information on dad1 was available in zhikong scallop yet. to bridge this gap, a novel dad1 gene (designated as cfdad1) has been identified and investigated in c. farreri, and the main objectives of the present study were (1) to characterize the molecular features of cfdad1 (2) to validate the spatial and temporal expression pattern of its mrna transcripts, and (3) to confirm its function via double strand rna (dsrna) mediated rna interference (rnai). material and method scallops, in vivo experimental infection and samples collection adult scallops (average 50 mm in shell length) were purchased from a local farm in qingdao, china, and maintained in aerated seawater at about 20 °c. approximately two hundred scallops were used for in vivo experimental infection assay according to our previous report (wang et al., 2017). animals were infected by bath exposure using different bacteria and yeast. being acclimated for two weeks, fifty scallops were transferred to tanks containing live bacteria vibrio splendidus strain jz6 at a final concentration of 1.0 × 108 colony forming units (cfus) per 1 ml, defined as gram-negative bacterial infection group. another fifty scallops were transferred and kept in the tanks containing live bacteria staphylococcus aureus strain 33025 (sanyao, china) at a final concentration of 1.0 × 108 cfus ml-1, defined as gram-positive bacterial infection group. the third fifty scallops were maintained in the fungi-containing tanks with live yeast yarrowia lipolytica strain n11b at a final concentration of 1.0 × 108 cfus ml-1, defined as fungi infection group. and the last fifty scallops were treated as the control group. five scallops from every group were randomly sampled at 0, 3, 6, 12, 24 and 48 h post stimulation, respectively. the hemolymphs were collected and centrifuged at 800 g, 4 °c for 10 min to harvest the hemocytes for rna isolation. hemocytes, muscle, mantle, gill, hepatopancreas and gonad from five untreated scallops (one animal was employed as one repeat for one tissue sample) were collected to determine the spatial expression pattern of cfdad1 mrna transcripts. rna isolation and cdna synthesis raw rna was isolated using rnaiso plus (9108, takara, japan). the superscript iv reverse transcriptase (18090010, thermo fisher scientific, usa) was employed to synthesize the first-strand cdna synthesis using rq1 dnase i (m6101, promega, usa) treated raw rna as template and adaptor primer-oligo (dt) as primer (table 1). the reaction were carried out at 55 °c for 1 h, terminated by heating at 80 °c for 5 min, then a homopolymeric tail was added with dctp (4028, takara, japan) and terminal deoxynucleotidyl transferase (tdt, 2230, takara, japan) and then stored at -80 °c till use. cloning the complete cdna sequence of cfdad1 in our previous works (wang et al., 2018b), a transcript sequence homologues to previous identified dad1s was generated via assembling and screening public available transcriptomic data and expression sequence tags (ests) in c. farreri. and this transcript sequence was selected for further cloning the complete cdna sequence of cfdad1. four gene-specific primers, cfdad1-race-r1/2 and cfdad1-race-f1/2, were designed based on this sequence for 5` and 3` rapid amplification of cdna ends (race) technique, respectively (table 1). all pcr amplification was carried out in a mj mini personal thermal cycler (bio-rad, usa), and pcr products were gel-purified using monarch dna gel extraction kit (t1020s, neb, usa), ligated into the pmd18-t simple vector (d103a, takara, japan), and then transformed into the competent cells escherichia coli strain dh5α (cb101, tiangen, china). the positive recombinants were identified via anti-ampicillin selection and verified by pcr using m13-47 and rv-m as primers (table 1). five of the positive clones were sequenced using a prism 3730xl automated sequencer (thermo fisher scientific, usa). bioinformatical analysis of cfdad1 cdna and deduced amino acid sequences the blast+ 2.7.1 was employed to perform search for protein sequence similarity. the deduced protein sequences of cfdad1 were analyzed by lasergene suite 7.1.0.44 using the editseq module. the presence and location of signal peptide and the function domains were predicted with signalp 4.1 and simple modular architecture research tool (smart) 7.0, respectively. multiple sequence alignments were generated using clustal omega 1.2.4 combined with sequence manipulation suite 2.0. a neighbor-joining (nj) phylogenic tree was generated using mega-x 10.0.1. bootstrap trials were replicated 1000 times to derive confidence value for the phylogeny analysis. quantitative real-time pcr analysis of cfdad1 mrna expression profiles the spatial and temporal mrna expression profiles of cfdad1 in hemocytes of scallops infected by various microbes were detected by quantitative real-time pcr (qrt-pcr). all qrt-pcr reactions 296 table 1 oligonucleotide primers used in the experiments primers sequence (5`-3`) brief information adaptor primer ggccacgcgtcgactagtac anchor primer for 3’ race adaptor primer-oligo (dg) ggccacgcgtcgactagtacg10hn anchor primer for 5’ race adaptor primer-oligo (dt) ggccacgcgtcgactagtact17vn olido (dt) for cdna synthesizing cfef-1α-qrt-f* atccttcctccatctcgtcct internal control for real-time pcr cfef-1α-qrt-r* ggcacagttccaatacctcca internal control for real-time pcr cfdad1-cds-f atgcctgatagtttattttccgtggtg gene specific primer for cds cfdad1-cds-r ctatccaatgaagttgataacaactaa gene specific primer for cds cfdad1-dsrna-basic-f ttatattctgtcattcacttc gene specific primer cfdad1-dsrna-basic-r tgtaaaataatatgagcaaaaataaag gene specific primer cfdad1-dsrna-t7-f ggatcctaatacgactcactatagggatccttatattctgtcattcacttc gene specific primer with t7 promoter cfdad1-dsrna-t7-r ggatcctaatacgactcactatagggatcctgtaaaataatatgagcaaaaataaag gene specific primer with t7 promoter cfdad1-qrt-f* cggctacaagtgaatccacag gene specific primer for real-time pcr cfdad1-qrt-r* tccttcccatcaccatttcca gene specific primer for real-time pcr cfdad1-race-f1 cattggatagtatgttatggaaatggt gene specific primer for race cfdad1-race-f2 attcttcaacagtacagatgtgtatat gene specific primer for race cfdad1-race-r1 ggatgacgcctgtcatcaggatgtaga gene specific primer for race cfdad1-race-r2 tttttgcaaaataaaatcaacgtggca gene specific primer for race egfp-dsrna-basic-f cgacgtaaacggccacaagt gfp primer egfp-dsrna-basic-r cttgtacagctcgtccatgc gfp primer egfp-dsrna-t7-f ggatcctaatacgactcactatagggatccgacgtaaacggccacaagt gfp primer incorporated with t7 promoter egfp-dsrna-t7-r ggatcctaatacgactcactatagggatccttgtacagctcgtccatgc gfp primer incorporated with t7 promoter m13-47 cgccagggttttcccagtcacgac sequencing primer rv-m gagcggataacaatttcacacagg sequencing primer * the efficiency of cfef-1α-qrt-f/r and cfdad1-qrt-f/r were 98% and 101%, respectively. were carried out with the sybr premix extaq (rr420, takara, japan) in a linegene k fqd-48a (a4) fluorescence quantitative pcr detection system (bioer, china) using 100 ng cdna as template. all the primer information for qrt-pcr was listed in table 1. the efficiency of qrt-pcr primers were checked using serial two-fold dilutions of cdna (pfaffl, 2001), and the efficiency of cfef-1α-qrt-f/r and cfdad1-qrt-f/r were almost the same (98% and 101%, respectively). the mrna expression of cfdad1 was normalized to that of elongation factor 1 α (ef-1α) for each sample. the relative mrna expression levels of cfdad1 were calculated using comparative ct method (2-δδct method) (schmittgen and livak, 2008) as mean ± s.d. (n = 5). the data were tested with one-way analysis of variance (anova) followed by a multiple comparison using ibm spss statistics 23.0.0.0, and the p values less than 0.05 were considered statistically significant. knock-down of cfdad1 gene in vivo via dsrna mediated rna interference and apoptotic assay t7 promoter adapted primers cfdad1-dsrna-t7-f/r and egfp-dsrna-t7-f/r (table 1) were used to amplify cdna fragments from cfdad1 and enhanced green fluorescent protein (egfp), and the pcr products were gel-purified and used as templates to synthesize dsrna. the dsrna products were synthesized via in vitro transcription according to the our previously reports (wang et al., 2011; wang et al., 2016a,b; wang et al., 2018a), and its concentration was quantified using nanodrop lite (thermo fisher scientific, usa) by the absorbance at 260 nm and adjusted to a final concentration of 1 mg ml-1. one hundred micrograms of cfdad1 dsrna was injected into the adductor of each scallop, and the control groups received an injection of 100 μg egfp dsrna or pbs, while the untreated scallops were employed as blank group. hemocytes from five randomly sampled scallops of each treatment were collected every 12 h (0, 12, 24, 36, 48, 60, 72, 84, 96, 108 and 120 h post dsrna injection) and used for total rna isolation and cdna synthesis. the effect of dsrna-mediate rnai for cfdad1 in scallops was verified via qrt-pcr, and 70% inhibition of mrna abundance after dsrna injection was a threshold for an effective rnai assay (krueger et al., 2007). five individuals were randomly sampled at 0, 48, 72 and 120 h post dsrna injection, and the hemolymphs were collected and centrifuged at 800 g, 4 °c for 10 min to harvest the hemocytes for apoptotic assay. the apoptosis level of scallops was assayed via caspase-3 colorimetric assay kit (kga204, keygen biotech, china). all data were given in terms of od450 as means ± s.d (n=5). the data were tested with one-way analysis of variance (one-way anova) followed by a multiple comparison (s-n-k) using ibm spss statics 23.0.0.0, and the p values less than 0.05 were considered statistically significant. 297 fig. 1 sequence features, multiple alignments and phylogenetic analysis of cfdad1. a: nucleotide and deduced protein sequences of cfdad1. the nucleotides and amino acids were numbered along the left margin. the function domain was in shade. the transmembrane segments were underlined. the stop codon was indicated by asterisks. b: multiple alignments of cfdad1 with previous known ones. the black shadow region indicated positions of same amino acid residues shared by all sequences. similar amino acid residues were shaded in grey. gaps were indicated by dashes to improve the alignment. c: consensus phylogenetic tree based on the amino acid sequences of different dad1s. neighbor-joining method was selected to infer the evolutionary history. the numbers at the forks stood for the bootstrap value (%). the sequences and their accession numbers are as follows: argopecten irradians, aax56947; gallus gallus, aac60276; haliotis discus, anf99571; haliotis diversicolor, aga92566; homo sapiens, aab58540; marsupenaeus japonicas, aon76443; mus musculus, caa73779; orseolia oryzae, alh18096; palaemon carinicauda, agj03553; penaeus monodon, abu54835; sus scrofa, baa13115 results the molecular feathers and phylogeny relationship of cfdad1 the complete cdna sequence of cfdad1 was obtained via 5` and 3` race, and then submitted to genbank with the accession number ku361830. it comprised 660 bp, containing a 78 bp 5` untranslated regions (utr), a 240 bp 3` utr with a poly a tail and a 342 bp open reading frame (orf). this orf encoded a mature protein of 113 amino acid residues with a theoretical molecular mass at about 12.66 kda and a calculated isoelectric point (pi) of 7.99. no signal peptide could be predicted in the deduced protein sequence. the deduced amino acid sequence of cfdad1 contained a dad domain (from l5 to g113). additionally, three transmembrane segments (from a30 to f51, from f56 to n78, and from a93 to i112) were also revealed (figure 1a). an alignment of the protein sequence of cfdad1 with those previously identified ones was shown in figure 1b. the deduced amino acid sequence of cfdad1 exhibited high similarity with previously identified ones, such as 99% identity with that of a. irradians (aax56947), 94% with mizuhopecten yessoensis (owf34780), and 81% with haliotis diversicolor (aga92566). a nj phylogenetic tree based on protein sequences from multiple dad1s was positioned separately into two main branches, sequences form invertebrates and vertebrates were separated. within the invertebrates, cfdad1 was closer to similar sequences from another mollusc such as bay scallop a. irradians and far from those from crustaceans (figure 1c). 298 fig. 2 spatial and temporal mrna expression patterns of cfdad1. a: spatial mrna expression pattern of cfdad1. the mrna expression level in hemocytes, mantle, gill, hepatopancreas and gonad of five adult scallops was normalized to that of muscle. b-d: temporal mrna expression patterns of cfdad1 in hemocytes at 0, 3, 6, 12, 24 and 48 h post microbe infection (b: v. splendidus, c: s. aureus, d: y. lipolytica). vertical bars represented mean ± s.d. (n = 5), and bars with different characters were statically significant (p < 0.05) the spatial and temporal expression profile of cfdad1 mrna transcripts the qrt-pcr was employed to detect the spatial and temporal mrna expression profiles of cfdad1 with ef-1α as internal control. the cfdad1 mrna was detectable in all the tested tissues. the maximum level was observed in hemocytes, which was 5.33-fold (p < 0.05) of that in muscle, followed by gill and hepatopancreas, which were 3.29-fold and 3.16-fold of that in muscle (p < 0.05), respectively. and the mrna expression levels in mantle and gonads were 1.05-fold and 1.12-fold of that in muscle (p > 0.05), respectively (figure 2a). the mrna expression levels of cfdad1 were all up-regulated post different in vivo experimental infection. the mrna expression level of cfdad1 significantly up-regulated at 3 h post v. splendidus infection (4.04-fold compared with the origin level, p < 0.05), and reached to the peak level at 12 h (14.08-fold, p < 0.05, figure 2b). in the s. aureus infection group, its mrna expression level was significantly up-regulated at 6 h post infection (5.36-fold, p < 0.05), reached the peak level at 12 h post infection (9.38-fold, p < 0.05), maintained at a high level at 24 h (5.71-fold, p < 0.05) and then decreased to the origin level at 48 h (figure 2c). being infected by y. lipolytica, its mrna transcripts were significantly induced at 6 h post infection (3.17-fold, p < 0.05) and reached the maximum level at 12 h post infection (5.98-fold, p < 0.05), maintained at a high level at 24 h (3.32-fold, p < 0.05) and then decreased to the origin level at 48 h (figure 2d). the apoptotic levels in cfdad1-suppressed scallops in the present study, the effect of dsrna-mediate rnai was verified via qrt-pcr, and the mrna expression level of cfdad began to decrease at 24 h post dsrna injection (0.66-fold, p < 0.05) and kept at a rather low level (less than 0.3-fold of the normal expression level) during 36 to 84 h post rnai (figure 3a). after dsrna injection, the apoptosis level of cfdad1-suppressed scallops lagged behind the expression change of cfdad1 and was obviously higher than those of other groups at 48, 72 and 120 h post injection (figure 3b). 299 fig. 3 rnai of cfdad1 and apoptosis level. a: the relative abundance of cfdad1 mrna during rnai. each values was shown as mean ± s.d. (n = 5), and bars with different characters indicated statically significant (p < 0.05). b: gene silencing of cfdad1 increased apoptosis level. each values was presented as od405 and shown as mean ± s.d. (n = 5), and bars with different characters indicated statically significant (p < 0.05) discussion dad1 was a negative regulatory protein to inhibit the apoptosis process (kelleher and gilmore, 1997; hong et al., 2000; lockshin, 2016; nagata, 2018). in the present study, the complete cdna sequence of cfdad1 was identified and characterized in c. farreri. its sequence feathers, high similarity and close phylogenetic relationship to previously identified ones collectively suggested that it was a novel member of invertebrate dad1 family and may have similar functions. dad1 it has been observed to be ubiquitously expressed in various tissues in transcriptional level in marine invertebrates (molthathong et al., 2008; zhu et al., 2008; zheng et al., 2016), and in the present study, the cfdad1 mrna could be also detected in all the tested tissues. the highest mrna expression level was found in hemocytes, followed with gill and hepatopancreas, and the variable spatial distribution of cfdad1 mrna transcripts was speculated to be related with tissue function. the low apoptotic activity in hemocytes compared to other tissues has been reported in other bivalves 300 such as the mytilus galloprovincialis (romero et al., 2011), which was consist with our observation, for the function of cfdad1, the higher gene expression the lower apoptotic activity. moreover, the hepatopancreas was believed to be the central immune related organ in crustaceans and mollusks (chai et al., 2010), while gill was regarded as the first defense line against invading microbes in fish and invertebrates (ellis, 2001; wang et al., 2016c). the high mrna expression level of cfdad1 in these three tissue indicated that dad1 might be involved in the innate immune responses of scallop. it has been reported that dad1 could respond to various stimuli in transcriptional level. for examples, mjdad1 showed both dose-dependent and time-dependent responses to nitrite stress in m. japonicas (zheng et al., 2016), while the mrna expression levels of aidad1 gene of hemolymph were significantly high after injury in a. irradians (zhu et al., 2008). in the present study, cfdad1 mrna transcripts in hemocytes could be significantly induced by the stimulation of three typical microbes. one potential speculation would be that the increase of cfdad1 could reduce the apoptotic activity in hemocytes, for the low apoptotic level could favor the phagocytic activity of hemocytes to engulf invading pathogens as more as possible before its dead (canesi et al., 2002). so, the rapid responses of cfdad1 to various invading pathogens indicated that apoptosis and its regulators could play pivotal role in the innate immune response of scallops. the response of dad1s to inhibit apoptosis has been well studied in an array of model organisms. for examples, mutants of dad1 could increase cell apoptosis during embryogenesis in mouse (hong et al., 2000), while the over expression of dad1 would inhibit developmental apoptosis in c. elegans (sugimoto et al., 1995). for neither gene mutation technique nor gene overexpression method was available in marine mollusk yet, dsrna-mediated rnai technique was applied to inhibit the expression of cfdad1 to investigate its potential effects. in our previous reports, this dsrna-mediated rnai technique has been successfully applied to inhibit the expression of several target genes in scallop. in the present study, this protocol was employed as a powerful tool to analyse more in detail the apoptosis. after dsrna injection, the apoptosis level of cfdad1-suppressed scallops was obviously higher than those of other groups, suggesting the negative regulating roles of cfdad1 during apoptosis in scallop. in conclusion, the complete cdna sequence of a novel dad1 gene was identified and characterized in c. farreri. its mrna expression levels significantly increased post microbe infection. the cfdad1-suppressed scallops exhibited higher apoptosis level. all these results indicated that cfdad1 was efficient negative regulator of apoptosis and involved in the innate immunity. acknowledgement this research was supported by the national natural science foundation of china (u1706209), aoshan innovation project in science and technology from qingdao national laboratory for marine science and technology (2016askj07), science and technology service network plan (sts) major deployment project (kfzd-sw-106) and sts regional centre project (fujian province) from chinese academy of sciences. we are grateful to dr. zhenming chi, college of marine life science, ocean university of china, for his kindly providing the yeast y. lipolytica, and dr. zhao lv, institute of oceanology, chinese academy of sciences, for his kindly providing the bacteria v. splendidus. we also thank dr. xungang tan, institute of oceanology, chinese academy of sciences, for his kindly providing the pcs2+egfp plasmid containing the full orf of egfp. we would like to thank the editorial office and the 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drosophila melanogaster. dev. biol. 420: 186-195, 2016. zheng jb, mao y, su yq, wang j. effects of nitrite stress on mrna expression of antioxidant enzymes, immune-related genes and apoptosis-related proteins in marsupenaeus japonicus. fish shellfish immu. 58: 239-252, 2016. zhu l, song ls, zhang h, zhao jm, li ch, xu w. molecular cloning and responsive expression to injury stimulus of a defender against cell death 1 (dad1) gene from bay scallops argopecten irradians. mol. biol. rep. 35: 125-132, 2008. the unfolded protein response signaling pathways in mollusca 183 isj 15: 183-196, 2018 issn 1824-307x review the unfolded protein response signaling pathways in molluscs y huang1,2,3, j sun1,3, l wang1,2,3, l song1,2,3* 1liaoning key laboratory of marine animal immunology, dalian ocean university, dalian 116023, china 2laboratory of marine fisheries science and food production processes, qingdao national laboratory for marine science and technology, qingdao 266235, china 3liaoning key laboratory of marine animal immunology & disease control, dalian ocean university, dalian 116023, china accepted may 14, 2018 abstract unfolded protein response (upr) as collective signal transduction pathways is essential for surviving the endoplasmic reticulum (er) stress in vertebrates and invertebrates. upon accumulation of unfolded proteins in the er lumen, upr increases the degradative and protein-folding capacities of cells and decreases global protein synthesis to maintain the cell homeostasis. because of their importance in cellular stress and protein folding process, upr signaling pathways receive increasing attentions, and their components and multiple regulation functions have been well characterized in mammals, fly, and worm etc. molluscs are widely distributed in various environments with high species diversity, which exhibit remarkable capacity for adaptation and survival upon diverse stressors. because of the homeostatic role in response to er stress, the knowledge about upr would be helpful for understanding the wide distributions, living habits and adaptability to the environment of molluscs. this review summarizes the upr signaling pathways in molluscs with the emphasis on recent research progresses about the characteristics of molluscan upr signaling pathway members and their expression profiles in response to various environmental stressors. key words: molluscs, unfolded protein response (upr), signaling pathway, molecular components, expression profile, stress response introduction unfolded protein response (upr) is a collection of phylogenetically conserved signaling pathways to monitor the conditions in endoplasmic reticulum (er), including insufficiency of er’s protein-folding ability and the excessive misfolding, and then initiates the consequent signal transduction pathways (walter and ron, 2011). when protein folding in the er is inhibited, upr increases the biosynthetic capacity and decreases the biosynthetic burden of the er to maintain the homeostasis of cell (schröder and kaufman, 2005). in mammals, upr is composed of three signaling pathways represented by three er transmembrane protein sensors, inositol-requiring enzyme 1α (ire1α), double-stranded rna-activated protein kinase (pkr)-like endoplasmic reticulum kinase (perk), and activating transcription factor 6 (atf6) ___________________________________________________________________________ corresponding author: linsheng song dalian ocean university 52 heishijiao street, dalian 116023, china e-mail: lshsong@dlou.edu.cn (cao and kaufman, 2012). the signal transduction mechanisms of the three signaling pathways are different: atf6 by regulating proteolysis, perk by translational control, and ire1 by nonconventional mrna splicing (fig. 1) (walter and ron, 2011). upr plays an important role in immunity and inflammation, and is involved in various prevalent diseases such as metabolic syndrome, neurodegenerative disorders, inflammatory bowel disease, and cancer (grootjans et al., 2016). the phylum mollusca is second only to the arthropods with numbers of more than 100,000 species (telford and budd, 2011). they distribute extensively in marine intertidal zone, freshwater and terrestrial ecosystems, and even in extreme environments such as deep-sea hydrothermal vents, 40 °c freshwater, and permanent ice areas (haszprunar and wanninger, 2012). molluscs have evolved major stress-response pathways such as oxidation or anti-oxidation, apoptotic pathways and upr signaling pathways to respond stresses and survive in extreme conditions (zhang et al., 2012; zhang et al., 2016a). the activation of upr pathways is considered to be crucial to respond mailto:lshsong@dlou.edu.cn 184 cellular stresses in er and maintains the cellular homeostasis under various stressors (walter and ron, 2011), such as temperature elevation (wang et al., 2012; zhang et al., 2012; li et al., 2017), air exposure (kawabe and yokoyama, 2010), as well as metal bioaccumulation (poynton et al., 2014). the aim of this review is to outline the molecular components of upr signaling pathways so far identified in molluscs, their expression profiles under various stressors, and the related downstream response pathways, as well as their possible significance in the stress response of molluscs. the molecular components of upr signaling pathway in molluscs recently, upr and its response to various stressors in molluscs have been paid increasing attentions. the core members in ire1, perk and atf6 signaling pathways have been described in several molluscan species (table 1; fig. 2) (mori, 2009; vinther et al., 2012; hollien, 2013; janssens et al., 2014; tanner et al., 2017). er chaperone bip (binding immunoglobulin protein) and other important er molecules including calreticulin (crt), calnexin (cnx), protein disulfide isomerase (pdi) and peptidyl-prolyl cis-trans isomerases (ppi) (kennedy et al., 1992; kawabe and yokoyama, 2010; leung et al., 2011; mu et al., 2015; clark et al., 2016) involved in molluscan upr also receive widespread attention. the molecular characteristics of ire1, perk and atf6 signaling pathway members as well as other important er members involved in molluscan upr are discussed in this section. bip (grp78) binding immunoglobulin protein (bip), also referred as 78 kda glucose regulated protein (grp78) (hendershot, 2004), is an er molecular chaperone of the heat shock protein hsp70 family (ting and lee, 1988; hendershot et al., 1994). it is well known for its activity to bind hydrophobic patches on nascent polypeptides within the er and its role in upr signaling pathways (quinones et al., 2008). structurally, bip is highly conserved across species (quinones et al., 2008) with two conserved functional domains, a nucleotide-binding domain (nbd) at the n-terminus and a substrate-binding domain (sbd) at the c-terminus. the nbd binds and hydrolyzes atp to adp, and the sbd binds hydrophobic polypeptides in an extended conformation as substrates (yang et al., 2015). the bip genes have been so far described in some molluscan species including intertidal gastropod littorina littorea (storey et al., 2013), sea hare aplysia californica (kuhl et al., 1992), sea snail nacella concinna, antarctic clam laternula elliptica (clark et al., 2008; clark et al., 2016), pacific oyster fig. 1 schematic diagram of upr 185 table 1 the upr members and expression profiles in mollusca species species stresses response tissues references bip (grp78) grp78 l. littorea 20 h anoxia exposure ↑ foot muscles storey et al., 2013 grp78 a. californica 3, 7, 18 h glucose starvation ↑ central nervous system kuhl et al., 1992 grp78 n. concinna 2 h heat shock for 10°c, 15°c or 20°c — feet clark et al., 2008 grp78 l. elliptica 2 h heat shock 15°c ↑ gills, mantles and siphons clark et al., 2008 grp78 l. elliptica 12 h acute heat treatment at 3°c ↑ siphons (younger animals compared with older animals) clark et al., 2016 grp78 c. gigas 24 h and 48 h heat treatment for 35°c ↑ gills and adductor muscles yokoyama et al., 2006 grp78 c. gigas 12 h pb treatment 500 μg/l ↑ gills g. zhang et al., 2012 grp78 c. gigas 6, 12, 24 h acute heat stress 25°c ↑ haemocytes yang et al., 2017 grp78 c. hongkongensis 1h heat treatment 37°c ↑ gills li et al., 2017 grp78 g. demissa 1 h acute heat stress 40°c one isoform↓ another isoform↑ gills fields et al., 2012a grp78 g. demissa 1 h heat stress for 45°c ↑ gills fields et al., 2016 grp78 m. trossulus 4 weeks chronic temperature stress at 19°c ↑ gills fields et al., 2012b grp78 v. lienosa 6 days heat treatment for 29±2°c ↑ adductor muscles, mantles, and gills w ang et al., 2012 grp78 p. viridis 24 h benzo(a)pyrene stimulation 400 μg/l ↑ embryos jiang et al., 2016 ire1–xbp1 signaling pathway ire1 c. gigas 12 h pb treatment 500 μg/l ↑ gills g. zhang et al., 2012 ire1 c. gigas 6, 12, 24 h acute heat stress 25°c ↑ haemocytes yang et al., 2017 ire1 v. lienosa 6 days heat treatment for 29±2°c ↑ adductor muscles, mantles, and gills w ang et al., 2012 xbp1 v. lienosa 6 days heat treatment for 29±2°c ↑ adductor muscles, mantles, and gills w ang et al., 2012 xbp1 o. edulis heavy bonamiosis ↑ haemolymph cells martín-gómez et al., 2014 xbp1 o. edulis heavy disseminated neoplasia (dn) ↑ haemolymph cells martín-gómez et al., 2014 xbp1 o. edulis light dn ↑ mantles and digestive organs martín-gómez et al., 2014 xbp1 o. edulis light dn ↓ gills martín-gómez et al., 2014 xbp1 m. edulis 2 weeks pb treatment 0.54 μm ↑ gills poynton et al., 2014 xbp1 m. edulis 2 weeks (pb and cd) mix-treatment 0.54 μm ↑ gills poynton et al., 2014 perk–eif2a–atf4–chop signaling pathway perk c. gigas 9 days air exposure ↑ gills g. zhang et al., 2012 perk c. gigas 6, 12, 24 h acute heat stress 25°c ↑ haemocytes yang et al., 2017 perk c. hongkongensis 1h heat treatment 37°c ↑ gills li et al., 2017 perk v. lienosa 6 days heat treatment for 29±2°c ↑ adductor muscles, mantles, and gills w ang et al., 2012 eif2α v. lienosa 6 days heat treatment for 29±2°c ↑ adductor muscles, mantles, and gills w ang et al., 2012 eif2α c. gigas 12 h heat treatment 35°c ↑ gills g. zhang et al., 2012 phospho-eif2α l. littorea 20 h anoxia exposure ↑ foot muscles storey et al., 2013 phospho-eif2α mercenaria 18 h anoxia treatment ↑ adductor muscles ivanina et al., 2016 atf4 l. littorea 20 h anoxia exposure ↑ foot muscles storey et al., 186 2013 atf4 v. lienosa 6 days heat treatment for 29±2°c ↑ muscles, mantles and gills w ang et al., 2012 bcl2 c. gigas 5 days air exposure ↑ muscles g. zhang et al., 2012 gadd34 l. littorea 20 h anoxia exposure ↓ foot muscles storey et al., 2013 gadd153 l. littorea 20 h anoxia exposure ↑ foot muscles storey et al., 2013 atf6 signaling pathway p90 atf6 l. littorea 20 h anoxia exposure — foot muscles storey et al., 2013 p50 atf6 l. littorea 20 h anoxia exposure ↓ foot muscles storey et al., 2013 others members crt a. californica 3, 7, 18 h glucose starvation ↑ central nervous system kennedy et al., 1992 crt c. gigas 7 days air exposure 20°c fluctuated adductor muscles, mantles, gills kawabe et al, 2010 crt c. gigas 15 days air exposure 4°c fluctuated adductor muscles, mantles, gills kawabe et al, 2010 crt c. gigas 12 h pb treatment 500 μg/l ↑ gills g. zhang et al., 2012 crt c. hongkongensis 1h heat treatment 37°c ↑ gills li et al., 2017 crt p. viridis 14 days cd treatment 0.5 ppm fluctuated hepatopancreas leung et al., 2011 crt p. viridis 24 h benzo(a)pyrene stimulation 400 μg/l ↑ embryos jiang et al., 2016 cnx c. gigas 7 days air exposure 20°c fluctuated adductor muscles, mantles, gills kawabe et al, 2010 cnx c. gigas 15 days air exposure 4°c fluctuated adductor muscles, mantles, gills kawabe et al, 2010 cnx c. gigas 12 h pb treatment 500 μg/l ↑ gills g. zhang et al., 2012 cnx c. hongkongensis 1 h heat treatment 37°c ↑ gills li et al., 2017 pdi p. diffusa 3 weeks chronic heat treatment 35°c ↑ hepatopancreas mu et al., 2015 pdi p. canaliculata 3 weeks chronic heat treatment 35°c ↑ hepatopancreas mu et al., 2015 pdi c. hongkongensis 1 h heat treatment 37°c ↑ gills li et al., 2017 pdi c.gigas 6, 12, 24 h acute heat stress 25°c ↑ haemocytes yang et al., 2017 pdi p. viridis 14 days cd treatment 0.5 ppm ↑ hepatopancreas leung et al., 2011 pdi p. viridis 24 h benzo(a)pyrene stimulation 400 μg/l ↑ embryos jiang et al., 2016 ppi g. demissa 1 h acute heat stress 40°c ↑ gills fields et al., 2012a ppi g. demissa 1 h heat stress for 45°c ↑ gills fields et al., 2016 ppi l. elliptica 12 h acute heat treatment at 3°c ↑ siphons (younger animals compared with older animals clark et al., 2016 ↑: up-regulation ↓: down-regulation —: no significant change crassostrea gigas (yokoyama et al., 2006; zhang et al., 2012; yang et al., 2017), oyster c. hongkongensis (li et al., 2017), atlantic ribbed mussel geukensia demissa (fields et al., 2012a; fields et al., 2016), blue mussel mytilus trossulus (fields et al., 2012b), freshwater mussel villosa lienosa (wang et al., 2012), and green mussel perna viridis (jiang et al., 2016). however, most of the recent researches on molluscan bip are mainly focused on the gene cloning and sequence analysis, and mainly from bivalve and gastropod. the protein sequences of molluscan bip are found to be relatively conserved among molluscs, and even comparing with other invertebrates and vertebrates (mamady and storey, 2006; song et al., 2015). for instance, the oyster bip (genbank: bad15288.1) shares 89%, 87% and 83% similarities of amino acid sequences with that from bivalve mizuhopecten yessoensis (accession: xp_021349015.1), gastropod a. californica (accession: np_001191581.1), and mammal homo sapiens (accession: np_005338.1), respectively (yokoyama et al., 2006). oyster bip contains an atpase domain and a peptide binding domain, which denotes the 187 protein translocation, er calcium stores, and unfolded protein response activation as mammalian bip (hendershot, 2004). under homeostatic conditions, the er-luminal domains of er stress sensors (perk and ire1, atf6) in upr signaling pathways are maintained in an inactive state through association with bip, and the sensors can be activated by releasing from bip when the misfolded proteins are accumulated in er lumen (cao and kaufman, 2012; grootjans et al., 2016). although many homologues of bip have been identified in molluscs, the knowledge about the detailed structure and function of molluscan bips are still limited, and further studies are needed to evaluate their regulation mechanism in upr to maintain the homeostasis under er stress. ire1–xbp1 signaling pathway members ire1–xbp1 (x-box binding protein 1) signaling pathway is the most conserved upr pathway in yeasts, nematodes, insects, and mammals (mori, 2009; cao and kaufman, 2012). the molecular components of ire1 signaling mainly include ire1 and its substrate xbp1 (liu and kaufman, 2003). ire1 is a stress receptor of er localized type i transmembrane protein with a serine/threonine kinase domain on its cytosolic portion and an endoribonuclease (rnase) domain (grootjans et al., 2016). similar to other two er stress sensors (perk and atf6), ire1 can be activated directly by bip dissociation. moreover, yeast ire1 can also directly bind to unfolded proteins to activate the upr (gardner and walter, 2011). so far, two ire1s have been identified from pacific oyster c. gigas (zhang et al., 2012; yang et al., 2017) and freshwater mussel v. lienosa (wang et al., 2012). molluscan ire1 owns low conserved protein sequence compared with the ones identified from invertebrate and vertebrate. for example, the deduced amino acid sequence of oyster ire1 (genbank: ekc40550.1) shares 52% and 48% identity with that from echinoderm acanthaster planci (xp_022103307.1) and mammal bos mutus (elr62219.1), respectively. conserved domain (marchler-bauer et al., 2014) alignments showed that the oyster ire1 contained a luminal domain, a catalytic domain of the serine/threonine kinase and an rnase domain, which are conserved from human to yeast ire1s (tirasophon et al., 1998). upr is initiated by er-localized transmembrane receptors with lumenal domains that sense misfolded proteins, and then the cytosolic effector domains pass the signal to the downstream components (d and sr, 2008). the presence of conserved lumenal and cytosolic effector domains in the molluscan ire1 suggested that it might play important roles in the initiation of upr in molluscs (zhang et al., 2012; marchler-bauer et al., 2014; yang et al., 2017). dual copies of ire1 (ire1α and ire1β) have been discovered in vertebrates such as mammals (mori, 2009) and xenopus embryos (yuan et al., 2008), whereas only one ire1 has been identified in c. elegans and d. melanogaster (mori, 2009). similar to other invertebrates, the unique isoform of ire1 was recently found in molluscs (wang et al., 2012; zhang et al., 2012; yang et al., 2017). xbp1 mrna is the substrate of ire1α and ire1β in mammals, which encodes a basic leucine zipper containing transcription factor. three xbp1 genes were recently identified in molluscs, including flat oyster ostrea edulis (martín-gómez et al., 2014), blue mussel m. edulis (poynton et al., 2014), and freshwater mussel v. lienosa (wang et al., 2012). although all the xbp1s from mollusc contain a basic leucine zipper (bzip) domain, their amino acid sequences display low conservation compared with that in other organisms. xbp1 from oyster c. gigas (genbank: ekc34044.1) shares 59%, 51%, 41% and 35% identity with that in mollusca bivalvia mytilus edulis (accession: aba43316.1) and mizuhopecten yessoensis (accession: owf42676.1), vertebrate fish danio rerio (accession: aai52196.1), and mouse cricetulus griseus (accession: egw12286.1), respectively. the splicing of xbp1 mrna is initiated by the rnase activity of ire1 to generate mature xbp1 mrna (liu and kaufman, 2003). xbp1 induces the expression of a wide range of genes that orchestrate er protein folding, secretion, quality control and endoplasmic-reticulum-associated protein degradation (erad), and activates phospholipid biosynthesis and er expansion upon er stress (cao and kaufman, 2012). the identification of ire1 and xbp1 in oyster and mussel suggests that ire1–xbp1 signaling pathway also exists in the molluscs. nevertheless, the detailed mechanisms of xbp1 mrna maturation and the induction of xbp1 to the downstream genes in molluscs still need further investigations. perk–eif2α–atf4–chop signaling pathway members the perk signaling pathway usually attenuates the initiation of translation by activating perk to phosphorylate α subunit of eukaryotic translation initiation factor 2 (eif2α) to reduce the er protein-folding burdens in mammals (walter and ron, 2011). perk, eif2α, the activating transcription factor 4 (atf4) and ccaat/enhancer binding protein homologous protein (chop) are identified as the main members of perk signaling pathway in mammals (cao and kaufman, 2012). perk is a type i transmembrane protein with a cytosolic serine/threonine kinase domain, which shares similar luminal domain and activation machinery with ire1α (grootjans et al., 2016). in response to er stress, the activation of perk is triggered by a large number of signals to phosphorylate the α subunit of eif2α, blocks the recycling of gdp to gtp, and finally leads to translation attenuation (liu and kaufman, 2003; donnelly et al., 2013). the recently reports about molluscan perk–eif2α–atf4–chop signaling pathway are mainly from bivalve and gastropod. three molluscan perks have been identified in oyster c. gigas (zhang et al., 2012; yang et al., 2017), c. hongkongensis (li et al., 2017), and freshwater mussel v. lienosa (wang et al., 2012). the oyster perks are low-conservative in comparison with their homologues from other invertebrates and vertebrates. the deduced amino acid sequence of oyster perk (genbank: ekc35408.1) shares 43%, 38% and 36% identity with that from mizuhopecten yessoensis (accession: 188 fig. 2 family tree of molluscan upr owf40340.1), arthropoda limulus polyphemus, and homo sapiens (aai26355.1). the oyster perk protein possesses luminal dimerization domain, and catalytic domains of serine/threonine-specific and tyrosine-specific protein kinases, indicating the conserved sensing and activation machinery of perk in molluscs. eif2α genes have also been discovered in molluscs, including intertidal gastropod l. littorea (storey et al., 2013), oyster c. gigas (zhang et al., 2012), clam mercenaria mercenaria (ivanina et al., 2016), and v. lienosa (wang et al., 2012). the amino acid sequences of eif2αs are relatively conservative in molluscs, other invertebrates and vertebrates. the deduced amino acid sequence of oyster eif2α (genbank: ekc31327.1) contains a conserved eukaryotic translation initiation factor 2 alpha subunit，which shares 86% and 88% identity with that in mollusca bivalve m. yessoensis (accession: xp_022341697.1) and gastropod a. californica (accession: xp_005105117.1), and 79% with human (accession: np_004085.1). phosphorylation/dephosphorylation of eif2α at ser-51 is the major regulatory activity site in protein synthesis of eukaryotic cells and this site in molluscan eif2α also deserves further study (nonato et al., 2002). in mammalian perk signaling pathway, atf4 induces the transcriptions of bip and grp94, and stimulates autophagy gene expression. chop is the downstream targets of eif2α–atf4, and it is an important transcription factor of er stress-induced apoptosis to increase the protein synthesis by inducing transcription of growth arrest and dna damage 34 (gadd34) (cao and kaufman, 2012; grootjans et al., 2016). the induced gadd34 recruits protein phosphatase 1 (pp1) to dephosphorylate eif2α-p and reverses the translational attenuation (liu and kaufman, 2003). atf4 has been identified in mussel v. lienosa under elevated temperature exposure (wang et al., 2012). atf4, chop and gadd34 were further identified in intertidal gastropod l. littorea, suggesting that perk sensor could mediate upr response under anoxia to suppress protein synthesis and elevate er chaperones, which would contribute to cell survival during prolonged anaerobiosis (storey et al., 2013). the increasing reports about the components in perk–eif2α–atf4–chop signaling pathway in different molluscs suggests that this signaling pathway may be evolutionarily conservative in molluscs, and play indispensable contribution in the adaptive evolution of diverse molluscan species (pomar et al., 2003; cao and kaufman, 2012; kitamura, 2013; krivoruchko and storey, 2013). the atf6 signaling pathway members in mammals, the activation of atf6 signaling pathway is to cleave inactive 90 kda atf6 to active 189 50 kda atf6. as a type ii er transmembrane protein, atf6 harbors a creeb/atf basic leucine zipper (bzip) transcription factor in its cytosolic domain (cao and kaufman, 2012). there are two closely related factors, atf6α and atf6β, in mammals. it is reported that atf6α but not atf6β plays important role in transcriptional control (adachi et al., 2008). upon accumulation of unfolded proteins in er, 90 kda atf6 is released from bip and transferred to the golgi apparatus to be cleaved into 50 kda atf6 by site-1 protease (s1p) and site-2 protease (s2p). 50 kda atf6 is then transported to nucleus to activate gene expression (grootjans et al., 2016). the oyster atf6 (accession: xm_022470698.1) and other molluscan atf6s exhibit low conservation compared with that in other invertebrates and vertebrates. the deduced amino acid of oyster atf6 exhibits 42% identity with that in bivalve mizuhopecten yessoensis (accession: xp_021375264.1), 34% identity with that in arthropod limulus polypohemus (accession: xp_013792454.1) and fish ctenopharynqodon idella (accession: akz87017.1). the basic region leucine zipper (bzip) domain of oyster atf6 protein mediates its sequence-specific dna-binding, which is required for dimerization of leucine zippers (hurst, 1994). the information about molluscan atf6 is extremely limited so far, and the inactive 90 kda and active 50 kda atf6 (produced via protease cleavage) in intertidal gastropod l. littorea are the only identified molluscan atf6 members. the relative expression levels of the inactive 90 kd atf6 and active 50 kd form under anoxia exposure were detected and they were found not involved in resisting anoxia exposure (storey et al., 2013). the presence of these two molecules suggests that molluscan atf6 may possess the similar mechanisms for protease cleavage and activation of gene expression as its homologues in vertebrates (ye et al., 2000; sommer and jarosch, 2000; shen et al., 2005b). there is one atf6 in c. elegans and d. melanogaster, while two atf6 (atf6α and atf6β) in mammals, mice and fish ( mori, 2009; ishikawa et al., 2013), suggesting that the er stress sensors/transducers have experienced structural and functional differentiation during evolution. the identification of atf6 in molluscs also provides vital clues to understand the evolution of these molecules as well as the upr pathways. crt/cnx/ppi/pdi some other important upr related molecules, such as calreticulin (crt), calnexin (cnx), protein disulfide isomerase (pdi) and peptidyl-prolyl cis-trans isomerases (ppi) have also been identified from molluscs. crt and cnx are lectin-like er molecular chaperones induced by various er stresses, and they are involved in er quality control. crt is a soluble protein, while cnx is a type i transmembrane protein in the er (kawabe and yokoyama, 2010). crt and cnx have been identified from oysters c. gigas (kawabe and yokoyama, 2010; zhang et al., 2012) and c. hongkongensis (li et al., 2017). there is a signal sequence at the n-terminus of oyster crt and cnx, while a kdel peptide motif and a transmembrane domain locate at the c-terminus of crt and cnx, respectively, which shares high similarity with vertebrate cnxs and crts. crt genes were also found in sea hare a. californica (kennedy et al., 1992) and green mussel perna viridis (jiang et al., 2016; leung et al., 2011). the protein disulfide isomerase (pdi) and peptidyl-prolyl cis-trans isomerases (ppi) are able to catalyze the reactions of protein folding (schröder and kaufman, 2005). the pdi have been found in freshwater apple snail pomacea canaliculata and freshwater snail p. diffusa (mu et al., 2015), oysters c. gigas (yang et al., 2017), c. hongkongensis (li et al., 2017), and p. viridis ( leung et al., 2011; jiang et al., 2016). two ppis have been discovered in atlantic ribbed mussel geukensia demissa (fields et al., 2012a; fields et al., 2016) and antarctic clam l. elliptica (clark et al., 2016). these findings suggest a rigorous regulation mechanism on the er quality control may exist in molluscs. the protein sequences in molluscan crt/cnx/ppi/pdi have been analyzed in comparison with their homologues in other animals. as expected, the molecular conservation between molluscs and other invertebrates is higher than that between molluscs and vertebrates. for instance, the crt in oyster c. gigas (accession: baf63639.1) shares slightly higher identity with that in other invertebrate than vertebrate. the same situations have also been found in oyster cnx (genbank: baf63638.1), ppi (accession: aby27347.1) and pdi (accession: ekc29663.1), which display 70%-80% identity with that in invertebrates and 50%-60% with that in vertebrates, respectively. these data indicate that crt/cnx/ppi/pdi in molluscs, especially in oysters, exhibit relatively evolutional conservation, which provides intriguing pointcut for further protein structure and biological function investigations in molluscan upr regulation. although several upr members have been described in molluscs, it is only an overview and the knowledge of the structural diversity of molluscan upr system is still quite meagre and not comparable with that in other invertebrate and vertebrate (chen et al., 2014). gratifyingly, there are numerous predicted isoforms available in the data base of ncbi, owing to the increasingly release of the molluscan genome (zhang et al., 2012; albertin et al., 2015; wang et al., 2017), which will shed lights for the further structure and function studies of molluscan upr pathways. the response of molluscan upr signaling pathway under various stressors as a master er surveillance system, the upr regulates essentially all aspects of er function, and continuously coordinates the activity and participation of the processing and degradation pathways for unfolded proteins (hampton, 2000). therefore, the upr is a paradigm to establish and maintain the cell homeostasis (walter and ron, 2011). the majority of researches on molluscan upr are focused on the expression profiles of key molecules in response against environmental stresses and immune challenges. different upr signaling pathways display different expression http://fanyi.baidu.com/### 190 patterns under various stressors, and they play different roles in the response to diverse stressors. here we present the expression patterns of molluscan upr signaling pathways under different stressors. expression profiles of ire1–xbp1 signaling pathway after heat, metal stresses, and immune stimulations ire1-xbp1 signaling pathway is the most well studied pathway, and it plays an important role in a wide spectrum of biological processes, including differentiation, metabolism, inflammation, tumorigenesis, and neurodegeneration (patil and walter, 2001; cao and kaufman, 2012; chen and brandizzi, 2013). previous reports in vertebrates indicate that ire1-xbp1 signaling pathway responds against different stresses. for instance, cadmium and ultraviolet a (uva) could activate ire1-xbp1 pathway by splicing xbp1 mrna (yokouchi et al., 2007; komori et al., 2012; kitamura, 2013). in mammalian cells, cadmium induced the activation of the ire1-xbp1 pathway, which played proapoptotic roles in cadmium-induced apoptosis (yokouchi et al., 2007). as the oldest and most-conserved branch of the upr in eukaryotes (d and sr, 2008), ire1-xbp1 signaling pathway has been identified in invertebrates, which responds against tunicamycin and/or dithiothreitol (dtt) treatments (travers et al., 2000; shen et al., 2001; plongthongkum et al., 2007). in ddt treated drosophila melanogaster cells, the ire1 mediating xbp1 mrna splicing mechanism was extremely conserved and exerted a critical role for modulating xbp1 protein synthesis (plongthongkum et al., 2007). in virus challenged shrimp litopenaeus vannamei, the expression profiles of ire1 and xbp1 were detected, which confirmed that the ire1-xbp1 pathway was important for l. vannamei environmental stress resistance (chen et al., 2012). the activation of ire1-xbp1 signaling pathway in response against thermal, metal stresses and immune challenges such as parasites and neoplasia has also been evidenced in molluscs. under heat treatment, a significant increase of bip mrna was observed in both gill and adductor muscle of c. gigas, and the mrna transcripts of bip, pdi and ire1 were also up-regulated in c. gigas hemocytes (yokoyama et al., 2006; yang et al., 2017). similarly, bip, ire1 and xbp1 were all up-regulated in muscle, mantle and gill of v. lienosa exposed to heat treatment (wang et al., 2012). in the metal bioaccumulation toxicology experiment, bip, cnx, crt and ire1 were up-regulated in c. gigas gills exposed to plumbum (pb) treatment (zhang et al., 2012). xbp from m. edulis also showed significant up-regulation in gills under pb or mixture of pb and cadmium (cd) treatments (poynton et al., 2014), suggesting that ire1, xbp1 and bip were involved in the response of molluscs against the environmental stressors. in flat oyster o. edulis, bonamiosis is a lethal haemocyte infection caused by bonamia genus protozoan parasites, and disseminated neoplasia (dn) is a malignant proliferation of circulating cells resembling leukaemia. the expressions of xbp1 in o. edulis with bonamiosis or dn were significantly increased in hemocytes, mantle, and digestive gland. remarkably, the expression of xbp1 was significant down-regulation in the gill under slight dn infection (martín-gómez et al., 2014). the previous reports about responses of molluscan ire1-xbp1 signaling are mainly focused on the heat stimulation at temperature range of 20-30 °c both in short term as several hours (yang et al., 2017) and in long term for a few days (wang et al., 2012). single metal stimulation with short time (zhang et al., 2012) and mixture metal for long term (poynton et al., 2014) are also concerned in oyster and mussels. the severe and slight immune treatments with parasites and neoplasia (martín-gómez et al., 2014) are another interesting research related to the response of ire1-xbp1 signaling (table 1). in general, the ire1-xbp1 signaling pathway is believed to respond rapidly to different stressors and functions as early stress indicator, and the regulation mode of ire1-xbp1 signaling pathway is suspected to be relatively conservative in molluscs. the fluctuations of ire1 and xbp mrna expression under various stressors indicates that ire1 activity-dependent xbp1 mrna splicing under er stress also exist in molluscs (calfon et al., 2002; back et al., 2005), but further assays are need to investigate the biology functions of activated and un-activated xbp1 and the detailed activation and regulation mechanisms of ire1-xbp1 signaling pathway in molluscs. the activation of perk–eif2α–atf4–chop signaling pathway under anoxia and heat stress perk-mediated eif2α phosphorylation contributes to transcriptional activation in upr and decreases global protein synthesis to reduce the er load (ron and walter, 2007). previous studies in mammals shows that perk–eif2α signaling pathway is dominant over the ire1-xbp1 and atf6 signaling pathway (oyadomari and mori, 2004) and it is activated by many kinds of viruses (he, 2006) and malnutrition like glucose (glc) deprivation (fernandez et al., 2002; kitamura, 2013). for instance, perk was activated by an increase of perk auto-phosphorylation when mammalian cells were infected with herpes simplex virus and it played a key role in limiting viral replication (he, 2006). perk was also responsible for the phosphorylation of eif2α induced by glucose deprivation and the translational regulation via eif2α phosphorylation was important in response to cellular stress (fernandez et al., 2002). additionally, er stress responses leading to eif2α phosphorylation and xbp1 splicing were detected in heat stressed rat cortex as well (liu et al., 2012). as for invertebrates, perk–eif2α signaling pathway has also been reported. in shrimp, perk–eif2α signaling pathway was activated by white spot syndrome virus (wssv) (xu et al., 2014; zhang et al., 2016b), tunicamycin and cycloheximide (aparna et al., 2003) by inducing eif2α phosphorylation. the promoter of pro-apoptotic protein endoplasmic reticulum oxidoreductin was activated by the key transcription factor atf4 of perk–eif2α pathway during wssv stimulation. wssv infection enhanced the phosphorylation of 191 eif2α and the perk–eif2α pathway activation was important for innate immune during wssv infection in shrimp. it also indicated that wssv could induce apoptosis via the perk–eif2α pathway (xu et al., 2014; zhang et al., 2016b). the tunicamycin and low concentrations of cycloheximide promoted eif2α phosphorylation in spodoptera frugiperda ovarian cells but without apoptosis. these findings therefore suggested that eif2α phosphorylation was not always necessary to induce apoptosis, but it was a characteristic hallmark of stressed cells and also of cells undergoing apoptosis (aparna et al., 2003). in molluscs, the main components of perk–eif2α–atf4–chop signaling pathway have been found to response against various stresses, such as anoxia and thermal stress. perk from c. gigas was up-regulated in gills with air exposure treatment (zhang et al., 2012). perk in oyster hemocytes as well as eif2α in gills were also up-regulated after heat treatment (zhang et al., 2012; yang et al., 2017). the mrna expressions of bip, cnx, crt, pdis and perk were up-regulated when c. hongkongensis was subjected to 37 °c heat treatment (li et al., 2017), suggesting there was a complex cellular response to thermal stress in this species. a transcriptome analysis of freshwater mussel v. lienosa indicated that perk, eif2α and atf4 were up-regulated in muscle, mantle and gill during thermal stress, and the kegg analysis revealed a nearly complete perk–eif2α pathway in canonical upr system (wang et al., 2012). it indicates that molluscs may evolve perk–eif2α signaling pathway to cope with thermal stress in high temperature living regions. the phosphorylation of eif2α has also been observed in molluscs responded upon the anoxia exposure. the expressions of bip, phosphorylated eif-2α and atf4, and gadd153 in foot muscles of l. littorea were significantly up-regulated after anoxia exposure, while down-regulations of gadd34 were observed at 20 h after anoxia exposure (storey et al., 2013). in m. mercenaria, the protein expression level of eif2α was not affected by hypoxia stress, but the phosphorylated eif2α increased at 18 h (ivanina et al., 2016). the significance of eif2α phosphorylation in the induction of upr is highlighted in mammals by a number of studies. phosphorylated eif2α is essential for optimal expression of many upr genes and also critical for the maintenance of cellular homeostasis (roy and lee, 1999; harding et al., 2000). the increased phosphorylated eif2α in m. mercenaria after hypoxia exposure showed a greater suppression of energy-consuming processes such as protein turnover, indicating that inactivation of eif2α might be essential for molluscan hypoxia tolerance (ivanina et al., 2016). in mammals, de-phosphorylation of eif2α mediated by gadd34, as a negative feedback loop, can promote recovery from translational inhibition in the upr (novoa et al., 2001). the up-regulation of phosphorylated eif-2α and down-regulations of gadd34 give a hint that the negative feedback mechanism may dedicate to the regulation of perk–eif2α signaling pathway in molluscs, as part of the adaptive response of cells to er stress. the knowledge about the activation and responses of molluscan perk–eif2α–atf4–chop signaling pathway is mainly from the investigations on air exposure (zhang et al., 2012), heat treatments (wang et al., 2012; li et al., 2017), and anoxia exposure (storey et al., 2013; ivanina and sokolova, 2016), from hours to days and 20 ℃ to 30 ℃ (table 1). the accumulated evidences indicate that the perk–eif2α signaling pathway plays crucial roles for molluscs to defend the environment stresses. and it is necessary to study the involvement and regulation mechanism of molluscan perk–eif2α signaling pathway in malnutrition and pathogen infection to compare with that in vertebrate. expression profiles of atf6 signaling pathway in molluscs as a transcription factor, atf6 is initially synthesized as an er-resident transmembrane protein bearing a large er-luminal domain. in response to er stress, atf6 is translocated from er to golgi apparatus and cleaved to the active form by site 1 and 2 proteases (s1p and s2p) in golgi apparatus (haze et al., 1999; okada et al., 2002; shen et al., 2005a; adachi et al., 2008; walter and ron, 2011). it has been reported that mammalian atf6 enters nucleus and activates the transcription of its target genes such as er chaperone genes to increases er protein folding capacity under stress (yoshida et al., 2000, 2001; namba et al., 2007). for example, shigella dysenteriae toxins treatment induced the activation of atf6 in human myelogenous leukaemia cell line, and the nuclear translocation of atf-6 increased highly in riboflavin-deficient cells (manthey et al., 2005; lee et al., 2008; kitamura, 2013). the stimulation of the myelogenous leukaemia cell line with purified shiga toxins could induce the er stress response and increased the activation of the er stress sensors ire1, perk, and atf6. the degradation of the atf6 90 kda proteins and decrease of immune-reactive atf6 proteins were observed in monocytic cells treated with shiga toxins (lee et al., 2008). nuclear translocation of atf6 was enhanced and associated with activation of the unfolded protein response in riboflavin-deficient cells (manthey et al., 2005). in shrimps, atf6 was found to be vital for wssv replication, and upr in shrimp could facilitate wssv infection (yuan et al., 2017; xu et al., 2018). the expression of atf6 increased in kuruma shrimp marsupenaeus japonicus after wssv challenge. when atf6 was knocked down by rna interference, the cumulative mortality of shrimp decreased with presence of wssv infection (yuan et al., 2017; xu et al., 2018). the previous researches on molluscan upr are mainly focus on gene cloning and mrna expression (kuhl et al., 1992; yokoyama et al., 2006). in foot muscles l. littorea, the relative level of the inactive 90 kda atf6 did not change significantly, while a significant decrease of active 50 kda atf6 level was observed under anoxia exposure (storey et al., 2013). on the contrary, the level of active p50 atf6 in mammals increased significantly under stress (haze et al., 1999). these results indicated that the atf6 signaling pathway might not be part of an anoxia-responsive activation of the upr in l. littorea 192 (storey et al., 2013). in c. elegans, ire1–xbp1 was demonstrated to play predominant role in upr signaling pathways, while atf6 was less important (ma and hendershot, 2001). it is suspected that ire1α–xbp1 signaling pathway is activated to response against thermal, metal and immune stressors, while perk–eif2α–atf4–chop signaling pathway is inferred to involve in the response against anoxia and thermal stress in molluscs. although the active/inactive forms of atf6 have been found in molluscs, there is no solid evidence for its involvement in molluscan upr (table 1). in model organisms, the upr is differently regulated in different cell types (walter and ron, 2011), the characters in various cell types and its underlying mechanism of atf6 signaling pathway in molluscs deserve to further study. the pathways co-expressed with molluscan upr and the possible downstream immune response the pathways co-expressed with molluscan upr in mammals, several elements in pleiotropic pathways such as p38 mitogen-activated protein kinase, nuclear factor nf-kappa-b (nf-κb) and c-jun n-terminal kinase (jnk) are found to be involved in ire1α–xbp1 and perk–eif2α signaling pathways (cao and kaufman, 2012; grootjans et al., 2016). multiple immune pathways, such as apoptosis and inflammatory response, have been retrieved in the heat-treated gastropod and bivalve (zhang et al., 2012; clark et al., 2016; fields et al., 2016; li et al., 2017; yang et al., 2017), and the heavy metal-treated bivalve (poynton et al., 2014). although there are some reports about the downstream immune pathways of upr, the enrichment terms are still partial due to the incomplete reference database and lack of comprehensive investigation. further investigations are urgently needed to reveal the correlations between upr and immune pathways in molluscs. the possible downstream immune response of upr the chronic or unresolved mammalian er stress, such as prolonged activation of ire1 and chop, can trigger cell apoptosis (tabas and ron, 2011). the molecular mechanisms of apoptosis in molluscs have also been studied under different stressors. for instance, arg–gly–asp (rgd) peptides could inhibit integrin–ligand interactions and active caspase-3 to induce cell death (buckley et al., 1999), and the apoptosis was triggered in hemocytes of the pacific oyster when it was treated with rgd peptides (terahara et al., 2005; kiss, 2010). the large-scale apoptotic and anti-apoptotic genes annotated in molluscan genome suggested that apoptosis and anti-apoptotic mechanisms might play important roles in molluscan adaptive survival (zhang et al., 2012; zhang et al., 2016a). recently, upr has been reported to involved in autophagy (yeganeh et al., 2015), central nervous system (cns) development (godin et al., 2016), ribosomal proteins ubiquitylation (higgins et al., 2015), cell transformation, tumor development and aggressiveness (dejeans et al., 2015), and inflammation (janssens et al., 2014; grootjans et al., 2016). in-depth study of the upr pathway and its mediated pathways especially apoptosis will provide important reference for better understanding of the diversity of molluscs and their evolutionary adaptation mechanisms, as well as the complicated physiological and biochemical principle. conclusion a preliminary map of molluscan upr signaling pathways has been outlined in the last decades with conserved constitution similar to that in other eukaryotes. the molluscan upr signaling pathways exhibit different response to stressors at mrna and protein levels, among which ire1α–xbp1 and perk–eif2α–atf4–chop signaling pathways are speculated to devote enormously in adaptive response of molluscan cells to er stresses. meanwhile, numerous response and pathways, including apoptosis, anti-apoptosis, metabolic pathways, cytoskeletal, protein de-phosphorylation and ubiquitination, tissue regeneration, dna repair, antigen presentation, complement cascades, defense and immune responses, have been discovered paralleling with upr signaling pathways in molluscs. at the same time, the function and detailed regulation mechanism of molluscan upr signaling and the crosstalk between upr and other signaling pathways still need further investigations. in future, synergistic approaches including engineering of genetic, cell, protein, enzyme, and biochemical will make molluscan upr a promising field for evolutionary stress response. acknowledgement the authors would like to thank the lab members for helpful discussion. the work referenced was supported by a grant from national science foundation of china (no. u1706204), aoshan talents cultivation program supported by qingdao national laboratory for marine science and technology (no. 2017astcp-os13), dalian high level talent innovation support program (2015r020), the distinguished professor of liaoning (to l. s.), and the research foundation for talented scholars in dalian ocean university (to l. s.). references adachi y, yamamoto k, okada t, yoshida h, harada a, mori k. atf6 is a transcription factor specializing in the regulation of quality control proteins in the endoplasmic reticulum. cell struct funct. 33: 75-89, 2008. albertin cb, simakov o, mitros t, wang zy, pungor jr, edsinger-gonzales e, et al. the octopus genome and the evolution of cephalopod neural and morphological novelties. nature. 524: 220-224, 2015. aparna g, bhuyan ak, sahdev s, hasnain se, kaufman rj, ramaiah kv. stress-induced apoptosis in spodoptera frugiperda 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chongqing 400042, p.r. china accepted april 7, 2018 abstract nhl protein family is evolutionarily conserved in either vertebrates or invertebrates. it has been extensively studied in mammals for the function of regulating cell proliferation and differentiation. brat, known as the homologue of trim in insects, may have the same function. in this paper, we identified and characterized bmbrat in silkworm. the gene expression profile indicated that bmbrat was expressed in all tissues and the expression was especially high in ovary and head at larval stage. the expression profile was also detected at different stages of embryo development, and reached a peak at 4th and 5th days of the embryonic period. subcellular localization of bmbrat in hemocytes revealed that it was specifically expressed in cytoplasm. over-expression of bmbrat in silkworm significantly reduced the rate of cell proliferation by brdu staining. this finding suggested that bmbrat played an important role in cell processes. key words: silkworm, bmbrat, gene expression profile, subcellular localization introduction the name nhl originates from three members, which were earlier discovered: ncl-1, ht2a and lin-41. these three factors have relationship with the rna metabolism, and may be involved in post-transcriptional regulation of gene expression (loedige et al., 2009). researches showed that family members of the nhl contain three conserved motifs: a ring, one or two b-box and a coiled-coil domain (petrera et al., 2012; tocchini et al., 2015). nhl domain plays a vital role in regulating the protein activity of brat (brain tumor) (marchetti et al., 2014). brat itself as a member of the nhl protein family is a kind of tumor suppressor genes, but it is also a candidate gene controlling cell proliferation (loedige et al., 2014). in the drosophila nervous system, the expression of brat largely limits the peripheral nervous system, ventral nerve cord and brain development during embryonic stage (bello et al., 2006). in stem cells development system, brat, prospero and numb work as cell fate determination factors, promoting the changes of cell fate (lee et al., ___________________________________________________________________________ corresponding author: hongjuan cui cell biology laboratory state key laboratory of silkworm genomics biology southwest university chongqing 400716, p.r.china e-mail: hongjuan.cui@gmail.com; hcui@swu.edu.cn 2006). numb is a membrane-bound protein involved in the regulation of notch signaling pathway (cotton et al., 2013). during neural precursor cell division, numb gathered to one end of the cell to combine with the membrane, distributed into the ganglion mother cells through asymmetric cell division (hutterer et al., 2005). then numb interacts with sanpodo, which is α-ubiquitin mediated protein activity regulating factor, to inhibit the notch protein (berdnik et al., 2002). the inhibition of notch signaling pathway, which plays a crucial role in proliferation and differentiation of neuroblastoma cells, would result in a cell fate transformation, which means self-renewing cells can gradually begin to differentiate (wang et al., 2011). prospero is a transcriptional regulatory protein, which exists both in the embryo and larvae neuroblast, and was continuously expressed in the differentiated glial cells (choksi et al., 2006; reichert, 2011). prospero functions as a downstream regulator of brat, and plays a key role in cell fate determination and cell proliferation regulation. thus studying brat in cell differentiation and proliferation can provide theoretical basis for differentiation of stem cells. the homolog of brat in human and mammalian is the trim protein family, and they all play a role in the development, differentiation, host cell antiviral defense and cancer. trim family is a subgroup of the nhl domain, such as trim2, trim3, trim32 150 fig. 1 complete cloning of silkwrom bmbrat. (a) pcr of bmbrat. (b) the diagram of the complete bmbrat cdna. (c) the location of bmbrat on chromosome 14 and trim71 in mammalian, brat, mei-p26, abba and wech in drosophila, and ncl-1, nhl-1, nhl-1, nhl-3, lin41 in caenorhabditis elegans (nisole et al., 2005; neumuller et al., 2008; sardiello et al., 2008). among these, trim3, known as a tumor suppressor, can inhibit cancer development by binding to cdk inhibitor p21waf1/cip1 to prevent the accumulation of cyclin d1-cdk4 (boulay et al., 2009; liu et al., 2014). reducing trim3 expression in mice can increase and accelerate the platelet-derived growth factor (pdgf) induced glioma incidence (boulay et al., 2009; chen et al., 2014). however, there is almost no report on the bmbart gene in silkworm, bombyx mori at present, and the exact function of bmbart gene remains unknown. in this study, we reported the identification, characterization, expression analysis and functional study of the bmbart gene in silkworm, b. mori, which is not only an important economical insect for silk production, but also an excellent research model of invertebrate. materials and methods biological materials bombix mori strain dazao (p50) used in our study is provided by the gene resource library of domesticated silkworm of southwest university. the larvae was fed with fresh mulberry leaves under standard conditions. different tissues were extracted in 1× depc-treated pbs, flash-frozen and stored at -80 °c until needed. the cultured silkworm embryo-derived cell line was established by our laboratory, and it was cultured in grace' medium supplemented with 10% (v/v) fbs. rna extraction and cdna synthesis total rna was extracted from the head, midgut, epidermis, silk gland, fat body, malpighian tubules, testis, ovary, haemocytes and embryo with trizol reagent referring to the protocol provided by the manufacturer. after being treated with rnase-free dnase i, the first-strand cdna was synthesized with m-mlv reverse transcriptase, and the products were stored at -20 °c. bioinformatic and phylogeny analysis the orf was verified with the orf finder software (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). the bmbrat protein domain was predicted using smart (http://smart.embl-heidelberg.de/). the deduced amino acid sequences of bmbrat was aligned using the clustal x program, and a phylogenetic tree was constructed by the neighbor-joining method (tamura et al., 2007). the sequences used in this study were downloaded from genebank (http://www.ncbi.nlm.nih.gov/). quantitative real-time pcr (qrt-pcr) for qrt-pcr analysis, a stepone plustm real-time pcr system (applied biosystems, usa) and the sybr master mix (promega, usa) were used under the following conditions: 95 °c for 10 min, followed by 40 cycles of 5 s at 95 °c and 30 s at 60 °c. the housekeeping gene bmgapdh was used as an internal control. all reactions were performed in triplicates in a total volume of 20 μl. sequences of qrt-pcr primers were designed as follows: bmbrat-f, 5’-cgtgcgttacttgtcgtgt-3’; bmbrat-r, 5’-gcagttggggcagaggt-3’; gapdh-f, 5’-cattccgcgtccctgttgctaat-3’; gapdh-r, 5’-gctgcctccttgaccttttgc-3’. relative mrna expression levels were calculated by using the standard 2-δδct method (livak et al., 2001). the online t-test software graphpad software (http://www.graphpad.com/quickcalcs/ttest1.cfm) was used to evaluate the statistical significance (p<0.05). 151 fig. 2 bioinformatic analysis of bmbrat. (a) multiple protein domain alignment of bmbrat in several species. bmbrat protein has a conserved ring domain, two b-box domain and a bbc domain. (b) multiple sequence alignment of bmbrat in several species, and bmbrat gene has high homology in these species 152 fig. 3 the phylogenetic tree of bmbrat. the phylogenetic tree of the bmbrat based on the full-length amino acid by the neighbor-joining method. the number closed to individual branches represents the percentage of 1000 bootstrap iterations supporting the branch, and values below 60% were omitted. the silkworm bmbrat are labeled with circles cell transfection and bmbrat overexpression the bmbrat-flag fragment for overexpression was generated by pcr using dazao (p50) head cdna template. then it was ligated into the psl1180-a4-dsred plasmid. 2×105 bme cells were seeded into 24-well plates for 24 h, and transiently transfected with psl1180-a4-dsred-bmbrat-flag vector with x-tremegene hp dna transfection reagent (roche). after 72h transfection, cells were processed for western blot and immunofluorescence assay. observation of the fluorescent signal was performed by using the confocal fluorescence microscope (olympus fv1000, olympus corporation, tokyo, japan). brdu staining after transfection with the overexpression vector, cells were grown on coverslips, and incubated with 10 μg /ml brdu (sigma) for 3 h, then washed with phosphate buffered saline (pbs) and fixed in 4% paraformaldehyde (pfa) for 20 min. subsequently, cells were pre-treated with 1 mol/l hcl, and blocked with 10% goat serum for 1 h, followed by a monoclonal rat primary antibody against brdu 153 (1:200, ab6326, abcam, cambridge, ma, usa) for 1 h and alexa fluorr® 488 goat anti-rat igg secondary antibody, (h+l; invitrogen). dapi (300 nm) was used for nuclear staining; the percentage of brdu was calculated at least from 10 microscopic fields (nikon 80i, nikon corporation, tokyo, japan). flow cytometry for cell cycle analysis, flow cytometry was performed in bme cells overexpressing bmbrat. 1×106 cells were harvested and fixed with 70% ethanol, stained with propidium iodide (pi) (bd biosciences), then incubated with rnasea for 30 min at room temperature. finally, the cell samples were analyzed by flow cytometry using a facs c6 (bd biosciences), and the data was analyzed with cellquest pro software. results cloning and identification of bmbrat in silkworm bmbrat full-length sequence was obtained and identified (fig. 1 a and b), which was located on chromosome 14 (fig. 1 c), containing 859 amino acid residues. there is a conserved ring domain, two b-box domain and a bbc domain in brat protein sequence of silkworm (fig. 2 a). the homology of brat from silkworm and other species was explored via multiple sequence alignment using clustal x (fig. 2 b). results of multiple protein domain alignment (fig. 2 a) showed some differences between silkworm and other species in the genetic structure, especially in the sequence similarity among species. although a relatively conserved nhl domain was found in the c-terminal region in all analyzed species, there were some differences between invertebrates and mammals. it was found that the sequence conservation of each domain was rather low, especially the conservation of the ring domain. however, the homology of the nhl domains in invertebrates and mammals, respectively, was very high. in addition, the cysteine in the bbox domain was highly conserved, the hydrophobic amino acid was relatively conserved in the coiled-coil domain. furthermore, phylogenetic analysis of bmbrat from several species using mega6 software generated a phylogenetic tree (fig. 3). and results showed that, in evolution, bmbrat had far relationship with vertebrate homologous gene family trim. however, danaua plexippus had the closest relationship with silkworm. bmbrat is highly expressed in ovary and head the mrna expression levels of bmbrat over the embryo period from day 1 to day 9 and in various tissues at day 3 of 5th instar larvaes (l5d3) were analyzed by qrt-pcr, the results of which were shown in fig. 4. we found that the bmbrat was highly expressed at day 4 and day 5, especially in day 5, and the expression level went down rapidly after day 5. we collected and compared most of organs in silkworm, including testis, ovary, epidermis, silk gland, malpighian tubule, midgut, head, fat body, and hemocytes. the data showed that bmbrat was expressed in various organizations, including the highest in the ovary, followed by the head. fig. 4 the expression profile of bmbrat. (a) the temporal and spatial expression profiles of bmbrat in silkworm embryos. cdnas were obtained from embryos at different stages. (b) tissue-specific expression of bmbrat in silkworm larvae. cdnas were obtained from the head, whole body, midgut, epidermis, fat body, silk gland, testis, ovary, malpighian tubule of l5d3 larvae. qrt-pcr was used to analyse bmbrat mrna expression levels, and bmgapdh was used as an internal control. all reactions were performed in triplicate bmbrat is specifically located in cytoplasm for further analysis of bmbrat in expression characteristics silkworm, full length expression vector psl1180-a4-bmbrat-flag-dsred was constructed (fig. 5a). then we overexpressed bmbrat in bme cell line, psl1180-a4-dsred empty vector was used as control. results showed that in the control group, the red signal of dsred evenly spread out in cytoplasm and nucleus. on the other hand in the experimental group, the bmbrat signal only existed in cytoplasm but not in nucleus. in addition, we observed that the position of bmbrat signal is different in the prophase, metaphase, early telophase, and late telophase (fig. 5c). this result suggested that the bmbrat was contributed in different positions in different period of cell division. 154 fig. 5 the over-expression and localisation of bmbrat in silkworm bme cells. (a) construction of the psl1180-a4-bmbrat-dsred vector. bmbrat cloned into psl1180-a4-dsred vector with flag tag. (b) western blot analyses of over-expression of bmbrat gene in bme cells. over-expression of bmbrat gene in bme cells was obtained by transfection of bmbrat-flag-dsred plasmid. (c) localisation of bmbrat protein in bme cells by immunofluorescence assay. flag (green) showed the distribution of bmbrat-flag; dsred (red) showed the distribution of bmbrat-dsred; dapi (blue) showed the cell nuclei; scale bar: 5 µm 155 fig. 6 over-expression of bmbrat inhibits cell proliferation in silkworm bme cells. (a) brdu staining was used to investigate cell proliferation in bmbrat up-regulation cells. immunofluorescence detected by anti-brdu antibody. scale bar: 20 µm. (b) statistical analysis of brdu positive cells ratio. statistical analysis were performed using two-tailed student’s t-test, *p < 0.01. (c) facs analysis were performed to investigate bmbrat overexpression influence cell cycle of bme cells over-expression of bmbrat inhibits the cell proliferation in bme cells in order to figure out the role of bmbrat in cell process, brdu staining was utilized after it was up-regulated in bme cell line. first we use anti-flag antibody to make sure that the bmbrat was successfully overexpressed (fig. 5b). the brdu staining result revealed that the brdu positive cells decreased significantly (fig. 6 a and b), which means the cell proliferation was surpressed after overexprssion of bmbrat. moreover, facs anaylsis was performed to check the changes of cell cycle in bme cells which were overexpressed of bmbrat. and the result shown in figure 6 c clarified that the cell cycle of bme cells was arrested in g1 phase, and the s phase was significantly decreased, when overexpression of bmbrat was performed. it is suggested that the function of bmbrat may be to inhibit cell proliferation. discussion nhl, particularity trim family, has been commonly studied in mammals for the function in cell proliferation especially in cancer development (petrera et al., 2012). bioinformatic analysis of bmbrat showed the gene encoding protein has four relatively conservative structure domain from the n terminal to c terminal, respectively a ring, two b-box and a bbc structure domain, which were typical nhl structure domains. indicating it might function in the cell proliferation and differentiation (tocchini et al., 2015). expression spectrum analysis of bmbrat in silkworm embryonic showed that the amount of gene expression reached the peak at 4th and 5th day and went down after that. silkworm eggs need about 10 days to incubate, and this period can be divided into divers stages according to the embryonic development process. the earlier 6 days of the incubation period is time for organ formation, and 4th and 5th day is the very summit of that, and the gene was highly expressed at this time. the organ development completed after the earlier 6 days, and the gene expression was downregulated significantly at the same time. this suggested that bmbrat might be associated with silkworm organ formation process in the embryonic development. l5d3 larvae 156 expression spectrum analysis illustrated that the gene was almost expressed in all tissues, especially in ovary and head, which means that the bmbrat was a tissue-specific gene and might have some certain functions that are still unknown. results of localization analysis showed that the gene expression in embryonic cells bmbrat exists in cytoplasm, as part of the cells to start dividing bmbrat protein in the cells due to the role of cell polarity factors together to the bottom, asymmetrical distribution in the daughter cells. symmetrical division of cells, the protein will average distribution into the daughter cells. to sum up, bmbrat genes in silkworm growth process plays a very important role. speculation bmbrat genes involved in cell division, and is associated with the cell cycle, and the inhibition of cell proliferation. it has been reported the brat has a variety of functions in drosophila differentiating germ cells. it is known the maintenance of germline stem cell is regulated by the dpp signaling pathway (xie et al., 1998; chen et al., 2003) and translation inhibitory factor nanos (nos)/pumilio (pum) (forbes et al., 1998). in the ovary, pum-nos adjust the mrna level of the brat, limiting its expression in the cystoblasts (muraro et al., 2008). up-regulation of dpp signal can promote the inhibition on brat mrna generation by pum-nos (li et al., 2009). when cells divide, dpp signal come into silence, the loss of dpp signal downregulated nos, which accelerate expression of the brat. in return, brat formed new compounds with pum to inhibit mrna levels of mad and dmyc which works in dpp signaling pathway (ohlstein et al., 1997), reducing the response capacity of cystoblasts to dpp signal, promoting cell differentiation (harris et al., 2011). therefore, whether bmbrat has the similar function in silkworm remains to be further studied. competing interests the authors disclose no potential conflicts of interest. acknowledgement our work was supported by the national natural science foundation of china (grant no. 81502574, 31672496), the chongqing research program of basic research and frontier technology (grant no. cstc2017jcyjax0304), the fundamental research funds for the central universities (grant no. xdjk2016c007 and swu118033), the natural science foundation of chongqing (grant no. cstc2016jcyja0425), and the chongqing university innovation team building special fund (grant no. cxtdx201601010). reference bello b, reichert h, hirth f. the brain tumor gene negatively regulates neural progenitor cell proliferation in the larval central brain of drosophila. development. 133: 2639-2648, 2006. berdnik d, torok t, gonzalez-gaitan m, knoblich ja. the endocytic protein 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development and disease. semin. cell. dev. biol. 47-48: 52-59, 2015. wang w, liu w, wang y, zhou l, tang x, luo h. notch signaling regulates neuroepithelial stem cell maintenance and neuroblast formation in drosophila optic lobe development. dev. biol. 350: 414-428, 2011. xie t, spradling ac. decapentaplegic is essential for the maintenance and division of germline stem cells in the drosophila ovary. cell. 94: 251-260, 1998. società italiana di immunobiologia comparata 9 isj 17: 9-23, 2020 issn 1824-307x report of meeting xxist scientific meeting of the italian association of developmental and comparative immunobiology (iadci), 12 14 february 2020, department of biotechnology and life sciences (dbsv), university of insubria, varese, italy organizers: annalisa grimaldi, magda de eguileor, gianluca tettamanti, roberto valvassori, nicolò baranzini, daniele bruno, aurora montali, laura pulze department of biotechnology and life sciences (dbsv), university of insubria, varese, italy this is an open access article published under the cc by license session 1. chairmen: maria rosaria coscia, cnr of naples, naples, italy and piero g. giulianini, university of trieste, trieste, italy fish lymphocytes as an equivalent of mammalian innate-type lymphocytes? g scapigliati, a miccoli, s picchietti department dibaf, university of tuscia, viterbo, italy the immune defence system of vertebrates in its molecular and cellular components is remarkably conserved from teleost fish, the more ancient extant representatives at the base of the evolutionary lineage that brings to mammals. multiple observations support the hypothesis that a layered system of immune responses accumulated among vertebrates over evolution, and lower layers behave as the immune system actually present in extant fish species. in this view, lymphocytes are classified as responsible of acquired responses, but recent evidences show that mammalian lymphocyte subpopulations may behave as innate cells, engaging non-self rapidly and without antigen presentation. innate-like lymphocytes i) maintain gut homeostasis and provide early responses to intestinal infections, ii) are involved in autoimmune diseases and cancer iii) are able to combat non-self in a mhc-independent fashion, iv) produce unbiased natural polyreactive antibodies, v) are associated to typical cytokine patterns. the main lymphocyte subpopulations displaying innate-like activities in mammals have been identified as b1-b cells, -t cells, mait cells, and nkt cells. our research focuses on the spatial and temporal origin of fish lymphocyte populations, on their in vitro activities, and on the molecular and morphological signatures of fish lymphocytes. the aim is to predict knowledge related to mammalian innate lymphocytes from fish lymphocytes and to model human diseases. this review will present evidences suggesting the similarities between fish lymphocytes and mammalian innate-like lymphocytes. acknowledgements department of excellence-2018” program (dipartimenti di eccellenza) of the italian ministry of education, university and research, dibafdepartment of university of tuscia, project “landscape 4.0 – food, wellbeing and environment”. t lymphocytes in the sea bass (dicentrarchus labrax) intestine s picchietti, f buonocore, a miccoli, pr saraceni, am fausto, g scapigliati department for innovation in biological, agrofood and forest systems, university of tuscia, viterbo, italy the european sea bass dicentrarchus labrax is a marine species in which lymphocyte distribution in the digestive tract (dt) was first described, and much knowledge on dt immuno-physiology is available, including gene expression profiling that revealed functional specialization along the dt. 10 anatomically, sea bass does not contain lymphoid aggregates in the intestinal mucosa, however, its gut harbors high numbers of cd3ε+, tcr + and cd8+ cells and rare cd4+ cells. among the intestinal t lymphocytes, a large population expresses -t receptors. these cells play an essential role in intestinal cell-mediated immunity, and it is postulated in teleosts they are important in tolerance or in attack against the microbiota. in sea bass, tcr mrna was constitutively expressed at high levels in dt, and intestinal leukocytes stimulated in vitro by poly i:c showed an increase in tcr transcript levels. in addition, we showed that the tcr chain undergoes spontaneous somatic recombination in the dt, together with an increase of rag-1 transcripts in its posterior segment. when juveniles were infected in vivo with a retrovirus (betanodavirus), expression of tcr was downregulated. recently, a polyclonal antiserum was obtained against peptides deduced from the sea bass tcr full length sequence. ihc showed the presence of tcr-bearing cells in the intestinal mucosa, mainly located in the lamina propria and rarely in the epithelium. immunogold in preembedding of intestinal leukocytes revealed the ultrastructural characteristics of tcr+ cells, which fit with the typical lymphocyte morphology. in mammals -t cells possess unique features; for instance, they do not display mhc restriction, and they distinguish unconventional antigens, such as phosphorylated microbial metabolites and lipid antigens. sea bass intestinal mucosa responds to different diets and although the interplay between nutrition and the immune system is well recognised in teleosts, the link between diet, gut microbiota and health in sea bass is only in its infancy. acknowledgements department of excellence-2018” program (dipartimenti di eccellenza) of the italian ministry of education, university and research, dibafdepartment of university of tuscia, project “landscape 4.0 – food, wellbeing and environment”. evolution, adaptation and immune functions of fish f-type lectins. the novelty of fbl from trematomus bernacchii (boulenger, 1902) m dara1, mg parisi1, c la corte1, d parrinello1 p giulianini2, c manfrin2, m cammarata1 1department of earth and marine sciences, marine immunobiology laboratory, university of palermo, palermo, italy 2department of life sciences, university of trieste, trieste, italy lectins are a protein family, present in almost all living organisms and involved in different biological pathways, such as immune responses. the fucose binding lectin (fbl), constitute the latest lectin family identified and characterized in fishes. the fbl family is constituted by a large number of proteins exhibiting multiples of the f-type motif, either tandemly arrayed or in mosaic combinations with other domains. in an early step a fbl has been isolated and characterized from serum of the antarctic fish trematomus bernacchii by affinity chromatography on fucose-agarose column. a clear bacterial agglutinating activity (ba) towards different bacteria strains (escherichia coli, kokuria rhizophyla and bacillus subtilis) and hemagglutinating activity (ha) toward rabbit erythrocytes was induced from the serum as well from the purified protein and thus confirm its involvement in host pathogen interactions. in sds-page analysis, the fbl exhibited an apparent molecular weight of 30 kda. this data is confirmed from the sequence of the f lectin recognised on the t. bernacchii transcriptome. the sequence shows a similar and coherent structure with a supposed mw 32.16 kda and an isoelectropoint of 5.21. furthermore, sequencing the n-terminus confirmed the identity of the sequence runned on sds page and blotted on pvdf membrane. the ha activity was analyzed at different temperatures and it was maintained also at the physiological living low temperatures of this fish habitat (close to 0 °c). therefore, in order to identify trends linked to cold adaptation in antarctic fish, we present our hypothesis on the conformational change determined by the aminoacidic substitutions respect the others fish fucolectins living in warmer water on the light of the general phylogenetic scenario including the new preliminary data on sharks fbl. investigations on igt genes of sub-antarctic fish shed light on the ch2 exon loss a ametrano1, s greco2, u oreste1, m gerdol2, mr coscia1 1institute of biochemistry and cell biology, cnr, naples, italy 2department of life sciences, university of trieste, trieste, italy the antarctic fish belong to the perciform suborder notothenioidei, which constitutes the major ichthyofauna living in the freezing waters surrounding the antarctic continent and represent one of the most successful examples of adaptive radiation in a marine environment. the development of the antarctic polar front, a thermal barrier that inhibits species from migrating between subantarctic and antarctic waters, has contributed as an important biological boundary to the diversification of notothenioidei into eight different families: five are mainly antarctic and three occur in the coastal waters of new zealand, australia, and high-latitude south america. in the past few years, we have isolated and characterized the gene, encoding the igt heavy chain constant region, from one or more members of each antarctic notothenioid family, disclosing that all of them share the loss of most heavy chain second constant domain (ch2). this finding prompted us to go backwards through the phylogeny of notothenioidei in an attempt to reconstruct the loss of the ch2 exon. to this end, we have focused on some species each representative of one of the three subantarctic notothenioid families: bovicthus diacanthus and cottoperca gobio (family 11 bovichtidae), eleginops maclovinus (family eleginopsioidae), pseudaphritis urvillii (family pseudaphritioidea). these species have proved to be crucial in our investigations on the molecular changes occurred in exon–intron regions through evolution of notothenioidei. interestingly, based on a comparative analysis at genomic level, we highlighted that p. urvillii and g. gobio retained the whole ch2 domain like b. diacanthus. however, both species showed also some deletions in close proximity to the ch2 exon similarly to e. maclovinus that we had previously found to be devoid of almost the entire ch2 domain. overall, our studies have shed light on key steps of genome modifications, occurred during notothenioid igt evolutionary history. too warm or not too warm… is the antioxidant system of antarctic fish ready to face climate changes? am tolomeo1, a carraro1, r bakiu2, s toppo3, m gerdol4, p irato5, d pellegrino6, f garofalo6, p bisaccia5, f corrà5, d ferro7, sp place8, g santovito5 1department of women's and children's health, university of padua, padua, italy 2department of aquaculture and fisheries, agricultural university of tirana, tirana, albania 3department of molecular medicine, university of padua, padua, italy 4department of life sciences, university of trieste, trieste, italy 5department of biology, university of padua, padua, italy 6department of biology, ecology and earth sciences, university of calabria, rende, italy 7department of molecular and cellular biology, university of arizona, usa 8department of biology, sonoma state university, usa antarctic fish are considered highly stenothermal with limited evolutionary potential to cope with climate changes, due to the shortand long-term stability of thermal conditions in the southern ocean over the last million years. recently, warm acclimation experiments performed on some notothenioid species have indicated a significant capacity for these fish to elevate their heat tolerance, over the environmental critical thermal maximum (ctmax), through acclimation. this result suggests that thermal plasticity may be universal throughout the antarctic ichthyofauna. however, the physiological and genetic bases of their heat tolerance has been poorly studied. in the present work we described the molecular characterization of mitochondrial peroxiredoxins (prdx) in the antarctic emerald rockcod trematomus bernacchii and gene expression of these antioxidant enzymes in various tissues, in response to shortterm thermal stress. the obtained data are the first on the molecular and functional characterization of the genes encoding prdx3 and prdx5 of antarctic fish, and constitute a further contribution to study these enzymes in specific ecological contexts such as antarctica. our expression analyses may be important for predicting climate change responses in this organism. in fact, the obtained results revealed rapid and specific responses of the antarctic emerald rockcod to wormer temperature. the presence of a prdx (prdx3 gene) whose expression is activated by increased temperatures, may be a condition that limits the stenothermy of t. bernacchii, making this species less vulnerable to moderate environmental temperature changes and other environmental perturbations associated with global climate changes. (supported by p.n.r.a. and m.i.u.r. grants) heat stress in trematomus bernacchii: bias of experimental design d rizzotti1, s greco1, m gerdol1, c manfrin1, g santovito2, a pallavicini1, pg giulianini1 1department of life sciences, university of trieste, trieste, italy 2department of biology, university of padua, padua, italy the studies on trematomus bernacchii suggest that this shallow water benthic species has reduced ability to acclimate and tolerate warmer conditions, since like the other antarctic marine organisms it adapted to the extreme and stable antarctic sea. understanding the effects of exposure of stenothermic species to prolonged sub-lethal heat stress is of extreme interest in the scenario of rising polar sea temperatures presented by climate change. this study evaluated the impact of a 1.5 °c temperature increase over 19 days on some hematological and transcriptomic parameters. blood samples were obtained from heart, fixed and embedded in either acrylic or epoxy resin for ultrastructural analysis. the morphology of erythrocytes in semithin section was evaluated through the imagej shape descriptors. the mean erythrocytes circularity in control animals decreased from 0.810 ± 0.034 to 0.672 ± 0.079 after 19 days. the mean circularity in experimental ones changed from 0.820 ± 0.030 to 0.626 ± 0.051. the erythrocytes became more elliptical over the time during the experiment, regardless of the temperature of the water. the pairwise controlexperimental comparisons of cells circularity are all not significant (p > 0.05), whilst difference of cells circularity is already significant (p < 0.05) by comparing freshly caught animals (0.911 ± 0.018) and the control ones after 1 week of acclimation at the beginning of the experiment. samples of gills, brain and muscle were also collected from specimens in all conditions in order to perform rnaseq analysis and study the response of t. bernacchii to temperature increase also from a transcriptomic point of view. a reference transcriptome was assembled with sequencing reads obtained from short read archive, yielding promising results as quality and completeness were high: n50 = 1650 and busco score against the actinopterygii ortholog database (82.9 % complete, 10 % fragmented and 7 % missing). differential expression analysis are now in progress. 12 these results highlight how biases in experimental design can affect the study of sensitive polar species physiology. therefore, in order to better understand the physiological responses to challenges of these species it is advisable to adopt an eco-physiological approach minimizing the effects of confinement during experiment. morevover, the awareness of these limits will be of great help in the analysis of transcriptomic data. glutathione peroxidases in the striped rockcod trematomus hansoni p bisaccia1, f corrà1, m gerdol2, p irato1, g santovito1 1department of biology, university of padua, padua, italy 2department of life sciences, university of trieste, trieste, italy a large number of special physiological features that allow the life in their extreme environment characterizes antarctic species. in particular, the low temperature and salt concentration are chemiophysical conditions that increase oxygen solubility and, consequently, the po2 and the rate of ros formation. with the aim to study the components of the antioxidant defense system in the antarctic teleosts, we have characterized three genes codifying glutathione peroxidases (gpxs) in the striped rockcod trematomus hansoni. gpxs are a family of antioxidant enzymes that are able to reduce hydrogen peroxide and organic hydroperoxides, thus representing an important protection against the risk of oxidative stress. in the genome of t. hansoni we have verified the presence of three of the six gpxs isoforms known in vertebrates: gpx1, gpx3, gpx4. multi-alignment analysis, performed with fish orthologous sequences, demonstrated high conservation of the amino acids involved in catalytic activity of the gpx isoforms of t. hansoni. however, some substitutions with polar amino acids are characteristics of gpx1 of antarctic species, probably related to a thermal adaptation. the gene transcriptions of gpx1 from various tissues (gills, heart, liver, spleen, and skeletal muscle) of t. hansoni has been measured by real-time qpcr. the gills are the organ in which the highest levels of gpx1 mrna accumulation is present. gpx activity have been measured in the same organs. the highest levels are present in heart and liver. the tissue-specific differences in the mrna and active protein accumulations are probably related to a peculiar regulation of gene expression and the physiological function characteristic of these organs. (supported by p.n.r.a. and m.i.u.r. grants). assessing immunological memory in the solitary ascidian ciona robusta d melillo1, r marino2, g della camera1, p italiani1, d boraschi1,2 1institute of biochemistry and cell biology (ibbc), national research council (cnr), naples, italy 2stazione zoologica anton dohrn, biology and evolution of marine organisms (beom), naples, italy available information on the immune defensive mechanisms active in the solitary ascidian ciona robusta includes the functional evaluation of phagocytic and encapsulating activity, largely brought about by phagocytic cells within the haemocyte population, the molecular and functional definition of complement components, and the genome-based identification of a number of immune-related genes and pathways, homologous to vertebrate counterparts. since c. robusta only displays highly conserved innate immune mechanisms, being devoid of an adaptive immune system, this organism is an excellent model for studying the features of innate memory, i.e., the capacity of the innate immune system to reprogramming its responsiveness to potentially dangerous agents upon repeated exposure. in this study, we have developed an in vivo model for assessing the establishment and molecular/functional features of innate memory, by sequentially exposing c. robusta to a priming stimulus (the gram-negative bacterial agent lps, or the gram-positive molecule lta), followed by a period of resting to return to basal conditions, and a challenge with the same agents in homologous or cross-stimulation. the endpoints of immune activation were a functional activity (phagocytosis) and the molecular profiles of immune-related gene expression. the results show that exposure of c. robusta to bacterial agents induces a reaction that primes animals for developing a different (expectedly more protective) response to subsequent microbial challenges, showing the effective establishment of an immune memory. this immune memory relies on the modulation of a number of different mechanisms, some of which are priming-specific, others that are challenge-specific, and others that are completely non-specific, i.e., are common to all priming/challenge combinations (e.g., up-regulation of the tnf and lbp genes). memory-dependent expression of the humoral immunity-related gene c3ar inversely correlates with memory-dependent variations of phagocytic rate, suggesting that complement activation and phagocytosis are alternative defensive mechanisms in c. robusta. conversely, memory-dependent expression of the cellular immunity-related gene cd36 directly correlates with variations of phagocytic rate, suggesting a direct involvement of this gene in the functional regulation of phagocytosis. stress granules in ciona robusta: molecular evolution of tiar and ttp and early evidence of their gene expression under stress conditions induced by metals l drago, l ballarin, g santovito department of biology, university of padua, padua, italy stress granules are non-membranous cytoplasmic foci composed of messengers (not translated), ribonucleoproteins, translation initiation components and other additional proteins, that represent a primary mechanism by which gene expression is rapidly modulated when cells are 13 subjected to adverse environmental conditions (kedersha et al., 2002; anderson et al., 2009; lavut et al., 2012; waris et al., 2014). very few works have been devoted to study the presence of molecular components of stress granules in invertebrate animals. in this work, we characterized, for the first time in the solitary ascidian ciona robusta, the genetic sequences of two important protein components of stress granules, tiar (tia-1 related to proteins) and ttp (tristetraprolin), and carried out the first studies on their gene expression. the sequences characterized for tiar and ttp genes have allowed to start a study on the molecular evolution of these proteins in animals: for tiar the obtained results are consistent with recent phylogenetic analysis that place tunicates as sister group of vertebrates, whereas the phylogenetic position of ttp remains still uncertain. the data on mrna expression, provided by qrt-pcr analysis, are absolutely the first obtained in non-mammalian animals. as expected, the exposure to each metal (cu, zn and cd) led to a generalized decrease in mrna expression levels for both tiar and ttp, suggesting that the metal accumulation induce acute stress and the inhibition of the transcription of tiar and ttp genes. the data presented here improved our knowledge about the molecular evolution anti-stress proteins in metazoans and emphasize the importance of the transcription of tiar and ttp genes, which represents an efficient physiological response allowing c. robusta to survive in the presence of metals in the marine environment (supported by m.i.u.r. grant). references anderson p., kedersha n. stress granules. curr. biol. 19: r397-398, 2009. kedersha n., anderson p. stress granules: sites of mrna triage that regulate mrna stability and translatability. biochem. soc. trans. 30: 963969, 2002. lavut a., raveh d. sequestration of highly expressed mrnas in cytoplasmic granules, pbodies and stress granules enhances cell viability. plos genetics. 8: e1002527, 2012. waris s., wilce m.c.j., wilce j.a. rna recognition and stress granule formation by tia proteins. int. j. mol. sci. 15: 23377-23388, 2014. session 2. chairmen: davide malagoli, university of modena-reggio emilia, modena, italy and gianluca tettamanti, university of insubria, varese, italy cocktails and long lasting immunity: evolution of innate defenses and bacterial resistance j rolff institute of biology, department of biology, chemistry and pharmacology, freie universität berlin, berlin, germany upon infection, insects produce a cocktail of immune effectors to combat pathogens. this result of natural selection is in contrast to medical applications of antimicrobials, where often only one drug is employed. here, i will first discuss interactions between antimicrobial peptides as a main group of immune effectors and what the evolutionary consequences are for both, host and pathogen. antimicrobial peptides are usually synergistic when tested in vitro, in vivo the situation is more complex and yields results that cannot be predicted from in vitro studies. also, i will report on how antimicrobial peptide resistant bacteria fare when exposed to an insect immune system. based on these findings i will then ask, how to explain the evolution of complex immune systems. finally, i will ponder if and how research on insect amps and bacterial resistance evolution against them can inform medical applications of amps and of antimicrobials more generally. horizontal gene transfer can drive the evolution of insect immune defences s caccia department of agricultural sciences, university of naples “federico ii”, portici (na), italy horizontal gene transfer (hgt), the accidental acquisition of genetic material by an organism, is extremely common in prokaryotes but represents an important mechanism for genome evolution and acquisition of new traits also in eukaryotes. in multicellular organisms this phenomenon is often mediated by transposable elements or symbionts and parasites. recently, an original mechanism of hgt has been described in insects. in particular, the analysis of genomes of different lepidopteran species revealed multiple dna sequences derived from polydnaviruses (pdv). pdv are symbionts of parasitic wasps attacking lepidopteran larvae, which are injected during the oviposition and trigger metabolic and physiological alterations in the host, including immune suppression, to ensure progeny survival and development. despite the frequency of this peculiar hgt, an in depth characterization of the function of acquired sequences in recipient lepidopterans is almost lacking. this work elucidates the functional role of sl gasmin, a pdv gene that has been transferred to an ancestor of the moth species spodoptera littoralis and domesticated. sl gasmin is highly expressed in circulating immune cells, the hemocytes, of s. littoralis larvae and sl gasmin transcripts significantly increase upon pathogen challenge. the functional characterization of sl gasmin, performed by rna interference, has elucidated its fundamental role in the phagocytosis of bacteria by hemocytes. moreover, proteomic analyses have allowed to clarify that sl gasmin protein is secreted by hemocyes into insect hemolymph and acts as an opsonization factor. indeed, the protein binds on bacteria surface and mediates their recognition by hemocytes. this study demonstrates that in insects, important immune functions do not necessarily originate from evolution of existing genes but can be acquired by hgt events, paradoxically mediated by their natural enemies that are specialized in evading insect immune responses. 14 analysis of cellular and humoral immune response in hermetia illucens larvae d bruno1, a montali1, m mastore2, m casartelli3, s caccia4, a grimaldi1, mf brivio2, g tettamanti1 1department of biotechnology and life sciences, university of insubria, varese, italy 2department of theoretical and applied sciences, university of insubria, varese, italy 3department of biosciences, university of milano, milano, italy 4department of agricultural sciences, university of naples “federico ii”, naples, italy the larvae of the black soldier fly, hermetia illucens, are well known bioconverters due to their ability to grow and develop on a wide range of organic waste substrates. despite the great interest on the use of these larvae for bioconversion, there is a lack of knowledge about their biology, in particular regarding their immune system. in the present study we investigated the mechanisms involved in cellular and humoral response in h. illucens larvae. first, we performed a morphological characterization of immune circulating cells (i.e., hemocytes), before and after the immune challenge, and evaluated their phagocytosis and encapsulation capacity. moreover, we analyzed the activity of key components of the humoral immune response, such as phenoloxidase, lysozyme, and antimicrobial peptides (amps). our results clearly show that h. illucens larvae are able to mount efficient immune responses, both at cellular and humoral level. hemocytes proliferation and responses are rapidly activated after infection and the humoral response mainly involves the increase in lysozyme activity and the recruitment of amps, while phenoloxidase seems to be inhibited in infected larvae. this study provides basic information about the h. illucens larval immune system and opens up the possibility to improve the quality of the larvae during mass rearing through a modulation of the immune response. innate immune response and antimicrobial peptides in the silkworm bombyx mori o romoli1, a saviane2, s mukherjee3, d brady1, e franzolin1, c rampazzo1, p d’antona4, a montali4, a squartini5, g tettamanti4, s cappellozza2, a bhunia3, f sandrelli1 1department of biology, university of padua, padua, italy 2crea-honey bee and silkworm research unit, padua seat, padua, italy 3bose institute, department of biophysics, p-1/12 cit scheme vii (m), kolkata, india 4department of biotechnology and life sciences, university of insubria, varese, italy 5department of agronomy, food, natural resources, animals and environment, university of padua, padua, italy the domesticated silkworm bombyx mori has been extensively studied as a model organism for lepidoptera genetics and for its economic value in silk production. since silkworm infectious diseases can cause crop losses of ~27–35 %, a great attention has been posed on the study of b. mori immune mechanisms. b. mori has an innate immune system, whose main effectors are the antimicrobial peptides (amps). silkworm strains are commonly grouped into four geographical types (japanese, chinese, european and tropical), characterised by a variable susceptibility to infections. recently we showed that silkworm strains originating from the four geographical areas were characterised by a different susceptibility to serratia marcescens and enterococcus mundtii bacteria, with the indian strain displaying the lowest sensitivity to s. marcescens and the european one to e. mundtii (romoli et al. 2017). the resistance of the indian strain to s. marcescens seemed to be associated with its capability to recognise the pathogen and promptly activate the systemic amp transcription. on the other hand, all the strains were able to activate the amp response against e. mundtii. however, the highest resistance of the european strain appeared to be related to the specific composition of its amp cocktail, containing more effective variants such as a peculiar cecropin b isoform, named q53 cecb. a further characterisation of the q53 cecb antimicrobial property and action mechanism showed that the peptide exerted an efficient membranolytic activity also against relevant human pathogens, such as pseudomonas aeruginosa. in addition, q53 cecb did not show any cytotoxic activity or haemolytic effects against human cell lines or erythrocytes (romoli et al. 2019;). taken together our data suggest that b. mori strains with distinct genetic backgrounds employ different strategies to counteract bacterial infections, whose efficacy appears to be pathogen dependent. the highest antimicrobial activity of the european q53 cecb variant against clinically relevant microorganisms represents an interesting feature for possible biomedical applications. use of silkworm as infection model for novel antibiotic screening and development a montali1, f berini1, m mastore2, a saviane3, s cappellozza3, mf brivio2, f marinelli1, g tettamanti1 1department of biotechnology and life sciences, university of insubria, varese, italy 2department of theoretical and applied sciences, university of insubria, varese, italy 3consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, centro di ricerca agricoltura e ambiente (crea-aa), laboratorio di gelsibachicoltura, padua, italy the development of new drugs requires a series of preclinical tests followed by the clinical trials needed for assessing the safety and efficacy of the compound. commonly, mammalian models (e.g., mice and rats) are used in preclinical tests. due to the bioethical issue, the time required for the analyses, and the high costs, in 15 the last few years, alternative animal models, as lepidoptera, have been proposed for the initial screening phase. in this study, we evaluated the response of the silkworm bombyx mori, infected with staphylococcus aureus, to the treatment with different antibiotics (vancomycin, teicoplanin, and dalbavancin). we monitored the survival rate of the larvae, analysed different cellular and humoral markers of the immune system, and evaluated the bacterial load in the hemocoel, performing experiments at 37 °c to reproduce human physiological conditions. while amp expression and bacterial load resulted to be inadequate to our purpose, we demonstrated that larval survival rate, hemocyte viability, and activity of prophenoloxidase system respond to the infection as reliable and reproducible markers. considering the limited costs of using bombyx mori as infection model, the quickness of analysis, and the easiness of use, the three selected markers might represent potential tools for accelerating the discovery and development of novel antimicrobial drugs. 20-oh-ecdysone and extracellular atp are not pro-autophagic factors for the lepidopteran fat body cell line, iplb-ldfb a ferrari1, r fiorino1, m montanari1, a accorsi2, r simonini1, m de eguileor3, d malagoli1 1department of life sciences, university of modena and reggio emilia, modena, italy 2howard hughes medical institute, stowers institute for medical research, kansas city, mo, usa 3department of biotechnology and life sciences, university of insubria, varese, italy autophagy is a conserved self-protective cell process that may also help the induction of type i programmed cell death (pcd-i, apoptosis) and, specifically during insect metamorphosis, it also represents the foundation of type ii programmed cell death (pcd-ii). the double role played by autophagy in insects has been mainly studied in drosophila melanogaster and bombyx mori, thus we investigated pcd-ii initiation in an alternative in vitro insect model, iplb-ldfb, a cell line from the lepidopteran pest lymantria dispar. iplb-ldfb derive from larval fat body, an organ encompassing metabolic as well as immunerelated functions that is deeply rearranged during metamorphosis. after a 2 h-incubation with the atp synthase inhibitor oligomycin a (oa, 10 μm), iplb-ldfb cells release in the conditioned medium (cm) uncharacterized pro-autophagic factors (pafs) able to induce pcd-ii in naïve iplb-ldfb. in this study, we looked for the identity of pafs and the signaling pathways they activate. size-exclusion centrifugation of cm evidenced that pafs are retrieved into the < 3 kda fraction. proteomic studies on cm did not provide < 3 kda paf candidates, thus we investigated the effects of 20-oh-ecdysone and extracellular atp as potential non-protein candidates. surprisingly, 20oh-ecdysone (0,1 ÷ 10 µg/ml) was ineffective on iplb-ldfb cells and did not promote neither pcdii nor other forms of cell death, after 72 hincubation. conversely, morphological, flow cytometry and tunel assays showed that extracellular atp (0,1 ÷ 5 mm) induced pcd-i in iplb-ldfb cells after a 6 h-exposure. accordingly, luminometric assays showed cm does not contain significant levels of atp, excluding also this molecule from the potential pafs released under oa treatment. in order to elucidate the signaling pathways elicited by the still unidentified pafs, we assessed whether single protein kinase (pk) inhibitors could block the cm effects. cell viability, morphological and flow cytometry analyses showed that all the inhibitors tested (namely h-89 for pka, calphostin c for pkc and wortmannin for pi3kinase) acted on cell size and cytoplasm organization, but they did not induce cell death and did not prevent cm lethal effects after 24 hexperiments. this suggests that the pafs in the cm may promote pcd-ii through multiple signaling pathways. our studies confirm the complexity of the pcd-associated processes also in an in vitro system, and suggest that, in spite of the high conservation of the basic mechanisms from the unicellular organisms to the metazoans, autophagy can be carried out under various stimuli and following multiple and diversified signaling pathways. discovery and characterisation of novel chhs involved in the immune response of the red swamp crayfish procambarus clarkii c manfrin, f capanni, l dolzani, a pallavicini, pg giulianini department of life sciences, university of trieste, trieste, italy crustacean hyperglycaemic hormone (chh) has many functions to regulate carbohydrate metabolism, ecdysis and reproduction including ion transport in crustaceans. furthermore, rapid mobilization of neuropeptides, including chhs, from the sinus gland in the eyestalks represent the primary response to stress. here we report the identification of two potential transcripts pertaining to the chh family in procambarus clarkii, thanks to a wholetranscriptome sequencing approach. these two members, named chhip (chh immune-related procambarus) and chhop (chh homologous procambarus) share the typical features of the chh (the presence of a chhprecursor-related peptides between the signal peptide and the peptide, and 6 conserved cysteines involved in the three typical disulfide bonds of the chh-superfamily) and they have been found upregulated following the knock-down of the main chh isoforms through rna interference. to shed light on their possible role in the immune response of p. clarkii two experiments have been performed to evaluate chhip and chhop relative expression in the eyestalk in response to a lipopolysaccharide (lps)and a staphylococcus aureus-challenges (1 µg/animal (body weight 40 g) of lps and s. aureus 3 x 108 cfu/100 µl per animal, respectively). 16 following the lps injection, only chhip increased significantly (7-fold at 2 h post injection−hpi− and 14-fold at 4 hpi, p ≤ 0.01). s. aureus injection triggered chhip at 6 and 12 hpi (p ≤ 0.05), but not at 24 hpi. conversely, chhop resulted up-regulated exclusively at 24 hpi of s. aureus (p ≤ 0.05). these preliminary findings suggest an early immunological response to bacterial infection driven by chhip followed by an up-regulation of chhop, with the nearly contemporary down-regulation of chhip, at 24 hpi. these results seem to support the existence of a combined action of chhip and chhop in response to lps and gram bacteria in a time-dependent manner and increase the spectrum of action of this family of hormones up to their direct involvement in the immune response. hemocyte depletion as a tool for studying immune cell dynamics and contribution to fundamental biological processes in the freshwater snail pomacea canaliculata g bergamini1, a accorsi2, d malagoli3 1department of chemical and geological sciences department of life sciences, university of modena and reggio emilia, modena, italy 2stowers institute for medical research, howard hughes medical institute, kansas city (mo), usa 3department of life sciences, university of modena and reggio emilia, modena, italy pomacea canaliculata is an invasive freshwater gastropod native to south america which spread all over asia, with severe consequences on ecosystems and public health. this animal has been recently retrieved also in eu and usa and it is listed among the “100 of the world’s worst invasive alien species”. because of the uncommon adaptive features of this snail, its immune system (is) has been investigated through cytological analyses, flow cytometry, immunohistochemistry, proteomic and molecular techniques. major attention has been paid to the characterization of the cellular component of p. canaliculata is, especially circulating hemocytes. although the morphology and ultrastructure of these cells have been described, their development, maturation and the distribution of hematopoietic sites still remain unclear. similarly, the roles played by circulating hemocytes in fundamental activities, like tissue repair, wound healing and organ regeneration are unknown. on these bases, we decided to study circulating hemocytes during hemolymph repopulation, after the application of either repeated hemolymph withdrawals or injection of a pro-apoptotic drug specifically targeting phagocytic cells in mammals and insects, i.e., clodronate liposomes. samples of withdrawn hemolymph were analysed with optical microscopy and via the imagestream®x-ii imaging flow cytometer, while hemolymph from clodronatetreated snails underwent cytocentrifugation and was stained with combined histochemical and fluorescent staining. the observation of hemolymph after multiple withdrawals showed limited variations in its composition and a complete deletion of circulating hemocytes has never been observed, suggesting the presence of significant hemocyte reservoirs in our model. our first data from clodronate-treated animals, instead, indicate that the drug could efficiently and temporarily remove circulating hemocytes in a short-time incubation, without affecting snail survival. on the whole, multiple hemolymph withdrawals and injections of clodronate liposomes followed by morphologic and/or cytofluorimetric analyses, represent diverse and valid approaches for in-depth and novel studies on hemocyte balancing, turn over and maturation in molluscs. moreover, collected information and tools may be extended to investigate the contribution of the is in other processes relevant for the snail survival, e.g., wound healing and organ regeneration. micrornas: possible new players in bivalve antiviral immunity u rosani1, b fromm2, cm bai3, p venier4, km wegner1 1alfred wegener institut, list auf sylt, germany 2stockholm university, stockholm, sweden 3chinese academy of fishery science, qingdao, china 4university of padua, padua, italy deep sequencing is contributing to enlarge the view of small non-coding rnas (sncrnas), a group of rna molecules not producing proteins while serving other roles. among the variety of sncrnas (short interfering rnas, extracellular rnas, small nuclear ribonucleoproteins, small nucleolar rnas and piwi-interacting rnas), micrornas (mirnas) have been widely studied because of their role in disease and as biomarkers. mature mirnas are short sequences (20-23 nt), which originated from stem-loop precursor rnas (pre-mirnas). mirnas mostly act as post-transcriptional repressors by targeting 3’ untranslated regions (3’-utrs) of messenger rnas, with paramount functions across various cellular and developmental processes such as immunity, cell behavior and host-microorganism interactions. evolutionary studies showed that mirna families have been continuously added along the evolution of bilaterians, and that mirnas are conserved under strict negative selection. because of these features, including also a conserved biogenesis mechanism and a small probability of convergent evolution, mirnas have been proposed as powerful markers for phylogenetics. we have previously demonstrated the conservation of the mirna complement genes in bivalves. however, the knowledge regarding bivalve mirnas are fragmentary with sncrna sequences available only for a few species (pinctada martensii, p. fucata, crassostrea gigas, c. hongkongensis, chlamys farreri, tegillarca granosa and ruditapes philippinarum). aiming to identify conserved and novel bivalve mirna families and to verify their contribution during viral infection, we produced sncrna sequencing data of two species, the blood clam (scapharca broughtonii) and the pacific oyster (c. gigas). using the corresponding genomes, we analyzed 167 and 151 bona-fide mirnas for clam 17 and oyster, respectively. by measuring their expression levels during ostreid herpesvirus (oshv-1) infection in both species, we reported specie-specific expression patterns involving different mirnas. several of the most modulated mirnas have been reported as involved in antiviral immunity also in shrimp, underpinning possible functional conservation. in conclusion, we provided a first comparative view of bivalve “mirnaome”, tracing commonalities and differences within invertebrates. evolution and molecular diversity of mytilin-like defense peptides in marine mussels s greco, m gerdol, a pallacivini department of life sciences, university of trieste, trieste, italy the cysteine –stabilized alpha/beta motif (csαβ) is a widespread structural scaffold found in several low-molecular weight cationic peptides with defense function. while many cs-αβ antimicrobial peptides have been described as major players in bivalve immune response, several aspects concerning their evolution are still unclear and a clear-cut definition of the relationship among defensins, mytilins, myticins and other structurally similar peptides is still lacking. here, we redefine the distribution of mytilin-like cs-αβ peptides thanks to a bioinformatic screening of the sequence resources available for mytilida. we highlight that, in spite of limited primary sequence similarity, these amps retain a nearly identical three-dimensional folding, stabilized by eight highly conserved cysteine residues. we discuss that the variable position of the c1-c5 disulfide bond has a significant effect on the structural flexibility of the mytilins identified in perna spp., as well as in a few novel mytilus spp. mytilins. we further describe the organization of the mytilus galloprovincialis mytilin gene cluster, reporting the presence of two pseudogenes in addition to the four canonical genes previously identified, and discuss the presence of additional dispensable mytilin genes subject to presence/absence variation. session 3. chairmen: adriana vallesi, university of camerino,camerino, italy and gianfranco santovito, university of padua, padua, italy earthworm cellular immunity: an old story reloaded from the nanomaterial’s point of view p engelmann1, k bodó1, b kokhanyuk1, p németh1, y hayashi2 1department of immunology and biotechnology, clinical center, medical school, university of pécs, pécs, hungary 2department of molecular biology and genetics, aarhus university, aarhus, denmark broad commercial and biomedical applications of silver nanoparticles (agnps) may result in unintentional release of the agnps and their byproducts into the environment posing potential impacts on various living beings. it is largely unexplored how immune system reacts against agnps, albeit those particles can be effectively recognized and engulfed by innate immune cells. in this regard, special attention should be paid to nano–immuno interactions. earthworm macrophage-like cells (coelomocytes) offer potentially sensitive and accessible resources of monitoring the complex interactions of nps with biological systems. previously, we studied the evolutionary conserved molecular and stress mechanisms in earthworm coelomocytes cross-referencing to human monocytic cells exposed to agnps. in our recent study, we have further focused around the species differences within the earthworm taxa, revealing fine differences of various immune and stress-related parameters from two closely-related earthworm species eisenia andrei and e. fetida following in vitro exposure to agnps and aunps. finally, we touch on the emerging aspects of nanoparticle-protein interactions in which earthworm-specific proteins may play an unexpected role in pattern recognition. acknowledgements this work was supported by pte-áok-ka 2017-04, efop-3.6.1.-16-2016-00004, ginop 2.3.2-15-2016-00050, the únkp-19-3-i new national excellence program of the ministry for innovation and technology to kb and the bolyai jános research scholarship of the hungarian academy of sciences to pe. immunomodulation of selected nps in mytilus hemocytes: a morphofunctional study m auguste1, c mayall2, m hocevar3, d drobne2, l canesi1 1department of environmental, earth and life sciences, distav, university of genoa, genoa, italy 2university of ljubljana, biotechnical faculty, ljubljana, slovenia 3institute of metals and technology (imt), ljubljana, slovenia the nanoparticle (np) industry is continuously increasing in production and creativity; producing np with diverse size or surface coating that change their properties and will likely also change their interactions with the immune system of organisms. in a reducing strategy for the amount of animal used in science, to test such amount of nps, alternative protocols are needed and in vitro experiments have shown to be promising for future testing strategy. in this line, the hemocytes of mytilus galloprovincialis were utilized in in vitro short time (30 min) exposure to screen the immunomodulatory effects of several nps of different size, material and surface modifications (metal based zero valent, nano-oxides, amino functionalized nanopolystyrenes). moreover, to evaluate the role and interactions of nps with proteins from the biological media, experiments were performed using np suspensions in either artificial seawater (asw) or hemolymph serum (hs). upon exposure, several functional immune 18 parameters (lysosomal membrane stability, phagocytosis, lysozyme and ros release) were evaluated. in addition, the effects of different nps of hemocyte morphology were investigated by scanning electronic microscopy (sem). the results on determination of immune parameters showed activation of immune defences specific to the nps type and underline the importance of exposure media in the experimental design. the images obtained at the sem contributed to understand the original hemocyte morphology in different media and their potential morphological changes upon nps exposure, showing peculiar cell activations. these data will contribute to build information on the role of size and functionalization of nps for mytilus hemocyte for both functional and morphological responses. effects of different nanoparticles types on benthic foraminifera ammonia parkinsoniana c ciacci1, l canesi2, mv grimmelpont3, i corsi4, e bergami4, d curzi1, d burini1, vmp bouchet3, p ambrogini1, p gobbi1, y ujiié5, y ishitani6, r coccioni7, jm bernhard8, f frontalini7 1department of biomolecular science, university of urbino, urbino, italy 2department of earth, environment and life sciences, university of genoa, genoa, italy 3faculté des sciences et technologies, université de lille, lille, france 4department of physics, university of siena, siena, italy 5center for advanced marine core research, university of kochi, nankoku, japan 6section center for computational sciences, university of tsukuba, tsukuba, japan 7department of pure and applied science, university of urbino, urbino, italy 8woods hole oceanographic institution, geology and geophysics department, woods hole, ma, usa benthic foraminifera, with their developed processes for cellular internalization, can take up nanoscale and microscale particles for example during seawater endocytosis or in sediments via reticulopodia, which are used for gathering food such as bacteria and algae. the potential adverse effects of nanoparticles (nps) including nanoplastics, in marine environments have recently attracted considerable attention, though remain relevant knowledge gaps, particularly on their effects on the benthos. nps are expected to make their way into the aquatic environment where sedimentation of particles will likely occur, putting benthic organisms at particular risk. in this work, the effects of exposure (1 mg/l, 24 h) to different types of nps (tio2, 25 nm; polystyrene 42 nm and sio2, 92 nm) were investigated in the benthic foraminiferal species ammonia parkinsoniana using a combination of techniques (tem; sem; esem; eds and cmls). the results show the internalization of nps in the cytoplasm of a. parkinsoniana, as well as the associated intracellular lipid accumulation, free radical production and the alteration of mitochondria. in particular, ntio2 induced the highest ros production. moreover, preliminary gene expression analysis revealed that ntio2 potentially affects the following molecular pathways: endocytosis, exocytosis, lysosome, ros and biosynthesis of unsaturated fatty acid. this latter in particular may have a pivotal role in the detoxification process. these are the first data on the effects of different nps in a benthic unicellular eukaryote. the results suggest that nps can have deleterious effects on benthic organisms, which are commonly neglected in the research despite representing an important link in the trophic web. the ability to internalize nps coupled with np persistence may also promote their transfer and biomagnification the foodchain. in light of the increasing occurrence of nps in marine environments, the evaluation of their cytotoxicity and the internalization capability of bottom-dwelling organisms is an essential step to predict future exposure scenarios. these benthic marine organisms may represent a new model by which to assess future np impact scenarios in the marine benthos. session 4. chairmen: giuseppe scapigliati, university of viterbo, viterbo, italy and giovanna parisi, university of palermo, palermo, italy identification of novel lumbricins in eisenia andrei earthworms – a missing link of annelids’ antimicrobial peptides k bodó1, á boros2, z lászló2, é rumpler3,4, l molnár4, p németh1, p engelmann1 1department of immunology and biotechnology, clinical center, medical school, university of pécs, pécs, hungary 2department of medical microbiology and immunology, medical school, university of pécs, pécs, hungary 3laboratory of reproductive neurology, hungarian academy of sciences, institute of experimental medicine, budapest, hungary 4department of comparative anatomy and developmental biology, faculty of sciences, university of pécs, pécs, hungary antimicrobial peptides (amps) are the structurally conserved ancient weapons of innate immunity from plants to human. to date only one particular amp, the lumbricin and its relatives were identified in certain earthworms, but not in the eisena andrei. hereby we report the identification of the mrna for lumbricin and -serendipitouslya novel lumbricinrelated peptide in e. andrei earthworms. the characterized mrna sequences for e. andrei lumbricin and lumbricin-related peptide are composed of 477 and 575 nucleotides. the prolinerich e. andrei lumbricin and the related peptide contain 63 and 59 amino acids, and the anticipated molecular weights are 7413.35 and 7066.84 da, respectively. the novel e. andrei lumbricin and 19 lumbricin-related peptide revealed the closest relationship with lumbricins from the leech, hirudo medicinalis and lumbricus rubellus earthworms by phylogenetic analysis. most prominent mrna expression occured in the foregut (pharynx, gizzard), while other organs had fair (body wall, midgut, ovary, metanephridium, seminal vesicles, ventral nerve cord) or low (coelomocytes) levels. by means of coelomocyte sorting, only the amoebocyte subpopulation has evidenced the mrna expression for both peptides. during embryogenesis, a gradually increasing expression pattern was observed in the various embryonic stages that were coincident with the mrna expression pattern during tissue restoration processes. following 48 h of in vivo staphylococcus aureus bacteria challenge both mrnas were significantly elevated in coelomocytes, while escherichia coli bacteria or zymosan stimulation had no distinguishable effects. since e. andrei earthworms are widely applied in various toxicological assays, these novel peptides can be monitored as potential molecular targets that may be modulated by environmental contaminants. acknowledgements this work was supported by pte-áok-ka 2017-04, efop-3.6.1.-16-201600004, ginop 2.3.2-15-2016-00050, the únkp-19-3-i new national excellence program of the ministry for innovation and technology to kb and the bolyai jános research scholarship of the hungarian academy of sciences to ep. characterisation of the complement system of a colonial protochordate: study of the expression of c3, cr1, c3ar and their role in nonself recognition a peronato, n franchi, l drago, l ballarin department of biology, university of padua, padua, italy the complement system is one of the most ancient immune effector mechanism of bilaterian metazoans. three complement-activation pathways are known in vertebrates: the classical, the alternative and the lectin pathways. in the compound ascidian botryllus schlosseri, a reliable model organism for the study of immunobiology, we demonstrated the presence of the lectin and the alternative pathways. all the complement components identified so far, are expressed by morula cells, the most abundant circulating hemocytes. in mammals, once the complement system is activated, c3 is cleaved to c3a and c3b, the former exerting a chemokine–like activity, the latter acting as opsonin and, ultimately, activating the lytic pathway. in the present work, we continued our analysis of the role of c3 in botryllus immunity by studying the modulation of bsc3 transcription during the colonial blastogenetic cycle and the effect of bsc3 knockdown on immune responses. in addition, we looked for putative complement receptors. in mammals, the best-known receptor for c3a is c3ar, whereas cr1 is the receptor, on the phagocyte surface, able to recognize and bind c3b. here, we describe, in b. schlosseri, a gene showing similarity with vertebrate c3ar and three genes with similarity to cr1 (two soluble forms and one transmembrane). we also studied their transcription in the course of the colonial blastogenetic cycle. results indicate that complement receptor mrnas are located in different immunocytes, suggesting the presence of a cross-talk between phagocytes and morula cells. only morula cells, and no other immunocytes type, were labelled by the antisense probe for bsc3ar and the soluble cr1s, whereas phagocytes and young, undifferentiated cells known as hemoblasts were the cells stained by the probe for the membrane-linked bscr1. both the bsc3ar and bscr1 genes are constitutively transcribed; however, a modulation of transcription occurs during the colonial blastogenetic cycle as the amount of bsc3ar mrna abruptly decreased at take-over, whereas no differences were observed when earlycycle and mid-cycle were compared. this is probably related to the renewing of circulating cells at to, when 20 30 % of hemocytes undergo cell death by apoptosis and are replaced by new, differentiating cells entering the circulation in the same period. evidence for the chimeric origin of a pheromone-coding gene in euplotes raikovi a vallesi, p luporini school of biosciences and veterinary medicine, university of camerino, camerino, italy ciliates, unique among eukaryotes, evolved two types of nucleus which are distinct in both structure and function. a diploid transcriptionally silent germline micronucleus (mic) with an orthodox chromosomic structure coexists in the same cytoplasm with a polyploid transcriptionally active somatic macronucleus (mac) showing a unique sub-chromosomic structure. this structure is acquired in coincidence with every sexual event, when the ex-conjugant (or ex-autogamic) cell initiates a new life cycle developing a new mac from a mitotic product of the synkaryon. in spirotrichous ciliates such as euplotes, the chromosomes of this product undergo impressive phenomena of polytenization, fragmentation in thousands of dna fragments known as ‘macronuclear destined sequences’ (mdss), and dna elimination. under the guide of noncoding rna templates synthesized by the old mac before being destroyed, these mdss are assembled into sub-chromosomic (‘gene-size’) dna molecules which, amplified to thousands of copies, compose the new mac. the way to a correct mds assembly may be crossed by errors, with the consequent generation of functional chimeric genes which can stably be integrated and expressed in the mac genome. one of these chimeric genes came to light by studying the genetic basis of the pheromonemediated self/not-self recognition mechanism in e. raikovi. the genome of type i cells secreting pheromone er-1 was found to contain two structurally distinct mac er-1 coding genes, both expressed via a mechanism of intron splicing responsible for the synthesis of the er-1 soluble 20 form and a membrane-bound er-1 isoform functioning as autocrine pheromone receptor. the sequence of one gene resulted unmistakably homologous throughout its length with the sequences of other members of the e. raikovi pheromone gene family. in contrast, the sequence of the second gene resulted unique at level of a 359-bp segment of the 5’ region destined to specify the cytoplasmic domain of the er-1 membranebound isoform. by showing that this segment arises from a wrong assembly of a mds destined to a 2417-bp gene with no relationship with the signaling pheromone system, we provide additional evidence that the generation of functionally active chimeric genes from not-programmed phenomena of somatic mds recombination is an effective and micindependent source of gene variants in the ciliate mac genome. the mediterranean anthozoan anemonia viridis (forsskål, 1775) for the study of inflammation and regeneration c la corte1, m cammarata1, d parrinello1, m dara1, a grimaldi2, n baranzini2, mg parisi1 1department of earth and marine sciences (distem), university of palermo, palermo, italy 2department of biotechnology and life sciences, university of insubria, varese, italy regenerative capability in anthozoans is an important adaptive strategy for their survival to environmental disturbance of natural and anthropogenic origin such as predation or anchoring, that can cause injuries or removal of entire parts of the animal body, and it can be also considered indirectly a further tool of innate immune system. in the context of “self”-“non self” recognition, is significant the interaction with the endosymbiont of the genus symbiodinium and the recognition of pathogens and foreign agents capable of invading the injured tissues. from these premises and the growing stressors that can cause injuries, it is significant to understand how species respond to physical damage and how they manage to recover and regenerate compromised tissues. our research team, studied in the mediterranean anthozoan anemonia viridis (forsskål, 1775), the natural seasonal variability of its morphology and enzymatic biomarkers involved in inflammatory process, the immune system response following injection of molecules varied in type and dimension. in particular, after the infection of two pathogenic bacteria escherichia coli and vibrio alginolyticus a particular and strong reaction was observed. these previous knowledge allowed us to examine the activity of enzymes such as proteases (sds-page on gelatin and fibrinogen substrate), peroxidase and alkaline phosphatase as biomarkers traditionally involved in wound healing event and in the rearrangement of extracellular matrix. the regenerative process, in this mediterranean species of anthozoan, was analyzed by subjecting groups of animals to differential tentacle cuts (n = 10, 20, 30) and observing and estimating the regenerative potential after 7, 14 and 21 days from cutting. a morphological and histological observation of the tentacular regrowth event was conducted in addition to the evaluation of the expression of proliferating cell nuclear antigen (pcna) using the immunoblotting technique. the future goal is to increase the knowledge of the processes that trigger the immune response in inflammation and regeneration, for the considerable interest in basic sciences and for the transferability of results in biotechnological field. morpho-functional characterization of the microenvironment in muscle cell development of hirudo verbana l pulze, f ferraro, n baranzini, a grimaldi, m de eguileor department of biotechnology and life sciences, university of insubria, varese, italy muscle regeneration is a process of great interest to the scientific community because is involved in a big number of disorders that are still without a cure. to study the mechanisms behind this process, the most used animal models have always been rodents, for their closeness to humans. nonetheless, the rising ethical awareness has encouraged a reduction in the use of vertebrates in the research, but at the same time the complex puzzle in which the different factors interact for muscle regeneration has not been clarified, so the contribution of the in vivo experiments is still fundamental. the leech is an inexpensive and easily manageable animal model and represents an invaluable and necessary alternative for these studies. in fact, in a simple body organization it is able to elicit complex processes (wound-healing, angiogenesis, immune response, and muscle regeneration) characterized by the same phases described for vertebrates. for all these reasons, taking also in consideration that regeneration recapitulates some of the processes employed during development and that no satellite cells were identified in leech, we aimed to study the structure and composition of the microenvironment where myogenic precursors mature during the body wall growth of the juvenile leech. in this regard, we performed morphological analyses of the adult and of the juvenile of hirudo verbana, focusing on the extracellular matrix (ecm) organization and on the cell’s level of differentiation. moreover, we also reconstructed one of the main pathways by which mechanical stimuli are integrated and transduced into transcriptional activity: the hippo signaling pathway. our data suggest that ecm has a pivotal role in controlling myocytes’ proliferation, migration and differentiation thanks to the control exerted by yap1. moreover, many cellular types, which concur in this development as stem-like cells of different lineages, were identified in these processes. these preliminary data confirmed that juvenile leech can be a good model for understanding how muscle fibres can growth and differentiate in relation to the 21 stiffness of ecm and to its conformations, to other mechanical, and chemical stimuli and to the interactions with different cell types. in silico characterization of the leech helobdella robusta’s matrisome f ferraro, l pulze, n baranzini, a grimaldi, m de eguileor department of biotechnology and life sciences, university of insubria, varese, italy leeches (phylum: annelida) have been extensively studied to elucidate sophisticated evolutionary-conserved biological processes that are similar to those of mammals. recently, the growing interest on extracellular matrix (ecm), owing to its multifaceted roles in physiological and pathological processes, has led the scientific community towards the definition of the matrisome as the list of all the structural ecm components, of those proteins affiliated to them, and of all secreted factors that the ecm is usually impregnated with. this annotated list is an invaluable tool to get a better insight on the role of a substantial component of every process and could help in the design and analysis of data coming from both classical and “omics” experiments. despite the human matrisome as well as those of several animal models have been already annotated, no specific information regarding annelids and leeches in particular is available. in this work, using a combination of in silico evolutionary conservation, gene onthology, and structural analyses we conduct a first annotation of the helobdella robusta’s matrisome. session 5. chairmen: magda de eguileor, university of insubria, varese, italy and loriano ballarin, university of padua, padua, italy t2 ribonuclease-mediated tumor suppression: an evolutionary conserved process involving a cross-talk between cancer cells and the immune system a de vito1, n baranzini1, e balza2, d scaldaferri1, l monti1, d baci1, e rosini1, a inforzato3, r taramelli1, l pollegioni1, d noonan1, a grimaldi1, l mortara1, f acquati1 1department of biotechnology and life sciences, university of insubria, varese, italy 2department of integrated oncological therapies, ircss ospedale policlinico san martino, genoa, italy 3department of immunology and inflammation, humanitas clinical and research institute irccs, milan, italy the link between cancer development and immune system dysregulation has long been established, and both the innate and adaptive immune systems are widely acknowledged to establish a complex crosstalk in vivo with cancer cells, which might culminate into a proor antitumorigenic response. in this context, cells belonging to the monocyte/macrophage lineage represent a key cellular component of the innate immune system involved in the control of the tumorigenic process, since advanced human tumors are frequently infiltrated with tumor-associated macrophages (tams) which actively contribute to tumor growth and dissemination. however, the long established functional plasticity displayed by macrophages can provide these cells with a marked anti-tumor properties as well. thus, experimental approaches aimed at promoting a macrophage shift in vivo from pro-tumor to anti-tumor phenotype represent a deeply investigated research field to develop immune system-mediated oncosuppresive therapies. the human rnaset2 oncosuppressor gene has recently emerged as a potential tool for macrophage-mediated tumor suppression. t2 rnases represent very ancient, pleiotropic and evolutionary conserved enzymes involved in a wide range of biological processes, among which host defense is frequently observed. a key role for human rnaset2 in the regulation of macrophage activity in both in vitro and in vivo experimental models has been recently reported. of note, the ability of rnaset2 to tune the macrophage phenotype has been recently observed in both vertebrate and invertebrate experimental model, thus pointing at a very ancent role for t2 rnases in the modulation of the innate immune system. moreover, recent reports suggest a functional role for rnaset2 in a broader range of immune cell types, pointing at t2 rnases as a putative regulators of several functional features within the immune system. the antibacterial role of hirudo verbana rnaset2: evaluation in vivo and in vitro n baranzini1, e martegani1, v t orlandi1, m reguzzoni2, m de eguileor1, g tettamanti1, f acquati1, a grimaldi1 1department of biotechnology and life sciences, university of insubria, varese, italy 2department of medicine and surgery, university of insubria, varese, italy recent data obtained in the medicinal leech hirudo verbana revealed that the enzyme rnaset2, belonging to t2 ribonucleases family, took part in the regulation and modulation of the innate immune system, by the direct recruitment of immunocompetent cells during bacterial infection. indeed, the injection of lps (lipopolysaccharides) and lta (lipoteichoic acid), principal components of the bacterial cell wall of gram-negative and grampositive respectively, into the leech body wall, triggered an increased expression of the endogenous leech enzyme hvrnaset2 in both macrophages and granulocytes. these results suggested a possible antibacterial role for this enzyme, as observed for the rnase 3 and the rnase 7 in vertebrates. in order to better clarify this aspect, here we conducted in vitro experiments by treating escherichia coli and staphylococcus aureus bacterial strains with the recombinant protein hvrnaset2. the effect on microorganisms was analyzed by light, transmission (tem) and scanning (sem) electron microscopy, after 3 and 24 h from 22 the incubation. the images showed an evident agglutination in both cases and immunogold assays indicated a direct interaction between hvrnaset2 and the bacterial cell walls. moreover, experiments performed both in vivo and by using the matrigel biomatrix (mg), confirmed the presence of s. aureus bacteria aggregates in the leech body wall, supporting the idea that, during leech innate immune response, hvrnaset2 triggers bacterial clumps formation to promote a more rapid phagocytosis by macrophages and to elicit a rapid and effective eradication of the infecting microorganisms from inoculated area recruiting phagocytic cells. hyaluronan, a new neuroimmune modulator of the microbiota-immune-gut-axis c giaroni department of medicine and surgery, university of insubria, varese, italy the gut saprophytic commensal flora has a fundamental role in the modulation of several local functions including regulation of host immune system and defense against pathogenic microorganisms. alterations in the symbiotic relationship between the microbiota and the enteric microenvironment underlays development of complex gut disorders such as chronic inflammatory disease (ibd). in recent years, we have focused on hyluronan (ha), an unbranched glycosaminoglycan (gag) component of the extracellular matrix, as a new molecular player involved in neuroadaptive changes of enteric neuronal circuitries in the inflamed gut. the gag is a key molecule mediating the host immune response to commensal and pathogenic bacteria by binding to toll-like receptors, tlr2 and 4. since both tlrs have been localized to enteric neurons and may regulate intestinal inflammation by controlling enteric nervous system (ens) structural and functional integrity, ha represents a potential molecular tool involved in development of myenteric neural plasticity by tuning adaptive signals at the intersection between the microbiota-innate immunity axis and ens. this hypothesis is innovative and opens new scenarios in the study of the molecular mechanism involved in the onset and severity of bowel diseases as well as for the development of new therapeutic agents for the treatment of diseases with clinical and social impact, all with underlying derangements of the microbiota-immune-gut axis, such as ibd. preliminary data on senescence in hemocytes of the colonial ascidian botryllus schlosseri l ballarin, f cima department of biology, university of padua, padua, italy senescence is a cellular response to damage that limits the proliferation of aged or effete cells and plays physiological roles as it is required for tissue homeostasis. colonies of the protochordate botryllus schlosseri, undergo cyclical generation changes or takeovers (tos) during which adult zooids are replaced by their buds reaching adulthood. the period of time between two tos is referred to as blastogenetic cycle. during the to, cells of adult zooid tissues die by apoptosis and are cleared by circulating phagocytes that, in turn, undergo phagocytosis-induced apoptosis and are cleared by new phagocytes in a recurrent, apparently endless, process. in the present work, we demonstrate that phagocytes, after the engulfment of effete phagocytes enter a senescence status and home, in the following mid-cycle, in the ventral islands, on both sides of the endostyle, where undifferentiated (stem) cells are also found. senescent cells remain in the homing site at both sides of the endostyle and contribute to form the dark masses representing the remains of the previous zooid generations. identification of a lps induced chemo attractive peptide from ciona robusta v longo1, a longo1, a martorana2, a lauria2, a azzolina1, m cervello1 and p colombo1 1institute for biomedical research and innovation, national research council, palermo, italy 2department of biological, chemical, and pharmaceutical sciences and technologies “stebicef”, university of palermo, palermo, italy previously published work demonstrated that in ciona robusta, the lps-induced inflammatory response leads to the overexpression of a truncated form of an immune-related mrna by means of a cr-apa whose biological activity has not be investigated so far. by 3d in silico modelling we observed a certain degree of homology to a protein domain of the human cancer-related signaling adaptor protein ct10 regulator of kinase (crk), a vertebrate gene that has been demonstrated to induce cytoskeletal reorganization during cell migration. starting from this observation, we postulated its possible involvement in cell migration mechanisms upon lps challenge. we decided to study the biological function of the derived peptide, named crcp (ciona robusta chemoattractive peptide) using a primary epidermal human cell line (hude). to evaluate the potential cytotoxic effect of crcp peptide on hude cells, mts assays were performed using different concentration of crcp peptide (from 200 nm to 3.2 μm) for 24 h. data show that crcp peptide do not impair the cell viability of hude cells. then, the crcp peptide (400 nm) was used in in vitro assays to study its involvement in cell motility events. by means of a wound healing and boyden chamber we demonstrated that the crcp peptide can induced hude motility (p = 0.0277). furthermore, by real time pcr, we investigated whether the crcp modulated the expression of genes involved in cell motility pathways. hude cells treated for 16 h with the peptide showed a statistical significant increase in matrix metalloproteinase-7 (mmp-7) (p = 0.0431) mrna expression and a significant reduction in ecadherin (cdh1) levels (p = 0.0277), compared to control cells. finally, western blot analysis demonstrated that the treatment with 400 nm crcp induced activation of nf-κb signaling pathway. in 23 conclusion we demonstrate that the lps-induced peptide crcp is capable of inducing cell migration of a human primary dermal cell line via a nf-κbdependent mechanism, leading to the modulation of the expression of relevant genes involved in reepithelialization restoring tissue architectures probably trough the loss of cell-cell contact allowing a migratory phenotype. tlr4 present and future perspective c rossetti department of medicine and surgery, university of insubria, varese, italy the mammalian tlrs act as key molecular patterns pathogenic associated (pamp) sensors, such as bacterial lps, lipopeptides and flagellins, which are present in microbial cells but not in host cells. it was therefore considered that the tlrs played a central role in the discrimination between "self" and "non-self". however, since the discovery of their microbial ligands, many studies have shown that even molecules derived from the host can act as tlr4 agonists. these endogenous tlr4 ligands tend to fall into the categories of released intracellular proteins, ecm components, oxidatively modified lipids and other soluble mediators. this review aims to summarize the evidence supporting the intrinsic capacity of tlr4 stimulation in some of these proposed endogenous ligands and showing an activity different from the tlr4 receptor agonism. 75 isj 17: 75-89, 2020 issn 1824-307x research report long-term and comparative impacts of combined sewers and municipal effluents to freshwater mussels c andré1, m-a vaudreuil2, s vo duy2, s sauvé2, f gagné1* 1aquatic contaminants research division, environment and climate change canada, 105 mcgill, montréal, québec, canada 2chemistry department, montreal university, montréal, québec, h2v 2b8, canada this is an open access article published under the cc by license accepted may 1, 2020 abstract excess rainfall events could lead to overflows and combined sewer overflows, which could threaten local mussel populations. this study sought to compare the longterm effects of combined sewer overflows and treated municipal effluents in caged elliptio complanata mussels. mussels were caged at 2 overflow sites, one downstream site of a major municipal effluent dispersion plume and a reference upstream site for 3 months during the summer. at the end of the exposure period, mussels were collected, analyzed for municipal contaminants (including pharmaceuticals), and effects biomarkers based on endocrine disruption (vitellogenin expression), xenobiotic detoxification (glutathione s-transferase and metallothioneins), oxidative stress/inflammation (cyclooxygenase and lipid peroxidation) and dna damage. the data revealed that surface waters contained less pharmaceutical products than the downstream site but atrazine and its metabolite were at higher levels in overflow sites. mussels contained elevated amounts of total heterotrophic bacteria, caffeine, acebutolol and venlafaxine at the downstream site relative to the upstream site where caffeine was higher at one of the overflow site. the levels of vitellogenin gene expression were significantly increased in both sexes of mussels caged at the downstream site only. multivariate analysis revealed that the biomarker responses were completely separated between upstream, overflow and downstream sites. the site discrimination was based on vitellogenin, metallothioneins, dna damage in gonad and digestive gland, gonad lipids/proteins reserves, lipid peroxidation, gonado-somatic index and condition factor. adverse outcome pathways analysis using the power law approach revealed that most changes in the biomarkers identified by discriminant function analysis were significantly scaled to gonad energy reserves, tissue/ mussel size and loss of weight following air emersion stress. in conclusion, the toxic effects of mussels caged at overflow sites generally displayed lower responses than mussels caged downstream a treated municipal effluent suggesting that these overflows pose a lower risk than the continuous exposure to treated municipal effluent. key words: elliptio complanata; combined sewers; municipal effluent; vitellogenin; oxidative stress introduction freshwater mussels constitute an important group of the invertebrate community and are ubiquitous in many lakes and rivers. because of their sessile lifestyle and high water filtering capacity for feeding, these organisms are susceptible to local sources of pollution such as municipal effluents and agriculture runoffs, which can be exacerbated by heavy rainfall events. this ___________________________________________________________________________ corresponding author: francois gagnè aquatic contaminants research division environment and climate change canada 105 mcgill, montréal, québec, canada e-mail: francois.gagne@canada.ca situation is likely to worsen by global warming which could bring low water levels thereby increasing the concentration of contaminants and drought punctuated by periods of intense precipitation (min et al., 2011). important rainfall events albeit occurring for short periods (hours to days) could overwhelm the wastewater treatment plants capacity to handle excess rain and sometimes combines with untreated wastewater before discharging in the nearby water bodies. from a wastewater risk management perspective, the comparative and cumulative effects of combined sewers effluent following rain events and the continuous release of treated municipal effluents should be better understood. https://creativecommons.org/licenses/by/4.0/legalcode 76 table 1 physico-chemical characteristics and occurrence of pharmaceutical products in surface waters parameter upstream ovf1 ovf2 downstream rainfall events (mm)1 open time (min)2 249 - 249 719 249 860 249 - ph 8.38 8.4 8.45 8.39 conductivity 291 ± 10 292 ± 25 283 ± 20 296 ± 20 tsm (mg/l) 9 ± 1 13 ± 1 3 ± 0.5 85 ± 10* doc (mg/l) 2.7 ± 0.5 2.8 ± 0.5 2.9 ± 0.5 3.4 ± 0.5 ammonia (mg/l) 0.005 ± 0.001 0.01 ± 0.001 0.004 ± 0.001 0.1 ± 0.01* heterotrophic bacteria mussels (counts/mussels) 30000 ± 1400 22000 ± 1500 27200 ± 1500 80800 ± 2000* caffeine –water (ng/l) 0.074 ± 0.067 0.039 ± 0.004* 0.034 ± 0.014* 0.864 ± 0.006* mussels (ng/g) 4.2 ± 2 3.5 ± 1 3.7 ± 1.6 4 ± 1.5 acebutolol-water (ng/l) 0.26 ± 0.005 0.43 ± 0.01 0.28 ± 0.04 2.7 ± 0.13* mussels (ng/g) 0.01 ± 0.007 0.01 ± 0.007 0.005 ± 0.003 0.06 ± 0.03* venlafaxine-water (ng/l) 1 ± 0.05 1.8 ± 0.03 1.2 ± 0.03 7.7 ± 0.3* -mussels (ng/g) 1 ± 0.6 1.1 ± 0.3 0.9 ± 0.4 2 ± 0.9 desvenlafaxine-water (ng/l) -mussels (ng/g) 4.2 ± 0.25 6.5 ± 4 7.5 ± 2 4.7 ± 3 5 ± 2 4.8 ± 3 16+2* 4.1 ± 2 ibuprofen-water (ng/l) -mussels (ng/g) 6 ± 0.9 <lod 7 ± 0.026 <lod 6.7 ± 0.95 <lod 81 ± 2* <lod estrone-water (ng/l) <lod <lod 0.45 ± 0.04* <lod <lod <lod 0.56 ± 0.05* <lod atrazine-water (ng/l) 58+0.9 <lod 82 ± 2* <lod 56 ± 0.2 <lod 59 ± 0.8 <lod desethylatrazine-water (ng/l) 64 ± 1 <lod 89 ± 1.3* <lod 60 ± 0.9 <lod 48 ± 5 <lod 1 sum of rainfall received in this area from july to september 2016 (mm). 2 valve open time of rainfall overflows (min). the star symbol * indicates significance from the upstream site (p < 0.05) the freshwater mussel elliptio complanata (e. complanata) is commonly found in lakes and rivers of the eastern part of north america (downing and downing, 1992). it is one of the most abundant species of freshwater mussels in the saintlawrence river system and was chosen as a representative bivalve of the invertebrate community to study the cumulative impacts of water quality and quantity of urban and agricultural areas. this species could live more than 10 years and are sexually dimorphic with distinct male and female gonads with low occurrence of intersex (van der shalie, 1970). the synthesis of vitellogenin, the egg yolk proteins for the developing embryo, occurs in follicular tissues in gonads tissues (pipe, 1987) and recent studies have shown that long-term exposure to estrogens and municipal effluents could lead to vitellogenesis and feminization (gagné et al., 2011). 77 indeed, wild e. complanata mussels collected downstream the municipal effluent plume had 85 % of females compared to upstream sites where males and females gonad expressed vitellogeninlike proteins in male individuals. exposure of caged e. complanata mussels for one year to a municipal effluent dispersion plume increased the proportion of females from 40 % to 70 % and vitellogenin-like proteins were increased in both sexes. long-term exposure to estrogens also induced vitellogenin in both males and females oysters (andrew et al., 2010). these effects are reversible when the estrogenic stimulus is stopped and from the capacity of mussels to change sex over its lifetime. cities are known to discharge a plethora of contaminants through discharges of treated wastewaters and combined sewer overflows following heavy rainfall events (holeton et al., 2011; haji-mohamad et al., 2014). these overflows also contain large amounts of contaminants of various industrial and consumer products origins such as caffeine, acetaminophen, deet, atenolol, progesterone and the persistent drug carbamazepine (haji-mohamad et al., 2014). because these rainfall overflows occur only during short periods of time following intense precipitations, it is difficult to compare the long-term ecotoxicological impacts of these overflows with the continuous release of treated municipal effluents. however, in the context of global warming, the increase of warmer temperatures (low water levels) punctuated with strong precipitations will led to a situation where mussels have to endure long periods of drought (low water levels with sites exposed directly to air) with increased output of rainfall overflows. the long-term consequence of these stressors are largely ununderstood at the present time. urban pollution are well known to produce many other ecotoxic effects to fish and invertebrates besides endocrine disruption (lacaze et al., 2013; marcogliese et al., 2014; petrie et al., 2015). municipal effluents are known to cause oxidative stress, inflammation, dna damage/decreased dna repair, reduced immunocompetence and neurotoxicity to invertebrates (gust et al., 2013; lacaze et al., 2013; gagné et al., 2015). these effects arise from exposure to treated effluents suggesting that many contaminants still passes through the wastewater treatment system. this could contribute to the observed general declines in mussels populations nearby urban and agriculture areas (lydeard et al., 2004; nobles and zhang, 2015). the purpose of this study was therefore to compare the long-term effects of combined sewers overflows and municipal effluents to freshwater e. complanata mussels. mussels were caged at sites downstream combined sewer overflows and the corresponding treated effluent for 3 months during the summer where water fluxes and quality are quickly changing. toxicity was examined at different scales i.e., the biochemical levels, energy reserves, tissue weights, mussel size and general health status in the attempt to understand not only the mode of action of these urban contaminants but to better understand adverse outcomes in individuals. an attempt was made to highlight toxic profiles associated to combined sewer overflows and treated municipal effluents in resident mussels. this information is of importance to decision making whether increasing wastewater overflows retention capacity or optimizing wastewater treatments should be prioritized together or separated. weight loss survival condition factor ups ovfl1 ovfl2 downs sites 0,0 0,5 1,0 1,5 2,0 r e la ti v e t o u p s tr e a m s it e * fig. 1 morphological parameters and air survival of caged mussels. the data for condition factor (mussel weight/shell length), air survival (days) and weight loss at time of death in air (%) were normalized to the upstream site. the data represent the mean with standard error. the star symbol * indicates significance at p > 0.05 78 table 2 correlation analysis of biomarker data significant correlations are highlighted in bold. abreviations: condition factor: mussel weight/shell length (cf), air survival (air), weight loss at time of death during the air survival test (wl), dna breaks in digestive gland (dnadg), digestive gland lpo (lpodg), arachidonate cyclooxygenase (cox), gst activity (gst), metallothioneins (mt), gonado-somatic index (gsi), gonad proteins (prot), gonad dna breaks or lpo (dnag, lpog), alkali-labile phosphates (alp), total heterotrophic bacteria (bact), vitellogenin gene expression (vtg), gst gene expression (gstmrna) materials and methods mussels collection and handling elliptio complanata mussels were collected in a pristine lake i.e., under no direct contact of anthropogenic activity in the laurentians (québec, canada) in the first week of june 2016. mussels were transported in a humidified container (wet paper towels with lake water) at 4 °c. at the laboratory, mussels were placed in aquariums containing uv-treated and charcoal-filtered tap water at 15 °c for at least 15 days. they were fed three times a week in the morning with a commercial feed of phytoplankton (phytoplex®) and laboratory-cultured pseudokirchneriella subcapitata algae (50-100 billion algae in 60 l aquarium). mussels (40 individuals per cage) were then placed in cylindric (1 m long x 0.5 m diameter) polyethylene nets (1 cm diameter mesh) attached to a 1 kg cement block. they were immersed at least in 1 m depth at 2 rainfall overflow sites: overflow site 1 (ovf1; 45°38'26.3"n; 73°29'15.6"w) and overflow site 2 (ovf2; 45°36'05.2"n; 73°30'33.6"w). one upstream (1.5 km) site of a major municipal effluent dispersion plume (45°39'28,5"n; 73°28'37,9"w), considered the reference site, and one downstream site located 8 km downstream the municipal effluent plume (45°44'23,9"n; 73°25'43,9"w). this site was previously shown to induce vitellogenin-like proteins in gonads and mussel feminization after one year exposure (blaise et al., 2003). hence, the study comprised 4 sites: one upstream site, one downstream site in the municipal effluent dispersion plume and 2 rainfall overflows. the mussels remained at these sites for 3 months during julyseptember of 2016. the cages were inspected every two weeks to ensure no important mussel mortality or cage disturbances. no significant mortality was observed between the sites. at the end of the exposure period, mussels were transported back to the laboratory and allowed to depurate overnight to rinse the mussel’s shells and tissues and empty the gut contents. a group of 10 mussels per site and surface waters (4l) at the time of cage retrieval were retained for chemical analysis of surface waters and mussel tissues using high performance liquid chromatography mass spectrometry (boisvert et al., 2012; darwano et al., 2014). the following compounds were determined in surface waters and mussels: methotrexate, carbamazepine, estriol, estradiol, estrone, ethinylestradiol, norethindrone, levonorgestrel, testosterone, progesterone, atrenogest, caddeine, acebutolol, (des)venlafaxine, atrazine (and its metabolite desehtylatrazine), sulfamethoxazole, diclofenac, clarithromycine, ibuprofen and carbamazepine. another group of mussels (10) were set aside to determine the stress on stress test (air surivival) as general indicator of mussel health. the mussels were placed in individual plastic cups and held at 20 °c in air saturated at 80 % humidity in an incubator. mussels weights were measured each day and death was measured based on shell opening after handling. the time of death were expressed as days and the percentage of weight loss were determined as follows: 100 x (weight beginning-weight at death /weight beginning). basic surface water characteristics were measured at the 4 sites after one month of cage deployment: ph, conductivity, total suspended solids, dissolved organic carbon and ammonia levels (awwa, 2017). lc-ms/ms analysis a method involving on-line solid-phase extraction (on-line spe) coupled to ultra-high performance liquid chromatography tandem mass spectrometry (uhplc-ms/ms thermo tsq 79 quantiva) was used to analyse the targeted contaminants. the on-line enrichment was performed on a thermo hypersil gold aq spe column, while compound separation was performed on a thermo hypersil gold analytical column. water samples were passed through glass fiber membrane filters for particle removal. an exact volume of 5 ml was transferred to an amber glass vial and isotope-labeled internal standards were added. a sample volume of 2 ml was injected to the on-line spe – uhplc/ms/ms workflow; negative mode and positive mode analytes were analysed within the same run using a heated electrospray ionization source operated in polarityswitching mode. further details can be found in the related analytical method developments (montielleon et al., 2018; goeury et al. 2019). the method has been validated for both wastewater and surface water matrices and the different criteria (recoveries, accuracy, precision and linearity) were compliant with us epa guidelines (us epa, 2007). each site was sampled in duplicate and an aliquot from each bottle was also analysed in duplicate; the values reported thus account for sampling and analytical variation. lod’s for targeted analytes in the water samples are in the order to low ng/l (or sub ng/l). for solid samples (mussel tissues), an solvent extraction method using ultrasonication was used, followed by a quechers clean-up protocol adapted from previous work (martinez bueno et al. 2013). freeze-dried samples were ground until homogenized. water and acetonitrile were added along with various quechers salts (na2so4, nacl, na3cit:2h2o and na2hcit:3h2o) and the tube was vortex-mixed. after extraction, a centrifugation proceeded and a fraction of the supernatant was transferred to a second tube containing clean up reagents (na2so4, psa, c18 and formic acid). 1 ml of supernatant was collected and evaporated to dryness, reconstituted in injection solvent, and analyzed by uhplc-ms/ms using a similar method as that for water samples, but using a lower injection volume and without on-line spe. wholemethod recoveries were between 60 and 120%. lod’s in mussel samples were in the order of pg/g to ng/g. a b female male ups ovfl1 ovfl2 downs sites -400 -100 200 500 800 1 100 1 400 1 700 2 000 v t g m r n a /r e fe re n c e g e n e * * female male ups ovfl1 ovfl2 downs sites 0,0 0,5 1,0 1,5 a lk a li -l a b il e p h o s p h a te s * * c gsi dgi ups ovfl1 ovfl2 downs sites 0,5 1,0 1,5 2,0 2,5 in d e x v a lu e * * fig. 2 gametogenesis in caged mussels exposed to rainfall overflows and municipal effluent. the levels of vtg mrna (a), alkali-labile phosphates (b) and the digestive gland index (dgi)/ gonado-somatic index gsi (c) were determined in mussels. the star symbol * indicates significance from the upstream site 80 biomarkers analyses a sample of 12 mussels per site were randomly selected, weighted, measured and the digestive gland and gonad were dissected on ice. mussels and tissue weights were determined and the shell length recorded. for each mussel, the digestive gland and gonad were dissected out and the gonad tissues were separated in 2 portions: one preserved for rna extraction (rnalater) for vitellogenin gene expression analysis and the other portion for biomarker analyses. the tissues were thawed on ice for 30 min and 4 volumes of the buffer containing 140 mm nacl containing 10 mm tris-acetate, ph 8, 1 mm edta, 1 mm kh2po4, 0,1 mm dithiothreitol and 1 µg/ml apoprotinin were added. tissue homogenates were prepared using a teflon pestle tissue grinder (5 passses) and a portion was centrifuged at 12000 x g for 30 min at 2 °c. the supernatant (s12 fraction) was collected for mt, gst and cox activity assessments. the samples were stored at -85 °c until analysis. energy reserves in the gonad homogenates were determined by determining total proteins, sugars and lipids. total proteins were determined according to the procedure of bradford (1976) using serum bovine albumin for quantitation. the homogenate was diluted 1/20 in 10 mm naoh and incubated for 1 hour at room temperature to allow protein denaturation. then, 5 µl of the dilution was tested for proteins in 200 µl in clear microplates. the data was expressed as mg proteins/g gonad or digestive gland and normalized against the upstream site which is the reference site in the present field study. total sugars were determined in the gonad according to the anthrone reaction (jermyn, 1975). the reaction was adapted to 96well microplates for rapidity and reduction of reagents (waste). glucose standards were used for calibration and the data were expressed as μg sugars or glucose equivalents/gonad weight and normalized against the upstream site. total lipids were also determined in the gonad according to the phosphovanilate reaction (frings et al., 1972) which was also adapted to 96-well microplates. standard solutions of triton x-100 were used for calibration. the data were expressed as μg of lipid equivalent/ gonad weight and normalized with the upstream (reference) site. the levels of vitellogenin-like proteins were also determined according to the alkali-labile phosphate in acetone-fractionated proteins with slight modifications (gagné, 2014a). the high molecular weight proteins were precipitated in 30 % acetone and the protein pellet were washed once in 5 0% acetone (and recentrifuged at 10000 x g for 5 min) before the naoh addition step. standards of rainbow trout vitellogenin and inorganic phosphate were prepared for quantitation and determined by the phosphomolybdate procedure (stanton, 1968). data were expressed as μg of alkali-labile phosphate/mg protein in gonad and normalized against the upstream site in females and males. lipid peroxidation (lpo) in gill and gonad tissues was determined according to the thiobarbituric acid methodology (wills, 1987). the assay was also adapted to 96-well microplate where 10 μl of homogenates were mixed with 90 μl of water, 50 μl of 10 % trichloroacetic acid containing 1 mmfeso4, and 100 μl of thiobarbituric acid (0.7 %). the mixture was heated for 70–75 °c for 5 min and allowed to cool down at room temperature. fluorescence was measured with a microplate reader at 540 nm excitation and 600 nm emission (synergy-4, biotek instruments, usa). standards of tetramethoxypropane (stabilized form of malonaldehyde) were used for calibration. the data were expressed as μg of thiobarbituric acid reactants per mg of total proteins and normalized against the upstream (reference) site. the levels of sugars lipids proteins ups ovfl1 ovfl2 downs sites 0 1 2 3 n o rm a li z e d v a lu e ( u p s tr e a m ) * * fig. 3 energy reserves in mussel gonad tissues. the levels of proteins, lipids and carbohydrates were determined in gonads. the data represent the mean with standard error. the star symbol * indicates significance from the upstream site 81 dna strand breaks were determined in the homogenates by the alkaline dna precipitation assay as described previously (debenest et al., 2012). the data were expressed as µg dna strand breaks/mg proteins in gonad or digestive gland and normalized against the upstream site. the levels of metallothioneins (mt) were determined in the digestive gland using the silver saturation assay using non-radioactive silver (gagné, 2014b). briefly, the s12 fraction was incubated with 2 mg/l of ag+ at ph 8.5 in 100 mm glycine for 15 min and the excess silver and heat sensitive proteins were removed by two additions of hemoglobin followed by heat denaturation. the remaining silver was determined by graphite furnace atomic absorption spectrometry with standards of rabbit mt and silver nitrate for calibration. a ratio of 17 moles of ag/mole of mt standard was used for validation. the data were expressed as µg mt equivalents/mg proteins and normalized against the upstream site. cox and gst activities were also determined in the s12 fraction by fluorescence and absorbance-based assays in 96-well microplates as already described (gagné et al., 2007). the enzyme activities were expressed as substrate change/min/mg proteins in the gonad or the digestive gland and normalized against the upstream site. gst and vitellogenin gene expression were determined in e. complanata using the recently developed transcriptomics data in gene bank (project id prjna575711). the primers used were as follows, for gst: forward primer 5'-tacccaggtctttcggtttcc3', reverse primer 5'-ccttcaccgcctcagttaca-3' and for vitellogenin: forward primer 5’gtgtcctggggctttatgct-3’ and reverse primer 5’gcgtttcatcattggggtgg-3’. total rna was extracted in the gonad after sexing the mussels using the commercial rneasy plus mini kit (quiagen, canada). rna concentration (a260) and purity (a260/a280) were estimated by uv scanning between 220-320 nm using the nanodrop 1000 instrument (thermo fisher scientific, on, canada) and rna integrity was checked by electrophoresis (experion™ automated electrophoresis system, bio-rad, on, canada). synthesis of cdna was produced also with a commercial kit (quantitect® reverse transcription kit, qiagen, canada). the cdna samples served as the template dna for amplification using quantitative real-time polymerase chain reaction (qpcr). the qpcr analyses were performed using ssofast™ evagreen® supermix and cfx96 real-time pcr detection system (bio-rad, mississauga, on, canada). calibration was achieved using serial dilutions of cdna (10 ng) with amplification efficiency values between 96 % and 102 %. the amplification reactions were carried out in the following conditions: 5 ng cdna, 6.5 µl of 2×sofast evagreen supermix (bio-rad), 300 nm of each primer and depc treated water (ambion) up to a total volume of 13 µl. cycling temperatures were 95 °c for 30 sec, then 39 cycles of 95 °c for 5 sec and 60 °c for 10 sec. amplification specificity was verified using melting curve analysis. three reference genes were tested: ribosomal 18s, 60s ribosomal protein l8 and hypoxanthine-guanine phosphoribosyltransferase and the one showing the least variance between samples was selected for normalization. the cq values (the cycle with a significant increase in fluorescence) and the cq for the reference gene was used for comparison between sites. the gene expression data was also normalized to the upstream site responses for intersite comparisons. data analysis the mussels were randomly collected using a shell length range between 5-8 cm to minimize outliers to ensure an homogenous distribution of mussel size across the sites. the data were analyzed for normality and homogeneity of variance using the shapiro-wilks and bartlett tests. in the case that the data were homogenously distributed and normal, the data was subjected to an analysis of variance (anova) followed by the least square difference (lsd) test to highlight significant changes relative to the upstream (reference) site in this caging experiment in the saint-lawrence river. in the case that the data were not homogenous or deviated from normality, the data was analyzed using rank anova followed by the conower-inman non-parametric test for comparisons relative to the upstream site. correlations between the data were determined by the pearson-moment test. principal component and discriminant function analyses were also performed to determine the most important biomarkers that could identify specifically rainfall overflow and downstream sites. the propagation of effects at different scales (molecular responses, energy reserves, organ and body sizes) was studied using the power law paradigm (west et al., 2002) to find potential adverse outcome pathways. the power law is defined by y(x) = cxa or log y(x) = alogx + log c where a is the scaling exponent and c a constant, y the biomarker responses at a lower scale and x the corresponding biomarker at a higher scale. the significance of the scaling exponent a was determined by linear regression on the log transformed data where only significant slopes were considered. for example, the expression of lpo in gonad (y) is examined in terms of lipids levels in the gonad or gonad size (x) using the above power relationship. significance was set at p<0.05 and all tests were performed using the systat software package (version 13, usa). results the surface waters physico-chemical characteristics and occurrence of some pharmaceuticals were determined in surface waters and mussels at the end of exposure time (table 1). during the 3 months exposure period, the sites received 290 mm of rainfall and the release valves at the overflow sites were opened for 719 and 860 min which represented 0.7 % of total time. the total suspended solids (tss) and ammonia were circa 10 times higher at the downstream site compared to the upstream and rainfall overflows sites. the water ph, conductivity and dissolved organic carbon (doc) were not significantly different. mussels accumulated significant amounts of total heterotrophic bacteria (includes coliforms) at the downstream site with 80800 counts/mussel compared to 30000 counts/mussels at the upstream 82 ups ovfl1 ovfl2 downs sites 0,00 0,75 1,50 2,25 3,00 m t ( n g /m g p ro te in s ) * mrna activity ups ovfl1 ovfl2 downs sites 0 1 2 3 g s t ( re la ti v e t o u p s tr e a m s it e ) * fig. 4 metal sequestration and xenobiotic biotransformation activity of mussels exposed to municipal effluent plume and rainfall overflows sites. xenobiotic biotransformation where determined by following mt levels (a) and gst at the enzyme and mrna levels (b) in the digestive gland. the star symbol* indicates significance at p<0.05 site. water caffeine levels at the downstream site (0.9 pg/l) were significantly higher than the upstream (0.07 pg/l) and rainfall overflows sites (0.035 pg/l), respectively. however, no significant changes in caffeine contents in mussels were observed. acebutolol levels in surface waters were circa 10 times higher at the downstream site (0.9 pg/l) compared to the upstream and rainfall overflows sites. mussels accumulated more acebutolol at the downstream (0.06 ng/g) compared to the upstream (0.01 ng/g) site. venlafaxine levels were also elevated at the downstream sites reaching 8 pg/l compared to the other sites. the mussels did not significantly accumulate more of the drug at the downstream site compared to the other sites. the levels in ibuprofen and estrone were also significantly increased in surface waters at the downstream sites compared to the upstream site. however, their levels in mussel tissues remained below the method reporting limit. the condition factor and resistance to air emersion were not significantly affected in cages mussels (figure 1). however, the weight loss was significantly lower in mussel caged at the downstream site which suggests that mussels were less able to loss weight during the air challenge. correlation analysis (table 2) revealed that weight loss was significantly correlated with bacterial a) b) 83 ups ovfl1 ovfl2 downs sites 5 000 10 000 15 000 20 000 25 000 c o x a c ti v it y ( f lu o /m in /m g p ro te iin s ) gonad digestive gland ups ovfl1 ovfl2 downs sites 0 1 2 3 4 l p o ( re la ti v e u p s tr e a m s it e ) * * fig. 5 oxidative stress and damage in mussels exposed to municipal effluent plume and rainfall overflows sites. cox activity (a) and lpo levels (b) were determined in caged mussels. the data represent the mean with standard deviation. the star symbol * indicates significance from the upstream site loadings (r = -0.70). gonad activity in caged mussels was studied by following changes in vtg gene expression, alp (vitellogenin-like proteitn) and gsi. mussels caged at the downstream site of a municipal effluent had elevated levels of vtg gene expression in both males and females (figure 2a). the rainfall overflows had no effects. the levels of alp revealed that only females had elevated levels at the downstream site and females at ovfl2 had decreased levels compared to females at the upstream site (figure 2b). mussels caged at the downstream site had significantly higher levels of gsi compared to the upstream site (figure 2c). correlation analysis revealed that vtg gene expression was correlated with condition factor (r = -0.51), weight loss during air survival (r = -0.56) a) b) 84 and bacterial loadings (r = 0.63) indicating that mussels weight/shell length and bacterial loadings tended to be higher at the downstream site were less able to lose weight during air challenge. the dgi was significantly correlated with the cf (r = 0.73). energy reserves in gonad tissues were also determined in caged mussels (figure 3). total proteins and lipids were significantly elevated in the gonad of mussels caged at the downstream site. no change was observed in mussels caged at the rainfall overflow sites. correlation analysis revealed that gonad proteins were correlated with bacteria levels (r = 0.9), weight loss (r = -0.85), which was correlated with total gonad lipids (r = -0.54). gonad lipids were significantly correlated with total gonad sugars (r = 0.84), bacteria levels (r = 0.57) and vtg gene expression (r = 0.9). gonad sugars were correlated with vtg gene expression (r = 0.68). xenobiotic biotransformation was determined by following changes in mt levels and gst enzyme activity and mrna levels. mt levels in the digestive gland were significantly increased at the downstream site compared to the upstream site but not in mussels exposed to the rainfall overflow sites (figure 4a). correlation analysis revealed that mt levels were significantly correlated with gonad proteins (r = -0.57) and bacteria levels (r = -0.58). the activity of gst in the digestive gland was also significantly increased at the downstream site only compared to upstream site (figure 4b). gst mrna levels were not affected in mussels. correlation analysis revealed that mt levels were correlated with total proteins in the gonad (r = -0.57) and bacterial loadings (r = -0.58). gst enzyme activity was correlated with total proteins in the digestive gland (r = 0.68). oxidative stress was determined by following changes in cox activity and lpo in tissues of caged mussels. cox activity was not significantly influenced by the sites in respect to the upstream site (figure 5a). the lpo levels were significantly increased and decreased at the downstream site in the gonad and digestive gland respectively (figure 5b). no changes were observed in mussels caged at the rainfall overflow sites. correlation analysis revealed that gonad lpo was significantly correlated with bacteria levels (r = 0.70) and gonad protein levels (r = 0.65). dna strand breaks were also determined in tissues. dna strand breaks were reduced in the digestive gland in mussels caged at one overflow site and reduced in the gonad of mussels caged at the downstream site (figure 6). a decreased in dna strands indicates that dna repair activity and turnover are reduced. correlation analysis revealed that dna strand breaks in the digestive gland were significantly correlated with lpo in the digestive gland (r = 0.65), gonad proteins (r = -0.68), bacteria levels (r = -0.54) and weight loss during air emersion (r = 0.64). dna breaks in gonad were significantly correlated with gonad proteins (r = -0.7), mt in the digestive gland (r = 0.74) and vtg gene expression (r = -0.52). in the attempt to gain a global understanding on the various responses in mussels caged at a downstream and rainfall overflow sites, a multivariate and effects scaling analyses were performed to determine the major biomarkers than gonad digestive gland ups ovfl1 ovfl2 downs sites 0 1 2 3 d n a b re a k s ( re la ti v e t o u p s tr e a m ) * * fig. 6 dna damage in mussels exposed to rainfall overflows and a municipal effluent plume. dna strand breaks were determined in the digestive gland and gonad tissues in mussels. the data represent the mean with standard deviation. the star symbol * indicates significance from the upstream site can discriminate between downstream and rainfall overflow sites and adverse outcome pathways, respectively. principal components revealed that 60% of the variance was explained by 3 factors (figure 7 a). the most important biomarkers (biomarkers with the highest factorial weights) associated to these 3 factors were gonad lipids, dna strands in digestive gland and gonads, vtg gene expression, mt, gonad lpo, gonad proteins, gsi and cf. discriminant function analysis was used to determine if the downstream, rainfall overflows and upstream sites have distinct toxicity profiles and which effects were involved for site discrimination (figure 7b). the analysis revealed that the downstream, overflows and upstream sites were completely distinct (100 % classification efficiency) from each other. the biomarkers involved for site discrimination were gst activity, air survival time, lpo and dna damage in the digestive gland, protein gonads and gsi. in the attempt to determine whether effects observed at the molecular level are manifest at higher levels of complexity (adverse outcome pathways), the biomarker data were analyzed using the power law paradigm as described in methods (table 3). the analysis revealed that some biochemical responses were significantly scaled to effects at higher levels of biological organization. mt, gonad lpo, gst activity, apa in both tissues, vtg gene expression and alp levels were significantly scaled to energy reserves in the gonad (proteins, sugars and lipids). 85 factor loadings plot -1,0 -0,5 0,0 0,5 1,0 fac tor( 1) -1 ,0 -0 ,5 0 ,0 0 ,5 1 ,0 factor(2) -0,5 0,0 0,5 1,0 f a c to r( 3 ) cf air wl dnadg lpodg cox gst mt prot dnag lpog lipids sugars alp vtg gstmrna gsi (lip>dnadg>vtg) (prot>mt>lpogon) (gsi>cf>dnagon) canonical scores plot ups ovfl downs -8 -3 2 7 factor(1) -8 -3 2 7 f a c t o r (2 ) component 1 (gst>air surv>lpodg) c o m p o n e n t 2 ( d n a d g > p ro tg o n > g s i) fig. 7 multivariate analysis of biomarker data. biomarker responses were analyzed using factorial (a) and discriminant function (b) analyses. digestive gland lpo (lpodg), air survival (air surv), proteins in gonads (protgon) a subgroup of these biomarkers was also scaled at higher levels: digestive gland lpo (to weight loss), gonad lpo (to digestive gland and gonad size), gst activity (to gonad size), gst mrna (to soft tissues mass) and vtg gene expression (to shell length). cox activity was also associated to weight loss during air stress. the scaling analysis also revealed that changes occurring at the energy reserves levels were significantly scaled to changes at higher biological level as well. indeed, gonad sugars levels were associated to mussel size and condition factor. gonad size was significantly scaled to mussel weight, shell length and condition factor and to soft tissues weight/mussel weight ratio. based on this analysis, an adverse outcome pathway is proposed as follows. the effects observed at the biochemical levels resonate at energy stores especially at total proteins and lipids levels, which in turn is associated to gonad weight level and it is the changes in gonad size that is associated to mussel size, condition factor and general health indicators (air survival and weight loss). a) b) 86 table 3 scaling of effects analysis for adverse outcome pathways identification 1. the annotation y → x (h scaling exponent) is modeled using the power law scaling of effects: y = kxh where y is an effect at a lower level of complexity and x the effect observed at higher level of complexity (usually body mass, organ size or health index). the scaling exponent h is calculated the log transformation where log y = h log x + log k (a constant). only significant slopes were used discussion the effective protection of water bodies in urban areas requires knowledge about the relative contamination loads and toxic effects of municipal wastewaters discharges and rainfall overflows. for example, a predictive model was developed to predict changes in chemical oxygen demand and total suspended solids based on the water depth and maximum intensity of rainfall (brzesinska et al., 2018). in the present study, the surface waters at the overflow sites contained significantly higher levels of caffeine, estrone, atrazine and its metabolite desethylatrazine relative to upstream waters, which suggests that these sites were partially contaminated by wastewaters. these levels were somewhat lower than the surface waters at the downstream site the municipal effluent plume (8 km) with the exception of atrazine and its metabolite, which were highest at the overflow sites. the levels of pesticides represented an important component of hazardous substances from urban wet weather discharges (gosset et al., 2017). it appears that stormwater is an important input of pesticides and pharmaceutical products in aquatic ecosystems downstream urban area. this suggests that the combined sewer overflows are not simply a diluted version of municipal effluent by stormwater. based on the effects measured in this study, only decreased dna strand breaks were significantly influenced in one of the overflow sites compared to the upstream site which suggests that long-term exposure to an episodic release of combined sewer during the summer months do not produce toxic effects as observed in mussels downstream the municipal plume. this was shown by discriminant function analysis which showed that the responses pattern of the overflow sites where closer to the upstream site compared to the downstream site (figure 7b). it will be interesting to study this further under different rainfall volume scenario over time. however, other effects were also involved to discriminate the overflow sites from the upstream and downstream sites: dna strand breaks in digestive gland, gonad proteins and gsi. decreased dna strand breaks (repair activity) was also observed in quagga mussels exposed to combined sewer discharges during a major release of untreated wastewaters (gagné et al., 2017). chronic toxicity testing of combined sewer overflows following rainfall events revealed some effects towards fish pimephales promelas and daphnids 87 cerodaphnia dubia (gooré et al., 2015). exposure to the combined sewer overflows for 6 and 7 days resulted in decreased survival and reproduction in daphnids at 31% v/v of the effluent and growth and survival in fish at 41% v/v effluent volume. although a continuous exposure a 6-7 days exposure to the combined sewer overflows is unlikely especially in dynamic river systems i.e., intense rainfalls rarely occurs continuously for long periods of time and the released effluents will be diluted by the river’s currents which is also increased during rain. this approach could be of value from the perspective that toxic combined sewer overflows should not be released directly in the receiving water to begin with based on the precautionary principle. combined sewer overflows are environmental source of hormones and other micropollutants (phillips et al., 2012). the study revealed that concentrations of estrogens (estradiol-17β), androgens (testosterone) and other contaminants such as caffeine and coprostanol could reach 10 times the concentrations found in effluent after treatment. however, this discharge is restricted in time compared to the continuous release of effluents but contribute to the global contamination picture of the urban space. in the present study, the overflows valves were open for only 860 min over the 3-month exposure period representing about 0.6 % of the total time. estrone was detected at similar concentrations in surface waters at 0.45 and 0.56 ng/l in one of the overflow site and downstream site but vitellogenin gene expression and alp levels (a measure of estrogenicity) were significantly increased at the downstream site only. at the overflow site, mussels were exposed to estrone only 0.6% of the times while at the downstream site, exposure to the effluents was continuous (all the times) albeit the levels could have change during the exposure period. this was similar with heterotrophic bacteria in mussel’s tissues, which was 3.3 times higher in tissues in mussels caged at the downstream site than in tissues of mussels from the upstream site albeit both combined sewers overflow and this primary treated municipal effluents contains bacteria (gibson et al., 2017). it was noteworthy that total bacteria count in mussels were strongly correlated with proteins in gonads and vtg gene expression (table 2) suggesting that other factors than estrogenicity was at play in caged mussels. vtg is usually expressed following the activation of vtg receptors by estrogens but recent studies revealed that vtg could be involved in the immune response against foreign bacteria (bouchard et al., 2009; li et al., 2019). vtg was shown to possess bacteriostatic properties, which could assist the phagocytes to remove bacteria in the hemolymph and tissues. adverse outcomes analysis based on power law scaling revealed that biochemical changes was involved at changes at higher scales (levels of biological organization) from energy reserves in gonads up to mussel condition, size and health status. this is consistent to the observation that local mussel populations are scarce downstream municipal effluent in this study based on field observations. this was also shown in previous study in another river system in canada (gillis et al., 2017). mussels surveys in the grand river (ontario, canada) revealed that seven species of mussels were found at sites upstream of municipal effluent discharges while no live mussels were found for 7 km downstream a municipal effluent discharges. this suggests that the continuous release of municipal effluents could produce harmful effects to mussels, which threaten maintenance of local populations. this is consistent with a previous study with e. complanata mussels where mussels collected downstream to cities in the mille-isles river (québec, canada) were feminized at 85 % with males showing increased vitellogenin-like proteins (gagné et al., 2011). clams and mussels were caged upstream and downstream a tertiary treated municipal effluent for 72 days in a small stream to find changes in survival, growth and condition (nobles and zhang, 2015). survival and growth to the non-native asian clam and growth and condition of three ridge mussel species were significantly lower in caged animals at downstream site of the wastewater treatment plant compared to mussels caged upstream. they also found an absence of native mussels at downstream site further supporting that municipal effluents have negative impacts to mussel abundance and diversity. it was found that corbicula fluminea clams accumulate rapidly pharmaceuticals at downstream sites of wastewater treatment plants (burk et al., 2019). indeed, a 42 days in situ exposure with caged clams, the following compounds were detected in tissues: acetaminophen, caffeine, carbamazepine, dilitiazem, diphenhydrame, fluoxetine, norfluoxetine, sertraline, desmethylsertraline and methylphenidate. sertraline had the highest values at 341 ng/g tissues. this drug is a selective serotonin reuptake inhibitor which could stimulate spawning in mussels (ram et al., 1993). in addition to serotonin, exposure of gonad tissues to a filtered (0.2 um membrane) municipal effluent from the same city in the present study also stimulated spawning in e. complanata mussels showing the serotonergic effects of this municipal effluent (gagné et al., 2004). this effect was measured at a threshold concentration of 3 % corresponding to a distance of 4-6 km downstream the plume which is close to the 8 km downstream site in this study. moreover, exposure for 45 days of mussels exposed to a static-renewal test to the same municipal effluent in this study lead to increased serotonin and dopamine in mussel gonad tissues with an increase in monoamine oxidase activity, an enzyme involved in the elimination of serotonin and dopamine. the increase levels in serotonergic drugs such as sertraline, venlafaxine and perhaps other (fluoxetine, paroxetine) is consistent with the reported serotonergic properties of the primary-treated municipal effluent. the increase levels in mt and gst in the digestive gland suggests that perhaps other compounds than pharmaceuticals (metals and polyaromatic hydrocarbons) were at play. the increased levels in mt and gst activity were also observed in e. complanata exposed to the same municipal effluent for 7 days in the laboratory (gagné et al., 2015). mt and gst could also be involved in the removal of reactive oxygen species leading to oxidative stress 88 and damage (lpo) (giannetto et al., 2017). an analysis of covariance was performed with mt or gst levels with site location and lpo as the grouping variable and covariate respectively and revealed that mt was mostly explained by oxidative stress not by sites. lpo levels had no significant effects on gst activity in the digestive gland. this suggests that mt was also involved in oxidative stress in mussels exposed to a municipal effluent as suggested previously (gagné et al., 2007) and gst was more associated to xenobiotic conjugation than oxidative stress. in conclusion, the study revealed that mussels caged 8 km downstream site of municipal effluent plume led to increased accumulation of heterotrophic bacteria, acetobutolol and somewhat with venlafaxine in tissues and produced a number of effects at the biochemical level such as endocrine disruption (vitellogenin), oxidative stress (lpo and mt), xenobiotic biotransformation (gst) and dna damage. these effects were also scaled at higher levels of biological organization such as gonad energy reserves, tissue and mussel size and health status as determined by resistance to air and weight loss before death 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(eds.), biochemical toxicology: a practical approach. oxford university press, oxford, uk, pp. 127–152, 1987. https://www.ncbi.nlm.nih.gov/pubmed/?term=gibson%20j%5bauthor%5d&cauthor=true&cauthor_uid=29124707 https://www.ncbi.nlm.nih.gov/pubmed/?term=drake%20j%5bauthor%5d&cauthor=true&cauthor_uid=29124707 https://www.ncbi.nlm.nih.gov/pubmed/?term=karney%20b%5bauthor%5d&cauthor=true&cauthor_uid=29124707 https://www.ncbi.nlm.nih.gov/pubmed/29124707 https://www.ncbi.nlm.nih.gov/pubmed/25315929 https://www.ncbi.nlm.nih.gov/pubmed/23333517 https://www.ncbi.nlm.nih.gov/pubmed/23333517 https://www.ncbi.nlm.nih.gov/pubmed/23333517 https://www.ncbi.nlm.nih.gov/pubmed/23333517 https://www.ncbi.nlm.nih.gov/pubmed/23333517 https://www.ncbi.nlm.nih.gov/pubmed/?term=aboulfadl%20k%5bauthor%5d&cauthor=true&cauthor_uid=25189851 https://www.ncbi.nlm.nih.gov/pubmed/?term=darwano%20h%5bauthor%5d&cauthor=true&cauthor_uid=25189851 https://www.ncbi.nlm.nih.gov/pubmed/?term=madoux-humery%20as%5bauthor%5d&cauthor=true&cauthor_uid=25189851 https://www.ncbi.nlm.nih.gov/pubmed/?term=gu%c3%a9rineau%20h%5bauthor%5d&cauthor=true&cauthor_uid=25189851 https://www.ncbi.nlm.nih.gov/pubmed/?term=sauv%c3%a9%20s%5bauthor%5d&cauthor=true&cauthor_uid=25189851 https://www.ncbi.nlm.nih.gov/pubmed/25189851 https://www.ncbi.nlm.nih.gov/pubmed/25189851 https://www.ncbi.nlm.nih.gov/pubmed/?term=nobles%20t%5bauthor%5d&cauthor=true&cauthor_uid=26042840 https://www.ncbi.nlm.nih.gov/pubmed/?term=zhang%20y%5bauthor%5d&cauthor=true&cauthor_uid=26042840 https://www.ncbi.nlm.nih.gov/pubmed/26042840 https://www.ncbi.nlm.nih.gov/pubmed/?term=phillips%20pj%5bauthor%5d&cauthor=true&cauthor_uid=22540536 https://www.ncbi.nlm.nih.gov/pubmed/?term=chalmers%20at%5bauthor%5d&cauthor=true&cauthor_uid=22540536 https://www.ncbi.nlm.nih.gov/pubmed/?term=gray%20jl%5bauthor%5d&cauthor=true&cauthor_uid=22540536 https://www.ncbi.nlm.nih.gov/pubmed/?term=kolpin%20dw%5bauthor%5d&cauthor=true&cauthor_uid=22540536 https://www.ncbi.nlm.nih.gov/pubmed/?term=foreman%20wt%5bauthor%5d&cauthor=true&cauthor_uid=22540536 https://www.ncbi.nlm.nih.gov/pubmed/?term=wall%20gr%5bauthor%5d&cauthor=true&cauthor_uid=22540536 https://www.ncbi.nlm.nih.gov/pubmed/22540536 https://www.ncbi.nlm.nih.gov/pubmed/?term=ram%20jl%5bauthor%5d&cauthor=true&cauthor_uid=8468545 https://www.ncbi.nlm.nih.gov/pubmed/?term=crawford%20gw%5bauthor%5d&cauthor=true&cauthor_uid=8468545 https://www.ncbi.nlm.nih.gov/pubmed/?term=walker%20ju%5bauthor%5d&cauthor=true&cauthor_uid=8468545 https://www.ncbi.nlm.nih.gov/pubmed/?term=mojares%20jj%5bauthor%5d&cauthor=true&cauthor_uid=8468545 https://www.ncbi.nlm.nih.gov/pubmed/?term=patel%20n%5bauthor%5d&cauthor=true&cauthor_uid=8468545 https://www.ncbi.nlm.nih.gov/pubmed/?term=fong%20pp%5bauthor%5d&cauthor=true&cauthor_uid=8468545 https://www.ncbi.nlm.nih.gov/pubmed/?term=kyozuka%20k%5bauthor%5d&cauthor=true&cauthor_uid=8468545 https://www.ncbi.nlm.nih.gov/pubmed/8468545 https://www.ncbi.nlm.nih.gov/pubmed/8468545 327 isj 15: 327-337, 2018 issn 1824-307x research report biochemical basis of conventional and novel mode of action insecticides resistance in field population of diaphorina citri collected from southern punjab, pakistan a naeem, s freed* department of entomology, faculty of agricultural sciences and technology, bahauddin zakariya university, multan, pakistan accepted august 19, 2018 abstract diaphorina citri kuwayama is a phloem-feeding pest of citrus worldwide and vectors the putative causal agent of citrus greening, “candidatus liberibacter asiaticus” (clas). baseline toxicity and activities of esterases (est), and glutathione-s-transferases (gst) sensitivity to bifenthrin, chlorpyrifos, thiamethoxam, acetamiprid, nitenpyram, chlorfenapyr and imidacloprid were established with field populations of d. citri. the highest calculated toxicity was observed in thiamethoxam treated d. citri with lc50 of 4.6 μg/ml and minimum in chlorpyrifos and imidacloprid with lc50 values of 96.9 and 91.9 μg/ml, respectively. the est activity recorded in d. citri treated with different insecticides ranged from 11.8 to 52.1 µmol/min/mg of protein, while gst activity ranged from 10.9 to 51.3 µmol/min/mg of protein which subsequently linked with insecticide resistance. the present results provide a specific baseline to study levels of detoxifying enzymes against commonly used insecticides in d. citri that’s indirectly correlated with level of resistance. key words: insecticides; hlb; esterase; gst; resistance; citrus psylla; detoxification introduction diaphorina citri kuwayama (hemiptera: sternorrhyncha: liviidae), asian citrus psyllid (burckhardt and ouvrard, 2012), a worldwide pest of citrus (sapindales: rutaceae) (halbert and manjunath, 2004; bové, 2006) and primarily important due to its role as fastidious vector of candidatus (ca.) liberibacter (l.) africanus, ca. l. asiaticus., and ca. l. americanus associated with “citrus greening” also known as asian huanglongbing (hlb) (pelz-stelinski et al., 2010; boina and bloomquist, 2015). citrus trees infected by this disease produce small, discolored, lopsided and misshapen fruit over a shortened life span (bové, 2006; gottwald et al., 2012; graftoncardwell et al., 2013) and infected trees can cause 33% yield loss of citrus fruits (salcedo et al., 2016). hlb management programs include the removal of infected trees, use of disease-free nursery stock and psyllid vector control by pesticide sprays (belasque et al., 2010; tiwari et al., 2010; graftoncardwell et al., 2013). ___________________________________________________________________________ corresponding author: shoaib freed faculty of agricultural sciences and technology bahauddin zakariya university multan, pakistan e-mail: sfareed@bzu.edu.pk the injudicious use of different insecticides puts d. citri under pressure and the insects use different mechanism (increased detoxification, reduced penetration, alterations of target sites and behavioral adaptations) for evolving resistance (brattsten et al., 1986; chen et al., 2006; sétamou et al., 2010) that may be found among d. citri populations. the enzymatic mechanism involves increased activities of esterases (est) and glutathione-s-transferases (gst) (hemingway et al., 2004; tiwari et al., 2011a,b,c). a positive correlation between level of insecticide resistance and detoxifying enzymes has already been documented in several insect pests (scott, 1999; maymo et al., 2002; srigiriraju et al., 2009). the insecticide resistance management involves reducing the frequency of resistance alleles within a field population so that the efficacy of a specific insecticide can be preserved (georghiou, 1994). est are detoxifying enzymes produced through biotransformation of xenobiotics and directly linked to insecticides resistance (devorshak and roe, 1998) est are known to detoxify organophosphate insecticides by hydrolyzing the ester moiety (oakeshott et al., 2005) and are studied in several insects like culex quinquefasciatus (diptera: culicidae) (sahgal et al., 1994), blattella germanica (blattodea: ectobiidae) (anspaugh et al., 1994), and 328 table 1 list of insecticides used for bioassays trichoplusia ni (lepidoptera: noctuidae) (ishaaya and casida, 1980). similarly, increased level of gst is correlated with insecticide resistance (li et al., 2007). the enhanced oxidative and hydrolytic enzyme activities of gst expression have been associated with organophosphate and synthetic pyrethroid resistance in musca domestica (diptera: muscidae) (clark et al., 1984; tang et al., 1990); helicoverpa armigera (lepidoptera: noctuidae) (srinivas et al., 2004), and spodoptera litura (lepidoptera: noctuidae) (armes et al., 1997; kranthi et al., 2001). the understanding of baseline activity of enzymes is necessary to suggest better management tactics and to minimize insecticide resistance in d. citri (boina et al., 2011; tiwari et al., 2011a). the present study intended to examine the activities of est and gst after application of conventional and noval mode of action insecticides in field populations of d. citri so that better management program involving insecticides can be devised. materials and methods insects the adult d. citri were collected from citrus orchard of khanewal, punjab, pakistan through an aspirator and released on small citrus plants in plexiglas cages 40×40×40 cm under laboratory conditions. insects were reared in laboratory of insect microbiology, department of entomology, faculty of agricultural sciences and technology, bahauddin zakariya university, multan, pakistan until specific numbers were obtained to be used in bioassays. the rearing conditions were maintained at 27 ± 2 ºc and 60-65% relative humidity with a (12: 12h) light: dark photoperiod. insecticides seven insecticides belonging to different toxicological groups were chosen on the basis of their current use in field (table 1). insecticidal bioassays the leaf dip method was used to determine the toxicity of different insecticides against d. citri. serial dilutions of insecticides with selected concentrations (through hit and trial method) were formed for bioassay with distilled water (naeem et al., 2016). fresh shoots/flushes of citrus were exposed for about 10 sec with different insecticide dilutions and were allowed to dry for 1 h at room temperature, while distilled water was used to treat control insects instead of insecticide dilution. in each treatment fifteen (5-7 days old) adult psyllids were released on treated citrus flushes within plastic jars (9×6×6 cm) covered with net from two sides for good aeration. each treatment was replicated three times under similar conditions as described earlier. the mortality was recorded after 48, 72 and 96 h for conventional, neonicotinoid and pyrrole insecticides, respectively. the treatment samples for further biochemical assay were collected after 24, 48 and 72 h during bioassay. biochemical assays subsequent to the insecticidal bioassay, collected alive adult d. citri of different treatment levels and control were processed for further biochemical assay. firstly, three to five psyllids were taken from each treatment and crushed by using the 0.15 m nacl in the eppendorf tube. for centrifugation the final volume was made 400 μl. the samples were spun at 3000 rpm for 10 mins at 5 ± 1 °c using the centrifuge machine (z216mk, hermle labortechnik, germany) to obtain the supernatants. the supernatants (d. citri homogenate) were further used to determine est and gst activities. determining the activities of est and gst determination of protein contents the protein contents were determined in d. citri homogenates through bradford (1976) procedure, using bovine serum albumin as standard at 25 °c with a spectrophotometer (uv3000, o.r.i. germany). insecticide trade name and manufacturer target site of action group of insecticide chlorpyrifos (lorsban®, 400 ec; dow agro sciences) acetyl cholinesterase inhibitors organophosphates bifenthrin (talstar®,10 ec; fmc) sodium channel modulators pyrethrins imidacloprid (confidor®, 20 sl; bayer crop sciences) agonists of nicotinic acetylcholine receptor neonicotinoids acetamiprid (mospilan®, 20 sp; arysta life sciences) agonists of nicotinic acetylcholine receptor neonicotinoids thiamethoxam (actara®, 25w g; syngenta) agonists of nicotinic acetylcholine receptor neonicotinoids nitenpyram (paranol®, 10 ec; kanzo, agrochemicals) agonists of nicotinic acetylcholine receptor neonicotinoids chlorfenapyr (pirate®, 360 sc; basf chemicals and polymers) uncouplers of oxidative phosphorylation via disruption of the proton gradient pyrroles 329 table 2 toxicity of conventional and novel mode of action insecticides against d. citri insecticide lc50 (μgml−1) (fd) slope χ2 df pa nb chlorpyrifos 91.88 (62.67-144.85) 1.35 ± 0.25 2.722 4 0.61 270 bifenthrin 40.14 (23.05-80.21) 0.95 ± 0.23 0.231 4 0.99 270 imidacloprid 96.91 (49.17-487.05) 0.70 ± 0.22 1.218 4 0.88 270 acetamiprid 67.42 (43.13-135.74) 1.18 ± 0.26 0.024 4 1.00 270 thiamethoxam 4.64 (3.156.61) 1.25 ± 0.22 1.195 4 0.88 270 nitenpyram 6.10 (4.259.67) 1.25 ± 0.23 1.599 4 0.81 270 chlorfenapyr 23.99 (16.58-37.89) 1.21 ± 0.22 0.363 4 0.99 270 ap-values are based on chi-square goodness of fit test. p values > 0.05 suggest goodness of fit of the model bnumber of d. citri used in the bioassay, including the control est activity est activity was measured by using 1 mm 4nitrophenyl acetate as a substrate (damayanthi and karunaratne, 2010). in each replicate, 90 µl of phosphate buffer (pbs, ph 6.5) and 100 µl of 0.6 m ana (or bna) was added to 10 µl of d. citri homogenate. after 30 min incubation, 100 µl of fast garnett bc solution was added to stop the reaction. the concentration of the final product was determined at 405 nm as an endpoint calculated from standard curves of aand b-naphtol, respectively. gst activity the gst activity was determined according to caballero et al. (2008). for this purpose, 1cdnb (chloro-2, 4-dinitrobenzene) (1 mm) was used as a substrate in ultraviolet (uv) semi-micro cuvettes (4 ml) with 5 mm reduced glutathione, 0.1 m tris buffer ph 8 and 30 µl of d. citri homogenates. enzyme activity was determined by monitoring continuous changes in absorbance at 340 nm for 4 min at 25 °c with a spectrophotometer. statistical analysis the mortality data was evaluated by probit analysis (finney, 1971) using the software package epa probit analysis version 1.5. the enzymatic activities were analyzed through split plot design. significance was accepted at α = 0.05. the enzymatic activities in d. citri homogenates were expressed by plotting graphs using graph prismpad 6.02. results effect of insecticides on d. citri the toxicity of two groups of insecticides (conventional and novel mode of action) was evaluated against d. citri and their lc50 are given in table 2. the highest percentage mortalities by chlorpyrifos, bifenthrin, imidacloprid, acetamiprid, thiamethoxam, nitenpyram and chlorfenapyr were 75.0, 66.0, 62.0, 59.0, 82.0, 73.0 and 71.0, respectively at their highest concentrations μgml−1 (table 3). however, thiamethoxam with lowest lc50 value of 4.6 (3.2-6.6) μgml−1 proved to be most potential insecticide against d. citri as compared to imidacloprid with lc50 value of 96.9 (49.2-487.1) μgml−1, chlorpyrifos 91.9 (62.7-144.8) μgml−1, acetamiprid 67.4 (43.1-135.7) μgml−1, bifenthrin 40.1 (23.0-80.2) μgml−1, chlorfenapyr 23.9 (16.637.9) μgml−1 and nitenpyram 6.10 (4.2-9.7) μgml−1, respectively. est activity activity of est in d. citri after the exposure to conventional insecticides in case of conventional insecticides bifenthrin showed maximum inhibition of est i.e., (3.3 ± 0.7) µmol/min/mg of protein (f= 278.0, df= 5, p= 0.0000) observed after 48 h of treatment, while chlorpyrifos (34.6 ± 2.3) µmol/min/mg of protein (f= 2118.7, df= 5, p= 0.0000) expressed highest est activity at 200 μgml−1 after 24 h (fig. 1). activity of est in d. citri after the exposure to novel mode of action insecticides the maximum inhibition of est was expressed in chlorfenapyr treated d. citri (2.8 ± 0.4) µmol/min/mg of protein (f= 391.6, df= 5, p= 0.0000), while maximum est activity was recorded in nitenpyram (52.1 ± 5.8) µmol/min/mg of protein (f= 45.2, df= 5, p= 0.0000) followed by imidacloprid, chlorfenapyr, thiamethoxam and acetamiprid. in case of imidacloprid maximum activity (24.4 ± 1.9) µmol/min/mg of protein (f= 703.2, df= 5, p= 0.0000) was expressed in 24 h treatment as compared to 48 h and 72 h. in case of acetamiprid highest activity (11.8 ± 1.5) µmol/min/mg of protein (f= 368.5, df= 5, p= 0.0000) was observed after 48 h and lowest in 72 h post treatment. on the other hand, in case of nitenpyram, chlorfenapyr and thiamethoxam highest activity (12.9 ± 1.5) µmol/min/mg of protein (f= 110.7, df= 5, p= 0.0000) was expressed in 24 h than 48 h and 72 h samples (fig. 1). 330 table 3 percent mortalities of d. citri against conventional and novel mode of action insecticides insecticides concentration (μgml−1) %mortality f-value p-value lsd chlorpyrifos 200 75.5 ± 8.4 a 24 0.000 23.9 100.0 44.4 ± 1.9 b 50.0 40.0 ± 3.3 bc 25.0 26.6± 3.3 bcd 12.5 17.7 ± 1.9 cd control 4.4 ± 1.9 d bifenthrin 100 66.6 ± 3.3 a 41.1 0.000 16.7 50 57.7 ± 3.8 ab 25 46.6 ± 3.3 b 12.5 28.8 ± 1.9 c 6.2 24.4 ± 1.9 c control 6.6 ± 1.9 d imidacloprid 150 62.2 ± 1.9 a 26.8 0.000 18.3 75 44.4 ± 3.8 ab 37.5 37.7 ± 3.8 b 18.7 28.8 ± 1.9 b 9.4 28.8 ± 5.1 b control 2.2 ± 1.9 c acetamiprid 100 59.9 ± 3.3 a 28.5 0.000 18.3 50 46.6 ± 5.7 ab 25 33.3 ± 3.3 bc 12.5 22.2 ± 1.9 cd 6.2 15.5 ± 1.9 cd control 4.4 ± 1.9 d thiamethoxam 20 82.2 ± 3.8 a 45.4 0.000 20.2 10 66.6 ± 5.7 ab 5 46.6 ± 3.3 bc 2.5 31.1 ± 3.8 c 1.2 28.8 ± 1.9 c control 2.2 ± 1.9 d nitenpyram 14 73.3 ± 8.4 a 31.9 0.000 21.1 7 46.6 ± 1.9 b 3.5 40.0 ± 3.3 bc 1.7 22.2 ± 3.3 cd 0.8 17.7 ± 1.9 de control 2.2 ± 1.9 d chlorfenapyr 60 71.1 ± 5.1 a 50.3 0.000 16.7 30 53.3 ± 3.3 b 15 39.9 ± 3.3 bc 7.5 26.6 ± 0.0 cd 3.7 17.7 ± 1.9 de control 2.2 ± 1.9 e gst activity activity of gst in d. citri after the exposure of conventional insecticides in case of conventional insecticides, maximum inhibition of gst i.e., (5.3 ± 0.9 µmol/min/mg) of protein was due to chlorpyrifos at 200 μgml−1 after 72 h of treatment (f= 2196.4, df= 5, p= 0.0000), while bifenthrin expressed highest gst activity (47.6 ± 3.6 µmol/min/mg of protein) (f= 122.1, df= 5, p= 0.0000) at 100 μgml−1, 24 h post treatment (fig. 2). activity of gst in d. citri after exposure of novel mode of action insecticides chlorfenapyr expressed maximum gst inhibition (2.7 ± 1.6 µmol/min/mg of protein) (f= 220.1, df= 5, p= 0.0000) followed by thiamethoxam and acetamiprid. in contrast, nitenpyram expressed maximum gst activity. the gst activities of novel mode of action insecticides were as follows: imidacloprid (10.9 ± 2.5 µmol/min/mg of protein), acetamiprid (24.2 ± 1.9 µmol/min/mg of protein) (f= 38.6, df= 5, p= 0.0000), nitenpyram 331 fig. 1 est specific activity expressed as mean in bars followed by the same letter within columns indicate no significant difference (p≤ 0.05) in a tukey’s test after the exposure of different insecticides in d. citri. the line in the bars represents the mean standard error 332 fig. 2 gst specific activity expressed as mean in bars followed by the same letter within columns indicate no significant difference (p≤ 0.05) in a tukey’s test after the exposure of different insecticides in d. citri. the line in the bars represents the mean standard error 333 fig. 3 linear regression analysis showing the relation between percent mortality and est enzymatic expression with respect to different hours (p≤ 0.05) (51.3 ± 1.7 µmol/min/mg of protein) (f= 95.7, df= 5, p= 0.0000), thiamethoxam (16.9 ± 2.6 µmol/min/mg of protein) and chlorfenapyr (14.0 ± 1.6 µmol/min/mg of protein) after 24 h of treatment (fig. 2). relation between percent mortality of d. citri and enzymatic expression the relation between percent mortality and est (fig. 3) and gst (fig. 4) was expressed by drawing regression lines activity in d. citri. the difference between the slopes showed significant values. discussion asian hlb has grown into a great risk to the world citrus industry (bové, 2006; hall et al., 2013) as the vector d. citri has developed resistance to a wide range of insecticides (naeem et al., 2016; chen and stelinski, 2017). detoxification enzymes like est and gst are described as the defense mechanism against foreign toxic compounds (li and liu, 2007) and play important roles in maintaining the normal physiological activities in the insect’s body (chen et al., 2017). for example, due to neurotoxic action of insecticides, insects release the 334 fig. 4 linear regression analysis showing the relation between percent mortality and gst enzymatic expression with respect to different hours (p≤ 0.05) detoxifying enzymes which correlate with insecticide resistance (bilal et al., 2018). the biochemical mechanisms of insecticide resistance in d. citri have received significant recent attention (liu et al., 2016; chen et al., 2018). tiwari et al. (2011a) reported high levels of detoxifying enzymes activity at higher toxicity level of insecticide (thiamethoxam, imidaclorpid, bifenthrin, chlorpyrifos and fenpropathrin) in d. citri populations. similarly, our result showed higher enzymatic function at higher toxicity level of insecticides as compared to control d. citri population. our results suggested that d. citri showed maximum est (52.1 ± 5.8 µmol/min/mg of protein) after 24 h of exposure to nitenpyram. in relation with percent mortality 73.3% regression showed highly significant correlation with est activity after 24 h of exposure (f= 124.3, p= 0.0004) however, our results indicated that acetamiprid (11.8 ± 1.5) 335 µmol/min/mg of protein induced reduced activity of est whereas, imidacloprid (10.9 ± 2.5) µmol/min/mg of protein induced a reduction in activity of gst. results declared some inner connection between enzyme activity, time, percent mortality and concentrations of insecticide. for example, in case of est activity for acetamiprid, chlorfenpyr and thiamethoxam slope was positively related with percent mortalities at specific time i.e., 24 h, 48 h and 72 h, respectively as compared to other insecticides. its means that increase in est enzyme release was directly linked with percent mortality, which might be a reason for resistance development at specific time of exposure of insecticide. whereas, gst had significantly negative slope between percent mortality and enzyme activity for all insecticides with respect to time. suppression of detoxification enzymes indicates that these enzymes play no role in the detoxification of tested compounds and may increase the susceptibility of insect pests to these insecticides (abd-elaziz and el-siad, 2009). previous studies revealed that gst enzymes contribute to pyrethroid resistance (grant and matsumura, 1989; tiwari et al., 2011a, c). our results revealed that bifenthrin expressed (47.6 ± 3.6) µmol/min/mg of protein gst activity which coincides with previous study. in contrast, bifenthrin revealed very low est activity i.e., 3.3 ± 0.7 µmol/min/mg of protein. current research showed that in vivo, metabolism of imidacloprid changed to the main metabolite 4/5-hydroxyimidacloprid, a product of oxidative degradation, in honeybee apis mellifera (suchail et al., 2004). similarly, the main metabolite of imidacloprid i.e., 5hydroxyimidacloprid was detected in imidaclopridresistant strains of bemisia tabaci (rauch and nauen, 2003). parallel to previous results of our studies, imidacloprid showed est and gst activities i.e., 24.4 ± 1.9 and 10.9 ± 2.5 µmol/min/mg of protein, which subsequently linked with imidacloprid resistance. in future, there is crucial need to reduce p450 activity which increase the susceptibility to imidacloprid through silencing of cyp4 genes in adult d. citri (killiny et al., 2014). insecticide resistance management becomes a serious threat due to insecticide failures in the field. by monitoring detoxifying enzymes of currently used insecticides we practiced a strategy to obtain optimal resistance management. use of enzyme inhibitors, such as piperonyl butoxide, diethyl maleate and triphenyl phosphate, for cytochrome p450, gst and carboxylesterase, respectively may be useful in cases where increased enzymatic detoxification is contributing to resistance (terriere, 1984; ishaaya, 1993). biochemical assays explain that the insensitivity of different insecticides is likely the biochemical basis of conventional and novel mode of action insecticides resistance in d. citri. a proactive approach has been taken to characterize the molecular and biochemical response of d. citri to insecticides for optimization of rotation schedules (tiwari et al., 2011a; coy et al., 2016). the data presented here on sensitivity of est and gst for inhibition and on specific activity of various detoxifying enzymes. the enzymatic levels for the tested insecticides were highly variables. the confirmation of resistance associated 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bombyx mori, in the aspects of structures and classification, functions and pathways. a brief discussion on the ilps from several other model insects, including drosophila melanogaster, aedes aegypti, and apis mellifera, was also included. key words: insects; insulin-like peptides; bombyx mori; bombyxin; signaling pathway introduction insulin is a well-known hormonal peptide in the human body that regulates carbohydrate metabolism, and malfunction in the regulation can result in type ii diabetes. there are also peptides that exhibit similar sequence and structural similarity to insulin, known as insulin-like peptides (ilps), which regulate many different biological processes and have certain physiological activities similar to that of insulin, but their secretion sites are different and possess distinct functions (okamoto et al., 2009). ilps are hallmarked by the presence of a conserved cysteine knot motif, and their precursor proteins share similar structure domains, i.e., signal peptide followed by the b, c, and a chain. ilps have in general about 50 to more than 200 amino acids in length, and are not only conversed in pretty much all vertebrates but also in most invertebrates and even unicellular organisms, and have thought to be emerged very early during the evolution of eukaryotes (souza and lópez, 2004). insects compose the largest and most diverse class in the arthropods and many are important economically and scientifically. starting 1960s, many insulin-like peptides (ilps) have been reported in insects, and the ilps from several model organism will be discussed in detail in this review. ilps have been demonstrated as key regulators in ___________________________________________________________________________ corresponding authors: keping chen and feifei zhu school of life sciences jiangsu university zhenjiang, jiangsu, 212013, china e-mail: kpchen@ujs.edu.cn and feifzhu@ujs.edu.cn the animal body for growth and development, survival and proliferation. insulin-like peptides in other arthropods, such as crustaceans, have also been characterized and found exclusively produced by a male-specific androgenic gland (cronin, 1947). significant research has been carried out on these crustaceans, and proved that the ilps of these species were crucial in sexual differentiation and spermatogenesis, which have been discussed in detail elsewhere (abdu et al., 2002; aflalo et al., 2006; barki et al., 2006; ventura and sagi, 2012). a more distant invertebrate, the nematode caenorhabditis elegans encodes a remarkable 40 ilps in its genome (pierce et al., 2001). these ilps were found primarily expressed in the nervous system, and differs in disulfide bond arrangement (matsunaga et al., 2017; zheng et al., 2018). in this review, various aspects of insect ilps will be discussed, including classifications, structures, functions and pathways in several model insects, including bombyx mori, drosophila melanogaster, aedes aegypti, and apis mellifera, with a more detailed discussion into b. mori. classification of insulin-like peptides in b. mori insulin-like peptide in b. mori, also known as bombyxin, was first identified in 1984 by nagasawa, who found that a 19 amino acids long peptide at the n terminus of 4k-prothoracicotropic hormone (4kptth) was highly homologous to insulin and insulin-like growth factors in vertebrates. this was the first time that insulin-like peptide was identified in invertebrates (nagasawa et al., 1984). up to now, 187 a total of 38 bombyxins belonging to 12 families have been identified in b. mori (yoshida et al., 1998). among them, 32 bombyxin genes have been successfully cloned, and based on their sequence similarities, have been divided into 7 families, namely families a-g (ingvarsson et al., 1988; ikuyo et al., 1997; tsuzuki et al., 1997; iwami et al., 2008; iwami et al., 1990; kawakami et al., 1989; kondo et al., 1996). familes a, b and c contain 10, 12, and 6 bombyxin genes, respectively, and families d, e, f and g have only one bombyxin gene in each family (table 1) (ingvarsson et al., 1988). another 5 bombyxin families, namely families v, w, x, y and z, were predicted by aslam et al. through analysis of the b. mori genome and then confirmed experimentally (table 1). family v contains two bombyxin genes, and families w to z have one gene in each family (aslam et al., 2011). in 2009, okamoto et al. purified an 8 kda insulin-like peptide from the hemolymph of b. mori (okamoto et al., 2009). sequencing analysis showed that the peptide was more similar to insulinlike growth factors (igfs) of vertebrates than to bombyxin (okamoto et al., 2009). it was found that ecdysone could stimulate the secretion of the peptide and the secreted peptide could further promote the growth of specific tissues in the adult. therefore, this ilp peptide was also named b. mori insulin-like growth factor peptide (bigflp). both ilps and igfs have the conserved cysteine knot motif, but igfs hold additional domains, e.g., a c domain between the a and b chains, and/or d, e domains at the c terminus (lecroisey et al., 2015). spatial distribution of insulin-like peptides in b. mori early studies by northern blot hybridization could only detect bombyxins in the brain tissue of the silkworm, but later rt-pcr analysis showed that they could also be expressed in other tissues, such as ganglia, epidermis, testis, ovary, fat body, silk gland, malpighian tubule, midgut and hindgut of b. mori, although the expression levels were much lower than that in the brain (iwami et al., 1996). bombyxin families a-g, as well as family v and w, were mainly expressed in four pairs of central nerves in the brain of b. mori, while the expression in other tissues was very low (table 1) (iwami et al., 1996). family x was mainly found in the fat body, family y was mainly found in the brain and ovary at the larval stages, and family z was highly expressed in follicular cells (aslam et al., 2011). our group has previously characterized the bmilp gene in b. mori and found it has identical sequence to bombyxin z1 and had the highest expression level in the ovary tissue, especially in the mated females, suggesting a role of bmilp in the regulation of egg maturation (chen et al., 2016). the bilgfp peptide was mainly expressed in the fat body of b. mori from the pupal to adult stage (okamoto et al., 2009). the fat body tissue of b. mori is functionally equivalent to the liver and adipocytes of vertebrates, and the liver was the main place where mammals secreted igfs. bigflp was also found expressed in other tissues of b. mori, such as brain, ovary and testis. the median table 1 characteristics of bombyxin genes* bombyxin gene family number of genes chromosome location primary expression cites notes a 10 b1, b2, b4, a2-a4 clustered on an unknown chromosome; rest all on chromosome 11 brain a6 and/or a7 encodes bombyxin ii b 12 brain c 6 brain d 1 brain e 1 unknown brain encodes bombyxin iv f 1 chromosome 11 brain g 1 chromosome 11 v 2 chromosome 9 brain w 1 chromosome 1 brain x 1 chromosome 11 fat body y 1 chromosome 1 brain z 1 chromosome 1 follicular cells * data summarized based on reports from mizoguchi and okamoto, 2013, aslam et al., 2011, and iwami, 2000 188 fig. 1 structure of insulin and insulin-like peptides. (a) ilp maturation process and (a) alignment of representative insulin-like peptides from different species. grey, orange, blue and purple bars above or below and amino acid sequence indicate the peptide domains for signal peptide, b chain, c chain, and a chain, respectively. s-s indicate disulfite bonds formed between the cysteine sites. most species have more than one identified ilps, and only one or two of the ilps from one species was selected for alignment and comparison. mus musculus (np_032412.3), homo sapiens (np_001278826.1), bos taurus (np_001172055.1), anopheles gambiae (xp_314565.2), bombyx mori (xp_021202224.1 and xp_021202225.1), apis mellifera (np_001171374.1), aedes aegypti (xp_001657487.1), drosophila melanogaster (np_648359.1 and np_996037.2). conserved sites were colored according to amino acid hydrophobicity neurosecretory cells (mncs) in the brain have been identified to produce both bigflp and bombyxins, although the time of secretion varies between the two. in vitro experiments showed that the secretion of the bigflp gene in the ovary and testis was induced by ecdysone. the b. mori nephrocytes, which are functionally equivalent to glomerular podocytes of vertebrate kidneys, have been detected with high bigflp activity during the pupal stage, but the expression of bigflp gene was not detected. this indicated that the circulating bigflp was taken up and degraded by nephrocytes (okamoto et al., 2011). structures of ilp genes and proteins compared to ilps in higher animals, insect ilp genes are often intron-less (mizoguchi and okamoto, 2013). the human insulin gene contains two introns in two relatively conservative sites which are respectively located in 5’ utr and c-domain (steiner et al., 1985), though sometimes the first intron can be inefficiently spiced (wang et al., 1997). whereas, there are no introns for bombyxin families a-g, w, x, and y. two genes v1 and v2 in the family v were found to contain one intron in the utr of each gene, and the gene in family z1 contained two introns that are similar to those ilps found in vertebrates (aslam et al., 2011). in b. mori, 25 of ilp genes were found clustered on chromosome 11, and 3 were located on chromosome 1, 2 were located on chromosome 9, and 1 on chromosome 11, while the locations for the rest ilps have not been identified yet (table 1) (mizoguchi and okamoto, 2013). to now, five structural forms of bombyxin proteins have been isolated from the head of b. mori, namely, bombyxins i, ii, iii, iv and v (nagasawa et al., 1986; jhoti et al., 1987; maruyama et al., 1988). the amino acid sequences of bombyxins ii and iv have been fully identified, which are the products of the bombyxin a6/a7 and 189 the e1 genes, respectively (table 1). the structures of bombyxins i, iii and v have not been fully analyzed, and the bombyxin genes encoding them have not been revealed. the five protein structural forms all contain heterodimers of a a chain and a b chain, which have 50 % and 30 % amino acid similarity to those of human insulin, respectively. the a chain and b chain are linked by disulfide bonds within and between two chains in exactly the same way as insulin (figure 1a) (iwami, 2000). during translation, a bombyxin precursor, which is composed of a signal peptide, a b chain, a c chain, and a a chain successively from the n-terminal to the c-terminal end, will first be formed (iwami, 2000). after that, the mature peptide is formed by proteolytic cleavage of the signal peptide and the c chain, and thereby leaving it with a heterodimer structure linked by disulfide bonds (figure 1a). the newly discovered bigflp has a similar precursor to that of the bombyxin. however, when the bigflp matures, c chain is not completely excised, and part of the sequence is retained, while the signal peptide is completely excised, thus the bigflp peptide has a different structural form that contains the b, c, and a domains consecutively as a monomeric structure (figure 1a) (okamoto et al., 2009). from an evolutionary point of view, the amino acid sequence of the ilps can be very divergent between distant species, but all possess conserved cysteine sites that form interand intrachain disulfite bonds (figure 1b), indicating a common phylogenetic origin (mcrory and sherwood, 1997; jin chan and steiner, 2000). the overall three-dimensional structure of bombyxin is spherical, similar to that of human insulin, which will form a t shaped crystal in solution (jhoti et al., 1987; nagata et al., 1995b). although, the c terminal structure of the b chain in bombyxin ii is significantly different from that of human insulin. b chain extends in helical form, similar to that of relaxin, a protein hormone that belongs to the insulin superfamily, and this form is unnecessary for the activity of bombyxin. on the other hand, the b chain of human insulin extends in the form of sharp turn and beta strands, and is indispensable for its activity (nagata et al., 1995b; nagata et al., 1995a). therefore, bombyxin is more similar to relaxin in this regard, and human insulin may have evolved different receptor recognition sites at the c terminal of the b chain and thus distinguishes itself from bombyxin and relaxin. human insulin is composed of dimers and hexamers, however, bombyxin ii is unlikely to form dimers or hexamers by structural prediction. instead, there is a hydrophobic region on the surface of bombyxin ii (mizoguchi and okamoto, 2013), which may be important for the binding of bombyxin ii to other proteins. physiological functions: growth-promoting effect although many insulin-like peptide genes have been identified in the genome of invertebrates, knowledge about the physiological functions of these gene products is still limited. it was found that bombyxin could reduce the concentration of the major sugar content trehalose in the hemolymph of b. mori and the effect was dose-dependent. trehalose is a major carbohydrate in insects for energy storage. in the midgut and muscle of b. mori, bombyxin can enhance the activity of trehalase and help hydrolyze trehalose in the hemolymph into glucose, and facilitate the transport of the sugars to other tissues (satake et al., 1997; satake et al., 1999). similar to insulin secretion in mammals, glucose in the silkworm could also stimulate the release of bombyxin into the hemolymph, and the titer of bombyxin in the hemolymph would decrease after starvation treatment of the silkworm (masumura et al., 2000). bombyxin can also reduce the glycogen content in the fat body and improve the activity of glycogen phosphorylase. however, these stated functions of bombyxin only exerted at the larval stage of the silkworm, and bombyxin does not affect the concentration of glucose in the adult hemolymph (satake et al., 1997; satake et al., 1999). bombyxin has been reported to promote cell proliferation in the hematopoietic organ of the silkworm. when the hematopoietic organs of the silkworm larvae were cultured in vitro, the addition of hemolymph to the culture medium could significantly improve the cell proliferation (nakahara et al., 2003). studies indicated that this promoting effect was mainly attributed to bombyxin in the hemolymph. when the hematopoietic organs from the first day of the fifth instar larvae were cultured with bombyxin-ii for 48h, and the number of blood cells in the culture medium increased in a dosedependent manner (nakahara et al., 2006), indicating that bombyxin-ii could promote mitosis. in addition, when bombyxin was added to silkworm ovary culture, increased meiosis of the ovary cells were observed. however, this might be an indirect effect, because bombyxin can stimulate the ecdysone production in the ovary cells, and ecdysone has been reported to induce the meiosis of cells at low concentration (orikasa et al., 1993). in another study, when synthetic bombyxin-ii was added to the silkworm ovary culture, a series of morphological changes of the cells were induced (volkman and goldsmith, 1982). most of the cells became larger, round and aggregate into clusters. some other cell lines became attached tightly to the bottom of the culture flask to be fibrous and spindlelike (tanaka et al., 1995). bombyxin was supplemented exogenously to precis coenia and found to promote the growth of the wing imaginal disc (nijhout and grunert, 2002). when the wing imaginal disc of p. coenia larva was removed and cultured in a standard nutrient-rich tissue culture medium, the wing imaginal disc ceased to grow. however, when 20hydroxyecdysone and hemolymph from silkworm larva with an appropriate concentration were added to the tissue culture, the wing imaginal disc started its normal growth (nijhout and grunert, 2002). therefore, the promoting effect may come from some biological factors in the hemolymph. additionally, when 20-hydroxyecdysone and bombyxin-ii were added to the tissue culture medium, the division rate of cell nucleuses was increased. however, when bombyxin-ii was preincubated with bombyxin antibody, this growthpromoting effect was inhibited. similar results were 190 observed in manduca sexta (nijhout et al., 2007). this indicated that bombyxin acted as a growth promoting factor to wing imaginal discs of other insects (nijhout and grunert, 2002). bombyxin, together with 20-hydroxyecdysterone, played a synergistic role in promoting the growth of imaginal discs of insect species in a dose-dependent manner. this effect was different from promoting cell proliferation described above because here bombyxin relies on 20-hydroxyecdysone as a synergistic compound, while it can function independently to promote proliferation of silkworm hematopoietic and ovary cells. insulin-dependent signaling pathways in b. mori, bombyxin could regulate the molting process through the insulin signaling pathway. it was found that 24 h after the larvae were injected with bombyxin, silkworm molting were accelerated (gu et al., 2015). an insulin-dependent signaling pathway in insects for development and growth was drawn to figure 2. bombyxin stimulates insulin receptor and serine/threonine protein kinase (akt), which were then phosphorylated to activate downstream signal molecule target of rapamycin (tor). the phosphorylation of serine/threonine protein kinase depended on phosphorylation of phosphatidylinositol-3 kinase (pi3k), indicating that pi3k was the upstream signal molecule of akt and the activated tor continues to phosphorylate the translation initiation factors 4e-binding protein (4ebp) and p70 ribosomal protein s6 protein kinase (s6k) of its downstream signal molecules in eukaryotic cells and ultimately stimulate the molting of b. mori. meanwhile, bombyxin could inhibit the phosphorylation of amp-activated protein kinase (ampk), but this inhibition was not dependent on pi3k, indicating that ampk and pi3k were two different signal pathways of the downstream of insulin receptor. chemical activators of ampk could partially alleviate the inhibition of ampk phosphorylation by bombyxin, indicating that ampk is also involved in the tor signal pathway and that pi3k/akt and ampk are two different downstream signal pathways of the insulin receptor and eventually converged on the tor signal molecule to affect the molting of b. mori. in b. mori, serine/threonine protein kinase (akt) have been identified, which consists of 493 amino acid residues, including a pleckstrin homology (ph) domain, a kinase domain and phosphorylation site that could be activated by bombyxin (nagata et al., 2008). fig. 2 insulin-dependent signaling pathway in bombyx mori. “p” in the blue circle refers to phosphorylation, and red and blue ovals refer to proteins that are activated and inhibited, respectively. black arrow and red bar indicate activation and inhibition. pi3k, phosphatidylinositol-3 kinase; ampk, amp-activated protein kinase; akt, serine/threonine protein kinase; tor, target of rapamycin; 4e-bp, 4e-binding protein; s6k, p70 ribosomal protein s6 protein kinase 191 there is species-specific regulation of ecdysone secretion. in m. sexta, when insulin and bombyxin ii were used to activate the phosphorylation of insulin receptor and akt, no stimulation of ecdysone was observed (smith et al., 2014). likewise, when pi3k inhibiting factor was used to inhibit the phosphorylation of akt and 4e-bp, ecdysone secretion was not affected either. however, when extracellular signal regulated kinase (erk) phosphorylation was inhibited, ecdysone secretion would be inhibited significantly (smith et al., 2014). this indicated that ecdysis was mainly regulated through the erk signal pathway. in b. mori, bombyxin could not activate the phosphorylation of erk in prothoracic gland, but ptth would (gu et al., 2010; gu et al., 2013). when the phosphorylation of erk in b. mori was inhibited, it would not affect ecdysone secretion, but when the phosphorylation of akt and 4e-bp was inhibited, ecdysone secretion would be affected. this indicated that silkworm molting was mainly regulated through the pi3k/akt signal pathway (gu et al., 2015). insulin-like peptides in other insect species: the dipteran insect fruit fly the studies on the insulin-like peptides in d. melanogaster (dilp) were much more detailed than in b. mori. to date, 8 insulin-like peptides (dilp1-8) have been discovered in d. melanogaster (brogiolo et al., 2001; colombani et al., 2012; garelli et al., 2012). among them, dilp1, dilp2, dilp3 and dilp5 were mainly expressed in the median neurosecretory cells (mncs) of the brain, similar to that of bombyxin (brogiolo et al., 2001; ikeya et al., 2002; rulifson et al., 2002; broughton et al., 2005; nässel, 2012). the protein products encoded by these genes were structurally similar, represented by a heterodimer linked by disulfite bonds. at the adult stage, dilp2, dilp3 and dilp5 continued to express in mnc, however, dilp1 gene expression was not detected (broughton et al., 2005). although dilp2, dilp3 and dilp5 were all expressed in the mncs, their temporal expression patterns were different. dilp 2 was expressed starting from the 1st instar larvae, while dilp3 was from the middle and later stage of the 3rd instar, and dilp 5 was from the 2nd instar (ikeya et al., 2002). when the larvae were starved, the transcriptional level of dilp3 and dilp5 would decrease, but the dilp2 transcription was not affected (ikeya et al., 2002). upon dietary restriction, the expression of dilp5 in adult drosophila was down-regulated, but dilp3 was not affected (min et al., 2008). no expression of dilp4 was detected in the adult d. melanogaster (grönke and partridge, 2010). genetic knockout of dilp1 and dilp2 resulted in a smaller body size of d. melanogaster. when dilp1-7 was overexpressed in d. melanogaster, the adult body size increased proportionally. among them, dilp2 had the strongest promoting effect on the growth. the overexpression of dilp2 gene not only increased the size of mnc cell but also increased the number of cells (brogiolo et al., 2001; ikeya et al., 2002). dilp2 was found down-regulated in a mutant d. melanogaster that had a prolonged life span, suggesting that dilp2 played a key role in regulating the life span of d. melanogaster (hwangbo et al., 2004; bauer et al., 2007; lee et al., 2008). however, some scholars knocked down the dilp2 gene by rna interference and found that the life span of d. melanogaster was not prolonged like expected, but trehalose storage in the adult d. melanogaster was increased, indicating that dilp2 was involved in sugar metabolism (broughton et al., 2008). dilp6 was mainly expressed in the fat body tissue, and was positively regulated when d. melanogaster metamorphosed from larva to pupa (okamoto et al., 2009). dilp7 gene was only expressed in specific neurons of the ventral nerve chain and a few neurons in the brain (miguel-aliaga et al., 2008; yang et al., 2008). dilp1-4 were clustered on chromosome 3 of d. melanogaster, while dilp5, dilp6 and dilp7 genes were located at two different sites on the x chromosome (ikeya et al., 2002). the dilp1-7 precursor peptides contained 107-156 amino acids, and their primary structure included a signal peptide, b chain, c chain and a chain, and the cleavage sites lay between a chain and b chain and were similar to those of insulin and relaxin. among the 8 dilps, dilp6 was more similar to insulin growth factor. when dilp6 formed a tertiary structure, its c chain would not be completely cleaved by the protease, and thus some sequences were retained. sequence similarity analysis showed that dilp2 had the highest amino acid sequence similarity with mature human insulin, reaching 35 % (brogiolo et al., 2001; ikeya et al., 2002). animals would constantly adjust their growth status, such as maturity or metamorphosis so as to adapt to the disorder (such as damage or tumor) occurred during the growth and development. such processes require the connection between the tissues and organs of the animal so as to maintain the normality and symmetry of their bodies. it was found that the imaginal disc of drosphila melanogaster could activate dilp8 spontaneously. when abnormality was detected during the growth and development of d. melanogaster, dilp8 would delay the metamorphosis of the body by inhibiting the synthesis of ecdysone and slowing down the growth of imaginal disc, and finally producing normal sized individuals (garelli et al., 2012). when dilp8 was silenced or mutated, d. would develop asymmetrical bodies, irregular body size, and need more time to mature. therefore, dilp8 appears to be a key regulatory factor to ensure the stability and robustness during the growth and development of d. melanogaster, however, this regulatory mechanism has not been clarified yet (garelli et al., 2012). the dipteran insect mosquito when a. aegypti ingested the mammalian blood, it would transmit the parasites that caused diseases to mammals. meanwhile, the mammalian blood was very important for the maturation of female a. aegypti eggs. some studies have shown that after ingesting the mammalian blood, mosquitoes could stimulate the median neurosecretory cells of the brain to secrete a neuropeptide that could promote 192 the production of ecdysone in the ovary (brown et al., 2008; attardo et al., 2005). it was identified that the neuropeptide was insulin-like peptide which could activate multiple metabolic processes necessary for egg maturation. up to now, 8 insulinlike peptides (aaegilp1-8) and 1 insulin receptor have been identified from the genome of a. aegypti (riehle et al., 2006). among them, aaegilp1, aaegilp3 and aaegilp8 were specifically expressed in the brain of female a. aegypti at the adult stage. moreover, the precursors of these 8 insulin-like peptides were also composed of a signal peptide, b chain, c chain and a chain. in the formation of mature peptides, aaegilp6 was similar to insulin growth factor peptides (igfs), whose c chain was not completely excised (graf et al., 1997; riehle et al., 2006). it was found that aaegilp3 could stimulate the oocyte’s uptake of yolk and the ovary’s production of ecdysone in low concentration, indicating that aaegilp3 was a key regulatory factor for the production of eggs in a. aegypti (brown et al., 2008). aaegilp3 also showed metabolic activity and could increase the storage of carbohydrate and lipid, and the function of aaegilp3 depended on the expression of mosquito insulin receptor (mir), which have been previously detected in mosquito ovaries (graf et al., 1997). the mature peptide mir is a 400 kda tetramer consisting of two 116 kda α subunits and two 95 kda β subunits. immunohistochemistry analysis showed that mir was located on the cytomembrane of follicle cells around egg cells and nutrient cells (riehle and brown, 2002). in the fat body, the insulin-like peptides of a. aegypti could also regulate the expression of yolk protein precursor gene. it was found in vitro that when 20-hydroxyecdysone and aaegilps were added to the fat body culture separately, they could not stimulate the expression of yolk protein precursor gene. however, when 20hydroxyecdysone and aaegilps were incubated simultaneously in the fat body tissue culture, they played a strong synergistic role in regulating the expression of yolk protein precursor gene (larsen et al., 2017; roy et al., 2007). in the genome of another mosquito species anopheles gambiae, 7 insulin-like peptide genes (agamilp1-7) have been identified so far (riehle et al., 2002; krieger et al., 2004). the arrangement of insulin-like peptide genes on the chromosomes of a. gambiae was similar to that of insulin. agamilp1-4 genes clustered on chromosome iii, about 23 kb away from agamilp 6 and agamilp 7, whereas agamilp 5 was located on chromosome ii. among the agamilps, the two pairs agam 3/agam 6 and agam 1/agam 7 show high sequence homology, which were 98.7 % and 95.5 %, respectively (riehle et al., 2002). the hymenopteran insect honeybee two insulin-like peptides (amilp1 andamilp2) and two insulin receptors (amir1 and amir2) have been identified within the genome of a. mellifera (wheeler et al., 2006). at the critical stage of gender determination, the expressions of amilp1, amilp2 and amir2 were significantly different between the queen and worker bees (de azevedo and hartfelder, 2008; wheeler et al., 2006). in queen larvae, the expression of amilp1 gene was particularly high, and such high expression depended on the ingested royal jelly. in contrast, the expression of amilp2 gene was higher in the worker bee larvae. compared with the mature worker bees, the expression level of amilp1, amir1 and amir2 in the heads of the mature queen bees was very low. in the worker bee, the expression level of amilp1 decreased with aging, while in the queen bee, the expression level of amilp1 increased with the aging process (corona et al., 2007), indicating that the insulin signal pathway is related to the life span of a. mellifera. the expression difference between the queen and worker bee was relatively conservative during evolution, and the same phenomenon was also found in the bee polistes metricus (toth et al., 2007). based on phylogenetic analysis by riehle et al., the ilps of a. aegypti, a. gambiae, and d. melanogaster have higher homology to each other than to those of b. mori, indicating highly diverged sequences between insect orders, whereas, within the same insect order, the distances between the ilp families were greater than the orthologues within that order (riehle et al., 2006). conclusion from insects to mammals, insulin and insulinlike peptides play central roles in regulating the organ development and establishing adult body size. the signaling pathways of insulin and insulin-like peptides in b. mori as well as many other insects have been proposed, however, many details regarding the pathway, such as the interacting proteins of ilps and the mechanisms of 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pearl mussel (hyriopsis cumingii) q liu1,2, b xu1,2*, t xiao1,2* 1hunan engineering technology research center of featured aquatic resources utilization, hunan agricultural university, changsha 410128, china 2collaborative innovation center for efficient and health production of fisheries in hunan province, changde 415000, china accepted september 12, 2019 abstract serine proteases play central roles in immune defense in invertebrates through innate immunity, and are particularly important complement system in molluscs because their susceptibility to infection due to lack of an adaptive immune ability. a gene encoding the serine protease homolog from the triangle shell pearl mussel (hyriopsis cumingii) was identified and designated as hc-sph in this study. protein sequence analysis revealed that hc-sph consists of a typical tryp_spc functional domain of serine protease of s1 family lead by a signal peptide, and the molecule shares a highly conserved sequence and structural organization with other members, including a cleavage site, 3 enzymatic active sites and 3 substrate binding sites, so that it was clustered into a trypsin-like serine protease subfamily of the s1 superfamily. semi quantitative analysis of the amplicons separated on agarose gel by comparing to the β-actin products revealed that the digestive gland had a strong expression while the gonads were seen as weak expression sites. infected by aeromonas hydrophila, the gene expression was significantly up regulated in the kidney at the 6 hours post challenge (hpc), stomach at 12 hpc and gills at 24 hpc while the expression maintained steadily unchanged in the digestive gland. however, up to 48 hpc, the expression levels in all four tissues reached significantly high, and also joined by a high level of expression in intestine that was down regulated before 24 hpc, to build up an enhanced immune defense. the complementary up regulation of the gene expression in these tissues suggested a temporal and spatial reinforce model for hc-sph in immune response. key words: tryp_spc domain; serine protease homolog; s1 superfamily; immune response; mollusk introduction serine proteases (sps), commonly recognized by their serine activity, are hydrolyse protein family of enzymes with wide distribution and important biological actions (ross et al., 2003; liu et al., 2019; wei et al., 2019). sps are molecularly characterized by a conserved tryp_spc domain together with the catalytic triad histidine (his, h), aspartic acid (asp, d) and the serine (ser, s) (li et al., 2012b; wang et al., 2014). inactive sps with key residue(s) of catalytic triads replaced by other amino acid are defined as sp homologs (sphs), which are similar to sps in amino acid sequence (liu et al., 2010) and classified ___________________________________________________________________________ corresponding authors: baohong xu tiaoyi xiao hunan engineering technology research center of featured aquatic resources utilization hunan agricultural university changsha 410128, china e-mail: xbht568@126.com; tyxiao1128@163.com as protease precursors. sphs assist sps as cofactors to exert their functions (wang and jiang, 2004; lee et al., 2018) by activating/inhibiting the original protease activities in variety of biological processes such as digestion, cell differentiation, tissue remodeling, angiogenesis and embryonic development as well as their involvement in cellular and humoral immune responses (rawlings and barrett, 1993; krem and di cera, 2001, 2002). up to date, sph was identified in many invertebrates and some of which were shown to play roles in different biological processes, including antimicrobial activity from horseshoe crab tachypleus tridentatus (kawabata et al., 1996), defense of pathogen challenge in chinese white shrimp fenneropenaeus chinensis (ren et al., 2009,2011), cell adhesion in crayfish pacifastacus leniusculus (huang et al., 2000) and the tiger shrimp penaeus monodon (liu et al., 2006), and activation of the prophenoloxidase (propo) system (kwon et al., 2000; charoensapsri et al., 2009; cui et al., mailto:xbht568@126.com 174 2010), which represent an important host natural immune system that recognizes foreign invasive substances by molecular pattern of proteins. the molecular characterization, gene cloning and expression profile of the sph were also abundantly studied in insect and other arthropods (jitvaropas et al., 2009; ren et al., 2011) while little is known in mollusks. the freshwater triangle shell pearl mussel hyriopsis cumingii is a mollusk species widely cultured in china for pearl production (ren et al., 2012). however, mussel aquaculture has been declining in the recent years because of the diseases by bacterial and viral infection. like other mollusks, the mussel lacks acquired immune system and mainly relies on innate immunity to defense pathogens invasion (wang et al., 2013). in this study, a gene (termed hc-sph) coding for sph (termed hc-sph for encoded proteins) was cloned and characterized from the h. cumingii. the tissue specific gene expression profiles of the gene before and after infection with the common infectious bacteria aeromonas hydrophila were analyzed to provide evidence of its potential role in innate immune responses as suggested from other species. the results reported here summarized the molecular identification and characterization of hc-sph, which contribute to general knowledge of sph from a mollusk and particularly provide important molecular data for further investigation of hc-sph towards a better understanding for its role in immune defense against infections. materials and methods experimental animal and immune challenge mussels (hyriopsis cumingii), 285.33 ± 55.18 g and 14.37 ± 1.28cm, were obtained from a commercial farm in changde, hunan province, china and cultured in freshwater at 24 28 °c. they were acclimatized for a week before being used for experiments. the healthy mussels were injected in axe feet with 109 cfu/ml (≈ 0.5 ml) of aeromonas hydrophila isolated and purified from dying mussels for challenging, and with an equal volume of phosphate buffer saline (pbs) as controls. three challenged and three control mussels were chosen for sample collection from the digestive gland, stomach, intestine, gill, heart, mantle, axe foot, adductor muscle, kidney and the gonad at the time points of 0 h, 3 h, 6 h, 12 h, 24 h, 48 h after injection. all samples were snap frozen in liquid nitrogen and immediately stored at 80 °c under rnase free conditions. rna extraction and cdna synthesis total rna samples were isolated using the rnaprep pure tissue kit (tiangen biotech, china) according to the manufacturer’s instruction, and treated with dnase i. rna samples were kept in depc treated water, qualified by gel electrophoresis and quantified using a biophotometer (eppendorf, germany). about 5 μg total rna from the digestive gland of healthy mussel was converted into 5′-race ready cdna and 3’-race-ready cdna by reverse transcription using the smartertm race (rapid amplification of cdna ends) cdna amplification kit (clontech, usa) for full length cdna cloning. about 2 μg total rna from each tissue sample was reverse transcribed by revertaidtm first strand cdna synthesis kit (fermentas, canada) for gene expression study. race and sequencing analysis the designing of gene specific primers used for this study was based on the expressed sequence tag (est, genbank accession number: fe968619) from a cdna library generated by suppression subtractive hybridization (xiao et al., 2009). the 5′-race ready cdna was synthesized using the smart ii™ a oligonucleotide and the 5′-race cds primer a (table 1); the 5′-race was performed using gene specific primer pairs upm/ hc-sph gspr out and upm/ hc-sph gspr in (table1) in nested pcrs. 3’-race-ready cdna was synthesized using the 3′ race cds primer a (table 1). the 3′-race was performed using gene specific primer pairs upm/hc-sph-gspf out and upm/ hc-sph gspf in (table 1) in nested pcrs. the pcr reactions were catalyzed by ex taq polymerase (takara, japan) and the amplified fragments were cloned into the pucm t vector (biobasic, canada) for sequencing analysis (sangon, china). the full length cdna coding for the sp homolog of h. cumingii was amplified and identified by its overlapping sequence to the est. dna and amino acid sequence analysis the cdna sequence of hc-sph was submitted and translated into amino acid sequence (http://www.vivo.colostate.edu/molkit/translate/), termed as hc-sph, which was further characterized for its molecular property and homology prediction. the protein sequence was submitted to blastp searching against various databases through uniprotkb/swissprot (http://www.uniprot.org/blast/) for a primary homology result and collected close related amino acid sequence for alignment. the sequence was used for a prediction of its functional domains and motifs by the online service of the eukaryotic linear motif (elm) resource for functional sites in proteins. the signalp 4.1 (http://www.cbs.dtu.dk/services/signalp/) was used to define the signal peptide and the cleavage site. the amino acid sequences related to hc-sph found by uniprotkb were retrieved for annotating the place of hc-sph, sequences clustered directly with hc-sph and the sequences with well-defined tryp_spc domains used for functional motif alignment were listed in table s1. the sequences were aligned and edited by clustalx 1.83 (thompson et al., 1997). phylogenetic analysis was performed with mega 6.0 using maximum likehood method with a bootstrap valve of 1000. the hc-sph gene expression by rt-pcr and rt-qpcr the expression profiling of hc-sph transcripts in the digestive gland, stomach, intestine, gill, heart, mantle, axe foot, adductor muscle, kidney and gonad from healthy groups were measured using rt-pcr. the quantity of hc-sph transcripts in the 175 table 1 primers designed for cloning and expression analysis of hc-sph primer name sequence smart ii™ a oligonucleotide 3′-race cds primer a(3′-cds) 5′-race cds primer a (5′-cds) 10×universal primer a mix (upm) long primer short primerrandom primer oligo (dt)20 hcshp-gspr-out hcsph-gspr-in hcsph-gspf-out hcsph-gspf-in hc-sph-pf hc-sph-pr β-actin -pf β-actin -pr 5'-aagcagtggtatcaacgcagagtacgcggg-3' 5'-aagcagtggtatcaacgcagagtac(t)30v n-3' * 5'-(t)25v n-3' * 5'-ctaatacgactcactatagggcaagcagtggtatcaacgcagagt-3′ 5'-ctaatacgactcactatagggc-3' 5'-(dn)9-3' * 5'-(dt)20-3' 5'-ggcgatgtcgtccttgagactggtg-3' 5'-cgctgaatcctggaccctcaaaatg-3' 5'-ttgaccttgacctcagacattggcag-3' 5'-atgcctctcatcactaccgtgttgga-3' 5'-tgagggtccaggattcagcgtat-3' 5'-ccccaaccagggaggtagcaaac-3' 5'-actcacaccgtccccatctat-3' 5'-cgatttctctttcagcagtgg-3' * n = a, c, g, or t; v = a, g, or c in primer sequences digestive gland, stomach, intestine, gill and kidney from both challenged and control groups were measured using rt-qpcr. the hc-sph gene specific primer pairs, hc-sph pf and hc-sph pr (table 1), was designated to amplify a fragment of 279 bp located between the position 326 and 603 of the gene. the house keeping gene β-actin was used as an internal control amplified by the gene specific primer pair β-actinpf/β-actin-pr (table 1) for an expected fragment of 148 bp. the rt-qpcr reaction was conducted using the transstarttm top green qpcr supermix (transgen biotech, china) on an abi 7300 real-time detection system (applied biosystems, usa) as instructed by the manufacturer. briefly, the final pcr reaction mixture (25 μl) in h2o consisted of 12.5 μl of 2 x transstarttm top green qpcr supermix, 0.5 μl of 50 × passive reference dye1, 1 μl cdna and 0.5 μl of each primer, was incubated at the denaturing temperature of 94 °c for 30 s first followed by 40 cycles of 5 s at 94 °c, 31 s at 61 °c. all pcr products were dissociated and confirmed a single amplicon from each sample by the system. a series of 10 fold diluted mixture of first strand cdnas from all selected tissues were used to construct two standard curves (chang et al., 2007) for hc-sph and β-actin respectively against the no cdna negative controls. each sample was run in triplicates. data collected were analyzed using the sequence detection system (sds version1.4, applied biosystems, usa). the relative expression of hc-sph was determined using the comparative threshold cycle (ct) method (2-(△△ct) method) as described previously (livak and schmittgen, 2001; jiang et al., 2019). the arithmetic mean ± standard deviation was calculated and used for determining the significance of differences by t test using the statistics package for social science (spss) 17.0. p < 0.05 means significant differences and p < 0.01 for most significant differences. results isolation of hc-sph, a full length cdna sequence coding for hc-sph a cdna fragment of 478 bp (est tag of sp from the triangle shell mussel, genbank accession number: fe 968644) was retrieved from the database, based on which a pair of gene specific primers were designed (gspf and gspr listed in table 1) for amplifying the full length cdna sequence (fig. 1a). a 344 bps 5′-race fragment and a 495 bps 3′-race fragment (fig. 1b) overlapped to the est fragment to give the rise of a full length cdna sequence of 1046 bp (fig. 1c). the hc-sph contains a 5′ terminal untranslated region (utr) of 73 bps, an 864 bps open reading frame (orf) encoding 287 amino acid residues and a 3′-utr of 109 bps with a canonical polyadenylation signal sequence aataaa and a labile motif attta (both underlined) followed by a poly (a) tail (fig. 1c). bioinformatic analysis of amino acid sequence of hc-sph protein full-length hc-sph cdna and its encoded sp in h. cumingii (hc-sph) were submitted to the genbank accession number: gu222695. the amino acid sequence from orf (303 aa) of the hc-sph was analyzed using uniprotkb and elm in conjunction with biological databases and found a typical tryp_spc (trypsin-like sp) domain of the sp family between r52 and i290 (fig. 2a). the n terminus (k1 i70) was recognized as a signal peptide (fig. 2a) for proteolytic cleavage around r52. highly conserved motifs important for proteolytic cleavage, typical enzymatic active sites, and substrate binding sites for the enzyme are shown along the molecule (fig. 2a). multiple sequence alignment of hc-sph with six other well-defined sps showed those conserved motifs (fig. 2b) around the 176 fig 1 the full length cdna cloning of hc-sph gene and its encoded amino acid sequence (hc-sph). the hc-sph est tag sequence from genbank was used for designing of the gene specific primers for race pcrs (a), which amplified specific products as single bands (sizes of molecular weight maker and the products were given as indicated by arrows) for 5’ and 3’ rcae respectively (b). the determined nucleotide sequence, contained a canonical polyadenylation signal sequence aataaa and a labile motif attta (both underlined) followed by a poly (a) tail, and the deduced amino acid sequence were given in single letter format (c). the translation initiation methionine (m) is labelled by +1 on top enzymatic important sites with the ranges numbered at the bottom. the numbers of non conserved amino acid residues before, between and after the motifs are given for each species respectively in brackets (fig. 2b). the uniprotkb blast search against databases revealed a high similarity of hc-sph to the tryp_spc superfamily, amongst which protein sequences with defined biological properties were retrieved for similarity analysis (fig. 3). a phylogenetic tree was generated to show the position and similarity relationship of hc-sph in the unrooted tree (fig. 3). hc-sph was clearly clustered with a group of proteins that have strong trypsin activities. the hc-sph expression in tissues and regulation in response to bacterial infection the presence of hc-sph gene transcripts in different tissues of healthy h. cumingii was detected by rt-pcr as strategized (fig. 4a) using a pair of gene specific primers, hc-sph-pf and hc-sph-pr (table 1). a fragment with the expected size (279 bp) was amplified from the tissues listed in all replicates in comparison with the relative amount of cdna template reflected by pcr amplification of the house 177 fig. 2 characterization of the molecular properties of hc-sph. a signal peptide sequence was identified at the n terminus with a proteolytic cleavage site, presented by signalp 4.1 (a). using uniprotkb blast conjunct with elm searches, a typical tryp_spc functional domain was detected (a), including a cleavage site (indicated by blue ▲), 3 enzymatic active sites (indicated by red ▲) and 3 substrate binding sites (indicated by black ▲). the conserved motifs containing the sites were aligned with the corresponding residues numbered at the bottom and the numbers of non conserved residues were given in brackets (b) keeping gene β-actin (fig. 4b). rt-pcr amplification result showed hc-sph gene expression in tissues of 2 years old mussels examined, including the digestive gland, stomach, intestine, gills, heart, mantle, axe foot, adductor muscle, kidney and gonads, by the gene-specific primers (fig. 4b). semi-quantitative analysis of the amplicons separated on agarose gel by comparing to the β-actin products revealed that the digestive gland had a strong expression while the gonads were seen as weak expression sites. in the time course rt-qpcr analysis of gene expression in five selected tissues at hours post challenge (hpc), the hc-sph expression in digestive gland was slightly increased at 6 hpc followed by a decrease at 12 hpc detected, then the expression kept rising to a highest (p < 0.01) level till 48 hpc (fig. 5a). the hc-sph gene expression in kidney (fig. 5e), stomach (fig. 5b) and the gills (fig. 5d) rather fluctuated with a highest expression detected at 6 hpc for kidney, 12 hpc for stomach and 24 hpc for the gills. while kidney expressed basal level of hc-sph after a surge at 6 hpc, the expression in the stomach and gills showed complementary expression pattern (fig. 5). the intestine showed a constant low levels of hc-sph expression (although expression was significantly high at 3 hpc (p < 0.01) than that at 0 hpc), particularly between 6 hpc and 24 hpc at a significantly lower level (p < 0.01) than 0 hpc, before an expression surge starting from 24 hpc and reach a peak (p < 0.01) at 48 hpc (fig. 5c). discussion hc-sph, a secreted trypsin-like sp in h. cumingii sps are enzymes widely conserved across species not only in their molecular structures but also in their biological activities (ross et al., 2003). a cdna library was established using suppression subtraction hybridization achieves an est tag for sp gene of the h. cumingii in our previous study (xiao et al., 2009), which is similar with the genes encoding the factors responded to immunogenic stimulation (lund and olafsen, 1999; gerwick et al., 2000,2002). in the present study, the information of the est tag was used for identify and clone the full-length cdna, which contends an orf coding for hc-sph. the uniprotkb blast analysis against databases 178 fig. 3 the phylogenetic analysis of hc-sph. the phylogenetic analysis showing the position of hc-sph as a member of serine proteases of s1 superfamily (brunch was highlighted with bold) and protein was clustered into the group of with trypsin-like proteins revealed hc-sph exhibited a typical molecular characteristic of a sp. a well-conserved tryp_spc domain of the sp family proteins was found in hc-sph by functional search, including enzymatic active sites for its processing and activities along the molecule (fig. 2a). the molecule was recognized as an inactive trypsin-like sp precursor. a cleavage site around i37 (fig. 2b) was found in the precursor that are the characteristics of enzymatic activities and also known as catalytic triad for sps (perona and craik, 1995), and 3 substrate binding sites at around e226, s252 and 3 s254 (fig. 2b) of the molecule, which bind to the substrates for the function. these conserved motifs are ensuring the actions (li et al., 2012a). however, key residues of catalytic triads were replaced by other amino acids (q82 for h82, l232 for s232), similar to sps in amino acid sequence, are classified as protease precursors (liu et al., 2010). furthermore, the numbers of the amino acid residuals in non conserved regions were very close between species (fig. 2b), which were significant in ensuring similar molecular conformations essential for their function. sphs assist sps as cofactors to exert their functions (wang and jiang, 2004) by activating/inhibiting the original protease activities, including cell differentiation, tissue remodeling, embryonic development and humoral immune responses in the cell (rawlings and barrett, 1993; krem and di cera, 2001, 2002). 179 fig. 4 the hc-sph gene expression in tissues of healthy hyriopsis cumingii. a pair of gene specific primers were designed to amplify a fragment of 279 bp between the nucleotide position 326 and 603 (a). the rt-pcr products were confirmed by agarose gel electrophoresis showing the right size (top, b) and the relative template concentrations were judged by the house keeping gene β-actin (bottom, b) signal peptide at the amino terminus of a nascent protein, mediating protein targeting to the membrane of the endoplasmic reticulum (blobel, 1980, 2000), directs the protein transportation and the secretory pathway, which is recognized by the signal recognition particle (srp) and cleaved by the signal peptidase (duffaud et al., 1985). these residuals are comprising a characteristic tripartite structure of a hydrophilic, usually positively charged n region, a central hydrophobic h region and a cleavage site (von heijne, 1985), which are represented by c-score (raw cleavage site score), s-score (signal peptide score) and y-score (combined cleavage site score) in the signalp prediction (petersen et al., 2011). the hc-sph protein sequence was analyzed by software and a clear signal peptide region of 31 amino acids (the translation initiation starts at the m17, see fig. 1c) with a cleavage site (d valve = 0.829 by setting up d-cutoff 0.450) between c31 and q32 of the short stretch of vlc-ql recognized (fig. 2a). the high raw c-score recognized this region as a signal peptide and enhanced by the y-score of combined cleavage site, which compares the c-score peak(s) against the steep slope of the signal peptide score (s-score). signal peptides are rather heterogeneous and interchangeable between different species, but the signal peptide specifies the protein secretion (von heijne, 1985; kober et al., 2013). hc-sph was therefore regarded as a secreted trypsin-like sp. in addition, the hc-sph was very similar to the complement factor d (cfad_human, p00746), which is also single domain sp. the factor d is a component of the alternative complement pathway. recent studies show that an ancient proto complement complex (c3, sp factor b and complement receptor cr) exists in bivalves (gorbushin, 2018, 2019). a complement -like activity is shown in bivalves mytilus edulis, mya arenaria, and sinonovacula constricta (klimovich and gorbushin, 2017; niu et al., 2018). however, homolog of the factor d cleaving the factor b to bb and ba is not described until now (gerdol et al., 2018). our results implied that the hc-sph was a potential candidate for this role, although it was still need to further confirmation. hc-sph is a member of the peptidases s1 family hundreds of sequences producing significant alignments with low expect values (e values) were found by both blastp (altschul et al., 2005) and uniprotkb (apweiler et al., 2012) searches. they share similar enzymatic functions and classified as proteins of the peptidases s1 family (goldman et al., 2006), the largest of all of the peptidase families, by both the number of sequenced proteins and the 180 fig 5 the hc-sph gene expression profiles in tissues of mussels challenged by a. hydrophila infection. the transcripts were quantified using rt-qpcr in time-course manner and indicated by hours post challenge (hpc). the amplicons detected by qpcr in digestive gland (a), stomach (b), intestine (c), gill (d) and kidney (e) were normalized by control value (0 h) and adjusted to the scale for summarizing the result. the relative abundancy of the transcripts in a tissue at a time of hpc was compared with the control (0 h) to rule out the difference statistically. the upand down-regulated expression levels of significant difference (p < 0.05, n=3) were indicated by single asterisk (*), and of highly significance (p < 0.01, n=3) by double asterisks (**) number of distinct peptidase activities (serine endopeptidases) (rawlings and barrett, 1993) fell into three main activity types: trypsin-like, chymotrypsin-like and elastase-like sps (cooley et al., 2001; ovaere et al., 2009; madala et al., 2010). the peptidases of the s1 family proteins are secretory precursors led by an n terminal signal peptide that is cleaved to form the active enzyme. the cleavage initiates a structural rearrangement to produce a “new” n terminus for binding. the phylogenetic tree of the s1 peptidase (fig. 3) clearly showed that the hc-sph was clustered to a group of trypsin-like type sps roughly flanked by a group of elastase-like sps at the bottom part and a large group of chymotrypsin-like proteases on top. gene expression in tissues regulated by bacterial infection suggested temporal and spatial active roles of hc-sph in immune response immune responses to infections are a series of cellular and molecular actions in invertebrate species (peteiro et al., 2007). understanding of the reaction process is particularly important in molluscs because their susceptibility to microbial infection and transmitting diseases (barcia and ramos-martinez, 2008) due to the lack of an adaptive immune system (wang et al., 2013). the mollusk defenses through a chemicophysical barriers preventing host invasion and the circulating hemocytes and secreted bio factors to initiate immune responses (hine, 1999; garcia-garcia et al., 2008), including the activation of phenoloxidase system (little et al., 2005) and the complement system capable of recognizing and eliminating invading pathogens (kardos et al., 2008). the sps are the central components of the complement system, specifically bind to and cleave the substrates in forms of super molecular complexes with other sps and non enzymatic proteins (harmat et al., 2004). the expression patterns of sp in tissues would be altered to activate immune response stimulated by infection. the gene expression in physiological conditions examined by rt-pcr in this study showed the presence of hc-sph gene transcripts in all detected tissues (fig. 4b). in the time course rt-qpcr analysis of gene expression in five selected tissues of a. hydrophila challenged mussels showed that the trends of the hc-sph expression were similar to the cfcubsp in chlamys farreri which indicated cfcubsp might be involved in immune response (yang et al., 2017). these results implied that a temporal and spatial reinforce model for hc-sph to response to foreign invaders that yet to be proved experimentally. the hc-sph gene expressed at high levels in all secreting tissues at 48 hpc onwards when an immune acting system recruited and built up. the relatively low level of expression after the challenging and mechanisms of down regulation before 24 hpc in the intestine remain to be further investigated. in summary, the full length cdna coding for the sph of the s1 family protein was cloned from h. cumingii. hc-sph possessed a typical functional domain of a tryp_spc with characteristics of signal 181 peptide cleavage, active and substrate binding sites of the protease, which was further classified as a member of trypsin-like sp of s1 superfamily. under the physiological conditions, the gene expression remained high in the digestive gland, but the expression showed a temporal complementary pattern of high levels in the digestive gland, kidney, stomach and low levels in the gills upon the regulation of bacterial infection. all tissues reached high level of the gene expression at 48 hpc to accomplish the recruiting of functional immune response. the molecular pathways in which the gene expression is regulated and the enzyme interact with its receptors to activate the immune-reactive network are to be further studied. acknowledgments this study was supported by the national natural science foundation of china (no.31402289 and no.31040083) and the science foundation of hunan agricultural university(13yj04). the authors are grateful to dr. shuliang cui, australian phenomics facility, john curtin school of medical research, australian national university, for reading and revising the first drafted manuscript. references altschul sf, wootton jc, gertz em, agarwala r, morgulis a, schaffer aa, et al. 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department of sciences, university of basilicata, via dell’ateneo lucano 10, 85100 potenza, italy accepted june 26, 2018 abstract insect parasitic factors of both maternal and embryonic origin allow the endoparasitoid progeny to elude the humoral and cellular immune system responses of their host. endoparasitoid wasps of lepidopteran larval stages inject, along with venom and ovarian proteins, polydnavirus particles acting in synergy with all other factors in host regulation for parasitism success. to date, the molecular mechanisms used by endoparasitoid to circumvent the host immune system are little known. neverthless, several of these strategies are conserved through the wasp parasitoid species. heliothis virescens is a noctuid moth, host of the endophagous parasitoid toxoneuron nigriceps. the first observed effect of parasitism is immune system suppression, as direct consequence of the array of host regulation factors, both of embryonic and maternal origin. this review describes the contribution of all the parasitic components during alterations of the host immune response observed after oviposition by t. nigriceps. key words: heliothis virescens, immunomodulation, endoparasitoid, polydnavirus, venom, teratocytes introduction the relationship between parasitoids and their hosts can be very complex, involving physiological interactions at different and multiple levels. this association is the result of a long coevolutionary history that in some cases led to a very intimate interaction and to fine regulation of the host physiology by the parasitoid. in the ectoparasitoids, whose larvae feed on the host body from outside, the parasitism is based on the use of venom, able to paralyse the host, allowing the larvae to benefit. in endoparasitoids, whose larvae feed within the host body, parasitism involves several factors, modulating the physiology of the host, in order to obtain a more suitable environment for the development of juvenile stages of their progeny (schmidt et al., 2001; federici and bigot, 2003; rivers et al., 2005; vinson, 2012). endoparasitoid insects, belonging to the hymenoptera order, have developed strategies to parasitise their hosts, through specialised mechanisms generated by long adaptive processes that occurred within host/parasitoid physiological ___________________________________________________________________________ corresponding author: patrizia falabella department of sciences university of basilicata via dell’ateneo lucano 10, 85100 potenza, italy e-mail: patrizia.falabella@unibas.it interactions (vinson and scott, 1974; vinson and iwantsch, 1980; godfray, 1994; quicke, 1997). the first evidence for the physiological changes observed in parasitised hosts is the inactivation of humoral and cellular defences, that prevents parasitoid egg melanization and encapsulation (beck et al., 2000; asgari et al., 2003a; zhang et al., 2006). the insect immune system is generally able to respond to the intruders within few minutes after injuries. parasitoids, as a consequence, have evolved their ability to alter the host defence with different strategies, some of which acting very fast. for this reason, during oviposition, parasitoid females introduce maternal factors such as ovarian fluid into the host body (asgari and schmidt, 1994; webb and luckhart, 1994, 1996; tanaka et al., 2002), venom (beckage and gelman, 2004; zhang et al., 2004; kohler et al., 2007) and proteins that cover the egg surface (asgari and schmidt, 1994). all of these factors, along with embryonic ones, necessarily mediate immunosuppression during the early stages of parasitism, contributing to the success of parasitism (strand and burke, 2015). among maternal factors, polydnaviruses (pdvs) are obligate symbionts of endoparasitoid wasps, attacking exclusively larval stages of their lepidopteran hosts (webb et al., 2000; webb and strand, 2005). pdvs have a double-stranded segmented circular dna, integrated into the mailto:patrizia.falabella@unibas.it 241 genome of the parasitoid. pdvs infect hosts and express their genes in tissues without any replication, inducing alterations in host physiology. these alterations allow parasitoid larvae survival and growth, to finally pupate in silken cocoons (strand and burke, 2014). pdvs replicate exclusively in wasp ovaries, where their circular genome is generated from linear dna copies of wasp chromosomes (varricchio et al., 1999, volkoff et al., 2010; dupuy et al., 2011) and vertically transmitted to the next wasp generation (strand, 2010). in several host/parasitoid systems the maternal and embryonic factors of parasitic origin can suppress the host immune system. the innate immune system of insects includes several mechanisms that sometimes are arbitrarily divided into humoral and cellular responses, interacting at different levels to provide defence against invading intruders, both pathogens and parasites. the first physical barrier is the tegument, with the cuticle and the epithelium beneath. when an external organism overcomes this barrier and reaches the haemocoel, humoral and cellular defence reactions are specifically and sequentially activated, such as soluble molecules and haemocytes circulating in the haemolymph (lemaitre and hoffmann, 2007). among the humoral responses, melanization is a very fast reaction due to the activation of serine protease cascade, induced by several types of elicitors (microbial surface molecules). this pathway regulates the coagulation or development of melanine both upon injury and on the surface of the foreign invaders. the production of antimicrobial peptides (boman et al., 1991; hoffmann et al., 1993; hultmark, 1993; cociancich et al., 1994; lowenberger, 2001) and reactive intermediates of oxygen or nitrogen (bogdan et al., 2000; vass and nappi, 2001) are also part of the humoral response. cellular responses are mediated by different types of haemocytes and include encapsulation against parasitic eggs and larvae, phagocytosis of bacterial and yeast cells and nodulations against grouped cells of pathogens, as bacteria or yeasts (schmidt et al., 2001; lavine and strand, 2002). a very common effect of parasitism is host immune response suppression. endoparasitoid wasps belonging to braconidae and ichneumonidae families induce significant immunosuppression of parasitised hosts. host immunosuppression is usually attributed to the wasp’s ability to avoid or suppress encapsulation which is the major host defence against parasite egg invasion (summers and dib-hajj, 1995). eggs and larvae of endoparasitoids are able to evade host immune defences either passively and/or by active suppression of the host immune system (schmidt et al., 2001; lavine and strand, 2002). host defence regulation by the parasitoid is mediated by female secretions injected during oviposition (venom, pdv, ovarian calyx fluid) and embryonic (teratocyte) factors that may act alone and/or in synergy. for example, the microplitis demolitor bracovirus (mdbv) alone inhibits encapsulation and suppresses other host immune defences, including phagocytosis, melanization of haemolymph and inducible expression of other humoral defence molecules (strand and pech, 1995 a, b; beck and strand, 2005; thoetkiattikul et al., 2005; strand et al., 2006). for the endoparasitoid campoletis sonorensis, edson et al (1981) showed that purified viable polydnavirus was responsible for suppressing the host’s (heliothis virescens) ability to encapsulate the wasp eggs. a cysteine rich gene from campoletis chlorideae polydnavirus is responsible for the disruption of encapsulation and haemocytes cytoskeleton degradation (zhang and wang, 2003). early genes of cotesia congregata bracovirus (ccbv) cause haemocyte inactivation and apoptosis (lavine and beckage, 1995; le et al., 2003). the venom of the braconid apanteles glomeratus (kitano, 1982) and accessory gland secretions produced by pimpla turionella and the cynipid leptopilina heterotoma, specifically inhibit host encapsulation (osman, 1978; rizki and rizki, 1984; parkinson et al., 2001). the venom protein vn50 of cotesia rubecula blocks melanization of its host pieris rapae (asgari et al., 2003b) and a layer of calyx fluid glycoproteins protects the developing wasp during embryogenesis (asgari and schmidt, 1994). also teratocytes (kitano, 1969, 1974; vinson, 1972) or the egg itself (kitano and nakatsuji, 1978) can produce soluble substances to suppress host immune system. this review provides an overview of the interaction between the host/parasitoid model system heliothis virescens/toxoneuron nigriceps, focusing on the immune system inhibition of h. virescens by t. nigriceps during parasitism. the host/parasitoid system: heliothis virescens/toxoneuron nigriceps toxoneuron nigriceps (hymenoptera: braconidae) is a solitary braconid endoparasitoid wasp of the tobacco budworm larval stages heliothis virescens (lepidoptera, noctuidae). during oviposition, t. nigriceps females inject into the host larva, along with the egg, the venom and the ovarian calyx fluid that include the ovarian proteins and a symbiotic polydnavirus (pdv) named t. nigriceps bracovirus (tnbv) (fig. 1). parasitism can occur at all host larval stages and parasitised caterpillars can reach the stage of mature larvae but they fail to pupate (lewis and vinson, 1968; pennacchio et al., 1993). as in other endoparasitoid wasps, the integration of parasitic factors of maternal origin (ovarian calyx fluid, pdv and venom) with embryonic origin factors (teratocytes) causes physiological alterations resulting in host developmental arrest, crucial for t. nigriceps larvae that grow and finally pupate in silken cocoons (lewis and vinson, 1968; vinson and iwantsh, 1980). tnbv t. nigriceps is associated with a polydnavirus (pdv), t. nigriceps bracovirus (tnbv), injected during oviposition together with the egg. the tnbv genome is composed of dna circles of different 242 fig. 1 heliothis virescens (lepidoptera, noctuidae) last (fifth) instar larvae, parasitised by toxoneuron nigriceps (hymenoptera: braconidae). during parasitisation (c), t. nigriceps female (b) injects into h. virescens (a https://commons.wikimedia.org/w/index.php?curid=1215079) the egg, stored in the ovaries (c-1), the ovarian calyx (c-2) fluid, that includes the ovarian proteins and a symbiotic polydnavirus named t. nigriceps bracovirus (tnbv), and the venom, stored in the reservoir (c-3) and released by venom glands (c-4). the alteration of the neuroendocrine and the immune systems of the host prevents its final pupation and promotes the parasitoid larva development. this strategy leaves the host alive since the mature parasitoid larva egression (d). figures b and d were kindly provided by dr. vinson lab. sizes, ranging from 3.4 to 13.3 kb (fig. 2). the coding sequences include open reading frames (orfs) encoding putative proteins that for the majority belongs to 4 gene families: ank (protein with ankyrin repeat motif) (falabella et al., 2007) ptp (protein tyrosine phosphatase) (provost et al., 2004; falabella et al., 2006), udp (glucose-6dehydrogenase and sugar transporter) and ben domain proteins (falabella et al., unpublished data). in synergy with other parasitic factors, the symbiotic pdv seems to play the major role in inducing the disruption of host physiology. in particular, the injection and active transcription of tnbv genes are responsible for the alteration of the host neuro-endocrine balance, through the inactivation of host prothoracic glands (pgs) (tanaka and vinson, 1991; pennacchio et al., 1997, 1998 a, b). the consequent block of ecdysteroidogenesis in the last instar larvae inhibits pupation. although the involvement of both phosphoinositide 3-kinase/protein kinase b/target of rapamycin (pi3k/akt/tor) and mitogen-activated protein kinase (mapk) pathways in prothoracicotropic hormone (ptth)-stimulated ecdysteroidogenesis in h. virescens pgs has been demonstrated (scieuzo et al., 2018), to date only the effect of tnbv infection on pi3k/akt/tor cellular signalling is known (falabella et al., unpublished data). the viral genes potentially involved in this process have been identified (falabella et al., unpublished data). moreover, through a transcriptomic approach, viral genes differentially expressed in pgs were identified. these genes could be involved in modulating ecdysteroidogenesis pathways. tnbv gene expression in haemocytes of h. virescens parasitised larvae is primarily responsible for inducing immunosuppression. the exact mechanism of host immune response inactivation by tnbv is not completely elucidated (varricchio et al., 1999; pennacchio et al., 2001; falabella et al., 2003; malva et al., 2004; provost et al., 2004; lapointe et al., 2005). indeed, the in vitro and in vivo investigation of the specific function of tnbv genes expressed in h. virescens immune cells is difficult. nevertheless, the functional https://commons.wikimedia.org/w/index.php?curid=1215079 243 fig. 2 viral genes expressed in haemocytes of heliothis virescens parasitised by toxoneuron nigriceps. gel profile of toxoneuron nigriceps bracovirus (tnbv) showing a segmented genome, with circular double-stranded dna molecules of different size ranging from 3.4 to 13.3 kilobases. tnbv genes expressed in haemocytes of parasitised heliothis virescens larvae are located on different viral circles (provost et al., 2004; falabella et al., unpublished data), conventionally named from 1 to 6, in order from the higher to the lower molecular weight annotation of several isolated tnbv genes (fig. 2) may indicate their possible role in disruption of haemocytes-mediated encapsulation reactions by the host (asgari et al., 1997; provost et al., 2004). among tnbv genes actively expressed in the haemocytes of parasitised larvae, the gene tnbv1 seems to be involved in the host immunosuppression (lapointe et al., 2005). in order to characterise the possible involvement of tnbv1 in immunosuppression of h. virescens parasitised larvae, this gene was heterologously expressed in various lepidopteran cell lines using different systems and the effects of gene expression on cell morphology and viability were evaluated. tnbv1 induced apoptosis when expressed in sf21 cells derived from spodoptera frugiperda and when expressed in high five cells derived from the lepidopteran tricoplusia ni (lapointe et al., 2005). however, when tnbv1-recombinant baculovirus was used to in vivo infect the host haemocytes, the expression of tnbv1 does not exert any apoptotic effect, leaving the role of tnbv1 expression in parasitised host larvae only partly understood. it is possible that during parasitism tnbv1 protein may interact with other viral or host proteins, inducing the apoptotic pathway in synergy with them. on the other hand, all the effects observed in two different cell lines, both derived from lepidoptera and expressing tnbv1 alone, overlap with general effects observed in haemocytes of parasitised host. ferrarese et al (2005) demonstrated that following parasitism the total numbers of haemocytes transiently decreased and among them granulocytes showed actin cytoskeleton disruption and loss of adhesion properties. all these evident morphological changes point towards an induction of apoptosis, at least in granulocytes of parasitised host larvae (lapointe et al., 2005). the same apoptotic effects were observed in a polyclonal drosophila s2 cell line stably expressing the tnbv viral protein coding by thetnbvank1 gene, and in haemocytes of h. virescens larvae, both after natural parasitism and after tnbvank1 in vivo transient transfection (salvia et al., 2017). tnbvank1 belongs to the tnbv gene family including ankyrin motif proteins (anks) (falabella et al., 2007). the ank gene family, together with ptp encoding genes, is the largest and most conserved bracovirus gene family (bitra et al., 2012). these proteins show a high levels of amino acid identity to 244 the ankyrin motif of the drosophila transcription factor nuclear -factorkappa-light-chain-enhancer of activated b cells (nf-kb) inhibitor (iκb)-related protein cactus (meng et al., 1999; falabella et al., 2007). ikb, a member of the toll and imd pathways, is part of the immune defence system of insects, and in presence of some key molecules belonging to bacteria and fungi, activates the transcription factor nf-kb, that regulates the expression of several genes involved in immunity, for example the antimicrobial peptides (amps) (dushay et al., 1996; lemaitre et al., 1996; han and ip, 1999; de gregorio et al., 2001; uvell and engstrom, 2003). three ankyrin-like open reading frames (orfs) were identified in the tnbv genome and were named tnbvank1, tnbvank2 and tnbvank3 (falabella et al., 2007). all the tnbvank genes contain the ankyrin motif but the phosphorylable serine residues at the n-terminus, essential for the ikb protein function, are absent (falabella et al., 2007). moreover, all of putative proteins encoded by the tnbvank genes lack the c-terminal pest domain, characteristic in all actives iκb like proteins and essential for protein degradation (ghosh et al., 1998, ghosh and karin, 2002; webb and strand, 2005) and for the protein turnover control (rogers et al., 1986). on the basis of the predicted structure of tnbvank genes, it was demonstrated that the tnbvank1 protein binds nf-kb/rel transcription factors of the tumor necrosis factor (tnf)/toll immune pathway altering the signal transduction cascade (falabella et al., 2007). indeed, in parasitised h. virescens larvae, after bacterial challenge, the nuclear import of nf-κb was inhibited (falabella et al., 2007). moreover, the expression of tnbvank1 gene in human hela cells reduced the efficiency of the tnf-α-induced expression of a reporter gene under nf-κb transcriptional control, strongly suggesting that tnbvank1 protein is involved in the suppression of the insect immune response (bitra et al., 2012; falabella et al., 2007; thoetkiattikul et al., 2005). the same function was demonstrated for ank family members of microplitis demolitor bracovirus (mdbv) which, competing with the host ikb for binding nf-kb/rel, reduce the expression of amps (bitra et al, 2012). the early expression of the tnbvank1 gene (three hours after parasitism) in haemocytes of h. virescens last instar larvae and the presence of transcripts several hours after parasitism, corroborate tnbvank1 involvement during early immune response suppression of parasitised host, also through the activation of a kind of cell death attributable to a process of apoptosis (falabella et al., 2007). as reported before (salvia et al., 2017), tnbvank1 induces cell death in polyclonal drosophila s2 cells, that stably express tnbvank1 and in haemocytes derived from both h. virescens parasitised larvae or in vivo transfected using tnbvank1. coimmunoprecipitation and coimmunolocalisation experiments in tnbvank1 polyclonal drosophila s2 cells showed that tnbvank1 interacts with alg-2 interacting protein x (alix). alix is a multifunctional protein and it was first characterised as interactor of apoptosis-linked gene protein 2 (alg-2), that is a ca2+-binding protein necessary for cell death (missotten et al., 1999). it was demonstrated that tnbvank1-alix interaction is required to induce apoptosis in all cells expressing this gene, both in vitro (s2 cells) and in vivo (h. virescens larval haemocytes). indeed, upon alix silencing by rna interference (rnai), tnbvank1 was no longer able to induce apoptosis (salvia et al., 2017). among the gene families present in bracoviruses of different braconid wasp subfamilies, the ptp gene family is probably an ancient component of the ancestral bracovirus of both microgastrinae and cardiochilinae subfamilies. ptps are enzymes that catalyse the phosphorylation of specific protein target at tyrosine residues. together with protein tyrosine kinase, ptps regulate the phosphorylation level of cellular proteins (neel and tonks, 1997). the functional characterisation of bracovirus ptps expressed in their hosts may mean that most of them have a role in the success of parasitism. although in some cases ptp genes do not codify for functionality active enzymes, they could contribute to host/parasitoid interactions performing different functions, for example binding phosphorylated proteins and inhibiting their activities (provost et al., 2004). bracovirus ptps are numerous and characterised by a high degree of diversity, regarding also the target substrate, at least for ptps characterised until now. this heterogeneousness indicates that they may act altering several cell pathways in parasitised host, especially those regulating the host immunity and endocrine balance (provost et al., 2004). in particular, regarding parasitised host immune suppression, it is possible to hypothesise that bracovirus ptps could alter the haemocyte cytoskeleton, strongly reducing their ability to encapsulate the intruders and losing their phagocytic ability. in this last case we could compare the activity of bracovirus ptps with the ability of some pathogens, bacteria in particular, in using ptps to alter the control of actin dynamics in infected host cells, resulting in inhibition of phagocytosis (bleves and cornelis, 2000; de vinney et al., 2000; ernst, 2000; cornelis, 2002; gruenheid and finlay, 2003; singh et al., 2003; mustelin et al., 2005). among bracoviruses, the mdbv suppresses spodoptera frugiperda immune system inactivating phagocytosis, encapsulation and melanization reaction of haemolimph (strand and pech, 1995a,b; beck and strand, 2005; thoetkiattikul et al., 2005; strand et al., 2006; suderman et al., 2008). the putative proteins encoded by mdbv include 13 members of ptp gene family, but only 5 ptps show a potentially active catalytic domain, while the other 8 members of mdbv ptps seem to encode functionally inactive enzymes, since their sequence analysis showed modification not compatible with tyrosine phosphatase activity (pruijssers and strand, 2007). among the potentially active mdbv ptps, ptp-h2 and ptp-h3 are preferentially expressed in host haemocytes and localise to focal adhesions, also 245 when they are expressed in haemocyte-like cells, such as trichoplusia ni high five and drosophila s2 cell lines. in these cases, they strongly contribute to disruption of adhesion and inhibition of phagocytosis, complementing the activity of glc1.8, another mdbv gene with antiadhesive and antiphagocytic properties (suderman et al., 2008). moreover, mdbv ptp-h2 also induces apoptosis of sf21 cells. the transient expression of ptp-h2 in this cell line induced the depolarisation of mitochondrial membrane and caspase-dependent apoptosis (suderman et al., 2008). on the basis of these results suderman and colleagues (2008) hypothesised that ptp-h2 contributes to immunosuppression of hosts defences also with this activity. the tnbv genome includes 13 genes encoding putative classical ptps (falabella et al., 2006). all identified ptp genes have a specific domain including 10 conserved motifs found in vertebrates (andersen et al., 2001). no relation was found with dual-specificity ptps present in baculovirus or poxvirus (provost et al., 2004), challenging the hypothesis of a baculovirus as possible progenitor of bvs (drezen et al., 2003; federici and bigot, 2003). sequence analysis of the 13 tnbv identified ptps showed that 8 ptp genes have a complete and potentially active protein tyrosine phosphatase domain. the other 5 ptps encode incomplete putative proteins or they are interrupted by a stop codon or by a frameshift (provost et al., 2004). among the tnbv gene families, ptp is the largest one. its members lack introns, similarly to the cotesia congregata bracovirus (ccbv) ptp family (provost et al., 2004). using a multiple approach 4 ptps were identified as expressed in haemocytes of h. virescens parasitised larvae (falabella et al., 2006; falabella et al., unpublished data) (fig. 2). all ptp cdnas from tnbv encode putative classical ptps with cytosolic localisation. the expression of these ptps in haemocytes of h. virescens parasitised larvae suggests their contribution towards host immune system suppression induced by tnbv. although no experimental data support this conclusion, we propose tnbv ptps as responsible for the alteration of cytoskeleton dynamics during the cellular immune response. in particular, this event could occur during encapsulation and phagocytosis, similar to the mechanism adopted by some pathogens that, expressing their ptps as virulence factors, alter the host cell signal transduction pathway, modifying the actin rearrangements thus inducing inhibition of phagocytosis (bleves and cornelis, 2000; de vinney et al., 2000; ernst, 2000; pruijssers and strand, 2007) although similar data were found in case of ptps belonging to other bracovirus, where ptps are actually involved in alteration of host immunity, this remains to be verified experimentally. venom the venom of endoparasitod wasps is a mixture of compounds, mainly proteins, produced by venom glands, organs located in the female reproductive system in hymenoptera (fig. 1) (billen, 1987; dorémus et al., 2013). venom proteins start to be synthesised during the wasp pupal stages in the venom glands, connected to a reservoir where the venom is collected and stored (jones and wozniak, 1991) (fig. 1). the reservoir is directly attached to the terminal part of the oviduct, where the venom is mixed with the ovarian calyx fluid produced in the swollen base of the ovary (fig. 1). this fluid is composed of a protein mixture (ovarian proteins) and in case of endoparasitoids of lepidopteran larval stages, of polydnavirus (pdv) particles (webb, 1998). venom is one of the parasitic factor of maternal origin, injected together with the egg and other factors (i.e. ovarian protein and/or pdv) into the host body during parasitism (moreau and asgari, 2015). in synergy with all the parasitic factors, venom acts to assure an adequate environment, allowing the growth of progeny (beckage and gelman, 2004; moreau and asgari, 2015). in endoparasitoids associated with pdvs, venom regulates host physiology and no paralytic or lethal effects are observed, in contrast to the effects of venom injected into hosts by ectoparasitoids or by endoparasitoid lacking pdvs (wharton, 1993; quistad et al., 1994; parkinson and weaver, 1999; moreau et al., 2002; nakamatsu and tanaka, 2003; webb and strand, 2005; asgari, 2006). in many host/parasitoid systems, venom complements the activity of pdvs (kitano, 1986; tanaka, 1987) and in several instances some venom components provide protection for the eggs against the host immune system during the period between oviposition and expression of pdv genes (webb and luckhart, 1994). in endoparasitoids, venom can induce developmental alterations, host castration (digilio et al., 1998, 2000) and almost always plays an immunosuppression role in the host (webb and luckhart, 1994; luckhart and webb, 1996; asgari et al., 1998). the venom of t. nigriceps plays a fundamental role for the success of parasitism, indeed it was demonstrated that, when venom glands were removed from female wasps, no parasitoids emerged from host larvae, h. virescens (tanaka and vinson, 1991). on the other hand, in vitro experiments on h. virescens prothoracic glands (pgs) demonstrated that the t. nigriceps bracovirus (tnbv) alone was able to induce the biosynthetic inactivation of pgs (pennacchio et al., 1998b). regarding parasitoid wasp venom composition, it has thus far been analysed in at least 20 species belonging to different families, using different approaches (vincent et al. 2010; asgari and rivers, 2011; doremus et al. 2013; colinet et al., 2013a,b; 2014; shaina et al., 2016; yan et al., 2016; cusumano et al., 2018). the most recent studies combine transcriptomics of venom glands and proteomics of venom to identify the proteins inject by wasps into the hosts during parasitism (colinet et al. 2013b). among the different proteins identified in parasitoid venoms, many share significant homology with proteins which functions are known. 246 simultaneously, no experimental data exist for the vast majority of parasitoid venom proteins, so their specific role in parasitism generally remains unknown. indeed, most of the available data focus on proteins that are highly abundant or structurally related to genes in databases with predicted functions, but, when venom proteins are studied in a non-model organism, the interpretation of the results is very difficult (poirié et al., 2014). some of these factors, including serine proteases, metalloproteases or esterases (asgari and rivers, 2011) are shared by many wasp species, while others are known from only one or a small number of species. some enzymes are also present in hosts at very low concentrations, indicating that focusing exclusively on abundant venom components can potentially lead to overlooking factors that could have important roles in parasitism. the mechanism of entry into the host cells and the mode of action of parasitoid venom proteins are unknown, except in a few species whose venoms contain some virus-like particles (vlps). the best known vlps belong to leptopilina boulardi. they are produced in the venom gland and injected into host larvae, drosophila melanogaster, contributing to host immunosuppression. following injection into host larvae, vlps target haemocytes, leading to apoptosis and/or morphological changes (poirié et al. 2014). the main venom protein components of t. nigriceps have been identified through the combination of next-generation transcriptome sequencing and bottom-up proteomics (laurino et al., 2016). this integrated proteomic and transcriptomic approach allows to identify proteins and biological processes also in non-model organisms (escoubas et al., 2006; tang et al., 2010; safavi-hemami et al., 2014; labella et al., 2015). beside proteins similar to known venom components or annotated proteins (asgari and rivers, 2011; doremus et al., 2013; moreau and asgari, 2015), novel proteins, with no similarity in databases, were identified in t. nigriceps venom. although no functional data are available, we hypothesise on possible involvement of t. nigriceps venom components in alteration of host physiology and in particular in host immune system suppression. a total of 31 different proteins were identified in t. nigriceps venom. some of these proteins resulted similar to already described proteins in venom from other parasitoids, while others resulted identified for the first time in a venom. among the known proteins, hydrolases, followed by transferases, oxidoreductases, ligases, lyases and isomerases, was the major family similarly to that reported for venom of other parasitoids (asgari and rivers, 2011). it was speculated that some of the identified proteins could be involved in the host immune suppression directly, in synergy to each other and/or to the other parasitic factors (table 1). based on the merops database (http://merops.sanger.ac.uk), the t. nigriceps venom metalloproteases belong to the m12 (m12b subfamily) and m13 families (laurino et al., 2016). these enzymes could be involved in several biological and disease-related processes (van goor et al., 2009). regarding immunosuppression, there seems to be a parallel between the function of different subfamilies of microbial metalloproteases and the venom metalloproteases. indeed, entomopathogenic microorganisms use metalloproteases in order to suppress the insect cellular defence, inducing the degradation of host protection molecules (griesch and vilcinskas, 1998; liehl et al., 2006). this observation may indicate that t. nigriceps venom metalloproteases might have similar functions in host immune suppression, in addition to their possible activity in host protein and tissue degradation. besides, serine protease homologue (sph) and calreticulin were also found in t. nigriceps venom (laurino et al., 2016). these well-studied proteins are distributed in a wide range of organisms including insects, and it has been reported that they inhibit host cell encapsulation and act as immune suppressor factors in insect haemolymph (choi et al., 2002; asgari et al., 2003a; asgari and schmidt, 2003; zhang et al., 2004; kanost and jiang, 2015). in particular, sphs have a serine protease catalytic domain but lack one of the amino acid of the catalytic triad, essential for the proteolytic activity (hedstrom, 2002). sphs are frequently present as components of parasitoid venoms (hedstrom, 2002; de graaf et al., 2010) where they block the phenoloxidase (po) cascade at different levels (zhang et al., 2004), inducing the inhibition of melanization. possibly the serine protease homologue identified in t. nigriceps venom could play a similar crucial role in suppression of host humoral defences. the identification of a calreticulin-like protein in the venom of t. nigriceps is notable. indeed calreticulins, molecular chaperones acting as lectin and ca2+-binding proteins, have been previously identified as a component of several venoms of other parasitoids (de graaf et al., 2010; zhu et al., 2010; asgari et al., 2003b). in particular, a calreticulin in the venom of cotesia rubecula blocked pieris rapae haemocyte diffusion, inhibiting the encapsulation response (zhang et al. 2006). the calreticulin present in t. nigriceps venom could bind the intracellular calcium modifying its balance and altering all the cellular pathways in which ca2+ is involved, such as apoptosis, inflammation and activation of hydrolytic enzymes. in addition, a ci-48 like protein was identified in t. nigriceps venom (laurino et al., 2016), similar to the venom protein ci-48 previously identified in chelonus inanitus (vincent et al., 2010) and in microplitis demolitor venom glands (burke and strand, 2014). it was speculated that, in all these cases, the protein could have functions in the early phases upon parasitism that generally involve the inhibition of the host's immune response. regarding the function of elongation factor 1alpha (ef1-alpha) like protein, recovered in t. nigriceps venom (laurino et al., 2016), although its putative function in host/parasitoid interaction is still unknown, a similar protein was found in the venom http://merops.sanger.ac.uk/ 247 table 1 proteins identified in the toxoneuron nigriceps protein database venom putatively involved in the alteration of immune system of the host heliothis virescens (from laurino et al., 2016) t. nigriceps protein involved in immunity suppression contig corresponding acc. n. ncbi protein indicated in annotations obtained by t. nigriceps custommade database corresponding acc. n. protein obtained by blastp search membrane metallo-endopeptidaselike1-like 4489 gij340723203jrefjxp_003399984.1j e2afa5 venom metalloproteinase 3-like 7042 gij665805846jrefjxp_008551135.1j f4x7t0 serine protease homolog 90 isoform x1 18408 gij315131321jembjcbm69269.1j a0a034v0k7 calreticulin 5341 gij665788653jrefjxp_008559929.1j q8is63 venom protein ci-48° 18596 gij665819695jrefjxp_008558721.1j e6zck2 elongation factor 1-alpha 4154 gij665799004jrefjxp_008547400.1j k7ivs1 spermine oxidase-like 14112 gij665801233jrefjxp_008548621.1j v9iis9 ovalbumin-related protein x-like 4452 q8is84 of parasitoid leptopilina heterotoma (colinet et al., 2013b). moreover, it was also found as a secreted product in leishmania protozoan parasites, where it seems to be involved in the induction of host macrophage deactivation (nandan et al., 2002). among other t. nigriceps venom components possibly involved in host immune suppression, the spermine oxidase-like (smo) is a fad-dependent enzyme able to induce the production of reactive oxygen species. it plays several roles in numerous cell functions, and some of them are compatible with an alteration of immune responses (cervelli et al., 2012). although its biological role in venom has not been elucidated yet, it is tempting to speculate a similar mechanism regarding the host immune system. the ovalbumin-related protein x-like is a member of ovalbumin family and belongs to the ovalbumin serine protease inhibitor family (ovserpin). the serpin are well known proteins in insects because of their role in inhibiting the activation of the po cascade and thus the melanization production (kanost and gorman, 2008). this function could also be performed in t. nigriceps venom by this protein (laurino et al., 2016). among the 31 identified proteins of t. nigriceps venom, the 8 annotated proteins mentioned above (table 1) likely play a key role in host immune system suppression. this is in agreement both with the function of several other endoparasitoid venoms and with previous researches reported in case of t. nigriceps venom by tanaka and vinson (1991). even this finding supports possible role of t. nigriceps venom in the regulation of host immune system, to confirm what has been speculated so far, functional characterisation of venom components is required. teratocytes teratocytes are specialised cells deriving from the dissociation of the extraembryonic membrane, named serosa, during parasitoid egg hatching in the host haemolymph (dahlman, 1990, 1991; dahlman and vinson, 1993; de buron and beckage, 1997; ali et al., 2013). teratocytes are produced only by endoparasitoid wasps in a restricted number of species belonging to braconidae, scelionidae, mymaridae, trichogrammatidae, aphelinidae and platygastridae families (strand et al., 1986; dahlman and vinson, 1993; quicke, 1997; hotta et al., 2001; basio and kim, 2005). all known teratocytes, once in the hemolymph, rapidly increase in size without undergoing any cell division and thus becoming highly polyploid. probably this characteristic optimises their secretory function, in order to provide nutrients to the parasitoid progeny larvae, directly or indirectly (strand and wong, 1991). teratocytes, indeed, release several molecules impacting physiology, development and nutritional suitability of the host. they are involved in several different functions, such as the manipulation of host development, through the inhibition of protein synthesis, or the disruption of the endocrine balance, which often modulates host biochemical changes that are nutritionally relevant for the developing parasitoid larvae. the nutritional role of the teratocytes is more direct and evident in host/parasitoid associations, where these cells perform an extra-oral digestion of selected host tissues, in order to allow the release of nutrients in a suitable form for the developing sister larvae (falabella et al., 2000; nakamatsu et al., 2002; falabella et al., 2005; grimaldi et al., 2006; falabella et al., 2009; caccia et al., 2012; grossi et al., 2016). 248 in addition, in several host/parasitoid systems, they contribute to host immunosuppression and/or in host immune system modulation for the success of parasitism (nakamatsu et al., 2002; rana et al., 2002; burke and strand, 2014; ali et al., 2015). in the cotesia kariyai and cotesia glomerata, teratocytes (taken at 4 days post parasitism) seem to play a role in preventing encapsulation during the early stages of parasitism by interfering with the phenoloxidase (po) cascade. nevertheless, in c. kariyai it is reported that they act in synergy with ovarian calyx fluid and venom: only when all 3 parasitoid factors were injected into unparasitised hosts along with first-instar parasitoid larvae, the encapsulation was inhibited (kitano et al., 1990; tanaka and wago, 1990). few works have addressed to the role of t. nigriceps teratocytes (dahlman and vinson 1993; pennacchio et al., 1992, 1994a,b; consoli et al., 2005, 2007; rossi et al., 2012). their functions may be nutritive, immunosuppressive, or secretory and may be involved in regulating host development (dahlman and vinson, 1993). it has been reported that they provide an important contribution to prevent h. virescens pupation. indeed, as previously reported in parasitised h. virescens last instar larvae, the inactivation of the biosynthetic activity of pgs, induced by toxonigriceps nigriceps bracovirus (tnbv) infection, results in an evident reduction in ecdysone titre in haemolymph (tanaka and vinson, 1991; pennacchio et al., 1998 a,b). the alteration of host ecdysteroid metabolism is mediated, at least in part, also by t. nigriceps teratocytes (pennacchio et al., 1994b). indeed, when teratocytes of t. nigriceps were injected into unparasitised h. virescens 1 day old last instar larvae, they inhibited host pupation (pennacchio et al., 1992). results suggested that teratocytes acted by affecting the haemolymph ecdysteroid titre, through inactivation of the 20hydroxyecdysone, which is converted into polar inactive metabolites (pennacchio et al., 1994b; bloch et al., 2002). to date, only 1 protein has been identified in t. nigriceps teratocytes: it is an isolated and functionally characterised putative chitinase (consoli et al., 2005, 2007; rossi et al., 2012). chitinases are well known enzymes that catalyse the hydrolysis of chitin, but they can also be produced as a defence mechanism, as it has been shown by the expression of chitinases in transgenic plants challenged with micro-organisms, nematodes, and insects (broglie and broglie, 1993; lin et al., 1995). it has been hypothesised that t. nigriceps chitinase, released by teratocytes, may either play a putative protective role preventing any possible microbial infection in the host haemocoel, since host-immune defences are suppressed. moreover, it could promote host cuticle digestion, allowing the parasitoid larva to emerge from the host body, since the parasitoid larva does not possess powerful mandibles at egression (lewis and vinson, 1968). recently, we established a de novo transcriptome of t. nigriceps teratocytes (falabella et al., unpublished data). the functional annotation and analysis of contigs allowed us to identify several transcripts coding for putative proteins possible involved in the success of parasitism, through the modulation of host immunity. in particular, similarly to the transcriptomic analysis of microplitis demolitor teratocytes, where the expression of several antimicrobial peptides (amps) was found (burke and strand, 2014), we identified amps, belonging to defensins, attacins and lysozyme families, putatively expressed by t. nigriceps teratocytes and potentially released in the host haemolymph (falabella et al., unpublished data). teratocytes might prevent host infection by pathogen intruders supporting the immunosuppressed host by the production of amps. moreover, the same analysis allowed us to identify putative proteins belonging to ovalbuminrelated x-like, serpin and rho gap families (falabella et al., unpublished data). transcriptome analysis of cotesia plutellae teratocytes reported that these cells express several putative immunosuppressive factors, including several serpins and rhogaps proteins, involved in the inhibition of the prophenoloxidase (ppo) cascade system, inducing host immunosuppression (ali and kim, 2015; kanost, 1999). taken together, this information may indicate that also t. nigriceps teratocytes may have a role in host immunosuppression. however, the specific functions remain to be elucidated requiring further studies. concluding remarks although insects have only an innate immune system, they have evolved several complex strategies to defend themselves against pathogens. on the other hand, hymenopteran parasitoids, especially endoparasitoid, have coevolved an arsenal of factors that allow the physiology of the host to be synergistically regulated, in order to ensure the complete development of their progeny. these parasitic factors induce diverse and complex reactions against the host defence mechanisms. to date, these intriguing processes are still poorly understood. this review reports an overall picture describing the strategy used by t. nigriceps in inhibiting the immune system of its host, the lepidopteran h. virescens (fig. 3). the findings reported in previous works and some preliminary results showed here, highlight the connection and the fine regulation performed by all parasitic factors in host immunity inactivation. almost all the identified t. nigriceps bracovirus (tnbv) genes are expressed in host haemocytes and, as consequence, can be considered putatively involved in the host immunity alteration (fig. 2). to date the genome of tnbv is being sequenced and this could lead to the identification of new genes. once tnbv genome will be functional annotated, we will obtain an overview on the possible roles of putative viral proteins expressed in host tissues will be available. however a functional characterization of all the viral genes will be required. the role of each viral gene will be studied by in vivo transfection in h. virescens haemocytes as previously reported for tnbvank1 (salvia et al., 2017). 249 fig. 3 overview of toxoneuron nigriceps parasitic factors and their effect on the host immune system. overall scheme of t. nigriceps parasitic factors of maternal and embryonic origin and their role in heliothis virescens immunosuppression. moreover, 8 out of 31 venom proteins are putatively responsible for host immune suppression (table 1). further analysis of these proteins are required to understand their function and their contribution to the success of parasitism and more specifically to immune suppression. possible strategies could be the injection of each purified venom protein into the host larvae (digilio et al., 1998, 2000) and/or with a more advanced approach, the silencing of their expression into t. nigriceps venom gland (lynch and desplan, 2006; colinet et al., 2014). at last, also the teratocytes, a parasitic factor of embryonic origin, release molecules clearly involved in the control of the immune response of the parasitised host. the functional annotation of the de novo transcriptome may indicate the involvement of antimicrobial peptides released by teratocytes in the regulation of host immunity. neverthless only the functional characterization of each of them, produced by chemical synthesis or recombinantly, according to their size, will provide most of the information about their role in the host immune system regulation. what emerges from the information reported in this review is that the regulation of the host immune system by parasitoids is not limited to its inhibition, but also to keep the host alive and in good condition, since it represents the environment and the nutritional resource for the adequate and complete development of the parasitoid offspring. concluding, t. nigriceps female 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mulberry genetic improvement, ministry of agriculture, sericultural research institute, chinese academy of agricultural science, zhenjiang jiangsu 212018, china accepted june 12, 2018 abstract despite the low diversity and high variability at intraand interspecific levels, gut microbiota of insect play important roles in digesting and absorbing nutrients, health and development, even in resistance and immunity. in this study, we investigate the composition and diversity of gut microbiota in the 5th instar of silkworm larvae since the mount of ingested mulberry leaves of this stage is about 85% of the total amount. intestinal contents were collected at 0, 12, 24, 48 and 72h in the 5th instar. 32, 21, 29, 24, 14 phyla and 386, 163, 292, 196, 144 genera were detected respectively in the samples described above. in general, the dominated phyla in the larvae gut were firmicutes, cyanobacteria, proteobacteria, actinobacteria and bacteroidetes; the dominated genera were enterococcus, staphylococcus, arthrobacter, pseudomonas, lactobacillus, bacteroides, paenibacillus and serratia, respectively. the range of phyla and genera was different and there were two peaks of microbial diversity at 0 and 24 h. compared with the intestinal bacteria of these time points, there were unique bacteria and 56 common ones. in these bacteria, the abundance of gram-positive bacteria including enterococcus and staphylococcus were the highest. our results provided foundation and reference on the research of intestinal microorganisms and resistance breeding in silkworm. key words: silkworm, gut microbiota, composition, pyrosequencing 16s rrna genes introduction the silkworm, bombyx mori, an important economical insects and model organisms of lepidopteran, has contributed enormously to the study of lepidopteran genetics and immunology (goldsmith et al., 2005; hou et al., 2011). the silkworm corporeity is closely related to sericulture production, the related factors of effected corporeity include climatic condition, quality of mulberry leaves, silkworm strains, digestion and absorption of nutrients. digestion, absorption and disease prevention of silkworm were closely related to the microbiota lived in the silkworm digestive tract (yuan et al., 2006). therefore, understanding the change of intestinal bacteria can help us improve the utilization of mulberry and health of the silkworm. ___________________________________________________________________________ corresponding author: chengxiang hou the key laboratory of silkworm and mulberry genetic improvement ministry of agriculture jiangsu university of science and technology zhenjiang 212018, jiangsu province, china e-mail: cxhou587@163.com the insects gut inhabits many non-pathogenic microorganisms, which is called a special growth environment of microorganisms. the microbial community structure of insect gut is affected by insect species, the morphology of digestive tract, feed, feeding habits, insect age, pathological conditions and the like (dillon and dillon, 2004). these intestinal bacteria play important roles in metabolism, health, reproduction, immunity and pesticide degradation (maslowski and mackay, 2011; simon et al., 2011; lee and brey, 2013). they are also involved in host-parasite interaction, thus influence evolutionary and ecological processes of insect by affecting host biology (feldhaar et al., 2011; aptedeshpande et al., 2014). therefore, it is necessary to access the composition and diversity of the intestinal bacteria, which can provide some new insights in development, immunity, new pesticide exploration, even evolution of insect. molecular markers had been widely used in analysis of silkworm traits (li et al., 2007; xiang et al., 2007; li et al., 2013). 16s rrna genes are important references to identify and analyze phylogenetic bacteria, which are common markers 224 of bacteria with highly conserved and special sequences (caporaso et al, 2011). the related study of intestinal microbiota in silkworm was few. some classical microbiological and molecular techniques had been used to investigate the bacterial composition of the silkworm gut, such as culture-dependent isolation methods and dgge fingerprinting (yuan et al, 2006; xiang et al., 2007). however, these relatively low-throughput techniques cannot provide enough information about the microbial composition and diversity in hosts. compared with these methods, the recent rapid development of high-throughput sequencing techniques provide new methods in analysis of 16s rrna genes, genome sequence and transcriptome (boissière et al., 2012; hou et al., 2011; 2014; li et al., 2016; chengxiang et al., 2017; zhang et al., 2017). we explored the bacterial compositional changes and diversity at different developmental times by sequencing 16s rrna genes to understand the host-bacterial relationships in silkworm gut. materials and methods silkworm strain the silkworm strain dazao was provided by the sericultural research institute, chinese academy of agricultural sciences. the silkworm larvae were reared by using fresh mulberry leaves at standard temperature and humidity. the newly exuviated 5th instar larvae were used for the experiments. collection of intestinal contents the newly exuviated 5th instar larvae were fed with mulberry leaves. the mid guts of 15 larvae were dissected in a sterile environment at five time points (0, 12, 24, 48 and 72 h after feeding with mulberry leaves) respectively. three repeats of each time points were collected. prior to dissection, the dissecting apparatus such as scissors and forceps were sterilized by autoclaving and uv treatment. the intestinal contents were immediately collected into the sterile eppendorf tubes, quickly frozen in liquid nitrogen and stored at -80 °c. dna extraction, pcr amplification, library construction and sequencing the genomic dna of the intestinal contents was extracted using sds method, then the extracted dnas were detected by agarose gel electrophoresis and fluorimeter, then the eligible dna samples were diluted to 1 ng/ul. 16s rdna amplification sequencing was amplified based on illumina hiseq sequencing platform and the paired-end sequencing method, using high-fidelity enzyme and specific primers with barcode (515f: 5ʹ-gtgccagcmgccgcggtaa-3ʹ, 806r: 5ʹ-ggactachvgggtwtctaat-3ʹ) (caporaso et al., 2011; mchardy et al., 2013). the amplified system was 50 μl contained 10 ng dna, 5 μl 10x pcr buffer，0.5 μl 10 mmol/l dntps and 5 u/μl plantium taq, 1 μmol/l 515f and 806r primer 5 μl, respectively. thermocycling conditions were: 94 °c for 3 min; 5 cycles of 94 °c for 30 s, 45 °c for 20 s, 65 °c for 30 s; 72 °c for 90 s; then 20 cycles of 94 °c for 20 s, 55 °c for 20 s, 72 °c for 30 s; finally 72 °c for 5 min. the pcr amplification action used the phusion® high-fidelity pcr master mix with gc buffer kit (new england biolabs, usa). the amplification product was tested by 2% agarose gel electrophoresis. the aimed amplicons were purified by qiaquick gel extraction kit (qiagen, german) according to the protocols and their concentrations were determined by fluorimeter. the library was constructed by truseq® dna pcr-free sample preparation kit (illumina, usa) and quantified by qubit and quantitative real-time pcr, then the qualified library was sequenced in hiseq2500 pe250. among the sequenced sample libraries, there were 3 repeats for each sample of 0, 12, 24, 48, and 72 h in the 5th star. acquisition of effective tags raw tags of samples were obtained from machine data by splitting barcode sequence and pcr amplification primers, then splicing by flash (v1.2.7, http://ccb.jhu.edu/software/flash/). raw tags must be conformed to the following procedure by strictly filtrating and initially getting the high-quality data (clean tags): 1) tags interception: the first low-quality base site was truncated in raw tags from continuous low quality value (the acquiescent quality threshold was ≤ 19 base) to the setting length (the acquiescent length was 3 base). 2) length filtration of tags: after interception, further filter tags in the continuous high quality base with length was less than 75% of tags length. 3) acquisition of effective tags: the remaining tags after interception and filtration need compare and detected chimera sequences in the gold database (http://drive5.com/uchime/uchime_download.html) and remove them (http://www.drive5.com/usearch/manual/chimera_for mation.html), finally obtained the effective tags. operational taxonomic units (otus) clustering, species annotation and diversity analysis all the effective tags of samples were clustered into otus by uparse software (uparse v7.0.1001, http://drive5.com/uparse/) and the threshold of acquiescent identity was 97%. the representative sequence of otus were filtered according to their emerging frequencies and analyzed their species annotation of these sequence by mothur method and the ssurrna database of silver (http://www.arb-silva.de/), then the information of species annotation (the acquiescent threshold was 0.8-1) and taxonomy were obtained, finally the community composition of sample was counted in each classification level (kingdom, phylum, class, order, family, genus, species). the multiple sequence alignment were confirmed by muscle software (version 3.8.31, http://www.drive5.com/muscle/) and the systematic relationships of otus representative sequence were obtained. finally, all the samples data were treated by homogenization procedure, the treatment was carried out using the least amount in the data as standard, the followed alpha and beta diversity analyses, cluster and phylogenetic analysis were based on the normalized data. diversity analysis included alpha and beta diversity analysis, alpha diversity analysis (http://scikit-bio.org/docs/latest/generated/skbio.dive http://ccb.jhu.edu/software/flash/ http://drive5.com/uchime/uchime_download.html http://www.drive5.com/usearch/manual/chimera_formation.html http://www.drive5.com/usearch/manual/chimera_formation.html http://drive5.com/uparse/ http://scikit-bio.org/docs/latest/generated/skbio.diversity.alpha.html 225 rsity.alpha.html) was performed to calculate index of ace, chao1, shannon and simpson using qiime software (version 1.7.0), draw rarefaction curves, rank abundance and species accumulation curves by r software (version 2.15.3). ace and chao1 were used to calculate the bacterial community richness, while shannon and simpson were used to calculate the bacterial community diversity and estimate the total number of species. the higher index value of ace, chao1, shannon and simpson meant the more community number and diversity of microbiota. the bacterial diversity was evaluated by rarefaction curves, rank abundance and species accumulation curves. rarefaction curve was a representative value of alpha diversity, it was used to evaluate whether the sequencing was sufficient to cover all taxa or not, which could reflect the abundance of species in the samples. venn diagrams were drawn with the online venn tool (http://bioinformatics.psb.ugent.be/webtools/venn/), and used to represent their intersecting relations. beta diversity analysis was performed to get the maps of the phylogenetic tree, nonmetric multidimensional scaling (nmds), principal components analysis (pca) and principal coordinate analysis (pcoa). the unifrac distance was calculated by qiime software (version 1.7.0) and analyzed the molecular variance to directly compare the structure differences of bacterial community between two samples (schloss, 2008), then the phylogenetic tree was constructed by the neighbor-joining method of mega6.0. the maps of pca and pcoa were drawn by r software (version 2.15.3). pcoa was used to compare bacterial structures based on weighted unifrac from each library. pca was utilized to explore the correlation between dominant genera. in the maps of pca and pcoa, the difference of community structure was determined according to the condition of aggregation and separation between samples. nmds is commonly used to compare the differences among the samples and evolutionary information. the difference of community composition was small if the distance between two points was close. phylogenetic analysis phylogenetic analysis was performed to get the evolutionary and similar relationships. the evolutionary relationships of genera top100 were acquired by multiple sequence alignment, the relative abundance of these genera was combined and integrated, finally the map of phylogenetic relationships were gained by graphlan software (http://huttenhower.sph.harvard.edu/graphlan) (asnicar et al., 2015). the similar relationships of different samples, unweighted pair-group method with arithmetic mean（upgma） was used to cluster and analyze samples, construct phylogenetic tree of different samples, finally solve classification problems. results analysis of the sequencing data after a series of data management, 651,441 effective tags were obtained from the samples by illumina hiseq sequencing platform. 99.43% (647,707) of effective tags was assigned to taxon and 3,121 otus were obtained (table 1). there were 1200 otus in the pc0 group, and 394, 823, 484 and 220 otus in pc1, pc2, pc3 and pc4 groups, respectively. among them, there were 109 shared otus (support fig 1). after quality controlled, the length of total tags were 18,220,501 bp, length ranging from 191 to 381 bp and the average length was 251.1 bp. the index values of alpha diversity were calculated by qiime software (version 1.7.0) (table 2). in the index values of ace and chao1 for calculating bacterial abundance, the values of pc0, pc1, pc2, pc3 and pc4 group were gradually decreased in the index of acachao1 for calculating bacterial abundance, which indicated that their bacterial abundance was also decreasing. this result was consistent with the data in table 1. in the analysis of alpha diversity, the rarefaction curve showed that bacterial richness in the gut contents was different in 5 samples and they approached to table 1 sequence data and bacteria in the contents of the healthy silkworm gut sample name raw pe effective tags taxon_tag otus phylum class order family genus pc0 147,772 139,041 137,271 1200 31 58 120 209 368 pc1 150,265 146,121 145,682 394 21 35 78 116 163 pc2 47,301 44,601 43,772 823 29 46 105 169 292 pc3 161,610 156,628 156,070 484 24 43 87 141 196 pc4 168,714 165,050 164,912 220 14 28 55 81 144 total 675,662 651,441 647,707 3,121 119 210 445 716 1,163 the samples of pc0, pc1, pc2, pc3 and pc4 groups were collected from the mid gut contents of five time points in the 5th instar silkworm. the five times points were 0, 12, 24, 48 and 72 h after feed with mulberry leaves http://scikit-bio.org/docs/latest/generated/skbio.diversity.alpha.html http://huttenhower.sph.harvard.edu/graphlan 226 table 2 the index values of richness and diversity in the samples sample name shannon simpson chao1 ace goods_coverage pc0 3.771 0.729 1028.908 1021.058 0.995 pc1 1.016 0.241 383.591 417.949 0.997 pc2 5.760 0.844 926.030 898.144 0.997 pc3 1.342 0.428 439.077 469.194 0.998 pc4 0.240 0.039 227.480 250.154 0.999 pc0, pc1, pc2, pc3 and pc4 are samples mentioned in table 1 the saturation plateau (fig. 1). this result indicated that sequencing depth has been mainly covered all species in the samples and the bacterial richness of these time points in the silkworm gut could be estimated. it also indicated that species abundance of samples and species abundance of pc0 group was higher than others. the results of rarefaction curve were consistent with the data in table 1. beta diversity analysis was used to measure the changes of species composition on temporal and spatial scales by pcoa, nmds and pca. the differences between individuals and/or groups can be observed by pca and pcoa. nmds is commonly used to compare the differences among the samples and evolutionary information. the difference of community composition was small if the distance between two points was close. assignation of otus and composition of intestinal microbes a total of 675, 662 raw pe, 651,441 effective tags and 3121 otus were obtained after splitting, splicing and filtering. eliminating tags representing chloroplast of mulberry leaves, effective tags with 97% identity were clustered into otus and these otus were assigned to phylum, class, order, family and genus by rdp classifier (cole et al., 2005). 119 phyla, 210 classes, 445 orders, 716 families and 1,163 genera were detected in the intestinal contents of 5th instar silkworm larvae (table 1). among of them, the most abundant phyla were firmicutes (51.41%), cyanobacteria (32.24%), actinobacteria (3.07%) and proteobacteria (6.85%), respectively. the predominant genera were enterococcus (49.11%), staphylococcus (6.66%), arthrobacter (3.45%), lactobacillus (1.04%), pseudomonas (1.07%), respectively. changes of intestinal microbes to understand the changes of the silkworm intestinal microbes, the changes of composition and abundance of microbes were analyzed at phylum and genus level. the abundance of firmicutes, cyanobacteria, proteobacteria, actinobacteria and bacteroidetes was significant in mid-gut as shown in the histogram (fig 2a). in the histogram, the abundance of firmicutes occupied 71.20%, 3.21%, 13.99%, 72.92%, 98.49% at 0, 12, 24, 48 and 72 h time points, respectively; it decreased during 0-12 h of the 5th instar, and increased during 12-72 h. at these five time points, the abundance of cyanobacteria, proteobacteria and actinobacteria was 21.44%, 87.32%, 40.04%, 23.56%, 0.3%, 7.68%, 8.49%, 22.87%, 1.88%, 0.82% and 12.92%, 0.50%, 6.52%, 1.00%, 0.02%, respectively. among of them, the abundance of cyanobacteria increased rapidly before 12h, then gradually decreased during 24-72 h; there were no obvious changes about the proteobacteria abundance before 12 h while actinobacteria decreased at this time point, then their abundances both increased before 24 h and decreased during 48-72 h. fig. 1 rarefaction curves of the different samples. pc0, pc1, pc2, pc3 and pc4 are samples mentioned in table 1. rarefaction curves of otus were clustered according to the acquiescent sequence identity (97%). in this curve, abscissa axis and vertical axis represent the randomly extractive sequences number and otus number that can be constructed based on these sequences number, respectively. this curve reflects species abundance and sequencing depth. in this curve, species abundance of pc0 group was higher than others, the quantity of sampling is reasonable for curve tends to be flat 227 fig. 2 the dominant phylum and genera of all the samples at each time point; pc0, pc1, pc2, pc3 and pc4 were samples mentioned in table 1. the map of a and b were the changes of relative abundance of different bacterial phylum and genera, respectively. sequences that could not be classified into the known group were assigned as “unidentified”. in this fig, the abundance of enterococcus in firmicutes varied greatly in five samples, furthermore its proportion was higher in pc0, pc3 and pc4 than those of others. the dominant phylum and genera number of pc0 and pc2 was more than other samples enterococcus, staphylococcus, arthrobacter, pseudomonas, lactobacillus, carnobacterium, bacteroides, paenibacillus, serratia, frondihabitans, brachybacterium and bacillus were significantly abundant (> 1%). among of them, enterococcus, staphylococcus, lactobacillus, paenibacillus, carnobacterium and bacillus belong to firmicutes phylum; arthrobacter, frondihabitans and brachybacterium are part of actinobacteria phylum; pseudomonas and serratia pertain to proteobacteria phylum; bacteroides belongs to bacteroidetes phylum. the abundance of enterococcus, staphylococcus and arthrobacter at five time points was 48.32%, 1.99%, 0.44%, 71.91%, 98.03%, 17.26%, 1.67%, 1.74%, 0.49%, 0.33% and 6.52%, 0.12%, 1.89%, 0.51%, 0.12%, respectively (fig 2b). in fig 2b, the abundance of enterococcus quickly decreased before 24 h, then rapidly increased in the latter time and reached peak at 72 h; the staphylococcus abundance was gradual downward; and the abundance of arthrobacter decreased before 12 h, then increased a little and decreased during 48-72 h. difference of intestinal microbes at different times the gut microbes of silkworm were different at five different time points of the 5th instar larvae. at phylum level, the number of detected phylum were 31, 21, 29, 24 and 14, respectively. among them, the number of phylum with significant abundance (> 1%) were 5, 3, 5, 4 and 1, respectively (fig. 2a). the abundant changes of firmicutes, cyanobacteria, proteobacteria and actinobacteria were significant. the abundance of firmicutes were approximate at 0 h and 48 h, their abundances were 22.18, 5.09, and 0.75 times higher than those of 12 h, 24 h and 72 h, respectively. the abundance of cyanobacteria increased before 12 h, then gradually decreased. its abundance of 12 h, 24 h and 48 h was 40.8, 18.68 and 10.9 times higher than that of 0h, while its abundance of 0h was 7.22 times higher than that of 72 h. the abundance of proteobacteria was basically similar at 0 h and 12 h; compared with the abundance of 0 h, it increased at 24 h and was 3.97 times higher than that of 0 h, then decreased during 48-72 h, and the abundance of 0 h was 2.88 and 6.23 times higher than that of 48 h and 72 h, respectively. the abundance of actinobacteria was less than the former three phyla, while its abundance changed more rapidly. it decreased before 12 h, increased at 24 h, then continued to decrease until 72 h. based on the abundance of actinobacteria at 12 h, the abundance of 0 h, 24 h, 48 h and 72h was 26.37, 13.27, 2.04 and 0.41 times higher than that of 12 h, respectively. the numbers of detected genera were 368, 163, 292, 196 and 144, and the number of predominant bacterial genera (> 1%) were 8-4-8-2-1 at five time 228 fig. 3 common and unique genera analysis of samples at different time points. the analyses results were showed by venn diagram; pc0, pc1, pc2, pc3 and pc4 were samples groups mentioned in table 1. (a) the venn diagram of pc0, pc1 and pc2 samples groups. (b) the venn diagram of pc1, pc2 and pc3 samples groups. (c) the venn diagram of pc2, pc3 and pc4 samples groups. (d) the venn diagram of all samples groups, including pc0, pc1, pc2, pc3 and pc4 samples groups points, respectively. unique genera and common ones exist at the samples of different time points (fig. 3). venn diagrams of common and unique genera numbers were constructed to understand genera changes in the 5th instar. compared with detected intestinal microbes among 0 h, 12 h and 24 h, there were 126 common bacteria, 43, 11 and 55 unique ones at each time, respectively (fig. 3a). there were 99 shared bacteria, 24, 84 and 16 unique ones, among 12 h, 24 h and 48 h (fig. 3b). 67 common bacteria, 101, 20 and 13 unique ones, were present in the samples of 24 h, 48 h and 72 h, respectively (fig. 3c). compared with all detected intestinal microbes at these five time points, there were 56 shared genera, the number of unique ones were 105, 10, 39, 12 and 16, respectively (fig. 3d). cluster analysis of intestinal bacteria in the 5th instar silkworm according to the phylogenetic relationships between otus, the unifrac distance between samples was calculated by qiime software (version 1.7.0), a heatmap displaying the bacterial similarity of different samples by hemi software and the analytical map of pcoa by r software (version 2.15.3) were constructed (fig. 4). all the samples were assigned to three clusters: (i) pc0.1, pc0.2 (two repeats of 0 h in the 5th instar) and pc2.2 (the sample of 24 h) were grouped into a cluster. (ii) pc1.1, pc1.2 (two repeats of 12 h) and pc3.1, pc3.2 (two repeats of 48 h) were grouped into a cluster. (iii) pc4.1 and pc4.2 (two repeats of 12 h) were grouped (fig. 4a). 229 fig. 4 heatmap and pcoa map of all samples. unifrac distance was calculated by qiime software and used to construct heatmap and pcoa map. (a) heatmap was constructed by hemi software. rows and columns represented 5 samples 9 repeats, the similarity represented by the values in the heatmap of microbiota in samples. (b) pcoa map was drawn and analyzed by r software, the distance between two dots represented the similarity of species structure between two samples in the results map of pcoa, a dot represented a sample. the closer the sample distance, the more similar the species composition structure was. our pcoa results showed that the samples were clearly separated (fig. 4b); contribution of the principal components to samples difference, 72.34% and 17.14%, were represented by pc1 and pc2 axis, respectively. pcoa result suggested that the different developmental stage could affect the distribution of the silkworm intestinal bacteria. it showed that the composition of pc0 and pc2 were approached, pc1 and pc3 were nearest. it was also showed that the analysis result of pcoa and heatmap were concordant. phylogenetic tree of predominant microbes phylum to understand the similarity of different samples, unweighted pair-group method with arithmetic mean (upgma) was used to cluster and analyze samples, construct phylogenetic tree of different samples, finally solve classification problems. 16s rdna gene sequences of predominant phylum for pc0, pc1, pc2, pc3 and pc4 were selected to blast and calculated the unifrac distance matrix and constructed upgma phylogenetic tree (fig. 5). the components (phylum) of the phylogenetic tree were similar but their abundances were different. compared the abundance of predominant microbes phylum, pc0, pc3 and pc4 were assigned to one cluster, pc1 and pc2 were assigned to another cluster. phylogenetic relationships of genera level the relationships of genera top100 were acquired by multiple sequence alignment, the relative abundance of these genera was combined and integrated, finally the map of phylogenetic relationships were gained by graphlan software (http://huttenhower.sph.harvard.edu/graphlan) (fig. 6). in this map, the evolutionary relationships of genera from different samples, information about the relative abundance and distribution were displayed. discussion gut is digestive organ of insect larva, it accounts for about 2/3 of the volume of the body cavity at its maximum in some insect including silkworm. insect intestinal microbes harbor in digestive tracts with feature of low diversity but high variability at intra and interspecific levels (mathieu et al., 2014), they also play important roles in metabolism, development, reproduction, protection, resistance and immunity (yuan et al., 2006; xiang et al., 2007; hedges et al., 2008; oliver et al., 2009; vorburger, 2014). in the same insect species, its composition of intestinal microbiota was affected by several factors such as age, genetics and environment, especially diet (yun et al., 2014; david et al., 2016). furthermore, the innate immunity and physical barriers of insects limited the diversity of gut microbes by managing bacteria and formation of specific organ to separate the microbes from the host http://huttenhower.sph.harvard.edu/graphlan 230 fig. 5 phylogenetic trees of predominant microbes phylum in different samples. the phylogenetic tree was constructed using mega6.0 software and the bootstrap value was 1000 replications. samples groups pc0, pc1, pc2, pc3 and pc4 were mentioned in table 1 tissues (mathieu et al., 2014). the related research about other insect was abundant, but few in silkworm. the information of microbial compositional variation in the silkworm gut can help us identify the key aspects of microbial composition, and provide references for silkworm breeding. in previous study, two kinds of methods had been used to study intestinal microbes, which were culture-dependent and culture-independent methods. the former was the traditional low-throughput methods, including the methods of isolation and culture-dependent, pcr and 16s rdnarflp; they often provide biased results and the isolated / detected genera were limited (tian et al., 2007; xiang et al., 2007). the latter based on the analysis of high-throughput sequencing and open database, they can provide adequate information (sun et al., 2016). furthermore, rarefaction analysis could judge whether the sequencing approach was sufficient or not by a plateau and whether the detected bacterial diversity was underestimated. in the identified intestinal microbes of different insects, there were some same phyla in the composition, such as firmicutes and proteobacteria; but there were also some differences with the effect of environmental habitat, developmental stage, phylogeny of host, especially diet (xiang et al., 2010; cirimotich et al., 2011). there were also some reports about the predominant genera intestinal microbes of silkworm. after the silkworm larvae of dongting × bibo strain reared with tricuspid cudrania and mulberry leaves, there were only four shared and dominant genera (brevundimonas, stenotrophomonas, enterobacter and staphylococcus) in their gut (xiang et al., 2010). in the monophagous c108 and the polyphagous scn2 strains, the shared predominant phyla and genera were proteobacteria and lactobacillales, enterococcus and thermus, respectively (xiang et al., 2007). in our study some predominant phyla included firmicutes, cyanobacteria, actinobacteria and proteobacteria, predominant genera included arthrobacter, staphylococcus, enterococcus, lactobacillus and pseudomonas were identified. compared with the former research, there were some same phyla and/or genera, such as staphylococcus and enterococcus. maybe like some polyphagous lepidopteran insect (david et al., 2016), there was a relative stable core microbiota in silkworm. we suspected that silkworm and their core intestinal microbes establish a symbiotic relationship of co-adaptation and co-evolution to maintain survival and reproduction in the evolutionary process. some gut microbiotas were related with the organism’s resistance, such as enterobacter bacterium (dillon et al., 2010; cirimotich et al., 2011). in the intestinal bacteria of silkworm, enterococcus is a high frequency microorganism and can reduce ph of digestive juice and inhibit some pathogens growth in intestinal environment through its products (takizawa et al., 1968). the functions of enterococcus complement each other. its antibacterial function is enhanced in the acidic condition, at the same time the ph abatement of intestinal digestive juice can protect hosts against attacks of toxins (mead et al., 1988; broderick et al., 2004; li et al., 2010). the appropriate supplementary of enterococcus faecalis in diets can increase immunity of organisms (shi et al., 2015). in the prevention and control of silkworm disease, it can be used as a possible probiotic for biological control since its secreta can strongly inhibit the germination of nosema bombycis spores in vitro (lu and wang, 2002). these research implied that the appropriate supplementary of enterococcus in vivo can enhance immunity of host. in our study, the number of enterococcus increased with the prolongation of feeding period. we suspected that this may be related with resistance and immunity of silkworm. the 5th instar is the key stage of silkworm development, the amount of ingested mulberry leaves of this stage is about 85% of the total amount in the whole larval stage. intake of mulberry leaves was continuously increased at the 5th instar before gluttonous stage, the numbers of detected genera were reduced while the percent of some genera were increased in this study, e.g. enterococcus. it 231 fig. 6 the map of phylogenetic relationships in genera level. inside circle was a phylogenetic tree of genera level which was constructed by the representative sequences of species. branch color represented the corresponding phylum (details see the top right corner of legend). outside circle of each genus represented the relative abundance in each group, the colors of relative abundance represented different groups. the thermodynamic maps according to the abundance of each genera were then drawn, their values were 10000 times of the original value then converted to the value of log 2 may be related to the resistant and metabolic function of the silkworm, ingestion and digestion substantial mulberry leaves to prepare for the development of silk gland and metamorphosis, meanwhile it also need increase its resistance to defend bad environment and pathogenic microorganism. furthermore, the last instar is also the critical period for the growth of symbiotic bacteria in host body, the food intake during this period provide the conditions for the growth of commensal bacteria, and this condition is propitious to the common development of insect and its symbiotic bacteria (mathieu et al., 2014). we thought the resistance and intake of mulberry leaves may be corresponding to the main and/or core microbes of silkworm gut. compared with the traditional low-throughput methods, much more information of coverage and diversity of the gut microbes in hosts was detected by high-throughput sequencing techniques (caporaso et al., 2011; boissière et al., 2012). 16s rrnas were used to evaluate the diversity of microbial communities in the gut by high-throughput sequencing techniques, microbial taxonomic groups were determined and compared by otus. the changes of proportional otus can be observed based on the differences of gut microbe. 16s rrna gene is not only a highly conserved and special common marker of bacteria, but also can be used to predict the functional microbes in the gut for some diseases which were associated with changes of microbial composition (turnbaugh et al., 2006; qin 232 et al., 2012). now some corresponding intestinal microbes were used to treat the specific diseases in medicine (zhang et al., 2013). microbial symbionts of insects are potential tools which can improve the innate immune homeostasis and contribute to the general insect wellbeing (crotti et al., 2013). in this research, we explored the richness and dominance of bacterial 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china 2medical research institute, southwest university, chongqing 400715, china 3college of animal and technology, southwest university, chongqing 400715, china accepted july 17, 2019 abstract the silkworm, bombyx mori has great economic and scientific value, as it has long been exploited as a primary silk producer and as a model system for lepidopterans and arthropod studies. this species is highly susceptible to microbial diseases that affect quality and quantity of silk, thereby causing huge economical losses. insects have developed efficient innate immune system to fight against microbial pathogens. the innate immune system plays a crucial biological role in the limitation of microbial infections by using different immune strategies such as antimicrobial peptides production (amps), reactive oxygen species generation and melanin formation. so far, many studies identified different biological factors, which are considered to be involve in the regulation of these biochemical processes in b. mori. here, we describe, current knowledge on the molecular patterns of various immune factors and also highlight their molecular mechanism of action in the limitation of viral, bacterial and fungal pathogens in b. mori. furthermore, we discussed different strategies to improve the immune responses of silkworm species. this review will be helpful to understand the molecular aspects of immune factors, and their regulatory mechanism to control microbial diseases in the economically important insect species, b. mori. key words: immunity; biological pathways; antimicrobial peptides; prophenoloxidase cascade; pattern recognition receptors introduction the silkworm, bombyx mori has great economic and scientific value, as it has long been exploited as a primary silk producer and as a model system for lepidopterans and arthropod studies (nagaraju and goldsmith, 2002). this species is largely cultured in many asian countries (e.g. china, japan, india) for silk production (faruki, 2005). however, silk industry is gradually declining due to microbial (viral, bacterial, fungal) diseases, which greatly affect the quality and quantity of silk (xu et al., 2015; abbas et al., 2017a). thus, researchers have paid attention to understand the defense system of b. mori for better management of this economically important insect species. invertebrates including insects lack the adaptive immune system, therefore they rely on an innate immune system to fight against invading microbial pathogens (dai et al., 2017; wu et al., 2017; chu et al., 2019). ___________________________________________________________________________ corresponding author: hongjuan cui state key laboratory of silkworm genome biology southwest university chongqing 400715, china e-mail: hcui@swu.edu.cn the immune system is further subdivided into cellular and humoral immune responses, which together provide an effective barrier to microbial infection (abbas et al., 2017a; zhu et al., 2019). when microbial pathogens pass through host's physical barriers (e.g. epithelium of midgut or cuticle) and reach the hemocoel, the host pattern recognition receptors (prrs) recognize pathogen associated molecular patterns (pamps) resided on the surface of the microbes and stimulate cellular and humoral immune responses (kanost et al., 2004; ishii et al., 2010; dai et al., 2018). the cellular responses are mediated by various types of immune cells, hemocytes (lavine and strand, 2002; zhou et al., 2017). while, humoral immunity largely stimulates the immune deficiency (imd) and toll pathway, which produce antimicrobial peptides (amps) through a signal transduction cascade, melanin and reactive oxygen species (ros) (kanost et al., 2004; kausar et al., 2017a). the silkworm b. mori, utilize innate immune system to fight against microbial pathogens during their life cycles. the larvae of this species are susceptible to viral, bacterial and fungal infection. the innate immune system comprising amps and 131 lysozymes, melanization and phagocytosis plays a crucial biological role in limiting microbial infections to a nonlethal level (zhang et al., 2017; kausar et al., 2018). over the past years, many researchers studied the innate immune system of b. mori and reported different immune associated factors, and also described their molecular mechanism of action in this species. in this review, we demonstrate the existing knowledge on the molecular patterns of b. mori immune system following viral, bacterial and fungal infection. further, this review will provide a comprehensive knowledge for researchers to further explore the immune system, and it will also help the industrialists for better management of silkworm. immune patterns against fungal pathogens fungi are one of the important groups of microbial pathogens that cause various diseases in silkworm and in other insect species (abbas et al., 2019b; zhu et al., 2019). the white muscardine is one of the most common disease among silkworm species, which is caused by entomopathogenic fungus, beauveria bassiana (chengxiang et al., 2017; lu et al., 2017; sun et al., 2018). the spores of this species germinate on the host’s integument, and penetrate into the hemocoel to obtain nutrients, and ultimately cause larval death (wang and wang, 2017). thus, it is highly important to understand the immune responses of b. mori for its better management. recent technologies (e.g. transcriptome analysis and suppression subtractive hybridization) have made it possible to investigate immune responses of this species against fungal infection (liu et al., 2015; yang et al., 2018). many researchers suggested that immune associated genes (cecropin b, moricin, lysozyme precursor, ubiquitin, and β-1,3-glucan recognition protein (βgrp)-3 precursor) greatly vary their expression after fungal infection (fig. 1) (hou et al., 2011; sun et al., 2017; wang and wang, 2017). it seems that these immune associated genes play a crucial biological role in the limitation of fungal infection, however the detailed molecular mechanism remained to define in b. mori. fig. 1 the putative toll and immune deficiency signaling pathway (immune pathways) involved in the immune responses against microbial (bacterial and fungal) infections in silkworm, b. mori 132 immune strategies of b. mori against fungal infection it has been shown that melanin formation and amps production are most effective strategies among insects to fight against fungal infections (kausar et al., 2017a). the melanin formation is initiated by ppo cascade, which is modulated by various proteins in insects (takahasi et al., 2009; kausar et al., 2017b). four βgrps (βgrp1, βgrp2, βgrp3 and βgrp4) have been identified and molecularly characterized in b. mori, which play a crucial biological role in the activation of ppo cascade in this species. for instance, the βgrp1 contains a β-1,3 glucan-binding domain and a glucanase domain; additionally, the α-chymotrypsin digestion analysis of this protein indicates that it contains two functional components: the first 20 kda (first 102 amino acid residues) component can bind to fungal β-1,3-glucan, while 43 kda (a glucanase like domain) can induce ppo cascade (ochiai and ashida, 2000; tanaka et al., 2008; chen et al., 2016). a recent study determined that fungal β-1,3 glucan fails to activate the ppo cascade in βgrp deficient plasma. however, it resumes its activity on the addition of recombinant βgrp-3 protein (takahasi et al., 2009). overall, both of these (βgrp-1 and βgrp-3) proteins are important modulator in the activation of the ppo cascade by recognizing β-1,3-glucan in b. mori. the ppo activation mechanism has been well-established in a model insect, e.g. m. sexta. briefly, β-1,3 glucans stimulate self-association of βgrp-2 proteins and forms a complex that provides a molecular platform for the subsequent events in the ppo activation. the c-terminal of this protein interacts with lowdensity lipoprotein receptor class a domains of hemolymph protease 14 (hp14) to recruit hp14 zymogens, which leads to the conversion of prohp21 to its active form, and also stimulates the ppo cascade (wang and jiang, 2007; dai et al., 2013; takahashi et al., 2015). however, the molecular mechanism of ppo activation still has not been completely understood in b. mori. to date, only few studies demonstrated the structural features of βgrps, and the molecular mechanism of ppo activation in this species. a recent study reported that hp6 and hp21 form a complex with serine protease inhibitor 5 (serpin 5) during the ppo activation (li et al., 2016a), however, the subsequent events still need to be elucidated. thus, to understand the detail mechanism of ppo activation in b. mori, future studies should focus to determine the events that occur following the detection of β-1,3-glucan by βgrps, the mechanism of interaction between βgrp/β-1,3-glucan complex and ppo cascade. although, some researchers described that pgrp-s5, βgrp-1, and βgrp-3 and hp6 and hp21 play a crucial biological role in the activation of ppo cascade. the pgs following β1,3-glucan recognition activate probaeease (probzargoetase) in the presence of ca2þ, and this ppo-activating protein can directly cleave ppo (satoh et al., 1999). the production of amps is another important strategy for the limitation of fungal infection in invertebrates (kausar et al., 2017a; min et al., 2017). the toll pathway has been reported to be involved in the production of amps in silkworm (abbas et al., 2017a; kausar et al., 2017b). this pathway comprises toll9 and baeease proteins, which are essential for its normal biological functions (jang et al., 2006; wu et al., 2010). in b. mori, pamps stimulate the conversion of the inactive probaeease to its active form, which seems to be a homolog of drosophila spatzleprocessing protein. this protein cleaves prospatzle to activate the toll signaling cascade in drosophila (jang et al., 2006). it is assumed that toll pathway in b. mori also follow the same patterns of activation like its counterpart, drosophila. growing evidence suggest that jak-stat pathway is also an important regulator of amps production (abbas et al., 2017a). c-type lectin 5 act as a receptor molecule especially for fungal infection in this pathway. the suppression of this gene by rna interference (rnai) can reduce the production of stat and hop. whereas, it enhances the expression of jak/stat inhibitors such as socs2. furthermore, depletion of jak/stat inhibitors enhance the survival and hemolymph fungi clearance activity (geng et al., 2016). abbas and his co-workers noted that downregulation of jak-stat inhibitor (socs2) stimulates the production of various amps in b. mori (abbas et al., 2017a). further, it has been reported that lebocin 5, defensin b, cecropin a, and gloverin 2) have strong anti-fungal activities and show variable expression patterns (kaneko et al., 2008; lu et al., 2016, ma et al., 2019). besides the toll and jak-stat pathways, some cuticle proteins (e.g. svwc) have also been reported to be involved in the limitation of b. bassiana infection (han et al., 2017). til-type protease inhibitors such as spi38 and spi39 (serine protease inhibitors/serpins) inhibit melanization caused by cdep-1, and also prevent the germination of b. bassiana spores (li et al., 2012; li et al., 2015). immune patterns against bacterial pathogens in insects, bacteria are most broadly studied group of microbial pathogens. they use host metabolic machinery for their growth and reproduction. further, they paralyze the host cellular and biochemical activities by producing pgs or by secreting proteases (karlsson et al., 2012; kong et al., 2015). to fight against infection, insects have developed innate immune system through the evolutionary period. the immune system initiates by the recognition of invading pathogen, and subsequently produce effectors to eliminate invading pathogens (ochiai and ashida, 2000; chen et al., 2016). in this section, we describe different strategies being utilized by b. mori to fight against bacterial infection. bacterial detection mechanism of silkworm the silkworm, b. mori contains a group of pattern recognition receptors for the recognition of bacterial pathogens. these receptors interact with pamps, subsequently initiate immune signaling in 133 table 1 the list of peptidoglycan recognition proteins s.no pgrps recognize source reference 1 pgrp-s1 pgs m. luteus yang et al., 2017 2 pgrp-s2 contribute in amp production chen et al., 2018 3 pgrp-s3 4 pgrp-s4 pgs b. subtilis, s. aureus, s. marcescens yang et al., 2017 5 pgrp-s5 pgs e. coli, b. megaterium, b. subtilis, m. luteus, s. aureus chen et al., 2014; 2016 6 pgrp-s6 7 pgrp-l1 8 pgrp-l2 9 pgrp-l3 10 pgrp-l4 11 pgrp-l5 12 pgrp-l6 pgs s. aureus, e. coli, b. subtilis tanaka and sagisaka, 2016 host cells (charroux et al., 2009; karlsson et al., 2012). it has been shown that gram-positive and gram-negative bacteria strongly stimulate immune responses in insects e.g. b. mori and drosophila (lemaitre and hoffmann, 2007; kausar et al., 2018). many in vivo and in vitro studies suggested that pamps (pgs and lps) induce the production of various amps (e.g. cecropin b and lebocin 3) in b. mori (hua et al., 2016; abbas et al., 2017b). the pgs are recognized by peptidoglycan recognition proteins (pgrps), so far, variety of pgrps have been identified and characterized in animals (christophides et al., 2002; tanaka et al., 2008; zhu et al., 2019). in b. mori first pgrp was identified from hemolymph during 1990s (yoshida et al., 1996). later, this number increases to twelve in this species (tanaka et al., 2008). as shown in table 1, the biological functions of only few pgrps have been described, suggesting these proteins are primarily used to detect pgs (chen et al., 2016; yang et al., 2017). however, subsequent events following recognition of pgs remained unclear. generally, lps-binding protein (lbp) recognize lps (a component of gram-negative bacteria) and initiates downstream signaling. for instance, the lps-lbp complex interacts with cd14, and delivered lps to toll-like receptors to stimulate downstream signaling in human (tapping and tobias, 2000; ranoa et al., 2013). in b. mori, lbp protein has also been described, and suggested to be involved in the clearance of bacteria (koizumi et al., 1999). but still the mechanism of action of this protein need to be illustrated. immune strategies of b. mori against bacterial infection the silkworm, b. mori fight against bacterial pathogens by utilizing different effective strategies such as reactive oxygen species (ros), amps, melanization and immune cells (tanaka et al., 2008; panthee et al., 2017). the production of ros (nitric oxide and hydrogen peroxide) play a crucial biological role in the limitation of bacterial infection in insects including b. mori (zhang and lu, 2015; kausar et al., 2018). nitric oxide can directly remove bacterial pathogens, or indirectly it stimulates immune signaling to produce anti-bacterial effectors (nappi et al., 2000; liu et al., 2019). a study showed that lps administration can induce the production of nitric oxide synthase 1, leading to nitric oxide generation that stimulate the amps (e.g. cecropin b) production (imamura et al., 2002). ros protect host from bacterial pathogens by preventing their cellular growth. however, the increase in its concentration may harm cellular components e.g nucleic acids, proteins, and lipids (abbas et al., 2019a; chu et al., 2019). to neutralize excessive level of ros, animals including b. mori have developed an antioxidant system (wu et al., 2017; dai et al., 2018; abbas et al., 2019a). the variation in production of antioxidant enzymes (e.g. peroxiredoxins, catalase and other antioxidants) after bacterial infection has been demonstrated by various authors in b. mori (e.g. shi et al., 2012; zhang and lu, 2015; wang et al., 2016), suggesting their involvement in the limitation of bacterial infection in this species. production of amps have also been considered an important immune strategy to control bacterial infection in insects. in d. melanogaster, it has been shown that the toll, imd pathways are activated and produce amps following bacterial invasion to limit infection (rutschmann et al., 2002; kaneko and silverman, 2005). in b. mori, many studies reported the enhancement of cecropin, attacin, gloverin, defensin, moricin, and lebocin after bacteria, pgs, and lps challenge (kaneko and silverman, 2005; tanaka et al., 2008; ma et al., 2019). these amps have strong antibacterial activities; however, their expression patterns vary with different type of bacterial strains. for instance, s. aureus strongly stimulate the expression of cecropin xj (xia et al., 2013), e. coli and b. subtilis enhance the production 134 of defensin b in fat body (kaneko et al., 2008). whereas, p. aeruginosa can stimulate the production of defensin, attacin, cecropin, lebocin, gloverin, and moricin in fat body. the production of amps is regulated by the coordination of cpt1 (tweed lecuticular protein), pgrp-s5 and lbp (liang et al., 2015; chen et al., 2016). huang and his co-worker (2009) demonstrated that b. bombyseptieus (grampositive bacteria) induce the expression of lebocin, attacin, enbocin, moricin, and gloverin in gut of b. mori (abbas et al., 2017; abbas et al., 2018). in addition, in vitro experiments on nises-bomo-cam1 cells showed that lps and other bacterial challenge can induce different amps expression (ishii et al., 2010; min et al., 2017). interestingly, isopropanol limits m. luteus infection by stimulating cecropin d, gloverin 3, and cecrop in fat body. along with the production of amps, regulatory mechanism is also activated to control their excessive production in b. mori. for example, serpin-5, serpin-6 and serpin-15 are important negative regulators of amps in this species. of which serpin-5 modulate the toll pathway by targeting hp6 and sp21 (fig. 1) (liu et al., 2015; li et al., 2017). overall, amps production is an important strategy to control the bacterial infection in b. mori. however, future studies should address the threshold level of bacteria and pamps, which is required to stimulate the production amps in b. mori. the melanin formation is used by insects as an important strategy to limit bacterial infection. pattern recognition receptors recognize the invading bacteria, subsequently stimulate the ppo cascade, leading to melanin formation. melanin is deposited on the bacterial surface to prevent their cellular growth and movement, and finally cause bacterial death. the ppo1 and ppo2 genes are activated after bacterial infection in b. mori (kawabata et al., 1995; clark and strand, 2013). additionally, in hemolymph of this species, po and its associated proteins form a complex, which is essential to initiate melanin formation (clark and strand, 2013). a recent study suggested that hindgut of b. mori express ppo gene, which activate the ppo cascade, leads to melanin formation and ultimately reduces the bacterial load (shao et al., 2012). furthermore, pgrp-s1, pgrp-s4, and pgrp-s5 also play a crucial biological role in the activation of the ppo cascade (chen et al., 2016; yang et al., 2017). to prevent excessive melanization process various negative regulators are also produced in insects. the negative regulators (serpin-5, serpin-6, and serpin-15) inhibit melanin formation by suppressing the activities of serine proteases in b. mori (liu et al., 2015; li et al., 2017). many studies demonstrated that hemocytes also play a key biological role in the suppression of bacterial infection in silkworm. b. mori contains five different types of hemocyte cells (plasmatocytes, prohemocytes, granulocytes, oenocytoids, and spherulocytes) of which plasmatocytes comprise phagocytosis activity and granulocytes play a role in the encapsulation of small particles (ling et al., 2003; zhang et al., 2014). however, the detailed mechanisms underlying encapsulation, phagocytosis, and nodulation remained unclear in this species. immune strategies of b. mori against viral infection viral infection is considered a serious threat to living organisms and their diseases cause approximately 20 % losses of b. mori cocoons each year. so far, there is no effective strategy to control viral infection in this species. thus, the viral studies have great importance for better management of silkworm species. a recent report suggests that use of transgenic silkworms with strong antiviral capacity to reduce its larvae mortality would provide new strains for sericulture (jiang, 2014; gupta et al., 2015). the b. mori viruses, especially nucleopolyhedrovirus (bmnpv) is a notorious pathogen in the silk industry. researchers fail to develop potential strategy to control this infectious agent in b. mori (hao et al., 2015; nie et al., 2017; wang et al., 2017; gao et al., 2018). it has been shown that the red fluorescent proteins (rfps) is an effective protein against bmnpv infection. this protein is specifically produced in the midgut of b. mori. this protein effectively disrupts the npv nucleocapsid or limits the npv multiplication or agglutinates the virus and is excreted along with fecal material. however, still there is need to explore exact biological antiviral mechanism of this protein (yao et al., 2006; gupta et al., 2015; zhang et al., 2018). many authors reported the involvement of serine proteases and lipases in viral immune response (e.g. ponnuvel et al., 2003). several studies described that lipases greatly contribute in the removal of viral pathogens. lipase-1, purified from the digestive juice of b. mori larvae was found to have great antiviral activity particularly against bmnpv. this gene (bmlipase-1) is produced only in the midgut of b. mori. ponnuvel and his co-workers (2003) examined the oral administration of pretreated bmnpv-odv (odv incubated with bmlipase) in 5th instar larvae of b. mori, these larvae displayed strong resistance to viral infection and successfully entered the pupal stage, suggesting it might be due to the suppression of viral proliferation by midgut lipase1 (ponnuvel et al., 2003). it has been shown that serine proteases modulate different defense responses such as amps production, melanization and hemolymph coagulation in invertebrates (gorman and paskewitz, 2001; lekha et al., 2015). the presence of serine protease in b. mori larvae display strong activity against bmnpv (nakazawa et al., 2004; kausar et al., 2017a). further, some recent studies suggested the enhancement of lepidopteran-specific amps (lebocin, gloverin-1, 2, 3, attacin, cecropin) and lysozyme after bmnpv infection silkworm (bao et al., 2009; ma et al., 2019). interestingly, gloverin4 has also been reported to be upregulated in b. mori and bmn cells suggesting it is specific biological role in the limitation of viral infection (bao et al., 2009). heat shock proteins (hsps) are a group of molecules, which are enhanced following stress conditions as we as they are involved in the folding and unfolding of proteins. hsp70 and hsp90 members of this family have been reported to 135 greatly express after bmbdv, bmnpv and bmcpv treatment (bao et al., 2009; yin et al., 2016). moreover, hsp19.5, hsp 23.7 and hsp 27 are strongly increased to limit viral infection (liang et al., 2007). therefore, it is supposed that hsps are involved in anti-viral immune responses and may activate the downstream signaling following detection of viruses. the pirna pathway has widely been studied in vertebrates and invertebrates. this pathway has been recognized as the crucial protection mechanism against the activity of transposable elements in genome of animals. the pirnas production is dicer-independent and depends on the piwi proteins activity, a subclass of the argonaute family (siomi et al., 2011). primary pirnas are processed from single stranded rna precursors that are usually transcribed from chromosomal loci primarily comprising remnants of transposable element sequences, named as pirna clusters (aravin et al., 2007). the processing of primary pirna precursors in d. melanogaster and production of mature pirnas have been linked to activity of zucchini endonuclease (ipsaro et al., 2012; han et al., 2015; mohn et al., 2015). the processed precursor is loaded into piwi family argonaute proteins piwi or aubergine and then cleaved by nuclease to reach its final length that range from 24 to 30 nucleotides, which vary in different insects such as fruit fly (25 nucleotides) mosquitoes (28 nucleotides). the trimmed pirnas undergo a final 3′ end 2′-o-methyl nucleotide modification induced by the methyltransferase hen1 (saito et al., 2007) to become mature pirnas. primary pirnas contain a 5′ uridine bias and are generally antisense to transposable element transcripts (saito et al., 2006). the cleavage of complementary active transposon rna by primary pirnas loaded into aubergine proteins starts the second biogenesis round and leads to the generation of secondary pirnas that are loaded in argonaute-3. during this amplification cycle, aubergine and argonaute-3proteins loaded with secondary pirnas mediate the cleavage of complementary rna to produce new secondary pirnas similar to the pirna that started the cycle. since target slicing by piwi proteins happens between 10 and 11 nucleotides, the complementary secondary pirnas have a 10 nucleotide overlap and comprise an adenine at position 10 (aravin et al., 2007). recently, it has been described that the pirna pathway is involved in in antiviral defense of insects. the pirna pathway antiviral defense activity was first reported in 2010, when small rnas of virus with the sequence length of pirnas were observed in d. melanogaster ovarian somatic sheet cells (wu et al., 2010). since then, this pathway involvement in the antiviral defense of insects has attained attention of researchers, and many studies on this subject has performed using mosquito-arbovirus experimental systems. in cell lines and aedes mosquitoes, an expanded family of piwi proteins is transcribed in somatic cells/tissues and viral-derived pirnas are generated from the genomes of many arboviruses (brackney et al., 2010; vodovar et al., 2010). furthermore, functional links among the pirna pathway, arbovirus replication, and vpirna generation have also been reported. suppression of piwi-4 protein has been found to increase replication of semliki forest virus [sfv; (+) ssrna, togaviridae] without interfering with vpirna expression in aag2 cells (schnettler et al., 2013), whereas both piwi-5 and argonaute-3 have reported to be needed for the biogenesis of pirnas from sindbis virus [sinv; (+) ssrna, togaviridae] in the same cell line (miesen et al., 2015). however, in vivo experimental information are scarce, and further studies are required to completely understand the extent to which the pirna pathway participate to antiviral defense in insects. collectively, in recent years, the pirna pathway has been widely studied in insects particularly in mosquitoes (e.g aedes aegypti) that broaden our knowledge on the complex picture of this pathway. this has resulted in the clarification of more and more details of this fascinating biological pathway and its variation and similarities to pirna pathways in other invertebrates and vertebrates, such as d. melanogaster. a more detailed knowledge of the pirna pathway in insects, particularly its potential participation in heritable immune system memory and likely effect on virus infection, will help us to understand the variations in vector competence among different species of insects and the spread of the pathogen. comparison of b. mori immune responses with other insect species researchers use virus, fungi, bacteria and its wall component (e.g. lps, pgn) as immune elicitors to understand immune responses in different insect species. many immune studies are available on various economically important insects such as b. mori, a. pernyi, actia selene and m. sexta etc, which have described the molecular mechanisms of immune responses (tokura et al., 2013; abbas et al., 2017a; kausar et al., 2017b). this section will provide a comprehensive overview of comparison of immune responses between b. mori and other insect species. the differences in the susceptibility of different insect species to viral, bacterial and fungal invasion may be because of their immune potencies (seyedtalebi et al., 2017). however, following microbial challenge, despite of the variation in experimental methods and species in the immune studies, different species approximately show similar immune response as innate immune system in insects remain conserved during evolutionary period (wang et al., 2019). however, some difference may exist that may occur in the molecular mechanism of the insect species. b. mori and m. sexta approximately follow the same mechanism of ppo activation following microbial infection. many of their molecules are similar in function with approximately same molecular mechanism. only difference has been reported at the final step of ppo activation in these species (sakamoto et al., 2011; tokura et al., 2013). in m. sexta serine protease homolog 1 and 2 are associated loosely with ppo and pap1 or pap3 to form a large complex. these proteins are also 136 needed for proteolytic cleavage to gain function that leads to their association into the active, high mr cofactor required in the molecular reaction with pap and ppo to produce high levels of po activity (gupta et al., 2005). interestingly, this molecular interaction seems to be not needed for b. mori ppae (wang and jiang, 2004), therefore further research is required to understand this interesting phenomenon. strategies to improve immunity of silkworm against microbial infection to improve the management of silkworm, attempts have been made to improve the immune response of silkworm species against microbial infection. the immune responses could be improved by optimizing and integrating antimicrobial strategies, improvement to antimicrobial silkworm strains, and generating transgenic silkworm species, which have increased resistance to microbial pathogens. using these strategies silkworm species can be generated that have improved immune responses against pathogens infection. the best example is the improvement antiviral immunity in silkworm species. rna interference and overexpression of antiviral proteins that efficiently targets viral genes are two greatly effective antiviral strategies. additionally, combining these methods using transgenic technique can further improves host resistance (jiang et al., 2013b). the silkworm strain sw-h is the first transgenic animal that have ability to reduce viral infection at its different stages. bmlipase-1 is regulated through the b. mori midgutspecific, highly activity p2 promoter in this transgenic species (jiang et al., 2013a) and double stranded rna for the tandem bmnpv genes such as gp64, ie-1, lef-2, lef-1, and dnapol is derived from hr3þie1p (jiang et al., 2013b). furthermore, by combining the different anti-viral strategies such as bmlipase-1 overexpression, suppression of different viral genes, hycu-ep32 overexpression, and rna interreference of bmpgrp2 could generate a transgenic silkworm species with greater antiviral resistance , which can suppress viral infection at initial stages of infection and affects the expression of viral genes and synthesis of proteins as well as host immune responses. additionally, several antimicrobial including antibacterial, antiviral and antifungal agents (e.g. seroin) have been described in silkworm that can be used as potent candidates for use in development of transgene‐based disease resistant silkworm strains (singh et al., 2014). conclusion and future perspectives in the past years, many researchers investigated the immune responses of b. mori against microbial pathogens. the whole genome sequencing of this species has enhanced the resource essential to systematically identify and characterize putative immune genes. to date many genes have been demonstrated to be involve in the immune responses of b. mori. our knowledge of b. mori antimicrobial immunity has also been greatly expanded. many studies suggested that the canonical immune signaling pathways are involved in antimicrobial immune responses of b. mori. however, there are still various questions that require to be addressed in the future studies. for instance, demonstrating the detailed molecular mechanism of anti-microbial (virus, bacteria, and fungus) immunity, and identifying new immune associated genes will be a greatly important field of future research. funding we are grateful for funding support from the national key research and development program of china (no. 2016yfc1302204 and 2017yfc1308600 to h. 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https://www.ncbi.nlm.nih.gov/pubmed/?term=luo%20y%5bauthor%5d&cauthor=true&cauthor_uid=20080648 https://www.ncbi.nlm.nih.gov/pubmed/?term=lu%20r%5bauthor%5d&cauthor=true&cauthor_uid=20080648 https://www.ncbi.nlm.nih.gov/pubmed/?term=lau%20n%5bauthor%5d&cauthor=true&cauthor_uid=20080648 https://www.ncbi.nlm.nih.gov/pubmed/?term=lai%20ec%5bauthor%5d&cauthor=true&cauthor_uid=20080648 https://www.ncbi.nlm.nih.gov/pubmed/?term=li%20wx%5bauthor%5d&cauthor=true&cauthor_uid=20080648 https://www.sciencedirect.com/science/article/pii/s105046481730637x https://www.sciencedirect.com/science/article/pii/s105046481730637x https://www.sciencedirect.com/science/article/pii/s105046481730637x microsoft word isj405   isj 13: 56-65, 2016 issn 1824-307x review coevolution of host-parasite associations and methods for studying their cophylogeny a filipiaka, k zającb, d küblerb, p kramarzb ainstitute of plant protection national research institute, władysława węgorka 20, 60-318 poznań, poland binstitute of environmental sciences, jagiellonian university, gronostajowa 7, 30-387 kraków, poland accepted march 2, 2016 abstract coevolution may be defined as the process of reciprocal, adaptive genetic change in two or more species. host-parasite interactions play an important role in the evolutionary ecology. the host phylogeny is independent, and the phylogeny of the parasite depends to some extent on the host. this review provides a description of several different methods for studying host-parasite relationships, along with a description of the underlying models and theoretical background for each. it also shows the possible applications of different methods and describes the advantages and drawbacks of different techniques. key words: coevolution; host-parasite interactions; cophylogenetic methods; event-based methods; global fit methods   introduction one of the most fascinating topics in evolutionary biology is the coevolutionary race between hosts and parasites, driven by antagonistic interactions which lead to reciprocal adaptations. most parasites have shorter generation times and are more numerous than their hosts, and therefore evolve faster, adapting to and increasing the selective pressure on the host. when parasites adapt to the most common host genotype, rare host genotypes become favored by selection, resulting in changes in genotype frequencies in host populations. this negative frequency dependent selection results in fluctuations in the relative frequencies of genotypes and also maintains genetic diversity. for example, decaestecker et al. (2007) studied the infectivity of the parasitic bacterium pasteuria ramosa to its host daphnia magna using resting stages of hosts and parasites from different layers of pond sediments. by exposing hosts to parasites from different layers, the researchers showed that bacteria from a given sediment layer were most easily able to infect hosts that originated from the same layer, indicating that the parasites were adapted to the most-frequently encountered host genotypes. knowledge about the influences of hosts on parasites, and vice versa, ___________________________________________________________________________ corresponding author: paulina kramarz institute of environmental sciences jagiellonian university gronostajowa 7, 30-387 kraków, poland e-mail: paulina.kramarz@uj.edu.pl becomes particularly important because of its relevance and applicability to many current problems in biology, such as controlling the spread of diseases, predicting the influence of expanding invasive species, or identifying and developing biological control agents (de vienne et al., 2013). the host-parasite association is also a representative model for studying adaptation. reconstruction of a parasite’s ancestry through the study of its ancestral host is far less complex than reconstructing the entire ecosystem for free-living species (paterson and banks, 2001). in interpreting relationships between coevolving lineages, however, it is important to adopt a phylogenetic perspective. because of this, in this kind of study it is necessary to reconstruct the patterns of relatedness between related hosts and their respective parasites. when we think about associations between hosts and parasites, we can find an analogy in vicariance biogeography. in this metaphor, the host can be considered an ‘area’ for the parasite to exploit, with host speciation as a ‘vicariance event’ (page and charleston, 1998). the relationship between host and parasite can also be seen as analogous to that between gene and species trees (page and charleston, 1998). in the past, parasites were more or less considered equivalent to host phenotypic characters and thus, the parasite’s phylogeny was believed to mirror that of the host (desdevises, 2007), a pattern known as fahrenholz’s rule (brooks 1985; huelsenbeck et al., 2000). this rule, formulated in 1913, helped to develop knowledge in the area of host-parasite 56   mailto:paulina.kramarz@uj.edu.pl   coevolution; however, further studies have shown that most host and parasite phylogenies are partly incongruent (brooks and mclennan, 1993; paterson and banks, 2001; desdevises et al., 2002). because, in general, a parasite’s phylogeny only imperfectly mirrors that of the host, paterson and banks (2001) proposed to reformulate farenholz’s rule. even when we take into account phylogenetic relatedness and phenotypic or ecological similarity, however, comparative analyses may still lead to confounding results because of limitations in the ability of current methodologies to model all possible evolutionary scenarios. during the common history of hosts and parasites, five basic categories of events can shape the coevolutionary process. these are: cospeciation, host switching, duplication of parasite lineages, sorting events, and inertia (page and charleston, 1998; paterson and banks, 2001; desdevises, 2007). cospeciation events occur when host and parasite species diverge together (e.g., during host isolation). in the majority of cases, it takes place in two-species systems, but there is some evidence for cospeciation involving three species, specifically, in a symbiotic system made up of a fly, a nematode, and a plant (nelson et al., 2014). host-switching occurs when a parasite establishes on a new host and diverges from the original form as selection favours adaptations to the new host (imber, 1985; brooks and mclennan, 1993; shaw, 1994; thompson, 1994; hoberg et al., 1997; paterson and gray, 1997; norton and de lange, 1999; ricklefs and fallon, 2002; susoy and herrmann, 2014). duplication takes place when only a parasite lineage, and not its host, speciates. sorting events may include extinction or ‘missing the boat’, in which speciation as the result of a founder effect leads to the loss of a parasite in a particular lineage of host (johnson et al., 2003). finally, ‘inertia’ refers to the reverse of duplication, when a parasite species remains the same despite host speciation. in order to take into account different evolutionary events when comparing host and parasite trees, researchers must make use of complex analytical and numerical methods. for example, many studies have attempted to identify instances of coevolution, including cospeciation between hosts and parasites, using phylogenies based on dna sequences (dowton and austin, 1995; huelsenbeck et al., 1997; huelsenbeck et al., 2000; dixon, 2002). relatively, several methods were developed to reconstruct hypothetical coevolutionary scenarios and to assess levels of cospeciation. the development of new methods along with advances in computing has brought an improvement in the power and precision of coevolutionary studies (charleston, 1998; page and charleston, 1998; cornell et al., 1999; paterson and banks, 2001; meier-kolthoff et al., 2007). this review provides a description of several different methods for studying host-parasite relationships, along with a description of the underlying models and theoretical background for each. it also shows the possible applications of different methods and describes the advantages and drawbacks of different techniques. theoretical background coevolution may be defined as the process of reciprocal, adaptive genetic change in two or more species (brooks, 1987; woolhouse et al., 2002; banks and paterson, 2005), and the study of this phenomenon relies heavily on the examination of phylogenies of eco-connected groups. in the case of hosts and parasites, the host phylogeny is independent, and the phylogeny of the parasite depends to some extent on the host (stevens, 2004). this system is unique among coevolving associations because of its intimate nature and the strong selective pressures that each population can exert on the other. the process of host-parasite coevolution plays an important role in maintaining genetic variation, which may lead to changes in patterns of biological diversity (thompson, 1994; gandon et al., 2002; woolhouse et al., 2002; tellier and brown, 2007; forde et al., 2008). it is important to note, however, that coevolution should not be confused with the concepts of cospeciation, codivergence, or cophylogeny, which differ from coevolution in several important aspects. cospeciation involves the joint speciation of two or more species that are ecologically associated (e.g., host-parasites). however, the speciation of symbionts may occur independently of host speciation, often through host shifts as the symbiont comes to occupy a new host environment in isolation from the ancestral lineage. cospeciation is expected to have congruent phylogenies but also to have similar divergence times. similar congruent topologies as seen in cospeciation could arise as a result of host switches followed by cospeciation events (or pseudocospeciation) but not have similar divergence times. some authors caution against the use of ‘coevolution’ as a synonym for cospeciation because of the implication that short-term dynamics contributes directly to cospeciation in the long term, although the rationale underlying this idea and its potential implications have never been fully articulated (page, 2003; smith et al., 2008; de vienne et al., 2013; herrera et al., 2016). codivergence is the parallel divergence of ecologically associated lineages within two distinct phylogenies, and is one predicted outcome of coevolution. when sustained codivergence is accompanied by multiple examples of parallel phenotypic change, reciprocal coevolution can be considered a more probable mechanism than, for example, secondary, one-sided evolutionary change by one species to match its co-mimic, as follows. codivergence may not prove coevolution in the strict sense. however, codivergence can be considered some of the strongest available evidence for coevolution (joron and mallet, 1998; page, 2003; hoyal cuthill and charleston, 2012). cophylogeny focuses on species associations (organisms tracking organisms, such as parasites and hosts or pollinators and flowering plants), molecular systematics (organisms or genes tracking genes) and historical biogeography (organisms tracking areas). studies on cophylogeny stem from the observation that the diversification patterns over evolutionary time of tightly associated organisms, such as parasites and their hosts, are seldom 57     independent. therefore, some degree of topological similarity, often termed congruence, between the phylogenies of the associated taxa is expected to occur. congruence quantifies the extent to which each node in a given tree maps to a corresponding position in the other tree and perfect congruence can be interpreted as evidence for cospeciation, which may or may not result from coevolutionary mechanisms (ronquist, 1997; jousselin et al., 2008; baum and johnson, 2010; balbuena et al., 2013). population genetics, natural selection in gene pools, adaptation, and isolating mechanisms have traditionally been considered by many biologists as significant factors in the evolutionary process. hosts and their symbionts are involved in intimate physiological and ecological interactions. the impact of these interactions on the evolution of each partner depends on the time-scale considered. coevolution might occur either in beneficiary relationships as mutualisms or symbioses, or in antagonistic relationships as host-parasite systems. symbiotic associations are widespread phenomenon and these have had a significant impact on the ecology and evolution of many prokaryotes and eukaryotes. for hosts that are obligately dependent on the symbiont for survival, acquisition of the symbiont is a critically important life-history event for all members of each new host generation. to date, the coevolution of hosts and microbial symbionts from various environments has been analysed to disclose their symbiotic history (paracer and ahmadjian, 2000; noda et al., 2007; dunlop et al., 2012; erler et al., 2012; de vienne et al., 2013). starr has developed a classification which was called the symbiotic continuum (starr, 1975). this classification of symbiotic categories is based on the potential fitness, or reproductive ability, of the symbionts. competition and mutualism are at opposite ends of the continuum, and neutralism is in the center. competition between interacting species produces detrimental outcomes to both species, whereas mutualistic relationships increase the potential fitness of the symbionts. thus, symbiosis means that two interacting organisms can influence each other's rates of survival and reproduction. other continua in starr's classification deal with the duration of the symbiosis, from transient to permanent; the physical contact between the symbionts, from incidental to close; and nutrition, from saprobic to biotrophic (paracer and ahmadjian, 2000). the popularly known symbiosis is lichen, which is a merger of algae or cyanobacteria and fungi. the cyanobacteria or algae provide the photosynthetic metabolism while the fungus can reorganize its membranes to sustain the lichen in extreme weather changes such as frozen tundra and desert rocks (hird, 2010). from an evolutionary perspective, host-parasite relationships may begin in conflict but eventually they move toward compromise, and the immune system plays a central role in the complex system of checks and balances. the basic protective strategy of an innate immune system is for the organism to constitutively produce generic receptors that recognize conserved patterns on different classes of pathogens to trigger an inflammatory response that limits pathogen invasion (read, 1994; cooper and alder, 2006). distinctions between self and non-self exist in all animals and show phylogenetic complexity and adaptive immune responses. mounting an immune response is metabolically expensive; a host may compromise between the available resources for its growth and development and for defence (behnke et al., 1992). a balance between the beneficial and potential harmful effects of immune responses to infection has to be considered in terms of a series of evolutionary trade-offs. for the host it involves resistance, pathology, and loss of resources, and for the parasite it involves reproduction, immunogenicity, and pathogenicity. compromise strategies have led to stable equilibria between many parasites and their hosts. genetic structure, evolution of microparasites, mechanisms of pathogenesis, and the evolution of immune response have consequences for public health, treatment, and prevention. the acquired immune response results in the recovery of the host from a disease and is followed by the host acquiring a specific memory, with which it responds vigorously to an infection by the same parasite (read, 1994; paracer and ahmadjian, 2000). parasitologists have often described the phenomenon of premunition, which is the immunity of a host to reinfection following recovery from disease. the exact mechanism of premunition is not understood. some parasites, however, have evolved novel strategies to counter a host's immune system and are able to establish long-term chronic infections (paracer and ahmadjian, 2000; cooper and adler, 2006). there are many methods for the study of the phylogeny of host-parasite associations, so called cophylogenetic analyses. generally, they are divided into two categories: "event-based methods" and "global fit methods". event-based methods apply the five coevolutionary scenarios described above (i.e., cospeciation, host switching, duplication of parasite lineages, sorting events, and inertia) to map the phylogenies of host and parasite. these methods are aimed at finding the most probable coevolutionary history of the associated taxa. moreover, event-based methods have strong appeal because they promise to deliver the coevolutionary history of the associated taxa. the process model and the cost assignments of the method reveal its properties. thus, it is straightforward to compare event-based methods and predict how they will perform when applied to particular problems. three challenges are important in their application. first, well resolved phylogenies are required to obtain reliable results and even with a small number of taxa the number of equally parsimonious solutions can be exceedingly high. then, event-cost methods are strongly dependent on a good estimation of the set of costs considered. and finally, given that not all the topological congruence between trees is necessarily a result of cospeciation, the precise reconstruction of coevolutionary history often requires additional data, such as the ages of the nodes, assumptions on the probability of the different events, consideration to the geological history of the areas involved and experimental evidence, such as reciprocal transplant experiments 58     (hypsa, 2006; de vienne et al., 2007; balbuena et al., 2013). in this category we find brooks’ parsimony analysis, reconciliation analysis, costbased methods, and probabilistic methods (cruaud et al., 2012; balbuena et al., 2013). global fit methods instead of focusing on individual coevolutionary scenarios assess the congruence between host and parasite trees and also specify particular host-parasite associations that can help in deciphering the cophylogenetic structure (desdevises, 2007). to some extent, the approach taken by global-fit methods is similar to statistical tests for congruence between two given trees. there is a clear need for this kind of methods because they afford large-scale cophylogenetic analyses for which the application of event-based counterparts becomes computationally prohibitive (balbuena et al., 2013). distance-based methods provide a good example of this approach. the advantages and disadvantages of each method are presented below. methods for studying cophylogeny 1. brooks’ parsimony analysis (bpa; brooks, 1981) one of the first numerical methods aimed at examining cospeciation events was brooks’ parsimony analysis, developed in the early ’80s. this method was proposed by wiley on the basis of ideas developed by brooks (morrone and crisci, 1995), and is based on the approach of mapping a parasite species and its phylogeny as characters and a transformation series, respectively, on the host phylogeny (brooks, 1988). the protocol of the analysis includes identifying congruent and incongruent parts of the parasite and host phylogenies. the second step is mapping the parasite phylogeny onto the host phylogeny and estimating the fit measures. among the many different phylogenies generated, the most parsimonious one is retained. in many cases, though, additional information is required to address incongruence in the host and parasite trees. that is the weakness of this analysis: although the number of overall ad hoc hypotheses is minimized, a large number of post hoc hypotheses is required. a suitable program for this method is hennig86; the bpa method, once widely used, has now been displaced by other methods. in historical biogeography and coevolutionary analysis, it became evident that homology and homoplasy, as revealed by bpa and component analysis, had no straightforward interpretation in terms of evolutionary events such as dispersals and host shifts (page, 1994; charleston, 1998). 2. reconciliation analysis (page, 1990; 1994; charleston, 1998) reconciliation methods use the same null hypotheses as bpa; however, these two methods differ in their essential approaches. reconciliation methods are based on conditions determined a priori by the researcher. the parasite phylogeny is mapped onto the host phylogeny, and the best scenario is chosen using the criteria of the minimum number of events inferred or the least cost (de vienne et al., 2013). the first software developed for use in reconciliation was component. it estimates the best reconstruction scenario by minimizing the number of extinctions and intrahost speciation events and maximizing the number of cospeciation events. however, it completely ignores host switching (page, 1994). the component performs several methods of comparison and can generate consensus trees, calculate the similarity between pairs of trees, and map one tree onto another. it has the ability to perform measurements such as partition metrics and quartet measures, which allow for the quantification of phylogenies between host and parasite. however, the biggest drawback of component, as mentioned above, is that it does not include host switching as a coevolutionary event (dowling et al., 2003). instead, treemap (page, 1994) includes all host switching scenarios, but does not model them correctly (charleston, 1998). this method tries to reconcile host and parasite phylogenies by maximizing the number of cospeciations and minimizing the number of host-shift speciations. there are no constraints on the numbers of intrahost speciations or extinctions or on numbers of parasites present on internal nodes, so the number of parasites infecting ancestral host species or number of intrahost speciations may be assumed to be unreasonably high (refrégier et al., 2008). the complexity of the problem of multiple host switches and temporal incongruence in the coevolution of hosts and parasites inspired charleston (1998) to provide a new algorithm, called jungle. this method was implemented in the program treemap2, and it takes into consideration each hypothesized past association individually in order to find globally optimal solutions. the algorithm considers the costs of different phylogenetic events that may affect a parasite-host association, including multiple host switches, and gives more realistic coevolutionary scenarios than treemap does. another useful tool for reconciliation analysis is tarzan, which allows the user to define the timing of the nodes in a parasite phylogeny and enables rapid analysis. unfortunately, it has some disadvantages: for example, the solution proposed by tarzan may not necessarily be optimal and sometimes it is not possible to find a solution even if it exists (merkle and middendorf, 2005). the program jane is slower than tarzan, but has many advantages. it not only allows the user to define the timing of the nodes in a parasite phylogeny (like tarzan does), but in a host phylogeny as well. in addition, the program enables the user to define different host-switch costs independently and the maximum permitted hostswitch distance (conow et al., 2010). 3. generalized parsimony or cost-based method (ronquist, 1995) ronquist (1995) proposed a method of reconstructing host-parasite associations based on costs or weights related to the likelihood of occurrence of different kinds of coevolutionary events. the method involves the conversion of a host phylogeny into a cost matrix and allows for different evolutionary events, including host 59     switches. after that transformation, the ancestral states (hosts) on the parasite tree are optimized. the weight of switching events is specified relative to tracking events. generalized parsimony algorithms are used to find the least-cost historical reconstruction of the host-parasite association. this method can be implemented in treefitter. it estimates the number of events of each type that could explain the observed congruence between the two phylogenies. then, treefitter associates each event with the probability that it arose by chance, calculated by permutations of the host and/or parasite leaves on the phylogeny (de vienne et al., 2013). its main advantage is that the costs of each event are set by the user (ronquist, 1995). the main disadvantages are that cospeciation is considered to be the most parsimonious hypothesis, and there is no possibility to set cospeciation as costlier than host-shift speciation. also, temporary non-compliance can appear, leading to erroneous conclusions. 4.probabilistic methods (huelsenbeck et al., 2000) to test whether host and parasite phylogenies are identical huelsenbeck et al. (2000) proposed two different null hypothesis tests. the first is based on maximum likelihood and apply a likelihood ratio test which evaluates the likelihood that the two phylogenies are identical versus the likelihood that they are not. the second is based on maximum posterior probability, uses bayesian inference to directly calculate the posterior probabilities of the host and parasite phylogenies. the maximum likelihood method uses standard statistical techniques for inferring probability distributions in order to assign probabilities to particular possible phylogenetic trees. it requires a substitution model to assess the probability of particular mutations; roughly, a tree that requires more mutations at interior nodes to explain the observed phylogeny will be assessed as having a lower probability. this is broadly similar to the maximum-parsimony method, but maximum likelihood allows additional statistical flexibility by permitting varying rates of evolution across both lineages and sites. in fact, the method requires that evolution at different sites and along different lineages be statistically independent (simmons, 2012). the bayesian method is based on a simple stochastic model of host switching by a parasite in which there is an assumption that host switching events occur at a constant rate. the posterior probability density of the parameters of the model is evaluated numerically using a markov chain monte carlo approach. the method is reliable, as it treats both host and parasite phylogenies as random variables and accounts for phylogenetic nonindependence. however, its main weakness is that it does not include some important biological processes it only considers host shifts and cospeciation. this analysis can be carried out using mrbayes (ronquist and huelsenbeck, 2003). although methods based on maximum likelihood and bayesian inference have been specifically designed to study the cophylogeny of host and parasites, the applicability of these methods to cophylogenetic studies is limited because they are primarily intended for one-to-one associations, something that rarely occurs in nature (balbuena et al., 2013). 5. distance-based methods another group of methods is based on statistical tests for congruence between host and parasite phylogenies. a comparison is made between the assumption that the two trees are independent (null hypothesis) and the probability of obtaining a certain level of congruence between the trees. in this kind of analysis there is the possibility of obtaining more reliable a posteriori interpretations. the large number of random trees that must be generated de novo for each new comparison of trees which is one of the weaknesses of these methods. to overcome this problem has been proposed a test of tree independence based on comparisons of the topological or genetic distances of the focal host-parasite association with a distribution of distances computed from a large number of randomly generated trees. tests of independence have also been used to evaluate temporal congruence in the speciation histories of hosts and parasites (kupczok and von haeseler, 2009; de vienne et al., 2013). the distance-based method described by hafner and colleagues (1994) uses alignments for specific loci from a host and a parasite to calculate distance matrices and then to test whether the host and the parasite have accumulated a similar number of genetic differences. after that analysis, a mantel test is conducted to assess the significance of the correlation between the two matrices. this test can verify statistical non-independence, but it does not handle testing for phylogenetic non-independence (hafner et al., 1994). in parafit (legendre et al., 2002), another distance-based method, either raw data or trees for a host and parasite can be converted into distance matrices. the parafit method adapts well to any kind of host-parasite association, including cases in which multiple parasites are associated with one host. it can then assess the contribution of each individual host-parasite association to the total congruence statistics (de vienne et al., 2013). however, the main drawback of this method is that it does not account for phylogenetic nonindependence (felsenstein, 1985). a different kind of permutation test was proposed by hommola and co-workers (2009). just as in the parafit method, this method tests the null hypothesis that a host and parasite have evolved independently of each other using their phylogenetic trees and host-parasite association links. both methods use permutation tests to determine whether cospeciation occurred. moreover, hommola’s permutation test is based on the calculation of pearson’s correlation coefficients between host distances and parasite distances, considering all pairs of interacting hosts and parasites. (hommola et al., 2009; de vienne et al., 2013). unlike parafit, this method does not evaluate the contribution of individual host-parasite links to the global cophylogenetic structure. in addition, this method differs from parafit in the 60   https://en.wikipedia.org/wiki/maximum_likelihood https://en.wikipedia.org/wiki/probability_distribution https://en.wikipedia.org/wiki/probability_distribution https://en.wikipedia.org/wiki/substitution_model https://en.wikipedia.org/wiki/mutation https://en.wikipedia.org/wiki/statistically_independent   randomization procedure to test the significance of the global-fit statistic (balbuena et al., 2013). schardl and co-workers (2008) proposed the mrcalink (mrca for most recent common ancestor) algorithm. this method identifies phylogenetically independent pairs between host and parasite trees and the reduced host and parasite matrices can then be compared. the method is less dependent on tree topologies, which often can be misleading, compared to tree reconciliation, and it crucially improves on phylogeny-independent methods such as parafit or the mantel test by eliminating an extreme (but previously unrecognized) distortion of node-pair sampling (schardl et al., 2008; de vienne et al., 2013). recently described method, paco (procrustes approach to cophylogeny) is based on procrustes analysis. this analysis is an extremely flexible technique used for displaying two or more multivariate datasets in their optimal superimposition. paco provides a superimposition plot enabling a graphical comparison of the fit of the host-parasite associations. this test can be carried out with any pair of distance or dissimilarity matrices, i.e., fully resolved host and parasite phylogenies are not required, and allows for multiple host-parasite associations and different number of hosts and parasites. paco is also similar to parafit in that it uses the same three data matrices as input and converts the phylogenies to principal coordinates (pco), and it is possible to assess the contribution of individual host-parasite associations to the global topological congruence. an important conceptual difference with the previous tests is that both parafit and test was proposed by hommola and co-workers (2009) compare the host and parasite distance matrices and test for random association between the host and parasite taxa, whereas paco explicitly tests the dependence of the parasite phylogeny upon the host phylogeny, because in the procrustean superimposition, the parasite matrix is rotated and scaled to fit the host matrix (balbuena et al., 2013). as shown above, different methods of studying coevolution can vary fundamentally from each other or only in details. it is possible to distinguish two main categories of methods according to the null hypothesis that they test: event-based methods, in which coevolution is considered the most parsimonious explanation for congruence between host and parasite trees, and distance-based methods, which test the independence or similarity between trees or alignments (de vienne et al., 2013). a very useful categorization was proposed by dowling et al. (2003). they distinguished two main classes of methods for studying host-parasite associations, a priori and a posteriori methods. their deliberations were inspired by van veller and colleagues’ (2002) suggestion that a priori and a posteriori methods for studying biogeographical patterns correspond to different research programs established on different ontological bases. by analogy, a priori and a posteriori methods for studying host-parasite associations, although they test the same null hypothesis, are based on different ontologies. a priori methods (e.g., reconciliation methods) modify the data to fit the null hypothesis of coevolution (parsimony is considered an a priori criterion) and therefore cannot be falsified. instead, a posteriori methods (e.g., bpa) provide the opportunity to falsify the null hypothesis when the data do not support it. in some cases a priori and a posteriori methods yield the same results and interpretations, but not always. if coevolution were influenced mostly by coevolution, an a priori method could be preferred. however, when we do not know if coevolution is the dominant event, a posteriori methods are superior; they avoid oversimplification of the results and false conclusions (dowling et al., 2003). moreover, de vienne et al. (2013) showed that convincing cases of coevolution are rare and that cophylogenetic methods often overestimate their occurrence. furthermore, based on a literature review and theoretical considerations, they concluded that parasite speciation by host shift is more common than coevolution (de vienne et al., 2013). thus, it is very important that cophylogenetic methods take host shifts and other evolutionary events into consideration. in addition, fahrenholz’s rule must be redefined as speciation via host shifts is at least as likely as coevolution (de vienne et al., 2013). moreover, incongruence between host and parasite phylogenies can occur despite a very intense cophylogenetic history. similarly, congruence is not evidence of coevolution congruence means little without additional supporting data (paterson and banks, 2001). it would be ideal if a testable method of reconstructing and interpreting all phylogenetic events existed; however, it is not available yet, and in studying hostparasite associations one has to make compromises. dowling and colleagues (2003) insisted that a priori methods such as reconciliation methods fail to satisfy three fundamental principles of phylogenetic systematics: the principle of total evidence (using all available evidence), the principle of maximum explanatory power (selecting the best a posteriori working hypothesis that best fits all available data) and the principle of falsification. in contrast, all of the above principles are satisfied by a posteriori methods (dowling et al., 2003). another advantage of a posteriori methods is that in the study of hostparasite associations, parasites are regarded as independent variables whereas a priori methods consider parasites to be characters of the host. that makes the results of a posteriori methods biologically more realistic. the ontological difference in perspective represented by a priori and a posteriori approaches reflect changing opinions about the nature of parasitism and the evolutionary independence of hosts and parasites (dowling et al., 2003). in the opinion of yang (2009), the most logical approach are event-based methods, provided that they include all scenarios. however, an excess of outcomes from a given analysis can lead to difficulty in finding the actual relationship. furthermore, both methods do not consider potential phylogenic sampling and reconstruction error. instead, global-fit statistical methods account for such error and provide a good overview of congruence in a 61     relationship. where they fall short, however, is in linking with specific coevolutionary scenarios. still all types of methods can be improved, and combining the advantages of existing techniques into a single approach will substantially help in future cophylogenetic analyses of all phylogenetic relationships (yang, 2009). molecular-based phylogenetic studies of hosts and parasites are increasingly common in the literature. phylogenetic comparisons of hosts and their symbiotic associates offer the potential for studies that extend well beyond simple documentation of cophylogeny. if data gathered about the host and its associate (and trees resulting from those data) are statistically independent, show significant cophylogeny, and are based on homologous molecular markers. then timing of cladogenetic events and possible differences in rate of molecular evolution in the hosts and associates can be estimated. such studies have the potential to elucidate broad evolutionary processes that influence rates of molecular evolution across distantly related taxa (hafner et al., 1994; light and hafner, 2007). for phylogenetic reconstruction at the species level, the use of several independent gene trees is recommended to overcome the effect of stochastic sorting of ancestral polymorphisms. in addition, a comparison of nuclear and mitochondrial (mt) dna genealogies can be a powerful tool for detecting hybridization (moore, 1995). molecular phylogenetic analysis based on multiple mitochondrial and nuclear loci would be especially useful when obvious incongruence is found between phylogenetic hypotheses from single molecular (e.g., mtdna) markers and non-molecular (e.g., morphological) evidence at an early stage of a phylogenetic study (normark and lanteri, 1998; sota and vogler, 2001). it is believed that on the basis of only one marker, we can not reliably reconstruct the phylogeny of the species. some studies revealed that mitochondrial dna (mtdna) has been found to be a ‘powerful subject for evolutionary studies’ (dowton and austin, 1995; huelsenbeck et al., 1997; dixon, 2002). this is due to the fact that the mitochondrial dna is more susceptible to introgression than the nuclear dna (coyne and orr, 2004). however, the other studies proved that mtdna haplotypes do not coincide with the morphological species. the phylogeny of the three nuclear markers, at least when combined in a simultaneous phylogenetic analysis, is more congruent with the morphologically recognized species than is the mtdna. importantly for the test of the type-switching hypothesis, the various nuclear genes also exhibit incongruence with one another, possibly suggesting an overall high amount of incongruence between the various parts of the genome (su et al., 1996; sota and vogler, 2001). page and colleagues (1996) suggested several basic pre-requisites for the study of host-parasite associations, among which they singled out: an adequate alpha-taxonomy of both host and parasites, accurate phylogenies of host and parasites, and exhaustive sampling of clades or molecular phylogenies based on comparable genes (page et al., 1996). unfortunately, accurate phylogenies of both hosts and parasites are not always available, but some of the methods described above (e.g., maximum likelihood methods) are able to generate robust data despite phylogenetic inaccuracies (huelsenbeck et al., 2000; paterson and banks, 2001). the disadvantage of maximum likelihood methods, though, is that they are limited to the study of molecular data and cannot take into consideration any other kind of data. nevertheless, molecular divergence itself is very useful in testing scenarios of phylogenetic events. it not only provides an opportunity to examine comparative rates of molecular evolution in host and parasites, but it also makes it possible to gain knowledge of the timing of different evolutionary events (paterson et al., 2000). as we have shown, the study of host-parasite interactions has benefited from some major advances in analysis. however, none of the existing methods is ideal. in reconstructing the phylogenetic history of a host-parasite association, the abovementioned methods can only yield possible scenarios of the common history of the studied species. researchers must keep the shortcomings of different methods in mind when interpreting the results and especially when comparing results and interpretations obtained by different methods. 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sonora, navojoa, sonora, méxico 2 universidad estatal de sonora, lic. nutrición humana, navojoa, sonora, méxico accepted june 18, 2019 abstract the white spot syndrome virus (wssv) is the most lethal virus that infect shrimps, generating economic losses for aquaculture industry. osmotic stress compromises the immune response in wssv-infected shrimp. betaine aldehyde dehydrogenase (badh e.c. 1.2.1.8) is an enzyme that participates in the regulation of osmotic stress, and that is up-regulated in shrimp exposed to changes in salinity concentration. however, different gene expression studies in shrimp during wssv infection focused in changes on the mechanisms of energy production and cellular defense genes, without considering genes sensitive to osmotic variation. in this work, we evaluate the mrna levels and activity of the badh enzyme, as well as glycine betaine (gb) accumulation, during wssv infection in white shrimp. shrimps were inoculated with wssv and samples from the hepatopancreas were collected at 24 and 48 h post-infection. our results demonstrate that wssv infection increases the expression of badh 1.5-fold at 48h and the enzyme activity increase 4.28and 4.59-fold at 24 and 48 h, respectively. also, higher accumulation of gb was observed during wssv infection. these results suggest that badh could participate in the cellular response during wssv infection in white shrimp. key words: betaine aldehyde dehydrogenase; white spot syndrome virus; glycine betaine; mrna expression; shrimp introduction twenty-six year ago, the white spot syndrome virus (wssv) emerged as one of the most severe shrimp pathogens, with high rates of mortality and prevalence in penaeid shrimp (oidtmann and stentiford, 2011). liu et al. (2009) summarized some of the genes and proteins that are upregulated and respond or interact with the wssv virus in crustaceans. most of the molecules described are related to inflammatory and adaptative response affecting survival rates, but little is known about the mechanism and interaction of impactful pathways during the disease. in addition, different aspects of the infection in invertebrates, and how the virus access the host cell is yet to be elucidated, and all the analyses available to this day are based on the response described in vertebrates. ___________________________________________________________________________ corresponding author. jesús alfredo rosas rodríguez departamento de ciencias químico-biológicas y agropecuarias universidad de sonora apartado postal 85880, navojoa, sonora, méxico e-mail: jesus.rosas@unison.mx several studies have shown that variations in salt concentration compromise the shrimp response to the wssv infection (liu et al., 2006, 2009; joseph and philip, 2007; vaseehara, et al., 2013; ramos-carreño et al., 2014; amrillah et al., 2015; raj et al., 2015; xhue et al., 2015; thuong et al., 2016; qianqian et al., 2017). however, to date, there are no studies that demonstrate the relation of osmotic regulation and wssv, or other viral, infections. therefore, the importance of the enzyme badh from white shrimp litopenaeus vannamei (lvbadh), which belongs to the aldh9 family in the invertebrate clade, arises, as it is involved in the synthesis and accumulation of the organic osmolyte glycine betaine (gb, n,n,n-trimethylglycine) (delgado-gaytán et al., 2017). gb is a watersoluble organic molecule with the ability to bind a significant amount of water molecules to keep hydration and therefore, stabilize proteins under stress conditions. also, gb can bind inorganic ions acting as bioprotectant to proteins (fedotova and kruchinin, 2017). most of the studies of gb has been done in mammals, where it has been reported that gb has a role in cell metabolism, in addition to mailto:jesus.rosas@unison.mx 114 fig. 1 relative expression of lvbadh during wssv-infection. the mrna levels were normalized with l8 as a constitutive gene. asterisks denote statistically significant (p < 0.05) difference from the control group. its functions as osmolyte or osmoprotector (figueroa-soto and valenzuela-soto, 2018). on the other hand, in white shrimp it has only been shown that nad-dependent aldehyde dehydrogenase is up-regulated by the wssv infection at high temperature (32 °c), and that aldh interacts with the 70 kda heat shock proteins (hsp70), which inhibits viral replication of the wssv (lin et al., 2011). recently, we demonstrated that lvbadh is expressed in a tissue-specific manner in white shrimp , and the activity of the lvbadh enzyme is modulated by changes in the salinity, suggesting that lvbadh is involved in mechanisms of stress adaptation (delgado-gaytán et al., 2015; delgadogaytán et al., 2017). fan et al. (2016) described an increase in the osmoregulatory system under wssv infection in crustacea cherax quadricarinatus. considering the role of lvbadh as a detoxifying and stress-response enzyme, in this work we evaluate the mrna levels and activity of lvbadh enzyme, as well as gb accumulation during wssv infection in white shrimp. materials and methods animal handling a total of 30 juvenile shrimp litopenaeus vannamei were collected from a farm located in the state of sonora, méxico. the shrimps were acclimated in individual systems for 4 weeks in 200 l aquaria, temperature 28-30 °c, 35 ppt of salinity, and were fed with commercial pellet (35% protein). the virus inoculum for experimental infection was obtained from white shrimp muscle of moribund shrimp infected with the wssv. the tissue was homogenized in buffer tris-nacl (20 mm tris ph 7.4, containing 400 mm nacl). the supernatant was obtained by centrifugation of the homogenate at 9,402 x g during 9 minutes at 4 °c and then filtered with 0.8 m pore filters. the inoculum was diluted with saline buffer (1.4 x 10 5 wssv copies total) and injected intramuscularly to shrimps for cumulative mortality reached 100% at 48 to 72 h posterior to wssv infection. the hepatopancreas (n =7) were collected from the three groups as follows: a) saline solution (ss) group at 24 h after starting the experiment, b) wssv group at 24 h post-infection, and c) wssv group at 48 h post-infection. mrna quantification total rna was isolated from individual hepatopancreas using the trizol reagent (invitrogen) following the manufacturer instructions. total rna concentration and integrity was evaluated by measuring the absorbance at 260/280 nm and by 1% agarose gel, while contamination of genomic dna was eliminated by digestion with rq1 rnase-free dnase (promega). total dna-free rna (1 μg) was used to synthesize one cdna, for each tissue sample collected, using oligo-dt and the goscript™ reverse transcriptase kit (promega). for lvbadh quantification we used the lvbadhf3 and lvbadhrv3 primers previously reported (delgado-gaytán et al., 2015). the quantitative pcr reactions were performed separately for lvbadh and normalized by the expression of the ribosomal protein l8 (mirandacruz et al., 2018). three pcr reactions for each sample were run (step one real-time pcr systems, applied biosystems) in a final volume of 15 μl containing 7.5 μl of ssoadvanced universal sybr green supermix (biorad), 6 μl of h2o, 0.25 μl of each primer (20 μm), and 1 μl of cdna 115 (equivalent to 125 ng of dna-free rna). after an initial denaturing step at 94 °c for 10 min, amplifications were performed for 40 cycles at 94 °c for 15 s and 63 °c for 1 min with a single fluorescence measurement, and a final melting curve program increasing 0.3 °c each 20 s from 60 to 95 °c. positive and negative controls were included for each gene. standard curves for lvbadh and l8 were run together with the cdna samples to determine the efficiency of amplification and the mrna concentration, which was calculated based on the concentrations of the standard curve dilutions (5e -4 to 5e −8 ng μl −1 of pcr fragments) by the stepone™ software v2.3 (applied biosystems). the expression level (ng/µl) of lvbadh was normalized to l8 and expressed as relative values (lvbadh/l8). activity assay activity of badh was evaluated following the plate-based spectrophotometric quantification of aldehyde dehydrogenases, reported by sreerama and sládek (2005) and adapted to the specific badh assay conditions reported by guzmanpartida and valenzuela-soto (1998). a 96-well flatbottom microtiter plate was set up with 180 l of the reaction solution (hepes 100 mm ph 8.0 with 1 mm nad, 0.5 mm betaine aldehyde (ba) and 14 mm -mercaptoethanol) and 20 l of the cytoplasmic extract from each animal tissue (samples were diluted at 1:2 ratio). the protein extracts from hepatopancreas of shrimp was obtained following the directions of the ne-per nuclear and cytoplasmic extraction reagents (thermo fisher scientific). the enzyme activity was evaluated by triplicate following the reduction of nad + at 340 nm for a total of 10 minutes. the rate of reaction (a/min) was multiplied by a factor of 1639 to obtain the enzyme activity as nmol of nadh/min/ml. the protein content of the extracts was determined using the quick start bradford protein assay (bio-rad) to express the activity as nmol/min/mg of protein. glycine betaine quantification gb concentration in the cytoplasmic extract was evaluated following the assay proposed by grieve and grattan (1983). the assay was adapted for the analysis using a microplate reader, considering the minimum time for the effective aqueous extraction and optimal acidity for periodide precipitation. the extracts were diluted 1:1 with 2n h2so4 prior addition of potassium triiodide and the glycine betaine crystals were obtained as previously described. a standard curve of gb (50-200 ug/ml) was prepared in 1-2-dichloroethane for analysis at 365 nm. statistics the normality of the data from each experiment was evaluated using the kolmogorov-smirnov test, and one-way anovas were used to determine statistical differences for each group using the tukey and fisher lsd means comparison; the noninfected shrimps were used as control (p<0.05). all data was analyzed using originpro 9.0 software originlab, northampton, ma, usa. results and discussion analysis of lvbadh mrna expression and enzyme activity the accumulation of the osmolyte gb is primordial for osmoregulation and protection of proteins to avoid denaturalization (timasheff, 1993; yancey, 2005). in this sense, the gb synthesis is regulated in a two-step irreversible pathway. the first step involves the oxidation of choline to betaine aldehyde by the action of choline dehydrogenase (cdh ec 1.1.99.1) and the second step the oxidation of betaine aldehyde to glycine betaine by the enzyme betaine aldehyde dehydrogenase (badh ec 1.2.1.8) (perrino and pierce, 2000; muñoz-clares and valenzuela-soto, 2008). although investigations of gb in crustaceans are limited, there is evidence of the use of organic osmolytes to virus capsid formation and reduce the viral infectivity (tafur et al., 2013). therefore, in this work we evaluate the mrna and activity of badh enzyme, as a crucial participant in the gb synthesis, as well as the accumulation of gb during wssv in white shrimp our results demonstrate that lvbadh is expressed in the hepatopancreas of non-infected shrimps, and that infection with the wssv increases the expression of mrna levels. the lvbadh expression increased 1.5-fold at 48 h post-infection with the wssv, compared to the control group, while non-statistical significance was found at 24 h post-infection (figure 1). in contrast, the activity of lvbadh increased 4.28and 4.59-fold at 24 and 48h post-infection with the wssv, respectively, compared to the control group (figure 2). in agreement with our results, a naddependent enzyme (aldh) was previously described as a crucial protein to suppress the wssv replication at high temperatures (32 °c). moreover, the knock-out of the aldh enzyme promoted a severe wssv infection in the k.o. shrimps. the authors suggest that the aldh enzyme detoxifies the aldehyde compounds, which inhibits dna-replication and protein synthesis, protecting the cell during wssv infection (lin et al., 2011), as these inhibitions prevent the virus from replicating. although the authors did not analyze the sequence of the aldh reported (genbank accession no. ck571503), it had 86% identity with a predicted aldh4a1, which is involved in the proline degradation pathway; and it is well known that proline has osmoprotective and osmoregulatory properties under stress conditions. it is known that expression and activity of badh are increased in response to stress conditions in several organisms from bacteria, mammals, and plants with a broad range of catalytic properties and roles against different stress conditions (mccue and hanson, 1992; legaria et al., 1998; velasco garcia et al., 2006; singh et al., 2013; delgado-gaytán et al., 2015; rosas-rodríguez et al., 2017). to the best of our knowledge, our study represents the first report of badh modulation under a viral infection in invertebrates. it has been described that the activity of antioxidant enzymes and membrane-bound atpases are reduced during wssv infection, which 116 fig. 2 lvbadh activity during wssv-infection. asterisks denote the statistically significant (p < 0.05) difference from the control group. exposes shrimp cells into a stress environment and alterations in ion concentration (rameshthangam and ramasamy, 2006). this is important for badh activity, previous studies from animals indicate that stress conditions and monovalent cations, mainly na + and k + ions, can enhance badh activity (valenzuela-soto et al., 2003). in our results, the activity of lvbadh increases at 24 and 48 h postinfection while mrna expression increases only at 48 h. these results suggest an effect from posttranslational regulation of lvbadh during wssv infection. analysis of glycine betaine accumulation the accumulation of gb was evaluated in the hepatopancreas of healthy and wssv-infected shrimps. the standard curve of gb (50-200 ug/ml) for the quantification of the osmolyte in all the standards presented a good correlation with an rsquare value of 0.9904. the gb is accumulated in the hepatopancreas of non-infected shrimps with a concentration of 37.05 ± 1.73 g/ml. the wssvinfection increased the gb accumulation in the hepatopancreas 1.49and 1.23-fold at 24 and 48h, respectively, compared to the control group (figure 3). to date, the accumulation of gb is related to the osmotic pressure and changes in salinity in crustacea, but little is known about gb response to biotic stress (moran and pierce, 1984; jahn et al., 2006; romano and zeng, 2012; freire et al., 2013). gonçalves-soares et al., 2012 described that hypoosmotic stress induces changes in the expression of genes involved in cellular defense and energy production, but the infection with wssv does not change the transcription profile of the mentioned genes. in this work, we demonstrate that gb responds to the wssv infection, probably as a defense mechanism of the shrimp against the virus. our results support studies where gb levels increase during wssv infection to balance osmotic pressure, altered by the virus, and balance the phosphorylcholine –related to choline metabolism and membrane biosynthesis–, which may benefit the synthesis of virus envelope and virus assembly (fan et al., 2016). pointing out that gb accumulation in l. vannamei during wssv infection could be regulated by lvbadh expression and activity. however, the increase on gb accumulation in hepatopancreas has not a direct correlation with the increase in the enzyme activity. this behavior was previously observed in shrimp, with a lack of correlation between mrna expression, activity and gb accumulation during salinity stress (delgadogaytan et al., 2015). we must consider that gb synthesis could take place in hepatopancreas to further migrate to other tissues and facilitate them with osmotic substrates/ions and further osmoprotection. also, it has been demonstrated that gb could migrate using membrane transporters (burg and ferraris, 2008), which strengthens the hypothesis beforementioned of gb migration after its synthesis in hepatopancreas. the mechanisms of response from shrimp to the wssv infection are not clear, while some evidence indicates that apoptosis is induced as a defense strategy during wssv infection, increasing the mortality in shrimp, some anti-apoptosis proteins are described in shrimp cells infected with wssv, suggesting that virus decreases the apoptotic signals https://www.sciencedirect.com/science/article/abs/pii/s0044848605006150#! https://www.sciencedirect.com/science/article/abs/pii/s0044848605006150#! 117 fig. 3 glycine betaine accumulation during wssv-infection. asterisks denote the statistically significant (p < 0.05) difference from the control group. (wang et al., 2004). dna damage, caspases family activation, and regulatory factors activate the apoptosis mechanism, which functions as innate host defense in the shrimp. however, some of the cells with apoptotic characteristics did not contain any wssv virons, suggesting that the apoptotic response might not be discriminated, targeting cells with or without the infection alike, maybe to prevent further spreading among neighboring cells to the ones infected, but further studies beyond the scope of this study are required to validate those suggestions (li et al., 2019). the results obtained in this study are important considering that regulation of cell volume and homeostasis by gb plays a crucial role in the apoptosis process (panayiotidis et al., 2006; zhao et al., 2018). in summary, we demonstrate that lvbadh expression and activity increase during wssv infection in shrimp in a time-dependent manner, along with gb concentration. our results suggest that badh is involved in the 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dpn/wvd motifs in apostichopus japonicus recognizes multiple microbes q zhao1,4,5, h wang1,4,5, w wang1,4,5, j li1,4,5, y liu1,4,5, z xue1,4,5, z liu1,4,5, l wang1,3,4,5, l song1,2,3,4,5* 1liaoning key laboratory of marine animal immunology, dalian ocean university, dalian 116023, china 2southern laboratory of ocean science and engineering (guangdong, zhuhai), zhuhai, 519000, china 3laboratory of marine fisheries science and food production process, qingdao national laboratory for marine science and technology, qingdao 266235, china 4liaoning key laboratory of marine animal immunology and disease control, dalian ocean university, dalian 116023, china 5dalian key laboratory of aquatic animal disease prevention and control, dalian ocean university, dalian 116023, china this is an open access article published under the cc by license accepted may 14, 2021 abstract c-type lectins (ctls) are a superfamily of ca2+ dependent carbohydrate-recognition proteins with diversified functions ranging from embryonic development to immune defense. in the present study, a ctl containing only one crd domain with new motifs asp-pro-asn (dpn) and trp-val-asp (wvd) in its ca2+ binding site 2 (designated as ajsjl-1) was identified from sea cucumber apostichopus japonicus. the deduced amino acid sequence of ajsjl-1 was homologous to ctls from other animals with the identities ranging from 19 % to 28.4 %. the mrna transcripts of ajsjl-1 were detected in all the examined tissues with varied abundance. the expression level of ajsjl-1 mrna in coelomocyte was up-regulated significantly at 12 h after vibrio splendidus challenge. the recombinant protein of ajsjl-1 (rajsjl-1) displayed significant binding activity to lipopolysaccharide, peptidoglycan, mannose and d-galactose in a ca2+-dependent manner. moreover, rajsjl-1 exhibited strong binding capability to v. splendidus but week to staphylococcus aureus, bifidobacterium breve, pichia pastoris and yarrowia lipolytica in the presence of ca2+. these results collectively suggested that ajsjl-1 with new dpn/wvd motifs served as a pattern recognition receptor in sea cucumber with the capability to bind broad-spectrum microbes and initiate the immune response against invaders. key words: c-type lectin; apostichopus japonicus; dpn/wvd motifs; binding capability; innate immunity introduction the c-type lectin (ctl) is a superfamily of ca2+ dependent carbohydrate-recognition proteins with carbohydrate recognition domains (crds) (wang et al., 2011). most c-type lectins contain one crd, but some have two or more crds (east and isacke, 2002). studies in invertebrates have implied that the clustering of multiple crds in one molecule endow c-type lectin with broader spectrum and higher affinity of binding pathogen-associated molecular patterns (pamps) (wang et al., 2011). for instance, cflec-3 from chlamys farreri with three crds can bind more pamps than cflec-1 and cfllec-2 with single crd (zhang et al., 2009). in addition, the key _________________________________________ corresponding author: linsheng song dalian ocean university dalian 116023, china e-mail: lshsong@dlou.edu.cn motifs in crd of ctls also affect the recognition range of carbohydrates. in vertebrate ctls, there are four ca2+-binding sites in crd, among which the ca2+-binding site 2 is known to be involved in the carbohydrate binding (zelensky and gready, 2005). in the ca2+-binding site 2, the first motif is always glu-pro-asn (epn) or gln-pro-asp (qpd), which displays specific affinity to mannose or galactose, respectively. the second motif is always trp-asn-asp (wnd), which can improve the binding affinity and specificity to carbohydrate (drickamer, 1992; zelensky and gready, 2005). in invertebrate ctls, there are many novel motifs such as glu-pro-asp (epd), gln-pro-gly (qpg), gln-pro-ser (qps), tyr-pro-gly (ypg), tyrpro-thr (ypt) for the first motif, and trp-ile-asp (wid), trp-ser-asp (wsd), trp-his-asp (whd), phe-ser-asp (fsd) and leu-ser-asp (lsd) for the 87 table 1 designations and nucleotide sequences of the primers were used in this study primer sequence(5'-3') ajsjl-1-p1 atggctctgtctctggtcactg ajsjl-1-p2 ttcaatacatggaagtttacaaatga ajsjl-1-m1 ggatccatggctctgtctctggtcactg ajsjl-1-m2 gaattcttcaatacatggaagtttacaaatga cytb-p1 cgtagttcagtttctcctt cytb-p2 aagggaaaaggaagtgaaag t7 taatacgactcactataggg t7-terminator tgctagttattgctcagcgg m13-47 cgccagggttttcccagtcacgac m13-48 gagcggataacaatttcacacagg rt-sjl-1-p1 tgcttcagtccattcaaccga rt-sjl-1-p2 ggtcatttccgcaccagttct second motif (wang et al., 2011). the existence of diversified motifs in invertebrate ctls suggests that there are flexible binding sites to recognize and bind various carbohydrates, which endow them with multiple immune functions (huang et al., 2015). for example, a ctl with epd/wfd motifs (cflec-2) from scallop c. farreri exhibited the pamp binding activity to lps, pgn, mannan, and zymosan (yang et al., 2010). epd motif containing lectin aictl-7 in scallop argopecten irradians was able to bind lps, pgn, mannan, yeast glucan and poly i:c in vitro, and involved in agglutination of fungi and bacteria (wang et al., 2011). fclec3 with eps motif identified from chinese white shrimp fenneropenaeus chinensis recognized muramic acid and peptidoglycan, and functioned in the recognition of bacterial and viral pathogens (wang et al., 2009). ptclec1 with ypd motif from swimming crab portunus trituberculatus bound many pamps, including lipopolysaccharides (lps), peptidoglycan (pgn) and glucan (glu), and numerous microorganisms (su et al., 2020). the information about the ctls with novel motifs as well as their functions in the immune response of invertebrates is helpful for the comprehensive understanding of the diversity and complexity of ctl family (wang et al., 2007; li et al., 2015b). sea cucumber apostichopus japonicus is a special group of marine invertebrates, which is commercially important aquaculture species in asian countries. recently, the frequently outbreaks of diseases have caused severe economic losses and threatens the healthy development of sea cucumber framing industry. as an invertebrate, sea cucumber lacks adaptive immunity and solely relies on innate immunity to defend against various invaders. ctl plays an important role in recognizing and binding to microorganisms as prr in invertebrates to initiate immune responses (drickamer and taylor, 1993). the knowledge about ctl family members in a. japonicus would provide new insights into the immune defense mechanisms of sea cucumber (wang et al., 2018). in the present study, a previously identified ctl from a. japonicus (ono et al., 2018) was further investigated with the objectives to (1) characterize new motifs in crd, (2) examine its mrna expression after vibrio splendidus challenge, and (3) determine its activity to bind various pamps and microbes. materials and methods sea cucumber and microbes sea cucumbers (apostichopus japonicus) approximately 100 ± 25 g in weight were collected from a local commercial farm (dalian, china) and maintained in aerated seawater at 18 ± 2 °c for 7 days before experiments. lipopolysaccharide (lps from escherichia coli 055:b5, sigma-aldrich, usa), d-galactose (sigmaaldrich, usa), peptidoglycan (pgn from staphylococcus aureus, sigma-aldrich, usa) and mannose (man from saccharomyces cerevisiae, sigma-aldrich, usa) were dissolved in tbs-ca2+ buffer (50 mm tris-hcl, 50 mm nacl, 20 mm cacl2, ph 7.4) or tbs buffer (50 mm tris-hcl, 50 mm nacl, ph 7.4) at the final concentration of 1 m, respectively. these solutions were stored at -20 °c for following experiment. gram-negative bacteria vibrio splendidus and vibrio anguillarum (laboratory preservation), grampositive bacteria streptococcus aureus (laboratory preservation), bifidobacterium breve (cellbio, shanghai), and fungi pichia pastoris and yarrowia lipolytica (laboratory preservation) were cultured in 2216e medium (solarbio, beijing) at 28 °c for 24 h, lb medium at 37 °c for 24 h, bs medium (hopebio, qingdao) at 37 °c for 24 h, ypd medium (solarbio, beijing) at 28 °c for 24 h, respectively. 88 tissue collection and immune challenge six unstimulated sea cucumbers were sacrificed, and the tissues including body wall, muscle, gonad, gut, tube-foot and respiratory tree were collected to investigate the mrna distribution of ajsjl-1. coelomic fluid was also collected and immediately centrifuged at 800 × g, 4 °c for 10 min to harvest the coelomocytes. all these tissues and coelomocyte samples were stored at -80 °c after addition of 1 ml trizol reagent (thermo fisher scientific, china) for subsequent rna extraction (ren et al., 2014; yan et al., 2018). a total of 72 sea cucumbers were randomly divided into two groups for soaking stimulation. thirty-six individuals in control group were soaked in 100 l sterile sea water. another 36 sea cucumbers in the stimulation group were soaked in sterile sea water adding with v. splendidus (1 × 109 cfu/ml, dissolved in seawater) (wang et al., 2018). then six sea cucumbers were randomly collected from two groups at 0, 3, 6, 12, 24 and 48 h post-challenge (hpc) for coelomocytes collection as previously described (jiang et al., 2014). rna extraction and cdna synthesis total rna was extracted from the different tissues of sea cucumber a. japonicus by using the easy pure rna kit (transgen, beijing, china). the sample was lysed in trizol reagent and total rna was extracted in cold chloroform and pelleted by centrifugation. then the rna was purified and washed twice in 70 % ethanol for following steps. according to prime script™ first strand cdna synthesis kit (takara, dalian, china), the firststrand of cdna was synthesized at 37 °c for 15 min, and terminated at 85 °c for 5 s. the cdna mix was stored at 80 °c for subsequent experiments. cloning of ajsjl-1 gene based on the published sequence (genbank accession no. lc214944.1), two primers ajsjl-1-p1 and ajsjl-1-p2 (table 1) were designed to clone the open reading frame (orf) of ajsjl-1 (ono et al., 2018). the pcr products were gel-purified, cloned into the pmd19-t simple vector (transgen, beijing, china). the recombinant plasmid was transformed into trans-5α chemically competent cell (transgen, beijing, china), and three positive clones were sequenced by sangon biotech (shanghai). sequence analysis of ajsjl-1 the sequence of ajsjl-1 was analyzed using the blast algorithm at the national center for biotechnology information. the amino acid sequence of ajsjl-1 and its theoretical molecular weight as well as the isoelectric point were predicated with an online translation tool (http://web.expasy.org/translate). the functional domains were revealed by the simple modular architecture research tool (smart) version 4.0 (http://smart.embl-heidelberg.de/). the presumed tertiary structure of the crd in ajsjl-1 was established using the swiss-model prediction algorithm (http://swissmodel.expasy.org/) and displayed by pymol according to previous reports (arnold et al., 2006; kiefer et al., 2009). multiple sequences alignment was created by the clustal x and performed by online tools (http://www.biosoft.net/sms/). a phylogenetic tree was constructed based on the deduced amino acid sequences of ajsjl-1 and other c-type lectins by the neighborjoining (nj) algorithm using the mega 4.1 software (kumar et al., 2004). to derive confidence value for the phylogeny analysis, bootstrap trials were replicated 1000 times. quantitative real-time pcr analysis of ajsjl-1 mrna expression pattern the expression patterns of ajsjl-1 in different tissues (coelomocyte, gut, muscle, gonad, tube-foot, body wall, and respiratory tree) were analyzed by using the abi quantstudio sequence detection system (applied biosystems, usa). the fragment of cytochrome b (cytb) gene (genbank accession no. 7802877) amplified with primers cytb-p1and cytb-p2 (table 1) from a. japonicus was served as the internal control (sun et al., 2010). the 2△△ct method was used to analyze the relative expression levels of ajsjl-1 in different tissues with the calibrator of coelomocyte (livak and schmittgen, 2001). preparation of recombinant ajsjl-1 and polyclonal antibody the orf fragment encoding ajsjl-1 was amplified with specific primers ajsjl-1-m1 and ajsjl-1-m2 (table 1). the pcr products with bamh i and ecor i sites were cloned into pet-22b expression vector with his-tag (transgen, beijing, china). the recombinant plasmid (pet-22b-ajsjl-1) was transformed into transetta (de3) chemically competent cell (transgen, beijing, china). after pcr screening with t7 primer (table 1), the positive clones were confirmed by nucleotide sequencing. the positive transformants were incubated in lb medium (containing 50 mg/ml ampicillin) at 37 °c with shaking at 220 rpm. when the culture medium reached od600 of 0.6-0.8, isopropyl β-d-1thiogalactopyranoside (iptg, sigma, usa) was added at the final concentration of 1 mm, and cultured at 16 °c, 100 rpm for additional 12 h. the recombinant protein (designated rajsjl-1) was purified through the ni-sepharose ff column, refolded, verified and quantified, and then stored at 80 °c as previous report (jiang et al., 2014). the rajsjl-1 was used to prepare polyclonal antibody according to the previous report (wei et al., 2015). briefly, 500 μl of rajsjl-1 (0.64 mg/ml) protein was emulsified with 500 μl complete freund's adjuvant (sigma, usa) to immunize 6-8 weeks old mice (bought from dalian medical university) by subcutaneous implantation. the second and third inoculations were performed on the 14th and 30th day with incomplete freund's adjuvant (sigma, usa). the anti-rajsjl-1 serum was harvested on the 40th day and obliquely placed at 4 °c overnight. the immune serum (containing antirajsjl-1 antibody) was collected after centrifugation at 4 °c, 3000 g for 30min, and stored at -80 °c before use. the specificity of polyclonal antibody against rajsjl-1 was verified by western blotting assay. the rajsjl-1 proteins were subjected to 12% sds-page, and the proteins in the gel were transferred to a 0.22 μm pore nitrocellulose membrane for western blotting http://smart.embl-heidelberg.de/ http://swissmodel.expasy.org/ http://www.bio-soft.net/sms/ http://www.bio-soft.net/sms/ 89 analysis. the anti-rajsjl-1 antibody (diluted at 1:1000) was used as the primary antibody, and the hrp-linked goat-anti-mouse igg (bbi, usa) (diluted at 1:1000) was used as the secondary antibody. the immune-blotted protein bands were visualized by ecl chemiluminescent substrate reagent kit (thermo fisher scientific, china) and developed by herosbio gel-imaging system (ge healthcare, usa). non-immunized mouse serum was used as negative control. pamps binding assay the pamps-binding ability of rajsjl-1 was examined by enzyme-linked immune sorbent assay (elisa) according to previous report with some modification (loizou et al., 1985; devi et al., 2010; yang et al., 2016; liu et al., 2021). briefly, 100 μl solutions of lps, pgn, man and d-galactose dissolved in tbs or tbs-ca2+ buffer at the final concentration of 0.1 mg/ml were used to coat 96-well plate, and the wells were blocked with 3% bsa (sangon biotech, china) in tbst (tbs solution containing 0.05 % tween-20, ph 7.4) at 37 °c for 1 h. after three times of washing with tbst, 100 μl rajsjl-1 with gradient dilution was added, and the reaction was incubated at 37 °c for 1 h. the same concentration tag protein (rtrx-his) expressed by the pet-32a null vector was added to the wells as negative group at the same time. meanwhile, the wells filled with 200 μl tbs were used as blank. the plate was firstly incubated with 100 μl of anti-ajsjl1 serum (1:1000 dilution in 3% bsa) at 37 °c for 1 h, followed by four times of washing with tbst, and then incubated with 100 μl of goat-anti-mouse igalkaline peroxidase conjugate (sangon biotech, china) second antibody (1:4000 dilution in 3% bsa). after the last washing, 100 μl of tetramethylbenzidine (tmb) single component substrate solution (solarbio, beijing) was added and incubated at 37 °c for 15 min. the reaction was stopped by adding 50 μl of 2 m hcl per well, and the absorbance was measured at 450 nm (tecan, switzerland). the light absorption values of rajsjl-1 group, rtrx-his group and tbs group were employed as sample group (p), negative group (n) and blank group (b), respectively. samples with p (sample) – b (blank)/n (negative) b (blank) > 2.1 were considered as positive. three replications were performed for each sample, and the data were presented as mean ± sd (n = 3). microbial binding assay microbial-binding activity of rajsjl-1 was measured according to previous report with some fig. 1 sequence features of ajsjl-1. a: nucleotide and amino acid sequences of ajsjl-1. the motif putative for ligand binding specificity is boxed in blue and the cysteine residues in the crd are marked with yellow boxes. b: the functional domains predicted by smart program. light red represents crd, and red represents signal peptide. c: the spatial structure of crd in ajsjl-1 predicted by swiss-model program. random coil, β-stands and αhelices are marked as pink, purple, and blue, respectively. there are four cysteines marked as yellow (c35, c52, c118, and c138) involved in forming disulfide bridges 90 fig. 2 the multiple sequence alignment and phylogenetic tree of ajsjl-1 with other ctls. multiple sequence alignment (a) and phylogenetic tree (b) of 13 ctl from 12 organisms were analyzed. ctls from various species include a. japonicus (lc214944.1; jn133520), p. crispus (kfq60316.1), c. canorus (kfo79987.1), h. sapiens (np_001243649.1), d. rerio (np_001315005.1), s. purpuratus (xp_030844747.1), c. gigas (ekc23119.1), drosophila melanogaster (adv37094.1), argopecten irradians (aee36500.1), penaeus chinensis (acj06431.1), eriocheir sinensis (aff59979.1), penaeus vannamei (adw08726.1) modification (yu et al., 2007). gram-negative bacteria (v. splendidus and v. anguillarum), grampositive bacteria (s. aureus and b. breve) and fungi (p. pastoris and y. lipolytica) were employed to detect the microbial-binding ability of rajsjl-1. the microbes were suspended in tbs or tbs-ca2+ buffer, and incubated with rajsjl-1 (300 μl, 0.64 mg/ml) under slight rotation at 4 °c overnight. after rinsed by tbs for five times, the bound proteins were dissociated from the microorganisms by loading buffer and analyzed by sds-page and western blot. rtrx (300 μl, 0.5 mg/ml) protein was employed as negative control. the anti-his monoclonal antibody (sangon biotech, china) was used for western blot to analysis the bacteria binding activity of rtrx. statistical analysis all data were given as means ± sd and subjected to one-way analysis of variance (one-way anova) followed by a multiple comparison (lsd). differences were considered significant at p < 0.05 (livak and schmittgen, 2001). 91 results molecular characters and predicted spatial structure of ajsjl-1 the orf sequence of ajsjl-1 was of 492 bp, which encoded a polypeptide of 163 amino acids with molecular mass of 17.74 kda and theoretical pi of 4.48 (fig. 1a). there were a signal peptide and only one crd domain predicted in ajsjl-1 by smart analysis (fig. 1b). two new motifs, dpn and wvd, were identified in crd domain of ajsjl-1 for carbohydrate binding specificity (fig. 1a). the potential tertiary structure of crd in ajsjl1 was established by swiss-model prediction algorithm based on the template 1egi.1.b. the crd adopted a typical long-form double-loop structure consisting of two parts, a lower part that was constituted of two α-helices and two β-strands, and an upper part that was composed of three β-strands. moreover, four conserved cysteines (c35, c52, c118, and c138) were predicated to form two disulfide bridges at the bases of the loops. the cys35 and cys138 linked the whole domain loop, while cys52 and cys118 linked the long loop region (fig. 1c). in addition, another two cysteine residues (cys157 and cys161) were identified at the n-terminus of the crd, indicating a long-form crd in ajsjl-1 (fig. 1a). homology comparison of ajsjl-1 blast homology analysis revealed that ajsjl1 shared 19% to 28.4 % identities with ctls from a. japonicus (jn133520) and other animals, such as pelecanus crispus (kfq60316.1), cuculus canorus (kfo79987.1), homo sapiens (np_001243649.1), danio rerio (np_001315005.1), and strongylocentrotus purpuratus (xp_030844747.1) (fig. 2a). a neighbor-joining tree was constructed based on multiple sequence alignment of ajsjl-1 and other ten ctls from different species. there were generally three groups in the phylogenetic tree. ajsjl-1 and the ctls from a. japonicus (ajctl) and s. purpuratus (spctl) were first clustered into the echinoderm group, which formed a sister group to the ctls from vertebrates. the ctls from insects, molluscs and crustaceans were well separated and clustered together into the other invertebrate group (fig. 2b). the distribution of ajsjl-1 mrna in tissues ajsjl-1 mrna transcripts were found to be distributed in all the examined tissues of sea cucumber a. japonicus, with the highest level in respiratory tree and the lowest level in coelomocytes. the expression levels were significantly higher in respiratory tree (27.26-fold, p < 0.01), body wall (10.38-fold, p < 0.01) and tube-foot (4.70-fold, p < 0.05) than that in coelomocytes (fig. 3a). temporal expression of ajsjl-1 in coelomocytes after the v. splendidus stimulation the mrna expression level of ajsjl-1 in coelomocytes was monitored post the stimulation with v. splendidus. it was significantly up-regulated at 6 h (5.01-fold of that in control group, p < 0.05), reached the highest level at 12 h (19.27-fold of that in control group, p < 0.01), down-regulated sharply at 24 h, and increased again at 48 h (9.22-fold of that in control group, p < 0.01) after stimulation (fig. 3b). the recombination protein of ajsjl-1 and the specificity of its polyclonal antibody. the recombinant plasmid (pet-22b-ajsjl-1) was transformed into transetta (de3). after iptg induction with shake (100 rpm) at 16 °c for 12 h, the whole cell lysate was analyzed by 12 % sds–page. a distinct band was revealed with a molecular mass of 26 kda, which was consistent with the predicted molecular mass of ajsjl-1 plus his-tag (fig. 4a). fig. 3 the temporal and spatial expression patterns of ajsjl-1 mrna. a: ajsjl-1 mrna expression in different tissues of sea cucumber a. japonicus. vertical bars represent the mean ± s.d (n =3). the different letters indicate significant differences comparing with other groups (p < 0.05, anova). b: the mrna expression levels of ajsjl1 in coelomocyte of a. japonicus after v. splendidus challenge. the cytb gene was used as an internal control to calibrate the cdna template for all the samples. each value is shown as mean ± s.d. (n =3). the different letters indicate significant differences comparing with other groups (p < 0.05, anova) 92 fig. 4 the recombinant protein of ajsjl-1 and the specificity for its polyclonal antibodies. a: sds-page analyzes of rajsjl-1. lane m: standard protein molecular weight marker; lane 1: blank control for rajsjl-1 (without plasmid pet-22b-ajsjl-1); lane 2: iptg-induced expression of rajsjl-1; lane 3: purified rajsjl-1. b: western blot analyzes of the specificity of polyclonal antibody. lane m: standard protein molecular weight marker; lane 4: western-bolt based on the sample of purified rajsjl-1 the purified rajsjl-1 was used to prepare antibody, and the specificity of the antibody against rajsjl-1 was assayed by western blotting. a clear reaction band with 26 kda was revealed (fig. 4b). no visible reaction band was found in the negative control (data not shown). carbohydrate binding capacity of ajsjl-1 the binding assay was performed to examine the pamps binding spectrum of ajsjl-1. the pamp binding capacity was recorded as p/n at 405 nm, and the samples with p/n > 2.1 were considered as positive. in the presence of ca2+, p/n values of rajsjl-1 toward lps, pgn, d-galactose and man were all higher than 2.1 (fig. 5a). rajsjl-1 exhibited relatively higher affinity to pgn and man, with the minimum concentration of 0.005 mg/ml. its affinity to d-galactose and lps was lower with the minimum concentration of 0.025 mg/ml. the values of p/n increased correspondingly with the increasing of rajsjl-1 concentration, which suggested that the binding activities of rajsjl-1 towards pgn, man, d-galactose and lps were of dose-dependence. in the absence of ca2+, the p/n values of rajsjl-1 toward four pamps were all less than 2.1 (fig. 5b). as control, the p/n values of rtrx for all the examined pamps were less than 2.1 (data not shown). microbial binding activity of rajsjl-1 the microbe binding assay was carried out to determine the ability of rajsjl-1 to bind gramnegative bacteria, gram-positive bacteria and fungi. in the presence of ca2+, a strong band was detected for v. splendidus, and shallow bands were detected for s. aureus, b. breve, p. pastoris and y. lipolytica. however, when microbes were incubated with rajsjl-1 in the absence of ca2+, there were no visible bands detected in all the lanes (fig. 6a). the result suggested that rajsjl-1 displayed no obviously binding ability towards the tested microbes without ca2+. as a control, no binding affinity was found in the group of rtrx-his (fig. 6b). discussion ctl is a key type of prr to play significant roles in pathogen recognition through binding to the terminal sugars of glycoprotein in a ca2+ dependent manner. in the past decades, an increasing number of ctls have been identified in various species (mcnulty et al., 2012; zhang et al., 2012; huang et al., 2014; zhang et al., 2017), which function in immune response either as cytomembrane receptors in immunocytes or as soluble proteins existing in tissue fluids (wang et al., 2012; huang et al., 2013a; hoving et al., 2014). 93 fig. 5 the interaction between rajsjl-1 and the pamps revealed by elisa analysis. a: the interaction between rajsjl-1 and the pamps with tbs-ca2+ buffer. b: the interaction between rajsjl-1 and the pamps with tbs buffer. each value is shown as mean ± s.d. (n =3) the ctls harbor at least one crd, and every crd has four ca2+-binding sites, in which ca2+binding site 2 is considered as the key site to determine the binding spectrum of ctl (drickamer, 1999; zelensky and gready, 2005; wang et al., 2011). in ca2+-binding site 2, there are two conserved motifs determining the crd binding ability, and the first one is always epn or qpd in vertebrates (weis et al., 1998; zelensky and gready, 2005). it is found that the motifs in the invertebrate ctls are more diversified than the vertebrate ones. ajsjl-1 was previously identified as a ctl in sea cucumber containing only one crd with 163 amino acid residues (ono et al., 2018). six conserved cysteine residues (cys35, cys52, cys118, cys138, cys157 and cys161) were found in the ca2+ binding site 2 of ajsjl1, among which cys157 and cys161 allowed the crd to adopt a typical long-form double-loop structure (drickamer, 1999). in the present study, a novel dpn/wvd motif was identified in the ca2+ binding site 2 of crd from ajsjl-1, which was different from the previously reported motifs of ajctl (epn/wtd) (han et al., 2012), ajctl-1 (epn/wnd) and ajctl2 (epn/wnd). in the phylogenetic tree, ajsjl-1 was closer to ctl-19s from vertebrate. these results indicated that there was functional differentiation among ajsjl-1 and other ctls from a. japonicus. dpn was only found in gal/galnac lectin from entamoeba histolytica, which was able to recognize galactose/n-acetylgalac-tosamine (weber et al., 2006; yadav et al., 2016). it is implied that ajsjl-1 might display similar recognition ability to gal/galnac lectin from e. histolytica. all these results indicated that ajsjl-1 was a member of the ctl with novel dpn/wvd motifs in sea cucumber a. japonicus. the distribution of ajsjl-1 mrna and the alternation of its expression level in coelomocyte after bacterial challenge were examined to understand its involvement in the immune response of sea cucumber. ajsjl-1 mrna transcripts were found to be widely expressed in water pipe system of sea cucumber, with the highest expression level in respiratory trees (ono et al., 2018). ajctl-1 was reported to be highly expressed in gonad and functioned as a prr in a. japonicus to recognize microbes (wei et al., 2015). ajctl-2 was highly expressed in coelomocyte, indicating that it might be an important molecule in the coelomocyte mediated immune defense (wang et al., 2018). the different expression profiles of those ctls in tissues indicated that they might execute different functions in the innate immunity of a. japonicus. respiratory tree is considered as a unique tissue for breathing in a. japonica. the higher expressions of ajsjl-1 in respiratory tree suggested that ajsjl-1 might play an important role in mucosal immunity of sea cucumber. coelomocytes are the main component of the immune system and play important roles in sea cucumber to resist pathogen invasion (wang et al., 2015). when the sea cucumbers were challenged by v. splendidus, the expression level of ajsjl-1 mrna in coelomocyte increased significantly (p < 0.01) at 12 h, suggesting it was induced by immune stimulation similar to the ctls in other invertebrates (wang et al., 2012; drummond and brown, 2013; huang et al., 2013a; huang et al., 2013b; li et al., 2015a; yang et al., 2015; zhou and sun, 2015). all these results indicated that ajsjl-1 was involved in the immune response against microbial infection. the protein-carbohydrate interaction mediated by ctls is benefited from their crds, and the conserved motifs at the ca2+-binding site 2 of crds determine their binding ability (kong et al., 2011; huang et al., 2013b; yang et al., 2015). in the present 94 fig. 6 the microbe binding activity of rajsjl-1 and rtrx revealed by western blotting. a: the microbe binding activity of rajsjl-1 with tbs-ca2+ or tbs buffer. b: the microbe binding activity of rtrx. lane m: pre-stained marker (kda); lane 1: v. splendidus; lane 2: v. anguillarum; lane 3: s. aureus; lane 4: b. breve; lane 5: p. pastoris; lane 6: y. lipolytica study, ajsjl-1 was found to bind all the tested pamps including lps, pgn, mannose and dgalactose in a ca2+ dependent manner. it is noteworthy that a new dpn/wvd motif was found in the crd of ajsjl-1. different from the previously identified gal/galnac lectin from e. histolytica with dpn motif to recognize galactose/n-acetylgalactosamine (weber et al., 2006; yadav et al., 2016), the new dpn/wvd motif gifted ajsjl-1 with the broadspectrum binding activity. it is a common feature for all the identified vertebrate and invertebrate crds that the second amino acid in the motifs of ca2+ binding site 2 is always “pro” (mitra and das, 2001; wei et al., 2012; de melo et al., 2014; drickamer and taylor, 2015; zhou and sun, 2015). structurally, the side chain of “pro” is essential to keep the stability and bind metal ion (zelensky and gready, 2005; yang et al., 2015). moreover, the first and third amino acid in the motifs of ca2+-binding site 2 affects the binding spectrum of crd (mullin et al., 1997; zelensky and gready, 2005; wang et al., 2012). in the present study, the dpn motif endowed ajsjl-1 with binding activity to various pamps, which was different from the specific binding capability of epn and qpd in vertebrate ctl. according to previous report that the third amino acid “asn” in the motif of ca2+-binding site 2 determined the binding specificity of ctl towards mannose (yang et al., 2015), the first amino acid “asp” in the motif of ajsjl-1 might be the key determinant for its binding activity to d-galactose (yadav et al., 2016). in addition, compared with ajctl-2, ajsjl-1 displayed stronger binding ability to various pamps, which was suspected to be determined by the wvd motif. the second motif has been reported to affect the affinity with carbohydrates (zelensky and gready, 2005; yang et al., 2015). ajsjl-1 lost its binding ability to all pamps in the absence of ca2+. these results collectively suggested that ajsjl-1 was a ca2+-dependent ctl with novel dpn/wvd motifs responsible for the recognition of diverse pamps in immune response of sea cucumber. the predominant microbe recognition and binding capability of ctls make them indispensable in the first defense line against pathogen infection (drummond and brown, 2013). in the present study, 95 ajsjl-1 was found to bind gram-negative bacteria, gram-positive bacteria and fungi in the presence of ca2+, which was consistent with its broad binding spectrum to pamps. the broad microbe binding spectrum manifested its indispensable roles in innate immunity (yu et al., 2007), which favored that ajsjl1 was involved in immune response against various pathogens as a prr. it was previously found that some ctls with a single crd identified from invertebrates could bind to limited spectrum of microorganisms. for instance, aictl-7 only aggregated p. pastoris (kong et al., 2011), and cgclec-2 was able to bind lps, pgn, mannan and zymosan rather than β-glucan (yang et al., 2010). considering that there was a novel dpn/wvd motif in the ca2+-binding site 2 of ajsjl-1, it was speculated that this new dpn/wvd motifs might endow ajsjl-1 with a broad recognition spectrum of microorganisms. in conclusion, ajsjl-1 was characterized as a new member of ctl family with novel dpn/wvd motifs in sea cucumber a. japonicus. ajsjl-1 mrna was highly expressed in the respiratory tree, and its expression level in coelomocyte increased significantly at 12 h after v. splendidus stimulation. it recognized and bound various pamps and microbes with broad spectrum. acknowledgement we are grateful to all the laboratory members for their technical advice and helpful discussions. this research was supported by national key r&d program (2018yfd0900606), the national natural science foundation of china (no. 31530059), the distinguished professor of liaoning (xlyc19020120), the scientific research project of liaoning province department of education 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of the cost action 16203: stem cells of marine/aquatic invertebrates: from basic research to innovative applications (maristem), october 20, 2021, palazzo bo, university of padova, italy organizer: l ballarin university of padova, italy the 4th maristem general meeting was supported by cost through the cost action 16203 studying stem cells and differentiation with acme dissociation j solana department of biological and medical sciences, faculty of health and life sciences, oxford brookes university, oxford, england, uk how do stem cells give rise to a myriad of different cell types? the study of stem cells and their differentiation processes is being largely enabled by single-cell transcriptomic approaches. planarians are an ideal model to answer this question, since they have pluripotent stem cells that give rise to all adult differentiated cell types constantly. thus, an adult planarian contains a snapshot of the entire process, with stem cells, all differentiated cells and every intermediate differentiating cell in between. to answer this question, we have developed acme (acetic-methanol) dissociation. single-cell sequencing technologies are limited by the need to dissociate fresh samples that can only be fixed at later stages. this renders profiling cell types of nonmodel organisms extremely challenging. our novel method, acme, is a cell dissociation approach that fixes cells as they are being dissociated. acmedissociated cells have high rna integrity, can be cryopreserved multiple times, can be sorted by fluorescence-activated cell sorting (facs) and are permeable, enabling combinatorial single-cell transcriptomic approaches. acme is based on affordable reagents, can be done in most laboratories and even in the field, and thus will accelerate our knowledge of stem cells and differentiated cell types across the tree of life. a hydra claudin cell-cell contact protein is involved in epithelial tissue dynamics, regeneration, and osmoregulation m-k eder, m achrainer, k grüner, a sandbichler, w salvenmoser, b hobmayer institute of zoology, university of innsbruck, innsbruck, austria claudins are major components of tight junctions in vertebrates, and they are involved in apical junctional complexes in some invertebrate bilaterians. it is unclear, when the function of claudins in cell adhesion and junction formation evolved in basal, pre-bilaterian metazoans, and to which degree stem cell based processes such as dynamic tissue replacement, morphogenesis, and regeneration require claudin proteins in those ancestral taxa. we identified 38 potential orthologues of the claudin and claudin-like gene familes in the cnidarian polyp model hydra. their single-cell transcriptome expression patterns suggest specific assembly patterns in apical septate junctions built between the various cell types. in order to approach the potential functions of claudin proteins in adult epithelial stem cells in hydra, we went on to carry out a detailed analysis of gene expression, protein localization, and functional interference of one selected claudin gene, hydra claudin1. in situ hybridization experiments showed that claudin1 is expressed uniformly at a moderate level throughout the ectodermal epithelial layer. it is, however, upregulated in the tip of the hypostome, at the basis of tentacles, and just distal to the basal disc. during bud formation and head regeneration, up-regulation of 38 claudin1 expression is associated with the formation of differentiated tentacle, hypostome and foot tissues. transgenic hydra expressing a claudin1gfp fusion protein clearly demonstrate specific protein localization in apical septate junctions throughout the entire polyp body. using an optimized sirna protocol, we were able to efficiently knockdown claudin1. these animals show disruptions of ectodermal epithelial organization and a disturbed ultra-structure in their septate junctions. they also exhibited strongly reduced capacities for head and foot regeneration. furthermore, preliminary data suggest that claudin1 knock-down reduces the ability of the ectodermal epithelial layer to maintain normal osmoregulation. in conclusion, our study is the first detailed functional characterization of an ancestral claudin cell-cell-contact protein acting in epithelial stem cells in maintaining tissue integrity and in dynamic morphogenetic processes. evolutionary dynamics of the myc gene family and functional conservation of an ancestral and structurally unique hydra myc protein m lechable, m kibet kitilit, b egger, m hartl, b hobmayer institute of zoology and centre of molecular biosciences innsbruck, university of innsbruck among stemness genes, the proto-oncogene myc has been intensively studied over the last decades, investigated primarily in vertebrate and cell culture systems. myc transcription factors are known to control fundamental cellular processes such as cell proliferation, cell cycle and stem cell maintenance. in addition, myc requires hetero-dimerization with the protein max to be functional, and both co-regulate thousands of target genes. however, the ancestral origins of myc and max proteins and their evolutionary dynamics throughout all metazoans remain poorly understood. in the present study, myc and max conservation across the metazoan tree will be shown. vertebrates possess a diversification of myc proteins commonly known as c-myc, l-myc and n-myc. in comparison, the freshwater polyp hydra encodes four distinct myc genes in its genome. structural and biochemical characterization unraveled hymyc1 and hymyc2 to share high similarities with c-myc of vertebrates, also in terms of acting in adult stem cell maintenance. an additional myc protein in hydra, hymyc3, however seems to be highly divergent, totally lacking the common nterminal domain containing four conserved mycboxes. single-cell transcriptome sequencing clearly showed that hymyc3 is active in a distinct population of interstitial precursor cells committed for nerve and gland cell differentiation. we currently speculate that it may act in these cells as a dominant-negative factor counteracting the stemness action of hymyc1 and hymyc2, and thereby allowing the implementation of a differentiation program. furthermore, in order to define whether principal functions of the oncogenic vertebrate myc are associated with this ancestral hymyc3 protein, we performed cell transformation assay in avian fibroblast cultures. here, hymyc3 showed an unexpected high potential for oncogenic transformation. these oncogenic properties and aspects of transcriptional activation will be discussed in relation to 3d structure modeling of hymyc3. keywords: stem cell; myc; oncogene; hydra, evolution marine collagens on the development of biomaterials for tridimensional culture of stem cells towards tissue regeneration th silva 3b’s research group, university of minho, barco – guimarães, portugal tissue engineering is established in the last decades as a strategy aiming the regeneration of tissues and organs, to change the paradigm to regenerative medicine. this strategy relies on the design of a temporary artificial extracellular matrix where cells are cultured in a tridimensional way, receiving appropriate cues to produce new tissues. many materials have been explored to produce those matrices – named scaffolds – and in recent years marine origin compounds are arising as a valuable, safe and sustainable alternative. in particular, marine origin collagens have been used on the development of tissue engineering scaffolds by freeze-drying, 3d printing and chemical crosslinking, among other techniques. fish collagens are widely studied, being produced from fish by-products as valorization approach, but marine invertebrates have been also explored, namely marine sponges and jellyfish. this talk discusses examples of scaffolds comprising fish collagens, shown to support the differentiation of human stem cells towards osteogenic and chondrogenic lineages. moreover, other biomaterials produced with collagens from marine sponges and jellyfish, combines with polysaccharides are also addressed, capable to support the culture and proliferation of different cell lines. one can hypothesize that these polymeric and composite structures can also offer templates for the culture of other cells, namely marine invertebrate stem cells, to support advanced marine biotechnology studies. sub-lethal 5-fluorouracil treatment promotes transcriptional profile changes in the piwia/soxp1-positive planarian stem cells a salvetti, g gambino, l rossi department of clinical and experimental medicine, university of pisa, pisa, italy planarians possess extraordinary biological performance, such as the possibility to regenerate any missing body part, thanks to the presence of an abundant population of stem cells, the so called neoblasts. although neoblasts share similar morphology, several findings indicate a complex transcriptionally heterogeneous population of cells. contrasting models picture them as a series of populations with a progressive fate restriction or as a single type of cells that encompass different transcriptional states through the cells cycle. challenging conditions such as sub-lethal x-ray treatment, where the depletion of stem cells is rescued by a repopulation process, are ideal model 39 systems to deepen the understanding of the mechanisms orchestrating neoblast specialization and potency. here, we identified a further challenging condition by exposing planarians to different concentrations of 5-fluorouracil (5-fu), a genotoxic drug. we identified a sub-lethal concentration of 5fu that induces depletion of stem cells and s-phase slowdown that triggers transcriptional changes in the piwia/soxp-1-positive neoblasts. at later time, some piwia-/sox-p1-positive neoblasts reappear at the ventral side of the animals, close to the nerve cords, and repopulate the body reconstituting the complex neoblast system. our data support the possibility that some neoblasts, in the earlier steps of commitment (or even post mitotic early progeny), could modulate their expression profile, so reacquiring a broader differentiative potential. analysis of cell hierarchies in the regenerative flatworm macrostomum lignano using single-cell transcriptomics and stem cell-specific transgenic lines s mouton, k ustyantsev, j wudarski, e berezikov european research institute for the biology of ageing, university medical center, groningen, the netherlands marine flatworm macrostomum lignano is an excellent model organism to study the mechanisms of stem cell regulation and regeneration. this animal can regenerate most of its body parts, it is easy to culture in laboratory conditions, and it is possible to create transgenic m. lignano lines. we use this model to understand how stem cells (neoblasts) in this animal make all other cell types and regenerate tissues and organs. towards this, we have generated several transgenic m. lignano lines labeling specifically neoblast by crips/cas9-mediated knock-in of various fluorescent protein-encoding genes in frame with a histone 2a variant gene that is expressed specifically in neoblasts. single cell transcriptomic analysis of facs-isolated neoblasts, as well as all cells of the animal allowed us to identify major cell types in m. lignano and establish relations between neoblasts and differentiated cells. these data prove instructive for generating experimental hypotheses for further in-depth investigation of stem cell regulation in m. lignano. absence of larval regeneration in the highly regenerative crinoid antedon mediterranea m sugni, g pria, f bonasoro, a barbaglio, md candia carnevali department of environmental science and policy, university of milan, milan, italy regeneration is a fundamental mechanism among metazoan, being present in most existing phyla. echinoderms are marine deuterostomes, therefore phylogenetically close to vertebrates, and they are characterized by extraordinary regenerative abilities, both in adults and larval stages. larval regeneration (and larval cloning) is well documented for all echinoderm classes, except crinoidea, the most basal taxon. therefore, the aim of this work was to assess if the larval stage (doliolaria) of the crinoid antedon mediterranea, whose adults are perfectly able to regenerate almost any tissue, can regenerate as well. in normal conditions, free-swimming a. mediterranea doliolaria attaches to the substratum and develops in a temporary post-metamorphic stalked stage (pentacrinoid) provided with an apical calix; this latter will eventually detach originating the free-swimming adult individual. adult specimens of a. mediterranea were collected at le grazie (la spezia gulf, sp, liguria). after hatching, doliolaria larvae were collected and transversally bisected with surgical blades, thus obtaining anterior and posterior halves. the fragments were monitored for 2-3 weeks and the survival rate was registered and compared to non-bisected doliolariae. we defined different “developmental” stages (seven for the anterior fragments and five for the posterior ones) through which both fragments go during the days postamputation. each stage was characterized by stereomicroscopy, scanning electron microscopy (sem), light microscopy and transmission electron microscopy (tem). results indicate that less than 50% of the bisected larvae survived after 3 weeks and none of the surviving halves was able to completely regenerate. rather, after a woundhealing phase each half continued its pre-determined development and the obtained post-metamorphic stage lacked structures deriving from the missing half: anterior fragments originated a stalk without a calix whereas the posterior halves produced a calix without a stalk. in terms of inner microscopic anatomy, each of the fragment properly developed the specific tissues normally present in the corresponding half of the larva (i.e. the stalk produced by the anterior part developed serial columnal ossicles and a well-defined longitudinal nerve). light microscopy and transmission electron microscopy were helpful tools to highlight the different cellular types involved in this development. these data suggest that: doliolaria cells are strictly committed to their original fate; cellular plasticity/dedifferentiation is temporary blocked and/or “stem cells” are missing or in a “stand-by” state. whatever the mechanism, this is “suddenly” and remarkably reverted just upon metamorphosis, as freshly metamorphosed individuals are already able to regenerate their tissues/structures. considering the basal phylogenetic position of crinoidea these results are particularly significant to better understand the evolutionary trajectories which led to gain or loss of (larval) regenerative abilities. more studies could also shed light on the correlation between regeneration in adults and regeneration in early stages. sweet tunicate blood cells: a glycan profiling of hemocytes in three ascidian species f zeng1, a peronato2, l ballarin2, u rothbächer1 1institute of zoology, university of innsbruck, innsbruck, austria 2department of biology, university of padova, padova, italy ascidians are invertebrate chordates and may reveal evolutionary origins of vertebrate traits 40 including cellular immunity, tissue rejection and selfrenewal, all functions executed by ascidian blood cells. understanding their individual properties, lineage homologies and functional plasticity is, however, limited by a lack of cytochemical and molecular markers. we performed a lectin-based glycan profiling of hemocytes in three ascidian species to distinguish different blood cell populations and mirror their relatedness. we found differing repertoires of species specific glycans for blood cells thought homologous in their function. within species, characteristic glycans or glycan combinations mark hemocyte types and support their hematopoietic relatedness or distinguish maturation stages. strikingly, ciona and phallusia hemoblasts have few carbohydrate decorations and drastically differ from differentiated cells, likewise phagocytes from cytotoxic cells as compared to botryllus, where a complex role of hemocytes in asexual self-renewal and allorecognition may involve carbohydrates. cytotoxic cells generally carry most decorations. within cell types, specific carbohydrates reside on the cell surface including amoeboid extentions while others are within granules possibly marking molecules important in cytotoxicity and crosslinking. taken together, these carbohydrate biosensors should further the molecular and functional characterization of the outstanding properties of the different hemocytes in genetically accessible ascidian species. keywords: tunicates; ascidians; hemocytes; blood cells; lectin staining; glycans; carbohydrates; ciona; phallusia; botryllus stem cells in the colonial ascidian botryllus schlosseri: contribution to asexual development and morphological characterization of their niches v vanni, f caicci, a peronato, f gasparini, s deppieri, l manni department of biology, university of padova, padova, italy colonial ascidians are the only chordates able to regenerate whole organisms from a small pool of circulating stem cells. among them, botryllus schlosseri can regenerate the entire colony, once all the zooids are removed, starting from circulating stem cells (scs). moreover, it constitutively develops individuals (zooids) by budding, from a small number of multipotent cells located in the body wall, throughout its entire life. it is still unclear whether the initial bud rudiment has the capability to form all the tissues of newly developing zooid, whether sc participate to organogenesis, and which molecular machinery governs pluripotency in this chordate. moreover, stem cell niches have been identified in this species, but their precise morphological characterization is still missing. we characterized sc niches by means of histology, 3d reconstructions and observation of whole mount-fixed specimens, verifying that candidate scs undergo mitosis and differentiation in the niches. we followed the contribution of candidate scs to bud organogenesis in-vivo, founding that candidate scs can proliferate and localize in different territories and epithelia, undergoing mesenchymal-epithelial transition. candidate scs sorted by facs and injected into compatible colonies confirmed these results. in conclusion, we carefully described stem cell niches and evidenced where and how candidate scs contribute to bud organogenesis. isolation of immune cells and hematopoietic stem cells from the tunicate model botryllus schlosseri b rosental1, o goldshtain1, s talice1, s eliachar1, o gershoni-yahalom1, il weissman2, a voskoboynik2 1the shraga segal department of microbiology, immunology, and genetics. faculty of health sciences, regenerative medicine and stem cell research center, ben gurion university of the negev, beer-sheva, israel 2institute for stem cell biology and regenerative medicine, stanford university school of medicine, stanford, ca, usa the mechanisms that sustain immunological non-reactivity are the basis for understanding the maintenance of tissue in syngeneic and allogeneic settings. while most transplantation rejection occurs due to the adaptive immune response, the proinflammatory response of innate immunity is necessary for the activation of adaptive immunity both in syngeneic and allogeneic settings. here we studied the hematopoietic and immune systems of botryllus schlosseri, a colonial tunicate that has vasculature, circulating blood cells, and interesting characteristics of stem cell biology and immunity. self-recognition between genetically compatible b. schlosseri colonies leads to the formation of natural parabionts with shared circulation, whereas incompatible colonies reject each other. by means of flow-cytometry in combination with screened antibodies by cytof, lectins, and fluorescent enzymatic reagents, we isolated 34 b. schlosseri cell populations. using whole-transcriptome sequencing of defined cell populations, and diverse functional assays, we identified hematopoietic stem cells (hsc), progenitors, immune-effector cells, and the hsc-niche. our study implies that the hsc and myeloid lineages emerged in a common ancestor of tunicates and vertebrates and suggests that hematopoietic bone marrow and the b. schlosseri endostyle niche evolved from the same origin (1-2). these findings taken together, make the b. schlosseri as a full model for hscs transplantation and immune system modulation model. interestingly, since the methods developed in the project for cell isolation and immune functional assays are species non-specific, we could translate this research to a variety of non-classical model organisms. this including fish, urchins, anemones and even corals (3-6). funding: the work of br was supported by israel science foundation (isf) number 1416/19. br has received funding from european research council (erc) under the european union’s horizon 2020 research and innovation program under grant 41 agreement no. 948476. br has received funding from a bsf grant number 2019647. rosental b, et al. complex mammalian-like haematopoietic system found in a colonial chordate. nature 564: 425-429, 2018. corey dm, et al. developmental cell death programs license cytotoxic cells to eliminate histocompatible partners. proc. natl. acad. sci. usa 113: 6520-6525, 2016. pavlov v, et al. hydraulic control of tuna fins: a role for the lymphatic system in vertebrate locomotion. science 357: 310-314, 2017. yakovenko i, et al. the diverse transformer (trf) protein family in the sea urchin paracentrotus lividus acts through a collaboration between cellular and humoral immune effector arms. int. j. mol. sci. 22: 6639, 2021. rosental b, et al. coral cell separation and isolation by fluorescence-activated cell sorting (facs). bmc cell biol. 18: 1-12, 2017. snyder ga, et al. functional characterization of hexacorallia phagocytic cells. front. immunol. 12: 2402, 2021. preliminary data on senescence in haemocytes of the colonial ascidian botryllus schlosseri f cima1, l drago1, a peronato1, n franchi2, o ben hamo3, l ballarin1 1department of biology, university of padova, padova, italy 2department of life sciences, university of modena and reggio emilia, modena, italy 3national institute of oceanography, haifa, israel senescence is a cellular response to damage that limits the proliferation of aged or effete cells and plays physiological roles as it is required for tissue homeostasis. colonies of the protochordate botryllus schlosseri, undergo cyclical generation changes or takeovers (tos) during which adult zooids are replaced by their buds reaching adulthood. the period of time between two tos is referred to as blastogenetic cycle. during the to, cells of adult zooid tissues die by apoptosis and are cleared by circulating phagocytes that, in turn, undergo phagocytosis-induced apoptosis and are cleared by new phagocytes in a recurrent, apparently endless, process. in the present work, we demonstrate that phagocytes, after the engulfment of effete phagocytes enter a senescence status and home, in the following mid-cycle, in the ventral islands, on both sides of the endostyle, where undifferentiated (stem) cells are also found. from these sites, senescent cells cross the peribranchial epithelium and are released in the peribranchial cavity where they will be expelled with the exhalant water. studies on turkish coastlines’ botryllid ascidians a karahan, fn oğul, e öztürk, be tohumcu middle east technical university, institute of marine sciences, erdemli, mersin, turkey tunicates are hermaphrodite, filter feeder invertebrate organisms that have an essential role in the coastal ecosystem and distributed worldwide oceans from tropic to polar regions and continue to spread via anthropogenic actions. they have both sexual and asexual reproduction and can undergo whole-body regeneration; those features make them perfect model organisms for stem cell-related studies such as regeneration, aging, development, allorecognition. despite their unique features, botryllid ascidians are understudied. so far, 55 botryllid ascidians species have been identified. recent studies showed that only morphological identification is not enough to discriminate the species since most botryllid ascidian species show similar morphologies. also, it is shown that the most recent mtdna (coi) analyses give preliminary results; however, since evolution is a gradual and continuous process, there should be more comprehensive studies for the species identification of the botryllid group. different methods should be combined to understand their biology better and benefit from them as model organisms. in ims-metu, we have been doing regular sampling and monitoring along the mediterranean coastline of turkey for the last five years. we simultaneously observe botryllid ascidian species diversity, distribution, and fluctuations over the year with environmental changes, along with barcoding efforts. we also culture them in our aquaculture conditions and detail the comparative blastogenic cycle and whole-body regeneration, monitoring each morphology of the same or different species. besides, we also collect ecologic, biologic, and genetic data of most botryllid species on a regional basis. among all the species that have been monitored in the region, botrylloides anceps was the ascidian species that grew nearly two years in the culture condition and the best model for our regeneration studies. for the future periods, it is planned to make sampling for botryllid ascidians from all coastline of turkey, including the mediterranean sea, the aegean sea, the marmara sea, and the black sea. in this context, the determination of botryllid ascidians by using classical taxonomic and dna barcoding methods aims to identify possible new species. in addition, it is aimed to determine the species that can be used as an indicator of water quality or pollution and investigate the potential effects of environmental factors on species diversity distribution. in the range of these studies, botryllid ascidians will be examined from an ecological and genetic perspective. also, this process aims to designate the distribution of botryllus schlosseri, which is of mediterranean origin and distributed worldwide, on the turkish coasts. despite the extensive documentation of the presence in all turkish seas, detailed genetic profiles of the species have not been studied. for these reasons, detecting single nucleotide polymorphism of populations using next-generation sequencing methods and interpretation of species’ adaptive features according to detected variants and investigating its evolutionary history is valuable on both local and global scales. 84 isj 16: 84-91, 2019 issn 1824-307x research report ecotoxicity of hallachrome, an unusual 1-2 anthraquinone excreted by the infaunal polychaete halla parthenopeia: evidence for a chemical defence? r simonini*, d iori, l forti, s righi, d prevedelli department of life sciences, university of modena and reggio emilia, modena, italy accepted may13, 2019 abstract polychaetes play a prominent role in marine systems, but little is known about their secondary metabolites compared with other benthic taxa. in the present study, we investigated the toxicity of hallachrome, an unusual 1-2 anthraquinone identified from the skin of some polychaetes, including the mediterranean infaunal species halla parthenopeia. under stress conditions, this worm releases a harmful purple mucus, whose noxious compounds were still unknown. we hypothesized that hallachrome also occurs in the purple mucus, giving rise to its color and toxicity. soon after the production of the purple exudate, h. parthenopeia also secretes a harmless, transparent mucus, which pushes away the toxic one, suggesting protective functions for the worm itself. lc-ms and 1 h-nmr analyses confirmed the presence of the pigment hallachrome in the purple mucus. the average concentration of the pigment in the purple mucus was about 310 mg l -1 . ecotoxicological bioassays on representative species of bacteria, protozoans, rotifers, crustaceans (artemia franciscana) and polychaetes (dinophilus gyrociliatus) revealed its severe toxic effects: lc50/ec50 values ranged from 0.11-5.67 mg l -1 . hallachrome showed higher toxicity for a. franciscana than other naturally occurring anthraquinones. tests on encapsulated embryos of d. gyrociliatus evidenced the ability of a mucus layer to limit hallachrome diffusion, confirming the protective role of the transparent mucus. given the information available on polychaetes anti-predator strategies, hallachrome cannot be considered a consumer deterrent. however its toxicity and wide range of activity suggest chemical defensive functions against potential competitors, parasites and/or pathogens. key words: anthraquinones; halla parthenopeia; marine invertebrates; soft-bottoms; ecotoxicology introduction polychaetes comprise more than 15,000 described species of marine annelids and represent the major group of invertebrates in soft bottom communities. they include the errantia (species with greater mobility) and most of the sedentaria (species with less mobile lifestyles, and echiurids, annelids that lost segmentation) (worms, 2019). given the wide range of body plans and life style and their prominent role in trophic interactions, only very few polychaetes are known to be toxic or venomous (struck, 2017; coutinho et al., 2018). most of them belong to the families glyceridae and amphinomidae, commonly known as “bloodworms” and “fireworms”, respectively (von reumont et al., 2014a; struck, 2017). bloodworms (glycera spp.) have an eversible pharynx tipped with four hollow ___________________________________________________________________________ corresponding author: roberto simonini department of life sciences university of modena and reggio emilia via campi 213/d, 41125 modena, italy e-mail: roberto.simonini@unimore.it fangs which inject a complex proteinaceous venom into their invertebrate prey (von reumont et al., 2014b). fireworms have tufts of white, sharp, harpoon-shaped chaetae to deter potential predators. in humans, stings of large-sized fireworms cause immediate painful burning sensation on contact, followed by the development of erythema, edema and paresthesia (smith, 2002; ottuso, 2013). it is still unknown whether the cause of these responses is mainly mechanical or chemical, but inflammation-inducing secondary metabolites (complanines) were isolated from the fireworm eurythoe complanata (nakamura et al., 2008; von reumont et al., 2014b). besides, three other polychaete species secrete toxic substances: the echiurid bonellia viridis (de nicola giudici, 1984), the lumbrinerid lumbrineris (lumbriconereis) heteropoda (hashimoto and okaichi, 1960), and the oenonid halla parthenopeia. b. viridis produces copious green mucus containing a pigment named “bonellin”, a unique type of alkylated chlorin. bonellin is highly toxic at a very low concentration 85 for both eukaryotes and prokaryotes and protects b. viridis against benthic and microbic organisms widespread in the interstices where it lives (de nicola giudici, 1984). nereistoxin (4-dimethylamino1,2-dithiolane) is a strong neurotoxin isolated from l. heteropoda which causes neuromuscular blockade. it has been shown to have insecticidal activity and three chemically related synthetic insecticides have been developed and used commercially (e.g. cartap and bensultap; coutinho et al., 2018). the genus halla (errantia: oenonidae) includes large infaunal burrowing polychaetes (up to 1 m long), with documented reports from the mediterranean, red sea and japan sea. the mediterranean specimens of h. parthenopeia live in soft bottom habitats at 10-50 m depth and is highly appreciated as fishing bait in france, spain, italy and egypt (see iori et al., 2014). h. parthenopeia feeds mainly on bivalve molluscs and, when undisturbed, produces large amounts of a colourless mucus which stabilizes and keeps open its tubes in the sediment, facilitating also locomotion (osman et al., 2010; iori et al., 2014). when disturbed, h. parthenopeia emits a purple mucus with severe toxic effects on representative species of bacteria, polychaetes, rotifers and brine shrimps (iori et al., 2014). to date, information on the biology of this species derives only from laboratory studies and none is available on the origin of the purple mucus toxicity. just after the emission of the purple mucus, h. parthenopeia quickly releases large amounts of a transparent mucus that pushes away from its body the purple one (iori et al., 2014). given that mucus secreted by marine invertebrate could display different protective properties (stabili et al., 2009), the transparent mucus of h. parthenopeia could have defensive function against its own harmful purple exudate (iori at al., 2014). in this paper we hypothesized that the purple mucus of h. parthenopeia contains a red pigment called hallachrome (7-hydroxy-8-methoxy-6-methyl1,2-anthraquinone, c16h12o4, molecular mass = 268.26; prota et al., 1971) as responsible for its colour and toxicity. hallachrome was identified from the skin of h. parthenopeia (prota et al., 1971) and was synthesized by cameron and collegues (1999), although its biological activity remains unknown. to isolate the purple mucus pigment, samples of exudate were submitted to extraction and purification, whilst the toxicity was assessed through six ecotoxicological assays. four of these had been previously used to test purple mucus effects (iori at al., 2014): microtox ® (with the bacterium vibrio fischeri), rotoxkit ® (with the rotifer brachionus plicatilis), artoxkit ® (with the crustacean artemia franciscana) and acute toxicity bioassays with juveniles of the polychaete dinophilus gyrociliatus. bioassays on a. franciscana are commonly used because oral acute toxicity and cytotoxicity of natural products are correlated to brine shrimp lethality (parra et al., 2001). d. gyrociliatus well represents small sized invertebrates and larvae/juveniles that may come into contact with the toxic purple mucus (simonini et al., 2011). in addition, the toxi-chromotest tm (with the bacterium escherichia coli) and an acute test with the ciliate euplotes crassus were performed to extend the result obtained with the microtox ® to other microbes. to investigate the transparent mucus function, we tested the ability of a mucous layer to limit the toxic effect of the purple mucus pigment exposing d. gyrociliatus embryos and juveniles to different concentrations of the pigment dissolved in seawater. since this polychaete develops within a protective mucous capsule, the hatching rate (i.e., the number of survived juveniles that emerge from capsules; simonini and prevedelli, 2003) was analyzed, expecting higher survivorship in protected embryos than in free-living juveniles. material and methods mucus collection detailed information on h. parthenopeia rearing procedures and mucus collection are reported by iori and collaborators (2014). briefly, the worms (2540 cm in length) were collected by professional divers from sandy-muddy sea bottoms in the ligurian sea (north-western mediterranean sea). they were transported to the laboratory inside refrigerated containers (12-17 °c) and maintained in large, aerated plastic tanks filled with artificial seawater (reef crystal, instant ocean, 30-35 salinity) and 7 cm of sand. during the laboratory rearing, worms were periodically fed with live clams. they were removed from their tank the day before mucus collection and gently transferred in pairs into smaller plastic tanks (40×60×12 cm) filled with seawater. the transparent mucus produced during the night was removed. then, worms were repeatedly stimulated (with plastic pliers or by transferring them to adjacent tanks) until they secreted the purple mucus, which was collected using plastic tweezers or pipettes and stored at 4 °c. the mucus was then centrifuged in 50 ml plastic vials at 4,000 rpm, and the clear supernatant water was discarded (iori et al., 2014). pigment extraction nearly pure hallachrome can be selectively extracted from h. parthenopeia by direct immersion of live specimens in chloroform (prota et al., 1971). accordingly, to isolate purple mucus pigment, aliquots of centrifuged purple mucus (50-100 ml) were placed in a separating funnel containing 150300 ml of chloroform (carlo erba reagents). the next morning, the resulting red chloroform extract was removed and the procedure was repeated three times until the solvent assumed a pale pink colour. the residual mucus was almost uncoloured with brownish debris: it was collected and placed for four hours in a rotary evaporator (rotavapor buchi r200) in order to remove chloroform. a test to assess the potential toxicity of the residual mucus was performed with the same procedure adopted for the fresh transparent mucus (iori at al., 2014). juveniles of d. gyrociliatus were exposed for two weeks to experimental solutions with concentrations ranging between 10 and 500 g×l –1 (1 and 50%) of residual mucus. the residual mucus was not toxic. d. gyrociliatus fed on the residual mucus, survived, 86 grew up to sexual maturity and reproduced successfully. red chloroform extracts were pooled, filtered and concentrated using a rotary evaporator, obtaining a dry, purple pigment. this one was chromatographed on a 3x40 cm glass column filled with silica gel (0.06-0.20 mm, carlo erba reagents) using toluene/ethyl acetate (60:40, v/v) as mobile phase. on concentration, the red band gave purple crystals, which were insoluble in distilled water and seawater. lc-ms/ms and ft-nmr analyses known aliquots of the purified pigment were dissolved in acetonitrile (hplc grade, carlo erba reagents) and analysed by an agilent 1200 series hplc (agilent technologies) consisting of a vacuum degasser, an autosampler, and a binary pump equipped with a rp c18 analytical column (4.6×150 mm, 5 μm particle size, agilent zorbax eclipse plus). acidified water (0.1% formic acid v/v) and acidified acetonitrile (0.1% formic acid v/v) were used as mobile phases a and b, respectively. the mobile phase was programmed as follows: column flow, 0.300 ml min −1 ; stop time, 22.00 min; post time, 8.00 min; timetable, 0 min, 3% b; 2min, 3% b; 12 min, 100% b; 20 min, 3% b; injection volume, 10.0 μl. for lc-ms/ms analysis, the agilent 1200 lc was coupled to an agilent 6310a ion trap equipped with an electrospray interface in negative and positive ion mode. in negative mode, the ion trap scanned over the 50–800 m/z range at 13,000 u×s −1 during separation and detection. the maximum accumulation time for the ion trap was set at 10 ms, the target count was set at 8,000, and the compound stability was set at 100%. the optimum values of the esi-ms parameters were as follows: capillary voltage, 3.5 kv; drying gas temperature, 320 °c; drying gas flow, 10.0 l×min −1 ; nebulising gas pressure, 32.0 psi. in positive mode, the ion trap scanned over the 50–800 m/z range at 13,000 u×s −1 during separation and detection. the maximum accumulation time for the ion trap was set at 6 ms, the target count was set at 60,000, and the compound stability was set at 100%. the optimum values of the esi-ms parameters were as follows: capillary voltage, 3.5 kv; drying gas temperature, 320 °c; drying gas flow, 1.0 l×min −1 ; and nebulising gas pressure, 32.0 psi. samples were further analyzed using agilent q-tof accurate mass g6520a system. nmr spectra were also obtained at 200 mhz using a ft-nmr instrument. nmr spectra were recorded in dmso-d6 solutions and chemical shifts reported are in δ (ppm) scale relative to tetramethylsilane as external standard. ecotoxicological bioassays known aliquots of the purified pigment were dissolved in the carrier solvent dimethylsulfoxide (dmso, carlo erba reagents) to obtain two concentrate solutions (10 4 and 10 3 mg×l -1 ). the experimental solutions employed in the bioassays were obtained diluting the two initial ones in artificial sea water (salinity 30-35), reaching different concentrations tested as treatments. in each test, at least five treatments were considered. the concentration of the solvent was adjusted and maintained the same in each treatment. the microtox ® liquid phase test is an acute toxicity bioassay based on the reduction of the bioluminescence activity of v. fischeri after its exposure (15 min) to a toxic matrix. the test was carried out by arpa daphne (a reference centre for environmental control of coastal marine ecosystem) using the microtox m500 analyser (modern water) according to iso 11348-3:2007 protocol, with concentrations of pigment ranging from 0.1 to 10 mg×l -1 . the concentration of dmso was 1‰, far below the no observed effect concentration (noec) (1% for microtox, pagnout et al., 2006). the toxi-chromotest tm (ebpi, 2016) is a microplate toxicity bioassay based on the ability of toxicants dissolved in liquid matrices to inhibit bacterial growth and de novo synthesis of the inducible enzyme β-galactosidase. the test is performed on a highly permeable mutant of e. coli, which is highly sensitive to a wide spectrum of toxic substances (reinhartz et al., 1987). after the exposure (incubation period 90 min), a chromogenic substrate that measures β-galactosidase activity is added. if the sample is toxic, bacteria will not grow, thus no synthesis of β-galactosidase and colour development will occur; if the sample is non-toxic, bacteria will grow, synthesize the enzyme and a distinctive blue colour will appear quickly. the toxicity of the sample can be assessed by a simple visual qualitative evaluation of the colour obtained, or quantitatively by spectrophotometry analysis using a plate reader at a wavelength of 610 nm (the peak of absorption of the chromogenic substrate). in this study, toxi-chromotest tm 96well plates were set up according to producer guidelines (ebpi 2016) and the quantitative method was used to asses the toxicity of the pigment (concentration ranging from 250 to 0.03 mg×l -1 prepared by serial dilutions; microplate reader: synergy htx htx multi-mode). preliminary analysis evidenced that also the purple pigment absorbs at 610 nm. consequently, appropriate blanks were prepared by diluting the pigment in the reaction mixture lacking bacterial suspension. the dmso concentration was 2%, which was lower than the ec5 (3.1%). the ec50 of hgcl2 (positive control) was 0.20 mg×l −1 (95% confidence intervals [c.i. 95%]=0.18-0.22 mg×l -1 ). e. crassus is a common inhabitant of healthy d. gyrociliatus cultures. the exposure to the purple mucus causes the slowdown of ciliary beat followed by cellular lysis and death (iori et al., 2014; simonini, unpublished). a motility test was set up starting from the procedure proposed by gomiero and collaborators (2013) for e. crassus mortality test. five ciliates were picked up from the culture using a micropipette and placed onto a glass slide containing a depression filled with the experimental solution. for each treatment, ten slides-replicates were prepared and maintained in a wet chamber to avoid excessive evaporation. after two hours of exposure, ciliates were checked individually and were considered motionless if they did not show any ciliary beat during 5 s. concentrations of pigment ranged from 0.06 to 2 mg×l -1 . the noec for dmso was 1‰ (1,000 mg×l -1 ), which was higher than the 87 concentration of the solvent used in the experiments (200 mg×l -1 ). the rotoxkit m ® bioassay uses newborns emerging from cysts of b. plicatilis that were exposed to concentrations of pigment ranging from 0.025 to 0.5 mg×l -1 . rotoxkit m ® was run for 24 h and was conducted according to the standard operating procedure (microbiotests inc) by arpa daphne. the concentration of dmso (50 mg×l -1 ) was considered non toxic for b. plicatilis (marcial et al., 2005). the artoxkit m ® test uses instar ii–iii nauplii of a. franciscana. the concentrations of pigment tested ranged from 0.6 to 40 mg×l -1 (dmso concentration = 5‰). at concentrations of 25 mg×l -1 or higher, the pigment precipitated in few hours after its addition to the seawater. the motility of a. franciscana was checked 24 h after the exposure to the experimental solutions, according to the artoxkit m ® standard operating procedure (microbiotests inc). the sensitivity of instars to dmso and k2cr2o7 (panreacquimica, analytical grade) was also assessed. the noec for dmso was 6.7‰ (6700 mg×l -1 ), which was higher than the concentration of the solvent used in the experiments. the 24-h ec50 for k2cr2o7 was 35.0 mg l -1 (c.i. 95%=30.5-40.2 mg×l -1 ), consistent with that certified by the supplier (microbiotests inc., 24-h ec50 = 30.9 ppm, c.i. 95%= 26.7-35.6 mg×l -1 ). d. gyrociliatus is a small annelid (max adult length 1 mm) with a short life cycle (5 days from zygote to hatching, 6 days from hatching to sexual maturity at 24 °c; simonini and prevedelli 2003) that can be easily cultured in the laboratory and is very sensitive to various toxic compounds (reish and gerlinger, 1997; marcheselli et al., 2010), including secondary metabolites (simonini et al., 2011). adult females reproduce semicontinuously and lay transparent mucous capsules containing small eggs, that develop into dwarf males, and larger eggs, from which females originate. only females give rise to free living populations and are used in bioassays. the capsule (wall thickness 2050 μm) limits the stage of dispersion, permits matings among siblings and protects developing embryos. indeed, under optimal conditions, hatching rates and juvenile survival (i.e., percentage of females which become sexually mature) are both very high, reaching 92-96% and 98-100% respectively (simonini and prevedelli, 2003; marcheselli et al., 2010). the acute toxicity (96h) bioassays with newborn d. gyrociliatus were performed according to the astm e1562-00 protocol, using the same strain of iori and collaborators (2014). for each experiment, about 500 juveniles collected approximately 8-12 h after hatching were randomly placed in 10 ml bowls and exposed to the experimental solutions, which contained the pigment in concentration ranging between 0.025 and 0.3 mg×l -1 . the concentration of the solvent was 300 mg×l -1 . the ability of the mucous capsule of d. gyrociliatus to limit pigment toxic effects was assessed starting from newly laid eggs, which were obtained as well as the acute test. after adults removal, eggs in each bowl were counted and randomly assigned to an experimental treatment. d. gyrociliatus egg capsules tenaciously adhere to the glass of bowls, so the culture seawater can be completely replaced with the experimental solutions at the same concentrations used in the test with juveniles (and vice versa). capsules were not touched or removed until the end of the test to avoid any mechanical stress. consequently, the number of eggs presents in each treatment was different, varying from 24 to 39. developing embryos were daily checked during the following 96 hours through transparent capsules. after 4 days, bowls content was again substituted with culture seawater. to obtain information on hatching rates in different treatments, emerging juveniles were counted and moved in another bowl for the subsequent 48 h. additional tests were performed to check the sensitivity of d. gyrociliatus juveniles to the solvent (dmso) and to cu(no3)2 (panreacquimica) as reference toxicant. the 96-h lc50 for dmso was 16,300 mg×l -1 (1.63%): no lethal effects were observed at the concentration of 2,000 mg×l -1 , which was about 7 times the concentration of the solvent used in the experiments. the 96-h lc50 value for copper was 0.10 mg×l -1 (c.i. 95%=0.090.11 mg×l -1 ), similar to that reported in literature (reish and gerlinger 1997; iori et al., 2014). data analysis ec50 values of tests with v. fischeri were calculated using microtox ® and toxi-chromotest tm calculation programs and procedures. the trimmed spearman-karber method was used to obtain lc50/ec50 (median lethal/effective concentration) values and their relative 95% c.i. in tests with e. crassus, b. plicatilis, a. franciscana and d. gyrociliatus. mortality correction was not necessary because the control mortality was always less than 10%. hatching rates of treatments and controls in experiments with d. gyrociliatus egg capsules were compared using fisher‟s exact test. results lc-ms and nmr analyses of the purified pigment overall, 230 ml of purple mucus were obtained from 18 specimens of h. parthenopeia. after chromatographic purification, the concentration of the red band obtained gave 71 mg of crystalline pigment. thus, the average concentration of the pigment in the purple mucus was about 310 mg×l -1 . in the positive-ion-mode, ms and ms/ms mass spectra of the pigment displayed predominant protonated molecular ion ([m+h] + ) at m/z 269 and major daughter ion at m/z 254. in the negative-ionmode, ms and ms/ms mass spectra displayed deprotonated molecular ion ([m-h] ) at m/z 267 and major daughter ion at m/z 252. esi-ms m/z: exact mass calculated for c16h11o4 [m-h] : 267.0657, found: 267.0654; esi-ms + m/z: exact mass calculated for c16h13o4 [m+h] + : 269.0814, found: 269.0816. the inspection of the 1 h-nmr spectrum of the pigment in dmso-d6 (fig. 1) showed the presence of characteristic signals of three different types of aromatic protons at δ 8.49, 7.90 and 7.59 ppm (each integrating for 1h). furthermore, signals of one aromatic oh were present at δ 9.83 ppm. 88 fig. 1 1 h-nmr spectrum of the purified pigment extracted from the purple mucus of h. parthenopeia, with the structure of hallachrome. additionally, the 1 h-nmr showed the presence of two cis olefinic protons (doublets at 7.76 and 6.38 ppm, j = 10.2 hz), and two signals each integrating for 3h at 3.87 (ar-och3) and 2.34 (ar-ch3) ppm respectively (fig. 1). bioassays the emitted luminescence of v. fischeri was reduced by exposure to the pigment (microtox ® , ec50 15 min=0.88 mg×l –1 ) as well as the synthesis of β-galactosidase in e. coli (toxi-chromotest tm , ec50 90 min=1.40 mg×l –1 ) (table 1). the pigment inhibited the ciliary beat of e. crassus (ec50 2 h=0.29 mg×l –1 ) (table 1). after two more hours, all cells were lysed at 0.5 mg×l –1 . b. plicatilis was the second most sensitive organism (rotoxkit®, lc50 24 h= 0.18 mg×l –1 ), while lethal effects on a. franciscana were observed for higher concentrations (artoxkit®, lc50 24 h=5.67 mg×l –1 [table 1]; lc100 24 h=10 mg×l –1 ). the pigment was very toxic for d. gyrociliatus juveniles (lc50 96 h = 0.11 mg×l -1 ) (table 1). the lc100 96 h was 0.20 mg×l -1 , while all juveniles survived in the control group. when encapsulated embryos of d. gyrociliatus were exposed to the pigment, no young hatched after 96 h of exposure to 0.30 mg×l -1 , but the percentage of hatching found in the treatment with 0.10 mg×l -1 of pigment (93%, n=28) was not significantly different from that of embryos (94%, n=36; fisher test: p>0.56) and juveniles (survivorship: 100%, n=50, fisher test: p>0.12) control groups. discussion lc-ms spectra and mass estimates, and results of 1 h-nmr analysis of the pigment purified from the purple mucus of h. parthenopeia are consistent with characteristics and structure of hallachrome reported by prota and collaborators (1971). bioassay results support the hypothesis that hallachrome is the secondary metabolite responsible for the toxicity of the purple mucus. the test battery evidenced the harmful effects of hallachrome on all the organisms considered and highlighted its broad-spectrum of activity on marine invertebrates. the mortality of d. gyrociliatus and b. plicatilis, and the ciliary motility of e. crassus were the most sensitive endpoints. the bioluminescence emission of v. fisheri and the β-galactosidase synthesis of e. coli were intermediate, while the mortality of a. franciscana was the least sensitive endpoint. this pattern of test sensitivity was observed also for the purple mucus (table 1). tests on d. gyrociliatus embryos, which develop within a mucous capsule, revealed that hallachrome concentration correspondent to the lc50 of new-born 89 table 1 median lethal/effective concentration (lc50/ec50) and confidence intervals (c.i. 95%) obtained for hallachrome in ecotoxicity biossays. the confidence intervals do not overlap. reference toxicity data for purple mucus (iori et al., 2014) are also provided in the last column. * test not performed in previous experiments (iori et al., 2014) taxon species hallachrome (present experiment) [mg×l -1 ] purple mucus lc50 or ec50 [mg×l -1 ] lc50 or ec50 c.i. 95% bacteria vibrio fischeri 0.88 0.40-1.90 16,200 bacteria escherichia coli 1.40 0.96-2.40 * ciliophora euplotes crassus 0.29 0.27-0.32 * rotifera brachionus plicatilis 0.18 0.16-0.20 8,100 crustacea artemia franciscana 5.67 4.06-7.93 76,000 polychaeta dinophilus gyrociliatus 0.11 0.10-0.12 700 free-living d. gyrociliatus had no effects on hatching. this suggests that a mucous layer could limit the diffusion of hallachrome, reducing its harmful effects. further experiments on the diffusion rate of the hallachrome through the transparent mucus produced by h. parthenopeia should be performed to confirm if its release represents a physiological defence of the worm against its toxic purple mucus. hallachrome was the only 1,2-anthraquinone natural product characterized until the late „90‟s. more recently three other 1,2-anthraquinones, the pigment sinapiquinone and rufoolivacins c and d, were extracted from mushrooms of the genus cortinarius, but they have a biaryl skeleton very different from the hallachrome structure (gao et al., 2010; bai et al., 2013). while 1,2-anthraquinones are rare, mono-aryl 9,10-anthraquinones are very common. they occur (both free or as glucoside) in a large number of plants, fungi (e.g. mueller at al., 1999), and in some animals, including polychaetes of the genus tomopteris (francis et al., 2014). some 9,10-anthraquinones show weak to moderate toxicity for a. franciscana (gao et al., 2010). for instance, the 24-h lc50 of the emodin estimated by bioassay on the brine shrimp is about 43 mg×l -1 (159 μm; ayo et al., 2007). moreover, the 24-h exposure to 10 mg×l -1 of physcion (35 μm), 1hydroxy-6,8-dimethoxy-3-methyl anthraquinone (34 μm) and 6,8-dimethoxy-citreorosein (34 μm) induced the mortality of 41%, 39% and 37% of brine shrimp nauplii, respectively (gao et al., 2010). our artoxkit tests suggest that these anthraquinones are less toxic than hallachrome (lc50 24 h = 21 μm) for a. franciscana. in the bioassay with d. gyrociliatus hallachrome (lc50 96 h = 0.41 μm) was more toxic than copper (lc50 96 h = 1.59 μm), which is among the most harmful metals for polychaetes (reish and gerlinger, 1997). in addition to the severe toxicity, hallachrome is also very abundant in the purple mucus, where it reaches a concentration 2-3 orders of magnitude higher than the one toxic for the organisms here assessed. even today the dermal contact with hallachrome is quite common among fishermen using h. partenopeia as bait (iori et al., 2014). yet, no cases of human harm after contact with h. parthenopeia have been reported, apart from strong and persistent stainings of the skin. in fact, given the difficulty with which hallachrome crosses the thin mucus capsule of d. gyrociliatus, it seems unlikely that it could overcome the cornified layer of human skin. however further experiments on the toxicity of hallachrome for mammals should be performed to exclude the possibility that this substance is harmful to humans. it would be advisable for those who manipulate these worms (including divers who collect h. parthenopeia from seabed and sellers) to wear disposable gloves as a precautionary measure to avoid excessive exposure to hallachrome toxicity. the ecological significance of this toxin is still unclear. despite the harmful effect and concentration of hallachrome in the purple mucus, this exudate did not deter fish feeding in palatability experiments with the ricefish oryzias melastigma, and it is normally ingested by the seabass dicentrarchus labrax and the gilthead seabream sparus aurata when worms are used as fish baits (iori et al., 2014). indeed, oenonids may avoid predators living in tubes within the sediment and feeding preferably during the night (saito et al., 2004). these strategies are often observed in palatable worms lacking chemical and mechanical deterrents against predators (kicklighter and hay 2006). this pattern indicates that hallachrome cannot deter predation. on the other hand, analogies in terms of modality of emission, high toxicity and range of activity with the bonellin produced by echiurids (de nicola giudici, 1984) suggest that hallachrome can defend h. parthenopeia from potential competitors, parasites and/or pathogens, including the microflora colonizing worms‟ tube and body wall. further experiments with test species that co-occur in the same habitat of h. parthenopeia (or actually 90 represent a threat to the worm) are required to confirm that this unusual 1-2 anthraquinone could act as a chemical defense. cimino and collegues (1985) found hallachrome also in the body of another popular fishing bait, the lumbrinerid scoletoma (lumbrineris) impatiens, which belongs to one of the most specious family of polychaetes (messina et al., 2005). both h. parthenopeia and s. impatiens contain also 1,2,7trihydroxy-8-methoxy-6-methylanthracene (tmma), which yields hallachrome by oxidation and is considered its biogenetic precursor (cimino et al., 1985). perhaps, hallachrome and related compounds may also occur in other mobile polychaete species, but their ecological role cannot be predicted from chemical characterization alone. interestingly, tmma has never been found in the purple mucus of h. parthenopeia, but it occurs in a relatively large amount in s. impatiens, which does not secrete purple exudates. the few toxic or venomous polychaetes known to date show different lifestyles and possess a wide range of diversely acting biomolecules (nakamura et al., 2008; von reumont et al., 2014b). these studies are relatively rare (coutinho et al., 2018), perhaps because sheltered lifestyles make them difficult to observe on the field. however, given the diversity of polychaete species future ecological, chemical and toxicological investigations supported by the application of novel methods (i.e. next generation sequencing and -omic analyses, von reumont et al., 2014) should likely 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(ed.). evolution of venomous animals and their toxins, springer dordrecht, the netherlands: pp. 399-413, 2017. worms (2019). polychaeta. accessed at: http://www.marinespecies.org/aphia.php?p=tax details&id=883 on 2019-02-26 ions score is -10*log(p), where p is the probability that the observed match is a random event 388 isj 14: 388-403, 2017 issn 1824-307x research report comparative proteomic analysis reveals that juvenile hormone binding protein and adenylate kinase may be involved in the molting process of silkworm, bombyx mori y yang, l chen, q tang, y zhang, h tang, p lü, q yao, k chen institute of life sciences, jiangsu university, zhenjiang 212013, china accepted october 13, 2017 abstract the molting is an essential part of the silkworm metamorphosis development. although previous studies have demonstrated that molting in silkworm is associated with prothoracicotropic hormone (ptth), molting hormone (mh), and juvenile hormone (jh), the changes of proteins and genes during silkworm molting, as well as the molecular mechanism about its generating and maintaining remains unclear. in this paper, the proteomic approaches were employed to investigate this issue. totally, 35 different proteins were successfully identified through mass spectrometry and database searches, among which 42 % proteins were involved in cell structure and 16 % proteins belonged to the metabolism group. meanwhile, vacuolar atp synthase, juvenile hormone binding protein precursor and adenylate kinase isoenzyme were found to be down-regulated at early, mid-molt stages, which were further confirmed by quantitative real-time polymerase chain reaction (qrt-pcr). taken together, our data suggests that juvenile hormone binding protein (jhbp) and adenylate kinase (ak) play a critical role in the process of silkworm molting, which may participate in the regulation of silkworm molting. key words: bombyx mori; molting; proteomics; juvenile hormone binding protein; adenylate kinase introduction insects are the most abundant organisms in the world and encompass nearly 80 % species of our planet, which provide a large amount of desirable material for us to investigate the molecular basis of physiological mechanisms (dahanukar et al., 2005; li et al., 2010). the silkworm (bombyx mori) has been fed as an important economic insect in sericulture industry. with the implementation and completion of whole genome sequencing program, silkworm has become a model insect of lepidoptera molecular biological research (xia et al., 2004, 2009). ___________________________________________________________________________ corresponding author: keping chen institute of life sciences jiangsu university 301 xuefu road, zhenjiang jiangsu province 212013, pr china e-mail: kpchen@ujs.edu.cn list of abbreviations: acn, acetonitrile; cbb, coomassie brilliant blue; ief, isoelectric focusing; maldi-tof ms, matrix-assisted laser desorption/ionization time of flight mass spectrometry; pmsf, phenylmethanesulfonyl fluoride; sds-page, sodium dodecyl sulphate polyacrylamide gel electrophoresis; tfa, trifluoroacetic acid; 2-de, two-dimensional electrophoresis molting is a common phenomenon in the developmental process of many organisms and is essential for the molecular, physiological and morphological rebuilding of living animals. in insect, molting is the regular shedding of the exoskeleton at specific points in its life cycle to let it grow. the previous studies have demonstrated that molting in silkworm is associated with their growth and other physiological processes and is a cascade process of gene expression and interaction (kinjoh et al., 2007; muramatsu et al., 2008; wang et al., 2013), which is endogenously controlled, involving in prothoracicotropic hormone (ptth), molting hormone (mh), and juvenile hormone (jh). these hormones are secreted by the endocrine system, including brain neurosecretory cells, corpora allata and prothoracic glands, among which brain neurosecretory cells are dominant and play a central role. although molting has been reasonably defined, the molecular regulation mechanism involved in generating and maintaining remains unclear. meanwhile, the changes of genes or proteins during silkworm molting have not yet been resolved and it is also uncertain about which genes and proteins are indeed involved in the regulation of silkworm molting. these issues are very important for understanding the molecular mechanism of silkworm molting. therefore, systematic studies are required to investigate these issues. mailto:kpchen@ujs.edu.cn 389 in recent years, the proteomic approaches, including two-dimensional electrophoresis and mass spectrometry, have been applied successfully to identify the specific proteins from different tissues and organs of silkworm, such as haemolymph (li et al., 2006; hou et al., 2010; liu et al., 2010), fat body (moghaddam et al., 2008), midgut (qin et al., 2012; feng et al., 2014; kannan et al., 2016), silk gland (zhang et al., 2006; jia et al., 2007; yi et al., 2013), endocrine organs, larval head (li et al., 2010; li et al., 2016; arunprasanna et al., 2017), and so on. these results from proteome provided important evidences and clues for understanding the growth and development of silkworm. the silkworm is metamorphic molts involving in complex hormonal regulation and replacement of cuticle types and can be considered a model organism for understanding molting and metamorphosis in holometabolous insects. the insect head is composed of important sensory systems, including the olfactory, visual, gustatory organs, and some important endocrine organs, which can receive environmental stimuli and regulate insect growth and development. therefore, insect head plays a crucial role in insect growth, reproduction, diapause, and metamorphosis processes (li et al., 2009; li et al., 2010; li et al., 2016; wang et al., 2016). in current study, the proteomic approach was employed to detect and identify differentially expressed proteins from silkworm larval heads, which allowed us to determine the proteins and genes associated with molting and decipher the molecular mechanism of silkworm molting. materials and methods experimental insects and developmental stage the hybrid strain jingsong×haoyue of the silkworm b. mori were used for this experiment, which was provided by sericultural research of institute, chinese academy of agricultural sciences, zhenjiang, china. all larvae were reared with the fresh mulberry leaves at 25 ± 1 °c and 75 ± 2 % relative humidity (photoperiod 16 h light: 8 h dark). the larvae of 1st 3rd instar were fed with the chopped tender leaves and 4th 5th instar larvae were fed with the matured leaves. the silkworm larval heads were used for proteomic analysis, which were collected at specific time points during the molt from the penultimate (4th) to the last (5th) larval instar. based on the time of head capsule slippage (hcs) occurring at the fourth molt period, the molting stage was determined, that is, hcs occurring (denoted by mq), 12 h and 24 h after hcs (denoted by m1 and m2), and newly molted fifth instar larvae (denoted by qc). the larval heads were collected on ice, immediately frozen in liquid nitrogen and stored at -70 °c for later use (at least 100 heads of each sample). the experiments were repeated three times. protein sample preparation the larval heads of silkworm were grounded into fine powder in liquid nitrogen and homogenized on ice for 5 min in pre-cooled extraction buffer (20 mm tris-hcl ph 7.5, 250 mm sucrose, 10 mm egta, 1 mm pmsf, 1 mm dtt, and 1 % triton (v/v) x-100) as described by cilia et al. (cilia et al., 2009). the homogenate was transferred into the 2.0 ml centrifuge tube and centrifuged (12,000g, 4 °c, 30 min). the supernatant was collected and equal-volume tris-saturated phenol was added and mixed. the phenol layer containing proteins was collected and incubated 10 ~ 12 h at -20 °c with methanol solution containing 100 mm ammonium acetate. after centrifugation (12,000g, 30 min, 4 °c), the supernate was discarded. the precipitate was washed again with methanol solution containing 100 mm ammonium acetate and then washed 4 ~ 5 times using ice-cold acetone containing 13 mm dtt, centrifuged (12,000g, 4 °c, 20 min/each time), and vacuum-dried. the dried powder was dissolved in lysis buffer (7 m urea, 2 m thiourea, 4 % w/v chaps, 2 % v/v bio-lyte, ph 3 10, 1 % w/v dtt), overnight at 4 °c. finally, the mixtures were centrifuged (12,000g, 20 min, 4 °c). the supernatant was collected and used for two-dimensional electrophoresis. the protein concentration was measured by bradford method (bradford, 1976). two-dimensional electrophoresis (2-de) ief was carried out through bio-rad protean electrophoresis system and 17 cm immobilized ipg dry gel strips with ph 4 7 liner range (bio-rad, usa) was used. about 1.5 mg protein samples were loaded by passive rehydration (room temperature, 11 ~ 12 h). then ief was performed at 300, 500, and 1,000 v, linear for 1 h, respectively, next 10,000 v, linear for 5 h, and then remained 10,000 v until a total voltage of 54,000 vh. subsequently, the gel strips were equilibrated for 15 min in equilibration buffer (0.05 m tris-hcl ph 6.8, 2.5 % w/v sds, 30 % v/v glycerol and 1 % w/v dtt) and then equilibrated for 15 min again (0.05 m tris-hcl ph 6.8, 2.5 % w/v sds, 30 % v/v glycerol and 2.5 % w/v iodoacetamide). the second dimension sds-page was carried out with a laemmli buffer system (15,% resolving gels) (laemmli, 1970). finally, the gels were dyed with 0.116 % cbb r-250 (0.116 % w/v cbb, 25 % v/v ethanol, and 8 % v/v acetic acid). image and data analysis the 2-de gels were scanned using the imagescanner iii (ge healthcare life sciences). the images were analyzed using imagemaster 7.0 software (ge healthcare life sciences). the optimized parameters were as follows: saliency = 3, smooth = 6, and minimum area = 60. the value of each protein spot was normalized (a percentage of the total volume in the whole set of gel spots). the protein spots with ratios ≥ 1.5 or ratios ≤ 0.67 and passing the student’s t-test (p < 0.05) were considered to be differentially expressed proteins. in-gel digestion and protein identification the differentially expressed proteins were manually cut down from the gels and washed using ultrapure water. the gel pieces were destained 2 ~ 3 times by ultrasonic with 50 μl destaining buffer (50 % acn, 25 mm nh4hco3) until the gel pieces became colorless. next, the gel pieces were rinsed using 25 mm nh4hco3, 50 % acn, and 100 % can (50 μl/each), respectively, and vacuum-dried. the 390 fig. 1 the 2-de profiles from head extracted proteins between mq and m1. note: (reference gel: mq). the differentially expressed protein spots are indicated by circles and labeled with arabic numerals. the upward and downward arrows respectively indicate up-regulated and down-regulated proteins. the protein spots with circles and plus symbol indicate that the protein spot is expressed only in this 2-de gel. the numbers shown on the left indicate the protein markers in kda. (figs 2 and 3 are same as fig. 1) dried gel pieces were soaked in 25 mm nh4hco3 with 10 μg/ml of trypsin (promega, usa) at 4 °c for 30 min. subsequently, 10 ~ 15 μl 25 mm nh4hco3 was added to the gel pieces and the gel pieces were digested at 37 °c overnight (about 12 h). peptides were collected and dried by vacuum (-50 °c) and then stored at -70 °c until mass spectrometry. maldi tof/tof samples were prepared by spotting 2 μl digested protein solution (dissolved with 5 μl 0.1 %tfa) and 1 μl matrix (-cyano-4-hydroxycinnamic acid, sigma, 10 mg/ml, dissolved in 50 % acn containing 0.1 % tfa) on the 600 μm anchorchip maldi probe (bruker daltonik, germany). after dryness at room temperature, the samples were analyzed through maldi tof/tof mass spectrometer (bruker daltonik, germany). all the acquired peptide mass finger prints of maldi-tof ms/ms data were analyzed through the biotools 3.0 program to search the protein database (ncbinr) using in-house mascot software (matrix science, uk). metazoa (animals) was selected as the taxonomic category. to ensure the confidence of identified results, the search parameters were set as follows: trypsin was selected as enzyme, one missed cleavage was allowed, carbamidomethyl was selected as fixed modification, gln->pyro-glu (n-term q) was chosen as variable modification, and peptide tolerance was set at ±100 ppm with a mh+ mass values. the proteins whose mascot scores were more than 55 were considered to be reliably identified. gene ontology (go) analysis to evaluate the major biological functions of the differentially expressed proteins among mq-m1, mq-m2, and mq-qc, gene ontology (go) analysis (http://www.geneontology.org/) was employed. the go ids of all identified proteins were obtained by interproscan searching (http://www.ebi.ac.uk/interpro/scan.html) using the amino acid sequences. following the ye’s method (ye et al., 2006), the annotation information of identified proteins was gathered and then uploaded as web gene ontology annotation plot (wego) native format into wego (http://wego.genomics.org.cn/cgi-bin/wego/index.pl). finally, the go plot was obtained and downloaded as jepg format. according to wego, all identified proteins was classified as molecular function, cellular component and biological process (ye et al., 2006). rna extraction and quantitative real-time pcr (qrt-pcr) using trizol reagent (invitrogen, usa), total rna was extracted and 1 μg rna was used for the first strand synthesis. the specific primers were shown in table s1. the qrt-pcr was performed in a total volume of 20 μl containing 2 μl of cdna (200 ng), 10 μl of sybr green master mix (vazyme biotech co., ltd, china), 0.4 μl of 50×rox reference dye i, 0.4 μl of primers (10 μm) and 7.2 μl of h2o. amplification was carried out using an abi7300 pcr thermocycler (applied biosystems, usa) as follows: 5 min at 95 c, followed by 40 cycles of 10 s at 95 c, 31 s at 60 c and one cycle of 15 s at 95 c, 60 s at 60 c, 15 s at 95 c. the α-tubulin gene (ncbi accession no. nm_001043419.1) of silkworm was amplified as a reference and the experiments were repeated three times. results differentially expressed proteins between mq-m1, mq-m2, and mq-qc to investigate the molecular mechanism of silkworm molting, we applied proteomic approach to http://wego.genomics.org.cn/cgi-bin/wego/index.pl 391 comprehensively identify and then characterize the differentially expressed proteins during the fourth molt of silkworm. as shown in figures 1, 2 and 3, most protein spots were mainly distributed in the range of ph 4 7 and mass weight 20 100 kda, indicating that protein samples were correctly extracted and most proteins from the larval heads of silkworm were obtained. totally, 842 ± 15, 825 ± 13, 827 ± 10, 857 ± 16 protein spots were detected in mq, m1, m2, and qc, respectively, in cbb r-250 stained gels. using imagemaster 7.0 software, 24, 20, and 18 different protein spots were ultimately determined between mq-m1, mq-m2, and mq-qc, respectively (supplementary file, table s2). through further analysis, these 62 differentially expressed proteins can be classified into 35 different proteins, which were numbered uniformly (table 1) and labeled in figures 1, 2 and 3. fig. 2 the 2-de profiles from head extracted proteins between mq and m2. fig. 3 the 2-de profiles from head extracted proteins between mq and qc. maldi-tof ms/ms identification of differentially expressed proteins and functional classification among 35 differentially expressed proteins, 34 proteins were successfully identified through maldi-tof ms/ms and database searches (mascot score > 55), and one protein was identified through maldi-tof ms and database searches (mascot score > 84) (table 1). according to bevan et al. (1998), these 35 proteins can be divided into the following nine categories: cell structure proteins, metabolism-related proteins, disease/defense-related proteins, transporter, transcription, molecular chaperone, secondary metabolism, protein synthesis, and unknown function proteins (fig. 4). as shown in figure 4, 42 % of the identified proteins were classified into the 392 table 1 identification of differentially expressed proteins by maldi-tof ms/ms spot no. protein name accession no. mr(kda)/pi a mascot scores sc b (%) amino acid function 1 heat shock protein 70-3 aei58998 72.82/5.12 138 25% 655 molecular chaperone 2 tubulin alpha-3 chain, partial kfq41598 43.57/5.79 197 55% 389 cell structure 3 beta-1 tubulin tbb1_manse 50.65/4.75 262 64% 447 cell structure 4 uncharacterized protein loc101738727 xp_012552185 49.91/5.29 129 31% 449 unknown function 5 vacuolar atp synthase subunit b np_001091828 54.67/5.25 187 55% 490 metabolism 6 antichymotrypsin-2 ach2_bommo 41.43/5.26 109 39% 375 metabolism 7 antitrypsin isoform 1 act36276 43.46/5.41 145 41% 392 metabolism 8 uncharacterized loc101739385 xp_004922152 37.46/6.07 91 32% 328 unknown function 9 annexin bab16697 36.11/4.89 138 43% 323 disease and defense 10 alpha-tocopherol transfer protein-like xp_004928929 35.28/5.56 146 47% 306 transporter 11 alpha-tocopherol transfer protein-like xp_004928929 35.28/5.56 183 50% 306 transporter 12 juvenile hormone binding protein brp-2095 precursor np_001036987 28.05/5.42 140 55% 249 secondary metabolism 13 cuticular protein rr-1 motif 34 precursor np_001166717 23.18/4.78 107 56% 207 cell structure 14 adenylate kinase isoenzyme 1 xp_004929167 25.29/5.70 108 57% 226 metabolism 15 tumor protein d54 isoform x3 xp_004930117 22.32/5.41 91 28% 206 cell structure 16 putative cuticle protein faa00454 21.66/5.39 91 45% 215 cell structure 17 h+ transporting atp synthase subunit d np_001093279 20.19/5.56 57 52% 179 metabolism 18 transcription factor btf3 homolog 4 xp_012551703 19.04/9.12 201 48% 174 transcription 19 triosephosphate isomerase np_001119730 26.93/5.67 168 64% 248 metabolism 20 odorant binding protein fmxg18c17 precursor np_001157372 26.48/6.23 121 28% 236 cell structure 21 glutathione s-transferase sigma 1 np_001037077 23.60/5.98 131 54% 206 disease and defense 22 cuticular protein rr-1 motif 42 precursor np_001166712 17.17/5.16 151 86% 159 cell structure 23 actin-depolymerizing factor 1 np_001093278 17.23/6.17 175 64% 148 cell structure 24 muscular protein 20 np_001040476 20.29/8.70 79 20% 184 cell structure 25 cuticular protein 66d np_729400 30.80/5.97 74 20% 270 cell structure 26 cuticular protein rr-2 motif 67 precursor np_001166691 18.72/6.16 223 43% 178 cell structure 27 uncharacterized protein loc101741978 xp_004930780 16.55/4.93 117 46% 144 unknown function 28 ribosomal protein p2 np_001037213 11.53/4.68 121 16% 112 protein synthesis 29 beta tubulin np_001036887 50.72/4.83 267 70% 447 cell structure 30 centromere protein f isoform x2 xp_004926842 33.22/4.84 157 30% 297 cell structure 31 t-complex protein 1 subunit epsilon-like xp_004933262 59.19/5.63 127 31% 542 molecular chaperone 32 cuticular protein rr-1 motif 3 precursor np_001166744 14.68/4.66 93 28% 137 cell structure 33 cuticular protein rr-1 motif 3 precursor np_001166744 14.68/4.66 64 26% 137 cell structure 34 thiol peroxiredoxin np_001037083 22.07/6.09 252 18% 195 disease and defense 35 ubiquitin-like protein smt3 np_001037410 10.36/5.29 105 23% 91 transcription a mw: molecular weight; pi: isoelectric point b sc: sequence coverage c the identified protein by maldi-tof ms 392 fig. 4 functional classifications of the identified proteins. cell structure group, 16 % proteins belonged to the metabolism group, which accounted for 58 % of the identified proteins and were the most abundant proteins in the heads of silkworm larvae. compared with hcs occurring stage, up-regulated proteins in early, mid-molt of the fourth molt period, and newly molted fifth instar larvae mainly involved in cell structure, but down-regulated proteins mainly involved in metabolism (supplementary file, table s1). in particular, four metabolism/secondary metabolism-related proteins were found to express regularly (fig. 5, spot nos. 5, 12, 14, 17). among these four proteins, spot no. 5, 12 and 14 highly expressed at hcs occurring stage, and then gradually declined at early, mid-molt stages, especially spot no. 5 and 12. at newly molted fifth instar larvae stage, the expression levels of these proteins increased again (fig. 5). as shown in table 1, these proteins were vacuolar atpase subunit b, juvenile hormone binding protein brp-2095 precursor, adenylate kinase isoenzyme 1, h+ transporting atp synthase subunit d, respectively. meanwhile, we also identified some stress and/or defense-related proteins, including annexin (table 1, fig. 1, spot no. 9), glutathione s-transferase sigma 1 (table 1, fig. 1, spot no. 21), thiol peroxiredoxin (table 1, fig. 3, spot no. 34). as shown in table 1 and figure 1, annexin was down-regulated at 12 h after hcs (m1), whereas glutathione s-transferase was up-regulated at 12 h after hcs (m1). the expression level of thiol peroxiredoxin had no obvious change among hcs (mq), 12 h after hcs (m1), and 24 h after hcs (m2), but its expression level was significantly down-regulated at newly molted fifth instar larvae (qc) stage (table 1, fig. 3, spot no. 34). go analysis all go annotations of differentially expressed proteins were summarized and used for go analysis. as shown in figure 6, these proteins mainly involved in cell, cell part, organelle, organelle part, binding, catalytic, structural molecule, cellular process, and metabolic process, which were consistent with its specific developmental stages. the go analysis can provide useful clues for subsequent studies of physiological roles and characteristics of these identified proteins. quantitative real-time pcr in current study, four proteins (spot no. 5, vacuolar atp synthase subunit b; spot no. 12, juvenile hormone binding protein brp-2095 precursor; spot no. 14, adenylate kinase isoenzyme 1; spot no. 17, h+ transporting atp synthase subunit d) were selected to investigate their expression patterns at transcript level. as shown in figure 7, vacuolar atp synthase subunit b (spot no. 5) and h+ transporting atp synthase subunit d (spot no. 17) displayed good correlation between transcript and protein levels at each time point. compared with 12 h and 24 h after hcs (m1 and m2), juvenile hormone binding protein brp-2095 precursor (spot no. 12) and 393 394 fig. 5 enlarged view of distribution of regularly changed proteins. changed proteins were indicated by black arrows, spot numbers were shown on the first column, and the quantitative changes of proteins were shown in the last column (vol %: spot relative volume). values are average of three replicates. adenylate kinase isoenzyme (spot no. 14) were both up-regulated at transcript and protein levels at mq and qc stages (fig. 7). the quantitative real-time pcr results demonstrated that the expression patterns of these proteins at transcript level were basically consistent with the proteomic results. discussion in this study, we applied proteomic approach to globally identify different proteins during the fourth molting of silkworm and evaluate proteomic dynamic changes and their roles in this specific period. taken together, 35 differentially expressed proteins were successfully identified through mass spectrometry and database searches. among these identified proteins, 42 % proteins were classified as the cell structure group, which were the most abundant proteins in silkworm larval heads of the fourth molting stage, indicating that cell components of the heads at this stage changed dramatically. moreover, down-regulated proteins at early, mid-molt, and newly molted fifth instar larvae mainly consisted of metabolism-related proteins, indicating the metabolic levels at these stages were very low, which were consistent with its specific developmental period. interestingly, the expression levels of three metabolism/secondary metabolism-related proteins were down-regulated significantly at early, mid-molt stages (fig. 5, spot nos. 5, 12, 14), which were further confirmed by quantitative real-time pcr. meanwhile, these three proteins had the mascot scores with 187, 140, and 108, and their sequence coverages were 55 %, 52 %, and 57 %, which strongly supported for their positive identification (table 1, supplementary files, figs s1, s2, s3). the vacuolar atpase (v-atpase) is a heteromultimeric protein complexes that consists of the peripheral v1 domain and the integral v0 domain, which is atp-driven proton pumps and can transport protons across the plasma membrane (beyenbach and wieczorek, 2006; forgac, 2007; 395 fig. 6 the gene ontology (go) analysis (web gene ontology annotation plot) for differentially expressed proteins. the left coordinate axis represents the proportion of proteins for every go annotation, and the right one indicates the number of proteins for each go annotation. collaco et al., 2013; lv et al., 2014). v-atpases are essential for ph regulation of the intracellular compartments, the extracellular space, and the cytoplasm and play a vital role in acidification of organelles within the lysosomal, endocytic, and secretory pathways (breton and brown, 2007; collaco et al., 2013). the juvenile hormone binding proteins (jhbps) are specific carriers of juvenile hormone (jh) and also the first member in the series of proteins participating in jh signal transmission (sok et al., 2005). as the key proteins in jh signaling, jhbps can not only transport jh to its target tissues where jh exerts a physiological effect (goodman, 1990), but also inhibit enzymatic degradation of jh through general hemolymph esterases (ritdachyeng et al., 2012). previous studies have pointed out that more than 99.8 % jh in hemolymph emerges in a complex with jhbps and the interaction between jh and its carrier partner protein is specific (ozyhar and kochman, 1987; touhara et al., 1993; ritdachyeng et al., 2012). it is well known that jh plays pivotal roles in regulating insect growth and development, especially in maintaining the larval state, which ensures growth of the larva and prevents metamorphosis (riddiford, 1994; vermunt et al., 2001). therefore, as jh signal transmitters and specific carrier proteins, information concerning jhbps not only provides an alternative approach to understand how jh regulates metamorphosis, but also affords important clues for us to investigate the molecular mechanism of silkworm molting. previous studies have shown that jh regulates metamorphosis by way of specific gene up-regulation and down-regulation (riddiford, 1994; truman and riddiford, 1999; gilbert et al., 2000). in current study, although jh was not detected, the precursor of its carrier protein decreased significantly during silkworm molting. these observations suggest that jh regulates metamorphosis by way of jhbp down-regulation, that is, jhbp indirectly participates in the regulation of silkworm molting and plays a crucial role during silkworm molting (fig. 8, a pathway). adenylate kinase (ak) is ubiquitous in a variety of organisms and can catalyze a reversible high energy phosphoryl transfer reaction between atp and amp to generate adp (noda, 1973; miura et al., 2001), which facilitates to regulate amp levels. the previous study has demonstrated that the blood amp levels are potential metabolic signals related to vital functions, including body energy sensing, sleep, hibernation and food intake (dzeja and terzic, 2009). 396 fig. 7 relative expression levels of spot nos. 5, 12, 14 and 17. x-axis represents different samples, y-axis represents the relative expression level of each protein. the energy from adenylate kinase-catalyzed phosphotransfer can regulate multiple extracellular and intracellular energy-dependent and nucleotide signaling processes, including cell and ciliary motility, excitation-contraction coupling, energetics of cell cycle, dna synthesis and repair, hormone secretion, nuclear transport, and developmental programming (dzeja and terzic, 2009). in current study, the expression levels of ak isoenzyme at hcs and newly molted fifth instar larvae stages were higher than that at early, mid-molt stages. as mentioned above, amp levels were associated with some crucial functions, such as body energy sensing, sleep and hibernation. therefore, we postulated that ak may function as a mediator that control developmental events such as diapause, molting and eclosion, which was firstly found that ak might be correlated with silkworm molting (fig. 8, b pathway). these findings described here not only improve our understanding of the molecular mechanism of silkworm molting, but also demonstrate that comparative proteomic approach can be conducive to identifying different proteins and investigating their roles in silkworm studies. conclusions in current study, proteomic method was employed to investigate and analyze the changes of different proteins during silkworm molting, which enabled us to better understand the molecular mechanism of molting. eventually, 35 different proteins were successfully identified, including juvenile hormone binding protein precursor and adenylate kinase isoenzyme. the identification of these two proteins suggests that jhbps and ak may take part in the regulation of silkworm molting. as a consequence, our results not only provide novel insights and references for the explanation of molecular mechanism of silkworm molting, but also provide candidate proteins and genes for the following investigations of their roles in silkworm molting. 397 fig. 8 the speculative molecular mechanism of silkworm molting. acknowledgments this work was supported by national natural science foundation of china (31572467), the priority academic program development of jiangsu 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proteomics 6: 2586-2599, 2006. 399 supplementary material table s1 primer sequences used for quantitative real-time pcr spot no.a protein name forward primer (5’-3’) reverse primer (5’-3’) 5 vacuolar atp synthase subunit b gcctaggctcacttacaagact gaccagaacgaagggttcca 12 juvenile hormone binding protein brp-2095 precursor tgttcaacacaaacgccgac acccttcgtaaactcaggcag 14 adenylate kinase isoenzyme 1 cccggatcaggaaagggaac ctctccgagccgcttttgac 17 h+ transporting atp synthase subunit d ctactgccgtatgaccagat gacatagtcgagctgctctt α-tubulin ctccctcctccataccct atcaactaccagccaccc a the spot number of identified proteins (see table 1). table s2 the differentially expressed proteins and their identification through maldi-tof ms/ms material expression level b spot no. protein name accession no. mr(kda)/pi c mascot scores scd (%) amino acid function fold change mq-m1 a up-regulated proteins 1 heat shock protein 70-3 aei58998 72.82/5.12 138 25% 655 molecular chaperone 1.7 4 uncharacterized protein loc101738727 xp_012552185 49.91/5.29 129 31% 449 unknown function 2.4 7 antitrypsin isoform 1 act36276 43.46/5.41 145 41% 392 metabolism 1.8 10 alpha-tocopherol transfer protein-like xp_004928929 35.28/5.56 146 47% 306 transporter 2.9 11 alpha-tocopherol transfer protein-like xp_004928929 35.28/5.56 183 50% 306 transporter 2.5 13 e cuticular protein rr-1 motif 34 precursor np_001166717 23.18/4.78 107 56% 207 cell structure 3.9 15 tumor protein d54 isoform x3 xp_004930117 22.32/5.41 91 28% 206 cell structure 1.9 16 putative cuticle protein faa00454 21.66/5.39 91 45% 215 cell structure 2.2 20 odorant binding protein fmxg18c17 precursor np_001157372 26.48/6.23 121 28% 236 cell structure 3.1 21 glutathione s-transferase sigma 1 np_001037077 23.60/5.98 131 54% 206 disease and defense 2.9 23 actin-depolymerizing factor 1 np_001093278 17.23/6.17 175 64% 148 cell structure 2.3 down-regulated proteins 2 tubulin alpha-3 chain, partial kfq41598 43.57/5.79 197 55% 389 cell structure 2.2 3 beta-1 tubulin tbb1_manse 50.65/4.75 262 64% 447 cell structure 2.0 5 vacuolar atp synthase subunit b np_001091828 54.67/5.25 187 55% 490 metabolism 2.0 6 antichymotrypsin-2 ach2_bommo 41.43/5.26 109 39% 375 metabolism 1.8 9 annexin bab16697 36.11/4.89 138 43% 323 disease and defense 2.0 12 juvenile hormone binding protein brp-2095 precursor np_001036987 28.05/5.42 140 55% 249 secondary metabolism 3.1 14 adenylate kinase isoenzyme 1 xp_004929167 25.29/5.70 108 57% 226 metabolism 1.9 17 h+ transporting atp synthase subunit d np_001093279 20.19/5.56 57 52% 179 metabolism 1.7 19 triosephosphate isomerase np_001119730 26.93/5.67 168 64% 248 metabolism 1.7 22 cuticular protein rr-1 motif 42 precursor np_001166712 17.17/5.16 151 86% 159 cell structure 3.0 399 unique proteins 8 uncharacterized loc101739385 xp_004922152 37.46/6.07 91 32% 328 unknown function 18 transcription factor btf3 homolog 4 xp_012551703 19.04/9.12 201 48% 174 transcriptio n 24 muscular protein 20 np_001040476 20.29/8.70 79 20% 184 cell structure mq-m2 a up-regulated proteins 1 heat shock protein 70-3 aei58998 72.82/5.12 138 25% 655 molecular chaperone 2.1 10 alpha-tocopherol transfer protein-like xp_004928929 35.28/5.56 121 28% 306 transporter 1.8 11 alpha-tocopherol transfer protein-like xp_004928929 35.28/5.56 183 50% 306 transporter 1.9 13 cuticular protein rr-1 motif 34 precursor np_001166717 23.18/4.78 107 56% 207 cell structure 3.4 15 tumor protein d54 isoform x3 xp_004930117 22.32/5.41 91 28% 206 cell structure 1.7 17 h+ transporting atp synthase subunit d np_001093279 20.19/5.56 57 52% 179 metabolism 2.0 23 actin-depolymerizing factor 1 np_001093278 17.23/6.17 175 64% 148 cell structure 1.7 25 cuticular protein 66d np_729400 30.80/5.97 74 20% 270 cell structure 2.5 26 cuticular protein rr-2 motif 67 precursor np_001166691 18.72/6.16 223 43% 178 cell structure 8.6 28 ribosomal protein p2 np_001037213 11.53/4.68 121 16% 112 protein synthesis 2.4 down-regulated proteins 5 vacuolar atp synthase subunit b np_001091828 54.67/5.25 187 55% 490 metabolism 2.3 6 antichymotrypsin-2 ach2_bommo 41.43/5.26 109 39% 375 metabolism 2.8 12 juvenile hormone binding protein brp-2095 precursor np_001036987 28.05/5.42 140 55% 249 secondary metabolism 11.1 14 adenylate kinase isoenzyme 1 xp_004929167 25.29/5.70 108 57% 226 metabolism 2.1 18 transcription factor btf3 homolog 4 xp_012551703 19.04/9.12 201 48% 174 transcriptio n 4.0 19 triosephosphate isomerase np_001119730 26.93/5.67 168 64% 248 metabolism 1.6 20 odorant binding protein fmxg18c17 precursor np_001157372 26.48/6.23 121 28% 236 cell structure 3.7 24 muscular protein 20 np_001040476 20.29/8.70 79 20% 184 cell structure 4.8 unique proteins 22 cuticular protein rr-1 motif 42 precursor np_001166712 17.17/5.16 151 86% 159 cell structure 27 uncharacterized protein loc101741978 xp_004930780 16.55/4.93 117 46% 144 unknown function mq-qc a up-regulated proteins 10 alpha-tocopherol transfer protein-like xp_004928929 35.28/5.56 146 47% 306 transporter 2.0 13 cuticular protein rr-1 motif 34 precursor np_001166717 23.18/4.78 107 56% 207 cell structure 3.2 17 h+ transporting atp synthase subunit d np_001093279 20.19/5.56 57 52% 179 metabolism 2.4 29 beta tubulin np_001036887 50.72/4.83 267 70% 447 cell structure 1.9 30 centromere protein f isoform x2 xp_004926842 33.22/4.84 157 30% 297 cell structure 1.7 32 cuticular protein rr-1 motif 3 precursor np_001166744 14.68/4.66 93 28% 137 cell structure 2.4 33 cuticular protein rr-1 motif 3 precursor np_001166744 14.68/4.66 64 26% 137 cell structure 2.5 down-regulated proteins 5 vacuolar atp synthase subunit b np_001091828 54.67/5.25 187 55% 490 metabolism 1.8 12 juvenile hormone binding protein brp-2095 precursor np_001036987 28.05/5.42 140 55% 249 secondary metabolism 4.0 14 adenylate kinase isoenzyme 1 xp_004929167 25.29/5.70 108 57% 226 metabolism 1.7 22 cuticular protein np_001166712 17.17/5.16 151 86% 159 cell 4.4 400 399 rr-1 motif 42 precursor structure 31 t-complex protein 1 subunit epsilon-like xp_004933262 59.19/5.63 127 31% 542 molecular chaperone 2.1 34 thiol peroxiredoxin np_001037083 22.07/6.09 252 18% 195 disease and defense 2.5 35 ubiquitin-like protein smt3 np_001037410 10.36/5.29 105 23% 91 transcriptio n 1.8 unique proteins 7 antitrypsin isoform 1 act36276 43.46/5.41 145 41% 392 metabolism 8 uncharacterized loc101739385 xp_004922152 37.46/6.07 91 32% 328 unknown function 26 cuticular protein rr-2 motif 67 precursor np_001166691 18.72/6.16 223 43% 178 cell structure 27 uncharacterized protein loc101741978 xp_004930780 16.55/4.93 117 46% 144 unknown function a mq: silkworm from head capsule slippage (hcs) occurring; m1: silkworm from 12 h after hcs; m2: silkworm from 24 h after hcs; qc: newly molted fifth instar larvae; breference gel: mq; c mw: molecular weight; pi: isoelectric point; dsc: sequence coverage; ethe identified protein by maldi-tof ms. fig. s1 the mass spectrogram of vacuolar atp synthase subunit b and the amino acid assignment of the protein from the samples. 401 399 fig. s2 the mass spectrogram of juvenile hormone binding protein brp-2095 precursor and the amino acid assignment of the protein from the samples. 402 399 fig. s3 the mass spectrogram of adenylate kinase isoenzyme 1 and the amino acid assignment of the protein from the samples. 403 33 isj 18: 33-45, 2021 issn 1824-307x research report the influence of surface waters on the bioavailability and toxicity of copper oxide nanoparticles to freshwater mussels j auclair, p turcotte, c gagnon, f gagné* aquatic contaminants research division, environment and climate change canada, 105 mcgill, montréal, québec, canada h2y 2e7 this is an open access article published under the cc by license accepted january 22, 2021 abstract the increased commercial use of copper oxide nanoparticles (ncuo) led to the release of nanoparticles in wastewaters potentially harming the aquatic biota. the purpose of this study was to determine the toxic action of ncuo and dissolved cu (ii) to dreissena bugensis freshwater mussels placed in 4 types of surface waters: aquarium, green (high conductivity), brown (high organic carbon) and 10 % municipal effluent (high conductivity and anthropogenic source of organic carbon). mussels were exposed to 50 µg/l of ncuo or cu (ii) for 96 h at 15 °c in the above waters. the results revealed that the total cu loadings were higher in mussels placed in organic-rich waters (brown and effluent) and exposed to either forms of cu. tissue cu contents were correlated with air-time survival, lipid peroxidation, protein-ubiquitin levels and dna strand breaks. both surface water types and cu forms influenced zn (ii) mobilization, glutathione s-transferase activity and protein turnover (ubiquitin binding). based on the surface water properties, cu (ii) was more influenced by the levels and origin of the organic carbon content while ncuo was more influenced by the total suspended solids. in conclusion the toxicity of ncuo could be influenced by surface waters properties especially when similar physiological targets are impacts by these treatments. key words: quagga mussels; surface waters; copper oxide nanoparticles; oxidative stress; ubiquitin introduction the exponential commercial application of nanoparticles have lead to the mobilization of nanoparticles in the environment. nanoparticles are currently used in various consumer products because of their new properties (lee et al., 2010). the global production of nanoparticles is estimated in the order 320 metric tons (keller and lazareva, 2013). the continuous release of metallic nanoparticles could reach levels in the environment that exceed the toxic threshold in aquatic organisms (garner et al., 2017). given their intrinsic properties, metal oxide nanoparticles that are produced at large scales are cerium, titanium, zinc and copper oxide nanoparticles (ncuo) where zinc oxide nanoparticles and ncuo are the most commonly used metal-based nanoparticles. these metal oxide nanoparticles are poorly soluble and subject to aggregation when the surface charges ___________________________________________________________________________ corresponding author: françois gagné aquatic contaminants research division environment and climate change canada 105 mcgill, montréal, québec, canada h2y 2e7 e-mail: francois.gagne@canada.ca are neutralised by salt or other conterions (liu et al., 2018). conversely, in the presence of natural organic matter (humic and fulvic acids), an increased proportion of monomeric nanoparticles could be observed (keller et al., 2010). the predicted environmental concentration of ncuo in municipal wastewaters was estimated between the µg/l to mg/l range based on modeling approaches (musee et al., 2011). the levels of metal oxides nanoparticles could reach values well above the mg/l range (walden and zhang, 2016). the toxicity of ncuo to aquatic residents such as bacteria, algae, invertebrates and fish has been examined in numerous studies (gomes et al., 2011; isani et al., 2013; adam et al., 2015). while the acute and chronic lethality of ncuo are in the mg/l range, which are not expected to occur often in the environment. effects at molecular level were shown to occur at concentrations in the µg/l range which is of concern in the context of long-term exposure to these nanomaterials (buffet et al., 2011; cuillel et al., 2014; giannetto et al., 2019). dissolved cu (ii) are more acutely toxic to rainbow trout (96 h lc50=15 µg/l) indicating that the nanoforms of cu oxides are much less toxic (marr et al., 1999). the 34 96 h toxicity of ncuo for the copepod tigriopus fulvus was at 12 mg/l with sublethal effects (copepod moult and sea urchin egg fertilization) at 2 mg/l (rotini et al., 2018). the study also found that in the presence of sea water (salt-rich), only 0.16 % of ncuo were in the dissolved phase. the toxicity also depends on the environmental conditions, which could influence the dissolution, aggregation, surface charge states of the nanoparticles. bivalves are species particularly at risk to suspended particle in the water column since they are filter-feeders and sessile organisms. the gills filter water to trap suspended material (nanoparticles and their aggregates) which are then directed to the digestive system for assimilation. the toxicity of nanoparticles in bivalves species has been extensively examined over the years (gagné et al., 2007; canesi et al., 2012; rocha et al., 2015). these studies revealed the release of elements from the nanoparticle is an important driver of toxicity but also by other characteristic properties of the nanoparticles such as size, form, surface charges and coatings. nanoparticles and their aggregates were shown to accumulate in the digestive gland and not only producing oxidative stress but inflammatory responses as well in addition to cellular injury to membrane, proteins and dna. however, it was not determined whether the nature of freshwater could also influence exposure and effects of nanomaterials in mussels. indeed, in high salt conditions, the surface charge (zeta potential) could be canceled out and favor aggregation mechanisms which could be trapped in the digestive gland and initiate inflammatory/immune responses. conversely, the presence of natural organic matter could act as surfactants and favor dissolution of nanoparticles which favor transfer in cells (dominigo et al., 2009). these co-occurring mechanisms complicate the prediction of nanoparticle state in surface waters, which could in turn, influence bioavailability and toxicity in organism. indeed, the influence of water conductivity, the nature (natural, municipal, agricultural) and levels of total organic carbon is less understood at the present time. a recent review was recently published solely on the influence of organic matter on the state of engineered nanoparticles (grillo et al., 2015). natural organic matter are mainly composed of humic and fulvic acids while organic matter from urban areas (effluents) is mainly composed of protein compounds from human/animal wastes. the purpose of this study was therefore to determine the influence of surface water properties and form of cu (ncuo or cu (ii)) on the bioavailability and toxicity in freshwater mussels dreissena bugensis. in this study, 4 types of freshwater were examined in mussels: aquarium (dechlorinated tap water), high conductivity/low total organic carbon (toc) water (green waters), low conductivity/high toc water (brown water) and a diluted municipal effluent in aquarium water as another source of organic matter. toxicity was examined by following changes in oxidative stress, inflammation, genotoxicity and turnover of damaged proteins in the soft tissues of mussels. an attention was given on the combined influence of cu forms (dissolved vs nanoparticles) and surface water properties towards bioavailability and toxic effects in freshwater mussels. materials and methods copper oxide nanoparticle a stock solution of copper oxide nanoparticle (ncuo) from us research materials (usa) was used in this study. according to the manufacturer’s specifications, the size distribution of ncuo suspension was between 25-55 nm according to the supplier information. for the exposure regime, quagga mussels (dreissena bugensis) were exposed to a nominal concentration of 50 µg/l total cu as either ncuo or cu (ii) as cuso4 in dechlorinated tap water (controls), and three other types of surface water as described below. this concentration range was selected based on previously reported total copper concentrations in municipal effluents (10-50 µg/l) (gagnon et al., 2014; dietler et al., 2019). the hydrodynamic diameter and zeta potential of ncuo were measured at least three times using a dynamic light scattering instrument (mobius instrument, wyatt technologies, santa barbara, ca, usa) operating with a laser at a wavelength of 532 nm. the instrument was previously calibrated with latex beads at 50 nm diameter (polyscience, usa). two types of natural water samples were sampled in the st. lawrence river: green and brown waters (20 l each). the aquarium water consisted of uv and charcoal-treated dechlorinated tap water and was considered as the reference water. a 10 % dilution of a physico-chemically (primary) treated municipal effluent from a city of 2 million inhabitants was also prepared in aquarium water to simulate a water sample with same total organic carbon content (toc) than green and aquarium waters but of different (anthropogenic) origin. in each of water samples, toc, total suspended solids (tss), ph and conductivity were determined according to standard methods (apha, 2010). green water is characterized by a relatively high conductivity (200-250 μs/cm) and tss, low toc (< 4 mg/l), and a slightly alkaline ph (ph > 7.2) (table 1). brown water is characterized by low table 1 surface water characteristics data water type toc (mg/l) conductivity µscm-1 total suspended solid (mg/l) ph aquarium 3.1 ± 1 297 ± 30 < 1 8.1 ± 0.5 green 3.4 ± 1 245 ± 20 19 ± 2* 8.3 ± 0.5 brown 7.3 ± 2* 108 ±10* 4 ± 0.5* 7.5 ± 0.5 effluent 3.2 ± 0.75 319 ± 30 2 ± 0.2* 8.2 ± 0.5 * significant from aquarium water 35 conductivity (100-160 μs/cm) and tss, higher toc levels (> 5 mg/l) and a slightly acidic ph (ph < 8). the surface waters (20 l) were collected 2-3 weeks before the onset of exposure; they were stored in the dark at 4 °c until the exposure experiments at 15 °c. the total levels of cu in the 4 surface waters (green, brown, 10 % municipal effluent and aquarium) were determined before and after 1 h of dissolution of ncuo using icp-ms spectrometry following acidification with 1 % v/v nitric acid (seastar grade bc, canada). quagga mussels (n = 30) were exposed to each type of water with and without 50 µg/l total cu as either ncuo or cu (ii) as cuso4 for 96 h at 15 °c under constant aeration. control mussels were exposed to each type of surface water only with no addition of either forms of cu. the exposure experiment was repeated 2 times. the surface waters were not renewed and the mussels were not fed during the exposure period. a subgroup of 10 mussels was kept aside for air-stress survival while the remaining 20 mussels were processed as follows. for cu assessments in mussels, a subgroup of 10 individuals were placed in clean aquarium water overnight as a depuration step to remove loosely bound cu at the surfaces of the mussels. the soft tissues were removed for total cu determination using icp-mass spectrometry as described above. the tissues were acid-digested in 10 % hno3 at 7080 °c for 12 h and diluted to 1 % with milliq water. for the biomarker analyses, the remaining group of 10 mussels were analyzed for shell length, total weight and soft tissues weight. the soft tissues were then stored at -85 °c with the homogenization buffer (at 20 % weight/volume). the homogenization buffer consisted of 100 mm nacl, 25 mm hepesnaoh, ph 7.4, 1 µg/ml apoprotin and 1 mm dithiothreitol. air survival test after the exposure period, 10 mussels were kept aside to determine the air-time survival as previously described (gagné et al., 2015). in brief, the mussels were weighed and the shell length determined, and they were placed individually into plastic containers under 80 % humidity at 20 °c. they were maintained as such and weighed each day until mortality (opened shells). the time of death in days was determined for each individual over the 12 treatment groups: 4 types of surface water alone, 4 types of surface water with ncuo and 4 types of surface water with ncu (ii). the weight loss during air emersion were also measured and the data were expressed as the ratio of weight loss: (initial weight-weight at given day)/initial weight. biomarker analyses the soft tissues were allowed to thaw on ice for 30 min and homogenized still in melting ice using a teflon pestle tissue grinder at 4 °c. a portion of the homogenate was set aside for lipid peroxidation (lpo), dna damage and total proteins. the remainder of the homogenate was centrifuged at 15000 x g for 20 min at 4 °c and the supernatant (s15) was removed for arachidonate cyclooxygenase (cox) evaluation, glutathione stransferase (gst), acetylcholinesterase (ache), labile zinc (zn) and protein-ubiquitin levels. total proteins were determined in the homogenate and s15 fraction using the protein-dye binding principle using standard solutions of serum bovine albumin for calibration (bradford, 1976). lipid peroxidation (lpo) was determined in soft tissue homogenates using the thiobarbituric acid method (wills, 1987). a volume of 25 µl of the homogenate was mixed with 175 µl of 10 % trichloroacetic acid containing 1 mm feso4 and 50 µl of 0.7 % thiobarbituric acid. the mixture was heated at 75 °c for 10 min. the mixture was cooled to room temperature and centrifuged at 10000 x g for 5 min to remove any precipitates. a 200 µl volume was transferred to a 96-well dark microplate, and fluorescence readings were taken at 540 nm excitation and 600 nm emission. standard solutions of malonaldehyde (tetramethoxypropane, sigma chemical company, on, canada) were made for calibration in the homogenization buffer. results were expressed as µg thiobarbituric acid reactants (tbars)/mg total proteins in the homogenate. the levels of dna strand breaks were also determined in the homogenate using the fluorescence dna precipitation assay (olive, 1988; gagné et al., 2008). briefly, 25 µl of the homogenate from each tissues were mixed with 100 µl of 50 mm naoh, 10 mm tris base, 10 mm ethylenediamine tetraacetate and 2 % sodium dodecyl sulphate (sds). after 5 min, one volume of 0.12 m kcl was added and heated at 60 °c for 10 min. the mixture was cooled on ice for 10 min and centrifuged at 8000 x g for 10 min to precipitate sds-associated proteins and genomic dna. the supernatant (dna strands) was mixed with syto green dye in 3 mm sodium cholate, 0.4 m nacl and 100 mm tris-acetate ph 8 to control for the traces of sds in the supernatant which could interference the fluorescence readings (bester et al., 1994). fluorescence was measured at 485 nm excitation and 530 nm emission (microplate, synergy-4, bioteck, usa) using standard solutions of salmon sperm dna for calibration. the data were expressed as µg supernatant dna/mg proteins. levels of labile zn were determined using a fluorescent probe methodology (gagné and blaise, 1996). briefly, a 20 µl sample of the s15 fraction was mixed with 180 µl of 50 µm of tsq probe in 20 % dmso, 140 mm nacl, 5 mm kh2po4 and 10 mm hepes-naoh, ph 7.4. fluorescence was measured at 370 nm excitation and 490 nm emission (synergy-4, biotek instuments, usa) using standard solutions of znso4 for instrument calibration. data were expressed as relative fluorescence units (rfu)/mg proteins. the activity of arachidonatedependent cyclooxygenase (cox) activity was determined in the s15 fraction of soft tissues using a fluorescence microplate reader (gagné, 2014). briefly, 25 µl of the s15 fraction was added to 175 µl of the assay mixture composed of 50 µm arachidonate, 2 µm of dichlrofluorescein and 0.1 µg/ml of horseradish peroxidase in 50 mm tris-hcl, ph 8.0 and 0.05 % tween-20. the reaction was allowed to proceed for 20 min at 20 °c and fluorescence readings were made at each 5-min interval at 485 nm excitation and 528 nm emission 36 in dark microplates (synergy 4, biotek instruments, usa). the data were expressed as the increase in relative fluorescence units/min/mg proteins in the s15 fraction. the activity of acetylcholinesterase (ache) activity was determined in the s15 fraction using acetylthiocholine as the substrate. the formation of thiocholine were determined using the ellman’s reagent according a previously published methodology (gélinas et al., 2013). standard solutions of reduced glutathione were used for calibration. the data were expressed as rfu/min/mg proteins. the activity of glutathione stransferase (gst) activity were determined in the s15 fraction using a microplate spectrometric assay (boryslawskyj et al., 1988). the activity was determined using reduced glutathione and 2,4dichlorodinitrobenzene as the chromophore substrate at 340 nm. the data were expressed as the increase in absorbance at 340 nm/min/mg total proteins in the s15 fraction. the levels of polyubiquitinylated proteins were determined by enzyme-linked immunosorbent assay (elisa) in the s15 fraction as described in a previous study (auclair et al., 2019). standards of polyubiquitin (ub2-7, k48-linked, enzo life sciences, farmingdale, ny) or the s15 fraction were used to coat the microplate wells (immulon-4). the antibody ubiquitin lys48-specific rabbit monoclonal antibody (clone apu2; emd millipore, billerica, usa) was diluted 1/2000 in phosphate buffered saline (140 mm nacl, 5 mm kh2po4 and 1 mm nahco3, ph 7.4) containing 0.5 % albumin and was added to each wells. after incubation for 60 min, the wells were washed with 0.5 % albumin and the secondary antibody (anti-rabbit igg-linked with to peroxidase; adi-sab-300, enzo, usa) diluted 1/5000 and incubated for another hour. after well washing, the activity of peroxidase was detected using a highly sensitive chemiluminescence assay kit (roche diagnostics, qc, canada). data were expressed as ng of polyubiquitin/mg proteins. data analysis the study design examines the influence of surface waters on two chemical forms of cu -(ncuo and cu (ii)in terms of toxicity in quagga mussels. there were 12 treatments: mussels exposed to each type of water: aquarium, brown, green, effluent 10 % alone (treatments 1-4); mussels exposed to 50 µg/l cu as ncuo in each type of water (treatments 5-8); and mussels exposed to 50 µg/l of cu as cuso4 in each type of water (treatments 9-12). in each treatment, 10 mussels were used for air-time surivival, biomarkers and chemical analysis for a total of n=30 mussels per treatment and the experiment was repeated twice. data normality and homogeneity of variance were verified using the shapiro-wilk and bartlett tests respectively. the influence of surface water types (aquarium, green, brown and 10 % effluent) and cu forms (ncuo and cu (ii)) were examined using 2-way factorial analysis of variance. critical differences between treatments were determined using the least square difference (lsd) test. relationships between the biomarker data were also analyzed using the pearson moment correlation test. the data were also analyzed by discriminant function analysis to determine which biomarkers could discriminate between the influence of surface waters and forms of cu in mussels. significance was set at p<0.05. all statistical analyses were performed with the sysstat software package (version 13.2, usa). results the surface water physico-chemical properties were determined (table 1). in respect to total organic carbon (toc), brown waters had the highest level at 7.3 mg/l compared to green (3.4 mg/l), diluted municipal effluent (3.2 mg/l) and aquarium waters (3.1 mg/l). although the levels of toc in green waters were similar to the diluted municipal effluents sample, the nature differed in the following manner: the toc in green water is of natural origin (coined natural organic matter) while of municipal wastewater origin in the diluted municipal effluent. in respect to conductivity, the municipal effluent and aquarium waters had the highest conductivity at 319 and 295 µs*cm-1 respectively compared to green (245 µs*cm-1) and brown waters (108 µs*cm-1). in respect to total suspended solids (tss), green waters had the highest value (19 mg/l) compared to brown (4 mg/l) and municipal effluent (2 mg/l). aquarium waters had no measurable levels of tss. the ph was statistically the same for all water types. based on these properties, the influence of the origin of toc could be resolved by comparing effects between green and diluted municipal effluent. effects caused by an increase in toc of natural origin or by a decrease in conductivity (effect in the levels of toc and conductivity) could be estimated between responses of green and brown waters. the influence of tss could be appraised by comparing the responses between aquarium and green waters. the behavior of ncuo in the various water types was also analyzed using dls (table 2). dilution in table 2 behavior of ncuo in the water samples water sample ncuo concentration mean diameter 1h (nm) zeta potential 1h (mvolts) mean diameter 48h (nm) zeta potential 48h (mvolts) milliq water 200 ug/l 79 ± 10 not analyzed 80 ± 10 not analyzed aquarium 200 ug/l 74 ± 5 -13 ± 3 132 ± 462 -19 ± 4 green water 200 ug/l 100 ± 36 -20 ± 3 133 ± 18 -14 ± 2 brown water 200 ug/l 69 ± 8 -14 ± 4 140 ± 66 -19 ± 4 effluent 10 % 200 ug/l 92 ± 26 -14 ± 4 220 ± 801,2 -14 ± 3 1. significant from milliq water; 2. significant compared to 1h 37 a qu ar iu m nc uo aq ua c u( ii) aq ua g re en nc uo gr ee n c u( ii) gr ee n b ro w n nc uo b ro w n c u( ii) b ro w n e ffl ue nt nc uo e ff c u( ii) e ff conditions (ug/l) 0,0 0,5 1,0 1,5 c u ( u g /g ) * * * * * fig. 1 cu levels in mussels exposed to cu forms and surface water types. total cu levels were measured in soft tissues of mussels following a 12 h depuration period in clean aquarium water. the data represent the mean and standard deviation expressed as ug metal/g dry weight. the letters a, b, c and d represents significance in the following: a difference between water control types with no added cu (water type effect); b difference between the forms of cu (cu (ii) and ncuo) from aquarium water (reference water); c difference between the forms of cu (ncuo or cu (ii)) from corresponding water type control (e.g., ncuo-green vs green); d difference between forms of cu within each water type (e.g., ncuo brown vs cu (ii) brown) milliq water produced no change in the mean diameter in respect to the supplier’s specifications. after dissolution for 1 h in the 4 types of water, the mean diameter (70 nm) did not significantly change between the water types although significantly more variation in the size distribution was observed especially for green and diluted municipal effluent waters. after 48 h, the mean diameter of ncuo in milliq water did not significantly changed. in the diluted municipal effluent, the mean diameter of ncuo was significantly higher than aquarium water and with the mean diameter after 1 h of dissolution in the same water. the mean diameter in aquarium water was also significantly increased at 48h (132 nm) compared to 1 h (74 nm) in aquarium water. no significant changes were observed for the zeta potential between the water types and between 1 and 48 h in solution. the total cu levels were determined in tissues of mussels following a 12 h depuration period (figure 1). in respect to water controls, cu-levels in tissues were significantly lower in mussels placed in brown and the diluted municipal effluent compared to aquarium water controls. the data revealed that total cu tissue levels were significantly increased in mussels exposed to cu (ii) and ncuo in brown water and effluent compared to the respective controls. the increase in cu in tissues were also higher than those in mussels exposed to ncuo or cu (ii) in aquarium water suggesting that bioavailability is increased in organic-rich waters (brown water and the diluted effluent). the levels of cu in tissues were significantly higher in mussels exposed green water and cu (ii) compared to mussels exposed to green water and ncuo. in green water, cu-tissue levels was significantly increased in mussels exposed to cu (ii) compared to green water controls. the estimated bioavaibility for ncuo/cu (ii) in green, brown and diluted effluent waters were not determined/20, 14/12 and 13/19 l/kg respectively. this suggests that the presence of organic matter readily increased ncuo bioavaibility and compares to cu (ii). the condition factor (mussel weight/shell length ratio) and the soft tissues weight ratio (soft tissue wet weight/mussel weight) were not significantly influenced by either surface water type or cu forms (2-way anova p>0.1 for cu forms and water types; 38 a qu ar iu m nc uo aq ua c u( ii) aq ua g re en nc uo gr ee n c u( ii) gr ee n b ro w n nc uo b ro w n c u( ii) b ro w n e ffl ue nt nc uo e ff c u( ii) e ff conditions 1 2 3 4 5 6 7 l e th a l ti m e ( d a y s ) b b c a qu ar iu m nc uo aq ua c u( ii) aq ua g re en nc uo gr ee n c u( ii) gr ee n b ro w n nc uo b ro w n c u( ii) b ro w n e ffl ue nt nc uo e ff c u( ii) e ff conditions 0,0 0,1 0,2 0,3 0,4 w e ig h t lo s s ( b e fo re m o rt a lit y ) b d b b c b c fig. 2 air-time survival in mussels exposed to cu forms and surface waters. the air-time survival a) and weight loss at day 3 b) are schown. the letters a, b, c and d represents significance effect in the following: a difference between water control types with no added cu (water type effect); b difference between the forms of cu (cu (ii) and ncuo) relative to aquarium water (reference water); c difference between the forms of cu (ncuo or cu (ii)) from corresponding water type control (e.g., ncuo-green vs green); d difference between forms of cu within each water type (e.g., ncuo brown vs cu (ii) brown data not shown). the lethal time was mostly influenced by water type and significant interaction was observed between cu forms and the type of surface waters (figure 2a). the lethal time was significantly reduced in mussels exposed to ncuo in aquarium water compared to aquarium water (controls). the lethal time was significantly increased in mussels exposed to cu (ii) in green water compared to controls and mussels exposed to ncuo in green water. the weight loss during the air survival time test was also determined as this endpoint occurs before mortality response. (figure 2). the data revealed that the cu forms had greater effects than surface water types but no significant interaction between the 2 treatments. weight loss at the 3rd day (i.e., the last day before appearance of mortality) was more important in mussels exposed to ncuo in aquarium water compared to controls (aquarium water) and mussels exposed to cu (ii) in aquarium water. the weight loss was lower in mussels exposed to cu (ii) in green water compared to aquarium water but marginally so in green water control. the weight loss was more important in mussels exposed to either forms of cu in the diluted effluent compared to mussels in aquarium water and the diluted effluent water controls. weight loss was significantly correlated with the lethal time (r = -0.69) where mussels losing weight more quickly (after 3 days) survive less to air emersion. oxidative stress was examined in mussels exposed to cu forms and surface waters by following changes in labile zn levels, cox activity and gst activity (figures 3a, 3b and 3c). factorial 2-way anova revealed a significant interaction between surface water types and forms of cu. in respect to water types, the levels of labile zn were significantly reduced in mussels placed in green and brown waters compared to aquarium water control. in the presence of cu forms, labile zn was significantly decreased in mussels exposed to ncuo and cu (ii) compared to aquarium water. labile zn were lower in mussels exposed to cu (ii) in aquarium water compared to ncuo in aquarium water. in mussels placed in green and brown waters, labile zn levels were decreased compared to aquarium water suggesting that brown water masked the effects of cu forms on labile zn levels. inflammation in mussels was measured by following cox activity in soft tissues (figure 3b). two-way anova revealed that cox activity was significantly influenced by the type of surface water (p = 0.01) and marginally so with the cu forms (p = 0.07). cox activity in mussels exposed to brown water control was significantly lower relative to mussels in aquarium water control. cox activity was significantly lower in mussels exposed to ncuo and cu (ii) in green water compared to mussels placed in green water alone. cox activity was significantly lower in mussels exposed to both forms of cu in brown water in respect to aquarium water but not so when compared to brown water control suggesting that the water properties was the main driver for this effect. cox activity in mussels exposed to ncuo placed in the diluted municipal effluent was conditions a) b) conditions 39 a qu ar iu m nc uo aq ua c u( ii) aq ua g re en nc uo gr ee n c u( ii) gr ee n b ro w n nc uo b ro w n c u( ii) b ro w n e ffl ue nt nc uo e ff c u( ii) e ff conditions 0 10 20 30 40 50 60 70 l a b il e z n ( r f u /m g p ro te in s ) a qu ar iu m nc uo aq ua c u( ii) aq ua g re en nc uo gr ee n c u( ii) gr ee n b ro w n nc uo b ro w n c u( ii) b ro w n e ffl ue nt nc uo e ff c u( ii) e ff conditions 10 000 20 000 30 000 40 000 50 000 60 000 70 000 80 000 c o x a c ti v it y ( r f u /m in /m g p ro te in s ) a a b b c d b b b b b b c d a b c b b b c a qu ar iu m nc uo aq ua c u( ii) aq ua g re en nc uo gr ee n c u( ii) gr ee n b ro w n nc uo b ro w n c u( ii) b ro w n e ffl ue nt nc uo e ff c u( ii) e ff conditions 50 60 70 80 90 100 110 120 g s t ( a b s /m in /m g p ro te in s ) b b c b a b b a b b fig. 3 oxidative stress in fish exposed to dissolved cu and ncuo. after the exposure period, the levels of labile zn and lpo were determined in the soft tissues. the letters a, b, c and d represents significance effect in the following: a difference between water control types with no added cu (water type effect); b difference between the forms of cu (cu (ii) and ncuo) from aquarium water (reference water); c difference between the forms of cu (ncuo or cu (ii)) from corresponding water type control (e.g., ncuo-green vs green); d difference between forms of cu within each water type (e.g., ncuo brown vs cu (ii) brown) marginally (p = 0.1) lower that aquarium water control and the diluted effluent control. correlation analysis revealed that cox activity was significantly correlated with weight loss (r = -0.24) and gst activity (r = 0.38) (table 3). a significant interaction between surface water and cu forms was obtained for gst activity (figure 4c). gst activity was reduced in mussels placed in brown and the diluted municipal effluent controls compared to aquarium control. in aquarium and green waters, the activity was significantly reduced by both ncuo and cu (ii). in brown and diluted municipal waters, neither forms of cu produced any significant changes. toxic effects of mussels exposed to forms of cu in different surface waters were examined by following damage at the level of proteins (ubiquitin tagging of damaged proteins), lipids (lpo) and the genetic material (dna strand breaks) (figures 4a, b and c). the levels of protein-ubiquitin were significantly influenced both surface water types and cu forms according to 2-way anova (figure 4a). protein-ubiquitin levels were significantly lower in a) b) c) 40 table 3 correlation analysis of biomarker data significant correlations are indicated in bold mussels exposed to brown and diluted effluent controls compared to aquarium water control. their levels were significantly reduced in mussels exposed to cu (ii) in aquarium water compared to aquarium water (control). in green waters, protein damage was significantly decreased in mussels exposed to ncuo and cu (ii). in brown waters, the levels of protein-ubiquitin in mussels exposed to ncuo were significantly lower compared to aquarium water controls but not with the brown water controls suggesting again that the driver of effects was from surface water type. in mussels exposed to the diluted effluent, the levels were decreased in mussels exposed to cu (ii) compared to the diluted effluent and aquarium water controls. correlation analysis revealed that protein-ubiquitin levels were significantly correlated with many endpoints: cox activity (r = 0.25), ache (0.33), gst activity (r = 0.53) labile zn levels (r = 0.22) and total cu levels in tissues (r = 0.25). factorial anova revealed that lpo levels were mainly influenced by the cu forms but not by the surface water types (figure 4b). the levels of lpo was significantly elevated only in mussels exposed to green water and cu (ii) compared to green water and aquarium water controls. correlation analysis revealed that lpo was significantly correlated with tissue cu levels (r = 0.34), ache (r = -0.23) and dna strand breaks (r = -0.58). the levels of dna damage (genotoxicity) was also examined in mussels (figure 4c). the data revealed that a marginal interaction between water types and cu forms was found. in green water, the levels of dna strand breaks were increased in mussels exposed to ncuo compared to green water controls. the levels of dna strand breaks were lower in mussels exposed to cu (ii) in brown water compared to brown water controls. correlation analysis revealed that dna damage was correlated with lpo (r = 0.57), tissues weight (r = 0.24) and gst activity (r = -0.25). the levels of ache activity was also determined in mussels to detect changes in neural activity (figure 5). analysis of the data revealed that the water types significantly influenced the activity while the cu forms had no effect. ache activity was significantly decreased in green, brown and diluted effluent waters compared to aquarium water control. the decreased did not differ from the corresponding water controls which indicates absence of cu effects regardless of the form. ache activitiy was significantly correlated with gst activity (r = 0.43). in the attempt to gain a more global understanding, a discriminant function analysis was performed to seek out relationships between the influence of cu forms in the different surface water types (figure 6). based on this analysis, 99 % of the variance was explained and the classification performance was at 25 % which indicates that some conditions were not always discriminated (i.e., similar effects were obtained). the biomarkers with the highest factorial weight, explaining the total variance were protein-ubiquitin, labile zn and gst activity. in respect on the water types only, the surface waters were all well separated from each other suggesting influence of water properties such as ph, conductivity, organic matter content and suspended solids on the biomarkers used in this study. in aquarium water, the addition of ncuo did not produced important changes compared to cu (ii), which were mostly in the direction of the x axis with labile zn and protein-ubiquitin levels and gst activity. in green water, the effects of cu forms markedly differ from controls but were rather similar with each other based on labile zn, protein-ubiquitin levels and gst activity. in brown water, the effects between brown water controls, ncuo and cu (ii) were the least discriminated showing the dampening influence of this organic matter-rich water on the toxicity of cu. in respect to the diluted municipal effluent, the effects of cu (ii) were markedly different than those produced by ncuo and controls which were more closely related with each other. in summary, mussels placed in green and the aquarium waters led to more important effects when mussels were exposed to cu (ii) and to ncuo to a lesser extent compared to mussels in brown water and the diluted municipal effluent. the influence of organic carbon origin could be evaluated by comparing the responses between green waters and diluted municipal effluent which 41 a qu ar iu m nc uo aq ua c u( ii) aq ua g re en nc uo gr ee n c u( ii) gr ee n b ro w n nc uo b ro w n c u( ii) b ro w n e ffl ue nt nc uo e ff c u( ii) e ff conditions 100 150 200 250 300 p ro te in -u b iq u it in ( r f u /m g p ro te in s ) a a b b c b b b b c d a qu ar iu m nc uo aq ua c u( ii) aq ua g re en nc uo gr ee n c u( ii) gr ee n b ro w n nc uo b ro w n c u( ii) b ro w n e ffl ue nt nc uo e ff c u( ii) e ff conditions 0 5 10 15 l p o ( u g t b a r s /m g p ro te in s ) a qu ar iu m nc uo aq ua c u( ii) aq ua g re en nc uo gr ee n c u( ii) gr ee n b ro w n nc uo b ro w n c u( ii) b ro w n e ffl ue nt nc uo e ff c u( ii) e ff conditions 0 50 100 150 d n a d a m a g e ( u g /m g p ro te in s ) b c c c fig. 4 tissue damage in mussels exposed to cu forms and surface waters. after the exposure period, tissue damage were studied at the protein (ubiquitinylation), lpo, dna damage levels in soft tissues. the letters a, b, c and d represents significance effect in the following: a difference between water control types with no added cu (water type effect); b difference between the forms of cu (cu (ii) and ncuo) from aquarium water (reference water); c difference between the forms of cu (ncuo or cu (ii)) from corresponding water type control (e.g., ncuogreen vs green); d difference between forms of cu within each water type (e.g., ncuo brown vs cu (ii) brown) contain relatively the same amount of organic carbon content but from different origin (natural vs municipal). the data revealed the influence of the organic carbon origin influence more the responses for dissolved cu (ii) than ncuo. the influence of total organic carbon (toc) levels could be evaluated by comparing the responses between green and brown waters which as different levels of natural toc content (brown waters have 2.1 times more toc than green waters) but the conductivity was also inversely influenced (green water have 2.2 times more conductivity than brown water). the analysis also revealed that the cu (ii) responses were more strongly influenced than with ncuo but this effect could be related to both toc and conductivity. the influence of total suspended solids (tss) could be evaluated by comparing the response of aquarium and green waters which contains the highest levels of tss for the latter. the analysis revealed that tss influenced more the responses of ncuo than cu (ii). hence, the effects of ncuo are more influenced by tss than toc and conductivity while the effects of cu (ii) are more influenced by the levels/origin of the toc and conductivity. c) b) a) 42 a qu ar iu m nc uo aq ua c u( ii) aq ua g re en nc uo gr ee n c u( ii) gr ee n b ro w n nc uo b ro w n c u( ii) b ro w n e ffl ue nt nc uo e ff c u( ii) e ff conditions 0,0 0,5 1,0 1,5 a c h e ( a b s /m in /m g p ro t. ) a b a b b a b b fig. 5 ache in mussels exposed to cu forms in different surface waters. after the exposure period, ache and gat activities were determined in soft tissues. the letters a, b, c and d represents significance effect in the following: a difference between water control types with no added cu (water type effect); b difference between the forms of cu (cu (ii) and ncuo) from aquarium water (reference water); c difference between the forms of cu (ncuo or cu (ii)) from corresponding water type control (e.g., ncuo-green vs green); d difference between forms of cu within each water type (e.g., ncuo brown vs cu (ii) brown) discussion the organic carbon content was the highest in brown water and is composed of natural organic matter such as humic and fulvic acids. the presence of organic matter was shown to favor nanoparticle suspension and increase bioavailability towards cu (ii) which was associated to lpo, decreased dna strand breaks (reduced dna repair activity), protein-ubiquitin levels. indeed, the bioavailability of ncuo was always lower than cu (ii) in green and diluted effluent suggesting the influence of the type and concentration of organic matter. interestingly in brown waters, rich in natural organic matter, ncuo and cu (ii) were equally bioavailable for mussels. this is in keeping with the dls analysis showing no significant increase in mean particle diameter after 48 h suspension in brown water. however, the dispersity of ncuo was higher (coefficient of variation of 46 % of the mean diameter) at 48 h compared to 12 % at 1 h in brown water (table 2). this suggests that the ncuo intereacted with the organic matter matrix of the brown water samples but not in a coherent manner (increased disorder in the distribution of size). in a former study, dissolution experiments in 5 different types of surface waters revealed that ncuo could form large aggregates over time (24 h) in lake water reaching 400 nm diameter (liu et al., 2018). although the organic matter content was 11 mg/l, which was higher that the brown water sample at 7 mg/l in the present study, aggregation was less apparent. exposure to low concentrations of cu (ii) at 50 µg/l as total cu only increased tissue cu loadings in musses placed in brown water and which suggests that ncuo did not accumulate in mussels although cu tissue levels were correlated with biomarkers cited above. it was shown that the presence of natural organic matter could influence the dissolution state of ncuo which could, in turn, influence bioavailability in the gulf killifish (jiang et al., 2017). indeed, increased solubilisation of ncuo by natural organic matter was shown to decrease its bioavailability. in another study with the invertebrate shredder allogamus ligonifer exposed to ncuo and humic acids mixtures, exposure to ncuo increased the feeding rate and this response was inversely proportional to the nanoparticle’s size (pradhan et al., 2015). the addition of humic acids alone decreased feeding activity but this was dampened 43 -2 -1 0 1 2 factor 1 (zn>ub>gst) -2 -1 0 1 f a c to r 2 ( g s t > z n > u b ) cu(ii)+eff cu(ii)+aqu cu(ii)+brown cu(ii)+green ncuo+green green aqu ncuo+aqu eff brown ncuo+eff ncuo+brown fig. 6 discriminant function analysis of mussels responses to dissolved and nanoparticulate cu in different surface waters. discriminant function analysis was performed with the biomarker responses. the canonical scores for the surface waters types ο, cu (ii) δ and ncuo are shown for clarity by the addition of ncuo again by smaller size nanoparticles (≤ 50 nm). in the water flea daphnia magna exposed to ncuo and nzno for 72h, decreased gst activity, increased metallothioneins and lpo was observed (mwaanga et al., 2014). these effects were also dampened when natural organic matter was added at a concentration of 0.5 mg/l which was below the organic matter content in the present study. this is consistent with the observation that the biomarker responses obtained in mussels in brown water (organic matter-rich) where close to those observed with brown water controls. the decrease in the toxicity of ncuo in the presence of organic matter to rice was also observed (peng et al., 2015). it was postulated that organic matter forms a coating on the nanoparticles which enhance electrostatic repulsion between nanoparticles and interaction at the surface of the contact tissues. in this respect, the addition of anionic charges at the surface of the nanoparticle could limit interaction at the surface of membranes, which normally contains negative charges. conversely, positively-charged coatings of nanoparticles are usually more toxic in organisms. this was seemingly the same situation with metallic silver nanoparticles (fuman et al., 2013; auclair et al., 2019). humic and fulvic acids limited nanoparticle aggregation where silver nanoparticles with positively-coated surface were more toxic to juvenile trout than negatively-coated nanoparticles. in the attempt to determine if the toxic properties of cu could be changed by organic matter i.e., not only by reduced uptake, a metabolomic study was undertaken in daphnia pulex (taylor et al., 2016). the study revealed that the natural organic matter readily decreased cu toxicity but although cu increased oxidative stress in these organisms (mode of action of cu), the presence of organic matter increased metabolic energy status which could have mitigated cu toxicity. it is noteworthy that cu tissue levels were significantly related to oxidative damage (lpo), decreased dna strand breaks and the removal of damaged protein by ubiquitin tagging. in addition to the binding of organic matter on the nanoparticles, ubiquitin and other proteins could form a stable corona on metallic nanoparticles which could disrupt the turnover of damaged/denatured proteins in cells (duran et al, 2015). ubiquitin tagging of proteins was one of the major responses based from discriminant function analysis in the present study. protein-ubiquitin levels were significantly decreased in green waters for ncuo while cu (ii) decreased the levels in mussels placed in aquarium and the diluted municipal effluent. in respect to organic carbon-rich brown water, protein-ubiquitin levels were not influenced by either ncuo or cu (ii). the levels were also lower in brown water controls compared □ 44 to aquarium water controls which, suggests a surface water-mediated effect. because protein ubiquitin levels were not correlated with lpo, protein turnover was not the results of oxidative stress caused by cu. however, the correlation with labile zn in the post-mitochondrial fraction suggests the displacement of zn(ii) by other cations or by oxidative stress, perhaps cu (ii) or reactive oxygen species. protein-ubiquitin levels were also decreased in the digestive gland of freshwater mussels elliptio complanata exposed to 20 nm nano-silver but were induced by 80 nm nano-silver (gagné et al., 2013). this suggests that the size of the nanoparticles could influence protein ubiquitin levels in mussels. if this holds true for ncuo, the size distribution of the nanoparticles should encompass the 20 nm diameter range. proteinubiquitin levels were also significantly related to ache indicating decreased neuro-muscular activity, however the effects were rather associated to a surface water effect than exposure to cu forms. indeed, exposure of mussels to brown and diluted municipal effluent had decreased ache activity and cu produced no significant effect in respect to each water controls. hence, protein turnover changes observed by cu forms is also influenced with surface water types which suggests interaction or combined effects of surface water properties and exposure to cu. this forms the basis of cumulative effects when the various environmental conditions (surface water types and cu contamination) acts at the same biochemical targets in cells. nevertheless, it was shown in a previous study with the marine mussel mytilus galloprovincialis, that ncuo and cu (ii) decreased ache in mussels (gomes et al., 2011).the mussels were exposed to 10 µg/l of cu as ncuo and cu (ii) for 15 days which was longer than the exposure time used in the present study (96 h). metallothioneins and lpo was induced which suggests the release of cu (ii) in mussels exposed to ncuo after 15 days. in conclusion, the toxicity of ncuo and cu (ii) could be influenced by the surface waters properties and the form of cu in freshwater mussels. protein-ubiquitin, labile zn and gst activity were the major effects of mussels exposed to ncuo and cu (ii) in 4 types of freshwater. both gst activity and labile zn levels were decreased by surface waters and forms of cu as with protein-ubiquitin levels as these were common targets from which cumulative effects could be obtained. the biomarkers of damage lpo and dna strand breaks, representing an irreversible damage compared tot the other sublethal effects, were only affected by cu forms and not by the surface water properties. the same was also observed at the individual level with the air survival time and weight loss. based on the responses obtained for ncuo and cu (ii) in the 4 types of surface waters, it appears that the toc origin (natural vs anthopogenic) influence more the response of mussels exposed to cu (ii) while the tss influence more the response of mussels exposed to ncuo. more research will be needed to better understand the interplay between biomarkers at different levels of biological organisation in the context of multiple environmental stressor. acknowledgements this work was funded by the chemical management plan of environment and climate change canada. the technical assistance of joanne kowalczyk for tissue preparation and biomarker analyses is dully recognized. references adam n, leroux f, knapen d, bals s, blust r. the uptake and elimination of zno and cuo nanoparticles in daphnia magna under chronic exposure scenarios. water res. 68: 249-261, 2015. apha (american public health association). standard methods for the examination of water and wastewater. 17th edition. washington dc, united states, http://hdl.handle.net/1969.3/24401, 2010. auclair j, turcotte p, gagnon c, peyrot c, wilkinson kj, gagné f. the influence of surface coatings on the toxicity of silver nanoparticle in rainbow trout. comp biochem physiol c toxicol pharmacol. 226: 108623, 2019. bester mj, potgieter hc, vermaak 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with the environment that we are capable of? what does our system of integrated neuro-immune interactions have in common with simpler organisms where barriers among these systems do not exist? these ancient questions have bewildered and often profoundly divided scientists for decades. during the last forty years, research performed by enzo ottaviani, provided significant insights to these old questions, which led to an innovative comprehension of how basic immune, environmental and more recently, genetic factors interact to give rise to different outcomes in individuals. he was a pioneer in the study of equivalent behaviors and underlying functions in both simple as well as complex systems, and proposed that behavior may actually be sustained by processes that are more similar than different in diverse population, from the simplest to the more complex. his early work was in the area of neuro-immune integration where he proposed that these systems were organized into structures innately present from birth and throughout evolution. his work brought him into contact with a wide range of scientists from all over the world, including immunologists, endocrinologists, evolutionists and biologists, all of whom passionately discussed his findings within their respective fields of competence. in more recent years, ottaviani turned his attention in particular to how his field of research might be linked to stress related disorders and inflammation, both because of their importance to human public health and as potential windows into the mechanisms that underlie human defense mechanisms and resistance to stress. in this context, i had the privilege of being the co-author of the last paper he published, a few days before he passed away. a generous scientist, ottaviani mentored many students and inspired them with his curiosity and appetite for science. he was loyal and supportive to his many friends and colleagues. he was also an excellent communicator and a fervent writer, author of many articles, expert reviews, and books that cover different levels of comprehension. we will all miss him immensely because, without any doubt, he left a void that will be hard to fill, as a scientist, colleague, teacher, mentor and friend. johanna maria catharina blom department of education and humanities university of modena and reggio emilia modena, italy 375 isj 14: 375-378, 2017 issn 1824-307x short communication evaluation of the effects of some insecticides based on neonicotinoids on entomopathogenic nematodes, steinernema feltiae and s. carpocapsae v kwizera, ia susurluk uludag university, faculty of agriculture, department of plant protection, 16059 nilufer-bursa, turkey accepted september 21, 2017 abstract used of entomopathogenic nematodes (epns) are one biological control way against some insect pests, which especially pass one stage of their life in the soil. there have been a lot of developments and new discoveries in the field of entomopathogenic nematology. in spite of developments in the field of biological control, pesticides are still widely used for plant protection. these pesticides have side effects on soil biotop such as epn species. in this study, we evaluated the effects of two insecticides (acetamiprid and imidacloprid) belong to neonicotinoid group on two epns (steinernema carpocapsae and steinernema feltiae). the present experiments were carried out in the laboratory. mortality rate of the epn species was performed at 24, 48 and 96 h, after treatment. inspired by the compatibility observed between the neonicotinoid insecticides and epns against insect pest larvae, it was evaluated the direct effects of those insecticides on epns. our results showed less mortality by imidacloprid and mortality rates are a little higher by acetamiprid. these results confirm the high compatibility of imidacloprid with epns and the low synergism with acetamiprid. despite their effects on pollinators and other useful organisms of the biotop, these insecticides can be used effectively against soil dwelling insect pests without high risk on epns. key words: entomopathogenic nematodes; neonicotinoid; steinernema feltiae; steinernema carpocapsae introduction entomopathogenic nematodes (epns) are natural agents that control some pest insects (gaugler and kaya, 1990). it was first discovered in 1923 and since then the domain of epn has made great progress (thomas and poinar, 1979; poinar and grewal, 2012). epns in the future may have great potential for farmers to control many harmful insects. epns are a group of nematodes used to control insect pests (tomalak, 2004, 2005; jacobson and martin, 2011). epns of the families steinernematidae and heterorhabditidae are symbiotically associated with bacteria in the genera xenorhabdus and photorhabdus, respectively (griffin et al., 2005). when an infective juvenile (ij) of epns enters the body cavity of a susceptible host, the bacteria are released, multiply and host death occurs within two days, hence the term, entomopathogenic (webster et al., 2002). the nematodes develop and reproduce within the insect ___________________________________________________________________________ corresponding author: i. alper susurluk uludag university faculty of agriculture department of plant protection e-mail: susurluk@uludag.edu.tr cadaver, feeding on the symbiotic bacteria and degraded host tissues (forst et al., 2002; poinar and grewal, 2012). epns are a potential alternative to the use of insecticides, which have side effects on the soil biotope. since their discovery in 1923, researches on epns have been intense and a lot of progress have been made like mass production and shipment of ijs (ehlers and shapiro-ilan, 2005; poinar and grewal, 2012). despite the promising sustainability of biological control, insecticides are largely used to control insect pests. beside pest control, these insecticides have side effects on useful insects. this is the case for the bees, which populations are dangerously reduced by nicotinoid insecticides (woodcock et al., 2016). altought there are many studied on effects of pesticides that largely used in agriculture on epns, the studies on neonicotinoids is very limited (ulu et al., 2016). the 20 th century was going to be a century of neonicotinoids, this is after the discover of nicotinoids, their wide range use and their substitution to many insecticides as they are successfully applied to control pests in a variety of agricultural crops (yamamoto and casida, 1999). the first nicotinoid to be on the market was imidacloprid (thyssen and machemer, 1999). neonicotinoids are a class of insecticides chemically http://en.wikipedia.org/wiki/neonicotinoid 376 fig. 1 effects of acetamiprid on steinernema carpocapsae (sc) and steinernema feltiae (sf) (f = 37,4948; df = 5, 18; p = < 0,0001). related to nicotine. the name literally means “new nicotine-like insecticides”. initially neonicotinoids were praised for their low-toxicity to many beneficial insects, including bees; however this claim has come into question. researches point to potential toxicity to bees and other beneficial insects (penelope et al., 2012; tjeerd blacquière et al., 2012; gennaro di prisco et al., 2013). neonicotinoids ere today recognized as the first bee killer (jennifer et al., 2012). despite their side effects on bees and other beneficial insects, there are some researches showing synergism of neonicotinoid insecticides with epns (pisa et al, 2014). but no previous research has evaluated the direct effects of neonicotinoids on epns. this paper will evaluate the direct effects of two neonicotinoids (imidacloprid and acetamiprid) on ijs of two epns (steinernema carpocapsae and steinernema feltiae). materials and methods imidacloprid and acetamiprid (neonicotinoid insecticides) and two epns steinernema carpocapsae and steinernema feltiae were used. the first step was to multiply the epn samples on galleria mellonella. multiplication of epns twenty-four well cell culture dishes were used for the multiplication of epns. a larva was placed in each well and 10 % moistened sand was added. after filling the sand, 2 μl of ij nematodes were given. it was then incubated at 27 °c. the infected larvae are removed after 48 h and placed in larger wells. wells were left at room temperature and new ijs were obtained after 10 days (kaya and stock, 1997). infection twenty-four well cell culture vessels which we used for the replication of epns were also used here. 70-100 ij micropipettes were placed in each well and the drug was applied. four replications were used for each drug. in addition to these, 4 controlled recurrences were applied. doses field dose of each insecticide was applied. in the fields, doses are normally diluted in 100 liters. in this application, the dose is calculated and diluted in 20 ml: 1.2 mg acetamiprid and 3 µl imidacloprid in 1 ml, respectively. tap water is used in control wells. dead epns counting counts were made at 24, 48 and 96 h. live ijs are moving. dead ijs are still and flat (ulu et al., 2016). two watches were used to count the deadliving distinction. after counting, the ijs in each well were recorded. statistical analysis the anova test was used to compare the mortality rates of the species with those of each other and themselves in drug efficacy trials. anova test and lsd were performed in the jmp® 7.0 program. results and discussion effects of acetamiprid according to the results, the mortality rate increases with time (24, 48 and 96 h) (fig. 1). the lowest mortality rate was significantly detected at 24 hours. time-dependent differences were observed. http://www.sciencemag.org/content/335/6076/1555.short http://www.sciencemag.org/content/335/6076/1555.short 377 fig. 2 effects of imidacloprid on steinernema carpocapsae (sc) and steinernema feltiae (sf) (f= 2,6041; df = 5, 18; p = 0,0610) effects of imidacloprid according to the results obtained, the mortality rate increases with time and the lowest mortality was observed at 24 h (fig. 2). it was determined that the mortality rate changed with time and dose. the lowest mortality rates were observed at 24 h, high mortality at 96 h. there is no direct numerical correlation between doses and mortality rates (fig. 2). mortality increases with time without direct numerical correlation with dose variation. since there were no previous studies evaluating the direct effects of insecticides on epns, this study focuses on the effects of the two insecticides on epns. comparing these results, it is observed that the acetamiprid has a higher mortality than imidacloprid. this confirms existing research evaluating the compatibility of epns and imidacloprid and acetamiprid to control of pests. according to those researchers, compatibility with imidacloprid is high. acetamiprid is less compatible. these results were reported by koppenhöfer et al. (2003). in previous green houses and field studies on control of five scarab larvae, neonicotinoid insecticide imidacloprid has been shown to interact synergistically with five entomopathogenic nematode species. two other neonicotinoids, thiamethoxamine and acetamiprid showed weaker interaction with nematodes in the scarab larvae. imidacloprid and epns have shown good synergy against harmful insects (farkhanda, 2012; sheykhnejad et al., 2024), and are consistent with this research; imidacloprid was found to have a mortality rate of 0,122 % to 14,25 % in this study. compared to the mortality of insects by these three insecticides which are 7.1 µg 8.09 µg/bee in contact application and 8.85 µg 14.52 µg in oral application (acetamiprid) and 0.0179 µg 0.243 µg/bee in contact application and 0.0037 0.081 µg in oral application (imidacloprid) (jennifer et al., 2012), these neonicotinoid insecticides proved to be a lower epn killer. these differences can be assessed in terms of physiological and nutritional aspects. the physiology of the insects on one side and the epn on the other side are very different (wharton, 1986; klowden, 2013). it is proposed to investigate the contribution of nematode physiology to resistance to different pesticides. references ehlers r-u, shapiro-ilan di. mass production: nematodes as biological control agents. cabi publishing, oxfordshire ox10 8de uk, pp 6578, 2005. farkhanda m. synergism of imidacloprid and entomopathogenic nematodes for the control of eastern subterranean termite, reticulitermes flavipes (isoptera: rhinotermitidae). pakistan j. zool. 44: 1397-1403, 2012. forst s, clarke d. bacteria-nematode symbiosis: entomopathogenic nematology. cabi publishing. cab int. wallingford, oxon ox10 8de uk, pp 57-77, 2002. goulson d, kreutzweiser dp, krupke c, liess m, mcfield m, morrissey c, et al. effects of neonicotinoids and fipronil on non-target invertebrates. environ. sci. pollut. res. 22: 68102, 2014. griffin ct, boemare ne, lewis ee. biology and behaviour: nematodes as biological control 378 agents. cabi publishing, oxfordshire ox10 8de uk, pp 47-64, 2005. jacobson r, martin g. a potential role for entomopathogenic nematodes within ipm of tuta absoluta (meyrick) on organic tomato crops. iobc/wprs bulletin, 68: 79-83, 2011. jennifer h, mace v, schepherd m, biddinger d, mader e, black sh, et al. are nicotinoids killing bees? the xerces society for invertebrate conservation. www.xerces.org. portland, 2012. kaya, hk, stock, sp. techniques in insect nematology. in: lacey, l.a, 17 editor. techniques in insect pathology. london, uk: academic press, pp 18 281-324, 1997. koppenhöfer am, grewal ps, kaya hk. 2000. synergism of imidacloprid and entomopathogenic nematodes against white grubs: the mechanism. entomol. exp. et appl. 94: 283-293, 2000. pisa lw, amaral-rogers v, belzunces lp, bonmatin jm, downs ca, poinar gojr, et al. history of entomopathogenic nematology. j. nematol. 44: 153-161, 2012. sheykhnejad h, ghadamyari m, koppenhöfer am, karimi j. interactions between entomopathogenic nematodes and imidacloprid for rose sawfly control. biocontrol sci. technol. 24: 1481-1486, 2014. thyssen j, machemer l. 1999. lmidacloprid: toxicology and metabolism. in: yamamoto i, casida je (eds), nicotinoid insecticides and the nicotinic acetylcholine receptor, pp 213-222, 1999. tomalak m. infectivity of entomopathogenic nematodes to soil-dwelling developmental stages of the tree leaf beetles altica quercetorum and agelastica alni. entomol. exp. et appl. 110: 125-133, 2004. tomalak m. potentials of entomopathogenic nematodes for biological control of selected pests insects infesting urban trees. bulletin oilb/scrop 28: 3-7, 2005. webster jm, chen g, hu k, li j. bacterial metabolites: entomopathogenic nematology. cabi publishing. cab int., wallingford, oxon ox10 8de, uk pp 99-114, 2002. woodcock ba, isaac njb, bullock jm, roy db, garthwaite dg, crowe a, et al. impacts of neonicotinoid use on long-term population changes in wild bees in england. nature communıcatıons 7 (12459) | doi: 10.1038/ncomms12459 | www.nature.com/naturecommunications, 2016. ulu tc, sadic b, susurluk ia. effects of different pesticides on virulence and mortality of some entomopathogenic nematodes. inv. surv. j. 13: 111-115, 2016. yamamoto i, casida je. nicotinoid insecticides and the nicotinic acetylcholine receptor. 299 p., 1999. http://www.xerces.org/ http://www.nature.com/naturecommunications 119 isj 18: 119-129, 2021 issn 1824-307x research report potential toxic effects of titanium dioxide nanoparticles and carbon nanotubes on land snail helix aspersa: use of oxidative stress as a reliable biomarker for ecotoxicology assessment ky abdel-halim1*, sr osman1, htm el-danasoury2, rm ziada1 1mammalian and aquatic toxicology department, central agricultural pesticides laboratory (capl), agricultural research center (arc), 12618-dokki, giza, egypt 2department of plant protection, faculty of agriculture, suez canal university, ismailia, egypt this is an open access article published under the cc by license accepted september 10, 2021 abstract oxidative stress represents a main regularly informed mechanism of nanoparticles (nps) toxicity. the present work was designed to evaluate the oxidative stress in haemolymph and digestive gland of snail helix aspersa after exposure to different dermal doses: 10.8 and 2.17 µg snail-1 for titanium dioxide nanoparticles (tio2nps), and 8.0 and 1.6 µg snail-1 for single-walled carbon nanotubes (swcnts) during 48 h. the results indicated that, both nanomaterials (nms) induced significant increases in malondialdehyde (mda) as biomarker for lipid peroxidation (lpo). however, swcnts induced significant decline in reduced glutathione (gsh) content compared with the control. catalase (cat) activity significantly increased for the all treatments greater than the control. on the other hand, activities of glutathione-s-transferase (gst) and glutathione peroxidase (gpx) slightly decreased for both nms. nanoparticles (nps) of tio2 increased activity of glutathione reductase (gr) in both haemolymph and digestive gland homogenates, while swcnts treatments exhibited activities did not exceed the value of the control (0.08 u mg-1 protein). the present findings indicate that, alterations of antioxidant enzymes activity and levels of mda and gsh are recognized to oxidative stress. consequently, the use of snail, h. aspersa can offer a respectable sentinel model to assess ecotoxicological effects of nms on the gastropods. key words: nanomaterials; oxidative stress; terrestrial; helix aspersa; dermal toxicity introduction the land snails turn out to be an economic serious pest in egypt. it origins severe economic damage, particularly in gardening and ornamental plants (goden, 1983). land snail, helix aspersa is one of the bio-indicators which used for ecotoxicological assessment (regoli et al., 2006). the preferred choice of this species is mainly due to its bioaccumulation capability for many metal pollutants, global distribution, reflecting its ability to adapt to habitats, soil and varied climates and ease rearing (viard et al., 2004). land snails have also been widely accomplished as sentinel species for assessing metallic pollution in the global ecosystems (notten et al., 2006; regoli et al., 2006; abdel-halim et al., 2013). as documented in the _________________________________________ corresponding author: khaled y. abdel-halim mammalian and aquatic toxicology department central agricultural pesticides laboratory (capl) agricultural research center (arc) 12618-dokki, giza, egypt e-mail: khaled_yassen68@yahoo.com literature, h. aspersa represents a suitable bioindicator of metal and organic soil contamination (gimbert et al., 2006). application of synthetic molluscicides stayed the greatest effective method, chiefly above great areas (heiba et al., 2002; radwan et al., 2008). permitting to current improvements in nanotechnology, exposure to micro-and nano-sized particles or debris has increased (veranth et al., 2007). nanotechnology is not impartial the size of very small things; it is the innovative science and skill employing substance at the microscopic or molecular scale. nowadays, metal oxide semiconductors e.g. titanium dioxide nanoparticles (tio2nps) and carbon nanotubes (cnts) are definite critical due to their various practices: environmentally remediators for the contaminants, disinfection and preventive of virus, protecting uv, preserve corrosion way and depigment (greenwood and earnshaw, 1997). the quickly developed field of nanotechnology, which is forming materials with size-dependent properties, is likely to become alternative source of mailto:khaled_yassen68@yahoo.com 120 0 5 10 15 20 energy (kev) 0 5000 10000 15000 counts ti ti fig. 1 characterization of nps, where (a) sem image of tio2 visualized at 35.000x, (b) image of swcnts visualized at 35.000x, eda pattern of (c) ti and (d) purity in prepared particles, (e) zeta potential distribution of tio2 and (f) zeta potential distribution of swcnts determined by using dls exposure to nanoparticles (nps). engineered nps (enps) including, cnts have an increased surface area greatly enhances the chemical/catalytic reactivity compared with normal-sized form of the same substance (liu, 2006). carbon nanotubes (cnts) are significant original class of technical materials that have numerous-useful application. it includes singlewalled cnts (swcnts) in which a single sheet of graphite is revolved forming a joined tube, and multi-walled cnts (mwcnts) in which a number of sheets are revolved forming concentric tubes (alexander, 2007). the increased practices of nps will fit proliferation their release into the environment getting up adverse outcomes. a great ideal displayed that there is strong association between environmental pollutants and stress-related disease conditions in animals (sindermann, 1993). oxidative stress displays the main regularly described mechanisms of nps toxicity (mocan et al., 2010). particles with metal atoms or ions can provide free radicals on their surface in the attendance of atmospheric oxygen or ozone (nel et al., 2006). reactive oxygen species (ros) created through exposure to nps can disrupt cellular key components and interact with the normal metabolism (nel et al., 2006; choi and hu, 2008), may bind with macro-molecules interpreting them dysfunctional (gogoi et al., 2006), may offer a basis of soluble metal ions, leading to disrupt the antioxidant system and damage of lipids, protein, a) d) c) f) e) a) a) b) 121 and dna (kelly et al., 1998). numerous studies verified the potential toxic effects of nps on bacteria, human cells, rodents, aquatic organisms and others (lin and xing, 2007). for example, khene et al. (2017) observed the oxidative stress responses of tio2nps in a single exposure on the gastropod, h. aspersa, where some biomarkers have been described in homogenates of digestive gland and kidney. the present study aims to assess the effectiveness of h. aspersa in the environmental monitoring as a bio-indicator of tio2nps and swcnts contamination through quantifying certain biomarkers (oxidative stress parameters) in haemolymph and digestive gland. materials and methods chemicals tested nps: tio2nps and swcnts were supplied by nano lab., dream land, 6th october city, egypt. for biochemical analysis, chemicals: thiobarbituric acid (tba) and sodium azide (nan3) were supplied by loba chemie pvt. ltd, mumbai-400005, india. phosphate buffer, sodium phosphate monobasic; dibasic and potassium phosphate monobasic; dibasic were supplied by j.t. baker chem. co, phillipsburg, n.j. 08865. trichloroacetic acid (tca; cl3c3cooh), hydrochloric acid (hcl) and hydrogen peroxide (h2o2) were obtained from research lab. fine chem. indust., mumbai 400002, india. ethylene diamine tetra acetic acid disodium salt (edta), ethanol (c2h5oh), 1-chloro 2, 4-dinitrobenzene (cdnb), reduced glutathione (gsh), 2-amino-2-hydroxy methylpropane-1, 3-diol (tris-hcl), β-nicotinamide adenine dinucleotide reduced form (β-nadph), oxidized glutathione (gssg), and bovine serum albumin (bsa) were obtained from sigma chem. co. p.o. box 14508 st. louis mo 63178, usa. characterization of nps nanoparticles (nps) of tio2 and swcnts were employed for scanning electron microscope (sem) observation (joel, model, jsm 5300, japan) with great perseverance filament gun of 80 kev. an aliquot of each material was layered on copper grid and visualized for its shape and dimension. on the other hand, tio2nps and swcnts were subjected to x-ray electron dispersive analysis (eda) using an x-ray oxford detector unit (model 6697, england) equipped with sem instrument to achieve the purity of nps. moreover, the prepared solutions of nps were subjected to dynamic light scattering (dls) (dts nano v 5.2; malvern zeta sizer nano zs, malvern instruments, uk) to obtain the charge for each one. tested animals healthy individuals of land snail, h. aspersa weighing 4.0 ± 0.7 g was collected from some gardens in ismailia governorate, egypt. the individuals were maintained for 14 d in wood aerated cages (40 x 40 x 40 cm; 100 individuals each) under laboratory conditions (25 ± 2 ºc; 63 ± 2% relative humidity and 12:12 h light/dark). the animals were fed on lettuce leaves ad libitum. acute toxicity the contact toxicity of the examined nps against h. aspersa snails was evaluated by using topical application method (hussein et al., 1994; radwan et al., 2008), to determine the median lethal and the sublethal doses. the tested doses of tio2nps were 10, 20, 40, 80, 160, 320, and 640 µg snail-1 and 10, 20, 40, 80, 160, 320, and 640 µg snail-1 for swcnts. for each treatment, three replicates (10 animals for each) were used in plastic boxes. the tested doses were gently applied once on the surface of the snail body inside the shell using micropipette containing 10 µl of vehicle (phosphate buffered saline ph 7.0). all boxes were sprayed with water to provide suitable humidity for snail activity. ld50 values were determined after 48 h of exposure by using probit analysis program (finney, 1971). sub-acute toxicity independent on acute ld50 values for tested materials, sublethal doses: 1/10 and 1/50 of ld50 values (10.8 and 2.17 µg snail-1 for tio2; 8.0 and 1.6 µg snail-1 for swcnts) were applied as described above in acute toxicity experiment. control group was injected with vehicle (as a reference group). three replicates were maintained for each treatment of the tested dose (each contained 10 individuals). after 48 h of dosage, the live animals were taken for analysis. the haemolymph was carefully collected by inserting under the shell from the hemocoel along the right side of the head. the fluid was withdrawn in anticoagulant's vails and stored at -20 °c until used. then, they were dissected to remove digestive glands and stored as described above. table 1 the relative toxicities of the examined nps on snail h. aspersa nps no. ld50 (µg snail-1) lower limit upper limit 1 2 index folds slope swcnts 1 79.6 66.39 96.54 * * 100 1 1.41 tio2nps 2 108.2 91.29 129.28 * * 73.5 1.36 1.42 122 biochemical quantifications sample preparation one g of digestive gland tissue was homogenized in 0.05 m potassium phosphate buffer ph 6.5 (1/10 w/v) using a polytron for 15 s. the samples were centrifuged at 5000 rpm for 10 min at 4 °c. the homogenate was used for lipid peroxidation (lpo) and reduced glutathione content (gsh) assays, while supernatant was used for catalase (cat), glutathione peroxidase (gpx), glutathione-s-transferase (gst) and glutathione reductase (gr) assays. an aliquot of haemolymph was diluted with the above buffer (1/10 v/v) before used for all the above assays. lpo the thiobarbituric acid reactive substances (tbars) were used as a technique according to rice-evans et al., (1991). spectrophotometric quantification of malondialdehyde (mda) content in tissue homogenate was done. an aliquot (250 µl) of homogenate was mixed with 1ml of 15% (w/v) trichloroacetic acid (tca) in 25 mm hcl. the mixture was boiled for 10 min, quickly cooled, and immediately centrifuged at 5000 rpm for 5 min. the developed colour was determined at 535 nm. mda level was estimated using an extinction coefficient of 156 mm-1 and expressed as nm g-1 tissue. gsh the method was designed on the reduction of 5, 5ˊ-dithiobis 2-nitrobenzoic acid (dtnb) with gsh to give a yellow composite which is measured at 405 nm (beutler et al., 1963). an aliquot (500 l) of enzyme source, was mixed with same volume of 500 mm tca, followed by centrifugation at 3000 rpm for 15 min. half ml of the supernatant was well mixed with 1 ml of each 100 mm of phosphate buffered saline (pbs), ph 7.4 and 1 mm dtnb. after 10 min, the absorbance was measured at 405 nm against blank. gsh level was expressed as nm mg-1 protein. cat catalase (cat) activity was measured independence on decrease of absorbance at 240 nm associated with hydrogen peroxide (h2o2) consumption (beers and sizer, 1952). the reaction mixture consisted of 1 ml of 12.5 mm h2o2 (substrate), 2 ml of 66.7 mm phosphate buffer, ph 7.0 and an aliquot of enzyme source. the activity was expressed as u mg-1 protein, where the unit of cat is the amount of enzyme which liberates half the peroxide oxygen from hydrogen peroxide solution of any concentration in 100 at 25 °c. gpx the enzyme activity was measured according to method of flohe and gunzler (1984). in cuvette, phosphate buffer solution (100 mm, ph 7.0), edta (50 mm), sodium azide (250 mm), h2o2 (10 mm) and enzyme were well mixed. the change in absorbance was recorded every 3 s for 40 s at 340 nm. the activity was expressed as mu gpx mg-1 protein, where one unit of gpx is defined as the amount of enzyme necessary to oxidize 1 µm of nadph per min. gst spectrophotometric method of habig and jakoby (1981) by using 1-chloro, 2-4 dinitrobenzene (cdnb) was used. an aliquot (250 µl) of enzyme source was mixed with 500 l of potassium phosphate buffer (50 mm; ph 6.5). then, the mixture was incubated at 25 ºc for 5 min, followed by adding of 100 l of 0.2 m cdnb and 150 l of 10 mm gsh. after 1 min, the change of absorbance was recorded every 30 s for 6 min at 340 nm. the enzyme activity was expressed as m mg-1 min-1. gr the activity of gr was measured independent on the decrease in the absorbance during nadph oxidation (goldberg and spooner, 1987). in each cuvette, 0.1 m potassium phosphate buffer, 3.4 mm edta, ph 7.6, 30 mm oxidized glutathione (gssg), 0.8 mm -nadph and 1.0% of bovine serum albumin (bsa) was mixed by inversion. then, 100 µl of enzyme was added. the absorbance was recorded at 340 nm for approximately 5 min. the activity was expressed as u mg-1 protein. one unit will reduce 1.0 µm of gssg per min at ph 7.6 and 25 °c. protein content protein level was determined according to the method of lowry et al., (1951). intensity of the developed blue colour was measured a 750 nm against the blank. bovine serum albumin (bsa) was used as a standard. statistical analysis: ld50 value was expressed as µg snail-1 with confidence limit (cl) and slope for tio2nps and swcnts which computed using probit analysis. all data and means were compared to significance by student-newman keuls at the probability of 0.05 (cohort software inc., 1985). results nanoparticles characterization titanium dioxide nanoparticles (tio2nps) exhibited characteristic spherical shape with size ranged from 16.00 to 55.00 nm as made-up in sem image (figure 1a). however, swcnts exhibited single tubes with size ranged from 11.0 to 62.0 nm (figure 1b). in addition, eda pattern for elemental analysis is plotted in figure 1c displaying the dominance of tio2 (100.0 %) of the total content and swcnts exhibited carbon percent over 97.0 % (figure 1d). the average of zeta potential of tio2nps and swcnts in their vehicle solutions as determined by dls were -20.3 and 5.2 mv, respectively (figure 1e and f). acute toxicity of nps the examined nps represented ld50 values on the investigated snail h. aspersa after 48 h to be 108.3 and 79.6 µg snail-1 for tio2nps and swcnts, respectively (table 1). the percent mortality in sublethal dose experiments did not exceed 10 % of the total treated individuals against control group. 123 fig. 2 a) mda level (nm g-1 tissue) and b) % of control in the digestive gland and haemolymph of h. aspersa treated with nps for 48 h. each value represents mean of three replicates ± se. the same letters indicate no significant difference at 0.05 levels oxidative stress the effect of 1/10 and 1/50 of ld50 treatments for tio2nps and swcnts after 48 h were investigated on antioxidant stress biomarkers in haemolymph and digestive gland homogenate samples. the mda and gsh levels and cat, gpx, gst and gr activities in the above samples were altered in treated snails, respect to untreated individuals (control). lpo levels of mda in both haemolymph or digestive gland of np-treated snails were significantly greater than control (at p < 0.05) (figure 2a and b). mda levels for 1/10 ld50 of tio2 treatment were 1.66 and 0.89 nm g-1 tissue in haemolymph and digestive gland displaying % of control 154.42 and 553.68 %. however, 1/50 ld50 of tio2 treatment exhibited levels: 1.94 and 1.60 nm g-1 tissue in the above samples displaying % of control 194.41 and 898.28 %, respectively. regarding swcnts, 1/10 ld50 treatment exhibited levels 1.43 and 2.26 nm g-1 tissue with % of control 143.12 and 1319.78 % in haemolymph and digestive gland samples. however, 1/50 ld50 treatment exhibited 1.54 and 1.68 nm g-1 tissue with % of control 155.32 and 938.49 % for the above samples. gsh the reduced gsh content in haemolymph and digestive gland of np-treated snail is illustrated in figure 3. in haemolymph, no significant difference (p < 0.05) was observed in gsh levels for 1/10 and 1/50 ld50 of tio2 treatments (27.76 and 27.90 nm mg-1 protein) against control group (27.90 nm mg-1 protein). however, swcnts treatments at the same manner decreased gsh content (24.80 and 26.03 nm mg-1 protein). regarding digestive gland, all treatments decreased gsh content than the control (37.81 nm mg-1 protein). treatments of 1/10 and 1/50 ld50 for tio2 exhibited levels: 22.41 and 13.84 nm mg-1 protein, while swcnts treatments 124 exhibited the levels: 23.22 and 29.12 nm mg-1 protein. fig. 3 gsh content (nm mg-1 protein) in the digestive gland and haemolymph of snail h. aspersa treated with nps for 48 h. each value represents mean of three replicates ± se. the same letters indicate no significant difference at 0.05 levels cat the activity of cat enzyme increased in both haemolymph and digestive gland in all treated snails compared with untreated group. however, its activity was greater in digestive gland than haemolymph (figure 4). in haemolymph, 1/10 ld50 of tio2 treatment exhibited the greatest activity (19.83 u mg-1 protein), followed by 1/50 ld50 treatment (16.23 u mg-1 protein). however, swcnts treatments exhibited the activities: 9.81 and 10.02 u mg-1 protein, respectively, against control (5.23 u mg-1 protein). regarding digestive gland, the treatments were in the following order: 1/10 tio2, 1/50 swcnts, 1/10 swcnts, and 1/50 tio2 with mean values: 50.91, 39.00, 22.93 and 16.33 u mg-1 protein, respectively. gpx the activity of gpx in digestive gland was significantly greater than haemolymph (at p < 0.05) (figure 5). in haemolymph, the all treatments decreased enzyme activity lower than control (1.92 mu mg-1 protein) in the following order: 1.12, 1.84, 1.32 and 1.54 mu mg-1 protein for 1/10 tio2, 1/50 tio2, 1/10 swcnts, and 1/50 swcnts, respectively. regarding digestive gland, 1/10 ld50 swcnts treatment exhibited the greatest activity (48.43 mu mg-1 protein), followed by 1/50 ld50 of tio2 treatment (27.12 mu mg-1 protein) and 1/10 ld50 of tio2 treatment (14.74 mu mg-1 protein), respectively. however, 1/50 ld50 of swcnts treatment exhibited enzyme activity equal that of control group (12.53 mu mg-1 protein). fig. 4 cat activity (u mg-1 protein) in the digestive gland and haemolymph of snails h. aspersa exposed to sublethal doses of tio2nps and swcnts. each value represents mean of three replicates ± se. the same letters indicate no significant difference at 0.05 levels 125 fig. 5 gpx activity (mu mg-1 protein) in the digestive gland and haemolymph of snail h. aspersa treated with nps for 48 h. each value represents mean of three replicates ± se. the same letters indicate no significant difference at 0.05 levels gst the activities of gst in haemolymph and digestive gland of treated animals and control (untreated) are illustrated in figure 6. in haemolymph, 1/10 and 1/50 ld50 of tio2 treatments exhibited activities: 0.14, and 0.21 mu mg-1 protein min-1 against control (0.22 μm mg-1 min-1). however, 1/10 swcnts treatment exhibited the greatest activity (0.26 μm mg-1 protein min-1), followed by 1/50 ld50 of swcnts treatment (0.21 μm mg-1 protein min-1). regarding digestive gland, all treatments exhibited activities lower than control as obtained in the following order: 19.92, 4.01, 8.13, 27.32 μm mg-1 protein min-1 for 1/0 tio2, 1/50 tio2, 1/10 swcnts, and 1/50 swcnts, respectively. gr the activities of gr enzyme in digestive gland samples were greater than in haemolymph (p < 0.05) (figure 7). in haemolymph, 1/50 ld50 of tio2 treatment exhibited the greatest activity (0.19 u mg-1 protein), followed by 1/10 ld50 of tio2 treatment (0.13 u mg-1 protein) against control which did not exceed 0.08 u mg-1 protein. however, swcnts treatments exhibited activities did not exceed the value of control (0.08 u mg-1 protein). discussion the present findings provide an ideal illustrating potential toxic effects of tio2nps and swcnts on the treated snails. so, the selected snail represents a reliable sentinel model to assess the impacts of these nps on the gastropods. considering of ld50 on this species is of credible importance, because it offers appropriate evidence independent on getting total amount of dosage to the whole body of animal. the obtained ld50 values of tio2nps and swcnts in the present work mean that, these particles are very toxic on the gastropods. the digestive gland was chosen as an object tissue for biochemical assays independent on its ability to absorb and accumulate the pollutants with 5-10 folds greater than other sites (gomot devaufleury and pihan, 2000; beeby and richmond, 2003). also, haemolymph was chosen, because it non-bold represents the main compartment for nutrients and xenobiotic distribution in the body's compartments. oxidative stress can arise liberated to an imbalance in the biological oxidant-to antioxidant ratio. this explanation obtains the damage which may be caused to cell components e.g. lipids, proteins, enzymes, and nucleic acids. this damage to cell organization provides a critical signal of organ dysfunction (el-demrdash, 2007). the mechanism refers to the mode of action of metallic nps is in employment to generation of ros resulting in direct interaction with the biological targets (ma and diamond, 2013). as documented by pamplona (2008), the shifts in function and physical integrity modified bio-molecules through oxidative stress result in broad spectrum of downstream functional magnitudes and explain the cause of some cellular dysfunctions and tissue damage. one the most frequently reported toxicity endpoints for cnts is the formation of ros. oxidative stress may be caused directly by cnt-induced ros in the vicinity or inside the cell or could arise more indirectly due to the effects of internalized cnt on mitochondrial respiration (xia et al., 2008) or in depletion of antioxidant species with the cell (ahmad et al., 2012). cnt-induced oxidative stress mediates important cellular developments including inflammation, cell injury, apoptosis, and activation of cellular pathways (bonner, 2002; iavicoli et al., 2012). 126 fig. 6 gst activity (µm mg-1 protein min-1) in the digestive gland and haemolymph of snail h. aspersa treated with nps for 48 h. each value represents mean of three replicates ± se. the same letters indicate no significant difference at 0.05 levels the obtained data also disclosed a correlation between the heightening of lpo and reduction of gsh content. in fact, gsh depletion after nps exposure may refer to its increased consumption via free radicals scavenging. decreased gsh content may be induced by free radicals produced by nps and/or by the direct binding of glutathione to the metal as described by barillet (2007). moreover, enms and ultrafine particles are categorized by their binding with thiol group, resulting complexes and/or macro-molecules (xiong et al., 2011; fahmy et al., 2014). for example, some gastropods were exposed to different concentrations of cu resulting in significant declines in gsh contents (regoli et al., 1997; canesi et al., 1999). also, chandran et al. (2005) stated declines in gsh content of gastropod, achatina fulica treated with cd and zn. a finding was conducted by ramsden et al. (2013) in some organ of zebrafish exposed to tio2 demonstrating the same concept. also, the same depletion in gsh content was noted by khene et al. (2017) on snail, h. aspersa exposed to tio2 microparticles. another finding was demonstrated by ali et al. (2015), where freshwater snail, lymnea luteola was exposed to different concentrations of tio2nps for 96 h. after that, significant depletion in gsh content and gst activity as well as increases in mda level and cat activity were induced. in addition, tio2nps are capable to activate the apoptotic mechanism in hemocytes of some snails as well as genotoxic effects which were documented by comet assay (ali et al., 2015). on the other hand, increased activity of cat in the present study is in accordance with the findings of almeida et al. (2004), who found that cat activity was increased in mussels after exposure to lead (pb). in another study, swcnts exhibited a significant reduction in gsh, gst, and gpx in hepatopancreas of l. luteola after exposure to different concentrations for 96 h (ali et al., 2013). moreover, gsh as non-enzymatic defense is being a necessary cofactor for gpx and gst activities which play a principal role in conserve cellular redox status and protective cells from oxidative damage (dickinson and forman, 2002). furthermore, part of the system is gst which enhances the conjugation of gsh to nucleophilic xenobiotics or cellular components broken by oxyradicals award causing of their detoxication (halliwell and gutteridge, 1989; singal et al., 1992). gr catalyzes the nadph dependent regeneration of gsh from the oxidized form (gssg) (kehrer and lund, 1994). decline of gsh levels in analyzed samples of the examined snail proved to be a good bio-indicator for nps impacts. this explains the high affinities of nps to gsh molecule. cat detoxifies h2o2 in the biological systems. hermis-lima (2004) indicated that, under oxidative stress, cat activity often increases due to the regulation by ros. in the present findings, gpx activity in np-treated snails increased in comparison with control. this concept is considered a result of the oxidative stress from nps conjugation and accumulation in the selected samples of haemolymph and digestive gland. on the other hand, swcnts exhibited an increase of gst activity, which may be due to the activation of the natural antioxidant defense system by these particles, however, the detoxication process versus the pro-oxidation forces was mediated by this organs 127 fig. 7 gr activity (u mg-1 protein) in the digestive gland and haemolymph of snail h. aspersa treated with nps for 48 h. each value represents mean of three replicates ± se. the same letters indicate no significant difference at 0.05 levels enzyme (elia et al., 2007). this concept was documented by canesi et al. (1999), where copper treatment increased gst activity in reflecting to increased utilization of gsh conjugation in lipid hydro peroxides and carbonyl compounds metabolism after peroxidation of cellular membranes by the metal. similarly, the present data are in accordance with that obtained by radwan et al. (2010) and abdel-halim et al. (2013), where gst activity increased in snails: teba pisana and h. aspersa exposed to heavy metals contamination in some urban regions of egypt. the increase in gr activity in the treated individuals of h. aspersa is in accordance with that obtained by regoli et al. (2006), who suggested that the increase of gr activity was reflected in more integrated unbalance of oxyradical metabolism. conclusion the results of this work provide potential toxic effects of tio2nps and swcnts on snail h. aspersa and show variations of both nms in relationship to their properties and toxicities on the snail. these findings could explain the ability of nps to penetrate into the cells and induce damage resulting in dysfunctions and generation of ros that initiated actions referring to oxidative stress. the use of oxidative stress parameters could be established as a reliable tool to assess the impacts of nps on terrestrial ecosystems. so, further investigations are required to ensure the safe use of such materials without any risks. funding information this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. compliance with ethical standards the experiments have been carried out in accordance with the european ethical guidelines (directive 2010/63/eu, 2010). declaration of conflicting of interest the authors declare that no potential conflicts of interest with respect to the research, authorship and/or publication of this article. supplementary data no supplementary data are provided. reference abdel-halim ky, el-saad aa, talha m, hussein a, bakry n. oxidative stress on land snail helix aspersa as a sentinel organism for ecotoxicological effects of urban pollution with heavy metals. chemosphere 93: 1131-1138, 2013. abdel-halim ky, osman sr, abdou gy. in vivo evaluation of oxidative stress and biochemical alteration as biomarkers in glass clover snail, monacha cartusiana exposed to zinc oxide nanoparticles. environ. poll. 257: 113120, 2020. ahmad j, dwivedi s, alarifi s, al-khedhairy aa, musarrat j. use of 𝛽-galactosidase (lacz) gene 𝛼-complementation as a novel approach for assessment of titanium oxide nanoparticles induced mutagenesis. mut. res. gen. toxicol. environ. mutag. 747: 246–252, 2012. alexander aj. carbon nanotubes structures and compositions: implications for toxicological studies. in: monteiro-riviere, n.a., iran, c.l. 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and their bulk counterparts on zebra fish: acute toxicity, oxidative stress, and oxidative damage. sci. total environ. 49: 1444-1452, 2011. 105 isj 19: 105-114, 2022 issn 1824-307x research report identification and characterization of a cyclophilin a gene from chinese shrimp fenneropenaeus chinensis: sequence features and expression profiles q-q zhou1, y wang1,3, j-j hu1,2,3, l zhang1, j-b li1, y-j xu1, m-q wang1,2,3 * 1moe key laboratory of marine genetics and breeding (qingdao 266003), and key laboratory of tropical aquatic germplasm of hainan province of sanya oceanographic institute (sanya 572024), ocean university of china, china 2laboratory for marine fisheries science and food production processes, and center for marine molecular biotechnology, qingdao national laboratory for marine science and technology, qingdao 266237, china 3hainan yazhou bay seed laboratory, sanya 572024, china this is an open access article published under the cc by license accepted june 27, 2022 abstract cyclophilin a (cypa), a key member of the immunophilin family, is the most abundantly expressed one among the 16 identified cyclophilins. besides acting as an intracellular receptor for cyclosporine a, cypa plays a vital role in various pathological responses such as inflammation and microbial infections. in the present study, the full-length cdna of fenneropenaeus chinensis cypa (fccypa) was cloned by race technique. the complete sequence of fccypa cdna contained a 5' untranslated region (utr) of 33 bp, a 3’ utr of 341 bp with a polya tail, and an open reading frame (orf) of 495 bp encoding a polypeptide of 164 amino acids with the predicted molecular weight of 17.70 kda. the amino acid sequence of cypa has a typical peptidyl prolyl cis-trans isomerase family tag sequence (ykgstfhrvipnfmcqgg) as those of other species. the mrna transcripts of fccypa were constitutively expressed in all the tested tissues, including eyestalk, gill, gonad, heart, hemocytes, hepatopancreas, intestine, muscle, nerve and stomach, with the highest expression level in hepatopancreas. the mrna expression profiles of fccypa in hemocytes and hepatopancreas could be significantly induced by the stimulation of vibrio anguillarum suspension and white spot syndrome virus (wssv) stock, which verifies that cypa has an effect on viral and bacterial infection in shrimps. these results indicate that cypa may be involved in the innate immunity, biological immune response and physiological stress response of chinese shrimps. key words: cyclophilin a; fenneropenaeus chinensis; innate immunity; vibrio anguillarum; white spot syndrome virus introduction generally, elementary conformational transitions in protein folding and protein restructuring take place spontaneously (schiene-fischer et al., 2011). peptidyl prolyl cis-trans isomerase domain (ppiase) basically catalyzes the inter conversion between cis and trans isomers of n-terminal amide bond of proline (davis et al., 2010), which can be divided into four structurally unrelated families: cyclophilins (cyps), fk506-binding proteins (fkbps), parvulins and the recently identified protein ser/thr ________________________________________ corresponding author: meng-qiang wang moe key laboratory of marine genetics and breeding (qingdao), and key laboratory of tropical aquatic germplasm of hainan province of sanya oceanographic institute (sanya) ocean university of china e-mail: wangmengqiang@ouc.edu.cn phosphatase 2a (pp2a) activator (ptpa). cyclophilins have been the subject of intense research because they are cellular targets for the clinically used immunosuppressive drugs cyclosporin a (csa), an immunosuppressant which was used to suppress rejection after organ transplantations (lu et al., 2007). cyclophilins, also known as immunophilins, are a family of highly conserved multifunctional proteins widely distributed in nature and have been found in mammals, plants, insects, fungi, and bacteria. (göthel and marahiel, 1999). so far, a variety of different cyclophilins have been identified, various cyclophilins perform different functions by targeting unique domains of specific organelles (lee, 2013). 16 different types of cyclophilins have been identified in human, and the main 7 cyclophilin subtypes include cyclophilin a (cypa), cyclophilin b (cypb), cyclophilin c (cypc), cyclophilin protein d (cypd), 106 cyclophilin e (cype), cyclophilin 40 (cyp40) and cyclophilin nk (cypnk). drosophila has 9 cyclophilins, while 8 are found in saccharomyces cerevisiae (kumari et al., 2013). among them, cypa is the most widely distributed, exists in almost all prokaryotic and eukaryotic cells, act in cytosol and nucleus, has a variety of biological activities, and plays an important role in the immune system (harikishore and sup yoon, 2015). with the increasing research efforts on cypa, its biological role is recognized steadily. it participates in many biological processes, such as protein folding (fruman et al., 1994), cell growth (obchoei et al., 2011), cholesterol metabolism (smart et al., 1996), protein trafficking in cells and regulation of immune function of the body (hoffmann and schiene-fischer, 2014; li et al., 2016). cypa is also a direct inflammatory cell chemotactic substance (dawar et al., 2017). at present, high expression of cypa has been detected in various inflammatory diseases, such as atherosclerosis (coppinger et al., 2004), rheumatoid arthritis (kim et al., 2005), sepsis and inflammatory heart disease (dear et al., 2007; seizer et al., 2013). additionally, accumulating evidences indicated that cypa plays an important role during viral infection (chatterji et al.,2009), cypa may either promote or inhibit virus replication, depending on the host cell type and the viral species. cypa can promote the secretion of host interferon (inf-β) and inhibit rotavirus replication, thereby reducing the risk of rotavirus infection (he et al., 2012). cypa is a component of dynamic actin-rich structures formed during bacterial infection and within cells in general and may be involved in pathogenic processes in several bacteria or membrane translocation of bacterial toxins (dhanda et al., 2018). studies have reported that cypa may be the potential receptor of mgpa, which mediates the adhesion and invasion of mycoplasma genitalium to human urothelial cells (deng et al., 2018). cypa also affects parasite infection in different ways, for example, tccyp19, a human cypa, may be part of a complex interaction between parasites and host cells (perrone et al., 2018). these demonstrate the unique role of cypa in the replication and infection of different viruses, pathogens, mycoplasmas and parasites. at present, cypa gene is one of the hotspots of current research, and it is of great significance to use cypa as a molecular target for new drugs, animal and plant stress resistance breeding, etc. the latest research shows that cypa may have immune stress function in some aquatic organisms. however, the basic and applied research of cyclophilin a in aquatic animals is still very limited and the value of this gene has not been fully exploited. the chinese shrimp f. chinensis is one of the most commercially important cultured shrimp species in china. it is mainly distributed in the yellow sea and bohai sea of china, and west and south coast of the korean peninsula (wang et al., 2017). with the development of intensive culture and environmental deterioration, various diseases caused by bacteria, viruses and rickettsia-like organisms had frequently occurred in cultured shrimp populations, which resulted in enormous losses to this aquaculture industry. a better understanding of shrimp immune defense mechanisms is necessary for shrimp health management, and the cypa gene plays an important role in microbial infections and body's innate immunity. the main objectives of this study were to (1) clone the full-length cdna of cypa from f. chinensis, (2) investigate the tissue distribution of fccypa transcripts, and (3) its temporal distribution after microbial challenge. table 1 oligonucleotide primers used in the experiments primer sequence (5’-3’) tm (°c) information fc-cypa-race-f1 ctcagaacattccgccttagccgc 66.2 1st pcr for 3’-race (forward) fc-cypa-race-f2 tataatctttgctgtattggcacttcagtg 65.9 2nd pcr for 3’-race (forward) fc-cypa-race-r1 gaatttgttgccgtagatggacttgcc 66.1 1st pcr for 5’-race (reverse) fc-cypa-race-r2 tcacgcggtggaagcacgagccct 68.2 2nd pcr for 5’-race (reverse) adaptor-oligo(dg) ggccacgcgtcgactagtacg10hn anchor primer for 5’-race adaptor-oligo(dt) ggccacgcgtcgactagtact18vn cdna synthesis for 3’-race fc-cypa-cds-f atgggcaatcccaaagtctttttcgac 66.6 gene specific primer for sequence identification fc-cypa-cds-r ttacagctggccgcagttggcgatcatcac 69.2 gene specific primer for. sequence identification fc-cypa-qrt-f ttacaaacctacgccaactgaacc 62.6 gene specific primer for real-time pcr fc-cypa-qrt-r ctccatgatgatcctgccaactg 64.1 gene specific primer for real-time pcr fc-18s-qrt-f tatacgctagtggagctggaa 55.7 internal control for real-time pcr fc-18s-qrt-r ggggaggtagtgacgaaaaat 57.6 internal control for real-time pcr m13-47 cgccagggttttcccagtcacgac 64.5 vector primer for seqvence rv-m gagcggataacaatttcacacagg 62.9 vector primer for seqvence 107 materials and methods experimental animals, microbes challenge and samples collection the chinese shrimp used in the present study were obtained from ruizi seafood development co. ltd., qingdao, china white shrimps, with body weight 8 12 g, were cultured placed in 640 l cylindrical tanks with 500 l air-pumped circulating seawater at 20 ± 1 c for two weeks before processing. hemolymph was extracted from the ventral sinus of at least three untreated shrimps using a sterile syringe preloaded with equal volume of anticoagulant buffer (nacl 510 mmol l-1, glucose 100 mmol l-1, citric acid 200 mmol l-1, tri-sodium citrate 30 mmol l-1 and edta·2na 10 mmol l-1, ph 7.3). then the hemocytes were collected by centrifugation at 800 g for 10 min at 4 c. tissues including eyestalk, gill, gonad, heart, hepatopancreas, intestine (mid gut), muscle, nerve and stomach were collected from at least three untreated shrimps, kept in rnalater (am7020, thermo fisher scientific, usa) and stored at -80 c until rna isolation. approximately 200 shrimps were employed for microbe stimulation assay, and they were randomly divided into three groups which contained about 60 70 individuals. the v. anguillarum suspension and wssv stock were prepared according to previous reports (sha et al., 2016; xia et al., 2015). the shrimps were received an injection at the abdominal segment with 100 μl phosphate buffered saline (pbs, ph 7.4, 10010023, thermo fisher scientific, usa), v. anguillarum suspension (1 × 104 cfus μl-1, in pbs) and wssv stock (1 × 104 copies μl-1, in pbs), respectively. after stimulation, samples were taken from each group at 0 h, 3 h, 6 h, 12 h, 24 h, 36 h and 48 h, with 5 replications at each time point, and each replication was a mixture of 3 individuals. the hemocytes and hepatopancreas from each sample was collected and stored at -80 °c for rna extraction. total rna isolation and cdna synthesis total rna of ten tissues was isolated with trizol reagent (15596026, thermo fisher scientific, usa) following manufacturer protocol and a dnase i (rq1, m6101, promega, usa) treatment was carried out to eliminate genomic dna contamination. rna degradation and contamination were monitored on 1% agarose gels. the concentration and purity of rna were measured using a nanodrop lite spectrophotometer (thermo fisher scientific, usa). rna samples and adaptor primer-oligo (dt) were used as template and primer to synthesize the first strand of cdna (for gene cloning), which carried out with promega m-mlv. the reaction mixture was incubated at 42 °c for 1 h, terminated by heating to 95 °c for 5 min, and then stored at -80 °c until analysis as template. cloning the full-length cdna of fccypa one est (bm302619) of 740 bp from the cdna library was homologous to previously identified cypas (zhang et al., 2010), and it was selected for further cloning of cypa cdna from f. chinensis (designated as fccypa). the 5′-terminalregion of cypa cdna was determined by 5′-race, primers fc-cypa-race-r1 were used for the first pcr and fc-cypa-race-r2 was used for the second pcr (table 1). the 3'-terminal-region of cypa cdna was determined by 3'-race, which primers fc-cypa-race-f1 were used for the first pcr and fc-cypa-race-f2 was used for the second pcr (table 1) ， which used for the synthesis of single-stranded cdna by reverse transcription. the pcr products were gel-purified using monarch dna gel extraction kit (t1020s, neb, usa) and cloned into the pmd18-t simple vector (d103a, takara, japan). after being transformed into the competent cells of escherichia coli strain dh5α (cb101, tiangen, china), the positive recombinants were identified through anti-amp selection and pcr screening with m13-47 and rv-m primers (table 1). then three positive clones were picked for sequencing. sequence analysis signalp 3.0 program was utilized to predict the presence and location of signal peptide (https://www.cbs.dtu.dk/services/signalp/). the glycoslanation were performed with the(http://www.cbs.dtu.dk/services/netnglyc/). the protein motif features were predicted by simple modular architecture reasearch tool (smart) version 7.0 (http://smart.embl-heidelberg.de/) and interpro (https://www.ebi.ac.uk/interpro/). multiple sequences alignment and phylogenetic analysis multiple sequence alignment of fccypa and other reported cyps was performed with clustalw multiple alignment program (http://www.ebi.ac.uk/tools/clustaw2/) and multiple alignment show program (http://www.bioinformatics.org/sms/). an unrooted phylogenic tree was constructed based on the deduced amino acid sequences of fccypa and other reported cyps by the neighbour-joining (nj) algorithm using the mega 7.0 software (http://www.megasoftware.net). to derive the confidence value for the phylogeny analysis, bootstrap trials were replicated 1000 times. the amino acid sequences information for homologous and phylogenetic analysis was shown in table 2. expression analysis of fccypa genes by rt-qpcr the mrna transcriptional levels of fccypa in different tissues and its temporal expression pattern in hemocytes and hepatopancreas of chinese shrimps stimulated with v. anguillarum suspension and wssv stock were determined by quantitative real-time pcr using a linegene k fqd-48a (a4) fluorescence quantitative pcr detection system (bioer, china). all qpcr reactions were performed with sybr premix extaq (tli rnaseh plus) (rr420, takara, japan) using about 100 ng cdna template and 0.2 μm of each primer. amplification conditions were as follows: 94 °c for 10 s followed by 40 cycles of 94 °c for 5 s, 60 °c for 30 s (to acquire fluorescence). the expression of fccypa was normalized to the expression of 18s rdna gene for each sample. all the primers for qrt-pcr were designed using perlprimer 1.1.21 and listed in table 1. https://www.cbs.dtu.dk/services/signalp/ http://www.cbs.dtu.dk/services/netnglyc/ http://smart.embl-heidelberg.de/ https://www.ebi.ac.uk/interpro/ http://www.ebi.ac.uk/tools/clustaw2/ http://www.megasoftware.net/ 108 table 2 species and accession number of cypas used in the homologous and phylogenetic analysis scientific query cover identity e-value accession fenneropenaeus chinensis 100% 100% 1e-123 anh58180.1 penaeus monodon 100% 98.78% 3e-116 ags46493.1 penaeus vannamei 100% 95.12% 4e-113 ady69343.1 penaeus japonicus 100% 94.51% 9e-113 xp_042880185.1 scylla paramamosain 100% 86.59% 2e-102 aen94575.1 eriocheir sinensis 100% 85.37% 1e-101 adf32017.1 danio rerio 100% 77.44% 3e-95 np_001315353.1 ictalurus punctatus 100% 78.66% 3e-96 np_001187167.1 azumapecten farreri 100% 76.22% 2e-98 aar11779.1 argopecten irradians 100% 76.22% 3e-97 abm92916.1 xenopus laevis 100% 71.95% 1e-89 aah97540.1 gallus gallus 99% 71.95% 1e-88 acx31829.1 homo sapiens 99% 75.61% 8e-93 aau13906.1 mus musculus 100% 75.00% 7e-93 aah99478.1 statistical analysis all data were given in terms of relative mrna expression as means ± standard deviation (sd). the expression level of fccypa mrna was estimated using the comparative ct method (2−δδct). gene expression data were subjected to two-factor analysis of variance (anova) using spss statistic 26.0 software where values of p < 0.05 were considered significantly different. results cloning and characterization of fccypa cdna the full-length cdna sequence of fccypa was obtained by 3’ race technique and deposited in genbank under the accession number ku361825. it comprised 869 bp, containing a 5’ untranslated regions (utr) of 33 bp, a 3’ utr of 341 bp with a poly a tail and an open reading frame (orf) of 495 bp encoding a polypeptide of 164 amino acids with the predicted molecular weight of 17.70 kda and theoretical isoelectric point of 8.55. it has 17 strong basic amino acids (arg + lys), 14 strongly acidic amino acids (asp + glu). in 3´utr (untranslated region), the sequence has polyadenylation signal (aataaa). there was no availability of a signal peptide revealed by signalp program analysis. the glycosylation prediction showed that fccypa is characterized by presence of six n-glycosylation sites (n3, n71, n106, n108, n149 and n160). a highly conserved signature sequences of peptidyl-prolyl cis-trans isomerase (48ykgcafhrvipnfmcqgg65) and a pro-isomerase domain (from f7 to q163) was identified in the amino acid sequence of fccypa (fig. 1). multiple alignment and phylogeny relationship of fccypa the deduced amino acid sequence of fccypa shared significant homology with other reported cypas, such as 98.78 % identity with p. monodon, 95.12 % with p. vannamei, 94.51 % with p. japonicus, and 85.37 % with e. sinensis (fig. 2). fourteen amino acid residues critical for the fundamental structure and function of the cypa ppiase activity, including r55, i57, f60, m61, q63, g72, a101, n102, a103, q111, f113, w121, l122 and h126, were all conserved in fccypa (fig. 2). the construction of nj phylogenetic tree from multiple tetraspanins was based on the amino acid sequences of 14 cypa and separated into four branches, fccypa was clustered with its homologue as one of branches, arthropod, other branches teleost, mollusc and vertebrate (avian, amphibian and mammalian). among them, it is most closely related to several species of shrimps and crabs, followed by fish and shellfish, and the most distant relatives of mammals (fig. 3). the tissue distribution of fccypa mrna the mrna expression level of fccypa in ten different tissues of f. chinensis was quantified by real-time pcr with the 18s rdna gene as an internal control. the fccypa mrna transcripts could be detected in all the tested tissues, and the relative expression level was from high to low in hepatopancreas, hemocytes, gill, nerve, intestine, 109 fig. 1 nucleotide and deduced amino acid sequence of fccypa. the nucleotides and amino acids were numbered along the left margin. the function domain was in shade. the start and codon was marked in bold, the asterisk indicated the stop codon and the classical polyadenylation signal in the 3’-utr is dotted underlined. the cyp-type peptidylprolyl cis-transisomerase signature was indicated in underline. the black boxes are showing the glycosylation sites.the amino acid sequence of fccypa has been submitted to genbank with the accession number ku361825 gonad, heart, stomach, eyestalk, muscle. the highest mrna expression level was found in hepatopancreas, which was 309.28-fold (p < 0.05) of that in muscle, followed by hemocytes, gill and nerve, which were 15.34-fold, 4.60-fold and 2.87-fold of that in muscle (p < 0.05), respectively (fig. 4). the temporal expression of fccypa mrna after microbe challenge the fccypa mrna expression levels of hemocytes and hepatopancreas were all up-regulated post the two kinds of microbe stimulation. the fccypa mrna expression level of hemocytes was significantly up-regulated at 6 h post v. anguillarum stimulation (4.25-fold compared with the origin level, p < 0.05), and the highest level was observed at 12 h (13.42-fold, p < 0.05, fig. 5a). while after stimulated with wssv stock, the fccypa mrna expression level of hemocytes significantly increased at 3 h post stimulation (2.30-fold, p < 0.05) and reached the peak at 24 h (3.94-fold, p < 0.05) and then decreased to the origin level at 36 h (fig. 5a). in the hepatopancreas group, the mrna transcripts of fccypa significantly increased at 6 h post v. anguillarum stimulation (2.71-fold, p < 0.05) and reached the maximum level at 12 h (3.61-fold, p < 0.05) and then down to the normal level at 24 h (fig. 5b). while the mrna expression level of fccypa was reached the peak at 6 h (6.08-fold, p < 0.05) post wssv stimulation (6.21-fold, p < 0.05) and then down to the normal level at 48 h (fig. 5b). discussion cyclophilin a is a highly conserved important protein that widely exists in prokaryotes to higher mammals. the two most important biological functions are currently known: one is ppiase activity, which can catalyze the process of protein folding and transport and play the role of molecular chaperone. the second is the immunosuppressive function, which can mediate in vivo. it is involved in a variety of physiological and biochemical reactions such as tumor proliferation, inflammation, virus infection, cell apoptosis, and various physical and biological environmental stress responses. in the present study, the full-length cdna sequence of fccypa was obtained by 3’ race technique. the full length of the sequence is 869 bp, including the 5'-end untranslated region (utr) of 33 110 fig. 2 multiple alignment of fccypa with other cypas deposited in genbank. thesame amino acid residues were shaded inblack and the similar amino acids wereshaded in grey. gaps were indicated bydashes to improve the alignment. the sequences and their accession numbers are as follows: fenneropenaeus chinensis (anh58180.1), penaeus monodon (ags46493.1), penaeus vannamei (ady69343.1), penaeus japonicus (xp_042880185.1), eriocheir sinensis (adf32017.1), danio rerio (np_001315353.1), azumapecten farreri (aar11779.1) fig. 3 neighbor-joining (nj) phylogenic tree of fccypa constructed based on the protein sequences from different organisms. to derive confidence value for the phylogeny analysis, bootstrap trials were 1000 replicates. the black round indicated the tetraspanin protein of fccypa the numbers at the forks indicated the bootstrap value 111 fig. 4 relative expression of fccypa gene in ten tissues. the mrna transcripts levels in muscle, eyestalk, stomach, heart, gonad, intestine, nerve, gill, hemocytes and hepatopancreas of three untreated shrimps were normalized to that of muscle. the 18s rdna gene was used as an internal control to calibrate the cdna template for each sample. each vertical bar represents the mean ± sd (n = 5), and different letters indicate significant differences (p < 0.05) bp and the 3'-end untranslated region (utr) of 341 bp. the length of the open reading frame is 495 bp, which can encode 164 amino acid residues. the 3'utr region contains typical polyadenylation tailed signal sequences aataaa and polya tail. the primary sequence structural features of the cypa gene of f. chinensis were basically the same as those of other species. the blast protein alignment showed that there were 14 highly conserved amino acid residues in the amino acid sequence of cypa of f. chinensis. like cypa of other known species, ppiase was found in the encoded amino acid sequence, and no signal peptide sequence was detected at its n-terminus. this site is very conserved in both invertebrates and vertebrates, and it is located between the 48th and 65th bases (ykgstfhrvipnfmcqgg). sequence analysis found that it has 8 amino acid residues, which is the binding site of cyclosporin a and the active center of ppiase. in the evolutionary analysis, the phylogenetic tree analysis results showed that it is most closely related to several species of shrimps and crabs, followed by fish and shellfish, and mammals are the most distant, which is in line with the evolutionary sequence of animals and has a high degree of conservatism. we obtained a new cypa gene from f. chinensis, and analyzed its sequence characteristics, laying a foundation for further study of its functional characteristics. in this study, the distribution and expression of fccypa in different tissues were detected, and it was found that it was expressed in various tissues, with the highest expression in hepatopancreas, followed by hemocytes, gill, nerve, intestine, gonad, heart, stomach and eyestalk, and the lowest expression in muscle. this is consistent with the widespread tissue distribution of cyclophilin a in other animals (tu et al., 2003; qiu et al., 2009; chen et al., 2011; wang et al., 2013; muhammad et al., 2017). the ubiquity of fccypa transcripts in different tissues indicated that it could be potentially involved in some important physiological processes and played important roles in the basal metabolism of shrimp cells. muhammad et al. found that the tissues with cypa expression from high to low in litopenaeus vannamei were muscle, gill, lymphoid organ and hepatopancreas in order (muhammad et al., 2017). in previous reports, the expression level of cypa was found to be highest expressed in hepatopancreas and haemolymph of shrimp penaeus monodon (qiu et al., 2009), while in gonad and gill of scallop chlamys farreri (song et al., 2009). there are also some studies on cypa transcriptional expression in fish, mollusks and amphibians, overall, no consistent pattern of cypa gene expression was found among tissues in different species (dorfman et al., 1997; tu et al., 2003; massé et al., 2004). in the present study, fccypa was highest expressed in hepatopancreas, and it was suspected that fccypa might play an important role in the immune defence system of shrimp, as hepatopancreas was regarded as the main immune center in crustaceans and many 112 fig. 5 relative expression of fccypa gene in hemocytes(a) and hepatopancreas(b) after microbe challenge v. anguillarum suspension and wssv stock. sampling times at 0, 6, 12, 24, 36, 7 and 48 hr.18s rdna gene was used as an internal control to calibrate the cdna template for all the samples. each vertical bar represents the mean ± sd (n = 5), and different letters indicate significant differences (p < 0.05) immune-related genes were high expressed in this tissue (gai et al., 2009; lu et al., 2012). the higher expression level of fccypa is hemocytes，which also play an important role in the immune defense system of animals, especially for invertebrates that have no adaptive immune system (yang et al., 2015; koiwai et al., 2018). it further proved the importance of cypa gene in innate immunity in f. chinensis. many studies have demonstrated that cypa is involved in a variety of pathological states, including infection and inflammation. in order to further explore the function of fccypa, we infected chinese shrimps with v. anguillarum suspension and wssv stock and measured the relative expression levels of mrna in the two tissues. in other studies, the relative mrna expression of cypa of p. monodon was up regulated after stimulated by lps (qiu et al., 2009), significant upregulation of relative mrna expression levels of eriocheir sinensis cypa was observed throughout the fungal challenge (wang et al., 2013), slightenhancement of cypa protein was observed in the liver, spleen, body kidney and head kidney of pelteobagrus fulvidraco infected with edwardsiella ictalurid (dong et al., 2015), after bacterial challenge, the expression level of cypa of c. farreri was almost unchanged in haemocytes, but up-regulated in gonad and increased to the peak at 4 h post-injection (song et al., 2009). these results indicated that cypa gene plays an important role in the innate immune system of aquatic organisms. in this study, under the infection of v. anguillarum suspension and wssv stock, the relative mrna expression of cpya gene of f. chinensis in hepatopancreas was significantly up-regulated between 6 h and 12 h, and gradually returned to the initial expression level from 12 h to 48 h. similarly, a) b) 113 the fccypa mrna expression highest level of hemocytes was observed at 12 h and 24 h after the stimulation of v. anguillarum suspension and wssv stock, respectively. it demonstrates that the mrna expression profiles of fccypa in hemocytes and hepatopancreas could be significantly induced by the stimulation of v.anguillarum suspension and wssv stock, further validating the important role of cypa in viral and bacterial infections in shrimps. all these results indicated that fccypa was a typical cypa member and was 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zhang j, li f, jiang h, yu y, liu c, li s, et al. proteomic analysis of differentially expressed proteins in lymphoid organ of fenneropenaeus chinensis response to vibrio anguillarum stimulation. fish shellfish immunol. 29: 186-194, 2010. 38 isj 20: 38-43, 2023 issn 1824-307x research report pathological changes in the main immune organs of silkworm infected with staphylococcus aureus y pan1, p lü2, m tang2, k chen2* 1the laboratory animal research center, jiangsu university, zhenjiang 212013, people’s republic of china 2school of life sciences, jiangsu university, zhenjiang 212013, people’s republic of china this is an open access article published under the cc by license accepted april 12, 2023 abstract silkworm bombyx mori (b. mori), a lepidopteran model organism, has become an important model for molecular biology. it also offers an excellent model to study the innate immunity because of its similarity to human beings. staphylococcus aureus (s. aureus) is a typical gram-positive pathogenic microorganism that causes serious pneumonia, meningitis, endocarditis and septicemia. in this study, silkworm was used as animal model to study the innate immune responses against the pathogenic bacterial infections. we investigated the median lethal dose (ld50) of s. aureus infection in the silkworm, and the pathological changes in their hemolymph and midgut after infection. the ld50 of s. aureus infecting the silkworms was 4.39 × 104 colony-forming units per milliliter (cfu/ml) after 72 h. the peritrophic membrane of the silkworm showed severe damage after 36 h. insect hemocytes can participate in various innate immune responses, such as encapsulation and nodule formation. our results imply plasmacytes of hemocytes can adhere to and spread over s. aureus in the hemolymph and may play an important role in the resistance of the silkworms to s. aureus infection. key words: bombyx mori; staphylococcus aureus; hemolymph; midgut introduction silkworm (bombyx mori) breeding has a long history, and silk is an indispensable textile, with an important part of the commodity trade. the silkworm not only has economic value, but is also used as a model organism in biological research. for instance, the toxicity of pesticides to silkworms is often used as an indicator of the effects of chemical pesticides on the ecological environment (wang et al., 2011). the growth of silkworms fed mulberry leaves steeped in a liquid suspension of genetically modified pollen has been used as a reference in studies of the effects of transgenic foods (li et al., 2002). the silkworm is an ideal insect for immune studies due to its well-characterized innate immune system in which many genes are known to have a role in controlling the immune response (tanaka et al., 2008). the immune system of the silkworm mainly involves humoral and cellular immunity. humoral immunity refers to the recognition of pathogens in the insect hemolymph, the subsequent _________________________________________ corresponding author: keping chen school of life sciences jiangsu university 301 xuefu road, zhenjiang 212013, p. r. china e-mail: kpchen@ujs.edu.cn cascade of proteases, the antibacterial peptides induced, phenoloxidase, the intermediate products of melanization, and other immune factors present in the body fluid. in insects, ‘cellular immunity’ refers to phagocytosis, nodulation, encapsulation, and other functions mediated by hemocytes (stokes et al., 2015; wang et al., 2017). there are 21 immune-related gene families or superfamilies in the silkworm genome, including 218 associated with pattern recognition, signal regulation, or effector molecules, which provide important references in the study of the immune systems of lepidopteran insects (tanaka et al., 2011; tanaka et al., 2018). staphylococcus aureus (s. aureus) is a gram-positive bacterium that is widely distributed in nature and is commonly present in air, soil, and water. it is an important pathogen in bacterial food poisoning and one of the pathogens that cause bacterial diseases in the silkworm. when the silkworm consumes mulberry leaves, microorganisms enter the intestine through the mouth with the food, causing intestinal epithelial cells to contact a large number of microorganisms, often causing infection (kurokawa et al., 2007; hiroki et al., 2019). when silkworm was infected with s. aureus, the expression of genes related to immunity was altered, including genes encoding peptidoglycan recognition proteins and the class c scavenger receptor bmsr-c (wang et al., 2016); the 39 fig. 1 state of silkworm infected with s. aureus expression of antimicrobial peptides in the midgut and fat body was increased (wu et al., 2010); and the concentration of nitric oxide (no) in the hemolymph was changed (zhang et al., 2015). in this study, after silkworms were fed s. aureus, we determined the median lethal dose (ld50) of s. aureus, and detected the pathological changes in the midgut and hemocytes of the silkworms after infection. methods silkworm rearing the silkworm strain qingsong × haoyue was used in this study. fertilized silkworm eggs were maintained in containers at 25 °c. hatched larvae were reared to the 5th instar on mulberry leaves at 25 °c and 80% humidity, under 12 h light/12 h dark cycle for their entire lifespans (pan et al., 2017). silkworm infection two-day-old 5th instar larvae (l5d2) were randomly divided into two groups (30 larvae per group, three experimental replicates), the first of which was injected with 5 μl of 1 × 1010 cfu/ml s. aureus (atcc 26085) and the second with phosphate-buffered saline (pbs), as the control. determination of ld50 the ld50 of s. aureus in the silkworm was determined by counting the number of deaths in each group of 15 silkworms 72 h after they were injected intraperitoneally with a serial l0-fold dilution of s. aureus suspension (l02-l010 cfu/ml). the ld50 was calculated with the reed–muench method. hemolymph smear hemolymph was collected from the larvae at 12, 24, and 36 h after infection with s. aureus and quickly spread on a glass slide. the hemolymph was allowed to dry naturally and was stained with wright-giemsa stain for 1 min. phosphate buffer solution (ph 6.4) was added, and the slide was shaken gently and left at room temperature for 5-10 min. the slide was washed, dried and observed with interference fluorescence microscopy. fig. 2 survival curve of silkworms infected with s. aureus 40 fig. 3 cell smears of silkworm hemolymph infected with s. aureus. there were several large cells in the silkworm hemolymph 36 h after infection with s. aureus electron microscopic analysis of silkworm hemolymph cells hemolymph (10 µl) was collected from the larvae at 12 h after infection with s. aureus and quickly spread on a glass slide. fixing solution (2.5% glutaraldehyde, ph > 7.2) was added dropwise and the slide incubated for 30 min. the slide was washed three times with 0.1 m phosphoric acid buffer, dehydrated with a graded series of alcohol, and placed in a vacuum dryer to dry the sample. the dried sample was then sprayed with gold, and observed and photographed with a hitachi s-3400n scanning electron microscope (hitachi, japan). paraffin sectioning at 12, 24, and 36 h after s. aureus infection, the midgut and fat body tissues of the silkworms were dissected, and fixed in 10% neutral-buffered formalin for 2 weeks. sections (6 μm) were cut, mounted on glass slides, dewaxed in xylene, rehydrated through a graded alcohol series, washed in distilled water, and stained with hematoxylin and eosin. all slides were examined and photographed under a leica dmi4000 b microscope (leica, wetzlar, germany). results ld50 of s. aureus infection in b. mori as the period of silkworm infection with s. aureus increased, the crawling speed of the silkworms slowed, their leaf consumption gradually decreased, and the body wall became grayish brown (fig. 1). when the survival rate after infection with s. aureus was determined, the results indicated that the silkworms began to die 24 h after infection, only 23.3% of silkworms were alive at 36 h after infection, and all the silkworms had died within 96 h of infection (fig. 2). the ld50 of s. aureus in silkworms at 72 h was calculated with the reed–muench method to be 4.39 × 104 cfu/ml. effect of s. aureus infection on b. mori hemocytes when the hemolymph of silkworms infected with s. aureus was subjected to a smear test, we found the number of large cells in the hemolymph had increased at 36 h after infection (fig. 3). the large cells were then identified by electron microscopy as plasmatocytes (account for 10% of total cells), which adhered to s. aureus with many pseudopodia (fig. 4). 41 fig. 4 electron micrograph of phagocytosis of s. aureus by a silkworm plasmatocyte. s. aureus indicated by red arrow, plasmatocytes indicated by blue arrow plasmatocytes adhere to and spread over foreign bodies and are the main capsule-forming hemocytes. we speculated that these plasmatocytes play an important role in the cellular immunity of silkworms infected with s. aureus. effect of s. aureus infection on b. mori midgut the peritrophic membrane of the silkworm midgut plays an important role in protecting the midgut and in the immune defense of the larva, and is an effective physical barrier in the silkworm. after the larvae had been infected with s. aureus for 36 h, the peritrophic membrane of the midgut was broken and dispersed, indicating that its protective function was abolished (fig. 5). discussion wound infection is one of the ways that s. aureus invades the body, and can cause skin infection, suppuration, and even sepsis. we prepared the infection model by the way of silkworm epidermal puncture to simulate the invasion of s. aureus into the body due to trauma, and the silkworm has a high mortality rate and an ld50 of 4.39 × 104 cfu/ml at 72 h. the hemolymph is an important component of the cellular immune system of the silkworm (garsin et al., 2003; tan et al., 2013). there are five kinds of cells in the hemolymph: prohemocytes, granulocytes, plasmatocytes, oenocytoids, and spherulocytes (liu et al. 2013; zhang et al. 2022.). many genes may be involved in the immune reaction process of silkworm hemolymph against the invasion of bacteria. such as bmsr-c could bind to both gram-positive bacteria in hemocytes (zhang et al. 2021), bmhdd1 could induced after injected with different types of bacteria, it was mainly generated by hemocytes and secreted into hemolymph (zhang et al. 2017). plasmatocytes is one of the most abundant cell types in hemolymph, can participate in various innate immune responses, especially in encapsulation and node formation. bmintegrin β3 of plasmatocytes may triggered by s. aureus infection of silkworm, it may relate to the extensibility and adhesion of plasmatocytes cells (zhang et al. 2017; 42 fig. 5 tissue sections of silkworm midgut after infection with s. aureus. red arrows indicate the peritrophic membranes. peritrophic membrane was cracked at 36 h after s. aureus infection. in the control group injected with pbs, the peritrophic membrane was intact zhang et al. 2022). by scanning electron microscopy, the plasmatocytes were identified to play an important role in the resistance of silkworms against s. aureus infection. they are large, with a diameter of 10-25 μm, and have filamentous and lamellar pseudopodia, it also showed more resistant than other hemocyte morphotypes to b. mori nucleopolyhedrovirus infection (hori et al., 2013). the midgut is an important immune organ of insects, and the peritrophic membrane of the silkworm larva is located in the midgut. it is a colorless, transparent tubular structure composed of protein, chitin, and acid mucopolysaccharide (mltsuhashi et al., 2007; yang et al., 2010). its main function is to protect the cells of the intestinal wall. when silkworm was infected with s. aureus for 36 h, the peritrophic membrane showed severe damage, and the larval survival rate was <20%, it indicates that s. aureus has infected midgut tissue. in conclusion, the silkworm is susceptible to s. aureus infection, with a low survival rate at 72 h after infection. s. aureus infection damages the silkworm midgut and causes peritrophic membrane rupture. the plasmacytes in the silkworm hemolymph can capture s. aureus, and may play an important immune role in b. mori against s. aureus infection. acknowledgements this work was financially supported by grants from the national natural science foundation of china [grant number 32002235, 31602008]. references garsin da, villanueva jm, begun j, kim dh, sifri cd, calderwood sb, et al. long-lived c. elegans daf-2 mutants are resistant to bacterial pathogens. science 300(5627): 1921, 2003. hori t, kiuchi t, shimada t, nagata m, katsuma s. silkworm plasmatocytes are more resistant than other hemocyte morphotypes to bombyx mori nucleopolyhedrovirus infection [j]. j. invertebr. pathol. 112(1): 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histological anomalies with variations in enzyme activities of the earthworms lampito mauritii and drawida willsi after short term exposure to organophosphate pesticides s samal¹*, c.s.k mishra¹, s sahoo² ¹department of zoology, college of basic science and humanities, odisha university of agriculture and technology, bhubaneswar-751003, india ²school of life sciences, sambalpur university, jyoti vihar, burla-768019, india this is an open access article published under the cc by license accepted june 5, 2020 abstract monocrotophos and glyphosate are two potent organophosphate pesticides used on agricultural farms in india to control insect pests and weeds, respectively. consistent application of these chemicals poses a risk of residual soil contamination with possible adverse implications on non-target organisms, like earthworms. the present study evaluates the impacts of these pesticides on the skin, muscles and certain biochemical parameters such as protein content, lipid peroxidation (lpx) level, activities of lactate dehydrogenase (ldh), acetylcholinesterase (ache) and catalase (cat) of two tropical earthworms drawida willsi and lampito mauritii. monocrotophos at 1.0, 2.0, 3.0 g/kg soil and glyphosate at 0.1, 0.15, 0.2 g/kg soil were used for the experiment. at high concentrations, both pesticides induced lesions and skin undulation in the earthworms. in l. mauritii, the postclitellar region indicated muscle disorganization with high concentrations of monocrotophos. the lowest protein level was recorded in d.willsi and l. mauritii with high concentrations of monocrotophos. l. mauritii exhibited maximum lpx at high concentrations of glyphosate. both the earthworms indicated the least ldh activity with high pesticide concentration. minimal ache activity in l. mauritii was observed with a high concentration of glyphosate. a high concentration of monocrotophos inhibited cat activity in l. mauritii. the variable response of the selected morpho-histological and biochemical parameters in the earthworms to different pesticide concentrations could be useful early warning biomarkers to evaluate soil residual toxicity. key words: earthworm; glyphosate; monocrotophos; soil contamination; biomarker introduction pesticide application has consistently increased over the years on a global scale to control pest and weed populations in agricultural fields. residual chemical toxicity from pesticides on several nontarget organisms have previously been reported (santos et al., 2010; walker et al., 2010). earthworms are abundantly found in agroecosystems and perform vital ecological functions such as facilitating the decomposition of organics and the mineralization of nutrients (bartlett et al., 2010; tiwari et al., 2016). earthworms are extremely sensitive to environmental changes, can uptake toxic substances through their permeable ___________________________________________________________________________ corresponding author: suryasikha samal department of zoology odisha university of agriculture and technology college of basic science and humanities bhubaneswar-751003, india e-mail: suryasikha.777@gmail.com skin and those substances can accumulate in the body; therefore, earthworms are used as important indicators of ecosystem perturbations (hartenstein et al., 1981; kavitha et al., 2008; samal et al., 2017; nayak et al., 2018; mishra et al., 2019; samal et al., 2019a; singh et al., 2019; mishra et al., 2020). however, an increase in soil pesticide concentration is likely to deleteriously impact these animals (gobi et al., 2004; garcia et al., 2011; santos et al., 2011; mohan and sajayan, 2015; nayak et al., 2018; samal et al., 2019a). monocrotophos and glyphosate are potent organophosphorus pesticides used in india’s agricultural fields to control insect pests and weeds, respectively. the recommended agricultural doses (rad) of monocrotophos and glyphosate in india are 2.0 g/kg soil and 0.15 g/kg soil, respectively (agronika, 2005). due to manual pesticide application practised in india, there is always a high risk of overdose beyond the recommended dose, which enhances the risk of residual soil toxicity. https://creativecommons.org/licenses/by/4.0/legalcode 118 bioaccumulation of pesticides in earthworms could have a deleterious impact on these animals (hackenberger et al., 2008; van gestel et al., 2011). several studies have substantiated the adverse effects of pesticides on earthworms. alterations in ureotelic and ammoniotelic activity were noticed in lamipito mauritii exposed to monocrotophos (patnaik and dash, 1991). a minimal concentration of monocrotophos is toxic enough to induce dermal and histological alterations in earthworm (abbiramy et al., 2018). reports are also available on ways herbicides could adversely impact the growth and reproduction of earthworms (helling et al., 2000; zhou et al., 2007; correia and moreira, 2010; chen et al., 2018; niemar et al., 2018). the negative impact of glyphosate on earthworms, octodrilus complanatus, lumbricus terrestris and aporrectodea caliginosa in vineyards in italy’s northeast region has been reported (stellin et al., 2018). significant histopathological and enzymatic changes in the earthworms glyphidrillus tuberosus and eudrillus eugeniae due to high concentration of phosphogypsum and pesticides in soil, respectively, have also been reported (nayak et al., 2018, samal et al., 2017, 2019a,b). drawida willsi (epigeic) and l. mauritii (anecic) are abundantly found in india’s crop fields and are likely to be exposed to variable concentrations of pesticides in soil. information about the impact of organophosphates on these earthworms are not adequate. therefore, this study was undertaken to observe the possible morphological, histopathological changes and alterations in certain biochemical parameters in these earthworms with 24h exposure to three different concentrations of monocrotophos and glyphosate in soil under laboratory conditions. materials and methods experimental setup monocrotophos (monokem) was obtained from sumimoto chemical (india) pvt. ltd and glyphosate (shriram dart plus 71) from shreeji pesticides pvt. ltd. both the soil and earthworms were selectively sampled from an organic field of the university research farm to ensure that both soil and the test animals were not previously exposed to chemicals. the earthworms were then allowed to acclimatize in laboratory conditions for 15 days in an earthen culture pot with 5 kg field soil. for the present study, rad of the chemicals was taken as the medium concentration along with one low and one high concentration. thus, three concentrations of monocrotophos—1.0 g/kg soil, low (t1); 2.0 g/kg soil, medium (t2); 3.0 g/kg soil, high (t3)—and three concentrations of glyphosate—0.1 g/kg soil, low (t1); 0.15 g/kg soil, medium (t2); 0.2 g/kg soil, high (t3) —along with a control (c) were used in triplicate for the study. the chemicals were applied to soil in treatment pots (t1, t2, t3) and mixed thoroughly. soil moisture was maintained at 50 % in all the pots. ten clitellated earthworms of approximately equal size (d. willsi: 56 ± 3 mm; l. mauritii: 152 ± 4 mm) and weight (d. willsi: 1.5 ± 0.4 g; l. mauritii: 2.09 ± 0.3 g) were transferred into the control and experimental pots for 24h exposure. three earthworms from each pot were collected randomly and the experiment was repeated 3 times. the pots were covered with cotton nets to protect the experimental animals. dermal and muscular study the earthworms were sampled randomly after 24h of exposure for morphological and histological studies. the animals were thoroughly washed with distilled water and then transferred to a wet filter paper to remove gut contents and then sacrificed. sections from postclitellar segment were taken for scanning electron microscopic studies after processing with 2.5 % glutaraldehyde and upgrading (30 %, 50 %, 70 %, 90 %, 100 %) ethanol. air-dried sections were observed under the scanning electron microscope and photographed. for histopathological studies, sections of earthworms from different regions (pre clitellar, clitellar, post clitellar) were taken with the help of a sharp-edged stainless steel blade. the sections thus obtained were washed with distilled water and fixed in bouin’s fluid for 24 h. the dehydration of the sections was carried out by upgrading ethanol followed by xylene exposure. sections were embedded in paraffin wax at 60 °c to prepare wax blocks. transverse paraffin sections of 5 μm thickness were obtained using a rotary semiautomated microtome (leica). deparaffinisation of stretched sections was done using xylene and downgrades of ethanol. the slides were stained in delafield’s haematoxylin and eosin. stained slides were mounted using dpx and were observed under a binocular microscope at 100x magnification and photographed using a digital camera (nikon). determination of protein, lpx and enzyme activity fresh tissue samples were cleaned and weighed quickly to avoid post-mortal tissue degradation. potassium phosphate buffer (0.05 m, ph 7.4) was used for tissue homogenization. homogenates were centrifuged for 15 minutes at 10,000 rpm with the help of a cooling tabletop centrifuge. aliquots of supernatant were collected in 1,5 ml plastic tubes and stored at -20 °c in a deep freezer. tissue protein content was measured by folin–ciocalteau method as per lowry et al. (1951) at 700 nm. bovine serum albumin was taken as a known standard to compare the amount of proteins present in the sample. for lipid peroxidation (lpx) estimation, an aliquot of 100 μl was added to the reaction mixture (900 μl) of 0.8 % of tba, 20 % of acetic acid, 8.1 % sds and 2 % bht. the mixtures were allowed to heat at 95 °c for 45 min before taking a reading at 532 nm. the thiobarbituric acid-reactive substances (tbars) were measured to determine the concentration of mda following ohkawa et al. (1979) and expressed in nmol/mg protein. lactate dehydrogenase (ldh) estimation was done by adding samples in a reaction mixture containing 0.05 m phosphate buffer, 7.5 mm nadh and 30 mm sodium pyruvate, and the assay quantified the amount of enzyme catalyzing the conversion of 1 119 μmol of nadh per 1 min at 340 nm (cabaud and wróblewski, 1958). the ellmann et al. (1961) procedure was followed to measure acetylcholinesterase (ache) activity at 412 nm, taking 2 mm dtnb and 1 mm acetylthiocholine iodide. a sample (25 μl) was added to the reaction mixture (1.975 μl) of phosphate buffer and hydrogen peroxide to initiate the catalase assay as per cohen et al. (1970). the enzyme was estimated by observing the decrease in hydrogen peroxide concentration per unit time at 340 nm. the enzymes were expressed in u per mg protein. statistical analysis of data for one-way analysis of variance (anova) and duncan’s multiple range test (dmrt) was done with spss-20.0 software to determine the significance of variations in biochemical parameters. the significance level was set at 5 %. results electron microscopic images (fig. 1) indicated that both the untreated (control) earthworm species have smooth dermal layers. no distinct anomaly was noticed in the worms at low and medium concentrations of the pesticides. however, high concentration of both test chemicals caused dermal undulations in the earthworms. the morphological alterations were seen in each treated species at high concentrations of test chemicals, which was 97.5 % of the total number for l. mauritii and 99 % for d. willsi. fig. 1 scanning electron micrographs (5.00 kv, 200x magnification) showing dermal anomalies of earthworms treated with monocrotophos and glyphosate. a-c d. willsi, d-f l. mauritii. iskin lesion, iifolding of dermal area, iiiroughness on dermal surface 120 fig. 2 photomicrograph showing transverse section (100x) of d. willsi stained with hematoxylin and eosin stain after exposure to 24 h of monocrotophos treatment from low to high concentration, respectively, passing through (a-d)preclitellar region ,(e-h)clitellar region (i-l)postclitellar region. i-ruptured epidermal layer, iidisintegrated muscle layer, iiivacuolated muscle, ivnecrosis transverse sections of untreated earthworms indicated a thick and intact epithelial layer with closer circular and longitudinal muscle layers (figs 2-5 a, e, j). the epithelial layers were ruptured in the preclitellar regions of d. wiilsi with increasing concentrations of monocrotophos (figs 2 b, c, d). vacuolated muscle layers with necrosis in the connective tissue were evident. in the clitellar region, epithelial layers were thin and there were fewer connective tissues. the intestinal epithelium suffered damage at high concentration of monocrotophos (figs 2 g, h). postclitellar regions indicated disintegrated epithelial tissue with an accumulation of cell debris. connective tissues were also not intact. at low concentration of glyphosate, cuticle and the epithelial layer of preclitellar region were found to be intact. however, the fusion of muscle layers was observed (fig. 3 b). in a worm exposed to high concentrations of glyphosate, thin epithelial layers with loosely packed connective tissues were seen in the preclitellar region. the longitudinal muscle layer drifted away from the circular muscle layer (fig. 3 d). in the clitellar region, both the epithelial layer and intestinal epithelium were intact at a low concentration of glyphosate while at a high concentration, epithelial layers were thin with less connective tissue and ruptured muscle layers (fig. 3 h). the disintegration of epithelial tissue was observed in d. wiilsi at high concentration of glyphosate in the postclitellar region (fig. 3 i). sections through the preclitellar region of l. mauritii with monocrotophos treatment indicated damaged and vacuolated muscle layers. the cuticle became disintegrated at every concentration of monocrotophos. in the clitellar region, gaps were observed in the epithelial layer at low concentration while at high concentrations, epithelial layers were thin with disintegrated muscles (fig. 4 h). necrosis of cells at high concentration of the pesticide was also 121 fig. 3 photomicrograph showing transverse section (100x) of d. willsi stained with hematoxylin and eosin stain after exposure to 24 h of glyphosate treatment from low to high concentration respectively passing through (a-d) preclitellar region, (e-h)clitellar region (i-l)postclitellar region. iidisintegrated muscle layer, iiivacuolated muscle, ivnecrosis, vmerged circular and longitudinal muscle layer noticed. a transverse section of l. mauritii through the postclitellar region exposed to a high concentration of the pesticide indicated damage to the muscles as well as epithelial layers (fig. 4 i). minor displacement of circular and longitudinal muscle layers was observed at a low concentration of glyphosate in the preclitellar region of l. mauritii (fig. 5 b). in the postclitellar region, the epithelial layers were intact. however, no visible anomaly in the intestine (fig. 5 j) was noticed. at a high concentration, epithelial layers were found to be thin and muscle layers were ruptured (fig. 5 i). a considerable amount of cell debris was also noticed. the clitellar region indicated a damaged epithelial layer and indistinct muscle layer. tissue protein levels were found to be at a minimum (80.7 ± 2.71; 83.52 ± 6.14 mg/g tissue) in d. willsi and l. mauritii respectively, with exposure to high concentrations of monocrotophos. the protein level variation in d. willsi in response to different concentrations of monocrotophos was found to be significant (p < 0.05). the highest protein level (59.31 ± 3.72 mg/g tissue) was found in d. willsi at high concentration of glyphosate. the protein level in l. mauritii increased when exposed to low and medium concentrations of glyphosate, but indicated a lower value at high concentration. however, the protein level in earthworms at all treatments were higher than in the control (fig. 6 a). the protein level variation in l. mauritii exposed to different concentrations of glyphosate was found to be significant (p < 0.05). lpx level increased consistently with the low and medium concentrations of pesticides, but indicated minimum values (0.095 ± 0.007; 0.192 ± 0.06 nmol/mg protein) at high concentration in d. willsi exposed to monocrotophos and glyphosate, respectively (fig. 6 b). in l. mauritii, the lpx level indicated an increasing trend with low and medium concentrations of monocrotophos, but the difference was not significant. the lpx level was found to be the highest (0.132 ± 0.018 nmol/mg protein) in l. mauritii exposed to a high concentration of glyphosate. the lpx level variation in l. mauritii was 122 fig. 4 photomicrograph showing transverse section (100x) of l. mauritii stained with hematoxylin and eosin stain after exposure to 24h of monocrotophos treatment from low to high concentration respectively passing through (ad)preclitellar region, (e-h)clitellar region (i-l)postclitellar region. iruptured epidermal layer, iidisintegrated muscle layer, iiivacuolated muscle, ivnecrosis found to be significant between concentrations in response to glyphosate exposure (p < 0.05). the lowest ldh activities (0.101 ± 0.009; 0.24 ± 0.03 u/mg protein) were observed in d. willsi exposed to a high concentration of monocrotophos and glyphosate, respectively. the variation of enzyme activity at high concentration of glyphosate was significant (p < 0.05). this enzyme activity was observed to be at the minimum (0.015 ± 0.007; 0.028 ± 0.005 u/mg protein) in l. mauritii in response to a high concentration of monocrotophos and glyphosate, respectively (fig. 6 c). ache activity in d. willsi increased with low and medium concentrations of monocrotophos and declined at high concentration, but the activity was at the minimum (0.069 ± 0.006 u/mg protein) at a high concentration of glyphosate. in l. mauritii, the activity was the highest (0.103 ± 0.005 u/mg protein) at a high concentration of monocrotophos, but subsequently decreased with low and medium concentrations of glyphosate (fig. 6 d). the variations in this enzyme activity in both the earthworms exposed to the pesticides were not significant. cat activity in d. willsi decreased with a low concentration of monocrotophos but subsequently increased with medium and high concentrations. the minimum activity (0.265 ± 0.04 u/mg protein) of this enzyme was observed at a high concentration of glyphosate. in l. mauritii, the minimum (0.125 ± 0.004 u/mg protein) enzyme activity was observed at high concentration of monocrotophos. however, the enzyme activity was at the maximum (0.301 ± 0.06 u/mg protein) at a high concentration of glyphosate (fig. 6 e). the variation in the enzyme activity in response to monocrotophos was not significant in d. willsi. however, significant variation in the enzyme activity was observed in l. mauritii at high concentration (p < 0.05). 123 fig. 5 photomicrograph showing transverse section (100x) of l .mauritii stained with hematoxylin and eosin stain after exposure to 24 h of glyphosate treatment from low to high concentration respectively passing through (a-d) preclitellar region, (e-h)clitellar region (i-l)postclitellar region.i-ruptured epidermal layer, iidisintegrated muscle layer, iiivacuolated muscle, vmerged circular and longitudinal muscle layer discussion soil toxicity can be evaluated using indicator organisms such as earthworms. in these animals, contaminants may be absorbed through the skin and then transported throughout the body (saxe et al., 2001; jager et al., 2003; vijver et al., 2003). various workers have noticed morphological aberrations such as swelling, lesions and skin discolouration in earthworms due to pesticide and heavy metal toxicity (rao et al., 2003; youn, 2005; yvan et al., 2005; reddy and rao, 2008; singh et al., 2019). reddy and rao (2008) reported morphological and histological alterations in eiesenia fetida exposed to the organophosphate profenofos. identical effects have been observed in e fetida with the herbicide glyphosate and 2,4-d (correia and moreira, 2010). mukherjee and parida (2015) have reported lindane induced toxicity and significant biomass loss in e eugeniae. nunes et al. (2016) studied the effect of abamectin on eisenia andrei and observed thinning and discolouration of the skin, constriction of the body and fragmentation of the posterior segment. singh et al. (2019) reported variable morphological changes in the earthworm e. eugeniae with sublethal concentrations of triazophos. our observations on dermal anomalies in d willsi and l mauritii corroborate the earlier reports above on different earthworm species, likely due to trauma caused by pesticide exposure, which induced damage in the muscles and dermis to variable degrees, depending on the concentration. reports are available on the effects of diverse groups of chemicals on the histology of earthworms, which are more or less similar to the results obtained in this study. the earthworms perionyx sansibaricus and e fetida suffered from muscle degeneration and cuticular rupture when exposed to the weedicide burachlor (muthukaruppan et al., 2005; reddy and rao, 2008; gobi and gunasekaran, 2009). significant muscular aberrations were reported in nsukkadrilus mbae after exposure to sublethal doses of atrazine (oluah 124 fig. 6 changes in certain biochemical parameters in d. willsi and l. mauritii exposed to monocrotophos and glyphosate. aprotein content, b lpx level, cldh activity, dache activity and ecat activity. mp monocrotophos, gpglyphosate, dwd.willsi, lm-l.mauritii. different letters above the same colour bar indicate significant differences between control and pesticide concentrations at p < 0.05 et al., 2010). effects of various weedicides, heavy metals and hydrocarbons on different earthworms have been tested, which indicated muscle anomalies (lourenço et al., 2011; sharma and satyanarayan, 2011; canbek et al., 2012; eseigbe et al., 2013; bangarusamy et al., 2014; enuneku et al., 2014; oluah and ochulor, 2014). high concentrations of pesticides could cause significant damage to muscle and intestinal epithelium of tropical earthworms g tuberosus and e eugeniae (nayak et al., 2018; samal et al., 2019a). in the present study, we have noticed that monocrotophos at each concentration and glyphosate at high concentration could bring about 125 severe muscle and cuticular disintegration. disorganization of the circular and longitudinal muscles, along with an accumulation of cell debris in the earthworms, could seriously impair their ecological functions. clitellar damage could impair these animals’ cocoon production and other reproductive functions. we also noticed significant alterations in the biochemical parameters in both the earthworms, which corroborate the findings of several workers. significant protein reduction in the earthworm lumbricus terrestris has been reported after exposure to high concentrations of the insecticides endosulfan and aldicarb (mosleh et al., 2003). the reduction in the animal’s protein level was significant between treatment concentrations. the identical result had been obtained by ismail et al. (1997) with a consistent reduction in the total protein content in the earthworm aporrectodea caliginosa in response to chlorfluazuron exposure. another study indicated that phorate, an organophosphate pesticide, could cause significant protein reduction in earthworms perionyx sansibaricus, l. mauritii and metaphire posthuma (tripathi et al., 2009). in our study, the lowest protein levels in both the earthworms studied were observed in animals exposed to a high concentration of monocrotophos, which is in agreement with the findings on other species of earthworms. it appears that these pesticides at high concentrations adversely impact protein synthesis and accumulation in d. willsi and l. mauritii. l. mauritii, when exposed to low and medium concentrations of glyphosate, exhibited higher protein content which was reduced in response to a high chemical concentration. this is likely due to possible accumulation of stress proteins in response to low and medium concentrations and hyper toxicity at high chemical concentration. peroxidation of lipids in animals under physiological stress is conventionally used as a marker to evaluate oxidative stress (paulina et al., 2011). lipid peroxidation is usually determined from the carbonylic compounds called malondialdehyde (mda). the mda is used as an index of lipid peroxidation (khessiba et al., 2001). the earthworm e eugeniae indicated significantly high levels of mda after exposure to hydrocarbons (eseigbe et al., 2013). in the present study, both the earthworms exposed to the pesticides indicated increased lipid peroxidation at low and medium concentrations. interestingly, at high concentration of glyphosate, d. willsi indicated lower and l. mauritii showed higher lipid peroxidation levels. animals, including earthworms, have protective mechanisms to scavenge peroxides generated due to lipid peroxidation, which is likely to vary in different species. the lower level of lipid peroxides at high concentration of these chemicals in d. willsi is likely due to the superior adaptive mechanisms in this worm to counter cell damage due to peroxides. ldh accelerates the anaerobic energy production process by catalysing the conversion of pyruvate to lactate. environmental changes could induce ldh activity. it is reported that the ldh activity declined with rising environmental temperature in the earthworms m. posthuma and e eugeniae (tripathi et al., 2011; mishra et al., 2018). a consistent decline in ldh activity with increasing concentration of the organophosphate pesticide phorate has been reported (tripathi et al., 2009). our observations of the enzyme activity in both the earthworms are in agreement with these earlier reports, and it appears that high concentrations of both chemicals negatively influence ldh activity. however, rico et al. (2016) postulated significantly increased ldh activity in e.fetida after exposure to tebuconazole, this finding contradicts our results. depleted ache activity is impaired with altered neural conduction in earthworms due to pesticide toxicity (patnaik and dash, 1992; pradhan and mishra, 1998; reddy and rao, 2008; samal et al., 2017; nayak et al., 2018; samal et al., 2019a). earlier studies by rao et al. (2003) indicated that chlorpyrifos had an anticholinergic effect in the earthworm e. fetida. in our study, worms exposed to glyphosate indicated a suppressed level of ache relative to the control, which is in agreement with these earlier reports. identical results have also been obtained by singh et al. (2019), who reported that the pesticide triazophos significantly inhibited ache activity in e eugeniae. however, contradictory results were obtained in worms exposed to monocrotophos, which indicated higher enzyme activity relative to the control. this was possibly due to the animals’ short duration (24h) of exposure to this pesticide. animals produce cat under enhanced physiological stress to scavenge free radicals. in all aerobic organisms, this is used as a preventive against cytotoxicity, mutagenicity or carcinogenicity, which might result due to a reduction in the level of antioxidants (mates, 2000). in l. mauritii, high levels of this enzyme at all concentrations of glyphosate relative to control indicate that this species can counter the physiological stress more efficiently in comparison to d. willsi, where the enzyme activity was lower. therefore, we conclude that both monocrotophos and glyphosate are toxic to the earthworms. it is also evident that these dermal, histological 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golden r, horst mn. morphologic effects of in vivo acute exposure to the pesticide methoprene on the hepatopancreas of a non-target organism, homarus americanus. ecotox environ safe. 73: 1867-1874, 2010. youn ja. assessing soil ecotoxicity of methyl tertbutyl ether using earthworm bioassay; closed soil microcosm test for volatile organic compounds. environ pollut. 134: 181-186, 2005. yvan c, rault m, costagliola g, mazzia c. lethal and sublethal effects of imidacloprid on two earthworm species (aporrectodea nocturna and allolobophora icterica). biol fert soil. 41: 135143, 2005. zhou sp, duan cq, hui fu, chen yh, wang xh, yu zf. toxicity assessment for chlorpyrifoscontaminated soil with three different earthworm test methods. j environ scie. 19: 854-858, 2007. isj004.pdf 34 isj 1: 34-37, 2004 issn 1824-307x short communications analysis of the expression pattern of the defensin gene in the lepidopteran mamestra brassicae f borsatti, l casarini, m mandrioli* department of animal biology, university of modena and reggio emilia, modena, italy accepted june 30, 2004 abstract southern blotting experiments performed on mamestra brassicae genomic dna after digestion with methylation-sensitive restriction enzymes indicated that defensin gene is methylated at cpg targets in the promoter region. however, defensin gene is actively transcribed despite the presence of methylation. experiments performed by genome demethylation indicated that demethylated defensin gene resulted in an altered expression after bacterial induction. in particular, no increase in gene expression was observed after induction with gram positive bacteria if defensin gene was demethylated. the present results are intriguing since they indicate that in m. brassicae dna methylation is not involved in gene silencing, but also that dna methylation could be essential to assure the expression of specific genes. on the whole, data here presented argue against an unifying and evolutionary conserved role of cytosine methylation from invertebrates to vertebrates. key words: epigenetics; dna methylation; defensin gene expression; lepidopteran introduction defensins are 4kda cationic peptides with a characteristic six cysteine/three disulfide bridge pattern and three domains consisting in a flexible aminoterminal loop, a central α-helix and a carboxy-terminal antiparallel β-sheet (hoffmann and hetru, 1992; hetru et al., 1998; bulet et al., 1999). several authors reported the isolation and description of defensins or defensin-like peptides in different insects (chalk et al., 1995; lowenberger et al., 1995; cho et al., 1996, 1997; miyanoshita et al., 1996; richman et al., 1996; dimopoulos et al., *corresponding author: mauro mandrioli department of animal biology, university of modena and reggio emilia, modena, via campi 213/d, 41100 modena, italy e-mail: mandriol@unimo.it 1997; gao et al., 1999; muller et al., 1999; eggleston et al., 2000), but just two papers reported the presence of defensin-like peptides in lepidoptera (lamberty et al., 1999; mandrioli et al., 2003). in particular, lamberty and colleagues identified in heliothis virescens a peptide, named heliomicin, that shows antifungal activity and shares similarities with antibacterial insect defensins (lamberty et al., 1999). mandrioli and colleagues (2003) isolated a defensin gene that results highly conserved at a sequence level. it has also been indicated that, even if a weak constitutive expression of the defensin gene can be detected, defensin gene expression was greatly increased by gram positive, but not by gram negative bacteria (mandrioli et al., 2003). the present paper analyses the methylation status of the defensin gene and the functional significance of methylation for defensin gene expression in the immunocytes of the cabbage moth mamestra brassicae. 35 materials and methods samples the izd-mb-0503 immunocyte cell line from the insect mamestra brassicae (lepidoptera) (atcc number: crl-8003) was used. the cells were cultured in ex-cell 405 medium (jrh biosciences, ks, usa) at 26 °c. presence of defensin molecules the presence of defensin molecules was detected in the izd-mb-0503 cell line by immunocytochemical procedure and in situ hybridisation according to mandrioli et al. (2003). pcr amplification of defensin probe, genome demethylation and southern blotting genomic dna extraction from the izd-mb-0503 cell line, electrophoresis and transfer of the dna from agarose gel to nylon membrane were performed following mandrioli (2002). pcr amplification of a portion of the defensin gene was carried out using two primers: f (5’-ccaaatgcctcgtcatct) and r (5’attagagtc aagctcaaaaggg). primers were selected according to m. brassicae defensin gene sequence (af465486) available in genbank (mandrioli et al., 2003). the amplification mix contained 100 ng of genomic dna, 1 µm of each primer, 200 µm dntps and 2u of dynazyme ii polymerase (finnzymes oy, finland). amplification was performed with a thermocycler at an annealing temperature of 55 °c for 30 sec and extension at 72 °c for 45 sec. the amplified portion of the defensin gene was labelled using the dig high prime kit (roche) according to manufacturer’s instructions. genome demethylation was obtained by treating in vitro m. brassicae cells with a 20 mm 5-azacytidine solution for 72 hours according to mandrioli and volpi (2003). southern blotting has been performed according to mandrioli (2002). analysis of defensin gene expression by northern blotting and rt-pcr rna extraction was carried out using the “sv total rna isolation kit” (promega) following the manufacturer’s protocols. rna was successively electrophoresed on formaldehyde gel, transferred onto a nylon membrane by capillary transfer and blotted with the digoxygenin (dig)-labelled defensin probe following standard techniques. northern blotting was carried out following mandrioli (2002). rt-pcr amplification was performed according to ottaviani et al. (2002). results and discussion southern blotting performed on m. brassicae genomic dna after digestion with methylationsensitive restriction enzymes (mspi, hpaii, mboi and sau3ai) clearly indicated that the defensin gene is methylated (fig. 1a-b). in particular, comparison of the restriction pattern of mspi and hpaii showed that methylation is confined to cpg targets. mspi and hpaii are, in fact, isoschizomers that recognize the same target sequence 5’-ccgg-3’, but while mspi cleaves its target irrespectively of the methylation of the inner cytosine of the target sequence, hpaii is inhibited by its methylation. the distribution of the mspi restriction targets inside the defensin gene sequence, together with the results of the southern blotting experiments, suggested that methylation could be limited to the 5’ promoter region of the gene. in order to verify the effect of methylation on gene expression we analyzed the presence of both defensin mrna and peptide. the presence of defensin transcripts has been verified using rt-pcr, and in situ hybridisation (figs 1, fig. 2a), whereas gene expression was assessed using in situ immuno-procedures (fig. 2b). all the applied techniques clearly indicated that defensin gene is constitutively expressed despite the presence of methylation at the promoter region of the gene. in order to verify if gene demethylation was related to alteration in defensin expression, m. brassicae genome was demethylated by treating cabbage moth cells with a 20 mm 5-aza-cytidine solution for 72 h. the relationship between level of methylation and degree of defensin gene expression has been evaluated both at a constitutive level and after induction with the gram positive bacteria staphylococcus aureus (atcc 6538). 1 2 3 4 5 fig. 1 southern blotting with the defensin probe on cabbage moth dna digested with mboi (lane 1), sau3ai (lane 2), mspi (lane 3) and hpaii (lane 4) evidenced the presence of methylated cpg upstream the defensin gene. rt-pcr experiments indicated that defensin gene is expressed despite gene methylation. 36 fig. 2. experiments of in situ hybridisation (a) and immuno-cytochemistry (b) indicated that defensin gene is constitutively expressed. rt-pcr and northern blotting experiments showed that the constitutive expression of the defensin gene was not altered by genome demethylation (fig. 3). on the contrary, genome demethylation completely abolish gene up-regulation after induction by gram positive bacteria. the present findings demonstrated that methylation of the cabbage moth defensin gene is not important for the constitutive expression of the gene, but is essential for its up-regulation after bacterial challenge (fig. 3). these results are very intriguing since they indicate not only that in m. brassicae dna methylation is not involved in gene silencing, but also that cytosine methylation could be essential to assure the expression of specific genes. these findings are in agreement with those reported in other insects, such as myzus persicae (hick et al., 1986; field et al., 1989; field, 2000) and planococcus citri (bongiorni et al., 1999; bongiorni and prantera, 2003), where methylated genes resulted to be highly expressed and dna methylation was fundamental to assure their expression. the above reported data argue against an unifying and evolutionary conserved role of cytosine methylation from invertebrates to vertebrates. in fact, it appears that the dna methylation/gene silencing correlation, which is typically reported in vertebrates (wolffe et al., 1999), is not true for insects. methylation in insects could be fig. 3. northern blotting experiments (a) and rtpcr amplifications performed using rna samples extracted from non-induced control cells (lane 1), demethylated non-induced cells (lane 2), demethylated induced cells (lane 3) and induced control cells (lane 4). essential to focus initiation of transcription on genuine promoters in order to guarantee the expression of s p e c i f i c g e n e s s u s c e p t i b l e t o t r a n s c r i p t i o n a l interference. in view of this assumption, insects methylate genes that have to be expressed in place of silent ones. in conclusion, we hypothesize the presence of a discontinuity in the functional role of methylation from invertebrates to vertebrates since it appears that the dna methylation/gene silencing correlation is not present in insects. acknowledgements this work was supported by a miur grant to mm. references bongiorni s, prantera g. imprinted facultative heterochromatization in mealybugs. genetica 117: 271279, 2003. bongiorni s, cintio o, prantera g. the relationship between dna methylation and chromosome imprinting in the coccid planococcus citri. genetics 151: 1471-1478, 1999. bulet p, hetru c, dimarcq jl, hoffmann d. antimicrobial peptides in insects; 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behavior; histology; enzyme activity; transcriptome introduction neptunea cumingii is a large cold water marine snail, and in china, it is mainly distributed in the subtidal zone of the bohai and yellow sea areas (zhou et al., 1995; gao et al., 2015). n. cumingii has a spindle-shaped exterior shell with a bulged center and pointy ends as well as a horny operculum. it is carnivorous and feeds on benthic shellfish and rotten meat. n. cumingii is a popular and highly valued edible species due to its delicious and nutrient-rich meat, which contains a variety of amino acids, glycogen, protein, and essential trace elements that are good for human health (cai, 2001). to date, studies of n. cumingii have focused on its shell, distribution, and genetic diversity. for example, _________________________________________ corresponding authors: yaqing chang and zhenlin hao key laboratory of mariculture & stock enhancement in north china’s sea ministry of agriculture dalian ocean university dalian, liaoning, 116023, p. r. china e-mail: yqchang@dlou.edu.cn; haozhenlin@126.com zhao et al. (2004) studied the structural characteristics and the relationship between the structure and properties of the n. cumingii shell and found that the shell of n. cumingii was composed of calcite and aragonite which were enchased in organic phase. gao et al. (2008) surveyed the stock distribution of n. cumingii using bottom trawlers in liaodong bay and found that the n. cumingii was mainly distributed in laotieshan sea area of lvshun, the relative biomass of n. cumingii and the bottom salinity had a positive correlation. yu et al. (2019) introduced the reproductive biology of n. cumingii and the progress of artificial breeding technology. azuma et al. (2009) studied the polymorphic microsatellite markers of n. cumingii in hokkaido japan, and isolated eight polymorphic microsatellite dna loci. their results suggested that the loci could be used as markers for population and kinship analyses for this species. hao et al. (2020) studied the copulation, egg laying, embryonic development and changes in amino acids and fatty acids in n.cumingii during embryogenesis to understand the embryo development process and nutritional 2 requirements in the early life phase. whose results showed that n.cumingii had direct development within the egg capsule and the development of embryos was classified into five stages. however, nothing is known about the effect of water temperature on the behavior, histology, immune enzyme activity, or transcriptome in the gill and kidney of n. cumingii. before large-scale farming of n. cumingii begins, it is critical to identify the optimal conditions for such farms. we therefore explored the impact of temperature on n. cumingii, specifically aiming to 1) identify the effects of temperature on ingestion and survival in n. cumingii; 2) identify morphological changes in the gills and kidney of n. cumingii in response to temperature; 3) identify alterations in total antioxidant capacity (t-aoc) levels, superoxide dismutase (sod) and catalase (cat) levels in the gills of n. cumingii in response to changes in temperature; 4) identify expression information of related functional genes in the gills and kidney of n. cumingii in response to temperature in transcriptome. our results will help to the cultivation and protection strategies for n. cumingii. materials and methods experimental materials neptunea cumingii were collected from the lvshun sea, dalian city, liaoning province, china, and transported to the key laboratory of mariculture and stock enhancement in north china’s sea (ministry fig. 1 n. cumingii feeding behavior when cultured at different temperatures for 6 h. different lowercase letters indicate significant differences in feeding rates of n. cumingii at different temperatures (p < 0.05) of agriculture, dalian ocean university, dalian, p.r. china) in insulated containers. in the laboratory, samples were cultured in five 300 lt aquariums for two weeks before the experiments commenced. each aquarium accommodated 95 n. table 1 effects of different temperatures on the behavior and mortality of n. cumingii time temperature (°c) 0 4 8 12 16 20 24 28 6h behavior 〇 mortality rate (%) 0 0 0 0 0 0 0 0 12h behavior 〇 ▲ mortality rate (%) 0 0 0 0 0 0 0 0 24h behavior 〇 * mortality rate (%) 0 0 0 0 0 0 0 16.8 ± 2.4 48h behavior 〇 * mortality rate (%) 0 0 0 0 0 0 0 66.1 ± 1.2 72h behavior 〇 * mortality rate (%) 0 0 0 0 0 0 0 100 notice: 〇: shrink -: normal ▲: desorption *: death shrink: the rhynchodaenm is in the body without extending, and the foot extension area is very small. normal: the foot extension area is larger than that in shrink condition, and the rhynchodaenm is out of the body. desorption: the absorption force of the foot becomes so weak that they often failed to hold onto the wall of the pool. death: the foot and operculum turn out of the shell without shrinking, even when they are touched with fingers 3 cumingii, and the average individual wet weight was 85.3 ± 3.2 g. the temperature was 16 ± 1 °c, and the salinity was 30 ± 1 ‰. enough unionidae (3 kg) were thrown into each aquarium for n. cumingii’s food at 08:00 every day, 6 hours later, the residual bait and feces were clean up. the water was changed twice a day, half of the water was changed each time. experimental design before the experiment, all n. cumingii were deprived of food for two days. the eight temperature levels in the experiment were 0, 4, 8, 12, 16 (control), 20, 24, and 28 °c. 432 healthy and active n. cumingii were randomly selected and equally placed into 24 tanks (each temperature contained 3 tanks forming three replicates with 18 animals per tank). to achieve each test temperature, the temperature was increased or decreased from 16 °c by 1 °c/d. when the desired temperature was reached, that temperature was maintained for 72 h prior to the relevant experiments. behavior to assess behavior, 80 active n. cumingii (10 per temperature) were placed in eight observable containers for 2 h, and then 1 kg of live unionidae were added to the container. the feeding behavior of n. cumingii was observed for 6 h. feeding rate was calculated as the followings: fr =(w1-w2)/w1 × 100 % where w1 and w2 are the initial and final wet weights of unionidae. histology three individuals per replicate were selected for histological and enzyme activity assays. fresh gill and kidney tissues were removed from n. cumingii and fixed in bouin’s fixative solution for more than 24 h. fixed specimens were preserved in 70 % alcohol, then individually embedded in paraffin blocks and sectioned in serial sagittal sections (4 μm) using a rotary microtome (leica, germany). tissue sections were permanently mounted, and the slides were stained with hematoxylin-eosin (h&e) for general histological observations. the sections were examined under leica dm500 compound microscope. microphotographs were taken with leica mc170 hd camera. enzyme activity the activities of catalase (cat) and superoxide dismutase (sod) and total antioxidant capacity (t-aoc) levels in the gill and kidney of n. cumingii were measured using assay kits purchased following the manufacturer’s protocols. enzyme determination was carried out at 25 ± 1 °c in an air-conditioned room. t-aoc was measured based on the generation of the fe2+-o-phenanthroline complex, as the overall reducing agents in the sample supernatant reduced fe3+ to fe2+, which reacted with the substrate o-phenanthroline. stable color of the fe2+ o-phenanthroline complex was measured at 520 nm at 37 °c. one unit of t-aoc was defined as the amount necessary to increase the absorbance by 0.01 at 37 °c (u mg–1 protein). the decomposition reaction of h2o2 by cat can be terminated immediately by adding ammonium molybdate, which reacts with the remaining h2o2 to form a faint yellow complex. cat activity was detected by measuring the decrease in absorbance resulting from h2o2 decomposition at 405 nm. one unit of cat activity was defined as the amount of enzyme that decomposes 1 µmol of h2o2 (u/mg protein). sod activity was determined at 550 nm using the xanthine and xanthine oxidase systems. one unit of sod activity was defined as the amount of enzyme required to cause 50 % inhibition of the xanthine and xanthine oxidase reaction in 1 ml of enzyme extract of 1 mg protein (u/mg protein). transcriptome of gill and kidney according to the previous results of behavior, histology and enzyme activity, we selected two significantly different treatment groups (16 °c and 28 °c) for transcriptome assay. the gill and kidney samples of n. cumingii at different test temperature (3 individuals per replicate) were collected and frozen in liquid nitrogen and stored in a refrigerator at -80 °c for later use. total rna from gills and kidneys tissue of n. cumingii was extracted using the trizol kit (invitrogen, usa). genomic dna was treated with dnase i (takara, dalian, p r china), after with 0.7 % agarose gel electrophoresis was used to assess rna integrity, with an agilent 2100 bio-analyzer (agilent technologies, usa) and nd-2000 (nanodrop technologies) platforms used to confirm rna purity and quantity. equal amounts of high-quality rna (od260/od280 = 2.04-2.07, od260/od230 ≥ 1.65-2.18, rin ≥ 8.4) from the gills and kidneys of snails were then pooled to construct a sequencing library. mrna was collected using oligo(dt) beads, followed by fragmentation buffer-mediated fragmentation. random hexamer primers were used to generate double-stranded cdna. after end repair, “a” base addition, and adapter connection, 200-300 bp cdna fragments were selected and purified via agarose gel electrophoresis, amplified by 15 pcr cycles, the library sequencing was conducted. functional annotation and classification were conducted blast2 go and wego respectively. degs were compared with the terms in go database to get the list and number of transcriptions belonging to the go function. then hypergeometric tests were used to identify go entries in which degs were significantly enriched compared to the entire set of transcripts. kegg is another leading public database about the pathways used for understanding high-level functions and utilities of the biological system. when degs were compared with the pathways in kegg database, hypergeometric tests were used to identify the kegg pathways in which degs were significantly enriched. these go terms and pathways that had q-values ≤ 0.05 were deemed significantly enriched. analysis based on go terms and pathway is helpful to further understand the biological functions of transcripts. r was utilized for all the expression data statistics and visualization. systematically analyze the data and compare the gills and kidneys of the snails. 4 statistical analysis spss 22.0 and originpro 2017 software were used to analyze the differences in snail feeding rate caused by temperature changes over a 6 h period. statistica6.0 software was used to analyze the enzyme activity data. the results were expressed as the mean ± standard error. significant differences between groups were analyzed by one-way analysis of variance (anova) and tukey’s honestly significant different test where appropriate. p < 0.05 was considered statistically significant (95% confidence interval). statistical analysis of transcriptome data was performed using excel 2016 and spss 19.0. all data were presented as the mean ± standard deviation. significant differences were analyzed via one-way anova, and p < 0.05 was considered statistically significant. pearson correlation analyses were used to assess the strength of the relationship between rna-seq data and rt-qpcr measurements. results behavior behavior of n. cumingii differed among the different temperature conditions (figure 1, table 1). at 0 °c, most of the n. cumingii shrank in size and did not eat during the first 6 h. at 28 °c, the snails also did not feed. however, death began to occur at 24 h, and all snails died within 72 in the 28 °c group. there was no significant difference in the behavior of the n. cumingii when the temperature was 4-24 °c, but food intake did differ significantly. the food intake at 16 °c was significantly higher than at the other temperatures, followed by 8 and 12 °c, which did not differ significantly from each other. histology of gills and kidneys in n. cumingii at the control temperature of 16 °c (figure 2e), a blood vessel was visible in the center of the gill filaments, and gill lamella were present on both sides of the gill filaments to allow gas exchange. mitochondria-rich cells were distributed at the base of the gill lamella. at 8 °c, the gill filaments were swollen, and numerous blood cells flowed out. at 4 °c, the epidermis of the gill lamella exhibited shedding, and the distance between adjacent gill lamellas was enlarged. at 0 °c, the morphology of the gill tissue was difficult to judge because the low temperature had destroyed much of the tissue. at 24 °c, edema was visible in the gill lamella, and at 28 °c the gill lamella were severely deformed, the distance between the gill capillaries and the surrounding area was enlarged, and the gill tissue was severely damaged (figure 2). fig. 2 histology of the gills of n. cumingii cultured at different temperatures. a: 0, b: 4 °c, c: 8 °c, d: 12 °c, e: 16 °c, f: 20 °c, g: 24 °c, h: 28 °c. gl: gill lamella, bc: blood corpuscle, sec: squamous epithelial cells, mc: mucous cells, gf: gill filament 5 fig. 3 histology of the kidney of n. cumingii cultured at different temperatures. a: 0 °c, b: 4 °c, c: 8 °c, d: 12 °c, e: 16 °c, f: 20 °c, g: 24 °c, h: 28 °c. ct: collecting tube, rt: renal tubule, m: muscle, v: villi, sce: simple columnar epithelium the kidneys of n. cumingii contained only tubules and collecting ducts. the kidney columnar cells in the 16 °c group were longer than those in the other groups; at both higher and lower temperatures, the columnar cells were shorter and more numerous (figure 3). t-aoc and activities of cat and sod figure 4-a shows the t-aoc of the gills and kidneys of n. cumingii cultured under different temperature conditions. at temperatures ranging from 4 to 24 °c, the t-aoc of the gill was maintained between 0.7 and 1.0 u/mg prot, and values did not differ significantly between the 16 and 20 °c groups (p > 0.05). at 0 and 28 °c, the t-aoc of the gills was low. there was no significant difference in t-aoc of the kidney among the 12, 16, and 20 °c groups (p > 0.05), but the values gradually decreased as temperature increased to 24 and 28 °c. additionally, t-aoc of the kidney increased from 0 to 4 °c and was highest at 8 °c. fig. 4 t-aoc, cat and sod of the gills and kidneys of n. cumingii cultured at different temperatures. letters indicate significant differences of t-aoc, cat and sod activities among the treatments (p < 0.05). * indicate significant differences on t-aoc, cat and sod activities between the gill and kidney (p < 0.05) 6 table 2 annotation information statistics for the gill and kidney transcriptome of n. cumingii nr note swissprot notes kegg notes ko notes eggnog notes go notes pfam notes number of genes 20763 13106 9070 11068 15224 12015 12293 proportion (%) 25.50 16.10 11.14 13.59 18.70 14.76 15.10 the cat activities of the gills and kidneys of n. cumingii cultured at different temperatures are shown in figure 4-b. at 0 °c, the cat activity of the gill was 0.59 u/mg prot, but it was lower (0.30 u/mg prot) in the 4, 8, and 12 °c groups and then higher (0.65 u/mg prot) in the 16, 20, and 24 °c groups. the activity was lowest (0.23u/mg prot) in the 28 °c group. cat activity of the gills was much lower than that of the kidney at all temperatures. the activity increased and decreased several times with increasing culture temperature, but the values did not differ significantly between the 0 and 4 °c groups, between the 8 and 12 °c, or between the 16 and 20 °c groups (p > 0.05). the lowest activity occurred in the 28 °c group. figure 4-c shows the sod activities of the gills and kidneys of n. cumingii cultured at different temperatures. as the temperature increased from 0 to 28 °c, the activity of sod in the kidney exhibited a w-shaped pattern. the sod activity of the gills was maintained above 150u/mg prot at all temperatures. in the kidney, the highest sod activity (120 u/mg prot) occurred in the 0 °c group. sod activity differed significantly among all temperature groups (p < 0.05) except for the 4, 8, and 20 °c, which did not differ from each other (p > 0.05). transcriptome analysis transcriptome data showed that the original data q30 of each sample were distributed in 91.64-92.25 %, the effective data volume was distributed in 6.12-6.89 g, and the average gc content was 45.04 %. in total, 81,429 unigenes were spliced together, with a total length of 67,628,134 base pairs and an average length of 830 base pairs. table 2 shows the unigene database annotation results. the comparison rate of reads to unigenes was 83.53-84.33 %. the numbers of differentially expressed genes detected were 4397 for gills and 4360 for kidneys. in total, 52,399 simple sequence repeats (ssrs), 29,251 unigenes containing ssrs, 12,391 unigenes containing more than 1 ssr, and 11,640 composite ssrs were predicted. additionally, 40,930 coding sequences were predicted (20,776 were predicted by the database comparison method and 20,154 were predicted by estscan). in kegg enrichment, analysis showed that the assembled unigenes participate in 42 functional pathways (figure 5) in the following six categories: cellular processes, environmental information processing, genetic information processing, metabolism, human diseases, and organic systems. the number of genes in the different metabolic pathways varied greatly. after initial go annotation of the unigenes, the successfully annotated genes were classified according to the next level of the three major categories of go, which were biological process, cell composition, molecular function (figure 5). the abscissa shows the go term of the next level of each of the three major categories of go and the ordinate shows the number of genes annotated to the term. there were relatively more annotated genes for cellular processes, metabolic processes, and single biological processes in the biological process category. for the cell composition category, cells, cell parts, and organelles had the most annotated genes, and for the molecular function classification, the most annotated genes were in the connection and catalytic activity categories. as shown in figures 6 to 8, 4397 genes in the gills of n. cumingii showed differential expression (2339 up-regulated and 2058 down-regulated), and 4360 genes in the kidney showed expression differences (2300 up-regulated and 2060 down-regulated). in go enrichment, degs of gill significantly enriched in protein folding，translation， ribosome，unfolded protein binding and structural constituent of ribosome terms. and degs of kidney significantly enriched in dna recombination, nuclear euchromatin, rna-directed dna polymerase activity and aryl sulfotransferase activity terms. |log2fc| > 1 and fdr < 0.05 were used as the selection standards for differential expression in kegg enrichment analysis. we selected the first 20 pathways with significant enrichment and found the degs of gill significantly enriched in ribosome, protein processing in endoplasmic reticulum, apoptosis, nod-like receptor signaling pathway and tnf signaling pathway. the protein processing in endoplasmic reticulum involved the most degs (24 degs) in the gill, which also involved hsp 70 and hsp 90. the degs of kidney significantly enriched in nf-kappa b signaling pathway, longevity regulating pathway − multiple species, apoptosis − multiple species, nod−like receptor signaling pathway and tnf signaling pathway. the most degs involved in tnf signaling pathway (12 degs). discussion behavior water temperature has significant effects on movement, feeding, reproduction, and behavior of 7 fig. 5 kegg annotation statistics chart (above) and go function classification diagram (under) for the gill and kidney aquatic animals. for example, keen et al. (1994) showed that the swimming speed of oncorhynchus mykiss increased proportionally with increasing temperature but then decreased when the temperature exceeded the optimum value. chen (2004) found that the food intake of cyprinus carpio first increased and then decreased with increasing culture temperature. in another study, armstrong et al. (2013) found that oncorhynchus keta spawning grounds were situated only in areas with suitable water temperature. in this study, we also found that temperature had a significant impact on the behavior of n. cumingii. when the temperature was very low (0 °c), the snails contracted, closed their shells, and did not eat. as temperature increased, food intake first increased, peaked at 16 °c, and then decreased. the highest average food intake was 16 gram per individual, which was consistent with the normal food intake of n. cumingii. (fujinaga et al., 1999; miranda et al., 2008). other studies have shown that an initial increase followed by a decrease in food intake as temperature increases is a common 8 fig. 6 differential gene comparison volcano map for the kidney (left) and gills (right). gray is non-difference unigene, red is up-regulated significant difference unigene, and green is down-regulated significant difference unigene; the x-axis is log2 foldchange, and the y-axis is log10pvalue phenomenon in aquatic animals (sun et al., 1982; xie et al., 2019). li (2006) reported that digestive enzyme reactions in the black owl body accelerated with the increase in temperature within a certain range, but after a certain value the speed of the reactions slowed down. we propose that the weakened feeding ability of n. cumingii that occurred at 28 °c may have been due to the decreased concentration of dissolved oxygen in the water at higher temperature. this would result in more energy being used to maintain resting metabolism and less energy available for food digestion and thus decreased food consumption by n. cumingii. similar results also occurred in mizuhopecten yessoensis (ben, 2013) and haliotis discus hannai (jiang, 2017). when the temperature was 28 °c, the snails had poor adsorption ability and gradually closed their shells within 12 h. death began to occur at 24 h, and all snails had died within 72 h. this result showed that n. cumingii was not able to tolerate heat stress. histology of the gills and kidney gills are the main respiratory organs of many aquatic animals, and they regulate osmotic pressure and excrete ammonia nitrogen (qu et al., 2018). andrew et al., (2011) found that in addition to salinity, environmental temperature can also affect the osmotic pressure balance and membrane permeability of the porgy fish (pagrus sp.). in the current study, when the temperature decreased below 16 °c, the osmotic pressure of the n. cumingii gill became imbalanced. regaining balance required a lot of energy, so the number of mitochondrial cells and red blood cells increased. as the temperature dropped again, the blood vessels in the gills were constricted, and the uneven distribution of red blood cells was more evident. at 8 °c, the blood vessels in the gill lamella gradually absorbed water and expanded, eventually swelling and flowing out of the red blood cells. this was similar to the results of chen's research on the gills of tegillarca granosa (chen et al., 2012). when the temperature dropped to 4 and then 0 °c, the contraction of the gill lamella became more intense, and the number of red blood cells decreased. similarly, the damage to gill cells in oreochromis niloticus increased with decreased water temperature (bin et al., 2015). n. cumingii cultured at 24 °c exhibited edema in the gill lamella. at 28 °c, the gill fragments were seriously deformed, and the distance between the gill capillary and the surrounding area was increased. these changes would have resulted in decreased gas exchange capacity and ultimately in damage to the snail. this result is similar to cheng’s (2019) research about the structural changes of gill in scallop under high temperature. the kidney is also an important excretory organ in the body of aquatic animals. its function is to remove metabolic wastes from the body and regulate osmotic pressure (shi et al., 2014). li et al., (2011) reported that the kidney of the snail pomacea canaliculata is composed of renal tubules and collecting ducts. in the current study, no obvious changes in the renal tubules and collecting ducts of the n. cumingii kidney were detected at different culture temperatures. however, the columnar cells were longest in the 16 °c group. when 9 fig. 7 go enrichment map for the kidney (left) and gills (right). the x-axis is the name of the go item, and the y-axis is -log10pvalue the temperature gradually decreased or increased, the columnar cells became shorter and more numerous. this experiment was an acute test, and the temperature decreases or increases over a short time period likely accelerated the metabolic rate of the n. cumingii body. t-aoc and activities of cat and sod t-aoc is a comprehensive index used to assess the functional status of the body's antioxidant system (de oliveira et al., 2004; tania et al., 2005; guan et al., 2010). the antioxidant enzyme system mainly consists of sod and cat, which can decompose o2– and h2o2. xie (2016) studied the antioxidant capacity of the liver of pampus argenteus under acute temperature stress and found that the t-aoc decreased, indicating that temperature stress caused a stress response in this species. in the current study, the t-aoc of the gills and kidneys of n. cumingii differed only slightly at temperatures between 4 and 24 °c. in the gill, t-aoc first increased, then decreased, and then increased again as the culture temperature increased. this pattern differed from the results of most acute temperature stress experiments, and we speculate that the metabolic rate of the snail was accelerated quickly within a short period, and excessive reactive oxygen species (ros) were produced, which intensified the oxidation reaction. as the temperature increased, t-aoc of the kidney first increased and then decreased, which is similar to the results reported by feng et al. (2012) for the chinese sturgeon acipenser sinensis. water temperature can affect the metabolic rate of fish and the physiological activities of aquatic animals (martinez et al., 2005). li et al. (2008) set culture temperatures at 12, 21, 26, and 31 °c and measured ros content and sod and cat activities in the serum of chinese sturgeons. all these parameters increased with increasing temperature, which showed that the temperature increase likely promoted the production of ros and the oxidation state of cell components, leading to reactions of related antioxidants and oxidase systems. in n. cumingii, the sod and cat activities of the gills first decreased and then increased with increasing temperature, which was similar to the activity of enzymes in the liver of gift nile tilapia (wang et al., 2012). however, as the temperature increased, the sod activity of the n. cumingii kidney showed a w-shaped pattern, which was inconsistent with most results of the effect of temperature on the antioxidant defense system of aquatic animals. we speculate that this result was due to the acute nature of this experiment. when the experimental snails were exposed to external high or low temperature stress, the oxidative stress reaction caused the body's metabolic rate to increase and excessive ros to be produced within a short period of time. too many ros likely induced increased activities of sod and cat to eliminate the excessive o2– and h2o2 in the body to maintain the balance of the snail’s antioxidant defense system. with increased temperature, the cat activity in the n. cumingii kidney increased first, then decreased and then increased again, which was similar to the pattern of sod and cat activities in the liver of gifford strains of nile tilapia juveniles (nurdiani et al., 2007). additionally, there may be a fact that when the enzyme activities were measured, the temperature was set at 37 °c according to the manufacture’s protocol, which exceeded the experimental temperature (0, 4, 8, 12, 16, 20, 24, and 28 °c), so further research was needed on whether the experimental results can truly reflect the physiological state in this condition. although the results obtained in this research were similar to the expected results, and many scholars also used kits to detect the enzyme activities of aquatic animals in their experiments (wang et al., 2012; song et al., 2015; zhan et al., 2018), the experimental results in this aspect should be used with caution. transcriptome analysis in recent years, the survival of aquatic animals has been greatly threatened by the increase of global temperature (brander, 2007), as heat stress is one of the most important environmental stress factors affecting aquatic animals (zhou et al., 2018). under stress conditions, the metabolism of aquatic animals depends greatly on environmental temperature. studies have found that changes in the metabolic rate of aquatic animals are related to the increase in environmental and body temperature 10 fig. 8 kegg enrichment map for the kidney (left) and gills (right). the x axis is the enrichment score. the larger the bubble, the more the number of different unigenes, and the color of the bubble changes from purple-blue-green-red. the smaller the enrichment p-value, the greater the degree of significance (vergauwen et al., 2010; jiang, 2013; qian et al., 2016). transcriptomics technology has provided a new way to study the effects of temperature on the immune response, growth, and development of aquatic organisms (luo et al., 2015). in this study, it was found that the gill and kidney tissues of n. cumingii were significantly different at 16 °c and 28 °c through the study of feeding behavior, histology and immunoenzyme activity of n. cumingii. therefore, sequencing was conducted at these two temperatures, the results showed that of the 4397 genes with differential expression in the gills of snails, 2339 were up-regulated and 2058 genes were down-regulated. of the 4360 differentially expressed genes in the kidney of n. cumingii, 2300 were up-regulated and 2060 were down-regulated. we speculate that high temperatures may have activated cell metabolism and responded to the damage caused by a high temperature stress environment. studies have shown that when the body is stressed, the structure and function of many enzymes and structural proteins change. for example, to protect the body from stress, it would stimulate the synthesis of heat shock proteins (hamdoun et al., 2003; jiang, 2017). the go analysis of the gills and kidneys of n. cumingii showed that the number of down-regulated genes in both the high-temperature and the normal temperature group was significantly higher than the number of up-regulated genes. there was little difference in the number of upor down-regulated genes in the cell composition and molecular function categories. this may be because when n. cumingii responds to high-temperature stress, its metabolism and energy consumption continue to increase, and the differentially expressed genes may respond to high-temperature stress through metabolic pathways. kegg annotation statistics revealed that transcriptome differences were concentrated in six categories and 42 major metabolic pathways, and the largest difference was in the signal transduction process in the environmental information process. studies have found that when the organism was under environmental stress, the function and structure of many enzymes and structural proteins will change. meantime, the organism protects itself against adversity by up regulating the synthesis of heat shock proteins. heat shock proteins played an important role in the synthesis, transport, and glycosylation of proteins (ryckaert et al., 2010; jayasundara et al., 2013). in this study, the genes involved in the protein processing in the endoplasmic reticulum pathway have the highest proportion in the gills. the up-regulated genes mainly included sec61, pdis, nef, hsp70 and hsp90. the function of sec61 is mainly to transfer the protein subunits or misfolded peptides to the cytoplasmic matrix for ubiquitination. pdis play an important role in cell differentiation and the maintenance of function and cell activity (liu, 2017). the up-regulating of hsp 70, hsp 90 and negative factor (nef) indicates the feature of heat shock proteins expression under high temperature stress. in addition to heat stress proteins, we also found the immune-related gene tnf in the kidneys. studies have shown that tnf is a multi-effect pro-inflammatory cytokine. meantime, tnf-α also participates in the regulation of cardiomyocyte apoptosis, and the signal transduction pathway it mediates had a bidirectional effect, which can not only promote cardiomyocyte apoptosis, but also inhibit it (zhou et al., 2010; naudè et al., 2011). therefore, we suggested tnf plays an important role in the immune defense of n. cumingii. the current study used high-throughput sequencing technology to sequence the n. cumingii transcriptome. through the annotation of genes, we obtained a preliminary understanding of gene functions and a list of biological processes and metabolic pathways involved in the response of this 11 snail to different culture temperatures. our results provide valuable data for functional gene cloning, genomics, disease and stress resistance research, genetic breeding, and resource restoration. conclusion in conclusion, n. cumingii was very sensitive to changes in temperature. high or low temperature could affect the tissue structure, immune enzyme activity and gene expression in gill and kidney tissues of n. cumingii. when the temperature was 8-16 °c, n. cumingii had the best state with good food intake and active exercise status. finally, we think the results obtained from this research can provide a good theoretical basis for the healthy culture and artificial reproduction of n. cumingii in the future. acknowledgments the authors wish to express thanks to the staffs of key laboratory of mariculture & stock enhancement in north china’s sea, ministry of agriculture, p.r. china for their help with the experiment. the authors are also 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shandong fisheries of china 8-10, 1995. isj008.pdf 66 isj 1: 66-71, 2004 issn 1824-307x research report biochemical and histological alterations of mytilus galloprovincialis digestive gland after exposure to okadaic acid and derivatives r auriemma, s battistella department of biology, university of trieste, trieste, italy accepted november 24, 2004 abstract electrophoretical and histological analysis were performed on mytilus galloprovincialis digestive gland samples, in order to detect the presence of a previously identified protein ca 30 kda mw, synthesized during dinophysis spp. blooms, and assess a possible correlation between the occurrence of this protein and okadaic acid (oa) exposure by ingestion. mussels were sampled monthly from july 2000 to november 2001 in the gulf of trieste (upper adriatic sea) and immediately processed. parallel samples were maintained in sea water plastic tanks and fed with marine invertebrate feed mixed with oa and derivatives at different concentration of toxins for each experimental group (25 µg, 50 µg, 100 µg). in tank reared mussels fed with oa, degeneration of digestive cells and appearance of 24.6 kda protein were observed, while in wild mussels, neither histological alterations nor presence of a 24.6 kda protein, were detected. a correlation between the toxins concentration and time of appearance was highlighted, to demonstrate this protein is synthesized in response to oa and derivatives exposure. about the identity of 24.6 kda protein, it could be an enzyme involved in detoxification reactions, probably glyoxalase i. key words: mussels; digestive gland; okadaic acid; dsp; mytilus galloprovincialis introduction the ingestion by mussels of dinophysis spp. (marine dinoflagellates) is the main consequence for mussel toxicity and it is responsible for diarrhetic shellfish poisoning (dsp), a human toxic syndrome, caused by the ingestion of contaminated shellfish. also in italy, the dsp causes public health concern and economical problems with sale blockage. the more common dsp toxins belong to the okadaic acid group: okadaic acid (oa), dinophysistoxin-1 (dtx-1), dinophysistoxin-2 (dtx-2), dinophysistoxin-3 (dtx-3), dinophysistoxin-4 (dtx-4) (yasumoto and murata, 1993). *corresponding author: dr silvia battistella department of biology, university of trieste, via giorgeri 10, 34127, trieste, italy. e-mail: battiste@univ.trieste.it in order to detect the presence of dsp toxins in mussels, italian government (health ministry decree 1/9/1990, modified on 31/7/1995) enforced the yasumoto mouse bioassay (yasumoto et al., 1984). this test provides only qualitative information on toxin presence, has a low sensitivity (limit detection 20 µg/100 gr of mussel digestive gland) and can give false positive results. in relation with this problem, others tests aimed to detect the dsp toxins were proposed in the last twenty years. monoclonal antibody elisa test detects oa and dxt-1 in toxic mussels, but it underestimates total toxin presence and its sensitivity is 10 µg/100 gr of shellfish digestive gland (uda et al., 1989; morton and tindall, 1996). hplc-fluorescence detection assay is the most universally used analytical technique for the determination of oa and dxt-1, it is more sensitive than elisa (detection limit 1 µg/100 gr) but it is an expensive test and not easy to apply (lee et al., 1987; nunez and scoging, 1997). liquid chromatography-electrospray ionization mass spectrometry (lc-esi-ms test) gives quantitative/qualitative information about dsp toxins 67 and it is more precise than hplc method but has the same applicative problems (lee et al., 1989; suzuki and yasumoto, 2000; goto et al., 2001). tissue culture assay is based on direct microscopic observation of toxin-induced morphological changes in buffalo green monkey cell cultures (croci et al., 1997). finally, the fluorescence protein phosphatase (pp2a) inhibition assay is based on knowledge that serine/threonine protein phosphatases are inhibited by dsp toxins. it detects oa and dtx-1 in mussels down to 1 µg/100 gr of digestive gland tissue (tubaro et al., 1996; mountfort et al., 2000). in the gulf of trieste (upper adriatic sea), avian et al. (1993) identified, by histological analysis, alterations in the digestive gland of the mussel mytilus galloprovincialis, during dinophysis spp. algal blooms. this histological study was later supported by a qualitative electrophoretical analysis of water–soluble proteins of digestive gland, in order to investigate a possible modification in protein pattern as a consequence of toxin presence (amirante et al., 1994; bonivento et al., 1993, 1995, 1997). in this analysis the presence of a low-molecular weight protein was detected, only in mussels during dinophysis spp. algal blooms, synthesized ex novo, which could counteract to metabolic alterations. in order to confirm this observation and to verify the relation between dsp toxins and the appearance of this protein and finally to identify its role, in controlled conditions, oa and derivatives mixed with marine invertebrate feed were given to mussels. material and methods samples in this study 470 samples of digestive gland of mytilus galloprovincialis (lmk., 1819) were analyzed. from july 2000 to november 2001, every month, 10 mussels (range: 45-65 mm length) were collected in the gulf of trieste. their digestive glands were analyzed, for both histological and biochemical studies. parallel mussels groups, were kept in sea water 25 liters plastic tanks, of which 25 individuals were fed with marine invertebrate feed as control, and 25 fed with marine invertebrate feed mixed with oa and derivatives (sigma-aldrich italy). a further assay was performed, treating 25 mussels with heavy metals. to prevent additional environmental stress in the mussels, the seasonal photoperiod and water temperature were maintained. sea water was changed weekly (unless otherwise stated) and water salinity controlled daily. the temporal relationship between the toxin ingestion and the presence of the low molecular weight protein was assessed every three days from the beginning of every administration. the digestive gland of treated and control mussels (n=5), was extracted and processed. one third of every mussel digestive glands treated with oa and derivatives was used for histological analysis and the rest for biochemical analysis. treatments to investigate possible histological and biochemical alterations due to maintenance in tanks, in the first assay mussels fed with marine invertebrate feed, were compared with wild mussels. in the second assay, mussels were fed with invertebrate feed mixed with oa, for a total amount of 25 µg administered in the time span of 30 days. to avoid the water insolubility of oa and derivatives, the chemicals were dissolved in ethanol, mixed with invertebrate feed and given to mussels after ethanol evaporation, on alternate days. feed mixed with ethanol only was administered to control mussels. in the two following assays, mussels were fed both with oa and dtx-1 (1:1). in third assay 25 µg of oa and 25 µg of dtx-1 (for a total toxin amount of 50 µg) were administered within a 30 day period. in the last one 50 µg of oa and 50 µg of dtx-1 (for a total toxin amount of 100 µg) were administered in the same time span. to investigate if the protein under study appeared also as a consequence of different toxicants, the mussels were exposed for 30 days to cu2+ (625 nm) and hg2+ (200 nm). metals were additioned as cucl2 and hgcl2 in sea water every two days changes. controls were kept in artificial uncontaminated sea water for the same period. protein analysis each sample of digestive gland (about 0.5 gr) was homogenized in the same volume of extraction buffer (tris-hcl 50 mm ph 7.5, β-mercaptoethanol 10 mm, edta 1 mm, 3 % sds, 8 % glycerol) and centrifuged at 4 °c for 20 min at 12.000 rpm. 10 µl of supernatant was added (1:1) to denaturant sample buffer (tris-hcl 1 m ph 6.8, βmercaptoethanol 10 mm, 10 % sds, 8 % glycerol, 0.1 % bromophenol-blue) and denaturated at 100 °c for 3 min. the determination of the relative molecular mass of extracted proteins was performed in 16 % sds-polyacrylamide gel according to schagger and von jagow (1987) using the low mw kit for molecular weights 14.400-94.000 and the high mw-sds calibration kit for mw 53,000-212,000 (amersham-pharmacia, germany). water soluble proteins were stained with coomassie brilliant blue g250 (sigma) staining solution. histological analysis for histological analysis, about 2 mm3 of digestive gland of each specimen, were fixed in 2 % glutaraldehyde in 0.1 m cacodylate buffer ph 7.25. the tissue samples were washed in a 0.1 m cacodylate ph 7.25 buffer three times and post-fixed for one hour in 1% osmium tetroxide in the same buffer. after another wash cycle, the samples were dehydrated in ascending ethanol and embedded, via propylene oxide, in embed812/araldite (electron microscopy sciences, fort washington, pa). for light microscopy, sections 1 µm thick were collected on slides, baked for 5 min at 75 °c, stained with 0.5 % toluidine blue in 0.1 % sodium carbonate solution at ph 11.1 at the same temperature. results and discussion in the first assay, mussels, fed with marine invertebrate feed only, did not show qualitative differences in the low mw proteins compared with 68 wild mussels in no algal bloom conditions, neither histological analysis showed significant alterations of the histology. in the second run of experimental rearing, mussels fed with 25 µg of oa, expressed a 24.6 kda protein, never present in control mussels. this protein appeared in treated m. galloprovincialis, 28 days after the beginning of the exposure and disappeared 8 days after the end of oa administration. in the third assay, the mussels, fed with 50 µg of toxins (25 µg oa and 25 µg dtx-1), expressed the 24.6 kda protein (never present in control mussels) (fig. 1), 16 days after the beginning of the administration and the protein disappeared within the same time of the previous test. during the last exposure experiment, performed with 100 µg of dsp toxins (50 µg oa and 50 µg dtx-1), the protein was detected 12 days after the beginning of the treatment, and disappeared within 8 days post exposure. in graph (fig. 2) is represented the percentage of mussels that presented the 24.6 kda protein during the three essays. it is evident the temporal relationship between toxin administration dose and the appearance of the protein. in the heavy metal assay, the 24,6 kda protein appeared neither in treated mussels nor in control ones. the parallel histological analysis on the same samples, confirmed a digestive gland alteration consequent to toxic treatment. in fact an increase of lipid vesicles both in digestive and basophilic cells was visible a week after the beginning of treatment. in some cases, the vesicles filled the cells cytoplasm with the progression of the toxin treatment (figs 3, 4). in addition partial or total degeneration of digestive cells was observed in treated mussels starting from the second week (fig. 5). however, lipid vesicles accumulation and digestive cells degeneration do not represent an anomalous condition in the physiological cycle of m. galloprovincialis digestive gland, but remarkable is its degree occurrence in treated mussels only. from the results obtained, in m. galloprovincialis it is clear the correlation between the exposure to oa and derivatives and 24.6 kda protein synthesis. in fact its presence was observed only in treated mussels. moreover the observation of a temporal relationship between the dose dependency of toxins and the advanced appearance of the protein, further confirm their relationship. these results are in agreement with previous studies on m. galloprovincialis in the gulf of trieste during dynophisis spp. algal bloom (amirante et al. 1994; bonivento et al., 1993, 1995, 1997). histological analysis also showed that the treated mussels present an initial increase of lipid vesicles, especially originated in those digestive cells that during the toxin administration degenerate, up to their lysis. the role of the 24.6 kda protein, was preliminarily supposed that it might be involved in detoxification reactions or that it could be an enzyme, accumulated for indirect causes consequent oa poisoning. the principal toxic effect of oa is due to its inhibiting effects on protein phosphatase 1 (pp1) and 2a (pp2a) in eukaryotic cells, causing changes on protein phosphorylation state of enzymes (bialojan and takai, 1988). fig. 1 sds-page electrophoretic pattern of exposed mussels (25 µg of oa and 25 µg of dtx-1) that present 24,6 kda protein (line2), of untreated sea water maintained control (lane 3 and 4), hmw and lmw markers (lane 1 and 5). oa toxic effect allows to suggest that 24.6 kda protein could be an enzyme correlated with glycogen synthase (gs) activity, because the inhibition of gs activity by the action of oa on pp1 and pp-2a, is demonstrated. (haystead et al., 1989; pugazhenthi et al., 1993). this hypothesis was rejected because following studies on mytilus edulis (svensson and fõrlin, 1998) demonstrated that gs activity is not affected by oa levels that naturally occur in blue mussels. on the basis of these results, the authors suggested that there may be a protective mechanism against harmful effects of oa in the blue mussels. on this point it is to note that digestive cells of mollusc digestive gland are particularly rich in lysosomes (owen, 1974). these can accumulate and sequester foreign compounds such as lipophilic xenobiotics by increased autophagic activity. such date prompted the hypothesis that 24.6 kda protein could be a detoxificant enzyme. most common detoxificant enzymes in eukaryotic cells are the family group of cytochrome p-450, that mainly transform liposoluble toxins in hydrosoluble compounds easily eliminated. studies performed on m. galloprovincialis by peters et al. (1998) demonstrated the presence of five different types of cytochrome p-450 (cyp1a, 2b, 2e, 3a, 4a), about 50 kda each, all involved in detoxificant reactions both of organic pollutants and heavy metals contamination. the identification of 24.6 kda protein with one of cytochrome p-450 was excluded, since it is half the weight of cytochrome monomers, detected in m. galloprovincialis. another system of detoxificant enzymes is glyoxalase group (glyoxalase i, gi, and glyoxalase ii, gii), glutathione-dependent. glutathione (gsh) is 69 fig. 2 linear graph of percentage of mussels that present the 24.6 kda protein in each assay. yellow line: assay at 25 µg toxins, red line: assay at 50 µg toxins, blue line: assay at 100 µg toxins. fig. 3 cross section of digestive tubules of an untreated sample. some digestive cells, that release fragmentation spherules, are evident (arrow). a ubiquitarian tripeptide and it is a cofactor of many enzymes catalyzing the detoxification and excretion of several toxic compounds; in particular it is a coenzyme of gi. the gi activity is demonstrated also in m. galloprovincialis (fitzpatrick et al., 1995). in addition regoli et al. (1996) performed a first purification and characterization of glyoxalase i from the digestive gland of the same species. from this study it results that the pure enzyme is a 48 kda protein with an heterodimeric quaternary structure and in denaturant conditions (sds-page) it is composed of 24 and 25 kda subunits. fig. 4 sample about one week after the beginning of treatment with 25 µg toxins. oblique section of a digestive tube. lipid vesicles (in greenish blue) inside digestive cells are evident (arrow). taking into account that: gi is an enzyme involved in several detoxification reactions, and 24.6 kda protein appears after toxin (oa, dtx-1) treatment; gi consists of 2 subunits of mw very close and similar to the protein detected in the present study, and finally the staining used to evidence low mw proteins in the present work (blue comassie staining), does not allow to discriminate two subunits running so close in sds-page, we could suppose that 24.6 kda protein is the gi. further analysis will be necessary to confirm whether it is actually the gi. if this hypothesis will be 0 20 40 60 80 100 120 0 10 20 30 40 50 60 time in days p e rc e n ta g e o f s a m p le s t h a t p re s e n t th e 2 4 .6 k d a p ro te in 70 fig. 5 sample 20 days after the beginning of treatment with 50µg of toxins (25 µg of oa and 25 µg of dtx-1). digestive cells are pyknotic and/or lytic (arrows). tested and proved, it will be the first connection between this enzyme and the oa contamination. this could be a result of particular interest, because the detoxificant mechanism against oa and derivatives in m. galloprovincialis results still unresolved (blanco et al., 2002). besides, it will be interesting to evaluate if the 24.6 kda protein synthesis is induced also by others dsp toxins, like yessotoxins and pectenotoxins. indeed, were this induction demonstrated, this protein may represent a useful biomarker in biomonitoring studies, as an indirect index of dsp risk for all dsp toxins 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gdańsk, ul. wita stwosza 63, 80-308 gdańsk, poland 2 witold stefański institute of parasitology of the polish academy of sciences, twarda 51/55, 00-818 warszawa, poland 3 laboratory of chemical environmental risks, department of environmental analysis, faculty of chemistry, university of gdańsk, gdańsk, poland accepted august 18, 2017 abstract the headspace solid phase microextraction gas chromatography-mass spectrometry (hs-spmegc/ms) method was used for the determination of the volatile compounds of dermestes maculatus and d. ater pupae. these beetles are of economic importance and they are a common pest of stored products and also serve as an intermediate host of the parasitic tapeworm, therefore an understanding of their biology is very important. analyses of the volatile compounds of d. maculatus and d. ater pupae revealed differences between the insect species in their chemical composition. sixteen volatile compounds of d. ater pupae, including 6 hydrocarbons, 5 fatty acids, 3 esters, and 2 aldehydes were identified. the major volatile compound in d. ater pupae was pentacosane. ten compounds were present in < 10 % concentrations. a further five were present in < 1 % concentrations. a total of 39 compounds were identified in the d. maculatus pupae, including 28 esters of fatty acid, 4 fatty acids, 6 hydrocarbons and 1 aldehyde. two volatile compounds were detected as major compounds: octadecadienoic acid methyl ester and octadecenoic acid methyl ester. a further eight were identified in smaller quantities (from 1.12 to 8.30 %) and the remaining volatile compounds were present in < 1 % concentrations. key words: dermestes maculatus; dermestes ater; headspace solid-phase microextraction; gc-ms introduction insects are of growing significance in agriculture, veterinary medicine, medicine and human healthcare. the black larder beetle, d. ater and the hide beetle, dermestes maculatus, belong to the dermestidae (coleoptera) family. they are a common pests of stored products. d. ater feeds on various plant and animal products such as raw skin and hide, stored meat, cheese, tobacco, dried fish, copra, silk, wool, milk and dried museum specimens (bujang and kaufman, 2010; siddaiah and kujur, 2016). these beetles also serve as an intermediate host of raillietina laticanalis and choanotaenia ___________________________________________________________________________ corresponding author: marek gołębiowski laboratory of analysis of natural compounds department of environmental analysis faculty of chemistry university of gdańsk ul. wita stwosza 63, 80-308 gdańsk, poland e-mail: marek.golebiowski@ug.edu.pl infundibulum in poultry (bujang and kaufman, 2010). the hide beetle, d. maculatus is a pest of the silk industry and it can damage stored animal products such as dried fish, cheese, hide, fur, bacon, and dog treats (rajendran and hajira parveen, 2005; shaver and kaufman, 2009; fontenot et al., 2015). d. maculatus damages the wood and insulation of poultry houses (cloud and collison, 1985), but they are commonly used to clean carcasses as a part of the skeletonization process (museumpest.net, 2012) and the adult beetles are used to estimate the post mortem interval (shaver and kaufman, 2009; de souza and linhares, 1997). the volatile compounds of insects can be analyzed using many analytical techniques. for example, aggregation pheromones of the black larder beetle dermestes haemorrhoidalis kuster were analyzed by headspace gas chromatographymass spectrometry (hs-gc/ms) and gaschromatography with electroantennographic detection (gc-ead) (korada and griepink, 2009). 304 organic compounds of the egg surface of queen diploid and haploid eggs and worker haploid eggs of the honeybee were extracted in dichloromethane and analyzed by gas chromatography-mass spectrometry (katzav-gozansky et al., 2003). the main components of the external lipids of adult aleyrodes singularis were extracted in chloroform and analyzed by gas chromatography-mass spectrometry (soroker et al., 2003). cuticular hydrocarbons in the ant cardiocondyla wroughtonii were analyzed by solid injection and gc-ms (turillazzi et al., 2002). the cuticular and internal nalkane composition of lucilia sericata larvae, pupae, male and female were separated by highperformance liquid chromatography with laser light scattering detector (hplc-llsd) and analyzed by gas chromatography-mass spectrometry with selected-ion monitoring (gc/ms-sim) (gołębiowski et al., 2012a). thanks to the use of a number of analytical techniques, it is possible to identify many of the volatile compounds in insects (cerkowniak et al., 2013). for example, hydrocarbons were identified in heliothis virescens pupae, helicoverpa zea pupae (buckner et al., 1996), aleyrodes singularis adult and exuviae (nelson et al., 1998), lucilia sericata larvae, pupae, male and female (gołębiowski et al., 2012a), chrysomya rufifacies larvae (zhu et al., 2006), calliphora vomitoria, calliphora vicina and protophormia terraenovae (roux et al., 2008), musca domestica (butler et al., 2009), and sitophilus granaries (nawrot et al., 2010). fatty acids were present in heliothis virescens pupae, helicoverpa zea pupae (buckner et al., 1996), eurosta solidaginis larvae (nelson and lee, 2004), melanoplus sanguinipes adult, melanoplus packardii adult (soliday et al., 1974) and diapause and non-diapause larvae of cydia pomonella (khani et al., 2007). aldehydes and alcohols were found in heliothis virescens pupae, helicoverpa zea pupae (buckner et al., 1996), aleyrodes singularis adult and exuviae (nelson et al., 1998), chorthippus brunneus males and females (gołębiowski et al., 2016), musca domestica larvae, pupae, male and female (gołębiowski et al., 2012b), scaptotrigona postica workers (poiani et al., 2015) and the nymphs and exuviae of bemisia argentifolii (buckner et al., 1999). esters were identified in calliphora vomitoria larvae, pupae, male and female (gołębiowski et al., 2013) and acanthoscelides obtectus (gołębiowski et al., 2008). organic compounds in the cuticular lipids serve various functions in insects. they are the primary energy source, and a structural component of membranes. they are often involved in chemical communication such as pheromones, and in defense as components of defensive secretions, and as antimicrobial agents (cakmak et al., 2007; khani et al., 2007; martins and ramalho-ortigão, 2012; mann et al., 2013; ottaviani, 2014; cerkowniak et al., 2015; kühbandner and ruther, 2015; nguyen et al., 2015). however, volatile compounds mainly serve as pheromones. kairomones play a very important role in insect life. kairomones are often used for host location, recognition and acceptance over shorter distances (vet and dicke, 1992; vinson, 1998). these compounds are usually identified in host eggs, larvae, pupal cuticle, frass, silk, cocoons and glandular secretions (afsheen et al., 2008). the role of hostrelated kairomones was investigated for example in the case of hyphantria cunea (drury), a host of the parasitoid chouioia cunea yang (zhu, 2016). the study demonstrated that c. cunea is attracted to volatile kairomones from h. cunea. moreover, it has been shown a significant positive response of mated female c. cunea to 1-dodecene. furthermore, the volatile compounds secreted by insects can be used to detect insects in stored grains. the vocs were identified e.g. in tribolium castaneum (red flour beetle) and cryptolestes ferrugineus (rusty grain beetle) by headspace analysis (senthilkumar et al., 2012). it was found that the amount of volatiles produced by t. castaneum adults increased with an increase in insect density in stored wheat. in our study, hs-spme-gc/ms was used for the determination of the volatile compounds of d. maculatus and d. ater. materials and methods rearing of d. ater and d. maculatus both dermestes species were kept at 25 o c with cyclic changes of light (l:d 12:12) in separate glass aquaria with a layer of wood shavings spread on the bottom and covered with mesh cloth to prevent insects escape. the beetles and larvae were fed beef meat ad libitum. fully grown final instar larvae which ceased feeding were regularly isolated from the basic colonies and kept in glass jars until pupation in order to avoid slaughtering of larvae immobilized before pupation and naked pupae by younger larvae. headspace solid-phase microextraction polydimethylsiloxane/divinylbenzene (pdms/dvb) fiber was used for the analysis of the volatile compounds extracted from d. maculatus and d. ater pupae insects belonging to the dermestidae family. for experiments,1-day-old pupae were used. five insects of each species of dermestidae (0.19 g d. maculatus and 0.17 g d. ater) were used for the analysis. the insects were placed in 4 ml glass vials prior to extraction. next was added 4.8 µl of the undecane as standard and the capsule was capped. the samples were heated in a heating block. the best conditions for the hs-spme-gc/ms were taken from literature (cerkowniak et al., 2017). the extraction time was 50 min, the extraction temperature -105 °c, desorption time 10 min at 230 °c. analyzes were repeated 3 times. gas chromatography/mass spectrometry the volatile compounds of d. maculatus and d. ater pupae were analyzed on a gc/ms qp-2010 se (shimadzu) equipped with a fused silica rtx-5 capillary column, 30 m x 0.25 mm i.d., and with a 0.25 µm thick film. the oven temperature of 80 °c was increased to 190 °c at a rate of 4 °c/min, isotherm at 190 °c for 10 min, then the temperature was increased from 190 °c to 300 °c at a rate of 6 °c/min. helium was used as the carrier gas at a 305 column head pressure of 60 kpa and electron impact was applied to the ionization (70 ev). the transfer line and injector temperatures were maintained at 300 and 230 °c, respectively. the ion source was kept at 200 °c. the analysis of volatile compounds was carried out by gc/ms in the total ion current (tic) mode. results seventeen and thirty-nine volatile compounds were identified in the pupae of d. ater and d. maculatus, respectively. figs 1 6 give examples of the mass spectra of esters of fatty acid, fatty acids, aldehydes and hydrocarbons. fig. 1 mass spectrum of octadecanoic acid, methyl ester. fig. 2 mass spectrum of octadecenoic acid, ethyl ester. fig. 3 mass spectrum of octadecanoic acid, butyl ester. 306 fig. 4 mass spectrum of hexadecanoic acid. fig. 5 mass spectrum of hexadecanal. fig. 6 mass spectrum of pentacosane. 307 table 1 chemical composition of the volatile compounds found in pupae of d. ater d. ater no relative content [%] compounds 1. 0.50 decanoic acid 2. 3.58 dodecanoic acid 3. 3.72 tetradecanoic acid 4. 2.24 hexadecanal 5. 0.94 tetradecanoic acid, 1-methylethyl ester 6. 8.45 hexadecanoic acid 7. 0.66 octadecanal 8. 0.62 octadecenoic acid 9. 0.41 hexadecanoic acid, butyl ester 10. 9.93 tricosane 11. 1.71 octadecanoic acid, butyl ester 12. 3.17 tetracosane 13. 1.34 pentacosene 14. 54.28 pentacosane 15. 1.50 hexacosane 16. 6.95 heptacosane chemical composition of the volatile compounds found in the pupae of d. ater table 1 lists the volatile compounds identified in d. ater pupae. among sixteen volatile compounds of d. ater pupae, 6 hydrocarbons, 5 fatty acids, 3 esters, and 2 aldehydes were present. the volatile compounds of d. ater pupae contained only 2 unsaturated compounds, among which were acid and hydrocarbon. the remaining compounds were saturated. the major volatile compound in d. ater pupae was pentacosane (54.28 %). the compounds occurring in smaller quantities (from 1.34 to 9.93 %) were: dodecanoic acid (3.58 %), tetradecanoic acid (3.72 %), hexadecanal (2.24 %), hexadecanoic acid (8.45%), tricosane (9.93%), octadecanoic acid butyl ester (1.71 %), tetracosane (3.17 %), pentacosanol (1.34 %), hexacosane (1.50 %) and heptacosane (6.95%). a further six were present in concentrations of <1%: decanoic acid, tetradecanoic acid 1-methylethyl ester, octadecanal, octadecenoic acid, hexadecanoic acid butyl ester and octadecenoic acid. chemical composition of the volatile compounds found in the pupae of d. maculatus table 2 lists the volatile compounds identified in d. maculatus pupae. thirty-nine volatile compounds were identified in d. maculatus pupae. among them, 28 esters, 4 fatty acids, 6 hydrocarbons and 1 aldehyde were present. the volatile compounds of d. maculatus pupae contained 14 unsaturated compounds, among which were 13 acids and only one hydrocarbon. among the esters, 25 methyl, 2 ethyl, and 1 butyl ester were present. two volatile compounds were detected as major compounds: octadecadienoic acid methyl ester (35.32 %), and octadecenoic acid methyl ester (26.74 %). a further eight were identified in smaller quantities (from 1.12 to 8.30 %): tetradecanoic acid methyl ester (2.80 %), tetradecanoic acid (1.12 %), hexadecenoic acid methyl ester (8.30 %), hexadecanoic acid methyl ester (7.67 %), hexadecanoic acid (2.26 %), octadecatrienoic acid methyl ester (5.80 %), eicosatetraenoic acid methyl ester (1.48 %) and pentacosane (2.55 %). the remaining volatile compounds were present in <1% concentrations. comparison of the volatile compounds found in the pupae of d. ater and d. maculatus analyses of the volatile constituents of d. maculatus and d. ater pupae revealed differences between the insect species in chemical composition. the following compounds present in d. ater larvae lipids were absent in d. maculatus larvae: decanoic acid, tetradecanoic acid 1-methylethyl ester, octadecanal, octadecenoic acid and hexadecanoic acid butyl ester. on the other hand, twenty eight compounds that were present in d. maculatus were absent in the lipids of the larvae of d. ater (tables 1 and 2). only eleven compounds were present in both insect species: dodecanoic acid, tetradecanoic acid, hexadecanal, hexadecanoic acid, tricosane, octadecanoic acid butyl ester, tetracosane, pentacosene, pentacosane, hexacosane, and heptacosane (tables 1 and 2). twenty five methyl esters were identified in the d. maculatus pupae, while only one ester was found in the d. ater pupae. the lipids of d. maculatus contained also four fatty acids, six hydrocarbons, two ethyl esters, one butyl ester and one aldehyde, while the lipids of d. ater contained also five fatty acids, six hydrocarbons, two butyl esters, two aldehydes and one ethyl ester. the major compounds in d. ater pupae were pentacosane (54.3 %), tricosane (9.9 %) and hexadecanoic acid (8.5 %), while the major compounds in d. maculatus pupae were octadecadienoic acid methyl ester (35.5 %), octadecenoic acid methyl ester (26.7 %), hexadecenoic acid methyl ester (8.3 %) and hexadecanoic acid methyl ester (7.8 %). 303 table 2 chemical composition of the volatile compounds found in pupae of d. maculatus d. maculatus no relative content [%] compounds 1. 0.02 decanoic acid, methyl ester 2. 0.02 nonanoic acid, 9-oxo-, methyl ester 3. 0.26 dodecanoic acid, methyl ester 4. 0.08 dodecanoic acid 5. 0.04 tridecanoic acid, methyl ester 6. 0.02 tridecanoic acid, 12-methyl-, methyl ester 7. 0.24 tetradecenoic acid, methyl ester 8. 2.80 tetradecanoic acid, methyl ester 9. 1.12 tetradecanoic acid 10. 0.08 pentadecanoic acid, methyl ester 11. 0.08 tetradecanoic acid, 12-methyl-, methyl ester 12. 0.05 hexadecanal 13. 0.22 pentadecanoic acid, methyl ester 14. 0.75 hexadecadienoic acid, methyl ester 15. 8.30 hexadecenoic acid, methyl ester, 16. 7.67 hexadecanoic acid, methyl ester 17. 0.23 hexadecenoic acid 18. 2.26 hexadecanoic acid 19. 0.24 hexadecanoic acid, 14-methyl-, methyl ester 20. 0.57 heptadecenoic acid, methyl ester 21. 0.07 heptadecanoic acid, methyl ester 22. 5.80 octadecatrienoic acid, methyl ester 23. 35.32 octadecadienoic acid, methyl ester 24. 26.74 octadecenoic acid, methyl ester 25. 0.72 octadecanoic acid, methyl ester 26. 0.23 octadecadienoic acid, ethyl ester 27. 0.17 octadecenoic acid, ethyl ester 28. 1.48 eicosatetraenoic acid, methyl ester 29. 0.70 eicosatrienoic acid, methyl ester 30. 0.10 eicosadienoic acid, methyl ester 31. 0.04 tricosane 32. 0.11 eicosenoic acid, methyl ester 33. 0.01 eicosanoic acid, methyl ester 34. 0.17 octadecanoic acid, butyl ester 35. 0.11 tetracosane 36. 0.17 pentacosene 37. 2.55 pentacosane 38. 0.09 hexacosane 39. 0.37 heptacosane discussion chemical analyses of the volatile compounds of d. maculatus and d. ater pupae showed us that the esters of fatty acid are major compounds. the volatile compounds of d. maculatus and d. ater pupae contained 28 and 3 esters, respectively. esters were identified in some other insect species. the cuticular lipids of calliphora vomitoria larvae, pupae, males and females contained 6, 7, 5 and 7 fatty acid methyl esters (fames) from c15:0 to c19:0, respectively (gołębiowski et al., 2013). the isopropyl esters, including isopropyl dodecanoate, isopropyl (z)-9-tetradecenoate, isopropyl tetradecanoate, isopropyl (z)-9-hexadecenoate and isopropyl hexadecanoate were detected in male abdominal extracts of the black larder beetle, dermestes haemorrhoidalis, which belongs to the dermestidae (coleoptera) family (korada and griepink, 2009). the cuticular extract of female diaphorina citri contained three esters: ethyl undecanoate, isopropyl tetradecanoate and 1methylpropyl dodecanoate (mann et al., 2013). fatty acids are present in many insect species. for example, in the larvae of the wheat blossom midge, sitodiplosis mosellana, 10 16 kinds of fatty acids, of which the predominant compounds were palmitic, oleic and linoleic acids, which were more than 95 % of the total fatty acids (jun-xiang et al., 2001), can be observed. the cuticular extract of 309 female diaphorina citri contained four acids: acetic acid, hexanoic acid, decanoic acid and pentadecanoic acid. the cuticular extract of male d. citri contained only two acids: dodecanoic acid and tetradecanoic acid (mann et al., 2013). in the surface lipids of pupae of c. vomitoria were 23 carboxylic acids from c8:0 to c24:0. the major compounds were: c18:1 (37.2 %), c16:1 (27.3 %) and c16:0 (23 %). interestingly, pupae and larvae of this species showed complete resistance to c. coronatus infection (gołębiowski et al., 2013). in our work, we stated that the cuticular lipids of d. ater pupae contained five fatty acids from c10 to c18, and those of d. maculatus pupae contained four fatty acids from c12 to c16. in our study, two saturated aldehydes: hexadecanal and octadecanal, were identified in d. ater and only one aldehyde (hexadecanal) was present in d. maculatus. aldehydes were identified in some other insect species. for example, the unsaturated aldehydes: (z)-7-tetradecenal and (e)11-hexadecenal, are major sex pheromone components of prays oleae (lepidoptera: yponomeutidae) and palpita unionalis (lepidoptera: pyralidae) (milonas et al., 2009). the saturated aldehydes: hexadecanal, (z)-9-hexadecenal and (z)-11-hexadecenal, were found in the gland extracts of helicoverpa armigera (wu et al., 1997). aldehydes are also present in plants. for example, nonanal, decanal and (e)-2-hexenal are released from maize (zea mays). gc-ead analyses revealed a response from the asian corn borer, ostrinia furnacalis male and female to (e)-2-hexenal and nonanal (huang et al., 2009). the following hydrocarbons from c23 to c27 were identified in d. ater and d. maculatus: tricosane, tetracosane, pentacosene, pentacosane, hexacosane and heptacosane, with the marked dominance of odd numbers of carbon atoms. cuticular hydrocarbons are involved in various chemical communications in insects (zhi-bin et al., 2000). they are typically found in many insect species. for example, the following homologous series were identified in the cuticular lipids of the western flower thrips, frankliniella occidentalis: nalkanes from c25 to c29, 3-methylalkanes with 26 and 28 carbon atoms, and branched monomethyl alkanes with 26, 28 and 30 carbon atoms (gołębiowski et al., 2007). saturated hydrocarbons with a carbon chain length of 21 31 and six kinds of alkenes were identified in chrysomya rufifacies larvae (zhu et al., 2006). the extracts of elytra of the root weevil, diaprepes abbreviatus contained four groups of homologous compounds: a group of normal hydrocarbons, a group of monomethylbranched alkanes, a group of dimethyl-branched alkanes and alkenes (lapointe et al., 2004). composition of volatile organic compounds was investigated in larvae and pupae of blowfly (frederickx, 2012). using the spme volatile collection and gc-ms analysis, approximately 90 compounds were identified in the three stages of larval development and the 10 stages of development of the pupae of calliphora vicina. it helped to evaluate the age of flies, to establish a postmortem interval (pmi) in medical investigations. especially because the composition of the volatile compounds of pupae and the compounds secreted by the larvae were significantly different the compounds secreted by larvae and the pupae were analyzed by an ascending hierarchical clustering (ahc). pupae volatile profiles allow three groups to be distinguished: pupae of 1 3 days old; pupae of 4 7 days old and pupae of 8 10 days old. in the older pupae, from 1 to 3 days old, were identified: methyldisulphanylmethane and 4,7,7 trimethylbicyclo[3.1.1]hept-3-ene (alpha-pinene). the pupae from 4 to 7 days old were grouped in cluster because they emitted 3methylbutanal, ethanol, methyldisulphanylmethane (dimethyldisulphide), methylsulphanyldisulphanylmethane (dimethyltrisulphide) and alpha-pinene. in vocs of c. vicina pupae of 4 and 5 days old found 3 methylbutan-1-ol as characteristic compound. 6-day and 7-day-old pupae were grouped thanks acetic acid. in the last cluster were 8to 10-day-old pupae. in this pupae as characteristic were identified: 2methylpropan-1-ol, 1-methoxy-3-methylbutane, ethyl 3-methylbutanoate, 3-methylbutyl acetate and methyldisulphanylmethane (frederickx, 2012). the composition of hydrocarbons in insect species is also used as an indicator of the postmortem interval. for example, n-alkanes, methyl-branched alkanes, and dimethyl-branched alkanes in chrysomya megacephala were identified (zhu, 2007). for most of the hydrocarbons with molecular weight below n-c26 the abundance decreased significantly with the weathering time, what can be useful in determine the pmi. conclusions the hs-spme-gc/ms method is simple and successful for the determination of the volatile compounds of d. maculatus and d. ater pupae. this technique allows the positive identification and quantitation of fatty acids, hydrocarbons, esters of fatty acids, and aldehydes. using hs-spmegc/ms, sixteen volatile compounds of d. ater pupae, including 6 hydrocarbons, 5 fatty acids, 3 esters, and 2 aldehydes were identified, while 39 compounds were identified in the pupae of d. maculatus, including 28 esters of fatty acid, 4 fatty acids, 6 hydrocarbons and 1 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juveniles of an invasive snail after challenged with lipopolysaccharide y-m xiong 1 , z-h yan 1 , j-e zhang 2,3,4 , h-y li 1 1 department of aquiculture, college of animal science, south china agricultural university, guangzhou, china 2 institute of tropical and subtropical ecology, south china agricultural university, guangzhou, china 3 key laboratory of agro-environment in the tropics, ministry of agriculture p. r. china, guangzhou, china 4 guangdong provincial engineering technology research center of modern eco-agriculture and circular agriculture, guangzhou, china accepted august 17, 2017 abstract invasion by pomacea canaliculata is an increasing problem worldwide. its rapid invasion and spread depends on the immune mechanism of efficient treatment of unfavorable factors and pathogenic microorganisms in the environment. transcriptome technology is widely applied in studying the interaction of molluscs and pathogenic microorganisms and the development of disease. in this study, transcriptome technology is used to detect the expression of immune-related genes after the p. canaliculata juvenile snails were challenged with lipopolysaccharide (lps) for 48 h. a total of 402 immune-related differentially expressed genes(degs) were identified in comparison with the control. these degs involved in antigen recognition gene, immune stress and detoxification. the results showed that genes associated with detoxification metabolism were significantly up-regulated, such as cytochrome c oxidase, cytochrome b oxidase, nadh dehydrogenase, sterol 17-alpha-hydroxylase and cytochrome p450. these data will provide fundamental information for subsequent studies of the immune defense mechanisms against pathogens in the juvenile snail of p. canaliculata. key words: immune response; apple snail; transcriptome; innate immunity; molluscs introduction pomacea canaliculata is the only freshwater snail listed among the 100 worst invasive species worldwide (yang et al., 2017). p. canaliculata originally distributed in south america, which now has invaded in north america, asia and europe (accorsi et al., 2017). its large size and taste for aquatic plants makes it a threat for causing ecological and economical damage in wetlands and crops fields(youens and burks, 2008; hayes et al., 2012). in addition to the impact on crops, p. canaliculata also is the intermediate host of the nematode angiostrongylus cantonensis which responsible for potentially lethal encephalitis to human being (lv et al., 2009; song et al., 2016). at present there are no accepted and efficient strategies for controlling the spread of p. canaliculata. p. canaliculata lack clear evidence of adaptive ___________________________________________________________________________ corresponding author: hai-yun li department of aquiculture college of animal science south china agricultural university guangzhou, china e-mail: hyli@scau.edu.cn immunity and yet thrive in freshwater, where is rich in pathogenic bacteria, this means the p. canaliculata should have a perfect and effective immune system to disposal pathogens. in the past, research on bacterial challenge mainly focused on adult molluscs (liu et al., 2017). moreover, pathogens infection can also have serious consequences on mollusc’s survival, especially during the juvenile stages (genard et al., 2013). for instance in oysters, severe juvenile mortality was induced when they were treated with a vibrio strain at the concentrations from 10 4 to 10 8 cfu individual (lacoste et al., 2001); while another vibrio strain, named tnemf6, was also demonstrated to be responsible for the unexplained mortalities of juvenile oyster c. gigas living in bay of morlaix (le et al., 2002). although some progresses have been made in the study of the damage caused by pathogens on mollusc juvenile, the mechanism of pathogens that cause great harm to mollusc juvenile remains need further study. in recent years, using transcriptome sequencing can facilitate understanding the immune responsemechanisms of aquatic animals during pathogen stimulation (dheilly et al., 2014), the technology has been used in pelodiscus sinensis (xu et al., 2016), biomphalaria glabrata (zhang et al., 2016) mailto:hyli@scau.edu.cn 295 fig. 1 the length distribution of unigenes. the x axis represents the length of unigene. the y axis represents the number of unigene. and crassostrea gigas (liu et al., 2017). the purpose of the study is to reveal the immune-related degs expression profiles of p. canaliculata juvenile snails between lps challenged group and control, which can help us understand the immune defense mechanisms of p. canaliculata juvenile snails. materials and methods the fertilized eggs incubation and juvenile snail collection eggs were cultured in an incubator at 25 ℃ (sun et al., 2010). the hatching snails were collected to 100 mm petri dish and washed with sterile water three times. lps purchased from sigma (l2630-25mg). experimental snails were bred in petri dish with 10 mg/ml lps (lps dissolve in sterile water), control snails cultured use sterile water. after 48 h cultured, juvenile snails were collected in the frozen tube and placed in liquid nitrogen rapidly. finally, the samples were stored at -80 ℃ refrigerator for subsequent experiments. transcriptome sequencing and de novo assembly total rna was extracted from juvenile snails in each group according to instruction of the manufacturer (trizol reagent, thermofisher). obtained samples of total rna and use dnase i to digest dna, after extracting total rna and treated with dnase i, oligo(dt) are used to isolate mrna. the mrna will fragment and then as templates to synthesize cdna. the synthesized cdna was subjected to end-repair and single nucleotide a (adenine) addition. after that, short fragments are connected with adapters. during the quality control steps, agilent 2100 bioanaylzer and abi steponeplus real-time pcr system are used in quantification and qualification of the sample library. then the library is sequenced using illumina hiseq 4000 platform.the cutadapt used to define raw reads for which containing low-quality, adaptor-polluted and high content of an unknown base reads additionally. clean reads of each sample were assembled use trinity software (haas et al., 2013) and with tgicl to cluster transcripts to unigenes. the clustered unigenes were aligned to silva databases to exclude those matching rrnas of the score above 60. unigenes functional annotation the unigenes were blast (altschul et al., 1990) to nt, nr, cog, kegg and swissprot to get the annotation function, use blast2go (conesa et al., 2005) with nr annotation to get the go annotation, and use interproscan5 (quevillon et al., 2005) to get the interpro annotation. software parameters settings are based on references. identification degs and analysis unigene expression analysis was clean reads mapped to unigenes using bowtie2 (langmead and salzberg, 2012). gene expression level was calculated with rsem according to former study (li and dewey, 2011). degs detected with possiondis according as audic s, et al. (audic and claverie, 1997). 296 295 table 1 summary of functional annotation results of unigene database numbers percentage annotated in nr 10788 30.86% annotated in nt 11729 33.55% annotated in swissprot 11830 33.84% annotated in kegg 23977 68.58% annotated in cog 12251 35.04% annotated in interpro 12544 35.88% annotated in go 6925 19.81% annotated in all databases 2561 7.32% total sequences number 34963 100% results and discussion rna-seq de novo assembly next-generation sequencing technology was used in the study to detect immune-related genes expression pattern of lps challenge, and generated about 17.87 gb bases in total after illumina hiseq4000 sequencing (sra accession number srx3029507). then assemble all samples together. the average length is 1,080 bp and n50 is 2,231 bp. these results demonstrated that the sequencing quality was quite fine and suitable for subsequent transcriptome analysis. length distribution of all unigenes is shown in figure 1. finally, rna-seq data had been submitted to the sequence read archive (sra) database of ncbi with the accession number for srx3029507. unigene functional annotation the unigenes were aligned with sequences recorded in the major functional databases (nr, nt, swissprot, cog, kegg, go and interpro). a total of 34,963 unigenes were been annotated and 2,561 (7.3 %) unigenes were annotated in all databases (table 1). in this study, the species distribution was statistically analyzed for unigenes which annotated in nr database (fig. 2), the highest proportion of the best blastx hit was found for aplysia californica (33.32 %), lottia gigantean (22.69 %) and crassostrea gigas (12.27 %). the result means more than 68.28 % of the unigenes best matched to molluscan species, despite the limited number of molluscan sequences in the public databases. using go and kegg database to analyze the functional classification of annotated sequences. unigenes were assigned to go database to determine their functional classifications, a total of 6,925 (19.81 %) unigenes were classified into three categories of molecular function, cellular component and biological process, comprised of 19, 19 and 25 subcategories (fig. 3). according to the results of the go analysis, immune-related genes involved in the response to the lps challenge belong to the go terms of “immune system process” and “response to stimulus” as well, and the result revealed 133 and 952 unigenes were belonged to the subcategory “immune system process” and “response to stimulus” respectively. fig. 2 species distribution of best matched in nr database 297 296 fig. 3 functional distribution of go annotation. x axis represents the number of unigenes. y axis represents the go functional category. to classify the biological pathways in the juvenile snail of p. canaliculata, the unigenes were mapped to the kegg database, which is a bioinformatics database for systematic analysis (kanehisa and goto, 2000). a total of 23,977 (68.58 %) unigenes were annotated in kegg and grouped into 32 level 2 terms (fig. 4). among the 32 predicted pathways, immune-related unigenes were grouped into the immune system cluster and divided into 11 immune-related pathways, including platelet activation, leukocyte transendothelial migration, hematopoietic cell lineage, chemokine signaling pathway, complement and coagulation cascades, nod-like receptor signaling pathway, toll-like receptor signaling pathway, cytosolic dna-sensing pathway, rig-i-like receptor signaling pathway, tnf signaling pathway and nf-kappa-b signaling pathway. immune-related degs analysis in order to obtain further details on the immune response of p. canaliculata challenged with lps, immune-related degs were analyzed according to go and kegg enrichment result. in the study, a total of 402 immune-related degs were obtained between the experimental group and the control group, including 189 genes up-regulated and 213 genes down. 54 immune-related degs were enriched in different functional classes of go, such as the immune system 298 295 fig. 4 functional distribution of kegg annotation. x axis represents the number of unigenes. y axis represents the kegg functional category. table 2 list of immune-related degs enrichment in kegg pathways item pathway id up-regulated down-regulated hematopoietic cell lineage ko04640 48 67 complement and coagulation cascades ko04610 27 33 platelet activation ko04611 111 73 leukocyte transendothelial migration ko04670 65 103 nf-kappa b signaling pathway ko04064 12 46 toll-like receptor signaling pathway ko04620 31 6 nod-like receptor signaling pathway ko04621 12 20 tnf signaling pathway ko04668 42 14 rig-i-like receptor signaling pathway ko04622 3 14 cytosolic dna-sensing pathway ko04623 10 5 chemokine signaling pathway ko04062 19 20 299 296 table 3 representative of significantly up-regulated immune related degs after challenge with lps contig description fold change p-value cl4201.contig1_all cytochrome c oxidase subunit ii 14.24 2.54e-204 unigene8090_all cytochrome b 12.23 1.36e-58 unigene48594_all nadh dehydrogenase subunit 4 10.90 6.46e-10 cl5717.contig1_all steroid 17-alpha-hydroxylase 10.45 1.29e-84 cl5310.contig6_all cytochrome p450 5.52 6.52e-28 unigene36519_all fibrinogen-related molecule 7.61 5.11e-05 cl3301.contig3_all tnf receptor-associated factor 4 7.61 1.24e-20 unigene23546_all tnf receptor-associated factor 3 8.18 2.90e-17 cl4150.contig2_all tnf-inducible gene 6 protein 7.20 4.44e-08 unigene25880_all latrophilin-2 4.57 1.18e-06 unigene24146_all peptidoglycan recognition protein s1l 4.39 0 cl2173.contig3_all cell division cycle protein 16-like protein 7.38 2.19e-08 unigene15602_all c-type lectin domain family 4 7.38 1.07e-284 unigene25286_all c-type lectin domain family 4 member e 9.29 1.42e-26 cl705.contig1_all complement c1q-like protein 2 5.30 8.52e-21 cl4389.contig2_all c1q domain protein 3.21 5.71e-54 unigene14048_all c1q domain profile 4.65 0 cl364.contig12_all nf-kappa-b inhibitor-like protein 1 6.75 7.45e-07 cl2261.contig3_all nf-kappa-b inhibitor cactus 3.15 0 unigene3864_all neurotrypsin 6.20 0 unigene14909_all hsp70 6.05 4.28e-19 unigene23401_all apoptosis 2 inhibitor 5.73 5.20e-156 cl4290.contig2_all inhibitor of apoptosis protein 2.26 3.52e-45 unigene757_all baculoviral iap repeat-containing protein 7 4.63 7.83e-43 unigene21173_all baculoviral iap repeat-containing protein 2 4.22 2.30e-109 unigene14450_all dermatopontin 2 5.33 4.76e-11 unigene5788_all dermatopontin 3 3.58 2.63e-24 unigene9600_all toll-like receptor 2 3.45 1.30e-10 unigene5098_all toll-like receptor 4 9.76 2.41e-13 cl7289.contig3_all collagen alpha-1 3.89 1.05e-14 cl6048.contig2_all protein ambp 2.95 9.84e-51 cl2116.contig6_all kunitz-like protease inhibitor 2.56 0 process(37), immune system development(3), response to stress(19), innate immune response(8), response to lipopolysaccharide(2). in addition, 402 immune-related degs were enriched into 11 kegg database metabolic pathway subcategories, including platelet activation(184), leukocyte transendothelial migration(168), hematopoietic cell lineage(115), chemokine signaling pathway(39), complement and coagulation cascades(60), nod-like receptor signaling pathway(32), toll-like receptor signaling pathway(37), cytosolic dna-sensing pathway(15), rig-i-like receptor signaling pathway(17), tnf signaling pathway(56) and nf-kappa-b signaling pathway(58) (table 2). notably, many immune-related degs were involved in more than one function owing to their multiple roles in regulation of immune response. so the sum of each term degs is larger than total immune-related degs. when p. canaliculata juvenile snails were challenged by lps, some genes related to the 300 296 immune defense system were significant up-regulated (>2 fold change), these genes including toll-like receptor, c-type lectin, c1q domain containing proteins, peptidoglycan-recognition proteins, fibrinogen-related proteins (freps), cytochrome c oxidase and tnf receptor-associated factor (table 3). in invertebrates, the innate immune system mainly relies on pattern-recognition receptors (prrs) to recognize pathogen-associated molecular patterns (pamp) (akira et al., 2006). toll-like receptors (tlrs) are very important prrs, which play a critical role to recognize pamps in innate immune (lu et al., 2016). in this study, the results showed that tlr2 and tlr4 genes were up-regulated after challenged by lps. lps is a component of the cell wall of gram negative bacteria, is a potent activator of innate immunity in mammals, which can recognise by tlr4 and leads to subsequent cell signaling pathway responses. ren et al. (2014) reported that tlr2 is involved in regulation of antimicrobial peptides (amp) gene expression of hyriopsis cumingii, but the mechanism of how tlr2 regulates amp expression is unclear. some lectin-like proteins also have been detected up-regulated in this study. lectin-like proteins play an important role in the defense of gastropods against pathogen infection (bulat et al., 2016). lectin proteins often recognize a specific carbohydrate moieties of the pathogen’s surface (vasta et al., 2015). these proteins are including galectin-4, calnexin, calreticulin, c-type lectins, i-type lectins and c1q domain containing proteins. inhibitors of nuclear factor kappa b (iκbs) are major control components of the rel/nf-κb signaling pathway, a key regulator in the modulation of the expression of immune-related genes in vertebrates and invertebrates (gao et al., 2016; oyanedel et al., 2016). in zhikong scallop chlamys farreri, the mrna expression of cfiκb and cfnf-κb genes increase expression after lps stimulation (wang et al., 2011). the present study revealed that cytochrome c oxidase was the most highly up-regulated degs after challenged with lps. indeed, increase expression of a number of genes associated with degradation or detoxification was observed, including cytochrome c oxidase subunit ii, cytochrome b, nadh dehydrogenase subunit 4, steroid 17-alpha-hydroxylase and cytochrome p450. the immune system of molluscs consists of blood cells (hemocytes), which are the main cellular components of the hemolymph that covers all soft tissue. hemocytes depends on a multi-enzyme (referred to nadph and cytochrome oxidase) production of cytotoxic molecules (like nitric oxide and hydrogen peroxide) for killing and elimination pathogen (humphries and yoshino, 2008). the production of nitric oxide (no) by hemocytes of bivalve molluscs has been reported in m. edulis (ottaviani et al., 1993), v. ater (franchini et al., 1995), and the carpet shell clam ruditapes decussatus (tafalla et al., 2003). the hemocytes of these species are able to produce no in response to various stimuli, such as lps, zymosan, and live bacterial challenge by vibrio tapetis (tafalla et al., 2003). in conclusion, the study first using high-throughput transcriptome technology analyzed the expression of immune-related genes after the p. canaliculata juvenile snails were challenged with lps. the work will provide a molecular resource for further functional studies of p. canaliculata. acknowledgements this research was supported by the national natural science foundation of china (no. u1131006). the authors report no conflicts of interest. the authors themselves are responsible for the content and writing of the paper. references accorsi a, benatti s, ross e, nasi m, malagoli d. a prokineticin-like protein responds to immune challenges in the gastropod pest pomacea canaliculata. dev. comp. immunol. 72: 37-43, 2017. akira s, uematsu s, takeuchi o. pathogen recognition and innate immunity. cell 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dev. com. immunol. 56: 25-36, 2016. 302 36 isj 17: 36-50, 2020 issn 1824-307x research report effects of the glutathione administration via dietary on intestinal microbiota of white shrimp, penaeus vannamei, under cyclic hypoxia conditions sy han1, l wang2,3,6, yl wang2, y chen2, bj wang 2, mq wang4,5, j lin1 1faculty of basic medicine, guangxi university of chinese medicine, nanning 530001, china 2cas key laboratory of experimental marine biology, institute of oceanology, chinese academy of sciences, qingdao 266071, china 3laboratory for marine biology and biotechnology, national laboratory for marine science and technology, qingdao 266237, china 4moe key laboratory of marine genetics and breeding, ocean university of china, qingdao 266003, china 5center for marine molecular biotechnology, national laboratory for marine science and technology, qingdao 266237, china 6cas center for ocean mega-science, chinese academy of sciences, qingdao 266400, china this is an open access article published under the cc by license accepted april 10, 2020 abstract environmental stress can impair the survival, growth performance, and intestinal environment of shrimp. the objective of this study was to investigate the effect of glutathione (gsh) on the survival, growth performance, intestinal oxidation, microbiota, and histology of shrimp under hypoxia. four treatments were used: (1) normoxia, (2) cyclic serious/medium hypoxia (csmh), (3) csmh and 75 mg kg−1 gsh, and (4) csmh and 150 mg kg−1 gsh. white shrimp (penaeus vannamei) in groups 3 and 4 were fed a commercial diet supplemented with 75 and 150 mg kg−1 gsh for a 28 day period, respectively, and they were cultured under csmh (0.8 – 3.5 mg l−1 dissolved oxygen) for the last 14 days of the experiment. p. vannamei supplemented with 75 mg kg−1 gsh showed significantly improved survival and growth performance under csmh compared with the csmh condition alone. the dose of 75 mg kg−1 gsh completely eliminated overproduction of reactive oxygen species and malondialdehyde to suppress serious histopathological lesions and improve bacterial diversity and the relative abundance of beneficial bacteria, such as rhodobacteraceae, thereby preventing pathogen (e.g., vibrio) invasion in the intestine of shrimp under csmh. however, the dose of 150 mg kg−1 gsh was excessive, as it led to serious impairment of survival and growth. these results indicate that 75 mg kg−1 gsh has the potential to control shrimp mortality and growth inhibition under csmh in the shrimp farm setting. key words: penaeus vannamei; glutathione; intestinal microbiota; reactive oxygen species; histology introduction during the course of normal growth of aquatic ___________________________________________________________________________ corresponding authors: bao-jie wang cas key laboratory of experimental marine biology institute of oceanology chinese academy of sciences qingdao 266071, china e-mail: wangbaojie@qdio.ac.cn meng-qiang wang moe key laboratory of marine genetics and breeding ocean university of china qingdao 266003, china e-mail: wangmengqiang@ouc.edu.cn jiang lin faculty of basic medicine guangxi university of chinese medicine nanning 530001, china e-mail: 17135525452@qq.com invertebrates, the intestine and its symbiotic bacteria play a key role in digestion, nutrient supply, and immune function and serve as a barrier between the external environment and internal structures to defend against invading pathogens (harris, 1993; garcia-garcia et al., 2013). however, variations in the environment that inevitably occur in aquaculture systems, such as pollution and changes in controlled conditions, may increase the risk of bacterial infection, leading to disease outbreaks (le and haffner, 2000; zhou et al., 2009). previous investigations of shrimp species such as homarus gammarus (kristensen, 2015), neocaridina denticulata (cheung et al., 2015), and penaeus monodon (rungrassamee et al., 2013) suggested that intestinal microbiota structures could be influenced by environmental, physiological, and 37 fig. 1 monitoring of do levels during the 28-day experimental period. experiments were set in order to obtain the lowest do in the early morning and the highest do in the afternoon. nutritional factors. xiong et al. (2015) reported that the dynamic changes of the bacterial community in the intestine of shrimp were closely related to the severity of shrimp disease. these findings suggested that the intestinal microbiota in shrimp interact with environmental stressors, and microbiota homeostasis in the intestine is beneficial for maintaining health and growth of shrimp under complex conditions (gómez and balcázar, 2008; cardona et al., 2016). as an environmental stressor, hypoxia refers to a state of dissolved oxygen (do) deficiency, and it is observed frequently in mariculture ponds (diaz and rosenberg, 2008). hypoxia is especially critical in rearing ponds that do not use aerators, where shrimp can be exposed to hypoxia as do levels drop from 3 mg l−1 to less than 1 mg l−1 due to respiration of organis-ms and decomposition of accumulated organic matter, which can lead to death of the shrimp (cheng et al., 2003; chantal et al., 2008). penaeus vannamei is one of the most important farmed shrimp species in the world (zeng et al., 2013). in a previous study, we created an experimental environment of cyclic serious/medium hypoxia (csmh, do 0.8 – 3.5 mg l−1) that simulated the practical shrimp culture conditions, and we found that csmh led to death, growth impairment, and histopathological lesions in the intestine of p. vannamei (han et al., 2018). when aquatic organisms encounter environmental stress, they are likely to produce elevated levels of reactive oxygen species (ros), and a series of cellular antioxidant defense mechanism is initiated to control excessive ros in order to maintain homeostasis (franzellitti, 2005; sheikhzadeh et al., 2012). the antioxidant defense system consists of enzymatic antioxidants and non-enzymatic small molecules. the multiple enzyme system includes superoxide dismutase, catalase, and glutathione peroxidase, whereas the non-enzymatic system includes glutathione (gsh), vitamins a, c, and e, and ceruloplasmin (trasviña-arenas et al., 2013). among these non-enzymatic compounds, the tripeptide gsh is very important for cellular defense against ros. as a carrier of an active thiol group in the form of a cysteine residue in living organisms, gsh can react directly with ros in non-enzymatic reactions (cooper et al., 2011). in this study we investigated (1) survival and growth performance, (2) intestinal microbiota, (3) intestinal ros and malondialdehyde (mda) content, and (4) intestinal histology of p. vannamei fed with gsh under csmh with do ranging from 0.8 to 3.5 mg l−1. the goal of this study was to determine if adding gsh to the shrimp diet is an effective strategy for controlling shrimp death and growth inhibition by protecting the intestinal environment under csmh. materials and methods experimental shrimp for this study, 1,200 healthy penaeus. vannamei postlarvae of similar size (mean weight 0.26 ± 0.01 g) were obtained from the ruizi seafood development co. ltd. (qingdao, china). shrimp were placed in 12 cylindrical tanks (640 l) equipped with net covers (n = 100 per tank). every 640 l cylindrical tank contained 500 l of aerated seawater (do 6.4 – 7.5 mg l−1). the initial seawater was unfiltered water directly from the ocean with ph 8.0 – 8.4, salinity 30–31 ‰, total ammonia 0.022 – 0.038 mg l−1, nitrite 0.015 – 0.032 mg l−1, and nitrate 0.120 – 0.205 mg l−1 at 28 – 32 °c. shrimp were acclimated for 2 weeks under a natural photoperiod (12 h light: 12 h dark). the shrimp were fed three times daily with a commercial diet (41.52 % crude protein, 7.42 % lipid, and 12.03 % crude ash, supplied by yantai dale feed co. ltd, shandong, 38 fig. 2 survival rate (a) and weight gain percentage (b) of shrimp during the 28-day experimental period. each bar represents the mean value from three replicates with standard error. values in the same time period with different letters differ significantly (p < 0.05) china) at 07:00, 11:00, and 17:00; this represented a daily feeding rate that was 10 % of the total weight of shrimp. the unconsumed feed and feces were removed with a siphon tube, and 35 % seawater was replaced once daily. unfiltered seawater was prepared in three 1000 l cylindrical tanks to use for daily water changes. experimental design for gsh test and csmh challenge following acclimation, the 12 cylindrical tanks were randomly divided into four groups. each group had three cylindrical tanks constituting three replicates: (1) normoxia (n, do 6.4 – 7.5 mg l−1), (2) csmh (do 0.8 – 3.5 mg l−1), (3) csmh and 75 mg kg−1 gsh added to the diet (csmh.gsh75, do 0.8 – 3.5 mg l−1 + 75 mg kg−1 dietary gsh), and (4) csmh and 150 mg kg-1 gsh added to the diet (csmh.gsh150, do 0.8 – 3.5 mg l−1 + 150 mg kg−1 dietary gsh). the experiment lasted for 28-day. the photoperiod, water exchange, and waste disposal were exactly as described for the acclimation period. each day, the n and csmh groups were fed with the commercial diet, whereas the csmh.gsh75 and csmh.gsh150 groups were fed the commercial diet supplemented with 75 mg kg−1 gsh and 150 mg kg−1 gsh, respectively. the gsh test diets were prepared by thoroughly mixing the commercial diet with an aqueous solution of 75 mg kg−1 gsh and 150 mg kg−1 gsh, respectively, and then thoroughly covering them with egg white. egg white inhibits the leaching of gsh in seawater, ensuring the accuracy of the experiment. the commercial diet for the n and csmh groups was also covered with egg white. these diets were air dried, sealed in plastic bags, and stored frozen at −20 c until used. a) b) 39 table 1 sampling depth and diversity indices in the shrimp intestines item n.1 n.2 n.3 csmh.1 csmh.2 csmh.3 csmh. gsh75.1 csmh. gsh75.2 csmh. gsh75.3 csmh. gsh150.1 csmh. gsh150.2 csmh. gsh150.3 sampling depth no. of sequence 40156 38963 39954 44328 41718 39736 41908 41849 41937 43886 41394 41371 otus (97 %） 2887 3180 2580 2513 3584 3839 3363 3642 3981 3120 2605 2185 phylum 648 713 573 574 803 872 780 830 921 722 589 495 class 647 711 571 570 801 870 775 829 916 718 587 492 order 635 696 563 549 792 857 759 818 899 691 571 496 family 611 669 543 501 764 809 686 779 835 633 545 431 genus 231 258 216 222 292 283 248 266 280 266 230 209 diversity indices chao1 649.21 714.00 574.00 676.15 811.32 875.00 782.25 832.56 925.00 862.39 614.16 530.96 ace 653.51 714.00 574.38 676.62 832.92 875.74 786.47 844.95 925.74 844.96 633.19 542.71 simpson 0.92 0.95 0.92 0.90 0.93 0.92 0.94 0.91 0.93 0.96 0.95 0.91 shannon 5.18 5.91 4.98 4.72 5.45 5.71 5.67 5.56 5.88 6.02 5.55 4.90 during days 0 – 14 of the experiment, the water in each tank was aerated enough to generate do 6.4 – 7.5 mg l−1 automatically. the daily do concentration was characterized by the lowest do in the early morning and the highest do in the afternoon and by exposure to do 6.4 – 7.0 mg l−1 and 7.0 – 7.5 mg l−1 for about 16 and 8 h, respectively, over every 24 h cycle. during days 14 – 28 of the experiment, the water in the n group tanks was still aerated enough to generate do 6.4 – 7.5 mg l−1 automatically. however, the water in the tanks of the other three groups was not aerated enough to generate do 0.8 – 3.5 mg l−1 automatically. thus, the do concentration in the three csmh groups was characterized by the lowest do in the early morning and the highest do in the afternoon and by exposure to do 0.8–2.0 mg l−1 and 2.0 – 3.5 mg l−1 for about 16 h and 8 h, respectively, over every 24 h cycle. the do levels were monitored four times a day during the experimental period using a ysi model 55 do meter (ysi incorporated, yellow springs, oh, usa) (fig. 1). measurement of survival and growth and sampling procedure the number of dead shrimp in each tank was recorded every 24 h during the experimental period. shrimp were considered dead when they failed to move even when gently stimulated with a glass pipette. dead shrimp were removed to prevent fouling. ten shrimp from each tank were randomly selected on days 14 and 28 during the experimental period, and each shrimp was weighed. survival and growth were evaluated in terms of survival rate (sr) and weight gain percentage (wgp) based on the following standard formulae: sr (%) = 100(cumulative dead shrimp number)/(initial shrimp number); wgp (%) = 100(final weight–initial weight)/initial weight. twelve other shrimp were randomly selected and removed from each tank on day 28, and the intestine of each shrimp was collected using sterilized scissors and forceps. nine intestines were immediately ground in liquid nitrogen; three of them were stored at −80 °c until use in the ros production and mda content assay, and six were saved in dry ice and sent to shanghai personal biotechnology co., ltd. (shanghai, china) for dna extraction and illumina sequencing. the remaining three intestines were immediately fixed in 10% formaldehyde for histological analysis. sequence analysis polymerase chain reaction (pcr) products were generated with the forward primer 338f (5’-actcctacgggaggcagca-3’) and the reverse primer 806r (5’-ggactachvgggtwtctaat-3’), which amplified the v3−v4 regions of the bacterial 16s rrna gene. pcr amplicons were purified with agencourt ampure beads (beckman coulter, indianapolis, in, usa) and quantified using the picogreen dsdna assay kit (invitrogen, carlsbad, ca, usa). the products of pair-end 2 × 300 bp sequencing were sequenced with the illumina miseq platform (illumina, san diego, ca, usa). the short reads that overlapped were assembled with flash (edgar, 2013), and the poor-quality assembled reads were filtered with quantitative insights into microbial ecology (qiime) (wang et al., 2007). in qiime, poor-quality sequences were defined as sequences with a length of less than 150 bp, average javascript:void(0); javascript:void(0); 40 fig. 3 length distribution of high quality sequences in the shrimp intestines after the 28-day experiment phred scores of less than 20, and containing ambiguous bases and mononucleotide repeats of more than 8 bp. after chimera detection, the remaining high-quality sequences were clustered into operational taxonomic units (otus) at 97% sequence identity by uclust (edgar, 2010). otu taxonomic classification ranging from phylum to species levels was conducted using the greengenes database (desantis et al., 2006). otu-level alpha diversity indices, such as the chao biodiversity index, ace index, shannon index, and simpson index, were calculated for each sample using the otu table in qiime. a beta diversity analysis was used to compare the microbial community compositions in the different samples, and results were visualized via nonmetric multidimensional scaling (nmds) (ramette, 2007). the taxonomy compositions and abundances were visualized using megan (huson et al., 2011) and graphlan (asnicar et al., 2015). ros and mda content in the intestines intestines of three shrimp from each tank were mixed at a 1:5 ratio (w/v) with chilled tris-hydrochloric acid buffer solution (ph 7.6, 10 mmol l−1) and homogenized under ice-chilled conditions. the homogenate was centrifuged at 10,000 g for 10 min at 4 °c, and the supernatant was then collected for sample analysis. ros content in the intestine was measured using the 2′,7′-dichlorofluorescein diacetate (dcfh-da) method (guo et al., 2017). the supernatant was incubated with dcfh-da for 30 min in darkness and then diluted with modified alsevier's solution to a final concentration of 1 × 106 cells ml−1, followed by flow cytometry analysis (becton, dickinson and company, franklin lakes, nj, usa). ros production was expressed as mean fluorescence of dcf. mda content was evaluated using commercial kits (nanjing jiancheng bioengineering institute, nanjing, china) according to the manufacturer's instructions. mda content was expressed as the relative concentration per milligram of soluble protein (nmol per mg protein). both ros production and mda content were analyzed with three replicates of each sample. measurement of intestinal histology the fixed intestines were dehydrated in an ascending alcohol series (50 % to 95 % concentration). dehydrated tissues were embedded in paraffin, sectioned into 5 μm thick sections, and stained with hematoxylin and eosin (he). sections were then examined with a light microscope (casado et al., 2001). statistical analysis data for different bacterial community composition in the shrimp intestines are all presented as mean ± standard error (se). statistical analysis was performed using spss (version 17.0) (ibm, armonk, ny, usa). one-way analysis of variance and the least significant difference test were used to analyze the differences among the different treatment groups. p < 0.05 was considered to be statistically significant. all images were generated with origin 8.6 software (originlab, northampton, ma, usa). results survival and growth of shrimp after the first 14 days of the experiment, all shrimp in the four groups had survived (fig. 2a). the average wgp values of n, csmh, csmh.gsh75, 41 fig. 4 rarefaction curves of the bacterial 16s rdna gene sequences in the shrimp intestines after the 28-day experiment and csmh.gsh150 shrimp were 71.26 %, 71.54 %, 83.62 %, and 106.34 %, respectively. the wgp values of n and csmh shrimp did not differ significantly, but those of csmh.gsh75 and csmh.gsh150 shrimp were significantly higher (p < 0.05) than the values for the n and csmh shrimp. additionally, the wgp of csmh.gsh150 shrimp was significantly higher (p < 0.05) than that of csmh.gsh75 shrimp (fig. 2b). at the end of the 28-day experiment, the average sr values of n, csmh, csmh.gsh75, and csmh.gsh150 shrimp were 96.97 %, 63.03 %, 84.24 %, and 61.21 %, respectively. the sr values of the csmh and csmh.gsh150 shrimp did not differ significantly, but the values of the n and csmh.gsh75 shrimp were significantly higher (p < 0.05) compared with those of csmh and csmh.gsh150 shrimp. (fig. 2a). the average wgp values of n, csmh, csmh.gsh75, and csmh.gsh150 shrimp were 684.23 %, 549.23 %, 695.77 %, and 531.54 %, respectively. the wgp values of the n and csmh.gsh75 shrimp did not differ significantly, and the wgp values of the csmh and csmh.gsh150 shrimp did not differ significantly. however, the wgp values of the n and csmh.gsh75 shrimp were significantly higher (p < 0.05) than those of the csmh and csmh.gsh150 shrimp (fig. 2b). overview of 16s rdna high-throughput sequencing analysis high-throughput sequencing of the 16s rdna gene amplicons was performed to determine bacterial diversity in intestines of n, csmh, csmh.gsh75, and csmh.gsh150 shrimp after the 28-day experiment (table 1). a total of 497,200 reads were obtained from 12 samples with an average read length of 438 bases (fig. 3). in the rarefaction curves, the number of otus almost reached the plateau phase with the increasing read number at 20,000 (fig. 4), which suggested sufficient sampling depth in all samples. the average number of otus was highest in the csmh.gsh75 group (3662) and lowest in the csmh.gsh150 group (2637) (fig. 5). relationships among the bacterial communities in the four groups we used nmds analysis to investigate the similarity of community structure among different samples. the results of the n group and csmh.gsh75 groups both showed good repeatability, and they had different bacterial communities. however, the csmh group and csmh.gsh150 group had similar bacterial communities (fig. 6). heat map was constructed at the genus level based on the clustering analysis of bacterial communities. the result was consistent with the nmds findings to some extent (fig. 7). bacterial community composition the composition and abundance of bacterial communities in different samples were investigated according to five classification levels (table 2). figure 8a shows the top 20 bacteria at the phylum level. proteobacteria were the predominant microflora in the four groups, accounting for more than 71 % of the bacterial communities, followed by actinobacteria and bacteroidetes. the relative abundances of bacteroidetes and verrucomicrobia javascript:gg('p__actinobacteria'); javascript:gg('p__bacteroidetes'); javascript:gg('p__bacteroidetes'); 42 fig. 5 venn diagram showing the unique and shared operational taxonomic units (otus) in the shrimp intestines after the 28-day experiment were significantly higher in the n group than in the other three groups (p < 0.05). the relative abundance of cyanobacteria was highest in the csmh.gsh150 group, and it was significantly higher than that in the n group (p < 0.05). at the class level, the relative abundance of gammaproteobacteria was significantly lower in the csmh.gsh75 group than in the other three groups (p < 0.05). the relative abundance of verrucomicrobiae was significantly higher in the n group than in the other three groups (p < 0.05). in figure 8b, it is shown that the top 20 bacteria at the order level. the relative abundance of rhodobacterales was significantly higher in the csmh.gsh75 group than in the other three groups (p < 0.05). the relative abundance of vibrionales was highest in the csmh group, and it was significantly higher than that in the csmh.gsh75 group (p < 0.05). the relative abundance of verrucomicrobiales was significantly higher in the n group than in the other three groups (p < 0.05). at the family level (fig. 8c), the relative abundance of rhodobacteraceae was significantly higher in the csmh.gsh75 group than in the other three groups (p < 0.05). the relative abundances of rhodobacteraceae in the n and csmh groups were significantly higher than that in the csmh.gsh150 group (p < 0.05). the relative abundance of verrucomicrobiaceae was significantly higher in the n group than in the other three groups (p < 0.05). the relative abundance of vibrionaceae was highest in the csmh group, and it was significantly higher than that in the n group (p < 0.05). the relative abundance of streptococcaceae was significantly higher in the csmh.gsh150 group than in the other three groups (p < 0.05). at the genus level, the relative abundance of streptococcus was significantly higher in the csmh.gsh150 group than in the other three groups (p < 0. 05). ros production and mda content of the intestines at the end of the 28-day experiment, ros production and mda content in the intestines of csmh and csmh.gsh150 shrimp were significantly higher (p < 0.05) than values in the n and csmh.gsh75 shrimp. additionally, ros production and mda content in the intestines of csmh.gsh150 shrimp were significantly lower (p < 0.05) compared with values in the csmh shrimp. (fig. 9a, b). histology assays of the intestines after the 28-day experiment, the intestinal villi and the lamina propria of n shrimp were arranged regularly. compared with n shrimp, intestinal villi were exfoliated completely and the lamina propria was cleaved in the csmh shrimp. the intestinal villi length appeared shorter and the intestinal villi connection appeared to be twisted in the csmh.gsh75 shrimp, whereas intestinal villi were exfoliated and the lamina propria was scattered in the csmh.gsh150 shrimp (fig. 10). javascript:gg('c__gammaproteobacteria'); javascript:gg('c__verrucomicrobiae'); javascript:gg('o__rhodobacterales'); javascript:gg('o__vibrionales'); javascript:gg('o__verrucomicrobiales'); javascript:gg('f__rhodobacteraceae'); javascript:gg('f__rhodobacteraceae'); javascript:gg('f__verrucomicrobiaceae'); javascript:gg('f__vibrionaceae'); javascript:gg('f__streptococcaceae'); javascript:gg('g__streptococcus'); 43 fig. 6 non-metric multidimensional scaling (nmds) showing bacterial community differences in the shrimp intestines after the 28-day experiment discussion intestinal bacteria in shrimp play important roles in maintaining the health of the host, such as promoting digestion and inhibiting growth of pathogenic bacteria (ringø et al., 2003; round and mazmanian, 2009; clemente et al., 2012). previous research indicated that alteration of intestinal microbiota of p. vannamei under high or low salinity was likely attributed to pathogenic bacteria, leading to poorer growth (zhang et al., 2016). decreased bacterial diversity and anti-stress bacteria in the intestine of p. vannamei also promoted pathogenic bacteria invasion under exposure to increasing concentrations of sulfide, leading to slower growth (suo et al., 2017). in the present study, we found that csmh induced impairment of survival and growth performance of p. vannamei and that the addition of 75 mg kg−1 gsh to the diet significantly improved survival and growth of shrimp under csmh. thus, we speculated that both csmh and gsh addition affected bacterial diversity and microbiota structures in the intestine of the shrimp. proteobacteria, bacteroidetes, and actinobacteria were predominately distributed in the intestine of p. vannamei under different environmental conditions, such as normal (huang et al., 2016), hyposaline, hypersaline (zhang et al., 2016), and high-concentration sulfides (suo et al., 2017). similarly, these phyla accounted for the majority of intestinal bacteria in the four groups tested in the present study. bacteroidetes may play a specialized role in biopolymer degradation and uptake of micromolecular organics (kirchman, 2002; williams et al., 2013). however, csmh significantly reduced the relative abundance of bacteroidetes in the intestine of p. vannamei in our study, as was also reported for p. vannamei and nile tilapia (oreochromis niloticus) facing hyposaline or hypersaline stress (zhang et al., 2016). cardman et al. (2014) reported that verrucomicrobiaceae were capable of degrading polysaccharides in aquatic animals, but we found that csmh significantly reduced the relative abundance of verrucomicrobiaceae at the phylum, class, order, and family level in p. vannamei. the diversity indices in the present study indicated that csmh could reduce bacterial diversity in the intestine of p. vannamei. thus, we surmised that csmh reduced bacterial diversity and the beneficial bacteria community, which might have suppressed digestion and uptake in the intestine of shrimp, thereby impairing survival and growth performance. prebiotics, probiotics, and synbiotics are reported to improve health status of fish and shellfish through modulation of the gastrointestinal 44 fig. 7 heat map showing bacterial community differences in the shrimp intestines after the 28-day experiment tract microbiome, thus reducing the incidence of diseases in aquaculture (merrifield et al., 2014; hoseinifar et al., 2016; sha et al., 2016). in the present study, the diversity indices indicated that dietary gsh addition could improve bacterial diversity in the intestine of p. vannamei under csmh. in particular, csmh.gsh75 shrimp had the highest bacterial diversity. cardona et al. (2016) reported that the rhodobacteraceae could have limited the survival of pathogenic bacteria in litopenaeus stylirostris reared with biofloc technology. this finding supports the premise that addition of 75 mg kg−1 gsh to the diet could significantly improve the relative abundance of rhodobacteraceae at the order and family levels in the intestine of p. vannamei under csmh. thus, we propose that adding 75 mg kg−1 gsh to the diet improved bacterial diversity and the beneficial bacteria community, which might have helped the shrimp resist pathogenic bacteria in the intestine under csmh, thereby enhancing survival and growth performance. generally, bacteria in the class gammaproteobacteria are more adaptable to oligotrophic marine environments, and previous studies reported that the relative abundance of members of this class in p. vannamei was higher in the diseased samples than that in the healthy samples (bowman and mccuaig, 2003; zheng et al., 2016). in the present study, csmh significantly increased the relative abundance of gammaproteobacteria in the intestine of p. vannamei, 45 table 2 bacterial community composition in the shrimp intestines from different groups classification n csmh csmh.gsh75 csmh.gsh150 phylum proteobacteria 71.57 ± 4.80b 81.17 ± 3.56a 76.27 ± 6.08ab 75.27 ± 4.23ab actinobacteria 4.73 ± 0.90b 9.47 ± 5.75ab 16.23 ± 6.23a 4.03 ± 2.31b bacteroidetes 15.97 ± 2.74a 5.30 ± 3.67b 2.70 ± 1.70b 7.70 ± 3.21b tenericutes 1.8 ± 1.15b 1.83 ± 0.65b 1.23 ± 0.58b 6.7 ± 1.95a firmicutes 0.03 ± 0.06b 1.07 ± 1.19b 1.8 ± 1.67b 4.43 ± 1.68a verrucomicrobia 5.33 ± 4.11a 0.43 ± 0.49b 0.57 ± 0.46b 0.23 ± 0.06b cyanobacteria 0.07 ± 0.06b 0.17 ± 0.15ab 0.3 ± 0.26ab 0.57 ± 0.31a class alphaproteobacteria 44.60 ± 4.08b 45.37 ± 3.91b 56.00 ± 7.33a 37.13 ± 2.30b gammaproteobacteria 26.8 ± 0.82a 30.57 ± 3.70a 15.63 ± 2.21b 30.30 ± 8.34a flavobacteria 15.77 ± 2.727a 4.837 ± 3.70b 1.93 ± 1.21b 6.93 ± 2.87b mollicutes 1.8 ± 1.15b 1.83 ± 0.65b 1.23 ± 0.58b 6.70 ± 1.95a verrucomicrobiae 5.33 ± 4.11a 0.40 ± 0.52b 0.53 ± 0.49b 0.23 ± 0.06b bacilli 0 ± 0b 0.2 ± 0.26b 0.17 ± 0.12b 3.77 ± 2.57a order rhodobacterales 44.13 ± 4.23b 44.1 ± 4.57b 55.3 ± 7.56a 31.1 ± 3.52c vibrionales 16.57 ± 2.37ab 25.63 ± 4.01a 13.23 ± 1.06b 24.43 ± 11.18ab flavobacteriales 15.77 ± 2.72a 4.83 ± 3.70b 1.93 ± 1.21b 6.93 ± 2.87b alteromonadales 10.03 ± 3.07a 4.2 ± 4.92b 2.1 ± 1.13b 2 ± 1.39b verrucomicrobiales 5.33 ± 4.11a 0.4 ± 0.52b 0.53 ± 0.49b 0.23 ± 0.06b rhizobiales 0.27 ± 0.12b 0.83 ± 0.40b 0.47 ± 0.15b 2.93 ± 1.07a family rhodobacteraceae 44.13 ± 4.23b 44.1 ± 4.57b 55.3 ± 7.56a 31.1 ± 3.52c flavobacteriaceae 15.33 ± 3.02a 4.23 ± 4.03b 1.77 ± 1.20b 3.3 ± 4.19b vibrionaceae 1.2 ± 0.35b 7 ± 4.36a 1.93 ± 0.35ab 6 ± 3.29ab verrucomicrobiaceae 5.33 ± 4.11a 0.4 ± 0.52b 0.53 ± 0.49b 0.23 ± 0.06b streptococcaceae 0 ± 0b 0.2 ± 0.26b 0.17 ± 0.12b 3.33 ± 2.31a genus psychroserpens 7.93 ± 2.76a 0.23 ± 0.06b 0.2 ± 0.26b 0.1 ± 0.17b nautella 0.43 ± 0.15b 1.1 ± 0.17b 4.17 ± 1.80a 1.43 ± 0.85b haloferula 4.07 ± 3.09a 0.37 ± 0.55b 0.50 ± 0.44b 0.13 ± 0.12b streptococcus 0 ± 0b 0.20 ± 0.26b 0.17 ± 0.12b 3.33 ± 2.31a but the addition of 75 mg kg−1 gsh to the diet significantly suppressed the relative abundance of this group under csmh. numerous studies have reported that vibrio, which is a major pathogenic bacteria affecting shrimp, caused stressed shrimp to be susceptible to infectious diseases (vandenberghe et al., 1999; austin and zhang, 2006). in our study, csmh significantly increased the relative abundance of vibrionaceae in the intestine of p. vannamei, but adding 75 mg kg−1 gsh to the diet significantly suppressed the relative abundance of vibrionales under csmh. therefore, we suggest that the csmh environment might have been oligotrophic, which created a suitable environment for opportunistic pathogenic bacteria, which in turn impaired survival and growth performance of shrimp. however, adding 75 mg kg−1 gsh to the diet improved the beneficial bacteria javascript:gg('p__proteobacteria'); javascript:gg('p__actinobacteria'); javascript:gg('p__bacteroidetes'); javascript:gg('p__tenericutes'); javascript:gg('p__firmicutes'); javascript:gg('p__verrucomicrobia'); javascript:gg('p__cyanobacteria'); javascript:gg('c__alphaproteobacteria'); javascript:gg('c__gammaproteobacteria'); javascript:gg('c__flavobacteriia'); javascript:gg('c__mollicutes'); javascript:gg('c__verrucomicrobiae'); javascript:gg('c__bacilli'); javascript:gg('o__rhodobacterales'); javascript:gg('o__vibrionales'); javascript:gg('o__flavobacteriales'); javascript:gg('o__alteromonadales'); javascript:gg('o__verrucomicrobiales'); javascript:gg('o__rhizobiales'); javascript:gg('f__rhodobacteraceae'); javascript:gg('f__flavobacteriaceae'); javascript:gg('f__vibrionaceae'); javascript:gg('f__verrucomicrobiaceae'); javascript:gg('f__streptococcaceae'); javascript:gg('g__psychroserpens'); javascript:gg('g__nautella'); javascript:gg('g__haloferula'); javascript:gg('g__streptococcus'); javascript:gg('f__vibrionaceae'); javascript:gg('o__vibrionales'); 46 fig. 8 bacterial community compositions at phylum (a), order (b), and family (c) levels in the shrimp intestines after the 28-day experiment a) b) c) 47 fig. 9 ros content (a) and mda content (b) in the shrimp intestines after the 28-day experiment. each bar represents the mean value from three replicates with standard error. values with different letters differ significantly (p < 0.05) community, which prevented pathogen invasion in the intestine of shrimp under csmh and enhanced survival and growth performance. ros, including superoxide anion, hydroxyl radical, and hydrogen peroxide, can damage important biomolecules, such as dna, proteins, and lipids (halliwell and gutteridge, 1999). as the main component of lipid peroxides, mda has strong biotoxicity and can damage tissue structure and function (freeman and crapo, 1982). at the tissue and cellular levels, environmental stresses are likely to produce elevated levels of ros (lushchak, 2011). in the present study, csmh induced overproduction of ros and mda in the intestine of p. vannamei, leading to serious histopathological lesions. han et al. (2018) found similar results when shrimp were exposed to low or high ph as environmental stressors. additionally, qi et al. (2017) reported that histopathological lesions in the intestine of p. vannamei could promote pathogen invasion due to impaired function of the intestinal barrier. in our study, csmh reduced the beneficial bacteria community and improved that of pathogenic bacteria in the intestine of p. vannamei. thus, we propose that excessive ros was the main reason why csmh promoted pathogen invasion. however, addition of 75 mg kg−1 gsh to the diet completely eliminated excessive ros and mda to suppress a) b) 48 fig. 10 photomicrographs of the shrimp intestines after the 28-day experiment. the pathologies observed were: dilatation (d); vacuoles (v); shorter intestinal villi (sv); twisting intestinal villi (tv); exfoliation (e); cleavage (c); scatter(s). scale bar = 100 μm. serious histopathological lesions, preventing pathogen invasion under csmh. although gsh plays an important role in scavenging ros, too much gsh can be a problem when it combines with a variety of compounds, such as aldehydes and alkenyl halide, as these toxic metabolites covalently combine with dna and produce ros or tumors (monks et al., 1990; thomas et al., 1998). in the present study, 150 mg kg−1 gsh, compared with 75 mg kg−1 gsh, induced significant overproduction of ros and mda in the intestine of p. vannamei under csmh, leading to serious histopathological lesions. jia et al. (2016) reported that a low abundance of cyanobacteria was beneficial to aquatic animal growth, but other studies showed that a high abundance of cyanobacteria produced hepatotoxic microcystins and cytotoxic lipopeptides, which can cause cell necrosis (howard, 2012; kang 2012). in our study, 150 mg kg−1 gsh significantly increased the relative abundance of cyanobacteria in the intestine of p. vannamei under csmh compared with normoxia. streptococci are the cause of an emerging disease in penaeid shrimp in the americas and in regions of east africa (hasson et al., 2009; lightner et al., 2009). no streptococci were detected in the intestine of p. vannamei under normoxia, but the addition of 150 mg kg−1 gsh to the diet significantly increased the relative abundance of streptococci at the family and genus level under csmh. thus, 150 mg kg−1 gsh was excessive supplementation for p. vannamei under csmh, as it induced excessive ros to promote pathogen invasion, leading to serious survival and growth impairment. conclusions this study was the first investigation of the effect of gsh on the survival, growth performance, intestinal microbiota, oxidation, and histology of p. vannamei under csmh. dietary p. vannamei supplementation with 75 mg kg−1 gsh completely eliminated overproduction of ros and mda to suppress serious histopathological lesions and improved bacterial diversity and the relative abundance of beneficial bacteria community such as rhodobacteraceae, which prevented pathogen (e.g., vibrio) invasion in the intestine of shrimp under csmh and enhanced survival and growth performance. however, supplementation with 150 mg kg−1 gsh under csmh was excessive and led to serious impairment of survival and growth. therefore, adding 75 mg kg−1 gsh to the diet could improve the health status of shrimp experiencing csmh by protecting the intestinal environment. this supplementation dose could be used to control shrimp mortality and growth inhibition under csmh in the shrimp farm setting. acknowledgments this research was supported by the national natural science foundation of china-joint fund of shandong people's government (u1706209) and the chinese academy of sciences sts regional center project (fujian province). we thank international science editing (www.internationalscienceediting.com) for editing this manuscript. we would like to thank the editor and the expert reviewers for their constructive suggestions and enlightening comments during the revision. references asnicar f, weingart g, tickle tl, huttenhower c, segata n. compact graphical representation of phylogenetic data and metadata with graphlan. peer j. 3: e1029, 2015. austin b, zhang x. vibrio harveyi: a significant pathogen of marine vertebrates and invertebrates. lett. appl. microbiol. 43: 119, 2006. bowman jp, mccuaig rd. biodiversity, community structural shifts, and biogeography of prokaryotes within antarctic continental shelf sediment. appl. environ. microb. 69: 2463-2483, 2003. cardona e, 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of gut microbiota to salinity change in two euryhaline aquatic animals with reverse salinity preference. aquaculture 454: 72-80, 2016. zheng y, yu m, liu y, su y, xu t, yu m, et al. comparison of cultivable bacterial communities associated with pacific white shrimp (litopenaeus vannamei ) larvae at different health statuses and growth stages. aquaculture 451: 163-169, 2016. zhou j, wang wn, wang al, he wy, zhou qt, yuan l, et al. glutathione s-transferase in the white shrimp litopenaeus vannamei: characterization and regulation under ph stress. comp. biochem. physiol. c 150: 224, 2009. 24 isj 17: 24-31, 2020 issn 1824-307x report of meeting 2nd workshop of the cost action 16203 maristem: omic approaches to identify and characterize marine/aquatic invertebrate stem cells, escola superior de turismo e tecnologia do mar, peniche, portugal organizers: a varela coelho (chair)1, f herrera1, s blanchoud2, sm3 leandro 1instituto de tecnologia química e biológica antónio xavier, universidade nova de lisboa, av. da república, 2780-157 oeiras, portugal 2department of biology, université de fribourg, germany 3escola superior de turismo e tecnologia do mar, peniche, portugal this is an open access article published under the cc by license identification and relative quantification of asterosaponins isolated from marthasterias glacialis coelomic fluid during arm-tip/radial nerve cord regeneration l gomes, r laires, v marques, f brigham, p lamosa, l gafeira, av coelho instituto de tecnologia química e biológica antónio xavier, universidade nova de lisboa, av. da república, 2780-157 oeiras, portugal regenerative potential is commonly observed in echinoderms. starfishes are well-known echinoderms, which are capable of reconstructing external appendages and internal organs often subjected to amputation. the coelomic fluid bathes the internal organs, it transports the circulating cells and signaling compounds. the objective of this work was to identify the molecular species present in the coelomic fluid that could play an important role in the regeneration process of the starfish marthasterias glacialis. aiming that, a protocol to extract compounds present in the cell free coelomic fluid (cff) from control and regenerating (2, 13 and 70 days postamputation) groups was optimized. the regeneration process was induced by amputation of 2 arm tips or partial removal of 2 radial nerve cords per starfish. the solid phase extraction (spe) chromatography with acetonitrile discontinuous gradient was used to elute cff compounds. spe eluted fractions were analyzed by esi-ms/ms in positive and negative mode. more intense m/z signals were detected for negative molecular ions with monoisotopic and mass losses characteristic of asterosaponins. these pentaglycoside or hexaglycoside sulfated steroids have several bioactive properties, namely at the antimicrobial, immunological, physiological and pharmacological levels. our results show reproducible asterosaponin profiles for each regeneration time point. suggesting their promising participation in the regeneration process. echinoderms are valid deuterostome marine invertebrate models to study repair phase events after arm injury c ferrario1,2, y ben khadra3, a czarkwiani4, a zakrzewski4, p martinez5,6, f bonasoro1, md candia carnevali1, p oliveri4, m. sugni1.2 1university of milan, department of environmental science and policy, milan, italy 2university of milan, center for complexity and biosystems, department of physics, milan, italy 3université de monastir, institut supérieur de biotechnologie de monastir, laboratoire de recherche, génétique, biodiversité et valorisation des bioressources, monastir, tunisia 4university college london, department of genetics, evolution and environment, london, united kingdom 5universitat de barcelona, departament de genètica, microbiologia i estadística, barcelona, spain 25 6institut català de recerca i estudis avancats, barcelona, spain echinoderms are often subjected to traumatic amputations that damage or remove whole body parts i.e. arms. after such severe injuries, the repair phase must be effective with rapid emergency reaction and re-epithelialization as well finely regulated extracellular matrix (ecm) remodeling to ensure subsequent arm regeneration. here, we used the brittle star amphiura filiformis (ophiuroidea) and the starfish echinaster sepositus (asteroidea) as valid deuterostome marine invertebrate models to study similarities and differences in the repair phase phenomena of these two echinoderm species and discuss them in comparison with those of animals with limited regenerative abilities (i.e. mammals). to achieve this goal, we used an integrated approach based on both microscopy and molecular analyses. we showed that in both echinoderm models, immediately after injury, emergency reaction and reepithelialization are extremely rapid and more efficient than those displayed by mammals. the remodeling and the formation of the ecm, mainly collagen, is ensured by delayed activation of ecm genes and protein deposition and, together with absence of fibrosis (i.e. over-deposition of ecm), seem to be advantageous for regenerationcompetent animals in comparison to mammals. overall, we found that the echinoderm species here studied show comparable repair events. the differences between regeneration-competent and non-competent animals suggest that rapid wound closure and delayed ecm deposition are necessary to ensure an effective regeneration of whole lost body parts. further molecular and functional analyses must be performed to confirm this hypothesis. complement components as markers of hemocyte differentiation in the colonial ascidian botryllus schlosseri a peronato, n franchi, l ballarin department of biology, university of padua the complement system is one of the immune modulator mechanism of metazoans. the complement system of vertebrates is a complex array of soluble and membrane proteins able to orchestrate ancient immune responses such as inflammation and phagocytosis. three complementactivation pathways are known in vertebrates: the classical, the alternative and the lectin pathways: all of them converge on the cleavage of c3. complement in invertebrates have been much less studied; however, c3 genes have been identified in representatives of all the major invertebrates phyla, starting from basal metazoans such as porifera. as an invertebrate, the compound ascidian botryllus schlosseri relies only on innate immunity for its defense and immunocytes (i.e., cells with defined roles in immunity) represent the great majority of the circulating hemocytes: they include cytotoxic morula cells and phagocytes. in the same species, we demonstrated the presence of the lectin and the alternative pathways. all the complement components identified so far (c3, bf, mbl, ficolin and masp), are expressed by morula cells, the most abundant circulating hemocyte, the other immunocytes being represented by phagocytes. my project aims to use c3 transcript as a signature of morula cells to study their differentiation from hematopoietic cells during ontogenesis and blastogenesis. in the first case, i will carry out in situ hybridization and pcr on larvae, whereas, in the case of blastogenesis, i will investigate the quantity of c3-positive cells in the pharyngeal niches recently identified in the various phases of the colonial blastogenetic cycle. stem cells roles in the aging marine colonial invertebrate model animal, botryllus schlosseri o ben-hamo1,2, b rinkevich1, r ben shlomo3, l ballarin4 1israel oceanographic and limnological research, national institute of oceanography, p.o. box 8030, tel shikmona, haifa 31080, israel 2department of evolutionary and environmental biology, university of haifa, israel 3department of biology and environment, faculty of natural sciences, university of haifa – oranim 4department of biology, university of padua, padua, italy the aging process of living beings is one of the most intriguing and less understood biological phenomena. stem-cells (scs) may participate as effectors in aging. botryllus schlosseri, a marine colonial invertebrate is an interesting model for the studying of aging since it ages both at the level of the entire colony, a process that takes months/years, and at the level of the temporary modules that live in a colony for three weeks and constantly replaced by younger asexually budded modules (20 ˚c, temperature-dependent). in botryllus cell-island (ci) niches of scs were discovered in proximity to the endostyle organ of the zooids (mature modules). in diverse sc niches in mice and human, their number rises throughout the course of aging, while their functionality declines, and they undergo inevitable exhaustion. our research aims to elucidate changes in hematopoietic stem cell (hscs) numbers/behaviours along two aging processes in botryllus. the research focuses on scs in the hemolymph and in the ci niches of botryllus. preliminary observations using electron microscopy (tem) on labelled scs in the ci niches in botryllus show that old colonies (8 months old) have 6 times more scs than young colonies (3 weeks old). regarding the level of the zooid, old zooids have 3.6 times more scs compared to young zooids. further observations should be carried out soon to delineate the pattern of scs in the hemolymph of old/young colonies and of zooids along the life cycle. transcriptomic profiling of the mussel mytilus trossulus with a special emphasis on integrinlike genes during development m maiorova1, n. satoh2, k. khalturin2 n. odintsova1 26 1national scientific center of marine biology; the far eastern branch of the russian academy of sciences, vladivostok; russia 2marine genomics unit, okinawa institute science & technology; okinawa; japan a prerequisite for the emergence of long-lived multicellular organisms was the evolution of intercellular adhesion mechanisms. a set of positively selected genes related to integrin complex was identified in the transcription profiles of the bivalve mollusk mytilus trossulus, and changes in expression of candidate genes involved in cell adhesion were determined. the study is based on the illumina rna-sequencing data obtained for a de novo assembly of the transcriptome from early developmental stages and some tissues and cells. our transcriptome dataset supplements to the genetic databases of non-model animals such as bivalves and represents the first characterization of expressed sequences during early development of m. trossulus from the sea of japan. a total of 200079 contigs were obtained, and based on the go terms, the number of annotated contigs was estimated to reach 19.96 %. the main findings include evidence that the predicted mussel β integrin-like protein sequences are most closely related to the integrins sequenced for all classes of mollusca, while the highest similarity is observed between mussel and oyster proteins. additionally, an analysis of the transcriptome revealed four fulllength transcripts that seemed to be isoforms of two genes encoding β integrin-like proteins. the present study provides a transcriptome that can serve as a reference for future studies of mytilus in the marine ecosystems. whole genome sequencing and gene editing via silencing rna in some botryllid ascidians fn oğul, a karahan institute of marine science, middle east technical university, mersin, turkey botryllid ascidians are the closest relative of vertebrates, they have notochord in their free swimming larval stage and by metamorphosis they lose their notochord and live as sessile near shoreline. they can reproduce both sexually and asexually. by asexual reproduction they form colonies from an individual (zooid) following sexual reproduction. during asexual reproduction each zooid regress in approximately one week, and next generation is formed by budding and this process is called blastogenesis. furthermore, it is known that they undergo whole body regeneration although closest relative vertebrates have limited capacity of regeneration at tissue or organ level. during both blastogenesis and whole body regeneration, inhibitor apoptosis proteins have a great importance in order to control apoptosis. we also know that inhibitor apoptosis proteins are related a variety of human diseases such as cancer, thus it is important to understand the mechanism of them. botryllid ascidians have an important evolutionary spot and they can be a great model organism for further understanding of vertebrate evolution. in the present study we will determine a model organism from botryllid ascidian species and conduct a whole genome sequencing in order to determine apoptosis related genes. once we determine the genes we will conduct a series of knockdown experiments by using sirna and crispr method. at the end of the study we believe that the study provides extensive insight on the signal mechanism and roles of inhibitor apoptosis proteins. dynamics of circulating coelomocytes populations during starfish regeneration b simões1, b oliveira1, s guatelli2, m sugni2, r zilhão3, av coelho1 1instituto de tecnologia química e biológica antónio xavier, universidade nova de lisboa, oeiras, portugal 2department of biosciences, university of milan, milan, italy 3faculdade ciências universidade lisboa, lisboa, portugal the potential for regeneration has its maximum expression in echinoderms. preliminary studies of regeneration in echinoderms were based on the determination of growth rates and on the morphological, histological and cellular basis of this phenomenon. more recently, some advances have been made in the characterization of the molecular mechanisms involved in the regeneration process of tissues and organs. studies that have been developed in our laboratory focus on a starfish species common in portuguese coast with high regeneration capability, m. glacialis. coelomic fluid, the main intra-tissue communicating medium in echinoderms, contains different types of cells, generally called coelomocytes, which are believed to participate in several functions such as, nutrient storage, gas exchange, production of connective tissue components, immune defense and tissue regeneration upon natural amputation. although there is a generally accepted morphological identification of five types of coelomocytes there is no uniform criteria until now on their classification, as well as a correlation between morphology and the above specified functions. despite, this lack of knowledge, coelomocytes are hypothesized as the most actively involved elements during the repair phase of asteroid arm regeneration. coelomocyte populations from m. glacialis were characterized by flow cytometry and microscopy in our group. these results were used to define sorting strategies by flow cytometry, in order to perform their individual transcriptome characterization. this characterization will be used to identify coelomocytes functional activities and to follow their circulating dynamics during regeneration. additionally, identified specific biomarkers for each population will allow to generate probes for flow cytometry and in situ microscopy localization of tissue localized coelomocytes. 27 weekly regeneration of central nervous system in the colonial tunicate botryllus schlosseri c anselmi, f gasparini, l manni university of padua, department of biology the study of neural stem cells and cns development, regeneration, and organization, is an active field of research aiming to identify neural stem cells, diverse neurons, and cns supporting cell types, to better understand the individual pathways and cell intrinsic factors leading to the formation of an adult brain. we used botryllus schlosseri, an invertebrate chordate with a simple cns and a robust regenerative capacity to investigate the possible presence and role of adult neural stem cells. unlike most species, where the body is long lived and maintained by cellular replacement, b. schlosseri regenerates new colonial units (buds) on a biweekly basis from stem cells that remain for life, replacing the previous generation zooids, which then die through massive apoptosis. this cycle of development includes the formation of all body organs, including the neural complex. the latter is composed of a cerebral ganglion (brain), associated to a neural gland and a dorsal organ. during bud development, the neural gland and dorsal organ rudiments produce the brain cells. we studied the brain organization during the adult life using immunolabeling and confocal microscopy, finding that its cell number changes with a specific trend. moreover, histological serial sections of adult neural complex at different stages of adult life indicated that the neural gland and the dorsal organ could represent a source of new neurons also during adult life. all these evidences suggest that b. schlosseri possesses adult stem cells involved in the neural complex maintenance. proteomic approaches for shell biominerals: insights into the biomineralization of mediterranean spiny oyster spondylus gaederopus j sakalauskaite1,2, b demarchi1, marin f2 1department of life sciences and systems biology, university of turin, italy; 2umr cnrs 5561 biogéosciences, university of burgundy-franche-comté, dijon, france. mollusca is one of the most diversified metazoan phyla and their ability to form an exoskeleton represents a true innovation from an evolutionary point of view. shell biominerals possess exceptional material properties (e.g. nacre is outstandingly resistant!) and long have been an inspiration for biomimetic materials. the current knowledge on shell biomineralization processes is mainly attributed to the development of ‘omics-based techniques which have completely revolutionized the field enabling to study the processes at the molecular level. yet, uncovering the ancient ‘biomineralization toolkit’ seems still too ambitious, owing to the great diversity of shell structural architectures, size of phylum and the lack of studies for non-model systems. spondylus gaederopus, an iconic shell of mediterranean, is such an example. spondylus diverged quite recently, but had a peculiar evolutionary pathway. the shell is very hard and it has a composite microstructure with more than three different layers (including the complex crosslamellar). the spine growth is very fast and shells are able to repair them rapidly, indicating the evolution towards energy-effective biomineralization. we focus the study on biomineralizing shell proteins, which result trapped inside the skeleton. by applying proteomic analysis on shells shellomics, we aim to characterize the shell protein repertoire controlling the biomineralization. here we present the preliminary data on spondylus, discussing possible methods for “big data” analysis and interpretation, given the paucity of comparative ‘omics datasets for non-model system. the work sheds light on the complexity and rapid evolution of molluscan skeletal matrices. botryllus schlosseri, a model ascidian for the study of stem cells v vanni, f gasparini, l manni department of biology, university of padua, padua; italy the colonial ascidian botryllus schlosseri emerged in 1950s as an important model organism for the study of developmental biology and comparative immunology. here, we highlight b. schlosseri potential for the study of stem cells in asexual reproduction and regeneration. b. schlosseri is a colonial tunicate that can reproduce both sexually and asexually. the asexual cycle is characterized by the initial formation of a thickened disc of somatic stem cells in the lateral body wall of the parent zooid. this disk gradually becomes a vesicle where organogenesis takes place, developing an adult blastozooid. stem cells are, therefore, essential for asexual development. when all the blastozooids and buds are surgically removed from a colony, leaving only the tunic with its vasculature, the remaining circulating cells are capable to regenerate new individuals through a process called vascular budding. here, some stem cells aggregate to form a vesicle, which undergoes morphogenesis regenerating the whole colony. colonies of b. schlosseri possess also the ability to fuse each other and create chimeras. chimeras share the circulatory system that allows blood cells to circulate between the fused partners. this capability was used to show that, within the chimera, somatic and germline stem cells compete to populate niches and regenerate tissue or germline organs. in the last decade, dedifferentiation capabilities were also questioned: when buds are in vitro cultured, they form epithelial monolayers and show de novo emerged stemness signature. altogether, these data show that b. schlosseri has great potential for 28 investigating stem cell involvement in developmental pathways. components of tgf-beta signaling pathway in the sponge halisarca dujardini. i borisenko1, av ereskovsky1,2 1saint-petersburg state university; saintpetersburg; russia 2institut méditerranéen de biodiversité et d'ecologie marine et continentale; marseille; france tgf-beta pathway is one of the major signaling mechanisms that orchestrate development of multicellular organisms. axial patterning in embryo development is the brightest example of its role. thus, asymmetric expression of tgf-beta superfamily ligands determines of dorso-ventral axis in bilateral animals. ligands from several families manage the different embryonic morphogenesis through regulation of proliferation, differentiation, apoptosis, cytoskeleton, adhesion and cell migration. functions of tgf-beta pathway established early in evolution of multicellularity as ligands or receptors not found in protista, whereas in cnidaria this pathway determines the directive axis of body. at the same time, tgf-beta expression data from sponges’ development tell us about involvement of tgf-beta pathway in axial patterning of porifera embryo. number of ligands may be as many as eight in demosponge amphimedon queenslandica or twenty-two in calcisponge sycon ciliatum so sponges are not simple in sense of ligand repertoire in comparison to bilateria. in this study we search through transcriptome and analyze ligands, receptors and cytoplasmic messenger proteins of tgf-beta pathway in sponge halisarca dujardini (demospongiae). eight ligand sequences were identified, and their primary structure and domain organization correspond to eumetazoan tgf-beta. three of eight identified ligands can be classified as tgf-beta sensu stricto when next five are not fall into any of eumetazoan family of tgf-beta. with hybridization in situ and quantitative transcriptomics shown that expression of some ligands restricted to oscular tube similar to wnt expression pattern. similar situation described for wnt pathway ligands in sponges, and it shows independent involvement end lineage-specific expansion of some signaling pathways members inside porifera phyla. study of structural and functional proteins in the sea anemone actinia fragacea (cnidaria) and potential biomedical interest m almeida1,2, m rocha1,2, th silva1,2, rl reis1,2,3 13b's research group, i3bs – research institute on biomaterials, biodegradables and biomimetics, university of minho, barco, guimarães, portugal 2icvs/3b’s–pt government associate laboratory, braga/guimarães, portugal; 3the discoveries centre for regenerative and precision medicine, university of minho, barco, guimarães, portugal marine biological resources provide a diverse source of proteins with application in several biotechnological fields due to their broad structural and biological properties. cnidarians are examples of marine animals with biotechnology interest. these soft body animals are distinct as they possess a unique organ specialized in the production of toxins. research has been performed in several species due to their ecological importance (e.g. jellyfish blooms; coral reefs), regenerative capacity and for their bioactive compounds. more recently, other biotechnology interests emerged including collagen of jellyfish as an alternative to mammal collagen and adhesives proteins of hydrozoans, in view of the development of biomimetic adhesives and antifouling compounds. their basic features, ecology and high diversity make cnidarians interesting models in different biotechnological fields and many are potential sustainable resources making important the investigation in different fields (e.g. chemical and biochemical composition, physical–chemical features, screening of bioactive molecules and microbioma). in this study we propose to perform molecular biology, proteomic tools and other protein characterization techniques in the pedal disc and tentacles of actinia fragacea to analyze the collagen and protein adhesive molecular features. this study could provide information of interest in the biomedical field, with focus on the development of biomaterials for tissue engineering, wound healing and drug delivery. recovery process in sponges: morphogenesis and cell sources ai lavrov1,2,3, fv bolshakov1,2, ie borisenko2, vs frolova4, db tokina5, av ereskovsky2,5 1pertsov white sea biological station, biological faculty, lomonosov moscow state university, moscow, russia; 2department of embryology, biological faculty, saint-petersburg state university, saint-petersburg, russia 3koltzov institute of developmental biology ras, moscow, russia 4department of embryology, biological faculty, lomonosov moscow state university, moscow, russia 5institut méditerranéen de biodiversité et d’ecologie marine et continentale (imbe), aix marseille university, cnrs, ird, avignon university, station marine d’endoume, marseille, france sponges (porifera) represent one of the most ancient metazoan lineages. they possess unique anatomical and tissue structure, making them promising models for evolutionary studies. the high plasticity of sponge tissue and cells provide them with outstanding recovery abilities, ranging from wound healing to re-building of a functional body from dissociated cells. we have combined several microscopy techniques to elucidate morphogenesis, cellular mechanisms and cell sources during reparative regeneration and cell reaggregation in five species from different clades: halisarca dujardinii (demospongiae), aplysina cavernicola (demosponigae), sycon sp. (calcarea), 29 leucosolenia variabilis (calcarea) and clathrina arnesenae (calcarea). the main mechanism of reparative regeneration in studied demospongiae is cell migrations and epithelio-mesenchymal transformations, involving archaeocytes and choanocytes, which are a cell source for the recovery of lost structures. in contrast, the reparative regeneration in calcarea occurs due to extensive remodeling of intact tissues near the wound through epithelial morphogenesis, accompanied by cell transdifferentiations. the cell re-aggregation in both demospongiae and calcarea involves mass cell dedifferentiation on the early stages of the process. during progressive development of multicellular aggregates, individual cell migrations and transdifferentiations ensure restoration of required cell types and intact anatomical structures. however, epithelial morphogenesis contributes to the development of calcareous sponge aggregates. thus, during recovery processes, sponges utilize diverse and complex morphogenetic mechanisms, with a particular importance of cell transdifferentiation. while all sponges demonstrate high recovery abilities, the morphogenesis and cell sources for the recovery of lost structures varies in different clades. phylogenetic and phylogeographic resolution of botrylloides leachii (savigny, 1816) in northeastern mediterranean b temiz, e ozturk, a karahan middle east technical university, institute of marine sciences; department of marine biology and fisheries; mersin; turkey globally distributed sea squirts, tunicates, are one of the most diverse taxons among chordates. two genera of colonial tunicates, botryllus thought to originated in mediterranean sea including 32 and botrylloides with 19 species, are botryllid ascidians spread among all of the world seas. the presence of 6 botryllid species in the eastern mediterranean sea particularly in israel, egypt and gulf of suez shore are confirmed which are botrylloides leachii, botrylloides niger, botrylloides anceps, botrylloides israliense, botryllus schlosseri and botryllus rosaceus. tunicates being the closest invertebrate relatives to the vertebrates are one of the widely used model organisms especially in developmental biology and immunology studies. considering their unique characteristics as including the only chordate group having the ability of whole-body regeneration, b. leachii has a pivotal role in these studies with another special feature by undergoing into hibernation when the ambient conditions are not favorable. in this study, we target to determine the evolutionary phylogenetic status of b. leachii by estimating its resolution and biodiversity in the north-eastern mediterranean sea at 8 spatial stations from antalya to hatay regions employing mitochondrial and nuclear dna markers (coi and h3) on over hundreds of samples. also the morphological investigation of the species is performed and blastogeny (an asexual reproduction type) is analyzed. besides, kızkalesi station selected as transient sampling residing is monitored through a year to understand the biodiversity characteristics combined with several ecological parameters such as salinity, temperature and tidal rhythms. deciphering target genes of the hydra head inhibitor sp5 l iglesias ollé, mc vogg, b galliot department of genetics and evolution, institute of genetics and genomics in geneva (ige3), faculty of sciences, university of geneva, switzerland in 1744, abraham trembley discovered one of the most fascinating characteristics of the hydra model organism: its amazing ability to regenerate from any missing body part. but how does this animal trigger regeneration? the head organizer, located in the most apical part of the hydra, produces two signals, the head activator and the head inhibitor, the latter preventing ectopic head formation in intact and regenerating conditions. even though hydra is used as a model system since more than 250 years, the hydra head inhibitor, a protein named sp5, was just discovered recently. the expression of sp5 is under the control of wnt/β-catenin signaling, is mainly expressed in the head and re-expressed during regeneration. sp5 rnai triggers multiple head formation in intact and regenerating animals and sp5 inhibits wnt/βcatenin signaling by repressing the expression of wnt3. in addition, sp5 has an activating effect on its own promoter, suggesting that sp5 acts as a transcriptional repressor and activator (vogg et al., 2019 nature commun). here we present an approach for the identification of sp5 target genes. finding sp5 target genes and deciphering their role will help to understand if sp5 works alone or in cooperation with other genes to restrict hydra head formation. stem cells identification in regenerating tissues of the scleractinian coral stylophora pistillata j levanoni1,2, b rinkevich1 1israel oceanographic and limnological research, national institute of oceanography; haifa; israel 2department of marine biology, leon h. charney school of marine sciences, university of haifa; haifa; israel stem cells in some cnidarian taxa were identified as far back as the 19th century. the first cells to be identified were the i-cells in hydra. since then, along with the constant increase in the number of tools in the histological and molecular toolbox, more data has been accumulated regarding stem cells and cell dynamics in the rest of the cnidarian classes. one class that has been solely neglected is the anthozoa, and specifically the scleractinian corals. coral stem cells weren’t documented so far, leading to the assumption that corals are lacking in somatic stem cells. therefore, the common theory is that their ectodermal and endodermal epithelial cells 30 are able of transdifferentiation between the various cell lineages, thus allowing prompt renewal of damaged tissues. a previous research has shown a peculiar cluster of cells at the edge of a growing coral nubbin featuring a morphology unlike any ectodermal or endodermal tissues. we suggest that these cells might be pluripotent stem cells. using histological methods as well as utilizing stemness markers, such as piwi, vasa and nanos expression, we try to distinguish between this cluster of cells and typical differentiated somatic cells and evaluate their properties. first record of non-indigenous botrylloides anceps (herdman, 1891) species along the turkish levantine coasts, confirmed by dna barcoding e öztürk, b temiz, a karahan middle east technical university, institute of marine sciences, erdemli, mersin, turkey there are nearly 3000 ascidian species identified and they are all found in seas. however, the studies on tunicates on turkish coasts are limited; thus, not much known about the ascidian fauna of turkish seas. in this study, presence of the botrylloides anceps (herdman, 1891) species along the mediterranean coast of turkey has been proven by application of dna barcoding tool for the first time. sampling was performed in coastal area of the hatay-konacık region on 26 september 2018. mitochondrial cytochrome oxidase i (coi) and nuclear h3 genes were used for identification. although b. anceps were observed in israel, australia, india, brazil and japan, only israel and australia provided gene sequences to the database. in the present study, one b. anceps (israel) species haplotype data was obtained from the genebank to compare with the present study sample. phylogeographic resolution of these 2 haplotypes was investigated by using network software. five mutation steps differences were observed between present study and previously studied (in israel) b. anceps sample for coi gene and four for h3 gene. this study is a very first record in terms of using dna barcoding tool on the b. anceps identification for the sample that was collected in turkish seas. it is being planned to do whole genome sequencing of the species as a next project. contributing of “omics” for the understanding of chondrosia reniformis collagen aggregation phenomenon ms rocha1,2, d fassini1,2, e martins1,2, al alves1,2, rl reis1,2,3, th silva1,2 13b's research group, i3bs research institute on biomaterials, biodegradables and biomimetics, university of minho, barco, guimarães, portugal 2icvs/3b’s–pt government associate laboratory, braga/guimarães, portugal 3the discoveries centre for regenerative and precision medicine, barco, guimarães, portugal collagen is the most abundant structural protein, being extremely similar in both vertebrates and invertebrates. currently, the main sources of collagen are bovine and porcine by-products, but risks of zoonosis and religious constraints have encouraged research for alternative collagen sources. in this sense, marine organisms have gained wide acceptability. although being promising biomaterials for biomedical applications, marine origin collagens have inferior mechanical properties than their mammalian counterparts. this can be mitigated by collagen crosslinking reactions, though these often involve cytotoxic chemicals; thus, a challenge for the biomedical community is the development of effective enzymatic crosslinking systems. marine sponges (phylum porifera) are a sustainable source of collagen which possess dynamic collagenous tissues (dct), a singular physiological adaptation that allows them to regulate the mechanical properties of the connective tissue matrix. this calcium-dependent phenomenon can reversibly stiffen or soften the tissues in a short time-span. in our model, chondrosia reniformis, this phenomenon is mediated by at least one stiffening factor which we recently acknowledged and partially purified, with the extract fraction interacting with collagen. however, neither the identity of the enrolled compound(s) nor the mechanisms behind the dct’s adaptability are fully comprehended. considering this, proteomic approaches are envisaged for identification and characterization of stiffening factor(s), fundamental for comprehending the dct phenomenon, aiming at the development of a novel collagen crosslinking method fitting for biotechnological applications. raveling cell type diversity and cell type regulation of the coral stylophora pistillata by whole-organism single-cell transcriptome s levy1, a sebe-pedros2, t mass1, a tanay2 1marine biology department, the leon h. charney school of marine sciences; university of haifa; haifa; israel 2department of computer science and applied mathematics and department of biological regulation; weizmann institute of science; rehovot; israel corals that are part of the phylum cnidaria, are among the earliest metazoans that possess an organized body structure. the indo-pacific scleractinian coral stylophora pistillata is a colonial zooxanthellate coral that is one of the main builders of the gulf of aqaba reefs. the coral body structure consists of two cell layers; an ectoderm and an endoderm, separated by a non-cellular gelatinous matrix called mesoglea. in this study we aim to reveal cell type diversity and the genes define each cell type. to do so we performed whole-organism single-cell transcriptome using massively parallel single-cell rna-sequencing (mars-seq) analysis. our main interest is to identify and characterize the calicoblastic cells, which are involved in calcification and skeleton formation. we are also interested in the cells that host the algal symbionts (symbiodinium), to better understand the role of the symbionts in coral 31 calcification. in addition, corals as most cnidarians have high regenerative capacity, however their stem cells population was not yet defined, therefore we would like to identify and characterize stylophora stem cells. our preliminary results show that stylophora cells are grouped into 24 cell clusters. further analysis needed in order to identify and characterize each cluster. 256 isj 15: 256-264, 2018 issn 1824-307x research report differentially expressed genes in the midguts of bmnpv-susceptible and resistant silkworm strains determined using suppression subtractive hybridization l gao1, y yang2, q yao2, k chen2* 1school of food and biological engineering, jiangsu university, zhenjiang, jiangsu province, china, 212013 2institute of life sciences, jiangsu university, zhenjiang, jiangsu province, china, 212013 accepted july 19, 2018 abstract bombyx mori nuclear polyhedrosis virus (bmnpv) has caused great economic losses to sericulture and considerable effort has been made to identify disease resistance genes in b. mori. we constructed differential expression gene libraries of the resistant near-isogenic line (nil) bc10 and susceptible strain 306 using the suppression subtractive hybridization technique. a total of 23 differentially expressed genes were obtained, of which 17 genes were upregulated in bc10, and 6 genes were upregulated in 306. these differentially expressed genes are involved in cell metabolism, transmembrane transport, cytoskeleton, protease, development and immunity. six bc10 upregulated genes were verified by real-time quantitative pcr, and the results were consistent with those of subtractive hybridization. the resistance trait of nil bc10 was inherited from the resistant strain nb, and its genetic background was 99.99% similar to that of recurrent parent 306. the upregulated genes in nil bc10 may be associated with silkworm bmnpv resistance. key words: bombyx mori, nuclear polyhedrosis virus, suppression subtractive hybridization, antivirus introduction the bombyx mori nuclear polyhedrosis virus disease is a common problem in silkworm breeding, and it causes great economic losses every year. sericultural scientists have focused their research on the pathogenesis, transmission routes, and control of the disease. screening for resistant genes is a key research area. in nature, a few strains of silkworm have natural antiviral ability to bmnpv infection. after screening 344 chinese local silkworm strains, chen et al. (1991) obtained a highly resistant strain nb, whose median lethal concentration was 1000 times higher than the susceptible strain 306. silkworm's resistance to npv disease was controlled both by dominant major genes on autosomes and modificator genes on sex chromosome z (zafar et al., 2013). liu et al. (2004) used 500 random primers to screen genomic dna of resistant strain nb, susceptible strain 306 and the nil bc8. the marker opf-072023 was found to be linked to the disease resistance gene. the segregation ratio of npv resistance molecular marker (r736) in bc1 and f2 ___________________________________________________________________________ corresponding author: keping chen institute of life sciences jiangsu university zhenjiang, 212013 jiangsu province, china e-mail: kpchen@ujs.edu.cn populations was highly consistent with the resistant trait (feng et al., 2012). these results will contribute to the location of resistant genes. it is considered that the difference of mrna and protein levels between resistant and susceptible strains is related to the mechanism of bmnpv resistance. using fluorescence differential display pcr (fdd-pcr), a high expression gene bmsop2 was screened from resistant strain nb (xu et al., 2010). using proteomic analysis, liu et al. (2010) found that the expression of beta-n-glucosidase in hemolymph of nb and bc8 was upregulated after oral administration of bmnpv. the high expression of this gene may interfere with the glycosylation of the budded virion (bv) gp64 protein that is essential for initiating secondary infections (okada et al., 2007). using proteomics, qin et al. (2012) found that the expression levels of caspase 1 and serine protease in f1 reciprocal hybrids and resistant parents nb were all higher than those in susceptible strain 306. using suppression subtractive hybridization, bao et al. (2009) analyzed the differential gene expression in the midgut and fat body of the bmnpv-resistant strain kn and susceptible strain 306 at 12-hour post-bmnpv infection. they obtained a number of upregulated genes in resistant strain. using cdna microarray, zhou et al. (2013) analyzed the differential expression of genes in the midgut of nil bc8 and 257 306 after bmnpv infection. compared with 306, the expression of 10 genes in bc8 midgut was upregulated. using genome wide microarray in resistant race (sarupat) and susceptible race (csr-2), lekha et al. (2015) found large number of differentially expressed protein. among them, sugar transporters were down regulated upon bmnpv infection, and strongly associated with bmnpv infection (lekha et al., 2016). by comparing the proteomes of infected and non-infected susceptible p50 and resistant bc9 silkworms, yu et al. (2017) identified 84 differentially expressed proteins potentially involved in resistance to bmnpv. similarly, the functions of these identified differentially expressed genes involve cytoskeleton, immune response, apoptosis, ubiquitination, transmembrane transport, protease and so on. all of these results have added to our understanding of molecular mechanism of silkworm antivirus. even so, the bmnpv resistance genes of the silkworm have not yet been completely identified. in addition to resistance genes, the genetic background of nil bc10 is exactly the same as 306. the use of nil bc10 facilitates screening of resistant genes. in this study, the suppression subtractive hybridization (ssh) method was used to construct a differentially expressed gene library of resistant strain nil bc10 and susceptible strain 306, and the genes were screened in relation to disease resistance. materials and methods silkworm strains and viruses bmnpv-resistant nil bc10 and bmnpv-susceptible silkworm strain 306 were used in this study. chen keping's method (2003) was used to select nil bc10. after eight generations of backcrossing and two generation self-crossing, the nil bc10 was developed, which has the anti-bmnpv characteristics of resistant strain nb. bmnpv (t3 strain) preserved in our laboratory was used in this study. oral exposure to the virus bmnpv (5×106 polyhedra/larva) was administrated to the newly exuviated 5th fifth instar larvae by oral inoculation. the treated silkworms were reared at standard temperature and under a photoperiod of 12 h of light and 12 h of dark. sample preparation and extraction of rna after oral administration, silkworm larvae were dissected at 0, 12, 24, and 48 hour postinfection (h pi). at each time, midgut samples from each one of 15 larvae were collected and pooled for each strain. and their midguts were cleaned with pbs and stored at -70 °c. rna was extracted by trizol reagent (invitrogen, usa) and rna was quantified on a spectrophotometer (nanodrop, usa). rna purity was tested on a260/280 and a260/230 and the integrity of the rna was tested using an agarose gel. construction of an ssh library at 24 h pi, the midgut rna was extracted and the mrna was purified from total rna using polya tract mrna isolation systems (promega, usa). a total of 2 µg mrna was transcribed to cdna using smartertm pcr cdna synthesis kit (clontech, usa). two subtractive cdna libraries were constructed using the pcr-select cdna subtraction kit (clontech, usa). the forward cdna library used 306 cdna as the driver and bc10 cdna as the tester to enrich the high expression gene in bc10. the reverse cdna library used bc10 cdna as the driver and 306 cdna as the tester, enriched the high expression genes in 306. the pcr products of the differentially expressed genes obtained by ssh were cloned into the pmd18t vector (takara, dalian, china) respectively, and the positive clones were screened by blue-white selection. table 1 primers used in real-time qpcr for confirmation of differentially expressed genes gene name length primer (5’-3’) ubiquitin binding enzyme e2 192 bp gaatctaccgaaaacatgcaacac cagggtttggttccaagaataaat death-related protein kinase 236 bp accgacgaaagttcctctctgt tttttaatgttggccccaattc trypsin-like serine protease 277 bp ggccgtcatttacctacccagtcc agggccaccggagtcaccac actin-binding protein 183 bp gcgcggatggatgtaatgcctaac gagcccgtcgtttgagttcgtt lectin 289 bp aggccgttgtgatgtcgtgctct gcgcggatggatgtaatgcctaac uncharacterized protein 254 bp ccccagttccactaacagagc ggtgagtttatgaaccgaagagt bm gapdh* 256 bp tgcccccatgtttgttgtg agtagaggcaggaatgatgttttg *gapdh: glyceraldehyde-3-phosphate dehydrogenase 258 dot-blot hybridization and sequencing 98 clear positive clones were selected from the forward and reverse ssh libraries for pcr. the equal amounts of pcr products were spotted on two nylon membranes at the same position after denaturation respectively. bc10 and 306 cdna were separately labeled using biotin random prime dna labeling kit (beyotime, china). after hybridization with the two different cdna probes, analysis of hybridization signals variance on the two membranes was performed to identify differentially expressed genes. we used actin a3 and gapdh as the internal control. the positive clones were sequenced using an abi 3100 dna analyzer (applied biosystems, usa). the nucleic acid sequences obtained were searched in the genbank database and then the blastx similarity was compared. efficiency of suppression subtractive hybridization 0.5 μl of cdna present in the subtracted and unsubtracted cdna pools were respectively used as templates. the efficiency of subtractive hybridization was verified by pcr and quantitative pcr using the gapdh specific primers. the recombinant plasmids (pmd18t-gapdh) were constructed and used as the standard plasmid for the efficiency analysis. using te buffer, 10-fold serial dilution series from 1.0×107 copies/μl to 1.0×101 copies/μl were prepared from the standard plasmid. the standard curve was prepared by using an abi 7500 fluorescence quantitative pcr instrument (applied biosystems, ca, usa). the abundances of gapdh in two samples were quantified by standard curve method (sebastião et al., 2015). all the reactions were performed in triplicates. real-time quantitative pcr six differentially expressed genes were selected and verified using relative quantitative real-time pcr. specific primers for the candidate genes were designed from est sequences (see table 1). rna samples were extracted from the midgut of the silkworm at 0, 12, 24 and 48 h pi. quantitative real-time pcr was performed using abi 7500 fluorescence quantitative pcr instrument based on sybr green fluorescent label. each sample was tested in triplicate. gapdh was used as a housekeeping control gene to normalize data among the samples. expression levels were analyzed using the 2-△△ct method (livak et al., 2001). student’s t test was conducted using spss 20.0. a significant difference was accepted at p< 0.05. results efficiency evaluation of the subtractive library to verify the efficiency of suppression subtraction, we used pcr to compare the abundances of the gapdh housekeeping gene in both the subtracted and the non-subtracted cdna pools. the results show that the gapdh gene was detectable in the 10th cycle of amplification in the unsubtracted cdna, but was still not detectable in the 25th cycle of amplification in the subtracted cdna (fig. 1a). the number of gapdh copies in unsubtracted cdna was 9.2×104 copies/µl by quantitative pcr. after subtractive hybridization, the number of gapdh copies in the subtracted cdna was 6.3×102 copies/µl (fig.1b). both results indicate successful subtraction efficiency. dot blot hybridization analysis a total of 98 clear positive clones were screened by blue white selection in the forward and reverse ssh libraries. after dot blot hybridization using forward and reverse cdnas probes, most clones showed the variations in the hybridization signals on the two membranes. the results showed that 20 clones had strong positive signals when hybridized with the bc10 cdna probe (fig. 2a) compared to their weak or no signals with the 306 cdna probe. on the contrary, 13 clones had strong positive signals when hybridized with the 306 cdna probe (fig. 2b). the positive clones verified by dot blot hybridization indicated that the differentially expressed genes were identified and could be sequenced for function analysis. fig. 1 evaluation of the subtraction efficiency of subtracted cdnas 259 fig. 2 dot blot hybridization of est clones from the forward and reverse ssh libraries. 98 colony pcr products from the forward and reverse ssh libraries were transferred to two nylon membranes a and b. a was hybridized with the biotin-labeled cdna probes from bc10. b was hybridized with the biotin-labeled cdna probes from 306. the arrows refer to clones with differential hybridization signals. the actin a3 (j2 point) and gapdh (j9 point) of b. mori housekeeping gene were used as internal control sequencing and homology analysis based on above results, the 33 differentially expressed clones were sequenced by commercial sequencing services. all sequencing results were analyzed by using blastx to identify homologous sequences in the ncbi database sequences. by comparison, a total of 23 clones were matched homologous sequences, include 17 sequences from the forward cdna library and 6 sequences from the reverse cdna library respectively (table 2). of these, 21 clones were similar to silkworm genes, and 2 genes were similar to genes of other species. according to the annotation of gene function, differentially expressed genes were divided into eight main categories, including substance metabolism, development, transmembrane transport, apoptosis, cytoskeleton, protease and immunity. the distribution of gene function in the two subtractive libraries was different. in 306, the upregulated genes were mainly involved in protein metabolism, carbohydrate metabolism and transmembrane transport. the upregulated genes in bc10 were widely distributed and involved a variety of functions. in addition, two high expression genes were identified in bc10. at present, gene function is not yet clear (fig. 3). ssh results verified by real-time quantitative pcr to verify the reliability of subtractive hybridization, we selected six genes from the forward subtractive library, including e2 ubiquitin ligase, long chain fatty acid coa ligase, trypsin like serine protease, actin binding protein, sialic binding immunolectin, and an unknown functional gene. qpcr was performed to analyze expression patterns of these genes in the midgut. there was no significant difference in the expression pattern of these genes in 306 strain and nil bc10 at 0 h pi. following bmnpv infection, increased transcript levels were observed in the two silkworm strains. in susceptible 306 strain, the transcripts of these gene were consistently detected at low levels throughout the time course. however, the expression levels of these genes were consistently higher in the resistant nil bc10 when compared to the susceptible 306 strain. remarkably, the transcripts of an unknown functional gene were consistently detected at extremely low levels in 306. conversely, the expression of this gene quickly increased and reached a peak in nil bc10 at 24 h pi (fig. 4). the results of quantitative pcr showed that the level of their expression in the resistant strain bc10 was significantly higher than that in 306, which verified the results of suppression subtractive hybridization (p<0.01). discussion the resistance of silkworm strains to bmnpv is controlled by a dominant single gene. this has been verified by previous experiments (feng et al., 2012). in bao’s subtractive experiments (2009), resistant strain kb and susceptible strain 306 were used for comparison. due to the interference of other genetic traits, the differentially expressed genes may not be related to disease resistance. in order to eliminate the interference of other genes, we successfully bred 306 near isogenic lines through the 8 generation backcross and 2 generation selfing. each generation was screened by exposure to 100% lethal dose bmnpv (2.5 × 108 polyhedral/larvae). in addition to resistance trait from nb, the genetic background of the nil bc10 had 99.99% similarity to the recurrent parent 306. in theory, the difference in expression of 260 table 2 annotation of differentially expressed genes induced by bmnpv in the midgut from nil bc8 and strain 306 isolated by ssh ssh est number protein name strain genbank id e-value bc10 up-regulated gene ssh-1 mitochondrial ribosomal protein l2 bombyx mori abf71567.1 4.00e-77 ssh-2 chromodomain helicase dna binding protein bombyx mori xp_004921642.1 1.00e-54 ssh-3 tata binding protein bombyx mori np_001037059.1 4.00e-129 ssh-4 reverse transcriptase bombyx mori aaa17752.1 2.00e-43 ssh-5 ubiquitin-conjugating enzyme e2m bombyx mori np_001040241.1 2.00e-89 ssh-6 acyl-coa dehydrogenase aedes aegypti abf18453.1 1.00e-167 ssh-7 long-chain-fatty-acid-coa ligase bombyx mori xp_012550652.1 3.00e-59 ssh-8 juvenile hormone binding protein bombyx mori np_001037074.2 2.00e-140 ssh-9 juvenile hormone epoxide hydrolase bombyx mori aaq87024.1 5.00e-33 ssh-10 serine carboxypeptidase bombyx mori xp_004929002.1 5.00e-98 ssh-11 trypsin-like serine protease bombyx mori np_001243950.1 3.00e-95 ssh-12 tpr repeat-containing protein bombyx mori xp_004928023.1 4.00e-16 ssh-13 actin-binding protein bombyx mori xp_004922082.1 1.00e-08 ssh-14 death-associated protein kinase bombyx mori np_001189466.1 1.00e-14 ssh-15 sialic acid binding ig-like lectin homo sapiens eax10517.1 4.00e-13 ssh-16 uncharacterized protein bombyx mori np_001139709.1 1.00e-86 ssh-17 uncharacterized protein bombyx mori np_001153678.1 3.00e-81 306 up-regulated gene ssh-18 translation elongation factor 2 bombyx mori abf71565.1 2.00e-54 ssh-19 60s ribosomal protein l18 bombyx mori np_001037217.1 8.00e-95 ssh-20 ribosomal protein l13 bombyx mori np_001037153.1 4.00e-117 ssh-21 alpha-glucosidase bombyx mori xp_004925073.2 4.00e-16 ssh-22 ubiquinol-cytochrome c reductase complex 14 kda protein bombyx mori np_001038957.1 2.00e-40 ssh-23 aminopeptidase n bombyx mori xp_012551671.1 0 mrna between bc10 and 306 is correlated with bmnpv resistance. this could provide a basis for screening resistance-related genes. the silkworm midgut is the frontline where silkworm initiates its defense against bmnpv infection. in this study, 23 differentially expressed genes were obtained from midgut cells. of these, 17 genes were up-regulated in bc10. the other 6 genes were up regulated in 306, which were related to susceptibility of silkworm virus disease. the six genes selected for real-time quantitative analysis were bc10 up-regulated genes with significant difference in expression. according to the results of other whole genome gene expression studies, the differentially expressed genes mainly focus on the functions of cytoskeleton, immune response, apoptosis, ubiquitination, transmembrane transport, protease and so on. of the six genes, 5 genes belong to these functions. their differential expression might account for the resistance of b. mori. the expression level of the ubiquitin-binding enzyme e2 in bc10 was higher than that in 306. ubiquitin binding enzyme e2 is an important component of the ubiquitin proteasome system (ups), which combines with ubiquitin ligase e3 for ubiquitination of target proteins and participates in the degradation of endogenous proteins (ciechanover et al., 2015). ups can selectively degrade viral proteins and limit viral growth in host cells (wang et al., 2007). in addition, the ups plays an important role in the regulation of signaling pathway during viral infection. it is generally accepted that the nf-κb pathway plays a critical role https://www.ncbi.nlm.nih.gov/protein/112983068?report=genbank&log$=protalign&blast_rank=78&rid=095fkk07016 261 fig. 3 functional classification of differentially expressed genes identified in two subtractive libraries in host anti-viral defenses. bmnpv invaded the midgut of the silkworm and stimulated nf-κb signaling pathway by interacting with the cell surface pattern recognition receptor. the activation of transcription factor nf-κb regulates downstream immune active substances, such as antimicrobial peptides and defensins (yokoi et al., 2012). in this process, the ubiquitination of nf-κb inhibitor (iκb) can abolish the inhibition of nf-κb nuclear translocation (kim et al., 2014). however, some viruses can also develop deubiquitination strategies to evade the innate immunity of the host. for example, ubiquitin-specific protease encoded by herpes simplex virus deubiquitinates iκbα and limits activation of nf-κb (ye et al., 2016). so, ubiquitination of proteins is an important mechanism used by insects to regulate immune responses. apoptosis is an important mechanism used by the lepidoptera to defend against baculovirus infection. by initiating apoptosis, the host insect actively scavenges infected cells, and this improves its ability to resist viral infection. apoptosis occurs through the activation of caspase through the apoptosis information pathway (wang et al., 2016). we have used proteomics to demonstrate that caspase-1 was highly expressed in the midgut of bmnpv-resistant silkworm larvae (qin et al., 2012). in the present study, we found high expression of the death-related protein kinase in the midgut of bc10. death-related protein kinase is a positive regulator of apoptosis and plays an important role in the apoptosis pathway induced by p19arf/p53. phosphorylated p19arf triggers the apoptosis pathway of p53 and induces the expression of specific apoptotic genes downstream, such as bax and apaf (kögel et al., 2003). in addition, death-related proteins kinase can also interact with the microtubule protein map1b without relying on the p53 mechanism to induce cell autolysis (isshiki et al., 2015). death-related protein kinase appears to be the upstream regulatory proteins of the apoptosis activation pathway. fibrous actin is the main component of the eukaryotic cytoskeleton. baculovirus infection of lepidoptera host cells requires the involvement of actin. after baculovirus enters the host cell, the actin of the host cell is aggregated to participate in the transport of the nucleocapsid from the cytoplasm to the nucleus (wang et al., 2007). the migration of actin from the cytoplasm to the nucleus is essential for viral replication and nucleocapsid assembly (newsome et al., 2015). cytochalasin d and microfilament depolymerizing agent a can bind actin to inhibit actin polymerization. in this case, the baculovirus does not produce progeny in the host cell (kasman et al., 2000). actin-binding protein can regulate the function of fibrous actin and inhibit the polymerization of fibrous actin. in this study, we found that the expression of actin-binding protein in bc10 was higher than that in 306. upregulation of actin-binding protein expression may alter the formation of f actin in cells, thereby affecting the transport and replication of baculovirus in the host cells. trypsin-like serine protease belongs to the serine protease family, and it is an important digestive enzyme in invertebrate digestive tract (wu et al., 2005). besides its digestive function, trypsin-like serine protease also participates in the activation of phenoloxidase-associated reaction (liu et al., 2017) and in the activation of the antiseptic defensin precursor invertase (hamilton et al., 2010). the expression of serine protease in the bmnpv-resistant strain nb was higher than that of 262 fig. 4 analysis of mrna expression by real-time qpcr. six genes were randomly selected for rt-qpcr verification of the ssh library. the relative expression levels of 6 genes at different time points were normalized to bm gapdh, the endogenous control. all samples were tested in triplicate. results are expressed as means ± sd strain 306 at the protein level (qin et al., 2012). in bao’s study (2009), the expression of serine protease in midgut cells of resistant strain was also confirmed to be higher than that in 306. these results showed that serine protease was indeed involved in silkworm immune defense against bmnpv infection. a possible mechanism is that serine protease inactivated the occlusion-derived virus (odv) at the initial infection site of the silkworm gut and prevented the odv from adhering to the midgut epithelial cells and then spreading to the hemolymph (nakazawa et al., 2004). lectin was highly expressed in nil bc10. two kinds of silkworm agglutinin have been identified, bmlbp and bmiml (kim et al., 2003). they belong to the c-lectin family. there are two glycosyl recognition domains in the molecular structure, which have strong adsorption capacity for bacteria and nematode surface lipid polysaccharides (zhou et al., 2017). in vitro experiments showed that the combination of recombinant silkworm agglutinin and inactivated micrococcus luteus cells could cause their aggregation in the blood cells of the silkworm (watanabe et al., 2006). recombinant immulectin were injected into the hemocoel of the fifth instar manduca sexta larvae and triggered encapsulation and melanization responses (yu et al., 2004). there are many genes similar to agglutinin in the silkworm genome. the newly discovered lectin proteins are likely to be involved in the early recognition of silkworm infection and activate the humoral immunity. we found two unidentified genes that were highly expressed in nil bc10. in susceptible strain 306, gene expression remained at a low level, which was 40 times different from that of bc10. through homology analysis, the sequence contains the conservative domain of the transcription-activating factor (mbf-2). mbf-2 stabilizes the binding of the dna-binding factor (ftz-f1) to dna and coregulates the transcription of the ftz-f1-dependent gene with ftz-f1 (liu et al., 2000). ftz-f1 is a nuclear receptor type transcription factor, which trigger gene expression and regulate molting of larvae. in drosophila melanogaster, loss-of-function mutation in dmftz-f1 caused severe ecdysis failure (cho et al., 2014). the enzyme encoded by the baculovirus egt gene result in the inactivation of molting hormone, which leads to the disruption of larval molting and pupation and facilitates the proliferation of the viral progeny (zhang et al., 2012). previous reports documented that sloughing of infected midgut cells prevented the virus from spreading beyond the midgut (washburn et al., 1998). the highly expressed mbf-2 in nil bc10 increased sensitivity to ecdysones and promoted intestinal developmental resistance against lethal baculovirus infection. in susceptible strain 306, the upregulated genes were mainly involved in protein metabolism, energy metabolism, and transmembrane transport. these functions may be related to the susceptibility of silkworm to viruses. in previous study, sf21 cells were infected by recombinant baculovirus expressing vesicular stomatitis virus rna dependent rna polymerase. the results showed that the host sf21 cell translation elongation factor could be tightly bound to the expressed protein subunit (das et al., 1998). the p6 protein of the cauliflower mosaic virus interacts with the host ribosomal proteins l13, l18, l24 and initial factor eif3, which initiate the transcription and translation of the virus gene (bureau et al., 2004). aminopeptidase n (apn) is a transmembrane glycoprotein abundant on the 263 apical membrane of intestinal epithelial cells. besides the functions of peptide metabolism, cell movement and adhesion, apn is also the receptor of the virus. in silkworm, there are few studies on the role of apn in npv infection. in pig, aminopeptidase n (papn) can promote the infection of porcine epidemic diarrhea virus (pedv) to host cells (shirato et al., 2016). overexpression of papn in mice confers its susceptibility to pedv infection (park et al., 2015). these studies indicate that bmnpv can completely embezzle proteins from host cells for viral entry and translation of their own components. the resistant strain nil bc10 is highly similar to the susceptible strain 306 in genetic background, but there are differences in bmnpv resistance traits. using them as analysis materials for gene expression difference analysis can eliminate the interference of other trait genes and isolate the resistant genes more efficiently. the differentially expressed genes screened in this study will help clarify the resistance mechanism of b. mori to bmnpv infection. acknowledgments this study was supported by the research innovation plan of graduate students in jiangsu province (cx07b_09x) and (cx08b_09x), 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https://www.ncbi.nlm.nih.gov/pubmed/?term=islam%20mt%5bauthor%5d&cauthor=true&cauthor_uid=27449937 https://www.ncbi.nlm.nih.gov/pubmed/27449937 https://www.ncbi.nlm.nih.gov/pubmed/27449937 404 isj 14: 404-413, 2017 issn 1824-307x research report molecular characterization and transcriptional analysis of a crustacean heat shock protein 10 gene in shrimp litopenaeus vannamei m-q wang1, b-j wang1, ke-y jiang1, m liu1, si-y han1,3, l wang1, 2 1key laboratory of experimental marine biology, institute of oceanology, chinese academy of sciences, qingdao 266071, china 2laboratory for marine biology and biotechnology, qingdao national laboratory for marine science and technology, qingdao 266237, china 3university of chinese academy of sciences, beijing 100049, china accepted october 20, 2017 abstract heat shock proteins (hsps) are the most abundant and ubiquitous soluble intracellular proteins which conserved phylogenetically in all living organisms from archaebacteria to humans. recent research achievements indicated that hsp10s might not only be involved in the responses to environmental stresses, but also play a pivotal role in the host defenses mechanism. in the present study, a cdna of 715 bp for the pacific white shrimp litopenaeus vannamei hsp10 (designated as lvhsp10) was cloned via rapid amplification of cdna ends (race) technique. the complete cdna sequence of lvhsp10 contained an open reading frame (orf) of 309 bp, which encoded a protein of 102 amino acids. the protein sequence of lvhsp10 shared over 80 % similarity with previously identified hsp10s. there were a cpn10 domain and a chaperonins hsp10/cpn10 signature in the protein sequence of lvhsp10. the mrna transcripts of lvhsp10 were constitutively expressed in all the tested tissues, including eyestalk, gill, gonad, heart, hemocytes, hepatopancreas, intestine, muscle, nerve and stomach, with the highest expression level in hepatopancreas. the mrna expression profiles of lvhsp10 in hepatopancreas could be significantly induced by the stimulation of vibrio parahaemolyticus, white spot syndrome virus (wssv), and low and high ph challenge. these results provided useful information of the potential roles of lvhsp10 in the defense mechanism of shrimp against various biological stimulations and multiple environmental stresses. key words: heat shock protein 10; hepatopancreas; litopenaeus vannamei introduction heat shock proteins (hsps) are the most abundant and ubiquitous soluble intracellular proteins which phylogenetically conserved in all living organisms from prokaryotes to eukaryotes (schlesinger, 1990). in recent years, hsps have attracted considerable interest among immunologists in the context of transcriptional regulation, evolution and innate immunity (feder and hofmann, 1999). according to the molecular mass, hsps could be classified into several families, such as hsp100s, hsp90s, hsp70s, hsp60s, hsp40s, low molecular mass hsps (small hsps) and so on (sørensen et al., 2003). to date, a large number of ___________________________________________________________________________ corresponding author: lei w ang key laboratory of experimental marine biology institute of oceanology chinese academy of sciences qingdao 266071, china e-mail: wanglei@qdio.ac.cn hsp members have been identified. among them, hsp10 was first discovered and identified in the serum of pregnant women and displayed immunosuppressive properties (morton et al., 1974). hsp10 is a highly conserved 10 kda protein (hartman et al., 1992), which co-chaperones with another heat shock protein hsp60 for protein folding as well as the assembly and disassembly of protein complexes to be involved in many biological processes, such as cell apoptosis (lin et al., 2001), cellular differentiation (cappello et al., 2005), cell proliferation (sasu et al., 2001) and innate immunity (chen et al., 1999). for example, mammalian hsp10 exhibited anti-inflammatory activity by inhibiting lipopolysaccharide (lps) induced toll-like receptor (tlr) signaling pathway via interactions with hsp60 (dobbin et al., 2005). moreover, hsp10 alone could be widely involved in protecting cells from stresses caused by infection, inflammation and so on (jia et al., 2011). up to the present, most studies of hsp10 are 405 focused on typical model organisms (sun and macrae, 2005). and relatively little of gene information regarding hsp10 has been obtained from marine animals, such as apostichopus japonicas (xu et al., 2014), lutjanus sanguineus (zhang et al., 2011), oryzias latipes (hirayama et al., 2006), penaeus monodon (shi et al., 2016), salmo salar (andreassen et al., 2009), scylla paramamosain (ding et al., 2013) and xenopus tropicalis (klein et al., 2002). a clearly time-dependent mrna expression pattern of hsp10 identified in lutjanus sanguineus indicated that hsp10, might co-chaperoned with hsp60, played a pivotal role in the host defenses mechanism of marine animals (zhang et al., 2011). however, information about hsp10s from marine animals is still few and fragmentary, and more research evidences are still needed to illustrate their exact roles in the innate immune system. the pacific white shrimp litopenaeus vannamei has been widely cultured in the world as an important commercial species (li and xiang, 2013a). however, in the past two decades, outbreaks of infectious disease associated with bacteria, such as vibrio parahaemolyticus, and viruses, such as white spot syndrome virus (wssv), have become a major constraint, resulting in mass shrimp mortality, reductions in farmed shrimp production and considerable economic losses (li and xiang, 2013b; zhang et al., 2016). moreover, as an aquatic livestock, white shrimp are also suffered from multiple environmental stress during culture, and some of them, such as ammonia/nitrite accumulation, hypoxia stress and ph challenge, could be harmful to the survival of shrimps (liang et al., 2016; han et al., 2017a, b). a better understanding of mechanisms of stress tolerance is necessary for the health management of shrimp farming. in the present study, a novel hsp10 genes have been cloned and investigated in white shrimp (designated as lvhsp10), and the main objectives of the present study were (1) to characterize the molecular feature of lvhsp10 genes, (2) to detect the tissue distribution of its mrna transcripts and (3) to investigate its temporal mrna expression pattern after invading microbes and environmental factors stimulation and compare it with the previously identified white shrimp hsp60 (designated as lvhsp60). materials and methods shrimp and tissues sample collection the white shrimps used in the present study were obtained from ruizi seafood development co. ltd., qingdao, china, and all the experiments were conducted in accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health (nih). the study protocol and all experimental design were conducted with approval from experimental animal ethics committee of institute of oceanology, chinese academy of sciences. white shrimps, with body weight 8 12 g, were cultured placed in 640 l cylindrical tanks with 500 l air-pumped circulating seawater at 20 ± 1 c for two weeks before processing. hemolymph was extracted from the ventral sinus of at least three untreated shrimps using a sterile syringe preloaded with equal volume of anticoagulant buffer (nacl 510 mmol l-1, glucose 100 mmol l-1, citric acid 200 mmol l-1, tri-sodium citrate 30 mmol l-1 and edta·2na 10 mmol l-1, ph 7.3). then the hemocytes were collected by centrifugation at 800g for 10 min at 4 c. tissues including eyestalk, gill, gonad, heart, hepatopancreas, intestine (mid gut), muscle, nerve and stomach were collected from at least three untreated shrimps, kept in rnalater (am7020, thermo fisher scientific, usa) and stored at -80 c until rna isolation. table 1 oligonucleotide primers used in the present study primer sequence (5`-3`) tm (c) brief information adaptor primer ggccacgcgtcgactagtac 60 anchor primer for 3` race adaptor primer-oligo (dt) ggccacgcgtcgactagtact17vn olido (dt) for cdna synthetize lvef-1α-qrt-f gtattggaacagtgcccgt 60 internal control for real-time pcr lvef-1α-qrt-r catctccacagactttacctcag 60 internal control for real-time pcr lvhsp10-cds-f atggctggtgctctgaagaggttt 64 gene specific primer for cds lvhsp10-cds-r ttactcggtcttcatcttggccaaaag 64 gene specific primer for cds lvhsp10-qrt-f ggttgctgttggagaggga 60 gene specific primer for real-time pcr lvhsp10-qrt-r tgacctttgtgccaccga 60 gene specific primer for real-time pcr lvhsp10-race-f1 ggcacaaaggtcaccctggaggagaag 64 gene specific primer for 3` race lvhsp10-race-f2 gggtcaattcgtaggctgatg 64 gene specific primer for 3` race lvhsp60-qrt-f attgtccgcaaggctatc 60 gene specific primer for real-time pcr lvhsp60-qrt-r atctccagacgcttccat 60 gene specific primer for real-time pcr m13-47 cgccagggttttcccagtcacgac 56 vector primer for sequencing rv-m gagcggataacaatttcacacagg 56 vector primer for sequencing 406 fig. 1 nucleotide and deduced amino acid sequences of lvhsp10. the nucleotides and amino acids were numbered along the left margin. the function domain was in shade. the asterisks indicated the stop codon. immune stimulation, ph challenge assay and sample collection approximately 200 shrimps were employed for microbe stimulation assay. the v. parahaemolyticus suspension and wssv stock were prepared according to previous reports (yi et al., 2014; xia et al., 2015; sha et al., 2016). the shrimps were randomly divided into three groups and each group contained about 60 70 individuals. the shrimps were received an injection at the abdominal segment with 100 μl phosphate buffered saline (pbs, ph 7.4, 10010023, thermo fisher scientific, usa), v. parahaemolyticus suspension (1×105 cfus μl-1, in pbs) and wssv stock (1×105 copies μl-1, in pbs), respectively. the injected shrimps were returned to seawater tanks immediately and the hepatopancreas of at least three individuals were randomly sampled from each group at 3, 6, 12, 24 and 48 h post injection, kept in rnalater and stored at -80 °c until rna isolation. approximately 200 shrimps were employed for ph challenge assay. the shrimps were randomly divided into three groups and each group contained about 60 70 individuals. two kind of seawater at ph 6.7 and ph 9.7 were prepared using 4 mol l-1 hcl or 4 mol l-1 naoh. two groups of shrimps were amongst the seawater at ph 6.7 and ph 9.7 after acclimation in normal seawater (ph 8.2), respectively. the ph values were measured daily using a ph meter (phb-4, yidian scientific, china). the hepatopancreas of at least three individuals in the control group and experiment groups were collected at 1, 3, 7, 14 and 28 d after the ph challenge, kept in rnalater and stored at -80 °c until rna isolation. rna isolation and cdna synthesis total rna was isolated from various tissues using trizol reagent (15596026, thermo fisher scientific, usa). the synthesis of first strand was carried out with promega m-mlv using the dnase i (rq1, m6101, promega, usa) treated total rna as template and adaptor primer-oligo (dt) as primer (table 1). the reaction mixture was incubated at 42 c for 1 h, terminated by heating to 95 c for 5 min, and then stored at -80 c. cloning the full-length cdna of lvhsp10 the partial length sequence of lvhsp10 cdna was obtained from the transcriptome database of white shrimp (zhao et al., 2017). two gene-specific primers, lvhsp10-race-f1/2, were designed using primer premier 5.00 based on this partial length sequence to clone the 3’ end of lvhsp10 cdna by rapid-amplification of cdna ends (race) technique. and the coding sequence (cds) of lvhsp10 was amplified and confirmed using another two gene-specific primers, lvhsp10-cds-f/r, which was also designed using primer premier 5.00. all pcr amplification was performed in a mj mini personal thermal cycler (bio-rad, usa), and the pcr products were purified using monarch dna gel extraction kit (t1020s, neb, usa) and cloned into the pmd18-t simple vector (d103a, takara, japan). after being transformed into the competent cells escherichia coli strain dh5α (cb101-03, tiangen, china), the positive recombinants were identified via anti-ampicillin selection and verified by pcr screening using m13-47 and rv-m primers (table 1). three of the positive clones were sequenced using a 407 prism 3730xl automated sequencer (thermo fisher scientific, usa). bioinformatical analysis of lvhsp10 cdna and protein sequences the search for protein sequence similarity was conducted with blastp 2.6.0. the deduced protein sequences of lvhsp10 were analyzed by the editseq module in lasergene program suite 14.0.0.88. the function domains of lvhsp10 were predicted with simple modular architecture research tool (smart) 7.0. multiple sequence alignments were performed with clustal omega 1.2.4 and visualized using multiple alignment show module in sequence manipulation suite 2.0. a neighbor-joining (nj) phylogenic tree was constructed with mega 7.0.21. to derive confidence value for the phylogeny analysis, bootstrap trials were replicated 1,000 times. expression pattern analysis via real-time quantitative rt-pcr the mrna transcripts of lvhsp10 and the previous identified lvhsp60 (zhou et al., 2010) in different tissues or their temporal expression pattern in hepatopancreas of shrimps stimulated with various microbes or ph challenge were investigated by quantitative real-time pcr (qrt-pcr) technique. all qrt-pcr reactions were performed with the sybr premix extaq (tli rnaseh plus) (rr420, takara, japan) using 100 ng cdna template in a linegene k fqd-48a (a4) fluorescence quantitative pcr detection system (bioer, china). all the primers for qrt-pcr were designed using perlprimer 1.1.21 and listed in table 1. the mrna expression levels of lvhsp10 and lvhsp60 were normalized to those of elongation factor 1 α (ef-1α) for each sample. the relative mrna expression levels of lvhsp10 and lvhsp60 were generated using comparative ct method (2-δδct method) (schmittgen and livak, 2008). the data were subjected to one-way analysis of variance (anova) followed by a multiple comparison using ibm spss statistics 23.0.0.0, and the p values less than 0.05 were considered statistically significant. results sequence features of lvhsp10 the full-length cdna sequence of lvhsp10 was obtained by 3’ race technique, and deposited in genbank under the accession number mf062460. it comprised 715 bp, containing a 5’ untranslated regions (utr) of 80 bp, a 3’ utr of 326 bp with a poly a tail and an open reading frame (orf) of 309 bp. the orf encoded a polypeptide of 102 amino acid residues with a calculated molecular mass of approximately 11.04 kda and a theoretical isoelectric point (pi) of 8.12. the deduced amino acid sequence of lvhsp10 contained a cpn10 domain (from r7 to m99, fig. 1). the deduced protein sequence of lvhsp10 exhibited high similarity with other previously identified hsp10s, such as 93 % identity with that of p. monodon (als05376) (shi et al., 2016) and 80 % with s. paramamosain (agi74966) (ding et al., 2013). an alignment of the protein sequence of lvhsp10 with those of previously identified hsp10s was shown in figure 2, and a chaperonins hsp10/cpn10 signature (from f8 to i32) was revealed. the nj phylogenetic tree based on protein sequences from multiple hsp10s was positioned separately into two main branches, and lvhsp10 were clustered with its homologue from the black tiger shrimp p. monodon (fig. 3). fig. 2 multiple alignments of lvhsp10 with previous known hsp10s. the black shadow region indicated positions where all sequences share the same amino acid residue. similar amino acids were shaded in grey. gaps were indicated by dashes to improve the alignment. the chaperonins hsp10/cpn10 signatures were boxed. the sequences and their accession numbers are as follows: daphnia magna, kzs03047; hyalella azteca, xp_018022648; nicrophorus vespilloides, xp_017783781; penaeus monodon, als05376 and scylla paramamosain, agi74966. 408 fig. 3 consensus neighbor-joining phylogenetic based on the protein sequences of hsp10s from different organisms. the evolutionary history was inferred using the neighbor-joining method. the bootstrap consensus tree inferred from 1,000 replicates was taken to represent the evolutionary history of the taxa analyzed. all positions containing gaps and missing data were eliminated. the numbers at the forks indicated the bootstrap value. the sequences and their accession numbers are as follows: apostichopus japonicas, aif71190; galeruca daurica, arr95803; gallus gallus, aab86581; homo sapiens, caa53455; ixodes pacificus, aat92186; lutjanus sanguineus, adm63094; monopterus albus, aav37068; musca domestica, aqy54358; oryzias latipes, cab40895; paracyclopina nana, adv59557; paralichthys olivaceus, abb76383; penaeus monodon, als05376; salmo salar, np_001133144; scylla paramamosain, agi74966; tigriopus japonicas, aca03519 and wuchereria bancrofti, ejw73405. tissue distribution of lvhsp10 the qrt-pcr was employed to detect the tissue distribution of lvhsp10 mrna transcripts with ef-1α as internal control. the lvhsp10 mrna transcripts could be detected in all the tested tissues, including eyestalk, gill, gonad, heart, hemocytes, hepatopancreas, intestine, muscle, nerve and stomach. the highest mrna expression level was found in hepatopancreas, which was 9.12-fold (p < 0.05) of that in muscle, followed by hemocytes, gill and intestine, which were 5.19-fold, 4.51-fold and 4.27-fold of that in muscle (p < 0.05), respectively (fig. 4). expression profiles of lvhsp10 and lvhsp60 post microbe stimulation the mrna expression levels of lvhsp10 and lvhsp60 were all up-regulated post the two kinds of microbe stimulation. the mrna expression level of lvhsp10 was significantly up-regulated at 6 h post v. parahaemolyticus stimulation (3.37-fold compared with the origin level, p < 0.05), and the highest level was observed at 12 h (5.99-fold, p < 0.05, fig. 5a). while after stimulated with v. parahaemolyticus, the mrna scripts of lvhsp60 significantly increased at 3 h post stimulation (2.34-fold, p < 0.05) and reached the peak at 12 h (7.10-fold, p < 0.05), kept at a high level at 24 h (2.27-fold, p < 0.05) and then decreased to the origin level at 48 h (fig. 5b). in the wssv stimulation group, the mrna transcripts of lvhsp10 significantly increased at 3 h post stimulation (1.97-fold, p < 0.05) and reached the maximum level at 12 h (8.15-fold, p < 0.05), kept at a high level at 24 h (4.19-fold, p < 0.05) and then decreased but still higher than the origin level at 48 h (2.17-fold, p < 0.05, fig. 5c). while the mrna expression level of lvhsp60 was significantly up-regulated at 6 h post stimulation (6.21-fold, p < 0.05) and reached the peak at 12 h (9.12-fold, p < 0.05), kept at a high level at 24 h (5.93-fold, p < 0.05) and then down-regulated but still higher than the origin level at 48 h (2.21-fold, p < 0.05, fig. 5d). expression profiles of lvhsp10 and lvhsp60 post high or low ph change in the high ph challenge experiments, the mrna expression level of lvhsp10 was significantly up-regulated at 1 d post high ph challenge (3.97-fold, p < 0.05) and reached the peak at 7 d (8.07-fold, p < 0.05), kept at a high level at 14 d (7.88-fold, p < 0.05) and then down-regulated to a 409 fig. 4 tissue distribution of lvhsp10 mrna transcripts detected by qrt-pcr technique. the mrna transcripts levels in eyestalk, gill, gonad, heart, hemocytes, hepatopancreas, intestine, muscle, nerve and stomach of three untreated shrimps were normalized to that of muscle. the ef-1α gene was used as an internal control to calibrate the cdna template for each sample. vertical bars represent mean ± sd (n = 3), and bars with different characters were significantly different (p < 0.05), while bars with same characters were not significantly different. lower level at 28 d (0.43-fold, p < 0.05, fig. 6a). during the high ph challenge experiments, the mrna transcripts of lvhsp60 significantly increased at 1 d post high ph challenge (3.39-fold, p < 0.05) and reached the peak at 7 d (9.10-fold, p < 0.05), kept at a high level at 14 d (8.87-fold, p < 0.05) and then decreased to a lower level at 28 d (0.39-fold, p < 0.05, fig. 6b). in the low ph challenge group, the mrna scripts of lvhsp10 significantly increased at 3 d post low ph challenge (3.87-fold, p < 0.05) and reached the peak at 7 d (5.75-fold, p < 0.05), kept at a high level at 14 d (3.62-fold, p < 0.05) and then decreased to the origin level at 28 d (fig. 6c). while the mrna expression level of lvhsp60 was significantly up-regulated at 1 d post low ph challenge (2.47-fold, p < 0.05) and reached the peak at 7 d (6.95-fold, p < 0.05), kept at a high level at 14 d (2.58-fold, p < 0.05) and was then down-regulated to the origin level at 28 d (fig. 6d). discussion hsps are the most abundant and ubiquitous soluble intracellular proteins which conserved phylogenetically in all living organisms, including archaebacterial, bacteria and eukaryotes (schlesinger, 1990). recent research achievements indicated that hsp10s might not only be involved in the responses to environmental stresses, but also play essential roles in the innate immune defenses mechanism (jia et al., 2011). however, information about hsp10s from marine animals is still few and fragmentary. in the present study, the full-length cdna of hsp10 was cloned from white shrimp l. vannamei. the deduced polypeptide of lvhsp10 consisted of 102 amino acids, and its calculated molecular weight was 11.04 kda, which was close to those from vertebrate and invertebrate. the protein sequence of lvhsp10 shared over 80 % similarities with other identified hsp10s. moreover, a cpn10 domain and a chaperonins hsp10/cpn10 signature were revealed from the amino acid sequence of lvhsp10. additionally, in the nj phylogenetic tree, lvhsp10 were clustered with its homologue from the black tiger shrimp p. monodon. the conserved function domain and signature sequence of lvhsp10, high similarity with other identified hsp10s and the phylogenetic relationship collectively suggested that lvhsp10 was a novel member of invertebrate hsp10 family, and it could have similar functions to those from vertebrates and other invertebrates. to investigate the potential function of lvhsp10 in shrimp, the distribution of its mrna transcripts in different tissues was detected by qrt-pcr technique. the mrna transcripts of lvhsp10 were observed to be constitutively expressed in all the detected tissues, including eyestalk, gill, gonad, heart, 410 fig. 5 temporal mrna expression profiles of lvhsp10 and lvhsp60 detected via qrt-pcr technique in white shrimp hepatopancreas at 0, 3, 6, 12, 24 and 48 h post microbe stimulation. a: temporal mrna expression profiles of lvhsp10 post v. parahaemolyticus stimulation; b: temporal mrna expression profiles of lvhsp60 post v. parahaemolyticus stimulation; c: temporal mrna expression profiles of lvhsp10 post wssv stimulation; d: temporal mrna expression profiles of lvhsp60 post wssv stimulation. the ef-1α gene was used as an internal control to calibrate the cdna template for each sample. vertical bars represent mean ± sd (n = 3), and bars with different characters were significantly different (p < 0.05), while bars with same characters were not significantly different. hemocytes, hepatopancreas, intestine, muscle, nerve and stomach. the ubiquity of lvhsp10 mrna transcripts indicated that it could be involved in many important physiological processes of shrimps. the highest mrna expression level of lvhsp10 was observed in hepatopancreas, which was as high as 9.12-fold of that in muscle, followed by hemocytes, gill and intestine. similar mrna transcripts distribution was also observed in hsp10 from the black tiger shrimp p. monodon, whose highest mrna expression level was also found in the hepatopancreas (shi et al., 2016). the hepatopancreas was considered as the main immune related organ in mollusks and crustaceans (liu et al., 2009; song et al., 2015), hemocytes are the major immune cells and respond to invaders mainly through phagocytosis (canesi et al., 2002), gill has been reported as the first defense line against invading pathogens in lower animals (ellis, 2001), while intestine was observed to be involve in immune responses with hepatopancreas via the ‘liver-gut axis’ (chang et al., 2012). the high mrna expression levels of lvhsp10 in these tissues confirmed the hypothesis that lvhsp10 could be involved in the innate immune system of shrimp. to further understand the immunological and physiological roles of lvhsp10, its temporal expression profiles in hepatopancreas post microbe stimulation or ph challenge was detected by qrt-pcr technique and compared with those of lvhsp60. in previous researches, a clear time-dependent expression pattern of hsp10 from humphead snapper l. sanguineus was observed after bacteria challenge (zhang et al., 2011), while hsp10 from the black tiger shrimp p. monodon could be induced after high ph exposure, but exhibited stable expressions at low ph challenge in the hepatopancreas (shi et al., 2016). in the present 411 fig. 6 temporal mrna expression profiles of lvhsp10 and lvhsp60 detected via qrt-pcr technique in white shrimp hepatopancreas at 0, 1, 3, 7, 14 and 28 d post high/low ph challenge. a: temporal mrna expression profiles of lvhsp10 post high ph challenge; b: temporal mrna expression profiles of lvhsp60 post high ph challenge; c: temporal mrna expression profiles of lvhsp10 post low ph challenge; d: temporal mrna expression profiles of lvhsp60 post low ph challenge. the ef-1α gene was used as an internal control to calibrate the cdna template for each sample. vertical bars represent mean ± sd (n = 3), and bars with different characters were significantly different (p < 0.05), while bars with same characters were not significantly different. study, lvhsp10 mrna transcripts could be significantly induced by the stimulation of v. parahaemolyticus and wssv, and also significantly increased in the high or low ph challenge experiment, suggesting that lvhsp10 play a pivotal role in the host defenses mechanism not only against the cute phase microbe infection but also the long term environmental stresses. moreover, it has been further observed that the change tendencies of lvhsp10 and lvhsp60 mrna transcripts were similar to each other in most cases, which is consist with the observation in previous reports (cappello et al., 2005; lin et al., 2009; xu et al., 2014) and further confirmed the hypothesis that hsp10 might co-chaperones with hsp60. in conclusion, the full-length cdnas of lvhsp10 were obtained from white shrimp, and its mrna expression profiles were detected when the shrimps were exposed to microbe stimulation and high/low ph challenge. lvhsp10 was found to be involved in responses to the cute phase microbe infection, as well as the long term environmental stresses. the results obtained from this study would provide useful information of the potential roles of lvhsp10 in the defense mechanism of shrimp against various biological stimulations and multiple environmental stresses. acknowledgement this research was supported by the key research program of the chinese academy of sciences (kfzd-sw-106) and the major projects of shandong province (2015zdzx05002). we would like to thank the expert reviewers for their constructive suggestions and enlightening comments during the revision. reference andreassen r, lunner s, høyheim b. characterization of full-length sequenced cdna inserts (flics) from atlantic salmon (salmo salar). bmc genomics 10: 502, 2009. 412 canesi l, gallo g, gavioli m, pruzzo c. bacteria-hemocyte interactions and phagocytosis in marine bivalves. microsc. res. 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shanghai ocean university, shanghai, 201306 accepted july 18, 2019 abstract the hard clam (meretrix meretrix) is a commercially and ecologically important benthic mollusk in south and southeast asia. growth is one of the most important and complex traits affecting clam breeding. therefore, identification of key growth factor genes and their association with growth traits are the primary prerequisites for in-depth study of the mechanisms of growth regulation and molecular marker-assisted selection of the hard clam. in the present study, we generated the full-length cdna sequences of the growth-related genes bmp2 and bmp7 in m. meretrix (mmbmp2 and mmbmp7) and then investigated their gene expression patterns and associations with growth traits. the full-length cdna of mmbmp2 was 2641 base pairs (bp), with a 1395 bp open reading frame (orf) encoding 464 amino acids. the full-length mmbmp7 cdna was 3161 bp, with a 1257 bp orf encoding 418 amino acids. in adult clams, expression of mmbmp2 and mmbmp7 were significantly higher in the mantle than in the other five tested tissues (p < 0.05). among the seven developmental stages of m. meretrix, expression of mmbmp2 was the highest in juveniles (p < 0.05), and expression of mmbmp7 was very low in 4-cell embryo and eyespot larva stages (p < 0.05). two hundred and sixty-three individuals from two populations were screened for mmbmp2 and mmbmp7 polymorphisms, and single nucleotide polymorphisms (snps) associated with growth traits in mmbmp2 was found in both populations. subsequent analysis revealed that two snps in mmbmp2 were shared between the populations. one was a726g, for which individuals with genotypes ag had significantly higher growth traits than those with genotype aa (p < 0.05). the other was a732t, for which individuals with genotype at had significantly higher growth traits than those with genotype aa (p < 0.05). these results suggest that bmp2 markers may have potential value in molecular selective breeding to improve clam growth. key words: meretrix meretrix; bmp2; bmp7; snp; growth traits introduction the transforming growth factor-beta (tgf-β) supergene family plays various roles in the regulation of cell growth, cell specialization, and morphogenesis (massague, 1998; derynck and akhurst, 2007). more than 40 members of tgf-β have been identified in mammals (li et al., 2011), including bone morphogenetic proteins (bmps), smads, and activin. bmps are multi-functional growth factors, and their major function is to induce the formation of both bone and cartilage in vertebrates (i.e. the process of biomineralization) ___________________________________________________________________________ corresponding author: yinghui dong college of biological & environmental sciences zhejiang w anli university 8 south qianhu rd, ningbo, zhejiang 315100, p. r. china e-mail: dongyinghui118@126.com (chen et al., 2004; xiao et al., 2007). thus, most bone-related molecular studies focus on bmp2, bmp4, and bmp7 (salazar et al., 2016). bmps also play a role in cell growth and differentiation, embryogenesis, hematopoiesis, and neurogenesis (chen et al., 2004; bragdon et al., 2011). bmps have been found in invertebrates, including echinoderms (hwang et al., 1994; shih et al., 2002), arthropods (wharton et al., 1991; fritsch et al., 2010), mollusks (nederbragt et al., 2002; iijima et al., 2008; miyashita et al., 2008), platyhelminths (freitas et al., 2009), cnidarians (reinhardt et al., 2004; zoccola et al., 2009), and poriferans (müller et al., 2011). among the members of the bmps family, bmp2 is the factor that can induce bone formation alone, inhibit myogenic differentiation, and promote skeletal cell differentiation (musgrave et al., 2001). during mailto:dongyinghui118@126.com 142 table 1 primers used for mmbmp2 and mmbmp7 genes in the experiments embryonic development, bmp2 not only promotes embryonic muscle growth by inducing the expression of myogenic genes, but also inhibits muscle growth by inducing apoptosis (amthor et al., 1998). bmp7 is a key factor in bone homeostasis and spinal development (cook and rueger, 1996). previous studies indicated that bmp7 is abundantly expressed in articular cartilage and synovial membrane obtained from human patients undergoing autologous chondrocyte implantation (schmal et al., 2012). furthermore, researchers discovered that bmp7 in mouse was expressed in a wide variety of tissues during development (lyons et al., 1995) and was strongly expressed in the developing myocardium of embryos (dudley and robertson, 2010). bmp7-deficient mice were found to exhibit defects in eye development, renal development, and skeletal patterning. the deletion of bmp7 gene results in lens development defects, affects the development of ribs and skulls, and causes polydactyly of hind limbs. (dudley et al., 1995; luo et al., 1995). because of their important roles in growth and development, bmp2 and bmp7 have become widely studied genes in mollusks. several researchers reported that bmps and their homologs are involved in the regulation of morphogenesis in the shells of mollusks (herpin et al., 2010; lelong et al., 2010). furthermore, dpp-bmp2/4 is expressed around the shell gland of the embryo in gastropods such as lymnaea stagnalis and patella vulgata, which suggests that it may be involved in determination of the boundary of the embryonic shell region and in shell formation (nederbragt et al., 2002). the high-level expression of pinctada fucata bmp2 in the inner layer of mantle tissue suggests that it plays an important role in the formation of nacre (miyashita et al., 2008). in pinctada martensii, the microstructure observed using a scanning electron microscope indicated a disordered growth status in the nacre and obvious holes in the prismatic layer in the dsrna-pm-bmp7-injected group, which suggests that bmp7 plays a crucial role in the nacre and prismatic layer formation process of the shell (yan et al., 2014). the hard clam (meretrix meretrix) is a commercially important bivalve species that is distributed in the intertidal zone of south and primer name sequence from 5’to 3’ application mmbmp2-f1 atggcgtgtcctctcagtttac 3’ race cloning mmbmp2-r1 atcactccccagcctgtccttcaatg 5’ race cloning mmbmp2-f2 gtgggcaggatttcactgtagg verifying the sequence of cdna mmbmp2-r2 gtagcattgaaggacaggctggggagt verifying the sequence of cdna mmbmp2-f3 ctaacagcagcgacagtgtgt snp mmbmp2-r3 ctggacttcatcttcgtggaa snp mmbmp2-f4 ccagtctacctacagaacagcc snp mmbmp2-r4 tccgtttagtccgtgataagc snp mmbmp2-f5 tatcacggactaaacggagca snp mmbmp2-r5 cgtaaactaccttgactggaagca snp real-2-f tcagtcgggtgtattttagcaga real time pcr real-2-r cattttttccagcaggtcaagtg real time pcr mmbmp7-f1 gtcaggtgagcgatgtagggtcgtttc 3’ race cloning mmbmp7-r1 caagtgtctgctggttcccgattcc 5’ race cloning mmbmp7-f2 aagttgtgataaaactgcgcg verifying the sequence of cdna mmbmp7-r2 aactatatatagcttgcatc verifying the sequence of cdna mmbmp7-f3 tttaggcaatttcccgagac snp mmbmp7-r3 ccttgtcgtgcctaagatgc snp mmbmp7-f4 ttccgcatcttaggcacgac snp mmbmp7-r4 gggtaaccttcaggggcgat snp mmbmp7-f5 attatcgcccctgaaggttac snp mmbmp7-r5 aaacgaccctacatcgctcac snp real-7-f gttttcgcctgttgtggtg real time pcr real-7-r gagtgtgcggtgttcgtgta real time pcr 18s-f ctttcaaatgtctgccctatcaact real time pcr 18s-r tcccgtattgttatttttcgtcact real time pcr 143 southeast asia (liu et al., 2006; tang et al., 2006). with the success of artificial breeding, clam culture has become an economically important aquaculture industry in china. although a selective breeding program for genetic improvement of growth traits of m. meretrix has begun (wang et al., 2011), association analysis between phenotype and genotype has still been relatively rare to date. however, molecular marker-assisted selection of growth traits would probably further increase selection efficiency and lead to rapid progress in improving the process of clam breeding (lande and thompson, 1990; hospital et al., 1997; moreau et al., 1998; xie and xu, 2010). therefore, identification of growth-related markers for further development of phenotype-selective breeding is very necessary. studies of growth-related functional genes of m. meretrix, including growth factor receptor-bound protein 2 (gao et al., 2015), cathepsin b (yao et al., 2011), sulfotransferase (wang et al., 2012), and fructose-1, 6-diphosphate aldolase 9 (wang et al., 2012), have been conducted. however, the m. meretrix bmp2 and bmp7 genes (mmbmp2 and mmbmp7) have not been studied to date. in the current study, we investigated mmbmp2 and mmbmp7 by cloning their full-length cdnas and examining their expression levels during embryonic development and in adult tissues. we also screened and analyzed associations between single nucleotide polymorphisms (snps) and growth traits. the results of this study will improve our understanding of the functions of mmbmp2 and mmbmp7 and provide a basis for molecular breeding programs for m. meretrix. materials and methods experimental materials and sample collection for cloning and gene expression analysis of mmbmp2 and mmbmp7, adult clams were obtained from the ningbo danyan aquaculture company, zhejiang, china. six tissues (digestive gland, mantle, adductor muscle, foot, siphon, and gills) were dissected, immediately frozen in liquid nitrogen, and stored at -80 °c. samples from seven developmental stages (unfertilized mature eggs, 4-cell embryos, blastulae, gastrulae, trochophores, eyespot larvae, and juvenile clams) were collected and preserved at -80 °c. a total of 263 individuals were randomly sampled at the same time from two populations: 136 individuals from the jiangsu population (js) and 127 individuals from wanli no. 2 strain (wl2). js was collected from the wild population located in jiangsu province, china. wl2, a new fast-growing variety with a dark gray shell color and zigzag patterns, had been selected for five generations by our research team. the following six growth index parameters, total weight, soft weight, shell weight, shell width, shell length, and shell height, were measured for each clam. the mantles of each clam were collected and stored at -80 °c. cloning the full-length cdna of mmbmp2 and mmbmp7 total rna was extracted from the mantle using trizol reagent (sangon, shanghai, china). rna integrity was examined by electrophoresis on a formaldehyde-denatured 1.2% agarose gel and staining with ethidium bromide, while the quality and quantity were assessed by ultraviolet spectrophotometry. first-strand cdna was synthesized using the simple modular architecture research tool (smart) rapid amplification of cdna ends (race) reagents (clontech, usa) according to the manufacturer’s instructions. based on the 454 cdna library of m. meretrix (genbank accession no. sra021052), the expressed sequence tags of bmp2 and bmp7 genes were retrieved. the primers for 3’-race (mmbmp2-f1, mmbmp7-f1) and 5’-race (mmbmp2-r1, mmbmp7-r1) were designed (table 1). the amplification was conducted as follows: 5 cycles of 94 °c for 30 s, 72 °c for 3 min; 5 cycles of 94 °c for 30 s, 70 °c for 30 s, and 72 °c for 3 min; 25 cycles of 94 °c for 30 s, 68 °c for 30 s, and a final extension at 72 °c for 3 min. the pcr products were purified using a gel extraction kit (tiangen, sichuan, china). the purified pcr products were cloned into the pmd18-t vector (takara), which was transformed into dh5α cells (tiangen) according to the manufacturer’s protocols, and the positive clones were sequenced. the full-length cdna sequence was determined by piecing together the sequences of the 3’ and 5’ race products. to confirm the accuracy of cloning and sequencing, the full-length cdna was re-amplified with high fidelity polymerase (takara) using gene-specific primers (mmbmp2-f2 and mmbmp2-r2, mmbmp7-f2 and mmbmp7-r2) (table 1) that were designed based on the above-mentioned cdna sequences. pcr products were cloned and sequenced following the procedures described above. sequence analysis the sequences were spliced using the blast algorithm in the national center for biotechnology information database (http://www.ncbi.nlm.nih.gov/blast/). the deduced amino acid sequences were analyzed using the smart tool (http://smart.embl-heidelberg.de) to predict conserved domains. the presence and locations of the signal peptide and cleavage sites in the amino-acid sequence were predicted using the signal p 4.0 server (http://www.cbs.dtu.dk/services/signalp/). protein transmembrane were analyzed using the tmhmm server (http://www.cbs.dtu.dk/services/tmhmm). multiple alignments of bmp2 and bmp7 domains structures from m. meretrix and other species were performed using the clustalw2 multiple alignment program (http://www.ebi.ac.uk/tools/msa/clustalw2/). a phylogenetic tree was constructed using the neighbor-joining method with mega 6.0. quantitative expression analysis of mmbmp2 and mmbmp7 the expression levels of mmbmp2 and mmbmp7 at seven developmental stages (n > 500, three sets of samples for each stage) and in six tissues from adults (four sets of samples for each tissue) were analyzed using real-time quantitative reverse transcription pcr (qrt-pcr), with three 144 technical repeats for each pcr reaction. total rna was extracted from the samples using the method described above. the primers for mmbmp2 and mmbmp7, real-2-f and real-2-r, and real-7-f and real-7-r (table 1) were designed. primers 18s-f and 18s-r (table 1) were used as the internal reference for qrt-pcr. pcr amplification was performed in a 20 μl reaction volume containing 10 μl of itaq universal sybr green supermix (bio-rad, ca, usa), 7.2 μl of deionized water, 0.8 μl of the first strand cdna, and 1 μl of forward and reverse primers. amplification was performed using the following thermal cycling conditions: incubation at 94 °c for 20 s, 40 cycles of 94 °c for 3 s, 60 °c for 15 s, and 72 °c for 10 s. all qrt-pcr analyses were performed using three biological replicates, and negative controls were run with each primer pair. the results of qrt-pcr analysis were based on the ct values of the pcr products, and the expression levels of mmbmp2 and mmbmp7 were analyzed using the comparative ct method. statistical analysis was performed using spss 20.0 (chicago, il, usa). differences in relative mmbmp2 and mmbmp7 mrna expression levels among different developmental stages and different adult tissues were compared by one-way analysis of variance (anova). multiple comparisons were conducted using the student-newman-keuls test. differences were considered significant at p < 0.05 and extremely significant at p < 0.01. snp identification and association analysis rna samples were extracted from all 263 clams. based on the cdna sequences of mmbmp2 and mmbmp7, six pairs of primers (2-f3, 2-r3, 2-f4, 2-r4, 2-f5, 2-r5, 7-f3, 7-r3, 7-f4, 7-r4, 7-f5 and 7-r5) were selected (table 1). pcr amplification was performed in 50 µl reaction volumes containing 2 µl of template cdna, 19 µl of dh2o, 25 µl of pcr mix, and 2 µl of each primer. the pcr conditions were as follows: 94 °c for 3 min; 35 cycles at 94 °c for 30 s, 58 °c for 1 min, and 72 °c for 2 min; and a final extension at 72 °c for 5 min. each amplification product was verified by electrophoresis on a 1.0% agarose gel and staining with ethidium bromide. the amplicons representing unique banding patterns were sent to sangon biotech (shanghai, china) for sequencing in both directions. the sequences were aligned using mega6.0 software. mutation sites were named according to the position of the initiation codon. total weight, soft weight, shell width, shell length, shell height, and shell weight of the clams were analyzed by one-way anova using spss 20.0. the association of snps with growth traits in the two populations was analyzed using the above method. snp markers with genotypes that showed a significant correlation with growth traits were studied by post hoc multiple comparison (duncan method). results cdna sequence analysis the full-length mmbmp2 cdna was 2641 bp (genbank accession no. kp250876) and contained a 1395-bp open reading frame (orf) encoding 464 amino acids. the calculated molecular mass of the deduced mature mmbmp2 was 52.906 kda, and the theoretical isoelectric point was 9.33. analysis with signalp revealed that the deduced amino acid sequence contained a signal peptide of 25 amino acids (mtpsyrvsilvlavllselitstyt). the full-length mmbmp7 cdna was 3161 bp (genbank accession no. kp250875) and contained a 1254-bp orf encoding 418 amino acids. the calculated molecular mass of the deduced mature mmbmp7 was 47.54 kda, and the theoretical isoelectric point was 7.81. analysis with tmhmm showed that the mmbmp7 protein contained a potential transmembrane domain that was located between amino acid residues 12 and 31. smart analysis showed that the deduced amino acid sequence of them both contained a tgf-β superfamily domain (table 2). a phylogenetic tree was constructed using the neighbor-joining method and the deduced amino acid sequences of mmbmp2 and mmbmp7 and bmps from some selected animals available in the ncbi database (fig. 1). the tree obtained showed that bmp2 and bmp7 were divided into two major groups. one group contained all mollusks, whereas the other group contained vertebrates. in the bmp2 mollusk group, azumapecten farreri clustered with table 2 comparison of cdna sequence between mmbmp2 and mmbmp7 cdna sequence information mmbmp2 mmbmp7 full-length 2641bp 3161bp orf 1395bp 1257bp number of amino acids 464aa 418aa 5’utr 546bp 431bp 3’utr 703bp 1476bp protein molecular mass 52.906kda 47.54kda tgf-β superfamily domain 363cys-464arg 317cys-418his 145 fig. 1 neighbor-joining phylogenetic tree of bmp2 and bmp7 between m. meretrix and other selected species using mega6.0 software. the abbreviation and the genbank accession numbers used to construct phylogenetic tree are given in table 3 table 3 species and genbank accession numbers of bmps used for multiple alignment and phylogenetic analysis species abbreviation genbank no. species abbreviation genbank no. meretrix meretrix mmbmp2 alg64479 meretrix meretrix mmbmp7 alg64478 azumapecten farreri afbmp2 agf68558.1 tegillarca granosa tgbmp7 afp57673.1 crassostrea gigas cgbmp2 ekc18750.1 crassostrea gigas cgbmp7 ekc34211.1 patella vulgata pvbmp2 aam33143.1 pinctada martensii pmbmp7 ags32053.1 carassius auratus cabmp2 ban17326.1 danio rerio drbmp7 aaf17558.1 danio rerio drbmp2 aac25595.1 ictalurus punctatus ipbmp7 ahh42433.1 paralichthys olivaceus pobmp2 bad16743.1 xenopus laevis xlbmp7 aai08478.1 xenopus tropicalis xtbmp2 aai70888.1 rattus norvegicus rnbmp7 edl85136.1 bos taurus btbmp2 aai42130.1 equus caballus ecbmp7 adk93983.1 mus musculus mubmp2 edl28371.1 mus musculus mubmp7 edl06610.1 pan troglodytes ptbmp2 jaa30020.1 homo sapiens hsbmp7 aah08584.1 homo sapiens hsbmp2 acv32596.1 146 fig. 2 (a) multiple alignment of the mmbmp2 tgf-β domain structures peptide sequence with other mollusc species, (b) multiple alignment of the mmbmp7 tgf-β domain structures peptide sequence with other mollusc species. identities are shaded very light red and similarities are shaded lightest red. the * indicate the shared cysteine residues in compared species, the letter box is additional cysteine residue in mmbmp7. (c) prediction of bmp2 tgf-β domains in mollusc species, (d) prediction of bmp7 tgf-β domains in mollusc species. blue boxes indicate transmembrane region, green box indicates coiled coil region, and pink and red boxes mean low complexity region crassostrea gigas and then with m. meretrix and p. vulgata. in the bmp7 mollusk group, p. martensii first clustered with c. gigas and then with tegillarca granosa and m. meretrix. mmbmp2 shared the highest identity (53.1%) with a. farreri, and mmbmp7 shared the highest identity (58.7%) with p. martensii. for the mature bmp2 protein of the compared mollusks, the tgf-β domain structure peptide sequences shared seven cysteine residues and the spacing between cysteine residues was well conserved (fig. 2). the bmp7 protein had seven cysteine residues in m. meretrix but six common cysteines in t. granosa, p. martensii and c. gigas (fig. 2). quantitative expression analysis the expression patterns of mmbmp2 and mmbmp7 in the embryos/larvae and adult tissues were analyzed using qrt-pcr. in adult clams, the expression of mmbmp2 in the mantle was extremely significantly higher than that in the other five tissues (p < 0.01) (fig. 3), suggesting that it might be involved in shell growth. among the seven developmental stages, mmbmp2 transcript levels were the highest in juvenile clams (p < 0.05), followed by unfertilized mature eggs, blastulae, gastrulae, and eyespot larvae, with the lowest expression in 4-cell embryos and trochophores (fig. 3). this result may be related to shell formation in the juvenile clams. in adults, mmbmp7 expression was also significantly higher in the mantle than in the other five tissues (p < 0.05) (fig. 3). the mantle is a shell-forming tissue, and its high expression of bmps is in agreement with its role in bone formation. among the developmental stages, mmbmp7 expression was significantly higher in unfertilized mature eggs, blastulae, gastrulae, trochophores, and juvenile clams than in the other two stages (p < 0.05) (fig. 3), which suggests that it may be involved in morphogenesis and formation of the hard shell. mmbmp2 and mmbmp7 were expressed to different degrees during development, which indicates that bmps might play a role in ontogeny. association of snps in mmbmp2 and mmbmp7 with growth traits the snp analysis revealed that eight mmbmp2 snps (t134c, a476g, c570t, t585c, a715t, a726g, a732t, and t786c) were present in the js population, of which three (t585c, a726g, and a732t) were associated with growth traits. five snps (g390a, c570t, a726g, a732t, and t786c) in exons of mmbmp2 were identified in the wl2 population, including two (a726g and a732t) that were potentially associated with growth traits. js individuals with genotype tc at position t585c grew faster than those with the tt genotype in terms of shell length, shell width, and shell height (p < 0.05). 147 fig. 3 analysis of expression differences of mmbmp2 and mmbmp7. (a) analysis of expression difference of mmbmp2 in six tissues, (b) analysis of expression difference of mmbmp7 in six tissues, (c) analysis of expression difference of mmbmp2 in seven developmental stages, (d) analysis of expression difference of mmbmp7 in seven developmental stages. the same letters indicate no difference in the level of expression, whereas different letters indicate significant differences in expression levels among various developmental stages (p < 0.05). **p < 0.01, *p < 0.05 clams with genotype ag at a726g exhibited faster growth in all growth traits except shell weight compared with aa individuals (p < 0.05). the snp a732t at genotype was also associated with significantly higher growth traits than the aa genotype (p < 0.05) (table 4). wl2 individuals with genotype ag at position a726g had significantly better growth traits compared with genotype aa (p < 0.05). multiple comparison analysis showed that individuals with the a732t at genotype grew faster than those with the aa genotype in all growth traits measured (p < 0.05) (table 4). comparison of the loci between the js and wl2 groups revealed that the populations shared two snps at a726g and a732t in the mmbmp2 coding region. association analysis showed that individuals with the ag genotype at locus 726 had significantly higher growth traits than those with the aa genotype in both groups (p < 0.05). additionally, individuals with the at genotype at locus 732 had significantly higher growth traits than those with the aa genotype in both groups (p < 0.05). snp a732t was significantly associated with better outcomes for all growth traits tested in both the js and wl2 populations (p < 0.05). the snp analysis revealed the presence of three mmbmp7 snps (c32t, c126g, and g144a) in the js population, including two (c32t and g144a) associated with growth traits. js individuals with genotype ct at position c32t grew faster than those with the cc genotype in terms of shell height, shell weight, soft weight, and shell weight (p < 0.05). clams with genotype ga at g144a exhibited faster growth in terms of shell width, 148 table 4 correlation analysis of snps in the mmbmp2 with growth traits in two groups note: bold parts are the snps associated with growth traits, p < 0.05; underline parts are the snps potentially associated with growth traits, p < 0.01 total weight, soft weight, and shell weight compared with gg individuals (p < 0.05) (table 5). three snps (c32t, c126g, and g306a) were found in exons of mmbmp7 from the wl2 population, but none was associated with growth traits. discussion in vertebrates, every mature bmp shares seven conserved cysteines (bragdon et al., 2011), six of which build a cystine knot by forming three intrachain disulfide bonds to resist heat, denaturants, and extreme ph levels. the remaining cysteine residue forms an interchain disulfide bond that links two monomers into active heteroor homodimers (kingsley, 1994). this unique cysteine is absent in some mollusks (t. granosa, c. gigas, and p. martensii) (yan et al., 2014). this absence raises the possibility that bmp7 in some mollusks might not form dimers or might form noncovalently linked dimers between the subunits, which could be dynamic and subject to regulation. however, both bmp2 and bmp7 in m. meretrix contained all seven cysteine residues. gene expression analysis showed that expression of mmbmp2 and mmbmp7 was the highest in the mantle, which suggests that they might be involved in shell growth in m. meretrix. in p. martensii, expression of bmp2 and bmp7 was also the highest in the mantle (miyashita et al., 2008; yan et al., 2014). previous studies showed that bmps of mollusks may play an important role in formation of the shell boundary. using northern blot and in situ hybridization assays, miyashita et al. (2008) found that expression of bmp2 occurred predominantly in the mantle, suggesting that bmp2 played a key role in nacre formation. zhou et al. (2010) reported that p. martensii bmp2 was involved in repair after shell incision. furthermore, the expression of bmp7b increased significantly 24 h after shell incision in p. martensii. in the current study, both mmbmp7 and mmbmp2 were expressed in all six tissues tested, which suggests that they may be involved in a wide range of biological processes. for example, mgdf, which has high similarity to bmp2, in c. gigas, along with other growth factors, is involved in differentiation of the digestive gland from stem cells into functional cells (lelong et al., 2010). expression of mmbmp2 and mmbmp7 was detected at every embryonic stage of m. meretrix during development, suggesting that they may be involved in differentiation of early tissues and organs. bivalves have a common process of shell formation that occurs at the very early stage of larval development. during the gastrula stage, the shell formative region sinks, evaginates and enlarges until group locus genotype sample percent (%) shell length (cm) shell width (cm) shell height (cm) total weight (g) soft weight (g) shell weight (g) jiangsu 585 t>c tt 121 88.97 4.11±0.61a 2.05±0.32a 3.32±0.48a 17.28±6.68 8.38±3.37 8.89±3.42 tc 15 11.03 4.52±0.57b 2.27±0.33b 3.65±0.47b 20.51±5.86 9.83±2.70 10.68±3.39 726 a>g aa 63 46.32 3.97±0.62a 1.97±0.33a 3.20±0.48a 15.50±6.32a 7.49±3.25a 8.01±3.16a ag 50 36.76 4.26±0.60b 2.14±0.32b 3.42±0.48b 18.35±6.21b 8.92±3.05b 9.43±3.29b gg 23 16.91 4.47±0.45b 2.22±0.24b 3.64±0.40b 21.92±6.34c 10.61±3.08 11.31±3.45c 732 a>t aa 70 51.47 4.01±0.63a 2.00±0.34a 3.24±0.49a 15.91±6.39a 7.68±3.25a 8.23±3.22a ta 50 36.76 4.29±0.59b 2.15±0.32b 3.44±0.47b 18.95±6.47b 9.27±3.19b 9.68±3.43b tt 16 11.76 4.40±0.47b 2.17±0.24 3.60±0.42b 21.04±6.52b 10.05±3.20 10.99±3.50b wanli 2 726 a>g aa 41 32.28 35.95±2.56a 17.28±1.34a 30.04±2.23a 11.35±2.70a 5.57±1.31a 5.78±1.44 ag 59 46.46 37.36±3.14b 17.96±1.68b 31.07±2.51b 12.71±3.43b 6.30±1.60b 6.42±1.88 gg 27 21.26 36.18±3.27 17.34±1.59 30.29±2.77 11.64±3.60 5.70±1.67 5.94±1.96 732 a>t aa 51 40.16 36.25±2.63a 17.40±1.38a 30.28±2.32 11.56±2.83a 5.70±1.33a 5.86±1.55a at 52 40.94 37.51±3.22b 18.09±1.70b 31.17±2.52a 12.95±3.51b 6.38±1.64b 6.57±1.91b tt 24 18.90 35.70±3.11a 17.05±1.46a 29.94±2.69b 11.15±3.36a 5.50±1.62a 5.65±1.77a 149 table 5 correlation analysis of snps in the mmbmp7 with growth traits in two groups note: bold parts are the snps associated with growth traits, p < 0.05; underline parts are the snps potentially associated with growth traits, p < 0.01 the whole embryo is encapsulated (kin et al., 2009). afterward, the conchiolin is secreted and calcified, and simultaneously the shell forms a hinged part in the back and divides into two pieces until bivalve stage i (prodissoconch i) is reached. during formation of the velum, the phase ii embryo shell (prodissoconch ii) continues to form (kakoi et al., 2008). miyazaki et al. (2010) reported that the shell of p. martensii begins to form prodissoconch i at the trochophore stage and then forms prodissoconch ii at the d-shaped larvae stage. in the current study, mmbmp2 and mmbmp7 were highly expressed at the trochophore stage, which suggests that they might play an important role in the formation of the protegulum. moreover, expression of mmbmp2 was the highest in the juvenile clam stage, which indicates that organs and shells grew fast and developed rapidly at this stage. molecular marker-assisted breeding can be used to select new varieties with certain traits at the molecular level and thereby greatly improve breeding efficiency. snps, as the third generation of molecular markers, are a powerful tool for assessing genetic diversity and conducting association analysis. in marine mollusks, association analysis between snps of candidate genes and economic traits such as growth and stress tolerance has been conducted. for example, guo et al. (2012) identified a growth-related snp locus in the 3'utr region of tgf-β receptor type 1 in chlamys farreri. in the exon region of myostatin in argopecten irradians, two snp loci associated with growth were identified (guo et al., 2011). bao et al. (2013) characterized three immune-related snp loci in the hemoglobin of t. granosa. in the current study, we identified three snps potentially associated with growth traits in mmbmp2 of the js population and two in the wl2 population, and two of them were common to the two populations. for mmbmp7, we found two snps potentially associated with growth traits in the js population and none in the wl2 population. the individuals with the ag genotype of snp a726g grew significantly faster than those with the aa genotype, and individuals with the at genotype of snp a732t grew significantly faster than those with the aa genotype in both populations. furthermore, these two snps were located within the coding region of mmbmp2 without amino acid changes (a726g encoding threonine, a732t encoding valine). many studies have shown that synonymous mutations regulate gene transcription and translation. greenwood and kelsoe (2003) found that the synonymous mutation of many genes in promoters and introns could affect transcriptional efficiency. kimchi-sarfaty et al. (2007) reported that the synonymous mutation of the mdr1 gene could change the spatial structure of the protein. capon et al. 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receptor. mar.biotechnol. 11(2): 260-269, 2009. 66 isj 15: 66-82, 2018 issn 1824-307x research report comparative proteomic analysis of the silkworm (bombyx mori l.) silk gland reveals yield heterosis y zhang, h tang, y yang*, p lü, q yao, k chen* institute of life sciences, jiangsu university, zhenjiang 212013, china accepted february 26, 2018 abstract heterosis is a prevalent phenomenon in nature and is widely found in animals and plants, but the molecular mechanism of heterosis is still unclear. as the model insect of lepidoptera, bombyx mori is an ideal material to carry out heterosis research. in current study, we employed proteomic and genetic cross approaches to globally identify differentially expressed proteins in parental silkworms jingsong and haoyue and their f1 hybrids. the results showed that there were significant differences between hybrids and their parents. in all, 28 differentially expressed proteins were successfully identified through maldi-tof-tof mass spectrometry and database searches. interestingly, three silk-related proteins were also obtained and identified, including fibroin l-chain, nd-sd mutant fibroin light chain and fibrohexamerin, which were further confirmed by quantitative real-time polymerase chain reaction (qrtpcr). consistent trends were also found in other genes. taken together, our work not only provides the theoretical basis for the study of molecular mechanism of heterosis, but also provides candidate proteins and genes for the improvement of yield of silkworm. key words: heterosis, bombyx mori, proteomics, qrt-pcr introduction heterosis was firstly proposed by shull in 1914, referring to a hybrid offspring with better productivity, growth, development, and resistance compared with its parents (shull, 1948). the theoretical hypothesis about heterosis mainly includes “dominance” model, “overdominance” model and epistasis theory. the dominant hypothesis emphasizes the interactions between dominant genes and recessive genes, suggesting that favorable dominant genes can cover up the effects of unfavorable recessive genes, and ______________________________________________________________ corresponding author: yanhua yang keping chen institute of life sciences jiangsu university 301 xuefu road 212013, zhenjiang, pr china e-mail: yanhuayang@126.com list of abbreviations: maldi-tof ms, matrix-assisted laser desorption/ionization time of flight mass spectrometry; pmsf, phenylmethanesulfonyl fluoride; egta, ethylene glycol tetraacetic acid; dtt, 1,4-dithio-dlthreitol; acn, acetonitrile; sds-page, sodium dodecyl sulphate polyacrylamide gel electrophoresis; tfa, trifluoroacetic acid lead to the improvement of f1 vigor. the overdominance model indicates that the heterozygous allele plays an important role in the formation of heterosis. the epistasis theory emphasizes nonadditive genetic effects based on the overdominance hypothesis and believes that nonalleles also play roles in heterosis. in recent years, many studies on heterosis have been carried out (kawamura et al., 2016; kwan et al., 2016; li et al., 2016a; li et al., 2016b; wu et al., 2016). the results from transcriptome showed that overdominance, dominance, low dominance and additive effects coexisted in bombyx mori heterosis, among which overdominance was the primary mode of gene action (wang et al., 2015). recent researches have also suggested that additional mechanisms may explain heterosis in arabidopsis and maize, including expression of small rna (srna) and dna methylation, respectively (hou et al., 2017; lauria et al., 2017). to verify these mechanisms, transcriptional or gene expression profiles have been completed in maize, rice, pufferfish, and silkworm. li et al. (2016a) used the transcriptomic method to analyze gene expression patterns and genetic variations between parents’ rubber trees and their productive hybrids, including single nucleotide polymorphisms (snps) and small insertions/deletions (indels) (li et al., 2016a). the results showed that the higher yield of f1 hybrids was positively correlated with their higher mailto:yanhuayang@126.com 67 fig.1 results of character investigation from parental silkworms js, hy and their f1 hybrids. (a, b): weight measurement of silkworm larva and silk gland at 96h of the fifth instar; (c): measurement of whole cocoon weight, cocoon shell weight and pupal weight; (d): cocoon shell percentage and dead worm cocoon rate; (e): weight measurement of moth and egg; (f): the number of silkworm eggs. a) e) f) c) d) b) 68 genomic heterozygosity, and the genome-wide genetic variations played a vital role in maintaining the heterosis of rubber tree. the research from maize illustrated that cell expansion played a major role in the heterosis of aba (abscisic acid)-mediated maize seed germination. the up-regulation of inactivation gene zmaba8ox1b also contributed to seed germination by promoting cell expansion (li et al., 2016a). zhu et al. suggested that cumulative small advantages of the single-locus effects and two-locus interactions could explain the genetic basis of seedling heterosis in f1 hybrid (zhu et al., 2016). until now, however, there are no consistent conclusions in the investigation of molecular mechanism of heterosis. as an important economic insect and model insect of lepidoptera, the bombyx mori (l.) is an ideal material for molecular biological research because of its some advantages, such as clear genome and genetic background, high breeding coefficient, and easy control of growth environment and so on. the silk gland of b. mori, including three functionally distinct regions: anterior silk gland (asg), middle silk gland (msg), and posterior silk gland (psg), is a special organ for synthesis and secretion of the silk fibroin and the sericin. as the matter of fact, the silk gland of silkworm is not developed before the 4th instar, which grows rapidly at the 5th instar and will become the largest organ of silkworm at the late 5th instar (lv, 2008). the silk from silkworm is a very good and extensively utilized textile material, whose yield and quality mainly depends on silk fibroin and sericin synthesized and secreted by the silk gland. therefore, in recent years, researchers have paid more and more attentions on the silk gland of silkworm (dhawan et al., 2003; ma et al., 2011; miller et al., 2015; chen et al., 2017; xue et al., 2017). xue et al. used dge-seq (digital gene expression sequencing) to analyze gene expression profile of silk gland and examine the effect of tio2 nanoparticles on the silk protein synthesis of silkworm (xue et al., 2017). the results showed that the addition of tio2 could promote the synthesis of silk protein. chen et al. found that bmdredd, a homologue of the starting apoptotic protease, played a key role in the apoptosis of silk gland (chen et al., 2017). ma et al. investigated the effect of ras1 oncogene overexpression on the production of silk protein. the result showed that the activation of ras1 could increase the size of silk gland cells, promote protein synthesis of silk gland, activate mrna translational ribosome biogenesis, and eventually result in the increase of silk yield (ma et al., 2011). however, these studies mainly focused on the genome, transcriptome and proteome of silkworm silk glands, the heterosis research at the protein level are still few. currently, the proteomic methods, including two-dimensional electrophoresis and mass spectrometry, have successfully been employed to identify the specific proteins from different tissues and organs of silkworm (moghaddam et al., 2008; hou et al., 2010; li et al., 2010; liu et al., 2010; qin et al., 2012; zhong et al., 2013; feng et al., 2014; kannan et al., 2016; li et al., 2016a). the results from proteome provided some helpful evidences and clues for understanding the developmental and physiological processes of silkworm. in this study, we employed proteomic and genetic cross approaches to globally identify differentially expressed proteins of silk glands between parental silkworms jingsong, haoyue and their f1 hybrids. our objectives were 1) to detect, identify and classify the different proteins between parental silkworms and their f1 hybrids, 2) to find out the heterosis-related proteins and genes and attempt to decipher the molecular mechanism of silkworm heterosis. materials and methods experimental materials parental silkworms jingsong (js, chinese line) and haoyue (hy, japanese line), and their direct cross (js-hy, d-f1), reciprocal cross (hy-js, r-f1) hybrids were used for this experiment, which were fed in the institute of life sciences, jiangsu university, zhenjiang, china. the larvae of 1st~3rd instar were fed with the chopped tender leaves and 4th~5th instar larvae were fed with the matured leaves. the larvae were dissected to obtain silk glands at 96h of 5th instar, immediately frozen in liquid nitrogen and stored at −70 °c refrigerator for later use (at least 50 silkworms of each sample). the experiments were repeated three times. protein sample preparation the silk glands were quickly grounded into fine powder in liquid nitrogen and homogenized on ice for 5 min in pre-cooled extraction buffer (20 mm tris-hcl ph 7.5, 250 mm sucrose, 10 mm egta, 1 mm pmsf, 1 mm dtt, and 1% triton (v/v) x-100). then the proteins were extracted by phenol method, as described by cilia et al (cilia et al., 2009). finally, the protein samples were vacuum-dried. the dry powder was dissolved with lysis buffer (7 m urea, 2 m thiourea, 4% w/v chaps, 1% w/v dtt and 2% v/v biolyte, ph 3-10), and overnight at 4 °c. the mixtures were centrifuged at 12,000g for 20 min, 4 °c. the protein concentration was measured by bradford method (bradford, 1976). two-dimensional electrophoresis (2-de) the isoelectric focusing (ief) was carried out through ettan ipgphor 3 electrophoresis system and 17 cm immobilized ph gradient (ipg) dry gel strips with ph 4-7 liner range (bio-rad, usa) was used. about 2 mg protein samples were loaded (passive rehydration, room temperature, 11~12 h). then ief was performed at 20 °c with a voltage gradient of 100 v for 1 h, 300 v for 1 h, 500 v for 1 h, 1000 v for 1 h (linear), next 10000 v, linear for 5 h, and then remained 10000 v until a total voltage of 40000 vh. subsequently, the gel strips were equilibrated for 15 min in equilibration buffer (50 mm tris-hcl ph6.8, 2.5% w/v sds, 30% v/v glycerol and 1% w/v dtt) and then equilibrated for 15 min again with the same equilibration buffer without dtt. the second dimension sds-page was performed with a laemmli buffer system (12% resolving gels) (laemmli et al., 1970). the electrophoresis was divided into two steps, that is, 5 w/gel, 50min, then 15 w/gel, 4~5h. at last, the gels were dyed with 0.116% coomassie brilliant blue (cbb) r-250 and scanned with a high precision scanner (imagescanner iii, ge healthcare life sciences) at a resolution of 300 dpi. 69 image analysis image analysis was carried out using imagemaster 7.0 software (ge healthcare life sciences). the optimized parameters were set according to the gel background. the corresponding spots in different gels was normalized and calculated. the protein spots with ratios ≥ 1.5 or ratios ≤ 0.67 and anova (p < 0.05) were considered to be different proteins. in-gel digestion and maldi-tof-tof analysis the protein spots were excised from the stained gels, washed three times with milli-q water, destained by sonication in 50 mm nh4hco3, 50% acn, and 100% can (50 μl/each), respectively, then dried in room temperature. the dried protein spots were incubated with 10~15 μl trypsin solution (10 μg/ml) at 37 °c for about 12 h. peptides were collected and 50% acn containing 0.1% tfa 20 μl were added to each spot to incubate at 60 °c for 10 min, then treated by sonication for 1 min, and dried in centrifugal concentrator. the dried sample was dissolved using 3 μl of α-cyano-4-hydroxycinnamic acid (chca) matrix. finally, the mixtures were analyzed through maldi tof/tof mass spectrometer (maldi-tof/tof™ 5800 system, ab sciex). protein identification maldi-tof/tof data were analyzed using mascot (matrix science, london, uk, http://www.matrixscience.com/). using the ncbi and swissprot databases, the primary and secondary mass spectra of the corresponding protein spots were obtained. the parameters were set as follows: metazoa (animals) was selected as the taxonomic category, trypsin was selected as enzyme, carbamidomethyl was chosen as fixed modification, deamdated (nq) and oxidation (hw) were selected as variable modification, the peptide tolerance is 100 ppm, and ms/ms tolerance is 0.6 da. totally, 28 proteins with significant differences were successfully identified. gene ontology (go) and kegg analysis according to the annotations of the identified proteins from uniprot knowledgebase (http://www.expasy.org/sprot/) (ye et al., 1970), the corresponding gene ontology (go) ids of these proteins were obtained. based on gene ontology database, the go classification of these proteins was conducted with wego (http://wego.genomics.org.cn/). the kegg results of the corresponding proteins were obtained from the ncbi database. the kegg maps were summarized according to the kegg network (http://www.genome.jp/kegg/pathway.html) to obtain the relevant pathways of the different proteins. rna extraction and quantitative real-time reverse transcription polymerase chain reaction (qrt-pcr) the silk glands were thawed, ground into powder, weighed (0.1 g) and transferred into the centrifuge tube. using trizol reagent (invitrogen, usa), total rna was extracted. the extracted rna was assayed with 1% agarose. at last, 1 μg rna was used for the first strand synthesis. reverse transcription was performed using a reverse transcription kit. the qrt-pcr was carried out in a total volume of 20 μl containing 2 μl of cdna (200 ng), 10 μl of sybr green master mix (vazyme biotech co., ltd, china), 0.4 μl of 50×rox reference dye i, 0.4 μl of primers (10 μm) and 7.2 μl of h2o. amplification was performed using an abi7300 pcr thermocycler (applied biosystems, usa). the α-tubulin gene (ncbi accession no. nm_001043419.1) of silkworm was selected as a reference and the experiments were repeated three times. results the economic characters of js, hy and their f1 hybrids in our study, the weights of larva and silk gland, whole cocoon weight, cocoon shell weight, pupal weight, cocoon shell percentage, dead worm cocoon rate, moth weight, egg weight and the number of eggs were investigated. as shown in figure 1, all traits of f1 generation were significantly superior to their parents, but the differences of most characters between d-f1 and r-f1 generations, including the weights of larva and silk gland, whole cocoon weight, cocoon shell weight, pupal weight, cocoon shell percentage and dead worm cocoon rate were not significant. as the matter of fact, the investigated traits, especially silk gland weight and whole cocoon weight, showed obvious over-dominant effect in f1 hybrid (table s1). although dead worm cocoon rate of f1 generation appeared negative over-parent heterosis, the lower dead worm cocoon rate is actually advantageous for silkworm. therefore, our data illustrated that the heterosis of silkworm is overdominant. unfortunately, as a result of the restriction of the feeding condition, the whole cocoon weight in our experiment was lower than other research laboratories. however, in current study, the experiments were biologically repeated three times and the data of the triplicates were very similar to each other. therefore, the conclusions from these results will still remain interesting and reliable. the detection and identification of differentially expressed proteins in current study, the genetic cross and proteomic approaches (2-de and ms) were employed to investigate the molecular mechanism of heterosis at protein level. in order to improve the compatibility of ms identification, we employed cbb staining for visualization of protein spots, and triplicates were performed for each sample. as shown in figure 2, the gels with high resolution and sensitivity were obtained, which provided enough information for our study. the gels were analyzed using imagemaster 7.0 software (ge healthcare life sciences). totally, 768 ± 12, 755 ± 16, 759 ± 13, 762 ± 22 protein spots were detected in js, hy, jshy, and hy-js, respectively, in cbb r-250 stained gels. totally, 28 protein spots with significant differences were successfully identified, and 14, 21, 16 and 5 differentially expressed proteins were ultimately determined between js/hy, js/js-hy, js/hy-js, and js-hy/hy-js, respectively (supplementary files, figure s1 and table s2). these http://www.matrixscience.com/ http://www.expasy.org/sprot/ 70 fig. 2 the 2-de profiles from silkworm silk gland of parental silkworms js, hy and their f1 hybrids. note: the differentially expressed protein spots are indicated by circles and labeled with arabic numerals. the numbers shown on the left indicate the protein markers in kda. differentially expressed proteins were numbered uniformly (table 1) and labeled in figure 2. as shown in table 1, some different spots were the same protein, e.g., spots 3, 4, and 5 corresponded to protein disulfide-isomerase like protein erp57 precursor, and spots 20, 21 and 22 were fibrohexamerin-like. therefore, these 28 differentially expressed proteins can be classified into 22 different proteins, involving in genetic information processing, metabolism, cellular process, environmental information processing related proteins and organismal system (table 1, figure 3). as shown in figure 3, most of the identified proteins participated in genetic information processing (33%), and 28% proteins belonged to the metabolism group, which accounted for 61% of the identified proteins. interestingly, three silk-related proteins were also identified, that is, nd-sd mutant fibroin light chain (figure 2, spot 11), fibroin l-chain (figure 2, spot 13) and fibrohexamerin (fhx) (figure 2, spots 20, 21 and 22). because the sequence similarity of fibroin light chain precursor (spots 12 and 14, 261 amino acids) with fibroin l-chain (spot 13, 261 amino acids) reached 99.24%, protein spots 12, 13 and 14 were the same protein. the fibroin l-chain, encoded by the fib-l gene on the 14th chromosome, its expression level in js was 21.6 times higher than that of hy (figure 2, spot 13). however, the difference between js and f1 hybrids was not significant. go analysis these 22 different proteins were used for go analysis, which can be divided into cell components, molecular functions and biological processes three categories, mainly involving in catalytic, cellular process, metabolic process, binding and extracellular region (figure 4). qrt-pcr validation in current study, the silk gland from three biological samples were used for qrt-pcr to further validate the proteomic results. eventually, 10 different proteins (spot nos. 1, 5, 11, 12, 13, 16, 18, 22, 26, 27) were selected for qrt-pcr. the gene sequences of these proteins were obtained from the silkworm genome sequences. the primer sequences used for qrt-pcr were listed in table s3. as shown in figure 5, the qrt-pcr expression results were basically consistent with the proteomic results, which further confirmed the reliability of the results. 71 table 1 the identification of 28 differentially expressed proteins through maldi-tof/tof note: a mw: molecular weight; pi: isoelectric point b kegg results protein spot number protein id protein name sequence coverage mw (kda)/pi a mascot score amino acid function b 1 gi|112983032 calreticulin 45% 46.1/4.49 636 398 calcium ion binding; carbohydrate binding; protein folding 2 gi|827542145 zonadhesin-like isoform x2 18% 73.9/4.87 204 621 sperm-specific transmembrane protein 3 gi|112983366 protein disulfide-isomerase like protein erp57 precursor 26% 55.4/5.31 793 491 protein disulfide isomerase activity, cell redox homeostasis 4 gi|112983366 protein disulfide-isomerase like protein erp57 precursor 30% 55.4/5.31 875 491 protein disulfide isomerase activity, cell redox homeostasis 5 gi|112983366 protein disulfide-isomerase like protein erp57 precursor 41% 55.4/5.31 885 491 protein disulfide isomerase activity, cell redox homeostasis 6 gi|827563326 serine protease inhibitor 18 isoform x1 50% 44.2/4.44 596 392 extracellular space 7 gi|226342906 serine protease inhibitor 22 precursor 38% 44.1/4.41 562 392 extracellular space 8 gi|512894896 uncharacterized protein loc101739721 46% 32.9/4.56 708 289 unknown function 9 gi|148298752 14-3-3 epsilon protein 62% 29.8/4.66 632 262 monooxygenase activity 10 gi|512892462 inorganic pyrophosphatase 62% 32.2/4.96 503 288 oxidative phosphorylation 11 gi|452392 nd-sd mutant fibroin light chain 24% 30.9/7.66 127 276 extracellular region; integral component of membrane 12 gi|112984494 fibroin light chain precursor 51% 27.9/5.23 405 262 extracellular region 13 gi|304443558 fibroin l-chain 62% 27.8/5.06 447 262 extracellular region 14 gi|112984494 fibroin light chain precursor 88% 27.9/5.23 513 262 extracellular region 15 gi|512891724 phosphatidylethanolaminebinding protein homolog f40a3.3-like 62% 20.2/5.02 729 185 construction and remodeling of biofilm; signal transduction, nervous system growth and development 16 gi|112983600 cellular retinoic acid binding protein 59% 15.0/5.66 471 132 lipid binding; transporter activity 17 gi|827559362 cuticular protein rr-2 motif 90 isoform x1 19% 22.9/5.94 87 408 structural constituent of cuticle 18 gi|404276811 farnesoic acid omethyltransferase 41% 33.0/4.88 446 312 physiological processes 19 gi|512917993 uncharacterized protein loc101743840 isoform x2 68% 26.4/6.31 873 238 unknown function 20 gi|512891160 fibrohexamerin-like 38% 27.8/4.81 491 241 maintain the integrity of silk fibroin complexes 21 gi|512891160 fibrohexamerin-like 31% 27.8/4.81 200 241 maintain the integrity of silk fibroin complexes 22 gi|512891160 fibrohexamerin-like 63% 27.8/4.81 510 241 maintain the integrity of silk fibroin complexes 23 gi|827548944 zinc finger protein 569-like 22% 23.9/8.41 59 204 cell differentiation; embryonic development 24 gi|512918988 lish domain-containing protein c1711.05-like, partial 54% 39.1/9.61 36 635 protein dimerization 25 gi|112983898 elongation factor 1 gamma 46% 48.6/5.83 632 423 glycolytic process; magnesium ion binding; phosphopyruvate hydratase activity 26 gi|148298800 enolase 65% 47.2/5.62 951 433 magnesium ion binding; phosphopyruvate hydratase activity; glycolytic process 27 gi|827541166 arginine kinase 42% 40.3/5.87 640 355 atp binding; kinase activity 28 gi|827562394 aldo-keto reductase akr2e4-like isoform x2 37% 35.8/5.55 430 310 oxidoreductase activity 72 fig. 3 the classification of differentially expressed proteins by kegg discussion the silkworm is not only a domestic insect for silk production, but also is a model system for research on developmental as well as physiological processes of lepidoptera. in current study, we selected the silk gland of silkworm varieties js and hy and their f1 hybrids as research materials to investigate the molecular mechanism of heterosis. the hybrid js-hy is the most popular silkworm variety for spring rearing in china because of its high yield, high quality, especially its excellent silk production. the silk fibroin, produced by psg, is comprised of a heavy chain (fibroin h-chain, fib-h), a light chain (fibroin l-chain, fib-l), and a glycoprotein, p25 (inoue et al., 2000). the sericins, produced by msg, are a protein family and can ensure the cohesion of the cocoon by sticking the silk threads together. according to our proteomic results, 28 significantly differentially expressed proteins were successfully identified, involving in 22 different proteins. go analysis showed that these proteins were mainly involved in catalytic, cellular process, metabolic process, binding and extracellular region. kegg analysis indicated that most of the identified proteins were involved in metabolic and genetic information processing, which accounted for 61% of the identified proteins, suggesting that metabolic and figure 4 go analysis of 28 differentially expressed proteins. 73 74 fig. 5 validation of 10 differentially expressed genes using qrt-pcr. x-axis represents different samples, y-axis represents the relative expression level of mrna genetic information processing related proteins may play an important role in the heterosis of f1 hybrids. studies have shown that the epigenetic modification of genes associated with circadian rhythms changed the expression of downstream genes in arabidopsis. they speculated that hybrids obtained advantages of circadian-mediated physiological and metabolic pathways and therefore promoted the improvement of vigor and the increase of biomass, consistent with our findings (ni et al., 2009). in current study, fibroin l-chain, nd-sd mutant fibroin light chain, and fhx were detected to be differentially expressed between parental silkworms and their f1 hybrids. the fib-l gene locates on the 14th chromosome, contains 7 exons and 6 introns, and encodes a total of 262 amino acids, of which the first 18 amino acids are signal peptides (kikuchi et al., 1992). the h-chain and l-chain of fibroins are linked by a single disulfide bond between cys-172 of lchain and cys-c20 (the twentieth residue from the c terminus) of h-chain. the h-l linkage is essential for the effective secretion of large amounts of fibroin. compared with h-chain, the amino acids of l-chain are highly conserved, even a very little variation also affect the function of silk, indicating the functional importance of fib-l gene in the performance of silk fibroin. studies have illustrated that the silkworm with nd-s or nd-sd mutation of the fib-l gene could not form the disulfide linkage with h-chain, leading to less than 1% fibroin of the normal level to be secreted (tanaka et al., 1999). previous research has shown that the cocoon of transgenic silkworm with antimicrobial peptide cecropin gene possessed antibacterial properties, and its silk performance can 75 be improved through the modification of fib-l gene (li et al., 2015). because of the specific function of fib-l gene in silk fibroin synthesis, it well explained the advantages of f1 generation in silk gland weight, whole cocoon weight, and cocoon shell weight. the nd-s gene is a dominant gene that controls the formation of silk cocoons (takemura et al., 2016). nd-sd is the allele gene of nd-s, and only one base change exists in the coding region of nd-s and ndsd genes. the mutation of nd-sd gene occurs on the third intron of fib-l gene, caused by a deletion from the third exon, which leads to the recombination of sequences in the third intron, and therefore affects the synthesis and secretion of silk fibroin (takeiet et al., 1987; mori et al., 1995). in fact, nd-sd homozygous mutants can form immature psgs, that’s because the lack of fib-l in the endoplasmic reticulum (er) inhibits the development of psg cells (takei et al., 1987). eventually, the nd-sd mutant only secretes less than 1% fibroin of the normal level, and produces very thin and naked-pupa cocoon (barbosa et al., 2008). fhx, a class of glycoproteins, is highly expressed in psgs but is inhibited in msg. studies have shown that fhx plays roles in maintaining the integrity of silk fibroin complexes and promoting the expression of secretory cell genes (julien et al., 2002; barbosa et al., 2009). the expression levels of fhx in f1 hybrids were significantly higher than that of in the parents, consistent with the measurement result of the silk gland weight, indicating that fhx indeed played important roles in promoting the synthesis of silk fibroin and maintaining the integrity of silk fibroin. meanwhile, we also identified two molecular chaperone-related proteins, including calreticulin (crt, spot 1) and erp57 protein (spots 3, 4 and 5). as shown in figure 2, the total expression level of erp57 are significantly up-regulated in f1 hybrids. the similar results were also obtained from silk glands at 24h, 48h of 5th instar silkworms, verifying the reliability of our work (data not shown here). calreticulin (crt) is a unique er protein and affects many cellular functions inside and outside the er (michalak et al., 1999). as a multifunctional lectin-like molecular chaperone, crt is involved in the synthesis of various molecules, including ion channels, surface receptors, integrins and transporters (martin et al., 2006). the erp57 is a member of the protein disulfide isomerase (pdi) family. as a soluble protein of er, erp57 can form discrete complexes with the er lectins, crt and calnexin (cnx). these complexes can interact specifically with newly synthesized glycoproteins and modulate glycoprotein folding within the er lumen. additionally, erp57 also acts as a thiol-disulfide oxidoreductase for proteins carrying n-linked glycans (frickel et al., 2002). the thiol-disulfide oxidoreductase erp57 can efficiently catalyze disulfide reduction, disulfide isomerization, and dithiol oxidation in substrate proteins (frickel et al., 2004). thereby, it can be speculated that the up-regulation of erp57 in f1 hybrids ensures the correct folding and prevents the degradation of proteins, and thus promotes rapid growth of f1 hybrids (figure 6). fig. 6 the possible explanation of silkworm heterosis 76 at the same time, the metabolism, and/or energy-related proteins, 14-3-3 protein (spot 9) and enolase (spot 26) were also identified. the 14-3-3 protein is not only an important signal transduction protein, but also a conserved regulatory molecule. the main feature of 14-3-3 protein is to combine various functional signal proteins, including kinases, phosphatases, and transmembrane receptors. consequently, 14-3-3 protein plays vital roles in a variety of important regulatory processes, such as mitotic signal transduction, apoptotic cell death, and cell cycle control (fu et al., 2000). enolase is a key enzyme in the glycolysis that can provide large amounts of energy for organisms. studies have shown that the enolase of insect differs from mammalian enolase, which has relatively low conservation and shows species specificity in function (kikuchi et al., 2017). in addition, enolase is involved in rna degradation, glycolysis/gluconeogenesis, biosynthesis of amino acids, carbon metabolism and other pathways. as shown in figure 2, the expression of enolase was down-regulated in hybrids, which was confirmed by the result of qrt-pcr, suggesting that the downregulation of enolase might lead to the decline of metabolic rate in f1 hybrids and therefore cause the accumulation of large amounts of energy. finally, the growth and developmental time of f1 generation was prolonged. as a result, body weight, cocoon shell weight, and other economic traits were improved accordingly (figure 6). these observations, in conjunction with the qrt-pcr and proteomic results, suggesting that these different proteins may play important roles the formation of silkworm heterosis. these findings described here not only provide candidate proteins and genes for the improvement of yield in silkworm but also demonstrate that comparative proteomic approach can be feasible in investigating molecular mechanism of heterosis at the protein level. it should be noted, however, that the power to detect heterosis using the above methods may be not enough. further investigations using other methods on the molecular mechanisms of heterosis should be carried out in the future. conclusion based on proteomic and genetic cross approaches, 28 differentially expressed proteins were successfully identified, including three silkrelated proteins, nd-sd mutant fibroin light chain, fibroin l-chain and fhx, which were further confirmed by qrt-pcr. in addition, erp57 and enolase were found to be involved in the heterosis of silkworm. taken together, our work not only provides novel insights and references for the understanding of molecular mechanism of silkworm heterosis, but also provides candidate proteins and 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immortalized f2 population. j. genet. genomics 43: 87-97, 2016. 79 fig. s1 spot volume analyses of differentially expressed proteins among js, hy and their f1 hybrids. the gels are shown on the left panel, and the corresponding spot volume analyses performed using imagemaster are shown on the right panel. table s1 results of character investigation and evaluation of heterosis characters f1 a hp b lp c mr d the evaluation of heterosis e silkworm weight (g) 3.32 2.92 2.48 2.7 over-parent heterosis silk gland weight (g) 1.35 0.62 0.53 0.58 over-parent heterosis whole cocoon weight (g) 1.34 0.90 0.76 0.83 over-parent heterosis cocoon shell weight (g) 0.38 0.27 0.25 0.26 over-parent heterosis pupal weight (g) 1.06 0.94 0.92 0.93 over-parent heterosis cocoon shell percentage (%) 28.3 21.05 20.98 21.02 over-parent heterosis dead worm cocoon rate (%) 2.33 8.66 7.3 7.98 negative over-parent heterosis moth weight (g) 0.49 0.36 0.36 0.36 over-parent heterosis egg weight (g) (500 eggs/group) 0.37 0.23 0.17 0.2 over-parent heterosis the number of eggs 510 365 354 360 over-parent heterosis note: a f1: the average value of the f1 hybrids; b hp: the average value of the parent with a high trait value; c lp: the average value of the parent with a low trait value; d mr: the average value of two parents; e f1 > hp, over-parent heterosis; mr < f1 < hp, mid-parent heterosis; lp < f1 < mr, negative mid-parent heterosis; f1 < lp, negative over-parent heterosis. 80 table s2 detailed information of the differentially expressed proteins from silk glands at 96h of the fifth instar material and expression level protein spot number protein id protein name sc a mw (kda)/pib mascot score aa c function d fold change e js and hy 3 gi|112983366 protein disulfideisomerase like protein erp57 precursor 26% 55.4/5.31 793 491 protein disulfide isomerase activity, cell redox homeostasis 3.7 up-regulated proteins in js 4 gi|112983366 protein disulfideisomerase like protein erp57 precursor 30% 55.4/5.31 875 491 protein disulfide isomerase activity, cell redox homeostasis 5.9 6 gi|827563326 serine protease inhibitor 18 isoform x1 50% 44.2/4.44 596 392 extracellular space 2.9 13 gi|304443558 fibroin l-chain 62% 27.8/5.06 447 262 extracellular region 21.6 19 gi|512917993 uncharacterized protein loc101743840 isoform x2 68% 26.4/6.31 873 238 1.6 21 gi|512891160 fibrohexamerin-like 31% 27.8/4.81 200 241 maintain the integrity of silk fibroin complexes 2.1 down-regulated proteins in js 5 gi|112983366 protein disulfideisomerase like protein erp57 precursor 41% 55.4/5.31 885 491 protein disulfide isomerase activity, cell redox homeostasis 20.2 18 gi|404276811 farnesoic acid omethyltransferase 41% 33.0/4.88 446 312 physiological processes 2.0 28 gi|827562394 aldo-keto reductase akr2e4-like isoform x2 37% 35.8/5.55 430 310 oxidoreductase activity 3.6 unique proteins in js 7 gi|226342906 serine protease inhibitor 22 precursor 38% 44.1/4.41 562 392 extracellular space 8 gi|512894896 uncharacterized protein loc101739721 46% 32.9/4.56 708 289 11 gi|452392 nd-sd mutant fibroin light chain 24% 30.9/7.66 127 276 extracellular region, integral component of membrane 12 gi|112984494 fibroin light chain precursor 51% 27.9/5.23 405 262 extracellular region unique proteins in hy 15 gi|512891724 phosphatidylethanola mine-binding protein homolog f40a3.3-like 62% 20.2/5.02 729 185 construction and remodeling of biofilm, signal transduction, nervous system growth and development js and js-hy 1 gi|112983032 calreticulin 45% 46.1/4.49 636 398 calcium ion binding, carbohydrate binding, protein folding 5.6 up-regulated proteins in jshy 2 gi|827542145 zonadhesin-like isoform x2 18% 73.9/4.87 204 621 sperm-specific transmembrane protein 3.8 3 gi|112983366 protein disulfideisomerase like protein erp57 precursor 26% 55.4/5.31 793 491 protein disulfide isomerase activity, cell redox homeostasis 3.2 5 gi|112983366 protein disulfideisomerase like protein erp57 precursor 41% 55.4/5.31 885 491 protein disulfide isomerase activity, cell redox homeostasis 13.1 6 gi|827563326 serine protease inhibitor 18 isoform x1 50% 44.2/4.44 596 392 extracellular space 3.2 7 gi|226342906 serine protease inhibitor 22 precursor 38% 44.1/4.41 562 392 extracellular space 3.4 8 gi|512894896 uncharacterized protein loc101739721 46% 32.9/4.56 708 289 2.4 9 gi|148298752 14-3-3 epsilon protein 62% 29.8/4.66 632 262 monooxygenase activity 1.8 10 gi|512892462 inorganic pyrophosphatase 62% 32.2/4.96 503 288 oxidative phosphorylation 3.6 81 16 gi|112983600 cellular retinoic acid binding protein 59% 15.0/5.66 471 132 lipid binding, transporter activity 4.2 18 gi|404276811 farnesoic acid omethyltransferase 41% 33.0/4.88 446 312 physiological processes 2.8 20 gi|512891160 fibrohexamerin-like 38% 27.8/4.81 491 241 maintain the integrity of silk fibroin complexes 3.2 21 gi|512891160 fibrohexamerin-like 31% 27.8/4.81 200 241 maintain the integrity of silk fibroin complexes 2.8 22 gi|512891160 fibrohexamerin-like 63% 27.8/4.81 510 241 maintain the integrity of silk fibroin complexes 1.8 23 gi|827548944 zinc finger protein 569like 22% 23.9/8.41 59 204 cell differentiation, embryonic development 2.4 24 gi|512918988 lish domain-containing protein c1711.05-like, partial 54% 39.1/9.61 36 635 protein dimerization 2.4 down-regulated proteins in jshy 11 gi|452392 nd-sd mutant fibroin light chain 24% 30.9/7.66 127 276 extracellular region, integral component of membrane 2.2 25 gi|112983898 elongation factor 1 gamma 46% 48.6/5.83 632 423 glycolytic process, magnesium ion binding, phosphopyruvate hydratase activity 3.0 26 gi|148298800 enolase 65% 47.2/5.62 951 433 magnesium ion binding, phosphopyruvate hydratase activity, glycolytic process 3.1 27 gi|827541166 arginine kinase 42% 40.3/5.87 640 355 atp binding, kinase activity 2.5 unique proteins in js-hy 15 gi|512891724 phosphatidylethanola mine-binding protein homolog f40a3.3-like 62% 20.2/5.02 729 185 construction and remodeling of biofilm, signal transduction, nervous system growth and development js and hy-js 2 gi|827542145 zonadhesin-like isoform x2 18% 73.9/4.87 204 621 sperm-specific transmembrane protein 1.8 up-regulated proteins in hyjs 5 gi|112983366 protein disulfideisomerase like protein erp57 precursor 41% 55.4/5.31 885 491 protein disulfide isomerase activity, cell redox homeostasis 11.9 6 gi|827563326 serine protease inhibitor 18 isoform x1 50% 44.2/4.44 596 392 extracellular space 3.0 7 gi|226342906 serine protease inhibitor 22 precursor 38% 44.1/4.41 562 392 extracellular space 2.9 10 gi|512892462 inorganic pyrophosphatase 62% 32.2/4.96 503 288 oxidative phosphorylation 3.8 18 gi|404276811 farnesoic acid omethyltransferase 41% 33.0/4.88 446 312 physiological processes 3.2 20 gi|512891160 fibrohexamerin-like 38% 27.8/4.81 491 241 maintain the integrity of silk fibroin complexes 3.0 21 gi|512891160 fibrohexamerin-like 31% 27.8/4.81 200 241 maintain the integrity of silk fibroin complexes 3.2 22 gi|512891160 fibrohexamerin-like 63% 27.8/4.81 510 241 maintain the integrity of silk fibroin complexes 2.0 23 gi|827548944 zinc finger protein 569like 22% 23.9/8.41 59 204 cell differentiation, embryonic development 2.1 24 gi|512918988 lish domain-containing protein c1711.05-like, partial 54% 39.1/9.61 36 635 protein dimerization 2.4 down-regulated proteins in hyjs 25 gi|112983898 elongation factor 1 gamma 46% 48.6/5.83 632 423 glycolytic process, magnesium ion binding, phosphopyruvate hydratase activity 2.8 26 gi|148298800 enolase 65% 47.2/5.62 951 433 magnesium ion 2.6 82 note: a sc: sequence coverage b mw: molecular weight; pi: isoelectric point c aa: amino acid d kegg results e p < 0.05 f up-regulated proteins in js-hy table s3 primer sequences used for quantitative real-time pcr spot no.a protein name forward primer (5’-3’) reverse primer (5’-3’) 1 calreticulin gcgacccagaggatgacaaa agaccttgaggtatccgcct 5 protein disulfide-isomerase like protein erp57 precursor cggcttccccacaatcttct tggcgttaccctttctgtcc 11 nd-sd mutant fibroin light chain tctcacgtgcatgggactac acccatactgttagcggctg 12 fibroin light chain precursor ctcacgtcgatgggactacg caccgctgatgcttgacttg 13 fibroin l-chain cgatactctgtcggaccagc aagtgagcggtgatgtaggc 16 cellular retinoic acid binding protein aagagttcgaagaggaccgc agactctggtgcaagtcacg 18 farnesoic acid omethyltransferase aaatcgaagtgccaccgact cagtcctgtcctcccacaac 22 fibrohexamerin-like ggcggatgaggtagactgtg gtactggccccagcgttatt 26 enolase atgattggtctgcatgggca ctctgttacgctgccgatct 27 arginine kinase ttggaggctggtttcagcaa aggagggtggatccgaatga note: a the spot number of identified proteins (see table 1) binding, phosphopyruvate hydratase activity, glycolytic process 27 gi|827541166 arginine kinase 42% 40.3/5.87 640 355 atp binding, kinase activity 2.1 unique proteins in hy-js 15 gi|512891724 phosphatidylethanola mine-binding protein homolog f40a3.3-like 62% 20.2/5.02 729 185 construction and remodeling of biofilm, signal transduction, nervous system growth and development unique proteins in js 8 gi|512894896 uncharacterized protein loc101739721 46% 32.9/4.56 708 289 js-hy and hyjs 1f gi|112983032 calreticulin 45% 46.1/4.49 636 398 calcium ion binding, carbohydrate binding, protein folding 6.2 16f gi|112983600 cellular retinoic acid binding protein 59% 15.0/5.66 471 132 lipid binding, transporter activity 3.2 unique proteins in js-hy 8 gi|512894896 uncharacterized protein loc101739721 46% 32.9/4.56 708 289 11 gi|452392 nd-sd mutant fibroin light chain 24% 30.9/7.66 127 276 extracellular region, integral component of membrane unique proteins in hy-js 19 gi|512917993 uncharacterized protein loc101743840 isoform x2 68% 26.4/6.31 873 238 98 isj 18: 98-107, 2021 issn 1824-307x research report transcriptomic analysis of strain-specific and gender-specific response of silkworm to bmnpv infection s he, j xu, y fan, f zhu, k chen* institute of life sciences; jiangsu university, zhenjiang 212013, china this is an open access article published under the cc by license accepted june 18, 2021 abstract bombyx mori nucleopolyhedrovirus (bmnpv) is one of the main pathogens causing serious economic losses in sericulture. however, the molecular mechanism of silkworm resistance to bmnpv is still largely unclear, and the differences in the anti-bmnpv response between silkworms of different genders have been rarely studied. in this study, bmnpv resistant strain nb and bmnpv sensitive strain 306 of different genders were used as experimental materials to inoculate bmnpv, and their transcriptomes were sequenced to analyze their response to bmnpv. eighteen genes specifically differentially expressed in nb after bmnpv inoculation were finally obtained through transcriptomic analysis, fourteen of which were up-regulated and four were down-regulated, suggesting that they might be related to bmnpv resistance. among them, the expression abundance of eight genes were higher in males than in females, and one gene was in the contrary. these genes suggested that there were certain differences in the anti-bmnpv response between silkworms of different genders. this study provided a new understanding of the molecular mechanism of silkworm resistance to bmnpv and the differences in the anti-bmnpv response between silkworms of different genders, and laid a foundation for future prevention and control of bmnpv. key words: bombyx mori; bmnpv resistance; transcriptome sequence; strain-specific response; gender-specific response introduction the silkworm, bombyx mori has been domesticated for more than 5000 years and is an important economic insect in many developing countries (goldsmith et al., 2005). it is also a good model lepidoptera insect that is often used in the study of insect genetics and immunology (tanaka et al., 2009; guo et al., 2015). bombyx mori nucleopolyhedrovirus (bmnpv) is a baculovirus that infects silkworms. although some silkworm strains with bmnpv resistance like nb and cvdar have been cultivated (chen et al., 1991; qing et al., 2019)，their cocoon yield and quality still do not meet the production requirements. so far, the main silkworm strains used for production like yuke9 and yuncan10 are bmnpv-sensitive strains (liu et al., 2013; yang et al., 2019). there is no effective method or drug to control this virus disease, which brings huge losses to silkworm industry every year. therefore it is very urgent and important to identify _________________________________________ corresponding author: keping chen institute of life sciences jiangsu university zhenjiang, jiangsu 212013, china e-mail: kpchen@ujs.edu.cn the bmnpv resistant mechanism of silkworm. there have been many researches on silkworm resistance to bmnpv, and some resistance genes have been successively identified. for example, the increased expression of suppressor of cytokine signaling 2 (bmsocs2) has been reported to be correlated with suppression of bmnpv replication in silkworm (yuan et al., 2020). arginine kinase (bmak) and trypsin (bmtryp) have also been reported to be involved in the antiviral process (kang et al., 2011; ponnuvel et al., 2012). however, the molecular mechanism of silkworm resistance to bmnpv has not been clearly interpreted yet. in nature, male and female individuals of many species have great differences in many aspects (matthews et al., 2019). for example, there is sexual dimorphism in genetic loci for anthropometric traits (randall et al., 2013). and the fungal infection could induce sex-specific transcriptional changes in silene latifolia (zemp et al., 2015). similarly, there are certain differences in the development, cocoon characters and disease resistance between silkworms of different genders (qin et al., 2014; chen et al., 2016). however, the differences in the anti-bmnpv response between silkworms of different genders have been rarely studied. 99 table 1 primers used in rt-qpcr gene forward primer reverse primer product length (bp) bgibmga004955 5' atcgagattcggtcacgagc 3' 5' ttcagctgatcgcgccaata 3' 169 bgibmga009799 5' acctggctaggactgaacga 3' 5' ggtggtgccacacctttgta 3' 220 bgibmga006251 5' gggaggttgcaacggtgat 3' 5' cggggctgatacatttgcct 3' 173 bgibmga004809 5' actcgaagtggctctcaacg 3' 5' agcttcaatagctgccgtgt 3' 205 bgibmga012486 5' agcggttgcagtttctacga 3' 5' cagttcgtgcataccccagt 3' 168 bgibmga010514 5' ttgcaagccgtttttgctgt 3' 5' tctccggcctccagtagtag 3' 188 bgibmga010863 5' tgctgcataagatgcggaga 3' 5' tgtagcacctggctacttgg 3' 219 bgibmga011868 5' tgccaggctaacacagacag 3' 5' ctctgagcctgcacttccat 3' 150 bgibmga009091 5' cagccagggttcggttgaaa 3' 5' tcaacgggtacgcattttcc 3' 180 bmrps3 5' cgattcaacattccagagca 3' 5' gaacaccatagcaagcacgac 3' 142 the primers used in rt-qpcr ,and their product length were listed above. bmrps3 was used as reference gene in this study in this study, we conducted transcriptome sequencing of nb (bmnpv-resistant strain) and 306 (bmnpv-sensitive strain) in a gender specific manner. through comparative transcriptomic analysis, differentially expressed genes in silkworms after bmnpv inoculation were obtained. and some candidate genes that may be related to bmnpv resistance were finally obtained according to venn analysis. this study provides a new understanding of the molecular mechanism of silkworm resistance to bmnpv and the differences in the anti-bmnpv response between silkworms of different genders. materials and methods virus and silkworm the bmnpv and silkworms were all preserved in our laboratory. the virus was obtained from the hemolymph of infected larvae and purified by repeated and differential centrifugation according to the method of rahman et al. (2004), and the virus concentration (ob/ml) was measured by the hemocytometer. the half lethal concentration (lc50) of nb reached 6.39×108 ob/ml, which was nearly 1000 times higher than that of 306. the first four instar larvae were reared with fresh mulberry leaves. on the first day of fifth instar, the pbs solution containing bmnpv polyhedrons (1×10^7 ob/ml) was evenly smeared on the surface of fresh mulberry leaves and fed to the experimental group. after 24 hours feeding with virus, the experimental group was refed with normal mulberry leaves. and the control group was set simultaneously. on the third day of the fifth instar, the whole silkworms nb♂-v, nb♀-v, 306♂-v, 306♀-v of the experimental group and nb♂-c, nb♀-c, 306♂-c, 306♀-c of the control group were stored in the -80 °c refrigerator for later use. rna extraction and transcriptome sequencing trizol method was used to extract total rna from the eight samples, with three biological replicates for each sample. the purity, concentration and integrity of rna were determined by nanodrop one/onec (thermo, usa), invitrogen qubit 4 (thermo, usa), agilent 2100 bioanalyzer (agilent technologies, usa) and agarose gel electrophoresis. after that, mrna was enriched with oligo(dt) magnetic beads to construct a cdna library. after the library was qualified with agilent 2100 bioanalyzer (agilent technologies, usa), transcriptome sequencing was performed using illumina novaseq 6000 (illumina, usa). transcriptome assembly, annotation and gene expression analysis the raw reads were filtered by removing reads with adaptor, reads with a n ratio of more than 10 % (n denotes bases that cannot be identified), and reads containing more than 50 % bases with a qphred ≤ 20 to obtain the clean reads. then the clean reads were aligned with the reference genome (http://metazoa.ensembl.org/bombyx_mori/info/inde x) by hisat2 to obtain the mapped reads for subsequent transcriptome assembly and gene expression analysis. stringtie software was used to assemble the mapped reads based on the reference genome. go, kegg, cog, nr, swiss-prot and pfam databases were used to annotate the assembled transcriptome. the expression abundance of the annotated genes was analyzed using rsem software. read counts data of genes was obtained from the alignment result and annotation file. standardized gene expression abundance was obtained by fpkm (fragments per kilobase of transcript per million fragments) transformation (mortazavi et al., 2008). http://metazoa.ensembl.org/bombyx_mori/info/index http://metazoa.ensembl.org/bombyx_mori/info/index 100 table 2 gene annotation results statistics database expressed gene number percent all gene number percent go 4388 32.54 % 4531 28.78 % kegg 7162 53.12 % 7501 47.64 % cog 12652 93.84 % 13372 84.93 % nr 13125 97.34 % 14097 89.53 % swiss-prot 9403 69.74 % 9609 61.03 % pfam 10083 74.78 % 10311 65.49 % total-anno 13187 97.80 % 14219 90.31 % total 13483 100.00 % 15745 100.00 % expressed gene number: numbers of expressed genes in this study in each database all gene number: numbers of all expressed genes of silkworm in each database total-anno: total expressed genes that have been annotated in all databases total: total expressed genes in all databases differentially expressed genes (degs) analysis gene expression abundance was compared between the experimental group and the control group using deseq2 software to obtain up-regulated degs and down-regulated degs. the comparison schemes were as follows: nb♂-v vs nb♂-c, nb♀-v vs nb♀-c, 306♂-v vs 306♂-c, 306♀-v vs 306♀-c. p-adjust < 0.05 and |log2fc| ≥ 1 were set as standard to screen degs. venn diagrams were constructed to obtain unique degs of nb♂-v and nb♀-v, as well as degs that were common to nb♂-v and nb♀-v but not to 306♂-v or 306♀-v. further analysis was made on the degs that were common to nb♂-v and nb♀-v but not to 306♂-v or 306♀-v to select genes that differentially expressed between nb♂-v and 306♂-v, as well as between nb♀-v and 306♀-v. genes with low expression abundance (fpkm<10) in all samples were not considered, and genes with a ratio ≥ 1.35 were considered to be up-regulated, genes with a ratio ≤0.74 were considered to be down-regulated. the genes differentially expressed in nb♂-v and nb♀-v were screened from these genes to analyze the differences in the anti-bmnpv response between nb of different genders. the functions of degs and candidate genes were classified by go analysis, including molecular functions, cellular components and biological processes. real-time quantitative pcr (rt-qpcr) analysis nine genes that might be related to bmnpv resistance were selected and their relative expression levels in nb-v, nb-c, 306-v and 306-c (mixed samples including male and female silkworms) were detected by rt-qpcr. and relative expression levels of 4 genes were detected in nb♂-v and nb♀-v as well. all the primers were listed in table 1. rt-qpcr reactions were prepared with the hiscript q rt supermix kit (vazyme, nanjing, china), following the manufacturer’s instruction. reactions were carried out in applied biosystems quantstudio 3 real-time pcr system (thermo fisher, nanjing, china). all samples were performed in triplicate. b.mori ribosomal protein s3 (bmrps3) gene was used as a reference gene. and the relative expression levels were calculated using the 2-∆∆ct method (livak et al., 2001). results overview of transcriptome sequencing results a total of 172.4 gb of clean reads were obtained for transcriptomic analysis. for all libraries, the gc content was about 46 %, and the q30 was all higher than 92.12 %, indicating that the sequencing data were of sufficient quality and accuracy for further analysis. most reads of all samples were mapped successfully with the reference genome, with a uniquely mapped ratio of about 80 % (s1 table). there was no significant difference in the sequencing results among the biological replicates, indicating that the library construction and sequencing results were qualified. table 3 degs statistics experiment control up-regulated down-regulated degs 306♀-v 306♀-c 741 1410 2151 306♂-v 306♂-c 251 363 614 nb♀-v nb♀-c 328 222 550 nb♂-v nb♂-c 644 279 923 total 3205 standard: p-adjust < 0.05 & |log2fc| ≥ 1 101 fig. 1 venn diagrams showing the degs of different samples. (a) venn diagram of up-regulated degs; (b) venn diagram of down-regulated degs gene annotation and expression analysis reference sequence alignment was performed in go, kegg, cog, nr, swiss-prot and pfam databases, and 13,187 of the 13,483 expressed genes were successfully annotated (table 2). and gene expression abundance was then analyzed. differentially expressed genes (degs) analysis a total of 3205 degs were obtained by comparing the gene expression of the experimental group and the control group (table 3). in both male and female, nb had more up-regulated degs than down-regulated degs after bmnpv inoculation, while 306 were on the contrary. and the number of degs was higher in nb male than in female, while that in 306 was higher in female than in male. in order to further analyze which degs were related to bmnpv resistance, venn diagrams were performed on the up-regulated and down-regulated degs respectively (fig 1). according to the venn diagrams, 588 specific degs were found in nb♂-v (s2 table), of which 474 were up-regulated and 114 were down-regulated. while nb♀-v had 305 specific degs (s3 table), of which 244 were upr egulated 102 fig. 2 gene ontology (go) functional annotation of specific degs. (a) go functional annotation of specific degs of nb♂-v; (b) go functional annotation of specific degs of nb♀-v. the x axis shows the number of genes, and the y axis shows the categories of gene functions, including molecular functions, cellular components, and biological processes and 61 were down-regulated. go analysis showed that specific degs of nb♂-v and nb♀-v had similar functional categories, both of which were mainly involved in binding, metabolic process, membrane, catalytic activity and others (fig 2). however, the number of specific degs involved in all functions of nb♂-v was about twice that of nb♀-v, which was consistent with the ratio of their total degs (nb♂-v : nb♀-v = 923 : 550). meanwhile, 68 degs (53 up-regulated and 15 down-regulated) that were common to nb♂-v and nb♀-v but not to 306♂-v and 306♀-v were also obtained according to the venn diagrams (s4 table). go analysis revealed that 8 genes were related to metabolic process, 5 genes were related to membrane part, 8 genes were related to binding, and 12 genes were related to catalytic activity (fig 3). by analyzing the expression abundance of these 68 genes, 18 genes that differentially expressed between nb♂-v and 306♂-v as well as between nb♀-v and 306♀-v were obtained. among them, 4 genes were down-regulated and 14 were up-regulated in nb males and females after bmnpv inoculation (table 4). these genes might be involved 103 fig. 3 gene ontology (go) functional annotation of 68 specific genes of nb males and females table 4 expression abundance of 18 genes gene id nr annotation 306♂-v fpkm 306♀-v fpkm nb♂-v fpkm nb♀-v fpkm nb♂-v vs 306♂-v ratio nb♀-v vs 306♀-v ratio bgibmga010563 juvenile hormone acid o-methyltransferase-like 26.31 51.06 2.29 3.82 0.09 0.07 down-regulated bgibmga004955 serine protease inhibitor 13 precursor 17.48 13.3 4.84 5.25 0.28 0.39 bgibmga002176 none 26.38 311.93 7.22 5.26 0.27 0.02 bgibmga001147 facilitated trehalose transporter tret1-like 20.3 17.44 1.19 1.2 0.06 0.07 bgibmga014348 uncharacterized protein loc106069352 3699.11 108.64 7868.94 3157.47 2.13 29.06 up-regulated bgibmga012002 sericin 3 precursor 5463.96 179.71 16584.36 6671.33 3.04 37.12 bgibmga001213 none 108.52 2.26 1161.74 112.25 10.71 49.67 bgibmga009261 uncharacterized protein loc105842476 0.1 0 94.66 9.37 916.06 bgibmga009799 aldo-keto reductase akr2e4-like 112.76 93.66 288.16 182.82 2.56 1.95 bgibmga006251 zonadhesin-like 8.47 3.61 16.05 8.16 1.89 2.26 bgibmga004219 uncharacterized protein loc101743399 7.81 8.29 48.88 47.61 6.26 5.74 bgibmga004809 uncharacterized protein loc105842986 70.93 66.23 121.04 90.48 1.71 1.37 bgibmga012486 cytochrome b5-related protein 20.06 42.64 44.91 95.09 2.24 2.23 bgibmga010285 uncharacterized protein loc101738995 4.13 7.83 18.49 24.06 4.48 3.07 bgibmga010514 glucose dehydrogenase [fad, quinone] isoform x1 1.36 0.81 10.95 1.72 8.05 2.14 bgibmga010863 aspartate--trna ligase, mitochondrial 6.5 6.33 53.93 40.12 8.29 6.34 bgibmga011868 uncharacterized protein loc101742906 1.53 1.67 17.83 17.47 11.63 10.48 bgibmga009091 fungal protease inhibitor f-like 925.76 186.12 1814.67 1295.84 1.96 6.96 the ratio represents the fold change of fpkm values: genes with a ratio ≥ 1.35 were considered to be up-regulated, genes with a ratio ≤ 0.74 were considered to be down-regulated 104 table 5 expression abundance of 9 genes gene id nr annotation nb♂-v nb♀-v nb♂-v vs fpkm fpkm nb♀-v ratio bgibmga012486 cytochrome b5-related protein 44.91 95.09 0.47 down-regulated bgibmga014348 uncharacterized protein loc106069352 7868.94 3157.47 2.49 up-regulated bgibmga012002 sericin 3 precursor 16584.36 6671.33 2.49 bgibmga001213 none 1161.74 112.25 10.35 bgibmga009261 uncharacterized protein 94.66 9.37 10.1 bgibmga009799 aldo-keto reductase akr2e4-like 288.16 182.82 1.58 bgibmga006251 zonadhesin-like 16.05 8.16 1.97 bgibmga010514 glucose dehydrogenase [fad, quinone] isoform x1 10.95 1.72 4.61 bgibmga009091 fungal protease inhibitor f-like 1814.67 1295.84 1.4 in the anti-bmnpv response of silkworm. by comparing the expression abundance of these 18 genes in nb♀-v and nb♂-v, nine genes were found to have different expression abundance, of which 8 genes had higher expression abundance in nb♂-v and 1 gene had higher expression abundance in nb♀-v (table 5). these genes denote that nb of different genders have certain differences in the anti-bmnpv response. rt-qpcr validation of degs the relative expression levels of 9 genes in nb-v, nb-c, 306-v, and 306-c were detected by rt-qpcr (fig 4a), as well as the relative expression levels of 4 genes in nb♀-v and nb♂-v (fig 4b). the rt-qpcr results were consistent with the transcriptome data, confirming the reliability of transcriptome sequencing result in this study. discussion in this study, we obtained some genes involved in the silkworm response to bmnpv infection through transcriptome sequencing, so as to study the mechanism of silkworm resistance to bmnpv and the differences in the anti-bmnpv response between silkworms of different genders. by comparing the gene expression between the experimental group and the control group, some differentially expressed genes that might be related to the bmnpv resistance were successfully identified. among them, bmtret1 has already been reported to be involved in the anti-bmnpv response (yang et al., 2016), but its resistance mechanism has not been clarified. in many non-mammals, tret1 can transport exogenous trehalose into cells and induce autophagy (sarkar et al., 2007), while the autophagy of silkworm cells is conducive to bmnpv infection (wang et al., 2017). therefore, we speculated that the down-regulation of bmtret1-like may play a role in the anti-bmnpv response by regulating autophagy. a variety of serine proteases have also been proved to have anti-bmnpv activity (nakazawa et al., 2004; li et al., 2017). among the candidate genes obtained in this study, both serine protease inhibitor 13 and fungal protease inhibitor f-like were the inhibitors of serine proteases (pham et al., 1996). and they might be involved in the anti-bmnpv process by interacting with serine proteases. viruses must rely on cellular proteins to complete replication in cells, so the protein metabolism of the host plays an important role in the game between host and virus (emmett et al., 2005). in this study, some candidate genes related to protein metabolism were also identified. for example, aspartate-trna ligase could regulate protein translation (ibba et al., 2000; ribas et al., 2000). cytochrome b5 could significantly regulate the function of cytochrome p450, thus extensively affecting the metabolism of exogenous and endogenous compounds (zhang et al., 2007; im et al., 2011). go analysis showed that loc105842986 might have lyase activity and participate in the transport and metabolism of amino acids, and loc101738995 might be involved in coenzyme binding and catalysis activity. the up-regulated expression of these genes suggested that they might influence the replication of bmnpv by regulating protein metabolism of silkworm. 105 fig. 4 a) rt-qpcr results of 9 genes. b) rt-qpcr results of 4 genes. the x axis represents different samples, and the y axis shows relative gene expression levels. the significance of changes in different gene expression levels was also indicated. samples used for qrt-pcr of nine genes were mixed samples of female and male silkworms, and samples used for qrt-pcr of four genes were female and male silkworm samples respectively ecdysone and juvenile hormone are the two most important hormones in insects, which can jointly regulate the growth, development and metamorphosis process of insects (herboso et al., 2015; riddiford et al., 2020). in this study, aldo-keto reductase-like (akr2e4-like) was up-regulated, and juvenile hormone acid o-methyltransferase-like (jhamt-like) was down-regulated in nb after bmnpv inoculated. akr2e4 could catalyze the production of ecdysone (yamamoto et al., 2017), while jhamt was the enzyme that catalyzes the last step of juvenile hormone biosynthesis in lepidopteran insects (shinoda et al., 2003). therefore, changes in their expression abundance may influence the biosynthesis of ecdysone and juvenile hormone. interestingly, in this study, the expression abundance of sericin 3 precursor and bgibmga009261 (contained fibroin p25 domain) in nb were up-regulated after bmnpv inoculation, which might be caused by the changed titer of ecdysone and juvenile hormone. in drosophila, steroid hormone signaling played a key role in regulating innate immunity and fighting bacterial infection (regan et al., 2013). ecdysone could closely regulate innate immunity to recognize and defend against bacterial infection by controlling the expression of pattern recognition receptor （pgrp-lc）in drosophila (rus et al., 2013). while juvenile hormone was an immunosuppressive factor that could strongly interfere with this ecdysone-dependent immune enhancement (flatt et al., 2008). however, these two hormones have not 106 been found to be involved in the immune system of silkworm. in this study, the expression abundance of akr2e4-like and jhamt-like were changed in nb after bmnpv inoculation, suggesting that juvenile hormone and ecdysone might participate in the regulation of immune response and the anti-bmnpv process in silkworm. the pupation time of nb males was about 24 hours earlier than that of females under normal rearing conditions, and this phenomenon still maintained in nb of different genders after bmnpv inoculation. therefore, a number of genes must be differentially expressed between nb of different genders after bmnpv inoculation. however, it is not clear whether there are differences in the anti-bmnpv response between silkworms of different genders. in this study, nb of different genders produced different numbers of degs and different specific degs after bmnpv inoculation, indicating that they did have different responses to bmnpv. although go analysis showed that the functional classifications of their specific degs were similar, there were differences in the genes involved in the anti-bmnpv response and their expression abundance. we screened nine genes that differentially expressed in nb of different genders after bmnpv inoculation. among them, the expression abundance of cytochrome b5-related protein was lower and fungal protease inhibitor f-like and akr2e4-like was higher in nb♂-v than those in nb♀-v. these genes have been discussed above.,and they might participate in the anti-bmnpv response by regulating protein metabolism, protein interaction or other ways. while the higher expression abundance of sericin 3 precursor, bgibmga009261 (contained fibroin p25 domain), glucose dehydrogenase [fad, quinone] isoform x1 and zonadhesin-like in nb♂-v might be related to the faster growth and development of nb males. these differences in genetic background between silkworms of different genders were still maintained after 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2b4 and competes with cytochrome p450 reductase for a binding site on cytochrome p450 2b4. j biol chem. 282(41): 29766-29776, 2007. zemp n, tavares r, widmer a. fungal infection induces sex-specific transcriptional changes and alters sexual dimorphism in the dioecious plant silene latifolia. plos genet. 11(10): e1005536, 2015. 11 isj 18: 11-18, 2021 issn 1824-307x research report a cold bath for a formalin-free laboratory: alternative fixative methods in early developmental stages of the sea urchin paracentrotus lividus (lamarck, 1816) a cannavacciuolo, a chiarore*, m munari department of integrative marine ecology, ischia marine centre, stazione zoologica anton dohrn, punta san pietro, 80077, ischia, naples, italy this is an open access article published under the cc by license accepted december 9, 2020 abstract fixatives are widespread in biological and medical research because they allow preserving specimens for a long time. historically, formaldehyde has been the most used fixative so far, but new solutions are needed because of its carcinogenicity. in this study, we tested alternative fixative methods to find a harmless, economic, and simple-to-use methodology to fix samples for larval morphological analysis in paracentrotus lividus. in two separate experiments, p. lividus embryos were fixed after 48 h post-fertilization by adding formalin free tissue accustaintm, naoh-buffered formalin free tissue accustaintm, glacial ethanol and denatured ethanol at different concentrations (from 10 % to 70 %) and by submerging the vials containing the larvae in seawater at 0 °c and maintained at 4 °c for 144 h. our results suggested that all the alternative fixatives tested do not guarantee a good quality of larvae for morphological purposes, while larvae that faced the thermal shock and were kept at 4 °c did not show any evidence of damage throughout time. the results of this study candidate this method as a good and safe substitute of formalin in studies that require morphological and taxonomic recognition and shed light on its use in other kinds of studies as well. key words: formalin; ethanol; alternative fixatives; larval development; sample preservation; sea urchin introduction fixation is a crucial step in life science and medical research since it allows to preserve specimens from decay and to analyse samples after a long time from the collection. since its invention, in 1859, formaldehyde has become very popular in research laboratories because of its low cost and high efficacy in fixation (chesnick et al., 2010). it is a gas which condenses forming a liquid known as ‘formalin’ usually available at the standard concentration of 37 % with a ph range from 2.8 to 4.0 (schander and halanych, 2003), however it can be buffered for specific purposes such as the preservation of calcareous organisms (prado et al., 2012; munari et al., 2016). in the liquid phase (from 4 % dilution) formalin is present as methyleneglycol, which can react with the -nh2 groups of proteins, forming methylene protein bridges during a process called ‘cross-linking’ (benerini gatta et al., 2012). historically, formaldehyde has been widely used for different categories of marine species: for ___________________________________________________________________________ corresponding author: antonia chiarore department of integrative marine ecology ischia marine centre, stazione zoologica anton dohrn punta san pietro, 80077, ischia, naples, italy e-mail: antonia.chiarore@szn.it example, in invertebrates research, it is used for the fixation of benthic species with pelagic early life stages (munari et al., 2016; oliva et al., 2016; foo et al., 2020), meiofauna (pusceddu et al., 2016; bertocci et al., 2019; rizzo et al., 2020), corals (calcinai et al., 2015), annelids (gravina et al., 2018), hydroids (fraschetti et al., 2002), colonizers on artificial panels used for the study of invertebrates (martell et al., 2018) and vertebrates like fishes (vacchi et al., 2007; meneghesso et al., 2013). formalin is also used to fix vegetal tissues of seaweeds (falace et al., 2005; pinna et al., 2020) and seagrasses (vasapollo and gambi, 2012). over the years the ‘formalin dogma’, considered an irreplaceable fixative, has started to be questioned (zanini et al., 2012) because of its carcinogenicity (european parliament, 2008), the shrinkage effect on some fish larvae (fowler and smith, 1983; morkert and bergstedt, 1990), and several difficulties for dna extraction protocols (schander and halanych, 2003). in recent years a lot of patented fixatives, that do not contain formalin, have been commercialized to replace formaldehyde (i.e., cellblock, cymol, finefix, greenfix, holland, lugol, notoxhisto, paga, rcl2, upm and zinc-based fixatives) in biological, clinical and pathology studies (acton et al., 2005; benerini gatta et al., 2012; 12 oselladore et al., 2012; zanini et al., 2012; yang et al., 2017). among them, to be mentioned is formalin free tissue accustaintm whose use has started for the preservation of marine invertebrates (regoli et al., 2019). a more classical method is represented by ethanol that has been historically used for the preservation of tissues and entire organisms such as fishes, crustaceans, nematodes, echinoderms (black and dodson, 2003; uthicke et al., 2004; fonseca and fehlauer-ale, 2012; la mesa et al., 2017). in studies concerning sea urchin species, glutaraldehyde is often used instead of formaldehyde, alone (ruocco et al., 2020) or mixed with acrolein (jubinville et al., 1967). however, formalin-free fixatives could cause collateral effects that can compromise several protocols from molecular to morphological ones (i.e. shrinking of fishes larvae) more than formalin (fowler and smith, 1983). moreover, depending on the number and the kind of species present in the sample, the chemical composition of the fixative may act differently (fiocca et al., 2014). for all of these reasons it is then difficult to find the ‘perfect fixative’ since each compound can be used for species-specific (fonseca and fehlauer-ale, 2012; yang et al., 2017) or aim-specific (acton et al., 2005; zanini et al., 2012) purposes. also, the possibility to observe small organisms under the microscope without a prior fixation of any kind is not always suitable since their movement in the media makes it difficult to conduct morphological and taxonomic observations. in this study we tested alternative compounds on the larval stages of paracentrotus lividus with the aim to find a fast fixative method that can be economic and harmless to the operator for morphological analyses, to be carried out in a short time. formalin free tissue accustaintm (fffa), glacial ethanol (ge) and denatured ethanol (de) at different concentrations were tested as a possible substitute to formaldehyde. furthermore, larvae were also maintained at a 4 °c degree in seawater as an alternative method to stop larvae swimming and preserve them over time. materials and methods animal collection and adult treatments paracentrotus lividus adults were collected in the marine protected area (mpa) ‘regno di nettuno’ along the coast of ischia island (40°44'47.9"n 13°56'39.3"e) by scuba divers at a depth of 2-5 m and immediately transported in the laboratory of the ischia marine centre (imc) of the stazione zoologica anton dohrn in cool boxes to avoid further stress. in the laboratory, adults were induced to spawn by injecting 1 ml of 0.5 m kcl solution into the coelom (byrne et al., 2008) and shaking the specimens to allow a homogeneous distribution of kcl solution. sperms were collected dry to avoid instantaneous activation and kept on ice until use. females were let to spawn upside down in beakers filled with 0.22 μm filtered seawater (fsw). before the fertilization, both sperms and eggs were checked for anomalies with an optic microscope (chiarore et al., 2020). fertilization was performed using a constant sperm:egg ratio (1250:1) (moschino and marin, 2002) and observed after 30 minutes to check the elevation of the jelly coat. embryos were successively maintained in 25 ml vials, for 48 h post-fertilization (hpf) at 22 °c, at a concentration of 50 larvae/ml. all experimental procedures on animals were done according to the guidelines of the european union (directive 609/86). larval fixation procedures after 48 hpf, in a set of vials, calculated volumes of fsw were removed with a modified plastic pasteur pipette equipped with 60 µm mesh to be sure to not lose larvae from the vial. successively, the volume was restored, and larval development was stopped by adding the chosen fixatives. in another set of vials, development was stopped by thermal shock, submerging the vials into a tank filled with seawater at 0 °c for 30 minutes at least. this method numbs larvae making them fall on the bottom, facilitating their collection for morphological analyses. two separate experiments were conducted to test the fixatives at different concentrations. in the first experiment, three non-formalin fixatives (ge, de and fffa) were tested. based on the results of the first experiment, in the second one, only two non-formalin fixatives (de, buffered fffa named bfffa) were used. bfffa was prepared by manually buffering the 4.76 ph of fffa adding 1 m naoh until a value of 7.22 ph units was reached. all of these fixatives were tested following concentrations in table 1. after fixation, samples table 1 fixatives and relative concentrations were calculated in % as volume of fixative per total volume of solution (v/v). ge= glacial ethanol; de= denaturated ethanol; fffa= ready to use formalin free fixative, accustain™; bfffa= naoh buffered formalin free fixative, accustaintm. 1ste = concentrations used in the first experiment. 2nde = concentrations used in the second experiment fixative concentration (% v/v) fixatives 10 20 30 50 70 ge 1ste 1ste 1ste de 2nde 2nde 1ste 1ste 1ste fffa 1ste 1ste 1ste bfffa 2nde 2nde 2nde 13 fig. 1 examples of sea urchin larvae considered in the analysis. a= tissue state: not damaged; transparency: transparent; spicules state: visible; precipitates: absent. b= tissue state: damaged; transparency: non transparent; spicules state: not visible; precipitates: present were preserved at 4 °c until microscope observation. each method was tested in three replicates and three times after fixation (named 24 h-afx, 72 h-afx and 144 h-afx) were considered to evaluate the efficiency of each method. larval developmental observation and classification a minimum of 100 larvae per replicate were observed and photographed in petri dishes under an optical macroscope (leica z16 apo), equipped with a leica dfc 300fx camera connected to a computer with the leica las program (leica application suite, version 4.5) for the three times after fixation. each vial was removed from the fridge just before larvae had to be photographed to avoid any degradation in particular for the fsw method. the different fixatives efficiency was evaluated through the analyses of four variables, necessary for the morphological analyses, as shown in figure 1 and table 2. statistical analysis the statistical analyses were performed with a non-parametric permutational multivariate analysis of variance, permanova (anderson, 2001) applied on the euclidean distance matrix of raw data. two different permanova designs were used to test differences between larvae treated with different fixatives. to test the effect of different fixatives on larval tissue morphology at three times after fixation, a two-factors design, time (3 levels) and fixatives (5 levels), was chosen. only 30 % concentration was taken into account since it was the only shared concentration among the different substances. to test the effect of different fixatives at low concentrations (10 %, 20 %, 30 %) on larval tissue morphology at three times after fixation, a two-factor design, considering the combined factor concentration x fixatives (named as condition, 7 levels in total) was performed evaluating effects of de, bfffa and fsw. the effects of high concentrations (50 % and 70 %) of fixatives were table 2 morphological variables analysed tissue state damaged: the tissue appeared to be degraded or the different anatomical structures were not clearly distinguishable not damaged: the tissue was intact, and the anatomical structures were clearly distinguishable transparency transparent: larvae were clear non transparent: larvae were opaque spicules state visible: it was not possible to distinguish the spicules because of degradation not visible: spicules were clearly visible precipitates absent: no precipitation of fixatives occurred present: salt, clumps, or a cloudy mixture was present at the microscopic observation 14 fig. 2 morphology of larvae 144 h after fixation. white (a), den 10 % (b), den 20 % (c), den 30 % (d), den 50 % (e) den 70% (f), eth 30% (g), eth 50 % (h), eth 70 % (i), fffa 30 %(j), fffa 50 %(k), fffa 70 % (l), fffb 10 % (m), fffb 20% (n), fffb 30% (o) excluded from the statistical analysis. the tissue and spicule states were analysed as a percentage, while transparency and precipitates as presence/absence. a non-metric multidimensional scaling (nmds) was also conducted to highlight the pattern of aggregation among the different methods of fixation. the software package primer 6 permanova plus (primer-e ltd, plymouth, uk) was used for all statistical analyses and the nmds. results the morphology of p. lividus larvae kept at the different fixation methods after 144 h is shown in figure 2. the results of the different fixatives efficiency at the three times after fixation on the morphological parameters are shown in table 3. first experiment permanova results of the effect of the interaction between the two factors are reported in table 4. table 3 results of considered variables for all the methods at the three times after fixation. tissue state: d= damaged, nd= not damaged; transparency: t= transparent, nt= non transparent; spicules state: v=visible, nv= not visible; precipitates: p= present, a= absent time 24 h-afx method fsw ge de fffa bfffa % v/v // 30% 50% 70% 10% 20% 30% 50% 70% 30% 50% 70% 10% 20% 30% tissue state nd d d d d d d d d d d d d d d transparency t nt nt nt nt nt nt nt nt nt nt nt nt nt nt spicule state v nv nv nv v v nv nv v nv nv nv nv v v precipitate a p p p a a a p p p p p a a a time 72 h-afx method fsw ge de fffa bfffa % v/v // 30% 50% 70% 10% 20% 30% 50% 70% 30% 50% 70% 10% 20% 30% tissue state nd d d d d d d d d d d d d d d transparency t nt nt nt nt nt nt nt nt nt nt nt nt nt nt spicule state v nv nv nv v v nv nv nv nv nv nv nv nv v precipitate a p p p a a p p p p p p a a a time 144 h-afx method fsw ge de fffa bfffa % v/v // 30% 50% 70% 10% 20% 30% 50% 70% 30% 50% 70% 10% 20% 30% tissue state nd d d d d d d d d d d d d d d transparency t nt nt nt nt nt nt nt nt nt nt nt nt nt nt spicule state v nv nv nv v v nv nv nv nv nv nv nv nv v precipitate a p p p a a p p p p p p a a a 15 table 4 permanova results. pseudo-f values and permutational p-values for all morphological variables analysed in p. lividus larvae throughout the maintenance of samples at five fixation methods (fsw; glacial ethanol 30 %; denatured ethanol 30 %; ready to use formalin free fixative, accustain™ 30 %; naoh-buffered formalin free fixative, accustaintm 30 %), at different times (24 h, 72 h and 144 h) after fixation are listed. significant results are in bold source df ss ms pseudo-f p-value fixatives (fi) 4 120940.0 30235.0 24.396 <0.001 time (ti) 2 282.2 141.1 0.114 0.892 fi x ti 8 1822.6 227.8 0.184 0.993 permanova highlighted a significant effect of the fixation method used on larvae preservation, while the time of maintenance and its interaction with the fixation method did not influence larval preservation (table 4). furthermore, the nmds evidenced a pattern of distribution with a high euclidean distance between fsw and all the other fixatives which instead showed a pattern of aggregation (figure 3). second experiment significant differences among different combinations of concentrations and fixatives were highlighted by permanova (table 5). the time of maintenance and its interaction with the different methods, did not show to influence larval preservation. results from the nmds analysis, calculated as the distance among centroids (figure 4), showed that there was a pattern of distribution with a high euclidean distance between the fsw and all the fixatives which instead showed a pattern of aggregation. results showed that spicules in fixed larvae were well visible and measurable only in the fsw at all times of maintenance. the same result has been obtained using de, but only after 24 h after fixation while after 144 h larvae were not so well maintained as shown in figure 2. furthermore, results showed that larval tissues appeared to be intact only in fsw at all times of maintenance, while all the remaining fixating methods did not succeed in preserving the integrity of the tissues (figure 2). discussions and conclusions the present study aimed to test the fixation efficacy of four no-formalin, alcohol-based substances for morphological purposes on sea urchin larvae. different concentrations of glacial ethanol, denatured ethanol, ready to use formalin free fixative accustain™, buffered formalin free fixative accustain™ were tested during 144 h after fixation. moreover, an alternative non-chemical method, based on numbing the larval with a cold temperature shock, was tested. some of the fixatives used in this study (ge, de and fffa) formed salt precipitates, as observed by neuhaus et al. (2017) for ethanol, and flocculates when used at concentrations of 50 % and 70 %. in the first experiment, the effects of the fig. 3 n-mds ordination plot of euclidean distances for all the data at the 30 % concentration 16 table 5 permanova results. pseudo-f values and permutational p-values for all morphological variables analysed in p. lividus larvae throughout the maintenance of samples at seven different conditions of fixation (fsw; denatured ethanol at 10, 20 and 30 %; naoh-buffered formalin free fixative, accustaintm at 10, 20 and 30 %), at different times (24 h, 72 h and 144 h) after fixation are listed. significant results are in bold source df ss ms pseudo-f p-value condition (co=concentration x fixative) 6 150000 24982.0 38.758 < 0.001 time (ti) 2 1134.2 567.1 0.880 0.428 co x ti 12 8812.0 734.3 1.139 0.363 common 30 % concentration of the four formalinfree substances and fsw on the larval development parameters were evaluated. permanova highlighted a significant difference among methods, in particular there was a pattern between fsw, and all the fixatives used as demonstrated by the nmds. this common pattern among fixatives could be explained considering that all of them are alcohol-based. in the same experiment, the permanova showed for each method of fixation that the time of maintenance was not a significant factor, at least for 144 h, being no differences among 48 h post-fertilization larvae observed at different times from fixation. however, the formalin-free fixatives used in the first experiment did not demonstrate to be good substitutes since already after the first observation time (24 h) deleterious morphological effects were evident. in order to reduce precipitates and flocculates, in the second experiment, larvae of p. lividus were fixated and maintained in de, bfffa at lower concentrations (10 %, 20 %, 30 %; kept at 4 °c) as well as in fsw (cold bath at 0 °c; kept at 4 °c) for 144 h. permanova highlighted a difference among the different conditions of maintenance with an evident pattern between fsw and compared to the others, similarly to the first experiment. in general, the fsw method showed to be the best in both experiments. in terms of larval calcareous structures, only in the fsw method, there was a 100 % of larvae with visible spicules during the 144 h of maintenance. a similar result was obtained at the lowest concentration (10 %) of the de. however, spicules visibility was the only variable in common with the fsw methods since larval tissues were as damaged as for the other conditions of preservation. indeed, results showed that only applying the fsw method, and at all the time considered, there were 100 % of notdamaged larvae, while fixatives at all concentrations considered had severe effects on larval tissues even after the first 24 h of fixation. it is important to mention that also bfffa induced a dramatic effect on larval quality, leading to the hypothesis that the low ph of fffa was not the main driver of the scarce success of the fixation observed in the first experiment, but probably the effect must be sought in the composition of this product. fig. 4 n-mds ordination plot of euclidean distances among centroids for fsw, de and bfffa at low concentrations (10 % 20 % 30 %) 17 in conclusion, our result showed that all the compounds tested are not suitable for all research based on sea urchin larval morphology, such as ecotoxicological investigations (chiarore et al., 2020; foo et al., 2020). on the contrary, applying a thermal shock close to 0 °c on larvae, and their maintenance at 4 °c, did not negatively influence larval morphological parameters for 144 h. this method could be considered very suitable for all laboratories, considering its cheapness and simplicity. an aspect that could be further investigated as development of this experiment could be assessing the mid and long-term fixation efficacy of the thermal-shock procedure. however, according to fonseca and fehlauer-ale (2012) fiocca et al. 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abstract the date fruit stalk borer, oryctes elegans prell, is a destructive pest on date palms in saudi arabia. we evaluated the immune response of the third instars (last instars), by the intrahemocoelic injection of lipopolysaccharide (lps) (a pathogen-associated molecular peptide, pamp), which significantly increased the total hemocyte count. phase-contrast light microscopy revealed the presence of five hemocyte types: prohemocytes, granulocytes, plasmatocytes, oenocytoids ,and spherulocytes. transmission electron microscopy demonstrated that, among these hemocytes, only the granulocytes and plasmatocytes phagocytosed latex beads. injection with lps also significantly decreased the number of oenocytoids. the antibacterial activity of plasma proteins of larvae injected with lps, measured using the agar well diffusion method, against gram-positive and gram-negative bacteria varied based on the bacterial strain, the total concentration of plasma protein, and time postinjection with lps. the results of the current study may be useful in the biological control of o. elegans. furthermore, new compounds with antibacterial activity that might be useful for the development of innovative drugs of natural origin can be identified in o. elegans. key words: oryctes elegans; total hemocyte count; differential hemocyte count; hemocyte morphology; phagocytosis; antibacterial activity introduction date palm (phoenix dactylifera l.) (arecales: arecaceae) is one of the most economically important fruit trees in saudi arabia and they cover approximately 155,118 ha and produced 991,546 tons of fruit in 2011 (al-ayedh and al dhafer, 2015). the date fruit stalk borer, oryctes elegans prell (coleoptera: scarabaeidae), is a destructive pest on p. dactylifera in saudi arabia and many other places globally (latifian and rad, 2012; al-ayedh and al dhafer, 2015; bedford et al., 2015). adults of o. elegans bore tunnels into the stalks of fruit bunches to feed, causing them to break off during windstorms. females oviposit in leaf axils and groups of larvae may tunnel inside the trunk causing the palm to topple (bedford et al., 2015). moreover, there have been indications that the feeding sites of o. elegans larvae may predispose palm trees to oviposition by the females of the red palm weevil, rhynchophorus ferrugineus (olivier) (coleoptera: ___________________________________________________________________________ corresponding author: eh shaurub department of entomology faculty of science cairo university giza, p.o. box 12613, egypt e-mail: shaurub@sci.cu.edu.eg curculionidae) (al-ayedh and al dhafer, 2015). since o. elegans is a concealed tissue borer, it is difficult to detect symptoms of its attack at an early stage of infestation. therefore, preventative measures are applied to manage o. elegans, which include physical control, trapping, host plant resistance, quarantine measures, and biological control (latifian and rad, 2012; bedford et al., 2015; atwa, 2018). the development of a biological control strategy for the successful integrated management of o. elegans requires the identification of its pathogens and more in-depth information about its immune system and defensive mechanisms against its pathogens. the immune response in insects differs fundamentally from that of vertebrates in the lack of cell specificity, immunoglobulins, and response memory. however, similar responses to immune memory in insects have been described as a phenomenon called "immune priming," where the insect exposed to a low dose of a pathogen becomes more resistant when later exposed to a high dose of the same pathogen (gálvez and chapuisat, 2014; sheehan et al., 2020). the innate immune system of insects consists of physical barriers, including the exoskeleton and peritrophic https://creativecommons.org/licenses/by/4.0/legalcode 148 fig. 1 total hemocyte count of o. elegans third instars 4 h and 24 h post-injection with lipopolysaccharide (lps). data are presented as the mean ± se. *, significant at p < 0.05 compared to the control, using studentʼs t-test membrane, cellular responses, and humoral responses (lavine and strand, 2002; shaurub, 2012; hillyer and strand, 2014; hillyer, 2016). when the barriers are breached, the cellular and humoral immune responses are activated (lemaitre and hoffmann, 2007; hillyer, 2016). the cellular immune responses are performed by hemocytes and include phagocytosis, nodulation, and encapsulation (lavine and strand, 2002; shaurub, 2012; browne et al., 2013; hillyer, 2016; dorrah et al., 2019). in most insects, granulocytes and plasmatocytes are the phagocytic cells (manachini et al., 2011; kwon et al., 2014; zhang and zhang, 2019). the humoral immune responses are based on the products of characterized immune genes induced by microbial infection and encode antimicrobial peptides (amps), which are synthesized predominantly in fat body and released into hemolymph (hoffmann, 1995; gillespie et al., 1997; nakatogawa et al., 2009; shia et al., 2009). these genes are either not expressed or are constitutively expressed at a low rate prior to infection (hoffmann, 1995; engström, 1998). insect amps could be categorized into the following major groups based on their secondary structure, amino acid sequence and antibacterial activity: linear amphipathic αhelix-forming peptides (e.g. cecropins), β-sheets or cystine-rich and cyclic amps (e.g. defensin), and proline-rich peptides and glycine-rich peptides (e.g. drosocin and coleoptericin) (trenczek et al., 1997; bulet, 2005; hull, 2012). the literature has described several coleopteran amps (hall et al., 2011; ntwasa et al., 2012). among them, cecropins, defensin, and attacins are not peculiar to this insect order, but coleoptericin, rhinocerocin, and holotricin have been described exclusively in coleoptera (ntwasa et al., 2012). amps identified in many insects operate synergistically against a wide range of grampositive and gram-negative bacteria. the onset of these molecules occurs after stimulation of the host with both viable and killed bacteria (cociancich et al., 1994; brivio et al., 2006; lemaitre and hoffmann, 2007) but also if larvae are immunized with a purified bacterial pathogen-associated molecular peptide (pamp) (e.g. lipopolysaccharide, lps) (mastore, 2015). in addition, humoral immune responses include activation of enzymic cascades that regulate coagulation and melanization of hemolymph, and production of reactive oxygen and nitrogen species (ros-rns) (gilespie et al., 1997; bogdan et al., 2000; nappi and vass, 2001; hoffmann, 2003; mavrouli et al., 2005; dorrah et al., 2019). 149 fig. 2 differential hemocyte count of o. elegans third instars 4 h (a), and 24 h post-injection (b) with lipopolysaccharide (lps). data are presented as the mean ± se. *, significant at p < 0.05 compared to the control, using studentʼs t-test most of the immunological studies on coleopterans that infest palm trees have been conducted on r. ferrugineus. for instance, hemocytes counts have been conducted, and phagocytosis (manachini et al., 2011) and antibacterial activity have been reported (mazza et al., 2011; mastore et al., 2015; sewify et al., 2017). however, few immunological studies have been conducted on oryctes spp., except for studies on antibacterial activity (rabeeth et al., 2012; veeramani et al., 2017). to the best of our knowledge, no studies have been conducted to elucidate the inducible immune response of o. elegans. therefore, this study aimed to elucidate i) total and differential hemocyte counts of third instars of o. elegans injected with lps, ii) in 150 vivo phagocytosis by hemocytes of larvae injected with latex, and iii) antibacterial activity of plasma proteins of larvae injected with lps against three gram-positive bacteria, staphylococcus aureus rosenbach (bacillales: staphylococcaceae), micrococcus luteus (schroeter) cohen (micrococcales: micrococcaceae) and bacillus subtilis (ehrenberg) cohen (bacillales: bacillaceae), and a gram-negative bacterium, escherichia coli (migula) castellani and chalmers (enterobacterales: enterobacteriaceae). materials and methods sampling the mature third instars (last instars) of oryctes elegans averaging 7.61 g in weight, 7.58 cm in length, and 5.23 cm in body circumference were collected during october and november 2012 and 2013 from the trunks of infested date palms in a private organic farm at haʼil province, saudi arabia (27°11'56"n, 42°59'13"e). the fallen and rotted palm trunks were open up to collect the third instars or they were directly collected underneath such trunks (atwa, 2018; pradipta et al., 2020). larvae were transported together with the soft frond bases of date palms in a well-ventilated container to the laboratory at 27 ± 2°c and 65 ± 5 % relative humidity. they were used for the immunological assays within 24 h of collection. hemolymph collection the third instars of o. elegans were rinsed three times with sterile distilled water to remove the soils and plant fibers. they were then surface sterilized with 75 % ethanol for a few seconds, and chilled on ice for 5–7 min. two groups of 10 larvae each were separately injected in the dorsal blood vessel with 20 ng ̸ 10 µl lps from e. coli serotype o26:b6 (sigma-aldrich, st. louis, mo, usa) using a hamilton syringe (25-gauge needle) according to mazza et al. (2011). hemolymph of the first and second groups was obtained from the dorsal blood vessel of larvae 4 and 24 h post-injection, respectively. a sample of 1 ml of hemolymph was collected from each larva and placed in sterilized 2ml eppendorf® tubes (sigma-aldrich, st. louis, mo, usa) containing an anticoagulant solution described by mazza et al. (2011). a parallel control group included non-injected larvae. each injected and non-injected group was replicated three times. the same procedures were also conducted 4 and 24 h post-injection with 50 µl of carboxylate-modified polystyrene latex beads with a diameter of 0.9 µm in a 10 % aqueous suspension (sigma-aldrich, st. louis, mo, usa). total hemocyte count, differential hemocyte count, and hemocyte morphology the total number of hemocytes was counted using a neubauer hemocytometer (dhc-no 1) (thermofischer scientific, waltham massachusetts, usa). the hemolymph that was obtained from o. elegans larvae 4 and 24 h post-injection with lps was drawn into a thoma white blood cell diluting pipette up to the 0.5 mark and then diluted 20 times with toissin’s solution as described by perveen and ahmad (2017). after discarding the first three drops, one drop was placed near the edge of a coverslip of a neubauer ruling and counting chamber, and it was automatically filled by capillary action. four whitecell squares from both the upper and lower chambers were counted. the total hemocyte count (thc) was calculated by using the formula suggested by jones (1962). hemocytes in a parallel control using hemolymph from non-injected larvae were also counted using the same approach. each treatment was replicated three times. the differential hemocyte count (dhc) was calculated in terms of the percentage of each hemocyte type in the total cells counted (jones, 1964), using an oil immersion phase-contrast microscope (olympus cx41, hamburg, germany) at 1000 × magnification. for the examination of the hemocyte morphology, 100 μl of the hemolymph that was collected from o. elegans larvae 4 and 24 h postinjection with lps was smeared on a sterilized glass slide, air-dried, and fixed for 2 min with absolute ethanol. blood films were then stained with freshly prepared 4 % giemsa stain (sigma-aldrich, st. louis, mo, usa) for 15 min and subsequently washed with distilled water. the slide was air-dried and mounted in canada balsam (sigma-aldrich, st. louis, mo, usa). the smears were examined using optical microscope (olympus bx51) with a digital camera (18-megapixel sc180, olympus) at 1000 × magnification. hemolymph of non-injected larvae served as the control experiment. each treatment was replicated three times. in vivo phagocytosis hemolymph that was collected from o. elegans larvae 4 and 24 h post-injection with latex was centrifuged at 3000 g for 10 min at 4 °c to precipitate hemocytes. then, the precipitated hemocytes were fixed in a 0.1 m cacodylate buffer with a ph of 7.2 with 2.5 % glutaraldehyde solution for 2 h. the fixed pellets were then post-fixed in cacodylate buffer with 1% osmium tetroxide solution for 2 h, dehydrated in a graded ethanol solution, and embedded in araldite® (sigma-aldrich, st. louis, mo, usa). the ultrathin sections, which were prepared using a leica ultracut r microtome (leica microsystems gmbh, wetzlar, germany), were stained with uranyl acetate and lead citrate. they were then examined using a jem-2100f transmission electron microscope (jeol, usa). a parallel control of ultrathin sections of hemocytes of non-injected larvae was also conducted. bacterial strains and culture conditions to characterize antibacterial activity, three gram-positive strains, s. aureus (atcc 6538), m. luteus (atcc 10240), and b. subtilis (atcc 6051) and a gram-negative strain, e. coli (atcc 10536), were assayed. all strains were maintained at -20 °c in brain heart infusion broth with 20 % v/v glycerol. before experimental use, each bacterial culture was propagated separately at 35 °c in a medium of 0.6 % bactopeptone, 0.4 % casein hydrolysate, 0.15 % beef extract, 0.3 % yeast extract, and 0.1 % glucose, ph 7.9 following bíliková et al. (2001). bacterial standard strains were purchased from the university boulevard manassas, va 20110 usa. https://en.wikipedia.org/wiki/friedrich_julius_rosenbach https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5478291/#b0085 151 agar well diffusion method hemolymph that was collected from o. elegans larvae 4 h and 24 h post-injection with lps was centrifuged at 3000 g for 10 min at 4 °c. the plasma supernatant was kept at -20 °c in eppendorf tubes containing an anticoagulant solution following mazza et al. (2011). four serial dilutions of plasma, corresponding to 2, 1, 0.5, and 0.25 mg proteins/ml, were prepared with phosphate-buffered saline with a ph of 7.2. each of the above-mentioned bacterial strains was inoculated into nutrient broth and incubated overnight at 37 °c until growth was 1.5 mg/ml at an optical density of 640 nm. muellerhinton agar plates were prepared by dispensing 25 ml of a sterile diagnostic sensitivity test agar (dehydrated, thermo scientifictm oxoidtm) into sterile petri dishes with a diameter of 10 cm. a growth inhibition zone assay was conducted, according to magaldi et al. (2004) and valgas et al. (2007). the solidified agar plates were streaked uniformly with one of each of the above-mentioned cultured bacterial strains. four equidistant wells, with a diameter of 5 mm, were made in the agar, and each well was filled with 50 µl of each dilution of plasma. the same procedures were applied to a parallel control using plasma of non-injected larvae. all plates were left for 1 h at room temperature for complete diffusion, after which they were incubated at 37 °c for 24 h. the diameters of the agar inhibition zones were measured in mm. each assay was replicated three times. total protein content in lps-injected plasma and that of non-injected plasma was quantified according to the methods of bradford et al. (1976). statistical analysis all experiments were conducted according to a completely randomized design. data were checked for normality prior to analysis using shapiro-wilk test and were symmetric. data are presented as the mean ± standard error (se) and were analyzed using studentʼs t-test between treatments and controls. comparison between the two times of treatments (4 and 24 h) was also analyzed using studentʼs t-test. the significance level was set at p ≤ 0.05, and all statistical analyses were conducted using ibm® spss® statistics, version 25 (ibm® corp. armonk, ny, usa). results total hemocyte count injection of o. elegans larvae with lps significantly increased the thc 4 h (36.74%; t = 20.84, df = 4, p < 0.001) and 24 h (33.23%; t = 13.45, df = 4, p < 0.001) post-injection compared to that of the control (fig. 1). the thc of o. elegans larvae 24 h was not significantly different (p > 0.05) from that 4 h post-injection with lps. differential hemocyte count phase-contrast light microscopy revealed that five hemocyte types were identified in o. elegans larvae: prohemocytes, granulocytes, plasmatocytes, fig. 3 light microscopic photomicrographs (1000 ×) of normal giemsa-stained hemocytes of o. elegans third instars (column a), and 4 h post-injection with lps (column b). a. prohemocytes. b. granulocytes. c. plasmatocytes. d. oenocytoids. e. spherulocytes 152 oenocytoids, and spherulocytes. four hours postinjection with lps, the number of oenocytoids in the hemolymph of o. elegans larvae was significantly decreased (df = 4, t = 5.47, p = 0.005) compared to that of the control (fig. 2a). after 24 h of exposure to lps, the number of each hemocyte type did not change (p > 0.05) in the hemolymph of the o. elegans larvae compared to that of the controls (fig. 2b). a variable change in the number of each hemocyte type 24 h compared to that 4 h postinjection with lps was observed: the prohemocytes (df = 4, t = 23.02, p < 0.001) and oenocytoids (df = 4, t = 25.58, p < 0.001) were significantly increased. in contrast, the plasmatocytes were significantly decreased (df = 4, t = 5.45, p = 0.005). hemocyte morphology a particular morphology was observed in each hemocyte type: the prohemocytes were round and small with a central nucleus and homogenous cytoplasm, the granulocytes were round and heavily granulated with a central nucleus, the plasmatocytes looked spindleshaped and had pointed ends and fine poorly stained granules, the oenocytoids contained a round and eccentric nucleus and homogeneous cytoplasm, and the spherulocytes were oval with cytoplasm containing large, round vesicles or spherules (figs 3a, 4a). four hours after the injection of o. elegans larvae with lps, some changes were observed in hemocyte morphology compared to those of the controls: the central nucleus of the prohemocytes occupied most of the cytoplasm (fig. 3b-a), and the nucleus of the granulocytes was darkly-stained (fig. 3b-b). after 24 h of exposure to lps, the cytoplasm in the prohemocytes formed a thin layer around the nucleus (fig. 4b-a), while the cytoplasm of the granulocytes was highly degranulated, and the nucleus was more central when compared to the control (fig. 4b-b). in addition, the plasmatocytes were swollen with degranulated cytoplasm (fig 4bc), the cytoplasm of the oenocytoids appeared transparent (fig. 4b-d), and the spherulocytes contained dense spherules (fig. 4b-e). in vivo phagocytosis transmission electron microscopy (tem) revealed that granulocytes and plasmatocytes were the phagocytic cells in o. elegans larvae. these granulocytes were round with many granules and a central nucleus (figs 5a-a, 6a-a), and the plasmatocytes were spindle-shaped with a large and round nucleus (figs 5a-b, 6a-b). four hours post-injection with latex beads, the granulocytes and plasmatocytes showed conspicuous phagocytic activity that was more pronounced in granulocytes: several phagosomes containing latex beads were observed. the granules fused with the phagosomes and discharged their contents inside them, making the space around the latex beads more electron-dense. multi-vesicular bodies were also present (fig. 5b-a). the phagocytic granulocytes showed degranulated cytoplasm filled with latex beads (fig. 5b-b), and the phagocytic plasmatocytes, which contained latex bead particles inside their phagosomes (fig. 5b-c), were irregular in shape, varying from round to very fig. 4 light microscopic photomicrographs (1000 ×) of normal giemsa-stained hemocytes of o. elegans third instars (column a), and 24 h post-injection with lps (column b). a. prohemocytes. b. granulocytes. c. plasmatocytes. d. oenocytoids. e. spherulocytes 153 fig. 5 a. transmission electron microscopy photomicrographs of normal hemocytes of o. elegans third instars 4 h without injection with latex beads. a. granulocyte with many typical granules (gr), lobated nucleus (n) with an evident nucleolus (nu), rough endoplasmic reticulum (r), mitochondria (m), golgi complex (g), round vesicles (asterisks), and electron-dense vesicles (v). b. oval-shaped plasmatocyte with few granules (gr), nucleus (n) with evident nucleolus (nu), and mitochondria (m). scale bar = 2 µm. b. transmission electron microscopy photomicrographs of hemocytes of o. elegans third instars 4 h post-injection with latex beads. a. granulocyte showing several phagosomes with latex beads (lb), a nucleus (n) with an evident nucleolus (nu), rough endoplasmic reticulum (r), cytoplasm showing golgi complex (g), many filopodia (f), mitochondria (m), and round vesicles (asterisks). b. degranulated granulocyte with many vacuoles containing latex beads (lb). c. spindleshaped plasmatocyte with many phagosomes containing latex beads (lb), a heterochromatic nucleus (n), rough endoplasmic reticulum (r), mitochondria (m), and filopodia (f) engulfing latex beads (lb) (inset). scale bar = 2 µm 154 fig. 6 a. transmission electron microscopy photomicrographs of normal hemocytes of o. elegans third instars 24 h without injection with latex beads. a. granulocyte with granules (gr), indented nucleus (n) with nucleolus (nu), golgi complex (g), filopodia (f), mitochondria (m), and round vesicles (asterisks). b. spindle-shaped plasmatocyte with filopodia (f), nucleus (n), mitochondria (m), and rough endoplasmic reticulum (r). scale bar = 2 µm. b. transmission electron microscopy photomicrographs of hemocytes of o. elegans third instars 24 h post-injection with latex beads. a. degranulated granulocyte, nucleus (n), mitochondria (m), and golgi complex (g). b. plasmatocyte showing many elongated golgi complex (g), an eccentric nucleus (n) with nucleolus (nu), mitochondria (m), rough endoplasmic reticulum (r), and phagosomes with latex beads (lb). scale bar = 2 µm elongated, with deeply indented and heterochromatic nuclei. after 24 h of exposure to latex, the granulocytes became highly degranulated (fig. 6b-a), and the plasmatocytes exhibited an eccentric nucleus, many elongated golgi complexes and phagosomes containing latex beads (figs 6bb). antibacterial activity four hours post-injection with lps, the diameters of the agar inhibition zones of s. aureus were significantly lower than those of the controls (non-injected) at the four tested concentrations of total plasma proteins (df = 4, t = 4.70, p = 0.009; df = 4, t = 4.43, p = 0.011; df = 4, t = 10.35, p = 0.001; df = 4, t = 10.98, p < 0.001 for 2, 1, 0.5, and 0.25 mg protein/ml, respectively) (fig. 7a), indicating the suppression of antibacterial activity. however, after 24 h of exposure to lps, the agar inhibition zone diameters of s. aureus were significantly larger than those of the controls at 0.25 mg protein/ml (df = 4, t = 15.01, p < 0.001) (fig. 7b), suggesting antibacterial activity. the agar inhibition zone diameters of s. aureus were significantly increased 24 h compared to those 4 h post-injection with lps at 2 mg protein/ml (df = 4, t = 2.68, p = 0.05), 0.5 mg/ml (df = 4, t = 4.10, p = 0.015) and 0.25 mg/ml (df = 4, t = 7.65, p = 0.002). the plasma proteins of lps-injected o. elegans larvae resulted in a significant increase in the diameters of the agar inhibition zones of m. luteus 4 h post-injection at the four tested concentrations of 155 fig. 7 agar inhibition zone (aiz) diameters of s. aureus growth 4 h (a), and 24 h post-injection (b) of o. elegans third instars with lipopolysaccharide (lps). data are presented as the mean ± se. *, significant at p < 0.05 compared to the control, using studentʼs t-test plasma proteins compared to those of the controls (df = 4, t = 2.06, p = 0.019; df = 4, t = 4.74, p = 0.009; df = 4, t = 5.0, p = 0.008; df = 4, t = 4.81, p = 0.009 for 2, 1, 0.5, and 0.25 mg protein/ml, respectively) (fig. 8a). whereas, after 24 h of exposure to lps, the diameters of the agar inhibition zones of m. luteus were significantly decreased compared to those of the controls at 0.5 mg protein/ml (df = 4, t = 4.36, p = 0.012) and 0.25 mg protein/ml (df = 4, t = 13.19, p < 0.001) (fig. 8b). the agar inhibition zone diameters of m. luteus were significantly decreased 24 h compared to those 4 h post-injection with lps at 1 mg/ml (df = 4, t = 5.08, p = 0.007), 0.5 mg/ml (df = 4, t = 8.60, p = 0.001) and 0.25 mg/ml (df = 4, t = 10.25, p = 0.001). the agar inhibition zone diameters of b. subtilis were significantly increased compared to those of the controls 4 h post-injection with lps at the four 156 fig. 8 agar inhibition zone (aiz) diameters of m. luteus growth 4 h (a), and 24 h post-injection (b) of o. elegans third instars with lipopolysaccharide (lps). data are presented as the mean ± se. *, significant at p < 0.05 compared to the control, using studentʼs t-test tested concentrations of plasma proteins (df = 4, t = 2.50, p = 0.05; df = 4, t = 2.71, p = 0.05; df = 4, t = 5.29, p = 0.006; df = 4, t = 11.69, p < 0.001 for 2, 1, 0.5, and 0.25 mg protein/ml, respectively) (fig. 9a), indicating antibacterial activity. in contrast, the agar inhibition zone diameters of b. subtilis were significantly decreased compared to those of the controls after 24 h of injection with lps at all the concentrations of plasma proteins tested, indicating the suppression of antibacterial activity (df = 4, t = 11.26, p < 0.001; df = 4, t = 76.70, p < 0.001; df = 4, t = 12.70, p < 0.001; df = 4, t = 10.04, p = 0.001 for 2, 1, 0.5, and 0.25 mg 157 fig. 9 agar inhibition zone (aiz) diameters of b. subtilis growth 4 h (a), and 24 h post-injection (b) of o. elegans third instars with lipopolysaccharide (lps). data are presented as the mean ± se. *, significant at p < 0.05 compared to the control, using studentʼs t-test mg protein/ml, respectively) (fig. 9b). the agar inhibition zone diameters of b. subtilis were significantly decreased 24 h compared to those 4 h post-injection with lps at the four concentrations of plasma protein tested (df = 4, t = 10.25, p = 0.001; df = 4, t = 11.11, p < 0.001; df = 4, t = 11.90, p < 0.001; df = 4, t = 11.41, p < 0.001 for 2, 1, 0.5 and 0.25 mg protein/ml, respectively). the diameters of the agar inhibition zones of e. coli were significantly smaller than those of the controls 4 h post-injection of o. elegans larvae with lps at 2 mg protein/ml (df = 4, t = 7.89, p = 0.001), 0.5 mg protein/ml (df = 4, t = 6.63, p = 0.003), and 0.25 mg protein/ml (df = 4, t = 5.81, p = 0.004) (fig. 10a). after 24 h of exposure to lps, the agar inhibition zone diameters of e. coli were significantly smaller than those of the controls at 2 mg protein/ml (df = 4, t = 7.37, p = 0.002), 1 mg protein/ml (df = 4, t = 13.25, p < 0.001), and 0.5 mg protein/ml (df = 4, t = 17.70, p < 0.001), whereas at 0.25 mg protein/ml the diameters of the agar inhibition zones of e. coli were significantly larger than those of the controls 158 fig. 10 agar inhibition zone (aiz) diameters of e. coli growth 4 h (a), and 24 h post-injection (b) of o. elegans third instars with lipopolysaccharide (lps). data are presented as the mean ± se. *, significant at p < 0.05 compared to the control, using studentʼs t-test (df = 4, t = 12.98, p < 0.001) (fig. 10b), indicating antibacterial activity. the agar inhibition zone diameters of e. coli were significantly decreased 24 h compared to those 4 h post-injection with lps at the four concentrations of plasma protein tested (df = 4, t = 9.15, p < 0.001; df = 4, t = 10.55, p < 0.001; df = 4, t = 14.36, p < 0.001; df = 4, t = 4.05, p = 0.016 for 2, 1, 0.5 and 0.25 mg protein/ml, respectively). discussion the present study represents the first report on the inducible immune response of o. elegans. the immune response of this insect was time posttreatment dependent. although the thc 24 h postinjection with lps was not significantly affected compared to that 4 h post-injection, the thc was significantly increased compared to the controls at each time treatment. patton (1961) implicated the hemocytes of insects in the detoxification of toxic xenobiotics. the number of prohemocytes and oenocytoids were significantly increased with the increase of time post-injection with lps from 4 h to 24 h. the prohemocytes are the progenitor stem cells that can differentiate into other hemocyte types for phagocytosis to combat biotic and abiotic foreign invaders or apoptotic bodies (hernandez et al., 159 1999; de silva et al., 2000; yamashita and iwabuchi, 2001; lavine and strand, 2002). dorrah et al. (2019) suggested that increased oenocytoids in the fleshfly, sarcophaga argyrostoma (robineaudesvoidy) (diptera: sarcophagidae) last instars 36 h post-treatment with the limonoid, azadirachtin, may have been an attempt by larvae to compensate the decrease in phenoloxidase (po) activity in the hemocytes of treated larvae. hemocyte morphology and phagocytosis in o. elegans larvae were also time dependent, with the greatest effect 24 h post-injection with lps and latex, respectively. a declining trend in antibacterial activity, 24 h after exposure to lps, was observed for the grampositive bacteria studied, except for s. aureus where the antibacterial activity was increased 24 h compared to that 4 h post-injection with lps. this finding suggests that the humoral immune system of o. elegans cannot recognize s. aureus 4 h postexposure to lps. nevertheless, this hypothesis needs future confirmatory experiments. four hours post-injection of o. elegans larvae with lps, the number of oenocytoids was significantly decreased compared to the control. the decrease in the number of oenocytoids suggests the immunosuppressive effect of lps, where these cells are known to release the precursor of po (prophenoloxidase, propo), which plays a role in the melanization of the hemolymph (ribeiro et al., 1996). the insect hemogram and the peculiar hemocyte combination in each developmental stage are important and serve as indicators of environmental adaptability (sharma et al., 2008). hemocytes, via a change in cell number, are frequently used to demonstrate the cytogenetic damage induced by toxic xenobiotics (wessel et al., 2007). our study identified five hemocyte types in o. elegans larvae: prohemocytes, granulocytes, plasmatocytes, oenocytoids, and spherulocytes. likewise, the same hemocyte types have been reported in r. ferrugineus larvae (manachini et al., 2011). the finding that granulocytes and plasmatocytes were the most abundant hemocytes in o. elegans agrees with results reported by manachini et al. (2011) and kwon et al. (2014). tem observations revealed that granulocytes and plasmatocytes were the phagocytic hematocytes in the hemolymph of o. elegans larvae. the higher phagocytic activity of granulocytes suggests that they were the primary phagocytes, and the phagocytic activity of granulocytes and plasmatocytes may explain their substantial proportion in o. elegans larvae. this is in line with findings for other insects on the involvement of granulocytes and plasmatocytes in phagocytosis (manachini et al., 2011; kwon et al., 2014; zhang and zhang, 2019). our findings of ultrastructural changes in granulocytes and plasmatocytes due to phagocytosis are also comparable to those reported for most insects (giulianini et al., 2003; lemaitre and hoffmann, 2007; borges et al., 2008; amaral et al., 2010; kwon et al., 2014; zhang and zhang, 2019). in addition, several studies have reported that phagocytosis of latex beads in insects has been regulated by signaling pathways that could be initiated from the phagosome including activation of the nuclear factor κb (nf-κb) and activation of mitogen-activated protein kinases (mapk) (fratti et al., 2001; lamprou et al., 2007) and involves both the zipper and trigger mechanisms (borges et al., 2008). phagocytosis of latex beads do not depend on propo activation (mavrouli et al., 2005; lamprou et al., 2007). phagocytosed macromolecules have also been broken down by hydrolase localized in the lysosomes of insect hemocytes (callewaert and michiels, 2010; wu and yi, 2015). the agar well diffusion method demonstrated that plasma proteins of o. elegans larvae in our study showed antibacterial activity against the bacterial strains studied. in agreement with our results, the antibacterial activity of hemolymph of r. ferrugineus and the coconut palm rhinoceros beetle, oryctes rhinoceros (l.) (coleoptera: scarabaeidae), against the same tested bacteria has also been reported (mazza et al., 2011; rabeeth et al., 2012; mastore et al., 2015). most amps exert their antibacterial effects by interacting with and destabilizing both plasma membrane and bacterial walls, eventually leading to cell death, and some amps can enter the cell and interact with cytoplasmic factors (ding et al., 2003; brogden, 2005; alves et al., 2010; mastore et al., 2015). evidence in the literature suggests that host amps could act in cooperation with other immunocompetent factors to modulate innate immune responses (hoffman and reichhart, 2002). overall, the three tested gram-positive bacteria, s. aureus, m. luteus, and b. subtilis, were more sensitive than the gram-negative bacterium, e. coli. this sensitivity may be attributed to the fact that gram-positive bacteria are monoderms and have a single lipid layer, whereas gram-negative bacteria are diderms and have two layers. the presence of the outer cell membrane in gramnegative bacteria is thought to play an important role as a protective mechanism against antimicrobial agents and antibiotic selection pressure (sewify et al., 2017). furthermore, some species of gram-negative bacteria, such as klebsiella spp., are encapsulated in prominent polysaccharide capsules that provide them more resistance and protection against antibiotics (podschun and ullmann, 1998). insects recognize pamps (e.g. lps) as a result of the interaction with endogenous pattern recognition receptors (prrs). the interaction of pamps and prrs elicits humoral and cellular defence, leading to the elimination of foreign pathogens (hoffmann et al., 1999; govind, 2008). the presence of microorganisms and their pamps inside insects’ hemocoelic cavity triggers various short-term immune processes, such as propo-po system activation (humoral encapsulation) or phagocytosis (mastore et al. 2015). however, these defence processes are only successful in the presence of a limited infection; when bacterial proliferation overcomes short-term defence, the onset of antibacterial responses, performed by amps and lysozymes, are the most effective https://www.ncbi.nlm.nih.gov/pmc/articles/pmc6826287/#bio045864c7 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc6826287/#bio045864c7 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc6826287/#bio045864c32 160 mechanism to prevent host septicemia (yeaman and yount, 2003). in conclusion, our results point to the importance of understanding the inducible immune response of o. elegans. this may open new perspectives for the biological control of this insect. furthermore, o. elegans could represent a kind of “living laboratory” in which to identify new compounds with antibacterial activity that might be useful for the development of innovative drugs of natural origin, which are able to counteract the antibiotic resistance that currently presents a serious threat to human and animal health. conflict of interest on behalf of all 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../../../../jln/appdata/local/microsoft/windows/inetcache/content.outlook/fml5h3jd/biol.%20open 83 isj 15: 83-93, 2018 issn 1824-307x research report combined effects of temperature and salinity on growth, survival, gill morphology, and antioxidant capabilities in the horse mussel modiolus modiolus y zhan, m yang, d cui, j li, j sun, j ning, z hao, w zhang, y chang key laboratory of mariculture & stock enhancement in north china’s sea, ministry of agriculture, dalian ocean university, dalian, liaoning, 116023, p. r. china accepted march 04, 2018 abstract temperature and salinity are critical to the reproduction, growth, and survival of shellfish. we investigated the combined effects of temperature (15 °c, 20 °c, and 25 °c) and salinity [20, 30, and 40 practical salinity units (psus)] on the horse mussel modiolus modiolus, which inhabits the northern chinese coast. the specific growth rate of m. modiolus increased with salinity at a low temperature (15 °c). the interaction between temperature and salinity with respect to m. modiolus survival was significant (two-way anova; p < 0.01). across all temperatures tested, m. modiolus specimens kept at low salinity (20 psu) had narrower gill filament lumens than those kept at moderate salinity (30 psu); the gill filament lumens of specimens kept at high salinity (40 psu) were wider than either of these. the combined effects of temperature and salinity on superoxide dismutase (sod) activity, total antioxidant capacity (t-aoc), and glutathione peroxidase (gpx) activity in the gills of m. modiolus were significant (two-way anova; p < 0.05). our results suggested that temperature and salinity significantly affect the survival, gill morphology, and antioxidant capabilities of m. modiolus. our findings not only enrich our knowledge of the interactions between temperature and salinity with respect to shellfish but also provide a framework for m. modiolus aquaculture development. key words: horse mussel; gill lumen; sod; t-aoc; gpx; superoxide dismutase; total antioxidant capacity; glutathione peroxidase; sgr introduction the horse mussel, modiolus modiolus (mollusca: bivalvia), is a benthic, sessile species distributed worldwide in coastal areas (hutchison et al., 2016). in china, m. modiolus is mainly found in the subtidal zones of the yellow and bohai seas (ning et al., 2015). m. modiolus modifies its habitat via bio-deposition, forming beds on various substrata (fariñas‐franco et al., 2016). it is therefore believed that m. modiolus has an important impact on the ecological equilibrium of the sedimentary environments of the shallow sublittoral (ragnarsson and burgos, 1984). m. modiolus is highly edible and grows rapidly to a large size (ning et al., 2016). this species has thus recently been proposed as a potential new aquaculture species in china (ning et al., 2015). ___________________________________________________________________________ corresponding author: yaqing chang key laboratory of mariculture & stock enhancement in north china’s sea ministry of agriculture dalian ocean university dalian, liaoning, 116023, p. r. china e-mail: yqkeylab@hotmail.com temperature and salinity are the two primary environmental factors affecting the reproduction, growth, and survival of shellfish (meryem et al., 2015). for example, increased water temperature and water quality were thought to contribute to mortality in the oyster crassostrea gigas (shelaghk et al., 2009), while high temperature significantly affected survival, oxygen consumption, behavior, ammonia-n excretion, and related immune indicators in the japanese scallop mizuhopecten yessoensis (hao et al., 2014). in addition, the restoration of normal antioxidant enzyme activity in the clam cyclina sinensis was increased after exposure to abnormal salinities; c. sinensis had a greater tolerance for low salinity than for high salinity (li et al., 2012). recently, the combined effects of temperature and salinity on shellfish have received more attention (samuel et al, 2015). for example, wang et al. (2017) showed that a temperature of 26 °c and a salinity of 30 practical salinity units (psu) were optimal for the culture of juvenile ark shells (anadara broughtonii). unfortunately, studies of the combined influence of temperature and salinity on shellfish remain scarce. mailto:yqkeylab@hotmail.com 84 fig. 1 effects of temperature and salinity on sgr in m. modiolus. s20, salinity of 20 psu; s30, salinity of 30 psu; s40, salinity of 40 psu. bars show the means of 3 replicates. error bars show sd. table 1 one-way and two-way anova of the effects of different temperatures and salinities on sgr in m. modiolus. values are means ± sd (n = 3). temperature (°c) salinity (psu) p-value duncan test temperature salinity interaction one-way anova (factor: temperature) 15, 20, 25 20 0.85ns -- 15 °c = 20 °c = 25 °c 30 0.96ns 15 °c = 20 °c = 25 °c 40 0.04* 15 °c > 20 °c = 25 °c one-way anova (factor: salinity) 15 20, 30, 40 - 0.02* - 40 psu > 30 psu = 20 psu 20 0.84ns 20 psu = 30 psu = 40 psu 25 0.91ns 20 psu = 30 psu = 40 psu two-way anova (fixed factors: temperature & salinity) 15, 20, 25 20, 30, 40 0.15ns 0.16ns 0.11ns 15 °c = 20 °c = 25 °c 20 psu = 30 psu = 40 psu *, value is significant based on the duncan tests (p < 0.05); ns, value is not significant based on the duncan test. although m. modiolus tolerates a wide range of environmental conditions (lesser and kruse, 2004), it has previously been shown that alterations in a single environmental factor (such as temperature, salinity, or ph) affect growth, survival, and feeding (lesser and kruse, 2004; ning et al., 2012). before large-scale farming of m. modiolus begins, it is critical to identify the optimal conditions for such farms. we therefore explored the combined impact of temperature and salinity on m. modiolus, specifically aiming to 1) identify the effects of temperature and salinity on growth and survival in m. modiolus; 2) identify morphological changes in the gills of m. modiolus in response to temperature and salinity; 3) identify alterations in superoxide dismutase (sod) levels, total antioxidant capacity (t-aoc), and glutathione peroxidase (gpx) levels in the gills of m. modiolus in response to changes in temperature and salinity. our results will help to determine the ideal nursery conditions and farming locations for m. modiolus. 85 materials and methods animal collection and treatments in november 2015, we collected 220 healthy adult modiolus modiolus specimens (mean shell height = 42.71 ± 3.08 mm; mean shell length = 96.09 ± 4.65 mm; mean shell width = 49.82 ± 3.21 mm; and mean mass = 13.39 ± 2.32 g) from the near-shore sublittoral zone of the north china sea near the ministry of agriculture key laboratory of mariculture & stock enhancement at dalian ocean university, dalian, china (121°33′47″ e, 38°51′55″ n). all specimens were kept in ~60 l recirculating seawater tanks; each tank was fitted with an automatic temperature control and monitoring system (dalian huixin titanium equipment development co., ltd, liaoning, china). seawater was sand filtered and continuously aerated. specimens were kept in natural light. all specimens were supplied with 0.25 g spirulina powder (dalian golden bay technology co., ltd, liaoning, china) daily. the amount of spirulina powder supplied exceeded the daily requirements of the specimens (ning et al., 2015) to ensure that our results were not biased by insufficient nutrition. we also judged that the spirulina was supplied in excess because extra powder remained in the water after a day of circulation. all specimens were acclimated to default laboratory conditions (13.95 ± 1.32 °c and 31.69 ± 0.35 psu) for one week before experiments began. experiments were conducted between november 2015 and january 2016. after acclimation, all specimens were dried with a paper towel and weighed with digital balance (0.01 g sensitivity; al204; mettler toledo, shanghai, china) to obtain initial mass (w1). we then divided mussels into 27 groups of three specimens each (three temperatures × three salinities with three replicates of each combination; table s1). each group was housed in a separate tank. to reach the desired temperature/salinity combinations, we removed half of the seawater from each tank every day, and replaced it with seawater at a different temperature/salinity. we changed the temperature of the new seawater such that the temperature of the entire tank did not increase by more than 1 °c per day; this was based on previous study on m. modiolus (ning et al., 2015) and on field survey data of the coastal waters of the yellow sea (zhang et al., 2014). we altered the salinity of the replacement seawater either by adding fresh filtered water or by adding synthetic sea salt (aqua salt; dalian salt industry co., ltd, liaoning, china). we altered the salinity of the new seawater such that salinity in the entire tank did not change by more than 2 psu per day (ning et al., 2015, hamer et al., 2008). we monitored the temperature and salinity in each tank both using the automatic temperature control, and with a water quality monitor (a329 portable meter; thermo scientific orion star, beijing, china). growth and survival in each treatment during the 30-day experimental period, dead m. modiolus [defined as loss of motility and lack of external stimulus response (ning et al., 2015)], were noted and removed immediately. at the end of the experiment, all living specimens were again dried and weighed to obtain a final mass (w2). we then calculated the specific growth rate (sgr; defined as the growth rate per day) and survival rate (sr) for each group as follows: sgr (% × day-1) = 100 × (lnw2 lnw1)/t sr (%) = 100 × (n2/n1) where n1 is the initial number of live specimens; n2 is the final number of live specimens; and t is the duration of the experiment in days. gill filament morphology and antioxidant analyses the gills of all living m. modiolus specimens were carefully removed at the end of the experimental period. gills were divided into two parts: one for morphological analysis, and the other for detection of sod, t-aoc, and gpx. to example gill filament morphology, we fixed gill tissue samples for 24 h in bonn fluid (75 ml saturated picric acid, 25 ml 40 % formaldehyde, and 5 ml acetic acid), following the procedures of wu et al. (2007). we then transferred the fixed samples into different concentrations of ethanol for gradient dehydration (han et al.,2014). dehydrated fixed tissues were embedded in paraffin wax, and 5 µm sections were stained with hematoxylin and eosin (qu et al., 2013). we then examined gill filament morphology and photographed the gill filament lumens using an optical microscope with attached camera (50i; nikon, japan). using dn-2 (dalian zhonghe company, liaoning, china) with our gill photographs, we measured the width of each gill filament lumen for each specimen, as it has been shown that lumen width is affected by changes in temperature and salinity (wang et al., 2016). to determine sod, t-aoc, and gpx, gill samples were dried with paper towels and homogenized immediately in an ice bath. gill homogenates were centrifuged at 3,500 rpm for 10 min at 4 °c. supernatants were stored at -80 ºc. we used commercial kits to determine sod, t-aoc, and gpx (nanjing jiancheng company, china; kit model used for sod: a001-1; for t-aoc: a015-1; for gpx: a005), following the manufacturer's instructions. sod activity, t-aoc level, and gpx activity were calculated in units per mg protein (u × mg protein-1) as follows: sod = (a2 a3)/0.5 × (v1/v0)/c0 t-aoc = (a3 a2)/0.01/30 × (v1/v0)/c0 gpx = (a2 a3)/(a1 a0) × c1 × 5/5/(v0 × c0) where a0 is the light absorption (o.d.) of the blank group; a1 is the light absorption (o.d.) of the standard group; a2 is the light absorption (o.d.) of the control group; a3 is the light absorption (o.d.) of the given experimental group; c0 is the protein concentration of the homogenate; c1 is the protein concentration of the standard (20 μg/l); v0 is the volume of the sample; and v1 is the volume of the total reaction. 86 the protein concentration of each gill homogenate was determined with the bradford method, using bovine serum albumin as the standard, on a multimode reader (spectramax i3x; molecular devices, shanghai, china). data analysis all statistical analyses were performed with spss 16.0 (ibm, shanghai, china). we first confirmed that our data were normally distributed and homogeneous with the shapiro-wilk test and with levene’s test. we then compared differences in sgr, survival rate, gill filament lumen size, sod activity, t-aoc level, and gpx activity among treatments with one-way anova (factor: temperature or salinity) and with two-way anovas (fixed factors: temperature and salinity). we considered p < 0.05 statistically significant. significant differences between pairs of treatments were identified with duncan’s multiple range test. results effects of temperature and salinity on growth and survival only m. modiolus specimens cultured at 15 °c in high salinity (40 psu) had an increased sgr (fig. 1; table 1, s2). we found that m. modiolus cultured in moderate salinity (30 psu) had relatively higher survival rates as compared to other salinity treatments at the same temperature (fig. 2). high survival rates (> 90%) were also observed at temperatures below 20 °c and salinities between 30 and 40 psu. m. modiolus specimens kept at 20 °c in low salinity (20 psu) had lower survival rates (68.75 ± 0.08 %). for m. modiolus specimens maintained at 25 °c, survival rate decreased as salinity increased (fig. 2). the interaction between temperature and salinity was significant with respect to survival (two-way anova, p < 0.01), and salinity had a more pronounced effect fig. 2 effects of temperature and salinity on survival in m. modiolus. s20, salinity of 20 psu; s30, salinity of 30 psu; s40, salinity of 40 psu. bars show the means of 3 replicates. error bars show sd. table 2. one-way and two-way anova of the effects of different temperatures and salinities on survival rate in m. modiolus. values are means ± sd (n = 3). temperature (°c) salinity (psu) p-value duncan test temperature salinity interaction one-way anova (factor: temperature) 15, 20, 25 20 0.06ns -- 15 °c > 25 °c > 20 °c 30 0.15ns 15 °c = 20 °c = 25 °c 40 0.004** 20 °c = 15 °c > 25 °c one-way anova (factor: salinity) 15 20, 30, 40 - 0.19ns - 20 psu = 30 psu = 40 psu 20 0.01* 40 psu = 30 psu > 20 psu 25 0.14ns 20 psu = 30 psu = 40 psu two-way anova (fixed factors: temperature & salinity) 15, 20, 25 20, 30, 40 0.02* 2.05×10-4**** 0.50×10-2* 15 °c = 20 °c > 25 °c 30 psu > 20 psu = 40 psu *, value is significant based on the duncan tests (*p < 0.05, **p < 0.01, ****p < 0.0001); ns, value is not significant based on the duncan test. 87 fig. 3 effects of temperature and salinity on gill filament morphology in m. modiolus. s20, salinity of 20 psu; s30, salinity of 30 psu; s40, salinity of 40 psu. on m. modiolus survival than the temperature (two-way anova, p < 0.01). it is interesting to note that the negative effects of low salinity (20 psu) on m. modiolus were somewhat alleviated at the low temperature (i.e., 15 °c) and the high temperature (25 °c) (one-way anova, p > 0.05; table 2). effects of temperature and salinity on gill filament morphology as salinity and/or temperature increased, the gill filament lumens of m. modiolus specimens widened (fig. 3). relatively narrow gill filament lumens were observed in m. modiolus specimens cultured at 20 °c in low salinity (20 psu). at moderate salinity (30 psu), gill filament lumen size did not vary obviously with incubation temperature. wider gill filament lumens were observed in m. modiolus specimens cultured at 15 °c in high salinity (40 psu). at 25 °c, gill filament lumens were significantly smaller in low salinity (20 psu) than in high salinity (40 psu; fig. 4; table 3). the interaction between temperature and salinity with respect to the width of gill lumen was significant (two-way anova, p < 0.01), and salinity had a more pronounced effect on gill filament morphology than did temperature (two-way anova, p < 0.01). high temperatures (25 °c) partially alleviated the negative effects of high salinity on the gill filament morphology of m. modiolus (one-way anova, p < 0.05; table 3). 88 fig. 4 effects of temperature and salinity on gill filament lumen width in m. modiolus. s20, salinity of 20 psu; s30, salinity of 30 psu; s40, salinity of 40 psu. bars show the means of 3 replicates. error bars show sd. table 3 one-way and two-way anova of the effects of different temperatures and salinities on gill filament lumen size in m. modiolus. values are means ± sd (n = 3). temperature (°c) salinity (psu) p-value duncan test temperature salinity interaction one-way anova (factor: temperature) 15, 20, 25 20 3.54×10-11**** -- 15 °c = 25 °c > 20 °c 30 1.51×10-4**** 15 °c = 20 °c > 25 °c 40 4.60×10-11**** 15 °c > 20 °c > 25 °c one-way anova (factor: salinity) 15 20, 30, 40 - 1.93×10-9**** - 40 psu > 30 psu > 20 psu 20 4.43×10-31**** 40 psu > 30 psu > 20 psu 25 0.10×10-2** 40 psu = 30 psu > 20 psu two-way anova (fixed factors: temperature & salinity) 15, 20, 25 20, 30, 40 3.27×10-13**** 2.09×10-40**** 1.57×10-10**** 15 °c > 20 °c = 25 °c 40 psu > 30 psu > 20 psu *, value is significant based on the duncan tests (**p < 0.01, ****p < 0.0001); ns, value is not significant based on the duncan test. effects of temperature and salinity on sod activity, t-aoc level, and gpx activity sod activity in the gills of m. modiolus specimens kept at 15°c declined significantly with increased salinity (p < 0.001); in specimens kept at 25°c, sod activity increased with increased salinity (fig. 5; table 4). at 20 °c, sod activity was significantly higher in specimens cultured in low salinity (20 psu) and in those cultured in high salinity (40 psu), as compared to those cultured in moderate salinity (30 psu). the interaction between temperature and salinity with respect to sod activity was significant (two-way anova, p < 0.001). the t-aoc of the m. modiolus gill was significantly affected by both temperature (one-way anova, p < 0.05) and salinity (one-way anova, p < 0.05; fig. 6; table 5). at each of the tested temperatures, the gills of m. modiolus specimens cultured in moderate salinity (30 psu) had lower t-aoc than those cultured in low (20 psu) and high salinities (40 psu; fig. 6). at each of the tested salinities, the gills of m. modiolus specimens kept at 89 fig. 5 effects of temperature and salinity on gill sod level in m. modiolus. s20, salinity of 20 psu; s30, salinity of 30 psu; s40, salinity of 40 psu. bars show the means of 3 replicates. error bars show sd. table 4 one-way and two-way anova of the effects of different temperatures and salinities on sod levels in m. modiolus. values are means ± sd (n = 3). temperature (°c) salinity (psu) p-value duncan test temperature salinity interaction one-way anova (factor: temperature) 15, 20, 25 20 8.49×10-6**** -- 15 °c = 25 °c > 20 °c 30 0.10×10-2** 20 °c > 25 °c > 15 °c 40 0.30×10-2** 20 °c = 25 °c > 15 °c one-way anova (factor: salinity) 15 20, 30, 40 - 2.53×10-6**** - 20 psu > 30 psu > 40 psu 20 3.03×10-5**** 20 psu > 30 psu = 40 psu 25 0.10×10-2** 40 psu > 30 psu = 20 psu two-way anova (fixed factors: temperature & salinity) 15, 20, 25 20, 30, 40 1.59×10-12**** 3.00×10-11**** 9.96×10-12**** 15 °c > 20 °c = 25 °c 40 psu > 30 psu > 20 psu *, value is significant based on the duncan tests (**p < 0.01, ****p < 0.0001); ns, value is not significant based on the duncan test. a moderate temperature (20 °c) had higher t-aoc than those maintained at low (15 °c) or high temperatures (25 °c; fig. 6). the interaction between temperature and salinity with respect to t-aoc in the m. modiolus gill was significant (two-way anova; p < 0.01). gpx activity in the gills of m. modiolus was significantly affected by both temperature (one-way anova; p < 0.0001) and salinity (one-way anova; p < 0.0001; fig. 7; table 6). at each of the tested temperatures, specimens kept at both low (20 psu) and high salinities (40 psu) had significantly less gpx activity than those kept at a moderate salinity (30 psu). at each of the tested salinities, the gills of m. modiolus specimens cultured at a moderate temperature (20 °c) had relatively higher gpx activity than those cultured at low (15 °c) and high (25 °c) temperatures (fig. 7). the interaction between temperature and salinity with respect to gpx activity in the m. modiolus gill was significant (two-way anova; p < 0.0001). discussion previous studies have indicated that shellfish growth is affected by both temperature and salinity (hao et al., 2014; çelik et al., 2015). hao et al. (2014) showed that exposing m. yessoensis to a low temperature (15 °c) for 30 days increased sgr significantly, while bashevkin and pechenik (2015) 90 fig. 6 effects of temperature and salinity on gill t-aoc in m. modiolus. s20, salinity of 20 psu; s30, salinity of 30 psu; s40, salinity of 40 psu. bars show the means of 3 replicates. error bars show sd. table 5 one-way and two-way anova of the effects of different temperatures and salinities on t-aoc in m. modiolus. values are means ± sd (n = 3). temperature (°c) salinity (psu) p-value duncan test temperature salinity interaction one-way anova (factor: temperature) 15, 20, 25 20 0.1×10-1* - 20 °c > 15 °c = 25 °c 30 0.1×10-2** 20 °c >25 °c > 15 °c 40 2.32×10-6**** 20 °c > 25 °c > 15 °c one-way anova (factor: salinity) 15 20, 30, 40 - 0.1×10-2** - 20 psu > 40 psu > 30 psu 20 4.26×10-4**** 40 psu > 20 psu = 30 psu 25 0.60×10-2** 40 psu > 20 psu = 30 psu two-way anova (fixed factors: temperature & salinity) 15, 20, 25 20, 30, 40 1.95×10-8**** 6.24×10-11**** 9.71×10-6**** 20 °c > 15 °c = 25 °c 40 psu > 20 psu > 30 psu *, value is significant based on the duncan tests (*p < 0.05, **p < 0.01, ****p < 0.0001); ns, value is not significant based on the duncan test. found that exposure to low temperature and low salinity significantly reduced both the larval and juvenile growth rates of the gastropod, crepidula fornicata. it has also been shown that salinity had the greatest effect on the growth rate of a. broughtonii (wang et al., 2017). here, we found that the sgr of adult m. modiolus specimens increased with salinity at a low temperature (15 °c). we therefore hypothesize that response to temperature and salinity in marine mollusks varies with species. adult m. modiolus specimens survived ten days at salinities of 20 and 40 psu and temperatures between 10 °c and 12 °c; at moderate salinity (~31 psu) and temperatures above 25 °c, survival decreased (ning et al., 2015). our results were consistent with this previous study. we also found that salinity alone, especially low salinity, was the main cause of reduced survival in m. modiolus. therefore, our data support the hypothesis that salinity maintenance should be the first concern of shellfish aquaculture (wang et al., 2017). as low salinity significantly reduced the feeding rate of c. fornicata juveniles (samuel and pechenik, 2015), we postulate that the reduced survival rate of m. modiolus might be due to a dramatic decrease in feeding rate. with respect to aquaculture management, it was worth noting that the negative effects of low salinity were somewhat alleviated by decreased or increased temperatures. 91 fig. 7 effects of temperature and salinity on gill gpx levels in m. modiolus. s20, salinity of 20 psu; s30, salinity of 30 psu; s40, salinity of 40 psu. bars show the means of 3 replicates. error bars show sd. table 6 one-way and two-way anova of the effects of different temperatures and salinities on gpx levels in m. modiolus. values are means ± sd (n = 3). temperature (°c) salinity (psu) p-value duncan test temperature salinity interaction one-way anova (factor: temperature) 15, 20, 25 20 1.31×10-10**** -- 20 °c > 25 °c > 15 °c 30 3.79×10-7**** 20 °c > 25 °c > 15 °c 40 2.33×10-4**** 20 °c > 25 °c = 15 °c one-way anova (factor: salinity) 15 20, 30, 40 - 5.59×10-8**** - 30 psu > 20 psu > 40 psu 20 1.40×10-7**** 30 psu > 20 psu > 40 psu 25 1.20×10-5**** 30 psu > 20 psu > 40 psu two-way anova (fixed factors: temperature & salinity) 15, 20, 25 20, 30, 40 1.89×10-17**** 1.30×10-18**** 1.17×10-6**** 20 °c > 25 °c > 15 °c; 30 psu > 20 psu > 40 psu *, value is significant based on the duncan tests (****p < 0.0001); ns, value is not significant based on the duncan test. the adverse effects of temperature and salinity on m. modiolus were also clear in our examination of gill morphology. gills are the primary organs of both respiration and defense in bivalves (fiddy et al., 2016). here, abnormal temperatures and salinities damaged gill filaments and altered gill filament lumen width. salinity affects the respiration and growth of larval and juvenile c. fornicata (samuel et al., 2015); thus, it is possible that the reduction in m. modiolus survival observed here might have been due to the effects of temperature and salinity on respiration, instead of feeding rate. alternatively, it has been shown that changes in gill filament lumen size indirectly demonstrate an imbalance in osmotic pressure (han et al., 2014). therefore, it might be that temperature and salinity affect the survival of m. modiolus by disrupting endogenous osmotic pressure. further work is necessary to assess the combined impact of temperature and salinity on respiration and osmotic pressure in m. modiolus. the generation of reactive oxygen species (ros) is a typical physiological phenomenon in aerobic organisms. it is clear that excessive accumulation of ros (such as o2, h2o2, and oh -) can cause oxidative damage to lipids, proteins, carbohydrates, and nucleic acids (khazri et al., 2016; shenaitirodkar et al., 2017). the maintenance of optimal ros levels is thus vital for endo-redox homeostasis and for the survival of aerobic species. 92 sod and gpx, two important enzymes in the antioxidant defense system, also serve as ros scavengers (levent et al., 2005); t-aoc typically reflects the balance of oxidants and antioxidants in aerobic organisms (fan et al., 2004). decreases in temperature (from 26 ºc to 15 ºc) significantly decreased sod activity in m. yessoensis at normal salinity (~31 psu; hao et al., 2014). in adult c. gigas, sod activity decreased and then increased as temperature increased from 24 ºc to 32 ºc, but in adult c. brasiliana, sod activity decreased steadily with the same temperature increase (moreira et al., 2017). here, we found that sod activity increased and then decreased as temperature rose from 15 ºc to 25 ºc. these results suggested that the effects of temperature on sod activity vary among bivalves; there may be species-specific mechanisms that manage temperature variations. at 15 °c, sod activity decreased significantly as salinity increased, but at 25 °c, sod activity increased significantly as salinity increased. this interesting result indicated that salinity might have a more profound impact on sod activity in the gill of m. modiolus than temperature. it was reported that elevated temperature increased the activity of gpx in the gills of the brown mussel perna perna (almeida et al.,2007). in contrast, gpx activity in the gills of m. modiolus at both low and high temperatures (15 °c and 25 °c) was lower at all tested salinities than gpx activity at 20 °c. this discrepancy might be due to differences in gpx function among species. in addition, the decreased gill gpx activity at both low and high salinities (20 and 40 psu) observed in m. modiolus might indicate that increased salinity interrupts the antioxidant system of this species by reducing the catalytic capacity of gpx. t-aoc levels in m. modiolus increased at both low and high salinities, suggesting that m. modiolus might compensate for the increased numbers of ros induced by abnormal temperatures and salinities by increasing t-aoc. further research is necessary to clarify the mechanism by which m. modiolus increases t-aoc. our results suggested that m. modiolus might be suitably cultured between 15 °c and 20 °c, and between 30 and 40 psu. our study provides a framework for the selection of suitable locations for m. modiolus aquaculture, as well as for the development of sustainable aquaculture methods. our data also increase our understanding of the impact of temperature and salinity on molluscan growth and survival. future studies should focus on identifying and testing additional ros markers to further clarify the effects of temperature and salinity on endo-redox homeostasis in m. modiolus. in addition, molecular studies (including differential gene expression, transcriptome comparison, 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http://xueshu.baidu.com/s?wd=author:(%c3%96zt%c3%bcrk,%20recep)%20sinop%20university%20faculty%20of%20fisheries%20and%20aquaculture%20sinop%20turkey&tn=se_baiduxueshu_c1gjeupa&ie=utf-8&sc_f_para=sc_hilight=person 90 isj 17: 90-98, 2020 issn 1824-307x review hirudo verbana as an alternative model to dissect the relationship between innate immunity and regeneration n baranzini, l pulze, f acquati, a grimaldi* department of biotechnology and life sciences, university of insubria, via j. h. dunant 3, 21100 varese, italy this is an open access article published under the cc by license accepted may 11, 2020 abstract given the key role of innate immunity in both defense against pathogens and tissue regeneration, innovative studies are becoming crucial to provide further information on how both processes are linked together and to clarify how immune cells perform the coordinated regulation of the aforementioned processes. the present review is mainly focused on two proteins that have been recently found to carry out critical functions in innate immune system regulation, i. e. the allograft inflammatory factor-1 (aif-1) and rnaset2, a protein belonging to the t2 ribonuclease family. their crucial role in both the activation and modulation of the inflammatory response and in the remodeling of connective tissue during grafts and wound repair have been thoroughly investigated in the medicinal leech and will pave the way for novel therapeutic approaches to control immune and systemic responses to disease, injury, and bacterial infection, based on the functionalities of these biomolecules. key words: medicinal leech; innate immunity; regeneration; aif-1; rnaset2 introduction the innate immune response serves not only to detect and eliminate pathogens following injury or infections, but it is also directly involved in regenerative processes related to maintaining homeostasis, modulating wound healing processes and preserving tissue functional integrity (martin, 1997; saltzman, 1999; frantz et al., 2005; muraille, 2013). indeed, recent data clearly suggest that infiltration of macrophages, essential cellular components of the innate immune system, play a predominant role in tissue regeneration by eliminating cellular debris, thus preventing the persistence of potentially toxic or immunogenic material in the tissue environment. moreover, tissue macrophages have also been involved in promoting the synthesis of growth factors and cytokines, thus favoring extracellular matrix (ecm) remodeling and tissue regeneration processes (godwin et al., 2013; aurora and olson, 2014; parisi et al., 2018). interestingly, inflammation-mediated tissue regeneration represents an evolutionarily conserved mechanism for tissue repair and several studies have been recently carried out to elucidate the corresponding author: annalisa grimaldi department of biotechnology and life sciences university of insubria via j. h. dunant 3, 21100 varese, italy e-mail: annalisa.grimaldi@uninsubria.it functional link(s) between the “classical” host defense features of the innate immune system and its role in the regenerative process. in this context, comparison of both regenerative processes and the cellular and molecular immune response effectors in different vertebrate and invertebrate model systems should represent a powerful tool in order to shed more light into this highly relevant topic (tasiemski and salzet, 2017; godwin et al., 2017; malagoli, 2018). it is now clear that successful tissue regeneration in pluricellular organisms requires the precise coordination of multiple processes, which include: 1) the recognition of both pathogens associated molecular pattern (pamps) and damageassociated molecular patterns (damps) that are produced and released during tissue damage in infectious and/or noninfectious inflammatory response (molteni et al. 2016) and 2) a rapid immune response stimulated by cytokines produced by activated tissue-resident inflammatory cells (eming et al., 2009; kawai and akira, 2010). in this context, specific pattern recognition receptors (prrs) (mahla et al., 2013), such as toll-like receptors (tlrs) are known to play a key role not only in host defence (by regulating both innate and adaptive immune responses) (takeda and akira, 2004; yang et al., 2008; girardello et al., 2019), but also in the regulation of the different phases of wound healing and regenerative processes. mailto:annalisa.grimaldi@uninsubria.it 91 indeed, recent studies revealed that macrophages and other immune cells, following activation of tlr4 and tlr2 signaling pathways, play a critical role in tissue regeneration (kluwe et al., 2009; wynn and barron, 2010; oishi and manabe, 2016) by producing and releasing a variety of soluble factors which stimulate the proliferation, differentiation and activation of many cell types involved in tissue regeneration, such as fibroblasts, epithelial, endothelial and stem cells (aurora and olson, 2014; wynn and vannella, 2016). of note, innate immune responses induced by tlrs are tightly controlled, since abnormal tlrs pathway activation could be harmful to the host by triggering an excessive inflammatory response (molteni et al., 2016). based on these premises, and given the fundamental role of innate immunity in both defense against pathogens and regeneration, innovative studies aimed at providing further information on how the innate immune response and the regenerative process are linked together would be of great interest, also to better clarify how immune cells perform the coordinated regulation of the aforementioned processes through the control mechanisms of tlrs-inducible genes. invertebrates as alternative animal models for studying the cross-talk between innate immune response and regeneration processes it is widely known that the use of animal models in biomedical research is crucial, as it provides a fundamental approach to study and extrapolate to humans the complex cellular and molecular interactions that occur in living tissues. however, the number of animal species that can be used for biomedical experimentation is currently undergoing extensive revision, due to ethical considerations, more stringent controls and legislative interventions aimed at improving animal welfare. indeed, the recent directive 2010/63/eu of the european parliament and council (available online: http://eur-lex.europa.eu/legal content/en/txt/pdf/?uri=celex:32010l0063&fro m=en) and the more restrictive italian dl 26/2014 act focused on the “protection of animals used for scientific purposes” strongly promote the use of alternative models in biological research. in the attempt to address the recent restrictions imposed by these directives on animal protection, many researchers have intensified the use of vertebrate cell lines and/or "primary" cell cultures as experimental models to tackle various research topics in the biomedical field. however, this approach suffers a considerable limitation due to the significantly reduced complexity of the experimental model used, which can greatly compromise the adequate and effective understanding of the biological phenomena under investigation. within this frame, some species of invertebrates have been increasingly used to successfully replace mammals or other vertebrates as experimental models in basic and applied biomedical research. indeed, despite their simpler body organization and the consequent lower genetic complexity, some higher invertebrate species show a level of cellular and molecular complexity quite similar to that of vertebrates and, above all, present biological processes and systems that have been highly conserved during evolution. moreover, the growing availability of “omics” dataset from many invertebrate species, combined with the increasing availability of recombinant proteins derived from the same species, has allowed to define and study in this organisms gene networks involved in complex biological phenomena (such as innate immunity) or biosynthetic pathways involving new molecules and bioactive metabolites, thus allowing further development of invertebrates as reliable model organisms in biological research (adams et al., 2000). invertebrate biotechnology therefore represents an emerging discipline that aims at using these organisms for an ever increasing number of biomedical and biotechnological applications. for instance, several invertebrates are currently being studied as potential sources of innovative products such as enzymes, biopolymers, bioactive compounds and secondary metabolites, which can find applications in various research fields related to human health and well-being, such as nutraceuticals, research on new antibiotics, development of anti-inflammatory drugs or anti fouling molecules and cosmetics. the use of a model organism in biomedical research raises the question of the most appropriate organism as an immediate issue. the main criterion for dealing with this choice clearly depends on the biological or biomedical problem of interest. in this review, we propose two species of medicinal leech (hirudo medicinalis and hirudo verbana) as alternative and innovative experimental models in the context of an important biomedical research branch. in particular, the medicinal leech, unlike long-established non-vertebrate models such as the nematode caenorhabditis elegans and the fruit fly drosophila melanogaster, display the fundamental feature of sharing with vertebrates not only the biological processes involved in innate immune response and tissue repair (inflammation, fibroplasia, angiogenesis, remodeling) but also the deployment of the same effector cell types (de eguileor et al., 2000b, a; grimaldi et al., 2006; grimaldi, 2016) and molecules (cytokines) for the regulation of the different phases of these processes (tettamanti et al., 2003, 2006; grimaldi et al., 2004, 2018). indeed, the recent release of both genomic and transcriptomic data from both h. medicinalis (macagno et al., 2010) and h. verbana (http://genomes.sdsc.edu/leechmaster/database/) have highlighted the occurrence of sequence homologs for several factors involved in both peripheral and neuroimmune leech systems (schikorski et al., 2009; macagno et al., 2010; tasiemski and salzet, 2017; girardello et al., 2019). finally, studies on the presence of nociception in this organism have not conclusively demonstrated its ability to experience the state of mental/emotional pain that is widely recognized in many vertebrate species (harvey-clark, 2011). based on these premises, the medicinal leech represents an excellent alternative experimental model to the currently used vertebrate models (such as mice, rats or rabbits) for biomedical studies http://eur-lex.europa.eu/legal-content/en/txt/pdf/?uri=celex%3a32010l0063&from=en http://eur-lex.europa.eu/legal-content/en/txt/pdf/?uri=celex%3a32010l0063&from=en http://eur-lex.europa.eu/legal-content/en/txt/pdf/?uri=celex%3a32010l0063&from=en http://genomes.sdsc.edu/leechmaster/database/) 92 focused on the innate immune system and tissue regenerative process. new insights into the role of allograft inflammatory factor -1 (aif-1) cytokine and rnaset2 ribonuclease in the medicinal leech several studies carried out in vertebrates have reported the presence of macrophages in all adult and embryonic tissues and, besides their long known role in host defense and apoptotic cells clearance, these reports have also unveiled unprecedented roles for this cell type in both trophic and regenerative functions. indeed, macrophages produce and secrete different molecules such as growth factors, cytokines and enzymes, which not only induce vessels and immune cells recruitment to injured/grafted or bacteria-infected tissues, but also promote tissue repair (ovchinnikov, 2008; mills, 2012). among these molecules, two macrophage derived interesting factors have been recently demonstrated to be involved in inflammatory responses and tissue regeneration: the cytokine allograft inflammatory factor 1 (aif-1) (alkassab et al., 2007; pawlik et al., 2008) and rnaset2, a member of ribonuclease t2 family (acquati et al., 2019). however, the mechanisms by which inflammatory responses and tissue regeneration are intimately connected and regulated by these molecules are still far from being fully elucidated. interestingly, both molecules are rapidly activated during medicinal leech response against different type of pathogen infections or damages and mechanical wounds (schorn et al., 2015b; baranzini et al., 2017, 2019; girardello et al., 2019), suggesting their involvement in the establishment of a functional cross-talk between the inflammatory response and the regenerative process. h. medicinalis homologous hmaif-1 expression regulates the cross-talk between innate immune response and tissue regeneration aif-1 is a 17 kda cytoplasmic cytokine (alkassab et al., 2007) originally identified in rat cardiac transplant subject to chronic rejection (utans et al., 1995). subsequently, several aif-1 like factors showing both a highly conserved aminoacid sequence and a well preserved functional role have been identified in other vertebrate species (deininger et al., 2000; watano et al., 2001; mentshel et al., 2002; autieri and chen, 2005) and in several invertebrates, including the genus hirudineae (kruse et al., 1999; de zoysa et al., 2010; zhang et al., 2011; ovando et al., 2012; li et al., 2013; drago et al., 2014; schorn et al., 2015b). in vertebrates, expression of this cytokine (mostly from the monocyte/macrophage lineage) is known to significantly increase after grafts, wounds or bacterial infections (utans et al., 1995; alkassab et al., 2007). since aif-1 is a ca2+-dependent factor, it has been hypothesized that this cytokine might have a crucial role in regulating cell-cell interactions during an inflammatory response and, as such, might act as a possible modulator of both immune response and tissue regeneration through macrophages activation (alkassab et al., 2007; pawlik et al., 2008). indeed, its ability to bind calcium leads to peculiar protein expression and cell cycle regulation patterns through distinct pathways of signal transduction (tanaka and koike, 2002). of note, aif-1 colocalizes with the cd45 transmembrane glycoprotein, expressed on the surface of vertebrate nucleated hematopoietic cells. cd45 is implicated in integrin-mediated adhesion of myeloid leukocytes (roach et al., 1997; zhu et al., 2011; st-pierre and ostergaard, 2013) and is known to modulate the responsiveness of monocytes and macrophages to chemoattractants, regulating chemokine receptor expression in these cells and in turn the mechanisms required to maintain adhesion and phagocytic activity (roach et al., 1997; mitchell et al., 1999). macrophage adhesion to the extracellular matrix is associated to their maturation, expression of the specific cd68 marker and response to environmental stimuli (trowbridge and thomas, 1994; roach et al., 1997). indeed, the hypothesis that both cd45 and aif-1 might be involved in macrophage maturation and activation stems from the observation that monocytes show high cd45 and aif-1 expression levels, whereas aif-1+ resident microglia barely express cd45 (jeong et al., 2013). the direct relationship between aif-1/cd45 expression and macrophage activation/migration during the early inflammation phase after injury, allograft or bacterial infection has been recently elucidated in leeches (schorn et al., 2015a, b). as already reported in vertebrates (mishima et al., 2008), aif-1 is constitutively expressed by leech resident macrophages in healthy tissues but its expression dramatically increases after injury, allograft or microbial infection (schorn et al., 2015a, b). furthermore, data obtained in leech demonstrated for the first time that hmaif-1 is a damp-like molecule since, once released from damaged tissue, it activates macrophages through the tlr4/cd14/myd88/tnf- pathway (girardello et al., 2019). thus, an aif-1enriched environment trigger chemotaxis in the stimulated areas of new infiltrating, tlr4+/cd45+/cd68+ macrophages, which by themselves produce and release a large amount of hmaif-1 to further recruit other macrophages in a positive feedback loop. indeed, following injection in the leech body wall, recombinant rhmaif-1 protein elicits a strong chemotactic activity towards macrophages-like cells co-expressing cd45, cd68 and aif-1 (schorn et al., 2015a), showing a very similar function to vertebrate’s aif-1. the maturation and functional responsiveness of macrophage-like cells to aif-1 is strictly dependent on cd45 since injection of recombinant rhmaif-1 together with an anti-cd45 polyclonal antibody reduces the migration of macrophage-like cells. moreover, injection of rhmaif-1 previously pre-incubated with specific anti-hmaif-1 antibody reduces the number of cd45+ and cd68+ infiltrating cells. interestingly, in autografts, macrophages show a low level of hmaif-1 and cd45 expression (schorn et al., 2015b). such difference in cd45 and hmaif-1 expression is due to the different role played by macrophages in response to autograft and to wound/allograft. indeed, as in vertebrates (mokarram et al., 2012), macrophages can display anti-inflammatory or pro-inflammatory features in 93 leeches as well. in allograft and wound healing, cd45+ and hmaif-1+ macrophages are mainly involved in inflammatory response. by contrast, these immune cells express low level of cd45 and hmaif-1 in autografts (schorn et al., 2015b) where they could be mainly involved in the regenerative process. indeed, macrophages support tissue repair by producing anti-inflammatory cytokines (tettamanti et al., 2003), which suppress the immune system-mediated destructive response and in turn stimulates angiogenesis, cell replacement and matrix remodeling. hvrnaset2 has a pleiotropic role in immune response and connective tissue remodelling ribonucleases (also called rnases) represent a wide family of enzymes whose common feature is the ability to process or degrade ribonucleic acids (kunitz, 1940; beintema and kleineidam, 1998; irie, 1999). although most rnases show a biochemically conserved housekeeping role, several peculiar features such as their different cellular localization pattern, their wide range of optimal ph for catalytic activity and their base specificity related to rna catabolism, have led to the ranking of these enzymes in different families and classes (d’alessio and riordan, 1997). in this context, a great attention has been focused in the last decades on an interesting group of ribonucleases, defined “transferase-type” rnases based on their mechanism of catalysis (kawata et al., 1990; deshpande and shankar, 2002; thompson and parker, 2009). this rnase subfamily has been in turn ranked into three subclasses showing different properties, namely a, t1 and t2 rnases, respectively (loverix and steyaert, 2001). transferase-type rnases are involved in an impressively wide range of biological processes and represent an excellent example of a multifunctional protein's family (lu et al., 2018). indeed, besides their main enzymatic activity, they have been shown to play a wide range of key biological functions (some of which in a ribonuclease-independent way) such as nutritional stress response, neural development, modulation of the immune response, cell death, cancer growth control and stress response are the most deeply investigated (luhtala and parker, 2010). strikingly, the ability to directly counteract different type of harmful agents, such as viruses or bacteria, turned out to be a common feature of both t2 rnase and a rnase families (acquati et al., 2011; lu et al., 2018; irie, 1999). however, unlike rnase a family members, which are present only in vertebrates (dehal et al., 2002; pizzo and d’alessio, 2007), and the alkaline t1 rnase family, reported only in fungi and bacteria (sato and egami, 1957), t2 rnases have been found in all phyla ranging from viruses to humans (luhtala and parker, 2010), suggesting a very ancient and evolutionary conserved role for this subclass of enzymes. accordingly, several studies have demonstrated that most biochemical and structural characteristics of t2 enzymes have been highly preserved from viruses to plants and vertebrates. remarkably, different t2 rnases family members have gained quite peculiar biological roles in different organisms as well, thereby showing a highly pleiotropic nature (deshpande and shankar, 2002). indeed, these enzymes play a key role in several biological processes in plants, such as regulation of self-incompatibility (mcclure et al., 1989; huang et al., 1994), phosphate scavenging (nürnberger et al., 1990; löffler et al., 1992) and nitrogen preservation (van damme et al., 2000). in other organisms, t2 rnases act to counteract harmful agents (irie, 1999) trigger cellular senescence (acquati et al., 2001), recruit reactive oxygen species during stressful condition (caputa et al., 2016), trigger cell apoptosis (wang et al., 2014) or cytotoxic events (huang et al. 1994) and regulate host immune response (acquati et al., 2001). an additional characteristic and biological feature of these enzymes is probably linked to their specific cellular localization. indeed, besides their canonical localization in the cytosol and nuclei, t2 rnases have been found in compartments where rna molecules are not usually found (macintosh, 2011) such as cytoplasmic vacuoles, and the extracellular environment (moriwaka, 1967; wiener and ashworth, 1970; nakamura et al., 1989). one of the first evidence in support of a role of t2 ribonucleases in immune response was reported for the human rnaset2 gene, which was shown to act as an oncosuppressor against different tumor types by triggering a marked innate immune response and by recruiting in vivo m1-polarized host macrophages endowed with tumor suppressive properties towards the tumor mass (acquati et al., 2011, 2013). the crucial role played by t2 rnases in host defense against different type of pathogens or after damages and mechanical wounds was later confirmed by recent data obtained in the medicinal leech h. verbana. indeed, an in silico search of a leech transcriptome database recently led our group to the identify and clone a cdna encoding a t2 rnase in h. verbana (hvrnaset2) (baranzini et al., 2020), thus confirming the extreme evolutionary conservation of t2 ribonucleases among distant taxa. of note, biochemical characterization of the recombinant hvrnaset2 protein confirmed its role as a functional ribonuclease with a low ph optimum for catalysis, a typical feature of t2 ribonucleases, strongly suggesting that hvrnaset2 represent the orthologous of vertebrate t2 rnases. in addition to the well-established role of hmaif-1 in triggering the innate immune response, during the very early phase of the inflammatory response another key cellular component of innate immunity (granulocytes) turned out to express and release t2 rnases from their granules, whose primary function is likely to help the innate immune system to kill infecting bacteria (baranzini et al., 2020a). moreover, as already observed in vertebrate murine models (acquati et al., 2013), the released hvrnaset2 was also deployed to recruit host macrophages, which migrate toward the infected or injured area and in turn produce further hvrnaset2 by themselves in order to strengthen the inflammatory state (baranzini et al., 2017, 2019 submitted). these data provided a further support to 94 the functional relationship between vertebrate and leech t2 rnases. of note, macrophage-secreted hvrnaset2 was also found to induce neocollagen synthesis and secretion by tissue fibroblasts, thus supporting a connective tissue remodeling role for this protein (baranzini et al., 2020b). the multifunctional activities (antibacterial activity, innate immunity stimulation and connective tissue remodelling) observed for leech’s t2 rnase not only confirm the pleiotropic function of this class of ancient and evolutionary conserved ribonucleases, but also suggest that they play a key role in establishing a functional cross-talk between the inflammatory response and the process of wound healing and tissue regeneration. aif-1 and rnaset2 interplay modulates the intimately connected immune response, wound healing and tissue regeneration processes collectively, the data obtained in leeches point strongly to the occurrence of a cellular "cross-talk" between granulocytes and macrophages, implicated not only in the defense against bacterial infection and in elimination of cellular debris accumulated following tissue damage, but also in connective tissue remodeling during wound healing and tissue regeneration (as schematically shown in fig. 1). granulocytes, by expressing the tlr4 receptor, usually represent the first cells to recognize "pamps" molecules expressed by pathogenic microbes (i.e. lps) or "damps" (i.e., aif-1) molecules mainly produced after injury and grafts (schorn et al., 2015b). the occurrence in leech of the highly conserved pathway based on tlr4/cd14 mediators (girardello et al., 2019) is a finding of key relevance, since it not only confirms a conserved functional role of tlrs in regulating the inflammatory response, but at the same time strongly suggest the existence of a functional link between tlrs and chemotaxis factors in both leech peripheral and in neuroimmune systems (schikorski et al., 2009). once activated, granulocytes release t2 rnase to trigger both an immediate antimicrobial activity and a massive recruitment of aif-1+ macrophages, whose role is to clear the area from bacteria or necrotic tissue and to produce further cytokines, such as fgfb and egf (tettamanti et al., 2006), that are involved in fibroblast proliferation. in turn, these cells promote wound healing and tissue regeneration through the deposition of new extracellular matrix. hmaif-1 and hvrnaset2 act therefore as key molecules in establishing such cellular "cross-talk" (baranzini et al., 2017, baranzini et al., 2020 a, b) and their high degree of conservation in higher vertebrates, including humans, further suggests their involvement in similar processes in humans as well. this is a fundamental premise for the extrapolation to humans of the experimental data obtained in leech and their possible use biomedical field. fig. 1 representation to explain the complementary roles of rnaset2 and aif-1 in innate immune response and connective tissue remodeling: 30 min after pamps or damps stimulation, activated tlr4+ granulocytes secrete rnaset2, whose first role is to carry out an antibacterial activity. after 3h-6h, the numerous macrophages, recruited in the infected/injured area by rnaset2, and secreting both aif-1 and rnaset2, maintain the inflammatory state by recruiting other macrophages. these phagocytic cells are involved not only in the clearance of cellular and bacterial debris, but also in inducing new collagen deposition by inducing fibroblast proliferation and activation 95 hmaif-1 and hvrnaset2: new targets for antifibrotic therapy despite the long established key role for tissue macrophages during most phases of the tissue healing process, it is not still clear how they stimulate tissue repair, fibrosis or full regeneration (larouche et al., 2017). indeed, whereas a limited inflammatory response might reduce the effectiveness of the wound healing process, an excessive inflammation could by contrast prevent normal wound resolution. in fact, several human clinical pathologies of scars, such as chronic wounds in which the skin layer cannot regenerate even one month after the wound and in which infections and microbial films often persist, or dermal fibrosis (i.e. keloids and hypertrophic scars) deriving from an increased ecm deposition and hyper proliferation of keratinocytes at the wound site, are the result of dysregulated inflammatory and immune response to the skin wound. of note, several data in literature have shown that chronic wounds are characterized by an imbalance between proand anti-inflammatory signals which alter the microenvironment and prevent the normal wound healing process (chen et al., 1999; eming et al., 2010). given that the pathogenesis of various immunoinflammatory diseases are related to chemotaxis of macrophages that, by releasing cytokines, are responsible for fibroblast recruitment, elucidating the exact mechanisms driving the expansion in vivo of anti-inflammatory/anti-fibrotic macrophages with pro-regenerative capacities may help to find novel regenerative strategies for promoting constructive tissue remodeling and regeneration of injured tissues and organs in adult mammals and humans as well. in this contest, our findings in leech clearly show a relationship between aif-1 and rnaset2 during immune response and wound healing and demonstrate that both these molecules not only promote the activation and the recruitment of macrophages, but, when overexpressed, also stimulate fibrosis and synthesis of new collagen (baranzini et al., 2020b). thus, a better understanding of the molecular mechanisms by which aif-1 and rnaset2 control the recruitment and activation of macrophages and fibroblasts during the wound healing process could facilitate the design of novel regenerative therapies, in which these molecules might represent new targets for antifibrotic therapy. indeed, a direct involvement of aif-1 in chronic fibroproliferative disorder (yamamoto et al., 2011), such as the systemic sclerosis (ssc) tissues (del galdo et al., 2006) in which a progressive substitution of tissue structure by collagen-rich extra cellular matrix causes functional impairment of affected organs, has been clearly demonstrated. concluding remarks taken together, the experimental data obtained in leech support a pleiotropic role for both rnaset2 and aif-1 in orchestrating an evolutionarily conserved "cross-talk" between inflammatory response and regenerative process, based on granulocyte activation and the concomitant recruitment/activation of macrophages and fibroblasts, which is in turn associated with a massive reorganization of the extracellular matrix and a rapid wound healing. the information derived from these studies in leeches will open new possibilities to exploit the functionalities of these biomolecules and lays the foundations for developing innovative therapeutic approaches to control immune and systemic responses to disease, injury, and bacterial infection. furthermore, the reported biological features of both molecules should attract a great interest from biotechnological pharmaceutical industries, given their potential to develop new topical treatments, using for example biomaterial and biologic-based strategies, aimed at 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are becoming crucial to provide further information on how both processes are linked together and to clarify how immune cells perform t... introduction concluding remarks acknowledgements references 453 isj 14: 453-463, 2017 issn 1824-307x short communication a novel macrophage migration inhibitory factor gene from the pacific white shrimp litopenaeus vannamei (lvmif2): comparative sequence and transcription analysis with lvmif1 m wang1, b wang1, m liu1, k jiang1, l wang1,2 1key laboratory of experimental marine biology, institute of oceanology, chinese academy of sciences, qingdao 266071, china 2laboratory for marine biology and biotechnology, qingdao national laboratory for marine science and technology, qingdao 266237, china accepted november 11, 2017 abstract macrophage migration inhibitory factor (mif) was an ancient cytokine and involved in innate immunity of vertebrates and invertebrates. in the present study, a novel mif homologue (designated as lvmif2) has been cloned from the pacific white shrimp litopenaeus vannamei via rapid amplification of cdna ends (race) technique. the full-length cdna sequence of lvmif2 was 555 bp and contained a 97 bp 5’ untranslated region (utr) and a 3’ utr of 110 bp, and an open reading frame (orf) of 348 bp which coded 115 amino acids. quantitative realtime pcr (qrt-pcr) analysis indicated that lvmif2 was constitutively expressed in all the tested tissues, including eyestalk, gill, gonad, heart, hemocytes, hepatopancreas, intestine, muscle, nerve and stomach, with the highest mrna expression level in hemocytes and hepatopancreas. after vibrio parahaemolyticus or white spot syndrome virus (wssv) challenge, the mrna expression levels of lvmif2 in hemocytes and hepatopancreas were all sharply upregulated. comparative sequence and transcription analysis between the previously identified lvmif1 and lvmif2 revealed that both lvmif1 and lvmif2 might play crucial and functionally differentiated roles in shrimp innate immune responses to bacterial and viral stimulation. key word: litopenaeus vannamei; macrophage migration inhibitory factor; innate immunity introduction macrophage migration inhibitory factor (mif) was first described as a cytokine 50 years ago, which was stemmed from activated t cells by its capacity to inhibit the migration of macrophages in vitro, and emerged in mammals as a mysterious cytokine with chemotactic, growth-promoting and pro-inflammatory activities (calandra and thierry, 2003). as an evolutionary ancient molecule, mif and its homologues have been found from prokaryotes to eukaryotes (sparkes et al., 2017). in addition, mif has recently gained substantial attention as a pivotal upstream regulator of both innate and adaptive immune responses (sparkes et al., 2017). in vertebrates, mif exhibits oxidoreductase activity and could utilize glutathione (gsh) as reductant to reduce insulin (blocki et al., 1993). such ___________________________________________________________________________ corresponding author: lei w ang key laboratory of experimental marine biology institute of oceanology chinese academy of sciences qingdao 266071, china e-mail: wanglei@qdio.ac.cn oxidoreductase activity plays a role in mif-mediated monocyte and macrophage function (mitchell et al., 2002). in invertebrates, especially marine invertebrates, recent research achievements suggest that mif is extensively involved in innate immune responses. for examples, in small abalone haliotis diversicolor supertexta, the mrna expression level of hdsmif was significantly upregulated in hepatopancreas post vibrio parahaemolyticus stimulation (wang et al., 2009). mif of snail biomphalaria glabrata (bgmif) participated in hemocytes activation to resist parasitic infection in vivo, and could stimulate cell proliferation to suppress no-induced apoptosis (garcia et al., 2010). in pearl oyster pinctada fucata, the mrna expression levels of pomif were significantly upregulated in digestive gland, gills, hemocytes and intestine, after pearl oyster was injected with vibrio alginolyticus, and the recombinant pomif (rpomif) exhibited oxidoreductase activity and could utilize dithiothreitol (dtt) as reductant to reduce insulin (cui et al., 2011). mif from zhikong scallop chlamys farreri (cfmif) was involved in innate immune responses mailto:wanglei@qdio.ac.cn 454 by promoting fibroblast migration (li et al., 2011a). the mrna transcripts of mif in chinese mitten crab eriocheir sinensis (esmif) were responsive to vibrio anguillarum challenge in hemocytes, hepatopancreas and gill (li et al., 2011b). two previous reports identified three mif in mud crabs scylla paramamosain (spmif, spmif1 and spmif2), and the mrna transcripts of spmif significantly increased after the crabs were challenge by v. parahaemolyticus (fang et al., 2013; huang et al., 2016). the mrna expression level of mif from the black tiger shrimp penaeus monodon (pmmif) was upregulated after bacterial infection, salinity challenge and heavy metals stress (xie et al., 2016). two mifs from starfish patiria (asterina) pectinifera, ppmif1 and ppmif2, could regulate larval immune cell chemotaxis (furukawa et al., 2016). the polymorphism of mif in clam meretrix meretrix (mmmif) was associated with the resistance/susceptibility to v. parahaemolyticus (zou and liu, 2016). in contrast to other animals, the mrna expression levels of mif from mussel mytilus galloprovincialis (mgmif) were always down regulated following microbe stimulation, including vibrio splendidus, v. anguillarum, micrococcus lysodeikticus, fusarium oxysporum and candida albicans (parisi et al., 2012). all these research achievements indicated that mif played a pivotal role in the innate immunity of marine invertebrates. the pacific white shrimp litopenaeus vannamei is one of the most important and leading farm crustacean species and takes a large amount of the total shrimp production in the world (li and xiang, 2010, 2013a, b). however, during the past two decades, outbreaks of infectious disease such as acute hepatopancreatic necrosis disease (ahpnd) or early mortality syndrome (ems) caused by v. parahaemolyticus and white spot disease (wsd) caused by white spot syndrome virus (wssv), have become a major constraint, resulting in mass shrimp mortality and considerable economic losses (lai et al., 2015; zhang et al., 2016; verma et al., 2017). as invertebrates, shrimps lack an adaptive immune system and rely on the ancient innate immune defenses (li and xiang, 2013a, b). a better understanding of the innate immunity of shrimp is necessary for the health management of shrimp farming (li and xiang, 2010). a previous research identified a homolog of mif in l. vannamei (designated as lvmif1), and the mrna expression level of lvmif1 in hepatopancreas was significantly upregulated after wssv injection, suggesting that lvmif1 may be involved in the innate immune response to viral infection in shrimp (zeng et al., 2013). in the present study, a novel mif gene (designated as lvmif2) have been cloned and investigated in white shrimp, and the main objectives of the present study were (1) to characterize the molecular features of lvmif2, (2) to detect the tissue distribution and temporal expression patterns after invading microbes stimulation of its mrna transcripts and (3) to compare these features with the previous identified lvmif1, in order to get a further understanding of the immunological roles of lvmif1 and lvmif2 in shrimp. materials and methods shrimp and tissues sample collection the white shrimps used in the present study were obtained from ruizi seafood development co. ltd., qingdao, china, and all the experiments were performed in accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health (nih). the study protocol and all the experimental design table 1 oligonucleotide primers used in the present study primer sequence (5`-3`) tm (°c) brief information adaptor primer ggccacgcgtcgactagtac 60 anchor primer for 3` race adaptor primer-oligo (dt) ggccacgcgtcgactagtact17vn olido (dt) for cdna synthetize lvef-1α-qrt-f gtattggaacagtgcccgt 60 internal control for real-time pcr lvef-1α-qrt-r catctccacagactttacctcag 60 internal control for real-time pcr lvmif2-cds-f atgcctttcgtaaccctgactacc 64 gene specific primer for cds lvmif2-cds-r tcatcccattagagccccattcac 64 gene specific primer for cds lvmif2-qrt-f gccagcggaagaattgacca 60 gene specific primer for real-time pcr lvmif2-qrt-r gttgaagtcatcccattagagccc 60 gene specific primer for real-time pcr lvmif2-race-f1 gctgggacccagtgtgtgaga 64 gene specific primer for 3` race lvmif2-race-f2 ctgaccgaactggcattcaacctgcaa 64 gene specific primer for 3` race lvmif1-qrt-f ttagtgaaaccattgggaaa 60 gene specific primer for real-time pcr lvmif1-qrt-r aactgcataaatcttagaag 60 gene specific primer for real-time pcr m13-47 cgccagggttttcccagtcacgac 56 vector primer for sequencing rv-m gagcggataacaatttcacacagg 56 vector primer for sequencing 455 table 2 sequence feature of lvmif1 and lvmif2 feature lvmif1 lvmif2 accession number kc513658 mf062461 cdna length (bp) 689 555 5’ utr length (bp) 101 97 3’ utr length (bp) 225 110 orf length (bp) 363 348 polyadenylation signal sites 0 1 polypeptide length (amino acid) 120 115 mif domain information from p2 to h115 from p2 to g115 conserved tautomerase activity sites p2 and k33 p 2 and k33 conserved oxidoreductase activity c57 c58 calculated molecular mass (da) 13442.61 12534.52 theoretical isoelectric point (pi) 7.034 7.116 were conducted with approval from experimental animal ethics committee of institute of oceanology, chinese academy of sciences. white shrimps, with body weight 8 12 g, were cultured in 640 l cylindrical tanks with 500 l air-pumped circulating seawater at 20 ± 1 °c for two weeks before processing. hemolymph was extracted from the ventral sinus of at least five untreated shrimps using a sterile syringe preloaded with equal volume of anticoagulant buffer (nacl 510 mmol l-1, glucose 100 mmol l-1, citric acid 200 mmol l-1, tri-sodium citrate 30 mmol l-1 and edta·2na 10 mmol l-1, ph 7.4). then the hemocytes were collected by centrifugation at 800 g for 10 min at 4 °c. tissues including eyestalk, gill, gonad, heart, hepatopancreas, intestine (mid gut), muscle, nerve and stomach were collected from at least five untreated shrimps, kept in rnalater (am7020, thermo fisher scientific, usa) and stored at -80 °c until rna isolation. immune stimulation and sample collection approximately 200 shrimps were employed for microbe stimulation assay. the v. parahaemolyticus suspension and wssv stock were prepared according to previous reports (yi et al., 2014; xia et al., 2015; sha et al., 2016). the shrimps were randomly divided into three groups and each group contained about 60 70 individuals. the shrimps were received an injection at the abdominal segment with 100 μl phosphate buffered saline (pbs, ph 7.4, 10010023, thermo fisher scientific, usa), v. parahaemolyticus suspension (1 × 105 cfus μl-1, in pbs) and wssv stock (1×105 copies μl-1, in pbs), respectively. the injected shrimps were returned to seawater tanks immediately and the hemocytes and hepatopancreas of at least five individuals were randomly sampled from each group at 3, 6, 12, 24 and 48 h post injection, kept in rnalater and stored at -80 °c until rna isolation. rna isolation and cdna synthesis total rna was isolated from different tissues using trizol reagent (15596026, thermo fisher scientific, usa). the synthesis of first strand was carried out with promega m-mlv (m1701, promega, usa) using the dnase i (rq1, m6101, promega, usa) treated total rna as template and adaptor primer-oligo (dt) as primer (table 1). the reaction was performed at 42 °c for 1 h, terminated by heating to 95 °c for 5 min, and then stored at -80 °c. cloning the full-length cdna of lvmif2 the partial length sequence of lvmif2 cdna was obtained from the white shrimp transcriptome sequencing database (qi et al., 2017). two gene-specific primers, lvmif2-race-f1/2, were designed with primer premier 6.00 based on this partial length sequence to clone the 3’ end of lvmif2 cdna by rapid amplification of cdna ends (race) technique. and the coding sequence (cds) of lvmif2 was amplified and confirmed using another two gene-specific primers, lvmif2-cds-f/r, which was also designed with primer premier 6.00. all pcr amplification was performed in a mgl96g peltier thermal cycler (longgene, china). the pcr products were purified using monarch dna gel extraction kit (t1020s, neb, usa) and then cloned into the pmd18-t simple vector (d103a, takara, japan). after being transformed into the competent cells escherichia coli strain dh5α (cb101, tiangen, china), the positive recombinants were identified via anti-ampicillin selection and verified by pcr screening using m13-47 and rv-m primers (table 1). three of the positive clones were sequenced using a prism 3730xl automated sequencer (thermo fisher scientific, usa). bioinformatical analysis of cdna and protein sequences the search for protein sequence similarity was conducted with blastp 2.6.0. the deduced protein sequences were analyzed by the editseq module in lasergene program suite 14.0.0.88. the function domains were predicted with simple modular architecture research tool (smart) 7.0. multiple sequence alignments were performed with clustal omega 1.2.4 and visualized using multiple alignment 456 fig. 1 nucleotide and deduced amino acid sequences of lvmif1 and lvmif2 (a: lvmif1, b: lvmif2). the nucleotides and amino acids were numbered along the left margin. the function domain was in shade. the conserved tautomerase activity sites (p2 and k33) are boxed. the conserved oxidoreductase activity site (c57/c58) is marked by circle. the asterisks indicated the stop codon. the single typical polyadenylation signal (aataaa) was double underlined. show module in sequence manipulation suite 2.0. a neighbor-joining (nj) phylogenic tree was constructed with mega 7.0.21. to derive confidence value for the phylogeny analysis, bootstrap trials were replicated 1,000 times. expression pattern analysis via real-time quantitative rt-pcr the mrna transcripts of lvmif2 and the previously identified lvmif1 in different tissues or their temporal expression patterns in hemocytes and hepatopancreas of shrimps stimulated with v. parahaemolyticus or wssv were investigated by quantitative realtime pcr (qrt-pcr) technique. all qrt-pcr reactions were performed with the sybr premix extaq (tli rnaseh plus) (rr420, takara, japan) using 100 ng cdna template in a linegene k fqd-48a (a4) fluorescence quantitative pcr detection system (bioer, china). all the primers for qrt-pcr were designed with perlprimer 1.1.21 and listed in table 1. the mrna expression levels of lvmif2 and lvmif1 were normalized to those of elongation factor 1 α (ef-1α) for each sample. the relative mrna expression levels of lvmif2 and lvmif1 were generated using comparative ct method (2-δδct method) (schmittgen and livak, 2008). the data were subjected to one-way analysis of variance (anova) followed by a multiple comparison using ibm spss statistics 23.0.0.0, and the p values less than 0.05 were considered statistically significant. 457 results sequence features of lvmif2 the full-length cdna sequence of lvmif2 was obtained by 3’ race technique, and deposited in genbank under the accession number mf062461. it comprised 555 bp, containing a 5’ untranslated regions (utr) of 97 bp, a 3’ utr of 110 bp with a poly a tail and an open reading frame (orf) of 348 bp. the orf encoded a polypeptide of 115 amino acid residues with a calculated molecular mass of approximately 12.53 kda and a theoretical isoelectric point (pi) of 7.12 (table 2). the deduced protein sequence of lvmif2 contained a mif domain (from p2 to g115), two conserved tautomerase activity sites (p2 and k33) and one conserved oxidoreductase activity (c58) (fig. 1). an alignment of the protein sequence of lvmif2 with those of previously identified mifs was shown in figure 2, and a mif family signature (from d55 to g69) was revealed. the deduced protein sequence of lvmif2 exhibited high similarity with other previously identified mifs, such as 52 % identity with that of spmif1 (akt09427) (huang et al., 2016). the nj phylogenetic tree based on protein sequences of multiple mifs was positioned separately into two main branches, the vertebrate one and the invertebrate one, and lvmif1 were clustered with its homologue from the black tiger shrimp p. monodon, pmmif, and located in the crustacean sub-branch, while lvmif2 were clustered with its homologue from zhikong scallop c. farreri, cfmif, and located in the mollusca sub-branch (fig. 3). fig. 2 multiple alignments of lvmif1 and lvmif2 with previous known mifs. the mif family signatures were boxed. the sites of mif tautomerase activity were indicated with ▼. the conserved oxidoreductase activity site was indicated with ★. the sequences and their accession numbers are as follows: cfmif, chlamys farreri, adf87941; mgmif, mytilus galloprovincialis, aen25591; mmmif, meretrix meretrix, akn56918; pfmif, pinctada fucata, adu19847; hddmif, haliotis discus discus, acj65690; hdsmif, haliotis diversicolor supertexta, abx76741; esmif, eriocheir sinensis, adm86239; spmif, scylla paramamosain, agb07600; spmif1, scylla paramamosain, akt09426; spmif2, scylla paramamosain, akt09428; pmmif, penaeus monodon, anz80588; ppmif1, patiria pectinifera, bar92638 and ppmif2, patiria pectinifera, bar92639. 458 fig. 3 consensus neighbor-joining phylogenetic based on the amino acid sequences of mifs from different organisms. the evolutionary history was inferred using the neighbor-joining method. the bootstrap consensus tree inferred from 1,000 replicates was taken to represent the evolutionary history of the taxa analyzed. all positions containing gaps and missing data were eliminated. the numbers at the forks indicated the bootstrap value. the sequences and their accession numbers are as follows: cfmif, chlamys farreri, adf87941; mgmif, mytilus galloprovincialis, aen25591; mmmif, meretrix meretrix, akn56918; pfmif, pinctada fucata, adu19847; hddmif, haliotis discus discus, acj65690; hdsmif, haliotis diversicolor supertexta, abx76741; esmif, eriocheir sinensis, adm86239; spmif, scylla paramamosain, agb07600; spmif1, scylla paramamosain, akt09426; spmif2, scylla paramamosain, akt09428; pmmif, penaeus monodon, anz80588; ppmif1, patiria pectinifera, bar92638; ppmif2, patiria pectinifera, bar92639; hsmif, homo sapiens, np_002406; pamif, pongo abelii, xp_009232451; csmif, carlito syrichta, xp_008064641; vpmif, vicugna pacos, np_001274126; ssmif, sus scrofa, np_001070681; hgmif, heterocephalus glaber, np_001266756; btmif, bos taurus, np_001028780; mumif, meriones unguiculatus, xp_021509432 and rnmif, rattus norvegicus, np_112313. tissue distribution of lvmif1 and lvmif2 the qrt-pcr was employed to detect the tissue distribution of lvmif1 and lvmif2 mrna transcripts with ef-1α as internal control. the lvmif1 mrna transcripts could be detected in all the tested tissues, including eyestalk, gill, gonad, heart, hemocytes, hepatopancreas, intestine, muscle, nerve and stomach. the highest mrna expression level of lvmif1 was found in hepatopancreas and hemocytes, which was 5.12-fold and 4.79-fold of that in muscle (p < 0.05), respectively, followed by heart, gill and intestine, which were 3.14-fold, 3.11-fold and 3.07-fold of that in muscle (p < 0.05), respectively (fig. 4a). the mrna transcripts of lvmif2 were also constitutively expressed in all the selected tissues of untreated shrimps, with the specific highest expression level in hemocytes and hepatopancreas, which was 8.93-fold and 8.13-fold of that in muscle (p < 0.05), respectively (fig. 4b). the mrna expression profiles of lvmif1 and lvmif2 post bacterial stimulation the qrt-pcr was performed to analyze the mrna expression patterns of lvmif1 and lvmif2 in hemocytes and hepatopancreas of shrimp challenged by v. parahaemolyticus. the mrna transcripts of lvmif1 in hemocytes significantly increased and reached the peak at 6 h post v. parahaemolyticus stimulation (2.97-fold compared 459 fig. 4 tissue distribution of lvmif1 and lvmif2 mrna transcripts detected by qrt-pcr technique (a: lvmif1, b: lvmif2). the mrna expression levels in eyestalk, gill, gonad, heart, hemocytes, hepatopancreas, intestine, muscle, nerve and stomach of five untreated shrimps were normalized to that of muscle. the ef-1α gene was used as an internal control to calibrate the cdna template for each sample. vertical bars represent mean ± sd (n = 5), and bars with different characters were significantly different (p < 0.05). with the origin level, p < 0.05), kept at a high level at 12 h (2.76-fold, p < 0.05), and then decreased to the origin level at 24 h (fig. 5a). after stimulated with v. parahaemolyticus, lvmif2 mrna transcripts in hemocytes significantly increased at 3 h post stimulation (2.33-fold, p < 0.05) and reached the peak level at 12 h (7.11-fold, p < 0.05), kept at a high level at 24 h (5.47-fold, p < 0.05) and then decreased but still higher than the origin level at 48 h (2.17-fold, p < 0.05, fig. 5b). the mrna expression profile of lvmif1 in hepatopancreas was similar with that in hemocytes, it also significantly increased and reached the peak at 6 h post v. parahaemolyticus stimulation (3.52-fold, p < 0.05), kept at a high level at 12 h (2.99-fold, p < 0.05), and then decreased to the origin level at 24 h (fig. 5c). while lvmif2 mrna transcripts in hepatopancreas significantly increased at 3 h post bacterial stimulation (3.18-fold, p < 0.05) and reached the peak level at 6 h and 12 h post stimulation (7.97-fold and 8.25-fold, respectively, p < 0.05), and then decreased but still higher than the origin level at 24 h and 48 h (2.97-fold and 3.37-fold, respectively, p < 0.05, fig. 5d). 460 fig. 5 temporal mrna expression profiles of lvmif1 and lvmif2 detected via qrt-pcr technique in white shrimp hemocytes and hepatopancreas post bacterial stimulation (a: lvmif1 in hemocytes, b: lvmif2 in hemocytes, c: lvmif1 in hepatopancreas, d: lvmif2 in hepatopancreas). the ef-1α gene was used as an internal control to calibrate the cdna template for each sample. vertical bars represent mean ± sd (n = 5), and bars with different characters were significantly different (p < 0.05). the mrna expression profiles of lvmif1 and lvmif2 post viral stimulation the qrt-pcr was performed to analyze the mrna expression patterns of lvmif1 and lvmif2 in hemocytes and hepatopancreas of shrimp challenged by wssv. after challenged by wssv, the mrna expression level of lvmif1 in hemocytes was significantly upregulated at 6 h and 12 h (1.98-fold and 1.85-fold, respectively, p < 0.05), downregulated to the origin level at 24 h, and then significantly upregulated again at 48 h (2.19-fold, p < 0.05, fig. 6a). during the wssv challenge, the mrna transcripts of lvmif2 in hemocytes significantly increased at 12 h and 24 h (2.11-fold and 2.47-fold, respectively, p < 0.05) and then decreased to the origin level at 48 h (fig. 6b). in hepatopancreas, lvmif1 mrna transcripts significantly increased at 3 h post stimulation (2.12-fold, p < 0.05) and reached the peak level at 6 h and 12 h (3.57-fold and 3.37-fold, respectively, p < 0.05), kept at a high level at 24 h (2.03-fold, p < 0.05) and then decreased to the origin level at 48 h (fig. 6c). while the mrna expression level of lvmif2 in hepatopancreas was significantly upregulated at 6 h and 12 h (2.57-fold and 2.65-fold, respectively, p < 0.05) and then decreased to the origin level at 24 h (fig. 6d). discussion for approximately half a century, mif has been considered as a mysterious cytokine (calandra and thierry, 2003). recently, mif has assumed an important role as a pivotal modulator of innate immunity (sparkes et al., 2017). in the present study, the full-length cdna of a novel mif homologue, lvmif2, was obtained from white shrimp l. vannamei. the deduced polypeptide of lvmif2 consisted of 115 amino acids, and its calculated molecular weight was 12.53 kda, which was close to the previously identified lvmif1. the protein sequence of lvmif2 shared 52 % similarities with the previously identified spmif1. moreover, a mif domain and a mif family signature were revealed from the amino acid sequence of lvmif2. the amino terminal proline residue (p2), invariant lysine residue 461 fig. 6 temporal mrna expression profiles of lvmif1 and lvmif2 detected via qrt-pcr technique in white shrimp hemocytes and hepatopancreas post viral stimulation (a: lvmif1 in hemocytes, b: lvmif2 in hemocytes, c: lvmif1 in hepatopancreas, d: lvmif2 in hepatopancreas). the ef-1α gene was used as an internal control to calibrate the cdna template for each sample. vertical bars represent mean ± sd (n = 5), and bars with different characters were significantly different (p < 0.05). (k33) and conserved oxidoreductase activity (c58), which are critical active sites of tautomerase activity in mammalian mif, were also revealed (xie et al., 2016). the conserved function domain and signature sequence of lvmif2 and high similarity with other previously identified mifs collectively suggested that lvmif2 was a novel member of invertebrate mif family, and it could have similar functions to those from vertebrates and other invertebrates. while in the nj phylogenetic tree, lvmif1 were clustered with its homologues from crustacean, and lvmif2 were clustered with its homologues from mollusca, indicating lvmif2 might be more ancient than lvmif1. previous researches showed that mif was ubiquitously expressed in various tissues (lue et al., 2002). in the present study, qrt-pcr analysis indicated that both lvmif1 and lvmif2 mrna transcripts were also constitutively expression in all the investigated tissues from healthy white shrimp, which were consisted with the previously observation in other crustacean (li et al., 2011b; fang et al., 2013; xie et al., 2016), and the ubiquity of mif mrna in all tissues in crustacean might be attributed to the open circulatory system (mcmahon and burnett, 1990). the highest mrna expression level of lvmif1 was found in hepatopancreas and hemocytes, followed by heart, gill and intestine, which was consistent with the previous results (zeng et al., 2013), and also confirmed that the qrt-pcr analysis in the present study were credible. while the highest expression level of lvmif2 mrna was also detected in hemocytes and hepatopancreas. similarly, in e. sinensis, esmif was highest expressed in hepatopancreas (li et al., 2011b). in s. paramamosain, spmif was highly expressed in hepatopancreas and hemocytes (fang et al., 2013). and in p. monodon, the highest level of pmmif mrna was detected in hepatopancreas, followed by gills and heart (xie et al., 2016). all the results collectively confirmed the hypothesis that hemocytes, hepatopancreas and gills might play pivotal roles in the innate immune system of crustacean (cerenius et al., 2010). mif could be released in response to bacterial stimulation (calandra and thierry, 2003). in order to 462 clarify the potential role of lvmif1 and lvmif2 in regulating shrimp innate immunity, their temporal expression profiles in both hemocytes and hepatopancreas post v. parahaemolyticus stimulation was detected and compared by qrt-pcr technique. the mrna expression levels of lvmif1 in both hemocytes and hepatopancreas were significantly upregulated at 6 h and 12 h post injection. compared with lvmif1, lvmif2 exhibited a fast and intense mrna expression pattern, and the lvmif2 mrna transcripts in both hemocytes and hepatopancreas sharply increased at 3 h post injection and reached the maximum at 12 h. likewise, in e. sinensis, the mrna expression level of esmif in hepatopancreas was sharply upregulated from 6 h post v. anguillarum challenge and reached the peak at 12 h (li et al., 2011b). the mrna expression level of spmif from s. paramamosain in hemocytes increased significantly after a 6 h challenge by v. parahaemolyticus and peaked at 8 h (fang et al., 2013). and in p. monodon, after the infection of v. harveyi, pmmif mrna expression level in hepatopancreas was sharply upregulated at 6 h post infection and reached the maximum at 12 h (xie et al., 2016). these results indicate that both lvmif1 and lvmif2 were involved in the innate immune system of shrimp. while as a fast releasing and intensive responsible defense molecule, lvmif2 might play a more important role in the defense to bacterial stimulation in the innate immunity of shrimp. to further understand the immunological roles of lvmif1 and lvmif2 in shrimp, their temporal expression profiles in both hemocytes and hepatopancreas post wssv stimulation was also detected and compared by qrt-pcr technique. after challenged by wssv, the mrna expression level of lvmif1 in hemocytes was significantly upregulated at 6 h, downregulated to the origin level at 24 h, and then significantly upregulated again at 48 h, while the mrna transcripts of lvmif2 in hemocytes significantly increased at 12 h and 24 h and then decreased to the origin level at 48 h. in hepatopancreas, lvmif1 significantly increased at 3 h post stimulation, reached the maximum at 6 h, kept at a high level at 24 h and then decreased to the origin level at 48 h, which was consistent with the previous results (zeng et al., 2013), while the mrna expression level of lvmif2 was significantly upregulated at 6 h and 12 h and then decreased to the origin level at 24 h. all the results collectively indicated that both lvmif1 and lvmif2 might participate in the shrimp immune response to viral infection, and lvmif1 might play a more important role than lvmif2. in conclusion, the full-length cdnas of a novel mif, lvmif2, were cloned from white shrimp, and its mrna expression profiles were detected and compared with the previously identified lvmif1, when the shrimps were exposed to bacterial and viral stimulation. these results of comparative sequence and transcription analysis suggested that both lvmif1 and lvmif2 may play crucial and functionally differentiated roles in innate immune responses to bacterial and viral stimulation. the results of this study would enrich the understanding of the shrimp immune immunity. acknowledgement this research was supported by the key research program of the chinese academy of sciences (kfzd-sw-106) and the major projects of shandong province (2015zdzx05002). we would like to thank the expert reviewers for their constructive suggestions and enlightening comments during the revision. reference blocki fa, ellis l, wackett lp. mif proteins are theta-class glutathione s-transferase homologs. protein sci. 2: 2095-2102, 1993. calandra t, thierry r. macrophage migration inhibitory factor: a regulator of innate immunity. nat. rev. immunol. 3: 791-800, 2003. cerenius l, 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modena and reggio emilia this is an open access article published under the cc by license accepted october 5, 2021 abstract in the compound ascidian botryllus schlosseri, we recently identified a novel c1q-domaincontaining (c1qdc) protein expressed by circulating immunocytes, called bsc1qdc. it has two globular c1q domains and a signal peptide and can act either as an opsonin and facilitate the phagocytosis of nonself particles or as a cytokine and stimulate the degranulation of cytotoxic cells. in the present work, we used a commercial antibody raised against human ctrp4 (hctrp4) to provide additional evidences of the involvement of this molecule in immune responses. the antibody was validated in immunoblot analysis and recognizes a band corresponding to the expected molecular weight inferred from the analysis of the amino acid sequence of bsc1qdc. the presence of the antibody in the culture medium in phagocytosis and degranulation assays significantly reduced the two responses. in addition, the relationships between complement c3 activation and bsc1qdc transcription was studied using the injection of c3ar agonist in the colonial vasculature. key words: colonial ascidians; botryllus; c1qdc; complement introduction c1q-domain-containing (c1qdc) proteins form an emerging family of proteins involved in immune responses in both vertebrates and invertebrates (kishore et al., 2004; ghai et al., 2007). they are characterized by the presence of globular c1q (gc1q) domain(s) (kishore and reid, 2000; ghai et al., 2007) sharing a typical jelly roll topology with five pairs of anti-parallel -strands organized in two parallel -sheets (gaboriaud et al., 2003). in invertebrates, acting as lectins and opsonins (gerlach et al., 2004; zhang et al., 2008; carland and gerwick, 2010; gerdol et al., 2011; li et al., 2011; yang et al., 2012; jiang et al., 2015; huang et al., 2017; wang, 2017; gorbushin, 2019). c1qdc proteins are involved in various responses, including microbial recognition (kong et al., 2010; wang et al., 2012a, 2015), agglutination (kong et al., 2010; wang et al., 2012b), phagocytosis promotion (wang et al., 2012b), and cell migration (tahtouh et al., 2009). they act as pattern recognition receptors and bind directly to pathogens by engaging a broad _________________________________________ corresponding author: loriano ballarin department of biology university of padova via ugo bassi 58/b, 35143 padova, italy e-mail: loriano.ballarin@unipd.it range of pamps (medzhitov, 2002; bohlson et al., 2007). most of the c1qdc proteins, identified in invertebrate, contain only one gc1q domain most of the c1qdc proteins have only a gc1q domain, but the presence of molecules with multiple tandem c1q domains has been reported in both invertebrates and vertebrates (gerdol et al., 2011; wang et al., 2012b; huang et al., 2017; gorbushin, 2019). in vertebrates, in addition to the complement component c1q, with only one gc1q domain, a protein with two c1q domains, called c1q/tnfrelated proteins 4 (ctrp4), has been described (ghai et al., 2007). the compound ascidian botryllus schlosseri is a cosmopolitan tunicate easily found in shallow waters of all the oceans and seas. a colony can reproduce by both sexual and asexual reproduction. three blastogenetic generations are usually present in a colony: the adult zooids, its palleal buds and budlets on buds. they share a common circulatory system in the form of a vessel network within the common tunic (brunetti, 1969; gasparini et al., 2007). a cyclical (weekly at 19 °c) generation change or takeover allows the replacement of adults zooids, that are progressively resorbed, by their buds. therefore, it is possible to define a blastogenetic cycle as the period of time between a takeover and the following one. the colonial developmental phases lying more than one day 130 from the previous and following to (lauzon et al., 1992; manni et al., 2007) are collectively called midcycle (mc). b. schlosseri is a reliable model organism for the study of immunobiology (franchi and ballarin, 2017). its immune defences rely mainly on circulating immunocytes i.e., hemocytes directly involved in immune responses. they constitute the majority of circulating hemocytes and are represented by granular morula cells and phagocytes. phagocytes can recognize and ingest foreign particles upon their recognition via surface receptors, such as toll-like receptors (peronato et al., 2020a), whereas morula cells, as a consequence of the recognition of nonself molecules, degranulate and trigger an inflammatory reaction involving various actors such as the enzyme phenoloxidase and the complement system. in addition, each immunocyte type can influence the activity of the other (franchi and ballarin, 2017). recently, in the same organism, we identified the transcript for a novel c1qdc protein, called bsc1qdc (peronato et al., 2021). it contains two gc1q domains and a signal peptide and resembles human ctrp4 (hctrp4); the bsc1qdc gene is transcribed by circulating immunocytes. the knockdown of bsc1qdc resulted in a reduction of phagocytosis and degranulation processes, so we can suppose that bsc1qdc is able to recognize and bind non-self molecules acting as both an opsonin, favoring phagocytosis, and a chemokine, activating the inflammatory reaction. (peronato et al., 2021). in the present work, we continued to study the expression of bsc1qdc and its role in botryllus innate immunity. using an antibody raised against human ctrp4 (hctrp4). the obtained results confirm what suggested by previous molecular studies and stress the important role of this molecule in botryllus immune responses. materials and methods animals colonies of botryllus schlosseri were collected in the lagoon of venice, near the marine station of the department of biology in chioggia. they were transferred to glass slides and kept in aerated aquaria in thermostatic rooms at the department of biology, university of padova. colonies were reared at 16 °c and fed with living unicellular algae (tetraselmis chuii) and phyto marine (oceanlife, bologna, italy). hemocyte collection to prevent haemocytes clumping, colonies were previously rinsed in 14.7 mm na citrate in filtered seawater (fsw), ph 7.5. then, the peripheral vessels were punctured with fine tungsten needles and the flowing hemolymph was collected with a glass micropipette. it was then centrifuged at 800 xg for 10 min and pelleted hemocytes were re-suspended in fsw to a final concentration of 106 cells/ml. primer design, rna extraction, cdna synthesis, cloning and sequencing total rna was isolated from colonies with the rna nucleospin rna xs kit (macherey-nagel, düren, germany) and its quality was determined by the a260/280 ratio. rna integrity was checked by the visualization of rrnas in midori green (nippon genetics, düren, germany)-stained 1.5 % agarose gels. the first strand of cdna was reverse transcribed from 1 μg of total rna at 42°c for 1 h in a 20 μl reaction mixture containing 1 μl of impromii reverse transcriptase (promega, madison, wi, usa) and 0.5 μl of random primers (promega, madison, wi, usa). pcr reactions were carried out in a 25-μl reaction volume containing 100 ng of cdna from b. schlosseri colonies, 2.5 μl of 10x incubation buffer (pcrbio classic taq, pcr biosystems, london, uk) with 15 mm mgcl2, 0.25 μm of each primer, 10 mm of each of the deoxynucleotide triphosphates, and 2 units of taq polymerase. pcr was performed on a mycycler (biorad, hercules, ca, usa) thermocycler as follows: 94 °c for 2 min, then 42 cycles of 94 °c for 30 s, 55-60 °c for 30 s, 72 °c for 40 s, and 72 °c for 10 min. amplicons were subjected to electrophoresis on 1.5 % agarose gel and the corresponding bands were purified with ultraprep agarose gel extraction miniprep kit (ahn biotechnologie, nordhausen, germany), ligated in pgem-t easy vector (promega, madison, wi, usa) and cloned in dh-5α escherichia coli cells (tang et al., 1994). positively screened clones were sequenced at eurofins genomics (ebersberg, germany) on a sanger sequencing with the abi 3730xl. qrt-pcr to compare the total amount of bsc1q mrna, mrna was extracted from control subclones and subclones injected with c3ar agonist. then, we carried out qrt-pcr with the sybr green method (qpcrbio sygreen mix separate rox, pcr biosystem, london, uk). specific primers (table 1) for bsc1qdc and for the elongation factor 1α (bsef1α) as housekeeping gene were synthesized by sigma-aldrich (st. louis, mo, usa). all the designed primers contained parts of contiguous exons, so to exclude contamination by genomic dna; a qualitative pcr was also carried out before table 1 primers used for qrt pcr analysis _______________________________________________________________ bsc1qdc-forward cgtcataaaggtcaccgtca bsc1qdc-reverse gtgagtcccgaaagtgcccc bsef forward gccgccatactctgaagc bsef reverse gtccaactggcactgttcc 131 qrt-pcr. furthermore, analysis of the qrt-pcr dissociation curve gave no indications of the presence of contaminating dna. qrt-pcr analyses were performed using an applied biosystem (foster city, ca, usa) 7900 ht fast real-time pcr system, using the following cycling parameters: 3 min at 95 °c (denaturation), 20 s at 95 °c plus 1 min at 60 °c, 45 times (extension/annealing), 15 s at 95 °c, 1 min at 60 °c. each set of samples was run three times and each plate contained cdna from three different biological samples (n = 3) and negative controls. the 2−δδct method (livak and schmittgen, 2001) was used to estimate the total amount of mrna. the levels of transcripts in different conditions were normalized to that of bsef1α to compensate for variations in the amounts of cdna. predicted 3d structure of bsc1qdc the structure of bsc1qdc was predicted as described in peronato et al. (2021). as already reported, the two gc1q domains have a typical jelly roll topology. immunoblot analysis in order to validate the anti-hctrp4 polyclonal antibody (thermofisher scientific, waltham, ma, usa), we carried out an immunoblot analysis on colony homogenates. briefly, colonies (5-6 systems in size) were transferred in lysis buffer (50 mm trishcl, 0.25 m sucrose, 1 % sds, 1 mg/ml pepstatin, 1 mg/ml leupeptin, 40 mg/ml pmsf, 2 mm naorthovanadate, 10 mm naf, 0.1 % np-40, 5 mm edta, 5 mm n-ethylmaleimide), in a 1.5 ml vial, disaggregated with a glass pestle, subjected to sonication at 4 °c in a branson (emerson corporate, london, uk) 1200 sonifier at 50 % duty cycles for.5 min, and centrifuged at 10,000 xg for 10 min. the protein content of the supernatants was determined according to bradford (1976). sds polyacrylamide (15 %) slab gel electrophoresis was performed according to the method of laemmli (1970). proteins were transferred to 0.45 mm electran nitrocellulose membrane (bdh, poole, uk) according to towbin et al. (1979), with 25 mm tris, 160 mm glycine, 20 % methanol, 0.7 mm sds as transfer buffer. after blotting, membranes were thoroughly washed in tris-buffered saline (tbs: 50 mm tris-hcl, 150 mm nacl, ph 7.4), incubated for 30 min in tbs containing 5 % powdered milk and probed overnight with 10 μg/ml anti-hctrp4 polyclonal antibody. after further extensive washing in tbs containing 0.05 % tween 20 (ttbs), membranes were overlaid for 1 h with goat antirabbit igg conjugated with peroxidase (biorad, hercules, ca, usa), 10 μg/ml in ttbs. immunogenic bands were revealed by incubation for 10 min in 0.025 % 3,3’-diaminobenzidine (dab) and 0.04 % h2o2 in tbs for 15 min. in controls, primary antibodies (20 μg/ml) were pre-incubated overnight with an equal volume of hemocyte lysate from colonies at mc as a source of bsc1qdc. the mixture was then used for immunoblot analysis. primary antibodies were replaced with rabbit preimmune serum, at the same dilution in negative controls. immunocytochemical assays sixty µl of hemocyte suspension were placed in the center of culture chambers prepared as described elsewhere (ballarin et al., 2008) and left to adhere to superfrost plus (menzer glaser) slides for 30 min at room temperature. they were then incubated for 60 min in fixed in in 4 % paraformaldehyde plus 0.1 % glutaraldehyde in 0.4 m cacodylate buffer containing 1.7 % nacl and 1 % sucrose, at 4 °c for 30 min. fixed haemocytes were incubated for 30 min in 3 % hydrogen peroxide in methanol, to block endogenous peroxidase, washed in pbs and treated for additional 30 min with 3 % powdered milk in pbs, to prevent unspecific binding. cells were then incubated overnight in polyclonal anti-human ctrp4 (thermofisher scientific; 50 µg/ml in pbs), washed and treated for 1 h with goat anti-rabbit secondary antibody, conjugated with biotin. they were finally incubated for 30 min in abc complex (vector) and exposed for 10 min to dab to reveal positive sites. phagocytosis assay hemocytes, collected as described above from colonies at mc, were left to adhere for 30 min on clean coverslips. they were then incubated for 60 min at room temperature with 100 µl of a suspension of yeast (saccharomyces cerevisiae) cells (yeast:hemocyte ratio = 10:1) in fsw in the presence or in the absence (control) of 50 µg/ml of anti-hctrp4 antibody (thermofisher scientific, waltham, ma, usa). anti-rabbit igg (50 µg/ml; calbiochem, san diego, ca, usa) was used as a negative control. slides were then washed several times in fsw to eliminate uningested yeast and cells were fixed in 4 % paraformaldehyde in 0.2 m na-cacodylate buffer containing 1 % nacl and 1 % sucrose, washed in pbs, stained with 10 % giemsa. finally, hemocyte monolayers were mounted on glass slides with acquovitrex (carlo erba, milano, italy). slides were thenobserved under the light microscope (lm), at 1250 x and the percentage of phagocytosing cells, i.e., cells with ingested yeast cells was evaluated. degranulation assays hemocytes were collected and as described above from colonies at mc and left to adhere for 30 min on clean coverslips. they were then incubated for 60 min at room temperature with 100 µl of a suspension of bacillus clausii (4 x 105 cells/ml) in fsw, in the presence or in the absence (control) of 50 µg/ml of anti-hctrp4 or anti-rabbit igg antibody, the latter used as negative control. previous experiments (peronato et al., 2021) demonstrated that, in the presence of b. clausii, most of morula cell rapidly degranulated and changed their morphology. effects of c3ar agonist on bsc1qdc transcription in another series of experiments, three colonies were split in three subclones of 3-4 systems each and, after an acclimation period of 5 days, one of them was injected with 5 μl of c3ar agonist (santa cruz biotechnology; 0.3 μm in dmso). the second one received 5 μl of dmso (control for c3ar 132 fig. 1 predicted 3d conformation of bsc1qdc. the colored part corresponds to the amino acid sequence corresponding to the epitope in hctrp4 used to raise the antibody agonist), whereas the third was injected with the same amount of fsw. after 24 h, mrna was extracted from treated and control subclones, reverse transcribed to cdna and the transcription of bsc1qdc and bsef1α was followed by qrt pcr. statistical analysis each experiment was replicated at least three times (n = 3) with three independent blood samples; data are expressed as mean ±sd. at least cells in 10 optical fields at 1250 were counted under the lm for each experiment. indexes were compared with the χ2 test. qpcr experiments were replicated three times (n = 3) with three independent samples. oneway anova was followed by the student-newmankeuls test to assess significant differences with respect to colonies injected with dmso in fsw. statistical analyses were performed with the primer statistical program. results analysis of the anti-hctrp4 antibody the commercial antibody used in our assays was develop against the epitope represented by the sequence of the amino acids form 32 to 61 of hctrp4. it corresponds to the amino acid sequence from 32 to 62 of bsc1qdc (genbank accession number: mt783425). the alignment of the two sequences shows 33 % of similarity, the full conservation of 7 amino acids and additional 5 with highly similar properties. in addition, according to our 3-d reconstruction, the sequence appears exposed to the external milieu (fig. 1), which supports our idea that the antibody raised against the corresponding human sequence can recognize our protein both in the native and denatured form. anti-hctrp4 antibody recognizes a band of 37 kda in immunoblot analysis and labels circulating immunocytes in immunoblot analysis of colony homogenates, the anti-hctrp4 antibody recognized a single band of 37 kda, in accordance with the predicted value of 37.19 kda (peronato et al., 2021). an additional band, of 75 kda, is clearly visible. in samples treated with the antibody pre-incubated with hemocyte lysate, the intensity of the 37-kda band was highly (67 %) reduced whereas the band was not visible when the preimmune serum was used (fig. 2). anti-hctrp4 antibody recognizes b. schlosseri immunocytes the anti-hctrp4 antibody, when used for immunocytochemical analyses, confirmed the result obtained with ish: only morula cells and phagocytes resulted immunopositive (fig. 3a,c), with a frequency of about 70 % of morula cells and 34 % of phagocytes, respectively (fig. 4a). anti-hctrp4 antibody inhibits yeast phagocytosis and morula cell degranulation when hemocytes were exposed to yeast cells under control conditions, the fraction of phagocytes able to ingest yeast cells (fig. 3c) amounted to 87 %. the incubation of the hemocytes with antihctrp4 antibody significantly (p < 0.01) decreased the number of cells with ingested yeast; no effects were observed in the presence of the anti-rabbit igg 133 antibody (fig. 4b). the percentage of degranulated morula cells (fig. 3e) amounted to 80 % in cells exposed to b. clausii; in the presence of antihctrp4, the extent of degranulated cells was (p < 0.001) reduced, although still significantly (p < 0.001) higher than in controls. the anti-rabbit igg antibody did not induce any inhibition of degranulation (fig. 4c). c3ar agonist influences bsc1qdc transcription in colonies injected with the c3ar agonist, a significant (p < 0.05) increase in the level of transcription of bsc1qdc was observed with respect to dmso-injected subclones (fig. 5). discussion according to our previous results (peronato et al., 2021), bsc1qdc is a novel soluble receptor, acting either as an opsonic lectin able to enhance the phagocytosis of nonself particles, and as a cytokine able to facilitate morula cell degranulation. the transcription of bsc1qdc is significantly enhanced by the injection of foreign microorganisms, such as b. clausii and saccharomyces cerevisiae, in the colonial circulatory system and its knock-down by rna interference influences both phagocytosis and degranulation (peronato et al., 2021). we also noticed the high predicted structural similarity and residue conservation of bsc1qdc and hctrp4, in particular of c-terminal gc1q domains (peronato et al., 2021). therefore, in the present work, we also used a commercial antibody raised against hcrtp4, to collect additional information on the expression and function of bsc1qdc. we are well aware that the antibody specificity can be directed towards other, still unrelated or undiscovered botryllus proteins; however, we were not able to find additional c1qdc proteins in our transcriptome and, although the antibody is not specific to our protein, it fig. 2 immunoblot analysis of lysates of colonies at mc. lane 1: pre-immune serum; lane 2: anti hctrp4 antibody; lane 3: pre-absorbed antihctrp4 antibody; lane 4: molecular weight standards. recognizes an electrophoretic band in colony homogenate with an estimated molecular weight around 37 kda, comparable to the expected molecular weight of bsc1qdc, that was greatly reduced after its pre-absorption with the lysate itself. fig. 3 staining of fixed hemocytes with anti-hctrp4 antibody. a: morula cells (dark arrowheads: labeled cells; white arrowheads: unlabeled cells); b: fixed phagocytes with (h) and without (i) ingested yeast cells. c-d: healthy (d) and degranulated (e) living morula cells. scale bars: 10 m 134 fig. 4 a: fraction of phagocyte and morula cells labelled by the anti-hctrp4 antibody. b: mean percentages of phagocytosis cells in hemocyte monolayers incubated with yeast cells in the absence and in the presence of either anti-hctrp4 or anti-rabbit igg antibody. c: degranulation of morula cells triggered by b. clausii in the absence or in the presence of either anti-hctrp4 or anti-rabbit igg antibody. significant differences with untreated cells are marked asterisks. **: p < 0.01; ***: p < 0.001 in addition, the human amino acid sequence against which the antibody was raised has a corresponding sequence in our protein which, in the 3-d reconstruction, appears exposed to the external environment so to justify its recognition by the antibody in both the native and denaturated form. in addition, the comparison of the human epitope used for immunization and the corresponding sequence of our protein shows 33 % of similarity and various conserved amino acids which can represent recognition sites for the antibody. the presence of a second band, of approximately 75 kda, is likely due to the presence of dimers: the presence of a putative multimerization surface was already suggested by the analysis of the predicted 3dstructure of bsc1qdc (peronato et al., 2021). in the hemolymph of b. schlosseri, immunocytes represent the majority of circulating hemocytes and are represented by phagocytes and granular morula cells. as suggested by their name, phagocytes can recognize and ingest foreign particles in order to clear them from the circulation. conversely, morula cells, upon the recognition of foreign molecules, trigger an inflammatory reaction consequent to their degranulation leading to cell recruitment and cytotoxicity (franchi and ballarin, 2017). among the vertebrate c1qdc proteins, the mammalian c1q is a pattern-recognition receptor involved in the recognition of antigen antibody complexes and in the consequent triggering of the complement classical activation pathway (carland and gerwick, 2010). however, it was demonstrated that it also binds directly to the surface of microbes and modulate their phagocytosis (bohlson et al., 2007). as demonstrated in our previous work (peronato et al., 2021), the transcription of bsc1qdc is significantly upregulated following the injection of nonself particles or molecules in the colonial circulation and both phagocytosis and morula cell degranulation are significantly impaired by the knock-down of the transcription. this suggests a role of the molecule as opsonin in phagocytosis and as cytokine in modulating morula cell degranulation. our results, showing that the anti-hctrp4 antibody recognizes the same circulating cells that actively transcribe bsc1qdc, i.e., phagocytes and morula cells, support the idea that immunocytes are the sites of synthesis of the bsc1qdc protein that, once released, can act in an autocrine or paracrine way to enhance phagocytosis and morula cell degranulation. indeed, the presence of the antibody in the incubation medium can significantly reduce yeast cell phagocytosis and degranulation of morula cells in the presence of b. clausii, a reliable inducer of degranulation (peronato et al., 2021). as for the relationships of bsc1qdc with the components of the complement activation pathways, we observed an increase in the transcription of bsc1qdc in colonies injected with the c3ar agonist. from previous experiments we know that the injection of c3ar agonist induces a significant increase in the transcription of bsc3 (peronato et al., 2020b), synthesized and released by morula cells (franchi et al., 2014). therefore, we can suppose that the observed results are the consequence of a positive autocrine loop between bsc3 and bsc1qdc so that the rising level of bsc3 135 fig. 5 effect of the injection, in the colonial vasculature, of fsw (control), dmso (negative control) and 3ar agonist on the transcription of bsc1qdc in the circulation fosters the synthesis and release of bsc1qdc as a strategy to reinforce the immune responses against the microbial invaders. a rise in human ctrp6, another c1qdc-protein was indeed observed in mice with rheumatoid arthritis, characterized by enhanced c3 activation (murayama et al., 2015). however, differently from what expected, we see a decrease of bsc1qdc at to, during which we previously reported an increase in the transcription of bsc3 (peronato et al., 2020c). we can suppose a delay in the synthesis of the bsc3 protein so that the activation of bsc1qdc transcription is postponed to the beginning of the new 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into the function of invertebrate c1qdc proteins. dev. comp. immunol. 52: 202-214, 2015. 137 397 isj 13: 397-410, 2016 issn 1824-307x research report a-type cpg odn with higher binding affinity to lvtoll1 could probably activate downstream ifn system-like antiviral response in shrimp litopenaeus vannamei j xu1,3, d zhao4, m sun1,3, l wang2, z jia1,3, r liu1, l wang1,3, l song2 1key laboratory of experimental marine biology, institute of oceanology, chinese academy of sciences, qingdao 266071, china 2key laboratory of mariculture & stock enhancement in north china’s sea, ministry of agriculture, dalian ocean university, dalian 116023, china 3university of chinese academy of sciences, beijing 100049, china 4dalian polytechnic university, dalian 116034, china accepted december 6, 2016 abstract cpg oligodeoxynucleotides (cpg odns) is a widely used immune adjuvant, which could activate various immune responses including antiviral response through interaction with toll-like receptor 9 (tlr9) in mammals. in the present study, four types of cpg odn (cpg-a, cpg-b cpg-c, and cpg-p) were synthesized and injected to the shrimp litopenaeus vannamei in order to evaluate their immune enhancement effect in shrimp. the copy numbers of white spot syndrome virus in the shrimps treated with different types of cpg odns were of 3.10×105 (cpg-a), 8.32×105 (cpg-b), 9.84×105 (cpg-c), and 8.12×105 (cpg-p) copies ng-1 dna respectively, which were significantly lower (p < 0.01) than that in pbs group (1.70×106 copies ng-1 dna). surface plasmon resonance (spr) assay revealed that the four types of cpg odn displayed different binding affinity to lvtoll1, lvtoll2 and lvtoll3, and the highest binding affinity was observed between cpg-a and lvtoll1. correspondingly, the mrna transcripts of lvtolls were up-regulated significantly in cpg-a stimulated shrimps， which was significantly higher than that in cpg-b, cpg-c and cpg-p groups (p < 0.01). the phagocytic rate and ros level of shrimp hemocytes in cpg-a and cpg-b groups increased significantly compared with that in other groups, which were 1.63-fold, 9.98-fold (p < 0.01) in cpg-a and 1.60-fold, 4.92-fold (p < 0.01) in cpg-b higher than those in pbs group, respectively. moreover, after cpg-a stimulation, the probable ifn level in shrimp plasma increased to 2.60-fold (p < 0.01) of that in pbs group, and the mrna expressions of ifn system-like antiviral genes (lvirf, lvvago4 and lvstat) were also significantly up-regulated in cpg-a group, displaying a stronger response than that in cpg-b, cpg-c and cpg-p groups. the results indicated that cpg-a could promote the cellular and humoral immunity in shrimp, and induce relatively higher antiviral immune response among the four cpg odns. it provided useful information to understand the stimulatory effects of cpg odns in shrimp, promoting the application of cpg odns in aquaculture. key words: litopenaeus vannamei; cpg odns; cpg-a; lvtolls; spr; ifn introduction cytosine phosphate guanine oligodeoxynucleotides (cpg odns) were originally defined for bacterial dna with unmethylated cpg dinucleotides in certain flanking sequences (cpg motifs), which had diverse stimulatory effects on the ___________________________________________________________________________ corresponding author: linsheng song key laboratory of mariculture & stock enhancement in north china’s sea ministry of agriculture dalian ocean university dalian 116023, china e-mail: lshsong@qdio.ac.cn, lshsong@dlou.edu.cn innate and adaptive immune system (krieg, 2002). there are four types of cpg odns with distinct structural and biological properties, including a-type, b-type, c-type and p-type (bode et al., 2011). a-type cpg odn (cpg-a) has a phosphodiester core flanked by phosphorothioate terminal nucleotides. it contains a single cpg motif flanked by palindromic sequences and poly g tails at the 3' and 5' ends. b-type cpg odn (cpg-b) and c-type cpg odn (cpg-c) are both composed entirely of phosphorothioate nucleotides, while they resemble cpg-a in containing palindromic cpg motifs and thus can form stem loop structures or dimmers. 398 p-type cpg odn (cpg-p) contains double palindromes that can form hairpins at the gc-rich 3' ends (shirota and klinman, 2014). these cpg odns mainly participate in activating intracellular signaling pathways and triggering the proliferation, maturation and differentiation of immune cells, consequently increasing the secretion of cytokines and chemokines with disease-resistant effects (klinman, 2004; krieg, 2006). cpg motifs are considered as a kind of pathogen-associated molecular patterns (pamps) due to their abundance in microbial genomes and rarity in animal genomes (krieg, 2006). the immunogenicities of cpg odns have been examined in numerous preclinical studies, demonstrating that cpg odns could enhance both humoral (tafaghodi et al., 2006; borges et al., 2008) and cellular (th1 cells and cytotoxic lymphocyte) (cho et al., 2000; shirota et al., 2000; shirota and klinman, 2011) immunity against pathogens in mammals. toll-like receptor 9 (tlr9) in mammals is proved to be the receptor of cpg odns (hemmi et al., 2000; bauer et al., 2001; takeshita et al., 2001). cpg odns could be recognized by tlr9 to promote the maturation of antigen presenting cells (apcs) and secretion of cytokines like type i interferon (ifn), interleukin (il) or tumor necrosis factor (tnf) (colonna et al., 2004). the immune responses triggered by different types of cpg odn have been widely reported in vertebrates and the mechanisms are diverse. for example, cpg-a and cpg-p could strongly induce type i ifn production, while cpg-b and cpg-c could trigger the production of tnf-and il-6, respectively (shirota and klinman, 2014). further studies have demonstrated that cpg-a could induce the ifn regulatory factor-7 (irf-7) dependent antiviral immunity by activation of the tlr9 in plasmacytoid dendritic cells in mice (honda et al., 2005). because of the effects in stimulation of immunity, many completed clinical trials have suggested a promising application of cpg odn as adjuvants (shirota and klinman, 2014). recently, cpg odns have also been reported to trigger humoral and cellular immune responses in invertebrates. in the bivalve mollusc, bacterial dna with unmethylated cpg motif could promote the antibacterial activity, lysozyme activity and prophenoloxidase production of hemolymph (hong et al., 2006). in crustacean, cpg-rich fragment containing tandem odns enhanced the immuno-protection efficiency and induced autophagy of chinese mitten crab, eriocheir sinensis (sun et al., 2013, 2015). cpg odns could also promote the generation of new hemocytes, increase the phagocytic ability and the reactive oxygen species (ros) level in the hemocytes of litopenaeus vannamei (sun et al., 2013). although cpg odns have been reported to induce immune response in crustacean, the mechanism of immune enhancement triggered by different type of cpg odn is still not well understood. it has been reported that cpg odns could activate the ifn-related antiviral pathway in mammals (levy et al., 2011) and teleosts (su et al., 2016), and poly (i:c)-induced antiviral immunity has also been reported in pacific white shrimp l. vannamei, which is similar to the ifn system in mammals (wang et al., 2013; li et al., 2015). in shrimp l. vannamei, the lvvago4 gene is considered to be a cytokine functionally similar to ifn, which could restricts virus infection through activating the jak-stat pathway; the irf-like gene lvirf showed similar protein nature to mammalian irfs and could be activated during virus infection. therefore, the vago system which is centered on the irf-vago-jak/stat axis plays a critical role in nucleic acid-induced antiviral immunity in shrimps, and exhibits similarity to the mammalian ifn system, suggesting an ifn system-like antiviral mechanism in shrimp (li et al., 2015). the shrimp l. vannamei is a prosperous commodity in the world, but its farming has been seriously disturbed by virus, especially white spot syndrome virus (wssv). even the immune defense mechanism of shrimp has been well documented (li and xiang, 2013), the effective approaches as well as novel therapeutic agents are in urgent to be developed for better controlling wssv in shrimp aquaculture. our previous studies have revealed that tandem cpg odns could induce antiviral immunity in shrimp (zhang et al., 2010). in the present study, four types of cpg odn (cpg-a, cpg-b cpg-c, and cpg-p) were employed to stimulate the shrimp l. vannamei with the objectives (1) to detect the copy number of wssv and the expression levels of lvtolls in the hepatopancreas of shrimps after the treatment of four types of cpg odns, (2) to evaluate the binding activities of lvtolls to various cpg odns by surface plasmon resonance (spr), (3) to investigate the cellular immune responses by detection of phagocytic rate, ros level in shrimps hemocytes, and humoral antiviral immunity via analysis of the ifn system-like related genes and the probable ifn level in plasma after the treatments with different cpg odns, and (4) to evaluate the immune enhancement effect of different cpg odns and provide theoretical guidance for the application of different cpg odns as immunopotentiator in aquaculture. table 1 names and sequences of the cpg odns used in this study name sequence (5’---3’) cpg 2216 (cpg-a) gggggacgatcgtcgggggg cpg 2006 (cpg-b) tcgtcgttttgtcgttttgtcgtt cpg 2395 (cpg-c) tcgtcgttttcggcgcgcgccg cpg 23617(cpg-p) tcgtcgacgatcggcgcgcgccg cpg-negative 2317 (cpg-n) tgctgcttttgtgcttttgtgctt javascript:void(0); javascript:void(0); javascript:void(0); javascript:void(0); javascript:void(0); javascript:void(0); javascript:void(0); 399 materials and methods shrimps and cpg odns shrimps, litopenaeus vannamei, about 10 cm in length, were collected from a commercial farm in qingdao, china, and cultured in tanks at 22 ± 1 °c for 14 days before processing. four types of cpg odns, including 2216 (a type), 2006 (b type), 2395 (c type) and 23617 (p type) (table 1), which had been demonstrated to be effective in mammalian and aquatic animals (chen et al., 2007; martinson et al., 2007; samulowitz et al., 2010; zhang et al., 2010), were employed in the present study. the dna sequence of the four types of cpg odns and the non-cpg odn (cpg-n) 2317 were synthesized by sangon biotech co. (china) with the purity above 99.9 %. virus preparation wssv was extracted and purified from gill tissue of wssv infected shrimps via the method of differential centrifugation described by xu et al. (2007) with some modification. briefly, l. vannamei shrimps with white spots on the cuticle were selected. one hundred milligram of the diseased shrimp gill were homogenized in 45 ml tne buffer (400 mmol l-1 nacl, 50 mmol l-1 tris-hcl, 5 mmol l-1 edta, ph 8.5) with 1 mmol l-1 pmsf (phenylmethanesulfonyl fluoride), and then centrifuged at 3,500g, 4 °c for 5 min. the supernatant was filtered through 0.45 μm membrane, and centrifuged at 30,000g for 30 min. the pellet was resuspended with tm buffer (10 mmol l-1 mgcl2, 50 mmol l-1 tris-hcl, ph 7.5) and centrifuged three times. finally, the pellet was dissolved with 1 ml tm buffer containing 0.3 % nan3. the copy number of wssv stock solution was quantified by the real-time pcr, then diluted with pbs (phosphate buffer saline, 0.1 mol l-1, ph 7.4) to a final concentration of 107 copies μl-1 for subsequent experiments (stored at -80°c). cpg odns treatment, wssv infection and sample collection a total of 432 shrimps were randomly divided into two groups (group i and group ii). in group i, the 162 shrimps were randomly separated to six sub-groups including cpg-a group, cpg-b group, cpg-c group, cpg-p group, cpg-n group and pbs group to survey the wssv copy numbers after virus infection, so the shrimp number in each treatment was 27. the cpg odns were diluted in sterilized pbs at a final concentration of 10 μg ml-1, and 40 μl of cpg odns was injected into each shrimp from the second abdominal muscle using a syringe. the dosage of cpg odns was selected according to the published papers (zhang et al., 2010; sun et al., 2013, 2015). in the cpg-n and pbs groups, the shrimps received the injection of same volume non-cpg dna (10 μg ml-1) or sterilized pbs. the shrimps were returned to the tanks and received an injection of 100 μl wssv stock (104 copies μl-1) at 12 h after the cpg treatment. nine shrimps were randomly sampled at first, third and fifth day post wssv challenge in each group, and the gill of every 3 shrimps in the same group was mixed as a single sample for the further dna extraction to measure the virus copy numbers. in group ii 270 shrimps were randomly separated into six sub-groups, and the cpg odns treatment was performed as descripted in the group i, and there were 45 shrimps in each treatment. nine shrimps in each group were sampled at 0, 3, 6, 12, 24 h post injection. tissues (hemocytes, plasma and hepatopancreas) from three shrimps in the same group at the same time point were mixed together as a single sample and there were three replicates for each group. the hemolymph (about 0.8 ml each shrimp) was collected from the pericardial cavity through the intersegmental membrane between the cephalothorax and the first abdominal segment using a syringe (2.5 ml) with 0.8 ml pre-cooled (4 °c) anticoagulant solution (115 mmol l-1 glucose, 27 mmol l-1 sodium citrate, 336 mmol l-1 nacl, 9 mmol l-1 edta·na2·2h2o, ph 7.4). the collected hemolymph was immediately centrifuged at 1,000g, 4 °c for 10 min to harvest hemocytes for the determination of phagocytosis and ros. the supernatants (plasma) were stored at -80 °c for subsequent enzyme activity examination. the hepatopancreas was collected and added into 1.5 ml microcentrifuge tube with 600 μl trizol reagent (takara, japan), stored at -80 °c for subsequent rna extraction. quantitative real-time pcr analysis of wssv copy numbers the qrt-pcr technique was employed to determine the wssv viral numbers according to the previous report (zhang et al., 2010) with some modification. briefly, the recombinant plasmid of a 154 bp wssv dna fragment was used as the standard dna for sybr green fluorescent qrt-pcr in an abi prism 7500 sequence detection system (applied biosystems, usa). the wssv copy numbers were estimated by the target amplicon in the plasmid, and the target amplicons were used to make 10-fold serial dilutions from 1×1010 to 1×102 copies μl-1. these dilutions were employed as absolute standards to make a curve for quantification. total genomic dna of gill tissue in infected shrimps was extracted following the instructions of tianamp marine animals dna kit (tiangen, china), and the dna concentration was determined by using nanodrop 2000 (thermo scienfic, usa). the wssv dna fragment in total genomic dna were examined by qpcr and the copy numbers of wssv were calculated with the standard curve. analysis of phagocytic rate of shrimp hemocytes the phagocytic activity of hemocytes was measured according to the previous description with some modification (wu et al., 2008). briefly, hemocytes from different groups were resuspended with cell resuspension (modified l-15 medium), and then incubated with the latexs beads (sigma, usa) under low speed of rotation at room temperature for 1 h. after three times of washing with cell javascript:void(0); javascript:void(0); javascript:void(0); 400 resuspension solution, the phagocytic rate of hemocytes was analyzed by facs arial ii flow cytometer (fcm) (becton, dickinson and company). measurement of intracellular ros level the reactive oxygen species assay kit (beyotime, china), based on the peroxide-sensitive fluorescent probe dcfh-da, was used to detect the intracellular ros level. the hemocytes (about 100 cell ml-1) from shrimps were harvested and incubated in the cell resuspension solution at room temperature for 20 min. after three times of washing with cell resuspension solution, the intracellular ros level in hemocytes was analyzed by facs arial ii flow cytometer (becton, dickinson and company). analysis of cpg odns binding activities of lvtoll-ecds by spr the recombinant extracellular domain (ecd) proteins of three verified toll-like receptors (lvtoll1 (abk58729.1), lvtoll2 (aek86516.1) and lvtoll3 (aek86517.1) in l. vannamei were employed for the spr assay. preparation of lvtoll-ecds was conducted according to the method from sun’s description (sun et al., 2014). cpg odns binding analysis of lvtoll-ecds (lvtoll1-ecd, lvtoll2-ecd, lvtoll3-ecd) was performed at 20 °c on a biacore t200 spr instrument (ge healthcare, usa). the anti-his tag antibody (ge healthcare, usa) was previously covalently immobilized onto the cm5 sensor chip surface with the included immobilization buffer and amine coupling kit. the recombinant lvtoll-ecds proteins with 6×his tag were diluted in hbs-ep (ge healthcare, usa), which was the running buffer containing10 mmol l-1 hepes (ph 7.5), 150 mmol l-1 nacl, 3 mmol l-1 edta and 0.005 % (v/v) surfactant p20. then they were injected to the loading tube and captured by the immobilized anti-his tag antibody to 240 (for rlvtoll-ecds) and 200 (for rtrx) response units (ru), respectively. the cpg odns (100 μmol l-1) were injected to the loading tube and passed over adjacent target. flow cells were controlled at a flow rate of 30 μl min-1 for 2 min. after 5 min dissociation, recombinant proteins and bound analyses were removed by a 60 s wash with 10 mmol l-1 glycine-hcl (ph 1.5) at a flow rate of 30 μl min-1. after subtracting the control values, the data were analyzed with a 1:1 langmuir binding model using the biacore t200 evaluation software. rna extraction, cdna synthesis and qrt-pcr analysis of immune related genes the rna extraction and cdna synthesis were conducted as the previous report (sun et al., 2015) with some modification. total rna was extracted from hepatopancreas using trizol reagent (invitrogen, usa). first-strand cdna synthesis was carried out based on promega m-mlv rt usage information with random hexamers primer (sangon, china). the mrna expression levels of immune related genes were detected by sybr green fluorescent qrt-pcr. gene specific primers for lvtolls, lvirf, lvvago4 and lvstat (primer p5-p16) (table 2) were used to amplify the corresponding products. the shrimp 18s rrna fragment, amplified with primers p3 and p4 (table 2), was chosen as reference for internal standardization. after the pcr program, data were analyzed by using the sds 2.0 software (applied biosystems). the relative expressions of related genes were calculated by the 2−δδct method. all the data were given in terms of relative mrna expressed as mean ± sd (n = 4) (zhou et al., 2012). table 2 summary of primers in this study primer sequence (5’---3’) p1 (wssv-154-rt-f) ccagttcagaatcggacgtt p2 (wssv-154-rt-r) aaagacgcctaccctgttga p3(18srrna-rt-f) tatacgctagtggagctggaa p4(18srrna-rt-r) ggggaggtagtgacgaaaaat p5(lvtoll1-rt-f) tcgaccatcccttttacacc p6(lvtoll1-rt-r) ttgcctggaaggtctgattc p7(lvtoll2-rt-f) catgcctgcaggactgttta p8(lvtoll2-rt-r) ggcctgagggtaaggtcttc p9(lvtoll3-rt-f) tcgtacaaccagctgacgag p10(lvtoll3-rt-r) atacttcaggtgggccacag p11(lvirf-rt-f) ctttcgctactgggctcttgc p12(lvirf-rt-r) ggtcgtagtgcttcggtttctc p13(lvvago4-rt-f) gcgagagggaaaaggaaaacagg p14(lvvago4-rt-r) ccagcacttcccggggtggtctg p15(lvstat-rt-f) agcccctgtctgagcgaaa p16(lvstat-rt-r) ggtgttctcttgtaaccttcatca 401 measurement of probable ifn level in plasma the probable ifn level in plasma was determined by using the commercial fish ifn elisa kit (jiangsu kete biological technology, china) following the manufacturer’s instructions. briefly, the 96 wells plates coated by the purified fish ifn antibody were incubated with 50 μl plasma (diluted 5-fold with sample diluent) from different groups at 37 °c for 30 min. after five times of washing, the plate was incubated with 50 μl horseradish peroxidase (hrp)-fish ifn antibody at 37 °c for 30 min. after the final washing of five times, chromogenic agent and stop buffer solution were added, and the absorbance was detected at 450 nm by a precision microplate reader (biotek, usa). data analysis for all experimental results, the data from three samples were recorded and analyzed by one-way analysis of variance (anova), followed by duncan’s multiple range tests using spss 16.0 program. differences were considered to be statistically significant at a p value of 0.05 or less. results the alternations of wssv copy numbers after cpg odn treatments the wssv copy numbers in the gill tissue of shrimps pretreated with different types of cpg odns were significantly lower than those in cpg-n and pbs-pretreatment shrimps on the third and fifth day post wssv challenge. on the first day after wssv challenge, no significant difference of the wssv copy numbers was observed between cpg-pretreatment groups and controls (fig. 1). on the third day, the wssv numbers in the shrimps pretreated with cpg-a, cpg-b, cpg-c and cpg-p were 2.20×105, 4.72×105, 6.09×105 and 4.40×105 copies ng-1 dna, whereas the numbers in cpg-n and pbs-pretreatment shrimps were 7.41×105 and 7.92×105 copies ng-1 dna. on the fifth day post wssv challenge, the wssv copy number of shrimps in cpg-a , cpg-b, cpg-c and cpg-p groups were 3.10×105, 8.32×105, 9.84×105 and 8.12×105 copies ng-1 dna respectively, which were significantly lower, approximately 18.19 %, 48.82 %, 57.75 % and 47.65 % (p < 0.01) respectively, than that in pbs group. moreover, the wssv copy numbers in cpg-a group was the lowest among the four types of cpg odn groups, especially on the fifth day post wssv challenge, the numbers in cpg-b, cpg-c and cpg-p groups was 2.68-fold, 3.17-fold and 2.62-fold (p < 0.01) higher than that of cpg-a group respectively. no significant difference was observed between the control groups (treated with cpg-n and pbs) throughout the experiment. fig. 1 the wssv copy numbers in gill tissue in cpg-a, cpg-b, cpg-c, cpg-p, cpg-n and pbs-pretreatment shrimps after wssv infection on the first, third and fifth day. data presented as mean ± s.d. (n = 4). the significant differences between the treated groups and control groups were subjected to one-way analysis of variance (one-way anova). 402 fig. 2 temporal expression of (a) lvtoll1, (b) lvtoll2 and (c) lvtoll3 mrna detected by real-time pcr in shrimp hepatopancreas after in vivo cpg-a, cpg-b, cpg-c, cpg-p, cpg-n and pbs injection at 3, 6, 12 and 24 h. comparison of the level of mrna (relative to 18s rrna gene) was normalized to 0 h. data presented as mean ± s.d. (n = 4). the significant differences between the treated groups and control groups were subjected to one-way analysis of variance (one-way anova). the mrna expressions of lvtoll1, lvtoll2 and lvtoll3 in hepatopancreas of shrimps after cpg odns treatment the mrna expression levels of three verified toll-like receptors (lvtoll1, lvtoll2 and lvtoll3) in hepatopancreas from shrimps were detected at 0, 3, 6, 12 and 24 h post the treatments with cpg odns. the expression levels of lvtolls in cpg-a and cpg-p groups were significantly up-regulated compared with those in cpg-n and pbs groups, while those in the cpg-b, cpg-c groups did not change dramatically. after cpg-a stimulation, the mrna expression levels of lvtoll1, lvtoll2 and lvtoll3 were all up-regulated at 3 h and peaked at 24 h (fig. 2), which were 28.62-fold (p < 0.01), 22.82-fold (p < 0.01) and 17.59-fold (p < 0.01) of that in pbs group, respectively (fig. 2). after the stimulation of cpg-p, the mrna transcripts of lvtoll1, lvtoll2 and lvtoll3 increased and reached the maximum level at 12 h, which were 12.52-fold (p < 0.01), 8.50-fold (p < 0.01) and 6.64-fold (p < 0.01) of those in pbs group, respectively (fig. 2). no significant difference was observed between the control groups (treated with cpg-n and pbs) throughout the experiment. in addition, the maximum mrna expression levels of lvtoll1, lvtoll2 and lvtoll3 in cpg-a group were 1.82-fold, 1.98-fold and 3.32-fold (p < 0.01) of that in cpg-p group, respectively, which were also significantly higher than that in cpg-b and cpg-c groups. the binding affinity of different cpg odns to lvtoll-ecds in order to determine the binding affinity of cpg odns to lvtolls, three lvtoll-ecds (lvtoll1-ecd, lvtoll2-ecd, and lvtoll3-ecd) and rtrx (the control protein) with his tag were captured by the chip-immobilized anti-his antibodies. cpg odns, including cpg-a, cpg-b, cpg-c and cpg-p, were screened by passing through the chip-immobilized proteins to examine the molecular interaction with cpg-n as control. cpg-a displayed a rapid association with lvtoll1-ecd on spr with javascript:void(0); javascript:void(0); 403 fig. 3 surface plasmon resonance measurement of the binding between different cpg odns and lvtoll-ecds. the sensorgrams of different cpg-odns binding to (a) lvtoll1-ecd, (b) lvtoll2-ecd, (c) lvtoll3-ecd and (d) rtrx. all data shown were reproducible and representative of at least three independent experiments. the binding signal of 35 ru, while other cpg odns showed no binding affinity to lvtoll1-ecd (fig. 3a). all the four types of cpg-odns exhibited low binding affinity to lvtoll2-ecd with the signals of 20, 7, 10 and 3 ru, respectively (fig. 3b). similarly, the signals of lvtoll3-ecd for cpg-a, cpg-b, cpg-c and cpg-p were 20, 5, 7 and 3 ru respectively (fig. 3c). no binding signal was observed in cpg-n and blank groups throughout the experiment. and all the four types cpg odns did not display any binding signal with rtrx (fig. 3d). cpg-a exhibited the strongest binding activity among the three lvtoll-ecd and it displayed a binding signal of 35 ru with lvtoll1-ecd, which was significantly higher than that of cpg-a with lvtoll2-ecd (20 ru) and lvtoll3-ecd (20 ru). the mrna expression of lvirf, lvvago4 and lvstat genes in hepatopancreas after cpg odns treatment lvirf (km277954), lvvago4 (hq541161.1) and lvstat (agt28261.1) were selected to investigate the induction of cpg odns on the activation of the ifn system-like response in shrimp. after stimulations of cpg-a and cpg-p, the mrna expression of the three genes increased significantly compared with that in cpg-n group and pbs group. the mrna expression levels of lvirf, lvvago4 and lvstat were significantly up-regulated and reached peak at 24 h after injection of cpg-a, which were 6.08-fold, 24.92-fold and 14.73-fold (p < 0.01) of those in pbs group, respectively (fig. 4). at 12 h post cpg-p injection, the mrna expression levels of lvvago4 and lvstat genes were also significantly up-regulated, which were 9.24-fold and 13.08-fold (p < 0.01) compared with those in pbs group (figs 4b, c). in addition, cpg-b could induce the up-regulation of lvirf at 6 h with 2.27-fold (p < 0.01) of that in pbs group and cpg-c could induce the up-regulation of lvstat at 3 h with 1.60-fold (p < 0.01) of that in pbs group, which were significantly lower than that in cpg-a and cpg-p group (figs 4a, c). no significant difference in the expression level of lvirf, lvvago4 and lvstat was observed between the control groups (treated with cpg-n and pbs) throughout the experiment. javascript:void(0); 404 fig. 4 temporal expression of (a) lvirf, (b) lvvago4 and (c) lvstat mrna detected by qrt-pcr in shrimp hepatopancreas after in vivo cpg-a, cpg-b, cpg-c, cpg-p, cpg-n and pbs injection at 3, 6, 12 and 24 h. comparison of the level of mrna (relative to 18s rrna gene) was normalized to 0 h. data presented as mean ± s.d. (n = 4). the significant differences between the treated groups and control groups were subjected to one-way analysis of variance (one-way anova). the probable ifn protein level in plasma after cpg odns treatment the probable ifn protein level in plasma of shrimps after cpg odn treatments was determined using the fish ifn elisa kit. cpg-a, cpg-b and cpg-p could induce the increase of probable ifn level in plasma. the concentration of probable ifn in cpg-a group was significantly up-regulated at 3 h and reached the peak (2.60-fold, p < 0.01) at 24 h (fig. 5). compared with the probable ifn protein level in pbs group (fig. 5), cpg-b group increased at 6 h (1.39-fold, p < 0.01) and cpg-p group also increased at 24 h (1.91-fold, p < 0.01) respectively, while they were both significantly lower than that in cpg-a group. in addition, there was a significant decrease in cpg-c group at 6h and an increase in cpg-n group at 3h in comparison to the pbs control (fig. 5). the phagocytic rate of shrimp hemocytes after cpg odns treatment the phagocytic rate of shrimp hemocytes was recorded by using the relative fluorescence intensity. after injections of cpg-a, cpg-b, cpg-c and cpg-p, the phagocytic rate of hemocytes increased in comparison with that from the shrimps treated with cpg-n and pbs. in cpg-a group, the phagocytic rate of hemocytes was up-regulated at 3 h and reached the peak at 6 h (1.63-fold of that in pbs group, p < 0.01) (fig. 6). in cpg-b group, the phagocytic rate increased to the maximum at 6 h (1.60-fold of that in pbs group, p < 0.01) (fig. 6). in cpg-p and cpg-c groups, the peak level of the phagocytic rate was detected at 6 h and 12 h, which were 1.40-fold and 1.15-fold (p < 0.01) of that in pbs group, respectively (fig. 6). the maximum levels of phagocytic rate in cpg-a group and cpg-b group were significantly higher than that in cpg-c and cpg-p groups. there was no obvious difference between the control (cpg-n and pbs) groups throughout the experiment. the ros level in hemocytes after injection of cpg odns the level of ros generation in hemocytes after cpg odns injection was recorded by using the relative fluorescence intensity. cpg-a, cpg-b and cpg-c could increase the ros generation in 405 fig. 5 determination of probable ifn in plasma after injection of cpg odns using the commercially fish ifn elisa kit. data presented as mean ± s.d. (n = 3). the significant differences between the treated groups and control groups were subjected to one-way analysis of variance (one-way anova). hemocytes compared with cpg-n and pbs. after injection of cpg-a and cpg-b, the ros levels increased significantly at 3 h and reached the peak at 6 h, which were 9.98-fold (p < 0.01) for cpg-a and 4.92-fold (p < 0.01) for cpg-b of those in pbs group, then they recovered to the original level at 24 h (fig. 7). in the cpg-c group, the ros levels also increased at 6 h, which were 2.22-fold (p < 0.01) compared with those in pbs group (fig. 7). no significant difference was detected among the cpg-p, cpg-n and pbs groups during the whole experimental period with exception of 6 h post injection, at which the ros levels in cpg-p and cpg-n groups were 1.88-fold and 1.92-fold (p < 0.01) of that in pbs group, respectively (fig. 7). discussion cpg odns with unmethylated cpg dinucleotides (cpg motifs) are one type of important immunomodulators which could induce various immune responses. four types of cpg odns with diverse characteristics exhibit different cellular and humoral immune effects in mammals (shirota and klinman, 2014). for example, a and p-type odns can trigger plasmacytoid dendritic cells (pdcs) to mature and secrete ifn-, whereas b-type odns mainly stimulate nfкb-mediated signaling pathway resulting in strong b cell activation (krug et al., 2001; vollmer and krieg, 2009). in our previous studies, tandem odns sequence has been observed to bind lvtolls, activate antiviral associated factors and enhance the survival rates of l. vannamei (zhang et al., 2010; sun et al., 2013; yi et al., 2014), but the exactly effects of a certain type of cpg odns is still not clear. in the present study, four types of cpg odns (cpg-a, cpg-b, cpg-c, and cpg-p) were employed to evaluate their immune enhancement effects. it is widely demonstrated that cpg odns have antiviral effects in multiple vertebrate species. in woodchuck, the combination therapy with cpg odns could induce early antiviral response and enhance the inhibition of viral replication in the hepadnaviral infection (meng et al., 2016). similarly, the antiviral dna vaccine containing multi-copy of cpg fragment showed increased immunogenicity to viral hemorrhagic septicemia virus (vhsv) in fish (martinez-alonso et al., 2011). recently, cpg odn has been applied as an immunopotentiator against pathogens in crustacean aquaculture and the previous studies have revealed that cpg odns could increase the survival rate after wssv injection (zhang et al., 2010; yi et al., 2014). it is generally believed that higher survival rate against virus should be associated with lower viral loads (jang et al., 2009). in the present study, all the four kinds of cpg odns were observed to be able to inhibit wssv copies in shrimps on the third and fifth day after treatment. cpg-a displayed the strongest inhibition on wssv, while cpg-b, cpg-c and cpg-p were less efficient. the results indicated that javascript:void(0); 406 fig. 6 hemocyte phagocytic rate of cpg-a, cpg-b, cpg-c, cpg-p, cpg-n and pbs treated shrimps at 3, 6, 12, 24 h post injection. data presented as mean ± s.d. (n = 3). the significant differences between the treated groups and control groups were subjected to one-way analysis of variance (one-way anova). cpg-a should play an important role in shrimp immune response against the invading of wssv. the biological properties of different types of cpg odn are diverse in mammals, and it is suspected that the difference of anti-wssv effects should be related to their involvements in the activation of shrimp immune system. cpg motifs are considered as one of pamps due to their abundance in microbial genomes and rarity in animal genomes (bauer and wagner, 2002). as a classical type of pamps, cpg odns could be recognized by some conserved tlrs in various species (keestra et al., 2010; kumagai et al., 2008). so far, three tolls (lvtoll1, lvtoll2 and lvtoll3) have been identified from l. vannamei (wang et al., 2012; yang et al., 2007). in the present study, the mrna expression levels of lvtolls in hepatopancreas of shrimps after cpg odns stimulation and the binding affinity between lvtoll-ecds and cpg odns were investigated. after treated with cpg-a and cpg-p, the expression levels of lvtolls were significantly up-regulated, while the treatments of cpg-b and cpg-c could not change the expression of lvtolls significantly. meanwhile, cpg-a exhibited the strongest activity to induce the expression of lvtolls, and the expression of lvtoll1 was highest after cpg-a treatment. it was speculated that the antiviral effects of cpg odns were associated with their promotion of lvtoll expressions. correspondingly, cpg-a exhibited the strongest binding activity towards lvtoll1-ecd in spr assay, which was significantly higher than that towards lvtoll2-ecd and lvtoll3-ecd, indicating that lvtoll1 should be the main receptor of cpg-a in shrimp. furthermore, ecd of lvtoll1 was found to share high identity with that of hstlr9 (56 %), the receptor of cpg odns in human (bauer et al., 2001; sun et al., 2014), providing convincing evidence to our conclusion that lvtoll1 was the receptor of cpg-a. the activation of the tlr signaling pathway by cpg odns could induce a series of cellular immune responses. for instance, the recognition of cpg dna by tlr could activate the expression of genes involved in phagocytosis in mice cells (doyle et al., 2004). as a powerful cellular response, phagocytosis plays a pivotal role in clearance of influenza virus and hepatitis c virus (hcv) in mammals (fujimoto et al., 2000; huber et al., 2001; bang and saito, 2015). similarly, phagocytosis also plays important roles in antiviral immunity in crustaceans (wang and zhang; 2008; liu et al., 2009). the accumulated evidences have proved that cpg odns could induce phagocytic activity in various aquatic animals (tassakka and sakai, 2002; zhao et al., 2016). in the present study, the phagocytic rate was significantly enhanced by cpg-a and cpg-b treatment, providing direct evidence for the resistance to wssv infection of shrimps immunized by cpg odns. it has been suggested that the enhanced phagocytosis might contribute to the partial protective functions of cpg javascript:void(0); 407 fig. 7 intracellular ros level of cpg-a, cpg-b, cpg-c, cpg-p, cpg-n and pbs treated shrimps at 3, 6, 12, 24 h post injection. data presented as mean ± s.d. (n = 3). the significant differences between the treated groups and control groups were subjected to one-way analysis of variance (one-way anova). odns in decreasing the virus load of wssv (zhang et al., 2010). apart from phagocytosis, cpg odns could also increase the ros generation in immune cells. for instance, cpg dna promoted the ros burst in murine b cells, which was linked to the activation of nfкb and then accelerated the leukocyte gene transcription and cytokine secretion (yi et al., 1998). as a product of cellular metabolism, ros is involved in the regulation of viral infection (skulachev, 1998; waris et al., 2005). in human, ros could disrupt the activity of hcv replication complexes in hepatoma cells to inhibit its replication (choi et al., 2004). moreover, ros was demonstrated to be essential in the defense against influenza a virus infection through ifn-related immune responses (kim et al., 2015). in the present study, the intracellular ros level in hemocytes of cpg-a and cpg-b stimulated shrimps increased significantly, which displayed a similar trend with the phagocytic rate. the boosted ros level was previously reported to contribute to the decreased wssv copies in cpg odns pre-treated shrimps (sun et al., 2013). the results suggested that the inhibition cpg-a and cpg-b on wssv might partly result from the increase of cellular immunity including the phagocytic rate and intracellular ros generation. apart from activation of cellular immunity, the recognition of cpg dna or single-stranded rna by pattern recognition receptors (prrs) also promotes humoral immunity including the secretion of proinflammatory cytokines, chemokines and type i ifns, which are the hallmarks of host innate immune system in defending against viral infections in mammals (sadler and williams, 2008; takeuchi and akira, 2009). in vertebrates, cpg dna could be recognized by tlr9 and then activated the downstream ifn regulation factor (irf) to promote the secretion of type i ifn (kumar et al., 2009; takeuchi and akira, 2009), and consequently activated jak/stat pathway via the interaction with the ifn receptor (sadler and williams, 2008). li et al. (2015) reported that shrimp might possess an irf-vago-jak/stat regulatory axis, which was similar to the irf-ifn-jak/stat antiviral axis of vertebrates，indicating that shrimp might possess an ifn system-like antiviral mechanism. the vago gene from arthropods encodes a viral-activated secreted peptide that restricts virus infection through activating the jak-stat pathway and it is considered as a cytokine functionally similar to ifn; as vago has been suggested to be an ifn-like protein and irfs to be conserved from invertebrates to mammals, it could be seen that the shrimp vago system exhibits similarity to the mammalian ifn system (li et al., 2015). in the present study, the temporal expression of ifn system-like related genes (lvirf, lvvago4 and lvstat) were detected after the treatments with different types of cpg odn. the mrna expression of lvirf, lvvago4 and 408 lvstat in hepatopancreas of cpg-a and cpg-p treated shrimps were dramatically up-regulated compared with that in the control groups and other cpg odns groups. moreover, the level of probable ifn in plasma was also increased significantly in cpg-a and cpg-p groups, indicating that cpg-a and cpg-p might promote the secretion of the probable ifn in shrimp. meanwhile, all the above responses were significantly higher in cpg-a group than that in cpg-p group. in vertebrates, type i ifns activate intracellular signaling pathways and regulate the expression of a set of genes, which are involved in eliminating viral components from infected cells and conferring resistance to viral infection on uninfected cells. therefore, the high antiviral effect of cpg-a was possible related to the downstream ifn system-like response. these results indicated that cpg-a might induce the irf-vago-jak/stat pathway in shrimp, and the cpg-mediated antiviral pathway might be evolutionarily conserved in crustacean. in conclusion, cpg-a can stimulate relatively higher antiviral effects in shrimp l. vannamei with effective 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wang l, kong p, qiu l, zhang h, gao y, et al. a gamma-aminobutyrate type a receptor-associated protein involved in the immune response of eriocheir sinensis. int. j. immunogen. 39: 46-54, 2012. 46 isj 18: 46-55, 2021 issn 1824-307x research report the temporal dynamics of bacteria in the coelomic fluid of sea cucumber apostichopus japonicus after evisceration c zhang1, z yu2,3,4,5, z xue2,3,4,5, h li3,4,5, j zhu1, l wang2,3,4,5, l song2,3,4,5* 1school of marine sciences, ningbo university, ningbo 315211, china 2laboratory of marine fisheries science and food production processes, qingdao national laboratory for marine science and technology, qingdao 266235, china 3liaoning key laboratory of marine animal immunology, dalian ocean university, dalian 116023, china 4liaoning key laboratory of marine animal immunology & disease control, dalian ocean university, dalian 116023, china 5dalian key laboratory of aquatic animal disease prevention and control, dalian ocean university, dalian 116023, china this is an open access article published under the cc by license accepted december 29, 2021 abstract sea cucumbers have been emerged as important models to study organ regeneration and development owing to the capacity to regenerate its organs quickly after evisceration. evisceration is a special defense mechanism for sea cucumber to eject all of internal organs when they encounter predators or adverse environmental conditions. however, little was known about the dynamics of bacterial community in coelomic fluid after evisceration. in the present study, evisceration was induced by intracelomic injection of 0.35 m kcl, and the significantly alternation of bacterial community in coelomic fluid of sea cucumber apostichopus japonicus was observed with lower diversity and total bacterial load at 7 dpe (days post evisceration) and 14 dpe. the bacterial community was tended to restore at 28 dpe. in particular, relative abundances of bacteroidetes and rubritaleaceae, which involved in degradation of polysaccharides and lipid, increased significantly at 7 dpe (p < 0.05), and returned to the original level at 28 dpe. in addition, the predicted functions of bacterial community indicated that the bacteria associated with metabolism pathways of amino acid, lipid and carbohydrate also increased significantly at 7 dpe. these results suggested that the bacterial community in coelomic fluid of a. japonicus was highly dynamic and could rebuild a stable community structure after evisceration. it was suggested that the enriched metabolic related beneficial bacteria at early stage played a role after evisceration in terms of decomposing polysaccharides and lipid to provide energy. key words: sea cucumber; evisceration; coelomic fluid; bacterial community introduction evisceration is a special physiological phenomenon in sea cucumbers that all the internal organs, including coelomic fluid, coelomocytes, intestinal tract and respiratory trees can be ejected when they encounter adverse environmental conditions or pathogenic infection (li et al., 2018). the evisceration can be induced artificially and the sea cucumbers display a capacity to regrow body parts and internal organs after evisceration (emson and wilkie, 1980), which is much greater than that of ___________________________________________________________________________ corresponding author: linsheng song liaoning key laboratory of marine animal immunology dalian ocean university, dalian 116023, china e-mail: lshsong@dlou.edu.cn sea stars and sea urchins, making them prime regeneration models (carnevali, 2006). in the past decades, there has been increasing reports on the mechanisms of intestinal regenerative processes in sea cucumbers, including morphological features, cell division, dedifferentiation, cell proliferation and migration, nerve regrowth, and molecular regulation (garcía‐arrarás et al., 1998; dolmatov and ginanova, 2009; sun et al., 2011; sun et al., 2013; li et al., 2017b; zhang et al., 2017). the regeneration of internal organs in sea cucumber is always accompanied with the reconstruction of host’s microbiota, and the quantity and structure of microbial community is suggested to be linked with the development of regenerating intestine (zhang et al., 2020). understanding the dynamics of microbial community after evisceration of sea cucumber is helpful to investigate the interaction 47 fig. 1 the growth curves of v. splendidus after incubation with coelomic fluids of different post-evisceration groups between microbiota and organ development in sea cucumber. accumulating evidence proves that microbiota play important roles in animal survival, homeostasis and development (mcfall-ngai et al., 2013). in mammals, the symbiotic microbiota have been considered to be a key element for intestinal regeneration and hematopoiesis. for example, the microbiota-intestinal stem cells dialog is a key element for intestinal regeneration in human (stedman et al., 2016), and the hematopoiesis can be promoted by microorganisms in mice (khosravi et al., 2014). the microbiota is also essential for organ regeneration in invertebrate. it has been proved that the dramatic change of microbiome in the coelomic of freshwater flatworm schmidtea mediterranea can cause the loss of its regenerative capacity (arnold et al., 2016), and the structure of microbial community was found to be linked with the development of regenerating intestine in a. japonicus (wang et al., 2018; zhang et al., 2019; zhang et al., 2020). however, the information about the changes of bacterial community in coelomic fluid after evisceration is very limited, and the data of coelomic fluid microbiota in sea cucumber are mainly obtained by means of culture-dependent methods. with the rapid development of modern biotechnology, some molecular fingerprinting methods have been applied to provide realistic estimates of community diversity and composition. next-generation sequencing (ngs) is commonly used for the detailed characterization of microbial community composition and dynamics, even for the rare phylotypes. the results of ngs can act as a seed bank to figure out the microbial community response to environmental change, and ngs method has been employed to characterize the bacterial communities in lower invertebrates, including sea cucumber gut microbiome (zhang et al., 2018). previous studies have demonstrated that the host can develop tolerance to microbial community and maintain a mutually beneficial relationship with the microbiota (knoop et al., 2017). adult microbiota are thought to be relatively stable over time, and this stability imparts resilience to various perturbations, ensuring the host’s continued function (koenig et al., 2011). in the present study, high-throughput sequencing of 16s rrna gene was employed to study the temporal dynamics of bacteria in coelomic fluid of a. japonicus after evisceration with the objectives to characterize the changes of bacterial community structure in coelomic fluid after evisceration, and predict the potential roles of coelomic fluid bacterial, which could help for the further evaluation on the roles of bacterial community after evisceration. materials and methods experimental design and sample collection healthy adult sea cucumbers about 100 ± 10 g in weight were obtained from a commercial farm in dalian, liaoning province, and cultured in natural seawater at 10 12 °c under natural illumination. evisceration was induced by intracoelomic injection of 1.5 ml of 0.35 m kcl. previous reports indicated that the lumen formation of the new intestine began at 7 dpe, and the intestine gradually developed to form a complete structure at 14-21 dpe (sun et al., 2011; sun et al., 2013), while the regeneration of coelomocytes was basically completed at 28 dpe (li et al., 2018). nine individuals of a. japonicus were sampled at four time points, including pre-evisceration (used as control group), 7 dpe (days post-evisceration), 14 dpe, and 28 dpe. for coelomic fluid collect, sea cucumber was allowed to stand on paper to desiccate the seawater accumulated in the body. the ventral surface of a. 48 japonicus was opened longitudinally and the coelomic fluid was collected in a petri dish. the coelomic fluid from three individuals was pooled together, and there were three replicates for each time point. the coelomic fluid was filtered through a 300-mesh bolting cloth under sterile conditions to remove turbid impurities, and then transferred to sterile microfuge tubes and stored at -80 °c before genomic dna extraction. antibacterial assay of coelomic fluid at different time post-evisceration the antibacterial activities of coelomic fluids against vibrio splendidus at different time after evisceration were tested as previously reported (jia et al., 2015). v. splendidus was cultured in 2216e liquid medium (5 g/l peptone and 1 g/l yeast extract in seawater). the culture in logarithmic phase was centrifuged, washed by tbs (20 mmol/l tris-hcl, 150 mmol/l nacl, ph 7.5) for three times, and then resuspended in tbs to the final concentration of 104 cfu/ml. fifty microliters of coelomic fluid were mixed with the same volume of v. splendidus resuspension and incubated at room temperature for 1 h. twenty microliters of the mixtures were added into a 96-well microliter plate with 200 μl 2216e culture medium. the plate was placed in a microplate reader (bioteka, usa) at 28 °c with a shake for every five seconds. absorbance value at od600 was measured every 30 minutes for 24 h to detect the growth curve. each group was repeated for three times. dna extraction and amplicon sequencing dna was extracted from 12 whole coelomic fluid samples with e.z.n.a soil dna kit (omega, usa) according to the manufacturer’s protocol. the concentration and purity of dna samples were examined by 1 % agarose gel electrophoresis and a nanodrop nd-2000 spectrometer (thermo, usa). the extracted dna was stored at -80 °c for further sequencing and real-time pcr analysis. the prokaryote--specific primers 515f and 806r (gtgycagcmgccgcggtaa, ggactacnvgggtwtctaa) (kozich et al., 2013) were used to amplify the v4 region of 16s rrna gene. to minimize reaction-level pcr bias, each sample was amplified in triplicate with the following reaction conditions: 25 cycles of denaturation at 95 °c for 30 s, annealing at 55 °c for 30 s and extension at 72 °c for 45 s, with a final extension at 72 °c for 10 min. equimolar amounts of pcr products (assuming that amplicons of the same size had a similar molar mass) from each sample were combined in a single tube for high-throughput sequencing on an illumina miseq platform (illumina, san diego, usa) in novogene company (beijing, china) which produced paired-ended reads. bioinformatics and statistical analyses the raw illuminafastq data were processed using mothur v.1.11.0 (http://www.mothur.org/) to eliminate the low quality and redundant reads. the sequences with ambiguous bases or truncated at any site of more than three consecutive bases receiving a phred quality score (q) < 20 were deleted. operation taxonomic units (otus) were clustered with 97% similarity by usearch (vsesion 7.0) against the silva database (release128) (edgar, 2013). chimeric sequence and a single sequence without duplication were removed (edgar et al., 2011). each 16s rrna gene sequence without chimera was analyzed by the ribosomal database project (rdp) classifier with a 70 % confidence threshold (version 2.2). the rarefaction curve was used to determine the sequencing depth by mothur software. the shannon, species richness and coverage indices were calculated for each sample using the methods of mothur (edgar et al., 2011). community composition barplot was drawn using r script. principal coordinates analysis (pcoa) was performed to evaluate the overall differences in bacterial community structure based on bray-curtis distance (clarke et al., 1993). real-time pcr data were analyzed by statistical package for social sciences (spss) 17.0 software. one-way analysis of variance (anova) was used to test the significant differences among all samples and the differences were considered to be significant at p < 0.05. real-time quantitative pcr to determine the bacterial load in the coelomic fluid of sea cucumbers at different time post-evisceration, absolute quantitative pcr was conducted according to the method previously reported (lu et al., 2017). 16s rrna gene were amplified by rtaq dna polymerase (takara, japan) using primers 27f/1492r (agagtttgatcmtggctcag, ggttaccttgttacgactt) (goodfellow and stackebrandt, 1991). standard plasmid was constructed by inserting the amplicon into pmd18-t vector (takara, japan) and transformed into escherichia coli trans5α (transgen, china). after verified by sequencing, the plasmids were extracted from e. coli with column plasmid preps kit (sangon, china). the concentration of recombinant plasmid was measured by nanodrop nd-1000 (thermo, usa), and the copy number of standard plasmid was calculated according to the method described previously (liu et al., 2016). the standard plasmid templates were diluted with a 1:10 gradient in depc water to the final copy numbers of 103 109. real-time pcr were performed using the serially diluted templates with primers 341f/534r (cctacgggaggcagcag, cgcggctgctggcacgta) (liu et al., 2011), and the threshold cycle (ct) values against the denary logarithms of copy number were recorded and analyzed to establish a standard curve for copy number. samples of genomic dna were adjusted to a final concentration of 10 ng/μl for real-time quantitative pcr. after real-time quantitative pcr detection with the primers 341f/534r, the ct value was recorded and analyzed. the total number of bacteria was calculated based on the standard curve and ct value. results the antibacterial activity of coelomic fluid at different post-evisceration times the antibacterial activity of coelomic fluid against v. splendidus was evaluated by detecting 49 the bacteria growth curve. there was no significant difference of od600 in the first nine hours among the coelomic fluid samples collected at different time points, but the values of od600 for the coelomic fluid at 7dpe and 14 dpe were higher than that at 28 dpe from 10 h to 24 h (fig. 1), indicating that the antibacterial abilities of coelomic fluid decreased firstly at 7 dpe and 14 dpe, and then recovered to the original level at 28 dpe. the abundance of total bacteria in coelomic fluid at different time points post-evisceration the abundance of total bacteria in different samples was examined by real-time quantitative pcr. the average copy number in ctrl group was 5.125 ± 0.76 × 106 copies/ml. after evisceration, the relatively lower copy numbers of bacterial loads were observed at 7 dpe and 14 dpe, which was about 50.7% (2.6 ± 0.5 × 106 copies/ml) and 72.3% (3.74 ± 0.89 × 106 copies/ml) of that in ctrl group, respectively. at 28 dpe, the bacteria load was almost the same as the control group (5.37 ± 0.37 × 106 copies/ml) (fig. 2). the high-throughput sequencing data of v4 region in 16s rrna gene high-throughput sequencing of the v4 region in 16s rrna gene was performed to determine bacterial diversity in coelomic fluid of a. japonicus at different time points post-evisceration. after the quality control of the raw sequences, a total of 901,496 clean tags with an average of 75,124 per sample were obtained. in the rarefaction curves, the number of otus almost reached the plateau phase with the increasing read number at 25,000, suggesting the sufficient sequencing depth in all samples (fig. s1a). after rarefaction to equal sequencing depth, there were 465 otus across all samples (fig. s1b). good’s coverage index was used to estimate the percentage of total bacterial otus in a sample. it was 0.99 in all the 12 samples, indicating the obtained sequences represented the majority of bacteria sequences in all the 12 samples. fig. 2 the 16s rrna gene copy number of the total bacteria by real-time quantitative pcr during different post-evisceration periods. means ± standard deviations were compared using one-way analysis of variance (anova). different lowercase letters indicate significant differences (p < 0.05) among groups bacterial community dissimilarity among the coelomic fluid at the different time points post-evisceration to estimate and compare the diversity of bacterial community among different samples, alphaand beta-diversity of bacterial communities were determined. alpha-diversity was estimated by the calculating shannon index and chao1 index. the shannon index decreased significantly at 7 dpe (0.79-folds to the ctrl group, p < 0.05), then increased slightly at 14 dpe (0.86-folds to the ctrl group, p > 0.05), and returned to the level of ctrl group at 28 dpe (0.92-folds to the ctrl group, p > 0.05) (fig. 3a). chao1 index also declined obviously fig. 3 comparison of shannon diversity (a) and chao1 index (b) among different post-evisceration groups. means ± standard deviations were compared using one-way analysis of variance (anova). * indicates significant differences (p < 0.05) among groups 50 fig. 4 principal coordinates analysis (pcoa) of the dissimilarities of a. japonicus coelomic fluid bacterial community among the four groups based on bray-curtis distance. (b) hierarchical clustering tree based on weighted unifrac distances for sequences derived from a. japonicus for each of the replicate ctrl, 7 dpe, 14 dpe and 28 dpe groups at 7dpe (p < 0.05), but recovered to level of ctrl group at 14 dpe and 28 dpe (fig. 3b). the beta-diversity of bacterial community among all the samples were explored by pcoa analysis (fig. 4a). the bacterial community replicates were clustered separately in different group, and the bacterial community structure at 28 dpe was more closely resembled the control group. the community structure at 7 dpe was more different from that of the control group. these patterns were further corroborated by the hierarchical clustering tree among groups (fig. 4b). taxonomic distribution and phylotypes of coelomic fluid bacterial community at different time points post-evisceration in the present study, 97.7 % of the sequences were classified into 10 phyla. in the ctrl group, the dominant phyla in coelomic fluid were proteobacteria (69.37 ± 1.02 %), followed by firmicutes (10.47 ± 2.15 %), verrucomicrobia (8.47 ± 1.29 %), bacteroidetes and actinobacteria (4.47 ± 1.75 %) (fig. 5a). the taxonomic distribution of coelomic fluid bacterial was companied large change at the phylum level in the ctrl and different post-evisceration periods. the relative abundance of bacteroidetes reached the highest level at 7 dpe (3.2-fold higher than that in ctrl group, p < 0.01), and then went down at 28 dpe, which was 1.6-fold higher (p > 0.05) than that in ctrl group (fig. 5b). in addition, the ratio of bacteroidetes and firmicutes increased significantly at 7 dpe and 14 dpe, which was 17.2-folds (p < 0.01) and 10.4-folds (p < 0.05) compared with ctrl, respectively (fig. 5c), and then decreased at 28 dpe (p > 0.05). significant changes of coelomic fluid bacterial assemblages at the family level after evisceration the bacterial community at family level generates the highest ecological potential to indicate host health status (xiong et al., 2014). in the ctrl group, the dominant family in coelomic fluid was colwelliaceae (21.37 ± 2.14 %), followed by rhodobacteraceae (11.47 ± 3.17 %), enterobacteriaceae (6.47 ± 1.09%), rubritaleaceae (5.42 ± 1.15 %) and vibrionaceae (3.17 ± 1.78 %) (fig. 6a). at 7 dpe, the dominant family in coelomic fluid was rubritaleaceae (17.05 ± 3.15 %), followed by hyphomonadaceae (10.47 ± 3.67 %), enterobacteriaceae (6.47 ± 2.13 %), rhodobacteraceae (5.40 ± 1.18 %) and vibrionaceae (4.17 ± 2.05 %) (fig. 6a). enterobacteriaceae was found to be the dominant family at 14 dpe (20.27 ± 5.16 %) in coelomic fluid, followed by rhodobacteraceae (10.20 ± 2.90 %), rubritaleaceae (8.91 ± 4.15 %), vibrionaceae (6.80 ± 4.10 %) and hyphomonadaceae (4.87 ± 1.24 %) (fig. 6a). at 28 dpe, the most abundant family in coelomic fluid was rhodobacteraceae (25.30 ± 5.12 %), followed by enterobacteriaceae (9.74 ± 1.17 %), rubritaleaceae (5.07 ± 1.29%), colwelliaceae (2.32 ± 1.12 %) and vibrionaceae (1.57 ± 1.71 %) (fig. 6a). specifically, the abundance of rhodobacterales also increased significantly from 14 dpe to 28 dpe (fig. 6a). while the relative abundance of rubritaleaceae increased significantly at 7 dpe (3.42-fold compared with ctrl, p < 0.01), then dropped to the similar level as ctrl group at 28 dpe (fig. 6b). prediction of bacterial community functions at different post-evisceration periods due to the lowest bacterial community diversity and maximum bacterial community structural change at 7 dpe, the functions the bacterial communities before evisceration as well as those at 7 dpe and 28 dpe were predicted by picrust. (langille et al., 2013). the metabolism related pathways were around the top-rated enriched pathways. the abundances of amino acid metabolism, lipid metabolism and carbohydrate 51 fig. 5 comparison of the relative abundances of major bacterial phyla. (a) histogram of species distribution. (b) relative abundance of bacteroidetes in different post-evisceration groups. (c) the ratio of bacteroidetes and firmicutes in different post-evisceration groups. means ± standard deviations were compared using one-way analysis of variance (anova). ** indicates significant differences (p < 0.01) among groups. * indicate significant differences (p < 0.01) among groups metabolism pathways all increased significantly at 7 dpe compared with ctrl group (fig. 7a). compared with that at 7 dpe, the abundance of amino acid metabolism pathway was upregulated while the abundance of carbohydrate metabolism pathway was downregulated, and lipid metabolism pathway showed no significant change at 28 dpe (fig. 7b). the nucleotide metabolism pathway decreased at both 7 dpe and 28 dpe (fig. 7a, b). discussion sea cucumber is considered as a good organism for studying organ regeneration and organogenesis for its ability to regenerate self-eviscerated organs within a few weeks (ortiz-pineda et al., 2009). microbiota exerts either positive or negative influences on the health status of aquatic animals, by symbiotic relationship or causing diseases, respectively (antunes et al., 2010). increasing evidences indicate the important roles of microbiota in organ regeneration of animals (zhang et al., 2020; stedman et al., 2016; khosravi et al., 2014; arnold et al., 2016; zhang et al., 2019; wang et al., 2018). however, the information about the dynamics of coelomic fluid microbiota after evisceration is very limited. in the present study, the bacterial community in coelomic fluid of a. japonicus after evisceration was investigated, and a significant variation was observed during early stage after evisceration. the interaction between microbial community and host is the main factor to maintain the halobiont homeostasis. echinodermata coelomic fluid contains abundant antimicrobial compounds (dybas et al., 1986), which can directly influence the growth of microbes in coelomic fluid. although the regeneration process of coelomocytes has been well described (li et al., 2018)，little was known about the variation of the antimicrobial ability during the process. in the present study, the antibacterial ability of the coelomic fluid was found to decrease firstly at 7 dpe and 14 dpe, and then recovered at 28 dpe. for the bacterial community, the total bacteria load was slightly increased from 7 dpe to 14 dpe and recovered to the original at 28 dpe. these results indicated that the lower antibacterial ability of coelomic fluid at 7 dpe and 14 dpe provided an ideal hotbed for the bacterial population. while the species richness (chao1 index) and diversity (shannon index) of coelomic fluid bacteria were significantly lower at 7 dpe. previous studies revealed a sharp decline in the species richness and diversity of intestinal bacteria in the early stage 52 fig. 6 comparison of the relative abundances of major bacterial family. (a) histogram of species distribution. (b) relative abundance of rubritaleaceae in different post-evisceration groups. ** indicates significant differences (p < 0.01) among groups of intestinal regeneration, and the diversity of intestinal bacteria then increased gradually (zhang et al., 2019). ecological evidence indicated that reduced bacterial amount and diversity would provide vacant niches for invading pathogens (mallon et al., 2015). in the current study, the reduced bacterial diversity and total bacteria load at 7 dpe might indicate the decrease of functional stability of bacterial community, which would increase the risk of developing diseases (xiong et al., 2017). and the higher antibacterial ability at 7 dpe (compared with that at 14 dpe) might contribute to inhibiting the thriving of harmful bacterial species at the early stage after evisceration. the dynamics of bacterial community after evisceration was determined by beta-diversity analysis. both pcoa and hierarchical clustering analyses indicated that the bacterial community structure at 7 dpe was significantly different from the normal level, and it began to restore at 28 dpe. the results were consistent with previous report on the bacterial community in intestine of a. japonicus that the intestinal bacterial community varied significantly and tended to be stable when the regeneration was accomplished (zhang et al., 2019). in the present study, the bacterial community in a. japonicus coelomic fluid was found to be dominated by proteobacteria, following by firmicutes, bacteroidetes and verrucomicrobia, which was consistent with the previous report (enomoto et al., 2012). interestingly, bacteroidetes increased significantly at 7 dpe and 14 dpe, and returned to pre-evisceration level at 28 dpe. previous study showed that the relative abundance of bacteroidetes in a. japonicus intestinal also increased in the middle stage of regeneration and returned to pre-evisceration levels in the end stage (zhang et al., 2019). bacteroidetes can ferment polysaccharides and indigestible carbohydrates to produce short-chain fatty acids (scfas) that beneficial for the host (faintuch and faintuch, 2019). the present results suggested that the bacteria in coelomic fluid might help to provide energy for the host before the intestine was fully grown. in addition, the ratio of bacteroidetes and firmicutes was found to increase significantly at 7 dpe and 14 dpe. it has been reported that that the ratio reduction of bacteroidetes and firmicutes in mouse intestine promoted fat storage (backhed et al., 2004). our result on the contrary indicated that the bacteria might promote the lipolysis, which helped to provide the energy. rubritaleaceae, confirmed to contribute to polysaccharide degradation (martinez-garcia et al., 2012), was also found to increase significantly at 7 dpe. previous study showed the abundance of rhodobacterales in intestinal increased significantly in samples from 14 dpe to 21 dpe in the intestinal regeneration process, and it was predicted to function as keystone taxa in intestinal community (zhang et al., 2019). in deriving germ-free a. japonicus, the abundance of rhodobacteraceae increased to promote the intestine regrowth rate of a. japonicus (zhang et al., 2020). in the present study, the abundance of rhodobacterales in coelomic fluid was also found to increase significantly from 14 dpe to 28 dpe. as rhodobacterales retains polyhydroxybutyrate (phb) metabolism genes to promote the growth of a. japonicus (yamazaki et al., 2016), it is also hypothesized to be a keystone taxa in coelomic fluid after evisceration. it was generally believed that the disruption of bacteria homeostasis can alter bacterial-mediated functions, which in turn affects the growth of animals (xiong et al., 2017). the synchronic changes in microbial composition 53 fig. 7 comparison of a. japonicus coelomic fluid bacterial kegg pathways between ctrl with 7 dpe (days post–evisceration) groups (a), and 7 dpe with 28 dpe groups (b) using the response ratio methods and function have been well demonstrated, especially the metabolic pathways of three major nutrients (catalan et al., 2018). in the present study, the pathways related to amino acid metabolism, lipid metabolism and carbohydrate metabolism were found to be more abundant in 7 dpe. intestine is the main digestive organ, but it has no digestive function until its regeneration is complete. all these results suggested that the coelomic fluid bacteria might decompose polysaccharides and lipid to provide energy after evisceration. in summary, the temporal dynamics of coelomic fluid bacterial community in a. japonicus was investigated after evisceration. the bacterial community structure changed greatly after evisceration and it gradually recovered and tended to be stable at 28 dpe. the coelomic fluid bacterial community was involved in the decomposition of polysaccharides and lipid before the recovery of intestinal digestive function from 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review tuning the host-pathogen relationship through evolution with a special focus on the echinoid sp185/333 system lc smith1, mr coscia2 1department of biological sciences, george washington university, washington dc, usa 2institute of protein biochemistry, national research council of italy, naples, italy accepted november 3, 2016 abstract diversification of immune genes in host organisms that are in deadly arms races with pathogens has resulted in a wide range of approaches by which the host survives. well known examples of adaptive immunity in vertebrates include somatic recombination of the immunoglobulin gene family and assembly of the variable lymphocyte receptors. the crispr-cas system in bacteria and archaea is also considered adaptive. for invertebrates that survive in the absence of adaptive immunity, innate immune diversity is accomplished based on functions of clusters of immune genes such as freps, vcbps, c1qs, tlrs, and r genes. single copy gene diversity can be accomplished through extensive alternative splicing or increases in alleles in populations. the sp185/333 gene family in the purple sea urchin has multiple levels in which to generate immune diversity. these include clustered sp185/333 genes, genomic instability in regions harboring the genes, predicted mrna editing, expression of broad repertoires of sp185/333 proteins, and post translational modifications. the sp185/333 proteins have anti-pathogen activities and an individual recombinant protein can bind to multiple foreign cells and molecular patterns. the underlying characteristics of gene clusters, many with repeats, that are present in unstable genomic regions is common to a number of these examples, and is likely of central importance for organisms that survive solely on innate immunity. key words: ig; tcr; vlr; dscam; freps; vcbp; tlr; fu/hc; r genes; sp185/333 introduction when alice went through the looking glass, she joined the red queen in the queen’s race. during the race when alice complained about running so hard for so long and getting nowhere, the queen said to her, “it takes all the running you can do, to keep in the same place” (carroll, 1871) (fig. 1). although this statement seems as crazy as both wonderland and the red queen appeared to be, the idea has been employed as a hypothesis for evolution. the red queen’s race is analogous to the immunological arms race or coevolution between the diversity of an immune system and its effectiveness in protecting the long-lived host vs. the continuously changing virulence of pathogens that have (most often) much shorter life spans (dawkins and krebs, 1979). this host-pathogen arms race is driven by the need to adapt constantly to survive for ___________________________________________________________________________ corresponding author: l. courtney smith department of biological sciences science and engineering hall suite 6000, 800 22nd st nw w ashington dc 20052 e-mail: csmith@gwu.edu both the host and the pathogen, which in turn shapes the virulence and success of the pathogens as well as the detection and effector mechanisms of the host immune system (haldane, 1949; van valen, 1973). the host-pathogen battle drives molecular evolution and affects all ranges of life including plants and animals and their pathogens, in addition to bacteria and their bacteriophages (sironi et al., 2015). an example of this process in action is illustrated by an elegant study illustrating the experimental evolution of the bacterium pseudomonas fluorescens sbw25 in the presence of its pathogen, bacteriophage φ2 (patterson et al., 2010). the outcome of this arms race demonstrates that evolutionary changes occur at higher rates when the host and pathogen coevolve together, and drives greater genotype diversification in both pseudomonas and the phage compared to the bacteria alone or to the phage after the host cells have been killed by the phage. as expected, the bacteria show greater genetic diversity, and remarkably the phage genes that function in infectivity are also selected to change more rapidly. because the number of genes that are involved both in pathogen virulence and host immunity is finite, 356 fig. 1 alice and the red queen in the red queen’s race. they both run as fast as they can but remain in the same place. illustration by sir john tenniel from “through the looking-glass” (carroll, 1871). different approaches to diversification are incorporated into a few genes for increasing pathogen virulence or are incorporated into host immune genes to gain an advantage over the adversary in the arms race. here, we review a range of examples of how a pathogen or a host changes or increases diversity of genes that function in the arms race. we end by focusing on the immune diversity in the sp185/333 system of the purple sea urchin. pathogen diversification for altered or increased virulence there is a wide range of how microbes generate genomic diversity to alter their phenotype to overcome a host immune system and/or to improve avoidance tactics to hide from host attack (deitsch et al., 2009). microbial pathogens adapt constantly to avoid mechanical clearance, circumvent barriers to invasion, avoid recognition and destruction by the host immune system, and/or alter or derail the host immune system with the aim of overwhelming the host response to result in a successful infection. the success in pathogen variation is measured by fitness or success in infection and proliferation leading to subsequent infection of other hosts. dna recombination is employed by both bacterial and eukaryotic pathogens, and can move coding regions into an area of the genome that allows expression (deitsch et al., 2009). this process can fuse intact genes or fragments of genes to generate chimeric sequences, or can move a promoter near a gene (or vice versa) that does not have a functional promoter, leading to altered expression and/or altered protein sequences. this can change not only whether a gene is expressed but can also change the level of expression. antigenic variation or hypervariability, which includes phase variation, is the expression of a particular antigen or set of antigens sequentially over time that alters the pathogen surface phenotype and results in temporary avoidance of the host immune response until the next surface antigen is expressed (fig. 2). antigenic variation can be accomplished by changes in gene expression in single or multi-copy genes that are members of expanded families. modifications to gene sequences within families can result from gene conversion or recombination among similar genes, and slipped strand mispairing followed by excision and repair (deitsch et al., 2009; oren et al., 2016). variation in expression among genes that are members of an expanded family that have variable sequences is a very effective means for avoiding or “out running” immune recognition by the host. furthermore, changes in gene expression within a gene family may be related to variations in the environment, and success in infecting a host that may be based on which genes within the family are expressed and under what circumstances. an example of this type of immune avoidance is the variable surface glycoprotein (vsg) that changes expression over time on the surface of trypanosome parasites, which are the pathogens that cause african sleeping sickness (horn, 2014) (fig. 2). using a large pool of mosaic vsg genes, the parasite is able to escape host antibody recognition and attack by continuosly changing epitopes on its protective extracellular coat. over time, expression switches to an antigenically different vsg by silencing the initially expressed gene and activating the transcription of a new vsg, or by recombining a vsg from another region in the genome into the active transcription site. 357 fig. 2 vsg gene expression in trypanosome parasites switches over time. this simplified scheme of antigenic phase variation over time in trypanosome infection illustrates changes in the version of vsg proteins (red, green, blue) on the parasite surface that enables it to avoid the immune response temporarily. once the host immune response is directed to the expressed vsg (dotted lines), parasites with a variant vsg expression survive, take over the population, and are the source of the relapsing parastaemia. this figure is modified from (horn, 2014). antigenic variation also occurs in bacteria and is characterized by multiple copies of genes that are slightly different, such as when variation is limited to a specific region of the gene sequence. changes in antigens can be controlled by 1) switch mechanisms that regulate independent expression, 2) the expression of a single gene while other members of the family are silenced, and 3) continuosly changing which gene copy is expressed (finlay and mcfadden, 2006). antigenic variation is also employed by viruses. the accumulation of mutations within viral genes that encode epitopes that are bound by antibodies is a process of antigenic drift followed by antibody-based selection, which gives rise to increased success in viral infectivity and is directly related to antigenic changes to the viruses. this is particularly frequent in the case of rna viruses in which antigenic variation depends mostly on the higher mutational frequency of rna replicases (lauring et al., 2013). the strategies of the human immunodeficiency virus (hiv) to escape the host immune system is a combination of a high mutation rate (3x10-5 nucleotides per viral replication cycle) plus retroviral recombination to alter viral recognition by host cytotoxic t lymphocytes and binding by antibodies (hamelaar, 2012). furthermore, conformational masking and glycan shielding of the viral envelope protein and its unfolding only upon entry into a cell serve to neutralize the host antibody response (kwon et al., 2015). antigenic variations are a good means by which to avoid, at least temporarily, the host immune response and are employed by a wide range of pathogens and parasites. a common feature of genes that function in immune avoidance is the expansion of genes in pathogens that encode effector proteins that disrupt the host immune response. this has been documented repeatedly for bacterial pathogens of higher plants (macho and zipfel, 2015) and is also the means by which shigella infects humans (ashida et al., 2015) and bacillus thuringiensis infects caenorhabditis elegans (schulte et al., 2013). for some plant pathogens and for shigella, microbes inject effector peptides into the host cell cytoplasm that disrupt or derail the innate immune response of the host. similarly, for both bacillus and shigella, the effector peptides block pathogen specific responses in nematodes and humans, respectively, and allow the pathogen to proliferate successfully. in addition to changes in genes encoded in the genome, prokaryotes also carry virulence genes on plasmids. a bacillus pathogen of c. elegans has a plasmid with variable numbers of crystal toxin genes with altered sequence diversity (schulte et al., 2010) in addition to increased gene diversity in the genome (schulte et al., 2013) in response to interactions with the host. genes encoding the effectors and other virulence genes are greatly expanded in the genomes of these pathogens when they are directly involved in the arms race and adapt to the co-adaptations of the host immune system. there is a wide range of means by which horizontal transfer can be used by microbes to change their genotypes and phenotypes that can be beneficial for increased virulence capabilities and proliferation success. mating, or bacterial conjugation, is a key mechanism for the horizontal transfer of dna, including virulence genes, among microbes (de la casa-esperon, 2012; cabazon et al., 2015). transfer is mediated by the formation of the type iv secretion system by gram negative bacteria and mating pair formation results in membrane fusion and the transfer of dna from the donor to recipient cell. this complex is employed in “double duty” activity because it is also used to inject virulence factors into eukaryotic host cells to increase bacterial survival and proliferation. nonmating horizontal or lateral dna transfer is another 358 means for altering the genome of a pathogen (de la casa-esperon, 2012). microbes can acquire dna either from other cells of the same species, or from cells of different species that can modify the means by which the altered microbe survives in the environment, in a host, or both. the outcome of dna acquisition can increase the pathogen diversity and alter the interactions between host and pathogen. natural competence to take up dna from the environment rather than directly from other cells can also transform the capabilities of microbes. horizontal dna transfer is frequent in marine vibrios not only among the cells but also based on the uptake of environmental dna (sun et al., 2013). acquired dna that includes genes encoding proteins with functions that improve fitness to survive a host immune response are likely to be selected for, maintained, and passed on to daughter cells. bacteriophage infection can also result in the acquisition of bacterial dna fragments that have been captured within the phage genome and can be transferred to other bacteria upon subsequent infections. “infections of infections” also occur in the form of mobile genetic elements that use phages for transfer from one bacterial cell to another and can influence the pathogenicity and evolution of microbes (penades et al., 2015). the wide variation and efficiency of microbial diversification in the arms race with their hosts, particularly given their short generation times, have driven the diversity mechanisms in the long-lived multicellular hosts. host immune diversification adaptive immune systems in vertebrates; v(d)j recombination the long-lived (relative to microbial pathogens) host response to constant genomic diversification and virulence changes in pathogens is to diversify the sequences of single copy genes in individual immune cells and/or to expand the copies of a gene followed by diversification of the sequences in the resulting gene family. both approaches have been well documented in animals and the second approach has been particularly well employed in higher plants. gnathosomes diversify their immunoglobulin (ig) family genes, which function in both pathogen detection and response, by somatic recombination of variable, diversity and joining (v(d)j) segments to generate antigen binding sites in the variable exon of the rearranged single copy ig and t cell receptor (tcr) genes (tonegawa, 1983; davis et al., 1984). the enzymes of central importance in this process are the recombinase activating genes (rags) that function in “cutting and pasting” segments of the ig gene family into a functional exon (teng and schatz, 2015) and the activation induced deaminase (aid) gene that is involved in class switching and affinity maturation of ig genes (muramatsu et al., 2000). however, other variations in ig family proteins are known that are not involved in v(d)j recombination and show sequence diversity in the constant region. for example, the hinge region in igm from an antarctic fish (trematomus bernacchii) is unique relative to the hinge regions of all other vertebrate ig isotypes because it is much longer, is encoded by a 3′ extension of the cµ2 exon, and is highly polymorphic fig. 3 the origins of the adaptive immune system in vertebrates inferred from the tm regions of immune receptors. preliminary evidence of a common origin is derived from the phylogenetic relationships among agnathan vlrs, gnathostome igμ, tcr β and δ chains based on the consensus of their transmembrane region nucleotide sequences. sequences were aligned by clustalx (ver. 1.8) and the phylogenetic tree was constructed by the neighbor-joining method with 100 bootstrap iterations. positions with gaps were excluded from the alignment used to generate the tree. among individuals in the population (coscia et al., 2012). because a longer hinge region contributes to greater flexibility of the antibody binding sites, this may improve antigen binding capacity and effectiveness for igm in this fish. changes in the size of the hinge region may result from variations in the splice sites that alter the mrna, or may be the result of non-synonymous substitutions at several specific sites that are under purifying selection compared to most other sites. consequently, because the polymorphism mostly affects the hinge, which is long enough to be solvent exposed, it may protect this region from cleavage by pathogen proteases, such as those of the nematode parasites that cause a high incidence of infections in antarctic fish (coscia and oreste, 1998; coscia et al., 2000). in addition to differences among antibodies outside of the variable regions, there are also conserved features in antibodies that are present across a wide range of vertebrates. one of these features is the motif fxxxfxxs/txxxs (x; any amino acid) in the ig transmembrane (tm) region that can be considered a universal attribute of ig molecules, and likely play a significant role in their function (varriale et al., 2010). interestingly, a 72 nucleotide sequence encoding the tm region includes 13 amino acids under purifying selection and show some identity with the tm regions from agnathan variable lymphocyte receptor (vlr)a, vlrb, vlrc (see below) (pancer et al., 2005), and murine tcr β and δ chains (coscia and oreste, unpublished) (fig. 3). nearly half of the positions in the vlr tm region are conserved including the 72 nucleotides. although vlrs are phylogenetically unrelated to igs, both classical antibodies from the gnathostomes and vlrs from agnathans show regions of diversity within the assembled or copied antigen binding regions, in addition to regions outside of the antigen binding sites. furthermore, certain regions are conserved likely because of essential functions perhaps including interactions with tm regions of other proteins on the lymphocyte surface. thus, the presence of a shared sequence 359 supports the notion that a common ancestor of the jawed and jawless vertebrates likely had both humoral and cellular based adaptive immunity. the apparent immunological “big bang” of adaptive immunity (bernstein et al., 1996) and the sudden appearance of somatic recombination of the ig gene family in higher vertebrates was thought to have arisen by a horizontal transfer of a transposon encoding recombinase, which was inserted into an ancestral ig superfamily gene during the early evolution of jawed vertebrates (bernstein et al., 1996; agrawal et al., 1998; hiom et al., 1998; schluter et al., 1999; flajnik, 2004). however, protorag genes with similarities to vertebrate rags and transib transposases with putative “cut and paste” activity have been found in a range of invertebrates including the purple sea urchin (strongylocentrotus purpuratus), hydra (hydra magnipapillata), a sea anemone (nematostella vectensis), and amphioxus (branchiostoma floridae) in addition to related transib transposons identified in the fruit fly (drosophila melanogaster) and the mosquito (anopheles gambiae) (reviewed in (fugmann, 2010). although these invertebrate gene sequences show similarities to protorag-like sequences, most are not affiliated with terminal inverted repeats, which are typical of transposases and are associated with binding and cutting activity. however, functional analysis of a transib transposase from the corn earworm (helicoverpa zea) demonstrated dna cleavage and ligation that is similar to vertebrate rag1 (hencken et al., 2012). in the purple sea urchin (s. purpuratus), the rag1 protein homologue, sprag1-like, associates with rag2 homologues including sprag2-like from the sea urchin and rag2 from the bull shark, and it also binds to the mouse one-turn recombination signal sequence (fugmann et al., 2006). furthermore, when associated with rag2, sprag1-like supports low levels of v(d)j recombination in vitro (carmona et al., 2016). the protorag genes in amphioxus, branchiostoma belcheri, are linked and oriented “head to head” as in other animals, are associated with terminal inverted repeats (unlike the sea urchin spragl genes), and support dna cleavage, transposition, and the ligation of terminal inverted repeats, albeit at low effeciency, which is similar to signal joint ligation in vertebrates (huang et al., 2016). the identification of rag gene pairs in basal, extant deuterostomes in addition to other invertebrates with transib transposase genes indicates that the origins of the rag enzyme mediated adaptive immune system in higher vertebrates may have resulted from vertical inheritance of the ancestral protorag transposases in invertebrates rather than by lateral transfer from a microbe to a basal vertebrate (fugmann, 2010). assembly of variable lymphocyte receptors the jawless vertebrates, hagfish and lampreys, have an alternative adaptive immune system that uses a completely different mechanism to diversify the vlr genes, which function as antibodies and tcrs in these fish (pancer et al., 2005; boehm et al., 2012). there are three vlr genes that encode vlra, vlrb and vlrc that have functions similar to αβtcr, ig, and γδtcr, respectively (reviewed in (flajnik, 2014) (table 1). the vlr genes are assembled in a “copy choice” or gene conversionlike mechanism that randomly selects leucine rich repeats (lrr) from flanking cassettes of lrr segments and copies them into and replaces the non-coding region of the single copy, incomplete, germline vlr gene (nagawa et al., 2007; rogozin et al., 2007; boehm et al., 2012). the assembly employs short stretches of nucleotides that match between different lrrs, which can be located at any place within an individual lrr. the assembly activity takes place in the thymoid regions at the tips of the gill filaments for vlra and vlrc (bajoghli et al., 2011) and in the hematopoietic regions of the typhlosole and kidney for vlrb genes (reviewed in boehm et al., 2012; kasahara, 2015). unlike the ig system in higher vertebrates, vlr gene assembly does not use rag encoded recombinases, but is mediated by two cytidine deaminases (cdas) in the lamprey (guo et al., 2009). expression of the cda1 gene is restricted to the tips of the gill filaments in association with vlra and vlrc expression, whereas expression of cda2 is associated with vlrb expression in the typhlosole (bajoghli et al., 2011). although the assembly results in a combinatorial set of full length lrrs in vlr genes that appears to maintain the reading frame (nagawa et al., 2007; rogozin et al., 2007), apoptotic lymphocytes are detected and selection of vlra and vlrc is suggested based on non-random numbers of lrrs (sutoh and kasahara, 2014). the astounding parallels between the ig/tcr system and the vlr system suggest the possibility of common origins of antigen diversification and mechanisms of lymphocyte functions in the jawed and jawless vertebrates that predates rag activities (table 1). table 1 parallels in the antigen receptors and types of lymphocytes in jawless and jawed vertebrates1 jawless vertebrates jawed vertebrates antigen receptor cell type antigen receptor cell type vlra on the surface of t-like lymphocytes αβtcr on the surface of αβ t lymphocytes vlrc on the surface of t-like lymphocytes γδtcr on the surface of γδ t lymphocytes vlrb on the surface of and secreted from blike lymphocytes bcr on the surface or and secreted from b lymphocytes 1modified from kasahara and sutoh (2014). 360 fig. 4 crispr-cas, the adaptive immune system in prokaryotes. the crispr locus is composed of crisprassociated (cas) genes followed by a start transcription site (bent arrow), a leader (l) and a series of repeats (black triangles) separated by spacers (rectangles of various colors). upon infection with a bacteriophage (shown at the top), a section of the phage dna (green) is incorporated as a new spacer at the beginning of the crispr array. upon subsequent infection by a phage with dna sequence that is the same as the spacer, the expression of the crispr (cr)rna that includes the “green” spacer results in a sequence match to the dna of the second phage infection and results in cleavage and destruction of the invading dna. this figure is from (barrangou and marraffini, 2014) and reprinted from molecular cell with permission from elsevier. crispr-cas adaptive immunity in prokaryotes a third version of adaptive immunity mediated by clustered regularly interspersed repeats (crispr) has been identified in about 50% of bacteria and most of the archaea (sternberg et al., 2016). the crispr system functions to protect bacteria from bacteriophage infection and lysis using activities with similarities to rna interference in higher animals and plants. the crispr region of the bacterial genome acquires and incorporates short stretches of bacteriophage dna called spacers, which act as a mechanism to recognize bacteriophage dna in subsequent infections (fig. 4). this sequence similarity is employed to cleave the foreign dna and rna using the crispr associated (cas) proteins (jiang and doudna, 2015; marraffini, 2015). although there are a range of variations in crispr systems in different bacterial and archaeal species, in general it is the prokaryote adaptive immune system that is effective in protection against bacteriophages and other foreign dna such as mobile genetic elements (barrangou and marraffini, 2014). because the spacers are incorporated into the genome, immunity to specific phages is heritable and adaptive, although it is lamarckian rather than darwinian and is not anticipatory. although it was long believed that higher vertebrates were the only group to function with adaptive immunity, the vlr and crispr/cas examples demonstrate that there are multiple mechanisms for adaptive self protection against the diversity of pathogens. diversity of innate immunity in invertebrates drosophila melanogaster most animals and all plants survive solely on their innate immune functions. drosophila melanogaster was one of the first model organisms that lent itself to analysis because of its well understood genetics, short life cycle, and easily modified genes for functional analysis to understand how the immune system responds to pathogenic threats and injury. there are four major immune signaling pathways in drosophila which are toll, imd, jak/stat and jnk (stokes et al., 2015; huang et al., 2016) that enable the fruit fly to translate the detection of pathogens to the production of anti-microbial peptides (amps) that control pathogens. the same signaling pathways are present in the common house fly, musca domestica, with similar numbers of genes encoding proteins that function in the signaling pathways 361 (armitage et al., 2015). however, compared to drosophila the house fly genome shows a great expansion in the numbers of immune genes encoding recognition and effector proteins with increased sequence diversity. clearly, the difference in life histories between the fruit fly and the house fly living on and eating fruit vs. the well-known preferred habitats of the house fly on which it walks, eats, and lays eggs is reflected in the diversity of the immune system in the house fly. for any given organism, the activities and diversity of immune function is the outcome of co-evolution with the types and diversity of the pathogens with which it shares the habitat, and its ability to keep pace with rapidly evolving microbes (reviewed in (ghosh et al., 2011). dscam in arthropods one aspect of the drosophila immune system, in addition to many other members of the arthropod phylum, is the gene encoding the down syndrome cell adhesion molecule (dscam). dscam is present in more advanced arthropods as a single copy gene with duplications of specific exons (fig. 5), or as a multicopy gene family in more basal arthropods that do not show exon expansions (armitage et al., 2015; brites and du pasquier, 2015). dscam proteins in vertebrates and arthropods have 10 ig domains and six fibronectin domains in the extracellular region, a transmembrane region, and a cytoplasmic tail that includes signaling motifs (schmucker and chen, 2009; ng et al., 2014). the single copy dscam gene in insects generates significant sequence diversity in the encoded proteins (armitage et al., 2015). diversity is based on extensive expansion of exons that encode the 5′ portions of ig domains 2 and 3 and all of ig 7, which show mutually exclusive alternative splicing of the duplicated exons in the primary transcripts that encode each of these ig domains (fig. 5). mutually exclusive alternative splicing of duplicated exons is based on insufficient distance between two duplicated exons, incompatible splicing signals located in regions between different sets of exons, secondary stem/loop structure of the dscam mrna, and trans-acting factors that regulate splicing (park et al., 2004; kreahling and graveley, 2005; schmucker and chen, 2009; hemani and soller, 2012; spoel and dong, 2012). the transcriptional outcome for this single copy gene is a theoretical 38,016 different proteins in drosophila, about half of which are expressed in neuronal tissue and function in axon guidance, while the rest are expressed in hemocytes (schmucker et al., 2000; watson et al., 2005; schmucker and chen, 2009). the resulting sequence diversity in the three ig domains defines the shape of the extracellular region that is based on interactions among the ig domains, and influences interactions with particular binding targets. the dscam response in mosquito and shrimp immunity in response to pathogen challenge is to mount the dscam proteins on the hemocyte surface and to secrete or release dscam proteins into the hemolymph (deitsch et al., 2009; dong and dimopoulos, 2009; ng et al., 2014). remarkably, the versions of dscam that are present in the hemolymph and on hemocytes after challenge show elevated specificity towards the infecting pathogen and function as opsonins to induce phagocytosis, clearance, and host protection, all of which lead to survival (dong et al., 2006; hung et al., 2013). dscam appears to be a major means by which the arthropods generate immune sequence diversity in response to pathogens. overall, there are several means for generating sequence diversity in essentially single copy or very small families of genes in animals. these include somatic recombination of duplicated gene segments for ig and tcr genes, copy choice or gene conversion assembly of duplicated lrr cassettes in the vlr genes, and extensive alternative splicing of duplicated exons in dscam genes in arthropods. fig. 5 the dscam gene structure and alternative splicing. the exons in the dscam gene in drosophila that encode the n-terminal portions of ig domains 2 (red) and 3 (blue), plus all of ig domain 7 (green) are duplicated in tandem in the gene (top). in addition, the exon encoding the transmembrane region (yellow) is duplicated. the duplicated exons undergo alternative splicing in the mrna (middle), to produce a highly diversified set of proteins (bottom) that are expressed in neurons and hemocytes. this figure is from (schmucker et al., 2000) and reprinted from cell with permission from elsevier. 362 fig. 6 diversity in the frep gene families and the encoded proteins. the frep genes (on the left) have one to seven exons and encode proteins (on the right) with one or two ig superfamily (igsf) domains or proteins with only the fibrinogen (fbg) domain, although some include the interceding region (icr). protein families listed to the right and are underlined produce mrnas that are alternatively spliced. color coding in the genes and the proteins is defined in the legend below; epidermal growth factor domain, egf. this figure is from (hanington and zhang, 2011) with minor modifications and is reprinted from the journal of innate immunity with permission from karger publishers. freps in gastropods another approach for generating sequence diversity in immune systems is through gene duplication that generates clustered families of similar genes followed by sequence variation among gene family members, which is driven by pathogen pressure. one example is the freshwater gastropod mollusc, biomphalaria glabrata, that is an intermediate host for trematode parasites and serves as a vector for terminal infection in birds or mammals including humans (gordy et al., 2015; macho and zipfel, 2015). snails have 14 families of fibrinogen related protein (frep) genes that are expressed in response to infection and result in wide arrays of freps in their hemolymph (adema et al., 1997). freps are composed of a signal sequence, one or two ig domains, an interceding region of variable length and sequence, and a conserved fibrinogen related domain (gordy et al., 2015) (fig. 6). the gene structure shows great diversity including variations in the ig exons, changes to the interceding domain exons, deletion of the fibrinogen domain exon, or the absence of all exons except for the fibrinogen domain exon (fig. 6). in addition to variations in the structure of the genes, frep2, 3, 4, and 12 gene families in the germ line dna of b. glabrata appear to be expanded and diversified in hemocyte dna (gordy et al., 2015). for example, the estimate for the number of germ line frep13 genes is about four, whereas the number of different sequences of the first ig coding region in hemocyte dna is increased by about 10 fold and may be expanded by somatic recombination (loker et al., 2004; zhang et al., 2004; hanington et al., 2010). the frep gene families are interesting examples of small gene families that are expanded and diversified in the snail hemocytes (adema, 2015), however the mechanism by which this occurs is not known. the freps highlight the concept that somatic diversification of genes encoding innate immune molecules can occur in (at least some) invertebrates, and therefore is not exclusive to vertebrates. 363 immune diversity from expanded gene families and/or from expanded alleles per locus variable region-chitin binding proteins a family of innate immune receptors, the variable region-containing chitin-binding proteins (vcbps), are present in the marine protochordates, branchiostoma floridae and ciona intestinalis, and encode proteins composed of a leader, two tandem ig v-type domains, and a chitin-binding domain (cannon et al., 2002, 2004; dishaw et al., 2010, 2011); reviewed in liberti et al. (2015). there are five vcbp genes (a-d) in c. intestinalis, that have little sequence diversity, however, two of the five genes in b. floridae, bfvcbp2 and bfvcbp5, show significant sequence diversity relative to the other three b. floridae genes and all of the vcbp genes in c. intestinalis. the diversity for bfvcbp2 and bfvcbp5 is based on hundreds of alleles in the population in which most of the diversity is in the nterminal ig v-type domain. however, there are also indels in non-coding regions and inverted repeats that may influence genomic instability in the region of these loci (dishaw et al., 2010). instability may drive sequence diversity that result from gene duplications, gene conversion, recombination and unequal crossing-over that would lead to significant diversity in allelic sequences in b. floridae populations. the v-type domains are likely the site of bacterial binding because both a full-length vcbp protein and a recombinant protein missing the chitin binding domain show similar binding characteristics (dishaw et al., 2011). none of the four vcbp genes in c. intestinalis nor three of the vcbp genes in b. floridae (bfvcbp1, 3, and 4) show sequence diversity, suggesting that each recognizes stable non-self molecules. vcpb proteins are variably expressed in association with the stomach and intestinal epithelium, are expressed by celomocytes, and appear to mediate immune activities (dishaw et al., 2011; liberti et al., 2014). interactions may result in gastrointestinal homeostasis between host and commensal gut microbes in addition to protection from pathogenic invasion. the vcbp gene family in the protochordates provides an example of immune detection genes that are both conserved without sequence diversity and others that show significant diversity. expanded gene families in plants and animals in many cases, genes encoding immune detection and response proteins are simply expanded in the genome of the host organism. this has been evaluated extensively for the variety of disease resistance (r) genes in higher plants (spoel and dong, 2012), and may in part be an outcome of shared sequences among r genes that encode the lrr region of the proteins that may drive recombination and meiotic mispairing (mcdowell and simon, 2008; joshi and nayak, 2013; oren et al., 2016) in addition to ectopic duplications and gene recombination (meyers et al., 2003; kuang et al., 2004; smith and hulbert, 2005). diversification of r genes also appears to be induced by contact with pathogens that activates mechanisms for nonhomologous recombination among the r genes to diversify the sequences with potential to create new recognition capabilities for r proteins in progeny of the infected plant (durrant et al., 2007). in addition to plants, significantly expanded gene copy numbers have also been identified in the genome of the pacific oyster, crassostrea gigas, particularly for complement homologues encoding c1q, fibrinogen domain containing proteins, and c-type lectin domain-containing proteins (zhang et al., 2015). gene family expansions for the tlr genes have been documented in sea urchins, b. floridae, and the annelid, capitella capitata (reviewed in (buckley and rast, 2012) (davidson et al., 2008)) in addition to expansions in a variety of other genes encoding pattern recognition receptors in a wide range or organisms (reviewed in (buckley and rast, 2015). sequence diversity of single copy genes in addition to expansions in the number of members of gene families, some immune genes in invertebrates are present in only a few copies or are single copy yet have significant sequence diversity, which is based on large numbers of alleles with slightly different sequences in the population. one example is the set of linked genes in the fusion/histocompatibility (fu/hc) locus in the compound tunicate, botryllus schlosseri. the fu/hc locus is composed of single copy genes that function in allograft recognition and rejection or fusion among individuals, which is an important life history trait for b. schlosseri. natural allograft reactions occur commonly when these sessile invertebrates grow into contact with one another resulting in either fusion of their vasculature or rejection and tissue separation (oka and watanabe, 1957; scofield et al., 1982). the responses to nonself are controlled by 1) two, very tightly linked candidate fuhc (cfuhc) genes that encode secreted and transmembrane fu/hc proteins, 2) fester and uncle fester that encode putative receptors in nonself detection, 3) hsp40-l that encodes a chaperone protein, and 4) the botryllus histocompatibility factor (bhf) that shows lower allelic polymorphism, encodes a cytoplasmic protein that is partially disordered and is associated with the cytoplasmic face of the plasma membrane (fig. 7) (voskoboynic fig. 7 the fu/hc locus in botryllus schlosseri. the fu/hc locus spans about 305 kb and the orientations and distances between the genes are indicated including the tight linkage between cfuhcsec and cfuhctm. additional genes are present in this region including nine genes that match to homologues in other organisms with known function and seven with unknown function. this figure is modified from (taketa and de tomaso, 2015). 364 et al., 2013, taketa et al., 2015; reviewed by (taketa and de tomaso, 2015)). diversity of the polymorphic genes in the fu/hc locus is based on up to hundreds of alleles in the population, of which the most polymorphic are the two cfuhc genes and fester, plus the hsp40-l region that encodes the cterminus of the protein. diversity for fester and uncle fester proteins can also be increased through alternative splicing. the examples of immune genes described above for both vertebrates and invertebrates illustrate multiple ways in which to achieve sequence diversity at immune loci, either through the assembly or copying of gene segments into a non-functional single copy gene rendering it functional, through the expansion of genes into clustered families of similar genes, through the presence of many alleles for single copy genes in a population, and/or through extensive alternative splicing. the sp185/333 system in echinoids sp185/333 mrnas the variety of highly variable gene families has shown that different groups of organisms often use different genes with different diversification mechanisms to attain the same biological outcome of host protection from pathogens (raftos and raison, 2008; ghosh et al., 2011). in the purple sea urchin, strongylocentrotus purpuratus, for which the genome sequence is available (sodergren et al., 2006), the numbers of immune genes and immune gene families with expanded members illustrate a surprising level of complexity for immune defense in this invertebrate (hibino et al., 2006; rast et al., 2006). significant expansions are present in the tlr family (buckley and rast, 2012), the nod and nalp families (hibino et al., 2006; rast et al., 2006; buckley and rast, 2015), the genes encoding scavenger receptors with cysteine rich domain (pancer, 2000; pancer, 2001), and small c-type lectin genes (sodergren et al., 2006). overall, the set of immune genes identified as homologues of those known from vertebrates indicates that the echinoid immune system is quite sophisticated (hibino et al., 2006; rast et al., 2006). another immune response gene family are the sp185/333 genes that are strongly upregulated in adult coelomocytes and larval blastocelar cells in response to challenge with marine bacteria and lipopolysaccharide (lps) (rast et al., 2000; nair et al., 2005; ho et al., 2016). the sp185/333 sequences identified from a cdna library screen of immune activated celomocytes using a subtracted probe (nair et al., 2005) matched to only two sequences in genbank; est333 [(accession number; r62081 (smith et al., 1996)] and dd185 [accession number; af228877 (rast et al., 2000)]. these two matches are the basis of a combined name for the gene family, sp185/333, because the sequence provides no prediction for putative function. the paradox of the result is that many of the partial cdna sequences do not match to each other, however, once alignments were undertaken by hand, patterns of recognizable short regions of sequence emerged called elements (fig. 8a) (terwilliger et al., 2006, 2007). although this appears reminiscent of alternative splicing as in dscam from arthropods from sets of duplicated exons (ng et al., 2014; brites and du pasquier, 2015), the sp185/333 genes are small with only two exons, which rules out the possibility of alternative splicing as a mechanism for sequence diversity (terwilliger et al., 2006). careful alignments of genes amplified from three sea urchins show that the element structure of the cdnas is entirely encoded by the second exon (fig. 8a) (buckley and smith, 2007). element patterns result from the mosaic of elements that are variably present and absent in the second exon, which impart significant sequence diversity to the mrnas and deduced proteins. although, individual elements are recognizable based on length and sequence, the same element from different genes may have slightly different sequences that adds to the diversity of genes and mrnas with the same element pattern. analysis of 121 unique sp185/333 genes from three sea urchins shows that none share sequence among animals, although sequences of some element are shared among genes. in addition to elements, the second exon has six types of repeats that are present as two to four imperfect tandem repeats in the 5′ end of the second exon plus interspersed repeats at the 3′ end (fig. 8a) (buckley et al., 2008a). computational predictions for the origins of the tandem repeats suggest three ancestral versions that underwent duplications, recombinations and deletions to generate the current structure of the 5′ end of the second exon. these characteristics that impart significant sequence diversity within the gene family plus increased expression in response to immunological challenge strongly suggest that these genes function in the immune response of the purple sea urchin (smith, 2012). it is generally assumed that transcription is a high fidelity process producing mrnas that reflect exactly the sequence of the coding regions in the genes that has likely been optimized by selection. consequently, the identification of early stop codons and missense sequences in the sp185/333 cdnas were assumed to be the result of pseudogene expression (terwilliger et al., 2007). however, comparisons among the mrnas and genes from each of three sea urchins show, very few identical matches (buckley et al., 2008b). the gene sequences show that pseudogenes (based on coding regions) are not present in the genome (buckley and smith, 2007) and that the early stop codons and frame shifts are only observed in the mrnas. alignments of genes and mrnas with the same element pattern from the same sea urchin show that most of the differences are in positions of cytidine in the genes that corresponded to uracil in the mrnas (buckley et al., 2008b). speculations on the basis for these changes include mrna editing by a cytidine deaminase for the c to u changes, and perhaps polymerase (pol)µ for other nucleotide changes. both cytidine deaminases and polµ genes are present in the sea urchin genome. it is noteworthy that the most common cdna sequence element pattern is e2, which is edited at the same nucleotide in many cdna sequences to generate a truncated protein (fig. 8a) (buckley et al., 2008b). 365 fig. 8 diversity of the sp185/333 system in sea urchins. a) the sp85/333 mrnas are encoded by the two exons in the genes that code for the leader (l) and the mature protein. the mrnas are composed of a mosaic of blocks of sequence called elements that are shown as colored rectangles in this cartoon alignment. the combination of elements defines the element pattern that is named according to the distinctive and highly diverse sequence of element 15 (terwilliger et al., 2006). element 25 has three versions that are defined by the position of the stop codon. a very common edit site for mrnas is in element 12 of the e2 element pattern (indicated) and changes a codon to a stop resulting in truncated proteins that are missing the his-rich region. repeats are shown at the bottom and correspond to the locations of imperfect tandem repeats towards the 5ʹ end of the second exon and the imperfect interspersed repeats in the 3ʹ half of the second exon. this figure is modified from (buckley and smith, 2007). b) the gene family locus structure has tightly clustered genes within 34 kb that is positioned 6.1 kb from the end of the bacterial artificial chromosome (bac) clone insert. distances between the genes are indicated below. each gene is surrounded by ga short tandem repeats (strs) and segmental duplications that include three d1 genes (yellow, green, blue) of nearly identical sequence are surrounded by gat strs. the size of the segmental duplications are indicated by brackets above. this figure is modified from (miller et al., 2010). c) the standard sp185/333 protein structure has an n-terminal leader (red), a gly-rich region (orange), a his-rich region (blue) and a c-terminal region (gray). the recombinant fragments employed for binding analyses are indicated below. this figure is modified from (smith and lun, 2016). 366 not only does this appear to be non-random, but edited e2 messages encoding truncated proteins are more commonly present prior to immune challenge, whereas after challenge, the preponderance of mrnas encoding full-length proteins increases (terwilliger et al., 2007; sherman et al., 2015). given the possibility of mrna editing, the set of mrnas expressed from a single gene may encode a set of similar but non-identical sp185/333 proteins. in general, the diversity of the sp185/333 gene family generates diverse mrnas that increase diversity by mrna editing to produce a broad array of sp185/333 proteins. the sp185/333 gene family the sp185/333 gene family structure shows tightly clustered genes that are spaced apart by 3 to 12 kb (miller et al., 2010). one cluster of six genes is present within 34 kb, with the peripheral genes oriented opposite relative to the internal genes (fig. 8b). all genes are surrounded by ga short tandem repeats (strs), or microsatellites, and the regions between the strs, which includes the genes and flanking regions of the genes, show increased sequence conservation compared to regions that are distal to the ga strs (miller et al., 2010). this has been interpreted as a result of gene duplication that is mediated by the ga strs. sequence conservation among regions bounded by the ga strs is also consistent with gene conversion in which sequence exchange is initiated by short regions of shared sequences within the coding regions of the genes and is terminated at the ga strs. this type of sequence exchange may be similar to observations reported in yeast (gendrel et al., 2000). the beneficial outcome of limited gene conversion would be sequence diversification of the genes while blocking sequence homogenization of entire gene clusters (miller et al., 2010). in addition to ga strs surrounding the genes, gat strs are positioned at the edges of three segmental duplications that are located in tandem, are of identical size, nearly identical sequence, and include duplicated genes of the d1 element pattern with almost identical sequence (fig. 8b). the level of sequence identity suggests very recent gat str-mediated duplications within the sp185/333 gene cluster, which correlates with d1 genes being the most common type based on random gene sequencing from three sea urchins (buckley and smith, 2007). overall, the basis for sp185/333 gene sequence diversity is likely based on 1) shared blocks of sequences among genes, 2) the presence of strs within the sp185/333 gene cluster, and 3) tight clustering of the genes, all of which may drive gene conversion, duplication, deletion and recombination (miller et al., 2010; oren et al., 2016). this complex family structure with a variety of repeats is very likely the basis for the poor assembly of the sp185/333 gene family in the sea urchin genome sequence in which only six genes are present in a single cluster when 50 ± 10 genes per genome have been estimated (smith, 2012). the sp185/333 proteins sequence diversity is a characteristic of the sp185/333 gene family, which appears to be increased by mrna editing, and infers great sp185/333 protein diversity. the edited mrnas encoding missense sequence has been suggested as another means for increasing protein diversity (smith, 2012) as missense amino acids in sp185/333 proteins are present in the sea urchin coelomic fluid (cf) as confirmed by mass spectrometry (dheilly et al., 2009). the sp185/333 proteins from the cf appear much larger than expected relative to sizes predicted from the cdna sequences suggesting the possibility of multimerization (brockton et al., 2008). this outcome is also observed for a recombinant sp185/333 protein that appears as monomers, dimers and multimers. sp185/333 proteins from all sea urchins show multimers, and the repertoires among individual sea urchins are quite different (brockton et al., 2008; dheilly et al., 2009; sherman et al., 2015). when sp185/333 proteins are evaluated by two dimensional (2d) western blots, significant diversity is noted, and most of the proteins migrate to the acidic region during isoelectric focusing (dheilly et al., 2009) (fig. 9a). however, the presence of many histidines in the cterminal region of the deduced protein sequences (see fig. 8c) (terwilliger et al., 2006) allows isolation of full-length sp185/333 proteins from the cf of sea urchins by nickel affinity (sherman et al., 2015; lun et al., 2016). consistent with the presence of multiple histidines, most of these nickelisolated proteins are present in the basic region of a 2d western blot (sherman et al., 2015) (fig. 9b). when arrays of sp185/333 proteins are compared among sea urchins both before and after challenges with a variety of microbes, there are no similarities among the arrays either among animals in response to the same microbe or in an individual animal responding to the same microbe over multiple challenges (sherman et al., 2015). clearly, the diversity of these proteins is very broad within sea urchins and the diversity within the population of sea urchins must be astounding. the characteristics of the sp185/333 immune response system in sea urchins are based on a number of attributes (smith, 2012). 1) the differences among the genes in different individual sea urchins (buckley and smith, 2007), the structure of the gene family with shared sequences among genes, their tight linkage, and the association with strs, is likely to promote swift sequence diversity among the genes (miller et al., 2010; oren et al., 2016). 2) mrna editing (buckley et al., 2008b) increases the sequence diversity of the proteins beyond the diversity encoded in the genes and generates truncated and missense proteins that appear in the cf (dheilly et al., 2009; smith, 2012). these truncated proteins may be functional given their propensity to be produced prior to immune challenge (sherman et al., 2015). 3) the arrays of sp185/333 proteins are very different among individual sea urchins, and show much greater diversity than expected (dheilly et al., 2009; sherman et al., 2015). the great variety of proteins may be due in part to post translational modifications that have been predicted from conserved sequence motifs for glycosylation (terwilliger et al., 2006; sherman et al., 2015). in 367 general, these results suggest that the diversity of the sp185/333 proteins may be highly protective for sea urchins that are in constant contact with microbes in the marine ecosystem. the sp185/333 proteins are expressed by the phagocyte class of coelomocytes the sp185/333 proteins are expressed by a subset of the phagocyte class of coelomocytes including the polygonal, discoidal and small phagocytes (brockton et al., 2008; majeske et al., 2014). subsets of each of these cell types have sp185/333 proteins in small cytoplasmic vesicles that are typically positioned near the nucleus. in addition, small phagocytes have sp185/333 proteins associated with the surface of the cell. expression of the sp185/333 proteins in the adult tissues before vs. after challenge with lps shows increases in coelomocytes, axial organ, esophagus, pharynx and intestine (majeske et al., 2013). sp185/333-positive cells are present in all tissues and significant increases in these cells are noted for coelomocytes and for the axial organ after challenge with lps (brockton et al., 2008; majeske et al., 2013). in larvae, which are free swimming, feeding members of the zooplankton, sp185/333 gene expression is limited to a subset of differentiated blastocoelar cells called phagocytic filopodial cells (ho et al., 2016) that have similar phagocytic functions as the discoidal and polygonal phagocytes in the adult. one of the tenets of an effective innate immune response is a quick response to the detection of pathogens before they have time to undergo significant proliferation that may overwhelm host immunity. this can be accomplished by swift upregulation of the host immune response genes and the production of the encoded effector proteins. this has been documented for the sp185/333 genes in both adult and larval immune cells responding to injected bacteria or microbes in the seawater (silva, 2000; nair et al., 2005; terwilliger et al., 2007; furukawa et al., 2009; ho et al., 2016). it follows that the multiple sp185/333 genes would be expressed simultaneously in cells to optimize the numbers of effector proteins that can be produced and secreted over time. however, evaluation of sp185/333 gene expression in single adult phagocytes shows mrnas of a single sequence per cell, inferring expression from a single gene (majeske et al., 2014). this result suggests a complex mechanism for selecting the gene that is expressed and for silencing the others that is quite unexpected for an invertebrate innate immune response. it is expected that mrna editing will result in a small set of proteins of the same element pattern but with slightly different sequence will be produced by single phagocytes. however, the wide range of sp185/333 protein diversity observed for individual sea urchins (see below) will require that large numbers of phagocytes express the sp185/333 genes. a recombinant sp185/333 protein has multiple antipathogen activities hundreds of different isoforms of the sp185/333 proteins are expressed by individual sea urchins in response to immune challenge, and they fig. 9 the diversity of the native sp185/333 proteins are characterized by 2d western blots. a) native sp185/333 proteins from the cf of s. purpuratus show the diversity, but also the preponderance of proteins in the acidic pi range. the figure is from (dheilly et al., 2009) and is reprinted from the journal of immunology with permission from the american association of immunologists, inc., copyright 2009. b) when fulllength, native sp185/333 proteins with sufficient histidines are isolated by nickel affinity, these proteins are generally present in the basic pi range. this figure is republished from (sherman et al., 2015). in both cases, the sizes of most of the proteins are larger than expected compared to sizes deduced from cdnas and genes. may function synergistically in response to the challenge. understanding the activities of these proteins is essential for understanding the sea urchin immune system, however this requires that individual sp185/333 isoforms be isolated away from other versions and from other proteins in the cf. consequently, a recombinant sp185/333 protein called rsp0032 with an e1 element pattern (see fig. 8a) was evaluated for predicted antimicrobial activities (lun et al., 2016). when rsp0032 is incubated with vibrio diazotrophicus, bacillus subtilis, b. cereus, or saccharomyces cerevisiae, the protein shows saturation binding to vibrio and saccharomyces, but no binding is observed for the bacillus species (fig. 10). affinity for vibrio and saccharomyces is high, binding sites are specific, and once bound rsp0032 cannot be dissociated from the target cells. mass spectrometry of protein 368 bands from gels used to analyze rsp0032 incubation with vibrio target cells shows an association between rsp0032 and flagellin indicating that binding may be mediated through pathogen associated molecular patterns (pamps). expanded pamp binding analysis shows that rsp0032 also binds tightly with flagellin from salmonella, lps from e. coli, and β,1-3,glucan from saccharomyces, but does not bind to peptidoglycan (pgn) from bacillus. this suggests that there are multiple types of foreign cells to which rsp0032 can bind, that it does not bind indiscriminately to all foreign cells, and that it uses several pamps as specific binding targets that have very different molecular characteristics. despite the diversity that has been documented for the full-length sp185/333 proteins, they have a standard structure with an n-terminal glycine rich (gly-rich) region, a central region that is the cterminal end of the gly-rich (c-gly) region that has an arginine-glycine-aspartic acid motif (which has not been tested for integrin binding), a more cterminal histidine rich (his-rich) region, and a cterminal region (fig. 8c). based on the significant differences in the amino acid content of the gly-rich and his-rich regions, in addition to observations that edited mrnas (buckley et al., 2008b) encoding truncated proteins missing the his-rich region are more prevalent prior to immune challenge (terwilliger et al., 2007; sherman et al., 2015), these two regions of the proteins are expected to have different activities or functions. consequently, when tested, the recombinant fragments of the fulllength rsp0032 (rgly-rich, rc-gly, rhis-rich fragments; fig. 8c) show differences in activities compared to rsp0032 (lun et al., 2016). all recombinants bind to all foreign target cells including bacillus species to which rsp0032 does not bind indicating a broadening of binding characteristics (fig. 9). furthermore, the rc-gly fragment multimerizes in the presence or absence of binding targets in agreement with the rgly-rich and rhis-rich fragments, which do not include the cgly region and do not multimerize. the binding specificity of the rhis-rich fragment for yeast is identical to that of the full-length protein, however, the rgly-rich fragment shows expanded binding compared to either the full-length protein or the rhisrich fragment. these results indicate several noteworthy aspects of the binding activities that include 1) rsp0032 binds specifically and with high affinity to quite different foreign cell targets, 2) rsp0032 binds to multiple pamps that have very different characteristics, 3) the central c-gly region mediates multimerization, 4) the gly-rich and his-rich regions of the protein have different activities, and 5) when all regions of the full-length protein function together, they show increased specificity for binding targets. the binding characteristics of the rgly-rich fragment predict broadened binding activities of truncated sp185/333 proteins that are not characteristic of either the his-rich region or the fulllength protein. this suggests interesting possibilities for truncated proteins, which are more likely to be present prior to challenge, and may be important for immune surveillance. fig. 10 multitasking binding characteristics of rsp0032 and the recombinant fragments to microbes and pamps. rsp0032 (to the left) is composed of a gly-rich region (orange) including the c-terminal end of the gly-rich region (red), a his-rich region (blue), and a c-terminal region (gray). the recombinant fragments that correspond to the regions of the full-length protein are shown to the right in corresponding colors. the binding targets against which rsp0032 and the recombinant fragments were tested are shown in the middle. colored arrows indicate binding, whereas black blocked lines indicate no binding. the major differences in binding is to bacillus and to peptidoglycan (pgn) from bacillus, which are highlighted with the red box. the recombinant fragments were not tested against the pamps. the rc-gly fragment also mediates dimerization and multimerization of the full-length protein. the figure was modified from (lun et al., 2016; smith and lun, 2016). 369 when the multitasking activities of rsp0032 are applied to the hundreds of native sp185/333 proteins that are expressed by coelomocytes, this has vast implications for the sea urchin immune response and its activities in host protection. if all or most of the sp185/333 proteins have multitasking binding activities with slightly different but perhaps overlapping targets to which they bind, and many or all of the isoforms function synergistically, the outcome is a highly diverse and flexible innate immune effector response directed at controlling attack and infection from foreign microbial cells. if the expressed arrays of sp185/333 proteins have broad binding capabilities, it is highly unlikely that microbes will be able to circumvent or defeat the sp185/333 system because it would likely require simultaneous changes to multiple characteristics of the microbes and their pamps. the sp185/333 system, including the sequence diversity noted in the members of the gene family, the (putative) directed editing of mrnas that are translated to truncated and missense proteins, and the extraordinarily diverse arrays of proteins that are expressed among individual sea urchins strongly suggests that this system represents the central immune diversification system for echinoids. conclusion the common theme reviewed here describes host immune responses to microbial pathogens and the abilities to drive diversity at one or more levels to produce broad sequence variations among immune proteins that function in detection of foreign cells and effector proteins that act to remove and/or kill the invaders. there are commonalities among animals for detecting foreign molecules including the tlr and nod proteins among others, however, the wide variety of effector proteins and their modes of action make different immune systems appear to function quite differently. the underlying commonality is that although mechanisms to generate immune diversity are vastly different, ranging from rag-induced somatic recombination and aid-mediated gene assembly in the vertebrate ig and vlr systems respectively (tonegawa, 1983; davis et al., 1984; nagawa et al., 2007; rogozin et al., 2007), to extensive alternative splicing of dscam genes in arthropods (schmucker and chen, 2009; ng et al., 2014; brites and du pasquier, 2015), to clusters of duplicated genes with shared sequences that drive gene recombination, deletion, duplication (oren et al., 2016), the outcome for effective immunity and survival of organisms is to stay ahead of the pathogens. the example of the sp185/333 system in echinoids illustrates that multiple levels of diversification may function simultaneously to generate diverse anti-pathogen protein isoforms, perhaps each with slightly different activities towards microbial invasions, that together act as effectively in host protection as the somatic rearrangement and gene assembly mechanisms that are employed in the immune systems of vertebrates. acknowledgements this work was supported by funding from the italian national program for antarctic research (pnra; pea2009/a1.12) to mrc, and the us national science foundation (ios-1550474) to lcs. references adema cm. fibrinogen-related proteins (freps) in mollusks. results and problems in cell 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biomédicas, unam, ciudad de méxico, méxico 3departamento de biotecnología, universidad autónoma metropolitana, unidad iztapalapa, 09340, ciudad de méxico, méxico 4unidad de bioimagen, centro de investigación y desarrollo en ciencias de la salud, universidad autónoma de nuevo león, monterrey, nuevo león, méxico 5laboratorio de peces, departamento de biología, universidad autónoma metropolitana, 09340, ciudad de méxico, méxico 6laboratorio de neuroendocrinología reproductiva, departamento de biología de la reproducción, universidad autónoma metropolitana, 09340, ciudad de méxico, méxico 7departamento de hidrobiología, laboratorio de ecotoxicología, universidad autónoma metropolitana, unidad iztapalapa, 09340, ciudad de méxico, méxico this is an open access article published under the cc by license accepted october 17, 2020 abstract vulnerability depends on the exposure and sensitivity levels of a system to a specific pressure, together with the capacity to cope, recover, or adapt to this pressure. we propose the use of wellknown tissular techniques to measure the components of vulnerability. immunohistochemistry and histopathology indicate the health status of living organisms and the environment. therefore, these techniques should provide the necessary information to determine the vulnerability of an organism. immunohistochemical analysis uses biomarkers to determine the presence of toxic compounds, reflecting the exposure level of an organism. histopathological analysis reveals the environmental impact of a given toxin, reflecting the sensitivity level of the organism to said toxin. here, we propose a strategy to use these techniques to assess the vulnerability of clams from tecolutla, veracruz. we developed categories for each vulnerability component using semi-quantitative scales. briefly, we calculated the exposure level based on the average number of positive immunohistochemical biomarkers among several organs of clams. then, we compared the prevalence of histological alterations with the exposure level to determine the sensitivity level. finally, to estimate the recovery capacity, we placed the control group in a clean environment for 40 days. these led us to observe the capacity of the clams to reverse the effects of environmental stress. clams showed a moderate level of exposure, a low sensitivity level, and an effective recovery capacity. in conclusion, these results indicate that clams have a low level of vulnerability. this proposal has the potential to guide future works assessing the vulnerability of organisms and later include them in the estimation of vulnerability from aquatic bodies. key words: polymesoda caroliniana; histopathological index; vulnerability; immunohistochemistry; environmental stress introduction coastal areas are considered one of the most vulnerable environments affected by pollutants. some studies have already proposed some vulnerability ___________________________________________________________________________ corresponding author: xochitl guzmán-garcía universidad autónoma metropolitana, unidad iztapalapa san rafael atlixco 186, vicentina, c.p. 09340, iztapalapa, ciudad de méxico, méxico e-mail: xgg@xanum.uam.mx parameters for coastal areas (ramírez and torres, 2011). however, none of these assessments included animal physiological traits that describe the health status of living organisms. environmental stress can cause measurable effects over the physiological state of different animals, compromising their health (bayne et al., 1976; baum et al., 1984; häder et al., 2020). diverse methodological approaches measure these effects at different biological levels. histopathological and 187 immunohistochemical analyses evaluate the effects of environmental stress at the tissue level (costa et al., 2013; cuevas et al., 2015). on one hand, histopathological analysis determines the physiological consequences of pollutants over organisms, through a characterization of tissue injuries (usheva et al., 2006; boscolo papo et al., 2014b). these toxic responses are semi-quantified through the histopathological index (ih), detailing the health status of an organism (costa et al., 2013; cuevas et al., 2015). on the other hand, immunohistochemical analysis determines cytological responses related to the presence of some pollutants. many biomarkers are used to this end, indicating several types of toxic compounds that affect living organisms (moraga et al., 2005; wang et al., 2010; zhang et al., 2012; boscolo papo et al., 2014a). consequently, the integration of histopathological and immunohistochemical approaches constitutes a valuable strategy to investigate the vulnerability of an organism to a specific environment (boscolo papo et al., 2014a). tecolutla is located in veracruz state, along the eastern coast of mexico (fig 1). the town of tecolutla surrounds a river that has associated estuaries, canals, and mangroves, giving this locale huge biodiversity. the most important and growing economic activity in the zone is tourism, hence the river receives many discharges of wastewater, agrochemicals, and hydrocarbons, which impact on the natural environment (lópez-portillo et al., 2009; arriaga-gaona, 2009; temino-boes et al., 2020). water samples taken from the tecolutla river exceed the limits of total and fecal coliform bacteria allowed according to mexican standards (arriagagaona, 2009). moreover, veracruz state shows a high vulnerability, according to impact studies conducted on fishery production and damage to the mangrove (ramírez and torres, 2011). nevertheless, none of these studies included physiological indicators of the health of living organisms. clams are bioindicators of the health status of an environment (markert et al., 2003; park et al., 2007; wu et al., 2013; boscolo papo et al., 2014a; zhang et al., 2014; carneiro et al., 2015; cuevas et al., 2015; santovito et al., 2015; vodopivez et al., 2015; delgado-alvarez et al., 2019). among the diversity of tecolutla, we found the clam polymesoda caroliniana, commonly named black clam. therefore, these organisms are potential sources of vulnerability data. vulnerability depends on the exposure and sensitivity levels of a system to a specific pressure, together with the capacity to cope, recover, or adapt to this pressure (villa and mcleod, 2002; stein et al., 2014; carantoña and hernández, 2017; ocaña and pech, 2018). these components are measured through indicators at different biological levels. here, we created an approach to measuring the three vulnerability components by assessing the health status of the clams. the proposed approach includes the use of different tissular techniques to fig. 1 map indicating the location of tecolutla in veracruz state, mexico 188 this end. to measure the exposure level, we used immunohistochemical analysis. immunohistochemical analysis reflects the level of exposure, indicating cytological responses to the presence of different pollutants. then, to measure the sensitivity level, we correlated the prevalence of histopathological injuries with the level of exposure. finally, we estimated the recovery capacity through a group of clams that depurated pollutants for 40 days in a clean environment. these organisms were shown to be sensitive enough to reflect that tecolutla is polluted but also resilient enough to resist and survive environmental stress. moreover, they showed an effective recovery capacity when introduced to a clean environment. here we proposed the integration of histopathological and immunohistochemical analyses through semiquantitative scales to assess the vulnerability of clams from tecolutla. we created categories for each component, proposing approaches to their measurement. in conclusion, we determined that the clam p. caroliniana has a low vulnerability, based on the fact that clams could recover fully in a clean environment. this first proposal is a guide for future investigations to determine the vulnerability of specific organisms. the vulnerability of several organisms based on physiological traits should be included in the determination of the health status and vulnerability of an environment. material and methods sampling area and sample collection the tecolutla river is located in the state of veracruz between parallels 96°59′849′′ w and 20°27′628′′n, on the eastern coast, facing the gulf of mexico (fig 1). prior to collection, we obtained physicochemical water parameters (temperature, salinity, dissolved oxygen, and ph) with a hach® model dr / 2000 direct reading spectrophotometer. the physicochemical parameters of tecolutla, veracruz characterize a typical estuarine environment: 22 °c, 2 ‰ salinity, 6.6 mg/l dissolved oxygen, and ph 7.7. these were later used to maintain the same conditions in the control group. local fishermen collected 100 adult polymesoda caroliniana clams (4 to 5.5 cm in length). the clams were divided into a control and an experimental group, each containing 50 individuals. transportation to the laboratory was in thermal boxes at 4 °c. once they arrived, all the clams were cleaned with 70% alcohol. then, all the clams were subjected to a macroscopic evaluation, and the following parameters were recorded: shell perforations and the presence of vermes or ectoparasites. macroscopic analysis of polymesoda caroliniana clams showed healthy organisms without shell distortion or perforations, but some of them had parasitic worms. immediately, the visceral mass of the experimental group was dissected and processed for histopathological and immunohistochemical analysis. the clams in the control group were maintained in seawater under the original physicochemical parameters (22 °c, 2‰ salinity, 6.6 mg/l dissolved oxygen, and ph 7.7) for 40 days. during the 40 days, the seawater was continuously table 1 percentage of dissemination degree assigned to each a value dissemination degree a value 0 – 9% 0 10 – 40% 2 50 – 70% 4 70 – 100% 6 aerated with partial water replacement every 72 h. the clams were fed with a suspension of 909,935 cells/ml microalgae culture of chlorella sp every day. after 40 days of maintenance, these clams were subjected to histopathological and immunohistochemical analysis. tissue preparation the visceral mass of all clams was fixed in 10% neutral buffered formalin for 48 h, dehydrated through a graded series of ethanol in a tissue processor (leica, model tp1020), and embedded in paraffin using a paraffin embedding station (leica, model eg1140-h). serial 5-µ-thick sections were cut in a microtome (microm, model hm3156). histopathological analysis histopathological slides were prepared by a standard technique, stained with hematoxylin-eosin (cuevas et al., 2015), and examined under light microscopy (zeiss, primostar) to observe the general morphology, presence of parasites, and histological alterations. for each clam, three slides were analyzed. for each slide, we observed six different optical fields, each one at three magnifications, 10x, 40x, and 100x. three different people analyzed the slides separately following the same criteria. each person observed and quantified the alterations to report the number of clams presenting each alteration in a prevalence matrix. the histopathological analysis included: i. observation and description of tissue alterations, ii. construction of an alteration prevalence matrix, and iii. pathological importance factor (w) assignment and the degree of dissemination value (a). these values created a prevalence matrix, used to calculate the histopathological index. histopathological index the histopathological index (ih) is part of a protocol for the evaluation of aquatic pollution, and it was calculated according to previous studies (bernet et al., 1999; costa et al., 2013; cuevas et al., 2015). first, each type of alteration was categorized with respect to reaction patterns. a reaction pattern is the set of alterations in the functional unit of the targeted organ. the reaction patterns were tubular alterations and intertubular alterations. then, we calculated the pathological importance factor (w) and the degree of dissemination (a). the pathological importance factor (w) is defined as the degree of damage or 189 compromise of an organ after an alteration on a scale of 0 to 3 (0 = absence, 1 = negligible, 2 = moderate, and 3 = severe pathological importance), according to the original work (cuevas et al., 2015; costa et al., 2013). parasites were weighted as w = 3, hemocytic infiltrations and atrophies as w = 2, and brown cells and lipofuscin aggregates were assigned w = 1, according to the original proposal for clams (costa et al., 2013; cuevas et al., 2015). the degree of dissemination (a) was the level of spread of a particular alteration in a functional unit or organ, in the range of zero to six. therefore, to assign a value, we used a prevalence matrix (table 1). each a value correspond to (0) unchanged, (2) mild occurrence, (4) moderate occurrence, and (6) severe occurrence, according to previous reports (bernet et al., 1999; cuevas et al., 2015). later, we calculated the ih using the following equation (costa et al., 2013) where wj is the pathological importance factor of each alteration, ajh is the degree of dissemination of an individual alteration, and mj is the maximum attributable value for the alteration, estimated from the maximum value of w times the maximum value of a. this denominator normalizes ih to a value between 0 and 1, thus permitting comparisons between distinct organs. immunohistochemistry for immunohistochemical staining, tissue slides were deparaffinized in xylene before hydration via a graded series of ethanol. to retrieve the heatinduced antigen, we used a recuperator (diva decloaker 20x dv2005, biocare medical) diluted 1:20 with distilled water in a digital electric pressure cooker (decloaking chamber, model dc2002, biocare medical) at 25 psi and 125 °c for 5 min. slides were washed with tbs (auto wash buffer, 40x, biocare medical) and then cooled down progressively at room temperature for 20 min. endogenous peroxidases were neutralized by incubating the slides with the blocker (endogenous peroxidase blocker px968g, biocare medical) for 5 min at room temperature in an incubator chamber (model rmiq105, biocare medical), to avoid natural temperature fluctuations. excess blocking solution was removed by washing in tbs. thereafter, all incubations were performed at room temperature. serial sections were incubated for 45 min with 100 μl of the primary antibodies inside the incubator chamber. for metallothioneins, we used mouse monoclonal metallothionein antibody [uc1mt] (genentex catalog number: gtx12228), dilution 1:30. for cytochrome p450, we used mouse monoclonal antibody cyp1a2 [15e2] (genentex catalog number: gtx84638), dilution 1:50. finally, for hsp70, we used a mouse monoclonal hsp70 antibody [3a3] (genentex® catalog number: gtx25439), dilution 1:50. tbs solution without primary antibody served as the control. after washing in tbs, sections were incubated with a polymer (envision+ system-hrp, labelled polymer (mouse) k4000, agilent dako) that contained secondary antibody and streptavidin for 45 min. then, we stained the slides with diaminobenzidine (dab), using 1 ml of substrate buffer and 50 μl of chromogen (dako liquid dab+ substrate chromogen system k3465, agilent dako) for 10 min. the slides were counterstained with hematoxylin (tacha’s automated hematoxylin, biocare medical) for 10 min and later dehydrated. samples were mounted with a 50% synthetic xylene resin solution. staining was performed in triplicate for each tissue of three clams. all slides were observed under a light microscope (zeiss®, primostar 176045) coupled to a digital camera (canon, powershot g10). immunolabeling was considered positive when the staining intensity was greater than the background observed in the negative control. the specificity of the immunostaining was verified by incubating the sections with pbs instead of the specific primary antibody. this validation was performed for all antibodies in each the tissues. all preparations were kept in a buffer solution to avoid drying. table 2 relations between the prevalence of histopathological alterations and level of exposure. the sensitivity value was calculated by subtracting the prevalence minus exposure level. these relations resulted in a categorization of sensitivity (very low = -2, low = -1, normal = 0, high = 1, very high = 2) exposure level value prevalence of histopathological alterations value sensitivity value sensitivity category minimal 1 low prevalence 1 0 normal moderate 2 1 1 high high 3 1 2 very high minimal 1 moderate prevalence 2 -1 low moderate 2 2 0 normal high 3 2 1 high minimal 1 high prevalence 3 -2 very low moderate 2 3 -1 very low high 3 3 0 low 190 fig. 2 digestive gland histological description of several organisms showing different dissemination degrees (a). a. digestive tubules showing semi-circular normal aspects. basophilic cells lining the lumen of digestive tubules. b. secretory vesicles (dashed arrows) derived from epithelium. c. digestive gland tubules showing few brown cells (a = 2, diamonds). d. digestive gland tissue showing an increase of brown cells (a = 4), and some lipofuscin aggregates (a = 2, square). e. tubules showing lipofuscin aggregates (square) with the highest dissemination degree (a = 6). f-g. digestive gland showing atrophy (solid arrows) of the tubule epithelium (a = 2), and hemocyte infiltration (a = 6, circles). h. digestive gland tissue showing inclusions of possible parasites (a = 2). 1. epithelium 2. lumen 3. connective tissue. hematoxylin-eosin staining 191 vulnerability parameters vulnerability is composed of the levels of exposure, sensitivity, and recovery capacity (ocaña and pech, 2018). exposure was measured through the number of immunohistochemical biomarkers identified. the immunohistochemical index (ii) is the average of biomarkers found in different organs of the organisms. this average was categorized into four groups: no exposure (ii = 0), minimal exposure (ii between 0.1 and 1), moderate exposure (ii between 1.1 and 2), and high exposure (ii between 2.1 and 3). moreover, the sensitivity is the level of response according to the level of exposure to certain stresses (ocaña and pech, 2018). therefore, the sensitivity relates the prevalence of histopathological alterations to the level of exposure, hence we assigned values to each category. the prevalence of histopathological alterations is a classification derived from the histopathological index, where 0 – 0.25 is low, 0.25 – 0.50 moderate, 0.50 – 0.75 high, and 0.75 – 1, very high (costa et al., 2013). we considered high and very high prevalence as the same category. all the organisms are sensitive to their environment to survive, thus we made four categories of sensitivity from very low to very high. these categories were assigned according to the following criteria. if an organism was exposed to a low level of exposure and had a low level of prevalence of alterations, then it had a normal sensitivity. however, if the organism was exposed to a low level of exposure but had a moderate or high prevalence of alterations, then the organism was highly sensitive (table 2). results clams from tecolutla had a low prevalence of histopathological alterations the digestive glands have been used to assess pollution effects on many organisms, as epithelial cells lining the digestive glands are very sensitive to environmental stress (usheva et al., 2006). on one hand, the digestive gland of p. caroliniana clams in the control group showed tubular structures with an epithelial lining and a central lumen (fig 2a, 2b). moreover, on this epithelium we observed secretory vesicles (fig 2b), which aid in the detoxifying processes of the clam (usheva and frolova, 2006). around the tubules we observed connective tissue, which provides support to the digestive gland and a medium for oxygen and nutrients to diffuse to cells. moreover, we found few hemocytes, brown cells and lipofuscin aggregates in the connective tissue, as expected. most of this histological morphology agrees with the description of digestive glands in other clam species (usheva and frolova, 2006; usheva et al., 2006; sıkdokur et al., 2020; bejaoui et al., 2020; costa et al., 2013). on the other hand, as compared with the control groups, digestive gland sections from the experimental group underwent a change in their structure. brown cells and lipofuscin aggregates in the slides from the experimental group increased (fig 2c, 2d, 2e), as did hemocytic infiltrations, atrophy, and parasites (fig 2f, 2g, 2h, and table 3). these responses were classified into tubular and intertubular alterations. we used this classification to associate a pathological importance factor (w) and degree of dissemination (a) (costa et al., 2013), to further determine the general health status of clams from tecolutla, veracruz. the clams from the experimental group presented alterations with diverse pathological importance factors (w); however, the most disseminated pathologies had a low w (table 4). these results suggest that the most prevalent pathologies are the least harmful for the organisms. to integrate both w and a, we calculated the histopathological indices for these clams. the average histopathological index (ih) for the control group was 0.01, while the average ih for the experimental group was 0.18. based on these results, we concluded that the clams from tecolutla, veracruz had a low prevalence of histopathological alterations, according to previous reports classifying the ih (costa et al., 2013; cuevas et al., 2015). clams from tecolutla are exposed to several pollutants besides histopathological analysis, other biomarkers are used to corroborate the impact of environmental stress. some of these biomarkers include p450 cytochromes (cyps), metallothionein proteins (mts), and heat-shock proteins (hsp) (moraga et al., 2005; boscolo papo et al., 2014a; b). we used these biomarkers to assess environmental table 3 pathological alterations and dissemination degree observed in clams from the experimental group. a total of 50 clams belonged to this group. clams from the control group lacked any of these alterations. dissemination degree (a) is the percentage of clams presenting each alteration alteration number of clams with the alteration dissemination degree (a) brown cells 47 95 lipofuscin aggregates 30 60 hemocytic infiltrations 20 40 atrophy 10 25 parasites 1 0.05 192 table 4 values of pathological importance factor (w) and degree of dissemination (a) of clams from the experimental group. at the end is the ih for this group reaction pattern alteration w a tubular alterations brown cells 1 2 lipofuscin aggregates 1 4 hemocytic infiltrations 2 0 atrophy 2 0 parasites 3 0 intertubular alterations browns cells 1 6 lipofuscin aggregates 1 2 hemocytic infiltrations 2 6 atrophy 2 6 parasites 3 2 histopathological index (ih) 0.18 stress effects on the clam of tecolutla (table 5). digestive gland slides of the control group lacked any immunoreactivity. however, digestive gland slides of the experimental group showed positive immunoreactivity to p450 cytochrome (cyp) and heat-shock protein 70 (hsp70), but not to mts. both cyp and hsp70 were observed on the cytoplasm of the epithelial tissue lining of the digestive tubules (fig 3a, 3b, 3c). these results suggest that injuries caused to the digestive gland by environmental stress are reversible, as the control group did not show immunoreactivity to these markers. nevertheless, the digestive system of clams is composed of other organs, such as a short esophagus, a stomach, and a gut. hence, we tested the same biomarkers on the rest of them. stomach and gut tissues showed immunoreactivity to hsp70 and cyp but not to mts (fig 3d). the immunohistochemical responses indicated that the tecolutla environment has many pollutants, but apparently the concentration of heavy metals is low, as the digestive system did not show any immunoreactivity. nevertheless, to obtain an overview about the overall health of the clams, we tested the same antibodies in other target organs (table 5). the gills, the gonads and the foot are widely used to assess the health of many mollusks through histopathological and immunohistochemical approaches (moraga et al., 2005; usheva et al., 2006; costa et al., 2013; boscolo papo et al., 2014a; cuevas et al., 2015; sıkdokur et al., 2020). in our clams, the gills showed immunoreactivity to hsp70 and cyp, but not to mts. meanwhile, the gonads showed immunoreactivity only to hsp70. finally, the foot showed immunoreactivity to all the biomarkers, hsp70, cyp, and mts (fig 3e, 3f). to observe the accuracy of the immunohistochemical test, we showed that in the absence of the antibody, the color of the slides changes dramatically (fig 3g, 3h). these organs have different levels of exposure to the environment, due to their diverse physiological functions. accordingly, the gills are highly related to water quality, while the foot is more related to sediment quality. however, the general response to immunohistochemical biomarkers indicated that the environment is affecting all the organs. furthermore, as the control group lacked immunoreactivity to any biomarker, we concluded that the health of our clams from tecolutla could be restored if the quality of the environment is improved. proposed approach to assess vulnerability using tissular analysis vulnerability is the degree to which a system is susceptible to adverse effects caused by environmental stress (villa and mcleod, 2002; stein et al., 2014; gauthier et al., 2014; carantoña and hernández, 2017; ocaña and pech, 2018). the strategies to estimate environmental vulnerability table 5 immunoreactivity responses in different organs of clams from the experimental group organ cyp hsp70 mts total digestive gland 1 1 0 2 stomach 1 1 0 2 gut 1 1 0 2 gills 1 1 0 2 gonad 0 1 0 1 foot 1 1 1 3 immunohistochemical index (ii) = 2 193 fig. 3 immunoreactivity of cyp, hsp70, and mts in different tissues of clam from tecolutla. all sections are counterstained with tacha hematoxylin. a-b. digestive gland showing an immunoreactivity to cyp. c. digestive gland showing an immunoreactivity to hsp70. d. intestine showing immunoreactivity to hsp70. e-f. foot showing immunoreactivity to mts. g-h immunoreactivity controls showing difference in the brown staining, both from foot tissue. negative control (g) processed with pbs instead of the hsp70 antibody and positive control (h) processed with the hsp70 antibody 194 include indicators categorized in anthropogenic, biological, geological, and meteorological components (villa and mcleod, 2002; skondras et al., 2011; karmaoui, 2015; sahoo et al., 2016; harik et al., 2017). however, these indicators do not include any physiological trait of the living organisms of each system. therefore, we propose the integration of the histopathological index and immunohistochemical approaches as valuable tools to assess the vulnerability of clams from tecolutla. the assessment of vulnerability includes the measure of exposure, sensitivity, and recovery capacity (villa and mcleod, 2002; ocaña and pech, 2018). to begin with, exposure was measured through the immunoreactivity of different biomarkers. we created a range based on the immunohistochemical data previously presented. the average number of biomarkers found in the organs was classified into no, low, medium, and high exposure, called the immunohistochemical index (ii) (table 5). the digestive glands of clams from tecolutla showed medium exposure, but very close to the high category (table 6). we recommend the use of at least three different immunochemistry biomarkers in three or more target organs to assess exposure. the gills, digestive gland, and foot constitute the preferred organs to evaluate this (moraga et al., 2005; usheva et al., 2006; boscolo papo et al., 2014a; cuevas et al., 2015). afterwards, the sensitivity of clams was assessed through a comparison between the histopathological index (ih) classification and the exposure level (table 2). we determined that an organism is sensitive if the level of exposure is lower than the category of the ih. in other words, an organism that shows a low prevalence of histopathological alterations in an environment with moderate exposure level has low sensitivity (table 2). therefore, clams from tecolutla had low sensitivity to the environment. finally, to assess recovery capacity, we observed both the ii and the ih of the control group. these clams depurated toxic molecules during 40 days in a clean environment, hence they constituted a good parameter to assess recovery capacity. clams from the control group showed an exposure level of zero, according to ii. furthermore, these clams had an ih of 0.01, meaning that the prevalence of histopathological injuries was reversed almost completely. in other words, these organisms have a good recovery capacity (table 6). however, another study is necessary to propose an approach that classifies the levels of recovery capacity in these organisms. for the integration of the three vulnerability components in a mathematical model, we needed a value of recovery capacity. however, recovery capacity regulates vulnerability through the modulation of exposure and sensitivity (adger et al., 2007; engle, 2011). therefore, we concluded that clams from tecolutla had a low vulnerability, as they reversed all the effects of environmental stress. in summary, these physiological traits and their semi-quantification should be included in the vulnerability measurements of aquatic bodies. these traits represented directly the health status of tecolutla, veracruz. briefly, these results indicate that the effects of pollutants in these organisms are still reversible. however, it is important to stop table 6 proposed semiquantification of parameters considered as indicators of vulnerability vulnerability factor indicator scale clams vulnerability exposure immunoreactivity index (ii) no exposure 0 moderate exposure minimal 0.1 1 moderate 1.1 2 high 2.1 3 sensitivity exposure vs prevalence of histopathological alterations (ii vs ih) very low -2 low sensitivity low -1 normal 0 high 1 very high 2 recovery capacity control group assessment low ? efficient recovery capacity moderate ? high ? 195 pollution on tecolutla so that organisms can improve their health. this work sets a health record for future monitoring references. the full quantification of histopathological indexes for the rest of the organs and the integration with the immunohistochemical part is a perspective of this work that contains valuable information about the vulnerability of these organisms. furthermore, it is important to develop an approach to classify the recovery capacity properly. additionally, the perspective of this work includes the application of this strategy in other indicator organisms to fully estimate the vulnerability of tecolutla. discussion vulnerability is the degree to which a system is susceptible to adverse effects caused by environmental stress (villa and mcleod, 2002; gauthier et al., 2014; stein et al., 2014; carantoña and hernández, 2017; ocaña and pech, 2018). environmental vulnerability indexes created along the last years, through many approaches, take into account indicators categorized into anthropogenic, biological, geological, and meteorological components (villa and mcleod, 2002; skondras et al., 2011; gauthier et al., 2014; karmaoui, 2015; sahoo et al., 2016; harik et al., 2017). however, these indicators do not include any physiological traits of the living organisms of each system. some approaches include water quality and plant distribution, while others include plant physiological traits (esperón-rodríguez and barradas, 2015; trevisan et al., 2020). the biological responses of organisms reflect the health status of a particular environment. recently, some vulnerability indexes analyzed responses based on specific biomarkers (gauthier et al., 2014; chalghmi et al., 2020). vulnerability depends on the levels of exposure and sensitivity of a system to a specific pressure, altogether with the capacity to cope, recover, or adapt to this pressure (villa and mcleod, 2002; stein et al., 2014; carantoña and hernández, 2017; ocaña and pech, 2018). these components can be assessed at different levels. here, we created an approach to measure the three vulnerability components through the assessment of the health status of the clams (table 6). the proposed approach includes the use of different tissular techniques to this end. exposure several human activities generate pressure on aquatic bodies. vulnerability cannot be assessed without exposure to a pressure. therefore, the first step is to determine the sources of pressure (ocaña and pech, 2018). environmental exposure is related to the presence and absence of immunohistochemical biomarkers (moraga et al., 2005; wang et al., 2010; boscolo papo et al., 2014a; santovito et al., 2015). these responses reflect the environmental stress that affects the organisms. the immunohistochemical approach determines the exposure to pollutants quickly, without the need for specific chemical procedures. the biomarkers used cover a broad range of pollutants. among them, we used p450 cytochromes (cyps), metallothionein proteins (mts), and heat-shock proteins (hsp) that are expressed after exposure to pesticides, hydrocarbons, metals, heat shock, and other toxic compounds (moraga et al., 2005; boscolo papo et al., 2014a; b). these biomarkers are widely used in different organisms to show exposure to environmental stress (moraga et al., 2005; wang et al., 2010; boscolo papo et al., 2014a; santovito et al., 2015). different target organs are used to this aim, such as the digestive gland, gills, foot, and gonads. to obtain an overview of the general exposure of the clams, we evaluated several target organs. these responses were compiled and averaged to create a range of different levels of exposure. although the immunochemical approach lacks quantification of pollutants, averaging the immune reactivity to different biomarkers in different organs provides an overview of the level of exposure. the target organs differed in their reactivity to the biomarkers, because they have different physiological functions. the physiological function of each organ and its anatomical position determine its interaction with the environment. for instance, the foot and the gills are in direct contact with sediment and water, making them more exposed, while the gonads are less exposed because they do not interact directly with the environment. our proposal weights equally the biomarkers exposed in different organs; however, it is necessary to determine if the organs should be weighted differently. nevertheless, our proposal, using an immunochemical approach, includes two types of exposure information. on one side, it evaluates a broad range of pollutants to which organisms are exposed. on the other side, it includes different target organs with distinct metabolic pathways that cope with the toxic compounds. sensitivity sensitivity is the degree to which a system or species is affected by environmental stress (stein et al., 2014). a description of what makes a system sensitive is necessary, keeping in mind that each sensitivity level is specific to each pressure (ocaña and pech, 2018). all living organisms are sensitive to their environment to survive; however, the level of responses should be related to an exposure level to determine if the system is sensitive. hence, to evaluate the organism’s sensitivity, we related two aspects. on one hand, we measured the types and dissemination of the responses to a stress, called the prevalence of histopathological alterations. this prevalence categorizes the values of the histopathological index (ih), for comparison among several organisms (costa et al., 2013; cuevas et al., 2015). besides, the prevalence correlates with the health status of a specific environment. on the other hand, we correlated the prevalences with the exposure level to determine the clam’s sensitivity to the pollutants present in tecolutla. histopathology is widely used to recognize the effects of environmental stress on the organisms (bernet et al., 1999; usheva et al., 2006; costa et al., 2013; 196 boscolo papo et al., 2014a; b; cuevas et al., 2015; sıkdokur et al., 2020). it has been used in several organisms to determine the health status of aquatic bodies (usheva et al., 2006; chalghmi et al., 2020; costa et al., 2013; boscolo papo et al., 2014a). therefore, in our proposal, sensitivity is the measure of responses (prevalence of histopathological alterations) related to the exposure level. if the exposure level is equal to the prevalence of histopathological alterations, then the sensitivity is normal. in case the exposure level is lower than the prevalence, then the sensitivity is low. in contrast, if the exposure level is higher than the prevalence then the sensitivity is high. as far as we know, this is the first approach giving a semiquantitative method to assess sensitivity through the evaluation of physiological responses to pollutants. recovery capacity the third component of vulnerability is adaptive capacity. adaptive capacity refers to the ability of a species or system to cope with environmental impact with minimal disruption (stein et al., 2014). however, adaptive refers to the evolution of a system in ecology, and it is usually assessed through evolutionary potentials, such as plasticity, dispersal ability and evolutionary potential (ocaña and pech, 2018). tecolutla lacks monitoring programs that assess health status through time, hence we required another strategy to evaluate this vulnerability component. we determined that the recovery capacity attribute could be used instead. recovery capacity is usually assessed through both extrinsic factors and intrinsic traits. to evaluate this capacity, we develop an approach to evaluate the ability of the organism to cope with the consequences of environmental stress. a control group of clams lived in a clean environment for 40 days. then, they were subjected to histopathological and immunohistochemical analysis to observe differences with the experimental group. we observed a full recovery of the control group. the control group did not show immunoreactivity to any exposure biomarker (immunochemical analysis). moreover, it had a lower histopathological index (ih = 0.01) compared with the experimental group (ih = 0.18). nonetheless, we lacked a categorization for this component. another essay is necessary to evaluate different levels of recovery capacity related to different levels of exposure. nevertheless, we concluded that, having the current level of exposure, the clams from tecolutla have sufficient recovery capacity to reverse the consequences of environmental stress. briefly, we employed different indicators to measure the three components of vulnerability. however, to integrate them in a mathematical model, standardization, response scaling, weighting, and aggregation are necessary. although we had semi-quantitative values for exposure and sensitivity levels, we lacked values for the recovery capacity component. nonetheless, knowing that the clams were able to recover with a moderate exposure level and a low sensitivity, we can estimate the vulnerability. recovery capacity was taken as a measure of adaptive capacity, hence both regulate vulnerability through modulation of exposure and sensitivity (adger et al., 2007; engle, 2011). this agreed with the method we used to assess recovery capacity, evaluating exposure and sensitivity levels through the same methods and then compare the results of the control versus the experimental group. as we observed that the clams reverted almost all the effects of environmental stress, we determined that the recovery capacity exceeded the exposure and sensitivity. therefore, these results indicated that clams from tecolutla showed a low vulnerability. higher recovery or adaptive capacity aids in reducing the effects of exposure and sensitivity and, in consequence, reduces the vulnerability of the system (art vulnerability & risk assessment report, 2012; stein et al., 2014; thomas et al., 2019). the basic role of recovery or adaptive capacity is accepted as a positive attribute to reduce vulnerability (engle, 2011; thomas et al., 2019). as previously mentioned, it is still necessary to develop an assay to identify the limits of the recovery capacity of clams at different exposures levels. in conclusion, adding the vulnerability components, exposure, sensitivity, and recovery capacity, we determined that the clams of tecolutla have a low vulnerability. these clams had a moderate exposure level, low sensitivity, and a high recovery capacity. assets with a higher adaptive capacity, or recovery capacity, and low sensitivity better tolerate impacts, and therefore have a lower vulnerability (engle, 2011; art vulnerability & risk assessment report, 2012). as far as we know, this is the first approach to semi-quantifying the vulnerability of an organism. besides, this proposal constitutes an appealing approach for organisms in aquatic bodies that lack monitoring programs. the perspective of this work is to create categories to divide the recovery capacities of several organisms and compare them. also, we propose to determine the vulnerability from several organisms, in order to estimate the general vulnerability of an environment. finally, along with other approaches we aim to determine in the future the vulnerability of tecolutla, veracruz. declaration of conflict of interest the authors declare no conflict of interests. acknowledgments this study is part of the graduate program on energy and environmental sciences (doctorado en energía y medio ambiente), universidad autónoma metropolitana iztapalapa (uam-i), méxico. r jerónimo juárez is a doctoral student in this program and has received conacyt fellowship 470845. this study was supported by uam-i through the project ecological integrity and environmental health indices (indicadores de integridad ecológica y salud ambiental), and by prodep-sep through the project enviromental diagnostic of the town of tecolutla, veracruz (diagnóstico ambiental del municipio de tecolutla veracruz). we thank miguel ángel león tapia (institute of biology, national university of mexico) for drawing the map. also, to dr. katya frank hoeflich the manuscript writing training team 197 (cemai-conacyt) for the 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pollutant phenanthrene in clam venerupis philippinarum using oxidative stress biomarkers. environ. toxicol. pharmacol. 37: 697-704, 2014. zhang l, gan j, ke c, liu x, zhao j, you l, et al. identification and expression profile of a new cytochrome p450 isoform (cyp414a1) in the hepatopancreas of venerupis (ruditapes) philippinarum exposed to benzo[a]pyrene, cadmium and copper. environ. toxicol. pharmacol. 33: 85-91, 2012. 69 isj 19: 69-84, 2022 issn 1824-307x report of meeting xxist scientific meeting of the italian association of developmental and comparative immunobiology (iadci), february 16-18 2022, didactic pole, department of biology, university of padua, italy organizers: l ballarin, v matozzo, f sandrelli, g santovito, p venier department of biology, university of padua, padua, italy this is an open access article published under the cc by license award “soci non strutturati” (best presentation and curriculum studiorum for members under 35) characterization and functional role of a novel c1qdc from a colonial ascidian a peronato1, n franchi2, m tabarelli3, l ballarin1 1department of biology, university of padua, padua, italy 2department of biotechnology and life sciences, university of modena and reggio emilia, modena, italy 3phd school in agricultural science and biotechnology, university of udine, udine, italy the complement system is present in all the metazoans as a complex array of soluble and membrane proteins able to orchestrate innate immune responses such as inflammation and phagocytosis. although the complement system of invertebrates has been much less studied than that of vertebrates, however, it is equipped with at least the alternative and the lectin activation pathways. the c1q-domain-containing (c1qdc) proteins are a large family of proteins, present in both vertebrates and invertebrates, characterized by one or more globular c1q (gc1q) domain(s) at the cterminus. c1qdc proteins are distinguished in c1qlike proteins, with a gc1q domain and a collagenlike region at the n-terminus, and globular head c1q proteins (ghc1q) with one or more gc1q domains and a short n-terminus with no defined domains. the latter can be further divided into proteins without a signal peptide (cellular ghc1qs or cghc1qs) and proteins endowed with a signal peptide (secreted ghc1qs or sghc1qs). the gc1q domain has a typical jelly roll topology of five pairs of anti-parallel β-strands creating two β-sheets, with eight conserved hydrophobic amino acids and can interact with a large variety of ligands, both self and non-self. the same topology is present in the tumor necrosis factor (tnf) domain of protein of the tnf family so that a c1q-tnf superfamily of proteins (c1q/tnf-related proteins or ctrps) has been defined. the mammalian complement component c1q, a subunit of the c1 complex of the classical complement activation pathway, has been the most thoroughly studied vertebrate c1qdc protein. in addition to activating c1r and c1s (and, as a consequence, in c3), c1q can also act as pattern recognition receptor (prr) as, through its qc1q domain, it can recognize and bind pathogenassociated molecular patterns (pamps) on the surface of microbes and modulate their phagocytosis. most of the c1qdc proteins have only a gc1q domain but the presence of molecules with multiple tandem c1q domains have been reported in both invertebrates and vertebrates; among the latter, ctrp4 is the only protein with two c1q domains described in mammals, birds, reptiles, amphibians and teleosts. the compound ascidian botryllus schlosseri is a chordate invertebrate that relies only on innate immunity for its defense. immunocytes (i.e., cells with defined roles in immunity) represent the great majority of the circulating hemocytes: they include cytotoxic morula cells and phagocytes. in this same species, we identified the key components of the lectin and the alternative pathways. all these complement components (c3, bf, mbl, ficolin and masp), are expressed by morula cells, the most abundant circulating hemocyte. in this study, we mined the available transcriptomes and identified, in b. schlosseri, a novel multidomain c1qdc protein (bsc1qdc). it belongs to the sghc1q proteins and contains two gc1q domains, a signal peptide and present high similarity with human ctrp4. we followed the expression of bsc1qdc during the colonial 70 blastogenetic cycle and in colonies injected with gram (+) bacteria and identified its mrna location by in situ hybridization (ish). the expression trends during the colonial blastogenetic cycle suggest the presence of checkpoints modulating the transcription of bsc1qdc. the protein is synthesized and released by morula cells and a minority of phagocytes. when we knocked down the gene, we observed a decrease in phagocytosis of target particles, probably related to the involvement of bsc1qdc in the opsonization of non-self, as well as a decrease in degranulation and is involved in. ongoing studies are trying to better clarify the role of bsc1qdc in botryllus immune modulation and its interplay with the other complement components such as complement control proteins. award “giovani laureati” (best presentation and curriculum studiorum for members under 29) immune contribution to tentacle regeneration in adult mollusc and cnidarian models g bergamini1, s sacchi2, m ahmad1, m cocchi1, s basu3, a ikmi3, d malagoli2 1department of chemistry and geology, university of modena and reggio emilia, modena, italy 2department of life sciences, university of modena and reggio emilia, modena, italy 3developmental biology unit, european molecular biology laboratory, heidelberg, germany adult regeneration is a fascinating process that consists in regrowth and regain of function of tissues and organs. the role and contribution of the immune system and immune-related pathways in adult vertebrate and invertebrate regeneration have been investigated for a long time, but important gaps remain. the freshwater snail pomacea canaliculata and the sea anemone nematostella vectensis are two phylogenetically distant organisms, with regenerative capabilities in adult life. these models present different innate immune components, and we focused on their involvement during tentacle regeneration. the two cephalic tentacles of p. canaliculata are sensory components used for food search, co-specific recognition and orienting. in n. vectensis, the numerous oral tentacles (4-18) are extensions of the diploblastic body, forming appendages that feed, defend and expand the surface area of the gastric cavity. histological studies focusing on the early cephalic tentacle regeneration in p. canaliculata, have demonstrated that wound closure and blastema formation took place within 24 h post amputation (hpa). a matlab® plugin allowed the semi-automated identification and quantification of a phagocytic hemocyte sub-population in the blastema. flow cytometry analysis showed that the injection of the phagocyte-specific drug clophosome® (45 µg/g snail) could transiently remove circulating hemocytes, that recovered the pre-treatment level within 24 h. consistently, histological experiment demonstrated that rare hemocytes were present in the early regenerating tentacles of clophosome®-injected snails. moreover, the hemocyte depletion impacted on regeneration time, and the blastema took twice as long to form, i.e., 24 h. this extended time overlaps with the time of recovery from clophosome® treatment, further suggesting a role for p. canaliculata hemocytes in the onset of tentacle regeneration. differently from molluscs, n. vectensis presents no specialized immune cells, though cells displaying phagocytic activity were recently identified. moreover, components of the main pathways of invertebrate humoral immunity are present. a transgenic line labelling a highly motile population of cells (mpc), similar in shape to vertebrate macrophages, enabled us to investigate their behaviours in homeostasis and regenerating conditions. because immunostaining showed an accumulation of mpc to the wound site at 6 hpa, a high-resolution live imaging method was developed to investigate in real time to validate their direct migration to the wound site. in vivo imaging showed that mpc are positive for soxb2, a neural precursor marker, which suggests that mpc originates from a neural cell lineage during development and differentiate in a migrating cell type. to define the molecular signature of mpc, we used facs to sort them in view to perform rna smart-sequencing and characterize their gene expression. in all, these data suggest a pivotal role for hemocytes during early stages of tentacle regeneration in p. canaliculata. similarly, our original data on n. vectensis suggest the presence of cells imitating the behavior of molluscan hemocytes during early stages of regeneration. intriguingly, mpc also seem to share their origin with neural cells, thus providing an example of the tight connection between immune and nervous systems also in diploblastic animals. plenary lecture i immune-microbiota interplay in oyster health and disease d oyanedel, a lagorce, g charrière, m-a travers, d destoumieux-garzón ihpe, university of montpellier, cnrs, ifremer, université de perpignan via domitia, montpellier, france. the presence of complex host-associated microbial communities is a characteristic shared by most animal species and the type of interactions that they establish with their host can range from mutualistic to pathogenic. the capacity for microbes to colonize a host depends on both host and microbial determinants. in the marine environment, bivalve mollusks constitute habitats for bacteria of the vibrionaceae family. vibrio belong to the microbiota of healthy bivalves, which have the ability to concentrate bacteria in their tissues and body fluids, including the hemolymph. the oyster immune system tolerates rather high amounts of vibrio in its hemolymph. however, vibrio can also proliferate in oyster tissues leading to mass mortalities of oysters. other microorganisms such as the oshv-1 virus have the ability to alter oyster immunity leading to 71 dysbiosis and oyster death. in this process oshv-1 and vibrio synergize to kill oysters. however, while some vibrio populations actively cooperate, others behave as cheaters. in this presentation, i will review the current knowledge on the complex immune-microbiota interactions at play in oyster health and disease. session 1. immune competence and immune response (part i). chairmen: paola venier, university of padua, padua, italy and annalisa grimaldi, university of insubria, varese, italy antiviral sensors and znfx1 in the innate immunity of invertebrates g blasi, e bortoletto, m gasparotto, f filippini, u rosani, p venier department of biology, university of padua, padua, italy double-stranded rnas (dsrnas) are commonly detectable in metazoan cells. in particular, dsrnas of viral origin can trigger the immune response in the host. sensors of nucleic acids (including dna, ssrna, or dsrna) have emerged across the three domains of life as a first step in activating specific and non-specific host defenses against viruses. sensors, such as toll-like receptors and cytosolic proteins from the broad family of dexd / h-box helicases, act alone or in combination with other proteins to promote and amplify the antiviral response in infected cells. the zinc finger-type nfx1 containing 1 (znfx1) is one of the early sensors of viral dsrnas. the human znfx1 is an outer mitochondrial membraneassociated helicase, capable of promoting a type i interferon-mediated response. we have traced the presence of znfx1 in metazoans and, based on 221 sequences, we obtained a polyphyletic tree generally consistent up to the phylum level. moreover, we examined the znfx1 expression profiles of selected invertebrate species for which transcriptomic data after viral infection are available. as a result, we found that znfx1 was overexpressed in mollusks (crassostrea gigas, scapharca broughtonii, and haliotis diversicolor) and in a lepidopteran arthropod (trichoplusia ni), but not in a hydrozoan of the genus millepora (acropora millepora), a decapod crustacean (litopenaeus vannamei) and in a nematode (caenorhabditis elegans). overall, our analyses support the role of znfx1 as an early antiviral sensor in different invertebrate species. first morpho-functional characterization of anemonia viridis amoebocytes: a light microscopy study j fabrello, m ciscato, d asnicar, mg marin, v matozzo department of biology, university of padua, padua, italy anthozoan, as other cnidarians have ameboid cells into their mesoglea. these cells, called amoebocytes can move through the tissues and aggregate near wound sites reacting also to grafts. for the first time, we evaluated morpho-functional characteristics of amoebocytes of the sea anemones anemonia viridis. for this purpose, we sampled cells from the mesoglea of sea anemones. under the light microscope, we recognized two subpopulations of amoebocytes: granulocytes and hyalinocytes. granulocytes showed a high number of cytoplasmatic granules, while hyalinocytes showed a cytoplasm with no or few granules. amoebocytes showed both round and spreading shapes and were divided in basophils and acidophils, in addition also neutrophils were observed. amoebocytes actively phagocytized yeast cells and produced intracellular superoxide anion. in addition, we evaluated the presence of hydrolytic enzymes in amoebocytes. we observed positive cells to acid phosphatase, acid esterase and nonspecific esterase, with no differences in term of positivity between granulocytes and hyalinocytes. other studies are needed to fully investigated amoebocytes features in this anemone species, including ultrastructural investigations. analysis of structural variants and associated gene presence-absence variation phenomena in the genome of the pacific oyster crassostrea gigas cf tucci, s greco, m sollitto, pallavicini, m gerdol department of life sciences, university of trieste, trieste, italy in recent decades the advancement of sequencing technology has given the opportunity to assemble very complex genomes on a chromosomal scale and to re-sequence the genome of several individuals belonging to the same species. through the re-sequencing of different individuals of the same population it has been observed that some genomic regions may be present in some specimens and not in others. this phenomenon is called gene presence absence variation (pav) and leads to the identification in a population of two sets of genes: the core genes, i.e., genes shared by all individuals, and the dispensable genes, i.e., genes that may be present in some individuals and absent in others. core genes are mostly linked to housekeeping functions, while dispensable genes are not essential for survival but may provide accessory functions useful for adaptation and survival under certain environmental conditions. pav appears to be widespread in nature and is likely fundamental to allow an enormous inter-individual diversity in many branches of the tree of life. in this work, a bioinformatics pipeline was developed for the identification of dispensable genes in the genome of diploid eukaryotic species and for the study of the pav phenomenon. the pipeline was tested and applied on the pacific oyster crassostrea gigas, demonstrating that pav phenomena also occur in the genome of this species. functional enrichment tests have shown that dispensable genes have specific functions related to survival, such as immune response and apoptosis. further analyses focused on the 72 functional characterization of some of the many molecular players involved in such processes will be necessary to clarify their involvement in pathogen recognition and elimination, and to define in particular the evolutionary advantages that may arise from their presence/absence in different individuals. understanding bivalve physiology through nmr metabolomics r frizzo1, e bortoletto1, t riello1, p venier1, s mammi2 1department of biology, university of padua, padua, italy 2department of chemical sciences, university of padua, padua, italy the increase of abnormal mortalities in marine aquaculture bivalves occurring in this time of global warming has brought more attention to the intricate relationships between host and pathogens. the study of metabolite profiles (relative concentration of small molecules, such as amino acids, carbohydrates and organic acids) in biological fluids and tissues is an expanding field of investigation, successfully applied to human, animal, and plant physiology and pathology. we obtained 1h 1d-nmr tissue-specific metabolic profiles of digestive gland, gills and hemolymph samples of mussels (mytilus galloprovincialis) recovering from functional anaerobiosis or responding to an injection of live vibrio bacteria at 18 °c and 25 °c. we detected many resonances, from 227 in total hemolymph lysates to 883 in the whole flesh samples and 69 82 % of the signals were assigned. the assigned signals mostly arise from free amino acids (faa), osmolytes, organic acids, and sugars, such as mytilitol. mussel acclimation after functional anaerobiosis led to a significant decrease of succinic acid in hemolymph spectra, accompanied by an increase of most faa and osmolytes, more pronounced at 18 °c than 25 °c. after injection of 200 μl of 108 cfu/ml of live vibrio splendidus, several faa, sugars and unassigned chemical species significantly decreased, with similar variations between 18 °c and 25 °c. these variations could match a simultaneous increase of faa in the hepatopancreas, as reported for perna canaliculus injected with vibrio bacteria (nguyen et al., 2019). overall, these results support the integration of nmr metabolomics with other ‘omics as a convenient tool to study marine bivalves. adar-editing in mollusc species: in between physiology and antiviral response e bortoletto1, u rosani1, c montagnani2, c-m bai3, p venier1 1department of biology, university of padua, padua, italy 2ihpe, university of montpellier, cnrs, ifremer, montpellier, france. 3chinese academy of fishery sciences, qingdao, china rna editing processes increase the molecular diversity of primary transcripts and can significantly alter the gene product function. the conversion of adenosine to inosine (a-to-i) is considered as the most common type of rna editing in metazoans and is catalyzed by members of the enzyme family of adenosine deaminases acting on double strand rna (adars). the adar-mediated rna-editing is crucial for the organism homeostasis, for instance the editing of proteins such as ion channels and neuroreceptors is critical in the development and functioning of nervous system. adar is also involved in host-virus interactions, and it can act as proviral or antiviral depending on virus-host combination. as regards mollusca, the physiological role of adar has been investigated only in cephalopoda. we analyzed 87 and 30 rna-seq datasets pertaining to crassostrea gigas and scapharca (anadara) broughtonii, respectively, both exposed to ostreid herpesvirus-1 (oshv-1). we compared the adar-editing levels on host and viral transcripts, and we traced the adar hyper-editing impact on the host genes. in contrast to the infected blood clam, oyster rnas were more hyper-edited than viral rnas and this could relate to the differential amount of oshv-1 dsrnas. a core set of genes were constantly hyper-edited in both bivalve hosts, a finding suggesting a physiological role of adar hyperediting. conversely, host genes involved in antiviral response, mirna maturation and epigenetic regulation were hyper-edited in specific infection phases only. tissue distribution of a rhamnose binding lectin in the colonial ascidian botryllus schlosseri and effects of microinjections of the specific antibody into the circulatory system g bovo, l ballarin department of biology, university of padua, padua, italy lectins are non-enzymatic and nonimmunoglobulin proteins, or glycoproteins, that bind carbohydrates with their carbohydrate recognition domains (crds). they are involved in various biological processes, including host-pathogen interaction and intercellular communication. they play pivotal roles in the immune system of invertebrates by binding pathogens directly and opsonizing them. botryllus schlosseri in a cosmopolitan ascidian, considered a reliable model organism for studies on the evolution of the immune system. b. schlosseri rhamnose-binding lectin (bsrbl) acts as a chemokine and opsonin by interacting with different cell types. although described in previous works, many aspects and roles of this lectin remain unknown. here we studied the changes in tissue distribution of bsrbl during immune responses using light and electron microscopy. in addition, following the hints from extant data, suggesting a possible role of bsrbl in the process of takeover, we investigated the effects of the removal of this protein, using a specific antibody, during the generation change, opening new queries on the roles of this lectin in botryllus biology. 73 phylogenetic and transcriptomic analysis of two putative stat genes in the colonial ascidian botryllus schlosseri and their involvement in immunity. f la torre, a peronato, f gasparini, v vanni, g martello, l manni department of biology, university of padua, padua, italy the jak/stat pathway is an evolutionary conserved signalling pathway triggered by diverse cytokines, interferons, grow factors and related molecules. this pathway represents a remarkably straightforward mechanism whereby extracellular factors control gene expression. 7 stat family members have been identified in mammals, while most of non-chordate invertebrates have one or two stat genes. in all species here investigated belonging to tunicates, which are the chordates considered the sister group of vertebrates, two stat proteins have been found, but their functions have not been elucidated yet. in this study we present the identification of two putative different stat transcripts in the transcriptome of the colonial tunicate botryllus schlosseri. these transcripts, found in silico, have been verified experimentally and sequenced, and their translation produces two proteins named stat1 and stat2. once we obtained the full sequences of these transcripts, we performed a maximum likelihood phylogenetic analysis to investigate the homology relationship between the stats in chordates. the analyses indicate that a duplication from a unique stat gene happened in the chordate ancestor, resulting in the ortholog to vertebrates stat5 and in an ortholog to all the other vertebrate stat gene for all chordate, followed by subsequent duplications from the second one in the vertebrate evolutionary lineage. the molecular expression of b. schlosseri stat genes was measured throughout the colonial asexual cycle. initially, this analysis has been done collecting cdna from whole colonies, and then extended collecting cdna from extracted haemocytes, showing that these genes are differentially expressed in the different colonial phases. as reported in literature, the jak/stat pathway is triggered by several interleukins in mammals, such as il17. three il17 genes have been found in the solitary tunicate ciona intestinalis and their expression is upregulated through lipopolysaccharide (lps) stimulation. the latter activates the immune response, producing the cytokine tnf-α. we hypothesized the potential activation of the jak/stat pathway via lps stimulation in b. schlosseri and we verified it throughout rt-qpcr, finding over expression of the stat genes and myc, an important gene for stemness, which has been proved to be a target of these transcription factors. in conclusion, the results suggest that the jak/stat pathway play a role in the innate immune responses of this colonial chordate. how can we define “self”? complement system and missing-self theory. n franchi department of life sciences, university of modena and reggio emilia, modena, italy the complement system, with its main protein c3, is the main humoral system of the innate immunity, that type of immunity possessed by almost all the animal kingdom. in the absence of regulatory proteins, uncontrolled activation of the system can also result in disruption of host cells, resulting in immune-mediated tissue damage. we know that vertebrate immune system is able to discriminate between self tissues from nonself as well invertebrates. furthermore, the former are able to have a rejection reaction against allogeneic transplants thanks to the adaptive part of their immune system. the latter, that totally lack of adaptive immune system, are able to discriminate between self body and body parts derived from conspecific organisms very similarly to vertebrates. how is it possible if they lack of adaptive immunity? now we know allorecognition machineries in invertebrate animals has nothing to do with those of vertebrates partially responding to the question if allorecognition in the animal kingdom are of monophyletic origin or evolved independently. cnidarian, annelids and protochordates, e.g., are able to allograft rejection with molecular machineries that has nothing in common with the mhcbased histocompatibility reactions of vertebrates, but (some of them) involved highly variable complement receptor-like protein. now i’m exploring the hypothesis that proteins controlling complement system might have an evolutionary ancient role not only in immune defense but also in histocompatibility. hypothesis that associates well with the so called “missing-self theory”. isolation and characterization of the polymeric ig receptor gene from the cold adapted teleost trematomus bernacchii a ametrano1, s picchietti2, l guerra2, s giacomelli1, u oreste1, mr coscia1 1institute of biochemistry and cell biology, national research council of italy, via p. castellino 111, 80131 naples, italy 2department for innovation in biological, agro-food and forest systems (dibaf), university of tuscia, largo dell'università snc, 01100 viterbo, italy the polymeric immunoglobulin receptor (pigr) plays a pivotal role in vertebrate immunity, as it mediates the transport of mucosal antibodies across epithelial layers into the external secretions. in recent years, much attention has been devoted to the function of pigr in teleost fish, however, information on its gene structure remains still limited. not even data are available on pigr from teleost species living under extreme conditions, such as notothenioidei (suborder 74 perciformes), the dominant group of fish inhabiting the extremely cold environment of antarctica. to enhance the current knowledge in this field, we characterized the structure of the pigr gene of the antarctic teleost trematomus bernacchii, through a comparative analysis built on genomic and transcriptomic databases available for multiple species belonging to five perciform suborders. we identified unexpected modifications in the antarctic pigr genes, e.g., intron lengthening, transposable element content and additional regulatory elements. furthermore, the full-length cdna and deduced amino acid sequence were obtained. multiple sequence alignment highlighted that several amino acid substitutions were exclusive to antarctic pigrs. among notable changes, some led to a gain of nglycosylation sites, strongly suggesting a putative role in the adaptive response to cold temperatures. expression analysis through q-pcr and in situ hybridization showed that pigr transcripts were constitutively expressed in the mucosal tissues and liver. overall, our work unraveled specific features of the pigr gene from cold adapted teleost species, providing additional information regarding the structure and organization of pigr genes in teleost fish. this research was funded by the italian national program for research in antarctica, grant number pnra18_00077 plenary lecture ii climate change and pollution effects on bivalves: cell culture models as promising tools? d mello1, c corporeau1, y even1, c brigaudeau2, h talarmin3, c lambert1, c dubreuil1, m smits1, s artigaud1, v pichereau1, g le blay1, c paillard1, s madec1 1laboratory of environmental marine sciences (lemaruniv brest, cnrs, ird, ifremer, umr6539) lemar, f-29280, plouzané, france. 2laboratory of b cells and auto-immunity (lbaiinserm u1227), university hospital center, 5 avenue foch, brest, france 3laboratory of optimization of physiological regulations (orphy-ea4324), medicine university, 22 rue camille desmoulins, brest, france an overarching aim of the laboratory of environmental marine sciences (lemar) from université de bretagne occidentale (ubo) in france is to better elucidate the interactions between marine organisms and their environment. a better understanding of organism adaptation to biotic and abiotic factors and the role of the microbiota in health and disease are crucial focal points for ecosystem sustainability on a changing planet. to address this challenge, our group is currently developing two key research strategies using two bivalve species: the pacific oyster crassostrea gigas and the manila clam ruditapes philippinarum. bivalves are well-recognized as excellent models to study environmental health and, most recently, host-microbe-environment interactions. our first strategy aims at better understanding the health impacts of pollution and climate change on aquatic invertebrates, by performing measurements at cellular level. cells integrate a multiplicity of signals to ensure organismal defense, survival and growth, thus disruption of cellular function can be detrimental often leading to decreased health or lifespan. it is therefore essential to improve culture protocols of bivalve cells. like the oyster cardiomyocyte model, a well-standardized and long-term hemocyte culture model would be really useful, as well as a toolbox of cellular tests for functional monitoring, particularly for ecotoxicology analyses. our group has recently successfully optimized the conditions to maintain c. gigas immune cells (hemocytes) viable and functional in culture for at least fifteen days, which is unprecedented for bivalve hemocytes. our second research strategy aimed to address the fundamental and timely issue of the manila clam's response to climate change, and encompasses a unique, multi-level and transdisciplinary experimental research plan. the recently accepted international climclam project, joins scientists from two laboratories, lemar and the department of comparative biomedicine and food science (bca) from university of padua (unipd) in italy. by combining in vitro and in vivo approaches, our group aims at unravelling environmentally relevant cellular mechanisms and organismal effects of biotic and abiotic factors in the context of global changes to support human, ecosystem, and animal health (one health). session 2. development and immunity. chairmen: giuseppe scapigliati, university of tuscia, viterbo, italy and davide malagoli, university of modena and reggio emilia, modena, italy the role of the immune modulator hvrnaset2 during development l pulze, n baranzini, a grimaldi university of insubria, department of biotechnology and life sciences, varese, italy the hirudo verbana protein hvrnaset2 is a pleiotropic enzyme, acting as a key molecule in inflammation, immune response and regenerative processes. indeed, following bacterial infection, hvrnaset2 induces macrophages recruitment in order to trigger and potentiate the inflammatory response. furthermore, hvrnaset2 is also involved in the correct progression of muscle tissue regeneration during wound healing process, by regulating the recruitment of the myoendothelial vessel-associated precursor cells, fibroplasia and synthesis of new collagen. taken together, these data strongly suggest that during wound healing and tissue regeneration, hvrnaset2 is involved in the restoration and maintenance of tissue homeostasis through extracellular matrix (ecm) remodeling. 75 recently, several data in literature have demonstrated a correlation between collagen synthesis, ecm organization and tissue morphogenesis during embryogenesis and postembryonic development. in order to shed light on the possible role of hvrnaset2 as modulator of collagen deposition during these processes, here we evaluated the expression of this enzyme during different stage of h. verbana development, starting from the embryos until the juvenile and adult leeches. defining the antarctic krill’s ontogenesis from a transcriptomic point of view a biscontin1, f muller2, i urso1, c bertolucci4, s kawaguchi5, b meyer2,3, c de pittà1 1department of biology, university of padua, padua, italy 2alfred wegener institute helmholtz centre for polar und marine research, bremerhaven, germany 3institute for chemistry and biology of the marine environment, university of oldenburg, oldenburg, germany 4department of life sciences and biotechnology, university of ferrara, ferrara, italy 5department of environment and heritage, australian antarctic division, kingston, tasmania, australia the antarctic krill (euphausia superba) has a key role in the southern ocean ecosystem. during the last 30 years, the abundance of krill in the southwest atlantic sector has constantly decreased and the reasons behind this decline are still unclear. the main bottleneck that affects the population abundance is the surviving to the larval life across the first winter. daily vertical migrations as well as an oscillatory oxygen consumption pattern have been recently observed in krill larvae and they likely take part in a complex behavioural and physiological strategy to increase the recruitment success. total rnas extracted from seven different krill’s larval stages, belonging to three out of 4 main developmental phases (metanauplius, calyptopis and furcilia), were used to produce and sequence seven stage-specific cdna libraries (illumina technology) in order to define the gene expression signature of each developmental stage. also, the juvenile stage which represents the adult stage that is not sexually mature was included in the analysis. we obtained a total of about 1.2 billions of 100 nt paired-end reads. an unsupervised hierarchical clustering analysis showed specific gene expression signatures for early developmental larval stages with respect to late larval stages and young adult (juvenile). interestingly, among differentially expressed genes between early and late larval stages we identified the circadian clock components as well as genes involved in light entrainment that could elucidate the role of the time-keeping system during larval development. dynamics of formation of stress granules during the colonial blastogenetic cycle of botryllus schlosseri l drago, g santovito, l ballarin department of biology, university of padua, padua, italy stress granules (sgs) are cellular ribonucleoprotein foci preserving mrnas for antistress proteins and so regulating stress responses. this is possible thanks to the presence of mrnabinding proteins such as tia-1 related nucleolysin (tiar), considered an important core component of sgs. botryllus schlosseri is a colonial ascidian easily found in the lagoon of venice, which undergoes weekly generation changes called take-overs (tos). a blastogenetic cycle is defined as the period between two successive tos. during the to, lasting 24-36 h, a diffuse apoptosis occurs in tissues of old zooids, which will be replaced by their primary buds representing the new generation. at to, an increase in oxygen consumption (respiratory burst) takes place with the consequent production of reactive oxygen species representing a stressful condition for the new zooid generation. we suppose that sgs can play a pivotal role in the protection from oxidative damages. to verify this hypothesis, in this work we used the tiar protein as marker to study the dynamics of formation of sgs during the colonial blastogenetic cycle of b. schlosseri. at first, we analyzed the modulation of mrna transcription levels for tiar by quantitative real time pcr (qrt-pcr) and the location of its transcript in the hemocytes through in situ hybridization (ish). then, we used an antibody specific for tiar on hemolymph monolayers, and on colony paraffin sections, to confirm the involvement of immunocytes in detoxification. our results agree with the idea that immunocytes represent the major detoxification system in ascidians, active in the control of tiar protein synthesis and, therefore, in sgs formation. brain and immunity in the teleost fish dicentrarchus labrax along development: a preliminary study v pianese, a miccoli, g scapigliati, f buonocore, am fausto. s picchietti department for innovation in biological, agrofood and forest systems, university of tuscia, viterbo, italy the central nervous system (cns) was traditionally considered an immune privileged anatomical region, unable to produce a proinflammatory immune response, as it would be hazardous for the brain tissue integrity. however, it has recently been found that multiple lymphocyte populations reside in and/or migrate to mammalian brain compartments under steady state conditions, playing fundamental roles in cognitive process, 76 neurogenesis and immunosurveillance. the european seabass (dicentrarchus labrax), an economically relevant marine teleost fish, is a wellknown attractive model for developmental, anatomical and functional comparative studies of the immune system (is). however, despite the recent advances, the link between the adaptive immunity and the cns is still unclear in this species, and the current knowledge remains very limited. thus, we report herein a preliminary study aimed at describing the relationship between is and cns along its development. two panels of monoclonal and polyclonal antibodies, currently available for d. labrax, were used for the identification and localization of t and b lymphocytes within the cns, both in larval stages and juveniles. moreover, the expression of several immune-related genes was evaluated through q-pcr in the brain tissue of juvenile specimens. finally, a novel rna-seq experimental approach is being used for an in-depth expression analysis, which has never been carried out before on early sea bass developmental stages. using the laser microdissection technology, we dissected the brain of sea bass larvae, and the transcriptomes obtained will soon be analyzed for the first expression of its kind of immune and neurorelated genes. session 3. organism interactions. chairmen adriana vallesi, university of camerino, camerino (mc), italy and valerio matozzo, university of padua, padua, italy structural and phylogenetic evidence supports a key role played by helix-3 of euplotes pheromone structure in autocrine and heterologous pheromone/receptor interactions c alimenti1, b pedrini2, p luporini1, a vallesi1 1school of biosciences and veterinary medicine, university of camerino, camerino (mc) italy 2paul scherrer institute, 5232 villigen psi, switzerland like many other organisms, ciliates rely on diffusible pheromones to socialize. in euplotes, these cell signals form species-specific families of structurally homologous disulfide-rich globular proteins which bind their receptors on target cells in competition with one another, eliciting cell growth or mating responses according to whether the binding occurs in autocrine, or heterologous fashion, respectively. in each cell type, the receptors’ extracellular ligand binding domain is structurally identical with the soluble pheromone, as a result of a common gene determination via an intron-splicing mechanism. given this structural context, the pheromone/receptor interactions were inquired by carrying out a comparative crystallographic analysis of pheromones er-1 and er-13 each specific to cells with strong mating compatibility. er-1 and er-13 crystals showed to markedly differ in their symmetry space groups, c2 and p43, respectively. nonetheless, both crystals equally result from a tight association of molecules into linear chains, in which each molecule (i) rigorously takes an opposite orientation with respect to its neighborhood (as expected to be the case for pheromone and receptor molecules interacting on the cell surface), and (ii) forms contact interfaces relying on aminoacid side-chains lying in the great majority on helix-3 of its three-helical fold. this identification of helix-3 as central functional element of the pheromone molecular structure receives strong support from the tight conservation of the backbone structure that this helix shows at both intraand inter-specific level and suggests a parsimonious explanation for the molecular mechanisms underlying the competitive, autocrine and heterologous, pheromone/receptor binding reactions. session 4. environmental stress and immunity. chairmen: maria giovanna parisi, university of palermo, palermo, italy and luigi abelli, university of ferrara, ferrara, italy first insights into the mechanisms involved in immune response in mytilus galloprovincialis towards the emerging pathogen arcobacter m auguste1, f ur rahman2, t balbi1, m leonessi1, c oliveri1, l vezzulli1, d furones2, l canesi1 1distav, dept. of environmental, earth and life sciences, university of genoa, genoa, italy 2irta-sant carles de la ràpita, sant carles de la ràpita, spain bacteria of arcobacter spp. are regarded as emerging foodborne zoonotic pathogens affecting both humans and animals. their presence has been increasingly reported in seafood, suggesting its increasing occurrence in seawater as a consequence of marine water pollution, this raising some environmental concern. more recently, the presence of arcobacter species has been reported in diseased oysters (crassostrea gigas) during mortality events or in stressed bivalve species. however, no data are available so far on the persistence of arcobacter in bivalves, its potential pathogenicity or interactions with the immune system of the host. in the present work two strains of the genus arcobacter, isolated from moribund oyster during a mortality event in 2019 in spain (r1 and r2) were investigated for their interactions with the immune system of mytilus galloprovincialis both in vitro, in isolated hemocytes, and in vivo, in injected mussels at 24 h p.i.. the results obtained suggest that mussels are able to mount an efficient immune response against these arcobacter strains. the most notable effects of arcobacter on mussel hemocytes both in vitro and in vivo was observed on lysosomal membrane stability, with the presence of swollen lysosomes and large vacuoles, and on the increase in extracellular ros production and lysozyme activity. these responses contribute to a significant bactericidal activity, with a large contribution of hemolymph serum alone. indeed, arcobacter observed within the lysosomal system showed signs of degradation over time. in contrast, preliminary experiments with c. gigas indicate that arcobacter did not induce ros production and lysozyme 77 activity by hemocytes, and the absence of bactericidal activity in hemolymph serum. the results provide a first insight on the immune responses of different bivalve species towards arcobacter strains and the underlying mechanisms. inflammation events occurring upon bacterial infection in mytilus galloprovincialis c la corte1, n baranzini2, m. dara1, a grimaldi2, mg parisi1 1department of scienze della terra e del mare, university of palermo, palermo, italy 2department of biotechnology and life science, university of insubria, varese, italy bivalves, and in particular the mediterranean mytilus galloprovincialis are important sources of food in several countries in the world. because of that, mussels farming has a strong economic impact. due to their status as sessile and filterfeeding animals, bivalves accumulate in their tissues environmental pollutants and a larger amount of microorganisms and between these, a multitude of infective bacteria for higher vertebrates and humans, such as vibrio species. several immunological responses of m. galloprovincialis were investigated and described after vibrio infection both, in vitro and in vivo conditions, such as hemocytes count and different cellular subpopulations. particularly, intracellular signaling pathways are activated to trigger the synthesis of antimicrobial effectors here, were investigated the modulation of immunological cellular markers of the mediterranean bivalve m. galloprovincialis in response to in vivo exposure with vibrio splendidus. the activation of inflammatory cascade was examined through immunolabeling with antibodies involved in the pathway: toll-like receptors 4 (tlr4), myeloid differentiation factor 88 (myd88), allograft inflammatory factor-1 (aif1) and ribonucleases rnaset2 (t2 family), that trigger the recruitment and activation of macrophages in vertebrates. results confirmed the activation of trl4 during bacterial infection and myd88 adapter suggesting a role in recognition and intracellular signaling. moreover, gram-negative bacteria determine the recruitment by the ribonuclease rnaset2 of haemocytes and a huge migration of aif-1+ cells. this approach is suitable to understand the molecular defense mechanisms in invertebrates during the exposure to possible pathogens, also in order to develop new technics and tools to evaluate mussel immunity response used in aquaculture to prevent mass mortality of these mollusks, economic loss and potential risks for consumers of seafood. investigating the role of viral infections in the population of the congeria kusceri a scapolatiello1, u rosani2, c manfrin1, s puljas3, a pallavicini1, m gerdol1 1department of life sciences, university of trieste, trieste, italy 2department of biology university of padua, padua, italy 3faculty of science, university of split, split, croatia congeria kusceri (bole, 1962), a highly endangered freshwater bivalve endemic to the dinaric karst, belongs to the only extant genus of cave-dwelling bivalves. this cave clam lives in a unique habitat, which has been subjected to minimal environmental changes over the past five million years. since the natural populations of c. kusceri have been quickly declining over the past few decades, a key question to be answered is whether the “living fossil” status of this species, i.e. the apparent lack of morphological and physiological changes compared with its fossil relatives, is linked with a scarce resilience to the impact of human activities on the subterranean environment. in particular, the alteration of the seasonal abundance of pathogens, linked with the modified influx of water in karst caves, might pose a severe threat to the survival of this species. here, through a transcriptomic approach, we describe the identification in the tissues of c. kusceri of 5 nearlycomplete genomes of rna viruses belonging to the picornaviridae family, which displayed a strong tissue preference and a significantly changes in abundance over the summer season. rna-seq data also allowed to investigate whether these viral infections had an impact on gene expression in different host tissues. since numerous reports have previously implicated picorna-like viruses in the mass mortality events observed in different bivalves, we suggest that the presence of the 5 identified viruses should be closely monitored, together with other biological and chemical parameters, to gather a better understanding of the causes underlying the quick decline of c. kusceri populations. ampylation: a new facet of host-virus crosstalk in mollusks u rosani1, r frizzo1, s kawato2 1department of biology, university of padua, padua, italy 2laboratory of genome science, tokyo university of marine science and technology, tokyo, japan fic-domain-containing enzymes (ficd) performed post-translational protein modifications from bacteria to humans, known as ampylations. as part of the toxin-antitoxin system, ficds of pathogenic bacteria occasionally induce ampylation to overcome host defenses, whilst vertebrates ficds drive the unfolded protein response (upr) and act during neurogenesis. mining genomic and transcriptomic data of protostome metazoans, with a focus on marine invertebrates and associated dsdna viruses, we traced a single-copy ficd gene transversally conserved in metazoans and viral pathogens, with structural and functional traits suggesting a preserved ampylation capacity. extranumeral ficd gene copies are present in rotifers, in some bivalves and in the isopod armadillidium vulgare genomes. less conserved protein features and no syntenic conservation suggested their recent 78 genome integration by horizontal gene transfers from endosymbiont or microbiome communities. analyzing dual rna-seq data of the only host-virus combinations both encoding a ficd gene, we revealed a time-dependent expression for white spot syndrome virus and ostreid herpesvirus-1 ficds, with higher expression levels than crab and bivalve genes. the frequent exchange of ficds and the shift of this enzymatic ability from bacteria to pathogenic dsdna viruses underlie complex hostpathogen-predator interactions in the marine environment and a possible role for ampylation at the edge of host-virus interactions in mollusks. protein extractions from amphistegina lessonii: a new approach for the evaluation of the effects of heavy metals on benthic organisms c ciacci1, m betti1, s abramovich2, m cavaliere3, f frontalini3 1department of biomolecular science, urbino university, urbino, italy 2department of earth and environmental sciences, ben gurion university of the negev, beer sheva, israel 3department of pure and applied sciences, urbino university, urbino, italy different stressing factors such as heavy metals, nanomaterials and others environmental contaminants can induce physiological and biochemical changes on foraminifera, unicellular organisms typically living in sediments. research on protein content of foraminifera is currently limited and no comparative analysis on the efficacy and reliability of the different assays has been conducted on foraminifera so far. the first purpose of this study is to compare the results of different methods of lysis, protein assays, and protein staining on amphistegina lessonii – a symbiontbearing foraminiferal species and presents a new protocol. this protocol could be applicable to other benthic foraminiferal species and represents a new experimental approach for the application of a. lessonii as a potential biomarker. the second part of this study is related to the evaluation of short-term effects of hg exposure (10 ppb, 24 h) on a. lessonii. benthic foraminifera have been mostly utilized as pollution bioindicators in marine and transitional marine environments and mercury contamination is a global issue due to its significant toxic effects on human health, and cytotoxicity being higher than many other heavy metals. in this work, we have evaluated the activity of different enzymes involved in antioxidant defence (sod, gst, gsr and gpx) and several proteins (hsp70 and p38mapk) are analysed by western blotting. the results show that the activities of the antioxidant enzymes increased in hg-exposed foraminifera, indicating that heavy metals can induce oxidative stress and stimulate the engagement of antioxidant enzymes as cellular defense mechanisms; moreover, hg treatment induces expression of hsp70 and activation of p38mapk. as previously demonstrated, these data support that heat shock proteins (hsp) play a crucial role in maintaining protein homeostasis under stress conditions in both prokaryotes and eukaryotes and the mitogen-activated protein kinase (mapk) pathway is an evolutionarily conserved signalling pathway existing in unicellular organisms to humans. the results can be used to obtain information on the environmental quality of marine sediments and the development of the cellular biomarkers might represent a complementary approach to the traditional biomonitoring. hirudo verbana as a freshwater invertebrate model to assess the effects of polypropylene nano and microplastic dispersion. n baranzini, l pulze, c bon, l izzo, a grimaldi department of biotechnology and life sciences, university of insubria, varese, italy plastics represent the most widely employed synthetic materials that, given their physical and chemical properties, are used to produce robust and above all economic objects. although from their discovery these materials became indispensable, their worldwide distribution caused an uncontrolled accumulation of waste products followed by an indiscriminate release in the environment. in particular, millions of tons are reversed in waters every year, making the aquatic ecosystems the most affected by plastics pollution. moreover, their degradation, due to biotic and abiotic events, leads to the formation of small-size particles, known as nano (nps) and microplastics (mps), that can persist inside organisms for a long time and can accumulate in the trophic chain. to evaluate the possible tissue accumulation and harmful effects in freshwater dispersion, we have exposed the leech hirudo verbana to fluorescent polypropylene nps and mps at different timings (1 and 6 hours, 1 week, 1 and 2 months). optical and fluorescent analyses demonstrate that these particles penetrate inside the organism both through the integument and food and induce epidermal mucous cell proliferation, angiogenesis, release of the pro-inflammatory molecules hmaif-1 and hvrnaset2, macrophage-like cell migration and amyloidogenesis. moreover, qpcr analyses reveal an increasing expression of the specific oxidative stress enzymes superoxide dismutase and glutathione s-transferase. taken together, these data suggest the medicinal leech h. verbana as an innovative environmental biomarker ideal for evaluating the possible harmful effects deriving from nps and mps on freshwater animals. study of immunotoxicity responses of sabella spallanzanii exposed to copper sulphate f bertini, c la corte, m dara, d parrinello, mg parisi department of earth and sea sciences, university of palermo, palermo, italy in the last decade, the growing use of heavy metals in human activities has inevitably altered the health of the aquatic environment. marine pollution due to heavy metals is an issue that has now a global dimension and has interested many researchers because of their ability to persist, 79 bioaccumulate and biomagnify in the trophic chain leading to adverse effect also on human health. copper sulphate is a very soluble xenobiotic, still used today into antifouling paint and in aquaculture like biocide, with high activity against algae, fungi and marine invertebrates. its main component is copper that at high concentrations have immunomodulating effect on marine organisms. in this study we have used the polychaete worm sabella spallanzanii to investigate its immune response after exposure to copper sulphate and the combinate effect with the inoculation of escherichia coli, to provide an overview of effect biomarkers for monitoring chemical and bacterial stressors. we aimed also to validate the species as model organism in marine-coastal biomonitoring and to investigate the modulation of tlr-4 as response useful to evaluate environmental alterations. polychaetes were subjected to five treatments: 1. control (naïve), 2. filtered sea water + tbs injection, 3. filtered sea water + e. coli injection, 4. copper sulphate + tbs injection, 5. copper sulphate + e. coli injection. the exposure to xenobiotic was chosen in relation to the specificity of the animals’ response and the bacterial injections were made after the exposure. the immune markers evaluated in the total body extract of the animals were inflammatory and antioxidant markers. therefore, were investigated the activity of: esterase (est), alkaline phosphatase activity (alp), cytotoxicity and detoxifying/antioxidant enzyme such as glutathione peroxidase (gpx). in addition, toll-like receptor (tlr), allograft inflammatory factor-1 (aif1), lysozyme (lys) and hemagglutinating and inhibition activity have also been considered to highlights possible modulations. the analysis of results indicated a significant activation of tlr-4 and aif -1 in specimens inoculated with bacteria and treated with combinate exposure factors. the results regarding lysozyme and peroxidase enzyme as well as the hemagglutination activity have well highlighted the differences between the different environmental stimulations. overall copper sulphate had an immunomodulating effect of the species under investigation, varying the immune response especially after bacterial inoculum. herbicide exposure alters the expression of antimicrobial peptide patterns in the mealworm beetle infected with the natural entomopathogen beauveria bassiana ml vommaro1,2, c zanchi2,3, a giglio1, j kurtz2 1department of biology, ecology and earth science, university of calabria, arcavacata di rende (cs), italy 2institute for evolution and biodiversity, university of münster, münster, germany 3institute for biology, freie universität berlin, berlin, germany herbicide treatments are an integral part of agricultural practice. however, repeated applications lead to soil, water, and food contamination and adverse effects on non-target organisms. in this study, a pendimethalin-based commercial formulation (pnd) was tested on the beetle tenebrio molitor linnaeus, 1758. the effects of herbicides on interspecific relationships, such as host-pathogen interaction are poorly studied. in the laboratory, adults were exposed to two different doses, the concentration corresponding to the contamination of the treated soil and the maximum residue level allowed by the eu in cereals. the beetles were then exposed to an inoculum with the entomopathogenic fungus beauveria bassiana (bb), commonly used as a bioinsecticide. survival, sporulation of the fungus in cadavers, and expression levels of antimicrobial peptides (amps) tenecin 1, 2, and 3 were examined. although the survival rate did not change significantly between control and herbicide-exposed beetles, an alteration in the expression pattern of inducible amps was recorded. pnd-treated beetles showed up-regulated tenecin 1 and tenecin 2 after inoculation with bb. in addition, a slight increase in bb sporulation on cadavers was recorded at the highest dose of the herbicide. our results showed a potential alteration of the host-pathogen interactions, raising the question of bioinsecticide compatibility with synthetic pesticides and the effects of herbicides on interspecific relationships in wild species. organ-specific accumulation of plga nanoparticles injected into pomacea canaliculata snails: preliminary in vivo observations a ferrari1, j duskey2, g bergamini3, a rinaldi2,4, r fiorino1, b ruozi2, g tosi1, d malagoli1 1department of life sciences, university of modena and reggio emilia, modena, italy 2nanotech lab, te.far.t.i., department of life sciences, university of modena and reggio emilia, modena, italy 3department of chemical and geological sciences, university of modena and reggio emilia, modena, italy 4clinical and experimental medicine phd program, department of biomedical, metabolic and neural sciences, university of modena and reggio emilia, modena, italy nanoparticle-delivered drugs are attracting growing interest for their potential to minimize side effects and create “patient personalized” therapeutic applications. currently, preliminary animal testing of nanodrug systems must be performed following the european directives and national laws, which are mainly centered on vertebrates. however, pomacea canaliculata, a freshwater amphibious snail from south america, is emerging as a possible substitute of vertebrates in preliminary bio-accumulation and bio-safety studies of nanodrug systems. p. canaliculata, which is easy to breed, manipulate, and presents a similar weight and longevity to mice, has already been proposed as a potentially model suitable for studying the distribution and organ accumulation of superparamagnetic iron oxide nanoparticles (spions, size 14-80 nm). here the accumulation of the cy5 fluorescently labeled fda approved polylactic-co-glycolic acid nanoparticles 80 (120-180 nm, z= ~-25 – -30 mv) (plganps-cy5), often used in the literature as np drug delivery systems, was assessed after injection in the snail’s foot (20 mg/kg) and incubation for 4 h, 24 h, or 1 week. the considered organs were the posterior (pk) and anterior kidney (ak), the digestive gland (dg), a functional analogue of vertebrate liver, the lung, and the cerebral-pedal ganglion ring (g). at the end of the incubation times, the organs were dissected and processed for paraffin embedding and laser scanning confocal microscopy (lscm) observations. lscm evidenced that plga-nps-cy5 accumulated in the pk more than the other target organs in all the experimental times, but the highest levels of accumulation were seen after 4 h. the accumulation within the pk was not homogeneous but concentrated in regions labeled as hemocyte islets. plganps-cy5 also accumulated in the ak at all the tested time intervals but, contrary to the pk, the maximal positivity was observed at 24 h postinjection (hpi). surprisingly, the dg did not seem to retain plga-nps-cy5, though the presence of autofluorescent pigments made the observation difficult. plganps-cy5 were seen in the lung only at 4 hpi and in the ganglia at 24 hpi. tissue distribution of fluorescent nps suggested that they did not massively enter the ganglia, but they remained in the peripheral tissue. further studies are necessary for detailing whether the plga-nps-cy5 enter the cells, but these preliminary results show that plga-nps-cy5 are distributed by the snail open circulatory system and are retained in a time and organ-specific fashion with an absence of suffering or mortality. these first results could pave the way for using invertebrate models such as pc in preliminary in vivo observations concerning np biotoxicity, distribution, and accumulation as a cheaper and more manageable first analysis before involving mammals or other vertebrate models. immunotoxic effects of a new-generation antifouling biocide in a compound ascidian r varello, f cima department of biology, university of padua, padua, italy dichlofluanid has long been employed as a fungicide in agriculture and has been massively introduced in antifouling paints for boat hulls over the last two decades. one of the most important toxic effects of antifoulants is represented by immunosuppression in marine invertebrates, which can be analysed in vitro with a number of short-term toxicity assays on haemocytes. among bioindicators, the colonial ascidian botryllus schlosseri is a useful candidate; it is a filter-feeding organism living in the water-sediment interface that is found worldwide and is sensitive to antifouling xenobiotics. dichlofluanid adversely affects both immunocyte lines (phagocyte and cytotoxic lines) after exposure to sublethal concentrations. at 0.05 μm (16.65 μg/l), dichlofluanid induced haemocyte apoptosis and cell shrinkage with a decrease in both motility and phagocytosis. at the lowest concentration (0.01 μm, 3.33 μg/l), inhibition of pivotal enzymatic activities of phagocytes and cytotoxic cells occurred. at the highest concentration (0.1 μm, 33.3 μg/l), dichlofluanid increased glutathione oxidation, leading to stress conditions. the effects of dichlofluanid on immune defence responses are similar to those of organometal-based antifoulants (i.e., organotin compounds and zinc pyrithione), and its use in coastal areas requires attention. effects of nanoand microplastics in the development of immune memory in the ascidian ciona robusta d melillo1, r marino2, p italiani1,2, d boraschi1,2,3 1istituto di biochimica e biologia cellulare, cnr, naples, italy; 2stazione zoologica anton dohrn, naples, italy; 3shenzhen institute of advanced technology, chinese academy of science, china microplastic contamination appears as one of the world’s main environmental concerns. small plastic fragments dispersed in the marine habitat can be consumed by different marine organisms and ultimately transferred to humans along the food chain. microplastics could compromise the health of marine organisms by interfering with the functionality of the digestive system when ingested, but there is concern that they could also interact with the immune system and affect the susceptibility/resistance of animals to the infections. recently, we have demonstrated the establishment of immune memory in the ascidian ciona robusta by priming and challenging animals with microbial agents. this immune memory relies on the modulation of different cellular and humoral immune mechanisms, aiming to develop a more protective response. in the present study, we analyzed the expression level of several immune-related genes in pharynx and gut of animals primed by shortor longterm exposure (2 and 18 h) to nanoand microplastics (nmps) and challenged seven days later with a prototypical inflammatory stimulus (bacterial lipopolysaccharide, lps). we aimed to determine (i) whether the nmps can induce an immune response in different tissues, (ii) whether the nmps could prime the animals and drive the development of innate memory, and finally (iii) to which extent filterfeeding animals could stand this kind of pollutants. transcription data suggest that animals primed with nmps develop an immune memory that potentiates the secondary response to lps and that such memory is tissue-specific and depends on both the length of the exposure period and the particle size. this study demonstrates that nmps can modulate the immune reactivity of marine invertebrates, which can influence their defensive fitness. how this may affect their health and survival capacity is still unknown. this work was funded by the h2020-mscaitn-2018-812661 project endonano and financed through the joint research centre of the european commission proof of concept project “nanoplastics”. 81 metallothionein gene expression in trematomus eulepidotus as a response to environmental variation of metal ion concentrations m carnera, s schumann, p irato, g santovito department of biology, university of padua, padua, italy. antarctic waters are characterized by a naturally high concentration of metal ions such as cu and cd. this peculiar condition has probably influenced the evolution of the organisms that populate this environment. indeed, those ions tend to bioconcentrate into the animals that occupy the higher levels of the trophic chain, like fishes. antarctic fishes are able to reduce negative effects caused by the excess of metal ions thanks to the presence of specific adaptations as the expression of two distinct metallothioneins (mts) isoforms, which are regulated by a gene expression control mechanism. mts are a family of cysteine-rich, low molecular weight proteins. they have the capacity to bind both physiological (such as zn, cu, se) and xenobiotic (such as cd, hg, ag, as) metals through the thiol group of its cysteine residues. in this work, we analyzed the gene expression of mts in the antarctic fish trematomus eulepidotus, experimentally exposed to an increase of cu and cd concentrations. analyses were performed in liver, white muscle, heart and kidney, using realtime pcr, and the expression levels were related to the physiological role of these organs and tissues. the data presented in this preliminary study improve our knowledge about the molecular and functional evolution of antarctic fishes and may be used as a starting point for using mts as biomarkers both for the exposition to cu and cd ions and oxidative stress, considered the antioxidant function of this proteins, using notothenoids as bioindicator organisms in biomonitoring campaign of antarctic marine habitat. supported by p.n.r.a. and m.i.u.r. grants. transcriptomic response of trematomus bernacchii to shortto medium-term mild heat stress and experimental design bias s greco, as gaetano, a pallavicini, pg giulianini, m gerdol department of life sciences, university of trieste, trieste, italy many stenotherm marine species live in the cold and stable environment of the antarctic ocean which could be impacted by climate change in the upcoming years. to investigate how antarctic fish would cope with this issue, gene expression analysis was carried out on trematomus bernacchii specimens caught near mario zucchelli station. brain, gill and muscle tissues were sampled from naïve (right after catch) animals and those kept in control (-1.7 °c) and experimental (-0.2 °c) tanks for six hours, seven and twenty days post acclimation. the brain was the most affected in terms of gene expression, showing a time dependent pattern. immune response was up-regulated at seven days of exposure, as the 114 upregulated genes (over a total of 117 differentially expressed genes, fdr < 0.05 and |logfc| > 2) were significantly enriched (fdr < 0.05) in gene ontology terms such as: endopeptidase inhibitor activity, complement activation, inflammatory response. in the same tissue the response to a 20 days heat stress consisted in 519 up-regulated and 490 downregulated genes. the up-regulated set was enriched in terms related to cell adhesion, synapse and glutamate receptor activity, while the downregulated genes were enriched in terms related to ribosome, mitochondrion, energy management, protein folding/turnover and cytoskeleton. interestingly, consistent reduction in expression levels of hspa9, hsp90aa1.2, hsp90ab1, hspa14 and hspa8b was observed. gill tissue showed a mild response after 20 days, with 17 up-regulated and 7 down-regulated genes: the enrichment test suggested that dna replication and negative regulation of apoptosis processes were perturbed in this tissue. a notable early-starting response to stabling was also observed across the entire experiment in brain (2879 degs) and gills (239 degs). expression pattern clustering analysis was performed, allowing the identification of trends in gene expression and a more thorough enrichment analysis of the identified clusters. in brain many synapse-related genes were down-regulated and energy-related genes were up-regulated early after transfer to the small experimental tanks, while several binding processes increased their expression level at the latest time point. in gills gene modulation response was milder and mostly involved cytoskeleton and glycolysis related genes in the early experimental phases. no significant change was observed in muscle. these results show that the brain is the tissue most affected by heat and confinement, demonstrating how sensitive it is to the smallest environmental changes and the importance of careful experimental design when working with captive wild organisms. the effect of group size on the stress level in an open-channel swimming flumeon telestes muticellus s schumann1, g mozzi2, c manes2, d nyqvist2, c comoglio2, e negrato3, p irato1, d bertotto3, a marion4, g santovito1 1department of biology, university of padua, padua, italy 2department of environment, land and infrastructure engineering, politecnico of turin, turin, italy 3department of comparative biomedicine and food science, university of padua, padua, italy 4department of industrial engineering, university of padua, padua, italy artificial barriers in rivers cause a substantial decline in endemic and migratory fish due to habitat fragmentation, changed reproduction environments and blocked migratory routes. fishways can ensure a safe passage over these barriers and sustain biodiversity in rivers. for this reason, it is essential to understand fish swimming performance and kinematics to build appropriate fish passages. high 82 velocities at passages, in which fish cannot consist or counterbalance, can result in physical stress. in schools, better performance is observed due to the hydrodynamic benefit of swimming near other individuals. a multidisciplinary research approach was tested to study interactions of group size at different hydraulic conditions. therefore, collective behavior was analyzed concerning physiological stress responses. wild vairone (telestes muticellus) was tested in a portable flume in 1, 2 and 6 fish groups. the stress response was studied through the analysis of the hypothalamic-pituitary-internal axis. the neuroendocrine stress response was evaluated by the cortisol level in the muscle tissue. additionally, oxidative stress was assessed by evaluating cell damage and gene expression of the protein components of the antioxidant defence system. lipid peroxidation was studied through the level of malondialdehyde (mda) in the muscle. further, non-enzymatic oxidative changes were studied by advanced oxidative proteins (aopp). preliminary results suggest that oxidative stress is lower in grouped fish than in single fish. supported by the european union horizon 2020 research and innovation programme under the marie sklodowska-curie actions, grant agreement no. 860800. preliminary data on physiological responses induced by pfas exposure in freshwater fish of the veneto region e piva1, s schumann1, s dotteschini1, m bonato1, d bertotto2, a marion3, p irato1, g santovito1 1department of biology, university of padua, padua, italy. 2department of comparative biomedicine and food science, university of padua, padua, italy. 3department of industrial engineering, university of padua, padua, italy. in recent decades, the interest towards perand polyfluoroalkyl substances has grown exponentially around the world, due to the toxic effects induced by these chemical compounds in humans, as well as in other animals and plant organisms. however, the knowledge related to the antistress responses that organisms can express when exposed to these substances is still lacking and therefore requires further investigation. for this purpose, this study was launched on the possible physiological responses that exposure to environmental concentrations of pfas can induce on squalius cephalus and padogobius bonelli, two freshwater fish species widely spread in the veneto region, a geographical area directly involved in what is considered one of the most significant cases of pfas pollution. specimens of the two species were sampled in three rivers of the vicenza area, characterized by three different levels of pfas pollution. several biomarkers of stress have been evaluated and the results obtained suggest an increase in the expression of mitochondrial antioxidant enzymes. conversely, no change in total antioxidant capacity was observed, suggesting that the effect of oxidative stress detected in the mitochondria does not correspond to a more extensive cellular response. for both species, various morphological indices were calculated with the aim of determining both the general well-being of the organisms and identifying possible variations in the size of the liver, spleen and gonads. the obtained results are preliminary and certainly require further analyses and investigations, but they suggest an interesting protective mechanism against damage to the protein component based on lipid vacuolation in the liver. supported by the european union horizon 2020 research and innovation programme under the marie sklodowska-curie actions, grant agreement no. 860800. the skin of mediterranean cetaceans as model to analyse accumulation and toxicity of pop. l abelli1, a mancia1, m baini2, g limonta2, c panti2, mc fossi2 1department of life sciences and biotechnology, university of ferrara, ferrara, italy 2department of physical sciences, earth and environment, university of siena, siena, italy stockholm convention (sc; 2001) on persistent organic pollutants (pop; http://www.pops.int/) aimed at eliminating production or lowering use of 12 long-lasting toxic chemicals, e.g. polychlorinated biphenyls (pcbs), dioxins and pcdh. production of ddt was not banned, but restricted to limited production, only to fight malaria in endemic regions of the world. later on same year, the sc added other 4 pop (comprising lindane), becoming operative since 2004 (with adhesion of 181 nations). the scheduled sc 2022 meeting is regarding also inclusion of pfoa and bpa in the annex a. due to the lipophilic nature of these chemicals, environmental concerns have arisen about bioaccumulation and biomagnification through the food chain, in both land and water ecosystems. due to large occurrence of lipids in the skin of cetaceans, these aquatic mammals represent a valuable tool to analyse accumulation and potential toxic effects of pop. even in males, large amounts of hypodermic blubber have adaptive roles, but are also sites of pop capture, storage and long-lasting slow systemic release. our previous epigenetic study in the mediterranean (ligurian sea) fin whale (balaenoptera physalus) skin biopsies (all males) revealed a relationship between levels of blubber contaminants (organochlorines and phthalate) and dna changes in gene methylation profiles. differentially methylated genes fell into six main pathways: wnt signaling, cadherin signaling, angiogenesis, endothelin signaling, axon guidance mediated by semaphorins, and nicotinic achr signaling. these results depicted a dystrophic condition of the skin. collection of new dart skin biopsies from free-ranging fin whales is now allowing a wider analysis about contaminants load and histopathological correlates. 83 plenary lecture iii linking stem cells and innate immunity research via data, information and knowledge integration d drobne department of biology, biotechnical faculty, university of ljubljana, ljubljana, slovenia stem cell biology and the immune system research should be integrated in order to provide a more holistic understanding of how organisms maintain and restore homeostasis. the scopes related to the interplay between stem cells and the immune system go beyond basic insights of organism’s physiology and include also regeneration research, (eco)toxicology, biotechnology, and medicine as well as regulatory and ethical aspects. traditionally, stem cells and immune cells are considered as parts of two branches of biological research with few interconnections between them. it is a challenge to integrate the conceptual frameworks of these two disciplines to address basic questions in biology in a new and innovative way. great opportunities for linking scientific disciplines are given in the data driven era. computation has been an important part of science for more than half a century, and the data explosion is making it even more central. but crossdisciplinary collaboration and data, information and knowledge sharing according to findability, accessibility, interoperability, and reusability (fair) data principle is a relatively new concept. the contribution of fair data will be discussed and aquatic invertebrates are taken as an example. how to integrate new and traditional approaches in an inclusive way will be emphasized. session 5. immune competence and immune response (part ii). chairmen: matteo cammarata, university of palermo, palermo, italy and piero g. giulianini, university of trieste, trieste, italy conservation and diversity in the inflammatory reaction of ciona robusta: hemocyte populational dynamics and molecules related to internal defense v longo1, d parrinello2, a longo1, mg parisi2, n parrinello2, p colombo1, m cammarata2 1institute for biomedical research and innovation, national research council, palermo, italy 2department of scienze della terra e del mare, university of palermo, palermo, italy ascidians are marine invertebrate chordates belonging to the earliest branch (tunicata) in the chordate phylum, therefore, they are of interest for studying the evolution of immune systems. due to the known genome, the noncolonial ciona robusta, previously considered to be c. intestinalis type a, is a model species for the study of inflammatory response and here we further explore the topic of hemocyte and molecules related to internal defense of ascidians involved in the inflammatory reaction. the internal defense of ascidians mainly relies on hemocytes circulating in the hemolymph and pharynx. hemocytes can be in vivo challenged by lps injection and various granulocyte and vacuolated cell populations differentiated to produce and release inflammatory factors. molecular biology and gene expression studies revealed complex defense mechanisms involving different inflammatory hemocytes. furthermore, cloning procedures allowed sequence analyses and molecular studies disclose immune-related gene families including toll-like receptors, galectins, ctype lectins, collectins, interlectins, pentraxine-like, peroxinectins, complement factors-like, tnfα-like, il-17-like, tgf-like, mif-like. these genes are promptly upregulated by the inflammatory stimulus and show a time course of transcription similar to each other. domains sequence similarity and phylogenetic relationships with the vertebrate counterparts are shedding some light on immunerelated gene evolution. selective bioassays as well as bioinformatic approaches have allowed the characterization of antimicrobial peptides and the identification of post transcriptional molecular mechanisms able of influencing dynamics of gene regulation are described. identification of potential hemocyte markers in the gastropod model pomacea canaliculata a ferri1, s sacchi2, g bergamini1, m nasi3, d malagoli2 1department of chemical and geological sciencesdepartment of life sciences, university of modena and reggio emilia, modena, italy 2department of life sciences, university of modena and reggio emilia, modena, italy 3department of surgery, medicine, dentistry and morphological sciences, university of modena and reggio emilia, modena, italy the immune system of non-model invertebrates remains poorly characterized, although it may contribute to a better knowledge of vertebrate innate immunity. the main cellular immune component of invertebrates is represented by circulating and tissue-infiltrating hemocytes. in absence of specific cell markers, hemocytes are usually classified on morpho-functional bases. in these respects, the investigation of molecules selectively expressed in hemocytes (i.e., hemocyte markers, hm) of the freshwater and invasive gastropod, pomacea canaliculata, would represent a valuable contribution to track hemocyte development, maturation, and distribution. taking advantage of the available genome and organ-specific transcriptomes, sequence analyses were performed, selecting allograft inflammatory factor-1 (pcaif1), hematopoietically expressed homebox protein (pchhex), hemocyanin (pchc), runt domain-containing protein (pcrunt) and transglutaminase-2 (pctg-ase-2) as potential hm candidates. single probe fluorescent in situ hybridization (spfish) evidenced all the potential hm in cytocentrifuged hemocytes, with a prevalence of pchc-positive cells. after dual probe fish for each combination of hm, double or single positive, and double negative hemocytes were observed, 84 confirming that similar morphologies can correspond to a different transcriptional pattern, then possibly to a different functional status or a diverse role. spfish enabled the specific identification of tissueresident hemocytes in two different histological contexts: a complex and potentially hematopoietic organ (i.e., the posterior portion of the kidney) and the blastema of early regenerating cephalic tentacle (rct) at 12 h post amputation (12 hpa). in order to corroborate the morphological observations with molecular data, rt-qpcr experiments investigated the hm expression levels in rct 12 hpa. the increased number of hemocytes observed by confocal microscopy was in agreement with rtqpcr data, that confirmed the expression of all hm analyzed and the significant induction of pchc, pctg-ase2 and pcrunt in rct 12hpa samples. in all, the combined application of in silico, microscopy and molecular techniques has led to the first identification of potential hm in p. canaliculata. while these markers are diversely expressed in circulating hemocytes, they evidenced the hemocyte islets in the kidney and also hemocyte-like cells in the blastema of rct. these markers could also be used for future experiments of cell sorting and for following hemocyte maturation in circulation or within hematopoietic organs. insight on the signal transduction pathways involved in morula cell degranulation in the colonial ascidian botryllus schlosseri a peronato, l ballarin department of biology, university of padua, padua, italy morula cells are granular cells constituting the majority of hemocytes of the hemolymph of botryllid ascidians. they are the first cells sensing nonself and, as a consequence of the recognition, they synthesize and release cytokines and trigger and inflammatory process by release their granular content through exocytosis (degranulation). they are directly involved in the formation of the necrotic points of rejection along the contact border between incompatible colonies. during this process, they are selectively recruited and gather in the ampullae (the blind endings of the colonial circulation) close to the contact region before crossing the vascular epithelium and entering the tunic where they degranulate releasing their granular content, in primis the cytotoxic enzyme phenoloxidase and its polyphenol substrata. the degranulation reaction can be mimicked in vitro by exposing hemocytes to cell-free hemolymph from genetically incompatible colonies of to microbial cells such as bacillus clausii cells. in the present research we induced in vitro degranulation to study the signal transduction pathways involved in the process using specific inhibitors of a series of kinases. preliminary results indicate the involvement, in morula cell degranulation, of the pathways mediated by pka, pkc and jnk. translational comparative immunology: from fish to bed, new antibiotics and sars-cov-1 immunodiagnostic g scapigliati, f porcelli, f buonocore, g zarletti, m tiberi, v de molfetta, am timperio, f gevi, g fanelli department for innovation in biological, agrofood and forest systems, university of tuscia, viterbo, italy available knowledge shows that a morphological and functional implant of immune defences is remarkably similar and conserved among vertebrate classes, as confirmed by the zebrafish model, widely employed for translational research applied to human health. the knowledge on fish immune defences may find application in biotechnology and, in this respect, we investigated whether natural antimicrobial peptides produced by the antarctic fish species chionodraco hamatus and trematomus bernacchii might be candidate molecules to be employed as novel antibiotics. the results have shown that a modified synthetic 22-mer peptide derived from chionodracine sequence shows important features such as being actively lytic against antibiotic-resistant bacterial strains of acinetobacter baumannii and klebsiella pneumoniae (both mic 1.55 µg/ml): the peptide does not induce lysis to human erythrocyte and is not cytotoxic for some human cells lines at the mic concentrations useful to kill bacteria. another antimicrobial peptide, trematocine, has been investigated for its antibacterial and antimicrobial activity and some mutants designed to be more active against bacterial cell walls are currently under screening. in vertebrates, the elispot assay is employed to measure and quantitate the presence of antigen-specific antibody-secreting memory b cells in vitro, but due to its complexity the assay can be solely performed in research laboratories. we previously described a simplified version of the elispot and described the in vitro presence of circulating memory b cells long after immunization of the fish sea bass against bacterial antigens. due to the necessity of information regarding the mounting of an antibody memory response against the sars-cov-2 spike-s1 protein, we applied our cellelisa platform (simplified elispot) to the screening of human blood samples, in order to achieve data on both serological igg presence and in vitro igg produced by memory b cells. the results have shown that in a cohort of 150 donors a significant number of serology-negatives patients resulted indeed positive to cellelisa, thus revealing an active immunization against spike-s1 even in the absence of circulating igg. the cellelisa assay showed the presence of an antibody memory 12 months after first virus encounter and suggested that blood leukocytes from sars-cov-2-infected patients display a modified metabolomic profile. 129 isj 17: 129-137, 2020 issn 1824-307x research report redefining operant conditioning of escape behaviour in lymnaea stagnalis c benatti1,2, v rivi3, c colliva1,2, g radighieri3, f tascedda1,2, jmc blom2,3* 1department of life sciences, university of modena and reggio emilia, modena, italy 2centre of neuroscience and neurotechnology, university of modena and reggio emilia, modena, italy 3department of biomedical, metabolic and neural sciences, university of modena and reggio emilia, modena, italy this is an open access article published under the cc by license accepted june 16, 2020 abstract the escape behaviour is one of the many behavioural responses that can be operantly conditioned in a stimulus-dependent manner in both vertebrates and invertebrates. by exposing the pond snail lymnaea stagnalis repeatedly to a negative reinforcement its natural tendency to explore its surroundings can be operantly conditioned in both adult and aged snails. when adult snails were trained with 100 mm of kcl their number of escapes was significantly decreased and the latency to first escape was significantly increased. our behavioural protocol allowed us to investigate memory acquisition, consolidation, and retrieval in preand post-training sessions over different days. from the 3rd day of training the learned response was strengthened: the number of the escapes in the post-test session remained significantly reduced even when animals were presented with distilled water. moreover, adult snails exposed to the negative reinforcement for at least 4 days started to escape significantly less than the control group also in the pre-test session. this effect became more pronounced in the following days and was accompanied by a significant increase in the latency to first escape at the beginning of the pre-test on day 6 and 7. aged snails, instead, showed selective deficiencies when operantly conditioned: memory retention appeared only after 7 days, while memory retrieval could not be induced. this redefined paradigm can help unravelling a variety of sophisticated cognitive phenomena in l. stagnalis and could be employed also to study the basis of memory impairment occurring during neuro-aging. key words: associative learning; memory; behaviour; pond snails; aging introduction escape behaviour is the result of a complex integration of information from sensory systems, internal states and expectations arising from experience and prior beliefs (evans et al., 2019). from a neuroscience perspective, escape is a behavioural response that can be operantly conditioned in a stimulus-dependent manner, allowing to study complex cognitive processes, such as learning, memory and decision-making not only in vertebrates, but also in invertebrate model systems (kemenes and benjamin, 1989a). in the last decades, in fact, the behavioural, cellular and molecular basis of associative learning and memory have been found to be basically similar throughout ___________________________________________________________________________ corresponding author: joan mc blom department of biomedical, metabolic and neural sciences university of modena and reggio emilia via campi 287, 41125, modena, italy e-mail: joan.blom@unimore.it the animal kingdom, suggesting that these processes are so fundamental that they appeared early in evolution and are fairly conserved over time (carew et al., 1981; kemenes and benjamin, 1989; rivi et al., 2020). this has led to the recognition of invertebrates as a more flexible model for the study of conserved basic and advanced cognitive processes (carew et al., 1981). among the invertebrate models, the pond snail lymnaea stagnalis has been extensively used to study memory and learning (kemenes and benjamin, 1989a; kojima et al., 1996; lukowiak et al., 1996; swinton et al., 2019; benjamin, 2000). in fact, thanks to the dynamicity of their behaviours, snails can be trained to respond to a wide variety of stimuli, acquiring different forms of associative learning, such as singleor multi-trials, aversion or reward, operant or classical conditioning (alexander et al., 1984; kojima et al., 1996; lukowiak et al., 1996; kemenes et al., 1997; andrew and savage, 2000; kawai, 2004; fulton et al., 2005; ito et al., 2013). https://creativecommons.org/licenses/by/4.0/legalcode 130 fig. 1 experimental groups and behavioural procedure of experiment 1 (a). escapes performed every 20 minutes and latency to first escape for each session were evaluated on day 1 (b), day 2 (c), day 3 (d), day 4 (e) in adult l. stagnalis. each snail was placed in a 35 mm water container filled with distilled water (dw) set on a filter paper, during training the filter paper was soaked with kcl 100 mm, kcl 50 mm, kcl 10 mm or dw while in the other sessions the filter paper was soaked with dw. data are expressed as mean ± sem. comparisons were made by one-way analysis of variance (anova) followed by tukey post-hoc test; ### p < 0.0001, ## p < 0.01, # p < 0.05 vs dw; *** p < 0.0001, ** p < 0.01, * p < 0.05 vs kcl_10mm; +++ p < 0.0001, ++ p < 0.01, + p < 0.05 vs kcl_50mm 131 in a preliminary study kobayashi and colleagues observed that the repeated exposure to an aversive stimulus suppressed the natural tendency of snails to explore their surroundings without eliciting a long lasting memory retention (kobayashi et al., 1998). the chosen aversive stimulus, potassium chloride (kcl) acted as a strong stressor stimulus eliciting the whole-body withdrawal response of the snails into their shells (kojima et al., 1996; ito et al., 2015). previous studies, in fact, demonstrated that the exposure to kcl 100 mm was more aversive than lower concentrations, causing snails to remain in their shells for a prolonged period of time (kojima et al., 1996). the purpose of this study is to extend kobayashi findings and investigate memory acquisition, consolidation, and retrieval and test the hypothesis if repeated exposure to a negative reinforcement results in a long-lasting behavioural alteration manifested even when kcl is not present in the external environment. moreover, lymnaea has been identified as valid model system for exploring age-related behavioural, cellular and molecular changes, including memory impairment and decline (kemenes et al., 2006; hermann et al., 2007, 2020; pirger et al., 2014; watson et al., 2012, 2013; fodor et al., 2020). so, we employed the paradigm of operant conditioning of escape behaviour in adult and aged snails to assess age-related differences in memory formation and retrieval. materials and methods ethics statement pond snails lymnaea stagnalis are very abundant in the northern continents of the world and are not endangered nor a protected species. experiments on pond snails are not subject to the approval of our ethics committee. nonetheless, every effort was made to maximise the wellbeing of the snails during the behavioural procedures. snails and colony maintenance laboratory-reared l. stagnalis (linnaeus 1758), originally derived from a stock generously donated by the vrije university in amsterdam (the netherlands), were used in this study. animals were maintained in aquaria at the university of modena and reggio emilia (italy) at 21-23 °c in well-aerated dechlorinated tap water on a 12/12 h light/dark cycle (lights on at 08:00 a.m.), animals were fed pesticidefree lettuce twice a week. sixand nine-month old snails having shell lengths of 20-25 mm were used in these experiments. because snails survive for 9-10 months in our breeding facility, we selected two age categories, 6 and 9 months old, based on the age categorization by hermann and co-workers (2007), to compare learning performance among these cohorts (hermann et al., 2007). we refer to these two groups as “adult” and “aged” snails in the rest of the text, but these terms should be interpreted as indicating middle and late stages of lymnaea’s life cycle under the current breeding conditions of our lab. the term “aged” as it is used here should not be generalized to mean the maximal chronological age this species can attain. twenty-four hours before the behavioural test the animals were food deprived, and 12 hours prior to start the behavioural test, animals were kept in 12 l tanks supplied with 6 l of distilled water (dw) and 6 l of water of aquaria (15-18 animals per tank). all experiments were performed between 10 am and 1 pm local time. conditioning paradigm snails were placed in the middle of a 35 mm petri dish (sarstedt, germany) with 2 mm depth of dw. that was enough for the snails to be fully submerged. in the pre-test session, the petri-dish lids containing lymnaea were set on a sheet of dwsoaked filter paper and every time snails escaped from the lids and touched the soaked paper with their lips, the experimenter moved the snail back into the lid. snails have been excluded from the study if the total number of escapes during the pretest on day 1 were < 12 (in 60 minutes). next, the lids containing lymnaea were transferred on a sheet of filter paper soaked with either dw or different concentrations of kcl for training. when snails escaped and touched the negative reinforcement with the lips, the withdrawal response of their bodies into their shells was elicited. in the post-test session, the petri-dish lids containing lymnaea were transferred on a new sheet of dwsoaked filter paper. for each session, the number of escapes performed in a 20-minute interval and the time necessary for the first escape to occur were annotated by an experimenter blind to the experimental condition. during the procedure, snails were kept wet minimalizing any form of suffering. at the end of the training procedure, snails were fed ad libitum with fresh lettuce until 12 hours prior to the start of the next-day procedure. experiment 1 effect of different concentration of kcl on escape conditioning one hundred and nine six-month old snails were used in this experiment and divided into 4 experimental groups: 1. kcl 10 mm: snails exposed to kcl 10 mm during the training session (n= 29); 2. kcl 50 mm: snails exposed to kcl 50 mm during the training session (n= 16); 3. kcl 100 mm: snails exposed to kcl 100 mm during the training session (n= 31); 4. dw: snails exposed to dw during the training session (n= 33). the conditioning procedure consisted in (1) a pre-test session lasting 60 minutes, (2) a training session lasting 20 minutes and (3) a post-test session lasting 60 minutes, repeated over 4 days. experiment 2 effect of massed training on escape conditioning while in the spaced procedure snails were exposed to kcl for 20 minutes a day repeated for 4 days, the exposure to the negative reinforcement in the massed training procedure occurred only in day 1 and lasted 80 minutes. 132 sixty-seven six-month old snails were used in this experiment, articulated over 3 days: day 1: (1) a pre-test session lasting 60 minutes, (2) a training session lasting 80 minutes and (3) a post-test session lasting 40 minutes. during the pre and post-test sessions, the filter paper was always soaked with dw. during the training session, the filter paper was soaked with a kcl 100 mm solution for the conditioning group (kcl group) (n= 31), and with dw for the control group (dw group) (n= 36). day 2 and 3: observational session lasting 60 minutes, where all snails were exposed to dw. experiment 3 effect of aging on the efficacy of escape conditioning thirty six-month old snails and 45 nine-month old snails were used in this experiment and divided into 4 experimental groups: 1. aged dw: nine-month old snails exposed to dw during the training session (n= 22); 2. aged kcl: nine-month old snails exposed to kcl 100 mm during the training session (n= 23); 3. adult dw: six-month old snails exposed to dw during the training session (n= 15); 4. adult kcl: six-month old snails exposed to kcl 100 mm during the training session (n= 15). the conditioning procedure consisted in (1) a pre-test session lasting 60 minutes, (2) a training session lasting 20 minutes and (3) a post-test session lasting 60 minutes, repeated over 7 days. statistics behavioural data were expressed as the mean number of escapes in a 20-minute interval or the latency to first escape from the water container (seconds) for each session ± s.e.m. (standard error of the mean). exclusion of extreme outliers was made prior to statistical analysis using the boxplot tool in spss (more than 3x the interquartile range outside of the end of the interquartile box). behavioural data were tested for significant differences by a univariate analysis of variance (anova) followed by tukey (with p < 0.05 significance level). analyses were conducted using spss for windows® v.26 (spss inc., chicago, usa). results in experiment 1, a spaced training procedure similar to that employed by kobayashi et al. (1998) was used: animals were divided in four groups with a similar mean number of escapes in the pre-test of day 1 and trained either with kcl 100 mm, kcl 50 mm, kcl 10 mm or dw for 4 days (fig. 1a). during training, from day 1 to day 4, snails exposed to 50 and 100 mm kcl performed less escapes and showed a longer latency to first exit than their dw and kcl 10 mm counterparts (see table 1a for a detailed summary of the main effects, one-way anova; fig. 1b-1e). in the day-1 post-test session the number of escapes and the latency to the first exit were restored after the negative reinforcement was removed, with the sole exception of animals exposed to kcl 50 mm whose performance remained impaired with respect to dw and kcl 10 mm groups (fig. 1b). no difference was observed between the performance of dw and kcl 10 mm either during training or during the other sessions throughout experiment 1 (fig. 1b-1e). table 1 summary of statistical analysis of experiment 1 (a), experiment 2 (b), and experiment 3 (c).main effects on escapes performed every 20 minutes and latency to first escape for each session were evaluated using one way analysis of variance (anova), all mean differences were considered statistically significant if p < 0.05 (bold). a) b) c) 133 fig. 2 experimental groups and behavioural procedure of experiment 2 (a). escapes performed every 20 minutes and latency to first escape for each session were evaluated in l. stagnalis (b). each snail was placed in a 35 mm water container filled with distilled water (dw) set on a filter paper, during training the filter paper was soaked with either kcl 100 mm or dw while in the other sessions the filter paper was soaked with dw. data are expressed as mean ± sem. comparisons were made by one-way analysis of variance (anova) ### p < 0.0001, ## p < 0.01; # p < 0.05 on the second day of training, some alterations in the behavioural parameters started to emerge only in animals exposed to the highest concentration of the negative reinforcement: the 100 mm kcl group performed less escapes in the final part of the pre-test and in the first twenty minutes of the post-test: when compared to their dw counterparts they showed a significantly increased latency to first exit after training (fig. 1c). this effect became more pronounced on day 3, where a significant decrease in the number of escapes was evident in kcl 100 mm-exposed animals during the whole pre-test session, while snails exposed to kcl 50 mm performed significantly less escapes than dw and kcl 10 mm-exposed animals only in the first 40 minutes of the pre-test session. after training the latency to first exit and the number of escapes of the 50 mm kcl group was restored, while in animals exposed to the highest concentration of the negative reinforcement the escape behaviour remained significantly compromised: snails exposed to 100 mm of kcl during training preformed significantly less escapes in the post-test and showed a higher latency to first exit with respect to all the other experimental groups (fig. 1d). in the 4th day, no difference was observed in the behaviour of the dw and kcl 10 mm exposed animals, while training with both the higher concentrations of kcl affected the performance of the animals both in the pre – and post-test sessions. the number of escapes of animals exposed to 50 or 100 mm kcl was significantly decreased with respect to dw or kcl 10 mm groups only in the first 40 minutes of the pretest, this effect was not present in the last interval of the session. after training, the kcl 100 mm group took significantly more time to first exit from the petri lid than the other groups and the number of escapes they performed remained significantly lower than their 50 mm, 10 mm and dw exposed counterparts for the first 40 minutes of the post-test session. in the last interval the performances of animals exposed to the higher concentrations of the negative reinforcement were not different from each other, while remaining significantly lower than the kcl 10 mm and dw groups. only training with kcl 100 mm for 4 days significantly affected the latency to first exit in the post-test session (tab. 1a, fig. 1e). in experiment 2 a readapted protocol of a massed training procedure was employed (fig. 2a, 2b): on day 1, lymnaea were operantly conditioned for 80 minutes using the highest concentration of kcl tested in experiment 1. initially, 67 snails were monitored for their exploratory activity in the pre-test session and were divided in two groups not statistically different from each other (see table 1b for a detailed summary of the main effects, one-way anova; fig. 2a). during the 80-minute long training, the number of escapes was significantly suppressed in kcl exposed animals with respect to the dw control group (fig. 2b). at the end of the training session, when the animals were re-exposed to dw-soaked filter paper, the number of escapes of the kcl-exposed group remained lower in the first 20 minutes and was recovered 40 minutes after the end of the training session. as observed in the spaced procedure, the latency to first exit at the beginning of the training session was significantly increased in animals exposed to negative reinforcement. a similar, but not statistically significant, trend was observed in conditioned snails 134 135 fig. 3 experimental groups and behavioural procedure of experiment 3 (a). escapes performed every 20 minutes and latency to first escape for each session were evaluated on day 1 (b), day 2 (c), day 3 (d), day 4 (e), day 5 (f), day 6 (g), day 7 (h) in six-month old (adult) or in nine-month old (aged) l. stagnalis. each snail was placed in a 35mm water container filled with distilled water (dw) set on a filter paper, during training the filter paper was soaked with either kcl 100 mm or dw while in the other sessions the filter paper was soaked with dw . data are expressed as mean ± sem. comparisons were made by one-way analysis of variance (anova) followed by tukey post-hoc test ### p < 0.0001, ## p < 0.01, # p < 0.05 vs dw; §§§ p < 0.0001, §§ p < 0.01, § p < 0.05 vs dw_aged; +++ p < 0.0001, ++ p < 0.01, + p < 0.05 vs kcl in the post-test session as well (fig. 2b). on the second and third day all snails were placed over dw-soaked filter papers: while no difference was observed in the latency to first exit between conditioned animals and the control group, the number of escapes of kcl-exposed animals remained significantly lower than their dw counterparts in the first 40 minutes of the observation period and then was restored completely on day 3 (fig. 2b). in experiment 3, 30 six-month old snails and 45 nine-month old snails were divided in two groups (not performing differently from each other in the pre-test of day 1). in day 1, the negative reinforcement significantly reduced the number of escapes and the latency to first escape during training in both aged and adult animals (see table 1c for a detailed summary of the main effects, one-way anova; fig. 3b). at the end of the session, the escape tendency was restored in kclexposed snails irrespective of their age (tab. 1c; fig. 3b). from day 2 to day 7, exposure to 100 mm kcl during training resulted in a significant decrease in the number of escapes coupled to an increase in the latency to first escape with respect to control animals in both adult and aged snails (fig. 3c-h). no difference was observed in the performance of aged and adult animals following the same procedure, with the sole exception of day 3 when older snails exposed to dw performed less escapes during training than their younger counterparts (fig. 3d). in the post-training session of day 2 the number of escapes was restored in kcl exposed snails and younger snails escaped more than both their older counterparts and dw controls (tab. 1c, fig. 3c). from day 3 onward, in the post-test session, younger snails exposed to kcl continued to escape significantly less than their dw-counterparts, while for aged snails a significant difference in the escape tendency was evident with respect to the dw group after exposure to the negative reinforcement on day 6 and 7 (tab. 1c, fig. 3g, 3h). as already observed in experiment 1, adult snails exposed to the negative reinforcement for 4 days started to escape significantly less than their dw counterparts also in the pre-test session. this effect became more pronounced on the following days and was accompanied by a marked increase in the latency to first escape at the beginning of the pre-test on day 6 and more distinctively on day 7 (fig. 3f-h). discussion the purpose of this study was threefold. first, we investigated the conditioning effect of different concentrations of kcl in l. stagnalis; second, we assessed the optimum concentration of kcl for memory retention, and third we established the number of days of training necessary for memory to occur. our results showed a concentrationdependent effect of kcl as negative reinforcement: training with either kcl 50 mm or 100 mm resulted in an immediate reduction of the number of escapes and increased latency to first escape, whereas the behavioural outcome induced by the exposure to kcl 10 mm were either transient or minor, possibly because the withdrawal response can be elicited only by concentrations of kcl higher than 10 mm (kojima et al., 1996; ito et al., 2015). the experience-derived changes in behaviour observed in snails exposed to the higher doses of kcl may represent a key-step for associative learning and memory acquisition: snails, in fact, associated the negative reinforcement with their escape behaviour, avoiding exiting from their lids. in the first two days of the procedure, once lymnaea recognized that the external environment was safe (i.e. without kcl), the memory of the negative past situation was extinguished and normal escape behaviour was recovered. from the third day of training on, in animals conditioned with kcl 100 mm, the learned response was strengthened: the number of escapes in the post-test session remained significantly reduced and the latency to the first escape was enhanced. re-exposure to the highest dose of the negative reinforcement consolidated memories and conditioned the tendency of snails to escape from their lids even if they were presented with dw. on the other hand, the behavioural effects elicited by exposure to 50 mm of kcl observed on day-1 and 3 were less consistent. moreover, on day-3 they were confined to the pre-test session, where they mirrored the effect of the highest concentration. it took 4 days of training with this intermediate concentration of kcl to observe a significant effect on the escape behaviour both in the preand the post-test session. while the latency to first exit in the post test was affected only by kcl 100 mm, the number of escapes was decreased in a concentrationdependent manner. in the paper by kobayashi et al. (1998) the acquisition of learning in the spaced training 136 procedure was very brief and extinguish quickly (kobayashi et al., 1998), whereas in our study we observed that the exposure to negative reinforcement for 4 consecutive days started to affect the behaviour of the snails even in the pretest session, indicating that the memory of the past situation was enough to impair the escape behaviour also in a safe external environment. this behavioural output suggests that the previously acquired memory has been retrieved. thus, memory retrieval made snails able to predict what and when stimuli are likely to occur avoiding, in that way, exiting even in the pre-test session. in our study, we also confirmed that memory persists longer with spaced training than it does with massed training. with the massed training experiments, in fact, we aimed to evaluate the effects of this procedure on the escape behaviour and to confirm what had been already observed by kobayashi et al., (1998). we observed that the reduction in the number of escapes induced by an 80-minute long training with kcl 100 mm did not persist for more than 24 hours and did not impact the latency to first exit when the aversive stimulus was removed. the spaced procedure was more effective in conditioning the escape behaviour of l. stagnalis. this is in accordance with several studies using both classical and operant conditioning paradigms in vertebrates and invertebrates models demonstrating that spaced training is superior at generating long-term memories (lukowiak et al., 1998; commins et al., 2003; takigami et al., 2014). finally, we demonstrated that memory retention appeared in aged snails after 7 days of training, three days later than in the younger ones. interestingly from day 5 to day 7 the escape behaviour of adult snails became more compromised also in the pre-test session. based on these data, we hypothesized that memory acquisition is not affected by aging, but its retention gradually declines in older lymnaea. because the same kcl-induced withdrawal responses have been observed in adult snails as in the aged ones, it seems that independent of their age, snails can detect the presence of kcl and modify their behaviour consequently. similar to what we observed, hermann and colleagues (2007) demonstrated that the acquisition of appetitive memory was not compromised by aging, but its retention and consolidation become progressively impaired with advancing age (herman et al., 2007). moreover, using the appetitive classical conditioning, they correlated the age-associated learning and memory deficits with a reduced electrophysiological excitability in key neurons controlling the feeding behaviour (herman et al., 2007). at this point of our research, future studies will be needed to explore which mechanisms may sustain our behavioural results: the impact of agerelated changes in electrophysiological activity, motor and/or chemosensory functions and the functionality of biochemical components of memory formation on escape behaviour in aging snails remains to be investigated. interestingly, pirger and colleagues (2014) demonstrated that the age-related memory decline in old snails can be reversed by administrating the pituitary adenylate cyclase activating polypeptide 38 (pacap38), a molecular key-factor involved in synaptic plasticity and memory processes in both vertebrates and in invertebrates (kiss and priger, 2013; pirger et al., 2014). other studies characterizing age-related changes in “learning ganglia” of the feeding and respiratory network used classical and operant conditioning, respectively (patel et al., 2006, 2010; yeoman et al., 2008; watson et al., 2012, 2013). lymnaea has been established as a powerful and suitable platform for the study of evolutionarily conserved age-related modifications (audesirk et al., 1982; patel et al.,2006; hermann et al., 2007, yeoman et al., 2008; deak and sonntag, 2012; 2020; watson et al., 2012, 2013; pirger et al., 2014; tascedda et al., 2015). our data support for this model a more prominent role in this field: the operant conditioning of escape behaviour in l. stagnalis, scarcely employed in the last twenty years, could help to unravel a variety of sophisticated cognitive phenomena that were previously thought to be restricted to vertebrates or humans, such as configural learning (swinton et al., 2019) and goaldirected decision-making (crossley et al., 2019). acknowledgements this research was supported by regione emilia-romagna "l’invertebrato l. stagnalis quale modello per la medicina traslazionale" l.r. n. 20/2002 progetti di ricerca sui metodi alternativi all'utilizzo di animali; and far 2016 department of life sciences, university of modena and reggio emilia. these sources of funding had no involvement in the study design, data collection, analysis, and interpretation, writing of the report, or in the decision to submit the paper for publication. the authors thank the conad 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610-620, 2013. yeoman ms, patel ba, arundell m, parker k, o’hare d. synapse-specific changes in serotonin signalling contribute to age-related changes in the feeding behaviour of the pond snail, lymnaea. j neurochem 106: 1699-1709, 2008 138 480 isj 14: 480-487, 2017 issn 1824-307x research report teratological changes on the prosoma of eratigena atrica spiders caused by alternating temperatures t napiórkowska, j templin department of invertebrate zoology, faculty of biology and environmental protection, nicolaus copernicus university, lwowska1, 87-100 toruń, poland accepted november 14, 2017 abstract environmental temperature has a tremendous impact on many areas of spiders' lifestyle. it can also be a strong teratogen. the study presents the results of teratological research involving embryos of eratigena atrica spiders. it was aimed at demonstrating a relationship between the temperature of embryo incubation and the mortality of embryos as well as the number of individuals with body deformities. the experiment was based on the application of alternating temperatures as a teratogenic agent. e. atrica embryos were exposed to temperatures of 14 and 32 ºc changed every 12 h for the first 10 days of their development. we observed that these alternating temperatures significantly influenced embryo mortality and the number of body deformities. embryo mortality was high and the experimental group contained 40 individuals with different anomalies of the prosoma: oligomely, schistomely, heterosymely, symely and bicephaly. four individuals were affected by so-called complex anomalies, (combination of several defects). complex anomalies of appendages on the prosoma were recorded for the first time in the history of our research. since each case was different, we analyzed and described each of the spiders separately. key words: alternating temperatures; complex anomalies; malformations; spiders introduction in arthropods collected in the natural environment deformities are reported frequently. in the majority of cases these anomalies affect external structures (particularly appendages on certain body segments) and the exoskeleton (shelton et al., 1981; spanò et al., 2003; mąkol and łaydanowicz, 2006; asiain and márquez, 2009; gregati and negreiros-fransozo, 2009; fernandes et al., 2010). more complicated cases include conjoined twins and duplicitas posterior, all observed in juveniles obtained from embryos collected in the wild and transferred to the laboratory (jara and palacios, 2001; rudolph and martinez, 2008; janssen, 2013). it is impossible to identify a factor/factors responsible for malformations in individuals found in nature; the most likely causes are therefore investigated (ćurčić et al., 1983; eeva and penttinen, 2009). however, deformities can also be induced in strictly controlled laboratory conditions by disturbing embryogenesis ___________________________________________________________________________ corresponding author: teresa napiórkowska department of invertebrate zoology faculty of biology and environmental protection nicolaus copernicus university lwowska1, 87-100 toruń, poland e-mail: tnapiork@umk.pl using a certain teratologic factor, e.g., humidity (buczek, 2000) or chemical compounds (itow and sekiguchi, 1979, 1980; buczek, 1992). temperature is yet another powerful teratogen. embryo incubation at temperatures significantly different from the species optimum may lead to a number of developmental deformities (juberthie, 1962, 1963a, b). among arthropods these are arachnids, including spiders, that are often used in teratological studies. similarly to other arthropods, spiders are poikilotherms. however, they are commonly referred to as ectotherms due to the fact that their body temperature is determined mainly by passive heat exchange with the surroundings (punzo, 2007). temperature has a huge impact on many areas of spiders' lifestyle including mating behavior (davis, 1989), escape speed (cobb, 1994), food intake and growth (aitchison, 1981), copulation duration (costa and sotelo, 1984), net casting (barghusen et al., 1997), nest site choice (hanna and cobb, 2006), and intervals between egg deposition (downes, 1988). according to li (1995, 2002) darymple (2005) hanna and cobb (2006), temperature is the main factor influencing egg hatching and the duration of embryogenesis and post-embryogenesis as well as the survival of juvenile stages. in the optimum temperature embryogenesis is undisturbed mailto:tnapiork@umk.pl 481 and embryo mortality is low. an increase in incubation temperature usually accelerates embryogenesis, while a decrease delays this process (pulz, 1987). the studies of jacuński (1969, 1971) and mikulska and jacuński (1970, 1971) demonstrate that the application of temperature higher than the thermal optimum (supra-optimal) (32 oc) of eratigena atrica (previously known as tegenaria atrica) may impair their morphogenetic processes, leading to body deformities. in subsequent experiments the exposure of embryos to temperature changed at regular intervals (lower and higher than the optimum) led to a higher number of individuals with anomalies and more complex changes in their prosoma and opisthosoma than when a single thermal shock was applied. these alternating temperatures (14 and 32 oc) used as a teratogenic agent were responsible for oligomely, heterosymely, polymely, schistomely, symely, and bicephaly in spiders (jacuński, 1984; jacuński and templin, 2003; jacuński et al., 2004; templin et al., 2009, 2009-2010; napiórkowska et al., 2010a, b, 2016; napiórkowska and templin, 2013). in addition, in some cases the structure of internal organs is investigated (especially the central nervous system) to assess changes corresponding to particular deformities (jacuński et al., 2002a, b, c, 2005; napiórkowska et al., 2010a, b, 2013, 2015). we put forward a hypothesis that the application of alternating temperatures (14 and 32 °c) would significantly affect the mortality of embryos and number of deformities in eratigena atrica. the experiment was aimed primarily at evaluating the effect of thermal conditions on spider embryonic development. another objective was to present several examples of developmental deformities in spiders in order to demonstrate changes in their morphology caused by temperature used as a teratogen. morphological changes analyzed in this study were identified for the first time in the history of teratology. materials and methods the study involved embryos of eratigena atrica (agelenidae). 43 sexually mature females and 17 males were collected during one breeding season in august 2015 near the towns of chełmża and toruń (poland) and transported to the laboratory, where each spider was put into a separate glass container with a capacity of 250 cm3. spiders were kept in a dark room at the temperature of 21 23 ºc and relative humidity of 70 % and fed twice a week larvae of tenebrio molitor linnaeus. water was provided regularly. three weeks after the culture was established, a male was introduced into the container with a female ready for fertilization. the procedure was repeated after two weeks. first cocoons were laid at the end of november. embryos were immediately removed from the containers, then counted and divided into two groups: the control group, maintained at the temperature of 22 ºc and the humidity of 70 %, in conditions optimal for the embryonic development of this spider species (jacuński and wiśniewski, 1997). the experimental group was exposed to temperatures of 14 and 32 ºc (both significantly deviating from the optimum) applied alternately every 12 h. this method was applied for the first time by jacuński (1984). the procedure continued for ten days, until the first metameres of the prosoma appeared on the germ band. subsequently, all experimental embryos were incubated under the same conditions as the control ones. hatching took place approximately 20 days after the eggs were laid. all control and experimental larvae were evaluated for developmental deformities. a relationship between incubation temperature and embryo mortality was tested using χ2 test for a contingency table. the same test was used for the number of anomalies, with yates correction for small abundances. results during one breeding season we obtained approx. 1,200 embryos, half of which constituted the control group. individuals from this group were not affected by any developmental abnormalities. embryo mortality was 5 % in the control group and about 30 % in the experimental group, due to the incubation temperature (χ2 = 129.9, df = 1, p < 0.0001). 40 individuals had leg anomalies. the number of teratogenically modified individuals was related to alternating temperatures (χ2 = 54.1, df = 1, p < 0.0001). the highest number of larvae were affected by oligomely, i.e., they had a reduced number of legs on the prosoma. teratological material also contained individuals with schistomely (bifurcation) of walking legs, heterosymely (fusion of legs lying next to each other on the same side of the prosoma), and symely (fusion of legs of the same pair, lying opposite each other on the prosoma). in addition, one individual was affected by bicephaly (presence of two heads) and four others, by complex anomalies, i.e., a combination of at least two deformities (table 1). since the majority of these table 1 kinds and frequency of anomalies in the prosoma in eratigena atrica kind of anomaly number of individuals % oligomely 28 70.0 heterosymely 2 5.0 schistomely 3 7.5 symely 2 5.0 bicephaly 1 2.6 complex anomalies 4 10.0 total 40 100.0 482 fig. 1 eratigena atrica larvae with developmental anomalies of the prosoma. a, b = larva with oligomely and a forked walking leg (ventral view): a, b = forked ends; c1, c2 = chelicerae; l = walking legs; p1, p2 = pedipalps, scale bar = 0.40 mm. c, d = larva with oligomely, heterosymely and shortening of walking leg (ventral view): a = fused coxae, c1, c2 = chelicerae, l = walking legs; p1, p2 = pedipalps, scale bar = 0.50 mm. e, f = larva with heterosymely of chelicerae and schistomelic pedipalps (ventral view): a, b = forked ends of pedipalps; c1, c2 = chelicerae; l = walking legs; p1, p2 = pedipalps, scale bar = 0.40 mm. g, h = larva with oligomely, double heterosymely, polymely and short stump (ventral view): c1, c2 = chelicerae; g1, g2 = gnathocoxae; p1, p2 = pedipalps; l = walking legs, s = stump, scale bar = 0.45 mm. 483 malformations (i.e., oligomely, schistomely, heterosymely) had already been discussed in previous papers (e.g., jacuński et al., 2005; napiórkowska et al., 2007, 2013), we decided to focus on four individuals with complex anomalies. these four cases differ significantly and are therefore analyzed separately. case 1. the spider in figure 1 (a, b) was affected by two anomalies observed on two sides of the prosoma. on the right side of the prosoma (ventral side) there were only three walking legs (l) (unilateral oligomely). the deformity on the opposite side was more complex and therefore more difficult to classify. the second walking leg was slightly shorter and thicker than the others and had two ends (a, b). the ends were relatively short and had the same length but one (a) was massive and had two small protuberances. the free ends had a uniform structure and there were no visible articular surfaces on them. this anomaly may be classified in two ways: 1) as schistomely (bifurcation) 2) as polymely with partial heterosymely (one supernumerary walking leg and incomplete fusion of two others). case 2. the complex anomaly in the spider in figure 1 (c, d) affected its walking legs and was very complicated. on the left side of the prosoma (ventral side) only two legs were fully developed (l) (oligomely). on the right side of the prosoma, apart from two well-developed walking legs (l), there were two appendages behind the pedipalp (p2). however, these two legs had the coxae fused into one large, massive structure (heterosymely) (a). the remaining part of one of these appendages was properly built, with six segments, but the other was teratogenically changed: it was significantly shorter, and consisted of only two narrow segments. unilateral lack of legs caused significant asymmetry of the prosoma. case 3. the spider in figure 1 (e, f) had anomalies in the anterior part of the prosoma. the anomalies affected its chelicerae and pedipalps. both chelicerae (c1 and c2) were partially fused (heterosymely) with schistomelic pedipalps (p1 and p2). deformities of the pedipalps were identical. forked ends of both pedipalps (a and b) were of equal length and width and consisted of three parts: patella, tibia and tarsus. the ends of the legs moved independently. these teratogenically changed pedipalps with well developed gnathocoxa merged with chelicerae. the fusions on the right and left part of the prosoma were identical. heterosymely affected half of the base part of each chelicera and the first three segments of each pedipalp. the remaining part of the chelicerae including claws were not fused. case 4. the spider in figure 1 (g, h) had several deformities affecting its walking legs: oligomely, double heterosymely, polymely. moreover, one leg was much shorter and another was not well-developed. on the right side of the prosoma (ventral side), there were only two fully developed legs with seven parts (l). before them, behind the pedipalp (p2), was a short stump (s), probably a considerably shortened walking leg. on the other side of the prosoma behind the pedipalp (p1) were five walking legs (l); one was supernumerary (polymelic). the first was shorter and thinner than the others. its first three segments (coxa, trochanter, and femur) were well-developed, while the remaining ones were teratogenically changed: they were considerably narrower, with indistinct segmentation. moreover, the coxa of this leg was fused with the coxa of the pedipalp (p1) (heterosymely). the coxae of the two next legs on this side of the prosoma were also fused (heterosymely), the remaining parts of these legs were properly developed. discussion the results of the experiment confirm the hypothesis that temperatures of 14 and 32 °c used alternately have a significant impact on the mortality of embryos of e. atrica and the number of morphological deformities. oligomely (absence of legs) was the most frequent. it was identified in 70 % of individuals affected by deformities. other anomalies, including complex ones (10 %), occurred with a significantly lower frequency. in total, specimens with morphologic changes constituted approx. 10 % of all individuals which hatched in this experiment. the number of individuals with anomalies would probably have been larger but the mortality of embryos was very high. we assume that the majority of embryos died in the early stages of embryogenesis due to extensive deformities. others, although successfully completed embryonic development, could not leave the egg shells because of developmental defects. in the teratological material we found heavily deformed embryos, which did not leave the chorion. it can thus be concluded that serious leg deformities prevented them from hatching. the question then arises why the remaining 90 % of individuals from the experimental group did not show any changes. the possible explanation lies in efficient repair mechanisms that can effectively eliminate at least some of the deformities. our results indicate that organs of locomotion are most susceptible to developmental abnormalities. it may be connected with the fact that they are less vital for survival than, for example, chelicerae. in addition, it is believed that the thorax, with its metameric structure, is more primal and more prone to teratogenic effects of temperature (jacuński, 1984; jacuński et al., 2004). moreover, simple leg anomalies are not life threatening for a spider. as indicated by jacuński et al. (2005), individuals without one or two legs can still build webs, hunt and molt, even though their fitness is severely impaired. similar observations were made by pechmann et al. (2011). it should also be noted that a majority of the anomalies consisted of one type of deformity. complex anomalies were identified only in four individuals. in two cases deformities affected only walking legs (figs 1 a, b and c, d). it seems that in one specimen (figs 1a, b) the defect was not very severe: only one leg was missing, and the other was slightly shorter and thicker than the remaining ones. two free ends in the distal part of this leg could indicate light schistomely. however, it cannot be ruled out that 484 these were actually two not fully fused legs. this would indicate the presence of two anomalies on one side of the prosoma: polymely and partial heterosymely. of these two possibilities, schistomely is a less severe option. the results obtained by napiórkowska et al. (2007) demonstrate that schistomely is subject to spontaneous processes of repair: after several molts the defect is eliminated. furthermore, after the bifurcated end of the appendage is amputated, the damage is repaired as a result of epimorphic processes. the affected spider can lead a normal life. however, in the second case (figs 1c, d) the defect seems more serious. a higher number of missing legs combined with heterosymely (fusion of legs) significantly impaired the symmetry of the prosoma and influenced molting of this individual. during the first post-larval molt the deformed leg detached. this led to a large loss of hemolymph and caused the death of the spider. it should be noted that the change which we identify as heterosymely resembles a biramous leg. in arthropods a biramous leg is considered primal, so, it can be viewed as a possible return to ancestry. similar conclusions were made by jacuński (1971, 1984, 2002) and templin et al. (2009-2010) who investigated larvae of e. atrica with appendages on petiolus (the first segment of the opisthosoma) or in place of book lungs as well as larvae with a change in the opisthosoma resembling postabdomen. according to these authors, alternating temperatures impairing embryonic development, caused a return to phylogeny and atavism (the appearance of traits belonging to ancestors). of the two remaining cases of complex anomalies, one affected only pedipalps and chelicerae (figs 1e, f), while the other affected walking legs and a pedipalp (figs 1g, h). case 3 is particularly interesting (figs 1e, f). this type of deformity was identified for the first time in the history of teratological studies. the deformity affected pedipalps and chelicerae on both sides of the prosoma in exactly the same way. before, jacuński et al. (2002a) reported one case of unilateral heterosymely of these appendages. histological analysis then revealed a change in the position of a venom gland, which was contained entirely within the fused appendages and shifted towards the pedipalp. the specimen in case 4 (figs 1g, h) had six abnormalities, which affected legs on the prosoma. although extremely rare, such anomalies have already been described by jacuński and napiórkowska (2000) and jacuński et al. (2004). it would be very interesting to understand why these abnormalities developed. according to rousseaux (1988) abnormal phenotypes are the results of the genetic constitution of an animal and the molecular, cellular, and histogenic environment in which they grow. for normal development, the critical cellular mass, correct cellular induction and movements must occur at the proper time and place. mutations, chromosomal aberrations, and environmental interactions with genes that produce abnormal development may initially produce abnormal cells and later a phenotypically abnormal individual. it could be assumed that anomalies that we observed in spiders result from changes in the movement or death of cells caused by alternating temperatures or displacement or dislocation of the cumulus in blastoderm stages. however, based on latest reports (e.g., damen et al., 1998; telford and thomas, 1998; pechmann et al., 2009; schwager et al., 2009; khadjeh et al., 2012) we suppose that these deformities result from changes in hox gene expression patterns. hox genes are able to alter the arthropod plan. hox genes encode transcription factors and are expressed in partially overlapping expression domains along the anterior-posterior body axis. these genes determine the presence or absence of legs in different parts of the body. one of them is antennapedia gene (antp), whose expression pattern in a. tepidariorum suggests a role in leg repression rather than in leg development (khadjeh et al., 2012). it can therefore be assumed that in the deformed spider shown in figs 1g, h there was, among other things, a variation in the expression pattern of this gene, and this led to the development of a supernumerary leg. another homeobox gene which is well known for its role in appendage formation is distal-less (dll) gene. the results of pechmann et al. (2011) suggest that the early expression of at-dll in the germ disc is required for the formation of l1 and l2 segments. the loss of early at-dll function leads to malformation of the l1 and l2 segments and a large portion of the cells in these malformed segments is later removed by cell death. moreover, the cell death apparently removes more cells in this region, resulting in the animals lacking both l1 and l2. therefore, in case of oligomely (absence of walking legs), identified in three individuals (figs 1a, b; c, d and g, h), there might have been a change in the expression of the hox (dll) gene responsible for the formation of segments and the entire appendages. without sufficient research we were unable to determine which ones were lost, and we therefore marked them with the letter l only. in the natural environment many factors may influence embryogenesis of spiders. body deformities and changes in the internal structure are common results. it should be stressed that temperature affects not only the embryogenesis, but also biology of spiders and other invertebrates. moreover, it determines the distribution and dynamics of entire populations (ahmad et al., 2016; elekcioğlu, 2017; khan and naveed, 2017). unquestionably, complex anomalies described above were induced by the application of alternating temperatures during the first ten days of embryonic development. deformities of this kind prevent spiders from leading a normal life not only in the environment, but also in the laboratory under the supervision of researchers. extensive changes in spiders' morphology can seriously impair their life processes, eventually leading to their death. they do not have a chance to reach sexual maturity and reproduce. it should be accentuated that climate change and extreme weather events may result in a growing number of serious deformities in spiders in the natural environment. 485 acknowledgments this work was supported by the faculty of biology and environmental protection 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(b) although some lectins such as galectins, may bind endogenous ligands and mediate critical functions in early development, others like cand f-type lectins bind sugars on the surface of potential pathogens. fucose-binding lectins are present in tissues and fluids from invertebrate and vertebrate species. well-characterized examples, such as the lectin cpl-iii from the tunicate clavelina picta and the fucose-binding mammalian collectins, clearly belong to the c-lectin type. others, such as fbp32 from the striped bass morone saxatilis, the agglutinin from anguilla anguilla (aaa), and serum “fucolectins” from a. japonica, lack a typical sequence motif present in any of the lectin families described so far. furthermore, because of their specificity for carbohydrate moieties present on potential microbial pathogens, and their inducibility upon infectious or inflammatory challenge, these lectins are considered as recognition factors in innate immunity. we have characterized the biochemical properties and primary structure of the carbohydrate recognition domain (crd) of fbp32 and by comparison with other related lectins, identified the sequence motif that defines a lectin family (f-lectins) which includes members present in organisms ranging from insects (drosophila melanogaster) to ectothermic vertebrates (xenopus laevis). f-lectins exhibit considerable diversity in organization, with crds present as single units, such as in aaa, organized in multiple homologous tandem repeats, or associated to other recognition domains. the crystal structure of the aaa enabled the characterization of a structural fold (f-lectin fold), which is shared not only with other lectins from this family, but also with a glycosidase, a glycooxidase, and a human clotting factor. the identification of novel recognition/effector factors that mediate innate immunity in fish opens new possibilities for their use as genetic markers for disease resistance/susceptibility, and the design and implementation of novel intervention strategies. (supported by grants mcb-00-77928 from the nsf and ro1 gm58593-01 from nih to grv) specificity of innate immune response: evidence from antimicrobial peptides p roch, m toubiana, v bonnichon, j-r bonami pathogens and immunity, umr cnrs ecolag, university of montpellier 2, cc 093, place e. bataillon, 34095 montpellier cedex 05, france antimicrobial peptides are potent humoral effectors of innate immunity. in acquired immunity, the counterpart would be the immunoglobulins characterized by their high target specificity. as small molecules, acting by disrupting the cell membrane integrity, coded by simple genes and 18 acting on many targets, antimicrobial peptides were generally considered as non specific. meanwhile, more and more data revealed they are not acting on all targets and not with the unique efficacy. the present lecture will focus on mollusk and crustacean antimicrobial peptides. molecular structures, both sequences and 3d, strongly suggest specificity. precise distribution among circulating cells and particular localization in sub cellular compartments, also suggest specificity. biological activities on bacteria, virus and protozoa, revealed differences in sensitivity of targets, kinetics and molecular structures involved. finally, gene structures, signal transduction pathways and regulation of gene expression under various experimental conditions, disclosed the specificity of the antimicrobial peptides. definite evidence will come from quantification of antimicrobial responses in situations closer to natural stressing conditions. antarctic teleost immunoglobulin heavy chain: evolution through genes and pseudogenes s giacomelli, m maglione, mr coscia, u oreste istituto di biochimica delle proteine, cnr, napoli, italy the diversification of antarctic teleost species was driven by the climatic history of the southern ocean. in fact their ecological success was depending on the capacity to altere the structural features of the molecules to optimize their function at low temperature (-1.86 °c). in this context the immunoglobulin molecule is an interesting model to investigate the adaptive evolution. because of the relatively fast climatic changes, the evolution used a peculiar molecular strategy to modify the genes. insertion/deletion of many bases, elongation using direct or inverted repeats, insertion of reverse complementary nucleotide segments occurred in coding regions very frequently, extensively modifying the aminoacid sequences. in addition gene duplication increased the number of the evolutive experiments. to approach this subject, we determined the sequence of cdna and genomic clones from the igh locus of different antarctic teleost. the selected species were: trematomus bernacchii, trematomus eulepidotus, trematomus pennelli, trematomus hansoni, trematomus newnesi, trematomus loennbergii, dissostichus mawsoni, gobionothoten gibberifrons, belonging to the family of nototheniidae; pagetopsis macropterus and chionodraco hamatus belonging to the family of channichthyidae; cygnodraco mawsoni and gymnodraco acuticeps belonging to the family of bathydraconidae; histiodraco velifer and pogonophrine scotti belonging to the family of artedidraconidae. the experimental procedures utilized were pcr or rt pcr, using as primers oligonucleotides reproducing nucleotide sequences of t. bernacchii ch1 and ch4 exons. the results obtained showed a wide diversity concentrated in specific regions as that connecting the ch2 and ch3 exons; differential usage of donor and acceptor splicing sites; different length of the introns. in addition pseudogenes, altered in the polyadenilation signal and in the length of the 3’ utr, were also found. nucleotide homology among gene and pseudogenes of different species was analyzed to determine their evolutive relationships. immunity in dicentrarchus labrax culture: in vivo effects of alginate immunostimulation l abelli1, n romano2, g scapigliati2, p-g tiscar3, m bagni4, g marino4 1dip. biologia, univ. ferrara; 2dip. scienze ambientali, univ. viterbo; 3dip. scienze biomediche comparate, univ. teramo; 4icram, roma, italy parameters of cellular and humoral immunity were analysed in dicentrarchus labrax to evaluate the modulatory activity of ergosan®, an algal extract containing 1% alginic acid. four cycles using normal fish feed formulation supplemented with 0.5% ergosan were performed at 60-days intervals (15-days treatment + 45days suspension). serum complement (c), lysozyme and total proteins and tissue hsp were measured 15, 30 and 45 days after the first 15-days feeding cycle (short-term) and 45 days after each feeding cycle over a 35-weeks period (long-term). the percentages of b and t lymphocytes in pbl and gut were measured over longterm trial using mabs against homologous ig and t cells. short-term alginic acid treatment raised serum c activity (p<0.05) at 15 days and serum c and lysozyme, gill and liver hsp (p<0.05) at 30 days. no significant differences with control group were found at 45 days. over the longterm period, innate and specific immune parameters, survival, growth performances and conversion index did not differ in treated and control fish. an experimental group received an oral vibrio anguillarum vaccine (avl, uk) according to the following administration protocol: 5-days vaccine and 0.5% ergosan diet, 5-days normal diet, 5-days vaccine and ergosan diet (vaccination cycle, total dose: 0.2 ml/fish). controls received the same dose of vaccine and were fed with normal diet throughout the cycle. total and specific serum ig, c activity and percentages of lymphocytes in pbl and gut were measured 30 days after the start of the vaccination cycle. oral vaccine raised significantly pbl ig+ cells (p<0.05) and gut t-cells (p<0.01) in the ergosan group. the titre of specific serum anti-vibrio ig was higher (p<0.05) in ergosan group compared with controls, while total ig were unmodified. these effects were accompanied by a rise of pbl (t-cells +12%, b-cells +27%), intestine lymphocytes (t-cells +17%) and serum c (+19%) compared with vaccinated controls. the present studies in the sea bass indicate very interesting immunostimulatory properties of alginate. this work was granted by mipaf projects 5c68 and 5c116. cloning and expression of tcr coreceptor cd8 αα in sea bass (dicentrarchus labrax) e randelli, f buonocore, s bird1, cj secombes1, gf wiegertjes2, g scapigliati 19 dip. scienze ambientali, università della tuscia, viterbo, italy; 1s.f.i.r.c, aberdeen, uk; 2dept. cell biol. immunol., university of wageningen, nl cytotoxic and helper t cells recognize endogenously and exogenously derived peptides presented by mhc class i and ii molecules via their α and β t cell receptors. this recognition process also involves the tcr coreceptor molecules, cd8 and cd4, which bind to class i and ii molecules, respectively and trigger helper and/or killer t-cell activities. in mammals, cd8 is a dimeric membrane-bound glycoprotein, present on cytotoxic t cells and consisting of either cd8 αα homodimers or cd8 αβ heterodimers. both chains are composed of a single extracellular ig superfamily v domain, a membrane proximal hinge region, a transmembrane domain and a cytoplasmic tail. recently, the cd8 gene has been found also in cartilaginous and teleost fish like shark, halibut and rainbow trout. in this work, degenerate primers corresponding to conserved region of the extracellular ig superfamily v domain, were used to identify a cd8 α homologous gene from sea bass thymocyte cdna. using these primers we obtained a product of 330 bp that, once sequenced and analysed by blastx search, corresponded to a fragment of cd8 alpha gene. from this fragment we designed sea bass specific primers that were used in 3’ and 5’ race pcr to complete the cdna sequence. an alignment was performed using available cd8 α amino acid sequences and a phylogenetic tree was generated with the putative amino acid sequences lacking the signal peptide. further, sea bass specific primers giving a 220 bp product, were used to analyse the basal cd8 α expression in organs as gills, brain, liver, intestine, head kidney, spleen, peripheral blood leukocytes and thymus. molecular characterization of an antimicrobial peptide isolated from leucocytes of the teleostean dicentrarchus labrax g salerno1, m cammarata1, n parrinello1, p roch2 1marine immunobiology laboratory, university of palermo, via archirafi 18, 90123 palermo, italy, 2 pathogens and immunity, umr cnrs ecolag, university of montpellier 2, cc 093, place e. bataillon, 34095 montpellier cedex 05, france endogenous antimicrobial peptides (amp) are widely distributed in nature and are considered as ancestral components in the evolution of innate immunity. a large variety of amps were described in all living creatures, and particularly among aquatic organisms. in most vertebrates, antimicrobial peptides are associated with peripheral blood leukocytes and mucosal surface such are skin, digestive tract and lung. the literature reports the presence of amps in many teleostean fish. in perciformes (euteleostei, acanthopterygii), two families of amps have been isolated: moronecidin from striped sea-bass, morone saxatilis, and from white bass, m. chrysops, and several hepcidin-related amps from white bass, m. chrysops, and red sea-bream, pagrus major. dicentrarchus labrax (perciformes, moronidae) is a valuable species subjected to intensive farming. the present study was undertaken to identify amps from d. labrax, starting from head-kidney leukocytes. consensus pcr probes were designed from moronecidins and a fragment of 282 bp was cloned in e. coli. the sequence contained an orf of 240 bp coding for 79 amino acids and representing the complete cdna. this sequence was named dicentrarcine (ay303949) and shows a strong homology with moronecidine and pleurocidine. the precursor is constituted by a signal peptide of 22 amino acids, followed by the mature amp of 22 amino acids, and a c-terminal extension of 35 amino acids. in situ hybridization was used for tissue location in both naïve fish and fish challenged by one injection of heat-killed gram negative bacteria, vibrio alginolyticus. results show that the dicentrarcine is express constitutively. the mrna is located in monocytes and granular cells from head-kidney leukocytes, and in macrophages and granular cells from peritoneal cavity leukocytes. protein fractions were separated in reverse phase-hplc of acidic extract from head-kidney leucocytes collected at the interface 34/46% of percoll. fractions collected from a c8 column were assayed for in vitro antibacterial activity against micrococcus lysodeitkus. active fractions were loaded onto a c18 column and eluted with 25-50% linear gradient of acetonitrile. only one fraction revealed a clear inhibition of bacteria growth, but was not in quantity enough for sequencing. recombinant il-1â directly affects intracellular ca2+ concentration on leucocytes of the sea bass (dicentrarchus labrax) s benedetti, m tiberi, e randelli, f buonocore, m mazzini, g scapigliati dipartimento di scienze ambientali, università degli studi della tuscia, viterbo, italy ca2+ is an important second messenger that mediates a large number of cellular processes, and under physiological conditions transient changes in intracellular ca2+ concentration may be related to the transmission of extracellular signals. in this work we further studied the biological activities of cytokines by investigating the effects induced on intracellular ca2+ of leucocytes by recombinant sea bass ril-1â. leucocytes from peripheral blood (pbl), head kidney, gills, spleen and gut, obtained by density gradient centrifugation were loaded with the calcium-binding fluorochrome fura 2-am (4ìm) at 16°c for 45 min, and then stimulated with various concentrations of ril-1â. positive controls have been made with 5 µm of a calcium ionophore (ionomycin), and emitted fluorescence read on a dual excitation fluorescence fluorimeter that allows simultaneous excitation of fluorescence at 355 and 460 nm. results showed that ril-1â induced a rapid rise in intracellular ca2+ concentration, and a subsequent decrease until 5 min after stimulation. the stimulating effect is dose-dependent with a maximal amount of ril-1â of 200 nm, and can be abolished by heath-treatment of ril-1â. the stimulating effect can be also affected in a dose-dependent fashion by treatment of leucocytes with 20 trypsin, thus suggesting a ril-1â-receptor involved in the binding. considering the difference with mammals, where ril-1â do not directly affects intracellular ca2+ concentration, experiments are in progress to verify if the rise in ca2+ concentration may activate cellular processes through tyrosine-kinase enzymes. imaquanim: an integrated approach to study immunoregolatory genes and molecules of teleosts and molluscs g scapigliati, e randelli, s meloni, f buonocore dipartimento di scienze ambienatli, università della tuscia, viterbo, italy imaquanim is an eu-funded integrated project involving the participation of 22 partners to study molecular and cellular responses of farmed fish and shellfish to pathogen invasion. in this project, sea bass (dicentrarchus labrax) and sea bream (sparus aurata) are the species subject of interest by our group and for which the creation of est libraries for immunomodulatory molecules is in progress. at present, for sea bass we have cloned the cdna coding for il-1â, tcrá, tcrâ, cyclooxygenase-2, cd8, gpcrk, and these sequences will be employed for homology cloning in sea bream. in progress is the cloning of interferon-induced mx protein and of mhc class i. other interesting sequences available in databases for sea bass are for ig light chain, rag-1, rag-2, hsp-70. these and other sequences will be employed in microarray screening of est from libraries obtained from pathogen-resistant strains of sea bass to monitor the expression of immunoregulatory molecules. in addition, we have established the continuos embryonic cell line dlec, and we are establishing a continuous headkidney leucocyte cell line. these cells will be transfected with interesting recombinant immunoregulatory genes for their expression in an homologous piscine system. immunity in dicentrarchus labrax culture: effects of oxygen, carbonic dioxide and nitrate n romano, g scapigliati, l abelli1, s picchietti, v de molfetta, l mastrolia, m mazzini department of environmental sciences, tuscia university, viterbo; 1department of biology, university of ferrara, italy both non-infectious and transmittable diseases commonly result from improper rearing management and stressful environments. critical changes of physical and chemical parameters have been associated with onset of disease. in particular, improper temperature, low oxygen or high ammonia and carbon dioxide concentrations could strongly affect the health of cultured fish. we evaluated the effects of variation of individual parameters: oxygen (from 6 to 12 ppm), no3 (from <50 to 200 ppm) and carbonic dioxyde (from <10 to 50 ppm) on specific immune response of the sea bass, dicentrarchus labrax (l.). in control and vibrio anguillarum-immunised specimens we analysed: a) percentage of t and b lymphocytes in the leucocyte fraction of blood and gut (by flow cytometry using specific mabs), b) anti-vibrio serum ig by captured-elisa method. oxygen effects gut t lymphocytes (54.5±1.9%, p<0.001) and b lymphocytes (2.3±1.5%, p<0.05) were significantly enhanced in hyperoxygenated (n=10) fish compared with normo-oxygenated group (n=12), while pbl t and b lymphocytes were not significantly different; whereas pbl did not show significative differences in t and b lymphocytes percentages in the tissues. elisa did not display statistic significative changes (n=25 per group). no3effects vaccination increased specific ig and circulating and intestinal t and b lymphocytes, while did not increase head kidney ig+ cells. high concentrations of no3 apparently enhanced per se pbl b-lymphocytes (n=8, for each group), without modifying serum ig in immunised groups (n=15 for each group). carbonic dioxyde effects high carbonic dioxide concentration decreased significantly (p<0.001) t and b lymphocytes (n=8) and at least by 50% of anti-vibrio ig (n=20), evidencing a strong effect in the circulating lymphocytes. this study remarks the sensitivity of sea bass to oxygen concentration or hypercapnia, evidenced by modification of parameters of specific immunity. sea bass culture under high-flow water recycling and high oxygen concentration and subjected to frequent vaccination boosts is therefore suggested. this project was financed by mipaf 4c047 and 5c176. lymphocyte activities "in vitro" of the sea bass dicentrarchus labrax s meloni, g zarletti, s benedetti, e randelli, f buonocore, g scapigliati dipartimento di scienze ambientali, università della tuscia, viterbo, italy in this work we studied some “in vitro” activities of sea bass peripheral blood leukocytes (pbl) against allogeneic pbl inactivated by irradiation. stimulator pbl were cultured with inactivated allogeneic pbl, and direct counting of lymphocytes was done after 2 and 4 weeks by immunofluorescence and flow cytometry using the mab dlt15 and dlig3 specific for t-cells and b-cells, respectively. results showed a marked increase of t lymphocytes after two weeks in a one way mixed leukocyte reaction (mlr), whereas b lymphocytes had values similar to control pbl. the proliferation of t-cells in mlr cultures was also confirmed by rt-pcr by analysing the expression of the t-cell receptor mrna. on the contrary, a significative decrease of t-cell proliferation was observed by adding to mlr 5 ìg/ml of cyclosporin a (csa). leucocytes from mlr cultures displayed an enhanced cytotoxic activity against xenogeneic target cells with respect to control pbl, suggesting the presence of cytotoxic t lymphocytes. cellular activation of pbl in a 2 weeks mlr was tested by measuring antibody-induced intracellular ca++ mobilization with fura-2 am, and resulted increased in mlr and inhibited by csa. this work represent the first direct quantitative determination of a “in vitro” t-cell activity in a teleost species. 21 developmental expression of tcr in lymphomyeloid tissues of sea bass, dicentrarchus labrax e caccia, f buonocore, r piergentili, l abelli1, g scapigliati, l mastrolia, s picchietti, n romano department of environmental sciences, tuscia university, viterbo; 1dept. biology, univ. ferrara, italy in jawed vertebrates, t cell receptor (tcr) is a membrane molecule composed by a couple of chains: á-â or ã-ä [nam et al., j.immunol. 170, 3081-3097,2003]. rna codifying for tcr chains is normally expressed throughout the entire tlymphocyte life, even on early t-cell that had not still rearranged the membrane protein. with the aim to study the appearance and distribution of t cells, sea bass tcrβ probes were prepared and used for in situ hybridization studies. immunohistochemical studies (abc-peroxidase with nickel enhancement) using the anti-t cell mab dlt15 were made in parallel. lymphocytes were first localised as dlt15+/tcrβcells in the thymic anlage around day 35 post fertilisation (pf). tcrβ+ cells are found from day 38 in thymus and day 41 in mucosae (e.g. gut, gills). five days later, tcrβ+ cells were scattered in head kidney and four days later in spleen, whereas dlt15+ cells were in head kidney already from day 38. it can be speculated that early t-cells (dlt15+/tcrβ -) found in the sea bass may be tcrγδ+, as observed in mammalian thymus [parham, the immune system, current trends/garland, 2001]. tcrβ+ cells are first rearranged in thymus, then sent to other tissues including the midgut. traffic of tcrβ+ cells via blood occurs from day 41 to day 80 pf; thereafter, these cells are mainly in the lymphoid organs. a similar traffic was hypothesised in mammals [lefrançois & puddington, immunol. today 16,16-21,1995]. when epithelial architecture is completed (from day 75 onwards), tcrβ+ thymocytes are confined mainly in the cortex/cortical-medullary border. this finding suggests occurrence and localization of positive/negative selection. expression and distribution of dlgr1 in the ontogeny of dicentrarchus labrax ml di bella, mi vazzana, a vizzini, m cammarata, n parrinello department of animal biology, university of palermo, palermo, italy in teleost fish cortisol is the major corticosteroid hormone released by the interrenal gland under the control of hypotalamus-pituitary-interrenal axis (hpi). it is considered the stress hormone, and it plays many functions including immunomodulatory and inflammatory ones. cortisol enters into the cells by passive diffusion and, once inside the cells, it binds to a glucocorticoid gr receptor. cdna of a gr isoform of dicentrarchus labrax from leucocytes separated from head kidney and peritoneal cavity (vizzini et al., submitted, 2003) has been identified and sequenced. in the present study we report the expression and distribution of dlgr1 in the larval stages of sea bass by in situ hybridation assay with a riboprobe. the ontogeny of lymphoid organs presents the same pattern than other marine species, and signals of the riboprobe were present in brain, gills, head kidney, trunk kidney, liver, spleen and anterior gut cells. positive cells were identified in brain cells 3 days post hatching, while in other examined organs the signal appeared later within 50 days. in particular positive cells in head kidney, spleen and gut were observed at 20-25 days post hatching. further research are in progress to examine dlgr1 modulation under stress conditions. stress and immunomodulation indicators in gilthead seabream (sparus aurata): preliminary data concerning the alterations due to the social behaviour m cammarata, d accardi, c romano, m vazzana, a mazzola, n parrinello dipartimento di biologia animale, università di palermo, palermo, italy fish are highly sensitive to stressful conditions that have an effect on the immune system increasing susceptibility to diseases. we examined social stress caused by aquaculture conditions. the hierarchic position (dominant/subordinate) of gilthead seabream (sparus aurata) specimens were identified by using the “feeding order” and “aggressiveness” parameters in specimen pairs. this hierarchic position appeared at 24-36 hours, and did not change during one year. to search for stressing effect of social relationship on natural immunity, leucocytes from peritoneal cavity were identified. phagocytosis assays with saccharomyces cerevisiae were performed by using a chemiluminescence test (cl) after zymosan stimulation of leucocytes and phagocitic index (pi) was calculated. blood samples and peritoneal cavity leucocytes were obtained avoiding the animal sacrifice. plasma cortisol level, glucose and lactate level were evaluated and agglutinating activity assayed. results showed that plasma levels of osmolarity, glucose, lactate and cortisol resulted higher in subordinate individuals than in dominant ones. in particular, cortisol reach 1.5 times values than controls. related to cortisol increase cl values and pi appeared to be lower in subordinate individuals 24 hours after a hierarchical relationship was established. cl and pi values increase at levels higher than the dominant animals after one month, reaching after 3-6 months. on the contrary, dominant animals maintain constant levels. finally, in vitro cortisol treatment affected the cl response of zymosan-stimulated cells showing a direct effect of this hormone on leucocytes. evaluation of biomolecular pattern in fish cell line exposed to chemical compounds c messina1, g catalano1, s manuguerra1, r vento2, a santulli1 1ist. di biologia marina, consorzio universitario di trapani. 2dip. di biologia cellulare e dello sviluppo, sez. biochimica, università di palermo, palermo, italy 22 interest in fish cell line utilization to evaluate potential environmental risks, determined by chemical pollutants, is growing rapidly. tributyltin (tbt), an organotin (iv) compound used in antifouling formulation, determines cytotoxicity in vitro e in vivo in a wide variety of marine organisms. we evaluated the effects of tbt on an established fish cell line, rainbow trout ovary cells (rto), by studying some proteins involved in triggering apoptosis. preliminary results indicated that tbt exposure produced a timeand dose-dependent cytotoxic effect, already evident at 0,1 µm tbt. light microscopy showed that, in comparison with control, after tbt exposure a large number of rto cells showed a reduction in size and condensed chromatin. this finding was confirmed by fluorescent microscopy, performed by dual staining with acridine orange and ethidium bromide. by this technique, a large number of cells appeared apoptotic, as revealed by the presence of condensed chromatin. presence of markers of apoptosis, such as activation by cleavage of caspase 8 and caspase-3 and parp in tbt exposed rto cells, was demonstrated by western analysis. we also evaluated the levels of p53, a well known tumour suppressor whose levels dramatically increase after various types of stress. observed increase of p53 levels suggests an involvement of p53, as consequence of cytotoxic effects induced by tbt. immune depression triggered in insects by the bacteria xenorhabdus nematophila and photorhabdus luminescens m brehelin ecologie microbienne des insectes et interactions hôtepathogène université montpellier ii, france enterobacteriaceae of the genus xenorhabdus and photorhabdus are potent pathogens of a large spectrum of insect species, some strains of which are toxic for immunocompromised human. insect larvae die in few days after infection. because xenorhabdus and photorhabdus septicemia arises in the insect body, it is obvious that these bacteria are able to escape defense reactions and especially the cellular ones that are early settled after pathogen penetration. the means by which entomopathogenic bacteria escape the defense reactions are totally unknown. in this review we show that different toxins are secreted by these bacteria and have the insect immunocytes (haemocytes) as main targets. there is a high redundancy in the kind of immunodepressive toxins and in their mode of action. kinase-mediated cell signalling in the response of mytilus hemocytes to bacterial challenge l canesi1, m betti1, c ciacci1, lc lorusso1, c pruzzo2, g gallo2 1istituto di scienze fisiologiche, università di urbino “carlo bo”, loc. crocicchia, 61029 urbino (pu); 2dipartimento di biologia, università di genova, 16132 genova, italy studies on host-pathogen interactions in mammalian systems have shown that certain bacteria can evade the bactericidal activity of the host cell through disregulation of the signalling pathways involved in the immune response; in particular, mapks (mitogen activated protein kinases) represent an important target for pathogenic microorganisms. in marine bivalves, persistence of different bacteria (mainly vibrionaceae and coliforms) largely depends on their sensitivity to the bactericidal activity of circulating hemocytes: however, the signaling pathways involved in bacteria-hemocyte interactions are still largely unknown. in the mussel mytilus, differences in interactions between hemocytes and different e. coli and v. cholerae strains (e. coli mg155, a wild type strain carrying type 1 fimbriae, and its unfimbriated derivative, aaec072 ∆fim; v. cholerae o1 el tor biotype strain n16961, carrying the mannose-sensitive hemagglutininmsha, and its msha mutant) lead to differences in bactericidal activity in the presence of serum. here we show that different bacteria induced distinct patterns of phosphorylation of mapks, in particular of the stressactivated p38 and jnk mapks, in mussel hemocytes. differences in pkc phosphorylation were also observed. the results support the hypothesis that, like in mammalian host cells, different bacteria can modulate the signaling pathways of mussel hemocytes. in particular, the lower bactericidal activity towards the mutant e. coli strain and wild type v. cholerae compared with that of wild type e. coli may be due to a reduced capacity of these strains of activating mapks. moreover, the mutant v. cholerae strain, that was the most resistant to the hemocyte bactericidal activity and showed the strongest cytotoxic effect, induced downregulation of both mapk and pkc signaling. these data suggest that certain bacteria could evade the bactericidal activity of mussel hemocytes through disruption of the host signalling pathways. effects of tnfαα in mussel hemocytes: role of kinase pathways m betti1, c ciacci1, lc lorusso1, b canonico2, l canesi1 1istituto di scienze fisiologiche, 2istituto di scienze morfologiche, università di urbino “carlo bo”, loc. crocicchia, 61029, urbino (pu), italy tnfα (tumor necrosis factor α) is a pleiotropic cytokine that plays a pivotal role in orchestrating innate immune responses as well as in apoptotic processes. tnfα triggers several intracellular pathways, including the mapk (mitogen activated protein kinase) cascade, which can control gene expression through modulation of transcription factors. invertebrate counterparts of tnfα signaling have been identified in drosophila; a cytokine network is also active in molluscs, and molluscan hemocytes are responsive to heterologous cytokines. in this work, the possible effects of tnfα on hemocyte signaling and function were investigated in the bivalve mytilus. the results demonstrate that tnfα induced lysosomal membrane destabilization and depression of phagocytic activity in mussel hemocytes. these effects were rapidly followed by irreversible cell damage, as evaluated by flow cytometric analysis, and were paralleled by persistent increases in phosphorylation of 23 the stress-activated mapks p38 and jnks and of the transcription factor stat1. however, in the presence of hemolymph serum, the cytotoxic effects of tnfα were significantly reduced. interestingly, a transient and reversible ps (phosphatidyl serine) exposure was observed; such an effect was associated to a rapid, large, but transient stimulation of the stress-activated mapks and of the transcription factor stat1. the results demonstrate the presence of conserved components of tnfα signaling in molluscan immunocytes; moreover, these data indicate that hemolymph serum components actively participate in modulating the pathways involved in maintaining the balance between survival/death stimuli. sessione 2. invertebrate immunity: cellular response anticipating innate immunity without a toll el cooper laboratory of comparative neuroimmunology department of neurobiology school of medicine at ucla university of california, los angeles, usa when we think of the immune system, we can now consider it from an evolutionary viewpoint. there is resurgence of interest in immune-defense systems of invertebrates, such as fruit flies and earthworms. their properties can be characterized as innate, natural, nonspecific, non-anticipatory, and non-clonal. innate immunity operates through leukocytes that are not components of the macrophage t and b systems that characterize vertebrate adaptive immunity whose properties can be categorized as adaptive, induced, specific, anticipatory, and clonal. in this review, we will focus on the earthworm system. earthworms, in particular, like other complex invertebrates, possess several leukocytes and the molecules that they synthesize and secrete. together they effect phagocytosis, encapsulation, agglutination, opsonization, clotting and lysis of foreign components. at least two major leukocytes: small coelomocytes, (sc) and large coelomocytes (lc) mediate lytic reactions against several targets. destruction of tumor cells in vitro reveals the dissociation of phagocytosis from natural killer cell responses. a third type, the chlorogogen cell (cc), synthesizes and sheds effector lytic molecules. among the lytic molecules, three have been identified and sequenced (fetidins, ccf-1, lysenin) and another has been discovered (eiseniapore) three others h1 h2 h3 share agglutinating and lysing functions and lumbricin i is the only small antimicrobial but non-lytic molecule. cellular and humoral components function to distinguish between self and not self, dispose of internal (cancer?), damaged components and external antigens (microbes). innate immunity evolved as an essential survival strategy in all living species. the innate immune system is capable of recognizing conserved microbial structures or products of microbial metabolism (pathogen associated molecular patterns [pamps]) through a set of germ line encoded receptors called pattern recognition receptors (prrs). the prrs of the innate immune system, particularly the family of toll-like receptors (tlrs) is responsible for initiating inflammatory response against invading pathogens. toll and toll-like receptor signaling is essential for phagocytosis and antimicrobial peptide production in insects and vertebrates. there is a need to examine the situation with respect to toll and tlrs in earthworms in the face of the above information. their presence or absence would be of enormous interest. effect of immunostimulants on immune gene expression in decapod crustaceans vj smith gatty marine laboratory, university of st andrews, st andrews, fife, scotland, uk the expansion of the crustacean aquaculture industry over the last two decades years has been accompanied, like all intensive farming enterprises, by the growing problem of disease in the stock animals. as the use of conventional vaccination programmes are inappropriate for shellfish, interest has focused on non-specific enhancement of the crustacean immune system, particularly by compounds known to activate the prophenoloxide activating system. several are now marketed commercially. whilst claims have been made by some authors for the success of these substances when included in the diet, other arguments have been forward that their use may be less than beneficial. indeed, recent work on lobsters has revealed that some may actually be cytotoxic for the haemocytes in vitro. in addition, molecular analyses with quantitative real-time pcr or other approaches are beginning to show that these compounds do not up-regulate immune gene expression, as might be expected. thus, the efficacy and value of immunostimulation in aquaculture remains questionable. this talk will discuss the effects of immune stimulants on the crustacean host defence system and consider whether or not immune-potentiating strategies are the best way forward for disease control in these animals. evolution of innate immunity. components of inflammatory reaction in ciona intestinalis n parrinello, f giaramita, a vizzini, m di bella, d parrinello, m vazzana, c ciancialo, v arizza, m cammarata dipartimento di biologia animale, università di palermo, palermo, italy there are several reasons for analyzing tunicate immune systems and phylogenesis. first, they can be established as primitive models, ancestors of vertebrates, for understanding fundamental immunological mechanisms by analyzing their cells and molecular products; inflammation is the first defence system that includes tissue response to foreign agents locally injected. second, molecular and morpho functional study coupled with molecular approach are requested to characterize molecules (e.g. cytokines) in a phylogenetic contest. third, tunicates are protochordates, pivotal in phylogenetic reconstruction. the body wall of ciona intestinalis mounts a defence inflammatory reaction to 24 lps. the kinetics of the response, cell components and histochemical aspects have already been described. in botryllus schlosseri the non fusion reaction shows inflammatory characters. the comparative study is of interest because these ascidians belong to distinct clade and present different life cycle. cytokine –like molecules have a basic role in inflammatory responses. we will examine cells and molecules, by using several approaches including immunochemistry and immunohistochemistry, protein purification methods, specific antibodies production, functional assay of purified proteins. protein and gene sequence data will contribute to prepare primers, cdna sequences and riboprobes to evaluate hemocyte and tissue expression and modulation (in situ hibridization, real time pcr). with the aim of verifying the cytokine-like role, functional approaches will be carried out to modulate immune responses as phagocytosis and cytotoxicity. here we show an opsonizing activity of c. intestinalis hemolymph that can be significantly decreased by absorption with antihuman-il1. (research granted by miur prin cofin 2004) molecular evolution of innate immunity and mitochondrial genome in ascidiacea c gissi, f iannelli, g pesole dipartimento di scienze biomolecolari e biotecnologie, università di milano, milano, italy systematic comparative analyses of complex gene "categories" in chordates, such as genes belonging to a given metabolic pathway or involved in a particular cellular process, can shed light on the evolutionary dynamics of functionally related genes and reveal specific evolutionary differences between vertebrates and tunicates. moreover, by comparing the evolutionary dynamics of many genes, suitable molecular phylogenetic markers can be identified to reliably reconstruct the chordate phylogeny. in general, a molecular phylogenetic tree depicts the relationships between the analysed sequences and is not necessarily coincident with the organism tree. conversely, congruent phylogenies obtained from several genes strongly suggest that the inferred gene trees reflect the underlying species phylogeny. therefore, the comparison of phylogenies derived from many unrelated genes, such as nuclear and mitochondrial genes involved in unrelated processes, will provide an opportunity for robust reconstruction of the tunicate/chordate phylogeny. we have recently started a project focused on molecular evolutionary and phylogenetic studies of unrelated cellular processes, the innate immunity and the mitochondrial system, in ascidiacea. the molecular phylogeny and evolutionary dynamics of innate immunity nuclear genes will be compared with those of mitochondrial genomes (mtdna). indeed, given its reliability as a phylogenetic marker, the mtdna will be used as reference frame for molecular phylogenetic studies. we want to use incongruencies between the inferred nuclear and mitochondrial gene phylogenies to identify and characterize genes evolving in vertebrates and ascidians under different evolutionary dynamics, as the effect of different selective constraints or change of functions. in order to increase the sample of ascidian mtdnas available for phylogenetic studies, the mtdna of phallusia mammillata and phallusia fumigata (phlebobranchia, ascidiidae) were completely amplified and sequenced. such genomes show extensive gene rearrangements not only compared to other chordate and ascidian mtdnas, but also at intra-genus level. the base compositional features, the shortness of rrna genes, and the absence of a main non-coding region indicate that the evolutionary dynamics of ascidian mtdna differ markedly from those of vertebrates. morula cells and non-fusion reaction in the compound ascidian botryllus schlosseri l ballarin, f parisotto, f cima dipartimento di biologia, università di padova, padova, italy morula cells (mc) are a common haemocyte-type in the compound ascidian botryllus schlosseri, their frequency ranging between 40 and 60% of the circulating blood cells. they are the effector cells of the non-fusion reaction, characterised by the appearance of necrotic foci along the contact border, which occurs when genetically incompatible colonies contact each other. we previously demonstrated that, in the course of this reaction, mc acquire immunopositivity to anti-cytokine (il-1-α and tnf-α) antibodies, degranulate and release the enzyme phenoloxidase which is responsible of the cytotoxicity observed both in vitro (when haemocytes are incubated with blood plasma (bp) from incompatible colonies) and in vivo (non-fusion reaction). subsequently, mc leave the facing marginal ampullae (sausage-like, blind endings of the colonial marginal vessels to reach the tunic, apparently attracted by soluble, diffusing factors, where they degenerate and contribute to the formation of the cytotoxic spots. in the present work, we focussed on the chemotactic recruitment of mc in the course of the nonfusion reaction. as a first approach, we studied the distribution of mc inside the facing marginal ampullae of both contacting colonies (either non-fusible or fusible) and solitary ones. results clearly indicates a significantly higher concentration of mc inside facing marginal ampullae of incompatible colonies with respect to compatible colonies; the latter concentration is, however, significantly higher than that inside ampullae from solitary colonies. in addition, we used transwell chambers to evaluate whether incompatible bp has chemotactic properties. we put haemocyte suspensions in filtered sea water (fsw) in the upper wells and bp from either incompatible or compatible colonies in the lower wells. we observed a significant increase migration of haemocytes, and of mc in particular, in the presence of incompatible bp. this migration was significantly decreased by the addition of anti-cytokine(il-1-α, tnf-α or il-8) antibodies, suggesting that molecules molecules recognised by these antibodies can be responsible of the chemotaxis observed. 25 responses of botryllus schlosseri immunocytes to exogenous cytokines: results and perspectives a menin, l ballarin dipartimento di biologia, università di padova, padova, italy we studied the effects of recombinant tnf-α, il-1-α and il-8 and of antibodies raised against mammalian cytokines (polyclonal anti-tnf-α and anti-il-1-α, monoclonal anti-il-8 and anti-il-12) on phagocytes of the colonial ascidian botryllus schlosseri. in particular, we analysed the ability of phagocytes to assume an amoeboid shape (expressed as amoebocytic index) and to phagocyte yeast cells (expressed as phagocytic index). rtnf-a and ril-1-a have no effects on both the amoebocytic and the phagocytic index, whereas ril-8 significantly increases both the indexes. the observed increase in the phagocytic index was absent in the presence of calphostin c 0.1 µm, an inhibitor of pkc, and of h89 1 µm, an inhibitor of pka, indicating the involvement of both the camp and ip3 pathways in signal transduction required for phagocytosis to occur. the il-8induced increase in amoebocytic and phagocytic index was not observed when haemocytes were pre-incubated in the presence of suramin 0.7 mm, a protein g inhibitor. antitnf-a, anti-il-1-a and anti-il-8 antibodies significantly reduce the above indexes; no effects were observed in the presence of anti-il-12. our results suggest the presence of molecules able to cross-react with mammalian antibodies, involved in the regulation of phagocyte behaviour. heliothis virescens/toxoneuron nigriceps: parasitoid strategies for succesful endoparasitic development m de eguileor, r ferrarese, m brivio, m mastore, a grimaldi, g tettamanti, r valvassori dipartimento di biologia strutturale e funzionale, università degli studi dell’insubria, varese, italy in the association host/parasitoid, heliothis virescens (lepidoptera, noctuidae) shows a combination of different associated mechanisms to kill its parasitoid toxoneuron nigriceps (hymenoptera, braconidae) which, in turn, has to escape encapsulation and melanization by using systems that interfere with host capacity for immunologic recognition. the possibility of recognizing the non-self is due, in heliothis, as for all insects, to humoral and cellular defence reactions. it is well known that eggs of parasitoid toxoneuron nigriceps co-injected with venom (i.e. ovarian calyx fluid) and polydnaviruses (pdv) directly into the host hemocoel are able to re-program the host behaviour, its reproductive potential and its immunity system (pennacchio et al., 1998). the data presented focus on the dramatic suppression of immune responses by parasitization occurring in heliothis larva at different times from wasp ovodeposition. we report the description of some temporary alterations of the immune defences of h. virescens during the very early parasitization stages of t. nigriceps. in addition, due to the fact that successful endoparasitic development requires, especially during the first phases of parasitization, numerous strategies enabling parasite to rapidly growth and to protect itself, we also describe the function of serosal membrane enveloping 1st instar larva of t. nigriceps. biological activities and cytotoxic effects of aqueous extracts from petrosia ficiformis cells p pagliara, l dini disteba-dipartimento di scienze e tecnologie biologiche e ambientali, università degli studi di lecce, lecce, italy many marine organisms represent an important source for bioactive natural products with pharmacological properties. it is well known that, also if potential biomedical activity and chemical defence cannot be equated, nevertheless they can be considered strictly correlated, being bioactive compounds often produced by invertebrates as elements of a chemical defence mechanism. some of these compounds resulted efficient anti-inflammatory, anti-tumor, immunosuppressive and cytotoxic agents. in this framework, an important role is played by sponges. they are characterised by a simple structural organization, lacking of tissue and organs, and posses cells with the peculiar ability to change shape and function, showing in this way an unusual totipotency capability. in this work we report our investigation on biological activity of the marine sponge petrosia ficiformis cell lysate. haemolytic, haemagglutinating and cytotoxic activities of these extracts were monitored. we found that cell extracts do not possess haemolytic or haemagglutinating activity against human blood erythrocytes. on the contrary, a cytotoxic effect on a fibroblast-like cell line was detected: the vitality of detroit 550 was drastically reduced after treatment with sponge extracts. the influence of cell aqueous extracts was also evaluated on brine shrimps vitality and on sea urchin development. sponge cell lisate strongly affected vitality of artemia salina brine shrimps, as well as the sea urchin development, that resulted in an accelerate process with an increase in abnormal embryos production. defensive response of the freshwater crayfish astacus leptodactylus following three different challenges in vivo s lorenzon, pg giulianini, s battistella, m bierti, ea ferrero dipartimento di biologia, università di trieste, trieste, italy this study assesses the in vivo response of the freshwater crayfish astacus leptodactylus (eschscholtz, 1823) following three different challenges (from a natural system to a completely artificial one) by controlling at the same time the thc (total hemocytes count) as immunological parameter and glycemia as a blood stress one. we investigated the effect of injection of 1) pseudomonas sp. different amounts (200 µl of 1x108, 1x107, 1x106 live bacteria ml-1), 2) different doses of lps (lipopolysaccharide) from pseudomonas sp ( 0.01, 0.001, 26 0.0001 mg g-1 of living weight), 3) three kinds of polystyrene latex beads (1.1, 3.0 µm in diameter and carboxylated polystyrene latex beads 0.9 µm in diameter) and 4) sterile pbs and bled only animals as controls. both injection of pbs and bled only experiments showed an increase in thc in the first 2h. injection of bacteria as well as of lps caused a dose related decrease of thc that lasted until 24h; the lowest doses of both treatments on the contrary stimulated an increase of thc. all polystyrene latex beads produced an increase of thc that resulted more evident in the case of carboxylated polystyrene latex beads. when considering glycemia, both pseudomonas sp. and lps induced again a dose related response. no significant variation of glycemia was induced by the response elicited by 1.1 µm in diameter polystyrene latex beads while carboxylated latex beads resulted the most effective in inducing hyperglycemia. high doses of bacteria and lps caused a loss in immunodefence and a stress response. on the contrary low concentrations of bacteria, lps and the three kinds of latex beads induced an activation of the immunosystem causing an increase of thc and, in particular, the carboxylated latex beads result the artificial system that best simulates the natural challenge. ultrastructural and functional characterization of circulating hemocytes from adults and larvae of carabus lefebvrei dejean 1826 (coleoptera, carabidae) a giglio1, s battistella2, ff talarico1, m romeo1, t zetto1, pg giulianini2 1dipartimento di ecologia, università della calabria, arcavacata di rende (cs); 2dipartimento di biologia, università di trieste, trieste, italy in the context of comparative studies on immunity defence mechanisms of adults and larvae of the coleopteran carabus lefebvrei dejean 1826, the circulating hemocytes of the third instar larval stage and adults has been investigated by means of transmission electron microscopy (tem). phagocytosis assays were performed in vivo by injection of polystyrene latex beads in order to identify the cell types involved in this cellular response. 50 µl of carboxylate-modified polystyrene latex beads 0.9 µm in diameter diluted 1:10 in 0.15 m phosphate buffered saline ph 7.4 (pbs) were injected with a 26-gauge needle in the abdomen of 5 larvae and 5 adults. parallel controls were run with untreated animals and with animals injected with 50 µl of sterile pbs alone. after 2 h individual animals were cold anesthetized and the last two abdominal segments laterally torn; a 26-gauge needle was inserted in the neck membrane and pbs slowly injected. the two first drops exiting through the tear in the abdomen were collected directly in 2.5% glutaraldehyde, 1% paraformaldehyde in 0.1 m cacodylate buffer ph 7.4 and, the hemocytes pelleted for 10 min. the pellets obtained from pooled hemolymph of the five animals were then post-fixed in 1% osmium tetroxide and embedded in embed812/araldite. sections were observed with a tem philips em 208. four types of hemocytes were found in the hemolymph of larvae and adults of c. lefebvrei and they were identified as prohemocytes, granulocytes, plasmatocytes, and oenocytoids. prohemocytes are the smallest circulating hemocytes with an oval/irregular profile and a high nucleus/cell surface ratio. granulocytes are oval, elongated cells with a maximum diameter up to 13 µm, they are characterized by electron dense granules (up to 15 per section) with a round to elliptical profile and a mean diameter of 465±118 nm (n=10). plasmatocytes are large, irregular cells with a maximum diameter up to 15 µm and they represent, after injections, about 70% of total circulating hemocytes, 10% of which presenting up to 11 phagocytized latex beads. they are characterized by numerous electron dense granules with a mean diameter of 214±74 nm (n=18). they present a large euchromatic nucleus with a prominent large nucleolus and a welldeveloped rough reticulum. it was demonstrated that the plasmatocytes are the only hemocyte types involved in phagocytic response of foreign particles both in larvae and adults. mussel response to anthropic pressures measured by hsp70 gene expression in q-pcr c cellura1, m toubiana2, n parrinello1, p roch2 1 marine immunobiology laboratory, university of palermo, via archirafi 18, 90123 palermo, italy; 2pathogens and immunity, umr ecolag, university of montpellier 2, cc 093, place e. bataillon, 34095 montpellier cedex 05, france coastal environment constitutes a fragile ecosystem submitted to several anthropic pressures. large variety of marine animals is living in coastal shallow water. among them, bivalve molluscs are generally considered as good sentinels to evaluate environmental changes. as a first approach to investigate the response of bivalves through quantification of key gene expressions, the present study focussed on experimental heat-shock effect on expression of hsp70 gene in mussel, mytilus galloprovincialis. adult mussels were submitted to a single heat-shock consisting in 90 minutes immersion in 30°c sea water. before heat-shock, and at 0, 3, 6, 9, 12, 15, 18 and 24 h after heat-shock, hemocytes were collected from posterior adductor muscle. total rna was extracted and converted into cdna using reversetranscriptase. as non available, we designed consensus primers to amplify a hsp70 cdna fragment of 296 bp. nucleotide sequence revealed identities of 99.4 % with m. edulis (af172607), 80.1 % with oyster ostrea edulis (aj318883) and 73.6 % with oyster, crassostrea gigas (aj271444). quantification of hsp70 gene expression was done by real-time pcr (or q-pcr) using the roche light cycler and sybr green. according to literature, we chosen 28s ribosome as house keeping gene, designed primers and considered its expression as constant through the experiment. expression of hsp70 gene improved during the first 6 h, and returned to baseline after 24 h. significance of both, the phenomenon and the calculations, will be discussed. early developmental stages of wssv in marsupenaeus japonicus va di leonardo1, n parrinello2, j-r bonamì1 27 1marine immunobiology laboratory, university of palermo, via archirafi 18, 90123 palermo, italy; 2pathogens and immunity, umr ecolag, university of montpellier 2, cc 093, place e. bataillon, 34095 montpellier cedex 05, france the white spot syndrome virus (wssv) is an highly pathogenic agent for marine and freshwater crustaceans, so it is highly dangerous for crustacean farming. we examed two shrimps, palaemon sp. and marsupenaeus japonicus treated per os with a wssv preparation from sick amimals. in addition treated animals, after five days, were furtherly infected through intramuscolar injection of suitable virus preparation. among the diagnostic tools we used, pcr and dot-blot were able to identify the virus present in the acute phase of injection, whereas in situ hybridization (ish) gave positive signals just at a un precocious stage of the infection. palaemon treated per os were not affected by wssv, but the syndrome was evident when they were furtherly injected. already, m. japonicus was sensitive to per os treatment, as reveled by ish on cells of intestinal epitelium, gills and oka lymphoid organ. finally, in m. japonicus were observed by tem, early developmental stages of wssv in oka organs and intestinal epitelium. virus development model is proposed. natural immunity in paracentrotus lividus: coelomocyte cooperation v arizza, f giaramita, m cervello, m cammarata, n parrinello dipartimento di biologia animale, università di palermo, palermo, italy paracentrotus lividus coelomic fluid contains several coelomocyte types that have been identified as amoebocytes, uncoloured spherulocytes (us), red cells and vibratile cells. previous studies on coelomocytes, revealed that amoebocytes show phagocytic activity and are able to encapsulate foreign particles, in addition they release haemoagglutinins; uss show in vitro cytotoxic activity against rabbit erythrocytes (re) as revealed by plaque forming assay. in this study, the coelomic fluid was fractionated through a discontinuous iodixinol gradient, and four cell bands(b1-b4) were obtained. each of them appeared to be enriched in a particular coelomocyte type: b1 mainly contained amoebocytes; in b2 are present vibratile cells and little amount of amoebocytes; b3 was enriched with uss, and b4 contained red cells. the reported results support the phagocytic activity of amoebocytes assayed with yeast cells, whereas the separated us population were not able to show their in vitro cytotoxic activity (plaque forming assay) against re. when amoebocytes (1 x 106ml-1) from the enriched fraction were added to uss (1:1), this activity was restored revealing a coelomocyte interaction. since supernatant from amoebocyte cultures, added to the reaction medium, was able in inducing us cytotoxicity, the interaction seems to be based on substances released from amoebocytes. western–blot assay with anti-human il1 á antibodies (sigma) showed that, in a short time, cultured amoebocytes release interleukin-like molecules. on the other side, protein preparations from us cell membranes cross-reacted with anti-human il1-r antibodies. further studies are requested to characterize the molecules that contain human il1 and il1-r epitopes. isj002.pdf 38 isj 1: 38-46, 2004 issn 1824-307x research report role of cathepsin b in leech wound healing a grimaldi1, g tettamanti1, l rinaldi1, g perletti2, r valvassori1, m de eguileor1* 1 department of structural and functional biology, university of insubria, varese, italy 2 department of structural and functional biology, university of insubria, busto arsizio, italy accepted june 30, 2004 abstract the wound healing process in leeches involves different types of cells like macrophages, nk-like cells and granulocytes. these cells that are involved in immune defence, can co-operate to attack and/or isolate the non self (de eguileor et al., 1999; de eguileor et al., 2000a; de eguileor et al., 2000b). in addition other types of cells, like fibroblasts and endothelial cells, are involved in the formation of new vessels. to exert their functional role, all these cells must infiltrate and migrate through extracellular matrix (de eguileor et al., 2001a; de eguileor et al., 2003). here we show, by histochemical and biochemical methods, that the cathepsin b peptidase is present and active in all migrating cells, involved in immune responses of leeches subjected to different stimuli. interstingly the cellular function of cathepsin b in invertebrates appear to be equivalent to that of vertebrates, where the secreted enzyme plays a role in basement membrane and matrix disruption operated by cells involved in angiogenesis, wound repair and immune defence. key words: leeches; cathepsin-b; immune cells; angiogenesis introduction both in invertebrates and vertebrates, inflammation is an acute reaction triggered by different types of lesions and aimed at preventing systemic infections. it is mediated by specific cells such as macrophages and neutrophiles that infiltrate the damaged tissue, removing tissue debris and controlling invading microorganisms. these cells synthesize different molecules such as growth factors and cytokines, inducing mesenchymal cell recruitment in the lesioned area (moulin, 1995). in addition, actively phagocytizing cells produce and secrete into the extracellular matrix a wide array of lysosomal enzymes, among which cathepsins play a pivotal role. the role of cathepsins as scavengers has been recently reconsidered due to *corresponding author: magda de eguileor department of structural and functional biology, university of insubria, j.h. dunant 3, 21100 varese, italy. e-mail: magda.deeguileor@uninsubria.it the increasing identification and functional characterization of new lysosomal cysteine proteases (bond and butler, 1987; kirschke et al., 1995; turk et al. 2000; turk et al. 2001). cysteine proteases were believed to be mainly involved in intracellular protein degradation, since they are optimally active in the slightly acidic, reducing milieu found in lysosomes; these endopeptidases are synthesized as inactive precursors and they are regulated by several inhibitors or by ph, to regulate their lytic effects (mach et al., 1994; lah et al., 1995; turk et al., 2000). recently, it has been demonstrated that besides their role within lysosomes, cysteine proteases can degrade proteins outside lysosomes, and can contribute to protein processing within cell cytoplasm (kos and lah, 1998; lah and kos, 1998, pierre and mellman, 1998, saftig et al., 1998, shibata et al., 1998). cathepsins have been shown to be involved in cancer progression (kos and lah, 1998; lah and kos, 1998), apoptosis regulation (shibata et al., 1998), mhc ii antigen presentation (pierre and mellman, 1998) and tissue remodeling (saftig et al., 1998). in particular, secreted cathepsins seem to have a role in degradation of basement membrane and extracellular matrix, 39 favouring migration of cancer (kos and lah, 1998) and immune (diment et al., 1988; young et al., 1991) cells. regarding invertebrates, cathepsin-like proteins have been characterized in several taxa like protozoa (judice et al., 2004), platyhelminthes (wong et al., 1997), molluscs (pipe, 1990), arthropods (deraison et al., 2004), while an involvement of cathepsin b in regulation of neo-angiogenesis has recently been hypothesized for the annelid hirudo medicinalis (tettamanti et al., 2004, in press). in addition lefebvre et al., (2004) have cloned a cystatin gene (tt-cysb) in the leech theromyzon tessulatum, suggesting that the proteinase inhibitor tt-cysb may regulate leech cathepsin b. in leeches the wound healing process is characterized by the same sequence of events described in vertebrates. in particular, during the inflammation phase, immune cells numerically increase and move toward the lesioned area, while during the granulation tissue stage angiogenesis and fibroplasia take place (tettamanti et al., 2003). during these events, different types of immune cells (macrophages, nk-like cells and granulocytes) that co-operate to attack and/or isolate the non self, as well as fibroblasts and endothelial cells, involved in the formation of new vessels, have to push their way through the extracellular matrix (de eguileor et al., 1999; de eguileor et al., 2000a; de eguileor et al., 2000b; de eguileor et al., 2001b; de eguileor et al., 2003). in the present study, utilizing histochemical and biochemical methods, it is shown that the peptidase cathepsin b is present in all migrating cells that are involved in a number of immune functions in leeches subjected to different stimuli. materials and methods leeches glossiphonia complanata (annelida, hirudinea, glossiphoniidae) were collected from the adda river, near milan, italy. they were kept in water at 18°c in aerated tanks and fed ad libitum with planorbidae snails. leeches were divided into two groups. group 1 included 5 unstimulated leeches as control animals; group 2 included 15 leeches stimulated by lipopolysaccharide (lps) injection. a solution of 10 mg/ml of lps was used for immune stimulation (de eguileor et al., 2000b). leeches were kept in water in separate tanks and sacrificed 30 min, 2 hr and 4 hr intervals after lps treatment. hirudo medicinalis, (annelida, hirudinea, hirudidae), purchased from ricarimpex, eysines, france, were kept in water at 22-23°c in aerated tanks, and were fed monthly with calf liver. prior to experiments, leeches had been kept fasting for a month. leeches were subdivided into two groups. group 1 included 5 unstimulated h. medicinalis, as control animals, used to document the normal body wall organization of this leech. group 2 included 25 h. medicinalis subdivided into subgroups a and b. subgroup a): 20 animals were subjected to lesions consisting of a tissue explant (about 2 mm × 2 mm x 2 mm) affecting the entire body wall. the tissue explant was surgically removed with microdissecting scissors, and the wounded leeches were observed at 3 and 5 hr intervals after surgical explantation. subgroup b): 5 animals were injected with 30 ìl of the growth factor gm-csf (10 ng/ìl) solution. injected leeches were observed at 5 hr intervals after growth factor injection. prior to surgical procedures and fixation, leeches of both species were anesthetized with a saturated solution of mephenesin (3-o-toloxy-1,2propanediol), a muscle relaxant. light and electron microscopy (tem) leeches were dissected and fixed in 2% glutaraldehyde in 0.1 m na-cacodylate buffer (ph 7.2) for 2 hr at room temperature, then washed in the same buffer and postfixed for 2 hr with 1% osmic acid in 0.1 m na-cacodylate buffer (ph 7.2) at room temperature. after standard steps of serial ethanol dehydration, specimens were embedded in an epon-araldite 812 mixture. semi-thin and thin sections were cut with a reichert ultracut e ultratome (leica, nussolch, germany). semi-thin sections (1 µm) were stained by conventional methods (crystal violet and basic fuchsin), and observed with a light microscope (olympus, tokyo, japan). thin sections (80 nm) were collected on 300 mesh copper grids, stained with uranyl acetate and lead citrate, and observed with a jeol 1010 ex electron microscope (jeol, tokyo, japan). immunocytochemistry anaesthetised leeches were dissected in a cold ringer solution (miller and aidley, 1973) into small blocks, which were immediately embedded in polyfreeze cryostat embedding medium (oct) (polyscience europe, eppelheim, germany), and stored in liquid nitrogen according to geiger and coworkers (1980). cryosections (10 µm thick) of unfixed leeches were obtained with a reichert-jung frigocut 2800; slides were immediately used or stored at -20°c. sections were incubated for 15 min with evans blue (de la lande and waterson, 1968) to reduce autofluorescence, washed with pbs, and incubated in a moist chamber for 1 h at 37 °c with a primary mouse anti-human cathepsin b monoclonal antibody (clone ca10, calbiochem, cambridge, ma, usa), diluted 1:20. after incubation with the primary antibody, specimens were washed and incubated with appropriate secondary antibody conjugated with fluorescein isothiocyanate (fitc) (1:50 dilution, jackson immuno research laboratories, west grove, pa, usa). incubations were performed for 1 hr in a dark moist chamber at 37 °c. double stainings were performed by firstly incubating samples with anti-human cathepsin b diluted 1:20 and fitc-conjugated secondary antibody. subsequently the same specimens were incubated with a primary mouse monoclonal antihuman cd68 antibody (clone pg-m1, diluted 1:20, dba, milan, italy) and with a tetramethylrhodamine (tritc)-conjugated secondary antibody. antibodies were diluted with pbs containing 2% bovine serum albumin (bsa). in control samples, primary antibodies were omitted, and sections were treated with bsa-containing pbs. coverslips were mounted in vectashield mounting medium for fluorescence (vector laboratories, burlingame, ca, usa); slides were 40 examined at 40x (na 1.30) magnification with a confocal laser microscope (laser 568 nm for rhodamine, laser 492 nm for fluoresceine; mrc 1024, bio-rad laboratories, hemel hempstead, uk). confocal images were superimposed using the photoshop 5.0 program; fluorescent images were then overlayed onto transmission images showing the corresponding tissue sections. biochemical procedures h. medicinalis tissues from the unstimulated body wall or from areas involved in surgical events were frozen in liquid nitrogen and then homogenized with a mortar. for sds-polyacrylamide gel electrophoresis (sdspage), leech homogenates were suspended in extraction buffer (2x laemmli's buffer in the presence of a protease inhibitor cocktail (sigma, milan, italy)); the particulate material was removed by centrifugation at 13000 rpm for 10 min at 4°c in a refrigerated eppendorf minispin microcentrifuge. supernatants were denatured at 100°c for 10 min. sds-page proteins were separated in analytical sds-page using 15% acrylamide minigels (running conditions 200v for 1h). molecular weights were determined by concurrently running broad range standards from biorad (bio-rad, richmond, ma, usa). gels were stained with 2.5% coomassie blue (bio-rad) in methanol:acetic acid:water 4:1:5. western blot proteins separated by sds-page were transferred onto bio-rad nitrocellulose filters. membranes were then saturated with 2% bsa in trisbuffered saline (tbs, 20mm tris-hcl buffer, 500mm nacl, ph 7.5) at room temperature for 2 hr, and incubated overnight at 4°c with a mouse anti-human cathepsin b antibody (1:250 dilution in 2% tbs-bsa). after washing the membrane three times with tbs, the immunocomplexes were revealed with an appropriate alkaline phosphatase-conjugated secondary antibody (1:1000) (sigma). immunoreactivity was detected with a sigma fast bcip/nbt system (sigma). results general characteristics of leech body wall in unstimulated and stimulated animals the body wall of leeches is virtually avascular (figs 1, 2) and is made of tightly-packed muscle fibers, grouped in fields and separated only by extracellular matrix or by dorsoventral fibers (figs 1, 2). after stimulation there is always a proliferation and movement of immune cells and macrophages, nk-like cells and granulocytes migrate towards the injured area (figs 3, 4, 5) forming a plug (fig. 6). while in glossiphoniidae proliferation and migration of immune cells are basic events, in hirudidae proliferation and migration are associated to important effects as angiogenesis and fibroplasia. thus in these leeches the formation of a network of blood vessels, spanning the entire body wall of the animal, occupies the space among the fields of muscle fibers (fig. 7) within the whole thickness of the body wall. cathepsin b detection in glossiphonia complanata cathepsin b immunolocalization was performed, both in control and stimulated leeches. in untreated animals anti-cathepsin-b antibody stained the cytoplasmic core of circomyarian helical muscle fibers (figs 8, 9). after 30’ from lps administration a marked signal was detectable not only in the basal lamina surrounding the muscle fibers of the treated area, but also in the cytoplasm of migrating cells localized among the muscle fields (fig. 10). double immunofluorescence localization with cd68 (a specific marker for vertebrate macrophages) and cathepsin b antibodies, showed that migrating cells, were macrophage-like cells since cd68 and cathepsin b signal co-localized in the same cell types (fig. 11). two and 4 hr after lps injection, cathepsin b signal was detectable in migrating cells: in particular the signal was mostly visible in cell basal lamina (fig. 12). double staining with anti-cd68 and anti-cathepsin b antibodies showed that 4 hr interval from lps treatment the cathepsin b signal was mainly localized in the basal lamina surrounding cd68+ macrophage-like cells (fig.13). cathepsin b detection in hirudo medicinalis in unstimulated animals cathepsin b was mainly expressed in the cytoplasm of muscle fibers (figs 14, 15). about 3 hr after a surgical lesion, cathepsin b immunoreactivity was detectable, as in g. complanata, in migrating cells localized in the connective tissue among muscle fibers (fig. 16). most migrating cells were macrophages, as demonstrated by co-localization of cathepsin b and cd68 (fig. 17). the two antibodies recognized both migrating cells in the injured area and cells filling the explanted area and forming the “plug” (figs. 6, 18). five hours after tissue explantation, cathepsin b and cd68 signals did not co-localize. in the plug area, cd68 stained macrophage-like cells, while cathepsin b was mainly found in the surrounding connective tissue (figs 19, 20). after 5 hr from injection of gm-csf growth factor, cathepsin b was detected in migrating cells as well as in endothelial cells lining the new vessels of stimulated animal (figs 21, 22). western blot analysis of cathepsin b in stimulated hirudo medicinalis the presence of cathepsin b in leeches was assayed in protein extracts from stimulated h. medicinalis body wall. tissues from area close to explant were taken 3 and 5 hr intervals after surgical lesion (fig. 23). western blot showed that the anticathepsin b antibody detected a single band of about 34 kda in protein extracts of leeches sacrificed 3hr after surgery, while in proteins extracts of leeches sacrificed 5hr after surgery two immunoreactive bands of about 34 kda and 31 kda were detected. 41 figs. 1, 2 semi-thin cross sections of unstimulated leeches g. complanata (fig. 1) and hirudo medicinalis (fig. 2). under the epithelium (e) muscle layers, circularly, obliquely and longitudinally oriented, are visible (m). helical muscle fibers are tightly packed and organized into groups sometimes separated by dorsoventral muscles (dm). bars = 80 µm and 100 µm respectively. figs 3,4,5. semi-thin cross section of stimulated g. complanata (fig 3). migrating cells (arrowheads) are visible in the extracellular matrix among muscle fields. e (epithelium), m (muscle fibers). bar = 80 µm. semi-thin (fig. 4) and thin (fig. 5) sections of surgically lesioned hirudo medicinalis. macrophage-like cells (arrowhead) are detected among muscle fibers (m) in the newly synthesized extracellular matrix. n (nucleus). bars = 20 µm, 4 µm. discussion as we reported previously, characterization and expression studies have stressed the existence of several similarities between leech and vertebrate immune responses. in leeches, processes in response to surgical wounds, grafts or injections of stimulating factors (lps, growth factors) are similar to equivalent responses in vertebrates and involve a wide range of sequential events triggered by inflammatory reactions (de eguileor et al., 2003, tettamanti et al., 2003, tettamanti et al., 2004). macrophage-like cells, nk-like cells and granulocytes migrate through the extracellular matrix from the inner regions of the body towards stimulated areas. contextually, as demonstrated for h. medicinalis, beside the migration of activated fibroblasts involved in the production of the scaffold, supporting the network of new capillaries (tettamanti et al., 2004), the formation of new vessels begins from the botryoidal tissue. in fact, within a short time interval after an angiogenic stimulus, botryoidal tissue cells forming a solid cord, proliferate and reshape into tubes, the new capillaries, that stretch towards the lesioned area by moving between muscle fiber fields through the ecm (de eguileor et al., 2001b). it is important to underline that all steps of these complex responses have the migration of cells as a common denominator. in fact either immune cells or endothelial cells or fibroblasts (involved directly or indirectly in neovascularization) need the degradation of extracellular matrix in order to move and change their spatial position in the leech body. 42 fig. 6 semi-thin cross section of surgically lesioned h. medicinalis. the area of lesion is infiltrated by a high number of migrating cells forming a “plug” (arrowheads). bar = 200 µm fig. 7 semi-thin cross section of surgically lesioned h. medicinalis. numerous new vessels (encircled areas) are visible among muscle fibers (m). e, (epithelium). bar = 100 µm. fig. 8 cryosection of unstimulated leech g. complanata. an anti-cathepsin b antibody stains the cytoplasmic core of helical muscle fibers (m). fig. 9 cryosection of stimulated g. complanata. negative control for cathepsin b staining. positivity is detected neither in muscle cells (m) nor in migrating cells (arrowheads). fig. 10 cryosection of stimulated g. complanata sacrificed 30 min after lps administration. cathepsin b is markedly expressed in the cytoplasm of migrating cells (arrowheads). the signal is detected also in the cytoplasm of a few helical fibers (m) and, widely, in their basal membrane (arrows). fig. 11 cryosection of stimulated g. complanata analyzed 30 min after lps administration. co-localization of cathepsin b (green) and cd68 (red) is indicated by yellow fluorescence in migrating cells (arrowheads) localised between muscle fibers (m). a signal for cathepsin b is also detected in basal lamina of muscle fibers (m). 43 fig. 12 cryosection of stimulated g. complanata, analyzed 2-4 hr after lps injection. cathepsin b signal is detected in the cytoplasm and in the basal lamina of macrophages (arrowheads) and muscle fibers (m). fig. 13 cryosection of stimulated g. complanata, analyzed after 2-4 hr from lps administration. double immunofluorescence shows that while anti-cd68 antibody (red) stains the cytoplasm of all macrophages, only some of them are positive also for cathepsin b (green) as demonstrated by co-localization (yellow fluorescence). fig. 14 cryosection of unstimulated h. medicinalis. cathepsin b is expressed in the cytoplasm of muscle fibers (m). fig. 15 cryosection of h. medicinalis. negative control for cathepsin b staining. no positivity is detected. muscle fibers (m). fig. 16 cryosection of stimulated h. medicinalis analyzed after 3 hr from surgical stimulation. cathepsin b is mainly located in the cytoplasm of migrating cells (arrowheads) localized among muscle fibers (m). figs. 17, 18 cryosections of h. medicinalis analyzed 3 hr after surgical stimulation. double staining using anticathepsin b (green) and anti-cd68 (red) antibodies shows that migrating cells (fig. 17) and those forming the plug are macrophages (fig. 18). 44 figs. 19, 20 cryosections of h. medicinalis analyzed 5 hr after surgical stimulation. double immunofluorescence evidences that in plug area (figs. 19, 20) macrophages (arrowheads) are cd68 positive (red stain) while extracellular matrix (encircled areas) is cathepsin b positive (green stain). figs. 21, 22 cryosections of stimulated h. medicinalis analyzed 5 hr after gm-csf injection. cathepsin b is detectable in cells migrating towards the injected area and in endothelial cells (encircled area) of neo-vessels (v) (fig. 21). in negative control for cathepsin b staining (fig. 22) no positivity is detected. in vertebrates cell migration involves the degradation of extracellular matrix through enzymatic digestion. cathepsin b, is a protease that can degrade components of ecm outside the cells. it is active against large substrate components such as laminin, fibronectin and collagen iv. in the animal model used in the present study, in addition to data previously described regarding immune responses, it is important to underline that the extracellular matrix is formed of the same components typical of vertebrates (de eguileor et al., 1999); for this reason we have hypothesized the involvement of cathepsin b in stimulated and lesioned g. complanata and h. medicinalis. in unstimulated leeches (both species), cathepsin b is detected in the cytoplasmic core of muscle fibers and in the very few migrating cells present, while there is a considerable change in the intensity and in the localization of the immunoreactivity against the enzyme in the period of time following any stimulating events. following surgical stimulation, the enzyme is not only detectable in muscle fiber cytoplasm, but also in the basement membrane of muscle fibers. in addition, a strong immunoreactive signal is detected predominantly in the cytoplasm of migrating cells, mostly cd68-positive macrophages, and, as previously demonstrated, in activated fibroblasts (tettamanti et al., 2004). local degradation of basement membrane followed by ecm digestion, is a crucial step in cellular migration. in h. medicinalis, 5 hr after surgical stimulation, cathepsin b is mainly localised in the connective tissue surrounding 45 fig. 23 western blot analysis of surgically stimulated h. medicinalis. the antibody anti-cathepsin b detects a single band of about 34 kda and 2 bands of 31 and 34 kda 3 and 5 hr after surgery, respectively. mwm (molecular weight marker), cat b (cathepsin b). numerous migrating cells as immune cells, fibroblasts and endothelial cells. these cells push their way among groups of muscle fibers. some of these cells are involved in the obstruction of the wound and in tissue repair, whereas cellular elements, involved in agiogenesis, create a “pipe system” to transfer nutrients, oxigen and immune cells in stimulated area (tettamanti et al. 2003, de eguileor et al., 2003). it is interesting to underline that the increased immunoreactivity towards cathepsin b that follows the surgical stimulation, has been also detected after activating the immune system by injection of lps (de eguileor et al., 2000b). in addition, in vertebrate it is well known that the growth factor administration may upregulate the expression and activity of cathepsin b (koblinski et al., 2000); thus it is suggested here that in leeches the same regulation might be present since vegf, egf, bfgf and gm-csf figure among the most potent angiogenic factors and since the formation of new vessels requires massive movements of cells (de eguileor et al., 2004, tettamanti et al. 2003), our hypothesis is supported by the markedly increased immunopositivity for cathepsin b in the stimulated areas by gm-csf administration. this growth factor released in the ecm, is generally produced by a variety of different cells such as macrophages, endothelial cells and fibroblasts. it must be emphasized that, to validate the immunocytochemical localization of cathepsin b, western blot analysis of this protease confirms a degree of similarity between hirudinean and vertebrate cathepsin b signal. immunostaining with antibody raised against cathepsin b resulted, in control animals (tettamanti et al., 2004, in press) and in stimulated leeches after short time elapsed from stimulation, in one band, while two bands that probably could correspond to the mature form and proform (takahiro et al., 1993) resulted after 5 hr from stimulation. in conclusion, the results reported here on the differentiated release of cathepsin b from unstimulated and variously stimulated leeches confirm, also in 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appendix leeches are invertebrates characterized by a relatively simple anatomy. the body wall of unstimulated, control leeches (glossiphonia complanata and hirudo medicinalis), contains several organs embedded in a loose connective tissue. it is composed of a monolayered epithelium surrounding three different layers of circular, oblique and longitudinal helical muscle fibers. in spite of the basic plan, several differences can be noted in the body organization of leeches belonging to different families. the differences that may be remarked vary from the degree of longitudinal muscle layer thickness to a reduction of blood system, to the presence of peculiar tissues, embedded in the connective tissue and localized between the gut and the body wall. the more primitive leeches like g. complanata have a reduced l o n g i t u d i n a l m u s c l e l a y e r , d i s t i n c t b l o o d v e s s e l s l y i n g w i t h i n c o e l o m i c s i n u s e s , a n d n o e v i d e n c e o f peculiar tissues. conversely h. medicinalis shows a very thick longitudinal muscle layer, it has completely lost all traces of blood vessels, and the blood circulates in coelomic sinuses. in addition, between the gut and the body wall muscle fibers, hirudo shows tissues characteristic of jawed leeches, called botryoidal and vaso-fibrous tissues (mann, 1962). the combination of the varied complexity of body organization and the different types of physical, biochemical or immune stimuli leads to modulated selected responses including production of antibacterial peptides, proliferation and migration of immune cells, new synthesis of extracellular matrix, phagocytosis, encapsulation of cumbersome foreign bodies and angiogenesis (characterised by a proliferation and migration of endothelial cells) (de eguileor et al., 1999, 2000a, 2000b, 2001a, 2003; lefebvre et al., 2004, salzet 2001, tettamanti et al, 2003). 115 isj 19: 115-125, 2022 issn 1824-307x research report molecular characterization and expression ananlysis of a qm protein gene from chinese shrimp fenneropenaeus chinensis l zhang1, y wang1,3, j-j hu1,2,3, j-b li1, y-j xu1, q-q zhou1, m-q wang1,2,3 * 1moe key laboratory of marine genetics and breeding (qingdao 266003), and key laboratory of tropical aquatic germplasm of hainan province of sanya oceanographic institute (sanya 572024), ocean university of china, china 2laboratory for marine fisheries science and food production processes, and center for marine molecular biotechnology, qingdao national laboratory for marine science and technology, qingdao 266237, china 3hainan yazhou bay seed laboratory, sanya 572024, china this is an open access article published under the cc by license accepted june 15, 2022 abstract based on lots of results, the qm protein was proposed to be a tumor suppressor, and facilitate in the innate immune responses, especially the prophenoloxidase (propo) activation system. in the present study, a cdna of 761 bp for the qm of chinese shrimp, fenneropenaeus chinensis (designated as fcqm) was cloned via rapid amplification of cdna ends (race) technique. the complete cdna sequence of fcqm contained an open reading frame (orf) of 663 bp, 5' untranslated regions (utr) of 42 bp and 3' utr of 56 bp, which encoded a polypeptide of 155 amino acid residues. the analysis of the peptide sequence of fcqm revealed that fcqm sequence contained a ribosomal_l16 domain, an sh3-binding motif, an n-acylation site, two putative antibiotic binding sites, two putative protein kinase c phosphorylation sites and two acylamidation sites, which shared over 80 % similarity with previously identified qm protein genes. quantitative real-time pcr (qrt-pcr) results showed that the transcript of fcqm was extensively distributed in all the tested tissues and most highly expressed in hemocytes and hepatopancreas. after challenged with vibrio anguillarum or white spot syndrome virus (wssv), the fcqm transcripts were significantly increased (p < 0.05) both in hepatopancreas and hemocytes. hence, the analysis of sequence and transcription suggested that fcqm might play crucial roles responses to bacterial and viral invasion in innate immunity of arthropods. this study is the first report of the qm protein gene from f. chinensis, which would enrich the understanding of the innate immune defense mechanism in shrimp. key words: fenneropenaeus chinensis; innate immunity; qm protein gene introduction chinese shrimp, fenneropenaeus chinensis, is one of the most important mariculture species in china. it is mainly distributed along the coast of yellow sea and bohai sea and the west coast of korean peninsula (meng et al., 2021). in recent years, outbreaks of diseases caused by bacteria, such as vibrio parahaemolyticus, and viruses, such as white spot syndrome virus (wssv) have resulted in mass shrimp mortality, seriously inhibiting the production and economic income of ________________________________________ corresponding author: meng-qiang wang moe key laboratory of marine genetics and breeding (qingdao), and key laboratory of tropical aquatic germplasm of hainan province of sanya oceanographic institute (sanya) ocean university of china e-mail: wangmengqiang@ouc.edu.cn the shrimp industry nationwide (liu et al., 2005; wang et al., 2013). the invasion of pathogens drives the fluctuations of physiological and behavioral status of shrimp, which could be harmful to the survival of shrimps (alfaro et al., 2021; he et al., 2022). due to the lack of adaptive immune system, shrimps only rely on innate immune system to protect the organism from pathogens (destoumieux-garzon et al., 2001). therefore, it is particularly important to better understand the innate immune defenses mechanism to promote the development of disease treatment, and then to find more effective ways to solve the problems. the qm protein gene was originally identified from human in the non-tumorigenic wlims’ cell line g401 using subtractive hybridization method (dowdy et al., 1991). initially, it was found that the qm protein gene was expressed at a higher level in non-tumorigenic wlims’ cells than in the 116 tumorigenic parental cells, speculating that the qm protein gene might be a tumor suppressor gene (chen et al., 2012). in addition, the qm protein gene encodes the ribosomal protein l10, which is required for the formation of the 80s ribosome (loftus et al., 1997). because of its presumed functions, the qm protein gene has been the subject of intensive study. homologues of qm have been identified in various species, include vertebrates and invertebrates and have been shown to play important immune roles in cell growth, tissue differentiation and apoptosis (green et al., 2000). the mouse qm protein gene is differentially expressed throughout the embryo, and the expression pattern of qm suggests that qm might be involved in the posttranslational protein processing, playing an important role in the differentiation of specific tissues during embryogenesis (mills et al., 1999). the homolog of qm in chicken (jif-1), a negative transcription regulator of c-jun, could bind to the protooncogene c-jun and suppressed ap-1 function, suggesting that qm protein gene may be involved in transcriptional regulation (imafuku et al., 1999). and in bombyx mori, qm protein gene interacted with jun protein and negatively regulated the expression of ap-1, which could affect some immune genes expression (zhou et al., 2019). accumulating evidence has demonstrated that qm protein are involved in regulating the immune response of organisms. in grass carp, qm protein was significantly up-regulated by aeromonas hydrophila and grass carp hemorrhagic virus (gchv) infection, suggesting that qm protein gene is associated with anti-pathogens inflammatory response (wen et al., 2005). following bacterial stimulation, qm protein gene transcription was significantly increased in the hepatopancreas of clams, demonstrating that qm transcriptional expression can be induced by pathogens challenge and play an active role in immune responses (cui et al., 2019). as one of the major invertebrate immune responses, prophenoloxidase activation system (propo-as) plays an important role in innate immunity (soderhall et al., 1994). through pathogens-associated molecular patterns (pamps) such as lipopolysaccharide (lps), peptidoglycan (pg) or β-1,3-glucan (bg), bind to specific shrimp humoral pattern recognition proteins (prps) and activate the propo system (sritunyalucksana et al., 1999; sritunyalucksana et al., 2000; cerenius et al., 2004). the qm protein in large yellow croaker could regulate the activity of phenol oxidase (po) (han et al., 2015). the qm protein in penaeus japonicus can interact with hemocyanin and myosin, thereby regulating the activity of po in the innate immunogen activation system of arthropods (xu et al., 2008). in shrimps, qm protein can significantly improve bacterial clearance and positively regulate po activity (liu et al., 2014). more interestingly, researchers reported for the first time that the qm protein was present in crustaceans as a potential peptidoglycan recognition protein (pgrp) in penaeus monodon and activated the propo response (udompetcharaporn et al., 2014). therefore, qm protein may play an important role in the innate immune response of shrimp. in this study, we cloned the full-length cdna of the fcqm gene and analyzed the sequence features and molecular characteristics of the fcqm protein. subsequently, the prawns were challenged with v. anguillarum and white spot syndrome virus (wssv) to explore the tissue-specific expression of the fcqm gene. finally, we analyze the immune response of qm protein gene to pathogens challenges. table 1 oligonucleotide primers used in the present study primer sequence (5’-3’) tm (°c) brief information adaptor primer ggccacgcgtcgactagtac 60 anchor primer for 3’ and 5’ race adaptor primer-oligo (dt) ggccacgcgtcgactagtact17vn oligo (dt) for cdna synthetize adaptor primer-oligo (dg) ggccacgcgtcgactagtacg10hn oligo (dt) for cdna synthetize fc-18s-qrt-f tatacgctagtggagctggaa 58 internal control for real-time pcr fc-18s-qrt-r ggggaggtagtgacgaaaaat 57 internal control for real-time pcr fcqm-cds-f atggggcgccgtccggcccgatgc 77 gene specific primer for cds fcqm-cds-r ttaagcaagaccagcaagttccaactg 64 gene specific primer for cds fcqm-qrt-f ggcttcaccaagttcgacc 58 gene specific primer for real-time pcr fcqm-qrt-r ttaagcaagaccagcaagttcc 59 gene specific primer for real-time pcr fcqm-race-f1 tcattgaggctctccgacgtgcca 68 gene specific primer for 3’ race fcqm-race-f2 tggtcgccttaggcctgatggtgt 67 gene specific primer for 3’ race fcqm-race-r1 cctaagtcatagatacgaatcttg 54 gene specific primer for 5’ race fcqm-race-r2 ttacagtaacggtagcatcgggccgga 69 gene specific primer for 5’ race m13-47 cgccagggttttcccagtcacgac 56 vector primer for sequencing rv-m gagcggataacaatttcacacagg 56 vector primer for sequencing 117 materials and methods experimental shrimps, immune challenge and sample collection in the present study, the shrimps weighing 8 12 g were obtained from a local shrimp farm in qingdao, china, and cultured in the laboratory for two weeks before processing. in this phase, shrimps were cultured in 640 l cylindrical tanks with 500 l air-pumped circulating seawater at 20 ± 1 °c. to study the tissues-specific expression of fcqm, the hemolymph and tissues, including eyestalk, gill, gonad, heart, hepatopancreas, intestine (mid gut), muscle, nerve and stomach were collected from at least fifteen untreated shrimps. to determine the immune response of fcqm, approximately 360 shrimps were employed for microbe stimulation assay. to determine the immune response of fcqm, approximately 360 shrimps were employed for microbe stimulation assay. the shrimps were randomly divided into three groups. the shrimps of control groups were received challenges at the abdominal segment with 100 μl phosphate buffered saline (pbs, ph 7.4, 10010023, thermo fisher scientific, usa). the shrimps of two treatment groups v. anguillarum suspension (1 × 104 cfus μl-1, in pbs) and wssv stock (1 × 104 copies μl-1, in pbs), respectively. the v. anguillarum suspension and wssv stock were prepared according to previous reports (yi et al., 2014; xia et al., 2015; sha et al., 2016). hepatopancreas and hemolymph samples were removed from shrimps of the bacteria-injected, wssv-injected and the control groups at 0, 3, 6, 12, 24, 36 and 48 hpi. the shrimp hemolymph was collected from the ventral sinus into a sterilized syringe with an equal volume of modified anticoagulant alsever solution (trisodium citrate 30 mm, nacl 510 mm, citric acid 200 mm, glucose 115 mm, and edta 10 mm, ph 7.4), and centrifuged at 800 × g for 10 min at 4 °c. all of the tissue samples were kept in rnalater (am7020, thermo fisher scientific, usa), which snap-frozen in liquid nitrogen and used for rna isolation. total rna isolation and cdna synthesis the total rna was extracted from hemolymph and tissues, including eyestalk, gill, gonad, heart, hepatopancreas, intestine (mid gut), muscle, nerve and stomach, using the trizol reagent (takara, dalian, china) following the manufacturer’s protocol. the rna concentration was determined by measured uv absorbance at 260 nm with a nanodrop lite. its completeness was examined by electrophoresis on a 1.5 % agarose gel. according to the manufacturer’s instructions, the synthesis of first-strand cdna was carried out with promega m-mlv (m1701, promega, usa). briefly, a mixture containing 1 µg of total rna, 2 µl of adaptor primer-oligo (dt) or random primers and 5 µl of rnase free ddh2o was incubated at 70 °c for 5 min. then, 4 µl of 5 × m-mlv buffer, 2 µl of dtt (100mm), 1 µl of dntp (10mm), 1 µl of ribonuclease inhibitor, 1 µl of m-mlv and rnase free ddh2o were added (total 20 µl). the mixture was incubated at 42 °c for 1 h and terminated at 95 °c for 5 min before cooling on ice. then, the synthesized cdna was stored at 80 °c for further use. molecular cloning of fcqm cdna sequence the partial length sequence of fcqm cdna was obtained from the shrimp transcriptome sequencing database (genbank accession number: bm302396) (zhang et al., 2010). two pairs of sequence-specific primers, fcqm-race-f1/f2 and fcqm-race-r1/r2 (table 1), were designed using primer premier 5.0. the sequence-specific primers and adaptor primers were used to clone the full-length cdna of fcqm by 5’ and 3’ race techniques. specific primers fcqm-race-f1, fcqm-race-f2 and adaptor primer-oligo(dt) were employed to obtain 3’ ends sequence of fcqm using a semi-nested pcr. then, specific primers fcqm-race-r1, fcqm-race-r2 and adaptor primer-oligo(dg) were employed to obtain 5’ ends sequence of fcqm using a semi-nested pcr. and the coding sequence (cds) of fcqm was amplified and confirmed using another two gene-specific primers, fcqm-cds-f/r (table 1). amplification was performed in 25 µl reaction volume, containing 2.5 µl of 10 × pcr buffer, 1.5 µl of mgcl2 (25 mm), 2.0 µl of dntp (2.5 mm), 1.0 µl of each primer (10 mm), 1.0 µl of template, 0.2 µl of ex taq (takara, japan) and 15.8 µl of rnase free ddh2o. the protocol of pcr was one initial denaturation cycle 94 °c for 5 min, followed by 35 cycles of 94 °c for 10 s, 55 °c for 30 s and 72 °c for 30 s, 5 min at 72 °c for the final extension. all pcr amplification were performed in a veritipro thermal cycler (thermofisher, usa). the pcr products were gel-purified and then cloned into the pmd-18t simple (takara, japan). after being transformed into the competent cells escherichia coli strain dh5α (tiangen, china). the positive recombinants were identified through anti-ampicillin selection and verified by pcr screening using vector primers, m13-47 and rv-m (table 1). the positive recombinants ware carried out using an abi 3730 sequencer (thermofisher, usa). bioinformatics analysis of fcqm cdna and protein sequences the fcqm nucleotide sequence was translated into the amino acid sequence using the danman 8.08 software package. the fcqm amino acid sequence was used for protein functional domains prediction and analysis using the simple modular architecture research tool (smart, smart.embl-heidelberg.de). and the calculated molecular mass and theoretical isoelectric point (pi) were predicted using the expasy molecular biology server (web.expasy.org/compute_pi). similar qm sequences of other species were searched in ncbi gene database using the blast program. and multiple alignments were performed using clustalw2. finally, an unrooted phylogenetic tree was constructed using amino acid neighbor-joining (nj) method with mega 10.0 software and tested for reliability with 1000 bootstrap replications. 118 fig. 1 nucleotide and deduced amino acid sequences of fcqm (genbank accession ku361824). the start codon (atg), the stop codon (taa), and the polyadenylation signal sequence (attaaa) are in bold and underlined. the predicted sh3-binding motif (rparcy) are in bold italics, two acylamidation sites (mgrr) and (lgrk) are underlined with dotted lines, two putative antibiotic binding sites (gri) and (nk) are shaded with a line, an n-acylation site (gmrgaf) are double-underlined and two protein kinase c phosphorylation sites (svr) and (srk) are boxed. in addition, a ribosomal protein l10 signature is in shaded quantitative real-time pcr analysis of fcqm mrna expression the transcripts of fcqm in various tissues from healthy f. chinensis and its temporal expression in hemocytes and hepatopancreas after v. anguillarum or wssv challenges were detected by qrt-pcr. two fcqm gene-specific primers, fcqm-qrt-f and fcqm-qrt-r (table 1), were designed using primer premier 5.0 to detect the temporal expression of the fcqm gene. 18s rdna was chosen as reference gene for internal standardization. two primers, fc-18s-qrt-f and fc-18s-qrt-r (table 1) were used to amplify the fragments of 18s rdna. qrt-pcr was carried out in a 20 µl reaction volume containing 10 µl of 2 × chamq sybr qpcr master mix (q311-02, vazyme, china), 0.4 µl of each primer (10 mm), 2 µl of the 1:10 diluted cdna and 7.2 µl of rnase free ddh2o. the protocol of qrt-pcr was one initial denaturation cycle 95 °c for 30 s, followed by 39 cycles of 95 °c for 10 s and 60 °c for 30 s. after each run, a dissociation step (95 °c for 15 s, 60 °c for 60 s, 95 °c for 15 s) was performed to generate a melting curve thermal profile to verify a single product. all qrt-pcr amplification were performed in an abi 7300 (thermofisher, usa). the mrna relative expression level of fcqm and 18s rdna were analyzed by comparative ct method (2-δδct method) (schmittgen et al., 2008; wang et al., 2011). all data were given in terms of the mrna relative expression of fcqm as the mean ± standard deviation and subjected to one-way anova followed by duncan's multiple range test by using ibm spss statistics 23.0.0.0. the p values less than 0.05 were considered statistically significant. results cloning and characterization of fcqm cdna to further characterize the functions of qm protein gene in shrimp immune response against pathogens infections, qm protein gene was cloned from f. chinensis. the full-length cdna (genbank accession number: ku361824) of fcqm was obtained by 5' and 3’ race techniques. as is shown in fig. 1, it comprised 761bp, containing an open reading frame (orf) of 663 bp, 5' 119 untranslated regions (utr) of 42 bp and 3' utr of 56 bp. the orf encoded a 155 amino acid polypeptide with a calculated molecular mass of approximately 25.53 kda and a theoretical isoelectric point (pi) of 10.08. smart indicated that fcqm contains a ribosomal_l16 domain (from c12 to f166), an sh3-binding motif (rpafcy), an n-acylation site (gmrgaf), two putative antibiotic binding sites (gri; nk), two putative protein kinase c phosphorylation sites (svr; srk), two acylamidation sites (mgrr; lgrk). in addition, it contains a ribosomal l10 signature motif. a polyadenylation signal (aataaa) was found upstream of polya. homology and phylogenetic analysis of fcqm homology and similarity analysis of the deduced amino acid of fcqm was performed by genedoc and dnaman software to determine the evolutionary conservation of characteristic motifs and active sites of qm proteins. the amino acid sequence homology of the qm protein of f. chinensis and p. vannamei was the highest, and the amino acid sequence homology of the qm protein of homo sapiens was the lowest. in addition, the amino acid sequence of fcqm was similar to that of homo sapiens (75 %), penaeus japonicus (92 %), eriocheir sinensis (92 %), danio rerio (77 %), pinctada fucata (78 %), drosophila melanogaster (79 %), bombix mori (80 %), and pediculus humanus corporis (81 %) had higher homology in fig. 2. the alignment results show that the n-terminal and internal region of the peptides are conserved. in addition, the study also found that sh3-binding motif (rparcy), ribosomal protein l10 signature (adrlqtgmrgawgkqgtvarv) and protein kinase c phosphorylation site (rparcy) were highly conserved during the evolution of qm protein. in order to study the molecular evolution of fcqm genes, a neighbor-joining (nj) phylogenetic tree based on the amino acid sequences of 16 different species was constructed to analyze the phylogenetic relationship of fcqm. as is shown in fig. 3, it was clearly to find that the qm group can be divided into mammalians (o. aries, b. taurus, h. sapiens, m. musculus), amphibians (x. tropicalis), fish (d. rerio, l.crocea, o. niloticus) , mollusks (h. diversicolor supertexta, h. discus discus, p. fucata, a. farreri, m. meretrix), arthropods (p. vannamei, f. chinensis, p. japonicus, p. clarkii, d. melanogaster, p. monodon, b. mandarina) four groups. further analysis showed that fcqm is the closest relative to lvqm and is located in the crustacean subclade. fig. 2 multiple alignments of fcqm with previous known qm. the sequences and their accession numbers are as follows: homo sapiens, aab27665.1; fenneropenaeus chinensis, ku361824; penaeus vannamei, aga16579.1; penaeus japonicus, abs45569.1; eriocheir sinensis, ato74510.1; danio rerio, aav34163.1; pinctada fucata, aan85578.1; drosophila melanogaster, aac16108.1; bombyx mori, aak73358.1 120 fig. 3 consensus neighbor-joining phylogenetic based on the protein sequences of fcqm from different organisms. the evolutionary history was inferred using the neighbor-joining method. the bootstrap consensus tree inferred from 1,000 replicates was taken to represent the evolutionary history of the taxa analyzed. all positions containing gaps and missing data were eliminated. the numbers at the forks indicated the bootstrap value. the sequences and their accession numbers are as follows: penaeus vannamei, aga16579.1; fenneropenaeus chinensis, ku361824; penaeus japonicus, abs45569.1; procambarus clarkii, aeb54638.1; drosophila melanogaster, aac16108.1; haliotis diversicolor supertexta, acj71721.1; haliotis discus discus, abo26700.1; pinctada fucata, aan85578.1; azumapecten farreri, akm12718.1; danio rerio, aav34163.1; larimichthys crocea, acs93602.1; xenopus tropicalis, np_001004965.1; ovis aries, abv64839.1; bos taurus, np_777185.1; homo sapiens, aab27665.1; mus musculus, caa53061.1; meretrix meretrix, ang56314.1; oreochromis niloticus, xp_003454286.1; bombyx mandarina, aac98301.1; penaeus monodon, acv72062.1 tissue-specific expression of fcqm the relative expression level of fcqm in various tissues including eyestalk, gill, gonad, heart, hemocytes, hepatopancreas, intestine, muscle, nerve and stomach was detected by qrt-pcr with 18s rdna as the internal control in fifteen untreated shrimps. as shown in fig. 4, fcqm genes were all expressed in the tested tissues, with the highest expression in hepatopancreas, higher expression in hemocytes, and less expressed gills, eye stalk, gonad, heart, intestine, muscle, nerve, stomach expression. the mrna expression profiles of fcqm post bacterial stimulation in order to understand the changes in the transcriptional response of fcqm gene after stimulation by v. anguillarum, qrt-pcr was used to detect the mrna relative expressions of fcqm in hemocytes and hepatopancreas at various time points (0, 3, 6, 12, 24, 36, 48 hpi). as shown in fig. 5, the relative expression level of fcqm in both tissues was up-regulated under the v. anguillarum stimulation, while the expression level of fcqm in the control group had no significant change. fig. 5a 121 fig. 4 tissue distribution of fcqm mrna transcripts detected by qrt-pcr technique. the 18s rdna gene was used as an internal control to calibrate the cdna template for each sample. vertical bars represent mean ± sd (n = 3), and bars with different characters were significantly different (p < 0.05), while bars with same characters were not significantly different shows that the relative expression of fcqm gene in hemocytes after v. anguillarum stimulation was significantly increased at 3 hpi and 6 hpi (p < 0.05) with the peak value of 6.41-fold higher than that in the control group at 3 hpi. the expression level of fcqm gene in the treatment group were not significantly different from that in the control group from 12 hpi to 48 hpi. fig. 5 b shows that the relative expression of fcqm gene in the hepatopancreas after immune stimulation. the expression level of fcqm were significantly increased at 12 hpi and 24 hpi after v. anguillarum challenges (p < 0.05) and reached a peak value at 24 hpi with the peak value of 7.87-fold higher than that in the control group, and recovered to 36 hpi at 36 hpi. normal level. the expression level of fcqm gene did not exhibit any significant changes in control group. the mrna expression profiles of and fcqm post wssv stimulation under wssv stimulation, the expression of fcqm in hemocytes and hepatopancreas was up-regulated. as shown in fig. 6 a, the relative expression of fcqm in hemocytes after wssv challenges was significantly up-regulated at 3 hpi and 6 hpi (p < 0.05) with the peak value of 6.58-fold higher than that in the control group at 6 hpi. after 6 hpi, the expression level of fcqm was the same as that of the control group. there was no significant difference, and gradually returned to normal level. fig. 6 b shows that under wssv stimulation, fcqm also showed a higher expression level. the highest expression level (2.9-fold) of fcqm transcripts was detected at 12 hpi after the challenge with wssv (p < 0.05). in addition, the expression level of fcqm gene was fluctuated at 48 hpi, representing 2.49-fold higher level than that in the control group. discussion since the first qm protein gene was identified from humans, the qm protein gene has been identified in a large range of organisms. recent research achievements indicated qm protein may play an important role in the innate immune defenses mechanism of shrimp (wen et al., 2005). in the present study, we first reported the identification and molecular characterization of 122 fig. 5 temporal mrna expression profiles of fcqm detected via qrt-pcr technique in shrimp hemocytes and hepatopancreas at 0, 3, 6, 12, 24, 36 and 48 hpi stimulation. a: temporal mrna expression profiles of fcqm post v. anguillarum stimulation in shrimp hemocytes; b: temporal mrna expression profiles of fcqm post v. anguillarum stimulation in shrimp hepatopancreas; the 18s rdna gene was used as an internal control to calibrate the cdna template for each sample. vertical bars represent mean ± sd (n = 3), and bars with different characters were significantly different (p < 0.05), while bars with same characters were not significantly different fcqm cdna sequence from the chinese shrimp, f. chinensis. the full-length cdna of fcqm was 761bp, including a 663 bp orf encoding a 155 amino acids polypeptide with a calculated molecular mass of approximately 25.53 kda and a pi of 10.08. the protein sequence of fcqm shared over 80 % similarities with other identified qms. the deduced amino acid sequence of fcqm was conformity to the conserved domains of those identified qms of marine invertebrates, which included an sh3-binding motif, an n-acylation site, antibiotic binding sites, protein kinase c phosphorylation sites, acylamidation sites and a ribosomal l10 signature motif, indicating that fcqm was an orthologue of qm family (liu et al., 2014; han et al., 2015; cui et al., 2019). the highly conserved signature sequences might suggest the important role of qm in the basic functions. phylogenetic analysis showed that no signal peptide identified from the deduced amino acid sequence of fcqm, pointed out that fcqm may not be a secretory protein. a study confirmed that qm localized to the cytoplasmic face of the rough er on mammalian cells and yeast (loftus et al., 1997). in the nj phylogenetic tree, fcqm is the closest relative to lvqm and is located in the crustacean subclade, which could have similar functions to those from other invertebrates. the sequence characteristics and the phylogenetic relationship of fcqm indicate that the fcqm gene could have similar functions to those from other invertebrates. previous research showed that qm was ubiquitously expressed in various tissues. interestingly, it was also reported that the qm protein gene participated in the innate immune response. in order to better understand the potential biological role of qm in shrimp, the tissue distribution of fcqm mrna transcripts was detected by qrt-pcr technique. the fcqm mrna transcripts were observed to be ubiquitously expressed in all the examined tissues, including eyestalk, gill, gonad, heart, hemocytes, hepatopancreas, intestine, muscle, nerve and stomach, which were consisted with the previously observation in other invertebrates (xu et al., 2008; li et al., 2016; cui et al., 2019). the ubiquity of fcqm mrna transcripts indicated that it could be involved in many physiological responses of shrimps. the highest expression level of fcqm mrna was found in hepatopancreas and hemocytes, followed by gill. similarly, in m. meretrix, mmqm was highest expressed in hepatopancreas and hemocytes, followed by gill (cui et al., 2019). the hepatopancreas was considered as the main immunity and detoxification organ (song et al., 2015). hemocytes as the major immune cells respond to pathogens mainly through phagocytosis (jiravanichpaisal et al., 2006). gill is part of the mucosal system, functioning as the first defense line against pathogens in lower animals (wang et al., 2019). therefore, the high expression level of fcqm in these tissues further confirmed that fcqm could participate in the innate immune system of shrimp. accumulating evidence has shown that qm protein genes have important immune functions in organisms. previous research have shown that lvqm mrna transcripts in hemocytes and hepatopancreas increased significantly in the early stage of the experiment after v. anguillarum stimulation, and rnai and bacterial clearance experiments showed that qm protein regulated the activity of po (liu et al., 2014). to further investigate the role of fcqm in immune responses, the present research detected the temporal expression profiles of qm in response to a bacterial 123 fig. 6 temporal mrna expression profiles of fcqm detected via qrt-pcr technique in shrimp hemocytes and hepatopancreas at 0, 3, 6, 12, 24, 36 and 48 hpi stimulation. a: temporal mrna expression profiles of fcqm post wssv stimulation in shrimp hemocytes; b: temporal mrna expression profiles of fcqm post wssv stimulation in shrimp hepatopancreas; the 18s rdna gene was used as an internal control to calibrate the cdna template for each sample. vertical bars represent mean ± sd (n = 3), and bars with different characters were significantly different (p < 0.05), while bars with same characters were not significantly different and wssv challenge. the expression level of fcqm gene after v. anguillarum and wssv stimulation was significantly increased at 3 hpi and 6 hpi in hemocytes and increased at 12 hpi and 24 hpi in hepatopancreas, which suggest the important immune regulation effect of fcqm in f. chinensis. the expression of fcqm in hemocytes is earlier than that in hepatopancreas. gill is the first defense line against pathogens in shrimps. however, once this barrier is passed, a series of complex innate humoral and cellular immune reactions is induced in both haemocoel and tissues, resulting in a fast elimination of pathogens (cerenius et al., 2010). this also reasonably explains that the differential expression level of fcqm in hemocytes is earlier than that in hepatopancreas in this study. in hemocytes, the induction expression level of fcqm mrna transcripts reached a peak value at 3 hpi with 6.41-fold after v. anguillarum stimulation and at 6 hpi with 6.58-fold after wssv stimulation. in hepatopancreas, the induction expression level of fcqm mrna transcripts reached a peak value at 24 hpi with 6.41-fold and 6.58-fold after v. anguillarum or wssv stimulation. researchers have reported that the results of qrt-pcr and western blot indicated that the pjqm gene was significantly upregulated in wssv-resistant penaeus japonicus (xu et al., 2008). in the chlamys farreri, the relative expression of cfqm sharply increased at 6 hpi and normalized at 48 hpi under v. anguillarum and acute viral necrobiotic virus stimulation, which suggested that cfqm can protect organisms against pathogens challenges (chen et al., 2015). bacterial challenge tests indicated that hdiqm gene expression was induced by the bacterial isolates from haliotis diversicolor, which implied that hdiqm might be an inflammatory stress-inducible gene associated with pathogen infection (li et al., 2016). the above results further verify that qm may play an essential role in the response to pathogens. therefore, qm protein gene have important immune functions in shrimps and other invertebrates, especially in process of innate immune defense. specially, the upregulation of the expression level of fcqm at 48 h after wssv stimulation, which is different from the other results, suggests that fcqm might be involved in multiple innate immune response processes in shrimp. in summary, we have successfully cloned the cdna sequence of fcqm from the chinese shrimp, f. chinensis. tissue expression analysis indicated that fcqm was detected in all examined tissues, and higher expressed in hemocytes, hepatopancreas and gills. the fcqm was significantly increased both in hemocytes and hepatopancreas after v. anguillarum and wssv stimulation. these results of sequence and transcription analysis suggested that fcqm may play crucial roles in innate immune responses to bacterial and viral stimulation. acknowledgments this research was supported by the project of sanya yazhou bay science and technology city management foundation (skjc-kj-2019ky01), and the startup fund of young talents project of ocean university of china. we would like to thank the expert reviewers for 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insect mol. biol. 28: 578-590, 2019. 1 isj 17: 1-8, 2020 issn 1824-307x research report evaluating the implications of moisture deprivation on certain biochemical parameters of the earthworm eudrilus eugeniae with microbial population and exoenzyme activities of the organic substrate csk mishra, s samal*, a rout, a pattanayak, p acharya department of zoology, odisha university of agriculture and technology, college of basic science and humanities, bhubaneswar-751003, india this is an open access article published under the cc by license accepted january 8, 2020 abstract reduction in moisture in the top soil and decomposing organic substrate is likely to influence the epigeic earthworms along with the microbial population and exoenzyme secretions. this study reports the results of the effects of consistent moisture reduction in semidecomposed organic substrate on the tissue protein, lipid peroxidation and catalase activity of the earthworm eudrilus eugeniae along with ph, organic carbon reduction, bacterial-fungal population, activities of exoenzymes, amylase, cellulase, invertase over an experimental period of 22 days. consistent depletion in tissue protein, increase in lipid peroxidation level and catalase activity was observed in the earthworm with moisture depletion. catalase activity indicated significant negative correlation with substrate moisture. significant differences in the carbon reduction, microbial population, exoenzyme activities in the substrate was observed with reduction of moisture and with respect to control. significant positive correlation was observed between percent substrate moisture with microbial population and activities cellulase. it was concluded that desiccation of decomposing organics could enhance physiological stress on the earthworm and adversely impact the microbial population, exoenzyme secretions, consequently impairing mineralization. key words: earthworm; exoenzyme; microbes; moisture deprivation; organic substrate introduction abiotic factors such as moisture and temperature considerably influence the life of organisms (acevedo-whitehouse and duffus, 2009; singh et al., 2014). soil organisms too are likely to be influenced by variations in temperature and moisture. rising global atmospheric temperature might result in deprivation of moisture from the surface soil thus adversely impacting major groups of biota. moisture is expected to considerably influence physiological state of organisms due to its role as a suitable solvent for uptake of nutrients by microbes and plants and cutaneous respiration and excretion in certain soil fauna (wall et al., 2008; blankinship et al., 2011; eisenhauer et al., 2012). water availability is a major determinant of the biotic community composition and functioning in soil. climate change induced alterations of soil water ___________________________________________________________________________ corresponding author: suryasikha samal department of zoology odisha university of agriculture and technology college of basic science and humanities bhubaneswar-751003, india e-mail: suryasikha.777@gmail.com content could occur as a consequence of interacting changes to precipitation and temperature (knapp et al., 2008; bell et al., 2010). below ground ecosystems are expected to be affected by soil moisture which could influence nutrient availability and decomposition rates (trofymow et al., 2002). earthworms, the major group of soil sub system have the tendency to live in moist soils (berry and jordan, 2001). earthworms in natural habitats may be subjected to periods of drought and in agricultural lands are frequently turned out of the soil and exposed to surface conditions. in the vermicomposting process the moisture level might be reduced drastically during summer. with consistent substrate desiccation, the worms are likely to suffer from oxidative stress. likewise, bacteria and fungi which play vital role in organic matter decomposition might be influenced considerably due to moisture variations. microbes in soil produce various enzymes which are constantly being synthesized, accumulated, inactivated and decomposed in soil and their activities are useful for assessing the functional diversity of the microbial communities or organic mass turnover (zhang et al., 2 2009). fluctuating physical parameters such as soil moisture could adversely impact microorganisms and their activities in soil and decomposing organic matter. information is rare on the impact of depleting moisture in the substrate on microbes and epigeic earthworms which facilitate decomposition. the epigeic earthworm eudrilus eugeniae, native to africa is globally used as an efficient vermicomposting agent for processing the solid organic wastes. therefore, the present study was intended to evaluate the effects of variable moisture levels in organic substrate on the tissue protein, lipid peroxidation and catalase activity of the earthworm with the microbial population and exoenzyme activities. materials and methods experimental set up for both microbial and earthworm studies, two different experimental setups were prepared. for control (c) and experimental (e) setups, rectangular plastic pots (30 cm x 30 cm) were taken in triplicates. each pot was filled with 4 kg air dried and powdered semi-decomposed cattle dung as substrate material. the water holding capacity of the substrate was measured gravimetrically. the maximum water holding capacity of the organic substrate was calculated to be 30 % which was taken as control. the substrate moisture level in both c and e were made up to 30 % level by adding distilled water. this moisture level was maintained in c throughout the experimental period of 22 days. in the experimental set, no water was added further, thus allowing moisture loss from the substrate over time. the percent moisture of the substrate in c and e sets were measured with a portable moisture meter (omegahsm50). the experiment was conducted at room temperature (maximum 28 ± 2.3 °c and minimum 21 ± 3.2 °c) and there was no significant variation in average temperature during the experimental period. the earthworms (eudrilus eugeniae) were obtained from the vermiculture unit of quality control laboratory of the government of odisha and were acclimatized in laboratory condition for 7 days. after acclimatization, 30 adult worms of approximately equal weight were inoculated in to each of the control and experimental pots. both substrate (3 samples from each replicate) and earthworm (3 samples from each pot) were collected at random on day 1 and subsequently at 2 day interval for analysis. biochemical analysis of earthworm tissue the gut contents of the worms were cleaned before biochemical analysis by keeping the worms submerged in distilled water kept in glass beaker for 15 min. the animals were rinsed well in clean distilled water and then sacrificed for obtaining the body wall tissue. the tissue samples were homogenized and centrifuged with potassium phosphate buffer (0.05 m, ph-7.4) at 4 °c and 10,000 rpm for 10 min using a table top refrigerated centrifuge (remi, india). the supernatant was collected and maintained in a chilled condition for further analysis. protein was estimated using folin and ciocalteu’s phenol method (lowry et al., 1951). the amount of protein was determined at 700 nm with uv-vis spectrophotometer (systronics, india) taking bovine serum albumin (bsa) as standard. lipid peroxidation (lpx) was measured in terms of malondialdehyde (mda) at 532 nm as per ohkawa et al. (1979). catalase assay was done as per cohen et al. (1970) taking both phosphate buffer and hydrogen peroxide. the absorbance was measured at 242 nm. evaluation of substrate chemical parameters, microbial load and enzyme activities the substrate ph was measured using digital ph meter. organic carbon (oc) was determined titrometrically as per walkley and black method (1934). bacterial and fungal populations were estimated by serial dilution and spread plate method taking nutrient agar and potato dextrose agar as nutrient media respectively (parkinson et al., 1971). three carbohydrate exoenzymes amylase, cellullase and invertase were assayed by spectrophotometric method (ross and robert, 1970) taking starch, carboxymethyl cellulose and sucrose as substrates respectively. the data were checked for normality prior to further analysis and were found to be normally distributed. one way anova was applied along with post hoc using spss software (ibm® spss® statistics, ver. 25) to determine the significance levels at α = 0.05, 0.01, 0.001. the significance in variation was checked between both the control and experimental sets. correlation analysis was done between percent substrate moisture and biological parameters. results biochemical studies consistent reduction in tissue protein level was observed in the earthworm in the experimental set with respect to control over the incubation period. the highest percent change in protein content was 70.72 % on day 13. the level of protein got reduced by 47.4 % on day 22 with respect to day 1 (fig 1a). a significant variation (f = 4.62, p = 0.001) in the percent tissue protein was observed between control and experimental sets. lpx level in experimental worms increased from day 1 to day 22. the highest percent change in lpx level (194.21 %) was noticed on day 22 with respect to control (fig 1b). a significant variation (f = 4.33, p = 0.021) was observed in the lpx level of the animal between control and experimental sets. the percent change in catalase activity in the experimental worms showed an increasing trend up to day 16 with the maximum increase on this day (604.7 %). on day 22, the activity increased up to 337.05 % with respect to control (fig1 c). the variation in percent change in catalase activity between control and experimental sets was found to be significant (f = 8.1, p = 0.001). correlation analysis indicated significant negative correlation between percent substrate moisture and catalase activity (r = -0.66, p < 0.05). 3 fig. 1 mean ± sd indicates percent changes in tissue protein, lpx level and catalase activity in eudrilus eugeniae with respect to control and substrate moisture. “*” indicates significant variation at p < 0.05 and “**” indicates at p < 0.01 between control and experimental sets, n= 9, c-control, e-experimental soil physico-chemical and microbiological study the percent moisture and values of the substrate chemical parameters at different days of measurement have been presented in table 1. the percent moisture in the experimental set decreased from the 30 ± 1.1 % on day 1 to 9.93 ± 0.9 % on day 22 indicating a reduction of 66.9 % over the experimental period. the variation in percent moisture over the days of incubation was found to be significant (f = 17.9, p = 0.0008). the mean substrate ph on day 1 was found to be 6.73 ± 0.12 in control and 7.05 ± 0.01 in experimental set. a minor variation in the ph value was observed over the experimental period. the substrate ph was 7.20 ± 0.01 in control and 7.41 ± 0.02 in experimental set on the day 22. a) b) c) 4 table 1 mean ± sd of substrate moisture (%), ph, oc (%) at different days of incubation (n=9). “*” indicates significant variation at p < 0.05, “**” at p < 0.01 and “***”at p < 0.001 between control and experimental sets a significant variation (f = 4.8, p = 0.041) in the oc (%) was observed over the days of the experiment. the maximum reduction in the oc was 9.7 % on day 10. it increased after day 16 and reached 14.1 % on day 22. in the control set the % oc reduction in the substrate was 28 % where as in the experimental it was 18.84 %. the noticeably lower % oc reduction in experimental set is likely due to significantly lower substrate moisture and microbial activity. the mean bacterial population on day 1 was found to be 34.67 x 105 cfu/g in control and 48.33 x 105 cfu/g in experimental samples. a variation in the bacterial population was observed over the days of incubation. the bacterial population decreased to 19.33 x 105 cfu/g and 11 x 105 cfu/g soil in control and experimental sample respectively on the day 22. the bacterial population increased by 65.3 % up to day 10. the maximum reduction (43 %) of the bacterial population in experimental set with respect to control was observed on day 22 (fig 2a). the fungal populations on day 1 was found to be 92 x 105 cfu/g and 99 x 105 cfu/g soil in control and experimental sets respectively which declined to 10 x 105 cfu/g soil in control and 8 x 105 cfu/g soil in experimental sets on day 22. the maximum increment in fungal count (63.77 %) was recorded on day 10 in the experimental set with respect to control. the fungal population indicated a reduction of 18.7 % in the experimental set on the day 22 (fig 2a). significant difference (f = 6.8, p = 0.02) in the bacterial population of control and experimental sets was observed over the days of incubation. however, the variation in fungal population was not significant. percent substrate moisture indicated significant positive correlation with both bacterial (r=0.9, p < 0.01) and fungal (r = 0.8, p < 0.01) populations. the maximum increase in amylase activity (96.5 %) in experimental sample with respect to control was noticed on day 10. the enzyme activity increased up to day 13 and subsequently decreased. the maximum reduction in activity (31.3 %) was observed on day 19 with 13 % moisture level (fig 2b). cellulase activity showed an initial increment with the maximum enzyme activity (28 %) on day 10 after which the activity declined. maximum reduction in enzyme activity (47.3 %) was observed on day 19 (fig 2b). this enzyme activity indicated significant variation between control and experimental sets (f = 5.66, p = 0.032). invertase activity too showed an identical trend. the enzyme activity increased by 38.4 % on day 10 with respect to control and then showed a reduction. minimum activity of this enzyme (27.5 %) was recorded on day 22 (fig 2b). significant positive correlation was observed between substrate moisture with cellulase (r = 0.6, p < 0.05) and invertase (r = 0.61, p < 0.05) activity. moisture (%) day 1 day 4 day 7 day 10 day 13 day 16 day 19 day 22 c 30.08 ± 1.3 30.01 ± 0.7 30 .07± 0.5 30 .02± 1.2 30.02 ± 0.7 30.03 ± 1.3 30 .02 ± 1.6 30.01 ± 0.9 e 30 .01 ± 1.1 ***26.7 ± 0.4 ***23.86 ± 1.3 ***20.30 ± 1.8 ***16.63 ± 2.3 ***14.86 ± 1.9 ***13.05 ± 0.8 ***9.93 ± 0.9 ph day 1 day 4 day 7 day 10 day 13 day 16 day 19 day 22 c 6.73 ± 0.12 7.56 ± 0.04 7.54 ± 0.02 7.46 ± 0.06 7.46 ± 0.07 7.63 ± 0.27 7.26 ± 0.03 7.20 ± 0.01 e *7.50 ± 0.01 *7.27 ± 0.02 *7.38 ± 0.18 7.32 ± 0.02 7.32 ± 0.11 **9.01 ± 0.21 *7.61 ± 0.02 7.4 ± 0.02 oc (%) day 1 day 4 day 7 day 10 day 13 day 16 day 19 day 22 c 3.25 ± 0.06 3.19 ± 0.12 3.16 ± 0.18 3.08 ± 0.24 2.40 ± 0.54 2.41 ± 0.14 2.38 ± 0.07 2.34 ± 0.30 e 3.29 ± 0.01 3.12 ± 0.17 *3.01 ± 0.69 *2.78 ± 0.12 2.3 ± 0.23 2.47 ± 0.25 *2.51 ± 0.14 *2.67 ± 0.24 5 fig. 2 mean ± sd indicates percent change in the bacterial-fungal populations and activities of amylase, cellulase and invertase in the substrate with respect to control and substrate moisture. c-control, e-experimental, bebacterial experimental, fefungal experimental, aeamylase experimental, cecellulase experimental, ie invertase experimental. “*” indicates significant variation at p < 0.05, n= 9 discussion studies on the effects of soil abiotic factors on the biomolecules in earthworms are limited (tripathi et al., 2011; mishra et al., 2018 a). bilalis et al. (2013) studied the effects of aluminium and moisture levels on the tissue protein content of the earthworm octodrilus complanatus. they have reported that although tissue protein content decreased with aluminium accumulation, it did not indicate much variation with moisture levels of 100 % and 60 %. mishra et al. (2018a) reported that low and high temperature exposures could significantly reduce tissue protein level in e. eugeniae. mishra et al. (2019) also observed non significant variation in tissue protein of the earthworm e eugeniae exposed to low intensity colour lights over a period of 21 days. in the present study, a consistent decrease in the protein level in the earthworm with reduction in soil moisture is consistent with these earlier reports. high lpx level in e eugeniae has been observed with exposure to different abiotic factors such as temperature and light (mishra et al., 2018a; mishra et al., 2019). reports are also available on the chemical stress induced lpx levels in earthworms (samal et al., 2017; nayak et al., 2018; samal et al., 2019). liu et al (2010) had observed increase in the lpx level in the earthworm eisenia fetida after exposure to rising concentrations of 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-ɣ-2-benzopyran. the consistent increase in the lpx level in e eugeniae with reducing substrate moisture in the present study corroborates these earlier results and indicates that drier substrate could cause cellular damage with enhanced lipid peroxidation in the earthworm. catalase counters the reactive oxygen species produced in organisms so as to minimize oxidative damage to cells. with increased stress conditions due to desiccation of substrate it is expected that the catalase activity would increase in the animal as has been observed in the present study. it has been reported earlier that with increasing temperature and decreasing moisture, the catalase activity was a) b) 6 induced in different organisms (khessiba et al., 2005; mishra et al., 2018a). however, hackenberger et al. (2018) observed reduction in catalase activity of e fetida in soil treated with different concentrations of propiconazole and chlorantranilpole at low moisture level. our results more or less are in agreement with the results of some of these earlier workers and with the hypothesis that enhanced lipid peroxidation due to environmental stress would enhance catalase activity. zhang and wienhold (2002) reported that under aerobic conditions, there was no significant alteration in soil ph with an increase in moisture level in agricultural soil. however, with an increase in moisture from 100 g/kg to 400 g/kg, the soil ph increased marginally from 6.2 to 6.6. in the present study, the marginal decline in ph of the substrate in the experimental set might be due to accumulation of organic acids due to reduced decomposition of organics in a moisture deprived environment. dan et al. (2016) during their studies on the effects of temperature and moisture on soil organic matter decomposition observed consistent rise in the rate of decomposition and carbon reduction with increase in soil moisture. mishra et al. (2018a, b) observed variation in the reduction of organic carbon content exposed to low intensity lights and temperature. with higher temperature and lower moisture levels the decomposition process slowed down. the results of the present study are in partial agreement with the findings of dan et al. (2016) indicating that lower substrate moisture level could adversely impact organic carbon degradation and decomposition because of reduced microbial population. barros et al. (1995) and schnürer et al. (1982) had reported positive correlation between soil moisture and microbial growth. stark and firestone (1995) observed a decline in nitrifying bacterial activity at low soil water content. however, chen et al. (2007) reported a non significant difference in the soil microbial biomass between 60 % and 80 % moisture levels. they reported that fungi in soil might adversely respond to over moisture supersaturation. baldrian et al. (2010) observed significant difference in microbial biomass in dry and wet patches in forest soil. more recently borowik and wyszkowska (2016) noticed an optimal soil moisture level of 20 % for maximal development of organotrophic bacteria and 40 % for azotobacter and actinomycetes in agricultural soil of poland indicating that different bacteria have different moisture requirement in the same habitat. it has been reported that moisture in soil plays vital role in sustaining the growth and activity of bacteria, fungi and other microorganism (ojeda et al., 2013; borowik and wyszkowska, 2016). majority of the above findings corroborate the results obtained in the present study which has shown significant reduction in bacterial and fungal population with depletion of moisture in the substrate. a reduced moisture level is likely to adversely impact microbial population and exoenzyme activities in the organic substrate. baldrian et al. (2010) have reported significant positive correlation between soil moisture and the activities of exoenzymes laccase, mn-peroxidase, endo-1,4-ßglucanase,endo-1,4ß–xylanase, cellobiohydrolase, acid phosphatase along with few other enzymes in hardwood forest ecosystem. steinweg et al. (2012) proposed that low moisture level can strongly limit in situ enzyme activity in soils, negating any positive effect of warming. however, contradictory result was obtained by geisseler et al. (2011) who studied the combined effects of soil moisture potential and plant residue addition and concluded that, residue quality and quantity modulate exoenzyme activities and moisture plays a relatively minor role. the findings on exoenzyme activities in the present study are in agreement with majority of the above reports and it is apparent that reduction of substrate moisture will slow down microbial growth, enzyme activities and consequently the decomposition of organics. surface feeding soil fauna like e. eugeniae are likely to be affected by moisture depletion from top soil in the natural habitat due to rising atmospheric temperature as a result of climate change. even during vermicomposting of organic substrate, these worms are likely to suffer from physiological stress in a desiccated environment which is likely to adversely influence their ecological functions. moisture deprivation in soil and organic substrate could reduce the population of microbes and their exoenzyme 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(anguinidae) on the four-eyed fir bark beetle polygraphus proximus: effects on the host's immunity and its susceptibility to beauveria bassiana ia kerchev, na kryukova, vyu kryukov, vv glupov institute of systematics and ecology of animals siberian branch of russian academy of sciences 630091, frunze str. 11, novosibirsk, russia accepted august 30, 2017 abstract this study for the first time demonstrated evidence of immunosuppressive influence of the entomoparasitic nematode sychnotylenchus sp. (anguinidae) on the four-eyed fir bark beetle polygraphus proximus. we detected significant decrease of phenoloxidase activity and non-specific esterases and an increase of glutathione-s-transferase rates in nematode-infected beetles. although infection by the nematode did not cause significant increase of mortality of the four-eyed fir bark beetle, it did enhance its susceptibility to infection by beauveria bassiana. our observations indicated synergetic effect between nematode and fungus infections. thus, it was shown that the nematode suppresses immunity of its host only to the extent required for effective implementation of phoresy within the bark beetle. key words: polygraphus proximus; immune suppression; bark beetle culture; nematodes; sychnotylenchus introduction recent invasion of the four-eyed fir bark beetle, polygraphus proximus (ffbb), a forest pest of far eastern origins, into siberian fir stands (russia) has had significant ecological and economic consequences (krivets et al., 2015, kononov et al., 2016). the most abundant fungal pathogens of this bark beetle in siberia are beauveria species (kerchev et al., 2017). however, the complex of natural enemies and pathogens of the ffbb in siberia remains poorly studied. nematodes are recognized as one of the major biotic factors affecting populations of aggressive bark beetles (massey, 1974). nevertheless, data on interactions of p. proximus with roundworms are entirely absent. the roundworms of the genus sychnotylenchus (anguinidae) are the predominant endoparasitic nematodes (figs 1a, b) in the west siberian populations of ffbb (kerchev and ryss, 2017 unpublished data) therefore, among other parasites they are of considerable interest. nematodes from the genus sychnotylenchus are always associated with bark beetles and their ___________________________________________________________________________ corresponding author: ivan a kerchev institute of systematics and ecology of animals siberian branch of russian academy of sciences 630091 frunze str. 11, novosibirsk, russia e-mail: ivankerchev@gmail.com free-living form feeds on fungi growing in the bark beetle frass (fortunes and maggenti, 1987). infection of the insect occurs at the pupal stage, when the nematode juvenile penetrates the host’s body cavity through the spiracles. despite the obvious traits associated with parasitism, these nematodes are not identified as parasites of insects (fortunes and maggenti, 1987). the nematodes can be found in the beetle body cavities (fig. 1c) from pupation of ffbb until oviposition and their presence does not lead to death of the host. nevertheless, the nematodes infesting the body of the host may instigate an immunomodulatory effect. for endoparasitic nematodes, one of the proven ways to suppress various systems of the host immune response is secretion of immunomodulatory molecules (cooper and eleftherianos, 2016). studies of the interaction of entomopathogenic nematodes with the host’s immune system are mainly focused on the effect that the parasite has on the cellular immunity and prophenoloxidaseactivating system (propo cascade) (cooper and eleftherianos, 2016). the detoxification pathway, however, remains unclear. the esterases (est) and glutathione-stransferase (gst) are important detoxification enzymes for insects to degrade the toxic substances produced by pathogens. it is known that gst takes part in metabolite removal and protection of tissues from damage by free radicals produced mailto:ivankerchev@gmail.com 324 fig. 1 rearing of the bark beetle culture and experimental infection by nematodes sychnotylenchus sp. on sandwich plates: a) sychnotylenchus sp. female; b) juvenile stage of nematode from bark beetle hemocel; c) dissection of polygraphus proximus infected by nematodes; d) piece of fir phloem mounted on glass and sandwich plate prepared for rearing of bark beetles; e) washed eggs of bark beetle in the phloem incision. during pathological processes (hemingway, 2000; wu et al., 2013). ests perform important functions by catabolizing the esters of higher fatty acids, by mobilizing lipids and by degrading inert metabolic esters, including various xenobiotics (wu et al., 2013). changes in these physiological parameters are associated with insect resistance to entomopathogenic fungi (e.g., yaroslavtseva et al., 2017). a synergistic effect between various pathogenic nematodes and fungi is broadly demonstrated (ansari et al., 2004; acevedo et al., 2007; jabbour et al., 2011). in addition, fungi can cause mass death of bark beetles under certain hygrothermal conditions and these are the same conditions that promote mass breeding of the endoparasitic nematodes inhabiting galleries of bark beetles (shimizu, 2013). however, the influence of nematodes of the genus sychnotylenchus on various parameters of host physiology and susceptibility to fungi has not been studied. thus, the aim of our work was to quantify the effects of sychnotylenchus sp. infection on ffbb immune and detoxificative system and to assess the impact of nematode infection on the susceptibility of ffbb to infection by the fungus beauveria bassiana. 325 324 materials and methods laboratory populations of the p. proximus and endoparasitic nematodes sychnotylenchus sp. were used in all our experiments. a nematode-free line of ffbb was obtained by sterilization of beetle eggs with 3 % solution of hydrogen peroxide. for initiation of nematode infected lines, we designed conditions similar to the natural situation of infection. larvae of nematodes from the body cavities of infected bark beetles were placed on moistened pieces of filter paper (0.5×0.5 cm) and put near slits in the fir phloem containing sterilized eggs of beetles (10 nematodes per 25 eggs) in sandwich plates (kerchev, 2014) (figs 1d, e). the plates were kept at 22 ºc for 60 days. verification of infection and purity of the lines was performed by dissecting randomly selected beetles under light microscopy. full details of obtaining nematode infected and nematode free ffbb lines are provided in the electronic supplementary material. infected and parasite-free p. proximus adults were used in experiments on the 55th day after the nematode juveniles had been injected into the fir bark containing ffbb eggs. the presence or absence of melanotic cell capsules around the nematodes was registered using insect dissection and light microscopy. the po est and gst activity in the hemolymph were estimated on the adult beetles. all measurements were taken following conventional techniques as follows. for the samples intended for measuring gst and est activity, phenylthiourea (ptu; 4 µg/ml) was added to each tube containing hemolymph to prevent melanization. po activity in the hemolymph plasma was determined spectrophotometrically at a wavelength of 490 nm, using solution of dl-dihydroxyphenylalanine (4 mg/ml pb) as a substrate (a modification of the method described by ashida and soderhall, 1984). the activity of gst was estimated following habig et al. (1974) and using 2-nitro-5-thiobenzoic acid (dntb) as a substrate. the concentration of 5-(2, 4dinitrophenyl)-glutathione produced by the reaction was estimated at a wavelength of 340 nm. est activity in the samples was determined spectrophotometrically by the asperen’s method (asperen, 1962) with minor modifications. the concentration of 1-naphthyl produced during the reaction was measured spectrophotometrically at the wavelength of 410 nm. enzyme activity was measured in units of transmission density (da) of the incubation mixture during the reaction for each 1 min and 1 mg of protein. the concentration of protein in the hemolymph was determined by the bradford (1976) method. bovine serum albumin was used for the calibration curve. for analysis of enzymatic activity 10 replicates (one replicate = 5 beetles) were used. for infection of insects, the culture of beauveria bassiana s.s. sar-31 from the collection of the institute of systematics and ecology of animals (isea), siberian branch, russian academy of sciences, was used. fungi were cultivated on sabouraud agar for three weeks. then conidia were washed from the surface of sporulating areal mycelium and suspended in water with added tween 20 (0.03 %). nematode-free and nematode infected beetles were treated with fungi by dipping in a conidia suspension (1×107 conidia/ml) for 10 s. the concentrations were counted in a hemocytometer. fifty insects in each treatment were used. the two control groups of insects, infected with nematodes and nematode-free lines, were dipped in water-tween solution. immediately after submersion, the beetles were placed on filter paper to remove extraneous suspension. then the insects were placed in 60 mm petri dishes (ten specimens per one dish). fir bark was returned to the same dishes from which the beetles were previously taken and moistened disks of filter paper were placed to support 100 % rh. after treatment, insects were incubated in the dark at constant temperatures of 18 20 °c. mortality was recorded every day for 15 days. the mannwhitney u test was used to estimate differences in enzyme activity in nematode-infected and nematode-free beetles. a log-rank test followed by holm-sidak correction was used for analysis of mortality dynamics. the differences between synergistic and additive effects were determined daily by comparing the expected and observed mortality using the χ2 criterion (tounou et al., 2008). results we analyzed activity of the po, est and gst in ffbb infected by parasitic nematodes of the genus sychnotylenchus sp. a suppressive effect of the nematodes on the immunological status of the infected beetles was evident from our results. melanotic cell capsules around the nematodes inside the ffbb were not detected which suggests that the immune system of the beetles was not able to recognize nematodes. po activity of nematodeinfected beetles was significantly reduced by 4.7 fold compared to nematode-free beetles (z = 2.74; p = 0.006) (fig. 2a). the activity of non-specific esterases in nematode-infected beetles was also significantly reduced by 3.02 fold (z = 3.38; p = 0.0007) (fig. 2b), while the gts rate was 2.1 fold higher in nematode-infected beetles (z = 2.73; p = 0.006) compared to nematode-free beetles of the control group (fig. 2c). nematode-infected beetles were more susceptible to beauveria bassiana infection compared to nematode-free beetles. median lethal time (lt50) was 9 ± 0.35 days for nematode-free beetles and 8.00 ± 0.32 days for nematode-infected beetles (χ2 = 13.44; p = 0.0002). a synergistic effect between fungal and nematode infections occurred from 9 to 15 day after treatment with fungus (χ2 > 3.97; p < 0.05) (fig. 2d). nematode infection alone causes sublethal effects. however, after 15 days, differences in survival between infected and nematode-free lines were not significant (χ2 = 3.41; p = 0.06) data not shown. discussion the absence of the capsule around nematodes and the decrease of the phenoloxidase activity 326 324 fig. 2 аctivity of immune and detoxificative enzymes in hemolymph of polygraphus proximus adults which were infected and non-infected with nematode sychnotylenchus sp., and influence of nematode infection on susceptibility to beauveria bassiana. a) phenoloxidase activity, b) non-specific esterases activity, c) glutathiones-transferase activity, d) survival dynamics after b. bassiana treatment. † significant differences between the lines infected and non-infected with nematode (z > 2.73; p < 0.006), * synergistic effect between nematode and fungus (χ2 > 3.97; p < 0.05). suggest that an active mechanism functions in these immune reactions inhibited. the immediate response of host beetles to nematodes is encapsulation, accompanied by the release of a toxic reactive oxygen species (ros) formed during melanization that leads to destruction of the pathogens (dowds and peters, 2002). there are a number of intermediates similar to ros linked with the melanotic cascade (quinones, hydrogen peroxide, o-semiquinone radicals, etc.) that are cytotoxic and destroy pathogenic microorganisms (nappi and ottaviani, 2000; komarov et al., 2009). on the other hand, active oxygen radicals are quite toxic to the host and subsequently detoxified by enzymes to prevent oxidative stress. reduced activity of po is the most common method to expose the parasite to the host's immune response. the inhibition of po activity may be due to secretion of immunosuppressive molecules into the hemocel of the insect by the parasite itself, and/or by its symbiont microorganisms (cooper and eleftherianos, 2016). steinernema carpocapsae secretes chymotrypsin proteases inhibiting the prophenoloxidase cascade to avoid encapsulation in the hemocel of galleria mellonella (balasubramanian et al., 2010). the nematode species s. feltiae induces prophenoloxidase inactivation in g. mellonella larvae by interaction of nematode cuticular components with the prophenoloxidase activating lps-binding proteins (brivio et al., 2002). the main detoxification enzymes in invertebrates are monooxygenases, est and gst. est participate in many biological functions and begin the metabolism of xenobiotics, lipids, acetylcholine and juvenile hormone (kumar et al., 2003; lozinskaya et al., 2004; li et al., 2007). our observed effect of sychnotylenchus sp. on the detoxification enzymes of ffbb is similar to the results of wu et al. (2013) and li et al. (2016). authors detected significantly dose-dependent enhancement of the gst activity in host larvae was detected along with a decrease of the carboxylesterase activity on the systems of g. mellonella larvae when infested with the entomopathogenic nematode heterorhabditis beicherriana n. sp. (rhabditida: heterorhabditidae) and on tenebrio molitor (coleoptera: tenebrionidae) larvae when infested with h. 327 324 beicherriana. we observed an increase in the gst activity and that can be both a result of the general depression of the po activity by toxic or mechanical damage to host cells and tissues by the nematodes or an adaptive response to neutralize ros induced by oxidative stress. the decrease of the est activity in hemolymph is probably because ones besides detoxification take a direct part in lot of physiological processes in insects. many physiological processes of insects are resource consuming and can go against the interests of the parasite; as a result, total count of enzymes can be depleted by nematode. depression in the functioning of the host's immune system induced by sychnotylenchus sp. contributes to an increasing susceptibility for entomopathogenic fungi. it has been confirmed that ascomycetes as the generalists are adapted to infect insects weakened by various ecological factors (boomsma et al., 2014) and in this case with immune systems suppressed by nematode infestation. from an ecological point of view, the ffbbsychnotylenchus sp. system is one of the examples of a stable host-parasite relationship, in which the nematode suppresses the host's immunity only as much as is required for the effective implementation of phoresy within the host. this is the first study to determine an influence of nematodes from the genus sychnotylenchus on its host’s physiological system. nematode infection causes sub lethal effects on p. proximus adults, but significantly modulates immune and detoxificative systems that lead to increase in susceptibility to fungal infection. acknowledgements the study was supported by the russian science foundation (grant № 15-14-10014). we thank alexander ryss (zoological institute russian academy of sciences) for nematode identification. references acevedo jpm, samuels ri, ribeiro mi, dolinski c. interactions between isolates of the entomopathogenic fungus metarhizium anisopliae and the entomopathogenic nematode heterorhabditis bacteriophora jpm4 during infection of the sugar cane borer diatraea saccharalis (lepidoptera: pyralidae). j. invertebr. pathol. 96: 187-192, 2007. ansari ma, tirry l, moens m. interaction between metarhizium anisopliae clo 53 and entomopathogenic nematodes for the control of hoplia philanthus. biol. control 31: 172-180, 2004. asperen k. van a study of housefly esterases by means of a sensitive colorimetric method. j. insect physiol. 8: 401-414, 1962. balasubramanian n, toubarro d, simões n. biochemical study and in vitro insect immune suppression by a trypsin-like secreted protease from the nematode steinernema carpocapsae. parasite immunol. 32: 165-175, 2010. boomsma jj, jensen a b, meyling nv, eilenberg j. evolutionary interaction networks of insect pathogenic fungi. annu. rev. entomol. 59: 467-485, 2014. bradford mm. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. anal. biochem. 72: 248-254, 1976. brivio mf, mastore m, pagani m. parasite-host relationship: a lesson from a professional killer. inv. surv. j. 2: 41-53, 2005. brivio mf, pagani m, restelli s. immune suppression of galleria mellonella (insecta, lepidoptera) humoral defenses induced by steinernema feltiae (nematoda, rhabditida): involvement of the parasite cuticle. exp. parasitol. 101: 149-156, 2002. cooper d, eleftherianos i. parasitic nematode immunomodulatory strategies: recent advances and perspectives. pathogens 5: 58, 2016. dowds bca, peters a. virulence mechanisms. in entomopathogenic nematodes, gaugler r (ed.), cabi: wallingford, uk, pp 79-98, 2002. dubovskiy im, slyamova nd, kryukov vyu, yaroslavtseva on, levchenko mv, belgibaeva ab, et al. the activity of nonspecific esterases and glutathione-s-transferase in locusta migratoria larvae infected with the fungus metarhizium anisopliae (ascomycota, hypocreales). entomol. rev. 92: 27-32, 2012. fortuner r, maggenti ar. a reappraisal of tylenchina (nemata). the family anguinidae nicoll, 1935 (1926). rev. nematol. 10: 163-176, 1987. habig wh, pabst mj, jakoby wb. glutathione-stransferases. j. biol. chem. 249: 7130-7139, 1974. hemingway j. the molecular basis of two contrasting metabolic mechanisms of insecticide resistance. insect biochem. mol. biol. 30:1009-1015, 2000. jabbour r, crowder dw, aultman ea, snyder we. entomopathogen biodiversity increases host mortality. biol. control 59: 277-283, 2011. kerchev ia, kryukov vyu, yaroslavtseva on, polovinko gp, tokarev yus, glupov vv. the first data on fungal pathogens (ascomycota, hypocreales) in the invasive populations of four-eyed fir bark beetle polygraphus proximus blandf. russian j. biol. invasions 8: 34-40, 2017. kerchev ia. on monogyny of the four-eyed fir bark beetle polygraphus proximus blandf. (coleoptera, curculionidae: scolytinae) and its reproductive behavior. entomol. rev. 94: 10591066, 2014. komarov da, ryazanova ad, slepneva ia, khramtsov vv, dubovskiy im, glupov vv. pathogen-targeted hydroxyl radical generation during melanization in insect hemolymph: epr study of probable cytotoxicity mechanisms. appl. magnetic resonance, 35: 495-501, 2009. kononov a, ustyantsev k, blinov a, fet v, baranchikov yun. genetic diversity of aboriginal and invasive populations of four-eyed fir bark beetle polygraphus proximus blandford (coleoptera, curculionidae, scolytinae). agr. forest. entomol., 2016. doi: 10.1111/afe.12161. krivets sa, bisirova em, kerchev ia, pats, en, chernova na. transformation of taiga 328 324 ecosystems in the western siberian invasion focus of four-eyed fir bark beetle polygraphus proximus blandford (coleoptera: curculionidae, scolytinae). russian j. biol. invasions 6: 94108, 2015. kumar s, christophides gk, cantera r, charles b, han ys, meister s, et al. the role of reactive oxygen species on plasmodium melanotic encapsulation in anopheles gambiae. proc. natl. acad. sci. usa 100: 14139-1414, 2003. li y, lopez p, durand p, ouazzani j, badet b, badet-denisot ma. an enzyme-coupled assay for amidotransferase activity of glucosamine-6phosphate synthase. anal. biochem. 370: 142146, 2007. lozinskaya yal, slepneva ia, khramtsov vv, glupov vv. change of the antioxidant status and free radicals production in hemolymph of galleria mellonella larvae in microsporidiosis. j. evol. biochem. physiol. 40: 119-125, 2004. massey cl. biology and taxonomy of nematode parasites and associates of bark beetles in the united states. forest service, us dept. of agriculture, washington, dc, washington, 1974. nappi a, ottaviani e. cytotoxicity and cytotoxic molecules in invertebrates. bioessays 22: 469480, 2000. tounou, ak, kooyman c, douro-kpindou ok, poehling hm. interaction between paranosema locustae and metarhizium anisopliae var. acridum, two pathogens of the desert locust, schistocerca gregaria under laboratory conditions. j. invertebr. pathol. 97: 203-210, 2008. wu h-d, liu q-z, li x-y, wang y-l, zhang h. activities of four enzymes in galleria mellonella larvae infected with entomopathogenic nematode heterorhabditis beicherriana n. sp. afr. j. agric. res. 8: 3245-3250, 2013. xingyue li, qizhi liu, edwin e. lewis, eustachio tarasco activity changes of antioxidant and detoxifying enzymes in tenebrio molitor (coleoptera: tenebrionidae) larvae infectedby the entomopathogenic nematode heterorhabditis beicherriana (rhabditida: heterorhabditidae). parasitol. res., 2016. doi 10.1007/s00436-016-5235-7. yaroslavtseva on, dubovskiy im, khodyrev vp, duisembekov ba, kryukov vyu, glupov vv. immunological mechanisms of synergy between fungus metarhizium robertsii and bacteria bacillus thuringiensis ssp. morrisoni on colorado potato beetle larvae. j. insect physiol. 96: 14-20, 2017. supplementary material obtaining nematode-free and nematode-infected lines of four eyed fir bark beetles the laboratory culture of four eyed fir bark beetles free from nematode parasites was produced from natural population from west siberia (tomsk region, near the settlement of zavarzino, 56° 27'45" n, 85° 05'25" e). in this stand, the infection of beetles by sychnotylenchus sp. reached 97 %. collected beetles were put in plastic containers on the freshly cut logs of siberian fir abies sibirica ledeb. on the 14th day after introduction of parent beetles, bark was removed from the logs. only unhatched eggs from the maternal galleries were used for rearing insect culture. to remove eggs or larvae of nematodes from the surface of the chorion, bark beetle eggs were put in a sieve with a cell size of 0.2 mm, and then placed for 30 sec. in 3 % solution of hydrogen peroxide followed by 10 min. washing in distilled water. this procedure was repeated twice. after washing, the eggs in batches of 25 pcs were placed on freshly-peeled fir phloem (15×15 cm) into 2.5 cm long incisions. the phloem tissue containing eggs was mounted on sandwich plates (kerchev, 2015). to prevent desiccation, the sides were covered with parafilm. a total of six sandwich plates were prepared each containing 150 bark beetle eggs free from nematodes. after 5 days, half of the plates were injected with nematode juveniles from the hemocoel of native p. proximus beetles. after preliminary identification of a taxonomical group of nematodes under a light microscope, nematode larvae, along with a drop of distilled water, were transferred from the slide in groups of 10 pcs onto a 0.5×0.5 cm filter paper, which was placed next to phloem with eggs on sandwich plates. plates with insects were kept for 60 days at 22 °c and relative humidity of 80 %. the presence of nematodes in larval galleries on sandwich plates and beginning of invasion of juveniles into bark beetles pupae was checked through glass. the infection of beetles was assayed on 20 randomly selected beetles per line. all insects in the nematode-infected line were invaded by larvae of sychnotylenchus sp. and the intensity of infection was 18.2 ± 6.4 (mean ± sd) per insect. 329 116 isj 15: 116-130, 2018 issn 1824-307x report of meeting xixth scientific meeting of the italian association of developmental and comparative immunobiology (iadci), 7 9 february 2018, department of earth, environment and life sciences (distav), university of genoa, genoa, italy organizers: l canesi, t balbi, m auguste, e grasselli, l vergani, i demori, r fabbri, m montagna, a voci department of earth, environment and life sciences (distav), university of genoa, genoa, italy session 1. chairmen: laura canesi, university of genoa, genoa, italy and giuseppe scapigliati, university of tuscia, viterbo, italy nanoparticles and the immune system evolution of innate immunity, lessons learned for assessing safety and efficacy of nanomaterials and nanodrugs d boraschi1, b swartzwelter1, d melillo1, r marino2, g della camera1, f barbero3, v puntes3, p italiani1 1institute of protein biochemistry, cnr, naples, italy 2stazione zoologica anton dohrn, naples, italy 3icn2, barcelona, spain innate immune defensive mechanisms are the major surveillance and effector mechanisms deputed to maintaining the integrity and functionality of a living organisms. from plants to human beings, innate immunity has conserved a number of defensive cells, molecules and pathways. innate immunity is the only type of immunity present in plants and invertebrates, at variance with higher vertebrates that also display slower but more specific adaptive immune functions. immunological memory, a feature though to characterise adaptive immunity, is clearly present in innate immunity although based on different mechanisms, and can be clearly displayed by phagocytic cells, which are rapid and efficient cells in recognising and destroying invading microorganisms or foreign particles. we have studied both innate immune responses and the development of innate memory in human blood monocytes in vitro in comparison with the marine tunicate ciona intestinalis, in which adaptive immunity is absent. the strong primary response to a bacterial challenge (e.g., lps) can prime cells in vitro or animals in vivo to respond differently to a subsequent challenge with the same or with a different stimulus. human cells primed with lps showed a significant modulation of their activation capacity in response to a second challenge (with lps or other stimuli), with a clear decrease in the capacity of producing tnfα. on the other hand, challenge with lps strongly increased the phagocytic ability of c. intestinalis haemocytes primed with either lps or lta, whereas challenge with lta decreased it. for human cells, strong donor-to-donor variations were evident, suggesting that previous in vivo exposure to diverse external agents may change the reactivity of blood monocytes to in vitro stimuli. although significant quantitative inter-individual differences were noted, in c. intestinalis the development of innate memory was more reproducible in terms of increase or decrease of secondary response, suggesting a limited or similar influence of the previous life conditions. the comparative study of innate memory is expected to generate information that will help us controlling and modulating memory development in 117 future personalised immunoprophylactic and immunotherapeutic approaches. human immune cells as target of gold nps (aunps): potential impacts on immune responses s michelini1, a prinelli2, m sarajlic1, a duschl1, j horejs-höck1 1paris lodron-universität salzburg, salzburg, austria 2avanticell science ltd, gibbsyard, auchincruive, ayr, scotland, uk gold nanoparticles (aunps) are becoming every day more part of our life. because of their unique optical and physical properties, their applications in different fields are dramatically increasing. particularly in medicine, they have been suggested as new delivery systems, diagnostic tools, vaccination adjuvants, as enhancer for radiation therapy and as novel contrast agents for computer tomography (ct). some of these approaches include high doses of intravenously and sometimes repetitively injected gold nps which then can spread throughout the body coming in contact with many different cell types including immune cells. since this is a branch of science in active development and still little is known about the potential effects of nps on the immune system, especially in the context of long term and chronic exposure with concomitant pathologies, there is an urgent need to better understand the possible functions of aunps on immune cells with phagocytic properties such as macrophages and dendritic cells (dcs). a matter of interest is also the extremely reactive surface of nps, because nps in fact are capable of binding proteins and molecules, changing their conformation and maybe, their activity. in order to identify common mechanisms/markers of cellular reactivity that can be predictive for determining risk or safety of these nano-objects, the current study investigates the role of aunps of different sizes and surface area, on modulating dendritic cell-activation induced by lps. we observed that treatment of human monocyte-derived dendritic cells with lps in combination with endotoxin-free 26nm-aunps, alters the expression of specific cell surface markers and modulates the release of certain cytokines. no similar effects were observed with aunps with a size of 4, 9 and 11 nm. this suggests that 26 nm aunps may have specific properties, maybe in terms of surface area, that contribute to their immune modulatory properties. our observation could also substantiate some earlier evidences that the medical use of colloidal gold might be beneficial to reduce the swelling and inflammation of joints in patients with rheumatoid arthritis. optimization of a cryopreserved immune cell product for nanosafety testing a prinelli, ma ewart, cj wilde avanticell science ltd, gibbsyard, auchincruive, ayr, scotland, uk although nanoparticles (nps) have always been on our planet, recently the impact of nanomaterials on health and the environment has received considerable attention. nps are defined as materials within nanoscale dimensions and nowadays nps are applied in various fields such as chemistry, electronics, consumer goods and medicine. there is now a pressing need for more predictive and efficient methodologies to examine nanoparticle immunosafety on humans, animals and the environment. therefore, the purpose of this project was to set up a panel of cell-based assays to measure the human innate inflammatory response to nps under analytical conditions and in formats representing industrial prototypes for commercial exploitation. the research objective was to establish and optimise a ready for use immune product in 96 well plate format, that was translatable to a 3d assay format, and which could assess the activation of immune cells after exposure to different kinds of np. the developed “plug and play” immune-cryotix™ format is based on the activation of human monocyte derived macrophages, which are essential as the first line of defence in many organisms, and the detection of cytokines secreted by these macrophages after inflammatory stimulation. briefly, peripheral blood mononuclear cells (pbmc) were isolated from samples of whole blood by immuno-magnetic selection. monocytes were then differentiated to m1 and m2 macrophages, the provenance of which was confirmed via flow cytometric analysis, and optimised cryopreservation protocols were established. cryopreserved plates can be stored at -150 °c, shipped to the customer via acs shipping container and thawed 24 h before the inflammatory screen is performed. recovered cryopreserved macrophages maintain high viability and display comparable morphology to unfrozen macrophages. inflammatory cytokine secretion, in unstimulated or lpsstimulated macrophages was similar for control and cryopreserved cells. experiments are ongoing to improve the performance of the immune-cryotix® product and also to establish standardise assay readouts for np testing. combining whole-organism toxicity endpoints, stress-related enzyme levels, and cellular & humoral immune system markers of porcellio scaber to evaluate nanoparticle toxicity, using gold nanoparticles as an example c mayall, d drobne biotechnical faculty, university of ljubljana, ljubljana, slovenia due to their stability and ease with which they can be synthesised, gold nanoparticles are popular nanoparticles (nps) in nano-bio interaction research. with the myriad of potential uses they 118 could have, it is inevitable that these nps will eventually reach the environment and therefore it is imperative that the possible side effects they may have be evaluated. it is thought that organisms may modulate their immune system after exposure to nps as they may recognise nps as foreign in much the same way they would recognise and respond to invading bacteria. the aim of this study was to investigate whether ingestion of gold nps alters the immune response of the terrestrial isopod, porcellio scaber, and how these changes will be reflected at higher levels of organisation such as feeding and mortality. as the immune system is an early responder to foreign matter, studying it in conjunction with traditionally used parameters of toxicology can give more information into the possible effects these particles may have. animals were fed gold nps for 14 days, during which time the feeding, defecation and survival rates of the animals were recorded, these are the traditional environmental toxicity endpoints. after 14 days, the hemolymph was removed and the number, viability and proportion of hemocytes were counted. along with cellular tests, the humoral side of the immune system was investigated by measuring the activity of the enzyme phenoloxidase in hemolymph, which is associated with melanisation. the levels of stress markers, glutathione s-transferase (gst) and soluble acetylcholinesterase (ache), were also assayed. the removal of hemolymph has no impact on the other measurements, meaning no extra animals have to be used while garnering this immunological information. animals injected with a non-lethal dose of lps acted as a positive control for the activated immune system. results so far indicate that the gold nps tested had no significant effect on the feeding, mortality or defecation rates of isopods. there were also no significant changes to the cellular immune system and preliminary results suggest no effects on the humoral immune system. these results suggest that gold nps at the selected, environmentally relevant exposure concentration are not toxic nor immunomodulating agents to p. scaber. we discuss the immune system as an additional endpoint in assessing the effects of nanomaterials on environmentally relevant organisms. "les liaisons dangereuses": do nanoparticles affect immune defense by modulating innate memory? b swartzwelter1, d melillo1, r marino2, g della camera1, am campos1, f barbero3, v puntes3, p italiani1, d boraschi1 1institute of protein biochemistry, cnr, naples, italy 2stazione zoologica anton dohrn, naples, italy 3icn2, barcelona, spain innate immunity is evolutionarily conserved across species and is dependent upon specialized phagocytic cells. these cells largely consist of monocytes and macrophages in vertebrates and haemocytes in invertebrates, and play a major role in the recognition and elimination of foreign threats. they are able to mount a defense response against foreign objects by secretion of inflammatory cytokines and chemokines, phagocytosis and, following elimination of the danger, they are also heavily involved in the resolution phase of inflammatory reactions. furthermore, recent studies have rejuvenated interest in the concept that, following resolution, innate immune cells display non-specific memory of previous stimulation, which alters subsequent innate defense responses. based upon this concept, known as innate immune memory, we have primed human monocytes in culture with either gold engineered nanoparticles (au enp) or lps. we have undertaken to treat cells in both 2d and 3d conditions, evaluating whether culture conditions affect memory responses. while lps priming initiated a strong inflammatory response, demonstrated by increased tnfα, il-8, il-1ra and il-6 production, au enp priming did not induce cytokine production in either culture condition. following a resting period of 5-7 days, cells were again challenged with lps or au enp. no cytokine production was noted in response to au enp challenge, while lps challenge of rested cells resulted in cytokine production that was lower than the initial response of fresh cells. of the two culture conditions, 3d culture resulted in elevated reactivity compared to 2d, both in the priming and challenge phases. in 2d culture, lps primed cells that were then challenged with lps demonstrated reduction of the cytokine response compared to unprimed control cells. interestingly, in 3d culture this modulation was lost or altered in the cases of il-1ra, il-8, and il-6, while in tnfα downregulation was retained. both the magnitude and the direction (increase or decrease) of the altered memory response appeared to be donordependent. au enp appeared to have a role in the memory response in 3d culture, a response that is also donor-dependent. these observations demonstrate a role for enp driven memory in innate immune responses, and that the conditions in which these responses are measured are critical. real time pcr and image-based cell profiling analyses evidence correlation between hematopoietic and ampulla cell populations in the gastropod pomacea canaliculata a accorsi1,2, a montanari3, m nasi4, s pecorini5, a box1, r peuß1, d malagoli3 1stowers institute for medical research, kansas city, mo, usa 2howard hughes medical institute, stowers institute for medical research, kansas city, mo, usa 3department of life sciences, university of modena and reggio emilia, modena, italy 4department of surgery, medicine, dentistry and morphological sciences, university of modena and reggio emilia, modena, italy 5department of biomedical, metabolic and neural sciences, university of modena and reggio emilia, modena, italy 119 pomacea canaliculata is a freshwater snail gaining visibility among the scientific community due to both its biology and environmental invasiveness. in previous studies, the hematopoietic tissue of this snail has been identified in the pericardial cavity and we hypothesized that the ampulla, a hollow organ directly connected to the heart and located into the pericardial cavity, may play a role in hemocyte maturation. in order to test this hypothesis, different approaches have been adopted. by real time pcr, we demonstrated that the hematopoietic tissue and the ampulla present similar levels of expression of the prokineticin-like protein, pc-plp. conversely, preliminary experiments suggested that the expression levels of a second prokineticin domain-containing protein, pc-astakine, are significantly different between hematopoietic tissue and ampulla. these molecular data have been correlated with those collected with imagestream® x mark ii imaging flow cytometer. this instrument, combining flow cytometry and microscopy technologies, rapidly collects images of thousands of cells and allows high-throughput analysis of cell morphologies and their frequency in different samples. freshly dissociated or fixed hematopoietic tissue and ampulla have been run. shared cell morphologies have been observed but the largest part of their frequencies have been shown to be significantly different between the two organs. our results suggest that, beyond the apparent differences, hematopoietic tissue and ampulla may share immune-related gene expression and cell populations. the availability of genome sequence and organ-specific transcriptomes for p. canaliculata and the recent optimization of in situ hybridization protocols, will help to further clarify these observations and to elucidate the role played by the ampulla in hemocyte maturation. effects of silver nanoparticles on mytilius galloprovincialis hemocytes and embryos m auguste1, m montagna1, c ciacci2, a brunelli3, e badetti3, a marcomini3, l canesi1 1department of earth, environment and life sciences (distav), university of genoa, genoa, italy 2department of biomolecular sciences, university of urbino carlo bo, urbino, italy 3department of environmental sciences, informatics and statistics (dais), ca' foscari university of venice, venice, italy silver nanoparticles (agnps) are more and more used in consumer products partly due to its biocidal action. as coastal sessile organisms, mussels are likely to be more impacted by nps in reason of their filter feeding ability. in this work, the effects of uncoated agnps (47 nm) on the marine mussel mytilus galloprovincialis were evaluated at the cellular and whole organism level utilizing both immune cells (hemocytes) and developing embryos. the effects were compared with those of ionic silver (agno3). hemocytes were exposed in vitro for 30 min to agnp suspensions in asw at several concentrations (1-100 µg/ml). several functional parameters were evaluated (lysosomal membrane stability-lms, extracellular ros production, phagocytosis). the effects of agnps on the actin cytoskeletal structures and mitochondrial membrane potential were also investigated using fluorescent dyes by confocal fluorescence microscopy. the embryotoxicity test was performed exposing fertilized eggs to different concentrations (0.0011000 µg/l) of agnp suspensions in 96-microwell plates, and the percentage of normal d-larvae was evaluated after 48 h. the results showed that agnp mainly targeted lysosomes, cytoskeleton and mitochondria of the hemocyte. toxicity of agnps was much lower than that of ag+, as indicated by the ec50 values obtained for lms (273.1 and 1.23 µg/µl, respectively). regarding the embryos, agnps induced a dose-dependent decrease in normal larval development, with concentrations > 80 µg/l inducing developmental arrest. also in the embryotoxicity test, the effects of agnps were lower than those of ag+ (ec50 23.71 and 1 µg/l, respectively). despite the formation of agglomerates in exposure medium, agnps were able to negatively interact with both hemocytes and developing embryos. these observations could be bothersome not only for the immune function of mussels but also for future mussel population establishment and potentially hinder their development until the adult stage. these results highlight the fast response of the mussel immune system and strengthen the suitable use of early embryos as sensitive targets for the adverse effects of nps in marine invertebrates. session 2. chairmen: loriano ballarin, university of padua, padua, italy and davide malagoli, university of modena and reggio emilia, modena, italy invertebrate immunity (1) myticalins: towards a link between sequence diversity and antimicrobial function m gerdol1, m mardirossian1, f spazzali1, v musso1, m benincasa1, s pacor1, a tossi1, p venier2, m scocchi1, a pallavicini1 1department of life sciences, university of trieste, trieste, italy 2department of biology, university of padua, padua, italy myticalins are a recently described family of linear cationic antimicrobial peptides taxonomically restricted to marine mussels (mytilus spp.). while our findings permitted to confirm a relatively broad spectrum of activity towards both gram-positive and -negative bacterial strains, the functional implications of the puzzling molecular diversity of the amps still remain unexplained. indeed, the “myticalin inventory” shows significant intraspecific variation, both in terms of gene number and presence/absence of particular sequence subfamilies, as confirmed from whole-genome resequencing data. in summary, each mussel is 120 endowed with a potentially unique set of myticalins. this surprising inter-individual diversity might be linked to benefits at the whole-population level, fitting with the concept of ecological immunity. the localized expression of myticalins in the gill tissue, a major interface with the external environment, suggests that these amps play a role as a first line of defense towards invading microbes acquired from the water column. unfortunately, classical microbiological assays can only provide preliminary indications about these microbial targets, as the vast majority of marine bacteria are non-culturable. we developed a new method to detect significant shifts in the composition of the gillassociated microbiota following myticalin treatment, based on the metabarcoding of bacterial 16s rrna. other unanswered questions concern the mode of action of myticalins. we demonstrate that, unlike other proline-rich amps, myticalins do not exploit the bacterial inner membrane transporter bac7 to enter target cells. on the other hand, myticalins display marked permeabilizing properties on eukaryotic cells at mic concentrations. this, together with the complete inhibition of the antimicrobial activity in marine broth, suggests that myticalins act intracellularly on phagocytosed bacteria, without adopting secondary structures, as evidenced by circular dichroism. myticalins: a novel multigenic family of linear, cationic antimicrobial peptides from marine mussel (mytilus spp.) a de poli1, m gerdol1, m mardirossian1,2, p venier3, a pallavicini1 1department of life sciences, university of trieste, trieste, italy 2genzentrum, ludwig maximilian universität, munich, germany 3department of biology, university of padua, padua, italy the application of high-throughput sequencing technologies to non-model organisms has brought new opportunities for the identification of bioactive peptides from genomes and transcriptomes. from this point of view, marine invertebrates represent a potentially rich, yet largely unexplored resource for de novo discovery due to their wide range of adaptation strategies to challenging habitats. thorough bioinformatics analyses of available genomic and transcriptomic data we succeeded to identify myticalins, a novel family of antimicrobial peptides (amps) from the mussel mytilus galloprovincialis, and a similar family of amps from modiolus spp., named modiocalins. their coding sequence encompasses two conserved n-terminal (signal peptide) and c-terminal (propeptide) regions and a hypervariable central cationic region corresponding to the mature peptide. myticalins are taxonomically restricted to mytiloida and they can be classified into four subfamilies. these amps are subject to notable interindividual sequence variability and possibly to presence/absence variation. functional assays performed on selected members of this family indicate a remarkable tissuespecific expression (in gills) and a broad spectrum of activity against both gram-positive and gramnegative bacteria. overall, we present the first linear amps ever described in marine mussels and confirm the great potential of bioinformatics tools for the de novo discovery of bioactive peptides in nonmodel organisms. a phylogenetic perspective of the cytokine mif in bivalves u rosani1, s domeneghetti1, m gerdol2, a pallavicini2, p venier1 1department of biology, university of padua, padua, italy 2department of life sciences, university of trieste, trieste, italy macrophage migration inhibitory factor (mif) is a key regulator of vertebrate immune systems, known to be involved in many other processes, from normal development to diseases. owing to its multiple roles and its capacity to finely tune several molecular pathways, mif has been renamed in the ‘most interesting factor’. however, the evolutionary history of this cytokine is not limited to vertebrates, given that mif-like genes have been identified in protostomes, plants and even in bacteria. notably, mif-based interactions of some parasites have been reported as a central node to hijack host immune system. only a few mif sequences have been identified so far in bivalves, for instance in mytilus galloprovincialis, magallana gigas and meretrix meretrix. with the aim to update the phylogenetic description of mif, we searched for mif-like sequences in genomic and transcriptomic data of bivalves, retrieving a total of 148 sequences (80 % of them were previously unknown). we demonstrated that, like vertebrates, most of the analyzed bivalve species encode two mif genes, namely one mif and one dd-t gene. moreover, the genomic expansion of mif genes was evident in two bivalve families, a condition found only in the genomes of obligate blood or lymph-feeding ectoparasites. to investigate the role of mif in marine mussels, we exploited rna-seq and qpcr data. in m. galloprovincialis, expression data only partially support the involvement of mif-like genes in immunity and suggest that mif and dd-t may be involved in different functional pathways. the ongoing recombinant production of m. galloprovincialis mif in pichia pastoris should provide a valuable tool to clarify its functional role(s) in an invertebrate model of study. molecular characterization and expression analysis of the first porifera tumor necrosis factor superfamily member and of its putative receptor in the marine sponge chondrosia reniformis m pozzolini1, s scarfì1, f mussino1, s ghignone2, l vezzulli1, c cerrano3, m giovine1 1department of earth, environment and life sciences (distav), university of genoa, genoa, italy 2institute for sustainable plant protection-turin unit, cnr, turin, italy 121 3department of life and environment sciences (disva), marche polytechnic university, ancona, italy tumor necrosis factor (tnf) is a proinflammatory cytokine involved in a number of cell signaling pathways such as immune function, inflammation, apoptosis, cell differentiation and proliferation. it belongs to a large family of structurally related proteins known as “tnf ligand superfamily” (tnfsf). in the present study, we report the first molecular cloning of a tnfsf member (chtnf) and of its putative receptor (chtnfr) in porifera, the oldest metazoan group still extant on our planet. full-length cdnas were isolated and cloned from the marine sponge chondrosia reniformis. the deduced chtnf amino acid sequence indicates that this sponge tnf-like is a type ii transmembrane protein containing the typical tnfsf domain. phylogenetic analyses reveal that it is related to abalone disk tnf. chtnf and chtnfr tissue expression profile was evaluated by qpcr indicating that they are constitutively expressed both in the ectosome and in the choanosome of the sponge, with higher levels in the ectosome. in order to establish the immunological role of the tnf-like protein and to evaluate the presence in sponges of a first ancient tnfr towards which the tnf-like protein may be directed to, the mrna expression level of the chtnf and of chtnfr were investigated after sponge tissue explants treatment with gram positive (enterococcus faecalis) or gram negative (vibrio alginolyticus and vibrio fluvialis) bacteria for 3, 6, 24 and 48 hours. the obtained results indicated that chtnf was significantly upregulated in gram positive-treated fragmorphs as compared to controls, while chtnfr was upregulated by both treatments. finally, to investigate the possible involvement of sponge chtnf in collagen biosynthesis, the expression levels of a fibrillar and of a non fibrillar collagen gene were evaluated after c. reniformis tissue explants treatment with specific tnf inhibitors and after gram positive bacterial infection. the obtained results indicate that the cytokine is involved in sponge collagen deposition and homeostasis as described in higher animals. allograft inflammatory factor-1 (aif-1) in the common sea urchin paracentrotus lividus: molecular and expression analysis p pagliara1, a barca1, j vizioli2, f drago2, t verri1 1disteba, university of salento, lecce, italy 2university lille, inserm, protéomique réponse inflammatoire spectrométrie de masse, prism, lille, france allograft inflammatory factor1 (aif1), alias ionized calcium-binding adapter molecule 1 (iba1), is a highly conserved ca2+-binding cytokine that has been identified as a key regulator of the immune response in vertebrates. aif1 is highly expressed in activated macrophages during inflammatory responses, thus representing an accurate indicator of macrophage activation in the body and a pathogenic factor in several inflammatory diseases. proteins of the aif1 superfamily are also present in invertebrates, from sponges to echinoderms. here, we describe the paracentrotus lividus aif1, which encodes a predicted protein of 151 amino acids with high similarity to vertebrate aif1. in the common sea urchin, molecular and immunocytochemical analyses showed the constitutive expression of aif-1 in the coelomocytes. aif-1 localizes in the perinuclear area of amoebocytes and inside the granules of red cells, but it is not present in vibratile cells and colorless spherula cells. moreover, significant increase of p. lividus aif-1 expression, at both mrna and protein level, are observed in coelomocytes after gram+ bacterial challenge. blast searches across echinoderm databases resulted in identification of orthologous proteins from 24 species (8 sea urchins, 1 brittle star, 12 starfishes and 3 sea cucumbers). among these, p. lividus aif-1 shared a high identity with several species, e.g., 85.4% with the sea urchin strongylocentrotus purpuratus, 60.9% with the brittle star ophiocoma echinata, 59.6% with the starfish achantaster planci, and 52.3% with the sea cucumber apostichopus japonicus. our study on p. lividus aif-1 will contribute to elucidate aif1 function along the evolutionary scale and to consolidate the key evolutionary position of echinoderms throughout metazoans with respect to the common immune paths. tlr2 expression in medicinal leech n baranzini1, r girardello1, v orlandi1, f acquati1, j vizioli2, m de eguileor1, a grimaldi1 1department of biotechnology and life science, university of insubria, varese, italy 2university lille, inserm, protéomique réponse inflammatoire spectrométrie de masse, prism, lille, france it is well known that toll-like receptors (tlrs) play a central role for innate immunity in both vertebrates and many invertebrates, by recognizing conserved pathogen-associated molecular patterns in order to trigger the innate immune response. in the case of gram-positive bacteria, tlr2 is the primary receptor to detect them and mainly senses lipoteichoic acid lta (a predominant surface glycolipid of gram+). in order to evaluate whether the recognition of gram+ bacteria involves tlr2 in leech as well, we performed immunofluorescent and western blot analysis on leech body wall lta injected. our morphological and histochemical results clearly indicate that lta induces immune cells migrating toward the stimulated area. the expression profile of tlr2 has been also evaluated by western blot analysis highlighting the presence of an immunoreactive product at about 100 kda, in according with that found in vertebrates. moreover, to characterize the migrating immune cells, we further performed immunofluorescent experiments 122 using antibodies against the aif-1 (allograft inflammatory factor-1) and rnaset2 (a member of the ribonuclease t2 family) factors. as we previously demonstrated, the expression of these two markers is enhanced after grambacterial lipopolysaccharides (lps) infection and is mainly located in activated macrophages. our results clearly show that after lta injection, numerous aif1+/rnaset2+ macrophages migrate towards the infected area and express tlr2. given these encouraging preliminary results, indicating that tlr2 may functions as lta receptor in leeches as well, our further research will be focused to better clarify tlr2 expression and signaling in the medicinal leech. tlr4 expression in medicinal leech r girardello1, n baranzini1, c rossetti1, m molteni1, j vizioli2, m de eguileor1, a grimaldi1 1department of biotechnology and life science, university of insubria, varese, italy 2university lille, inserm, protéomique réponse inflammatoire spectrométrie de masse, prism, lille, france given the enormous vastness and diversity of pathogens, one of the fascinating problems that immunology has to understand is how organisms detect the presence of infectious agents and destroy the invader without damaging the tissues of the organism itself. in this study, we take into consideration toll-like receptor 4 (tlr4), which plays a key role in the innate immune response, by using an invertebrate animal model, the medicinal leech (hirudo verbana). one of the advantages of using this model is found in its innate immune system that is very similar to that of vertebrate, but lacking the complex cross-talk typical of adaptive immunity. these features are promising to gain novel insight into the basic mechanisms of the innate immune response. here, we evaluate the expression of tlr4 and its co-receptor cd14, after injection in the leech body wall of rhmaif-1, an endogenous cytokine able to recruit macrophages, or lps, an exogenous molecule known to induce a strong inflammatory response. our results indicate that immune cells migrating toward the body wall of treated animals are macrophages (hmaif-1+) and granulocytes (cd11b+) and express both tlr4 and cd14. moreover, the expression profile of tlr4 has been also evaluated by western blot analysis highlighting the presence of immunoreactive products at about 130 and 110 kda. we hypothesize that, according to literature, the presence of different molecular masses of tlr4 probably depends on its glycosylation state. furthermore, functional studies both in vivo and in vitro were carried out by using cyp, a cyanobacterium selective tlr4 antagonist, which inhibited the secretion of the pro-inflammatory cytokines, such as tumor necrosis factor alpha. by stimulating with cyp and lps (grambacterial lipopolysaccharides) or lta (gram+ lipoteichoic acid), we found that only tlr4 pathway was blocked, while the immune response caused by lta treatment was not affected. our results are consistent with literature on vertebrates indicating that tlr4 functions as lps receptor while the recognition of gram+ bacterial products involves other pathways, such as tlr2. further research is needed to better clarify tlr4 expression and signalling in the medicinal leech. aif-1 and rnaset2 play different roles in the modulation of leech innate immune response n baranzini1, r girardello1, l monti1, f acquati1, j vizioli2, m de eguileor1, a grimaldi1 1department of biotechnology and life science, university of insubria, varese, italy 2university lille, inserm, protéomique réponse inflammatoire spectrométrie de masse, prism, lille, france in recent years, several studies have demonstrated that the aif-1 (allograft inflammatory factor-1) and rnaset2 (a member of the ribonuclease t2 family) factors are involved in the activation and modulation of inflammation processes, both in vertebrates and in invertebrates. in particular, it has been demonstrated that both aif-1 and rnaset2 have a chemotactic activity for macrophages and that their expression significantly increases after bacterial infections. however, the details on the mechanisms by which these evolutionarily conserved proteins regulate the innate immune response are still poorly defined. in order to better understand the specific role of these two factors, we report here the effect of grambacterial lipopolysaccharides (lps) injection on aif-1 and rnaset2 expression in an invertebrate model, the medicinal leech. this animal has been chosen for its very simple anatomy and for its notable similarities during inflammatory processes with those of vertebrates. western blot assays demonstrate that aif-1 and rnaset2 have a different temporal expression profile. in fact, while rnaset2 has two expression peaks after 30 min and 6 h from lps injection, aif-1 shows a peak after 30 min and its expression remains high after 24 h from stimulation. furthermore, double immunostaining coupling antiaif-1 or anti-rnaset2 to an antibody directed against the common granulocyte marker cd11b demonstrates that these two factors are expressed by two different types of cells, whose amount varies over time after lps stimulation. rnaset2 is mainly localized in the granulocytes, the first cells migrating towards the stimulated area, aif-1 is instead expressed by macrophages. taken together, our results clearly suggest that aif-1 and rnaset2 play a different role in the initial phase of the inflammatory response. we suggest that rnaset2 is at first released by granulocytes in order to induce a massive recruitment of macrophages. once chemoattracted to the stimulated area, activated macrophages highly express aif-1 to sustain the recruitment of further macrophages whose role is to rid the area of bacteria. 123 preliminary data on a toll-like receptor from the colonial ascidian botryllus schlosseri a peronato, l ballarin department of biology, university of padua, padua, italy toll-like receptors (tlrs) represent a wellknown family of conserved pattern recognition receptors the importance of which, in non-self recognition, was demonstrated in both vertebrates and invertebrates. tunicates represent the vertebrate sister group and, as invertebrates, they rely only on innate immunity for their defense. as regards tlrs, two transcripts have been described and characterized in the solitary species ciona intestinalis, referred to as citlr1 and citlr2. using the ciona tlr nucleotide sequences, we examined the genome and the available transcriptomes of botryllus schlosseri looking for similar sequences. we were able to identify a sequence, with similarity to citlr2 and, through in silico transduction and subsequent sequence analysis, we studied the domain content of the putative protein. the sequence, called bstlr, has a tir and a transmembrane domain, four llr and two lrr-ct domains. in addition, we analized bstlr expression in vivo and in vitro, under various experimental conditions and in different phases of the botryllus blastogenetic cycle. our data show that, in different phases, there is a change in gene expression and mrna location, according to the blastogenetic phase. session 2. chairmen: magda de eguileor, university of insubria, varese, italy and paola venier, university of padua, padua, italy invertebrate immunity (2) the case of ostreid herpesvirus 1 (oshv-1) and the antiviral host’s countermeasures u rosani1, m abbadi2, s domeneghetti1, g arcangeli2, a pallavicini3, p venier1 1department of biology, university of padua, padua, italy 2istituto zooprofilattico sperimentale delle venezie, legnaro and adria, padua, italy 3department of life sciences, university of trieste, trieste, italy prevention and mitigation of infective bivalve diseases is the central objective of the current european project vivaldi (2016-2020, research and development programme h2020) coordinated by the institut français de recherche pour l'exploitation de la mer (ifremer, i. arzul). here, we present an overview on a current problem and the results achieved so far with the support of european grants (bivalife, vivaldi). herpesviruses infecting marine molluscs have been associated to recurrent mortality outbreaks worldwide and, still today, cause serious production losses. described in france since 1991-1993, the acute and anomalous mortality occurring in hatchery larvae as well as in spat and juveniles of pacific oyster magallana gigas is usually detected at the increasing temperatures of spring-summer transition in association with the infection with a herpes-like virus. possibly triggered by multiple causes, the oyster mortality rates can reach 80100% in less than a week. hence, this is an issue somewhat different from the summer mortality of adult oysters more often interpreted as a physiological disorder related to overgrowth and gonad over-maturation. electronic transmission microscope (tem) observation of herpesvirus-like particles in gill and mantle tissues of muribund oysters and positive pcr-based diagnosis pointed to a viral etiology already in the early ‘90s in france. a herpesvirus purified from naturally infected m. gigas larvae originated the first genome sequence (a dsdna of about 207 kbp, genbank accession number ay509253) and permitted the classification of ostreid herpesvirus type 1 (oshv-1) within the malacoherpesviridae family from the herpesvirales order (davison et al., 2009). more virulent oshv-1, called microvariants because of a microdeletion and other nucleotide changes, emerged in 2008-2010 in france, europe and subsequently could be diagnosed in farmed m. gigas worldwide. in addition to the precise knowledge on diagnostic sequence regions, two microvariant oshv-1 genomes from french and irish oysters are now available and more ones will likely appear. molecular data resulting from natural and experimental oshv-1 infections and from the transcriptome analysis of oshv-1-infected host will be discussed as knowledge basis for prevention and mitigation strategies. molecular basis of melanization in bivalves s domeneghetti, u rosani, p venier department of biology, university of padua, padua, italy melanization is part of the ancestral defense systems of invertebrate phyla such as arthropods and mollusks and commonly participate to wound healing and encapsulation of invading microbes or parasites. melanin synthesis and deposition has been associated to pigmentation, exoskeleton production or biomineralization and it is relevant during larval development. the chemical reactions leading to melanization are based on the sequential activity of phenoloxidases (po such as tyrosinases, laccases or catecholases) and dopachrome converting enzymes (dce or dct). here, we review the candidate molecules possibly involved in the melanization process in bivalves. recent studies indicate a lineage-specific expansion of bivalve tyrosinase-like genes. their gene expression patterns, together with the neofunctionalization of some paralogs, support the dual role of tyrosinases in immune defense and shell biomineralization. tyrosinase enzymes might enable the biosynthesis of melanin starting from the precursors dihydroxyindole (dhi) or dihydroxyindole-2-carboxylic acid (dhica). however, the nature of the enzymes involved in the generation of these molecules from l-dopachrome is presently unknown in bivalves, due to the absence of both dct and dce-like sequences. 124 we report the expansion of d-dopachrome tautomerase genes in bivalves. moreover, their mantle-specificity and inducibility upon bacterial challenge, together with their enzymatic activity suggest an involvement in melanin biosynthesis. however, the activity of these enzymes to promote the conversion of l-dopachrome to dhi or dhica in the melanin pathway still remains to be established. survival and immunological competence of procambarus clarkii after x-ray irradiation c manfrin1, a giglio2, p fabbrizio1, a beorchia3, r vidimari3, pg giulianini1 1department of life sciences, university of trieste, trieste, italy 2department of biology, ecology and earth sciences, university of calabria, cosenza, italy 3azienda sanitaria universitaria integrata di trieste, s.c. fisica sanitaria, trieste, italy the louisiana's red swamp crayfish (procambarus clarkii), native to the southern united states and currently present all over the world, competes with native species and is a vector of crayfish plague caused by the water mold aphanomyces astaci. p. clarkii is also reported among the 100 worst invasive species (dasie) and among the 9 ones with the worst impact on more than 4 threatened species (genovesi et al., 2015). the sterile male release technique (smrt) represents one of the newest controlling methods for invasive crustaceans and in combination with intensive trapping has been proved to be particularly effective in the lake of casette (pordenone, friuli venezia giulia, italy) with a reduction of about 87% of p. clarkii population after two years of activity (aquiloni and zanetti, 2014). in a previous study, short-term effects of x-ray have been obtained and analysed after 20 days from the irradiation of p. clarkii males (giglio et al., 2018). the present study investigates the long-term effects of x-ray irradiation of a dose of 40 gy on 19 males after about 6 months from the exposure. immune competence has been evaluated by analyzing total hemocyte counts (thc), and activity of basal and total plasmatic prophenoloxidase. glycemia, as a generic stress index, and total plasmatic proteins were also measured. hemolymph withdrawals were performed at 5, 12, 28, 35, 65, 99, 132 and 193 days post treatment. testicular damages, at the end of experiment, were assessed by means of measurements of acini in semithin sections of fixed testis. the mean death days after treatment were 44.21 ± 4.15 for irradiated animals and 82.71 ± 19.47 for controls. the survival model with constant hazard estimates an average death age for irradiated animals of 113.14 days and for controls of 165.43. irradiated and control groups do not differ in survival rate (p= 0.33) and activity of plasmatic prophenoloxidase (p> 0.12). the thcs of irradiated animals are significantly lower than those of untreated animals 5, 12, 28, 65, and 99 days after treatment (p< 0.05). basal plasmatic po activities of irradiated animals are significantly lower at 132 and 196 days after irradiation. our findings document that despite a significant damage to the immune cell component, the humoral prophenoloxidase activity do not differ between irradiated and control animals. surprisingly, whilst after 1 month from irradiation an extensive gonadal damage was described (piazza et al., 2015), after 6 months in surviving crayfish no significant differences in acini diameters were recorded. our data confirm the validity of smrt for managing p. clarkii in confined basins with the indication of radiating large males a few days before the reproductive season considering life expectancy of about 80 days after treatment. furthermore, our data demonstrate that cytological damage in gonadal tissues could be repaired in 6 months after irradiation with a functional recovery. amyloid and immune responses in the colonial ascidian botryllus schlosseri l ballarin1, n franchi1, a peronato1, a grimaldi2, r girardello2, m de eguileor2 1department of biology, university of padua, padua, italy 2department of biotechnology and life science, university of insubria, varese, italy increasing evidences indicate that functional, non-toxic amyloid is widely distributed among living organisms. in invertebrates and vertebrates, functional amyloid is involved in inflammatory reactions and modulation of immune responses. in the present study, we investigated the occurrence of functional amyloid in tunicates, the closest living relatives of vertebrates. previous studies indicate that orthologous genes for the amyloid precursor protein (app), involved in amyloid synthesis in vertebrates, are present in solitary ascidians of the genus ciona. in addition, colonies of compound ascidians of the genus botryllus, respond to contacting genetically incompatible colonies with an allorecognition reaction that leads to the formation of necrotic, melanic spots along the contact border. our data provide evidence that functional amyloid is involved in immune responses of botryllus schlosseri and indicate that, both the circulating immunocyte types, i.e., the cytotoxic morula cells and phagocytes, can produce amyloid using two different proteins: bsp102 and bsapp. bsp102 forms the amyloid functioning as scaffold to store mc granular content and, once released upon mc degranulation, the support where phenoloxidase and melanin are deposited, thus limiting the diffusion of cytotoxicity. bsapp is released by phagocytes and contribute to the formation of extracellular nets that entrap microbes and prevent their diffusion within the organism. to the best of our knowledge, this is the first report of functional amyloidogenesis in protochordate immunity. purification of galactose binding lectin from the mucus of sabella spallanzanii (gmelin, 1791) and interaction of arsenic with bacterial agglutinating activity m dara1, g benenati1, mg parisi1, d piazzese1, l stabili2,3, m cammarata1 1department of heart and marine science, university of palermo, palermo, italy 125 2institute for coastal marine environment, u.o.s. of taranto, cnr, taranto, italy 3disteba, university of salento, lecce, italy lectins are present in almost all living organisms and are involved in several biological processes, including immune responses. in the present study, a galactose binding lectin (ssgbl) exhibiting an apparent mw of 43 kda has been characterized and purified from the mucus of the polichaete sabella spallanzanii by using both affinity chromatography and high-pressure liquid chromatographic methods. its agglutinating activity towards rabbit erythrocytes was significantly modified by the addition of calcium or edta. the activity was optimal at temperature values comprised between 4 °c and 37 °c, was partially retained after exposure at 50 °c, and was depleted at 90 °c. the ssgbl was able to agglutinate bacteria. the strongest activity was observed towards v. alginolyticus and e. coli, by contrast ssgbl at lesser extent agglutinated the gram positive micrococcus lysodeikticus, suggesting its possible involvement in host pathogen interactions. chemical analysis of animal tissues shows high concentrations of arsenic, in the branchial crown of animals (bio accumulation factor: 1,869) respect to sea water. we then investigated the arsenic effect and according to the branchial crown accumulation the mucus bacterial agglutinating activity results show that the presence of arsenic determines a modification but not complete inhibition also at the higher used concentration (baf ranging from 200 to 20,000), whereas methylmercury totally depletes the mucus agglutinant activity. mytilus galloprovincialis hemocytes activity as potential biomarker of marine pollution: in vitro effects of organic mercury mg parisi, j pirrera, d parrinello, m cammarata department of heart and marine science, university of palermo, palermo, italy hemocytes found in hemolymph, constitute a heterogeneous population of immune cells involved in defences against pathogens including non-self recognition, encapsulation and generation of cytotoxic molecules. these immunocompetent cells are also functionally and morphologically responsive to various xenobiotics present in the aquatic environment. toxic metals, such as mercury, contribute substantially to anthropogenic pollution in many coastal environments. animals living in those environments, particularly invertebrate filter feeders like bivalves, can be used as bioindicators. in an attempt to identify cellular markers for revealing pollution, this study examined in vitro the effects of different concentrations of methyl mercury on mytilus galloprovincialis hemocytes activities and morphology. as immunological parameter of cellmediated immune response, hemocytes mortality and morphology change, within the permissible physiological limit, were evaluated adding three concentrations of mehg in ms to the cultured cells. to evaluate the physiological status of the organisms, responses at the cellular and molecular biological organization were measured through in vivo cytochemical method. the neutral red uptake in lysosomes test was performed on withdrawn hemocytes and cytotoxic activity was evaluated toward various target by spectrophotometric measurement of the released hemoglobin and plaque-forming cell assay. in particular, hemocytes exposed to the xenobiotic presented a significantly decrease in the phagocytic activity toward yeast. the cytotoxic activity of m. galloprovincialis hemocytes toward erythrocytes and plaque lysis activity was not altered by suitable methylmercury concentrations in the medium. in both the responses cell-target contacts could be affected by methylmercury. the harvested hemocytes that were exposed to the metal had a significant mortality, cellular count and morphometric alterations. these findings provided evidence of mehg immunotoxic effects resulting in hemocyte death and morphological changes induced by cytoskeleton alterations. thus, a morphometric cellular parameter, such as spreading ability, was used as a complementary method and results confirm that mehg is toxic for m. galloprovincialis hemocytes, causing immunosuppression either by cell death or morphological changes. involvement of the multixenobiotic resistance (mxr) system in the physiological chemoresistance of haemocytes in marine mussels s franzellitti, p valbonesi, e fabbri department of biological, geological and environmental sciences (bigea) – ravenna unit, university of bologna, ravenna, italy in bivalves, the immune response is mainly supported by haemocyte cells found in the open hemolymphatic circulatory system. haemocytes are responsible for cell-mediated immunity through phagocytosis and different cytotoxic reactions, and are targets for the effects of several environmental pollutants. therefore, haemocytes are biological models to explore pollutant effects on bivalve immunity, and to infer the role of protective mechanisms triggered in these cells under immunotoxicological reactions. this study investigated functional and transcriptional modulation of the multixenobiotic resistance (mxr) system as a cytoprotective mechanism contributing to the physiological chemoresistance of haemocytes in the mussel mytilus galloprovincialis. the mxr system allows aquatic organisms to cope with their habitat despite high pollution levels. atp-binding cassette (abc) transport proteins are integral part of the mxr system, as they perform a crucial step in detoxification processes, i.e., the active efflux of metabolites and xenobiotics over the cell membrane, which results in lower intracellular concentrations and lower toxic potential. basal transport activity was assessed using the model substrate rhodamine 123 and specific 126 inhibitors for the mxr-related transporters pglycoprotein (abcb mrna) and multidrug resistance-related protein (abcc mrna). results showed that mxr activity in mussel haemocytes was mainly supported by the mrp-mediated efflux. in agreement, abcc was expressed at higher levels than abcb. activation of the cyclic-amp (camp) dependent protein kinase a (pka) resulted in increased rhodamine efflux, which was counteracted by the selective pka inhibitor h89. in vitro experiments treating haemocytes with physiological agonists (noradrenaline and serotonin) and pharmacological modulators (prop, forskolin, dbcamp, and h89) of the camp/pka system showed that serotonin (5-ht) acts as a physiological modulator of abcb transcription in haemocytes. although under these experimental conditions overall mxr activity was not affected, the environmental pharmaceuticals fluoxetine, propranolol, and carbamazepine, which interact in different ways with the adrenergic and serotoninergic pathways, acted as modulators and substrates of mxr-related transporters and affected cell viability, highlighting the potential of these pharmaceuticals to induce immunotoxicological effects as part of their adverse outcomes in marine mussels. in vitro effects of different ganoderma lucidum extracts on mytilus hemocytes c ciacci1, r saltarelli1, l potenza1, r cuppini1, l canesi2 1department of biomolecular sciences, university of urbino carlo bo, urbino, italy 2department of earth, environment and life sciences (distav), university of genoa, genoa, italy aquaculture is one of the fastest growing foodproducing sectors worldwide. however, during intensive farming practices, a major problem is represented by development of various infectious diseases. in this regard, the use of medicinal plant and mushroom extracts that can act as immunomodulators has been proposed to control to prevent and control fish and shellfish bacterial, viral, and parasitic infections. many species from the genus ganoderma p. karst (basidiomycotina, polyporales, ganodermataceae) include a group of fungi that have been widely used in traditional medicine in asian countries. in particular, the most studied species is g. lucidum, that has beneficial health properties due to a wide variety of bioactive components, such as polysaccharides, phenolic compounds, triterpenes, sterols, lectins and some proteins, with different biological activities. in this work, preliminary data are presented on the possible effects of ethanolic and aqueous extracts of g. lucidum on the hemocytes of the marine bivalve mytilus, an important aquacultured species worldwide. the aqueous extract, containing polysaccharides, induced increases in lysosomal membrane stability, phagocytic activity and oxidative burst, indicating immunostimulatory effects. dose-dependent effects were observed in the µg/ml range. in contrast, the ethanolic extract, containing polyphenolic compounds, induced lysosomal membrane destabilization, inhibition of phagocytosis, and oxidative stress. the results represent the first data on the effects of medicinal mushroom extracts on immune parameters of aquacultured bivalve species. studies are in progress to evaluate the immunostimulatory effects of ganoderma extracts and fractions in mussels. session 3. chairmen: luigi abelli university of ferrara, ferrara, italy and maria rosaria coscia, institute of protein biochemistry, cnr, naples, italy fish immunity fish cytokines: molecular characterization and biological activity of il-2 and il-2l in sea bass (dicentrarchus labrax l.). f buonocore1, v stocchi1, e randelli1, m gerdol2, a pallavicini2, cj secombes3, t wang3, g scapigliati1 1department for innovation in biological, agro-food and forest systems (dibaf), university of tuscia, viterbo, italy 2department of life sciences, university of trieste, trieste, italy 3scottish fish immunology research centre, school of biological sciences, university of aberdeen, aberdeen, uk interleukin-2 (il-2) is a fundamental immunomodulatory cytokine, secreted mainly by thelper 1 cells, involved primarily in the proliferation, activation and differentiation of t cells. in teleost fish, the first evidence for the existence of an il-2 gene was reported about ten years ago and only in rainbow trout (oncorhynchus mykiss) the il-2 bioactivity has been studied quite in detail. in our work, we have found in a sea bass (dicentrarchus labrax l.) gills transcriptome two sequences related to fish il-2, a proper il-2 gene and a il-2l (il-2 like) transcript. these two transcripts that have been mapped on the available sea bass genome and the sequences have been confirmed by cloning from a sea bass gills cdna. basal expression analysis by real-time pcr revealed that il-2 was mainly expressed in gut, with lower expression in brain and spleen, whereas il-2l in gut. moreover, we investigated the expression of the il2 and il-2l in sea bass head kidney leukocytes after in vitro stimulation with the t cell mitogen agent pha and after in vivo infections with nodavirus and vibrio anguillarum. finally, we produced sea bass il2 and il-2l as recombinant proteins in e. coli and we tested their action in vitro on the modulation of different immune-related genes expression after stimulation of leukocytes from head kidney. this is a first preliminary analysis of the biological activity of the il-2 cytokine in sea bass and it will highly contribute to a broader understanding of the evolution of t-cell immunity in this species. 127 first evidence of t cell restricted intracellular antigen (tia) protein gene expression in antarctic fish e nicorelli1, m gerdol2, f buonocore3, a pallavicini2, g scapigliati3, l guidolin1, p irato1, f corrà1, g santovito1 1department of biology, university of padua, padua, italy 2department of life sciences biology, university of trieste, trieste, italy 3department for innovation in biological, agro-food and forest systems, university of tuscia, viterbo, italy stress granule (sg) formation is a primary mechanism through which gene expression is rapidly modulated when the eukaryotic cell undergoes cellular stresses (including heat, oxidative, viral infection, starvation). the importance of sgs is seen in several disease states in which sg function is disrupted. fundamental to sg formation are the t cell restricted intracellular antigen (tia) proteins (tia-1 and tia-1 related protein (tiar)), that both directly bind to target rna and self-associate to seed the formation of sgs. the only tia proteins of antarctic fish so far present in the ncbi database (three variants of tia-1 and one tiar) were those of the black rockcod notothenia coriiceps. with the aim to increase the knowledge on these proteins in antarctic teleosts, we have characterized the genes codifying for tia-1 protein variant 2 (tia-1b) in the emerald rockcod trematomus bernacchii and in the crocodile icefish chionodraco hamatus. in the transcriptome of t. bernacchii we have verified the presence of tia-1b. for c. hamatus, a partial cdna sequence of this gene was obtained by rt-pcr technique, with primers designed after cross analyses between ncbi and t. bernacchii transcriptome databases. multi-alignment analysis, performed with fish orthologous sequences, demonstrated high conservation of amino acids. the gene transcription of tia-1 from various tissues (gills, heart, liver, spleen, and skeletal muscle) of both t. bernacchii and c. hamatus has been measured by quantitative real time pcr. the tissue-specific differences in the tia-1 mrna accumulation are probably related to the physiological function characteristic of these organs (supported by p.n.r.a. and m.i.u.r. grants). evolutionary analysis of immunoglobulin t genes from antarctic fish a ametrano, m vitale, mr coscia institute of protein biochemistry, cnr, naples, italy in 2015, our group isolated igt heavy chain constant region gene from the antarctic teleost trematomus bernacchii (family notothenidae), disclosing a unique feature: the loss of most heavy chain second constant domain (ch2). t. bernacchii belongs to the perciform suborder notothenioidei which represents the main component of antarctic fish fauna. this teleost group has acquired peculiar features to adapt to the extremely cold antarctic environment, providing an extraordinary model system for investigating the relationship between evolutionary genomic changes and environmental adaptation. notothenioidei comprise five antarctic families, channichthydae, bathydraconidae, artedidraconidae, nototheniidae, and harpagiferidae, and two non-antarctic families, eleginopidae and bovichtidae. in an attempt to reconstruct the loss of ch2 through phylogeny of notothenioids we isolated and characterized igt genes from two more species belonging to family nototheniidae, one representative of two other antarctic fish families, (family bathydraconidae and family artedidraconidae). moreover, one representative each of the two non-antarctic families was included in our studies for comparison: eleginops maclovinus (family eleginopidae), and bovichtus diacanthus, (family bovichtidae). the former was proposed as the closest sister group to all the rest of notothenioids, the latter was chosen for comparison since it represents the phyletically basal lineage of notothenioid species that inhabited non-antarctic more temperate waters before antarctica became isolated from other continents. genomic studies have been carried out in an attempt to define some events, such as insertion of transposable elements, alternative splicing or other molecular mechanisms, involved in leading to the lack of the most ch2 domain. a comparative analysis at genomic level has highlighted that the presence of a remnant ch2 domain is shared to all antarctic fish families analyzed in the present work. it is worth of note that the loss of most ch2 is shared also by e. maclovinus. this result may be viewed as a preadaptation sign accompanying the lineage radiation. session 4. chairmen: pierangelo luporini, university of camerino, camerino, italy and piero giulianini, university of trieste, trieste, italy recognition and immunomodulation insights into the molecular basis of self/non-self recognition in the ciliate euplotes from the determination of pheromone crystal structures b pedrini1, a finke1, m marsh1, a vallesi2, c alimenti2, p luporini2 1paul scherrer institute, villigen, switzerland 2university of camerino, camerino, italy species-specific families of protein pheromones, each encoded by one of a series of co-dominant alleles at the same genetic locus (the mat locus), are constitutively secreted by species of euplotes and used as autocrine (autologous, or self) signals that promote the cell vegetative growth and paracrine (heterologous, or non-self) inducers of cell-cell unions in mating pairs. in e. raikovi, the structures of a number of pheromones (designated er-1, er-2, er-3 and so forth) have been determined by solution nmr spectroscopy, and the structure of pheromone er-1 has additionally been solved also by x-ray crystallography. in each cell type, the soluble pheromone finds a structural counterpart 128 with the extracellular ligand binding domain of its membrane receptor, both the molecules being encoded by the same gene through a mechanism of intron-splicing. given this genetic context, the protein-protein interactions in the crystals can be assumed to mimic those underlying the pheromone/receptor interactions on the cell surface. we used non-conventional ab initio crystal structure determination methods, that exploit the highresolution of collected diffraction data, to compare the crystal structures of two pheromones, er-1 (structure re-determined at a resolution of 0.7 å) and er-13 (structure de novo determined at a resolution of 1.4 å), that are secreted by two strongly mating compatible cell types. in spite of sharing the same disulphide bond pattern and the same up-down-up three-helix fold, er-1 and er-13 differ markedly in the arrangement of the molecules in the crystals. the resulting intermolecular contacts assign er-1 and er-13 to distinct crystallographic space groups (c2 and p41, respectively), suggesting that the autocrine pheromone/receptor interactions on the cell surface are likely regulated by the capability of each pheromone to homooligomerize with an its own specific pattern. in addition, although adopting distinct crystal structures, er-1 and er-13 make a common use of their helix 3 to stabilize the inter-molecular crystal contacts, which implies that this helix likely plays a central role in allowing cells to establish paracrine pheromone/receptor interactions. silica-induced fibrosis: from a physiological response in the early metazoans to the wellknown pathological outcomes of higher vertebrates s scarfì, m pozzolini, l gallus, m giovine department of earth, environment and life sciences (distav), university of genoa, genoa, italy exposure to fine crystalline silica particles (quartz) causes silicosis, an occupational disease leading to an overproduction of collagen in the lung. on the other hand, in the invertebrate world, the marine sponge chondrosia reniformis is able to incorporate and partially dissolve crystalline silica grains without toxic effects by means of high concentrations of ascorbic acid (aa) able to erode the crystalline silica surface. in fact, quartz erosion in c. reniformis seems to stimulate a physiological process of collagen deposition probably due to the necessity to strengthen the body structure since the animal does not produce spicules by itself. this primitive process is specific for the crystalline silica, the amorphous one, even if equally incorporated by the sponge, remain untouched. this could be the cause of the subsequent abnormal, pathological response to quartz in higher metazoa, namely mammals, which retain the ability to interact and engulf the quartz particles through macrophage activation but have lost the ability to dissolve them. this unresolved process leads to a chronic inflammation which finally causes the development of silicosis. our studies point out as, in mammalian macrophages, aa-pretreated quartz, mimicking the sponge erosion process on the crystalline particles, acquires a high cytotoxic as well as proinflammatory potential leading to a significant increase of inflammatory mediators such as tnf, cox2 and pge2 production as well as ros and lipid peroxide generation. these effects are due to the abundance of oxygen radicals generated on the crystalline silica particle surface after ascorbic acid interaction. also, primary human fibroblasts show an abnormal response to aa-pretreated quartz particles. in fact, in fibroblasts, the abundance of surface radicals on quartz crystals significantly enhances cell proliferation, ros production, nf-kb nuclear translocation, smooth muscle actin, fibronectin, bcl-2 expression and collagen production. these in vitro results may be relevant to the comprehension of the in vivo onset of silicainduced pathologies in the lung since ascorbic acid, as in c. reniformis marine sponge, is present in significant amounts in the lung epithelium surfactant and could indeed participate to the chronicization of quartz toxicity in this organ renewing the particle surface radicals ad libitum and thus perpetrating the inflammatory response which ultimately leads to the development of the silicotic disease. immunoregulatory and pro-tumoral effect of circulating dna t altosole1, f ferrera1, s tardito1, c bernardi1, l arpesella1, c marini1, a parodi1, f kalli1, g nasi1, d fenoglio1,2,3, g filaci1,2,3 1centre of excellence for biomedical research, university of genoa, genoa, italy 2department of internal medicine, university of genoa, genoa, italy 3irccs azienda ospedaliero universitaria san martino ist istituto nazionale per la ricerca sul cancro, genoa, italy circulating dna (cdna) is detectable at low concentration in the plasma of healthy subjects and at high concentrations in the peripheral blood of pregnant women and patients affected by tumors and vasculitis. dna is considered a danger signal mediating pro-inflammatory reactions after binding to dna sensors. however, this view conflicts with recent data on dna-sensors function and with the presence of elevated cdna concentrations in tumor patients, where processes related to immune tolerance generally prevail, leading to variable degrees of immunodeficiency. hence, the biologic role of cdna is still controversial. previous data from our group suggested that dna may exert in vitro immunoregulatory functions interacting with hla class ii molecules. here, we show that exogenously administered cdna mediates immunoregulatory functions in vivo. in particular, it protects lupus-prone mice from disease progression and favors tumor growth in tumor-challenged mice. interestingly, cdna was found associated only to cells expressing mhc class ii molecules and its binding to mouse mhc class ii molecules was demonstrated. some of the mechanisms underlying cdna immunoregulatory functions were elucidated and consisted in regulatory t cell (treg) expansion, 129 increased monocyte production of ccl22 (a chemotactic factor for treg) and inhibition of antigen-specific t cell proliferation. hence, this study unveils unprecedented biologic functions of cdna that may have pathogenic relevance in cancer. impact of marine contaminants of emerging concern on the cetacean transcriptome a mancia1, d lunardi1, c panti2, mc fossi2, l abelli1 1department of life sciences and biotechnology, university of ferrara, ferrara, italy 2department of physical sciences, earth and environment, university of siena, siena, italy contaminants of emerging concern (cecs) are widely distributed in the environment, but their occurrence and potential toxicity are only now being evaluated. cecs are increasingly being detected in the waters and many act as endocrine disruptor compounds (edcs), causing a variety of effects on health. the worldwide distributed perfluorooctanoic acid (pfoa) and bisphenol a (bpa) are cecs falling in the edcs category. skin samples from the bottlenose dolphin (tursiops truncatus), a top predator that spends its entire life in the water and therefore subject to accumulation and magnification of contaminants, were collected to analyze the impact that environmentally relevant concentrations of cecs may have on global gene expression. we combined transcriptomic analysis and ex vivo assays using small skin slices cultured and treated for 24 h with pfoa or bpa or vehicle. rna from dolphin biopsies was labeled and hybridized to a species-specific oligomicroarray. the skin transcriptome displayed changes related to contaminant exposure, potentially predictive about long-term effects on health, being the genes affected involved in immune modulation, response to stress, lipid homeostasis, and development. within the genes differentially expressed in the transcriptome after cecs treatment, 4 were tested as potential gene markers of anthropogenic contaminants exposure on skin samples from wild cetaceans. rna from 12 individuals, including the species stenella coeruleoalba, t. truncatus, and grampus griseus were sampled in 3 areas (adriatic, ionian and tyrrhenian seas). three out of the 4 genes tested showed higher expression in the samples collected from the adriatic sea. the transcriptomic signature of a dolphin skin could be relevant as classifier for a specific contaminant whilst giving information of the specific geographic location where the marine mammal spent its life, due to the different impact on gene expression exerted by different contamination levels. amphibian peptides for skin protection and healing i demori1, s salvidio1, d burkart2, l queirolo1, l rovegno1, a catenazzi2,3, l canesi1, e grasselli1 1department of earth, environment and life sciences (distav), university of genoa, genoa, italy 2department of zoology, southern illinois university, carbondale, il, usa 3department of biological sciences, florida international university, miami, fl, usa background: amphibians are currently suffering a dramatic decline worldwide, mainly due to chytridiomycosis, a skin infection caused by the pathogenic fungus batrachochytrium dendrobatidis (bd). an important natural defense of amphibian skin is the production of antimicrobial peptides (amps) by granular glands in the dermis. amps collected from several species of frogs successfully inhibit the growth of bd in vitro. besides their antimicrobial and anti-fungal activities, amps have been shown to exert other biological effects such as antiviral, anti-tumor, anti-oxidant, immunomodulating and wound healing. aim: we intended to test the efficacy of amps as cutaneous defenses in frog species either resistant or susceptible to bd. methods: 3 frog species, gastrotheca nebulanastes (gn), g. excubitor (ge) and hypsiboas gladiator (hg), were collected in montane scrub, cloud forest and high elevation grassland habitats near manu national park in southeastern peru. amp secretion was stimulated by injection of norepinephrine into the dorsal lymph sacks. amps were then purified by chromatographic techniques. the human endothelial cell line hecv was treated with amp concentrations ranging from 0.005 to 50 µg/ml. cell viability was verified by mtt test. wound healing properties were analyzed by scratch wound assay. amp inhibition strength against bd growth was measured in vitro by incubating bd zoospores with different concentrations of amps. results: treatment with amps secreted from gn, ge and hg did not affect hecv cell viability at any concentration tested. no significant differences in cell migration rate were observed in hecv cells scratched and treated with gn and ge amps. only hg peptides showed wound healing properties as well as strong bd growth inhibiting ability. conclusions: stimulation of wound healing mechanisms and inhibition of bd growth by skin amps might both contribute to hg resistance to chytridiomycosis. understanding the role of skin defenses may lead to the development of novel bd mitigation strategies. possible applications of amphibian amps in skin medicine deserve attention and further studies. characterization of cd3+ t lymphocytes of sea bass dicentrarchus labrax l. s picchietti, f buonocore, l guerra, e randelli, am fausto, g scapigliati department for innovation in biological, agro-food and forest systems, university of tuscia, viterbo, italy cd3 is an important cell surface marker of t 130 lymphocytes and it is essential for t cells activation in higher vertebrates. here, we report expression analysis and quantitative distribution of the cd3ɛ+ t lymphocytes in sea bass dicentrarchus labrax. from a whole sea bass gills transcriptome, we identified and successively cloned a complete cdna sequence of cd3ɛ. the basal expression of sea bass cd3ɛ chain in different organs has been analysed by real-time pcr. in vitro stimulation with the t-cell mitogen pha, resulted in a significant increase of the cd3ɛ expression level compared to control cultures. the cd3ɛ transmembrane and cytoplasmic tail region has been identified and used to select three peptides for the immunisation of rabbits in order to obtain antisera against cd3ɛ (ra cd3ε1). flow cytometry, ihc and iif were conducted to address the normal distribution and number of cd3ε+ lymphocyte population in the lymphoid organs (thymus, head kidney and spleen) and mucosal tissues (intestine and gill), with relatively high percentages of these cells identified in thymus, head kidney and spleen, while lower in gills and intestine. at the microscope, the iifpositive cells had the morphology of lymphocytes and the presence of uniquely stained cd3ɛ+ igm subset cells fits the expected profile of t-cells. the increase of cd3ε expression level found in head kidney leukocytes in response to vibrio anguillarum vaccination suggested that cd3ɛ+ t lymphocytes might play important roles in the protection against bacterial infections, as in mammals. seasonal changes in immune parameters in mytilus galloprovincialis farmed at different sites of the gulf of la spezia, ligurian sea, and their relationship with other biomarkers of the health status t balbi1, m auguste1, r fabbri1, m montagna1, l serracca2, l canesi1 1department of earth, environment and life sciences (distav), university of genoa, genoa, italy 2izs istituto zooprofilattico sperimentale piemonte liguria e valle d'aosta, la spezia, italy marine mussels (mytilus spp.) are worldwide utilized in marine biomonitoring by a multi-biomarker approach. however, for a correct interpretation of different biomarker responses, information is needed on their natural seasonal variability due to environmental stressors/physiological factors. this particularly applies to immune-related biomarkers, that are not currently measured in biomonitoring programs. the gulf of la spezia (ligurian sea, italy) is an intensive rearing area for mytilus galloprovincialis. seasonal variability of 7 different biomarkers, from subcellular to whole organism level, including immune biomarkers, has been recently investigated over 1 year (2015-2016) on a monthly basis in mussels grown at 3 different sites of the aquaculture plant of la spezia in comparison with a reference site. the data represent the first baseline information on the health status of mussels in this farming area (balbi et al., 2017). in this work, we report data collected within a project of the italian ministry of health, carried out in collaboration with the izs of la spezia*. mussels were sampled every 3 months (from nov 2016 to jul 2017) from the same 4 sites. a set of immune, oxidative stress and inflammation related parameters were evaluated: lysosomal membrane stability-lms and phagocytic activity of circulating hemocytes, nitric oxide production in the gills as a marker of tissue inflammation, antioxidant enzyme activities (gst, catalase) in both gills and digestive gland. the results were related to determination of mussel survival in air (stress on stress-sos response) as a biomarker of general health condition at the whole organism level. the results confirm the seasonal trend previously observed in different biomarkers in mussels sampled at different sites. high immunocompetence was generally observed throughout the year. differences observed among sites in inflammatory and oxidative stress parameters were mainly due to differences in the gametogenic cycle. the results underline the importance of lms and sos as core descriptors of the mussel health status in relation to seasonal variations in temperature and reproduction. isj006.pdf 72 isj 1: 72-81, 2004 issn 1824-307x review molecular genetics of oogenesis in drosophila melanogaster s gigliotti1*, v cavaliere2, g gargiulo2, f graziani1, c malva1 1institute of genetics and biophysics "adriano buzzati traverso", cnr, napoli, italy 2dipartimento di biologia evoluzionistica sperimentale, universita' di bologna, italy accepted december 09, 2004 abstract gonadal development requires complex differentiation programs leading to the production of functional female and male gametes. upon fertilization, while the male germ cell contributes to the newly formed zygote only its genetic material, the female germ cell also supplies its cytoplasmic components, including fundamental molecular cues on which early embryonic development will rely. unravelling the mechanisms employed by animal species for building up their eggs is therefore a challenging task in developmental biology. as demonstrated by the impressive body of data produced in recent years, drosophila melanogaster is a useful model system for attempting a step by step dissection of the whole oogenesis process. remarkable opportunities for comparative analyses are in turn expected to be provided by these studies, since it is becoming evident that conserved themes underlie oogenesis in all animal species. in this review, we focus on few key differentiation events occurring during egg chamber development in drosophila, outlining our interest in the mechanisms leading to egg polarity establishment, transfer of information between nuclear and cytoplasmic cell compartments and germ cell apoptosis. key words: oogenesis; drosophila; egg polarity; nuclear pore; apoptosis. introduction the production of functional gametes is essential for the propagation of all sexually reproducing metazoan species. the central biological importance of this process has for centuries stimulated a remarkable scientific interest towards the mechanisms underlying female germline establishment and development. an unanticipated aspect of these studies is the emerging evidence that evolutionarily distant animals make eggs using common strategies (for review, see matova and cooley, 2001). in higher insects, oogenesis starts with the formation of a group of interconnected cells known as germline cist, originated from a single germline progenitor cell that undergoes synchronous divisions followed by incomplete cytokinesis (buning, 1994). germ cell clusters have been more recently described *corresponding author: dr. silvia gigliotti istituto di genetica e biofisica, consiglio nazionale delle ricerche, area della ricerca napoli 1, via pietro castellino 111, 80131 napoli, italia e-mail: gigliott@igb.cnr.it also in primitive insect and vertebrate females, suggesting that early steps of germ line development involve a cyst stage also in these animals (for review, see pepling et al., 1999). a second conserved feature in oogenesis is programmed cell death. this process initiates in the germ cells when they are interconnected and requires the activation of a conserved intracellular program, according to a predictable temporal and spatial pattern. the assembly of ovarian follicles, where gonadal somatic cells envelope female germ cells to create a special compartment for their growth and differentiation, is a third characteristic shared by a number of species throughout the animal kingdom. finally, in many invertebrates and vertebrates, the establishment of one or more body axes is already set up during oogenesis (gerhart and kirschner, 1997) and is based on common principles, such as cortical localization of key mrnas and proteins (palacios and st johnston, 2001; pellettieri and seydoux, 2002; vinot et al., 2004). in this frame, the study of oogenesis in model organisms that have the entire genome sequenced and annotated can be viewed as a promising tool for identifying conserved players of basic developmental programs leading to germ cell differentiation and growth. a particularly useful 73 paradigm for understanding these processes is provided by the drosophila egg chamber. this structure is composed by one oocyte and 15 nurse cells surrounded by a monolayer of somatic follicle cells and represents the fundamental morphological and physiological unit where oogenesis takes place (king, 1970). egg chambers are organized in linear arrays termed ovarioles. at the tip of each ovariole, in a specialized region called germarium, a germline stem cell divides asimmetrically to produce one cell that maintains the characteristics of stem cell and a cystoblast. this cell undergoes four rounds of synchronous mitotic divisions with incomplete cytokinesis, giving rise to a cluster of 16 cells interconnected by intercellular bridges called ring canals. only one cell within a cyst adopts the oocyte fate, while the remaining 15 develop into nurse cells, which provide the synthetically quiescent oocyte with rnas and proteins. after encapsulation of the germline cyst by follicle cell precursors originating from the asymmetric division of somatic stem cells, egg chamber assembly is complete (margolis and spradling, 1995; spradling et al., 1997). this new individualized structure buds off the germarium and undergoes maturation while moving toward the posterior of the ovariole. in this review we will focus on few key aspects of drosophila oogenesis, summarizing our studies on the differentiation events leading to: 1) definition of the egg chamber anterior-posterior polarity; 2) integration of nuclear and cytoplasmic developmental processes during egg chamber maturation; 3) activation of apoptosis in germline cells. for a better understanding of each single topic, a brief outline including the most relevant and recent contributions given by the scientific community, is reported below together with our results. establishment of the egg chamber anteriorposterior polarity: the hold hup mutation the anterior-posterior polarity of the drosophila oocyte is established by a multi-step process involving a complex sequence of interactions between somatic and germline cells. an essential early event taking place in the germarium is oocyte positioning at the posterior pole of the germline cyst, mediated by its association with posterior follicle cells (gonzales reyes and st johnston, 1994). this association involves e-cadherin-based cell adhesion mechanisms and relies on a cascade of inductive signals that are transmitted from the older posterior egg chamber (godt and tepass, 1998; gonzales-reyes and st johnston, 1998a; torres at al., 2003). these signals are believed to trigger only a temporary polarization of the follicle epithelium, required to induce e-cadherin upregulation in posteriorly located follicle cells. once anchored at the posterior end of the germline cyst, the oocyte will become the source of a new inductive signal, driving the adjacent terminal follicle cells to adopt a posterior fate (gonzales-reyes et al., 1995; roth et al., 1995). the two central players of this cell signaling event are the tgf-α like protein gurken (grk) produced by the oocyte and the egf receptor (egfr) homologue torpedo (top), that is expressed throughout the follicular epithelium, but is selectively activated only in the follicle cells that contact the oocyte and are therefore exposed to the grk signal (fig. 1a). at the time when this signal is produced, the follicle cells located at both termini of the egg chamber appear to be equivalent (gonzales-reyes and st johnston, 1998b). two pairs of so-called polar cells, one at each end of the egg chamber, have been induced to differentiate through delta signaling from the germline cyst and have in turn acquired a distinctive organizer function. by generating a gradient of activation of the janus kinase (jak) pathway in adjacent follicle cells, polar cells are in fact able to specify their fate (grammont and irvine, 2001; lopezschier and st johnston, 2001; grammont and irvine, 2002; xi et al., 2003). therefore, as soon as it is triggered by grk at the posterior pole of the egg chamber, egfr signaling defines posterior terminal follicle cell identity overriding the default anterior fates specified by jak activity alone (xi et al., 2003). oocyte to follicle cell signaling is then followed by an unidentified back signal that induces an overall reorganization of the oocyte microtubule cytoskeleton. this eventually allows proper localization of the two key determinants of embryonic anterior-posterior axis formation: bicoid (bcd) and oskar (osk) mrnas (theurkauf et al. 1992; pokrywka and stephenson, 1995) (fig. 1b, c). grk mrna is in turn relocated, first, transiently, to the anterior margin of the oocyte and finally to the anterior corner where the nucleus has in the meantime migrated (mac dougall et al., 2003) (fig.1a). this process is tightly regulated by several factors and is coupled to translational control, ensuring that, when a second round of gurken signaling is elicited, it will interest only the group of follicle cells that are next to the oocyte nucleus. these cells will acquire a dorsal identity, defining the egg chamber dorsal-ventral axis (neuman-silberberg and schupbach, 1993). even if significant progresses have been made in the last years, a complete picture of the mechanisms defining the anterior-posterior polarity of the drosophila egg is still missing. since the whole process requires a tight integration between differentiation events taking place in somatic and germline cells, the identification of mutants displaying phenotypic alterations in both cell types might be useful in the search for gene functions involved in egg chamber polarization. on the basis of these considerations we have focused our studies on the hold up (hup) mutation (sandler, 1977). a detailed phenotypic characterization carried out in our laboratory has in fact shown that this female sterile mutation causes several characteristic defects in oocyte and follicle cell behaviour and has a striking impact on egg chamber anterior-posterior axis formation (rotoli et al., 1998). the earliest morphologically visible alteration detectable in hup mutant egg chambers is oocyte mislocalization. in 15% of the germline cysts produced by females bearing the hup mutation in trans with a non-complementing chromosomal rearrangement, the oocyte was in fact randomly positioned. in fig. 2a, where a hup mutant ovariole is shown, one of the egg chambers displays an anteriorly localized oocyte and is direcly joined to the following, older egg chamber. the absence of an intervening stalk suggests that the primary defect responsible for oocyte mispositioning in hup mutant 74 fig. 1 anterior-posterior and dorsal-ventral polarization of the drosophila oocyte. the localization of key molecules involved in axes formation was visualized in wild type egg chambers by in situ hybridization. (a) triphasic grk mrna localization: at the posterior end of the oocyte in early egg chambers, at the anterior margin of the oocyte at stage 8 (thin arrow) and at the anterior-dorsal corner of the oocyte at stage 9. (b) bcd mrna localization at the anterior margin of the oocyte (arrow). (c) osk mrna localization at the posterior pole of the oocyte (arrow). egg chambers might reside in the chain of events leading to somatic cell fate specification in the germarium. as mentioned above, oocyte positioning at the posterior pole of a newly formed egg chamber involves in fact a series of inductive signals that originate in the adjacent more mature follicle and eventually lead to the formation of a separating stalk. hup gene function is required also for later somatic cell differentiation processes taking place during egg chamber development. even when supporting correct oocyte positioning, nearly half of the mutant egg chambers were in fact unable to fully specify posterior follicle cell fates and displayed ectopic expression of anterior follicle cell markers in posteriorly located follicle cells. this striking defect had profound consequences on oocyte polarization and resulted in the abnormal distribution of several localized mrnas, as shown in fig. 2b, where bicoid (bcd) mrna appears to be partially mislocalized to the posterior of the oocyte (rotoli et al., 1998). these results indicated that appropriate cross-talk mechanisms between oocyte and posterior follicle cells are not established in hup mutant egg chambers. in fig. 2 defective anterior-posterior axis formation in hup mutant egg chambers. in situ hybridization was used to visualize the localization of selected mrna molecules in the oocyte. in (a) the position of the oocyte is marked by the accumulation of osk mrna during previtellogenic stages of oogenesis. one of the egg chambers shows oocyte mislocalization to the anterior tip of the germline cyst (arrow). in (b) bcd mrna is ectopically found also at the posterior pole of the oocyte in a stage 10 egg chamber (arrow). agreement with this hypothesis, we found that hup genetically interacts with top. interestingly, in 10% of double homozygous mutant egg chambers follicle cells formed multiple posterior layers and were in some cases engaged in a centripetal migration process, displaying a behaviour characteristic of anterior follicle cells. the finding that hup cooperates with top for the correct specification of posterior follicle cell identity suggested that it might be a crucial component of the molecular machinery that leads to the establishment of polarity in both the follicle cell layer and the oocyte. in this frame, two lines of evidence indicated that the primary defect due to the hup mutation does not reside in the oocyte. first of all, grk mrna always accumulated at the posterior pole of the oocyte in hup mutant egg chambers where this cell was correctly localized. second, mitotic recombination experiments demonstrated that hup mutant germline clones did not exhibit the hup mutant phenotype. up to now the molecular nature of the hup gene is not identified because only ems-induced hup mutant 75 alleles are available and several screenings performed in our laboratory to isolate p element-induced hup mutations failed. to identify the hup gene we are focusing on the genes that have been annotated in the drosophila genome sequence in the region where hup was genetically mapped. p element-induced transformation experiments with overlapping fragments containing these genes are currently in progress in the attempt to rescue the hup mutant phenotype. integration of nuclear and cytoplasmic differentiation events during egg chamber maturation: the tulipano mutation and the nup154 nucleoporin gene in the drosophila ovary, germline derived nurse cells differentiate while undergoing 10-12 cycles of endoreplication. this process is characterized by morphologically visible changes in both chromosome architecture and chromatin configuration (dej and spradling, 1999). during the first four endocycles, homologous chromosomes are paired and progressively condense acquiring the banding pattern characteristic of polytene chromosomes. during the fifth endocycle homolog pairing loosens but chromatin compaction increases. five chromatin masses corresponding to individual chromosome arms transiently appear as a sort of “blobs” inside nurse cell nuclei. at the end of this endocycle, each polytene chromosome dissociates into 32 chromatid pairs. their chromatin becomes uniformly distributed throughout the nucleus, acquiring a decondensed structure that is maintained also during the following endocycles, when each chromatide pair is used as template to generate new polytene chromosomes (fig. 3a). the mechanisms responsible for the transition from polytene to dispersed chromosomes in the nurse cells are poorly understood. it has been proposed that this fig. 3 abnormal nurse cell chromosome dispersal in tlp mutant egg chambers. the egg chambers were stained with dapi to visualize the dna. (a) wild type ovariole, showing homogeneous dna distribution in the nurse cells of post-stage 5 egg chambers. (b) tlp mutant ovariole displaying persistent “blob like” nurse cell chromosome conformation throughout oogenesis. process takes place during a mitosis-like phase and is therefore carried out through the control of crucial cell cycle regulators (dej and spradling, 1999). this hypothesis has been corroborated by the finding that the activity of the anaphase-promoting complex/cyclosome is required for nurse cell polytene chromosome breakdown (kashewski et al., 2002). it is however not known how cell cycle regulation can be affected by developmental cues. appropriate genetic programs must be in fact responsible for triggering nurse cell chromosome dispersal in a spatially and temporally regulated manner. several putative members of these genetic programs have been identified by phenotypic characterization of female sterile mutations altering nurse cell chromosome morphology. these mutations, called rhino (rhi), half pint (hfp), hrb27c, squid (sqd), ovarian tumor (otu) and cup cause the persistence of “blob like” chromosomes in late stage egg chambers (king, 1970; king and storto, 1988; keyes and spradling, 1997; volpe et al., 2001; van buskirk and schüpbach, 2002; goodrich et al., 2004). a similar phenotype is shown by the tulipano (tlp) mutation, isolated in our laboratory (gigliotti et al., 1998). tlp female sterile alleles displayed striking defects in both nurse cell chromatin organization and egg morphology and have been grouped into two general classes. stronger alleles caused egg chamber developmental arrest during mid-oogenesis, while weaker alleles sustained egg chamber maturation till complete egg assembly (gigliotti et al., 1998; kiger et al., 1999). morphological features characteristic of early developmental stages persisted in nurse cell nuclei of both types of mutant alleles, even if with different degrees of penetrance (fig. 3b). we have cloned the gene affected by the tlp mutation and identified its protein product as the drosophila homolog of yeast nucleoporin nup170 and nup157 and mammalian nup155 (radu et al., 1993; aitchison et al., 1995; zang et al., 1999). these proteins are structural components of the nuclear pore complexes, the gated channels that perforate the nuclear envelope and mediate nucleo-cytoplasmic transport in all eukaryotes. accordingly, the drosophila protein, named nup154 with reference to its deduced molecular weight, was found to be localized at the nuclear periphery in both somatic and germline cells of the ovary (fig. 4). the nup154 gene was also shown to be expressed in other tissues and at all developmental stages (gigliotti et al., 1998; kiger et al., 1999). this finding, together with the identification of strong loss of function lethal alleles suggested that nup154 is required in all cell types, confirming that nuclear pore complex components play essential roles in different aspects of cell physiology (fahrenkrog et al., 2004). however, the peculiar ovarian phenotype of tlp hypomorphic alleles indicated that nup154, probably in association with other ovarian proteins, might also play cell-type specific functions. one possibility is that nup154 is directly implicated in the nurse cell chromosome dispersal process. it has in fact been proposed that, serving as anchorage sites for chromatin, several nucleoporins might be involved in chromatin organization (ishii et al., 2002; feuerbach et al., 2002). it is also conceivable that cytoplasmic and/or nuclear components of the molecular pathway 76 fig. 4 immunolocalization of the nup154 protein during oogenesis. confocal microscope images of wild type ovaries stained with anti-nup154 antibodies (a,d) and the toto-3 nucleic acid dye (b,e). nup154 is present throughout egg chamber development (a) and is localized in both germline and follicle cells all along the perimeter of the nuclei (b), as best seen in the merged image (c). in (d,e merged in f) high magnification of a germarium shows that nup154 is already detectable during germline cyst formation. regulating nurse cell chromosome dynamics during oogenesis require nup154 function for moving in and/or out of the nuclei. a possible list of candidates includes the proteins coded by the genes listed above, whose mutant alleles closely resemble the tlp mutant phenotype. interestingly, cup, that is a cytoplasmic protein, is transiently localized at the nuclear envelope of the nurse cells as long as their chromosomes remain condensed (keyes and spradling, 1997) and is able to shuttle between the nucleus and the cytoplasm, at least in transfected schneider cells (zappavigna et al., 2004). hrb27c and sqd belong to the heterogeneous nuclear ribonucleoprotein (hnrnp) a/b family of rna-binding proteins, involved in various aspects of rna metabolism, including nuclear transport (for review, see dreyfuss et al., 2002). the phenotype induced by tlp mutations not only affected nurse cell chromosome dispersal, but also the dorsal-ventral patterning of the eggs, which, when produced by weak alleles, displayed fused or missing dorsal appendages (fig. 5). remarkably, dorsalventral defects are also shown by mutations in hrb27c, sqd and otu (goodrich et al., 2004). genetic and biochemical data have suggested that the protein products of these genes can form a complex regulating the localization and/or the translation of specific mrna targets. one of these targets is grk mrna: it has been proposed that the association of hrb27c and sqd with this transcript already occurs in the oocyte nucleus and persists during its translocation to the cytoplasm. here, the two proteins are involved together with otu in grk mrna localization and translation (goodrich et al., 2004). we are currently testing the hypothesis that nup154 can participate to this process and preliminary results indicated that both grk mrna localization and translation are affected also in our mutant (unpublished results). in conclusion, nup154 is required, probably in association with some of the gene products listed above, for both nuclear and cytoplasmic differentiation events taking place during oogenesis. this dual role could be explained by postulating that nup154 is able to mediate the nucleocytoplasmic transport of specific regulatory factors independently involved in nurse cell chromosome dispersal and dorsal-ventral polarity establishment. to test this hypothesis we are currently performing genetic interaction tests of tlp with cup, otu, hrb27c and sqd and employing overexpression approaches aimed at investigating the effects induced by nup 154 overproduction and/or mislocalization. 77 fig. 5 altered egg morphology in tlp mutant females. wild type egg with two chorionic appendages that mark the dorsal surface. in eggs laid by tlp mutant females, these structures are reduced, fused (b) or completely missing (c). apoptosis in the germ cells: analysis of dumpless mutants programmed cell death during drosophila oogenesis occurs at distinct stages and is triggered by both developmental and environmental stimuli. at late stages of oogenesis, developmentally regulated cell death is responsible for the removal of the nurse cells after their function of supplying nutrients to the oocyte has been fulfilled, and is necessary for the proper development of every oocyte. during the first ten stages of oogenesis, a flux of cytoplasm gradually streams from the nurse cells into the oocyte through the ring canals. starting from stage 10b, a fast phase of cytoplasmic transfer occurs and outcomes of this transport into the oocyte are the regression of the nurse cell cluster and the doubling of the oocyte volume. the cytoplasmic dumping is preceded by dramatic rearrangements of the actin cytoskeleton. during stage 10b, filamentous actin forms bundles extending from the plasma membrane toward the envelope of nurse cell nuclei (fig. 6a-c), preventing the movement of these nuclei during the dumping of the cytoplasm. coincident with actin bundle formation, the nurse cell nuclei begin to break down, lose their lamin staining, show gaps in their envelope and become permeable (smith and fisher, 1989; cooley et al., 1992; mccall and steller, 1998; matova et al., 1999). following the massive cytoplasm dumping, that has been attributed to the myosin-based contraction of subcortical actin (wheatley et al., 1995), the nurse cells dye by apoptosis (cavaliere et al., 1998; foley and cooley, 1998; mccall and steller, 1998). the nurse cell remnants are phagocitized by the follicle cells that in turn, at the completion of oogenesis, show sign of apoptosis and are reported to be engulfed by epithelial cells of the oviducts (nezis et al., 2000). the programmed cell death of nurse cells shows distinct differences compared to cell death in other drosophila tissues. the formation of actin bundles, the cytoplasmic transfer to the oocyte and the protection of the connected oocyte from the death process of nurse cells are cellular events not seen in apoptotic death occurring in other drosophila cells. in addition, germline cell death at late-oogenesis does not require the three pro-apoptotic proteins reaper, hid and grim that promote caspase activation in the vast majority of cell deaths occurring during drosophila development (foley and cooley, 1998). the upstream signals that induce developmentally regulated cell death are still largely unknown (for review, see mccall, 2004). some evidences point to the involvement of the ecdysone signaling pathway in controlling nurse cell death at late-oogenesis. injection of 20-hydroxyecdysone, the biologically active ecdysteroid, in virgin females leads to premature nurse cell death suggesting that ecdysone may normally act to promote nurse cell death (soller et al., 1999). more than one signaling pathway may contribute to the regulation of nurse cell death. genetic analyses of the decapentaplegic (dpp) signaling pathway suggest that this pathway is involved in nurse cell death. conditional mutations in the dpp gene, which encodes a tgf-β family member, give rise to small eggs at non permissive temperature and mutations in saxophone, a gene encoding a tgf-β receptor also cause defects in nurse cell dumping and nurse cell death (twombly et al., 1996; myster et al., 2000; royzman et al., 2002). recently, it has been reported that lipid composition may influence the timing of nurse cell death (buszczak et al., 2002). mutations in the midway gene, which encodes a diacylglycerol acyltransferase, result in premature nurse cell death and degeneration. we have studied the nature of the death process of the nurse cells after their function has been fulfilled. our studies started with the analysis of nuclear dna integrity, since dna fragmentation is a diagnostic hallmark of apoptotic death. we have analyzed dna from egg chambers isolated at relevant steps of oogenesis and from whole ovaries of wild-type females and found that dna extracted from late-stage egg chambers is fragmented into nucleosomal-size 78 fig. 6 morphological changes associated with rapid cytoplasm transport from nurse cells to oocyte. the egg chambers were stained with fitc-conjugated phalloidin to visualize the actin cytoskeleton. in stage 10a egg chambers (a) filamentous actin localizes to the cortex of nurse cells (see arrow). at stage 10b a network of actin filaments polymerizes around the nurse cell nuclei (b, see arrow) and actin bundles extend from the plasma membrane to the internal region of nurse cells (c) containing the nuclei. fig. 7 in situ detection of dna fragmentation by tunel labeling. the tunel technique labels the 3’-end of dna witnessing its fragmentation. in wild type ovaries tunel staining starts to appear at stage 12 (a) and two darkly stained nuclei are visible at the ventral side of the egg chamber (see arrowheads in higher magnification b). at stage 13 extensive tunel labeling is observed in nurse cells (c). differing from the wild type egg chambers at a comparable degree of maturation, egg chambers from chickadee (d), quail (e) and singed (f) mutants show few apoptotic nuclei, visible as darker brown spots (see arrowheads) in the nurse cell compartment. 79 fragments (cavaliere et al., 1998). the tunel technique labels the 3’-end of dna, witnessing its fragmentation (gavrieli et al., 1992). thus we have analyzed wild-type egg chambers by this assay and we found that starting from stage 12, the nurse cell nuclei become tunel positive (fig. 7a-c) indicating that cell death by apoptosis is responsible for the removal of the nurse cells at the end of oogenesis. in order to gain insights into the signals that induce nurse cell death, we investigated if the dumping process per se could trigger the apoptotic program and we extended our analyses to dumping defective mutants. mutations in chickadee, quail and singed genes cause a defect of the dumping process due to the absence of actin filament bundles (cooley et al., 1992; cant et al., 1994; mahajan-miklos and cooley, 1994). as a consequence, the nurse cell nuclei lodge into the ring canals, block nurse cell cytoplasm transport and the resulting dumpless egg chambers contain oocytes smaller than normal. total dna extracted from quail and singed mutant ovaries showed the typical oligonucleosomal dna ladder (cavaliere et al., 1998). tunel analysis of egg chambers from chickadee, quail and singed mutants showed that the apoptotic process is activated and proceeds even if not all nuclei appeared to be in an apoptotic state (fig. 7d-f), differently from the wildtype egg chambers at a comparable degree of maturation. the tunel positive nuclei were found to be well surrounded by a relevant amount of cytoplasm (fig. 7d, f). this indicates that the apoptotic process program in the nurse cells can be activated and proceeds even if their cytoplasm is retained. in conclusion our results showed that, during the normal development of the egg chamber, the exhausted nurse cells dye by apoptosis at the late stage of oogenesis. in wild type egg chambers the timing of nurse cell death follows the massive cytoplasm transport into the oocyte, but the finding of apoptotic nuclei in chickadee, quail and singed egg chambers indicated that apoptosis is not initiated by cytoplasm dumping. conclusions and perspectives advances in drosophila research have placed this organism in a unique position to contribute a detailed understanding of the cellular and molecular mechanisms underlying oogenesis. many important insights have been provided by the analysis of female sterile mutants, used as invaluable tools for the identification of essential gene functions involved in egg chamber development. more recently this forward genetic approach has been joined by other experimental strategies such as cell specific gene inactivation and rna interference. our knowledge of the overall complexity of the developmental pathways involved in oogenesis is therefore expected to greatly increase in the next future. this will in turn provide remarkable opportunities for studying comparative aspects of oogenesis. in this frame, the availability of the complete sequence of the drosophila genome will contribute an essential basis for the identification of homologous proteins in different species. investigating the existence of functionally conserved roles of these molecules is therefore the next, intriguing perspective. acknowledgments we thank s. andone, our stock curator, m.r. grimaldi for performing genetic transformation experiments and a. bellopede for technical assistance. we thank the regione campania for supporting the entire re-equipment of our laboratory that was fully destroyed by the flood of september 2001, thus giving us the possibility to start again our work on drosophila oogenesis, despite the loss of a large amount of unpublished data. also various mutants isolated by our groups, such as tegamino, tondo, palla and others, have been lost and are no longer in our stock collection. 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haemocyte types; haemocyte morphometry; haemocyte cytometry; haemocyte ploidy; haemocyte identification key introduction free circulating cells in the insect haemocoel are called haemocytes. these cells are major players in immunity and homeostasis, conducting _________________________________________ corresponding author: christoph-rüdiger von bredow applied zoology department of biology technische universität dresden zellescher weg 20 b, 01217 dresden, germany e-mail: christoph.von.bredow@mail.de list of abbreviations: β-1,3-glucan recognition protein 1, βgrp-1; 4',6diamidino-2-phenylindole, dapi; fluorescein isothiocyanate, fitc; granular cell, gr; length-towidth-ratio, l:w; nucleus, nuc.; nucleus-tocytoplasm-ratio, n:c; oenocytoid, oe; plasmatocyte, pl; spread granular cell, sprgr; spread plasmatocyte sprpl; peanut agglutinin, pna; pnapositive cell membrane, pnam+; prohaemocyte, prohc; round plasmatocyte, rpl; spherule cell, sp; tetramethylrhodamine isothiocyanate, tritc; trisbuffered saline, tbs cellular immune responses such as phagocytosis and encapsulation (horohov and dunn, 1982; wiegand et al., 2000; lavine and strand, 2002) and participate in extracellular matrix deposition and degradation (nardi and miklasz, 1989; kuschegullberg et al. 1992; bunt et al., 2010; sánchezsánchez et al., 2017). basically, the haemocyte types or classes are classified based on morphological characteristics (jones, 1962; price and ratcliffe, 1974; gupta, 1979; horohov and dunn, 1982). functional analyses revealed specialised actions of distinct haemocyte types. the oenocytoids of lepidopteran insects for example synthesize and release prophenoloxidase, the key component of the melanisation reaction necessary to fight large pathogens (iwama and ashida, 1986; jiang et al., 1997), plasmatocytes and granular cells phagocytose or encapsulate foreign bodies (pech and strand, 1996; levin et al., 2005) and prohaemocytes are thought to divide and differentiate to form other haemocyte types (yamashita and iwabuchi, 2001) and can therefore be seen as either progenitor cells or stem cells with limited differentiation potential. by developing 14 table 1 marker molecules used for haemocyte determination primary antibody epitope and/or cell type specificity host / type ig type / subtype inventor / references / rrid dilution mab ms7 granules in granular cells, unknown epitope mouse mab igg willot et al., 1994 / beetz et al. 2004, 2008, this paper hybridoma supernatant mab ms13 β-integrin expressed on m. sexta plasmatocytes and haematopoietic organs mouse mab igg2b willot et al., 1994 / wiegand et al., 2000; nardi et al., 2003; beetz et al., 2004, 2008; levin et al., 2005 / ab_2618094 hybridoma supernatant mab ms77 plasmatocyte membrane, unknown epitope mouse mab igm willot et al., 1994 / beetz et al., 2008 hybridoma supernatant anti-esterase ephestia kuehniella haemolymph esterase binds to cytoplasm of m. sexta spherule cells rabbit immune serum generous gift from g. mann (mann, 1992) / von bredow et al., 2021 1:1,000 anti-b. mori prophenoloxidase (ppo) bombyx mori prophenoloxidase, oenocytoids (m. sexta, b. mori) rabbit immune serum iwama and ashida, 1986 / iwama and ashida, 1986; ashida et al., 1988; von bredow et al., 2021 1:1,000 anti-βgrp-1 3a β-1,3-glucan recognition protein from m. sexta oenocytoids rabbit immune serum ma and kanost, 2000 / von bredow et al., 2021 1:500 lectin cell type carbohydrate specificity supplier / cat. no. references dilution arachis hypogaea lectin (pna) tritc, 2mg/ml granules in granular cells granules and occasionally membrane of oenocytoids membrane of prohaemocytes gal, galβ13galnac of nglycans and glycolipids sigma-aldrich, st. louis, mi, usa / l3766 nardi 2004; kobayashi et al. 2014, y. von bredow et al., 2020; c. von bredow et al., 2021 1:2,000 secondary antibody conjugate host supplier cat. no. / rrid dilution anti-mouse igg/igm fitc goat dianova gmbh, hamburg, germany 115-095-044 / ab_2338593 1:200 anti-rabbit igg dylight 488 goat vector laboratories, burlingame, usa di-1488 / ab_2336402 1:2,000 15 monoclonal antibodies against haemocytes, it turned out that the morphologically distinct haemocyte classes also differently express certain epitopes which confirmed the earlier classifications in lepidopterans, e.g. manduca sexta (willot et al., 1994), pseudoplusia includens (gardiner and strand, 1999), bombyx mori (nakahara et al., 2009). in m. sexta, as in most lepidopteran insects, five classes of haemocytes were described based on morphological and ultrastructural criteria (horohov and dunn, 1982) which later was partly confirmed by antibodies labelling specific haemocyte types (willot et al., 1994; nardi et al., 2001; beetz et al., 2004; levin et al. 2005). moreover, horohov and dunn (1982) provided information about approximate size of the haemocyte types and nuclear-to-cytoplasm ratio, but detailed values or ranges for each haemocyte type were missing. since specific markers are not always accessible it is often necessary to distinguish haemocyte types by light microscopy or easily accessible standard staining procedures. however, even trained researchers may encounter difficulties in haemocyte type determination due to similarities between cell types and variable size and shapes within the same cell type. therefore, we present a set of morphological and cytometric criteria which can be used to identify the main haemocyte classes in m. sexta larvae by light microscopy and nuclear staining methods. methods insects manduca sexta of the colony maintained at the institute of general zoology and developmental biology, justus-liebig-universität gießen, germany, were reared under standard conditions as outlined elsewhere (beetz et al., 2004). briefly, eggs were deposited on nicotiana rustica plants, collected and incubated at 25 °c in polystyrene boxes containing a small block of artificial diet (modified after yamamoto, 1969). larvae were reared individually under long day light regime (16/8h light/dark). haemocyte collection and preparation of haemocyte monolayers two-day-old fifth-instar larvae (l5d2) were cleaned and anaesthetised by chilling on ice. an incision was made at a proleg or the abdominal horn and the haemolymph was collected in 5 ml ice cold anticoagulant saline (ac; 3.9 mm nacl, 40 mm kcl, 146 mm sucrose, 0.1 % (w/v) polyvinylpyrrolidone 40, 8 mm edta, 9.5 mm citric acid, 27 mm trisodium citrate, 1.7 mm pipes, ph 6.5). haemolymph in ac was then centrifuged at 200 x g, 4 °c for 15 min. after discarding the supernatant, the haemocyte pellet was resuspended in 5 ml fresh ac and the procedure was repeated twice. the haemocyte pellet was then resuspended in approx. 1 ml insect cell culture medium (tc-100, gibco ™ thermo fisher scientific inc., waltham, ma, usa), and 15 µl cell suspension per well seeded on a 10-well glass slides (thermo scientific™ diagnostic slide, cat. number x1xer308b#mnz, thermo fisher scientific inc., waltham, ma, usa). after allowing the cells to settle for 45 min at room temperature in a humid chamber, the supernatant was removed and the cells were fixed by adding 3.5 % paraformaldehyde dissolved in calcium and magnesium free m. sexta saline (ms-; 3.9 mm nacl, 40 mm kcl, 146 mm sucrose, 0.1 % (w/v) polyvinylpyrrolidone 40, 1.7 mm piperazine-n,n′-bis(2-ethanesulfonic acid) (pipes), ph, 6.5) for five min. after several rinses with tris-buffered saline (tbs; 500 mm nacl, 20 mm tris, ph 7.5) the slides were stored at -20 °c. nucleus staining and antibody labelling we used a toolset of haemocyte markers comprising monoclonal antibodies raised against larval haemocytes and immune sera against different proteins to identify the haemocyte types. additionally, we combined labelling with these markers with the galactose-specific lectin pna that has previously been shown to label different haemocyte types (nardi, 2004; von bredow et al., 2021). all markers, their specificity and known epitopes are listed in table 1. haemocytes were thawed and blocked with 5 % (v/v) normal goat serum (ngs), 3 % (w/v) bovine serum albumin (bsa), 0.05 % (w/v) sodium azide in tbs for one hour at room temperature. incubation with primary antibodies (plasmatocyte specific antibodies: mab ms13 (anti-plasmatocyte specific β-integrin, willot et al., 1994, beetz et al. 2004, 2008; levin et al., 2005), or mab ms77 (unknown epitope), (willot et al., 1994; beetz et al. 2008); granular cell specific antibody: mab ms7 (unknown epitope), (willot et al., 1994; beetz et al., 2004, 2008); oenocytoid binding antibody: rabbit immune serum against m. sexta β-1,3-glucan recognition protein 1 (βgrp-1), diluted 1:500 (ma and kanost, 2000), generous gift from m. kanost; spherule cells specific antibody: rabbit immune serum against ephestia kuehniella haemolymph esterase, diluted 1:500 (mann, 1992), generous gift from g. mann, was performed overnight at 4 °c, followed by three rinses with tbs. secondary antibody diluted in 5 % ngs, 3 % bsa, 0.05 % sodium azide in tbs, goatanti-mouse igg/igm fitc-labelled, dianova, hamburg, germany, diluted 1:200 or goat-antirabbit igg dylight 488-labelled, vector laboratories, burlingame, ca, usa, diluted 1:2,000, respectively, together with lectin from arachis hypogaea (peanut agglutinin, pna, tritc-labelled, sigma-aldrich, st. louis, mi, usa, diluted 1:2,000) was added for one hour at room temperature, followed by two rinses with tbs and dna-labelling by incubation with 360 nm 4',6-diamidino-2phenylindole (dapi) in tbs for 15 min. after washing the cells with tbs for 10 min, the slides were mounted with fluoromount g (southern biotech, birmingham, al, usa) and analysed with a bx60 fluorescence microscope with mounted camera (altra 20 or xc10, both olympus, tokyo, japan). determination of cell size, nucleus size and nucleus-to-cytoplasm ratio cell diameter, nucleus area and cytoplasm area of haemocyte types were selected manually from 16 photomicrographs and measured with the function “measure” of the open source software imagej 1.50e (abràmoff et al., 2004, https://imagej.nih.gov/ij). haemocyte or nucleus length was determined as the highest dilatation of a single cell, cell width as the distance between cell boundaries orthogonally to the length-axis. length-to-width ratio of haemocytes or nuclei was calculated by dividing length by width. the nucleus-to-cytoplasm ratio was calculated as nucleus area divided by cytoplasm area (cell area minus nucleus area). this method was only used for non-spread haemocytes due to the high degree of polymorphism of spreading haemocytes. the relative dna-content was calculated by measuring the integrated density and area of dapistained nuclei with imagej 1.50e (abràmoff et al., 2004). for each photomicrograph, five non-dividing, typical granular cells, distinguished by morphology and pna-positive granular inclusions were measured to receive a baseline intensity of diploid cells (nardi et al., 2003). background fluorescence was subtracted and fluorescence intensity was calculated according to the method described by mccloy et al. (2014). relative dapi-fluorescence intensity was calculated by dividing dapi-intensity of sampled cells by mean dapi-intensity of diploid granular cells. each analyses was performed on at least three independent biological replicates. statistical analysis tests for significance were performed with r version 3.6.2 (r core team, 2014). the cytometric values cell diameter (length and width), nucleus diameter, cell area, nucleus area, cell length-towidth-ratio, and nucleus length-to-width-ratio were tested for differences between the cell types using one-way-anova with post-hoc pairwise comparison (tukey-test). differences were classified as significant at p ≤ 0.05. the relative dapi-intensities and the nucleusto-cytoplasm-ratios were compared between the haemocyte types using mann-whitney-u-test, with p ≤ 0.05 considered as significant difference. results morphology of haemocyte types and specific labelling by antibodies and the lectin pna binding characteristics of the antibodies and the lectin pna on haemocytes are shown in fig. 1, and summarised in table 1. granular cells were specifically labelled by the mab ms7 (fig. 1 a’), plasmatocytes were specifically bound by the mab ms13 (fig. 1 b), and the mab ms77 (fig. 1 e’), respectively. oenocytoids were the only haemocyte type labelled by anti-βgrp-1 immune serum (fig. 1 c’, table 1). the immune serum against e. kuehniella haemolymph esterase (anti-esterase) is the only known available antibody binding exclusively to spherule cells but no other haemocyte type (fig. 1 d’). granular inclusions of granular cells (fig. 1 a’’) and few granules in oenocytoids were pna-positive (fig. 1c’’, 1ei’). furthermore, some haemocytes were labelled with pna at the cell surface. these cells were spherule cells, which had a slightly labelled cell surface (fig. 1 d’’). more extensively stained were non-spread plasmatocytes (fig. 1 e’’), oenocytoids and putative prohaemocytes (fig. 1 eii’, pnam+). small spread haemocytes which were similar in shape to spread plasmatocytes did not share most key characteristics of plasmatocytes. the nuclei of small spread haemocytes were small and had a round shape, whereas plasmatocytes had large and mostly irregularly formed nuclei (compare the nuclei of sprgr to the nuclei of rpl or sprpl in fig. 1). antibody labelling revealed no antigenic similarities between the small spread cells and plasmatocytes. plasmatocytes were labelled by ms13 and ms77, whereas small spread haemocytes were not (compare labelling pattern of sprgr to labelling pattern of rpl or sprpl in fig. 1 b’, and sprgr with rpl in fig. 1 e’). furthermore, the granular cells labelling mab ms7 bound to small spreading cells (fig. 1a’), and pna labelled granular inclusions, too, as opposed to sprpls (fig. 1 a’’, b’’). oenocytoid markers (anti-βgrp-1, fig. 1 c’; antiprophenoloxidase, data not shown) and the spherule cell marker anti-esterase (fig. 1 d’) did not label the small spread haemocytes. additionally, the relative dapi intensity fitted to granular cells rather than plasmatocytes or other cell types (fig. 3 a), indicating that these small spread cells resemble a pseudopodia forming, spread granular cell population, which we termed spreading granular cells (sprgr). confusingly, oenocytoids and round plasmatocytes sometimes share the same size and shape (compare appearance of oes with appearance of rpls in fig. 1), and might therefore be misidentified. two characteristics were identified to reliably identify each haemocyte type: first, the oenocytoid cytoplasm does sometimes appear in light microscopy more granulated (a weak characteristic), and second, the size and shape of the nuclei (fig. 1, fig. 2 d, e), but not of the dnacontent (fig. 3 a), differed largely between oenocytoids and round plasmatocytes. oenocytoid nuclei were round while plasmatocyte nuclei were most often irregularly-oval formed (compare the nucleus of an oe to the nucleus of rpls in fig. 1 c’’’ and fig. 1 b’’’), and normally a larger portion of the cytoplasm is visible in oenocytoids than in round plasmatocytes, the latter possess a larger nucleus and only sparse cytoplasm (fig. 3 b). size and shape of the haemocyte types for the morphological description of the haemocyte types, the cell area, length (maximum distance) and width (minimum distance) and the length-to-width ratio were determined. all data are shown in fig. 2 (cell and nuclei diameter and length to width ratios), fig. 3 (relative dapi-intensity of the nuclei and nucleus-to-cytoplasm-ratios), fig. 4 (typical appearance of haemocyte types and nuclei) and supplementary table s1. granular cells occur in two different morphs, either and most abundant as unspread, round cells (abbreviated as gr as it is the common form), or, as evidenced in this work, as spreading cells (sprgr). the round granular cells (gr) ranged in diameter from 5.0 µm to 10.1 µm, with a mean 17 fig. 1 antibodyand pna-labelling of haemocyte types and corresponding nuclei morphology. a-a’’’: granular cells and spread granular cells have ms7and pna-positive cytoplasmic inclusions (granules). nuclei of round granular cells are round, nuclei of spread granular cells are round to oval. b-b’’’: plasmatocytes can be round or spread, both forms labelled by ms13. plasmatocytes contain no pna-positive granules. nuclei of both spread and round plasmatocytes are eccentric or oval in shape. c-c’’’: oenocytoids are positive for β-grp-1 and may contain pna-positive granules. nuclei are round and larger than nuclei of granular cells. d-d’’’: anti-e. kuehniella haemolymph esterase immune serum labels spherule cells, pna slightly labels their cell surface. spherule cells are round (lower sp) to oval or rod-shaped (upper sp), their nuclei are mostly elongated or irregularly formed (d’’’). e-e’’’: a round plasmatocyte (labelled by ms77) exhibits labelling of the cell surface by pna. ei-ei’’: oenocytoid with pna-positive granules and pna cell surface staining. eii-eii’’: a round, small haemocyte labelled with pna at the cell surface. note the lesser granulation of the cytoplasm when compared to the granular cell and the absence of pna-positive granules. the nucleus is round in shape and slightly larger than that of the granular cell. abbreviations: gr, granular cell; oe, oenocytoid; pl, plasmatocyte; pnam+, haemocyte with pna-positive cell membrane; rpl, non-spread plasmatocyte; sp, spherule cell; sprgr, spread granular cell. the scale bar represents 10 µm 18 fig. 2 size, length-to-width-ratio and area of different haemocyte types and their nuclei. a: haemocyte length and width of each haemocyte type. b: haemocyte length-to-width ratio depicting the differences in shape of the haemocyte types. c: area occupied by each haemocyte type. the y-axis (area) is logarithmized. d: nucleus length and width of each haemocyte type. e: nucleus length-to-width ratio depicting the differences in nucleus shape of the haemocyte types. f: area occupied by the nuclei of each haemocyte type. bars depict mean values, whiskers depict the standard deviation. statistical analysis (anova with post-hoc tukey-test) is summarised in supplementary table s2 and s3. abbreviations: gr, granular cell; oe, oenocytoid; sprgr, spread granular cell; sprpl, spread plasmatocyte; pnam+, haemocyte with pna-positive cell membrane; rpl, non-spread plasmatocyte; sp, spherule cell diameter of 6.1 µm, sd 0.9 (width) and 6.7 µm, sd 0.9 (length). the area covered by these cells ranged from 20.7 µm2 to 59.9 µm2, with a mean of 36.3 µm2, sd 8.8. with a range of length-to-width-ratio (l:w) between 1.0 and 1.3, and a mean l:w of 1.1, these cells are best described as round in shape. the spread granular cells (sprgr) differed from the round form by projecting numerous pseudopodia, and in consequence by their altered cytometric values. they were larger in their length (i.e. maximum diameter) and occupied a larger area (between 32.6 µm2 and 64.8 µm2, mean 46.1 µm2, sd 7.9) than round granular cells. their diameter measured between 4.3 µm and 12.8 µm, with mean diameter of 6.3 µm, sd 0.9 (width) and 9.3 µm, sd 1.4 (length). the l:w was between 1.0 (evenly spread cells without polar extensions) and 2.9 (highly polar spread cells projecting cytoplasmic protrusions preferably along a single axis), with a mean l:w of 1.5, sd 0.3. numerous granular inclusions were frequently visible in the cytoplasm of both round and spread granular cells by differential interference contrast microscopy, many of them were labelled by the lectin pna and the mab ms7 (fig. 1 a’, a’’). spherule cells (sps) were the smallest cell type with diameter between 3.8 µm and 6.5 µm, with a mean diameter of 4.7 µm, sd 0.4 (width) and 5.4 µm, sd 0.6 (length). the cell area occupied by these cells was between 15.5 µm2 and 39.5 µm2, with a mean area of 23.7 µm2, sd 5.3. their shape ranged from round (l:w of approx. 1.0) to elongated (l:w of ≥ 1.3). the mean l:w of 1.16 indicates the morphological variability. large spherules, eponymous for this haemocyte type, were found in the majority of the spherule cells, but were not 19 always clearly visible by means of light microscopy (cf. the upper sp with large spherules and the lower sp lacking obvious spherules in fig. 1 d). plasmatocytes occur in two main forms, round (non-spread) or spread. intermediate forms can also occur, which are characterised by a large round cell body containing the nucleus and pseudopodia projecting from there, indicating a transition from the round to the spread form (not shown). the round plasmatocytes (rpl) were relatively large round haemocytes with diameter ranging from 6.5 µm to 10.4 µm, with a mean diameter of 7.75 µm, sd 0.8 (width) and 8.36 µm, sd 0.9 (length). the cell area covered between 35.3 µm2 and 75.3 µm2, mean area 53.1 µm2, sd 10.1. the l:w ranged between 1.0 and 1.2, mean l:w 1.1, sd 0.1, which mirrors their round shape. the spread plasmatocytes (sprpl) differed in their dimensions from the unspread form, with a diameter between 7.5 µm and 51.1 µm, mean diameter 20.1 µm, sd 9.2 (width) and 31.2 µm, sd 8.8 (lenght). the cell area was between 133.5 µm2 and 1,787.1 µm2, mean area 550.5 µm2, sd 378.6. spreading as a result of pseudopod projection led to l:w ranging between 1.1 (isotropic spread plasmatocytes, which appear nearly round, “fried egg shape”) and 3.7 (highly polarised, anisotropic spread cells, with pseudopods projecting mainly to one or two directions along a single axis). oenocytoids (oes) were relatively large, round cells, with a diameter measuring between 6.2 µm and 12.8 µm. the mean diameter was 8.2 µm, sd 1.1, (width) and 8.7 µm, sd 1.3 (length). oenocytoids covered areas between 34.2 µm2 and 123.3 µm2, with a mean of 57.6 µm2, sd 17.6. owing to their round to oval appearance, the l:w ranged between 1.0 and 1.2, with a mean l:w of 1.1, sd 0.1. putative prohaemocytes (prohc), small round haemocytes which were identified by labelling of the cell membrane with pna (pnam+) and a relatively large area occupied by the nucleus, measured between 4.6 µm and 8.4 µm, with a mean diameter of 5.8 µm, sd 0.8 (width) and 6.1 µm, sd 0.7 (length). they covered between 19.7 µm2 and 54.9 µm2, with a mean area of 30.3 µm2, sd 8.0. the l:w was between 1.0 and 1.3, with a mean l:w of 1.1, sd 0.1, mirroring their overall round shape. length and width of each of the round haemocytes types did not differ largely within individual cells, resulting in a length-to-width ratio close to 1 (fig. 2 a, b). this characteristic was found in granular cells, round plasmatocytes, oenocytoids and putative prohaemocytes. spherule cells exhibited a slightly elevated length-to-widthratio. spread granular cells as well as spread plasmatocytes showed a remarkable difference between their maximum expansion and minimum expansion, resulting in a mean length-to-width-ratio above 1.5 (fig. 2 b). nuclear morphology, relative dna content and nucleus-to-cytoplasm-ratio of haemocyte types haemocytes exhibited haemocyte type specific differences of size, shape and dna content of their respective nuclei. as a rule of thumb, round haemocytes had a similarly round nucleus, while spread or otherwise deformed haemocytes had oval or irregularly formed nuclei. an exception were the round plasmatocytes with mostly irregularly shaped nuclei. all data can be found in fig 2 d f, fig. 3, and supplementary table s1. nuclei of the round granular cells measured between 3.1 µm and 5.3 µm in diameter, with a mean of 3.8 µm, sd 0.4 (width) and 4.1 µm, sd 0.4 (length). the nucleus area occupied between 8.4 µm2 and 19.1 µm2, mean area 13.0 µm2, sd 2.6. the nuclear l:w ranged between 1.0 and 1.4, mean 1.1, sd 0.1, which defines the nuclei as mostly round. the relative dapi-intensity was between 0.7fold and 2.3-fold of diploid granular cells, with a median of 1.1, interquartile range (iqr) 0.5. nuclei of the spread granular cells had a similar range of relative dapi-intensity as round granular cells, ranging from 0.6-fold to 2.2-fold of diploid granular cells, median 1.3, iqr 0.7. the nucleus-tocytoplasm-ratio (n:c) was not determined for this cell type. nuclei of the spherule cells had a remarkably low dapi-intensity at the minimum level measured with 0.34-fold intensity, the highest intensity, however, was 2.0-fold compared to granular cells. the median dapi-intensity also was lower than expected at 0.76, iqr 0.3. the n:c ranged between 0.46 and 2.51, with a median n:c of 1.0, iqr 0.6. nuclei of the round plasmatocytes exhibited always an elevated relative dapi-intensity, from 1.4fold to 6.3-fold, median 3.1, iqr 0.6. this implies a relatively high dna-content compared to diploid granular cells. the n:c measured between 0.45 and 2.73, median 1.2, iqr 0.5. spread plasmatocytes had an even higher relative dapi-intensity between 1.8-fold and 7.1-fold, median 3.8, iqr 0.7. the n:c was not determined for this cell type. the nuclei of the oenocytoids had a relative dapi-intensity between 1.1 and 7.1, median 3.0, iqr 1.1. their n:c ranged between 0.4 and 1.4, median 0.9, iqr 0.4, which indicates that the area occupied by the nucleus was mostly smaller than the area of the cytoplasm. putative prohaemocytes (pnam+ cells) exhibited nuclei with a relative dapi-intensity between 0.7 and 2.6, median 1.3, iqr 0.5. their n:c was between 1.0 and 2.8, with a median n:c of 1.6, iqr 1.0. therefore, these cells had a nuclear area exceeding the area covered by the cytoplasm. dichotomous identification key for m. sexta haemocyte types from the data presented in this paper, we derived a key to each haemocyte type in m. sexta larvae, which can be applied to fixed adhering haemocytes labelled with standard dna-stains like dapi and light microscopy, preferentially using differential interference contrast. please note that some characteristics such as granulation of the cytoplasm may be obscured depending on the microscopy technique used. an overview of typical haemocyte type shape and nuclei is given in fig. 4. a graphical short-key is shown in fig. 5. we recommend to refer to the detailed description presented in the main text below, and summarised in supplementary table s1 to enhance the accuracy of determination. 20 fig. 3 relative dapi-intensity and nucleus-to-cytoplasm-ratio of different haemocyte types. a: the dapi-intensity of the different haemocyte types in relation to the intensity of typical diploid granular cells. granular cells (gr), spread granular cells (sprgr), spherule cells (sp) and pna cell membrane positive putative prohaemocytes (pnam+) do seldom exceed a two-fold granular cell dapi-intensity, whereas plasmatocytes, both spread (sprpl) and round (rpl) as well as oenocytoids (oe) regularly contain nuclei with a more than two-fold higher dapi intensity, indicating polyploidy of these cell types. b: the nucleus-to-cytoplasm area ratio of non-spread haemocyte types. horizontal bars within each box depict the median, upper and lower quartile are depicted by the upper and lower borders. whiskers represent the 1.5-fold interquartile range, outliers are depicted by empty circles. different lower-case letters depict significant differences at p ≤ 0.05, mann-whitney-u-test. these data were also used in another context in von bredow et al. (2021) to clarify the identity of haematopoietic organ cells. abbreviations: gr, granular cell; oe, oenocytoid; sprgr, spread granular cell; sprpl, spread plasmatocyte; pnam+, haemocyte with pna-positive cell membrane; rpl, non-spread plasmatocyte; sp, spherule cell cell spread, with pseudopods (1); cell not spread (2). 1) maximum cell diameter > 13 µm (3), smaller (4) 2) cell not spread. cell shape oval, nucleus round with smooth edge (5), other (6) 3) spread plasmatocyte (sprpl). large spread cell (diam. 7.5 – 51 µm), nucleus large (diam. 4.7 – 11 µm) and mostly oval or irregular (nuc. l:w 1.0 – 1.9), polyploid (rel. dapi-intens. 1.8 – 7.1-fold of diploid granular cells). 4) spread granular cell (sprgr). small spread cell (diam. 4.3 – 12.8 µm), nucleus small (diam. 2.8 – 5.1 µm) and round to oval (nuc. l:w 1.0 – 1.5), diploid or tetraploid (rel. dapi-intens. 0.6 – 2.2-fold of diploid granular cells). 5) oenocytoid (oe). medium to large sized cell (diam. 6.2 – 12.8 µm), with round to oval shape (cell l:w 1.0 – 1.2) and smooth edge, nucleus round to slightly oval (nuc. l:w 1.0 –1.2), mostly polyploid (rel. dapi-intens. 1.1 – 7.1), cytoplasm area mostly larger than nuclear area (n:c 0.4-1.4). 6) cell shape irregularly, elongated, comma-shaped or vermiform, often with large cytoplasmic inclusions (7), other (8) 7) spherule cell (sp). small, irregularly formed cell (cell diam. 3.8 – 6.5 µm, cell l:w 1.0 – 1.4), often with visible spherules. nucleus small, irregularly shaped (sickle, triangle, elongated, irregularly oval; diam. 1.6 – 4.9 µm, nuc. l:w 1.0 – 3.9) with mostly larger cytoplasm than nucleus (n:c 0.5 – 2.5). diploid to tetraploid (rel. dapi-intens. 0.3 – 2.0). 8) nucleus irregularly shaped or oval (9), nucleus round (11) 9) max. nucleus diameter 3 – 5 µm, max. cell diameter 4 – 6.5 µm (7), max. nucleus diameter 5 – 10 µm, max. cell diameter > 6.5 µm (10) 10) round plasmatocyte (rpl). large, mostly irregular-round (cell diam. 6.5 – 10.4 µm, cell l:w 21 fig. 4 typical appearance and nucleus shape of the larval haemocyte types in m. sexta. differential interference contrast microscopy merged with fluorescence microscopy of the nuclei (red). numbers in square brackets correspond to the numbers in the dichotomous identification key (text based, and fig. 5). upper row from left to right: a typical prohaemocyte (prohc, [13]) with small nucleus and sparse cytoplasm and a putative prohaemocyte with a large nucleus; a spherule cell (sp, [7]) with slightly deformed nucleus and typical large cytoplasmic inclusion (spherule, solid arrowhead); a granular cell (gr, [12]) with granulated cytoplasm, the empty arrow marks the nucleolus; an oenocytoid (oe, [5]) with typical round nucleus; a round plasmatocyte ([10] rpl) with ovoid nucleus. lower row from left to right: a spread granular cell (sprgr [4]), the empty arrowhead marks a typical cytoplasmic protrusion; a spread plasmatocyte (sprpl [3]) with a large, irregularly shaped nucleus, the empty arrowhead marks a thin cytoplasmic protrusion. the scale bar equals 20 μm 1.0 – 1.2) cell with smooth edge. nucleus large (nuc. diam. 3.0 – 9.2 µm), excentric to elongated (nuc. l:w 1.0 – 2.9), with uneven edge. polyploid (rel. dapi-intens. 1.4 – 6.3), nuclear area mostly exceeding cytoplasmic area (n:c 0.5 – 2.7). 11) a) cytoplasm granulated, n:c < 1.0, nucleus diameter 3-5 µm, cell diameter typically 6 – 8 µm, 2n or 4n (12); or b) n:c < 1.0, nucleus diameter 4 – 8 µm, cell diameter 6 – 13 µm, typically > 2n (5); or c) n:c > 1.0, nucleus diameter 3 – 6 µm, cell diameter 4-9 µm, 2n or 4n (13) 12) granular cell (gr). small to medium sized (cell diam. 5.0 – 10.1 µm) round to oval (cell l:w 1.0 – 1.3) cell with granulated cytoplasm, often with uneven edge. nucleus small (nuc. diam. 3.1 – 5.3 µm) and round (nuc. l:w 1.0 1.4), diploid to tetraploid (rel. dapi-intens. 0.7 – 2.3). cytoplasm area most often exceeds nuclear area (n:c 0.3 – 1.5). 13) prohaemocyte (prohc). small to medium sized round cell (cell diam. 4.6 – 8.4 µm, cell l:w 1.0 – 1.3) with smooth edge. nuclear diameter between 3.1 and 6.0 µm, more or less round (nuc. l:w 1.0 – 1.2), diploid to tetraploid (rel. dapi-intens. 0.7 – 2.6), with a nuclear area exceeding the cytoplasm area (n:c 1.0 – 2.8). discussion albeit there exist numerous definitions of haemocyte types in insects, and it is consensus that five main haemocyte types exist in lepidoptera (arnold, 1982), a clear definition of the haemocyte types in m. sexta based on cytometric data has not been published yet. with a defined tool set of monospecific markers for each haemocyte type, foremost monoclonal antibodies raised against whole larval haemocytes (willott et al., 1994), immune sera against specific proteins (iwama and ashida, 1986; mann, 1992; ma and kanost, 2000), and the lectin pna, we were able to clearly distinguish each haemocyte type and determined their key characteristics. we found that many haemocyte types exhibit overlapping characteristics, but when these are combined, a definitive separation of each type was possible. the most obvious difference occurred between spreading cells, which expand numerous pseudopodia, and unspread cells lacking pseudopodia. initially it was unclear whether the small spreading cells, which may occur on shorttime cultured haemocyte monolayers, represent small plasmatocytes or another haemocyte type. due to the unambiguous labelling pattern with granular cell markers labelling the small but not the large spread haemocytes, and the unambiguous labelling pattern with plasmatocyte labelling antibodies labelling only the large spread haemocytes, it turned out that the small spreading haemocytes represent a spread form of granular cells. this finding was later confirmed by both the size and shape of the nuclei, which fits to granular cells quite good, and moreover by the dna-content, which did not exceed that of the typical round granular cells. a clear cut between spread granular cells and spread plasmatocytes could be made by the maximum cell diameter: spread plasmatocytes 22 fig. 5 simplified dichotomous key to circulating larval m. sexta haemocytes. the main determination criteria are given as yes/no questions (grey boxes). the cell types appear in colour-coded boxes, where same colour indicates same cell type. cell types occuring in different morphs (spread and non-spread) are indicated by same colour. numbers correspond to the text-based dichotomous key presented in the main text. please verify determination by referring to fig. 4 and the detailed characteristics listed in the main text and supplementary table s1 always exhibited a maximum diameter of ca. 18 µm or more, while spread granular cells were never longer than 13 µm. this characteristic, however, could vary, e.g. when the cells are cultured over a shorter or longer period of time before fixation or when other substrata than glass slides are present (e.g. poly-l-lysin-coated slides). therefore, these data should be carefully validated separately by each researcher before it can be applied. the ability of adhering granular cells to spread on glass surfaces and forming lamellipodia is known from several lepidopteran insect species. for example bombyx mori (yamashita and iwabuchi, 2001), pseudoplusia includens (lavine and strand, 2002), mythmina unipunctata (ribeiro and brehélin, 2006), galleria mellonella (i̇zzetoğlu, 2012), but we found no literature describing granular cell spreading in m. sexta. owing to the overall seldom appearance of granular cells showing spreading behaviour, which does neither occur in a high number of granular cells nor in each preparation of haemocytes (data not shown), we conclude that these cells may have been overlooked or ignored, or misinterpreted as small plasmatocytes in previous works. which mechanisms underlies the spreading behaviour of granular cells is largely unknown. the plasmatocyte spreading peptide does not influence spreading of granular cells (eleftherianos et al., 2009). it is, however, imaginable that spreading of granular cells is a consequence of degranulation, which leads to a decrease of the cell volume of the granular cells and could promote cell shape changes. additionally, spreading could be influenced by the immune status of the animal. within the non spreading haemocyte types, i.e. round granular cells, round plasmatocytes, spherule cells and oenocytoids, each cell type exhibited a unique combination of characteristics allowing a determination without the use of specific markers. with simple dna-staining and light microscopy, each cell type can be determined based on its combination of cytometric characteristics. while traditional characteristics like “granulated cytoplasm” or ”spherules in the cytoplasm” may be misleading, characteristics like size and shape of cell and nucleus, combined observation of dnacontent and nucleus-to-cytoplasm-ratio allow a relatively reliable determination of each cell. by comparison of each cell to be analysed with the dichotomous key provided in this paper it is possible to determine each haemocyte type. it must be taken care whether the guide is suitable to compare haemocyte types of stages other than l5, if the culturing conditions influence the shape of the haemocytes to be compared, or if 23 physiological conditions such as immune stimulation or starvation may influence the haemocyte appearance. therefore, we recommend for each haemocyte type determination under other conditions than stated in this paper to assess the morphometric data to ensure comparable results. highlights cytometric analyses of m. sexta larval haemocyte types allow their identification a dichotomous identification key for m. sexta haemocytes was created ploidy levels of haemocyte types were identified by relative dapi-intensity nucleus-to-cytoplasm ratio was measured for each unspread haemocyte type funding this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. competing interests the authors declare that they have no competing interests. acknowledgements we wish to express our gratitudes to yvette m. von bredow for drawing figure 5, valuable discussions concerning the data and help in conducting the experiments. we thank michael kanost for providing anti-βgrp-1 immune serum, masaaki ashida for providing anti-b. mori ppo immune serum, graciella mann for providing anti-e. kuehniella haemolymph esterase immune serum, and sabine wagner for technical assistance. the three anonymous reviewers provided very valuable suggestions that helped us to improve the manuscript, and make it more intelligible. therefore we wish to thank the reviewers for their help and detailed comments. authors contributions cvb conceived the study, performed experiments and data analyses, and wrote the manuscript; tet provided lab utilities, antibodies and edited the manuscript. references abràmoff md, magalhães, pj, ram, sj. 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immunochemical identification of insect hemocyte populations: monoclonal antibodies distinguish four major hemocyte types in manduca sexta. eur. j. cell biol., 65, 417-423, 1994. yamamoto r. mass rearing of the tobacco hornworm. ii. larval rearing and pupation. j. econ. entomol., 62, 1427-1431, 1969. yamashita m, iwabuchi k. bombyx mori prohemocyte division and differentiation in individual microcultures. j. insect physiol., 47, 325-331, 2001. 25 supplementary table s1 cytometric data for each m. sexta haemocyte type gr sprgr sp rpl sprpl oe pnam+ min max mean±sd min max mean±sd min max mean±sd min max mean±sd min max mean±sd min max mean±sd min max mean±sd cell l [µm] 5.54 10.11 6.67±0.92 6.86 12.84 9.34±1.38 4.12 6.49 5.43±0.60 6.73 10.37 8.36±0.91 17.88 50.96 31.19±8.79 6.47 12.77 8.66±1.33 4.99 8.41 6.13±0.72 cell w [µm] 5.00 8.33 6.14±0.71 4.25 8.37 6.27±0.91 3.76 5.58 4.72±0.43 6.52 9.30 7.75±0.80 7.48 42.13 20.08±9.21 6.21 10.80 8.16±1.13 4.61 8.36 5.80±0.83 cell l:w 1.01 1.29 1.09±0.08 1.04 2.88 1.52±0.33 1.01 1.37 1.15±0.11 1.00 1.19 1.08±0.05 1.05 3.68 1.76±0.65 1.00 1.19 1.06±0.05 1.00 1.26 1.06±0.08 cell a [µm2] 20.65 59.98 36.30±8.81 32.60 64.79 46.06±7.91 15.54 39.49 23.72±5.32 35.34 75.27 53.05±10.14 133.52 1,787.14 550.49±378.63 34.21 123.26 57.56±17.57 19.66 54.93 30.30±8.00 nuc. l [µm] 3.53 5.27 4.07±0.37 3.43 5.09 4.29±0.41 3.09 4.85 3.79±0.41 5.30 9.23 6.73±0.84 7.45 14.32 9.74±1.51 4.67 7.70 5.80±0.71 3.25 6.03 4.44±0.71 nuc. w [µm] 3.14 4.39 3.75±0.36 2.78 4.51 3.72±0.44 1.58 3.88 2.95±0.50 3.00 7.42 5.01±0.99 4.69 11.17 7.69±1.66 4.28 7.13 5.54±0.66 3.07 5.38 4.11±0.59 nuc. l:w 1.00 1.43 1.09±0.10 1.02 1.47 1.16±0.11 1.00 2.09 1.33±0.26 1.02 2.85 1.39±0.33 1.02 1.92 1.30±0.24 1.00 1.24 1.05±0.05 1.00 1.21 1.07±0.06 nuc. a [µm2] 8.44 19.09 13.00±2.60 9.33 20.28 13.94±2.66 7.73 14.69 10.16±1.55 19.97 40.54 28.37±5.12 21.84 103.79 55.40±20.06 16.42 44.97 26.38±6.80 10.73 29.09 15.95±4.33 gr sprgr sp rpl sprpl oe pnam+ min max mdn±iqr min max mdn±iqr min max mdn±iqr min max mdn±iqr min max mdn±iqr min max mdn±iqr min max mdn±iqr rel. dapiint. 0.73 2.32 1.06±0.51 0.61 2.21 1.28±0.67 0.34 2.03 0.76±0.27 1.44 6.34 3.11±0.59 1.80 7.07 3.81±0.69 1.13 7.06 3.00±1.07 0.65 2.63 1.25±0.46 n:c 0.32 1.45 0.58±0.34 n.d. n.d. n.d. 0.46 2.51 1.02±0.61 0.45 2.73 1.17±0.47 n.d. n.d. n.d. 0.37 1.39 0.85±0.37 0.97 2.81 1.64±1.04 abbreviations: a, area; l, length; l:w, length-to-width ratio; nuc., nucleus; w, width; mean±sd, mean ± standard deviation; mdn±iqr, median ± interquartile range; n:c, nucleus-to-cytoplasm-ratio. numbers of cells and animals analysed for cell length, width and cell l:w: gr cells n = 36, animals n = 3, sprgr cells n = 47, animals n = 3, rpl cells n = 33, animals n = 3, sprpl cells n = 33, animals n = 3, oe cells n = 33, animals n = 3, sp cells n = 27, animals n = 3, pnam+ cells n = 21, animals n = 4. numbers of cells and animals analysed for cell area: gr cells n = 48, animals n = 4, sprgr cells n = 53, animals n = 4, rpl cells n = 35, animals n = 4, sprpl cells n = 32, animals n = 4, oe cells n = 34, animals n = 4, sp cells n = 36, animals n = 4, pnam+ cells n = 17, animals n = 4. numbers of cells and animals analysed for nucleus length, width and nucleus l:w: gr cells n = 35, animals n = 3, sprgr cells n = 52, animals n = 3, rpl cells n = 41, animals n = 3, sprpl cells n = 31, animals n = 3, oe cells n = 35, animals n = 3, sp cells n = 34, animals n = 3, pnam+ cells n = 17, animals n = 4. numbers of cells and animals analysed for nucleus area: gr cells n = 44, animals n = 4, sprgr cells n = 50, animals n = 4, rpl cells n = 33, animals n = 4, sprpl cells n = 38, animals n = 4, oe cells n = 36, animals n = 4, sp cells n = 38, animals n = 4, pnam+ cells n = 18, animals n = 4. numbers of cells and animals analysed for nucleus-to-cytoplasm-ratio (n:c): gr cells n = 62, animals n = 5, rpl cells n = 71, animals n = 3, oe cells n = 55, animals n = 7, sp cells n = 120, animals n = 5, pnam+ cells n = 33, animals n = 5. numbers of cells and animals analysed for dapi-intensity: gr cells n = 86, animals n = 3, sprgr cells n = 112, animals n = 5, rpl cells n = 76, animals n = 5, sprpl cells n = 60, animals n = 5, oe cells n = 66, animals n = 5, sp cells n = 174, animals n = 5, pnam+ cells n = 46, animals n = 5 26 supplementary table s2 comparison of cell dimensions and statistical analysis (p-values) of the cytometric data shown in fig. 2, one-way-anova and post-hoc pairwise analysis (tukey-test) haemocyte length [µm] haemocyte width [µm] gr sprgr sp rpl sprpl oe gr sprgr sp rpl sprpl oe sprgr 0.0110398 sprgr 0.9999984 sp 0.7975833 0.0001074 *** sp 0.7088514 0.5545922 rpl 0.4080174 0.8757483 0.0221555 * rpl 0.503945 0.5338877 0.0213787 * sprpl 0 *** 0 *** 0 *** 0 *** sprpl 0 *** 0 *** 0 *** 0 *** oe 0.2151145 0.9772673 0.0074831 0.999851 0 *** oe 0.2280589 0.2351917 0.0048936 ** 0.9992203 0 *** pnam+ 0.9975917 0.0092327 ** 0.9928197 0.2488556 0 *** 0.1284124 pnam+ 0.9998618 0.9988498 0.9447655 0.4480311 0 *** 0.2191943 haemocyte length/width haemocyte area [µm2] gr sprgr sp rpl sprpl oe gr sprgr sp rpl sprpl oe sprgr 0 *** sprgr 0.9996648 sp 0.976258 0.0000101 *** sp 0.9995434 0.9846875 rpl 0.9999999 0 *** 0.963466 rpl 0.9977688 0.9999938 0.969715 sprpl 0 *** 0.0062833 ** 0 *** 0 *** sprpl 0 *** 0 *** 0 *** 0 *** oe 0.9997856 0 *** 0.8927594 0.9999716 0 *** oe 0.9921326 0.9998405 0.9418144 0.9999994 0 *** pnam+ 0.9999321 0.0000002 *** 0.9409127 0.9999922 0 *** 1 pnam+ 0.9999987 0.9993978 0.9999982 0.997557 0 *** 0.9935715 level of significance indicated by asterisks: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. for sample size, please refer to supplementary table s1 27 supplementary table s3 comparison of nuclei dimension and statistical analysis (p-values) of the data shown in fig. 2, one-way-anova and post-hoc pairwise analysis (tukey-test) nucleus length [µm] nucleus width [µm] gr sprgr sp rpl sprpl oe gr sprgr sp rpl sprpl oe sprgr 0.8774779 sprgr 0.9999988 sp 0.7310479 0.0647721 sp 0.0016233 ** 0.0008057 *** rpl 0 *** 0 *** 0 *** rpl 0 *** 0 *** 0 *** sprpl 0 *** 0 *** 0 *** 0 *** sprpl 0 *** 0 *** 0 *** 0 *** oe 0 *** 0 *** 0 *** 0.0000073 *** 0 *** oe 0 *** 0 *** 0 *** 0.0833951 0 *** pnam+ 0.676252 0.9912137 0.0726404 0 *** 0 *** 0.0000002 *** pnam+ 0.7670549 0.6423261 0.0000905 *** 0.0047555 ** 0 *** 0.0000005 *** nucleus length/width nucleus area [µm2] gr sprgr sp rpl sprpl oe gr sprgr sp rpl sprpl oe sprgr 0.7104003 sprgr 0.9983098 sp 0.0000509 *** 0.0051948 ** sp 0.7499775 0.3874797 rpl 0 *** 0.0000021 *** 0.768254 rpl 0 *** 0 *** 0 *** sprpl 0.0004703 *** 0.0298539 0.9996884 0.5241554 sprpl 0 *** 0 *** 0 *** 0 *** oe 0.9707772 0.1490822 0.0000006 *** 0 *** 0.0000092 *** oe 0 *** 0 *** 0 *** 0.9611964 0 *** pnam+ 0.9999959 0.7894768 0.0010933 0.0000034 *** 0.0047575 ** 0.99811 pnam+ 0.8813264 0.9790609 0.2210812 0.000029 *** 0 *** 0.0006936 *** 63 isj 17: 63-74, 2020 issn 1824-307x research report study of the proliferative activity of hemolymph cells in pulmonate molluscs as tokmakova1, mk serebryakova2, ee prokhorova1, gl ataev1* 1laboratory of experimental zoology, department of zoology, faculty of biology, herzen state pedagogical university of russia, st. petersburg, russia 2department of immunology, institute of experimental medicine, st. petersburg, russia this is an open access article published under the cc by license accepted may 7, 2020 abstract hemocytes, the cell of the hemolymph, play a key role in the immune response of pulmonate molluscs to various pathogens including trematode infection. the number of hemocytes is known to increase after immunization but the mechanism of their multiplication remains debatable, proliferative capacity being the stumbling block. some scientists consider that hemocytes may proliferate, while others think that their multiplication is only possible in special hematopoietic centres. in this work we studied the proliferative activity of hemocytes in five species of pulmonate molluscs: biomphalaria glabrata, planorbarius corneus, planorbis planorbis, lymnaea stagnalis and succinea putris. imagestream technique was used for the study of the hemocyte populations of these molluscan species for the first time. the hemocytes of all the studied species were represented by two main types, granular cells and hyalinocytes. microscopic and flow-cytometric study of the hemocytes with the use of edu revealed some edu-positive cells. however, the analysis of the cell cycle of the hemocytes showed that the amount of dna in these cells was not increased. thus, it remains unclear whether the cells of the hemolymph retain the capacity to multiplication. key words: pulmonate molluscs; hemocytes; proliferation; edu; flow cytometry; imagestream introduction hemolymph cells of pulmonate molluscs, hemocytes, are usually considered as the major effectors of molluscan defence reactions to various pathogens including trematode invasion (sminia et al., 1974; lie and heyneman, 1976; van der knaap and loker, 1990; ataev and coustau, 1999; connors, 2003; pila et al., 2016). hemocytes are generally divided into two main types: granular cells (the term granulocytes is also widely used to refer to these cells) and hyalinocytes (see discussion). despite intensive studies of morphology and functional activity of hemocytes, there is still no consensus about the nature and the mechanism of hemopoesis in pulmonate molluscs. many authors consider that the an amoebocyte-producing organ (apo), located between the pericardial and the mantle epithelium, is a common hemopoetic centre (pan, 1958; jeong et al., 1983; joky et al., 1983; sullivan, 1988). immunization of molluscs with various pathogens is shown to intensify the ___________________________________________________________________________ corresponding author: gennady l. ataev department of zoology herzen state pedagogical university of russia 191186, moyka emb. 48, saint-petersburg, russia e-mail: ataev@herzen.spb.ru proliferation of its cells (lie and heyneman, 1976; sullivan, 1988; ataev and prokhorova, 2013). other scientists think that the origin of hemocytes is polycentric and that they may also form from the cells of the molluscan connective tissue (souza and andrade, 2006). according to a third hypothesis, the proliferation of circulating cells of the snail hemolymph is also possible (sminia et al., 1983; monteil and matricon-gondran, 1991; portet et al., 2019). the proliferative potential of cells is often assessed with the use of brdu (dolbeare, 1995; terry and white, 2001; rodriguez et al., 2018 et al.) but this technique has limitations. one of them is that dna denaturation, which is necessary for revealing the label, also destroys most protein epitopes. these complications can be avoided by using a technique based on 5-ethynyl-2deoxyuridine (edu) (buck et al., 2008; cappella et al., 2008). being an analogue of thymidine, edu allows one to register the instances of dna synthesis in the nuclei. this technique has mostly been used for the study of cells of vertebrates (salic and mitchison, 2008; lin et al., 2009; chehrehasa et al., 2009; zeng et al., 2010; fabrice et al., 2015) but portet et al. (2019) successfully used it to determine proliferative activity of hemolymph cells in molluscs 64 fig. 1 hemocyte morphology in pulmonate molluscs b. glabrata, p. corneus, p. planorbis, s. putris, l. stagnalis. fluo – fluorescence images of phalloidin-rhodamin (red) and hoechst (blue) stained hemocytes, ph – phase contrast microscopy images of living cells. bar = 10 μm biomphalaria glabrata. they showed that edu accumulated in the nuclei of some hemocytes and interpreted this observation as an evidence of the possible proliferation of the circulating cells (portet et al. 2019). however, the accumulation of edu may also indicate some other processes such as reparation. we agree with the hypothesis that hemocytes of pulmonates multiply only in special centres such as аро. these centres probably contain stem hematopoietic cells, which can divide and differentiate into the hemolymph cells. at the same time, there are occasional reports about the proliferative capacity of the circulating cells of gastropods, including pulmonates. to check this capacity, we conducted a complex study with the use of cytometric and microscopic methods of five species of pulmonate molluscs: freshwater snails b. glabrata, planorbarius corneus, planorbis planorbis, and lymnaea stagnalis and a land snail succinea putris. 65 fig. 2 hemocyte populations of molluscs b. glabrata, p. corneus, p. planorbis and l. stagnalis identified by flow cytometry based on forward (fsc) and side (ssc) scatter. population a corresponds to hyalinocytes, while populations в and с are subpopulations of granular cells materials and methods snails freshwater snails planorbarius corneus l., 1758, planorbis planorbis l., 1758, lymnaea stagnalis l., 1758 and land snails succinea putris l., 1758 used in this study were collected in the leningrad region of russia. snails biomphalaria glabrata say, 1818 (laboratory strain) were obtained from the “interactions hôtes-pathogènesenvironnements” laboratory of the perpignan university (france) in 2011. the strains of p. corneus have been kept in the laboratory since 2010 (prokhorova et al., 2015). the snails of all species were kept in aerated water tanks on a 12l/12d cycle at a temperature of 22 23 °с. the snails collected in natural water bodies were brought to the laboratory and placed into glass jars. the jars were screened for the presence of cercariae, which are shed by snails infected with trematodes. the hemolymph of the seemingly uninfected snails was sampled, and the snails were dissected to ascertain the lack of infection. only the hemolymph of uninfected snails was used for the study. all the hemolymph samples were analysed immediately after sampling. land snails s. putris (prokhorova et al., 2020). were kept in containers with moist soil (temperature 22 23 °c, air humidity 60 70 %). snails collected in the nature were kept in the laboratory for not more than 1 2 before the experiment. all snails were fed on lettuce leaves once in two days. hemolymph collection, incubation and sample preparation нemolymph was collected from the pericardial region of the snail using glass pasteur pipettes (sminia, baredsen, 1980). morphological analysis of hemocytes was performed on temporary and fixed preparations. for in vitro studies, freshly sampled 66 table 1 relative number of cells of different populations in the hemolymph of molluscs b. glabrata, p. corneus, p. planorbis, l. stagnalis based on cytofluorimetric analysis. snail species (sample size) hemocyte populations based on forward and side scatter a b c biomphalaria glabrata (n = 20) 54.09 (45.97; 63.68)*,** 28.80 (20.67; 32.03)*, ^^ 13.98 (11.58; 18.59)*,^^ planorbarius corneus (n = 18) 44.14 (37.15; 55.26)* 35.66 (26.13; 41.36)*,^^ 18.49 (13.10; 26.84)*,^ planorbis planorbis (n = 19) 48.03 (38.58; 59.59)* 16.11 (12.07; 23.30)* 28.39 (20.14; 38.62)* lymnaea stagnalis (n = 22) 11.56 (8.06; 20.74) 83.03 (72.01; 87.46) 4.66 (3.59; 7.35) data are given as percentage from the total number of analysed single cells in the sample as median values and interquartile range (%, ме [q25; q75]). * р < 0.001 when compared with lymnaea stagnalis, ** р < 0.05 when compared with planorbarius corneus, ^ p < 0.05 when compared with planorbis planorbis, ^^ p < 0.005 when compared with planorbis planorbis hemolymph was smeared on object slides with an adhesive covering and analysed using phase contrast. for phalloidin staining, smears of hemocytes were fixed in 4 % paraformaldehyde in pbs and incubated for 10 min. after washing twice in pbs, cells were preincubated in pbs with 0.1 % tritonx-100 for 5 min and then incubated with phalloidin-rhodamin (thermo fisher scientific) diluted with pbs for 30 min at room temperature. the nuclei of hemocytes were counterstained with hoechst (thermofisher scientific). to study proliferative activity of hemocytes, the hemolymph was incubated with 20 mm edu for 30 min on object slides in a moist chamber. edu label was exposed with the use of click-it edu alexa fluor 594 imaging kit according to the manufacturer’s protocol (thermofisher scientific). the nuclei of hemocytes were counterstained with hoechst (thermofisher scientific). the preparations were analysed using a fluorescent microscope leica dmi8. the images were processed with the help of lasx software. flow-cytometric assay the composition of the population of hemolymph was studied using flow cytometer bd accuri c6 (bd bioscience, usa). for this, freshly drawn hemolymph was diluted with chernin buffer (chernin, 1968) in the proportion of 1 : 1 (for p. corneus and b. glabrata) and 1 : 4 (for p. planorbis). hemolymph of l. stagnalis was studied without dilution. hemolymph samples were not pooled, and the hemolymph of each individual was analysed separately. the composition of the hemocyte population was determined based on forward and side scatter (see: prokhorova et al., 2018a, b). the debris was removed based on cell staining with a vital stain syto62 (invitrogen, usa) specific to nucleic acids. the stain was added to the freshly sampled hemolymph (final concentration, 250 nm), incubated for 10 min and immediately analysed using the flow cytometer. syto62-negative events were considered as debris and excluded from the analysis zone. the aggregates were excluded successively based on the proportion of the peak (h) and the integral signals (a) registered for the parameters of the forward and the side scatter. at least 5,000 events were analysed for each hemolymph sample of b. glabrata; at least 20,000 events for p. corneus, at least 30,000 events for l. stagnalis. for the analysis of proliferative activity of hemocytes with the help of flow cytometry, the hemolymph was incubated with 20 mm edu for 30 min in cytometric tubes (sarstedt, germany). edu was exposed as described above. at the last stage of the sample preparation, dapi dna-binding stain was added to the samples for the subsequent analysis of the distribution of the circulating hemolymph cells across the cell cycle stages (firsanov et al., 2017). monocytic leukemia thp-1 cells, for which the amount of proliferating cells in the culture is about 30 %, were used as positive control. samples for thp-1 were prepared according to the protocol used for the preparation of hemocytes. hemocytes not incubated with edu but subjected to all the previous stages of the protocol were used as negative control. the samples were analysed with the use of a navios 10/3 flow cytometer (beckman coulter, usa). not less than 5000 dna-containing events were analysed for each sample. imagestream assay we also used an amnis® imagestream® imaging flow cytometer (luminex, usa), which makes it possible to obtain cytometric data about a hemolymph sample and simultaneously to visualize events on a cytogram. signals characterising cell morphology (ch01, ch06) and fluorescence syto62 by ch11 channel were registered. not less than 200,000 events, including gauge particles, were analysed for all the samples. the data obtained with the help of imagestream technology were used to construct a two-parameter histogram, where the values of cellular complexity (intencity mc ch06) were plotted along the x-coordinate and the values of cell area (area m01) were plotted along the y-coordinate. area m01 is the parameter closest to the forward scatter of the classical flow cytometer. 67 fig. 3 populational analysis of circulating hemocytes of molluscs b. glabrata and p. corneus performed with the help of amnis® imagestream imaging flow cytometer. hemolymph histograms (x-coordinate – cellular complexity, y-coordinate – cell area) and visualised events are shown. а, в, с — hemocyte populations based on forward and side scatter, agr — aggregates. bar = 7 μm statistical analyses the cytometric data were processed using kaluza™ v.2.0 software (beckman coulter, usa). cell cycle parameters were calculated in the automatic mode with the help of cellcycle module incorporated in kaluza™ v.2.0. statistical treatment was conducted with the use of ms excel (microsoft, usa), statistica 8.0 (statsoft, usa) and graphpad prism 6.01 (graphpad software inc, usa). the normality of distribution in the samples was assessed with the help of kolmogorov-smirnov’s test. the results were represented as a median (ме) and an interquartile range (q25, q75). results morphological types of hemocytes in micrographs obtained with the help of light and fluorescent microscopes, the hemocytes of all studied molluscs could be divided into two types: granular cells and hyalinocytes (ataev et al., 2016; prokhorova et al., 2018 a,b). all hemocytes have a well-developed actin cytoskeleton ensuring shape maintenance (fig. 1). granular cells are larger, contain numerous granules, form filopodia and spread out easily on the substrate. hyalinocytes are agranular, rounded and may form lobopodia. 68 fig. 4 edu incorporation in nuclei of hemocytes of pulmonate molluscs. ph -phase-contrast microscopy. fluo – fluorescence photomicrographs of hemocyte nuclei stained with hoechst (blue) and edu (red) pools of hemocytes detected by flow cytometry based on the results of the cytofluorimetric analysis of the hemolymph, the hemocytes were divided into two main populations: а and в (fig. 2, table 1). the values of forward and side scatter indicated that the cells of population a were small and had a relatively simple structure. the cells of population в were located further along both xand ycoordinate, that is, were larger and more complexly organised because of the presence of numerous granules and vesicles. depending on the snail species, the hemolymph contained from 5 to 29 % of heterogeneous hemocytes, which were, on the average, somewhat smaller than granular cells of population b (table 1). based on the results of the cytometric analysis, we denoted these as a separate subpopulation с (fig. 2). this group of cells was especially noticeable in p. planorbis (table 1). in general, the proportion of different cell populations in the hemolymph varied in molluscs of different species. for instance, more than 80 % of the 69 fig. 5 distribution of molluscan hemocytes across cell cycle phases based on cytofluorimetric analysis of dapitreated hemolymph hemocytes of l. stagnalis were granular cells, which means this snail differs statistically significantly (p < 0.0001) from the other snail species, in which hyalinocytes predominated. the hemolymph of molluscs b. glabrata (n = 10) and p. corneus (n = 10) was also analysed with the help of imagestream technique (fig. 3). the results confirmed the presence in the hemolymph of two main cell types, hyalinocytes and granular cells (population а and в). however, it can be seen on the cytograms that subpopulation с includes small strongly vacuolated cells that look like cells at the terminal stages of the apoptosis, as well as cell debris. in this way, the results of imagestream analysis made it possible to correct the gating tactics during the analysis of the cytograms obtained with the use of flow cytometer and to exclude the cells of population с from the analysis. proliferative activity of the hemocytes and cell cycle to study the proliferative activity of the hemocytes, we conducted microscopic (fig. 4) and cytometric analysis (fig. 5-8) of edu incorporation into dna of molluscan hemocytes at the background of their distribution across the cell cycle phases. we found that edu accumulated in the nuclei of some hemocytes of molluscs b. glabrata (n = 42), p. corneus (n = 45), p. planorbis (n = 10), l. stagnalis (n = 29) and s. putris (n = 12). these results in general confirmed the data obtained on b. glabrata by portet et al. (2019). at the same time, the analysis of the cell cycle showed that most of the hemocytes were in the phase g0/g1 (fig. 5). the percentage of hemocytes in this phase from the total number of these cells made up 91.82 (86.60; 94.01) % for b. glabrata, 92.77 (92.00; 93.94) % for p. corneus and 93.98 (92.92; 94.88) % for l. stagnalis. the percentage of hemocytes in the synthesis phase was 5.92 (4.93; 9.08), 1.37 (0.79; 2.45) and 1.55 (0.93; 2.51) % for b. glabrata, p. corneus and l. stagnalis, correspondingly. dna amount of other hemocytes corresponded to phase g2 and mitosis. the number of such cells was 1.31 (0.31; 4.12), 5.51 (4.33; 6.19) and 4.36 (3.65; 5.44) % for b. glabrata, p. corneus and l. stagnalis, correspondingly. cytometric analysis of edu incorporation into the dna of hemocytes showed that most of the edu-positive cells (fig. 6a) were located in the upper right part of the fs/ss histogram (fig. 6b). however, an important aspect of cytometric study of proliferative activity of any cells is the exclusion of aggregates from the analysis. we identified single cells based on the proportion of the peak and the integral signal of dapi fluorescence and showed that most of the edu-positive events were situated in the area of aggregates and cell debris containing a lowered amount of dna (fig. 6c). monocytic leukemia thp-1 cells, for which the amount of proliferating cells in the culture is about 30 %, were used as positive control (fig. 7). after the exclusion of cell aggregates and cell debris from the analysis zone, edu-positive hemocytes in b. glabrata made up 5.8 % (5.57; 6.21) of the total number of single cells (fig. 6c). most of these cells, 4.02 % (3.45; 4.94), were located in the area of g0/g1 peak based on the amount of dna (fig. 8). in p. corneus the percentage of edu-positive hemolymph cells was lower, making up 2.89 % (1.59; 3.53) of the total number of single hemocytes. more than a half of them, 2.03 % (0.78; 2.72), were located in the area of g0/g1 peak (fig. 8). however, for l. stagnalis the number of stained cells in the samples incubated with edu did not exceed the corresponding value for negative control, and so no conclusions about edu incorporation into the dna of the hemocytes could be made (fig. 7). discussion classification of hemocytes in pulmonates remains debatable. from one to several tens of types of hemolymph cells have been described. however, most authors (sminia, 1972; cheng, 1975; jourdane and cheng, 1987; ataev et al., 2016; pila et al., 2016) identify two main populations of hemocytes based on morphological characters: hyalinocytes (agranular cells) and granular cells (easily spreading cells, whose cytoplasm contains numerous granules and vesicles). the difference between hemocyte types has been shown both at the light and at the electron-microscopic level (joky et al., 1983; ottaviani and franchini, 1988; cueto et 70 fig. 6 cytofluorimetric analysis of edu incorporation into hemocytes of molluscs b. glabrata а) identification of edu-positive events from all events in a sample. x-coordinate –edu fluorescence intensity, y-coordinate – number of cells b) localization of edu-positive events (red) on the histogram, where ss axis is side scatter and fs axis is forward scatter. all registered events are presented on the histogram c) identification of single cells based on dapi incorporation, exclusion of aggregates and debris. dapi int axis — integral signal of dapi fluorescence intensity, dapi peak axis — peak signal of dapi fluorescence intensity. edu-positive events are shown in red d) localization of edu-positive events (red), where ss axis is side scatter and fs axis is forward scatter. only single cells are shown in the histogram al., 2015; prokhorova et al., 2018b and others). however, morphological characters of hemocytes of different types have never been clearly formulated because of their polymorphism. this might be the reason why additional types (subpopulations) of hemolymph cells of snails have often been described (joky et al., 1983; cavalcanti et al., 2012; cueto et al., 2015; rodriguez et al., 2018). in our earlier studies we also described four subpopulations of cells with different functional characteristics in each hemocyte population in the hemolymph of b. glabrata, p. corneus and p. planorbis based on cytometric analysis with the use of specific fluorescent dyes, a lysosome dye (lysotracker) and a nucleic acid dye (syto62) (prokhorova et al., 2018a,b). fig. 7 histograms showing superimposition of fluorescence signals of cells incubated with edu (green) and negative control – cells without incubation with edu (red) 71 fig. 8 cytofluorimetric analysis of hemocyte distribution across cell cycle phases with the use of dapi and edu. a) negative control without edu incubation, b) experiment. dapi int axis — integral signal of dapi fluorescence intensity, edu_fl3_int axis — integral signal of edu fluorescence intensity. edu-positive events corresponding to g0/g1 phase are shown in red the results of this study confirmed that the circulating cells in of hemolymph of b. glabrata, p. corneus, p. planorbis, l. stagnalis and s. putris were represented by two main populations: granular cells and hyalinocytes. these cells differ in their morphology, adhesive capacity and functional activity (ataev et al., 2016; prokhorova et al., 2018 a,b). as noted above, the classification of hemocytes based on morphological characters may not fully reflect the functional features of these cells, which may be expressed during their specialization. for instance, at first, with the help of flow cytometric analysis of the hemolymph of b. glabrata, p. corneus, p. planorbis and l. stagnalis, we revealed subpopulation с represented by small granular cells (fig. 2). however, the use of imagestream technology showed that this population was represented by degranulating cells and/or cells undergoing apoptosis. as a result, we excluded these cells from the further analysis. 72 thus, the use of imagestream technique in general confirmed the results obtained by flow cytometry. based on the size and signal intensity assessing the cellular complexity, hemocytes were represented by two main populations (fig. 3). they evidently corresponded to population a (hyalinocytes) and population b (granular cells) identified based on morphological and cytometric analysis (fig. 1, 2). the study of the hemolymph of b. glabrata with the use of edu confirmed the results of portet et al. (2019) that edu is incorporated into hemocyte nuclei. these authors have reported the relative number of these cells based on the results of microscopic analysis: 1.16 % in uninfected snails; 2.57 % in snails with sympatric infection; 5.19 % in snails with allopatric infections. these results are difficult to comment upon since some hemocytes are lost in the process of making the preparations (especially during washing). however, the authors also report the results of flow cytometry of the hemolymph with the use of edu. their data generally show that the number of edu-positive cells increased up to 2 % in molluscs with sympatric infection and up to 4.2 6.8 % in molluscs with allopatric infection. we cannot provide a reliable percentage of edu-positive cells based on microscopic analysis because such counts may be biased (see above). based on cytometric analysis, the proportion of such cells in uninfected b. glabrata and p. corneus varies but also does not exceed several per cent. moreover, the study of hemolymph of these molluscs with a simultaneous use of edu and dapi also revealed a small percentage of cells in phase s and phase g2/m. however, it is the edu-positive cells that were found among hemocytes in phase g0/g1 (fig. 8). the incorporation of edu into the nuclei of the circulating cells may be associated with the dna repair in them. to the best of our knowledge, no studies of this kind have been conducted on molluscan hemocytes but there are such data for vertebrates (kim et al., 2019; wiley et al., 2019). in this case, the number of edu-positive hemocytes may increase after immunization and, in particular, after infection of molluscs with trematodes. another explanation of this phenomenon is also possible. in vertebrates, bone marrow is the central organ of hemopoesis but differentiated and extremely specialised cells, the macrophages, can proliferate in tissues (lin and stewart, 1973; luo et al., 2008; robbins et al., 2013). this mechanism, alongside with the recruitment of monocytes, is considered as an alternative means of increasing the numbers of tissue macrophages in damaged tissues during inflammation (mlcochova et al., 2017). besides, clonal expansion of lymphocytes occurs in vertebrates during antigen-dependent differentiation (fox and smith, 1986; janeway et al., 2001; zink et al., 2017). a similar mechanism may also be at work in pulmonates. in such a case, prohemocytes that have left the hemopoetic centre (apo) before differentiation into hemocytes may retain the ability to divide. in our opinion, it is also possible that a small percentage of hemocytes retain proliferative capacity that may be activated under certain conditions. for instance, local proliferation of snail hemocytes may be stimulated by cytokines secreted by the cells of damaged tissues as well as metabolic products of pathogens. acknowledgements the research was supported by rfbr according to the research project no. 20-54-15003. imagestream study was 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wide web newsgroup. we felt that speed and facilitated distribution, together with an absence of publishing costs, were important elements. at the same time, the reliability and academic rigour of the published items must be guaranteed. consequently, we have decided to launch an online journal that will allow rapid publication of papers without distribution expenses, while also offering additional important advantages such as linking to referenced articles, database entries, supplier information, etc. moreover, we have preferred a free and online journal in order to guarantee unlimited access to the published papers and attract the largest number of readers. the only element that could affect the publication time is the peer-review of articles, but this procedure is imperative to guarantee the quality level of the contributions. however, as an online journal, all steps from submission through reviewing and editing to final formatting are managed through the isj' web server, so avoiding the need to transmit texts and figures by electronic or standard mail. enzo ottaviani isj editor 414 isj 14: 414-422, 2017 issn 1824-307x research report cloning and expression analysis of a stomatin gene from the sea cucumber apostichopus japonicas s cheng, y chen, y chang, k li, x zhang, s shang, g li, l li key laboratory of mariculture & stock enhancement in north china’s sea, ministry of agriculture, dalian ocean university, dalian 116023, pr china accepted october 25, 2017 abstract stomatin was the first member of the stomatin-prohibitin-flotillin-hflc/k (spfh) superfamily proteins to be studied. it is also known as band 7 integral membrane protein. in this study, a stomatin gene in the sea cucumber apostichopus japonicus (designated as ajsto) was identified and characterized. its cdna was 2085 bp in length including 131 bp of 5’-utr, 1117 bp of 3’-utr, and 837 bp of open reading frame (orf) encoding a putative protein of 279 residues with a spfh domain of band 7 family, a predicted molecular mass of 30.5 kda and a theoretical pi of 5.25. protein structure prediction and phylogenetic analysis showed that ajsto is highly conserved as compared to those from other vertebrate and invertebrate species. analysis of ajsto expression in the tissues of a. japonicas showed that the respiratory tree and body wall had the highest expression, followed by the intestine, celomocytes, tube feet, and longitudinal muscle. time-course analysis of ajsto expression in the celomocytes revealed obvious and significant inhibition of expression following vibrio splendidus challenge, with a 0.18-fold reduction after 6 h of exposure to the bacteria compared to the control, but the expression was up-regulated by 2.12-fold after 72 h of exposure. these results suggested that ajsto might play critical roles not only by acting as the major integral protein of erythrocyte lipid rafts, but may also involved in the innate immune defense against bacterial infections. key word: stomatin; apostichopus japonicus; tissue distribution; temporal expression introduction stomatin was first identified in human erythrocyte in 1991 and was also termed band 7 membrane protein (hiebl-dirschmied et al., 1991a). stomatin plays an important role in the modulation of k+ and na+ permeability in red blood cells. absence or partial deficiency of the stomain gene can cause overhydrated hereditary stomatocytosis, which is a form of autosomal dominant hemolytic anemia (lande et al., 1983; hiebl-dirschmied et al., 1991b; salzer et al., 1993). stomatin belongs to the superfamily of stomatin-prohibitin-flotillin-hflc/k (spfh) proteins, which also includes prohibitin, flotillin and hflk/hflc (gehl and blatt, 2009; lapatsina et al., 2012; chi and hu, 2016). members of this superfamily possess a representative spfh domain that is involved in regulating targeted protein ___________________________________________________________________________ corresponding author: yaqing chang key laboratory of mariculture & stock enhancement in north china’s sea dalian ocean university 52 heishijiao road dalian, liaoning province 116023, p. r. china e-mail: yaqingchang@hotmail.com turnover between stomatins and other membrane-associated proteins (tavernarakis et al., 1999; browman et al., 2007). as a member of the spfh superfamily, stomatin is the major membrane-bound protein that participates in the regulation of membrane-associated proteins. stomatin in particular, has a single hydrophobic domain that plays a critical role in the regulation of ion transport (stewart, 1997; chen et al., 2005). furthermore, proteins of the stomatin family contain a conserved core stomatin-domain spanning a region of 150 amino acids, and this domain defines a family of proteins that is found in an ancient duplication event which occurred early on in the evolution of prokaryotes (green and young, 2008). five main members of the mammalian stomatin family have been found, and they include stomatin, stomatin-like protein 1(slp-1), slp-2, slp-3 and podocin (lapatsina et al., 2012; chi and hu, 2016). moreover, the spfh superfamily includes another branch named mechanosensory protein 2 (mec-2). the mouse mec-2 protein is similar to stomatin from caenorhabditis elegans, as these two proteins share over 65 % sequence identity in the stomatin domain. at least nine stomatin-like proteins have been 415 identified in c. elegans (lapatsina et al., 2012). all the available data show that members of the spfh superfamily are all homologous and the stomatin domains in these proteins are remarkably conserved. stomatin is widely expressed in various types of cells, such as erythrocyte, diseased cells including cancer cells and tumor cells, and nerve cells. stomatin deficiency will lead to overhydrated hereditary stomatocytosis. (hiebl-dirschmied et al., 1991a; fricke et al., 2000; salzer and prohaska, 2001; arkhipova et al., 2014; chen et al., 2016). existing data show that stomatin is closely linked to diseases, but the precise function of stomatin remains unclear. chi and hu found that stomatin-like protein 2 of turbot play a vital role in host immune defense against bacterial and viral pathogens (chi and hu, 2016). structurally, the protein contains a closed n-terminus and a single hydrophobic domain (gehl and blatt, 2009). in addition, it also as an independently organized higher order oligomer, which acts as a separate scaffolding component at the cytoplasmic face of erythrocyte lipid rafts (salzer and prohaska, 2001; green and young, 2008; lapatsina et al., 2012). functionally, stomatin binds to glucose transporter-1 (glut1) and modulates the rate of glucose uptake, a function that may involve the interaction with membrane-bound scaffolding protein modulating transport proteins (zhang et al., 1999; rungaldier et al., 2013). stomatin exerts the most prominent effect on acid-sensing ion channels (asics), and it achieves this by inhibiting the acid-evoked current. asics as h+ gated channels are involved in the sensing of acidosis associated with painful conditions such as skin and muscle inflammation (brand et al., 2012; moshourab et al., 2013). although the wide distribution of stomatin indicates that it has an important role, its physiological function in invertebrates as well as the mechanisms associated with the diseases that it causes in these animals have never been studied before. it was necessary to explore the properties of stomatin in sea cucumber, an invertebrate of high economic value. since 2004, diseases like skin ulceration syndrome (sus) have already caused mass mortalities and resulted in serious economic losses for the sea cucumber farming industry (yan et al., 2014). the sea cucumber apostichopus japonicas is an economically important aquaculture species in china, japan, south korea and russia. however, the outbreak of sus has severely limited the sustainable development of the industry (chang et al., 2009). analysis of the bacterial strain isolated from the lesions of sea cucumbers suffering from sus revealed similarity to vibrio splendidus. although the main pathogenic bacterium responsible for sus has been confirmed, there has been no effective measure to prevent the occurrence of sus (deng et al., 2009). many innate immune genes of sea cucumber have been characterized, like mitogen-activated protein kinase kinases, tnf receptor associated factors and thioredoxin (cheng et al., 2016; wang et al., 2016; yang et al., 2016), but so far there has been no study on the stomatin gene regarding its role in the immune responses. in this study, we described the identification and characterization of a stomatin gene from a. japonicus and analyzed its expression in six different tissues of healthy adult a. japonicus individuals and in the celomocytes of a. japonicus individuals that had been challenged with v. splendidus. materials and methods samples preparation and bacterial challenge experiment healthy apostichopus japonicus individuals (body weight 68 ± 4.59 g) were collected from dalian and kept at 16 17 ℃ in our laboratory for one week. three animals were sacrificed and their tissues, including the body wall, intestine, respiratory tree, body wall, tube feet, celomocytes, and longitudinal muscle were extracted and subjected to spatial expression analysis. to harvest the celomocytes, the celomic fluid was collected and centrifuged immediately at 1,000g for 5 min at 4 ℃. the celomocytes were immediately snap-frozen in liquid nitrogen and stored at -80 ℃. table 1 primer sequences used for ajsto cloning and expression analysis primers sequences (5′-3′) application melting temperatures ajsto-5’-out caatgaccttcgcacgggctt 5’-race 56℃ ajsto-5’-in cctggtccgttgtctttcgcc 5’-race 56℃ ajsto-3’-out tatttcccctttgcttttgcc 3’-race 56℃ ajsto-3’-in ggggtttgcttattacgctgg 3’-race 56℃ ump-1 taatacgactcactatagggcaagcagtggtatcaacgcagagt race 56℃ ump-2 ctaatacgactcactatagggc race 56℃ ajsto-f cacgcttcccttctctctgttct qpcr 60℃ ajsto-r gaaggagtcaatgcagggtagtatg qpcr 60℃ cytb-f tgagccgcaacagtaatc reference gene 60℃ cytb-r aagggaaaaggaagtgaaag reference gene 60℃ 416 total rna extraction and cdna synthesis total rna was extracted from a. japonicus using an rnaprep pure tissue kit (tiangen, china) according to the instructions of the manufacturer. the quality and quantity of the rna were assessed by 1 % agarose gel electrophoresis and uv spectrophotometry, respectively. uv spectrophotometry was performed on a nanophotometer (munich, germany). the first strand cdna was synthesized in a 10-μl reaction mixture containing 1 g of total rna, 2 μl of 5× primerscript buffer, 0.5 μl of oligo dt primer (50 μm), 0.5 μl of random 6 mers (100 μm), and 0.5 μl of primerscript rt enzyme mix (primerscripttm rt reagent kit, takara, japan) and rnase free dh2o. the sample was incubated at 37 ℃ for 15 min, followed by heating at 85 ℃ for 5 s to denature the reverse transcriptase. all cdna samples were stored at -20 ℃ until used. gene cloning and sequencing analysis the partial cdna sequence of stomatin was acquired from our transcriptome assembly data (unpublished data). gene specific primers for stomatin were designed by primer premier 5.0. all primers are listed in table 1. 5’and 3’-race by the smarter®race 5’/3’ kit (takara,japan) were performed according to the manufacturer’s instructions. the polymerase chain reaction (pcr) was performed in a 25-μl reaction mixture containing 2.5 μl of 3’-race-ready cdna, 1 μl of gene specific primer (gsp, 10 μm),1 μl of 10×upm, 12.5 μl of 2×transstart® fastpfu pcr supermix (transgen biotech, china) and 8 μl of ddh2o. the pcr conditions were as follows: an initial denaturation step at 94 ℃ for 3 min followed by 35 cycles of denaturation at 94 ℃ for 30 s, annealing at 55 ℃ for 30 s and extension at 72 ℃ for 1 min, and a final extension step at 72 ℃ for 10 min. the pcr product was detected by 1.0 % agarose gel electrophotesis and purified using the easypure quick gel extraction kit (transgen biotech, china). the purified pcr product was ligated to peasy®-1 cloning vector (transgen biotech, china) and trans1-t1 phage resistant chemically competent cells (transgen biotech, china) were transformed with the ligation products. positive transformants were verified by colony pcr using m13 primers (transgen biotech, china). three independent clones were subjected to dna sequencing to confirm the presence of the correct insert. bioinformatics analysis of ajsto the full-length cdna sequence of the a. japonicas stomatin gene was analyzed using basic local alignment search tool (blast) (http://www.ncbi.nlm.nih.gov/blast) program. open reading frame (orf) was determined by orf finder (http://www.ncbi.nlm.nih.gov/projects/gorf/orfig.cgi). the amino acid sequence encoded by the open reading frame was analyzed by the online expert protein analysis system (http://www.expasy.org/). the molecular weight of the encoded polypeptide chain was calculated with the expasy compute pi/mw tool (http://www.expasy.org/), and the signal peptide was predicted by signaip 4.1 server (http://www.cbs.dtu.dk/services/signalp/). multiple sequence alignment of amino acid sequences was performed with dnaman program, while a phylogenetic tree constructed was by the neighbor-joining (nj) method using the mega 7 program. domain structure of the protein was analyzed using the simple modular architecture research tool (smart) program (http://smart.embl-heidelberg.de/) and interpro: protein sequence analysis & classification (http://www.ebi.ac.uk/interpro/). transmembrane region was predicted by tmhmm (http://www.cbs.dtu.dk/services/tmhmm-2.0) and tmpred (http://www.ch.embnet.org/software/tmpred_form. html). the secondary structure and three dimensional (3d) structure of ajsto protein were predicted using psipred v3.3 software (http://bioinf.cs.ucl.ac.uk/psipred/) and the swissmodel workspace (https://swissmodel.expasy.org/) which was evaluated by swiss-pdbviewer (version 4.1). bacterial challenge experiment vibrio splendidus d4501 was obtained from our laboratory and cultured at 28 ℃ with shaking at 200 rpm for overnight. the bacterial cells were harvested at the following day by centrifugation at 4,000g for 1 min. for the v. spendidus challenge experiment, twenty sea cucumbers were immersed in a tank of sea water containing v. splendidus at a concentration of 1×107 cfu/ml. the same number of sea cucumbers were immersed in sea water without v. splendidus as controls. three individuals from each group were removed at 0, 6, 12, 24, 48 and 72 h post-immersion, and their celomocytes were extracted for further experiment. expression analysis of a. japponicus stomatin gene the expression profile of the stomatin gene in the celomocytes was analyzed by quantitative real time pcr (qrt-pcr), which was carried out using the applied biosystem 7500 real-time system (applied biosystems, usa). the cytochrome b (cytb) gene was used as a reference gene (yang et al., 2010). primers of the qrt-pcr assay are listed in table 1. quantitative rt-pcr was performed in a 20-μl reaction sample containing 1 μl cdna, 10 μl of 2× sybr green master mix (takara, japan), 0.4 μl of rox reference dyeii, 7 μl pcr grade water and 0.8 μl (10 mm) of each primer (table 1). amplification was carried out under the following conditions: 95 °c for 30 s, 40 cycles of 95 °c for 5 s, 60 °c for 32 s. at the end of the amplification, pcr melting curve analysis was conducted to confirm the presence of a single pcr product. the relative expression level of the stomatin gene was determined by the comparative 2-δδct method. the concrete formula was: δδct = [ct (sample) ct (internal reference)] [ct (control) − ct (internal reference)]. all bacterial challenge experimental data were expressed as mean values ± standard deviations. differences in stomatin expression among the various tissues were analyzed by one-way anova and bacterial infection was tested by t-test contained in the spss software (version 16.0) package. http://blast.ncbi.nlm.nih.gov/blast http://www.expasy.org/ http://www.cbs.dtu.dk/services/signalp/ http://smart.embl-heidelberg.de/ http://www.ebi.ac.uk/interpro/) http://www.cbs.dtu.dk/services/tmhmm-2.0) 417 fig. 1 nucleotide and deduced amino acid sequences of a. japonicus stomatin cdna. start codon (atg) is boxed, and asterisk represents the stop codon. the transmembrane domain is underlined. the spfh domain of band 7 family is shaded. the polyadenylation signal (attaaa) is double-underlined. result and discussion sequence analysis of ajsto cdna the complete cdna sequence of the a. japonicus stomatin gene (designated as ajsto, and genbank accession no. mg209701) was obtained through the assembling of est from the transcriptome database and two amplified fragments, one (44 bp) from 5’-race and the other (60 bp) from 3’-race. the full-length cdna of ajsto was 2085 bp, with an 837-bp open reading frame (orf) encoding a 278-amino acid polypeptide with a predicted molecular mass of 3.5 kda and a theoretical pi of 5.25. in addition, the gene also contained 131 bp of 5’ untraslated region (utr) and 1117 bp of 3’ utr (fig. 1). no signal peptide was found in the amino acid sequence, but signalp 4.0 detected a discriminating signal peptide within the transmembrane region. the transmembrane region comprised 29-51 amino acids, and it was connected 418 fig. 2 secondary structure of ajsto protein. black lines (c), yellow arrows (e) pink cylinders (h) and blue column chart represent coils, strands, helices and confidence of prediction, respectively. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article) by an intracellular n-terminus (52-278 amino acid) and an extracellular c-terminus (1-28 amino acid). a typically representative spfh domain of band 7 family (residues of 52-225) was found in polypeptide sequence of ajsto. the secondary structure of ajsto protein analysis showed that 8 helices and 3 strands were located among amino acid positions 27-275 (fig. 2). the result is different from pyrococcus horikoshii stomatin that included 7 helices and 9 strands, which were regarded as the major contributors to dimeric interaction in n-terminal of p. horikoshii stomain (yokoyama et al., 2006). blastp analysis showed that the amino acid of ajsto had 79 %, 77 %, 74 % and 62 % sequence identity with the sequences of stomatin from strongylocentrotus purpuratus (xp_780332.3), branchiostoma.belcheri (xp_019618408.1), crassostrea gigas (xp_011417141.1) and homo sapiens (eaw87494.1), respectively. band 7 domain was found in all aligned orthologs, illustrating the conservation of stomatin family proteins in both vertebrates and invertebrates. 419 fig. 3 3d structure prediction of ajsto protein (a) and mouse stomatin protein (b). nand c-termini are marked. 3d molecular modeling and phylogenetic analysis 3d molecular modeling of ajsto was generated using mouse stomatin (pdb accession no. 4fvf) as the template (fig. 3). the rate of consistency on the basis of sequence identity between ajsto and the template was 69.53 %. according to the result of ajsto protein 3d molecular modeling analysis, the core of the sea cucumber stomatin domain is very similar to that of mouse stomatin. nand c-termini are located at opposing sides of the molecular (brand et al., 2012). phylogenetic analysis of the various stomatin sequences assigned ajsto to the stomatin sub-family, with ajsto and s. purpuratus stomatin grouped into the same branch of the phylogenetic tree (fig. 4). the result demonstrated that ajsto exhibited a closer relationship with s. purpuratus stomatin, corresponding to the result of traditional taxonomy. taken together, ajsto is highly conserved as compared to those from other vertebrate and invertebrate species. therefore, we named this novel gene as ajsto. tissues distribution of ajsto the spatial expression pattern of the ajsto gene obtained from qrt-pcr showed that ajsto may be a ubiquitous gene. the order of relative ajsto expression levels in the various a. japonicas tissues from high to low was respiratory tree > body wall > intestine > celomocytes > tube feet > longitudinal muscle (fig. 5). for comparison purpose, the transcript level of ajsto in intestine (taken as 1) was compared with the transcript levels in the other tissues. the highest expression level was detected in the respiratory tree, which accounted for 2.4-fold, while the lowest expression, 0.06 fold, occurred in the longitudinal muscle. there has been no reported study on the stomatin protein in deuterostome, but slp-2 from the turbot scopthalmus maximus has been studied and shown to be involved in host immune defense against bacterial and viral pathogens (chi and hu, 2016). however, in human, stomatin participates in immunoreaction and its immunoreactivity has been explored in the ciliated a) b) 420 homo sapiens eaw87494 mus musculus edl08650 aphyosemion striatum sbp28883 nothobranchius furzeri sbp48989 ▲apostichopus japonicus strongylocentrotus purpuratus xp 011680205 mus musculus np 081218 sus scrofa xp 001928425 gallus gallus np 001280135 austrofundulus limnaeus xp 013859108 bos taurus daa26810 mus musculus aag53404 oncorhynchus kisutch xp 020313469 salmo salar np 001135208 homo sapiens cab83216 mus musculus aal06146 xenopus laevis xp 018114018 danio rerio aax89381 fig. 4 consensus neighbor-joining tree based on the amino acid sequences of spfh superfamily members from other species. the phylogenetic tree was constructed by the neighbor-joining method using mega 7 software. the numbers at the forks indicate the numbers of bootstraps. the acronyms including sto, slp-1, slp-2 and pod represent stomatin, stomatin-like protein 1, stomatin-like protein 2 and podocin, respectively. the complete name of the species and their genbank accession numbers are the following: h. sapiens sto (homo sapiens eaw87494), m. musculus sto (mus musculus edl08650), a. striatum sto (aphyosemion striatum sbp28883), n. furzeri sto (nothobranchius furzeri sbp48989), ▲a. japonicus sto (apostichopus japonicus mg209701), s. purpuratus sto (strongylocentrotus purpuratus xp_011680205), m. musculus slp-1 (mus musculus np_081218), s. scrofa slp-1 (sus scrofa xp_001928425), g. gallus slp-1 (gallus gallus np_001280135), a. limnaeus slp-1 (austrofundulus limnaeus xp_013859108), b. taurus slp-2 (bos taurus daa26810), m. musculus slp-2 (mus musculus aag53404), o. kisutch slp-2 (oncorhynchus kisutch xp_020313469), s. salar slp-2 (salmo salar np_001135208), h. sapiens pod (homo sapiens cab83216), m. musculus pod (mus musculus aal06146), x. laevis pod (xenopus laevis xp_018114018) and d. rerio pod (danio rerio aax89381). cells of human airway epithelin (fricke et al., 2003). based on currently available data, ajsto may be associated with immunologic function in a. japonicus. temporal expression pattern of ajsto in celomocytes after bacterial challenge vibrio splendidus is a gram-negative bacterium and the main pathogen of skin ulceration disease in a. japonicas (zhang et al., 2006). one hundred and seven immune-related genes have been characterized from a. japonicus celomocytes after bacterial challenge (dong et al., 2014; zhang et al., 2014). although the differential types distribution of cell in different animals can modify the gene expression and influence the results, celomocytes are essential cells for exploring immune related genes because they are a fundamental component of the innate immune system in echinoderm animals. therefore, to verify the immune function of ajsto, we further tested the expression profile of ajsto in the celomocytes of a. japonicus in response to bacterial challenge. ajsto in the celomocytes was found to display a dynamic expression profile in response v. splendidus challenge at six time points (0, 6, 12, 24,48 and 72 h) following exposure to the bacteria (fig. 6). ajsto expression was significantly depressed 6 h after exposure to v. spendidus, resulting in a 0.18-fold decrease compared to the control. however, ajsto expression was up-regulated 72 h after exposure to the bacteria, yielding a 2.16-fold increase. fig. 5 relative expression of ajsto in different tissues. each vertical bar represents the mean ± sd (n = 3). stomatin stomatin like protein 1 podocin stomatin like protein 2 421 obviously, all data in this study suggested that ajsto might involve in innate immune response of a. japonicus against bacterial infection. the presence of the highly conserved spfh domain in ajsto further suggested that stomatin might have the same function in both vertebrates and invertebrates. our study was the first to describe the stomatin gene and its corresponding protein in a marine organism. temporal expression levels of ajsto also have been emerged in the organism in response to bacterial infection. stomatin has been widely studied in human cancer and tumor, where its expression was found to decrease in non-small cell lung cancer and breast cancer (chen et al., 2012; arkhipova et al., 2014). a possible explanation is the decrease in stomatin expression an favorable factor for bacterial challenge. in summary, a full-length of cdna sequence of a stomatin gene from a. japonicas was cloned and characterized. the encoded protein shared a number of conserved structural features characteristic of the stomatin family proteins. although the gene was expressed in all the a. japonicas tissues examined, the pattern of expression did vary to some extent, suggesting a preference in certain tissue type, the respiratory tree in this case. further analysis of its transcript level following bacterial challenge revealed expressional changes that were dictated by infection time, suggesting that the stomatin gene characterized in a. japonicus may well be linked to immune response. studying the stomatin gene of a. japonicus could fill the vacancy in marine organism research. further study will seek to clarify the immune pathway of stomatin and the specific mechanism governing its regulation of the innate immunity in a. japonicus. acknowledgments the authors thank the reviewer who provided helpful comments. this work was supported by grants for chinese outstanding talents in agricultural scientific research (for yaqing chang). references arkhipova ka, sheyderman an, laktionov kk, mochalnikova vv, zborovskaya ib. simultaneous expression of flotillin-1, flotillin-2, stomatin and caveolin-1 in non-small cell lung cancer and soft tissue sarcomas. bmc cancer 14: 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370-377, 2014. 464 isj 14: 464-476, 2017 issn 1824-307x research report enhanced activities of acetylcholinesterase, acid and alkaline phosphatases in helicoverpa armigera after exposure to entomopathogenic fungi m bilal, s freed, m zubair ashraf, s muhammad department of entomology, faculty of agricultural sciences and technology, bahauddin zakariya university, multan, pakistan accepted november 11, 2017 abstract the toxicity of entomopathogenic fungi, metarhizium anisopliae (isolates ma-11.1 and ma-4.1), isaria fumosorosea (isolates if-02 and if-2.3) and beauveria bassiana (isolates bb-01 and bb-08) to helicoverpa armigera was assessed by measuring the activity of acetylcholinesterase (ache) and acid/alkaline phosphatases after 48 h of exposure till 168 h. the results showed high ache activity in the hemolymph, intestine and fat bodies samples of h. armigera exposed to higher concentrations of i. fumosorosea (if-02 and if-2.3), m. anisopliae (ma-4.1), b. bassiana (bb-08) between 72 -168h at 7×108 spores/ml concentration, while m. anisopliae (ma-4.1) exhibited maximum acid phosphatases (acp) activity at similar concentration in the hemolymph and intestine samples with similar duration of exposure. on the other hand, isolates if-2.3, ma-4.1 and bb-08 showed maximum acp activity during the same exposure duration. the results showed similar high alkaline phosphatases (alp) activity in all body samples of h. armigera when treated with ma-4.1 (7×108 spores/ml), while, isolate bb-08 at similar concentration showed maximum alp activity after 72 and 120 h of exposure. the increased production of these detoxification enzymes in this economic insect pest can possibly lead to the development of resistance against entomopathogenic fungi. key words: acetylcholinesterase; acid phosphatases; alkaline phosphatases; body tissues; helicoverpa armigera; hemolymph introduction helicoverpa armigera (lepidoptera: noctuidae) generally known as american bollworm or cotton bollworm is a serious noctuid pest which causes severe economic losses to agricultural crops (talekar et al., 2006). about 180 crop species including sorghum (sorghum bicolor), tomato (lycopersicon esculentum), corn (zea mays), cotton (gossypium hirsutum), chickpea (cicer arietinum), sunflower (helianthus annuus) and soybean (glycine max) are attacked by this pest (czepak et al., 2013). the infestation of h. armigera is mainly controlled by the use of insecticides and about 30 % of chemicals used all over the world are being applied against h. armigera, which becomes the major cause of resistance to insecticides and bacillus thuringiensis endotoxins(ahmad et al., 1995; akhurst et al., 2003; gao et al., 2009). ___________________________________________________________________________ corresponding author: shoaib freed department of entomology faculty of agricultural sciences and technology bahauddin zakariya university multan, pakistan e-mail: sfareed@bzu.edu.pk entomopathogenic fungi (epf) are mostly used for the control of several insect pests such as galleria mellonella and myzus persicae (yang et al., 2014). approximately 1000 species of insect pathogenic fungi are known to cause death in pests. most common epf are metarhizium anisopliae, beauveria brongniartii, isaria fumosorosea, and beauveria bassiana (de faria and wraight, 2007). these fungi are the environment friendly alternatives to majority of insecticides being applied in the field against insect pests (federici et al., 2008). insects normally defend themselves from xenobiotics by producing certain detoxification enzymes such glutathione s-transferases (gst), esterase, acid and alkaline phosphatases which are commonly present in the insects for their defense (serebrov et al., 2006; zibaee et al., 2009). in insects acetylcholinesterase (ache) which catalyzes the hydrolysis of acetylcholine, a neurotransmitter conducts messages in the nerve impulses throughout the synapses (wang et al., 2004). in many insect species the alteration of ache is also a cause of resistance against insecticides belonging to carbamates and organophosphates. the information about these enzymes in evaluating the mailto:sfareed@bzu.edu.pk 465 comprehensive effect of insect pathogenic during exposure is not much explored. the objective of the current work was to evaluate the effect of entomopathogenic fungi on detoxification enzymes activities in insects as well as the role of these enzymes in the development of resistance to epf. materials and methods helicoverpa armigera rearing helicoverpa armigera larvae collected from fields were reared on fresh brassica oleracea var. botrytis leaves individually in (5cm) petri dishes. uneaten leaves and feces were cleaned and leaves were changed daily. the pupae were placed in plastic cages (30×30×30cm) for the emergence of moths. the newly emerged male and female adults 5:5 were shifted in plastic jars for egg laying with provision of 10 % sugar solution soaked in cotton swab. the 3rd instar larvae of f1 generation were used for the treatment. entomopathogenic fungi the already maintained laboratory culture of entomopathogenic fungi i.e., b. bassiana (bb-01, 08), m. anisopliae (ma-11.1, 4.1) and i. fumosorosea (if-02, 2.3) were further propagated by the method of freed et al. (2012). the spores of all isolates were individually collected in 0.1 % tween80 solution and final concentration was determined by hemocytometer. the stock solutions of different isolates were serially diluted to obtain the desired concentrations for bioassay. insect pathogenic fungi exposure experiments with the selected epf consisted of six treatments including a control. there were four replicates for each treatment. third instar larvae of h. armigera were immersed individually in various mycological concentrations 7×108 spores/ml (highest concentration) followed by 6×108, 5×108, 4×108, 3×108 and 2×108 spores/ml (lowest concentration) for twenty seconds. each fungal isolate was used in same high and low concentration to observe the efficiency against h. armigera. larvae of control set were treated with distilled water containing 0.1 % tween 80 only. the extra moisture of treated larvae was soaked on tissue paper. the individual larva was placed in plastic petri dish having cauliflower leaves. the treated larvae were kept under temperature 26 ± 2 ºc and humidity 75 ± 5 % maintained for complete treatment. forty larvae were used in each treatment and half of the alive larvae from each treatment were sampled for the enzyme activity analysis. sample preparation for enzyme activity analysis the activities of detoxification enzymes in h. armigera samples were evaluated according to methods of serebrov et al. (2006) with slight changes. the hemolymph was collected into eppendorf tubes by making a small cut on the mid abdominal prolegs and few crystals of ascorbic acid were added to avoid coagulation. in about 100 µl of 0.15 m nacl, intestine and fat bodies free from debris were put in eppendorf tubes for crushing and later the final volume was adjusted to 400 µl per sample for centrifugation. the body tissue samples were centrifuged at 3,000 and 1,500 rpm for 10 min (5 ± 1 °c). the supernatants were used for determining enzyme activity and the samples were made for three durations (72, 120 and 168 h) after 48 h of treatment to allow epf infection in the treated h. armigera larvae. determination of protein concentrations in samples bradford (1976) assay was used for the determination of protein in hemolymph and body tissues homogenates by using bovine serum albumin as standard on a spectrophotometer (uv3000, o.r.i. germany). acetylcholinesterase (ache) assay the activity of acetylcholinesterase was measured as explained by ellman et al. (1961) using 0.075m acetylcholine iodide as substrate. the variation in absorbance at λ of 412 nm was recorded for 4 min. alkaline and acid phosphatases assay the alkaline and acid phosphatases activities were determined by the method of serebrov et al. (2006), while 0.23 mm 4-nitrophenyl disodium orthophosphate was used as substrate. at 30 ºc the samples were incubated for 2 h and 400 µl of 0.05 m naoh was added for color development. the change in absorbance at λ of 410nm was noted. statistical analysis all statistical analyses were performed with the statistical software statistix (version 8.1) and microsoft excel 2010.the activity levels of enzymes were measured and the graphs were plotted, while differences between treatments were compared using least significant difference (lsd) test. results the exposure of the different concentrations of epf caused mortalities of h. armigera larvae (un published data). in this study it was observed that ache, acp and alp activities in the tested body tissues of h. armigera were positively correlated with increasing concentrations and the fungal isolates. acetylcholinesterase (ache) activity acetylcholinesterse activity was observed in hemolymph, intestine and fat body samples of h. armigera treated with different isolates of epf on all three days post infection. the results showed that significant ache activity was observed in hemolymph samples (72-168h), when treated with ma-4.1 and if-02 at higher concentration of 7×10⁸ spores/ml (figs1a-c) as compared to the control. the data showed considerable changes in ache activities in ma-4.1 and if-2.3 treated intestine samples (72 and 120 h), whereas similar trend was observed in 168 h samples, which depicted isolate ma-4.1 to enhance the enzyme activity at concentrations 7×10⁸ and 5×10⁸ spores/ml (figs 2a-c). as far as, the enzyme activity in the fat bodies treated samples is concerned, statistically significant results were recorded for all three days with different isolates (figs 3a-c). 466 fig. 1 activity of ache enzyme in hemolymph samples of h. armigera on (a) 72h (b) 120h (c) 168h after treated with m. anisopliae (ma-11.1, 4.1), i. fumosorosea (if-02, 2.3) and b. bassiana (bb-01, 08). *represents significant difference between treatments at 0.05 % level of significance. acid phosphatases (acp) activity acid phosphatases activity was noted in hemolymph and body homogenates samples of h. armigera on all three days post infection. at higher concentration of 7×10⁸ spores/ml, maximum significant activity was observed in ma-4.1 treated 72 168 h samples, while statistically significant results were recorded in hemolymph samples at concentration of 4×10⁸ and 2×10⁸ spores/ml after treated with bb-08 (figs 4a, b) however, no significant activity was observed on 7th day (fig. 4c). in case of intestine samples, the maximum changes in acp activity were observed in all three samples (72 168 h) of ma-4.1 at concentrations 7×10⁸, 5×10⁸ and 2×10⁸ spores/ml, while significant results were also noted in bb-08 (7×10⁸ and 2×10⁸ spores/ml) treated insect samples after 72 and 120 h of infection (figs 5a-c). similarly, statistically significant variations in acp activities were also recorded for all sampling durations after treatment with various isolates as compared to the control (figs 6a-c). 467 fig. 2 activity of ache enzyme in intestine samples of h. armigera on (a) 72 h (b) 120 h (c) 168 h after treated with m. anisopliae (ma-11.1, 4.1), i. fumosorosea (if-02, 2.3) and b. bassiana (bb-01, 08). *represents significant difference between treatments at 0.05 % level of significance. alkaline phosphatases (alp) activity in the current investigations statistically significant change in alp activities were observed in hemolymph samples treated with ma-4.1 at various concentrations (4-7×10⁸ spores/ml) as compared to the control (figs 7a-c), while similar trend was observed in case of intestine samples (figs 8a-c). on the other hand, as far as the fat bodies treated samples are concerned, maximum change in alp was noted only in 168h treated samples of ma-4.1 at concentration of 7×10⁸ spores/ml (fig.9c). 468 fig. 3 activity of ache enzyme in fat body samples of h. armigera on (a) 72 h (b) 120 h (c) 168 h after treated with m. anisopliae (ma-11.1, 4.1), i. fumosorosea (if-02, 2.3) and b. bassiana (bb-01, 08). *represents significant difference between treatments at 0.05 % level of significance. 469 fig. 4 activity of acid phosphatases enzyme in hemolymph samples of h. armigera on (a) 72 h (b) 120 h (c) 168 h after treated with m. anisopliae (ma-11.1, 4.1), i. fumosorosea (if-02,2.3) and b. bassiana (bb-01, 08). *represents significant difference between treatments at 0.05 % level of significance. 470 fig. 5 activity of acid phosphatase enzyme in intestine samples of h. armigera on (a) 72 h (b) 120 h (c) 168 h after treated with m. anisopliae (ma-11.1, 4.1), i. fumosorosea (if-02, 2.3) and b. bassiana (bb-01,08). *represents significant difference between treatments at 0.05 % level of significance. 471 fig. 6 activity of acid phosphatases enzyme in fat body samples of h. armigera on (a) 72 h (b) 120 h (c) 168 h after treated with m. anisopliae (ma-11.1,4.1), i. fumosorosea (if-02, 2.3) and b. bassiana (bb-01,08). *represents significant difference between treatments at 0.05 % level of significance. 472 fig: 7 activity of alkaline phosphatases enzyme in hemolymph samples of h. armigera on (a) 72 h (b) 120 h (c) 168h after treated with m. anisopliae (ma-11.1,4.1), i. fumosorosea (if-02,2.3) and b. bassiana (bb-01,08). *represents significant difference between treatments at 0.05 % level of significance. 473 fig. 8 activity of alkaline phosphatase enzyme in intestine samples of h. armigera on (a) 72 h (b) 120 h (c) 168 h after treated with m. anisopliae (ma-11.1,4.1), i. fumosorosea (if-02,2.3) and b. bassiana (bb-01,08). *represents significant difference between treatments at 0.05 % level of significance. 474 fig. 9 activity of alkaline phosphatase enzyme in fat body samples of h. armigera on (a) 72 h (b) 120 h (c) 168 h after treated with m. anisopliae (ma-11.1,4.1), i. fumosorosea (if-02,2.3) and b.bassiana (bb-01, 08). *represents significant difference between treatments at 0.05 % level of significance. 475 discussion in insects, development of resistance against many microorganisms is mainly by the removal of disease causing microbes from the body and escaping their attack (lord and howard, 2004). outcomes of recent work support the findings of previous research on the effect of epf on detoxifying enzymes of insects (serebro et al., 2001). the previous studies illustrate that infectious diseases play a pivotal role in detoxification of pathogens with the help of enzymatic actions in insects. the enzymes such as ache, acp and alp detoxify the toxic metabolites, depict the insect’s response to body intoxication and development of resistance (serebro et al., 2006). ache an important enzyme, hydrolyses acetylcholine (ach), a neurotransmitter present in the nervous system of insects (wang et al., 2004). inhibition of ache results in increase level of ach at the synapses causing the post-synaptic membrane in a state of long lasting stimulation, ultimately leading to ataxia and insect death (singh and singh, 2004). in our research, the treatment of h. armigera larvae by epf changed the activities of ache, acp and alp in hemolymph, intestine and fat bodies homogenates. these results (increased activity of detoxification enzymes) coincide with prior research work. previously, it revealed that ache activity decreased in sunn pest, eurygaster integriceps after the treatment of b. bassiana (zibaee et al., 2009). according to shafeek et al. (2004) ache activity was observed in various parts of the nervous system such as brain, terminal ganglion, nerve cord and muscles of periplaneta americana (linnaeus) after exposure to azadirachtin, while there was no significant activity of ache as compared to the control. similarly, application of neem extracts inhibited the ache activity in blatella germania and musca domestica (khan et al., 2003), while increased ache activity was also observed when tribolium castaneum was treated with ambush and dimilin(saleem and shakoori, 1987). in the current study the highest ache activity was observed in hemolymph samples (120h) treated with b. bassiana (bb-08), while lowest was noted in i. fumosorosea (if-2.3) treated samples for all three durations, whereas,m. anisopliae (ma-4.1) and i. fumosorosea (if-2.3)treated intestine and fat bodies samples showed the highest ache activity. less infectious epf strains are commonly present in natural insect populations that not necessarily kill the insects. hence, detoxification enzymes are generally affected by epf as well as other pathogenic microbes. moreover, detoxification enzymes have several functions and can facilitate repair procedures, metabolism of biologically active compounds and intoxication of toxic products. the variations in activities of enzymes can develop resistance in insects and may have impact on the insect’s body that makes them adaptable to environment (serebro et al., 2001, 2006). in the previous studies, the treatment of spodoptera exigua larvae with nuclear polyhedrosis virus showed high activity of acp in fat bodies with respect to days and high activity was observed on third day until the death of the larvae, while alp activity was three times less than acp in the fat bodies samples (sujak et al., 1978). earlier, maximum acp activity was observed in the hemolymph of desert locust, schistocerca gregaria on the third day after exposure to m. anisopliae (xia et al., 2000). parallel activities of acp and alp in hemolymph, intestine and fat bodies samples treated with m. anisopliae (ma-4.1), b. bassiana (bb-08) and i. fumosorosea (if-2.3) for different days were observed in the current study. enzymes activities sharply increase degradation in infected insects after exposure with xenobiotics. this enhances the adaptation ability of insect body and decreases their sensitivity against insecticides. inhibition of detoxification enzymes abruptly increases the insect death from fungal contamination, which ultimately confirms the participation of detoxification enzymes in insect resistance development to epf and opens novel opportunities for the development of proficient biological products on the basis of enzyme inhibitors and epf. acknowledgements this research was supported by the higher education commission, pakistan under research project no.2263. references ahmad m, arif im, ahmad z. monitoring insecticide resistance of helicoverpa armigera (lepidoptera: noctuidae) in pakistan. j.econ. 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nosema bombycis; pebrine; loop-mediated isothermal amplification; ncbi introduction sericulture is a main source of livelihood for subsistence with farmers engaged in silk production. like other economically important insects, silkworm is also prone to many diseases caused by pathogens. these pathogens are responsible for significant economic loss in the sericulture industry. among them, nosema bombycis, a microsporidian causes pebrine disease in silkworms, bombyx mori; not only horizontally transmitted in the silkworm larva, pupa, and moth, but also vertically to the next generation through the silkworm eggs; adversely affecting silk yield and quality of cocoons resulting in huge losses to sericulture. pebrine disease is the only microbial infection, listed under quarantine items with regard to silkworm egg production (fu et al., 2016). pasteur (1870) discovered that the parasite (n. bombycis) could transmit from mother moths to _________________________________________ corresponding author: vankadara sivaprasad silkworm breeding laboratory silkworm crop improvement division central sericultural research and training institute berhampore 742101, india e-mail: siva.nsso@gmail.com progenies via transovarial transmission. based on this observation, the mother moth microscopic examination method to eliminate eggs laid by infected moths and produce n. bombycis-free eggs for the sericultural industry was developed. from then, sericultural countries have adapted light microscopy examination for the detection of n. bombycis spores (fujiwara, 1984). even though, microscopy is inexpensive and easy to use, it has several limitations such as late detection (microsporidian spores are visible only after 3-8 days of onset of infection), detects only spore stage and only infections leading to a spore production above 1 x 106 spores/g tissue (yan et al., 2014). therefore, a low intensity infection could easily be missed as the infection remains undetected and spores represent only a fraction of an ongoing infection; the other life stages remain undetectable. advancement in pcr-based methods could overcome these drawbacks as they can detect parasitic dna of all life-cycle stages and allow discrimination of parasites via specific primers (refardt and ebert, 2006). mother moth and hatched larvae examination for pebrine does not completely prevent the development of pebrine disease. but, the crop 67 should be monitored strictly for pebrine at each stage of silkworm rearing to prevent the infection of n. bombycis by external sources like mulberry leaf, contaminated rearing materials etc. this is possible only by adapting the detection methods which are field applicable. even though, advancement in the molecular diagnostic approaches like conventional pcr has made great progress in enhancing the sensitivity and specificity in the detection of n. bombycis compared to visual identification by microscopic examination, these techniques are not suitable for conducting tests in the field conditions as they require sophisticated instruments, expertise and longer duration. therefore, there is a demand to innovate specific, accurate and early detection method which could be utilized at the field level for effective pebrine disease management among sericulture farmers and silkworm seed production centres. this method still demands for costly instrumental set-up like thermal cycler and uv transilluminator for the detection of pcr products and yet might not be feasible for field conditions. an approach combining low-cost dna extraction process and pcr detection for high-throughput screening could be the ultimate goal for pebrine detection in sericulture. notomi (2000) discovered loop-mediated isothermal amplification (lamp) technique in 2000. since then, lamp has been improved constantly and widely used for the detection of pathogenic microorganisms (njiru et al., 2008; jiang et al., 2012; singh et al., 2013). yan et al. (2014) found the main hindering factor affecting the sensitivity of microsporidian inspection is the recovery of genomic dna. furthermore, he has successfully applied the lamp method for detection of n. bombycis using fta cards for genomic dna extraction. to make the current invention, a practically effective and field-oriented diagnostic system for silkworm pebrine disease, lamp detection method was reoriented using special lysis buffer to detect n. bombycis infection; which requires less sophisticated instruments for amplification of the target gene and the product could be detected by chromogenic system. to our knowledge, it is the first attempt of its kind to simplify the dna extraction method from microsporidia infected tissues and combine it with lamp assay to develop field-friendly diagnostic method (ff-lamp assay) for the detection of pebrine in silkworms. materials and methods silkworm race and microsporidia the bivoltine silkworm race, csr2 and purified spores of nosema bombycis (nik-1s) maintained at central sericultural research and training institute, mysuru, india were used for this experiment. n. bombycis spores were propagated through periodical inoculation of spores to silkworms (ii instar 1st day) followed by collection of infected moths. the n. bombycis spores were isolated following tissue homogenization and purified by percoll centrifugation. preparation of n. bombycis infected silkworm larvae/tissue silkworm larvae after hatching and after each moult were fed with mulberry leaves that were uniformly smeared with n. bombycis spores at different doses (101-108) separately. from there, the onset of infection was examined every day by homogenizing the host tissues in physiological saline and observing under phase-contrast microscope (600x). similarly, the infections at different developmental stages of silkworm viz. egg, pupa and moth were also examined. the experiment was performed in 5 replications containing 100 silkworm larvae /replication /dose of spores. samples were collected from each replication on daily basis and were preserved at -20 °c until use. five batches of silkworms were inoculated with different concentrations of n. bombycis spores from 101 to 108 immediately after brushing and after each moult, respectively. the inoculated silkworm samples were collected every 24 h intervals. samples were collected from 1st instar 2nd day to moth emergence and eggs. samples were stored at -20 °c for further use. dna extraction from n. bombycis the spores of 107 were suspended in equal volume of solution containing 100 mm nacl, 200 mm sucrose, 10 mm edta and 30 mm tris-hcl (ph 8.0) along with 0.45-0.50 mm glass beads for disruption of spores. the suspension was vortexed followed by centrifugation at 5000 rpm for 10 min. supernatant was added with ¼th volume of 2.5 % sds, 250 mm edta and 500 mm tris-hcl (ph 9.2) along with 100 µg/ml of proteinase k followed by table 1 list of primers used for conventional pcr and ff-lamp assay primer sequence (5'-3') length amplicons (bp) method reference ssu-f accaggttgattctgcctga 20 794 pcr ravikumar et al 2011 ssu-r gttgagtcaaattaagccg 19 794 f3 gcggcttaatttgactcaa 19 124 pcr & ff-lamp yan et al 2014 b3 acctgttttaatcctctcct 20 124 fip gccatgcaccactatcatgatcgcggggtaatttaccag 39 ff-lamp bip gtttccaatggatgctgtgaagttcatatgtatcactacatctgtct 47 68 table 2 comparison of light microscopy and ff-lamp results in the detection of n. bombycis sampling place no. of samples tested presence of n. bombycis spores microscopy ff-lamp + + p4bsf (hassan) 451 1 450 6 445 p3bsf (mysuru) 607 70 537 138 469 p2bsf (ambuga) 4 0 4 0 4 p2bsf (dharmapura) 11 0 11 0 11 p2bsf (horsely hills) 6 0 6 0 6 p2bsf (krishnagiri) 28 0 28 1 27 p2bsf (madakasira) 4 0 4 0 0 p2bsf (palakkad) 10 0 10 0 0 sspc-mysuru 2 0 2 0 0 sspc-kr nagar 2 0 2 0 0 sspcdharmapuri 145 1 144 1 144 csrti-mysuru 12 12 0 12 0 ssbs-coonoor 66 0 66 0 66 sspc-tumkur (dos-ka) 25 0 25 0 25 total 1373 84 1289 158 1197 incubation at 55 °c for 1 h. the proteins and sds were removed by precipitation with 1m potassium acetate by incubating for 1h at 4 °c. after centrifugation at 5000 rpm for 20 min, the dna was precipitated by 2 volumes of cold ethanol. the dna pellet was dissolved in the milliq water and stored at -20 °c for further use. the dna was quantified using spectrophotometer (amersham biosciences, hongkong, china). direct-pcr (dpcr) ethanol pre-soaked (30 min) n. bombycis infected host tissues were homogenized in 200 μl of lysis buffer containing 10 mm tris-hcl, 1 mm edta buffer (ph 8) and 10 mm nacl (ph 8.0). the homogenized tissues were centrifuged at 10000 rpm for 10 minutes. proteinase k (200 μg/ml) was added to the supernatants and mixed gently. then the supernatants were incubated at 55 °c for 30 min followed by incubation at 95 °c for 5 min. the resulting supernatant was diluted in 1:25 ratio and used as template for performing pcr reactions. before optimization of the lysis buffer, the chemical ingredients with varying concentrations were tested. as the detergent inhibits pcr reaction, the same were not utilized in formulating the lysis buffers. the pcr reaction was performed to amplify the 16s small subunit ribosomal rna (ssu-rrna) of n. bombycis by using the pcr cycle conditions as described by ravikumar et al. (2011). the reaction was carried out in a total volume of 10 μl containing 1x taq buffer, 0.25 mm dntps, 0.50 μm of each primer, 2.5 mm mgcl2, 1u taq dna polymerase and 2 μl of pre-diluted extract/ template. all the pcr components were from thermo fisher scientific, usa. the amplified products were resolved on 1.3 % agarose gel and visualized by ethidium bromide staining under uv transillumination. all the reactions were performed on ptc 100 (mj research, usa). polymerase chain reaction (pcr) primers previously designed and successfully used by yan et al. (2014) for n. bombycis‘s small ribosomal subunit gene (accession no. ay259631) were used for this study. for optimising the lamp assay, conventional pcr was performed using f3 and b3 primers (table 1). initially, the pcr conditions were optimized for annealing temperature (45 °c and 47 °c) and different mgcl2 concentrations (1 mm to 6 mm). at optimal pcr conditions, the f3 and b3 primers were tested for non-specific amplifications against pebrine free silkworm dna along with n. bombycis‘s dna and a negative control. the pcr conditions constituted initial denaturation step at 94 °c for 5 min followed by 35 cycles comprising of 94 °c for 30 s of denaturation, 47 °c for 30 s of primer annealing and 72 °c for 1 min of extension with final extension step at 72 °c for 10 min. 69 fig. 1 conventional pcr amplification of dna extracted from pebrine infected silkworms using lysis buffer. m 100 bp ladder, lane 1 to 3 and 9 to 11 proteinase k treatment for 30 min, lane 4 to 6 and 12 to 14 proteinase k treatment for 60 min, lane 7 and 15 positive control, lane 8 blank without dna the pcr reaction was performed in a 25 μl reaction mix containing template dna, 1x pcr buffer, 4.0 mm mgcl2, 250 μm dntp mix, 10 μm of each primers and 1u taq polymerase). the pcr product was analysed on 2 % metaphor agarose gel electrophoresis stained with ethidium bromide and photographed under uv transilluminator. the pcr product was sequenced by sanger dideoxy method and the data was submitted to ncbi. extraction of genomic dna from n. bombycis infected tissues for lamp assay n. bombycis infected silkworms were briefly homogenized in mortar and pestle using lysis buffer (ph 8.0), the mixture was heated for 10 min at 95 °c, centrifuged for 10 min at moderate speed. 2 μl of the supernatant was directly used as the dna template for the lamp assay. optimization of lamp assay the lamp reaction was optimized for mgso4 and dntps concentration, and temperature; mgso4 concentrations (1-6 mm), dntps at 1.4 mm and 2.8 mm, the inner (fip and bip) to outer primers (f3 and b3) concentration at 2:1, 4:1, 6:1 and 8:1, and reaction temperature at 60-65 °c. ff-lamp reaction system the ff-lamp reaction for the detection of n. bombycis was performed in a total volume of 25 µl containing 20 mm tris-hcl (ph 8.8), 10 mm kcl, 6 mm mgso4, 10 mm (nh4)2so4, 0.1 % triton x-100, 1.4 mm dntps, 1.6 µm (each) fip and bip primers, 0.2 µm (each) f3 and b3 primers, 0.4 % glycerol, 8u bst dna polymerase, template dna (crude homogenate 2µl), 120 µm hydroxy naphthol blue (hnb) and distilled water. the mixture was incubated at 63 °c for 45 min in a water bath. detection of ff-lamp amplification ff-lamp amplification was detected by chromogenic assay by adding hnb dye prior to the reaction. the initial color of hnb at ph 8.8 will be maroon/violet. after the reaction, the color would turn to sky blue (in presence n. bombycis dna), if the result is positive; otherwise, there would be no change in colour indicating negative result (in presence of no template or silkworm dna). these results can be observed by naked eye under daylight without using any instruments. amplified products were also confirmed by electrophoresis in ethidium bromide stained 2 % agarose gels under uv light. fig. 2 optimization of pcr conditions for detection of n. bombycis using f3 and b3 primers. m 100 bp ladder, lane 1-6 1 mm to 6 mm mgcl2 and annealing temperature of 45 °c, lane a-f 1 mm to 6 mm mgcl2 and annealing temperature of 47 °c 70 validation of the ff-lamp assay a total of 1373 samples collected from different sericulture organizations of central silk board, india as listed in table 2 were subjected to ff-lamp assay. all the test samples were also inspected simultaneously for n. bombycis infection by regular lamp and light microscopy method. independent sample t test was applied to understand the effectiveness of microscopy and ff-lamp assay in pebrine detection. all statistical analyses were performed using spss 11.5 statistical package. results and discussion genomic dna was isolated from pure n. bombycis spore, pebrine-free silkworm, and also from n. bombycis infected silkworm using different isolation methods. dna isolated from pebrine infected silkworm using different buffers was utilized for direct pcr. dna extracted from the lysis buffer was amplified by dpcr and the amplification product (794 bp) for ssu-f and ssu-r primers, that targets the small ribosomal subunit of n. bombycis was observed in agarose gel electrophoresis (fig. 1). dna isolated from pure n. bombycis spore was utilized to check the efficiency of the selected primers (f3 and b3) using conventional pcr. the f3 and b3 primers form the outer primers in lamp reaction could be utilized for conventional pcr. the amplification product (fig. 2) could be seen in all the tested conditions of mgcl2 (1-6 mm) and annealing temperatures (45 °c and 47 °c). pebrine-free silkworm dna was utilized as negative control. non-specific amplification was observed with silkworm dna and thus the pcr product (~120 bp) was subjected to sequencing (fig. 3). the analysis of sequencing showed hits to an extent of 120 bp of the pcr product. the sequencing of n. bombycis spore pcr product and nucleotide blast showed 100 % identity with nosema species; whereas the sequenced product of non-specifically amplified pebrine-free silkworm dna didn’t show any identity with any organism. these results indicate that f3 and b3 primers are specific to n. bombycis ssu rdna and the sequence of n. bombycis f3 and b3 region was submitted to ncbi (accession no. mg831719). the pair-wise alignment of n. bombycis ssu rdna fig. 3 agarose gel electrophoresis of pcr products for detecting n. bombycis. m 100 bp ladder, lane 1 and 2 blank (no dna), lane 3 and 4 pebrine-free silkworm dna, lane 5 and 6 n. bombycis dna (accession no. ay259631) as the query sequence with that of f3 and b3 primer amplified product as subject sequence had 100 % identity (fig. 4). further, the dna isolated from n. bombycis infected silkworm was utilized for lamp assay using inner primers fip and bip along with f3 and b3. the sequence, amplicon size of all the primers employed in this study is detailed in table 1. the loci of each lamp primer in the small ribosomal subunit of n. bombycis dna (accession no.ay259631) are shown in fig. 5. ff-lamp assay the tissue samples (egg, larvae, pupae, and moth) were homogenized in lysis buffer for breaking the chitin layered n. bombycis spores and heated at 95 °c to facilitate release of dna into the buffer system. then the sample was centrifuged and 2 µl of supernatant that contains dna was sampled as template for the ff-lamp assay in pcr tubes (0.2 ml tubes or 96 well plates). the optimization of fflamp assay for different parameters showed that fig. 4 pairwise alignment of n. bombycis ssu rrna (query) and pcr amplified f3 and b3 region of pebrine infected sample (subject) 71 fig. 5 the locus of each primer in the dna sequence of n. bombycis small ribosomal subunit the effective target amplification could be obtained with 6 mm mgso4 concentration, dntps concentration at 1.4 mm, the inner to outer primer ratio at 16:1 and reaction temperature at 63 °c. the optimal combination of mgso4, dntps and inner and outer primers concentrations not only achieves the target amplification, but helps in maintaining the maroon/violet color at the start of the reaction (ph 8.8) with hnb. the optimization of lamp reaction helps to distinguish easily between the positive and negative reactions. hnb showed more effective color contrast between the positive and negative samples as compared to the other dyes used in this study (data not shown). hnb at ph 8.8, at the start of the reaction will be in violet color and turns sky blue color, if the samples are positive for n. bombycis infection. to summarize, no colour change (remains violet) is observed in blank and negative samples; while the colour changes to sky blue in the samples positive for n. bombycis infection by the ff-lamp assay. the non-specific amplification observed does not affect the results of lamp assay as non-specific amplified product from silkworm dna was less and in turn could not alter the color of the reaction. in the lamp assay, as the nucleic acid polymerize, the solution becomes acidic by the release of protons (h+) which is detected by chromogenic assay by adding hnb dye prior to the reaction (shunbi et al., 2014; yan et al., 2014). therefore, non-specifically amplified product from silkworm dna is not a reliable readout enough to change the initial color of hnb dye. to further confirm the obtained results, agarose gel electrophoresis was run to find the ladder like band appearance (fig. 6). silkworm larvae fed on n. bombycis were detected 12, 24, 48, and 72 h post-inoculation by the ff-lamp method. at 48 h post-inoculation, the results showed that the amplification reaction was positive by the ff-lamp method and negative by microscopy (fig. 7). in contrast, at 72 h postinoculation, both the methods detected a positive amplification i.e., n. bombycis infection. this experiment illustrates that the ff-lamp technology shifts the diagnosis to 24 h earlier than that obtained through conventional microscopic method, even before the proper spore formation. specificity of ff-lamp assay the n. bombycis and silkworm dna was tested utilizing the ff-lamp method established in this study. the results showed that amplification of n. bombycis dna occurred only; whereas all the amplifications were negative for silkworm dna (fig. 8), indicating that the lamp primers used in this experiment cannot amplify the genome of the silkworm and is very specific for n. bombycis. the primers used in this study have no specificity to silkworm genes as evidenced by primer blast. fig. 6 agarose gel electrophoresis of ff-lamp products. m 100 bp ladder, b no dna template, lane 1 2 µl of n. bombycis dna, lane 2 2 µl of crude extract of pebrine infected sample 72 fig. 7 agarose gel electrophoresis of ff-lamp products at different intervals of post-inoculation. m 100 bp ladder, lane a 24 h, lane b 48 h and lane c 72 h validation of ff-lamp assay samples collected from various basic seed farms and silkworm seed production units of central silk board and other agencies were tested for n. bombycis infection by using ff-lamp assay and microscopic method in coordination with scientific personnel from respective units. totally, 1373 samples were subjected for pebrine detection (table 2), of which 84 (6.11 %) samples were positive and 1289 samples were negative for n. bombycis infection by microscopy method; whereas, 158 (11.51 %) samples were positive and 1197 samples were negative for n. bombycis infection by lamp method. independent sample t test performed on the field samples tested by microscopy and ff-lamp assay resulted in nonsignificant differences [t(54) = 0.019, p = 0.985] indicating that ff-lamp assay is as effective as the traditional microscopy method. in addition to its superiority in terms of specificity and early detection of n. bombycis, ff-lamp assay showed increased number of positive samples (5.4 %) than microscopy; which could not detect due to lower sensitivity of microscopy. the samples tested positive for infection by ff-lamp assay was further confirmed by running agarose gel electrophoresis, where the typical ladder-like bands appeared. there are many other methods such as normal pcr and loop mediated isothermal amplification (lamp) that are targeting the amplification of ssurrna gene sequence for early diagnosis and identification of silkworm microsporidia (kawakami et al., 1995, 2001; hatakeyama and hayasaka, 2001, 2003). though, pcr based methods can diagnose n. bombycis even at low spore concentration than microscopic method, but the methods are not being used for screening pebrine in seed production centres and field level as these modern techniques require highly sophisticated instruments and involves extensive processing of samples and technological interventions. these methods majorly involve dna extraction and targeted gene amplification. pan et al. (2005) reported an extraction method for microsporidian dna from infected eggs, which had been treated with 30% koh at 37 °c. a couple of researchers (ravikumar et al., 2011; gourab et al., 2017) used commercially manufactured kits or standard protocols (sambrook, 1989) for extraction of dna and agarose gel for detecting pcr products. the liu et al. (2004a and 2004b) optimized the pcr conditions to inspect simulated ‘‘infected’’ silkworm eggs with the minimum detection of 299 spores, and suggested that silkworm egg contains some substances that may inhibit the pcr amplification. ff-lamp method achieved sensitivity equal to conventional pcr with lesser steps and resources. yan et al. (2014) applied lamp assay for the 73 fig. 8 agarose gel electrophoresis of ff-lamp products. m 100 bp ladder, b no dna template, lane 1 pebrinefree silkworm dna (negative control), lane 2 and 3 pebrine infected samples detection of n. bombycis using fta cards and glass beads method to extract microsporidian dna, but it is not cost effective as compared to ff-lamp assay. further, attempts were made by researchers to simplify the lamp technique with several detection measures like in lamp-ph and lampgold nanoparticle (lamp-aunp) methods. the former involves amplification of n. bombycis polar tube protein 1 gene and direct detection by measuring the released hydrogen ions during lamp reaction using a ph meter (shunbi et al., 2014); whereas, the lamp-aunp assay involves extraction of n. bombycis dna by fta card fta card method followed by visualization of the amplified product via hybridization at 63 °c for 5 min with a ssdnalabelled nanogold probe (weijiang et al., 2019). esvaran et al. (2018) employed phenol chloroform method for extraction of n. bombycis dna followed by amplification of polar tube protein 1 gene through lamp method and detection of amplification by colour change. these methods are time consuming, require sophisticated instruments and skilled personnel. extraction of dna for pcr amplification requires dna with high purity, involves timeconsuming processes and other factors such as salt ionic concentration etc. in the final sample interfere with pcr amplifications. in this study, lamp assay was combined with single step dna extraction method using special lysis buffer formulated to isolate n. bombycis dna, which could be utilized for n. bombycis-ssu rrna gene amplification successfully like direct-pcr (dpcr) method in the crude homogenate by ff-lamp method. moreover, this study shows that n. bombycis infection could be diagnosed 24 hours prior to that of light microscopy with limited resources; therefore, the lamp assay described could be an effective alternative for light microscopy; which is simple, less time consuming, cost-effective and amenable to high throughput for pebrine inspection in silkworm. references daia w, qia j, chena h, zhanga z, shanga r, zhanga y, et al. rapid and sensitive detection of nosema bombycis using loop-mediated isothermal amplification and colorimetric nanogold. science asia 45: 268-274, 2019. esvaran vg, gupta t, mohanasundaram a, ponnuvel km. development of isothermal amplification assay for detection of nosema bombycis infection in silkworm bombyx mori targeting polar tube protein 1 gene. inv surv j 15: 352-361, 2018. fu z, he x, cai s, liu h, he x, li m, et al. quantitative pcr for detection of nosema bombycis in single silkworm eggs and newly hatched larvae. j. microbiol. meth. 120: 72-78, 2016. fujiwara t. studies on the mass pebrine inspection of mother moth. technical bulletin of sericulture experiment station (japanese), 120: 113-160, 1984. 74 gourab r, kalidas m, ravikumar g. pcr-based 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633-638, 2012. liu jp, cao y, smith je. studies on the application of pcr molecular diagnosis to silkworms with simulated pebrine disease. sci. agric. sin. 37: 1925-1931, 2004. liu jp, cao y, smith je. studies on the application of pcr molecular diagnosis to silkworm eggs and moth with simulated pebrine disease. sci. agric. sin. 30: 367-370, 2004. njiru zk, mikosza as, armstrong t, enyaru jc, ndung'u jm, christopher thompson andrew richard. loop-mediated isothermal amplification (lamp) method for rapid detection of trypanosoma brucei rhodesiense. plos negl. trop. dis. 2: e147, 2008. notomi t, okayama h, masubuchi h, yonekawa t, watanabe k, amino n, et al. loop-mediated isothermal amplification of dna. nucleic acids res. 28: e63, 2000. pasteur l. etudes sur la maladie des vers à soie: 2.: notes et documents. gauthiervillars, 1870. pan zh, zheng xj, xue zy et al the method for extraction of nosema bombycis dna from silkworm eggs with pebrine. sci. seric. 31: 486489, 2005. refardt d, ebert d. quantitative pcr to detect, discriminate and quantify intracellular parasites in their host: an example from three microsporidians in daphnia. parasitology 133: 11-18, 2006. ravikumar g, urs sr, prakash nv, rao c, vardhana k. development of a multiplex polymerase chain reaction for the simultaneous detection of microsporidians, nucleopolyhedrovirus, and densovirus infecting silkworms. j. invertebr. pathol. 107: 193-197, 2011. sambrook j, fritsch ef, maniatis t. molecular cloning: a laboratory manual, second ed. cold spring harbor laboratory press, cold spring harbor, ny, 1989. singh p, mirdha br, ahuja v, singh s. loopmediated isothermal amplification (lamp) assay for rapid detection of entamoeba histolytica in amoebic liver abscess. world j. microbiol. biotechnol. 29: 27-32, 2013. singh rn, magadum sb, kamble ck, daniel agk. silkworm disease forewarning model – an analysis. indian silk 9: 12-14, 2006. xie s, yuan y, song y, zhuo y, li t, chai y, et al. using a ubiquitous ph meter combined with loop mediated isothermal amplification method for facile and sensitive detection of nosema bombycis genomic dna ptp1. chem comm. 100: 15825-16001, 2014. yan w, shen z, tang x, xu l, li q, yue y, xiao s, fu x. detection of nosema bombycis by fta cards and loop-mediated isothermal amplification (lamp). curr. microbiol. 69(4): 532-40, 2014. zhang fy, shi yh, jiang k, xu z, ma l. sensitive and rapid detection of two toxic microalgae alexandrium by loop-mediated isothermal amplification. acta oceanol. sin. 31: 139-146, 2012. 423 isj 14: 423-431, 2017 issn 1824-307x research report molecular characterization and expression of ajnlrp3 in the antibacterial host defense of the sea cucumber (apostichopus japonicus) j ding*, lx li*, xh liu, h wang, sy ding, ls han, yq chang key laboratory of mariculture & stock enhancement in north china's sea, ministry of agriculture, dalian ocean university, dalian, liaoning, 116023, p.r. china *these authors contributed equally to this work. accepted october 24, 2017 abstract the nod-like receptor proteins (nlrp) is an important genes primarily involved in innate immunity. in this study, we identified a novel nlrp gene designated as ajnlrp3 (genbank accession number mf663701) in apostichopus japonicus using transcriptome sequencing and the rapid amplification of cdna ends approach. the full-length of ajnlrp3 is 3642 bp and a putative open reading frame of 2424 bp encodes a polypeptide with 807 amino acid residues. the predicted molecular mass of the protein sequence predicted for ajnlrp3 is 93.03 kda and its theoretical pi is 6.14. ajnlrp3 contains two low complexity regions, two internal repeat regions, and a nacht domain. spatial distribution expression analysis detected ajnlrp3 in all of the tissues tested, with greater expression levels in the body wall, moderate expression in the respiratory tree, and weaker levels in the intestine, tube feet, celomocytes, and longitudinal muscle. the expression levels of ajnlrp3 increased by 2.60-fold in the celomocytes and decreased by 0.87-fold in the respiratory tree of diseased sea cucumbers compared with those in healthy sea cucumbers. time-course expression analysis under bacterial challenge in celomocytes showed that the expression of ajnlrp3 was significantly decreased by 0.23-fold at 8 h and by 0.20-fold at 24 h. in conclusion, this study showed that the ajnlrp3 protein found in the sea cucumber has a similar structure and biological function to that in other organisms, where it appears to be involved with the innate immune responses against bacterial infection. key words: ajnlrp3; apostichopus japonica; cloning and expression; antibacterial experiment introduction animal immune mechanisms include innate immunity and acquired immunity, where both mechanisms protect the body from invasive bacteria, fungi, viruses, and other pathogens. previously, it was considered that complex molecular immune responses only occur in higher vertebrates, but recent studies have shown that echinodermata also possess many immune gene families and various immune responses. in general, immune cells move to chemical irritants along a concentration gradient. thus, when the source of an invasive pathogen is located in the body, the damaged cells release certain chemical substances to attract large inflammatory cells and induce the ___________________________________________________________________________ corresponding author: yaqing chang key laboratory of mariculture & stock enhancement in north china’s sea dalian ocean university dalian, 52 heishijiao rd.,liaoning 116023, p. r. china e-mail: yaqingchang@hotmail.com inflammatory response. subsequently, large numbers of inflammatory cells infiltrate the damaged tissue. studies have shown that this type of response also occurs in the coelom cells of members of the echinodermata. indeed, pisaster ochraceus possesses an analogue of interleukin-1, which is found in higher animals (meng et al., 2009), and this analogue participates in phagocytosis. the sea cucumber, apostichopus japonicus, can also respond to pathogens via cell-mediated immunity and humoral immunity, although the mechanism of action is not clear. in recent years, some genes related to immunity in a. japonicus have been identified using expressed sequence tag (est) libraries, gene chips, proteomics, and cloning, including agglutinin, antimicrobial peptide, muramidase, immune-related enzymes, clotting protein, pattern recognition receptors (prrs), toll-like receptors, complement c3, and other serum factors (sun et al., 2013; ji et al., 2014; yang et al., 2016). most of the mechanisms related to immune molecules have not been elucidated in a. japonicus. 424 inflammation refers to the initial response of hosts to external challenges via changes in the local circulation (i.e., hyperemia and increased vessel permeability) and the recruitment of immune cells (i.e., granulocytes, lymphocytes, and macrophages) to infected sites, and the eventual elimination of aggressors and the promotion of tissue repair (cone, 2001). proinflammatory cytokines are produced in this process and released into the extracellular milieu. proinflammatory cytokines have proinflammatory effects where they activate the inflammatory response by binding to the extracellular domains of ubiquitous inflammatory cytokine receptors. the cleavage of proinflammatory cytokine precursors is strictly controlled by inflammasomes, which comprise a family of proteins that were first described in 2002 (martinon et al., 2002). inflammasomes were initially identified based on their roles in marginal or benign pathologies, such as periodic fevers and gout, but they are now considered key factors in most inflammatory diseases (hawiger, 2001; brigati et al., 2002; lin et al., 2014; dutartre, 2016). cytoplasmic receptors of the nucleotide binding domain-like receptor (nlr) family are key components of inflammasomes, where nlrp3 plays vital roles in initiating the inflammatory process (davis et al., 2011). a. japonicus (selenka) belongs to echinodermata, holothuroidea, aspidochirotida, stichopodidae, apostichopus, and it is one of the most economically important aquatic animals in china (chang, 2004). in recent years, outbreaks of diseases such as skin ulcer syndrome (wang et al., 2006), stomach atrophy (deng et al., 2008), red body disease (hao et al., 2013), and bad side disease (zhang et al., 2010) have severely affected the development of sea cucumber aquaculture. sea cucumbers are lower marine animals and like other invertebrates, they can only rely on the innate immune system to resist infection by pathogenic bacteria. therefore, by studying the characteristic nonspecific immune signal transduction pathways in the sea cucumber, we can determine the immune functions of key genes and their characteristic responses in important cell signaling pathways, thereby clarifying the molecular mechanisms of nonspecific immune responses to facilitate disease prevention, molecular therapy, and the development of new drugs for sea cucumbers. materials and methods preparation of animals and collection of samples healthy sea cucumbers (body weight 68.00 ± 4.59 g) were obtained from dalian heshengfeng marine product farm and maintained at 16 °c 17 °c in our laboratory for 1 week (the sea cucumbers did not feed and change water from the 1st to 3rd day, and start feeding and changing water every two days from the 4th to 7th day). to examine the spatial expression of ajnlrp3, six tissues comprising the intestine, respiratory tree, tube feet, coelomocytes, body wall, and longitudinal muscle were dissected carefully from five healthy animals. the vibrio splendidus d4501 was initially isolated from a skin ulceration dieased a. japonicus in our laboratory. the v. splendidus was grown in 2216e liquid medium at 28 ℃ with shaking at 200 rpm for 12 h and the cultures was centrifuged at 5,000 rpm for 10 min to harvest the bacteria. the experiment employed a control group and a bacterial challenge group. each group was divided into seven replicates containing five individuals. the sea cucumbers in the bacterial challenge group were immersed in a suspension of v. splendidus d4501 at a density of 107 cfu/ml, whereas the other group was not treated with v. splendidus. the celomic fluids were collected at 0, 4, 8, 12, 24, 48, and 72 h post-immersion. in order to harvest coelomocytes, the coelomic fluid was centrifuged immediately after collection at 1,000 rpm and 4 °c for 5 min. all of the tissues were snap frozen immediately in liquid nitrogen and stored at -80 °c. total rna extraction, cdna synthesis, and cloning of the full-length ajnlrp3 total rna was extracted from all of the tissues collected from a. japonicus using trizol (ambion) according to the manufacturer’s protocol. the quality and quantity of the isolated rna were measured table 1 the primers used in this research primer sequence (5'→3') purpose ajn3 5-1 tatcagggtattcgtcaaatccatcc 5'race pcr ajn3 5-2 ctctcagttgtcttagccgcaggta 5'race pcr ajn3 3-1 cctgaccatggccttcaaggagtg 3'race pcr ajn3 3-2 gaatgtctgctgcctctttcgcc 3'race pcr ump-1 taatacgactcactatagggcaagcagtggtatcaacgcagagt 3'&5' race ump-2 ctaatacgactcactatagggc 3'&5' race m13f tgtaaaacgacggccagt colony pcr m13r caggaaacagctatgacc colony pcr ajnlrp3-f ctgagaaaacgacttggggatg qrt-pcr ajnlrp3-r gataatcagcaatgtagtgggca qrt-pcr cytb f tgagccgcaacagtaatc qrt-pcr of cytb cytb r aagggaaaaggaagtgaaag qrt-pcr of cytb 425 fig. 1 nucleotide and deduced amino acid sequences of ajnlrp3 in sea cucumber. the termination codon is marked with an asterisk. the low complexity regions are boxed. the putative nacht domain is highlighted in gray. the internal repeat regions are underlined. 426 with 1.0 % agarose gel electrophoresis and using a spectrophotometer (nanophotometer, munich, germany). first strand cdna was synthesized in a 20-l reaction mixture comprising 1000 ng total rna, 4 l of 5 primerscript buffer, 1 l oligo dt primers (50 mm), 1 l random 6-mers (100 mm), 1 l primerscript rt enzyme mix i (primerscript™ rt reagent kit, takara, japan), and rnase-free double-distilled h2o (ddh2o) was added to make up the remaining volume. the reaction mixture was incubated at 37 c for 15 min, and then at 85 c for 5 s to denature the reverse transcriptase. all of the cdna samples were stored at -20 c until use. the partial cdna sequence of ajnlrp3 was obtained from our transcriptome assembly data (unpublished data). based on the partial sequence, nested pcr with rapid amplification of cdna ends (race) was performed to obtain the full length of ajnlrp3 using universal primers and the specific primers nlrp3-5race1/2 (for 5race) and nlrp3-3race1/2 (for 3race). the cdna template for race-pcr was prepared using a smart race cdna amplification kit (takara). the pcr protocol was as follows: 94 °c for 3 min, 30 cycles at 94 °c for 30 s, 65 °c for 30 s, and 72 °c for 2 min, with a final extension at 72 °c for 10 min. nested pcr was conducted using the first pcr products as templates and nlrp3-5race or nlrp3-3race with nup as primers. the pcr protocol was the same as that used in the first round of pcr. the specific products were purified with a gel extraction kit (qiagen), before cloning into the t1 vector (tran, china) and transforming into trans1-t1 phage-resistant chemically competent cells (tran). the positive recombinants were identified by colony pcr using m13 primers (tran). three independent clones were sequenced to confirm the sequence. the primer sequences are shown in table 1. bioinformatics analysis the blast program (http://www.ncbi.nlm.nih.gov/blast/) was used to analyze the nucleotide sequences and to search for protein sequences in other species via the ncbi website (http://www.ncbi.nlm.nih.gov/). signal peptides were predicted using signalp 4.1 (http://www.cbs.dtu.dk/services/signalp/). potential n-glycosylation sites were identified using the netnglyc 1.0 prediction server. multiple sequence alignments were generated by clustalw2 (http://www.ebi.ac.uk/tools/clustalw2). the features of protein motifs were predicted with the simple modular architecture research tool (smart; http://smart.emblheidelberg.de/). a neighbor-joining phylogenic tree was constructed based on the deduced amino acid sequences using the mega 7.0 program. bootstrap sampling was repeated 1,000 times. expression analysis of ajnlrp3 six types of tissues comprising the intestine, respiratory tree, tube feet, celomocytes, body wall, and longitudinal muscle were obtained from the healthy sea cucumbers for rna extraction. total rna extraction and real-time reverse transcription pcr (rt-pcr) analysis were performed as described previously. rt-pcr was conducted in a 20-l reaction mixture comprising 2 l of the original cdna diluted at 1:10, 10 l of 2 sybr green master mix (sybr primescript™ rt-pcr kit ii, takara), 0.4 l of rox reference dye ii, 0.8 l (10 mm) of each primer (table 1), and 6 l ddh2o. the quantitative rt-pcr (qrt-pcr) parameters were as follows: 95 c for 30 s, followed by 40 cycles at 95 c for 5 s and 60 c for 32 s. the specific amplification of the pcr products was confirmed by melting curve analysis. the relative ajnlrp3 mrna levels were analyzed using the 2–△△ct method. the expression quantities of each sample were analyzed by using spss statistics 19.0 software. the cytochrome b (cytb) gene was used as the reference gene. bacterial challenge experiment in the bacterial challenge experiment, 70 healthy sea cucumbers were split between the treatment and control groups, where the treatment group was soaked in 1107 cfu/ml v. splendidus and the control group did not receive the bacterial treatment. the two groups were kept in water at the same temperature. after soaking for 0, 4, 8, 12, 24, 48, and 72 h, the body cavity fluid was collected with five biological replicates in each group. the fluid samples were centrifuged at 1,000 rpm for 5 min, and the body cavity cells were collected and frozen quickly in liquid nitrogen. all of the samples were stored at -80 c for later use. results sequence analysis of ajnlrp3 the full-length ajnlrp3 (genbank accession number mf663701) gene comprises 3642 bp encoding 807 amino acids, where leucine is the most abundant and tryptophan is the least abundant. the predicted molecular mass of the protein deduced from ajnlrp3 (c4178h6596n1104o1220s38) is 93.03 kda and the theoretical pi of 6.14. the amino acid sequence contains two low complexity regions (amino acids 67 78 and 397 413) and two internal repeat sequences (amino acids 483 601 and 639 756), where amino acids 84 239 comprise a nacht domain. the amino acid sequence contains no tailing signal, transmembrane structure, or signal peptide sequence (fig. 1). analysis of the amino acid composition of ajnlrp3 we analyzed the amino acid hydrophilicity/hydrophobicity of the full-length ajnlrp3 cdna using protscale online. the fatty acids coefficient for ajnlrp3 is 97.24, the maximum hydrophilic coefficient is 2.52, the minimum hydrophilic coefficient is -3.089, and the total average hydrophilic coefficient is -0.239, which indicates that the protein is hydrophilic (fig. 2). homology and evolutionary analysis based on the amino acid sequence of ajnlrp3 we used orf finder to identify the open reading frame of the ajnlrp3 gene in the sea cucumber and then compared it with other nlrp3 427 fig. 2 the hydrophilicity/hydrophobicity results of ajnlrp3. horizontal indicated the position of amino acid, vertical coordinates indicated the score. genes using smartblast. the results showed that the amino acid sequence predicted for ajnlrp3 shared 24 % similarity compared with the amino acid sequence of nlrp3 in homo sapiens, while the shared similarity with that in mus musculus was 25 % and that in danio rerio was 23 %. in order to determine the phylogenetic relationships between ajnlrp3 and other nlrp3s, we constructed a phylogenetic tree based on the deduced amino acid sequences of ajnlrp3 and different members of the nlrp3 superfamily using the neighbor-joining method with 1,000 replicates, and the results are shown in figure 3. the phylogenetic tree showed that the sequence in a. japonicus clustered with those in the echinoderm subgroup and it had a close evolutionary relationship with the sequence in strongylocentrotus purpuratus. the sequences in a. japonicus and s. purpuratus had high genetic distances from those in vertebrates. tissue distribution of ajnlrp3 the spatial expression pattern of ajnlrp3 mrna was investigated using qrt-pcr with the cytb gene as the internal control and the results are shown in figure 4. the results show relative expression in arbitrary units normalized to longitudinal muscle expression. we found that the ajnlrp3 gene was expressed in all of the tissues tested, with a significantly higher expression level in the body wall compared with the other tissues (p < 0.01), whereas the lowest expression level was found in the longitudinal muscle. there were no significant differences in the expression levels in the respiratory tree, intestine, tube feet and body cavity cells. expression of the ajnlrp3 gene in diseased sea cucumbers we examined the expression of the ajnlrp3 gene in the intestine, respiratory tree, and coelomocytes in healthy sea cucumbers and those with skin ulcer syndrome. the results show relative expression in arbitrary units normalized to healthy sea cucumber expression. the expression level of the ajnlrp3 gene was increased significantly by 2.6-fold in celomocytes in the infected sea cucumbers compared with those in the control group. in the respiratory tree, the expression level was reduced to 0.87-fold and it differed significantly from that in the control group. there was no significant difference in the intestine tissues (fig. 5). expression of ajnlrp3 in celomocytes after bacterial challenge the effects of challenge with v. splendidus on the transcriptional expression levels of the ajnlrp3 gene were compared in celomocytes by rt-pcr (fig. 6). the results show relative expression in arbitrary units normalized to healthy sea cucumber expression. the results showed that the expression level of the ajnlrp3 gene did not differ significantly from that in the control group at 4 h, but the expression level decreased significantly by 0.23-fold at 8 h. after 12 h, the difference between the two groups was not significant. at 24 h, the expression level of the gene was decreased significantly by 0.20-fold compared with the control group, but it then increased to the level in the control. 428 myotis brandtii epq01414.1 bos taurus aai47937.1 homo sapiens aai43360.1 mus musculus aai16176.1 gallus gallus agz03665.1 xenopus tropicalis xp 004911151.1 maylandia zebra xp 014268508.1 cyprinus carpio xp 018921775.1 apostichopus japonicus ajnlrp3 strongylocentrotus purpuratus xp 011680412.1 fig. 3 consensus neighbor-joining tree based on the amino acid sequence ofajnlrp3from other species. the evolutionary history was inferred using the neighbor-joining method. the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches evolutionary analyses were conducted in mega7.0. discussion in this study, we characterized and analyzed the expression of a cdna for the ajnlrp3 gene in a. japonicus. a 3642-bp nucleotide sequence representing the complete cdna sequence of ajnlrp3 was obtained by overlapping an est from the a. japonicus cdna library and 5/3race products. domain and motif analysis showed that the ajnlrp3 gene contains two low complexity regions, two internal repeat regions, and a nacht structural domain. nacht is a common domain structure in the members of the nlr family, where it participates in the innate immune recognition process, as well as apoptosis and the major histocompatibility complex transcriptional activation process (orlowski et al., 2016). blastp analysis showed that the deduced amino acid sequence of ajnlrp3 shares 24 % identity with nlrp3 from homo sapiens (aac37547.1), 25 % with that from mus musculus (aaa37375.1) and 23 % with that from danio rerio (aaq97764.1). multiple comparisons showed that ajnlrp3 and other nlrp3s share low homology. in addition, the relationships in the phylogenetic tree showed that a. japonicus has a closer evolution relationship with s. purpuratus. the cdna sequence did not contain the same protein structural domain and repeated leucine domain that are found in all vertebrate nlrp3s. our analysis of nlrp3 genes in vertebrates using the ncbi database showed that some vertebrate nlrp3 genes also lack the same protein structural domain and leucine repeated domain, thereby suggesting effects due to gene duplication during their evolution. our results demonstrated that ajnlrp3 is a novel member of the nlr family. to better understand the role of ajnlrp3 in innate immunity in the sea cucumber, we analyzed the tissue distribution of ajnlrp3 by real-time pcr. ajnlrp3 was distributed in all of the tissues that we tested, which indicates that it has important roles in physiological processes. nlrp3 regulates various processes in inflammation and thus it is distributed in a wide range of tissue (tohno et al., 2011; huang et al., 2014; ye et al., 2015). the mrna highest expression level of ajnlrp3 was found in the body wall tissues. seawater contains a large number of pathogenic microorganisms and the body wall of the sea cucumber is the first line of defense against infection by pathogens via direct contact with seawater, which may explain why the highest mrna expression level of ajnlrp3 was detected in the body wall. the relatively high mrna expression levels of ajnlrp3 in the respiratory tree, intestine, tube feet, and celomocytes indicate that it is involved in the immune response of a. japonicus (motta et al., 2015). there were no significant differences in the expression levels among the different tissues, but the lowest expression level was found in the longitudinal muscle. the expression profiles indicate that ajnlrp3 is involved in important physiological functions. prrs play vital roles in the recognition of pathogenic microorganisms and the initiation of the natural immune response. the four known categories of prrs comprise toll-like receptors and c-type lectin receptors in the cell membrane, and retinoic acid-inducible gene-i-like receptors and nod-like receptors (nlrs) in the cytoplasm. studies have shown that nlrs have important roles in the inflammatory response (kate et al., 2010; tee et al., 2013). ajnlrp3 is a member of the nlr genes family. as reported previously, celomocytes are the main cellular components of the immune system as freely circulating cells in sea cucumbers. v. splendidus is regarded as a major pathogen that affects sea cucumbers in aquaculture (wang et al., 2007; wang et al., 2009; zhao et al., 2011). thus, to 429 fig. 4 relative expression of ajnlrp3 in different tissues. the results show relative expression in arbitrary units normalized to longitudinal muscle expression.each vertical bar represents the mean ± sd (n = 3), cytb used as a reference gene. different roman letters above the bars indicate significant differences in different tissues at p < 0.05. better understand the role of ajnlrp3 in the response of a. japonicus to bacterial challenge, we tested the effect of treatment with v. splendidus as a potential pathogen. we considered that the expression level of the ajnlrp3 gene in the celomocytes after bacterial challenge might provide insights into the defensive mechanisms and regulatory processes in the sea cucumber after infection. we found that infection with v. splendidus caused changes in the expression level of the ajnlrp3 gene, where the expression level of ajnlrp3 decreased significantly from 8 to 24 h after the bacterial challenge, thereby indicating that the transcription of ajnlrp3 was suppressed before tissue damage occurred. in the nod signal transduction pathway, multiple tissue proteases act as upstream regulatory genes to promote the synthesis of the interleukin-1β precursor and the secretion of interleukin-1β is mediated by nlrp3 (fujisawa et al., 2007; hirota et al., 2011; morishige et al., 2010). similar regulatory effects may occur in the sea cucumber. the expression level of the ajnlrp3 gene was decreased significantly in the respiratory tree tissues of diseased sea cucumbers compared with the control group. the expression level was also significantly higher in the body cavity cells of diseased sea cucumbers compared with the control group, thereby indicating that this gene is involved in the response to infection by pathogens. fig. 5 relative expression of ajnlrp3 in coelomocytes after challenged with v. splendidus. the results show relative expression in arbitrary units normalized to healthy sea cucumber expression. each vertical bar represents the mean ± sd (n = 3). the asterisks (*) and double asterisks (**) above the bars represent differences at p < 0.05 and significant differences at p < 0.01, respectively. 430 fig. 6 relative expression of ajnlrp3 in celomocytes after challenged with v. splendidus. the results show relative expression in arbitrary units normalized to healthy sea cucumber expression.each vertical bar represents the mean ± sd (n = 5).the asterisks (*) and double asterisks (**) above the bars represent differences at p < 0.05 and significant differences at p < 0.01, respectively. inflammatory response is an immune defense process caused by cell infection or cell damage and plays an important role in the process of wound repair and anti-infection. the cytoplasmic receptor of the nucleotide binding receptor (nlr) family is one of the key components of the inflammatory body, and in many animals and sea cucumber genome sequencing, there are nlrs, a large family of genes. inflammatory small bodies induced by nlrp3 are the most important types of inflammatory complexes (kanneganti et al., 2007; franchi et al., 2009). unlike other known inflammatory reactions, nlrp3 inflammation is induced by various types of activators, including pathogenic bacteria, portotoxins, environmental irritants and some internal molecules. the inflammatory response can be induced when the transcriptional level of nlrp3 exceeds the threshold. the high and low levels of the threshold are associated with the type of cell, such as the lower threshold of dendritic cells compared to phagocytes. as the inflammatory response worsens, the host cell can synthesize mir-223. the transcription products of nlrp3 were degraded by the 3 'non-coding region of nlrp3 (bauernfeind et al., 2012; haneklaus et al., 2012). nlrp3 genes have been detected in homo sapiens (gross et al., 2009; hise et al., 2009), mus (bauer et al., 2010; heneka et al., 2013), vitulus (zimin et al.,2009), porcorum (tohno et al., 2016), pullum (ye et al., 2015), and other vertebrates, but few studies have investigated them in invertebrates, and thus the results obtained in the present study provide important new insights into the role of the nlrp3 gene in invertebrates. acknowledgments this work was supported by grants from science & technology project of liaoning province (2015203003) and the elitist of agricultural scientific research and innovative team of ministry of agriculture (2014). references bauer c, duewell p, mayer c, lehr ha, fitzgerald ka, dauer m, et al. colitis induced in mice with dextran sulfate sodium (dss) is mediated by the nlrp3 inflammasome. gut 59: 1192, 2010. bauernfeind f, rieger a, schildberg fa, knolle pa, schmid-burgk jl, hornung v. nlrp3 inflammasome activity is negatively controlled by mir-223. j. immunol. 189: 4175-4181, 2012. brigati c, noonan dm, albini a, benelli r. tumors and inflammatory infiltrates: friends or 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stage and analysis of reservoir of pathogens acta microbiol. sinica 50: 687-693, 2010. zhao y, ma h, zhang w, ai q, mai k, xu w, et al. effects of dietary -glucan on the growth, immune responses and resistance of sea cucumber, apostichopus japonicus against vibrio splendidus infection. aquaculture 315: 269-274, 2011. zimin a v, delcher a l, florea l, kelley dr, schatz mc, puiu d, et al. a whole-genome assembly of the domestic cow, bos taurus. genome biol. 10: r42, 2009. http://www.medsci.cn/sci/submit.do?id=3aeb2443 http://www.medsci.cn/sci/submit.do?id=3aeb2443 42 isj 19: 42-52, 2022 issn 1824-307x research report characterization of a novel c-type lectin against oshv-1 infection in scapharca broughtonii d wang1,2,3, b huang2,3, j yu2,3,4, c bai2,3, c li2,3, c wang2,3, l xin2,3*, h zhou1* 1hainan university, haikou, 570228, pr china 2function laboratory for marine fisheries science and food production processes, qingdao national laboratory for marine science and technology, qingdao 266071, pr china 3qingdao key laboratory of mariculture epidemiology and biosecurity, key laboratory of maricultural organism disease control, ministry of agriculture, yellow sea fisheries research institute, chinese academy of fishery sciences, qingdao 266071, pr china 4dalian ocean university, dalian 116023, pr china this is an open access article published under the cc by license accepted march 2, 2022 abstract as important pattern recognition receptors (prrs), most c-type lectins (ctls) are a class of ca2+-dependent carbohydrate-binding proteins that are found to be involved in non-self-recognition and antiviral process. in this study, a new ctl, named sbctl, was identified from ark clams, scapharca broughtonii. the amino acid sequence of sbctl consisted of a predicted crd (carbohydrate recognition domain) structural domain (including 102 amino acid residues). the amino acid sequence of sbctl shared 28 % 39 % similarity with other ctls. there were two potential ca2+ binding sites in sbctl. the expression of sbctl mrna was detected in all selected tissues, with the highest expression in the gills. expression of sbctl mrna increased significantly (p < 0.05) and total vibrio content increased significantly (p < 0.05) in ark clam infected with oshv-1 at 72 h post-infection. the binding activities of recombinant sbctl (rsbctl) to various pamps with or without ca2+ were analyzed by elisa, rsbctl showed especially high binding activity to lps in a ca2+-independent manner. rsbctl also functioned on sheltering ark clams from oshv-1 infection in vivo, there were less mortality occurred in the rsbctl treated group than the control. in all, it suggests that sbctl, could served a critical role in the immune response against intruders in ark clams. key words: scapharca broughtonii; c-type lectin; pamp binding; oshv-1 infection introduction pattern recognition receptor-mediated recognition of pathogen-associate molecular patterns (pamps) is a key defense component in invertebrate innate immunity (vasta et al., 2004). as a family of pattern recognition receptors, c-type lectins (ctls) could execute recognition and binding activity to a variety of carbohydrate ligands in immune defense which would regulate the expression of _________________________________________ corresponding authors: lusheng xin function laboratory for marine fisheries science and food production processes qingdao national laboratory for marine science and technology qingdao 266071, pr china e-mail: xinls@ysfri.ac.cn hailong zhou hainan university haikou 570228, pr china e-mail: zhouhl@hainu.edu.cn signaling molecules and inflammatory responses, further induce apoptosis or other immunological effect on pathogen killing and clearance (shi et al., 2004; eisen, 2010). in red swamp crayfish, a ctl, pclec6, was found to motivate crayfish defense system against pathogen invasion by initial recognition of pathogens and up-regulation of immune effectors such as anti-lipopolysaccharide factor (alf) (zhang et al., 2018). in marsupenaeus japonicus, two ctls, ldlrlec1 and ldlrlec2, were found to protect shrimp from white spot syndrome virus (wssv) infection by binding to wssv vesicle protein vp28 and inhibiting viral replication (qian et al., 2012). another ctl of penaeus vannamei (lvctld) is involved in shrimps against the invasion of yellow head virus (yhv) by means of recruiting hemocytes to the site of virus infection and activating the prophenoloxidase cascade system (propo system) (junkunlo et al., 2012). ctls commonly contain one or more carbohydrate recognition domains (crds). a single crd consists of 110-130 amino acids, featured by internal and external bilayer structures. its 43 three-dimensional structure is reinforced by two pairs of highly conserved disulphide bonds at the base layer (zelensky and gready, 2005). there are usually four ca2+-binding domains in each crd. ca2+ plays an important role in crd structure maintaining the ligand binding activity. the carbohydrate binding sites of crds are diverse, such as epn, qpd, qpe, epg, npr (zhang et al., 2009a; qian et al., 2012b). crds entitled ctls the activities of recognition and binding to the polysaccharides of intruders (zelensky and gready, 2005; luo et al., 2007). ctls with a single crd have greater specificity, while those with multiple crds show broader recognition spectrum (zhang, et al., 2009; wang, et al., 2012). in oysters, cgclec-3 containing a single crd showed the strongest binding activity to pgn among tested pamps (song et al., 2019). in the silkworm bombyx mori, a ctl named bmmbp, containing two crds, each one had dissimilar binding spectra to sugars. crd1 bound to teichoic acid and mannan, crd2 bound to teichoic acid, peptidoglycan, and mannan, pamps of gram-positive bacteria and yeasts. these properties contribute to the multi-recognition characteristic of bmmbp (watanabe et al., 2006). in chlamys farreri, a ctl named cflec-4 with four crds showed strong binding activity to a variety of pamps including lps, pgn, mannan and glucan, resulting in that cflec-4 could bind to diverse microorganisms including gram-negative bacteria, gram-positive bacteria and yeasts (wu et al., 2012; wang et al., 2013). ark clam, scapharca broughtonii, is a large marine benthic bivalve and mainly distributes in the offshore water of northern asia (zhao et al., 2018)， it is susceptible to ostreid herpesvirus-1 (oshv-1) infection as oysters and other bivalves. mass mortalities of ark clams and other culture bivalves are frequently occurred due to oshv-1 infection (ren et al., 2013). thus, much attention has been attracted on bivalve molecular immune mechanisms in order to achieve effective prevention and control of the oshv-1 disease. bivalve ctls are multitudinous and conservatively execute initial non-self recognition. however, it is largely unknown how bivalve ctls would function in response to oshv-1 infection. in this study, a novel c-type lectin sbctl was characterized from ark clam, s. broughtonii, with the aim of exploring the molecular functions of sbctl, investigating its protective effects on ark clams against oshv-1 in vivo. the results of this study will deep our understanding on the role of c-type lectins in bivalve innate immunity. materials and methods ark clams, experimental infection and sample collection ark clams were obtained from a local aquaculture farm in qingdao, china and were acclimated in air-pumped seawater under temperature 16 ± 2 °c, do 5 mg/l, salinity 30 ± 1 ‰, ph 7.5-8.5 for two weeks prior to the following experiments. different tissues, including hepatopancreas, hemocytes, gill, mantle, adductor muscle and foot, were dissected from six healthy ark clams as parallel samples to investigate the mrna distribution of sbctl. for experimental infection, oshv-1 inoculum was prepared as described by schikorski (sorvillo et al., 2011) with modification. briefly, the mantle tissues from oshv-1 positive ark clams were split and homogenized. then mantle homogenate was centrifuged at 1000 r/min for 15 min at 4 °c, the precipitate was discarded and the supernatant was filtered through a 0.22 μm pore size filter. meanwhile, the mantle tissues from oshv-1 negative ark clams were parallelly treated to prepare oshv-1 negative inoculum for the control group. a total of 100 ark clams were randomly selected for virus inoculation by injection with 100 μl prepared oshv-1 inoculum (~105 copies of viral dna/μl). another 100 ark clams received an injection with the same volume of oshv-1 negative inoculum as the control group. nine individuals of each group were randomly sampled at 0, 6, 12, 24, 48 and 72 h post injection. three hemocyte samples were prepared for each treatment by centrifugation of the mixed hemolymph of three random individuals. rna extraction and cdna synthesis total mrna was extracted from hemocytes using trizol reagent (invitrogen) according to the manufacture's protocol. the first-strand cdna synthesis was carried out based on m-mlv rt usage information using the dnase i (promega) treated total mrna as template and oligo (dt)-adaptor primer (table 1). the reaction mixtures were incubated at 50 °c for 1 h, terminated by heating at 85 °c for 5 min. the cdna mix was diluted to 1:20 and stored at -80 °c for subsequent quantitative real-time pcr (qrt-pcr) analysis. cdna clone and sequence analyses of sbctl gene a highly homologous sequence to ctl was identified in the transcriptome data of s. broughtonii. its complete open reading frame (orf) was predicted by orf finder in the national center for biotechnology information (http://www.ncbi.nlm.nih.gov/). a pair of specific primers sbctl-f and sbctl-r (table 1) were designed to clone the orf sequence of sbctl using primer 5 according to the sequence information of the transcriptome assembly results. pcr products were gel-purified and verified by sequencing. the expert protein analysis system (http://www.expasy.org/) was used to deduced the amino acid sequence of sbctl. the conserved protein domain of sbctl was revealed by the simple modular architecture research tool (smart) version 4.0 (http://www.smart.emblheidelberg.de/). the presumed tertiary structure of sbctl was established using iterative threading assembly refinement (i-tasser, https://zhanggroup.org/i-tasser/) and presented by rasmol version 2.7.5. the clustal omega multiple http://www.ncbi.nlm.nih.gov/ http://www.expasy.org/ http://www.smart.emblheidelberg.de/ https://zhanggroup.org/i-tasser/ 44 table 1 designations and nucleotide sequences of the primers were used in this study primer sequence (5′-3′) oligo (dt) ttttttttttttttt sbctl-f atgctttattttttgttcctagtg sbctl-r ctaaagacgtaccggtctctcac bf gtcgcatctttggatttaacaa b4 actgggatccgactgacaac bp fam-tgcccctgtcatcttgaggtatagacaatc-bhq t7 taatacgactcactataggg t7-terminator tgctagttattgctcagcgg rt-ef-f agtcaccaaggctgcacagaaag rt-ef-r tccgacgtatttctttgcgatgt sbctl-f-c2 ctctttaccatgaagatacccacc sbctl-r-c6 gtgcacggcttaccattttt sbctl-bamhⅰ-f cgcggatccgctttattttttgttcctagtg sbctl-notⅰ-r aaggaaaaaagcggccgcaaagacgtaccggtctctcac tf ggcgtaaagcgcatgcaggt tr gaaattctacccccctctacag vf cgccagggtttcccagtcacgac vr cacacaggaaacagctatgac alignment program (http://www.ebi.ac.uk/clustalox/) was used to perform the multiple sequence alignment. an unrooted phylogenetic tree of various ctls containing single crd was constructed based on the sequence alignment by the neighbor-joining (nj) algorithm using the mega 5.0 program (song et al., 2019). to derive confidence values for the phylogenetic analysis, the bootstrap test was repeated 1000 times. the expression and purification of recombinant sbctl protein recombinant sbctl (rsbctl) were expressed with pet-30a system (novagen). the coding region of sbctl was amplified by the primers sbctl-bamhi-f and sbctl-noti-r, bamhi and noti endonuclease digestion sites were added at their 5’ end, respectively (table 1). the pcr products were digested with bamhi and noti endonucleases, gel-purified, then ligased to linearized pet-30a that was digested and recycled in the same way, the recombined expression vector pet-30a-sbctl was verified by dna sequencing. finally, the recombined pet-30a-sbctl was transformed into transetta (de) (transgen biotech). the positive colony was incubated and induced by 1 mm isopropyl-β-dthiogalactoside (iptg). then rsbctl was purified by a ni2+ chelating sepharose column, eluted by 500 mm imidazole under denatured condition (8 m urea). the purified proteins were re-natured in gradient urea-tbs glycerol buffer (50 mm tris-hcl, 50 mm nacl, 15 % glycerol, 2 mm reduced glutathione, 0.2 mm oxide glutathione, a gradient urea concentration of 6, 4, 3, 2, 1, 0 mm, ph 7.6; dialyzed under each gradient at 4 °c for 12 h). protein concentration was determined by bca method (walker, 1994). pamps binding assay the pamps-binding activity of rsbctl was assessed by enzyme-linked immune sorbent assay (elisa) according to previous report with modification. briefly, 100 μg of lps, pgn, man and cpg diluted in 100 μl carbonate-bicarbonate buffer (35 mmol l-1 nahco3, 15 mmol l-1 na2co3, ph 9.6) were used to coat a 96-well plate. the wells were blocked with 5 % bsa (sangon biotech, china) in tbst at 37 °c for 1 h, and then washed with tbst for three times. 100 μl of gradient diluted rsbctl (containing 0 mm, 10 mm and 100 mm cacl2) was added sequentially to the wells and incubated for 1 h at 37 °c. the same concentration of rtrx was used as negative control. after three times washing with pbst and then the bound protein was detected immunochemically. firstly, 100 μl of hrp-labeled goat-anti-hrp-conjugated mouse anti-his-tag mab (sangon biotech, china, diluted 1:2000 in 5 % bsa) was added into each well and incubated at 37 °c for http://www.ebi.ac.uk/clustalox/ 45 fig. 1 nucleotides and deduced amino acid sequences of sbctl. nucleotides and amino acids are labelled with numbers on the left of each line, with the green and red boxes representing the two predicted ca 2+ binding sites, respectively. the functional domains predicted by the smart program are marked with different colours: red for signal peptides and dark pink for crd 1 h. after another three times washing, 100 μl tmb single component substrate solution (solarbio, beijing) was added and incubated at 37 °c for 15 min. the reaction was stopped by adding 50 μl of 2 m termination solution per well, and the absorbance was measured at 405 nm (tecan, switzerland). the wells with 100 μl tbs were used as blank. the non-immunized mouse serum was employed as negative control. three replications were performed for each sample, and the data were presented as mean ± s.d. (n = 3). samples with p (sample)-b (blank)/n (negative) b (blank) > 2.1 were considered as positive. quantification of oshv-1 the samples of hemocytes were collected at 0 h, 24 h, 48 h and 72 h post the injection of oshv-1 in ark clams. tissue genomic dna was extracted using the dneasy blood and tissue kit (qiagen) following the manufacturer's instructions. dna concentration and quality were assessed by spectrophotometer (nanodrop2000, thermo-scientific). to reduce individual differences, each dna sample was prepared from three individuals. then dna samples were taken for real-time quantitative pcr (qrt-pcr) with the pair of primers bf/b4 (table 1) and the probe bp. the reaction system was 20 μl and amplification was performed using cfx connect from bio-rad, usa under the following condition: 1 cycle 95 °c for 10 min, followed by 40 cycles of 95 °c for 10 s, 60 °c for 30 s (segarra et al., 2010). the oshv-1 quantitation was calculated from the standard curve, which was created from a 10-fold dilution series (107-101 copies µl-1) of plasmid containing the target sequence. quantification of total vibrio dna by qpcr the qpcr assay based on sybr® green described by julien (de lorgeril et al., 2018) was used to quantify total vibrio with modification. tissue samples and dna extraction were processed as quantification of oshv-1, differently, each tissue 46 sample was mixed with equal escherichia coli (10 µl, od600 1.0) containing plasmid puc18 as the reference. the primer pairs tf and tr (table 1) targeting 16s rdna were designed to relatively quantify vibrio content by the 2−δδcq method with the measured threshold cycle values of the reference genes in e. coli using the primer pairs vf and vr (table 1). real-time pcr amplification was carried out in an abi 7500 real-time thermal cycler according to the manual (eppendorf, hamburg, germany). quantitative real-time pcr analyses of sbctl mrna expression sbctl mrna expression level was measured by sybr® green fluorescent quantitative real-time pcr. two specific primers of sbctl, sbctl-f-c2 and sbctl-r-c6 (table 1), were designed for rt-pcr amplification. a pair of primers for elongation factor, rt-ef-f and rt-ef-r (table 1), were chosen for pcr amplification as an internal control. real-time pcr amplification was carried out in an abi 7500 real-time thermal cycler according to the manual (eppendorf, hamburg, germany). dissociation curve analysis of amplification products was performed at the end of each pcr to confirm the specificity. after the pcr program, the 2−δδcq method was used to analyze the expression level of sbctl. survival rate of s. broughtonii ark clams were divided into two groups (with 100 ark clams in each group). one group was injected with 1 μg/μl sbctl (100 μl), and the other group with the same volume of sterile sea water as control. after 1 h, 100 μl of oshv-1 inoculum was injected into each ark clams of both groups. the number of surviving ark clams in each group was counted every 12 h post oshv-1 infection. the survival rate was equal to (surviving ark clams/100) × 100 %. survival curves were generated by graphpad prism 8.0. statistical analysis data was analyzed using origin 8.1 (origin lab) and statistical package for social sciences (spss) 23.0. significant differences for each assay were tested by one-way analysis of variance (anova). if significant differences were indicated at the 0.05 level, then a post hoc multiple-comparisons (tukey’s) test was used to examine significant differences among treatments using spss. difference was considered significant at p < 0.05 and extremely significant at p < 0.01 results cdna clone and sequence analyses of sbctl a coding sequence of 465 bp was amplified with specific primers sbctl-f and sbctl-r (table 1). it encoded a polypeptide of 154 amino acids (predicted molecular mass of 17.453 kda and pi was 4.64). the red colour region showed the signal peptide (position 1st to 17th amino acid). the purple region showed the single carbohydrate recognition structural domain (crd) of sbctl with 102 amino acid residues. the crd of sbctl has two calcium binding sites, one ca2+ binding site linked to tyr62, asn87, asn89, glu93, glu149 and the other to ser121, glu136, ser137 (fig. 1). multiple sequence alignment and phylogenetic analysis of sbctl blast homology analysis showed that sbctl has 28 % 39 % homology with ctls from the following species (fig. 2): eudyptes pachyrhynchus fig. 2 multiple sequence alignment of sbctl with other ctls, including eudyptes pachyrhynchus (kaf1598677.1); channa argus (kaf3707004.1); perca fluviatilis (xp_039677198.1); anabas testudineus (xp_026205614.1); a. testudineus (xp_026205615.1); a. testudineus (xp_033181795.1); falco naumanni (xp_040469685.1); crassostrea virginica (xp_022311951.1); c. virginica (xp_022307439.1). red arrows indicate four conserved cysteine sites 47 fig. 3 phylogenetic tree of ctls containing a single crd. mega 5.0 program was used to construct the tree by neighbor-joining (nj) algorithm based on the multiple sequence alignment. the optimal tree is shown. the reliability of the branching was tested by bootstrap re-sampling (1000 pseudo-replicates). the ctls used in phylogenetic tree were listed as the following: homo sapiens (np_987099.1)：homo sapiens (caa04230.1); crassostrea gigas (np_001292233.1); strongylocentrotus purpuratus (np_999766.1); canis lupus familiaris (xp_005637254.1); danio rerio (xp_005172687.1); litopenaeus vannamei (dq858900.1); penaeus indicus (adv17348.1); portunus trituberculatus (ahk59786.1); oplegnathus fasciatus (acy66647.1); lutjanus sanguineus (agt37609.1); penaeus monodon (aaz29608.1); rattus norvegicus (xp_006237382.1); ruditapes philippinarum (azr37772.1); macrobrachium rosenbergii (qjs38721.1); scylla paramamosain (udm59627.1); toxocara canis (aov81587.1); sinonovacula constricta (qha94921.1); saxidomus purpuratus (bbe43064.1); saxidomus purpuratu (bbe43065.1); penaeus japonicus (ahf21000.1); ruditapes philippinarum (acu83213.1); penaeus merguiensis (acr56805.1) (kaf1598677.1); channa argus (kaf3707004.1); perca fluviatilis (xp_039677198.1); anabas testudineus (xp_026205614.1); a. testudineus (xp_026205615.1); a. testudineus (xp_033181795.1); falco naumanni (xp_040469685.1); crassostrea virginica (xp_022311951.1); c. virginica (xp_022307439.1). the highest similarity was found with a. testudineus at 39.19%. similarity with c. argus was higher at 37.5%. four cysteines (cys48; cys78; cys120; cys148) were conservatively existed in all selected ctls as the red arrow shown, these four conserved cysteine residues are involved in the formation of disulfide bonds within the crd. to evaluate the molecular evolutionary relationships between sbctl and other ctls containing a single crd, a phylogenetic tree was constructed using the nj algorithm (1000 bootstrap replicates) based on the amino acid sequences of 18 ctls (fig. 3). of the selected species, homo sapiens, canis lupus familiaris and rattus norvegicus belong to the chordata phylum mammalia. danio rerio, oplegnathus fasciatus and lutjanus sanguineus belong to the chordata phylum pisce. strongylocentrotus purpuratus belongs to the echinodermata phylum echinoidea. c. gigas, saxidomus purpuratus, sinonovacula constricta and ruditapes philippinarum belong to the mollusca phylum bivalvia. in the phylogenetic tree, the sbctl is clustered in a branch with the ctl of a bivalve species, s. constricta, and is most closely related to other bivalve ctls. the predicted three-dimensional structure of sbctl the potential three-dimensional structure of sbctl was established by the i-tasser. the crd of sbctl composed of two α-helices and five β-strands forming a bilayer structure. two potential ca2+ binding sites were shown in crd. the two ca2+ binding sites are located at tyr3 and glu129. a man binding domain were also indicated with five amino acids (ser127, glu136, gly138, thr106, ser125) linked together (fig. 4). the purified protein and pamp-binding specificity of rsbctl sds-page showed a distinct band with a molecular mass of ~25 kd in the lane of purified 48 fig. 4 the i-tasser program predicts the structure of sbctl. random coil marked as white, β-stands marked as yellow and α-helices marked as red. the grey and blue spheres forming the cyclic structural domain are man binding sites rsbctl, which was consistent with the predicted molecular mass of sbctl (fig. 5). the pamp-binding capacity of rsbctl was recorded as p/n at 405 nm, and the samples with p/n > 2.1 were considered as positive. in the presence of ca2+ or not, the p/n values for lps and man were greater than 2.1, with the concentration of 100 μg/ml to 1.5625 μg/ml respectively. rsbctl showed the highest affinity to lps, lower affinity to man, and almost no affinity to pgn and cpg. the p/n values for lps and man showed inapparent change with different ca2+ concentration. (fig. 6). the load change of oshv-1 in infected ark clams the quantification of oshv-1 was performed by real-time pcr based on taqman probe method. the oshv-1 dna load in these tissues sharply increased to ~107 copies per ng total dna within 48 h. after that, oshv-1 copies tended to sustain at this level without ongoing increase (around 107 copies/ng total dna). (fig. 7). total vibrio quantity post oshv-1 infection the total vibrio in ark clam hemocytes at 0, 24, 48 and 72 h after oshv-1 infection (fig. 8) was further examined. after oshv-1 stimulation, vibrio content in hemocytes decreased at 24 h, then increased before reaching peak levels at 72 h (4.25-fold that in 0h, p < 0.05) at 72 h. the mrna expression pattern of sbctl the distribution of sbctl mrna was investigated in tissues, including hemocytes, mantle, foot, gill, hepatopancreas and adductor muscle. the transcripts of sbctl mrna could be detected in all tested tissues. the highest transcript levels of sbctl fig. 5 sds-page of rsbctl. lane m: protein molecular standard; lane 1: lysate of e. coli transetta cells transformed with pet-30a with iptg induction; lane 2: purified rsbctl protein 49 fig. 6 elisa analysis of rsbctl binding activity to pamps, including lps, pgn, man and cpg. the value of p/n > 2.1 was considered positive. 10mm and 100mm indicate the concentration of cacl2. results represent the mean of three replicates ± s.d. mrna were found in the gills (p < 0.05), while the lowest in adductor muscle. there were no significant differences of sbctl mrna expression in hemocytes, mantle, foot and hepatopancreas (p > 0.05). (fig. 9a). the mrna expression of sbctl in response to oshv-1 infection was analyzed in hemocytes. at the early stage of oshv-1 infection, oshv-1 dna copies increased sharply (fig. 7), while the level of sbctl mrna showed inapparent change post 48 h infection (p > 0.05). later oshv-1 dna copies reached a plateau (~107 copies per ng total dna), the level of sbctl mrna increased significantly post 72 h infection compared to the prior time points (fig. 9b). the protection role of rsbctl in vivo the survival rates of oshv-1 infected ark clams were recorded in both rsbctl treated group and control group. the control group was set with the oshv-1 infection only, and the experimental group was pretreated with rsbctl prior to oshv-1 infection. the results showed that the survival rate of the control group began to decline post 48 h infection, while no death was simultaneously found in the rsbctl treatment group. after 72 h of infection, the survival rate of the rsbctl treatment group decreased to 85 %, meanwhile the control group decreased to 48.14 %. after 108 h infection, the survival rate of the experimental group was 30 % and that of the control group was only 5.55 %. survival curve comparation of both groups showed significant difference (p < 0.05) using the method of log-rank (mantel-cox) test. (fig. 10) discussion ctls, as members of the prrs, are crucial in the initial non-self recognition by the binding between their crds and specific carbohydrate structures on the surface of microorganisms. a single and multiple crds-containing ctls conservatively exist in invertebrates, recognize various pamps and activate intracellular immune fig. 7 the load changes of oshv-1 at different time points in the hemocytes of ark clams. oshv-1 load was expressed as the oshv-1 copies in per ng total tissue dna. each value was shown as mean ± s.d. (n = 3) 50 signal pathways against pathogens (wang et al., 2007; zhang et al., 2009b; kong et al., 2011). in contrast to vertebrate ctls, the immunological function of invertebrate ctls defects in comprehensive understanding. in this study, a new member of the ctls containing a single crd, sbctl, was identified from ark clams, s. broughtonii. the sequence characteristics and biological activities of sbctl were investigated in order to gain more understanding of their role in ark clam innate immunity. sbctl was highly conserved in sequential and structural features with other ctls. a signal peptide at the n-terminal end of sbctl revealed that sbctl could function as a secreted ctl. four cysteine residues involved in the formation of the internal disulfide bridges were well conserved in the crd of sbctl. according to available studies, there were commonly four ca2+ binding sites in crds of ctls. the recognition activity and binding specificity of ctls depends on the location of the proton donor and proton acceptor at ca2+ binding site (zelensky and gready, 2005). two ca2+-binding site was also predicted in the crd of sbctl, suggesting ca2+ might be needed for sbctl binding to carbohydrates. however, ca2+ binding site is not necessary for all ctls, such as two ctls, spl-1 and spl-2, that were capable of binding to acetamide-containing carbohydrates in the absence of ca2+ in s. purpuratus (unno et al., 2019; unno et al., 2020). fig. 8 the change of total vibrio post oshv-1 infection. the total vibrio in ark clam hemocytes after oshv-1 infection was detected by real-time pcr at 0, 24, 48 and 72 h. each value was shown as mean± s.d. (n=3). the significant difference among groups is indicated by dissimilar letters (p < 0.05) the recognition and binding to pamps is one of the most important properties of ctls, which was achieved by the crds of ctls. multiple-crds containing ctls possess broader recognition spectrum than ctls containing a single crd. conversely, a single crd containing ctls are fig. 9 real-time pcr analyses of sbctl. a) sbctl mrna expression levels in different tissues of ark clam s.broughtonii. sbctl mrna expression levels in mantle, gills, hemocytes, foot, muscle and hepatopancreas were all normalized to that of muscle. vertical bars represent the mean ± s.d. (n = 6). the significant difference among groups is indicated by dissimilar letters (p < 0.05). b) at 0, 6, 12, 24, 48 and 72 h after oshv-1 challenge, hemocytes were collected and the mrna transcription patterns of sbctl were detected by real-time pcr. the cytb gene was used as an internal control to calibrate the cdna template for all the samples. each value was shown as mean ± s.d. (n = 3). the significant difference among groups is indicated by dissimilar letters (p < 0.05) b) a) 51 relatively more specific. sbctl contains a single crd, showed dissimilar binding activity to various pamps. sbctl performed the strongest binding activity to lps, lower to man, and almost none to pgn and cpg, implying that sbctl might focus on the recognition of bacteria. ca2+ binding site seems not necessary for the binding activity of sbctl to pamps, though there were two ca2+ binding sites predicted in sbctl. even without ca2+, sbctl also showed high binding activity to lps and man. death of bivalves caused by oshv-1 infection went through two stages. during primary oshv-1 infection, viral rapid replication leads to the host entering an immune-compromised state. then opportunistic bacteria were liable to invade the oshv-1 infected bivalves, which constituted secondary bacterial infection (de lorgeril et al., 2018). in healthy ark clams, there were constitutive expression of sbctl in tissues. during primary oshv-1 infection, oshv-1 dna copies increased sharply, sbctl might not sense nor directly binding to oshv-1, it showed extremely low binding activity to nucleic acid pamps. sbctl was not mobilized during the primary oshv-1 infection, the mrna expression of sbctl kept stable until the next stage. after infection with oshv-1, the vibrio content in ark clam reached a peak at 72 h (fig. 8). this phenomenon was consisted with the expression pattern of sbctl (fig. 9b) in hemocytes post oshv-1 infection. thus, it was speculated that the delayed up-regulation of sbctl mrna might contribute to protect hosts from secondary bacteria infection (fig. 11). in summary, a c-type lectin (sbctl) with a signal peptide and a crd structure was identified from s. broughtonii. sbctl showed especially high binding activity to lps in a ca2+-independent manner. sbctl constitutively expressed in tissues and could respond to oshv-1 infection at the late stage. rsbctl pretreatment in vivo showed a significant protection effect against oshv-1 infection. fig. 10 survival rate of ark clams after oshv-1 infection. rsbctl presents the group with rsbctl treated in vivo, control presents the group treated with the same volume of filtered seawater. the number of survival individuals were recorded at 24, 46, 60, 72, 84, 96 and 108 h, respectively to sum up, we presumed that sbctl served a protection role in the innate immunity of s. broughtonii by recognizing and binding to pamps of intruders. acknowledgements we are grateful to all the laboratory members for their technical support and helpful comments. this research was financially supported by the national natural science foundation of china (grant number: u1706204, 31902400), the special scientific research funds for central non-profit institutes, yellow sea fisheries research institutes (project no. 20603022020007 and 20603022021012), key laboratory of healthy mariculture for the east china sea (grant number: 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zagreb, croatia 4 laboratory for ichthyopathology-biological materials, rudjer bošković institute, bijenička cesta 54, 10000 zagreb, croatia accepted september 25, 2017 abstract the aim of this study was to determine interspecies and gender differences in the lipid metabolism and hemolymph oxidation stability in intermoult period of the european spider crab (m. squinado), the warty crab (e. verrucosa) and the european spiny lobster (p. elephas). in hemolymph samples activities of superoxide dismutase (sod), glutathione peroxidase (gsh-px) and paraoxonase 1 (pon 1), as well as hemolymph cholesterol, triacylglycerol and non-esterified fatty acid (nefa) concentrations were determined. the hemolymph of m. squinado showed the highest levels of sod activity, cholesterol and triacylglycerol and the lowest levels of pon 1 activity compared to values determined in the hemolymph of e. verrucosa and p. elephas. gender differences were determined only in m. squinado samples. males had significantly higher activity of pon 1 and lower nefa values compared to females. m. squinado hemolymph lipid metabolism and oxidation stability in intermoult period could be attributed to their biological, metabolic characteristics and environmental conditions in which these species live. key words: crustaceans; hemolymph; antioxidant enzymes; lipids introduction decapod crustaceans include many species that are widely used as food and represent economically important resource for fisheries. over recent decades research and development activities have been directed to various aspects of their biology including reproduction, nutrition, disease control, rearing in captivity and interaction between environmental changes and life cycle. large numbers of decapod crustacean species inhabit the adriatic sea (kirinčić and števčić, 2008) some of which are the european spider crab (maja squinado), the warty crab (eriphia verrucosa) and the european spiny lobster (palinurus elephas). the european spider crab, m. squinado (decapoda, majidae) is a migratory species distributed in the north-east atlantic ocean, ___________________________________________________________________________ corresponding author: blanka beer ljubić internal diseases clinic faculty of veterinary medicine heinzelova 55, hr-10000 zagreb, croatia e-mail: bljubic@vef.hr including the mediterranean and adriatic sea (neumann, 1998). it feeds on seaweeds and molluscs in winter and on many types of echinoderms and sea cucumbers in summer. this species shows seasonal and migratory variation in relation to the sea depth; in summer spider crab lingers along the coast, at sea depth of 30 meters; in winter this species can be found at a depth of 120 meters. m. squinado life cycle is 5 8 years, growing during 2 3 years by moult taking place in spring, while reproductive activity can last for 6 years (de kergariou, 1984). hatching takes place in spring and early summer. warty crab, e. verrucosa (decapoda, xanthidae) is widespread in the eastern atlantic ocean from britany to mauritania, mediterranean and adriatic sea. warty crab lives on rocky shores in shallow waters (flores and paula, 2001) and hatching takes place from june to august (erkan et al., 2008). this species feeds mainly on molluscs (da silva-castiglioni et al., 2007). european spiny lobster p. elephas (decapoda, palinuridae) can be found in the eastern atlantic ocean, from southern norway to morocco and in http://www.interna.vef.unizg.hr/ http://en.wikipedia.org/wiki/seaweed http://en.wikipedia.org/wiki/mollusca http://en.wikipedia.org/wiki/holothuroidea http://en.wikipedia.org/wiki/atlantic_ocean http://en.wikipedia.org/wiki/brittany http://en.wikipedia.org/wiki/mauritania http://en.wikipedia.org/wiki/mediterranean_sea http://en.wikipedia.org/wiki/atlantic_ocean http://en.wikipedia.org/wiki/atlantic_ocean http://en.wikipedia.org/wiki/norway http://en.wikipedia.org/wiki/morocco 380 the mediterranean and adriatic sea. this species lives mainly at depths of 30 to 80 meters, but it can be found at depths of 160 meters. growth lasts about 4 years, males moult twice a year (spring and fall), and adult females only in the spring (slavica et al., 2004). hatching takes place from autumn to early spring. european spiny lobster feeds on a variety of echinoderms, shellfish, snails, fish, and dead organisms. monitoring of biological and physiological status in these crustacean species is important for investigation of rearing in captivity and possibilities to use these species as biomarkers of chemical pollution of the sea (lorenzon, 2005; bowen and depledge, 2006; vidal-lińán et al., 2010) and littoral sediment pollution (martin-diaz et al., 2009). research of hemolymph biochemical profile showed that some biochemical parameters vary depending on the physiological processes and development stage (gonads development, moulting and reproduction), indicating changes of the environment in which crabs live (travis, 1955; dove et al., 2005; čolak, 2012). during growth and development of crustaceans and under the influence of environmental changes reactive oxygen species (ros) and other prooxidants are produced in metabolic processes. these molecules are removed by mechanisms of cell antioxidant defence, consisting of enzymes and non-enzymatic molecules with antioxidant activity (halliwell and guteridge, 1999). increase in the activity/concentration of antioxidants in the body can be caused by changes in temperature, oxygen concentration (zenteno-savin et al., 2006), salinity (paital and chainy, 2010), ph (wang et al., 2009) and pollutants (livingstone, 2001; lavarias et al., 2011). superoxide dismutase (sod), glutathione peroxidase (gsh-px) and catalase (cat) are the most important antioxidant enzymes. sod catalyses the dismutation of superoxide radicals to hydrogen peroxide, which is removed by gsh-px and catalase. moreover, gsh-px catalyzes hydrolysis and degradation of lipid hydroperoxides. the effects of gsh-px are mainly reflected in/on the cell (brigelius-flohe, 1999), while paraoxonase 1 (pon 1) acts on the lipid peroxides bound to serum lipoproteins (halliwell and guteridge, 1999; livingstone, 2001). lipids are particularly susceptible to oxidation caused by ros. the oxidative stability of a tissue depends on the concentration and composition of the fatty substances. lipids are essential for the crustacean growth, moulting and reproduction. juvenile crustacea have lower body lipid levels than adult animals that accumulate body lipids over the life in the form of triacylglycerol (correia et al., 2003). lipids in this form provide the main source for reproductive energy (wouters et al., 2001). cholesterol is essential for crustaceans (ravid et al., 1999; yepiz-plascencia et al., 2000). this molecule is important in the cell membranes structure and for gonad development and reproductive cycle (wouters et al., 2001). the adriatic sea is a shallow sea, average salinity of 38 ‰ (very salty sea), and oxygen concentration is higher than the average (krstulović et al., 2012). according to specific features of adriatic sea, the aim of this study was to determine interspecies differences in the lipid metabolism and oxidation stability of hemolymph of m. squinado, e. verrucosa and p. elephas in intermoult period. materials and methods animals m. squinado was sampled from commercial catches using the crab netting in the novigrad sea at the beginning of march. after the catch, animals were kept for 24 h in outdoor pools with a flow of fresh sea water with temperature of 11 °c. animals were transported in portable refrigerators to the laboratory. e. verrucosa was sampled at three locations in the central adriatic sea (starigrad, vrsi and ţdrelac) from june to september, at a temperature of 20 °c and harvested manually at night hunt. after the catch, animals were transported in a portable refrigerator to the laboratory. p. elephas was sampled from commercial catches (lobsters netting and foldable traps) from the vis archipelago from august to september. sampling sites are presented on figure 1. animals were delivered in cold storage in zadar where they were fig. 1 sampling sites (hydrographic institute of the republic of croatia http://www.hhi.hr). croatia adriatic sea novigrad starigrad, vrsi, ždrelac vis http://en.wikipedia.org/wiki/mediterranean_sea 381 table 1 biometric data of three crustacena species (m. squinado, e. verrucosa, p. elephas) kept in pools with fresh sea water for three days at the temperature of 18 °c, and then transported in portable refrigerators to the laboratory. all crabs were exposed to air for about nine hours due to transport to laboratory. number of sampled animals and the biometric data are shown in table 1. hemolymph sampling for biochemical analysis during hemolymph sampling in the lab all the animals had hard shell without signs of upcoming moulting. hemolymph of m. squinado was sampled from the ventral sinus through the articular membrane at the base of the 5 th pereiopod, of e. verrucosa from the pericardial cavity, and of p. elephas from the ventral abdominal artery. hemolymph samples were centrifuged at 12000g/95 seconds (12,000g attained to 15 sec) at room temperature and supernatants were stored at the temperature -80 °c. antioxidant enzyme activities: gsh-px, sod and pon 1, and the concentration of triacylglycerol, cholesterol and nefa were determined on the biochemistry analyzer saba 18 (ams, italy). paraoxonase 1 activity in hemolymph supernatant was assayed by modified method hydrolysis of paraoxon described by charlton-menys (2006) and enzyme activity was presented in u/l (1 mol p-nytrophenol formed/min/l serum). activities of gsh-px and sod and nefa concentrations were measured using commercial kits ("randox", ireland). the concentration of cholesterol and triacylglycerol were determined using commercial kits (herbos dijagnostika, croatia). gsh-px activity was expressed in u/l and sod activity was presented in u/ml of hemolymph supernatant. statistical analysis all statistical analyses were performed with statistica 9 (statsoft tm ) computer software. the normality of distribution was checked using kolmogorov-smirnov and shapiro-wilk's w tests. descriptive statistics were done and results are presented as median with lower and upper quartile values. significant interspecies differences were detected by kruskall-wallis anova and sum of rank. gender differences were tested by mannwhitney u test. correlation of the measured variables was analysed using the spearman rank order correlation. significant differences were established at p < 0.05 level. table 2 antioxidative enzyme activities and lipid concentration in the hemolymph of three crustacena species (m. squinado, e. verrucosa, p. elephas) indices m. squinado e. verrusosa p. elephas median (lower quartile-upper quartile) sod (u/ml) 95.22a (84.53 108.98) 7.78b (6.85 10.59) 13.38b (6.63 19.10) pon 1 (u/l) 0.84a (0.25 2.19) 3.55b (3.03 4.79) 2.80b (2.71 2.88) gsh-px (u/l) n.d. 123 (108 645) 111 nefa (μmol/l) 9.22a (6.30 12.78) 42.30b (30.90 72.40) 21.90ab (15.70 22.50) cholesterol (mmol/l) 0.86a (0.57 1.08) 0.29b (0.24 0.42) 0.43b (0.13 0.59) triacylglycerols (mmol/l) 0.23a (0.15 0.30) 0.09b (0.07 0.12) 0.10b (0.04 0.13) triacylglycerols/nefa ratio 30.34a (17.24 41.15) 1.93b (1.39 3.14) 4.57b (2.54 7.68) a, b = rows with different letters showed a significant interspecies differences; kruskall-wallis anova and multiple comparisons of mean ranks nd. = not detectable; (-) = measurable only in one sample measured parameters m. squinado (n = 63) e. verrucosa (n = 44) p. elephas (n = 15) weight (g) 277-1021 28-210 308-888 carapace lenght (cm) 9.8-15.2 4.1-6.9 3-13.2 carapace width (cm) 8.2-13.1 5.0-8.2 total lenght (cm) 21.5-30.69 382 m f m f f maja squinado eriphia verrusosa p alinurus elephas 0 20 40 60 80 100 120 140 160 s o d ( u /m l ) median 25%-75% non-out lier range m f m f f maja squinado eriphia verrusosa p alinurus elephas 0 1 2 3 4 5 6 7 8 9 p o n 1 ( u /l ) median 25%-75% non-out lier range out liers fig. 2 hemolymph sod (upper panel) and pon 1 (lower panel) activity in male (m) and female (f) of m. squinado, e. verrusosa and p. elephas; *p < 0.05. results antioxidant enzyme activities hemolymph supernatant antioxidant enzymes activities of m. squinado, e. verrucosa and p. elephas are shown in table 2, and gender differences are shown in the figures (figs 2 4). the highest of sod activity (table 2) was observed in the samples of m. squinado and significantly higher (p < 0.05) than the values recorded in the e. verrucosa and p. elephas. according to gender, sod activity between males and females were uniform in hemolymph supernatant of m. squinado (95.82 u/ml male, 97.40 u/ml female respectively) and e. verrucosa (8.39 u/ml male, 7.88 u/ml female, respectively). all p. elephas samples were from males and median value in the hemolymph was 13.39 u/ml (fig. 2). the lowest pon 1 activity was observed in m. squinado (table 2) and were significantly lower (p < 0.05) compared to the values obtained in the hemolymph supernatant of other crustaceans (table 2). activity of pon 1 in the hemolymph supernatant of male m. squinado was significantly higher than the values in females (0.84 u/l v. 0.39 u/l, p < 0.05; fig. 2b). * 383 m f m f f maja squinado eriphia verrusosa p alinurus elephas 0.0 0.1 0.2 0.3 0.4 0.5 tr ia c y lg ly c e ro ls ( m m o l/ l ) median 25%-75% non-out lier range out liers m f m f f maja squinado eriphia verrusosa p alinurus elephas 0.0 0.3 0.6 0.9 1.2 1.5 1.8 c h o le st e ro l (m m o l/ l ) median 25%-75% non-out lier range out liers fig. 3 hemolymph triacylglycerls (upper panel) and cholesterol (lower panel) concentrations in male (m) and female (f) of m. squinado, e. verrusosa and p. elephas. lipid content of hemolymph supernatant the highest values of cholesterol and triacylglycerols concentration were found in the hemolymph supernatant of m. squinado and these values were significantly higher than those determined in e. verrucosa and p. elephas (p < 0.05; table 2). the concentrations of cholesterol and triacylglycerols showed no gender differences (figs 3a, b). the lowest nefa concentration was determined in the hemolymph supernatant of m. squinado (table 2) and this value was significantly lower compared to e. verrusosa (p < 0.05; table 2). analysis according to gender showed significantly (p < 0.05) lower values of nefa concentrations in the hemolymph supernatant of m. squinado males (7.94 mmol/l) compared to females (15.55 mmol/l; fig. 4a). also, the highest concentration of nefa in the hemolymph supernatant was determined in e. verrucosa males (57.30 μmol/l males 32.60 μmol/l females, respectively), but was not statistically significant (p > 0.05; fig. 4a). 384 m f m f f maja squinado eriphia verrusosa p alinurus elephas 0 20 40 60 80 100 120 140 n e f a ( m m o l/ l ) median 25%-75% non-out lier range out liers m f m f f maja squinado eriphia verrusosa p alinurus elephas 0 15 30 45 60 75 90 tr ia c y lg ly c e ro l/ n e f a r a ti o median 25%-75% non-out lier range out liers fig. 4 hemolymph nefa concentrations (upper panel) and triacylglycerol/nefa ratio values (lower panel) in male (m) and female (f) of m. squinado, e. verrusosa and p. elephas; *p < 0.05. significantly higher median values of triacylglycerol/nefa ratios were determined in m. squinado hemolymph supernatant compared to other two species (p < 0.05; table 2). significant (p < 0.05) gender differences were found in m. squinado, the value for males was 34.56 and 17.80 for females. antioxidant parameters and lipid concentrations in the hemolymph supernatant of crustaceans showed significant correlation of triacylglycerols and cholesterol in hemolymph of all three species (p < 0.05), whereas significant correlation of pon 1 and triacylglycerols was found only in the hemolymph of e. verrucosa (p < 0.05; table 3). discussion biomarkers of the antioxidant status of animals living in the sea and fresh water are used in environmental monitoring (sroda and cossuleguille, 2011). sod activity depends on the concentration of superoxide radical and increased concentrations are related to oxidative stress. it has been found that some crustacean species have cytoplasmic isoenzyme dependent on manganese (mnsod) (brouwer et al., 2003) whereas mammals have isoenzyme dependent on copper and zinc (brouwer and hoexum brouwer, 1998). in mammals mnsod is predominantly located in the mitochondria * * 385 table 3 correlation between triacylglycerols and cholesterol and pon1 and triacylglycerols in the hemolymph of three crustacena species (m. squinado, e. verrucosa, p. elephas) triacylglycerols – cholesterol r p m. squinado 0.90 0.05 e. verrusosa 0.88 0.05 p. elephas 0.64 0.05 pon 1 triacylglycerols e. verrusosa 0.46 0.05 r = correlation coefficient; p = statistical significance and activity correlates with the number of these organelles in the tissues (halliwell and gutteridge, 1999). depending on the concentration of superoxide radicals, dna transcription of both cytosolic and mitochondrial mnsod is induced in a short time (lin et al., 2010). in this study, the highest activity of the total sod was found in the hemolymph supernatant of m. squinado and the lowest in e. verrucosa, which probably reflects adaptation to different oxygen concentrations (spicer et al., 1990). further, in our study crustaceans prior to sampling were exposed to the air for approximately 9 h and likely reacted to the reduced level of oxygen in their organism. the oxidation intensity and antioxidant systems are tissue specific. hemolymph, like blood, shows the state of metabolism and oxidation processes in the whole organism. in mammals, the most important components of antioxidant protection are sod, gsh-px and catalase. in the hemolymph supernatant samples in which the gsh-px activity could be determined (7 % of samples of e. verrucosa and p. elephas) values were very low. similarly, the animal plasma gsh-px activity is low, since it is located in erythrocytes (gradinskivrbanac et al., 2002). low and undetectable activities obtained by methods applied in this study are probably due to a different composition of hemolymph supernatant. another possible reason for such a low gsh-px activity is the presence of catalase that eliminates hydrogen peroxide. namely, paital and chainy (2010) found about 3.5 times more catalase activity in the hepatopancreas and muscle tissue of mud crab (scylla serrata), and 7.5 times in the gills compared to the gsh-px. also, correia (2003) in gammarus locusta homogenates found higher activity of catalase and authors attribute a great importance of catalase in hydrogen peroxide removal. gsh-px values in this paper suggest that hemolymph supernatant perhaps is not the best sample for determination of activity of this enzyme. lipids are the main source of energy involved in the processes of growth, moulting and reproduction (wouters et al., 2001). in crustacean hepatopancreas, muscles and female gonads, lipids are stored (marques et al., 2010; da silvacastiglioni et al. 2007) due to lack of the body fat (o'connor and gilbert, 1968). as in serum, lipids in the hemolymph are transported bound to lipoprotein complexes (yepiz-plascencia et al., 2000). lipoproteins have an important role in cholesterol and triacylglycerol transport (yepiz-plascencia et al. 2000). in present study, the concentration of triacylglycerols in the hemolymph supernatant was very low. that is in accordance with studies of sommer vinagre (2007) in hemolymph of ocypode quadrata and yepiz-plascencia (2000) in penaeid shrimp. the highest triacylglycerol concentrations in this study were observed in the hemolymph of m. squinado, and the lowest in the hemolymph of e. verrucosa, suggesting interspecies variations. in our research, we did not determine differences in the concentration of triacylglycerols according to gender in any of the investigated species (fig. 2a), which is in correspondence to cheng et al. (2001). cholesterol is essential for crustaceans (van den oord, 1964). the concentrations of cholesterol in hemolymph of some crustaceans show seasonal and gender differences (da silva-castiglioni et al., 2007; sommer vinagre et al., 2007). in the lobster, those changes are caused by fluctuations in temperature and exposure to air (lorenzon et al., 2007). in our study, the highest cholesterol concentration was found in the m. squinado and the lowest in the e. verrucosa hemolymph supernatant and there were no gender differences. our results showed a high correlation between the concentration of cholesterol and triacylglycerols in the hemolymph supernatant of all three crustacean species. this correlation can be interpreted by lipid mobilization from hepatopancreas and other tissues (yepiz-plascencia et al., 2000; da silva-castiglioni et al. 2007; marques et al., 2010) in the hemolymph due to harvesting, handling and sampling. the nefa concentration in blood is an indicator of negative energy balance and increased mobilization of triacylglycerols. in our study, nefa concentration in e. verrucosa was twice as high as in p. elephas and five times as high as in m. squinado. these results reflect interspecies variations during intermoult period. we also determined a significantly higher nefa concentration in the female of m. squinado compared to the males (fig. 4a) while ruiz-verdugo (1997) found no gender differences in white shrimp. 386 paraoxonase 1 is a calcium-dependent esterase bound to high-density lipoproteins (hdl) (costa et al., 2005). in the blood this enzyme removes lipid peroxides within low density lipoproteins (ldl) and hdl (halliwell and gutteridge, 1999; livingstone, 2001). changes of pon 1 activity are used as biomarkers of sea chemical pollution (galloway, 2006). in this study, very low activities of pon 1 in the crustacean hemolymph supernatant were determined, whereas e. verrucosa had the highest and m. squinado the lowest values. in m. squinado pon 1 activity was significantly higher in males (fig. 2b). in mammalian blood, age, species, physiological and pathological condition and gender differences of pon 1 activity were also identified, but with significantly higher values in females (costa et al., 2005). in e. verrucosa correlation of triacylglycerols and pon 1 were determined. the activity of pon 1 increases to protect triacylglycerols when concentrations of triacylglycerols in the hemolymph rise, and crustaceans live in a stressful intertidal zone. most previous related studies were performed on certain tissues or homogenates of individuals, which comprises killing of animals. this research was made on hemolymph supernatant, wherein if qualified personnel do the sampling all individuals survive and samples are of good quality. the results obtained by such sampling could be used for periodic monitoring of mentioned species, and hemolymph indicators could be bioindicators of contamination of certain parts of the sea. in conclusion, in this study on hemolymph supernatant of m. squinado, the highest sod activity, cholesterol, triacylglycerols and ratio of triacylglycerols/nefa were determined, as well as the lowest pon 1 activity in comparison to the other crustaceans. in hemolymph of male m. squinado higher pon 1 activity and the ratio of triacylglycerols/nefa were determined, as well as lower nefa concentration compared to the females. although gender differences in the e. verrucosa hemolymph supernatant were present, significant differences were not determined, probably due to the great sample variability in e. verrucosa in this research. the observed differences can be attributed to the species, their biological and metabolic characteristics and different environmental conditions. further research should be focused on determination of specific mechanisms of antioxidant defence. furthermore, it is necessary to standardize these indicators as health biomarkers of the animals themselves, but also as 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ahuejote-sandoval m. superoxide radical production in response to environmental hypoxia in cultured shrimp. comp. biochem. physiol. 142c: 301-308, 2006. 387 488 isj 14: 488-493, 2017 issn 1824-307x review various roles of β-glucan in invertebrates v vetvicka1, p sima2 1university of louisville, department of pathology, 511 s. floyd, louisville, ky 40202, usa 2institute of microbiology, czech academy of sciences, videnska 1083, 142 20 prague 4, czech republic accepted november 15, 2017 abstract glucans have a long history as immunomodulators; their effects confirmed in every species tested-from bees to humans. in invertebrates, glucan binding receptors are involved mainly in starting of the prophenoloxidase system, representing one of the first defense mechanisms that evolved in phylogeny. our review summarizes the current knowledge of the glucan and lipopolysaccharidebinding proteins in invertebrates and offers a new possibility of using these proteins in human medicine. key words: invertebrates; glucan, receptors; gbp: lipopolysaccharide introduction various glucans have a long history as immunomodulators. however, for several decades, the focus of investigations was almost exclusively oriented towards vertebrates. glucans, and most of all their β-1,3 configurations, are structurally complex homopolymers of glucose, isolated from yeast, grain, seaweed, and fungus. more than 20,000 scientific papers describing the biological activities of glucans exist. strong immunostimulating effects of β-glucans have been demonstrated in all tested animal species including earthworms (beschin et al., 1998), bees, (mazzei et al., 2016) shrimp (duvic et al., 1992), fish (anderson 1992), mice, rats (feletti et al., 1992), guinea pigs (ferencik et al., 1986), sheep, pigs (benkova et al., 1992), cattle (buddle et al., 1988), and humans. currently, there are at least 80 clinical trials underway in numerous countries. clearly, glucan belongs the oldest molecules with significant immunomodulating processes, and its activity found throughout the evolutionary scale. from an evolutionary point of view, three main principles (i.e., recognition, processing, and elimination) are common in all invertebrates, despite the fact that they might be based on varying molecular or biochemical backgrounds. defense strategies of invertebrates lacking lymphocytes and antibodies are based entirely on innate immunity. the major defensive reactions involve phagocytosis, ___________________________________________________________________________ corresponding author vaclav vetvicka university of louisville department of pathology 511 s. floyd, louisville, ky 40202, usa e-mail: vaclav.vetvicka@louisville.edu wound healing, graft rejection, production of various factors (e.g., agglutinins, precipitins, opsonins, clotting factors, lysozyme and many others), and humoral defense (sima et al., 1990). one of the major defensive mechanism of invertebrates is the use of distinctive molecular patterns, including βglucans. prophenoloxidase system invertebrates lack immunoglobulins and their immune system relies on the innate ability initiated by pattern recognition receptors and pattern recognition proteins. these receptors are involved in microbial recognition and initiate protein-ligand interaction. these proteins are able to bind to a variety of microbial cell wall components. in crustaceans alone, at least 15 different types of pattern recognition receptors are known. in general, arthropods, molluscs, and deuterostomian tunicates recognize the microbial surface determinants that are conserved and ubiquitous among microorganisms but not present in the eukaryotic host. these structures-mainly lipopolysaccharide, peptidoglycan, mannan, β-1,3 glucan, gram-negative-binding protein, and c-type lectin-are recognized by means of a group of germline encoded receptors, usually termed pattern recognition receptors. lipopolysaccharide and glucan-binding protein (lgbp) genes involved in the activation of prophenoloxidase (propo) system were identified in almost every invertebrate species studied. the expression of propo and lgbp genes is different in individual cell hemocyte types (yang et al., 2015a). these pattern recognition proteins participate in both humoral and cellular aspects of defense reactions by facilitating pathogenic 489 recognition of pathogen-associated molecular patterns (pamp) and by subsequent triggering of a cascade of reactions such as phagocytosis, production of antibacterial peptides, activation of clotting, and propo cascade (lai et al., 2011). recognition of pamp provides an essential step for the activation of the propo cascade (amparyup et al., 2008). using a highly selective recognition process, signaling cascades are activated. these cascades regulate production of defense substances, agglutinins, opsonins, nonagglutinin factors, inducible or constitutive antibiotic peptides, and the components of the propo complex in the host (ratcliffe et al., 1979). specific nonself-recognition mechanisms of the propo system, as a basic part of immune defense of invertebrates, are involved during a row of hierarchized processes like cell cooperation and communication in the course of phagocytosis, nodule and capsule formation, melanin and sclerotization, hemocyte locomotion, and coagulation of blood (vetvicka et al., 2004). although the mechanism of the propo system has been determined, the precise contribution of βglucan-binding protein (gbp) integration with glucan for the activation remains to be fully elucidated. some studies show that the gbp can be specifically degraded following the activation of propo with glucan, suggesting that the variations in the gbp levels after specific challenge are an important regulation mechanisms to immune response (zhang et al., 2016). glucan-binding protein invertebrates are using innate immune mechanisms which are well conserved throughout the evolution of immunity. a heterologous group of hemolymph proteins serves as a surveillance mechanism by binding to the surface of invading microbes. one member of this group is gbp, which plays a critical role in triggering the innate immune response by detecting glucan found on the surface of microbes. gbp comprises of proteins that share sequence homology to β-glucanase of bacteria (juncosa et al., 1994) and of sea urchins (bachman et al., 1996). the first family of gbp was discovered in the hemolymph of bombyx mori (ochiai et al., 1988). subsequently, proteins binding to the βglucan have been identified in numerous invertebrate species; their activity is usually stimulating the propo activation cascade. subsequent purification and identification revealed similar properties-proteins containing carboxylterminal glucanase-like domain without any enzymatic activity. glucan is bound via less conserved amino-terminal domain. older review studies of the gbp-protein binding are available (vetvicka and sima, 2004). interestingly, in earthworms, the coelomic cytolytic factor shares strong homology with gbp isolated from numerous invertebrates (bilej et al., 2000). glucan-binding protein isolated from mangrove crab episesarma tetragonum and its interaction with pathogens was evaluated. molecular recognition showed the specific binding affinity towards βglucan molecule. this bindings triggers the innate immunity inside the host (sivakamavalli et al., 2014). a molecular cloning of the lgbp isolated from chinese mitten crab eriocheir sinensis showed significant homology with the same protein in shrimp. additional experiments showed that the highest level of expression is in hemocytes, but the protein is also expressed in hepatopancreas, muscles, gills, stomach, and intestines. the expression is upregulated after bacterial infection (zhao et al., 2009). subsequent study showed that the recombinant lgbp triggers the whole hemolymph-dependent melanization and stimulates the propo cascade (zhang et al., 2016). additional study of gbp isolated from river crab paratelhupsa hydrodromus confirmed anti-inflammatory, antioxidant, and antibiofim properties (iswarya et al., 2017a). in shrimp, gbp plays the vital role in the recognition mechanisms against pamp found in the membrane of fungi. recognition of pamp leads to the widespread innate immune activation including cellular and humoral components of defense (sritunyalucksana et al., 2000). formation of gbpglucan complex induces degranulation of hemocytes and activation of the propo system. as the gene encoding gbp is abundant in white spot syndrome virus (wssv)-resistant shrimp, it can be hypothesized that gbp is involved in antiviral response. purification, characterization, and functional analysis of gbp from green tiger shrimp penaeus semisulcatus showed it has a bifunctional role in propo-enhancing activity and agglutinating activity. this gbp not only recognizes pamp, but also induces intracellular signaling (sivakamavalli et al., 2013). lgbp from shrimp fenneropenaeus chinensis was cloned. sequence analysis and comparison revealed a high identity of 94 %, 90 %, 87 %, and 72 % with paneaus monodon β-1,3-glucan binding proteins, litopenaeus stylirostris lgbp, marsupenaeu japonicus β-1,3-glucan binding proteins, and homarus gammarus β-1,3-glucan binding proteins, respectively (liu et al., 2009). in all cases, the transcription of lgbp increased at 6hrs postinfection, suggesting that this gene is not only a constitutive expression gene, but and an inducible acute-phase expression gene, the product of which is necessary to amplify the activity of propo. subsequent study named this peptide fcβgbp-hdl and showed that the full-length cdna of 6713 bp has an open reading frame with two glucanase-like motifs and one arginylglycylaspartic acid motif. the expression was upregulated upon infection, but the level of changes was different in each tissue, suggesting that this protein performs its role differently in different tissues (lai et al., 2011). similar homology between individual prawn species was found between lgbp genes from macrobranchium nipponense and m. rosenbergii (89 % identity), m. japonicus (76 %), and fenneropaneus chinensis (74 %). again, expression of lgbp mrna was elevated in the hepatopancreas (xiu et al., 2014). similar data were found for lgbp gene from indian white shrimp f. inducus (valli et al., 2012) and giant freshwater prawn m. rosenbergii (yeh et al., 2009). a recent study not ony cloned and characterized lgbp from fenneropeaeus merguiensis, but showed its wide 490 specificity towards both gram-positive and gramnegative bacteria and yeast (chaosomboon et al., 2017). similar studies performed in f. chinensis, however, showed binding activity towards gramnegative bacteria only and expression of lgbp mrna in hemocytes only (du et al., 2007). in crayfish astacus leptodactylus, there exists three apolipoproteins, all translated as a large precursor. cleavage at the furin-type side results in high density lgbp (stieb et al., 2014), which is directly involved in innate immunity of crustaceans (schmidt et al., 2010). a gbp isolated from hemocytes of blue swimmer crab portunus pelagicus was shown to have a multifunctional role in defence reactions including agglutination, propo-enhancing activity, phagocytosis, and encapsulation. in addition, gbp reaction product exhibited antibacterial and antibiofilm activity against both gram-positive and gram-negative bacteria (anjugam et al., 2016). molecular cloning and characterization of gbp from plutella xylostella showed significant similarities with β-glucan recognition proteins of other insects. the transcription levels were found upregulated by microbial challenges in all life stages; tissue distribution was mainly expressed in fat body (huang et al., 2015). rather different effects of gbp were found in experiments using zno nanoparticles coated by the crustacean gbp. these particles had significant antibacterial activities and showed cytotoxicity against hepg2 cancer cells (iswarya et al., 2017b), but the mechanisms remain unclear. rather similar and more detailed data were found using peptides derived from gbp isolated from pacific abalone haliotis discus hannai (nam et al., 2016). lectins and opsonins were routinely found in molluscs, but only limited information about gbp is available. the first study found and isolated a gbp from the plasma of marine mussel perna viridis. further characterization showed a 510 kda protein with ability to activate the propo cascade via inherent serine protease activity (jayaraj et al., 2008). in scallop chlamys farreri, the lgbp has significant polymorphism, enhancing the binding activity of lipopolysaccharide and glucan, this protein has a direct association with disease resistance (siva et al., 2012). in zhikong scallop chlamys farreri, mrna expression of lgpb in hemocytes was strongly upregulated by stimulation of lipopolysaccharide and β-glucan and moderately stimulated by peptidoglycan. a recombinant lgbp showed strong agglutination activity towards escherichia coli, bacillus subtilis, and pochia pastoris (yang et al., 2010). these results suggest that lgbp plays not only a significant part in the response against gram-negative bacteria as in other invertebrates (lee et al., 1996), but also against infection with gram-positive bacteria. lgbp with vastly diverse specificities might function as a nonclonal effector for animal immune system. an excellent review of lgbp molecules in bivalves was published in 2015 (allam et al., 2015). one group managed chromosomal localization and molecular marker development of the lgbp gene in the zhikong scallop chlamys farreri (huan et al., 2010). molecular characterization and gene expression analysis of lgbp was also successfully achieved in the hard clam, meretrix meretrix. the expression was observed in six different tissues, the highest levels in the gill and digestive gland tissue (liu et al., 2014). direct effects of glucan in invertebrates in earthworms injected with glucan, an increase in celomic cytolytic factor and lysozyme-like activity was reported. is seems that this action is caused by direct binding of glucan to hemocytes (kohlerova et al., 2004; vetvicka and sima 2004). diet supplementation of sea cucumber apostichopus japonicus with glucan resulted in strong enrichment of intestinal-dominant classes and in increased proliferation of the rhodobacteraceae and verrucomicrobiaceae families. in addition, glucan addition had significant impact on immune response of the intestine via nfκb signaling pathway (yang et al., 2015b). in scallops, glucan treatment increased the expression of cf/tep, leading to higher survival of those infected by vibrio (xue et al., 2017). one of the most common targets of glucan are shrimp, as the negative impact of wssv can be commercially significant. a detailed study revealed how the pattern recognition protein binds to glucan and subsequently activates the propo system (amparyup et al., 2012). besides general immunostimulating activity, various glucans were found to offer protection in penaeus monodon post larvae against wssv infection by changing the immune gene expression (wilson et al., 2015). a study (bae et al., 2012) of flesh shrimp f. chinensis showed that glucan supplementation in absence of pathogen challenge increased total hemocyte counts. single administration of glucan prior to the wssv challenge resulted in strong activation of the propo cascade and reduced shrimp mortality up to 50 % (thitamadee et al., 2014). however, the second application of glucan led to a significant increase in mortality, most probably through the combination of wssv infection and overproduction of reactive oxygen species. these data suggest that with prolonged application of glucan in shrimp aquaculture, caution should be prudent as more is not always better (wang et al., 2013). conclusions many of the genes for gbp and lgbp are highly expressed in the digestive glands, particularly in bivalves, suggesting that the filter-feeding life might show biased pattern recognition towards digestive system as a first line of defense (allam and raftos, 2015). clearly, the importance of these binding molecules in invertebrates is extremely high, with some authors even suggesting that the functional diversity of these molecules is comparable to antibodies in vertebrates (fisher et al., 1991). however, the current rush to clone and characterize the lgbp in various invertebrates is reminiscent of the golden age of competitive immunology, when the scientists happily evaluated phagocytosis or other immune attributes in one 491 species after another. a plethora of reports on lgbp in invertebrates concludes that this protein plays an important role against infection in crustaceans. although the detailed characterization of this protein in individual species adds to the mosaic of our knowledge of the immune reaction in 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its transcription in relation to foreign material injection and the molt stage. fish shellfish immunol. 27: 701-706, 2009. zhang x, zhu yt, li xj, wang sc, li d, li ww, et al. lipopolysaccharide and beta-1, 3-glucan binding protein (lgbp) stimulates prophenoloxidase activating system in chinese mitten crab (eriocheir sinensis). dev. comp. immunol. 61: 70-79, 2016. zhao d, chen l, qin c, zhang h, wu p, li e, et al. molecular cloning and characterization of the lipopolysaccharide and β-1,3-glucan binding protein in chinese mitten crab (eriocheir sinensis). comp. biochem. physiol. 154b: 1724, 2009. 163 isj 17: 163-174, 2020 issn 1824-307x research report discovery and functional analysis of a new gene (bm123) in silkworm (bombyx mori) l sun, l gao, f zhu, p lü, c li, y yuan, k chen* school of food and biological engineering; institute of life sciences; jiangsu university, zhenjiang 212013, china this is an open access article published under the cc by license accepted august 3, 2020 abstract previously our research group used the microarray analysis and suppression subtractive hybridization technologies to find a bombyx mori resistance related gene (ncbi id: np_001153678.1) to b. mori nucleopolyhedrovirus (bmnpv) and the gene was named bm123. but there are no more confirmatory studies about bm123. in this study, bmnpv resistant strain nb, susceptible strain 306, hybrid group 306♀×nb♂ (resistant strain) and nb♀×306♂ (resistant strain) were analyzed by transcriptomic sequencing and weighted gene co-expression network work analysis (wgcna) to verify the new gene bm123 function. correlation analysis between the wgcna data and phenotype showed that bm123 is a gene in me turquoise module. this module has a strong correlation with disease resistance phenotype (correlation coefficient is 0.753, p value is 0.0047), indicating that bm123 is a correlated gene with anti-bmnpv. the full length of bm123 gene was 691 bp, which is not similar with any sequences of other species in ncbi database. but the bm123 protein contained the transcriptional activator (multiprotein bridge factor 2, mbf2) domain in the 34 to 122 amino acid sequence, closely to tribolium castaneum by the evolutionary relationship analysis. the bmnpv resistance function, developmental expression pattern and tissue expression pattern of bm123 were analyzed by using silkworm resistant strain bc10 (screened by eight backcross and two generation of nb and 306 through hybridization and selfing method, each generation is constructed from the feed by adding bmnpv), nb and sensitive strain 306. it was found that after infection with orally bmnpv, the mrna and protein levels of bm123 were up-regulated in the midgut of bc10 and nb, and almost not expressed in 306, indicating that bm123 was a gene associated with resistance to bmnpv. bm123 protein expression in various tissues of silkworm (fat body, hemolymph, midgut, epidermis, testis, ovary, malpighian tubule and silk gland) was analyzed. it was found that bm123 was highly expressed in the midgut and malpighian tubule, while the expression in other tissues was lower. analysis of bm123 expression in different development stages of silkworm (eggs, 1st to 5th instar larvae, pupae and moth) found that the expression level of bm123 increased in the 3rd, 4th and 5th instar. the expression level of bm123 decreased during the pupae and moth stages. it was speculated that the expression of bm123 was related to the evolution of resistance genes in silkworm. in situ hybridization showed that the bm123 gene of bc10 was localized in the nucleus of columnar epithelial cells of the midgut, suggesting that bm123 protein interacts with bmnpv in the silkworm cell nucleus. key words: bombyx mori; transcriptomic analysis; wgcna; bm123; mbf2 introduction silkworm (bombyx mori) is one of the most important economic insects in china. however, the silkworm diseases seriously threaten sericulture development. b. mori nucleopolyhedrovirus (bmnpv) in particular has a more serious impact on ___________________________________________________________________________ corresponding author: keping chen school of food and biological engineering institute of life sciences jiangsu university zhenjiang, jiangsu, 212013, china e-mail: kpchen@ujs.edu.cn silkworm production, accounting for about 70 % of the loss of cocoons caused by the whole silkworm disease (singh et al., 2019). sericulture researchers have reported pathogenesis, transmission routes, and prevention and treatment methods of the disease, especially the screening of disease resistance genes has been the key research direction. by investigating the 344 silkworms sources, our research team found a unique resistant strain (chen et al., 1991), and proved that the resistance of silkworms to bmnpv was controlled by a dominant single gene (feng et al., 2012). red fluorescent protein (rfp) in the anterior midgut of 164 table 1 the reaction process of bm123 gene expression by northern blot composition dosage bm123 pcr amplified fragment 50 ng random primer (0.2 μg/μl) 4 μl sterilized water add to 10 μl incubate at 70 °c for 5 min, quickly place on ice dntp mix (2.5 mm each) 2 μl bovine serum albumin (bsa) (10 mg/ml) 2 μl 10×buffer 2 μl klenow enzyme (5 u/μl) 1 μl α-32p-dctp 1 μl sterilized water add to 20 μl incubation was done at 37 °c for 1 h. silkworm can inactivate bmnpv (hayashiya, 1978). the b. mori lipase-1 (bmlipas-1) isolated from the digestive juice of silkworm has a strong anti-bmnpv activity (ponnuvel et al., 2003). the bms3a gene expression level was changed in resistant strains of silkworm, indicating bms3a was related to resistance of silkworm to bmnpv (xu et al., 2008). different protein expression between resistant and susceptible strains of silkworms revealed that β-n-acetylglucosaminidase and aminoacylase (liu et al., 2010a), serine protease-4 and caspase-1 (qin et al., 2012) were related to the resistance of silkworm to bmnpv. it is also reported that arginine kinase and polyprotein are related to silkworm resistance to viruses in the fat body proteome of three silkworm strains (nb, 306 and bc9) (liu et al., 2010b). b. mori serine protease 142 (bmsp-142), isolated from the digestive juice of silkworm, has anti-bmnpv activity (li et al., 2017). phosphoenolpyruvate carboxykinase mitochondrial subunit (pepck-m) can enhance the expression of autophagy genes (atgs) to inhibit bmnpv proliferation (guo et al., 2019). however, the nuclear hormone receptor (bmnhr96) of silkworm promotes bmnpv entering into the body of silkworm, which is a gene promoting virus infection (yang et al., 2017). autophagy gene (bmatg13) can promote proliferation and replication of bmnpv (xiao et al., 2019). there have been many reports on the resistance related genes of silkworm to bmnpv, but there is no evidence to prove the main gene, and its disease resistance mechanism has been unclear. previously, our research group used microarray analysis (zhou et al., 2013) and suppression subtractive hybridization technology (gao et al., 2018) screened different expression gene in midgut infected by bmnpv of silkworm resistant strain near-isogenic line bc10 (screened by eight backcross and two generation of nb and 306 through hybridization and selfing method, each generation is constructed from the feed by adding bmnpv), resistant strain nb, and susceptible strain 306. this research found that bm123 (ncbi id: fj770215) was up-regulated in the two resistant strains as a resistance related gene. however, there is no study reported the bmnpv antiviral characteristics of bm123. in this study, first, bmnpv resistant strain nb, susceptible strain 306, hybrid group 306♀×nb♂ (resistant strain) and nb♀×306♂ (resistant strain) were analyzed by transcriptomic sequencing to verify the new gene bm123 function. further, resistant functional analysis of bm123 were executed using bc10, nb and 306 infected by bmnpv. materials and methods silkworm strains and virus silkworm highly resistant strain bc10, nb, nb♀×306♂, 306♀×nb♂ and susceptible strain 306 of bmnpv were used in this study. bc10 is screening by eight backcross and two generation of nb and 306 through hybridization and selfing method, each generation is constructed from the feed by adding bmnpv screening. the nb♀×306♂ strain was obtained by breeding male nb and female 306, and the 306♀×nb♂ strain was obtained by breeding male 306 and female nb. the bmnpv t3 viral strain was used. virus feeding and tissue collection the silkworms were reared in the clean silkworm room, and samples were taken at the egg, 1st, 2nd, 3rd, 4th and 5th instars, pupae and moth stages, and ten samples were collected at each stage. when the silkworm reached the 5th instar, each silkworm was fed with 1 × 106 bmnpv per os. after 6, 12, 24, 36, 48, and 72 h of virus feeding, the silkworms were dissected to collect fat body, hemolymph, midgut, epidermis, testis, ovary, malpighian tubule and silk gland tissues of silkworms, and stored at -70 °c. 165 fig. 1 (a) volcano plot showing 1007 degs between 306♀×nb♂ and 306; among which bm123 (transcriptome id: bgibmga004287) was an up-regulated gene. (b) wgcna module analyzes the correlation between differential genes and phenotypic traits; (c) visualization analysis of bm123 in me turquoise transcriptomic sequencing and weighted gene co-expression network work analysis (wgcna) to screen resistant genes 1) sequencing reagents and instruments the cdna library construction kit (superscript ii reverse transcriptase) was purchased from invitrogen.the truseq rna sample prep and truseq pe cluster were purchased from illumina company. instruments: nucleic acid protein analyzer (nanodrop, thermo); bioanalyzer (agilent 2100 bioanalyzer, inc.); genome analyzer ii (illumina). 2) transcriptomic sequencing process nanodrop, qubit 2.0 and aglient 2100 were used to detect the purity, concentration and integrity of rna samples to ensure the quality of samples for transcriptomic sequencing. after the sample was qualified, the library was constructed. eukaryotic mrnas were first enriched with magnetic beads containing oligo (dt), mrna was randomly interrupted by fragmentation buffer. secondly, the first cdna strand was synthesized using mrna as template and random hexamers. then buffer, dntps, rnase h and dna polymerase i were added to synthesize the second cdna strand. ampure xp beads were used to purify cdna. purified double stranded cdna were then repaired, added a tail and connected for sequencing. then ampure xp beads were used to select the fragment size. finally, cdna library was obtained by pcr enrichment. after the library was constructed, qubit 2.0 and agilent 2100 were used to detect the library concentration and insert size, respectively. q-pcr method was used to accurately quantify the effective concentration of the library to ensure the quality. hiseq 2500 was used for high-throughput sequencing and the sequencing reading length was pe125. the raw data were obtained. joint sequences and low quality reads of raw data were filtered and removed to obtain high quality clean data. clean data were sequentially assembled to obtain the unigene library of the species. based on unigene library, the expression quantity analysis and gene structure analysis are carried out, and the 166 differential expression analysis was carried out according to the gene expression quantity in different samples or different sample groups. 3) screening and analysis of differentially expressed genes (degs) in the process of analysis of differentially expressed genes(degs), the accepted effective benjamini-hochberg method have been used to correct the original significance p values (p-value), and finally the adjusted p values (false discovery rate, fdr) were used to screen significantly degs. in the screening process, fdr ≤ 0.01 and fc (fold change) ≤ 2 were used as screening criteria. fc represented the ratio of the expression quantity between two samples groups. 4) correlation analysis between phenotypes and genotypes of degs weighted correlation network analysis (wgcna) was used to analyze the relationship between biological characteristics and degs to detect bmnpv resistant genes. wgcna (v1.47) was used to construct an unsigned co-expression network on the transcription expression matrix (langfelder and horvath, 2008). the nodes were defined as genes and grouped into different gene networks with similar expression levels. the wgcna algorithm first assumes that the gene network follows a scale-free distribution and defines the relevant time of gene co-expression (wang et al., 2017a). the correlation module diagram of genotype and phenotype was constructed through wgcna analysis. cutreedynamic and mergeclosemodules were used as network construction and module detection methods. the relationship between modules and specific phenotype was analyzed. if the module correlation coefficient is close to 1, most of the modules are related to the corresponding characteristics. in this study, the relationship between transcripts and sample characteristics was investigated, and important modules related to the characteristics were identified. cytoscape (v3.5.0) was used to visualize the co-expression network (shannon et al., 2003). sequence characteristics and evolutionary analysis of bm123 the sequence of silkworm bm123 was obtained by transcriptomic analysis. bm123 amino acid and protein analysis used ncbi-blast (http://www.ncbi.nlm.nih.gov/blast). protein blast in the ncbi database revealed that bm123 contained multiprotein bridge factor 2 (mbf2) domain. mbf2 gene of several species was downloaded from ncbi, and the protein sequences of bm123 and other species were compared. the 13 sequence names and sequence numbers were as follows: culex tarsalis (jav33098.1); drosophila persimilis (edw32147.1); drosophila grimshawi (edw01064.1); drosophila mojavensis (edw09458.1); drosophila willistoni (edw85625.2); drosophila sechellia (edw56946.1); drosophila pseudoobscura (q290w7); samia cynthia (baa34219.1); danaus plexippus plexippus (owr45174.1); drosophila virilis (edw62015.1); tribolium castaneum (xp 001811691.1); sergentomyia schwetzi (qho60785.1); nyssomyia neivai (jav05291.1). multiple sequence alignment of bm123 was performed using danman 8.0, and evolutionary tree analysis was performed using nj method of mega 5.2. identification of bm123 gene expression using northern blot a random primer method was used to synthesize the silkworm bm123 specific probe and α-32p-dctp purchased from beijing furui corporation, china. the reaction process is as in table 1. after oral administration of bmnpv 48 h, the midgut of silkworm strains, including nb, 306 and bc10 were collected. total rna was extracted using the trizol method. non-deformable polyacrylamide gel electrophoresis gel was used to separate rna and transfer to the membrane, and prehybridize. after prehybridization, the probe α-32p-dctp-bm123 was added to continue hybridization for 12 h to 16 h. and then the membrane was wrapped with plastic film and placed in a cassette. then, the x-ray film was pressed tightly. the cassette was placed at -70°c for 3 d to develop autoradiographs. identification and quantification of bm123 by reverse transcription pcr (rt-pcr) and quantitative real-time pcr (qrt-pcr) the eggs, larvae, pupae, moth and various tissues of silkworm 5th instar (fat body, hemolymph, midgut, epidermis, testis, ovary, malpighian tubule and silk glands) of silkworm bc10, nb and 306 strain were collected. rt-pcr and qrt-pcr were performed with bm123 specific primers, including 5'ccccagttccactaacagagc'3, 5'ggtgagtttatgaaccgaagagt'3. the housekeeping gene was ribosomal protein l8 (bmrpl-8), the primers: 5'ttccgcgatccatacaagttc'3, 5'cgacctctatcacccattttctct'3. bm123 expression analysis using western blot the silkworm bc10 and 306 strains were collected, and the midgut of eggs, larvae, pupae, moth and 5th instar tissues (fat body, hemolymph, midgut, epidermis, testis, ovary, malpighian tubule and silk gland) were cut and placed. the sample were added liquid nitrogen and ground into powder. 5 ml ripa lysate was added and ground for 10 min. the homogenate was transferred to an ep tube and centrifuged at 10,000 rpm for 10 min. the supernatant was transferred to a new ep tube as wb samples. sds-page electrophoresis was performed, and the membrane was transferred. after transmembrane, samples were incubated at room temperature for 1 h by the primary antibody of bm123 (anti-rabbit antibody, self-made by our research group). after washing three times with pbst (phosphate-buffered saline with tween), anti-rabbit secondary antibody were added and incubated at room temperature for 1 h. after washing the membrane three times with pbst, the color was developed, and the picture was taken. in situ hybridization analysis of silkworm midgut after 48 h of bmnpv infection, fresh midgut of silkworm nb and 306 strains were collected and fixed 167 table 2 partial transcriptomic differential expression genes between 306♀×nb♂ and 306 with 4 % paraformaldehyde. an in situ hybridization analysis was performed. the overnight fixed samples were embedded, sectioned and incubated with 100% methanol containing 3 % peroxidation hydrogen at room temperature for 10 min to block endogenous peroxidation activity. after washing with phosphate-buffered saline (pbs), the slides were incubated in citric acid buffer (1.8 mm citric acid and 8.2 mm sodium citrate) at 95 ℃ for 10 min. and then the slides were incubated with blocking buffer (10 % bovine serum albumin and pbs) at room temperature for 1 h. the sections were incubated with α-32p-dctp-bm123 for 1 h. after washing with pbs, the slides were incubated with anti-rabbit igg-hrp for 30 min followed by diaminobenzidine (dab) substrate solution. then the picture were got. statistical analysis all data were analyzed using originpro 8.5.1 and graphpad prism 6. the t-test between the two groups was used to determine the statistically significant of different sample by spss. the data were triplicate. statistical significance was defined as *p < 0.05, **p < 0.01, n = 3. results transcriptomic sequencing and wgcna analysis according to the transcriptomic analysis of nb, 306 and their positive and negative hybrid strains, 1007 degs between 306♀×nb♂ and 306 were purchased, including up-regulated 783 genes and down-regulated 224 genes. bm123 belonged to the up-regulated genes database and its id was bgibmga004287 in transcriptomic data (fig 1a). partial degs data in table 2 showed that fc (306_nb/306) and log2fc (306_nb/306) of bm123 were 95.53 and 6.578, indicating it was a significantly up-regulated gene. in order to screen bmnpv resistant genes, the relationship between degs and biological phenotypic characteristics was analyzed through wgcna and the relationship correlation module was established. the results showed that there were six modules represented by six colors, namely me green, me yellow, me blue, me brown, me red and me turquoise (fig 1b). among them, the genes of me green and me yellow were negatively correlated with phenotypic trait of resistance, and these of me blue, me brown, me red and me turquoise were positively correlated with resistance. the correlation coefficient with bmnpv resistance phenotype in me turquoise modules is 0.753 (p value 0.0047), followed with silk gland phenotype (coefficient 0.389, p value 0.211). however the coefficients with silkworm pupae weight, cocoon layer, cocoon weight, midgut and moth weight were 0 in this module (fig 1b). these results indicated this module is most closely correlated with bmnpv resistance phenotype, yet not correlated with other phenotypes. visualization analysis of degs in me turquoise found bm123 (bgibmga004287) existed in this module (fig 1c), indicating bm123 is a bmnpv resistant related gene. bm123 gene sequence analysis bm123 gene obtained from transcriptome analysis data was compared in ncbi-blast (ncbi id fj770215), but no gene with high indentity consistency was found. the full length of this gene is 691bp, including open read frame (orf) 372bp, 5 'non-translated region 45bp, 3' non-translated region 274bp.the orf encoded a polypeptide chain of 123 amino acids (fig 2a). the translated amino acid sequences were analyzed by blastp similarity comparison in ncbi to obtain other insect proteins with similar sequences. the amino acid sequence similarity between these proteins and bm123 protein is 40 % ~ 50 %, respectively. there is no high similarity between silkworm and other insects both in the gene sequence and the protein sequence, indicating that bm123 is a newly discovered gene. but gene_id gene description fc(306_nb/306) log2fc(306_nb/306) pvalue significant regulate bgibmga003323 putative membrane protein 3827.188 11.90206916 4.37888e-46 yes up bgibmga014524 serine protease 9968.006 13.28308917 2.1291e-29 yes up bgibmga010640 lipase 2125.396 11.05351565 9.29749e-21 yes up bgibmga004287 hypothetical midgut protein bm123 95.534 6.577938594 1.6705e-06 yes up bgibmga002905 acyl-coa binding protein 35.65 5.155814899 6.29306e-23 yes up bgibmga005781 heat shock protein 25.4 3.229 1.691120945 0.004663434 yes up bgibmga012031 carboxylesterase 0.419 -1.255001364 1.08571e-16 yes down bgibmga000029 nucleolar protein family a member 2 0.284 -1.815229193 3.41449e-13 yes down bgibmga009073 serine protease inhibitor 0.344 -1.539012289 5.66688e-09 yes down bgibmga008116 ctp synthase 0.434 -1.204447268 2.48365e-07 yes down bgibmga007371 bm8 interacting protein 2d-2 0.35 -1.516173143 0.003090896 yes down 168 fig. 2 (a) full length of bm123 gene of silkworm; (b) bm123 protein domain prediction and analysis; (c) multi-sequence alignment analysis of bm123 protein sequences with mbf2 proteins of 13 species; (d) evolutionary tree analysis of bm123 protein sequence and mbf2 protein of these species,culex tarsalis (jav33098.1); drosophila persimilis (edw32147.1); drosophila grimshawi (edw01064.1); drosophila mojavensis (edw09458.1); drosophila willistoni (edw85625.2); drosophila sechellia (edw56946.1); drosophila pseudoobscura (q290w7); samia cynthia (baa34219.1); danaus plexippus plexippus (owr45174.1); drosophila virilis (edw62015.1); tribolium castaneum (xp 001811691.1); sergentomyia schwetzi (qho60785.1); nyssomyia neivai (jav05291.1) 169 fig. 3 bm123 levels analysis in the midgut of silkworm infected by bmnpv. (a) northern blot verification of bm123 gene differentially expressed in the midgut of silkworm bc10, nb and 306 infected by bmnpv 48 h; (b) qrt-pcr analysis of different time expression patterns of bm123 gene in bc10 and 306 midgut of silkworm infected by bmnpv; (c and d) wb detected the different time protein expression of bm123 in bc10 and 306 of silkworm midgut infected by bmnpv. p* < 0.1, p** < 0.05, n = 3 the amino acid sequence of bm123 protein 34 ~ 120 contains a transcription activator mbf2 domain. comparing of seven species on ncbi, drosophila mojavensis (edw09458.1); tribolium castaneum (xp 001811691.1); drosophila virilis (edw62015.1); drosophila grimshawi (edw01064.1); drosophila willistoni (edw85625.2); drosophila sechellia (edw56946.1); drosophila pseudoobscura (q290w7), the mbf2 domain was found to be conserved (fig 2b), suggesting that bm123 protein was related to transcriptional activation. multiple sequence alignment analysis between bm123 and 13 mbf2 related genes of other species found that the domain of bm123 protein was consistent with that of mbf2 related proteins of other species (fig 2c). then, the mbf2 related protein sequences of these species were selected for the evolutionary tree analysis with the bm123 orf region (fig 2d). it was found that the bm123 protein of silkworm was most similar to that of tribolium castaneum in terms of its evolutionary relationship and was subsequently classified into a branch with culex tarsalis. identification of the bm123 differential expression by northern blot to further identify the differential expression of the bm123 gene, total rna from the midgut tissue of silkworm resistant strains (nb and bc10) and susceptible strain (306) were extracted before and after feeding virus, and northern blot analysis was performed. bm123 specific cdna labeled with α-32p-dctp was used as a probe named α-32p-dctp-bm123, and 18srrna was used as an internal reference. as shown in fig 3a, the bm123 hybridization band of bc10 and nb strains was slightly stronger than that of the strains without virus feeding, and the hybridization signal was not observed in 306 strain. these results indicated bm123 levels were higher in nb and bc10 strains than that in 306. analysis of bm123 mrna level by qrt-pcr the expression phase of bm123 in midgut tissues was detected by qrt-pcr. the expression level of bm123 was low at 0 h of feeding virus, while increased significantly from 6 h to 36 h infected by bmnpv, and the mrna expression level reached and remained the highest level after 36 h. however, no change in the bm123 expression level was observed in the midgut of 306 strain, and the expression level was very low (fig 3b). the above results indicated that bmnpv up-regulated the mrna expression of bm123 gene in resistant silkworm strain. analysis of bm123 protein expression level by western blot western blot analysis of bm123 protein in midgut tissue showed that the expression level of bm123 protein of bc10 strain was significantly higher 170 fig. 4 rt-pcr and western blot analysis of bm123 gene expression in bc10 tissues and at various developmental stages.(a and b) expression level of bm123 at various developmental stages (egg, 1st, 2nd, 3rd, 4th and 5th instars, pupae and moth stages) of midgut; (c and d) expression level of bm123 in different tissues (fat body, hemolymph, midgut, epidermis, testis, ovary, malpighian tubule and silk gland ). p* < 0.05, p** < 0.01, n = 3 than that of 306 strain. the protein expression level reached the highest at 24 h after feeding bmnpv, and the expression level decreased slightly after 72 h. there was no obvious expression of bm123 protein in the midgut of 306 strain (fig 3c and d). this result was consistent with the analysis of rna expression analysis (fig 3a and b), indicating that the difference in bm123 protein expression in the midgut of bc10 and 306 strains was very obvious. expression levels of bm123 protein at various development stages and tissues of silkworms to further analyze the expression of bm123 gene in silkworms, different development stages of bc10 and different tissues of bc10 5 th instar larvae were collected, bm123 expression levels were detected from mrna and protein levels. during the development of silkworm from eggs, 1st, 2nd, 3rd, 4th, 5th instar larvae, pupae to moth, bm123 mrna and protein expression levels were different. from eggs to 2nd instar larvae, the expression level of bm123 was low. from 3rd instar, the expression level of bm123 rose, and the expression level of bm123 reached the highest in the 5th instar. the expression level of bm123 decreased during the pupae and moth stages (fig 4a and b). bm123 was expressed in all tissues of the silkworm, but the expression level was different in different parts. the expression of bm123 was the highest in the midgut and malpighian tubule, and the expression in other tissues was lower (fig 4c and d). the results of western blot were similar with rt-pcr results, which indicates that there are tissue differences in bm123 expression at both the mrna and the protein levels, and bm123 expression level is controlled by development regulation. positioning of bm123 gene in midgut bm123 gene was localized by in situ hybridization in bc10 and 306 midgut. the midgut tissues of bc10 and 306 strains were selected after 171 fig. 5 in situ hybridization detection of bm123 gene localization in the midgut. a and b were bc10 midgut tissue;c and d were 306 midgut tissues.the red box shows the nuclei, and the arrows shows positive signal.the bar = 50 μm bmnpv infection. the color reaction generated by immunological binding of α-32p-dctp-bm123 specific label determined the expression position of bm123 gene in midgut cells. at low magnification, there was a clear positive hybridization signal in the midgut tissue of bc10 strain (fig 5a). under high magnification, bm123 was mainly located in the nucleus of columnar epithelial cells in the midgut (fig 5b). no positive signals were observed in 306 strain (fig 5c and d). discussion the resistance gene of silkworm strains to bmnpv is controlled by major dominant genes (watanabe, 1986). comparative genomics, transcriptomics and proteomics analyses revealed that many genes and proteins might be involved in bmnpv resistance (bao et al., 2010; zhou et al., 2013; wang et al., 2017b; yu et al., 2017; wu et al., 2019), such as v-atpase (lü et al., 2013) and bm-sp142 (li et al., 2017). however, are these genes associated with resistance to bmnpv the dominant genes? it's still hard to determine. in this study, transcriptomic sequencing was used to identify the degs of four silkworms strains, including nb, 306, nb♀×306♂, and 306♀×nb♂. the correlation between bmnpv resistance phenotype and degs of 306♀×nb♂ and 306 was analyze by wgcna to further identify the bmnpv resistance characteristics of bm123. wgcna is the most widely used method for disease studies and genes related to traits identification (chen et al., 2017; huang et al., 2017; liu et al., 2017a; liu et al., 2017b; 172 wang et al., 2017a). through wgcna analysis, me turquoise module represented the high correlation of genotype and bmnpv resistance phenotype was found (fig 1b). bm123 in this module, meant a bmnpv resistance related gene (fig 1c), and bm123 was significantly up-regulated (table 2). both microarray analysis technology (zhou et al., 2013) and suppression subtractive hybridization technologies (gao et al., 2018) showed that bm123 was an significantly up-regulated bmnpv resistance related gene. these results were consistent with our research. in order to study the function of bm123, the full length of bm123 protein was amplified and analyzed, and its protein domain was predicted. through protein comparison analysis, bm123 sequence contained the mbf2 conserved domain. mbf2 is a transcription activated positive co-factor isolated from silkworm, combined with ftz-f1 (fushi tarazu factor-1) to jointly activate gene transcription (li et al., 1997; liu et al., 1998). evolutionary analysis shows that mbf2 gene is insect-specific and can resist bacterial invasion (zhou et al., 2016a). in situ hybridization of this study showed that bm123 gene of bc10 was localized in the midgut columnar epithelial nucleus, and bm123 was not present in 306 (fig 5). as known, ftz-f1 is a nuclear hormone receptor transcription factor, mostly present in the nucleus (sultan et al., 2014). therefore, we hypothesized that mbf2 binds to ftz-f1 in the nucleus. in addition, mbf2, bmftz-f1 and tata binding protein (tbp) form complexes (li et al., 1994), and tbp was also found to interact with viral proteins (lin and green, 1991; scholer et al., 1991). it is known that bmnpv enters the host nucleus and begins to replicate and proliferate.therefore, we speculated that bm123 protein could be involved in transcriptional regulation and virus interaction by binding ftz-f1 and tbp in the nucleus, accelerating the transformation of silkworm larva into pupae (cruz and martin, 2007; konopova et al., 2011), to escape from the infection of bmnpv, which may also be the original cause of the evolution of resistance of silkworm. the near-isogenic lines bc10, showed the same bmnpv resistance feature with nb strain, but its genetic background was 99.99 % similar to the susceptible strain 306, indicating the expression differences gene between bc10 and 306 may be mostly related with resistant phenotype. so next, bc10, nb and 306, were used for bm123 resistance performance analysis. the increasing expression of bm123 dna, mrna and protein levels were detected by northern blot, qrt-pcr and western blot, indicating that bmnpv improved bm123 activity in midgut in nb and bc10 strains. however, virus has no effect on the bm123 activity in the midgut of 306 strain. further study on the expression pattern of bc10 strain bm123 at developmental stage found that the protein expression of bm123 reached its peak in the third to fifth stages of the larva, interestingly the gene expression increased with the increase amount of eating mulberry leaves (fig 4a and b). it is a meaningful result. bmnpv usually exists on mulberry leaves, which are eaten by silkworm, so the silkworm can get easily infected. in the egg and pupal stage, silkworm does not eat mulberry leaves, but eat a lot of mulberry leaves in the larva stage, which is the main stage of silkworm oral infection of bmnpv. therefore, we supposed that the high expression of resistance-related genes in the larval stage may be a major defense mode formed in the long-term evolution of silkworm. previous studies have shown that mulberry leaves containing bmnpv can activate the disease resistance gene of silkworm (aikawa, 1962; feng et al., 2013). but resistance to the virus of silkworm is weaker in later larval stages (teakle et al., 1986; kirkpatrick et al., 1998). recent studies on the silkworm infected with nosema bombycis also showed that long-term species evolution would lead to differences in disease-resistant genes among silkworm in different regions (hassan et al., 2020), and this evolutionary nature of species supported our suppose. in addition, the expression level of mbf2 gene in silkworm was the highest before the 4th instar molting and then gradually decreased (zhou et al., 2016b), and mbf2 was detected on the second and third days of the 3rd, 4th and 5th instars (liu et al., 2000). these results indirectly confirm our previous speculation that bm123 can activate transcription and participate in hormone regulation, promote the growth of silkworm larvae, and regulate virus infection, but how to regulate is still unknown. the bm123 tissue expression pattern of bc10 strain found that the expression level was relatively higher in the midgut and malpighian tubule. silkworm midgut is an important barrier for resisting bmnpv infection (hath-stapleton et al., 2003). up-regulation of several resistant genes (bmlippase-1, bmnox and bmserine protease-2) was also detected in the midgut of four bmnpv resistant strains (p50, a35, a40 and a53) (cheng et al., 2014). hence, disease-resistant genes of silkworm strain should be highly expressed in the midgut, which is consistent with the results of this experiment. malpighian tubule of lymantria dispar has certain immune characteristics (pannabecker et al., 1995). in addition, from the perspective of embryonic development, both the malpighian tubule and the midgut originate from the endoderm, so the malpighian tubule also has the characteristic of high expression of resistance genes. this study also found that bm123 was highly expressed in the malpighian tubule of resistant silkworm strain. by detecting the gene expression pattern of bmnpv, some studies have found that the virus has obvious viral tissue tendency in host larvae (hikida et al., 2018). the conclusion of this study can also explain the tissue tendency of gene expression. this research verified the bmnpv resistance related function of the new gene bm123 based on transcriptional sequencing and wgcna of silkworm. the function and expression characteristics of bm123 gene associated with anti-bmnpv revealed that bm123 is a gene with transcription activator (mbf2) domain which located in cell nucleus of silkworm midgut. further analysis of the resistance function of bm123 as well as the development expression pattern and tissue expression pattern showed that bm123 had obvious specific period and tissue expression characteristics as a bmnpv resistance gene. 173 acknowledgments this work was supported by fund from chinese postdoctoral science foundation (2018m642185); the general project of natural science foundation of the jiangsu higher education institutions (19kjb240001); guangdong south china sea key laboratory of aquaculture for aquatic economic animals, guangdong ocean university (kfkt2019yb09); national natural science foundation of china (31861143051, 31900359, 31872425 and 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resistance-related genes to nucleopolyhedrovirus. genomics. 101, 256-262, 2013. 94 isj 15: 94-103, 2018 issn 1824-307x research report characteristics of α-amylase gene and its association with growth traits in meretrix meretrix y dong, x gao, w sheng, h yao, z lin* zhejiang key laboratory of aquatic germplasm resources, zhejiang wanli university, ningbo, zhejiang, 315100, pr china accepted march 17, 2018 abstract α-amylase is a major digestive enzyme in phytophagous animals that catalyzes the hydrolysis of starch into sugars so that it affects the carbohydrate metabolism and growth of many species. in the present study, the full-length cdna and intron sequences of α-amylase (mmamy) were investigated by rapid amplification of cdna ends (race) method. the genomic dna sequence of mmamy is 6689 bp, which contains 7 exons and 6 introns. the cdna of mmamy is 1783 bp, with a 1569 bp of open reading frame (orf) encoding 522 amino acids. no expression was detected in the early stages of unfertilized mature eggs, fertilized eggs, 4-cell embryo, blastula, gastrulae, trochophore, whereas it was detected after d-shaped larva showing the highest expression in umbo larvae. the quantitative expression of six different tissues in adults showed that expression of mmamy was enriched in visceral mass, while no expression was detected in other tissues. furthermore, gene expression and enzyme activity of mmamy in visceral mass were highly correlated with the growth rates of four shell color strains. the positive correlation between mmamy and growth traits suggested that α-amylase gene could be used as a candidate gene in the molecular breeding programs to improve clam growth. key words: meretrix meretrix; α-amylase; cloning; expression; activity; growth traits introduction α-amylases are enzymes that catalyze the hydrolysis of the α-(1,4) glycosidic linkages in starch and related compounds. because of their main functions in carbohydrate metabolism, α-amylase enzymes are important for the utilization of energy sources and growth (sellos et al., 2003). in marine bivalves, activities of digestive enzymes, especially α-amylase, have been considered to control absorption efficiency for their corresponding substrates, which is one of the principal factors explaining growth variation (prudence et al., 2006). therefore molecular characterization of genes encoding α-amylase is a prerequisite for further understanding their genetic contributions to growth of bivalves. so far, in mollusks, α-amylase genes have been reported in some species, such as pecten maximus (le moine et al., 1997), and later from corbicula fluminea (da lage et al., 2002), crassostrea gigas (sellos et al., 2003), pinctada maxima (pan, 2011), pteria penguin (pan, 2011), ___________________________________________________________________________ corresponding author: dr. zhihua lin college of biological & environmental sciences zhejiang w anli university 8 south qianhu rd., ningbo, zhejiang, 315100, p.r. china e-mail: 911761480@qq.com haliotis discus hannai (kumagai et al., 2013) and pinctada fucata (huang, et al., 2016). furthermore, in the pacific oyster c. gigas, two α-amylase genes (amya and amyb) have been characterized (sellos et al., 2003), and their associations between α-amylase gene polymorphism and growth have been established (prudence et al., 2006; huvet et al., 2008). in the razor clam sinonovacula constricta, liu et al. (2017) investigated the gene structure and expression of α-amylase gene, and identified one growth-associated single nucleotide polymorphism (snp), suggesting the amylase markers was considerably valuable in selective breeding. although α-amylases belong to the glycoside hydrolase superfamily, their gene structure was unique to the distinct species. for example, the number of introns is different in various species. no intron is identified in drosophila melanogaster (boer et al., 1986), but 14 introns in daphnia pulex (da lage et al., 2011). similarly, some differences were also found in the promoter region. no functional pancreatic transcription factor-1 (ptf-1) binding site is identified in seabass (lates calcarifer) amylase promoter, which is responsible for pancreas-specific transcription in higher vertebrates. instead a hepatocyte nuclear factor 3 (hnf-3) binding site is found to modulate the amylase mailto:911761480@qq.com 95 fig. 1 phenotypes of four shell color strains in m. meretrix. df: dark fringe, tc: thin checkered, ws: white shell, rs: red shell promoter expression (ma et al., 2004). although α-amylases from diverse sources have different primary structures, they are homologous in three-dimensional structure (maurus et al., 2005). the protein has been reported with 3 domains: a a (beta/alpha) 8-barrel; b a loop between the beta 3 strand and alpha 3 helix of a and c the c-terminal extension characterized by a greek key (chen, 2007). the hard clam (meretrix meretrix) is extensively distributed along the eastern coastal area of asia. it has been one of the most important farmed shellfishes in china. in recent years, several genes involved in growth, such as sulfotransferase-like gene (wang et al., 2012a), grb2 (gao et al., 2015), hdac1 (gao et al., 2016), have been cloned and analyzed (wang et al., 2012b; dai et al., 2015; zou et al., 2015). moreover, several studies showed that the shell color of hard clam was varied and some of them were associated with their growth traits. in nodipecten nodosus (alfonsi and perez, 1998; jessica et al., 2012), argopecten purpuratus (wolff et al., 1991), and p. fucata (zou et al., 2014), shell colors and decorative patterns have been used as genetic markers to assist selective breeding. recently, the relationships linking α-amylase, shell color and growth have been reported. in chlamys nobilis, growth traits and amylase activity are both associated with shell color strains (deng et al., 2008). to investigate the regulation of mmamy, the full-length cdna and introns were cloned, and the expression profiles were detected during embryonic/larvae development stages, in adult tissues and in visceral mass of different shell color stains. and the activity of α-amylase was also tested. the results may contribute us to understand genetic mechanism in clam growth and offer candidate gene and markers for breeding programs in this species. materials and methods experimental animals and sample collection for cloning and gene expression of mmamy, adult clams were obtained from the danyan nursery field in yinzhou district of ningbo, china. six tissues, including mantle, adductor muscle, visceral mass, foot, gill and siphon were dissected, immediately frozen in liquid nitrogen and then stored at -80 °c. embryos/larvae of ten developmental stages (unfertilized mature eggs, fertilized eggs, 4-cell embryo, blastula, gastrulae, trochophore, d-shaped larva, umbo larva, eyespots larva, juvenile clams) were collected and preserved at -80 °c. in order to detect possible difference of mmamy activity and gene expression in four shell color strains, thin checkered (tc), white shell (ws), dark fringe (df) and red shell (rs) (fig. 1), the parents of four strains were collected from ningbo yongsheng shellfish hatchery, ningbo, china. the breeding offspring was cultured in the same growing environmental conditions and the main growth traits (shell length, shell width, shell height and total weight) were measured every six month. samples of visceral mass of four strains were collected from 18-month clams, for the analysis of mmamy activity and gene expression. cloning the full-length cdna of mmamy total rna was extracted from the visceral mass using trizol reagent (sangon, china). rna integrity was examined by electrophoresis on a formaldehyde-denatured 1.2% agarose gel staining with ethidium bromide, and the quality and quantity were assessed by ultraviolet spectrophotometry. the first-strand cdna was synthesized according to the manufacturer’s instruction of smart race reagent (clontech, usa). on the basis of 454 cdna library of m. meretrix (genbank accession no. sra021052), est sequences of mmamy gene were retrieved. primers for 5′-race (mmamy-f1) and 3′-race (mmamy-r1) were designed (table 1). the amplification was performed as follows: 5 cycles of 94 °c for 30s, 72 °c for 3 min; 5 cycles of 94 °c for 30 s, 70 °c for 30 s and 72 °c for 3 min; 25 cycles of 94 °c 30s, 68 °c 30s and a final extension at 72 °c for 3 min. the pcr products were purified using gel extraction kit (tiangen, china) and then cloned into the pmd18-t vector (takara). the vector was transformed into dh5α cells (tiangen, china) according to manufacturer’s protocols, and the positive clones were sequenced. the full-length cdna sequence was determined by piecing together the sequences of the 3′ and 5′ race products. to confirm the accuracy of cloning and sequencing, the full-length cdna was re-amplified with high fidelity polymerase (takara), using a pair of gene specific primers, mmamy-f2 and mmamy-r2 96 table 1 oligo nucleotide primers used in the experiments primer name sequence from 5′ to 3′ application mmamy-f1 gggtggggaaccaataaaaatgactga 3′ race mmamy-r1 agcatactcgctcgcaggggaaacc 5′ race mmamy-f2 taaagcagagtgcgagag verifying the sequence of cdna mmamy-r2 tgacggaagtccagtatt verifying the sequence of cdna mmamy-f3 ccaacccacggcaagcagacaa cloning of intron mmamy-r3 ataacttacaggctggtatctctc cloning of intron mmamy-f4 taaccaatccaaaccgccc cloning of intron mmamy-r4 tgatttcccccagtcccaa cloning of intron mmamy-f5 ctgggtcttactctggcattg cloning of intron mmamy-r5 tcagtcatttttattggttccc cloning of intron mmamy-f6 tagacattggtgttgctgggat cloning of intron mmamy-r6 atcacctcccccaccatctc cloning of intron mmamy-f7 aaagaggagatggtgggggag cloning of intron mmamy-r7 cacagtaatcgcctgacggaa cloning of intron mmamy-f8 cttgttggtgatgacagttcctta cloning of intron mmamy-r8 taacagatcagtacttgtcttgcgt cloning of intron real-a-f ggaaggaccaccacataacg qrt-pcr real-a-r acagacccaatcaccgctac qrt-pcr 18s-f ctttcaaatgtctgccctatcaact qrt-pcr 18s-r tcccgtattgttatttttcgtcact qrt-pcr (table 1), which were designed based on the above mentioned cdna sequence. pcr products were cloned and sequenced following the procedures described above. cloning the introns of mmamy genomic dna from adductor muscle tissue was extracted with phenol/chloroform/isoamyl alcohol (25:24:1), and then re-extracted by chloroform/isoamyl alcohol (24:1). the other steps were the same as the procedure described by sambrook et al (2001). according to the full-length cdna, three primer pairs (mmamy-f3, mmamy-r3; mmamy-f4, mmamy-r4; mmamy-f5, mmamy-r5, table 1) were designed to detect the introns. pcr conditions were as follows: an initial denaturation (94 °c , 5 min), followed by 35 cycles of amplification (94 °c, 30s; 55 °c, 30 s; 72 °c, 2 min), and a final extension (72 °c, 10 min). pcr products were cloned and sequenced following the procedures described above. sequence and phylogenetic analysis the sequences were spliced using blast algorithm at the national center for biotechnology information database (http://www.ncbi.nlm.nih.gov/blast/). the deduced amino acid sequence was analyzed using the simple modular architecture research tool (smart) (http://smart.embl-heidelberg.de) to predict conserved domains. the presence and location of the signal peptide and the cleavage sites in the amino acid sequence were predicted using signalp 4.0 server (http://www.cbs.dtu. dk/services/signalp/). the multiple alignment of α-amylase proteins of m. meretrix and other species was performed using the clustalw2 multiple alignment program (http://www.ebi.ac.uk/tools/msa/clustalw2/). a phylogenetic tree was constructed using the neighbor-joining (nj) method with mega 6.0. quantitative analysis of mmamy the expression profiles of mmamy at different developmental stages (n>500, 3 sets of samples for each stage), in different adult tissues (4 sets of samples for each tissue) from four randomly sampled clams were analyzed using real-time quantitative reverse transcription pcr (qrt-pcr), with three technical repeats for each pcr reaction. total rna was extracted from the samples using the same method described above. 97 fig. 2 analysis of sequence and structure of mmamy gene. (a) the full length cdna and deduced amino acid sequence of mmamy. the letters boxes are the start codon, the stop codon and the polyadenylation signal sequence, the * represents the end of the protein translation, shaded gray part is the signal peptide, green letters are calcium binding residues, cyan letters are chloride binding sites, yellow letters are active site residues. (b) structure of mmamy and selected amy genes from model species. blue boxes are introns and lines are exons. the number of introns in the amys is significantly different in different species, no introns in dmamy and 9 introns in hsamy. (c) neighbor-joining phylogenetic tree of amy between m. meretrix and other species using mega6.0 software. the abbreviation of amys and the genbank accession numbers used to construct phylogenetic tree are given in table 2. the primers real-a-f and real-a-r (table 1) located in the exon 4 and exon 5, respectively, were used to amplify a 104bp fragment of mmamy from cdna template. primers 18s-f and 18s-r (table 1) were used to amplify 186 bp products of 18srrna, as the internal control for qrt-pcr. pcr amplification was performed in a 20 μl reaction volume containing 10 μl of itaq universal sybr green supermix (bio-rad), 7.2 μl deionized water, 0.8 μl of the first strand cdna and 1 μl of forward and reverse primers. amplification was performed using the following thermal cycling conditions: incubation at 94 °c for 20s, 40 cycles of 94 °c for 3 s, 60 °c for 15s and 72 °c for 10s. all qrt-pcr was performed in three biological replicates with one replicate for each clam, and negative controls were set with each primer pair. to compare the mmamy expression levels in the viscera of clams with the different shell colors, six individuals of each shell color strains were selected. total rna was extracted from the viscera of 24 clams and qrt-pcr was performed as described above. assay for α-amylase activities to assay the activities of α-amylase, viscera of four shell color strains (6 sets of samples for each shell color strain) were homogenized 10min in ice bath. the homogenate was centrifuged at 11000 r/min for 10 min at 4 °c. the supernatant of enzyme crude extracts was preserved at 0~4 °c and analyzed within 24h. α-amylase activities were measured using the colorimetric iodine starch and total protein contents were measured 98 table 2 species and genbank accession numbers of amys sequence used for multiple alignment and phylogenetic analysis species abbreviation type genbank no. size meretrix meretrix mmamy kp250879 522 crassostrea gigas cgamy caa69658.1 520 pinctada fucata pfamy agn55419.1 522 hyriopsis cumingii hcamy agw45296.1 523 pinctada maxima pmamy aei58897.1 518 pteria penguin ppamy aei58894.1 523 haliotis discus hannai hdamy bam74656.1 511 saccostrea forskali sfamy aha11414.1 520 colobus angolensis caamy abw02886.1 511 ctenopharyngodon idella ciamy acx35465.1 512 marsupenaeus japonicus mjamy ahn91844.1 512 sus scrofa ssamy aaf02828.1 511 bombyx mori bmamy act64133.1 500 salmo salar saamy abd13895.1 505 drosophila melanogaster dmamy caa28238.1 494 lithobius forficatus lfamy aca34374.1 539 homo sapiens hsamy aaa52280.1 511 mus musculus msamy caa24099.1 511 lates calcarifer lcamy aal84163.1 509 using coomassie blue staining according to the manufacturer’s instruction of kits (nanjing jiancheng, china). one unit of α-amylase activity is defined as: per milligram of protein in tissues at 37 °c and substrate for 30 min, the amount necessary to hydrolyze 10 mg starch. statistical analysis the results of qrt-pcr analyses were based on the ct values of the pcr products and the comparative ct method was used to analyze the expression level of mmamy. statistical analysis was performed using software spss 19.0. one way analysis of variance (anova) was used to compare the differences of relative mmamy mrna expression in different developmental stages, in various adult tissues and in different shell color strains. multiple comparisons were conducted using the student newman keuls test. the comparisons of growth traits and α-amylase activities of four shell color strains were performed as above. the significance level of difference is p < 0.05. results cdna and intron sequence analysis of mmamy the full-length mmamy cdna was comprised of 1783 bp (genbank accession no. kp250879) and contained a 1569 bp open reading frame (orf) that encoded 522 amino acids. the cdna also contained a 5’ untranslated region (utr) of 49 nucleotides, a 3’utr of 117 nucleotides that included a stop codon (tag), a putative polyadenylation consensus signal (aataaa) and a polya tail (fig. 2a). the calculated molecular mass of the deduced mature mmamy was 57.58 kda, and the theoretical isoelectric point was 6.72. sequences analysis indicated that mmamy has a signal peptide of 19 amino acids (mltpiaftvgiifprvvlg), 11 cysteine residues, 3 active site residues (asp216, glu252, asp318), 4 calcium binding residues (asn121, arg177, asp186, his220) and 3 chloride binding sites (arg214, asn319, arg354) (fig. 2a). analysis with smart revealed that the deduced amino acid sequence contained 8 stranded alpha/beta barrel that contains the active site, also known as domain a (asp32-arg416). the domain a was interrupted by a 70 amino acid calcium-binding domain b protruding between beta strand 3 and alpha helix 3, and a carboxyl-terminal greek key beta-barrel domain c (asn425-lys512). three pairs of primers pcr amplification produced fragments of 1555 bp, 682 bp, 424 bp, 367 bp, 2041 bp and 1850 bp, respectively. after assembly, seven exons and six introns were revealed. all six introns were located within the orf (fig. 2b). the maximum length of mmamy intron is 1563 bp, while the minimum intron length is 272 bp. the other introns ranged from 311 bp to 1268 bp. all exon–intron junctions follow the consensus rule of the splice acceptor –ag/gt– splice donor for splicing. a phylogenetic tree was constructed with the deduced amino acid sequences of all mollusk amys 99 fig. 3 analysis of expression difference of mmamy gene in different developmental stages of m. meretrix. the same letters indicate no difference in the level of expression, whereas different letters indicate significant differences in expression levels among various developmental stages (p < 0.05, based on anova, n>500). and some model animals’ amys available from the ncbi database using the neighbor-joining method (fig. 2c). the abbreviation of amys and the genbank accession numbers used to construct phylogenetic tree are shown in table 2. the obtained nj tree showed that amys were divided into two major groups. one group is comprised of all mollusk amys, while the other group contains mammals, fish and insects. in the mollusk group, mmamy first clusters with hd-amy, then with the others mollusk. multiple alignment indicated that mmamy shared the highest identity (73.6%) with c. gigas and 57%~72.3% with other species. quantitative expression analysis of mmamy for the different tissues of adults, mmamy was found to express at moderated level in visceral mass, but no expression was detected in other five tissues. for the developmental stages, mmamy was not expressed in the early stages, such as unfertilized mature eggs, fertilized eggs, 4 cells, blastula, gastrulae, and trochophore. in contrast, it was expressed after the stage of d-shaped larva, showing the highest expression in the umbo larvae. no significant difference was found among d-shaped larva, eyespots larva and juvenile clams (fig. 3). mrna and enzyme activity of mmamy associated with growth traits of four shell color strains the results of growth traits, such as shell length, shell width and shell height, showed that strain df had a significantly higher growth rates than strain rs. consistently, the comparison in total weight between the four lines showed the same result as size measurements, where the highest mean value was observed in strain df, and the lowest mean is in strain rs (fig. 4). the expression of mmamy in the visceral mass of four shell color strains indicated that the highest expression was observed in stain df and the lowest expression was found in rs. however, no significant difference was detected between strain df and ws (fig. 5a). the α-amylase activity of four shell color strains indicated that the highest activity was observed in strain df and lowest in strain rs (fig. 5b). no significant difference among other color strains. this result is similar to that of growth traits and the expression in four shell color strains of m. meretrix, which suggest the potential connection among α-amylase activity and growth traits. discussion analysis of genomic structure in this study, the full-length mmamy cdna sequence is 1,783 bp, with an orf of 1,569 bp encoding 522 amino acid, which was within the range of amys length in most animals. the calculated molecular mass of the mature mmamy protein was 55.948 kda, and the range of animal amys was 45–67 kda (zółtowska, 2001; sellos et al., 2003). 100 fig. 4 comparison of growth parameters (a. shell length; b. shell height; c. shell width; d. total weight) in four shell color strains. the same letters indicate no difference in the growth trait, whereas different letters indicate significant differences in the growth trait among four shell color strains (p < 0.05, based on anova, n>500). like other animals’ amys, mmamy had all the conserved motifs, such as the active sites, calcium biding sites and chloride binding sites (janecek, 1997). amys of all animals are chloride-dependent enzymes that require chloride for full activity (strobl et al., 1997; qian et al., 2005). like most animals, the three amino acid residues in the hard clam were involved in the chloride binding of amys, including two arginine (r) residues and an asparagine (n) residue. all animal amys contain eight conserved cysteine residues that form four disulfide bridges (janecek, 1993). however, the number of cysteine residues is not usually the same among different species. for example, 12 residues of amys were found in silkworm bombyx mori (ngernyuang et al., 2011), whereas 10 residues were observed in the shrimp penaeus vannamei (wormhoudt and sellos, 1996). in mollusk, 10 and 11 residues of amy were detected in indian rock oyster saccostrea forskali (thongsaiklaing, 2014) and pearl oyster p. maxima (pan, 2011), respectively, which are comparable to 11 cysteine residues of mmamy in this study. the genomic sequence of mmamy contains 7 exons and 6 introns, and their junctions obey the rule of “gt ... ag”. it is well known that the number of intron-extron in the amys is highly variable between taxa (da lage et al., 2002). for example, no intron was found in d. melanogaster, while 14 introns were observed in d. pulex. by comparing mmamy with other 6 mollusks (patella vulgate, p. maximus, c. gigas, s. constricta, p. fucata and lottia gigantea), the maximum number of 8 introns occurred in p. fucata (huang, et al., 2016), 7 introns in c. gigas (sellos and van wormhoudt, 2002) and the minimum of 5 introns in l. gigantean and s. constricta (liu et al., 2017). this may be more likely related to factors linked to splicing mechanisms and requirements, and to recognition of introns and exons, or to more extrinsic factors (da lage et al., 2011). quantitative expression analysis of mmamy no expression of mmamy was detected at embryonic stages probably because they have yolk as nutrients and don’t live on food from surroundings. though trochophore started into the larval stage they 101 fig. 5 analysis of expression difference of mmamy gene (a) and α-amylase activity difference (b) in different shell color strains of m. meretrix. the same letters indicate no difference in the level of gene expression or enzyme activity, whereas different letters indicate significant differences in the level of gene expression level or enzyme activity among four shell color strains (p < 0.05, based on anova, n=6). df: dark fringe, tc: thin checkered, ws: white shell, rs: red shell. still retained the yolk for nutrients. d-shaped larva began to filter and digest food resulted in the increased expression levels of mmamy after this stage, which is similar to the situation in s. constricta (liu et al., 2017). the highest expression level occurred in umbo larvae was possibly connected with the increased food intake and fast growth. in contrast, eyespot larva, an important stage of metamorphosis and attachment for bivalve molluscs, showed the decreased expression, probably due to the declined amount of food intake (wang et al., 2008). a similar profile of amy mrna expression was observed in h. discus hannai, with the lowest or even no expression in early embryos and higher expression during the larval stages (he et al., 2011). these similar results suggest that the nature of α-amylase may be served as the main digestive enzyme in mollusks. in addition, amylase expression also had a similar trend of profiles that transcripts were first detected at 5 dph, peaked at 20 dph and then decreased during metamorphosis in the winter flounder, pleuronectes americanus (susan et al., 2000). there were mainly salivary amylase and pancreatic amylase in mammals, and the two types of enzymes were expressed in tissue specific pattern (nishide et al., 1986). since there was no salivary gland in fish, the amylase were main expressed in hepatopancreas, and also intestine, even with high activity (tomita, 1987). for mollusks, in c. gigas, the main expression of the two amylase gene was in digestive gland leading to high amylase activities, in accordance with the digestive function, however, trace mrna was observed in all five tissues (huvet et al., 2003). in this study, the specific expression of mmamy in visceral mass showed a similar profile with that in h. discus hannai, in which amylase was only expressed in digestive gland (kumagai et al., 2013). correlation analysis of the activity of amylase and growth traits the shell color of marine bivalves may have been previously dismissed as mere excretory products (cain et al., 1988). as evidenced, shell color is not only related to ecology and habits (beukema and meehan., 1985), but also associated with phenotypic traits such as growth and survival in some species (alfonsi and perez, 1998; zhang et al., 2009). digestive enzyme activity was an important index to reflect the physiological function of the digestive system, which determined the ability of the nutrient digestion and absorption, and the speed of the growth and development (pan et al., 2006).to accommodate their fast growth, larger clams need more nutrients to maintain their metabolic activity, which required higher activity of digestive enzyme. in this study, the positive correlation among growth traits, shell color, α-amylase activity in different strains were also observed in some other shellfish species such as p. fucata (zou et al., 2014) and c. nobilis (deng et al., 2008). this might be explained by pleiotropic effects, which inferred that the expression of shell color genes may closely link with genes determining growth traits (sokolova and berger, 2000). however, further study is necessary to shed light on their molecular mechanisms. 102 acknowledgments this work was financially supported by national natural science foundation of china (31372527), zhejiang provincial natural science foundation (y16c190015), national infrastructure of fishery germplasm resources programme （2018dka30470）, ningbo project of agricultural science and 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isj 17: 99-107, 2020 issn 1824-307x review the potential immune alterations in insect pests and pollinators after insecticide exposure in agroecosystem a zibaee1*, d malagoli2 1department of plant protection, faculty of agricultural sciences, university of guilan, rasht, iran 2department of life sciences, university of modena and reggio emilia, modena, italy this is an open access article published under the cc by license accepted may 11, 2020 abstract agroecosystems are the habitat of pests and beneficial insects from different orders, which are exposed to agro-practices, especially treatments with chemicals. insecticides are a wide group of chemicals used in agroecosystems that affect insect ecology and physiology in different ways. among physiological components affected by insecticides, the immune system (is) is an important one, enabling insects to resist against invading microorganisms and parasitoids thanks to the action of hemocytes and humoral components. so the determination of any immune alterations should be considered as a critical issue in insecticide application within agroecosystems. insecticides of synthetic or natural origin, e.g. insect growth regulators (igrs) and botanicals, are frequently cytotoxic and alter hemocyte morphology and number, impairing cellular-based immune responses in addition to humeral responses. exposure of pollinators to neurotoxin insecticides like neonicotinoids may inhibit the immune-related transcription factor, nfb, with a negative impact on the expression of antimicrobial peptides, melanization and clotting. in contrast, some igrs may have enhancing effects on hemocyte spreading mainly plasmatocytes and cellular-based immune responses. chemical insecticides have several impacts on the physiology of insects in which immune modulation is one of the most important cases because any alteration may alter their ability to respond toward invading pathogens and directly their survival. this is more severe once pollinators are in contact with chemicals because of the presence of several pathogenic agents that directly influence their performance. key words: antimicrobial peptide; hemocyte; immune response; insecticide; pollinator introduction the widespread distribution of agricultural pests and climatic changes are the major threats to global food security, a topic that creates comprehensible concern in view of the growth of the human population. nowadays agricultural pests are widespread in almost all agroecosystems, heavily influencing the economy and food resources worldwide. it has been estimated that more than 60000 species cause damages to agricultural products of which 50000 species are plant pathogens, 8000 species are weeds and 9000 species are insects and mites (zhang et al., 2011). the latter reduces the global harvest of about 14 %, while a further 26 % is lost in consequence of plant ___________________________________________________________________________ corresponding author: arash zibaee faculty of agricultural sciences university of guilan rasht, iran, 416351314 e-mail: arash.zibaee@gmx.com; arash.zibaee@guilan.ac.ir pathogens and weeds (pimentel, 2009a, b; zhang et al.,2011). several control procedures try to limit the insect-driven damages, and among these chemical insecticides and biological control agents attract the attention of the experts. a loss of almost 78 % of fruit production, 54 % of vegetables and 32 % of cereals is estimated in absence of pesticides (liu et al., 2002; cai, 2008; zhang et al., 2011; zhang, 2018). like other pesticides, also chemical insecticides negatively affect non-target organisms they leave polluting residues, display direct or indirect toxicity on humans, and pose the risk of a resurgence of resistant pests (chandler et al., 2011; zhang, 2018). a common approach to reducing undesirable side effects of insecticides in agroecosystems is to use selective or low-persistent insecticides once the most sensitive developmental stage of target insect is available in the field (dent, 2000). the chemical insecticides most frequently distributed in agroecosystems have different modes https://creativecommons.org/licenses/by/4.0/legalcode 100 fig. 1 an overview on the immune reactions of insects toward infectious objectives of action. organochlorine insecticides like cyclodienes as well as pyrethroids have neurotoxic and metabolic effects acting on the different ligand and voltage-gated ion channels (yu, 2008; timbrell, 2009). organophosphates and carbamates, inhibitors of acetyl cholinesterase, slowly dispatched from the enzymes and cause repeated nervous pulses in the synaptic cleft. nicotine and neonicotinoid insecticides block acetylcholine receptors on the post-synaptic membrane causing an influx of sodium ions and the generation of abnormal action potential. insect growth regulators (igrs) constitute a wide group of insecticides designed on growth-regulating hormones like juvenile hormone (jh), ecdysone and chitin synthesis inhibitors. igrs disrupt insect development by intervening in molting, as well as on the synthesis of chitin, a major constituent of insect integument. finally, plant-derived insecticides (botanicals) may act as antifeedants, development and reproduction inhibitors (yu, 2008). besides the most relevant and known effects, chemical insecticides may also cause widespread discrepancies on intermediary metabolism and immunity of insects (zibaee and bandani, 2010; zibaee et al., 2011; zibaee et al., 2012; mirhaghparast et al., 2015a, b; mirhaghparast et al., 2016). intermediary metabolism is referred to all biochemical and cellular processes which provide sufficient energy for the biological demands of insects (klowden, 2007; nation, 2008). insect immune system (iis) is a highly efficient component that confers protection against invading pathogens and tumors (park and lee, 2012). once an invader passes non-specific defensive barriers like integument and alimentary canal, the immune system raises several rapid and efficient responses (nehme et al., 2007; tsaka and marmaras, 2010). cellular (e.g., phagocytosis, nodulation and encapsulation) and humoral (e.g., antimicrobial peptides, lectins, coagulation and melanization) reactions act synergistically to remove the infection from insect hemolymph and body cavity (lavine and strand, 2002; nation, 2008; park and lee, 2012). detailed immune reactions of insects toward infections have been illustrated in figure 1. in consideration of the concerns related to the usage of chemical pesticides, in the last four decades, there has been increasing attention towards the integrated pest management (ipm) to achieve sustainable agroecosystems. ipm aims to combine a responsible usage of chemicals, biological knowledge and cultural techniques in order to keep the populations of insect pests at acceptable levels. efforts have been made in order to find the best synergistic combinations in the control of insect species. insecticides and entomopathogen organisms are two elements that may prove highly efficient in ipm programs when used simultaneously (zibaee, 2019). therefore, it becomes crucial to assess also in laboratory conditions the combined effects of different insecticides (e.g., synthetic, botanical or igrs) and entomopathogens. indeed, a pesticide alone could be ineffective in keeping the insect pest at an acceptable level, but it could prove highly efficient in weakening the iis, acting as an enhancer for the entomopathogen organisms. recently, this issue has been successfully addressed by exploring the fundaments of bacillus thuringiensis activity (caccia et al., 2016) and by testing the combination of an entomopathogenic fungus with a pesticide against 101 leptinotarsa decemlineata (coleoptera: chrysomelidae) (tomilova et al., 2016). this review focuses on the effects of the different classes of insecticides on the iis and provides a useful recapitulation of the insecticides more suitable for the combined use with entomopathogenic organisms. effects of synthetic conventional insecticides on iis as mentioned earlier, synthetic conventional insecticides (si) from almost all classes impair nervous or hormonal systems leading to behavioral disability and subsequent death of insects (chandler et al., 2011). the connections between nervous and immune functions are well-known in all metazoans, including insects. for instance, after single or repeated pathogen exposures, reproducible changes of behavioral fever, illness-induced anorexia, decreased learning ability and increased egg-laying have been reported (adamo, 2008). so, it is conceivable that the changes in insect neural function following sub-lethal treatment with si may also affect iis. most frequently, insecticides play inhibitory effects on iis, although the increased performance of iis has been found in some cases (zibaee et al., 2011; zibaee et al., 2012; mirhaghparast et al., 2015a, b; mirhaghparast et al., 2016). in order to infer how the insecticides could influence the iis, delpuech et al. (1996) designed an experiment to evaluate the effects of synthetic insecticides on host-parasitoid interactions. the authors treated topically the eggs of the susceptible and non-susceptible strains of drosophila melanogaster (diptera: drosophilidae) with sublethal concentrations of chlordimeform, oxydemeton-methyl, propoxur, endosulfan, lindane, and dieldrin. the larvae hatched from these eggs were then reared on the media containing lc30 of each insecticide till adult emergence. the eggs laid by insecticide-treated adults (both sexes) were finally exposed to female parasitoid, leptopilina boulardi (hymenoptera, eucoilidae). the results demonstrated that endosulfan and dieldrin significantly decreased the encapsulation rate in the treated embryos, while the total encapsulation rate decreased only after treatment by endosulfan. the other insecticides used in this study had no significant effects. the authors proposed that the adverse effects of endosulfan and dieldrin on d. melanogaster is might be due to inhibition of monoamine oxidases responsible for melanization and, indirectly, for encapsulation efficiency (delpuech et al., 1996). a different point of view has been offered by studying the effect of si, imidacloprid, on the immune response of the termite reticulitermes flavipes (isoptera: rhinotermitidae) against the entomopathogenic fungus beauveria bassiana (boucias et al., 1996). the exposure to lethal and sub-lethal concentrations of imidacloprid did not modify the phagocytic competence of the treated termites. further studies by using lipopolysaccharides, heat-killed or live bacteria revealed that r. flavipes may lack or have negligible cellular or humoral immune response in contrast to other insects (boucias et al., 1996). this experiment suggests that while the iis is a good target for control strategies, this might not hold true for all the species. a 10 to 20 day-treatment with monocrotophos, dimethoate, methylparathion, quinalphos or endosulfan increased the hemocyte count in the assassin bug rhynocoris kumarii (hemiptera: reduviidae) (caccia et al., 2016). the results on endosulfan could have been related to its effects on insect diuresis and hemocyte dilution in the hemolymph. the increase in total hemocyte count corresponded to an unbalance in the hemocyte population, with a significant increase of granulocytes and a decrease of prohemocytes and plasmatocytes after 10 days of exposure (george and ambrose, 2004). recently, it has been observed that sublethal hexaflumuron exposure caused a concentration-dependent increase in plasmatocyte number, while the total hemocyte number and the phagocytic activity declined in the armyworm mythimna separate (lepidoptera: noctuidae) (huang et al., 2016). changes in the hemocyte population may not be directly linked to changes in the response versus a specific pathogen, but as different hemocytes play diverse roles, changes in cell population usually reflect changes in immune efficiency (smith et al., 2016). a gain in immune response was observed in the wax moth galleria melllonella (lepidoptera: gelechidae) and the potato beetle l. decemlineata after exposure to lc50 or lc30 of the phosphorothioate pirimiphos-methyl. the pesticide promoted a dosedependent increase in the total hemocyte count and elevated the encapsulation rate toward nylon (dubovskiy et al., 2013). while the molecular basis for the effect is still unclear, the authors proposed that this organophosphate insecticide could suppress the neuro-hormonal stress reaction that in untreated control limited the immune response. as it will be further detailed below, in the section on the effects of insecticides on pollinators, in some cases the effects of si on the iis can be considered as an undesirable side-effect. in the silkworm philosamia ricini (lepidoptera: saturnidae) the pyrethroid insecticide cypermethrin presented genotoxic effects and induced apoptosis in hemocytes at very low concentrations (kalita et al., 2017). effects of insect growth regulators (igrs) on iis because of their structure, mimicking that of natural compounds, igrs present more selective effects on insect pests than the previously mentioned si (merzendorfer, 2013). by regulating developmental and metabolic processes, the endocrine system has relevant interconnections with the immune response of insects, and hormones may act on both hemocytes and immune-related organs like the larval fat body (adamo, 2008). on the other side, hemocytes directly intervene in tissue remodeling during molting and metamorphosis (gillespie et al., 1997; zibaee et al., 2012). in these respects, izzetoglu and karacali (2003) made an in vitro experiment assessing the effects of a three-day treatment with different concentrations of the steroid hormone 20hydroxyecdysone (20-e) on the total and 102 differentiated hemocyte counts in g. mellonella larvae. the authors reported an increase in total hemocyte count until the third day of 20-e treatment when the number of total hemocytes significantly decreased at all tested concentrations. a similar trend was observed for plasmatocytes and granulocytes, but the latter increased more significantly for low concentrations of 20-e (izzetoglu and karacali, 2003). in agreement with these observations, it has been observed in larvae of the grey flesh fly, neobellieria bullata parker (diptera: sarcophagidae) that 20-e and its synthetic agonists, rh2485, rh5849 and rh0345, increase laminarin-induced nodulation in a dose-dependent manner. the effects were observed either after injection or topical administration and were more evident for 20-e than its agonists. conversely, juvenile hormone (jh) ii and its analogs fenoxycarb and pyriproxyfen reduced laminarin-induced nodulation (franssens et al., 2006). the stimulatory action of 20-e and the inhibitory effect of jhi and ii were observed also in the beet moth, where the response towards the plasmatocyte-spreading peptide was enhanced or inhibited by the two jhs, respectively (kim et al., 2008). differently, form 20e, the action of jh family members is mediated by extracellular receptors (kim et al., 2008). several studies agreed on the hemocyte activating action of 20-e and the inhibiting activity of jh, which could play a physiological role in balancing the proliferation, differentiation and dispersal of hemocytes involved in immune competency (rantala et al., 2003; sorrentino et al., 2002). the igrs tested against agricultural pests have shown effects on iis partly similar to those described for 20-e and jh. metyrapone, an igrs and jhas analog decreased the number of granulocytes but it increased plasmatocyte densities in the treated larvae of spodoptera littoralis (lepidoptera: nooctuidae) (gelbič et al., 2005). abd el-aziz and awad (2010a) demonstrated the effects of b. thuringiensis and dimilin against the larvae of agrotis ipsilon hufnagel (lepidoptera: noctuidae). they reported signiifcant increase in the numbers of plasmatocytes, granulocytes and spherulocytes but the number of prohemocytes showed a significant decrease in the treated larave compared to control after 12 and 24 h. in another experiment, the authors found a significant decrease of phenoloxidase activity in the treated larvae compared to control in addition to changes in protein patterns of hemolymph in electrophoresis (abd el-aziz and awad, 2010b). zibaee et al. (2012) injected the adults of eurygaster integriceps (hemiptera: scutelleridae) with methoxyfenozide, an activator of the ecdysteroid signaling cascade, or pyriproxyfen, four hours prior to injection of b. bassiana spores. methoxyfenozide increased the number of total and differentiated hemocyte counts after 12 and 24 hours of treatment, whereas the opposite effects were observed with pyriproxyfen. similar trends were observed for nodulation, especially at the highest concentrations used for pyriproxyfen (zibaee et al., 2012). similar effects on hemocytes but no effects on phenoloxidase were observed in the 4th instar larvae of ephestia kuehniella zeller (lepidoptera: pyralidae) (rahimi et al., 2013). a course study of the effects of pyriproxyfen on the striped rice stemborer chilo suppressalis (lepidoptera: crambidae) infected with b bassiana, revealed a complex pattern of response, depending on both the time and the dosage of the pesticide (mirhaghparast et al., 2015a). in this case, hemocyte counts revealed a time-dependent fluctuation in plasmatocyte number and the increase of granulocytes only after 3 h incubation. also, nodule formation was significantly increased at specific pyriproxyfen concentrations (mirhaghparast et al., 2015a). similarly, the chitin-synthesis inhibitor hexaflumuron, increased hemocyte number and phenoloxidase activity of c. suppressalis larvae either alone or in combination with b. bassiana (mirhaghparast et al., 2015b). shaurub and sabbour (2017) determined the effects of pyriproxyfen and flufenoxuron on thc and dhc of a. ipsilon larvae. thc in the treated larvae significantly decreased compared to control. no significant changes were observed in the number of prohemocytes but pyriproxyfen significantly decreased the number of plasmatocytes. both igrs significantly decreased the number of granulocytes in the treated larvae. yu et al. (2018) determined the effects of oral administration of tebufenozide on the immunity of the asian corn borer, ostrinia furnacalis (lepidoptera: crambidae). the igr tebufenozide is an ecdysone agonist and belongs to diacylhydrazine molt-inducing insecticides which bind to ecdysteroid receptors into insect cells and mimics the effect of molting hormones. tebufenozide increased both the total hemocyte count and the activity of phenoloxidase compared to control larvae. moreover, the treated larvae displayed an enhanced ability to clear the bacterial infection from their hemolymph so that an increased survival rate was observed after 33 hours of bacterial challenge (yu et al., 2018). botanical insecticides (bi) plants developed various compounds during their evolutionary processes, in order to be well equipped against insect pests. these compounds are known as secondary metabolites and they may have several functions, including the defense against herbivore attacks (wink, 2018). due to the adverse effects connected with the usage of synthetic insecticides, the plants-derived compounds, further classified into the two categories of plant extracts and essential oils are promising candidates against insect pests. today, botanical insecticides have shown significant effects with no or negligible destructive effects on the environment and less prone to be inactivated by resistant insects (enane, 2001; hardin et al., 2009; pavela and benelli, 2016). as far as their target is concerned, botanical insecticides (bi) may affect digestion, reproduction, intermediary metabolism, oxidative stress, cell death, octopaminergic system and immune system. recognized molecular targets include acetylcholine esterase, p450 cytochromes, octopamine and gamma-aminobutyric acid (gaba) receptors (abdelgaleil et al., 2009; zibaee, 2011, lukasik et al., 2011; senthil-nathan, 2013; 103 kumrungsee et al., 2014; wei et al., 2015; tak and isman, 2016; pavela and benelli, 2016; shahriari et al., 2017; shahriari et al., 2018). the observed effects of bi on iis include abnormal hemocyte morphology, altered total and differentiated hemocyte counts, reduced phagocytosis, nodulation as well as inhibition of phenoloxidase (zibaee, 2011). pioneer studies on the limonoid bi azadirachtin, revealed, besides the antifeedant effects, immune depression in the nymphs of the kissing bug rhodnius prolixus l. (hemiptera: reduviidae) subjected to bacterial challenge. effects of azadirachtin on the iis involved hemocyte count, nodule formation and antibacterial and lysozyme activities. interestingly, no effects were reported on the prophenoloxidase-activating system in the azadirachtin-fed nymphs (azambuja et al., 1991). ayad et al. (2001) reported the higher thc and oenocytoid counts in the azadirachtin treated larvae of parasarcophaga surcoufi (diptera: sarcophagidae) after 40 h while the numbers of plasmatocytes and granulocytes decreased after 10 and 40 h, respectively. moreover, the authors recorded the significant hemocyte deformities as lysis of plasmatocytes and releasing of cytoplasmic components of granulocytes (ayad et al., 2001). relationships between bi effects and the iis were observed also after treating target insects with neem gold and artemisia calamus. these vegetable extracts provoked significant damage in hemocytes, which resulted in almost disrupted by the treatment (sharma et al., 2003, 2008). after feeding e. integricpes with artemisia annua., it was observed a significant decrease in phagocytic rate and nodule formation. at all-time intervals, the a annua extract decreased the activity of phenoloxidase in the treated hemipterans and inhibited the enzyme activity by reducing vmax and increasing km. the authors concluded that a. annua may affect ligandreceptor complex formation on the plasma membrane of e. integricpes hemocytes, thus reducing or impairing immune reactions of phagocytosis and nodulation (zibaee and bandani, 2010). shaurub et al. (2014) found that the larvae of s. littoralis treated by azadirachtin had the less thc compared to control. also, significant changes in morphology of plasmatocytes and granulocytes were observed as the presence of rough endoplasmic reticulum filled with fibrous materials, looped, vacuolated and swollen mitochondria with the specific appearance of autophagic lysosomes in granulocytes and ruptured cell membrane and folded nuclear envelope in plasmatocytes (shaurub et al., 2014). similar results were observed also in g. mellonella larvae treated with azadirachtin and in s. littoralis exposed to essential oils of castor and camphor (er et al., 2017). in contrast to the abovementioned studies, dhivya et al. (2018) reported an increase of total hemocyte counts and an augmented activity of phenloxidase in s. litura larvae exposed to prosopis juliflora extract. individual and combined effects of a bacterium, micrococcus luteus, and azadirachtin were evaluated on the immune system of sarcophaga argyrostoma (diptera: sarcophagidae) larvae (dorrah et al., 2019). azadirachtin decreased the number of total hemocyte count 12 and 24 h posttreatment while a set of various changes were observed in differentiated hemocyte counts. oenocytoids, spherulocytes, granulocytes and prohemocytes presented an irregular trend, with their number increased or decreased at different time points. plasmatocytes significantly decreased at all time intervals after azadirachtin treatment except for 60 h when no significant difference was observed in comparison to control. the injection of larvae with bacterium and azadirachtin significantly reduced the numbers of formed nodules, phenoloxidase and lysozyme activity compared to individual bacterial injection. these effects were somehow similar in fat bodies, hemocyte and plasma preparations, and were attributed to a potential mutagenic action of azadirachtin (dorrah et al., 2019). however, the full explanation of these data has not been provided, yet. effects of insecticides on the immune system of pollinating bees pollinator insects are one of the most important organisms in agroecosystems as they engaged in pollinating about 240,000 species of flowering plants (mcgregor, 1976; dafni, 1992). these plants are not only directly fed by humans, but they are used also as food sources for livestock. well-known estimates proposed that 30 – 50 % of total human food may directly or indirectly depend on pollinator insects as they influence the quality and quantity of agricultural products (mcgregor, 1976). the agricultural techniques implemented in the last decades and the expansion of the food demand in the developing countries make it difficult to refine these estimates but still, the fundamental contribution of pollinators is out of the question (aizen et al., 2009). in addition to agricultural products and plant reproduction, the biodiversity of animals feeding on fruits and seeds also depends on pollinator activity, and in this sense the precise relevance of pollinators on the global ecosystem is hardly quantifiable (eardley et al., 2006). unfortunately, agrochemicals in general, and si in particular, frequently affect life history, reproduction, motility, learning and physiological functions, including immunity, of pollinators beside those of insect pests (desneux et al., 2007; domingues et al., 2017) and sublethal doses of them can represent a chronic stress leading to colony failure of colonial pollinators such as bees, the principal pollinators in human agriculture (brandt et al., 2016). several recent studies have highlighted the adverse effects of insecticides on immune responses of bees, especially the western honeybee apis mellifera (hymenoptera: apidae). in the case of neonicotinoids, it has been reported that these sis up-regulate an inhibitor of the immunerelated transcription factor, nf-kb, thus paving the way to the replication of the deformed wing virus (di prisco et al., 2013). such inhibition could also suppress other forms of immune defenses, e.g., expressions of antimicrobial peptides, hemocyte clotting and melanization (di prisco et al., 2013; brandt et al., 2016). simultaneous exposure to a neonicotinoid and an immune challenge increases 104 the insecticide-mediated immune suppression, thus in open field, the action of sublethal doses of neonicotinoid or other pesticides could be even more aggressive than that measured in laboratory by exposing bees only to a pesticide (lopez et al., 2017; walderdorff et al., 2018). this observation agrees with previous results indicating that the coexposure to the si imidacloprid and the entomopathogenic microsporidia nosema apis has a significant impact on mortality while leaving apparently unaffected hemocyte count and phenoloxidase activity (alaux et al., 2010). in these respects, the oral administration of a mixture containing the si diazinon, malathion, profenofos and chlorpyrifos did not impact on bee survival after short-term exposure, but it affected the expression of the antimicrobial peptide hymenopteran (al naggar et al., 2015). in the class of neonicotinoids, compounds such as thiacloprid and imidacloprid can affect immune parameters at concentrations similar to those used on the field, whereas the clothianidin can influence total hemocyte count only at nonrealistic concentrations but it does affect encapsulation at all the concentrations assessed in a. mellifera (brandt et al., 2016). these discrepancies are probably to be related to the chosen compound, the doses applied and the selected immune parameters. the information collected on a. mellifera is consistent with data collected on other bees. in apis bursata five commercial formulations of the si, endosulfan, bifenthrin, diafenthiuron, imidacloprid and ethofenprox affect bee hemocyte number, shape and integrity (perveen and ahmad, 2017). in the bumblebee bombus impatiens cresson (hymenoptera: apidae) field-realistic 6-day pulses of imidacloprid revealed that the immune response to a non-lethal immune challenge can be maintained only in control bumblebees and in those exposed to the lowest concentration the animals exposed to the highest concentration did not sustain the immune response and had a higher chance to die after the immune challenge (czerwinski and sadd, 2017). in this way, it is confirmed that both the insecticide and the immune challenge act as a stressor and they could present additive or synergistic effects, highly deleterious for the pollinators (czerwinski and sadd, 2017). walderdorff et al. (2018) designed a study to determine the effects of separated and simultaneous exposure of a. mellifera to the si imidacloprid and the gram-negative escherichia coli. results demonstrated that imidacloprid alone decreased phagocytosis and production of hydrogen peroxide and nitric oxide, while e. coli slightly increased these immune reactions compared to sole insecticide exposure. finally, the authors reported that imidacloprid had more severe effects on the unspecific oxidants hydrogen peroxide and nitric oxide than on phagocytosis. hernández lópez et al. (2018) reported that sublethal dose of dimethoate, an organophosphate si, led to the increase of totaland differentiated hemocyte (granulocytes and oenocytoids, but not plasmatocytes) counts in a. mellifera larvae while ld50 concentration decreased total hemocyte count compared to control although no statistical differences were observed in case of differentiated hemocyte count. the si clothianidin, a neonicotinoid, increased total hemocyte count and activated cellular responses but it had no effect on differentiated hemocyte count. the pyrethroid fluvalinate showed no statistical effects on hemocyte counts and cellular immunity compared to control. the authors also demonstrated synergistic and negative effects of demethoate and clothianidin on bees simultaneously exposed to the bacterium paenibacillus larvae and concluded that fluvalinate might be the less aggressive insecticide for a. mellifera larvae. recently, santos et al. (2018) designed the series of experiments to determine the effects of cymbopogon martinii essential oil and its major constituent, geraniol (a bi), along with the si imidacloprid on a. mellifera. at first, the authors investigated the oral and contact toxicities of the two compounds against adult bees. second, the ld20 concentration of the compounds separately was exposed to evaluate the encapsulation and foraging behavior of a. mellifera. the oral exposure was more toxic than contact exposure, but c. martini essential oil and geraniol toxicity were lower than that of imidacloprid. no significant difference was observed in encapsulation and foraging behavior of the individuals treated the bis and the si, while negative effects were observed in the average distance traveled, light orientation and movement in the same direction among the orally treated honeybees, especially for bees treated with imidacloprid. the authors attributed the lower toxicity of the essential oil and geraniol to their efficient metabolic degradation thanks to the activity of glutathione s-transferase, esterase and cytochrome p450-dependent monooxygenase. the higher toxicity of the nicotinoid imidacloprid is correlated to its neurotoxic action which is not counteracted by specific metabolic activities. conclusion this overview of the action of different class insecticides, si, igrs and bi highlight the relevance of the effects that all these compounds have on the iis. the effects are in some cases controversial because there is not a unique effect in terms of immune activation or inhibition, but in general, the immune functions are altered in a way that makes the iis less efficient and responsive. while these can be useful for controlling insect pests, it may also represent a double-edged sword, because immune impairment extends also to beneficial pollinators. the development of new generation and ecofriendly pesticides, especially those of natural origin or based on pathogens, specifically targeting the immune system of the insect pest under treatment, may represent a valid alternative. however, longterm effects, persistence in the environment and side effects on beneficial insects must be carefully evaluated for all the products that will be developed in the future. this represents a further obstacle because for several users the cost, availability and environmental persistence are considered advantageous points in favor of present-day chemical/synthetic pesticides. 105 acknowledgements results collected from the authors and mentioned in this review derived from far grants (university of modena and reggio emilia) to dm. references abd el-aziz nm, awad hh. changes in the haemocytes of agrotis ipsilon larvae (lepidoptera: noctuidae) in relation to dimilin and bacillus thuringiensis infections. micron 41: 203-209, 2010a. abd el-aziz nm, awad hh. immune response in agrotis ipsilon (lepidoptera; noctuidae) induced by bacillus thuringiensis and dimilin. egypt. j. biol. pest control 20: 7-13, 2010b. abdelgaleil sam, mohamed mie, badawy mei, elarami saa. fumigant and contact toxicities of monoterpenes to sitophilus oryzae (l.) and tribolium castaneum (herbst) and their inhibitory effects on acetylcholinesterase activity. j. chem. ecol. 35: 518-525, 2009. adamo sa. bidirectional connections between the immune system and the nervous system in insects. in: insect immunology. beckage, n. e. 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feeding habits; whole-genome sequencing; gene family cluster analysis; venn analysis; positive selection analysis introduction bombyx mori (b. mori) is an important economic insect, and its diet plays an important role in silkworm cocoon production. therefore, it is of great significance to the economic development of our country to study the molecular mechanism of feeding habits, cultivating polyphagous silkworm strains and developing artificial feeds that are more conducive to the growth of silkworms. silkworm, as an oligophagous insect, only eats mulberry leaves under normal conditions. mulberry leaves contain all the nutrients needed for silkworm growth and development (fraenkel, 1959). silkworm feeding on mulberry leaves is due to the presence of volatile feeding factors in mulberry leaves that can stimulate the feeding desire of silkworms (xue, 2009), such as α,β-hexenal (watanabe et al., 1958), _________________________________________ corresponding author: keping chen school of life sciences jiangsu university 301 xuefu road, zhenjiang 212013, p. r. china e-mail: kpchen@ujs.edu.cn citral and linalool, etc., they act on the chemoreceptors of silkworms (hsiao, 1969), causing positive feeding responses in silkworms. in addition, there are also contain biting factors such as β-sitosterol (hamamura et al., 1961) and swallowing factors such as cellulose. isoquercitrin, quercetin, morin (horie et al., 1962), polyphenolic acid (hamamura and naito, 1961), choline (hayashiya, 1965), chlorogenic acid (naito et al., 1963, 1965), certain vegetable oils and fatty acids (kato et al., 1966), vanillin (yamada et al., 1966) and so on in mulberry leaves can also promote the feeding of silkworms. the monocular structure of silkworm larvae prevents silkworms from recognizing food (chen, 2009), so silkworms do not rely on vision for feeding. animals require a complex and powerful system of taste and smell to assess their food environment (mack and zhang, 2021). the colfactory atractan acting on the olfactory system can cause the silkworm to seek and recognize plants (hsiao, 1969; wilde, 2011), and the phagostimulant acting on the taste sensory system can affect the amount of food 137 table 1 genes used for gene family clustering in each species species name number of genes number of gene families sfr spodoptera frugiperda 22086 19805 aya antheraea yamamai 13610 11923 bmj bombyx mori jiangsu 13053 11923 bmo bombyx mori_jp 16815 15776 pxy plutella xylostella 17441 15287 pra pieris rapae 12064 11578 har helicoverpa armigera 13258 12634 bma bombyx mandarina 12520 12118 dpl danaus plexippus 14514 12286 lde leptinotarsa decemlineata 12671 10771 tca tribolium castaneum 12786 11586 ame apis mellifera 9881 9340 aga anopheles gambiae 12311 10896 aae aedes aegypti 14535 13356 cel caenorhabditis elegans 20060 14320 lhu linepithema humile 11428 10811 dme drosophila melanogaster 13554 10774 nvi nasonia vitripennis 12834 11086 eaten by silkworms (fraenkel, 1959; chapman, 2003). therefore, silkworm food selection is based on the use of taste and smell-related chemical feedback from the nervous system to guide feeding behavior, ingest nutritious food that the body needs, and avoid toxic food (zhang et al., 2011; kumar, 2018). taste receptors can detect nutrients in the environment, and cellular sensors can monitor the levels of nutrients needed by cells. when certain substances are deficient, animals choose to contain corresponding food sources (kim et al., 2021). in addition, an animal's decision to accept or reject an expected food is also influenced by palatability, including smoothness and grit, the latter being influenced by particle size. insects have the ability to discern the size of particles in their food and use this information to decide whether the food is attractive (li and montell, 2021). as the most species and huge number of animals, insects are closely related to the development of human society, and the research of entomomics has been paid more and more attention. as an important data resource for insect molecular biology research, the genome sequences of various species have attracted much attention in current biological research. the rapid development of sequencing technology has brought entomology into the genome era, and the sequenced insect genome data are stored in various large-scale comprehensive databases. since 2000, drosophila melanogaster (dem), apis cerana fabricius (ace), b. mori, danaus plexippus (dpl), acyrthosiphon pisum harris (api), plutella xylostella (pxy), nilaparvata lugens (nlu), locusta migratoria (lmi) and other insect genomes have been deciphered, and the sequencing of insect genomes has shown an explosive growth trend in recent years. in 2004, the genome of the silkworm dazao strain was firstly sequenced by shotgun. in 2008, the silkworm genome data was updated. comparative genomics can be used to compare and analyze the genome sequences of different individuals of the same species and related species in terms of gene family, collinearity or gene structure, and explore the origin and evolution of genes in different species, and the amplification and loss of gene families, and analyze the genetic mechanism of important traits of species. the current research on the molecular mechanism of silkworm's feeding preference for mulberry leaves is insufficient. with the development of sequencing technology and the improvement of various protein databases, it can help find the genes that determine the feeding habits of the silkworm. in this study, the genome of bombyx mori strain jiangsu was sequenced and assembled using next-generation sequencing (ngs), third-generation sequencing (tgs), and hi-c technology. through comparative genomics analysis, gene structures and 138 fig. 1 the distribution of gene families in different species and estimation of species divergence time. (a) the horizontal axis showed 18 species, and the vertical axis was the number of corresponding gene families. (pink) the number of gene families in the single-copy orthologs. (yellow) the number of gene families in multiple-copy orthologs. (dark yellow) the number of unique gene families in related species. (green) the number of gene families in other orthologs. (b) estimation of the divergence time between bombyx mori jiangsu and other 17 species. the number of the node position represented the divergence time of the species in millions of years, and the brackets indicated the confidence range of the divergence time 139 fig. 2 venn diagram of the gene family. (a) bmj, bmo, bma, sfr, har, aya gene family venn diagram; (b) bmj, bmo, bma, dpl, pxy, pra gene family venn diagram.venn analysis was uesd to screen the gene families shared by bmj, bmo and bma and remove all gene families of sfr, har, aya, dpl, pxy and pra functions were annotated in order to find genes that affect silkworm feeding, particularly genes that affect the gustatory and olfactory processes of the silkworm. these results will provide essential information for future research of the silkworm genome. they will be of great significance for further research on the feeding habits of the silkworm and the promotion of the development of the sericulture industry. materials and methods sample preparation and whole-genome sequencing bombyx mori strain nb was raised in the laboratory of the school of life sciences, jiangsu university. the posterior silk glands of the fifth-instar third-day larvae were taken and rinsed with pbs. the silkworm genome resequencing used ngs and tgs technology. ngs used dnasecure plant kit (tiangen) to extract dna. neb next® ultra dna library prep kit (neb, usa) was used to construct a library of qualified dna samples, and index codes were added to each sequencing sample. the dna was purified using the ampure xp system (beckman coulter, beverly, usa), the pcr products were purified, and the library quality was evaluated on the agilent bioanalyzer 2100 system. the illumia cluster kit was used to cluster the qualified libraries on the cbot cluster generation system. after cluster generation, the library preparation was sequenced on the illumina hiseq platform, and 125/150/250 bp paired-end sequencing was generated. the dna extraction of tgs adopted the sds extraction method, and the qualified dna samples were 140 randomly broken into fragments of about 20 kb, and the sticky ends of the fragments were turned into blunt ends. the two ends were connected to the single circular strand, and the two ends of the single strand were connected to the double strand's positive and negative strands. the two ends of the single strand were respectively connected to the positive and negative strands of the double-strand connected to the single circular strand. the two ends of the single strand were connected to the double strand's positive and negative strands to obtain a dumbbell-like structure, which completed the preparation of the third generation 20 kb library (smrtbell dna library). the constructed library was sequenced through the pacbio sequel platform. after self-correcting the third-generation data, falcon (branch 3.1) was used to perform a pure three-generation genome assembly. the corrected long reads were compared, and the string graph was generated according to the overlap connection, and the primary contig was formed. in order to find heterozygosity differences, falco-unip analysis was used to reassemble 'haplotype fused' contigs into haplotigs and obtain the updated primary contigs and haplotigs that constitute diploids genome assembly then phased and classified these heterzygosity differences. finally, for the assembly results of the previous step, the third-generation data was used to correct the assembly results based on quiver software, and then the second-generation data was used to perform secondary corrections based on pilon software to improve the accuracy of the assembly results and finally we obtained a high-quality consensus sequence. the obtained 10×genomics linked-reads data was aligned with the contig sequence assembled from the third-generation data. for contigs that were relatively close in actual distance, there were many linked-reads that support their connection relationships; for contigs that were relatively far away, they lacked linked-reads support, it could not be connected. fragscaff software (version 140324) was used to extend contig and get the preliminary version of scaffold. we compared the hi-c data to the genome draft, performed comparison analysis, and performed clustering, sorting, and orientation according to the comparison results to assist the initial version of the genome to be upgraded to the chromosome level. hi-c clean data was compared with the initial version through burrows-wheeler-alignment (bwa) software. the contig/scaffold of the same kind was sorted and accurately oriented according to the mutual intensity of the two contigs and the position of the comparison of the mutual read. according to the link relationship provided by the restriction region and hi-c data, the map was composed and the weight was calculated to obtain the final upgraded version. finally, core eukaryotic genes mapping approach (cegma) (http://korflab.ucdavis.edu/dataseda/cegma/) (parra et al., 2007) combined with softwares such as tblastn, genewise and geneid and benchmarking universal single-copy orthologs(busco) (http://busco.ezlab.org/) (simão et al., 2015), augustus and hmmer were used to evaluate the assembled genome to ensure its consistency and integrity. gene family cluster analysis and species divergence time estimation the orthomcl (http://orthomcl.org/orthomcl/) process (li and l., 2003) was used to analyze bombyx mori jiangsu (bmj), aedes aegypti (aae), dme, anopheles gambiae (aga), apis mellifera (ame), nasonia vitripennis (nvi), tribolium castaneum (tca), leptinotarsa decemlineata (lde), pieris rapae (pra), antheraea yamamai (aya), bombyx mori_jp (bmo), bombyx mandarina (bma), spodoptera frugiperda (sfr), helicoverpa armigera (har), dpl, linepithema humile (lhu), pxy, and caenorhabditis elegans (cel) for gene family clusters. the gene information of each species was obtained from national center for biotechnology information search database (https://www.ncbi.nlm.nih.gov/), gigadb (http://gigadb.org/), and silkbase (http://silkbase.ab.a.u-tokyo.ac.jp/cgi-bin/index.cgi). the gene set of each species was filtered. when there were multiple alternatively spliced transcripts for a gene, only the transcript with the longest coding region was retained for further analysis; genes encoding proteins less than 30 amino acids were excluded. treefam software was employed to compare and cluster the results and 1.5 was used for the expansion coefficient (li et al., 2006). mcmctree in the paml (yang, 2007) software package (http://abacus.gene.ucl.ac.uk/software/paml.html) was used to estimate the divergence time, where the time calibration point was, aya and bmo: 50-100 mya; dpl, tca: 271.9-293.8 mya; pxy, bmj: 97-190 mya, time calibration point was taken from reference 26 (tingcai cheng et al., 2017). venn analysis in order to obtain genes that affect the feeding of silkworms, gene families from 9 species were selected, including bmj, pra, aya, bmo, bma, sfr, har, dpl, and pxy, for comparison. among them, bmj, bmo, and bma eat mulberry leaves. pra, aya, sfr, har, dpl, and pxy do not eat mulberry leaves. according to the clustering results, the gene families shared by the three mulberry leaf-eating species were screened out through venn analysis, and all gene families of the remaining six species were removed. the screened silkworm shared and unique gene families may contain genes that affect silkworm feeding. since a venn diagram can only count 6 species at most, we divided the 9 species into two groups, one group included bmj, bmo, bma, sfr, har, and aya; the other group had bmj, bmo, bma, dpl, pxy, and pra. the gene ontology (go) and kyoto encyclopedia of genes and genomes (kegg) enrichment analysis of these gene families was carried out to find the pathways related to feeding habits and then determine the genes that may affect the eating habits of the b. mori. kegg information refered to https://www.kegg.jp/kegg/kegg1.html, the permission was provided by the kanehisa laboratory (kanehisa and goto, 2000). positive selection analysis positive selection means that in a single-copy gene family, a particular gene is affected by environmental or human factors during the evolution 141 process, and non-synonymous mutations occur at the amino acid level in order to adapt to changes in the environment. the probability of receiving a positive selection can be detected by calculating ka/ks through the maximum likelihood ratio. among them, ka/ks refers to the ratio of non-synonymous mutation rate to synonymous mutation rate. if ka/ks>1, it is considered to have a positive selection effect; if ka/ks=1, it is considered that there is neutral selection; if ka/ks <1, it is considered to have purifying selection effect. we selected the species combination of bmj, bmo, and bma in the foreground, pra, aya, sfr, har, dpl, and pxy in the background. muscle software was used to perform multiple sequence comparisons of the protein sequences of the two species combinations. the two sets of protein sequence alignment results were filtered through gblocks software to remove low-quality alignment regions. then the filtered protein sequence alignment results were used as templates to generate multiple sequence alignment results corresponding to cds (castresana, 2000). the branch-site model (yang and rasmus, 2002; zhang et al., 2005) in the codeml tool in paml (yang, 1997) for each gene family was used to conduct maximum likelihood analysis. the selection pressure analysis was carried out by pairwise comparison of the nested model of the null hypothesis and the alternative hypothesis. model pairs were tested by the likelihood ratio test (lrt) to determine whether the models have a significant difference. if p-value <0.05, it was considered that there was a significant difference between the two models. the empirical bayes method (beb) was used to calculate the posterior probability of the detected positive selection sites. the sites with the posterior probability> 95% when p<0.05 were considered to be credible positive selection sites. the go and kegg enrichment analysis of positive selection genes were performed to find pathways related to feeding habits. kegg information refered to https://www.kegg.jp/kegg/kegg1.htm, the permission was provided by the kanehisa laboratory (kanehisa and goto, 2000). the amino acid sites where positive selection occurred in related genes in the pathways were analyzed to determine genes that may affect silkworm eating mulberry leaves. results genome sequencing and assembly results we sequenced and assembled the genome of the bmj by using ngs, tgs, and hi-c technology. according to the specific characteristics of the silkworm genome, the dna insert is 350 bp. paired-end sequencing was performed on both ends of these fragments on ilumina hiseq to obtain 64.81g whole-genome sequencing data (139.41×). the hi-c strategy platform obtained a total sequencing volume of 83.78g, with a coverage depth of 180.21×; at the same time, the pacbio platform was used for sequencing with a total sequencing volume of 50.08g and a coverage depth of 107.72× (table s1). the silkworm genome was assembled using the 198.67g sequencing data of the silkworm genome. the contig n50 of the silkworm genome reached 3.75mbp, and the scaffold n50 reached 17.26 mbp (table s2). the gc fig. 3 go and kegg enrichment analysis of common genes in bombyx-specific species. (a) go enrichment analysis of common genes in bombyx-specific species; (b) kegg enrichment analysis of common genes in bombyx-specific species. kegg information refered to https,//www.kegg.jp/kegg/kegg1.htm, the permission was provided by the kanehisa laboratory 142 table 2 go enrichment analysis resuits of common genes in bombyx-specific species go term go class q value genes extracellular region cc 0.001869189 low molecular mass 30 kda lipoprotein 21g1-like; low molecular mass 30 kda lipoprotein 19g1-like; low molecular mass 30 kda lipoprotein 19g1-like; alcohol dehydrogenase precursor; uncharacterized protein; uncharacterized protein loc101740086; uncharacterized protein loc101739948; serine protease inhibitor 21 isoform x3; phospholipase a2-like; microvitellogenin-like; cuticular protein rr-2 motif 58 precursor; seroin 1 precursor; uncharacterized protein loc101742341; rna exonuclease 4-like; uncharacterized protein loc101746057; uncharacterized protein loc101736844; actin cytoskeleton-regulatory complex protein pan1; microvitellogenin-like; low molecular 30 kda lipoprotein pbmhp-12-like; glow molecular mass 30 kda lipoprotein 19g1-like precursor; low molecular 30 kda lipoprotein pbmhpc-19-like precursor; plasma kallikrein-like; uncharacterized protein loc101743393; uncharacterized protein loc105843019; uncharacterized protein loc101742472; spherulin-2a-like; low calcium response v antigen transferase activity, transferring glycosyl groups mf 0.080585849 udp-glycosyltransferase ugt33r1 precursor; udp-glycosyltransferase ugt33r2 precursor; udp-glycosyltransferase ugt33r2 precursor; udp-glucosyltransferase precursor; udp-glycosyltransferase ugt46a1; phospholipase a2-like isoform x1; cuticular protein glycine-rich 26 precursor; beta-1,4-galactosyltransferase 7-like transferase activity, transferring hexosyl groups mf 0.120013148 udp-glycosyltransferase ugt33r1 precursor; udp-glycosyltransferase ugt33r2 precursor; udp-glycosyltransferase ugt33r2 precursor; udp-glucosyltransferase precursor ; udp-glycosyltransferase ugt46a1; phospholipase a2-like isoform x1 cell outer membrane cc 0.202397931 uncharacterized protein loc101742377; sericin 2 isoform 1 precursor; uncharacterized protein loc101737731; glow molecular mass 30 kda lipoprotein 19g1-like precursor lipase activity mf 0.237835251 lipase member h-a-like; phospholipase a2-like; phospholipase a2-like isoform x1; pancreatic triacylglycerol lipase-like content is 38.31%, and the proportion of n is 0.000037%, which is an acceptable range (<10%) (table s3). the comparison rate of all small fragment reads to the genome is about 98.92%, and the coverage rate is approximately 99.83%, indicating that the reads and the assembled genome have good consistency (table s4). samtools (http://samtools.sourceforge.net/) was used to sort the bwa comparison results by chromosome coordinates, duplicate reads were removed, single nucleotide polymorphisms (snp) calling was performed, and the original results were filtered. the final snp statistics showed that the heterozygous snp ratio of the silkworm genome was 0.0528%, and the homozygous snp ratio was 0.0001%, indicating that the assembly has a high single-base accuracy rate (table s5). the cegma evaluation found that 223 (89.92%) of 248 core eukaryotic genes were complete (table s6), and the busco evaluation statistics of the b. mori genome showed that 1658 orthologous single-copy genes assembled 98.0% of the entire copy genes (table s7), indicating that the assembly was relatively complete. the repetitive sequence library predicted by de novo was integrated with the homologous repetitive sequence database repbase, and the bmj genome was annotated with repeatmasker software. the results showed that the jiangsu silkworm genome contained 56.88% repeat sequences (table s8). a total of 11,509 genes were predicted from the bmj. the protein sequence obtained by gene structure prediction was compared with the known protein library, and 99.8% of the genes were able to predict the function (table s9). gene family cluster analysis and species divergence time estimation in order to reveal the genome characteristics of the bmj, a genome-wide phylogenetic tree combining the sequenced genome of bmj and published genomes of 17 other insect species was constructed, including aae, dme, aga, ame, nvi, tca, lde, pra, aya, bmj, bma, sfr, har, dpl, lhu, pxy, and cel. the number of genes involved in the 143 table 3 kegg enrichment analysis results of common genes in bombyx-specific species maptitle q value genes insect hormone biosynthesis 0.0000971 calexcitin-2-like; uncharacterized protein loc101742377; uncharacterized protein loc101745533; uncharacterized protein loc101743036; farnesol dehydrogenase-like isoform x1; uncharacterized protein loc101738236 retinol metabolism 0.000202086 udp-glycosyltransferase ugt33r1 precursor; udp-glycosyltransferase ugt33r2 precursor; udp-glycosyltransferase ugt33r2 precursor [bombyx mori] udp-glucosyltransferase precursor; udp-glycosyltransferase ugt46a1; 17-beta-hydroxysteroid dehydrogenase 14-like; 17-beta-hydroxysteroid dehydrogenase 14-like metabolism of xenobiotics by cytochrome p450 0.001730997 udp-glycosyltransferase ugt33r1 precursor; udp-glycosyltransferase ugt33r2 precursor; udp-glycosyltransferase ugt33r2 precursor; udp-glucosyltransferase precursor; udp-glycosyltransferase ugt46a1; carbonyl reductase [nadph] 3-like chemical carcinogenesis 0.001730997 udp-glycosyltransferase ugt33r1 precursor; udp-glycosyltransferase ugt33r2 precursor; udp-glycosyltransferase ugt33r2 precursor; udp-glucosyltransferase precursor; udp-glycosyltransferase ugt46a1; carbonyl reductase [nadph] 3-like starch and sucrose metabolism 0.001730997 udp-glycosyltransferase ugt33r1 precursor; udp-glycosyltransferase ugt33r2 precursor; udp-glycosyltransferase ugt33r2 precursor; tpa, putative cuticle protein; udp-glucosyltransferase precursor; udp-glycosyltransferase ugt46a1 comparison of each species is shown in table 1. the gene family cluster analysis of protein-coding genes from these 18 species showed that there were 21904 gene families in the 18 species, and there were 650 single-copy gene families shared by all species (fig. 1a). it could be seen from fig. 1b that the divergence time of the clades of the bmj and bmo was about 3.0 million years. to be precise, the divergence time of bmj and bmo was far less than this length. this may be due to the result of artificial selection in the evolution process. venn analysis results according to the venn diagram (fig. 2), the gene families of spr, har, aya, dpl, pxy, and pra that do not eat mulberry leaves are excluded. there are a total of 217 gene families shared by bmj, bmo, and bma that eat mulberry leaves. go enrichment analysis found that these 217 gene families had a total of 135 functional annotations (table s10). the top five with the highest-ranking were extracellular region, transferase activity, transferring glycosyl groups, transferase activity, transferring hexosyl groups, cell outer membrane, and lipase activity (fig. 3a). the genes involved are shown in table 2. kegg enrichment results showed that these 217 gene families have 111 significant enrichment results (table s11), of which insect hormone biosynthesis, retinol metabolism, metabolism of xenobiotics by cytochrome p450, chemical carcinogenesisand starch and sucrose metabolism had higher enrichment levels (fig. 3b), the genes involved were shown in table 3. positive selection analysis results bmj, bmo, and bma were set as foreground branches, spr, har, aya, dpl, pxy, and pra as background branches for positive selection analysis, and a total of 62 positive selection genes were identified. the functional enrichment analysis of genes subject to positive selection revealed a total of 19 terms in go enrichment results (table s12), but no terms related to eating habits were found (fig. 4a). there were 22 valid results in kegg (table s13). the pathways related to eating habits included olfactory transduction, glycolysis / gluconeogenesis, and galactose metabolism (fig. 4b). the genes involved in the related pathways are shown in table 4, and the amino acid positions where positive selection occurs are shown in table s14. discussion this study used ngs, tgs, and hi-c technology to sequence and assembled the bmj genome and annotated the gene structures and functions. bioinformatics tools were used to compare the gene family information of bmj, bmo, bma, spr, har, aya, dpl, pxy, and pra. it was found that udp-glycosyltransferase ugt46a1 (ugt46a1), 144 fig. 4 go and kegg enrichment results of positive selection genes in bombyx mori jiangsu. (a) go enrichment results of positive selection genes in bombyx mori jiangsu; (b) kegg results of positive selection genes in bombyx mori jiangsu. kegg information refered to https,//www.kegg.jp/kegg/kegg1.htm, the permission was provided by the kanehisa laboratory udp-glycosyltransferase ugt33r1 (ugt33r1), and udp-glycosyltransferase ugt33r2 (ugt33r2) only existed in species that ate mulberry leaves. go and kegg enrichment analysis showed that ugt46a1, ugt33r1 and ugt33r2 were involved in the most food-related pathways and were ranked high, including metabolism of xenobiotics by cytochrome p450, starch, and sucrose metabolism, and pentose and glucuronate interconversions, etc (table s13, s14). udp-glycosyltransferases (ugts) are a superfamily of enzymes present in all life including plants, animals, fungi and bacteria, and even some viruses (bock, 2016). ugt catalyzes the combination of uridine diphosphate (udp) with various exogenous substances such as pollutants, food additives or drugs, and endogenous substances such as hormones, bilirubin, and bile acids, so that the receptor molecules are converted from hydrophobic to hydrophilic to facilitate their clearance (tephly and burchell, 1990). exogenous metabolic enzymes (xmes) such as gt, cytochrome p450 (cyp), carboxylesterase (coe) and glutathione transferase (gst) are involved in the detoxification of exogenous and endogenous active molecules and their removal from the body by catalyzing their biotransformation in inactive metabolites. phase i enzymes (cyp, ce, etc.) catalyze functionalized chemical groups (-oh, -cooh, etc.) to form xenobiotics, and phase ii enzymes (gst, ugt, etc.) catalyze polar groups (glutathione, glucuronic acid, etc.), and their presence and activity against volatile odorant substrates have also been demonstrated in olfactory tissues (nagashima and touhara, 2010). a growing body of evidence demonstrated the primary function of these odor metabolizing enzymes (omes) in olfactory physiology. ome catalyzed odorant metabolism, removed odorants from the receptor surrounding environment to terminate the signal to maintain the highest sensitivity of detection and participated in the synthesis of metabolites, generating additional stimulatory signals that may be modulated (nagashima and touhara, 2010). rane et al. compared the detoxification gene families (p450, gst, and carboxylesterases (cce)) in 65 insect genomes and found a clear relationship between the number of p450, cce, and gst genes and eating preferences. the size of the gene family related to the xenotoxic detoxification in insects may be related to the complexity of their diet and tendency to develop resistance to insecticides (rane et al., 2016). then they examined the genomes of 160 insect species and found that omnivores and herbivores have more detoxification genes, while species that eat simpler tissues such as sap, nectar, and blood have relatively fewer detoxification genes (rane et al., 2019). in olfactory tissues, ugt has been demonstrated to have high glucuronic acid-binding activity for different odorants (jedlitschky et al., 1999) and to respond to a variety 145 table 4 kegg enrichment analysis of positive selection genes in silkworm map id map title q value genes map04740 olfactory transduction 0.34567298 cyclic nucleotide-gated cation channel subunit a-like map00010 glycolysis / gluconeogenesis 0.34567298 aldose 1-epimerase-like map00052 galactose metabolism 0.381133633 aldose 1-epimerase-like of plant allelochemicals (luque and o'reilly, 2002). it was first demonstrated in spodoptera littoralis that odor exposure modulates ugt expression, demonstrating that ugt has a specific role in olfaction (bozzolan et al., 2014). the enzymatic activity of ugt has been detected in insect brain, olfactory tissue, fat body, midgut and other tissues (heydel et al., 2010). these shreds of evidence all showed that ugt plays an important role in various dietary-related aspects such as olfactory, detoxification, and starch and sucrose metabolism in silkworms. therefore, we speculate that ugt46a1, ugt33r1, ugt33r2 may greatly affect the feeding habit of silkworm. through positive selection analysis and go and kegg functional enrichment, it was found that cyclic nucleotide-gated cation channel subunit a-like (cnga) is the most likely gene in positive selection genes to affect the feeding habits of the silkworm. cyclic nucleotide-gated (cng) channels are non-selective cation channels, which were first identified in retinal photoreceptors (haynes and yau, 1985) and olfactory sensory neurons (nakamura and gold, 1987). the natural cng channel is a heterotetramer formed by cnga (a1, a2, a3, a4) and cngb (b1, b3) subunits. cnga plays an essential role in many signal transduction pathways, especially in the visual and olfactory sensory systems. after the surface receptors of competent cells receive light signals or chemical signals, the level of cyclic nucleotides in the cells will change, which in turn regulates the opening or closing of cng channels (varnum and zagotta, 1996). the electrical signals generated by cng channels activate downstream neurons so that mammals can sense changes in light and odor from the outside world (bradley et al., 2005). cng channels are also involved in the transduction cascade of invertebrate photoreceptors and olfactory receptors (baumann et al., 1994). a cdna encoding a putative cng channel has been cloned from drosophila. the n-terminal half of the predicted protein (cngl) has a high degree of sequence similarity with the known cng channel protein. northern blot analysis showed that messenger rna (mrna) corresponding to the size of the cloned cdna was expressed in the head of drosophila. immunolocalization studies have demonstrated that cngl is expressed in the brain. these results indicate that cngl channels may play a role in visual and olfactory information processing in the drosophila nervous system (miyazu et al., 2010). olfactory signal transduction is confirmed to include the following steps: after olfactory stimulation activates the binding of olfactory receptors, the tertiary structure of olfactory receptor proteins changes and the olfactory g protein (golf) α subunit golf is activated. the activation of golf dissociates the β and γ subunits in the g protein and changes the intracellular atp to cyclic adenosine monophosphate (bakalyar and reed, 1990). intracellular cyclic adenosine monophosphate (camp) acts as a second messenger, opening cng channels and causing extracellular ca2+ influx and intracellular ca2+ concentration to increase. this process opens ca2+/clgated channels and causes clefflux, causing the potential difference between the inside and outside of the olfactory receptor neuron membrane, producing membrane depolarization and the formation of nerve impulses (sands and palmer, 2008; restrepo et al., 2015). many odorants have been shown to cause an increase in camp in olfactory neurons (breer, 1993). compounds that activate olfactory cng channels will enhance the sense of smell and can be used to prepare foods that are more palatable to individuals with reduced olfactory function. conversely, compounds that inhibit olfactory cng channels will inhibit smell and can be used to block modulators (zoller et al., 2002). cng is indispensable in the process of olfactory signal transduction, so cng may play an important role in the process of silkworm feeling the attraction of mulberry leaves. it is speculated that cng may affect the silkworm olfactory information processing, thereby affecting the silkworm's feeding. it is known that mutations of phosphorylation sites in cng can interfere with short-term adaptation and change odor sensitivity (o'halloran et al., 2017). we used netphos 3.1 server to predict phosphorylation sites in silkworm cnga and found that the presence of phosphorylation sites had a positive selection, which may be one of the reasons why silkworms changed their sensitivity to odor recognition and were attracted by the unique odor of mulberry leaves and thus eat mulberry leaves. aldose 1-epimerase-like is a crucial enzyme of galactose metabolism (map00052). it is found in plants that may be involved in plant growth and control of defense responses against pathogen invasion and carbohydrate metabolism (sheshukova et al., 2017). however, this enzyme has not been studied in insects, and there are many positively selected amino acid sites, so it is not yet possible to judge whether it has an effect on the feeding habit of the silkworm. all the above inferences need further experimental verification. 146 conclusions this study compared the gene families of sfr, har, aya, dpl, pxy, and pra that did not eat mulberry leaves and bmj, bmo, bma that ate mulberry leaves through resequencing and comparative genomics and found that ugt46a1, ugt33r1, and ugt33r2 are genes unique to the silkworm, and may be involved in the process of silkworm odor recognition. cnga is a positive selection gene, which may be involved in the process of silkworm olfactory transmission. ugt46a1, ugt33r1, ugt33r2 and cnga may be an important factor affecting silkworm feeding. these findings provide important materials and directions for in-depth analysis of the 'silkworm's feeding habits. availability of data and materials all genome data is accessible on-line on the ncbi database through accession number prjna721561 (https://www.ncbi.nlm.nih.gov/bioproject/prjna721 561/). acknowledgements we would like to thank beijing novogene technology co., ltd for assistance in genome sequencing, assembly, annotation and technical support for this article. this work was supported by the national natural science foundation of china (no. 31861143051, 31872425 and 31702186). references ahmad sa, hopkins tl. b-glucosylation of plant phenolics by phenol b-glucosyltransferase in larval tissues of the tobacco hornworm, manduca sexta (l.). insect biochem 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total 455,441,876 455,458,976 241 70 max 12,311,683 21,717,036 number>=2000 241 70 n50 3,751,416 17,263,684 42 12 n60 2,941,879 16,129,543 57 15 n70 2,487,228 15,544,768 73 18 n80 1,833,732 14,680,109 95 21 n90 1,088,538 12,534,500 126 24 ** contig after scaffolding table s3 statistics of base content in silkworm genome number (bp) % of genome a 140500920 30.85 t 104438610 30.83 c 87251660 19.16 g 87250686 19.16 n 17100 0.000037% total (bp) 455458976 gc 174502346 38.31 * gc content of the genome without n table s4 statistics of silkworm genome reads coverage percentage reads mapping rate(%) 98.92 genome average sequencing depth 123 coverage(%) 99.83 coverage at least 4x(%) 99.73 coverage at least 10x(%) 99.62 coverage at least 20x(%) 99.41 note: （1）mapping rate: the ratio of reads compared to the genome; （2）average sequence depth: the average depth of each base on the genome covered by reads; （3）coverage: the percentage of genome covered by reads; （4）coverage at least nx(%): the percentage of genome covered by nx reads. 149 table s5 snp statistics of silkworm genome number percentage(%) all snp 239135 0.0529 heterozygosis snp 238736 0.0528 homology snp 399 0.0001 table s6 cegma assessment results of silkworm genome species complete complete+partial #prots %completeness #prots %completeness bymbox mori 214 86.29 223 89.92 note: （1）complete: core gene >70% assembled; （2）complete + partial: partially assembled core gene; （3）#prots: the number of assembled core genes; （4）% completeness: the percentage of assembled core genes in the core gene library. table s7 busco assessment results of silkworm genome species busco notation assessment results bymbox mori c:98.0%[s:97.2%,d:0.8%],f:0.5%,m:1.5%,n:1658 note: c: complete single-copy buscos ; d: complete duplicated buscos ; f: fragmented buscos ; m: missing buscos ; n: total. table s8 statistical results of repeated sequences type repeat size(bp) % of genome trf 7,535,223 1.65 repeatmasker 252,412,824 55.42 proteinmask 57,221,315 12.56 total 259,068,541 56.88 table s9 statistical results of gene function annotation number percent(%) total 11,509 swissprot 8,809 76.50 nr 11,204 97.30 kegg 8,474 73.60 interpro 11,399 99.00 go 10,541 91.60 pfam 8,523 74.10 annotated 11,482 99.80 unannotated 27 0.20 150 table s10 go enrichment analysis of common genes in bombyx-specific species go_term go_class p value genes extracellular region cc 0.00000956 low molecular mass 30 kda lipoprotein 21g1-like [bombyx mori],low molecular mass 30 kda lipoprotein 19g1-like [bombyx mori], low molecular mass 30 kda lipoprotein 19g1-like [bombyx mori], alcohol dehydrogenase precursor [bombyx mori], uncharacterized protein loc101745266 [bombyx mori],uncharacterized protein loc101740086 [bombyx mori],uncharacterized protein loc101739948 [bombyx mori],serine protease inhibitor 21 isoform x3 [bombyx mori],phospholipase a2-like [bombyx mori],microvitellogenin-like [bombyx mori],cuticular protein rr-2 motif 58 precursor [bombyx mori],seroin 1 precursor [bombyx mori],uncharacterized protein loc101742341 [bombyx mori],rna exonuclease 4-like [bombyx mori],uncharacterized protein loc101746057 [bombyx mori],uncharacterized protein loc101736844 [bombyx mori],actin cytoskeleton-regulatory complex protein pan1 [bombyx mori],microvitellogenin-like [bombyx mori],low molecular 30 kda lipoprotein pbmhp-12-like [bombyx mori],glow molecular mass 30 kda lipoprotein 19g1-like precursor [bombyx mori],low molecular 30 kda lipoprotein pbmhpc-19-like precursor [bombyx mori],plasma kallikrein-like [bombyx mori],uncharacterized protein loc101743393 [bombyx mori],uncharacterized protein loc105843019 [bombyx mori],uncharacterized protein loc101742472 [bombyx mori],spherulin-2a-like [bombyx mori],low calcium response v antigen [bombyx mori] transferase activity, transferring glycosyl groups mf 0.002081246 udp-glycosyltransferase ugt33r1 precursor [bombyx mori],udp-glycosyltransferase ugt33r2 precursor [bombyx mori],udp-glycosyltransferase ugt33r2 precursor [bombyx mori],udp-glucosyltransferase precursor [bombyx mori],udp-glycosyltransferase ugt46a1 [bombyx mori],phospholipase a2-like isoform x1 [bombyx mori],cuticular protein glycine-rich 26 precursor [bombyx mori],beta-1,4-galactosyltransferase 7-like [bombyx mori] transferase activity, transferring hexosyl groups mf 0.005827085 udp-glycosyltransferase ugt33r1 precursor [bombyx mori],udp-glycosyltransferase ugt33r2 precursor [bombyx mori],udp-glycosyltransferase ugt33r2 precursor [bombyx mori],udp-glucosyltransferase precursor [bombyx mori],udp-glycosyltransferase ugt46a1 [bombyx mori],phospholipase a2-like isoform x1 [bombyx mori] cell outer membrane cc 0.010454439 uncharacterized protein loc101742377 [bombyx mori],sericin 2 isoform 1 precursor [bombyx mori],uncharacterized protein loc101737731 [bombyx mori],glow molecular mass 30 kda lipoprotein 19g1-like precursor [bombyx mori] lipase activity mf 0.017171493 lipase member h-a-like [bombyx mori],phospholipase a2-like [bombyx mori],phospholipase a2-like isoform x1 [bombyx mori],pancreatic triacylglycerol lipase-like [bombyx mori] carboxylic ester hydrolase activity mf 0.019188704 lipase member h-a-like [bombyx mori],phospholipase a2-like [bombyx mori],phospholipase a2-like isoform x1 [bombyx mori],pancreatic triacylglycerol lipase-like [bombyx mori] homoiothermy bp 0.019327415 antifreeze protein[bombyx mori],angiomotin-like [bombyx mori],vitelline membrane protein vm26ab-like [bombyx mori],ice-structuring glycoprotein-like [bombyx mori],calpain-b [bombyx mori] ice binding mf 0.019327415 antifreeze protein[bombyx mori],angiomotin-like [bombyx mori],vitelline membrane protein vm26ab-like [bombyx mori],ice-structuring glycoprotein-like [bombyx mori],calpain-b [bombyx mori] response to freezing bp 0.019327415 antifreeze protein[bombyx mori],angiomotin-like [bombyx mori],vitelline membrane protein vm26ab-like [bombyx mori],ice-structuring glycoprotein-like [bombyx mori],calpain-b [bombyx mori] phospholipase a2 activity mf 0.019410108 phospholipase a2-like [bombyx mori],phospholipase a2-like isoform x1 [bombyx mori] phospholipase activity mf 0.023901444 lipase member h-a-like [bombyx mori],phospholipase a2-like [bombyx mori],phospholipase a2-like isoform x1 [bombyx mori] tachykinin receptor signaling pathway bp 0.025561348 uncharacterized protein loc101737931 [bombyx mori],cuticle protein 6.4 [bombyx mori] organonitrogen compound biosynthetic process bp 0.02817645 15-hydroxyprostaglandin dehydrogenase [nad(+)]-like [bombyx mori],collagenase-like [bombyx mori],uncharacterized protein loc105842596 [bombyx mori],uncharacterized protein loc105841445 [bombyx mori],tpa: putative cuticle protein [bombyx mori],uncharacterized protein loc101747020 [bombyx mori],putative cuticle protein cph45 precursor [bombyx mori], uncharacterized protein loc101744973 [bombyx mori] acid phosphatase activity mf 0.028894576 prostatic acid phosphatase-like [bombyx mori],multiple inositol polyphosphate phosphatase 1-like [bombyx mori] 151 interleukin-6 receptor binding mf 0.033333743 alcohol dehydrogenase precursor [bombyx mori] lactose synthase activity mf 0.033333743 phospholipase a2-like isoform x1 [bombyx mori] lactose biosynthetic process bp 0.033333743 phospholipase a2-like isoform x1 [bombyx mori] ferrous iron binding mf 0.033333743 extradiol ring-cleavage dioxygenase-like [bombyx mori] lipid metabolic process bp 0.041910497 lipase 1-like [bombyx mori],lipase 1-like [bombyx mori],lipase member h-a-like [bombyx mori],lipase member h-a-like [bombyx mori],lipase member h-a-like [bombyx mori],phospholipase a2-like [bombyx mori],short-chain dehydrogenease/reductase-like protein [danaus plexippus],phospholipase a2-like isoform x1 [bombyx mori],pancreatic triacylglycerol lipase-like [bombyx mori] structural constituent of cuticle mf 0.054802939 cuticular protein rr-2 motif 60 precursor [bombyx mori],cuticular protein rr-2 motif 58 precursor [bombyx mori],cuticular protein rr-1 motif 9 precursor [bombyx mori],cuticular protein rr-2 motif 124 precursor [bombyx mori] lipid catabolic process bp 0.056445567 phospholipase a2-like [bombyx mori],phospholipase a2-like isoform x1 [bombyx mori] pathogenesis bp 0.06174495 uncharacterized protein loc101740086 [bombyx mori],evm.model.bomo_chr11.201,phospholipase a2-like [bombyx mori],uncharacterized protein loc101742341 [bombyx mori],rna exonuclease 4-like [bombyx mori],uncharacterized protein loc101746057 [bombyx mori],low molecular 30 kda lipoprotein pbmhpc-19-like precursor [bombyx mori],uncharacterized protein loc105843019 [bombyx mori],uncharacterized protein loc101742472 [bombyx mori],low calcium response v antigen [bombyx mori] cell envelope cc 0.064680839 alaserpin-like [bombyx mori],uncharacterized protein loc101742377 [bombyx mori],sericin 2 isoform 1 precursor [bombyx mori],uncharacterized protein loc101737731 [bombyx mori],glow molecular mass 30 kda lipoprotein 19g1-like precursor [bombyx mori] external encapsulating structure part cc 0.064680839 alaserpin-like [bombyx mori],uncharacterized protein loc101742377 [bombyx mori],sericin 2 isoform 1 precursor [bombyx mori],uncharacterized protein loc101737731 [bombyx mori],glow molecular mass 30 kda lipoprotein 19g1-like precursor [bombyx mori] thrombin receptor activity mf 0.0649584 uncharacterized protein loc105842978 [bombyx mori],putative uncharacterized protein ddb_g0282133 [bombyx mori],uncharacterized protein loc101743399 [bombyx mori] thrombin receptor signaling pathway bp 0.0649584 uncharacterized protein loc105842978 [bombyx mori],putative uncharacterized protein ddb_g0282133 [bombyx mori],uncharacterized protein loc101743399 [bombyx mori] adenosine kinase activity mf 0.065561692 collagenase-like [bombyx mori] purine ribonucleoside salvage bp 0.065561692 collagenase-like [bombyx mori] dna transport bp 0.065561692 uncharacterized protein loc101739948 [bombyx mori] pericentriolar material cc 0.065561692 calexcitin-2-like [bombyx mori] cell volume homeostasis bp 0.065561692 uncharacterized protein loc101744296 [bombyx mori] 152 table s11 kegg enrichment analysis of common genes in bombyx-specific species maptitle pvalue genes insect hormone biosynthesis 0.000000952 calexcitin-2-like [bombyx mori],uncharacterized protein loc101742377 [bombyx mori], uncharacterized protein loc101745533 [bombyx mori], uncharacterized protein loc101743036 [bombyx mori] ,farnesol dehydrogenase-like isoform x1 [bombyx mori] ,uncharacterized protein loc101738236 [bombyx mori] retinol metabolism 0.00000396 udp-glycosyltransferase ugt33r1 precursor [bombyx mori] udp-glycosyltransferase ugt33r2 precursor [bombyx mori], udp-glycosyltransferase ugt33r2 precursor [bombyx mori] udp-glucosyltransferase precursor [bombyx mori], udp-glycosyltransferase ugt46a1 [bombyx mori], 17-beta-hydroxysteroid dehydrogenase 14-like [bombyx mori] , 17-beta-hydroxysteroid dehydrogenase 14-like [bombyx mori] metabolism of xenobiotics by cytochrome p450 0.0000601 udp-glycosyltransferase ugt33r1 precursor [bombyx mori], udp-glycosyltransferase ugt33r2 precursor [bombyx mori], udp-glycosyltransferase ugt33r2 precursor [bombyx mori] ,udp-glucosyltransferase precursor [bombyx mori], udp-glycosyltransferase ugt46a1 [bombyx mori] ,carbonyl reductase [nadph] 3-like [bombyx mori] chemical carcinogenesis 0.0000716 udp-glycosyltransferase ugt33r1 precursor [bombyx mori] ,udp-glycosyltransferase ugt33r2 precursor [bombyx mori], udp-glycosyltransferase ugt33r2 precursor [bombyx mori], udp-glucosyltransferase precursor [bombyx mori] ,udp-glycosyltransferase ugt46a1 [bombyx mori] ,carbonyl reductase [nadph] 3-like [bombyx mori] starch and sucrose metabolism 0.0000849 udp-glycosyltransferase ugt33r1 precursor [bombyx mori] ,udp-glycosyltransferase ugt33r2 precursor [bombyx mori] ,udp-glycosyltransferase ugt33r2 precursor [bombyx mori], tpa: putative cuticle protein [bombyx mori],udp-glucosyltransferase precursor [bombyx mori], udp-glycosyltransferase ugt46a1 [bombyx mori] steroid hormone biosynthesis 0.000102613 udp-glycosyltransferase ugt33r1 precursor [bombyx mori], udp-glycosyltransferase ugt33r2 precursor [bombyx mori] ,udp-glycosyltransferase ugt33r2 precursor [bombyx mori], udp-glucosyltransferase precursor [bombyx mori], udp-glycosyltransferase ugt46a1 [bombyx mori] ascorbate and aldarate metabolism 0.000172821 udp-glycosyltransferase ugt33r1 precursor [bombyx mori], udp-glycosyltransferase ugt33r2 precursor [bombyx mori], udp-glycosyltransferase ugt33r2 precursor [bombyx mori] ,udp-glucosyltransferase precursor [bombyx mori], udp-glycosyltransferase ugt46a1 [bombyx mori] porphyrin and chlorophyll metabolism 0.000251556 udp-glycosyltransferase ugt33r1 precursor [bombyx mori], udp-glycosyltransferase ugt33r2 precursor [bombyx mori] ,udp-glycosyltransferase ugt33r2 precursor [bombyx mori], udp-glucosyltransferase precursor [bombyx mori], udp-glycosyltransferase ugt46a1 [bombyx mori] drug metabolism cytochrome p450 0.000417474 udp-glycosyltransferase ugt33r1 precursor [bombyx mori], udp-glycosyltransferase ugt33r2 precursor [bombyx mori] ,udp-glycosyltransferase ugt33r2 precursor [bombyx mori], udp-glucosyltransferase precursor [bombyx mori] ,udp-glycosyltransferase ugt46a1 [bombyx mori] pentose and glucuronate interconversions 0.000655238 udp-glycosyltransferase ugt33r1 precursor [bombyx mori], udp-glycosyltransferase ugt33r2 precursor [bombyx mori] ,udp-glycosyltransferase ugt33r2 precursor [bombyx mori] ,udp-glucosyltransferase precursor [bombyx mori], udp-glycosyltransferase ugt46a1 [bombyx mori] drug metabolism other enzymes 0.002091395 udp-glycosyltransferase ugt33r1 precursor [bombyx mori], udp-glycosyltransferase ugt33r2 precursor [bombyx mori] ,udp-glycosyltransferase ugt33r2 precursor [bombyx mori], udp-glucosyltransferase precursor [bombyx mori], udp-glycosyltransferase ugt46a1 [bombyx mori] fat digestion and absorption 0.002525354 phospholipase a2-like [bombyx mori] ,1-acyl-sn-glycerol-3-phosphate acyltransferase alpha-like [bombyx mori] ,scavenger receptor class b member 1 like protein 15 [bombyx mori], phospholipase a2-like isoform x1 [bombyx mori] tropane, piperidine and pyridine alkaloid biosynthesis 0.003151203 short-chain dehydrogenease/reductase-like protein [danaus plexippus] ,dehydrogenase/reductase sdr family member 2, mitochondrial-like [bombyx mori] 153 ether lipid metabolism 0.006849896 phospholipase a2-like [bombyx mori] ,rrna 2'-o-methyltransferase fibrillarin-like [bombyx mori], phospholipase a2-like isoform x1 [bombyx mori] arachidonic acid metabolism 0.007969487 phospholipase a2-like [bombyx mori] ,phospholipase a2-like isoform x1 [bombyx mori] ,carbonyl reductase [nadph] 3-like [bombyx mori] amoebiasis 0.014034042 serine protease inhibitor 21 isoform x3 [bombyx mori], alaserpin-like [bombyx mori] sco-spondin [bombyx mori], retrotransposon gag domain-containing protein 1-like [ficedula albicollis] linoleic acid metabolism 0.01781794 phospholipase a2-like [bombyx mori], phospholipase a2-like isoform x1 [bombyx mori] transcriptional misregulation in cancer 0.024704244 15-hydroxyprostaglandin dehydrogenase [nad(+)]-like [bombyx mori] ,alcohol dehydrogenase precursor [bombyx mori] ,15-hydroxyprostaglandin dehydrogenase [nad(+)]-like [bombyx mori] ,15-hydroxyprostaglandin dehydrogenase [nad(+)]-like [bombyx mori] glycerophospholipid metabolism 0.026306467 phospholipase a2-like [bombyx mori], rrna 2'-o-methyltransferase fibrillarin-like [bombyx mori], 1-acyl-sn-glycerol-3-phosphate acyltransferase alpha-like [bombyx mori] ,phospholipase a2-like isoform x1 [bombyx mori] rna polymerase 0.028505803 cuticular protein rr-2 motif 60 precursor [bombyx mori], cuticular protein rr-2 motif 58 precursor [bombyx mori], cuticular protein rr-2 motif 124 precursor [bombyx mori] steroid biosynthesis 0.032255476 lipase 1-like [bombyx mori], lipase 1-like [bombyx mori] herpes simplex infection 0.034283226 cuticular protein rr-2 motif 60 precursor [bombyx mori] ,cuticular protein rr-2 motif 58 precursor [bombyx mori] ,caspase-8-like [bombyx mori] .cuticular protein rr-2 motif 124 precursor [bombyx mori] alpha-linolenic acid metabolism 0.034595265 phospholipase a2-like [bombyx mori] .phospholipase a2-like isoform x1 [bombyx mori] salivary secretion 0.065217254 g-protein coupled receptor moody-like [bombyx mori] ,osteopontin ,sericin 1-like isoform x8 [bombyx mori] amino sugar and nucleotide sugar metabolism 0.080679888 nose resistant to fluoxetine protein 6-like [bombyx mori], n-acetylneuraminate lyase-like [bombyx mori],n-acetylneuraminate lyase-like [bombyx mori] vascular smooth muscle contraction 0.086856672 phospholipase a2-like [bombyx mori], g-protein coupled receptor moody-like [bombyx mori] ,phospholipase a2-like isoform x1 [bombyx mori] glycosaminoglycan biosynthesis chondroitin sulfate / dermatan sulfate 0.113961615 beta-1,4-galactosyltransferase 7-like [bombyx mori] betalain biosynthesis 0.133264342 extradiol ring-cleavage dioxygenase-like [bombyx mori] toxoplasmosis 0.135497312 caspase-8-like [bombyx mori] ,cuticular protein glycine-rich 19 precursor [bombyx mori] 154 table s12 go enrichment analysis of positive selection genes in silkworm go_term go_class p value genes signal recognition particle, endoplasmic reticulum targeting cc 0.004766285 signal recognition particle 14 kda protein-like protein[bombyx mori] endoplasmic reticulum signal peptide binding mf 0.004766285 signal recognition particle 14 kda protein-like protein[bombyx mori] ribonucleoside-diphosphate reductase activity, thioredoxin disulfide as acceptor mf 0.004766285 ribonucleoside-diphosphate reductase large subunit [bombyx mori] peptidyl-diphthamide biosynthetic process from peptidyl-histidine bp 0.004766285 diptheria toxin resistance protein[bombyx mori] rna-dna hybrid ribonuclease activity mf 0.009510249 ribonuclease h2 subunit a[bombyx mori] regulation of anion transport bp 0.014231995 26s proteasome non-atpase regulatory subunit 6[bombyx mori] 7s rna binding mf 0.023609241 signal recognition particle 14 kda protein-like protein[bombyx mori] voltage-gated anion channel activity mf 0.028206301 26s proteasome non-atpase regulatory subunit 6[bombyx mori],isovaleryl coenzyme a dehydrogenase[bombyx mori] cellular metal ion homeostasis bp 0.028206301 uncharacterized loc101746868[bombyx mori],flavin-dependent monooxygenase fmo3 precursor[bombyx mori] extrinsic component of intraperoxisomal membrane cc 0.028264944 calcium and integrin binding protein cib[bombyx mori] steroid hormone mediated signaling pathway bp 0.031114487 heparin sulfate o-sulfotransferase[bombyx mori],apterous a splicing isoform type e[bombyx mori] srp-dependent cotranslational protein targeting to membrane bp 0.032898834 signal recognition particle 14 kda protein-like protein[bombyx mori] sulfate transport bp 0.032898834 prestin isoform x2[bombyx mori] sulfate transmembrane transporter activity mf 0.032898834 prestin isoform x2[bombyx mori] regulation of mapk cascade bp 0.037511014 uncharacterized loc101746868[bombyx mori] chitinase activity mf 0.037511014 chitinase domain-containing protein 1[bombyx mori] chitin catabolic process bp 0.037511014 chitinase domain-containing protein 1[bombyx mori] anion transmembrane transporter activity mf 0.038144162 26s proteasome non-atpase regulatory subunit 6[bombyx mori],low quality protein: protein clec16a[bombyx mori],isovaleryl coenzyme a dehydrogenase,prestin isoform x2[bombyx mori] chromosome cc 0.038588406 chromodomain-helicase-dna-binding protein 1[bombyx mori],signal recognition particle 14 kda protein-like protein[bombyx mori],peptidyl-prolyl cis-trans isomerase-like 4[bombyx mori],aldose 1-epimerase-like[bombyx mori] 155 table s13 kegg enrichment analysis of positive selection genes in silkworm maptitle pvalue q value genes glycosaminoglycan biosynthesis heparan sulfate / heparin 0.065807145 0.34567298 heparin sulfate o-sulfotransferase[bombyx mori] protein export 0.086813143 0.34567298 signal recognition particle 14 kda protein-like protein[bombyx mori] spliceosome 0.091743986 0.34567298 u2 small nuclear ribonucleoprotein auxiliary factor 2 [bombyx mori]，inner centromere protein a[bombyx mori] mineral absorption 0.097147109 0.34567298 zinc transporter 1[bombyx mori] insect hormone biosynthesis 0.114123927 0.34567298 cytochrome p450[bombyx mori] notch signaling pathway 0.124163983 0.34567298 protein numb isoform x1[bombyx mori] dna replication 0.137382666 0.34567298 ribonuclease h2 subunit a [bombyx mori] phototransduction fly 0.140657599 0.34567298 sn1-specific diacylglycerol lipase beta[bombyx mori] valine, leucine and isoleucine degradation 0.143920718 0.34567298 isovaleryl coenzyme a dehydrogenase[bombyx mori] olfactory transduction 0.150411676 0.34567298 cyclic nucleotide-gated cation channel subunit a-like[bombyx mori] proteasome 0.163253593 0.34567298 26s proteasome non-atpase regulatory subunit 6[bombyx mori] retrograde endocannabinoid signaling 0.163253593 0.34567298 esn1-specific diacylglycerol lipase beta[bombyx mori] glycolysis / gluconeogenesis 0.166435138 0.34567298 aldose 1-epimerase-like[bombyx mori] galactose metabolism 0.197624847 0.381133633 aldose 1-epimerase-like[bombyx mori] glutathione metabolism 0.230652047 0.415173684 ribonucleoside-diphosphate reductase large subunit[bombyx mori] aldosterone synthesis and secretion 0.270821318 0.452136217 sn1-specific diacylglycerol lipase beta[bombyx mori] tight junction 0.284678359 0.452136217 flavin-dependent monooxygenase fmo3 precursor[bombyx mori] ubiquitin mediated proteolysis 0.350292258 0.482989658 f-box/wd repeat-containing protein 7 isoform x1[bombyx mori] regulation of actin cytoskeleton 0.35528663 0.482989658 rho guanine nucleotide exchange factor 7[bombyx mori] rap1 signaling pathway 0.357770117 0.482989658 flavin-dependent monooxygenase fmo3 precursor[bombyx mori] ribosome 0.393949097 0.497747803 ribosomal protein s7[bombyx mori] pyrimidine metabolism 0.405572284 0.497747803 ribonucleoside-diphosphate reductase large subunit[bombyx mori] 156 table s14 positive selection sites of genes related to feeding habit of bombyx mori jiangsu gene id type p value positive selection site u2 small nuclear ribonucleoprotein auxiliary factor 2[bombyx mori] positive 0.000110954 2 g 0.991**;3 e 0.972*;5 k 0.614;382 c 0.775;404 f 0.933;406 e 0.980*; pdz domain-containing protein 8 isoform x1[bombyx mori] positive 4.37e-11 17 l 0.576;20 a 0.785;34 v 0.779;39 i 0.779;46 k 0.658;52 d 0.586;61 p 0.784;212 t 0.753;215 r 0.696;266 i 0.793;272 v 0.763;295 r 0.802;346 a 0.903;348 g 0.692;355 h 0.508;515 m 0.706;568 s 0.568;642 i 0.776;666 k 0.654;672 e 0.963*;696 w 0.850;732 p 0.666;805 e 0.556;819 r 0.786;839 v 0.999**;840 r 0.884;842 h 0.970*;866 n 0.859;886 i 0.652;923 e 0.790;929 d 0.793;960 c 0.688;971 i 0.811;972 h 0.969*;975 r 0.826;986 f 0.961*;987 a 0.920;988 a 0.998**;989 c 0.999**;990 a 0.998**;991 s 0.995**;992 t 0.813;993 w 0.998**;994 l 1.000**;995 a 0.986*;996 g 0.933;997 g 0.999**;998 g 0.996**;1002 l 0.566;1010 a 0.999**;1012 p 1.000**;1013 p 0.832;1014 d 0.740;1015 a 0.999**;1016 l 0.981*;1017 f 1.000**;1019 a 0.998**;1020 a 0.998**;1021 r 1.000**;1022 d 0.998**;1023 s 1.000**;1024 a 0.796;1026 q 0.974*;1037 h 0.971*;1038 e 0.998**;1039 m 0.929;1040 l 0.993**;1041 l 0.991**;1042 a 0.803;1044 r 0.974*;1045 e 0.997**;1048 s 0.854;1054 c 0.794;1055 a 0.841;1073 f 0.506;1082 c 0.999**;1089 v 0.835;1090 q 0.783;1091 q 0.835;1092 r 0.999**;1093 a 0.995**; flavin-dependent monooxygenase fmo3 precursor[bombyx mori] positive 0.000567658 5 r 0.995**;6 v 0.993**;7 c 0.830;8 v 0.993**;9 i 0.721;11 a 0.796;12 g 0.999**;13 a 0.755;14 a 0.827;15 g 0.999**;16 l 0.991**;17 c 0.746;18 a 0.997**;19 a 0.999**;20 r 0.798;21 h 0.611;22 l 0.708;23 l 0.992**;24 v 0.774;25 e 0.605;26 p 0.995**;28 v 0.749;29 s 0.945;30 q 0.944;31 v 0.995**;32 d 0.529;33 i 0.992**;34 l 0.991**;35 e 0.999**;36 q 0.995**;37 a 0.997**;38 d 0.718;39 q 0.693;40 l 0.952*;41 g 0.999**;42 g 0.790;43 t 0.804;44 w 0.867;45 v 0.859;51 g 0.996**;52 y 0.822;53 d 0.999**;54 d 0.983*;55 f 0.991**;56 g 0.991**;57 l 0.951*;59 i 0.994**;60 h 0.886;61 s 0.997**;62 s 0.840;65 k 0.993**;66 s 0.821;68 l 0.942;99 a 0.736;109 g 0.991**;112 k 0.562;115 k 0.592;121 q 0.993**;137 t 0.884;212 i 0.578;244 v 0.818;251 a 0.740;256 g 0.992**;260 e 0.699;282 d 0.576;284 v 0.990*;328 v 0.691;331 m 0.693;356 a 0.819;372 e 0.738;373 k 0.990*;376 a 0.997**;379 a 0.833;382 a 0.780;385 r 0.767;389 s 0.845;391 l 0.611;393 k 0.988*;395 r 0.953*;396 e 0.986*;397 e 0.658;399 t 0.512;400 i 0.999**;401 r 0.727;404 n 0.608;407 k 0.903;408 d 0.844;411 q 0.992**;415 k 0.716;416 i 0.812;420 n 0.607;421 t 0.999**;422 y 0.805;423 a 0.894;424 i 0.993**;427 i 0.998**;428 s 0.997**;429 n 1.000**;430 g 1.000**;431 r 0.998**;432 q 0.951*; calcium and integrin binding protein cib[bombyx mori] positive 0.000173765 89 q 0.975*;94 r 0.668;166 p 0.787;171 t 0.764;211 q 0.943;212 y 0.999**;213 v 0.954*;214 0.959*;216 1.000**; serine/threonine-protein kinase mig-15 isoform x2[bombyx mori] positive 1.29e-11 332 g 0.965*;556 p 0.882;570 g 0.960*;593 0.661;822 k 1.000**;929 l 0.885;1299 0.975*;1338 0.999**;1387 0.995**;1392 0.862;1410 0.989*;1411 0.966*;1413 0.997**;1419 0.999**;1420 0.999**;1423 0.965*;1425 0.999**; gene12186 positive 1.11e-16 196 a 0.989*;199 d 0.988*;200 n 0.920;201 s 0.889;203 k 0.799;204 p 0.685;206 k 0.999**;207 k 0.974*;407 t 0.888;690 m 0.761;693 s 0.677;737 n 0.691;804 i 1.000**;805 s 0.752;806 l 0.994**;807 f 0.986*; chromodomain-helicase-dna-binding protein 1[bombyx mori] positive 0.001355458 83 s 0.641;99 s 0.652;441 q 0.839;851 i 0.528;1043 m 0.637;1359 q 0.806;1392 n 0.527;1429 k 0.515;1432 k 0.634;1595 k 0.706;1628 a 0.769;1670 e 0.683;1683 0.652;1858 p 0.867; ataxin-10 isoform x1[bombyx mori] positive 1.75e-06 56 t 0.619;82 r 0.801;89 s 0.711;90 y 0.713;95 l 0.623;140 s 0.793;184 t 0.745;214 v 0.702;299 s 0.828;377 a 0.608;394 n 0.599;396 p 0.660;435 n 0.665;439 v 0.669;456 k 0.535;457 f 0.690;461 i 0.887;466 n 0.551;469 g 0.628;477 p 0.819;478 i 0.947;485 l 0.594;488 v 0.556;504 r 0.645;537 n 0.519;559 l 0.619;564 r 0.698;586 l 0.755;587 k 0.538;609 t 0.580;622 v 0.515;663 a 0.526;675 n 0.641;677 l 0.775;678 p 1.000**;679 s 0.999**;680 v 1.000**;681 k 0.960*;682 i 0.997**;684 s 1.000**;685 p 0.817;687 s 0.976*;688 q 0.997**;695 i 0.800;703 a 0.779;721 a 0.980*;727 a 0.867;730 c 0.879;731 w 0.876;732 k 0.996**;733 n 157 0.998**;734 q 0.839;736 n 0.967*;737 k 0.999**;738 k 0.999**;739 q 1.000**;742 e 0.696;744 e 0.627;772 n 0.936;785 s 0.867;787 m 0.950;792 p 0.804;793 v 0.998**;794 d 0.783;795 n 0.995**;796 e 0.611;799 q 1.000**;801 m 0.958*;802 g 0.845;804 t 0.998**;805 l 1.000**;806 h 1.000**;807 t 0.998**;808 d 0.651;809 p 0.999**;810 q 0.699;811 g 1.000**;812 n 0.997**;813 t 0.999**;814 i 1.000**;815 k 0.998**;816 m 0.926;817 v 0.993**;820 s 0.784;822 g 1.000**; 26s proteasome non-atpase regulatory subunit 6[bombyx mori] positive 1.51e-06 39 q 0.958*;60 p 0.660;220 e 0.918;221 r 0.998**;222 h 0.993**;223 a 0.994**;224 l 0.823;225 q 0.798;228 l 0.999**;229 r 0.695;230 r 0.999**;231 q 0.611;232 g 1.000**;233 a 0.998**;234 a 0.760;235 v 0.753;236 q 1.000**;237 a 0.749;238 l 0.814;239 r 0.997**;240 s 0.773;242 f 0.998**;243 p 0.977*;244 e 0.994**;245 l 0.838;246 r 1.000**;248 l 0.999**;251 s 1.000**;253 h 0.991**;254 e 0.738;255 c 1.000**;262 k 0.640;263 s 0.998**;264 l 0.680;265 a 0.999**;278 r 0.917;307 n 0.542;309 a 0.996**;310 d 0.923;311 t 0.999**;312 f 0.933;313 g 0.931;315 t 0.999**;319 v 0.745; transmembrane protein 136-like[bombyx mori] positive 2.40e-09 23 f 0.832;28 y 1.000**;30 w 0.698;32 v 0.579;33 e 0.847;34 e 0.510;35 a 1.000**;74 t 0.880;106 n 0.507;111 c 0.678;149 t 0.685;156 g 0.569;159 e 0.939;165 f 0.870;188 y 0.824;232 s 0.551;233 r 0.732;236 d 0.979*;239 n 0.876;240 l 0.988*;241 c 0.974*;242 n 0.765;243 v 0.609;264 i 0.951*; solute carrier family 35 member c2[bombyx mori] positive 0.005766172 5 k 0.959*;7 e 0.975*;9 l 0.973*;10 s 0.995**;22 n 0.609;29 v 0.585;40 l 0.975*;41 s 0.971*;42 l 0.838;43 i 0.758;44 a 0.977*;45 l 0.844;46 y 0.998**;49 l 0.995**;50 s 0.912;51 i 0.764;52 g 0.967*;61 l 0.999**;62 r 0.711;66 f 0.676;94 g 0.991**;102 f 0.639;105 c 0.834;106 l 0.608;115 v 0.721;186 v 0.535;189 n 0.684;233 s 0.753;235 l 0.648;266 s 0.750;341 r 0.828;355 d 0.733; diptheria toxin resistance protein[bombyx mori] positive 1.11e-06 2 1.000**;4 1.000**;5 0.865;6 0.999**;7 0.783;8 0.991**;9 1.000**;11 0.928;12 0.999**;14 1.000**;15 0.999**;16 1.000**;23 1.000**;24 0.888;26 0.883;27 0.978*;28 0.718;30 0.988*;31 1.000**;33 0.961*;34 0.990*;35 0.912;36 0.866;37 0.999**;38 0.998**;39 0.995**;40 0.875;44 0.900;45 0.815;46 0.822;48 0.547;49 0.999**;50 0.999**;51 0.992**;52 0.999**;55 0.998**;56 0.956*;57 0.951*;58 0.846;59 1.000**;60 0.999**;61 0.999**;62 0.959*;63 0.975*;65 0.999**;66 0.997**;67 0.948;68 0.932;69 0.984*;70 0.994**;71 0.888;72 0.746;73 0.993**;74 0.712;75 0.999**;76 0.783;77 0.993**;78 0.998**;79 0.998**;80 0.628;81 0.992**;82 0.994**;84 0.995**;85 0.836;86 0.855;87 0.999**;91 0.998**;92 1.000**;94 0.994**;95 0.786;96 1.000**;97 0.783;98 0.986*;99 1.000**;100 1.000**;101 0.999**;116 0.629;125 g 0.848;127 a 0.880;128 v 0.807;158 m 0.575;163 e 0.942;185 k 0.528;190 k 0.824;204 a 0.551;226 h 0.618;228 f 0.582;231 d 0.653;255 i 0.777;256 a 0.734;258 s 0.818;269 k 0.509;272 r 0.945;294 v 0.547;295 v 0.625;299 v 0.564;305 d 0.839;316 q 0.625;335 v 0.987*;369 y 0.807;375 v 0.656;384 y 0.676;385 f 0.998**;403 c 0.506;407 h 0.871;412 t 0.746;420 k 0.855;424 t 0.813;427 d 0.504;434 k 0.696;439 k 0.574;446 s 0.991**;450 n 0.807;470 r 0.794;479 p 0.780;486 p 0.855; sentrin-specific protease-like [bombyx mori] positive 4.62e-09 2 k 0.763;3 s 0.987*;4 v 0.803;5 v 0.974*;6 a 0.691;76 n 1.000**;78 r 1.000**;83 i 0.998**;118 l 0.712;120 s 0.574;149 v 0.941;153 e 0.934;164 w 0.699;233 d 0.523;253 s 0.913;435 r 0.547;461 q 0.535;486 i 0.988*;510 t 0.552;578 r 0.612;645 s 0.910;714 a 0.638; signal recognition particle 14 kda protein-like protein [bombyx mori] positive 0.002525675 5 a 0.602;14 t 0.667;16 l 0.998**;17 f 0.898;18 q 0.962*;19 k 0.997**;20 a 0.997**;21 r 0.966*;22 p 0.778;23 t 0.998**;24 g 0.927;25 s 0.998**;26 v 0.747;27 t 0.965*;28 m 0.993**;29 t 0.713;30 m 0.910;31 k 0.991**;36 r 0.999**;37 t 1.000**;40 q 0.997**;57 i 0.627;79 s 0.951*; leucine-rich repeat-containing protein aac1 isoform x1 [bombyx mori] positive 0 3 y 1.000**;5 f 0.610;6 n 0.991**;7 1.000**;8 y 0.992**;11 g 0.897;12 p 0.853;14 n 0.626;16 f 0.997**;17 t 0.992**;30 t 0.783;32 i 0.981*;33 p 0.992**;34 p 0.649;35 p 0.998**;36 0.985*;37 0.967*;75 0.913;96 0.753;97 0.984*;98 p 0.994**;99 p 0.994**;100 p 0.997**;101 p 0.994**;103 f 0.979*;104 f 0.908;105 i 0.765;106 p 0.997**;107 s 0.989*;108 n 0.963*;109 0.993**;111 i 0.851;112 f 0.982*;113 s 0.968*;114 q 0.997**;115 e 0.701;116 v 0.970*;117 t 0.977*;120 e 0.996**;121 f 1.000**;123 q 0.991**;124 t 0.998**;125 158 f 0.999**;127 r 0.607;128 i 0.973*;129 i 0.808;130 p 0.995**;131 p 0.990**;132 d 0.991**;133 q 0.971*;140 e 0.586;142 0.521;143 i 0.993**;144 s 0.539;145 i 1.000**;146 s 1.000**;147 q 0.873;148 v 1.000**;149 f 0.985*;150 h 0.797;152 l 0.997**;153 k 0.783;154 s 0.965*;155 i 1.000**;156 v 0.840;157 t 0.949;159 i 0.780;160 k 0.987*;162 l 0.999**;163 k 0.994**;164 d 0.999**;165 k 0.807;166 n 0.996**;167 e 0.914;168 i 0.909;171 q 0.981*;172 e 0.998**;177 s 0.983*;178 s 0.974*;179 i 0.627;180 s 0.959*;182 e 0.920;183 e 0.682;208 g 0.917;209 t 0.721;306 n 0.707;321 e 0.992**;324 a 0.506;330 n 0.763;333 k 0.953*;405 d 0.699;406 v 0.943;414 i 0.617;423 v 0.829; uncharacterized protein loc101737699 isoform x1 [bombyx mori] positive 0.000362583 11 d 0.533;30 t 0.580;34 t 0.686;47 h 0.775;108 s 0.715;109 s 0.609;111 s 0.694;114 h 0.652;136 h 0.897;141 n 0.532;145 d 0.982*;146 s 0.994**;154 r 0.803;155 s 0.650;157 t 0.995**;158 s 0.676;161 p 0.811;174 q 0.519;212 a 0.730;221 v 0.688;249 g 0.732;250 l 0.785;278 s 0.824;287 p 0.655;371 f 0.995**;372 s 0.665;373 g 0.812;374 s 0.596;375 s 0.994**;376 s 0.997**;379 s 0.999**;380 y 0.994**;385 r 0.991**;386 r 0.916;399 a 0.898;400 y 0.780;402 t 0.744;403 r 0.991**;404 g 1.000**;405 a 0.746;407 v 0.997**;408 r 0.838;409 i 0.999**;411 g 0.989*;412 a 0.776;413 d 0.998**;417 a 0.806;419 c 0.998**;420 s 0.790;421 r 0.997**;422 e 0.998**;423 a 0.956*;425 s 0.765;426 p 0.755;428 s 0.994**;429 s 0.834;430 s 0.997**;431 c 0.994**;433 d 0.997**;434 a 0.995**;436 t 0.640;437 t 0.999**;439 f 0.891;440 w 0.995**;441 n 0.997**;442 k 0.998**;443 k 0.988*;444 s 0.999**;445 w 0.998**;446 k 0.993**;447 k 0.722;448 i 0.994**;449 s 0.995**;451 f 0.998**;452 s 0.805;454 s 0.849;455 n 0.997**;456 s 0.699;457 i 0.996**;458 n 0.992**;459 k 0.990*;461 g 0.995**;462 l 0.792;463 t 0.930;464 g 0.524;498 0.599;499 0.623;501 l 0.983*;502 l 0.993**;503 v 0.991**;505 y 0.995**;506 t 0.703;507 f 0.998**;508 r 0.721;509 r 0.871;510 k 0.993**;511 s 0.988*;512 g 0.984*;513 s 0.779;514 q 0.994**;515 g 0.976*;516 s 0.803;517 q 0.990*;519 k 0.992**;520 r 0.973*;521 r 0.974*;523 e 0.573;524 p 0.831;526 k 0.827;527 c 0.996**;528 d 0.986*;529 a 0.629;530 c 0.788;531 l 0.747;532 s 0.765;534 r 0.673; chitinase domain-containing protein 1 [bombyx mori] positive 1.01e-09 2 k 0.874;4 i 0.827;7 i 0.964*;8 l 0.997**;9 q 1.000**;10 v 1.000**;12 f 0.827;13 l 0.999**;18 v 1.000**;19 c 0.999**;44 n 0.817;48 d 0.674;49 r 1.000**;50 k 0.699;51 l 1.000**;52 v 0.891;53 d 0.819;54 e 0.732;55 a 0.998**;56 p 0.874;57 l 1.000**;58 a 0.899;59 k 0.999**;61 i 0.975*;62 i 0.831;63 k 1.000**;64 y 0.999**;65 h 0.986*;66 a 1.000**;67 t 1.000**;68 y 1.000**;69 h 0.999**;70 q 0.999**;71 d 0.999**;72 v 1.000**;73 t 0.986*;74 t 1.000**;75 r 0.999**;76 n 0.757;77 f 0.901;79 n 1.000**;81 v 1.000**;82 l 1.000**;95 v 0.541;98 t 0.583;124 s 0.739;138 k 0.516;142 s 0.593;146 l 0.646;153 l 0.847;159 a 0.825;221 n 0.992**;224 d 0.514;234 f 0.678;243 f 0.689;280 r 0.695;296 r 0.751;312 a 0.772;326 l 0.997**;351 t 0.723;352 s 0.784;374 k 0.566;385 l 0.645; putative wd repeat domain phosphoinositide-interacting protein [danaus plexippus] positive 0.000110226 65 s 0.964*;205 m 0.981*;251 a 0.978*;269 r 0.809;313 g 0.854;502 g 0.971*;504 l 0.746;512 l 0.515;524 p 0.608;664 r 0.979*; inner centromere protein a [bombyx mori] positive 7.15e-13 2 1.000**;3 1.000**;4 1.000**;5 1.000**;6 0.894;7 1.000**;9 0.971*;10 1.000**;11 1.000**;12 0.985*;13 0.967*;14 1.000**;15 0.935;16 0.999**;17 0.997**;18 1.000**;19 0.987*;20 0.959*;21 0.749;22 0.875;23 0.954*;25 m 1.000**;26 s 1.000**;27 h 0.999**;28 l 1.000**;29 y 1.000**;31 p 1.000**;32 w 1.000**;33 g 1.000**;34 t 0.995**;35 g 0.992**;36 e 0.947;37 k 0.868;38 y 1.000**;39 s 0.903;40 n 1.000**;46 q 0.898;47 t 0.995**;48 r 1.000**;49 n 0.863;50 q 0.993**;51 f 0.970*;52 a 1.000**;53 s 0.997**;54 n 1.000**;55 l 1.000**;58 h 0.940;60 h 1.000**;61 a 0.999**;62 s 1.000**;63 y 1.000**;65 y 0.957*;71 e 1.000**;73 g 0.956*;74 q 0.999**;86 r 0.997**;87 l 0.912;88 r 0.989*;89 s 1.000**;90 p 1.000**;91 s 0.890;92 l 0.960*;93 p 0.964*;94 p 1.000**;95 i 0.878;96 r 0.927;97 n 0.998**;98 v 0.993**;100 e 0.975*;101 t 0.982*;102 e 0.939;104 p 0.701;105 k 0.998**;107 e 0.998**;109 r 1.000**;134 a 0.983*;135 p 0.937;136 v 1.000**;137 l 1.000**;138 n 0.863;139 r 1.000**;140 n 1.000**;141 i 1.000**;143 s 159 1.000**;144 a 1.000**;145 k 0.958*;166 v 0.711;193 e 0.845;194 n 0.604;203 s 0.909;204 n 0.533;209 m 0.826;212 g 0.998**;276 t 0.876;281 v 0.860;321 i 0.871;327 n 0.550;337 s 0.878;349 r 0.907;369 q 0.954*;374 e 0.685;377 l 0.839;399 t 0.819;405 e 0.592;423 k 0.697;425 s 0.694;435 v 0.541;442 k 0.533;465 i 0.930;466 k 0.542;476 a 0.900;479 k 0.833;492 t 0.867;506 l 0.887;512 s 0.664;513 v 0.894;520 a 0.935;521 d 0.614;532 g 0.934;538 e 0.648;539 i 0.806;541 0.600;555 q 0.758;559 n 0.988*;563 s 0.877;564 v 0.888;565 s 0.887;566 l 0.879;570 n 0.602;574 e 0.569;578 t 0.800;579 n 0.566;589 s 0.531;601 d 0.623;610 t 0.666;611 d 0.764;612 k 0.721;614 i 0.687;616 t 0.957*;628 s 0.794;640 h 0.579;660 e 0.783;690 i 0.691;697 q 0.922;698 a 0.991**;716 a 0.507;730 n 0.998**;739 s 0.529;744 q 0.733;763 f 0.937;766 s 0.782;774 n 0.870;776 n 0.877;812 d 0.674;815 s 0.771;816 e 0.570;825 d 0.643;831 k 0.514;838 t 0.822;842 e 0.913;847 k 0.551;862 k 0.692; uncharacterized protein loc101739083 isoform x2 [bombyx mori] positive 0 1 m 0.533;2 p 0.577;3 a 0.574;5 s 0.568;6 0.670;7 i 0.530;8 i 0.522;9 t 0.575;10 y 0.592;11 0.670;12 f 0.571;13 q 0.585;14 d 0.548;15 t 0.547;16 r 0.535;17 n 0.545;19 f 0.558;21 a 0.547;22 v 0.550;23 k 0.609;24 p 0.568;25 0.547;26 s 0.614;27 l 0.534;28 c 0.591;29 t 0.558;30 e 0.519;31 i 0.575;32 a 0.510;33 q 0.556;34 p 0.584;35 v 0.670;36 g 0.548;37 q 0.552;38 c 0.591;39 h 0.545;41 i 0.569;42 h 0.544;43 i 0.532;44 s 0.592;45 t 0.551;47 c 0.559;48 y 0.572;49 y 0.545;50 g 0.566;51 y 0.559;52 h 0.517;53 s 0.521;54 t 0.570;56 t 0.600;57 a 0.628;58 e 0.557;59 n 0.568;60 v 0.524;61 q 0.545;62 n 0.545;63 n 0.568;64 i 0.561;66 c 0.571;67 e 0.554;68 m 0.533;69 p 0.592;70 e 0.508;71 t 0.584;72 p 0.583;73 r 0.542;74 v 0.522;75 v 0.553;76 n 0.521;77 g 0.609;78 s 0.524;79 s 0.570;80 m 0.579;81 l 0.568;82 0.670;83 0.670;84 q 0.670;85 a 0.568;86 g 0.559;87 s 0.556;88 r 0.535;90 r 0.535;91 c 0.512;92 a 0.547;94 n 0.511;95 s 0.548;96 d 0.574;97 y 0.546;98 p 0.577;99 q 0.585;100 s 0.551;101 k 0.542;102 r 0.572;103 i 0.566;104 r 0.608;105 e 0.508;106 e 1.000**;107 g 0.987*;108 1.000**;109 1.000**;110 0.944;111 1.000**;112 0.942;113 0.959*;114 0.670;115 1.000**;116 0.999**;117 0.556;118 0.947;119 1.000**;120 0.980*;121 0.939;122 0.999**;123 m 1.000**;124 v 1.000**;125 p 0.937;126 s 0.999**;127 t 0.994**;128 t 1.000**;129 h 0.981*;130 y 1.000**;131 0.670;132 0.670;133 n 1.000**;134 p 1.000**;135 c 1.000**;136 l 1.000**;137 l 0.994**;138 r 1.000**;139 p 1.000**;140 t 0.985*;141 h 0.990**;142 n 1.000**;143 n 1.000**;144 p 0.991**;145 d 0.995**;146 i 1.000**;147 s 1.000**;148 r 1.000**;149 c 1.000**;150 l 0.999**;151 m 0.999**;152 v 0.999**;153 h 1.000**;154 m 1.000**;155 i 0.999**; eh domain-containing protein 3 isoform x2 [bombyx mori] positive 0 141 p 0.539;323 g 0.795;488 0.961*;489 0.951*;490 0.918;491 0.995**;492 0.970*;493 l 0.916;494 l 0.963*;496 y 0.979*;497 k 1.000**;498 m 1.000**;500 s 1.000**;501 n 0.992**;502 1.000**;503 l 0.940;504 l 1.000**;505 l 1.000**;506 f 0.999**;507 w 0.970*;508 e 0.992**;510 l 0.999**;511 l 0.995**;512 a 0.913;513 s 0.809;514 v 0.991**;515 0.914;516 1.000**;517 1.000**;518 0.991**;519 0.960*;520 0.999**;521 0.981*;522 0.999**;523 0.997**;524 0.912;526 1.000**;527 0.916;529 0.896;530 1.000**;531 0.988*;532 1.000**;533 0.924;534 1.000**;536 0.996**;603 1.000**;605 1.000**;606 1.000**;607 0.511;608 0.916;609 1.000**;610 0.953*;611 0.584;612 1.000**;613 0.957*;614 0.927;617 0.545;618 1.000**;619 0.999**;620 1.000**; protein lin-28 homolog [bombyx mori] positive 0.00076189 25 g 0.987*;26 g 0.994**;27 n 0.515;28 a 0.989*;29 v 0.994**;30 p 0.980*;31 s 0.993**;109 i 0.810;140 s 0.784;143 s 0.995**;144 a 0.782;145 m 0.910;148 q 0.529;151 h 0.533;158 k 0.927;198 i 0.639;216 s 0.983*;217 s 0.688;223 s 0.529;224 q 0.996**;225 p 0.774;227 n 0.974*;229 p 0.589; cyclic nucleotide-gated cation channel subunit a-like [bombyx mori] positive 0 36 0.945;38 0.951*;39 0.962*;40 0.966*;42 0.799;43 0.987*;44 0.938;45 0.955*;46 0.642;114 0.626;115 0.998**;116 0.944;117 0.988*;118 0.761;119 0.998**;516 k 0.569;633 a 0.961*;664 0.965*;730 r 0.660;749 s 0.979*;750 m 0.897;752 i 0.895;753 t 0.677;754 s 1.000**;755 m 0.987*;764 i 0.991**;765 r 0.967*;766 e 0.967*;767 r 1.000**;770 s 1.000**;771 l 0.997**;772 c 1.000**;773 v 0.966*;774 e 0.970*;775 r 0.999**;776 t 1.000**;777 p 160 0.998**;778 s 1.000**;779 l 0.972*;780 l 1.000**;781 h 0.996**;782 i 0.691;783 a 1.000**;784 p 0.998**;785 e 0.961*;786 p 1.000**;787 r 0.977*;788 s 0.997**;789 k 0.997**;790 s 1.000**;791 l 0.970*;792 r 1.000**;793 l 1.000**;794 k 0.997**;795 r 0.978*;796 v 0.660;811 k 0.706; ribosomal protein s7 [bombyx mori] positive 1.90e-13 140 d 1.000**;184 d 1.000**;185 t 0.977*;186 f 0.963*;187 q 1.000**;188 s 1.000**;194 v 0.980*;195 y 0.961*;196 k 0.997**;199 t 0.998**;200 g 1.000**;201 r 1.000**;202 e 1.000**;203 v 1.000**;204 t 0.958*;205 f 0.991**;206 e 1.000**;207 f 0.970*;208 p 1.000**;209 e 1.000**;210 p 1.000**;211 y 0.972*;212 l 1.000**; uncharacterized loc101746868 [bombyx mori] positive 5.17e-13 1 0.582;2 0.582;3 0.591;4 m 0.583;5 t 0.579;6 s 0.586;7 t 0.577;8 v 0.577;9 i 0.575;10 k 0.575;11 y 0.578;12 v 0.578;13 g 0.578;14 r 0.576;15 t 0.579;16 t 0.577;17 d 0.578;18 f 0.578;19 k 0.575;20 g 0.578;21 k 0.575;22 s 0.580;23 l 1.000**;24 w 1.000**;25 e 1.000**;26 i 1.000**;27 v 0.995**;28 g 0.994**;31 k 0.989*;33 c 0.999**;35 v 1.000**;37 r 1.000**;38 i 0.577;39 i 0.576;40 v 0.580;41 r 0.576;42 s 0.579;43 v 0.578;44 f 0.579;45 d 0.583;46 r 1.000**;47 y 1.000**;48 p 1.000**;49 e 0.988*;50 p 1.000**;51 s 1.000**;52 f 1.000**;53 m 1.000**;54 k 1.000**;55 i 0.998**;56 v 0.996**;57 k 0.999**;58 v 1.000**;59 e 1.000**;60 t 1.000**;61 c 1.000**;62 p 1.000**;63 d 1.000**;64 e 0.996**;80 r 0.750;85 i 1.000**;95 p 0.756;103 n 0.644;106 a 0.863;107 k 0.548;112 a 0.506;114 d 0.530;117 t 0.824;135 k 0.842;148 q 0.998**;149 m 0.995**;150 t 0.998**;151 s 0.579;152 l 0.579;153 s 0.592;154 m 0.575;155 p 0.580;156 l 0.580;157 s 0.576;158 y 0.578;159 n 0.575;160 h 0.794;161 s 0.576;162 p 0.578;163 n 0.575;164 r 0.576;165 t 0.581;166 k 0.698;167 r 0.817;168 i 0.575;169 a 0.577;170 v 0.577;171 g 0.615;172 d 0.774;173 e 0.576;174 k 0.744;175 p 0.580;176 n 0.577;177 v 0.735;178 q 0.815;179 f 0.578;180 s 0.581;181 m 0.575;182 g 0.578;183 l 0.579;184 g 0.578;185 k 0.575;186 p 0.578;187 v 1.000**;188 s 1.000**;189 p 1.000**;190 s 0.995**;191 l 0.999**;192 y 0.578;193 e 0.734;194 g 0.578;195 i 0.580;196 p 0.579;197 l 0.578;198 n 0.582; solute carrier family 35 member f5 isoform x2 [bombyx mori] positive 0.000504278 130 v 0.913;183 h 0.842;355 c 0.973*;410 s 0.948;412 q 0.969*;424 l 0.559;498 s 0.952*;499 s 0.994**; ribonuclease h2 subunit a [bombyx mori] positive 5.70e-06 97 s 0.905;123 h 0.533;318 p 0.963*;320 n 0.772;332 i 0.974*;333 s 0.974*;336 f 0.723;337 a 0.952*;338 p 0.969*;339 k 0.822;342 n 0.914;343 s 0.915;346 a 0.996**;429 k 0.847;432 k 0.986*;489 r 0.965*;490 h 0.866;492 t 1.000**;493 t 0.992**;575 n 0.999**;589 a 0.709;591 e 0.998**; nudc domain-containing protein 1 [bombyx mori] positive 4.86e-05 18 h 0.608;205 y 0.987*;332 p 0.693;333 s 0.698;436 m 0.949;534 a 0.999**;535 e 0.999**;536 l 0.982*;537 i 0.579;551 l 0.506;581 0.748;586 0.979*; uncharacterized protein loc101741203 [bombyx mori] positive 1.68e-05 1 0.758;2 m 1.000**;3 m 1.000**;4 n 0.751;5 s 1.000**;6 f 1.000**;7 r 0.978*;8 n 1.000**;9 t 0.996**;10 v 1.000**;11 k 1.000**;12 n 0.755;13 0.758;14 0.758;15 0.758;16 0.758;17 0.758;18 0.758;19 0.758;20 0.758;21 0.758;22 0.758;23 0.758;24 0.758;25 l 0.756;26 r 0.755;27 l 0.976*;28 l 0.852;29 n 0.752;30 f 0.855;31 t 0.753;32 n 0.754;33 v 0.950;34 v 0.755;35 k 0.921;36 s 0.775;37 v 0.756;38 t 0.753;39 e 0.996**;40 r 0.888;41 k 0.761;42 0.757;43 p 0.755;44 i 0.752;45 s 0.757;46 i 0.753;47 i 0.757;48 d 0.756;49 t 0.815;50 p 0.756;51 k 0.751;52 c 0.757;53 k 0.751;54 n 0.753;55 e 0.754;56 i 0.756;57 i 0.790;58 s 0.987*;59 n 0.882;60 v 0.759;61 g 0.754;62 i 0.758;63 l 0.756;64 s 0.758;65 f 0.754;66 a 0.761;67 p 0.755;68 r 0.755;69 s 0.874;70 i 0.782;71 g 0.755;72 v 0.869;73 e 0.753;74 i 0.753;75 s 0.756;76 e 0.753;77 k 0.753;78 p 0.755;79 l 0.757;80 n 0.757;81 s 0.754;82 k 0.758;83 v 0.756;84 k 0.752;85 q 0.753;86 d 0.754;87 p 0.755;88 i 0.754;89 p 0.755;90 y 0.754;91 v 0.756;92 p 0.755;93 i 0.752;94 v 0.968*;95 n 0.751;96 p 0.755;97 r 0.755;98 s 0.756;99 i 0.753;100 l 0.755;101 p 0.755;102 l 0.754;103 i 0.790;104 d 0.946;105 s 0.758;106 g 0.756;107 w 0.756;108 r 0.831;109 k 0.752;110 d 0.963*;111 e 0.753;112 i 0.753;113 g 0.755;114 l 0.755;115 p 0.755;116 s 0.756;117 v 0.756;118 r 0.758;119 q 0.753;120 d 0.753;121 e 0.753;122 i 0.755;123 q 0.753;124 a 0.754;125 a 0.756;126 r 0.753;127 l 0.756;128 i 0.753;129 v 0.754;130 i 0.753;131 r 0.753;132 k 0.753;133 k 0.751;134 k 0.752;135 m 0.750;136 r 0.752;137 k 161 0.751;138 h 0.752;139 q 0.753;140 r 0.753;141 k 0.753;142 k 0.752;143 l 0.754;144 w 0.756;145 k 0.752;146 k 0.755;147 m 0.750;148 r 0.753;149 y 0.762;150 r 0.756;151 w 0.756;152 a 0.756;153 r 0.753;154 i 0.754;155 k 0.752;156 q 0.753;157 r 0.755;158 r 0.755;159 q 0.755;160 q 0.753;161 i 0.756;162 k 0.751;163 e 0.753;164 k 0.751;165 i 0.767;166 f 0.754;167 q 0.753;168 k 0.756;169 e 0.753;170 l 0.754;171 l 0.755;172 a 0.755;173 l 0.755;174 i 0.754;175 k 0.936;176 q 0.778;177 a 0.754;178 n 0.751;179 e 0.753;180 f 0.755;181 s 0.756;182 a 0.754;183 e 0.753;184 a 0.774;185 y 0.754;186 v 0.754;187 n 0.783;188 d 0.772;189 k 0.752;190 i 0.753;191 q 0.756;192 r 0.923;193 a 0.755;194 n 0.752;195 h 0.947;196 t 0.755;197 p 0.755;198 l 0.972*;199 p 0.755;200 t 0.754;201 r 0.756;202 w 0.756;203 r 0.753;204 h 0.754;205 k 0.752;206 r 0.753;207 l 0.756;208 p 0.755;209 e 0.753;210 f 0.755;211 i 0.753;212 i 0.752;213 r 0.755;214 q 0.753;215 l 0.755;216 l 0.754;217 g 0.755;218 i 0.753;219 d 0.754;220 k 0.752;221 k 0.751;222 i 0.754;223 n 0.751;224 y 0.755;225 n 0.752;226 h 0.752;227 s 0.754;228 d 0.752;229 t 0.758;230 y 0.755;231 k 0.751;232 a 0.755; fragile x mental retardation syndrome-related protein 1-like [bombyx mori] positive 1.35e-11 61 v 0.965*;189 q 0.750;295 k 0.512;346 v 0.657;351 r 0.895;376 a 0.891;426 g 0.994**;427 r 0.968*;428 r 0.952*;429 m 0.793;430 t 0.980*;431 p 0.988*;432 e 0.958*;433 l 0.995**;434 g 0.994**;435 e 0.936;436 e 0.988*;437 r 0.982*;438 g 0.791;439 e 0.906;441 g 0.970*;447 y 0.594;449 p 0.849;451 g 0.722;453 r 0.968*;458 n 0.620;486 h 0.987*; cytochrome p450 [bombyx mori] positive 2.41e-05 43 s 0.904;44 a 0.731;49 a 0.592;88 p 0.783;100 k 0.989*;153 d 0.998**;156 r 0.953*;158 h 0.996**;159 q 0.996**;164 d 0.994**;166 n 0.998**;167 n 0.994**;319 n 0.535;388 a 0.524;495 s 0.733;541 s 0.587;551 e 0.746; low quality protein: protein clec16a [bombyx mori] positive 5.95e-06 234 a 0.806;353 s 0.617;634 p 0.640;787 r 0.758;822 a 0.574;1007 r 0.605;1115 n 0.993**;1116 d 0.994**;1129 l 0.567;1131 k 0.993**;1132 d 0.851;1165 s 0.734;1168 e 0.934;1169 d 0.975*;1174 n 0.929;1177 r 0.996**;1189 p 0.857;1191 p 0.678;1192 q 0.962*;1195 p 0.855;1196 0.982*;1198 t 0.750;1199 s 0.989*;1200 s 0.734;1204 v 0.576; uncharacterized protein loc101735700 [bombyx mori] positive 0 46 0.999**;47 0.997**;48 1.000**;49 1.000**;50 1.000**;51 1.000**;52 0.999**;53 0.995**;54 1.000**;55 1.000**;56 1.000**;57 0.963*;58 0.873;60 0.986*;61 1.000**;63 0.989*;64 0.999**;65 1.000**;66 1.000**;73 0.572;165 0.642;233 0.646;246 0.752;256 0.521;394 0.731;588 v 0.600;750 s 0.755;1068 r 0.722;1122 r 0.592;1185 a 0.958*;1217 y 0.698; ring finger protein 113a [bombyx mori] positive 0.006139895 10 s 0.760;52 q 0.915;53 p 0.915;63 a 0.764;70 v 0.566;72 i 0.636;73 d 0.922;112 r 0.671;138 v 0.510;210 s 0.998**;211 d 0.994**;212 y 1.000**;214 h 0.679;215 g 0.858;216 w 0.977*;217 q 0.712;218 l 0.883;219 e 0.670;220 r 0.653;231 a 0.526;241 v 0.616;253 i 0.840;256 n 0.655;257 n 0.752;259 t 1.000**;260 d 0.964*;261 p 0.844;263 v 0.799;264 t 0.996**;265 k 0.711;273 k 0.886;276 l 0.998**;277 d 0.576;278 n 0.975*;280 k 0.944;281 k 0.999**;282 s 0.996**;283 t 0.984*;284 r 0.998**;286 f 0.834;325 d 0.876; retinoblastoma-binding protein 5 homolog [bombyx mori] positive 0 1183 r 0.976*;1331 t 0.998**;1332 t 0.999**;1333 w 1.000**;1334 k 0.968*;1335 k 0.992**;1342 g 1.000**;1343 e 0.977*;1344 y 1.000**;1345 i 1.000**;1346 c 1.000**;1347 a 1.000**;1348 g 1.000**;1349 s 0.987*;1350 a 0.985*;1361 s 1.000**;1362 i 0.998**;1365 l 0.988*;1366 v 1.000**;1367 k 0.972*;1368 i 1.000**;1369 l 0.992**;1370 h 0.981*;1371 g 0.984*;1372 t 1.000**;1373 k 0.999**;1379 d 1.000**;1381 v 1.000**;1382 w 0.999**;1383 h 0.982*;1384 p 0.991**;1385 i 0.979*;1387 p 0.986*;1388 i 0.990**;1389 i 1.000**;1390 a 1.000**;1391 s 1.000**;1392 i 0.989*;1393 s 1.000**;1394 a 1.000**;1431 f 0.981*;1433 v 1.000**;1435 d 1.000**;1436 e 1.000**;1437 d 1.000**;1438 r 1.000**;1439 s 0.985*;1440 i 0.983*;1441 d 1.000**;1442 q 1.000**;1443 t 0.994**;1445 e 0.999**;1446 s 0.991**;1447 r 1.000**;1448 n 0.988*;1449 d 0.999**;1450 e 1.000**;1451 e 1.000**;1453 e 0.989*;1460 v 1.000**;1461 d 0.992**;1462 v 1.000**;1463 t 0.985*;1464 s 1.000**;1465 c 1.000**;1466 e 1.000**;1467 p 0.999**;1468 v 1.000**;1469 a 1.000**;1470 a 1.000**;1471 f 1.000**;1473 s 0.986*;1474 s 162 1.000**;1476 e 1.000**;1477 e 0.998**;1478 g 1.000**;1480 d 1.000**;1481 e 1.000**;1482 n 1.000**;1483 m 0.989*;1485 l 0.850;1486 f 1.000**;1487 l 0.989*;1488 p 0.986*;1489 i 0.989*;1490 a 1.000**;1491 p 0.985*;1492 e 0.992**;1493 i 1.000**;1494 e 0.998**;1495 d 0.999**;1496 p 0.991**;1498 d 1.000**;1500 w 1.000**;1501 a 1.000**;1502 a 0.986*;1503 t 0.985*;1504 q 1.000**;1506 t 0.999**;1507 v 0.987*;1508 t 0.999**;1509 p 0.991**;1510 t 1.000**;1511 e 1.000**;1512 t 1.000**;1513 p 1.000**;1514 e 0.988*;1515 k 0.999**;1516 m 1.000**;1517 e 0.998**;1518 p 1.000**;1519 a 0.991**;1520 a 1.000**;1521 k 1.000**;1522 r 0.999**;1523 p 0.985*;1524 k 0.999**;1525 s 0.990**;1526 k 1.000**;1527 t 1.000**;1528 y 1.000**;1529 d 0.999**;1530 i 0.999**;1531 s 1.000**;1532 l 0.991**;1533 k 1.000**;1534 i 1.000**;1535 a 0.995**;1537 p 0.984*;1538 e 1.000**;1539 q 0.999**;1540 p 0.991**;1541 l 0.999**;1542 a 0.997**;1543 f 1.000**;1555 k 1.000**;1556 n 0.991**;1557 k 1.000**;1558 q 1.000**;1559 a 1.000**;1560 a 1.000**;1561 g 1.000**;1562 s 1.000**;1563 k 0.976*;1565 v 0.987*;1566 a 0.986*;1567 g 1.000**;1568 r 1.000**;1569 p 1.000**;1570 r 0.992**;1571 k 0.991**; iq domain-containing protein g-like [bombyx mori] positive 5.54e-05 66 0.572;69 0.531;75 0.566;76 0.517;87 0.799;88 0.561;89 0.732;91 0.960*;92 0.872;93 0.942;95 0.929;97 0.664;98 0.545;101 0.920;102 0.906;104 0.690;105 0.924;106 0.555;108 0.559;110 0.877;113 0.553;114 0.573;118 0.535;119 0.562;125 0.510;126 0.946;127 0.658;141 0.571;145 0.518;146 0.539;152 0.952*;156 0.957*;164 0.605;168 0.705;172 0.573;175 0.753;179 0.933;194 0.514;217 v 0.680;222 i 0.669;252 y 0.794;253 d 0.662;271 e 0.998**;273 i 0.693;274 k 0.993**;275 m 0.864;277 e 1.000**;278 l 0.996**;281 k 1.000**;292 q 0.665;294 y 0.987*;300 n 0.764;314 i 0.700;348 a 0.882;377 k 0.873; leucine-rich repeat protein shoc-2 [bombyx mori] positive 2.48e-05 2 v 0.999**;3 a 0.999**;4 h 0.691;5 k 0.922;6 y 0.983*;7 a 0.936;9 a 0.935;10 a 0.925;11 a 0.934;12 l 0.988*;14 c 0.999**;15 a 0.999**;16 l 0.925;17 a 0.923;18 g 1.000**;29 a 0.925;30 m 0.978*;31 r 1.000**;32 r 0.994**;33 e 0.897;34 i 0.928;35 s 0.999**;36 p 0.999**;37 c 0.940;38 t 0.999**;40 r 1.000**;41 r 0.999**;43 d 0.893;44 a 0.998**;46 t 0.934;47 g 0.935;48 a 1.000**;49 i 0.998**;50 l 0.949;51 v 0.981*;52 v 1.000**;53 c 0.986*;56 i 0.915;57 n 0.814;58 s 0.920;61 e 0.999**;62 i 0.981*;63 s 0.999**;65 a 0.991**;66 l 0.999**;67 s 0.997**;68 n 0.924;69 k 0.999**;72 p 0.949;90 d 0.899;117 d 0.819;142 m 0.978*;177 v 0.903;189 k 0.838;192 r 0.895;198 a 0.761;211 s 0.881;236 d 0.832;241 i 0.910;246 i 0.731;247 t 0.694;261 k 0.997**;265 q 0.928;270 e 0.897;284 g 0.900;285 a 0.892;291 k 0.885;306 a 0.892;307 e 0.834;325 q 0.885;328 s 0.930;349 l 0.817;372 k 0.890;376 i 0.736;379 n 0.807;380 i 0.878;423 l 0.883;425 h 0.879;427 s 0.935;432 i 0.925;486 v 0.548;493 q 0.878;560 v 0.853;568 v 0.847;569 i 0.878;571 q 0.739;577 v 0.912;584 g 0.921;586 g 0.887;589 m 0.564;592 r 0.888;595 v 0.568;596 d 0.724;603 p 0.884;614 g 0.998**;615 t 0.737;618 g 0.928;623 e 0.816;628 l 0.886;629 p 0.921;631 v 0.751;632 l 0.865;633 e 0.823;636 v 0.911;640 p 0.910;643 t 0.937;644 n 0.830;651 d 0.826;653 r 0.972*;655 m 0.760;657 e 0.830;658 h 0.629;670 d 0.809; ovarian serine protease isoform x1 [bombyx mori] positive 1.63e-09 74 c 0.778;218 k 0.550;236 d 0.649;310 h 0.783;336 g 0.946;369 p 0.613;456 e 0.957*;532 n 0.943;901 m 0.651;1017 p 0.904;1078 n 0.775;1209 t 0.536;1229 e 0.794;1346 e 0.954*;1497 l 0.730;1498 e 0.863;1516 r 0.636;1592 k 0.749;1598 k 0.852;1604 g 0.822;1674 n 0.780;1723 r 0.794;1867 e 0.971*;1884 y 0.840;1900 d 0.935;1933 c 0.940;2045 h 0.833;2055 k 0.949;2072 i 0.548;2125 m 0.675;2138 g 0.800; f-box/wd repeat-containing protein 7 isoform x1 [bombyx mori] positive 0.000126936 134 0.944;135 0.620;342 k 0.906;468 y 0.792;519 a 0.598;561 s 0.891;878 v 0.875; ribonucleoside-diphosphate reductase large subunit [bombyx mori] positive 0.0005413 2 m 1.000**;3 v 1.000**;4 g 0.920;5 k 0.890;6 t 0.995**;7 n 1.000**;8 k 0.999**;11 v 0.999**;13 k 1.000**;14 r 0.965*;15 d 0.933;55 i 0.926;107 s 0.823;110 y 0.805;111 n 0.722;113 i 0.651;121 s 0.658;126 d 0.529;132 i 0.636;134 k 0.544;135 n 0.739;153 y 0.905;229 i 0.605;307 i 0.616;353 n 0.621;355 n 0.739;357 s 0.896;373 e 0.687;377 a 0.874;381 k 0.810;384 q 0.557;386 e 163 0.596;415 y 0.827;446 p 0.683;464 n 0.612;472 n 0.901;483 q 0.813;504 m 0.984*;521 v 0.732;530 e 0.996**;571 q 0.877;582 t 0.553;590 a 0.584;595 k 0.682;607 l 0.841;662 d 0.575;682 k 0.810;735 a 0.849;767 g 0.517;769 v 0.609;778 g 0.821;795 q 0.901; zinc transporter 1 [bombyx mori] positive 5.12e-05 2 a 0.999**;3 m 0.998**;4 k 0.847;5 e 0.976*;6 w 1.000**;7 l 0.661;8 q 0.998**;9 w 0.999**;27 s 0.926;29 r 0.925;30 l 0.984*;32 a 1.000**;33 s 0.999**;35 f 0.970*;37 h 1.000**;38 s 1.000**;42 l 0.583;43 v 0.954*;44 d 0.999**;45 t 0.908;47 h 0.999**;48 s 0.999**;49 l 0.999**;50 c 1.000**;51 r 0.925;52 l 0.899;53 v 0.955*;62 y 0.939;67 a 0.876;71 a 0.717;96 v 0.955*;99 a 0.999**;120 q 0.935;138 l 0.849;184 t 0.999**;185 s 0.827;190 m 0.692;196 s 0.804;200 p 0.750;203 a 0.822;220 i 0.804;248 a 0.951*;257 l 0.520;282 s 0.830;289 s 0.809;317 l 0.693;325 a 0.578;328 d 0.806;339 a 0.651;344 a 0.811;357 t 0.730; aldose 1-epimerase-like [bombyx mori] positive 1.28e-06 157 0.528;159 0.803;163 m 0.986*;164 a 0.654;165 d 0.847;166 n 0.659;171 0.654;172 0.558;173 0.782;174 0.894;176 0.591;178 0.840;181 0.652;184 0.886;188 0.639;194 0.534;205 0.533;210 0.527;211 0.819;212 0.746;420 s 1.000**;427 i 0.583;428 k 0.649;430 e 0.601;431 e 0.994**;432 v 0.965*;434 e 0.844;435 0.513;436 0.511;438 p 0.827;439 e 0.988*;441 0.685;442 p 0.998**;443 k 0.552;453 p 0.851;454 d 0.989*;455 v 0.997**;456 e 0.548;457 l 0.839;461 i 0.924;463 d 0.575;464 g 0.834;465 f 0.834;466 g 1.000**;467 f 0.996**;468 m 0.997**;469 p 0.998**;470 k 0.554;526 0.515;530 e 0.896;583 s 0.998**;584 g 0.858;587 k 0.567;588 k 1.000**;591 k 0.523;593 l 0.619;594 n 0.626;595 e 0.994**;619 a 0.810;620 k 0.621;621 s 0.898;622 p 0.861;623 c 1.000**;624 a 1.000**;625 s 0.852;626 s 1.000**;627 t 0.996**;629 i 1.000**;630 d 0.556;631 i 0.999**;632 v 0.997**;633 r 0.992**;634 r 0.974*;635 y 0.996**;636 t 0.813;644 t 0.995**;646 q 1.000**;649 t 0.860;736 r 0.801;783 t 0.830;793 n 0.769;807 e 0.940;841 a 0.980*;857 i 0.657;858 p 0.801;868 i 0.696;879 q 0.632;891 l 0.588;893 v 0.805;900 m 0.823;954 e 0.989*;976 e 0.766;980 v 0.663;989 g 0.566;996 y 0.878;1029 r 0.884;1030 n 0.626;1031 f 1.000**;1032 p 0.998**;1042 y 0.801;1043 i 0.960*;1044 h 0.742;1046 i 0.993**;1048 y 0.769;1052 i 0.994**; rho guanine nucleotide exchange factor 7 [plutella xylostella] positive 3.61e-09 63 a 0.736;403 0.940;510 k 0.842;511 y 0.963*;513 r 0.826;514 n 0.732;517 0.519;524 0.951*;525 0.798;526 0.621;760 n 0.984*;765 i 0.915;766 p 0.974*;768 r 0.792;772 l 0.511;773 n 0.879;775 d 0.902;779 r 0.631;780 r 0.803;781 s 0.960*;791 t 0.890;794 k 0.985*;797 g 0.950;799 r 0.849;801 q 0.847;802 r 0.749;803 m 0.972*;804 s 0.974*;811 r 0.990*;813 q 0.822;817 e 0.991**;819 c 0.960*;821 r 0.987*;822 s 0.961*;823 l 0.975*;824 c 0.976*;826 s 0.818;828 r 0.630;832 s 0.979*;834 v 0.991**;835 v 0.956*;836 g 0.955*;839 n 0.993**;840 l 0.813;844 d 0.967*;846 q 0.948;847 e 0.977*;859 n 0.986*;861 s 0.985*;864 q 0.962*;865 e 0.969*;866 k 0.593;874 s 0.513;877 n 0.876;878 l 0.695;879 s 0.983*;880 h 0.780;885 n 0.554;890 k 0.968*;894 s 0.982*;895 e 0.607;899 e 0.970*;900 t 0.973*;901 f 0.923;902 e 0.985*;907 c 0.879;909 s 0.948;910 p 0.983*;913 s 0.977*;914 t 0.985*;915 a 0.545;916 r 0.956*;917 t 0.964*;919 c 0.971*;920 v 0.518;921 t 0.527;922 s 0.975*;923 v 0.984*;924 t 0.967*;925 s 0.981*;927 n 0.958*;928 s 0.957*;929 l 0.966*;932 i 0.944;933 d 0.977*;935 s 0.980*;942 t 0.938;943 f 0.991**;944 a 0.864;945 a 0.975*;947 p 0.518;949 l 0.732;950 s 0.566;951 t 0.546;953 q 0.795;956 r 0.628;958 s 0.989*;959 i 0.514;972 i 0.735;973 k 0.991**;974 p 0.982*;975 k 0.998**;976 p 0.976*;977 p 1.000**;978 p 1.000**;979 r 1.000**;980 i 0.992**;982 r 0.998**;983 k 0.911;985 s 1.000**;986 t 0.977*;987 h 0.941;988 l 0.837;989 e 1.000**;990 i 1.000**;991 p 0.997**;992 k 1.000**;993 n 1.000**;994 v 0.982*;995 r 0.999**;996 h 0.999**;997 q 0.987*;1009 e 0.848;1017 y 0.939;1022 y 0.963*;1023 r 0.982*;1025 n 0.851;1027 e 0.976*;1030 s 0.793;1031 m 0.767;1032 k 0.642;1033 r 0.992**;1036 r 0.533;1038 n 0.903;1042 n 0.975*;1095 h 0.945;1097 w 0.960*;1098 m 0.635; apterous a splicing isoform type e [bombyx mori] positive 0 1 0.628;2 0.628;3 0.628;4 0.628;5 0.628;6 0.628;7 0.628;8 0.628;9 0.629;10 0.628;11 0.628;12 0.628;13 0.628;14 0.628;15 0.628;16 0.628;17 0.628;18 0.628;19 0.628;20 0.628;21 0.628;22 0.628;23 0.628;24 0.628;25 0.628;26 164 0.628;27 0.628;28 0.630;29 0.628;30 0.628;31 0.628;32 0.628;33 0.628;34 0.628;35 0.628;36 0.628;37 0.628;38 0.628;39 0.628;40 0.628;41 0.628;42 0.628;43 0.628;44 0.628;45 0.628;46 0.628;47 0.628;48 0.628;49 0.628;50 0.628;51 0.628;52 0.628;53 0.628;54 0.628;55 0.628;56 0.628;57 0.628;58 0.628;59 0.628;60 0.628;61 0.628;62 0.628;63 0.628;64 0.628;65 0.628;66 0.630;67 0.628;68 0.628;69 0.628;70 0.628;71 0.630;72 0.628;73 0.630;74 0.628;75 0.628;76 0.628;77 0.628;78 0.628;79 0.628;80 0.628;81 0.628;82 0.628;83 0.628;84 0.628;85 0.628;86 0.628;87 0.628;88 0.628;89 0.628;90 0.628;91 0.628;92 0.628;93 0.628;94 0.628;95 0.628;96 0.628;97 0.628;98 0.628;99 0.628;100 0.628;101 0.628;102 0.628;103 0.628;104 0.628;105 0.628;106 0.628;107 0.628;108 0.628;109 0.628;110 0.628;111 0.628;112 0.628;113 0.628;114 0.628;115 0.628;116 0.628;117 1.000**;118 0.628;119 1.000**;120 1.000**;121 0.997**;122 0.628;123 0.628;124 0.628;125 0.628;126 0.628;127 0.628;128 0.997**;129 0.997**;130 0.628;131 0.628;132 0.628;133 0.630;134 0.628;135 0.628;136 0.628;137 0.628;138 0.628;139 0.628;140 0.628;141 0.628;142 0.628;143 0.628;144 0.628;145 0.628;146 0.628;147 0.628;148 0.628;149 0.628;150 m 0.513;151 r 0.570;152 a 0.571;153 r 0.546;155 l 0.529;156 v 0.578;157 f 0.528;159 v 0.537;160 h 0.537;161 c 0.572;162 f 0.528;163 s 0.531;164 c 0.529;165 a 0.529;166 l 0.590;167 c 0.529;168 s 0.581;169 t 0.531;170 p 0.522;171 l 0.581;172 n 0.544;173 k 0.550;174 g 0.530;176 t 0.531;177 f 0.596;178 g 0.530;179 i 0.524;180 r 0.570;182 s 0.620;183 a 0.523;184 v 0.532;185 y 0.576;186 c 0.529;187 r 0.532;188 l 0.577;189 h 0.537;190 y 0.528;191 e 0.508;192 t 0.575;193 m 0.513;194 p 0.570;195 e 0.545;196 y 0.528;197 g 0.581;198 a 0.570;199 h 0.537;200 m 0.513;201 a 0.524;202 v 0.537;203 p 0.527;204 g 0.577;205 p 0.522;206 p 0.527;207 q 0.543;208 m 0.513;209 c 0.572;210 p 0.573;211 g 0.577;212 p 0.566;213 y 0.528;214 0.628;215 s 0.529;216 g 0.581;217 p 0.527;218 p 0.573;219 a 0.527;220 g 0.534;221 p 0.566;223 y 0.576;224 p 0.522;225 p 0.527;226 y 0.528;227 p 0.527;228 s 0.551;229 p 0.527;231 f 0.528;232 s 0.531;233 r 0.576;234 v 0.532;236 t 0.534;237 d 0.540;238 v 0.537;239 p 0.567;240 k 0.550;241 g 0.530;242 p 0.527;243 f 0.645;244 f 0.569;246 g 0.530;247 g 0.527;248 s 0.572;249 a 0.529;250 p 0.522;251 p 0.573;252 p 0.527;253 r 0.588;254 q 0.543;255 k 0.550;256 g 0.581;257 r 0.527;258 p 0.570;259 r 0.588;260 k 0.550;261 k 0.550;262 k 0.514;263 p 0.570;264 k 0.514;265 d 0.540;266 q 0.543;268 i 0.524;269 m 0.513;270 t 0.526;271 a 0.571;273 l 0.545;274 d 0.628;275 l 0.628;276 n 0.628;277 a 0.628;278 e 0.635;279 y 0.628;280 l 0.628;281 e 0.628;282 m 0.628;283 g 0.628;284 f 0.628;285 r 0.628;286 g 0.628;287 g 0.628;288 g 0.628;289 g 0.679;290 l 0.628;291 g 0.628;292 t 0.628;293 t 1.000**;294 s 0.996**;295 r 1.000**;296 t 0.575;297 k 0.996**;298 r 0.997**;299 m 0.999**;300 r 0.985*;301 t 1.000**;302 s 1.000**;303 f 1.000**;304 k 1.000**;305 h 1.000**;306 h 1.000**;307 q 1.000**;308 l 1.000**;309 r 1.000**;310 t 1.000**;311 m 1.000**;312 k 0.996**;313 s 1.000**;314 y 0.997**;315 f 0.997**;316 a 1.000**;317 i 0.996**;318 n 1.000**;319 h 0.628;320 n 0.628;321 p 1.000**;322 d 1.000**;323 a 1.000**;324 k 1.000**;325 d 1.000**;326 l 1.000**;327 k 1.000**;328 q 1.000**;329 l 0.994**;330 s 0.626;331 q 1.000**;332 k 0.998**;333 t 0.999**;334 g 1.000**;335 l 1.000**;336 p 0.996**;337 k 0.996**;338 r 1.000**;339 v 1.000**;340 l 1.000**;341 q 1.000**;342 v 0.996**;343 w 0.542;344 f 0.996**;345 q 0.628;346 n 0.628;347 a 0.628;348 r 0.628;349 a 0.628;350 k 0.628;351 w 0.542;352 r 1.000**;353 r 0.996**;354 m 0.999**;355 v 0.556;356 t 1.000**;357 k 1.000**;358 q 1.000**;359 e 0.997**;360 n 0.995**;361 k 0.628;362 m 0.996**;363 a 0.999**;365 k 1.000**;366 c 1.000**;367 s 0.997**;368 p 1.000**;369 d 0.997**;370 g 0.996**;371 s 1.000**;372 l 0.554;373 e 1.000**;374 m 1.000**;375 d 0.997**;376 m 1.000**;377 y 1.000**;378 h 1.000**;379 g 1.000**;380 p 1.000**;381 m 0.513;382 g 1.000**;383 s 1.000**;384 i 165 1.000**;385 q 0.999**;386 s 1.000**;387 l 1.000**;388 p 0.628;389 p 0.628;390 h 0.628;391 s 0.628;392 p 0.628;393 p 0.628;394 y 0.628;395 s 0.628;396 v 0.628;397 m 0.628;398 g 0.628;399 g 0.628;400 p 0.628;401 p 0.628;402 s 0.628;403 p 0.628;404 n 0.628;405 s 0.628;406 l 0.628;407 d 0.648;408 c 0.572;409 p 1.000**; e3 ubiquitin-protein ligase ring1 [bombyx mori] positive 0.000101585 390 r 0.703;443 t 0.936;446 v 0.687;494 s 0.988*;567 r 0.672;578 a 0.587;587 k 0.547;647 y 0.997**;648 l 0.770;664 y 0.995**;667 l 0.963*;668 n 0.914;669 f 0.691;671 i 0.995**;677 p 0.997**;682 p 0.738;693 n 0.700;699 v 0.739;700 n 0.913;701 k 0.991**;703 l 0.757;704 e 0.997**;705 m 1.000**;706 y 0.995**;712 t 0.987*; sn1-specific diacylglycerol lipase beta [bombyx mori] positive 2.22e-16 3 0.808;4 0.995**;5 0.994**;6 0.805;7 0.913;8 1.000**;9 0.816;10 1.000**;11 0.999**;13 0.905;15 1.000**;16 0.991**;17 0.939;18 1.000**;20 1.000**;21 1.000**;22 0.896;23 1.000**;25 0.999**;26 0.922;29 1.000**;30 0.987*;31 0.999**;33 1.000**;36 0.999**;291 e 0.866;369 r 0.609;385 s 0.735;430 a 0.924;481 f 0.585;487 a 0.949;619 v 0.563;635 k 0.962*;662 r 1.000**;663 m 0.999**; gene3463 positive 2.97e-05 2 0.978*;3 1.000**;4 1.000**;5 0.971*;6 0.724;7 0.783;8 0.999**;9 0.996**;10 0.822;11 0.894;12 0.825;14 0.787;16 0.999**;17 0.998**;18 0.999**;19 1.000**;20 0.998**;21 0.837;22 0.958*;23 0.998**;24 0.981*;39 0.660;40 0.988*;41 0.839;42 0.848;44 0.907;45 0.999**;47 1.000**;102 0.797;105 0.784;106 0.997**;140 0.942;141 0.999**;142 0.997**;143 0.997**;206 e 0.682;251 t 0.994**;276 q 0.743;281 m 0.911;289 i 0.506;294 s 0.779;302 s 0.748;365 k 0.546;366 a 0.575;371 l 0.826;378 k 0.918;379 c 0.996**;380 r 0.994**;396 p 0.786;415 t 0.653;435 l 0.570;439 h 0.817;469 n 0.507;477 d 0.697;478 t 0.770;484 v 0.687; peptidyl-prolyl cis-trans isomerase-like 4 [bombyx mori] positive 0.00025426 64 q 0.857;333 h 0.643;337 e 0.948;464 0.759;612 0.753;720 0.660;738 s 0.806;747 * 0.707;748 0.999**;751 0.741;756 0.843;766 0.540; tetratricopeptide repeat protein 14 homolog isoform x2 [bombyx mori] positive 0.000105176 91 l 0.901;98 t 0.745;174 t 0.657;576 p 0.757;648 g 0.826;788 s 0.847;789 p 0.883;814 s 0.512;815 v 0.826;830 r 0.627;846 p 1.000**;847 s 0.953*;848 p 0.809;856 s 0.976*;858 r 0.867;859 r 0.996**;860 a 0.981*;1030 0.937;1112 0.847;1230 0.588; lim/homeobox protein lhx3 isoform x4 [bombyx mori] positive 0.000284942 7 y 0.995**;8 p 0.997**;9 g 0.835;10 v 0.802;11 e 0.886;13 a 0.816;15 l 0.994**;31 d 0.994**;33 r 0.996**;34 v 0.828;35 p 0.998**;36 p 0.771;37 i 0.828;38 q 0.996**;39 l 0.997**;40 e 0.998**;47 l 0.795;49 e 0.601;51 f 0.758;63 s 0.743;104 m 0.771;106 s 0.777;126 k 0.739;133 a 0.988*;297 m 0.697;327 e 0.608;340 g 0.791;370 a 0.738;385 l 0.934;386 v 1.000**;387 y 1.000**;436 p 0.676;437 h 0.622;438 h 0.989*;440 s 0.997**;441 p 0.784;442 r 0.513; transmembrane protein 14c [bombyx mori] positive 2.75e-10 3 v 0.852;6 l 0.885;19 i 0.922;74 m 0.996**;88 r 0.954*;89 y 1.000**;90 y 1.000**;91 n 1.000**;92 g 1.000**;93 r 1.000**;94 k 0.997**;103 c 1.000**;104 l 0.988*;105 s 0.982*;106 v 1.000**;107 g 0.980*;108 m 0.962*;109 i 0.998**;111 k 1.000**;112 l 0.977*;113 l 1.000**;114 v 1.000**;116 n 1.000**;118 g 0.969*;119 s 0.978*;121 s 0.994**;122 p 0.989*;123 v 0.969*;124 p 1.000**;125 v 0.973*;126 k 1.000**;127 t 0.997**;128 g 0.991**; tpa: putative cuticle protein [bombyx mori] positive 0 1 0.732;2 0.732;3 0.732;4 m 0.659;5 q 0.570;6 s 0.612;7 m 0.717;8 i 0.946;9 f 0.564;11 a 0.647;15 c 0.549;18 q 0.569;19 a 0.625;23 a 0.690;26 p 0.630;29 i 0.564;30 q 0.587;32 s 0.552;33 p 0.630;34 d 0.546;35 g 0.596;36 k 0.646;42 p 0.630;43 e 0.522;44 v 0.641;47 a 0.606;48 k 0.629;51 h 0.584;52 y 0.662;54 a 0.647;56 a 0.647;57 q 0.587;59 s 0.612;60 s 0.608;61 g 0.625;62 h 0.584;64 a 0.647;65 w 0.662;66 a 0.677;68 0.732;69 0.732;70 0.732;71 0.732;72 0.732;73 g 0.649;74 a 0.523;76 y 0.732;77 l 0.732;78 g 0.732;79 a 0.732;80 g 0.732;81 v 0.732;82 a 0.706;84 g 0.762;85 a 0.686;86 g 0.557;87 a 0.647;90 g 0.732;91 d 0.732;93 a 0.606;94 p 0.610;95 g 0.636;97 g 0.997**;98 l 1.000**;99 l 1.000**;100 k 1.000**;101 y 1.000**;102 g 1.000**;103 p 1.000**;104 a 0.629;105 p 0.997**;106 l 0.994**;107 a 0.997**;108 h 0.651;109 d 0.732;110 g 0.521;111 r 0.631;112 v 0.997**;113 v 0.997**;114 d 0.732;115 t 1.000**;116 p 1.000**;117 e 0.999**;118 v 1.000**;119 a 0.997**;120 166 h 1.000**;121 l 0.998**;122 k 1.000**;123 a 1.000**;124 a 0.997**;125 h 0.995**;126 i 1.000**;127 s 1.000**;128 a 1.000**;129 l 1.000**;130 s 0.996**;131 a 0.998**;132 a 1.000**;133 h 0.995**;134 a 0.998**;135 s 0.996**;136 a 1.000**;137 s 0.997**;138 h 0.999**;139 g 0.732;140 a 0.732;141 0.732;142 0.732;143 0.732;144 0.732;145 0.732;146 l 0.732;147 a 0.732;148 h 0.732;149 a 0.732;150 g 0.732;151 a 0.800;152 h 0.734;153 a 0.732;154 p 0.732;155 l 0.732;156 a 0.732;157 y 0.732;158 a 0.732;159 a 0.753;160 p 0.732;161 l 0.732;162 g 0.732;163 h 0.732;164 g 0.732;165 a 0.992**;166 g 0.995**;167 y 0.954*;168 g 0.744;169 a 0.996**;170 g 0.596;171 a 1.000**;172 g 0.732;173 y 0.732;174 a 0.732;175 a 0.732;176 g 0.732;177 y 0.732;178 g 0.732;179 k 0.646;180 w 0.662;181 t 1.000**;182 g 1.000**;184 q 1.000**;185 a 0.996**;186 h 0.996**;187 i 0.998**;188 q 1.000**;189 l 0.732;190 t 0.732;191 h 0.732;192 d 1.000**;193 g 1.000**;194 q 1.000**;195 f 1.000**;196 v 0.626;197 v 1.000**;198 d 1.000**;199 t 0.536;200 p 0.539;201 e 0.998**;202 v 1.000**;203 q 1.000**;204 h 1.000**;205 a 0.998**;206 r 0.732;207 a 0.998**;208 s 0.993**;210 l 1.000**;211 s 0.994**;212 q 0.999**;213 y 1.000**;214 a 0.924;215 a 0.991**;216 a 0.997**;218 h 1.000**;219 a 1.000**;220 a 0.995**;221 a 1.000**;222 a 1.000**;223 a 1.000**;224 0.732;225 0.732;226 0.732;227 0.732;228 p 0.539;229 e 1.000**;230 e 1.000**;231 p 1.000**;232 w 0.999**;233 a 1.000**;235 h 1.000**;236 g 0.996**;237 0.732;238 0.732;239 0.732;240 h 0.732;241 0.732;242 0.732;243 g 0.998**;244 w 1.000**;246 0.732;247 0.732;248 0.732;249 0.732;250 0.732;251 0.732; uncharacterized protein loc105381380, partial [plutella xylostella] positive 0.002199021 11 0.540;13 0.994**;14 0.998**;15 0.724;16 0.995**;18 0.557;19 0.671;48 0.831;50 0.808;51 0.994**;52 0.992**;54 0.811;82 0.548;94 0.529;103 0.923;104 0.945;106 0.528;108 0.811;135 0.528;136 0.827;137 0.922;138 0.713;139 0.739;140 0.994**;141 0.997**;142 0.901;143 0.996**;145 0.995**;147 0.740;148 0.998**;151 0.997**;152 1.000**;284 m 0.685;315 d 0.972*;344 g 0.611;346 c 0.995**;347 s 0.999**;348 p 0.827;349 k 0.733;358 l 0.681;359 p 0.993**;360 p 0.996**;363 0.989*;386 0.996**;387 0.606;388 0.989*;389 0.997**;399 0.503;438 d 0.997**;439 i 0.715;440 r 0.996**;442 g 0.997**;443 l 0.756;444 m 0.994**;446 m 1.000**;447 g 0.997**;449 d 0.993**;450 g 0.984*;451 v 0.995**;453 g 0.994**;454 p 0.783;455 f 0.792;456 g 0.995**;457 d 0.997**;458 d 0.993**;464 d 0.989*;466 a 0.993**;467 r 0.962*;468 g 0.995**;469 l 0.815;470 s 0.826;482 e 0.980*;484 d 0.552;486 e 0.996**;496 a 0.806;499 f 0.742;500 q 0.845;507 t 0.996**;512 n 0.995**;513 d 0.556;515 r 0.999**;517 e 0.647;518 g 0.993**;519 i 0.768;520 i 0.725;521 s 0.995**;533 p 0.750;535 r 0.988*;537 g 0.996**;538 l 0.992**;539 i 0.984*;540 i 0.754;545 p 0.997**;546 y 0.991**;548 t 0.704;549 t 0.998**;550 v 0.795;551 i 0.990*;555 t 0.818;556 a 0.991**;557 r 0.995**;558 d 0.673;560 e 0.995**;561 s 0.641;562 g 0.996**;563 d 0.977*;576 l 0.824;577 s 0.996**;578 g 0.808;580 s 0.847;584 n 0.899;585 r 0.923;586 s 0.995**;587 e 0.513;589 d 0.987*;591 r 0.993**;592 r 0.726;593 g 0.996**;595 r 0.979*;596 q 0.843;597 m 0.999**;598 s 0.993**;599 g 0.990**;600 y 0.707;601 r 0.963*;618 e 0.968*;619 s 0.770;620 e 0.968*;621 t 0.846;622 d 0.996**;623 s 0.879;624 q 0.732;625 h 0.663;626 i 0.715;627 a 0.996**;628 t 0.995**;630 h 0.727;631 k 0.999**;633 a 0.989*;634 e 0.998**;635 e 0.944;636 c 0.997**;637 e 0.997**;638 g 0.822;640 s 0.994**;641 n 0.723;660 l 0.660;662 q 0.994**;663 e 0.968*;664 r 0.572;665 g 0.794;666 p 0.842;667 e 0.715;668 0.903;669 g 0.718;670 s 0.513;671 e 0.981*;672 r 0.997**;673 r 0.973*;674 t 0.894;675 s 0.998**;676 g 0.992**;677 d 0.537;679 a 0.996**;680 f 0.770;681 p 0.994**;682 c 0.997**;683 s 0.999**;684 n 0.998**;685 s 0.977*;714 e 0.971*;725 s 0.713;726 l 0.726;727 l 0.993**;728 v 0.989*;729 p 0.834;730 q 0.843;731 m 0.989*;732 l 0.870;733 c 0.770;734 r 0.992**;735 a 0.870;736 r 0.995**;737 a 0.813;738 l 0.772;739 v 0.996**;740 d 0.673;741 y 0.727;743 p 0.806;745 i 0.660;746 y 1.000**;758 i 0.995**;759 i 0.822;760 e 0.647;761 v 0.735;762 i 0.731;763 n 0.525;765 n 0.764;766 a 0.806;767 s 0.998**;768 g 0.976*;797 s 0.999**;798 r 1.000**;800 s 0.884;801 k 0.996**;831 m 0.537;834 a 0.761;854 y 0.771;855 l 167 0.999**;856 g 0.818;857 i 0.618;858 m 0.887;861 e 0.990*;862 h 0.993**;864 t 0.994**;867 l 0.723;868 a 0.997**;869 a 0.712;870 v 0.998**;878 c 0.904;881 g 0.897;882 e 0.823;888 g 0.996**;890 s 0.999**;891 s 0.995**;892 s 0.995**;893 e 0.515;894 g 0.995**;898 p 0.960*;899 f 0.770;901 r 0.809;903 q 0.872;904 f 0.727;936 h 0.857;962 i 0.779;963 e 0.997**;964 r 0.981*;965 y 0.998**;966 p 0.997**;977 s 0.630;999 k 0.994**;1000 f 0.770;1001 v 0.784;1002 a 0.800;1004 g 0.621;1005 e 0.524; isovaleryl coenzyme a dehydrogenase [bombyx mori] positive 2.71e-11 212 p 0.600;380 g 0.951*;391 n 0.938;392 g 0.993**;394 i 0.959*;395 n 1.000**;396 d 1.000**;397 y 0.945;398 p 0.954*;399 t 1.000**;402 g 1.000**;403 r 1.000**;405 l 0.946;406 r 0.928;407 d 0.917;408 a 0.949;409 k 0.960*;410 l 0.997**;411 y 1.000**;418 e 1.000**;420 g 0.948;421 a 1.000**;422 g 0.968*;423 t 0.959*;425 e 1.000**;426 v 0.991**;427 r 0.947;428 r 1.000**;429 m 0.997**;430 l 0.992**;431 i 0.992**;435 l 0.992**;436 n 1.000**;437 s 0.866;440 k 0.945; methyltransferase-like protein 16 homolog isoform x1 [bombyx mori] positive 7.05e-05 61 c 0.837;71 e 0.657;156 k 0.830;189 q 0.998**;234 k 0.985*;307 v 0.558;417 d 0.999**;418 v 0.736;479 p 0.626;505 g 0.840; prestin isoform x2 [bombyx mori] positive 0 20 e 0.703;21 g 0.878;25 t 0.984*;26 p 0.984*;27 d 0.699;29 e 0.991**;31 e 0.990*;32 k 0.782;33 l 0.957*;34 n 0.998**;35 p 0.998**;37 g 0.911;39 w 0.763;276 l 0.678;309 l 0.653;431 a 0.692;482 s 0.630;714 s 0.944; protein numb isoform x1 [bombyx mori] positive 0 127 v 0.685;242 g 0.924;243 l 0.995**;244 t 0.828;279 a 0.506;280 d 0.631;281 a 0.616;340 a 0.504;366 f 0.929;367 a 1.000**;369 l 1.000**;370 n 1.000**;371 n 0.946;372 s 0.994**;391 p 1.000**;392 s 1.000**;395 e 1.000**;396 r 0.706;397 q 0.980*;398 r 0.780;399 a 0.909;403 g 1.000**;404 s 1.000**;424 l 0.866;425 p 0.997**;427 v 0.658;428 v 0.594;429 k 0.995**;430 p 0.936;431 v 0.858;435 0.999**;436 0.987*;437 0.916;438 1.000**;439 0.950;440 0.884; calcium uptake protein 1 homolog, mitochondrial-like [bombyx mori] positive 3.67e-09 2 y 0.741;83 l 0.543;147 r 0.768;148 s 1.000**;155 i 0.805;157 r 0.560;162 v 0.940;163 k 0.733;164 l 1.000**;165 t 1.000**;167 g 1.000**;169 n 0.657;170 t 1.000**;172 i 0.614;182 a 0.926;183 i 0.506;187 m 0.995**;402 n 0.604; probable hydroxyacid-oxoacid transhydrogenase, mitochondrial [bombyx mori] positive 1.83e-08 8 v 0.999**;9 f 1.000**;12 l 0.997**;13 r 0.997**;14 t 1.000**;17 v 0.910;29 g 0.974*;31 i 0.996**;32 q 1.000**;33 v 0.952*;34 s 1.000**;35 n 0.944;36 a 0.967*;37 t 1.000**;38 p 0.967*;39 i 0.985*;40 k 1.000**;41 d 0.948;42 y 0.961*;43 a 1.000**;44 f 0.968*;45 e 1.000**;82 v 0.890;114 s 0.940;121 k 0.579;134 v 0.844;151 s 0.950*;170 p 0.933;173 v 0.871;175 l 0.911;253 y 0.941;296 y 0.835;299 d 0.931;313 m 0.947;374 d 0.622;393 e 0.794;397 k 0.880;402 t 0.995**;405 k 0.999**;410 m 0.945;437 d 0.959*;438 r 0.965*;439 l 1.000**;441 k 0.971*;442 i 0.996**;446 s 0.704;448 t 0.943;458 d 0.789; heparin sulfate o-sulfotransferase [bombyx mori] positive 5.36e-06 3 f 0.653;5 v 0.753;9 c 1.000**;10 l 0.992**;11 f 1.000**;12 s 0.878;13 c 1.000**;14 i 0.950;16 i 0.997**;17 i 0.999**;18 v 0.999**;19 l 0.978*;20 i 0.860;21 s 0.866;23 y 1.000**;29 a 0.953*;31 l 0.935;36 r 0.998**;37 v 0.995**;38 r 0.994**;40 y 0.755;43 l 0.537;44 q 0.546;45 s 0.622;46 h 0.945;61 e 0.775;64 d 0.920;66 d 0.989*;85 v 0.637;123 k 0.684;227 q 0.514;251 l 0.753;275 s 0.667;276 n 1.000**;277 k 0.998**;278 s 0.999**;279 h 0.996**;280 l 0.999**;281 r 0.973*;282 q 0.997**;285 s 0.771;286 s 1.000**;287 k 0.576;288 i 0.996**;289 d 1.000**;290 p 0.826;291 i 0.920;293 a 0.847;294 t 0.999**;296 e 0.935;297 k 0.999**;299 q 0.994**;300 q 0.996**;301 s 0.855;302 i 0.594;303 v 0.869;307 w 0.998**;308 k 0.999**;309 m 0.525;310 e 0.699;312 e 0.606;313 l 0.957*;314 y 0.996**;315 e 0.603;317 a 0.834;318 l 0.891;319 e 0.997**;320 q 0.998**;321 f 0.997**;323 f 1.000**;372 k 0.962*;373 r 0.978*;374 k 0.773;375 l 0.858;377 v 0.809;378 k 0.780;379 e 0.820;380 a 0.998**;381 n 0.989*;382 n 0.774;383 v 0.811;385 q 0.790;386 i 0.931;400 y 0.754;403 e 0.626;404 k 0.576;405 i 0.891;406 r 0.955*;408 k 0.925; sex combs on midleg [bombyx mori] positive 1.87e-07 263 p 0.716;530 k 0.836;538 s 0.807;604 a 0.968*;673 k 0.999**;674 n 0.995**;676 r 0.998**;677 h 0.963*;678 y 0.981*; fibrinogen c domain-containing protein 1-b-like [bombyx mori] positive 0 15 i 0.534;16 f 0.910;17 s 0.691;18 l 0.953*;20 v 0.964*;22 n 0.982*;25 k 0.943;26 k 0.542;27 e 0.706;29 a 0.528;30 r 1.000**;31 i 0.725;32 q 0.991**;33 k 0.959*;35 i 0.716;37 d 0.934;38 e 0.845;40 e 0.980*;200 l 0.911;204 t 0.980*;205 g 0.696;206 r 0.565;207 g 0.964*;208 a 0.847;209 d 0.649;217 0.540;222 e 0.819;225 i 168 0.590;233 m 0.587;234 s 0.928;241 m 0.503;242 i 0.774;247 r 0.970*;248 k 0.915;251 h 0.982*;255 t 0.937;257 h 0.986*;260 t 0.960*;261 n 0.850;264 t 0.985*;266 y 0.987*;267 l 0.788;268 t 0.843;274 l 0.996**;277 s 0.945;280 s 0.990**;281 0.549;282 e 0.995**;283 a 0.988*;284 l 0.987*;286 d 0.827;287 a 0.978*;290 i 0.681;293 s 0.623;296 d 0.998**;300 h 0.847;301 s 0.567;302 v 0.996**;303 i 0.869;304 e 0.995**;305 r 0.918;307 e 0.869;308 h 0.964*;310 p 0.865;311 n 0.581;397 e 0.980*;508 h 0.806;618 n 0.574;720 s 0.621; *indicates that the posterior probability >0.95; **indicates that the posterior probability >0.99. 363 isj 14: 363-374, 2017 issn 1824-307x research report molecular cloning and functional characterization of a calreticulin gene from the sea cucumber apostichopus japonicus s cheng * , y chang * , y wang, g li, y chen, j ning, k li key laboratory of mariculture & stock enhancement in north china’s sea, ministry of agriculture, dalian ocean university, dalian 116023, p.r china *these authors contributed equally to this work. accepted september 18, 2017 abstract calreticulin (crt) plays crucial roles in the regulation of ca 2+ homeostasis, immune responses and molecular chaperon. in this study, a new member of the crt gene (denoted as ajcrt) was cloned and characterized from the sea cucumber apostichopus japonicus. the full-length of the gene was of 3316 bp, which consisted of 204 bp 5’ untranslated region (utr), 1870 bp of 3’-utr and a putative 1242-bp open reading frame encoding a 48-kda polypeptide (ajcrt) with a theoretical pi of 4.19. ajcrt had 59 % 65 % sequence identity with crts from other species. it contained two crt domains (residues 94 109 and 126 134) with a ring finger domain. ajcrt was ubiquitously expressed in all tissues examined, but was highly expressed in the coelomocytes. temporal transcriptional levels in the coelomocytes revealed significant upregulation of ajcrt after the animal was challenged with vibrio splendidus, reaching 4.97-fold the level of control at 4 h, but then decreased to 2.56-fold the level of control at 72 h. ajcrt knockdown decreased the expression level of the binding of ca 2+ to protein gene about 50 %. at the same time, intracellular concentration of ca 2+ also increased by 1.86-fold and 1.94-fold compared to that of the control. taken together, the results suggested that ajcrt may be associated with the immune response against bacterial infection, probably through participating in the regulation of intracellular ca 2+ homeostasis in sea cucumber. key words: calreticulin; apostichopus japonicus; spatial expression; time-course expression; ajcrt knockdown; ca 2+ concentration introduction apostichopus japonicus is a sea cucumber that belongs to the phylum echinodermata. it is widely distributed in china, japan, korea and russia, and is one of the most important aquaculture animals in north china (chang et al., 2004). however, increasing demand for sea cucumber and over-farming have resulted in the frequent occurrence of epidemic diseases in both juvenile and adult sea cucumber during the last decade, especially skin ulceration syndrome (sus), which has caused massive mortality for cultured sea cucumbers (deng et al., 2009; wang et al., 2009; ma et al., 2013), resulting in serious economic losses and limiting the sustainable development of this industry. to control epidemic diseases in sea cucumber, ___________________________________________________________________________ corresponding author: yaqing chang key laboratory of mariculture & stock enhancement in north china’s sea dalian ocean university dalian, 52 heishijiao rd., liaoning 116023, p. r. china e-mail: yaqingchang@hotmail.com antibiotics and chemicals are frequently used in aquaculture. however, indiscriminate use of these drugs has resulted in drug residues in the animals and environment, which is considered as a form of water pollution (bonnie and stuart, 2011; yang et al., 2015), further limiting the healthy and sustainable development of sea cucumber industry. as a marine invertebrate which lacks adaptive immune system, a. japonicus completely relies on innate immunity to combat pathogen infection (iwanaga and lee, 2005). in recent years, several immune-related genes such have been found to play crucial roles in the defense against bacterial, fungal and viral pathogens. some of these genes (wang et al., 2011, 2015a, b; lu et al., 2013; sun et al., 2013; ji et al., 2014; jiang et al., 2014; zhang et al., 2015; shao et al., 2015a, b, 2016; yang et al., 2015, 2016; lv et al., 2016) are listed in this study. therefore, identifying and characterizing more immune-related genes in a. japonicus will enable us to better understand the immune responses of sea cucumber and find a new way to ensure the development of a more robust and healthier aquaculture industry. 364 table 1 primer sequences used in the cloning of the ajcrt gene calreticulin (crt) was first isolated from the sarcoplasmic reticulum (sr) of rabbit by ostwald and maclennan in 1974. it is a unique endoplasmic reticulum (er) luminal resident protein, and it plays a crucial role in the regulation of ca 2+ homeostasis, immune responses and molecular chaperoning (thomas and david, 1974; michalak et al., 1999). structurally, crt contains three functionally distinct domains: n-domain, p-domain and c-domain. the n-domain forms a stable core which is resistant to proteolysis in the presence of calcium. the p-domain contains a high-affinity calcium-binding site and participates in either substrate binding or protein-protein interactions. the c-domain has a large number of negatively charged residues (ellgaard et al., 2001; corbett et al., 2009; michalak et al., 2009) crt is also a long-existing and highly conserved protein that has a variety of functions and is found in a wide range of species (wang et al., 2012). initially known as a high capacity calcium-binding protein in er that participates in the folding of newly synthesized sr proteins, crt is now known to associate with many intracellular and extracellular processes, including lectin-like chaperone activity, ca 2+ storage and signaling, wound healing, inhibition of tumor growth and c1q-dependent complement activation, as well as in the regulation of gene expression, cell adhesion, cancer and autoimmunity (johnson et al., 2001; gelebart et al., 2005; michalak et al., 2009; ayoola and miodrag, 2010). calreticulin is also a major calcium-binding/storage chaperone residing in the er lumen, where it plays important roles in molecular chaperoning function and in the response to viral infection (michalak et al., 2002). intracellular ca 2+ is associated with various biological processes, such as apoptosis and phagocytosis (may et al., 2001). in addition, crt and the binding of ca 2+ protein gene are likely to be critical for host-pathogen interaction (zhang et al., 2014). in human, crt plays vital roles in t-cell development and in the initiation of immune cell response (zhang et al., 1998; bryce et al., 2007). in marine organisms, crt has been identified in dicentrachus labrax (rute et al., 2007), exopalaemon carinicauda (duan et al., 2014), fenneropenaeus chinensis (luana et al., 2007) and trypanosoma carassii (zhao et al., 2011), where it can be induced by bacterial or viral challenge. however, no study on the crt gene in a. japonicus has been reported. furthermore, the connection between crt and the binding of ca 2+ to protein is still poorly understood (according to our knowledge). therefore, by studying the crt gene of a. japonicas, we could gain more insight into the role of this gene in sea cucumber and at the same time, obtain further information on its regulation of intracellular ca 2+ homeostasis. materials and methods animals preparation and samples collection healthy sea cucumber (body weight 68 ± 4.59) were collected from dalian heshengfeng marine product farm and maintained at 16 17 °c in our laboratory for one week. the intestine, respiratory tree, tube feet, coelomic fluid, body wall and longitudinal muscle were then carefully removed from three healthy animals. to harvest the coelomocytes, the collected coelomic fluid was immediately centrifuged at 1,000g/4 °c for 5 min. all of the other tissues were immediately snap-frozen in liquid nitrogen and stored at -80 °c. bacterial challenge experiment vibrio splendidus d450 was cultured and harvested as previously described by yang et al. (2015), and the cell pellet was resuspended in primers sequences (5’-3’) application melting temperatures ajcrt-5’-out cttctgctcgtgcttgactgta 5’-race 56 °c ajcrt-5’-in ggtggtaacacgcactccctgg 5’-race 56 °c ajcrt-3’-out tagcaggtccacagtacatgccca 3’-race 56 °c ajcrt-3’-in atcgctttgtttacataaggaata 3’-race 56 °c ump-1 taatacgactcactatagggcaagcagtggtatcaacgcagagt race 56 °c ump-2 ctaatacgactcactatagggc race 56 °c ajcrt-f tcctccactctgacaacacctacg qpcr 60 °c ajcrt-r agtcctctggtttcttggcttcgg qpcr 60 °c cytb-f tgagccgcaacagtaatc reference gene 60 °c cytb-r aagggaaaaggaagtgaaag reference gene 60 °c ajcrt sirna gcaaguggguucauccuautt auaggaugaacccacuugctt ajcrt silence negative control (nc) sirna uucuccgaacgugucacgutt acgugacacguucggagaatt nc for ajcrt sirna interference 365 table 2 primer sequences used for the analysis of the binding of ca 2+ to protein gene phosphate buffered saline (pbs, 0.1 mm, ph 7.4) to a final concentration of 10 7 cfu/ml. sea cucumbers were divided into two groups: control group and a bacterial-challenged group. the control group (a total of 25 individuals) was immersed in a tank containing pbs only whereas the bacterial-challenged group (also 25 individuals) was immersed in a tank containing pbs plus v. splendidus d4501 at a concentration of 10 7 cfu/ml. the celomic fluids were collected from the sea cucumbers at 0, 4, 8, 12, 24, 48 and 72 h post-immersion, and three individuals were randomly selected at each time point. total rna extraction and cdna synthesis total rna was isolated from each of the six different tissues using trizol (ambion) according to the manufacturer’s instructions. then, the quantity and quality of extracted rna were assessed by uv spectrophotometry (nanophotometer, munich, germany) and agarose gel electrophoresis. the cdna was synthesized using a primerscript tm rt reagent kit (takara, japan) for real-time pcr (rt-pcr), diluted to 1:10 and stored at -20 °c until used. cloning and sequencing of ajcrt a partial cdna sequence of ajcrt was obtained from our transcriptome assembly data (unpublished data). based on the partial sequence, 5’ and 3’-rapid amplification of cdna ends (race) was conducted using the smarter ® race 5’/3’ kit (takara, japan). all primers used are listed in table 1. pcr product from race was detected in 1.2 % agarose gel and purified using easypure quick gel extraction kit (transgen biotech, china). the purified product was then ligated to the peasy ® -1 cloning vector (transgen biotech, china). trans1-t1 phage resistant chemically competent cells (transgen biotech, china) were immediately transformed with the resulting construct. positive transformants were verified by colony pcr using m13 primers (tran, china), and three independent clones were subjected to dna sequencing to confirm the sequence of the insert. bioinformatics analysis of ajcrt the full-length of the ajcrt gene was analyzed using basic local alignment search tool (blast) (http://www.ncbi.nlm.nih.gov/blast) program. open reading frame (orf) was identified by orf finder (http://www.ncbi.nlm.nih.gov/projects/gorf/orfig.cgi). the deduced amino acid sequence was analyzed online using the expert protein analysis system (http://www.expasy.org/), and the molecular weight of the predicted polypeptide was calculated using the expasy compute pi/mw tool (http://www.expasy.org/). signaip 4.1 server (http://www.cbs.dtu.dk/services/signalp/) was used to predict the signal peptide sequence of ajcrt whereas simple modular architecture research tool (smart) program (http://smart.embl-heidelberg.de/) was used to analyze its domain structure. phylogenetic tree was conducted by the neighbor-joining (nj) method using mega 5.2 program. multiple sequence alignment was performed by the dnaman. quantification analysis of ajcrt expression spatial and temporal expression levels of ajcrt were analyzed by qrt-pcr using the applied biosystem 7500 real-time system (applied biosystem, usa). the sample contained 2 μl of 1:10 diluted original cdna, 10 μl of 2 × sybr green master mix (sybr primescript tm rt-pcr kit ⅱ, takara, japan), 0.4 μl of rox reference dye ⅱ, 0.8 μl (10μm) of each primer and 6 μl ddh2o in a total volume of 20l. the condition of the amplification consisted of the followings: a holding step at 95 ℃ for 30 s, followed by 40 cycles of 95 ℃ for 5s, and 60 ℃ for 32 s. the specific amplification of ajcrt was confirmed by melting curve. the gene for cytochrome b was selected as the reference gene (yang et al., 2010). the sequences of all the primers used are shown in table 1. the relative mrna levels of ajcrt were quantified using the 2 -△△ct method and the results were given as relative expression patterns (means ± s.d., n = 3), and the differences were obtained by one-way analysis of variance (anova). primary coelomocytes culture and ajcrt silencing in vitro primary celomocytes were cultured as described by yang et al. (2016) and lu et al. (2015). in brief, the washed cells were re-suspended in leibovitz's l-15 cell culture medium (invitrogen, usa) containing penicillin (100 u/ml), streptomycin sulfate (100 µg/ml) and gentamycin sulfate (100 µg/ml). the final concentration of the cells was 10 6 cells/ml. the osmotic pressure of the cell suspension was adjusted with 0.39 m nacl, and 500 l aliquots were then dispensed into a 24-well plate and incubated at 17 ℃ for 12 h before the ajcrt knockdown experiment. the ajcrt targeting small interfering rna (ajcrt sirna) and a negative control sirna (nc sirna) were designed and primers sequences (5’-3’) application melting temperatures ca 2+ -f tcctccactctgacaacacctacg qpcr 60 °c ca 2+ -r agtcctctggtttcttggcttcgg qpcr 60 °c http://blast.ncbi.nlm.nih.gov/blast http://www.ncbi.nlm.nih.gov/projects/gorf/orfig.cgi http://www.expasy.org/ http://www.cbs.dtu.dk/services/signalp/ http://smart.embl-heidelberg.de/ 366 synthesized by genepharma (shanghai, china). the sequences of these sirnas are shown in table 1. for ajcrt knockdown, 3 μl of ajcrt sirna (20 µm) or nc sirna was mixed with an equal volume of lipotap liposomal transfection reagent (beyotime biotechnology, china) and 500 μl of the cultured coelomcytes prepared earlier was transfected with this sirna sample. twenty-four hours after the transfection, the cells were harvested and used in subsequent experiments. analysis of the binding ca 2+ to protein gene after ajcrt knockdown total rna harvested from the celomocytes following the ajcrt knockdown was used to synthesize the cdna. then, the expression levels of ajcrt and the binding ca 2+ to protein gene were conducted as described above. the primers of the binding ca 2+ to protein are listed in table 2. measurement of intracellular ca 2+ level intracellular ca 2+ concentration ([ca 2+ ]i) was measured according to ding et al. (2008) and girard et al. (2002) with little modification. briefly, 500 μl coelomocytes at the final concentration of 10 6 cells/ml was mixed with 1.3 μl of 2 μm fura-2/am and 0.8 μl 0.05 % (w/v) of pluronic-f127, and then incubated in 37 ℃ for 30 min. following incubation, the coelomocytes were collected by centrifugation at 1000g and 4 °c for 5 min, and then washed thrice with hanks’ balanced salt solution (hbss) without calcium chloride (sangon, china). the washed cells were re-suspended in 250 μl hbss. [ca 2+ ]i was automatically recorded with a hitachi f-4500 fluorimeter at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. statistic analysis data were represented as mean ± standard error (se). difference in relative expression of ajcrt and the binding of ca 2+ protein was analyzed by one way anova or independent-t-test on spss 13.0. the asterisk (*) and double asterisks (**) above the bars represent difference at p < 0.05 and significant difference at p < 0.01, respectively. results sequence analysis of ajcrt the full-length cdna of ajcrt (genbank accession no. mf960913) was obtained by 5’and 3’-race according to an est obtained from an a. japonicus cdna library. the gene was 3316 bp long, containing 1242-bp of open reading frame, 204-bp of 5’-untranslated region (utr) of and 1870-bp of 3’-utr, which included a putative polyadenylation consensus signal (aataaa) and a poly a tail (fig. 1) the open reading frame was found to encode a polypeptide of 480 amino acids with a predicted molecular mass and a theoretical pi of 48 kda and 4.19, respectively. domains and motifs analysis revealed two conserved domains reminiscent of the crt family proteins: lys 94 -phe 109 and lle 126 -gly 134 ng finger domain (residues 119 160). three crt family repeat motifs (dwd), one putative er targeting motif hdel as well as a coiled coil (residues 346 383) were also found in ajcrt. crt was also found to contain a putative signal peptide of 16 amino acids (mkflvalailcytasa). blastp analysis revealed 69 75 % sequence identity between ajcrt and crts from other species, including strongylocentrotus purpuratus (xp 006824896), haliotis discus discus (aly 11013), danio rerio (np 956007) and sus scrofa (np 001167604). multiple alignment and phylogenetic analysis the deduced amino acid sequence of ajcrt and the corresponding crt sequences of invertebrates and vertebrates were aligned using the dnaman program (fig. 2). ajcrt was found to have significant percentages of sequence identity (> 50 %) with the crt sequences from other species, e.g., 59 % with homo sapiens crt and 65 % with s. purpuratus crt (fig. 3). moreover, phylogenetic analysis showed that ajcrt was clustered into the invertebrate subgroup with closer evolution relationship with s. purpuratus. figure 3 depicts the phylogenetic analysis of crts, which was constructed by the neighbor-joining method. the relationships among the 10 crts shown by the phylogenetic tree were consistent with traditional taxonomy. tissue distribution and mrna expression pattern of ajcrt in coelomocytes after v. splendidus challenge ajcrt transcript was detected in all the different tissues examined, suggesting that it was ubiquitously expressed in a. japonicus (fig. 4), but the expression varied, depending on the tissue. for comparison purpose, the transcript level of ajcrt in the intestine was taken as 1 and the transcript levels in the other tissues were compared against that of the intestine. such comparison yielded the highest ajcrt transcript level in the cleomocytes (3.2 fold the level found in the intestine), followed by tube feet (1.24-fold), respiratory tree (1.05-fold), body wall (0.92-fold), and lastly, the longitudinal muscle (0.3-fold). the different levels of ajcrt expression in the different tissues suggested that ajcrt may be involved in important physiological functions. a significant (p < 0.01) increase in the expression level of ajcrt was found in the coelomocytes of a. japonicas following v. splendidus challenge. the expression of ajcrt was 4.97-fold and 2.56-fold the expression of non-infected a. japonicas 4 h and 72 h, respectively, after the bacterial challenge (fig. 5). ajcrt knockdown affects the expression of the binding of ca 2+ to protein gene [ca 2+ ]i knockdown of ajcrt by sirna in the primary celomocytes was found to affect the expression of the binding to ca 2+ protein gene in the cells. the level of ajcrt transcript in the cells transfected with the ajcrt-specific sirna was 0.46 fold and 0.44 fold the levels ajcrt in the cells transfected with a negative control sirna or in the non-transfected cells (fig. 6a). at the same time, the transcript level of a binding of ca 2+ to protein gene in the ajcrt-knocked down celomocytes was also reduced to about 0.52-fold the level found in the negative control cells (fig. 6b). 367 fig. 1 nucleotide and deduced amino acid sequences of the crt gene cloned from a. japonicus. the start codon (atg) and the putative polyadenylation consensus signal (aataaa) are boxed. the asterisk represents the stop codon. the signal peptide is underlined. the two crt family signature motifs (kheqkidcgggyvkvf and imfgpdicg) are shaded. the three crt family repeat motifs (dwd) and the putative er targeting motif hdel are underlined and shaded. 368 fig. 2 multiple alignment of the deduced amino acid sequence of ajcrt with crts from other organisms. h. discus (aly11013), l. gigantea (xp_009056939), s. purpuratus (np_999643), d. rerio (ajg06014), c. semilaevis (xp_008336669), x. tropicalis (np_001001253), m. musculus (np_031617) and h. sapiens (np_004334). the two crt family signature motifs are indicated by two boxes. the three crt family repeat motifs (dwd/e) are underlined. 369 homo sapiens bad96780 oryctolagus cuniculus np 001075704 sus scrofa np 001167604 mus musculus np 031617 xenopus tropicalis np 001001253 cynoglossus semilaevis xp 008336669 danio rerio ajg06014 ▲apostichopus japonicus strongylocentrotus purpuratus np 999643 lottia gigantea xp 009056939 59 25 26 19 8 6 4 2 fig. 3 consensus neighbor-joining tree constructed from the amino acid sequences of crts from other species. the phylogenetic tree was constructed according to the neighbor-joining method using mega 5.2 software. the numbers at the forks indicate the bootstrap. intracellular ca 2+ concentration ([ca 2+ ]i) in the primary celomocytes increased following the knockdown of ajcrt. a [ca 2+ ]i of 61.83 ± 2.79 nm was obtained for cells transfected with the control sirna, 59.51 ± 2.65 nm for non-transfected cells, and 115.2 ± 2.25 nm for cells transfected with ajcrt-specific sirna. thus knockdown of ajcrt in primary coelomocytes increased the level of intracellular ca 2+ by almost 2 fold compared to cells without ajcrt knockdown (fig. 6c). discussion sea cucumbers lack an adaptive immune system and therefore they rely completely on an innate system for protection against potential pathogens. the vital roles involved in immune responses from the calcium-related proteins were identified in a. japonicus, such as calumenin, annexin, calreticulin and phospholipase c-gamma (andrew et al., 2007; zhang et al., 2014). ca 2+ acts fig. 4 relative expression of ajcrt in different tissues. each vertical bar represents the mean ± sd (n = 3). 370 fig. 5 temporal expression of ajcrt in the coelomocytes after infection of the a. japonicus by v. splendidus. each vertical bar represents the mean ± sd (n = 3). significant difference between the challenge group and the control group are indicated by asterisk (* represented p < 0.05, ** represented p < 0.01). as a diffusible second messenger and plays a crucial role in the metabolism and physiology of eukaryotes. in addition, the maintenance of intracellular ca 2+ homeostasis is essential in the metabolic processes of and physiology of eukaryote. intracellular ca 2+ concentration can be affected by various external signals, such as drugs, stress, light and pathogens (carafoli et al., 1999; chamilani et al., 2010). therefore, the modulation of calcium-related proteins is critical for intracellular ca 2+ homeostasis. calreticulin (crt) is a highly conserved protein that modulates calcium binding to proteins and storage (wang et al., 2012; zhang et al., 2014). in order to better understand the role of crt in the processes of immune responses and the maintenance of intracellular ca 2+ homeostasis, we identified and characterized a crt gene from a. japonicus. as a result, a total of 3316 bp nucleotide sequences represented the complete cdna sequence of ajcrt. typical domains of crt proteins were found in the deduced ajcrt protein sequence, suggesting that ajcrt might perform similar functions as other crts from other animals, including invertebrates and vertebrates. similar results of crt domains have also been reported in plenty of species, such as trypanosoma carassii, pieris rapae, exopalaemon carinicauda, et al., which further supporting the notion that ajcrt being an ancient and highly conserved protein (johnson et al., 2001; gelebart et al., 2005; ayoola and miodrag, 2010; wang et al., 2012). in the white prawn e. carinicauda, crt could be detected in all the tissues examined, including hemocytes, gill, hepatopancreas, muscle, ovary, intestine, stomach and eyestalk, with the highest expression level in hepatopancreas, whereas the lowest was found in eyestalk (duan et al., 2014). in this study, ajcrt was distributed in all the tested tissues, including celomocytes, tube feet, respiratory trees, intestine, body wall and longitudinal muscle. the highest expression level of ajcrt mrna was detected in the celomocytes, cells that are regarded as the main cellular component of the immune system and the metabolic center for reactive oxygen species (ros) production in sea cucumber. similar to the hemocytes of vertebrates, celomocytes are freely circulating cells and they play an irreplaceable role in the immune responses of sea cucumber (wang et al., 2007; wang et al., 2009; cheng et al., 2016). thus ajcrt may act as an immune-related gene and its function may be to defend the animal against bacterial, fungal and viral pathogens (duan et al., 2014; zhang et al., 2014). in addition, the ubiquitous distributing pattern of ajcrt suggested that it may have a multifunction in many cellular processes, both intracellular and extracellular processes, such as calcium-binding in er and folding of newly synthesized protein (thomas and david, 1974; michalak et al., 1999). infection by pathogen can induce the generation of excessive 371 fig. 6 ajcrt knockdown in a. japonicas coelomocytes and its effect on intracellar ca 2+ and ca 2+ -binding protein expression. (a) ajcrt knockdown. effect of ajcrt knockdown on intracellular ca 2+ concentration (b) and ca 2+ -binding protein expression (c). data are the means  sds (n = 3). significant difference between the ajcrt sirna group and the negative control group is indicated by asterisk (*represented p < 0.05, **represented p < 0.01). ros, which could cause mass mortality in marine organisms (siripong et al., 2014). vibrio splendidus was regarded as the major pathogen of sea cucumbers (zhao et al., 2011). to better understand the role of ajcrt in the immune response, the animals were challenged with v. splendidus d450 to simulate a state of infection. pathogens infection can elicit variation in the concentration of intracellular ca 2+ in eukaryotes. maintenance of intracellular ca 2+ homeostasis is essential to the processes of metabolism and physiological function (chamilani et al., 2010). as the result, ajcrt expression was significantly up-regulated at 4 h after v. splendidus challenge, which indicated that ajcrt expression was quickly induced upon bacterial infection. at the same time, our data also showed that the concentration of intracellular ca 2+ increased when ajcrt was knocked down, further suggesting that ajcrt was involved in the homeostasis of intracellular ca 2+ directly. however, the second peak of ajcrt expression was observed at 72 h post bacterial challenge, and a possible explanation for the emergence of this peak could be due to the momentarily disruption of the innate immune system of a. japonicus, caused by the growing number of bacteria inside the animal. similar results have been observed in exopalaemon carinicauda after wssv challenge and in the hepatopancreas fenneropenaeus chinensis following wssv infection (duan et al., 2014; luana et al., 2014). this further demonstrated that a role for ajcrt in the immune response against bacterial infection. crt is initially thought to be responsible for the high calcium-binding capacity in er and the folding of newly synthesized proteins (michalak et al., 1999). a growing number of studies have focused on unravelling the function of crt since its discovery in mammals (ostwald and maclennan, 1974). it is now known that crt has many functions, including lectin-like chaperone activity, ca 2+ storage and signaling, and regulation of gene expression, cell adhesion, wound healing, cancer and autoimmunity 372 (corbett et al., 2000; ellgaard et al., 2001; johnson et al., 2001; wang et al., 2012). the role of crt in immune reaction has been reported in different invertebrates (luana et al., 2007; duan et al., 2014). in this study, the connection between crt and the binding of ca 2+ to protein gene was investigated by looking at the effect of ajcrt knockdown on the level of intracellular ca 2+ and the expression of a binding of ca 2+ to protein gene. the binding of ca 2+ to protein gene known as ef hand domain-containing calcium regulation protein plays important roles in signal transduction and calcium binding to proteins (ikura et al., 1996). the modulation of the binding of ca 2+ to protein gene is considered to be vital during thermal stress (zhang et al., 2013). the level of the binding of ca 2+ to protein gene in the celomocytes of a. japonicus decreased when ajcrt in these cells was knocked down. this basically consistent decreasing of ajcrt and the binding of ca 2+ to protein gene after ajcrt knockdown maybe suggested that ajcrt and the binding of ca 2+ to protein gene are involved in the general physiological processes of a. japonicus. intracellular ca 2+ was regarded as critical for various biological events. particularly, increase in intracellular ca 2+ is associated with many defense responses elicited by the host cell during an infection (michalak et al., 2002; zhang et al., 2013). significant increase in intracellular ca 2+ concentration resulting from the knockdown of ajcrt in a. japonicus celomocytes indicated that ajcrt was directly affected intracellular ca 2+ homeostasis. a possible perspective of ajcrt involved in different biological events like intracellular ca 2+ homeostasis. in conclusion, we have cloned and characterized a full-length crt gene from a. japonicus and analysis of its spatial and temporal expression pattern suggested that this gene might be involved in the immune response, perhaps by mediating the regulation of intracellular ca 2+ homeostasis in sea cucumber. acknowledgments this work was supported by grant from the national natural science foundation of china (no. 31572608). references andrew f, adam p. invertebrate immune systems-specific, quasi-specific,or nonspecific? j. immunol. 179: 7209-7214, 2007. ayoola o, miodrag b. trypanosoma carassii calreticulin binds host complement component c1q and inhibits classical complement pathway-mediated lysis. dev. comp. immunol. 34: 396-405, 2010. bonnie m, stuart b. food animals and antimicrobials: impacts on human health. clin. microbiol. rev. 24: 718-733, 2011. bryce w, doo 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mediche, università di padova, padova, italy accepted january 25, 2005 abstract in the ascidian botryllus schlosseri, we studied the effects of hemocyte incubation with foreign cells, such as bacteria, bacterial spores and yeast. in the presence of yeast and bacterial spores, morula cells, a common cell type in botryllid ascidians, changed their morphology, release phenoloxidase in the medium, thus causing an increase in cytotoxicity, and express molecules recognised by anti-il-1-αand anti-tnf-α-antibodies. these effects were not observed when hemocytes were incubated with both gram-positive (staphylococcus aureus) and gram-negative (escherichia coli) bacteria. considering that morula cells are the main source of molecules recognised by anti-cytokine-antibodies we suggest an immunosurveillance role of these cells, which may influence immune responses such as phagocytosis. key words: ascidians; botryllus; cytotoxicity; morula cells; phenoloxidase; immunomodulation introduction two types of circulating immunocytes are present in the colonial ascidian botryllus schlosseri: phagocytes and morula cells (mc). phagocytes include hyaline amoebocytes and macrophage-like cells. the former are amoeboid cells, 5-10 µm in size, capable of active movements and phagocytosis, whereas the latter are larger (10-15 µm in diameter) roundish cells with at least one vacuole containing ingested material, deriving from hyaline amoebocytes having engulfed foreign cells or particles (ballarin et al., 1993, 1994). mc are large, vacuolated cells which represent the most abundant blood cells, their frequency ranging from 20 to 60% of circulating hemocytes (ballarin et al., 1995). they are the mediators of the inflammatory *corresponding author: loriano ballarin università di padova, dipartimento di biologia, via u. bassi 58/b – 35100 padova, italy e-mail: loriano.ballarin@unipd.it (rejection) reaction which occurs, in the form of a series of necrotic spots, along the contacting borders, when genetically incompatible colonies are juxtaposed. mc induce cytotoxicity in the contacting tissues through the release of the enzyme phenoloxidase (po), probably stored as a pro-enzyme inside their granules (ballarin et al., 1995, 1998). we have recently demonstrated that, in b. schlosseri, mc synthesise molecules recognised by antibodies raised against the mammalian inflammatory cytokines il-1-α and tnf-α upon the recognition of soluble foreign molecules, such as mannan (ballarin et al., 2001) and allogeneic humoral factors diffusing from one colony into the blood of the alien colony (cima et al., 2004) in the course of the rejection reaction. in an attempt to better understand the role of mc in botryllus immunobiology, we studied the behaviour of mc when hemocytes are incubated in vitro with foreign cells, such as bacteria, bacterial spores and yeast. results indicate that mc can recognise bacterial spores and yeast cells and, as a consequence of this recognition, degranulate and release the enzyme po that, in turn, increases in vitro cytotoxicity among hemocytes. in addition, mc acquire 2 immunopositivity to anti-il-1-αand anti-tnf-α antibodies. such responses are not observed when hemocytes are incubated with bacteria. we suggest that the synthesis and release of molecules immunopositive to anti-cytokine-antibodies by mc can influence phagocyte activity. materials and methods animals colonies of b. schlosseri from the lagoon of venice were used. they were kept in aerated aquaria, attached to glass slides and fed with liquifry marine (liquifry co., dorking, england). hemocyte collection and culture blood samples were collected with a glass micropipette by puncturing, with a fine tungsten needle, the tunic marginal vessels of colonies previously rinsed in 0.38 % na-citrate in filtered sea water (fsw), ph 7.5, as anti-clotting agent. they were then centrifuged at 780 x g for 10 min and pellets were finally resuspended in fsw to give a concentration of 5 x 106 cells/ml. sixty µl of hemocyte suspension were placed in the centre of culture chambers prepared as described elsewhere (ballarin et al., 1994) and left to adhere to coverslips for 30 min at room temperature (rt). morula cell morphology after the adhesion of hemocytes to the coverslips, the debris-containing fsw was discarded and replaced with an equal volume of one of the following cell suspensions in fsw: escherichia coli (200 x 106 cells/ml), staphylococcus aureus (50 x 106 cells/ml), bacillus clausii spores (200 x 106 spores/ml) or ordinary baker’s yeast (50 x 106 cells/ml) in fsw (fsw alone in controls). after 60 min of incubation at rt, the morphology of living mc was observed under a leitz dialux 22 light microscope at a magnification of 1200 x. immunocytochemistry after the incubation with foreign cells, hemocyte monolayers were fixed for 30 min at 4°c in 4 % paraformaldehyde plus 0.1% glutaraldehyde in saline buffer (sb: 0.2 m na-cacodylate buffer, ph 7.4, plus 1.7% nacl and 1% sucrose), rinsed in phosphatebuffered saline (pbs: 1.37 m nacl, 0.03 m kcl, 0.015 m kh2po4, 0.065 m na2hpo4, ph 7.2) and permeabilised with 0.1% triton x-100 in pbs for 5 min. immunoreactivity against rabbit polyclonal antihuman-il-1-α (santa cruz biotech) and goat polyclonal anti-human tnf-α (santa cruz biotech) (1 µg/ml, according to manufacturer’s suggestions) was revealed by immunoperoxidase staining using the avidin-biotin-peroxidase complex method for signal enhancement (hsu et al., 1981) and 3,3’diaminobenzidine (fluka) as substrate. endogenous peroxidase was blocked by incubation for 30 min in a solution of 6 % h2o2 in methanol. in control preparations, primary antibodies were either substituted with non-immune sera or absorbed with homologous antigen, i.e., recombinant human il-1-α and tnf-α (peprotech). moreover, a rabbit polyclonal anti-botryllus agglutinin (ba) antibody (ballarin et al., 2000) was used for specificity control. cells were observed under the light microscope to determine the frequencies of positive haemocytes. po assay in each experiment, 400 µl of hemocyte suspensions (5 x 106 cell/ml) were incubated, in 1.5 ml vials, with bacterial spores (final concentration: 200 x 106 spores/ml), bacterial cells (final concentration: 200 x 106 and 50 x 106 cells/ml for e. coli and s. aureus, respectively), yeast cells (final concentration: 50 x 106 cells/ml) or fsw (reference control) at rt for 60 min. cells were then centrifuged at 780 x g for 10 min, and the po activity of supernatants was determined spectrophotometrically. briefly, 20 µl of supernatant were added to 440 µl of pbs and 440 µl of a saturated solution of dihydroxyphenyl-l-alanine (l-dopa) in pbs and the time course of the reaction was read at 490 nm for 5 min. one relative unit (ru) of po activity was defined as an increase in absorption of 0.001/min in the reaction mixture (söderhäll and smith, 1983). protein concentrations of the supernatants were determined according to bradford (1976) and results were expressed as ru/mg protein. cytotoxicity assay cytotoxicity among hemocytes was assessed by trypan blue exclusion, after 60 min of incubation of hemocytes with foreign cells or spores. blood cells were exposed to 0.25 % trypan blue in fsw for 5 min. the cytotoxicity index, i.e., the percentage of haemocytes positive for trypan blue staining, was then calculated. in the experiments with yeast, the effects of the addition of the po inhibitors nabenzoate, phenylthiourea (ptu) and tropolone (20 mm, 1 mm and 2 mm, respectively) to fsw were also evaluated. statistical analysis at least 300 cells, in ten optical fields at a magnification of 1250 x, were counted in each type of experiment. each experiment was carried out in triplicate. data are expressed as means ± sd and were compared with the χ2 test. po activities were compared with duncan’s test. results mc change their morphology in the presence of foreign cells in unexposed hemocyte, living mc appeared as large, slightly mulberry-shaped cells, with the cytoplasm almost completely occupied by round, uniform vacuoles, 2 µm in diameter (fig. 1a). after aldehyde fixation, the granules reduced in size and show a yellowish content (fig. 1b). in the presence of either bacterial spores or yeast cells, living mc changed their morphology: some of their granules reduced their size, others coalesced into a few, large granules which appeared devoid of their contents (fig. 1c). no changes the morphology of mc were observed in the presence of either e. coli or s. aureus. mc acquire immunopositivity to anti-cytokine antibodies in the presence of foreign cells following incubation with bacterial spores or 3 fig. 1 morula cell morphology in various experimental conditions. (a, c) living cells incubated in absence (a) or presence (c) of foreign cells; (b, d) aldehyde-fixed cells, immunostained for anti-cytokine antibodies, previously incubated in absence (b) or presence (d) of foreign cells. scale bar: 15 µm. yeast cells, a significant increase (p < 0.001) in the amount of cells showing immunopositivity to anti-il-1αand anti-tnfα-antibodies was observed. most of the immunopositive cells were mc, accounting for more than 95% of labelled cells. immunopositivity was located in the cytoplasm of mc (fig. 1d) and the frequency of mc recognised by anti-cytokine antibodies shifted from about 10 % in unexposed cultures to more than 80 % in the presence of either microbial spores or yeast cells. no significant increase of labelled mc was observed when haemocytes were incubated with e. coli or s. aureus (fig. 2). no labelling of mc was observed with anti-ba antibodies. influence of foreign cells on po activity in the culture medium and cytotoxicity when hemocytes were incubated in the presence of either bacillus spores or yeast cells, po activity in the medium significantly increased (p < 0.001 and p < 0.01, respectively) with respect to controls. no significant increase was reported in the presence of either e. coli or s. aureus cells (table 1). a significant increase in the cytotoxicity index was observed when haemocytes were incubated in the presence of bacterial spores or yeast cells (p < 0.05 and p < 0.001, respectively). in experiments with yeast, no significant increase in cytotoxicity was measured when na-benzoate, ptu or tropolone were added to fsw. e. coli and s. aureus did not induce any significant increase in cytotoxicity (table 2). discussion many different types of circulating hemocytes are present in the blood of the colonial ascidian b. schlosseri (ballarin et al., 1993). among them, mc are the most frequent cells and play a key role as effectors of the rejection reaction which occurs when genetically incompatible colonies of the ascidian b. schlosseri contact each other. in the course of this reaction, mc crowd inside the facing ampullae (blind, sausage-like endings of the marginal vessels) and cross the ampullar epithelium to reach the tunic (sabbadin et al., 1992; rinkevich et al. 1998). in the meantime, they degranulate and release the contents of their vacuoles, mainly po and its polyphenol substrata (ballarin et al., 1998; cima et al., 2004). the enzyme, in turn, induces cytotoxicity that appears in the form of a series of necrotic spots along the contact border. this sequence of events is triggered by recognition of allogeneic humoral factors that diffuse through the partially fused tunics from one colony to the alien one and it can be mimicked in vitro when hemocytes are exposed to blood plasma from incompatible colonies (ballarin et al., 1994, 1998, 2002). here, we demonstrate that a change in the morphology of mc occurs in response to the recognition of foreign cells, such as bacterial spores or yeast cells: it is similar to what observed when hemocytes are incubated with blood plasma from genetically incompatible colonies (ballarin et al., 1998) and, therefore, ascribable to a degranulation event. this indicates that mc can readily “sense” the presence of particular foreign molecules or cells in the environment (both in vivo and in vitro) and activate themselves upon their recognition, thus acting as “sentinel” cells which degranulate as a consequence of their activation. mc degranulation is followed by an increase in both po activity in the culture medium and hemocytes cytotoxicity among hemocytes. the two events are closely related as demonstrated in previous reports (ballarin et al., 1998) and in the present paper, since the addition of po inhibitors prevents cytotoxicity. fig. 2 percentage of mc showing immunopositivity to anti-il-1α (dark grey) and anti-tnf-α (light grey) antibodies after incubation of hemocyte cultures in fsw without (c = control) or with foreign cells. asterisks: significant differences with respect to controls. *** p < 0.001. c b. clausii s. cerevisiae e. coli s. aureus 100 80 60 40 20 0 *** *** *** *** % i m m u n o p o s it iv e m c 4 table 1 po activity in culture medium of hemocytes incubated in fsw in presence of b. clausii spores, s. cerevisiae, e. coli and s. aureus with respect to controls (incubation in fsw alone). significant differences are marked by asterisks. *** p < 0.001; * p < 0.05. po activity (ru/mg protein) control (fsw) 10.73 ± 0.96 b. clausii spores 16.89 ± 0.67 *** s. cerevisiae cells 15.73 ± 1.55 * e. coli cells 11.83 ± 0.66 s. aureus cells 9.73 ± 0.45 we can, therefore, state that, through the release of po, mc induces an increase in hemocyte cytotoxicity, which represents a first, relatively rapid response towards cells non-self cells. the above assumption is further supported by the finding that bacterial cells do not induce any mc degranulation: the absence of mc activation is associated with the lack of both the increase in po activity in the culture medium and hemocyte cytotoxicity. mc cannot recognise e. coli and s. aureus cells: this probably reflects the difference in cell surface with respect to bacterial spores and yeast cells and the absence of molecules able to activate mc and trigger a degranulation event. the observation that e. coli lps does not induce any degranulation of mc (our unpublished data) fits the above hypothesis. analogously, hata et al. (1998) found that po is not released by hemocytes of the solitary ascidian h. roretzi in the presence of β1-3 glucan. recent evidences indicate that activated mc synthesise molecules recognised by anti-mammaliancytokine antibodies. this occurs when hemocytes are exposed to either soluble microbial surface molecules or allogeneic blood plasma (ballarin et al., 2001; cima et al., 2004). the above-presented results indicate that mc can synthesise these molecules even as a consequence of the recognition of non-self particulate material, such as foreign cells. the presence of a low percentage of immunopositive mc in controls is probably to be ascribed to their activation during blood collection. again, the absence of a significant increase in immunopositive mc in the presence of bacteria, is the consequence of the lack of recognition of these cells by mc. it is our opinion that these molecules can modulate immune responses. therefore, in our hypothesis, mc act as “surveillance” cells able to readily recognise foreign molecules or cells and, as a consequence of this recognition, they: i) release po and induce cytotoxicity; ii) synthesise and release cytokine-like molecules which induce, in a paracrine way, either the activation of other mc or phagocyte stimulation. our preliminary (unpublished) data indicate that activated mc release factor(s) able to stimulate phagocytosis. this could explain why e. coli cells that do not activate mc, are engulfed less actively than yeast cells by b. schlosseri phagocytes (ballarin et al., 1994). in addition, agreement with the above roretzi, po table 2 cytotoxicity index in hemocyte cultures incubated in fsw, in absence and presence of foreign cells. yeast cells were also incubated in the presence of po inhibitors. asterisks mark significant differences with respect to controls. *** p < 0.001; * p < 0.05. cytotoxicity index control (fsw) 3.93 ± 1.11 b. clausii spores 6.02 ± 1.60 * s. cerevisiae cells 7.17 ± 1.69 *** s. cerevisiae cells + na-benzoate 5.07 ± 2.00 s. cerevisiae cells + ptu 5.00 ± 1.10 s. cerevisiae cells + tropolone 3.44 ± 1.07 e. coli cells 3.33 ± 1.44 s. aureus cells 3.69 ± 1.19 activity in the culture medium and phagocytosis are closely related (hata et al., 1998). moreover, in c. intestinalis, lysates of mc from hemocyte cultures previously exposed to yeast cells can positively influence phagocytosis (smith and peddie, 1992) this observation was considered as evidence of the synthesis of opsonins by ascidian mc, but the absence of data directly proving the synthesis of opsonins by mc and the evidence of the production of such molecules by phagocytes (ballarin et al., 2000) fit the idea that it can be the consequence of the synthesis of cytokine-like molecules by activated mc, able to enhance phagocytosis. further investigations are in progress to better elucidate this point. acknowledgements the authors wish to thank mr m. del favero for technical help. this work was supported by the italian miur. 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science and food production processes, and center for marine molecular biotechnology, qingdao national laboratory for marine science and technology, qingdao 266237, china 3hainan yazhou bay seed laboratory, sanya 572024, china this is an open access article published under the cc by license accepted october 18, 2022 abstract reactive oxygen species (ros) is a product of normal metabolism of aerobic cells, but excess ros is harmful to the organism. copper/zinc superoxide dismutase (cu-zn sod) is a metalloenzyme for scavenging ros. in this study, two extracellular cu-zn sod genes (designated as lvecsod1 and lvecsod2) were cloned by rapid amplification of cdna ends (race) technique. the cdna length of lvecsod1 is 801 bp, with an open reading frame (orf) of 555 bp, encoding a peptide of 184 amino acids. the cdna length of lvecsod2 is 934 bp with an orf of 678 bp, encoding a peptide of 225 amino acids. the predicted amino acid sequences of the two lvecsod both contained conserved four cu2+ binding sites and four zn2+ binding sites. the mrna scripts of lvecsod1 and lvecsod2 were widely detectable in the eyestalk, gill, gonad, heart, hemocytes, hepatopancreas, intestine, muscle, nerve, and stomach of litopenaeus vannamei. both lvecsod1 and lvecsod2 exhibited the highest expression levels in hemocytes and hepatopancreas. after the white shrimp was stimulated by white spot syndrome virus (wssv) or vibrio parahaemolyticus, the mrna expression levels of these two genes were up-regulated to varying degrees. the relative expression level of lvecsod2 was significantly increased after stimulation by two pathogenic microorganisms, which was generally later than that of lvecsod1. these results indicated that the two genes are both involved in the innate immunity of l. vannamei with different functions. key words: litopenaeus vannamei; extracellular cu-zn sod; innate immunity introduction reactive oxygen species (ros) are produced by aerobic organisms in the process of cellular oxygen metabolism, which includes superoxide anion (o-2), hydroxyl radical (oh) and hydrogen peroxide (h2o2) (nosaka and nosaka, 2017). previous studies have shown that ros were involved in various cell signaling pathways and played an important role in the elimination of harmful pathogens (zelko et al., 2002; yang et al., 2019). however, overproduction of ros, also known as oxidative stress, may lead to damage to ________________________________________ corresponding author: meng-qiang wang moe key laboratory of marine genetics and breeding (qingdao 266003), and key laboratory of tropical aquatic germplasm of hainan province of sanya oceanographic institute (sanya 572024) ocean university of china, china e-mail: wangmengqiang@ouc.edu.cn cell structure, such as dnas, proteins and lipids (lushchak, 2014). to keep ros at a normal concentration, aerobic organisms have evolved an antioxidant enzyme defense system composed of various enzymes, such as superoxide dismutase (sod, ec: 1.15.1.1), catalase, glutathione peroxidase and so on. among them, sod is an important enzyme for eliminating ros by converting superoxide radicals into hydrogen peroxide and oxygen (sheng et al., 2014). based on the metal co-factor, sods in eukaryotes could be classified into three isoforms: the cytosolic copper-zinc dimeric form, known as sod1 or cytcuznsod; the mitochondrial tetrameric manganese superoxide dismutase, as sod2 or mnsod; and the extracellular tetrameric cu-zn superoxide dismutase, as sod3 or ec-sod (mondola et al., 2016). among them, the two forms of cu-zn sod have high sequence and structural similarity to each other. ec-sod with an 127 n-terminal signal cleavage peptide for secretion which directs this enzyme exclusively to extracellular spaces is found in the extracellular matrix of tissues and the nucleus of human cells (lin et al., 2008). while cytcuznsod without a signal peptide is mainly found in the intracellular space, and it was also noted to be in the intermembrane space of mitochondria and in nuclei (okado-matsumoto and fridovich, 2001; zelko et al., 2002). ec-sod was first detected in human lymph, plasma, ascites, and cerebrospinal fluids (marklund et al., 1982). the expression pattern of ec-sod varies widely in different cell types and tissues, and its activity may exceed that of cytcuznsod and mnsod in some cells (zelko et al., 2002). subsequently, ec-sod gene has been reported in many species such as hydra vulgaris (dash et al., 2007), procambarus clarkia (meng et al., 2013), argopecten irradians (bao et al., 2009), phaedon cochleariae (gretscher et al., 2016), tribolium castaneum (ferro et al., 2017) and scylla serrata (lin et al., 2008). previous studies have shown that the relative mrna expression level of ec-sod in the hemocytes, hepatopancreas, and gills of procambarus clarkii significantly increased after being challenged by pathogenic microorganisms, suggesting that ec-sod plays an important role in the innate immune response of p. clarkia (meng et al., 2013). however, information on ec-sod from marine animals is still rare and more research is needed to demonstrate its potential roles. the pacific white shrimp (litopenaeus vannamei) is a worldwide farmed aquaculture species. the annual production of the pacific white shrimp reaches 4 million tons and accounted for 70% of the world's total shrimp production (shen et al., 2022; yin et al., 2023). however, with the expansion of aquaculture scale and the continuous increase of aquaculture intensity, the ecological environment of aquaculture is also deteriorating. the diseases of shrimp are becoming more and more serious and have caused great economic losses. white spot syndrome virus (wssv) and vibrio parahaemolyticus have been reported to be the main pathogenic bacteria causing large mortalities of shrimp and the shrimp farming industry has lost billions of dollars due to these two pathogens (zhang et al., 2022; zhou et al., 2022). as for invertebrate, the innate immune system is almost the only important defense line for shrimp against pathogenic microorganisms (han et al., 2020). as an enzyme related to the innate immune system in many invertebrates (lin et al., 2008; bao et al., 2009; ferro et al., 2017), ec-sod in the pacific white shrimp is still insufficient in research. in this study, we cloned two ec-sod genes from l. vannamei, examine the mrna expression of the two ec-sods in various tissues and investigated their expression profiles upon stimulation by the microorganisms wssv and v. parahaemolyticus, which would provide new insight into the function on this important, widespread and functionally diverse enzyme. materials and methods shrimp culture, tissues sample collection and rna isolation about 480 white shrimps with body weight 8-12 g, were obtained from ruizi seafood development co. ltd., qingdao, china, and acclimated at 20 ± 1 °c in 640 l cylindrical tanks with 500 l air-pumped circulating seawater (salinity 30 ‰) for two weeks before processing. tissues, including eyestalk, gill, gonad, heart, hemocytes, hepatopancreas, intestine (mid gut), muscle, nerve and stomach, were collected from at least fifteen shrimps and each sample was a mixture from three individuals. hemolymph was extracted from the abdominal sinuses of shrimps using a sterile syringe with equal volume of anticoagulation buffer (nacl 510 mmol l-1, glucose 100 mmol l-1, citric acid 200 mmol l-1, tri-sodium citrate 30 mmol l-1 and edta·2na 10 mmol l-1, ph 7.3), and then hemocytes were collected using centrifugation at 800 g for 10 min at 4 °c. wssv and v. parahaemolyticus challenge and sample collection wssv and v. parahaemolyticus were prepared according to the previous reports (yi et al., 2014). 480 shrimps were divided into three groups and each group of shrimps was cultivated in separate tanks. different groups of shrimps were injected with 100 μl of phosphate buffered saline (pbs, ph 7.4, 10010023, thermo fisher scientific, usa), v. parahaemolyticus suspension (1 × 104 cfus μl-1, in pbs) or wssv stock (1 × 104 copies μl-1, in pbs). at each time point of 0h, 3 h, 6 h, 12 h, 1 d, 2 d, 3 d, 4 d and 5 d post injection, hepatopancreas and hemocytes samples were extracted from fifteen shrimps and each sample was a mixture from three individuals. all samples were immediately stored in rnalater and stored at -80 °c until rna isolation. cloning the full-length cdna of lvecsod1 and lvecsod2 total rna was extracted from different samples using trizol reagent (15596026, thermo fisher scientific, usa). the synthesis of first strand was conducted by the promega m-mlv using the dnase i (rq1, m6101, promega, usa) treated total rna as template and adaptor primer-oligo (dt) as primer (table 1). partial length sequences of lvecsod1 and lvecsod2 cdnas was obtained from a transcriptome database of pacific white shrimp (qi et al., 2017; zhao et al., 2017). all primers were designed using primer premier 5.00 and all pcr amplification was performed in a miniamp thermal cycler (thermo fisher scientific, usa). the 3' end of lvecsod1 and lvecsod2 cdnas were obtained using rapid-amplification of cdna ends (race) technique. gene-specific primers, lvecsod1-race-f1/2 and lvecsod2-race-f1/2 (table 1), and adaptor primer-oligo (dt) were used in the semi-nested pcr for cloning the 3' end of lvecsod1 and lvecsod2. the coding sequence (cds) of lvecsod1 and lvecsod2 were amplified by 128 table 1 primer sequences used in this study name sequence (5’-3’) tm (°c) brief information adaptor primer ggccacgcgtcgactagtac 60 anchor primer for 3` race adaptor primer-oligo (dt) ggccacgcgtcgactagtact17vn olido (dt) for cdna synthetize lvecsod1-race-f1 tatcattttagacacgactgccattgt 64 gene specific primer for 3` race lvecsod1-race-f2 tatcagtgacaggcagcaccttgctgc 72 gene specific primer for 3` race lvecsod2-race-f1 gtcataggtcacgcttgagggatgtca 70 gene specific primer for 3` race lvecsod2-race-f2 tgacctgtaatgaagaacgaaacaaacgac 69 gene specific primer for 3` race lvecsod1-cds-f atgatgttggctggactcctgtgcctctca 76 gene specific primer for cds lvecsod1-cds-r ttaatagtattttgtttgtgtgtatcgctgggcttg 73 gene specific primer for cds lvecsod2-cds-f atgggactgatcacaccgttgcta 65 gene specific primer for cds lvecsod2-cds-r tcaagcgtgacctatgaccccaca 68 gene specific primer for cds lvef-1α-qrt-f gtattggaacagtgcccgt 60 internal control for real-time pcr lvef-1α-qrt-r catctccacagactttacctcag 60 internal control for real-time pcr lvecsod1-qrt-f cggacacttcaaccctctc 55 gene specific primer for real-time pcr lvecsod1-qrt-r gaataaggagatgcgagcca 58 gene specific primer for real-time pcr lvecsod2-qrt-f ggcaacgacgagagtttgaagac 63 gene specific primer for real-time pcr lvecsod2-qrt-r tcacatttgacctctgacatccctc 64 gene specific primer for real-time pcr m13-47 cgccagggttttcccagtcacgac 56 vector primer for sequencing rv-m gagcggataacaatttcacacagg 56 vector primer for sequencing gene-specific primers, lvecsod1-cds-f/r and lvecsod2-cds-f/r (table 1). the pcr products were gel-purified, ligated into the pmd18-t simple vector (d103a, takara, japan), and transformed into the competent escherichia coli dh5α (cb101-03, tiangen, china). positive clone was identified via anti-ampicillin selection and verified by pcr screening using m13-47 and rv-m primers (table 1) and then sequenced using a prism 3730xl automated sequencer (thermo fisher scientific, usa). bioinformatical analysis similarities to other ec-sod nucleotide and protein sequences was identified by the blast web server with default settings (http://www.ncbi.nlm.nih.gov/blast). the function domains of lvecsod1 and lvecsod2 were predicted by the smart program (http://smart.embl-heidelberg.de/) using default pattern definitions. the search for signal peptide was carried out by signalp 5.0 server (https://services.healthtech.dtu.dk/service.php?sig nalp-5.0/). the prediction of the potential n-glycosylation sites was performed by netnglyc 1.0 server (https://services.healthtech.dtu.dk/service.php?net nglyc-1.0/). euk-mploc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/euk-multi-2/) was employed to predict the subcellular localization of proteins. based on the selected cu-zn sod sequences (table 2), mega 7.0.21 was used for multiple sequence alignment and construction of neighbor-joining (nj) phylogenetic trees, in which bootstrap trials were replicated 1000 times. expression pattern analysis by quantitative real-time pcr quantitative real-time pcr (qpcr) technique was used to investigate the mrna expression patterns of lvecsod1 and lvecsod2 in different tissues and after stimulated with various microbes. all the primers for qpcr were designed using perlprimer 1.1.21 and listed in table 1. the mrna expression levels of lvecsod1 and lvecsod2 were normalized to those of elongation factor 1α (ef-1α) and processed using comparative ct method (2-δδct method) (schmittgen and livak, 2008; wang et al., 2011). the data were subjected to one-way analysis of variance (anova) followed by a multiple comparison using ibm spss statistics 26.0.0.0, and the p values less than 0.05 were considered statistically significant. results sequence analysis of lvecsod1 and lvecsod2 cdnas the full-length cdna sequences of two cu-zn sods were obtained by 3’ race technique and submitted to the genbank database under the accession number mf318887 and mf318886. the nucleotide and predicted amino acid sequences of lvecsod1 and lvecsod2 cdnas are shown in figure 1. lvecsod1 comprised 801 bp, containing a 5’ untranslated regions (utr) of 42 bp, a 3’ utr of 204 bp with a poly a tail and an open reading frame (orf) of 555 bp. its orf encodes a 184 129 table 2 sequences used in the alignment and phylogenetic tree accession no. species name type of cuznsod abbreviation bap28202 penaeus japonicus extracellular pjecsod efa10685 tribolium castaneum extracellular tcecsod abc25025 hydra vulgaris extracellular hvecsod acg80589 argopecten irradians extracellular aiecsod agh30392 procambarus clarkia extracellular pcecsod np_035565 mus musculus extracellular mmecsod np_001106630 xenopus tropicalis extracellular xtecsod np_037012 rattus norvegicus extracellular rnecsod 2jlp homo sapiens extracellular hsecsod qoe76459 rimicaris exoculate extracellular reecsod xp_001332758 danio rerio extracellular drecsod xp_015141186 gallus gallus extracellular ggecsod np_000445 homo sapiens cytoplasmic hsicsod np_777040 bos taurus cytoplasmic bticsod np_571369 danio rerio cytoplasmic dricsod aaw59361 salmo salar cytoplasmic ssicsod aci28282 cristaria plicata cytoplasmic cpicsod abd58974 azumapecten farreri cytoplasmic aficsod acm48346 argopecten irradians cytoplasmic aiicsod np_476735 drosophila melanogaster cytoplasmic dmicsod aap93581 apis mellifera ligustica cytoplasmic amicsod amino acid sequence with a calculated molecular mass of approximately 19.39 kda and theoretical isoelectric point (pi) of 7.72. four zn2+ binding sites (h-82, h-90, h-99 and d-102) and four cu 2+ binding sites (h-65, h-67, h-82 and h-141) were found in the amino acid sequence. the smart program revealed that the lvecsod1 possessed a conserved cu-zn sod domain at position 45-164. the full-length cdna of the lvecsod2 was 934bp, containing a 5’ utr of 48bp, a 3’ utr of 208 bp with a poly a tail and a 678bp orf encoding a 225 amino acid sequence with a predicted molecular mass of approximately 23.24 kda and theoretical pi of 6.22. the lvecsod2 protein possessed two potential n-linked glycosylation sites (n110lsp and n171itd). four zn2+ binding sites (h-136, h-144, h-153 and d-156) and four cu 2+ binding sites (h-119, h-121, h-136 and h-193) were found in the amino acid sequence. signalp and targetp predicted that lvecsod1 contained a putative 17 amino acid signal peptide and lvecsod2 contained a putative 21 amino acid signal peptide. these results combined with the prediction of euk-mploc 2.0 indicated that these sods are two extracellular enzymes. multiple sequence alignment and phylogenetic analysis multiple sequence alignment revealed that lvecsod1 and lvecsod2 share high similarity with previously identified ec-sods and are conserved in zn2+ and cu2+ binding sites (fig. 2). in order to clarify the evolutionary positions of lvecsod1 and lvecsod2, a phylogenetic tree was constructed based on the amino acid sequences of ec-sod and cytcuznsod from different species using the neighbor-joining method (fig. 3). these results showed that ec-sod of different species, including lvecsod1 and lvecsod2, were clustered together, and cytcuznsod was clustered in other groups. in the subgroup of ec-sod, lvecsod1 was clustered with ec-sod from the kuruma shrimp penaeus japonicus and lvecsod2 was clustered with ec-sod from the hydrothermal vent shrimp rimicaris exoculata. tissue distribution of sod expression as shown in figure 4, lvecsod1 and lvecsod2 was widely detectable in the eyestalk, gill, gonad, heart, hemocytes, hepatopancreas, intestine, muscle, nerve and stomach of l. vannamei. the highest mrna transcripts level of lvecsod1 was detected in hemocytes, which was 410.9-fold of that in muscle (p < 0.05), followed by hepatopancreas, which was 46.72-fold of that in muscle (p < 0.05). the highest expression of the lvecsod2 was detected in hepatopancreas, which was 1026.39-fold of that in muscle (p < 0.05), then in hemocytes, which was 72.39-fold of that in muscle (p < 0.05). 130 fig. 1 nucleotide and deduced amino acid sequences of lvecsod1 (a) and lvecsod2 (b), the translational start codon and stop codon are bolded. the signal peptide is underlined and the italic and bold letters represent the amino acids required for cu2+ and zn2+ binding. the cu-zn sod family signature sequence is boxed and the predicted n-link glycosylation sites are double underlined 131 fig. 2 multiple sequence alignment of ec-sod proteins with those from other animals. taxa information are shown in table 2. the same amino acid residues are shaded in black and similar amino acids are shaded in grey. zn2+ and cu2+ binding site are marked by (*) lvecsod1 and lvecsod2 mrna expression analysis post wssv and v. parahaemolyticus stimulation the mrna expression levels of lvecsod1 and lvecsod2 mrna were investigated in hemocytes and hepatopancreas after stimulation with two pathogenic microorganisms. after being stimulated by v. parahaemolyticus, the mrna expression levels of lvecsod1 and lvecsod2 in hemocytes were significantly up-regulated after 3h and 6h (8.28-fold and 20.70-fold compared with time 0 of the control group, p < 0.05), and reached the highest level at 6h and 12h (38.09-fold and 168.53-fold, p < 0.05). in hepatopancreas, the mrna transcripts of lvecsod1 were significantly up-regulated and reached the highest level after 3h (7.82-fold, p < 0.05), while the mrna transcripts of lvecsod2 was significantly up-regulated after 12h (14.83-fold, p < 0.05) and at 1d reached to the peak (63.87-fold, p < 0.05). after being stimulated by wssv, the mrna expression levels of lvecsod1 in both hemocytes and hepatopancreas were significantly up-regulated after 6h (5.14-fold and 18.38-fold, p < 0.05), and reached the highest at 12h (17.95-fold and 59.99-fold, p < 0.05). as for lvecsod2, the mrna expression levels in hemocytes and hepatopancreas were significantly up-regulated and reached to the peak at 1d and 2d (11.91-fold and 5.11-fold, p < 0.05). the transcripts of these two ec-sod mrnas dropped back to the original level in all tissues after 3d. no significant change in the control group was observed throughout the experiment (fig. 5). discussion sod enzymes play an important role in both controlling ros damage and regulating ros signaling in aerobic organisms (wang et al., 2018). in this study, we characterized and compared two cu-zn sod genes in an important aquaculture species, l. vannamei, and then analyzed their mrna expression profiles of them. based on the signal peptide prediction by signalp and targetp software and subcellular localization prediction by euk-mploc 2.0, the two sod enzymes were determined to be two extracellular enzymes. these two genes all have the conserved cu2+ binding sites and zn2+ binding sites, indicating that they have cu-zn sod family characteristics. high similarity with other identified ec-sod and the phylogenetic relationship collectively suggested that lvecsod1 and lvecsod2 were two novel members of invertebrate cu-zn sod family, and it could have similar functions to those from other invertebrates. the lvecsod1 and lvecsod2 exhibited only 37.94 % and 33.36 % similar to a previously studied lvecsod, which indicates the diversity of ec-sod genes in l. vannamei (tian et al., 2011). two ec-sod genes were also identified in marsupenaeus japonicus and they have different functions against pathogenic microorganisms wssv and v. parahaemolyticus challenge (hung et al., 2014). we deduced that the redundancy of gene products for ec-sods in l. vannamei might have contributed to the functional specialization in the innate immunity system, which could increase their survival during microbial infections. 132 fig. 3 consensus neighbor-joining phylogenetic based on the protein sequences of ec-sod and cytcuznsod from different animals. taxa information are shown in table 2. to derive the confidence value for the phylogeny analysis, bootstrap trials were conducted 1000 replicates. the numbers at the forks indicate the bootstrap value the results of qpcr showed that lvecsod1 and lvecsod2 were expressed in various tissues of shrimps, and the highest relative expression levels were observed in hemocytes and hepatopancreas, which was significantly different from other tissues. similarly, in p. clarkii, ec-sod was expressed highest in hepatopancreas and hemocytes (meng et al., 2013). previous studies have shown that hepatopancreas and hemocytes are considered to be the main immune organ and immune cells in crustaceans (wang et al., 2013; wang et al., 2017). moreover, the hepatopancreas was also regarded as the main organ in which multiple oxidative reactions and antioxidant defenses occur with high metabolic activity (wang et al., 2015). the higher mrna expression level of lvecsod1 and lvecsod2 in hemocytes and hepatopancreas implied that they might play important roles in the innate immunity and detoxification system in pacific white shrimps. additionally, the basal mrna expression level of lvecsod1 in hemocytes was higher than that of lvecsod2 by approximately five times, which indicated that lvecsod1 might play a more routine role in the physiological activity of pacific white shrimps. fig. 4 tissue distribution of lvecsod1 (a) and lvecsod2 (b) mrna transcripts detected by qpcr technique. the mrna transcripts levels in eyestalk, gill, gonad, heart, hemocytes, hepatopancreas, intestine, muscle, nerve and stomach of three untreated shrimps were normalized to that of muscle. the ef-1α gene was used as an internal control to calibrate the cdna template for each sample. vertical bars represent mean ± sd (n = 3), and bars with different characters were significantly different (p < 0.05), while bars with same characters were not significantly different 133 fig. 5 temporal mrna expression profiles of lvecsod1 in hemocytes (a) and hepatopancreas (b), and lvecsod2 in hemocytes (c) and hepatopancreas (d), which were detected via qpcr technique at 0h, 3h, 6h, 12h, 1d, 2d, 3d, 4d and 5d post white spot syndrome virus (wssv) and vibrio parahaemolyticus stimulation. the ef-1α gene was used as an internal control to calibrate the cdna template for each sample. vertical bars represent mean ± sd (n = 3), and bars with different characters were significantly different (p < 0.05), while bars with same characters were not significantly different it has been reported that v. parahaemolyticus and wssv could activate the shrimp's antioxidant defense mechanisms, which include sod scavenging of reactive oxygen species (ji et al., 2011). the depletion of sod makes the host express more sod to compensate, so the relative expression of mrna will increase significantly (jiravanichpaisal et al., 2006). in the present study, both two ec-sod gene expressions were significantly up-regulated after shrimp stimulation with v. parahaemolyticus and wssv pathogens. after stimulated by wssv, the expressions of both two genes started to be up-regulated were later than stimulated by v. parahaemolyticus. different invading microorganisms induce different toxicity and produce different amounts of oxygen-derived products in the host. in chlamys farreri, the temporal expression patterns of cytcuznsod challenged by listonella anguillarum, micrococcus luteus and candida lipolytica were different (ni et al., 2007). the relative expression of lvecsod2 was significantly increased after stimulation by two pathogenic microorganisms, which was generally later than that of lvecsod1, which shows that the two different ec-sods might have specific functions in shrimp innate immunity. ros have the effect of killing pathogenic microorganisms in the phagosome and play an important role of immune signaling pathway in innate immune system (o'neill et al., 2015; martinvalet and walch, 2021). therefore, a small amount of ros may be beneficial for the innate immunity of l. vannamei, appropriately increase the concentration of ros to allow them to survive the attack of pathogens, and then secrete sod to remove ros when the concentration of ros is too high, which might be the reason for the slower response of lvecsod2. the role of sod’s can be viewed as regulators of ros, the difference in expression of two ec-sod genes in different tissues or in response to different pathogenic stimuli reflects the precise control of ros by sod. our research further verified that two lvecsods participated in the immune response as a part of response against the pathogen. 134 in conclusion, we cloned and characterized the full-length cdnas of 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y, zhou q, et al. molecular characterization and expression ananlysis of a qm protein gene from chinese shrimp fenneropenaeus chinensis. invert surviv j. 19: 115-125, 2022. zhou q, wang y, hu j, zhang l, li j, xu y, et al. identification and characterization of a cyclophilin a gene from chinese shrimp fenneropenaeus chinensis: sequence features and expression profiles. invert surviv j. 19: 105-114, 2022. 2 isj 15: 2-13, 2018 issn 1824-307x research report molecular cloning of the heat shock protein 90 gene in scallop mizuhopecten yessoensis and the effects of temperature stress on gene expression j ding1*, h wang1*, c yin1, xw zhao2, x sun2, xh liu1, ls han1, yq chang1 *equal contributors 1key laboratory of mariculture& stock enhancement in north china's sea, ministry of agriculture, dalian ocean university, dalian, liaoning, 116023, p.r. china 2zhangzidao group co. ltd (zoneco), dalian, liaoning, 116001, p.r. china accepted november 24, 2017 abstract mizuhopecten yessoensis, one of the most highly favored scallops on the international market, has suffered from massive summer mortalities. water temperature is an important environmental stressor which has significant effects on the physiological and biochemical response of scallops. heat shock protein 90 (hsp90) plays a key role in defense against various environmental stresses that could damage the cellular and molecular structure of cells. in this study, molecular characterization and expression of hsp90 in m. yessoensis (designated myhsp90) were analyzed as an indicator to understand the mechanisms of heat shock response in m. yessoensis under temperature stress. the full-length sequence of myhsp90 cdna (genbank accession no. mf196912) is composed of 2639 base pair encoding a 726-amino acid polypeptide with a predicted molecular mass of 83.26 kda. tissue expression analysis of myhsp90 genes revealed ubiquitous expression in each tissue examined, and showed a temperature-dependent response, and the expression level was up-regulated significantly in all the six tissues tested (p < 0.05) except gonad. the results will be useful in furthering the understanding of the massive summer mortalities in m. yessoensis under temperature stress. additionally, myhsp90 could be used as a potential biomarker in practice to monitor environmental changes in future studies. key words: mizuhopecten yessoensis; scallop; heat shock protein 90; cloning and expression; temperature stress introduction scallops are one of the most valuable marine resources in china. the scallop mizuhopecten yessoensis (or patinopecten yessoensis) is a cold-water species and found around the northwestern pacific coast of china, korea, japan and russia (hou et al., 2011). it is one of the most highly favored scallops on the international market, and the global capture production of m. yessoensis is 366 kilotons with a total value over us $1.5 billion in 2014 (fao, 2014) while the aquaculture production for this species is195 kt. m. yessoensis was introduced from japan to china by liaoning ocean and fisheries research institute in 1982 ___________________________________________________________________________ corresponding author: yaqing chang key laboratory of mariculture& stock enhancement in north china's sea ministry of agriculture dalian ocean university dalian, liaoning, 116023, p.r. china email: yaqingchang@hotmail.com (wang, 1984), and aquaculture of this species has been growing rapidly since its introduction because it has a larger size and a higher market price than other native scallops. it is cultured only in the northern province, liaoning, and the north side of the shandong peninsula (shumway and parsons, 2015), and it has become a major aquaculture industry in these areas. however, this species has difficulties with the high summer temperatures found in the bohai sea of china, which often reaches 25 °c – 28 °c, and it has been suffering massive summer mortalities since 1998, which causes a 37 % decline in scallop production (mac, 1999). water temperature is an important environmental stressor which could have significant effects on the survival, growth and reproduction of marine organisms (bruc et et al., 2012; bian et al., 2014), as well as effect relevant gene expression in molluscs (farcy et al., 2007; fearman and moltschaniwskyj, 2010; jiang et al., 2016). high temperatures, especially, affects the mortalities of cold-water scallop species m. yessoensis as 3 described previously. therefore, it is important to investigate the mechanisms of the heat shock response in m. yessoensis under temperature stress, and the current study focuses on heat shock proteins. heat shock proteins(hsps) are a family of proteins that were discovered in drosophila after exposure to high temperatures caused the genes to appear as chromosome puffs, hence the name heat shock in 1962 (ritossa, 1962). subsequently, many hsps and their genes have been characterized (fehrenbach and niess, 1999; lindquist 2003; sørensen et al., 2003). hsps are highly conserved proteins which are found in all organisms from bacteria to plants and animals (kiang and tsokos, 1998; sørensen et al., 2003). as molecular chaperones, hsps are evolutionary important and play a role in the protection of various environmental stresses which could damage cellular and molecular structures in cells (sørensen et al., 2003; peng et al., 2016). hsps are a superfamily that are often classified according to their molecular weight, such as the hsp70 family (molecular weight approximately 66 78 kda), the hsp90 family (molecular weight 83 110 kda), the hsp33 family, the hsp60 family, and the small molecular weight family (molecular weight 12 43 kda) (sørensen et al., 2003; li et al., 2004). among the hsp family, hsp70has been shown to be one of the most sensitive to thermal stress (uhlinger et al., 1998; hamdoun et al., 2003). in invertebrates, hsp70 has been cloned from different species of molluscs (boutet et al., 2003; franzellitti and fabbri, 2005; farcy et al., 2007; gunter and degnan, 2007; zhang and zhang, 2012; liu et al., 2015), and in scallops, such as argopecten irradians, placopecten magellanicus and patinopecten yessoensis, heat stress induces the over-expression of hsp70, (brunet al., 2008, jiang et al., 2016). hsp90 is another highly conserved member (csermely et al., 1998), weighing roughly 90 kda. hsp90 is highly conserved and expressed in a variety of different organisms from bacteria to all branches of eukarya (chen et al., 2006). unlike with hsp70, only a few studies have demonstrated that thermal stressors stimulate hsp90 expression in molluscs (choi et al., 2008; liu et al., 2015), including two species of scallop azumapecten (chlamys) farreri and a. irradians (gao et al., 2007, 2008). however, the hsp90 response has not been investigated in m. yessoensis under high-temperature stress. to investigate the mechanisms of heat shock response in m. yessoensis under temperature stress, it is important to realize the existence of hsp90 in this species first. therefore we cloned and characterized the full-length sequence of hsp90cdna in m. yessoensis (designated myhsp90), and studied the expression pattern of myhsp90 in each tissue, as well as defining the molecular regulation of myhsp90 mrna expression under temperature stress. materials and methods animals and rearing conditions the scallops m. yessoensis were bred at dalian ocean university, china. they were grown around zhangzi island for two years. healthy m. yessoensis, averaging 8.0 ± 2.0cm in shell length, were transported back to the laboratory of dalian ocean university and acclimated in culture tanks containing aerated sand-filtered seawater (salinity: 30 ‰, ph 8.2) at 15 ± 1 °c for 7 days. before the test, the scallops were fed a powder of spirulina and sargassum thunbergii, and seawater was changed completely twice a day. temperature stress test and sample collection the scallops were randomly divided into five groups of 10 individuals, and each group was treated at 15 (as control), 20 °c, 22 °c, 24 °c and 26 °c for 7 days, respectively, while each temperature was replicated three times (n = 3). three scallops from each replicated group were sampled at each test temperature. then the tissues (including gonad, adductor muscle, mantle, blood/hemocytes, gill, and kidney) were excised and stored at −80°c until use. scallop blood/hemocytes (hemolymph) was collected from the adductor muscle using a 20 g-needle and syringe, and then centrifuged at 800g, 4 °c for 10 min to obtain the hemocytes. the blood/hemocytes pellets were immediately stored at −80 °c until use. table 1 nucleotide sequences of primers used in this study primers sequence (5'→3') usage 3’, 5’ race myhsp90-f1 acatggctgccaagaaacatctagaga 3’ race primer myhsp90-r1 gagccaccagcagatgattcccagat 5’ race primer ump-1 taatacgactcactatagggcaagcagtggtatcaacgcagagt 3’/5’ race primer ump-2 ctaatacgactcactatagggc 3’/5’ race nested primer m13f tgtaaaacgacggccagt colony pcr m13r caggaaacagctatgacc colony pcr qpcr myhsp90-f2 tctgggaatcatctgctgg qpcr primer myhsp90-r2 gattgggtaaccgatgaactg qpcr primer cytb-f attcggattcaggaagtggc reference primer cytb-r atttggtccagcatgtc reference primer race: rapid amplification of cdna ends; qpcr: quantitative real-time polymerase chain reaction; cytb: cytochrome b. 4 table 2 the homology analysis between mizuhopecten yessoensis and other representative species for hsp90 species genbank accession no. identity (%) molluscs (14) mizuhopecten yessoensis mf196912 azumapecten farreri aar11781.1 97.66 argopecten irradians abs50431.1 94.75 crassostrea gigas ekc25687.1 85.87 crassostrea hongkongensis adl59936.1 85.46 scapharca broughtonii alz42089.1 83.75 ruditapes philippinarum ahy27548.1 83.33 cellana toreuma agh32328.1 80.47 hyriopsis cumingii alm87690.1 80.17 laternula elliptica acf35426.1 80.72 mytilus coruscus all27016.1 83.37 mytilus galloprovincialis caj85741.1 82.96 paphia undulata afz93093.1 82.37 corbicula fluminea amm04544.1 81.96 insects (3) plutella xylostella aha36864.1 77.27 mythimna separata aby55234.1 65.27 bemisia tabaci ado14474.1 75.56 nematodes (2) bursaphelenchus mucronatus adk26462.1 76.41 heterodera glycines aao14563.2 73.37 crustaceans (3) penaeus monodon abm54577.1 78.06 eriocheir sinensis aha61463.1 77.99 macrobrachium nipponense adk66920.1 76.31 teleosteans (4) danio rerio np_571385.2 75.17 salmo salar aad30275.1 75.48 oncorhynchus mykiss cdq59193.1 76.11 megalobrama amblycephala agi97008.1 75.76 reptiles (2) pelodiscussinensis xp_006120052.1 76.72 alligator mississippiensis np_001274200.1 76.58 aves (1) gallus gallus np_996842.1 77.24 mammals (3) homo sapiens np_031381.2 77.21 mus musculus np_032328.2 77.21 bos taurus np_001073105.1 77.49 total rna extraction and reverse transcription total rna was extracted from six tissues of m. yessoensis with a rnaprep pure kit (for tissue) (tiangen biotech, beijing, china) according to the manufacturer’s protocol. rna quantity and purity were performed at 260/280 nm and 260/230 nm absorbance ratios using a nv3000micro-spectrophotometer (vastech inc., wilmington, de, usa), while the integrity was verified with electrophoresis on 1 % agarose gels. sample concentrations were determined to ensure sufficient rna for complementary dna (cdna) synthesis. the cdna was synthesized from total rna from each sample using smarter race 5′/3′ kit (clontech laboratories, inc., palo alto, ca, usa). the cdna was diluted to 1:50 and stored at −20°c for subsequent cloning and expression analysis. cloning the full-length cdna of myhsp90 using rapid-amplification of cdna ends (race) pcr partial sequences of myhsp90 cdna were obtained from the splicing transcriptome of the scallop m. yessoensis established by our laboratory (ding et al., 2015), and the gene-specific primers were designed according to the partial sequences. the nucleotide sequences of all the primers used in this study are shown in table 1, and the primers were obtained from sangon biotech (shanghai, china). the myhsp90 cdna from the gill of m. yessoensis was used as a template for cloning. the 5 pcr was performed using a gradient mastercycler (eppendorf ag, hamburg, germany) in a total volume of 50 μl, comprising 25 μl of 2 ×seqamp buffer, 2.5 μl of cdna template, 5 μl of 10×upm, 1μl of myhsp90-f1 or myhsp90-r1 (10 μm), 1μl of seqamp dna polymerase and 15.5 μl distilled water. the pcr program was as follow: 30 cycles of 94°c for 30s, 65 °c for 30 s, and 72 °c for 1 min; the nested pcr program was as follow: 30 cycles of 94 °c for 30 s, 55 °c for 30 s, and 72 °c for 30 s. the pcr products were analyzed with electrophoresis on 1.0 % agarose gel, and the expected bands were purified using easypure quick gel extraction kit (transgen biotech, beijing, china). the target amplified cdna fragments were subcloned into the peasy-t1 cloning vector (transgen biotech) and transformed into trans-t1 phage resistant chemically competent cells (transgen biotech) following the manufacturer’s protocol. the positive clones were identified with colony pcr with m13 primers (m13f and m13r, table 1), and sequenced at sangon biotech (shanghai, china). bioinformatic analysis the nucleotide sequence of the full-length myhsp90 cdna sequence and deduced amino acid (aa) sequence were analyzed in the basic local alignment search tool (blast) program at the national center for biotechnology information (https://www.ncbi.nlm.nih.gov/blast/). the open reading frame (orf) was identified with orf finder (https://www.ncbi.nlm.nih.gov/orffinder/). the deduced aa sequence information was analyzed with the expert protein analysis system (http://www.expasy.org/). the physical and chemical parameters of the deduced protein were computed using the protparam tool (http://web.expasy.org/protparam/), and the computed parameters included the molecular weight, theoretical isoelectric points (pi), instability index, and grand average of hydropathicity (gravy) (gasteiger et al., 2005). the presence and location of the signal peptide cleavage site in the aa sequence were predicted with signalp 4.1 server (http://www.cbs.dtu.dk/services/signalp/) (petersen et al., 2011). the functional domain and important sites of the protein were predicted by interpro (http://www.ebi.ac.uk/interpro/).due to the absence of the x-ray crystal structure of scallop hsp90 in the protein data bank (pdb, http://www.rcsb.org/pdb/home/home.do), homology modeling was performed to find the three-dimensional (3d) structure of the protein from its primary sequence. the template protein was human hsp90 (pdb code: 5fwp chain a (verba et al., 2016) with a high resolution (3.9 å). the homology structure models of the scallop hsp90 were generated using the program swiss-model, automated protein structure homology-modeling server (https://www.swissmodel.expasy.org/) with all default parameters (arnold et al., 2006; guex et al., 2009; biasini et al., 2014). multiple sequence alignment and phylogenetic analysis multiple sequence alignment of the deduced aa sequence of myhsp90 was performed using the clustalw program (http://www.ch.embnet.org/software/clustalw.html). the phylogenetic tree was constructed according to aa sequences of the selected hsp90 (table 2) using the neighbor-joining (nj) method in mega 6.0 software and bootstrap analysis (1,000 times) to evaluate the reliability of the phylogenetic trees (saitou and nei, 1987). quantitative analysis of myhsp90 mrna expression total rnas were pooled from three m. yessoensis scallops, and the relative levels of myhsp90 mrna transcripts in different tissues of the scallop were analyzed with quantitative real-time pcr (qpcr), which was conducted with the applied biosystems 7500 real-time system (applied biosystems, foster city, ca, ca, usa). the reaction conditions were 94 °c for 5 min, followed by 28 cycles of 94 °c for 30 s, 60 °c for 30 s, and 72 °c for 30 s. the final extension step was at 72 °c for 5 min. three independent biological replicates were carried out, and the dissociation curve of the amplicon was analyzed to confirm that there was only pcr product in each reaction. the cytochrome b gene (cytb) was used as the internal reference gene. after being normalized to the cytb gene, the relative expressions of myhsp90 mrna were calculated with the 2−δδct method (livak and schmittgen, 2001). statistical analysis all data were expressed as the mean± sd. all analyses were performed using the analysis of variance (anova) of the spss software (version 16.0). p < 0.05 was considered statistically significant. results molecular cloning and characterization of myhsp90 cdna in m. yessoensis the nucleotide and deduced aa sequence of myhsp90 cdna are shown in figure 1. the cdna sequence of myhsp90 was submitted to genbank (accession no. mf196912). the full-length of myhsp90cdnawas 2639 base pair (bp), containing a 54-bp 5′-terminal untranslated region (utr), a 2178-bp orf, and a 406-bp 3′-utrwith a poly(a) tail. the orf encoded a 726-aa polypeptide with a predicted molecular mass of 83.26kdaand a theoretical pi of 4.81. the instability index was computed to be 39.78, which classified the protein as stable. the grand average of hydropathicity of this protein was −0.698. the deduced aa sequence of myhsp90 lacked a typical signal peptide as predicted by signalp 4.1 server. interpro program analysis revealed that there was a highly conserved hsp90 site (ysnkeiflre) and hsp90 family signature motifs (meevd) at the c-terminus in the myhsp90 sequence. a typical histidine kinase-like atpase domain was located from position 14-228 aa. a ribosomal protein s5 domain and hsp90 c-terminal domain was located from position 258-540 aa and 564-685 aa, respectively (fig. 1). https://www.ncbi.nlm.nih.gov/blast/ https://www.ncbi.nlm.nih.gov/orffinder/ http://www.expasy.org/ http://web.expasy.org/protparam/ https://www.swissmodel.expasy.org/ 6 fig. 1 nucleotide sequence of the myhsp90 gene and its deduced amino acid sequence from mizuhopecten yessoensis (genbank accession no. mf196912). the amino acid sequence is shown with one letter codes below the nucleotide sequence in blue. hsp90 conserved site (ysnkeiflre) and hsp90 family signature motifs (meevd) are indicated in underline. histidine kinase-like atpase, atp-binding and hsp 90 n-terminal domain are shaded in gray. ribosomal protein s5 domain and hsp90 c-terminal domain are indicated in wavy lines and double underlines, respectively. the primary sequence alignment of the aa showed that the rate of consistency between the target (myhsp90) and the template (human hsp90β) protein was 77.21 % (fig. 2a). the 3d structure ofmyhsp90 was modeled using swiss-model workspace at the expasy server (fig. 2b). multiple sequence alignment and phylogenetic analysis the deduced aa sequence of myhsp90 shared high homology with those of other representative speciesincluding14 molluscs, three insects, two nematodes, three crustaceans, four teleosteans, two reptiles, one bird and three mammals. the species 7 fig. 2 (a) alignment of the sequence of template human heat shock protein 90 (hsp) (pdb id: 5fwp) chain a with the target protein heat shock protein 90 of mizuhopecten yessoensis. the sequence identity is 77.21%. conserved residues are represented by asterrisk (*), strong and weak conserved amino acids are denoted by (:) and (.), respectively. gaps are represented by (-); (b) the three-dimensional (3d) structure of myhsp90 in m. yessoensis, modeled using swiss-model a) b) 8 fig. 3 neighbor-joining phylogenetic tree of myhsp90 amino acid sequences and other representative species. the representative species names and the genbank accession numbers are the same as those in table 2. numbers on the nodes indicate the confidence level of the bootstrap analyses name, the genbank accession numbers, and identity are listed in table 2. searching for sequence similarities revealed that myhsp90 shared more than 73 % similarity in all the matches except with mythimna separata. similarity with the hsp90s of other molluscs was over 80 %, myhsp90 presented the highest identity with two other species of scallops, sharing 97.66 % identity with a. farreri (aar11781.1) and 94.75 % with a. irradiants (abs50431.1), respectively. in mollusks, myhsp90 displayed high similarity to other species and all the levels of identity were higher than 80 %. multiple sequence alignment of myhsp90 with other hsp90s showed that they were highly conserved, especially in the regions of hsp90 family signatures. to examine the relationship between various hsp90, phylogenetic trees were generated with the mega 6.0 nj methods (fig. 3). the resulting phylogenetic treeswere composed of two major large clusters, including the eight major groups: mammals, aves, reptiles, teleosteans, crustaceans, nematodes, insects, and molluscs. myhsp90 was divided into the mollusc cluster. 9 fig. 4 relative expression level of myhsp90 mrna in different tissues by quantitative real-time pcr analysis. the tissues include gonad, adductor muscle, mantle, blood, gill, and kidney. after normalization to cytochrome b (cytb), relative expression of myhsp90 mrna were calculated with 2-δδct method. bars represent the mean ± sd. different letters indicated significant differences (p < 0.05) relative expression of myhsp90 mrna in different tissues myhsp90 mrna expressions were quantified in different tissues with qpcr with cytb as an internal reference gene and expressed values with the mean of the gonad set as 1 (fig. 4). myhsp90 mrna was expressed in each of the six tissues examined, and the relative expression level was from high to low in kidney > gill > gonad > mantle > blood > adductor muscle. there was strong expression in both the gill and kidney, which was much higher than other tissues (p < 0.05), and there was no significant difference among the gonad, adductor muscle, mantle and blood (p > 0.05). expression level of myhsp90 mrna under temperature stress myhsp90 mrna expression levels in different tissues responded differently to temperature stress (fig. 5). myhsp90 mrna expression was temperature-dependent following temperature stress. in the gill, myhsp90 expression was the highest level at 26 °c, and the maximal expression level was 130 times higher than that of the control temperature (15 °c) (p < 0.05). in the kidney and mantle, the maximal expression levels at 26 °c were 100 times and 70 times higher than the control. compared to other tissues, myhsp90 mrna expression was lowest at 15 °c – 26 °c in the gonad, though the maximal was 10-fold that of the control group, no significant differences were found between the heat shock treatment and the control treatment (p > 0.05). at 24 °c, the expression level peaked in gonad and muscle, 10-fold and 40-fold of the control group, respectively. discussion in the present study, we obtained and characterized the full-length cdna sequence of the myhsp90 gene from the gill of m. yessoensis using race methodology, and studied the expression pattern of myhsp90 in different tissues in order to investigate the mechanism of heat shock response under temperature stress. this is the first time the expression of myhsp90 has been investigated under temperature stress. the full-length of myhsp90 cdna was 2639 bp and encoded a 726-aa protein. unlike the hsp70 protein which contains three hsp family signature sequences, there was only one signature sequence in the hsp90 protein. similar to other hsp90 family members, myhsp90 contained highly conserved sequences, an atp-binding domain in the n-terminal, a family signature sequence (meevd) in the c-terminal and a ribosomal protein s5 domain, as well as major structure and functional domains (gupta,1995; caplan, 1999), which is consistent with other studies (prodromou et al., 1997; gao et al., 2007, 2008).based on the presence of the c-terminal meevd sequence (ahn et al., 2003), the myhsp90 was concluded to belong to the hsp90 family. multiple sequence alignment and homology analysis revealed that the deduced aa sequence of myhsp90 shared high similarity with thehsp90 proteins of other eukaryotic organisms (65.27 % 97.66 %). gao et al. (2007, 2008) obtained the full-sequence of hsp90 cdna from the zhikong scallop a. farreri and bay scallop a. irradians. myhsp90 shared higher identities with a. farreri 10 fig. 5 myhsp90 mrna expression levels in different tissues under various temperatures stress. each group of mizuhopecten yessoensis was treated at 15 (as control), 20, 22, 24 and 26 °c for 7 days respectively, and each temperature was replicated three times (n = 3). after normalization to cytochrome b (cytb), the relative expressions of myhsp90 mrna were calculated with 2-δδct method. vertical bars represent the mean ± sd (97.66 %) and a. irradians (94.75 %). the sequence alignment and phylogenetic analysis further suggested that the myhsp90 was a member ofthehsp90 family. there are two different subtypes of hsp90 in vertebrates (hsp90α and hsp90β), which are different in the structure of glutamine-rich sequence (qtqdq) at the n-terminus, a site of phosphorylation by a dsdna-dependent kinase (lees-miller and anderson, 1989; theodoraki and mintzas, 2006; manchado et al., 2008). hsp90α plays an important role in cell growth, apoptosis, and the cell cycle. hsp90β is involved in maintaining normal physiological cell function through its role in cell differentiation and maintenance of cell structure and defense. in contrast, there is only one hsp90 gene in most invertebrates. myhsp90 lacks the qtqdq sequence at the n-terminus and shared higher similarity with hsp90β. these facts suggest that myhsp90 is more closely related to the vertebrate hsp90β isoform. there is presently no crystal structure of scallop hsp90 deposited in pdb, hence, in this study, a homology model was performed to determine the 3d structure of myhsp90. using blast search, homo sapiens hsp90was selected as the template (pdb id: 5fwp), which had an identity of 77.21 %. the cartoon representation of myhsp90 is depicted in fig. 2. all alpha-helices and beta-sheets, and the backbone structure resembling the same alignment that had been found in the template structure. based on the present knowledge of the hsp90 structure in other organisms (e.g., human, yeast), hsp90 is an intact dimeric chaperone which forms homodimers, and each monomer has three structural domains: a c-terminal domain (ctd) responsible for dimerization; a middle domain (md) implicated in client binding; and the n-terminal domain (ntd) that binds atp (meyer et al., 2003). in the present study, tissue expression analysis of the myhsp90 gene revealed ubiquitous expression in each tissue examined, indicating that myhsp90 was synthesized under control conditions (15 °c). myhsp90 mrna expression was detected in all tissues tested in m. yessoensis, though there was no significant difference among the tissues examined in this study (p > 0.05). the myhsp90 gene was expressed at a higher level in the gill and kidney, and at a lower level in the adductor muscle and blood (or hemocytes) (fig. 4), which is consistent with previous studies in abalone haliotis diversicolor hsp90 (huang et al., 2014) and clam ruditapes philippinarum (liu et al., 2015). in contrast, the hsp90 gene expression level was highest in ovary when compared with other tissues tested (hepatopancreas, hemocytes, gill and muscle) in shrimp and crab (jiang et al., 2009; zhang et al., 2009; li et al., 2012). myhsp90 expressed in scallop gonad, which implied that hsp90 might have a certain relationship with genital organ development and maturation. 11 heat shock stress could cause denaturation and aggregation of proteins, disrupt the integrity of essential organelles, and inhibit vital processes such as transcription and mrna translation (richardson et al., 2011). however, as a general molecular chaperone, the function of hsp90 is in promoting proper folding or refolding and preventing potentially damaging interactions or protein aggregations, and in assisting the disassembly of already formed protein aggregates (pratt and toft, 1997; wang et al., 2008). m. yessoensis is a cold-water shellfish with an optimum growth temperature of 15 °c. the metabolic activity and feeding behavior of this species decreased greatly when the ambient environment temperature is above 23 °c (chang 2007). the scallops were able to adapt to temperatures between 15 °c and 22 °c and maintain a survival rate >82 % (hao et al., 2014). after the scallop was exposed to heat shock treatment (15 °c – 26 °c) for 7 days, the expression level of myhsp90 mrna in all the six tissues tested except gonad was up-regulated significantly (p < 0.05) (fig. 5). the different myhsp90 mrna expression levels in these tissues may be caused by different sensitivity to temperature stress. the expression level showed a significant increase in the gill (15 °c – 26 °c), which was 130 times higher than that at the control temperature (fig. 5). the gill is an important organ for respiration in the scallop and presents a large surface area in direct contact with the ambient environment, which would act as a first response to seawater temperature changes (jiang et al., 2016). in the gonad, there was no significant difference between the expression level under heat shock and the control. this could further imply that the myhsp90 gene expressed stably in scallop gonad might have other functions in organ (gonad) development and maturation. conclusion we identified full-length hsp90 cdna in the scallop m. yessoensis (myhsp90). this is the first time that the expression of myhsp90 has been investigated under temperature stress (15 °c -26 °c).in the environment, water temperature changes had a strong effect on myhsp90 mrna expression level, and this gene was up-regulated significantly in gill, kidney, adductor muscle and mantle of scallop but not in the gonad. these results will be useful in understanding the mechanism of heat shock response in m. yessoensis under temperature stress and in understanding the massive summer mortalities of this species. these results are also an 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and malachite green. gene 497: 172-180, 2012. 352 isj 14: 352-362, 2017 issn 1824-307x research report toxicological properties of a major release of untreated wastewaters into the st. lawrence river to quagga mussels dreissena bugensis f gagné, c andré, m pilote, p turcotte, c gagnon aquatic contaminants research division, environment and climate change canada, 105 mcgill, montreal, qc, canada accepted september 19, 2017 abstract before maintenance work could be carried out on the sewage system in the city of montreal (quebec, canada), 5 billion litres of untreated wastewater had to be released directly into the st. lawrence river over a five-day period in november 2015. the purpose of this study was to examine the toxicity of untreated wastewaters on quagga mussels. water samples were collected at various points from the most densely populated downtown area and downstream points: 0 km, 5 km, 12 km, 16 km and 19 km. a river water sample was collected at the opposite shore as a reference site, and aquarium (dechlorinated tap) water was used for controls. mussels were exposed for four days at 15 o c to these wastewaters, then examined for biotransformation (cyp1a1 and glutathione s-transferase activities), energy expenses (mitochondria activity and triglycerides), estrogenicity (alkali-labile phosphates) and damage (lipid peroxidation and dna strand breaks). the data revealed that exposure to the released wastewaters produced changes to all the above biomarkers. cyp1a1 activity, dna damage and triglyceride levels were the most responsive biomarkers for optimal site classification as determined by discriminant function analysis. cyp1a1 activity, lipid peroxidation and alkali-labile phosphate levels were significantly correlated with distance from downtown, suggesting that population density influenced more directly those effects. in conclusion, exposure to untreated wastewaters could lead to adverse toxic effects in quagga mussels. mussels could be at risk from the release of untreated wastewaters events given that high-intensity precipitation could also result in the release of untreated wastewaters in these times of climate change. key words: quagga mussels; untreated wastewater; cytochrome p4501a activity; oxidative damage; dna damage; energy expenses introduction in november 2015, the city of montreal (quebec, canada) had to release billions of litres of untreated wastewaters from its sewage system’s southeast interceptor in order to empty it before upgrading the interceptor and a snow collection chute. this resulted in the release of 5 billion litres of untreated wastewater into the st. lawrence river over a five-day period. although no evidence of acute toxicity was observed (e.g., from high ammonia or low dissolved oxygen content during the spills), concerns were raised about possible long-term chronic effects on resident sessile organisms such as local mussel populations. urban ___________________________________________________________________________ corresponding author: gagné françois aquatic contaminants research division environment and climate change canada 105 mcgill, montreal, qc, canada e-mail: francois.gagne@canada.ca wastewaters are sources of many contaminants, ranging from metals, polyaromatic hydrocarbons and pesticides to pharmaceuticals, personal care products and nanomaterials (holeton et al., 2011). these wastewaters contain many endocrinedisrupting compounds, especially those from the estrogen signaling pathways, such as 17α-ethynylestradiol, estradiol-17β, nonylphenol (surfactants) and bisphenol a (chen et al., 2006). these substances are known to induce egg yolk proteins (vitellogenins) in fish and bivalves, leading to feminization of the organisms (aravindaksham et al., 2003; blaise et al., 2003; tetreault et al., 2011). recent evidence also revealed that toxins in municipal effluents lead to oxidative stress/inflammation, serotonergic and genotoxic effects in fish and bivalves (gagné et al., 2006, 2010, 2011). the release of municipal effluent and untreated wastewater during rain overflow events could pose a risk to local mussel populations. mailto:francois.gagne@canada.ca 352 moreover, under climate change conditions, more intense precipitation could lead to increased rainfall events (min et al., 2011) that could overwhelm the capacity of existing wastewater treatment plants to handle these inputs of rainfall-driven wastewater. that would lead to increased releases of untreated wastewater at sites where local mussels thrive, and the impacts are unknown. in this respect, the november 2015 major release of untreated wastewater for the purpose of upgrading sewage infrastructure presented a unique opportunity to examine the toxicological properties of released untreated wastewaters in local dresseinid mussels in the st. lawrence river. bivalves are at particularly high risk to aquatic contaminants, given that they are sessile organisms and filter feeders. they are thus exposed to both dissolved and particulate-bound contaminants. dresseinid mussels are small and formidable aquatic invaders found in many freshwater bodies in europe and north america. quagga mussels (dresseina burgensis) are similar to zebra mussels in appearance, but quagga mussel shells are paler toward the hinge and somewhat larger (15 25 cm wide). the quagga mussel usually feeds on phytoplankton, algae and other suspended materials in the water column. it is dioecious and a prolific breeder. fertilization occurs in the water column. a fully mature adult could produce 0.5 million to 1 million eggs per year, depending on food availability and ambient temperature. the veligers and adults adhere to surfaces with byssal threads. dresseinids were previously used to determine the toxic effects of municipal effluents and urban pollution. increased expression of metallothioneins and cytochrome p4501a activity, markers of exposure to heavy metals and polyaromatic hydrocarbons respectively, was used to identify the impacts of urban pollution (de lafontaine et al., 2000). changes in lipid peroxidation (lpo) as a marker of oxidative damage were also monitored in local mussel populations. dresseinid mussels have previously been used to measure estrogen and a related product, nonylphenol, in municipal effluents (quinn et al., 2004, 2006). exposure to a tertiarytreated municipal effluent for 112 days resulted in increased levels of egg yolk proteins, as indirectly determined by alkali-labile phosphates (alp) and gel electrophoresis. exposure to nonylphenol to 5 µg/l and 500 µg/l for 112 days also resulted in increased alp and cholesterol levels in mussels. dna damage in whole tissues of zebra mussels was also monitored in dresseinids using the alkaline precipitation assay (de lafontaine et al., 2000; ács et al., 2016). the purpose of this study was therefore to examine the toxicity of released untreated wastewater to quagga mussels in the st. lawrence river (quebec, canada). the untreated wastewaters were collected during the major release in november 2015. quagga mussels were exposed to the undiluted wastewaters for four days and analyzed for toxic effects through monitoring of changes in energy metabolism, xenobiotic biotransformation, oxidative stress and dna damage. fig. 1 map of study sites. the surface waters were collected at five discharge sites located at increasing distances from downtown (0, 5, 12, 16, 19 km). a simulated dispersion plume is shown on the map. the river water samples were collected in the st. lawrence river near the south shore, which was outside the plume formed by the released untreated water along the north shore of the river. the st. lawrence river’s direction of flow is from the lower left to the upper right of the map. source: google maps. river 16 19 12 5 0 353 352 materials and methods study sites and mussel exposure on the third day of the five-day release of untreated waters, surface water samples (40 l) were collected at the mouths of five sewage overflows. the sewage overflows selected were located at increasing distances from montreal’s downtown, starting at 0 km (the most densely populated area) and gradually moving northeast 5 km, 12 km, 16 km and 19 km from downtown (fig. 1). river water from outside the dispersion plume of the released untreated wastewater was also collected along the south shore. surface water (five 4-l samples) at each sampling location was collected using an immersion pump lined with teflon tubing and was stored in 4-l plastic containers that had been washed with 1 % hcl. quagga mussels were collected along on the south shore of the st. lawrence river (10 km upstream from the city of montreal) a location which was not in direct contact with sources of contamination. the mussels were collected by hand in clumps (held together by byssal threads) and transferred back to the laboratory in coolers (kept at 4 o c in the dark). at the lab, they were placed in a 20-l aquarium at 15 o c with an 8/12-h light and dark cycle and fed daily with commercial algal feed. they were kept in those conditions for at least 30 days. the mussels were then delicately separated from each other with a scalpel, and mussels of similar size (0.7 to 1.3 g/shell length) were selected for analysis. the mussel gonads were in the late or early gametogenesis stages (sex was indeterminate). twelve mussels were placed in 4-l containers filled with each of the surface water samples described above (0 km, 5 km, 12 km, 16 km and 19 km distance, plus the reference river sample taken at 19.5 km on the other side of the st. lawrence river near the south shore). the control was aquarium water, i.e., uv/charcoal-filtered tap water. the mussels were kept for 96 h at 15 o c under constant aeration, and water samples were changed after 48 h. water chemistry surface water characteristics (ph, conductivity, total suspended solids, salinity, redox potential and dissolved oxygen) were measured using standard methodology (hach laboratories, usa), and total fecal coliform plate counts (apha, 2012) were performed. elemental analysis was performed in the dissolved fraction (0.45 µm filtration) by icp-mass spectrometry (xseries 2, thermoscientific, usa). the following elements were measured: cd, co, cr, cu, fe, li, na, ni and zn. the data were expressed in µg/l. toxicity assessment at the end of the exposure period, mussels were measured (shell length) and weighed. the soft tissues were dissected out on ice, weighed and immediately placed in the four volumes of homogenization buffer (10 mm hepes-naoh, ph 7.4, 100 mm nacl, 1 mm edta, 1 mm dithiothreitol and 0.1 μg/ml apoprotinin). the whole body tissues were homogenized with a teflon pestle tissue grinder (five passes at 4 o c), and a sub-sample of the homogenate was kept aside for lipid peroxidation (lpo), dna damage, total triglycerides (lipids) and total protein assessments. the other portion was centrifuged first at 1,500g for 10 min, then the supernatant was centrifuged at 10,000g for 30 min at 2 o c. the pellet contained the crude mitochondria fraction for mitochondrial electron transport (met) activity and total protein assessments and was resuspended in 0.25 ml of 100 mm nacl, 1 mm kh2po4, 1 mm edta and 10 mm hepes-naoh, ph 7.4, before storing at -85 o c. table 1 physico-chemical characteristics of released untreated waters sites distance from downtown (km) ph conductivity (µs*cm -1 ) total suspended solids (mg/l) salinity (mg/l) redox (mvolt) o2 (mg/l) fecal coliforms (counts/100 ml) 1 0 7.3 505 0.26 0.32 119 8.2 750,000 2 5 8.1 280 0.13 0.18 118 11.1 10,000 3 12 8.1 331 0.16 0.21 137 10.6 10,000 4 16 8.2 308 0.15 0.20 140 10.8 69,000 5 19 7.9 290 0.14 0.18 237 12.6 8,400 municipal effluent 22 6.8 800 2-4 na na 4-5 300,000 to 500,000 reference 19.5 8 286 0.14 0.18 211 12.7 5,800 na: not available. 354 352 table 2 spearman correlation of water quality properties distance ph cond. tss salinity redox o2 fecal coliforms distance 1 0.49 -0.61 -0.58 -0.63 0.84 0.81 -0.81 ph 1 -0.90 -0.91 -0.88 0.13 0.64 -0.93 cond. 1 0.99 0.99 -0.46 -0.89 0.98 tss 1 0.99 -0.42 -0.87 0.98 salinity 1 -0.50 -0.91 0.98 redox 1 0.80 -0.42 o2 1 -0.85 cond.: conductivity; redox: redox potential; tss: total suspended solids. the 10,000g supernatant (s10) was similarly stored for 7-ethoxyresorufin o-deethylase (erod), glutathione s-transferase (gst) and akali-labile phosphates (alp) and total proteins. total proteins were determined using the bradford (1976) method, and serum bovine albumin was used for calibration. cellular energy allocation was determined by monitoring changes in met activity and total triglyceride levels. met activity was determined using a previously described procedure (andré and gagné, 2017) based on the reduction of marker dye (frings and packard, 1975). briefly, the isolated mitochondria (100 µg/ml total proteins) were mixed in 150 μl of 0.1 m tris-hcl (ph 8.5) containing 5 % polyvinylpyrrolidone, 0.1 mm mgso4 and 0.1 % triton x-100 for 5 10 min on ice. then, 1 mm nadh and 0.2 mm napdh were added and the reaction was initiated with 1 mm p-iodonitrotetrazolium. the reaction was allowed to proceed at 20 o c and absorbance at 520 nm was measured at 5-min intervals for 30 min. the data was expressed as change in absorbance/min/mg proteins. total triglyceride contents were determined in soft tissue homogenate using an adipored fluorescent assay (lonza; walkersville, md, usa): 5 µl of adipored reagent was added to 100 µl of homogenate in a 96-well black microplate. the plate was shaken gently to ensure uniform mixing and incubated in the dark for 10 min. the fluorescence was then measured at 485 nm excitation and 535 nm emission in a microplate reader (synergy 4 microplate reader, biotek, vt, usa). the data were expressed as µg triglycerides/mg total proteins. xenobiotic biotransformation activity was characterized by the activities of erod (cytochrome p4501a) and gst. erod activity was determined in the s10 fraction in the presence of 10 µm 7-ethoxyresorufin, 100 mm nadph in 50 mm tris-hcl, ph 7.4, containing 0.1 mm mgcl2. the reaction was allowed to proceed for 40 min at 30 o c, and fluorescence readings for the formation of product 7-hydroxyresorufin at 540 nm for excitation and 590 nm for emission were taken using a microplate reader (synergy 4 microplate reader, biotek, vt, usa). standard solutions of 7-hydroxyresorufin (0.5 5 µm) were used for instrument calibration. the data were expressed as fluorescence units formed/min/mg proteins. gst activity was determined in the s10 fraction using the spectrophotometric method (boryslawskyj et al., 1988). briefly, the s10 fraction (50 µl) was mixed with 150 µl of 0.1 mm of 1-chloro2,4-dinitrobenzene and 0.1 mm of reduced glutathione. absorbance readings at 340 nm were taken every 5 min for 30 min in clear microplates (synergy 4, microplate reader). data were expressed as the decrease in absorbance/min/mg proteins. the levels of alkali-phosphates were determined in the s10 fraction as a marker of estrogenic properties in dresseinid mussels (quinn et al., 2004), with slight modifications. the s10 fraction was mixed with acetone at 35 % for 5 min on ice and centrifuged at 10,000g for 5 min. the pellet was washed in 50 % acetone, centrifuged and resuspended in 1 m naoh. the mixture was incubated at 60 o c for 30 min. the levels of inorganic phosphates were then determined using the phosphomolybdate methodology. the naoh sample was centrifuged at 15,000g for 5 min if turbidity was found. the data were expressed as mg phosphate/g soft tissues. biomarkers of tissue damage were determined by monitoring changes in lipid peroxidation (lpo) and dna breaks (db). lpo was determined in tissue homogenates using the thiobarbituric acid reactants (tbars) methodology as described elsewhere (gagné, 2014). the levels of tbars were determined by fluorescence at 520 nm for excitation and 590 nm for emission (bioscan, usa). standard solutions of tetramethoxypropane were used for calibration. the data were expressed as µg tbars/mg proteins. the levels of dna strand breaks were determined using the alkaline precipitation assay (benchalgo et al., 2014): 25 µl of the homogenate was mixed with 175 µl of 2 % sds containing 40 mm naoh, 10 mm edta and 10 mm tris base. after mixing by inversion, 200 µl of 0.12 m kcl was added and placed at 60 o c in a water bath for 10 min and cooled on ice for 30 min. the mixture was centrifuged at 8,000g for 5 min and the supernatant was collected for dna analysis. 355 352 dna levels in the supernatant were determined using sybr green dye in the presence of 0.4 m nacl, 4 mm sodium cholate and 0.1 m tris-acetate, ph 8.5, to limit interference with traces of sds in the supernatant (bester et al., 1994). fluorescence readings were taken at 485 nm excitation and 520 nm emission, and standard solutions of salmon sperm dna were used for calibration. the data were expressed as µg dna/mg proteins in the homogenate. data analysis mussels were exposed to the untreated waters in triplicate and n = 10 mussels were analyzed for biomarker analyses. data normality and homogeneity of variance were verified using shapiro-wilk and bartlett tests respectively. the data were log-transformed if proved not normal or presented heterogeneity of variance. the data were then subjected to analysis of variance, and critical difference between controls was determined using the least squares difference test. correlation analysis was performed using the pearson-moment procedure. the biomarker data were analyzed using discriminant and factorial analysis to determine differences in the toxicity profile of untreated waters collected at increasing distance from downtown. results and discussion from among the numerous (150) release points for rainwater and wastewater overflows, we selected locations at increasing distance from downtown montreal, where the population density is highest. the river water ph was slightly more acidic at the closest (0 km) site (ph 7.1) than at the sites farther downstream (ph 7.9 8.2) (table 1). this value was similar to that of municipal effluent that had undergone primary physical-chemical treatment. water conductivity was also higher at the site closest to downtown (505 µscm -1 ) than at the sites farther downstream and in the river water (331 280 µscm -1 ); this value represented 63 % of the conductivity of the treated municipal effluent (which is in the order of 800 µscm -1 ). the same pattern was found with suspended solids (0.26 mg/l at 0 km, compared to 0.13 0.16 mg/l at the downstream sites), salinity (0.32 mg/l at 0 km compared to 0.18 0.21 at the downstream sites), dissolved oxygen (8.2 mg/l at 0 km compared to 10.6 12.7 mg/l at the downstream sites). the redox potential was 119 mvolt at 0 km and gradually increased, reaching 237 mvolt at 19 km from downtown, which suggests the presence of electron scavengers in the downtown area. the distance from downtown was significantly correlated with redox potential (r = 0.84; p < 0.01), dissolved oxygen (r = 0.81; p < 0.01) and total fecal coliforms (r = -0.81; p < 0.01) (table 2). the total levels of some metals were also determined in the dissolved fraction of collected surface waters (table 3). in general, the levels of na, cd, cr, fe, co, ni, cu and zn were higher at the site closest to downtown (0 km) than at the downstream sites. distance from downtown was negatively significantly correlated with levels of cr (r = -0.93, p < 0.01), cu (r = -0.94, p < 0.01), zn (r = -0.83, p < 0.05) and marginally correlated with levels of ni (r = -0.75, p = 0.08) and fe (r = -0.78, p = 0.06). correlation analysis with general physical and chemical properties revealed that ph was negatively correlated with ni (r = -0.93; p < 0.01), co (r = -0.85; p < 0.05), cu (r = -0.94; p < 0.01) and zn (r = -0.94; p < 0.01) levels. conductivity was positively correlated with all metals (r > 0.9; p < 0.01) and sodium (r = 0.97; p < 0.001). total suspended solids were also positively correlated with all the metals tested (r > 0.88; p < 0.01). the redox potential was positively correlated with distance from downtown and not correlated with any of the metals (r < 0.63; p > 0.1). quagga mussels were exposed for 96 h during the five-day release of billions of liters of untreated wastewaters. no changes in the mussel weight-toshell-length ratio were observed, nor were there any trends based on distance from downtown (results not shown). changes in the energy status of mussels exposed to the released untreated waters were examined by monitoring met activity and total triglycerides in mussels (fig. 2). met activity was significantly reduced at 0 km, 5 km and 12 km, and in the river samples compared to the control (aquarium water) (fig. 2a). the lower met activity in the reference site compared to control aquarium laboratory suggests the presence of agents or contaminants that exists at this urban area site. the maximum decrease, 70 %, was found at the 5 km and table 3 selected dissolved elements of released untreated waters sites na 1 cd cr fe co ni cu zn 1 41.5 0.031 0.22 34 0.068 0.82 8.5 10.8 2 14.4 0.006 0,14 21 0.023 0.57 0.97 1.6 3 13.7 0.005 0.11 17 0.021 0.57 1.1 0.87 4 13 0.007 0.12 22 0.026 0.55 0.83 0.98 5 14 0.006 0.10 17 0.012 0.54 0.82 0.90 reference 13.2 0.005 0.10 15 0.03 0.55 0.76 0.78 1 expressed as the mean from n = 3 determinations in ug/l; coefficient of variations between 5 % and 15 % of the mean. 356 352 0 5 12 16 19 riv er control distance from downtown (km) 0,75 0,80 0,85 0,90 0,95 1,00 1,05 1,10 1,15 1,20 1,25 m it o c h o n d ri a l e le c tr o n t ra n s p o rt a c ti v it y (a b s o rb a n c e /m in /m g p ro te in s ) mean stand. error a a,b a b a a 0 5 12 16 19 riv er control distance from downtown (km) 1,1 1,2 1,3 1,4 1,5 1,6 1,7 1,8 1,9 t o ta l tr ig ly c e ri d s ( g /m g p ro te in s ) trigly c erids : f(6;31) = 1,6629; p = 0,1635 mean stand. error a,b a,b b fig. 2 energy status of mussels exposed to the untreated wastewaters. mussels were exposed to the water samples for 96 h. mitochondrial electron transport activity (a) and total triglycerides (b) were determined in mussel soft tissues. the letter a indicates significance when compared to control water; the letter b indicates significance when compared to river water at p < 0.05. 12 km sites. however, when compared to the river water outside the area of untreated wastewater release, met activity was significantly increased at 0 km and 19 km. this suggests that an initial suppression in met activity is observed with the water samples compared to control aquarium water but this is mitigated when compared to river water. energy reserves were significantly lower at 16 km and 19 km from downtown compared to both control and river water (fig. 2b). the maximum decrease was 70 % of the control values. taken together, the increase in met activity at 0 km and 19 km was associated with lower lipid values when compared to river water, although triglyceride levels for the 0 km site were not significantly different from river water, due to higher variability. however, these changes appeared to be site-specific, since no significant trends based on distance were observed. in a previous study (gagné et al., 2007), met activity and gonad lipids were higher in mussels exposed to treated wastewater from montreal. those effects were reversed following ozonation with 10 20 mg/l ozone. 357 352 0 5 12 16 19 river control distance from the downtown (km) 0,08 0,10 0,12 0,14 0,16 0,18 0,20 0,22 0,24 e r o d a c ti v it y (f lu o re s c e n c e /m in /m g p ro te in s ) mean stand. error a,b a,b* a,b a,b a,b a 0 5 12 16 19 river control distance from downtown (km) 2,2 2,4 2,6 2,8 3,0 3,2 3,4 g s t a c ti v ti y (a b s o rb a n c e /m in /m g p ro te in s ) mean stand. error a,b b a b a,b a,b b fig. 3 biotransformation activity of mussels exposed to the untreated wastewaters. mussels were exposed to the water samples for 96 h. phase i and ii biotransformation activities were determined by erod (a) and gst (b) activities respectively. the letter a indicates significance when compared to control water; the letter b indicates significance when compared to river water at p < 0.05. biotransformation activity was determined in mussels by measuring changes in erod and gst activities. erod activity was significantly increased at all sites compared to both river and control waters (fig. 3a). the maximal response was found at 0 km, where it was 2.2 times greater than the control/river water samples. erod activity was significantly correlated with distance from downtown (r = -0.95; p < 0.001) indicating increased polyaromatic hydrocarbons offshore from at the most densely populated area of the city. gst activity was significantly increased at all sites when compared to the river water, reaching 1.4 times greater at 16 km. the river water sample showed lower gst activity than the laboratory control water, indicating a suppression effect of “natural” waters, as observed for met activity. it is possible that lower metabolic activity (met) contributed also to reduce gst activity given its role in the scavenging of reactive oxygen species during respiration. there was no significant correlation with distance from downtown. primary cultures of rainbow trout hepatocytes were exposed to 12 wastewaters before and after six different treatment processes and had elevated cyp1a1 mrnas (responsible for erod activity) nearly 70 % of the time (gagné et al., 2013). gst gene expression was induced by 70% of the various treated effluents and was significantly related to the expression profiles obtained with the untreated effluent, which suggests that the cyp1a1-inducing properties of the effluent were not produced by the treatment process per se. 358 352 0 5 12 16 19 river control distance from downtown (km) 500 600 700 800 900 1000 1100 1200 1300 1400 a lk a li -l a b il e p h o s p h a te s ( g /g t is s u e s ) mean stand. error a a a,b fig. 4 changes in alkali-labile phosphates in quagga mussels exposed to the untreated wastewaters. mussels were exposed to the water samples for 96 h. the levels of vitellogenin-like proteins were determined by the alkaliphosphate method. the letter a indicates significance when compared to control water; the letter b indicates significance when compared to river water at p < 0.05. the levels in alp were also examined in mussels exposed to the released wastewaters (fig. 4). alp levels were significantly higher at 0 km, 5 km and 12 km than in the controls. there was no significant difference between river water and control water samples, but variability was higher for the river samples. in the river water, only the 12-km site had significantly higher alp levels. interestingly, alp levels were significantly correlated with distance from downtown (r = -079; p < 0.05). the alp levels in dresseinids were associated with egg yolk proteins and were shown to be significantly induced in tertiary-treated municipal effluents (quinn et al., 2004). this suggests that releasing of untreated wastewater from a highly populated area produces estrogenic effects in this species of mussel. nonyphenol, a common estrogenic compound found in municipal wastewaters, was shown to increase alp levels in zebra mussels (quinn et al., 2006). the levels of oxidative damage (lpo) and damage to the genetic material (dd) in mussels were examined. lpo levels were significantly higher at 0 km than in river and control waters (figure 5a), reaching 1.5 times the control values. there was a significant correlation between distance from downtown and lpo (r = -0.72; p < 0.05). this is consistent with previous studies on the oxidative stress/inflammatory effects of municipal effluents on mussels and fish (gagné et al., 2007; jasinska et al., 2015). caged fathead minnows exposed for four weeks downstream from the city of edmonton’s municipal effluent dispersion plume had elevated levels of oxidized glutathione and higher antioxidant enzyme and glutathione s-transferase activity. mussels exposed to physico-chemically treated effluent had elevated levels of lpo and dd in gills. for dd, a more complex pattern was observed. the levels of dd in the river water were significantly lower than in the aquarium control water, which suggests a suppressive effect of river water on dna turnover and repair activity. when compared with river water, dd was significantly induced at 0 km, 12 km and 19 km from downtown while dd was significantly reduced at 16 km from downtown. caged mussels exposed for one month to waters downstream from montreal’s municipal discharge had elevated dd as determined by the comet assay (lacaze et al., 2011). genotoxicity was apparent up to 20 km distance and its intensity was similar to that found in the municipal effluent (< 200 m). this suggests that even with physicochemical treatment, genotoxic compounds are still released and persist at 20 km downstream from the plume. reduced levels of dna strand breaks were also observed previously in mussels exposed to primary-treated effluent (the same treatment for these wastewaters) (gagné et al., 2011). a biphasic response was observed, with an initial decrease in dna strand breaks at a low effluent concentration (3 % v/v) followed by a decrease in dna breaks at higher concentrations (> 10 % v/v). the initial decrease in dna strand breaks could be associated with the decrease in dna repair activity, such as reduced precursors for purine synthesis by inhibition of dehydrofolate reductase inhibitors, leading to reduced dna strand breaks. decreased dna repair activity would therefore result in increased dna damage and increased alkali-labile 359 352 0 5 12 16 19 river control distance from the downtown (km) 0,007 0,008 0,009 0,010 0,011 0,012 0,013 0,014 l p o ( g t b a r s /m g p ro te in s ) mean stand. error a,ba 0 5 12 16 19 river control distance from the downtown (km) 1,2 1,4 1,6 1,8 2,0 2,2 2,4 2,6 2,8 3,0 d n a s tr a n d b re a k s ( g d n a / m g p ro te in s ) mean stand. error a,ba a b b b b fig. 5 oxidative damage and genotoxicity in mussels exposed to the untreated wastewaters. mussels were exposed to the water samples for 96 h. the levels of lpo (a) and dna strand breaks (b) were determined in whole tissues of quagga mussels. the letter a indicates significance when compared to control water; the letter b indicates significance when compared to river water at p < 0.05. sites in dna at higher effluent concentrations. a previous study (gagné et al., 2007) showed strong inhibition of dihydrofolate reductase activity in elliptio complanata mussels exposed to the same primary-treated municipal effluent and suggested that the dehydrofolate reductase-inhibitors such as the bacteriostatic agent trimethoprim and other similar compounds (methotrexate and pyrimethamine) present in municipal effluents accounted, at least in part, for the observed inhibition. the reported concentration of methotrexate is in the order of 60 ng/l in untreated wastewater from montreal (garcia-ac et al., 2009). however, methotrexate was present at concentrations between 20 ng/l and 4,000 ng/l in hospital wastewater effluents (isidori et al., 2016). the concentration of methotrexate required for inhibition in vitro is in the order of 0.5 g/l to 1 g/l. this suggests that mussels are able to accumulate this compound or that other inhibitors are also present in municipal effluents and act cumulatively in mussels. recent evidence suggests that genotoxic compounds are impervious to wastewater treatment even after anoxic-oxic process (zhang et 360 https://en.wikipedia.org/wiki/pyrimethamine 352 -6 -4 -2 0 2 4 6 8 component 1 (64%) (dd>gst>triglycerides) -4 -3 -2 -1 0 1 2 3 4 5 c o m p o n e n t 2 ( 1 5 % ) (e r o d > a l p > t ri g ly c e ri d e s ) 0 km 5 km 12 km 16 km 19 km river control total variance: 79% mean classification: 90% fig. 6 discriminant function analysis of biomarker data. mussels were exposed to the water samples for 96 h. biomarkers were analyzed using discriminant function and factorial analyses to determine site similarities and the most important biomarkers. al., 2013). even though the acute toxicity of municipal wastewater was effectively removed by anoxic/oxic treatment, the dna-damaging properties (micronucleus and comet assays) were not affected in zebrafish. thus, treated and untreated wastewaters represent continuous sources of genotoxic compounds in the environment. moreover, under climate change conditions where increased precipitation events are likely (min et al., 2011), more frequent rainwater overflows can be expected, given the limited capacity of some wastewater treatment plants to handle more intense rainfall events. discriminant function analysis was performed to identify differences between the samples and determine the most responsive biomarkers in achieving site discrimination (fig. 6). the analysis revealed that 79 % of total variance was explained with a mean classification efficiency of 90 %. water samples from river and aquarium water were somewhat similar and formed a distinct group from the untreated water samples. the farthest site (19 km) was closely related to the river water sites. water samples at 0 km, 5 km, 12 km and 16 km formed clusters distinct from the farthest and the river water sites. these were mainly explained by dd, gst and triglyceride levels. the site closest to downtown (0 km) differed from the other untreated waters and from river water in erod activity, alp levels and triglycerides (on the y axis). interestingly, the untreated water samples generally differed from each other from 0 km to 16 km. this suggests some heterogeneity in the water samples which is dependent on distance from downtown. this is consistent with the data showing correlation of distance with erod, lpo and alp endpoints. in conclusion, exposure of quagga mussels to released untreated waters for four days led to toxic effects. the strongest response was increased erod activity, for which the intensity in activity was proportionally related to the distance from the 0 km point (downtown, the most densely populated part of the city). dd showed a biphasic response with either increased or decreased dna strand breaks at the release sites when compared with river water outside the area where untreated wastewater was released. other changes included potential estrogenic effects (alp), altered energy metabolism and oxidative damage. exposure to untreated wastewater for relatively brief periods (four days) could negatively impact local mussel populations. acknowledgements this study was supported by the st. lawrence action plan of environment and climate change canada. joana kowalczyk’s technical assistance with biomarker analyses was much appreciated. references ács a, vehovszky á, győri j, farkas a. seasonal and size-related variation of subcellular biomarkers in quagga mussels (dreissena bugensis) inhabiting sites affected by moderate contamination with complex mixtures of pollutants. environ. monit. assess.188: 426439, 2016. american public health association, american water works association, and water environment federation 2012. in standard methods for the examination of water and wastewater, 22nd edition, isbn: 9780875530130. 361 https://www.ncbi.nlm.nih.gov/pubmed/?term=%c3%81cs%20a%5bauthor%5d&cauthor=true&cauthor_uid=27329477 https://www.ncbi.nlm.nih.gov/pubmed/?term=vehovszky%20%c3%81%5bauthor%5d&cauthor=true&cauthor_uid=27329477 https://www.ncbi.nlm.nih.gov/pubmed/?term=gy%c5%91ri%20j%5bauthor%5d&cauthor=true&cauthor_uid=27329477 https://www.ncbi.nlm.nih.gov/pubmed/?term=farkas%20a%5bauthor%5d&cauthor=true&cauthor_uid=27329477 https://www.ncbi.nlm.nih.gov/pubmed/27329477 352 andré 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https://www.ncbi.nlm.nih.gov/pubmed/15129770 https://www.ncbi.nlm.nih.gov/pubmed/15129770 https://www.ncbi.nlm.nih.gov/pubmed/15129770 19 isj 18: 19-32, 2021 issn 1824-307x research report evolutionary insights on a novel mussel-specific foot protein-3 gene family e bortoletto1, p venier1, a figueras2, b novoa2, u rosani1,3* 1department of biology, university of padua, padua, italy 2institute of marine research (iim), spanish national research council (csic). vigo, spain 3coastal ecology section, awi alfred wegener institute helmholtz centre for polar and marine research, list auf sylt, germany this is an open access article published under the cc by license accepted december 18, 2020 abstract silky byssus threads enable a number of marine and freshwater bivalve mollusks to attach themselves to hard substrates. byssus production is an energy-costly process, which accompany the switch from planktonic to sessile life. pointing the attention to a small foot protein (fp-3α) first identified in perna viridis and abundantly secreted during the bissogenesis, we report the presence of a fp-3α gene family in species of the mytilus complex, byssogenic bivalve mollusks mostly inhabiting marine waters. in the genome of mytilus galloprovincialis we identified twelve fp-3α genes showing differences in exon-intron organization and suggesting that, as in the case of arthropod and mollusk defensins, exon shuffling could have played an important role in the evolution of this gene family. also, the different tissue expression patterns of these mussel genes support their functional diversification. all predicted fp-3α proteins curiously possess a csαβ three-dimensional motif based on 10 highly conserved cysteines and exhibit structural similarity to invertebrate defensins. the role of these small cysteine-rich proteins in supporting the byssus-mediated mussel adhesion or their action as host defence peptides remains to be established with further study. key words: byssogenesis; dopa-containing proteins; foot proteins; fp-3α; mytilida; mytilus galloprovincialis introduction the class of bivalvia comprises many thousands of marine and fresh water mollusk species (worms world register of marine species, 2020), including relevant aquaculture bivalves such as mussels, oysters and clams (wijsman et al., 2019). bivalves are found in a variety of aquatic habitats throughout the world, from cold-water seas to freshwater basins and even in deep anoxic vents (darrigran and damborenea, 2011; karatayev et al., 2015; bielen et al., 2016). many bivalves live attached to hard substrates ___________________________________________________________________________ corresponding author: umberto rosani department of biology university of padova viale giuseppe colombo 3, 35131 padova, italy e-mail: umberto.rosani@unipd.it list of abbreviations: amino acid, aa; expressed sequence tag, est; open reading frame, orf; rna sequencing, rnaseq; transcriptome shotgun assembly, tsa; untranslated transcript region, utr thanks to the byssus, a bunch of silky threads ending with a tiny adhesion plaque (waite, 2017). the byssus threads with their terminal plaques are secreted by composite glandular tissues, possess an outer cuticle and span 2-6 cm in length. such a byssus-mediated anchoring allows the aggregation of mussels in dense intertidal beds and also prevents the physical damage consequent to wave motion (bennett and sherratt, 2019). the byssus was postulated to appear during the devonian period (350-400 mya), following a modification of the gastropoda pedal glands (waite, 1992). although the anchoring process is essential to both post-larval dispersal and settlement (sigurdsson et al., 1976; de blok and tan-maas, 1977), the byssus structure is retained throughout life in several species (figure 1). the byssus production is a very common life strategy in bivalves, excepting subclasses of protobranchia. actually, byssus is detectable in all orders of pteriomorphia with the exception of ostreida, in unionida (a monophyletic order of freshwater mussels), and in all orders of heterodonta, although this bivalve subclass mostly includes species adapted to a burrowing life style (canapa et al., 2001). 20 fig. 1 phylogenetic tree representing main orders of bivalvia (based on gonzález et al., 2015; combosch et al., 2017; lee et al., 2019). a cross and a red background indicate orders whose known species do not produce byssus. the orders written in bold against a green background include at least one byssus-producing species. the byssus formation has been widely studied in mytilus spp. and more than 20 proteins known as mussel foot proteins (mfps) have been identified (waite, 2017). this process begins with the mussel foot searching for a suitable substrate and the subsequent creation of a watertight vacuum between foot and substrate (ponder et al., 2020). the three types of glandular tissue involved in the byssogenesis are named core, cuticle and plaque glands in agreement to the final destination of the secreted proteins. the latter are released in secretion vesicles and move along the ventral foot grove to the different parts of each byssal thread (harrington et al., 2018). it is worth noticing that the plaque gland was formerly called phenol gland, owing to its l-3,4-dihydroxyphenylalanine (dopa)rich proteins. the core of byssus threads, covered by a protective cuticle, is constituted by pre-polymerized collagen (precol) assembled with matrix proteins (tmps) (sagert and waite, 2009) whereas the secondary structure of the core vesicle proteins, as resolved by raman spectroscopy, resembles that of type i collagen (priemel et al., 2017). in the case of kcl-induced byssogenesis, mfp1-containing vesicles released by the cuticle gland progressively aggregate in the ventral foot grove and, finally, the discharged proteins form a thin layer on the assembled thread core (priemel et al., 2017). dopa residues present in the primary structure of mfp1 mediate the formation of metal coordination complexes, which confer hardness and stiffness to the cuticle (harrington et al., 2010). the adhesive plaque is composed by at least 6 different mfps. its formation occurs as a temporally regulated process in which the proteins involved in the adhesion are secreted first, namely mfp3 and mfp5, followed by the other proteins (petrone et al., 2015). the adhesive plaque proteins are rich in dopa residues, which easily autoxidize thus impairing the dopa adhesion potential. this risk is prevented by the formation of disulphide bridges and cysteinyl–dopa bonds involving cysteine-rich proteins such as mfp6 (yu et al., 2011) and, in the case of perna viridis, the foot protein-3α (fp-3α) protein (petrone et al., 2015). amazingly, the byssus threads are initially gel-like but quickly coagulate after the contact with the water.(ponder et al., 2020). notably, byssogenesis and under-water adhesion inspired many studies aimed at the 21 development of novel adhesive and extensible materials (lee et al., 2007; lee et al., 2011; zhong et al., 2014; harrington et al., 2018). in this study, we focused the attention on a small protein (fp-3α), firstly identified by amino acid sequencing in the asian green mussel p. viridis (guerette et al., 2013; petrone et al., 2015). the considerable amount of this protein in p. viridis byssus threads and the fact that it was never reported in other species, initially indicated fp-3α as a perna-exclusive gene product. following genomic and transcriptomic data analyses, we present fp-3α sequences identified in some bivalve species of the order mytilida. accordingly, we report the taxonomic distribution fp-3α proteins and, in particular, we discuss the fp-3α gene family detected in mytilus galloprovincialis in the light of evolution. table 1 fp-3α genes retrieved from the m. galloprovincialis genome. for each of the eight fp-3α gene types, the transcript id, species of origin, presence (y) or absence (n) of the related genes in the m. galloprovincialis genome (with gene id, length, and exon-intron number) are reported. fp-3α type transcript id species mg gene [y/n] gene id length exons/introns fp3α -1 pvir_29208 p. viridis y mgal10a052660, mgal10a002276 2.98 4.64 4 exons/ 3 introns 3 exons/2 introns fp3α -2 medu_01889 m. edulis y mgal10nca009345, mgal10a009044 3.79 1.71 3 exons/ 2 introns 3 exons/ 2 introns medu_32159 m. edulis mcal_11206 m. californianus mtro_29948 m. trossulus mtro_29950 m. trossulus medu_62051 m. edulis mtro_86325 m. trossulus mtro_86326 m. trossulus medu_62050 m. edulis medu_81265 m. edulis mgal_12191 m. galloprovincialis medu_62048 m. edulis fp3α -3 mgal_36283a m. galloprovincialis y mgal10nca069861, mgal10a001553, mgal10nca009347 mgal10nca073074 0.45 3.44 0.26 1.60 1 exon/0 introns 3 exons/2 introns exon/0 introns 3 exons/2 introns mgal_36283b m. galloprovincialis mgal_36283c m. galloprovincialis mtro_41977 m. trossulus mtro_41976 m. trossulus mtro_66797 m. trossulus fp3α -4 medu_44249 m. edulis y mgal10nca031470 3.46 4 exons/3 introns mgal_6748 m. galloprovincialis mcor_91378 m. coruscus mcal_60322 m. trossulus thir_34375 t. hirsuta svir_10851 m. virgata fp3α -5 medu_59989 m. edulis y mgal10a048479 3.55 3 exons/2 introns mgal_91363 m. galloprovincialis mgal_52154 m. galloprovincialis medu_60453 m. edulis mtro_48177 m. trossulus mtro_48176 m. trossulus fp3α -6 mcor_37770 m. coruscus y mgal10a031169 mgal10a001023 3.21 4.34 3 exons/2 introns 4 exons/3 introns medu_63946 m. edulis mcor_6509 m. coruscus medu_12581 m. edulis fp3α -7 pvir_01678 p. viridis n pvir_37518 p. viridis pvir_02094 p. viridis fp3α -8 pvir_18700 p. viridis n pvir_03227 p. viridis pvir_22782 p. viridis pvir_29362 p. viridis 22 material and methods retrieval and preliminary analysis of bivalve sequences the available bivalve genomes and the corresponding gene models were retrieved from public repositories. basically, we considered 14 bivalve genomes sequenced up to now, including argopecten irradians (liu et al., 2020), bathymodiolus platifrons (sun et al., 2017), crassostrea gigas (zhang et al., 2012), crassostrea virginica (gómez-chiarri et al., 2015), dreissena polymorpha (mccartney et al., 2019), limnoperna fortunei (uliano-silva et al., 2018), mizuhopecten yessoensis (wang et al., 2017), modiolus philippinarum (sun et al., 2017), pecten maximus (kenny et al., 2020), pinctada fucata (du et al., 2017), ruditapes philippinarum (yan et al., 2019), saccostrea glomerata (powell et al., 2018), sinonovacula constricta (ran et al., 2019), venustaconcha ellipsiformis (renaut et al., 2018). moreover, unassembled rna-seq reads referring to 20 bivalve species were retrieved from the ncbi sra archive (atrina pectinate, bathymodiolus platifrons, dreissena rostriformis, limnoperna fortunei, loripes orbiculatus, mytilisepta virgata, mytilus californianus, mytilus coruscus, mytilus edulis, mytilus galloprovincialis, mytilus trossulus, paphia undulata, perna viridis, pinctada fucata, pinna nobilis, pyganodon grandis, tegillarca granosa, trichomya irsuta, uniomerus tetralasmus, villosa lienosa). raw rna-seq data in .sra format were converted into .fastq using the fastq-dump utility of the ncbi sra toolkit (staff, 2011) and the reads were trimmed for the presence of adaptor sequences and for quality using trimgalore! (babraham bioinformatics-trim galore, 2019), allowing a maximum of two ambiguous bases and a quality threshold of phred20. high quality reads were de-novo assembled with the clc assembler (clc genomic workbench v.20, qiagen, germany) with word and bubble size parameters set to ‘automatic’ and a minimal contig length of 200 bp. the obtained contigs were subjected to open reading frame (orf) prediction using transdecoder tool (trinity suite (grabherr et al., 2011)), modifying the minimal orf length to 150 nucleotides (-m 50 parameter). identification of fp-3α homologs and phylogenetic analysis putative fp-3α sequences were extracted from the above mentioned bivalve datasets using phmmer (eddy, 2011), with p. viridis fp-3α (agz84285.1) as initial query. since no recognizable pfam domains are present on this protein, we built a hmm profile (hmmbuild) based on the alignment of all the identified fp-3α proteins, then using it to scan against the protein dataset with hmmsearch (eddy, 2011) and applying an e-value cut-off of 0.05. all the resulting protein sequences were aligned with muscle (edgar, 2004). modeltest-ng v0.1.2 (posada and crandall, 1998) was used to assess the best-fitting model of molecular evolution for the multiple sequence alignment (identified as the wag+i+g). bayesian phylogenetic analysis was performed using mrbayes v3.2.6 (ronquist et al., 2012). in detail, markov chain monte carlo analysis were run until reaching the convergence cutoff of 0.01, with a sampling frequency of 1,000 and a burn-in removal of 50% of the sampled trees. the convergence of parallel runs was estimated by reaching an average standard deviation of split frequency <0.05 and of a potential scale reduction factor equal to 1. adequate posterior sampling was evaluated by reaching an effective sample size > 200 for each of the estimated parameters using tracer v1.6 (drummond et al., 2012). the final majority-rule consensus tree was visualized and edited using figtree v1.4.3 (rambautfig tree, 2012). identification of m. galloprovincialis fp-3α genes in order to identify the possible fp-3α genes, we searched the fp-3α hmm profile in the m. galloprovincialis genomic contigs translated into the six lecture frames. according to the mussel genome annotation, we retrieved all annotated fp-3α proteins and we compared them with the transcriptomederived ones, correcting the former in case of errors. to predict intron-exon boundaries, fp-3α transcript sequences have been back mapped on the m. galloprovincialis genomic contigs, using the clc splice-aware aligner (clc large gap mapping tool) and applying 0.8 of similarity on 0.2 of transcript length. mussel tissue sampling, rna extraction and highthroughput sequencing adult mussels (m. galloprovincialis, shell length 5-7 cm) were collected near the chioggia lagoon outlet (45° 14.227’n, 12° 16.706’e) during springsummer 2019 in order to assess levels and trends of fp-3α gene transcripts. at the arrival to the laboratory, mussels were cleaned from epibionts and let to acclimatize in reconstituted seawater at 35 ‰ (instant ocean, aquarium systems, mentor, usa; 2 l/mussel) with constant aeration and feeding them once a day (coral food xpro, aquatic nature, oostnieuwkerkesteenweg, belgium) at 18 °c for two days. mussel hemolymph was individually withdrawn from the adductor muscle with a 23g disposable needle and, following dissection, two gill samples and two hemolymph samples were selected, placed in trizol (1 ml/50 mg tissue, 0.5 ml/pellet from 1 ml hemolymph) thermofisher, bremen, germany) and stored at −80 °c. total rna was extracted with rneasy micro kit (qiagen, hilden, germany). rna quantity and quality were checked using a nanodrop instrument (thermofisher) and an agilent rna6000 nanochip (agilent technologies, santa clara, ca, usa), respectively. one microgram of high-quality rna per sample was used to construct libraries using the truseq stranded mrna kit (illumina, california, usa) according to the kit instruction. amplified libraries were checked for size and quality and run with a 2 × 150 read layout in a nextseq500 instrument. gene expression analysis based on rna-seq data we also used available rna-seq datasets of m. galloprovincialis to compute the expression profiles of fp-3α genes in different tissues, for a total of 30 23 fig. 2 mussel fp-3α genes. a. the gene structure of the 12 m. galloprovincialis fp-3α genes (continuous and interrupted arrows describe the complete genes and gene exons, respectively). notice that mgal10a009044 has two splicing variants. b. detail of mgal10a009044 (arrows next to contig id represent the orfs) available samples (prjna525609, prjna295512, prjna230138, prjna88481, prjna484309) plus the 4 samples produced for this work. clean rna-seq reads were mapped on the m. galloprovincialis genome annotated with the predicted gene models, using the clc read mapper, setting length and similarity fractions to 0.8 and 0.8, respectively, whereas mismatch/insertion/deletion penalties were set to 3/3/3. the number of unique mapped reads of each dataset were counted and used to calculate digital expression values as transcripts per million (tpm), to ensure the comparability of different datasets (wagner et al., 2013). fp-3α protein structure prediction molecular weight and isoelectric point were calculated for each fp-3α sequence with the expasy compute pi/mw tool (gasteiger et al., 2005), whereas average hydrophobicity and hydrophilicity (gravy), aliphatic and instability index were calculated with expasy protparam (gasteiger et al., 2005). the secondary structure of fp-3α proteins was predicted with psspred (yan et al., 2013). then, the protein sequence was aligned to protein data bank (pdb) (berman et al., 2000) templates using muster (wu and zhang, 2008) and the alignment was refined with cysbar (shafee et al., 2016). conserved cysteines were imposed as 24 distance constraints for i-tasser modeling (roy et al., 2010). the cysteine connectivity was predicted from i-tasser models and imposed before refinement based on molecular dynamics (md) as previously described (franzoi et al., 2017). results identification of fp-3α transcripts and genes in order to investigate the distribution of fp-3α genes among bivalves, we used the only available fp-3α sequence record (p. viridis, agz84285.1) to search similar sequences in the different ncbi databases (nucleotide, est, protein and tsa). as a result, we retrieved few hits, namely two ests from m. galloprovincialis and m. edulis (fl496987.1 and am881265.1) and two tsa datasets (belonging to p. viridis). to expand the dataset, we assembled rna-seq data of 20 byssogenic bivalve species belonging to mytilinae, unionoida, bathymodiolinae and pteriidae families. as a result, we retrieved 43 fp-3α transcripts belonging to m. galloprovincialis (7), m. edulis (11), m. trossulus (9), m. californianus (3), m. coruscus (3), trichomya hirsuta (one incomplete sequence), mytilisepta virgata (1) and p. viridis (8). among the 14 bivalve genomes already sequenced, we could identify fp-3α genes only in m. galloprovincialis. in detail, we searched for fp-3α genes in the m. galloprovincialis genome translated into six reading frames by using hmmer. this allowed the identification of 12 fp-3α gene regions, of which 7 and 5 were annotated as coding and non-coding genes, respectively (table 1). in order to reconstruct exon-intron boundaries of the mussel fp-3a genes, we mapped all fp-3α transcripts on the m. galloprovincialis genome using a splice-aware aligner. as a result, we could map 20 out of 43 sequences, including hits of m. galloprovincialis, m. edulis and m. trossulus: most of the transcripts mapped to the mgal10a009044 (12) and mgal10nca009345 fp-3α genes (3). the twelve fp-3α genic sequences identified in m. galloprovincialis generally displayed a small size (< 4 kb) and a structure based on three exons. in detail, the first exon contains the 5’ utr, the second one encodes for the remaining part of 5’-utr and the main part of the signal peptide, whereas the third exon contains the rest of the coding sequence, including a short c-terminal region. however, some fp-3α genes displayed a single exon or an additional exon between those encoding for the signal peptide and the mature peptide (figure 2a). fig. 3 tissue-specific expression levels in transcripts per million (tpm) for twelve genes encoding fp-3 proteins in m. galloprovincialis. the heatmap reports the average of the expression values per tissue 25 fig. 4 alignment of 43 predicted fp-3α proteins from mytilida rna-seq data. the segmented line over the multiple alignment indicates the signal peptide, mature peptide and the c-terminal region. the contig names are joined with a curly bracket per each fp-3a isoform. the conservation percentage at each amino acid position is also reported under the multiple alignment of the fp-3α protein sequences moreover, we identified two splicing isoforms for mgal10a009044, with the first exon located slightly upstream in m. edulis and m. trossulus, compared to m. galloprovincialis, and thus resulting in a longer intron (519 bp compared to 392 bp). while m. trossulus and m. galloprovincialis possess only one mgal10a009044 isoform, m. edulis has both the splicing isoforms (figure 2b). m. edulis has two different splicing isoforms also for mgal10nca009345. expression levels of mussel fp-3α genes based on rna-seq data to investigate the expression patterns of the 12 m. galloprovincialis fp-3α genes, we used 30 carefully selected rna-seq datasets generated from different tissues of naïve mussels, plus the rna-seq data from two gill samples (128 milions reads) and from two hemolymph samples (74 million reads) produced for this study from m. galloprovincialis. among the six fp-3a genes expressed in mantle, mgal10a009044 showed the highest average expression, namely 52 transcripts per kilobase million (tpm) (range: 20-118 tpms) whereas mgal10a031169 was also expressed in gills with 27 and 20 tpm, respectively (figure 3). conversely, mgal10a002276, mgal10nca031470 and mgal10a001023 were selectively expressed in digestive gland, hemolymph and gills (average expression values: 81, 42 and 27 tpms, respectively). the expression levels of the three remaining fp-3α genes was considered negligible (< 5 tpms). inferences on mussel fp-3α proteins the virtual translation of the fp-3 orfs resulted in proteins of 58-90 aa residues, including a signal peptide (15-23 aa in length) and no recognizable interpro or pfam domains (figure 4). the mature peptide region (length: 40-44 aa) included 10 highly conserved cysteines organized as follows: c(n)c(n)c(n)c(n)c(n)c(n)cccxxc. some of these mature peptide sequences displayed an n terminal extension long up to 25 aa. even if very short, not more than 8 aa, some c-terminal region included peculiar motifs, like ggygk or kppkry. fp-3α phylogenesis and classification the bayesian phylogenetic tree resulting from the multiple alignment clearly divided p. viridis fp-3α sequences from the others (upper branch in figure 5). the remaining sequences split into two main clades. clade 1 included two different subclades and one outlier m. californianus sequence, whereas clade 2 grouped three different subclades: one subclade included sequences from different bivalve species (mytilus sp., mytilisepta virgata, trichomya hirsuta) whereas the other two subclades included only sequences from mytilus spp. based on the primary protein sequences and phylogenetic analysis, we could distinguish eight different fp-3 types in mytilida: fp3-1, -7, -8 typical of p. viridis and fp3-2, -3, -4, -5, -6 occurring in other species (table 1). six of these fp-3 types are represented by one or more genes in the m. galloprovincialis genome (see table 1). the most 26 fig. 5 bayesian phylogenetic tree of 43 virtually translated fp-3α transcript sequences. the bayesian phylogenesis was run for 3 million generations with a sampling frequency of 1,000 and a burn-in removal of 50% of the sampled trees. in the sequence names, the uppercase letter indicates the genus, the lower case letter indicates the species whereas numbers identify the contigs. the node labels report the bootstrap values as probability percentage. the scale bar at the bottom indicates the branch length representing 0.3 as amount of genetic change abundant protein type in the above described bayesian tree was fp3α-2 (12 sequences). since m. galloprovincialis possesses two fp3α-1 genes, this protein type is not exclusive of p. viridis and could be reminiscent of the common mytilida ancestor molecule. structural model of the m. galloprovincialis fp3α-2 protein the 43 fp-3α proteins derived from the rnaseq data are characterized by a low molecular weight, commonly between 4-6 kda. the predicted isoelectric point varied from 4.17 to 10.2, whereas the gravy score, indicative of average hydrophobicity and hydrophilicity, ranged from -0.75 to +0.67. the stability index of these fp-3α proteins varied from -1.9 to 69.4. the predicted secondary structure computed for mgfp3α-2 (mgal_12191) indicated the presence of three β-strands and one α-helix motif between the 1st and the 2nd β-strands. within the cysteine array typically found in the fp-3α proteins, a stretch of three cysteine residues at the c-term of the mature peptide region is curiously located in one predicted β-strand. in the absence of pdb protein homologs, we generated a fp3α-2 draft structure and then refined it by imposing the disulfide bonds. as regards the mature peptide, disulfides 9-31, 16-37 and 20-39 resulted to be highly conserved among the muster hits (figure 6a) and were imposed for model generation with i-tasser. the resulting structures supported cysteine bonds also between residues 25-42 and 3-38, which were then imposed for the further refinement of the protein model. hence, the final fp3α-2 model was characterized by three β-strands between aa 5-9, 30-33 and 37-40 (including an antiparallel β-strand) and a α-helix motif between aa 17-22 (figure 6b). the quality of the fp3α-2 model was supported by ramachandran plot (lovell et al., 2003), since all the residues dihedrals were in the allowed regions. with a prosa ii score of -5.16, the final model fell in the range of native conformations (wiederstein & sippl, 2007). during the dm simulation, the c-terminal region showed a considerable displacement, indicative of an intrinsic flexibility and consistent with the prdos analysis (ishida and kinoshita, 2007; richardson et al., 2009). dali structural alignment revealed a high similarity with several different toxins and defensins (table 2). discussion the byssus production has been studied in both marine and freshwater mussels and more than 20 different proteins have been involved in this process (lee et al., 2011). crucially, the adhesive 27 fig. 6 structural modelling of m. galloprovincialis foot protein 3α (mgfp-3α) related to the plant defensin raphanus sativus antifungal protein 1 (rs-afp1) which showed the highest z score among other defensin proteins (see table 2). a. multiple alignment of mgfp-3α with the best fitting hits from protein data bank (pdb); the mgfp-3α ribbon model is coloured according to the values of root mean square fluctuation index (rmsf) (blue and red indicate low and high rmsf, respectively). b. the mgfp-3α and rs-afp1 alignment makes evident a different pattern of disulphide bonds, although the rs-afp1 and mgfp-3α ribbon models are rather similar plaque formation depends on a time-regulated secretion of different foot-associated proteins (guerette et al., 2013) as the secretion of dopacontaining proteins is immediately followed by that of other cysteine-rich proteins, which likely prevent the oxidation of dopa residues through redox interactions. in mytilus spp., the cysteine-rich foot protein 6 (mfp6) has been considered largely responsible for the reducing activity within the terminal plaque (yu et al., 2011; waite, 2017) and a similar role has been outlined for the foot protein 3 alpha (fp-3α) of p. viridis (petrone et al., 2015). searching into genomic and transcriptomic data of byssogenic bivalve species, we identified 43 fp3α-like transcript sequences. in addition to p. viridis, we could trace these sequences in m. virgata, t. hirsuta, and mytilus sp. (brachidotinae septiferinae and mytilinae subfamilies, respectively). the absence of fp-3α sequences in the subfamilies bathymodiolinae, modiolinae, and limnoperninae would locate a primordial fp-3α gene after the division of the mytilidae family in its two main clades (lee et al., 2019). notably, we could describe 12 fp3α genes in the genome of m. galloprovincialis, which is the only available genome for a species having fp-3α genes. the tentative reconstruction of the evolutionary history of fp-3α genes suggests the duplication of an ancestral gene similar to fp3α-1 before the divergence of the asian green mussels from the mediterranean mussels. the fp-3α genes exhibit a very diversified gene organization, based on 1, 3 or 4 exons. such a situation could be explained by exon-shuffling events involving the exon encoding for the mature peptide (froy and gurevitz, 2003). exon shuffling consists in an intron-mediated recombination of exons from existing genes (long et al., 2003). to corroborate such an hypothesis for the fp-3α gene family, the mature peptide region has to be encoded by a single exon, with phase i flanking introns, and it should display folding authonomy (patthy, 1999; froy and gurevitz, 2003). and this appears to be the case. possibly favoured by exon shuffling, we also reported two different m. edulis splicing isoforms for the mgal10a009044 and mgal10nca009345 genes. three different fp-3α mussel genes possess 4 exons and 3 introns, with the third exon encoding for the n-terminal extension. irrespective of the exon number, the nine remaining fp-3α mussel genes are devoid of the exon with the n-terminal extension, which could possibly have been lost via exon shuffling and perhaps not essential to the protein function. a similar phenomenon has been reported for crassostrea gigas β-defensins, which likely originated from an ancestral big defensin by loss of 28 table 2 best hits from dali server for fp-3α. protein data bank (pdb) code, name, class, organism of origin and function are reposted (z, confidence index; rmsd, root mean square deviation between fp-3α and the structural homolog; lali, length of the alignment, % id, percentage of common residues) pdb code name class organism function z rmsd lali %id reference 2i61 lqhit2 toxin leiurus quinquestriatus hebraeus sodium channel activator 3.6 3.1 46 20 (karbat et al., 2007) 1agt alpha-ktx 3.2 toxin leiurus quinquestriatus hebraeus potassium channel inhibitor 3.3 1.9 36 19 (krezel et al., 1995) 1fh3 lqh3 toxin leiurus quinquestriatus hebraeus sodium channel activator 3.2 3.3 45 18 (krimm et al., 1999) 1npi ts1 toxin tityus serrulatus sodium channel activator 3.2 3.7 46 20 (pinheiro et al., 2003) 2uvs alpha-ktx 3.1 toxin androctonus mauretanicus potassium channel inhibitor 3.1 2.5 37 22 (pentelute et al., 2009) 2k4u alpha-ktx 3.6 toxin mesobuthus martensii potassium channel inhibitor 3.1 2.5 36 19 (yin et al., 2008) 1ayj rs-afp1 defensin raphanus sativus fungicide 3 2.5 40 23 (fant et al., 1998) 4he7 brazzein defensin pentadiplandra brazzeana sweet-taste receptor binding peptide 3 3.4 40 23 (nagata et al., 2013) 2lj7 lc-def defensin lens culinaris antimicrobial 2.9 2.3 40 23 (shenkarev et al., 2014) 3psm spe-10 defensin pachyrhizus erosus antimicrobial 2.9 2.5 39 21 (song et al., 2011) the n-terminal domain via exon shuffling (loth et al., 2019). conversely, the fp-3α genes having a singleexon (mgal10nca069861, mgal10nca009347) could be explained by a retrotransposition mechanism, which basically consists in the reintegration of a mrna back into genomic dna (navarro and galante, 2015). as regards the genomic location of mussel fp3α genes, the m. galloprovincialis genome is assembled to scaffold level and, therefore, the distance between these genes can be established with certainty only if they fall in the same scaffold. actually, only mgal10a001023 and mgal10a031169, both encoding for fp3α-6 type proteins and expressed at similar levels in the mussel gills, are in close vicinity in the same scaffold, thus supporting a recent duplication event. from a functional point of view, only 9 of the 12 fp-3α genes resulted to be expressed at and variable, not negligible, level in the analysed transcriptomic datasets derived from multiple tissues and populations of m. galloprovincialis. five of them were uniquely expressed in the mantle, while the remaining four were expressed in gills or digestive gland or hemolymph (figure 3). regarding the foot tissue, no transcriptome data were available for m. galloprovincialis but rna-seq data specifically obtained from the three foot-associated glands of m. californianus (demartini et al., 2017) indicated an almost exclusive expression of mgal10nca069861, mgal10a009044 and mgal10nca009347 in the accessory (cuticle) gland. mapping the bivalve transcript sequences (listed in materials and methods) on the mussel genome, we could associate the fp3α-2 and fp3α-3 protein types (those composing clade 1 in the bayesian tree) to the fp-3α genes expressed either in the accessory (cuticle) gland or in the mantle edge, tissues which contain mfp1, a dopa-rich protein (waite, 2017), and periostracin, another dopa-rich protein (waite et al., 1979), respectively. reasonably, these two fp-3α protein types could have a dopa-protective role, preventing its spontaneous oxidation and supporting dopamediated cohesion processes (nicklisch and waite, 2012). conversely, the fp3α-4, -5 and -6 (those composing clade 2 in the bayesian tree) are mainly expressed in other mussel tissues. curiously, these fp-3α protein types possess a signal peptide and a conserved csαβ folding (including a cys-x-gly pattern in the γ-core) similarly to csαβ antimicrobial peptides (yeaman and yount, 2007). with no doubt, their possible host defence role requires a dedicated investigation. if this was the case, it would corroborate the hypothesis of an ancestor mussel fp-3α gene evolved by gene duplication and functionally diversified through a neofunctionalization or a subfunctionalization mechanism (innan and kondrashov, 2010). as reported in p. viridis and mytilus spp., native foot proteins start from a disordered state and assume elongated conformations still including disordered sequence tracts (olivieri et al., 1997; hwang and waite, 2012; mirshafian et al., 2016). similar to defensins and scorpion toxins, the five disulphide bridges and the csαβ motif of mussel fp3α proteins (here modeled for fp3 α-2) are expected to improve the resistance to protease degradation and protein stability at non-physiological values of temperature and ph (tam et al., 2015; koehbach, 2017). moreover, the tyrosine residue present in the 29 flexible c-terminal of the mature peptide region in fp3α-2 transcripts (subterminal in some other fp-3α types) could be modified to dopa, as demonstrated for the p. viridis homolog (petrone et al., 2015). overall, these findings suggest that at least the cterminal of fp3α-2 proteins can be involved in binding activity, for instance with other proteins involved in the byssogenesis. in conclusion, the comparison between 43 transcript-derivedand 12 genome-derivedfp-3α protein sequences demonstrated that m. galloprovincialis possesses at least one gene for each of the six fp-3α types detected in the mytilus sp. complex, including fp3α-1 which is expressed in p. viridis. likely expanded by several duplication events, the twelve members of the mussel fp-3α gene family show peculiar tissue expression patterns indicative of functional divergence at least between the two main gene clades recognized by bayesian phylogenesis. while confirming the role of some fp-3α proteins in the maintenance of dopa properties, the antimicrobial activity reported for canonical defensin might be demonstrated in the future at least for some of these genes. actually, the defensin-like character of mussel fp-3α proteins is supported by the highly conserved cysteine residues, exon-intron 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tissue biomarkers as vulnerability indicators in the clam polymesoda caroliniana [17: 186-197, 2020] jr jerónimo-juárez1, ml matadamas-guzman2, i guerrero legarreta3, jc segoviano-ramírez4, m del rocío zarate-hernández5, m arteaga-silva6, x guzmán-garcía7* 1posgrado en energía y medio ambiente, universidad autónoma metropolitana, 09340, ciudad de méxico, méxico 2doctorado en ciencias biomédicas, unam, ciudad de méxico, méxico 3departamento de biotecnología, universidad autónoma metropolitana, unidad iztapalapa, 09340, ciudad de méxico, méxico 4unidad de bioimagen, centro de investigación y desarrollo en ciencias de la salud, universidad autónoma de nuevo león, monterrey, nuevo león, méxico 5laboratorio de peces, departamento de biología, universidad autónoma metropolitana, 09340, ciudad de méxico, méxico 6laboratorio de neuroendocrinología reproductiva, departamento de biología de la reproducción, universidad autónoma metropolitana, 09340, ciudad de méxico, méxico 7departamento de hidrobiología, laboratorio de ecotoxicología, universidad autónoma metropolitana, unidad iztapalapa, 09340, ciudad de méxico, méxico in the above article table 2 was reproduced incorrectly, the table and caption should have appeared as below: table 2 relations between the prevalence of histopathological alterations and level of exposure. the sensitivity value was calculated by subtracting the prevalence minus exposure level. these relations resulted in a categorization of sensitivity (very low = -2, low = -1, normal = 0, high = 1, very high = 2) exposition level value prevalence of histopathological alterations value sensitivity value sensitivity category minimal 1 low prevalence 1 0 normal moderate 2 1 -1 low high 3 1 -2 very low minimal 1 moderate prevalence 2 1 high moderate 2 2 0 normal high 3 2 -1 low minimal 1 high prevalence 3 2 very high moderate 2 3 1 high high 3 3 0 normal ___________________________________________________________________________ corresponding author: xochitl guzmán-garcía universidad autónoma metropolitana, unidad iztapalapa san rafael atlixco 186, vicentina, c.p. 09340, iztapalapa, ciudad de méxico, méxico e-mail: xgg@xanum.uam.mx 85 isj 19: 85-90, 2022 issn 1824-307x short comunication stimulation effect of probiotic bacteria bacillus spp. and inactivated yeast on the honey bees apis mellifera physiology and honey productivity e sokolova1,2,3*, s mager2, e grizanova1,3, g kalmykova2, n akulova2, i dubovskiy1,2,3* 1novosibirsk state agrarian university, laboratory of biological plant protection and biotechnology 2siberian federal scientific centre of agro-biotechnologies of the russian academy of sciences 3tomsk state university, tomsk, russia this is an open access article published under the cc by license accepted may 11, 2022 abstract in order to find effective and safe ways to prevent the weakening and death of honey bee colonies from various stress factors, it is necessary to focus on the stimulation of physiological processes in the bee’s body, activating their own mechanisms of resistance. bacteria bacillus subtilis and bacillus licheniformis produce important digestive enzymes, which have antimicrobial and detoxification effects, and also stimulate metabolic processes in the organism. the inactivated yeast saccaromyces cerevisiae was used as a protein and vitamin component of the supplement. it was found that the addition of the studied supplement to the bees' feeding increased the activity of proteases 3.32-fold in the gut, non-specific esterases 2.16-fold in the fat body, glutathione-s-transferases 2.64-fold in the gut and 1.69-fold in the fat body in the apis mellifera. application of the supplement in the field has shown that the honey productivity per family increases 1.5-fold compared to the control. key words: probiotics; apis mellifera; bacillus; saccaromyces cerevisiae; supplement; detoxification system introduction over the last twenty years, both technical and scientific progress and the growth of beekeeping have led to a significant increase in honey production, with an average annual growth of 35,000 tonnes since 2000, amounting to a total of 1.8 mt of honey produced in 2016 worldwide (pippinato et al., 2020). however, in some years, the world production of honey has reduced compared to the previous one (for example, in 2007, 2009, 2018, 2019) (faostat). high mortality in honey bee colonies has been reported worldwide in recent decades without definitive identification of the causes (benaets et al., 2017). nevertheless, recent investigations have established some of the most important factors contributing to honey bee losses, in particular, pests and diseases, bee management, including bee keeping practices and breeding, the change in climatic conditions (potts et al., 2010), agricultural practices, and the use of pesticides (gill et al., 2012; hristov et al., 2020). the decline in honey bee populations causes serious damage not only to the production of honey, but also to pollination _________________________________________ corresponding authors: elina sokolova ivan dubovsky novosibirsk state agrarian university laboratory of biological plant protection and biotechnology st. dobrolyubova, 154, 630039 novosibirsk, russia e-mails: elinq.98@mail.ru; dubovskiy2000@yahoo.com of plants affecting the functioning of natural and agricultural ecosystems (klein et al., 2007; van engelsdorp et al., 2008), therefore the attention of the international community is focused on this phenomenon. in the early spring period, with a meager flow, as well as during wintering, when environmental conditions become unfavorable for the vital activity of bees, they are most susceptible to the influence of various pathogenic factors (becsi et al., 2021). to provide bees with the necessary amount of nutrients and to activate metabolic processes during these periods, beekeepers use various feedings. they can activate the defense systems of bees and increase the amount of obtained honey (brodschneider and crailsheim, 2010). to prevent and protect the honey bees from infections and parasitosis (such as varroatosis, acaripidosis), beekeepers generally use a variety of antibiotics and insecticides, which imposes restrictions on the production of organic beekeeping products (ruoff and bogdanov, 2004; luttikholt, 2007) and their contamination may carry serious human health hazards (al-waili et al., 2012). most importantly, it leads to the accumulation of a stockpile of resistance capabilities in the microbiota of a healthy gut, providing a source of resistance genes for pathogens themselves (tian et al., 2012). therefore, in order to find effective and safe ways to prevent the weakening and death of honey bee 86 colonies, it is necessary to focus on the stimulation of natural physiological processes in the body of bees, activating their own mechanisms of resistance. bacteria bacillus subtilis and bacillus licheniformis are related species of gram-positive bacteria. as antagonists of pathogenic and opportunistic microorganisms (staphylococcus sp, salmonella sp, shigella sp) (moore, 2013), they stimulate the growth of normal intestinal microbiota (mazkour et al., 2019). reproducing in the intestinal lumen, these bacteria produce all the main digestive enzymes (proteases, amylases, lipases, pectinases, cellulases), stimulate metabolic processes in the microorganism (suva et al., 2016). in addition, there are more than two dozen known antibiotics produced by b. subtilis (kudriashova et al., 2005; stein, 2005). it should be noted that multi-strain mixture of these microorganisms is able to mutually reinforce and complement each other's biological activity (cutting, 2011). probiotic bacteria are not only antagonists of opportunistic microbiota in the bee’s gut, but also a constant source of microbial protein. this is especially important during intensive brood growth the sensitivity of adult honey bees to pesticides directly depends on the amount and quality of protein consumed in the first 10 days after hatching (wahl and ulm, 1983). because bees fed a high protein diet were better able to survive insult with interacting stressors (archer et al., 2014) the studied feeding included the inactivated yeast saccaromyces cerevicaea as a protein and vitamin component, since it contains all the essential amino acids (abbas, 2006) and is easily digested by honey bees (abbasian and ebadi, 2002). honey bees and other insect pollinators utilize detoxification enzymes such as carboxylesterases and glutathione-s-transferases (gsts) to mitigate the toxic effects of xenobiotics such as plant defense compounds and pesticides (panini et al., 2016). glutathione-s-transferases (gst) are the principal phase ii (conjugation of products of phase i) enzymes, although they can also function in phase i during which the toxin structure alters enzymatically (berenbaum and johnson, 2015). indeed, esterase enzyme activity positively correlates with pesticide tolerance in many insect species, including bees of all stages of development (milone et al., 2020). total protease activity in honey bee midgut is also an important parameter related to protein digestion (li et al., 2012). a decrease in its activity is not only associated with a low level of protein in the diet, it may also be related to inhibitory effects of various infections and parasitosis. the inhibition of the activity of the host's proteolytic enzymes by the parasite often occurs during endoparasitosis (zółtowska et al., 2005). beekeeping is still done mainly to produce honey (crane, 2009). it was found that honey production is governed by the interaction of three primary factors: average daily brood production, length of worker life and individual productivity of workers (woyke, 1984). the health and, consequently, the productivity of honey bee colony depends on abiotic factors such as pesticides, management, weather conditions (abou-shaara et al., 2017), biotic factors such as mites, viruses, bacteria and fungi as well as on the nutrition profile (steinhauer et al., 2018). the aim of this study was to examine the effect of bacteria b. subtilis and b. licheniformis and inactivated yeast culture on the detoxifying and digestive enzymes of honey bees apis mellifera, as well as on their honey productivity in the field. material and methods experimental design adult worker bees of the middle russian race were collected from one hive of a medium-strong family (novosibirsk region, 54.759912, 82.633827). the bees were taken from the surface of the combs and transported to the laboratory of novosibirsk state agrarian university. the bees were kept under laboratory conditions in shaded cages at a temperature of 28 30 °c and a relative humidity of 60 – 65 % with approximately 200 bees in each variant. experimental diet was performed with 60 % sugar syrup with the addition of bacteria with yeast additive (5 g/l of a mixture of bacillus subtillis and bacillus licheniformis and 30 g/l of inactivated yeast). in the control variant, no components were added to the sugar syrup. the syrup was prepared and replaced daily. enzymes activity in the fat body and midgut 10 days after the start of the feeding with experimental diet, the honey bees (n = 30) were placed on ice and their fat body and gut were dissected at 4 °c (carreck et al., 2013). each organ was homogenized by ultrasound in 100 µl of 0,1 м na-phosphate buffer ph 7.2 (pbs). the homogenates were centrifuged for 15 min, 10,000 g at 4 °c. the supernatant was used for the analysis of enzyme activity. the activity of glutathione-s-transferases was determined spectrophotometrically at 340 nm, calculating the rate of increase in the concentration of 5(2,4-dinitrophenyl)-glutathione, which is a reaction product of dinitrobenzene and reduced glutathione (habig et al., 1974). incubation was carried out at a temperature of 28° c for 5 min with the following composition of the reaction mixture: 205 μl of pbs with the addition of 150 mm nacl, 1 mm glutathione, 1 mm o-dinitrobenzene, and 5 μl of the supernatant of the studied tissue (grizanova et al., 2018). nonspecific esterase activity was estimated by spectrophotometric analysis of the pnitrophenylacetate hydrolysis rate (prabhakaran and kamble, 1993). five microliters of the supernatant were incubated with 200 μl phosphate buffer with the addition of 0.54 mm 1-naphthyl acetate in darkness for 5 min at 28 °c, and then the transmission density was measured at 410 nm. the method for determining the total proteolytic activity was to measure the rate of hydrolysis of 0,3 % azocasein (sigma) by bee intestinal proteinases with some modifications (alarcón et al., 2002). 30 μl of intestinal supernatant was added to 210 μl of 0.3 % azocasein in pb, after which the reaction mixture was incubated at 37 °c for 1. the reaction 87 was stopped by adding 200 μl of a 30 % tca solution and subsequent incubation at -18 °c for 30 min. then the resulting mixture was centrifuged for 10 min, 10000 g, and 150 μl of the supernatant was taken from it. after adding 70 μl of 1m naoh to the resulting mixture, the optical density was measured at 440 nm. enzyme’s activity was measured in units of optical density (δa) of incubation mixture per 1 min and 1 mg of protein. the total protein concentration in all of the samples was determined according to bradford (bradford, 1976). standard curves to estimate protein concentration in the samples were prepared using bovine serum albumin (bsa). microscopic analysis of the fat body fat bodies of 30 bees from the control and treated groups were assessed according to the developmental index (a scale from one to five, with five being the best developed structure) proposed by maurizio (1954), examining the inner surfaces of tergites using a binocular microscope (fliszkiewicz et al., 2012). honey productivity three honey bee colonies with one year-old sister queens were selected per variant (one of these colonies was used in laboratory tests of bees enzymes activity in the fatbody and midgut), feeding was carried out three times (every three weeks) during the spring of 2021. bee colonies of the control variant received sugar syrup without additives, the experimental group of bees received the supplement with the studied additives 10 g of inactivated yeast and 2 g of a mixture of b. subtilis and b. licheniformis per colony. the honey was pumped out twice in july and in september. colony honey production was determined by weighing the honey supers before and after extraction, after which the mass of honey per one bee family was calculated (nelson and gary, 1983). statistic the data were analyzed using graphpad prism® ver. 8 (graphpad software, usa). the results are reported as the mean values ± sd. a kolmogorov-smirnov normality test was used to check the normal distribution of the data. the data of visual scoring of the fat body of bees was analyzed by a mann whitney test. two-way anova (with dunnett’s multiple comparison test) was used to assess differences between activities of ferments in the insect midguts and fat bodies. results and discussion it was shown that adding bacteria b. subtilis and b. licheniformis with inactivated yeast to the honey bees' diet significantly increased the activity of gst both in the gut (p < 0.001) and in the fat body (p <b0.05), and esterases in the fat body (p < 0.001) compared to the control after ten days of feeding the diet with supplement (fig. 1). in addition, the diet with supplement significantly (p < 0.0001) increased the activity of proteolytic enzymes in the gut of honey bees compared to the control on the tenth day of the experiment (fig. 1). fig. 1 increased activity of non-specific esterases and glutathione s-transferases in the fat bodies and glutathione s-transferases and proteases in the guts of honey bees fed with probiotic bacteria and inactivated yeast. the detoxifying enzymes activity was assessed in the fat bodies and the guts of bees. proteolytic ferments were assessed in the guts. treatment (bacteria with yeast additive) contained sugar syrup with 5 g/l of b. subtilis and b. licheniformis and 30 g/l of inactivated yeast culture; control contained sugar syrup without additives. (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 compared with control group bees) 88 fig. 2 increased degree of the fat body development of honey bees fed with probiotic bacteria and inactivated yeast (treatment). treatment contained sugar syrup with 5 g/l of b. subtilis and b. licheniformis and 30 g/ of inactivated yeast culture; control contained sugar syrup without additives. (***p < 0.001 compared with control group bees) increasing activity of gst both in the intestine and in the fat body and esterases in the fat body of honey bees indicate the stimulation of the detoxifying system when bacteria b. subtilis and b. licheniformis and inactivated yeast culture were added to bees’ diet. it is important to note that the development of insect resistance to pesticides often has a metabolic basis (berenbaum and johnson, 2015; heckel, 2018) and can be microbe-mediated (itoh et al., 2018). therefore, an increase in the activity of detoxifying enzymes can provide protection from the harmful effects of chemical pesticides. an increase in the activity of proteolytic enzymes in the intestine of bees can be caused not only by an increase in the level of protein in the diet (li et al., 2012), but also directly by proteases produced by bacillus bacteria itself (connelly et al., 2004). the fat body of insects is the most metabolically active tissue and plays a crucial role in storing and utilizing energy and in detoxification processes (skowronek et al., 2021). it is responsible for the synthesis of most of the hemolymph proteins, including antimicrobial peptides (arrese and soulages, 2010). fat body assessment showed that the average value for the group, which diet was supplemented with probiotic bacterial strains and inactivated yeast, was 4.43 ± 0.63 points and was significantly higher (***p < 0.001) as compared with the control group 3,37 ± 0.61 points (fig. 2). well-developed fat body of honey bees fed with receiving b. subtilis, b. licheniformis and inactivated yeasts suggest a higher level of metabolism and activity of internal defense mechanisms, coinciding with studies of the development of fat body when feeding commercial probiotics with added protein (kazimierczak-baryczko and bozena, 2006). in addition, evans and lopez have showed that mix of bacterial spores from species in the genera bifidobacterium and lactobacillus induced as strong immune response (in antimicrobial peptide abaecin level) as a bee pathogen paenbacillus larvae when ingested (evans and lopez, 2004). the average honey productivity for the 2021 season was 27.4 ± 1.04 kg per hive in the control group and 40.1 ± 1.44 kg per hive in the experimental group. accordingly, the use of inactivated yeast s. cerevicaea and bacteria b. subtilis and b. licheniformis increased the final honey productivity by 1.5 times compared to the control. this can be caused by the normalization of the microbiota of the honey bee (kaznowski et al., 2005; vásquez et al., 2012), the suppression of pathogenic microorganisms (baffoni et al., 2016), the stimulation of the production of antimicrobial peptides in the honey bee's body (evans and lopez, 2004), as well as a decrease in the absorption of pesticides by probiotic bacteria (trinder et al., 2016). in addition, according to many authors, the lack of protein in bee feed negatively affects ovarian activation and brood rearing, which in the long term affects the amount of honey (herbert et al., 1977; pirk et al., 2010). from the presented data it can be assumed, that inactivated yeast mixed with b. subtilis b. licheniformis as a bee supplement has properties of 89 stimulating the detoxifying and digestive systems of bees, and also affects the honey productivity of honey bee colonies. the use of this feeding option can help to cope with the weakening of bee colonies increasing the quantity of obtained honey. additional experiments using a larger set of apiaries will be performed to further support the present suggestions. at the same time, honey bee populations of different subspecies from other regions will be tested to determine the supplement’s effects on colony performance, changes in the gut microbiome of treated honey bees and resistance to bacterial diseases. funding details this work was supported by the ministry of science and higher education of the russian federation in accordance with agreement № 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parazytol. 51: 4347, 2005. 28 isj 19: 28-36, 2022 issn 1824-307x research report identification of five picorna-like viruses associated with the endangered cavedwelling bivalve congeria kusceri (bole, 1962) a scapolatiello1, u rosani2, c manfrin1, s puljas3, a pallavicini1, m gerdol1* 1university of trieste, department of life sciences, trieste, italy 2university of padova, department of biology, padova, italy 3university of split, faculty of science, split, croatia this is an open access article published under the cc by license accepted november 16, 2021 abstract congeria kusceri is a bivalve mollusk species endemic to the dinaric karst, which displays unique adaptations that have allowed its survival in the subterranean environment with small morphological changes compared with its fossil relatives. anthropic activities have recently impacted the surface flow of the neretva river, impairing the seasonal water cycle that has characterized the habitat of this species for hundreds of thousands of years. the lack of an adequate water supply, together with pollution from agricultural and farm water runoff, are posing a serious threat to c. kusceri, as evidenced by the sharp population decline observed in several locations during the past few decades. due to the limited knowledge available about the basic biology of this filter-feeding species, the precise factors that may affect its health status and reproduction and therefore represent a hazard for its conservation are still unclear. here, through a transcriptomic approach, we describe the nearlycomplete genomes of five c. kusceri-associated rna viruses belonging to the picornaviridae family and phylogenetically related with picorna-like viruses previously described in other mollusca. although it is presently unknown whether these viruses may have a detrimental effect on bivalve health, we observed a significant increase of viral load during the summer season. key words: virome; karst; subterranean; picornaviridae; transcriptome; rna-sequencing introduction congeria kusceri (bole, 1962) is a freshwater bivalve endemic to the dinaric karst, a vast region geologically characterized by the presence of carbonate rocks and located in the balkan peninsula, between the adriatic sea and the pannonian basin (zupan hajna, 2019). c. kusceri is currently listed as a vulnerable (vu) species in the european red list of non-marine mollusks (cuttelod et al., 2011) and as a critically endangered (cr) species in the croatian red list of subterranean fauna (bilandžija and jalžić, 2009). a member of a once widespread genus of freshwater bivalves that inhabited the paratethys sea, c. kusceri is a living fossil which maintained an apparent morphological stasis since the end of miocene (approximately 5.3 mya). the development of several unique adaptations, such as a k-selected reproductive strategy, larval brooding, and a long _________________________________________ corresponding author: marco gerdol university of trieste department of life sciences via giorgieri 5, 34127 trieste, italy e-mail: mgerdol@units.it lifespan (morton and puljas, 2013), allowed the survival of this species in the subterranean environment to the present day (stepien et al., 2001; bilandžija et al., 2013). c. kusceri, whose current range of distribution includes just eight known sites in caves in to the neretva river basin in croatia and bosnia and herzegovina, was long thought to be the only member of a single pandinaric genus. however, recent molecular studies have established that two other reproductively isolated congeneric species survive in the dinaric karst, i.e. congeria mulaomerovici and congeria jalzici (bilandžija et al., 2013). the adaptation of c. kusceri to a highly stable cave environment poses a serious threat for its survival, since this species is not expected to be able to withstand significant long-term environmental alterations. in particular, the annual cycles which typically characterize the waters influx in karst caves play an important role in regulating shell growth, reproduction and other important aspects of congeria’s life cycle (morton and puljas, 2013; puljas et al., 2014; jovanović glavaš et al., 2016). the building of dams, hydropower plants and channels for agricultural land use over the past few 29 decades have undoubtedly modified the water regime of the neretva river basin, with a severe impact on the amount of water that seasonally floods the cave systems where c. kusceri lives. these alterations are now threatening the survival of this species, which has already disappeared from some of the caves where large populations had been historically documented and is quickly declining in others (bilandžija et al., 2021). moreover, agriculture and farm runoff can lead to organic and inorganic pollution in the subterranean environment (sasakova et al., 2018). this, together with the progressive salinization of the lower parts of the neretva basin due to seawater infiltration, poses a further threat for the unique habitat of congeria, which is thought to have remained unchanged for hundreds of thousands of years (vranješ et al., 2013). such environmental changes could have a significant impact on the cave microbial communities, as well on the congeriaassociated microbiota, primarily consisting of chemolithoautotrophic bacteria (rigonni et al., 2021). it is presently unknown whether alterations in the microbiome and in the virome found in karst subterranean waters can pose a threat for c. kusceri survival and no scientific literature is available on this subject. nevertheless, alterations of the bacterial and viral communities associated with bivalve tissues have been clearly linked with mass mortality events and with a general decline of the health of marine bivalve populations (barbosa solomieu et al., 2015; lasa et al., 2019). picornaviridae are among the most frequently described viruses associated with bivalve mollusks. even though their detection is often the result of bioaccumulation from ingested food particles or pathogens of bivalve associated parasites (crespogonzález et al., 2008), some of them can infect mollusks and replicate in their tissues (renault, 2016). in particular, ultrastructural studies have identified on several occasions picorna-like viral-like particles (vlps) in bivalves affected with granulocytomas ( rasmussen, 1986; jones et al., 1996) and numerous reports have suggested that picorna-like viruses may be the main responsible or contributors to mass mortality events observed in different species of marine bivalves (carballal et al., 2003; renault and novoa, 2004). it is however still unclear whether these rna viruses should be just considered as bivalve pathogens or rather as a component of the virome associated with these filter-feeding organisms under normal physiological conditions, considering their frequent detection though metatranscriptomic approaches in absence of detectable disease (moreira et al., 2012; rosani and gerdol, 2017; rosani et al., 2019). the information available about picornaviridae infections in freshwater bivalves is extremely limited. high viral loads of different picorna-like viruses have been previously observed in association with mass mortality events of actinonaias pectorosa in the clinch river (usa), even though they were unlikely to be the cause of the pathology (richard et al., 2020). although other picornaviruses have been also tentatively indicated as potential pathogens in north american freshwater bivalves (goldberg et al., 2019), on some occasions they have been linked with concurrent infections by parasitic trematodes (ip and desser, 1984). considering the role potentially played by picornaviridae as bivalve pathogens and the high susceptibility of cave-dwelling animals to environmental alterations, the detection of these viruses in c. kusceri may represent a potential threat for the conservation of this highly endangered species. materials and methods viral genome identification and characterization the transcriptome of congeria kusceri was generated through the de novo assembly of rnasequencing data obtained from multiple tissues (gills, mantle, gonads, digestive gland and adductor muscle). all tissues were carefully dissected with the aid of a scalpel, following the severing of the adductor muscle which allowed valve opening, from a total of ten individuals sampled between july and september 2018. the full methodology is described in detail elsewhere (scapolatiello et al., 2021). this sequence dataset was screened for the presence of contigs encoding proteins containing a rnadependent rna polymerase (rdrp) domain. in brief, the viral rdrp hmm profiles belonging to the clan rdrp (cl0027) were obtained from pfam (finn et al., 2009) and used as queries for hmmer v.3.3.2 (finn et al., 2011) against the virtuallytranslated proteome of the species (obtained with transdecoder, https://github.com/transdecoder/). positive matches were identified with an e-value threshold of 1x10-5. the reliability of the selected contigs was evaluated by the visual inspection of the uniformity of mapped illumina reads along the full length of the contigs and double checked through the comparison with an alternative de novo assembly obtained with metaspades v.3.10 (nurk et al., 2017), which fully confirmed the sequences obtained. the presence of open reading frames (orfs) was evaluated with the clc genomics workbench 20 (qiagen, hilden, germany) and the encoded proteins were: (i) used as queries for a blastp search (altschul et al., 1990) against the ncbi nr protein database, looking for significant homologies with sequences from previously described viruses; (ii) screened with interproscan v.5 to detect conserved protein domains (jones et al., 2014); (iii) analyzed with hhpred, looking for structural similarities not detectable using the two methods described above (söding et al., 2005). this approach allowed the identification of five viral genomes, whose completeness was assessed based on the pairwise comparison with the sequences of the closest available relatives detected in public databases. phylogenetic analyses since the five picorna-like viruses identified in c. kusceri displayed a different genome architecture and were all incomplete, the most conserved protein domain in picornaviridae, i.e. the rdrp, was extracted to create a multiple sequence alignment (msa) with muscle v.3.8.31 (edgar, 2004). the regions corresponding to the rdrp domain were similarly extracted from known viruses displaying a 30 significant homology with the five genomes under scrutiny, based on the detection of a significant blastp and pairwise identity at the amino acid level, higher than 40%. the previously published transcriptome of the closely related species dreissena rostriformis bugensis (péden et al., 2019), deposited in the ncbi transcript shotgun assembly database under the accession id ghix00000000.1, was similarly screened to detect other uncharacterized viruses. redundant sequences (i.e. those displaying a pairwise similarity higher than 90%) were removed with cd-hit (li and godzik, 2006). the msa was trimmed with gblocks v.0.91b in order to remove highly divergent and phylogenetically uninformative regions (talavera and castresana, 2007). then, this sequence set was analyzed with modeltest-ng v.0.1.3 (darriba et al., 2019) to identify the most appropriate model of molecular evolution according with the corrected akaike information criterion (cavanaugh, 1997), which was found to be a lg model (le and gascuel, 2008), with a proportion of invariable sites and a gamma-distributed rate of variation among sites. phylogenetic inference was carried out with mrbayes v.3.2.7a (huelsenbeck and ronquist, 2001), using two independent monte carlo markov chain (mcmc) analyses in parallel running for 250,000 generations each, sampling trees each 1,000 generations. the first 25% the obtained trees were discarded during the burn-in process and the convergence of all the estimated parameters of the model was evaluated with tracer v.1.7.1 (rambaut et al., 2018). quantification of viral abundance the trimmed illumina reads obtained from the 10 samples available were mapped against the five reference viral genomes with the clc genomics workbench v.20, using stringent parameters (length fraction = 0.75, similarity fraction = 0.98) to avoid non-specific matches. based on the number of reads mapped on each viral genome, the average read coverage per base was calculated for each virus and each sample, obtaining a normalized measure of abundance that could allow a direct comparison among samples. in detail, the five sampled tissues were gills, mantle, gonad, digestive gland and adductor muscle, which were taken from individuals collected in the pit in predolac in the neretva river basin (croatia) in july and september 2018, i.e. at the beginning and the end of summer, two critical time points for the life cycle of c. kusceri, as well as for the annual water cycle of the karst subterranean environment. each sample derived from a pool of five different individuals. the sequencing data analyzed in this study have been deposited in the sequenced read archive (sra) under the bioproject prjna704250 (srr13767952-srr13767961). results and discussion viral genome organization we identified five rna viruses characterized by the presence of a complete orf encoding a protein with a rna-directed rna polymerase (rdrp) domain. upon further scrutiny, and based on the homology detected with other known picornaviridae, all five genomes were found to be partial, as they were missing a region of variable length at the 5’end. since the library preparation protocol used a poly-a selection step, this result was expected, due to the enrichment of illumina reads towards the 3’end of poly-adenylated mrnas (love et al., 2016), including those of viral origin (rosani et al., 2019). the five viruses were named “congeria picorna-likevirus”, followed by a progressive number (from 1 to 5), thereafter abbreviated as cplv1, cplv2, cplv3, cplv4 and cplv5. overall, while the genomes of all five viruses encoded, as expected, the presence of a rnadirected rna polymerase (rdrp), a picornain 3c peptidase and a number of structural proteins, their organization largely varied. in line with previous reports about other picornaviridae isolated from invertebrates (shi et al., 2016), structural and nonstructural proteins were organized in polyproteins. these could be either encoded by a single or by two distinct orfs and they could be interchangeably encoded either at the 5’ or the 3’ ends of the viral genome. however, the regions encoding the picornain and rdrp domains were always detected in pair (with the peptidase located in a 3’ position compared with rdrp). in detail, the sequence of congeria picorna-like virus 1 (cplv1) was 6,133 bp long and harbored two orfs. based on the comparison with its known close relatives, the assembled viral genome was expected to lack approximately 3 kb at its 5’ end, corresponding to the n-terminal region of the replicative polyprotein, which contained the picornain and rdrp domains. the second complete orf encoded the structural polyprotein (fig. 1). unlike cplv1, the 5,614 bp genome of congeria picorna-like virus 2 (cplv2) included a single partial orf, which was predicted to lack about 3 kb of coding sequence. the architecture of the encoded polyprotein included a picornain/rdrp domain pair in the n-terminal part and the structural proteins of the capsid at the c-terminal end (fig. 1). the genome of congeria picorna-like virus 3 (cplv3) was 7,585 bp long and was predicted to lack approximately 1 kb of sequence at its 5’ end, likely making it the most complete out of the five viral genomes identified in the present work. the first incomplete orf encoded the structural polyprotein, whereas the second complete orf encoded a polyprotein which included the picornain/rdrp domain pair at the c-terminal end (fig. 1). in addition, a portion of this polyprotein showed structural homology with the 2c non-structural protein of other picornaviridae, which bears atpase and rna helicase activities (cheng et al., 2013) and is thought to be involved in the inhibition of the tnf-α-mediated activation of nf-κb in the host (li et al., 2016). the assembled genome of congeria picornalike virus 4 (cplv4) was the shortest out of the five genomes identified in this study, with a length of 3,817 bp. the only detectable orf encoded the picornain/rdrp domain pair in its c-terminal part. similar to cplv3, cplv4 had a region with structural homology with the 2c non-structural protein (fig. 1). it is presently unknown whether the capsid proteins 31 fig. 1 schematic representation of the five partial viral genomes identified in the present study. the serrated margins at the 5’ end indicate missing sequence. the main detectable viral domains (i.e. the capsid proteins, the rna-directed rna polymerase, the 3c peptidase and the 2c atpase/helicase) are indicated. white regions indicate the spacer regions located between orf1 and orf2 and the 3’utr in this virus are encoded by the n-terminal part of the same polyprotein or by a second orf located in the 5’ missing end of the viral genome. the genome of congeria picorna-like virus 5 (cplv5) was 5,230 bp long and it was predicted to lack ~3 kb of sequence at its 5’ end. the first, incomplete orf displayed the usual picornain/rdrp domain pair at the c-terminal end, whereas the second complete orf encoded the structural polyprotein (fig. 1). phylogenetic placement of the congeria picorna-like viruses although all the five congeria picorna-like viruses were novel, cplv3 was placed in a long branch of the phylogenetic tree, distantly related with most known picornaviridae, but displayed a very high sequence identity (75-90% at the amino acid level) with a small group of viruses previously described in freshwater environments and occasionally found associated to mollusca (fig. 2). in detail, cplv3 was phylogenetically related with a few nearly-identical viruses, placed in a single cluster by cd-hit and indicated by “freshwater picorna-like virus cluster 1” in figure 2. this included the trichosanthes kirilowii picorna-like virus, the forsythia suspensa picorna-like virus (yang et al., 2021), the snail beihai picorna-like virus 105/niflavirus (ng et al., 2012; shi et al., 2016) and with the freshwater mussel clinch dicistro-like virus 1 (richard et al., 2020). on the other hand, cplv1 belonged to a large monophyletic and heterogeneous group of picornaviridae (fig. 2). the closest know relative to cplv1 included the beihai picorna-like virus 70, 71 and 72, as well as the wenzhou picorna-like virus 26 and atrpec virus 1, which displayed sequence homology at the amino acid in the range of ~40% for the structural polyprotein and ~50% for the replicative polyprotein. these viruses have been reported in different invertebrates, including cnidaria and crustacea, but also in mollusca (i.e. the beihai picorna-like virus 70 and wenzhou picorna-like virus 26) (shi et al., 2016). intriguingly, albeit quite distantly related with cplv1, we found that another virus detected in d. rostriformis bugensis (genbank accession id: ghix01007600.1), one of the closest extant relatives of congeria spp. (stepien et al., 2001; bilandžija et al., 2013;), was placed in the same large clade of picornaviridae (fig. 2), which may therefore show some degree of host specificity to mollusca. cplv4 was placed in a different, well distinct group of picornaviridae (fig. 2) and only showed weak sequence homology with known picorna-like viruses isolated from several different invertebrates, which most notably included mollusca, such as in the case of the beihai mollusks virus 1 and 2 (shi et al., 2016). interestingly, albeit distantly related, two viruses detected in d. rostriformis bugensis (genbank accession ids: ghix01044252.1 and ghix01014380.1) also belonged to this clade, which 32 fig. 2 unrooted phylogenetic tree displaying the evolutionary relationship between the five congeria picorna-like viruses (indicated by red dots), other picorna-like viruses identified in the d. rostriformis bugensis transcriptome (indicated by blue dots) and other known picornaviridae. the tree is represented as a 50% majority consensus tree and posterior probability support values are only shown for major nodes. the scale bar indicates the number of substitutions per site suggests that these viruses may be part of the regular virome of dreissenidae and possibly of other bivalves (fig. 2). cplv2 and cplv5 did not show any significant match with known picornaviridae, only showing sequence homologies with less than 30% identity at the amino acid level. they were therefore both placed as orphan taxa in long branches of the phylogenetic tree, which nevertheless created a highly supported monophyletic clade with the cplv3 and the freshwater picorna-like virus cluster 1 (fig. 2). tissue and temporal distribution of the five c. kusceri picorna-like viruses overall, the five viruses displayed a very similar pattern of relative abundance, both across tissues and between the two sampling time points, even though it was not possible, due to the pooling strategy used prior to rna-sequencing, to evaluate whether such abundances were influenced by significant inter-individual differences. all viruses could be detected in c. kusceri in july 2018, albeit at relatively low levels. higher viral loads were identified in the mantle tissue, followed by gonads and digestive gland, with little or no detectable signal in the gills and adductor muscle (fig. 3), suggesting that the viruses were poorly replicating in bivalve tissues. in september 2018, higher viral loads were recorded in all tissues, with an increase by several orders of magnitude in the digestive gland (by ~50x for cplv5 and by > 100x for the other 4 viruses). cplv3 was the most abundant in this tissue, reaching an average read coverage close to 800x. the relative abundance of cplv3 was ~3.7 higher than cplv1, ~5.5x higher than cplv2, ~13.1x higher than cplv4 and ~17.4x higher than cplv5. four out of the five viral sequences were identified as differentially expressed transcripts in the statistical analysis for differential gene expression (a kal’s z-test on proportions (kal et al., 1999) carried out between the september 2018 and july 2018 digestive gland samples (scapolatiello et al., 2021). in detail, differential expression was 33 fig. 3 relative abundance of the 5 congeria kusceri picorna-like viruses in five different tissues in june and september 2018, calculated based on the number of reads mapped on the reference genomes supported by false discovery rate-corrected pvalues equal to 0 (cplv3), 1.82 x 10-12 (cplv1), 4.26 x 10-9 (cplv2) and 8.31 x 10-3 (cplv5). the significant viral loads recorded in the digestive gland may have different interpretations. in absence of ultrastructural observations, the precise location of the viral particles cannot be defined, leaving three alternative explanations open for the detection of viral rna: (i) bioaccumulation of the virus from exogenous sources by filter-feeding; (ii) association of the virus with parasites or symbionts of c. kusceri; (iii) active viral replication in the digestive gland of c. kusceri. the bioaccumulation of picornaviridae by filterfeeding has been documented in bivalves on several occasions, in particular for what concerns wastewater-borne human enteric viruses, even though such viruses are likely unable to infect bivalves and replicate in their tissues (hansman et al., 2008). it is therefore possible that the five picorna-like viruses described in this work are the result of influx of surface waters containing virusassociated food particles in the caves, with no direct impact on the health of c. kusceri. nevertheless, this hypothesis is challenged by relatively poor influx of water from the external environment which occurs during the summer season at the jama u predolcu (croatia). the possibility that the detection of these five viruses was linked with the presence of other parasitic infections, as previously reported in some marine species (crespo-gonzález et al., 2008), would certainly deserve further study. although several freshwater bivalve species have been previously identified as viable trematode hosts (brian and aldridge, 2019; taskinen, 1998; marszewska and cichy, 2015; müller et al., 2015), no scientific literature is available on this subject for c. kusceri. as of note, no 18s/28s rrna or coi markers suggestive of the presence of eukaryotic parasites was detected in the assembled transcriptome. future studies should also aim at investigating possible host-virus links, such as the presence of endogenous viral elements (eves) (blair et al., 2020), or at mapping crispr spaceromes (shmakov et al., 2020) in the genome of c. kusceri, once this resource will become available. concerning the possibility of active viral replication in the digestive gland, it needs to be remarked that histological lesions linked with picorna-like vlps have been previously observed in this tissue in other bivalve species, supporting the idea that the digestive gland may serve as a preferential viral replication site also in c. kusceri (hine and wesney, 1997). conclusions the five different picorna-like viruses identified in c. kusceri represent novel additions to the poorly known group of bivalve-associated rna viruses and provides some insights in the virtually unexplored viral landscape associated with subterranean waters. moreover, our bioinformatics-based detection approach confirms the usefulness of analyzing rna-sequencing datasets for the identification of uncharacterized rna viruses in bivalve hosts (rosani and gerdol, 2017; rosani et al., 2019). 34 it is worth noting that cplv2, cplv3 and cplv5 belonged to a well-supported monophyletic clade that only comprised a few known viruses previously isolated from freshwater sources, which may therefore represent an understudied group of picorna-like viruses associated with freshwater bivalves, and possibly with other freshwater invertebrates. all viruses displayed a marked increase in abundance throughout the summer season, which clearly indicates that this threatened bivalve species can be exposed to high viral loads in its natural environment. while it is presently unknown whether these viruses may have a detrimental effect on the health of this species, their phylogenetic relatedness with other picornaviridae found in different molluscan species suggest they may be not the product of bioaccumulation by filterfeeding of food particles. further ultrastructural investigations could clarify whether the presence of vlps in the tissues of congeria are linked with tissue lesions, helping to define whether alterations of the virome associated with this species should be considered as an additional risk factor to be monitored for the conservation of this endangered cave-dwelling bivalve. in light of the previous experience built in the conservation of freshwater bivalves living in surface waters (brian et al., 2021), we believe that an improved characterization of the potentially pathogenic microorganisms associated with congeria and its unique habitat, should be considered as a priority for a better planning of future translocation-based conservation efforts. acknowledgements this work was supported by the microgrants funding program of the university of trieste transcriptomic investigation of the endemic cavedwelling “living fossil” bivalve congeria kusceri. references altschul sf, gish w, miller w, myers ew, lipman dj. basic local alignment search tool. j mol biol. 215: 403-410, 1990. barbosa solomieu v, renault t, travers ma. mass mortality in 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(eds.), encyclopedia of caves (third edition). academic press, pp. 353-362, 2019. research report isj 13: 1-10, 2016 issn 1824-307x research report inheritance of heat stable esterase in near isogenic lines and functional classification of esterase in silkworm bombyx mori sm moorthy1, n chandrakanth1, n krishnan2 1silkworm breeding laboratory, central sericultural research and training institute, mysore 570 008, india 2department of biochemistry, molecular biology, entomology and plant pathology, mississippi state university, mississippi state, ms 39762, usa accepted december 30, 2015 abstract esterases are ubiquitous in living organisms and perform multiple functions in animals, plants, insects and microorganisms. insect esterases broadly perform physiological and defense functions. the present work aims towards identifying heat stable esterase in the hemolymph of near isogenic lines (nils) and their parents, and further to classify esterases based on substrate-inhibition reactions in silkworm, bombyx mori. five different α-esterases viz. est-1, est-2, est-3, est-4 and est-5 were observed in this study. of them, est-1 and est-2 were monomorphic and, est-3, est-4 and est-5 were polymorphic. heat stability studies revealed est-2 and est-3 as heat stable and, est-1, est-4 and est-5 as heat liable. through inhibition analysis, est-1 was identified as cholinesterase and est-5 as carboxylesterase because their activity was totally inhibited, respectively, by eserine sulphate and phenylmethylsulfonyl fluoride (pmsf) while est-3 was inhibited by both the inhibitors. the est-2 and est-4 were unaffected by both pmsf and eserine sulphate. the isozyme patterns of breed specific as well as heat stable esterases supports the variations in the survival percentage of silkworm breeds at high temperatures. this study enhances the knowledge on esterase-mediated thermotolerance in b. mori. key words: bombyx mori; hemolymph; carboxylesterase; cholinesterase; thermotolerance   introduction esterases (ec 3.1.1.x) are ubiquitously present in all living organisms. they form a group of hydrolases that perform multiple functions in plants, animals, insects and microbes. in particular, insect esterases are involved in digestion of nutritional material and mediation of insecticide resistance (oakeshott et al., 1993; shiotsuki and kato, 1999; amanullah et al., 2010). furthermore, they also participate in degradation of pheromone and hydrolysis of the juvenile hormone (jh) at different life-stages of insects (taylor and radic, 1994). the specificity between esterase and substrate varies in insects, therefore insect esterases are classified based on their reactions with different substrates as αor β-esterases (specific esterases) and nonspecific esterases (α and β). the esterases ___________________________________________________________________________ corresponding author: s. manthira moorthy silkworm breeding laboratory silkworm crop improvement division central sericultural research and training institute srirampura, manandavadi road mysore 570008, india e-mail: moorthysm68@gmail.com can be detected by staining acrylamide gels electrophoresed by samples in presence of αor βnapthylacetate (for specific esterases) or together (for nonspecific esterases) (simms, 1965). occurrence of esterases in numerous isoforms at distinct genetic locus, codominant inheritance and high degree of variability in banding pattern has made esterases a reliable marker for studying genetic variability within and among populations (eguchi et al., 1965). intraand inter-breed genetic variability in silkworms has been reported by analysing polymorphism based on esterase isozymes (staykova et al., 2003; moorthy et al., 2007a, b; staykova, 2008). moreover, ontogenetic changes in the esterase profiles of different tissues viz. hemolymph, silk gland, fat body, mid gut, genital organ, ovaries and mature eggs of silkworm have also been investigated (staykova et al., 2003). eguchi et al. (1965) conducted genetic and electrophoretic studies on blood esterases of silkworm and described four (besa, besb, besc and beso) fundamental types of blood esterases controlled by codominant alleles and studied their inheritance. gamo (1978) linked bes (blood 1 table 1 morphological characters of silkworm breed esterase) genes on 11th chromosome with reference to phenotypic markers. among the blood esterases of b. mori, besb, a major esterase has been purified and characterized as carboxylesterase (arai et al., 2000). despite the use of esterases as markers for genetic diversity studies (staykova et al., 2008) they are also known to participate in thermotolerance mechanism in insects (moorthy et al., 2007c, 2008; chattopadhyay et al., 2001). positive relationship has also been found between the activity of heat stable esterase of mid gut and thermotolerance in temperate silkworm breeds (wu and hou, 1993). heat stable esterase has also been identified in hemolymph of tropical silkworm breeds (chattopadhyay et al., 2001). the mulberry silkworm, bombyx mori is an insect with economic importance, domesticated for more than 5000 years within a narrow range of rearing temperature of 25 28 °c. therefore, silkworms are vulnerable to above or below this temperature range. particularly, rearing silkworms at higher temperatures have adverse effects on their survivability and silk yield (kumar et al., 2012). thermotolerance in b. mori is influenced by genetic and environmental factors (kumar et al., 2012). moreover, the thermotolerance in b. mori is measured in terms of survival rate (kumar et al., 2012), expression of heat shock proteins (velu et al., 2008), activity of catalase enzyme (nabizadeh and kumara, 2011) and synthesis of heat stable esterases at high temperature conditions (moorthy et al., 2008). while several studies are linked with detection, profiling and quantifying activity of heat stable esterases in b. mori (chattopadhyay et al., 2001; moorthy et al., 2008; somasundaram et al., 2009; patnaik et al., 2012; neerati et al., 2013), far few studies are associated with their classification (velu et al., 2008). furthermore, based on substrate-inhibitor specificities, esterases are classified as arylesterases, carboxylesterases and cholinesterases (yoo et al., 1996). therefore, this study is aimed to identify the heat stable α-esterase and classify the hemolymph esterases including the heat stable esterase of b. mori through inhibition studies. materials and methods silkworm breeds and high temperature treatment six silkworm breeds comprising of two multivoltines (nistari and cambodge), two bivoltines [d6(p) and sk4] and their near isogenic lines (nils) [d6(p)n and sk4c] were considered for this study. pure lines of all the breeds for this study were obtained from central sericultural research and training institute (csrti), berhampore, west bengal, india. d6(p)n and sk4c are near isogenic lines of d6(p) and sk4, respectively. d6(p)n was developed by using d6(p) as recurrent parent and nistari as donor parent, similarly, sk4c was developed by using sk4 as recurrent parent and cambodge as donor parent (moorthy et al., 2007b). the details of morphological characters of the selected silkworm breeds are presented in table 1. selected silkworm breeds were reared from hatching to 2nd day of 5th instar at 25 ± 1°c as recommended by krishnaswami et al. (1978). three replicates with 300 larvae each were reared. on 3rd day of 5th instar, the larvae were exposed to five different temperature regimes viz. 25 ± 1, 32 ± 1, 34 ± 1, 36 ± 1 and 38 ± 1 °c in a sericatron (chamber for temperature and humidity control) for 6 h a day to till they started spinning. the larvae were fed twice a day with mulberry leaves. matured larvae were picked and mounted on plastic mountages to spin cocoons. cocoon harvesting was carried out on the 7th day of spinning and defective ones were removed. the number of larvae that survived high temperature treatment, formed healthy cocoons and able to metamorphose to pupa, such cocoons were considered and counted as survival percentage. data on economically important quantitative traits viz., fecundity, cocoon yield/10,000 larvae by weight (kg), single cocoon weight (g), single shell weight (g) and shell percent (%) were collected at 25 ± 1 °c as described by krishnaswami et al. (1978). data on survival percentage of selected silkworm breeds at different high temperature regimes was also collected. breeds egg colour larval marking cocoon colour cocoon shape type nistari yellow marked yellow spindle with one end pointed nondiapausing cambodge light yellow plain yellow spindle nondiapausing d6(p)n light yellow marked white dumbbell diapausing sk4c yellow marked white dumbbell diapausing d6(p) light yellow plain white dumbbell diapausing sk4 colour less marked white dumbbell diapausing 2 table 2 quantitative traits of the silkworm breeds reared at 25 ± 1ºc presented as mean ± sd. means sharing different letters are significantly different breeds fecundity larval weight (g) cocoon yield / 10,000 larvae by weight (kg) single cocoon weight (g) single shell weight (g) shell percent nistari 312 ± 9.12a 23.28 ± 0.45a 9.89 ± 0.16a 1.18 ± 0.05a 0.168 ± 0.002a 14.25 ± 0.026a cambodge 345 ± 14.26b 25.66 ± 0.71b 9.641 ± 0.33b 1.23 ± 0.02a 0.179 ± 0.002b 14.54 ± 0.402a d6(p)n 485 ± 19.50c 33.57 ± 1.65c 13.587 ± 0.04c 1.53 ± 0.03b 0.298 ± 0.003c 19.36 ± 0.220b sk4c 512 ± 12.50d 34.42 ± 0.50c,d 14.087 ± 0.18c,d 1.61 ± 0.01c 0.333 ± 0.001d 20.51 ± 0.173c d6(p) 560 ± 15.50e 36.82 ± 1.94c,e 14.362 ± 0.04c,e 1.62 ± 0.01c,d 0.334 ± 0.003d,e 20.58 ± 0.152c,d sk4 520 ± 16.20d,f 36.95 ± 0.49c,f 13.820 ± 0.66c,f 1.57 ± 0.05b,c,e 0.307 ± 0.003c,f 19.50 ± 0.195b,e hemolymph collection hemolymph was collected separately from the larvae of selected silkworm breeds reared at 25 ± 1 °c on the 5th day of 5th instar by cutting a proleg and bleeding into a pre-chilled microfuge tubes with 1 mg of phenylthiourea to avoid the activity of prophenol oxidase leading to melanization of hemolymph. each sample was pooled from five larvae to minimize variations. the hemolymph was centrifuged at 1500g for 10 min at 4 °c and the supernatant was stored at -20 °c until use. polyacrylamide gel electrophoresis hemolymph samples were electrophoresed on 7.5 % polyacrylamide gels with tris-glycine electrode buffer of ph 8.3 under non-denatured conditions using dual vertical gel electrophoresis system (omega) connected with thermo-controlled water bath (pharmacia lkb multitemp ii). the procedure was followed as described by harris and hopkinson (1970). the haemolymph samples were electrophoresed under constant voltage of 110 v at 4 °c until the tracking dye reached the bottom of the gel. following electrophoresis, gels were soaked in a boric acid (0.5 m, ph 5) for 30 min at 4 ºc. traces of boric acid were removed by washing gels for two times with ice cold distilled water. the gels were stained with α-esterase substrate (2 % or α-napthyl acetate in acetone), 50 mg of fast blue bb salt and 100 ml of 0.1 m phosphate buffer ph 7.0 for 1 2 h at room temperature by following the procedure of simms (1965). gels were photographed and relative mobility (rf values) of each esterase band was calculated by using the formula: rf = distance of protein migration / distance of dye migrated. esterase bands were designated as est-1, est-2, est-3 and so on from the anodal migration to cathode. identification of heat stable esterases for identification of heat stable esterases, the gels were incubated at 60 ± 1 ºc for 15 min in a water bath prior to addition of α-napthyl acetate. following incubation, the gels were stained for esterase activity by following aforesaid procedure. esterase bands that were able to retain the activity at higher temperatures were considered as heat stable esterases. inhibition of esterases phenylmethylsulfonyl fluoride (pmsf) and eserine sulphate were used as inhibitors in this study. for inhibition analysis, gels were incubated in 50 ml of phosphate buffer (0.1 m, ph 7.0) containing 200 µl of inhibitor (final concentration of 0.2 mm) for 1 h at room temperature. following incubation, the gels were stained for esterase activity by following aforesaid procedure in presence of inhibitor. esterase bands that were able to retain the activity were considered to be unaffected by the inhibitors while the esterase bands which were unable to retain their activity were considered to be affected by the inhibitors. statistical analysis data on fecundity, cocoon yield/10,000 larvae by weight, single cocoon weight, single shell weight, shell percent and survival percentage of selected silkworm breeds at 25 ± 1 °c were collected. additionally, survival percentages of the selected silkworm breeds at different high temperature regimes were also collected. the collected data on 3 table 3 one-way anova on each quantitative trait of silkworm different quantitative traits were subjected to one way analysis of variance (anova) with silkworm breeds as factor. prior to analysis the data was tested for normal distribution (shapiro-wilk test) and homogeneity of variances (levene test) otherwise, after appropriate transformations to meet the criteria of parametric analysis. tukey hsd post hoc test was conducted for detecting significant differences (p < 0.05) between means. all statistical analyses were performed using spss 11.5 statistical package. results variations in the quantitative traits at 25 ± 1 °c generally, multivoltine breeds had low values than bivoltine breeds for all the quantitative traits studied. nils had intermediate values compared to their parents that is of higher than their multivoltine parents and lower than bivoltine parents (table 2). moreover, the performance of nils was almost equal to their bivoltine parents than multivoltine parent. fecundity was highest and lowest in d6(p) (560 ± 15.50) and nistari (312 ± 9.12), respectively. highest and lowest larval weight was noted in sk4 (36.95 ± 0.49 g) and nistari (23.28 ± 0.45 g), respectively. highest and lowest cocoon yield/10,000 larvae by weight was noted in d6(p) (14.362 ± 0.04 kg) and nistari (9.641 ± 0.33 kg), respectively. d6(p) (1.62 ± 0.01 g) and nistari (1.18 ± 0.01 g) displayed highest and lowest single cocoon weight. single shell weight and shell percent were highest and lowest in d6(p) (0.334 ± 0.003 g, 20.58 ± 0.152 %) and nistari (0.168 ± 0.002 g, 14.25 ± 0.026 %), respectively. one-way anova revealed significant differences (p < 0.05) in all the quantitative traits studied (table 3). survival percentage of selected breeds at high temperatures in order to understand level of thermotolerance in selected silkworm breeds, they were exposed to different high temperatures viz. 32 ± 1, 34 ± 1, 36 ± 1 and 38 ± 1 °c and survival percentages of all breeds were collected. the larvae reared at 25 ± 1 °c were considered as normal and had better survival. survival percentage was decreased as the rearing temperature was increased in all the breeds. multivoltine breeds (nistari and cambodge) exhibited higher survival followed by nils [d6(p)n, sk4c] and bivoltine breeds [sk4 and d6(p)]. of the multivoltine breeds, nistari displayed highest survival percentages at each tested temperatures with overall mean of 82.91 %. similarly, among nils and bivoltine breeds, sk4c and sk4 displayed highest survival percentages at each tested temperatures with overall mean of 70.03 % and 48.57 % respectively. the overall means of survival percentage of cambodge, d6(p)n and d6(p) were 79.34 %, 65.99 % and 41.80 % respectively. interestingly, nils had survival percentages higher than their bivoltine parents and lower than their multivoltine parents at high temperatures. significant differences (p < 0.05) were observed between survival percentages of nils and their parents at each tested temperatures (fig. 1). esterase isozyme polymorphism five different isoforms of α-esterases were observed in this study. starting from the anode end the esterase isoforms were designated as est-1 (rf 0.425), est-2 (rf -0.346), est-3 (rf -0.295), est-4 (rf -0.247) and est-5 (rf -0.217). of the five esterases, two were monomorphic (est-1 and est-2) and three were polymorphic (est-3, est-4 and est-5) between quantitative traits df mean square p value fecundity 5 30900.089 0.0001 larval weight 5 0.131 0.0001 cocoon yield / 10,000 larvae by weight 5 9.762 0.0001 single cocoon weight 5 0.121 0.0001 single shell weight 5 0.319 0.0001 shell percent 5 25.815 0.0001 survival percentage at 25°c 5 21.173 0.0001 survival percentage at 32°c 5 12821307.59 0.0001 survival percentage at 34°c 5 870.309 0.0001 survival percentage at 36°c 5 1283.602 0.0001 survival percentage at 38°c 5 28.387 0.000 i 4 fig. 1 survival percentages of different silkworm breeds reared at different high temperature regimes. data represented in mean ± sd. means sharing different letters are significantly different. the selected silkworm breeds accounting for 60 % polymorphism. est-1 and est-2 were expressed in all the selected breeds. est-3 was expressed in nils [d6(p)n and sk4c] and multivoltine parents (nistari and cambodge) but was not expressed in the bivoltine parents. est-4 was expressed only in d6(p). est-5 was expressed in nil, sk4c and its bivoltine parent, sk4. the highest number of esterases was expressed (four) in sk4c, while three esterases were expressed in each of the other breeds. the esterase isozyme banding pattern of nils (d6(p)n and sk4c) was very similar to their multivoltine parents (nistari and cambodge) with an exception of est-5 in sk4c, which was inherited from its bivoltine parent sk4 (fig. 2). identification of heat stable esterases acrylamide gels electrophoresed with hemolymph samples were incubated at 60 ± 1 ºc for 15 min in a water bath following staining with αnapthyl acetate as said before. while the heat liable esterases disappeared, heat stable esterases retained their activity. in this study, est-1, est-4 and est-5 were found to be heat liable and, est-2 and est-3 were identified as heat stable (fig. 3). classification of esterases inhibition studies using pmsf and eserine sulphate as substrate inhibitors were carried out to classify the hemolymph α-esterases. the inhibition studies on hemolymph α-esterases showed that est-1 and est-5 were totally inhibited by eserine sulphate and pmsf respectively, while est-3 was totally inhibited by both inhibitors. but est-2 and est4 were unaffected by both pmsf and eserine sulphate (figs 4a, b). discussion esterases represent a large, diverse and multifunctional group of hydrolytic enzyme systems that possess the property of overlapping substrate specificity, hydrolysing both endogeneous and exogenous esters of widely differing structures leading to hindrance of identification and classification (dixon and webb, 1979; walker and mackness, 1983). in silkworm, most of esterase isozyme polymorphism studies have done with bivoltine breeds and very little work has been conducted on multivoltine breeds (chattopadhyay et al., 2001). our study concentrated on multivoltines, bivoltines and their nils. this study revealed expression of five esterases, of which three were polymorphic and two were breed specific. esterase with rf -0.247 (est-4) was specific to d6(p) and another esterase with rf -0.295 (est-3) was expressed only in nils and multivoltine breeds. another esterase with rf -0.217 (est-5) was expressed in sk4 and its near isogenic line sk4c. est-3 was specific to multivoltine silkworm breeds and same was inherited by their nils. while developing nils, est-3 was used as marker to track the multivoltine blood characterized with higher survival. therefore, nils had higher survival than their bivoltine parents (moorthy et al., 2007a). heat stable esterases have been reported in plants (pandey, 1973), fishes (okumura et al., 1981) and in model insects such as drosophila 5 (cochrane, 1976). wu and hou (1993) also observed one heat stable α-esterase band (i.e., est5) which tolerated 60 °c and was found abundant in the mid gut tissue of temperate silkworm breed. chattopadhyay et al. (2001) observed a heat stable β-esterase and an est-3 in the haemolymph of tropical silkworm breed. in our earlier study (moorthy et al., 2007c), we reported the differential expression of esterases (est-1 and est-3) in the mid gut and fat body tissues of nistari breed at different temperature regimes ranging from 0 ºc to 32 ºc. however, the differences in the esterases of fat body tissue were not significant, while in the mid gut the expression of est-1 was high at 32 ºc followed by 0 ºc, 25 ºc and 15 ºc. in case of est-3, the expression was comparatively higher at 0 ºc, than 32 ºc and 15 ºc. recently, patnaik et al. (2012) reported a non-specific esterase (est-3) and a specific α-esterase (est-3) as heat stable esterases in the haemolymph of b. mori. in this experiment, two esterases viz. est-2 and est-3 were found to be heat stable while est-1, est4 and est-5 were heat liable. though, est-2 was heat stable, it was monomorphic and was expressed in all the studied silkworms. therefore, it had little effect on the specific thermotolerance with respect to breeds. contrary to it, est-3 was expressed in multivoltines and nils but not in bivoltines, which were sensitive to high temperatures. thus, it can be opined that the heat stable est-3 inherited from multivoltine parents to the developed nils followed directional selection (selection based on est-3) during the course of breeding. generally, characters under stringent selection tend to be conserved and inherited in populations, while those under less stringent selection vary highly. this mode of selection was made conserved and heritable in the successive generations by strictly selecting larvae that are tolerant to 33 ºc with the esterase banding pattern similar to multivoltine parents for breeding successive generations. according to staykova et al. (1998) ‘biochemical marker’ is a term used for biochemical macromolecules, which are able to differentiate between two species or different biotypes of the same species. such biochemical markers are also used to identify the breeds resistant to pesticides. hemolymph is a rich source of biochemical compounds in b. mori, of which enzymes are less changeable with respect to their genetic structure compare to other biochemical compounds, proving that they are good biochemical markers. further, better breeding program in the silkworm can be designed by identification of biodiversity (etebari et al., 2005). in this study, we have identified one (est4) breed specific esterase, two esterases inherited to nils, one each from bivoltine (est-5) and multivoltine parent (est-3). this result reveals that the esterases identified can differentiate between the silkworm breeds under study, more clearly between bivoltine and multivoltine silkworm breeds. est-3, inherited from multivoltine parents to their respective nils was heat stable and was the only marker tracking the multivoltine blood in each generations of nil development. presence of breed specific esterases in d6(p) (est-4) and sk4 (est-5) fig. 2 esterase isozyme pattern in the haemolymph on 5th day of 5th instar larvae of silkworm breeds reared at 25 ± 1°c. lane 1 and 2 are nils [d6(p)n and sk4c], 3 and 4 are bivoltine parents [d6(p) and sk4] and 5 and 6 are multivoltine parents (nistari and cambodge). and absence of (est-3) did not increase survivability in bivoltines, but their respective nils had increased survivability at high temperatures, which had heat stable esterase (est-3) inherited from their multivoltine parents. therefore, est-3 is a prominent esterase that was able to differentiate the thermotolerant and thermo susceptible breeds under study. hence, est-3 can be employed as a biochemical marker to classify the silkworm breeds based on their thermotolerance ability inherited from their multivoltine parents. est-3 is characteristic feature of thermotolerant multivoltine breeds integrated in nils. therefore, selection of parents based on such biochemical markers could be an effective approach for successfully breeding high temperature tolerant silkworm breed. the presence of two breed specific and one multivoltine specific esterases observed in this study supports that esterases can be used as reliable marker for breed identification. furthermore, screening of the entire silkworm germplasm for the esterases would generate valuable information that can serve to identify the silkworm breeds. patnaik et al. (2012) suggested that characterization of candidate heat stable esterases will enlighten its suitability as markers towards heat resistance and could be explored in the future breeding programs. in this direction, the characteristics of each esterase isozyme can be determined by the addition of specific inhibitors during the process of enzymatic staining of gels. based on substrate-inhibitor specificity, esterases are classified as arylesterases, carboxylesterases and cholinesterases (yoo et al., 1996). in this study, the inhibitors, pmsf and eserine sulphate were used to classify esterases. inhibition studies revealed that pmsf totally inhibited est-5 activity but it was unaffected by eserine sulphate. pmsf inhibits carboxylesterases. the pmsf is a serinehydrolase inhibitor suggests that inhibited esterases contain serine residues in their active sites. thus, these observations suggest that the est-5 might be 6 carboxylesterase. eserine sulphate totally inhibited est-1 indicating that it might be cholinesterase. cholinesterases are important regulatory enzymes that are responsible for controlling the neural transmission on synapses by hydrolyzing acetylcholine, the excitatory of neurotransmitter (yu et al., 2009). also cholinesterases are targeting on organo-phosphorus and carbamate insecticides, as these toxic compounds readily inhibit those (baffi et al., 2005). carboxylesterases also serve a protective role for the target cholinesterases during organophosphate intoxication because the carboxylesterases are alternative phosphorylation sites (watson and chambers, 1996). carboxylesterases are also associated with physiological processes of the cuticular wall synthesis and regulation of juvenile hormone levels (sparks et al., 1979). the inhibition pattern for est2, est-3 and est-4 were complex. est-3 (heat stable esterase) was inhibited by both pmsf and eserine sulphate which putatively indicates that it represents cholinesterase activity and has serine in its active site. cholinesterases also have a proteolytic activity in addition to their cholinergic activity, acting as proteases in regulating cell growth and development (small, 1990). therefore, cholinesterases (est-3) might be responsible for higher survival of nils and their multivoltine parents than bivoltines at high temperatures as est-3 was not expressed in bivoltines. est-2 and est-4 were unaffected by both the inhibitors, therefore it is assumed that they may belong to arylesterases group, but empirical studies are needed to prove this assumption. arai et al. (2000) characterized an esterase (besb) in the hemolymph as carboxylesterase using substrate-inhibition studies and enzyme kinetics methods. yu et al. (2009) identified 76 putative carboxylesterases on newly assembled b. mori genome through in silico analysis. murthy et al. (1996) purified and characterized a carboxylesterase from the mid gut of b. mori by inhibition studies in conjunction with other molecular techniques. recently, neerati et al. (2013) classified the esterases from silk gland of b. mori as carboxylesterases by inhibition studies. qualitative (chattopadhyay et al., 2001; moorthy et al., 2008; somasundaram et al., 2009) and quantitative analysis (velu et al., 2008; patnaik et al., 2012) of heat stable esterases has also been carried out by many researchers in b. mori. accepting the calls from patnaik et al. (2012) to characterise the heat stable esterase, in this study, in addition to the identification of heat stable esterase (est-3) we have also found through inhibition studies that it represents cholinesterase activity with serine in its active site. hemolymph is a liquid open circulatory system with tissues like mid gut, fat body and silk gland embedded in it. consequently, several proteins including nonsecretory proteins synthesized in the fat body are carried in the hemolymph to specific subcellular sites. these factors are important in the acquisition of thermotolerance (kampinga, 1993). in this regard, the presence of heat stable esterases in the hemolymph of b. mori can be considered as a desirable feature in conferring thermotolerance to fig. 3 heat stable esterases in haemolymph on 5th day of 5th instar larvae of silkworm breeds. lane 1 and 2 are nils [d6(p)n and sk4c], 3 and 4 are bivoltine parents [d6(p) and sk4] and 5 and 6 are multivoltine parents (nistari and cambodge). the larvae. therefore, hemolymph was selected as suitable tissue for detecting heat stable esterases. this study also delineates the relationship between multivoltine and bivoltine breeds, and their nils with reference to quantitative traits. except survivability, bivoltine breeds had higher values for all quantitative traits than the multivoltine breeds. since, bivoltines are originated from temperate regions and multivoltines from tropical regions, they have different genetic backgrounds. consequently, the bivoltines produce high quality and quantity of silk but sensitive to high temperatures, whereas, multivoltines can tolerate high temperatures but produce low quality and quantity of silk (moorthy et al., 2007a; kumar et al., 2012). but nils, sk4c and d6(p)n showed intermediate values nearer to their bivoltine parents, sk4 and d6(p) (table 2). these nils were developed by backcrossing females of bivoltine parents with f1 males (♀ bivoltine × ♂ multivoltine) for six generations followed by two generations of self-crossing. in each generation, larvae were screened at 33 °c and esterase banding pattern similar to multivoltine parents (moorthy et al., 2007b). therefore the variability in quantitative traits can be explained by genetic differences in the breeds because the quantitative traits discussed are mainly controlled by genetic factors (ahsan et al., 2010; singh et al., 2011). it is clear that the studied traits are quantitative in nature and are controlled by many quantitative trait loci (qtls) present on multiple chromosomes. most probably, during the development of nils most of the qtls for the studied traits are inherited from the recurrent bivoltine parents by their respective nils. thus, the nils performed almost equally to their respective bivoltine parents. exception to this result is survival percentage which is directly associated with thermotolerance trait. in case of survival percentage, nils had higher survival percentages than their bivoltine parents and lower than their multivoltine parents because thermotolerance is also controlled by many quantitative trait loci (qtl) 7 a) b) fig. 4 a) lnhibition of esterases by pmsf in haemolymph on 5th day of 5th instar larvae of silkworm breeds. lane 1 and 2 are nils [d6(p)n and sk4c], 3 and 4 are bivoltine parents [d6(p) and sk4] and 5 and 6 are multivoltine parents (nistari and cambodge). b) lnhibition of esterases by eserine sulphate in haemolymph on 5th day of 5th instar larvae of silkworm breeds. lane 1 and 2 are nils [d6(p)n and sk4c], 3 and 4 are bivoltine parents [d6(p) and sk4] and 5 and 6 are multivoltine parents (nistari and cambodge). present on multiple chromosomes. it is obvious that all the qtls for thermotolerance are not inherited by nils from their multivoltine parents. because the multivoltine parents are involved in a single cross that is in the development of f1 hybrid resulting in partial inheritance of qtls linked to thermotolerance from their multivoltine parents. this partial number of qtls (but not all) were activated and retained in the successive generations by rearing and exposing the larvae to high temperature of 33 °c and selecting the tolerant larvae for successive generations. hence, nils had intermediate values for survival percentages compared to their parents. therefore, esterase polymorphism at distinct genetic loci might also be linked to the changes observed in the quantitative traits between nils and their parents. the heat stable est-3 which is present in the nils and their multivoltine parents might be responsible for their higher survival percentages than the bivoltines at tested high temperatures. nevertheless, the introgression of est-3 in the nils during breeding might be one of the major factors that led to the increase in its thermotolerance capacity than their bivoltine parents. the overall means of survival percentage at all the tested temperatures of d6(p)n and sk4c (nils) were 1.58 and 1.44 times more than d6(p) and sk4 (bivoltine parents), respectively and 0.8 and 0.88 times lower than nistari and cambodge (multivoltine parents), respectively. the integration of est-3 in the nils during breeding coupled with heat selection indicates the retained thermotolerance character from the multivoltine breed in each generation because expression of est-3 increased survivability in nils as well as in multivoltines but expression of other esterases did not increased or decreased the survivability in bivoltine breeds. this selection phenomenon was adopted from wu and hou (1993), they also proved that heat stable esterase activity was increased with thermotolerance by observing the heat stable esterase activity for 7 generations. in drosophila species also, the survival was increased from 35 % to 64 % after selecting 10 generations at 40 °c for 30 min. recently, heat stable est-1 was successfully integrated in the nils of csr2, a productive bivoltine breed. this marks the introgression of multivoltine thermostable factors into the bivoltine silkworm breed ‘csr2’ leading to improved survivability measured in terms of pupation percentage (das et al., 2013). though thermotolerance in b. mori is controlled by multiple genetic factors, this study shows that expression of heat stable esterases is one of the major factors among them. furthermore, complete transcriptome analysis in multivoltines and bivoltines with their nils at high temperature conditions is required to understand the molecular mechanism involved in thermotolerance in b.mori. this study confirms that the nils [d6(p)n and sk4c] have inherited most of the qtls linked to silk quality and quantity from their bivoltine parents [d6(p) and sk4] and that of thermotolerance from their multivoltine parents (nistari and cambodge). similarly, chatterjee et al. (1993) and chatterjee and datta (1992) reported that digestive juice amylase had a positive correlation with survivability and a negative correlation with larval weight, larval duration, single cocoon weight and single cocoon shell weight. alkaline phosphatase and invertase also had positive roles in the expression of yield attributes. however, further investigation is needed to correlate the variations in the quantitative traits with esterase activity. 8 results of this study indicate that esterase can be used as a reliable biochemical marker for diversity studies and breeding programs associated with thermotolerance in b. mori. this work also describes the classification of heat stable hemolymph esterase (est-3) through inhibition studies in b. mori. the information generated from this study would be useful for understanding of molecular and functional mechanisms involved in esterase-mediated thermotolerance in b. mori. acknowledgement we thank the anonymous reviewer, whose 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(in japanese) jpn. seric. sci. 34: 95-101, 1965. yu q, lu c, li w, xiang z, zhang z. annotation and expression of carboxylesterases in the silkworm, bombyx mori. bmc genomics 10: 553, 2009. 10 494 isj 14: 494-504, 2017 issn 1824-307x research report association of α-amylase gene with growth traits in the razor clam sinonovacula constricta c liu, d lin, y dong, q xue, h yao, z lin key laboratory of aquatic germplasm resource of zhejiang, zhejiang wanli university, ningbo, zhejiang, 315100, pr china accepted november 24, 2017 abstract the razor clam sinonovacula constricta is a commercially and ecologically important benthic mollusk. in the present study, we investigated the full-length cdna sequence of the s. constricta α-amylase gene (scamy) using expressed sequence tags and rapid amplification of cdna ends. the genomic dna sequence of scamy is 5086 bp, which contains 6 exons and 5 introns. the full-length scamy cdna was 2196 bp, with a 2085 bp open reading frame encoding 694 amino acids. scamy expression was very low before the d-shaped larvae stage, with the highest expression levels in juvenile clams. expression levels of scamy were significantly higher in the digestive gland compared with other tissues in adults (p < 0.01). analysis of the scamy gene in the digestive gland in starved clams indicated that both gene expression and enzyme activity increased before decreasing, reaching its highest expression on the second day. gene expression and amylase activity both increased gradually after refeeding. these results demonstrated that starvation and refeeding increased amylase activity in razor clams. association analysis identified one shared single nucleotide polymorphism, c1503t, for which individuals with genotypes tt and ct had significantly higher growth traits than those with genotype cc (p < 0.05). this study suggests the potential value of amylase markers in selective breeding to improve clam growth. key words: sinonovacula constricta; α-amylase; cloning and expression; snp; enzyme activity; growth traits introduction α-amylase enzymes comprise a superfamily of structurally related proteins that hydrolyze α-1, 4-glycosidic bonds in starch to produce oligosaccharides and small dextrin molecules (kuriki et al., 1999; yu et al., 2013). its role in controlling carbohydrate metabolism means that α-amylase is regarded as one of the main factors affecting the growth of many aquatic animals (sellos et al., 2003). amylase is one of the most important digestive enzymes for phytophagous animals, especially in several bivalves. the α-amylase gene, amy, has been identified in several bivalves, including pecten maximus (le et al., 1997), crassostrea gigas and pinctada maxima (pan et al., 2013). α-amylase is the main digestive enzyme in shellfish, and thus has an important effect on growth. amylase, lipase and cellulase activities in perna viridis were shown ___________________________________________________________________________ corresponding author: yinghui dong college of biological & environmental sciences zhejiang w anli university 8 south qianhu road, ningbo, zhejiang 315100, p.r. china e-mail: dongyinghui118@126.com to increase with the growth of individuals, but interestingly, activities of amylase, pepsin and lipase decreased with the growth of potamocorbula rubromuscula (huang et al., 2003). the razor clam sinonovacula constricta is a benthic marine bivalve that is naturally distributed along the western pacific coasts of china, japan and korea. it has been a popular seafood and important mariculture species in china for hundreds of years. molecular research into growth-related genes in the clam is thus of great significance in relation to marker-assisted selection. previous studies of this species have cloned and analyzed several genes associated with growth, including the genes for insulin-like peptide (niu et al., 2016), igfbp-like (xie et al., 2013) and β-actin1 (feng et al., 2011). in this study, we investigated the regulation of the s. constricta α-amylase gene (scamy) by cloning the full-length cdna and introns, examining its expression levels during embryonic development and in adult tissues. we also analyzed the gene expression and α-amylase activity in the digestive gland during starvation and refeeding. we additionally screened and analyzed associations 495 between single nucleotide polymorphisms (snp) of the scamy gene and growth-related traits. the results of this study improve our understanding of the function of the scamy gene, and provide a basis for molecular breeding programs of s. constricta. materials and methods experimental animals and sample collection adult clams were collected from danyan farm, yinzhou district of ningbo, china. mantle, adductor muscle, digestive gland, foot, gills, and siphon in adult clams were dissected, frozen immediately in liquid nitrogen and preserved at −80 °c. unfertilized mature eggs, fertilized eggs, 2-cell embryos, 4-cell embryos, morulas, trochophores, d-shaped larvae and juvenile clams were obtained by independent spawning and artificial insemination and stored at −80 °c. a total of 215 individuals were randomly sampled at the same time from two razor clam populations: yl ("yongle no.1", a fast-growing strain, selected for four generations by our team from changle population, fujian province, china; n = 110) and sm (wild population from sanmen county, zhejiang province, china; n = 105). six growth index parameters, including body weight, soft-tissue weight, shell weight, shell width, shell length and shell height, were measured. digestive glands were collected and stored at -80 °c. we explored the possible effects of starvation and refeeding on scamy gene expression levels and enzyme activities using adult clams with an average shell length of 6.0 cm. clams were kept in seawater at a salinity of 20 22 and temperature of 19 21 °c, and acclimated to the experimental conditions for 1 week before use. during this period, the clams were fed nannochloropsis oculata. the clams were randomly allocated to experimental groups. experiments were conducted in three replicates; each replicate included 60 clams. groups were then starved for five days (s1 s5) and then fed with n. oculata for 4 days, until satiety (f1 f4). four randomly selected clams from each replicates were sampled at 10 am every day. cloning of full-length scamy cdna total rna was extracted from the digestive gland using trizol reagent (comwin, beijing, china). rna integrity was examined by electrophoresis on a 1.0 % agarose gel and staining with ethidium bromide, and the quality and quantity of rna were assessed by ultraviolet spectrophotometry. first-strand cdna was synthesized using smart rapid amplification of cdna ends (race) reagents, according to the manufacturer’s instructions (takara, otsu, shiga, japan). we retrieved expressed sequence tag (est) sequences of the scamy gene from the 454 cdna library of s. constricta (genbank accession no. galb00000000) and designed primers for 5′-race (scamy-f1) and 3′-race (scamy-r1) (table 1). table 1 primers and sequences used in this study primer sequence (5′→ 3′) application scamy-f1 attggtatggtgccgaggctggg 3′race scamy-r1 atagttgccctgcccagcctcgg 5′race scamy-f2 ttgctattcgtttgcgggg verifying the sequence of cdna scamy-r2 gacctcccacaataatcgcaagta verifying the sequence of cdna scamy-f3 tggaagcactggatgatg cloning of intron scamy-r3 aacagcccgacacctatt cloning of intron scamy-f4 aataggtgtcgggctgtt cloning of intron scamy-r4 gtccacgaatacaaatgc cloning of intron scamy-f5 gttctatcacgaagtcatc cloning of intron scamy-r5 cctcccacaataatcgcaagt cloning of intron scamy-f6 acttgcgattattgtgggagg cloning of intron scamy-r6 acctcctggtctgttagtagtcc cloning of intron scamy-f7 ccgtgtggactactaacagacc cloning of intron scamy-r7 ccacatctatggtattgggttt cloning of intron scamy-f8 atcaaggcagtggtgaatggc cloning of intron scamy-r8 catccacatctatggtattgggttt cloning of intron scamy-f9 ttgctattcgtttgcggg snp scamy-r9 tcattccccagccaaagt snp scamy-f10 actttggctggggaatga snp scamy-r10 gcattgtctcgcatagggata snp scamy-f11 tatccctatgcgagacaatgc snp scamy-r11 accagccgttgtcagtccga snp real-a-f1 tgttgactttggctggggaa qrt-pcr real-a-r1 gataagcggttgccatcctgta qrt-pcr 18s-f ctttcaaatgtctgccctatcaact qrt-pcr 18s-r tcccgtattgttatttttcgtcact qrt-pcr 496 polymerase chain reaction (pcr) amplification was performed as follows: five cycles of 94 °c for 30 s and 72 °c for 3 min; five cycles of 94 °c for 30 s, 70 °c for 30 s and 72 °c for 3 min; 25 cycles of 94 °c for 30 s and 68 °c for 30 s and a final extension at 72 °c for 3 min. the pcr products were purified using a gel extraction kit (tiangen, beijing, china), cloned into the peasy-t1 vector (transgen, beijing, china), and transformed into trans1-t1 phage-resistant cells (transgen, beijing, china) according to the manufacturer’s protocols. positive clones were selected and sequenced, and the full-length cdna sequence was determined by piecing together the sequences of the 3′ and 5′ race products. to confirm the accuracy of the cloning and sequencing, the full-length cdna was re-amplified with high-fidelity polymerase (takara, otsu, shiga, japan), using a pair of gene-specific primers, scamy-f2 and scamy-r2 (table 1), designed based on the above-mentioned cdna sequence. pcr products were cloned and sequenced following the procedures described above. cloning the introns of scamy genomic dna from adductor muscle tissue was extracted with phenol/chloroform/isoamyl alcohol (25:24:1), and then re-extracted by chloroform/isoamyl alcohol (24:1). the other steps were the same as the procedure described by sambrook et al. (2001). according to the full-length cdna, six primer pairs (f3, r3, f4, r4, f5, r5, f6, r6, f7, r7, f8 and r8) were designed to detect the introns (table1). pcr conditions were as follows: an initial denaturation (94 °c, 5 min), followed by 35 cycles of amplification (94 °c, 30 s; 55 °c, 30 s; 72 °c, 2 min), and a final extension (72 °c, 10 min). pcr products were cloned and sequenced following the procedures described above. sequence analysis of scamy the sequences were spliced using the blast algorithm in the national center for biotechnology information database (http://www.ncbi.nlm.nih.gov/ blast/). the deduced amino acid sequence was analyzed using the simple modular architecture research tool (smart) (http://smart.embl-heidelberg.de) to predict conserved domains. the presence and locations of the signal peptide and cleavage sites in the amino acid sequence were predicted using the signal p 4.0 server (http://www.cbs.dtu.dk/services/signalp/). multiple alignments of α-amylase proteins from s. constricta and other species were performed using the clustalw2 multiple alignment program (http://www.ebi.ac. uk/tools/msa/clustalw2/). a phylogenetic tree was constructed by the neighbor-joining method with mega 6.0. quantitative expression analysis of scamy expression levels of scamy were determined at different developmental stages (unfertilized mature eggs, fertilized eggs 2-cell embryos, 4-cell embryos, morulas, trochophores, d-shaped larvae and juvenile clams; n > 500, three sets of samples for each stage), and in different adult tissues (mantle, adductor muscle, digestive gland, foot, gills and siphon; four sets of samples for each tissue), using real-time quantitative reverse transcription pcr (qrt-pcr), with three technical repeats for each pcr reaction. total rna was extracted from the samples as described above. a 142-bp fragment of scamy was amplified from the cdna template using the primers real-a-f and real-a-r (table 1), and 186-bp products of 18s rrna were amplified as an internal control for qrt-pcr using primers 18s-f and 18s-r (table 1). pcr amplification was performed in a 20-μl volume containing 10 μl itaq universal sybr green supermix (bio-rad, ca, usa), 7.2 μl deionized water, 0.8 μl first-strand cdna and 1 μl forward and reverse primers. amplification was performed using the following thermal cycling conditions: incubation at 94 °c for 20 s, 40 cycles of 94 °c for 3 s, 60 °c for 15 s and 72 °c for 10 s. we compared scamy expression levels between starved and refed clams based on four individuals from each replicates selected at random every day throughout the experiments. total rna was extracted from the digestive glands of 120 clams, and qrt-pcr was performed as described above. α-amylase assay we assayed α-amylase activity in digestive glands extracted from starved and refed clams (twelve samples per day). after homogenization for 10 min in an ice bath at 4 °c, samples were centrifuged for 10 min at 10,000g, and the supernatant was used as the crude enzyme extract. all samples were analyzed within 24 h. α-amylase activity and total protein content were measured using kits from shanghai yuanye (china) and nanjing jiancheng (china), respectively, according to the manufacturers’ instructions. α-amylase activity was measured using the microplate iodine starch method and total protein content was measured using coomassie blue staining. one unit of α-amylase activity was defined as the amount necessary to hydrolyze 10 mg starch per milligram of protein at 37 °c for 30 min. snp identification and association analysis of scamy gene exons rna samples were extracted from 215 clams, as described above. according to the cdna sequence of the scamy gene, three pairs of (f9, r9, f10, r10, f11 and r11) primer sets were selected (table 1). pcr amplification was performed in 50-µl volumes containing 2 µl template cdna, 19 µl dh2o, 25 µl pcr mix and 2 µl of each primer. the pcr conditions were as follows: 3 min at 94 °c; 35 cycles of 30 s at 94 °c, 1 min at 58 °c and 2 min at 72 °c; and a final 5 min extension at 72 °c. each amplification product was verified by electrophoresis on a 1.0 % agarose gel and staining with ethidium bromide. the amplicons representing unique banding patterns were sent to beijing genomics institute (beijing, china) for sequencing in both directions. the sequence was aligned using mega6.0 software. mutation sites were named according to the position of the initiation codon. univariate analysis was performed according to the general linear model procedure in spss 20.0 software. 497 statistical analysis the results of qrt-pcr analysis were based on the ct values of the pcr products and the expression levels of scamy were analyzed using the comparative ct method. statistical analysis was performed using spss 19.0 (chicago, il, usa). differences in relative scamy mrna expression levels among different developmental stages and different adult tissues, and between starved and refed clams were compared by one-way analysis of variance (anova). multiple comparisons were conducted using the student-newman-keuls test. growth traits and α-amylase activities in starved and refed clams were compared as above. differences were considered significant at p < 0.05. body weight, soft weight, shell width, shell length and shell height of the clams were analyzed by one-way anova using spss 20.0. the effects of snps on growth traits in the two populations were analyzed using the above method. snp markers with genotypes that showed a significant correlation with growth traits were studied by post hoc multiple comparison (duncan method). results cdna sequence analysis of scamy the full-length scamy cdna comprised 2,196 bp (genbank accession no. kx197931.1) and contained a 2,085 bp open reading frame encoding 694 amino acids. the cdna contained a 5′ untranslated region of 41 nucleotides, a 3′ untranslated region of 73 nucleotides including a terminator codon (tag), a putative polyadenylation consensus signal (aataa) and a poly(a) tail (fig. 1). the calculated molecular mass of the deduced mature protein was 76.02 kda, and its theoretical isoelectric point was 5.64. sequence analysis suggested that the protein contained a signal peptide but had no transmembrane region and was a hydrophilic protein. smart analysis revealed that the deduced amino acid sequence contained an a domain (27-386) and a c domain (395-473). a phylogenetic tree of α-amylase was constructed using the neighbor-joining method, based on the deduced amino acid sequences of α-amylase from molluscs and some other animals for which the sequences were available in the ncbi database (fig. 2). the obtained tree showed that α-amylases were divided into two major groups: one group comprised mollusc and crustacean α-amylases, and the other contained mammal and fish α-amylases. in the mollusc group, s. constricta α-amylase clustered with α-amylases from cerastoderma edule and corbicula fluminea, and then with others molluscs. multiple alignment indicated that the s. constricta α-amylase shared the highest sequence identity (78.6 %) with c. fluminea α-amylase and 41.4 % 71.6 % identity with other species (fig. 3, table 2). introns analysis of scamy six pairs of primers pcr amplification produced fragments of 432 bp, 408 bp, 534 bp, 594 bp and 922 bp, respectively. after assembly six exons and five introns were obtained. all five introns were located within the orf (fig. 4). the maximum intron fig. 1 full-length cdna sequence and deduced amino acid sequence of the scamy. the three letters boxes are the initiation codon, the terminator codon and polyadenylation signal sequence, the * represents the end of the protein translation, the single underlined is the signal peptide of protein, the bold shaded part is a domain, the double underlined part is c domain and the coarse underlined part is polya. 498 fig. 2 phylogenetic analysis of scamy and other known α-amylases. the neighbor-joining tree was generated with mega6.0. species and the accession number of each species are shown in table 2. table 2 species and genbank accession numbers of amys sequence used for multiple alignment and phylogenetic analysis species genbank no. size (bp) identity (%) sinonovacula constricta kx197931.1 694 crassostrea gigas ekc28393.1 697 64.2 corbicula fluminea aao17927.2 699 78.6 litopenaeus vannamei aij02080.1 719 61.4 macrobrachium rosenbergii akl71614.1 706 65.5 mytilus edulis aca34372.1 660 64.9 haliotis discus discus abo26611.1 694 71.6 colobus angolensis abw02886.1 511 41.2 ctenopharyngodon idella acx35465.1 512 39.3 astacus leptodactylus aiw65942.1 696 65.0 sus scrofa aaf02828.1 511 40.7 bombyx mori act64133.1 500 41.0 salmo salar abd13895.1 505 40.9 drosophila ananassae aac79122.1 494 41.1 homo sapiens aaa52280.1 511 41.4 mus musculus caa24099.1 511 41.0 cerastoderma edule aca34380.1 460 54.4 499 fig. 3 amino acid sequence alignments of amy between s. constricta and other species. identities are shaded dark and similarities are shaded gray. was found to have the length of 922 bp, compared to the minimum length of 408 bp. the other introns ranged from 432 bp to 594 bp. all exon-intron junctions followed the consensus rule of the splice acceptor -ag/gtsplice donor for splicing. quantitative expression of scamy the expression profiles of scamy in embryos/larvae and in adult tissues were analyzed by qrt-pcr. in adult clams, scamy was mainly expressed in the digestive gland and was barely expressed in the other tested tissues (mantle, adductor muscle, foot, gills and siphon) (p < 0.01) (fig. 5). among the eight developmental stages, scamy transcripts were expressed at low levels before the trochophore stage, and increased from the d-shaped larva stage, with the highest expression levels in juvenile clams. there were significant differences in scamy expression levels between juvenile clams and other developmental stages (p < 0.01) (fig. 6). quantitative expression of scamy in starved and refed clams we also analyzed the expression patterns of scamy in starved and refed clams by qrt-pcr. during starvation, scamy expression initially increased up to the second day (s2), but then fell sharply on s3. scamy mrna levels on the last two days of starvation (s4 s5) were lower than the level before starvation. scamy expression then increased after refeeding, and was slightly reduced on the fourth day after refeeding (f4), but remained higher than before starvation (fig. 7). 500 fig. 4 the structures of scamy gene.ⅰ-ⅴ are the five introns of scamy gene, 1-6 are the six exons of scamy gene. they are shown relative to their lengths in the cdna sequences obtained. amylase activity assay amylase-specific activity initially increased and then decreased significantly during starvation of s. constricta. activity increased rapidly on s2 and decreased sharply on s3, and then stabilized during late starvation. amylase activity rose progressively after refeeding, and reached a higher level than before starvation, with a peak at f4 (fig. 8). association between scamy snps and growth traits twenty-one snps were found in the yl population, including seven associated with growth traits. nineteen snps in exons of scamy were identified in the sm population, including three potentially associated with s. constricta growth traits. yl individuals with genotype ac at position c952a grew faster than those with the cc genotype in terms of all of the measured growth traits (p < 0.05). clams with genotype ct at c963t had faster growth in all the growth traits compared with tt individuals (p < 0.05). snp t1371c ct genotype was also associated with significantly higher growth traits than the tt genotype (p < 0.05). in addition, genotype tt at position c1503t had a significant positive effect on all the growth traits compared with genotype cc (p < 0.05) (table 3). t1527c was significantly associated with shell width and shell length in the sm population sm, but not in the yl population (p < 0.05). multiple comparison analyses showed that individuals with c1503t tt genotype grew faster than those with cc genotype in terms of shell width, shell height, body weight and soft weight (p < 0.05) (table 4). comparing the loci in the two populations identified one shared snp at c1503t in the amylase coding region (fig. 9). on further analysis, it was found that the snp c1503t was synonymous. association analysis showed that individuals with the tt genotype at locus 1503 had significantly higher growth traits than those with the cc genotype in both populations (p < 0.05). discussion α-amylases from various species have been crystallized and analyzed by x-ray diffraction. their structure comprises three domains: a tim barrel (domain a); a long loop region inserted between βa3 and αa3 (third β-strand and α-helix in the a domain), known as domain b; and a c domain at the end of the sequence (gerard et al., 2001). the predicted s. constricta amino acid sequence aligned well with these conserved regions, suggesting that its primary structure had features typical of other α-amylases. the deduced amino acid sequence of s. constricta α-amylase shared 41.4 % 71.6 % identity with α-amylases from other animals. it was thus confirmed to belong to the α-amylase family and to have similar biological functions to α-amylases in other species. phylogenetic analysis showed distinct fig. 5 mrna expression levels of scamy gene in different tissues (adductor muscle, siphon, foot, gills, mantle, digestive gland,). **p < 0.01. 501 fig. 6 mrna expression levels of scamy gene in different developmental stages(unfertilized mature eggs, fertilized eggs, 2-cell embryos, 4-cell embryos, morulas, trochophores, d-shaped larvae, juvenile clams). **p < 0.01. fig. 7 analysis of expression difference of scamy gene in starvation and refeeding (s0.before starvation, s1-s5.starvation, f1-f4.refeeding). **p < 0.01. boundaries in terms of α-amylase structures among crustaceans insects, mammals, fish and molluscs, with s. constricta α-amylase grouped in a subcluster with molluscs, forming a branch with c. fluminea, suggesting that s. constricta and c. fluminea are closely related. scamy mrna was mainly expressed in the digestive gland, as demonstrated by qrt-pcr, consistent with its function. similar results have been observed in c. gigas (huvet et al., 2003) and p. fucata (huang et al., 2016), and α-amylase was also expressed only in the digestive gland in saccostrea forskali (thongsaiklaing et al., 2014). scamy mrna levels were higher in the digestive gland than in the mantle, adductor muscle, foot, gills or siphon, indicating that the digestive gland is the main digestive organ in bivalves, given that α-amylase is the main digestive enzyme (le moine et al., 1997). further studies of the α-amylase gene are needed to elucidate the key factors regulating its expression. 502 table 3 effect of seven snps in the scamy gene on growth traits in population yl note: bold parts are the snps associated with growth traits, p < 0.05; underline parts are the snps potentially associated with growth traits, p < 0.01. in this study, scamy mrna appeared at the beginning of embryonic development, and its expression level was increased in d-shaped larvae, by which stage the digestive gland had formed. this is similar to the situation in grass carp, in which amylase gene expression was shown to increase obviously in line with the development of the hepatopancreas and digestive tract (tang et al., 2015). among mollusks, low expression of α-amylase has been observed in early embryos and higher expression during the larval stages in haliotis discus hannai (he et al., 2015) and meretrix meretrix (unpublished data), consistent with its role as the main digestive enzymes in mollusks. amylase gene expression peaked in juvenile clams, suggesting that expression of the scamy gene may have been promoted by increased ingestion in juvenile clams. we analyzed the effects of starvation and refeeding on scamy expression, and showed that expression initially increased and then decreased during starvation, followed by an increase during refeeding. this was in accord with another study that demonstrated decreased amylase mrna levels from day 29 of starvation in dicentrarchus labrax (péres et al., 1998). the results of the current study suggested that starvation stress may enhance amylase activity to boost energy, indicating that the body can improve the metabolic activity of various enzymes to meet the energy requirements imposed by different physiological situations. however, the ability to respond to starvation stress decreased with prolonged starvation, and amylase activity decreased and then stabilized during late starvation. similar phenomena have been found in other aquatic animals, such as litopenaeus vannamei (meng et al., 2006), megalobrama pellegrini (zheng et al., 2015) and ruditapes philippinarum (li et al., 2016). previous studies showed that refeeding significantly improved digestive enzyme activity in aquatic animals, but that the degree of recovery differed among different species. amylase activity increased in rutilus rutilus caspicus after refeeding (abolfathi et al., 2012), while amylase and cellulose activities in r. philippinarum improved to different degrees after refeeding, and reached the levels seen fig. 8 mrna expression levels of scamy gene in starvation and refeeding (n = 4). s0 = before starvation, s1-s5 = starvation, f1-f4 = refeeding. snp geno type n* frequency (%) shell length (mm) shell width (mm) shell height (mm) body weight (g) soft weight (g) 420 aa 12 11.11 51.20±4.26a 12.85±0.90a 17.45±1.61a 7.91±2.12a 5.79±1.87a ga 32 29.63 54.51±3.67b 14.41±1.62b 18.81±1.23b 10.19±2.62b 7.28±2.11b gg 64 59.26 54.10±5.03b 13.87±1.85 18.52±1.71b 9.54±2.84 6.96±2.29 952 cc 16 15.24 51.76±4.22a 13.13±1.15b 17.72±1.48a 8.40±2.17a 6.14±1.84a ac 40 38.10 55.21±4.53b 14.60±1.65a 19.03±1.43b 10.58±2.72b 7.63±2.24b aa 49 46.67 53.67±4.71 13.63±1.80b 18.33±1.66a 9.14±2.77a 6.64±2.22a 963 cc 44 43.56 53.56±4.84 13.58±1.77a 18.31±1.72a 9.14±2.78a 6.62±2.21a ct 40 38.10 55.14±4.58a 14.56±1.72b 19.01±1.44b 10.54±2.79b 7.60±2.28b tt 17 16.19 51.83±4.10b 13.12±1.11a 17.78±1.45a 8.37±2.10a 6.08±1.80a 1287 cc 59 56.73 54.43±4.85a 13.89±1.82 18.61±1.67a 9.57±2.81 6.96±2.27 ct 34 32.69 54.55±3.40a 14.40±1.61a 18.76±1.26a 10.24±2.61a 7.33±2.12 tt 11 10.58 50.86±4.34b 12.81±0.84b 17.52±1.67b 8.03±2.23b 5.96±1.99 1371 cc 60 54.55 54.20±5.11a 13.86±1.84 18.55±1.72a 9.48±2.87 6.90±2.30 ct 34 30.91 54.56±3.60a 14.40±1.61a 18.76±1.26a 10.24±2.61a 7.33±2.12a tt 16 14.55 50.83±3.51b 12.92±1.31b 17.64±1.38b 8.08±2.10b 5.94±1.83b 1503 cc 24 23.53 51.98±3.84a 13.23±1.34a 17.97±1.38a 8.42±2.08a 6.09±1.75b ct 54 52.94 53.30±5.17 14.41±1.97 18.20±1.71 9.97±2.70 6.47±2.10 tt 24 23.53 55.13±4.57b 14.45±1.58b 18.96±1.55b 10.36±2.86b 7.50±2.33a 1737 tt 49 45.37 53.28±4.53b 13.65±1.92a 18.26±1.60b 9.12±2.73b 6.65±2.17b ct 45 41.67 55.13±4.61a 14.44±1.68b 18.97±1.55a 10.46±2.81a 7.58±2.33a cc 14 12.96 51.34±3.68b 13.11±1.20a 17.49±1.35b 8.23±2.09b 6.00±1.80b 503 fig. 9 the sequencing maps of snp for scamy gene at the position of c1503t on the first and second days, respectively, within 3 days (li et al., 2016). amylase activity in macrobrachium nipponense increased slightly and then decreased upon refeeding (li et al., 2007). our results showed a progressive increase in amylase activity to a much higher level than that before starvation, with a peak after refeeding for 4 days. these results indicated that starvation and refeeding could increase amylase activity, thus providing a physiological basis for compensatory growth in razor clams (zhang et al., 2010). further studies are needed to develop the optimal starvation and refeeding model to maximize growth. recent research has focused on correlations between gene polymorphisms and growth of animals. two α-amylase gene snps were highly correlated with growth traits in haliotis diversicolor supertexta (unpublished data), and one site was found in litopenaeus vannamei (glenn et al., 2015), but its relationship with growth was not significant, probably because of the small sample size. in the current study, we identified seven snps potentially associated with growth traits in the yl s. constricta population and three in the sm population, including one snp in the scamy exon that was common to both populations and potentially associated with clam growth. s. constricta individuals with the tt genotype of snp c1503t grew significantly faster than cc individuals in both populations. we therefore hypothesize that clams with the c1503t tt or ct genotype are favorable for breeding. furthermore, this scamy snp could influence growth performance and may be a suitable marker for marker-assisted selection in this species. table 4 effect of three snps in the scamy gene on growth traits in population sm snp geno type n* frequency (%) shell length (mm) shell width (mm) shell height (mm) body weight (g) soft weight (g) 1098 cc 85 82.52 35.97±3.06a 8.23±0.94 12.02±1.09 2.36±0.67 1.84±2.35 ct tt 16 2 15.53 1.94 34.27±2.63b 35.03±3.97 7.91±0.91 7.73±0.35 11.49±1.06 11.86±1.19 2.10±0.57 2.18±0.66 1.65±0.44 1.71±0.51 1503 cc 36 35.29 35.23±3.25 7.91±0.92a 11.69±1.09a 2.16±0.61a 1.68±0.45a ct 56 54.90 35.67±2.89 8.22±0.88 11.95±1.10 2.34±0.67 1.84±0.53 tt 10 9.80 36.99±3.61 8.80±1.08b 12.53±1.17b 2.63±0.68b 2.08±0.55b 1527 cc 95 92.23 35.84±3.04 8.23±0.92a 12.01±1.09a 2.35±0.65 1.83±0.50 ct 6 5.83 33.91±2.71 7.41±0.70b 10.89±0.80b 1.86±0.54 1.48±0.44 tt 2 1.94 33.74±3.82 7.88±1.73 11.52±0.88 2.09±0.92 1.68±0.70 note: bold parts are the snps associated with growth traits, p < 0.05; underline parts are the snps potentially associated with growth traits, p < 0.01. 504 acknowledgments this work was financially supported by zhejiang major program of science and technology (2016c02055-9); modern agro-industry technology research system (cars-49); national infrastructure of fishery germplasm resources programme (2015dka30470); ningbo natural science foundation (2016a610230); innovation project of the graduates and outstanding undergraduates of zhejiang provincial top key discipline (cx2015012). references abolfathi m, hajimoradloo a, ghorbani r, zamani a. effect of starvation and refeeding on digestive enzyme activities in juvenile roach, rutilus rutilus caspicus. j. comp. biochem. physiol. a 161: 166-173, 2012. feng bb, zhong ym, niu dh, chen h, lin gw, li jl. molecular characteristics and expression analysis of β-actin 1 gene from sinonovacula constricta. j. fish. china 35: 650-659, 2011. pujadas g, palau j. evolution of α-amylases: architectural features and key residues in the stabilization of the (β/α)8 scaffold. mol. biol. evol. 18: 38-54, 2001. glenn kl, grapes l, suwanasopee t, harris dl, li y, wilson k, et al. snp analysis of amy2 and ctsl genes 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clam (sinonovacula constricta). j. fish. china 34: 1106-1112, 2010. 10 isj 20: 10-20, 2023 issn 1824-307x research report induction of antioxidant and detoxifying systems of chilo suppressalis walker after exposure to entomopathogenic fungi m shahriari1, a zibaee1*, mf dinan2, a armand1, m tabari3, h hoda3 1department of plant protection, faculty of agricultural sciences, university of guilan, rasht, iran 2department of medical entomology and vector control, school of public health and health sciences research centre, mazandaran university of medical sciences, sari, iran 3rice research institute of iran, mazandaran branch, agricultural research, education and extension organization (areeo), amol, iran this is an open access article published under the cc by license accepted january 10, 2023 abstract the current study aimed to determine antioxidant and detoxifying responses of chilo suppressalis walker (lepidoptera: crambidae) to beauveria bassiana (strains bbrr1, bbal1, bbln1, bbln2), metarhizium anisopliae and hirsutella subulata. the interactions of insect humoral immune responses with the entered conidia of entomopathogenic fungi in addition to nodule formation and melanization caused the production of several reactive oxygenate species (ros), such as hydrogen peroxidase (h2o2), hydroperoxides (rooh), superoxide radicals (o2−), and hydroxyl radical (oh−). the highest activity of catalase was recorded by bbrr1 and bbal1, treatment after 48 to 96 h while the larvae treated by bbrr1 showed the highest peroxidase activity. both ascorbate peroxidase and glucose-6phosphate dehydrogenase showed the highest activity in the larvae treated by bbrr1 after 48-96 h. the highest concentration of malondialdehyde (mda) reported in the larvae treated by bbrr1, bbal1 and bbln1, after 48 hours. the highest activity of general esterases was recorded in the larvae treated by bbrr1 after 48-96 hours. similar results were recorded in the activity of glutathiones-transferase but the enzyme had also the highest activity in the larvae treated by bbal1 and bbln2 after 48 hours. the larvae treated by bbrr1 and bbln1 showed the highest activity of acid phosphatase (acp) after 72 and 96 hours while the highest activity of alkaline phosphatase (alp) was obtained in the larvae only treated by bbrr1 after 48-96 hours. the results clearly revealed that bbrr1 significantly and severely induced antioxidant and detoxifying systems of c. suppressalis larvae implying on virulence and immune induction of bbrr1 against the larvae. key words: entomopathogen; antioxidant; chilo suppressalis; detoxification introduction entomopathogenic fungi are among the most successful microbial agents to combat insect pests that are exist in most aquatic and terrestrial habitats. they cause enzootic and epidemics in insect populations and serve as one of the alternatives to chemical insecticides in agroecosystems because of their adaptations and interactions with environmental factors such as ph, salinity, organic and inorganic materials as well as imposition of ecological and physiological costs on host insects (lacey et al., 2015; zibaee and ramzi, 2018). _________________________________________ corresponding author: arash zibaee department of plant protection faculty of agricultural sciences box 41635-1314, rasht, iran e-mail: arash.zibaee@gmx.com; arash.zibaee@guilan.ac.ir moreover, entomopathogenic fungi are the unique microbial agents that infect their host by penetrating thorough integument rather than bacteria or viruses that require to be ingested. therefore, insect exposure to the fungal conidia in the contaminated environment will cause the onset of infection (zibaee and ramzi, 2018). pathogenicity by entomopathogenic fungi initiates following attachment of conidia to the insect cuticle, passing through by germination, and proliferating after arrival into host hemocoel. eventually, the entered fungal cells acquire yeast-like forms known as hyphal body that sequentially utilize food resources of host body and cause cellular death by producing secondary metabolites (brownbridge et al., 2001). cell death imposed to different tissues of insects following penetration of fungal hyphae and the production of secondary metabolites causes lipid peroxidation and production of reactive oxygen 11 fig. 1 changes in superoxide dismutase, catalase and peroxidase activities of c. suppressalis larvae treatments by different isolates of entomopathogenic fungi. different letters indicate statistical differences at p < 0.05 in each time interval. b. bassiana includes bbrr1, bbal1, bbln1 and bbln2 strains as well as one isolate of m. anisopliae (masa) and h. subulata (hsla) 12 fig. 2 changes in ascorbate peroxidase and glucose-6-phosphate dehydrogenase activities of c. suppressalis larvae treatments by different isolates of entomopathogenic fungi. different letters indicate statistical differences at p < 0.05 in each time interval. b. bassiana includes bbrr1, bbal1, bbln1 and bbln2 strains as well as one isolate of m. anisopliae (masa) and h. subulata (hsla) species (ros) (gulpov et al., 2003; karthi et al., 2018; shamakhi et al., 2020). these molecules are involved in cell signaling and stimulate the genes engaged in host defense. the increased concentrations of ros inflict damages to cell structure under a series of reactions under oxidative stress (dalton et al., 1999; kamata and hirata, 1999; shamakhi et al., 2019, 2020). moreover, the interactions of insect humoral immune responses with the entered conidia of entomopathogenic fungi mainly nodule formation and melanization caused the production of several (ros), such as hydrogen peroxidase (h2o2), hydroperoxides (rooh), superoxide radicals (o2−), and hydroxyl radical (oh−). these molecules act bilaterally to disable the pathogenic infliction of invading organism and to impose some harmful effects on insect tissues including dissociation of cell membrane, apoptosis and dna damage that led to suppress developmental and reproductive disorders (robinson and badwey, 1994; whitten and ratcliffe, 1999; monaghan et al., 2009). therefore, insects have recruited several components of antioxidant systems to regulate ros level within body. these enzymatic and nonenzymatic molecules include catalase (cat), peroxidase (pod), superoxide dismutase (sod), asocorbate peroxidase (apox), glucose-6phosphate dehydrogenase (gpdh), ascorbic acid, thiols, and α-tocopherol (wang et al., 2001; dubovskiy et al., 2008; shamakhi et al., 2020). there are several reports to imply induction of detoxifying system of insects after microbial 13 infections that may trigger insect resistance to chemical insecticides (sokolova and sundukov, 1999; xia et al., 2000, 2001; zibaee et al., 2009). glutathione-s-transferase (gst) and general esterases (ests) are the multifunctional and the important enzymes in detoxification of xenobiotics in insects. gst conjugates the reduced glutathione to the electrophilic center of xenobiotics and it may engage in both detoxification and cellular antioxidant responses (substrate, 2002; li et al., 2007). ests catalyze the esters of higher fatty acids within metabolic process of insects and xenobiotic hydrolyzation (li et al., 2007). nevertheless, these enzymes have been reported to engage in degradation of toxic molecules synthesized during fungal penetration (serebrov et al., 2006; dubovskiy et al., 2008; zibaee et al., 2009). understanding the physiological interaction of entomopathogenic fungi with their hosts is critical to success of agricultural pest control. these interactions can vary depending on the fungal virulence and the host's immune as well as biochemical responses. in the previous study, the cellular and humoral immunity of chilo suppressalis walker (lepidoptera: crambidae) to the different entomopathogenic fungi was investigated by evaluating the changes in hemocyte count and the expression of antimicrobial peptide genes at different time intervals (shahriari et al., 2021a). these studies are of interest because c. suppressalis is an economically important rice pest in north of iran. the larvae severely fed on stems causing “dead heart” and “white head” of rice plants. such deficiencies significantly decrease rice yield of imposed field requiring insecticide treatment. hence the current study was done to determine the effects of four isolates of beauveria bassiana, one isolate of metarhizium anisopliae and hirsutella subulata on the antioxidant and detoxification system of c. suppressalis to elucidate other aspects of fungiinsect interactions. materials and methods insect rearing the field collected adults were transferred to the laboratory and allowed to lay eggs on rice leaves of hashemi variety. the hatched larvae were gently transferred on rice stems and kept in sterile containers at 28 ± 2 °c, 80% relative humidity (rh) and 16h light:8h dark (ld 16:8). fresh stems were provided daily until the fourth instars. two generations were reared on laboratory to have a cohort with the least environmental stresses (zibaee and malagoli, 2014). entomopathogenic fungi culture four isolates of b. bassiana including bbrr1, bbal1, bbln1 and bbln2 in addition to one isolate of m. anisopliae (masa) and h. subulata (hsla) were selected based on the results of our previous studies, and cultured on potato dextrose agar at 25 ± 2 °c. after 14 days, conidia were gently gathered and added onto a 0.01% solution of tween 80 (sigma aldrich, usa) to prepared desirable concentration. larval treatment and sample preparations the fourth instar larvae of c. suppressalis were randomly immersed into 105 conidia/ml concentration of each fungal isolate (each containing 30 specimens in the three replicates of 10) while the control larvae were dipped in tween80 (0.02%) alone. after 24, 48, 72 and 96 h of posttreatment, the total bodies of the larvae were separately homogenized in distilled buffer by a glass pestle in a 1.5 ml of microtubes then centrifuged at 20000 ×g, 4 °c for 20 min. the supernatants were stored at -20 °c and used in the biochemical experiments. antioxidant enzymes catalase (ec 1.11.1.6) briefly, 50 μl of enzyme sample was added into 500 μl of hydrogen peroxide (1%) and incubated at 28 °c for 10 min. then, the absorbance was read at 240 nm (wang et al., 2001). superoxide dismutase (ec 1.15.1.1) the method of mccord and fridovich (1969) was used to measure sod activity. briefly, 50 μl of enzyme sample was added into 500 μl of a solution containing 70 μm of nbt, 125 μm of xanthine, dissolved in pbs (20 mm, ph 7.1). then, 100 μl of xanthine oxidase solution added by 10 mg of bovine serum albumin was mixed with the earlier medium. finally, 100 μl of xanthine oxidase (5.87 units/ml) was added and incubated at darkness, 28 °c for 20 min. at the end of incubation time, the absorbance was read at 560 nm. peroxidase (ec 1.11.1.x) the reaction mixture contained 50 μl of enzyme sample, 250 μl of buffered pyrogallol (0.05 m pyrogallol in 0.1 m phosphate buffer [ph 7.0]) and 250 μl of h2o2 (1%). the absorbance was continuously recorded every 30 s for 2 min at 430 nm (addy and goodman, 1972). ascorbate peroxidase (ec 1.11.1.11) the enzyme assay was done by preparing a reaction mixture containing 50 μl of sample, 150 μl potassium phosphate buffer (67 mm, ph 7.0), 70 μl ascorbic acid (2.5 mm) and 200 μl h2o2 (30 mm). the absorbance was read every 40 s for 5 min at 290 nm (asada, 1992). glucose-6-phosphate dehydrogenase (ec 1.1.1.49) the enzyme assay was done by mixing 100 μl tris-hcl (100 mm, ph 8.2), 50 μl nadp (0.2 mm) and 30 μl mgcl2 (0.1 m). after 1 min, 50 μl water, 50 μl of the sample and 100 μl gpdh (6 mm) was added to the earlier mixture. finally, the absorbance was read at 340 nm after 5 min (balinsky and bernstein, 1963). malondialdehyde (mda) the mda concentration was measured by mixing 100 μl of 20% trichloroacetic acid and 50 μl of supernatant. the mixture was centrifuged at 15,000 g for 10 min at 4 °c. then, 100 μl of 0.8% tba reagent was added into the gained supernatant 14 fig. 3 changes in malondialdehyde content of c. suppressalis larvae treatments by different isolates of entomopathogenic fungi. different letters indicate statistical differences at p < 0.05 in each time interval. b. bassiana includes bbrr1, bbal1, bbln1 and bbln2 strains as well as one isolate of m. anisopliae (masa) and h. subulata (hsla) and incubated at 100 °c for 60 min before to read absorbance at 535 nm. the mda concentration was calculated using a molar extinction (bar-or et al., 2001). detoxifying enzymes general esterase (ec 3.1.1.1.) seventy-five microliters of the two substrates, α-naphthyl acetate and β-naphthyl acetate (10 mm) was separately added into 75 μl fast blue rr salt (1 mm). then, 50 μl of the enzyme solution was added to the reaction mixture and the absorbance was read at 450 nm w at intervals of 10 s for 1 min (han et al., 1995). glutathione-s-transferase (ec 2.5.1.18) twenty microliters of cdnb (20 mm) or dcnb (40 mm) were separately was added into 100 μl of enzyme solution. then the absorbance was read at 340 nm at intervals of 9 s in 1 min (oppenoorth et al., 1979). alkaline(ec 3.1.3.1) and acid (ec 3.1.3.2) phosphatase the enzyme assay was done based on the method of bessey (1954). the buffered substrate, p-nitrophenol phosphate in tris-hcl (20 mm) ph 8 and (ph 5) was separately used to assay alkaline and acid phosphatase, respectively. twenty microliter of the enzyme assay was added into the buffered substrate separately and incubated for 5 min before reading the absorbance at 450 nm. protein assay the protein content of the samples in both control and fungal treated was assayed by the method of lowry et al. (1951) (recommended by ziest chem. co., tehran-iran). statistical analyses the date of antioxidant and detoxification experiments were compared by one-way analysis of variance (anova) using tukey’s test (sas 9.3 2010). the statistical differences were marked by different letters at a probability less than 5%. results and discussion exposure of c. suppressalis larvae to the six native isolates of b. bassiana, m. anisopliae and h. subulata significantly enhanced antioxidant and detoxifying activities in the fourth instar larvae c. suppressalis. the activities of sod, cat and pod in the larvae treated by bbrr1, bbal1 and bbln1 significantly increased at 48 and 72 h of posttreatment while no significant difference was observed between fungal treatments and control larvae after 24 h (pr>f: 52.88; df= 6; p<0.0001, fig. 1). moreover, the activities of sod, cat and pox in the larvae significantly elevated at 96 h posttreatment for all isolates (pr>f: 71.23; df= 6; p<0.0001, fig. 1). entomopathogens and their toxins have been recognized as one of the important exogenous resources that generated free radicals (jia et al., 2016; karthi et al., 2018; 15 fig. 4 changes in general esterase activity of c. suppressalis larvae treatments by different isolates of entomopathogenic fungi. different letters indicate statistical differences at p < 0.05 in each time interval. b. bassiana includes bbrr1, bbal1, bbln1 and bbln2 strains as well as one isolate of m. anisopliae (masa) and h. subulata (hsla) shamakhi et al., 2020). overproduction of ross by biological and chemical toxic compounds, apart from its nature of origin, resulted in oxidative stress that eventualy leads to lipid peroxidation and damage of dna (dubovskiy et al., 2008). several reports have shown involvements of sod, cat, pod, gpdh, apox and nonenzymatic components (e.g., mda and α-tocopherol) in the antioxidant defenses of insects (dubovskiy et al., 2008; rahimi et al., 2018; shahriari et al., 2019; shamakhi et al., 2019, 2020). however, there are few reports regarding the effects of entomopathogenic fungi on antioxidant induction of insect pests. sod has an important role to catalyze o2radicals into h2o2. then, h2o2 changed to h2o and oxygen by the activities of cat and pod (dubovskiy et al., 2008). because the activities of cat and pod significantly increased in the larvae treated by the all six isolates, it seems that metabolites produced from fungal conidia may induce contents of superoxide radicals and hydrogen peroxide which subsequently elevated activation of the antioxidant enzymes. moreover, the enhanced activities of cat and pod may be attributed to the higher activity of sod which generates hydrogen peroxides in the larvae of c. suppressalis. similar results were reported regarding the effects of b. bassiana on c. suppressalis larvae (shamakhi et 16 fig. 5 changes in glutathione s-transferase activity of c. suppressalis larvae treatments by different isolates of entomopathogenic fungi. different letters indicate statistical differences at p < 0.05 in each time interval. b. bassiana includes bbrr1, bbal1, bbln1 and bbln2 strains as well as one isolate of m. anisopliae (masa) and h. subulata (hsla) al., 2020). karthi et al. (2018) demonstrated that infection of spodoptera litura (fabricius) (lepidoptera; noctuidae) by aspergillus flavus conidia caused the higher activities of sod, cat and pod. jia et al. (2016) reported that m. anisopliae caused the higher activities of sod, cat, and pod in locusta migratoria (meyen) (orthoptera: acrididae). the highest activity of antioxidant enzymes in c. suppressalis larvae was observed in the larvae treated by bbrr1, bbal1 and bbln1. in our previous study, bbrr1, bbal1 and bbln1 showed the highest virulence in the shortest time against the larvae of c. suppressalis (shahriari et al., 2021b). moreover, the authors demonstrated the highest activities of the extracellular enzymes in bbal1, bbrr1 and bbln1 therefore the conidia of these isolates pass through the cuticle of larvae in a faster time than other isolates (shahriari et al., 2021b). these differences may be correlated to the presence of different molecules in the cell membrane of conidia and production of toxins by the isolates which impose oxidative stress in the larvae. exposure of bbrr1, bbal1, bbln1 and bbln2 on c. suppressalis larvae increased the activities of apox and gpdh after 48 and 72 h so that these enzymes showed the highest activities compared to control at 96 h post-treatment (pr>f: 65.23; df= 6; p<0.0001, fig. 2). gpdh and apox are the two antioxidant enzymes which engaged in detoxification of pro-oxidant compounds within insects (asada, 1992). apox eliminates h2o2 in 17 fig. 6 changes in acidand alkaline activities of c. suppressalis larvae treatments by different isolates of entomopathogenic fungi. different letters indicate statistical differences at p < 0.05 in each time interval. b. bassiana includes bbrr1, bbal1, bbln1 and bbln2 strains as well as one isolate of m. anisopliae (masa) and h. subulata (hsla) chloroplasts, cytoplasm, and mitochondria, while gpdh involved in deleting oxidant compounds in cytosol via transduction of nadph to nadp+ (asada, 1992). similar to our results, the larvae of galleria mellonella l. (lepidoptera: pyralidae) infected by bacillus thuringiensis showed also the higher activities of apox and gpdh compared to control (dubovskiy et al., 2008). shamakhi et al. (2020) demonstrated significant higher activities of apox and gpdh in c. suppressalis injected with b. bassiana conidia. the higher gpdh activity led to elevation of nadph synthesis to remove products of apx activity. moreover, nadph reduces the toxic effects via transporting electrons to free radicals (barbehenn, 2002; shamakhi et al., 2020). content of mda in the larvae treated by bbrr1, bbal1 and bbln1 were significantly induced after 48 h while other isolates caused the mda elevation in the larvae of c. suppressalis after 72 and 96 h (pr>f: 67.23; df= 6; p<0.0001, fig. 3). mda is an oxidative stress indicator to show enhancement in radical oxidative stresses. the 18 increased content of mda indicated occurrence of oxidative stress following lipid peroxidation (rahimi et al. 2018). on the other hand, ross led to catalysis of polyunsaturated acids, generation of mda and elevation of toxic compounds within cells (wang et al., 2001). results of our study demonstrated that lipid peroxidation was higher in the larvae treated by fungal isolates than control so it can be concluded that conidia proliferation and secretion of secondary metabolites increased lipid peroxidation in hemocoel that leads to cytotoxicity. some research reported a direct correlation between fungal infection and lipid peroxidation in insects. for example, the induction of mda under mycoses showed in c. suppressalis larvae injected by b. bassiana (shamakhi et al., 2020), as well as s. litura larvae infected by a. flavus (karthi et al., 2018). activity of general esterase significantly increased after 72 and 96 h for all isolates except for bbrr1, bbal1 and bbln1, while no significant difference was observed among isolates and control after 24 h (pr>f: 49.73; df= 6; p<0.0001, fig. 4). similar results were obtained for gsts activity in which the exposure of all isolates led to enhanced activity of gsts at 72 and 96 h of larval posttreatment (pr>f: 49.18; df= 6; p<0.0001, fig. 5). in spite of efforts to develop chemical and biological pesticides, insects are also looking for ways to neutralize the effects of these pesticides. ests and gsts are the two important groups of detoxifying enzymes that involve in the breakdown and neutralization of toxic compounds entering insect body (zhu et al., 2011). findings of our study support results of earlier investigations regarding the effect of fungal conidia on detoxifying enzymes of insects (xia et al., 2000, 2001; serebov et al., 2001, 2006; dubovskiy et al., 2008; zibaee et al., 2009; fan et al., 2013; petlamul et al., 2019). in our study, treatment of c. suppressalis larvae by conidia of the entomopathogenic fungi increased activities of ests and gsts especially in the larvae treated by bbrr1, bbal1 and bbln1. this could be a nonspecific response to integument damage. in fact, tissue damage is the most common feature of infection by entomopathogenic fungi causing secretion of detoxifying enzymes which could be the usual response of larvae to infection irrespective of the agent nature (xia et al., 2001; serebov et al., 2006; zibaee et al., 2009). acp activity in the larvae treated by all isolates significantly increased at 96 h of post-treatment while no significant difference was observed among isolates and control after 24 and 48 h (pr>f: 34.12; df= 6; p<0.0001, fig. 6). activity of alp significantly induced after 96 h for all isolates except for bbrr1, bbal1 and bbln1 isolates but no significant difference was observed among isolates and control after 24 h of treatment (pr>f: 47.26 df= 6; p<0.0001, fig. 6). acp and alp are the two hydrolytic enzymes responsible for removing phosphate groups from some molecules like proteins, nucleotides and alkaloids in acidic and alkaline conditions, respectively (ramzi et al., 2014). these enzymes also are able to support, modulate and accelerate phagocytosis (karthi et al., 2018). in the present study, the activities of acp and alp significantly increased in the larvae treated by all isolates. the higher phosphatase activity was found in the larvae exposed to conidia of bbrr1, bbal1 and bbln1. our findings are parallel to the research of karthi et al. (2018), who demonstrated acp and alp activities increased in s. litura after exposure to a. flavus. similarly, b. bassiana and m. anisopliae significantly increased the activities of acp and alp in s. littoralis (mirhaghparast et al., 2013). in contrast, shaurub et al. (2020) reported that the ld50 of b. bassiana did not affect alp activity in the 4th-instar larvae of s. litura. these findings are expected because enzyme activity is dependent on several factors, such as pathogen strain, dose, host insect and etc. conclusions results of our research reported the induction of antioxidant system in the larvae of c. suppressalis exposed to several isolates of entomopathogenic fungi that may be the result of ross production after larval immune responses and damages to tissues caused by entomopathogenic fungi infection. also, larval infection by the isolates increased the activities of detoxifying enzymes. therefore, it may be concluded that the antioxidant and detoxifying systems of c. suppressalis is a part of larval defense mechanisms against fungal infection in addition to humoral or cellular responses which have been previously reported. acknowledgments the research leading to these results received funding from the iran national science foundation 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s. cuticle-degrading proteases of entomopathogenic fungi: from biochemistry to biological performance. arch. phytopathol. plant protect. 51 (13-14): 779794, 2018. zibaee a, malagoli d. immune response of chilo suppressalis walker (lepidoptera: crambidae) larvae to different entomopathogenic fungi. bull. entomol. res. 104(2): 155-163, 2014. 108 isj 18: 108-118, 2021 issn 1824-307x research report activity of detoxification enzymes in rhynchophorus ferrugineus (olivier) (coleoptera: curculionidae) after exposure to beauveria bassiana (balsamo) r ahmed, s freed*, a naeem, m akmal department of entomology, faculty of agricultural sciences and technology, bahauddin zakariya university, multan, punjab, pakistan this is an open access article published under the cc by license accepted august 27, 2021 abstract rhynchophorus ferrugineus is a devastating pest of palms worldwide. an integrated management strategy largely depends on chemical insecticides but due to concerns about human health risks and environmental pollution, it’s essential to emphasize on the integrated pest management (ipm). in the current research the activities of detoxification enzymes esterases (est), alkaline phosphatases (alp), acid phosphatases (acp), glutathione s-transferases (gst), and acetylcholinesterase (ache) in r. ferrugineus collected from punjab, baluchistan, sindh and khyber pakhtunkhwa (kpk) provinces of pakistan were estimated after infection of beauveria bassiana on 3rd-, 5thand 7th-day posttreatment. the insects were exposed by immersion method with different concentrations of b. bassiana. the significant increase in activities of alp (6.09), acp (2.51), ache (21.28) and est (8.61) μmol/min/mg protein was observed in kpk population, while a significant increase in the activity of gst (5.23 μmol/min/mg protein) was recorded in baluchistan population on 7thday. the detection of elevated activities of detoxification enzymes showed the possibility of the resistance development against b. bassiana in r. ferrugineus. key words: date palm; rhynchophorus ferrugineus; entomopathogenic fungi; biocontrol; biochemical; detoxification enzymes; resistance mechanism introduction date palm (phoenix dactylifera) is probably the oldest tree cultivated by humans and its production in pakistan ranks at sixth position (tavakolian et al., 2013; fao, 2014). among notable insects damaging date palm, red palm weevil, rhynchophorus ferrugineus (coleoptera: curculionidae) appears to be one of the cryptic insect (molet et al., 2011; arab and el-deeb, 2012). r. ferrugineus infestation was recorded in 50 % of date producing countries (suma et al., 2014; wakil et al., 2015). the native range of r. ferrugineus is melanesia and south asian countries and dispersal occurs due to transportation of ornamental palms across all continents (el-mergawy and al-ajlan, 2011). r. ferrugineus prefers to attack young palm which are less than the age of 20 years because stem of young palm is juicy, soft, and easily penetrated by the insects. a single female of red palm weevil can give rise to more than approximately _________________________________________ corresponding author: shoaib freed department of entomology faculty of agricultural sciences and technology bahauddin zakariya university multan, punjab, pakistan e-mail: sfareed@bzu.edu.pk half billion of grubs in three generations. moreover, r. ferrugineus is reflected as very disparaging insect of coconut palms (ferry and gomez, 2002). despite huge efforts have been done to protect palm trees via synthetic chemicals, quarantine and other traditional methods (abd-elgawad, 1996), r. ferrugineus has proved to be stronger than these control measures and it has been entitled as the aids (acquired immune deficiency syndrome) of palm tree (hanounik, 1998). the growing demand of farmers to reduce chemical insecticides in agriculture, along with the environmental pollution and increased resistance to insecticides has provided huge impetus for the development of alternative control. an entomopathogenic fungus is alternative to the use of chemical insecticides (sandhu et al., 2012). entomopathogenic fungi can penetrate the host cuticle and can be transmitted by contact with fungal spores or infected insects (klein and lacey, 1999), these are the main insect pathogens infecting beetles, because bacterial and viral diseases are rare among beetles (hajek and st. leger, 1994). the entomopathogenic fungi are usually host specific and are known to cause many physiological and biochemical changes in the host that alter the rate of growth, development and food utilization of 109 table 1 median lethal time of b. bassiana virulence against r. ferrugineus at the highest tested concentration provinces lt50 (days) (95 % fl) slope χ2 df p n punjab 2.849 2.497-3.203 2.20 ± 0.22 6.472 6 0.372 80 sindh 3.166 2.769-3.586 2.02 ± 0.22 1.314 6 0.970 80 baluchistan 3.599 3.162-4.099 1.98 ± 0.22 3.972 6 0.680 80 kpk 3.027 2.753-3.300 3.17 ± 0.25 10.381 6 0.109 80 fl=fiducial limits p-values are based on chi-square goodness of fit test. n=number of larvae used in the treatment including control. the host (butt et al., 2016). b. bassiana is potential fungi against r. ferrugineus (gindin et al., 2006; güerri‐agulló et al., 2010; ricaño et al., 2013). insects routinely deal with many toxic substances that may be chemicals or microbial agents. insects use enzymes including acetylcholinesterase (ache), esterase (est), alkaline phosphatases (alp), acid phosphatases (acp) and glutathione s-transferases (gst) as their defense mechanism to xenobiotic agents (zibaee et al., 2009a). xenobiotic agents are compounds which might penetrate insect body and then enzymes enable insects to escape from these agents. the toxic chemicals are degraded by these enzymes without showing their action (bogwitz et al., 2005). the entomopathogenic fungi infected insects elevate the expression of gst, est and ache. the activation of detoxifying enzyme after fungal infection initiates its rapid degradation, catalyzation, hydroxylation and finally excretion (wang et al., 2004). the activities of est and gst increased post treatment with entomopathogens in dendrolimus tabulaeformis tsai and liu (fan et al., 2013) and elevated est and gst in eurygaster integriceps puton were also detected post-treatment with b. bassiana (zibaee et al., 2009a). the ache activity also increased in nilaparvata lugens (stål) after treatment with fungal metabolites and botanical insecticides (nathan et al., 2008). for the management of r. ferrugineus, previous studies just focused on the use of insecticides. however, ecofriendly b. bassiana can challenge the voracious damage against r. ferrugineus (qayyum et al., 2020), but the role of detoxifying enzymes after its infection remains under explored. thus, the present study was conducted to assess metabolic resistance development after treating r. ferrugineus with b. bassiana, as a baseline to suggest better management tactics. materials and methods insect collection and rearing the adults and larvae of r. ferrugineus were collected from all four provinces of pakistan i.e., baluchistan, punjab, khyber pakhtunkhwa (kpk) and sindh. the insects were later on shifted to sterile cages (60×60×30cm) covered with muslin cloth. saccharum officinarum was used as diet for adult r. ferrugineus that was refreshed after two days. the larvae were reared on artificial diet made by following the method described by ahmed and freed (2021a) which was refreshed after three days. the rearing conditions were maintained at 27 ± 2 °c temperature, 70 ± 5 % relative humidity and 12/12 hours l/d photoperiod. beauveria bassiana the isolate of b. bassiana tested was bb-01 and had been maintained in laboratory culture prior to the beginning of the study. fungal bioassay 3rd instar larvae of r. ferrugineus were subjected to bioassays. for this individual larva was dipped for 10-15s in concentrations of b. bassiana i.e., 3 × 108, 2 × 108, 1 × 108, 1 × 107 and 1 × 106 spores/ml. all concentrations were prepared in 0.1 % tween 80 solution following the methodology of alkhaibari et al. (2017). eighty larvae were treated for each concentration and each concentration was replicated four time. while a total of 480 larvae were treated with different concentrations including a control which was treated with tween 80 solution only. the larvae after treatment were shifted in petri plates (2.5 cm diameter) with an artificial diet. the data on enzymatic activity was recorded on 3rd-, 5thand 7th-days post treatment. the pathogenicity of b. bassiana against kpk, punjab, sindh and baluchistan were statistically non-similar (95 % fls did not overlap) to each other. nevertheless, lowest lc50 (1.3×107 spores/ml) was noted in the kpk samples, while samples from punjab, sindh and baluchistan had lc50 values of 1.5 × 107, 5.3 × 107 and 1.02 × 108 spores/ml, respectively (ahmed and freed, 2021b). sample preparation for determining the enzyme activities the samples (n= 4) were taken from the aforementioned assays to further assess the enzymatic levels in b. bassiana-treated r. ferrugineus on 3rd-, 5thand 7th-days as described by serebrov et al. (2006). third instar larvae were crushed in 80 µl of 110 table 2 mean (± se) enzyme activities in the r. ferrugineus after infection with b. bassiana across different concentration (spores/ml), three post infection times and four different locations in pakistan beauveria bassiana treatment gst ache acp alp est 1×106 1.83 ± 0.08d 3.53 ± 0.39e 0.49 ± 0.02e 2.19 ± 0.09e 2.72 ± 0.21e 1×107 2.15 ± 0.11d 4.76 ± 0.57d 0.94 ± 0.03d 2.65 ± 0.12d 3.27 ± 0.23d 1×10⁸ 2.81 ± 0.14c 5.81 ± 0.68c 1.27 ± 0.04c 3.64 ± 0.11c 4.13 ± 0.27c 2×10⁸ 3.67 ± 0.15b 7.81 ± 1.05b 1.51 ± 0.03b 4.15 ± 0.14b 4.64 ± 0.32b 3×10⁸ 4.17 ± 0.17a 9.51 ± 1.34a 1.92 ± 0.04a 4.76 ± 0.14a 5.37 ± 0.33a control 1.36 ± 0.04e 1.45 ± 0.04f 0.34 ± 0.02f 1.53 ± 0.05f 1.41 ± 0.03f location baluchistan 2.90 ± 0.23a 5.35 ± 0.76b 1.08 ± 0.07b 2.99 ± 0.15bc 3.41 ± 0.27bc kpk 2.64 ± 0.21a 5.83 ± 0.73a 1.25 ± 0.09a 3.67 ± 0.21a 4.16 ± 0.29a punjab 2.21 ± 0.19b 5.22 ± 0.77b 0.95 ± 0.07c 2.74 ± 0.16c 3.23 ± 0.25c sindh 2.90 ± 0.22a 5.51 ± 0.73ab 1.03 ± 0.07bc 3.21 ± 0.17b 3.55 ± 0.26b day 3rd day 2.26 ± 0.10c 2.34 ± 0.10c 0.98 ± 0.07c 2.83 ± 0.14b 2.48 ± 0.13c 5th day 2.62 ± 0.14b 3.05 ± 0.14b 1.07 ± 0.06b 3.21 ± 0.15a 2.97 ± 0.15b 7th day 3.11 ± 0.16a 11.05 ± 0.75a 1.19 ± 0.07a 3.43 ± 0.16a 5.31 ± 0.26a means with similar alphabets within columns, for each tested variable, are not significantly different (tukey’s hsd test, p > 0.05) 0.15 m nacl with a mortar and pestle. the final volumes were adjusted to 900 µl per replication for centrifugation. the samples were spun at 10,000 rpm for 10 min, and supernatants were used to determine enzyme activities. protein determination protein contents in b. bassiana-treated larval samples of r. ferrugineus were measured by following the bradford (1976) procedure. enzyme assays the activity of ache was measured as explained by ellman et al. (1961) using acetylcholine iodide (0.075 m) as a substrate. the samples were incubated in 0.1 mm of edta, 100 mm phosphate buffer (ph 7.2), 10 mm of 5,5′dithiobis (2-nitrobenzoic acid), and 100 mm of acetyl-choline at 30 °c for 30 min. the variation in absorbance was recorded at λ of 412 nm for 4 min at 30 s interval. alp and acp activities were determined by following the method of serebrov et al. (2006) with slight modification. the samples were mixed with 2.3×10-4 m p-nitrophenylphosphate in 0.05 tris-hcl, ph, 8.8 for alp, 0.05 m citrate phosphate buffer, ph, 5.0 for acp and incubated for 2 h at 30 °c. 500 μl (0.05 m naoh) was added for color development. the change in absorbance was noted at 410 nm for 4 min and 30 s intervals. gst activity was measured by using chloro-2, 4dinitrobenzene1 mm with 5 mm reduced glutathione and 0.1 m tris buffer ph 8.0 (caballero et al., 2008). the activity of the enzyme was evaluated by monitoring continuous changes in absorbance at 340 nm for 4 min at 25 °c. the extinction coefficient of cdnb (0.0096) was used to determine the total gst’s activity (rizvi et al., 2018). est activity was recorded by using 1 mm p-nitrophenyl acetate and 50 mm phosphate buffer as substrate (damayanthi and karunaratne (2005). in each replicate, 100 μl of 0.6 m ana (or bna) and 100 μl of phosphate buffer (ph 6.5) were added to 10 μl of r. ferrugineus homogenate. after 30 min incubation, 100 μl solution of fast garnett bc was mixed to stop the reaction. the changes were determined at λ of 405 nm as an end point calculated from standard curves of aand bnaphtol. following rizvi et al. (2018), extinction coefficient of pnpa (176.47) was used to measure est activities. statistical analysis the mortality data of b. bassiana treated r. ferrugineus were examined by polo plus software which yielded lt50 values, 95 % confidence limits 111 table 3 anova results for release activities of detoxification enzyme in the r. ferrugineus after infection with b. bassiana across different concentration (spores/ml), three post infection times and four different locations in pakistan enzyme activity against beauveria bassiana(μmol/min/mg) sources df ache gst acp alp est f p f p f p f p f p treatment (t) 5 614.2 <0.001 119.31 <0.001 394.87 <0.001 162.85 <0.001 206.92 <0.001 location (l) 3 7.58 <0.001 15.85 <0.001 26.24 <0.001 24.69 <0.001 25.14 <0.001 day (d) 2 3390.04 <0.001 36.1 <0.001 23.38 <0.001 19.56 <0.001 466.65 <0.001 t × l 15 0.68 0.7975ns 1.73 0.051 1.25 0.2419 ns 1.92 0.0255 2.11 0.0123 t × d 10 286.22 <0.001 2.23 0.019 0.67 0.7503 ns 0.75 0.6778 ns 20.76 <0.001 l × d 6 4.65 <0.001 1.54 0.1693 ns 1.59 0.1534 ns 1.11 0.3579 ns 1.05 0.3941 ns t × l × d 30 0.73 0.8376 ns 0.46 0.9929 ns 0.2 1.0000 ns 0.3 0.9999 ns 0.33 0.9996 ns error (df) 144 ns labelled values are showing non-significant results (p > 0.05) (fl), chi-square values and slope ± se. statistical analyses were undertaken with the linear model using a factorial analysis of variance (anova) considering location, concentration effects and postinfection time and their interaction as factor against the dependent responses (i.e., enzyme activity). further, concentration effects were compared across districts for each post-infection time. the significant (p < 0.05) means for above analyses were compared using tukey’s honestly significant difference (hsd) multiple comparisons test. graphs were prepared by using graph pad prism, version 6.02. results median lethal time of b. bassiana virulence against r. ferrugineus the infectivity of b. bassiana on r. ferrugineus and its lt50 values were calculated. the lowest lt50 value (2.849 days) was noted in punjab population, while populations of kpk, sindh and baluchistan had values of 3.027, 3.166 and 3.599 days, correspondingly at highest concentration (table 1). enzymatic response in r. ferrugineus post infection with b. bassiana the results indicated the significant effects for concentration, location, and post-infection time towards ache, gst and est activities in b. bassiana treated r. ferrugineus (table 2, 3). the activities of enzymes in b. bassiana treated r. ferrugineus increased in a highly concentrationdependent as well as time-dependent manner, i.e., enzyme activities increased after each concentration and post-infection time increase. ache, gst, est, acp and alp activities were highest for kpk and lowest for punjab populations. an effect for treatment × day was typically significant towards ache, gst and est activities. however, the location × day interaction was typically significant towards ache activity. ache, gst, acp, alp and est post infection responses to b. bassiana in b. bassiana treated r. ferrugineus, the postinfection activities of ache, gst, acp, alp and est increased with increasing post-infection time. the releases were highest for seventh-days postinfection time and typically for the highest exposure concentration (i.e., 3 × 108 spores/ml) (figure 1-5). ache the kpk population of r. ferrugineus treated with b. bassiana showed the maximum ache activities on the seventh-day i.e., 21.28 ± 0.78 μmol/min/mg protein (f = 186.78, df =23, p < 0.001) followed by sindh, baluchistan and punjab populations with maximum ache activities i.e., 20.95 ± 0.45, 20.61 ± 0.23 and 19.95 ± 0.34 μmol/min/mg protein, respectively, at the highest exposure concentration (figure 1). acp the kpk population of r. ferrugineus infected by b. bassiana showed the maximum activity of acp on the 7th-day i.e., 2.51 ± 0.39 (f = 30.19, df =23, p < 0.001) at the highest concentration in 3×108 spores/ml followed 1.98 ± 0.03, 1.85 ± 0.08 and 1.86 ± 0.06 μmol/min/mg protein in baluchistan, punjab and sindh, respectively (figure 2). alp the b. bassiana treatment on r. ferrugineus showed maximum activity of alp on 7th-day in kpk population i.e., 6.09 ± 0.32 μmol/min/mg protein 112 b a l o c h i s t a n k p k p u n j a b s i n d h 0 1 2 3 4 5 3 r d d a y 1 × 1 0 6 1 × 1 0 7 1 × 1 0 8 2 × 1 0 8 c o n tr o l 3 × 1 0 8 a a b a -c a -e b e d e a -c a -d a -e b e d e d e a b a -d a -e c e d e e a -e b e a bc e d e e b a l o c h i s t a n k p k p u n j a b s i n d h 0 2 4 6 8 5 t h d a y a a b a -e a -g b -h g h a f a -h a -h c -h d -h g h a -e b -h c -he -h f -h h a b a -c a -d a -h c -h f -h b a l o c h i s t a n k p k p u n j a b s i n d h 0 5 1 0 1 5 2 0 2 5 a b c d d f g h a b c d c e f g h a b c c d f g h a b c d d g e g h 7 t h d a y p r o v i n c e s a c h e a c t iv it y ( µ m o l/ m in m g p r o t e in ) fig. 1 mean ( ± se) activities of ache in b. bassiana treated r. ferrugineus across three post-infections times for populations from different provinces of pakistan. se denotes standard error. figure panels are showing postanova statistics for concentration effects according to 3rd, 5th and 7th day of treatment by location interaction. bars within each panel labelled with similar letters are not significant from one another (f = 12.78, df =23, p < 0.001) at the highest exposure concentration followed by sindh, punjab and baluchistan populations with maximum ache activities i.e., 5.26 ± 0.32, 5.01 ± 0.51 and 4.51 ± 0.16, respectively (figure 3). est b. bassiana-treated r. ferrugineus showed a significant increase in est activity. the maximum activity of est was recorded in kpk population at the highest exposure concentration i.e., 8.61 ± 0.48 μmol/min/mg protein (f = 40.13, df =23, p < 0.001) on 7th-day followed by 7.94 ± 0.52, 7.28 ± 0.54 and 7.27 ± 0.19 μmol/min/mg protein in baluchistan, sindh and punjab, respectively (figure 4). gst these results showed maximum gst activity in baluchistan population 5.23 ± 0.38 μmol/min/mg protein (f = 11.77, df =5, p = p < 0.001) on 7th-day at the highest exposure concentration followed by sindh, kpk and punjab populations with maximum 113 b a l o c h i s t a n k p k p u n j a b s i n d h 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 a b a -e d g e i h -j ij a a -d e -h e g h -j h -j a -c c gd g g j ij j a -d c g d g f g h -j j 3 r d d a y b a l o c h i s t a n k p k p u n j a b s i n d h 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 5 t h d a y a b a -d d f e -h g j ij a a -d b e e g g j h -j a -c c f e g f i h -j j a -d b ec f e -h h -j j b a l o c h i s t a n k p k p u n j a b s i n d h 0 1 2 3 4 a b b e c g f g h i i a b c b d d g g i h i b d b f e -h f i h i i b d b e b f e -h h i i 7 t h d a y a c p a c t iv it y ( µ m o l/ m in m g p r o t ie n ) p r o v i n c e s fig. 2 mean ( ± se) activities of acp in b. bassiana treated r. ferrugineus across three post-infections times for populations from different provinces of pakistan. se denotes standard error. figure panels are showing postanova statistics for concentration effects according to 3rd, 5th and 7th day of treatment by location interaction. bars within each panel labelled with similar letters are not significant from one another ache activities i.e., 5.17 ± 0.26, 4.87 ± 0.56 and 4.71 ± 0.51 μmol/min/mg protein, respectively (figure 5). discussion the enzymatic system is activated prior to infection by entomopathogens and maintains the regular physiological activities of an insect (jun et al., 2003). in the current study, treatment of larvae of r. ferrugineus with b. bassiana resulted in a significant increase in activities of the enzymes alp, acp, ache, and est in the kpk population only. in the baluchistan population, only the activity of the gst enzyme was increased. the increased activities of detoxifying enzymes in insects against fungal infection may be due to activation of the immune response (moorhouse et al., 1993). the results of our research are consistent with those of bilal et al. (2018) showing amplified gst and est 114 b a l o c h i s t a n k p k p u n j a b s i n d h 0 2 4 6 3 r d d a y 1 × 1 0 6 1 × 1 0 7 1 × 1 0 8 2 × 1 0 8 c o n tr o l 3 × 1 0 8 a a b b f f g d g g a -da -e b g e g f g g a -d a -e b f e ge g g a -c a -d b f c g d g g b a l o c h i s t a n k p k p u n j a b s i n d h 0 2 4 6 8 5 t h d a y a -d b db e e -he -h h a a b a -c b e d -h g h b d b f c g f -h g h h a -c b d b d d -h e -h g h b a l o c h i s t a n k p k p u n j a b s i n d h 0 2 4 6 8 7 t h d a y a -e a -ga f d i e i i a a -c a f b i e i i a -d b g c i f i h i h i a b a -d b -h d i g i h i a l p a c t iv it y ( µ m o l/ m in m g p r o t e in ) p r o v i n c e s fig. 3 mean ( ± se) activities of alp in b. bassiana treated r. ferrugineus across three post-infections times for populations from different provinces of pakistan. se denotes standard error. figure panels are showing postanova statistics for concentration effects according to 3rd, 5th and 7th day of treatment by location interaction. bars within each panel labelled with similar letters are not significant from one another levels in helicoverpa armigera hübner after b. bassiana infections. similar results were reported by serebrov et al. (2006) in galleria mellonella l. in which the activity of gst and est increased post fungal infection. similarly, naeem et al. (2020) reported increased activity of est and gst in diaphorina citri (kuwayama) post fungal infection. the results of our research also relate to farooq et al. (2018) who showed maximum gst activity in musca domestica l. against the combined treatment of b. bassiana and imidacloprid. enzymes enable insects to escape from infection of microbial agents. the toxic chemicals are degraded by the detoxification enzyme prior to show their effectiveness (bogwitz et al., 2005). our results showed that the application of different concentrations of b. bassiana to r. ferrugineus caused a significant increase in est and gst 115 b a l o c h i s t a n k p k p u n j a b s i n d h 0 2 4 6 8 3 r d d a y 1 × 1 0 6 1 × 1 0 7 1 × 1 0 8 2 × 1 0 8 c o n tr o l 3 × 1 0 8 b c b e c e c e d e e b c b e b e c e c e e b d b e b e c e d e e a a b b c b e c e d e b a l o c h i s t a n k p k p u n j a b s i n d h 0 2 4 6 8 5 t h d a y a -d b g b g d g d g g a -c a -d b f c g d g e g a -d a -e b g c g d g g a a b a -d b g c g f g b a l o c h i s t a n k p k p u n j a b s i n d h 0 2 4 6 8 1 0 7 t h d a y a -d b f c g e g g h a b a -d b f e g g h a a -c b e e gf g h a -d a -d b f d g g h p r o v i n c e s e s t a c t iv it y ( µ m o l/ m in m g p r o t e in ) fig. 4 mean ( ± se) activities of est in b. bassiana treated r. ferrugineus across three post-infections times for populations from different provinces of pakistan. se denotes standard error. figure panels are showing postanova statistics for concentration effects according to 3rd, 5th and 7th day of treatment by location interaction. bars within each panel labelled with similar letters are not significant from one another activities. similar results were reported in e. integriceps which showed increased activities of est and gst due to post-treatment with b. bassiana (zibaee et al., 2009a). similarly, enhancement of gst and est activities in locust was observed after fungal infections by dubovskiy et al. (2012). in the current study, ache activity increased after infection by b. bassiana. our results are quite similar to the findings of vidhya et al. (2016) who described elevated activity of ache in spodoptera litura (fabricius) after the treatment of b. bassiana. the results of our study are also consistent with bilal et al. (2018) who reported increased ache activity in h. armigera post fungal infections. contrary to this cao et al. (2016) reported inhibiting activities of ache in locusta migratoria l. after fungal infection. insects use detoxification enzymes to show resistance against xenobiotics (zibaee et al., 2009b). detoxification enzymes e.g., alp and acp 116 b a l o c h i s t a n k p k p u n j a b s i n d h 0 2 4 6 3 r d d a y 1 × 1 0 6 1 × 1 0 7 1 × 1 0 8 2 × 1 0 8 c o n tr o l 3 × 1 0 8 a -d a -e b f d f e f f a -e d f b f f a a b b f c f d f a -d c f a -c b f b f c f d f f f b a l o c h i s t a n k p k p u n j a b s i n d h 0 2 4 6 a -da -e a f e fe f f a f a f b f e f f f a a b a f b f e f f a a -c a -e b f d f f 5 t h d a y b a l o c h i s t a n k p k p u n j a b s i n d h 0 2 4 6 a b a b a -d b e c e d e a b a -e c e c e c e d e a a b a a b a -c b e c e c e c e e a -e d e 7 t h d a y p r o v i n c e s g s t a c t iv it y ( µ m o l/ m in m g p r o t ie n ) fig. 5 mean ( ± se) activities of gst in b. bassiana treated r. ferrugineus across three post-infections times for populations from different provinces of pakistan. se denotes standard error. figure panels are showing postanova statistics for concentration effects according to 3rd, 5th and 7th day of treatment by location interaction. bars within each panel labelled with similar letters are not significant from one another hydrolyze phosphomonoesters under alkaline and acidic conditions. in the current study, application of different concentrations of b. bassiana on r. ferrugineus showed increase in alp and acp activities. similar enhanced expression of alp and acp as a defense mechanism was also reported by bilal et al. (2017) in h. armigera after treatment with b. bassiana. our results are also quite similar to the results of vidhya et al. (2016) who showed an increased activity of acp and alp in b. bassianatreated larvae of s. litura. moreover, similar results were reported in schistocerca gregaria post fungal infections (xia et al., 2000). in conclusion, current study has described that r. ferrugineus infection with b. bassiana sharply increased detoxification enzyme activities mediating 117 detoxification and degradation of b. bassiana. this consequently increased the adaptation ability of insect body, particularly by decreasing their sensitivity to entomopathogenic fungi. this research provided novel options to develop very effective biocontrol agents based on entomopathogenic fungi and their effect on r. ferrugineus due to the activities of enzymes. references abd-elgawad m. the indian red palm weevil: modernization of the methods for the pest management. agric. and develop. in the arab homeland. 15: 36-45, 1996. abe f, hata k, sone k. life history of the red palm weevil, rhynchophorus ferrugineus (coleoptera: dryophtoridae), in southern japan. fla. entomol. 92: 421-425, 2009. ahmed r, freed s. biochemical resistance mechanisms against chlorpyrifos, imidacloprid and lambda-cyhalothrin in rhynchophorus ferrugineus (olivier)(coleoptera: curculionidae). crop prot. 143, 105568, 2021a. ahmed r, freed s. virulence of beauveria bassiana balsamo to red palm weevil, rhynchophorus ferrugineus (olivier) (coleoptera: curculionidae). egypt. j. biol. pest control. 3: 1-4, 2021b. alkhaibari a, carolino a, bull j, samuels r, butt t. differential pathogenicity of metarhizium blastospores and conidia against larvae of three mosquito species. j. med. entomol. 54: 696704, 2017. arab ya, el-deeb hm, the use of endophyte beauveria bassiana for bioprotection of date palm seedlings against red palm weevil and rhizoctonia root-rot disease. sci. j. king faisal univ. 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entomopathogenic fungus metarhizium anisopliae. j. insect physiol. 46: 1249-1257, 2000. zibaee a, bandani ar, tork m. effect of the entomopathogenic fungus, beauveria bassiana, and its secondary metabolite on detoxifying enzyme activities and acetylcholinesterase (ache) of the sunn pest, eurygaster integriceps (heteroptera: scutellaridae). biocontrol sci. technol. 19: 485-498, 2009a. zibaee a, jalali sendi j, ghadamyari m, alinia f, etebari k. diazinon resistance in different selected strains of chilo suppressalis (lepidoptera: crambidae) in northern iran. j. econ. entomol. 102: 1189-1196, 2009b. 75 isj 18: 75-85, 2021 issn 1824-307x research report immunological and oxidative responses of the lesser mulberry pyralid, glyphodes pyloalis by an aqueous extract of artemisia annua l. z afraze1, jj sendi1,2* 1department of plant protection, faculty of agricultural sciences, university of guilan, rasht, iran 2department of silk research, faculty of agricultural sciences, university of guilan, rasht, iran this is an open access article published under the cc by license accepted may 17, 2021 abstract in this search for affordable and locally available biological substances both to farmers and environment, an aqueous extract of artemisia annua l. was investigated for the first time against the lesser mulberry pyralid, glyphodes pyloalis walker a serious pest in mulberry orchards. the lc10, lc30 and lc50 values were estimated 12.82 %, 20.6 % and 27.35 % (w/v) respectively. the extract adversely affected oviposition, impaired immunity through reduced granulocytes and phenoloxidase activity. the increased activity of detoxifying enzymes including esterases and glutathione stransferase (gst) were also observed. the enhanced antioxidant system including peroxidase (pox), catalase (cat), glucose 6-phosphate dehydrogenase (gpdh) and superoxide dismutase (sod) were also observed. the results of the present study may provide a very safe way to control this pest in mulberry orchard and deserve further studies. key words: antioxidant enzymes; aqueous extract; artemisia annua; detoxifying enzymes; immune response; oviposition introduction nowadays, increasing populations coupled with increased food demand has been resulted in a disaster for both human health and the surrounding environment (sparks and lorbasch, 2017; isman, 2020; ali et al., 2021); natural substances extracted from plants can be an alternative remedy to chemical pesticides (isman, 2000; govindarajan et al., 2016; verma et al., 2021). artemisia anuua, also known as sweet wormwood or annual wormwood, is a medicinal plant in many parts of the world and is now of great economic importance due to its biological activity against various pests (khosravi et al., 2011; seixas et al., 2018; liu et al., 2019; oftadeh et al., 2021). this plant has long been known in ancient chinese medicine for its antimalarial activity of artemisinin against plasmodium (meshnick et al., 1996; tu, 2016; salehi et al., 2018; shahrajabian et al., 2020). moreover, it is used to treat fever, summer heat wounds, jaundice, lice, scabies, tuberculosis, hemorrhoids, dysentery, while in iran is used as an _________________________________________ corresponding author: jalal jalali sendi department of plant protection faculty of agricultural sciences university of guilan rasht, iran e-mail: jjalali@guilan.ac.ir antispasmodic, sedative remedy for children (sadiq et al., 2014; septembre-malaterre et al., 2020; feng et al., 2020; trendafilova et al., 2021). the bioactivities of a. annua against pests have attracted the attention of many scientists, especially the research that has been done and focused in our laboratory since 2008 (shekari et al., 2008; hasheminia et al., 2011; zibaee and bandani, 2011). the mulberry pyralid has become a major pest of mulberry plantations in orchards of northern iran where the leaves are harvested for use in sericulture (lalfelpuii et al., 2014). this insect is suspected as an intermediate host of densoviruses and picornavirus to the silkworm (watanabe et al., 1988). the use of chemical insecticides is strictly prohibited because the fresh leaves are provided daily to silkworm. farmers have their plantations adjacent to their homes which this prevents the use of synthetic chemicals (afraze et al., 2020; oftade et al., 2020) and also breed domestic animals on a smaller scale. therefore, they preferably do not use any chemical insecticide. therefore, plant-based products can be considered as a safe and inexpensive option for their health and that of domesticated animals. the immune system in insects includes cellular and humoral immune responses. cellular immunity occurs with changes in hemocyte counts, coagulation, 76 table 1 the lc values, from oral toxicity of a. annua aqueous extract on fourth instar larvae of g. pyloalis aqueous extract time* lc10 lc30 lc50 slope ± se x 2 (df=3) p value artemisia annua 48 12.82 (3.04 -18.98) 20.06 (9.40 27.06) 27.35 (18.13 39.15) 3.895 ± 0.577 5.348 1.761 *48 h after treatments; lc: lethal concentration (% w/v for oral toxicity); x2: chi-square value; df: degrees of freedom micro accumulation, increased phenoloxidase enzyme activity and melanin formation around external factors. while in humeral immunity, antimicrobial peptides produced by fat body cells play an effective role in eliminating toxic (hoffmann, 2003; beckage, 2011; krautz et al., 2014; dubovskiy et al., 2016; baghban et al., 2018; ebrahimi and ajamhassani, 2020). much research has been done on the use of plant pesticides, including extracts and essential oils of a. annua plant against insect immune system (padmaja and rao, 2000; zibaee and bandani, 2010; ali and ibrahim, 2018; ramírez-zamora et al., 2020; oftadeh et al., 2020). in addition, antioxidant enzymes are considered as part of the immune response against causative agents (beutler, 2004; iwanaga and lee, 2005). these enzymes are responsible for controlling ros (reactive oxygen species) produced by biotic and non-biotic stresses in insects (pavlick et al., 2002; kang et al., 2015; nasi et al., 2020). ros contains oxygen ions, free radicals, and organic and inorganic molecules. the activity of these molecules increases under the influence of external factors and causes damage to the structure of cells and tissues in the insects (lyakhovich et al., 2006; wan et al., 2014). the results obtained by many researchers show that the antioxidant system act as a defense mechanism against the production of ros produced by external factors (dhivya et al., 2018; lin et al., 2018; manjula et al., 2020; magierowicz et al., 2020). various studies have shown a reasonable control by a. annua extracts and essential oils. however, the solvent or essential oil extractions are time consuming tasks. this is our first attempt to use the aqueous extract of this precious plant against the mulberry pyralid, which is easily available inside to meet the needs of farmers. in this way, it reduces the costs and environmental side effects of solvents. materials and methods insect rearing different instar larvae of glyphodes pyloalis were collected from infected mulberry orchards rasht (37.1936° n, 49.6410° e.), northern iran. the larvae were feed on fresh mulberry leaves (ichinose var.) at 24 ± 2 °c, 75 ± 5 % of relative humidity, and 16:8 (l:d ) h photoperiod, in plastic boxes. adults were kept in plastic receptacles (18 × 15 × 7 cm). then a piece of cotton soaked in 10 % water and the honey solution was fed to the moths and fresh leaves were placed in containers to oviposition. preparation of aqueous extract the leaves of a. annua were collected from the area around the faculty of agricultural sciences, the university of guilan, rasht 37.1936° n, 49.6410° e. then leaves were boiled with a proportionate amount of distilled water for 1 h. after passing through a strainer, the resulting solution was placed in an oven at 50 °c for 48 h. then the residue was mixed with distilled water and used as a stock and concentrations were made from it. oral toxicity of aqueous extract of a. annua in order to determine the toxicity of the extraction on fourth instar lesser mulberry pyralid larvae, leaf disc immersion method was used (horowitz et al., 2004). for this purpose, 5 concentrations (10, 20, 30, 40 and 50 % w / v) of aqueous extract were evaluated. this experiment was performed with four replications and each replication with ten larvae. the mortality was recorded after 48 h. the lc10, lc30 and lc50 were estimated using polo-plus (2002) software. oviposition deterrence this experiment was performed using a choice bioassay. for this purpose, newly emerged adults were kept in the mating cage for 24 h and used for oviposition bioassay at next day. the oviposition cage consisted of a rectangular plastic container measuring (45 cm × 20 cm × 20 cm). at the top of the cage, there was a window (10 cm × 10 cm) covered with a piece of mesh cloth to release the moths inside. four 30 cm long glass-plastic tunnels are attached to each wall of the cage. the opening of each tunnel was opened into the cage and the end is blocked with a small fan for air circulation. in this method, leaf discs treated with aqueous extract of a. annua and the control treated with distilled water were placed in each tunnel. five female moths were mated and released into the cage. after 24 h, the leaves were collected and the number of eggs laid was counted. this experiment was performed with five replications and each replication with 5 moths. the oviposition deterrent index (odi) was calculated using the following formula (huang and renwick, 1994). odi = cn−tn / cn + tn × 100 where, cn and tn represents number of eggs laid on control and treated leaves, respectively. 77 table 2 ovipositional responses of g. pyloalis females to different concentration of aqueous extracts of a. annua in choice bioassay concentration time* control lc10 lc30 lc50 f p df odi 24 91.4 ± 3.044a 36.4 ± 6.738b 2.4 ± 1.197c 0c 277.40 0.0001 3,19 *24 h after treatments; means (±se) followed by the same letters in a row indicate no significant difference (p < 0.05) according to the tukey test immunological assay total hemocyte count (thc) hemolymph of fourth instar larvae were prepared from the first abdominal pro leg, after 24 and 48 h. for thc a neubauer hemocytometer (hbg, germany) was used, then larval hemolymph (10 μl) was mixed with 290 μl of anti-coagulant solution (0.017 m edta, 0.041 m citric acid, 0.098 m naoh, 0.186 m nacl, ph 4.5) (amaral et al., 2010). differential hemocyte count (dhc) for this purpose, the first abdominal proleg was dissected and 5 μl of hemolymph was placed on to a clean slide and then smeared using another slide. after the smear was dried, the slide was stained using diluted giemsa's stain (1:9) for 12 minutes. they were subsequently differentiated in dilute lithium carbonate solution for red staining structures and then in hcl acidified distilled water for blue staining structures. the slides were washed in distilled water and mounted in canada balsam (merck, germany). then counting 200 hemocytes randomly from 4 corners and a central part in the slides (wu et al., 2016), these cells were identified based on morphological features under a microscope (leica light-microscope) (rosenberger and jones, 1960). phenoloxidase activity assay to measure phenoloxidase activity, the method of catalan et al. (2012) was used with some modification. 10 μl of hemolymph was dissolved in 90 μl of phosphate buffer and l-dopa (3, 4 dihydroxyphenylalanine) (10 mm) was used as a substrate. the samples were centrifuged at 4 °c for 5 minutes at 5000 rpm. 50 μl of the solution was mixed with 150 μl of the substrate. enzyme activity was calculated per mg of hemolymph protein, which was also measured by the lowry method (lowry 1951). the specific activity of the enzyme was read at 490 nm with a micro plate reader. enzymatic assays sample preparation the 4th instar larvae were homogenized in 1 ml of ice-cold 50 mm pbs with 10 % glycerol. sample mixtures were centrifuged at 13000 rpm for 15 min in 4 °c. the collected supernatant was used for the enzymatic assay. gst activity assay glutathione s-transferase activity was measured using the method habig et al. (1974) with two substrates of 1-chloro-2,4-dinitrobenzene (cdnb) and 3,4-dichloronitrobenzene (dcnb). 50 μl of sample, 135 μl of phosphate buffer (ph 7), 50 μl of reagent and 100 μl of reduced glutathione were mixed together. after 5 minutes of incubation at 25 °c, the absorbance was read at 340 nm. general esterase activity assay general esterase activity was measured according to the method of han et al. (1998). the larval midgut was homogenized with 1000 µl of 0.1 mm phosphate buffer and triton x-100 (0.01 %), and centrifuged at 4 °c for 10 minutes. then 10 μl of each substrate of alpha naphthyl acetate and beta naphthyl acetate (10 mm) separately with 5 μl of rr-salt blue salt with 40 μl of phosphate buffer (20 mm) and 5 μl enzyme samples were mixed. after 5 minutes of incubation at 25 °c, the light absorption was read at 450 nm. cat activity assay the method of wang et al. (2001) was used to measure the cat activity. 500 microliters of 1 % hydrogen peroxide were added to 50 μl of sample (treated and control). the reaction mixture was incubated for 10 minutes at 28 °c. the absorbance was read at 240 nm. sod activity assay the method of mccord and fridovich (1969), has been used to measure the sod activity of treated and control larvae. 500 μl of sod solution (70 μm nbt and 125 μm xanthine prepared in phosphate buffer (ph 7). then, it was mixed with 100 μl of xanthine oxidase solution including 100 μl of xanthine oxidase (5.87 units/ml) and 10 mg of bovine serum albumin dissolved in 2 ml of pbs) were added to 50 μl of sample. the reaction mixture was. incubated at 28 °c for 20 min in darkness. the absorbance was read at 560 nm. pox activity assay 50 μl of sample was added to 250 μl of buffered pyrogallol (0.05 m pyrogallol in 0.1 m phosphate buffer (ph 7.0)) (250 μl) and 250 μl of h2o2 (1 %). the absorbance was recorded for every 30 s up to 2 minutes at 430 nm (addy and goodman, 1972). 78 gpdh activity assay the procedure of balinsky and bernstein (1963) was adopted to calculate gpdh amount in treated and control larvae. we used 100 μl of tris-hcl (100 mm, ph 8.2), 50 μl of nadp (0.2 mm) and 30 μl of mgcl2 (0.1 m) were mixed to initiate the reaction. 100 μl of gpdh (6 mm) was added to the mixture and od raise was measured at 340 nm. protein assay protein content was determined by the method of lowry et al. (1951) and using ziest chem's biochemical kit (ziest chem. co., tehran-iran). 100 μl of reagent along with 20 μl of enzyme extracts were incubated at 25 °c for 30 minutes and the adsorption was read at 545 nm using a micro plate reader. statistical analyses determination of mortality and lethal concentrations were done by polo-plus (2002) software. the least significant among treatments were compared using tukey analysis (sas institute, 1997). differences among means were considered to be significant at p ≤ 0.05. all the data in relation to immune system analyzed by t-test. results bioassay of a. annua aqueous extract on g. pyloalis larvae the aqueous extract of a. annua caused mortality in g. pyloalis larvae after 48 h. the lc50 value was 27.35 % w/v. the rate of mortality in treated larvae was dose-dependent. the confidence limits (cl) and the slope of regression are shown in table 1. effect of a. annua aqueous extract on g. pyloalis oviposition the oviposition rate in female moths decreased significantly compared to the control after 1 d (f = 277.40, df t,e = 3, 19, p = 0.0001) table 2. fig. 1 total hemocyte count (thc) following treatment with lc30 with a. annua aqueous extract compared to control (c) in fourth instar larvae of g. pyloalis after 24 and 48 h. statistical differences have been marked asterisks (p ≤ 0.05). according to a t-test effect on thc and dhc the number of total hemocytes in treated larvae of g. pyloalis increased significantly after 24 h compared to the control (t = 7.03, df = 4, p= 0.002) (fig. 1), while no significant reduction was observed after 48 h (t = 2.34, df = 4, p = 0.079). the number of plasmatocyte (t = 2.95, df = 4, p = 0.041) and granulocyte (t = 2.82, df = 4, p = 0.047) followed the thc trend i.e. significantly increased after 24 h (fig. 2). whereas a significant decrease in the number of granulocytes was detected compared to the control (t = 2.89, df = 4, p = 0.044) (figs. 2). the activity of phenoloxidase increased significantly after 24 h (t = 4.90, df = 4, p = 0.008) (fig. 3), and the reduced activity was significant compared to the control at 48 h (t = 3.31, df = 4, p = 0.029) (fig. 3). fig. 2 the effect of lc30 of a. annua aqueous extract on percentages of plasmatocytes and granular cells in g. pyloalis after 24 and 48 h. statistical differences have been marked with asterisks (p ≤ 0.05) according to t-test 79 effect of a. annua aqueous extract on activity of detoxifying enzymes in g. pyloalis larvae general esterase activity in larvae treated with lc50 and lc30 aqueous extracts of a. annua increased after 24 and 48 h for both alpha and betaacetyl naphthalene as substrate table 3. while no significant difference was observed between lc10 and control. the overall results of the effect of a. annua aqueous extract on gst activity increased enzyme activity in treated larvae respectively (f = 20.60, df t,e = 3, 8, p = 0.0004), (f = 20.35, df t,e = 3, 8, p = 0.0004), (f = 24.47, df t,e = 3, 8, p = 0.0002) and (f = 34.99, df t,e = 3, 8, p = 0.0001) table 3. the lowest activity was observed in the controls. effect of a. annua aqueous extract on antioxidant enzymes of g. pyloalis larvae the antioxidant enzymes including po, cat, gpdh and sod in the treated larvae at lc50 of a. annua aqueous extract after 24 and 48 h showed enhanced level of activity compared to control, while the lc30 and lc10 treated larvae given time showed no significant changes compared to control (table 4). discussion many plants with insecticidal properties should be considered worthy as insect control strategies due to the excellent availability, viability and costeffectiveness of plant resources (idoko et al., 2020; isman, 2020). plant extracts, due to their biodegradable nature, are an environmentally friendly approach to pest control (vurro et al., 2019; damalas and koutroubas, 2020). in this study, the aqueous extract of the a . annua leaves caused dose-dependent mortality in g. pyloalis larvae. based on studies conducted by many researchers, it has been determined that the insecticidal activity of a. annua extract can be due to the presence of fig. 3 the effect of lc30 of a. anuua aqueous extract on (po) activity in g. pyloalis after 24 and 48 h. statistical differences have been marked asterisks (p ≤ 0.05) according to a t-test terpenes. these compounds have insect repellent properties and also affect the insect biology as antifeedants, fumigants, ovicides and contact toxicants (khosravi et al., 2010; hasheminia et al., 2011; deb and kumar 2019; oftadeh et al., 2020). to investigate the repellent effects of a . annua aqueous extract, we measured the oviposition deterrent index. based on results, the rate of oviposition in females decreased by increasing the concentration of the extract, this may be due to the presence of repellent compounds in the extract (milano et al., 2010; abdelgaleil et al., 2019; couto et al., 2019; vats et al., 2019). table 3 detoxifying enzyme activities in 4th instar larvae of g. pyloalis after treatment with a. annua aqueous extract detoxifying enzymes time* treatment p f df control lc10 lc30 lc50 glutathione s-transferase (dcnb) 24 48 0.049 ± 0.0052c 0.070 ± 0.0072c 0.093 ± 0.0028b 0.099 ± 0.0058bc 0.101 ± 0.0048b 0.102 ± 0.0057b 0.127 ± 0.0047a 0.151 ± 0.0080a 0.0004 0.0002 20.60 24.47 3,11 glutathione s-transferase (cdnb) 24 48 0.069 ± 0.0075c 0.048 ± 0.0058c 0.070 ± 0.0047bc 0.074 ± 0.001b 0.071 ± 0.0035b 0.086 ± 0.0014b 0.101 ± 0.0052a 0.115 ± 0.0069a 0.0001 0.0004 20.35 34.99 3,11 α-naphtyl acetate 24 48 0.038 ± 0.0069b 0.039 ± 0.0043c 0.039 ± 0.0062b 0.041 ± 0.0036c 0.068 ± 0.0024a 0.070 ± 0.0046b 0.074 ± 0.0056a 0.091 ± 0.0040a 0.0029 0.0001 11.45 34.36 3,11 β-naphtyl acetate 24 48 0.053 ± 0.0075b 0.054 ± 0.0038c 0.054 ± 0.0038b 0.070 ± 0.0029bc 0.073 ± 0.0060ab 0.085 ± 0.0073ab 0.093 ± 0.0057a 0.100 ± 0.0071a 0.0039 0.0022 10.39 12.46 3,11 unit: (u/mg protein); * 24 and 48 h after treatments; means (±se) followed by the same letters in a row indicate no significant difference (p < 0.05) according to the tukey test 80 table 4 antioxidant enzyme activities in forth instar larvae of g. pyloalis after treatment with a. annua aqueous extract antioxidant enzymes time* treatment p f control lc10 lc30 lc50 df pox 24 48 0.001 ± 0.0003b 0.001 ± 0.0001c 0.001 ± 0.0003b 0.002 ± 0.0002bc 0.002 ± 0.0002ab 0.003 ± 0.0004ab 0.003 ± 0.0004a 0.004 ± 0.0005a 0.0087 0.0007 7.98 17.39 3,11 cat 24 48 0.096 ± 0.0061c 0.096 ± 0.0083c 0.113 ± 0.0157bc 0.132 ± 0.0061bc 0.146 ± 0.0087ab 0.167 ± 0.0122b 0.182 ± 0.0109a 0.218 ± 0.0116a 0.0025 0.0002 11.91 27.07 3,11 sod 24 48 0.032 ± 0.0009b 0.033 ± 0.0010b 0.033 ± 0.0022b 0.042 ± 0.0025ab 0.045 ± 0.0035a 0.046 ± 0.0023a 0.047 ± 0.0014a 0.050 ± 0.0040a 0.0022 0.0080 12.4 18.18 3,11 gpdh 24 48 0.060 ± 0.0019b 0.061 ± 0.0022c 0.061 ± 0.0036b 0.061 ± 0.0020c 0.068 ± 0.0036b 0.075 ± 0.0021b 0.083 ± 0.0019a 0.092 ± 0.0022a 0.0022 0.0001 12.44 44.14 3,11 unit: (δod/min/mg protein);* 24and 48 h after treatments; means (±se) followed by the same letters in a row indicate no significant difference (p < 0.05) according to the tukey test the effect a. annua extract on immune system of g. pyloalis larvae has been investigated. innate immune system of insects is a major defense factor against pathogens and other external factors with important effect on insect survival. this system consists of two parts, cellular and humoral, which prevent the spread of infection (mandrioli et al., 2003; malagoli et al., 2007). cellular immunity includes phagocytosis, encapsulation and nodule formation through hemocytes and humoral immunity includes antimicrobial peptides and the prophenoloxidase system (bulet and stöcklin, 2005). given the role of hemocytes in insect cellular immune responses to external factors (lavine and strand, 2002), changes in their number significantly affect the ability of the immune system against invading organisms (bergin et al., 2003). according to our results, the aqueous extract of a. annua increased thc as well as plasmatocytes and granulocytes after 24 h. there are many reports showing an increase in the total number of hemocytes affected by plant insecticides (shaurub et al., 2014; el-sheikh, 2016; shaurub and sabbour, 2017; ghoneim et al., 2018; dhivya et al., 2018). in this study, based on differential hemocytes count, the percentage of granulocytes was significantly reduced compared to the control after 48 h. there are also same results reported by some authors (azambuja et al., 1991; suyog et al., 2012; hassan et al., 2013; er and keskin, 2016; asiri, 2017; er et al., 2017; manjula et al., 2020). sharma et al. (2008) reported that rhizome extract of acorus calamus caused morphological changes in plasmatocytes and granulocytes of spodoptera littura larvae and reduced the differential hemocytes count in larvae. in other study, zibaee and bandani (2010) reported depression in thc, dhc, in eurygaster integriceps fed on a. annua extract. decreased insect hemocytes count can be due to the antimitotic effects of plant extracts (huang et al., 2011). it has been reported that the insect hemocyte count are affected by the mitotic division of the circulating hemocytes (er et al., 2010). the plant extract seems to disrupt hematopoietic organs and inhibits cell division and proliferation in which leads to decreased insect hemocytes. on the other hand, the cytotoxicity effect of some plant extract has also been reported (ghoneim, 2018). this phenomenon may be another factor in hemocyte depression in g. pyloalis larvae that is treated by the extract of a. annua. phenoloxidases are important factors in insect cellular system which are involved in the coagulation process of hemolymph, melanization processes in nodules and capsules, as well as, wound healing (chapman, 2013). the activity of this enzyme increased in larvae treated with a. annua aqueous extract after 24 h. however, 48 h later, phenoloxidase activity was significantly reduced compared to the control. some authors have reported reduced phenoloxidase activity in insects treated by plant extracts (liu et al., 2009; zibaee and bandani, 2010; zibaee and bandani, 2012; shayegan et al., 2019). the initial increase in the enzyme activity may be due to the insect immune response to the introduction of plant extract. it seems that decreased phenoloxidase activity after 48 h of larval treatment is due to the cytotoxicity effect of the plant extract which disrupts hemocytes and is responsible for enzyme production. esterases and glutathione s-transferase are two components that play important role in the detoxification process in insect. due to their high substrate activity, these enzymes reduce the toxic effects of the compounds entering the insect body and eventually lead to resistance against pesticides (alias, 2016). general esterases are type of hydrolase enzymes that breaks down many compounds, including aliphatic and aromatic esters, 81 choline esters and organophosphate (ramsey et al., 2010; chapman, 2013). in this research, the activity of these enzymes increased significantly after 48 h at concentrations of lc30 and lc50 of a. annua aqueous extract which seems to be due to the excretion of compounds in the extract. other studies have reported an increase in enzymes by some plant extracts (senthil-nathan, 2013; yazdani et al., 2013; chen et al., 2019; murfadunnisa et al., 2019; wang et al., 2020). glutathione s-transferase is an important enzyme in detoxification process and insect antioxidant systems. this enzyme is responsible for removing the products from lipid peroxidation and hydroperoxidase from cells (park and tak, 2016). another part of the detoxification process is done by antioxidant enzymes. enzymes such as sod, pox, and cat, in combination with other non-enzymatic antioxidants, are involved in the removal of toxic free radicals produced in response to exposure to toxic substances. (nappi et al., 2005; krych et al., 2014). the results show that the activity of cat, sod, gpdh and pox increased in g. pyloalis larvae treated by aqueous extract of a. annua. this increase was dose-dependent and may be due to the production of ros in the insect body, which is produced under the influence of plant extracts in the body of herbivorous insects. the resultant oxidative stress activates the antioxidant system in the body of the insect (chapman, 2013). many studies have shown that the activity of these enzymes is increased by plant compounds (xiong et al., 2016; pandey and singh 2017; lin et al., 2018; rahimi et al., 2018; dhivya et al., 2018; magierowicz et al., 2020). this study was conducted to control the disastrous mulberry pyralid in accordance with the provision of a safe, secure and cost-effective method, especially for ordinary farmers. in this study, we showed that an aqueous extract of a. annua leaves not only causes moderate mortality in g. pyloalis larvae but 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ocean university, dalian 116023, china 2functional laboratory of marine fisheries science and food production processes, qingdao national laboratory for marine science and technology, qingdao 266235, china 3liaoning key laboratory of marine animal immunology and disease control, dalian ocean university, dalian 116023, china 4dalian key laboratory of aquatic animal disease prevention and control, dalian ocean university, dalian 116023, china this is an open access article published under the cc by license accepted april 8, 2021 abstract microalgae such as dinoflagellate and diatom are the major food source of bivalve species, and sufficient food intake contributes to the immunity and the growth of bivalves. in the present study, a monoamine oxidase gene (named as cgmao), which is the rate-limiting enzyme of norepinephrine (ne) biosynthesis, was cloned from c. gigas. after the oysters were fed with a diet rich in diatom for 21 and 40 d, the ne contents in oyster serum, as well as the mrna expression of cgmao in oyster haemocytes, increased significantly compared with control group. besides, the mrna expression of cytokines cgtnf-1 and cgil17-5 in haemocytes and the activities of immune-related enzymes (sod and lyz) in oyster serum also increased significantly after diatom feeding. these results collectively suggested that sufficient microalgae intake might significantly enhance the antibacterial capacity in oyster by prompting the biosynthesis of ne and triggering the subsequent antibacterial processes modulated by ne. key words: crassostrea gigas; antibacterial capacity; microalgae; monoamine oxidase; norepinephrine introduction most of the bivalve molluscs live in a microbe-rich environment and are under a persistent threat of infection by resident pathogenic microbes. due to their filter-feeding habit, they concentrate a rich and diverse bacterial commensal microbiota, composed of various species belonging to different genera like vibrio, pseudomonas, acinetobacter, photobacterium, moraxella, aeromonas, micrococcus and bacillus (kueh and chan, 1985). most bacterial diseases of bivalves are caused by a large range of vibrio species (v. alginolyticus, v. splendidus, and v. anguillarum), pseudomonas, and aeromonas (garnier et al., 2008; guo and ford, 2016; zannella et al., 2017). for example, brown ring disease (brd) induced by vibrio tapetis affected both juveniles and adults, killing up to 100 % of clams if the bacterium was able to penetrate soft _________________________________________ corresponding author: zhaoqun liu dalian ocean university dalian 116023, china e-mail: liuzhaoqun@dlou.edu.cn tissues (allam et al., 2002). in recent years, the excessive host farming densities, as well as a lack of bait algae in seawater, might have contributed to bacterial diseases in marine bivalves. a deep investigation on the interactions between hosts and bacterial pathogens will contribute to the sustainability of bivalve farming industry. the resistance of bivalve molluscs to bacterial challenge can be seen as a sort of stress response which is a set of coordinated physiological reactions enhancing host’s capability for homeostasis maintenance (ottaviani and franceschi, 1996). hormones and neurotransmitters are crucial for the regulation of responses to external and internal stress (chrousos, 2009). among the neurotransmitters responsive to stressors, catecholamines (cas), consisting of dopamine (da), norepinephrine (ne) and epinephrine (e), play important roles in stimulating responses to a perceived threat (ottaviani and franceschi, 1996; ottaviani, 2011). for example, ne concentration in the haemolymph of scallop chlamys farreri increased significantly after bacteria challenge, and high concentration of ne repressed the activities of immune-related enzymes in haemolymph (zhou et al., 2011b; zhou et al., 2012). moreover, ne is able 57 to regulate both cellular and humoral immunomodulation in oyster c. gigas through a “nervous-haemocyte” neuroendocrine immunomodulatory axis-like pathway (liu et al., 2017). monoamine oxidase (mao) is the key rate-limiting enzyme of ne metabolism, which catalyzes the oxidative deamination of biogenic and vasoactive amines to their corresponding aldehydes (tipton et al., 2004). in humans, there are two types of mao: maoa and maob. maoa and maob are widely distributed in tissues such as neurons, liver, pulmonary vascular endothelium, gastrointestinal tract and blood platelets (shih and chen, 2004). the deamination catalyzed by mao is always associated with the immunomodulation of monoamines and the production of intracellular h2o2, which influences the immune competence of immunocytes ( shih et al., 1999; ou et al., 2006; shih, 2018). recently, homologues of mao have been characterized from bivalves. a mao gene (cfmao) was cloned from scallop c. farreri, which was widely expressed in tissues including haemocytes, hepatopancreas and adductor muscle. the mrna expression of cfmao in scallop haemocytes was activated upon v. anguillarum challenge to modulate the immune response of scallops through the deamination of monoamines such as ne, da and serotonin (zhou et al., 2011a). a deeper investigation is expected to reveal the antibacterial mechanism mediated by mao and the monoamine neurotransmitters. marine bivalves filter water-suspended particles, such as microalgae, bacteria, organic debris and micro-zooplankton, and the microalgae provide dominant and highly variable nutritional value for the growth and immunity in bivalves (yang et al., 2017). cell size, shape and biochemical composition of microalgae determine their nutritive quality and utility as food (yaakob et al., 2014; beal et al., 2018). many studies have shown that the quality of the algal diet affects the growth and development of molluscs such as the great scallop pecten maximus, c. gigas, o. edulis and r. philippinarum (sanina et al., 2004). specifically, diatoms and haptophytes (prymnesiophytes) are nutritious microalgae that are frequently used as feed for oysters (coutteau, 1992). the prymnesiophytes isochrysis sp. and p. lutheri are rich sources of docosahexaenoic acid (dha, 22:6n-3) comprising 8-10% total fatty acids (volkman, 1989), while diatoms are a rich source of eicosapentaenoic acid (epa, 20:5n-3) (dunstan, 1994). mixed microalgal diets of prymnesiophytes and diatoms are common in bivalve hatcheries, and considered as highly nutritious in terms of requirements for essential n-3 polyunsaturated fatty acids (pufas) (knuckey, 2002). when the energetic requirements of oysters are not satisfied by microalgae intake, the animals lose weight as they use energy reserves. for instance, poorly fed c. gigas use protein for metabolism, while other species such as o. edulis use lipids. consumption of tissue reserves eventually result in physiological stress, leaving oysters more susceptible to other stressors, that is to say susceptible to infectious agents (camargo-cely and collin, 2019). and, delaporte et al. reported that arachidonic acid (ara, 20:4n-6) supplementation derived from sufficient microalgae uptake led to an increase in hemocyte numbers, phagocytosis, and production of reactive oxygen species by hemocytes of oyster c. gigas (lawrence, 2006). therefore, the algal diet is closely correlated to the physiological condition of oysters, and also to their resistance to bacterial infections. the pacific oyster c. gigas is one of the most important maricultural bivalves and contributes weightily to the aquaculture industry in china. microalgae, especially dinoflagellates and diatoms are their major food source. however, owing to the intensive mariculture of bivalve molluscs in recent years, the structure of microalgae community in nearshore area has changed dramatically, which in turn severely restricts the shellfish farming industry. in the present study, diets with different proportions of dinoflagellates and diatoms were fed to adult c. gigas, and the antibacterial activities mediated by mao were explored to (1) illustrate the molecular features of oyster mao gene; (2) understand the correlations between algal intake and antibacterial capacity in oyster; and (3) investigate if a diet rich in diatoms contributes more to the antibacterial activity than a diet rich in dinoflagellates. materials and methods oysters, microalgae feeding and sample collection oysters crassostrea gigas (about 3 years old, averaging 150 mm in shell length) were collected from a local oyster farm in dalian, liaoning province, china, and maintained in the aerated seawater at 20 °c for two weeks before processing (zheng et al., 2020). oysters were fed with microalgae powder (prorocentrum micans and nitzschia closterium f. minutissima, commercially purchased respectively from the shanghai guangyu biotechnology co., ltd and the ocean university of china), and the seawater was replaced every day. the current experiment was performed in spring from march to may, which was not the breeding season for c. gigas. the gonad of the oysters in the present study was rarely matured during the feeding procedure. thus, the current results were believed to be convincing. oysters in the dinoflagellate dominant group were fed with a mixed diet of 80 % prorocentrum micans and 20 % nitzschia closterium f. minutissima, while oysters in the diatom dominant group were fed with a mixed diet of 80 % nitzschia closterium f. minutissima and 20 % p. micans. all the oysters were fed once a day with a total amount of 10-12 × 1010 cells algae. oysters without any feeding treatment were employed as the control group. the abundance of microalgae in seawater was measured every day, and each measurement was repeated three times. nine oysters from each group were sampled randomly at 0, 21 and 40 d. tissues including hepatopancreas, muscle, labial palp, gonad, gill and mantle were collected for tissue distribution assay, and the haemolymph was centrifuged at 800 xg, 4 °c for 15 min to harvest haemocytes and serum. samples for quantitative real-time pcr analysis was added with 1 ml trizol reagent (invitrogen) for subsequent rna extraction and stored immediately at -80 °c. table 1 sequences of the primers used in the experiment https://en.wikipedia.org/wiki/monoamine_oxidase_a https://en.wikipedia.org/wiki/maob http://xueshu.baidu.com/s?wd=paperuri%3a%28ac54eaeebee229d2524a14e0485a8a23%29&filter=sc_long_sign&sc_ks_para=q%3d%e5%af%b9%e5%b0%8f%e6%96%b0%e6%9c%88%e8%8f%b1%e5%bd%a2%e8%97%bb%28nitzschia%20closterium%20f.minutissima%29%e5%88%86%e7%b1%bb%e5%9c%b0%e4%bd%8d%e7%9a%84%e9%87%8d%e6%96%b0%e8%ae%a4%e8%af%86&sc_us=18162504800843460590&tn=se_baiduxueshu_c1gjeupa&ie=utf-8 http://xueshu.baidu.com/s?wd=paperuri%3a%28ac54eaeebee229d2524a14e0485a8a23%29&filter=sc_long_sign&sc_ks_para=q%3d%e5%af%b9%e5%b0%8f%e6%96%b0%e6%9c%88%e8%8f%b1%e5%bd%a2%e8%97%bb%28nitzschia%20closterium%20f.minutissima%29%e5%88%86%e7%b1%bb%e5%9c%b0%e4%bd%8d%e7%9a%84%e9%87%8d%e6%96%b0%e8%ae%a4%e8%af%86&sc_us=18162504800843460590&tn=se_baiduxueshu_c1gjeupa&ie=utf-8 http://xueshu.baidu.com/s?wd=paperuri%3a%28ac54eaeebee229d2524a14e0485a8a23%29&filter=sc_long_sign&sc_ks_para=q%3d%e5%af%b9%e5%b0%8f%e6%96%b0%e6%9c%88%e8%8f%b1%e5%bd%a2%e8%97%bb%28nitzschia%20closterium%20f.minutissima%29%e5%88%86%e7%b1%bb%e5%9c%b0%e4%bd%8d%e7%9a%84%e9%87%8d%e6%96%b0%e8%ae%a4%e8%af%86&sc_us=18162504800843460590&tn=se_baiduxueshu_c1gjeupa&ie=utf-8 javascript:; http://xueshu.baidu.com/s?wd=paperuri%3a%28ac54eaeebee229d2524a14e0485a8a23%29&filter=sc_long_sign&sc_ks_para=q%3d%e5%af%b9%e5%b0%8f%e6%96%b0%e6%9c%88%e8%8f%b1%e5%bd%a2%e8%97%bb%28nitzschia%20closterium%20f.minutissima%29%e5%88%86%e7%b1%bb%e5%9c%b0%e4%bd%8d%e7%9a%84%e9%87%8d%e6%96%b0%e8%ae%a4%e8%af%86&sc_us=18162504800843460590&tn=se_baiduxueshu_c1gjeupa&ie=utf-8 http://xueshu.baidu.com/s?wd=paperuri%3a%28ac54eaeebee229d2524a14e0485a8a23%29&filter=sc_long_sign&sc_ks_para=q%3d%e5%af%b9%e5%b0%8f%e6%96%b0%e6%9c%88%e8%8f%b1%e5%bd%a2%e8%97%bb%28nitzschia%20closterium%20f.minutissima%29%e5%88%86%e7%b1%bb%e5%9c%b0%e4%bd%8d%e7%9a%84%e9%87%8d%e6%96%b0%e8%ae%a4%e8%af%86&sc_us=18162504800843460590&tn=se_baiduxueshu_c1gjeupa&ie=utf-8 58 primer sequence (5’-3’) sequence information p1 (forward) agacaactgatggagtgacggtg real-time cgmao primer p2 (reverse) tccaaaaaggggtcttgtagtagc real-time cgmao primer p3 (forward) ggtaataacgaaaggaaacgaag real-time cgdbh primer p4 (reverse) caccgataacttcccgacac real-time cgdbh primer p5 (ef-rtf) agtcaccaaggctgcacagaaag real-time cgef primer p6 (ef-rtr) tccgacgtatttctttgcgatgt real-time cgef primer p7 (forward) cttctcgtctgcggcttcttt real-time cgtnf-1 primer p8 (reverse) cagggctgcggtctttcc real-time cgtnf-1 primer p9 (forward) cgtccttgccttactgactaga real-time cgil17-5primer p10 (reverse) tgtcgttgtcctctaccatgat real-time cgil17-5 primer p11 (forward) aacacatgactatggaggaagatcagct cgmao specific primer p12 (reverse) gtttccaccaataaagacacagtccc cgmao specific primer rna isolation, gene cloning, sequence analysis and quantitative real-time pcr analysis total rna was isolated from the oyster haemocytes using trizol reagent according to the standard protocol. the synthesis of the first-strand cdna was carried out with promega m-mlv rt with oligo (dt)-adaptor priming according to the manufactory's protocol. the full-length cdna sequence of cgmao (cgi_10022845) (boutet et al., 2004) was cloned from the cdna library using specific primers (table 1). the sequence analysis was performed as the description of zhou (zhou et al., 2011a). the quantitative real-time pcr was carried out on an abi prism7500 sequence detection system. cgdbh, cgmao and the immune-related genes including cgtnf-1 (cgi_10018786) and cgil17-5 (cgi_10004922) were amplified using the corresponding primers, and a fragment (168 bp) of oyster elongation factor (cgef, cgi_10012474) was employed as endogenous control (table 1). dissociation curve analysis of amplification products was performed to confirm that only one pcr product was amplified and detected. all data were given in terms of relative mrna expression using the 2−δδct method (zhang et al., 2008). quantification of ne in oyster serum the concentration changes of ne in oyster haemolymph after microalgae fed were determined by norepinephrine elisa kit (abnova, ka1891) (jiang et al., 2014). briefly, ne was extracted from samples using a cis-diol-specific affinity gel, acylated and then derivatized enzymatically. after the samples were equilibrated, free ne and free ne-antibody complexes were removed by three rounds of washing with wash buffer. the antibody bound to the solid phase was detected by using an anti-rabbit igg-peroxidase conjugated with tmb as substrate. the product of the enzyme-substrate reaction forms a blue-colored complex. finally, a stop solution was added to stop the reaction, which would then turn the solution yellow. the reaction was monitored by a microtiter plate reader (biotek, usa) at 450 nm. quantification of samples was achieved by comparing their absorbance with a reference curve (n = 6). measurement of key immune-related enzymes activity the activities of three immune-related enzymes including superoxide dismutase (sod), catalase (cat) and lysozyme (lyz) in the oyster serum were measured by the kits (nanjing, jiancheng, a001-1, a007 and a050-1) according to the protocol. total sod activity was determined by the hydroxylamine method. one sod activity unit was defined as the enzyme amount causing 50% inhibition in 1 ml reaction solution. total cat activity was determined by using the spectrophotometry to measure yellowish complex compound yielded from the reaction between hydrogen peroxide and ammonium molybdate (kono, 1978). as for the detection of lyz activity, 20 μl serum was added to 200 μl bacteria suspension (0.25 mg ml-1 in bacterial buffer supplied from the kit). the mixture was incubated at 37 °c for 15 min, and then bathed in ice for 3 min. the reduction of absorbance at 530 nm was measured at room temperature using a microtiter plate reader (biotek, usa). the total lyz activity was measured by comparing the turbidimetry of the samples with that of lyz standard solution (2.5 mg ml-1, offered by the kit) (jiang et al., 2013). antibacterial assay of oyster serum in the antibacterial assay, 50 µl of serum was mixed with 10 µl of alive v. splendidus, and then were pipetted into each well of a flat bottom 96-well plate, incubating at 18 °c for 3 h with gentle agitation. then, 200 µl of 2216e medium was added into the plate, and the plate was incubated at 28 °c. the od value of the mixture was continually read using a microtiter plate reader for 24 h at intervals of 30 min. the od value measured at 600 nm was used to determine the antibacterial ability of serum. the differences were acquired when the absorbance 59 among each group reached the maximum (zhou et al., 2013). statistical analysis all the data were given as means ± s.d, and analyzed by statistical package for social sciences (spss) 20. significant differences between treatments for each assay were tested by one-way analysis of variance (anova), and considered significant at p < 0.05 (a, b, c, etc.). results molecular characteristics of cgmao the nucleotide sequence of cgmao contained an orf of 1566 bp, encoding a polypeptide of 521 amino acids with the molecular mass predicted as 58.83 kda. the theoretical isoelectric point of cgmao was 6.13. two fad building domains, one nad building domain and one amino oxidase domain (from leu18 to leu456) were identified from cgmao by smart program analysis. multiple sequence alignment and phylogenetic analysis the sequence similarity of cgmao with other maos was analyzed by blast algorithm (fig. 1a). it shared 68.8 % similarity with that from danio rerio, 78.3 % similarity with that from mizuhopecten yessoensis, 19 % similarity with that from xenopus tropicalis, 17.7 % similarity with that from oryzias latipes, 54.5 66 % with maoas and 66.3 67.2 % with maobs from vertebrates (table 2). specifically, cgmao exhibited 66.0 % sequence similarity with maoa from h. sapiens, and 66.9 % sequence similarity with maob from h. sapiens. thus, it was very hard to tell whether cgmao belonged to maoa subtype or maob subtype, maybe because the mao gene was the ancestor gene and not fully differentiated. an unrooted phylogenetic tree was constructed using neighbor-joining method with 1000 bootstrap test based on the multiple sequence alignment of cgmao and maos from other vertebrate and invertebrate species. cgmao was clustered into the invertebrate group with the closest match of scallop m. yessoensis. in the vertebrate group, maoas were clustered together and formed a sister branch to the branch of maobs (fig. 1b). tissue distribution of cgmao mrna transcripts the quantitative real-time pcr was performed to investigate the distribution of cgmao transcripts in different tissues with cgef as the internal control. the mrna transcripts of cgmao were detected in all the tested tissues including haemocytes, gonad, mantle, labial palp, gill, muscle and hepatopancreas (fig. 1c). it was relatively abundant in hepatopancreas and haemocytes. the highest cgmao expression level was detected in haemocytes, which was 1029.2-fold (p < 0.05) of that in gill. the expression of cgmao mrna in hepatopancreas was 309.5-fold of that in gill (p < 0.05). fig. 1 (a) multiple sequences alignment of cgmao with maoas from other species. the black shadow region indicates positions where all sequences shared the same amino acid residue, while the similar amino acids are shaded in grey. gaps are marked by dashes to improve the alignment. the species and the genbank accession numbers were as follows: maoas from human homo sapiens (np 000231), zebra fish danio rerio (np_997992), and scallop mizuhopecten yessoensis (xp_021339456). (b) consensus neighbor-joining tree based on the sequences of cgmao and maos from other species. the protein sequences used for phylogenetic analysis included mao from zebra fish danio rerio (np_997992), scallop mizuhopecten yessoensis (xp_021339456), medaka oryzias latipes (xp_023806277), xenopus xenopus tropicalis (np_001120572); maoas from human homo sapiens (np_000231), cattle bos taurus (np_851357), mouse mus musculus (np_776101), pig sus scrofa (np_001001640), orangutan pongo abelii (np_001124913), horse equus caballu (np_001075301); and maobs from human homo sapiens (aab27229), cattle bos taurus (aaf23179), mouse mus musculus (aai13183), pig sus scrofa (aat06259), orangutan pongo abelii (np_001124895), horse equus caballus (np_001075302) . (c) tissue distribution of the cgmao mrna transcript detected by sybr green real-time pcr. cgmao relative mrna expression level in gonad, mantle, labial palp, mantle, muscle, haemocytes and hepatopancreas was normalized to that of gill. each group values were shown as mean ± sd, and bars with different letters were significantly different (p < 0.05) table 2 the monoamine oxidase (maos) from various species javascript:; javascript:; javascript:; https://www.ncbi.nlm.nih.gov/protein/np_997992.2?report=genbank&log$=prottop&blast_rank=1&rid=f4027jxp014 https://www.ncbi.nlm.nih.gov/protein/xp_021339456.1?report=genbank&log$=prottop&blast_rank=1&rid=f417n35t014 https://www.ncbi.nlm.nih.gov/protein/np_997992.2?report=genbank&log$=prottop&blast_rank=1&rid=f4027jxp014 javascript:; https://www.ncbi.nlm.nih.gov/protein/xp_021339456.1?report=genbank&log$=prottop&blast_rank=1&rid=f417n35t014 https://www.ncbi.nlm.nih.gov/protein/xp_023806277.1?report=genbank&log$=protalign&blast_rank=1&rid=yjfbk51r013 https://www.ncbi.nlm.nih.gov/protein/np_001087039.1?report=genbank&log$=prottop&blast_rank=1&rid=f41rn9xg01n 60 name organism accession number similarity identity maoa homo sapiens np_000231 66 55.5 maoa bos taurus np_851357 65.8 54.7 maoa sus scrofa np_001001640 65.8 55.3 maoa rattus norvegicus np_387502 65.4 56 maoa mus musculus np_776101 54.5 55.2 maob homo sapiens aab27229 66.9 55.8 maob bos taurus aaf23179 66.5 55 maob sus scrofa aat06259 66.3 54.9 maob rattus norvegicus aaa41566 67.2 56.2 maob mus musculus aai13183 67.4 57 mao danio rerio np_997992 68.8 57.6 biosynthesis of ne after oysters were fed with different algal diet the concentration of ne in oyster serum kept increasing after algae fed at different time points. at 21 d, ne concentration in diatom dominant group was higher than that of other two groups (p > 0.05), which was 1.21-fold of that in the dinoflagellate dominant group and 1.23-fold of that in the control group (fig. 2a). at 40 d, ne concentration in either dinoflagellate dominant group or diatom dominant group was higher significantly (p < 0.05) than that in control group. meanwhile, the mrna expression of cgdbh and cgmao, two rate-limiting enzymes for ne biosynthesis, was detected with real-time pcr (fig. 2b and 2c). after the oysters were fed with microalgae for 21 d, the mrna expression levels of cgdbh decreased significantly, which were 0.45-fold and 0.5-fold of that in the control group (p < 0.05). at 40 d post feeding, the mrna expression level of cgdbh in the diatom dominant group was 3.75-fold of that in the control group, which was dramatically higher than that in control group (p < 0.05). besides, the mrna expression of cgmao in the diatom dominant group decreased significantly on both 21 and 40 d post feeding, which was 0.7and 0.29-fold of that in the control group (p < 0.05). but, once oysters were fed with a diet rich in dinoflagellates for 40 d, the expression of cgmao was significantly prompted (2.23-fold) comparing with control group (p < 0.05). changes of cgtnf-1 and cgil17-5 mrna expressions in oyster haemocytes after microalgae feeding after the oysters were fed with microalgae for 21 d, the mrna expression of cgtnf-1 increased significantly, which was 2.43-fold (dinoflagellate dominant group) and 2.20-fold (diatom dominant group) of that in the control group (p < 0.05). at 40 d post feeding, the expression levels in the dinoflagellate dominant group and diatom dominant group were also dramatically higher than that in the control group, which were 3.60-fold and 7.1-fold of that in the control group, respectively (p < 0.05, fig. 3a). as for cgil17-5, its expression level increased significantly after oysters were fed with diet rich in diatom for 40 d (p < 0.05) which was 2.0-fold of that in the control group, while its expression level was obviously lower (0.33-fold, p < 0.05) in dinoflagellate dominant group comparing with that in the control group (fig. 3b). fig. 2 the norepinephrine (ne) concentration in oyster serum (a), as well as the mrna expression of cgdbh (b) and cgmao (c) in haemocytes, after the oysters were feed with diets with different proportion of dinoflagellate and diatom for 21 and 40 d javascript:; javascript:; 61 fig. 3 the mrna expression level of cgtnf-1 (a) and cgil17-5 (b) in haemocytes after the oysters were feed with diets with different proportion of dinoflagellate and diatom for 21 and 40 d the enzyme activity of three key immune-related enzymes including sod, cat and lyz was detected post microalgae feeding (fig. 4). after the oysters were fed with a diet rich in diatom, the sod activity increased significantly comparing with the control group (1.14-fold, p < 0.05, fig. 4a). the cat activity in the dinoflagellate dominant group increased significantly at 21 d, but was obviously lower than that in the control group at 40 d (p < 0.05, fig. 4b). as for lyz, its activity increased significantly in the diatom dominant group at both 21 and 40 d (p < 0.05, fig. 4c). in the dinoflagellate dominant group, the cat activity increased significantly at 21 d post feeding, which was 1.32-fold of that in the control group (p < 0.05). antibacterial activity of oyster serum the antibacterial activity of oyster serum post microalgae feeding was determined. at 40 d after feeding, the antibacterial activity of serum increased significantly (p < 0.05, fig. 5a) comparing to that in the blank group. serum in the diatom dominant group exhibited the highest antibacterial activity, whereas the lowest was observed in blank group. the absorbance of bacteria from the blank, dinoflagellate dominant and diatom dominant group was shown in fig. 5b, when the absorbance difference between the blank and diatom dominant group reached the maximum at 21.5 h after the incubation with the serum. discussion bivalve molluscs, such as clams, mussels, oysters and scallops, are relevant bred species, and their global farming maintains a high incremental annual growth rate, representing a considerable proportion of the overall fishery activities. bivalve molluscs are filter-feeding species. they bio-accumulate large amount of microorganisms, in which bacteria can be a great threat, while microalgae such as dinoflagellate and diatom are their major food source. also, much previous research has evidenced that sufficient food supply was of great importance to the antibacterial immunity by offering adequate energy for the immune activity. so far, great progress has been made on the study of immune system and energy metabolism in bivalves. but, the comprehensive interpretation of the nutritional immunology in bivalves is still in urgent need. in the present study, correlation between microalgae intake and the antibacterial immune capacity mediated by norepinephrine (ne) in the pacific oyster c. gigas was investigated, aiming to better illustrate how food intake affects the antibacterial immune response in oyster, and what roles the neuroendocrine system play in such process. according to our previous research, the monoamine neurotransmitter ne was able to modulate the antibacterial immunomodulation via a simple “nervous-haemocyte” neuroendocrine-immune axis to promote the synthesis of cytokines and the apoptosis of haemocytes in oyster (liu et al., 2016a). monoamine oxidase (mao) is the crucial enzyme for the metabolism of ne, which catalyzes the oxidative deamination of biogenic and vasoactive amines to their corresponding aldehydes (slotkin, 1999). thus, in the present study, the full-length sequence of a previously identified oyster mao gene (cgmao) was cloned from c. gigas (boutet et al., 2004). the nucleotide sequence of cgmao contained an orf of 1566 bp, encoding a polypeptide of 521amino acids. there were two fad building domains, one nad building domain, and one amino oxidase domain (from leu18 to leu456) in cgmao. cgmao shared high sequence similarity with maos identified from vertebrate species, and its mrna transcripts were highly expressed in oyster tissues including hepatopancreas and haemocytes. results of phylogenetic analysis showed that cgmao was javascript:; javascript:; javascript:; 62 fig. 4 the activities of key immune-related enzymes including sod (a), cat (b), lyz (c) in serum after the oysters were feed with diets with different proportion of dinoflagellate and diatom for 21 and 40 d first clustered into the invertebrate group with the closest match of scallop m. yessoensis, and then gathered with maos from vertebrate species. these results showed cgmao was the homologue of maos in model creatures, and could perform as the rate-liming enzyme of ne biosynthesis. besides, the mrna transcripts of cgmao were widely expressed in oyster tissues, with the highest expression levels detected in haemocytes and hepatopancreas. haemocytes are the most significant immunocytes in oysters, and hepatopancreas is the most important immune-related tissues since it accounts for the synthesis of several neurotransmitters such as ne and acetylcholine (ach) in oyster (liu et al., 2018). these results indicated that cgmao might be related to the immune activities in oyster. dietary conditioning is extremely important for the growth and health of farmed oysters, which basically live on the microalgae in the seawater. in recent years, the oyster farming has suffered severe mortality owing to the biomass shortage and community imbalance of microalgae caused by the increasing farming density (pernet et al., 2016). based on our latest research, dinoflagellate and diatom are the major components of the microalgae community in the bohai sea and the north yellow sea (unpublished data). in this study, diet with different proportion of dinoflagellate and diatom were fed to the oysters for as long as 40 d, and the antibacterial parameters were determined. after oysters were fed with microalgae for 40 d, ne contents in both dinoflagellate dominant group and diatom dominant group were slightly higher than that in the control group. meanwhile, at 40 d post feeding, the mrna expression of cgdbh was significantly higher in the diatom dominant group, and the mrna expression level of cgmao was also dramatically increased in the dinoflagellate dominant group. dbh is a copper-containing enzyme that uses molecular oxygen and ascorbate to catalyze the addition of a hydroxyl group on the beta-carbon of dopamine to form norepinephrine (kaufman and friedman, 1965), while mao is crucial enzyme for the degradation of ne (franco et al., 2007; malagoli et al., 2017). these results indicated that sufficient microalgae supply could promote the biosynthesis of ne, which might then enhance the antibacterial immune process. in order to validate the above hypothesis, a series of parameters related to the antibacterial immunomodulation were determined. after the oysters were fed with microalgae for 40 d, the mrna expression of cgtnf-1 and cgil17-5 increased significantly. at 40 d post feeding, the enzyme activities of sod and lyz in the diatom dominant group were obviously higher than those in the control group, while the enzyme activity of cat was significantly higher than control group at 21 d post feeding. in addition, at 40 d after feeding, the antibacterial activity of serum increased significantly, and the diatom dominant group exhibited the highest antibacterial activity. interestingly, the increase of mrna expression level of cytokines, as well as the enzyme activity was obviously higher in the diatom dominant group than in the dinoflagellate dominant group. our previous research demonstrated that ne could regulate the antibacterial activity mediated by oyster haemocytes by binding to the transmembrane receptor cga1ar-1, and up-regulate the activity of sod and lyz in oyster serum (liu et al., 2016b). cgtnf-1 was reported to be involved in the modulation of immune response including apoptosis and phagocytosis of haemocytes upon lps stimulation (sun et al., 2014). zheng et al. reported that the mrna transcripts of cgtnf-2 were highly expressed in oyster hemocytes and could be induced by lps and pgn stimulation. cgtnf-2 exhibited inhibitory effects on the growth of a549 cell, and it could promote the lysozyme activity and synthesis of no to regulate the antibacterial response in c. gigas (zheng et al., 2020). also, cgil17-5, as an ancient inflammatory cytokine, could not only activate signal transduction for the release of other cytokines, but also mediate the javascript:; javascript:; 63 fig. 5 the effects of microalgae feeding on the antibacterial activity of oyster larvae. (a) the growth curve for bacteria v. splendidus exposed to oyster serum from the control, dinoflagellate dominant and diatom dominant group at 40 d after feeding. bacterial growth was recorded as absorbance at 600 nm. (b) the absorbance of bacteria from the control, dinoflagellate dominant and diatom dominant group, when the absorbance different between the control and diatom dominant group reached the maximum. each group value is shown as mean ± sd, and bars with asterisks are significantly different (p < 0.05) clearance of extracellular bacteria in oysters (xin et al., 2015). cgil17-5 was reported to promote nuclear factor nf-кb pathway and mitogen-activated protein kinase (mapk) pathway to play an important role in antibacterial immunity (roberts et al., 2008). these results inferred that sufficient microalgae intake could significantly enhance the antibacterial immune response level in oyster by activating ne and cytokine production, elevating the activity of immune-related enzyme, and up-regulating the antibacterial activity of oyster serum. moreover, oysters fed with a diet rich in diatom exhibited significantly higher antibacterial capacity comparing with those fed with a diet rich in dinoflagellate, which implied that diatom might be the more preferable food for oysters than dinoflagellate. acknowledgements we are grateful to all the laboratory members for their technical advice and helpful discussions. this work was funded by the grants (nos. 31802339, 32072946) from national science foundation of china, the national key research and development program of china (2018yfd0900606), key r & d program of liaoning province (2017203004, 2017203001), earmarked fund (cars-49) from modern agro-industry technology research system, the fund for outstanding talents and innovative team of agricultural scientific research, the research foundation for talented scholars in dalian ocean university and the distinguished professor of liaoning (xlyc1902012), aoshan talents cultivation program supported by qingdao national laboratory for marine science and technology (no. 2017astcp-os13), fund from liaoning department of education (ql201901), and liaoning baiqianwan talents program. 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li m, zhang y, zong y, et al. a novel tumor necrosis factor in the pacific oyster crassostrea gigas mediates the antibacterial response by triggering the synthesis of lysozyme and nitric oxide. fish shellfish immunol. 98: 334-341, 2020. zhou z, jiang q, wang m, yue f, wang l, wang l, et al. modulation of haemocyte phagocytic and antibacterial activity by alpha-adrenergic receptor in scallop chlamys farreri. fish shellfish immunol. 35: 825-832, 2013. zhou z, wang l, gao y, wang m, zhang h, wang l, et al. a monoamine oxidase from scallop chlamys farreri serving as an immunomodulator in response against bacterial challenge. dev. comp. immunol. 35: 799-807, 2011a. zhou z, wang l., shi x, yue f, wang m, zhang h, et al. the expression of dopa decarboxylase and dopamine beta hydroxylase and their responding to bacterial challenge during the ontogenesis of scallop chlamys farreri. fish shellfish immunol. 33: 67-74, 2012. zhou z, wang l, shi x, zhang h, gao y, wang m, et al. the modulation of catecholamines to the immune response against bacteria vibrio anguillarum challenge in scallop chlamys farreri. fish shellfish immunol. 31: 1065-1071, 2011b. isj005.pdf 46 isj 1: 46-59, 2004 issn 1824-307x report of meeting vith scientific meeting of the italian association for developmental and comparative immunology (iadci), 12 and 13 february 2004, university of padova, padova, italy organizers: l ballarin, p burighel, g zaniolo dipartimento di biologia, università degli studi di padova, padova, italy session 1. immunity and soluble factors cloning and biotesting of cytokines in fish j zou, s bird, c secombes scottish fish immunology research centre, school of biological sciences, university of aberdeen, aberdeen ab24 2tz, uk fish est and genome projects have advanced at a tremendous speed in the last few years. to date, there exists nearly 1 million fish ests and sequence data is still accumulating daily. the third draft of the whole fugu genome has been published and the zebrafish genome is nearly finished. all this makes it easier than ever before to search for elusive fish genes, especially those with low sequence homology with known molecules in higher vertebrates such as cytokine genes. in this report, by blast searching the fugu genome database with known cytokine sequences or with the conserved genes known to be adjacent to cytokine genes in the human genome, a number of cytokines including il-6, il-12, il-15, il-18, il-19, il-20, and interferon, have been successfully found and their relationship to the mammalian counterparts investigated. comparative studies have revealed that synteny of some cytokine genes is conserved between fugu and humans. to characterise their biological functions, trout homologs of some of the identified molecules have been cloned by est database analysis or homology cloning and expressed in bacterial or mammalian cells. the recombinant proteins have been purified and their bioactivities tested. wound repair in hirudo medicinalis: phases and response modulation m de eguileor1, g tettamanti1, a grimaldi1, t congiu2, m raspanti2, g perletti, r valvassori1 1dipartimento di biologia strutturale e funzionale, università dell'insubria, varese, italy 2facoltà di medicina e chirurgia, università dell'insubria, varese, italy in leeches, different antigens and immunisation conditions lead to responses that may vary both quantitatively and qualitatively. different antigens can selectively stimulate both proliferation and migration of immune cells that can be classified as macrophagelike cells, nk-like cells or granulocytes (not only for their morphological aspect but also for the expression of specific cd antigens). defence responses can vary in relation to the dimension of the non-self. leeches are able to phagocyte small spheres or yeast, but also can incapsulate and subsequently melanize larger non-self, such as parasites. all these responses towards the non-self, show that leeches can use a variety of selected defence systems. in addition hirudo medicinalis can react to large and deep explantation through wound healing process that can be divided, as in vertebrates, in three different stages: inflammation, granulation tissue and scar tissue remodelling. in leeches, the granulation tissue stage is characterized by a massive angiogenesis and fibroplasia: -the formation of new vessels is a complex process based on a finely regulated network of cellular and molecular events, involving a great variety of ecm proteins and cytokines; -the production of connective tissue, with the maturation of collagen fibrils, leads to the formation of new solid extracellular matrix network, which can be used as a scaffold not only for tissue reconstruction, but also for migration of immune cells and for directing new vessels growth. we have used different approaches (light microscopy, scanning and transmission electron microscopy, atomic force microscopy, immunocytochemical studies and selective enzymatic digestions) to evaluate how in leeches angiogenesis and production of new collagen occur in response to surgical lesions. the first cytokine from antarctica: il-1ββ from the icefish chionodraco hamatus f buonocore1, s bird2, f paderi1, e randelli1, m mazzini1, cj secombes2, g scapigliati1 1dipartimento di scienze ambientali, università della tuscia, viterbo, italy 47 2fish immunology research centre, university of aberdeen, aberdeen, scotland, uk over 200 species of fish from various groups are known to live in antarctica, the most abundant species of which belonging to the suborder notothenioidei, family perciformes. fishes are estimated to have adapted to polar conditions 20-25 million years ago when there was a physical water barrier established between the antarctic continent and the temperate oceans. these fish have diversified and developed several peculiar features, among which a protein that is produced which prevents tissues freezing, the lack of a swim bladder and the absence of haemoglobin and functional blood erythrocytes, which is found only in the so called icefishes, members of the family channichthyidae. recently, the immune system of icefish has been investigated to study its morphological and functional organisation, to evidence the presence of specific immune humoral responses and to analyse immunoglobulin genes. in the presented study, primers designed to conserved regions of interleukin-1β (il-1β) were used for the homology cloning of the icefish chionodraco hamatus il-1β gene, the first cytokine gene sequenced in an antarctic teleost. the full length c. hamatus il-1β cdna consists of 1289 nucleotides that translated in a single reading frame to give a predicted 250-amino acid il-1β molecule. the c. hamatus il-1β sequence had highest nucleotide identity (75.7%) and amino acid similarity (69.8%) and identity (63.2%) with turbot il-1β, followed by european seabass and gilthead seabream. the closer relationship between the turbot, seabass and seabream il-1β with c. hamatus il-1β was also apparent in the phylogenetic tree obtained using amino acid data. studies of il-1β expression in c. hamatus indicated that the il-1β molecule is induced by bacterial lipopolysaccharide (lps) and these data raised questions about antarctic fish immune responses to gram-negative bacteria. similar to the other perciformes il-1β genes, seabass and seabream, the c. hamatus il-1β gene is organised in five exons and four introns. sea bass (dicentrarchus labrax) recombinant interleukin-1â: purification and biological activities e randelli1, f buonocore1, m forlenza2, s meloni1, s benedetti1, d prugnoli1, cj secombes3, j zou3, g scapigliati1 1dipartimento di scienze ambientali, università della tuscia, viterbo, italy 2department of immunology, university of wageningen, the netherlands 3fish immunology research centre, university of aberdeen, uk studies on the immune system of teleost fish are important for the evolution of defence mechanisms within vertebrates and may have relevance in biotechnology. the sea bass dicentrarchus labrax is the major aquacultured seawater fish species in the mediterranean sea and its health control is a subject of intense research. interleukin-1 (il-1) is a cytokine playing pivotal roles in modulating immune responses in vertebrates and it has been preliminarily employed as immunoadjuvant in fish vaccination experiments. our group recently cloned the sea bass il-1β and characterized its genomic and molecular structure. the putative mature peptide deduced from the sequence has been expressed as a recombinant protein in e. coli by using the plasmid pqe-30. this plasmid is able to add 6xhis tag at the n-terminus of the protein of interest, allowing the purification of the protein on ni-nta matrices. in addition, anti-his antibodies can be used for detection of the recombinant protein. the bioactivity of the purified molecule has been studied in some biological assays. recombinant sea bass il-1β (ril-1β) induced the proliferation of murine th1 cells d10.g4.1, although with less activity with respect to human il-1β. ril-1β was able to increase phagocytic activity and phagocytic index of head kidney leukocytes in a dose-dependent fashion. sea bass ril-1β was also able to increase the expression levels of il-1β gene both “in vitro” and “in vivo”. stimulatory doses of ril-1β were typically >10 ng/ml. interestingly, ril-1β seems to induce sea bass thymocyte proliferation also in the absence of a costimulatory factor. moreover, the effect of ril-1β as immuno-adjuvant has been tested in sea bass vaccination experiments using a model sea bass pathogen (vibrio anguillarum) as antigen of immunisation. the preliminary results seem to show an increase of the specific antibody production in the fish treated with the recombinant molecule. differential lymphocyte proliferation during in vitro allogeneic and xenogeneic reactions in the sea bass dicentrarchus labrax s meloni, g zarletti, f buonocore, m mazzini, g scapigliati dipartimento di scienze ambientali, università della tuscia, viterbo, italy in the sea bass dicentrarchus labrax, t lymphocytes and b lymphocytes can be recognised by the specific markers dlt15 and dlig3, respectively. apart their identification in several species, the immunobiology of teleost lymphocytes is almost unknown, and to study some basical features of these cells, we setup some in vitro experimental models using sea bass peripheral blood leucocytes (pbl). these models were an allogeneic mixed leucocyte reaction (mlr), an allogeneic reaction (ar), and a xenogeneic reaction (xr), and lymphocytes were detected by indirect immunofluorescence (iif) and counted by flow cytometry employing mab dlt15 and dlig3. mlr was performed by incubating donor pbl with irradiated (600 rads) or mitomycin-treated target pbl for 3 and 6 weeks in l15 medium at 18 °c. the mean content of t-cells in pbl was 3.1±0.8%, in the mlr this percent raised to 8.9±4.3% after 3 weeks (n=9, max value 16.7%), and remained almost unchanged at 6 weeks with a value of 8.0±4% (n=9, max value 20.5%). the mean content of immunoreactive b-cells 48 in pbl was 21.1±3.8%, in the mlr this percent decreased to 6.71±3.18 after 3 weeks (n=9, max value 8.9%), and to 3.0±1.6 at 6 weeks (n=9, max value 6.32%). these mlr data showed the increased proliferation of t-cells and the decrease of b-cells, and were confirmed by rt-pcr experiments using specific primers for ig (light chain) and tcr (vβ). these data also showed the high individual variability between animals. ar was performed against the mitomycin-treated cells of the sea bass embryonic cell line dlec, and results are under evaluation. xr was performed against mitomycin-treated sea bream fibroblast cell line saf-2, and after several attempts, the incubation time of the reaction was fixed at 4 and 7 days. each sample (n=10) was tested with respect to the same untreated pbl at same incubation time. the mean content of t-cells in pbl was 3.4±1.8% at 4 days and raised to 6.5±3 in the xenoreaction, after 7 days the mean percent of t-cells was 4.8±2 in controls and 3.9±2.2 in the xenoreaction. the mean content of immunoreactive bcells was 13.2±5.5 at 4 days and raised to 19.8±6.5 (min value 6.9%, max value 35.9%) in the xenoreaction, after 7 days the mean content of immunoreactive b-cells was 13.4±7.3% in controls and 16.6±5.5 (min value 7.3%, max value 28.3%) in the xenoreaction. these xr data indicated that t-cells may have a fast reaction and, surprisingly, that b-cells are involved in this reaction. studies are in progress to assess the presence and to evaluate effects of non-specific cytotoxic cells in the xr, and to assess the effects induced by the tcell function inhibitor cyclosporin in the mlr. humoral immune activities in vivo and in vitro in sea bass dicentrarchus labrax g scapigliati, s benedetti, s meloni, m mazzini dipartimento di scienze ambientali, università della tuscia, i-01100 viterbo, italy during the last years, vaccination has become an important way to prevent infectious diseases in farmed fish and the control of fish diseases is the sole method to prevent mass mortality; this control can be carried out by means of vaccination with different antigens. in this work, we used three different antigens: dnp-klh (administered intraperitoneally). vibrio anguillarum (administered intraperitoneally and by immersion to juvenile sea bass) and photobacterium damselae spp. piscicida that is mainly a mucosal bacterium. the elisa assay was used to detect the serum specific antibody production. after 30 days post-immunization, a strong serum antibody response was observed against dnp-klh, with a serum titer of ca. 1:13000; is interesting that, by using the molecule carrier klh alone as antigen, we have measured a poor antibody response. fish vaccinated i. p. with vibrio anguillarum were sampled at day 37 after stimulation and this antigen induced an evident production of serum antibody. by employing p. damselae, a single immersion vaccination wasn't sufficient to elicit a detectable serum antibody response after 30 days. consequently, a boosting in the same conditions was performed, and this treatment resulted in the appearance of serum antibody in fish with a titer of ca. 1:3000. the elispot modified assay was used to detect in vitro the presence of ig-producing b cells for the antigen. in every experiment a control was performed by adding the drug cycloexhimide to the same number of leucocytes to block de novo protein synthesis. after 30 days post-immunization with dnpklh the production of ig by head-kidney leucocytes was detected; cells from immunized animals, when plated in wells coated with dnp-klh, gave a 173-fold higher response with respect to cells coming from control fish. a similar pattern was obtained when using klh alone; these data suggest the in vivo presence of b-t cells cooperation. the results of experiments with vibrio anguillarum clearly showed that, with respect to controls, leucocytes from fish of 2 g in weight immersion-vaccinated once with vibrio vaccine one year before had memory b-cells for the antigen, since they were able to produce specific ig against vibrio when re-exposed to immunization antigen. by using p. damselae bacterin in immersion vaccination of juvenile sea bass, it was observed the presence of memory b cells in hk leucocytes in vaccinated fish from 51 days after boosting onwards. proliferations experiments with leucocytes from hk, pbl and spleen of fish vaccinated with vibrio anguillarum suggested that pbl were the more effective leucocyte population in this process. it can be concluded that the humoral immune system of sea bass can be differentially modulated by the type of antigen and the administration protocol. expression of t-cell receptor in sea bass, dicentrarchus labrax n romano1, f buonocore1, a moschetti1, l abelli 2, s picchietti1, g scapigliati1, l mastrolia1 1department of environmental sciences, tuscia university, viterbo, italy; 2department of biology, university of ferrara, italy t cell receptor (tcr), responsible in jawed vertebrates for the recognition of self and foreign cells and molecules is recently demonstrated in teleost fish, as composed, like in mammals, by αβ or γδ chains [nam et al., 2003, j. immunol.,170, 30813089]. in this study, rna probes of sea bass tcrβ were prepared and used for rt-pcr and in situ hybridization. immunohistochemical studies (abcperoxidase with nickel enhancement) were made in parallel using the anti-t cell monoclonal antibody dlt15 [scapigliati et al.,1995, fish & shellfish immunol., 5, 393-405]. in sea bass thymus, dlt15+ cells (70±15 % of cells) were localised mainly in the cortex, while the reactive tcrβ+cells were numerous at the corticalmedullary border, less in the cortex and a few in the medulla (60±10 % of cells). in the spleen, tcrβ+cells (7±3,1 % of cells) appeared less numerous compared with the dlt15+cells (10±4,5 % of cells), grouped in white pulp areas. in the head kidney, tcrβ+ cells 49 (10±3,2 % of cells) and dlt15+cells (15±4 % of cells) were scattered or grouped close to blood vessels. sparse tcrβ+cells were more numerous in the midgut (8,3±1,5 % of cells) localised in the lamina propria, while dlt15+ cells distinctly increase in number toward the hindgut (from 11,2±1,2 % in anterior portion to 19±1,5 of cells in the posterior portion. the localisation of intestinal dlt15+ cells was in the basolateral epithelium and lamina propria. these findings suggest that numerous t cells in lymphomyeloid organs express the tcrβ, while a in the gut t-cells do not express tcrβ, but possibly another tcr (γ/δ?). tcrβ+cells (from head kidney and pbl) are significative involved in activities of specific immune response such as the proliferation processes induced by mitogen (pha) or xenogenic cells (sea bream leucocytes). in allograft rejection, the tcrβ+cells are clearly involved because their percentage (13±2,5) is relevant respect the counting leucocytes. biological activity, tissue distribution and preliminary molecular characterization of a serum fucolectin from the sea bass (dicentrarchus labrax) m cammarata1, g salerno1, g benenati1, m vazzana1, d parrinello1, e w odom2, gr vasta2, n parrinello1 1 laboratorio di immunobiologia marina,dipartimento di biologia animale, università di palermo, via archirafi18, palermo, italy 2 center of marine biotechnology, university of maryland biotechnology institute, 701 e. pratt street, baltimore md, usa sugar binding proteins (lectins) and free or cell surface-bound sugars constitute an evolutionary conserved recognition system involved in innate immunity. lectins are widely distributed in both vertebrates and invertebrates. in those taxa endowed of both innate and adaptive immunity, lectins mediate rapid recognition and effector functions that precede adaptive immunity. fucose-binding lectins are present in tissues and fluids from invertebrate and vertebrate species. wellcharacterized examples, such as the lectin cpl-iii from the tunicate clavelina picta and the fucosebinding mammalian collectins, clearly belong to the clectin type. others, such as dll34 from the sea bass dicentrarchus labrax (cammarata et al., biochim. biophys. acta, 2001, 1528: 196-202), the serum “fucolectins” from anguilla japonica (honda et al j biol chem, 2000. 275: 33151-33157) and the fbp32 of morone saxatilis lack a typical sequence motif present in any of the lectin families described so far. furthermore, because of their specificity for carbohydrate moieties present on potential microbial pathogens, and their inducibility upon infectious or inflammatory challenge, these lectins are considered to function as recognition factors in innate immunity. a fucose-binding lectin from serum of the sea bass d. labrax was purified and partially characterized. in sds-page, the lectin has an apparent molecular size of 34 kda. in this report, studies on the d. labrax lectin structural aspects, iological properties, tissue distribution and ontogenetic aspects are described. these results suggest that the d. labrax lectin is involved in innate immunity. in addition, the primary structure (nucleotide and amino acid sequence) indicate the presence of a tandem 2-crd fucolectin that confirm the d. labrax lectin can be a component of the recently identified f-lectin family (vasta et al., isdci meeting 2003, scotland). [supported by grants miur ex 60% and cori to np and mc, and by a national science foundation grant (mcb-00-77928) to grv] hepato-biliary transport of immunoglobulin in the antarctic teleost trematomus bernacchii l abelli1, mr coscia2, a de santis2, c zeni1, u oreste2 1dipartimento di biologia, sezione di anatomia comparata, università di ferrara, italy 2cnr-istituto di biochimica delle proteine, napoli, italy presence of immunoglobulins (ig) in the liver of trematomus bernacchii was investigated by biochemical and immunochemical assays with polyclonal antisera raised in rabbits against purified t. bernacchii serum ig heavy (igh) and light chains (igl). bile ig were quantified by elisa and purified. western blot analysis of sds-page separated bile proteins, performed using ighor igl-specific antisera, revealed two igh bands (76 and 66 kda) and the igl band (25 kda). similar results were obtained when extracted liver proteins were analysed by the same method. sds-page under non-reducing conditions of bile proteins revealed multiple bands (ranging from 200 to 830 kda) resulting from different ig polimerization forms. finally, ief analysis of the bile ig showed a wide range of pi values. immunohistochemistry detected ighand iglreactivity in the plasma of hepatic sinusoids, in cells extravasated in the perisinusoidal space, in bile canaliculi and pre-ductules. these findings strongly indicate that ig, derived from blood and/or activated bcells, could be transported across the hepatocytes to be secreted into the bile. in the anterior intestine, the intraluminal mucus retained a significant igimmunoreactivity, while the mucosa housed a limited density of ig-bearing cells. in addition, extravasated plasma cells accumulated within identifiable portal tracts and close to the liver capsule that, in turn, was evenly coated by ig molecules at the peritoneal surface. these peculiar features could be explained in light of liver defence system against ascending infections and/or colonization by nematode parasites and suggest that the ig could protect the intestinal epithelium via the hepato-biliary transport route. immunoglobulin light chain isotypes from the antarctic teleost trematomus bernacchii mr coscia, l furino, u oreste institute of protein biochemistry, cnr, naples, italy immunoglobulin light chains (igl) have been sequenced in eleven different teleost species belonging to order siluriformes, gadiformes, 50 salmoniformes, gasterosteiformes, perciformes, and cypriniformes. in each species two or three different isotypes have been described. to identify igl in the antarctic teleost trematomus bernacchii a cdna expression library was constructed in zap express vector from spleen poly (a)+ rna and immunoscreened with rabbit igg specific for t. bernacchii igl chain. several immunopositive clones were isolated and sequenced. in an attempt to yield additional clones encoding t. bernacchii igl, a pcr approach was chosen. first-strand cdna was synthesized by reverse transcriptase using head kidney total rna. to accomplish pcr amplification a multiple alignment of igl sequences from different teleost species was used in the design of two oligonucleotide primers complementary to the most conserved part in fr2 (sense) and in the terminus of cl (antisense), respectively. the resulting pcr products were cloned into pgem-t easy vector and recombinant clones were isolated and sequenced. three different isotypes were identified by calculating percent of identity among the nucleotide sequences, and referred to as trbecl1 trbecl2 and trbecl3 based on comparison with the isotypes definied in other teleosts. by southern blot analysis different dna fragments were labeled by isotype specific probes. a multiple alignment of cl sequences from t. bernacchii and other vertebrate species was obtained with clustal x and a phylogenetic tree was constructed. session 2. immunity: applicative aspects immunity and vaccination in aquaculture g bovo, a manfrin, l selli istituto zooprofilattico sperimentale delle venezie, viale dell’ università, 10 35020 legnaropd, italy first reports on fish vaccination date back to the thirties while only since the seventies, following the appearance of antibiotic resistance in trout industry, studies on vaccination reached a renewed interest. during the last twenty years new acquisitions have been obtained on fish immunology and different commercial vaccines have been registered. several pharmaceutical products against nearly 15 fish diseases are available up today on the international market and vaccines against vibriosis, furuncolosis and red mouth disease are routinely applied in salmonid industry quite often as polyvalent formulations . first generation vaccines obtained by formalin inactivation of bacterial whole cells still represent a consistent and very efficient product ; they are usually administered to young fish by immersion. alternatively, when 10-15 g or bigger fish must be vaccinated, i.p. inoculation with or without adjuvants is suggested. oral vaccination seems to be a very promising system to protect fish mainly due to reduction of handling stress and manpower costs but in spite of substantial research efforts no efficient commercial product, with the exception of a vaccine for edwardsiella ictaluri, have been registered jet. besides these unsophisticated products, a few innovative vaccines have been investigated during the recent years and an ipn recombinant vaccine has recently been registered in norway and chile. more recently positive results have been published on experimental dna vaccines particularly against salmonid rhabdoviruses (ihnv and vhsv). the evaluation of vaccine efficacy is measured by the rps index which reflects the relative% protection of vaccinated groups in comparison with control fish. experimental results may be affected by several parameters like age of fish, challenge dose, pathogenicity of challenge strains, administration route, environment temperature and fish susceptibility; moreover field trials may be affected by further variables and strict statistical methods are suggested in order to evaluate final results . the reduction of antibiotic treatments achievable when vaccination strategies are correctly applied , represents an important result not only from an economical point of view but in addition with regard to environment protection and food safety which, during the recent years, became an important growing concern for many people: in norway following the adoption of routine vaccination the use of antibiotics has been significantly (98%) reduced in ten years . in conclusion vaccination should be regarded in the future as a fundamental tool for the control of infectious diseases . the appearance of new and severe viral diseases, hard to control by eradication methods, particularly in the marine environment , like salmon anemia and encephalopathy and retinopathy, suggests that an integrated control approach in which vaccination plays a fundamental role, should represent the ideal strategy to avoid disease spreading in aquaculture. rearing density may influence seabass (dicentrarchus labrax, l.) stress related protein gene expression r gornati, e papis, g bernardini dipartimento di biologia strutturale e funzionale, università dell’insubria, varese, italy the worldwide decline of ocean fisheries stocks has provided a rapid growth in fish, shellfish farming and aquaculture and the problems connected to animal welfare in aquaculture gained importance. in this context, we have looked for molecular markers among those genes whose expression could reasonably result modified by the different farming conditions. with this purpose, we have evaluate, in liver and brain sea bass, grown for two months at different biomass density (<10 kg/m3, 80 kg/m3 and 100 kg/m3), the expression of those genes coding for proteins related to stress such as heat shock proteins (hsps), metallothioneins (mt) and cytochrome p4501a (cytp4501a). mt and cyp4501a mrna resulted induced in liver of animals reared at 80 and 100 kg/m3. hsp70 appeared significantly over expressed only at the biomass of 100 kg/m3, while apparently, no induction was detectable for hsp90. in brain tissue instead, mt and hsp90 were inducted already at 80 kg/m3, cytp4501a was influenced only at the higher population density of 100 kg/m3, while we have not obtained any results for hsp70. 51 in the last three decades, there has been an exponential increase in the interest concerning the description, classification and functional significance of stress related proteins, in particular hsps. these proteins represent precious molecular biomarkers able to detect the welfare conditions when they are still recoverable. here we wish to underline that to detect their mrna by pcr is fast, easy and relatively unexpensive, therefore we propose this method as a good alternative to monitor fish welfare. sequence diversity of antarctic fish igtm exons m maglione, s giacomelli, mr coscia, u oreste institute of protein biochemestry, cnr, naples, italy the secreted (igh) and membrane-bound forms (migh) of the immunoglobulin heavy chains are encoded by a single gene, and alternate pre-mrna processing determine which mrna is expressed. in teleost the splicing mechanism is different from that occurring in mammals; in fact the ch4 exon is totally excluded in the teleost rearranged migh mrna. the tm exon encodes the c-terminal sequence of the migh comprehensive of the cytoplasmic, the membrane crossing, and the extracellular spacer regions. the region crossing the cell membrane is known to be highly conserved during the evolution. the aim of the present study is to sequence mighs from different antarctic teleost species was in an attempt to identify specific features accounting for the evolutionary adaptation. rt-pcr was performed on rna extracted from head kidney of trematomus pennelli, trematomus newnesi, notothenia coriiceps, pagetopsis macropterus, and gymnodraco acuticeps using trematomus bernacchii oligonucleotides as primers. migh cdna were synthesized, cloned and the sequence determined for each of the above mentioned species. in addition the t. bernacchii genomic sequence comprising the ch3 and tm exons was completely determined by a rt-pcr approach using testis dna. the deduced amino acid sequences of migh from the antarcic species were aligned with those from other teleost species reported in data banks, by clustal x and a phylogenetic tree was constructed. several specific features of the antarctic migh sequences were found identified: while the cytoplasmic and membrane crossing regions were highly conserved in antarctic as well as in all the examined species, the extracellular regions were found to be very different in length and composition. in addition migh of several antarctic species showed the lack of the ch3 exon and the presence of an extracellular spacer region about 40 amino acid residue longer than usual. this tm exon elongation may be arisen from a particular evolutive mechanism involving the multiple repeats of a short sequence which is reverse complementary to a portion of the ch3 exon. no in the developing gut of dicentrarchus labrax: an early immune role? l mola, a pederzoli, a gambarelli, d tagliazucchi*, a conte* dipartimento di biologia animale, *dipartimento di scienze agrarie, università di modena e reggio emilia, italy it is known that nitric oxide (no) plays an important role in the immune-neuroendocrine communications. in this issue, we have examined the appearance and distribution of nitric oxide sinthase (nos) histochemically, immunohistochemically and biochemically during development of the sea bass gut. in 4 and 24-day-old larvae the nos activity evaluated by nadph-diaphorase was strong in all epithelial cells and in cell and fibers of intestinal wall, at all gut levels. in the same larval stages and localizations immunoreactive material to antibodies against nnos and inos was present. in the 5-monthold adult gut both enzimatic activity for nadphdiaphorase and immunoreactivity (ir) to anti-nnos disappeared from epithelium remaining in the gastroenteric nervous system. the ir to anti-inos completely disappeared. western blot analysis showed that neuronal (about 150,000 mol. wt band) and inducible (about 135,000 mol wt band) nos-immunoreactive proteins are present in 24 day-old larvae gut. in the 5 monthold adult gut nnos and inos ir disappeared in the soluble fraction of crude gut homogenates. a small amount of nnos ir was present in particulate gut fraction. our data show that both the calcium-calmodulin dependent nnos and calcium-independent inos are present in the larval gut of sea bass. in this species the maturation of cell-mediated immune responses and humoral immune system takes place respectively around the first and second month post hatching (scapigliati et al., 2002). the presence of inducible nos in the same regions of the sea bass gut in which the galt will differentiate, may suggest for no a role in early defence mechanisms, before of establishment of immune responses in galt. investigation on the phagocytosis operated by the mussel (mytilus galloprovincialis) hemocytes using the 1,2,3-dihydrorhodamine as fluorescent probe f mosca, l gioia, v narcisi, pg tiscar dipartimento di scienze biomediche comparate, università degli studi di teramo, piazza a. moro 45, 64100 teramo, italy different fluorescence techniques, such as flow cytometry, micromethod fluorimetry and confocal laser scanning microscopy have been applied in the study of cellular defense system of mytilus galloprovincialis. the phagocytosis stimuli was represented by the yeast cells (zymosan), whereas the fluorescent probe utilised for the measuring the synthesis of the reactive oxygen species (ros) was the 1,2,3dihydrorhodamine (dhr). flow cytometry distinguished three hemocyte sub-populations in mussel hemolymph, showing also the relation between hemocyte size, granularity and phagocytosis ability. micromethod assay confirmed the respiratory burst produced by the hemocytes whereas the use of superoxide dismutase (sod) and l-ng-monomethyl 52 arginine citrate (l-nmma) increased fluorescence intensity. in addition, the confocal microscopy assay permitted to valuate the oxidation of dhr, due to ros synthesis, inside the cellular organelles involved in phagocytosis. in conclusion, these innovatory techniques could represent useful tools for valuating the immune system of marine invertebrates, both in research approach and in applied studies. hemocyte circularity (hc) and heat shock protein 70 kda (hsp 70) expression as stress parameters in mytilus galloprovincialis f mosca, lm spadaccini, v narcisi, c palmieri, pg tiscar dipartimento di scienze biomediche comparate, facoltà di medicina veterinaria, università degli studi di teramo, piazza a. moro 45, 64100 teramo, italy the present study aim of investigating, in mussel (mytilus galloprovincialis) specimens exposed to sublethal thermic stress, the variations of two parameters such as the hemocyte circularity (hc) and the mantle hsp 70 expression. a cell viability analyzer has been applied for valuating the hemocyte concentration, the viability and the morphological characteristics, such as circularity and diameter. the assays have been conducted on mussel groups maintained in aquaria at 18°c, then placed for different times at 40°c and finally put back to the original conditions. the heat stress determined a similar increase in the hc in the different groups whereas the recovery to the homeostasis condition showed some differences: the specimens more exposed to the heat stress, indeed, exhibited a slower ability to recovering their hc value to the control condition. furthermore, mussel specimens maintained at refrigeration conditions displayed a slow progressive increase in the hc. concerning the hsp 70 induction, the group exposed to the heat treatment for the shorter time developed a subsequent increase compared with the hc variations whereas. one day after the treatment, the hsp 70 induction was still evident. the cold treatment has demonstrated any hsp 70 induction. in conclusion, hemocytes circularity and mantle hsp 70 expression could represent two important stress parameters, morphological and biochemical, immediate and late, respectively. the markers investigated, therefore, could find application in the valuating the general status of the mussel health and, particularly, in studies concerning both the mollusc immunity and their response to environmental stress. effects of estrogens on mytilus hemocytes l canesi1, c ciacci1, lc lorusso1, m betti1, b marchi2, g gallo2 1istituto di scienze fisiologiche, università di urbino, italy 2dipartimento di biologia sperimentale ambientale ed applicata-dibisaa ,università di genova, italy estrogens affect the functioning of several nonreproductive tissues, the immune system in particular. in mammalian immunocytes, 17β-estradiol (e2) has both doseand celltype specific effects that seem to be mediated by rapid, non genomic mechanisms; these may be initiated at either membrane or cytosolic locations, and can result in both direct local effects, such as modification of ion fluxes, and regulation of gene transcription secondary to activation of different kinase cascades, including mitogen activated protein kinases (mapks). in this work we investigated the possible rapid effects and mechanisms of action of estrogens in the immunocytes of the bivalve mollusc, the mussel mytilus galloprovincialis lam. data are reported show that the natural estrogen e2 induced both morphological (as evaluated by sem) and functional changes (such as extracellular release of hydrolytic enzymes, lysosomal membrane destabilisation, stimulation of the bactericidal activity) within 10-30 min from addition. e2 (25 nm) caused a rapid and significant increase in cytosolic [ca2+]; lower concentrations (5 nm) showed a smaller, not significant effect. both e2 concentrations increased the phosphorylation state of the components of tyrosine kinase-mediated signal transduction mapkand stat(signal transducers and activators of transcription) -like proteins within 5-15 min from e2 addition. experiments with specific kinase inhibitors confirmed that rapid kinase cascades, in particular those leading to p38 mapk activation, are involved in mediating the effects of e2. western blotting revealed the presence of immunoreactive erαand erβlike proteins in hemocyte extracts. overall, our data support the hypothesis that the rapid effects and mechanisms of action of 17β-estradiol are extremely conserved and that they may play a crucial role in endocrine-immune interactions in invertebrates. the results are compared to those obtained with synthetic estrogens and estrogenic chemicals. the results address to the importance of the role of kinasemediated pathways in the action of endocrine disrupters in invertebrate systems. gene identification and expression profiling in mytilus galloprovincialis: a new tool for basic and applied research p venier1, c de pittà1, f marsano2, a pallavicini3, a viarengo2, c romualdi1, g lanfranchi1 1 department of biology, university of padua, italy 2department of science and advanced technology, university of piemonte orientale, 15100 alessandria, italy 3department of biology, university of trieste, italy mussels of the genus mytilus constitute an interesting model of study from different points of view. however, the number of available mussel sequences is small and functional studies refer to a few genes only. messenger rna was purified from multiple mussel tissues and a 3’-end-specific primary library was prepared in plasmid vector. massive sequencing of the cdna inserts (3’ fragments of transcribed genes or expressed sequence tags) allowed us to build a collection of 5664 3’ests. such experimental approach allows rapid and large-scale identification of protein coding genes and can provide information on the relative abundance of the most common mrnas. 53 putative identification of the consensus sequences has been published in part (gene, 2003) whereas the complete set of 1731 consensus sequences deriving from the clustering of rigorously selected ests is under refined annotation. in addition, one representative est for each cluster have been selected and the related cdna clones used to print on slide such genome-wide collection. preliminary analysis of gene expression in selected tissues of normal and stressed mussels will be discussed. the obtained results indicate the great potential of the mussel gene catalogue to increase in size and to become an essential tool for answering specific biological questions. commercial transport stress in homarus americanus (crustacea: decapoda) investigated by hematological parameters s lorenzon1, m bierti1, m martinis1, d mazzoni2, t scovacricchi3, ea ferrero1 1dipartimento di biologia, università di trieste, trieste, italy 2ecoblu scarl, ravenna, italy 3cnr,-ismar,castello1364/a, venezia, italy markets for live crustaceans are global and may require shipments over long distance and time, exposing animals to high stressful conditions. impact of transport condition, emersion and handling were studied on live, commercial homarus americanus imported from north america at the arrival and during the stocking period (3h and 12h) at warehouse. lobsters maintained in laboratory tanks (in controlled conditions) were used as control and for simulated emersion and handling stress. hemolymph sampling was followed by total and differential hemocyte counts (thc and dhc, as immunological indicator of contamination and impaired defence responses) on fresh as well as fixed material, whereas the physiological state of lobsters was studied in vivo by measuring ph, glucose (as general stress indicator) and lactate (as indicator of tissue hypoxia) concentration in the hemolymph. moreover the physiological parameters were related to the vigour index and physical damage of the animals. at the arrival animals show alterated parameters with a low number of circulating haemocytes (compared with the control) and that may cause a loss in immunocompetence; a highly disturbed metabolic state is revealed by elevated concentrations of lactate and glucose profile and alteration of the hemolymph ph. undamaged animals start to recover 12 h after reimmersion and cell counts return to control values within 48 h. host-parasite relationship: toward a role for entomoparasitic nematodes body surface mf brivio, m moro, m mastore dept. of structural and functional biology, university of insubria, varese, italy recent advances in understanding how insects eliminate pathogens and parasites have led to the realization that innate immunity plays a vital role in protecting from infection. this work examines some feature of humoral and cellular defenses involved in innate immune responses, how they act to control parasites and if their engagement or counteracting can explain many immune features characteristic of parasitic infections. aim of this work is to examine the interaction between the humoral and cellular defences system of an insect host model (galleria mellonella) and a pathogenic parasitic nematode (steinernema feltiae). particularly we are studying the involvement of the parasite body surface in two main strategies leading to immunoevasion/immunodepression mechanisms: molecular disguise and host proteins sequestering. our data suggested that the parasite body surface plays a role in the inhibition of host propo system, interfering with the activation of the proteases cascade involved in prophenoloxidase activity; moreover parasites were not recognized by host immunocompetent cells. finally, as confirmed by our preliminary data, steinernema seems to be able to interfere also with the host antibacterial peptides synthesis. immunomodulation and apoptotic events in the clam tapes philippinarum after exposure to 4nonylphenol v matozzo, l ballarin, mg marin dipartimento di biologia, università di padova, padova, italy nonylphenol (np) is used in the production of nonylphenol ethoxylates (npes), np phosphites and insecticide sprays. npes are a large group of nonionic surfactants employed in plastics, latex paints, lubricating oils, emulsifiers, household and industrial detergents and paper and textile industries, whereas np phosphites are commonly used as stabilisers and antioxidant agents in both rubber and plastic industries. np is known to be an endocrine disruptor, being able to alter hormonal functions in various aquatic organisms. in this study, specimens of the clam tapes philippinarum were exposed for 7 days to various sublethal np concentrations (0, 0+acetone, 0.025, 0.05, 0.1 and 0.2 mg/l) and their effects on the uptake of the vital dye neutral red (as index of cell membrane alteration), superoxide dismutase (sod) and lysozyme activities, the number and volume of haemocytes were evaluated. the capability of np to induce apoptotic events in haemocytes was also studied. exposure of clams to 0.2 mg/l np increased significantly (p<0.05) neutral red uptake when compared with controls, suggesting that np may cause alterations in cell membrane stability. significant (p<0.01) decreases with respect to controls in both sod and lysozyme activities were observed at concentration higher than 0.05 mg /l np. a significant (p<0.001) increase in apoptotic index (i.e., the percentage of haemocytes showing positivity to the tunel reaction) was also recorded in the same 54 concentration range. apoptotic haemocytes generally showed a shrinkage of cell volume and a spherical shape. alterations in both circulating haemocyte number and volume, consequent to np exposure, were recorded using a coulter counter. in this case, we observed both significant increases (p<0.05, for clams exposed to 0.2 mg /l np) in total number of circulating haemocytes and different size frequency distribution. our results, showing a relationship between np exposure and alterations in functional responses of haemocytes, demonstrated that np can influence immune responses in t. philippinarum. immunotoxicity of new antifouling compounds, alternative to tbt, on tunicate haemocytes f cima, a menin, p burighel, l ballarin dipartimento di biologia, università di padova, padova, italy xenobiotics, which cause severe alterations of the immune responses, can provoke the death of individuals and the local disappearance of the involved species. organotin compounds were massively introduced since the second half of 1960s in the formulation of antifouling paints for the preservation of submerged structures from the settlement of various aquatic sessile organisms. they resulted highly immunotoxic to benthic marine invertebrates, in particular filter-feeding ones, and most of them persist for a long time in the environment. after their ban, industries turned their attention to new biocidal combinations. about twenty new substances are at present in commerce in italy in various paint formulations, in which the biocidal compounds play various roles, i.e. as alternative substances to tbt or as boosters to increase the toxic performance of the main biocides towards a wider spectrum of fouling organisms. we carried out assays of acute toxicity on shortterm cultures of haemocytes of the colonial ascidian botryllus schlosseri to evaluate the alterations of the immune responses, as described by a series of biomarkers, by sublethal concentrations of seven active ingredients employed in the formulation of new antifouling paints. results indicate severe and irreversible effects on immunocyte morphology and functionality with mechanism of action sometimes similar to that of tbt, i.e. induction of apoptosis, cytoskeletal protein disassembly, inhibition of both phagocytic and cytotoxic ability, negative interactions with mitochondrial oxidative phosphorylation, cytosolic ca2+ homeostasis and gsh content. the comparison of our results suggests the following order of immunotoxicity: tbt ~ cu(i) ~ znp > sea-nine 211 ~ chlorothalonil > tcms pyridine > diuron > irgarol 1051. owing to the immunosuppressive effects of these compounds on tunicates, which make organisms more vulnerable to both pathogenic agents and other xenobiotics, we remark that more assays of acute and chronic toxicity should be necessary before leading new potentially pollutants into the market, in order to prevent the repetition of the irreversible errors on coastal biocoenoses already occurred with tbt. [this work was supported by grants of co.ri.la.] session 3. inflammation inflammation in ascidians n parrinello(1), a vizzini(1), c chinnici(1,2), m vazzana(1), v arizza(1), r de santis(3), r pinto(3), m cammarata(1) 1laboratorio di immunobiologia marina,dipartimento di biologia animale, università di palermo, italy 2department of pathology and laboratory medicine, university of pennsylvania, philadelphia, usa 3stazione zoologica a. dohrn, napoli, italy inflammatory responses in solitary ascidians include cell migration, phagocytosis, encapsulation of larger particles, tissue injury, and wound repair. in encapsulation responses in the tunic of ciona intestinalis, an increased expression of type iv-like collagen and elastin-like molecules have been found, apparently produced by the epidermis. inflammatory cells have been identified as amoebocytes, univacuolar cells, unigranular refringent cells (urg) and morula cells. we show the involvement of a large amount of urgs following lps injections. these cells contain polyphenols and, in vitro, showed a phenoloxidase-dependent cytotoxic activity. probably, urgs migrate through the epithelium from tissue lining the lacunae under the tunic. chemotactic stimuli, that induce migration into the inflamed area, could be due to a c3-like molecule while an immunohistochemical study shows that molecules containing interleukin-1-like epitopes are expressed (2-4 hours) by endothelial tissue lining the pharyngeal wall. an il-1-like functional activity may be indicated by the increased number in the lacunae as a result of the cell proliferation response. accordingly, we found il-1-receptor epitopes in cell nodules of the pharyngeal bars ansae. the recently elucidated genome of ciona intestinalis did not reveal il-1-like genes whereas an il-1-receptor was found. however, human il-1 traits can be observed by examining the ciona genome sequence. finally, the expression of a phenoloxidase component could be stimulated by inflammatory stimuli. inflammation in ascidians presents invertebrate and vertebrate characteristics. [supported by grants miur ex 60% and cori to np] the ascidian ciona intestinalis as experimental model for the study of the complement system inflammatory pathway in deuterostome invertebrates mr pinto*, cm chinnici†, y kimura‡, d melillo*, r marino*, la spruce‡, r de santis*, n parrinello†, jd lambris‡ *laboratory of cell biology, stazione zoologica “a. dohrn”, napoli, italy †department of animal biology, university of palermo, palermo, italy ‡protein chemistry laboratory, department of pathology and laboratory medicine, university of pennsylvania, philadelphia, usa one of the most challenging problems in immunology is the identification of the molecules that regulate the defence systems and the cross-talk 55 between innate and adaptive immune systems. a key approach to these issues is the study of how immunity is exploited by and evolved in invertebrates. many molecules belonging to the arms of the innate immune system have been found scattered in invertebrates at different levels of the phylogenesis. a major breakthrough has been the identification in the echinoderms, in the urochordates and in the cephalochordates of gene homologs of c3, a multifunctional protein that plays a central role in the complement system. however, neither the functions nor involvement of c3 in the inflammation processes have been proved in deuterostome invertebrates. recently, we have isolated two c3-like genes (cic3-1 and cic3-2) from ciona intestinalis blood cell total rna and shown that these genes are constitutively expressed in only one type of blood cells. a major contribution to the otherwise scanty panorama of the mechanisms of inflammation in invertebrates, has been provided by our group with the finding that cic3-1a, the anaphylatoxin peptide generated by the activation of the complement system, exerts a chemotactic activity on c. intestinails blood cells. to investigate the presence of complement-mediated chemotaxis in the ascidian c. intestinails, as a means of verifying the presence of the inflammatory pathway of the complement system in the deuterostome invertebrates, we have expressed in e. coli the fragment of c. intestinails c3-1 (rcic31a) corresponding to mammalian complement c3a and assessed its chemotactic activity on c. intestinalis hemocytes. we found that the migration of c. intestinalis hemocytes toward rcic3-1a was dosedependent, peaking at 500 nm, and was specific for cic3-1a, being inhibited by an anti-rcic3-1a-specific antibody. as it is true for mammalian c3a, the chemotactic activity of c. intestinalis c3-1a was localized to the c-terminus, since a peptide representing the 18 c-terminal amino acids (cic31a59-76) also promoted hemocyte chemotaxis. furthermore, the cic3-1a terminal arg was not crucial for chemotactic activity, since the desarg peptide (cic3-1a59-75) retained most of the directional hemocyte migration activity. the cic3-1a-mediated chemotaxis was inhibited by pre-treatment of cells with pertussis toxin, suggesting that the receptor molecule mediating the chemotactic effect is g protein-coupled. immunohistochemical analysis with anti-rcic3-1a-specific antibody and in situ hybridization experiments with a riboprobe corresponding to the 3'-terminal sequence of cic3-1, performed on tunic sections of lps-injected animals, showed that a majority of the infiltrating labelled hemocytes were granular amoebocytes and compartment cells. our findings indicate that cic3-1a mediates chemotaxis of c. intestinalis hemocytes, thus suggesting an important role for this molecule in inflammatory processes. eosinophilic granulocytes of sparids: cytochemical characterization and possible role in inflammatory response b contessi, d volpatti, p beraldo, m galeotti dipartimento di scienze della produzione animale, facoltà di medicina veterinaria, università di udine, italy in mammals eosinophilic granulocytes take part in defence against parasites and hypersensitivity reactions whereas the neutrophilic granulocytes are specialised into phagocytosis and killing of microorganisms. basophils contain vasoactive substances and in tissues are known as mast cells. in teleosts three types of granulocytes, neutrophilic, acidophils and basophils have been reported (hine et al., 1992). however, there are enormous variations amoung the teleosts in both relative abundance and staining reaction of granulocytes. most of the information on the teleosts eosinophils refers to cells found in association with different tissues, mainly in headkidney. eosinophils are the most abundant circulating granulocytes in gilthead seabream, as occurs in cyrprinus carpio, tinca tinca, and salmo gairdneri (lopez-ruiz et al, 1992). eosinophilic granular cells (ecgs) are a peculiar kind of granulocytes observed in the blood and in many tissues (gills, heart, liver, skin, spleen and kidney) of a variety of fish; in the gut ecgs are located in stratum granulosum and lamina propria. some cytochemical and functional characteristic suggested that they are analogous to mammalian mast cells, but other authors have considered them to be akin to eosinophils (sire & vernier, 1995; reite, 1998). sparids affected by "winter syndrome" showed a severe infiltration of granulocytes involving gastrointestinal tract, pancreas and adipose tissue surrounding these organs. also in gilthead seabream (s. aurata) affected by nephrocalcinosis is evident a considerable presence of these cells in the kidney. cytochemical analysis of this infiltrate by means of specific and enzymatic staining, and immunocytochemistry for lysozyme, showed that the granules of these cells are acidophilic. they contain peroxidase and acid phosphatase proteins and reveal lysozyme positivity only in few cells. literature refers that ecgs of gilthead seabream are peroxidase and acid phosphatase negative (noya et al., 1996), whereas in atlantic salmon (salmo salar l.), the granules of ecgs show strong immunoreactivity for lysozyme. on the basis of morphological, histochemical and enzymatic properties of these cells, we conclude that they could enter into tissue from circulating blood, in loco proliferate and act as mature cells. so probably they represent an evolutive stage of eosinophilic granulocytes. the proliferative mechanisms and their role in inflammatory response is under investigation. a glucocorticoid receptor identified in cells of sea bass innate immunity a vizzini, m vazzana, m cammarata, n parrinello dipartimento di biologia animale, università di palermo, palermo, italy in fish, cortisol is the major corticosteroid produced by the interrenal glands that acts as a component of the neuroendocrine circuit known as the hypothalamo-pituitary-interrenal (hpi) axis. 56 in dicentrarchus labrax confinement experiments increase in plasma cortisol levels, has been related to inhibition of cytotoxic activities by eosinophilic granule cells (egcs), were observed. in in vitro experiments, using head kidney and peritoneal cavity cells an increased zymosan-induced respiratory burst activity after 1 and 24 hours of incubations with hydrocortisone was increased at 10-100 ng/ml whereas it was reduced after cell incubation with 1000 ng/ml cortisol. immunohistochemical studies revealed that exogen cortisol can be found in the leukocytes. we cloned and sequenced a glucocorticoid receptor (gr) from leukocytes (dlgr1). that present homologies with fish, xenopus, and human gr. in particular, 80% homology between the sea bass and hapochromis burtoni gr1 receptor was found. in situ hybridization study demonstrated that mrna dlgr1 was expressed in head kidney and peritoneal cavity macrophages and neutrophils. gr expression was never observed in eosinophilic granule cells. present results support a direct effect of cortisol on phagocytic leukocytes containing dlgr1 whereas suggest that an alternative modulation mechanism could be invoked to explain the suppressed peritoneal cytotoxic activity of eosinophils. research is in progress to isolate and to sequence the isoforms of dlgr1. identification, characterization and “phylogenomic” analysis of toll-like receptor genes in the gilthead seabream (sparus aurata l.) r franch1, j antonello1, b cardazzo1, t patarnello1,2, l bargelloni2 1dipartimento di biologia, università di padova, padova, italy 2dipartimento di sanità pubblica, patologia comparata e igiene veterinaria, università di padova, padova, italy toll-like receptor (tlr) family consists of at least ten proteins encoded by distinct genes in man and mouse. each tlr protein recognizes in a specific manner one or more pathogen-associated molecular patterns typical of different group of pathogens and activates the immune system. the aim of the present work was to study tlr proteins in a marine teleost fish, s. aurata. all available tlr sequences from the human, mouse and pufferfish genome were aligned and gene-specific primers were designed on conserved regions and used in a rt-pcr approach, obtaining partial cdna sequences of seabream tlr 2 and tlr 9. subsequently, the entire sequence for both proteins was obtained using the 5’ and 3’ race method. expression analysis performed from different adult tissues and from whole larvae at different stages of development provided evidence of broad spatial and temporal distribution of tlr 2 gene, and of the presence of at least two splice variants of the tlr 9 gene, which appear to be differentially regulated. extensive analysis of sequence similarity and data mining were carried out to obtain all the available tlr proteins in the teleost fishes. all teleost tlrs, including the two seabream genes, were aligned with all human and murine tlrs, and a gene genealogy was reconstructed. phylogenetic analyses of tlr evolution suggested that in the common ancestor of all gnathostomes were present at least ten tlr genes. successively, lineage-specific deletions and/or duplications contributed to determine the present gene configuration, eight orthologues and two mammalian specific tlrs in mouse and man, and eight orthologues and four/five teleost specific tlrs in pufferfish and zebrafish, respectively, as confirmed from analysis of completly sequenced genomes. thymus response to algal yessotoxin a franchini, e marchesini, e ottaviani department of animal biology, university of modena and reggio emilia, modena, italy mice thymus responses to algal yessotoxin (ytx) were examined by histochemical and immunocytochemical procedures. immunoreactivity for different mw cytokeratins (ck) and for cytokines (il1α, il-6, il-8) was analyzed. modifications of parameters such as cell proliferation and cell death were also studied. thymus from male swiss cd1 mice intraperitoneally injected with lethal (420 µg/kg) and not-lethal (10 µg/kg) doses of ytx were examined after 2 and 24 hours, respectively. histological studies revealed morphological modifications with both ytx doses. lethal treatment provoked changes in the cortex region that appeared less compact with light areas containing a reduced number of thymocytes and large pale epithelial cells. an increased number of mitotic as well as apoptotic phenotypes was also observed. more severe damages were observed with the lower ytx dose and after 24 h of treatment. indeed, an increased number of apoptotic cells was observed mainly in cortico-medullary junction and in medulla. groups of flattened medullary epithelial cells formed single or clustered round structures that resembled hassall’s corpuscles and contained heterogeneous secretory material and necrotic nuclei. the medullary epithelial cells were the most affected cell population. these cells were arranged in a regular reticulum of stellate cells immunoreactive (ir) to high mw cks, whereas after ytx treatment some cells decreased their ir and some others withdrew cytoplasmic projections modifying to strongly ir round cells. the core of the newly formed medullary structures and true hassall’s corpuscles was also strongly ir to higher mw cks. with regard to cytokine response, changes were observed in both experimental treatments in comparison to controls. an higher number of cells ir to il-6 located at the corticomedullary junction and medulla was found, and they were mostly dendritic cells, while il-1 a and il-8 ir cells, observed in the cortex, decreased. the present findings, in disagreement with others reporting little or no toxic effects, indicate that ytx provokes severe morphofunctional damage to thymic microenvironment. procrh in the catfish ameiurus nebulosus: gene structure and tissue-specific responsiveness to lps 57 d malagoli, e ottaviani university of modena and reggio emilia, department of animal biology, via campi 213/d, 41100 modena, italy the procorticotrophin-releasing hormone (procrh) gene from the teleost ameiurus nebulosus was cloned by direct and inverse pcr-based technologies and characterized. sequence similarity with the procrh coding sequences in oreochromis mossambicus (tilapia) and homo sapiens is 97.7 % and 78 %, respectively. constitutive expression of the gene was assessed by rt-pcr approach. western blot experiments performed with an anti-human crh (1-41) polyclonal antibody revealed the presence of an immunoreactive molecule with an approximate mw of 18 kda, value comparable to that of the putative catfish procrh peptide. western blot and immunocytochemical experiments showed modification in procrh immunoreactivity in the central nervous system (cns), in the head kidney and in the pancreatic gland after catfish exposure for 15 and 120 min to lps. an increase in procrh immunoreactivity in the cns after 15 min but not after 120 min exposure to lps was observed, while the increased immunopositivity is detectable throughout the entire period of exposure in the head kidney and only after 120 min of treatment in pancreatic cells. our findings indicate that cns responds to the altered conditions for a shorter time with respect to the peripheral organs suggesting a hierarchical and time-regulated response to a persistent stressor. stem cell maintenance and committment: the planarian model l rossi1, r batistoni2, a salvetti1, a lena1, p deri2, g rainaldi3, t locci4, m evangelista3, v gremigni1 1dipartimento di morfologia umana, sezione biologia e genetica, università di pisa, italy 2dipartimento di fisiologia e biochimica, laboratorio di biologia cellulare e dello sviluppo, università di pisa, italy 3laboratorio di terapia genica e molecolare, istituto di fisiologia clinica, cnr, pisa, italy 4dipartimento di patologia sperimentale, biotecnologie mediche, infettivologia e epidemiologia, università di pisa, italy stem cells are defined by a unique capacity of self-renewal and broad differentiation plasticity. although the knowledge of the fundamental properties of these cells is the focus of an expanding field of the scientific research, almost nothing is known about the molecular nature of the regulatory mechanisms that determine whether a daughter of a stem cell remains a stem cell or commits to differentiation. the cellular mechanisms that govern these stem cell fate decisions have been explored in a variety of models, including invertebrates. planarian flatworms, well known for their exceptional regenerative capability, maintain a stable population of totipotent stem cells (neoblasts) throughout their life. neoblasts are involved not only in regeneration, but also ensure the physiological turnover of all cell types. pumilio and piwi have recently emerged as regulatory factors involved in asymmetric stem cell division at the posttranscriptional level in drosophila. in order to define the regulatory pathways involved in the rapid events of neoblast fate decisions, we have isolated the planarian homologs of pumilio and piwi genes of drosophila. pumilio and piwi expression pattern is partially overlapping in planarians. in fact both genes are expressed in the same sub-population of neoblasts, with a different antero-posterior gradient. pumilio mrna is also present in the central nervous system. knock-down experiments, performed by the rnai technique, suggest that both these genes are involved in neoblast maintenance. indeed, planarians injected with dsrna pumilio and dsrna piwi were unable to regenerate. in addition, analyses performed by facs, in situ hybridization, confocal microscopy as well as tem observations, revealed a dramatic reduction of the neoblast number in the treated animals, indicating that these genes are fundamental for their maintenance. ultrastructure of the hemocytes of astacus leptodactylus (eschscholtz, 1823): in vivo phagocytosis assays m bierti, pg giulianini, s lorenzon, ea ferrero, s battistella dipartimento di biologia, università di trieste, trieste, italy in the context of comparative studies of immunity cellular defence mechanisms of different taxa of arthropods, the ultrastructure and the in vivo phagocytosis of the circulating hemocytes of adults of the crustacean decapod astacus leptodactylus has been investigated by means of light and transmission electron microscopy (tem). four types of hemocytes were found in the hemolymph and they were identified as: large-granule cells, medium-granule cells, smallgranule cells, and hyaline cells. in order to identify the phagocyte cell, phagocytosis assays were performed in vivo by injection of: 1) sterile phospate buffered saline, ph 7.4; 2) 0.9 µm carboxylate-modified polystyrene latex beads; 3) 1.1 and 3 µm unmodified latex beads; 4) pseudomonas fluorescens. 200 µl hemolymph samples (about 1.08x104 hemocytes) were drawn at 0-0.5-1-2-4 hrs from the dorsal vessel of each animal; the hemocytes were pelleted by 14.000 rpm centrifugation, fixed, post-fixed and embedded in epoxy resin for lm and tem sectioning. differential cell counts were made from 2 µm semithin transverse sections of the full pellet thickness stained with toluidine blue. the in vivo treatments induce changes in relative percentages of hemocyte types. the decrement of hyaline cells compared to the control was concomitant with the increase of the largegranule cells percentage in all treatments. the smallgranule cells of a. leptodactylus were the main hemocyte type involved in phagocytic response reaching a maximum of 3.6% of the total hemocytes. the percentages of small-granule cells decreased in all treatments suggesting their lysis after the phagocytic activity. the 3 µm latex beads were the only particle not phagocyted at 30 min, advocating that the size of the ingested particle is important for the phagocyting mechanism. 58 first evidences of toll–like receptor on paracentrotus lividus coelomocytes v arizza, f giaramita, m cervello, m cammarata, n parrinello dipartimento di biologia animale, università di palermo, via archirafi, 18 90123 palermo, italy microbial pathogens use a variety of complex strategies to subvert host defences and to ensure their multiplication and survival. the innate immunity system detects and eliminates invading pathogenic microorganisms by discrimining between self and nonself. the host organism can determine the presence of non-self by recognizing a limited number of conserved structures only produced by pathogen organisms and not by multicellular hosts, called pamps (pathogen-associated molecular patterns). some of these molecules are lipopolysaccharides (lps), peptidoglicans, lipoteichoic acid, lipoarabinomannan acid, lipopeptides and bacterial dna. receptors that recognize this molecular pattern have an important role in connecting pathogen detection with innate immune-response. toll like receptors seem to be the main candidates to perform this function. in the echinoid paracentrotus lividus, we already characterized phagocytose and cytotoxic activities. until now, it is not clear the molecular mechanisms for activating these cell responses, and if an inducible system toll dependent, like to that found in drosophila, may be involved. to assess the presence of these receptors, polyclonal antibodies against human tlr4 (htlr iv), were used. immunoprecipitation and immuno-blotting experiments revealed epitopes that were identified by antyhtlr iv on protein preparation from coelomocyte membranes, and immunofluorescence reactions showed that they are distributed on coelomocytes. using a known dna sequence of strongilocentrotus purpuratus of tlr1.1 receptor, degenerated primers were designed. a 350 bp fragment was amplified by prc using a coelomocyte cdna, indicating the presence of a toll-like mrna. searches are in progress to clone the fragment and establish the complete sequence and the homologies with the known toll-like receptors sequences. allorecognition in botryllus schlosseri: ultrastructural study of fusion between genetically compatible colonies g zaniolo, a sabbadin, f caicci, l ballarin dipartimento di biologia, università di padova, italy when colonies of the ascidian botryllus schlosseri contact each other, they can either fuse and anastomise their circulatory systems if genetically compatible, or reject and produce a series of cytotoxic spots along the contact border in the case of incompatibility. although many studies have been devoted to the analysis of rejection, few data on fusion are available. in order to fill this gap, we started a preliminary study on the morphology of the various steps of the fusion reaction at both light and electron microscope. we were able to distinguish at least five different stages in the fusion process. in stage 1 the tunics of the facing colonies contact each other and epithelial cells of the ampullar tips appear cylindrical in shape with a cytoplasm rich in rer with enlarged cisternae containing homogeneous, finely dispersed material. in stage 2, the tunics are strictly juxtaposed and the cuticular papillae are tightly intermingled, but the two tunics are still distinguishable. in this stage, cells of the ampullar tips contain numerous membrane bound granules, with homogeneous electron-dense material, in the supranuclear (“pad”) region. stage 3 is marked by the dissolution of the two cuticles and the local fusion of the tunics in front of the facing ampullae. less granules are now present in the ampullar pad and some haemocytes leak out from the circulation through the ampullar tips. in stage 4, the pads of the two facing epithelial adhere and new junctional complexes are formed. basal lamina still delimitate the ampullar lumen and appear highly folded. in stage 5 the juxtaposed epithelia open, thus permitting the communication between the vessels of the two colonies. cell pads are progressively resorbed and cells of the ampullar tips, now lining a new vessel, return to a cubic shape. future studies will investigate the occurrence of apoptotic events in the process of ampullar fusion in b. schlosseri. cell cooperation among immunocytes of the compound ascidian botryllus schlosseri l ballarin, e chemello, a menin, f cima dipartimento di biologia, università di padova, italy two different immunocyte types are present in the blood of the colonial ascidian botryllus schlosseri: phagocytes and morula cells (mc). the latter are cytotoxic cells, involved in the inflammatory reaction which occurs when genetically incompatible colonies contact each other. upon the recognition of non-self molecules, mc release of the proenzyme prophenoloxidase, readily converted to active phenoloxidase which is directly responsible for the induction of cytotoxicity observed along the contacting colonial borders in the forms of a series of pigmented, necrotic spots. we have recently demonstrated that mc respond to the recognition of non-self molecules from the microbial surface through the synthesis of molecules recognised by antibodies raised against mammalian il-1-α and tnf-α.here, we demonstrate that mc synthesise the above-reported molecules as a consequence of the recognition of both bacteria and non-self factors from incompatible blood. the positivity is located in the cytosol. in addition, since lysates of mc, previously exposed to bacteria, have been claimed to increase phagocytosis in the solitary ascidian ciona intestinalis, we investigated whether anti-il-1-αand anti-tnf-α-antibodies and recombinant il-1-α and tnf-α can influence the activity of phagocytes. data obtained clearly show a 59 significant (p < 0.01) decrease of yeast phagocytosis in the presence (1 µg/ml) of anti-cytokine antibodies whereas, a significant (p<0.01) increase is reported when phagocytosis occurred in the presence (10 ng/ml) of the recombinant cytokines. the results support the hypothesis of a role of il-1-αand tnf-αimmunopositive molecules in communication among immunocytes. 53 isj 19: 53-68, 2022 issn 1824-307x research report host-pathogen interactions of the two native isolates of beauveria bassiana to a predatory coccinellid, cryptolaemous montrouzieri mulsant (coleoptera: coccinellidae) s aghaeepour1, a zibaee1*, s ramzi2, h hoda3 1department of plant protection, faculty of agricultural sciences, university of guilan, rasht, iran 2tea research center, horticulture science research institute, agricultural research, education and extension organization (areeo), lahijan, iran 3iranian research institute of plant protection, agricultural research, education and extension, amol, iran this is an open access article published under the cc by license accepted march 21, 2022 abstract fungi are among the most important microorganisms affecting population dynamics of insects. although they are used as biocontrol agents for several decades but their interactions to insect pests, predators and parasitoids are still interesting in case of virulence, host physiology and environmental persistence. understanding the possible synergistic or antagonistic interactions of entomopathogenic fungi with other biocontrol agents mainly predators is a critical factor to achieve a successful pest control program. in the current study, effects of the two native isolates of beauveria bassiana (am-118 and bb3) were studied on survival, cellular immunity and antioxidant system of cryptolaemous montrouzieri mulsant. bioassay results showed that both am-118 and bb3 caused significant mortality on the third instar larvae and the adults of c. montrouzieri. moreover, they increased total and differential hemocyte counts and significantly induced phenoloxidase activity and nodule formation at 48, 72 and 96 h post-treatment. a considerable increase was also observed in the activities of antioxidant enzymes at 72 and 96 h post-treatment. although the isolates caused mortality on both stages, induction of immune and antioxidant systems protect c. montrouzieri against infective conidia. key word: cryptolaemous montrouzieri; beauveria bassiana; virulence; immune response; antioxidant system introduction mealybugs are the economically important pests of citrus and tea orchards that cause direct and indirect losses in production of well-quality crops. the chemical sprays to combat mealybugs may have no satisfactory efficiency because of their structural and behavioral capabilities to neutralize pesticide toxicity. these compounds may also endanger beneficial organisms like predators and parasites in cropping systems due to direct mortality or reproductive deficiencies (rabindra and ramanujam, 2007). biological control procedures through pathogenic microorganisms and predators are much appreciable to control mealybugs in order to maintain sustainable production in orchards (ramanujam et al., 2017). the entomopathogenic fungus, beauveria bassiana (balsamo) vuillemin _________________________________________ corresponding author: arash zibaee department of plant protection faculty of agricultural sciences box 41635-1314, rasht, iran e-mail: arash.zibaee@gmx.com; arash.zibaee@guilan.ac.ir (ascomycota: hypocreales) and the predatory coccinellide, cryptolaemus montrouzieri mulsant (coleoptera: coccinellidae) may be used simultaneously because of varying degrees of success in managing the mealybugs of citrus and tea orchards (mani and krishnamoorthy, 2008; maqsoudi et al., 2018). c. montrouzieri is an important natural enemy in integrated pest management programs of orchards because it utilizes different life stages of mealybugs including pseudococcus viburni (signoret), maconellicoccus hirsutus green, planococcus citri (risso), ferrisia virgata outbreaks and phenacoccus solenopsis tinsley (thungrabeab and tongma, 2007; jiang et al., 2009; ibrahim et al., 2011; scorsetti et al., 2012). moreover, b. bassiana is a successful entomopathogenic fungus naturally exists in soils and even lives as endophytes. it is extensively used as a mycoinsecticide to control different insect pests of lepidopterans, coleopterans, and dipterans (toledo et al., 2014; maistrou et al., 2018; shahriari et al., 2021a). b. bassiana has several positive characteristcs containing high potential mortality on target pest population, high genetic variation of the 54 table 1 lc30 and lc50 concentrations of the two isolates of b. bassiana against c. montrouzieri after 14 days of exposure life stage isolates lc30 (cl 95 %) conidia/ml lc50 (cl 95 %) conidia/ml x2 (df) slope±se 3rd larvae am-118 4×104 (4.3×103 1.5×105) 6.4×105 (1.8×105 2×106) 0.619 (3) 0.438 ± 0.082 bb3 2×104 (2.3 ×103 7×104) 2.2×105 (6.2×104 6.3×105) 0.527 (3) 0.497 ± 0.087 adults am-118 1.3×105 (2.3×104 4.4×105) 2.6×106 (6.6×105 7.3×106) 0.262 (3) 0.450 ± 0.082 bb3 8.1×104 (1.1×104 2.7×105) 1.2×106 (3.7×105 4×106) 0.438 (3) 0.446 ± 0.082 note: calculations were carried out by polo-plus software isolates, infection of different developmental stages of host insect, vertical and horizontal dispersal capacity depending on the host environment (wraight et al., 2010; jaronski, 2014). insects have adapted themselves to colonize different habitats through developing a range of defense mechanisms. immune system of insects inflicts invading pathogens by cellular and humoral responses. upon distinguishing the pathogen entrance, cellular immune functions are triggered by hemocyte proliferation, micro-aggregation, nodulation and encapsulation followed by melanin deposition (lavine and strand, 2002). additionally, a series of humoral responses including antimicrobial peptides, phenoloxidase cascades and generation of reactive oxygen species are simultaneously activated to fully disable pathogen dispersal and proliferation in body (tsakas and marmaras, 2010; liu et al., 2013). antioxidant enzymes are the other defense responses that act non-specifically against invading pathogens. these enzymes control concentration of reactive oxygen species (ros) in insects (karthi et al., 2018). in details, ros including, hydroperoxides (rooh), superoxide radicals (o2−), hydrogen peroxidase (h2o2) and hydroxyl radical (oh−) are produced under exposure to pathogens damaging cell structure (dubovskiy et al., 2008). oxidative stress finally induces lipid peroxidation that disturbs cell membrane liquidity, dna damage and apoptosis (monaghan et al., 2009). hence, regulating the content of ross is necessary to decrease their harmful effects and to modulate proper cell signaling pathways toward their concentrations (shamakhi et al., 2020). insect antioxidant system includes enzymatic and non-enzymatic components such as catalase (cat), superoxide dismutase (sod), peroxidase (pox), ascorbate peroxidase (apx), glucose-6-phosphate dehydrogenase (gpdh), glutathione peroxidase, thiols, ascorbic acid and α-tocopherol (dubovskiy et al., 2008; shamakhi et al., 2020). table 2 lt50 concentrations (days) of the two isolates of b. bassiana against c. montrouzieri after 14 days of exposure life stage isolates lt50 (cl 95 %) days x2 (df) slope±se 3rd larvae am-118 8.12 (7.18 – 9.03) 3.237 (4) 5.433 ± 0.693 bb3 8.23 (6.44 – 9.89) 6.414 (4) 5.664 ± 0.720 adults am-118 9.90 (7.83 – 12.12) 6.280 (4) 4.845 ± 0.646 bb3 9.72 (6.78 – 12.83) 11.569 (4) 5.264 ± 0.686 note: calculations were carried out by polo-plus software 55 fig. 1 changes of the total hemocytes counts in c. montrouzieri infected by 105 conidium/ml of the two isolates of b. bassiana (am-118 and bb3), larvae (a), adults (b). different letters indicate significance at p < 0.05 in each time interval integrative use of entomopathogenic fungi and predators is essential to increment the role of biological agents to alleviate population outbreaks of pests with the least influence to the ecosystem (wu et al., 2018). on the other hand, the successful use of entomopathogenic fungi in integrated pest management requires not only high virulence against insect pests, but also possible low or selective virulence against natural enemies (portilla et al., 2017). synergetic interactions between entomopathogenic fungi and insect predators may increase control efficacy, while antagonistic interactions lead to significant deficiency in pest control and impose financial and environmental costs (roy et al., 1998). our previous study revealed appropriate virulence of the two native isolates of b. bassiana against tea mealybug, pseudococcus viburni signoret (hemiptera: pseudococcidae) (maqsoudi et al., 2017). in case, it is important to investigate the effects of entomopathogenic fungi on c. montrouzieri to increase efficiency of mealybug control program. this would lead to implement a safe and sustainable pest control scheme. therefore, the present study was done to increase our knowledge on side-effects of b. bassiana against c. montrouzieri from virulence and physiological aspects. briefly, the third instar larvae and the adults of c. montrouzieri were treated with am-118 and bb3 isolates of b. bassiana, then the sub-lethal concentration of the isolates was treated to determine immune and antioxidant responses. 56 materials and methods rearing of prey and predator cryptolaemous montrouzieri were provided from the iranian research institute of plant protection, agricultural research, education and extension, amol, iran and reared at the laboratory conditions of 25 ± 2 °c, 70 ± 5 % rh, and 16l:8d photoperiod, in the glass jars (15 × 20 cm), on potato sprouts infested by pseudococcus viburni. the mealybugs population built-up on newly infested potato sprouts for two weeks and then transferred to beetle-rearing glass jars twice a week. both larvae and adults were fed on the mealybugs but they kept at separate containers to avoid cannibalism (aghdam and malekshah, 2019). fungal isolates and culture the isolates of beauveria bassiana (am-118 and bb3) were cultured at 25 ± 2 °c on potato dextrose agar (pda) supplied with yeast extract (1 %). the cultures were kept for three weeks, and a hemocytometer was used to determine conidia concentration for stock and intended suspensions (maqsoudi et al. 2018). bioassay dipping method was used to assay the effects of fungal isolates against the larvae and the adults c. montrouzieri. at first, a series of primary tests were done to estimate the concentration range causing 15 to 85 % mortality. then, five concentrations were prepared at logarithmic intervals of 104, 105, 106, 107, and 108 conidia/ml within 0.02 % solution of tween-80. the third instar larvae and adults were randomly dipped into the prepared concentrations of each isolate although the control insects were dipped in an aqueous solution of 0.02% tween-80 alone. three replicates including 10 individuals were considered for the experiment (n = 180). after two weeks, mortality was recorded and values of lc30 and lc50 were calculated by the probit analysis using polo-plus software. also, lt50 value was determined by exposing different groups of the larvae and the adults to 108 conidia/ml concentration of each isolate. mortality recorded for eight days to death of the last individual. cellular immunity insect treatment, hemolymph collection, and hemocyte counts the third instar larvae and the adults of c. montrouzieri were randomly dipped into 104 conidia/ml concentration of am-118 and bb3 isolates while the control insects were immersed in tween-80 (0.02 %) alone. the hemolymph of treated and control individuals was separately collected at intervals of 24, 48, 72 and 96 h by cutting the last thoracic leg with a microscissor. hemolymph was transferred into the ice-cold anticoagulant buffer (0.01m ethylenediaminetetraacetic acid, 0.1m glucose, 0.062 m nacl and 0.026 m citric acid, ph 4.6) in the ratio of 3:1. the total and differential hemocyte (granulocyte and plasmatocyte) counts as well as the number of nodules were specified using a neubauer hemocytometer (chemkind co. china). in each time intervals, sixty insects were used in six replicates. assay of phenoloxidase activity po activity was measured at all-time intervals for both control and fungi-treated specimens as described by leonard et al. (1985). the obtained hemolymph was added into the anticoagulant buffer and centrifuged at 10,000 × g for 8 min. the supernatant was discarded, and the pellet was washed with phosphate buffer for three times (0.02 m, ph 7.1). the current samples were homogenized in 100 μl of phosphate buffer after a night incubation in -20 °c and centrifuged at 12,000×g for 15 min. the final mixture for po assay contained 30 μl of dihydroxyphenylalanine (10 mm), 50 μl of phosphate buffer (ph 7.1), and 15 μl of the supernatant. the absorbance was recorded at 492 nm following 5 min of incubation at 30 °c. assay of antioxidant components sample preparation the third instar larvae and the adults of c. montrouzieri were exposed to 104 conidia/ml concentration of each isolate to evaluate fungal effects on enzymatic and non-enzymatic antioxidant components. after 24, 48, 72 and 96 h of posttreatment, total bodies of the insects were homogenized in distilled water, and centrifuged at 12000 g for 15 min at 4 °c. then, the supernatant was used in the biochemical experiments. superoxide dismutase (sod) activity of superoxide dismutase (sod) was measured by suppression of declined level of nbt (nitro blue tetrazolium) by the superoxide anion to be produced after xanthine oxidation (mccord and fridovich, 1969). enzyme solution (60 μl) was added into 500 μl of the reaction solution (125 μm of xanthine; 70 μm of nbt; both dissolved in pbs) and 20 μl of xanthine oxidase solution [10 mg of bovine albumin; 100 μl of xanthine oxidase (5.87 units/ml); dissolved in 2 ml of pbs]. the reaction mixture was maintained in darkness at 25 °с for 20 min. finally, the absorbance was recorded at 560 nm and reported as δa 560 nm/min/mg protein. catalase (cat) activity of cat was determined by the decomposition amount of hydrogen peroxide (h2o2) (wang et al., 2001). fifty microlitre of enzyme solution was added into 500 μl h2o2 in pbs (1 %) and kept at 25 °с for 10 min. activity of cat was recorded as the δa at 240 nm/min/mg protein. peroxidase (pod) as described by addy and goodman (1972), a reaction mixture was prepared containing buffered pyrogallol [80 μl, 50 mm pyrogallol in 100 mm phosphate buffer (ph 7.0)], solution of 1 % of h2o2 (80 μl) and enzyme solution (20 μl). alteration in absorbance was read every 30 seconds at 430 nm for 2 min and reported as δa 430 nm/min/mg protein. ascorbate peroxidase (apox) the apox activity was assayed based on the procedure of asada (1984). the reaction mixture was 57 fig. 2 changes of granulocyte counts in c. montrouzieri infected by 105 conidium/ml of the two isolates of b. bassiana (am-118 and bb3), larvae (a), adults (b). different letters indicate significance at p < 0.05 in each time interval 200 μl of h2o2 (30 mm), 50 μl of enzyme solution, 70 μl of ascorbic acid (2.5 mm) and 150 μl of phosphate buffer (100 mm, ph 7.0). the absorbance was continually recorded for 5 min at 290 nm and reported as δa at 290 nm/min/mg protein. glucose-6-phosphate dehydrogenase (gpdh) gpdh was assayed based on the method of balinsky and bernstein (1963) which 15 μl of mgcl2 (100 mm), 70 μl of tris-hcl (100 mm, ph 8) and 30 μl of nadp (0.2 mm) were poured together as the stock mixture. then, 70 μl of gpdh (6 mm), 20 μl of distilled water and 20 μl of the enzyme were mixed with stock mixture before reading the absorbance changes at 340 nm. activity was reported as δa at 340nm/min/mg protein. malondialdehyde content (mda) the amount of mda was assayed based on the method of bar-or et al. (2001). a solution containing 20 μl of the sample solution and 80 μl of trichloroacetic acid (20 %) was mixed and centrifuged at 15000 g for 10 min at 4 °c. afterward, supernatant was carefully added into 100 μl of 2thiobarbituric acid solution (tba, 0.8 %) and maintained for 60 min at 100 °c. absorbance of the samples was measured at 535 nm. the molar extinction coefficient was 1.56×105 m-1 cm-1 to determine mda concentration per mg protein. 58 fig. 3 changes of plasmatocyte counts in c. montrouzieri infected by 105 conidium/ml of the two isolates of b. bassiana (am-118 and bb3), larvae (a), adults (b). different letters indicate significance at p < 0.05 in each time interval total protein content the content of protein in the samples was determined by the lowry et al. (1951) procedure. the reaction contained 20 µl of the sample mixed with 100 µl of reagent. the incubation was done for 30 min prior to read the absorbance at 545 nm. statistical analysis probit analysis calculated lc30, lc50 and lt50 values at the corresponding 95 % confidence interval (ci) values using polo-plus software. a one-way analysis of variance (anova) followed by tukey’s test was used to compare biochemical and immunological data. the statistical differences were marked by different letters at a probability less than 5 %. results bioassay the effects of am-118 and bb3 against the third instar larvae and the adults of c. montrouzieri have been shown in tables 1 and 2. the lc30 and lc50 concentrations after larval treatment were 4×104 and 6.4×105 conidia/ml for am-118 as well as 2×104 and 2.2×105 conidia/ml for bb3, respectively (table 1). moreover, the lc30 and lc50 values on the adults were obtained 1.3×105 and 2.6×106 conidia/ml for am-118 as well as 8.1×104 and 1.2×106 conidia/ml for bb3, respectively (table 1). the lt50 values of am-118 and bb3 isolates were calculated 8.12 and 8.23 days on the larvae (table 2) as well as 9.90 and 9.72 days on the adults, respectively (table 2). 59 fig. 4 changes of nodule counts in c. montrouzieri infected by 1 × 105 conidium/ml of two isolates of b. bassiana (am-118 and bb3), larvae (a), adults (b). different letters indicate significance at p < 0.05 in each time interval cellular immunity the two native isolates of b. bassiana considerably increased the total and differential hemocyte counts, nodule formation and po activity in the third instar larvae and the adults of c. montrouzieri. the total hemocyte count in the larvae were significantly enhanced at 48, 72 and 96 h of posttreatment (f = 25.38, df = 4, p: 0.0001; f = 64.66, df = 4, p: 0.0001; f = 25.83, df = 4, p: 0.0001) while no significant difference were recorded at 24 h posttreatment (f = 2.34, df = 4, p: 0.139) (figure 1). the highest hemocyte counts were recorded after 72 and 96 h by treating bb3 conidia (figure 1). moreover, the number of total hemocytes in the adults significantly increased at 72 and 96 h post-treatment (f = 35.69, df = 4, p: 0.0001; f = 62.65, df = 4, p: 0.0001) although no significant different was observed between am-118 and bb3 isolates (figure 1). a considerable increase of granulocytes counts was observed following larval treatment after 48, 72 and 96 h (f = 26.34, df = 4, p: 0.0001; f = 52.98, df = 4, p: 0.0001; f = 18.39, df = 4, p: 0.0001) (figure 2). after 72 and 96 h, the numbers of granulocytes significantly elevated in the treated adults compared to control (f = 28.84, df = 4, p: 0.0001; f = 61.46, df = 4, p: 0.0001) (figure 2). after 48, 72 and 96 h of exposures, the number of plasmatocytes at all treated larvae significantly increased compared to control (f = 11.48, df = 4, p: 0.002; f = 66.15, df = 4, p: 0.0001; f = 11.78, df = 4, p: 0.001) but the number of plasmatocytes significantly increased after 72 and 96 h for the adults (f = 20.85, df = 4, p: 0.0001; f = 17.64, df = 4, p: 0.0001) (figure 3). the larval treatment by am-118 and bb3 significantly increased the number of nodules after 48, 72, and 96 h (f = 4.75, df = 2, p: 0.030; f = 16.60, df = 2, p: 0.0001; f = 91.60, df = 2, pr>f: 0.0001) although the adults showed the highest number of nodules for all time intervals (f = 4.0, df = 2, p: 0.047; f = 14.0, df = 2, p: 0.001) (figure 4). in both larvae and adults, the highest number of nodules was recorded after 96 h following fungal treatments (figure 4). 60 fig. 5 changes of phenoloxidase activity in c. montrouzieri infected by 105 conidium/ml of the two isolates of b. bassiana (am-118 and bb3), larvae (a), adults (b). different letters indicate significance at p < 0.05 in each time interval phenoloxidase activity the activity of po in the larvae treated by both isolates significantly increased at 72 and 96 h of post-treatment with the highest activity by bb3 treatment after 96 h (f = 54.74, df = 4, p: 0.0001; f = 215.21, df = 4, p: 0.0001) (figure 5). similarly, the treated adults by both isolates demonstrated the highest po activity after 96 h (f = 124.61, df = 4, p: 0.0001) (figure 5). antioxidant components sod activity in the larvae and the adults treated by am-118 and bb3 significantly induced after 72 and 96 h (f = 55.27, df = 4, p: 0.0001; f = 70.55, df = 4, p: 0.0001) while no significant difference was recorded among the isolates and the control after 24 and 48 h (f = 0.09, df = 4, p: 0.917; f = 0.42, df = 4, p: 0.664) (figure 6). similar results obtained for catalase activity (figure 7). after 72 and 96 h, a significant increase of catalase activity (f = 26.94, df = 4, p: 0.0001; f = 66.95, df = 4, p: 0.0001; f = 8.62, df = 4, p: 0.005; f = 23.84, df = 4, p: 0.0001) observed in the treated individuals (figure 7). moreover, the pod activity of the larvae (f = 49.43, df = 4, p: 0.0001; f = 39.39, df = 4, p: 0.0001) and the adults significantly increased after 72 and 96 h although the highest pod activity was recorded in the treated individuals by bb3 (f = 9.93, df = 4, p: 0.003; f = 25.43, df = 4, p: 0.0001) (figure 8). 61 fig. 6 changes of superoxide dismutase activity in c. montrouzieri infected by 105 conidium/ml of the two isolates of b. bassiana (am-118 and bb3), larvae (a), adults (b). different letters indicate significance at p < 0.05 in each time interval apod activity in the larvae treated by the isolates significantly increased after 96 h (f = 47.92, df = 4, p: 0.0001) (figure 9). similarly, the adults treated by both isolates showed the highest apod activity at 96 h post-treatment compared to control (f = 25.43, df = 4, p: 0.0001) (figure 9). gpdh demonstrated the highest activity in the treated larvae and adults compared to control after 96 h (f = 215.21, df = 4, p: 0.0001) (figure 10) but the statistically highest activity between isolates was recorded in the larvae and the treated by bb3 (f = 122.34, df = 4, p: 0.0001) (figure 10). the exposure of am-118 and bb3 led to the highest content of mda of the larvae and the adults after 72 and 96 h (f = 47.01, df = 4, p: 0.0001; f = 132.70, df = 4, p: 0.0001) (f = 70.74, df = 4, p: 0.0001; f = 53.53, df = 4, p: 0.0001), respectively (figure 11). discussion in the current study, both native isolates of b. bassiana demonstrated virulence on the larvae and the adults of c. montrouzieri, although the results highlighted the higher virulence bb3 than am-118 with lower lc50 and lt50 values. different virulence of the isolates may be attributed to the initial host and the origin of their collection. bb3 was collected from soil where it was associated with many organisms, while am-118 was cultured from body mycelium of rice stem stripped borer. therefore, bb3 may develop more mechanisms to cause pathogenicity on hosts while it seems am-18 may more specialized to narrow host specificity. moreover, both isolates were capable to kill the larvae with a lower conidia concentration and in a shorter time compared to the adults. less 62 fig. 7 changes of catalase activity in c. montrouzieri infected by 105 conidium/ml of the two isolates of b. bassiana (am-118 and bb3), larvae (a), adults (b). different letters indicate significance at p < 0.05 in each time interval susceptibility of the adults compared to larvae can be correlated with the hard covering of their cuticles and the slower penetration of conidia into the body. maqsoudi et al. (2018) reported that am-118 and bb3 were lethal on the tea mealybug, p. viburni, by the lc50 values of 1.8 × 105 and 2 × 103 conidia/ml, while the lt50 of the isolates were obtained to be 6.63 and 3.66 days, respectively. so it may be mentioned bb3 as the more virulent isolate on both tea mealy bug and c. montrouzieri. several studies have shown virulence of the numerous isolates of entomopathogenic fungi against coccinellide predators although the displayed differences in the virulence depend on the lethal concentrations and the time of exposure (thungrabeab and tongma, 2007; er et al., 2008; ibrahim et al., 2011; scorsetti et al., 2012; trizelia et al., 2017). insect immunity is a fundamental process to protect insects against entomopathogens (lavine and strand, 2002). previous investigations demonstrated that hemocytes have the major roles in immune responses of insects to entomopathogenic fungi (russo et al., 2001; zibaee et al., 2014; aghaee pour et al., 2021; shahriari et al., 2021b). cellular immune reactions depend on circulating hemocytes such as prohemocyte, granulocyte and plasmatocyte (zibaee et al., 2014). granulocytes and plasmatocytes are the two important hemocyte involved in immune reactions through phagocytosis, nodule formation, and encapsulation to ensnare and kill conidia of pathogens (borges et al., 2008). nodulation occurs rapidly after microbial infection by micro-aggregation of hemocytes and assembling of additional hemocytes (zibaee et al., 2014; aghaee pour et al., 2021; shahriari et al., 2021b). in the current study, the numbers of total hemocytes, plasmatocytes, granulocytes, and nodules increased in the larvae 63 fig. 8 changes of proxidase activity in c. montrouzieri infected by 105 conidium/ml of the two isolates of b. bassiana (am-118 and bb3), larvae (a), adults (b). different letters indicate significance at p<0.05 in each time interval and the adults of c. montrouzieri after fungal exposure and time intervals of 48, 72 and 96 h, while no significant difference was recorded between treatments and control after 24 h. although, both isolates caused some fluctuations in the total and differential hemocyte counts, the highest elevation in the hemocyte counts was determined at 72 and 96 h post-treatment in the larvae and the adults, respectively. there were nosignificant differences in the hemocyte counts between treatments and control after 24 h which may be attributed to the lack of pathogen entrance into the hemolymph. the subsequent increase in hemocyte counts reported in our study may result from recognizing the conidia in hemolymph, inducing hematopoietic organs to produce more prohemocytes and to trigger the hemocyte adherence on body mass (zibaee et al., 2014; shahriari et al., 2021b). several studies revealed a direct correlation between entomopathogen infections and number of hemocytes in insects. for example, the increase of hemocyte and nodule counts under mycoses was reported in the spodoptera littoralis boisduval (lepidoptera: noctuidae) when injected by b. bassiana and metarhizium anisopliae (mirhaghparast et al., 2013), in addition to glyphodes pyloalis walker (lepidoptera: pyralidae) infected by b. bassiana (am-118) (aghaee pour et al., 2021). meshrif et al. (2011) demonstrated no significant alteration in the number of hemocytes after s. littoralis larvae treatment by b. bassiana until 48 h post treatment although however an increase was observed after 70 h. 64 fig. 9 changes of glucos-6-phosphate dehydrogenase activity in c. montrouzieri infected by 1 × 105 conidium/ml of the two isolates of b. bassiana (am-118 and bb3), larvae (a), adults (b). different letters indicate significance at p < 0.05 in each time interval phenoloxidase is one of the important components in immune system of insects that plays a significant role in the conversion of phenols to quinones, and subsequently to melanin (gorman et al., 2008). in insects, melanin has a key role in cuticle sclerotization, wound healing, coagulation process of hemolymph (zdybicka-barabas et al., 2014). the higher po activity was observed in the hemolymph of the individuals treated with both fungal isolates after 72 and 96 h. since po is produced and stored within hemocytes (mak and saunders, 2006), increased level of po activity may be attributed to the higher hemocyte counts during fungal treatment as observed in our research. several studies have been reported an increment of hemocyte counts leading to the higher activity of po in insects (gillespie et al., 2000; mirhaghparast et al., 2013; zibaee et al., 2014; shahriari et al., 2021b). the higher po activity increases coagulation and melanization of hemolymph that subsequently causes the higher resistance of insects to entomopathogens mainly due to toxicant effects through generation of the toxic free radical against invading agents (leger et al., 1988). secondary metabolites produced by entomopathogens have been recognized as the one of main exogenous resources that lead to production free radicals after tissue afflictions which imposes oxidative stress (lukasik, 2007; dubovskiy et al., 2008; jia et al., 2016; karthi et al., 2018; shamakhi et al., 2020). oxidative stress eventually leads to peroxidation of lipid and dna damage (dubovskiy et al., 2008). cat, sod and pod are the major antioxidant enzymes to protect insect 65 fig. 10 changes of ascorbat proxidase activity in c. montrouzieri infected by 105 conidium/ml of the two isolates of b. bassiana (am-118 and bb3), larvae (a), adults (b). different letters indicate significance at p < 0.05 in each time interval tissues against oxidative stress. sod has a key role in catalyzing dismutation of toxic superoxide radicals into hydrogen peroxide and oxygen. afterward, hydrogen peroxide is converted to h2o and oxygen by cat and pod (dubovskiy et al., 2008). in the present study, the activities of sod, cat and pod significantly induced in the larvae and the adults treated by am-118 and bb3 isolates compared to control. similar results were obtained regarding the effects of m. anisopliae on locusta migratoria (meyen) (orthoptera: acrididae) (jia et al., 2016). shamakhi et al. (2020) demonstrated that infection of c. suppressalis by b. bassiana caused the higher activities of sod, cat, and pod. karthi et al. (2018) revealed that aspergillus flavus caused the higher activities of sod, cat, and pod in the third instar larvae of s. litura. similarly, our findings revealed the higher activities of sod, cat and pod in the larvae and the adults of c. montrouzieri infected by the conidia of pathogenic fungi, which may be connected with the higher generation of ros. glucose-6-phosphate dehydrogenase (gpdh) and ascorbate peroxide (apod) are the two antioxidant components which involved in detoxification of pro-oxidant compounds in different tissues (asada, 1984). apod removes hydrogen peroxide in cytoplasm, chloroplasts, and mitochondria, while gpdh engaged in eliminating oxidant compounds within cytosol through transduction of nadph to nadp+ (asada, 1984; nation, 2008). in our study, the higher activities of 66 fig. 11 changes of malondialdehyde content in c. montrouzieri infected by 105 conidium/ml of the two isolates of b. bassiana (am-118 and bb3), larvae (a), adults (b). different letters indicate significance at p < 0.05 in each time interval apox and gpdh found in the larvae and the adults exposed to conidia of am-118 and bb3 isolates. a similar result was observed in apox and gpdh activities when c. suppressalis were injected by b. bassiana conidia so fungi may act as stress factors to induce generation of ross in the treated insects (shamakhi et al., 2020). dubovskiy et al. (2008) reported the significant higher activities of sod and cat in g. mellonella exposed to bacillus thuringiensis. the higher gpdh activity caused enhancement of nadph generation to eliminate products of activity of apox. moreover, nadph decreases possible virulent effects through shifting electrons to free radicals (barbehenn, 2002; dubovskiy et al., 2008). lipid peroxidation is another marker indicating ross damages to cellular membranes. mda have been known as a significant by-product of lipid peroxidation so its content demonstrates destruction and elevation of cell membrane dissociation (dubovskiy et al., 2008). our results indicated that lipid peroxidation was higher in the larvae and the adults exposed to am118 and bb3 than in control. our results are similar to previous findings like s. littura infection to a. flavus (karthi et al., 2018) and c. suppressalis to b. bassiana (shamakhi et al., 2020). conclusions in a successful biocontrol program, it is very crucial to recognize the indirect effects of biopesticides on natural enemies like predators because such a program requires biopesticides with low toxicity to natural enemies. the current study revealed the direct and indirect effects of the two isolates of entomopathogenic fungi, b. bassiana (am-118 and bb3) on c. montrouzieri through mortality on the larvae and the adults as well as on 67 some physiological processes. overall, bioassay results demonstrated that am-118 and bb3 isolates were less virulent to c. montrouzieri than to mealybugs and more specifically, am-118 has a lesser virulence than bb3. moreover, both larvae and adults were able to induce immune and antioxidant systems to protect themselves against infective conidia. therefore, it may be concluded that am-118 may be a better candidate to be used along with c. montrouzieri in integrative management of tea mealybug. acknowledgement the research was supported by a grant from university of guilan (15p/135209). references addy sk, goodman rn. polyphenol oxidase and peroxidase in apple leaves inoculated with a virulent or an avirulent strain for ervinia amylovora. ind. phytopathol. 25: 575-579, 1972. aghaeepour s, zibaee a, rostami mg, hoda h, shahriari m. mortality and immune challenge of a native isolate of beauveria bassina against the larvae of glyphodes pyloalis walker (lepidoptera: pyralidae). egypt. j. biol. pest control 31: 1-9, 2021. aghdam hr, malekshah sam. estimation of the lower temperature threshold and thermal requirement of cryptolaemus montrouzieri mulsant (coleoptera; coccinelidae) using degree-day and ikemoto-takai linear models. j entomol soc iran 39: 343-358, 2019. asada k. chloroplasts: formation of active oxygen species and its scavenging. meth. enzymol. 105: 422-429, 1984. barbehenn rv. gut-based antioxidant enzymes in a polyphagous and a graminivorous grasshopper. j. chem. ecol. 28: 1329-1347, 2002. balinsky d, bernstein re. the purification and properties of glucose-6-phosphate dehydrogenase from human erythrocytes. biochim. biophys. acta 67: 313-315, 1963. bar-or d, rael lt, lau ep, rao nk, thomas gw, winkler jv., et al. an analog of the human albumin n-terminus (asp-ala-his-lys) prevents formation of copper-induced reactive oxygen species. biochem. biophys. res. comm. 284: 856-862, 2001. borges ar, santos pn, furtado af, figueiredo rcb. phagocytosis of latex beads and bacteria by hemocytes of the triatomine bug rhodnius prolixus (hemiptera: reduvidae). micron 39: 486-494, 2008. dubovskiy im, martemyanov vv, vorontsova yl, rantala mj, gryzanova ev, glupov vv. effect of bacterial infection on antioxidant activity and lipid peroxidation in the midgut of galleria mellonella l. larvae (lepidoptera, pyralidae). comp. biochem. physiol. toxicol. pharmacol. c 148: 1-5, 2008. gillespie jp, burnett c, charnley ak. the immune response of the desert locust schistocerca gregaria during mycosis of the entomopathogenic fungus, metarhizium anisopliae var acridum. j. insect physiol. 46: 429-437, 2000. gorman mj, an c, kanost mr. characterization of tyrosine hydroxylase from manduca sexta. insect biochem. mol. biol. 37: 1327–1337, 2007. er mk, tunaz h, isikber aa, satar s, mart c, uygun n. pathogenicity of entomopathogenic fungi to coccinella septempunctata (col.: coccinellidae) and a survey of fungal diseases of coccinellids. j. king saud. univ. eng. sci. 11: 118-122, 2008. jaronski st. mass production of entomopathogenic fungi: state of the art: 357-413, 2014. in morales-ramos j. 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(lepidoptera: crambidae) larvae to different entomopathogenic fungi. bull. entomol. res. 104: 155-163, 2014. zdybicka-barabas a, mak p, jakubowicz t, cytryńska m. lysozyme and defense peptides as suppressors of phenoloxidase activity in galleria mellonella. arch. insect. biochem. physiol. 87: 1-12, 2014. 175 isj 17: 175-185, 2020 issn 1824-307x research report investigating the effect of starvation and various nutritional types on the hemocytic profile and phenoloxidase activity in the indian meal moth plodia interpunctella (hübner) (lepidoptera: pyralidae) m ebrahimi, m ajamhassani * department of plant protection, faculty of agriculture, shahrood university of technology, shahrood. iran this is an open access article published under the cc by license accepted august 17, 2020 abstract the defense mechanisms of the insects are based on involvement of the hemocytes and phenoloxidase. hemocytes are the basic component of the cellular immunity and phenoloxidase as the part of prophenoloxidase (ppo) cascade is the component of both humoral and cellular defense. nutrition as well as starvation and attack by any organisms can modify these parameters of the innate immunity. in the current study, the effects of the stresses imposed by the starvation or different types of diets were investigated on the important immunity aspects of the indian meal moth larvae. results showed a decline in the total hemocyte count in hemolymph with the increase in the starvation duration. in the first test, 5th instar larvae were starved for three time intervals including 24, 48, and 72 h and then, the changes in hemocyte number and phenoloxidase activity were studied. in the second experiment, the larvae bred on four diets including diet (a) walnut, diet (b) pistachio, diet (c) pea and raisin, and diet (d) artificial diets were used. the total number of the hemocytes and percentage of each hemocyte were also considered. larvae were kept in an incubator set at a temperature of 25 ± 1 °c with 45 % of relative humidity (rh), and a constant photoperiod of 14:10 h (l:d) during the tests. the number of the plasmatocytes, one the main immune cells was sharply decreased with prolongation of the starvation duration and finally, their number reached by 134.04 ± 25.25 mm3 of hemolymph. the number of the granulocytes was also decreased significantly 72 h post-starvation than other treatments. the prohemocytes as the stem cells were initially increased within 24 h, and they were decreased later. the oenocytoids as the key cells involved in the phenoloxidase activity were initially increased significantly within 24 h of starvation compared to the control, but they were decreased significantly after 48 and 72 h reaching the same amount as the controls. results revealed that the types of the consumed diet influenced the number of cells and phenoloxidase activity. the highest total hemocyte count was related to the diet (c) pea and raisins (2158.18 ± 172.5 mm3), and the lowest was observed in the larvae fed on the pistachios (924 ± 78.33 mm3). the number of plasmatocytes, granulocytes, and oenocytoids was the highest in those larvae fed on the diet (c) pea and raisin and diet (d) artificial diet, respectively but the lowest numbers were observed for other treatments. the number of prohemocytes in the larvae fed on different diets did not differ significantly. the phenoloxidase activity was significantly reduced in the fifth instar larvae following starvation. the highest activity of phenoloxidase in feeding treatments was observed in those larvae fed on the artificial diet while the lowest activity was observed in the pistachiofed larvae. thus, the amount and type of the diet and the stresses including starvation can determine the immune response of the insects against the entomopathogens. key words: indian meal moth; immunity; diet; starvation; phenoloxidase introduction indian meal moth, as a cosmopolitan and polyphagous insect is the pest particularly affecting ___________________________________________________________________________ corresponding author: maryam ajam hassani department of plant protection, faculty of agriculture shahrood university of technology, shahrood. iran e-mail: shahroodm@gmail.com the dried fruits. the larvae spin a net and feed from the inside, which then includes the larvae and their exudates. they usually exude an unpleasant smell. the contaminants in addition to the direct loss inflict indirect costs, such as reduced quality of the grains and dried fruits (mohandes et al., 2007), which are considered as the main hosts of the indian meal moth in the world (rahrrabe et al., 2020). 176 fig. 1 light microscopy pictures of p. interpunctella hemocytes stained with giemsa. pr (prohemocyte: large nucleus covering whole cell and small cytoplasmic area (arrows). pl (plasmotocyte spherical with short pseudopodia). oe (oneocytoid). sp (spherulocyte), gr (granulocyte). scale bar = 10 µm biological success of the insects is due to their strong immune system, and the first defensive barrier against the exogenous agents is related to the insect's cuticle, the peritrophic membrane, a curtain around the food, the midgut epithelium, and trachea (stanley and miller, 2006; hillyer, 2016). understanding the physiological defense features of the insects is considered as an effective step to characterize the genotoxic, physiological and biochemical effects of the infections (yeh et al., 2005). the immune response of the insects is an important indicator of their sensitivity to various types of contamination caused by the influx of the foreign agents, such as spores of the fungi and bacteria, toxins, diapause, molt, starvation stress, environmental conditions, dietary changes and gender (mowlds et al., 2008). the cellular immunity is associated with participation of various types of hemocytes to phagocytize, form the nodules, or even encapsulate the invaders depending upon their types (stanely and miller, 2006). humoral immunity includes the production of antimicrobial peptides, reactive oxygen and nitrogen derivatives, as well as coagulation and melanization of hemolymph (buyukguzel et al., 2007). nutrition has been shown to play an important role in the insect growth, development, and immunity (siva-jothy et al., 2002; kang et al., 2011; myers et al., 2011; triggs and knell, 2011; maggini et al., 2012; le gall and rehmer, 2014). variation in the usage of energy sources including proteins and carbohydrates could change the immune reactions 177 fig. 2 effect starvation durations on total hemocyte count of 5th instar larvae of p. interpunctella (different letters show significance using tukey’s test at p < 0.05) and physiological function in the insects (mason et al., 2014; vogelweith et al., 2016). if the insects are fed by richer sources of nutrition, they will possess more defensive ability and on the contrary, those that do not have sufficient nutrition or are starved will experience a significant reduction in their number of hemocytes and phenoloxidase enzyme activity making them susceptible to the pathogens (manjula et al., 2020). larvae of samia cynthia fed on the diet containing cassava leaf have been shown to have an activated cellular defense as evidenced by a higher total hemocyte count (thc) in comparison with the larvae fed on the artificial diet alone (tungitwitayakul and tatun, 2017). confirming that the leaf of cassava plant possesses a high percentage of proteins, lipid and carbohydrates (manjula et al., 2020). low content of protein in the diet has been shown to significantly influence the immunological challenges and defense power against the foreign agents in the bumblebees (roger et al., 2017). angela et al. (2011) in a study reported a significant difference in the immune reaction of grammia incorrupta fed on the diets containing different contents of iridoid glycoside. results of a study about the diet effects on the honeybee showed that the larvae starved for 7 days had reduced concentration of antimicrobial peptides in the hemolymph (alaux et al., 2010). also, results of a research regarding the effects of starvation on the immune characteristics of the 5th instar larvae of manduca sexta (l.) indicated that the amounts of glucose and trehalose were increased. the total hemocytes count was also increased (adamo et al., 2016). starvation for 24 and 48 h has been shown to cause significant changes in the count of plasmatocytes and granulocytes and phenoloxidase activity in the larvae of yponomeuta malinellus. (ajamhassani and mahmoodzadeh, 2020). results of the study about the effect of food deprivation on the amount of contamination in the galleria mellonella by candida albicans showed a significant decrease in the density of the hemocytes (banville et al., 2012). studies on the flour beetle, tribolium castaneum showed that the susceptibility to the beauveria bassiana was increased by increasing the days of malnutrition (lord, 2010). similar results have been reported in other invertebrates e.g., garden snail after starvation on its immunity (alrawadeh, 2010). hence, nutrition plays a key role in maintaining the immunity of the garden snail, and continuous starvation for 3 weeks has been found to significantly reduce the total hemocyte count and the number of phagocytic cells, and decrease the activity of the phenoloxidase enzyme. based on the reports, macronutrient contents and protein: carbohydrate ratios influence on the life-history traits including pupal and adult weight, and fecundity of the adults in plodia interpunctella (littlefair and knell, 2016). also, larvae of p. interpunctella raised on a good-quality diet have been reported to have substantially higher hemocyte count and po activity in high density (triggs and knell, 2011). thus, it seems that starvation periods or food limitation could influence on the life history-traits and immune function in the natural population of pests, such as p. interpunctella. it has been proved that the food depletion could influence on the horizontal transmission of the pathogens by increasing the larval movement (beisner and myers, 1999). in addition, the starvation could trigger the virus into an 178 fig. 3 effect starvation on plasmatocyte count of 5th instar larvae of p. interpunctella (different letters show significance using tukey’s test at p < 0.05) active form and initiate its activity at high densities of malacosoma pluvial in the field myers et al, 2011). in contrast, findings of kang et al. (2011) have shown that the starvation of larvae of bombyx mori at the time of baculovirus exposure had a negative effect on the virus transmission and pathogenesis. also, no relationship has been found between the po activity in the hemolymph of the starved larvae of lymantria dispar and larval susceptibility to the baculovirus (kasianov et al., 2017). different species act differently while being subjected to the stresses and in particular starvation. variations in the feeding habit will also cause different effects in various species. taking into account this theory, the current study was conducted in order to investigate the immune reaction of the indian meal moth after administrating various diets and starvation periods. but, comprehensive research is needed in this respect to provide new aspects regarding the physiological defense of this important stockpile pest. understanding the interactions between the insect,s immune system and the attacking pathogen is of great importance. habrobracon hebetor and venturia canescens are known to attack and successfully develop within the larvae of several lepidopterous pests of the stored products, mainly pyralids (e.g., p. interpunctella) in the stored wheat (schöller, 1998; heinlein et al., 2002; ghimire and phillips, 2010). in addition, plodia larvae are susceptible to the infection by the p. interpunctella granulovirus from genus baculovirus (sait et al., 1994). so, determining the susceptibility or resistance of the larvae to these parasitoides or pathogens is somehow influenced by the starvation, diets, and other environmental factors. materials and methods insect rearing adults of plodia interpunctella hübner (lepidoptera: pyralidae) were procured from plant protection research organisation of iran (tehran, iran). the adults were released in boxes (20 × 15 × 10 cm) for mating in an incubator set at 25 ± 1 °c with 45 % rh, 14:10 l:d. the eggs were collected and the hatched larvae divided into 2 groups. the first group was bred on artificial diet (malts 160 g, wheat germ powder 800 g, glycerol 200 ml and honey 200 ml) for hemocyte profile light microscopy studies, total hemocyte count, po activity and the starvation assessments. second group were bred on four diets including diet (a) walnut, diet (b) pistachio, diet (c) pea and raisin and diet (d) artificial diets for the analysis of immune factors affected by various diet. hemocyte profile light microscopy studies a clean slide was used to make a hemolymph smear after excising one of the larval pro-legs with the help of a microscissor. the slide was left to dry at room temperature for 20 min and then stained with giemsa (merck kgaa, germany) (diluted 9 times with distilled water and filtered before use) for 25 min. it was differentiated in saturated solution of lithium carbonate (merck kgaa, germany) for 30 seconds and then washed in distilled water. after drying, permanent microscopy slides were prepared using canada balsam. hemocytes were observed under a light microscope (olympus bh2) at 40 x magnification (ghasemi et al., 2013). total hemocyte counts (thc) the total hemocyte count was done by collecting the hemolymph of 5th instar larva after 179 cutting one of the prolegs and diluted with tyson solution (nacl2 72 mm, na2so4 9 mm, glycerol 43 mm, methyl violet 0.06 mm, distilled water) (mahmood and yusaf, 1985). the thc was counted using a standard neubauer hemocytometer under a light microscope at 40x magnification. the number of total hemocytes per cubic millimeter (mm3) was calculated using the following formula of jones (1962): dilution = 10 times depth factor of the chamber = 10 no. of squares counted = 5 the effect of starvation on hemocyte number in order to carry out this experiment, 5th instar larvae (2 old-days) that reared on artificial diet, were starved for three time intervals including 24, 48 and 72 h. each treatment and control included 30 larvae and in total 120 larvae. each of the larvae experienced starvation individually in a separate petri (total starvation method was used here). the total numbers of hemocytes were counted. data were analyzed in a complete randomized design with sas software and the means were compared using tukey's test (p < 0.5). the experiment included four treatments a control and three treatments i.e. 24, 48 and 72 h starved larvae. the effect of diet type on hemocyte number for this purpose, larvae were bred on four diets including diet (a) walnut, diet (b) pistachio, diet (c) pea and raisin and diet (d) artificial diets were used. (second group of larvae that described above). hatched larvae was transferred individually to separate food-bearing petri. for each treatment, 20 replicates were used (5th instar larvae 2 olddays). (in total 80 larvae). the volume of food in each peti was almost equal about 20 gr. fresh food was provided for larvae, daily. the total number of hemocytes and percentage of each hemocyte was recorded. data were analyzed in a complete randomized design with sas software and averages were compared with tukey's test (p < 0.05). phenoloxidase enzyme activity (po) to determine the effect of starvation and diet types on the activity of phenoloxidases in hemolymph, hemocyte lysate method was used (leonard et al., 1985). in this method, for each treatment, 5th instar larval (2 old-days) hemolymph were collected (~ 150 µl hemolymph related to 30 larvae) and centrifuged (sigma, germany) at 4 °c and 10,000 g for five minutes. the supernatant was removed and remaining pellet was mixed with 100 μl of phosphate buffer (ph 7) and then homogenized. the solution was centrifuged again at 4 °c and 12.000 g for 15 min, and supernatant was used in po assay. then, 25 μl of the supernatant were added to 50 μl of l-dopa (l-dihydroxy phenyl alanine), a specific substrate of the phenoloxidase enzyme and 50 μl of phosphate buffer. phenoloxidase activity was measured at 490 nm wavelength (elx800, usa). results as seen in fig. 1 we could identify prohemocytes, plasmatocytes, granulocytes, spherulocytes, and oenocitoids in p. interpunctella. the smallest cells were round, oval, or elliptical with large nucleus filling the entire cell. the cells with various shapes and sizes and irregular plasma membrane were the plasmatocytes whose nuclei were, with centrally located. the granulocytes were mostly spherical or oval with a peculiar granular packed cytoplasm and a relatively small and centrally located nucleus. the spherulocytes were typically filled with spherules with small centrally located nucleus. the oenocytoids were mostly oval and eccentric nucleus. fig. 4 effect starvation periods on granulocyte count of 5th instar larvae of p. interpunctella (different letters show significance using tukey’s test at p < 0.05) 180 fig. 5 effect starvation periods on prohemocyte and oenocytoid count of 5th instar larvae of p. interpunctella (different letters show significance using tukey’s test at p < 0.05) effect of starvation on hemocyte number there was no significant changes on hemocyte number at 24 h starvation duration compared to controls (f = 34.21, df t,e = 3, 112, p < 0.0001). however, the cell number significantly decreased after 48 h of starvation. while the total hemocyte count was 647.53 ± 61.78 mm3, it was significantly reduced to 306.5 ± 34.29 mm3. the same trend was observed after 72 h of starvation (199.4 ± 29.29 mm3) (fig. 2). the plasmatocyte numbers after 48 h reduced significantly (174.15 ± 20.85 mm3) and more significantly after 72 h (134.04 ± 25.25 mm3) whereas in control it was (450 ± 46.65 mm3). (f = 19.34, df t,e =3, 112, p < 0.0001). in other words, plasmatocytes were significantly reduced in larvae that had a longer starvation than the controls (fig. 3). of the with the same trend of reduction was also seen in the number of granulocytes (f = 11.32, df t,e= 3, 112, p < 0.0001). the number was as low as 75.33 ± 6.02 mm3 after 72 h starvation (fig. 4). on the other hand the number of prohemocytes and oenocytoids behaved somehow differently where it was initially increased after 24 of starvation (f = 74.5, df t,e=3, 231, p < 0.0001), but decreased gradually after 48 h and reaching the amount of control after 72 h. (fig. 5). effect of diet types on hemocyte number diets showed a significant effect on all hemocyte numbers except prohemocytes. the type of food provided to the larvae could affect the number of hemocytes of indian meal moth larvae. the highest total hemocyte count (f = 20.60, df t,e= 3, 72, p < 0.0001) was related to diet (c) pea and raisins (2158.18 ± 172.5 mm3). the total hemocyte counts in those larvae that were provided with diet (d) artificial diet, diet (c) walnuts and diet (b) pistachios were 1078 ± 128.1, 963 ± 116 and 924 ± 78.33 mm3, respectively (table 1). table 1 effect of diet on hemocyte number of 5th instar larvae of p. interpunctella dietm thcn pl gr oe pr a 963 ± 116.5 b 552.7 ± 78 b 332 ± 53.6 b 16.5 ± 4.4 b 13.2 ± 6.4 a b 924.5 ± 87.4 b 521.3 ± 52 b 377 ± 57.6 b 25.3 ± 5.8 b 10 ± 3.7 a c 2158 ± 172.6 a 1463 ± 152.6 a 555.5 ± 64.6 a 71.5 ± 12.3 a 20 ± 5.5 a d 1078.4 ± 128.4 b 506 ± 72.5 b 497.5 ± 71.4 a 34 ± 7.7 b 6.6 ± 4 a dietm: a= walnut, b=pistachios, c= pea and raisin, d= artificial diet thcn= all hemocyte numbers are in mm3 hemolymph (different letters in each column show significance using tukey’s test at p < 0.05) 181 fig. 6 effect of starvation periods on phenoloxidase activity of 5th instar larvae of p. interpunctella (different letters show significance using tukey’s test at p < 0.05) as it is shown in fig. 6. the highest total number of plasmotocytes (f = 23.17, dft,e = 3, 72, p < 0.0001) and oenocytoids (f = 8.79, dft,e = 3, 72, p < 0.0001) were observed in those larvae that fed on diet (c) pea and raisins. the number of these cells in the larvae fed on other diets were not different significantly. (table 1). taking the number of granulocytes into consideration the diet (c) pea and raisin and diet (d) artificial diet significantly (f = 66.5, dft,e = 3, 72, p < 0.0001) increased its number. however, there was no differences between other diet types as far as granulocytes were concerned (table 1). effect of starvation and diets on the activity of phenoloxidase enzyme our results indicated that by prolonging the starvation time, significant reduction in the activity of the phenoloxidase enzyme are expected (f = 25.4, df t,e = 3, 112, p < 0.0001). (fig. 6). we observed that the nutrition can affect the activity of the phenoloxidase enzyme. the highest po activity in larvae fed on diet (d) artificial diet (f = 32.61, df t,e = 3, 112, p < 0.0001) was significantly higher than that in larvae fed on other diets. (fig. 7). discussion beeman et al., (1983) in a research studied the hemocytes of p. interpunctella. they identified six different types of hemocytes: prohemocytes, plasmatocytes, granulocytes, oenocytoids, spherulocytes, and granulocytophagous cells. in the present research, five various types of hemocytes were found in the hemolymph of p. interpunctella. existence of these hemocytes (prohemocytes, plasmatocytes, granulocytes, oenocytoids, and spherulocytes) has already been reported by other researchers in the hemolymph of more insects particularly lepidoptera. e.g., b. mori, (liu et al., 2013), eupholidoptera smyrnensis (ozturk et al., 2018) and zeuzera pyrina (ajamhassani, 2019) and many other lepidopteran species. physicochemical properties of the insect,s hemolymph are significantly influenced by the stress conditions (mowlds et al., 2008; duarte et al., 2020). once there is a weakness in the immunity of the insects there will be a chance of more susceptibility to the routine control practices of the pests (yeh et al., 2005; ajamhassani, 2015). the hemocytes or insect,s blood cells are the most important hemolymph factors that undergo the environmental changes ,such as temperature, population density, starvation, nutrition, gender and the effects by foreign agents e.g., pollutants, insecticides ,and spore of entomopathogenic fungi and bacteria lee et al, 2008, siva-jothy and thompson 2002, ghasemi et al., 2014; pourali and ajamhassani, 2018). it has been proved that the immune potential is parallel to the hemocyte number (li et al., 2019). so, new hemocytes as the key factors in the innate immunity are always produced to replace the damaged hemocytes in order to maintain the homeostasis that is essential in the hemolymph (nakahara et al., 2003). the decreased number of hemocytes can be deadly to the insects, as the immune system weakens and makes them helpless against any control methods including the microbiological and chemical controls (zhu et al., 2012; ajamhassani et al., 2013). on the other hand, different insects show varied immune levels, which is based on the type and 182 fig. 7 effect of diet on phenoloxidase activity of 5th instar larvae of p. interpunctella (different letters show significance using tukey’s test at p < 0.05) percentage of the infection. the insects physiology can act in its best condition if the nutrition is available to them adequately (both quantitatively and qualitatively). regarding the importance of feeding for the organisms including the insects, as well as, the existence of differences among the species, herein, it was attempted to show the role of feeding in causing these differences. hence, in the current study, two sets of experiments were performed that may influence the immune system of our model. hence, the starvation was administered in different time intervals to assess its effects on the insect, hemocyte count in one set of experiments. it was observed that the nutrition is very important in the studied insect, as both the cell counts and the production of phenoloxidase were significantly influenced. thus, it seems that the activity of ppo cascade is related to the number of hemocytes. however, it is assumed that the feeding rapidly increases the level of phenoloxidase activity and hemocyte density, which is related to the energy obtained from nutrition (siva-jothy et al, 2002). as we know, the immune responses by the insects are dependent on the amount of energy received through feeding that is essential for homeostasis and immune activities (banville et al., 2012). our results indicated that despite experiencing short-term starvation for 3 days, the larvae of p. interpunctella showed significant immune responses to this stress. it was indicated that all the larvae survived after overcoming the starvation and continued the feeding until they became pupa and adult (the data were observational and not statistical). similar to our results, siva-jothy et al., (2002) indicated that short-term nutrient deprivation influenced the immune function in tenebrio molitor l. starvation causes a significant decrease in the phenoloxidase activity and on the contrary, it increases immediately after access to the food. also, 24 h exposure to a supplemented diet has been shown to increase the immune capacity of drosophila melanogaster larvae in the face of parasitoid infusion into hemocoel (vass and nappi, 1998). in our study, starved larvae survived and fed on the diet 72 h after the starvation. similar results have also been observed in dendrolimus pini larvae characterized by the ability to survive without food for up to one month (lukowski et al, 2020). in contrast, food limitation has been shown to sharply reduce the larval survival, development rate, larval and pupal size, fecundity rate, and phenoloxidase activity in the m. pluvial (myers et al, 2011). regarding the effect of different diets on the immune function, it was found that the changes in the number of hemocytes and phenoloxidase activity were sharply increased by feeding on the diets c and d. this is probably due to the presence of the considerable amounts of macromolecules involved in the growth and immunity, namely carbohydrates, proteins, and lipid in these diets compared to other diets. it has been well established that the carbohydrates are an important macronutrient necessary for the activities with highenergy needs, such as movement, somatic maintenance, and growth (maklakov et al, 2008). in addition, proteins are essential for different developmental stages and reproduction of the adults (bowen et al., 1995; crutz and hay, 2000; simpson and raubenheimer, 2012). increased 183 dietary protein has been reported to elevate the level of hemocytes and enzymes in the hemolymph leading to the high immune level (lee et al, 2008, graham et al, 2014). fatty acids as a rich source of energy are critical for the ecdysis. also, they are essential in the insects that have non-feeding adult stages, such as some lepidopteran species (stockhoff, 1993). for investigating the effect of various diets on the immune function, we must define what is variation in the quantity of macronutrients in each diet, but it is noteworthy that the exact contents of different macronutrients in the diets (e.g., carbohydrates, proteins, and lipids) are also unknown. on the other hand, the effects of the diet types should also be considered with respect to their total calories (littlefair and knell, 2016). similar results have been reported in the larvae of hyposidra talaca. they showed significantly higher thc when reared on the artificial diet (containing a percentage of protein) in comparison with the natural diet (tea leaf) (ghosh et al., 2018). shikano et al., (2010) found that trichoplusia ni larvae fed on a good quality host plant showed increased hemocyte number in their hemolymph and were more resistant against the nucleopolyhedral virus. in the larvae of spodoptera littoralis, immune parameters, such as lysozyme-like antibacterial activity were higher following feeding on the high-protein diet (casein) compared to the larvae grown with poor quality proteins (zein). these larvae also had a more melanized cuticle, and thus nutrition was also effective as a key factor in the melanization (lee et al., 2008). in addition, the macronutrient composition and zinc supplementation in the diet have been shown to have positive effect on the immune-tolerance in spodoptera littoralis larvae exposed to the entomopathogenic nematode of mesorhabditis belari, symbiotic bacteria, and its metabolites (manjula et al., 2020). in a study to evaluate the changes in immune function of the eupoecilia ambiguella larvae under the effects of diets, it was found that the larvae fed on high-sugar grapes showed a higher immunity against bacillus cereus, beauveria bassiana and serratia marcescens (vogelweith et al., 2016). in conclusion, results of the present study and studies by several authors including alaux et al., (2010) on the honey bees indicated that the immunity of the insects is dependent on the quality and quantity of the foods they receive. therefore, depriving the pest insects from obtaining enough nutrition alters their immunity function as a result of which the microbial agents can efficiently kill them. therefore, if any degradable material and in-minute quantities are included in our microbial control measures to weaken the immune system of the pests, then their efficiency will be certainly enhanced. references adamo sa, dvies g, easy r, kovalko i, turnbull kf. reconfiguration of the immune system network during food limitation in the caterpillar manduca sexta. exp biol. 219: 706-718, 2016. ajamhassani m, sendi jj, zibaee a, askary h, farsi mj. immunological responses ofhyphantria cunea (drury) (lepidoptera: arctiidae) to entomopathogenic fungi beauveria bassiana (bals-criy) and isaria farinosae (holmsk) fr. plant prot res. 53: 110-118, 2013. ajamhassani m. cellular immune reactions of spodoptera litura (fabricus) (lepidoptera: noctuidae) against entomopathogenic fungi beauveria bassiana. plant pests res. 4: 59-68, 2014. 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chitin synthesis inhibitor, hexaflumuron, on the development and hemolymph physiology of the cutworm, spodoptera litura. j insect sci 12: 113, 2012. isj092.pdf 6 isj 2: 6-16, 2005 issn 1824-307x review occurrence and structural characterization of heparin from molluscs n volpi department of animal biology, university of modena and reggio emilia, modena, italy accepted february 25, 2005 abstract several invertebrate species contain variable amounts of one or more types of sulfated glycosaminoglycans (gags). at present it is well known the existence of a species-specific sulfated gags composition based on the relative amount and type of chondroitin sulfates, heparan sulfate and heparin. heparin is a sulfated polysaccharide belonging to the family of gags with numerous important biological activities, such as anticoagulant and antithrombotic properties that derive from its interaction with diverse proteins. unusual heparin samples for molecular mass, fine structural organization and anticoagulant activity, are isolated and characterized from molluscs. variable presence of the trisulfated disaccharide [∆ua2s(1->4)-α-d-glcn2s6s] and significant modifications of the disaccharides bearing non-sulfated iduronic and glucuronic acids, [->4)-α-l-idoa(1->4)-α-dglcnac6s(1-> and ->4)-α-l-idoa(1->4)-α-d-glcn2s6s(1->] and [->4)-β-d-glca(1->4)-α-dglcn2s6s(1->], and oligosaccharide sequences bearing part of the atiii-binding region, [∆ua2s(1->4)-α-d-glcn2s6s(1->4)-β-d-glca(1->4)-α-d-glcn2s3s6s] and [∆ua2s (1->4) -α-dglcn2s6s (1->4)-α-l-idoa (1->4)-α-d-glcnac6s (1->4)-β-d-glca (1->4)-α-d-glcn2s3s6s], are detected and measured in heparin samples derived from different clam species. this review more specifically deals with structural and biologically important aspects of heparin in invertebrates with special emphasis on the heparin from molluscs. furthermore, the fine characterization of heparin from tapes phylippinarum and callista chione is reported. keywords: glycosaminoglycans; polysaccharides; heparin; molluscs; anticoagulant drugs introduction the presence of sulfated glycosaminoglycans (gags) in some taxa of invertebrates is now well documented (cassaro et al., 1977; hovingh and linker, 1982; pejler et al., 1987; nader et al., 1984; dietrich et al., 1985; jordan et al., 1986; chavante et al., 2000; medeiros et al., 2000; cesaretti et al., 2004; luppi et al., 2005). a comprehensive survey of different classes of invertebrates has shown that heparan sulfate (hs)-like and/or heparin-like compounds, besides chondroitin sulfate, are present in many species (medeiros et al., 2000). *corresponding author: nicola volpi university of modena and reggio emilia, dept. animal biology, via campi 213/d, 41100 modena, italy e-mail: volpi.nicola@unimore.it previous studies have also shown that heparin is present in several species of molluscs. a compound from the clam mercenaria mercenaria (jordan et al., 1986) exhibits several structural similarities to heparin. heparins with high anticoagulant activity have been isolated from the molluscs anomalocardia brasiliana (pejler et al., 1987; dietrich et al., 1985) tivela mactroides (pejler et al., 1987) and tapes phylippinarum (cesaretti et al., 2004). due to our knowledge of the sulfated polysaccharides in vertebrates and invertebrates, is now possible to draw a phylogenetic tree of the distribution of sulfated gags in the animal kingdom. heart and vascular diseases, also including thrombosis, are the leading causes of death in the united states and europe (arias and smith, 2003) even if after the introduction of antithrombotic agents, particularly heparin and its derivatives, deadly heart diseases have decreased substantially (about 30 %) when compared to malignant cancer, 7 they are still the main cause of death (arias and smith, 2003). this explains the efforts to discover and develop specific and more potent antithrombotic agents. commercial manufacture of heparin relies on either porcine or bovine intestinal or bovine lung tissue as raw material. the apparent link between bovine spongiform encephalopathy and the similar prion-based creutzfeldt-jakob disease in humans (schonberger, 1998), has limited the use of bovine heparin. moreover, it is not easy to distinguish bovine and porcine heparins, thus making it difficult to ensure the species source of heparin (linhardt and gunay, 1999). furthermore, porcine heparin also has problems associated with religious restrictions on its use. non-animal sources of heparin, such as chemically synthesized, enzymatically synthesized, or recombinant heparins are currently not available for pharmaceutical purposes. these concerns have motivated to look for alternative, non-mammalian sources of heparin. structure of heparin heparin is a linear polysaccharide consisting of 1->4 linked pyranosyluronic acid (uronic acid) and 2-amino-2deoxyglucopyranose (d-glucosamine, glcn) repeating units (linhardt, 2003). the uronic acid usually comprises 90 % l-idopyranosyluronic acid (l-iduronic acid, idoa) (fig. 1a) and 10 % d-glucopyranosyluronic acid (dglucuronic acid, glca). heparin, with its high content of sulfo and carboxyl groups, is a polyelectrolyte, having the highest negative charge density of any known biological macromolecule. at the disaccharide level, a number of structural variations exist (fig. 1b), leading to sequence microheterogeneity within heparin. gag heparin is polydisperse with a molecular mass range of 5-40, an average molecular mass of ~12 kda, and an average negative charge of about -75, making it an extremely challenging molecule to characterize. heparin's complexity extends through multiple structural levels. at the proteoglycan level, different numbers of gag chains (possibly having different saccharide sequences) can be attached to the various serine residues present on the core protein. heparin chains are biosynthesized attached to a unique core protein, serglycin, found primarily in mast cells. on mast cell degranulation, proteases act on the heparin core protein to release peptidoglycan heparin, which is further processed by a β-endoglucuronidase into gag heparin (linhardt, 2003). the chemical, physical, and biological properties of heparin are primarily ascribed to gag structure (or sequence), saccharide conformation, chain flexibility, molecular weight, and charge density. hs, while structurally related to heparin, is much less substituted with sulfo groups and has a more varied structure (or sequence) (fig. 1b) (gallagher et al., 1992). d-glucuronic acid predominates in hs, and it is polydisperse, having an average molecular mass of ~30 ranging from 5 to 50. hs chains also often contain domains of extended sequences having low or high sulfation (gallagher et al., 1992). interaction of heparin with proteins with the discovery of increasing numbers of heparinbinding proteins (capila and linhardt, 2002), there was a need to characterize the molecular properties, within the proteins and heparin, responsible for specific recognition (table 1). the biological activities of heparin and hs primarily result from their interaction with hundreds of different proteins. by using modelling, cardin and weintraub (1989) demonstrated that some heparin binding proteins had defined motifs corresponding to consensus sequences, giving the first evidence for the general structural requirements for gag-protein interactions. their results suggested that if the xbbbxxbx (b is a basic and x is a hydropathic amino acid residue) sequence was contained in an α-helical domain, then the basic amino acids would be displayed on one side of the helix with the hydropathic residues pointing back into the protein core. heparin binding sites, commonly observed on the external surface of proteins, correspond to shallow pockets of positive charge. thus, the topology of the heparin binding site is also an important factor in heparin binding consensus sequences. structural analysis of the heparin binding sites in acidic fibroblast growth factor (fgf1), basic fgf (fgf-2), and transforming growth factor β-1 (tgfβ-1) implicated a txxbxxtbxxxtbb motif (t defines a turn) (hileman et al., 1998). by screening peptide libraries the conservation of amino acids in heparin binding domains and the importance of spacing between basic amino acids in heparin binding was demonstrated. peptides enriched in arginine and lysine and polar hydrogenbonding amino acids were observed to bind heparin with highest affinity (capila and linhardt, 2002). studies on the role of the pattern and the spacing of the basic amino acids in heparin binding domains showed that heparin interacted more tightly with peptides containing a complementary binding site of high positive charge density while the less sulfated heparan sulfate interacted more tightly with a complementary site on a peptide that had more widely spaced basic (linhardt, 2003). anticoagulant activity the anticoagulant action of pharmaceutical heparin (120-180 usp u/mg) is the most thoroughly studied of its activities. anticoagulation occurs when heparin binds to atiii, a serine protease inhibitor (serpin). atiii undergoes a conformational change and becomes activated as an inhibitor of thrombin and other serine proteases in the coagulation cascade (fig. 2). fig. 1 structure of heparin: a) major trisulfated dlsaccharide repeating unit (x = sulfo or h, y = sulfo, ac or h); b) undersulfated structural variants (modified from linhardt, 2003). 8 table 1. characteristics of selected heparin-binding proteins (modified from linhardt, 2003). [a] hs: high sulfation; is: intermediate sulfation; ls: low sulfation. heparin-binding protein physiological/pathological role kd oligosaccharide size sequence features [a] function proteases/esterases at iii coagulation cascade serpin ca. 20 nm 5-mer glcns6s3s enhances slpi inhibits elastase and cathepsin g ca. 6 nm 12-mer to 14-mer is enhances c1 inh inhibits c1 esterase ca 100 nm hs enhances vcp protects host cell from complement nm unclear growth factors fgf-1 cell proliferation, differentiation, morphogenesis and angiogenesis nm 4-mer to 6-mer idoa2s-glcns6s activates signal transduction fgf-2 same as fgf-1 nm 4-mer to 6-mer idoa2s-glcns same as fgf-1 chemokines pf-4 inflammation and wound healing nm 12-mer hs/ls/hs inactivates heparin il-8 pro-inflammatory cytokine ca 6 µm 18-mer to 20-mer hs/ls/hs promotes sdf-1α pro-inflammatory mediator ca 20 nm 12-mer to 14-mer hs localizes lipid-binding proteins annexin ii receptor for tpa and plasminogen, cmv and tenascin c ca. 30 nm 4-mer to 5-mer hs unclear annexin v anticoagulant activity; influenza and hepatitis b viral entry ca. 20 nm 8-mer hs assembles apoe lipid transport; ad risk factor ca. 100 nm 8-mer hs localizes pathogen proteins hiv-1 gp120 viral entry 0.3 µm 10-mer hs inhibits cypa viral entry inhibits tat transactivating factor, primes cells for hiv infection ca. 70 nm 6-mer hs antagonizes hsv gb and gc viral entry into cells inhibits hsv gd viral entry and fusion glcnh23s inhibits dengue virus envelope protein viral localization ca. 15 nm 10-mer hs inhibits malaria cs protein sporozoite attachment to hepatocytes ca. 40 nm 10-mer hs inhibits adhesion proteins selectins adhesion, inflammation and metastasis µm > 14-mer hs with glcnh2 blocks vitronectin cell adhesion and migration µm removes fibronectin adhesion µm 8-mer to 14-mer hs with glcns reorganizes hb-gam neurite outgrowth in development ca. 10 nm 16-mer to 18-mer hs mediates ap in amyloid plaque µm 4-mer hs assembles a major breakthrough in the study of heparincatalyzed anticoagulation resulted from the separation of distinct heparin fractions differing markedly in affinity for atiii. low-affinity atiii binding heparin comprises about two-thirds of porcine intestinal heparin and has a low anticoagulant activity (typically <20 u/mg). in contrast, high atiii affinity heparin comprises the remaining third of porcine intestinal heparin and has a high anticoagulant activity (typically ~300 u/mg). rosenberg et al. (1979) and lindahl et al. (1979) examined the atiii binding site by performing a partial chemical and enzymatic depolymerization of heparin and then purified the products using affinity chromatography on immobilized atiii. the isolation of 3-o-sulfatase from human urine, capable of desulfonating 3-osulfoglucosamine residues provided the crucial clue to the structure of the atiii binding site (fig. 3). nmr studies by several groups proved the presence of the 3-o-sulfo group within the atiii binding site, and chemical synthesis of a pentasaccharide containing the 3,6-di-o-sulfo group substantiated these findings (torri et al., 1985). the atiii binding sequences found in certain hss are partially responsible for the blood compatibility of the vascular endothelium (marcum and rosenberg, 1987). the atiii pentasaccharide is sufficient to catalyze the atiii-mediated inhibition of factor xa, 9 fig. 2 convergence of intrinsic/extrinsic pathways of the coagulation cascade leading to fibrin generation. a critical serine protease in the coagulation cascade. to catalyze the atiii-mediated inhibition of thrombin, 16-18 saccharide units are required (sinay, 1999). thus, the structure-activity relationship of thrombin inhibition has been more difficult to establish because it relied on the synthesis of oligosaccharides substantially larger than atiii for pharmacological evaluation. recent studies show that a relatively nonspecific but highly charged thrombin binding domain in heparin, localized on the nonreducing side of the heparin's atiii binding site, is required to form a ternary complex. success in understanding the structure-activity relationship of heparin's inhibition of thrombin has resulted in a new class of potent, synthetic, but still experimental thrombin inhibitors. other heparin binding proteins, such as tissue factor pathway inhibitor (tfpi) and annexins, can also play important roles in anticoagulation. occurrence of heparin in vertebrates and invertebrates an updated phylogenetic tree of the distribution of sulfated gags in the animal kingdom is shown in fig. 4 (medeiros et al., 2000). whereas hss are ubiquitous components of all tissue-organized metazoan, heparin has shown a very peculiar distribution in mammalian and other vertebrate tissues as well as invertebrates. chondroitin sulfate also has a widespread distribution (fig. 4). since the earlier studies from a variety of mammals, it has been found that lung, intestine, and liver were the organs richest in heparin (nader et al., 2004) (table 2). except for rabbit tissues, heparin’s presence was demonstrated in lung, skin, ileum, lymph nodes, thymus, and appendix of all species studied. the absolute content of heparin varied depending on different tissues. the lack of heparin in rabbits was correlated with the absence of mast cells in the species (nader et al., 1980). a large variation of the concentration of heparin among species is evident. thus, bovine and dog tissues contain the highest amounts of heparin. generally, in non-mammalian vertebrate tissues the amount of heparin is considerably lower (nader et al., 2004). in invertebrates, heparin is found in few taxa, namely molluscs, crustacean, annelida, echinoderma and fig. 3 antithrombin iii pentasaccharide binding site. the anionic groups in bold are critical (95 % loss in binding energy on removal), and those in italics are important (25-50 % loss in binding energy on removal) for interaction with atiii (modified from linhardt, 2003). cnidaria (table 3). as observed for vertebrate heparin samples, the anticoagulant activity and molecular mass varied according to the species analyzed. furthermore, no correlation between molecular mass and anticoagulant activity of the heparins is evident. all these results imply that heparins have a large structural variation depending on their origin (nader et al., 2004). biological role of heparin in molluscs the biological function of the clam heparins and their apparently specific atiii-binding regions is unclear at the moment. molluscs do not possess any blood coagulation system similar to that of mammals, yet their heparins are capable of dramatically accelerating the inactivation of mammalian coagulation enzymes by the mammalian protease inhibitor, atiii. it is possible that the bivalve heparin is designed to interact with an endogenous antithrombin-like protease inhibitor acting on serine protease target enzymes. the existence and function of such an enzyme system remain to be established. in mammals heparin is released from the mast cells in response to specific inflammatory agents such as ige antibodies or complement fragments (anaphylatoxins). since the discovery of mast cells by paul ehrlich (1879) and after the demonstration that their metachromatic properties when stained with basic dyes was related to heparin, the question whether this compound was only confined to the mast cells or not has been a matter of controversy. studies on the concentration of heparin and its content in different fetal and adult bovine tissues (nader et al., 1982) have shown that a good correlation between the mast celi number and heparin concentration could be obtained in ali tissues analyzed. studies on mast-cell deficient mice of the genotypes w/wv and s1/s1d established that mast cells originate from hematopoietic stem cells. these experiments also demonstrated that heparin is present in appreciable amounts in the skin of the breeders and of the normal progeny. on the other hand, no heparin was detected in the skin of the w/wv genotype, which are deficient in mast cells. no significant differences in the relative amounts of 10 table 2. distribution of heparin in mammalian and other vertebrates (modified from nader et al., 2004). bov = bovine. tissue rabbit guinea pig rat dog cat pig bov human bony fish shark µg/g dry tissue lung <1 70 67 217 63 211 300 8 0.03 liver <1 <1 <1 141 1 <1 50 <1 1.32 0 ileum <1 27 1 400 87 113 1015 32 0 0 kidney <1 4 <1 2 6 <1 26 <1 0.29 aorta <1 <1 9 102 <1 2 150 <1 brain <1 <1 <1 <1 <1 <1 <1 <1 0 0 muscle <1 <1 36 9 <1 5 2 <1 0 0 spleen <1 <1 <1 11 <1 <1 19 <1 11.9 skin <1 <1 175 15 63 2 108 39 0 0 lymph <1 11 5 160 74 242 180 41 thymus <1 112 20 20 10 286 35 appendix <1 17 38 20 47 branchia 0.03 0 fig. 4 distribution of sulfated glycosaminoglycans in the animal kingdom (modified from medeiros et al., 2000). 11 table 3. distribution of heparin in invertebrates (from nader et al., 2004 updated). the molecular mass and the anticoagulant activity is reported. class and species average m.m. (kda) anticoagulant activity (usp) molluscs ciprinia islandica nd 95 mactrus pussula nd 100 mercenaria mercenaria 18 348 (anti-iia) anomalocardia brasiliana 32 320 donax striatus 20 220 tivela mactroides 25 180 tapes phylippinarum 14 350 callista chione 11 97 crustacea ucides chordatus nd 60 dedrocephalus brasiliensis 10 52 penaeus brasiliensis 9 60 annelida aphrodite longicornis nd nd hermodice carunculata nd nd echinoderma mellita quinquisperforata 12 50 cnidaria physalia sp. nd nd mnemiopsis sp. nd nd sipuncula nudu nd nd the other sulfated gags, namely hs, dermatan sulfate and chondroitin sulfate were observed among the breeders analyzed. this suggests that heparin is not replaced by other sulfated gags in the animals that lack heparin. these results clearly indicate that heparin is related to the presence of mast cells (kitamura and go, 1979; marshall et al., 1994). furthermore, in mammals, the heparin-containing mast cells are accumulated in lymphoid organs and in tissues exposed to the external milieu (skin, lungs, intestine) and one suggested role for this polysaccharide in mammalian is to fight external parasites (nader et al., 2004). heparin and other sulfated gags as well as histamine were found and quantified in various organs of the mollusc anomalocardia brasiliana. the heparin was present in granules inside the cytoplasm of mast-like cell (pejler et al., 1987; dietrich et al., 1985). a good correlation between heparin and histamine content was found in the labial palp, intestine, ctenide, mantle, and foot tissues. some conclusions can be derived from these studies: 1) heparin seems to be present exclusively in mast cells of vertebrates or mast-like cells in the case of molluscs; 2) its primary biological activity is not related with antithrombotic activity since molluscs, which do not possess a coagulation system, contain heparin and rabbits that possess this system are devoid of heparin; 3) many indirect evidences suggest that in mammals and molluscs heparin and its mast cells are involved in defense mechanisms independently of the immune system and support the hypothesis that this macromolecule could function as a mechanism for the surveillance of these organisms against certain pathogens. extraction, purification and characterization of heparin from tapes phylippinarum gags extracted from t. phylippinarum by defatting and proteolytic treatments, were successively fractionated on an anion-exchange resin and eluted with a linear nacl gradient at increasing molarity. agarose-gel electrophoresis analysis of single fractions eluted from the anionexchange resin showed low amounts of chondroitin sulfate besides heparin. after treatment with endonuclease and chondroitinase, and precipitation with organic solvent, t. phylippinarum yielded approx. 2.1 mg heparin/g of dry animals (volpi, 1993; volpi and maccari, 2002). fig. 5a illustrates the agarose-gel electrophoresis of the mollusc heparin showing the two components, the slow moving species having high-molecular mass and charge density and the fast moving heparin, possessing a lower molecular mass and sulfate groups amount. the densitometric scanning of t. phylippinarum heparin showed 22 ± 6.8 % of the slow moving component and 78 ± 5.4 % of the fast moving species (cesaretti et al., 2004). fig. 6a illustrates the page analysis of t. phylippinarum heparin showing an average molecular mass of 13.600 calculated on a calibration curve of oligosaccharide standards of 12 fig. 5. agarose-gel electrophoresis of heparin (4 µg) purified from a) tapes phylippinarum (from cesaretti et al., 2004) and b) callista chione (from luppi et al., 2005). the gel was dried and stained according to the sequential procedure by using toluidine blue (volpi and maccari, 2002). the quantitative analysis of the (1) slow moving heparin component and of the (2) fast moving heparin species was performed by specific calibration curves obtained from purified heparin species. o = origin known molecular mass prepared from bovine mucosal heparin. qualitative and quantitative oligosaccharide mapping (linhardt et al., 1988, 1989, 1990, 1992; rice and linhardt, 1989) was performed by depolymerizing t. phylippinarum heparin (fig. 7a) with heparinase (ec 4.2.2.7) and then separating the resulting unsaturated oligosaccharides by sax-hplc. mass balance close to 91-95 % (table 4) was excellent for t. phylippinarum heparin, confirming its purity. important differences between pharmaceutical bovine and mollusc heparin were the higher content of the [∆ua2s(1->4)-α-dglcn2s6s] disaccharide and the lower percentage of the 3b, 4 and 6 oligosaccharide sequences in the clam heparin (table 4). in contrast, a striking higher amount (more than 130 % than standard pharmaceutical heparin) was calculated for the oligosaccharide sequence 8 bearing part of the atiii-binding region, [∆ua2s (1->4) -α-d-glcn2s6s (1->4) -α-l-idoa (1->4) α-d-glcnac6s (1->4)-β-d-glca (1->4)-α-dglcn2s3s6s] in the t. phylippinarum heparin (table 4). this unusual sequence contains sulfation at position 3 of the glucosamine residue, characteristically found in the atiii-binding site. the results of these analyses were used to calculate the disaccharide composition. as expected from the oligosaccharide compositional analysis, heparin from t. phylippinarum is a more sulfated polysaccharide, as also indicated by the presence of a greater mol % of the trisulfated disaccharide [∆ua2s(1->4)-α-d-glcn2s6s], with respect to bovine mucosal heparin (73.5 %), and, very interestingly, also with respect to porcine mucosal (72.8 % of the trisulfated disaccharide) and human heparin (71.0 % of the trisulfated disaccharide) calculated with the same methodological approach. furthermore, there is a significant increase (+ 35 %) of the disaccharide bearing the sulfate group in position 3 of the nsulfo-glucosamine 6-sulfate part of the atiii-binding region. the increased number of atiii binding sites in t. phylippinarum heparin is accompanied by an increased anticoagulant activity. higher aptt (350 ± 56 iu/mg) and atiii-mediated antifactor xa (320 ± 48 iu/mg) activities were observed for the clam heparin in comparison with pharmaceutical bovine mucosal heparin (linhardt et al., 1992). isolation and fine structural characterization of heparin from callista chione fig. 5b illustrates the agarose-gel electrophoresis of the mollusc heparin (approx. 1.9 mg heparin/g of dry animals) showing the two components, the slow moving species (15 ± 1.3 %) and the fast moving heparin (85 ± 7.6 %). figure 6b illustrates the page analysis of c. chione heparin showing an average molecular mass of 10.950. the 1h-nmr spectrum, the agarose-gel and the page, showed c. chione heparin free from impurities (luppi et al., 2005). c. chione heparin was treated with heparinase (ec 4.2.2.7) and the resulting unsaturated oligosaccharides then separated by sax-hplc (figure 7b). mass balance close to 88 % (table 4) was calculated for c. chione heparin mainly due to the presence of approximately 10 % not identified oligosaccharides signed in the fig. 7b with x. important differences between pharmaceutical grade and mollusc heparin were the lower (approximately 37 %) content of the [∆ua2s(1->4) -α-d-glcn2s6s] disaccharide and the lower percentage of the 4 and 6 oligosaccharide sequences in the c. chione heparin (table 4). on the contrary, a strong increase was calculated for the oligosaccharides 1, 2, 3b, 5 and 6a. no substantial quantitative modifications were found for the sequence 8 bearing part of the atiii-binding region, [∆ua2s (1->4)-α-d-glcn2s6s (1->4)-α-lidoa (1->4)-α-d-glcnac6s (1->4) -β-d-glca (1->4) -α-d-glcn2s3s6s] in the c. chione heparin (table 4). the results of these analyses were used to calculate the disaccharide composition. as expected from the oligosaccharide compositional analysis, heparin from c. chione is a less sulfated polysaccharide, due to the presence of a lower mol % of the trisulfated disaccharide [∆ua2s(1->4)-αd-glcn2s6s]. furthermore, there is a significant decrease of the specific sulfatation in position 2 of the iduronic acid unit with the strong increase of the disaccharides bearing non-sulfated iduronic acid, [->4)-α-l-idoa(1->4)-α-d-glcnac6s(1->] and [->4)α-l-idoa(1->4)-α-d-glcn2s6s(1->], and a strong decrease of the sulfatation in postion 2 of the gluronic acid with a greater percentage of the disaccharide [->4)-β-d-glca(1->4)-α-d-glcn2s6s (1->]. no significant quantitative modification of the disaccharide bearing the sulfate group in position 3 of the n-sulfo-glucosamine 6-sulfate part of the atiii-binding region (table 4) was evident. the anticoagulant activity of the clam heparin was next evaluated. heparin isolated from c. 13 fig. 6 page analysis of the molecular mass of the a) tapes phylippinarum (from cesaretti et al., 2004) and b) callista chione (from luppi et al., 2005) heparins (hep). 40 µg of the purified polysaccharides were layered on the gel and the calibration curve was constructed by using oligosaccharide standards of known molecular mass prepared from bovine mucosal heparin. 1. 13,500; 2. 7,560; 3. 6,300; 4. 4,560; 5. 3,640; 6. 2,820; 7. 1,620. m = bromophenol blue. table 4. oligosaccharide analysis of clam heparins. total mol % of the oligosaccharides is calculated by summing the mol % for the oligosaccharides in each column. an error of ± 0.1 mol % is possible in the measurement of each oligosaccharide. the 2.5 (tapes phylippinarum heparin, from cesaretti et al., 2004) and 10.0 (callista chione heparin, from luppi et al., 2005) mol % correspond to unknown oligosaccharides. oligosaccharide t. phylippinarum c. chione 1 n.d. 11.8 2 3.3 9.5 3 71.0 33.7 3a 2.0 1.7 3b 0.4 6.4 4 0.9 1.9 4a 0.5 n.d. 5 7.4 13.5 6 2.8 1.8 6a 0.4 4.3 7 1.1 1.4 8 5.1 2.0 total (1-8) 94.9 88.0 n.d. = not detected. 1, ∆ua(1->4)-α-d-glcn2s6s; 2, ∆ua2s(1->4)-α-d-glcn2s; 3, ∆ua2s(1->4) )-α-d-glcn2s6s; 3a, ∆ua2s(1->4)-α-d-glcn2s(1->4)-α-l-idoa2s(1->4)-α-d-glcn2s; 3b, ∆ua2s(1->4)-α-d-glcn2s(1->4)-β-d-glca(1->4)-α-d-glcn2s6s; 4, ∆ua2s(1->4)-α-d-glcn2s6s(1->4)-α-l-idoa2s(1->4)-α-d-glcn2s; 4a, ∆ua2s(1->4)-α-d-glcn2s6s(1->4)-α-l-idoa(1->4)-α-d-glcn2s6s; 5, ∆ua2s(l->4)-α-d-glcn2s6s(1->4)-β-d-glca(1->4)-α-d-glcn2s6s; 6, ∆ua2s(1->4)-α-d-glcn2s6s(1->4)-α-l-idoa2s(1->4)-α-d-glcn2s6s; 6a, ∆ua2s(1->4) -αd-glcn2s6s(1->4) -αl-idoa(1->4)-α-dglcnac6s (1->4) -β-d-glca(1->4) -αdglcn2s6s; 7, ∆ua2s(1->4)-α-d-glcn2s6s(1->4)-β-d-glca(1->4)-α-d-glcn2s3s6s; 8, ∆ua2s(1->4) -α-d-glcn2s6s (1->4)-α-l-idoa(1->4)-α-d-glcnac6s(1->4)-β-d-glca(1->4)-α-d-glcn2s3s6s. 14 fig. 7 sax-hplc chromatograms of heparin lyase-treated a) tapes phylippinarum (from cesaretti et al., 2004) and b) callista chione (from luppi et al., 2005) heparin samples. 10 µg of polisaccharides were analyzed. the major peaks corresponding to oligosaccharides 1-8 are indicated and they were assigned by coinjection with standards (see their structures in table 4). 15 chione in our laboratory showed a lower anti-factor xa activity of approximately 60 % (52 ± 7.4 ui/mg) and a decrease of the aptt activity of 33 % (97 ± 12.1 ui/mg) in comparison with standard pharmaceutical grade heparin. conclusions the study of heparin will certainly extend well into this new century with so many questions left still unanswered. some focal points in the near future include: a) improved preparation and synthesis of heparins; b) new heparin-based anticoagulants with improved properties; c) new therapeutic applications for heparins; d) new heparin mimetics; (e) new biomaterials and (f) development of an improved understanding of physiology and pathophysiology through glycomics. the preparation of heparin from mammalian tissues creates concern particularly after the recent appearance of bovine spongiform encephalopathy in europe. bovine tissues are now rarely used in heparin production, and there are growing concerns about porcine tissues. heparins prepared by alternative routes, such as defined, recombinant mammalian cell lines capable of being cultured in large-scale fermentations, or non mammalian tissues, offer an exciting alternative to the present preparations. new anticoagulants based on heparin’s structure might offer enhanced specificity targeting one or selected groups of coagulation proteases and avoiding undesired interactions with other proteins, thus decreasing the side effects associated with heparins use. furthermore, new therapeutic applications might include the use of heparin to treatment of infectious diseases, inflammation, and control of cell growth in wound-healing and cancer. these new activities will require the elimination of heparin’s anticoagulant activity, the engineering of appropriate pharmacokinetics and pharmacodynamics, and optimally oral bioavailability. a concern about the application of heparins to promote wound healing is that they might simultaneously promote cancer. thus, wound healing applications will probably require localization of the drug at the site of action possibly through the application of polymers or gels. these new potential applications of heparin may be strictly associated with molecules possessing peculiar and unusual structure, as those isolated from invertebrates. finally, one of the most important future directions of heparin research is driven by the recent sequencing of the human genome and the field of genomics. while much attention is currently focused on the proteome encoded by the genome and the rapidly developing field of proteomics, the glycome has garnered little attention. glycomics is the study of the structure and function of the glycome, the most important and complex of the post-translation modifications that proteins undergo. an improved understanding of the glycome should be beneficial in better understanding genetic diseases, offering new therapeutic approaches to treat these very serious pathologies. moreover, improved knowledge of glycomics, in vertebrate and invertebrate species, should lead to a better understanding of physiology and pathophysiology, offering new approaches to drug development. acknowledgments research partially supported by the “cassa di risparmio di modena” (2003) to n volpi. references arias e, smith bl. deaths: preliminary data for 2001. national vital statistics reports. 51: 1-44, 2003. capila i, linhardt rj. heparin-protein interactions. angew. chem. int. ed. 41: 390-412, 2002. cardin ad, weintraub hj. molecular modeling of protein-glycosaminoglycan interactions. arterio sclerosis 9: 21-32, 1989. cassaro cm, dietrich cp. distribution of sulfated mucopolysaccharides in invertebrates. j. biol. chem. 252: 2254-2261, 1977. cesaretti m, luppi e, maccari m, volpi n. isolation and characterization of a heparin with high anticoagulant activity from the clam tapes phylippinarum. evidence for the presence of a high content of antithrombin iii-binding site. glycobiology 14: 1275-1284, 2004. 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"fast moving" and "slow moving" heparins, dermatan sulfate, and chondroitin sulfate: qualitative and quantitative analysis by agarosegel electrophoresis. carbohydr. res. 247: 263278, 1993. volpi n, maccari f. detection of submicrogram quantities of glycosaminoglycans on agarose gels by sequential staining with toluidine blue and stains-all. electrophoresis 23: 4060-4066, 2002. the abbreviations used are: ac, acetate; aptt, activated partial thromboplastin time; atiii, antithrombin iii; fgf, fibroblast growth factor; gags, glycosaminoglycans; glca, β-d-glucopiranosyluronic acid; glcn, 2-deoxy-2amino-α-d-glucopyranose; hplc, high-performance liquid chromatography; hpsec, high-performance sizeexclusion chromatography; hs, heparan sulfate; idoa, α-l-idopyranosyluronic acid; page, polyacrylamide gel electrophoresis; s, sulfate; sax, strong anion exchange; tfpi, tissue factor pathway inhibitor; tgfβ, transforming growth factor β; ua, uronic acid. 340 isj 14: 340-351, 2017 issn 1824-307x research report bacterial communities in gills and intestines of yesso scallop (patinopecten yessoensis) and its habitat waters in changhai (dalian, china) g lu 1 , f wang 1 , z yu 1 , m lu 1 , y wang 1 , c liu 1 , z xue 1 , y wu 1 , l wang 1,2 , l song 1,2 1 liaoning key laboratory of marine animal immunology and disease control, dalian ocean university, dalian 116023, china 2 laboratory for marine fisheries science and food production processes, qingdao national laboratory for marine science and technology, qingdao 266071, china accepted september 5, 2017 abstract yesso scallop is a marine bivalve mollusc of economic importance in the coastline of northern china. the frequently outbreak of various diseases has heavily threatened the sustainable development of the scallop industry. the information about the bacterial communities inside and outside the body of yesso scallop will provide insights into disease prevention, probiotic application and health aquaculture. in the present study, the diversity of bacterial communities in intestines, rectum and gills of yesso scallop and its habitat waters were investigated. the bacterial diversity and richness in waters were higher than that in intestines, rectum and gills. the microbiota from intestines, rectum, gills of scallop and waters were clearly separated into four clusters by non-metric multidimensional scaling and hierarchical cluster analysis, suggesting a microbiota selection of scallop at organ scale. venn diagram suggested that the bacterial community in the body of scallop was more specialized than that in waters. high-throughput sequencing revealed that the main bacterial communities in intestine, rectum and gill were firmicutes, tenericute and proteobacteria, chlamydiae, proteobacteria and firmicutes, and proteobacteria and firmicutes, respectively. while the bacterial communities in waters mainly included proteobacteria, bacteroidetes and cyanobacteria. real-time pcr for bacterial load showed that the total bacterial abundance in waters was significantly higher than that inside the scallop body. the abundance of bacteroides fragilis in rectum was significantly higher than that in intestines and gills. the results about the bacterial community inside and outside the body of yesso scallop are helpful to better understand the relationship between the symbiotic bacteria of yesso scallop and their habitat, and also provided critical information to develop strategies of disease prevention and probiotic application in scallop aquaculture. key words: patinopecten yessoensis; bacterial diversity; habitat waters; gills; intestines; high-throughput sequencing; real-time pcr introduction microbiota refers to the microbial community harbored in a specific ecosystem, and the commensal microbiota associated with a host plays a critical role in the health of the host organism (ruby et al., 2004). the interaction of host-microbiota is essential to many processes of normal physiology, including immune homeostasis, metabolic activity and host development (rawls et ___________________________________________________________________________ corresponding author: linsheng song liaoning key laboratory of marine animal immunology and disease control dalian ocean university dalian 116023, china e-mail: lshsong@dlou.edu.cn al., 2004). with the great contribution of the interaction between host and microbiota, scientists are becoming increasingly aware of the coevolution of animals and microorganisms and the relationship between host health and commensal microbiota (xu and gordon, 2003; lai et al., 2009; goffredi et al., 2014). there are more and more reports on the classification and functional diversity of the microbial community, especially in marine invertebrates whose mortality is an important global concern for marine ecosystems (lafferty et al., 2004; plowright et al., 2008; ngangbam et al., 2015; nielsen et al., 2015). the composition of microbial communities could be strongly controlled and regulated by the external environments (fierer and jackson, 2006). 341 table 1 characteristics of sampling sites the microbiota in marine invertebrates has been extensively studied, and the formation of microbiota was believed to be affected by many factors, such as host habitat (klaus et al., 2005), individual host characteristics (cardenas et al., 2014), host health level (lu et al., 2013), organ compartmentalization (meisterhans et al., 2016), diet and development stages (rungrassamee et al., 2013; miyake et al., 2015). there is a selective association between invertebrates and the related microbes. for instance, the composition of bacterial communities in intestine and habitat environment of shrimp (litopenaeus vannamei) was reported to be influenced by habitat environment (peng et al., 2006; luo et al., 2009). the dominant bacteria members in crab (eriocheir sinensis) intestines were tenericutes and proteobacteria, while actinobacteria, proteobacteria and bacteroidetes were dominant in its habitat waters (zhang et al., 2016). the bacterial diversity in the habitat surface sediment and intestine contents also showed significant difference in sea cucumber (apostichopus japonicas) (gao et al., 2014). the accumulating evidences suggest that the composition of aquatic microbial communities in habitat waters has a strong influence on the symbiotic microbes of farmed aquatic animals (peng et al., 2006). however, the information about the relationships between the symbiotic microbiota and the habitat of yesso scallop is still very limited. the host microbiota structure is always different among organs, indicating that there may be a selection at organ scale (meisterhans et al., 2016). for example, the microbial compositions in coral surface mucus layer and skeleton were significantly different (sweet et al., 2011). the symbiotic microbiota in crab (eriocheir sinensis) was found to be body site-specific in intestine and gill (zhang et al., 2016). comparison of microbial diversity along human intestinal tract suggested that the microbial community in jejunum was different from those in ascending colon, rectum and distal ileum (mei et al., 2005). in easter oyster (crassostrea virginica), microbial community in intestine also differed from that in stomach and harbored a relatively multiple assemblage of phylotypes (king et al., 2012). in adult yellow grouper (epinephelus awoara), the autochthonous microbiota was variable in different parts of the digestive tract (zhou et al., 2009). these results suggested that the variability of microbiota among organs was associated with their respective physiological roles (meisterhans et al., 2016). however, the symbiotic microbiota in yesso scallop especially in intestine is still not clear. yesso scallop (patinopecten yessoensis) is a low-temperature bivalve, which mainly distributes in the coastline of northern japan, northern korean peninsula and far east of russian (hou et al., 2011). it has become one of the most important farmed marine molluscan species in northern china since it was introduced into china in 1980s (wang, 1984). however, the frequent outbreaks of infectious diseases have caused massive mortalities and heavy economic losses in the past years (li and xue, 2005; yuan et al., 2010). there are many factors contributing to the high mortality of cultured scallops such as pathogens, pollutants, elevated temperature, aquaculture practices and physiological stress (xiao et al., 2005). the symbiotic microbiota and habitat environment have been recognized as the most important factors impacting on the fitness of aquatic animals (li et al., 2007b; liu et al., 2011;yan et al., 2012; ye et al., 2013). characterizing the bacterial community in yesso scallop and its habitat waters is an important step to learn the function of symbiotic bacterial in disease prevention and probiotic application for scallop culture. in the present study, high-throughput sequencing analysis of 16s rdna gene was employed to study bacterial diversity in intestine, rectum and gill of yesso scallop and its habitat waters in january from three parallel sites in changhai, dalian, china. the abundance of the total bacteria and bacteroides fragilis load were examined by real-time quantitative pcr. our objectives were (1) to find the difference of bacterial community at organ scale, (2) to understand the relationship between the symbiotic bacteria of yesso scallop and their habitat, and (3) to provide theoretical basis for the development for disease prevention and probiotic application in yesso scallop farming. materials and methods sample collection yesso scallops (patinopecten yessoensis) and seawater were collected from three parallel sampling sites (table 1) in a yesso scallop farm in changhai (dalian, china) in january 2017. there were three parallel sample sites with average distance of 0.2 km. the temperature, salinity, dissolved oxygen (do) and ph content of the sampled seawater were measured using a portable meter (hach, usa). about two liters of surface seawater was collected per site longitude latitude temperature (°c) salinity (‰) do (mg/l) ph a 39°17′23″n 122°44′18″e 12.36±0.45 32.11±0.34 8.27±0.39 8.26±0.19 b 39°17′21″n 122°44′59″e 12.34±0.31 32.04±0.29 8.78±0.67 8.37±0.08 c 39°17′15″n 122°45′12″e 12.04±0.42 32.00±0.19 8.80±0.55 8.42±0.07 342 fig. s1 rarefaction curves of the bacterial 16s rdna gene sequences from intestines, rectum, gills of yesso scallop and its habitat waters. rarefaction curves of operational taxonomic units (otu) were clustered at 97 % sequence similarity level across different samples. sampling site and filtered through a 0.22 μm polycarbonate membrane (sangon, china) under sterile conditions. the membrane was transferred to sterile microfuge tubes and stored at -80 °c before bacterial genomic dna extraction. twelve yesso scallops of 1-year-old with an average weight of about 10 g were collected from each parallel sampling site. the shell surface of scallops was washed with sterile seawater for three times and disinfected with 70 % ethanol to wash away debris attached on the surface. after the shells were opened, scallops were washed with sterile water for three times. intestines (in), rectum (re) and gills (gi) were aseptically isolated according to the previous reports (prosser et al., 1965; fritsch et al., 1976), and stored at -80 °c before bacterial genomic dna extraction. genomic dna extraction and high throughput sequencing the same tissue obtained from each site was mixed together prior to dna extraction. total bacterial genomic dna was extracted from the samples according to the manufacturer’s instructions of e.z.n.a.water dna kit (for seawater samples) and e.z.n.a.soil dna kit (for tissue samples) (omega, usa). the quality and quantity of the genomic dna were examined by 1 % agarose gel electrophoresis and measured by uv/visible spectrophotometer at 260 and 280 nm. the extracted dna was stored at -20 °c for further sequencing and real-time pcr analysis. high throughput sequencing of v3-v4 region of 16s rdna gene was performed by majorbio co., ltd (shanghai, china) based on the illuminamiseq sequencing platform. real-time quantitative pcr in this study, absolute quantitative pcr was conducted to determine the number of total bacteria and 10 b. fragilis group species. 16s rdna genes of bacteria and b. fragilis were amplified by rtaq dna polymerase (takara, japan) using primers 27f/1492r (goodfellow and stackebrandt, 1991) and bfrf/bfrr (liu et al., 2003), respectively. standard plasmid was constructed by inserting the amplicon into pmd18-t vector (takara, japan) and transformed into escherichia coli trans5 (transgen, china). after verified by sequencing, the plasmids were extracted from e. coli with column plasmid preps kit (sangon, china). the concentrations of recombinant plasmids were determined by nanodrop nd-1000 (thermo, usa), and the copy number of standard plasmid was calculated according to the method as previously reported (liu et al., 2016). standard templates (10 3 10 9 copies of target genes) were diluted at a ratio gradient of 1:10 in depc water according to the copy number. the dissociation curve of real-time pcr with each diluted templates was used as quantification standards for samples. the threshold cycle (ct) values against the denary logarithms of 343 the copy numbers were recorded and analyzed to set up copy number standard curve. primers 341f/534r (liu et al., 2011) and bfrf/bfrr were used to specifically estimate the total bacteria and b. fragilis load, respectively. bacterial genomic dna of different samples was adjusted to the final concentration of 10 ng/μl for real-time quantitative pcr. the ct values were recorded and analyzed after real-time quantitative pcr assay. the abundance of the total bacteria and b. fragilis were calculated based on standard curve and ct values. bioinformatics and statistical analyses the raw illuminafastq data were processed using mothur v.1.11.0 (http://www.mothur.org/) to eliminate the low quality and redundant reads. a window with 50 bp was set to filter reads tail mass value of < 20 bp. bases were cut at the end of the window with the average quality score of < 20. operation taxonomic units (otus) were clustered with 97 % similarity by usearch (vsesion 7.0; http://drive5.com/uparse/) against the silva database (release128; http://www.arb-silva.de) (edgar, 2013). chimeric sequence and a single sequence without duplication were removed (http://drive5.com/usearch/manual/singletons.html) (edgar et al., 2011). each 16s rrna gene sequence without chimera was analyzed by the ribosomal database project (rdp) classifier with a 70 % confidence threshold (version 2.2 http://sourceforge.net/projects/rdp-classifier/). sequencing depth was determined using rarefaction curve by mothur software. the shannon, chao and coverage indices were calculated for each sample using the methods of mothur (shannon, 1948; chao and lee, 1992; edgar et al., 2011; larsen et al., 2014). community composition barplot and pieplot were drawn using r script. non-metric multidimensional scaling (nmds) and hierarchical clustering trees were constructed with weighted unifrac distance. venn diagram was plotted based on the otus in all samples from intestines, rectum, gills and waters. real-time pcr data were analyzed by statistical package for social sciences (spss) 17.0 software. one-way analysis of variance (anova) was used to test the significant differences among all samples and the differences were considered significantly at p < 0.05. the figures were pictured by origin 8.0. results general information on sampling sites and overview of 16s rdna high-throughput sequencing analysis three parallel sampling sites in yesso scallop farm in changhai were used in this study. the temperature, salinity, dissolved oxygen and ph was 12.04 12.36 °c, 32.00 32.11 ‰, 8.27 8.80 g/l and 8.26 8.42, respectively (table 1). high-throughput sequencing of the 16s rdna gene amplicons was performed to determine bacterial diversity in intestines, rectum and gills of yesso scallop as well as its habitat waters. in the rarefaction curves, the number of otus almost reached the plateau phase with the increasing read number at 30,000 (fig. s1), suggesting the sufficient sampling depth in all samples. a total of 447,232 sequences and 692 distinct operational taxonomic units (otus) were obtained from 12 samples with an average read length of 445 bases. there were 475, 386, 188 and 553 otus in rectum, intestine, gill and seawater samples, respectively (table 2). the number of bacteria genera was highest in waters (234 genus), whereas lowest in gill (67 genus) (table 2). bacterial community dissimilarity among different samples to estimate and compare the bacterial diversity among different samples, the proportion of otus was used to calculate community richness and diversity indices. good’s coverage index was used to estimate the percentage of total bacterial otus in a sample. it was 0.99 in all the 12 samples, indicating the obtained sequences represented the majority of bacteria sequences in all the 12 samples. table 2 sampling depth and diversity indices for rectum, intestine, gill and water bacteria from three sites rectum intestine gill water re-a re-b re-c in-a in-b in-c gi-a gi-b gi-c w-a w-b w-c sample depth no. of sequences 30,482 40,648 37,461 40,814 35,101 39,903 44,315 32,564 33,113 412,48 35,483 36,100 otus (97%） 422 389 383 298 288 225 125 140 88 438 434 461 phylum 23 20 24 20 17 18 11 12 12 23 23 23 class 45 39 44 37 32 31 22 23 18 46 47 45 family 134 127 129 124 114 101 63 70 49 151 149 157 genus 200 192 191 170 160 146 93 102 67 230 221 234 diversity indices chao 1 462.23 432.83 416.73 365.68 318.84 286.29 168.16 224 128.62 500.63 509.62 529.65 shannon 3.63 2.85 2.89 2.13 2.84 2.24 0.74 1.12 0.46 3.91 4.05 4.21 good's coverage 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99 http://www.mothur.org/ http://drive5.com/uparse/ http://www.arb-silva.de/ http://drive5.com/usearch/manual/singletons.html http://sourceforge.net/projects/rdp-classifier/ 344 fig. 1 bacterial community dissimilarity among intestines, rectum, gills of yesso scallop and its habitat waters. (a) non-metric multidimensional scaling (nmds) showing bacterial community difference in samples. the distances were determined using the weighted unifrac method with relative abundance of otus. circles indicate samples from intestines, triangles samples from rectum, diamonds samples from gills and squares samples from water. (b) hierarchical clustering tree based on weighted unifrac distances for sequences derived from yesso scallop for each of the replicate intestines, rectum, gills and waters. the bacterial diversity and richness were estimated by the shannon index and chao 1. the bacterial diversity in water group with shannon index of 4.21 was higher than that of intestine, rectum and gill group (shannon index = 2.84, 3.63 and 1.12, respectively). chao 1 values were 462.23, 365.68, 168.16 and 529.7 in rectum, intestine, gill and water group, respectively (table 2). it indicated that the bacterial richness was highest in the scallop inhabit waters. alpha-diversity analysis showed that the bacterial diversity and richness in water sample were higher than that of intestines, rectum and gills, indicating the water harbored a remarkable diversity of bacterial communities. the similarities and dissimilarities of bacterial community composition among all samples were explored by a nmds (fig. 1a) and hierarchical cluster analysis (fig. 1b). the bacterial communities in intestine, rectum, gill and water replicates were clustered separately in different group on both axes in the nmds analysis. the gill and water bacteria were separated on nmds axis 2, but the intestine and rectum bacteria were clustered together. the clustering pattern in the samples did not change significantly among the sampling sites (fig. 1a). in the hierarchical clustering tree based on bacterial community profiles at otus level, the bacterial communities from intestine, rectum, gill and water replicates formed four distinct clades. intestine group was firstly clustered with rectum group and formed a sister group to the gill groups. the water group was separated from the scallop organ group and shared a closer relationship with gill groups (fig. 1b). venn diagram was constructed to evaluate the distribution of otus among different samples (fig. 2). 108 otus were shared by all the samples. the number of specific otus in intestine, rectum, gill and water groups was 12, 32, 7 and 166, respectively. fig. 2 venn diagram analysis of the different samples. venn diagram showing the shared and unique otus among intestines, rectum, gills of yesso scallop and its habitat waters. 345 fig. 3 bacterial community compositions of intestines, rectum, gills of yesso scallop and its habitat waters. relative abundance of bacterial composition on phyla level. ‘other’ represented a combination of species whose abundance was below 1 %. bacterial community compositions the composition and abundance of bacterial communities in different samples was shown in figure 3. at phylum level, the dominant bacteria in intestine were firmicutes (54.28 %), tenericutes (21.60 %) and proteobacteria (10.87 %), which accounted for 86.75 % of total abundance (fig. 3a). in rectum, three phyla of bacteria including chlamydiae (31.83 %), proteobacteria (24.12 %) and firmicutes (23.44 %) were dominant, which accounted for 79.39 % of total abundance (fig. 3b). proteobacteria (87.73 %) and firmicutes (9.05 %) were the dominant phyla in gill-associated bacteria, which accounted for 96.78 % of total abundance (fig. 3c). proteobacteria (57.47 %), bacteroidetes (21.77 %) and cyanobacteria (14.59 %) were dominant phyla in water accounting for 93.83 % of total abundance (fig. 3d). further classification at family level indicated that bacterial communities varied considerably among different samples (fig. 4). for instance, the intestine bacteria were dominated by bacillaceae and mycoplasmataceae belonging to the phyla firmicutes and tenericutes, respectively. bacillaceae and chlamydiales in the phyla firmicutes and chlamydiae were dominated in rectum. hahellaceas and bacillaceae were detected in all samples, but hahellaceas showed more abundant in gill than that in the other groups. the proportions of rhodobacteraceae, cyanobacteria and flavobacteriaceae, belonging to the phyla proteobacteria, cyanobacteria and bacteroidetes, varied in waters, and rhodobacteraceae was only detected in waters. the abundance of the total bacteria and b. fragilis the abundance of the total bacterial and b. fragilis of samples was examined by real-time quantitative pcr. it showed a high linear relationship between the ct (threshold cycles) values and logcn (denary logarithm of the copy numbers) in the standard curves of total bacteria load and b. fragilis (fig. s2). the copy numbers of total bacterial load in waters ranged from 1.42×10 7 to 1.99×10 7 copies per nanogram of dna, which was significantly higher than that in other groups. the relatively lower copy numbers of total bacterial load were observed in rectum, which was about 22.28 % of total bacterial load in waters (fig. 5a). whereas the copy numbers of the b. fragilis in rectum ranged from 5.67×10 3 to 6.43×10 3 copies per nanogram of dna, which was significantly higher than eleven times and two times that in intestine and gill, respectively (fig. 5b). discussion the yesso scallop is a filter-feeding marine bivalve, which filter and accumulate large numbers of microbe from the harvesting water (antunes et al., 346 fig. 4 bacterial community compositions at family level in intestines, rectum, gills of yesso scallop and its habitat waters. ‘other’ represented a combination of species whose abundance was below 5 %. 2010). bacteria can exert positive or negative influences on the health status of aquatic animals, either by the construction of symbiotic relationships or by causing diseases, respectively (antunes et al., 2010). the symbiotic microbiota has been investigated in many marine invertebrates, such as easter oyster, scallop, black tiger shrimp and sea cucumber (king et al., 2012; coton et al., 2013; rungrassamee et al., 2013; gao et al., 2014). however, the information about the symbiotic microbiota in p. yessoensis and the microbiota present in its habitat waters is still very limited. in the present study, bacterial composition inside and outside the body of yesso scallop was analyzed. the objectives of this work were to identify microbiota diversity in different functional organs (intestine, rectum and gill) of p. yessoensis and to assess the relationship between the environmental microbiota and symbiotic microbiota, which could contribute to better understanding about the microbiota structure inside and outside the body of yesso scallop also, and also provided critical information to develop strategies of disease prevention and probiotic application in scallop aquaculture. in aquatic ecosystems, aquatic animals are in direct contact with the environment water. therefore, they are in continual contact with a complex and dynamic microbiota, and some of them are host symbiotic microbiota (gatesoupe, 1999). the temperature of the environmental waters was a key driver of bacterial community, the higher temperatures harbored remarkably higher diversity and richness in bacterial composition when compared to the lower temperatures (tang et al., 2014). marine microbial have seasonal dynamic changes in the south sea of korea (suh et al., 2015). during the sampling period, temperature recorded values close to 12 °c in the surface layers, and the lower water temperature may be affected the bacterial community inside and outside the body of yesso scallop. although intestinal bacteria of aquatic animals may mainly originate from the culture waters (han et al., 2010; wu et al., 2012), the symbiotic microbiota of some aquatic animals showed significant difference from that in the surrounding environment (suh et al., 2015; zhang et al.,2016). for example, clear differences were seen between the bacterial community associated with great scallop (pecten maximus) and in the water from the different aquaculture systems (sandaa et al., 2003). in the present study, proteobacteria was found to be the dominant phyla in the organs of yesso scallop and its habitat waters, which was consistent with previous reports (wu et al., 2012; zhang et al., 2016). but the bacterial community structures in waters and organs of yesso scallop were not exactly the same. the bacteria belonging to rhodobacteraceae family was dominant in water but almost absent in intestine, rectum and gill samples, suggesting that there were specific microbiota for organs and habitat waters of aquatic animal. it was suggested that bivalve could select bacteria through filtration by gills and remove bacteria through their immune system (antunes et al., 2010; meisterhans et al., 2016). for example, the wood-eating marine bivalve shipworms can select 347 fig. s2 standard curve of the 16s rdna gene copy number of the total bacteria (a) and b. fragilis load (b), showing the linear relationship between the threshold cycles (ct) and copy number. endosymbiotic bacteria by gills to produce wood degrading enzymes (o’connor et al., 2014). anodonta cygnea has the ability to filter and eliminate escherichia coli from the surrounding environment, and establish a commensal relationship with vibrio metschnikovii and aeromonas sobria through phagocytic process conducted by heamolymph circulating cells without causing disease and mortality (antunes et al., 2010). the persistence of some bacteria in the bivalve tissue depends in part on their resistance to the bactericidal activity of the hemolymph (canesi et al., 2001; zampini et al., 2003). in addition, the bacterial diversity and abundance of the inhabit waters of yesso scallop were significantly higher than that of its intestines, rectum and gills, which was similar to the previous reports in scallop, fish and sea cucumber (sandaa et al., 2003; gao et al., 2014; miyake et al., 2015), suggesting that most of the bacterial species in aquatic animal intestines might live as specialized members of symbiotic microbiota rather than free-living environmental bacteria. bivalves depend on their immune system to eliminate the invading bacteria and decrease the microbial load inside their bodies (antunes et al., 2010). the symbiotic microbiota of aquatic animalsis derived from the habitat waters. the difference between the symbiotic microbiota and environmental bacteria suggests a mechanism of selection, adaptation and regulation between the host and the microbe. the microbiota structure differed among organs indicated that host have body site specific microbiota (zhang et al., 2016). the different organs in yesso scallop (intestine, rectum and gill) represent bacterial niches with unique feature. recently, the microbiota associating with great scallop (pecten maximus) gonads (lasa et al., 2014) and the bacterial diversity in the mantle of healthy and incised symptoms of yesso scallop (p. yessoensis) (ding et al., 2014) have also been studied. in manila clam, the bacterial communities among organs were different, which was associated with the respective physiological functions of organs (meisterhans et al., 2016). the differences in intestine regional structures of adult mussel (mytilus edulis), such as intestinal epithelium and mucous cells, contribute to the functional differences (fritsch et al., 1976). in norway lobster (nephrops norvegicus), the digestive system consists of the foregut, midgut and hindgut. the midgut is the main absorption organ, and no absorption occurs in the short hindgut (yonge, 1924; miyake et al., 2015). previous studies revealed the functional differences between midgut and hindgut of the eriocheir sinensis, which affacted the diversity and abundance of bacteria in different intestinal regions (chen et al., 2015). in the present study, significant differences were observed in the rectum and intestines associated bacteria, which was similar to the autochthonous microbiota in different parts of adult yellow grouper digestive tract (zhou et al., 2009). these results suggested that the bacterial niche with unique features in different intestinal structures might affect physiological functions, and the microbiota in rectum and intestines should experience district selective processes for their microbial community. the variations of intestine microbiota can indicate the alternations of food supply for marine invertebrates (meziti et al., 2010; miyake et al., 2015). gills can perform physical and physiological functions to selectively exclude microbe, therefore, the microbiota communities in gills and intestines are always different (wildish, 1998). it has been reported that the microbe associated with bivalve organs might be partially selected through phagocytosis mechanisms (pruzzo et al., 2005). in the present study, the different microbiota patterns among organs also indicated a selection of scallop microbiota at organ scale. b. fragilis is a gram-negative anaerobic bacterium found in the intestinal tract of most animals and human (betteken et al., 2015). it is the major component of the endogenous human bowel flora, commonly related to a variety of human infections, such as bacteremia and wound infection (cheng et al., 2009; martin et al., 2009), and contributes significantly to the morbidity and even mortality (hadano et al., 2013). in mub crabs, b. fragilis 348 fig. 5 the 16s rdna gene copy number of the total bacteria (a) and b. fragilis load (b) by real-time quantitative pcr inintestines, rectum and gills of yesso scallop and its habitat waters with three parallel sites. was present in the intestine of both healthy and diseased individuals (lie et al., 2012). bacteroidetes was also found to be common in wild and pond-raised crab intestines, but the abundance of total bacterial and b. fragilis load in the pond-raised crabs were about four-to-ten times higher than that in wild crabs (li et al., 2007a). bacteroidetes were dominant bacteria in the adductor muscle of sick yesso scallop (p. yessoensis), but barely in the adductor muscle of healthy scallop, indicating it might be related to the disease of scallops (dou et al., 2016). b. fragilis has been reported to activate the tlr pathway in t lymphocytes to establish host microbial symbiosis (round et al., 2011). in the present study, the abundance of bacteroidetes and b. fragilis in rectum was much higher than that in gills and intestines, indicating the abundance of b. fragilis in rectum might attribute to the scallop intestinal disease. it is suggested that a small amount of b. fragilis cannot cause scallop rectum disease, and there should be a threshold of intestinal microbiota imbalance for the outbreak of intestinal disease. in addition, the proportion of bacteroides decreased significantly from spring to summer, and increased during autumn and highest in winter in the south sea of korea (suh et al., 2015). the bacterial community composition in the digestive diverticula of scallops (chlamys farreri) was significantly seasonal variation (yang et al., 2012). the bacterial community changed significantly at different development stages of yesso scallop larvae (p. yessoensis) (sun et al., 2016). to a certain extent, the high abundance of b. fragilis in the rectum may be affected by the water microbiota, seasons, scallop development stages. in conclusion, intestine, rectum and gill of yesso scallop and its habitat waters have distinct microbiota. the bacterial diversity and richness in waters were higher than that in intestines, rectum and gills, suggesting that the bacterial community in the body of scallop was more specialized than that in its habitat waters. the different microbiota patterns among scallop organs indicated that there was a selection of microbiota at organ scale. the results provided useful information for the disease prevention and probiotic application in scallop aquaculture. acknowledgement this study was supported by earmarked fund (cars-48) from modern agro-industry technology research system, a grant from national science foundation of china (no. 31530069), dalian high level talent innovation support program (2015r020), and the 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aquaculture 286: 184-189, 2009. isj001.pdf 2 isj 1: 2-4, 2004 issn 1824-307x visions and perspectives the mollusc as a suitable model for mammalian immune-neuroendocrine investigations e ottaviani department of animal biology, university of modena and reggio emilia, modena, italy accepted june 30, 2004 abstract the same or relatively similar molecules seen in molluscan defense responses are also found in mammals, where their functions remain basically similar. the mollusc immunocytes are able to recognize a variety of stimuli and to set up correspondingly complex responses, in which primitive, but very efficient, forms of immune and neuroendocrine responses are intermixed. thus, invertebrates could represent an ideal alternative in studying mammalian complexity from an immunoneuroendocrine point of view. key words: immune-neuroendocrine responses; molluscs; mammals immune responses molluscs are characterized by a coelomatic cavity, which makes it possible to distinguish a well-defined cellular and humoral component in the immune system (for review, see ottaviani, 1992). in the majority of molluscs, there are two circulating immunocytes, which control the main immune responses, i.e. phagocytosis, cell shape changes (the expression of cell motility), chemotaxis (the expression of cell migration), and cytotoxicity. the humoral factors are represented by agglutinins, lectins, nitric oxide, cytokine-like molecules (lm), corticotropin-releasing hormone (crh)-lm, enkephalin-lm, pro-opiomelanocortin (pomc)-derived peptide-lm, e.g. adrenocorticotropin hormone (acth), α-melanocyte-stimulating hormone (α-msh) and β -endorphin (for reviews, see stefano et al., 1989; ottaviani, 1992; ottaviani et al., 1997). there is a close relationship between the two components, and it has been observed that the exogenous humoral factors increase cell motility, cell migration corresponding author: enzo ottaviani department of animal biology, university of modena and reggio emilia, modena, via campi 213/d, 41100 modena, italy e-mail: ottaviani.enzo@unimo.it and phagocytosis (for reviews, see stefano et al., 1989; ottaviani et al., 1997). exogenous molecules, such as crh, acth, interleukin (il)-8, platelet-derived growth factor (pdgf)-ab and transforming growth factor (tgf)-β1, induce cell shape changes in the immunocytes via an adenylate cyclase/camp/protein kinase a pathway, as well as the activation of protein kinase c (sassi et al., 1998; malagoli et al., 2000; for review, see ottaviani et al., 2001). as other invertebrates, molluscs are able to discriminate between self and not-self. in a freshwater mollusc, an autograft was accepted, while an allograft and xenograft were rejected. these latter elicited an initial inflammatory reaction followed by the encapsulation of the foreign tissue (ottaviani and vergine, 1990). humoral and cellular experiments, bacterial clearance studies, and the specific responses found in molluscan transplantations suggest the presence of a memorytype response of short duration (ottaviani et al., 1986; ottaviani, 1990; ottaviani and vergine, 1990). with regard to cytotoxicity, a natural killer (nk)-like activity has been observed which is modulated by il-2 (franceschi et al., 1991). hubert et al. (1997) isolated in the mussel a cytotoxic protein complex able to kill eukaryotic cells, tumor cells and protozaon parasites. altogether, the molluscan immune system presents the three functional components identified by hildemann et al. (1979) as minimal criteria for immunological competence, i.e. specificity, cytotoxicity and memory. 3 neuroendocrine responses it is well known that crh and acth are the main mediators of stress response in vertebrates. this phenomenon involves several organs. the release of acth by the pituitary, which is modulated by hypothalamic crh, guides the glucocorticoids by means of the adrenal gland, which, in turn and together with the sympathetic nervous system, induces the release of catecholamines from adrenal medulla. molluscs present a stress response superimposed on that observed in mammals, in which the key mediators are the same, i.e. crh and acth, and the series follows the same order and pattern, i.e. crh > acth > biogenic amines. together with pomc-products and crh-lm, the presence of biogenic amines and cortisollm has also been reported in molluscs (ottaviani et al., 1998; for review, see ottaviani and franceschi, 1996). however, unlike in vertebrates, the stress response in invertebrates does not require the intervention of numerous organs and cells. it is rather concentrated in a single immune-neuroendocrine cell, which is also able to perform fundamental immune functions (for review, see ottaviani and franceschi, 1996). invertebrates and mammals: similarities in the immune and neuroendocrine mechanisms the data reported above demonstrates that similarities exist in both the immune and neuroendocrine mechanisms of invertebrates and mammals (fig. 1). these extraordinary parallelisms are also highlighted by the fact that the mechanisms appear to use the same signal molecules. indeed, the history of all the biological sciences demonstrates the significance and contribution of invertebrate models. in brief, cytokine-lm are present in the molluscan hemolymph, immunocytes and nervous system, and they seem to act in concert with similar effects both in molluscan and human immunocytes. another parallelism between mammals and invertebrates regards the stress response. in general, invertebrates, as mammals (for review, see blalock, 1989), present a bidirectional interaction between the immune and neuroendocrine systems. indeed, the various peptides found in invertebrates derive from neural tissue (for review, see stefano, 1989), endocrine tissue (for review, see roeder, 1995) and immunocytes, and act as messengers between the two systems, playing an important role in the autoregulatory communication between immunocompetent cells. thus, invertebrate organisms represent an ideal alternative to mammals, since they mirror in a simplified way the immuno-neuroendocrine activities in vertebrate cells. stefano’s group pioneered the use of invertebrate models to analyse the complex biological mechanisms in mammals. similar mechanisms of acth action have been found both in molluscan and in human immunocytes in schistosomiasis (duvaux-miret et al., 1992). the parasite schistosoma mansoni is able to avoid the immune response of the intermediate (the mollusc biomphalaria glabrata) and the final host(man). pomc products such as acth and βendorphin are released from the worm. during infection, the neutral endopeptidase 24.11 converts fig. 1. scheme of an unitarian immuno-neuroendocrine interaction during evolution. acth into α-msh which, in turn, deactivates the immunocytes of the intermediate and final hosts. it is also known that α-msh exhibits an immunosuppressive effect on invertebrates and mammalian immunocytes (stefano et al., 1991; van epps et al., 1992). another example of acth immunomodulation with the same characteristics in both molluscan immunocytes and human granulocytes has been demonstrated in human immunodeficiency virus (hiv) (smith et al., 1992). hiv induces the production of acth and msh by h9 t-lymphoma cells. these peptides provoke the deactivation of granulocytes in 2 h by acth and in 20 min. by msh. the longer period required by acth is because the immunosuppressive action is observed after its conversion to msh, an action modulated by nep. similar experiments performed on molluscan immunocytes revealed a superimposed response by acth and msh (smith et al., 1992). the addition of a synthetic peptide fragment of hiv gp120 blocks the movement of spontaneously active human granulocytes and mussel immunocytes, while stimulates monocyte spontaneous cell motility. moreover, when gp120 is added together with the chemotactic substances d-ala2-d-met5 enkephalinamide (dama) or il-1, a slow and not directional cell migration of both human granulocytes and mussel immunocytes is observed, indicating that gp120 reduces the chemotatctic effect of both the opioid dama and il-1. the effect provoked by gp120 seems to be due to its irreversible binding to the calcium channel (stefano et al., 1993). this would represent that an inhibitor mechanism has been conserved in molluscan and human immunocytes. 4 acknowledgement this study was supported by miur (italy) grant to e.o. references blalock je. a molecular basis for bidirectional communication between the immune and neuroendocrine systems. physiol. rev. 69: 1-32, 1989. duvaux-miret o, stefano gb, smith em, dissous c, capron a. immunosuppression in the definitive and intermediate hosts of the human parasite schistosoma mansoni by release of immunoactive neuropeptides. proc. natl. acad. sci. usa 89: 778-781,1992. franceschi c, cossarizza a, monti d, ottaviani e. cytotoxicity and immunocyte markers in cells from the freshwater snail planorbarius corneus (l) (gastropoda, pulmonata): implications for the evolution of natural killer cells. eur. j. immunol. 21: 489-493, 1991. hildemann wh, johnson is, jokiel pl. immunocompetence in the lowest metazoan phylum: transplantation immunity in sponge. science, 204: 420-422, 1979. hubert f, cooper el, roch p. structure and differential target sensitivity of the stimulable cytotoxic complex from hemolymph of the mediterranean mussel mytilus galloprovincialis. biochim. biophys. acta 1361: 29-41, 1997. malagoli d, franchini a, ottaviani e. synergistic role of camp and ip3 in corticotropin-releasing hormone-induced cell shape changes in invertebrate immunocytes. peptides 21: 175-182, 2000. ottaviani e, aggazzotti g, tricoli s. kinetics of bacterial clearance and selected enzyme activities in serum and haemocytes of the freshwater snail planorbarius corneus (l.) (gastropoda, pulmonata) during the primary and secondary response to staphylococcus aureus. comp. biochem. physiol. 85c: 91-95, 1986. ottaviani e, franceschi c. the neuroimmunology of the stress response from invertebrates to man. prog. neurobiol. 48: 421-440, 1996. ottaviani e, franchini a, franceschi c. presence of immunoreactive molecules to crh and cortisol in invertebrate haemocytes and lower and higher vertebrate thymus. histochem. j. 30: 61-67, 1998. ottaviani e, franchini a, franceschi c. pro-opiomelanocortinderived peptides, cytokines and nitric oxide in immune responses and stress: an evolutionary approach. int. rev. cytol. 170: 79-141, 1997. ottaviani e, franchini a, kletsas d. pdgf and tgf-β in invertebrate immune and neuroendocrine interactions: another sign of conservation in evolution. comp. biochem. physiol. 129c: 295-306, 2001. ottaviani e, vergine c. allo-implant in the freshwater snail planorbarius corneus (l.) (gastropoda, pulmonata). i. histological and histochemical study. zool. jb. physiol. 94: 261-267, 1990. ottaviani e. immunocytochemical study on bacterial elimination from the freshwater snail planorbarius corneus (l.) (gastropoda, pulmonata). zool. jb. anat. 120: 57-62, 1990. ottaviani e. immunorecognition in the gastropod molluscs with particular reference to the freshwater snail planorbarius corneus (l.) (gastropoda, pulmonata). boll. zool. 59: 129-139, 1992. roeder t. octopamine in invertebrates. prog. neurobiol. 59: 533-561, 1999. sassi d, kletsas d, ottaviani e. interaction of signalling pathways in acth (1-24)-induced cell shape changes in invertebrate immunocytes. peptides 19: 1105-1110, 1998. smith em, hughes tkjr, hashemi f, stefano gb. immunosuppressive effects of corticotropin and melanotropin and their possible significance in human immunodeficiency virus infection. proc. natl. acad. sci. usa 89: 782-786,1992. stefano gb, smith de, smith em, hughes tk. msh can deactivate both tnf stimulated and spontaneously active immunocytes. in: kits ks, boer hh, joosse j (eds), molluscan neurobiology, north holland, amsterdam, pp 206-209, 1991. stefano gb, sawada m, smith em, hughes tk. selective effects of human immunodeficiency virus (hiv) gp120 on invertebrate neurons. cell. mol. neurobiol. 13: 569-577, 1993. stefano gb. role of opioid neuropeptides in immunoregulation. prog. neurobiol. 33: 149-159, 1989. van epps de, mason mm. modulation of leukocyte migration by α-melanocyte stimulating hormone. in: florey e, stefano gb (eds), comparative aspects of neuropeptide function, pergamon press, oxford, pp 335-345, 1992. 432 isj 14: 432-442, 2017 issn 1824-307x review characterization and roles of lysozyme in molluscs w jielian1,2, h baoqing1 , w chungen1, y peipei1 1school of life science, education ministry key laboratory of poyang lake environment and resource utilization, nanchang university, nanchang 330031, china 2jiangxi science＆technology normal university, nanchang 330013, china accepted october 31, 2017 abstract lysozyme can hydrolyse the β-1.4-glycosidic linkage between the n-acetylmuramic (nam) and n-acetylglucosamine (nag). the three major categories of lysozymes in molluscs are goose-type, chicken-type and invertebrate-type lysozymes. the function of lysozymes is served as an innate immune protection against exogenous microbial invasion. the tyoical c-, gand i-type mollusc lysozymes are secreting type, have signal peptide and eight, six and fourteen cysteine residues, respectively. the cand g-type lysozymes are highly expressed in hepatopancreas, hemocytes and gills, and weakly expressed in foot and goand tissues of muscle. the i-type lysozyme gene is high expression in different tissues. the three type lysozymes exhibit antibacterial and digestive activity, and i-type lyaozyme also has antifungi activity. furthermore, this review includes current knowledge regarding to the genomic structure, tissue distribution of mollusc lysozyme, the antimicrobial function and mechanism. the evolution of three type lysozymes in molluscs is also discussed. these lysozymes research may help to understand the basic knowledge and to use it in the production of molluscs. key words: c-type; g-type; i-type; mollusc; antimicrobial introduction molluscs possess as much as approximately 200,000 species, which widely distribute in various ecosystem, including terrestrial, freshwater and marine environments (ponder and lindberg, 2008), and rely on innate immune systems to mediate cellular and humoral components for defense against pathogens (loker et al., 2004). in recent years, mollusc aquaculture has been facing a set back due to challenges emanating from pathogenic infections. haliotis discus hannai suffers from abnormal deaths, and results in the considerable reduction of abalone output throughout the world (zhang et al., 2004; sawabe et al., 2007). the effector of mollusc immune is crucial to better understand the immune defense mechanisms and provides the potentially feasible solutions for disease control. the innate immune system is of great importance to protect invertebrate against a wide ___________________________________________________________________________ corresponding author: w en chungen school of life science education ministry key laboratory of poyang lake environment and resource utilization nanchang university, nanchang 330031, china e-mail: cgwen@ncu.edu.cn range of microbial pathogens and encompasses a complex array of defense reactions, in which mainly focusing on immune recognition, signal transduction and effector synthesis involved in cellular and humoral immunity in the field of mollusc immunity. lysozyme is identified a classic mollusc immune effector in innate immune (wang et al., 2013), which is originally found to dissolves bacterial cell walls in human saliva and tears (haug et al., 2004), and was subsequently described in other vertebrates and invertebrate (zhao et al., 2007; whang et al., 2011; he et al., 2012; wang et al., 2012; umaasuthan et al., 2013). the enzyme is a ubiquitous bacteriolytic enzyme, which is produced by diverse groups of organisms, ranging from bacteria and bacteriophages to fungi, plants and animals (bathige et al., 2013), is characterized by their ability to bacterial peptidoglycan between two amino sugars, n-acetylmuramic acid and n-acetylglucosamine and cause bacterial cell lysis (chipman and sharon, 1969; prager and jollès, 1996), and has bactericidal and digestive ability (dobson et al., 1984; itoh and takahashi, 2007). besides antimicrobial activity, lysozymes have also proved to perform many other functions, such as growth 433 stimulation, digestion, antiviral, anti-inflammatory, and even association with tumors (irwin, 2004; wang and zhang, 2010; lee et al. 2015; xin et al., 2015), which are regarded to play important roles in the innate immunity and physiological activities, is a first line defensive protein that acts as a barrier to resist bacterial pathogen invasion in innate immune systems of invertebrates, and is widespread in many tissues and secretions (bachali et al., 2002; liu et al., 2006). extensive studies have been devoted to their structure, catalytic mechanism, relationship between structure and activity, phylogeny, immunology, and genetics (jollès, 1996). the types of lysozymes are different in amino acid sequences, biochemical and tissue distribution. the present review attempts to mainly focus on classification, distribution and function of mollusc lysozyme. it will help to improve the current knowledge about lysozyme of molluscs. classification and charecteristics of mollusc lysozyme lysozyme (ec 3.2.1.17) catalyzes the hydrolysis of 1, 4-beta-linkages between n-acetyl-dglucosamine (nag) and n-acetylmuramic acid (nam) in peptidoglycan heteropolymers of prokaryotic cell walls, and leads to the breakdown of bacterial cells (fleming, 1922; jollès and jollès, 1984a). the enzyme are generally classified into six types based on differences in structural, catalytic and immunological characteristics, including chicken-type (c-type), goose-type (g-type), plant, bacteria, t4 phage, and invertebrate-type (i-type) lysozymes (inouye et al., 1970; matthews et al., 1981; joskova et al., 2009). these types of lysozymes have been described in organisms (jollès and jollès, 1984). three types lysozymes, c, g and i-type, have been recorded in molluscs (wang et al. 2013; guo et al., 2014; zhu et al., 2016). the distribution and properties of lysozyme in molluscs are shown in table 1. table 1 the tissue distribution and characteristics of three types mollusca lysozymes species type distribution lysozyme gene accession number(s) number of amino acids haliotis discus discus c pallium, muscle, gill, digestive gland hdlysc adr70995 146 haliotis discus discus c pallium, muscle, gill, digestive gland hdlysc adr70996 146 ruditapes philippinarum c pallium, gill, hepatopancreas vpclyz-1 ago06638 156 ruditapes philippinarum c pallium, gill, hepatopancreas vpclyz-2 ago06639 153 mytilus galloprovincialis g crystalline, digestive gland mglyz1 aff18185 206 mytilus galloprovincialis g crystalline, digestive gland mglyz2 aff18186 206 argopecten irradians g pallium, gill, hepatopancreas ay788903 200 physella acuta g hepatopancreas palysg adv36303 198 chlamys farreri g hepatopancreas, gill cflysg abb53641 200 mizuhopecten yessoensis g gill, pallium, hemocytes mylysog aey77130 201 meretrix meretrix i hepatopancreas, gill mmelys adl27913 146 cristaria plicata i pallium, gill, hemocytes cplyz2 afn66526 161 cristaria plicata i pallium, gill, hemocytes cplyz1 afn66527 160 crassostrea gigas i digestive gland, basophil cgl baf48044 142 crassostrea gigas i gill, hemocytes cglys bad19059 137 haliotis discus discus i pallium, muscle, gill, digestive gland ablysg agq50336 131 crassostrea virginica i digestive gland, hemocytes, cv-lysozym e 3 bae93114 135 ruditapes philippinarum i pallium, gill, hepatopancreas tj-lysozym e bac15553 136 ruditapes philippinarum i mantles, gill, hepatopancreas rpilyz-2 ams37097 156 434 the c-type lysozyme is originally isolated from chicken-egg (itoh et al., 2007b), and subsequently is reported in other vertebrates and invertebrates, including amphibians, reptiles, mammalia, insect, crustacean and mollusc ( jollès et al., 1996; ito et al., 1999; miyauchi et al., 2000; olsen et al., 2003; liu et al., 2006). the c-type lysozymes have two 2 catalytic residues (glu53 and asp70) and 8 cysteine residues that can form 4 disulfide bonds to stabilize the protein structure (hikima et al., 2001; jimènez-cantizano et al., 2008; ye et al., 2008). several c-type lysozymes have recently been determined in mytilus galloprovincialis (wang et al., 2013), abalone haliotis discus hannai (umasuthan et al., 2013), and manila clam venerupis philippinarum (yang et al., 2017). comparison with c-type lysozyme of vertebrates, that of mollusc counterparts have not been well characterized. the c-type lysozyme of h. discus hannai is firstly described, the full-length cdna of hdlysc is 586 bp, and contains an open reading frame of 441 bp encoding a 147-amino acid protein with a calculated molecular mass of 15.64 kda, an isoelectric point being 4.87, and a polyadenylation signal (aataa). the genomic length of hdlysc is 2865 bp, and has four exons interrupted by three introns, 2 catalytic residues (glu53 and asp70), as well as the 8 cysteine residues involved in disulfide bond formation (ding et al., 2011). the homologous structure of c-type lysozymes also exists in the genome of v. philippinarum and m. galloprovincialis (wang et al., 2013; yang et al., 2017). the genome of c-type lysozyme possess 4 exons interspaced by relatively large introns in vertebrate (hikima et al., 2000), which do 3 exons separated by relatively smaller introns in invertebrate (liu et al., 2006), and even is lost in drosophila (kylsten et al., 1992). the typical genomic structures of c-type lysozymes, number and size of both exons and introns, which exist in chicken, amphioxus, mosquito and silk moth, is also found in abalone h.discus hannai, and seem difference due to the changes of the introns length (ding et al., 2011). the results indicate that the c-type lysozyme gene must have undergone unknown evolutionary events, e.g., a recombination, insertion or deletion in different lineages during evolution (larsen et al., 2009). the lysozymes could usually be divided into the calcium binding and the noncalcium binding lysozymes according to the presence/absence of conversed calcium binding residue asp (nitta et al., 1987), which of birds and mammals belong to calcium binding lysozyme (lemos et al., 1993), which of fish has not yet found calcium bindin lysozyme (saurabh et al., 2008). due to lack of calcium binding asp residue, manila clam v. philippinarum is also categorized into the non-calcium binding lysozymes family (yang et al., 2017). the g-type lysozyme is initially identified from egg of the whites embden goose (canfield, 1967), many of which is recently described from birds and fishes (nakano and graf; 1991; thammasirirak, 2001; larsen et al., 2009). the enzyme of molluscs is originally detected in argopecten irradians (zou et al. 2005; zhao et al., 2007), and is subsequently reported in other molluscs (he et al. 2012; wang et al., 2012; zhang et al., 2012; guo et al., 2014). the g-type lysozymes in birds and mammals are secreting type, and have four conserved cysteine in signal peptide that can make secreted proteins to form a more stable three-dimensional structure (jollès and jollès, 1975). all of known g-type lysozymes from argopecten irradians, chlamy farreri, mytilus edulis, physa acuta and h. discus hannai contain signal peptides, have similar three active center with (glu82, asp97, asp108), and share one conserved cysteine that also exists in birds and mammals. the six conserved cysteines are observed in mollusc g-type lysozymes, except the lysozyme of oncomelania hupensis that contains eight conserved cysteine. comparison with other g-type lysozymes of scallops, that of o. hupensis has two additional cysteines (zhang et al., 2012), and shares some features with other g-type lysozymes, such as the substrate binding sites, a signal peptide, the catalytic residues critical for the fundamental structure and function of g-type lysozymes (nakai et al., 2005, 2007). p. acuta can survive better in polluted water environment than other snails. a better understanding the immune mechanisms of p.acuta may be lead to important advances in the innate immune system of invertebrate. comparison with g-type lysozyme of other molluscs, the lysozyme of p. acuta shares the same substrate binding sites, the catalytic residues and the same six cysteines (hikima et al., 2001; zhao et al., 2007; itoh et al., 2009). the same six cysteines also appear in p. acuta，which possibly constitute disulphide bridge to result in a compact structure, and are specific in molluscs (zhao et al. 2005, 2007). the i-type lysozyme is originally described from starfish asterias rubens (jollès and jollès,1975), which is identified in phylogenetically diverse organisms of invertebrates, including porifers, molluscs, annelids, nematodes, echinodermates, hemichordates, and arthropoda (ito et al., 1999; van herreweghe and michiels, 2012). the first i-type enzyme is described in marine shellfish (hikima et al., 2001), and recently identified in other molluscs, including chlamys islandica, mytilus edulis, m. galloprovincialis, crassostrea gigas, crassostrea virginica, ruditapes philippinarum (nilsen et al. 1999; olsen et al., 2003; yue et al., 2011; zhu et al., 2016). the complete amino acid sequence of the enzyme is cloned from tapes japonica (nilsen and myrnes, 2001). the enzymes of t. japonica and c. islandica contain the same as fourteen cysteine residues (ito et al., 1999; nilsen et al., 1999), that of meretrix meretrix, r. philippinarum and c. gigas have a signal peptide and fourteen conservative cysteine residue, and all structure domains are destabilase (naoki et al., 2007; xin et al., 2011; yang et al., 2017). the typical g-type lysozyme of molluscs has six cysteine residues (zou et al. 2005), which of numbers vary ranging from zero to ten in different species (irwin and gong 2003; nilsen, 2003). the typical c-type lysozyme of molluscs has eight cysteine residues, which also exist in digestive organ (xin et al., 2011; yang et al., 2017). the high content of cysteine residues of g-type and i-type lysozymes 435 in molluscs are proposed to maintain more stable proteins that can possess a compacter structure in high osmolarity seawater and in the digestive (ito et al., 1999; zhao et al., 2007). meanwhile, the three type lysozymes of molluscs are secreting type, and have signal peptide (ito et al., 1999). the genetic structure of i-type lysozyme has is similar to that of c-type lysozyme, and both have 4 exons and 3 introns (nilsen et al., 1999, 2001; paskewitz et al., 2008). however, the lysozyme with 5 exons and 2 exons was also found in mytilidae. the i-type lysozyme genome of m. edulis comprises 5 exons instead of the classical 4 exons of the c-type lysozyme gene (bachali et al., 2002; paskewitz et al., 2008). the maximum number of exons in g-type lysozyme genome possess 7 exons from human (irwin and gong, 2003), and chicken and mice have 6 exons (nakano and graf, 1991). the g-type lysozyme of abalone h. discus discus has 7 exons and 6 introns (ding et al., 2011), and that of m. galloprovincialis do 6 exons and 5 introns (hui et al., 2008). therefore, it is suggested that i and c-type lysozyme may originate from the same ancestor. the genetype of lysozyme is more than 2 in molluscs (li et al., 2008; wang et al., 2012; wen et al., 2015; yang et al., 2017). three hypotype of the g-type lysozymes are firstly found in one species of molluscs (zhu et al., 2016), and c-, iand phage-type lysozymes are described in r. philippinarum (zhao et al., 2010; ding et al., 2014). however, relatively little is known about the hypotype of lysozymes in vertebrates. phylogenetic analysis showed that the major lysozyme genes were clustered into two main clades (fig. 1) that include g-, cand i-type lysozyme sequences. it is indicated that cand i-type lysozyme belong to the near-edge parallel macromolecules, and the cand g-type lysozyme is a parallel evolution. except for abalone h. discus discus, the cand i-type lysozymes were clustered into two main clades in molluscs. phylogenetic analysis of lysozyme gene also showed that the i-type lysozymes were clustered into main clades. chlamys islandica and calyptogena sp were clustered to the corresponding subgroup in the phylogenetic tree (fig. 2). antimicrobial protection and mechanism lysozyme is antibacterial and digestion of bacteria in the major functions, and widely distribute in the tissues or secretions of vertebrates and invertebrates (hultmark et al., 1996; irwin et al., 1996). the transcript expression of c-type lysozyme is obvious in kidney, spleen, brain and ovary tissues from paralichthys olivaceus (hikima et al., 2001). the g-type lysozyme gene replication is common in vertebrates, except for cartilaginous fish (irwin, 2014). the expression of the g-type lysozyme is a high level in the kidney of oncorhynchus (miyauchi et al., 2000), by liquidchromatography tandem mass spectrometry (lc-ms/ms), that of c-type lysozyme increase significantly in the blood cells and blood lymphocytes of biomphalaria glabrata (mollusca) after stimulated by the live bacillus megaterium (cheng et al., 1978). the activity of mollusc lysozyme is detected in the hepatopancreas, hemolymph, gills, mantles, and digestive organs, by transcripts were detected inall tissues tested (he et al., 2012; wang et al., 2012; wen et al., 2015). the distribution of the lysozymes in molluscs is shown in table 1. the c-type lysozyme transcripts are highly expressed in hepatopancreas, hemocytes and gills from v. philippinarum and h. discus hannai (yu et al., 1999; yang et al., 2017). the g-type lysozyme from the fig. 1 phylogenetic tree constructed by the neighbor-joining method in mega software based on the c, g, i-type lysozyme sequences. bootstrap support values for the nj tree are shown at the nodes (out of 1000 replicates). ruditapes philippinarum ago06638.1 ruditapes philippinarum ago06639.1 cyclina sinensis aeg19518.1 mytilus galloprovincialis ajq21538.1 ruditapes philippinarum akn35212.1 haliotis discus discus adr70995.1 crassostrea gigas baf48044.2 haliotis discus discus agq50336.1 mytilus galloprovincialis ajq21515.1 meretrix meretrix adl27913.1 ruditapes philippinarum acu83237.1 chlamys islandica cac34834.1 haliotis discus discus agq50335.1 mytilus galloprovincialis aff18185.1 physella acuta adv36303.1 mizuhopecten yessoensis aey77130.1 azumapecten farreri abb53641.1 argopecten irradians aax09979.1 56 75 58 56 100 69 30 53 97 98 60 93 96 55 56 0.2 i-type lysozyme g-type lysozyme c-type lysozyme 436 fig. 2 phylogenetic tree constructed by the neighbor-joining method in mega software based on the i-type lysozyme sequences. bootstrap support values for the nj tree are shown at the nodes (out of 1,000 replicates). mizuhopecten yessoensis is the highest expression in the hepatopancreas, gills and mantle (he et al. 2012). the i-type lysozymes of meretrix meretrix and octopus ocellatus mainly present in hepatopancreas, blood cells and gills (hultmark et al., 1996; zhao et al., 2010), that of c. virginica mainly exists in digestive gland and hemolymph (xue et al., 2007), that of r. philippinarum is the highest expression in mantle (zhu et al., 2016). the expression of i-type lysozyme in mantle is higher than that in gills, digestive glands and hemocytes from crassostrea virginica, and is abundant in the tissues of gills, hepatopancreas and haemocytes from v. philippinarum (itoh et al., 2007). the cand g-type lysozymes are highly expressed in hepatopancreas, hemocytes and gills, and are weakly expressed in the tissues of muscle, foot and goand (zou et al., 2005; zhao et al., 2007; ding et al., 2011; wang et al., 2013; umasuthan et al., 2013; guo et al., 2014; yang et al., 2017). the expression pattern of i-type lysozyme gene in different tissues probably indicate that the different biological functions of the enzyme occur during their evolution, that of gand c-type lysozymes in different organs/tissues also suggested that they may serve as some extent reflect their functional role. the major biological role of lysozymes can act as antibacterial and immune-modulating agents (hikima et al., 2001). the mrna of lysozymes from mizuhopecten yessoensis, h. discus hannai and m. galloprovincialis predominately express and execute its antibacterial activity in hepatopancreas, gills and mantle (nilsen et al., 1999; li et al., 2008; wang et al., 2011; he et al., 2012). the expression of c-type lysozyme from c. farreri is in the hepatopancreas, gill and gonad, and the higher expression level in gills may contribute to the clearance of bacteria (zhao et al., 2007). the g-type lysozyme possess combined features of the immune and digestion, and also gain the lytic activities to inhibit gram-positive and gram-negative bacteria in vitro, the g-type lysozyme s of c. farreri, m. galloprovincialis and m. yessoensis can inhibite micrococcus lysodikicus, that of physa acuta is beyond restraint to s. aureus (zhao et al., 2007; wang et al., 2013). the g-type lysozyme gene of o. hupensis is mainly expressed in hepatopancreas, and antibacterial activity was stronger than the c-t ype lysozyme (zhang et al., 2012). the i-type lysozymes are detected in hemocytes from ruditapes decussatus and r. philippinarum (yue et al., 2011). the activity of i-type lysozyme in hemocytes from mytilus edulis is higher than that from r. decussatus and r. philippinarum (pipe, 1990; carballal et al., 1997; lopez et al., 1997). the gills often face to the invasion of all kinds of pathogens, which construct of only a single layer of fragile cells and covered with a thin layer of protective mucus, were constantly flushed with water that contained pathogens (callewaert and michiel, 2010). the antimicrobial activities of two lysozymes from v. philippinarum (rvpclyz-1 and rvpclyz-2) are investigated against staphyloccocus aureus, micrococcus luteus, vibrio anguillarum, enterobacter cloacae. rvpclyz-1 displays broad spectrum antibiotic activities, and they possess strong microbicidal activities against m. luteus and v. anguillarum. rvpclyz-2 has strong inhibitory activity against all detected bacteria, but is less effective against p. pastoris km71. the turbidimetric assay is also performed to measure thelysozyme activity of rvpclyzs against m. luteus and v. anguillarum (yang et al., 2017). the recombinant cplyz1 has bacteriolytic activity against e. coli dh5a, a. chlamys islandica cac34834.1 calyptogena sp. sb2001_1;aan16211.1 calyptogena sp. sb2001_1;aan16212.1 ruditapes philippinarum acu83237.1 meretrix meretrix adl27913.1 meretrix lusoria p86383.1 crassostrea virginica bae47520.1 mytilus galloprovincialis ajq21515.1 haliotis discus discus agq50336.1 ruditapes philippinarum ams37097.1 crassostrea gigas baf48044.2 crassostrea virginica bae93114.1 cyclina sinensis aeg19518.1 100 96 83 75 61 48 50 62 40 55 0.2 437 hydrophila, staphyloccocus aureus, streptococcus sp. and staphylococcus epidermidis, and the bacteriolytic activity of cplyz1 against b. subtilis is the strongest, while the relative activity is 50 %. its relative activity against e. coli dh5 a, a. hydrophila, s. aureus and streptococcus sp. is 19 % 28 %, and against s. epidermidis is only 16 %. the bacteriolytic activity of standard lysozyme against a. hydrophila, s. aureus, b. subtilis, streptococcus sp. and s. epidermidis are higher than the recombinant cplyz1, but its bacteriolytic activity against e. coli dh5 a is lower than the recombinant cplyz1(wu et al., 2013). therefore, the lysozyme in gills of v. philippinarum shows strong antibacterial activity against gram positive and gram negative bacteria. the high expression level of mollusc lysozyme in gills implies that it has a significant contribution in prevention of microbial exploitation (matsumoto et al., 2006). however, some i-type lysozymes from venerupis philippinarum and ruditapes decussates also express in haemocytes, and exhibit antibacterial activity against gram-positive bacteria and gram-negative bacteria (lopes c, 1997; itoh et al., 2007). besides killing bacteria, the c-type lysozyme of r. philippinarum shows high antimicrobial activities, and the i-type lysozyme of v. philippinarum also has antifungi activity (goto et al., 2007). most lysozymes exhibit muramidase activity, and also do chitinase activityenzymatic hydrolysis of chitin to produce nacetyl glucosamine (yang et al., 2017; bathige et al., 2013). the result is probably the similarity between peptidoglycan (heteropolymer of β-1,4 linked nacetylmuramic acid and n-acetylglucosamine), the natural substrate of lysozymes, chitin (homopolymer of β-1,4 linked n-acetylglucosamine), and the natural substrate of chitinases. besides warding off pathogenic bacteria infections, the lysozymes have also other clear function of the chitinase activity, which of v. philippinarum, tapes japonica and crassostrea virginica are reported to possess chitinase activity, (mchenery and birkbeck, 1982; ito et al. 1999; nilsen et al. 1999; miyauchi et al. 2000; xue et al. 2004). the quaternary structure in vp-ilys crystal is revealed dimer formation by venerupis philippinarum lysozyme (vp-ilys) molecules, which is assumed to result from the dissociation of the vp-ilys dimer at high ionic strength with a high salt concentration (≥ 133 mm nacl), thereby increasing chitinase and muramidase activity (goto et al., 2007). the activity of lysozyme originated from glycosidic hydrolases is powerful to hydrolyze pgn and chitin (takeshita et al., 2003; goto et al., 2007; callewaert and michiels, 2010). the degradation of pgn and chitin in bacterial cell wall may lead to rapid killing of bacteria and fungi (elmogy et al., 2015). the lysozymes serve as the function of important digestive enzymes in some animals (dobson et al., 1984; stewart et al., 1987; lemos et al., 1993; kornegay et al., 1994; hultmark et al., 1996; prager, 1996). while the enzymes are present in a high concentration, they are a major digestive enzyme in the true stomach of ruminants (dobson et al., 1984; jollès and jollès, 1984; irwin, 1996). three-type lysozymes of molluscs are detected in digestive systems, and are regarded as digestive lysozymes (nilsen et al., 1999; olsena et al., 2003; zhao et al., 2007). digestive gland has an important lymphoid site in molluscs, and the hepatopancreas may act as a major site for the production of lysozymes (mchenery et al., 1979; jollés et al., 1996; tan et al., 2007). the i-type lysozyme of c. gigas plays complementary role in digestive organs, it has been reported that the basophil cells have an intense enzyme activity, demonstrating that lysozyme is synthesized in the digestive tubule basophil cells. the i-type lysozyme genes in the hepatopancreas of hyriopsis cumingii are down-regulated, which can inhibite bacteria to attack the host immune organs, and also promote the acid digestion of bacteria in molluscs (zhang et al., 2010). therefore, bacteria may protect themselves from lysozyme-induced digestion by down-regulating i-type lysozyme genes. the nutriments of molluscs are harvested to produce by autotrophic bacteria, the cand i-type lysozyme of c. farreri are detected to serve as digestive lysozymes in digestive tract (nilsen et al., 1999; olsena et al., 2003; zhao et al., 2007). it is postulated that lysozymes of deep-sea bivalves are similar to that of ruminants in digestive function (jollès et al., 1996). the lysozyme of m. edulis is also involved in digestion, since lysozymes from the digestive gland-associated crystalline style are believed to be purified from the digestive gland (olsen et al., 2003). in two i-type lysozymes (cv-ilys1, 2) of eastern oyster c. virginica, cv-ilys2 is mainly found in the digestive gland, which is lower amounts in the crystalline style, and is expressed in basophil cells of digestive tubules. in contrast, cv-ilys1 is mainly found in lips and mantle, and is lower amounts in gills, style sac, midgut, digestive gland and gonads (zobel et al., 1938; mchenery et al., 1985; langdon et al., 1990). the molluscs are also ability to utilize bacteria as food. the deepwater molluscs rely on symbiotic bacteria in gills for nutrition (jollès et al., 1996). the biochemical and molecular information about mollusc lysozymes is obtained from digestive systems (mchenery et al., 1979; jollès et al., 1996; ito et al., 1999; miyauchi et al., 2000; olsen et al. 2003; liu et al., 2006). the lysozymes of molluscs not only possess combined features of immunity and digestion, but also can inhibit gram-positive and gram-negative bacteria. therefore, it is suggested that the digestive lysozymes apparently evolve from parallel in different species, and acquire the ability to function in highly acidic and protease-rich environments (jollès et al., 1984; stewart et al., 1987; kornegay et al., 1994; prager, 1996; regel et al., 1998). the lysozyme can also induce regulation of the synthesis and secretion of other immune factors in vivo of animal software (zobel et al., 1983), and involve in digestion, promoting reproduction, stimulating growth, and cancer related functions, besides the common function of lysis of bacterial and fungal cell wall (irwin, 2004; zhang et al., 2005; kanda et al., 2007). the lysozyme of o. hupensis not only has the function of resisting the removal of foreign pathogenic microorganisms, but also does the function of hydrolyzing fibrin. other potential activities include isopeptidase activity and perhaps chitinase activity that is detected in both c-type (chipman and sharon, 1969; callewaert and 438 michiels, 2010) and i-type lysozymes (jollès and jollès, 1984; takeshita et al., 2003; goto et al., 2007; xue er al., 2007). however, molluscs constantly encounter various potential pathogenic microorganisms in their living environment, and the content of lysozyme is affected by a variety of environmental factors and pathogens (irwin et al., 1996). the lysozyme of m. meretrix shows strongly antibacterial activity against gram-positive and gram-negative bacteria, and the gene expression of lysozyme increases following vibrio parahaemolyticus challenge, the recombinant g-type lysozyme shows strong antibacterial activity against micrococcus luteus (xin et al., 2011). the expression levels of c-type lysozymes increase after bacterial (vibrio anguillarum) stimulation from v. philippinarum, h. discus hannai and cyclina sinensis, and the recombinant lysozyme also shows bacteriolytic activity against both gram-positive and gram-negative bacteria (goto et al., 2007; yang et al., 2017). the two lysozymes are identied from v. philippinarum, the recombinant proteins of lysozymes (rvpclyz-1 and rvpclyz-2) possess strong microbicidal activities against m. luteus and fungi. comparison with rvpclyz-1 and rvpclyz-2, the lysozyme from chicken egg-white shows lower activity against m. luteus (yang et al., 2017). the mrna expression of i-type lysozymes from m. galloprovincialis can be induced by vibrio anguillarum (hui et al., 2008). the lysozymes of r. philippinarum are designed as rpilyz-1, rpilyz-2, the expression of rpilyz-1, 2 are induced after vibrio anguillarum stimulation, vplyz mrnas are down-regulated sharply from 6 to 12 h post-infection. then, the expression level increase to the peak at 72 h, and recover to the original level at 96 h (yang et al., 2017). therefore, mollusc lysozymes have obvious antibacterial activity against v. anguillarum (bassem et al., 2006; pan et al., 2010; yue et al., 2011). while o. hupensis is infected by schistosome, the g-lysozyme gene expression significantly increase (zhu et al., 2016), p. acuta (palysg) possess to inhibite capacity against m. lysodikicus, and c. farreri ( cflysg) can not inhibite s. aureus (zhao et al., 2007). these results reveal that the c-type lysozyme is involved in the non-specific immune of molluscs. the external environment parameters, such as ph, temperature, and ion strength, can influence on the lytic activity of lysozymes (ye et al., 2010). generally, the optimal ph of the lytic activity is below 7 from mollusc lysozymes, c-type of m. galloprovincialis and r. philippinarum, g-type of o. hupensis, i-type of crassostrea virginica (umasuthan et al., 2013; wang et al., 2013). while ph is less than 7, the lytic activity of g-type mollusc lysozymes changes to follow ph (huang, 2014). however, the optimal ph of the lytic activity is generally ranging from 7 to 10 from c-type lysozymes of mammal and chicken (hui et al., 2017; yang et al., 2017). moreover, high lytic activities are detected at ph 9.5 10. similar phenomenon is also observed in lysozyme from chicken egg white with high activity at both ph 6.2 and 9.2 (davies et al., 1969). the existence of a wide range of optimal conditions for the activity of c-type lysozyme is suggested that these conditions are perhaps species-specific (bathige et al., 2013). the antibacterial activity of lysozyme in o. hupensis is examined. while the temperature is less than 50 ℃, the activity of lysozyme changes to follow temperature. therefore, the optimum temperature of lysozyme activity was 50 ℃, and the optimum ph was 7.0 (saurabh et al., 2008; ye et al., 2008). at temperature ranging from 15 ℃ to 50 ℃, while the temperature increased, the bacteriolytic activity of i-type lysozyme from cristaria plicata gradually increased. the relative activity declined when the temperature was above 50 ℃. the effect of ph on the enzyme of cristaria plicata between ph 4.5 8.5 shows that ph of the highest activity was 5.5. the optimal ph and temperature for the enzyme activity of c. plicata were 5.5 and 50 °c (wu et al., 2013; dai et al., 2015). meanwhile, the activity of i-type lysozyme from v. philippinarum is high in low temperature, and the optimal temperature is 20 ℃. the lysozyme of v. philippinarum has activity at low temperature, which is in agreement with the characteristic of coldblooded aquatic animals (yang et al., 2017). the expression profiles of mollusc lysozymes further indicate the coexistence of multiple types of lysozymes in molluscs. the most known function of lysozyme is antibacterial activity by catalyzing the hydrolysis of bacterial cell walls, and can kill bacteria using non-enzymatic bactericidal domains (dobson et al., 1984; stewart et al., 1987; lemos et al., 1993). meanwhile, the mechanisms of action are different for gram-positive bacteria and gram-negative bacteria, the cell walls of gram-positive bacteria are exposed so that lysozyme can act directly on the cell walls and cause lysis of cell walls, and the cell wall components of gram negative bacteria, such as lipopolysaccharide (lps), plii and mlic/plic, affect the cell wall of bacteria (callewaert et al., 2008; vanderkelen et al., 2011). therefore, lysozyme should be combined with other components of the immune system in order to lysis the cell wall structure of gram negative bacteria, resulting in bacterial lysis death (cheetham et al., 1992). the lytic activity of lysozyme against bacteria and fungi is suggested to be associated with the muramidase and chitinase activities. the c-type lysozymes typically possess muramidase activity that cleaves the -1, 4-glycosidic bond of peptidoglycan (pgn) in microbial cell walls, and cause the lysis of bacteria (vocadlo et al., 2001; supungul et al., 2010). the lysozyme is also served as a model for studies on enzyme structure and function (peters et al., 1989; prager and jollès, 1996). typical i-type lysozymes exhibit muramidase activity and generate bactericidal activity by hydrolyzing the cell wall, which show bacteriolytic activity against both gram-positive and gram-negative bacteria (zhao et al., 2010; zhou et al., 2017). conclusion and perspective lysozymes are present in variety of organisms, ranging from viruses to plants and animals. although all lysozymes perform the same enzymatic function, and exhibit overall similarity in three dimensional (3d) structures, the primary amino acid sequences of these lysozymes is rarely the same. it is speculated that the 3d structure and function of the enzymes 439 are analogous, and the genes of the enzymes are not homologous. the various types of lysozymes are generated by convergence during evolution, and can coexist in the same taxon. for example, the cand g-type lysozymes are in vertebrates. the cand i-type lysozymes are present in arthropod, the c-, i and g-type lysozymes exist in molluscs. the question of evolutionary relationship is raised among different types of lysozymes. the phylogenetic tree analysis shows that i-type lysozyme is more closely related to c-type one than g-type one in molluscs. the partial sequence of i and c-type lysozyme gene is homology. the central exon of lysozyme genome from m. galloprovincialis is homologous to the second exon of that from chicken, and both belong to the c-type lysozyme (wang et al., 2013). it is suggested that cand i-type lysozyme belong to the near edge parallel macromolecules, and is believed that cand i-type lysozyme gene evolved from a single complete gene. the i-type of c. gigas, g-type of h. discus discus and m. galloprovincialis, was also clustered to the corresponding subgroup in the phylogenetic tree. however, the other evolutionary relationship of three type lysozymes also is supposed (jollès and jollès, 1984; bachali et al., 2002). some studies assumed that i-type lysozymes were more closely related to g-type lysozymes, and suggested that c-type was basal (implication ancestral) to gand i-type lysozymes (hikima et al., 2003). the iand g-type lysozymes diverge from an ancestor of c-type. others believe the g-type lysozyme is considered as the common ancestor to cand i-type ones (thunnissen et al., 1995). the i-, gand c-type lysozymes are detected in molluscs, and this may provide some clues to clarify the relationship of the three types of lysozyme (xin et al., 2011). these results consist with the notion that the three type lysozymes diverge from a common precursor, and c-type lysozyme is closed to the ancestor. further, the molluscs encounter a greater range of bacterial strains or species in the marine environment, and the varied composition and structure of the bacterial cell wall may promote a type of ‘substrate-induced evolution’ of lysozymes (jollès and jollès, 1984). two conserved amino acid glu54 and asp70 are critical for the c-type lysozyme lytic activity to bacterial cell wall, and the motif that flanking asp70 is also conserved in the c-type lysozyme (vocadlo et al., 2001). these results indicate that the mature c-type lysozyme of molluscs may possess the antimicrobial activity as well as that of other species. other potential activities, such as isopeptidase activity and perhaps chitinase activity, are detected in c-type lysozymes (chipman and sharon, 1969; callewaert and michiels, 2010). in conclusion, the c-type lysozymes are characterized from some molluscs, and their expression profiles and antimicrobial activities are also investigated. these results provide helpful evidence for further understanding the innate immunity of molluscs. more investigation should be directed to understand the interaction mechanisms of c-type lysozymes with membranes or cell walls of bacteria. o. ocellatus has three conservative enzyme activity center (glu40, glu49, ser52) and 12 conserved cysteines that form 4 pairs of protein disulfide bonds and the stable conformation. the characteristics of i-type mollusc lysozyme structure possess two catalytic domains exhibiting muramidase and isopeptidase activities (jollès and jollès, 1984b; ito et al., 1999; takeshita et al., 2003; xue er al., 2007; goto et al., 2007). although lysozyme research is described in 1960s, the data about lysozyme is increasingly abundant. so far, 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cells of marine/aquatic invertebrates: from basic research to innovative applications (maristem), february 5-6, 2018, marine biology station, national institute of biology, piran, slovenia organizers: a ramšak national institute of biology, marine biology station piran, slovenia expression study of molecular markers involved in staminality and differentiation in the colonial ascidians botryllus schlosseri f ballin, n franchi, a peronato, l ballarin department of biology, university of padova ascidians are invertebrate chordates, members of the subphylum tunicata that represents the sister group of vertebrates. they offer the opportunity to investigate and compare the behaviour of both embryonic and adult stem cells. morphological data suggest the presence of undifferentiated haemocytes (haemoblasts) able to proliferate and give rise to terminally differentiated cells. relevant studies were also carried out in the neural lineage, in which neural progenitor cells regenerate the brain after extirpation. in b. schlosseri, during the cyclical generation change, bud primordial cells, probably deriving from a pool of long-living stem cells, are able to give rise to the neural complex. we screened the b. schlosseri genome and transcriptome, looking for transcripts/genes showing similarity to vertebrate molecular markers of haematopoietic and neural stem cells. four sequences, orthologous to mammalian transcripts considered markers of haematopoietic progenitor cells, were identified in b. schlosseri. they are: bsabcg2, bscd133, bsgata1/2/3 and bsgata4/5/6. in situ hybridization on haemocyte monolayers and colony sections, resulted in labelling of cells in the sub-endostylar haemolymph lacunae. this results matches previously morphological data that identified the endostyle as a stem cell niche. quantitative real time pcr (qrt-pcr) highlighted the over-expression of the considered genes in the mid-cycle phase of the blastogenetic cycle. during this phase, there is the formation of new secondary buds emerging from the primary buds. the high expression levels of bsabcg2, bscd133, bsgata1/2/3 and bsgata4/5/6 genes in the mid-cycle phase reflect the presence of undifferentiated cells involved in proliferative and differentiation events required for giving rise to the new blastogenetic generation. for the neural lineage, we identified and characterised two transcripts orthologues of vertebrate neural stem cell markers (bssox2 and bsmsi2). we also studied the expression, during the blastogenetic cycle, of a panel of genes already known to be involved in ascidian larvae neurogenesis, i.e., orthologues of pax2/5/8, hox1 and hox3. ish with riboprobes for bssox2, bsmsi2, bspax2/5/8, bshox1 and bshox3 revealed a common labelling in the endostyle niche. the presence of bssox2, bsmsi2, bspax2/5/8, bshox1 and bshox3 transcripts in the cells of the region known to be a stem cell niche, led us to conclude, not only that our probes identified undifferentiated cells but even that in b. schlosseri are probably present a single population of pluripotent stem cells that could differentiate into haematopoietic or neural cells. the qrt-pcr, showed an high expression level in the mid-cycle phase of all the putative neural markers considered. in this phase new secondary buds are produced from primary buds. each new bud needs its own neural complex and this requires the proliferation of undifferentiated cells to originate neural gland rudiment and cerebral ganglion. bssox2, bsmsi2, bspax2/5/8, bshox1 and bshox3 increased their expression associated with these neurogenesis events and this support their involvement in neural stem cell differentiation. 105 on all these sequences, we also performed a phylogenetic analysis that, always, returned us the tunicate relevant position, within the protochordates cluster, of vertebrate sister group. studying colonial ascidians in a landlocked country s blanchoud department of biology, university of fribourg, switzerland tunicates belong to the chordata phylum, phylogenetically positioned between the more basal cephalochordata and the higher vertebrata, of which they are considered the closest relatives. these organisms include a wide range of reproductive methods, regenerative abilities, developmental strategies and life cycles. in addition, and despite a drastically different body plan during their adulthood, tunicates have a tissue complexity related to that of vertebrates. consequently, the study of these organisms offers a unique evolutionary perspective into the emergence of clade specific traits and the function of conserved molecular mechanisms. in particular, colonial ascidians are established models for important biological processes including allorecognition, immunobiology and angiogenesis. furthermore, these sessile compound organisms are the only known chordates that can undergo whole-body regeneration, whereby a fully functional adult is restored from a minute portion of their vascular system. identifying and investigating the shared regulatory mechanisms and signalling pathways required for successful regeneration in the closest relatives of the vertebrates is of interest to regenerative medicine and ageing research. however, the current paucity in breeding infrastructures and technics limits the study of ascidians to coastal regions and thus hinders their wider scientific spreading and popularity. our group is interested in dissecting wholebody regeneration in the viviparous styelidae botrylloides leachii. to enable such research in switzerland, we will develop a custom recirculating husbandry setup and identify optimal rearing parameters for the long-term culture of b. leachii. the designed equipment will be highly flexible to adapt to the needs of additional colonial ascidian species, as well as to those of other tunicates. the establishment of a standalone system for controlled breeding will provide the necessary platform for the worldwide spreading of colonial ascidians as model organisms. our goal is to promote research in botrylloides and thus offer a unique evolutionary perspective into chordate processes and into regeneration in particular. mitochondria inheritance and germ line formation in bivalves a burzyński institute of oceanology, polish academy of sciences doubly uniparental inheritance of mitochondria, a phenomenon known discovered in several bivalve families, defies the common rule of strictly maternal mitochondrial inheritance, commonly seen in animals. under dui system, paternal mitochondrial dna is retained in male offspring, leading to heteroplasmy in males. the two emerging mitochondrial lineages are usually significantly divergent and easily distinguishable. neither the mechanics nor evolutionary context of this phenomenon are fully understood, however the mechanism of paternal mitochondria retention in male zygotes involves sorting of sperm mitochondria at early stages of larval development, in all examined cases. thus, the process of germ line formation in bivalves is intimately linked with dui. therefore, both stem cell research and dui research will benefit from developing cellular models of bivalvian origin. genomic controls of skeleton development and regeneration in the brittle star amphiura filiformis a czarkwiani1, vd dylus1, l piovani2, b cambiaghi2, m sugni2, p oliveri1,3 1department of genetics, evolution and environment, university college london, gower street, london, wc1e 6bt, uk 2department of biosciences, university of milan, italy 3ucl centre for life’s origins and evolution (cloe), ucl, gower street, london, wc1e 6bt, uk regeneration of adult body parts can be considered a by-product of development, however to which extent development and regeneration use the same molecular mechanisms and regulatory program is still an open question. echinoderms are well known for their extensive regenerative abilities and well-characterized embryonic development. the aim of our study is to understand at which level the regulatory program governing the production of biomineralized skeleton is conserved between the developing embryo and the regenerating adult arm of the brittle star amphiura filiformis. to understand the cellular and molecular aspects of skeletogenesis in these two developmental contexts, we used candidate and differential transcriptome approaches in conjunction with histological and high-resolution spatio-temporal gene expression analyses. we also dissected the role of the fgf signalling pathway in these two processes using a well established signalling inhibitor. we found that 23 embryonic skeletogenic genes (transcription factors, signaling receptors and downstream differentiation genes) are also expressed in mesenchymal cells in the dermal layer of the adult regenerating arm, where skeletal spicules form. this indicates a very similar molecular signature of embryonic and regenerative sclerocytes. fgf signalling perturbation using the su5402 inhibitor interferes with skeleton formation during both embryonic development and adult regeneration of this brittle star. a comparison of 115 genes affected by su5402 in adult arm regeneration and during embryonic development revealed a large conservation of molecular function of fgf signalling 106 between those two developmental contexts. taken together these data suggest the re-usage of the developmental regulatory program for skeleton development during regeneration in amphiura filiformis. however, important differences are revealed in the early developmental genes that are not expressed in adult regeneration. future work will aim at identifying the source of sclerocytes during regeneration and the initial factor(s) responsible of reactivating the skeletogenic program during regeneration. pocilloporid corals as laboratory models for research on somatic stem cells in scleratinians i domart-coulon mnhn mcam laboratory, umr7245 cnrs mnhn, sorbonne-université, 57 rue cuvier (cp54) 75005 paris scleractinian corals are evolutionary ancient, long-lived marine animals with high regeneration abilities, which can provide perspectives to research on stem cells, aging and regeneration processes. reef-building colonial species are formed of a multitude of polyps connected together by coenosarc tissue. this thin (few millimeters to centimeters) tissue forms a hard calcareous exoskeleton which continuous accumulation over the years provides structural framework for many other reef species, sustaining rich marine biodiversity. complex microbial communities are hosted within the animal tissue and skeleton, contributing an additional layer of complexity, with a major trophic role for example for the photosynthetic dinoflagellate symbionts. despite their slow growth rates, the process of continuous coral extension via clonal budding of new polyps provides opportunities to study the involvement of somatic stem cells in tissue morphogenesis, and their commitment to various differentiated coral cell lineages. however, to this day fundamental processes in coral cell biology remain relatively underexplored. laboratory models are needed to identify coral stem cells and investigate the balance controls between coral cell proliferation and differentiation. pocilloporids are widespread indo-pacific species with a branched growth form, and the pocillopora genus contains key pioneer species for the colonization of new reef substrates, prior to the successive installation of other corals. the pocillopora damicornis species complex provides laboratory models which are easy-to-grow in aquarium microcosms, and for which there exist a large pool of publically available datasets on ecology, physiology (symbiosis, biomineralization), reproductive biology, immune system and disease susceptibility. these models are frequently selected for establishing primary cell cultures, which are used for applications in short-term (few days to few weeks) in vitro physiology and ecotoxicology studies. regarding ‘omics’ resources for pocillopora damicornis, although a reference genome has not yet been released, the pocilloporabase transcriptome database can be searched for gene transcripts, and very recent metabolomics datasets exist. we selected pocillopora damicornis type beta, sensu schmidt-roach et al. 2014 (= pocillopora acuta lamarck 1916) as a model species, propagated in long-term aquarium cultures (at aquarium tropical du palais de la porte dorée in paris and oceanopolis brest), and genotyped its dominant symbiodinium clade c1 dinoflagellate endosymbionts and ostreobium (ulvophyceae) microborers. pocillopora acuta produces brooded planula larvae, emitted periodically few days before full moon (variable number of released larvae across colonies of the same size). we used aquariumgrown, biofilm-covered larval settlement substrates, for which settlement success rates are over 75%, allowing us to study biomineralization processes and cellular turnover during larval metamorphosis and in the forming primary polyp. regular budding of polyps in the rapidly spreading juvenile colonies (few days to 1 month post-settlement) offer a window into morphogenetic processes, from the accumulation of cells into a locally raised tissue area to the opening of a slit around which tentacle start budding, forming the new oral disk. this evolutionary ancient and ecologically important metazoan model should benefit from comparative approaches with more established marine or freshwater cnidarians models with advanced cellular and molecular tools, to provide new insights into the evolution of stem cell systems. mechanisms of cell recruitment in echinoderm regeneration: pluripotent versus dedifferentiated cells c ferrario1,2, f bonasoro1, md candia carnevali1, m sugni1,2 1dipartimento di scienze e politiche ambientali, università degli studi di milano, milano, italia 2center for complexity & biosystems, dipartimento di fisica, università degli studi di milano, milano, italia regenerative abilities are remarkably widespread among echinoderms. indeed, at all life stages these marine deuterostomes are capable of regenerating both external and internal body parts following self-induced or traumatic mutilations. although two different mechanisms are usually employed to describe the regenerative processes, namely epimorphosis and morphallaxis, in the case of echinoderm regeneration the origin and fate of the involved cells are still unclear. an up-to-date overview of the cell recruitment during this process in all the five echinoderm classes is here provided in order to clarify the state of the art on this topic and therefore highlight the necessary future steps to cover this gap of knowledge. among stellate echinoderms, crinoids are the only group clearly displaying the recruitment of morphologically undifferentiated cells stocked in the stump tissues (i.e. coelom and brachial nerve) and their active migration to form a true blastema where they massively proliferate and differentiate to regenerate the lost tissues. dedifferentiation occurs only in specific cases, such as basal arm amputations or stress situations. 107 both brittle stars and starfish do not show a true regenerative blastema, apparently mainly relying on dedifferentiation phenomena with subsequent cell re/trans-differentiation. in starfish, dedifferentiation is massively employed in muscle tissues. additionally, scarcely differentiated cells are apparently recruited via epithelial-mesenchymal transition (emt) from distant sources (i.e. coelomic epithelium, pyloric caeca). the same occurs for brittle star cell recruitment with an important contribution of the coelomic epithelium as source of progenitor-like cells after emt. sea cucumbers are studied mainly for nervous system and gut regeneration. in the former, the absence of “stemness” marker in the transcriptome suggests that radial nerve cord regeneration depends on dedifferentiation of the supporting cells that re-differentiate in both the same cytotype and new neurons. massive myocyte dedifferentiation is employed during gut regeneration. in sea urchins, damaged test and broken spines are reformed through dedifferentiation of stump cells with only minor local cell proliferation, whereas totally removed spines are regenerated via undifferentiated (pluripotent) cells. overall, echinoderm regeneration mainly relies on dedifferentiation phenomena rather than recruitment of pluripotent cells already stocked in the stump tissues but the precise origin and fate of the involved cells are still largely unknown. echinoderm tissues, especially coelomic epithelium and muscles, show a high level of plasticity and cell proliferation, migration, and emt play key roles in this process. cell tracking, and coupled molecular and microscopy approaches will be strongly necessary to define the main challenge of echinoderm (and, in general, animal) regeneration, namely the understanding of the origin and fate of the recruited cells. hydra, a model for studying the impact of autophagy on stem cell behaviour b galliot1, q schenkelaars1, n suknovic1, y wenger1, s austad2, s tomczyk1 1department of genetics and evolution, ige3, university of geneva, geneva, switzerland 2department of biology, university of alabama at birmingham, usa background: hydra vulgaris (hv) exhibit a negligible senescence as their epithelial stem cells (escs) and interstitial stem cells (iscs) continuously divide. by contrast h. oligactis (ho) that undergo gametogenesis upon transfer to cold, develop an aging phenotype and die within four months (brien, 1953; yoshida et al., 2006). to investigate the mechanisms of aging and resistance to aging in hydra, we characterized two ho strains, one cold-resistant (ho_cr) where animals adapt and remain healthy, another cold-sensitive (ho_cs) where animals age (tomczyk et al., 2015). thus the mechanisms that lead to resistance to aging can be dissected in hydra. results: we first show that gametogenesis, which leads to a dramatic depletion in interstitial somatic derivatives, is actually not necessary for aging in ho_cs animals. indeed when we treat ho_cs animals maintained at room temperature with hydroxyurea, as such as they lose their iscs in the absence of gametogenesis, these animals also rapidly develop an aging phenotype. we previously showed that in h. vulgaris escs adapt to isc loss by up-regulating a large subset of genes, including genes involved in neurogenesis and neurotransmission (wenger et al., 2016). therefore we reasoned that aging-sensitive animals (ho_cs) might not tolerate isc loss as a consequence of a lack of epithelial adaptation. in fact, we first found that in ho_cs but not in ho_cr, escs irreversibly lose their self-renewal potential within the first six weeks of aging. we have also investigated epithelial autophagy and found it strongly deficient in ho_cs, with limited autophagosome formation during starvation, a poor inducibility of the autophagy flux measured in vivo upon mg132 or rapamycin treatment, and an accumulation of p62/sqstm1 levels. also kinase assays show a constitutivelyrepressed ulk1 activity in ho_cs. chronic rapamycin exposure delays aging by sustaining epithelial self-renewal, but surprisingly this treatment stimulates epithelial phagocytosis without rescuing autophagy. finally, we were able to induce aging in aging-resistant hv animals by inhibiting autophagy through the silencing of an early component of the autophagy machinery wipi2. conclusions: this study highlights the essential role of autophagy on epithelial stem cell renewal to maintain low senescence in hydra. this longevity mechanism, novel in early-branched eumetazoans, values the hydra model for aging studies. platynereis dumerlilii, a new model to study the involvement of stem cells during growth and regeneration e gazav1, a planque2, j male2, p alvarezcampos2, m vervoort1 1institut jacques monod, cnrs, umr 7592, paris france 2université paris diderot, sorbonne paris cité, f75205 paris france stem cells are the subject of intense research in biology and medicine. in many species, various categories of stem cells are present at postembryonic stages and participate to processes such as growth and regeneration. the main experimental model of our team is the marine annelid worm platynereis dumerilii, an emerging developmental biology model that has proven to be very useful for large-scale evolutionary developmental comparisons. platynereis worms continuously grow during most of their life (a process called posterior elongation) and possess important regeneration abilities, two features that are widespread in animals, but not found in classical developmental biology models. we evidenced the presence of two populations of putative pluripotent/multipotent stem cells located in a posteriorly-localized growth zone (gz) responsible for the continuous elongation of the body. these cells express a molecular signature 108 composed of about 20 genes, such as piwi, vasa and nanos, whose orthologs are known to be expressed in pluripotent somatic stem cells and primordial germ cells in other animals. platynereis has also extensive posterior regeneration abilities: after amputation of their posterior part, including the pygidium (terminal part of the worm), the gz and many segments, the worms will efficiently regenerate, from a regeneration blastema (a mass of undifferentiated progenitor/stem cells that forms at the amputation site), the pygidium and the gz, which will in turn allow the elongation of the body. preliminary data indicate that cells of the blastema share at least a part of the molecular signature of the stem cells of the posterior gz. using gene candidate and transcriptomic approaches, we currently expand with additional genes the molecular signature of the gz stem cells and blastemal cells. our data provide new insights about the evolution of stem cells and their involvement in growth and regeneration in animals. germinal stem cells in their niche in the oyster magallena gigas (crassostea gigas) c heude berthelin1, m cherif feildel1, c lelong1, n elie2, d goux2, b adeline1, k kellner1 1umr borea, biology of aquatic organisms and ecosystems, university of caen, normandy, esplanade de la paix, 14 032 caen, france. 2cmabio3, microscopic centre applied to biology, university of caen, normandy, esplanade de la paix, 14032 caen, france. at the origin of the germline, there are the germinal stem cells (gsc) and the study of the gsc is essential in animal organism in order to understand the sexual reproduction. our work is interested in the gsc study in a mollusk, the pacific oyster magallana gigas (ex-crassostrea gigas). our original model presents some essential benefits for our researches as the genome availability and the fact that the gonad constitutes 80% of the animal weight during the sexual maturity period. to identify and locate the germinal stem cells in the oyster, we used some conserved and classical criteria of the gsc. firstly, we used the location criteria because gsc are found at the edge of the gonad structures, equivalent to gonadal tubules in oyster. secondly, we combined morphological and molecular criteria with the observation of the chromatin decondensation and the labeling of a specific germ cell protein named vasa. the chromatin appears decondensed in gsc nucleus, that denotes a low mitotic activity specific in quiescent cells as gsc (chiarini-garcia and russell, 2001, 2002). concerning the molecular criteria, the protein vasa is known as a specific marker of the germinal cells including the gsc in several organisms from invertebrates to vertebrates (juliano et al., 2010). vasa is present in oyster in germ lineage and we used a specific antibody developed against the oyster vasa protein oyvlg (fabioux, 2004, 2009). these cellular and molecular criteria are not sufficient to identify for sure the putative gsc in oyster. that’s why we developed an additional approach of quantitative screening on tissue sections (quantitative histology). this approach allowed us to visualize a subpopulation of putative gsc and gave us information about the nucleus shape of these cells. looking at the ultrastructure of these cells, we identify two types of putative gsc by transmission election microscopy (with two nucleus shapes) or more probably two differentiation stages of gsc. we also became interested in the description of the specific microenvironment, named germinal niche, of gsc in the gonad. this last part will be helpful for a better identification of cells neighbouring gsc and that could be implied in the regulation of stemness and differentiation of gsc pool. finally, to study the functioning of the germinal niche, we develop in vitro tools of enrichment of populations in gsc or associated somatic cells. these enrichments were performed by differential adhesion of the cell fractions of by cell sorting based on aldehyde dehydrogenase activity. action of beta-catenin and myc signaling in the hydra interstitial stem cell system b hobmayer1, s glasauer1, b artes1, k bister2, m hartl2 1institute of zoology, university of innsbruck, austria 2institute of biochemistry and center for molecular biosciences, university of innsbruck, austria myc factors are known for their roles in stem cell maintenance, cell cycle regulation, and cancer. they are in fact evolutionarily old: they evolved at the transition from pre-metazoan colonies to true metazoans and have since not dramatically changed their protein structure. cnidarians are the only metazoan phylum together with vertebrates to show ancestral diversification of the myc gene family. cnidarian genomes commonly encode at least four myc paralogs. in hydra, two of the four myc factors are structurally and functionally similar to mammalian c-myc. two hydra myc factors, however, exhibit a unique and derived n-terminal protein structure that has no homology hit in structural data bases. we have meanwhile studied structure and function of three hydra myc factors. analysis of gene expression patterns demonstrates that the three corresponding myc genes are activated in interstitial stem cells and their proliferating derivatives. two of these three genes seem to be directly regulated by wnt-beta-catenin signaling. unexpectedly, myc1 shows pronounced down-regulation in beta-cat-transgenic and alsterpaullone-treated polyps. this is different to mammals, where beta-catenin is one of the bestknown upstream regulators strongly activating cmyc. myc1 repression by hydra beta-catenin also occurs in reporter assay experiments in vertebrate cell culture. we have started to approach functional interference with beta-catenin and myc1 by using transgenic approaches, sirna, and small molecule inhibitors, and we find effects on decision making in the interstitial stem cell system. a model how beta-catenin and myc factors affect stem cell dynamics, self renewal and differentiation is discussed. 109 metagenomics of the northeastern mediterranean; cave and marine habitats a karahan, s küçükavşar, k gökdağ, ae kıdeyş, b temiz, e öztürk middle east technical university, institute of marine sciences, department of marine biology and fisheries, mersin, turkey next generation sequencing (ngs) technology provides great amount of information about marine biodiversity as well as functional diversity and active metabolism. metagenomic analyses using ngs technology have revealed numerous previously unrecognized microorganisms. in the present study, bacterial community of four different depths samples (0, 25, 150, 200 m) from a north-eastern mediterranean offshore station (200 m) and a surface water sample from a seaside cave lake were studied using metagenomics amplicon sequencing technique. targeted marine samples were micro-plastic particle-attached microorganisms and cave sample was both free-living and particle attached organisms. about 30,000 operational taxonomic units (otus) from 695,000 sequences were observed for all the depths of marine samples and 13,000 otus from 162,000 sequences for the cave sample. pseudomonadales (proteobacteria) was the most dominant order in the particle attached marine environment, which the members of taxa are capable of degrading polycaprolactone (pcl). another common bacterium for marine environment was phenylobacterium that degrades the chloridazon herbicide. on the other hand nitrosporia, which found as the most abundant group for the cave lake, plays a role in the nitrogen cycle by performing nitrite oxidation in the second step of nitrification. this is a first study using a culture-independent approach for identify the northeastern mediterranean free-living and particleattached marine and cave habitats microorganisms. how to identify and isolate bivalve cells for in vitro applications: tools, first results and perspectives k kellner, c heude berthelin, c lelong, b adeline, d goux, n elie, a franco, r travert, h koechlin umr boreabiology of aquatic organisms and ecosystemcnrs-mnhn-umpc-unicaen-irdua, university of caen-normandie-esplanade de la paix cs 14032caen cedex 5 france our laboratory has a quite long history in investigating the neuroendocrine control of various processes in molluscan species. we have developed specific bioassays in mytilus edulis and crassostrea gigas allowing to evaluate physiological processes of isolated cells maintained in primoculture for several days (dna and protein synthesis, glycogen metabolism…). the presentation aims to propose a situational analysis of our background regarding the bivalve cell culture assays. one of the major difficulties concerns frequent contaminations (bacteria, fungi and protozoa). the use of antibiotics and antifungal substances in the suspensions of dissociated cells may be combined with density gradient allowing to separate cells from contaminants. dissociation processes consist in a combination of mechanical and enzymatic treatments. cell enrichment in one cell type relies on the possibility to identify some of these cell fractions using specific markers (antibodies, transcriptomic probes, enzymatic activities…). three examples of cell fractionation are illustrated: 1enrichment of oyster germinal cells of the male lineage using staput bsa gradient associated to cell identification based on ultrastructural characteristics of collected cells combined with flow cytometry (dna quantity and mitochondrial labelling). 2germinal stem cells enrichment based on differential adhesion in flask assessed by expression of stemness markers (klf4, sox) 3germinal stem cells enrichment based on facs cells sorting (aldh activity). proliferation and viability of cell are currently checked using trypan blue, mtt or brdu incorporation tests. the impedance measurement is also an interesting tool for adherent cells (xcelligence system, ozyme), allowing a real time, label free, sensitive cellular analysis of cell number, adhesion, viability and morphology. first attempts on c.gigas hemocytes provided low cell index regarding to vertebrate tumoral cells, potentially due to reduced cell size, low cell adhesion and restricted proliferative activity. cellular dynamics during clytia medusa regeneration l leclère villefranche developmental biology lab (lbdv), sorbonne universités-upmc-cnrs, villefranchesur-mer, france. marine metazoans show remarkable regenerative capabilities. cnidarians, in particular, have long been shown to possess very efficient repair mechanisms. research has so far mainly focused on the regeneration of the polyp form, with studies on hydra, nematostella and hydractinia. in contrast, medusae were thought to have lower regenerative capacities, due to their anatomic complexity: striated muscle, a well-organized neurosensory system, and well defined organs (manubrium – feeding organ, tentacle bulbs and gonads), connected by a system of radial and circular canals. the hydrozoan clytia hemisphaerica has recently emerged as a model in evolution, cell and developmental biology. its complex life cycle, including a planula larva, colonial polyps and pelagic medusae, is controlled in the lab, and tools for functional analyses, as well as assembled genome and transcriptomes, are now available. we could demonstrate that clytia medusae also possess remarkable regenerative capacities, being able to restore both their organs and body form. mouth, gonads and tentacle bulbs harbor pools of multipotent stem cells, and show high levels of cell proliferation. dissection and grafting experiments – each organ can survive as an autonomous unit – are allowing us to address the interplay between the stem cell pools. i will present our current advances 110 in the characterization of the cellular and molecular processes during clytia medusa regeneration, offering a fresh perspective on our understanding of cnidarian regeneration and medusa patterning. gabab signaling regulates metamorphosis, neurogenesis and regeneration in the sea anemone nematostella vectensis s levy, v brekhman, t lotan marine biology department, the leon h. charney school of marine sciences university of haifa, haifa, 31905, israel gaba has multiple functions in mammalians during early development and in the adult, promoting neurogenesis progenitor proliferation as well as synaptically inhibiting neurons. we found that in the basal sea anemone nematostella vectensis, gaba play roles in planula-to-polyp transformation and during regeneration, which are mediated by gabab receptor. the effects of chronic application of gabab agonist on the developing nervous system as well on the physiology of developing planulae will be discussed. arachidonic acid metabolism in marine invertebrates h lõhelaid, t teder, n samel laboratory of lipid research, department of chemistry and biotechnology, tallinn university of technology, akadeemia tee 15, 12618 tallinn research goals: our research group focuses on discovering novel lipid mediators (eicosanoids and other oxylipins) in marine organisms, identification of their biosynthetic routes, and characterization of genes and proteins involved in the lipid metabolism. oxylipins, oxygenated lipid mediators formed from various polyunsaturated fatty acids, are well-established stress mediators, synthesized mainly by lipoxygenase (lox) and cyclooxygenase (cox) in vertebrates, and by loxdependent pathways in plants. in soft corals, along with cox and lox enzymes, the initial oxidation of arachidonic acid (aa) is also catalyzed by a catalase-related allene oxide synthase-lipoxygenase (aos-lox) and hydroperoxide lyase (hpl-lox) fusion proteins. techniques used: we are specialists in biochemistry and molecular biology of fatty acid dioxygenases. in our experiments we routinely use bioinformatics for evolutionary and structural studies, clone and express enzymes in the bacterial, yeast and insect expression systems, conduct sitedirected mutagenesis, purification and kinetic characterization of recombinant proteins, define their structures (x-ray crystal studies) and analyze enzymatic reactions; separate and analyze the formed products by hplc, mass-spectrometry (lcms and gc-ms) and uv-visible spectroscopy and identify novel compounds by nmr. in addition, we perform gene expression profiling by rt-qpcr. we also study biomolecular interactions by surface plasmon resonance, fluorimetry and calorimetry. maintaining of the marine aquarium and providing a suitable environment for cultivating and propagating corals is also essential, as part of our work includes experiments in vivo. main findings: we have unraveled eicosanoid biosynthetic routes in corals (varvas et al., 1999), amphipods and red algae. specifically, we have discovered and characterized (i) 15r-specific cox from caribbean coral plexaura homomalla (valmsen et al., 2001) and the first algal cox from gracilaria vermiculophylla (varvas et al., 2013) (ii) lipoxygenase (lox) with a unique arachidonate 11r-specificity from arctic coral gersemia fruticosa, (iii) catalase-related allene oxide synthaseand hydroperoxide lyase-lipoxygenase (aos-lox and hpl-lox) fusion protein pathways in soft corals (koljak et al., 1997; teder et al., 2015). furthermore, we have defined the x-ray structure of 11r-lox (eek et al., 2012), analyzed the oxylipin metabolism in corals in vivo and shown the involvement of oxylipins in the stress response of soft coral capnella imbricata (lõhelaid et al., 2014, 2015), and determined oxylipin profiles of multiple stony corals, acropora millepora, a. cervicornis and galaxea fascicularis (lõhelaid and samel, 2018). our results suggest that although the profiles of lipid mediators vary among species, the enzymes involved in oxylipin synthesis are highly conserved from invertebrates to vertebrates providing an excellent tool to study complicated mammalian signaling pathways in invertebrates as model organisms. the tunicate botryllus schlosseri: a model for developmental and evolutionary studies l manni department of biology, university of padova, italy. ascidians are marine, filter-feeding chordates, belonging to the subphylum tunicata, which are considered the sister-group of vertebrates. in the last years, several solitary ascidians (such as ciona intestinalis and halocynthia roretzi) have emerged as model organisms to study the molecular control of embryogenesis and cell lineage. their genome has been partially or fully sequenced and different molecular tools are now available. colonial ascidians are less well known at molecular level. however, they offer the opportunity to study the development of clonal individuals during asexual reproduction and to compare, in the same organism and at various levels (molecular, biochemical, morphological), sexual and asexual reproduction. botryllus schlosseri is a colonial ascidian whose genome has been sequenced and the anatomical and developmental ontology is available. fertilisation is internal and embryos develop inside the parent attached to placental cups. at 18 °c, larvae develops in a week and then hatch. they swim for few hours searching a substrate on which to metamorphose, in order to found a new colony. colonies are formed of numerous, genetically identical individuals (blastozooids) undergoing cyclical generation changes: weekly, in laboratory conditions, adult zooids die and are replaced by their maturing buds (primary buds). buds grow on the lateral walls of their parent and produce 111 secondary buds (or budlets). at generation change, budlets are ready to become primary buds and to produce a new generation of budlets. budding occurs continuously within a colony, in an orderly and synchronized fashion, so that three blastogenic generation coexist: the adult zooids, the primary buds, and the secondary buds. since embryogenesis and blastogenesis lead to formation of morphologically similar individuals, this species is interesting from a developmental and evolutionary point of view to verify whether steps in embryogenesis are repeated during blastogenesis, or if the latter is a completely new type of development. multipotency of adult neural stem cells in the rodent brain s martín-suárez1,2, r valcárcel-martín1,2, o pastor-alonso1,2, i durá1,2, e rueda-alaña1,2, f garcía-moreno1,2,3, jr pineda-martí1, jm encinas1,2,3 1achucarro basque center for neuroscience, leioa, bizkaia,spain. 2university of the basque country (upv/ehu). leioa, bizkaia, spain. 3ikerbasque, the basque science foundation. bilbao, bizkaia, spain. neural stem cells (nscs) persist in the hippocampus of most mammals and are able to generate neurons through adulthood, a process known as adult neurogenesis. adult neurogenesis is important for spatial memory and learning, pattern separation and responses to stress and anxiety. rnscs are more multipotent than previously thought and generate more copies of themselves and astrocytes. in pathophysiological conditions such as epilepsy they can generate reactive astrocytes that participate in the neuroinflammatory response. nscs can also generate oligodendrocytes after genetic manipulation. the type of cell division (symmetric versus asymmetric) and the differentiation path is tightly regulated by neuronal activity with gamma aminobutyric acid (gaba) being a main mediator. in normal conditions neurogenic asymmetric cell division is predominant with a low percentage of symmetric cell division. in conditions of neuronal hyperactivation such as epileptic seizures nscs become reactive (hypertrophic and with massive mitotic activation) and switch to symmetric cell division generating more copies of rnscs that will differentiate into reactive astrocytes, thus abandoning their neurogenic potential and participating in the neuroinflammatory response. we are currently exploring the molecular pathways linking neuronal hyper excitation and the induction of rnsc. small undifferentiated cells from starfish asteria rubens l.: candidates to the role of progenitor cells o petukhova, n sharlaimova, s shabelnikov, d bobkov, m martynova, o bystrova institute of cytology, russian academy of sciences, saint-petersburg, russia the study aims to characterize cells and protein factors involved in regeneration of tissues and organs of starfish asterias rubens. starfishes regenerate at a much slower rate than other echinoderms. we focussed on the study of coelomocytes origin, an immune/haematic system of adult a. rubens which is able to rapid renewal. two types of experimental traumatic treatment, puncture wound and vast blood loss (maximal coelomic fluid draining off followed by washing the coelomic cavity with sea water) were used to stimulate coelomocyte production. the small morphologically undifferentiated cells with high nuclear-cytoplasmic ratio were proposed to be the progenitors of coelomocytes. they are comprised up to 50% of the subpopulation of cells weakly attached on the surface of the coelomic epithelium (ce) and were named ce-w. their characteristic is the proliferative activity in vivo and in vitro. the presence of morphologically similar cells was found in other tissues of starfish. two types of small cells with high nuclear-cytoplasmic ratio, with densely stained nuclei and discretely stained nuclei were found in coelomic cavity, ce, axial organ, tiedemann bodies, pyloric caeca and near-anal сaeca. stomach and rectal glands possess their own type of cells with high nuclear-cytoplasmic ratio. mitotic activity at extremely low level was typical of two types of cells: small cells with high nuclearcytoplasmic ratio and larger ones possessing the visible cytoplasm. stomach and rectal glands have their own type of mitotic cells. these data suggest that small cells with high nuclear-cytoplasmic ratio can perform their functions in different organs of starfish. the irregular distribution of cells on the surface of the ce was demonstrated. ultrastructural analysis of the ce revealed the localisation of small cells under the layer of ciliated cells and in the connective tissue and showed cells migration from ce into the coelomic cavity. the proteomic study of cell-free coelomic fluid was performed by mass-spectrometry. ninety-one proteins were identified. proteins were classified into 12 categories according to putative molecular function. the largest fraction of proteins was classified under “pattern recognition receptor activity”, including “carbohydrate binding”. two other highly represented categories were “signal transducer activity” and “peptidase inhibitor activity”. domain organisation of proteins was presented. the comparison of two types of damage was done. proteins, specific for each type of injury, were identified. quantitative evaluation of injury stimulated changes was performed and downregulated and up-regulated proteins were defined. the data give us an understanding of molecular processes which take place in the cf of a. rubens in response to injury. to obtain molecular markers for coelomocytes, ce and ce-w, enriched with undifferentiated cells, a comparative proteomic study is in progress. proteins common and unique for each cell population were identified (preliminary data). percoll density gradient centrifugation was performed to enrich heterogeneous cell 112 populations with specific cell types before performing proteomic analysis. we obtained 70% enrichment with undifferentiated small cells, significant separation of a mass of ciliated cells and the functional fractionation of coelomocytes. this study was supported by the russian foundation for basic research № 15-04-07798. the experimental work was performed on the base of white sea biological station of the zoological institute, ras, cape kartesh. molecular and cellular aspects of neurogenesis in the anthozoan nematostella vectensis f rentzsch sars centre for marine molecular biology, university of bergen, norway in many bilaterians, the generation of nerve cells involves symmetric and asymmetric divisions that eventually lead to a set of functionally and morphologically diverse neurons. we are studying neurogenesis in the anthozoan nematostella vectensis, a member of the cnidaria, the sister group of bilaterians. while their phylogenetic position can be informative for reconstructing early steps in the evolution of neurogenesis, their broad neurogenic potential and high regenerative capacity makes cnidarians also interesting models for understanding general aspects of nervous system development. using transgenic reporter lines, gene knockdown and genome editing, we are trying to analyze the molecular and cellular basis of neurogenesis in nematostella. we have identified a population of soxb(2)-expressing neural progenitor cells which can give rise to all major neural cell classes and a population of unipotent progenitor cells that generates a particular type of putative sensory cells. by comparing the gene expression profiles of embryos with increased and decreased neurogenesis, respectively, we are now moving beyond candidate gene approaches to obtain a detailed picture of neural development in nematostella. stem cells in marine biotechnology g romano stazione zoologica anton dohrn, naples, italy marine biotechnology is becoming progressively more central to delivering benefits from the sea to human health and wellbeing. the seas and oceans represent indeed a unique environment with the potential to contribute enormously to the sustainable supply of food, energy and biomaterials. it is estimated that 25% of the total number of species on earth correspond to marine species (mora et al., 2011). these have evolved mechanisms to thrive in an extremely different and hostile environment compared with land, leading to an high biodiversity reflected by the myriad of secondary metabolites (or natural products) that marine species produce to defend themselves against predators, to locate mates and to compete for resources. many of these compounds have no terrestrial counterparts and are unique in terms of chemical structure and biological activity. marine organisms thus provide an enormous potential for exploration of bioactive molecules for biotechnological applications. in fact, notwithstanding the high number of compounds isolated so far from marine organisms (now exceeds 28,000), hundreds of new compounds are discovered every year (blunt et al., 2015). major source of bioactive natural products are marine invertebrates among which sponge, cnidarian and mollusks are the most productive. despite the high number of natural products isolated so far, those that have either been marketed or are under development are relatively few. bottlenecks to overcome to reach the market are still present, as for example difficulties in harvesting organisms, low amount of natural product in producing organisms, problems in obtaining a sustainable supply of the compound, difficulties in isolation and purification procedures, ecological impact on natural populations, and insufficient investment by pharmaceutical companies (torjesen, 2015). notwithstanding these difficulties there has been a ‘renaissance’ in marine drug discovery in the last decade due to technological developments and the use of marine microbial genomics to provide biosynthetic pathways for the production of marine natural products (glaser and mayer, 2009). further improvement in the biodiscovery pipeline may come from study and utilization of stem cells for biotechnological applications, e.g. for bioactive metabolite production, which may solve one of the major bottleneck represented by sustainable supply of sufficient amount of bioactive compounds to support all phases of clinical trials for new drug delivery to the market. tissue engineering inspired in marine organisms: current biomaterials and future persprctive on stem cell role th silva1,2 13b’s research group – biomaterials, biodegradables and biomimetics, university of minho, headquarters of the european institute of excellence on tissue engineering and regenerative medicine, avepark – parque de ciência e tecnologia, zona industrial da gandra, 4805-017 barco guimarães, portugal 2icvs/3b’s pt government associate laboratory, braga/guimarães; portugal marine organisms are growingly recognized as an attractive source of inspiration for scientists and engineers. diverse chemical compounds are being produced, from polysaccharides to biologically active metabolites, with a few examples being in the market as components of anti-tumour and antimicrobial drugs. additionally, the morphological features of marine glass sponges influencing architectural trends and materials processing endeavours, as well as mussel foot proteins with fundamental role on the development of new wet adhesives are striking examples of marine biomimetics. within an extensive work being developed in our research group on tissue engineering and 113 regenerative medicine (term), combining human stem/progenitor cells with biodegradable 3d polymeric matrices towards the establishment of invitro tissue constructs, several models of marine inspiration are being explored in our lab. most efforts are on the development of biomaterials for term, as well as other advanced therapies to address diseases as cancer and diabetes, following both the route of marine origin materials and the marine biomimetic approach. in this regard, examples of different marine origin polymers will be discussed, exploring different processing technologies towards the proposal of scaffolds and membranes for cell culture under term approaches, namely addressing: (i) marine origin collagens, isolated from skins of different fish species or squids, marine sponges and jellyfish, on the production of hydrogels and composite scaffolds; (ii) squid chitosan, with higher deacetylation degree, on membranes or porous scaffolds; (iii) seaweed sulfated polysaccharide fucoidan, not only as potential drug to tackle breast cancer but mainly as structural component of hydrogels for cell encapsulation (iii). moreover, marine biomimetic concepts will be also explored, namely by using the marine sponges porous skeletons as nature made scaffolds or as inspiration for the development of hierarchical structures by combining 3d printing of a support material with microfibers made of a cytocompatible biopolymer aiming to influence cell fate, as cell differentiation into a specific lineage. in this regard, the comparison of the behaviour of human and marine invertebrate stem cells may give important cues to trigger the new tissue formation and ultimately the design of advanced regeneration therapies. the amphioxus regeneration model iml somorjai1,2 1biomedical sciences research complex, north haugh, university of st andrews, ky16 9st, uk 2gatty marine laboratory, scottish oceans institute, east sands, university of st andrews, ky16 8lb, uk the cephalochordate amphioxus is emerging as a promising invertebrate chordate model for studies of the evolution of development (“evo-devo”) and genome organisation, particularly at the invertebrate-vertebrate transition. it is also beginning to provide insight into the evolution of regeneration mechanisms: unlike most vertebrates, amphioxus shows considerable regenerative ability of all major structures (notochord, neural tube and axial musculature) even as an adult. in an effort to begin to elucidate the molecular and cellular processes underlying tail regeneration in amphioxus, we have generated transcriptomic and proteomic resources. unexpectedly, this has uncovered a number of candidates, notably gene duplications, with interesting evolutionary histories. here, i will present some of these new data and discuss their possible implications for the evolution of embryonic and regenerative processes. i also highlight current and future avenues of research that will improve the amphioxus model system. ciona robusta as model system for regeneration and in vitro cell culture a spagnuolo, m francone, f ristoratore department of biology and evolution of marine organism, stazione zoologica anton dohrn napoli (italy) ascidians, as ciona robusta, belong to the phylum chordata, subphylum tunicata and are characterized by a larval stage and an adult stage as result of a dramatic metamorphosis. their larval stage thus retains pluripotent cells as a reservoir for extensive changes that occur in these animals during metamorphosis. in this regard, a study has been conducted in order to address how much and which part of the larval cns contributes to form the adult cns during metamorphosis, by tracing cells in ciona larval central nervous system (cns). the data demonstrated that most parts of the ascidian larval cns are maintained during metamorphosis and recruited, as stem-like cells, to form the adult cns (horie et al., 2011). in particular, this study highlighted an important role of ependymal cells as reservoir of neural stem-like cells in order to reconstruct the adult nervous network during chordate metamorphosis. ciona has also retained the capacity to regenerate, although more limited compared to colonial ascidians, and this process potentially involves mechanisms of cell dedifferentiation. ciona adults, indeed, can rapidly and robustly regenerate their oral siphons (os) as well as their central nervous systems (jeffery, 2015a, 2015b). the stem cells involved in os replacement are located in lymph nodes lining the transverse vessels of the branchial sac; they initiate proliferation in response to distal injuries and invade the wounded areas to form the blastemal. recently, microarray and rna sequencing approaches have permitted to characterize mrna and mirna expression profiles of stage-matched samples during os regeneration of c. robusta (hamada et al., 2015; spina et al., 2017). furthermore, in the attempt to develop cell cultures from ciona larva, a simple method has been devised that allows the maintenance of dissociated neuronal cells, together with other cell types, from ciona in primary culture for 2 weeks (zanetti et al., 2007). this opens the possibility that this method, further improved and refined, could be used in a wide range of experiments on this animal model, including studies of the biochemical, molecular and biophysical properties of individual cells in the larval nervous system of ciona. effects of biological extracts on terminal differentiation of hl60 & nb4 leukaemia cell lines s suleiman anatomy department, university of malta cancer is the second leading cause of death 114 worldwide (22.8%) following heart disease (26.6%). unlike normal cells, cancer cells show a block in differentiation leading to uncontrolled proliferation, eventually invading surrounding tissues and organs. this can lead to metastases as the cancer cells spread to other parts of the body through the haemopoietic and lymphatic systems. the aim of this study is to cause terminal differentiation of cancer cells using extracts from the axolotls (ambystoma mexicanum), a highly regenerative organism able to regenerate complex structures. axolotl extract (axe) was tested against hl60 & nb4 leukaemia cell lines. axe was also tested in combination with two histone deacetylase inhibitors namely belinostat and bml-210. following treatment of different cell lines, axe was evaluated for its ability to induce granulocytic differentiation using the nbt test. its anti-proliferative and cytotoxic effects were tested using the mtt assay, and trypan blue was used to determine the number of live and dead cells. hl-60 cells were also tested for cell surface antigens cd11b and cd14, cell cycle analysis, and degree of apoptosis. key results: axe had a differentiation effect on both nb4 and hl60 cells. both cell lines exhibited granulocytic differentiation that was detected using the nbt test and cd11b surface marker. conclusions and implications: these results indicate that axe merits further investigation to elucidate the pathways in which they are implicated and also isolate and identify any active compounds that can be used in cancer therapy cnidarian primary cell culture as a tool to investigate the effect of thermal stress at cellular level p ventura1, g toullec1, c fricano1, l chapron1,2, v meunier1, e röttinger3, p furla1, s barnayverdier1 1sorbonne universités, upmc université paris 06, université antilles, université nice sophia antipolis, cnrs, laboratoire evolution paris seine, institut de biologie paris seine (eps-ibps), paris, france 2sorbonne universités, upmc université paris 06, cnrs, laboratoire d'ecogéochimie des environnements benthiques (lecob), observatoire océanologique, banyuls/mer, france 3université côte d’azur, cnrs, inserm, institute for research on cancer and aging (ircan), nice, france in the context of global change, symbiotic cnidarians are largely affected by seawater temperature elevation leading to symbiosis breakdown. this process, also called bleaching, is triggered by the dysfunction of the symbiont photosystems causing an oxidative stress and cell death to both symbiont and host cells. in our study, we wanted to elucidate the intrinsic capacity of isolated animal cells to deal with thermal stress in the absence of symbiont. in that aim, we have characterized an animal primary cell culture form regenerating tentacles of the temperate sea anemone anemonia viridis. we first compared the potential of whole tissue tentacle or separated epidermal or gastrodermal monolayers as tissue sources to settle animal cell cultures. interestingly, only isolated cells extracted from whole tentacles allowed establishing a viable and proliferative primary cell culture throughout 31 days. the analysis of the expression of tissue specific and pluripotency markers defined cultivated cells as differentiated cells with gastrodermal origin. the characterization of the animal primary cell culture allowed us to submit the obtained gastrodermal cells to hyperthermal stress (+ 5°c and + 8°c) during 1 and 7 days. though cell viability was not affected at both hyperthermal stress conditions, cell growth drastically decreased. in addition, only a + 8°c hyperthermia induced a transient increase of antioxidant defences at 1 day but no ubiquitin or carbonylation protein damages. these results demonstrated an intrinsic resistance of cnidarian gastrodermal cells to hyperthermal stress and then confirmed the role of symbionts in the hyperthermia sensitivity leading to bleaching. metabarcoding of symbionts in the mediterranean stony coral (cladocora caespitosa): preliminary work d stanković, a ramšak national institute of biology, marine biology station piran, slovenia the foundation of coral reef biology is the coral holobiont – an entire community of living organisms that make up a healthy coral head: the coral animal itself, its endosymbiotic algae or zooxanthellae (dinoflagellate genus symbiodinium), and other resident microbes (bacteria, fungi and green algae). anthropogenic disturbances, especially thermal stress, can strongly affect this dynamic symbiosis mainly by decreasing the photosynthesis/photosynthetic efficiency of the symbiotic zooxanthellae, which most often leads to their expulsion from the coral host commonly known as coral bleaching. bleaching events are associated with nutritional depletion of the coral, its impaired reproduction and increased susceptibility to disease and mortality. while responses to thermal stress have been well investigated in tropical corals, responses of their counterparts inhabiting temperate waters is less studied. mediterranean stony coral (cladocora caespitosa) is the only coral reef building scleractinian coral living in the mediterranean sea, where it is spread in almost all biogeographic areas. mass bleaching events in c. caespitosa colonies are observed regularly in the adriatic since 1997 with even subsequent mass mortality in some cases. however, it appears that populations inhabiting north adriatic could be more resistant to bleaching events. during the project “response of biogenic formations to heat stress and the consequences for ecosystem services” funded by the slovenian ministry of education, science and sport, the european regional development fund (5442-15/2016/18 researchers at the beginning of their careers 2.0) and the slovenian research agency (p1-0237 coastal sea research) shifts in the host–microbiota associations due to the thermal stress will be investigated using a common garden http://www.cancer.gov/common/popups/popdefinition.aspx?term=lymph&version=patient&language=english 115 experimental approach. corals from different sources will be exposed to an increase in temperature over a longer and shorter period and the diversity of their holobiont will be assessed with a metabarcoding approach. as most suitable common metabarcoding markers have low phylogenetic resolution and can miss a large portion of the biodiversity a multi-marker approach will be used for identification of both prokaryotic and eukaryotic organisms. coral cell culture and their use in biomineralization study t mass1, jl drake2, p falkowski3 1university of haifa, department of marine biology, the leon h. charney school of marine sciences, mt. carmel, haifa 3498838, israel 2department of ecology and evolutionary biology, university of california, los angeles, ca 90095, usa 3rutgers university, department of marine and coastal sciences, new brunswick, nj 08901, usa coral biomineralization is important at the organismal, ecosystem, and global scales, yet the biological component has not been well understood. in particular, identities, roles, and environmental susceptibility of the proteins retained in coral skeleton were previously unknown. understanding the cellular and molecular responses of stony corals to ocean acidification is key to predicting their ability to calcify under projected high co2 conditions. of specific interest are the links between biomineralization proteins and the precipitation of new calcium carbonate (caco3), which potentially can provide a better understanding of the biomineralization process. to address this, we developed a novel coral tissue cultures to investigate the biophysical mechanism of calcification in corals. our goals were to (a) establish an experimental system in which calcification is facilitated at the cellular level, while simultaneously allowing in vitro manipulations of the calcifying fluid, (b) to test the effects of increased co2 on the calcification process at the cellular and molecular levels, and (c) to study the mineral initiation mechanism in corals. viable cell cultures of the hermatypic, zooxanthellate coral, stylophora pistillata, have been maintained for 6 to 8 weeks. using an enriched seawater medium with aragonite saturation state which mimiks open ocean surface waters ( arag ~4). we have shown that within 72 hr after isolation, cultures of separated coral cells aggregate into proto-polyps and form an extracellular organic matrix (ecm) and precipitate aragonite crystals at a rate comparable to the intact organism and with geochemical properties similar to parent skeleton. in addition, our results suggest that compensatory molecular adjustments to deal with ocean acidification are successful only up to a point, beyond which these mechanisms cannot compete with local chemical conditions unfavorable to biomineralization. we further suggest that calcium is concentrated in intracellular pockets that are subsequently exported from the cell where a nucleation process leads to the formation of extracellular aragonite crystals. 136 isj 19: 136-149, 2022 issn 1824-307x review current research on the effects of plastics pollution in marine and freshwater aquatic invertebrates v pirillo, n baranzini* department of biotechnology and life sciences, university of insubria, via j. h. dunant 3, 21100 varese, italy this is an open access article published under the cc by license accepted october 18, 2022 abstract plastics pollution in the aquatic environments represents one of the most critical worldwide issue. every year, million tons of waste products are reversed both in marine and freshwaters, persisting for long timings and determining serious effects to living organisms. here, these synthetic materials are fragmented in small particles, known as microand nanoplastics, under the effects of both biotic and abiotic factors. due to their characteristics, smaller fragments are easier accumulated inside animal tissues and organs, risking to enter in the trophic chain. to date, despite the current situation, only a small amount of research has been conducted, especially on aquatic invertebrates, which can represent a suitable model for better analyzing the possible plastics dangerous effects. for this reason, in the present review we aim to collect the recent information about micro and nanoplastics effects on both marine and freshwaters invertebrates. in particular, we do not only focus the attention on the obtained results, but also, we report the main experimental methods and particle types used. regardless of the heterogeneity present in literature, the actual data result fundamental for setting up the future research. key words: plastic pollution; invertebrates; microplastics; nanoplastics; plastics uptake introduction plastics are lightweight, versatile, resistant, and cheap materials. thanks to their qualities, these polymers can be easily manufactured for a variety of civil and industrial applications. since the middle of the last century, their peculiar physical and chemical characteristics made these synthetic materials hardly replaceable and, for this reason, employed in almost all industrial areas, such as food and textile industries, healthcare, transports, electronics, and telecommunications (gourmelon, 2015). moreover, due to their multiple use and the low costs, the total amount has significantly increased over the past 70 years, passing from 1,5 million tons in 1950 to 359 million tons in 2020 (tournier et al., 2020). however, the wide distribution and the indiscriminate use, results into threatening consequences, making the environmental plastics pollution one of the most serious ecological problems at the global level (guzzetti et al. 2018; alimba and faggio 2019; prokić et al. 2019). in particular, most of the monomers that constitute _________________________________________ corresponding author: nicolò baranzini department of biotechnology and life sciences university of insubria via j. h. dunant 3, 21100 varese, italy e-mail: nicolo.baranzini@uninsubria.it these polymers derive from fossil hydrocarbons resulting not biodegradable and less than the 20 % are recycled, while most of them are burned, stored in landfill sites or environmental dispersed (gourmelon, 2015). furthermore, plastics not only are extremely resistant, persisting for a long time inside the ecosystems, but also result harmful for the living organisms with which interact. in this context, the aquatic environments are considered the most affected, in which waste products deriving from the human consumption or from the industrial processing rapidly spread transported by currents and winds (barnes et al. 2009; galgani, 2015; jambeck et al. 2015). of the 360 million tons produced, approximately between 4 to 12,7 million are reversed into oceans every year, constituting a significant part of the marine waste, even if this value is considered lower (andrady, 2011; tramoy et al., 2020). unfortunately, with this rate it is estimated that the total amount of dispersed plastics may soon exceed that of numerous species, leading to a great loss for many industrial sectors, not only in terms of biodiversity but also in the economic perspective (letcher, 2020). interestingly, plastics have been already recognized as a possible environmental problem for aquatic systems since 1960s, when the first studies have been conducted focused on their ecological impact on marine species. however, at the time any eventual considerations 137 have been rejected, due to the inability to forecast whatever future trends (letcher, 2020). larger plastics, known as macroplastics, induce different problems both in vertebrate and invertebrate aquatic species when ingested, blocking the digestive tract, damaging organs, and leading to a decrease in food intake (taylor et al. 2016; burgos-aceves et al. 2018; faggio et al. 2018; aliko et al. 2022; lombardo et al. 2022). many organisms remain entangled in these debris that cause injuries and limit movements, also preventing the animal to feed on (gregory, 2009). however, macroplastics represent only a small part of the plastics pollutants. in fact, when released in marine or freshwater environments, these products are subjected to biotic and abiotic processes, which lead to the formation of small particles. microplastics (mps) are in a range between 5 mm and 1 µm, while nanoplastics (nps) show a lower diameters comprised between 1 µm and 1 nm and due to their size these particles are easier ingested or assimilated by animals, accumulating inside tissues and entering in the trophic chain (arthur et al., 2009; xu et al., 2020). both types have been observed from sediment to the surface, differently floated into the water column (van cauwenberghe et al., 2013; lusher et al., 2015; mistri et al., 2017; honoratozimmer et al., 2021). furthermore, the different chemical properties and the diverse density make the comprehension of their fate highly complex (haegerbaeumer et al., 2019). to date, mps and nps not only have been found in drinking water and in 200 edible species, whose consume through the diet can expose humans to an inevitable absorption. due to the ability in crossing the biological barriers and penetrating inside tissues, many studies showed as mps and nps are stored in biotic samples, leading to the activation of many different processes that tend to reduce vitality, promote metabolic disorders and decrease the reproductive fitness. their assimilation is associated with many toxic responses that occur in organisms such as oxidative stress, activation of the immune system, inflammations and growth inhibition (alimba et al. 2021; burgos-aceves et al. 2021a, b). other research conducted in mammals reveal that the uptake of some mps induces dysbiosis, hepatic and metabolic disorders in mice (luo et al., 2019). moreover, another important aspect is determined by the fact that, although these materials should not be considered reactive for their biochemical structures, plastics particles can act as carrier for other pollutants, which are easier transported inside organisms producing further harmful effects. at any rate, the plastic degradation increases the nature availability of these synthetic polymers with severe impacts on living organisms, especially in marine and freshwaters ecosystems (tiwari et al., 2020). thus, it results essential to collect and extend the current knowledge to successfully understand their real impact and prevent the potential risks. for this reason, in this review we reported the most recent and relevant data available in literature on the mps and nps effects both in marine and freshwaters environments. in detail, we aim to underly how plastics are produced and used for the experimental applications and describe the effects both at organism and cellular level in aquatic invertebrate species. this work sets out to bring new insights and provide useful information for the future research. types of plastics despite, several emerging methods are developing for a more sustainable recycling of plastic (e.g., based on the enzymatic and microbial biodegradation (tournier et al., 2020; lu et al., 2022; pirillo et al., 2022; sonnendecker et al., 2022), there are a few critical steps for the removing process of high-density plastics from the environment and many bench-scale experiments are not enough for the application in large-scale process (patil et al., 2022). indeed, due to their low biodegradability, the plastic materials are high recalcitrant in several environments and the deriving debris widespread vary in terms of chemical e physical properties (e.g., chemical composition and density). starting from their initial dimension, the engendering process that leads to formation of mps and nps can be classified in primary and secondary sources . with primary sources are identified all the discarded particles that derive either from the industrial production or that are generated by the fragmentation of common objects used in everyday life (e.g., fibers of synthetic textile products during washing, personal care products, or cleaning applications). instead, as secondary sources are considered the total amount of microand nanofragments that originate directly from a slow decomposition of the former, which result in a fragmentation in smaller size particles as effect of the environmental exposition to several both biotic (i.e., living organisms that influences its environment) and abiotic factors (i.e., uv rays, temperature, salinity, atmospheric events or ocean currents, and also microbial degradation), enhancing the persistence in natural ecosystems. moreover, oxidation can be also the cause of plastics physical abrasion (arthur et al., 2009) and some polymers result highly inclined to this type of chemical process, which impairs the strong molecular bonds between chains (tiwari et al., 2020). from packaging to the manufacture of singleused items, polyethylene terephthalate (pet), highdensity (hdpe) and low density (ldpe) polyethylene, polyvinyl chloride (pvc), polypropylene (pp), and polystyrene (ps) are the polymers which possess a major industrial relevance and are the most common identified in the investigated marine environments (table 1) (auta et al., 2017; danso et al., 2019). the particles found in nature are characterized by different both sizes and shapes (e.g., spheres, fiber, film, irregular) (chubarenko et al., 2020; patil et al., 2022), but also their density is an important aspect to be considered for determining their specific localization and their fate in the in the water column. in fact, debris with a density higher than seawater (1.020-1.029 g ml1) (e.g., pet, pvc, and ps) would sink, on the contrary polymers less dense (e.g., hdpe, ldpe, and pp) would float on the surface (table 1) (brignac et al., 2019). considering all these aspects, 138 table 1 the most diffused synthetic plastics in the aquatic environments. for each type, resin code, abbreviation, commonly use, decomposition rate for marine debris, density and toxicity levels are reported. athe reported chemical density referred to the pure plastic compounds in absence of additives (e.g., plasticizer, pigments, stabilizers) as reported by brignac et al., 2019 it results necessary shed light on the presence of different types of mps and nps and their effects on living organisms in freshwater and marine ecosystems. overview on the mps and nps production and potential applications in literature are reported several types of mps and nps for evaluating the in vivo effects that are based on a chemical-mechanical production or are commercially available (fig. 1). several techniques of synthesis are reported by lee et al., 2016 to produce mps and nps, that can be divided into four categories: (i) emulsion-based methods, (ii) precipitation-based methods, (iii) direct compositing methods, and (iv) new approaches including microfluidic technique. however, emulsion and precipitation are the most used to easily produce mps and nps. for examples, (grillo et al., 2021) reported a simple emulsion/solvent extraction synthesis method in chloroform (1 % m/v) and polyvinyl alcohol (pva, 0.025 % m/v), producing a range of ps microparticle sizes, where the 78 % present an average diameter < 5 µm and a spherical shape. similarly, straightforward protocols are available to produce pet nps based on a dissolution/precipitation method with water-soluble solvent hexafluoro-2-propanol (pirillo et al., 2021) or two step of trifluoroacetic acid solution (tfa, 90 % v/v and 20 % v/v) (rodríguez-hernández et al., 2019; pirillo et al., 2021), starting from commercial pet (e.g., drink bottle, granulate, films, or fibers); then, thanks to a filtration-step, it is possible to obtain a particulate size that is usually in the 50to 300-nm range. moreover, the nps so produced can be colored with different dye, such as the nile red, which has been proposed by maes et al., 2017 for pet microparticles identification and exploited for cell internalization experiments and ecotoxicological studies (rodríguez-hernández et al., 2019). beyond the chemical synthesis that allow to produce all spherical and in the same scale range particles, easier protocol for mps and nps production are also available based on a mechanical processing. as proposed by (romeroblanco et al., 2021), mps can be obtained by pulverization of ps tube-test, with a size ranged between 10 nm and 514 µm. likewise, other reported protocols are based on the cutting and fragmentation of commercially plastics fibers or row pellets (jemec et al., 2016; kim et al., 2021). the major advantage of this method is to produce irregular fragments that are more similar to those found in nature, in order to better simulate the environmental conditions (baranzini et al., 2022 accepted). regarding commercial mps and nps, several modified particles exhibit fluorescence properties polymer name polyethylene terephthalate high-density polyethylene polyvinyl chloride low-density polyethylene polypropylene polystyrene resin code abbreviation pet hdpe pvc ldpe pp ps use water bottles, medicine jars, clothing and carpet fiber detergent bottles, plastic bags, toys credit cards, window and door frames plastic wrap, bread bags, squeezable bottles bottle caps, potato chip bags, packing tape, drinking straws food boxes, watering cans, storage bins decomposition rates for marine debris up to 450 years up to 450 years up to 450 years 500-1000 years 20-30 years 900 years density (g ml-1)a 1.37−1.41 0.94−0.98 1.38−1.45 0.89−0.93 0.85−0.92 1.04−1.06 toxicity level high low high low high low 139 and can be functionalized with carboxyl (-cooh) or amino (-nh2) groups on their surface. the most used fig. 1 scheme representing environmental plastics dimensions, correlated to their production methodologies for in vivo studies research and example of affected organisms. ft-ir, fourier-transform infrared spectroscopy; sem: scanning electron microscopy; tem: transmission electron microscopy fluorescent mps and nps are those of ps, in particular available as blue-dyed (345 nm excitation and 435 nm emission, phosphorex), red-dye (552 nm excitation and 580 nm emission, micromer®redf), and yellow-green microspheres (441 nm excitation and 486 nm emission, fluoresbrite® plain yg) (della torre et al., 2014; canesi et al., 2015; bergami et al., 2017; gambardella et al., 2017; capolupo et al., 2018; liu et al., 2019; rist et al., 2019; cappello et al., 2021; gonçalves et al., 2022). thanks to their easy detection, fluorescently labeled and functionalized ps plastics are the most common used in ecotoxicological studies, toxicity bioassays and for the evaluation of the aquatic organisms uptake. however, independently from how have been produced, mps and nps behavior is extremely variable and an in-depth characterization is required. a dynamic light scattering (dls) analyses combined with a zetasizer nano series software (zs) should be necessary for determining several key parameters, such as z-average (nm), polydispersity index (pdi, dimensionless) and zeta (ζ-) potential (mv), which describe mps and nps behavior in relation to the environmental media (bergami et al., 2017). in fact, salinity or ph can alter the plastics bioavailability and distribution in the water column, leading to polymers aggregation and changing many physicochemical properties (surface change or coating). moreover, to evaluate the potential effects, other aspects must be taken into consideration. concentration of mps or nps used in the experimental conditions do not always reflect the exposure scenario and the real environmental conditions (e.g., using extremely high concentrations). in this context, it is reported in literature the needed to mimic as close as possible the natural state, to obtain more accurate data on the real toxicity of plastics dispersion. moreover, considering that the fate and the bioavailability of mps and nps depends on size, shape, and charge, it is important to have comparable methodologies and approaches, to obtain a deeper knowledge that considers all the physicochemical variables that can interfere with interactions between plastics and organisms (oliveira and almeida, 2019). mps and nps effects on marine invertebrate organisms phylum of cnidaria cnidarians are benthic invertebrates that were able to adapt to many different habitats. living fixed 140 on substrates in contact with sediments or floating in the water column, these animals represent one of the main targets for environmental pollutants. for this reason, many studies have been conducted on hydrozoan, scyphozoan and anthozoan marine models to evaluate toxic effects deriving from different compounds, such as chemicals or metals (muñoz-vera et al., 2015; lozano-bilbao et al., 2018). the presence of waste products can induce morphological changes, affect vitality and alter the expression levels of those gene related to metabolism and oxidative stress (kalsom and mehman, 2020). due to their ability in the incorporation of various anthropogenic materials, both medusoid and polypoid forms are important bioindicators of mps and nps impact in marine ecosystems (macali and bergami, 2020). moreover, representing one of the first step of the trophic chain, the comprehension about their ability to accumulate synthetic polymers is extremely relevant (devereux et al., 2021). in nature, the presence of plastic fragments has been detected in different cnidarians species, such as cyanea capillata, c. lamarckii and aurelia aurita (schyphozona), cosmetira pilosella (hydrozoan) and coelogorgia palmosa (devereux et al., 2021; vencato et al., 2021), confirming as these invertebrates are directly interested by mps and nps pollution. in the scleractinia coral species porites porites, ps mps absorption was evaluated using chemically produced spherical particles presenting a diameter inferior to 5 μm, which were dispersed in seawater in a range between 1 and 1000 mg/l. the analyses conducted using both histological and enzymatic techniques, in which the levels of catalase have been evaluated, revealed that although the particles uptake occurred in every condition, no significant toxic effects on organisms behavior have been registered in short time (96 h) of exposition. based on the initial concentration, ps microsphere have been stored in gastrovascular tissue, mesenterial filaments and coral tissue without affecting viability or inducing bleaching and stress response (grillo et al., 2021). contrariwise, long-term treatments (17 days) with ps cause potential harmful effects both in polyps and ephyrae of the anthozoan jellyfish sanderia malayensis. independently from the dimensions, fluorescent fitc-conjugated microbeads were able to instantly interact (already after 24 h) with both epidermis tissue and digestive cavity, in which they were clearly detectable. moreover, mps can accumulate and persist until 52 days, altering feeding behavior and fitness. after 17 days, animals reproduction appeared reduced, revealing as prolonged exposures to plastics, although do not affect survival, can interfere with polyps budding (eom et al., 2022). the effects of pe were evaluated in the coral stylophora pistillata, analyzing the as mps inhibit photosynthetic capacity of scleractinian zooxanthellae symbionts. after 4 weeks, chlorophyl reduced activity was analyzed by pulse amplitude modulated (pam) fluorometry method, indicating a condition of stress. although this effect seemed to be due to a direct contact between algae and mps, given the physical inability of this interaction, authors suggested that mps produced a signaling interference between symbionts and hosts. moreover, by means of nuclear magnetic resonance (nmr) analyses, the metabolism of different important metabolites has been detected (lanctôt et al., 2020). pe mps effects were also investigated in the aurelia sp. ephyra stages using fluorescent particles at different concentrations (from 10 µg/l to 10 mg/l). by means of confocal and tomographic analyses, it was observed as mps attached around mouth surface or were stored into the digestive tract. moreover, already after 24 h from the initial exposition, in juveniles jellyfishes the survival rate, behavior and radial symmetry resulted significantly altered. although after 72 h a total recovery is restored in absence of mps, these data also confirmed a direct impact on larval immobility and frequency of pulsations (afp) that impair animals viability (costa et al., 2020). coral reef cnidarians were also used to test the effects of pvc mps. in the species zoanthus sociatus, commercial mps, possessing an irregular size, were dispersed in the aquarium under continuously moving water condition, in which two different pvc concentrations (1 and 10 mg/l) were assessed. plastics immediately adhered to epidermis or gut cavity, stimulating stress response and inducing photosynthetic events. as regards the macroscopic effects, no variations in survival or behavior have been detected, also after long term exposure (rocha et al., 2020). phylum of mollusca given their commercial and ecologically importance, many studies were conducted on several molluscs species, in which the effects of different plastic types have been tested with different aims. in particular benthic and sedentary organisms, such as bivalves, are the main models employed in toxicological research (pagano et al. 2020; stara et al. 2020, 2021; curpan et al. 2022). filtrating waters or directly feeding from the substrate, they are excellent candidates for investigating potential toxic effects, analyzing plastics bioaccumulation and morphological or behavioral changes (rittschof and mcclellan-green, 2005; jaeschke et al., 2015). spherical and colored ps mps, with a diameter of 3 µm, were administered to the marine mussel mytilus galloprovincialis and the several metabolic parameters were analyzed by nmr-based metabolomics spectra. filtered mps deposit into the digestive gland and, especially after 48 h up to 72 h, alter the production of various metabolites, osmolytes and antioxidants. the levels of several amino acids, lactate, glycogen, taurine, hypotaurine and glutathione increased, suggesting a potential toxicity for ps mps (cappello et al., 2021). significant change in the expression levels of different genes was also observed after 3 µm-size spherical mps uptake in m. galloprovincialis larval stages. although plastics were assimilated and retained, bioaccumulating inside the digestive tract and risking persisting in the trophic chain, their presence did not cause visible morphological modifications during embryonic development up to 192 h. however, the upregulation of several genes https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/aurelia-aurita 141 related both to shell biogenesis and immune response or the downregulation of those involved in lysosomal activity were evident, highlighting as ps plastics mostly act at the cellular level (capolupo et al., 2018). a similar result was also observed in the mytilus edulis larvae, in which although mps were faster stored into embryo tissues after ingestion, the developmental stages were not altered, except under extreme conditions (high concentration and long exposition timings) (rist et al., 2019). interestingly, similar potentially dangerous effects were also detectable in mussels cells after nps chemical modifications. indeed, cationic nps (ps-nh2) affected cellular processes and influenced molecular intracellular pathways. based on studies conducted in sea urchin embryos and mammalian cells, canesi and colleagues demonstrated as the addition of positive charges not only impacted on the plastics uptake, but also on their final biological targets. indeed, m. galloprovincialis hemocytes exposed to 50 nm ps-nh2 particles showed both a reduced phagocytic activity and the increase of lysozyme in a dose-dependent manner, together with reactive oxygen species (ros) and nitric oxide (no) production. in parallel, the apoptotic rate and the loss of mitochondrial potential were evaluated by means of flow cytometry (canesi et al., 2015). the ps nps ability in impairing immune system was also confirmed by electron microscopy (tem) and molecular analyses. the presence of numerous laminar cytoplasmatic expansions and the diverse modification of the p38 mapk and pkc phosphorylation state represented a symptom of cellular stress in mussels hemocytes. in addition, once entered inside tissues and cells, mps and nps can be also conditioned by the chemical interactions established with different proteins, known as corona-protein, that interfere with plastics behavior. the formation of a ps-nh2-corona proteins complexes was investigated in the extracted hemolymph, combining electrophoresis method and hplc-ms/ms techniques. interestingly, the results showed a specific binding with a c1q domain protein, suggesting that although this interactions makes more difficult the comprehension of plastics fate, are essential to better assess their potential impact (canesi et al., 2016). nps toxic effects can be also calculated using the integrated biomarker response (ibr) index that allows to compare and analyze the relations between biomarkers and pollutants levels. this tool has been used in m. galloprovincialis to better interpretate the obtained results, in which a concentration of 10 µg/l of ps particles caused a chronic response characterized by a tissue-specific genotoxicity. among all the organs involved, gills resulted the main affected. ps nps aggregate and interfere with the cell biological functions, damaging dna and impacting on cell viability. moreover, the antioxidant defenses, based on different enzymes, result ineffective and after 14 days from the beginning of exposure. the levels of ros were so high that lipid peroxidation (lpo) occurred, leading to the disruption of cell membranes and causing a chronic response (gonçalves et al., 2022). although the ps registered effects are different in diverse mussel species, the ibr index should represent a valuable tool to simplify and clarify data interpretation. to date, although numerous investigations have been performed on ps particles, only few were conducted on the other plastic types. among them, the potential role of hdpe was analyzed in the pacific oyster crassostrea gigas larvae, in which develop, morphological modifications and swim ability were assessed. after 24 h from the initial exposition, the smallest hdpe microparticles, proposed at three different concentrations (100 µg/l, 1 and 10 mg/l), caused severe malformations and arrest larval growth. moreover, although the speed during swim was not completely reduced, trajectories appeared significantly altered. in the 70 % of oyster embryo, the interaction with mps produced more circular movements, decreasing rectilinear proceeding. all these consequences on their behavior not only interfere with the capacity of larvae to feed or escape from predators, but also with the possibility to naturally colonize new substrates (bringer et al., 2020). phylum of arthropoda among all the invertebrate phyla, those of arthropods is certainly the most numerous. indeed, it contains a great variety of species that, thanks to their characteristics, conquered different types of habitats and colonized quite all the ecological niches (kremen et al., 1993; verma and prakash, 2020). as for bivalve molluscs, the marine taxon of crustaceans possesses a large importance both in economic and biological terms and also recently acquired a certain relevance in scientific and ecotoxicological field (anger, 2006). therefore, it results fundamental to investigate the plastics toxic effects in these invertebrates, analyzing as mps and nps could bioaccumulate inside tissues, not only altering the development and life cycle, but also risking to consequently enter in the tropic chain. this the case of several classes such as copepoda, cladocera or branchiopoda, composed by small crustaceans that form zooplankton and that represent the main prey of fishes and other invertebrates. given that the chronic presence of plastics in these organisms can be extremely dangerous, numerous studies have been focused proper on these animals. in the brine shrimp artemia salina, chronic toxicity induced by ps mps was determined using both labeled and not-labeled plastics particles at different concentrations (from 1 to 100 mg/l). moreover, the employ of greenly fluorescent mps better allowed to follow plastics fate and body localization. although mps accumulate inside tissues in all the developmental stages, organisms growth was considerably reduced only with the highest concentration. in this condition, a considerable modification of the midgut cell morphology was detected. in addition, to characterize the way by which plastics influence cells biology, transcriptome analyses have been conducted in combination with gene ontology (go) analyses, confirming a change in the expression levels of those genes involved both in metabolic and catalytic activities, as already observed in other arthropods species (suman et al., 2020). 142 the ps plastics ingestion followed by harmful effects was detected also in another crustacean of the genus artemia: artemia franciscana. the exposure to various mps concentrations and sizes revealed a variation in the activity of specific proteins and enzymes, such as heat shock protein 70 (hsp70), catalase, superoxide dismutase and acetylcholinesterase, considered important markers of the oxidative stress. however, the most relevant data was represented by the increase in shrimps mortality after 30 days of treatment. mps impaired survival rate in a dose-depending manner, by inducing chronic and acute responses at different biological levels (eom et al., 2020). a. franciscana brine shrimps have been also treated in combination with the green microalgae dunaliella tertiolecta to assess as charged ps nps could induce diverse effects depending on the associated chemical group. interestingly, if on one hand anionic carboxylate (ps-cooh) particles did not impact on organisms health until highest concentrations, although are easier internalized, on the contrary the anionic ps-nh2 plastics induced both the inhibition of d. tertiolecta algae growth and the increase of a. franciscana shrimps mortality. all these results are extremely important to better understand the ps plastics role also in zooplankton organisms that occupy a relevant place in the trophic chain (bergami et al., 2017). phylum of annelida thanks to the possibility to amputate body segments without compromise their survival, the class of marine polychaeta constitutes an important phylogenetic group to assess mps and nps effects on tissues regeneration. moreover, being prey of many other animals and living directly on the sediments, these invertebrates are continuously exposed to environmental pollutants, representing one the main channels for the entrance of anthropogenic materials in the trophic chain (pires et al., 2022). however, despite their ecologic relevance, only few studies have been performed to determine how plastics could impact not only on polychaetes life cycle and behavior, but also interfere with the ability to regenerate tissues. two different species hediste diversicolor and perinereis aibuhitensis were treated with different concentrations of ps mps and nps, in which behavioral change were determined after plastics treatment. in detail, h. diversicolor worms exposed to different concentration of ps nps showed a reduced burrowing ability. this parameter is probably connected with a decrease in the cholinesterase activity, a fundamental enzyme that regulates muscular functions. furthermore, in the 50 % of the samples exposed to a nps concentration of 50 mg/l also the antioxidant defenses resulted significantly reduced. however, lpo levels remained lower than control groups, suggesting that in h. diversicolor mechanisms of membrane repair could be activated to cope with ps adverse effects (silva et al., 2020). whereas leung and chan analyzed the impact ps mps during regeneration in the polychaeta p. aibuhitensis. authors demonstrated as the smallest microparticles (8-12 µm) increased mortality and affected the ability to regenerate removed segments (leung and chan, 2018). moreover, these studies also revealed that, in particular smaller beads, aggregated and consequently compromised samples physiology and behavior, acting at the cellular level by influencing the expression of specific antioxidant enzymes. h. diversicolor has been also used to investigate the potential effects of other environmental frequently found plastic types. pp and pe mps were added at different concentrations into water (10 and 100 μg of mps/l) and sediments compartments (10 and 50 mg of mps/kg), demonstrating a different accumulation inside worms tissues after 96 h. by means of flow cytometry and enzymatic assays, the coelomocytes viability, the phagocytic activity and the levels of phenoloxidase (po) and acid phosphatase (acp) were analyzed. both a slight reduction of both cells viability and po and acp were observed, suggesting as different plastic types can interfere with organisms integrity (revel et al., 2020). a similar result has been recently obtained mixing and using together more type of mps. indeed, a mixture of pe, pp, hdpe, ldpe, polyamide (pa) and polyethylene/ethylene combined with vinyl acetate copolymer (peva) was tested in h. diversicolor, revealing that plastics accumulate, affecting animals survival and growth rate at high concentration when exposed for long time (missawi et al., 2021). phylum of echinodermata several studies have been also conducted in echinoderms, in which the attention has been mainly focused on the gametes formation or on the embryonic and larvae phases. thanks to the higher sensitivity to pollutants then adults, juvenile stages represent a perfect model to test possible dangerous effects that can compromised the development (nobre et al., 2015). in both eggs and spermatozoa of the sand dollar scaphechinus mirabilis, ps mps induced a significant dna damage, in which more than the 20 % of the sequences resulted shattered. by means of dna comet assays, the genome loss was effectively observed already after 1 h after 105 particles/l exposure. however, despite this evident effect, spermatozoa did not lose their capacity to fertilize eggs (mazur et al., 2021). ps toxicity was also evaluated in the embryonic stages of the sea urchin paracentrotus lividus, in which, as observed for a. franciscana brine shrimps, positive and negative charged nps showed different properties. the structures and dimensions were evaluated by means of tem and dynamic light scattering (dls) analyses. although fluorescently ps-cooh-labeled particles resulted more aggregated and were easily accumulated inside embryos gut, no particularly severe effects were observed. on the contrary, positive ps-nh2 modified plastics, despite appearing more water dispersed, caused serious developmental deficiencies. this outcome is probably due to the activation of apoptotic pathways, given that after 24 h from exposure, nps lead to a significant upregulation of the caspase 8 gene (della torre et al., 2014). interestingly, these data represent a further confirm of how any plastics chemical 143 modification could not completely change the fate of these synthetic polymers, but also lead to diverse responses both at organism and cellular level. mps and nps effects on freshwater invertebrate organisms it is estimated that a considerable part of the plastics presents in seas and oceans arise from freshwaters, in which a large amount of waste products is reversed every year. lakes and rivers very often are the main environments affected by plastics pollution, being in direct contact with urbans and rural locations (strungaru et al., 2019). here, mps and nps float in the water column or settle on the sediments, entering in contact with living organisms. notwithstanding it results necessary to better comprehend their impacts also in these ecosystems, nowadays only few studies have been performed compared to those conducted on marine species (imhof and laforsch, 2016). moreover, freshwater benthic invertebrates are considered useful biomarkers, thank to their extremely vulnerability to pollutant, and can represent a fundamental instrument to easily decode the ecological conditions of a specific environment. phylum of cnidaria as marine cnidarians, also the freshwaters one are an important source of food for many aquatic species. moreover, due to their anatomical and physiological characteristics, these invertebrates are extremely sensitive to chemical environmental pollutants, representing a valid indicator of the water quality (beach and pascoe, 1998). the presence of toxic substances could have severe effects on the body morphology and animals vitality. among the major critical outcomes, hydrozoans are subjected to tentacles loss and impediment in food uptake. for these reasons, also the toxic effects of ps nps have been tested on these invertebrate models, mainly focused the attention on regenerating processes (auclair et al., 2020). in detail, the freshwater hydra attenuata was treated for 96 h with different concentrations of ps fluorescent particles (from 1.25 to 80 mg/l 50 and 100 nm in size), whose diameter has been evaluated by dls analyses. despite nps accumulation, depending on the administered dose, following 24 h of depuration severe loss of biological mass occurred. moreover, an important sign of oxidative stress condition was determined by the fluorescent measuring of both lpo and lipid-like liquid crystal (lcs) formation inside cells cytoplasm. these data revealed as 100 nm particles were most effective then the smaller one and highlighted as lipids metabolism could play an important role in determining plastics toxicity in freshwater cnidarians (auclair et al., 2020). phylum of mollusca the effects of synthetic polymers have been assessed in the mud snail potamopyrgus antipodarum, recreating the gastropods environmental conditions. in detail, freshwater was combined with river sands, in which mps and nps, presenting sizes between 10 nm and 514 μm and concentrations from 100 to 4000 mg/kg, have been added. after 31 days, no critical impacts on both mortality and reproduction rate were observed. however, adverse behavioral responses were detected analyzing animals immobilization, reaction time and distribution at different conditions. interestingly, the highest concentrations of mps did not cause negative effects compared to the control, while all the other treatments tent to increase the p. antipodarum reaction time. although different hypotheses were developed, this factor is probably closely related to the plastics intake, which risks to block the digestive tract or to damage tissues. nevertheless, these organisms seem to well tolerate plastics presence, independently from mps and nps concentrations (romero-blanco et al., 2021). similar results were obtained by imhof and laforsch, which examined the potential plastics effects in p. antipodarum using a mixture of five different common non-buoyant polymers (pa, pet, pc, ps and pvc), directly added to food in two different doses. the main focus was to analyze possible morphological change in both adults and juvenile stages, but no significant modifications have been observed. moreover, the shell formation was not affected by the presence of polymers also after 8 weeks at the higher concentration, suggesting that the juvenile development was not compromised. however, it must be considered that in the present work large particles have been used and the lack of effects should be due to plastics size and shape (imhof and laforsch, 2016). phylum of arthropoda among freshwaters arthropods, daphnia spp. are considered the main conventional model used in freshwater ecotoxicological studies, recognized at an international level (taylor et al., 2018). filtering nutrients in a not-selective manner, these crustaceans come into direct contact with numerous contaminants, and, thanks to their simple body anatomy, they allow to easily detect any possible morphological or physiological modification. in daphnia pulex, fluorescent ps nps immediately entered inside digestive tract after 48 h from initial exposure, showing lethal effects at the concentration of 76.69 mg/l. moreover, not only survival rate was significantly reduced, but also both the reproductive capacity and growth. the analyses of the expression levels of several gene involved in oxidative stress showed, as for other aquatic invertebrates, a significant increase depending on particles concentration. in particular, superoxide dismutase (sod), glutathione transferase (gst) and hsp90 genes resulted more expressed than in control samples. also after long-term treatments, a chronic response was visible, in which ps nps influence the reproduction, hatching time and future offspring (liu et al., 2019b). a comparable result was also obtained in daphnia galeata, in which 5 mg/l of ps nps caused aberrant development and a fitness reduction after 5 days (cui et al., 2017). plastics particles aggregated with lipid droplets located in the adult ovary, which play a fundamental role in regulating crustacean embryos development (cai et al., 2019). although the nps effects were also transient and did not impact on future offspring, by means of nile red staining assay, cui and colleagues demonstrated that their presence led to 144 a decrease in lipid storage (cui et al., 2017). these variations revealed as nanoplastics could modify metabolic pathways, becoming an effective barrier to embryonic development. as regards other synthetic materials, the effects related to pet microfibers ingestion have been examined in daphnia magna after 48 h of treatment and following 24 h of recovery. pet fibers were characterized by ftir spectrometer in atr mode comparing the results with spectral databases. although plastics found in the digestive tract possessed a mean dimension of about 300 μm, also particles with large dimensions (1400 μm) were found in the gut. d. magna mortality appeared significantly increased in no prefeeding animals, while no effects were recorded in daphnids fed before treatment. subsequent experiments, in which tissues were digested with h2o2 and the gut contents were analyzed by means of scanning electron microscopy (sem), confirmed a considerable amount of pet fibers in d. magna tissues (jemec et al., 2016). also pp particles accumulated in d. magna digestive tract, in a similar concentration, though these polymers caused a less response and minor lethal effects after 96 h after the initial exposure (kim et al., 2021). phylum of annelida differently from all the other invertebrates, freshwater annelids that belong to the oligochaeta and hirudinea classes are considered extremely tolerant to waters contamination and very often their population benefit of these compounds compared with those of non-tolerant organisms (sharma and chowdhary, 2011). indeed, especially oligochaete benthic species are generally the dominant in muddy sediments of lakes or in the swampy areas, able to exploit all the waste materials that decompose on the bottom (abubakr, 2018). despite the low number of studies, thank to their high resistance, these organisms should be considered extremely useful to understand how much plastics could be dangerous. in fact, any effect caused by these synthetic polymers represent a significant sign of their potential toxicity. in the oligochaete aquatic worm allonais inaequalis, the role of pe mps was investigated. particles possessed a size between 40 and 48 μm and were provided both in normal temperature (24 °c) and thermal stress (19 °c and 29 °c) conditions. independently from temperatures, after 96 h mps were ingested by worms without affecting animals survival. moreover, also chronic response did not induce particular effects in terms of mortality and reproductivity. however, as suggested by the authors, although no specific effects were recorded, exposition time, particle size, age and plastic type could also produce different responses. nevertheless, thanks to its resistance properties, a. inaequalis can represent a suitable model for deepening microplastics effects in freshwaters animals (castro et al., 2020). the resistance of oligochaete species to contaminants was also confirmed in tubifex spp., which inhabit areas in which the pollution levels are extremely high (sharma and chowdhary, 2011). it was demonstrated that in the species tubifex tubifex, mps ingestion did not affect life cycle and reproduction. as confirmed by ftir analyses, a wide range of synthetic polymers were found, located in the digestive system and presenting different sizes and concentrations. although no effects were registered, this study specifically suggests as the bioaccumulation of mps into tissues and organs, make these oligochaetes a potential risk for the other organisms in relation to the trophic transfer (hurley et al., 2017). as concern hirudinea, leeches are considered a valid bioindicators, whose ability to accumulate pollutants is comparable only with that of oligochaetes. is has been discovered that the concentrations of contaminants in different species of leeches were higher than in other analyzed organism, vertebrates included. for example, in erpobdella punctata the levels of mirex, a chlorinated hydrocarbon used in the past as insecticide and nowadays banned due to its significant environmental impact, were higher than in other invertebrates and fishes. whereas leeches of the glossiphoniidae family collected in the tahoe lake (usa) presented a greater quantity of ddt than filter-feeding clams of the genus pisidium (metcalfe and carey, 1984). in addition, leeches not only are considered a suitable model for studying innate immunity and regeneration (grimaldi et al., 2009, 2010; baranzini et al., 2020, 2021), but also they have been already used to investigate nanoparticles effects (girardello et al., 2015, 2017; bodó et al., 2020). for all these reasons, the potential role of mps and nps pp has been analyzed in the freshwater leech hirudo verbana, focused the attention on the possible inflammatory response activation. to better follow particles fate, mechanical-obtained fluorescent plastics were administered to water at the concentration of 400 mg/l and the effects were analyzed both after short and long exposure timings (1 h 6 h, 1 week, 1 month and 2 months). by means of microscopy and dls analyses, the size and the fluorescent properties of manually created pp fragments have been evaluated. morphological, histoenzymatic and molecular assays confirmed as these synthetic polymers not only entered inside tissues, passing the external cuticle, but also lead to the activation of angiogenetic processes and macrophage activation. moreover, despite an initial protection by mucous cells, their presence caused an increase of the expression levels of important pro-inflammatory markers, such as hmaif-1 and hvrnaset2 (baranzini et al., 2019). gene upregulation was also observed for sod and gst antioxidant enzymes, suggesting as pp mps and nps induced oxidative stress and confirming their potential harmful effects, also at the cellular level (fig. 2) (baranzini et al., 2022, accepted). concluding remarks plastic mps and nps, deriving from waste materials or directly released during industrial manufacturing, are widely present both in marine and 145 fig. 2 representation of pp plastics uptake and effects in the medicinal leech h. verbana. leeches were exposed to water resuspended plastic fragments that are able to pass external cuticle and accumulate inside tissues. once entered, the main effects immediately relevant induced by pp mps and nps involved angiogenesis, macrophage recruitment and activation, and oxidative stress freshwater aquatic ecosystems. the consequent pollution represents a significant problem at the global level, due to the potential risks not only for organisms that inhabit a specific environment but also for human health. in this context, invertebrates are essential for better comprehend these aspects. living in all the ecological niches, they are often the primary organisms that enter in contact with plastics particles, also representing an entrance for these synthetic materials in the trophic chain. however, compared with the actual situation, the number of studies present in literature is still low and investigating plastics effects must be considered crucial also in these models. moreover, although the highest percentage of research is focused on ps mps and nps, less is known about other plastics and the different administering methods, particle types, timings and the diverse concentrations used make complicated to find uniformity in these results. 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innate and intrinsic immunity in planarians l gao1#, a li1#, n li1,2, x liu1,2, h deng1,2, b zhao2, q pang1,2 1anti-aging & regenerative medicine research institution, school of life sciences, shandong university of technology, zibo 255049, pr china 2laboratory of developmental and evolutionary biology, school of life sciences, shandong university of technology, zibo 255049, pr china #l gao and a li contributed equally to this work. accepted november 4, 2017 abstract planarians in the phylum platyhelminthes occupy a peculiar phylogenetic position and have a strong regenerative capacity of their adult tissues, which has aroused general attention. planarians are spontaneously exposed to various pathogens (microbes and harmful chemicals), but typically survive these challenges. therefore, these animals can provide useful insights into the evolution of the innate immune system. this review mainly focuses on immune tissues (epidermis, pharynx, and intestine), immune cells (phagocytic reticular cells) and immune genes of planarians. in addition, we provide an overview of the critical proteins in the innate immune system for example, pattern recognition receptors, complement system proteins, anti-microbial peptides and antioxidant enzymes. in particular, the effectors of the signaling pathways activated upon planarian infection are reviewed. key words: planarian; innate immune system; immune-related genes introduction animals are exposed to microbes from the environment and require defense systems for protection from infectious agents. in the process of survival and evolution, animal immune systems have been improved by the invasion and attack of various exotic pathogens and endogenous harmful substances. the immune system generally includes immune tissues and organs, immune cells and immune molecules (franchi and ballarin, 2017). when a pathogen invades an organism, a series of responses occur through these molecules, cells and organs to eliminate the invader. in vertebrates, immunity includes both innate and acquired immunity (nyholm and graf, 2012). acquired immunity, which is stimulated by infection and vaccination, exhibits antigen specificity, diversity, immunological memory, and non-self recognition that is mediated by activated b and t cells. conversely, the innate immune system is an evolutionarily older defense strategy, and is an ubiquitous immune system found in every species of organisms studied so far (medzhitov and janeway, 2000b). ___________________________________________________________________________ corresponding author: qiuxiang pang school of life sciences shandong university of technology 266 xincun w road, zibo 255049, p.r. china e-mail: pangqiuxiang@163.com invertebrates, which are distributed in all parts of the world, do not produce specific antibodies to recognize and cope with multiple invasive pathogens; they rely on innate immune recognition and have the capacity to recognize self and non-self (chu and mazmanian, 2013). upon the invasion of non-self, the innate immune system exploits two defense mechanisms: constitutive defenses and inducible defenses (medzhitov and janeway, 2000a). constitutive defenses effectively prevent the non-self pathogens from entering the body through inherent barriers, e.g., surface barriers, mucous membranes of the gastrointestinal tract, respiratory tract and reproductive tract, antimicrobial peptides secreted by epithelial cells, antimicrobial enzymes and lysozymes in body fluids. the vast majority of the defense responses upon infection are through inducible defenses. in these processes, the exotic pathogens are recognized by immune cells, triggering a cascade of immune responses and high expression of related proteins. finally, pathogens are eliminated through phagocytosis, encapsulation and nodulation by phagocytes (browne et al., 2013). in addition, cytotoxicity by ros production is another way to kill microorganisms widely distributed among invertebrates (jiang et al., 2007; xu et al., 2017). the recognition mentioned above, also referred to as pattern recognition, is mediated by pattern recognition receptors (prrs) and pathogen-, http://link.springer.com/search?facet-author=%22lili+gao%22 http://link.springer.com/search?facet-author=%22qiuxiang+pang%22 mailto:pangqiuxiang@163.com 444 microbialor damage-associated molecular patterns (pamps, mamps or damrs) (wenger et al., 2014). a number of prrs have been reported in invertebrates, including toll-like receptors, nod-like receptors, rig-i-like receptors, c-type lectin (ctl), scavenger receptors, mannose receptor, peptidoglycan recognition proteins (pgrps), thioester-containing proteins, gram-negative binding proteins and so on (gao et al., 2017a). prrs specifically recognize the common and highly conservative molecular structures on the surface of pathogenic microorganisms to remove these pathogens. planarians (platyhelminthes, free-living rhabditophorans, tricladida) are of great interest to scientists because of the special evolutionary status and the prominent capacity of regeneration. planarians are exposed to a wide range of pathogenic microorganisms and harmful chemicals, and sometimes they feed on detritus, fungi, and bacteria (gonzalez-estevez, 2009; abnave et al., 2014). what is noteworthy is that there is little wound infection in regenerating planarians, suggesting that they may possess an efficient immune system in order to prevent infections. therefore, the study of planarian immunity is important to shed light on the relationship between the immune system and the regenerating capacity. here, we review the limited studies in this field, encompassing all levels of immune defenses to expound the innate and intrinsic immunity of planarians, and attempt to provide support for future immune response studies. the sites of immune responses planarians are naturally exposed to various pathogens within their habitat. epidermis, pharynx, intestines and reticular cells, are the most effective weapons to defend against pathogen invasion, by detecting and removing pathogens with different defense mechanisms. epidermis in planarian, as in other organisms, the epidermis not only prevents the leakage of internal molecules but also acts as the first barrier against invading pathogens and parasites in the environment. epidermal cells are constantly replacing themselves to maintain homeostasis and normal function. the planarian epidermis is a simple and unilaminar epithelium, which mainly consists of two types of cells: true, ciliated epidermal cells and microtubule-lined necks of the rhabdite-forming gland cells, whose cellular bodies lay in the parenchymal cell layers (skaer, 1965; rieger, 1991; hayes, 2017). the body surface of planarians is covered by a layer of secreted mucus mainly derived from specialized secretory granules known as rhabdites (hayes, 2017). the ejected slime structures are rich in sulphated glycosaminoglycans (sgags), and can be divided into two broad categories: a large one presenting in the outermost epidermal layer, and a small and highly striated one produced by rhabdite-forming gland cells (hayes, 2017). the viscous slime produced by these unique rhabdites not only lubricates the interface and prevents damage to the soft body, but also contributes to adhesion to the substratum. previous studies suggest that the slime is implicated in ciliary gliding, locomotion, predator avoidance, prey capture, innate immunity, and substrate adhesion (bocchinfuso et al., 2012; hayes, 2017). interestingly, some entrapped bacteria or ‘outer membrane’ embedded in the complex fibrous sgag-containing nets formed by ruptured rhabdites have been observed (hayes, 2017), suggesting that external mucous coating exerts a strong influence in response to environmental stimuli. this hypothesis has been confirmed through proteomic analysis of mucous secretions of the planarian, schmidtea mediterranea (bocchinfuso et al., 2012). proteomics data reveal that 119 planarian mucous proteins appear to be orthologs or significantly similar to mucous proteins in nasal mucus, olfactory mucus, cervical mucus, and tear fluid in human (bocchinfuso et al., 2012). among these annotated proteins, we discovered several immune-associated proteins, including 14-3-3 protein zeta (lu et al., 2017), alpha-2 macroglobulin (armstrong and quigley, 1999; ponprateep et al., 2017), dj-1 (tsushima et al., 2012), syntenin-1 (gordon-alonso et al., 2012; liu et al., 2015), peroxiredoxin-6 and superoxide dismutase (bocchinfuso et al., 2012). pharynx planarian pharynx is the exclusive food entrance towards the internal cavity, referred to as pharyngeal pouch or pharynx cavity, in the middle portion of the body. the highly extensible pharynx is a complex muscular organ consisting of multiple cell types, e.g., epithelial cells, muscle cells, neuronal cells and secretory cells (kobayashi et al., 1999; shimoyama et al., 2016). this cylindrical organ is also a channel that connects the intestine duct through the esophagus at the proximal end of the pharyngeal cavity (scimone et al., 2016) and the outside world rich in pathogenic microorganisms and harmful chemicals. pharynx epithelia had been recognized as a site of microbiota colonization and the first line of defense against pathogens in food and environment. therefore we speculate that it houses a large number of symbiotic species. recently, adler et al. discovered many genes required for pharynx regeneration through microarray technologies. among these genes, 356 are significantly up-regulated after amputation (adler et al., 2014). our follow-up study shows that many of these genes are immune-associated genes, e.g., placenta specific protein 8 (plac8), low-density lipoprotein receptor-related protein (lrp), and beta-defensin 114. our previous study found that plac8 of dugesia japonica inhibits the growth of the gram-negative bacteria in pharynx (pang et al., 2017). similarly, d. japonica lrp was found to be expressed in pharynx using an in-situ hybridization technique (unpublished data). in addition, in amphioxus, bjlrp, a novel pattern recognition protein, is capable of identifying and interacting with invading bacteria (gao et al., 2017c). beta-defensin, a member of amps, also possesses antimicrobial activities by resisting a broad spectrum of microorganisms, including gram-positive bacteria, gram-negative bacteria and fungi (pero et al., 2017). 445 it is not difficult to find that all of these immune-associated genes, located in pharynx, can identify and restrain exogenous microbes, indicating that pharynx, as the early immune defense organ, plays a pivotal role in immune responses. intestines the planarian intestinal duct is the organ managing nutrient digestion and metabolite distribution, it can be easily visualized by whole-mount in-situ hybridization with the innexin1 riboprobe (nogi and levin, 2005). anatomically, the gut can be divided into three main branches; one anterior, located in the head and prepharyngeal regions, which is linked together with the anterior end of the pharynx, and two posterior branches located in the pharyngeal and tail regions (forsthoefel et al., 2011). virtually, the branches can extend to the lateral margins of the body, and the gut can reach almost all positions of the body except the anterior region of the head. the intestinal epithelium, or gastrodermis, is composed of a single columnar layer surrounded by a thick basement membrane and muscle fibers (bueno et al., 1997), and consists mainly of two cell types, namely, absorptive phagocytes and secretory goblet cells (forsthoefel et al., 2011). phagocytes play a crucial role in the elimination of apoptotic cells and necrotic tissues deriving from injury and immune responses. in addition, phagocytes also ingest and breakdown invading pathogens and parasites (peiris et al., 2014; franchi and ballarin, 2017). phagocytosis is one of the main processes of bacterial elimination in planarians. one study demonstrates that the labeled bacteria, legionella pneumophila and staphylococcus aureus, in intestinal tissues shows a descending trend on day 6 post-feeding (abnave et al., 2014). in addition, whole-mount in situ hybridization reveals that 18 genes, which are highly involved in the elimination of l. pneumophila and s. aureus, are primarily expressed in the intestinal tissues in response to infection of bacteria (abnave et al., 2014). in summary, these findings underscore the remarkable ability of planarian intestine in the elimination of a broad range of bacterial strains. reticular cells all animals must maintain the bodies functional by eliminating wastes, repairing tissues, and resisting pathogens and foreign invaders. in these events, phagocytes play a pivotal role. phagocytes are common to all invertebrates; they can move freely in the circulating body fluids, where foreign materials and their own cells need to be recognized and obliterated, either actively or passively (morita, 1991). morita et al. have demonstrated that a special type of cells, which is similar to the fixed parenchymal cells, migrates to the wounded tissues (morita and best, 1974). they named this type of cells the reticular cell. planarian reticular cells play a significant role in nutrient transportation, homeostatic regulation of cells, and surveillance systems (morit, 1995). but beyond that, planarian reticular cells can be considered a sort of primitive mobile cells, equivalent to circulating cells involved in immune responses (immunocytes) in other invertebrates (kounatidis and ligoxygakis, 2012; taffoni and pujol, 2015; franchi and ballarin, 2017). structural and specific functional characteristics of planarian reticular cells have been investigated by light and electron microscopy; numerous glycogen granules, lipid droplets, some lysosomes and a nucleus with an irregular shape have been discovered in the cytoplasm (morit, 1995). the defense mechanism of reticular cells against invading pathogenic microorganisms was explored by injecting heat-killed bacteria into an incision and then examining the tissues around the incision (morita, 1991). as early as 10 h after the injection, foreign intruders are found in the phagosomes of reticular cells. by 12 h, a large amount of bacteria is encapsulated by the cytoplasmic processes of reticular cells, and by 24 h, the encapsulated bacteria are eliminated in the intestine (morit, 1995; morita, 1991). subsequent investigation in d. japonica and s. mediterranea has also confirmed the strong capacity of planarians to cope with infection of any of the 16 bacterial strains that are pathogenic to humans, caenorhabditis elegans and/or drosophila melanogaster (abnave et al., 2014). these findings indicate that foreign invaders can be recognized, phagocytized and encapsulated by reticular cells and expelled into the intestinal cavity to be digested and degraded. it is thus conceivable that reticular cells act as the main immunocytes involved in the defense against pathogens. in addition to participating in the immune response, reticular cells also take part in the process of tissue regeneration. immediately after the amputation, the cut surface lacking the epidermis and basement membrane is totally exposed to the environment which contains various pathogenic bacteria, fungi and harmful chemicals (morita and best, 1974). one hour after the amputation, the pre-existing epidermal cells around the wound surface stretch out their own bodies and maintain a close connection with each other to make a thin basement membrane (morita and best, 1974). six to eight h after the amputation, the reticular cells appear on the entire cut surface underneath the epidermal film, and then, some of them appear to extend their cytoplasmic processes and contact with one another to form the regeneration blastema or a specific meshwork (morita and best, 1974; morit, 1995). approximately 16 h after the amputation, the entire wound surface is covered by the basement membrane, after which, the thinly stretched epidermal film begins to thicken and the neoblasts begin to differentiate (morita and best, 1974). in the late stages of the wound regeneration, the epidermal film is broken down, and differentiating cells infiltrate the interstices of the meshwork of reticular cells. during this period, reticular cells phagocytize the debris of the damaged or degenerating cells. the reticular cells, which appear early in the cut surface, play a very important role in both its own cell clearance and pathogen scavenging during the regeneration process. immune-related genes in the innate immune responses, prrs have an essential role in identifying specific components of foreign pathogens and triggering a series of cascade 446 table 1 expression of innate immune candidate genes of planarian upon challenge with bacteria. the table is adopted from the whole-transcriptome analysis performed by abnave et al. (2014) protein name pfam domain sequence numbers trafficking protein particle complex subunit 1 (trappc1) sybindin domain (pf04099) seq51130 progestin and adipoq receptor family member 3 (paqr3) hemolysin-iii-related domain (hly iii) (pf03006) seq53353 trafficking protein particle complex subunit 2-like (trappc2l) sedlin n domain (pf04628) seq74013 membrane occupation and recognition nexus (morn) repeat-containing-2 (morn2) cog4642 superfamily domain (pf02493) seq74746 atox1 heavy-metal-associated (hma) domain (pf00403) seq75036 hydroxyacylglutathione hydrolase (hagh) lactamase b domain (pf00753) seq78560 cuta cuta1 domain (pf03091) seq76275 dusp19 dual-specificity phosphatase (dusp) domain (pf00782) seq31603 hypothetical protein dynamin n domain (pf00350) seq39446 dynein light chain roadblock-type (dynlrb) roadblock/lc7 domain (pf03259) seq75092 reactions through the downstream adaptor proteins and effector molecules to eliminate the invading microbes. planarians are exposed to a wide range of pathogens and harmful chemicals. however, infections which lead to death rarely occur, suggesting that they possess an effective immune system. abnave et al. (2014) discovered that planarians can resist infections by multiple bacterial species pathogenic to humans, drosophila and nematode. they identified 18 genes expressed in the intestine of the infected planarians that actively contribute to the elimination of l. pneumophila and s. aureus. table 1 provides the detailed information of the ten genes that contain clear domains recorded by pfam. other studies have also reported that immune-related genes play an essential role in different aspects of the immune responses. it should be noted that planarian immune-related genes and immune response pathways are also involved in other physiological processes. in addition, peiris et al. (2014) revealed that planarians contain many potential homologs of the mammalian innate immune system that are activated during injury and repair of adult tissues. furthermore, tsoumtsa et al. (2017) uncovered that smed-tim, a homolog of the mammalian clock gene tim, is an indispensable protein for anti-microbial activities against s. aureus infection during the light/dark cycle. table 2 includes a collection of candidate genes that possess potential immune defense functions. details about these candidate genes are provided below. pattern recognition receptors (prrs) in animals, especially invertebrates, the innate immune system plays a crucial role in the defense against causative agents. the innate immune responses rely on the specific pattern recognition, in which, prrs identify pamps, such as bacterial cell wall composition, dsrna, and some obsolete components released by damaged and dead cells. in the absence of the adaptive immunity, the number and diversity of prrs may provide an advantage for animals living in pathogen-rich environments. our analysis of previous reports of planarians identified some prr genes, including toll-like receptors (tlrs), rig-i-like receptors (rlrs), ctl/selectins, mannan binding lectin, peptidoglycan recognition protein-1 (pgrp1) and g-protein-coupled receptor (gpcr). 447 table 2 summary of the innate immune candidate genes reported so far in planarians. a brief description of the domain, data sources and reference is also reported protein types protein name domain data sources references receptor toll-like receptors leucine rich repeat domain (lrr); toll/interleukin-1 receptor domain (tir) smedgd peiris et al., 2014 rig-i-like receptors helic domain; dexd domain dj-bstd pang et al., 2016 c-type lectin/ selectins c-type lectin like domain dj-bstd pang et al., 2016; peiris et al., 2014 mannan binding lectin (mbl) c-type lectin like domain dj-bstd pang et al., 2016 peptidoglycan recognition protein-1 pgrp superfamily domain afj24867.1 wenemoser et al., 2012 g-protein-coupled receptor 7tm_gpcrs superfamily domain ab495373 nishimura et al., 2009 anti-microbial peptides/ defense antimicrobial peptide resistance and lipid a acylation protein pagp pagp superfamilu domain; om_channels superfamily domain smedgd peiris et al., 2014 beta-defensin 114 defensin_beta_2 superfamily domain sm-prtd adler et al., 2014 lysozyme lysozyme_like superfamily domain dj-bstd pang et al., 2016 membrane attack complex component /perforin/c9 macpf superfamily domain smedgd altincicek et al., 2008 complement system alpha 2-macroglobulin a2m domain dj-bstd pang et al., 2016; peiris et al., 2014 complement cub domain; c1q domain smedgd peiris et al., 2014 properdin thrombospondin type 1 domain smedgd no reference complement factors or integrins von willebrand factor type a domain smedgd peiris et al., 2014 antioxidant enzyme catalase (cat) catalase_clade_3 domain sm-mpd bocchinfuso et al., 2012; zhang et al., 2014; zhang et al., 2016 superoxide dismutase (sod) soda domain sm-mpd bocchinfuso et al., 2012; zhang et al., 2014; zhang et al., 2016 glutathione peroxidase (gpx) grx_grxh_1_2_like domain smedgd zhang et al., 2014; zhang et al., 2016 peroxiredoxin prx5_like domain sm-mpd bocchinfuso et al., 2012 thioredoxin peroxidase (tpx) prx_typ2cys domain smedgd no reference various immune associated proteins phenoloxidase (po) tyrosinase domain dj-bstd pang et al., 2010; lapan et al., 2011 lps-induced tnf-alpha factor litaf-like zinc ribbon domain smedgd peiris et al., 2014 traf family proteins traf-type zinc finger; math domain dj-bstd peiris et al., 2014; pang et al., 2016 low-density lipoprotein receptor-related protein (lrp) ldla; egf_ca; ly domain sm-prtd adler et al., 2014 placenta specific protein 8 (plac8) plac8 superfamily domain alj10579 pang et al., 2017 phospholipid scramblase lor superfamily domain apl96720.1 han et al., 2017 syntenin pdz domain sm-mpd bocchinfuso et al., 2012 448 c-jun n-terminal kinase (jnk) stkc_jnk domain dj-bstd pang et al., 2016 mitogen-activated protein kinase (mapk) pb1 domain; catalytic domain; serine/threonine kinase dj-bstd pang et al., 2016 p38 serine/threonine kinase; catalytic domain dj-bstd pang et al., 2016 traf family member-associated nf-κb activator-binding kinase 1 (tbk1) pkc_like superfamily; s_tkc domain dj-bstd no reference interferon regulatory factor (irf) irf-3 domain; irf superfamily domain dj-bstd no reference mx p-loop_ntpase; dynanin_m domain dj-bstd no reference γ-ifn-inducible lysosomal thiol reductase gilt superfamily domain dj-bstd no reference timeless (tim) timeless domain sm-pmtd tsoumtsa et al., 2017 toll-interacting protein (tollip) c2 domain; cue domain dj-bstd pang et al., 2016 tdj-bstd: dj-bs transcriptome database (https://www.ncbi.nlm.nih.gov/sra/?term=srx1479355) sm-prtd: s. mediterranea pharynx regeneration transcriptome database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse56181) sm-mpd: s. mediterranea mucous proteomic database (http://www.mcponline.org/content/11/9/681/suppl/dc1) smedgd: s. mediterranea genome database (http://smedgd.neuro.utah.edu/) ddbj: dna data bank of japan (http://www.ddbj.nig.ac.jp/) sm-pmtd: s. mediterranea transcriptome database planmine (http://planmine.mpicbg.de/planmine/begin.do) the transmembrane tlrs belong to type i transmembrane receptors, composed of the c-terminal toll/interleukin-1 receptor (tir) domain and n-terminal leucine rich repeat (lrr) domain. peiris et al. identified several gene segments that contain the lrr domain and tir-like domain, respectively (peiris et al., 2014). in the dj-bs (clonal strain d. japonica collected from boshan district of china) transcriptome database, we also found many sequences that contain different combinations of lrr domains, but no tir domain was found in these sequences. it is unknown whether these lrr repeats have tlr-like activities. interestingly, toll-interacting protein (tollip) and ifn-stimulated genes (isgs), such as mx and γ-ifn-inducible lysosomal thiol reductase (gilt), were identified in the databases. subsequently, we need to do more research on the tlr signaling pathway of planarians. rlrs play an important role in viral infection by recognizing and binding the double stranded rnas and 5'-triphosphate single stranded rnas in the invading virus (xu et al., 2005). after binding with viral rnas, rlrs interact with mavs/visa/cardif/ips-1 and form a protein complex to recruit tumor necrosis factor receptor associated factor 3 (traf3) and traf6. then, a number of transcription factors, including nuclear factor-κb (nf-κb) and interferon-regulated-factor (irf), are activated, and type i interferons (ifn-α/β) are successfully expressed (kawai et al., 2005; xu et al., 2005). in our previous study, we revealed that the mrnas of rig-i, traf3, traf6, and p38 involved in the rig-i-like receptor signaling pathway are significantly up-regulated after being induced by lps, peptidoglycan, and poly (i:c) as well as gram-negative and gram-positive bacteria; the results show that these factors may be involved in planarian immune responses (pang et al., 2016). the other two proteins, traf family member-associated nf-κb activator-binding kinase 1 (tbk1) and irf in this signaling pathway, have also been identified. but the adaptor protein mavs/visa/cardif/ips-1 was not found in the annotated proteins of the databases (fig. 1). additional experiments are required to investigate whether there is a novel adaptor protein in planarians. ctl, selectins and mbl have the same conserved c-type lectin (ctl)/c-type lectin-like (ctld) domain (clect domain), homologous to the carbohydrate-recognition domains (crds) of the ctl. the clect of these lectins can recognize specific carbohydrates on the surface of pathogens, allergens, necrotic or apoptotic cells, and then mediate the functions associated with phagocytosis and elimination. recently, we identified a novel c-type lectin-like protein, which participates in the immune response to bacteria infection (gao et al., 2017a) (fig. 1). in the dj-bs transcriptome database, we identified several gene segments that contain different amounts of the crd domain. this phenomenon has also been reported in scallops (chlamys farreri), where three crd-containing genes, cflec-1, cflec-2 and cflec-4, have been identified, and their relative immune functions and binding mechanism have been elucidated (yang et al., 2010; yang et al., 2011; huang et al., 2013). gpcrs, a large family of seven transmembrane domain receptors, are involved in multiple signaling pathways. they are directly or indirectly related to many important physiological processes, such as https://www.ncbi.nlm.nih.gov/sra/?term=srx1479355 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse56181 http://www.mcponline.org/content/11/9/681/suppl/dc1 http://smedgd.neuro.utah.edu/ http://www.ddbj.nig.ac.jp/ http://planmine.mpicbg.de/planmine/begin.do 449 fig. 1 overview of the supposed intracellular immune signaling pathway activated by pamp binding. only parts of the identified components are shown. proteins not yet identified are marked by a question mark. the proposed but yet to be verified interaction between proteins is indicated using a dashed arrow. nerve impulse transmission, cell metabolism, immune defense, and cell differentiation (hollenstein et al., 2013). in invertebrates, numerous pieces of evidence point to an important role of gpcrs in host defense through different signaling pathways (reboul and ewbank, 2016). in d. japonica, a novel gpcr (djser-7) has been identified, but its specific immunological functions have yet to be identified (nishimura et al., 2009). similarly, the preliminary study of pgrp of planarians is also limited. four pgrp-like receptors, namely, smed-pgrp-1, -2, -3, and -4, have been identified in s. mediterranea (arnold et al., 2016). recently, torre et al. discovered that primo-infection of s. aureus leads to the expression of smed-pgrp-2, which, in turn, promotes smed-setd8-1 methyltransferase signaling and increases the lysine methylation level in neoblasts to sensitize anti-bacterial gene responses during re-infection (torre et al., 2017) (fig. 1). in future, more members of gpcrs and pgrps of planarians and immune response mechanisms need to be explored. antimicrobial peptides peptides with a variety of broad-spectrum antimicrobial activities are important components of non-specific defense systems. these small peptides are characterized by the evolutionarily conserved clusters of positively charged and hydrophobic amino acids (pasupuleti et al., 2012). the antibacterial mechanism of amps is mainly through dissolving bacterial cell membranes or altering the permeability of cell walls. meanwhile, a few amps can kill bacteria by inhibiting the synthesis of bacterial dna, rna and proteins (pasupuleti et al., 2012). besides amps, in vertebrates, membrane attack complex component/perforin/c9 also contributes to the elimination of pathogens, which is one of the important effector molecules in immune responses and immune defenses. undeniably, antibacterial peptides and perforin play an indispensable role in the innate immune system of animals. we describe four proteins, beta-defensin 114, lysozyme, antimicrobial peptide resistance and lipid a acylation protein pagp and membrane attack complex component/perforin/c9, which were discovered in different databases of planarians. these proteins are all functional proteins at the bottom of the signaling pathway activated after external pathogen invasion. how these critical proteins are activated during infection remains to be ascertained. 450 complement system in vertebrates, the complement system, as one of the original innate immune systems, is a complex protein reaction system composed of serum proteins, regulatory proteins and membrane receptors (ghebrehiwet, 2016). when responding to pathogen and inflammation, the host employs the activation pathways of complements, including the classical pathway, the activating pathway and the lectin pathway, to form a membrane attack complex and finally give rise to immune cytolysis and immune haemolysis. in this process, complement component 3 (c3) is the central node of these pathways and locates in a position not to be ignored in immune surveillance and immune responses (ricklin et al., 2016). as an important and highly conserved component of the innate immune system, the complement system plays a significant role in the process of host defense against infections. planarians appear to have some conservative complement system associated domains that may resist invading pathogens, but their exact functions need to be confirmed and further studied. only a few homologous domains of complement proteins and factors have been discovered in the s. mediterranea genome database for example cub domain, c1q domain and von willebrand factor type a domain (peiris et al., 2014). these domains are associated with more than one protein family, so, without knowing the full length of a gene, we could not assign it to a complement protein. similarly, in the dj-bs transcriptome database and many other databases, we did not find any annotated complement protein either. although planarian c3 is absent, we cloned the full length of a2m which possesses almost exactly the same structural domain as c3 from d. japonica. a2m is an evolutionary conservative element of the innate immune system which is also considered as a broad-spectrum protease inhibitor (armstrong and quigley, 1999). in recent years, a large number of a2m in invertebrates have been identified, which are involved in a variety of immune responses in the case of pathogen invasion (pathirana et al., 2016; ponprateep et al., 2017). properdin, also known as p factor, has also been discovered in planarians. as a serum glycoprotein, properdin can initiate and positively regulate the alternative pathway activity (blatt et al., 2016). however, planarians lack the necessary complement system proteins, so we hypothesize that properdin may engage in immune responses by other mechanisms. in amphioxus, recombinant bjfp (factor p of branchiostoma japonicum) is capable of interacting with both gram-negative and gram-positive bacteria as well as lps and lipoteichoic acid (gao et al., 2017b). antioxidant enzymes organisms have evolved a variety of mechanisms to protect themselves from the toxic effects of heavy metals, harmful chemicals and microorganisms that cause oxidative stress. reactive oxygen species (ros) is derived from one-electron reduction of oxygen molecule mainly in mitochondria (bernard et al., 2015). ros production can be associated with phagocytosis and lead to killing of phagocytes, but abnormal ros levels can lead to damage to the animal body (tan et al., 2016). in order to minimize oxidative damage, organisms have developed antioxidant defenses composed of antioxidant enzymes, including catalase (cat), superoxide dismutase (sod), glutathione peroxidase (gpx), thioredoxin peroxidase (tpx) and other peroxiredoxin (bernard et al., 2015). an induction of the antioxidant defenses can be considered as an adaptation of the organisms to overcome insecure environments and to prevent toxicity (zhang et al., 2014). coincidentally, these antioxidant enzymes are also present in planarians. bocchinfuso et al. (2012) identified several antioxidant enzymes, including peroxiredoxins, cat and sod, in the mucus secreted by s. mediterranea. in addition, studies have shown that copper (zhang et al., 2014) and 1-octyl-3methylimidazolium bromide ([c8mim]br) (zhang et al., 2016) can induce oxidative stress in planarians, which, in turn, activates the corresponding antioxidant enzymes. in these studies, the activity changes of the enzymes also indicate that they participate in the corresponding immune responses in facing the external environment stresses. how these antioxidant enzymes are activated, when animals land in harsh environments, remains to be ascertained. concluding remarks planarian is traditionally a favored animal model in regeneration and development, while recently, its immune system has also attracted wide attention from biologists. the complex living environment, containing various pathogens, and high survival rates of planarians suggest that they have an effective innate immune system to prevent infections. surface barriers and mucous membranes from epidermis, pharynx and intestine of planarian effectively prevent pathogens from entering the internal body by constitutive defense mechanisms. in addition, some of the pathogens that evade the constitutive defense system and enter the planarian body are eliminated by inducing defense mechanisms, in which, prrs, e.g., rlrs, ctl, gpcr and pgrp1, recognize the pamps on the surface of those non-self foreign pathogens, leading to a high expression of related proteins and a series of immune responses. in conclusion, the reports on the planarian immune system indicate that more studies need to be carried out to expound the strong survivability of planarians. moreover, although numerous transcriptome and proteome studies on planarians have been reported, a large number of sequences are not spliced or annotated. hence, one important step in the study of the planarian immune system is to acquire the complete genome 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university, dalian 116023, china 3dalian key laboratory of aquatic animal disease prevention and control, dalian ocean university, dalian 116023, china this is an open access article published under the cc by license accepted may 16, 2022 abstract yesso scallop (patinopecten yessoensis) is one of the most important cultured mollusc in china. however, the diseases of yesso scallops, especially abscess disease, occurred frequently and led to massive mortality in recent years. in the present study, 20 bacterial strains were isolated and characterized from the adductor muscle of moribund yesso scallops in the north yellow sea to identify the potential pathogens, and 14 of them were grouped into genus vibrio. vibrio neocaledonicus (m-08 strain) was found to be the most dominant strain, and it was able to survive and exhibit haemolytic activity from 8 °c to 36 °c with the highest activity at 32 °c. the pathogenicity of m-08 in yesso scallops was further investigated by intravalvar injection and immersion challenges, and the cumulative mortality rates were calculated to be 93 % and 53 %, respectively. the activities of superoxide dismutase (sod) and catalase (cat) increased after m-08 intravalvar injection, and peaked at 3 h and 6 h, respectively. the mda contents increased and were significantly higher than that in the control group at 3 h after m-08 intravalvar injection. immediately afterwards, immune response and haemocyte apoptosis were observed in yesso scallops. therefore, v. neocaledonicus is the pathogen of abscess disease in the yesso scallops, which was able to activate the antioxidant system and immune response, and cause haemocyte apoptosis, tissue damage and death sequentially. the information would provide helpful contribution to the prevention and control of abscess disease in yesso scallop aquaculture. key words: yesso scallop; isolation and identification; pathogenicity; bacterial infection; vibrio neocaledonicus introduction the yesso scallop (patinopecten yessoensis) is a cold-water bivalve species naturally distributed in northern japan, northern korean peninsula and far east of russian. since it was introduced to china in 1982, yesso scallop has become one of the major farmed marine species in northern china, and the processed product contributes to the worldwide aquatic protein supply (fao, 2014; pan et al., 2018). in the past decades, mollusc aquaculture has been greatly plagued by repeat episodes of bacterial diseases all over the world (romalde et al., 2014; _________________________________________ corresponding authors: lei gao linsheng song dalian key laboratory of aquatic animal disease prevention and control dalian ocean university dalian 116023, china e-mail: gaolei@dlou.edu.cn; lshsong@dlou.edu.cn cheikh et al., 2016). massive mortalities caused by frequent outbreaks of bacterial diseases, especially during the summer, have seriously limited the development of yesso scallop aquaculture industry. the abscess disease is one of the most direct triggers causing high mortalities of the cultured yesso scallops (teng et al., 2012; liu et al., 2013). it was reported that the abscess disease with apparent lesions on adductor muscle of yesso scallops had occurred for three consecutive years from 2009 to 2011 in northeastern china (teng et al., 2012; liu et al., 2013). the mortality of the cultured yesso scallops was up to 80 % in the summer of 2009 and 2010 in changhai county, mainly caused by abscess disease (teng et al., 2012). the main symptoms of abscess disease were the presence of abscess and lesions on adductor muscle of yesso scallops, and several species in genus vibrio, such as v. splendidus and v. chagasii, were preliminarily 92 identified to be involved in the onset of abscess disease (teng et al., 2012; liu et al., 2013). however, the pathogens and the pathogenesis for abscess disease are still not well understood, as various pathogenic bacterial species were speculated to be involved and the pathogenic process was opportunistic and complex. it is very important to identify the pathogens and investigate their pathogenesis in abscess disease. the genus vibrio consists of more than 100 species grouped into 14 clades, which are widely distributed in aquatic environments such as estuarine, coastal waters and sediments (romalde et al., 2014). some species of vibrio spp. have been reported to cause high mortalities of farmed molluscs (gómez-león et al., 2005; jones et al., 2012), and vibriosis is considered as one of the most prevalent bacterial diseases in aquaculture. the pathologies caused by vibrio spp. in bivalves have been described since the 1960s (karaolis et al., 1998; beaz-hidalgo et al., 2010). most of the pathogenic vibrio strains are opportunistic and strongly thermo-dependent, and many species thrive in warm water exceeding 17 °c (destoumieux-garzón et al., 2020). some species of genus vibrio, such as v. splendidus and v. harveyi, were often reported as pathogens in various invertebrate species (lacoste et al., 2001; austin and zhang, 2006; liu et al., 2013). v. neocaledonicus is a newly reported vibrio species with high similarity to v. alginolyticus and v. natriegens. the extracellular products secreted by v. neocaledonicus exhibited corrosion inhibition effect on marine materials (moradi et al., 2015a; moradi et al., 2015b). v. neocaledonicus was reported to be a pathogenic bacterium for echiuroid worm (urechis unicinctus) (yu et al., 2019a) and pacific white shrimp (litopenaeus vannamei) (wang et al., 2021). however, there is no report about the pathogenicity of v. neocaledonicus in molluscs. in the present study, a five-year survey was conducted on the yesso scallop aquaculture industry in northeastern china from 2016 to 2020 (yu et al., 2019b; yu et al., 2019c). a batch of moribund yesso scallops in abscess disease were collected to identify the potential pathogens. v. neocaledonicus was found to be most dominant among the culturable bacteria isolated from lesions, and it was further characterized in order to (1) investigate the physiological and biochemical characteristics of the strain, (2) preliminarily determine its pathogenicity, and (3) examine the pathological and physiological alternation of yesso scallops caused by the strain. materials and methods pathogenic bacteria isolation and culture scallops (average shell length of 65 ± 4 mm, average weight of 34 ± 7 g) with lesion-like niduses on the adductor muscle were collected from a raft farming area located at n39°18’10” latitude and e122°44'0" longitude in the north yellow sea. the sample was processed in the superpurgative working table, and the specific process was as follows: the surface of the shells and the soft tissues were washed using sterile seawater, the niduses on the adductor muscle were cut from the adductor muscle using sterilizing equipment and homogenised in 5 ml sterile seawater. the homogenized solution was centrifuged at 800 g (4 °c, 5 min), and the supernatant was plated on zobell 2216e agar (code no. hb0132, hopebio biotech, china) (liu et al., 2013; yu et al., 2019c). twenty single colonies were randomly selected from zobell 2216e agar according to the colony size, shape, edge, gloss, texture, color and transparency. dna extraction, 16s rdna and gyrb gene amplification single colonies were incubated in zobell 2216e broth at 25 °c for 24 h to obtain bacterial suspension. the genomic dna was extracted from the bacterial suspension by following methods: bacterial culture was centrifugated at 8,000 g (4 °c, 5 min), washed once with ultrapure water, resuspended with ultrapure water according to the amount of precipitation after centrifugation under the same conditions, and heated in a metal bath at 100 °c for 10 min. after a centrifugation at 1,000 g for 3 min, the final supernatant containing the bacterial genomic dna was verified by 1 % agarose gel electrophoresis and used for 16s rdna and gyrb gene amplification (xia et al., 2014). the primers of 16s rdna and gyrb gene amplification are listed in table 1. the 16s rdna amplification was carried out in a total volume of 50 μl, containing 2 μl of genomic dna, 25 μl of pcr mastermix, 2 μl of each primer and 19 μl of pcr grade water. the genomic dna template was substituted with pcr-grade water in the negative control. the pcr reaction was performed at 94 °c for 5 min, 30 cycles of 94 °c for 1 min, 55 °c for 30 s, and 72 °c for 1.5 min. then the reaction was completed by extension at 72 °c for an additional 10 min. the gyrb amplification was carried out in a total volume of 25 μl, containing 2 μl of genomic dna, 12.5 μl of pcr mastermix, 0.5 μl of each primer and 9.5 μl of pcr grade water. the genomic dna template was substituted with pcr-grade water in the negative control. the pcr reaction was performed at 94 °c for 2 min, 30 cycles of 94 °c for 30 s, 55 °c for 45 s, and 72 °c for 30 s. then the reaction was completed by extension at 72 °c for an additional 10 min. the amplified products were purified using an agarose gel recovery kit (code no. 9762, takara bio, china) and were sequenced by sangon biotech company (shanghai, china). bacterial 16s rdna sequences were analyzed on the rdp (http://rdp.cme.msu.edu/classifier/classifier.jsp), ezbiocloud (www.ezbiocloud.net) and ncbi (www.ncbi.nlm.nih) database. gyrb sequence was analyzed on the ncbi (www.ncbi.nlm.nih) database. the phylogenic trees were constructed by the neighbour-joining (nj) method and maximum likelihood (ml) method using the mega 6 software based on the 16s rdna and gyrb gene sequences of m-08 and nine other vibrio spp. species phenotypic analysis of m-08 the most dominant strain was named as m-08 according to the isolated tissue and the isolation http://www.ncbi.nlm.nih/ http://www.ncbi.nlm.nih/ 93 table 1 the primers used in this study name sequence（5'-3'） comment 27f agagtttgatcctggctcag for pcr 1492r ggttaccttgttacgactt for pcr gyrb-f gargtggtrgataactcwattgatgaagc for pcr gyrb-r cggtcatgatgatgatgttgt for pcr nf-κb-f tgcccgtgttgtggtaaccttgg for qrt-pcr nf-κb-r cgtgagagagttttgtccgccctt for qrt-pcr pytnfr-1-f ggatgtcagcaatgtagagaaggc for qrt-pcr pytnfrr-1-r catatcgtgtcacgtgtaggggta for qrt-pcr qβ-actin-f agtcccaatctacgaaggttatg for qrt-pcr qβ-actin-r ccagtgatgaggaggaagcag for qrt-pcr number, and chosen for subsequent experiments. m-08 was cultured in zobell 2216e broth at 25 °c for 24 h, and the bacterial culture was centrifuged at 8,000 g for 10 min. the pellet was resuspended and washed twice using sterile seawater to eliminate interference from the medium. the morphological structure of m-08 was observed by a transmission electron microscope (tem). the physiological and biochemical characteristics of m-08 were tested by api 20ne (bio mérieux, french) and tcbs agar. the susceptibility of m-08 to antibiotics was determined by a disk-diffusion method using commercially antibiotic disks (hangzhou microbial reagent co., ltd., china). a total of 14 types of common antibiotics (amoxicillin, penicillin, florfenicol, chloramphenicol, erythromycin, neomycin, streptomycin, tetracycline, doxycycline, rifampin, sulfamethoxazole, sulfisoxazole, enrofloxacin, norfloxacin) were dotted on the zobell 2216e agar that uniformly plated by 100 μl suspensions of m-08. the plates were incubated at 25 °c for 24 h to check if m-08 zone existing around the chemical sheet. haemolytic activity of m-08 the haemolytic activity of m-08 was tested using the sheep blood agar (zobell 2216e agar with 5 % sheep blood). m-08 culture was dotted (2 μl for each dot) on the sheep blood agar and incubated at different temperatures (8 °c, 16 °c, 24 °c, 32 °c and 36 °c) for one week. the presence of a clear zone on the blood agar was defined as positive haemolysis (liu et al., 2013). the haemolytic circle around the drop of m-08 was analyzed both by observation of naked eyes and measurement of the diameter. the challenge test of scallop with m-08 a total of 300 scallops (average shell length of 64 ± 3 mm, average weight of 33 ± 5 g) were employed for the challenge experiments, and they were acclimatized at 15 °c in 30-liter tanks with aeration for one week. the water temperature was raised by 1 °c a day to 18 °c and kept for a week for the challenge test. there were two challenge experiments using the bacterial suspension of m-08, including intravalvar injection experiment and immersion challenge experiment. in the intravalvar injection experiment, 120 scallops were randomly assigned to four groups (30 scallops for each group) including three challenge groups and one control group. in the three challenge groups, 100 µl of m-08 suspension was inoculated into the adductor muscle of each scallop with three concentration levels of 3×102 cfu ml-1, 3×104 cfu ml-1 and 3×106 cfu ml-1. in the control group, each scallop was inoculated with 100 µl of sterile seawater. another 120 scallops were employed for the immersion challenge experiment, and they were also randomly assigned to four groups (30 scallops for each group). in the three challenge groups, m-08 suspension was added to the seawater at three final concentrations of 3×102 cfu ml-1, 3×104 cfu ml-1 and 3×106 cfu ml-1. no treatment was conducted for the control group. the mortality was checked and recorded every day during the 16-day challenge experiment. behrens-kärber method was used to calculate the ld50 (zhang et al., 2015). according to koch’s postulates, the bacteria were reisolated from the gaping or dying scallops with intravalvar injection and immersion challenge. the adductor muscle tissues were homogenized under aseptic condition and cultured on zobell 2216e agar to isolate and purify bacteria as described above. the bacterial colonies inoculated on tcbs agar plus penicillin (1 µg ml-1) were harvested for dna extraction and 16s rdna sequencing as described above. histopathologic analysis of the challenged scallops the adductor muscle, hepatopancreas, gill and mantle samples of the dying challenged individuals and the control individuals were dissected and fixed with bouin's fixative for 24 h. after embedding in paraffin, the samples were cut with a microtome (leica, china). the samples were processed as following: (1) deparaffinize as followed: xylene i for 20 min; xylene ii for 20 min; 100 % ethanol i for 5 min; 100 % ethanol ii for 5 min; 75 % ethanol for 5 min; rinsing with tap water; (2) stain sections with 94 table 2 the bacterial strains isolated from the moribund yesso scallops note: the bold highlights the dominant strains hematoxylin solution for 3 min; rinse with tap water; then treat the section with hematoxylin differentiation solution; rinse with tap water; treat the section with hematoxylin scott tap bluing; rinse with tap water; (3) 85 % ethanol for 5 min; 95 % ethanol for 5 min; finally stain sections with eosin dye for 5 min; (4) dehydrate as followed: 100 % ethanol i for 5 min; 100 % ethanol ii for 5 min; 100 % ethanol iii for 5 min; xylene i for 5 min; xylene ii for 5 min; finally seal with neutral gum; (5) observe with microscope inspection, image acquisition and analysis. the analysis of antioxidant response after intravalvar injection challenged with m-08 at the concentration of 3×104 cfu ml-1 that was closet to ld50 among the three challenge concentrations used in the challenge test, the activities of superoxide dismutase (sod) and catalase (cat), and the contents of malondialdehyde (mda) in serum of control and challenged scallops were analyzed using the corresponding assay kits (jiancheng, nanjing, china; product code: a001-3-2, a007-1-1, and a003-1-2, respectively) according to the manufacturer’s guidelines. the international unit of enzyme activity was defined as the amount of enzyme required to convert 1 μmol of substrate. the analysis of immune response after intravalvar injection challenged with m-08 at the concentration of 3×104 cfu ml-1 that was closet to ld50 among the three challenge concentrations used in the challenge test, the contents of lysozyme (lzm) in serum of control and challenged scallops were analyzed using the corresponding assay kit (jiancheng, nanjing, china; product code: a050-1-1), and the total rna was extracted from the samples using trizol™ reagent according to the manufacture's protocol. rna concentration was measured by a nanodrop 2000 (saveen & werner aps, denmark), and the integrity and purity of rna were examined by electrophoresis analysis in 1.0 % agarose gel. the first strand of cdna was synthesized using total mrna (treated with dnase i) as the template and oligo (dt)17-adaptor as primer according to the protocol of manufacturer (takara, japan). the synthesis reaction was performed at 42 °c for 5 min and terminated by heating at 85 °c for 5 s. the cdna mix was diluted ten times by pcr-grade water and used as the template. temporal expression of pynf-κb and pytnfr-1 mrna in haemocytes of control and challenged scallops were measured by quantitative real-time pcr (qrt-pcr). pynf-κb and pytnfr-1 gene-specific primers (table 1) (li et al., 2015; xing et al., 2016) were used to amplify the product from cdna, and the pcr product was sequenced to verify the specificity of qrt-pcr. the expression of β-actin was used as the internal control to verify the successful transcription and to calibrate the cdna template for the corresponding scallop samples. all reactions were conducted in an abi prism 7500 detection system (applied biosystems®, usa). the assay of haemocyte apoptosis after intravalvar injection challenge with m-08 at the concentration of 3×104 cfu ml-1 that was closet to ld50 among the three challenge concentrations used in the challenge test, the scallop haemocytes were processed using annexin v-fitc/pi detection kit (beyotime biotechnology, china), and the apoptosis was assessed by a flow cytometry (bd biosciences, usa). the haemocytes from the unchallenged scallops were regarded as the blank group. all the haemocyte samples were resuspended in a 195 μl 1×binding buffer solution at a final concentration of 1-5×105 cells ml-1 and then bacterial names number proportion ( %) vibrio spp. 14 70 vibrio xuii 1 5 vibrio europaeus 1 5 vibrio campbellii 1 5 vibrio neocaledonicus 8 40 vibrio chagasii 3 15 pseudoalteromonas spp. 6 30 pseudoalteromonas shioyasakiensis 2 10 pseudoalteromonas espejiana 3 15 pseudoalteromonas carrageenov 1 5 95 fig. 1 the phylogenetic tree constructed using m-08 and other nine vibrio species. (a) 16s rdna; (b) gyrb. the numbers 1 and 2 represented the neighbor-joining method and maximum likelihood method respectively. the phylogeny was tested by bootstrap based on 1000 replicates stained with 5 μl annexin vfitc and 10 μl pi at room temperature for 30 min in dark. the cell suspensions were analyzed by the flow cytometry. statistical analysis the statistical analysis was performed using one-way anova by statistical package for social sciences (spss) version 20.0, and the data were presented as means ± standard deviations with three parallel replicates. the differences were considered statistically significant at p < 0.05 labeled with “*” and extremely significant at p < 0.01 labeled with “**”. results bacterial identification a total of 20 bacterial strains were isolated from the lesions of moribund scallops. after identified through 16s rdna sequencing, 14 strains of vibrio spp. and six strains of pseudoalteromonas spp. were obtained (table 2). notably, eight of the 14 vibrio strains were found to be v. neocaledonicus by 16s rdna sequence alignments. therefore, v. neocaledonicus was suspected to be the potentially pathogenic species. among the eight strains of v. neocaledonicus, the most dominant strain was named as m-08 according to the isolated tissue and the isolation number, and chosen for the subsequent experiments. for further confirmation of its taxonomic relationship, first, 16s rdna and gyrb sequences of m-08 (genbank accession numbers: ol584447 and ol656098, respectively) was aligned on the rdp, ezbiocloud and ncbi databases, and v. neocaledonicus was the most hit objective species. second, phylogenic trees were constructed using both 16s rdna and gyrb sequences, and m-08 was clustered into the branch consisting of the typical v. neocaledonicus strain (fig. 1). third, phenotypic characteristics of m-08 were identified as following. after incubated at 25 °c on tcbs agar for 24 h, the green colonies of m-08 were round, and the surface is raised, smooth, not easy to pick (fig. 2a). according to the tem observation, m-08 was regularly rod-shaped in the uniform size of approximately 1.5 μm long and 1 μm wide. a microcapsule-like structure and a single polar flagellum that was grew at one or both ends of the thallus were observed on the surface of the cell (fig. 2b). in the api 20 ne test, negative reactions were observed for urea and p-nitro-β-d galactose, while all the other tested biochemical activities of m-08 were recorded positively (table 3). for the test of its susceptibility to antibiotics, m-08 was found to be insensitive to penicillin and amoxicillin, and sensitive to chloramphenicol et al. (table 4). based on the above results of sequence alignments, phylogenic trees construction and phenotypic characteristics identification, m-08 was identified to be v. neocaledonicus (table 5). haemolytic activity of m-08 clear zones were observed around the colonies of m-08 on the sheep blood agar after cultured at 8 °c, 16 °c, 24 °c, 32 °c and 36 °c for 24 h. different sizes of β-haemolytic circles were observed at different temperatures. the haemolytic circles gradually enlarged from 8 °c to 32 °c and reached biggest at 32 °c and decreased at 36 °c (fig. 3). the pathogenicity of m-08 for the 3×102 cfu ml-1 challenge group in the intravalvar injection experiment, the first dead scallop was observed on the 6th day after the challenge, and the mortality kept increasing until the 10th day. the mortality rate of the challenge group 96 fig. 2 morphological observation of m-08. (a) the colonies of m-08 on medium tcbs; (b) transmission electron microscope photograph of m-08 reached 33 % at the end of the experiment, which was 6 % in the control group. for the 3×104 cfu ml-1 challenge group in the intravalvar injection experiment, the first dead scallop was observed on the 5th day after the challenge, and the mortality kept increasing until the 11th day. the mortality rate of the challenge group reached 47 % at the end of the experiment, which was 6 % in the control group. in the 3×106 cfu ml-1 challenge group of the intravalvar injection experiment, the first dead scallop was observed on the 4th day after the challenge, and the mortality kept increasing until the 11th day. the mortality rate reached 93 % at the end of the experiment, which was 6 % in the control group (fig. 4a). in the infection experiment, the half lethal dose (ld50) of m-08 to yesso scallop was 3.54×104 cfu ml-1. for the 3×102 cfu ml-1 challenge group in the immersion experiment, the first dead scallop was observed on the 7th day after the challenge, and the mortality kept increasing until the 8th day. the mortality rate of the challenge group was 13 % at the end of the experiment, which was 6 % in the control group. for the 3×104 cfu ml-1 challenge group in the immersion experiment, the first dead scallop was observed on the 6th day after the challenge, and the mortality kept increasing until the 12th day. the mortality rate of the challenge group was 33 % at the end of the experiment, which was 6 % in the control group. the concentrations of 3×104 cfu ml-1 and 3×106 cfu ml-1 could cause the abscess symptom, while the precise bacterial concentration that induces abscess symptom needs further study. in the 3×106 cfu ml-1 challenge group of the immersion experiment, the first dead scallop was observed on the 5th day after the challenge, and the mortality kept increasing until the 10th day. the mortality rate was 53 % at the end of the experiment, which was 6 % in the control group (fig. 4b). for koch’s postulates, all the reisolated bacterial strains (18 strains) were confirmed to share both the same 16s rdna fragment sequences and the same phenotypic characters (green colonies on tcbs agar and anti-penicillin) with m-08. table 3 results of the api 20ne test of m-08 note: “+” for positive; “-” for negative the histopathological variation the histopathological features of adductor muscle, hepatopancreas, gill and mantle tissues of the control and challenged dying scallops were examined by paraffin section and hematoxylin-eosin. the adductor muscles of challenged scallops were test result potassium nitrate (no3) + tryptophan (trp) + glucose (glu) + arginine (adh) + urea (ure) horse leaf spirit (esc) + gelatinase (gel) + p-nitro-β-d galactose (pnpg) glucose (glu) + arabinose (ara) + mannose (mne) + mannitol (man) + n-acetylglucosamine (nag) + maltose (mal) + gluconate (gnt) + capric acid (cap) + adipic acid (adi) + malic acid (mlt) + citrate (cit) + phenylacetic acid (pac) + cytochrome oxidation c (ox) + 97 table 4 drug sensitivity of m-08 note: “s” for sensitive; “r” for resistant; “i” for intermediate sensitive scattered with increased muscle septum compared to that in the control individuals (fig. 5a). the cell membranes of the liver tubule epithelial cells of challenged scallops were ablated, with broken cell structure, condensed nucleus and increasing chromocyte (fig. 5b). the gill lamellaes of challenged scallops were bigger than that of control individuals, and the epithelial cells of the gill filament were necrotic and disintegrated, while the blood vessels were broken and disappeared (fig. 5c). the mantles of challenged scallops were swollen, with broken epithelial cells and condensed nucleus (fig. 5d). the activities of sod and cat and the contents of mda the activities of sod and cat in the challenge groups increased after m-08 challenge, and peaked at 3 h and 6 h, respectively, which were all significantly higher than that in the control group (p < 0.05) (fig. 6a and 6b). the mda contents also increased after challenge, which were significantly higher than that in the control group at 3 h after m-08 challenge (p < 0.05) (fig. 6c). the variation of the contents of lzm and the temporal expressions of pynf-κb and pytnfr-1 mrna in haemocytes the contents of lzm in the challenge groups increased gradually and peaked at 12 h, which were significantly higher than that in the control group (p < 0.05) (fig. 6d). the temporal expressions of pynf-κb and pytnfr-1 mrna in haemocytes were quantified by qrt-pcr with β-actin as the internal table 5 the sequence similarity of the alignments for all the strains id top-hit taxon top-hit strain similarity ( %) 1 vibrio neocaledonicus nc470 99.36 2 vibrio neocaledonicus nc470 99.43 3 vibrio neocaledonicus nc470 99.57 4 vibrio neocaledonicus nc470 99.57 5 vibrio neocaledonicus nc470 99.86 6 vibrio neocaledonicus nc470 99.58 7 vibrio neocaledonicus nc470 99.79 8 vibrio neocaledonicus nc470 99.93 9 vibrio chagasii r-3712 99.77 10 vibrio chagasii r-3712 99.04 11 vibrio chagasii r-3712 99.47 12 vibrio xuii lmg 21346 98.5 13 vibrio europaeus pp-638 99.31 14 vibrio campbellii caim519 99.44 15 pseudoalteromonas shioyasakiensis se3 99.09 16 pseudoalteromonas shioyasakiensis se3 98.82 17 pseudoalteromonas espejiana ncimb 2127 99.42 18 pseudoalteromonas espejiana ncimb 2127 99.05 19 pseudoalteromonas espejiana ncimb 2127 99.78 20 pseudoalteromonas carrageenov iam12662 99.43 antibiotics diameter/mm s i r result amoxicillin 14 ≥ 18 14~17 ≤ 13 i penicillin — ≥ 28 20~27 ≤ 19 r florfenicol 34 ≥ 19 15~18 ≤ 14 s chloramphenicol 23 ≥ 18 14~17 ≤ 12 s erythromycin 23 ≥ 23 14~22 ≤ 13 s neomycin 33 ≥ 18 14~17 ≤ 13 s streptomycin 30 ≥ 15 12~14 ≤ 11 s tetracycline 32 ≥ 19 12~14 ≤ 11 s doxycycline 29 ≥ 14 11~13 ≤ 10 s rifampin 23 ≥ 20 17~19 ≤ 16 s sulfamethoxazole 22 ≥ 17 13~16 ≤ 12 s sulfisoxazole 22 ≥ 17 13~16 ≤ 12 s enrofloxacin 34 ≥ 23 17~22 ≤ 16 s norfloxacin 32 ≥ 17 13~16 ≤ 12 s 98 fig. 3 haemolysis of m-08 at different temperatures (8 °c, 16 °c, 24 °c, 32 °c and 36 °c) control. the mrna expression level of pynf-κb in haemocytes decreased firstly at 3 h (p < 0.05), then increased and peaked at 12 h (p < 0.05), and returned to normal at 24-72 h (fig. 7a). the mrna expression level of pytnfr-1 in haemocytes exhibited an increasing trend compared to that in the control from 12 h to 72 h after exposure to m-08. significant up-regulation of pytnfr-1 expression occurred at 24 h and 72 h (p < 0.05) (fig. 7b). the apoptosis of haemocytes after challenged with m-08 the apoptosis of haemocytes was assessed by annexin v-fitc and pi staining followed by the analysis of flow cytometry (fig. 8a). the percentage of apoptotic haemocytes in the blank was 7.07 %. the percentage in the sterile water injection groups and m-08 challenge groups were 2.60 % and 8.23 % at 12 h, 5.03 % and 15.47 % at 24 h, respectively. significant differences were observed between the two groups at both 12 h and 24 h (p < 0.05) (fig. 8b). discussion abscess disease is a severe disease in cultured yesso scallop, which has caused huge economic losses in the aquaculture industry. however, the knowledge about the pathogenesis of abscess disease is still very limited. in the present study, v. neocaledonicus was isolated and identified as the potential pathogen in the abscess disease of yesso scallops by the physiological and pathogenical and immunological analysis. haemolytic activity is considered as one of the most crucial characters for pathogenicity evaluation of vibrio spp. (austin and zhang, 2006). in the present study, v. neocaledonicus was found to display β-haemolytic activity at different temperatures from 8 °c to 36 °c, with the highest activity at 32 °c. the temperature for the highest haemolytic activity of v. neocaledonicus is much higher than that for some other pathogens of scallops. for instance, v. splendidus exhibited haemolytic activity from 4 °c to 32 °c, with the highest activity at 10 °c, v. splendidus is a psychrotolerant bacterium (liu et al., 2013). the reason for the difference of haemolytic activity temperature might due to the differences of the characteristics between the two bacteria species. in addition, the haemolytic effect of m-08 on sheep blood, usually used for the risk assessment for mammals, indicated that m-08 was not only the potential pathogen of aquatic animals such as scallops but also a danger for the health of mammals (liu et al., 2013). in the api 20 ne test, the results were consistent with the reported phenotypic characteristics of v. neocaledonicus (table 3) (yu et al., 2019a). drug sensitivity test showed chloramphenicol might be the appropriate drug to use in the control of this disease. fig. 4 accumulative mortality of yesso scallops treated by intravalvar (a) and immersion (b) challenges with strain m-08 at 18 °c 99 fig. 5 histological sections of the normal and necrotic tissues of yesso scallops dyed with hematoxylin-eosin. the letters a, b, c, d stand for adductor muscle, hepatopancreas, gill and mantle tissue, respectively; the numbers 1 and 2 represent normal and necrotic samples, respectively. pc: pigment cell; cv: cell vacuolization; te: tubular epithelial cells; bv: blood vessel; ha: haemocyte; ct: connective tissue; ec: epithelial cell 100 fig. 6 the variation of the activities of (a) sod, (b) cat and (c) mda and (d) lzm content in the serum of yesso scallops after m-08 challenge. significant difference between blank sample and treated samples is indicated by an asterisk (*: p < 0.05; **: p < 0.01). vertical bars represent the mean ± s.d. (n = 3). sw indicates sterile water; v. n indicates v. neocaledonicus pathogenic bacteria challenge test is widely used to evaluate the virulence of pathogenic bacterial strains, especially in aquatic animal disease and immunology researches (ren et al., 2009). in the present study, both intravalvar injection and immersion experiments were used for the challenge to comprehensively investigate the pathogenicity of m-08. in the intravalvar injection experiment, the first dead scallop was observed on the 4th day (100 μl × 106 cfu ml-1 of m-08 suspension for each scallop), with the cumulative mortality rate as high as 93 % at the end of the challenge, indicating the strong pathogenicity of m-08. strong virulence of v. neocaledonicus was also reported in echiuroid worm with the first dead individual observed on the 2nd day (100 μl × 108 cfu ml-1 injection for each individual) with the cumulative mortality rate of 100 % (yu et al., 2019a). in the immersion infection experiment of the present study, the relatively high cumulative mortality rate of 53 % was also observed, which was lower than that in the intravalvar injection experiment. this is mainly because injection operation could cause more serious infection than immersion operation. in summary, the above results suggested that v. neocaledonicus was an important pathogen involved in yesso scallop abscess disease. the phenomenon shows that different strains of the same bacteria have different virulence. the antioxidant system consists of both enzymatic and non-enzymatic components, which are interdependent and work synergistically to prevent oxidative damage (chaudière and ferrari-iliou, 1999). sod, cat and other antioxidant enzymes are non-specific immune factors to resist the invasion of pathogens. the lipid peroxidation product mda resulted from oxidative stress is always used to indirectly reflect the damage degree of tissue peroxidation (géret et al., 2002). in the present study, the activities of sod and cat increased quickly at 3 h and 6 h after m-08 challenge, respectively. after the increase of antioxidant enzyme activities, the contents of mda peaked at 6 h, indicating that the oxidative damage reached the highest at 6 h, which was generally consistent with the previous reports. for example, sod activity and mda content in the serum of zhikong scallop (chlamys farreri) peaked at 3 h and 6 h after v. 101 fig. 7 the mrna expressions of (a) pynf-κb and (b) pytnfr-1 in haemocytes of yesso scallops after m-08 challenge. significant difference between blank sample and treated samples is indicated by an asterisk (*: p < 0.05). vertical bars represent the mean ± s.d. (n = 3). sw indicates sterile water; v. n indicates v. neocaledonicus anguillarum stimulation, respectively (wang et al., 2012), which were consistent with the results of sod activity and mda content after m-08 challenge. the sod activity increased significantly in mussel (mytilus crassitesta) at 24 h after the injection with v. splendidus (liang et al., 2018; liang et al., 2021), which was later than the result of sod activity after m-08 challenge. in crab charybdis japonica, cat activity in the serum increased significantly at 4 h and reached the highest at 24 h after infection with v. parahaemolyticus (wang et al., 2010), which was earlier than the result of sod activity after m-08 challenge. different vibrio strains activated host antioxidant enzyme activities within 24 h after stimulation. the results demonstrated that m-08 challenge could active the antioxidant system as an acute response in yesso scallops to keep homeostasis (wang et al., 2012). the innate immune system is important for most organisms to defend against the invasion of pathogens (li et al., 2015; xing et al., 2016). it has been reported that pathogenic infection can induce lzm activity and activate the expression of immune-related genes including nf-κb, tnfr and toll-like receptors in bivalves such as yesso scallop and pacific oyster (crassostrea gigas) (zhang et al., 2013; li et al., 2015; xing et al., 2016). in the present study, pynf-κb and pytnfr-1 were chosen as representatives to investigate the expression variation of immune-related genes, as they were proved to be involved in the antibacterial immune response to protect scallops against fig. 8 the apoptosis of haemocytes after m-08 challenge. (a) the apoptotic haemocytes detected by annexin v-fitc/pi staining after 12 h and 24 h of m-08 challenge; (b) the percentages of apoptotic haemocytes. significant difference between blank sample and treated samples is indicated by an asterisk (*: p < 0.05; **: p < 0.01). vertical bars represent the mean ± s.d. (n = 3). sw indicates sterile water; v. n indicates v. neocaledonicus 102 fig. 9 graphical abstract pathogenic invasion (li et al., 2015; xing et al., 2016). lzm is one of the most ubiquitously distributed antibacterial factors in invertebrate species (wei et al., 2018). in the present study, the lzm contents in challenge groups were found to peak at 12 h, and the mrna expression levels of pynf-κb and pytnfr-1 were generally up-regulated from 12 h to 72 h. however, the expression patterns of pynf-κb and pytnfr-1 were some different from other reports. for example, the mrna expression levels of pynf-κb and pytnfr-1 in the haemolymph of yesso scallop were significantly up-regulated at 3 h and 6 h after v. anguillarum challenge, respectively (li et al., 2015; xing et al., 2016). the differences are suspected to be caused by the use of different pathogenic bacterial species. the results indicated that the innate immune system of yesso scallops was activated after m-08 challenge to protect the organism from sustained oxidative damage. apoptosis is a process of programmed cell death and also a defense mechanism in immune reactions (liang et al., 2021). in the present study, obvious apoptosis of haemocytes was observed, indicating that m-08 challenge could cause apoptosis in yesso scallops. it has been reported that some pathogenic or opportunistic bacteria were able to induce or inhibit host cell apoptosis (lancellotti et al., 2009). for instance, v. harveyi haemolysin could induce apoptosis in the erythrocytes and the gill cell lines of flounder (paralichthys olivaceus), the fibroblast of black sea bream (mylio macrocephalus) and the fibroblast cell lines of silver sea bream (sparus sarba) (bai et al., 2010; deane et al., 2012). the extracellular metalloprotease and the haemolysin produced by v. vulnificus were reported to induce apoptosis of human cells (lee et al., 2008; sun et al., 2012; lee et al., 2014; lee et al., 2018). in the present study, the apoptosis of haemocytes was checked at 12 h and 24 h after m-08 challenge, when the immune system was activated. significant differences of apoptosis rate were observed, and the apoptosis rate at 24 h in the challenge group was extremely significantly higher than that in the control group, indicating that apoptosis occurred after m-08 challenge. furthermore, severe damage of the four tissues of yesso scallops was observed after the challenge, such as increased muscle septum, ablated cell membranes of the liver tubule epithelial cells and broken cell structure, which were consistent with the reported symptoms of abscess disease (liu et al., 2013). the sustained apoptosis was speculated to lead to tissue damage and scallop death after v. neocaledonicus infection (sawant et al., 2014; liu et al., 2018; nash and rahman, 2019). conclusion v. neocaledonicus m-08 was isolated and identified as a pathogen of abscess disease. the m-08 colony was round, with raised surface and a single flagellum. m-08 could ferment glucose, etc., and was insensitive to penicillin, etc. m-08 challenge 103 could cause severe tissue damage and massive mortalities of yesso scallops (fig. 9). these results indicated the pathogenicity of m-08, which provided helpful information for better understanding of the pathogenicity of v. neocaledonicus in abscess disease and the development of strategy to prevent and control the abscess disease in yesso scallop aquaculture. acknowledgments we are grateful to all the laboratory members for their technical advice and helpful discussions. this research was supported by national key r&d program (2018yfd0900501), grants (nos. u1706204, 41961124009) from national science foundation of china, china agriculture research system of mof and mara, the fund for outstanding talents and innovative team of agricultural scientific research from mara, liaoning climbing scholar, the distinguished professor of liaoning (xlyc1902012), key r&d program of liaoning province (2017203004, 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v vetvicka1*, p sima2 1department of pathology, university of louisville, louisville, usa 2institute of microbiology, czech academy of sciences, videnska, czech republic accepted november 8, 2004 abstract β−glucans, as biologically active polysaccharides, have been used for decades but only recently have become a focus of evolutionary studies. as β−glucans are shown to be active in all animal species studied from earthworms to humans we can safely conclude that β−glucan immunostimulation is one of the first defensive mechanisms active across the entire evolutionary spectrum. the recognition of β−glucans as major components of yeast, fungal, and bacterial cell walls belongs to the first defense mechanisms that evolved during phylogenetic processes. key words: glucan; invertebrates; immunity; ppo introduction polysaccharides and particularly glucans have a long history as immunomodulators. interest in glucans has increased after experiments showing that zymosan stimulates the macrophages via the activation of complement system. β-1,3 glucans are structurally complex homopolymers of glucose, usually isolated from yeast and fungi. the number of individual glucans is almost as great as the number of sources used for isolation. different physicochemical parameters, such as solubility, primary structure, molecular weight, branching, and polymer charge, influence the biological activities of β-1,3 glucans. it is not surprising that β-glucans have been extensively studied for their immunological and pharmacological effects. more than 600 papers describing the biological activities of glucans exist. up until now, strong immunostimulating effects of β-1,3 glucans have been demonstrated in all tested animal species including earthworms (beschin et al., 1998), shrimps (duvic and söderhäll, 1990), fish (anderson, 1992), mice, rats (feletti et al., 1992), rabbits, guinea pigs (ferencik et al., 1986), sheeps, *corresponding author: vaclav vetvicka university of louisville, department of pathology, 511 s. floyd, louisville, ky 40202, usa. e-mail: vetvickavaclav@netscape.net pigs (benkova et al., 1991), cattle (buddle et al., 1988) and humans. the immunomodulating effects of β-glucan are well established during the development of immune reactions. at the same time, it is easy to understand why glucan, forming part of the bacterial and fungal cell walls, is an important molecule to be recognized by the defense system. however, the importance for the invertebrates, often less concerned with the sterility of the inner milieu, is not as clear. in invertebrates, as in all metazoans, each phylum represents an appropriate fundamental morphological pattern according to the evolutionary history of the phylum and adaptation to the environmental conditions in which it has radiated. however, from the defense standpoint, three major principles, which may well be based on varying molecular and biochemical backgrounds, are common to all of them. the animals are capable of recognition, processing and elimination of non-self. invertebrates have evolved a wide variety of active defense mechanisms enabling them to use their highly effective innate defense pathways to protect themselves against invading pathogens despite the absence of an adaptive immune system based on lymphocytes or antibodies. at the same time, microorganisms possess distinctive molecular patterns, including β-glucans. pattern recognition proteins binding to β-glucan have been implicated in the activation of the innate defense reactions. 61 phenoloxidase invertebrates do not use molecules of the immunoglobulin superfamily as receptors for recognition of non-self structures. regardless of this, they exhibit considerable selectivity in their defense reactions (ratcliffe and rowley, 1979; vìtvièka and šíma, 1998). in general, arthropods, molluscs, and deuterostomian tunicates recognize non-self material, the microbial surface determinants that are conserved and ubiquitous among microorganisms but not present in the eukaryotic host. these structures mainly lipopolysaccharide (lps), peptidoglycan (pgn), mannan, and β-1,3 glucan are recognized by means of a group of germ-line encoded receptors, usually termed pattern recognition receptors (prr). using highly selective recognition processes, signaling cascades are thus activated. these cascades regulate production of defense substances, agglutinins and poisonings (generally lectins), and non-agglutinin factors, inducible or constitutive antibiotic peptides and the components of the prophenoloxidase (ppo) complex in the host (ratcliffe, 1991; hoffmann et al., 1999; andreu and rivas, 1998; hoffmann, 2004). the ppo of tyrosinase type, a cu-containing enzyme, is widely distributed both in prokaryotes and eukaryotic organisms. it was hypothesized that the cu-binding domains of the ppo evolved from similar regions of an ancestral hemocyanin molecule of arthropods (fujimoto et al., 1995; kawabata et al., 1995; van holde et al., 2001). specific non-self recognition mechanisms of the ppo system, as a basic part of immune defense of invertebrates, are involved during a row of hierarchized processes like cell cooperation and communication in the course of phagocytosis (smith and söderhäll, 1983; söderhäll et al., 1990), nodule and capsule formation (kobayashi et al., 1990), melanin synthesis (melanization of foreign bodies) and sclerotization (cuticle tanning and hardening) (lackie, 1988; marmaras et al., 1996; söderhäll and cerenius, 1998), hemocyte locomotion (takle and lackie, 1986) and coagulation of blood (durliat, 1991). participation of the ppo during melanization, a characteristic defense reaction of arthropods, was first documented in grasshopper eggs in 1941 (bodine and allen, 1941). the ppo catalyzes two key steps in the melanin synthesis (mason, 1955, 1956). both cellular and humoral immunity are commonly associated with melanin deposits (rattcliffe, 1991) which formation could be very rapid: in galleria melonella the entrapped microbes melanize during 5-60 min (rattcliffe and gagen, 1977). similarly, the ppo can control terminal differentiation of blood cells. in crayfish pacifastacus leniusculus the administration of β-1,3 glucan induces a rapid decrease of hemocytes followed by an accelerated increase of maturation of new cells expressing ppo transcript and their release into circulation (söderhäll et al., 2003). some activities of the ppo system resemble reactions of vertebrate lectin pathway of the complement system (c), e.g. cell lysis, opsonization, and substance release (söderhäll, 1982; cerenius and söderhäll, 2004; ma et al., 2004). both the lectin pathways of c and the ppo are proteolytic and comprise prr proteins, serine proteases activation and their inhibitors, generation of opsonins and ca2+ presence (ashida and dohke, 1980; aspán et al., 1990; söderhäll and cerenius, 1998; cerenius and söderhäll, 2004). the role of ca2+ during the β-1,3 glucan-dependent ppo activation has been repeatedly documented but it is still unclear (ashida et al., 1983; lee et al., 2004). in addition to the substances of microbial origin mainly pps, pgn, mannans, and β-1,3 glucans various physical and chemical stimuli such as temperature, ph, detergents, denaturing agents or proteases activate the ppo cascade (söderhäll and unestam, 1979; ashida and yoshida, 1988; dunphy, 1990; brivio et al., 1996). the ppo is discharged from cellular granules in an inactive form (pro-enzyme). after the conversion into its active form, the ppo cascade molecules are released from hemocytes into the hemolymph pool (preston and taylor, 1970; ashida et al., 1983; durlay and lackie, 1985). when the ppo is activated at the surfaces of microbial cell, it generates highly reactive and toxic quinone intermediates (johanson and söderhäll, 1989). the reaction consists of active catalyzation of the oxygenation of monophenols to odiphenols and their further oxidation to o-quinines (aspán et al., 1995; chase et al., 2000). β-1,3 glucans are first recognized by prr which activate serine proteases of ppo system (see below). the ppo system activating factors (similar to drosophila easter-type serine proteases) cleave ppo to phenoloxidase (po). by means of oxidation of phenols to melanin, the po produce toxic antimicrobial substances, which process is accompanied by sclerotization of cuticle (lee et al., 1998, 2004; satoh et al., 1999). the smallest structure able to activate ppo cascade is a laminaripentose as has been demonstrated in crayfish ppo system (söderhäll and unestam, 1979). it is remarkable that the invertebrate peptidoglycan and β-1,3 glucan prr triggering the ppo cascade are non-enzymatic homologues of bacteriophage t7 lysozyme and bacterial β-1,3 glucanase (ochiai and ashida, 1999; zhang et al., 2003). moreover, some prr recognizing pgn can also function to recognize β-1,3 glucan and induce the ppo cascade (lee et al., 2004). in a preliminary study, the β-1,3 glucans isolated from yeast cell walls exhibited a significant stimulation of the ppo system activity in hemocytes in vitro and in hemolymph in vivo of black tiger shrimp, penaeus monodon (suphantharika et al., 2003). the complex of ppo and il-1 like molecule found in manduca sexta (tobacco hornworm) could be regarded as an evolutionary novelty in invertebrate type defense (beck et al., 1996). glucan-binding protein invertebrates are using innate immune mechanisms conserved throughout the animal kingdom. a heterologous group of hemolymph proteins, among others, serves as a surveillance mechanism by binding to the surface of invading microbes. one member of this group is so called βglucan-binding protein (gbp). 62 proteins binding to the β-glucan have been identified in numerous arthropod species. their activity is usually to stimulate the ppo activation cascade. subsequent purification and identification revealed similar properties: proteins containing carboxyl-terminal glucanase-like domain without enzymatic activity. glucan is bound via less conserved amino-terminal domain. in m. sexta, two different gbps are 57% identical in amino acid sequence (kanost et al., 2004). both these gbp differ with respect to their presence and up regulation upon the immune challenge. in both cases, they are involved in potentiation of ppo activation and in agglutination of bacteria and yeast. gbp, sometimes also named as glucan-receptor, was also isolated from plasma of a silkworm, bombyx mori (yoshida et al., 1986). later studies showed that these molecules are 30 kda lipoproteins (ujita et al., 2002). for a detailed study of a gbp from b. mori, see (ochiai and ashida, 2000). glucan binding proteins are commonly found in crustaceans. they usually have a size app. 100 kda and besides binding glucan, bacteria and hemocytes, they have strong ability to act as opsonins (cerenius et al., 1994). even as numerous proteins with similar properties (such as mannan-binding, lps-binding or factor g) have been found (for review see söderhäll and cerenius, 1998), the gbps are probably the most important. there are suggestions that these proteins might develop from a primitive glucanase and later evolved into glucan-binding molecules without any enzymatic activity. in addition, crayfish gbp is identical to the shrimp protein lp1, which is involved in lipid transport to the ovary. both molecules react to the glucan binding by binding to the surface of the hemocytes, probably via an arg-gly-asp motif (holmblad et al., 1997). a different, high-density glucan-binding lipoprotein has been found in the white shrimp panaeus vannamei, having only significant similarity to the gbp from the crayfish (romofigueroa et al., 2004) and to ovarian vitellin (garciaoroyco et al., 2002). söderhäll’s group isolated and characterized a gbp from the crayfish pacifastatus leniusculus and found that this 40 kda protein has a strong similarity to bacterial glucanases and to the gbps from eisenia foetida. this protein bound both linear and branched glucans as well (lee et al., 2000). a detailed study evaluated cdna cloning, purification, properties and functions of a gbp from a moth, plodia interpunctella. the report showed that the protein contains an open reading frame that encodes 488 amino acids of which the first 17 residues comprised the secretion signal peptide. carboxyl-terminal domain was similar to other invertebrate gbps as well as β-glucanases from bacteria and sea urchin. in addition, this gbp was constitutively expressed in all life stages and no microbial challenged changed its expression (fabrick et al., 2003). subsequent study showed that the amino-terminal domain consists primarily of an α-helix secondary structure with only minor β-structure. functional data revealed that this gbp bound only to the 1,6-branched glucans (as it bound to laminarin, but not to curdlan). hence, this gbp has two binding domains separated by a putative linker region, one for glucan and the second for the stimulation of the ppo cascade (fabrick et al., 2004). a surprising observation has been made by bilej’s group. these authors found that the cytolytic factor 1, present in the coelomic fluid of e. foetida earthworms, has significant homology with the catalytic region of β-1,3-glucanase and strongly binds β-1,3-glucan. in addition, this molecule also participates in activation of the ppo cascade (beschin et al., 1998). other effects of ββ -glucan immunostimulating effects of glucans have also a significant commercial potential. β-glucans have successfully been used to increase the resistance of shrimp panaeus japonicus against vibriosis (itami et al., 1994), further studies using p. monodon showed protection against vibriosis, white spot syndrome virus and vibrio damsela (su et al., 1995; song et al., 1997) and also enhancement of survival and immunity during brood-stock rearing (chang et al., 2000). all these effects were caused by direct impact on haemocytes via stimulation of phagocytosis, cell adhesion and superoxide anion (chang et al., 2000) and superoxide dismutase production (chang et al., 2003). surprisingly, the glucan-induced resistance was maternally transmitted (huang and song, 1999). in addition, glucan injection reduced expression of peroxinectin (sritunyalucksana et al., 2001). surprising data was obtained in the freshwater crayfish, p. lenieusculus. the injection of glucan caused a short-term severe loss of hemocytes, followed by a rapid recovery due to the accelerated release of cells from the hematopoietic organs (söderhäll et al., 2003). when compared to other effects of glucan in invertebrate, this function on stem cells is the only one, which is completely comparable to vertebrates including humans (patchen and macvittie, 1983). another potentiation of defense reaction by glucan was documented in earthworms. a study of e. foetida showed that earthworms responded to the glucan challenge by increase in coelomic cytolytic factor and lysozyme-like activity and that these effects were caused by direct binding of glucan to hemocytes (kohlerova et al., 2004). in mosquito anopheles gambiae, injection of glucan leads to the induction of hemolymph proteins. when compared with the effects of lps or escherichia coli, the study showed that each stimulus induced different proteins, suggesting that mosquitoes have the ability to discriminate between elicitors (han et al., 1999). previous experiments using a drosophila cell lines showed strong in vitro induction of cecropin genes by addition of algal glucan (samakovlis et al., 1992) suggesting the role of hemocytes in glucan-induced effects. the means of how invertebrates recognize glucan are still not completely understood. a novel approach was used during studies of an insect apolipoprotein. it has been shown that insect apolipoprotein, homologous to mammalian apoe, strongly binds β-glucan and probably acts as a pattern recognition molecule (whitten et al., 2004). 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42072-42079, 2003. isj659 21 isj 20: 21-37, 2023 issn 1824-307x report of meeting xxiii scientific meeting of the italian association of developmental and comparative immunology (iadci), february 13-15, 2023, dadom darwin dohrn museum, villa comunale, naples, italy organizers: mr coscia1, d melillo1, a ametrano1, r marino1, d malagoli2,3, mg parisi4 1institute of biochemistry and cell biology, national research council of italy, naples, italy 2department of life sciences, university of modena and reggio emilia, modena, italy 3nbfc, national biodiversity future center, palermo, italy 4marine immunobiology laboratory, department of earth and marine sciences ed. 16, university of palermo, palermo, italy this is an open access article published under the cc by license award “soci non strutturati” (best presentation and curriculum studiorum for members under 35) a bombyx mori infection model for testing antimicrobial compounds against staphylococcus epidermidis infection a montali1, f berini1,2, a saviane3, s cappellozza3, f marinelli1,2, g tettamanti1,2 1department of biotechnology and life sciences, university of insubria, varese, italy 2interuniversity center for studies on bioinspired, agro-environmental technology (bat center), university of naples federico ii, portici, italy 3research centre for agriculture and environment (crea-aa), council for agricultural research and economics, padua, italy there is an urgent need to develop new antimicrobial molecules due to the increasing spread of antibiotic resistant bacteria. mammalian models used for preclinical tests during drug discovery and development raised serious ethical problems in the last decades. as a consequence, after the european parliament directive 2010/63/eu on animal’s welfare, many restrictions have been posed on the use of mammals for research purposes. in addition, trials are time consuming and very expensive. to overcome these issues, the traditional approach of drug screening must to be reconsidered. in particular, researchers are focusing attention on alternative invertebrate models. in this context, insects are increasingly considered a promising tool, able to accelerate preclinical tests of new drugs. furthermore, the use of insect infection models can decrease the costs of the experimentation and the number of animals used, according to the 3r principle. in last years, we focused our efforts on developing a silkworm infection model to test antibiomicrobial compounds against staphylococcus aureus. to this purpose, we assayed cheap and easy to perform markers for evaluating three glycopeptide antibiotics (gpas, i.e., vancomycin, teicoplanin and dalbavancin) (montali et al., 2020). with these analyses, we were able to assess the different efficacy of the three tested antibiotics in curing bacterial infections. to validate this silkworm infection model, in the present work we tested, for the first time, the three gpas against another important gram positive, nosocomial pathogen, i.e., staphylococcus epidermidis. after the injection of bacteria in the hemocoel, larvae were reared at 37 °c to reproduce the human physiological conditions, and different immunological markers were used to monitor the infection. in addition to the survival of the larvae, cellular and humoral immune responses were assessed through the analysis of the viability of hemocytes, and the activity of phenoloxidase system and lysozyme. all the three antibiotics proved to be effective, curing infected larvae, increasing their survival rate. moreover, the administration of gpas blocked the activity of the immune response. in conclusion, this study lays the foundations for using bombyx mori as a trustable infection model to test novel antimicrobial compounds with therapeutic potential against staphylococci. 22 award “giovani laureati” (best presentation and curriculum studiorum for members under 29) how can climate change affect antarctic fish physiology? a study on the effects of increasing seawater temperatures on fish heart rates e piva1, s schumann1, s pacchini1, e brasola2, v stoilova3, p irato1, d pellegrino4, g santovito1 1department of biology, university of padua, padua, italy 2department of mathematics, university of padua, padua, italy 3department of environmental and life sciences, university of karlstad, karlstad, sweden 4department of biology, ecology and earth sciences, university of calabria, rende (cosenza), italy it is well known that the oceans and marine organisms are suffering the consequences of climate change. increasing seawater temperatures are causing a severe threat to the survival of living organisms. global climate change is expected to cause an increase in water temperatures, especially in polar regions, but it is unclear how this will affect marine fish species in general. the southern ocean, which surrounds antarctica, is a central component of the global absorption of ocean heat. the surface waters within and north of the antarctic circumpolar current and the abyssal waters are experiencing a gradual temperature increase. temperature can influence many intracellular dynamics, such as the movement of molecules, the activity of enzymes and the function of ion channels. fish may respond to these changes in various ways, including behavioural strategies like avoidance tactics, molecular modifications like changes in protein or lipid biosynthesis, and physiological responses that impact the entire organism, like changes in cardiovascular function. this study aims to understand the physiological changes that might occur in an antarctic fish species and whether these peculiar stenothermal fish can adapt to these changes. it is known from previous studies that various circumstances, including oxygen availability, swimming, activity, feeding, stress and temperature, influence the heart rate of fish. to analyse the effect of temperature, we exposed adult specimens of trematomus bernacchii, a fish species endemic to antarctica, to different temperature conditions. the control group of fish was exposed to 0°c, while another group experienced a rising temperature range from +1 °c to +3 °c. the exposure time for each experimental group was 15 days. to measure the heart rate, dst micro-hrt loggers, developed by star-oddi (iceland), were implanted on four t. bernacchii specimens for each experimental group before the experiment started. the dst micro-hrt uses innovative technology, simultaneously measuring the target animal's long-term heart rate and body temperature. the logger is ideal for a wide range of investigations, such as studying behaviour and stress response in laboratory animal research, wildlife research, animal welfare, and fish physiology. understanding the physiological reactions to such changes may help determine whether these creatures can withstand future global changes and point out any restrictions (including energy investment and stress) on how they can respond to further warming in the southern ocean. we analysed the data collected after the experiment to obtain some preliminary findings that offer interesting information regarding how variations in water temperatures may affect heartbeat frequency. two comparisons have been performed: one between the tank that was maintained at 0 °c and the tank that experienced temperature increases, and the other between the various temperature increases that the fish experienced. the heartbeat rates of the fish in the control group did not change statistically during the trial. still, our analysis revealed a statistically significant increase in heart rates between +1 °c and +3 °c in the tank where the temperature was manually increased. furthermore, a subsequent correlation analysis showed a strong positive correlation between temperature and heart rate, confirming that the heart rate also increases when the temperature increases. this presumably represents a response to increased atp and oxygen demand at the tissue level in relation to increased metabolic activity. the present study, therefore, demonstrates that the application of tags provides a powerful analysis tool to measure the heart rate in fish. it also provides a unique insight into the physiological responses of t. bernacchii exposed to increasing temperatures. for example, when t. bernacchii specimens are exposed to an acute temperature increase from 0 °c to +3 °c, they tend to show tachycardia. this, in principle, may result in unforeseen energy investment for these organisms. keynote lecture novel insights on fish immunoglobulins and b cell subsets c tafalla animal health research center (cisa), national institute for agricultural and food research and technology (inia), spanish research council (csic), madrid, spain although teleost fish constitute the first animal group in which all the elements of acquired immunity are found, the many structural and functional differences of these elements with those of mammals strongly condition how b cells and immunoglobulin (ig) production are regulated in these species. one of the main differences when compared to higher vertebrates is the fact that fish only express three antibody isotypes, namely igm, igd, and the teleost-specific igt. interestingly, igt+ b cells only express this ig and constitute a b cell linage that apparently is regulated independently of other b cell subsets and seems to play a major role in mucosal immunity. nevertheless, systemic igt responses have also been broadly reported and there are still many aspects of igt regulation that remain unknown. on the other hand, b cells co23 expressing igm and igd on the cell surface (igm+igd+ b cells) constitute the major b cell subset in systemic compartments and seem to correspond to naïve mature b cells. these fish b cells down-regulate igd after encountering antigen as mammalian b cells do, once they start a differentiation program towards plasmablasts/ plasma cells and become igm+igd− b cells. finally, igd+igm− b cells have also been detected in some species such as rainbow trout (oncorhynchus mykiss) or catfish (ictalurus punctatus), being this subpopulation numerous in some specific mucosal tissues such as intestine or gills. the precise role of this b cell subset is still lacking in teleosts as in mammals, yet some important discoveries have been made in the past years. in this talk, we will go through the most recent discoveries regarding the functionality of all these fish b cell subsets. session 1. characterization of immune genes. chairpersons: umberto rosani, university of padua, padua, italy and adriana vallesi, university of camerino, camerino (mc), italy how does gene presence/absence variation shape the repertoire of defense molecules in marine mussels? the case of crp-i, cysteine rich peptides belonging to the knottin superfamily n gualandi1, d fracarossi2, d riommi2, m sollitto2, s greco2, m mardirossian2, s pacor2, t hori3, a pallavicini2, m gerdol2 1international school for advanced studies, trieste, italy 2department of life sciences, university of trieste, trieste, italy 3atlantic aqua farms ltd, vernon bridge, pe, canada we have recently demonstrated that marine mussels (mytilus spp.) have an unusual pangenomic architecture, characterized by widespread hemizygosity and gene presence-absence variation (pav). this phenomenon does not randomly affect all genes, but disproportionately targets multigene families involved in immune response and survival, including those encoding antimicrobial peptides and other small effectors. as a result, each individual mussel is endowed with a unique repertoire of defense molecules. however, the lack of a reference chromosomescale genome assembly has so far prevented to gather a complete understanding of the architecture of these genomic loci, preventing an accurate ascertainment of the orthology/paralogy relationships among variants. the analysis of a fully-phased genome assembly of mytilus edulis allowed to fill this knowledge gap, with the identification of over 50 paralogous genes and pseudogenes belonging to the crp-i family, located in a small genomic region within chromosome 5, characterized by extreme structural variation. we provide evidence that crp-i genes are subjected to massive gene pav within the mytilus species complex, display a complex evolutionary history and a peculiar pattern of expression, and belong to the knottin structural superfamily, according to alphafold prediction. although most knottins either act as toxins, antimicrobial peptides or protease inhibitors, the functional role of crp-i remains elusive. functional assays, carried out on the synthetic peptide scrp-i h1, do not support neither a role as an antimicrobial agent, nor as a protease inhibitor, suggesting on the other hand that it may be toxic towards invading eukaryotic parasites. comparative gene expression analysis reveals adaptation mechanisms in the antarctic scallop adamussium colbecki s greco1, as gaetano2, g voltarel1, pg giulianini1, a pallavicini1, m gerdol1 1department of life science, university of trieste, trieste, italy 2department of chemical and pharmaceutical sciences, university of trieste, trieste, italy antarctica is the most extreme continent of earth, with strong winds, freezing temperatures on land, and ocean temperatures constantly below 0 °c. nonetheles the antarctic ocean is home to an astounding diversity of living organisms, that adapted to the multiple challenges posed by such an environment thanks to a diverse set of evolutive traits. thanks to the recent advancements in sequencing technologies, it was possible to discover many of the molecular bases of such adaptations in antarctic fish, while little is known about invertebrates and in particular for bivalves. in this preliminary study, we tried to address this knowledge gap using transcriptomic data to obtain insights into some of the adaptations that made adamussium colbecki the only antarctic pectinid species existing in our era. we used a transcriptome assembly and rnaseq data we generated for a. colbecki and retrieved the reference genomes and rnaseq data from the pectinids pecten maximus, mizuhopecten yessoensis, argopecten irradians and azumapecten farreri. in particular, samples from gill, mantle, and digestive gland tissues were selected. with the reciprocal best hit method, we built a dataset of 461 ortholog genes that were used as reference for rnaseq read mapping. the gene expression data was then aggregated and used to perform differential gene expression analysis, identifying the genes with an increased expression in a. colbecki compared to the other species. to functionally characterize the obtained gene set, we performed gene ontology, superfamily and pathway enrichment analysis and string interaction analysis. our results revealed that, specially in gills, a. colbecki displays higher expression of several genes primary involved in transcription, alternative splicing and protein degradation via ubiquitination. additionally, genes encoding for membrane-bound proteins are also included in this set. although with some limitations, our analyses are indicative of adaptive traits that compensate for a reduced efficiency of transcription and mrna 24 splicing at low temperature, resulting in an increased need for the degradation of degenerated or misfolded protein products. such adaptations poorly overlap those of antarctic fish, and represent a novel insight in the survival strategies of a. colbecki, that will be expanded with the everincreasing availability of high quality data. ficd genes in invertebrates: a tale of transposons, viruses and proviruses u rosani1, s de felice1, r frizzo1, s kawato2, m wegner3 1department of biology, university of padua, padua, italy 2laboratory of genome science, tokyo university of marine science and technology, tokyo, japan 3alfred wegener institute helmholtz centre for polar and marine research, list auf sylt, germany many genes are shared across the tree of distantly related species because of horizontal gene transfers (hgts). however, the frequency of hgts varies strongly between gene families and biological realms suggesting differential selection pressures and functional bias. one gene family with a wide distribution are fic-domain containing enzymes (ficds). ficds catalyze ampylation, a posttranslational protein modification consisting in the addition of adenosine monophosphate to accessible residues of target proteins. in humans, ampylation plays a role in neuro-development and neurodegeneration and similar ficd activities have been reported for drosophila and caenorhabditis elegans ficds. moreover, it has been shown that bacterialpromoted ampylation can induce cytotoxicity in the infected cells by targeting host proteins of the rho gtpase family. beside the known conservation of ficds in deuterostomes, we report the presence of a conserved ficd gene ortholog in a large number of protostomes and basal eukaryotes, with structural and functional traits suggesting a preserved ampylation capacity. we also discovered additional ficd gene copies in the genomes of some rotifers, parasitic worms, bivalves and isopods. a few dsdna viruses of these invertebrates, including white spot syndrome virus, cherax quadricarinatus iridovirus, ostreid herpesvirus-1 and the beetle nudivirus, carry copies of possibly functional ficds. phylogenetic analysis suggested a common origin of the ficd copies of these viruses and the duplicated ficds of their invertebrate hosts, and the strong conservation between wssv and oshv-1 ficds indicated an evolutionary advantage in maintaining this gene. hgts and gene duplications possibly mediated by endogenous viruses or genetic mobile elements seem to have contributed to the transfer of ampylation ability from bacteria and eukaryotes to pathogenic viruses, where this pathway could have been hijacked to promote viral infection. investigating the role of a-to-i rna editing in bivalves physiology e bortoletto1, bm kaczmarek2, c bernecky2, u rosani1, p venier1 1department of biology, university of padua, padua, italy 2institute of science and technology austria, klosterneuburg, austria enzymes of the adenosine deaminase acting on dsrna (adar) family can perform posttranscriptional modifications on structured rnas, by converting adenosine in inosine (a-to-i editing), eventually diversifying transcriptomes and proteomes. adar-mediated editing is involved in the neural development, autoimmune disorders and it has been reported to exert proor antiviral effects depending on virus-host combination. however, except for a few species, the extension and functional roles of rna editing have been poorly investigated across the tree of life. our research aims to reveal the presence of adar-mediated editing in bivalves, developing appropriate pipelines to detect genuine editing sites, characterizing the involved enzymes and revealing editing patterns conserved among species possibly underpinning functional significance. accordingly, we phylogenetically characterized the gene complements involved in rna editing and we recombinantly produced the crassostrea gigas adar1 to verify its editing potential, efficiency and specificity on known human targets. in parallel, we exploited paired dnaand rna-sequencing data to develop an editing index based on bivalve repeats, recalling the alu index used for humans. we tested this novel bivalve editing index in samples collected during an experimental infection with ostreid herpesvirus-1 carried out in scapharca (anadara) broughtonii. finally, we characterized the rna editing in mytilus galloprovincialis, s. broughtonii and c. gigas and we compared their targets and distribution to highlight the conserved and unique editing sites among these organisms. as results, we demonstrated that c. gigas radar is able to edit dsrna structures even if it shows some difference in the rna editing targets comparing to the human adar proteins. the editing index based on bivalves repeats is able to rapidly estimate the rna editing levels in bivalve samples, indeed in the dataset obtained from a time course experiment performed with s.broughtonii injected with oshv-1 and monitored up to 72 hours both rna editing frequency in several editing sites and the newly defined editing index significantly increased in parallel along the time course. in conclusion, we validated the presence of a ato-i editing also in bivalves confirming again the conservation of this mechanism in metazoans and the pivotal role of the adar mediated rna editing in organism physiology. 25 molecular evolution of euplotes pheromones and pheromone-coding genes a vallesi, c alimenti, y jiang, p luporini school of biosciences and veterinary medicine, university of camerino, camerino (mc), italy in the ciliate euplotes, species-specific families of water-borne protein pheromones regulate self/not-self recognition phenomena which are responsible for the cell decision to switch between vegetative (mitotic) growth and sexual mating. the knowledge of the pheromone and pheromone-gene structures has recently been widened to a number of species that localize in different positions of the euplotes phylogenetic tree and thrive in different environments, making it possible to seek into how the structures of these molecules evolve in relation to speciation. the development of one or more (usually gly-rich) random-coil segments is a major trait of the evolution of the pheromone structure, which is basically determined by a common tightly conserved, disulfide-rich helical fold. by determining sites of local flexibility of the molecular backbone, these segments come to serve the double function of greatly improving the pheromone adaptive plasticity and capability to interact with other proteins. in parallel, the pheromone-gene structural evolution primarily involves the inclusion of multiple intron sequences within the 5’-leader region or, more rarely, within the coding region. by determining a mechanism of alternative splicing, these sequences make each pheromone gene (which is expressed in the somatic genome of the cell macronucleus) capable of synthesizing multiple mrnas in addition to the pheromone-specific transcript. lost igt gene in icefish: another chapter of the evolutionary tale of antarctic fish a ametrano1, m gerdol2, s picchietti3, v pianese3, u oreste1, mr coscia1 1institute of biochemistry and cell biology, national research council of italy, naples, italy 2department of life sciences, university of trieste, trieste, italy 3department for innovation in biological, agro-food and forest systems (dibaf), university of tuscia, viterbo, italy notothenioidei is a monophyletic lineage that accounts for the majority of teleost fish fauna living in the freezing sea of the southern ocean. they are an amazing example of adaptive radiation and an interesting model for the study of cold adaptation. during their evolutionary history, antarctic fish have undergone significant genome alterations, as also highlighted in a previous work on the gene encoding the igt heavy chain, lacking most of the second constant exon (ch2). a reconstruction across notothenioid phylogeny revealed that the partial loss of ch2 was shared by representative species from four antarctic families along with the nearest nonantarctic sister species eleginops maclovinus. the present work is aimed at investigating channichthyidae, since remaining the only family apparently lacking the entire igt gene. the survey of available genomes and trascriptomes for this family revealed a heterogeneous situation, with some species (most notably chionodraco, cryodraco and chaenodraco spp.) having entirely lost the igt gene and others displaying a remnant pseudogene carrying only tm exons and a part of the upstream intron. since the igt gene was present and complete in all the other closely related taxa (bathydraconinae, cignodraconinae and gymnodraconinae), the timing of the gene loss can be inferred to be coincident with the loss of red blood cells and hemoglobin occurred in the family. to evaluate how the two other evolutionary conserved isotypes can functionally compensate for the loss of igt, tissue-specific expression of c. hamatus igm and igd was performed by qpcr, including trematomus bernacchii for comparison. igm was found to be the predominant isotype expressed in icefish mucosal tissues. in particular, abundant igm expressing cells were found in the lamina propria of the posterior intestine, as indicated by in situ hybridization analysis. conversely, igd transcripts were found to be predominantly expressed in the lymphoid organs. of note, the expression levels of igm in the intestine and of igd in head kidney of c. hamatus were respectively 6 and 20-fold higher than in t. bernacchii tissues. as expected, igt was the highest expressed isotype detected in mucosal tissues of t. bernacchii. overall, the preliminary results presented here pave the way for completing the reconstruction of the evolutionary history of the igt gene in antarctic fish, culminated with its loss in icefish. this research was supported by the national programme for antarctic research (pnra), project number pnra18_00077 main lecture reconstructing the evolutionary history of the antibody molecule u oreste, mr coscia institute of biochemistry and cell biology, national research council of italy, naples, italy antibodies, otherwise called immunoglobulins (ig), are among the best-known molecules. a notable issue is the origin and evolution of antibody. here, an integrate view of the emergence of antibody in evolution, based on a literature survey across a wide range of prokaryotic and eukaryotic organisms, is provided. the ig molecular architecture relies on the very ancient ig domain. its chemical features permit the formation of multiple domain chains, self dimerization or association with different partners. the amino acid sequence of the ig domain determines a compact structural core and allows a great conformational variability of flexible loops carrying out recognition functions. this key structural role accounts for its occurrence in numerous recognition molecules. the ig domains that are found in different taxa, are distinguished in four types, igv, igc 1, igc2 and igi, based on different numbers and arrangements of the occurring b-strands. 26 ig-like domains, defined as big domain, have been detected on prokaryotic cell surface, and a role in host cell adhesion has been hypothesized. whether the similarity between big and eukaryotic ig is related to genetic evolution or to the physicochemical properties of amino acid motives driving the domain assembly is still under debate. igv appears to be the oldest ig domain since it has been found in an adhesion molecule of a living fossil sponge. igv domain has been widely used by the recognition molecules of earlier vertebrates. however, only in the jawed vertebrates the v domains have gained an important structural role in forming the antigen-binding site. the sequence variation arose from the encounter between igv domain encoding genes and the evolutionary lines leading to the enzymes involved in the mechanisms of somatic recombination and hypermutation. while igc2 and igi domains are present in both invertebrates and vertebrates, igc1 domain is limited to jawed vertebrates and has been found only in the molecules of adaptive immunity. this observation supports the idea that key actors of the adaptive immune response, all using the novel igc1domain type, emerged at the same time during the so-called immunological "big bang". various antibody classes, each containing a different heavy chain isotype, differentiated during vertebrate evolution and acquired distinct functions in mucosal and systemic immunity. in mammals, in addition to heavy chain isotypes, subisotypes are distinguished on the basis of their functions. the evolution of immunoglobulins can thus be considered as a paradigmatic example of how diversity and specificity of molecular interactions between proteins can increase. session 2. from comparative immunological studies to biotechnological applications. chairpersons: nicolò baranzini, university of insubria, varese, italy and maria rosaria coscia, national research council of italy, naples, italy investigation of the igm heavy chain gene from antarctic fish inspired a novel engineered monoclonal antibody a ametrano1, b miranda2, l de stefano2, u oreste1, mr coscia1 1institute of biochemistry and cell biology, national research council of italy, naples, italy 2institute of applied sciences and intelligent systems, national research council of italy, naples, italy immunoglobulin m (igm) is the major circulating ig isotype in teleost fish. unique features in crucial parts of the igm molecule have been uncovered in antarctic fish species that have experienced a special evolutionary history. the most striking structural characteristic is an extraordinary long hinge region, located between the second and third heavy chain constant domains. it can be viewed as a result of adaptive evolution to enhance the functionality of the molecule under very extreme environmental conditions. this finding prompted the idea to modify the heavy chain constant region (igh) of a murine monoclonal antibody (mab) by replacing its hinge with that from antarctic fish igm by using the crispr-cas9 system. this technology has been recently proposed as an rna-guided dna targeting platform and widely used as a powerful tool for precise gene editing. given its simplicity and flexibility, the crispr-cas9 system has been successfully used also in the field of immunology to edit mouse and human ig genes. a stepwise approach was chosen for targeted genome editing of a hybridoma cell line secreting igg mab. the first step was the creation of a targeted dna doublestranded break at the hybridoma igh gene locus to be modified. homology-directed repair was then used to insert the “antarctic” hinge sequence through recombination of a dna donor template with the target locus. the correct sequence insertion was assessed by using a fluorescent protein as selection marker. a preliminary characterization of the antigen binding activity of the engineered mab was performed by the localized surface plasmon resonance. the association constant k of the engineered mab was found to be three-fold higher than that of the murine counterpart, suggesting an enhanced ability of the “antartized” mab to recognize its target antigen, when immobilized on a rigid substrate. overall, these results may open a new frontier in the field of antibody engineering by using an innovative and versatile crispr-based method. medicinal leeches: a promising source of cell lines with multiple biotechnological applications g marcolli1, l pulze1,2, l monti1, n baranzini1,2, f acquati1,2, a grimaldi1,2 1department of biotechnology and life sciences, university of insubria, varese, italy 2ilfarm s.r.l., varese, italy stem cells represent one of the most dynamic research fields in biology and biomedicine. however, the vast majority of research is carried out in mammalian models, which represent only 0.4% of extant metazoans. aquatic invertebrates show the greatest biodiversity and the widest phylogenetic radiation on earth, but cell lineages are currently available form only few of them (in particular molluscs and crustaceans, while there are none from annelids). in the last decades, we have identified and characterized different cell types in the medicinal leeches hirudo verbana and hirudo medicinalis, such as hemopoietic precursor cells, monocytes/macrophages, granulocytes, natural killers, fibroblasts, telocytes, myofibroblast and myoendothelial cells, all sharing the same morphofunctional and molecular features with vertebrates. in addition, by means of an innovative system developed in our laboratory, based on the injection into the leech body wall of the matrigel biopolymer (mg) supplemented with selected cytokines, we were able to isolate, expand and differentiate in vitro 27 hemopoietic precursor cells into muscle and myofibroblasts cells. these studies enabled us to clarify some key processes concerning the role of cytokine signaling on progenitor cells differentiation, muscle regeneration and wound healing. starting from these promising results, we propose the medicinal leech, whose experimental use is not subjected to legislative restrictions, as an emerging experimental model for the production of invertebrate cell lines endowed with innovative biotechnological potential and in support of vertebrate cell lines-based research. to this aim, using the consolidated mg technique and combining it with the cytokines pdgf (platelet-derived growth factor) and egf (epidermal growth factor), we have isolated different leech cell populations and cultured them in vitro in a medium containing fibroblast growth factor 2 (fgf2), transforming growth factor-𝛽 (tgf-𝛽) or hvrnaset2 enzyme. the cell responses have been then evaluated by both morphological and immunocytochemical assays. concurrently, an expression vector (pegfpn1) containing the leech’s actin 1 minimal promoter was created in order to perform, for the first time, transfection experiments aimed at producing immortalized leech cell lines which, in addition to the relative scientific interest due to their sheer diversity, will provide the potential for multiple applications, such as assays for ecotoxicological analyses. session 3. model organisms for basic and translational immunology. chairpersons: nicola franchi, university of modena and reggio emilia, modena, italy and daniela melillo, national research council of italy, naples, italy the multiple potentialities of anthozoans: analyses and comparisons between animal models c la corte, l bisanti, f bertini, m dara, d parrinello, m cammarata, mg parisi marine immunobiology laboratory, department of earth and marine science ed. 16, university of palermo, palermo, italy anthozoans are the richest class of species of the phylum cnidaria. they are a candidate group for studying the evolution of mutualisms and immunity and despite their morphological simplicity exhibit a repertoire of immunological components with large genomes and gene families similar to those of the bilateria. like other invertebrates, anthozoans immunity is based on self/non-self recognition mechanisms and allorecognition responses, therefore, maintaining their integrity and responding actively to selection pressures. highlight and investigate the link between innate immunity, homeostasis maintenance, inflammation, tissue remodelling and regeneration in anthozoa could be useful to elucidate the adaptive capability features to different stress factors. we have carried out studies demonstrating that all these processes are highly conserved among the anthozoans species. we have compared the inflammatory responses and the morpho-functional aspects related to regeneration in different species of mediterranean anthozoans using histological, cellular and molecular technical approaches on organisms, maintained in aquaria under environmental and pathogenic stressful conditions. this approach appears to be a useful tool from baseline studies in immunology and anthozoans result valid models able to respond to environmental stress conditions. important results have been obtained with potential biotechnological transferability in pharmacology. the protochordate ciona robusta as an experimental system for studies of gut microbial immune interactions a liberti1, lj dishaw2, 3 1stazione zoologica anton dohrn, biology and evolution of marine organisms (beom), naples, italy 2university of south florida, morsani college of medicine, department of pediatrics, tampa, fl, usa 3children’s research institute, division of molecular genetics, st. petersburg, fl, usa the recognition that animals exist as metaorganisms suggests that attention should be focused on defining host-microbe interactions in not just pathogenic conditions, but during health as well. for example, the gut microbiota serves vital roles in various aspects of animal life, including development of the immune system and influence of host physiology. the gut immune system, and specifically the innate immune components, is at the forefront of the crosstalk between host and microbes, where colonization by commensal microbes is tolerated while pathogens are resisted. this dialogue is evolving and is shaped by encounters with a continuum of microbial species. establishing diverse experimental systems is essential for understanding the fundamental rules legislating these ecological interactions. ciona robusta, a marine invertebrate belonging to the subphylum of protochordate, a sister taxon to vertebrates, represents an ideal experimental system for such studies: it is a highly tractable model that engages with a complex environment using only innate immunity and develops into transparent juveniles with a digestive tract that is easy to stage, dissect and study the steps shaping microbial colonization dynamics. our group is establishing this model for defining critical host-microbiota interactions, combining approaches of microbiology, molecular biology, biochemistry, and functional assays. we have identified and characterized some components of the ciona gut environment, and these include the presence of a gut epithelium layered with chitin-rich mucus, secreted immune effectors, namely the immunoglobulin-like variable region-containing chitin-binding proteins (vcbps) and a stable gut microbiome in adults that includes abundant and diverse bacteriophages. a large catalog of cultured bacteria and fungi, from which cultured juveniles 28 can be colonized for experimental manipulation and study, have been isolated and characterized. we have observed that vcbps are able to interact with distinct elements of the microbiome, mediating transkingdom interactions and shaping biofilm formation and colonization dynamics among microbes. finally, we have developed approaches to rear aseptic juveniles to facilitate future studies interrogating each component of the symbiosis, including the role(s) of microbes in animal development. thus, ciona is a valuable experimental organism that may help to define conserved and unique innate immune adaptations shaping the gut ecosystem and its homeostasis. comparing immune responses of mytilus galloprovincialis to different bacterial isolates from oyster mortality outbreaks m auguste1, m leonessi1, c oliveri1, l vezzulli1, d furones2, f frontalini3, l canesi1, c ciacci4 1distav, department of environmental, earth and life sciences, university of genoa, italy 2irta-sant carles de la ràpita, sant carles de la ràpita, spain 3dispea, department of pure and applied sciences, university of urbino “carlo bo”, italy 4disb, department of biomolecular science, university of urbino “carlo bo”, italy marine bacteria of the vibrio genus are widely distributed in estuaries and coastal waters and sediments. they include species pathogen for humans (e.g., vibrio cholerae) and for aquatic animals (e.g., vibrio splendidus, vibrio aestuarianus). several vibrio species have been repeatedly associated with oyster mortality outbreaks in europe. host-pathogen interactions have been investigated in cultured and wild populations of bivalves susceptible to infection by certain vibrio spp. and strains, showing that different vibrios can elicit distinct immune responses in bivalve hemocytes. v. aestuarianus (v. a.) is a common bivalve pathogen detected in samples from crassostrea gigas mortality episodes since 2001, suggesting a strong link between their presence in oyster tissues and development of disease. most studies on the interactions with the host immune system were carried out with the v. a. 01/032 isolate. this strain can secrete extracellular products that inhibit phagocytosis by oyster hemocytes and enable bacterial proliferation. in contrast, the mussel mytilus galloprovincialis can activate efficient immune responses to challenge with v. a. 01/032, by a combination of cellular and soluble defences. during a mortality event in 2019 in spain, different strains of malaciobacter marinus were isolated from moribund oyster together with v. a. strains. we have recently shown that m. galloprovincialis hemocytes efficiently responds to challenge with m. marinus strains. in this work, immune responses of mussel hemocytes to the v. a. strain 106 isolated from the same mortality event were investigated. the results of in vitro experiments, carried out in the absence and presence of hemolymph serum hs, show that this strain induced strong lysosomal destabilization without stimulating extracellular defences (ros production, lysozyme release). however, vibrio a. 106 was apparently internalized in hemocytes: accordingly, significant bactericidal activity was observed, that was stimulated by hs. the results obtained so far suggest that v. a. 106 may be more pathogenic to m. galloprovincialis with respect to v. a. 01/032. moreover, in the presence of m. marinus, v. a. 106 may significantly affect the health status of mussels over longer period. further in vivo experiments are in progress to investigate the responses of m. galloprovincialis to co-infection with these two bacteria. bacterial oral infections: exploring the role of the circadian clock in the modulation of pathogen sensitivity in drosophila melanogaster m battistolli, f sandrelli department of biology, university of padua, padua in almost all organisms, circadian clocks regulate physiological and behavioral rhythms, showing a periodicity of 24 h in the absence of any external cue. moreover, they are synchronized by environmental stimuli (such as light), enabling organisms to adapt to environmental changes in phase with the 24 h day. in drosophila melanogaster, sensitivity to systemic infection and some immune response components show daily variations in 12 h light: 12 h dark cycles (12:12 ld) and in constant darkness (dd), suggesting these phenotypes are circadian controlled. these data refer to flies exposed to a pathogen via an injection route, while a possible role of the circadian clock during oral infections is unclear. here we show our first experiments analyzing the role of the circadian clock in the modulation of flies’ pathogen sensitivity during oral infections. using a capillary feeder assay, we determined the 24 h feeding profile in wildtype and clock mutant flies, with and without gut microbiota, in ld and dd conditions. we identified the proper moments of the day to orally infect flies with the same amount of pathogen during daytime and nighttime, in different conditions. the first results about the daily sensitivity to oral infection with providencia rettgeri will be shown. main lecture biotechnology meets aquaculture needs: a promising support for fish health management c buonocore, v cassella, a coppola, d coppola, g della sala, f palma esposito, c melchiorre, c ragozzino, p tedesco, ga vitale, l vitale, d de pascale marine biotechnology department, stazione zoologica anton dohrn, naples, italy seas and oceans are the largest resource of the planet and are of fundamental importance for developing sustainably a strategic asset for a country 29 with 8700 km of coasts and possessing ca 15% of the surface of the mediterranean sea. however, their potential is still largely unexploited. in order to expand the marine biotechnology field, the stazione zoologica anton dohrn has recently opened a new premise: the department of marine biotechnology. the philosophy beyond this department is to have a direct access to the marine organisms and to valorise their potential in many fields of industrial sectors, therefore the location of the department is directly on the sea, at molosiglio in naples. the marine life offers a plethora of novel opportunities, either in terms of seafood, nutraceutical, and bioactive molecules, which are of extraordinary importance in the field of pharmacology, biomaterials and bioremediation. the development of new technologies and biotechnologies can unlock this underexploited potential with positive impacts in several industrial sectors and with positive impact on national economy. the department of ecosustainable marine biotechnology represents an international strategic implementation of the biotechnological research and is planned to enhance the research potential in specific industrial sectors like pharmaceuticals, nutraceuticals, and aquaculture. the mission of the marine biotechnology department is to conduct and promote scientific research regarding the possible applications of marine natural products in the biomedical and environmental sectors. we also focus on the isolation and characterization of marine microorganisms, including bacteria and fungi, which can resist and degrade pollutants such as heavy metals and polycyclic aromatic hydrocarbons. bioremediation and bioaugmentation studies are aimed at validating new technologies for the recovery of marine polluted coastal areas, such as in the aquaculture sector. new and innovative approaches are being developed for the restoration of such sites using seagrasses and corals. to foster the blue economy in our country, we need a tight cooperation between research and industry capable of identifying smart solutions. in fact, from one side, we need to develop tools for the sustainable use of marine resources, and, from the other side, we need create new occupational opportunities. session 4. new promising sources of bioactive compounds. chairpersons: marco gerdol, university of trieste, trieste, italy and simona picchietti, university of tuscia, viterbo, italy nk-lysin homologue from the antarctic teleost trematomus bernacchii and its ala mutant: focus on antibacterial and anticancer activities s picchietti1, l guerra1, ar taddei2, pr saraceni1, m gerdol3, f porcelli1, f bugli4, r rosato4, a miccoli1, am fausto1, g scapigliati1, f buonocore1 1department for innovation in biological, agro-food and forest systems (dibaf), university of tuscia, viterbo, italy 2center of large equipments, section of electron microscopy, university of tuscia, viterbo, italy 3department of life sciences, university of trieste, trieste, italy 4department of basic biotechnological sciences, intensive and perioperative clinics, catholic university of the sacred heart, rome, italy antimicrobial peptides (amps) are small molecules naturally produced by all living organisms, with a great diversity in amino acid composition, structural organization and mechanism of action. given their broad-spectrum activity against multiple classes of pathogens in addition to their immunomodulatory and anticancer activities, amps have been proposed as promising candidates for pharmacological applications. several families of amps have been identified from teleost fish to date. among them, nk-lysin (nkl), predominantly characterized in mammals, garnered scientists’ attention due to antimicrobial, immunomodulatory and anticancer activities. in this study, a nkl sequence belonging to the saposin-like protein superfamily was identified in silico from the head kidney transcriptome of the antarctic teleost trematomus bernacchii. the sequence is rich in positively charged amino acids and contains six cysteine residues that form three disulphide bridges. tissue-specificity qpcr analysis indicated predominant nkl expression in t. bernacchii gills and spleen, followed by head kidney, gut, liver and brain. a putative biologically active 27-residue long peptide, containing 2 cysteines, was identified by multiple sequence alignment of the t. bernacchii nkl amino acid sequence with teleost homologues. a nkl mutant (nkl-mut) was designed by replacing cys-10 and cys-20 of the wild type peptide with ala residues, with the aim of verifying the functional importance of the disulfide bridge. the nkl wild type (nkl-wt) and nkl-mut peptides were synthesized and their bioactivity was evaluated in vitro: they significantly increased membrane permeability in bacterial models and inhibited the growth of clinical isolates with known resistance profiles. notably, nkl-wt and nkl-mut exhibited bactericidal activity against the human pathogenic bacteria enterococcus faecalis and acinetobacter baumannii, respectively. of note, nkl-mut possessed improved selective cytotoxicity and pro-apoptotic activity compared to nkl-wt towards the melanoma cell line b16f10, without affecting the viability of fb789, a primary human fibroblast cell line. neither peptide showed any significant haemolytic activity against mammalian erythrocytes. these results provide interesting perspectives on the possible application of such peptides as antimicrobial and/or anticancer agents. this research was supported by the national programme for antarctic research (pnra), project number pnra18_00077. botryllin, a novel antimicrobial peptide from the colonial ascidian botryllus schlosseri n franchi1, l ballarin2, f cima2 1department of life science, university of modena and reggio emilia, modena, italy 2department of biology, university of padua, padua, italy 30 by mining the transcriptome of the colonial ascidian botryllus schlosseri, we identified a transcript for a novel styelin-like antimicrobial peptide, we named botryllin. the gene is constitutively transcribed by circulating cytotoxic morula cells (mcs). the synthetic peptide, obtained from in silico translation of the transcript, shows robust killing activity to bacterial and unicellular yeast cells causing breakages of both the plasma membrane and the cell wall. specific monoclonal antibodies were raised against the epitopes of the putative amino acid sequence: they label the mc granular content. upon mc degranulation induced by the presence of nonself, the antibodies recognise the extracellular nets with entrapped bacteria nearby mc remains. the obtained results suggest that the botryllin gene carries the information for the synthesis of an amp involved in the protection of b. schlosseri from invading foreign cells. red cells of the black sea urchin arbacia lixula: a promising source of anti-inflammatory compounds s quarta1, e scoditti2, v zonno1, l siculella1, f damiano1, p pagliara1 1department of biological and environmental sciences and technology, university of salento, lecce, italy 2institute of clinical physiology (ifc), national research council of italy (cnr), lecce, italy many echinoderms’ species are regarded as an important source of promising bioactive compounds. among them, sea urchins are increasingly investigated for their potential pharmacological benefits, producing compounds with several biological properties, such as antibacterial, anticoagulant, antifungal, anti-inflammatory, antitumor, and antiviral activities. the globose body of sea urchins possess a cavity filled with a circulatory medium (coelomic fluid) containing a heterogeneous population of cells (coelomocytes) involved in immune defense. among coelomocytes, also red spherule cells play a role in immune response being able to accumulate around injuries and sites of infection. red cells contain echinochrome a, a naphthoquinone which gives the cells their characteristic red color. echinochrome a is released from red cells in the presence of bacteria or virus and has antimicrobial properties against both gram-positive and gram-negative bacteria. an antioxidant property of echinochrome a from sea urchins a. crassispina and s. mirabilis has been also reported. in this work, we investigated the antiinflammatory properties of the red cells methanolic extracts from the black sea urchin arbacia lixula. human microvascular endothelial cells (hmec1) stimulated with the pro-inflammatory cytokine tnf-α were used to assess the anti-inflammatory effect of the methanolic extracts. the total phenol content of the samples was estimated by folinciocalteau assay, and trolox equivalent antioxidant capacity (teac) assay has been performed to evaluate the antioxidant activity of the extracts. treatment of hmec-1 with 100 µg/ml of red cell methanolic extract significantly reduced the tnf-αinduced expression of inflammatory genes, including adhesion molecules (vcam-1), chemokines and cytokines (mcp-1, il-6). moreover, the activation of the transcription factor nuclear factor k-b (nf-kb), responsible for the induction of inflammatory genes, was significantly inhibited by red cell extracts. these genomic effects translated into a reduced adhesion of monocytes to inflamed endothelial cells treated with methanolic extracts. these data contribute to characterize the biological activities of the red cells from the sea urchin a. lixula and point to beneficial role of its red cells extracts against inflammatory response in vascular endothelial cells, with potential implications for the modulation of inflammatory diseases. antimicrobial and antibiofilm activity of a synthetic amp derived from a bombyx mori cecropin b natural variant i varponi1, s ferro2, l menilli1, a grapputo1, f moret1, o marin2, f sandrelli1 1department of biology, university of padua, padua, italy 2department of biomedical science, university of padua, padua, italy a natural variant of the antimicrobial peptide cecropin b derived from the silkworm bombyx mori showed an effective antimicrobial activity against the opportunistic pathogen pseudomonas aeruginosa. this isoform, named q53 cecb, also displayed a high stability to ph and temperature variations, as well as low toxicity against human cells. importantly, q53 cecb maintained an antipseudomonas activity at a high saline concentration, a typical condition of the respiratory tract of cystic fibrosis patients, which are often affected by multi-drug resistant p. aeruginosa infections. however, q53 cecb was sensitive to enzymatic degradation. protease degradation represents one of the main limitations in the use of amps as antimicrobial drugs. to overcome this drawback, we produced a synthetic q53 cecb-derived variant, carrying damino acids in its sequence. we are currently characterizing this peptide, evaluating its activity and mechanism of action against both planktonic and biofilm forms of p. aeruginosa, sensitivity to enzymatic degradation, and toxicity against human cells. halla parthenopeia and its defense systems: first steps towards and annelid model in ecoimmunology and blue biotechnologies a ferri1, s sacchi2, r iseppi3, c sabia2, r simonini2*, d malagoli2,4* 1department of chemical and geological sciences, university of modena and reggio emilia, modena, italy 2department of life sciences, university of modena and reggio emilia, modena, italy 3department of biomedical, metabolic and neural sciences, university of modena and reggio emilia, modena, italy 31 4nbfc, national biodiversity future center, palermo, italy *co-senior authors halla parthenopeia is a marine annelid which colonize soft bottom of the mediterranean sea. due to its endemicity, ability to regenerate and to colonize habitats richly populated by pathogens it has a great potential as a model of study in different fields, including eco-immunology and blue biotechnologies. the latter aim to the identification of new compounds from marine organisms useful for the human health. during feeding, locomotion and predator defense, h. parthenopeia produces different types of mucus which are rich of bioactive compounds, with at present unknown biological roles and applications. in order to observe its behaviour, to study its biology and to obtain a significant amount of mucus for bioactive compound purification, the worm husbandry was optimized in ad hoc designed aquarium systems. the easily collectable defense mucus, secreted by h. parthenopeia, contains a toxic anthraquinone known as hallachrome. this compound showed a minimal inhibitor concentration (mic) of 0.12 mm and 0.06 mm for gram positive bacteria and for candida albicans, respectively. given the effect of hallachrome on human pathogens, it could be used by animals as a defense against pathogens present in the sediment, or to control the microbial environment into the tunnels. antimicrobial peptides (amps) represent molecules that could integrate the action of hallachrome. however, as for many non-model animals, studies on h. parthenopeia suffer from the lack of omics data and optimized experimental protocols. the whole-body rna extraction protocol was optimized to obtain good quality rna for transcriptomic studies. the de novo transcriptome, in association with tissue-specific proteomics, will serve as database for h. parthenopeia blue biotechnology-oriented studies. while developing transcriptomic data, the purified mrna was used for rt-pcr analysis aimed at identifying immune-related soluble factors constitutively expressed by the worm. first positive reactions included the amp bactericidal/permeability-increasing protein (bpi)like transcript and the immune mediator lipopolysaccharide binding protein (lbp)-like transcript, immune-related factors already observed in other annelids. in conclusion, this study presents the first evidence on the humoral defense of h. parthenopeia with the aim to offer a new research organism to the fields of eco-immunology and blue biotechnologies. actinins as novel broad-spectrum amp isolated from the tentacle of anthozoan actinia equina (linnaeus, 1758) m cammarata, m dara, c la corte, l bisanti, v catania, d parrinello, mg parisi marine immunobiology laboratory, department of earth and marine science ed 16, university of palermo, palermo, italy capturing activities and defense mechanisms of cnidarian are strongly associated with toxins and peptide with antimicrobial properties. amp are an important component of many organisms’ innate immune system with a good inhibitory or killing effect against invaders pathogens. we investigated the amp activity of acid extracts obtained from tentacle and body of actinia equina (cnidaria, anthozoa) against gram positive (micrococcus lysodeikticus) and gram negative (escherichia coli, vibrio alginolyticus) bacteria. the peptide fractions showed interesting minimum inhibitory concentrations (mic) values (concentrations up to 0.125 µg/ml) against tested pathogens. tentacle acid extracts exhibiting a good antimicrobial activity, were further investigated, characterized and the peptides purified by reverse phase chromatography on solid phase sep-pak c8 column followed by several hplc runs on c18 column. a broad-spectrum antibacterial peptides activity was detected in 40 % acetonitrile fractions. the peptide 6.2 has a molecular weight of 2612.91 da and is composed of 27 amino acids (actinin a); while peptide 7.3 has a molecular weight of 4323.07 da and is composed of 35 amino acids (actinin b). the two peptides were completely sequenced and their aa sequence revealed similarity with the already described amps identified in amphibians and fish, with anti-gram+ & gram-, antifungal, candidacidal, antimethicillin-resistant staphylococcus aureus (mrsa) activity actinins a and b were chemically synthesized and tested in vitro against the above-mentioned bacterial pathogens. the analysis identified the peptide actinin b which showed an interesting antibacterial and can be considered good candidates for new therapeutic applications. session 5. embryogenesis, development and regeneration. chairpersons: annalisa grimaldi university of insubria, varese, italy and jacopo vizioli, university of lille, lille, france hemocytes, motile cells and immune-related mediators in adult sensory organ regeneration: evidence from the evolutionary distant models pomacea canaliculata (mollusca, gastropoda) and nematostella vectensis (cnidaria, anthozoa) g bergamini1, s sacchi2, s basu3, m ahmad1, a ferri1, m cocchi1, a ikmi3, d malagoli2,4 1department of chemical and geological sciences, university of modena and reggio emilia, modena, italy 2department of life sciences, university of modena and reggio emilia, modena, italy 3developmental biology unit, european molecular biology laboratory (embl), heidelberg, germany 4national biodiversity future center (nbfc), palermo, italy 32 the apple snail pomacea canaliculata and the sea anemone nematostella vectensis possess different body organization, but they share outstanding regeneration capability in adult life. p. canaliculata and n. vectensis tentacles are analogue sensory appendages, with a different anatomical organization. our aim was to search for common cellular and molecular immune-related events during the early regeneration of these two evolutionary distant models. confocal laser-scanning microscopy demonstrated that during p. canaliculata tentacle regeneration, the hemocytes accumulated at the wound site and infiltrated the forming blastema. by combining flow cytometry and microscopy, it was shown that the transitory reduction of circulating hemocyte number, secondary to the injection of the phagocyte-targeting drug, clophosome®, corresponded to a delay in the blastema formation. after the number of circulating hemocytes was autonomously restored by the snails, the regeneration process took place regularly, with numerous hemocytes accumulating in the forming blastema. as these results suggested a proregenerative role for the phagocytic hemocytes of p. canaliculata, their potential involvement was also investigated by qpcr experiments on immune-related genes (irgs), e.g., pchemocyanin, pc-aif-1, pc-tgase and pc-runt, and on genes associated with regeneration and cellproliferation, e.g., pc-wnt1, pc-jagged-1, pcfgf18 and pc-pcna. our data demonstrated a significant increase in the expression of irgs during hemocyte accumulation in the blastema, and a modified gene expression profile for regeneration markers following clophosome® treatment, indicating a plausible role of hemocytes in modulating regeneration-associated gene expression. high-resolution live-imaging method applied to control and regenerating specimen of n. vectensis, allowed the identification of a highly motile population of cells (mpc), that was found to accumulate at wound site within 6 h after oral tentacle amputation. after facs sorting, rna smart-sequencing was performed on the mpc, demonstrating that they express irgs such as aif1, c2, c5, dscam, elf-1, jagged-1, lta4h, prxl2b, and the macrophage marker mpeg. this further supports the hypothesis that the mpc could represent an original immunocompetent cell population in cnidarians, with a cell behaviour similar to that of snail hemocytes, during tentacle regeneration. in all, by combining advanced microscopy and molecular strategies, it has been demonstrated that phylogenetically distant animals such as snails and sea anemones, present similar cell behaviour profile during early regeneration phases, with the accumulation irgs-expressing motile cells, supporting the view of an ancient and proactive role of the immune system in the onset of sensory organ regeneration. role of the extracellular matrix during the postembryonic development of the medicinal leech hirudo verbana l pulze1,2, n baranzini1,2, f acquati1,2, a grimaldi1,2 1department of biotechnology and life sciences, university of insubria, varese, italy 2ilfarm s.r.l., varese, italy the extracellular matrix (ecm) is a 3d noncellular network, composed of collagens, proteoglycans/glycosaminoglycans and various glycoproteins, which regulate different cellular functions. interestingly, several evidence in literature have demonstrated that cell-ecm physical interactions, and thus the mechanical forces, are the most important factors involved in the maintenance of stem cell phenotype, differentiation, and cell behavior. the remodeling of the ecm and the physical interactions between cell and ecm are two important aspects that could also be involved in muscle development; however, still today, little information is available on this topic. to obtain new insights on how a fine tuning of the 3d ecm microenvironment can drive both cellular and tissue fates and their spatial organization during post embryonic development, we used the invertebrate hirudo verbana as a valuable inexpensive and easy manipulable experimental model characterized by a simple anatomical organization reproducing many aspects of the basic biological processes of vertebrates and by post-embryonic stages of development easily to stage, to manipulate and whose morphology is well described. overall, our data show that during leech developmental stages, ecm remodelling leads to a stiffness gradient that regulates both the migratory cell pathway and the cell fate of the developing body wall. these cells respond to changes in the 3d ecm network by activating the hippo pathway epigenetic mechanism, of which one of the main players is yap (yes-associated protein 1), evolutionarily conserved in the medicinal leech as well. stress granules-related genes regulation during embryogenesis of an invertebrate chordate l drago1, a. pennati2, u. rothbächer2, g. santovito1, l. ballarin1 1department of biology, university of padua, padua, italy 2department of zoology, university of innsbruck, innsbruck, austria stress granules (sgs) allow eucaryotic cells to regulate stress responses and face adverse environmental conditions. their formation occurs in the cytoplasm with the over-expression of mrna binding proteins, the aggregation of which blocks the translation of mrnas for anti-stress proteins and prolong their stability. 33 the protection from stress represents a critical issue during embryogenesis and is important for survival of the organisms and the perpetuation of the species. the aim of this research was to investigate the transcriptional regulation of the genes for three important protein components of sgs, during early stages of development of the solitary ascidian ciona robusta, the role of which in stress defense was already demonstrated in adult: tia1 cytotoxic granule associated rna binding protein like (tiar), tristetraprolin (ttp) and gap sh3 domain-binding protein (g3bp). electroporation experiments on embryos were carried out with constructs for reporter gene (lacz) expression, containing the promoter region for tiar, ttp or g3bp. the gene reporter assays allowed us to study level, time and cellular specificity of the production of tiar, ttp and g3bp, which reflects the action of the regulatory sequences, especially occurring in notochord and mesenchyme cells of larval stages under normal physiologic conditions and in response to metalinduced stress. morphological and functional characterization of hemocytes in hermetia illucens larvae d bruno1, a montali1, m casartelli2,3, g tettamanti1,3 1department of biotechnology and life sciences, university of insubria, varese, italy 2department of biosciences, university of milan, milan, italy 3interuniversity center for studies on bioinspired agro-environmental technology (bat center), university of naples “federico ii”, portici (naples), italy understanding the physiology of saprophagous larvae that are used for waste management is fundamental to monitor the health status of the insect during the bioconversion process. in particular, information on the immune system could provide new insights on the ability of these insects to survive in rearing substrates that are characterized by high bacterial loads and potential pathogenic microorganisms. in the present study, we analyzed the cellular immune response of hermetia illucens, known as black soldier fly (bsf), which represents the most promising ecological decomposer of organic waste substrates. in particular, although some information on the humoral and cellular response of these larvae to bacterial infections is present in the literature, little is known about their circulating cells. in fact, a characterization of the hemocyte populations of this dipteron has never been performed, nor the processes carried out by these cells (i.e., phagocytosis, nodulation and encapsulation) have been investigated so far. our results demonstrate that, in addition to prohemocytes, five hemocyte types mount the cellular immune response in these larvae (i.e., plasmatocytes, lamellocyte-like cells, granulocytes, crystal cells, and adipohemocytes). moreover, we shed light on their behavior, role, and morphofunctional changes in response to bacterial infection and injection of chromatographic beads. noteworthy, differently from other insects, plasmatocytes represent the only circulating phagocytes in the hemolymph of these larvae. in combination with granulocytes and lamellocyte-like cells, they also take part in nodulation and encapsulation, by establishing a starting core for nodule/capsule formation around non-self agents. these processes are supported by the action of crystal cells, which release melanin precursors, and adipohemocytes, which could trophically support the cellular mechanisms through nutrient reserve mobilization. this work was supported by fondazione cariplo (grant number 2020-0900). response of mytilus galloprovincialis ovaries to different environmental pressures t chianese, m prisco, r scudiero, l rosati department of biology, university of naples “federico ii”, naples, italy the mediterranean mussel (mytilus galloprovincialis) is a marine invasive species cultured all over the world. mussels are an appreciated resource in local aquaculture enterprises, become increasingly economically and ecologically relevant. being sessile and filtering animals, they have been used for decades in programs to monitor chemical contamination in the marine environment. although no cases of massive mortalities have been reported, the effects of different environmental pressures and water quality can alter the morphology and functionality of the organs, including the reproductive capacity of these organism. the objective of this study was to investigate bioaccumulation abilities and biomarker responses in m. galloprovincialis ovaries. analysis was carried on specimens from two different coastal areas of the gulf of naples, one graded as a-area and another as b-area. such areas were differently classified according to escherichia coli levels and other chemical parameters established from regulation (ec) no. 854/2004 of the european parliament; mussels from a-area can be sold without purification, whereas mussels from b-area must be purified before the sale. we determined the accumulation in the ovaries of the bisphenol a (bpa), a chemical compound used to synthetize polycarbonate plastic and, mimicking the estrogen activity, able to modifies gonadal development and reproduction. then, we investigated variables as condition and gonadsomatic indices (ci and gsi), the morphology of female gonad, the presence of apoptosis, the expression of estrogen receptors (ers) genes, both in the non-reproductive (july) and reproductive (october) periods. significant differences in bpa content and in the expression of ers genes, particularly for er2, were observed between the individuals collected in the different areas during the two periods. statistically significant differences were observed also in ci and gsi, as well as in degeneration events affecting the structural organization of the ovary, leading to a considerable 34 increase of apoptotic cells in gonads of specimens from the b-area, no matter the period. the collected data demonstrate that mussels are excellent sentinel organisms also for the assessment of endocrine disruptors, and point out the importance of water quality parameters on health status of mussel specimens. understanding the vulnerability of mussel beds to specific contaminants could inform and improve the management of mussel farms. session 6.1. environmental challenges and the immune response. chairpersons: piero g. giulianini, university of trieste, trieste, italy and gianfranco santovito, university of padua, padua, italy bio-plastic recognition by mussels hemocytes m dara1, n torregrossa2, c la corte1, mg parisi1, d piazzese2, m cammarata1 1marine immunobiology laboratory, department of earth and marine sciences ed. 16, university of palermo, palermo, italy 2department of earth and marine sciences, university of palermo, palermo, italy the growing use of bio-polymers derivatives poses an increasingly pressing problem regarding their environmental sustainability. in particular, it should be still ascertained the claimed absence of direct and indirect influence on ecosystems and the health of living organisms, including humans. our goal was about assessing the potential effects of poly-lactates and polyhydroxyalkanoates, the most widely used bio polymers classes with promising different applications for replacing conventional plastics on natural aquatic environments. we chose m. galloprovincialis as sentinel species since their extensive filter-feeding activity. when it is exposed to microparticles can bioaccumulate them in soft tissues and organs. in the immunobiological investigation, to highlight if bio-polymers can influence the marine ecosystems, in vitro exposure assays on bivalve mussel have been carried out, and their impacts have been explored, by evaluating the cellular response of hemocytes referred to their phagocytic and/or encapsulation activity. preliminary evidences have shown that bioplastic particles behave in a very similar way to fossil plastic triggering the immuno-system and activating the elimination of non-self particles via cellular response. as future perspectives, although it is widely recognized that in vitro testing is an effective method for defining the effects of emerging pollutants, the in vitro test will be further deepened with in vivo experiments. the medicinal leech as a valuable model to evaluate the effect of polypropylene micro and nanoplastics on innate immune response activation c bon1, n baranzini1,2, l pulze1,2, l izzo1, a grimaldi1,2 1department of biotechnology and life sciences, university of insubria, varese, italy 2ilfarm s.r.l., varese, italy among the different types of plastic, polypropylene (pp) is one of the most widespread, widely used in the food and textile industries for disposable packaging and to produce surgical masks. due to the enormous distribution and the consequent abundant presence of pp waste products, it is necessary to investigate the possible toxicity on living organisms. in particular, successful regeneration requires precise coordination of multiple processes, such as clearance of cellular debris, progenitor cell activation and proliferation, immunomodulation, angiogenesis, and granulation tissue formation. the presence of pp micro (mps) and nanoplastics (nps) could hinder regenerations as their ingestion depletes energy reserves, reduces nutrition, survival and immune response. here we demonstrate that the medicinal leech hirudo verbana, considered as a substitute method not subject to legislative restrictions (legislative decree 26 /2014), is a useful and promising model to elucidate the effects of pp on the inflammatory response. fluorescent pp, in which a probe was introduced into the carbon chains of the polymer, has been used to better follow the plastic fate in tissues and cells of leeches exposed to water dispersed pp mps and nps and to evaluate their potential effect on the innate immune response stimulation as compared to not exposed leeches. data here presented demonstrate that pp debris entering leech tissues cause morphological changes in body wall organization and increase both inflammatory and fibrotic responses, altering proper extracellular matrix and collagen deposition. cytotoxicity of ether perfluoro carboxylic acid pfas congeners in earthworm granulocytes d rotondo, a calisi, d gualandris, f dondero department of scienze e innovazione tecnologica, università del piemonte orientale, alessandria, italy soil pollution has enormously increased in the last decades. from different type of pollutants perand polyfluoroalkyl substances (pfas) are found in the soil due to their various industrial uses (e.g., anti-fire foams, fluoropolymer resins such as teflon; separation processes; textiles; cosmetics) and their high persistence. most emerging pfas congeners do not have toxicity data that would allow an environmental assessment. biological approaches to soil monitoring, such as the measurement of biochemical and cellular responses to pollutants (i.e., biomarkers) on organisms living in the soil, have become of major importance for the assessment of the quality of soil. aim of this work was to investigate the effect of these substances from an eco-toxicological point of view on non-target species, using pfas as a reference standard. for this purpose, our study focused on assessing the potential cytotoxicity and genotoxicity of four different pfas congeners (pfoa, hfpo-da, pf4moba, pf3mopra) on immune system cells 35 (coelomocytes) of sexually mature earthworm eisenia foetida in a range concentration of 0.6-229 microm. toxicity tests were carried out according to oecd test no 207 (by filter paper test) to assess the cytotoxic and genotoxic effect in earthworms coemolocytes. coelomocytes were collected from the coelomatic fluid using an insulin syringe prefilled with 0.25 ml saline solution. coelomocytes morphometric alterations were determined by diff quick staining; oxidative stress alterations and micronuclei frequency were determined respectively by h2dcfda staining for ros and dapi coupled with fluorescence microscopy. results showed significant alteration of the investigated patterns. an increased enlargement of granulocytes was usually observed in exposed earthworms with respect to control group. a hormetic pattern was observed. the enlargement was quantified by measuring the area of 2d digitalized granulocyte images. a decrease of oxidative burst and an increase of micronuclei frequency were also seen. further investigation of impaired cell-mediated immune function is ongoing. the results of this study will lead to the construction of an ecotoxicological database on numerous alternative pfas congeners allowing a weight of evidence risk assessment analysis of these substances. these data can contribute to regulation and restriction of pfas at national and european level. antioxidant enzyme gene expression in trematomus newnesi from ross sea (antarctica) experimentally exposed to pfoa s pacchini1, e piva1, s schumann1, p irato1, d pellegrino2, g santovito1 1department of biology, university of padua, padua, italy 2department of biology, ecology and earth sciences, university of calabria, rende (cosenza), italy thanks to its geographical and climatic isolation, antarctica is the continent with the lowest local human impact, yet it is still vulnerable to contaminants from external sources. emerging pollutants, like pfas, pose an increasing threat to this environment and therefore require more in-depth investigations to understand their environmental fate and biological impacts. this research is part of the "antagps" project, funded by the pnra, which aims to make antarctica a global pollution sensor, using its endemic organisms as bioindicators. the project’s purpose is to study the antioxidant defence components of antarctic fish and how this system can be influenced, at the biomolecular level, by exposure to some chemical stress factors. all aerobic organisms have evolved metabolic strategies to reduce the toxicity of reactive oxygen species (ros), natural by-products of aerobic metabolism and continuously produced by cells, both at the cytoplasmic and mitochondrial levels. furthermore, several studies confirm the correlation between toxicity induced by xenobiotics and an increase in ros formation, with a consequent more significant risk of oxidative stress. for these reasons, variations in the content and the activity of antioxidant enzymes can be used as biomarkers for contaminant-mediated oxidative stress in several marine organisms. specifically, this research focuses on the expression analysis at the transcriptional level of genes coding for four antioxidant enzymes (sod1, sod2, gpx1, gpx4) in 2 different organs of an antarctic fish species, trematomus newnesi. the kidney showed a higher expression level than the liver of wildlife specimens for each antioxidant enzyme (but the most expressed is sod1). the mrna levels were assessed in fish exposed to 1.5 μg/l of pfoa for ten days. in the liver, the treatment induced an increase in gene expression for all the considered enzymes, while in the kidney, it induced a general decrease (especially for sod1). the obtained results can contribute significantly to the prediction of the physiological responses of these organisms to environmental changes, which can improve the general understanding of the molecular and functional evolution of antarctic fishes. furthermore, our gene expression analysis may provide the basis for using antioxidant enzymes as indicators of oxidative stress and pfoa exposure. main lecture ascidian cytotoxic cells: from zero to hero l ballarin department of biology, university of padua, padua, italy ascidian cytotoxic cells are multivacuolated cells, variable in morphology, abundantly represented in the circulation, playing important roles in ascidian immunosurveillance. the interest of researchers towards these cells arose three decades ago when it was realized that, in colonial species, they were the effectors of the rejection reaction between contacting, genetically incompatible colonies. indeed, they are the first cells to sense nonself and, upon the recognition of foreign molecules, are selectively recruited to the infection site where they release the content of their vacuoles. their cytotoxic activity is closely linked to the activity of the enzyme phenoloxidase (po), a copper-containing enzyme widely distributed in invertebrates, contained inside their vacuoles together with its polyphenol substrata. recent data indicate that ascidian cytotoxic cells synthesize and release the majority of the complement factors of both the alternative and lectin pathways. in addition, they are also the main source of antimicrobial peptides codified by the ascidian genome. therefore, these cells, once neglected, are now drawing the attention of researchers for their multiple roles in immune defense. 36 session 6.2. environmental challenges and the immune response. chairpersons: maria giovanna parisi university of palermo, palermo, italy and loriano ballarin, university of padua, padua, italy immune response of the ascidian ciona robusta to abiotic and biotic stressors d melillo1, r marino1,2, d boraschi1,2,3, p italiani1,2 1institute of biochemistry and cell biology, national research council of italy, naples, italy 2stazione zoologica anton dohrn, naples, italy 3shenzhen institute of advanced technology, chinese academy of science, china environmental changes provoke huge stresses on sessile marine organisms making them more or less susceptible to infections, and in turn organisms develop complex response mechanisms to keep homeostasis for surviving. the immune system of the solitary ascidian ciona robusta relies on two organs, the pharynx and the gut, and encompasses a wide array of immune and stress-related genes which are involved in the response to environmental challenges and modulated in adapting to changing conditions. recently, we have demonstrated that c. robusta immune response can be modulated after priming and challenging animals with microbial agents. the establishment of “immune memory” relies on the activation of different cellular and humoral immune mechanisms aimed to develop a more protective response. in the present study, we assessed the primary response in the gut and pharynx of c. robusta in terms of expression of several immune-related genes (c3-1, c3ar, il17-1, il17-2, il17r, tnf, tgfb, lbp, tlr-2, tlr13, cd36), pharynxand gut-specific genes involved in mucosal immunity (vcbp-b and vcpb-c) and oxidative stress-related genes (soda, gst, gr) upon exposure to hypoxia/starvation (h/s) for 2 or 18 h and in presence of polystyrene nanoplastics (nps) or not. we also evaluated how a previous exposure to abiotic stresses could influence the ability of c. robusta immune organs to react to a subsequent bacterial challenge (lps). our preliminary data suggest that i) the immune response to stress vary greatly between the two organs, ii) the presence of nps attenuate the gene expression induced by h/s in both organs, iii) animals previously exposed to h/s stress when challenged with lps shown a “tolerance” to lps and this h/s stress-induced memory response is only partially modulated by the presence of nps. in conclusion, while gut and pharynx differently react to abiotic and biotic stresses, the concurrence of abiotic stress seems to reduce the primary response in both organs, and nps only partially affect the h/s stress-dependent induction of innate memory. tyre wear particles effects on growth and some immune parameters in the terrestrial isopod armadillidium pallasii g torreggiani1, c manfrin1, a dissegna2, c chiandetti1, p giotta1, m renzi1, a babczyńska3, pg giulianini1 1department of life sciences university of trieste, trieste, italy 2center for mind/brain sciences (cimec), university of trento, italy 3faculty of natural sciences, university of silesia in katowice, poland about 1.3 million tons of tyre wear are generated on europe's roads each year and are probably the most important source of polymerbased plastics entering the environment. therefore, itis of particular interest to know the impact of this material on soil organisms living near busy roads (baensch-baltruschat et al., 2020). armadillidium pallasii (brandt, 1833) was collected in gradisca d'isonzo (gorizia), italy and grown for several months in containers with soil certified for organic farming, ph 7 (bioterril, geotec), in an experimental room with controlled photoperiod (12:12, light:dark) at a mean temperature of 25 °c. 75 a. pallasii (weight: 0.223±0.036 g, length: 17.6±0.9 mm) were evenly distributed in 15 glass terrariums (12x12 cm): 3 replicates for 4 different concentrations of 10%, 5%, 2.5% and 1.25% (tyre particles/soil, w/w) and 3 control replicates without tyre particles were tested for 30 days. the tyre dust was obtained in laboratory by abrasion of the tyre tread with a grinding stone. haemolymph from a. pallasii was collected from described by dolar et al. (2021). for determination of total haemocyte count (thc), one drop was placed in the buerker counting chamber and about 10 μl were centrifuged and immediately stored at -20 °c for analysis of phenoloxidase (po)like activity. after 30 days of treatment, animals in soil containing 5% and 10% tyre particles are significantly shorter (5%: 17.23±1.00 mm; 10%: 17.4±0.06 mm) than at the beginning of the experiment (5%: 17.60±0.76 mm; 18.20±0.04 mm). average weights decrease during the experiment, but no significant differences were found between animals raised in soils with different proportions of tyre particles. mean thc levels are highest in animals raised in soil containing 5% (5.44x106±3.14x106 haemocytes/ml) and 10% (4.36x106±1.87x106 haemocytes/ml), but no significant differences were observed. po activities of animals showed higher values in animals treated with soil containing 1.25%, 5% and 10% tyre particles, compared to the control animals but no significant differences were found. since the literature emphasises the stronger effect of tyre dusts together with road abrasion dusts, a second experiment was conducted to evaluate the effects of 37 these dusts collected in poland also on key behavioural abilities of isopods such as their locomotor activity, anxiety-like responses and a form of learning. evaluation of stress biomarkers in the european nightcrawler dendrobaena veneta exposed to pfas under controlled conditions e pietropoli1, s pacchini2, e piva2, s schumann2, g santovito2, p irato2 1department of comparative biomedicine and food science, university of padua, padua, italy 2department of biology, university of padua, padua, italy polyand perfluoroalkyl substances (pfass) are a broad family of synthetic compounds widely used by industries and subsequently released in the environment. several studies on the effects of pfas were carried out on animals. these studies showed the danger of these substances to the world community. to investigate the effects of pfass, an in vivo laboratory study was set up. the nightcrawler dendrobaena veneta has been used as a model organism for an in vivo exposure experiment under controlled laboratory conditions. d. veneta has been chosen as an experimental model since earthworms are recognised among the 5 key bioindicators for the ecotoxicological evaluation of persistent environmental pollutants. earthworms were exposed to three different concentrations of pfas mixtures: the concentrations applied resulted in average concentrations of soil samples in the veneto region. the results underlined that, in the short term (30 days), these compounds induce an increase in ros levels in the mitochondria with a consequent increase in genomic damage. the antioxidant system of these organisms consists mainly of low molecular mass antioxidant molecules such as gsh, metallothionein (mt) and vitamins. no significant differences in the total antioxidant capacity (tac) between the different treatments were shown, indicating the activation of the physiological stress response induced by xenobiotics. the oxidation of low molecular mass antioxidant molecules mainly drives the depletion of tac. however, there was a trend in the tac according to the increasing concentration of pfass. furthermore, it was observed that the genomic damage assessed by the comet test reported dna damage in the control group and the pfas-exposed groups. of course, laboratory conditions may affect the overall health assessment of organisms compared to in situ experiments. furthermore, it has been reported that exposure to pfass enhances both transcriptional and translational levels of mts, implying their possible involvement in response to oxidative stress in mitochondria. molluscs as a models for translational medicine 477 isj 14: 477-479, 2017 issn 1824-307x letter to editor lymnaea stagnalis as model of neuropsychiatric disorders f tascedda department of life sciences and center for neuroscience and neurotechnology university of modena and reggio emilia, modena, italy accepted november 14, 2017 to the editor this paper describes the advantages of adopting a molluscan model for studying the biological basis of some central nervous system pathologies affecting humans. in particular, i will focus on the freshwater snail lymnaea stagnalis, which is already the subject of electrophysiological studies related to learning and memory, as well as ecotoxicological studies (il-han et al., 2010; bavan et al., 2012; ivashkin et al., 2015; benatti et al., 2017; ito et al., 2017). understanding psychiatric and neurological disorders is a major medical and scientific challenge. these pathologies are extremely widespread and contribute in an important way to the high cost of public health (nestler et al., 2010; tascedda et al., 2015; kaiser et al., 2015). unfortunately, the developing or mature human brain are not open to direct molecular observation, and experimental manipulations are clearly not ethical. this makes it necessary and urgent to develop new and reliable animal models for the study of brain disorders. while the research community has accepted the value of rodent models for the study of human pathology and treatment, there is less awareness of the utility of other small vertebrate and invertebrate animal models. however, in recent years, the neuroscientists are increasingly turning to smaller, non-rodent models to understand molecular physiopathological mechanisms related to neurological or psychiatric disorders. although they can never replace clinical research, these species offer flexible genetic tools that can be useful to validate the function of specific genes, or their role in more complex functions. although, animal models, of any origin, size or complexity, can never summarize the full phenotype of a human clinical disorder, in particular neuropsychiatric ones, different small animals, such as, worms, flies, bees and fish offer new important and innovative tools for the neuroscientist. (burne et al., 2011; curran et al., 2012; tascedda et al., 2015). ___________________________________________________________________________ corresponding author: fabio tascedda department of life sciences university of modena and reggio emilia via campi 287, 41125 modena, italy e-mail: fabio.tascedda@unimore.it indeed, by using a range of models of different complexity, together with a number of different experimental approaches, researchers can divide complex phenotypes into simpler neurobiological correlates of clinical syndromes (nestler et al., 2010). as mentioned above, normally, for these studies, rats and mice have been used (vinet et al., 2003; vinet et al., 2004; blom et al., 2006; alboni et al., 2011; benatti et al., 2011), but this approach may not be always effective and is accompanied by many ethical and economical problems (tascedda et al., 2015). many researchers have attempted to solve the problem by using in vitro cell systems (alboni et al.; 2013, 2014; caraci et al., 2016) that have many important advantages (tascedda et al., 2015). unfortunately, the results are often inconclusive and fail to elucidate the basis of disease (alberts, 2010). given of these considerations, the need to identify alternative and reliable models that have fewer ethical restrictions is both important and urgent. invertebrates, thanks to their relatively simple nervous system structure are fast becoming a useful tool for the study of neuronal physiology and for better disease process characterization (kaang et al., 1993; ottaviani et al., 2013; tascedda et al., 2015). above all, molluscan gastropods, such as aplysia, hermissenda, limax, and so forth, have been widely recognized as useful animals with which to study the molecular and cellular mechanisms underlying complex human pathologies such as neurological and psychiatric disorders (kandel et al., 1965; gelperin 1975; nestler et al., 2010; burne et al., 2011). furthermore, numerous studies suggest that the pond snail l. stagnalis could be an innovative useful model in genetic and translational research for the study of the molecular basis of human brain diseases and for the development of new therapeutic strategies (tascedda et al., 2015, ito et al., 2017, benatti et al., 2017). l. stagnalis, an aquatic pulmonate gastropod with a central nervous system (cns) consisting of ≈20,000 neurons organized in a ring of interconnected ganglia, has proven to be an extremely interesting and accessible model to study 478 fundamental aspects of cns function such as synaptic plasticity and associative memory (sadamoto et al., 2004, 2010; ito et al., 2017). compared to d. melanogaster and c. elegans, lymnaea has many benefits due to the large size of its neurons, results from electrophysiological studies, and its already characterized neuronal circuit (andrianov et al., 2015). lymnaea also offers the possibility of performing behavioral tests [ito et al., 2017]. more importantly, while d. melanogaster and c. elegans have a life cycle of 2 3 weeks, lymnaea has an average life span of 9 12 months. this last factor becomes particularly interesting and useful in studies on chronic human pathologies, especially neurodegenerative diseases such as alzheimer's, parkinson's or chronic psychiatric diseases such as major depression, schizophrenia or bipolar disorder. in this context, l. stagnalis offers, to the neuroscientist involved in translational medicine, a powerful new tool to study neuropsychiatric diseases and allow the identification of new molecular targets for the development of innovative therapeutic strategies. using an interdisciplinary approach, studying the appropriate animal model, passing from the more simple ones to the most complex, while combining different methods and expertise that include fields as evolution, ecology, and life history theory with physiology, pathology, neuroscience, genetics, molecular biology, and ultimately behaviour it will be possible to open new 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